MXPA97003039A - Protein kinase inhibitors for the treatment of neurologi disorders - Google Patents

Abstract

The invention includes novel derivatives of K-252a, for example (IL-4), as well as novel substituted BIS-N derivatives of staurosporine of the formula (I). The invention also includes a method for treating diseased neuronal cells involving the administration of the novel staurosporine derivatives or specified functional derivatives of K-252a. In the formula (I): [STAU] -N (CH3) -wN (CH3) - [STAU] represents a residue of the formula (a) and W represents a radical of the formula - (C (= Y) -NH -W-NH-C (= Y), where W is a hydrocarbylene radical of 2-20 carbon atoms and Y is = or S. In the formula (IL-4), R1, R2, Z1 and Z2 are each H , X is CH2OH, and R is OC

Description

INHIBITORS OF PROTEIN QUINABA FOR THE TREATMENT OF NEUROLOGICAL DISORDERS BACKGROUND OF THE INVENTION Protein kinases are a broad class of enzymes that act to chemically modify many cellular proteins, by phosphorylating amino acids. Inhibitors of protein kinases are li? structurally varied, and have variable (and sometimes contradictory) effects on the nervous system and other tissues. A given protein kinase inhibitor can influence more than one protein kinase. For example, it was originally reported that K-252a, a material in the form of an alkaloid isolated from the culture broth of Nocardiopsis sp. and Actino adula sp. , is a protein kinase C inhibitor, but was subsequently found to also inhibit protein kinases A and G, myosin light chain kinase, and trk (a tyrosine kinase 0 activated by growth factor). nervous (NGF), the latter being a neurotrophic protein that promotes the survival of peripheral, sensory, and sympathetic neurons). Consistent with this latter effect, K-252a 5 blocks the neurotrophic actions of nerve growth factor on PC-12 cells (chromaffin cells of rat adrenal medullary tumors, feocro ocitomas), and promotes the survival of ganglionary neurons of dorsal root and hippocampal neurons. However, it has been found to be cytotoxic in a wide range of concentrations, leading some researchers to conclude that it has limited utility in vivo. A microbial alkaloid related to K-252a, staurosporine, also has a variety of effects on different protein kinases and cell types. It was discovered that staurosporine has effects in the form of nerve growth factor on PC-12 cells, and protects the gerbil hippocampus from post-ischemic injury. It can reverse the damage to cholinergic neurons in the rat basal forebrain. K-252a and staurosporine have been proposed as tumor inhibitors. Staurosporine has been offered as an insecticide. Staurosporine derivatives have been made with a hydrocarbyl radical or an acyl radical substituted on the methylamine nitrogen, and have been proposed for the following uses: tumor inhibition, inhibition of inflammation, immunomodulation, and treatment of diseases of the cardiovascular and central nervous systems.

SUMMARY OF THE INVENTION The invention provides, in one aspect, novel bis-N-substituted derivatives of staurosporine, represented by the formula: [Stau] -N- (CH3) -W-N (CH3) - [Stau] (I) where [Stau] represents a residue of the formula: H and represents a radical of bis (carbamyl) or bis (thiocarbamyl), -C (= Y) -NH-W -NH-C (= Y) - wherein W is a hydrocarbylene radical of 2 to 20 carbon atoms, and Y is 0 or S. In a preferred aspect, the invention provides, for example: 1, 6-hexamethylene-bis- (carbamyl-staurosporine) (HBCS); p-phenylene-bis- (carbamyl-staurosporine) (PBCS). The invention also provides a novel derivative of K-252a, represented by the formula (II-4): (II-4) wherein R1, R2, Z1, and Z2 are each independently H; X is hydroxymethyl (CH20H); and R is 0CH3. The invention also provides a novel derivative of K-252a, represented by the formula: wherein R1, R2, Z1, and Z2 are each independently H; X os CH2-NH-SerH; and R is OH. Also included in the compound invention: represented by the following formula (11-49): (11-49) wherein R2, Z1, and Z2 are each H; R is OH; R1 is CH2S02C2H5; and X is C02CH3. Also included in the invention are compounds represented by the following formula (11-38): X? -J o) wherein R1, R2, Z1, and Z2 are each H; R is OH; and X is CH2NHC02C6H5, Compounds represented by the following formula (11-45) are also included in the invention: wherein R1 and R2 are each Br; R is OH; Z1 and Z2 are each H; and X is CONHC6H5. Also included in the invention are compounds represented by the following formula (11-57): wherein R1, R2, Z1, and Z2 are each H; R is OH; and X is CH2NHC02CH3. Also included in the invention are compounds represented by the following formula (11-72): (11-72) wherein R1 is CH2S (CH2) 2NH2; X is C02CH3; R is OH; and R2, Z1, and Z2 are each H. Compounds represented by the following formula (11-75) are also included in the invention: .:? - 75; N / - where R1 is CH = N-N is C02CH3; R is OH; \ - and R2, Z1, and Z2 are each H.

Also included in the invention are compounds represented by the following formula (11-79) "CI-79) wherein R1 is CH2S (CH2) 2NH n-C4H9, X is C02CH3; R is OH; and R2, Z1 and Z2 are each H. Compounds represented by the following formula (11-80) are also included in the invention: CI-30) wherein R1 IS CH2S (CH2) 2N (CH3) 2; R2 is CH2S (CH2) 2N (CH3) 2; X is C02CH3; R is OH; and Z1 and Z2 are each H. Compounds represented by the following formula (V) are also included in the invention: (V) wherein X represents C02R5 (wherein R5 represents lower alkyl) or CH2NHC02R6 (wherein R6 represents lower alkyl or aryl); R1 represents hydrogen or CH2S02R7 (wherein R7 represents lower alkyl), provided that the combination of X = C02R5 and R1-hydrogen is excluded. In the definitions of the groups of the formula (V), lower alkyl means a straight or branched chain alkyl group having from 1 to 6 carbon atoms, preferably from 1 to 3 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, tertiary butyl, pentyl, neopentyl, and hexyl. Aryl means an aryl group having from 6 to 10 carbon atoms, such a phenyl and naphthyl cone. Also included in the invention are compounds (VI-i, represented by the following formula (VI): X (VI-I) wherein X is C02CH3; R is OH; each R1, R1, Z1 and Z2 is H; and R8 is NHCONHC2H5. Also included in the invention are compounds (VI-2) represented by the formula (VI): (VI-2) where X is C02CH3; each R2 and Ra is NH2; R is OH; and each R1, Z1, and Z2 is H.

The compounds of the invention may be in the form of pharmaceutically acceptable salts, including acid addition salts, metal salts, ammonium salts, organic amine addition salts, and pharmaceutically acceptable amino acid addition salts.

Examples of the pharmaceutically acceptable 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, and citrate. Examples of the pharmaceutically acceptable metal salts are alkali metal salts, such as sodium salt and potassium salt, alkaline earth metal salts, such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of the pharmaceutically acceptable ammonium salts are ammonium salt and tetraethyl ammonium salt. Examples of the pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of the pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine, and phenylalanine.

In another aspect, the invention provides a method for improving the function of cholinergic neurons, striatal neurons, basal forebrain neurons, and sensory neurons, eg, dorsal root ganglion neurons, by administration to a mammal. , for example, a human being, of a therapeutic amount of one of the novel bis-substituted derivatives of staurosporine.

The therapy can be given in conjunction with a trophic factor, preferably a member of the neurotrophin family, and more preferably nerve growth factor (NGF). As used herein, a "trophic factor" is a molecule that directly or indirectly affects the survival or function of a cell that responds to the trophic factor. The neurotrophin family is a group of proteins with significant homology to nerve growth factor, and includes, in addition to nerve growth factor, neurotrophic factor derived from the brain (BDNF; Leibrock et al., Nature. 341: 149-152, 1989; neurotrophin-3 (NT-3; Hohn et al., Nature 344: 339-341, 1990); and neurotrophin-5 (NT-4/5; Berkemeier et al., Neuron 7: 857-866, 1991).

In another aspect, the invention provides a method for protecting nerve cells of a mammal, eg, a human being, from degeneration induced by excitatory amino acids, by administering to the mammal a therapeutic amount of one of the derivatives novel bis-substituted of staurosporine. The conditions in which this degeneration can occur include Alzheimer's disease; motor neuron disease, for example, amyotrophic lateral sclerosis; Parkinson's disease; cerebrovascular disease, for example, ischemic conditions; dementia due to AIDS; epilepsy; Huntington's disease; and concussive or penetrating injuries to the brain or spinal cord. The therapy can be given in conjunction with a neutrophic factor, preferably a member of the neutrophil family, more preferably nerve growth factor (NGF).

In another aspect, the invention provides a method for improving the function of cholinergic neurons, striatal neurons, neurons of the basal forebrain, and / or sensory neurons, eg, dorsal root ganglion neurons, in a mammal, for example, a human being, by administering to the mammal a therapeutic amount of a functional derivative of K-252a, represented by the formulas: l -.- .. (IV) H (V) (VI) with any of the substitutions shown in Table 1 below. Preferably, the method for improving the function and / or survival of a cholinergic neuron, a striated neuron, a basal forebrain neuron, and / or a sensory neuron, eg, a dorsal root ganglion neuron, in a mammal , involves administering an effective amount of, for example, Compound II-3, 11-20, 11-30, 11-33, 11-38, 11-49, 11-51, 11-65, 11-69, 11 -72, 11-73, 11-79, 11-80, VI-1, or VI-2 of Table 1, to the mammal. More preferably, the method for improving the function and / or survival of a cholinergic neuron, a striated neuron, a basal forebrain neuron, or a sensory neuron in a mammal, involves administering an effective amount of Compound 11-51: Table 1 • 7 1 (- 'I Compound R * X R - 2 : I-I H CH-.N - OH:? - 2 Í. HC0NHC5H ¡H C0: CH, OH H:? - 3 CH: S0C: Hs H co2c: -: 2 OH H:? - 5 C0NHC: H «OH H:? - 6 CK» N; H- 'OH 11-43 HC0: CH3 H -. W 1 V-Hl OH H 11-44 3rd 3r OH 11-45 3rd Sr v -.- .. h .6Hs OH H 11-46 Br 3r CCí.HCH2CH2OH OH H 11-47 CH2OC2H5 H Ji ..I1 OH H 11-48 CH2N (CH3) 2 H w 1 Crí ^ CH H 11-49 CH-, SO.C-, Hs H C02CH3 OH H 11-50 CH2S- > H Cs-- > 2 Hi OH H 11-57 H H CH2NHC02CH3 OH H 11-58 Br H C0NH2 OH H 11-59 H H CH2SC5H5 OH H 11-60 H H CH: S- (?> OH H 11-61 H H CH2SOCsH5 OH H 11-62 H H C02n- hexyl OH H 1-63 OH H CO-CH, CH • J :: - 64 0 n-pr opilo C3:: CH -_ • 11-65 CH: SCH2CH: N (CH3) 2 H -.-.-.-. p 3 CH and 11-66 H H -... 2 --- i2 H u 11-57 H rt H CC..HCH J, OH H 11-68 CH: S-fQ | H CC2CH, OH H 2. 1-72 CH2S (CH2) 2 H2 H OH -: 11-73 CH-.S ^ '.- H H ::: CH: OH -: p- CH = NNH-C. = NH) ..H- - ZZ-Zr .. ^ u • -. 1-7 or: H = N- H C02CH OH 1-79 H-.S (CH -.), NH- or CC-CH CH H n-C4H9: -β CH-.S- CH, S (CH,) 2- C3-.CH, OH (CH2) 2N (CH3) 2 * (* CH3) 2 II- 81 CH2SCH (CH3) 2 Cf, SCH (CH,), CO-.CH, OH H II- 82 CH2S (CH2) 2CK, CK: S (CH :): CH, CO-CH, OH; -j 83 CH2S (CH2), CH: CH: S.CH :) 2CH, CO, CH, CH H - "84 CH2OCH3 CH; OCH3 C02CH3 OH H - .X" 85 CH.OCH, CH; OC2H5 CO: CH, OH H :? - 86 CH20H HCONHCH. CO, CH, OH H • t_87 CH2SC- > He N? CCNHC2H5 CO2CH3 OH .w. 88 CH. CH3 C02CH3 OH H II-89 CH2SC2HS CK2S (0) C2H5 CO- .CH-, OH H 90 CHnOH CH2 OH CH2 OH OH H -91 CH (-5CH2CH: S- NO: CO: CH, CH H -92 CK (-SCK-.CH, S-) S'HCONHCH, CO.CH, H • _¡ II -2 D '- 1' "* '•' H H .2 < S) 7-3 < 6 > H • _4l3- °) .- 1 •? -? Li: > C3: CH. OH CO-CH. OH (1) Z1 and Z2 are both hydrogen, or both combine to represent oxygen, where indicated. (2) NH-amino acid linkage is an amide bond through the carboxyl group of the amino acid. (3) X and R combine with each other to form the linking group. (4) R3 is CH2CH = CH2; R4 is H. (5) R3 and R4 are each H. (6) R3 and R4 are each CH2CH = CH2. (7) The compound is in the hydrochloride form. (8) R3 is H and R4 is CH2CH = CH2. (9) IV-1 and IV-4 are a mixture of 1.5 to 1.0 of the two components. (10) R3 = R4 = CH2CH2CH2OH (11) R3 = CH2CH2CH2-N? ° '* R4 = - (12) R8 = NHCONHC2H5. (13) R8 = NH2.

The therapy can be given in conjunction with a trophic factor, preferably a member of the neurotrophin family, more preferably nerve growth factor (NGF).

In a preferred aspect, the invention provides a method for improving the function of a dorsal root ganglionic nerve cell, by administering to a mammal, e.g., a human, a therapeutic amount of a functional derivative of K-252a, represented by the formula (II) or (III): (II) (III) where the following substitutions are made: Table 2 C) Compound (i) R * I -l H OH u II-2 NHCCNHC6H5 C02CH3 OH H SI-3 CH - * SOC- > rí e C02CH3 OH H II-4 H CH20H OCH3 H II-8 H CON (CH3) 2 OH H II-9 < 3 »H -CH, NHC0: - H 11-10 3rd C0: CH3 OH H 11-11 H C0NH2 OH H 1-12 H CH; OH OH H SII-1 - - - H 11-13 - H CONHC3H7 OH H 11-19 H CH = NHC = NH) H; OH u II-201.1 '3r C0-CH3 OH 0 1 - . 1 -21 u CC;; H (CH :) OH OH H III-2 - - - 0 1-23 H H OH H 11-24 H »Hsm? FlC? NH? OH 11-25 H ZH ZCCCH2 OH and 11-30 CH-, S C -. H i - ^ C '? CH H 11-32 3rd ZZ2CH: OH - (1) R2 is hydrogen, except for compounds II-; 11-32 where R ~ = Br, (2) Z1 and Z are both hydrogen, or both combine to represent oxygen, where indicated. (3) X and R combine with each other to form the linking group.

The therapy can be given in conjunction with a neurotrophic factor, preferably a member of the neurotrophin family, more preferably nerve growth factor (NGF). In a preferred aspect, the invention provides a method for improving the function of cholinergic neurons of a mammal, for example, a human, by administering to the mammal an amount of K-252a, represented by formula (II): (I wherein R1 and R2 are each H # X is C02CH3, R is OH, and Z1 and are each H. Therapy may be given in conjunction with a trophic factor, preferably a member of the neurotrophin family, more preferably nerve growth factor (NGF). In a preferred aspect, the invention provides a method for improving the survival and / or function of a striatal nerve cell, by administering to a mammal, e.g., a human, a therapeutic amount of K-252a or a derivative Functional K-252a, represented by the formulas (II), (III), or (IV): II IV where the following substitutions are made: Table 3 t2 (1) Compound R1 R 'z1 Br H CH2OH OH H Br 3r C02CH-OH - H H CH, SC6HS; j CO-CH, OH H Cl "Ci C?" CH, CH (1) Z1 and Z2 are both hydrogen, or both combine to represent oxygen, where indicated. (2) R3 is CH2-CH = CH2; R4 is H.

In another aspect, the invention provides a method for improving the survival and / or function of a nerve cell of the basal forebrain, by administering to a mammal, eg, a human, a therapeutic amount of K-252a or a functional derivative of K-252a, represented by the formula (II) II where the following substitutions have been made: Table 4 Compound X R_ Y2, 71 .11 ..-: ---- H II co: cp, OH II II - 3 CH.SOC? Lj II coc? 3 OH II II - 3 II II CONHC ^ II, OH H l - 10 Br HC ?: CH 3 OH H 11-20 Or Ur CO: CH, OH 0 l! -2¡H II CONHíCH,). : OH OH H ii- :: H II C?: CMj OH 0 11-30 CH ~ SC.il. 1. C ?: CHj OH H II-J: Dr Hr CO-C1I- OH M 11-51 CH: SC, II5 CII-SC-H, CO: CIIj OH rl 11-62 H H C?; N-hex. ilo OH H I1-6.Í OH II CO.CI.J OH H 11-64 O n- propi lo H CO; CHj OH H 11-65 CH: SCH CH; .MCHjK H CO: CH3 OH H (1) Z1 and Z2 are both hydrogen, or both combine to represent oxygen, where indicated.

The therapy can be given in conjunction with a trophic factor, preferably a member of the neurotrophin family, more preferably nerve growth factor. Other characteristics and advantages of the invention can be seen from the following description of the preferred embodiments thereof, and from the claims.

Description of Preferred Modes First, the drawings are described.

Drawings Figure 1 is a graph illustrating the effect of the derivatives of K-252a: 1,6-hexamethylene-bis- (carboamyl-taurosporine) (HBCS) and staurosporine on the activity of basal ornithine decarboxylase (ODC) in cells PC-12. Figure 2 is a graph illustrating the effects of staurosporine, HDCS, and K-252a on the activity of ornithine decarboxylase stimulated by nerve growth factor in PC-12 cells. Figure 3 is a graph illustrating the enhancing effect of the nerve growth factor of the HDCS on the activity of ornithine decarboxylase in PC-12 cells. Figure 4 is a graph illustrating the effect of K-252a on the specific activity of choline acetyltransferase (ChAT) in rat embryonic spinal cord cultures. Figure 5 is a graph illustrating the time course of the effect of K-252a on choline acetyltransferase activity in rat embryonic spinal cord cultures. Figure 6 is a graph illustrating the effect of K-252a on the survival of chicken embryonic dorsal root ganglion neurons. Figure 7 is a graph illustrating the effect of the functional derivatives of K-252a on the survival of chicken embryonic dorsal root ganglion neurons. Figure 8 is a graph illustrating the effect of the functional derivatives of K-252a on the activity of choline acetyltransferase in rat embryonic spinal cord cultures. Figure 9 is a graph illustrating the effect of K-252a on the damage induced by kainate to rat hippocampus. Figure 10 is a graph illustrating the effect of K-252a on the speculin proteolysis induced by kainate in rat hippocampus. Figure 11 is a graph illustrating the effect of HBCS on the damage induced by kainate to the hippocampus. Figure 12 is a graph illustrating the effect of the functional derivatives of K-252a on the speculin proteolysis induced by kainate in the rat hippocampus. Figures 13a, 13b, and 13c are tables showing the relative activity of the K-252a derivatives on choline acetyltransferase activity in rat spinal cord cultures. Figure 14 is a table showing the relative activity of the K-252a derivatives on neuronal survival in chicken dorsal root ganglion cultures. Figure 15 is a graph illustrating the survival of striatal neurons in the presence of K-252a. Figure 16 is a graph illustrating the time course of the survival of striatal cells in the presence of K-252a. Figure 17 is a pair of photomicrographs of striated neurons cultured in the presence or absence of K-252a. Figure 18 is a table showing the relative activity of the K-252a derivatives on neuronal survival in rat striated cultures. Figure 19 is a table showing the relative activity of the K-252a derivatives on the survival of low-density basal forebrain neurons. Figure 20 is a bar graph demonstrating that Compound 11-51 prevents motoneuron death programmed in in ovo development.

Figure 21 is a photographic demonstration that Compound 11-51 prevents the loss induced by axotoraphy of choline acetyltransferase immunoreactivity in the adult hypoglossal nucleus. 5 Figure 22 is a diagram showing the synthesis of the Compound H from Compound of Compound C. Figure 23 is a diagram showing the synthesis of Compound 11-45 from Compound of Compound J. _-. Figure 24 is a diagram showing structure 0 of Compound P, Compound Q, and Compound R. Figure 25 is a diagram showing the synthesis of Compound IV-6 from Compound of Compound S. Figure 26 is a diagram that shows the chemical structure of the compounds (AA), (BB), (CC), (DD), and (EE). Figure 27 is a diagram showing the chemical structure of Compounds (FF), (GG), (HH), and (JJ). 0 Staurosporin Derivatives The present invention relates to novel bis-N-substituted staurosporine derivatives, and to their use as therapeutics for neurological diseases, especially those diseases characterized either by neuronal cells that are injured, compromised, suffering from axonal generation, or with greater risk of dying, or by an affected cholinergic activity. These diseases include those induced by excitation amino acids. The therapeutic use of these novel derivatives includes the use of the derivatives alone, and the use of the combined derivatives with an exogenous administration of neurotrophic factors (preferably members of the neurotrophin family, more preferably nerve growth factor). The compounds within the scope of this invention can be represented by the formula: [Stau] -N- (CH3) - -N (CH3) - [Stau] (I) where [Stau] represents a residue of the formula: and W represents a radical of bis (carbamyl) or bis (thiocarbamyl), -C (= Y) -NH-W'-NH-C (= Y) - and wherein W is a hydrocarbylene radical of 2 to 20 carbon atoms, and Y is 0 or S. W is preferably an alkylene radical of 2 to 10 carbon atoms, unsubstituted or substituted by 1 to 3 alkyl groups of 1 to 3 carbon atoms; an arylene radical of 6 to 12 carbon atoms, unsubstituted or substituted by 1 to 3 alkyl groups of 1 to 3 carbon atoms, chlorine or bromine. W is especially preferably hexamethylene and 1,4-phenylene. And it is preferably O. The compounds of the formula (I) can be prepared by processes known in the art for the preparation of carbamates and thiocarbamates. Preferably, the compounds are prepared by the reaction of bis-diisocyanate or a bis-diisothiocyanate with staurosporine, to give a compound of the formula (I) wherein Y = 0 or Y = S, respectively. Intermediate bis-diisocyanates and bis-diisothiocyanates suitable for use include: 1,6-diisocyanatohexane toluene-2,6-diisocyanate benzene-1,2-diisocyanate 2-methyl-1, 5-diisocyanatopentane naphthalene-2,6-diisocyanate 1,6-diisothiocyanatohexane 1,4-diisothiocyanatobutane toluene-2, 4 -diisocyanate benzene, 1,4-diisocyanate 1,2-diisocyanatoethane naphthalene-1,5-diisocyanate 1,5-diisocyanatopentane benzene-1,4-diisothiocyanate 2-methyl-1, 5-diisothiocyanatopentane for reviews of the preparation of isocyanates and isothiocyanates, see Richter and Ulrich, pages 619-818, in The Chemistry of Cvanates and Their Thio Derivatives. Part 2, (Patai, ed.) Wiley, New York, 1977. The compounds are preferably prepared by the reaction of phosgene (Y = 0) or thiophosgene.

(Y = S) with the corresponding diamine. Alternative methods of preparation may also be employed. For example, 1,2-diisocyanatoethane can be prepared by the reaction of ethyleneurea with phosgene, followed by heating.

Derivatives of K-252a The present invention also relates to the use of specific functional derivatives of K-252a, as therapeutics in certain neurological diseases or disorders characterized by neurons that are injured, compromised, suffer from axonal generation, or at risk of dying . Functional derivatives can be administered alone or in conjunction with a neurotrophic factor (preferably a member of the neurotrophin family, more preferably nerve growth factor, NGF). A "functional derivative" of K-252a is defined as a modified form of that molecule, which possesses the desired biological activity, defined herein as a neuroprotective activity, for example the ability to promote the survival of nerve cells, or promote the growth of nerve fibers (eg, axonal), or to improve the function of the cholinergic nerve cell, or to improve the function of sensory cells, for example, ganglionic nerve cells of the dorsal root, or to improve the function and / or survival of striated neurons, or to improve the function and / or survival of the neurons of the basal forebrain. These molecular modifications can improve solubility, absorption, transport (for example, through the blood-brain barrier and cell membranes), the biological half-life of the molecules, and so on. Alternatively, or in addition, some fractions may decrease the toxicity of the molecule, or eliminate or attenuate any undesirable side effects of the molecule. Compounds within the scope of the invention may be represented by the formula (II) [hereinafter referred to as compound (II)], of the formula (III) [hereinafter referred to as compound (III)], formula (IV) [hereinafter referred to as compound (IV)], of formula (V) [hereinafter referred to as compound (V)], and of formula (VI) [hereinafter referred to herein] as compound (VI)], following: (II) .III) (IV) (V) (v; 0 with the substitutions of Table 5 below. The functional derivatives of K-252a of the invention can be prepared de novo by chemical synthesis, using methods known to those skilled in the art. For example, the procedures employed for the preparation of Compound II are described by Murakata et al. (U.S. Patent No. 4,923,986), incorporated herein by reference. The procedures used for the preparation of (Compound III, are described by Moody et al, J Org Chem 57, 2105-2114 (1992), Steglich et al., Angew Chem. Int. Ed. Engl. 459-460 (1980), Nakanishi et al., J. Antibiotice 39: 1066-1071 (1986), and Japanese Patent Application Number 60-295172 (1985). Other methods for compounds II-1, 9, 12 are described. , and 15 in Japanese Patent Application Number 60-295172 (1985), for compounds II-2, 2, 4, 24, 25, and 26 in Japanese Patent Application No. 62-327858 (1987); compounds 11-20 in Japanese Patent Application Number 62-327859 (1987), and for compounds 11-10 in Japanese Patent Application Number 60-257652 (1985) by Meiji Seika Kaisha Ltd.

Table 5; Functional Derivatives of K-252a (12) -1 (1) Compound R4 R II-l H H CH: H3 OH H II-2 NHCONHC6H5 H C02CH3 OH H II-3 CH2SOC: H5 H C02CH3 OH H II-4 H H CH20H OCH3 H II-5 H H CONHC2H5 OH H II-6 H H CH-NNH- ^} OH H H_7 (2.7 'H H CH2NH-Gly OH H 11-10 Br H C02CH3 OH H 11-11 H H C0NH2 OH H 1-12 H H CH: 0H OH H :: - i3 H H C0KHC3H7 OH H II-14 (2 'H H CH; NH-Ser OH H 1-15 H H CH-, SOCH, OH H 11-16 H H CH-NOH OH H:? -? S < 2- '"> H u CH2NH-? Rs OH H 1-19 or H CH» NNKC (- H) NH2 OH H 11-20 3rd C02CH, OH 0 1-21 H H CONH (CH2) 2OH CHH 1-22 H u C0: CH, OH 0 11-23 • H H H OH u -24 H H CH-NNHCONH * OHH 11-25 H H CH: OCOCH 3 OH H II-26 < 3 > H H -CH20C (CH3) 2"H 11-29 NHCCNHC: H5 H CO: CH3 OH H 1-30 CH? SC2HC H C02CH3 OH H 1-31 Br H CH2OH OH H 11-32 Br Br C02CH3 OH H 11-33 CH2SC6H5 H C02CH3 OH H 11-34 Cl Cl C02CH3 OH H 11-36 H H C0NHC6HS OH H 1-37 H H CH: SO-? OH H 11-38 H H CH2NHC02C6H 5 OH H 11-39 NHCONHC2H5 NHCONHC2H5 CO: CH3 OH H 11-40 N (CH3) 2 H C02CH3 OH - H 11-41 CH3 H C02CH3 OH H 11-42 CH20C0NHC2H5 H C02CH3 OH H 11-43 NHC02CH3 H C02CH3 OH H 1-44 Br Br CH20H OH H 1-45 Br Br C0NHC6H5 OH H 1-46 Br Br CONHCH-.CH -.OH OH H - -. - < CH-.OC.Hc H CO: CH, OH K I Z - S CH2? (CH3) 2 H C0: CH3 OH n II-49 CH2S02C2H5 H CO: CH3 OH 1-50 CH: S- ^ > H CO: CH3 OH 1-51 CH2SC2H5 CH2SC: H5 CO: CH3 OH H 1 -52 CH-NNH-f H C0: CH3 OH H 1-53 CH, S- < ® * H C02CH3 OH H 1-54 CH2S (0) - '? H CO CH, CH u 11-55 CH2S (0) - < or > H CO: CH, OH II-5Ó CH - .. SC4..He3 CH ,, OH C02CH3 OH H I -.- 57 U: -: CH2NHCO: CK2 CH H 1-53 a «- -_; CONH: CH: -: :: - 5s' u; -: CH.SC_.KS CH: -: -60 H H cH2S- 0 > OH H -61 H H CH2SOC6H5 OH H -62 H H C02n-hexyl OH H -63 OH H C02CH3 OH H -64 0 n-propyl H C02CH3 OH H -65 CH2SCH2CH2N (CH3) 2 H C02CH3 OH H -66 H H CH2NH2 OH H -72 CH2S (CH2) 2NH2 H C02CH3 OH H -73 CH, S -? H H C02CH3 OH H -74 CH "NNH-C (-NH) NH 2 H C02CH3 OH H -75 CH- -N < 2 H C02CH3 OH H -76 HNN 0 H C02CH3 OH 'H -77 CH "NN (CH3) 2 H C02CH3 OH H -78 CH» NN ^^ NCH3 H C02CH3 OH H -79 CH-.S (CH2) 2NH- H C02CH3 OH H -n-C4H9 -80 CH-.S- CH2S (CH2) 2- C02CH3 OH H (CH2) 2N (CH3) 2 N (CH3) 2 -31 CH2SCH (CH3) 2 CH2SCH (CH3) 2 C02CH3 OH H -82 CH2S (CH2) 2CH3 CH2S (CH2) 2CH3 C02CH3 OH H -33 CH2S (CH2) 3CH3 CH2S (CH2) 3CH3 OH2CH3 OH H -84 CH2OCH3 CH2OCH3 C02CH3 OH H-35 CH: OC2H5 CH2OC2H5 'C02CH3 OH H -36 CH20H NHCONHC2H5 C02CH3 OH H -37 NHC0NHC2Hs C02CH3 OH H -38 CH, C02CH, OH H-39 CH: S (0) C: H «CO: CH2 OH? -90 CH-.0K CH-CH CH-.0H OH 11-91 CH (-SCH2CH, 5- N02 C02CH3 OH H 11-92 CH (-SCH2CH: S- HCONHC2H5 C02CH3 OH H III-1 - - - H III-2 - - - - 0 IV-1 < 4'9 'H H - - H IV-2 < 5 > Br H - - H IV-3 (6 > H H - - H IV - 4 (8'9) H H - - H IV-5 < 10 > H H - - H IV-6 (7 ':: »H H - - H VI-1 (13) H H C02CH, OH H VI-2 (14) H NH2 C02CH3 CH H (1) Z1 and Z2 are both hydrogen, or both combine to represent oxygen, where indicated. (2) The NH-amino acid bond is an amide bond through the carboxyl group of the amino acid. (3) X and R combine with each other to form the linking group. (4) R3 is CH2CH = CH2; R4 is H. (5) R3 and R4 are each H. (6) R3 and R4 are each CH2CH = CH2. (7) The compound is in the hydrochloride form. (8) R3 is H and R4 is CH2CH = CH2. (9) IV-1 and IV-4 are a mixture of 1.5 to 1.0 of the two components. (10) R3 = R4 = CH2CH2CH20H. (11) 0; R4 = H. (12) For K-252a itself, R1 = R2 = H, X = C02CH3, R = 0H, and Z1 and Z2 are each H. (13) R8 = NHCONHC2H5. (14) R8 = NH2.

The invention also involves a method for improving the function of cholinergic neurons, by administering a therapeutic amount of K-252a, represented by formula (II) given above, and the substitutions shown in Table 5 (note 12) . This compound is prepared by the methods described in the art (see Matsuda et al., U.S. Patent No. 4,554,402; Kase et al., J .. Antibiotics 37: 1059-1065, 1986). "Improving the function of cholinergic neurons" means promoting the survival of cholinergic nerve cells, and / or the growth of nerve fibers (eg, axonal), and / or improving the cholinergic function of nerve cells. K-252a can be administered with or without a trophic factor, preferably a member of the neurotrophin family, more preferably nerve growth factor (NGF).

Uses of the Compounds As described more fully below, the present invention provides novel uses of functional derivatives of K-252a or compounds of the formula I, either alone or in combination with neurotrophic factors such as nerve growth factor, as therapeutic for neurological diseases, especially those diseases characterized by neuronal cells that are injured, compromised, that suffer from axonal generation, or with greater risk of dying, or by a damaged cholinergic activity. These diseases include those induced by excitation amino acids. The bioactivity of the compounds of the invention, including the combination with a neurotrophic factor, can conveniently be tested by an ornithine decarboxylase assay of cultured PC-12 cells, a choline acetyltransferase assay of spinal cord or cultured basal forebrain. , a survival trial of cultured dorsal root ganglion neurons, a survival assay of cultured striated neurons, a survival trial of cultured basal forebrain neurons, an in ovo model of motor neuron death programmed in development, a model of adult hypoglossal axotomy in vivo, or an in vivo excitotoxin neuroprotection assay, for example, an excitotoxic lesion of the nucleus basalis. These tests are all described in detail below. Accordingly, the compounds of this invention are useful for administration to humans or other mammals suffering from diseases or neurological disorders characterized by an increased risk of death of neuronal cell dysfunction. These diseases and neurological disorders include, but are not limited to: Alzheimer's disease; motor neuron disease including amyotrophic lateral sclerosis; Parkinson's disease; concussion or other ischemic injuries; Huntington's disease; dementia due to AIDS; epilepsy; concussive or penetrating injuries of the brain or spinal cord; and peripheral neuropathies. The compounds provided herein can be formulated into pharmaceutical compositions by mixing with pharmaceutically acceptable non-toxic excipients and vehicles.

As noted above, these compositions can be prepared for parenteral administration, particularly in the form of liquid solutions or suspensions.; for oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols. The composition can conveniently be 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, PA, 1980). Formulations for parenteral administration may contain as common excipients, sterile water or whey, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. In particular, the following may be useful excipients for controlling the release of the active compounds: lactide polymer, lactide copolymer (glycolide, or biocompatible and biodegradable polyoxyethylene-polyoxypropylene copolymers) Other potentially useful parenteral application systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes The formulations for administration by inhalation contain, as excipients, for example, lactose, or they can be aqueous solutions containing, for example, ether polyoxyethylene-9-lauryl, glycocholate and deoxycholate, or oily solutions to be administered in the form of nasal drops, or as a gel to 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 materials of this invention can be used as the sole active agent in a pharmaceutical product, or they can be used in combination with other active ingredients, for example, other growth factors that can facilitate neuronal survival or axonal growth in diseases or disorders. neurological, for example, peripheral neuropathy. The concentrations of the compounds described herein in a therapeutic composition will vary depending on a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds of this invention can be provided in an aqueous physiological pH buffer solution containing from about 0.1 to 10 weight percent / volume of the compound for parenteral administration. Typical dosage scales are from about 1 microgram / kilogram to about 1 gram / kilogram of body weight per day; A preferred dosage scale is from about 0.01 milligrams / kilogram to 100 milligrams / kilogram of body weight per day. The preferred dosage of the drug to be administered possibly depends on variables such as the type and degree of progress of the neurological disease, the overall health status of the particular patient, the relative biological efficacy of the selected compound, the formulation of the excipients of the compound, and its route of administration. The present invention will be further illustrated by the following examples. These examples should not be construed as limiting the scope of the invention, which should be determined exclusively by the appended claims.

Example 1 1, 6-Hexamethylene-bis- (carbamyl-staurosporine) (HBCS) A solution of 1.0 milligrams (2.15 micromoles) of staurosporine (Ka iya Biochemical Company, Thousand Oaks, CA) in 1.00 milliliters of ethyl acetate (dried over anhydrous magnesium sulfate, was treated with 17 microliters (1.08 micromoles) of a solution of 10.75 milligrams of hexamethylene bis-isocyanate in 1.0 milliliter of dry ethyl acetate The reaction mixture was allowed to stand in an amber glass reaction flask at room temperature for 2 days A crystalline deposit weighing 600 micrograms was removed. Its composition was verified as HBCS by mass spectroscopy with fast atom bombardment (FAB-MS).

M + H + Calculated = 1102 M + Na + Calculated = 1124 Found = 1102 Found = 1124 This product and all the staurosporine derivatives subsequently described were stored in non-actinic glass bottles.

Example 2 p- £ enylene-bis- (carbamyl-staurosporine) (PBCS) A solution of 1.0 milligrams of staurosporine (2.15 micromoles) in 1.00 milliliters of dry ethyl acetate was treated with 45 microliters (1.08 micromoles) of a solution prepared from 3.83 milligrams of p-phenylene diisocyanate (Trans World Chemicals P1586-1) in 1.00 milliliters of dry ethyl acetate. The reaction mixture was allowed to stand overnight. A white precipitate was deposited. Then 0.5 milliliters of petroleum ether was added. The mixture was filtered on a vacuum-dried sintered glass funnel. A total of 0.90 milligrams of a crystalline product was collected, and was identified as p-phenylene-bis- (carbamyl-staurosporine) by mass spectroscopy with fast atom bombardment.

M + H + Calculated = 1093 Found = 1093 Preparation A N-Phenylcarbamyl-taurosporine (PCS) Reference: U.S. Patent Number 5,093,330. A solution of 2.0 milligrams of staurosporine (4.30 micromoles) in 1.50 milliliters of dry ethyl acetate was treated with 468 microliters (4.30 micromoles) of a solution of 10 microliters of phenylisocyanate in 0.990 milliliters of dry ethyl acetate. The solution was allowed to stand overnight, and 3 milliliters of hexane were added in portions. Colorless crystals that weighed 2.39 milligrams were obtained. After recrystallization of this product from 1 milliliter of ethyl acetate and 2 milliliters of petroleum ether, 1.75 milligrams of a crystalline product were isolated. From a similar preparation, the composition of the product as N-phenylcarbamyl-taurosporine, was verified by mass spectroscopy with fast atom bombardment.

M + H + Calculated = 586 Found = 586 Preparation B N-phenylthiocarbamyltaurosporine (PTCS) A solution of 1.0 milligrams (2.15 micromoles) of staurosporine in 1.00 milliliters of ethyl acetate was treated with 26 microliters of a 10 microliter supply solution of phenylisothiocyanate in 1.00 milliliters of ethyl acetate. This aliquot contained 290 micrograms (2.15 micromoles) of phenylisothiocyanate. The reaction mixture was kept at 25 ° C overnight, and then 2.0 milliliters of hexane were added.

The resulting crystalline product was filtered, washed with hexane, and dried with a stream of argon gas. FAB-MS Calculated: M + H + = 602 Found = 602 Preparation C N-ethylcarbamyltaurosporine (ECS) A solution of 0.9 milligrams (1.93 micromoles) of staurosporine in 900 microliters of ethyl acetate was treated with 1.93 micromoles (30.2 microliters of a delivery solution of 9.05 milligrams of ethyl isocyanate in 2.00 milliliters) of dry ethyl acetate (ethyl isocyanate) The reaction mixture was kept at 25 ° C overnight, and 2.0 milliliters of hexane was added.The crystalline product was separated and dried.

FAB-MS Calculated: M + H + = 538 M + NA + = 560 Found = 538 = 560 Example 3 Compound II-4 Compound A (962 milligrams, 2 mmol) was dissolved in a mixture of 30 milliliters of tetrahydrofuran and 10 milliliters of methanol, and then 760 milligrams of sodium borohydride (20 mmol) was added under ice cooling , followed by stirring at the same temperature for 4 hours, and also at room temperature for 12 hours. After 3N hydrochloric acid was added thereto, the solution was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate, followed by evaporation of the solvent. The residue was purified by silica gel column chromatography (chloroform / methanol = 98/2) to give 882 milligrams (97 percent yield) of Compound II-4. Melting point: 130-140 ° C. ^ -NMR (DMS0-d6) i (ppm): 2.032 (1H, dd, = 5.0, 13.9HZ), 2.231 (3H, s), 2.967 (3H, s), 3.609 (1H, dd, J * 7.6, 13.4HZ), 3.S59 (2H, p), 5.000 (2H, s), 5.268 (1H, t, J-5.3HZ), 7.065 (1H, dd, J-4.9, 7.3Hz), 7.254-3.038 ( 7K, m), 8.565 (1H, s), 9.206 (1H, d, J = 7.8HZ) Compound A Example 4 Compound 11-14 Compound B (393 milligrams, 0.9 mmol) was dissolved in 25 milliliters of tetrahydrofuran, and then 3 milliliters of tetrahydrofuran containing 309 milligrams of carbobenzoxy-L-serine (1.35 millimoles), 156 milligrams of N were added. -hydroxysuccinimide (1.35 millimoles), 0.1 milliliters of 4-methylmorpholine (0.9 millimoles) and 279 milligrams of dicyclohexylcarbodiimide (1.35 millimoles), under cooling with ice, followed by stirring for 12 hours. The reaction mixture was filtered, and the solvent was evaporated. The residue was purified by silica gel column chromatography (chloroform / methanol = 99/1), to give 429 milligrams (72 percent yield) of Compound C. Melting point: 188-193 ° C SIMS (m / z): 660 (M + l) + Compound C (399 milligrams) was dissolved in 10 milliliters of dimethylformamide, and then 300 milligrams of palladium on carbon at 10 percent was added, followed by stirring at 50 ° C for 7 hours in a stream of hydrogen. The reaction mixture was filtered through celite, and the solvent was evaporated. The residue was purified by silica gel column chromatography (chloroform / methanol / 28 percent ammonium hydroxide = 90/10/1), and the product obtained was dissolved in 5 milliliters of tetrahydrofuran, followed by the addition of 5 ml. milliliters of 1.7N hydrogen chloride / ethyl acetate and 10 milliliters of diethyl ether. The precipitate was separated from the solution by filtration to give 234 milligrams (69 percent yield) of Compound 11-14. Fusion Point: > 300 ° C l-mm (DMSO-d5-D20) S (ppm): 1.92-2.28 (1H, a), 2.20 (3H, s), 2.84-3.12 (7H, m),. 0-4.20 (5H, ra), 5.04 (2H, s), 6.98 (1H, ra), 7.24-8. 0 (7H, ra), 3.76 (1H, brs), 9.22 (1H, d, J = 8Hz) SIMS (m / z): 527 (M + 2) * Compound B Compound C CbZ: carbobenzoxy .--, Example 5 PC-12 cells are a clonal population that occurs from a rat adrenal medulla tumor, and it has been proven to be an extremely useful and widely studied model, 5 to study the actions of the patient. nerve growth factor (Guroff, Cell Culture in the Neurosciences, Plenum Publishing Corporation, pages 245-272, 1985). A particularly important effect of nerve growth factor on these cells is a rapid stimulation of ornithine decarboxylase (ODC) activity, an effect reported to be blocked by 200 nM K-252a (Koizumi et al. 1988). In the experiments of this example, PC-12 cells (obtained from Dr. G. Guroff, National Institute of Health, Bethesda, MD) were cultured in 48-well dishes, at a density of 6 × 10 4 cells / square centimeter, and incubated with a drug vehicle (0.5 percent dimethyl sulfoxide), K-252a, -.- staurosporine, or HBCS. K-252a and staurosporine are commercially available from Kamiya Biomedical. Four hours after the addition of the drug, the cells were harvested for the ornithine decarboxylase assay, as described by Huff et al. (J. Cell Biol. 88: 189-198, 1981). All three compounds produced an induction (ie, an increase) in ornithine decarboxylase activity, but there were considerable differences in potency and efficacy (Figure 1). K-252a produced a dose-dependent induction of ornithine decarboxylase activity, with detectable effects at 2 nM, and increasing to a maximum at 200 nM (36.3-fold induction). In the same way, the effects of staurosporine were detectable at 2 nM, but reached their peak at 20 nM (induction 34.7 times), and declined considerably at 200 nM. HBCS (Example 1) induced similarly at 2 nM, but higher concentrations failed to give a greater effect, such that the maximum efficacy was much lower than that of the other two compounds (6.5-fold induction). In another experiment, the effects of PTCS, PCS, and ECS (Example 2) on the activity of ornithine decarboxylase of PC-12 cells were compared with those of K-252a. At concentrations of 200 nM, which express the activity of K-252a as 100 percent, the PTCS exhibited 71.4 percent of the activity of K-252a, while the PCS and the ECS exhibited 88.9 percent and 61.9 percent. percent of the activity of K-252a, respectively. However, the protein kinase C H-7 inhibitor did not induce ornithine decarboxylase activity at 30 μM, a concentration known to inhibit the activity of protein kinase C (Nakadate et al., Biochem, Pharmacol. : 1541-1545, 1988). The ability of K-252a, staurosporine, and HBCS to enhance and / or inhibit the bioactivity of nerve growth factor was assessed by adding 10 nanograms of nerve growth factor per milliliter of cell culture medium, in the absence or in the presence of the above compounds at the concentrations indicated above, followed by an ornithine decarboxylase assay of the cells as described above (Figure 2). This concentration of nerve growth factor was selected to provide an intermediate level of induction, such that the effects of potentiation or inhibition of the compounds could be detected. K-252a in 200 nM inhibited the induction of the nerve growth factor of ornithine decarboxylase, -r- as reported by Koizumi et al. (1988), but, in a surprising way, potentiated induction at lower concentrations (2 nM and 20 nM). Staurosporine, at 2 nM, also potentiated induction by nerve growth factor, but 5 this effect was lost at higher concentrations (20 and 200 nM). The HBCS, in contrast, potentiated the effects of nerve growth factor in all concentrations tested. This surprising effect is shown in relation to the modest ornithine decarboxylase inducing effects of HBCS alone 0 in Figure 3.

Example 6 The effect of K-252a on the activity of choline acetyltransferase (ChAT) in dissociated spinal cord cultures prepared from fetal rats was tested by conventional methods (see below). Choline acetyltransferase is an enzyme that catalyzes the synthesis of the neurotransmitter acetylcholine, and is a specific biochemical marker for cholinergic neurons. In the spinal cord, the vast majority of cholinergic neurons are motor neurons. The assay of this enzyme, therefore, can be used as an indication of the effects of a factor (or factors) on the survival of cholinergic neurons and / or the regulation of this enzyme. K-252a was added at the indicated concentrations to the cultures, after being incubated for 2 to 3 hours, after coating to allow the cells to bind to the substrate. The activity of choline acetyltransferase was measured after 48 hours of culture. K-252a in spinal cord cultures, resulted in a dose-dependent increase in choline acetyltransferase activity, with maximum efficacy (2 to 3 fold increase) reached at 200-300 nM (Figure 4) . Higher concentrations resulted in a decrease in choline acetyltransferase activity (Figure 4). The longer incubation times of the culture, up to 7 days, resulted in 4 to 5 fold increases in choline acetyltransferase activity (Figure 5) due to the decreased baseline level of choline acetyltransferase activity. In this culture system, increasing numbers of motor neurons degenerate and die over time under basal conditions (control) (McMana an et al., Developmental Biol. 125: 311-320, 1988). The results shown in Figures 4 and 5 are the result of a single application of K-252a on the day of initiation of the culture, indicating a prolonged effect on the survival of cholinergic neurons of the spinal cord and / or the regulation of the enzyme itself. Experiments were performed with cultures dissociated from fetal rat spinal cord cells in general as described (Smith et al. J. Cell Biol. 101: 1608-1621, 1985). Dissociated cells were prepared from dissected spinal cords of 14-day old brionic rats by conventional techniques known to those skilled in the art, using tissue dissociation with trypsin (Smith et al., Supra). The cells were seeded (coated) in 6x105 cells / square centimeter in plastic tissue culture wells coated with poly-1-ornithine in a serum free N2 medium, and incubated at 37 ° C in a humidified atmosphere of the 5 percent C02 / 95 percent air (Bottenstein et al., Proc. Nati, Acad. Sci. USA 76: 514-517, 1979) for 48 hours. Choline acetyltransferase activity was measured using modifications of the Fonnum method (J Neurochem 2?: 407-409, 1975) according to Ishida and Deguchi (J. Neurosci., 3: 1818-1823, 1983), and McManaman and collaborators, supra (1988). The activity was normalized to the total protein measured by the bicinconic acid / Cu ++ reaction (BCA protein assay reagent, Pierce, Rockland, IL).

Example 7 More than 100 functional derivatives of K-252a were tested in the spinal cord choline acetyltransferase assay, to determine its relative effectiveness. The data in Figure 8 show that, of the original functional derivatives tested at 300 and 30 nM, 28 resulted in choline acetyltransferase activity significantly increased by 300 nM. A functional derivative, compound 11-21, was also active at 30 nM (30 percent improvement in choline acetyltransferase activity above baseline levels). This compound was more potent than K-252a or the remaining analogs, since none of these actively enhanced choline acetyltransferase activity by 30 nM. Figure 13 shows the ability of the original 28 K-252a derivatives shown to significantly increase the choline acetyltransferase activity in rat spinal cord cultures, as well as 30 additional derivatives (Compounds 11-29 to 11-34) , 11-36 to 11-56, and IV-1 to IV-3, including all). Figure 13B shows the ability of the derivatives of K-252a 11-66-80, IV-5, IV-6, VI-1, and VI-2 to significantly increase the activity of choline acetyltransferase in cultures of Rat spinal cord. Figure 13C shows the ability of the 12 additional K-252a derivatives to significantly increase choline acetyltransferase activity in rat spinal cord cultures.

Example 8 K-252a, as well as more than 50 functional derivatives, were evaluated for their ability to promote the survival of dorsal root ganglionic neuronal cells. Cell survival was measured by recovery of calcein AM, a viable dye analogue, fluorescein diacetate. Calcein is recovered by viable cells and dissociates intracellularly in fluorescent salts that are retained by the intact membranes of viable cells. The microscopic counts of the viable neurons correlate directly with the relative fluorescence values obtained with the fluorimetric viability assay. Accordingly, this method provides a reliable and quantitative measurement of cell survival in the total cell population of a given culture (Bozyczko-Coyne et al., J Neur., Meth 50,205-216, 1993). The dorsal root ganglia of 8-day-old chicken embryos were dissected, and dissociated cells were prepared by the subsequent dissociation of Dispase (neutral protease, Collaborative Research). The neurons were seeded in a low density (1.8xl04 cells / square centimeter) in 96-well plates coated with poly-L-ornithine and laminin. The cells were cultured for 48 hours in serum-free N2 medium (Bottenstein and Sato, 1979) at 37 ° C in a humidified atmosphere, with 5 percent C02 / 95 percent air. Cell survival was evaluated at 48 hours using the viable fluorometric assay described above. The survival of the dorsal root ganglionic neurons was improved by K-252a in a concentration-dependent manner. The maximum activity was observed at approximately 100 nM (Figure 6). 24 of the 50 analogues tested were active to promote the survival of dorsal root ganglion neurons, 22 of which are shown in the Figure 7. All of these analogues were also active to increase choline acetyltransferase activity of the spinal cord (see Example 5, Figure 8). The original 22, as well as the 2 additional active analogues (11-30, 11-32) are shown in Figure 14. Microscopic examination of dorsal root ganglion neurons stimulated with the 24 active functional derivatives indicated better growth of nerve fibers too.

Example 9 Infusion of the amino acid excitation cainic acid (cainate) directly in the ventricles of a rodent brain, results in neuronal degeneration of pyramidal cells of the hippocampus. This neuronal death is characterized by a marked increase in the proteolysis of the cytoskeletal protein, spectrin. The spectrin decomposes the products that can be measured in the hippocampal homogenates within 24 hours following the administration of the kainate. The magnitude of spectrin proteolysis is highly correlated with the magnitude of neuronal death in hippocampal pyramidal cells (Siman et al., J Neurosci, 9: 1579-1590, 1989), and therefore, spectrin proteolysis is an excellent biochemical marker of neuronal degeneration induced by arousal amino acids. Excessive release of endogenous arousal amino acids has been implicated as an etiology in numerous diseases and neurological disorders, including concussion and other ischemic lesions; Alzheimer's disease, motor neuron disease, including amyotrophic lateral sclerosis; Parkinson's disease; Huntington's disease; dementia due to AIDS; epilepsy; and concussive or penetrating lesions of the brain or spinal cord. The results shown in Figures 9 to 12 were generated according to the following methods: Cainate Infusion Regime: The effect of K-252a or its derivatives on neurite damage induced by kainate was evaluated. Male Sprague-Dawley rats or adult females (175-250 grams) were anesthetized with Nembutal (50 milligrams / kilogram, intraperitoneally). Each rat was given a test compound (in a total of 5 microliters) before and after treatment with kainate (5 microliters) by intracerebroventricular infusion. This was done using a dose and infusion schedule as indicated for the previous individual cases. The control animals received a vehicle instead of the cainate and the infusion of the drug. For the anatomical studies, intracerebroventricular infusions were applied through a cannula (Plástic One, Roanoke, VA) implanted approximately 1 week before the infusions of the drug, and placed in the stereotactic coordinates: anterior-posterior in bregma, 1.5 mm lateral to bregma, and 4.4 mm ventral from the top of the skull. The results of this treatment regimen were evaluated two weeks later, using the anatomical analysis described below. In studies to evaluate the effect of K-252a or its derivatives on the kaolin-induced spectrin proteolysis, the anesthetized rats received a 5 microliter intracerebroventricular infusion of the drug or the vehicle, simultaneously with kainate, through a Hamilton syringe. 10 microliters placed in the stereotactic coordinates described above. These rats were sacrificed 24 hours later, and subjected to biochemical analysis as described below. Anatomical and Biochemical Analysis: The anatomical analysis was performed as follows. The rats were sacrificed by decapitation two weeks following the treatments, and the brains were quickly removed and frozen on dry ice. A series of coronal sections mounted on slides of each brain were stained with thionin and examined microscopically. Damage to the hippocampus was quantified by adding the total number of four anatomically defined regions of the hippocampus (CA1-4 according to the classification of Lorente and Novas described by Shepard, 1979, The Synaptic Organization of 0 the Brain, Oxford, page 310, incorporated herein by reference), both on the left and right sides of the brain, which suffered a loss of pyramidal cells. The biochemical analysis was performed as follows: Calpain I-sensitive proteolysis of brain 5 spectrin (fodrin) in hippocampal homogenates was evaluated using an immunoblot analysis described by Siman et al. (1988, Neuron, I: 279-287, incorporated in the present as a reference). Briefly stated, the rats were sacrificed by decapitation 24 hours following treatment, and the dorsal 0 hippocampus was rapidly dissected from the brain, and homogenized in 20 mM Tris-HCl (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride. . Proteins from the aliquots of each homogenate were separated by SDS-PAGE, and an immunoblot analysis was used to quantify the amount of decay spectrin induced by kainate in each sample. Figure 9 shows the effect of K-252a on neuronal degeneration induced by kainate in the hippocampus. Male and female cannulated Sprage-Dawley rats received 0.4 micrograms of K-252a, or of a vehicle, 30 minutes before, and at approximately 3 and 24 hours following the injection of kainate (0.6 micrograms) directly into the lateral cerebral ventricles of the brain (icv). Two weeks later the brains were separated, frozen, sectioned, and stained for histological analysis, as described below. The data shown is the average number of sub-regions of the hippocampus damaged for each group, + Standard Error of the Average (S.E.M.). The K-252a significantly reduced the number of damaged areas within the hippocampus from 3.86 + 0.78 (in the absence of K-252a) to 1.18 ± 0.4 in the presence of K-252a). Figure 10 shows the effect of K-252a on the decay of specimen induced by kainate in the hippocampus. Female Sprague-Dawley rats received 0.4 micrograms of K-252a, or of a vehicle, along with a neurotoxic dose of kainate (0.6 micrograms), by intracerebroventricular infusion. The simulated control animals received vehicle infusions, but not kainate or K-252a. Twenty-four hours later, dorsal hippocampal homogenates were analyzed by spectrin decomposition products as described below. The magnitude of the spectrin proteolysis is expressed as a percentage increase in the spectrin decomposition products for each group on the simulated control values. The data shown is the increase in the average percentage in the products of decomposition of spectrin for each group (simulated = 100 percent + Standard Error of the Average.) The infusion intracerebroventricular of K-252a significantly reduced the degree of proteolysis of spectrin , from about 140 ± 15 percent (in the absence of K-252a) to about 102 + 10 percent (in the presence of K-252a) of the simulated values Figure 11 shows the effect of HBCS on induced neuronal degeneration In the hippocampus, the female Sprague-Dawley rats received 0.8 micrograms of HBCS, or vehicle, 40 minutes before, and approximately 4 hours after the cinate (0.6 micrograms) by intracerebroventricular infusion. The brains were separated, frozen, sectioned, and stained for histological analysis, as described below. average number of sub-regions of the hippocampus damaged by each group, ± standard error of the average. The HBCS significantly reduced the number of damaged areas within the hippocampus from approximately 2.5 + 0.6 (without treatment with HBCS) to 1.3 ± 0.5 (with HBCS treatment).

Figure 12 compares the effect of three functional derivatives of K-252a on the decay of specimen induced by kainate in the hippocampus. Female Sprague-Dawley rats received 0.4 micrograms of K-252a, or compounds III-1 or 11-21, or vehicle, together with a neurotoxic dose of kainate (0.6 micrograms), by intracerebrovicular infusion. The simulated control animals received vehicle infusions, but nothing of kainate derivative K-252a. Tw-four hours later, homogenates of the dorsal hippocampus were analyzed by the spectrin decomposition products as described below. The magnitude of the spectrin proteolysis is expressed as a percge increase in the products of decomposition of spectrin by each group on the simulated control values. The data shown is the average percge increase in the products of decomposition of spectrin for each group (simulated = 100 perc + Standard Error of the Average. Intracerebrovicular infusion of K-252a reduced the degree of spectrin proteolysis, from approximately 128 + 9 perc(vehicle treatm to approximately 104 + 9 perc(in the presence of K-252a) from the simulated values. The derivatives of K-252a III-1 and 11-21 failed to prevthe proteolyysis of specimen induced by kainate.

Example 10 K-252a was tested for its ability to promote survival in striated cultures. Striae from 17-day rat embryos were dissected, and the cells dissociated by Dispase (neutral protease, Collaborative Research). Neurons were seeded at 5x104 cells / well (1.5 x 105 / cm2) in 96-well dishes, the cavities having been previously coated with poly-1-ornithine and laminin. Cells were cultured in serum-free N2 medium containing 0.05 percbovine serum albumin (Bottenstein and Sato, 1979) at 37 ° C in a humified atmosphere, 5 percC02 / 95 percair. Cell survival was tested 5 days after sowing, using the calcein viable cell fluorometric assay described in Example 8. The survival of striatal neurons was improved by K-252a in a concation-dependmanner. Maximum activity was found with 75 nM K-252a, which produced an efficacy of 3 to 4 times more than the control (Figure 15). In the control cultures, 90 percof the neurons coated on day zero died in 5 days, whereas the cultures treated with K-252a, 50 percof the neurons survived (Figure 16). The effect of survival in striated neurons occurred after 3 days in culture, and was sustained for at least 7 days in culture. These results are from a single application of K-252a on the day of initiation of the culture, and indicate that the effect on the survival of neurons is sustained. Figure 17 is a pair of photomicrographs taken from control cultures or cultures treated with K-252a at 75 nM. There was an increase in cell survival and an overgrowth of neurites in these cultures in the presence of K-252a at 75 nM.

Example 11 31 functional K-252a derivatives were tested for their potency and efficacy in the striated cell survival assay of Example 10. Figure 18 shows data on 18 K-252a derivatives that promoted the survival of striated neurons .

Example 12 Compounds of the invon were evaluated for their ability to promote survival and increase in choline acetyltransferase activity in basal forebrain cultures. The activity of choline acetyltransferase in these cultures is a biochemical marker for cholinergic neurons (less than 5 percof cells in culture), which represthe largest cholinergic input to the formation of the hippocampus, the olfactory nucleus, the interpeduncular nucleus, to the cortex, to the tonsils, and to parts of the thalamus. The representative compounds of the invention not only increased the activity of choline acetyltransferase, but in addition, increased the overall survival of the neurons in the basal forebrain cultures. The basal forebrain was dissected from either 17- or 18-day rat embryos, and the cells were dissociated with Dispasa ™ (neutral protease, Collaborative Research). Neurons were coated at 5 x 10 4 cells / well (1.5 x 10 5 cells / square centimeter) in the wells of 96-well dishes previously coated with poly-1-ornithine and laminin. Cells were cultured in serum free N2 medium containing 0.05 percent bovine serum albumin (BSA) (Bottenstein et al., Eupra) at 37 ° C in a humidified atmosphere of 5 percent C02 / 95 percent air . The activity of choline acetyltransferase was measured in vitro on the sixth day, using a modification of the Fonnum procedure (supra) according to McManaman et al (supra), and Glicksman et al. (J. Neuroschem 61: 210-221). , 1993). Cell survival was evaluated 5 days after coating, using the calcein AM fluorimetric assay described by Bozyczko-Coyne et al. (Supra). The culture medium was partially aspirated at the time of the assay to leave 50 microliters per cavity. Then an 8 μM AM calcein supply was added to 150 microliters of Dulbecco's phosphate buffered serum (DPBS); Jibco BRL), to give a final concentration of 6 μM, in 200 microliters per cavity, in a 96-well dish. Plates were incubated for 30 minutes at 37 ° C, followed by four serial dilutions with 200 microliters of DPBS. The relative fluorescence of each cavity was measured using a plate reading fluorometer (Cytoflur 2350, Millipore) at an excitation wavelength of 485 nanometers, an emission wavelength of 538 nanometers, and a sensitivity position of # 3 . The average fluorescence background calculated from 6 cavities that received calcein AM, but did not contain cells, was subtracted from all values. The linearity of the fluorescence signal was verified during the incubation time of the substrate of 30 minutes for the range of cell densities found in these experiments. The K-252a, as well as at least twelve derivatives of K-252a (II-3, II-5, 11-10, 11-20, 11-21, 11-22, 11-30, 11-32, 11- 51, 11-62, 11-63, 11-64, 11-65) promoted the survival of basal forebrain neurons (Figure 19).

EXAMPLE 13 The following tests were conducted to evaluate the effect of Compound 11-51 on cortical cholinergic function, when subjected to nucleus basalis rats. Cholinergic neurons that originate from the basal forebrain, and project to the hippocampus via the septo-hippocampal path, or to the cortex through the corticobasal path, suffer profound degeneration during the course and progress of the Alzheimer disease. There is some degree of correlation between the loss of these neurons, and decreases in cognitive and memory function in individuals afflicted with this disorder (Fibiger, II, Trends in Neurosci 14: 220-223, 1991). Several models of cholinergic dysfunction have been proposed that show the loss of biochemical markers, as well as behavioral deficits. These models are parallel to the progress of Alzheimer's disease (Olton, D. and collaborators "Dementia: animal models of the cognitive impairments produced by degeneration of the basal forebrain cholinergic system" in Meltzer, H., (Ed.) Psychopharmacology: The Third Generation of Progress, Raven Press, NY, 1987, pages 941-953, Smith, S. Brain Res. Rev. 13.103-118, 1988). For example, a model of cholinergic degeneration is the excitotoxic lesion of the nucleus basalis (Wenk, G. et al., Exp. Brain Res. 56: 335-340, 1984). Lesions in cholinergic neurons within the basal forebrain result in the loss of neuronal cell bodies in this region, and the subsequent loss of cholinergic terminal markers in the frontal and parietal cortex (Dunnett, S. et al., Trends Neurosci 14: 494-501, 1991). Using the following methods, Compound 11-51 was shown to increase cortical cholinergic function in rats that underwent lesions of the nucleus basalis. Male Sprague-Dawley rats (225 to 275 grams) were used for all experiments. Unilateral ibonic lesions of the nucleus basalis magnocelularis were produced by methods known to those skilled in the art (see, for example, Wenk et al., Supra), with the modifications described below. The rats were anesthetized with 50 milligrams / kilogram of pentobarbital, and 5 micrograms of ibotenic acid (in 1 microliter of PBS) was injected, unilaterally, into the nucleus basalis magnocelularis. The coordinates used were from the Paxinos and Watson brain atlas (posterior 1.5 millimeters, 2.8 millimeters lateral, 7.2 millimeters dorso-ventral). The injections took place over a period of 6 minutes. Dye injections indicated that the injections went directly to the nucleus basalis. Compound 11-51 was dissolved in Solutol ™ at 30 percent in concentrations of 0.01 to 0.3 milligrams / milliliter. The compound (or the Solutol ™ vehicle) was administered subcutaneously 1 day after, or 6 hours before, inducing lesions in the nucleus basalis, and every 48 hours thereafter. The doses were 0.01, 0.03, 0.10, and 0.30 milligrams / -kilogram. The experiments were completed 4 to 21 days after inducing the lesions. The activity of choline acetyltransferase was measured in tissue homogenates of the frontal-parietal cortex by the Fonnum method (supra). The activity of choline acetyltransferase in the frontal cortex, ipsilateral to the side of the lesion, was compared and normalized with the activity of choline acetyltransferase in the contralateral side (the side without injury). The activity of choline acetyltransferase is expressed as the ratio of ipsilateral to contralateral. Data were analyzed by ANOVA, and differences between treatments were determined by Tukey post-hoc test. In animals in which lesions were made in the nucleus basalis, there was a time-dependent decrease in cortical choline acetyltransferase activity, presenting the maximum loss between 3 and 7 days after the injury (Table 6). The route of administration, the doses, and the dosing schedule were based on preliminary data showing the effects of Compound 11-51 on choline acetyltransferase levels in the basal forebrain of adult rats. To evaluate the effects of Compound 11-51 on the levels of choline acetyltransferase (ie, on cholinergic function) in animals with lesions, the drug was administered 1 day after inducing the lesions for 14 to 21 days, or hours before surgery for 4 days.

Table 6 Cortical Hill Acetyltransferase Activity Loss Time After Inducing Basalis Lesions in the Nucleus Basalis Magnocellularis8 ChAT activity Time of the (Injury Ratio (hours) Injection Ipsi / Contra) Control without injury 96 + 8 8 hours ibotenic acid (5μg) 97 ± 14 24 hours ibotenic acid (5μg) 105 + 11 72 hours ibotenic acid (5μg) 74 + ll1 168 hours ibotenic acid (5μg) 70 + (7 days). a Unilateral lesions were induced in the NBM of rats. The frontal cortex was assayed for the activity of choline acetyltransferase at the indicated time after the injury. Significantly different from the control without injury (p <0.05).

Dose response studies were conducted with compound 11-52, in doses of 0.01, 0.03, and 0.10 milligrams / kilogram (Table 7). Subcutaneous injections of compound 11-51 were given on alternating days for 21 days, starting 1 day after inducing lesions with ibotenic acid. The results showed that, with a dose as low as 0.03 milligrams / kilogram, compound 11-51 was effective in attenuating the disease in cortical choline acetyltransferase activity (Table 7). Table 7 Effects of Systematically Administered Compound 11-51 on Cortical Hill Acetyltransferase Activity in Injured Rats NBM: Dosage Response Study ChAT Activity Dose Treatment (Ipsi Ratio / - Compound Lesion 11-51 Against) Without injury 98.4 ± 4.5 With injury Vehicle 67.4 ± 7.2b With injury 0.01 mg / kg QOD 70.1 ± 11.2 With injury 0.03 mg / kg QOD 93.8 ± 14.9C With injury 0.10 mg / kg QOD 87.9 + 11.6C a Unilateral lesions were induced in the NBM of rats. Twenty-four hours after inducing the lesions, subcutaneous administration of Compound 11-51 began. Twenty-one days after the lesions, the animals were sacrificed, and the activity of cortical choline acetyltransferase was assayed. b Significantly different from the control (p <0.05). c Significantly different from the lesion alone.

The systemic administration of Compound 11-51 attenuated the decrease in cholinergic function in the frontal cortex measured at 4, 14, and 21 days after inducing the lesions (Table 8). In rats with unilateral lesions, the activity of choline acetyltransferase on the contralateral side remained unchanged, suggesting that Compound 11-51 only affected neurons with lesions. Table 8 Effects of Systematically-Administered Compound 11-51 on Cortical Hill Acetyltransferase Activity in Injured Rats NBM: Time Response Study 3 ChAT Activity Time of Dose of (Injury Ratio (days) Compound 11-51 Ipsi / Contra) Control without injury 99 + 6 4 days Vehicle 77 + 61 0.1 mg / kg (QOD) 96 + 12c 14 days Vehicle 72 + 8b 0.1 mg / kg (QOD) 94 + 6C 21 days Vehicle 66 _ + 8b 0.1 mg / kg (QOD) 87 + 7 (a Unilateral lesions were induced in the NBM of rats.) Six hours before, or one after, inducing the lesions, began Subcutaneous administration of Compound 11-51 The frontal cortex was tested for choline acetyltransferase activity at the indicated time after injury b Significantly different from control (p <0.05) c Significantly different from lesion + vehicle at the same point of time.

An in ovo model can be used to assess the ability of a compound to influence the death of programmed motor neurons in development. In the chicken, the somatic motor neurons suffer a death that occurs naturally between embryonic days 6 and 10 (E6 and ElO) (Chu-Wang et al., J. "Comp.Neurol., 177: 33-58, 1978; Hamburger , J. Comp.Neurol., 160: 535-546, 1975; McManaman et al., Neuron 4: 891-898, 1990.) During this period, the number of motor neurons on both sides of the lumbar spinal cord of embryos of chicken in development, it is reduced from approximately 40,000 to 23,000. Chicken embryos (E6-E9) were treated with vehicle (Solutol ™ HS 15 at 5 percent, BASF Aktiengesellschaft), or the concentrations of Compound 11-51 described. The treatments (50 microliters) were applied to the vascularized chorioallantoic membrane through a window in the cover by the method of Oppenheim et al. (Science 240: 919-921, 1988). The embryos were sacrificed in the ElO, and the spinal cords were removed, fixed in Carnoy's solution (10 percent acetic acid, 60 percent ethanol, 30 percent chloroform), embedded in paraffin, sectioned in sections of 8 microns, and stained with thionin as described previously (Oppenheim et al., supra). The motor neurons (identified by morphology and position) were counted in each tenth section according to previously established criteria (Oppenheim et al., J .. Comp.Neurol. 210: 174-189, 1982; Oppenheim et al., "Comp.Neurol., 246: 281-286, 1986.) Daily application of Compound 11-51 to the chorioallantoic membrane of chickens E6 to E9 in ovo, resulted in a dose-dependent increase in the number of surviving lumbar motor neurons (Figure 20) At the lowest effective dose tested (1.17 micrograms / egg), there was a 16 percent improvement in motor neuron survival, maximum effect was achieved at a dose of 2.3 micrograms / egg, resulting in a 40 percent increase in the survival of the motor neurons of the treated embryos versus the control ones treated with vehicle In 7 micrograms / egg, there was no greater increase in the survival of the motor neurons, indicating that, in this situation, a maximum response of 2.3 micrograms / egg had been reached.

Example 15 Motor neurons in the hypoglossal nucleus innervate the tongue by means of the hypoglossal nerve. In adult rats, transection of the hypoglossal nerve results in a dramatic loss of choline acetyltransferase activity in the motor neurons of the hypoglossal nucleus, without affecting the number of cells. The loss of choline acetyltransferase activity serves as a model to invert an immature phenotype. The left hypoglossal nerve was cut under the digastric muscle of the neck of each adult female Sprague-Dawley rat (120 to 180 grams) under anesthesia with Nembutal. 50 microliters of compound 11-51 were applied in Solutol ™ 10 percent (HS 15, BASF Aktiengesellschaft) or vehicle only, to a piece of gel foam, and then the proximal end of the transected nerve was wrapped together with the foam gel, in a paraffin film. At the end of 7 days, the rats were perfused under deep anesthesia with 4 percent paraformaldehyde in Sorenson pH buffer (0.1 M NaP04, pH 7). The brainstem was removed, and it was immersed in fixative for 24 hours, then rinsed well, and placed in 30 percent sucrose for cryoprotection before being sectioned. Coronal sections of 40 micras were cut from the brain, and stored in Sorenson pH buffer at 4 ° C, until they were stained. The hypoglossal nucleus yielded 40 to 48 sections, and each fifth section was processed for immunohistochemistry using the anti-ChAT mouse monoclonal antibody, 1E6, as described previously (Chiu et al., J. Comp.Neurol., 328: 351 -363, 1993). The neurons immunoreactive to choline acetyltransferase were visualized in sections by avidin-biotin amplification, with the Vectastain Elite ABCMR kit (Vector Laboratories, Burlingame, CA). Each fifth section of the hypoglossal nucleus was processed, and the immunoreactive cells of the control (non-injured) were counted, and the axotomized sides of each animal were counted. The results are expressed as the percentage of neurons immunoreactive to choline acetyltransferase on the axotomized side in relation to the number of neurons immunoreactive to choline acetyltransferase on the non-injured side. The application of 100 micrograms of Compound II-51 to the cut end of the hypoglossal nerve resulted in a significant number of neurons immunoreactive to choline acetyltransferase (33.7 percent + 9.9 (average + Standard Error of the Average)) (Figure 21) . In contrast, there were very few neurons immunoreactive to choline acetyltransferase (8.07 percent + 2.9 (average + Standard Error of Average)) in control animals treated with vehicle. Preparation Methods Example 16: Compounds (V) The processes for producing the compounds (V) are described below.

Process 1 The compound (V-l) (examples of Compound (V) wherein R1 is CH2S02R7 and X is C02R5) can be prepared by the following reaction step: (R5 represents lower alkyl or CH2NHC02R6 wherein R6 represents lower alkyl or aryl; R7 represents lower alkyl). The starting compound (A) is described in Japanese Published Unexamined Patent Application Number 295588/88 (incorporated herein by reference). The compound (V-1) can be obtained by treating the compound (A) with 1.5 equivalents of an oxidant. An example of the oxidant is m-chloroperbenzoic acid.

As the reaction solvent, a halogenated hydrocarbon such as methylene chloride, chloroform, or ethylene dichloride, or the like is used. The reaction is finished in 0.1 to 1 hours at -20 ° C to 30 ° C.

Process 2 Compounds of the formula (V-2) (examples of the compound (V) wherein R1 is hydrogen and X is CH2NHC02R5), can be prepared by the following reaction step: (B) V-2) R represents lower alkyl or aryl. The starting compound (B) is described in Japanese Published Unexamined Patent Application Number 155285/87 (incorporated herein by reference). The compound (V-2) can be obtained by the reaction of the compound (V) with 1 to 3 equivalents of C1C02R6, in the presence of 1 to 3 equivalents of a base. An example of the base is triethylamine. As a solvent for the reaction, a halogenated hydrocarbon, such as methylene chloride, chloroform, or ethylene dichloride, or the like is used. The reaction is finished in 0.5 to 3 hours from -10 ° C to 30 ° C.

Example 17 Compound 11-49 Compound (A; R5 = CH3 and R7 = C2Hb) (27 milligrams, 0.05 mmol) was dissolved in 1 milliliter of chloroform, and then 10 milligrams (0.06 millimoles) of low m-chloroperbenzoic acid were added. cooling with ice, followed by stirring at the same temperature for 45 minutes. After dilution with chloroform, the mixture was washed successively with an 8 percent aqueous solution of sodium thiosulfate, a saturated aqueous solution of sodium bicarbonate, water, and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol = 95/5), to give 17.7 milligrams (62 percent yield) of compound II-49.

: H-NMR (DMSO-d6) 5 (ppm): 1298 (3H,, J = 7.5Hz), 2.037 (1H, ad, -5.0, 14.1Hz), 2.153 (3H.s), 3.096 (2H, q , = 7.5Hz), 3.266 (2H, 5), 3.929.3H, S), 4.985 (1H, ú, -17.0HZ), 5-043 (lH, d, J-17.0HZ), 6.348 (1H, s ), 7.147 (1H, dd, J = .9, 7.1HZ), 7345-8.070 (6H, m) 8.612 (1H, s), 9.232 (1H, d, J = 1.5Hz) FAB-MS (m / z) ): 574 (M + ll * EXAMPLE 18 Compound 11-57 Compound (B) (43.8 milligrams, 0.1 mmol) was dissolved in 1 milliliter of tetrahydrofuran, and then 9.3 microliters (0.12 mmol) of methyl chloroformate were added. microliters (0.2 mmol) of triethylamine, followed by stirring for 50 minutes under ice-cooling, after dilution with tetrahydrofuran, the mixture was washed with a saline solution, and dried over sodium sulfate, after evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol = 99/1), to give 32.6 milligrams of compound 11-57.

-H-NMR (CDCl -) 'ppr.-. : 2 .099 (H, sj,: .579 (1H, m), 3.204 (1H, dd, J = 6.7m 13.8Hz), 3.837 (3H, s), 4.446 (1H, d, J = 17.3HZ) , 4,634 (1H, d, J-17.6HZ), 5,497 (lH, brs), 6,591 (1H, brs), 7,010-8,037 (7H, p.), 8,592 (1H, d, J = 6.6Hz) FAB- MS (m / z): 497 (+ l) * Example 19 Compound 11-38 Substantially the same procedure as in Example 18 was repeated, using 43.8 milligrams (0.1 millimoles) of compound (B), and 15 microliters of phenyl chloroformate, to give 27.8 milligrams (50 percent yield) of the compound (11-38).

-H-NMR (CDC13) S (ppm): 2.111 (3H, s), 2.890 (1H, brd, J-13.7HZ), 3.262 (1H, dd, J = 7.5, 13.9Hz), 3.742 (1H, d , J-L3.4HZ), 3.967 (1H, d, J = 12.9Hz 4.582 (1H, d, J = 15.3HZ), 5.342 (1H, brs), 5.906 (1H, brs), 5.550 (1H, brs) , 7.005- 8.042 (12H, m), 3.596 (1H, d, J = 7.6Hz) FA3- S (m / z): 559 (M * l) ' Example 20 (Figure 22 shows the synthesis of compound H from compound C). Compound 11-39 Compound (C) (Japanese Published Unexamined Patent Application Number 295588/88, incorporated herein by reference) (20 milligrams, 0.035 millimole) was dissolved in 1 milliliter of chloroform, and then 14.6 microliter was added (0.105 millimoles) of triethylamine and 13.9 microliters (0.175 millimoles) of ethyl isocyanate, followed by stirring at room temperature for 2 hours. To the solution was added 1 milliliter of methanol, followed by dilution with chloroform. The mixture was washed successively with water and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol = 98/2) to give 21 milligrams (84 percent yield of compound (D)).

^ -NM (CDCl-,) d (ppm): 1,195 (3H,, J- = 7.2Hz), 1222 (3H, -, J = 7.2HZ), 1,664 (3H, s), 2,194 (3H, S) , 2.555 (3H, s), 3.346 (4H, q, J = 7.2Hz), 3.820 (1H, d, J-7.5, 14.6Hz), 3.938 (3H, s), 5.036 (1H, d, --17 .-HZ), 5.125 (1H, d, J = 17.2Hz), 6.745 (1H, dd, J = 4.3, 7.4Hz), 7.260-7.398 (5H, m), 8.690 (1H, d, J-1.9HZ ) FAB-MS (m / z): 724 (Mt-1) * Compound (D), (9 milligrams, 0.012 mmol) was dissolved in a mixture of 0.2 milliliters of tetrahydrofuran and 0.2 milliliters of methanol, and then 2 microliters of 28 percent sodium methoxide / methanol was added., followed by stirring at room temperature for 10 minutes. To the solution was added 0.1 milliliters of a 5 percent aqueous solution of citric acid, followed by dilution with chloroform. The mixture was washed successively with water and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol = 9/1), to give 8 milligrams of compound 11-39.

-H-NMR (DMSO-d, 'ppm): 1.086 (3H, Z, = 7.lHz), 1.099. H,. -7.1HZ), 1948 (1H, dd, J = 4.3, 14.1HZ), I. IC '? H, 3),; .158 (4H, n), 3.910 (3H, s), 4.830': .- :, i. * 17 .- * Hz), 4.931 (1.4, d, J = 16.9HZ). "123 íl-i, dd, J = 5.0, ~ .1H.Z)," .3 2- 3.287 (5H. -. 333 f 1 H.-, J = 2.1HZ) FAB-MS (m / z ): -0-l, "Example 21 Compounds 11-51 v 11-56 Compound (E) (Japanese Unexamined Patent Application Publication Number 295588/88, supra) (60.7 milligrams, 0.1 millimole), was dissolved in a mixture of 5 milliliters of chloroform and 1 milliliter of methanol, and then 11 milligrams (0.3 millimoles) of sodium borohydride were added under ice-cooling, followed by stirring at the same temperature for 15 minutes, after dilution with chloroform, The mixture was washed successively with water and a saline solution, and dried over potassium carbonate.After evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol / triethylamine = 98/2 / 0.5. ) to give 36 milligrams (yield of 59 percent) to give 36 milligrams (yield of 59 percent) of compound (F).

: H-NMR (DMS0-d6) S (ppm): I.650 (3H, s), 2.027 (1H, dd, J-4.9, 4.5Hz), 2.12 = .: K. s :. 3.843 (1H, dd, J = .4, 14.5Hz). 3.891.3H. s •. 4.607 (2H, S), 4.673 (2H.S). i. Z Z d 'Z r. . 5 . 7.099 (IK, dd, J-5.0, 7.2HZ ,, ".4 -" .. •: "i r.), 3.S12 (1H, d, J = 0.3Hz, FAB-MS (n / Z): 612 (M * i; " Compound (F) (159 milligrams, 0.26 millimoles) was dissolved in 15 milliliters of chloroform, and then 0.8 milliliters (10.4 millimoles) of ethanethiol and 24 milligrams (0.104 millimoles) of camphorsulfonic acid were added, followed by stirring at room temperature for 12 hours. The solution was washed successively with a saturated aqueous solution of sodium bicarbonate, water, and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (ethyl acetate / toluene = 1/9 = chloroform / methanol = 99/1) to give 43 milligrams of the compound (G) and 75 milligrams of the compound (H). Compound (G) : H- MR (CDC13) d (ppm): 1292 (3H, t, J = 7.4Hz), 1297 (3H, r, J = 7.4HZ), 1.799 (3H, s), 2.141 (1H, dd, J -5.0, 14.5HZ), 2.256 (3H, s), 2.532 (2H, q, J = 7.4HZ), 2.553 (2H, q, J = 7.4Hz), 2.869 (3H, s), 3.971 (1H, dd , J = 7.5, 14.5Hz), 3.992 (2H, s), 4.005 (3H, s), 4.02K2H, S), 5.416 (1H, dd, J = 17.5HZ), 5.459 (1H.d, J = 17.4 Hz), 6,989 (1H, dd, J-5.1, 7.4Hz), 7.509-7.963 (5H, m), 9.134 (1H, d, J-1.2HZ) FAB-MS (m / z): 700 (M + l) * Compound (H) ^ -NR (CDCI3) S (ppm): 1294 (3H,, J-7.4HZ), 1799 (3H, s), 2.149 (1H, dd, J = 5.0, 14.6Hz), 2.273 (3H, s), 2.533 (2H, q, J-7.4HZJ, 2.813: 3K. S), 2.S72 (1H, dd, J = 7.4, 14.6HZ), 4.00S.3H. 31. 4.C15 (2H, s), 4.951 (2H, s). 5.; 77; : H, -. '*: "., H; .5,418 (1H, d, J = 17.4Hz), 6,973 (1H, dd, -:.;. _, H-;. 7.431-8.037 (5H, n), 9.093Í1H , d, J = 1.2HZ; FAB-MS (m / z): 656 (M + l) * Substantially the same procedure was repeated as in Example 20, using 34 milligrams of compound (G), to give 18.7 milligrams of the compound 11-51.

: H-NMR (CDCl, 5 (ppm): 1300 (3H, -, J = 7.4Hz), 1.325 (3H, ", J = 7. HZ), 2.135 (3H, s), 2.514CK. J = 4, 14.5HZ), 2.540 (2H, q, J = 7.4Hz), 2.555 (2H, q, J = 7.4Hz), 3.384 (1H, dd, J = 7.5, 14.5HZ), 3941 (2H , s), 3,976 (2H, s), 4,094 (3H, s), 4,836 (1H, d, J = 16.4Hz), 4,910 (1H, d, J-16.3HZ), 5,781 (1H, S), 6,845 (1H, dd, J = 4.8, 7.5Hz), 7.371-7.343 (5H, m), 8.998 (lH, s) FAB-MS (m / z): 616 (M + l) " The same procedure was repeated substantially as in Example 20, using 30 milligrams of compound (G), to give 20.4 milligrams of compound 11-56.

XH-NMR (CDC13) d (ppm): 1280 (3H, t, J = 7.4Hz), 2.144 (3H, S), 2.391 (1H, dd, J = 4.9, 14.5Hz), 2.517 (2H, q, J = 7.4Hz), 3.320 (1H, dd, J-7.4, 14.5Hz), 3.885 (2H, s), 4.069 (3H, S), 4.521 (1H.D, J-16.3HZ), 4.631 (1H, d, J-16.7HZ), 4.804 (2H,: S), 5.769 (1H, S), 6.830 (1H, dd, J »4.8, 7.4Hz), 7.375-7.771 (5H, ra), 8.934 (1H, s) FAB-MS (m / z): 572 (M +) * Example 22 Compound IV-3 Compound (II). (Z1, Z2 = H, R1 = Br, R2 = H, R = 0H; X = C02CH3) (Japanese Published Unexamined Patent Application Number 120388/87, incorporated herein by reference) (50 milligrams, 0.09 millimoles), was dissolved in a mixture of 0.5 milliliters of trifluoroacetic acid and 50 microliters of 3N HCl, and the solution was stirred at room temperature for 2 days. The precipitates were collected by filtration, and subjected to high performance liquid chromatography (Unisil 5C18; methanol / water = 8/2), to give 8.4 milligrams of the compound (IV-2). : H-NMR. ' MSO-d ^ ,;; "(ppm): 4,947 (2H, s), 7,300-3,310 (6H, m), 3,249 (1H, s), 9.26Ó (1K d, J = 2.C Hz¡ FAB-MS (n / z ): 390 (M + l) " Example 23 Compound 11-45 can be prepared by the reaction steps shown in Figure 23. The starting compound (J) is described in Japanese Published Unexamined Patent Application Number 120388/87 (incorporated herein by reference). ). compound II-4S Compound (J) (200 milligrams) was dissolved in 1 milliliter of dimethylformamide, and then 0.25 milliliters of an aqueous solution of 23.5 milligrams of sodium hydroxide was added, followed by stirring at room temperature for 4 hours. Then IN hydrochloric acid was added to adjust the pH of the solution to 1-2, and the precipitates were collected by filtration to give 1.78 milligrams (91 percent yield) of the compound (K).

• H-NMR (DMS0-d6) d (ppm): 1.965 (1H, dd, J = .S, 14. OHZ), 2.184 (3H, s), 2.364 (1H, d, J = 7.5, 14.0Hz) , 5.029 (1H, d, J-ld.lHz), 5.071 (1H.J- 13.0Hz :, 7.132 (1H. dd, J = 4.9, ~. Z r. Z;, ".3139 (5K.m) , 3.733 (1H, s), 9.398 (11- :, d, J = 2. Hz) Compound (K) (168 milligrams) was dissolved in 3 milliliters of pyridine, and then 0.44 milliliters (4.7 millimoles) of acetic anhydride was added, followed by stirring at room temperature for 4 days. After evaporation of the solvent, 4 milliliters of IN hydrochloric acid was added to the residue, and the precipitates were collected by filtration to give 182 milligrams (quantitative yield) of the compound (L).

• H-NMR (DMSO-d) 5 ppmj: 1.634 (3H, s), 2.135 (1H, dd, = 4.9, 14.4Hz), 2.252 (3H, s), 3.865 (1H, dd, = 7.6, 14.5Hz ), 5.063 (2H, s), 7.255 (1H, dd, .7 = 4.9, 7.5Hz), 7.612-8.582 (5H, m), 3.760CH, 3), 9.339 (1H, d, J = 2.1Hz) Compound (L) (172 milligrams) was suspended in thionyl chloride, followed by stirring at 90 ° C for 4.5 hours. After evaporation of the solvent, diethyl ether was added to the residue, and the precipitates were collected by filtration to give 180 milligrams of the compound (M). The compound (M) (67 milligrams, 0.1 mmol) was dissolved in 2 milliliters of ethylene dichloride, and then 180 microliters of aniline in tetrahydrofuran was added under ice-cooling, followed by stirring at the same temperature for 1 hour. After evaporation of the solvent, the residue was dissolved in a mixture of 2 milliliters of tetrahydrofuran and 0.5 milliliters of methanol, and then 1 milliliter of IN NaOH was added, followed by stirring at room temperature for 3 hours. To the solution was added INN hydrochloric acid (1.2 milliliters for neutralization, followed by dilution with tetrahydrofuran) The mixture was washed with a saline solution, and dried over sodium sulfate After evaporation of the solvent, the residue was subjected to to silica gel column chromatography (chloroform / methanol = 98/2), to give compound 11-45 (13 milligrams from 56 milligrams of isolated compound N).

-H-NHR (DMS0-? 5) S (ppm): 2.110 (1H, dd, J »4.9, 13.9Hz;, 2,175 (3H, s), 5,019 (1H, d, J-lß.lHz] 5,083 ( 1H. D, J = 13.0H?), 5.837 (1H, s), 7.119- 3.201 (11K, r.), 3.-11 (1H.S), 9.391 (1H, d, J = 2.2HZ,, 10.0"L1 H, 3; FAB-MS (rn / Z .: 637 • y. - I)" Example 24 Compound 11-65 R1 = CH2SCH2CH2N (CH3) 7 (Q) (11-65) The starting compound (Q) is described in Japanese Published Unexamined Patent Application Number 295588/88. The compound (Q) (50 milligrams, 0.0861 millimoles) was dissolved in 3 milliliters of chloroform, and then 200 milligrams (1.41 millimoles) of 2-dimethylaminoethanethiol hydrochloride and 49 milligrams (0.21 millimoles) of (+) - 10-camphorsulfonic acid were added, followed by stirring to the room temperature for 12 hours. The reaction mixture was washed successively with a saturated aqueous solution of sodium bicarbonate, water, and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to thin layer chromatography of preparation (chloroform / methanol = 99/1), to give 56.3 milligrams (98 percent yield) of compound 11-65 N, 0-diacetylated. FAB-MS (m / z): 668 (M + 1) +.

Compound 11-65 N, O-diacetylated (36.6 milligrams, 0.0548 millimoles) was dissolved in a mixture of 6 milliliters of chloroform and 3 milliliters of methanol, and then 18 microliters (0.09 millimoles) of 5.1N sodium methoxide was added, followed by stirring at room temperature for 20 minutes. Amberlyst® 15 ion exchange resin (100 milligrams) was added to the reaction mixture, followed by stirring for 1 hour, and the insoluble material was separated by filtration. After evaporation of the solvent, the residue was subjected to thin layer chromatography (chloroform / methanol = 97/3) to give 28.4 milligrams (89 percent yield) of compound 11-65.

: H-HMR (DMSO-d6) d ppm): 2.011 (1H, d, J = .9, 14.1Hz), 2.142 (9H.s), 2.460-2.584 (4H, n), 3.404 (1H, dd , -7.3.14.1HZ), 3.923 (3H, 3), 2.950 (2H.s), 4.951-5.054 (2H, m), 6.336 (1H, s>, -.1 1 (IH, dd, J = 4.9, 7.3HZ,, ..338-3,000 (5K.n), 3.595 (1H, S), P.137: H, d, J = 1.3Hz) FAB-MS (m / z): 585? M * l) * Example 25 Compound 11-66 Compound 11-66 is prepared, for example, according to a method of Japanese Published Unexamined Patent Application Number 155284/87 (incorporated herein by reference).

Example 26 Compound 11-75 Compound (P) Japanese Patent Application No Examined Published Number 295588/88) (100 milligrams, 0.173 millimoles) and 4-amino-1,2,4-triazole (17.4 milligrams, 0.207 millimoles) were dissolved in a mixture of 4 milliliters of chloroform and 1.5 milliliters of tetrahydrofuran, and then 0.05 milliliters of 3N hydrochloric acid was added, followed by stirring at room temperature for 3.5 hours. After ethyl acetate was added thereto, the insoluble matter was collected by filtration, and subjected to silica gel column chromatography (chloroform / methanol = 95/5), to give 71.9 milligrams (64% yield). percent) of compound II-75 N, 0-diacetylated. FABS-MS (m / z): 646 (M + l) + Compound 11-75 N, 0-diacetylated (37.5 milligrams, 0.058 millimole), was dissolved in a mixture of 2 milliliters of 1,2-dichloroethane and 0.6 milliliters of methanol, and then 11 microliters (11,058 millimoles) of methoxide were added. of sodium 5. IN in methanol, followed by stirring at room temperature for 20 minutes. Amberlyst® 15 (50 milligrams) was added to the reaction mixture, followed by stirring for 30 minutes, and the insoluble matter was filtered. The insoluble material was washed well with dichloromethane / methanol / ammonium hydroxide (8/2 / 0.5), and the combined filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, to give 26.8 milligrams (82 percent yield) of compound 11-75.

* HN R (DMSO-di) 3 (ppm .: 2-105 (1H, dd, J-5.0, 14.1 HZ), 2.1 = "; :: - :. s),. (Ip, ± á, .7 = 7.5, 14.1 Hz), 2.9-3 CH.5), 5.C20 1H, d, J-17.2HZ), 5.076 (1H, d, J-17.2HZ), 6,399 (1H, s), 7,226 (1H , dd, J = 5.0, ~ .5HZ), 7.366-3.114 (6H, m), 3.708 (1H, 5), 9.219 (2H, s), 9.260 (1H, 5), 9.701 (1H, i, * L .5Hz) FAB-MS (ra / z): 5O2 (Ml) - Example 27 Compound 11-79 Compound (Q) (Japanese Patent Application No Examined Published Number 295588/88) (50 milligrams, 0.0861 millimoles) and 2- (butylamino) ethanethiol (0.127 milliliters, 0.361 millimoles), were dissolved in chloroform, and then 300 milligrams (1.29 millimoles) of camphorsulfonic acid was added, followed by stirring at room temperature for 4 days. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the organic layer was washed with an aqueous solution of sodium chloride and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 95/5), to give 34.6 milligrams (58 percent yield) of compound 11-79 N, 0 -diacetylated. FAB-MS (m / z): 697 (M + l) + The same procedure was repeated substantially as in Example 26, using 31.1 milligrams (0.0447 millimoles) of the 11-79 N, 0-diacetylated compound, to give compound 11-79 (52 percent yield).

: H-NMR (DMSO-d5) i .ppm): 0.855 (3H, -, J-7.4HZ), 1286 (2H, n), 1.510 (2H, p), 2007 (1H, dd, J = 4.9, 14.1HZ), 2.143 (2H, s), 2.721 (2H, m), 2.843 (2H.r-., 7.12Ó (2H, rr.), 2.339 (1H, dd, J = 7.4, 14.1Hzr.: .927 CH.S), 4.022 (2H, S) .9,937 (1H, d,: = 1-.5H?), 5.030 (1H, d, J = 17.ÓHz!. -... 45 1H, 3 ), ~ .125 (1H, dd, .7 = 4.; - 7.4HZ), -.252--3.2Ó7 'SH, .1), 3,614 (1H, s), 9,151 (1 H. 3, FAB) -MS (m / Z): -113 ^ -1, * Example 28 Compound 11-80 Compound (F) (WO94 / 02488, incorporated herein by reference) (6.19 grams, 10.1 mmol), was dissolved in a mixture of 300 milliliters of 1,2-dichloroethane and 100 milliliters of methanol, and then 0.5 milliliters (2.55 millimoles) of 5.1N sodium methoxide in methanol was added, followed by stirring at room temperature for 35 minutes. it was poured into ice water, and the insoluble matter was collected by filtration to give 4.95 grams (93 percent yield) of a comp since it had bis (hydroxymethyl) in place of bis (dimethylaminoethylthiomethyl) of compound 11-80. FAB-MS (m / z): 528 (M + 1) +. The same procedure was repeated substantially as in Example 27, using 22.1 milligrams (0.0419 millimoles) of the compound having bis (hydroxymethyl) in place of bis (dimethylaminoethylthiomethyl) of compound 11-80, and 59.4 milligrams (0.419 millimoles) of hydrochloride of 2- (dimethylamino) ethanethiol, to give 13.1 milligrams (45 percent yield) of compound 11-80. -u K - .N »MR (DMS0-d5) -« pppij: 1999 (1H, dd, J-4.9, 14.2HZ), 2134 CH, Sj, 2.143 (6H, s), 2.149 (6H, 3; , 2.451-2.535 '3H, rr.j, 3.37S (iH, dd, -7.,, 14.2KZ), 3.922 ^ H, s), 3.950 (2H, s), 2.933' 2 K. 3,,, .95 1 K, z. -7 = 17. Hz) 5 01"'(1H, d, J = 17.7HZ), 6322 (1H, S), 7.108 (1H, dd, J = 4.9, 7.2Hz)," .444-7.952 (4H , m), 3,616 (1H, s), 9,133 CH. d, J = 1.4Hz) FAB-MS (m / Z): 702; M * 1) * EXAMPLE 29 Compound 11-72 Substantially the same procedure was repeated as in Example 27, using 50 milligrams (0.0861 mmol) of the compound (Q), and 97.8 milligrams (0.861 millimoles) of 2-aminoethanethiol hydrochloride, to give 49.6 milligrams (90 percent yield) of compound 11-72 N, 0-diacetylated. FAB-MS (m / z): 641 (M + 1) +. Substantially the same procedure was repeated as in Example 26, using 39.5 milligrams (O.i.i./limo) of compound 11-72 N, O-diacetylated, to give 30.2 milligrams (88 percent yield) of compound 11-72 .

; H-MMR (DMS0-d6) d (ppm): 2.014 (1H, dd J-4.5, 14.1HZ), 2.146 (3H, s), 2.519 CH. -. -7 - .2HZ .. 2.748 (2H,, J-7.2HZ). 2.336 1H. zd. _---;;. 14.1HZ), 3.925 (3H, s), 2.936 CH. s; ,.? 75 (1H, d, J-17.0HZ), 5,029 (1H, d, J-17.ZH.ZJ, 6,330 (1H, S), 7.1H (1H, dd, J-4.9, -.5Hz ), 7.344-3.059 (6H, .71), 3600 CH, s), 9.131 (1H, d, J = »1.5HZ) FAB-MS (m / Z): 557 (Ml)" Example 30 Compound VI-1 Compound (R) (J. Antibiotic, 38: 1437, 1985, Figure 24) (1 gram, 1.81 millimoles), was dissolved in 50 milliliters of 1,2-dichloroethane, and then 0.17 milliliters (3.60 millimoles) of nitric acid were added dropwise in vaporization at 0 ° C, followed by stirring at the temperature environment for 20 minutes.

After the reaction mixture was diluted with chloroform, a saturated aqueous solution of sodium bicarbonate was added, and the The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. After evaporation of the solvent under reduced pressure, 40 milliliters of dimethylformamide and 600 milligrams of Pd / C were added at 10 times. * - cent to the residue, followed by stirring at 60 ° C for 1 hour in an atmosphere of hydrogen. The insoluble material was filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (ethyl acetate / toluene = 20/80), to give 130.8 milligrams (13 percent yield) of an amine derivative. 15 FAB-MS (m / z): 567 (M + 1) +.

The amine derivative (23.9 milligrams, 0.0422 millimoles) was dissolved in 2 milliliters of chloroform, and then 9.2 microliters (0.0660 millimoles) of triethylamine and 87 milligrams were added. microliters (1.10 mmol) of ethyl isocyanate, followed by stirring at room temperature for 2 days. Water, methanol, and chloroform were added to the reaction mixture to terminate the reaction, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and was dried over sodium sulfate. After evaporation of the solvent under reduced pressure, the residue was subjected to silica gel column chromatography (chloroform / methanol = 98/2), to give 21.4 milligrams (80 percent yield) of compound VI-1 N, 0-diacetylated. The same procedure was repeated substantially as in Example 26, using 21.4 milligrams (0.0336 millimoles) of the N, 0-diacetylated compound VI-1, to give 17.0 milligrams (91 percent yield) of compound VI-1.

: H-.MR (DMSO-d6) or (ppm): 1.129 (3H.Z, J = 7.1Hz), 2.086 (3H, S), 2.110 (1H, dd, J = 5.5, 14.3Hz), 3.180 ( 2H, m). 3.237 (1H, dd, J => 7.4, 14.3Hz), 3.892 (3H, s), 4.934 (1H, d, J-17.0HZ), 5.030 (1H, d, J = * 17.0Hz), 6.359 ( 1H, s), 6.457 (1H, t, J = 5.7HZ), 7.157-7.230 (2H, -a), 7.272 (1H, dd, J = 5.5, 7.4HZ), 7.344-3.058 (4H,), 3.135 (1H, s), 3616 (1H, s), 9.243 (1H, dd, Jl.3, 7.3Hz) FAB-MS (m / z): 554 (M + l): EXAMPLE 31 Compound VI-2 Substantially the same procedure as Example 30 was repeated, using 5 grams (9.07 millimoles) of the compound (R; Figure 24), to give 259 milligrams (5 percent yield) of a diamine derivative. FAB-MS (m / z): 582 (M + 1) +.

Substantially repeated the same procedure as in Example 26, using 24.5 milligrams (0.0422 millimoles) of the diamine derivative, to give 3.8 milligrams (18 percent yield) of compound VI-2. lH-NMR (DMS0-d5) or (ppm): 1952 (1H, dd, J-5., 13.9HZ), 2.062 (3H, s), 3.894 (3H, s), 4.813- 5.339 (6H, n) , 6.198 (1H, s), 6.326-7.207 (4H, ra), -.507 'i'? , dd, J-5. , 7.3Hz), 7.630 (iH, d, J-8.3HZ), 3.443 (1H, S), 3.773 (1H, dd, -7 = 1.2, 7.3HZ) FA3- S .rn / Z): 493 'Ml ) " Example 32 Compound IV-6 Compound (S; Figure 25) [J. Chem. Soc. Perkin. Trans. 1, 2475 (1990)] (5.15 grams, 13.0 mmol), was dissolved in a mixture of 30 milliliters of dimethylformamide and 60 milliliters of toluene, and then 1.45 grams (12.9 mmol) of potassium tertiary butoxide at -20 ° was added. C in an argon atmosphere, followed by stirring at room temperature for 30 minutes. After cooling the reaction mixture to -20 ° C, 1.12 milliliters (12.9 millimoles) of allyl bromide was added, and the mixture was stirred at 0 ° C for 2 hours. The solvent was evaporated under reduced pressure, and water was added to the residue, followed by extraction with tetrahydrofuran. The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (ethyl acetate / toluene = 1/15), and triturated with dichloromethane, to give 898.4 milligrams (16 percent yield) of the compound (Tl) as a single regioisomer. -H-NTCR (DMS0-d6) d tppm): 1.56-1.61 CH. m), 1.73-1.37 (2H, ~), 2.00-2.14 (2H, m), 3.63-3.69 (1H, m), 3.99-4.02 (1H, m), 4.747 (??, dd, Jl.5, 17.1Hz), 5.053 (1H, dd, Jl.5, 10.4Hz), 5.084 (1H, d, J = 17.3HZ), 5.133 (1H, d, J-17.3HZ), 5.462 (1H. dd, J- 2.0, 11.0Hz), 5,593 (2H, d, J-4.6HZ), 6,173 (1H, ddt, J-4.6, 10.4 17.1HZ), 7,242 (1H, ddd, J-0.9, 7.0,7.9Hz), 7,368 (1H, dd, J-7.2, 7.8Hz), 7.455 (1H, ddd, Jl.2, 7.0, 3.2HZ), 7.542 (1H, ddd, Jl. I, 7.2, 8.3HZ), ".711, 1H , dd, J-0.9, 3.2HZ), -762 (1H.D, J-3C: -: z) 3.17- (IH.d, J = 7.3HZ), 9.3C5 (1H, d, J- 7.9Hz;, 11.5-3 (1H, S) FAB-MS (m / Z): 436 (M + l) * The compound (T-l) (1.44 grams, 3.30 mmol), was dissolved in 50 milliliters of tetrahydrofuran, and then 4.05 grams (33.2 millimoles) of 9-borabicyclo [3.3, 1] nonane (9-BBN) (dimer) was added, followed by stirring at the temperature environment for 3 hours in an argon atmosphere. After cooling the reaction mixture to 0 ° C, 6 milliliters of IN sodium hydroxide and 6 milliliters of a 35 percent aqueous solution of hydrogen peroxide were added, followed by stirring for 45 minutes. After dilution of the reaction mixture with water, the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 100/1), to give 875.5 milligrams (58 percent yield) of the compound (J-1).

: H-NMR (DMS0-d6) d (ppm): 1.5-1.6 (2H, brm), 1.7-1.9 (2H, brm), 2.0-2.2 (2H, brm), 2.08-2.14 (2H,), 3.49 -3.53 (2H, ra), 3.62-3.63 (1H, m), 3.99-4.02 (1H, m), 4.962 (2H, Z, J-6.9HZ), 5.072 (1H, d, J-17.2HZ). 5.081 (1H, t, J-4.7HZ), 5.123 (1H, d, J-17.2HZ), 5.458 (1H, dd, J-2.0, 11. OHZ), 7.251 (1H, ddd, J-0.9, 7.0 , 7.9HZ) 7.353 (1H, dd, J-7.2, 7.8Hz), 7.463 (1H, ddd, Jl.2, 7.0, 3.2HZ), 7.555 (1H, ddd, Jl., 7.2, 3.3HZ), 7.696 (1H, d, J-8.2HZ), 7.825 (1H, d, J-8.3HZ, 3.162 (1H, d, J-7.3HZ), 9.311 (1H, d, J-7.9HZ), 11.684 ( 1H, s) FAB-MS ~ / Z): 454; > '.-!) * The compound (Ul) (178.5 milligrams, 0.394 millimoles) was dissolved in 10 milliliters of dimethylformamide, and then 309.5 milligrams (1.18 millimoles) of triphenylphosphine and 0.060 milliliters (1.2 millimoles) of bromine were added at 0 ° C under an argon atmosphere. , followed by stirring at room temperature for 3 hours. After water was added to the reaction mixture to terminate the reaction, the mixture was extracted with ethyl acetate, and the organic layer was washed successively with water and an aqueous solution of sodium chloride, and dried over magnesium sulfate. . After evaporation of the solvent, the residue was subjected to silica gel column chromatography (ethyl acetate / toluene = 1/8), to give 134.6 milligrams (66 percent yield) of the compound (W).

^ -NMR (C0C13) d (ppm): 1.68-2.10 (6H, m), 2.13-2.18 (2H, m), 3.542 (2H, t, J-5.7HZ), 3.80-3.86 (1H, / - m ), 4.14-4.20 (1H, m), 4.658 (2H, Z, J-7.5HZ), * * 4.674 (1H, d, J-16.6Hz), 4.830 (1H, d, J-16.6HZ), 5.611 (1H, d, J-2.5, 10.6 Hz), 7.13-7.52 (6H, m), 7.746 (1H, d J = 7.6HZ), 3.884 (1H, s), 9.294 (1H, d, J-8.0HZ ) FAB-MS (m / Z): 516 (M + l) * Compound (W) was dissolved in 5 milliliters of dimethylformamide, and then 0.045 milliliter (0.52 millimole) of formolin was added, followed by stirring at 80 ° C for 3 hours in an argon atmosphere. After cooling the reaction mixture to room temperature, ice water was added, and the formed precipitates were collected by filtration. The precipitates were dried under reduced pressure, and subjected to thin layer chromatography (chloroform / methanol = 25/1). The obtained product was dissolved in 10 milliliters of tetrahydrofuran, and then 8 milliliters of 4N sulfuric acid was added followed by stirring at 60 ° C for 12 hours.

After cooling the reaction mixture to room temperature, ice was added, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with water and an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography. (ethyl acetate / toluene = 1/2). The obtained product was dissolved in a mixture of chloroform and ethyl acetate, and then 0.88N hydrochloric acid in ethyl acetate was added, followed by stirring at room temperature for 1 hour. The formed precipitates were collected by filtration, washed with ethyl acetate, and dried under reduced pressure, to give 35.0 milligrams (19 percent yield) of the compound (IV-6). lH-NMR- (DM = 0-d6) d (ppm): 2.29-2.34 (2H, m), 2.96-3.04 (2H, m), 3.30-3.40 (4H, m), 3.66-3.72 (2H, ), 3.56-3.90 (2H, ra), 4.972 (2H, s), 5.093 (2H, t, J-7.1HZ), 7.245 (1H, ddd, J-0.9, 7.0, 7.9HZ), 7.370 (1H, dd, J-7.0, 7.9Hz), 7.458 (1H, ddd, JL.2, 7.0, 3.2Hz). 7.565 (1H, ddd, Jl.2, 7.0, 3.2 Hz), 7.799 (1H, d, J-8.2HZ), 7.884 (1H, d, J-8.2HZ) 3.071 (1H, d, J-7.9HZ) , 3.516 (1H, S), 9.345 (1H, d, J-7.9HZ), 10.4-10.6 (1H, brs), 11.323 (1H, s) FAB-MS (m / z): 439 (-l) " Example 3 Compound IV-5 The compound (S) (J. Chem. Soc. Perkin Trans. 1: 2475, 1990) (823 milligrams, 2083 millimoles) was dissolved in 20 milliliters of dimethylformamide, and 166.4 milligrams (4.16 millimoles) of sodium hydride were added ( 60 percent) under cooling with ice, followed by stirring at the same temperature for 10 minutes. Allyl bromide (0.45 milliliters, 5.2 mmol) was added, and the solution was stirred for 2 hours under cooling with ice. After dilution with chloroform, water was added, and the organic layer was separated, washed with a saline solution, and dried over magnesium sulfate.

After evaporation of the solvent, the residue was subjected to silica gel column chromatography (ethyl acetate / toluene = 1/15), to give 735.0 milligrams (74 percent yield) of the compound (T-2).

: H- MR (DMS0-d) d (ppm): 1.56 -2.154 (6H, m), 3.657 (1H, n), 4.003 (1H, rn), 5.044-5.473 (11H, m), 6.153CH, m ), 7.240-7.640 (6H, m), 3.167 (1H, d, J-7.8HZ), 9.415 (1H, d, J-7.3HZ) FAB-MS (m / Z): 476 (MC) ~ Sodium borohydride (77.7 milligrams, 2. 05 millimoles) in 20 milliliters of tetrahydrofuran, and 231.0 milligrams (1.82 millimoles) of iodine were added at 0 ° C, under an argon atmosphere, followed by stirring at the same temperature for 15 minutes. Compound (T-2) (136.7 milligrams, 0.287 mmol) was added at the same temperature, and the mixture was stirred at room temperature for 4.5 hours. After the reaction mixture was cooled to 0 ° C, 3.7 milliliters of IN sodium hydroxide and 3.7 milliliters of a 35 percent aqueous solution of hydrogen peroxide were added, followed by stirring for another 30 minutes. The reaction mixture was diluted with water, and extracted with ethyl acetate. The ethyl acetate layer was washed successively with water and a saline solution, and dried over magnesium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol = 15/1), to give 88.9 milligrams (61 percent yield) of the compound (U-2). lH-NMR (CDClj) d (ppm): 1.60-2.11 (10H, m), 3.129 (2H, t, J-5.9HZ), 3.192 (2H, Z, J-5.9HZ), 3.798 (1H, dt, J-2.3, 11.7HZ), 4.09-4.15 (1H, m), 4.723 (2H, Z.J-7.2HZ), 4.307 (2H, Z, J-7.2HZ), 4.943 (1H, d, J-16.6 HZJ, 5.107 (1H, d, J-16.6HZ), 5.652 (1H, dd, J-2., 10.5Hz), 7.15-7.13 (1H, m), 7.313 (1H.Ddd, Jl.l, 7.3, 3.0Hz;, 7.33-7.39 (1.4, m), 7.461 (1.4, ddd, J = 1.2, 6.3, 3.0Hz), -.519 (1.4, dd, Jl.2, 3.0Hz), 7.610 (1H, d , J-ß.OHz), 7.95K1H, d, J-ß.OHz) 9.490 (1H, d, J-ß.OHz) FAB-MS (m / Z): 512 (M + l) * The compound (U-2) (88.9 milligrams, 0.174 millimoles) was dissolved in 10 milliliters of tetrahydrofuran, and 8 milliliters of 4N sulfuric acid was added, followed by stirring at 60 ° C for 24 hours. After the reaction mixture was cooled to room temperature, ice was added, followed by extraction with ethyl acetate. The ethyl acetate layer was washed successively with water and a saline solution, and dried over magnesium sulfate. After evaporation of the solvent, the residue was subjected to thin layer chromatography (chloroform / methanol = 15/1), to give 37.6 milligrams (51 percent yield) of compound IV-5.

-H-NMR (DMSO-d5) d (ppm): 1.59-1.65 (2H, m), 1.70-1.82CH,), 3.03-3.27 (2H, m), 3.09- 3.14 (2H, m), 4.371CH , Z, J-5. OHz). 4.419 (1H, t, J-5.0HZ), 4,730 (2.4, Z, J-7.3HZ), 4,813 (2H, Z, J-7.4HZ), 4,972 (2H, s), 7,283 (1.4, ddd. -0.3, TO 7.3HZ), 7.370CH, Z, J-7.2HZ), 7.501 (1H, ddd, Jl.2, 7.0, 3.2Hz), 7.563 (14, ddd, Jl. I, 7.2, 3.3HZ) , 7.779 (1.4, d, J-8.2HZ) 7.348 (1.4, d, J-8.2HZ), 3.043 (1H, d, J-7.2HZ), 9.412 (1H, dd, J-0.3, ".8Hz) FAB-MS (m / Z): 428 (* l) ' Example 34 Compound 11-68 Compound (Q) (50.1 milligrams, 0.0862 millimoles) was dissolved in 3 milliliters of chloroform, and then 129.5 milligrams (0.862 millimoles) of 2-mercaptobenzimidazole and 49 milligrams (0.21 millimoles) of acid were added ( +) - 10-camphorsulfonic, followed by stirring at room temperature for 12 hours. The reaction mixture was washed successively with a saturated aqueous solution of sodium bicarbonate, water, and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to thin layer chromatography of preparation (chloroform / methanol = 99/1), to give 46 milligrams (75 percent yield) of compound 11-68 N, 0-diacetylated. FAB-MS (m / z): 714 (M + 1) +.

The same procedure was repeated substantially as in Example 25, using 33.4 milligrams (0.0468 millimoles) of the 11-68 N, 0-diacetylated compound, to give 17.5 milligrams (59 percent yield) of compound 11-68.

-H-NMR, DMS0-d6) or (ppm): 2.995 (! K, dd, J-4.9, 14.1.4Z), 2.139 (2H, s), 3.914 (3H.3), 4.799 (2H, s) , 4.979 (lH, d, J-17.2HZ), 5.023 (1H, d, J-17.3HZ), 6.342 (1H, s), 7.101 (1H, dd, J-4.9, 7.3HZ), 7.123-3.056 ( 10H, m), 3.617 (IH, s), 9.273 (1.4, z?) FAB-MS, 'm / z): 630 (Ml) * Example 35 Compound 11-69 Substantially the same procedure was followed as in Example 25, using 50 milligrams (0.0861 millimoles) of Compound Q, and 0.0868 milliliters (0.0861 millimoles) of furfuryl mercaptan, to give 36.0 milligrams (62% yield) ) of the compound 11-69 N, 0-diacetylated. FAB-MS (m / z): 678 (M + 1) +.

The same procedure was repeated substantially as in Example 25, using 22.7 milligrams (0.0335 millimoles) of the compound 11-69 N, 0-diacetylated, to give 17.7 milligrams (89 percent yield) of compound 11-69.

-H-NMR (CDClj) 5 (ppm): 2,209 (3H, s) 2,607 (1H, dd, J-4.9, 14.5HZ), 3.401 (1H, dd, J-7.5, 14.5Hz), 3.671CH, 3 ), 3,357 (2H, s), 4,103 (3H, s), 4,532 (1H, brs), 4,789 (1H, d, J-lß.lHz), 4,373CH, d, J-15.1HZ), 5,690 (1H) , s), 6,373 (1.4, dd, Jl.9, 3.2Hz), Ó.41Ó (1H, dd, J-0.6, 3.2Hz), 6,846 (1H, dd, J-4.3, ".5Hz), 7 - ~ .932 (7H, p), 3.96K1H, ra) rAB-MS (m / Z): 593 (M) * Example 36 Compound 11-70 Compound (P) (100 milligrams, 0.173 mmol) was dissolved in 4 milliliters of chloroform, and then 34.0 milligrams (0.277 millimoles) of 1-aminopyrrolidine hydrochloride was added, followed by stirring at room temperature During 4 hours. After evaporation of the solvent under reduced pressure, the residue was subjected to silica gel column chromatography (chloroform / methanol = 99/1), to give 100.5 milligrams (90 percent yield) of compound 11-70 N, 0-diacetylated. FAB-MS (m / z): 648 (M + 1) +.

The same procedure was repeated substantially as in Example 25, using 40 milligrams (0.0618 millimoles) of the 11-70 N, 0-diacetylated compound, to yield 30 milligrams (86 percent yield) of compound 11-70.

'"LH-MR (DMSO-d5 d' ppm): 1. 10-1.937 (4H,), 2.031 (1H, dd, J-4.9, 14.1HZ), 2.142 (3H, s), 2.329-2.635 (4H , m), 3,395 (1.4, dd, J-7.3, 14.1Hz), 3,925 (3H, S), 4,981 (1H, d, J-17.0HZ), 5,030 (1H, d, J-17.0HZ), 7,110 (1H, dd, J-4.9, 5 7.3HZ), 7.345-3.057 (6H, m), 7.425 (1H, s), 3.596 (1H, s), 9.210 (1H, d, J-1.4HZ) FAB- MS- (m / z): 564 (M + l) * Example 37 Compound 11-71 Compound (P) (49 milligrams, 0.0846 millimole) was dissolved in 3 milliliters of chloroform, and then a solution of 15.8 milligrams (0.145 millimoles) of 2-hydrazinopyridine in chloroform and 49 milligrams ( 0.21 mmol) of (+) - 10-camphorsulfonic acid, followed by stirring at room temperature for 12 hours. The reaction mixture was washed successively with a saturated aqueous solution of sodium bicarbonate, water, and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to thin layer chromatography of Preparation (chloroform / methanol = 99/1), to give 35.8 milligrams (64 percent yield) of compound 11-71 N, 0-diacetylated. FAB-MS (m / Z): 671 (M + 1) +.

The same procedure q-ie was repeated substantially in Example 25, using 24.6 milligrams (0.0367 millimoles) of compound 11-71 N, 0-diacetylated, to give 11.8 milligrams (55 percent yield) of compound 11-71.

: H- / M (DMS0-d) 5? Ppm¡: 2.039 (1H, dd, J-5.0, 13.9HZ), 2.153 (3H, s), .413 and 1K, d, J-7.2, 13.9HZ) , 3.933 CH, 3), 5.001 (1H, d, J-17.5HZ). 5.057 (1H, d, J-17.5HZ), OR.35Ó iH, 3), 6.743 (1H, m), 7.164 (1H, dd, J-5.0, 7.2HZ), 7.301-3.120 (9H, m), 3.242 (1H, s), 3.656 (1H, S), 3.56 (1.4, s), 9.368 (1H) s, 10.738 (1H, s) FAB-MS (m / Z): 537 (MH) " Example 38 Compound 11-73 Substantially the same procedure was followed as in Example 25, using 30 milligrams (0.0516 millimole) of compound (Q), and 52.2 milligrams (0.516 millirale) of 1H-1,2,4-triazole-3 -thiol, to give 31.4 milligrams (92 percent yield) of compound 11-73 N, 0-diacetylated. FAB-MS (m / z): 665 (M + l) + The same procedure was repeated substantially as in Example 25, using 15 milligrams (0.0226 millimoles) of the 11-73 N, 0-diacetylated compound, to give the crude compound 11-73. Chloroform / methanol (90/10) was added, followed by stirring, to give 10.9 milligrams (83 percent yield) of compound 11-73 as a precipitate.

-H- MR SC- ^,. ' , ppra :: 2.0C6 (1H, dd, J-4.9, 13.9Hz), 2.144CH, s), 3.375 (14, d, J-7.3, 13.9HZ), 7.921 (3H, s), 4.559CH, brs ), 4.977 (1H, d, J-17.4HZ), 5.033CH, d, J-17.4HZ), 6,332 (14, 3), 7,106 (1H, d, J-4.9, 7.3Hz), 7341-3.062 ( 64, ra), 3,614 (1H, s). 9.202 (14, d, J-1.5HZ) FAB-MS (m / Z): 531 (M * l) * Example 39 Compound 11-74 Compound (P) (97.5 milligrams, 0.168 millimoles) was dissolved in 4 milliliters of tetrahydrofuran, and then an aqueous solution of 25.1 milligrams (0.0950 millimoles) of aminoguanidine sulfate was added, followed by stirring to the room temperature for 3 hours. Ethyl acetate was added, followed by stirring, and the insoluble matter was collected by filtration, and subjected to silica gel column chromatography (chloroform / methanol = 85/15), to give 87.1 milligrams (82 percent yield ) of compound 11-74, 0-diacetylated.

FAB-MS (m / z): 636 (M + 1) +.

The same procedure was repeated substantially as in Example 25, using 69.6 milligrams (0.110 millimoles) of the N, 0-diacetylated compound 11-74, to give 37.2 milligrams (62 percent yield) of compound 11-74.

LH-MR (DMS0-d) 5 (ppm): 2.046 (14, d, J-4.9, 14.2Hz), 2.143CH, s), 3.406 (1H, dd, J-7.5, 14.2HZ), 2.929; 34, s), 4,933 (14, a, J-17.2H.Z). 5.045 (1.4, - :, J-17.3HZ), 5.637-6.129 (4.4, ra), 0.350 (14, 3), -.156 (14, d, J-4.9, _.5Hz), "'.345 -3.292 (64, a), 3.206 (1H, s), 3.603 (1.- :, s), 9.271 (14, d, J-1, HZ) FAB-MS (m / z): 552 (M + l) " Example 40 Compound 11-76 Compound (P) (103.8 milligrams, 0.179 mmol) was dissolved in a mixture of 6 milliliters of chloroform and 3 milliliters of methanol, and then 0.5 milliliters of an aqueous solution of 0.020 milliliters (0.207 millimoles) were added. ) of 4-aminomorpholine and 0.05 milliliters of 3N hydrochloric acid, followed by stirring at room temperature for 3 hours. The reaction mixture was washed successively with a saturated aqueous solution of sodium bicarbonate and a saline solution, and dried over sodium sulfate. After evaporation of the solvent, the residue was subjected to silica gel column chromatography (chloroform / methanol = 90/100), to give 82.8 milligrams (70 percent yield) of compound 11-76 N, 0-diacetylated . FAB-MS (m / z): 663 (M + 1) +.

The same procedure was repeated substantially as in Example 25, using 50.6 milligrams (0.0763 millimoles) of the 11-76 N, 0-diacetylated compound, to give 36.4 milligrams (82 percent yield) of compound 11-76.

LH-NMR (DMS0-d6) S (ppm): 2.042 (1.4, dd, J-4.3, 14.3HZ), 2.144 (3H, s), .139-3.163 (4H, ra), 3.404 (1H, dd, J-7.5, 14.3HZ), 2,792-3,315 (4H, ra), 3,927 (34, s), 4,984 (14, d, J-17.3HZ), 5,040 (14, d, J-17.3HZ), 6,352 ( 14, s), -122 (14, dd, J-4.3, ".5HZ), 7344-3.OßS.? Tr - • • .97 (14, S;, 3,610 (IH, 3), 9,216 (14, d, J-1.7Hz) Example 41 Compound 11-77 Substantially the same procedure was followed as in Example 40, using 100 milligrams (0.173 millimoles) of compound P, and 16.7 milligrams (0.173 millimoles) of 1,1-dimethylhydrazine hydrochloride, to give 52.3 milligrams ( 49 percent yield) of the 11-77 N, 0-diacetylated compound.

FAB-MS (m / z): 622 (M + 1) +.

The same procedure was repeated substantially as in Example 25, using 38.4 milligrams (0.0618 millimoles) of compound 11-75 N, 0-diacetylated, to give 10.9 milligrams (33 percent yield) of compound 11-75. : 4-NMR (DMS0-d6) < S (ppm): 2.037 (1H, dd, J-5.0, 14.1HZ), 2.142 (3H, s), 2.939 (6H, s), 3.399 (1H, dd, J-7.5, 14.1HZ), 3,926 (3H , s), 4,981 (1H, d, J-17.7HZ), 5,037 (1H, d, J-17.7HZ), 6342 (1H, S), 7.118 (1H, dd, J-5.0, 7.5HZ), 7342 - 3.063 (6H, m), 7.533 (1H, S), 3.601 (14, s), 9.258 (1H, S) FAB-MS (ra / z): 533 (M + l) " Example 42 Compound 11-78 Substantially the same procedure as in Example 40 was followed, using 99.5 milligrams (0.172 millimoles) of compound (P), and 42.4 milligrams l-amino-4-methylpiperazine, to give compound 11-78 N , O-diacetylated. Then, substantially the same procedure was repeated as in Example 25, using the above compound 11-78 N, 0-diacetylated, to give 19.4 milligrams [yield from compound (P) of 19 percent] of compound 11-78 . : 4-NMR (DMS0-d6) ippm): 2.040 (1H, dd, J-5.0, 14. OHZ), 2.144CH, s), 2.263 (3H, s), 2.553 (4H, m), 3.1O7 (4H, m). 2.401 (1H, dd, J-7.2, 14.0Hz). 3.927 (3H, 3), 4.932CH, d, J-17.1HZ), 5.038 (1.4, d, J-17.1HZ), 6.345 (14, S), 7.128 (1H, dd, J-5.0, 7.2HZ) , 7.343-3.065 (6H, m), 7.827 (1H, s), 8.609 (14, 3), 9.299 (14, d, J-1.2HZ) FAB-MS (m / Z): 5 3, M-1 ) Example 43 Compound 11-81 Compound (AA), a compound having bis (hydroxymethyl) in place of bis (dimethylaminoethylthiomethyl) of compound 11-80 (described in Example 28) (53.9 milligrams, 0.102 millimoles) was dissolved in 2.5 milliliters of dichloromethane. Then 0.18 milliliters (2.0 millimoles) of 2-propanothiol and 0.03 milliliters (0.2 millimoles) of trifluoroacetic anhydride were successively added, followed by stirring at room temperature in a stream of argon for 3 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 99/1), to give 52.6 milligrams (80 percent yield) of compound 11-81.

: H-NKR (DMS0-dd) or (ppm): 1.259 (3H, d, J-6.6HZ), 1266 (? H, d, J-6.5HZ), 1993 (1H, dd, J-5.0, 14.1 HZ). 2.121C4, s), 2.831 (2H,), 2.375 (14, dd, J-7.2, 14.14;.), 3,920 (24, s), 3,963 (24, s), 4,002 (24, 3), 4,952 ( 14, d, J-17.14Z), 5.016 (14, d, J-17.14Z !, 7.093 (14, dd, J-5.0, 7.3Hz), 7.440-7.473 (24, ra), 7.332 (1H. , J-8.1HZ), 7.877 (1.4, d, J-8.3HZ), -, 959 (1H, d, J-1.7HZ), 3.592 (14, 3), 9.139 (14, d, J-lCHz) FAB-MS (m / Z): 643 (M) *, 644 (4 * 1) "Example 44 Compound 11-82 Substantially the same procedure was repeated as Example 43, using 51.9 milligrams (0.0958 millimoles) of the compound (AA ), 0.17 milliliters (1.9 millimoles) of 1-propanothiol, and 0.03 milliliters (0.2 millimoles) of trifluoroacetic anhydride, to give 52.3 milligrams (83 percent yield) of compound 11-82.

^ -NMR (DMS0-d6 d (ppm): 0.944 (3H, t, J-7.3HZ), 0.951 (3H, t, J-7.3HZ), 1.557-1.656 (H, ra), 1.995 (1H, dd , J-4.8, 14.1Hz), 2.132 (3H.s), 2.462 (2H, t, J-7.3HZ), 2.470 (2H, Z, J-7.34;;, 3.378 (1H, dd, J-7.4, 14.1Hz), 3.921 (34, sr 3.957 (2H, S), 4.951.CH, d, J-lT.lKz: 5.013 (1H.D, 17.1HZ), 7.102 CH.D.J.-4 .: 7.4HZ), 7.430-7.462CH, ra), -.336.14, z.J-8.3HZ), 7.880CH, d, J-ß.éH?;. -.94 :. IH. . . J-1.SHZ), 8.599CH, s), 9.122 (14. d, J-l.iHz) FAB-MS (ra / Z): 643 (M) ", 644 (M + l) * Example 45 Compound 11-83 Substantially the same procedure was repeated as in Example 43, using 49.4 milligrams (0.0937 millimoles) of the compound (AA), 0.20 milliliters (1.9 millimoles of 1-butanediol, and 0.03 milliliters (0.2 millimoles) of anhydride trifluoroacetic, to give 51.7 milligrams (82 percent yield) of compound 11-83.

: - ^ (DMSO-d, S: ppra, r 0.365 (3H, Z, J = 7, HZ) 0-877 (3H. "-, J-7, HZ) .328-1.409 (44, - ,,, '1,535-1,500 (4H, ra), 1,995 (14. dd, J-4.9, 14.1HZ), 2,132 (24, 3), 2,480 (2H, Z, J = 7.4Hz), 2,491 (2H, z, J-7.4HZ), 3,377 (iHf dd, J-7.5, 14.1HZ), 3,921 (54. s), 3,958 (2H, s), 4,952 (14, d, J-16.9HZ), 4,997 (1H , d, J = 16.94z), 6.314 (14, s), 7.101 (14, dd, J-4.9, 7.4HZ), 7.432-7.458 (2H, ra), 7.334 (1.4, d.J-8.4HZ) , 7.380CH, d, J-8.7HZ), 7.942 (14, d, Jl.SHz), 3.599 (1H.'S), 9.123 (14, d, J-1.4HZ) FAB-MS (m / Z) : 571 (M) " Example 46 Compound 11-84 Compound (AA) (45.3 milligrams, 0.0860 millimole) was dissolved in a mixture of 0.2 milliliters of methanol and 2 milliliters of chloroform, and then 20 milligrams were added. (0.086 mmol) of camphorsulfonic acid, followed by stirring at room temperature for 17 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 99/1), to give 23.1 milligrams (48 percent yield) of compound 11-84.

'H-NMR (DMSO-d5;' (ppm): 2.010 (1.4, dd, J-4.9, 14.1HZ), 2.142 (34, s), 3.341 (3H, s), 3.364 (3H, s), 2.333 (14, dd, J-7.4, 14.1Hz), 3,925 (3,4, 5) 4,583 (24, s, -,. 622 (2.4, s), 4,982 (14, d, J-16.9HZ),? .023CH , d, J-16.9HZ), 5,320 (14. s), -.127 (14, cd. J = 4.9, 7.4HZ), 7,441-7,464 (2.4, ra), -.372 (14, d, J -3.4HZ), -.917 (14 d, J-8.7HZ), -.972 (14, d, JL.iHz), 3.611, 14 s), 9.165 (14, d, Jl. OHz) "AB- S (m / Z): 555 (M) ' Example 47 Compound 11-85 Substantially the same procedure as in Example 46 was repeated, using a solution of 51.3 milligrams (0.0973 millimoles) of the compound (AA), in a mixture of 0.2 milliliters of ethanol and 2 milliliters of chloroform, to give 24.1 milligrams (42 percent yield) of compound 11-85.

H-MR (DMS0-d6) i (ppm): 1,139 (3H, Z, J-7.0HZ), 1199 (3H, Z, J-7.0HZ), 1999 (14, dd, J-4.9, 14.1HZ) , 2.142CH, s), 3.335 (1.4, dd, J-7.4, 14.1HZ), 2.547 (2H, q, J-7.0HZ), 3.563 (2H, q, J-7.0HZ), 3.925CH, s) . 4.622 (2H, s), 4.661 (2.4, s), 4.980 (14. d, J-16.9HZ), 5.032 (1H, d, J-16.9HZ), 6.325CH, s), 7.124 (14, dd, J-4.9, 7.4HZ), 7. 47-7.467 (2H, m), 7.864 (14, d, J-8.3HZ), 7.911CH, d, J = 8.74z), 3.602 (1.4, 3), 9.162 CH, d, Jl. OHZ) FAB-MS (m / Z): 533 (M) 'Example 48 Compound 11-86 Compound (BB) (Japanese Published Unexamined Patent Application Number 295588/88) (978 milligrams, 1.69 millimoles), was dissolved in 70 milliliters of 1,2-dichloroethane, and then 0.17 milliliters (3.8 millimoles) of nitric acid were added dropwise under vaporization under ice cooling, followed by stirring at room temperature for 30 minutes. The reaction mixture was diluted with chloroform, and a saturated aqueous solution of sodium bicarbonate was added. The insoluble material was collected by filtration and dried. The filtrate was washed with an aqueous solution of sodium chloride, and dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue and the insoluble material were combined to give 946 milligrams (90 percent yield) of the compound (CC) as a crude product. FAB-MS (m / z): 625 (M + 1) +.

The compound (CC) (640 milligrams, 1.03 millimoles) was dissolved in 30 milliliters of 1,2-dichloroethane, and then 0.3 milliliters (3.58 millimoles) of 1,2-ethanedithiol and 0.2 milliliters (2.0 millimoles) were added dropwise. boron trifluoride ether complex at 0 ° C, followed by stirring for 30 minutes. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to column chromatography on silica gel (chloroform), to give 579 milligrams (81 percent yield) of the compound (DD). FAB-MS (m / z): 701 (M + 1) +.

The compound (DD) (579 milligrams, 0.827 mmol) was dissolved in 56 milliliters of N, N-dimethylformamide, and then 400 milligrams of palladium / carbon was added, followed by stirring at 60 ° C under a hydrogen atmosphere for 2 hours . The insoluble material was filtered, and the solvent was evaporated under reduced pressure from the filtrate. The residue was subjected to silica gel column chromatography (chloroform / methanol = 98/2) to give 193 milligrams (35 percent yield) of the compound (EE). FAB-MS (m / z): 671 (M + 1) +.

The compound (EE) (193 milligrams, 0.288 millimoles) was dissolved in 10 milliliters of chloroform, and then 0.1 milliliters (0.7 millimoles) of triethylamine and 0.2 milliliters (2.5 millimoles) of ethyl isocyanate were added, followed by stirring at the temperature environment for 20 hours. After water was added, the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 96/4), to give 211 milligrams (99 percent yield) of the compound (FF). FAB-MS (m / z): 742 (M + 1) +. The compound (FF) (211 milligrams, 0.285 millimole) was dissolved in a mixture of 6 milliliters of ethanol and 6 milliliters of chloroform, and then 171 milligrams (1.01 millimoles) of silver nitrate was added at 50 ° C, followed by stirring during 20 minutes. After the reaction was finished, the insoluble material was filtered. The filtrate was washed with a saturated aqueous solution of sodium bicarbonate and an aqueous solution of sodium chloride, and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography. (chloroform / methanol = 97/3), to give 118 milligrams (62 percent yield) of the compound (GG). FAB-MS (m / z): 666 (M + 1) +. The compound (GG) (100 milligrams, 0.150 millimoles) was dissolved in a mixture of 4.5 milliliters of chloroform and 0.72 milliliters of methanol, and then 8.7 milligrams, = .23 millimoles) of sodium borohydride were added at 0 ° C, followed by by agitation for 45 minutes. The reaction mixture was poured into water, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 95/5) to give 101 milligrams (100 percent yield) of the compound (HH). FAB-MS (m / z): 668 (M + 1) +. The compound (HH) (21.7 milligrams, 0.0325 mmol) was dissolved in a mixture of 1 milliliter of 1,2-dichloroethane and 0. 3 milliliters of methanol, and then 6 microliters (0.03 millimoles) of a methanolic solution 5. IN of sodium methoxide was added, followed by stirring for 1 hour. The reaction mixture was poured into water, and the mixture was extracted with a mixture of chloroform and methanol (9/1). The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography. (chloroform / methanol = 90/10), to give 14.9 milligrams (79 percent yield) of compound 11-86. lH-NMR (DMSO d6; d (ppm): 1.097 (3H, Z, J-7.2HZ), 1.968 (1H, dd, J-4.9, 13.9Hz), 2.113 (3H, s), 3.170 (24, dq , J-5.6, 7.2Hz), 3,359 (1H, d, J-7.3, 13.9Hz), 3,915 (3H, s), 4,664 (2H, s), 4,887 (1.4, d, J-16.9HZ), 4,947 (1H, d, J-16.9HZ), 6,081 (1H, Z, J-5.6HZ), 6,273 (1H, s), 7,090 (14. dd, J-4.9, 7.3HZ), 7,364 (1H, dd, J-2.0, 9.0Hz), .455 (1H.D, Jl., 3.5Hz), 7.732 (14.D, J-9.04Z), 7.326 (14.D, J-8.5HZ), 3.139 (14, d, J-2.C4Z !, 3,493 (14, s), 3,537 (14, s), 9,127 (14, d, J-1.3HZ) FAB-MS (m / z): 534 (M + l) " Example 49 Compound 11-87 Substantially the same procedure was repeated as in Example 43, using 29.8 milligrams (0.0511 millimoles) of compound 11-86, and 0.14 milliliters (1.6 millirales) of ethanethiol, to give 24.2 milligrams (yield 76 per cent). percent) of compound 11-87.: H-NMR OMSO-c, Z (p? m): 1.097 (34, Z, J-7.14Z,, 1.230 (34.Z.J-7.2HZ), 1982 (1H , d, J-5.0, 14.1HZ), 2.111 (24, s), 2.437 (2H, dq, J-5.6, 7.1HZ), 2.937.'24, q, J-7.3HZ), 3.362 (14, dd , J-7.5, 1 HZ), 3,914 (3H, s), 3,939 (24, s). 4.333 (14, d, J-17.2HZ), 4950 (1H, d, J = 17.24z), 6.083 (14, z, J-5.6HZ), 6.235 (1H, s), 7.033CH, dd, J- 5.0, 7.5HZ), 7.370 (1H, dd, J-2., 9.0HZ), 7.436 (1H, dd, Jl.6, 3.5HZ), 7.783 (1.4, d, J-9.0HZ), 7.325CH , d, J-8.5HZ), 8.138 (1H, d, J-2.1HZ), 3.496 (1.4, 3), 8.532 (1H, S), 9.116 (14, d, J-1.64Z) FAB-MS ( m / z): 527 (4) " Example 50 Compound 11-88 Compound (AA) (50.4 milligrams, 0.0956 millimoles) was dissolved in 0.7 milliliters of dichloromethane, and then 0.09 milliliters (0.56 millimoles) of triethylsilane and 0.73 milliliters (9.5 millimoles) of trifluoroacetic acid were added in succession. cooling with ice, followed by stirring at room temperature for 10 minutes. The reaction mixture was neutralized with an aqueous IN sodium hydroxide solution, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (chloroform / methanol = 90/10), to give 20.7 milligrams (44 percent yield) of compound 11-88.

: H-NMR (DMS0-dd (ppm): 1.963 (14, dd, J-4.9, 13.9Hz), 2.116 (34, s), 2.510 (3H, s, 2.529 (34 S.), 3.353 (14, dd, J-7.2, 12.9HZ), 3914 (34, s, 4.955 (14, d, J-17.24Z), 5.007 (1H, d, J-17.2HZ, 5.273 (14, 5), -.074 ( 14, d, J-4.9, 7.3Hz), ".237-" .212 (24. - '), ".754 (1.4, d, J-8.3HZ), 7.803 (14, d, J-8.5HZ ), -.223 (14, 3), 3.575 (14, S), .006 (14, S) FAB-MS (m / Z): 496 (M + -1) * Example 51 Compound 11-89 Compound (AA) (4.3 grams, 8.16 mmol) was dissolved in 215 milliliters of dichloromethane, and then 12.1 milliliters (163 millimoles) of ethanethiol and 2.5 milliliters (17.7 millimoles) of trifluoroacetic anhydride were successively added, followed by stirring at room temperature for 12 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with an aqueous solution of sodium chloride, and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography (ethyl acetate / toluene = 3/7), to give 4 milligrams (yield of 0.08 percent) of compound 11-89. ^ -NMR (DMS0-d6) d (ppm): 1.233 (3H, Z, J - .2HZ: 1.253 (3H, dd, J-7.6, 3.3Hz), 2.0C "14. zd. J-4.6 , 1 CHZ), 2.139CH, S), 2.492 24., J-7.3HZ), 2.622-2.710 (14, ra), 2. 35 -2.? 7".14. m), 3.384 (1H, dd, J-7.4, 14.24Z), 2,926 (24 s), 3,979 (2H, S), 4,106 (1.4 .. d, J-1I.9HZ), 4,235 (14, d) , J-12.9HZ), 4.961CH, d, J-17.9HZ), 5.025 (14, d, J-17.9HZ), 6.325CH, S), 7.132 (14, dd, J-4.8, 7.4HZ), 7.433-7.473CH, a), 7.887 (1H, d, J-8.6HZ), 7.902 (1H, d, J-8.3HZ), 3.625 (1H, s), 9.147 (1H, s) FAB-MS (m / Z): 632 (M + l) " Example 52 Compound 11-90 Compound (JJ) (Japanese Patent Application No Examined Published Number 295588/88) (18.5 grams, 30.5 millimoles) was dissolved in a mixture of 900 milliliters of chloroform and 145 milliliters of methanol, and then 3.42 grams (90.4 millimoles) of sodium borohydride were added under ice cooling, followed by stirring for 25 minutes. The reaction mixture was poured into ice water, and the insoluble material was collected by filtration, washed with water, and dried under reduced pressure. The insoluble material was dissolved in a mixture of 555 milliliters of 1,2-dichloroethane and 185 milliliters of methanol, and then 0.925 milliliters (4.72 millimoles) of a 5.1N ethanolic solution of sodium methoxide was added, followed by stirring for 1.5 hours. . The reaction mixture was poured into water, and the insoluble material was collected by filtration, dried under reduced pressure, and subjected to silica gel column chromatography (chloroform / methanol = 8/2), to give 0.350 grams ( 2.3 percent yield) of the ligo compound. ^ -WMR (DMS0-d6) d (ppm): 1909 (1H, dd, J-4.9, 1J.4HZ), 2.148 (3H, s), 3.134 (1.4, dd, J-7.3, 13.4HZ), 3.757 (1H, dd, J-6.1, 11.3Hz), 3.83K1H,? Á, J-5.5, 11.3Hz), 4.662 (24, d, J-5.6HZ), 4.704 (2H, d, J-5.6HZ) , 4.944 (1H, d, J-17.0HZ), 5.007 (1H, d, J-17.0HZ), 5.093 (1.4, dd, J-5.5, 6.1Hz), 5.123 (1H, t, J-5.6HZ) , 5.139 (1H, Z, JS.6HZ), 5.346 (1H) s, 5.942 (1H, dd, J-4.9, 7.3HZ), 7.393-7.459 (2H, m), 7.722 (1H, i, J- 8.3HZ). 7.911 (1H. d, J-8.8HZ), 7.952 (1H, d, J-0.974Z), 3.538 (1.4, s), 9.129 (1H, -; FAB-MS (m / Z): 499 (M) ", 500 (4 * 1)" Example 53 Compound II-91 Compound (DD) (28.6 milligrams, 0.0266 millimole) was dissolved in a mixture of 1.5 milliliters of 1,2-dichloroethane and 0.5 milliliters of methanol, and then added 5 microliters (0.026 mmol) of a 5.1N methanolic solution of sodium methoxide, followed by stirring for 1 hour. The reaction mixture was poured into water, and the mixture was extracted with a mixture of chloroform and methanol (9/1). The organic layer was washed with an aqueous solution of sodium chloride, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography. (chloroform / methanol - = 95/5), to give 7.0 milligrams (43 percent yield) of compound 11-91. lH-MM (DMS0-d6) d (ppm): 2.017 (1H, dd, J-4.9, 14.4HZ), 2.133CH. s), 3.408-3.452 (2H, m), 3.588-3.651CH, m), 3.940 (3H, s), 5.122 (1H.d, J-18.1HZ), 5.175CH, d, J-13.1HZ), 5.943 (1H, S), 6.549 (1H, S), 7.189 (1H, dd, J-4.9, 7.3HZ), 7.739 (1H, dd, Jl.9, 3.7HZ), 7.917 (1H, d, J- 8.7HZ), 3.125 (1.4, d, J-9.4HZ), 3.373 (1.4, dd, J-2.2, 9.4HZ), 3.733 (1H, s), 3.343 (14, d, J-2.2HZ), 9.353 CH, d, J-1.9HZ) FAB-MS (ra / z): 617 (M + l) * Example 54 Compound 11-92 Substantially the same procedure was repeated as in Example 53, using 23.3 milligrams (0.0314 millimoles) of the compound (FF), to give 14.7 milligrams (71 percent yield) of compound 11-92.

"H-MMR (DMS0-d5) d (ppm): 1.097 (24, c.J-7.1HZ), 1.98 (IH, dd, J-4.9, 14. OHZ), 3.170 (2H, dq, J-5 .S. 12.7Hz), 3,359 (14, dd, J-7.4, 14.0Hz), 3,401-3,464 (24, -, 3,532-3,645 (24, ra), 3,914 (34, 3), 4,391 (14, d, J-17.54Z), 4,956 (14, d, J- 17.6Hz), 5,930 (14 s), 6,081 (14, z, J-5.6HZ), 6,237 (1H, s), 7,091 (1H, dd, J-4.9, 7.4HZ), 7,379 (1H, dd, J-2.2, 9.0Hz), 7,633 (14, dd, Jl., 3.54Z), 7,783 (14, d, J-9.0HZ), 7,850 (1H, d, J-8.5HZ), 3.133 (1H, d, J-2.2HZ), 3.499 (1H, s), 3.534 (1H, s), 9.296 (1H, d, J-1.7HZ) FAB- MS (ra / z): 653 (M + l) * Although the invention has been described in considerable detail, the invention disclosed herein should not be limited to the actual description, but should be given the full scope of the appended claims and all their equivalents. Other embodiments are within the following claims.

Claims (67)

1. CLAIMS 1. A composition of the formula [Stau] -N (CH3) -W-N (CH3) - [Stau] (I) where [Stau] represents a residue of the formula H and represents a radical of the formula -C (= Y) -NH-W -NH-C (= Y) -where W is a hydrocarbylene radical of 2-20 carbon atoms and Y is O or S.
2. 2. A composition of the formula (II -4): where R1, R2, Z1 and Z2 are each H; X is CH2OH; and R is OCH3
3. 3. A composition of the formula (11-14): 11-14 wherein R1, R2, Z1 and Z2 are each H; X is CH2-NH-Ser; and R is OH.
4. 4. A composition of the formula (11-49): 11-49 where R2, Z1 and Z2 are each H; R is OH; R1 is CH2S02C2H5; and X is C02CH3.
5. 5. A composition of the formula (11-38): 11-38 where R1, R2, Z1 and Z2 are each H; R is OH; and X is CH2NIIC02C6H5.
6. 6. A composition of the formula (11-45): 11-45 where R1 and R2 are each Br; R is OH, Z1 and Z2 are each H; Y X is CONHC6H5.
7. 7. A composition of the formula (11-57): 11-57 where R1, R2, Z1 and Z2 are each H; R is OH; and X is CH2NHC02CH3
8. 8. A composition of the formula (11-72): where R1 is CH2S (CH2) 2NH2; X is C02CH3; R is OH; and R2, Z1 and Z2 are each H.
9. A composition of the formula (11-751 where R1 is OH, and R2, Z1 and Z2 are each H.
10. 10. A composition of the formula (11-79) 11-79 where R1 is CH2S (CH2) NH n-C "H9; X is C02CH3; R is OH; and R2, Z1 and Z2 are each H.
11. 11. A composition of the formula (11-80): 11-80 wherein R1 is CH2S (CH2) 2N (CH3) 2; R2 is CH2S (CH2) 2N (CH3) 2; X is C02CH3; R is OH; and Z1 and Z2 are each H.
12. 12. A composition of the formula (V): where: X represents C02R5 or CH2NHC02R6; R1 represents hydrogen or CH2S02R7; R5 represents lower alkyl; R6 represents lower alkyl or aryl; and R7 represents lower alkyl; with the proviso that when X = C02R5, R1 is not hydrogen.
13. 13. A composition of the formula (VI-1): where X is C02CH3; one H; and R6 is NHCONHC2H5.
14. 14. A composition of the formula (VI -2) where X Z1 and Z are each H.
15. 15. A method for improving the function of cholinergic, striatal, basal forehead and sensory neurons in a mammal, said method comprising administering to said mammal a therapeutic amount of the composition of the claim.
16. The method of claim 15, wherein said sensory neurons are neurons of dorsal root ganglia.
17. 17. A method for treating cellular degeneration in nerves induced by exciting amino acids, said method comprising administering to a mammal a therapeutic amount of the composition of claim 1.
18. 18. The method of claim 17, wherein said cellular generation in nerves is associated with Alzheimer's disease.
19. 19. The method of claim 17, wherein said cellular degeneration in nerves is associated with neuronal motor disease.
20. 20. The method of claim 19, wherein said neuronal motor disease is amyotrophic lateral sclerosis.
21. The method of claim 17, wherein said cellular degeneration in nerves is associated with Parkinson's disease.
22. 22. The method of claim 17, wherein said cellular degeneration in nerves is associated with cerebrovascular disease.
23. 23. The method of claim 22, wherein said cerebrovascular disease is ischemic.
24. 24. The method of claim 17, wherein said cellular degeneration in nerves is associated with AIDS dementia.
25. 25. The method of claim 17, wherein said cellular degeneration in nerves is associated with epilepsy.
26. 26. The method of claim 17, wherein said cellular degeneration in nerves is associated with concussive lesions of the brain.
27. 27. The method of claim 17, wherein said cellular degeneration in nerves is associated with concussive lesions of the spinal cord.
28. 28. The method of claim 17, wherein said cellular degeneration in nerves is associated with penetrating injuries of the brain.
29. 29. The method of claim 17, wherein said cellular degeneration in nerves is associated with penetrating injuries of the spinal cord.
30. 30. The method of claim 17, wherein said cellular degeneration in nerves is associated with Huntington's disease.
31. 31. A method for improving the function of a neuron in a mammal, wherein said neuron is selected from the group consisting of sensory, cholinergic, basal forehead and striatal neurons, said method comprising administering to said mammal a therapeutic amount of a derivative functional K-252a, said functional derivative represented by the (IV) (V) (VI) where the following substitutions are made, • 1 (1) COMPOSITE R1 R 'R II-l H H CH2N3 OH H II-2 NHCONHC6H5 H C02CH3 OH H II-3 CH2SOC2H5 H C02CHj OH H II-4 H H CH20H OCH3 H II-8 H H CON (CH3) 2 OH H II-9 »3» H H -CH2NHC02- H 11-10 Br H C02CH3 OH H 11-11 H H C0NH2 OH H 11-12 H H CH20H OH H 11-13 H H CONHC3H7 OH H II-14 < 2 > H H CH2NH-Sßr OH H 11-15 H H CH2SOCH3 OH H 11-16 H H CH-NOH OH H II-18 (2 '7> H H CH2NH-Pro OH H 11-19 H H CH-NNHC (»NH) NH: OH H 11-20 'Br Br C02CH3 OH 0 11-21 H H CONH (CH-,) -.OH OHH 11-22 H H CO, CHj OH 0 11-23 H H H OH H 11-24 H H CH-NNHCO H2 OHH 11-25 H H CH: 0C0CH3 OH H II-26'3 »H H -CH2OC (CH3); , 0- H 11-29 NHCONHC2H5 4 C02CHj OH H :? - 3rd CH-.SCTH 4 C02CH? OH 4 11-31 3r 4 CH-.0H OH t-r re - or r x x -r: x or x x re X X X x X X X X x or H 11-66 H H CH2NH2 OH H 11-67 H rt H CONHCHj OH H 11-68 H C02CH3 OH H 11-69 CH2SCH2JQl H C02CH3 OH H 11-70 CH = N-N] H C02CHj OH H 11-72 CH2S (CH2) 2NH2 H C02CH3 OH H 11-73 CH2S - ^ * NH H C02CH3 • OH H 11-79 CH2S (CH2) 2 H- H C02CH3 OH H -p-C4H9 11-80 CH2S- CH2S (CH2) 2- C02CII3 OH H (CH2) 2N (CH3) 2 N (CH3) 2 11-81 CH2SCH (CH3) 2 CH2SCH (CH3) 2 C02CIf3 OH 4 11-82 CH2S (CH2) 2CH3 CH2S (CH2) 2CH3 C02CH3 OH H 11-83 CH2S (CH2) jCH3 CH2S (CH2) 3CH3 CO: CH3 OH 4 11-34 CH, OCH, CH, OCH3 CO, CH3 OH H 11-85 CH2OC2H5 CH, 0C, Hc CO, CH3 OH H 11-86 CH, 0-t NHCONHC2H5 C02CH3 OH 4 11-87 CH- > SC-H NHCONHC2H5 C02CH3 OH H 11-88 CH3 CH3 C02CH3 OH H 11-89 CH- »SC- > He CH2S (0) C2H5 C02CH3 OH H 11-90 CH2OH CH, 0H CH2OH OH H 11-91 CH (-SCH2CH2S-) NO, C02CHj OH H 11-92 CH (-SCH2CH: S-). NHCONHC2Hs C02CH3 OH H III-l - - - H 111-2 - - - O IV-i < 4, 9 | " 4 _-. 4 IV-2 < 5 > Br H - - - H IV-3 < 6 > H H - - H rv- (3'9) H H - - H IV-5 < 10 > H H - - H IV-ói7 '::' 4 H __ - H VI-1 (12) H H C02CH3 OH H VI-2 < 1"H NH, C02CH3 OH H (1) Z1 and Z2 are both hydrogen, or both are combined to represent oxygen, where indicated. (2) The NH-amino acid link is an amide bond through the carboxyl group of the amino acid. (3) X and R are combined to form the linking group. (4) R3 is HjCH-CHj R4 is H. (5) R3 and R4 are each H. (6) R3 and R4 are each CH2CH = CH2. (7) The compound is in the hydrochloride form. (8) R3 is H and R4 is CH2CH = CH2. (9) IV-1 and IV-4 are a 1.5 to 1.0 mixture of the two components. (10) R3 = R4 = CH2CH2CH2OH. (11) R3 = CH2CH2CH2-N < ^ ~); R4 = H. (12) R8 = NHCONHC2H5. (13) R8 = NH2.
32. 32. The method of claim 31, wherein said functional derivative is compound II-3.
33. 33. The method of claim 31, wherein said functional derivative is compound 11-20.
34. 34. The method of claim 31, wherein said functional derivative is compound 11-30.
35. 35. The method of claim 31, wherein said functional derivative is compound 11-33.
36. 36. The method of claim 31, wherein said functional derivative is compound 11-38.
37. 37. The method of claim 31, wherein said functional derivative is compound 11-49.
38. 38. The method of claim 31, wherein said functional derivative is compound 11-51.
39. 39. The method of claim 31, wherein said functional derivative is compound 11-65.
40. 40. The method of claim 31, wherein said functional derivative is compound 11-69.
41. 41. The method of claim 31, wherein said functional derivative is compound 11-72.
42. 42. The method of claim 31, wherein said functional derivative is compound 11-73.
43. 43. The method of claim 31, wherein said functional derivative is compound 11-79.
44. 44. The method of claim 31, wherein said functional derivative is compound 11-80.
45. 45. The method of claim 31, wherein said functional derivative is compound VI-1.
46. 46. The method of claim 31, wherein said functional derivative is compound VI-2.
47. 47. The method of claim 31, wherein said neuron is a cholinergic neuron.
48. 48. The method of claim 31, wherein said sensory neuron is a dorsal root ganglion neuron, and said functional derivative is represented by formula (II) or (II) (III) The following substitutions are made: -r l. 2 ) COMPOUND (1) R1 X R -r Z. 11-23 H H OH H 11-24 H CH-NNHCONH, OH H 11-25 H CH2OCQCH2 OH H 11-30 CH, SC, Hc CQ2C 3 OH H 1 -32 Br "C02CH3 OH (1) R2 is hydrogen, except for compound 11-20 and compound 11-32, where R2 = Br. (2) Z1 and Z2 are both hydrogen, or both are combined to represent oxygen together, where indicated. (3) X and R are combined together to form the linking group.
49. 49. The method of claim 15, wherein said composition is administered in conjunction with a trophic factor.
50. 50. The method of claim 17, wherein said composition is administered in conjunction with a trophic factor.
51. 51. The method of claim 31, wherein said functional derivative is administered in conjunction with a trophic factor.
52. 52. The method of claim 48, wherein said functional derivative is administered in conjunction with a trophic factor.
53. 53. The method of any of claims 49-52, wherein said trophic factor is a member of the neurotrophin family.
54. 54. The method of claim 53, wherein said member of the neurotrophin family is a nerve growth factor (NGF).
55. 55. The method of claim 31, wherein said neuron is a cholinergic neuron, and said functional derivative is represented by the formula (II): where R1 and R2 are H; X is C02CH3; R is OH; and Z1 and Z2 are each H.
56. 56. The method of claim 31, wherein said neuron is a striatal neuron, and said functional derivative is represented by formula (II), (III) or (IV): (II) (III) (IV) where the following substitutions are made .2 <; ComDOund X -252a CO, CH- OH n III-l 4:? -? CH- OH (1) Z1 and Z2 are both hydrogen, or both are combined to represent oxygen together, where indicated. (2) R3 is CH2-CH- = CH2; R4 is H.
57. 57. The method of claim 31, wherein said neuron is a basal neuron of the forehead, and said functional derivative is represented by formula II: where the following substitutions are made AND II-J CH: S0C: H5 H CO CH3 OH H ll-} H II CONHC, HJ OH \\ 11-10 Br H CO: CH3 OH H 11-20 Br Ur CO, CH3 OH or ?? - :? HH CONW (CH), OH OH H ii- :: H II C?, CH3 OH 0 11-30 CH; SC-, Hj HC?, CH3 011 H 11-31 Or Or C ?: CH3 OH H ll- JI CH-.Yes.- Hj CH -SC, II j CO: CH3 OH H ?? - 6: H to CO-.n-ne-rvi OH H 11-63 OH HC? CHj OH H 11-64 O n-propyl H CO, CHj OH H 11-65 CH-.SCH.CH H C?: CH 3 OH H (1) Z1 and Z2 are both hydrogen, or both are combined to represent oxygen together, where indicated.
58. 58. The method of any of claims 45, 55 or 56, wherein said method is used in the treatment of Iluntington's disease.
59. 59. A composition of the formula (11-51): 11-51 where R1 and R2 are CH2SC2H5; X is C02CH3; R is OH; and Z1 and Z2 are H.
60. 60. A composition of the formula (11-48): 11-48 where R1 is CH2N (CH3) 2; X is C02CH3; R is OH; and R2, Z1 and Z2 are II.
61. 61. A composition of the formula (11-50): 11-50 where R1 is CH2S- < ? >; X is C02CH3; R is OH; and R2, Z1 and Z2 are H.
62. 62 A composition of the formula (11-52) 11-52 where R1 is CH = NNH- < J; X is C02CH3; R is OH; and R2, Z1 and Z2 are H. H
63. 63. A composition of the formula (11-53): 11-53 where R1 is CH2S- (-5> X is C02CH3; R is OH; and R2, Z1 and Z2 are H. ti ~
64. 64. A composition of the formula (11-54): 11-54 where R1 is CH2S (0) - ^ "^; X is C02CH3; R is OH; and R2, Z1 and Z2 are H.
65. 65. A composition of the formula (11-55) 1 where R1 is CH2S (0) - (> X is C02CH3; R is OH; and R2, Z1 and Z2 are 'w H.
66. 66. A composition of the formula (11-58) '11-58 where R1 is Br; X is CONH2; R is OH; and R2, Z1 and Z2 are H.
67. 67. A composition of the formula (IV-6): IV-6 where R1, R2, Z1 and Z2 are H; and R3 is CH2CH2CH2-N or.