MXPA01002020A - Modulating multiple lineage kinase proteins - Google Patents

Abstract

Methods for identifying compoundswhich modulate activity of a multiple lineage kinase protein and promotes cell survival or cell death comprising the steps of contacting the cell containing the multiple lineage kinase protein with the compound, determining whether the compound decreases activity of the multiple lineage kinase protein, and determining whether the compound promotes cell survival are provided. Methods for identifying compounds which may be useful in the treatment of neurodegenerative disorders and/or inflammation are also provided. Methods for modulating the activity of a multiple lineage kinase protein comprising contacting the protein or a cell containing the protein with an indeno- or indolo- compound of the invention are also provided. Methods of treating neurodegenerative disorders and/or inflammation are also provided.

Description

PROTEINS OF MULTIPLE MODELING LINE FIELD OF THE INVENTION The present invention is directed, in part, to methods for modulating members of the multiple lineage kinase (MLK) family, methods for identifying compounds that modulate a multiple lineage protein and both promote cell survival and promote cell death, methods for identifying compounds that identify which may be useful in the treatment of neurodegenerative disorders and / or inflammation, and methods for treating neurodegenerative disorders with compounds that inhibit a multiple lineage kinase protein.

BACKGROUND OF THE INVENTION The MLK family comprises a group of proteins in which the protein sequence of the kinase domains of the family members closely resemble the MAPKKKs, but they have a greater similarity to each other than to other MAPKKKs. Members of the MLK family comprise a portion of very complex kinase cascades such as, for example, the stress signaling cascade, which involves modulation, inter alia, of the kinase c-Jun terminates (JNK), which at its it modulates, among other things, transcription factors including c-Jun, ATF2, and ELK-1. JNK is described in the patents of US Pat. No. 5,534,426, 5,593,884, 5,605,808 and WO 95/03324, each of which is incorporated herein by reference in its entirety. The MLK family includes, in part, the following groups: 1) multiple lineage kinase 1 (MLK1); 2) multiple lineage kinase 2 (MLK2); 3) multiple lineage kinase 3 (MLK3); 4) leucine zipper carrier kinase (LZK); 5) kinase carrying double leucine closure (DLK); and 6) multiple lineage kinase 6 (MLK6). MLK1 has a catalytic domain similar to both kinases specific for Tyr and Ser / Thr. Dorow, et al., Eur. J. Biochem., 1993, 213, 701-710. MLK2 also has a catalytic domain similar to both kinases specific for Tyr or Ser / Thr. Dorow, et al., Eur. J. Biochem, 1993, 213, 701-710. Also MLK2 is known as MST. Katoh et al., Oncogene, 1995, 10, 1447-1451. MLK3 comprises a protein which, in addition to the kinase domain, contains 2 leucine seals with an adjacent carboxy-terminal base region, and a proline-rich region. Ing, and others, Oncogene, 1994, 9, 1745-1750. The MLK3 is also known as SPRK. (Gallo et al., J. Biol. Chem., 1994, 269, 15092-15100), and PTK1 (Ezoe, et al., Oncogene, 1994, 9, 935-938). LZK is a kinase that carries a leucine closure. Sakuma, et al., J. Biol. Chem., 1997, 272, 28622-28629. DLK has a kinase domain and two putative leucine closure motifs. Holzman, et al., J. Biol. Chem., 1994, 269, 30808-30817. DLK is also known as ZPK (Reddy et al., Biochem Biophys, Res. Comm., 1994, 202, 613-620) and MUK (Hirai et al., Oncogene, 1996, 12, 641-650). Members of the MLK family are also described in, for example, the patents of U.A. 5,676,945, 5,554,523, WO 93/15201, Canadian Patent 2,148,898, Diener, et al., Proc. Nati Acad. Sci. USA, 1997, 94, 9687-9692, DeAizpurua, et al., J. Biol. Chem., 1997, 272, 16364-16373, Tung, et al., Oncogene, 1997, 14, 654-659, Sells, and others, Trends in Cell Biol., 1997, 7, 161-167, Mata, et al., J. Biol. Chem., 1996, 271, 16888-16896, and others, J. Biol. Chem., 1997, 272, 15167-15173, Fan, et al., J. Biol. Chem., 1996, 271, 24788-24793, Blouin, et al., DNA and Cell Biol., 1996, 15, 631-643, Pombo, et al., Nature, 1995, 377, 750-754, Kiefer, et al., EMBO J., 1996, 15, 7013-7025, Hu, et al., Genes & Dev., 1996, 10, 2251-2264, Su, et al., EMBO J., 1997, 16, 1279-1290, and Dorow, et al., Eur. J. Biochem., 1995, 234, 492-500. Recently, another MLK-related kinase was identified in the EST database. The DNA sequence of this clone, MLK6, is described by seven overlapping entries. Their clone identification numbers are: 1007489, 1460085, 510915, 666323, F5555, 482188 and 178522, the sequences of each of which is incorporated herein by reference in its entirety. Each of the references cited in the present paragraph is incorporated herein by reference in its entirety. Recently, stable expression of ZPK has been shown to reduce the proliferative capacity of NIH 3T3 fibroblasts as measured by a colony formation assay. Bergeron and others, Biochem. Biophys. Res. Comm., 1997, 231, 153-155. However, Bergeron and others failed to provide data showing that ZPK modulated the activity of a ZPK substrate or whether ZPK promoted cell death. It has been shown that the expression of a construct encoding Myc-MLK2 in Swiss 3T3 cells leads to apoptosis approximately 20 hours after injection. Nagata et al., EMBO J., 1998, 17, 149-158. Applicants have developed numerous indole and indene compounds which, among other things, inhibit cell growth associated with hyperproliferative states and inhibit death in a variety of embryonic cultures, such as dorsal root ganglia, upper cervical, striatal ganglia, and motor neurons. U.S. Patents 5,475,110, 5,591,855, 5,594,009, 5,461,146, 5,621,100, 6,621,101, 5,705,511 and 5,756,494, each of which is assigned to the assignee of the present application, and each of which is hereby incorporated by reference in its entirety. The compounds illustrated in US patent 5,705,511 having the formula G are referred to in the present application as having the formula I. Applicants have also shown that motor neuron apoptosis is inhibited by a K-252a derivative, an indolocarbazole which also modulates the voltage signaling cascade. Maroney et al., J. Neurosci., 1998, 18, 104-111, which is incorporated herein by reference in its entirety. Due to the inadequacies of classification of compounds that modulate members of the voltage signaling cascade and promote both cell death and cell survival, it continues to be a need to have new selective methods for classifying compounds. In addition, classification trials for therapeutic aspects that may be useful in the treatment of inflammation and neurodegenerative disorders remain a necessity. The present invention is directed to these, as well as to other important purposes.

COMPENDIUM OF THE INVENTION The present invention provides methods for identifying compounds that modulate the activity of a multiple lineage kinase protein and promote cell survival, comprising the steps of contacting the cell containing the multiple lineage kinase protein with the compound, determining whether the compound decreases the activity of the multiple lineage kinase protein, and determining whether the compound promotes cell survival. The present invention also provides methods for identifying compounds that modulate the activity of a multiple lineage kinase protein and promote cell death, comprising the steps of contacting the cell containing the multiple lineage kinase protein with the compound , determining whether the compound increases the activity of the multiple lineage kinase protein and determining whether the compound promotes cell death. The present invention also provides methods for identifying compounds that may be useful in the treatment of neurodegenerative disorders, comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound, and determining whether the compound decreases the activity of the multiple lineage kinase protein. The present invention also provides methods for identifying compounds that may be useful in the treatment of inflammation, comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound, and determining whether the compound decreases activity. of the multiple lineage kinase protein. The present invention also provides methods for the treatment of a mammal having or suspected of having a neurodegenerative disorder, comprising administering to said mammal a compound that inhibits or reduces the activity of multiple lineage kinase protein. The present invention also provides methods for treating a mammal having inflammation, comprising administering to said mammal a compound that inhibits or reduces the activity of multiple lineage kinase protein. The present invention also provides methods for modulating the activity of a multiple lineage kinase protein, comprising contacting the protein to a cell containing the protein with a compound having the formula II: p wherein: ring B and ring F, independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: an unsaturated 6-membered carbocyclic aromatic ring wherein from 1 to 3 carbon atoms can be replaced by nitrogen atoms; an unsaturated 5-membered carbocyclic aromatic ring; and an unsaturated 5-membered carbocyclic aromatic ring wherein: a carbon atom is replaced with an oxygen, nitrogen or sulfur atom; two carbon atoms are replaced with one atom of sulfur and one of nitrogen, one atom of oxygen and one of nitrogen, or. three carbon atoms are replaced with three nitrogen atoms; R1 is selected from the group consisting of: H, substituted or unsubstituted alkyl having from 1 to 4 carbons, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl; -C (= O) R9, wherein R9 is selected from the group consisting of alkyl, aryl and heteroaryl; -OR10, wherein R10 is selected from the group consisting of H and alkyl having from 1 to 4 carbons; -C (= O) NH2), -NR11R12, - (CH2) PNR11R12, - (CH2) pOR10, -O (CH2) pOR10 and -O (CH2) pNR11R12, wherein p is from 1 to 4; and wherein either: R11 and R12 each independently is selected from the group consisting of H and alkyl having from 1 to 4 carbons; or R11 and R12 together form a linking group of the formula - (CH2) 2-X1- (CH2) 2-, wherein X1 is selected from the group consisting of -O-, -S-, and -CH2-; R2 is selected from the group consisting of H, alkyl having 1 to 4 carbons, -OH, alkoxy having 1 to 4 carbons, -OC (= O) R9, -OC (= O) NR11R12, -O (CH2 ) pNR11R12, -O (CH2) pOR10, substituted or unsubstituted arylalkyl having 6 to 10 carbons, and substituted or unsubstituted heteroarylalkyl; R3, R4, R5 and R6 are each independently selected from the group consisting of: H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF3, -NO2, -OH, -OR9, -O (CH2 PNR11R12, -OC (= O) R9, -OC (= O) NR2R7, -OC (= 0) NR11R12, -O (CH2) pOR10, -CH2OR10 ,. -NR11R12, -NR10S (= O) 2R9, -NR10C (= O) R9; -CH2OR1, wherein R14 is the residue of an amino acid after the hydroxy group of the carboxyl group is removed; -NR 0C (= O) NR 11 R 12, -CO 2 R 2, -C (= O) R 2, -C (= O) NR 11 R 12, -CH = NOR2, -CH = NR9, - (CH2) PNR11 R12, - (CH2) PNHR14, or -CH = NNR2R2A, wherein R2A is the same as R2; -S (O) and R 2 - (CH 2) pS (O) and R 9, -CH 2 S (O) and R 14, wherein y is 0, 1 or 2; alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons, and alkynyl having from 2 to 8 carbons, wherein: each alkyl, alkenyl or alkynyl group is unsubstituted; or each alkyl, alkenyl or alkynyl group is substituted with 1 to 3 groups selected from the group consisting of aryl having 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy hydroxy alkoxy, alkyloxy alkoxy, hydroxyalkylthio, alkoxy alkylthio, F, Cl Br , I, -CN, -NO2, -OH, -OR9, -X2 (CH2) PNR11 R12 -X2 (CH2) pC (= O) NR11R12, -X2 (CH2) pOC (= O) NR11R12, -X2 (CH2) pCO2R9 -X2 (CH2) pS (O) and R9, -X2 (CH2) PNR10C (= O ) NR 11 R 12, OC (=) R 9, OCONHR 2 -O-tetrahydropyranyl, -NR 11 R 12, -NR 10 C (= O) R 9, -NR 10 CO 2 R 9 -NR 10 C (= O) NR 11 R 12, -NHC (= NH) NH 2, NR 10 S (O) 2 R 9, -S (O) and R9, -CO2R2 -C (= 0) NR11R12, -C (= O) R2, -CH2OR10, -CH = NNR2R2A, -CH = NOR2, -CH = NR9, -CH = NNHCH (N = NH) NH2, -S (= O) 2NR2R2A, -P (= O) (OR10) 2, -OR14, and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently either unsubstituted or it is replaced by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons; X2 is O, S, or NR10; R7 and R8 are each independently selected from the group consisting of H, alkyl having 1 to 4 carbons, alkoxy having 1 to 4 carbons, substituted or unsubstituted arylalkyl having 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl , - (CH2) PORlu, - (CH2) pOC (= 0) NR R1¿, and - (CH2) PNR? 1 '11 D R12.

R7 and R8 together form a linking group of the formula -CH2-X3-CH2- wherein X3 is X2 or a bond; m and n each is independently 0, 1 or 2; Y is selected from the group consisting of -O-, -S-, -N (R10) -, -N + (O -) (R10) -, -N (OR10) -, and -CH2-; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, -C (= O) -, -CH (OR10) -, -N (R10) -, -N ( OR10) -, CH (NR11R12) -, -C (= O) N (R17) -, -N (R17) C (= O) -, N (S (O) and R9) -N (S (O) and NR 11 R 12) -, -N (C (= O) R 17) -, -C (R 15 R 16) -, -N + (O ') (R 10) -, -CH (OH) -CH ( OH) -, and -CH (O (C = O) R9) CH (OC (= O) R9A) -, where R9A is the same as R9; R 15 and R 16 are independently selected from the group consisting of H, -OH, -C (= O) R 10, -O (C = O) R 9, hydroxyalkyl and -CO 2 R 10; R17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl; A1 and A2 are selected from the group consisting of H, H; H, OR2; H, -SR2; H, -N (R2) 2; and a group wherein A1 and A2 together form a portion selected from the group consisting of = O, = S, y = NR2; B1 and B2 are selected from the group consisting of H, H; H, -OR2; H, -SR2; H, -N (R) 2; and a group wherein B1 and B2 together form a selected portion of the group consisting of = O, = S, y = NR2; provided that at least one of the pairs of A1 and A2, or B1 and B2, forms = O. The present invention also provides methods for modulating the activity of a multiple lineage kinase protein, comprising contacting the protein or a cell containing the protein with a compound having the formula III: m where: Zi is H and Z2 is H or Zi and Z2 together form = O; Ri is selected from the group consisting of H, Cl, CH2SO2C2H5, Br, CH2S (CH2) 2NH2, CH2S (CH2) 2N (CH3) 2, CH2S (CH2) 2NH2 n-C4H9, NHCOCHC6H5, NHCONHC2H5, CH2SC2H5, CH2SC6H5, N (CH3) 2, CH3, CH2OCONHC2H5, NHCO2CH3, CH2OC2H5, CH2N (CH3) 2, OH, On-propyl, CH = NNH-C (= NH) NH2, CH = NN (CH3) 2, CH2S (CH2) 2NH- n-C4H9, CH2OCH2OCH2CH3, CH2S [3- (1, 2,4-triazine)], CH2CH2SCH3; R2 is selected from the group consisting of H, Br, Cl, I, CH2S (CH2) 2N (CH3) 2, NHCONHC2H5, CH2SC2H5, CH2OCH2OCH2CH3, CH2S [3- (1,2,4-triazine)], CH2CH2SCH3, and CH2OH; X is selected from the group consisting of H, CH2OH, CH2NH-serineH, CO2CH3, CONHC6H5, CH2NHCO2C6H5, CH2NHCO2CH3, CH2N3, CONHC2H5, CH2N H-glycine, CON (CH3) 2, -CH2NHC02-, CONH2, CONHC3H7, CH2NH-serine , CH2SOCH3, CH = NOH, CH2NH-proline, CH2CH2 (2-pyridyl), CH = NNHC (= NH) NH2, CONH (CH2) 2OH, CH = NNHCONH2, CH2OCOCH3, -CH2OC (CH3) 2O-, CH2SC6H5, CH2SOC6H5, CO2n-hexyl, CONHCH3, and CO2 (CH2) 4CH3; or one of the following formulas: R is selected from the group consisting of OH, and OCH3. The present invention also provides methods for modulating the activity of a multiple lineage kinase protein, comprising contacting the protein or a cell containing the protein with a compound having the formula IV: rv where: Zi is H and Z2 is H or Z, and Z2 together form = O; Ri is H or Br; R2 is H; R3 is H, CH2CH = CH2, CH2CH2CH2OH, R4 is H, CH2CH = CH2 or CH2CH2CH2OH.

BRIEF DESCRIPTION OF THE DRAWINGS For the purposes of illustrating the embodiments of the present invention certain features are shown in the drawings. However, it should be understood that this invention is not limited to the precise embodiments shown. Figure 1 is a schematic drawing showing a general preparation of bridged indenopyrrolocarbazoles. Figure 2 is a schematic drawing showing a general preparation of bridged indenopyrrolocarbazoles. Figure 3 is a schematic drawing showing a general preparation of bridged indenopyrrolocarbazoles. Figure 4 is a schematic drawing showing a general preparation of bridged indenopyrrolocarbazoles. Figure 5 is a schematic drawing showing the preparation of intermediate V. Figure 6 is a schematic drawing showing the preparation of bridged indenopyrrolocarbazoles using method A. Figure 7 is a schematic drawing showing the preparation of bridged indenopyrrolocarbazoles using the method B. Figure 8 is a schematic drawing showing the preparation of bridged indenopyrrolocarbazoles bridged with ring B. Figure 9 is a schematic drawing showing the derivatization of the ring E of bridged ndenopyrrolocarbazoles.

Figure 10 shows a graph of two separate experiments illustrating the number of neuronally viable, differentiated PC-12 cells, remaining after 5 days of culture in the absence of NGF. The results are expressed as a percentage of the control of NGF within each group (vector control in the absence of NGF, n = 12, all other groups, n = 3). The difference between vector control and stable combinations of cells expressing a dominant negative MLK-3 mutant in the absence of NGF is statistically significant as determined by a two-sided T test (p <0.05). Figure 11A shows phosphorylation of GST-SEK-1 from killed kinase through FLAG-MLK-3 expressed by baculovirus (mixture of the full-length domain and kinase) using a radioactive gel-based assay. Figure 11B shows the basic protein product of 32P-labeled phosphorylated melanin formed as a result of a kinase reaction catalyzed by FLAG-MLK-3 expressed by baculovirus (mixture of full-length domain and kinase) or GST-domain. MLK-3 kinase. Figure 12 is an immunostaining analysis showing the phosphorylation of GST-SEK-1 from dead kinase through FLAG-MLK-3 expressed in baculovirus (mixture of full-length domain and kinase) as detected by a SEK-1 antibody phospho-specific. Figure 13 shows the phosphorylation of myelin basic protein through the bacterially expressed GST-MLK-3 kinase domain using (or) the multiple-screen trichloroacetic acid precipitation assay, or the (*) phosphocellulose membrane method. Figure 14 shows a saturation binding curve of [3H] K252a incubated with lysate from insect cells infected with the MLK-3 baculovirus. Figure 15A shows the amount of 32 P-labeled c-jun in an immunoprecipitation / kinase reaction from cells overexpressing MLK-3, MLK-2 or DLK and treated with either 0.025% DMSO (control) or 500 nM of K-252a. Figure 15B shows a graph quantifying the percentage of activity remaining in immunoprecipitate / kinase reactions of samples described in Figure 15A. The columns represent the average of samples in duplicate, where the error bar indicates the scale of the mean. Figure 15C shows the amount of 32 P-labeled c-jun in an immunoprecipitation / kinase reaction from cells overexpressing HA-JNK1 alone or with MEKK1 at varying amounts of cDNA as indicated and treated with either 0.025% ) of DMSO (control) or 500 nm of compound III-3 (see Table 3). The columns represent the average of samples in duplicate, where the error bar indicates the scale of the mean. Figure 16 shows that compound III-3 promotes neuronal survival in a concentration-dependent manner. The dissociated neurons were calculated from sympathetic ganglia (SG) (A), dorsal root ganglia (DRG) (B), ciliary ganglia (CG) (C), and motor neurons (MN) (D), in the presence or absence of indicated trophic factors. Cells were counted 48 hours after plating as described in materials and methods. The data represent +. SD average of determinations in triplicate or in quadruplicate. One of the three experiments is shown. Figure 17 shows microphotographs of phase contrast of cultures E12 DRG (A, E), sympathetic E9 (B, F), ciliary neurons E8 (C, G) and motor neurons E5.5 (D, H) after 48 hours in culture (24 hours for ciliary neurons, in the presence of the respective neurotrophic factor (20 ng / ml of NGF for sympathetic and sensory neurons, 10 ng / ml of CNTF for ciliary neurons, 30 μg / ml of muscle extract (MEX) for motoneurons (AD) or in the presence of 1 μM of compound III-3 (EH) Bar = 200 μm Figure 18 shows a photomicrograph of in vitro dorsal root ganglia explants The explants of DRG (E9) chickens were placed on plates in a medium with 96-well plates containing 0.05% BSA After a 2-hour binding period, additions were made: (A) DMSO control; (B) 20 ng / ml of NGF; (C) 250 nM of Compound II-3. Forty-eight hours later the medium was removed and the explants were fixed with 4% parafolmadehyde in saline regulated at its pH with phosphate. Figure 19 shows the number of chicken lumbar motor neurons that survive in E10 after daily treatment (E5-9) with the specific doses of compound III-3. The data presented are + _ S.D. average of 5-6 animals / treatment group. The reported experiment was repeated twice. The data is from a representative experiment and represents one side of the lumbar spine. * p < 0.01, ** p < 0.001, Student t test between compound lli-3 and control groups with Bonferroni correction. Figure 20 shows the number of motoneurons in the female rat spinal nucleus of the cavernous bulb (SNB) surviving in PN10 or PN60 after daily treatment (PN1-5) with compound III-3, or control vehicle (5% of Solutol ™). In PN10 (A, B) or PN 60 (B), the rats were sacrificed and the region of the spinal cord containing SNB was separated and processed for hydrology; the motoneurons stained with Cresylecht violet were then counted and then counted in serial section of the 5-sacral 1 spinal cord lumbar region as described previously (Wingfield, et al., Steroids, 1075, 26, 311-327). The experimental data are + .S.E.M. of 4-8 animals / treatment group. Figure 21 shows the loss of ChAT immunoreactivity after hypoglossal axotomy in the adult rat after treatment with compound III-3. Photomicrographs of the hypoglossal nucleus after transection of the hypoglossal nerve and treatment with (a) vehicle solution alone (5% Soluto ™) and (B) 200 μg of compound III-3 applied at the transection site. (C) number of immunoreactive hypoglossal motoneurons ChAT after treatments described in (A) and (B) above. The results are expressed as a percentage of immunoreactive motoneurons with ChAT with 100% defined as that number of immunoreactive motor neurons ChAT in the contralateral hypoglossal nucleus, untreated. Figure 22 shows inhibition of the MLK-3 pathway demonstrating in vivo efficacy and blocking of downstream phosphorylation events. Figure 22A shows an increase in immunoglobulin tyrosine hydroxylase neurons of substantia nigra after MPTP injury after the routine administration of compound III-3. Figure 2-2B is a representative immunostaining showing increases induced by MPTP at levels of phosphorylated MKK4. Figure 22C illustrates a representative immunostaining and ELISA showing the attenuation of phosphorylated MKK4 with induced MPTP in the presence of compound III-3. Figure 23 shows the induction of IL-2 in Jurkat cells.

Figure 23A shows the time course of IL-2 induction. Figure 23B shows the inhibition of the induction of IL-2 by the compound MI-3. Figure 23C shows the inhibition of IL-2 induction through compound III-3 and compound I-4.

DETAILED DESCRIPTION OF THE INVENTION As used above and through the description, the following terms, unless otherwise indicated, should be understood as having the following meanings. "Apoptosis" refers to a specific morphological form of cell death characterized by fragmentation of cells and their nuclei to membrane bound particles. Apoptosis can be presented as an objective by, for example, treatment with apoptosis-inducing compounds such as etoposide, staurosporine, tumor necrosis factor, ceramide, and the like, or through conditions such as x irradiation. The term "cell death" refers to the death of cells through apoptosis, necrotic means or other means well known to those skilled in the art. "Cell death" can be characterized, for example, as a decrease in the total cell numbers of the cells or a decrease in cell viability compared to populations of untreated control cells. Compounds that "promote cell death" result in a decrease in cell numbers or a decrease in cell viability compared to control populations. In contrast, compounds that "promote cell survival" result in an increase in cell numbers or cell viability, or that reduce or decrease the rate of cell death.

The terms "selectively reacts" or "specifically binds" describe compounds that physically or chemically interact directly with the MLK protein. In contrast, compounds that do not "selectively react" or "bind specifically" can affect proteins downstream or upstream of the MLK protein, and thus can affect the activity of MLK proteins, but not physically or chemically interact in direct form with an MLK protein. The term "modulates" refers to increasing or decreasing an activity of a particular protein or its substrate. The present invention is directed, in part, to methods for identifying compounds that modulate the activity of an MLK protein and promote either cell survival or cell death. Compounds that result in increased MLK protein activity can promote cell death, whereas compounds that result in reduced MLK protein activity can promote cell survival. The MLK protein can be any protein identified as belonging to the MLK protein class. Preferably, the MLK protein is selected from the group consisting of MLK1, MLK2, MLK3 (SPRK, PTK1), LZK, DLK (ZPK, MUK), and MLK6, which were described above. In preferred embodiments of the invention, the methods identify compounds that directly interact or bind to the MLK protein as determined by binding assays, kinase assays, or other equivalent assays. In order to identify compounds that modulate the activity of MLK protein and promote cell survival beyond the cell, a cell or cells containing the M LK protein contacts the test compound. . Contact can occur in pH regulators or media well known to those skilled in the art. Alternatively, the contact can be presented in vivo, in an animal, such as, for example, a mouse or other suitable animal known to those skilled in the art, is contacted by administering a pharmaceutical composition comprising the compound of test and a pharmaceutically acceptable salt, carrier or diluent. further, variable numbers of cells and concentrations of test compounds can be used. It is determined whether the test compound increases or decreases the activity of the MLK protein. In addition, it is also determined whether the test compound promotes cell survival or cell death. The cells that are contacted with the test compounds can be any mammalian cell. Preferably, the cell is a neuronal cell. Preferably, the cell is involved in a neurodegenerative disease. For purposes of the present invention, a "neurodegenerative disease", a "neurodegenerative disorder" and a "neurodegenerative condition" are interchangeable and are used to describe any disease or disorder involving neuronal cells or cells involved in the neuronal system, including, but not limited to, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntington's disease, and disturbance or penetration damage to the brain or spinal cord. The activity of the MLK protein can be determined through a number of techniques, for example, the MLK activity can be determined by measuring the activity of a substrate of the MLK protein. Such substrates are well known and readily discernible by those skilled in the art. Preferably, the substrate is a member of the mitogen-activated protein kinase kinase family or the mitogen-activated protein kinase family or additional substrates that are current under the path which include, but are not limited to, a protein selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3, and also general substrates of Ser / Thr protein kinases such as myelin basic protein (MBP). Reagents and methods for measuring the activity of the substrates are also known to those skilled in the art. The presence of MLK can also be determined by measuring the amount of the MLK protein or mRNA that encodes the MLK protein. Reagents, including antibodies and oligonucleotide probes, as well as methods for measuring the amount of DNA or protein, including Northern and Western stains, are well known to those skilled in the art. The activity of the MLK protein can also be determined through an in vitro kinase assay. In vitro kinase assays are well known to those skilled in the art. Other techniques for measuring protein activity are known to those skilled in the art and are intended to be covered by the present invention. In this way, one skilled in the art can determine if the test compound modulates, ie, increases or decreases the MLK protein activity. In different ways it can be determined whether or not the test compound promotes cell survival or cell death. Preferably, the promotion of cell survival or cell death is determined using cells at risk of dying and comparing the number of cells that were contacted with the test compound and remain alive with the amount of cells that were not placed in the cell. contact with the test compound and remain alive. Preferably, the cells are primary embryonic motor neurons, which are pre-programmed to die. Primary embryonic motor neuron cells are described by Maroney et al., J. Neurosci., 1998, 18, 104-111, which is incorporated herein by reference in its entirety. Primary embryonic motor neuron cells will die unless rescued by the test compound. Thus, a large number of living motor neuron cells in the motor neuron cell population treated with the test compound as compared to the number of motor neuron cells in the population of motor neuron cells that were not treated with the test compound is indicative of a test compound that promotes cell survival. By contrast, a smaller number of living motor neuron cells in the population of motor neuron cells treated with the test compound as compared to the number of living motor neuron cells in the population of motor neuron cells that were not treated with the test compound is indicative of a test compound that promotes cell death. In another embodiment of the invention, normal cells, or wild-type cells, are converted to cells that are at risk of dying by expressing the MLK protein as described below in the examples, and then contacting the test compound Cells that overexpress MLK proteins can die unless they are rescued by the test compound. Over-expression of MLK protein can be achieved by using vectors capable of expressing the particular protein within a cell. Expression vectors are well known to those skilled in the art. In addition, methods for preparing expression vectors are also well known to those skilled in the art. Expression vectors expressing any of the MLK proteins can be prepared in a manner similar to that described in the examples. In contrast, a smaller number of living cells in the over expression cell population treated with the test compound as compared to the number of living cells in the over expression cell population that were not treated with the test compound is indicative of a test compound that promotes cell death. In another embodiment of the invention, the promotion of cell survival is determined observed or by measuring a reduction in apoptosis. Cytoplasmic shrinkage and nuclear condensation are associated with apoptosis. In this way, a person skilled in the art can measure a decrease in apoptosis by measuring or observing a reduction in cytoplasmic shrinkage and / or nuclear condensation. In addition, one skilled in the art can measure apoptosis using conventional staining techniques. In other embodiments of the invention, normal wild type neuronal cells can be used to identify compounds that promote cell death. Normal neuronal cells will survive unless they are induced to die by the test compound. A smaller number of living cells in the population of normal cells treated with the test compound as compared to the number of living cells in the population of normal cells that were not treated with the test compound is indicative of a test compound that promotes cell death In contrast, a greater or equal number of living cells in the population of normal cells treated with the test compound as compared to the number of living cells in the population of normal cells that were not treated with the test compound is not indicative of a test compound that promotes cell death. The present invention is also directed, in part, to method for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having the formula G (denoted herein as a formula) I) set forth in US Patent 5,705,511, which is assigned to the assignee of the present application and is hereby incorporated by reference in its entirety. The present invention is also directed, in part, to methods for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having the formula III which is presented below: m where: Zt is H and Z2 is H or Z, and Z2 together form = O; Ri is selected from the group consisting of H, Cl, CH2SO2C2H5, Br, CH2S (CH2) 2NH2, CH2S (CH2) 2N (CH3) 2, CH2S (CH2) 2NH2 n-C4H9, NHCOCHC6H5, CH2SC2H5, CH2SC6H5, N (CH3 ) 2, CH3, CH2OCONHC2H5, NHCO2CH3, CH2OC2H5, CH2N (CH3) 2, OH, On-propyl, CH = NNH-C (= NH) NH2, CH = NN (CH3) 2, CH2S (CH2) 2NH-n- C4H9, CH2OCH2OCH2CH3, CH2S [3- (1, 2,4-triazine)], CH2CH2SCH3; R2 is selected from the group consisting of H, Br, Cl, I, CH2S (CH2) 2N (CH3) 2, NHCONHC2H5, CH2SC2H5, CH2OCH2OCH2CH3, CH2S [3- (1,2,4-triazine)], CH2CH2SCH3, and CH2OH; X is selected from the group consisting of H, CH2OH, CH2NH-serineH, C02CH3, CONHC6H5, CH2NHCO2C6H5, CH2NHCO2CH3, CH2N3, CONHC2H5, CH2NH-glycine, CON (CH3) 2, -CH2NHCO2-, CONH2, CONHC3H7, CH2NH-serine, CH2SOCH3, CH = NOH, CH2NH-proline, CH2CH2 (2-pyridyl), CH = NNHC (= NH) NH2, CONH (CH2) 2OH, CH = NNHCONH2, CH2OCOCH3, -CH2OC (CH3) 2O-, CH2SC6H5, CHzSOCeHs, CO2n-hexyl, CONHCH3, and CO2 (CH2) 4CH3; or one of the following formulas: R is selected from the group consisting of OH, and OCH3. In preferred embodiments of the invention, Z and Z2 are H, X is CO2CH3, R1 is NHCONHC2H5, R2 is CH2CH2 (2-pyridyl), and R is OH. In other preferred embodiments of the invention, Z and Z2 are H, X is CO2CH3; R and R2 are CH2OCH2OCH2CH3, and R is OH; or Z, and Z2 are H, X is CO2CH3, R and R2 are CH2SCH2CH3, and R is OH; or Z Z2, R1t and R2 are H, X is CO2CH3; and R is OH; or Z ,, Z2, R ,, and R2 are H, X is CO2 (CH2) 4CH3, and R is OH; or Z Z2, and R1, are H, R2 is CH2OH, X is CO2CH3, and R is OH; or Z1t and Z2 are H, R-, and R2 are H2S [3- (1, 2,4-triazine)], X is CO2CH3, and R is OH; or Z ,, and Z2 are H, R ^ is Br, R2 is I, X is CO2CH3; and R is OH; or Z ,, and Z2 are H, R, and R2 are CH2CH2SCH3, X is CO2CH3, and R is OH; or Z Z2, R1f and R2 are H, X is CO2CH3, and R is OCH3; or Z, and Z2 together form = O, R-y and R2 are Br, X is CO2CH3 and R is OH. The present invention is also directed, in part, to methods for modulating the activity of an MLK protein, comprising contacting the protein or a cell containing the protein, with a compound having the formula II presented below : p wherein: ring B and ring F, independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring where 1 at 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring wherein: 1) any carbon atom is replaced with an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced with one atom of sulfur and one of nitrogen, one atom of oxygen and one of nitrogen, or two atoms of nitrogen; or 3) three carbon atoms are replaced with three nitrogen atoms; R1 is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbons, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl; b) -C (= O) R9, wherein R9 is selected from the group consisting of alkyl, aryl and heteroaryl; c) -OR10, wherein R10 is selected from the group consisting of H and alkyl having from 1 to 4 carbons; d) -C (= O) NH 2), -NR 11 R 12, - (CH 2) PNR 11 R 12, - (CH 2) p OR 10, -O (CH 2) POR 10 and -O (CH 2) p NR 11 R 12, wherein p is from 1 to 4; and wherein either: 1) R11 and R12 each independently is selected from the group consisting of H and alkyl having from 1 to 4 carbons; or 2) R11 and R12 together form a linking group of the formula - (CH2) 2-X1- (CH2) 2-, wherein X1 is selected from the group consisting of -O-, -S-, and -CH2 -; R2 is selected from the group consisting of H, alkyl having 1 to 4 carbons, -OH, alkoxy having 1 to 4 carbons, -OC (= O) R9, -OC (= O) NR11R12, -O (CH2 ) pNR11 R12, -O (CH2) pOR10, substituted or unsubstituted arylalkyl having 6 to 10 carbons, and substituted or unsubstituted heteroarylalkyl; R3, R4, R5 and R6 are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF3, -NO2, -OH, -OR9, -O (CH2) PNR11R12, -OC (= O) R9, -OC (= O) NR2R7, -OC (= O) NR 11 R 12, -O (CH 2) POR 10, -CH 2 OR 10, -NR 11 R 12, -NR 10 S (= O) 2 R 9, -NR 10 C (= O) R 9; b) CH2OR 14? wherein R is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; c) -NR10C (= O) NR11R12, -CO2R2, -C (= O) R2, -C (= O) NR11R12, -CH = NOR2, -CH = NR9, - (CH2) PNR11 R12, - (CH2) PNHR14, or -CH = NNR2R2A, wherein R2A is the same as R2; d) -S (O) and Rz- (CH2) pS (O) and Rs, -CH2S (O) and R14, wherein y is 0, 1 or 2; e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons, and alkynyl having from 2 to 8 carbons, wherein: 1) each alkyl, alkenyl or alkynyl group is unsubstituted; or 2) each alkyl, alkenyl or alkynyl group is substituted with 1 to 3 groups selected from the group consisting of aryl having from 6 to 10 carbons, heteroaryl, arylalkoxy, heterocycloalkoxy, hydroxy alkoxy, alkyloxy alkoxy, hydroxyalkylthio, alkoxy alkylthio, F, Cl, Br, I, -CN, -NO2, -OH, -OR9, -X2 (CH2) PNR1 R12, -X2 (CH2) pC (= O) NR 1R12, -X2 (CH2) pOC (= O) NR11R12 , -X2 (CH2) pCO2R9, -X2 (CH2) pS (O) and R9, -X2 (CH2) PNR10C (= O) NR11R12, OC (=) R9, OCONHR2, -O-tetrahydropyranyl, -NR11R12, -NR10C ( = O) R9, -NR10CO2R9, -NR10C (= O) NR11R12, -NHC (= NH) NH2, NR10S (O) 2R9, -S (O) and R9, -CO2R2, -C (= O) NR11R12, -C (= O) R2, -CH2OR10 , -CH = NNR2R2A, -CH = NOR2, -CH = NR9, -CH = NNHCH (N = NH) NH2, -S (= O) 2NR2R2A, -P (= O) (OR10) 2, -OR14, and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently either unsubstituted or is replaced by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons; X2 is O, S, or NR10; Rt and R8 are each independently selected from the group consisting of H, alkyl having 1 to 4 carbons, alkoxy having 1 to 4 carbons, substituted or unsubstituted arylalkyl having 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl , - (CH2) pOR10, - (CH2) pOC (= O) NR11 R12, and - (CH2) PNR11 R12; or R7 and R8 together form a linking group of the formula -CH2-X3-CH2- wherein X3 is X2 or a bond; m and n each is independently 0, 1 or 2; Y is selected from the group consisting of -O-, -S-, -N (R10) -, -N + (O -) (R10) -, -N (OR10) -, and -CH2-; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, -C (= O) -, -CH (OR10) -, -N (R10) -, -N ( OR10) -, CH (NR11R12) -, -C (= O) N (R17) -, -N (R17) C (= O) -, -N (S (O) and R9) -, -N (S (O) and NR 11 R 12) -, -N (C (= O) R 17) -, -C (R 15 R 16) -, -N + (O) (R 10) -, -CH (OH) -CH (OH) -, and -CH (O (C = O) R9) CH (OC (= O) R9A) -, wherein R9A is the same as R9; R15 and R16 are independently selected from the group consisting of H, -OH, -C (= O) R10, -O (C = O) R9, hydroxyalkyl and -CO2R10; R17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl; A1 and A2 are selected from the group consisting of H, H; H, OR2; H, -SR2; H, -N (R2) 2; and a group wherein A1 and A2 together form a portion selected from the group consisting of = O, = S, y = NR2; B1 and B2 are selected from the group consisting of H, H; H, -OR2; H, -SR2; H, -N (R2) 2; and a group wherein B1 and B2 together form a selected portion of the group consisting of = O, = S, y = NR2; provided that at least one of the pairs of A1 and A2, or B1 and B2, forms = O. The present invention is also directed, in part, to methods for modulating the activity of an MLK protein comprising contacting the protein or a cell containing the protein with a compound having the formula IV presented below: IV where: Z, is H and Z2 is H or Z (and Z2 together form = O; Rt is H or Br; R2 is H; R3 is H, CH2CH = CH2, CH2CH2CH2OH, or R4 is H, CH2CH = CH2 or CH2CH2CH2OH. In preferred embodiments of the invention, R1t R2I R4) Z and Z2 are H and R3 is CH2CH = CH2. In other embodiments of the invention, R1 is Br and R2, R3, R > Z1 and Z2 are H; or RL R2, Z and Z2 are H and R3 and R4 are CH2CH = CH2; or R R2, R3, Z and Z2 are H and R4 is CH2CH = CH2; or R ,, R2, Z ,, and Z2 are H, and R3 and R4 are CH2CH2CH2OH; or R ,, R2, R4, Z? and Z2 are H, and R3 is: * 36 The present invention also provides methods for identifying compounds that may be useful in the treatment of neurodegenerative disorders, comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound and determining whether the compound decreases activity of the multiple lineage kinase protein. The cells, and the extracts thereof, include those described above. The compounds that were found by the methods of the present invention (ie, those compounds that inhibit or reduce the activity of a multiple lineage kinase protein) may be useful for treating neurodegenerative disorders. The protein is preferably selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, leucine closure carrying kinase, double leucine closure kinase, and lineage kinase 6. multiple. The cells are contacted in vivo or in vitro. Preferably, the activity of the protein is determined by measuring the activity or phosphorylation status of a substrate of said protein. Preferably, the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38oc, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3, and also general Ser / Thr substrates, such as, for example, myelin basic protein (MBP). The activity of the protein can also be determined by measuring the activity of a substrate of the protein, amount of a substrate of the protein, or mRNA according to the substrate of the protein. The activity of the protein can also be determined through an in vitro kinase assay or binding assay. The cells are preferably primary embryonic motor neuron cells, overlying cells expressing a multiple lineage kinase protein, or a neuronal cell, but can be any cell or extract thereof. Preferably, the compounds that directly bind the multiple lineage kinase protein are identified, as described above. The present invention also provides methods for identifying compounds that may be useful in the treatment of inflammation, comprising contacting a cell or cell extract containing a multiple lineage kinase protein with the compound and determining whether the compound decreases the activity of the compound. the multiple lineage kinase protein. The cells, and the extracts thereof, include those described above. The compounds that were found by the methods of the present invention (ie, those compounds that inhibit or reduce the activity of a multiple lineage kinase protein) may be useful in treating inflammation. The protein is preferably selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, leucine closure carrying kinase, double leucine closure kinase, and lineage kinase 6. multiple. The cells are contacted live or in vitro. Preferably, the activity of the protein is determined by measuring the activity or phosphorylation status of a substrate of said protein. Preferably, the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38a, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3, and also general Ser / Thr substrates, such as, for example, myelin basic protein (MBP). The activity of the protein can also be determined by measuring the activity of a substrate of the protein, amount of a substrate of the protein, or mRNA according to the substrate of the protein. The activity of the protein can also be determined through an in vitro kinase assay or binding assay. The cells are preferably primary embryonic motor neuron cells, cells that overexpress a multiple lineage kinase protein, or a neuronal cell, but can be any cell or extract thereof. The cells also include, but are not limited to, those involved in inflammation such as, for example, lymphocytes, macrophages and other white blood cells well known to those skilled in the art. Preferably, the compounds that directly bind the multiple lineage kinase protein are identified. The present invention also provides methods for treating a mammal having or suspected of having a neurodegenerative disorder, comprising administering to the mammal a compound that inhibits or reduces the activity of multiple lineage kinase protein. A compound that inhibits or reduces the activity of multiple lineage kinase protein includes, but is not limited to, compounds having the formulas I, II, III and IV. Preferred compounds include those described above with respect to the method for classifying compounds that modulate the activity of a multiple lineage kinase protein and either promote cell survival or cell death. A preferred mammal is a human. An individual may be suspected of having a neurodegenerative disease if the individual has symptoms of a particular neurodegenerative disease, and is in a high-risk group, or has a family history of a neurodegenerative disease. The present invention also provides methods for treating a mammal having inflammation, comprising administering to said mammal a compound that inhibits or reduces the activity of multiple lineage kinase protein. A compound that inhibits or reduces the activity of multiple lineage kinase protein includes, but is not limited to, compounds having the formulas I, II, III and IV. Preferred compounds include those described above with respect to the method for classifying compounds, which modulate the activity of a multiple lineage kinase protein and promote both cell survival and cell death. A preferred mammal is a human. Contact with the compounds having the formulas I-IV can occur in pH or media regulators, which are well known to those skilled in the art. Alternatively, the contact can be presented through the administration of a pharmaceutical composition containing the test compound and a pharmaceutically acceptable salt, vehicle or diluent to a suitable animal or mammal, such as, for example, a mouse or other suitable animal known by those skilled in the art. In addition, variable numbers of cells and concentrations of compounds can be used. The cells that are contacted with the test compounds can be any mammalian cell. Preferably, the cell is a neuronal cell. Preferably, the cell is involved in a neurodegenerative disease, such as, for example, Alzheimer's disease, motor neuron disease, amyotrophic lateral sclerosis, Parkinson's disease, cerebrovascular disease, ischemic conditions, AIDS dementia, epilepsy, Huntington's disease, and damage of concussion or penetration in the brain or spinal cell. The compounds having the formula I, and the methods for making them, are described in the patent of US Pat. No. 5,705,511, which is incorporated herein by reference in its entirety. Compounds having formula III, and methods for making them, are described in U.S. Patents 5,741,8098, 5,621,100, 5,621,101, 5,461,146, and 5,756,494, and WO 97/46567, each of which is incorporated herein by reference. reference in its entirety. Compounds having formula IV, and methods for making them, are described in U.S. Patents 5,741,8098, 5,621,100, 5,621,101, 5,461,146, and 5,756,494, and WO 97/46567, each of which is incorporated herein by reference. by reference in its entirety. Compounds having the formula II include diastereomers and enantiomers around the carbon atoms to which the R2 substituents are attached., R7, and R8. Preferred bridge indenopyrrolocarbazoles are represented by formula II: p In some preferred embodiments of the compounds of the formula II R1 is H. In other preferred embodiments, R2 is H, hydroxyl or substituted or unsubstituted alkyl.

In other preferred embodiments, R, R4, R5 and R6 are independently H, substituted or unsubstituted alkyl, halogen, substituted or unsubstituted alkoxy, substituted or unsubstituted amino, substituted or unsubstituted aryl. In other preferred embodiments, R7 and R8 are independently H, or substituted or unsubstituted alkyl. In some preferred embodiments, Y is O. In other preferred embodiments Z is a bond, O, S, or substituted or unsubstituted N. In other preferred embodiments, m and n are independently 1 or 2. In some especially preferred embodiments, Y is O, Z is a bond or O, and m and n are independently 1 or 2. In other preferred embodiments, A1A2 and B1B2 are = O or H, H . In some especially preferred embodiments, R1, R4, R6 and R7 are each H, Y is = O, n is 1, A1A2 and B1B2 are = O or H, H, R2 is H, OH, or lower alkyl, R3 is H or substituted alkyl, R5 and R8 are each H or alkoxy, with methoxy being very preferred, Z is a bond or O, and m is 1 or 2. Some especially preferred embodiments of the compounds of formula II are compounds 11-1 , II-2, II-3, ll-4a, ll-4b, II-5, II-6, ll-7a, ll-7b, II-8, II-9, 11-10, 11-11 and 11 -12, established in Table 1, infra. The compounds represented by formula II hereafter are referred to as compound (II). As used herein, the term "carbocyclic" refers to cyclic groups wherein the ring portion is composed solely of carbon atoms. The terms "heterocycle" and "heterocyclic" refer to cyclic groups wherein the ring portion includes at least one heterogeneous atom, such as O, N or S. As used herein, the term "alkyl" represents a straight or branched chain cyclic alkyl group having from 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, hexyl, octyl, cyclopropyl and cyclopentyl. The alkyl portion of the alkyl-containing groups, such as the alkoxy, alkoxycarbonyl and alkylaminocarbonyl groups, has the same meaning given above to alkyl. The lower alkyl groups, which are preferred, are alkyl groups as defined above containing from 1 to 4 carbon atoms. The term "alkenyl" is intended to include straight or branched chain hydrocarbon chains having at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl and propenyl groups. As used herein, the term "alkynyl" is intended to include straight or branched chain hydrocarbon chains having at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl and propynyl groups. The acyl portion of acyl-containing groups such as acyloxy groups is intended to include a straight or branched chain alkanoyl group having from 1 to 6 carbon atoms, such as formyl, acetyl, propanoyl, butyryl, valeryl, pivaloyl or hexanoyl. As used herein, the term "aryl" represents a group having from 6 to 12 carbon atoms such as phenyl, biphenyl and naphthyl. Preferred aryl groups include unsubstituted or substituted phenyl and naphthyl groups. The term "heteroaryl" as used herein, denotes an aryl group in which one or more carbon atoms in the ring are replaced by a heterogeneous (i.e., non-carbon) atom such as O, N or S. Preferred heteroaryl groups include pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl, triazolyl, quinolyl, isoquinolyl, benzoimidazolyl, thiazolyl, pyrazolyl, and benzothiazolyl groups. The term "aralkyl" (or "arylalkyl") is intended to denote a group having from 7 to 15 carbons, consisting of an alkyl group bearing an aryl group. Examples of aralkyl groups include benzyl, phenethyl, benzhydryl and naphthylmethyl groups. Alkyl groups and alkyl portions contained within substituent groups such as aralkyl, alkoxy, arylalkoxy, hydroxyalkoxy, alkoxy alkoxy, hydroxy alkylthio, alkoxy alkylthio, alkylcarbonyloxy, hydroxyalkyl and acyloxy groups may be substituted or unsubstituted. A substituted alkyl group has 1 to 3 independently selected substituents, preferably hydroxy, lower alkoxy, lower alkoxy-alkoxy, substituted or unsubstituted arylalkoxy-lower alkoxy, substituted or unsubstituted heteroarylalkoxy-lower alkoxy, substituted or unsubstituted arylalkoxy, substituted heterocycloalkoxy or unsubstituted, halogen, carboxyl, lower alkoxycarbonyl, nitro, amino, mono or lower dialkylamino, dioxolane, dioxane, dithiolane, dithione, furan, lactone or lactam. The substituted aryl, substituted heteroaryl and substituted aralkyl groups each have from 1 to 3 independently selected substituents which are preferably lower alkyl, hydroxy, lower alkoxy, carboxy, lower alkoxycarbonyl, nitro, amino, mono or dialkylamino lower, and halogen. Heterocyclic groups formed with a nitrogen atom include pyrrolidinyl, piperidinyl, piperidino, morpholinyl, morpholino, thiomorpholino, N-methylpiperazinyl, indolyl, isoindolyl, imidazole, imidazoline, oxazole, triazole, thiazoline, thiazole, pyrazole, pyrazolone, and triazole groups. Heterocyclic groups formed with an oxygen atom include furan, tetrahydrofuran, pyran and tetrahydropyran groups. The "hydroxyalkyl" groups are alkyl groups having a hydroxyl group appended thereto. Halogens include fluorine, chlorine, bromine and iodine. As used herein, the term "heteroarylalkyl" represents an arylalkyl group that contains a heterogeneous atom. The term "oxy" denotes the presence of an oxygen atom. In this manner, the "alkoxy" groups are alkyl groups that are linked through an oxygen atom, and the "carbonyloxy" groups are carbonyl groups that are linked through an oxygen atom. The term "heterocycloalkoxy" means an alkoxy group having a heterocycle group attached to the alkyl portion thereof, and the term "arylalkoxy" represents an alkoxy group having an aryl group attached to the alkyl portion thereof. The term "alkylcarbonyloxy" represents a group of the formula -O-C (= O) -alkyl. As used herein, the term "alkyloxy-ahoxy" denotes an alkoxy group that contains an alkyl substituent attached to its alkyl portion. The term "alkoxy-alkylthio" represents an alkylthio group (ie, a group of the formula-S-alkyl) that contains an alkoxy substituent attached to its alkyl portion. The term "hydro-alkylthio" represents an alkylthio group (ie, a group of the formula-S-alkyl) that contains a hydroxy substituent attached to its alkyl portion. As used herein, the term "monosaccharide" has its usual meaning as a singular sugar. As used herein, the term "amino acid" denotes a molecule that contains both an amino group and a carboxyl group. Examples of amino acids include α-amino acids, ie, carboxylic acids of the general formula HOOC-CH (NH 2) - (side chain). The side chains of amino acids include portions of natural existence and non-natural existence. The amino acid side chains of non-natural (ie, non-natural) existence are portions that are used in place of naturally occurring amino acid side chains in, for example, amino acid analogues. See, for example, Lehninger, biochemistry, 2nd. Edition, Worth Publishers, Inc., 1975, pgs. 73-75, incorporated herein by reference. Preferred amino acids include glycine, alanine, proline, glutamic acid and lysine, having the configuration D, the configuration L or as a racemate. The additional representative α-amino acid side chains are shown below in Table 1.

C uad ro 1 CH3- HS-CH2- HO-CH H02C-CH (NH2) -CH, -S-S-CH2- HO-CA-CHj- CH3-S-CH2-CH2- H02C-CH2-NHC (= O) -CHr D- NH2C (= O) -CH2-CH2- (CH3) 2-CH- (CH3) rCH-CH2-CH3-CH2-CH2- J? XH * - H2N-CH2-CH CH2- H2N-C (= NH) - NH-CH2-CH2-CH2- H2N-C (= O) -NH-CHrCH2-CH2- In some preferred embodiments, the substituent groups of the compounds of the formula il include the residue of an amino acid after the removal of the hydroxyl portion of its carboxyl group, ie, groups of the formula -C (= O) -CH ( NH2) - (side chain). The functional groups present in the compounds of the formula II may contain protecting groups. For example, the amino acid side chain substituents of the compounds of the formula II can be substituted with protecting groups such as benzyloxycarbonyl or t-butoxycarbonyl groups. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render said functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups can be employed with the present invention. One of these protecting groups is the benzyloxycarbonyl group (Cbz; Z). Other preferred protecting groups according to the invention can be found by Greene, T. W. and Wuts, P.M., "Protective Groups in Organic Synthesis" 2o. Edition, Wiley & Sons, 1991. Bridged indenopyrrolocarbazole compounds have important functional pharmacological activities evidenced, which find utility in a variety of fixations, including both research and therapeutic areas. These derivatives are useful as therapeutic agents. The activities of the compounds show positive effects on the function and / or survival of cells sensitive to trophic factor. The effect of the function and / or survival of the trophic factor-sensitive cells, e.g., cells of a neuronal lineage, has been demonstrated using any of the following assays: (1) choline-acetyltransferase assay of the cultured spinal cord ( "ChAT"); or (2) ChAT activity assay of cultured basal forebrain neuron. As used herein, the term "effect" when used to modify the terms "function" and "survival" means a positive or negative alteration or change. An effect that is positive can be referred to herein as an "improvement" or "improvement", and an effect that is negative can be referred to herein as "inhibition" or "inhibiting". As used herein, the terms "improve" or "improvement" when used to modify the terms "function" or "survival" represent that the presence of a bridged indenopyrrolocarbazole compound has a positive effect on function and / or survival of the cell sensitive to trophic factor, compared to a cell in the absence of the compound. For example, and not in the form of limitation, with respect to the survival of, for example, a cholinergic neuron, the compound can evidently improve the survival of a cholinergic neuronal population at risk of dying (due, for example, to , a disease condition, a degenerative condition or natural progression), when compared to a cholinergic neuronal population not presented with said compound, if the treated population has a comparatively greater period of functionality than the untreated population. As used herein, "inhibit" and "inhibition" means that a specific response of a designated material (eg, enzymatic activity) is comparatively reduced in the presence of a bridged indenopyrrolocarbazole compound. As used herein, the term "trk" refers to the family of high affinity neurotrophin receptors that actually comprise trkA, trkB, trkC, and other membrane associated proteins to which a neurotrophin can be attached. As used herein, inhibition of VEGFR implies utility in, for example, diseases wherein angiogenesis plays an important role, such as cancer of solid tumors, endometriosis, diabetic retinopathy, psoriasis, hemangioblastoma, as well as other ocular diseases and cancers . The inhibition of trk implies utility in, for example, diseases of the prostate such as prostate cancer and benign prostatic hyperplasia, and the treatment of inflammatory pain. Inhibition of the Platelet Derived Growth Factor Receptor (PDGFR) implies utility in, for example, various forms of neoplasia, rheumatoid arthritis, pulmonary fibrosis, myelofibrosis, healing of abnormal wounds, diseases with cardiovascular endpoints, such as atherosclerosis, restenosis , restenosis after angioplasty, etc. As used herein, the terms "cancer" and "cancerous" refer to any malignant proliferation of cells in a mammal. Examples include prostate cancer, benign prostatic hyperplasia, ovarian, breast, brain, lung, pancreatic, colorectal, gastric, stomach, solid tumors, head and neck, neuroblastoma, renal cell carcinoma, lymphoma, leukemia, and other recognized diseases of hematopoietic systems, and other recognized cancers. As used herein, the terms "neuron," "neuronal cell line," and "neuronal cell" include, but are not limited to, a heterogeneous population of neuronal types that have single or multiple transmitters and / or unique functions or multiple; preferably, there are cholinergic and sensory neurons. As used herein, the phrase "cholinergic neuron" represents neurons of the Central Nervous System (CNS) and Peripheral Nervous System (PNS), whose neurotransmitter is acetylcholine; examples are basal, striatal, and spinal cord neurons. As used herein, the phrase "sensory neuron" includes neurons sensitive to environmental issues (e.g., temperature, movement) of, for example, the skin, muscles and joints; examples are a neuron of the dorsal root ganglion. A "trophic factor responsive cell" as used herein, is a cell that includes a receptor to which a trophic factor can be specifically linked; examples include neurons (e.g., cholinergic and sensory neurons) and non-neuronal cells (e.g., monocytes and neoplastic cells). The bridging indenopyrrolocarbazole compounds described herein find utility in both research and therapeutic aspects to, for example, inhibit enzymatic activity. For example, in a research setting, the compounds can be used in the development of assays and models to further improve understanding of the roles that inhibition of serine / threonine or tyrosine protein kinase (eg, PKC, kinase) tyrosine trk) plays on the mechanical aspects of the associated disorders and diseases. In a therapeutic aspect, the compounds, which inhibit these enzymatic activities, can be used to inhibit the harmful consequences of these enzymes with respect to disorders, such as cancer. As the examples below demonstrate the inhibition of the enzymatic activity using the bridged indenopyrrolocarbazole compounds can be determined using, for example, the following assays: 1. Inhibition assay of Tyrosine kinase activity trkA; 2. Inhibition of trk phosphorylation stimulated by NGF in a whole cell preparation; 3. Vascular Endothelial Growth Factor Receptor (VEGFR) Kinase Inhibition Assay; 4. PKC activity inhibition assay; and 5. PDGFR inhibition assay. The bridged indenopyrrolocarbazole compounds described can be used to improve the function and / or survival of neuronal lineage cells in a mammal, for example, a human. In these contexts, the compounds can be used individually or with other fused pyrrolocarbazoles and / or indolocarbazoles, or in combination with other beneficial molecules that also have evidence of the ability to effect the function and / or survival of a designated cell. A variety of neurological disorders is characterized by neuronal cells, which die, are damaged, functionally compromised, undergo axonal degeneration, are at risk of dying, etc. These disorders include, but are not limited to: Alzheimer's disease; motor neuron disorders (eg, amyotrophic lateral sclerosis); Parkinson's disease; cerebrovascular disorders (eg, seizure, ischemia); Huntington's disease; dementia due to AIDS; epilepsy, multiple sclerosis; peripheral neuropathies (for example, those that affect DRG neurons in peripheral neuropathy associated with chemotherapy) including diabetic neuropathy; disorders induced by excitation amino acids; and disorders associated with concussion damage or penetration of the brain or spinal cord.

ChAT catalyzes the synthesis of the neurotransmitter acetylcholine, and is considered an enzymatic marker for a functional cholinergic neuron. A functional neuron is also capable of surviving. The survival of neurons is analyzed through the quantification of the specific uptake and enzymatic conversion of a dye (for example, calcein AM) by living neurons. Due to their varied utilities, the compounds described herein, including those compounds identified by the methods described herein, find utility in a variety of aspects. The compounds can be used in the development of in vitro models of neuronal cell survival, function, identification or for the classification of other synthetic compounds, which have activities similar to those of the compounds described here, or compounds identified by the methods here described. The compounds described herein, as well as those identified using the methods described herein, can be used in a research environment to investigate, define and determine molecular targets associated with functional responses. For example, through radiolabeling a bridged indenopyrrolocarbazole compound, a compound identified by the methods described herein, associated with a specific cellular function (eg, mitogenesis), the target entity to which the derivative is bound can be identified, isolated, and purified for characterization. The compounds, those described herein as well as those identified using the methods described herein, are useful, among others, not only in improving the activities induced by the trophic factor of trophic sensitive cells, e.g., cholinergic neurons, but can also function as survival promoting agents for other neuronal cell types, for example, dopaminergic or glutamatergic. The growth factor can regulate the survival of neurons by signaling cascades downstream of the small GTP binding proteins including, but not limited to, ras, rae, and cdc42 (Denhardt, Biochem, J., 1996, 318, 729) . Specifically, ras activation leads to the phosphorylation and activation of extracellular receptor-activated kinase (ERK) which has been linked to biological growth and differentiation processes. The stimulation of rac / cdc42 leads to an increase in the activation of JNK and p38, responses that are associated with tension, apoptosis and inflammation. Although the growth factor responses are mainly through the path of ERK, performing these subsequent processes can lead to alternative mechanisms of neuronal survival that can mimic the survival properties of growth factor enhancement (Xia et al., Science, 1995, 270, 1326). The compounds can also function as survival promoters for neuronal and non-neuronal cells through mechanisms related to, but also different from, growth factor-mediated survival, eg, inhibition of JNK and p38 trajectories, which can lead to survival through the inhibition of apoptotic cell death processes. The compounds herein are useful in the treatment of disorders associated with reduced ChAT activity or death, damage to spinal cord motor neurons, and also have utility in, for example, diseases associated with apoptotic cell death of the central nervous system. and peripheral, immune system and inflammatory diseases. The compounds described herein also find utility in the treatment of disease states involving proliferation of malignant cells, such as many cancers. Pharmaceutically acceptable salts of the compounds described herein, as well as those compounds identified by the methods herein, include pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, and addition salts of amino acid. Examples of the acid addition salts are 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 the 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 the ammonium salts are ammonium salt and tetramethylammonium salt; examples of the organic amine addition salts are salts with morpholine and piperidine; and examples of the amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. The compounds provided herein, including those identified by the methods of the present invention, can be formulated in pharmaceutical compositions by mixing with non-toxic, pharmaceutically acceptable excipients and vehicles. Said compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions, or oral administration, particularly in the form of tablets or capsules; or intranasally, in particular in the form of powders, nasal drops or aerosols; or dermally, through, for example, transdermal patches. The composition can be conveniently administered in a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton. , 1980). Formulations for parenteral administration may contain as common excipients sterile or saline water, polyalkylene glycols such as polyethylene glycol, oils and of vegetable origin, hydrogenated naphthalenes and the like. In particular, the biodegradable, biocompatible lactide polymer, the lactide / glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers can be useful excipients for controlling the release of the active compounds. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for administration by inhalation contain, excipients, for example, lactose, or they may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions to be administered in the form of nasal drops, or as a gel that will be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration. The formulations for transdermal patches are preferably lipophilic emulsions. The compounds of this invention can be employed as the sole active agent in a pharmaceutical composition. Alternatively, these may be used in combination with other active ingredients, for example, other growth factors that facilitate neuronal survival or axonal regeneration in diseases or disorders. The compounds of the invention and their pharmaceutically acceptable salts can be administered orally or non-orally, for example, as an ointment or an injection. The concentrations of the compounds of this invention in a therapeutic composition may vary. The concentration will depend on factors such as the total dose of the drug to be administered, the chemical characteristics (eg, hydrophobicity) of the compounds used, the route of administration, age, body weight and symptoms of a patient, etc. . The compounds of this invention are typically provided in an aqueous physiological pH buffer solution containing about 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 around 0.1 to 20 mg / kg once four times a day. A preferred dose of the drug to be administered is likely dependent on variables such as the type and degree of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the selected compound and the formulation of the excipient of the compound , and its administration route. The compounds of the invention, including the test compound and compounds identified by the methods of the present invention and their pharmaceutically acceptable salts can be administered alone, or in the form of various pharmaceutical compositions, in accordance with the pharmacological activity and the purpose of administration. The pharmaceutical compositions according to the present invention can be prepared by uniformly mixing an effective amount of a compound or a pharmaceutically acceptable salt thereof, as an active ingredient, with a pharmaceutically acceptable carrier. The vehicle can take a wide variety of forms according to the forms of composition suitable for administration. It is desired that said pharmaceutical compositions be prepared in a unit dosage form suitable for oral or non-oral administration. Forms for non-oral administration include ointments and injections. Tablets can be prepared using excipients such as lactose, glucose, sucrose, mannitol and methylcellulose, disintegrating agents such as starch, sodium alginate, calcium carboxymethylcellulose and crystalline cellulose, lubricants such as magnesium stearate and talc, binders such as gelatin , polyvinyl alcohol, polyvinyl pyrrolidone, hydroxypropyl cellulose and methyl cellulose, surfactants such as sucrose fatty acid ester and sorbitol fatty acid ester, and the like, in a conventional manner. It is preferred that each tablet contains 15-300 mg of the active ingredient. The granules can be prepared using excipients such as lactose and sucrose, disintegrating agents such as starch, binders such as gelatin and the like in a conventional manner. Powders can be prepared using excipients such as lactose and mannitol, and the like, in a conventional manner. The capsules can be prepared using gelatin, water, sucrose, gum arabic, sorbite, glycerin, crystalline cellulose, magnesium stearate, talc, and the like, in a conventional manner. It is preferred that each capsule contains 15-300 mg of the active ingredient.

Syrup preparations can be prepared using sugars such as sucrose, water, ethanol, and the like in a conventional manner. The ointments can be prepared using ointment bases such as petrolatum, liquid paraffin, lanolin and macrogol, emulsifiers such as sodium lauryl lactate, benzalkonium chloride, sorbitan monohydric acid ether, sodium carboxymethylcellulose and gum arabic, and the like in a conventional way. Injectable preparations can be prepared using solvents such as water, physiological saline, vegetable oils (e.g., olive oil and peanut oil) ethyl oleate and propylene glycol, solubilizing agents such as sodium benzoate, sodium salicylate and urethane , isotonicity agents such as sodium chloride and glucose, preservatives such as phenol, cresol, p-hydroxybenzoic ester and chlorobutanol, antioxidants such as ascorbic acid and sodium pyrosulfite, and the like in a conventional manner. The invention is further illustrated by way of the following examples, which are intended to present the invention. These examples are not intended, nor will they be constructed as limiting the scope of the description.

EXAMPLES Example 1 General Description of Processes and Synthetic Examples The general synthetic route used to prepare the bridged indenopyrrolocarbazoles of this invention having the formula II, is shown in Figures 1 and 2. The general procedures for the synthesis of the indenopyrrolocarbazoles (III) / (VIII) may be made as described in US Patent 5,705,511, the disclosure of which is hereby incorporated by reference in its entirety. When R1 is H, the lactam-nitrogen of the indenopyrrolocarbazoles (III) / (VIII) is protected with an appropriate protecting group leading to (IV) / (IX). The protected compounds are treated with an appropriate base in anhydrous organic solvents, which results in the generation of a dark red solution, which is believed to be carbanion. The reaction of carbanion with a bi-functional reagent (V) results in the electrophilic addition to the C = Y bond of (V) leading to the initial intermediate (VI) / (X). The treatment of intermediates (VI) / (X) and / or (VI I) / (XI) either with a sulfonic acid or a Lewis acid, for example boron trifluoride etherate, provides the indepirrolocarbazoles in bridging (l) / (ll). The lactam-nitrogen protection strategy (shown in Figures 3 and 4) can be performed either through a process catalyzed by acid base. The acid-catalyzed reaction can be performed with a reagent bound to the resin allowing immobilization of the indenopyrrolocarbazole (III) / (VIII) to a polymeric support, such as a Rink acid resin with polystyrene base (XII) (Figure 3) , providing (XIII). Alternatively, the acid catalyzed reaction can be carried out with a soluble reagent to produce a compound (XIV) (Figure 4). The silyl protected compound (XV) is produced under base catalysis (Figure 4). Figure 5 describes various methods for preparing the intermediate (V). The procedure (a) describes the transformations of the various acetals (XVI) / (XVII, Z = bond). For example, the ester-acetal / ketal (XVI, D = COOR) is completely reduced to the corresponding alcohol and subsequently oxidized (eg, Swern or Dess-Martin oxidation) to the aldehyde-acetal / ketal (XVII, R8 = H) . Alternatively, the ether-acetal / ketal (XVI, D = COOR) is partially reduced with DIBAL to provide the aldehyde (XVII, R8 = H, directly) Similarly, the reduction of the nitrile-acetal (XVI, D = CN) with DIBAL gives the aldehyde (XVII, R8 = H) .The keto-acetals / ketal are prepared through the addition of Gringnard reagents to amide-acetal / Weinreb ketal (XVI, D = CON (Ome) Me). The intermediate (XVII, Z = bond) can also be obtained through a two-step procedure presented in procedure (b). The addition of the organometallic reagent (XIX) to acetal / ketal (XVIII) gives the alkene (XX), which after ozonolysis followed by a reductive process offers keto-acetal / ketal (XVII). The preparation of the intermediate (XVII, Z = heterogeneous atom) through a two-step procedure is presented in process (c). The coupling of acetal (XXII) with alkene (XXI) followed by ozonolysis (with a reductive process) of the resulting alkene yields keto-acetal / ketal (XVII). Alternatively, the intermediate (XVII, Z = heterogeneous atom) is prepared through a two-step procedure presented in procedure (d). The reaction of the compound (XXIV) with acetal / ketal (XVIII) provides (XXV), which is transformed to keto-acetal / ketal (XXVII) through the methods described in process (a). Condensation of keto-acetal / ketal (XVII) with hydroxylamines, hydrazines, N-alkyl-N-alkoxyamines, and amines provides the intermediate (XXVI) carrying an electrophilic C = N functionality. The resin bound indenopyrrolocarbazole (XIII) (Figure 6, method A) is treated with an excess of a Grignard reagent as a base, which results in the generation of a dark red carbanion solution. The subsequent reaction with (V) leads to products derived from the electrophilic addition to the C = Y group. The aqueous process and the splitting of the products with dilute acid (1% TFA in methylene chloride) from the resin results in the isolation of the compound (s) (XXVII) and / or (XXVIII). The treatment of intermediate (s) (XXVII) and / or (XXVIII) with either a sulfonic acid or a Lewis acid for example, boron trifluoride etherate, provides the bridged indenopyrrolocarbazoles (II). A similar strategy is employed for the reaction of the protected intermediate with soluble lactam, for example (XV) (Figure 7, method B). However, in this case the intermediate (XV) is treated with triton B in pyridine as a base instead of the Grignard reagent. Intermediate (s) (XXIX) and / or (XXX) can be isolated with the intact lactam protecting group, which is subject to chromatographic purification. As in method A (Figure 6), treatment with a Lewis acid (such as boron trifluoride etherate) provides the bridged indenopyrrolocarbazoles (II), wherein R1 = H. The introduction of the groups R3, R4, R5 and R6 can be carried out as described in the patents of US Pat. Nos. 5,705,511 and 4,923,986, the descriptions of which are hereby incorporated by reference in their entirety. A substituent R3 may otherwise be introduced after the construction of the bridged indenopyrrolocarbazoles, as shown in Figure 8. The 3-position of the B ring is brominated with NBS to provide the compound (XXXI). Subsequently, a carbon fragment is introduced using Stille, Suzuki, Heck, Kumada or Castro-Stephens catalysts catalysed by palladium to provide compounds of type (XXXll), (XXXIII), etc. In addition, the compound (XXXI) can provide access to compounds wherein the bromine group is displaced with a heterogeneous atom, for example, an amine base group through the use of amination chemistry catalyzed by Buchwald palladium. Through an oxidizing process, an oxygen bound group can be introduced into the indene-carbon of the E ring, as shown in Figure 9, compound (XXXIV). This chemistry also results in the oxidation of the methylene group of the lactam (ring A) to provide an imide derivative, as shown.

Example 2 Preparation of intermediates bound to the Rink resin: (Xlll-A), (Xlll-B) and (Xlll-Ch (Figure 3) Example 2A A three-necked round bottom flask equipped with an upper mechanical stirrer and a Dean-Stark trap was sequentially loaded with a Rink XII acid resin (10.00 g, 064 mmol / g), 80 ml of 1-methyl- 2-pyrroidinone, 350 ml of benzene, 3.00 g of VI I IA (A1, A2 = H2, B \ B2 = O, R3 = R4 = R5 = R6 = H)) and 1.00 g of p-toluenesulfonic acid. The reaction mixture was heated to reflux for 20 hours and then filtered. The resin was washed with THF (5 x 175 ml) and the filtrate was separated. The resin was then sequentially washed with DMSO (4 x 100 ml), 2% aqueous NaHCO3 (4x100 ml), water (4 x 100 ml), DMSO (2 x 200 ml), THF (4 x 100 ml) and ethyl acetate (4 x 100 ml). The resin was dried under vacuum (24 hours) to provide 11.70 (0.47 mmoles / g) of VI I I-A bound to the resin (Xlll-A).

The original THF washes were evaporated, the residue was diluted with 750 ml of water, and the resulting precipitate was filtered and sequentially washed with water, 2% aqueous NaHCO3 (4 x 100 ml), and water (4 x 100 ml). ). After drying under vacuum, Vlll-A (1.28 g) was recovered.

Example 2-B In a similar way, 0.5 g of Vlll-B (A1, A2 = O, B1, B2 = H2, R3 = R4 = R5 = R6 = H) were coupled to 1.52 g of the Rink XII acid resin. to provide 1.58 g of Vlll-B bound to the resin (Xlll-B).

Example 2-C In a similar way, 1.02 g of Vlll-C (A1, A2 = H2, B1, B2 = 0, R3 = R4 = R5 = H, R6 = 10-OMe) were coupled, to 3.12 g of the Rink XII acid resin to provide 3.70 (0.46 mmole / g) of the Vlll-C compound bound to the resin (Xlll-C) together with the recovered Vlll-C compound (0.44 g).

Example 3 Preparation of Compound (11-1), Compound (II-2). Compound (II-3). Compound (ll-4a). Compound (ll-4b). Compound (ll-ß) and Compound (II-8) (Method A, Figure 6) Example 3-AA a suspension of 1.25 g of (Xlll-A), in 24 ml of THF a solution of 1.0 M EtMgBr (6.25 ml in THF) was added and the reaction was stirred for 1 hour before the addition of 5.0 ml of HMPA. After stirring for 10 minutes, 3.0 g of diethoxybutyraldehyde (which was prepared according to the literature procedure of Paquette, et al., J. Am. Chem. Soc, 1997, 119, 9662-71) was added, and the The reaction was stirred for 20 hours. The reaction was quenched with 5 mL of 10% aqueous NH4CI and filtered. The resin was successively washed with 10% aqueous NH4CI (3 x 10 ml), water (3 x 10 ml), THF (3 x 10 ml), DMF (3 x 10 ml), water (3 x 10 ml), THF (3 x 10 mL), and ether (3 x 10 mL). The resin was dried under vacuum, taken up in 15 ml of methylene chloride, and treated with 0.15 ml of trifluoroacetic acid. After stirring for 1 hour, the reaction was filtered and the filtrate was evaporated. The resulting residue was taken up in 20 ml of methylene chloride and treated with 50 mg of pyridinium tosylate, and the resulting solution was stirred for 4 hours. At that time, the reaction was washed with saturated aqueous NaHCO3 and brine, and dried over MgSO4. After filtration and evaporation of the solvent, the residue was purified by preparative HPLC (Zorbax RX-8, 4 x 25 cm, eluted with MeCN / 60% water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO3 and extracted into methylene chloride 3 x 50 ml) and dried over MgSO. After filtration and evaporation of the solvent, 70.2 mg of compound 11-1 was obtained as a white powder, which had the following characteristics: 13C NMR (DMSO-d6) d 171.8, 143.3, 142.4, 141.4, 140.1, 140.0, 136.6, 129.2, 127.9, 127.4, 127.1,126.8, 124.1 (2C), 122.7, 121.6, 121.5, 118.3, 112. 1.88.1, 79.2, 56.6, 45.6, 33.4, 24.8; 1H NMR (DMSO-d6) d 9.21 (d, J = 7.5, 1H), 8.62 (s, 1H), 7.98 (d, J = 7.7, 1H), 7.86 (d, J = 8.3, 1H), 7.71 ( d, J = 7.3, 1H), 7.49 (dd, J = 7.9, 7.4, 1H), 7.41 (dd, J = 7.5, 7.4, 1H), 7.36-7.27 (m, 2 H), 6.86 (d, J = 6.0, 1H), 5.63-5.58 (m, 1H), 4.91 (s, 2H), 4.53 (d, J = 3.3, 1H), 2.23-2.14 (m, 1H), 1.96-1.92 (m, 1H) , 0.96-0.88 (m, 1H), 0.60-0.57 (m, 1H); MS m / z (M + H) cale. 379, obsd 379. Compound II-2 (0.5 mg) was also isolated by HPLC from the reaction product mixture, which had the following characteristics: 1H NMR (DMSO-d6) d 9.17 (d, J = 8.1, 1H), 8.62 (s, 1H), 7.98 (d, J = 7.0, 1H), 7.85 (d, J = 6.8, 1H), 7.57 (d, J = 6.8, 1H), 7.49 (dd) , J = 7.9, 7.4.1H), 7.44-7.26 (m, 3H), 6.81 (d, J = 6.0, 1H), 5.43-5.33 (m, 1H), 4.43 (s, 2H), 2.23-2.14 ( m, 1H), 1.96-1.92 (m, 1H), 1.45-1.55 (m, 2H), 0.96-0.88 (m, 1H), 0.60-0.57 (m, 1H), 0.29 (t, J = 7.0, 3H ); MS m / z (M + H) cale. 407, obsd 407.

Example 3-B In a similar manner, as described above for compound 11-1, the resin (Xlll-A) (70.3 mg) was treated with 0.75 ml of 1,1-diethoxy-2-pentanone (which is prepared according to the literature procedure of Sworin et al., J. Org. Chem., 1988, 53, 4894-6), to provide 3.5 mg of compound II-3, which was isolated through TLC preparation ( silica gel, eluted with 50% EtOAc / toluene) and had the following properties: 1H NMR (DMSO-d6) d 9.42 (d, J = 8.2, 1H), 8.58 (s, 1H), 7.95 (d, J = 7.4, 1H), 7.79 (d, J = 8.3, 1H), 7.71 (d, J = 7.1), 7.50-7.20 (m, 4H), 6.81 (d, J = 5.9, 1H), 4.90 (s, 2H), 4.46 (s, 1H), 2.35-2.20 (m, 1H), 1.98 (s, 3H), 1.75-1.60 (m, 1H), 1.25-1.00 (, 1H), 0.35-0.15 (m, 1H) ); MS m / z (M + H) cale. 393, obsd 393.

Example 3-C In a similar manner, 74.3 mg of (Xlll-A) was treated with 1,1-diethoxy-2-hexanone (which was prepared according to the literature method of Brenner, J. Org. Chem. , 1961, 26, 22-7) (0.75 ml) to provide 2.10 mg of compound 11 -4a and 1.06 mg of compound ll-4b, which were individually isolated through preparative HPLC (Zorbax RX-8, 4 x 25 cm, MeCN / water 65% w / 0.1% trifluoroacetic acid). Compound ll-4a had the following properties: 1H NMR (DMSO-d6) d 9.30 (d, J = 8.3, 1H), 8.55 (s, 1H), 7.97 (d, J = 7.2, 1H), 7.65 (d , J = 8.5, 1H), 7.59 (d, J = 7.5), 7.48 (dd, J = 7.8, 7.2, 1 H) 7.39-7.15 (m, 3H), 6.31 (dd, J = 5.9, 5.5, 1H ), 5.02 (s, 1H), 4.88 (s, 2H), 0.88 (s, 3H), other aliphatic signals lost under the solvent peaks; MS m / z (M + H) cale. 407, obsd 407. Compound 11-4b had the following properties: 1H NMR (DMSO-d6) d 9.43 (d, J = 8.1, 1H), 8.59 (s, 1H), 7.99 (d, J = 7.3, 1H ), 7.75-7.65 (m, 2H), 7.49 (dd, J = 7.0, 6.4, 1H), 7.43 (dd, J = 8.2, 8.1, 1H), 7.36-7.25 (m, 2H), 6.75 (s, 1H), 4.91 (s, 2H), 4.50 (s, 1H), 1.95 (s, 3H) other aliphatic signals were lost under solvent peaks; MS m / z (M + H) cale. 407, obsd 407.

Example 3-D In a similar manner, 1.00 g of (Xlll-C) was treated with 3.65 g of diethoxyburataldehyde to provide 87.8 mg of compound II-6 which was isolated through HPLC (Zorbax RX-8, 2.5 x 25 cm, MeCN / 65% water for 0.1% trifluoroacetic acid) and had the following properties: 1H NMR (DMSO-d6) d 9.09 (d, J = 8.6, 1H), 8.60 (s, 1H), 7.95 ( d, J = 7.4, 1H), 7.84 (d, J = 8.3, 1H), 7.47 (dd, J = 7.2, 7.0, 1H), 7.35 (s, 1H), 7.29 (dd, J = 7.0, 7.0, 1H), 6.98 (dd, J = 8.6, 1.9, 1H), 6.83 (d, J = 6.0, 1H), 5.65-5.55 (m, 1H), 4.88 (s, 2H), 4.48 (d, J = 3.9 , 1H), 3.82 (s, 3H), 2.25-2.10 (m, 1H), 2.08-1.85 (m, 1H), 0.96-0.75 (m, 1H), 0.65-0.50 (m, 1H); MS m / z (M + Na) cale. 431, obsd 431.

Example 3-E In a similar way, 153.2 mg of the resin (Xlll-B) was treated with 1.5 ml of diethoxyburataldehyde to provide 3.6 mg of compound II-8, which was isolated through preparative HPLC (Zorbax RX- 8, 2.5 x 25 cm, MeCN / 65% water w / 0.1% trifluoroacetic acid) and had the following properties: 1H NMR (DMSO-de) d 9.09 (d, J = 7.9, 1H), 8.81 (s, 1H), 7.81-7.73 (m, 3H), 7.48-7.35 (m, 3H), 7.24 (dd, J = 7.6, 7.5, 1H), 6.85 (d, J = 6.2, 1H), 5.63-5.59 (m , 1H), 4.86 (s, 2H), 4.61 (d, J = 3.6, 1H), 3.82 (s, 3H), 2.21-2.13 (m, 1H), 1.96-1.90 (m, 1H), 0.87-0.79 (m, 1H), 0.61- 0.56 (m, 1H); MS m / z (M + H) cale. 379, obsd 379.

Example 4 Preparation of Compound 11 -7a and Compound ll-7b (Method A. Figure 6) Example 4-A Preparation of (1,1-diethoxyethoxy) acetone To a cold (0 ° C) suspension of NaH (2.68 g, 60%) in 150 ml of THF was added a solution of 1,1-diethoxyethanol, which was prepared from according to the literature procedure of Zirkle et al., J. Org. Chem., 1961, 26, 395-407), (9.00 g) in 20 ml of THF, and the reaction mixture was stirred at room temperature for 1 hour before adding 8.0 ml of methallyl chloride. The reaction mixture was heated to reflux overnight, cooled and filtered through a plug of Celite. The solvent was removed through rotary evaporation, and the residue was purified by column chromatography (silica, 20% ether / hexane) to give 1,1-diethylethylmethyl ether (11.5, 90%). Ozonolysis of a cold solution (-30 ° C of this ether (6.00 g) in 80 ml of EtOAc was carried out until no starting material was detected by TLC (1 hour). purged with oxygen, treated with 150 mg of Pd (Pd (OH) 2 and stirred under a hydrogen atmosphere overnight.) The catalyst was filtered, and the filtrate was concentrated by rotary evaporation. through column chromatography (silica gel, EtOAc / hexane) 20%) to provide the title compound (4.53 g, 82%).

Example 4-B According to Method A (Figure 6), 230.2 mg of the resin (Xlll-A) was treated with 1.25 ml of EtMgBr followed by 1.2 ml of (1,1-diethoxyethoxy) acetone (Example 3A). After processing and unfolding of the resin, a portion of the crude reaction product mixture (10.5 mg) was taken up in 20 ml of methylene chloride and treated with 20 μl of BF3 etherate. After stirring for 2.5 hours, the solution was washed with saturated aqueous NaHCO3 and brine before being dried over MgSO. After stirring and removing the solvent, the resulting residue was purified by HPLC (Zorbax RX-8, 4 x 25 cm, MeCN / 65% water w / 0.1% trifluoroacetic acid) to provide 2.34 mg of compound II -7a and 1.34 mg of compound ll-7b. The compound (ll-7a) had the following properties: 1H NMR (CDCl 3) d 9.35-9.20 (m, 1H), 7.87 (d, J = 7.6, 1H), 7.62 (d, J = 7.0, 1H), 7.60 -7.45 (m, 1H), 7.49 (dd, J = 7.7, 7.5, 1H), 7.40 (d, J = 8.1, 1H), 7.37-7.26 (m, 3H), 6.22 (s, 1H), 5.20- 4.85 (m, 1H), 4.47 (s, 1H), 3.67 (d, J = 12.7, 1H) 3.52 (d, J = 11.8, 1H), 3.40 (d, J = 12.7, 1H), 3.38 (d, J = 11.8, 1H), 1.91 (s, 3H); MS m / z (M + H) cale. 409, obsd 409. Compound ll-7b had the following properties: 1H NMR (CDCl 3) d 9.58-9.22 (m, 1H), 7.82 (d, J = 7.4, 1H), 7.60-7.40 (m, 3H), 7.37-7.27 (m, 3H), 7.21 (d, J = 8.1, 1H), 5.81 (s, 1H), 5.21 (s, 1H), 5.10-4.80 (m, 1H), 4.59 (d, J = 13.5 , 1H), 4.38 (dd, J = 13.5, 5.3, 1H), 4.21 (d, J = 13.1, 1H), 3.82 (d, J = 13.2, 1H), 1.13 (s, 3H); MS m / z (M + H) cale. 409, obsd 409.

Example 5 Preparation of Compound 11-5 (Figure 8) To a solution of 8.1 mg of compound 11-1 in 2 ml of THF was added 4.6 mg of NBS, and the reaction was stirred overnight. An additional 4.5 mg of NBS was added and the reaction was stirred for 2.5 hours. The insoluble material was filtered and the filtrate was concentrated by rotary evaporation. The resulting residue was purified by column chromatography (C-18, MeCN / water al 65% p / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO3 and extracted with methylene chloride (3 x 20 ml) and dried over MgSO4. After filtration and evaporation of the solvent, 5.1 mg of compound II-5 was obtained as a white powder, which had the following characteristics: 1H NMR (DMSO-dβ) d 9.22 (d, J = 7.4, 1H), 8.67 (s, 1H), 8.14 (s, 1H), 7.86 (d, J = 8.7, 1H), 7.72 (d, J = 7.0 , 1H), 7.63 (d, J = 7.8, 1H), 7.42 (dd, J = 7.5, 7.3, 1H), 7.35 (dd, J = 7.3, 7.2, 1H), 6.86 (d, J = 6.0, 1H), . 63-5.58 (m, 1H), 4.94 (s, 2H), 4.54 (d, J = 3.1, 1H), 2.30-2.14 (m, 1H), 2.00-1.82 (m, 1H), 0.96-0.88 (m, 1H), 0.62-0.50 (m, 1H); MS m / z (M + H) cale. 457/9 (1: 1), obsd 457/9 (1: 1).

Example 6 Preparation of Intermediate XV (Figure 4) To a solution of 1.05 g of Vlll-A [A1, A2 = H2, B1, B2 = O, R3 = R4 = R5 = R6 = H] in 25 ml of DMF were added 0.75 ml of triethylamine and 0.65 g of t-butyldimethylsilyl chloride (TBS-C1).

After stirring for 3 hours, the reaction was quenched with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with water and brine and dried over MgSO4. After filtering and evaporating the solvent, the resulting residue was titrated with ether to give compound XV (848 mg). The washings were evaporated to leave a residue that was purified through column chromatography (silica, 1% EtOAc / CH 2 Cl 2) and gave the additional product (502 mg, 94% combined yield) which had the following spectral properties: 1H NMR (DMSO-d6) d 11.94 (s, 1H), 9.32 (d, J = 7.6, 1H), 8.03 (d, J = 7.7, 1H), 7.64 (d, J = 7.2, 1H), 7.58 (d , J = 8.1, 1H), 7.44 (dd, J = 7.7, 7.6, 1H), 7.39 (dd, J = 7.7, 7.6, 1H), 7.32 (d, J = 7.3, 1H), 7.25 (dd, J = 7.6, 7.3, 1H), 5.00 (s, 2H), 4.14 (s, 2H), 0.99 (s, 9H), 0.46 (s, 6H); MS m / z (M + H) cale. 425, obsd 425.

Example 7 Preparation of Compound 11-1 through Method B (Figure 7) A solution of Triton B in pyridine (0.45 M) was prepared by dissolving a 40% solution of Triton B in 10 ml of metal in 10 ml of pyridine. The solvent was removed under reduced pressure (20 mm Hg) to a final volume of about 8 ml. The residue was diluted with pyridine to 50 ml, it was filtered and stored under nitrogen. A solution of 20.3 mg of XV in 2.0 ml of pyridine was washed with argon and treated with 300 μL of Triton B (0.45 M in pyridine) and 50 μL of dietoxybutaraldehyde. After stirring for 2 hours, the reaction was extracted into EtOAc, washed with 1 N aqueous HCl, brine and dried over MgSO. After filtration and evaporation of the solvent, the adduct was taken up in 10 ml of CH 2 Cl 2 and treated with 10 μl of BF 3 etherate. After stirring for 2.0 hours, the solution was washed with saturated aqueous NaHCO3 and brine before drying over MgSO4. Removal of the solvent through rotary evaporation gave a residue that was purified through preparative HPLC (Zorbax RX-8, 2.5 x 25 cm, MeCN / 65% water w / 0.1% trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO3 and extracted into methylene chloride (3 x 20 ml) and dried over MgSO. After filtration and evaporation of the solvent, 11.8 mg of 11-1 (65% yield) were obtained whose spectra of 1H NMR and MS and HPLC retention time were identical to the material prepared and isolated through method A, described in Example 3-A.

Example 8 Preparation of Compound II-9 (Figure 8) To a suspension of 6.2 mg of bromine compound II-5 in 4.0 ml of 1-propanol was added 3.8 mg of 3-aminophenylboronic acid. After stirring for 0.25 hours, Pd (OAc) 2 (2.0 mg) Ph3P (4.8 mg), Na2CO3 (2.8 mg), and water (2.0 ml) were added sequentially. The mixture was heated to reflux for 0.74 hours, cooled, extracted into CH2Cl2 and washed with water and brine. The organic layer was dried over MgSO4, and the solvent was removed by rotary evaporation to give a residue that was purified through HPLC (Zorbax RX-8, 2.5 x 25 cm, MeCN / 50% water w / 0.1% of trifluoroacetic acid). The appropriate fractions were neutralized with NaHCO3 and extracted into methylene chloride (3 x 20 ml) and dried over MgSO4. After filtration and evaporation of the solvent, compound II-9 (3.1 mg, yield 49%) was obtained and had the following spectral properties: 1 H NMR (DMSO-de) d 9.22 (d, J = 7.5, 1H ), 8.66 (s, 1H), 8.00-7.25 (m, 8H), 7.12 (dd, J = 7.1, 7.0, 1H), 6.95-6.80 (m, 3H), 6.53 (d, J = 6.0, 1H) , 5.63-5.58 (m, 1H), 4.99 (s, 2H), 4.55 (s, 1H), 2.25-2.10 (m, 1H), 1.95-1.90 (m, 1H), 0.98-0.88 (m, 1H) , 0.65-0.57 (m, 1H); MS m / z (M + H) cale. 470, obsd 470.

Example 9 Preparation of Compound 11-10 (Figure 9) To a solution of 5.0 mg of compound 11-1 in 1 ml of DMSO was added 4.3 mg of NaCN and the mixture was heated at 145 ° C for 1 hour. The mixture was cooled, extracted into EtOAc, and washed with water (3 x 20 ml) and brine. The organic layer was dried over MgSO 4, filtered and evaporated to give a residue which was purified through preparative HPLC (Zorbax RX-8, 2.5 x 25 cm, MeCN / 55% water w / 0.1% trifluoroacetic acid ). The appropriate fractions were neutralized with NaHCO3 and extracted into methylene chloride (3 x 20 ml) and dried over MgSO4. After filtration and evaporation of the solvent, compound 11-10 (2.7 mg, 50% yield) was obtained and had the following spectral properties: 1H NMR (DMSO-de) d 11.4 (s, 1H), 8.86 ( d, J = 7.9, 1H), 8.79 (d, J = 7.6, 1H), 7.90 (d, J = 8.3, 1H), 7.62-7.55 (m, 2H), 7.49 (dd, J = 7.6, 7.4, 3H), 7.40 (dd, J = 7.4, 7.3 1H), 7.35 (dd, J = 7.5, 7.4, 1H), 6.86 (d, J = 6.0, 1H), 6.03 (s, 1H), 5.40-5.30 ( m, 1H), 2.25-2.14 (m, 1H), 2.03 -1.90 (m, 1 H), 1.10-0.98 (m, 1H), 0.82-0.77 (m, 1H).

Example 10 Preparation of Compound 11-11 (Method A, Figure 6) According to Method A, 150.2 mg of the resin (Xllla) was reacted with 1.0 mL of EtMgBr followed by 1.5 mL of ethyl 2,5-dioxopentanoate. (Schmidt, et al., Syntgesis, 1993, 809). After the process and splitting from the resin, the crude reaction product mixture was taken up in 20 ml of methylene chloride and treated with 20 μl of BF3 etherate. After stirring for 2.5 hours, the solution was washed with saturated aqueous NaCO3 and brine before drying over MgSO4. After filtration and removal of the solvent, the resulting residue was purified by preparative HPLC (Zorbax RX-8, 4 x 25 cm, gradient MeCN / water 55% -75% w / 0.1% trifluoroacetic acid) to provide 6.4 mg of compound 11-11, which had the following properties: 1H NMR (DMSO-d6) d 9.36 (d, J = 7.7, 1H), 8.68 (s, 1H), 8.00 (d, J = 7.7 , 1H), 7.83 (d, J = 8.3, 1H), 7.58-7.15 (m, 5H), 6.97 (d, J = 5.9.1H), 4.93 (s, 2H), 4.82 (s, 1H), 4.48 (q, J = 7.1, 2H), 2.42-1.91 (m, 2H), 1.37 (t, 314, J = 7.1), 1.25-0.63 (m, 2H).

Example 11 Preparation of Compound 11-12 A solution of 3.4 mg of compound 11-11 in 2 ml of THF was treated with a 2M solution of LiBH4 (1.0 ml in THF) and the reaction was stirred for 1.5 hours. The reaction was quenched through the addition of 4 ml of 1N aqueous HCl. After stirring for 20 minutes, 15 ml of 10% aqueous NaOH was added and the mixture was extracted with methylene chloride (3 x 10 ml). After drying over MgSO 4, the mixture was filtered and the solvent was evaporated to provide 0.32 mg of compound 11-12, which had the following properties: 1 H NMR (DMSO-d 6) d 9.35 (d, J = 7.7, 1 H) , 8.62 (s, 1H), 7.98 (d, J = 7.7, 1H), 7.83 (d, J = 8.2.1H), 7.75 (d, J = 8.2.1H), 7.50-7.25 (m, 4H), 6.84 (d, J = 7.7, 1H), 6.11 (s, 1H), 4.91 (s, 2H), 4.71 (s, 1H), 4.50-4.40 (m, 1H), 4.30-4.20 (m, 1H) 2.42 -1.91 (m, 2H), 1.25-0.63 (m, 2H); MS m / z (M + H) cale. 409, obsd. 409 Example 12 Improvement of the ChAT Activity of the Spinal Cord ChAT is a specific biochemical marker for functional cholinergic neurons. Cholinergic neurons represent a major cholinergic entry in the formation of the hippocampus, olfactory nucleus, interpeduncular nucleus, cortex, tonsil, and parts of the thalamus. In the spinal cord, motoneurons are cholinergic neurons containing ChAT (Phelps, et al., J. Comp.Neurol., 1988, 273, 459-472). The activity of ChAT has been used to study the effects of neurotrophins (for example, NGF or NT-3) on the survival and / or function of cholinergic neurons. The ChAT assay also serves as an indication of the regulation of ChAT levels within cholinergic neurons. Methods: Fetal rat spinal cord cells were dissociated, and experiments were performed as described by Smith et al., J. Cell Biology, 1985m 101, 1608-1621; Glicksman, et al., J. Neurochem, 1993, 61, 210-221). Dissociated cells were prepared from spinal cords extracted from rats (embryonic days 14-15) through standard trypsin dissociation techniques (Smith et al., Supra.). The cells were placed at 6 x 10 5 cells / cm 2 on plastic tissue culture cavities coated with poly-1-ornithine in a serum-free N 2 medium supplemented with 0.05% bovine serum albumin (BSA) (Bottenstein et al. others, Proc. Nati, Acad. Sci. USA, 1979, 76, 514-517). 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 procedure (Fonnum, Neurochem, 1975, 24, 407-409) according to McManaman et al., And Glicksman, et al., / McManaman, and others. , Develop. Biol., 1988, 125, 311-320; Clicksman, et al., J. Neurochem, supra). The compounds with the formula II described in the examples are listed in Table 2. The values for R1, R4, R6, and R7 are H; Cast; and n is 1.

Table 2 Example 13 PCDNA3-EE-MLK3. pcDNA3-EE-MLK3 (K144R) MLK3 was cloned as described (Lee, et al., Oncogene, 1993, 8, 3403-3410; Ezoe et al., Oncogene, 1994, 9, 935-938). CDNA was prepared from 200 ng of polyadenylated melanocyte mRNA and 5% of the reaction was used as a template to amplify a repertoire of PTK cDNAs using mixtures of either 2 or 4 highly degenerate oligonucleotide primers derived from the consensus sequences of the Vib and IX subdomains conserved from known PTKs: PTK1, 5'- CGGATCCACMGIGAYYT-3 '(SEQ ID NO: 1); PTK2, 5'- GGAATTCCAWAGGACCASACRTC-3 '(SEQ ID NO: 2); PTK3, 5'CGGATCCRTICAYMGIGAYYTIGCIGCIMGIAA-3 '(SEQ ID NO: 3); PTK4, 5'-GGAATTIAYIGGAWAIGWCCAIACRTCISW-3 '(SEQ ID NO: 4). 40 cycles of PCR were performed using Taq DNA polymerase (AmpliTaq, Perkin-Elmer / Cetus) and an automatic DNA thermal cycler; each cycle consisted of 40 seconds at 94 ° C, 2 minutes at 37 ° C and 3 minutes at 63 ° C. The products of 8 PCRs were combined, treated with DNA polymerase (Klenow), separated with BamH1 plus EcoR1 and electrophoresed on a 5% polyacrylamide gel. Staining with ethidium bromide identified a predominant band of 200-230 bp which was separated, eluted and cloned into M13mp18. In one experiment, part of the cDNA amplified by PCR was not separated, but rather was cloned into a shaved end in M13mp18 separated with Smal. The nucleotide sequences were determined through the chain termination sequencing method. A cDNA, identified as PTK1, was used as a probe to classify collections of human melanoma and melanocyte cDNAs. One clone, designated as PTK1-3.2, included the entire open reading frame of 2541 nt, coding for a protein of 847 amino acids. This cDNA was cut with Ncol, shaved at the end with DNA polymerase (Klenow), cut again with EcoRI and ligated to the pCDNA3-EE vector cut with BamHI., shaved at the end and then cut with EcoRl. The vector pCDNA3-EE was constructed by inserting into the HindIII / BamHI site an oligo coding for a start codon followed by an EE epitope, MEEEEYMPME (SEQ ID NO: 5) (Grussenmeyer et al., Proc. Nati. Acad. Sci. USA 1985 , 82, 7952-7954). The kinase death version of MLK3 was performed by making the K144R mutation using PCR using a previously published technique (Chen et al., Biotechniques, 1994, 17, 657-659). The first mutagenic oligo was 5'-GTGGCTGTGCGGGCAGCTCGCCAG-3 '(SEQ ID NO: 6) and the second oligo was 5'-GAGACCCTGGATCTCGCGCTT-3' (SEQ ID NO: 7). Using MLK3 as a template, these oligos were used in PCR to generate an 806 bp fragment and were used in a second PCR reaction using a T7 primer as the other amplimer and MLK as the template to generate a 1285 bp fragment. The fragment was separated through agarose gel electrophoresis, isolated, cloned into pGEM-5 (Promega) and sequenced. The fragment was separated with Hindlll and Hpal, and inserted into pCDNA3-EE-MLK3 cut with HindIII and Hpal. An additional spot mutation was detected at nucleotide 1342. To correct this, a Pf1M1 fragment (nt 1093-1418) was separated from wild type MLK3 and used to replace the identical fragment in MLK3 mutated with K144R.

Example 14 PFB-FLAG-MLK3 To obtain the MLK3 protein, the cDNA was cloned into the baculoviral expression vector pFB-FLAG. The MLK3 was separated from PTK1-3.2 through digestion with Ncol, was shaved at its end with DNA polymerase (Klenow), cut again with Notl and ligated into pFB-FLAG with Stul and Notl. PFB-FLAG was derived from pFB (Life Technologies) and has the coding sequence for the FLAG epitope (Hopp, et al., Biotechnology, 1988, 6, 1205-1210) with a start codon, MDYKDDDDK (SEQ ID NO: 8), added to the polylinker at the BAMH1 site.

Example 15 PFB-GST-MLK3ÍKD) Baculoviral expression of the MLK3 kinase domain was achieved by separating the MLK3 fragment from pGEXKG-MLK3 (KD) using EcoRI and Xhol and ligating it to a pFB vector cut with EcoRI and Xhol, wherein the coding sequence for glutathione S-transferase (GST) had been cloned upstream.

This was achieved by obtaining the GST coding sequence and the polylinker of the vector pGEXKG through PCR using the vector as a template (Guan, et al., Anal. Bioch., 1991, 192, 262-267). The 5 'oligo for PCR created a Bg12 restriction site at the 5' end of the fragment. This isolated fragment was then digested with Bg12 and HinD3 and ligated into pFB digested with BamHI and HinD3.

Example 16 pGEXKG-MLK (KP) A cDNA fragment that included both the MLK3 kinase domain and a portion of the leucine lock (nt 736-1791) was obtained by PCR using the PTK1 cDNA. The isolated fragment was digested with the restriction enzymes EcoRI and Xhol, the sites that were included in the oligos PCR, and cloned in pGEX-KG digested with EcoRI and Xhol. This fragment in pGEX-KG was then shortened through PCR to include only the kinase domain (nt 736-1638).

Example 17 PKH3-MLK2. pKH3-MLK2 (KA) MLK2 was cloned using degeneration PCR (Dorow, et al., Eur. J. Biochem., 1993, 213, 701-710; Dorow, et al., Eur. J. Biochem., 1995, 234 , 492-500). The cDNA segments encoding their catalytic domains of protein kinases expressed in Coló 6 epithelial tumor cell line were amplified through RNA by reverse transcriptase PCR. The degeneracy PCR primers were based on sequences encoding conserved motifs in the Vib and VIII subdomains of the kinase catalytic domains of the epidermal growth factor receptor family. The sequences of the primers were as follows: starter, 5'-CGGATCCGTG (A) CACC (A) GT (CG) G (A) ACC (T) T-3 '(SEQ ID NO: 9), reverse primer , 5'-GGAATTCACCA (G) TAA (G) CTCCAG (C) ACATC-3 '(SEQ ID NO: 10). Several PCR products were cloned in M13 and sequenced using a T7 Super-Base sequencing kit (Bresatec). A 216 bp PCR product was used as a probe to classify a human gt11 DNA colony collection (Clontech, catalog # HL10346). The fragment was randomly labeled by initiator, hybridization was performed at 65 ° C and the filters were washed at a severity of 0.2X NaCl / Citrate (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.5% SDS at 65 ° C. The filters were autoradiographed for 16 hours at -70 ° C on a Kodak XAR-5 film. Four clones were isolated and the longest, 1.2 kb was used to replace a probe in the same collection using the same conditions. Four more clones were selected and one of these clones represented a 1034 bp fragment of MLK2. This clone was used to rapidly heat and cool a human brain gt10 collection. Approximately 500,000 clones were classified and a clone of 3454 bp was isolated, representing the entire coding region of MLK2. MLK2 was cloned, from ATG to the polyA endpoint, in the pKH3 vector between the BamHI and EcoRI sites in two steps since there is a BamHI site in the middle of the MLK2 sequence. The pKH3 vector was constructed by inserting three copies of the HA epitope tag followed by a BamHI site between the Xbal and EcoRI sites of the polylinker pRK7. To make the mutagenized version, K125A, the 5'BamHI fragment of MLK2 was cloned into the PromegapAlter vector and mutated as recommended by the manufacturer. The fragment was cloned into the pKH3 vector of MLK2.

Example 18 PCDNA3-HA-JNK1 JNK1 cDNA was obtained as described (Coso et al., Cell, 1995, 81, 1137-1146). The cDNA was obtained by PCR using as human sperm muscle cDNA template (Invitrogen) and cloned into the Bg12 / Sal1 sites of pcDNA3-HA, a modified pcDNA3 expression plasmid encoding the HA epitope (Wilson, and others, Cell, 1984, 37, 767-778). This was then separated from pcDNA3, including the HA epitope and ligated to pGEX-473 (Pharmacia). The JNK1 cDNA was separated from the pGEX-473 construct as a Bg12 / Sal1 fragment and ligated to pcDNA3-HA, a vector with the HA epitope added to the HinD3 / BamHI site of pcDNA3.

Example 19 pFLAG-DLK DLK was cloned into the pcDNA3 expression vector with the FLAG epitope added as described (Holzman et al., J. Biol. Chem., 1994, 269, 30808-30817). A fragment of the cDNA for DLK was isolated through cloning of PCR based on degenerating oligonucleotide. Total RNA was extracted from embryonic kidneys from day 13.5 (32 organs) and embryonic kidneys from day 17.5 (16 organs) using a guanidine phenol / isothiocyanate reagent method commercially prepared according to the directions of the manufacturer (TRIzol Reagent, Life Technologies, Inc.). After digestion with Dnasel, Rnase-free total RNA was reverse transcribed with reverse transcriptase-Rnase H (Superscript, Life Technologies, Inc.) from an oligo (dT) synthetic oligonucleotide primer to DNA-structure cDNA. individual chain. The degenerate oligonucleotide primers corresponding to the protein tyrosine kinase subdomains of Vib and IX protein originally designated by Wilks (Wilks, Proc. Nati, Acad. Sci. USA, 1989, 86, 1603-1607) were modified in the EcoRI and Hind 111 5 'sites, respectively, (5'-ATAATTC (GT) GC (TAGC) GCCA (GA) GTC (TAGC) CGGTC-3' (SEQ ID NO: 11), 5'- ATAAGCTTCC (TC) ( AG) T (GC) AAGTGGA (TC) (GC) GC (AGC) CC (CT) GA-3 ') (SEQ ID NO: 12). 40 PCR cycles were performed for 1.5 minutes at 94 ° C, 2 minutes at 37 ° C and 3 minutes at 63 ° C. Fresh reagents were added and an additional 40 cycles were completed before the final extension of 10 minutes at 72 ° C. The resulting 200-210 bp DNA amplification product was isolated by gel, subcloned into the plasmid pGEM7zf (+) (Promega), and transformed into Escherichia coli. A minipreparation of plasmid DNA was prepared from transformed bacteria and a portion was digested with restriction endonucleases EcoRI and HindIII; the clones containing the grafts were sequenced. The 195 bp DLK cDNA fragment obtained from the degeneracy PCR was radiolabelled and used to sort approximately 1 x 10 6 recombinants from a collection of adult mouse brain cDNA initiated with Uni-ZAP II (Stratagene, La Jolla, CA) , oligo (dT) (Holzman et al., Mol.Cell.Biol, 1990, 10, 5830-5838). The filters were hybridized in a pH buffer consisting of 50% formamide, 5 x SSC, 3 x Denhardt's solution, 0.25% SDS, 1 mg / ml polyadenylic acid, and 200 mg / ml salmon sperm DNA at 42 ° C. The filters were washed once at room temperature in 2 x SSC, 0.2% SDS and twice for 30 minutes at 65 ° C. We identified 25 unique clones; 10 clones were purified to homogeneity, in vivo they were separated according to the manufacturer's protocol and restriction maps were made. The two largest clones (3401 and 3697 bp, respectively, differing only in their 5 'terms) were sequenced along both structures over their entire length.

The full-length Notl-Xhol DLK cDNA fragment (3401 bp) was subcloned into the cytomegalovirus promoter based on pcDNA3 of the eukaryotic expression vector (Invitrogen, San Diego, CA), (construction designated pcDNA3-DLK). Then, an activated construction of the epitope NH4-Met FLAG (DYKDDDDK) (SEQ ID NO: 13) (SEQ ID NO: 13) (pFLAG-DLK) was made. PCR was used to amplify cDNA fragments encoding the 5 'Hindlll site, the consensus sequence of DLK's Kozak's including ATG of initiation, the FLAG epitope and the open reading frame sequence of DLK cDNA extending from nucleotide 88 to an internal EcoRI site at nucleotide 758. (HPLC purified synthetic oligonucleotides used in equimolar quantities: 5'- ATAAAGCTTCCAGAGGCCATGGACTACAAGGACGACGATGACAAGGC-CTGCCTCCATGAAACCCGAACA-3 '(SEQ ID NO: 14) for the construction sense primer FLAG and 5'-GACAGGGCGGCCGGCTCT-3' (SEQ ID NO: 15) for the antisense primer). The amplified fragments digested with Hindll and EcoRl- and gel-purified were subcloned into the HindIII-EcoRI site to prepare plasmid pcDNA3-DLK. The constructs were sequenced along both structures to ensure the fidelity of Taq polymerase and the maintenance of the reading frame.

Example 20 PCDNA3-MLK1 The 5 'portion of MLK1 was obtained from the EST database (Accession No. AA160611). This clone was a fusion between MLK1 and another cDNA of unknown identity. It contained the previously unpublished 5 'sequence of MLK1 together with part of the previously published kinase domain of MLK1 (Dorow, et al., Eur. J.

Bíochem., 1993, 213, 701-710). The cDNA sequence of MLK1 from EST clone is as follows: GAATTCGGCA CGAGAGGACT CGCAGGTGTC CGGCGACGAG GGCTGGTGGA CCGGGCAGCT GAACCAGCGG GTGGGCATCT TCCCCAGCAA CTACGTGACC CCGCGCAGCG CCTTCTCCAG CCGCTGCCAG CCCGGCGGG AGGACCCCAG TTGCTACCCG CCCATTCAGT TGTTAGAAAT TGATTTTGCG GAGCTCACCT TGGAAGAGAT TATTGGCATC GGGGGCTTTG GGAAGGTCTA TCGTGCTTTC TGGATAGGGG ATGAGGTTGC TGTGAAAGCA GCTCGCCACG ACCCTGATGA GGACATCAGC CAGACCATAG AGAATGTTCG CCAAGAGGCC AAGCTCTTCG CCATGCTGAA GCACCCCAAC ATCATTGCCC TAAGAGGGGT ATGTCTGAAG GAGCCCAACC TCTGCTTGGT CATGGAGTTT GCTCGTGGAG GACCTTTGAA TAGAGTGTTA TCTGGGAAAA GGATTCCCCC AGACATCCTG GTGAATTGGG CTGTGCAGAT TGCCAGAGGG ATGAACTACT TACATGATGA GGCAATTGTT CCCATCATCC ACCGCGACCT TAAGTCCAGC AAC (SEQ ID NO: 16). This is translated to: NSAREDSQVS GDEGWWTGQL NQRVGIFPSN YVTPRSAFSS RCQPGGEDPS CYPPIQLLEI DFAELTLEEI IGIGGFGKVY RAFWIGDEVA VKAARHDPDE DISQTIENVR QEAKLFAMLK HPNIALRGV CLKEPNLCLV MEFARGGPLN RVLSGKRIPP DILVNWAVQI ARGMNYLHDE AIVPIHRDL KSSN (SEQ ID NO: 17). The 3 'portion of MLK1 was initially cloned through degeneration PCR as previously published (Dorow et al., Eur. J. Biochem., 1993, 213, 701-710). The protocol for cloning the 3 'portion of MLK1 was as described above for MLK2 with the following exceptions. Of the four clones obtained from the additional classification of the collection with the 1.2 kb clone, three of the four clones represented MLK1. None of the clones included the kinase domain, which was obtained through PCR. The phage of 1 ml aliquots of amplified collections, normal human colonic epithelium and human T84 colonic carcinoma cell line cDNA in Uni-ZAPXR (Stratagene, cat # 937204) was analyzed by suspending 20 ml of water and freezing. A 5 ml sample of the lysate phage was used as a PCR template in two reactions for each collection. The primers representing the vectors were taken from nucleotide sequences flanking the cloning sites. In the case of the T84 colonic cell line collection, the sequencing primers T3 and T7 (Promega) were used. In each reaction, one primer was from the 3'-5 'chain structure of the MLK1 gene, approximately 100 bp from the 5' end of the known sequence. The second initiator was one of the two vector primers. The PCR reactions contained 1X of PCR buffer, 2.5 mM of magnesium chloride, 1U of Taq polymerase (All of Bresatec), 0.2 mM dNTP and 0.4 mM of each primer in a total of 50 ml. The reaction conditions were 60s at 95 ° C, 90s at 52 ° C, 90s at 72 ° C for 30 cycles with an extension time of 15 minutes in the final cycle. The PCR products were cloned and sequenced as described above. The longest clone of the collection classification and a PCR fragment that included an additional MLK1 sequence were ligated together to create a 1.08 kb MLK1 cDNA in pUC18. The MLK1 clone of the EST database was provided in the vector pBluescript (Stratagene). The MLK1 cDNA from the colonic collection was ligated to the EST clone by digestion of the first with EcoRI, shaved at its end with Klenow, then cut with Aflll. This isolated fragment was cloned into the MLK1 cDNA of the EST database cut with Xhol, shaved at its end with Klenow, and cut with Aflll. This new construction was then separated from pBluescript by digestion with Notl and Apal and ligated to pcDNA3-EE also cut with Notl and Apal. All cloning junctions were verified in sequence.

Example 21 Expression of E. coli from GST-MLK? PGEXKG (KD) was transformed into E. coli strain BL21 by electroporation. The bacteria containing the plasmid were inoculated with an Applikon fermenter of 15 liters in a volume of 10 liters of the following enriched medium: 1.95 g / L K2HPO4, 0.9 g / L KH2PO4, 0.1 g / L ampicillin, 0.3 g / L (NH4) 2 SO4, 0.92 g / L MgSO47H2O, 42.7 mg / L sodium citrate, 21.8 mg / L FeSO47H2O, 0.5 ml trace metal Pichia (Higgins, et al., Methods Molecular Biology, 1998, 103, 149-177), 20 g / L of casamino acids, 40 g / L of glycerol, 25.5 mg / L CaCl2. Bacteria were grown overnight at 800 rpm / 68% dissolved oxygen / 30 ° C until the culture reached OD600 = 4.4. The production of recombinant protein was induced by the addition of 1 mM isopropyl-β-D-thiogalactoside, with continuous fermentation at 25 ° C for 6 hours. The bacteria were then recovered through centrifugation and the cell paste was stored frozen at -20 ° C until purification.

Example 22 Purification of GST-MLK3 p Partially purified GST-MLK3 D was prepared by applying sound to 100 g of bacterial cell paste in 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol (DTT), pH 7.5 (pH regulator A). The solution was made at 1% with Triton X-100, then stirred on ice for 1 hour. The solution of the supernatant after centrifugation for 45 minutes at 20,000 xg was mixed for 1 hour on ice with 10 ml of Sepharose 4B glutathione resin (Pharmacia) equilibrated in the pH regulator A. The pelletized resin was washed twice. times with 12.5 x volume buffer pH A, then eluted with 20 ml of 199 mM Tris-HCl, 150 mM NaCl, 5 mM DTT (buffer B), containing 20 mM glutathione pH 7.5. The protein was dialyzed overnight against pH B regulator and stored in aliquots at -80 ° C.

Example 23 Baculoviral Expression of FLAG-MLK3 v GST-MLK3 * r. Recombinant baculoviruses were produced by expressing FLAG-MLK3 and GST-MLK3KD from their respective transfer vectors, pFB-FLAG-MLK3 and pFB-GST-MLKKD, using the BAC-TO-BAC system (Life Technologies) according to the manual Instructions. Sf 21 cell suspension cultures (Vaughn, et al., In Vitro, 1977, 13, 213-217) were developed at 27 ° C / 129 rpm in a supplemented Grace medium (Hink, Nature, 1970, 226, 466- 467) with 10% heat inactivated fetal bovine serum (FBS). To produce FLAG-MLK3, recombinant, Sf21 cells were infected at a density of 1.5 x 10 6 / ml of supplemented Grace's medium containing 5% FBS, with an infection count (MOI) of 3.1 and harvested at 39 hours after infection . To produce recombinant GST-MLK3KD, Sf 21 cells were infected at a density of 1.5 x 106 cells / ml of Grace's medium supplemented containing 5% FBS with MOI of 2 and harvested at 41 hours after infection. In both cases, pelleted cells were resuspended in pH buffer composed of 10 mM HEPES, 50 mM NaCl, 0.5 mM Pefabloc SC, 5 μM pepstain, 10 μg / ml aprotinin, 10 μg / ml leupeptin, pH 7.4. The supernatant solution after centrifugation for 1 hour at 147,000 x g was re-adjusted to a pH of 7.4 with 3 M of Tris base and then stored at -70 ° C before purification.

Example 24: Purification of Baculoviral GST-MLK3 GST-MLK3 Baculoviral D was prepared, partially purified by glutathione affinity chromatography. For 10 ml of the cell extract (26.6 mg of total protein), 1 ml of Sepharose 4B glutathione resin (Pharmacia) equilibrated in 10 mM HEPES, 150 mM NaCl, pH 7.4 (buffer pH C) and the protein were added. it was allowed to bind for 45 minutes at 4 ° C. The resin was then washed in a column format with 30 column volumes of the pH C regulator, then eluted with 5 column volumes of the pH C buffer containing 20 mM glutathione. The combined final product was dialyzed overnight against pH C regulator and stored in aliquots at -70 ° C.

Example 25 Purification of baculoviral FLAG-MLK3 Baculoviral FLAG-MLK3 partially purified was prepared by antibody affinity chromatography. The 15 ml protein of the extract (19.5 mg of total protein) with additional 0.1 M NaCl was ligated onto a 0.25 ml column of the monoclonal FLAG peptide antibody M2 coupled to the agarose resin (Sigma) via repeated loading (a total of 3 times). The resin was equilibrated with a 5 column volume wash of 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS), a 3 column wash of 0.1 M glycine, pH 3.5, followed by another washing of 5 column volumes with TBS, before chromatography. The recombinant protein was mainly eluted with 5 column volumes of 0.2 mM of the FLAG peptide (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) (SEQ ID NO: 18), in TBS. The protein was stored in aliquots at -80 ° C before analysis.

Example 26 Dominant Negative Mutant: a dominant negative mutant of the MLK family blogs death in differentiated PC12 cells after the removal of nerve growth factor The PC12 cell line derived from a rat pheochromocytoma tumor was used extensively as a Neuronal cell model to examine molecular events leading to neuronal death (for review, see Troy et al., Adv. Neurology, 1997, 103-111). Nerve growth factor (NGF) induces PC-12 cells to differentiate into a sympathetic neuronal phenotype (Greene, Cell Biol., 1978, 78, 747-755). The PC-12 cells differentiated by NGF are dependent on NGF for survival and undergo a morphologically described apoptotic death after removal of NGF from the culture medium. A cell system was developed to determine the effect of members of the mixed lineage kinase family on PC-12 cell death after NGF withdrawal. PC-12 cells were transfected with cDNA encoding a dominant negative (DN) mutant of MLK-3 using the Pfx lipid transfer system as recommended by the manufacturer (Invitrogen, Carisbad, CA). A stable combination of DN-MLK-3 expressing the transfectant was selected using G418 sulfate (Mediatech Inc., Herndon, VA). Approximately 30% of the cells in these combinations express DN-MLK3 as determined by immunohistochemistry. Combinations of cells stably expressing the mutant kinase were plated in a 96-cavity culture tissue format coated with polyornithine / laminin (10 μg / ml each in phosphate buffered saline) at a density of 2. x 104 cells / well and treated with 100 ng / ml of NGF for 7 days. The medium containing the NGF was removed, the cell monolayer was washed with saline regulated at its pH with phosphate and the medium containing the neutralizing NGF antibody (cat. # N6655; Sigma, St. Louis, MO) at a final dilution of 1: 1000 was replaced for 1-5 days. Cell viability was quantified through a cell viability assay using the conversion of the tetrazolium salt, MTS, to a color formaan, which was read at an absorbance of 570 nm on a CytorFluor 2350 (Millipore, Bedford , MA) as recommended by the manufacturer (Promega, Madison, Wl). Stable combinations expressing DN-MLK-3 were partially rescued from cell death caused by the removal of NGF (Figure 10).

Example 27 Assay for Enzymatic Activity of Recombinant MLK Protein In order to demonstrate that the MLK protein expressed in its baculovirus or bacterial expression system is enzymatically active, various assay formats can be used. The MLK protein can be a full-length construct or a kinase domain expressed either in a baculovirus or bacterial expression system. The assay can be antibody-based such as the enzyme-linked immunosorbent assay (ELISA), time resolved fluorescence (TRF) or fluorescence polarization (FP). The antibody can be monoclonal or polyclonal with reactivity to phosphoserine, phosphothreonine, or a phospho-specific substrate. Alternatively, a non-antibody-based method such as a radioactive gel-based assay (see FIG. 11), multiple-classification trichloroacetic acid (TCA) precipitation assay (FIG. 13), scintillation proximity assay ( SPA), instant vaporization plate method, or a phosphocellulose filter assay format (Figure 13). The assay can be designed to verify direct phosphorylation of a substrate or a coupled assay system using the kinases downstream in the signaling path. The substrate can be a specific substrate such as SEK-1 or a relatively non-specific substrate such as a basic myelin protein (MBP).

Example 28 Kinase Assays (1) Radioactive Gel-based Kinase Assay. MLK-3 kinase activity was analyzed by checking the 32P incorporation of [? -32P] -ATP to a MLK substrate (eg, kinase killing SEK-1, myelin basic protein). The 50 μl assay mixture contained the pH regulator A (20 mM MOPS, pH 7.2, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol), mM MgCl2, 100 μM ATP, 10 μCi [? -32P] -ATP, and 0.1 μg substrate of SEK-1 kinase killing (Stressgen, Inc., S-transferase-SEK-1 (GST-SEK-1) of bound glutathione was released from glutathione-agarose beads with 10 mM glutathione, pH 8.0, or 25 μg MBP (Sigma Chemical Co.). The reaction was initiated by adding MLK protein (kinase domain or preparation containing both full length and kinase domain) or control protein.The mixture was incubated for 30 m inutes at 30 ° C. At the end of the reaction, the pH regulator of the 2 × reduction sample was added.The mixture boiled for 5 minutes, it was loaded either on a 12% SDS-PAGE gel (using MBP as substrate) or 8% gel (SEK-1 as a substrate) After the electrophoresis, the gel was dried. Phosphate poration in the substrate, SEK-1, was performed in a Molecular Dynamics Phosphorimager apparatus (Sunnyvale, CA). The results of the experiments designed to show the enzymatic activities of MLK-3 expressed in baculovirus (full length tagged with FLAG or kinase domain marked with GST) using GST-SEK-1 kinase kill or MBP as substrate are shown in the Figures 11A and 11B. (2) Western stain analysis. The kinase activity of MLK-3 expressed in baculovirus was examined through immunostaining analysis. The 20 μl assay mixture contained the pH A regulator, 15 mM MgCl 2, 100 μM ATP, and 0.1 μM substrate of SEK-1 kinase killing. The reaction was allowed to proceed for 30 minutes at 30 ° C, then was quenched with 10 μl of 4 x pH regulator sample reduction. The proteins were separated on an 8% Tris-glycine gel and electrophoretically transferred to a PVDF membrane of Immobilon. The membrane was incubated with the phospho-specific antibody SEK-1 (Thr223) (New England Biolabs, Inc.), followed by goat anti-rabbit IgG labeled with horseradish peroxidase (Bio-Rad). The detection of the immunoreactive bands was performed through improved chemiluminescence (Amersham). The phosphorylation of GST-SEK-1 kinase death through the protein (FLAG-MLK3 (baculovirus preparation containing both full length and kinase domain) is illustrated in Figure 12. (3) Multiple Classification Trichloroacetic Acid (TCA) Precipitation Test. The kinase activity of the bacterially expressed GST-MLK-3 kinase domain was classified using the Millipore Multiscreen "in plate" trichloroacetic acid (TCA) assay as described by Pitt et al. J. Biomol. Screening, 1996, 1, 47-51. The assays were performed on 96-well Multiscreen Durapore plates (Millipore). Each 50 μl assay mixture contained 20 mM Hepes, pH 7.4, 20 mM MgCl2, 20 mM MnCl2, 2 mM DTT, 0.1 mM Na2VO4, 1 μCi [? -P32] ATP and 30 μg of the MBP substrate. The reaction was started by adding the MLK protein and allowed to proceed for 15 minutes at 37 ° C. The reaction was stopped with 25 μl of 50% TCA. The plates were allowed to equilibrate 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 the Wallac MicroBeta 1450 PLUS scintillation counter. The protein dose response against the formation of 32 P-labeled MBP is shown in Figure 13. (4) Phosphocellulose Filter Test. The kinase assay was performed in a 50 μl reaction mixture containing 20 mM Hepes, pH 7.4, 20 mM MgCl2, 20 mM MnCl2, 2 mM DTT, 0.1 mM Na3VO4, 1 μCi [? -P32] ATP and 30 μg MBP . The reaction was started by adding the MLK protein and allowed to proceed for 15 minutes at 37 ° C. The reaction was stopped with 75 μl of 75 mM phosphoric acid. An aliquot of the extinguished solution was loaded directly onto the phosphocellulose membrane (Pierce). Alternatively, the 96-cavity phosphocellulose multiple classification plate (Millipore) can be used. The membranes were washed with 75 mM H3PO4. Phosphorylated and labeled 32P-labeled MBP was eluted in collection tubes by adding 1 M sodium hydroxide. Radioactivity was determined by Cerenkov count in a scintillation counter (Somerset, NJ). The formation of phosphorylated MBP with an increasing concentration of the bacterially expressed GST-MLK-3 kinase domain is shown in Figure 13.

Example 29 Assay to determine the binding of compounds to the family of recombinant MLK K-252a (Compound HI-3, see Table 4), a metabolite of indolocarbazole of the species Nocardia, binds to a variety of serine / threonine kinases and tyrosine (Angeles et al., Anal. Biochem, 1996, 236, 49-55; Knight et al., Anal. Biochem., 1997, 247, 376-381). A ligand of titrated K-252a was used to determine binding to human recombinant full length MLK-3 from a crude preparation of baculovirus infected cells. [3H] K-252a was specifically labeled with tritium at positions 3 and 9 through contact with products from NEN Research (Billerica, MA) and had a specific activity of 40 Ci / mmoles. Binding reactions were performed in 1 ml in a 96-well plate. The reaction mixture contained 50 mM MOPS, as pH buffer, pH7, 150 mM NaCl, 5 mM MnCl 2, 1 mg / ml BSA, 1% DMSO and 0.25 nM [3H] K252a. The samples were made in triplicate with a concentration of 5 μg / ml of MLK-3 derived from crude baculovirus. A non-specific binding was defined as binding in the presence of 1.2 uM of unlabeled K252a and was subtracted from the total binding to give the specific binding. At this dilution, 12-15% of the total accounts were not specifically bound to the protein, and 75-85% of these accounts were specifically bound to MLK-3 (Figure 14). All experiments were carried out for 2 hours at 4 ° C. Complexes of [3H] K252a / MLK-3 were collected on GF / C Whatman filters using a Brandel harvester washed with cold pH buffer of MOPS / NaCl and counted in a Wallac Micro Beta counter. A saturation binding experiment was performed to obtain a Kd for K252a. An example of the results of one of these experiments is shown (Figure 14). A Kd of 0.89 nM (Confidence Limits: 0.2 to 1.5 nM) was obtained.

Example 30 Intact Cell Assays (A) Cos Overexpression System 7 Materials K-252a and derivatives of this compound were provided by Kyowa-Hakko Kogyo Co. Ltd. (Tokyo, Japan) (Kaneko et al., 1997).

The compounds were dissolved in dimethyl sulfoxide (DMSO) of cell culture grade and stored in the dark at 4 ° C.

All dilutions of the compounds were made in Dulbecco's modified Eagle medium (DMEM) containing 1% bovine serum albumin. The Hemagglutinin antibody was purchased (HA) at BabCO (Richmond, CA). Substrate AP-1 (c-jun) was purchased from Promega (Madison, Wl). [? -32P] ATP (6000 Ci / mmol) was purchased from Amersham (Arlington Heights, IL).

Cultivation of Cos7 Cell. Cos7 cells were obtained from Mono Mono Riñon ATCC, Rockville, Maryland (CRL 1651) and were maintained in DMEM containing 10% bovine serum, 2 mM glutamine, 1 mM p-ruvate, 50 U / ml penicillin / streptomycin at 37 ° C in 10% CO2, 90% air atmosphere. The Cos7 cells were separated for the step by adding 0.25% trypsin. (1) Over-expression of members of the MLK family and JNK1 in Cos7 cells were plated at 80% confluence and were transfected with 2 μg each of cDNA constructs using lipofectamine as recommended by the provider (Gibco BRL , Gaithersburg, MD). A full-length cDNA of human MLK-3, MLK-2, or mouse DLK or a partial human MLK-1 as described above, and human JNK1 labeled with full-length hemagglutinin A, amicably provided by J. Silvio Gutkind ( NIH, Bethesda, MD), were subcloned into the pcDNA3 vector (Invitrogen, San Diego, CA). After 48 hours of transfection, the cells were treated with 0.025% DMSO or 500 nM of the indicated compounds for 2 hours followed by lysis in a 0.4 ml Triton pH regulator (1% Triton X-100, 50 mM of sodium chloride, 10 mM Tris (pH 7.6), 0.1% bovine serum albumin, 30 uM sodium pyrophosphate, 50 mM sodium fluoride, 20 ug / ml aprotinin, 1 mM phenylmethylsulfonyl fluoride , 1 mM sodium vanadate). The JNK activity of lysate was analyzed and analyzed by an immunoprecipitation / kinase assay as described below. (2) Immunoprecipitation and Kinase Assay from Whole Cells The Cos7 cell lysate was measured for protein concentration using the Pierce micro BCA kit (Rockford, IL) and equal amounts of protein were immunoprecipitated with the HA antibody for 1 hour. hour at 4 ° C. The immunoprecipitates were pelleted through centrifugation in a microfuge centrifuge for 20 seconds. They were resuspended in Triton's pH buffer, washed through centrifugation twice more, followed by a final wash in kinase pH buffer (20 mM Hepes pH 7.4, 20 mM MgCl 2, 2 mM dithiothreitol, 0.1 mM, 0.1 mM sodium vanadate). The immunoprecipitate was resuspended in a kinase pH buffer containing 1 μM ATP and 5 μCi [? -32 P] ATP and substrate (1 μg / sample of AP-1) and incubated for 15 minutes at 30 ° C. The kinase reaction was stopped by the addition of the pH regulator of the reduction sample (Laemmli, Nature 1970: 227; 680-685). The samples were heated at 80 ° C for 5 minutes and loaded onto 10% SDS-polyacrylamide gels. The proteins were separated through electrophoresis. The gel was dried and the quantification of radioactivity on the AP-1 substrate was performed in a Molecular Dynamics Phosphorimager (Sunnyvale, Ca). The results of the experiments in which MLK-3, ML-2 and DLK are co-expressed with HA-JNK1 and incubated in the absence or presence of K-252a, are shown in Figures 15A and 15B. In contrast, a derivative of the compound of origin K-252a termed compound III-3 (see Table 4), which is a more selective kinase inhibitor, did not interfere with the JNK pathway activated by another MAPKKK upstream of JNK, MEKK1 (Figure 15C).

(B) Whole Cell Reporting Assay for JNK Activated by MLK Attempts to derive clones constitutively expressing the MLK family has not been successful, suggesting that MLK overexpression can affect cell survival (Bergeron et al. Biochem Biophys, Res. Commun., 1997, 231, 153-155; Nagata, et al., EMBO J., 1998, 17, 149-158). Therefore, in the development of a whole-cell assay for tracking biochemical events induced by MLK, a cell line containing an expression system capable of genetically engineered induction of the kinase of interest may be required. For example, a PC-12 cell line was transfected with a transactivator controlled by tetracycline. When the cells were additionally transfected with a gene of interest activated by the promoter capable of teto induction, the expression of that gene is highly controlled by tetracycline in the medium (Shocken, et al., Proc. Nati. Acad. Sci. USA, 1995, 92, 6522).

To quantitate MLK activation, phosphorylation of downstream substrates such as MEK4, JNK or c-jun can be measured in multiple assay formats as described above. Another aspect to quantify the activation of MLK in whole cells is to use a reporter enzyme activity such as the c-jun luciferase report system commercially available through the PathDetect ™ system (Stratagene, La Jolla, CA). In this system, the cell line capable of tetracycline induction is transfected with two plasmids. A plasmid constitutively expresses a fusion of the transactivation domain of terminal NH2 of cJun with the DNA binding domain of GAL4 of yeast (fusion protein (cJun-DBD) .The other plasmid carries the coding sequences for firefly luciferase driven by five tandem repeats of the GAL4 binding site After activation of MLK, the substrate underneath the JNK fusion protein cJun-DBD is phosphorylated, binds to the GAL4 binding sites, and induces transcription of the luciferase gene. Luciferase is easily analyzed in cell lysates through the addition of its substrate (Promega, Madison, Wl) and chemiluminescence measurement.

Example 31 Association of Inhibition of MLK Family Members with Motoneurons and Cortical Survival Survival of Rat Spinal Cord Cultures Enriched for Motoneurons Spinal cords were separated from rat fetuses of Sprague-Dawley (Charles River Laboratories, Wilmington, MA) embryonic (E) 14.5-15. Cells were dissociated only from the ventral portion of the spinal cord, and were further enriched for motor neurons through centrifugation in a gradient of 6.5% step metrizamide, as previously described (Henderson et al., 1993), and analyzed for purity through staining with a low affinity neutrophin receptor antibody (IgG-192, Boehringer-Mannheim) (data not shown). The cells were seeded on 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 (Bottenstein, and others, 1979, supra). In order to separate the binding of survival effects, the addition of compounds to cultures was made after an initial binding period of 1-3 hours. Neuronal survival was analyzed after 4 days using calcein AM (Molecular Probes, Eugene, OR) in a fluorometric viability assay (Bozyczko-Coyne, et al., 1993, supra). The microscopic accounts of neurons were directly correlated with relative fluorescence values. Briefly, the culture medium was serially diluted in DPBS (saline regulated at its pH with Dulbecco's phosphate) and a final concentration of 6 uM of calcein A supply material was added after each 96 wells. Plates were incubated for 30 minutes at 37 ° C, followed by serial dilution washes in DPBS. The fluorescent signal was read using a Millipore plate reading fluorometer (Cytofluor 2350) at an excitation of = 485 nm and an emission of = 538 nm. For each plate, the average antecedent derived from cavities that receive calcein AM but that do not contain cells, was subtracted from all the values. The linearity of the fluorescence signal was verified for the concentration and incubation time for the scale of cell densities in these experiments. An example of the survival percentage above the motor neuron control in the presence of test compounds at 250 nM is shown in Table 3.

Survival of Cortical Neurons Brain cortices were dissected from embryonic rat fetuses for 18 days and digested enzymatically to obtain an individual cell suspension. Cells were seeded at a density of 1.56 x 10 5 / cm 2 on 96-well tissue culture plates coated with poly-ornithine / laminin in a serum-free neural basal medium containing B27 supplements. The layers were covered with a poly-ornithine / laminin solution (8 μg / ml each) made in PBS for at least 2 hours at 37 ° C. On days 5-7 in vitro, cortical neurons were exposed to Ab25-35 (20 uM) either in the presence or absence of the test compounds. The supply solutions (1 mM) of Ab25-35 (Sigma, St. Louis, MO) were prepared in sterile deionized-distilled H2O. The relative neuronal survival was determined 48 hours after the addition of peptide using lactate dehydrogenase (LDH) released as an indicator of plasma membrane integrity / cell viability. LDH was measured using the cytotoxicity detection kit Cytotoxicity Detection Kit (Boehringer-Mannheim, Indianapolis, IN) according to the manufacturer's instructions. The data are expressed as percentage of inhibition of LDH released in relation to cultures treated only with Ab25-35.

Table 3 1 The Compound has the formula III wherein Z1t Z2, Ri and R2 are H; X is CO2CH3; and R is OH. 2 The compound has the formula III, wherein Z ^ and Z2 are H; X is CO2CH3; R, and R2 are CH2SCH2CH3; and R is OH. 3 The compound has the formula I wherein Ai, A2, R ,, R3, R5 and Re are H; Bi and B2 together represent O; R2 is CH2CH2Oac; R 4 is CH 2 CH 2 (2-pyridyl); and X is CH2. The compound has the formula I, wherein AL A2, RI, R3, R5, and Re are H; B- and B together represent O; R2 is H; R 4 is CH 2 CH 2 (2-pyrimidinyl); and X is CH2.

Example 32 Immunoprecipitation of Endogenous JNK Activity from Motoneuron Cultures in Absence or Presence of Fused Indolocarbazoles or Pyrrolocarbazoles The purified motoneurons were plated at a density of 6 x 10 4 cells / cm 2 in cavities with a diameter of 16 mm. The cells were allowed to bind for 2 hours before treatment. The cells were treated with either 0.0125% DMSO or 500 nM of the compound for 2 hours in a defined N2 medium. The cells were then rinsed with pH regulated saline with ice-cooled phosphate followed by lysis in 0.4 ml of triton pH regulator as described above in Example 30. The lysate from the motor neuron cultures was normalized to a number of cell and immunoprecipitated with a JNK1 antibody (cat. # sc-474) sold by Santa Cruz Biotechnology (Santa Cruz, CA). The JNK activity of the immunoprecipitates was analyzed in the presence of 32 P-ATP and c-jun substrate as described above. The profile of the inhibitory activity of the four test compounds was compared in motor neurons and in Cos7 cells overexpressing either DLK, MLK-1, MLK-2 or MLK-3 (Table 3).

Example 33 Correlation between the inhibition of JNK induced by MLK3 in Cos7 cells and activity of choline acetyl transferase in primary embryonic cultures To determine if the inhibition of the pathway of JNK regulated by these kinases was correlated with neurotrophic compounds, the effect of compounds on JNK activity in Cos7 cells over-expressing Ha-JNK and MLK3. After a transfection period of 48 hours, the cells were incubated with compounds at 500 nM for 2 hours followed by cell lysis. The lysate was immunoprecipitated and the kinase activity was measured as previously described. The results are reported as percentage of inhibition of the control sample, where the control is the JNK activity in the presence of DMSO. As can be seen in Table IV, most of the compounds that were active in the activity of spinal cord ChAT and / or basal forebrain were potent inhibitors of activation by JNK MLK-3.

Table 4 Effect of Indolo- and Idedeno-carbazoles on the activity of JNK 1 Compound having the formula III wherein Z ^ and Z2 are H; X is CO2CH3; R-t is NHCONHC2H5; R2 is CH2CH2 (2-pyridyl); and R is OH. 2 Compound having the formula III wherein Z ^ and Z2 are H; X is CO2CH3; i and R2 are CH2OCH2OCH2CH3; and R is OH. 3 Compound having the formula III wherein Z and Z2 are H; X is CO2CH3; RI and R2 are CH2SCH2CH3; and R is OH. 4 Compound having the formula I wherein AL A2, RI, R3, and R4 are H; B! and B2 together represent O; R2 is CH2CH2OH; R5 and R6 are OCH3; and X is CH2. 5 Compound having the formula I wherein Ai, A2, Ri, R3, Rs, and Re are H; B ^ and B2 together represent O; R2 is CH2CH2OAc; R is Br, and X is CH2. 6 Compound having the formula I wherein A (l A2, R1t R3, R5, and Re are H; B- and B2 together represent O; R2 is CH2CH2OAc; R4 is CH2CH2 (2-Pyridyl); and X is CH2 7 Compound having the formula I wherein Ai, A2, R-, R3, R4, R5, and R6 are H, B1 and B2 together represent O, R2 is CH2CH2OH, and X is CH2 8 Compound having the formula I wherein Ai, A, R1, R3, R, R5, and R6 are H; B1 and B2 together represent O; R2 is CH2CH2CH2OH; and X is CH2. 9 Compound having the formula I wherein A ,, A2, R ,, R2, R3, R4, R5, and Re are H; B1 and B, together represent O; and X is S. Compound having the formula I wherein Ai, A2, R1t R3, R4, R5, and R6 are H; B, and B together represent O; R2 is CH2CH2CH2NHCO (4- (OH) Ph); and X is CH2. 11 Compound having the formula III wherein Z-1, Z 2, R 1, and R 2 are H; X is CO2 (CH2) 4CH3; and R is OH. 12 Compound having the formula III wherein Z \, Z2, and R1, are H; R2 is CH2OH; X is CO2CH3; and R is OH. 13 Compound having the formula MI wherein Z and Z2 together form = O; R? and R2 are Br; X is CO2CH3; and R is OH.

Compound having the formula I wherein A ,, A2, R ,, R3, R5l and R6 are H; Bi and B2 together represent O; R2 is H; R 4 is CH 2 CH 2 (2-pyrimidinyl); and X is CH2. Compound having the formula III wherein Zi and Z2 are H; ^ is Br; R2 is 1; X is CO2CH3; and R is OH. 16 Compound having the formula III wherein Z1t Z2, R (, and R2 are H; X is CO2CH3; and R is OH.17 Compound having the formula lll wherein Z ,, and Z2 are H; R1 and R2 are CH2CH2SCH3; X is CO2CH3; and R is OH 18 Compound having the formula I wherein A1t A2, R1t R2, R3, R5, and R6 are H; Bi and B2 together represent O; R4 is CH2CH2 (2-Pyridazinyl) and X is CH2 19 Compound having the formula I wherein Ai, A2, R ,, R3, R5, and R6 are H; B and B2 together represent O; R: is H; R, is CH2CH2 (2- Pyridyl), and X is CH2 Compound having formula III wherein Z, Z2, R ^ and R2 are H, X is CO2CH3, and R is OCH2.1 Compound having the formula I wherein A ,, A2, R (, R3, R4, R5, and Rβ are H; -iy B2 together represent O, R2 is (CH2) 3-NH-C (= O) -3,5-dihydroxyphenyl, and X is CH2. Compound having the formula I wherein An, A2, Ri, R3, R4, R5, and R6 are H, B1 and B2 together represent O, R2 is benzoyl, and X is CH2. 23 Compound having the formula I wherein Ai, A2, R1, R2, R3, R5, and Rβ are H; B1 and B, together represent O; R is CH = CH-C = N; and X is CH2.

Example 34 Gel shift assay for MLK activation Activation of MLKs can lead to the induction of c-jun transcription resulting in an increased protein of c-Jun. The increased amount of c-Jun protein can be measured through a standard assay, identified as a gel shift assay. Garner et al., Nucleic Acids Res., 1981, 9, 3047-3060, which is incorporated herein by reference in its entirety. The radiolabelled double-stranded DNA structure oligomers, coding for a c-Jun DNA binding site, were incubated with a nuclear cell extract followed by acrylamide gel electrophoresis and the radiolabeled DNA shifted to a mobility was quantified slower. This represents the portion of the DNA that is bound to the c-JUN protein and is directly proportional to the amount of c-Jun protein in the extract. Activation of MLKs can also induce phosphorylation of c-Jun. This can be detected using antibodies that specifically recognize the phosphorylated form of the protein in protection system such as, for example, Western or ELISA stains.

Example 35 Survival of Embryonic Chicken Neurons Materials The Leibovitz L15 media, glucose, sodium bicarbonate, trypsin and antibiotics were from Gibco. A muscle extract was prepared as described (Henderson et al., Nature, 1983, 302, 609-611, which is incorporated herein by reference in its entirety). All other reagents were from Sigma, unless otherwise indicated.

Cell culture Motoneurons were isolated (embryonic days 5.5) with an immunological method according to the procedure established by Bloch-Gallego, et al., Development, 1991, 111-221-232, which is hereby incorporated by reference in its entirety, with modifications as described by Weng et al., NeuroReport, 1996, 7, 1077-1081, which is incorporated herein by reference in its entirety. The purified motoneurons were seeded onto 35 mm tissue culture dishes (Nunc) precoated with poly-DL-ornithine and laminin (1 g / ml, Upstate Biotech). The culture medium was L15 with sodium bicarbonate (22.5 mM), glycosa (20 mM), progesterone (2 x 10"8M), sodium selenite (3 x 10" 8M), with albumin (0.1 mg / ml), insulin (5 g / ml), penicillin-streptomycin, and horse serum inactivated with 10% heat. The muscle extract was supplemented at 30 μg / ml. Compound III-3 was prepared as a 4 mM supply solution in DMSO and stored protected from light at 4 ° C. The final concentration of DMSO in treated and control cultures was 0.125%. Paraventral sympathetic ganglia (SG, embryonal day 12 (E12)), dorsal root ganglia (DRG, E9), and ciliary ganglia (CG; E8) of chicken embryos were dissected on the embryonic day indicated as described by Lindsay et al. , Dev. Biol., 1985, 112, 319-328, which is incorporated herein by reference in its entirety. After trypsinization and dissociation, the nerve cell suspensions were plated onto culture dishes coated with polyornithine-laminin in Ham's F14 culture medium, supplemented with 10% horse serum. Immediately after plaque placement, survival factors were added at the following concentrations: nerve growth factor (NGF), 20 ng / ml; ciliary neurotrophic factor (CNTF), 10 ng / ml. The cultures were maintained at 37 ° C and 5% CO2 in a humid environment.

Cell count Neurons were plated on 35 mm culture plates with gratings (Nunc). The selected areas of each dish together comprise approximately 10% of the surface scanned by the presence of bright phase cells immediately after plating and again after 48 hours to determine survival percentages.

Cell survival was confirmed by vital staining with trypan blue (not shown).

DRG Intact Lymph nodes were placed in 96-well plates previously coated with poly-1 -ornithine and lamina (5 μg each / ml saline regulated at pH with phosphate) in a serum-free N2 medium (Bottenstein et al., Proc. Nati, Acad. Sci. USA, 1979, 76, 514-517, which is incorporated herein by reference in its entirety), containing 0.05% bovine serum albumin (BSA) and maintained for 48 hours at 37 ° C. and 5% CO2 in a humid environment. The treated lymph nodes received either 250 nM of compound III-3 or 20 ng / ml of NGF 2 hours after plating.

Compound III-3 supports the survival of chicken embryonic peripheral neurons in a concentration-dependent manner. Removal of NGF from dissociated cultures of sensory neurons of dorsal root ganglion E9 (DRG) and sympathetic ganglion neurons E12 (SG) caused them to undergo PCD in 48 hours. This was prevented through the addition of compound III-3 to the culture medium at the time of NGF removal. At 1 μM, compound III-3 maintained 94% of SG neurons and 890% of DRG neurons alive after 48 hours (control treated with NGF: SG 65%, DRG 66%). Similarly, compound IM-3 promoted the survival of 76% of ciliary ganglion neurons (GC) dependent on CNTF after 24 hours (control treated with CNTF 67%). In the presence of 10% serum, the survival effects of compound III-3 were concentration dependent, with a plate attained around 1 μM for the three neuronal populations (Figure 16A-C). Neurons that survived showed further development of extensive neurite with thicker and more curved neuritis as compared to control cultures (Figure 17E-H). After 4 days, the survival promotion activity was still intact: DRG; compounds III-3, 52%, control treated with 41% growth factor; SG: compound III-3, 83%, control treated with NGF 55%; CG: compound III-3, 58%, control treated with CNTF 50%). Under optimal conditions, the cultures can be maintained with compound III-3 for 1 week or more (not shown).

Compound III-3 supports the survival of embryonic chicken motoneurons in a concentration-dependent manner. Cultured chicken motoneurons can survive and extend processes in the presence of muscle extract, although these die quickly in their absence. In the experiments, after 48 hours, 65% of the motor neurons survived in the presence of muscle extract, in contrast to 14% of untreated controls. In serum-free conditions, the survival effect of compound 111-3 was maximum at 300 nM and was slightly higher (79%) than that induced by the muscle extract.

The concentration dependence of the survival effect of compound III-3 in this system is different than in peripheral neurons, since the concentrations of compound III-3 above 300 nM, showed a progressively reduced effect (Figure 16A). This may indicate a particular sensitivity of the motor neurons to a certain activity aspect of compound III-3. Morphologically, motoneurons rescued with compound III-3 exhibited bright phase cell bodies and were able to extend long neurites, which appear slightly thicker than those induced by the muscle extract (Figure 17). After 4 days in culture, 56% of the motor neurons were alive with the III-compound, compared with 42% with the muscle extract. At 300 nM, neurons treated with compound III survived in vitro for at least one week (not shown). Compound III-3 promotes neurite growth from intact dorsal root ganglia. The results of the above experiments demonstrate that compound III-3 not only promotes the survival of embryonic neurons of the peripheral and central nervous systems, but also results in robust neurite growth. Many of these extensions appeared to be thicker than those produced in the presence of growth factors (compare Figure 17 A-D with Figure 17 E-H). This effect was also observed in the neuritic growth produced from intact embryonic dorsal root ganglia cultured in the presence of 250 nM of compound III-3 (Figure 18C). Neurites grew in response to both NGF (Figure 18B) and compound III-3; that were produced through NGF and were thinner and more branched than those developed in the presence of compound III-3 that appeared thick and possibly fasciculated.

Example 36 Treatment In vivo Motoneuronal Death Regulated by Development in the Chicken Embryo This example is described in detail by Glicksman et al., J.

Neurobiol., 1998, 35, 361-370, which is incorporated herein by reference in its entirety. In E6, a window was made in the eggshells of chicken eggs (Spafas, Preston, CT) and as a vehicle (5% Solutol ™ HS 15, polyethylene glycol hydroxy stearate 660, BASF Aktiengesellschaft, Ludwighafen, Germany (salt regulated in its pH with phosphate, pH 7.2)) or the specified dose of compound lli-3 in the vehicle, was applied directly on the vascularized chorioallantoic membrane once a day from E6 to E9 as described by Oppenheim et al., Science, 1991, 251 , 1616-1617, which is hereby incorporated by reference in its entirety. The embryos were sacrificed in E10 and their spinal cords were removed, fixed in a Carnoy solution (10% glacial acetic acid, 60% absolute ethanol, 30% chloroform), processed for paraffin serial sections, and It was stained with thionin. Every 20a. section of lumbar segments 1-8 were counted according to previously described criteria (Clarke et al., Methods in Cell Biology: Cell Death, 1995, Schwart &Osborne, Eds., Academic Press, New York, pp. 277-321 , which is incorporated herein by reference).

Motoneuronal Death Regulated by Development in the Neonatal Rat Obravid, premature Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis), IN) The female rat pups were injected daily, subcutaneously (SC) over the target perineal muscles, with compound III-3 in Solutol ™ HS 5% 5 or a vehicle together with the start of the day of birth (P1) and continuing for 5 days (P5). In P10 or P6, the offspring were decapitated, the blood was collected in heparinized capillary tubes, and the region of the spinal cord containing the sexually dimorphic spinal nucleus of the cavernous bulb (SNB) and the perineal area containing the cavernous bulb (BC) and Elevating muscles (LA) were dissected after perfusion of animals with saline / formalin. The region of the spinal cord containing the SNB was subsequently affixed, embedded in Paraplast, sectioned at 10 μm, and stained with Cresyiecht violet (Nordeen et al., Science, 1985, 229, 671-673, which is incorporated herein by reference. In its whole). The motoneurons were counted at X500 in serial sections from the lumbar region 5 to the sacral region 1 of the spinal cord as previously described (Nordeen et al., Supra). A microscopic enumeration was made in the sections coded by an observer who did not see the treatment groups. The motor neuron counts for cell size and section thickness were corrected (Konisgs.ark, Contemporary Research Methods a Neuronatomy, Nauta &; Ebbesson, Eds., 1970, Springer-Verlag, New York, p. 315-340, which is incorporated herein by reference in its entirety) and the statistical analysis was an analysis of a direction of variation (ANOVA). The perineal musculature was subsequently fixed, decalcified, Paraplast embedded, sectioned at 10 μm and stained with Milligan's trichrome. Using bright field microscopy (X250), BC and LA muscles in normal females and females treated with compound III-3 (405 animals / group) were positively identified both by their location and by the presence of striated fibers. The muscle tissue profile was traced from alternate sections using a projection microscope (62.5), and the sectional area was measured using a digitizing pad and a computer-based morphometric system (Sigmascan, Jandel Scientific). Muscle volume was calculated by talking about the total transverse area and multiplying it by the section thickness, and corrected for the percentage of the structure sampled. The collected blood was centrifuged for 5 minutes at room temperature; then, the plasma was removed and frozen at -20 ° C. Serum testosterone levels were measured (6-7 animals / group) through radioimmunoassay following the procedures established by Winfield et al., Steroids, 1975, 26, 311-327, which is incorporated herein by reference in its entirety. .

Motoneuronal Differentiation Induced by Axotomy in the Adult Rat The left hypoglossal nerve was cut transversely in the neck of female Sprague-Dawley rats, adults (120-180 g) under anesthesia with Neumbutol, and 50 μl of compound III-3 or its vehicle (Solutol ™ HS 5% 5) to a piece of Gelfoam ™ (AJ Buck, Owings Mills, MD), then wrapped around the proximal end of the transversely cut nerve. After 7 days, the animals were anesthetized and perfused with 4% paraformaldehyde in Sorenson pH buffer, 0.07 M phosphate, pH 7.2. The brain stem was removed and coronary sections were cut in series with a thickness of 40 μm on the cryostat (Chiu, et al., NeuroReport, 1994, 5, 693-696, which is incorporated herein by reference in its entirety). Each fifth section was processed for ChAT immunohistochemistry as previously described (Chiu, et al., J. Comp.Neurol., 1993, 329, 351-363, which is incorporated herein by reference in its entirety) using a dilution of 1. : 350 of an anti-ChAT monoclonal antibody obtained from Chemicon. Cells that were clearly stained above the background were counted in stained sections; the number of cells enumerated was expressed as the ratio of the number of immunoreactive cells to ChAT on the axotomized side of the hypoglossal nucleus against the number of immunoreactive cells on the control side (undamaged).

Compound III-3 rescued rat embryo motoneurons from apoptotic death in vitro and inhibited a signaling path resulting in the activation of JNK1 in these cells (Maroney et al., J. Neurosci., 1998, 18, 104-111 , which is hereby incorporated by reference in its entirety). To determine the potential activity in vivo, compound III-3 was analyzed in two models of programmed motoneuronal death regulated by development and in the dedifferentiation model induced by axotomy in adult motor neurons. In chickens, approximately 50% of the spinal cord motor neurons suffer PCD during E5-10 (Hamburger, et al., J. Neurosci, 1982, 1, 38-55, Purves, and others, Body and Brain: A Trophic Theory of Neural Connections, 1988, Harvard University Press, Cambridge, MA, both incorporated herein by reference in their entirety). The application of compound III-3 to the chorioallantoic membrane during this period prevented motoneuronal death in a dose-dependent manner (Figure 19). 50% of motoneurons that can normally die were rescued at the two highest doses tested (2.3 and 7 μg / day), while 25% of motoneurons were rescued at lower doses (1.2 and 1.8 μg). / day) (Figure 19). During the early perinatal life of female rats (final embryonic stage until the day after birth (PN) 4) more than 50% of the motor neurons in SNB were eliminated through PCD (Breedlove, J. Neurobiol, 1986, 17, 157 -176, which is hereby incorporated by reference in its entirety). In males, motor neurons in these striated penis muscles of nucleus invertebrates involved copulatory I reflexes. The testicular secretion of androgenic steroids reduces motoneuronal SNB death in males and prevents much of the atrophy of BC / LA muscles innervated by neurons. . The administration of testosterone to female offspring resulted in a fully male number of motor neurons SNB (Nordeen et al., Supra) and prevented BC and LA muscle atrophy (Waiman, et al., Endocrinology, 1941, 29, 955-978, which was incorporated here by reference in its entirety). Daily SC administration of compound III-3 (PN 1-5) to female rats significantly attenuated motoneuronal death (Figure 20A). The rescue of motoneuronal death SNB by compound III-3 occurred at two doses (0.5 and 1 mg / kg per day). At the maximum effective doses of 0.5 and 1 mg / kg per day, administration of compound III-3 resulted in a 70% improvement in motoneuronal survival that equals the effect of testosterone (Figure 20A). Compound III-3 did not alter the testosterone levels in the plasma of treated females. The radioimmune measurement of testosterone levels in the plasma in the group of 1 mg / kg per day resulted in no significant difference when compared to the vehicle control group (0.016 + 0.008 ng / ml and 0.029 + 0.015 ng / ml, standard error of the mean (S.M.), respectively). To determine whether treatment with compound HI-3 was effective in a long-term maintenance of motor neuron survival, females were treated with compound III-3 (0.5 and 1 mg / kg per day) for the same period PN (1-5). Half of the animals in the vehicle and treatment groups were sacrificed in PN10. The remaining animals were then maintained without further treatment of compound III-3 until sacrificed in PN60. As previously observed (Figure 20A), Treatment with compound III-3 resulted in a 70% improvement in motoneuronal survival (Figure 20B). In addition, 100% of these rescued motoneurons were identifiable morphologically 55 days after the last treatment with compound III-3 (Figure 20B). The inhibition of motoneuronal death of compound III-3 during the neonatal period allowed motoneuronal survival in adults. Despite the clear demonstration and devastating effects of motor neuron loss in adult human diseases such as adult amyotrophic lateral sclerosis motoneurons in most animal models of motor neuron damage are resistant to death. However, axonal damage results in morphological changes (Oppenheim, et al., Supra) as well as biochemicals (Oppenheim, et al., Supra).; Rende, and others, J. Comp. Neurol., 1992, 319, 285,298, which is incorporated herein by reference in its entirety; Chiu et al., J. Comp. Neurol., 1993, 328, 351-363, which is hereby incorporated by reference in its entirety, into adult motoneurons that may resemble the preceding death to a degenerative change in motor neuron diseases of degeneration. An example of this type of change results form the axotomy of the hypoglossal nerve that innervates the tongue. The unilateral transection of this nerve in the adult rat resulted in the loss of 95% of the hypoglossal ChAT-immunoreactive motoneurons in the ipsilateral nucleus after 7 days (Chiu, et al., NeuroReport, 1994, 5, 693-696, which is incorporated herein by reference in its entirety). The loss in the immunoreactivity of ChAT was not permanent. Four days after the axotomy, 100% of the motor neurons recovered control levels of ChAT immunoreactivity (Borke et al., J. Neurocytol., 1993, 22, 141-153, which is incorporated herein by reference in its entirety ). The immunoreactivity of ChAT in the contralateral hypoglossal motor neurons was not affected (Chiu, et al., Supra) (Figure 21 and Table 5). When applied in Gelfoam ™ at the end near the hypoglossal nerve, the dose of compound III-3 significantly attenuated the reduction in ChAT immunoreactivity in ipsilateral hypoglossal motoneurons analyzed 7 days after taxotomy. The maximum effective dose (50 μg) resulted in 40% more motoneurons immunoreactive to ChAT as compared to the untreated axotomized control (Figure 21B and Table 5). There was a bell-shaped dose dependency with both lower and higher doses resulting in greater survival than the untreated control, but lower than that achieved at 50 μg. As true with the SNB model, no associated weight loss, mortality, or gross tissue damage in these animals was observed at any of the doses tested. In three separate models of motor neuron degeneration in vivo, compound III-3 demonstrated neuroprotective activity: PDC regulated by development of motoneurons of spinal lumbar spine in embryos (Figure 19), androgen-sensitive death of postnatal SNB motoneurons (Figure 20) and loss induced by axotomy of a functional marker, ChAT, in adult hypoglossal motoneurons (Figure 21 and Table 5). Compound III-3 was effective when administered peripherally by injection, applied locally at the cutting end of a nerve, or directly on the chick embryo chorioallantoic membrane. In contrast to the parent molecule K252a, compound III-3 was approximately 5 times more potent to mediate survival in motoneuron-rich cultures (data not shown) and exhibited no inhibitory activity against trkA tyrosine kinase and several threonine kinases of serine (Maroney et al., supra; Kaneko et al., J. Med. Chem., 1997, 40, 1863-1869, which is incorporated herein by reference in its entirety).

Table 5 Effect of Compound III-3 on Choline-Acetyltransferase Immunoreactivity in Axotomized Hypoglossal Motoneurons Compound III-3 or vehicle in gel foam was added to the near end of the hypoglossal nerve immediately after its cross section. After 7 days, the animals were sacrificed and serially sectioned through the hypoglossal nucleus, and each fifth section was immunostained with anti-ChAT antibodies. ChAT-positive neurons were counted on the ipsilateral (experimental) and contralateral (control) sides of the nucleus. * p < 0.05, statistically significant compared to animals treated with control vehicle.

A pathway inhibitor of MLK-3 demonstrates in vivo efficacy and blocks phosphorylation events downstream of MLK-3 in the MPTP Model. MPTP was administered at a dose (40 mg / kg) which results in the loss of striatal dopaminergic terminals and cell bodies in the substantia nigra. Tyrosine hydroxylase was used as a marker for dopaminergic nerve terminals in the black substance. Compound III-3 was systematically administered and attenuated the loss of black substance tyrosine hydroxylase immunoreactive neurons after the MPTP lesion (Figure 22a, Saporito et al., 1999). Since compound III-3 is a known inhibitor of MLK3, the activity of a substrate below MLK3 was measured in mice treated with MPTP. Phosphorylated MKK4 levels were measured using a specific antibody in phospho-MKK4 (New England Biolabs, Beverly, MA) which recognizes the monophosphorylated form of MKK4 either through immunostaining (Figure 22b) or ELISA (Figure 22c). The administration of MPTP raised the levels of phosphorylated MKK4 in the substantia nigra up to 5 times over the control levels (Figure 22b). The peak elevations occurred 4 hours after the administration of MPTP and coincided with MPP + levels in the central nervous system, peak. MPTP-mediated phosphorylation of MKK4 was attenuated through pretreatment with 1-deprenyl, indicating that these phosphorylation events were mediated by MPP + (Figure 22c). In addition, the phosphorylation of MKK4 was partially inhibited with the pretreatment of compound 111-3 at a dose (1 mg / kg) that produces protection against MPES-induced black-dopaminergic loss (Figure 22c). These data demonstrate that MPTP (MPP +) activates MKK4, a substratum underneath MLK3. In addition, these data demonstrate that a known inhibitor of MLK3 inhibits the activation of this kinase pathway in vivo.

Example 37 Inflammation The induction of IL-1 and TNF-a by LPS in THP-1 cells and the effect of indolocarbazoles and pyrrolocarbazoles in their induction. Cells of the immune system were selected, since many kinases are involved in the regulation of numerous immunological functions, for example, the induction of cytokine synthesis and the induction of a cytokine biological response. A recent report (Hambleton, et al., Proc. Nati, Acad. Sci. USA, 1996, 93, 2774-2778, which is incorporated herein by reference) showed that the treatment of cell lines derived from monocyte with LPS caused a Rapid activation of JNK activity. When monocytes are contacted with bacterial endotoxins such as lipolisaccharide (LPS), they produce the inflammatory cytokines, IL-2 and TNF-a. The inhibition of the production of these two cytokines can be a useful treatment for certain inflammatory disorders of the immune system. These cytokines can be easily measured through commercial types of ELISA. Experiments were designed to determine, (1) whether the indole- and pyrrolocarbazole fused can inhibit the synthesis of IL-1 and TNF-a in the monocyte cell line THP-1, (2) if JNK is activated by LPS in cells THP-1, and (3) if the activation of JNK by LPS can be inhibited through indolo- and fused pyrrolocarbazoles.

Experimental Procedures THP-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. The LPS (E. coli, serotype 0111, B4, TCA) was purchased from sigma and dissolved in PBS. ELISA kits for analyzing IL-1 and TNF-a were purchased from Boerhinger-Mannhein, and assays in the THP-1 culture medium were performed as directed by the manufacturer. Standard curves according to directions were obtained with each test. Experiments were performed in 12 cavity culture plates with either 1 or 2 ml of THP-1 cells at 4 x 10 5 cells / ml. IL-1 and TNF-α were induced through the addition of LPS to the culture medium and the medium was collected at various times afterwards for cytokine assay. The cells were removed by filtration and the supernatants were frozen at -70 ° C until assay. To minimize costs, experiments were performed in duplicate cultures and the supernatants in duplicate were combined after centrifugation. Each combined supernatant was analyzed in duplicate. The supply solutions of indolo- and pyrrolocarbazoles fused in 100% DMSO were diluted to the desired concentrations either in a medium containing 10% fetal bovine serum or in a medium containing 0.5 mg / ml of BSA. Unless otherwise indicated, the compounds were added to the THP-1 cells 1 hour prior to the addition of LPS. Assays for JNK activity were performed after immunoprecipitation of the JNK protein from an extract of THP-1 cells. The THP-1 cells in pellets were used on ice for 15 minutes in 500 μl of pH-regulator Frac (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 30 μM sodium pyrophosphate, 1 mg / ml BSA, 1% triton-X-100). The extract was centrifuged for 10 minutes at 14 K and 5 μl of the JNK antibody (Santa Cruz) was added to the supernatant. The extract was rotated for 60 minutes at 4 ° C, 75 μl of washed protein A Sepharose (20% w / v Frac) was added and the extract was rotated for another 30 minutes to bind the antibody complex to protein A Sepharose . Protein A Sepharose was washed twice with the Frac regulator, once with 20 mM Hepes, pH 7.6 20 mM MgCl 2, 2 mM DTT, then incubated for 15 minutes at 30 ° C in 30 μl pH buffer of kinase (20 mM hepes, 20 mM MgCl, 2 MM DTT, 1 μg of recombinant c-jun, and 2 μM ATP -? - 32P, 2 μCi). The reaction was terminated by the addition of 10 μl of 4X SDS gel charge buffer, heated for 3 minutes at 80 ° C and the proteins were analyzed in a 10% SDS gel. The gel was dried, exposed to a Phosphorimager plate, and the radioactive bands were analyzed in a Phosphorimager imager. The results of initial experiments indicated that LPS at 2 μg / ml gave the maximum yield of IL-1 and this concentration of LPS was used in all subsequent experiments. The minimum time after the addition of LPS for maximum cytokine performance was determined by taking aliquots of the medium for analysis at varying times after the addition of LPS. The first experiment indicated both IL-1 and TNF-a obtained a maximum yield at least 5 hours after the addition of LPS. Since the earliest collection time was 2.4 hours in the first experiment, a second experiment was performed with media collections beginning 15 minutes after the addition of LPS. The results of this experiment were only that TNF-a was analyzed and showed that it obtained a maximum yield 3 hours after the addition of LPS. No significant TNF-a was found in the medium until 90 minutes after the addition of LPS. The rapid achievement of maximum yield indicated a tight regulation of the synthesis of the two cytokines, rapid synthesis and rapid regulation, cell cultures were treated for 30 minutes before the addition of LPS either with actinomycin D an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis. The medium was collected 3 hours after the addition of LPS and the TNF-α was analyzed. Both the new RNA synthesis and the new protein synthesis are required for the induction of TNF-a since no TNF-α was found in the medium of cells treated with any inhibitor. The following experiments were performed to determine if compound III-3 can inhibit the induction of IL-1 and TNF-α. Compound I and I-3 inhibited the induction of both IL-1 and TNF-α with IC50 values of 267 nM and 139 nM, respectively. The results of these experiments were obtained with cells in a medium containing 10% fetal bovine serum. Since the tests with spinal cord tissue and basal forebrain tissue for the neurotrophic activity of compounds were performed in a serum-free medium (500 μg / ml BSA), it was of interest to determine IC50 values for IL inhibition. -1 and TNF-a in a serum-free medium. When the THP-1 cells were treated with compound III-3 in a serum-free medium (500 μg / ml BSA), the IC 50 was reduced 10-fold from 269 nM to 23 nM. Unless otherwise indicated, all experiments performed hereafter were done in a serum-free medium. Inhibition of compound III-3 from the induction of 1L-1 and TNF-a in THP-1 cells suggests that the compound Hl-3 may be useful as a therapeutic agent in the treatment of pathological conditions caused by the production of normal amounts previous of these cytokines. Septic shock is one of these conditions. Septic shock is caused by the growth of gram-negative bacteria in the circulation, which in turn releases large amounts of the endotoxin, LPS. LPS then stimulates mainly monocytes and macrophages to produce large amounts of IL-1 and TNF-a, which can then cause massive tissue damage and in many cases death.

Several compounds were tested for their activity to inhibit TNF-α and compared with the ability to inhibit JNK. The results are shown in Table 6.

Table 6 Effect of Compound III-3 on the induction of IL-2 in Jurkat cells. Experiments were performed to determine if compound III-3 inhibited the induction of IL-II in Jurkat cells.

Experimental Procedures Jurkat cells were developed in the RPMl 1640 medium with 10% fetal bovine serum. The TNF-a was from Promega and the anti-CD3 and anti-CD28 antibodies were from Pharmigen. The Jurkat experiments were performed in 200 μl in a 96-well plate. IL-2 was measured with an ELISA kit purchased from Boehringer Mannheim. The antibodies for CD3 and CD28 were allowed to bind to the plastic of the 96-well plate (18 hours in PBS) before the addition of the Jurkat cells. The cells were treated with the compounds 1 hour before being added to the antibody coated plate. The antibodies for CD3 and CD28 were used to activate the T cell receptor and induce IL-2. IL-2 was released from Jurkat cells between 6 hours and 24 hours after the start of induction (Figure 23A). Constitutively no IL-2 was made (Figure 23A CNT). Then, the effect of compound III-3 (1 hour of treatment with compound III-3 before induction) on the induction of IL-2 was analyzed (Figure 23B). A concentration of compound 111-3 of 500 nM inhibited the induction of IL-2 by more than 80% (Figure 23B). A more extensive dose response experiment was performed with compound III-3 and with compound I-4, which produced IC50 values of 139 nM for compound III-3 and 207 nM for compound I-4 (Figure 23C). It is intended that each of the patents, applications and printed publications mentioned in this patent document are hereby incorporated by reference in their entirety. As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit thereof. It is intended that all these variations fall within the scope of the invention.

LIST OF SEQUENCES < 110 > Maroney, Anna Walton, Kevin Knightm Ernest Glicksman, Marcie Dionea, Craig Neff, Nicola < 120 > Methods to Modulate Multiple Lineage Kinase Proteins and Classify Compounds that Modulate Multiple Lineage Kinase Proteins < 130 > CEPH0431 < 140 > < 141 > < 160 > 18 < 170 > Patenln Ver. 2.0 < 210 > 1 < 211 > 17 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 1 Cys Gly Gly Ala Thr Cys Cys Ala Cys Met Gly lie Gly Ala Tyr Tyr 1 5 10 10 Thr < 210 > 2 < 211 > 23 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 2 Gly Gly Wing Wing Thr Thr Cys Cys Wing Trp Wing Gly Gly Wing Cys Cys 1 5 10 15 Ala Be Ala Cys Arg Thr Cys 20 < 210 > 3 < 211 > 33 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 3 Cys Gly Gly Wing Thr Cys Cys Arg Thr lie Cys Wing Tyr Met Gly lie 1 5 10 15 Gly Ala Tyr Tyr Thr lie Gly Cys lie Gly Cys lie Met Gly lie Wing 20 25 30 Wing < 210 > 4 < 211 > 30 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 4 Gly Gly Ala Ala Thr Thr lie Ala Tyr lie Gly Gly Ala Trp Ala lie 1 5 10 15 Gly Trp Cys Cys Ala lie Ala Cys Arg Thr Cys lie Ser Trp 20 25 30 < 210 > 5 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 5 Met Glu Glu Glu Glu Tyr Met Pro Met Glu 1 5 10 < 210 > 6 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 6 gtggctgtgc gggcagctcg ccag 24 < 210 > 7 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 7 gagaccctgg atctcgcgct 21 < 210 > 8 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 8 Met Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 < 210 > 9 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 9 cggatccgtg acaccagtcg gaacctt 27 < 210 > 10 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 10 ggaattcacc agtaagctcc agcacatc 28 < 210 > 11 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 11 ataattcgtg ctagcgccag agtctagccg gtg 33 < 210 > 12 < 211 > 39 < 212 > AND < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 12 ataagcttcc tcagtgcaag tggatcgcgc agcccctga 39 < 210 > 13 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 13 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 < 210 > 14 < 211 > 69 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 14 ataaagcttc cagaggccat ggactacaag gacgacgatg caaggcctg cctccatgaa 60 acccgaaca 69 < 210 > 15 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 15 gacagggcgg ccggctct 18 < 210 > 16 < 211 > 583 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 16 gaattcggca cgagaggact cgcaggtgtc cggcgacgag ggctggtgga ccgggcagct 60 gaaccagcgg gtgggcatct tccccagcaa ctacgtgacc ccgcgcagcg ccttctccag 120 ccgctgccag cccggcggcg aggaccccag ttgctacccg cccattcagt tgttagaaat 180 tgattttgcg gagctcacct tggaagagat tattggcatc gggggctttg ggaaggtcta 240 tcgtgctttc tggatagggg atgaggttgc tgtgaaagca gctcgccacg accctgatga 300 ggacatcagc cagaccatag agaatgttcg ccaagaggcc aagctcttcg ccatgctgaa 360 gcaccccaac atcattgccc taagaggggt atgtctgaag gagcccaacc tctgcttggt 420 catggagttt gctcgtggag gacctttgaa tagagtgtta tctgggaaaa ggattccccc 480 agacatcctg gtgaattggg ctgtgcagat tgccagaggg atgaactact tacatgatga 540 ggcaattgtt cccatcatcc accgcgacct taagtccagc aac 583 < 210 > 17 < 211 > 194 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 17 Asn Ser Wing Arg Glu Asp Ser Gln Val Ser Gly Asp Glu Gly Trp Trp 1 5 10 15 Thr Gly Gln Leu Asn Gin Arg Val Gly lie Phe Pro Ser Asn Tyr Val 20 25 30 Thr Pro Arg Ser Ala Phe Ser Ser Arg Cys Gin Pro Gly Gly Glu Asp 35 40 45 Pro Ser Cys Tyr Pro Pro lie Gln Leu Leu Glu lie Asp Phe Wing Glu 50 55 60 Leu Thr Leu Glu Glu lie lie Gly He Gly Gly Phe Gly Lys Val Tyr 65 70 75 80 Arg Ala Phe Trp lie Gly Asp Glu Val Wing Val Lys Wing Wing Arg Hís 85 90 95 Asp Pro Asp Glu Asp He Ser Gin Thr lie Glu Asn Val Arg Gln Glu 100 105 110 Wing Lys Leu Phe Wing Met Leu Lys His Pro Asn lie Wing Leu Arg 115 120 125 Gly Val Cys Leu Lys Glu Pro Asn Leu Cys Leu Val Met Glu Phe Wing 130 135 140 Arg Gly Gly Pro Leu Asn Arg Val Leu Ser Gly Lys Arg Pro Pro 145 150 155 160 Asp He Leu Val Asn Trp Wing Val Gln He Wing Arg Gly Met Asn Tyr 165 170 175 Leu His Asp Glu Wing He Val Pro He lie His Arg Asp Leu Lys Ser 180 185 190 Ser Asn < 210 > 18 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Novel Sequence < 400 > 18 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims (130)

1. CLAIMS 1. - A method for identifying a compound that modulates the activity of a multiple lineage kinase protein and promotes cell survival, comprising the steps of: (a) contacting said cell containing the multiple lineage kinase protein with said compound; (b) determining whether the compound decreases the activity of the multiple lineage kinase protein; and (c) determining whether the compound promotes cell survival.
2. 2. The method according to claim 1, wherein the protein is selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, kinase carrying a leucine closure, kinase which carries a double leucine closure, and multiple lineage kinase 6.
3. 3. The method according to claim 2, wherein the cell is contacted in vitro.
4. 4. The method according to claim 2, wherein the cell is contacted in vivo.
5. 5. The method according to claim 2, wherein the activity of the protein is determined by measuring the activity or phosphorylation status of a substrate of said protein.
6. 6. The method according to claim 5, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38a, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 ( SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homolog of AEX-3.
7. 7. The method according to claim 2, wherein said protein activity is determined by measuring the activity of a substrate of said protein, the amount of a substrate of said protein or the mRNA encoding said substrate of the protein.
8. 8. The method according to claim 2, wherein the protein activity is determined through an in vitro kinase assay or binding assay.
9. 9. The method according to claim 2, wherein the promotion of cell survival is determined using cells at risk of dying and comparing the amount of living cells that are contacted with the compound with the amount of living cells. that are not in contact with the compound.
10. 10. The method according to claim 9, wherein the cells are primary embryonic motor neurons.
11. 11. The method according to claim 9, wherein said cells overexpress the multiple lineage kinase protein.
12. 12. The method according to claim 2, in the promotion of cell survival is determined by observing a decrease in apoptosis.
13. 13. The method according to claim 2, wherein the cell is a neuronal cell. 14. - The method according to claim 2, wherein the cell is involved in a neurodegenerative disease. 15. A method for identifying a compound that modulates the activity of a multiple lineage kinase protein and promotes cell death, comprising the steps of: (a) contacting said cell containing the multiple lineage kinase protein with said compound; (b) determining whether the compound increases the activity of the multiple lineage kinase protein; and (c) determining whether the compound promotes cell death. 16. The method according to claim 15, wherein the protein is selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, kinase carrying a leucine lock, kinase carrying a double leucine closure, and multiple lineage kinase 6. 17. The method according to claim 16, wherein the cell is contacted in vitro. 18. The method according to claim 16, wherein the cell is contacted in vivo. 19. The method according to claim 16, wherein the activity of the protein is determined by measuring the activity or phosphorylation status of a substrate of said protein. 20. The method according to claim 19, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38a, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homologue of AEX-3. 21. The method according to claim 16, wherein said protein activity is determined by measuring the activity of a substrate of said protein, the amount of a substrate of said protein or the mRNA encoding said substrate of the protein. 22. The method according to claim 16, wherein the protein activity is determined through an in vitro kinase assay or binding assay. 23. The method according to claim 16, wherein the promotion of cell survival is determined using cells at risk of dying and comparing the amount of living cells that are contacted with the compound with the amount of living cells. that are not in contact with the compound. 24. The method according to claim 23, wherein the cells are primary embryonic motor neuron cells. 25. The method according to claim 23, wherein said cells overexpress the multiple lineage kinase protein. 26. The method according to claim 16, in promoting cell survival is determined by observing a decrease in apoptosis. 27. The method according to claim 16, wherein the cell is a neuronal cell. 28. The method according to claim 16, wherein the cell is involved in a neurodegenerative disease. 29. A method for modulating the activity of a multiple lineage kinase protein comprising contacting said protein or a cell containing said protein with a compound having the formula: wherein: ring B and ring F, independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring where 1 at 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring wherein: 1) a carbon atom is replaced with an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced with one atom of sulfur and one of nitrogen, one atom of oxygen and one of nitrogen, or two atoms of nitrogen; or 3) three carbon atoms are replaced with three nitrogen atoms; R1 is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbons, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl; b) -C (= O) R9, wherein R9 is selected from the group consisting of alkyl, aryl and heteroaryl; c) -OR10, wherein R10 is selected from the group consisting of H and alkyl having from 1 to 4 carbons; d) -C (= O) NH2), -NR1 R12, - (CH2) PNR11 R12, - (CH2) pOR10, -O (CH2) POR10 and -O (CH2) PNR 1R12, where p is 1 to 4; and wherein either: 1) R11 and R12 each independently is selected from the group consisting of H and alkyl having from 1 to 4 carbons; or 2) R11 and R12 together form a linking group of the formula - (CH2) 2-X1- (CH2) 2-, wherein X1 is selected from the group consisting of -O-, -S-, and -CH2 -; R2 is selected from the group consisting of H, alkyl having 1 to 4 carbons, -OH, alkoxy having 1 to 4 carbons, -OC (= O) R9, -OC (= O) NR11R12, -O (CH2 ) pNR11 R12, -0 (CH2) pOR10, substituted or unsubstituted arylalkyl having from 6 to 10 carbons, and substituted or unsubstituted heteroarylalkyl; R3, R4, R5 and R6 are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF3, -NO2, -OH, -OR9, -O (CH2) pNR11R12, -OC (= O) R9, -OC (= O) NR2R7, -OC (= O) NR 1R12, -O (CH2) pOR10, -CH2OR10, -NR11R12, -NR10S (= O) 2R9 , -NR10C (= O) R9; b) -CH2OR? , wherein R14 is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; c) -NR10C (= O) NR11R12, -CO2R2, -C (= O) R2, -C (= O) NR11R12, -CH = NOR2, -CH = NR9, - (CH2) PNR11 R12, - (CH2) PNHR14, or -CH = NNR2R2A, wherein R2A is the same as R2; d) -S (O) and R 2 - (CH 2) p S (O) and R 9, -CH 2 S (O) and R 14, wherein y is 0, 1 or 2; e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons, and alkynyl having from 2 to 8 carbons, wherein: 1) each alkyl, alkenyl or alkynyl group is unsubstituted; or 2) each alkyl, alkenyl or alkynyl group is substituted with 1 to 3 groups selected from the group consisting of aryl having 6 to 10 carbons, heteroaryl, arylalkoxy heterocycloalkoxy, hydroxyalkoxy, alkyloxy alkoxy, hydroxyalkylthio alkoxy alkylthio, F, Cl , Br, I, -CN, -NO2, -OH, -OR9, -X2 (CH2) PNR11 R12 -X2 (CH2) pC (= O) NR11R12, -X2 (CH2) pOC (= O) NR11R12, -X2 (CH2) pCO2R9 -X2 (CH2) pS (O) and R9, -X2 (CH2) PNR10C (= O) NR11R12, OC (=) R9, OCONHR2-O-tetrahydropyranyl, -NR11R12, -NR10C (= O) R9, -NR10CO2R9 -NR10C (= O) NR 11 R 12, -NHC (= NH) NH 2, NR 10 S (O) 2 R 9, -S (O) and R 9, -CO 2 R 2 -C (= O) NR 11 R 12, -C (= O) R 2, -CH 2 OR 10, -CH = NNR R2A, -CH = NOR2 -CH = NR9, -CH = NNHCH (N = NH) NH2, -S (= O) 2NR2R2A, -P (= O) (OR10) 2 -OR14, and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently either unsubstituted or is replaced by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons; X2 is O, S, or NR10; R7 and R8 are each independently selected from the group consisting of H, alkyl having 1 to 4 carbons, alkoxy having 1 to 4 carbons, substituted or unsubstituted arylalkyl having 6 to 10 carbons, heteroaryl substituted or unsubstituted , - (CH2) pOR10, - (CH2) pOC (= O) NR11R12, and - (CH2) PNR 1R12; or R7 and R8 together form a linking group of the formula -CH2-X3-CH2- wherein X3 is X2 or a bond; m and n each is independently 0, 1 or 2; Y is selected from the group consisting of -O-, -S-, -N (R10) -, -N + (0") (R10) -, -N (OR10) -, and -CH2-; Z is selected of the group consisting of a bond, -O-, -CH = CH-, -S-, -C (= O) -, -CH (OR10) -, -N (R10) -, -N (OR10) - , CH (NR1 R12) -, -C (= O) N (R17) -, -N (R17) C (= O) -, -N (S (O) and R9) -, -N (S (O) and NR 11 R 12) -, -N (C (= O) R 17) -, -C (R 15 R 16) -, -N + (O ") (R 10) -. -CH (OH) -CH ( OH) -, and -CH (O (C = O) R9) CH (OC (= O) R9A) -, wherein R9A is the same as R9; R15 and R16 are independently selected from the group consisting of H, -OH , -C (= O) R 10, -O (C = O) R 9, hydroxyalkyl and -CO 2 R 10; R17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl; A1 and A2 are selected from the group consisting of H, H; H, OR2; H, -SR2; H, -N (R2) 2; and a group wherein A1 and A2 together form a portion selected from the group consisting of = O, = S, y = NR2; B1 and B2 are selected from the group consisting of H, H; H, -OR2; H, -SR2; H, -N (R2) 2; and a group wherein B1 and B2 together form a selected portion of the group consisting of = O, = S, y = NR2; provided that at least one of the pairs of A1 and A2, or B1 and B2, forms = O. 30. A method for modulating the activity of a multiple lineage kinase protein, comprising contacting said protein to a cell containing the protein with a compound having the formula: where: Zt is H and Z2 is H or Z | and Z2 together form = O; RT is selected from the group consisting of H, Cl, CH2SO2C2H5, Br, CH2S (CH2) 2NH2, CH2S (CH2) 2N (CH3) 2, CH2S (CH2) 2NH2 n-C4H9, NHCOCHC6H5, NHCOCHC2H5, CH2SC2H5, CH2SC6H5, N (CH3) 2, CH3, CH2OCONHC2H5, NHCO2CH3, CH2OC2H5, CH2N (CH3) 2, OH, On-propyl, CH = NNH-C (= NH) NH2, CH = NN (CH3) 2, CH2S (CH2) 2NH- n-C4H9, CH2OCH2OCH2CH3, CH2S [3- (1, 2,4-triazine)], CH2CH2SCH3; R2 is selected from the group consisting of H, Br, Cl, I, CH2S (CH2) 2N (CH3) 2, NHCONHC2H5, CH2SC2H5, CH2OCH2OCH2CH3, CH2S [3- (1,2,4-triazine)], CH2CH2SCH3, and CH2OH; X is selected from the group consisting of H, CH2OH, CH2NH-serineH, CO2CH3, CONHC6H5, CH2NHCO2C6H5, CH2NHCO2CH3, CH2N3, CONHC2H5, CH2N H-glycine, CON (CH3) 2, -CH2NHC02-, CONH2, CONHC3H7, CH2NH-serine , CH2SOCH3, CH = NOH, CH2NH-proline, CH2CH2 (2-pyridyl), CH = NNHC (= NH) NH2, CONH (CH2) 2OH, CH = NNHCONH2, CH2OCOCH3, -CH2OC (CH3) 2O-, CH2SC6H5, CH2SOC6H5, CO2n-hexyl, CONHCH3, and CO2 (CH2) 4CH3; R is selected from the group consisting of OH, and OCH3. 31. The method according to claim 30, wherein Z and Z2 are H; X is CO2CH3; R, is NHCONHC2H5; R2 is CH2CH2 (2-pyridyl); and R is OH. 32. The method according to claim 30, wherein Z? and Z2 are H; X is CO2CH3; R1 and R2 are CH2OCH2OCH2CH3: and R is OH. 33. The method according to claim 30, wherein Zi, and Z2 are H; X is CO2CH3; Ri and R2 are CH2SCH2CH3; and R is OH. 34.- The method according to claim 30, wherein Z ,, Z2, R, and R2 are H; X is CO2CH3; R, is OH. The method according to claim 30, wherein Z ,, Z2, R, and R2 are H; X is CO2 (CH2) 4CH3; and R is OH. 36. The method according to claim 30, wherein Z Z2 and Ri are H; R2 is CH2OH; X is CO2CH3; and R is OH. 37.- The method according to claim 30, wherein Z and Z2 are H; R, and R2 are H2S (3- (1, 2,4-triazine)); X is CO2CH3; and R is OH. 38.- The method according to claim 30, wherein Z? and Z2 are H; R, is Br, R2 is I; X is CO2CH3; and R is OH. 39.- The method according to claim 30, wherein Z] and Z2 are H; R, and R2 are CH2CH2SCH3; X is CO2CH3; and R is OH. 40.- The method according to claim 30, wherein Zi, Z2, R, and R2 are H; X is CO2CH3; and R is OCH3. 41. The method according to claim 30, wherein Zi and Z2 together form = O; Ri and R2 are Br; X is CO2CH3; and R is OH. 42. - A method for modulating the activity of a multiple lineage kinase protein comprising contacting the protein or a cell having said protein with a compound having the formula: wherein: Z, is H and Z2 is H or Zi and Z2 together form = O; R, is H or Br; R2 is H; R3 is H, CH2CH = CH2, CH2CH2CH2OH, or R4 is H, CH2CH = CH2 or CH2CH2CH2OH. 43.- The method according to claim 42, wherein Ri, R2, R4, Z1 and Z2 are H and R3 is CH2CH = CH2. 44. The method according to claim 42, wherein RT is Br and R2, R3, R4, Z1 and Z2 are H. 45. The method according to claim 42, wherein R ,, R2, Z and Z2 are H and R3 and R4 are CH2CH = CH2. 46. The method according to claim 42, wherein R ,, R2, R3, Zi and Z2 are H and R4 is CH2CH = CH2. 47. The method according to claim 42, wherein R1, R2, Z1 and Z2 are H, and R3 and R4 are CH2CH2CH2OH; or R ,, R2, R4, Z and Z2 are H, and R3 is: 48. - A method for identifying a compound that may be useful in the treatment of a neurodegenerative disorder, comprising contacting a cell or cell extract containing a multiple lineage kinase protein with a compound, and determining whether said compound reduces the activity of the multiple lineage kinase protein. 49.- The method according to claim 48, wherein the protein is selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, kinase carrying a leucine closure, kinase which carries a double leucine closure, and multiple lineage kinase 6. 50.- The method according to claim 49, wherein the cell is contacted in vitro. 51. The method according to claim 49, wherein the cell is contacted in vivo. 52. The method according to claim 49, wherein the activity of the protein is determined by measuring the activity or phosphorylation status of a substrate of said protein. 53. The method according to claim 52, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38a, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homologue of AEX-3. 54. The method according to claim 49, wherein said protein activity is determined by measuring the activity of a substrate of said protein, the amount of a substrate of said protein or the mRNA encoding said substrate of the protein. 55. The method according to claim 49, wherein the protein activity is determined through an in vitro kinase assay or binding assay. 56. The method according to claim 49, wherein the cells are primary embryonic motor neurons. 57. The method according to claim 49, wherein the cells over-express the multiple lineage kinase protein. 58. - The method according to claim 49, wherein the cell is a neuronal cell. 59. The method according to claim 49, wherein the cell is involved in a neurodegenerative disease. 60.- A method for identifying a compound that may be useful in the treatment of inflammation, comprising contacting a cell or cell extract containing the multiple lineage kinase protein with a compound, and determining whether said compound decreases activity of said multiple lineage kinase protein. 61.- The method according to claim 60, wherein the protein is selected from the group consisting of multiple lineage kinase 1, multiple lineage kinase 2, multiple lineage kinase 3, kinase carrying a leucine closure, kinase which carries a double leucine closure, and multiple lineage kinase 6. 62. The method according to claim 61, wherein the cell is contacted in vitro. 63. - The method according to claim 61, wherein the cell is contacted in vivo. 64. The method according to claim 61, wherein the activity of the protein is determined by measuring the activity or phosphorylation status of a substrate of said protein. The method according to claim 64, wherein the substrate is selected from the group consisting of JNK1, JNK2, JNK3, ERK1, ERK2, p38a, p38β, p38 ?, p38d, MEK1, MEK2, MKK3, MKK4 (SEK1), MEK5, MKK6, MKK7, jun, ATF2, ELK1, and the mammalian homologue of AEX-3. 66. The method according to claim 61, wherein said protein activity is determined by measuring the activity of a substrate of said protein, the amount of a substrate of said protein or the mRNA encoding said substrate of the protein. 67.- The method according to claim 61, wherein the protein activity is determined through an in vitro kinase assay or binding assay. 68.- The method according to claim 61, wherein the cells are primary embryonic motor neurons. 69. The method according to claim 61, wherein the cells over-express the multiple lineage kinase protein. 70. - The method according to claim 61, wherein the cell is a neuronal cell. 71. The method according to claim 61, wherein the cell is involved in a neurodegenerative disease. 72. - A method for treating a mammal having a neurodegenerative disorder, comprising administering to said mammal a compound that inhibits a multiple lineage kinase protein or a pharmaceutically acceptable diluent. 73.- The method according to claim 72, wherein the compound has the formula: wherein: E and E2 independently, each together with the carbon atoms to which they are attached form either: an unsaturated 6-membered carbocyclic aromatic ring wherein from 1 to 3 carbon atoms can be replaced by nitrogen atoms; or an unsaturated 5-membered carbocyclic aromatic ring wherein either a carbon atom is replaced with an oxygen, nitrogen or sulfur atom; or two carbon atoms are replaced with a sulfur and nitrogen atom, or an oxygen and nitrogen atom; A1 and A2 together represent O, and B1 and B2 together represent O; R1 is H, alkyl of 1 to 4 carbon atoms (inclusive), aryl, arylalkyl, heteroaryl, and heteroarylaxy; COR9, wherein R9 is alkyl of 1 to 4 carbons (inclusive), or aryl, preferably phenyl or naphthyl; -OR10, wherein R10 is H or alkyl of 1 to 4 carbons (inclusive); -CONH2, -NR7R8, - (CH2) nNR7R8, wherein n is an integer of 1-4 (inclusive); or -O (CH2) nNR7R8; and either R7 and R8 independently are H or alkyl of 1 to 4 carbons (inclusive); or R7 and R8 are combined together to form a linking group of the general formula - (CH2) 2-X1- (CH2) 2-, wherein X1 is O, S or CH2; R2 is H, -SO2R9; -CO2R9, -COR9, alkyl of 1 to 8 carbons (inclusive) preferably an alkyl of 1 to 4 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), preferably an alkenyl of 1-4 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive), preferably a alkynyl of 1-4 carbons (inclusive); or a monosaccharide of 5-7 carbons (inclusive), wherein each hydroxyl group of the monosaccharide independently is either substituted or is replaced by H, alkyl of 1-4 carbons (inclusive), alkylcarbonyloxy of 2-5 carbons (inclusive) or 1-4 carbon alkoxy (inclusive); and either each alkyl of 1-8 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive), is unsubstituted; or each alkyl of 1-8 carbons (inclusive); alkenyl of 1-8 carbons (inclusive) or alkynyl of 1-8 carbons (inclusive) independently is substituted with 1-3 aryl of 6-10 carbons (inclusive), preferably phenyl or naphthyl; heteroaryl, F, Cl, Br, I, -CN, -NO2, OH, -OR9, -O (CH2) nNR7R8, -OCOR9, -OCONHR9, O-tetrahydropyranyl, NH2, -NR7R8, -NR10COR9; -NR10CO2R9, R10CONR7R8, -NHC (= NH) NH2, -NR10SO2R9, -S (O) and R11, wherein R11 is H or alkyl of 1-4 carbons, aryl of 6-10 carbons, preferably phenyl or naphthyl, or heteroaryl and is 1 or 2; -SR11, -CO2R9, -CONR7R8, -CHO, COR9, -CH2OR7, -CH = NNR11 R12, -CH = NOR11, -CH = NR9, -CH = NNHCH (N = N H) N H2, -SO2NR12R13, -PO (OR11) 2 or OR14 wherein R14 is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; and either R12 and R13 independently are H, alkyl of 1-4 carbons (inclusive), aryl of 6-10 carbons, preferably phenyl or naphthyl, or heteroaryl; or R12 and R13 combine together to form a linking group preferably - (CH2) 2-X1- (CH2) 2; each R3, R4, R5 and R6, independently is H, aryl, preferably an aryl of 6-10 carbons (inclusive), most preferably phenyl or naphthyl; heteroaryl; F, Cl, Br, I, -CN, -NO2, OH, -OR9, -O (CH2) nNR7R8, -OCOR9, -OCONHR9, NH2, -CH2OH, -CH2OR14, -NR7R8, -NR10COR9, -NR10CONR7R8, - SR11, -S (O) and R11 where y is 1 or 2; -CO2R9, -COR9, -CONR7R8, -CHO, -CH = NOR11, -CH = NR9, -CH = NNR11R12, - (CH2) nSR9, where n is an integer of 1-4 (inclusive), - (CH2 ) nS (O) and R9, -CH2SR15 wherein R15 is alkyl of 1-4 carbons, (inclusive); -CH2S (O) and R14, - (CH2) nNR7R8, - (CH2) nNHR14, alkyl of 1-8 carbons (inclusive), preferably alkyl of 1-4 carbons (inclusive); alkenyl of 1-8 carbons (inclusive), preferably alkenyl of 1-4 carbons (inclusive); alkynyl of 1-8 carbons (inclusive), preferably 1-4 carbon alkynyl (inclusive); and either each alkyl of 1-8 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive) is unsubstituted; or each alkyl of 1-8 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive) is substituted as described in d) 2), above; X is either an unsubstituted alkylene of 1-3 carbons (inclusive), or X is a alkylene of 1-3 carbons (inclusive) substituted with a group R2, preferably OR10, -SR10, R15, wherein R15 is an alkyl of 1-4 carbons (inclusive), phenyl, naphthyl, arylalkyl of 7-14 carbons (inclusive), preferably benzyl; or X is -CH = CH-, -CH (OH) -CH (OH) -, -O-, -S-, -S (= O) -, -S (= O) 2, -CR10) 2- , C (= O) -, -C (= NOR11) -, C (OR11) (R11) -, C (= O) CHR15) -, -CHR15) C (= O) -, -C (= NOR11) CHR15) -, -CHR1d) C (= NOR11) -, -CH2Z-, -Z-CH -, -CH2ZCH2-, where Z is C (OR11) (R11), O, S, C (= O), C (= NOR11), or NR11; or A1 and A2 together are each independently H, H; H, -OR11; H, -SR11; H, -NR 11 R 12; or together they represent = S o = NR11; B1 and B2 together represent O; and each of R1, R2, R3, R4, R5, R6 and X are as defined in c), d), e) and f), above; A1 and A2 together represent O, and B1 and B2 together are independently H, H; H, -OR11, H, -SR11, H, -SR11, H, -NR11R12, or together represent = S or = NR11; and each of R1, R2, R3, R4, R5, R6 and X are as defined in c), d), e), and f), above. 74. The method according to claim 73, wherein AL A2, R ?, R3 and R4 are H; ^ and B2 together represent O; R2 is CH2CH2OH; R5 and R6 are OCH3; and X is CH2. 75.- The method according to claim 73, wherein i, A2, Ri, R3, R5 and R6 are H; B- and B2 together represent O; R2 is CH2CH2Oac; R4 is Br, and X is CH2. 76.- The method according to claim 73, wherein Ai, A2, R R3, R5 and R6 are H; Bi and B2 together represent O; R2 is CH2CH2Oac; R4 is CH2CH2 (2-Pir); and X is CH2. 77. The method according to claim 73, wherein i, A2, R1t R3, R5 and R6 are H; Bi and B2 together represent O; R2 is H; R 4 is CH 2 CH 2 (2-pyrimidinyl); and X is CH2. 78.- The method according to claim 73, wherein i, A2, Ri, R3, R5 and R6 are H; B ^ and B2 together represent O; R2 is H; R4 is CH2CH2 (2-Pir); and X is CH2. 79. The method according to claim 73, wherein Ai, A2, Ri, R2, R3, R5 and R6 are H; Bi and B2 together represent O; R is CH2CH2 (2-pyridazinyl); and X is CH2. 80. - The method according to claim 73, wherein AL A2, RL R3, R, R5 and R6 are H; Bt and B2 together represent O; R2 is CH2CH2OH; and X is CH2. 81. The method according to claim 73, wherein AL A2, RL R3, R, RS and Re are H; Bi and B2 together represent O; R2 is CH2CH2CH2OH; and X is CH2. 82.- The method according to claim 73, wherein L A2, RL R2, 3. R4, Rs and Re are H; B ^ and B2 together represent O; and X is S. 83. The method according to claim 73, wherein L A2, RL R3, L R5 and e are H; B1 and B2 together represent O; R2 is CH2CH2CH2NHCO (4- (OH) Ph); and X is CH2. 84.- The method according to claim 73, wherein AL A2, RL R3, R4 > R5 and Re are H; B and B2 together represent O; R2 is CH2CH2OH; and X is CH2. 85.- The method according to claim 72, wherein the compound has the formula: wherein: ring B and ring F, independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: a) a 6-membered carbocyclic aromatic ring saturated in which 1 to 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring wherein: 1) a carbon atom is replaced with an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced with one atom of sulfur and one of nitrogen, one atom of oxygen and one of nitrogen, or two atoms of nitrogen; or 3) three carbon atoms are replaced with three nitrogen atoms; R1 is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbons, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl; b) -C (= O) R9, wherein R9 is selected from the group consisting of alkyl, aryl and heteroaryl; c) -OR10, wherein R10 is selected from the group consisting of H and alkyl having from 1 to 4 carbons; d) -C (= O) NH2), -NR11R12, - (CH2) PNR11 R12, - (CH2) pOR10, -0 (CH2) pOR10 and -0 (CH2) pNR, 1R12, wherein p is 1 to 4; and wherein either: 1) R11 and R12 each independently is selected from the group consisting of H and alkyl having from 1 to 4 carbons; or 2) R11 and R12 together form a linking group of the formula - (CH2) 2-X1- (CH2) 2-, wherein X1 is selected from the group consisting of -O-, -S-, and -CH2 -; R2 is selected from the group consisting of H, alkyl having 1 to 4 carbons, -OH, alkoxy having 1 to 4 carbons, -OC (= O) R9, -OC (= O) NR11R12, -O (CH2) pNR11 R12, -O (CH2) pOR10, substituted or unsubstituted arylalkyl having from 6 to 10 carbons, and substituted or unsubstituted heteroarylalkyl; R3, R4, R5 and R6 are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF3, -NO2, -OH, -OR9, -O (CH2) pNR11R12, -OC (= O) R9, -OC (= O) NR2R7, - OC (= O) NR11R12, -O (CH2) pOR10, -CH2OR10, -NR11R12, -NR10S (= O) 2R9, -NR10C (= O) R9; b) -CH2OR? , wherein R14 is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; c) -NR? i1? 0rC / (- = n Ou) NiDR'111 DR1122, CO2R '-C (= O) R: C (= O) NR11R12, -CH = NOR2, -CH = NR9, - (CH2 ) PN R11 R12, - (CH2) PNH R14, or -CH = NNR2R2A, wherein R2A is the same as R2; d) -S (O) and R 2 - (CH 2) p S (O) and R 9, -CH 2 S (O) and R 14, wherein y is 0, 1 or 2; e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons, and alkynyl having from 2 to 8 carbons, wherein: 1) each alkyl, alkenyl or alkynyl group is unsubstituted; or 2) each alkyl, alkenyl or alkynyl group is substituted with 1 to 3 groups selected from the group consisting of aryl having 6 to 10 carbons, heteroaryl, arylalkoxy heterocycloalkoxy, hydroxyalkoxy, alkyloxy alkoxy, hydroxyalkylthio alkoxy alkylthio, F, Cl , Br, I, -CN, -NO2, -OH, -OR9, -X2 (CH2) PNR11 R12 -X2 (CH2) pC (= O) NR1 R12, -X2 (CH2) pOC (= O) NR11R12, - X2 (CH2) pCO2R9-X2 (CH2) pS (O) and R9, -X2 (CH2) PNR10C (= O) NR11R12, OC (=) R9, OCONHR2-O-tetrahydropyranyl, -NR11R12, -NR10C (= O) R9 , -NR10CO2R9 -NR10C (= O) NR11R12, -NHC (= NH) NH2, NR10S (O) 2R9, -S (O) and R9, -CO2R2C (= O) NR11R12, -C (= O) R2, -CH2OR10, -CH = NNR2R2A, -CH = NOR2 -CH = NR9, -CH = NNHCH (N = NH) NH2, -S (= O) 2NR2R2A, -P (= O) (OR10) 2 -OR14, and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently either unsubstituted or is replaced by H, alkyl having from 1 to 4 carbons, alkylcarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 ca rbonos; X2 is O, S, or NR10; R7 and R8 are each independently selected from the group consisting of H, alkyl having 1 to 4 carbons, alkoxy having 1 to 4 carbons, substituted or unsubstituted arylalkyl having 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl , - (CH2) pOR1u, - (CH2) pOC (= O) NR11 R12, and - (CH2) PNR11 R12; or R7 and R8 together form a linking group of the formula -CH2-X3-CH2- wherein X3 is X2 or a bond; m and n each is independently 0, 1 or 2; Y is selected from the group consisting of -O-, -S-, -N (R10) -, -N + (O -) (R10) -, -N (OR10) -, and -CH2-; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, -C (= O) ~, -CH (OR10) -, -N (R10) -, -N ( OR10) -, CH (NR11R12) -, -C (= O) N (R17) -, -N (R17) C (= O) -, -N (S (O) and R9) -, -N (S (O) and NR 11 R 12) -, -N (C (= O) R 17) -, -C (R 15 R 16) -, -N + (O ') (R 10) -, -CH (OH) -CH ( OH) -, and -CH (O (C = O) R9) CH (OC (= O) R9A) -, wherein R9A is the same as R9; R15 and R16 are independently selected from the group consisting of H, -OH, -C (= O) R10, -O (C = O) R9, hydroxyalkyl and -CO2R10; R17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl; A1 and A2 are selected from the group consisting of H, H; H, OR2; H, -SR2; H, -N (R2) 2; and a group wherein A1 and A2 together form a portion selected from the group consisting of = O, = S, y = NR2; B1 and B2 are selected from the group consisting of H, H; H, -OR2; H, -SR2; H, -N (R2) 2; and a group wherein B1 and B2 together form a selected portion of the group consisting of = O, = S, y = NR2; provided that at least one of the pairs of A1 and A2, or B1 and B2, forms = O. 86.- The method according to claim 72, wherein the compound has the formula: wherein: Z (is H and Z2 is H or Z, and Z2 together form = O; Ri is selected from the group consisting of H, Cl, CH2SO2C2H5, Br, CH2S (CH2) 2NH2, CH2S (CH2) 2N (CH3 ) 2, CH2S (CH2) 2NH2 n-C4H9, NHCOCHCeHs, NHCOCHC2H5, CH2SC2H5, CH2SC6H5, N (CH3) 2, CH3, CH2OCONHC2H5, NHCO2CH3, CH2OC2H5, CH2N (CH3) 2, OH, On-propyl, CH = NNH- C (= NH) NH2, CH = NN (CH3) 2, CH2S (CH2) 2NH-n-C4H9, CH2OCH2OCH2CH3, CH2S [3- (1, 2,4-triazine)], CH2CH2SCH3; R2 is selected from the group consisting of H, Br, Cl, I, CH2S (CH2) 2N (CH3) 2, NHCONHC2H5, CH2SC2H5, CH2OCH2OCH2CH3, CH2S [3- (1,2,4-triazine)], CH2CH2SCH3, and CH2OH; X is selected from the group consisting of H, CH2OH, CH2NH-serineH, CO2CH3, CONHC6H5, CH2NHCO2C6H5, CH2NHCO2CH3, CH2N3, CONHC2H5, CH2NH-glycine, CON (CH3) 2, -CH2NHCO2-, CONH2, CONHC3H7, CH2NH-serine, CH2SOCH3, CH = NOH, CH2NH-proline, CH2CH2 (2-pyridyl), CH = NNHC (= NH) NH2, CONH (CH2) 2OH, CH = NNHCONH2, CH2OCOCH3, -CH2OC (CH3) 2O-, CH2SC6H5, CH2SOC6H5, CO2n-hexyl, CONHCH3, and CO2 (CH2) 4CH3; R is selected from the group consisting of OH, and OCH3. 87.- The method according to claim 86, wherein Z, and Z2 are H; X is CO2CH3; R, is NHCONHC2H5; R2 is CH2CH2 (2-pyridyl); and R is OH. 88. The method according to claim 86, wherein Z? and Z2 are H; X is CO2CH3; R, and R2 are CH2OCH2OCH2CH3: and R is OH. 89.- The method according to claim 86, wherein ZT and Z2 are H; X is CO2CH3; R, and R2 are CH2SCH2CH3; and R is OH. 90. The method according to claim 86, wherein ZL Z2, R-i and R2 are H; X is CO2CH3; R, is OH. 91.- The method according to claim 86, wherein ZL Z2, R, and R2 are H; X is CO2 (CH2) 4CH3; and R is OH. 92. The method according to claim 86, wherein ZL Z2 and Ri are H; R2 is CH2OH; X is CO2CH3; and R is OH. 93.- The method according to claim 86, wherein Zi and Z2 are H; R- and R2 are H2S (3- (1, 2,4-triazine)); X is CO2CH3; and R is OH. 94.- The method according to claim 86, wherein Z-i and Z2 are H; Ri is Br, R2 is I; X is CO2CH3; and R is OH. 95.- The method according to claim 86, wherein Zt and Z2 are H; Ri and R2 are CH2CH2SCH3; X is CO2CH3; and R is OH. 96.- The method according to claim 86, wherein ZL Z2, RI and R2 are H; X is CO2CH3; and R is OCH3. 97. - The method according to claim 86, wherein Z-i and Z2 together form = O; R- \ and R2 are Br; X is C02CH3; and R is OH. 98. - The method according to claim 72, wherein said compound has the formula: where: Z-, is H and Z2 is H or Z- and Z2 together form = O; R, is H or Br; R2 is H; R3 is H, CH2CH = CH2, CG2CH2CH2OH, or R4 is H, CH2CH = CH2 or CH2CH2CH2OH. 99.- The method according to claim 98, wherein RL R2, R4, ZI and Z2 are H and R3 is CH2CH = CH2. 100. The method according to claim 98, wherein Ri is Br and R2, R3, R4, Z and Z2 are H. 101. The method according to claim 98, wherein R1t R2, ZI and Z2 are H and R3 and R4 are CH2CH = CH2. 102.- The method according to claim 98, wherein RL R2, R3, ZI and Z2 are H and R4 is CH2CH = CH2. 103. The method according to claim 98, wherein RL R2, ZI and Z2 are H, and R3 and R4 are CH2CH2CH2OH; or RL R2, R4, Zi and Z2 are H, and R3 is: / \ CH2CH2CH2- N O 104. A method for treating a mammal having inflammation, comprising administering to said mammal a compound that inhibits a multiple lineage kinase protein in a pharmaceutically acceptable salt or diluent. 105. The method according to claim 104, wherein the compound has the formula: wherein: E1 and E2 independently, each together with the carbon atoms to which they are attached form either: an unsaturated 6-membered carbocyclic aromatic ring wherein from 1 to 3 carbon atoms can be replaced by nitrogen atoms; or a 5-membered carbocyclic aromatic ring saturated where either a carbon atom is replaced with an oxygen, nitrogen or sulfur atom; or two carbon atoms are replaced with a sulfur and nitrogen atom, or an oxygen and nitrogen atom; A1 and A2 together represent O, and B1 and B2 together represent O; R1 is H, alkyl of 1 to 4 carbon atoms (inclusive), aryl, arylalkyl, heteroaryl, and heteroarylalkyl; COR9, wherein R9 is alkyl of 1 to 4 carbons (inclusive), or aryl, preferably phenyl or naphthyl; -OR10, wherein R10 is H or alkyl of 1 to 4 carbons (inclusive); -CONH2, -NR7R8, - (CH2) nNR7R8, wherein n is an integer of 1-4 (inclusive); or -O (CH2) nNR7R8; and either R7 and R8 independently are H or alkyl of 1 to 4 carbons (inclusive); or R7 and R8 are combined together to form a linking group of the general formula ~ (CH2) 2-X1- (CH2) 2-, wherein X1 is O, S or CH2; R2 is H, -SO2R9; -CO2R9, -COR9, alkyl of 1 to 8 carbons (inclusive) preferably an alkyl of 1 to 4 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), preferably an alkenyl of 1-4 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive), preferably a alkynyl of 1-4 carbons (inclusive); or a monosaccharide of 5-7 carbons (inclusive), wherein each hydroxyl group of the monosaccharide independently is either substituted or is replaced by H, alkyl of 1-4 carbons (inclusive), alkylcarbonyloxy of 2-5 carbons (inclusive) or 1-4 carbon alkoxy (inclusive); and either each alkyl of 1-8 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive), is unsubstituted; or each alkyl of 1-8 carbons (inclusive); alkenyl of 1-8 carbons (inclusive) or alkynyl of 1-8 carbons (inclusive) independently is substituted with 1-3 aryl of 6-10 carbons (inclusive), preferably phenyl or naphthyl; heteroaryl, F, Cl, Br, I, -CN, -NO2, OH, -OR9, -O (CH2) nNR7R8, -OCOR9, -OCONHR9, O-tetrahydropyranium, NH2, -NR7R8, -NR10COR9; -NR10CO2R9, R10CONR7R8, -NHC (= NH) NH2, -NR10SO2R9, -S (O) and R11, wherein R 1 is H or alkyl of 1-4 carbons, aryl of 6-10 carbons, preferably phenyl or naphthyl, or heteroaryl and y is 1 or 2; -SR11, -CO2R9, -CONR7R8, * -CHO, COR9, -CH2OR7, -CH = NN R11 R12, -CH = NOR11, -CH = NR9, -CH = NNHCH (N = NH) N H2, -S02NR, 2R13, -PO (OR11) 2 or OR14 wherein R14 is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; and either R12 and R13 independently are H, alkyl of 1-4 carbons (inclusive), aryl of 6-10 carbons, preferably phenyl or naphthyl, or heteroaryl; or R12 and R13 combine together to form a linking group preferably - (CH2) 2-X1- (CH2) 2; each R 3, R 4, R 5 and R 6, independently is H, aryl, preferably an aryl of 6-10 carbons (inclusive), most preferably phenyl or naphthyl; heteroaryl; F, Cl, Br, I, -CN, -NO2, OH, -OR9, -O (CH2) nNR7R8, -OCOR9, -OCONHR9, NH2, -CH2OH, -CH2OR14, -NR7R8, -NR10COR9, -NR10CONR7R8, - SR11, -S (O) and R11 where y is 1 or 2; -CO2R9, -COR9, -CONR7R8, -CHO, -CH = NOR11, -CH = NR9, -CH = NNR11R12, - (CH2) nSR9, where n is an integer of 1-4 (inclusive), - (CH2 ) nS (O) and R9, -CH2SR15 wherein R15 is alkyl of 1-4 carbons, (inclusive); -CH2S (O) and R14, - (CH2) nNR7R8, - (CH2) nNHR14, alkyl of 1-8 carbons (inclusive), preferably alkyl of 1-4 carbons (inclusive); alkenyl of 1-8 carbons (inclusive), preferably alkenyl of 1-4 carbons (inclusive); alkynyl of 1-8 carbons (inclusive), preferably 1-4 carbon alkynyl (inclusive); and either each alkyl of 1-8 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive) is unsubstituted; or each alkyl of 1-8 carbons (inclusive), alkenyl of 1-8 carbons (inclusive), or alkynyl of 1-8 carbons (inclusive) is substituted as described in d) 2), above; X is either an unsubstituted alkyiin of 1-3 carbons (inclusive), or X is a alkylene of 1-3 carbons (inclusive) substituted with a group R2, preferably OR10, -SR10, R15, wherein R15 is an alkyl of 1-4 carbons (inclusive), phenyl, naphthyl, arylalkyl of 7-14 carbons (inclusive), preferably benzyl; or X is -CH = CH-, -CH (OH) -CH (OH) -, -O-, -S-, -S (= O) -, -S (= 0) 2, -CR10) 2-, C (= O) -, -C (= NOR11) -, C (OR11) (R11) -, C (= O) CHR15) -, -CHR15) C (= O) -, - C (= NOR11) CHR15) -, -CHR 5) C (= NOR 1) -, -CH2Z-, -Z-CH2-, -CH ZCH2-, where Z is C (OR11) (R11), O, S, C (= O), C (= NOR11), or NR11; or A1 and A2 together are each independently H, H; H, -OR11; H, -SR11; H, -NR 11 R 12; or together they represent = S o = NR11; B1 and B2 together represent O; and each of R1, R2, R3, R4, R5, R6 and X are as defined in c), d), e) and f), above; or A1 and A2 together represent O, and B1 and B2 together are independently H, H; H, -OR11, H, -SR11, H, -SR11, H, -NR11R12, or together represent = S or = NR11; and each of R1, R2, R3, R4, R5, R6 and X are as defined in c), d), e), and f), above. 106. - The method according to claim 105, wherein L A2, RL 3 and 4 are H; B-t and B2 together represent O; R2 is CH2CH2Oac; R5 and R6 are OCH3; and X is CH2. 107. - The method according to claim 105, wherein AL A2, RL R3, R5 and Rβ are H; Bt and B2 together represent O; R2 is CH2CH2Oac; R4 is Br, and X is CH2. 108. - The method according to claim 105, wherein AL A2, RL R3, R5 and Re are H; B ^ and B2 together represent O; R2 is CH2CH2Oac; R4 is CH2CH2 (2-Pir); and X is CH2. 109. The method according to claim 105, wherein AL A2, RL R3, Rs and Re are H; B1 and B2 together represent O; R2 is H; R 4 is CH 2 CH 2 (2-pyrimidinyl); and X is CH2. 110. - The method according to claim 105, wherein AL A2, RL R3, Rs and Re are H; B1 and B2 together represent O; R2 is H; R4 is CH2CH2 (2-Pir); and X is CH2. 111. - The method according to claim 105, wherein AL A2, RL R2, R3, R5 and Re are H; B, and B2 together represent O; R 4 is CH 2 CH 2 (2-pyridazinyl); and X is CH2. 112. - The method according to claim 105, wherein AL A2, RL R3, R4, Rs and Rβ are H; B and B2 together represent O; R2 is CH2CH2OH; and X is CH2. 113. - The method according to claim 105, wherein AL A2, RL R3, R4, R5 and Rβ are H; B ^ and B2 together represent O; R2 is CH2CH2CH2OH; and X is CH2. 114. The method according to claim 105, wherein AL A2, RL R2, R3, R, Rs and Rβ are H; B1 and B2 together represent O; and X is S. 115. The method according to claim 105, wherein AL A2, RL R3, R4, Rs and Rβ are H; B1 and B2 together represent O; R2 is CH2CH2CH2NHCO (4- (OH) Ph); and X is CH2. 116. - The method according to claim 105, wherein AL A2, RL R3, R4, R5 and Re are H; B ^ and B2 together represent O; R2 is CH2CH2OH; and X is CH2. 117. - The method according to claim 104, wherein the compound has the formula: wherein: ring B and ring F, independently, and each together with the carbon atoms to which they are attached, are selected from the group consisting of: a) an unsaturated 6-membered carbocyclic aromatic ring where 1 at 3 carbon atoms can be replaced by nitrogen atoms; b) an unsaturated 5-membered carbocyclic aromatic ring; and c) an unsaturated 5-membered carbocyclic aromatic ring wherein: 1) a carbon atom is replaced with an oxygen, nitrogen or sulfur atom; 2) two carbon atoms are replaced with one atom of sulfur and one of nitrogen, one atom of oxygen and one of nitrogen, or two atoms of nitrogen; or 3) three carbon atoms are replaced with three nitrogen atoms; R1 is selected from the group consisting of: a) H, substituted or unsubstituted alkyl having from 1 to 4 carbons, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl; b) -C (= O) R9, wherein R9 is selected from the group consisting of alkyl, aryl and heteroaryl; c) -OR10, wherein R10 is selected from the group consisting of H and alkyl having from 1 to 4 carbons; d) -C (= O) NH2), -NR11R12, - (CH2) PNR11 R12, - (CH2) pOR10, -0 (CH2) pOR10 and -O (CH2) pNR11R12, wherein p is from 1 to 4; and wherein either: 1) R11 and R12 each independently is selected from the group consisting of H and alkyl having from 1 to 4 carbons; or 2) R11 and R12 together form a linking group of the formula - (CH2) 2-X1- (CH2) 2-, wherein X1 is selected from the group consisting of -O-, -S-, and -CH2 -; R2 is selected from the group consisting of H, alkyl having 1 to 4 carbons, -OH, alkoxy having 1 to 4 carbons, -OC (= O) R9, -OC (= O) NR11R12, -O (CH2 ) pNR11 R12, -O (CH2) pOR10, substituted or unsubstituted arylalkyl having 6 to 10 carbons, and substituted or unsubstituted heteroarylalkyl; R3, R4, R5 and R6 are each independently selected from the group consisting of: a) H, aryl, heteroaryl, F, Cl, Br, I, -CN, CF3, -N02, -OH, -OR9, -O (CH2) pNR11R12, -OC (= O) R9, -OC (= O) NR2R7, -OC (= O) NR11R12, -O (CH2) pOR10, -CH2OR10, -NR11R12, -NR10S (= O) 2R9, -NR10C (= O) R9; b) -CH2OR1, wherein R14 is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; c) -NR10C (= O) NR11R12, -CO2R2, -C (= O) R2, C (= O) NR 11 R 12, -CH = NOR 2, -CH = NR 9, - (CH 2) PNR 11 R 12, - (CH 2) PNHR 14, or -CH = NNR 2 R 2 A, wherein R A is the same as R 2; d) -S (O) and R 2 - (CH 2) p S (O) and R 9, -CH 2 S (O) and R 14, wherein y is 0, 1 or 2; e) alkyl having from 1 to 8 carbons, alkenyl having from 2 to 8 carbons, and alkynyl having from 2 to 8 carbons, wherein: 1) each alkyl, alkenyl or alkynyl group is unsubstituted; or 2) each alkyl, alkenyl or alkynyl group is substituted with 1 to 3 groups selected from the group consisting of aryl having 6 to 10 carbons, heteroaryl, arylalkoxy heterocycloalkoxy, hydroxy alkoxy, alkyloxy alkoxy, hydroxyalkylthio alkoxy alkylthio, F, Cl , Br, I, -CN, -NO2, -OH, -OR9, -X2 (CH2) PNR11R12 -X2 (CH2) PC (= O) NR11R12, -X2 (CH2) pOC (= O) NR1 R12, -X2 (CH2) pCO2R9 -X2 (CH2) PS (O) and R9, -X2 (CH2) pNR10C (= O) NR11R12, OC (=) R9, OCONHR2-O-tetrahydropyranyl, -NR11R12, -NR10C (= O) R9, -NR10CO2R9, -NR10C (= O) NR11R12, -NHC (= NH) NH2, NR10S (O) 2R9, -S (O) and R9, -CO2R2, -C (= O) NR11R12, -C (= O) R2, -CH2OR10 , -CH = NNR2R2A, -CH = NOR2, -CH = NR9, -CH = NNHCH (N = NH) NH2, -S (= O) 2NR2R2A, -P (= O) (OR10) 2, -OR14, and a monosaccharide having from 5 to 7 carbons, wherein each hydroxyl group of the monosaccharide is independently either unsubstituted or is replaced by H, alkyl having from 1 to 4 carbons, alkycarbonyloxy having from 2 to 5 carbons, or alkoxy having from 1 to 4 carbons; X2 is O, S, or NR10; R7 and R8 are each independently selected from the group consisting of H, alkyl having 1 to 4 carbons, alkoxy having 1 to 4 carbons, substituted or unsubstituted arylalkyl having 6 to 10 carbons, substituted or unsubstituted heteroarylalkyl , - (CH2) pOR10, - (CH2) POC (= O) NR11R12, and - (CH2) PNR11R12; or R7 and R8 together form a linking group of the formula -CH2-X3-CH2- wherein X3 is X2 or a bond; m and n each is independently 0, 1 or 2; Y is selected from the group consisting of -O-, -S-, -N (R10) -, -N + (O) (R10) -, -N (OR10) -, and -CH2-; Z is selected from the group consisting of a bond, -O-, -CH = CH-, -S-, -C (= O) -, -CH (OR10) -, -N (R10) -, -N ( OR10) -, CH (NR11R12) -, -C (= O) N (R17) -, -N (R17) C (= O) -, -N (S (O) and R9) -, -N (S (O) yNR11R12) -, -N (C (= O) R17) -, -C (R15R16) -, -N + (O) (R10) -, 9A -CH (OH) -CH (OH) -, and -CH (O (C = O) Ra) CH (OC (= O) RyA) -, where R is the same as R9; R15 and R16 are independently selected from the group consisting of H, -OH, -C (= O) R10, -O (C = O) R9, hydroxyalkyl and -CO2R10; R17 is selected from the group consisting of H, alkyl, aryl, and heteroaryl; A1 and A2 are selected from the group consisting of H, H; H, OR2; H, -SR2; H, -N (R2) 2; and a group wherein A1 and A2 together form a portion selected from the group consisting of = O, = S, y = NR2; B1 and B2 are selected from the group consisting of H, H; H, -OR2; H, -SR2; H, -N (R2) 2; and a group wherein B1 and B2 together form a selected portion of the group consisting of = O, = S, y = NR2; Provided that at least one of the pairs of A1 and A2, or B1 and B2, forms = 0. 118. The method according to claim 104, wherein the compound has the formula: where: Zi is H and Z2 is H or Zi and Z2 together form = O; Ri is selected from the group consisting of H, Cl, CH2SO2C2H5, Br, CH2S (CH2) 2NH2, CH2S (CH2) 2N (CH3) 2, CH2S (CH2) 2NH2 n-C4H9, NHCOCHC6H5, NHCOCHC2H5, CH2SC2H5, CH2SC6H5, N (CH3) 2, CH3, CH2OCONHC2H5, NHC02CH3, CH2OC2H5, CH2N (CH3) 2, OH, On-propyl, CH = NNH-C (= NH) NH2, CH = NN (CH3) 2, CH2S (CH2) 2NH-n-C4H9, CH2OCH2OCH2CH3, CH2S [3- (1, 2,4-triazine)], CH2CH2SCH3; R2 is selected from the group consisting of H, Br, Cl, I, CH2S (CH2) 2N (CH3) 2, NHCONHC2H5, CH2SC2H5, CH2OCH2OCH2CH3, CH2S [3- (1,2,4-triazine)], CH2CH2SCH3, and CH2OH; X is selected from the group consisting of H, CH2OH, CH2NH-serinaH, CO2CH3, CONHC6H5, CH2NHCO2C6H5, CH2NHCO2CH3, CH2N3, CONHC2H5, CH2NH-Glycine, CON (CH3) 2, -CH2NHCO2-, CONH2, CONHC3H7, CH2NH-Serine, CH2SOCH3, CH = NOH, CH2NH-proline, CH2CH2 (2-pyridyl), CH = NNHC (= NH) NH2, CONH (CH2) 2OH, CH = NNHCONH2, CH2OCOCH3, -CH2OC (CH3) 2O-, CH2SC6H5, CH2SOC6H5, CO2n-hexyl, CONHCH3, and CO2 (CH2) 4CH3; R is selected from the group consisting of OH, and OCH3. 119. The method according to claim 118, wherein Z and Z2 are H; X is CO2CH3; R is NHCONHC2H5; R2 is CH2CH2 (2-pyridyl); and R is OH. 120. The method according to claim 118, wherein ZT and Z2 are H; X is CO2CH3; R ^ and R2 are CH2OCH2OCH2CH3: and R is OH. 121. The method according to claim 118, wherein Z1 and Z2 are H; X is CO2CH3; R ^ and R2 are CH2SCH2CH3; and R is OH. 122. The method according to claim 118, wherein ZL Z2, RI and R2 are H; X is CO2CH3; R is OH. 123. The method according to claim 118, wherein ZL Z2, RI and R2 are H; X is CO2 (CH2) 4CH3; and R is OH. 124. The method according to claim 118, wherein ZL Z2 and R1 are H; R2 is CH2OH; X is CO2CH3; and R is OH. 125. The method according to claim 118, wherein Z1 and Z2 are H; R ^ and R2 are H2S (3- (1, 2,4-triazine)); X is CO2CH3; and R is OH. 126. The method according to claim 118, wherein Z? and Z2 are H; R-i is Br, R2 is I; X is CO2CH3; and R is OH. 127.- The method according to claim 118, wherein Z-i and Z2 are H; RT and R2 are CH2CH2SCH3; X is CO2CH3; and R is OH. 128. The method according to claim 118, wherein ZL Z2, R, and R2 are H; X is CO2CH3; and R is OCH3. 129. The method according to claim 118, wherein Z? and Z2 together form = O; R ^ and R2 are Br; X is CO2CH3; and R is OH. 130. The method according to claim 104, wherein said compound has the formula: where: Z, is H and Z2 is H or Z | and Z2 together form = O; R, is H or Br; R2 is H; R3 is H, CH2CH R4 is H, CH2CH = CH2 or CH2CH2CH2OH. 131. The method according to claim 130, wherein Ri, R2, R4, Z1 and Z2 are H and R3 is CH2CH = CH2. 132. The method according to claim 130, wherein Ri is Br and R2, R3, R4, Z and Z2 are H. 133. -The method of claim 130, wherein Ri, R2, Z, and Z2 are H and R3 and R are CH2CH = CH2. 134. The method according to claim 130, wherein RL R2, R3, ZI and Z2 are H and R4 is CH2CH = CH2. 135. The method according to claim 130, wherein RL R2, Zi and Z2 are H, and R3 and R4 are CH2CH2CH2OH; or RL R2, R4, T and Z2 are H, and R3 is: / \ CH2CH2CH2- N O