PROTOBERBERINE DERIVATIVES WHICH INHIBIT ACTIVITY OF THE MITOGEN-ACTIVATED PROTEIN KINASE

Abstract: The present invention relates to protoberberine derivatives which inhibit the activity of mitogen-activated protein kinase. More specifically, the present invention is directed to protoberberine derivatives which inhibit the activity of Wis1 and Spc1 kinase in intracellular signal transduction cascade wherein mitogen-activated protein kinase (MAPK) is involved.

Description: PROTOBERBERINE DERIVATIVES WHICH INHIBIT ACTIVITY OF THE MITOGEN-ACTIVATED PROTEIN KINASE Technical field The present invention relates to protoberberine derivatives which inhibit the activity of mitogen- activated protein kinase. More specifically, the present invention is directed to protoberberine derivatives which inhibit the activity of Wisl and Spcl kinase in the intracellular mitogen-activated protein kinase (MAPK) signal transduction cascade. Background art The intracellular signal transduction system regulates cell growth and differentiation crucial for maintenance of life. Particularly, signal transduction systems transduce extracellular stimuli including growth factors, cytokines or hormones to the cell through kinase cascade in which activated upstream kinase by mitogens in turn activate downstream kinases . The signal transduction system transduce extracellular signals ultimately to the nucleus through intracellular intermediate messengers and thereby activate transcription regulatory factors and other target proteins, which leads to cell growth and differentiation. This mechanism is maintained in various organisms ranging from yeasts to mammalian cells [Davis, R.J., et al., TIBS , 19, 470 (1994)]. It has been known that there are three types of mitogen-activated protein kinase signaling pathways that recognize environmental change and then transmit the signal into the cell, thereby maintainig cell shape in the fission yeast Schizosaccharomyces pombe . In S. pombe , various mutants in mitosis and signal transduction are accumulated and molecular genetic approach is available. The signal is transmitted through the pathways wherein mitogen-activated protein kinase kinase kinase (MAPKKK) , mitogen-activated protein kinase kinase (MAPKK) and mitogen-activated protein kinase (MAPK) are activated sequentially [Nemin,A., TIGS, 9, 390(1993) : Marshall, C. J. , Cell, 80, 179(1995)]. There is a ras signaling pathway by which cells responds to nutrient exhaustion in media and to mating pheromone and thus transmit the signal to the cytosol, thereby inducing cell shape change. Another pathway is a mitogen-activated protein kinase signaling pathway by which extracellular stress including osmotic shock is transmitted to the cytosol employing Wisl and Spcl kinase. The other is Spml mitogen-activated protein kinase signaling pathway which responds to extracellular stimuli such as osmotic shock, and thereby regulates cell wall regeneration and cytokinesis. The malfunction of genes in mitogen-activated protein kinase singal transduction pathways has no effect on cell growth in normal condition. However, the change in environmental conditions cause inhibition of cell growth, excess mitosis, or cell shape change, since cell can not respond to environmental changes. Protoberberine derivatives of the present invention are synthesized from berberine compound, and exhibit lethal or growth inhibition activity against fungi such as Aspergillus or Candida including cutaneous filamentous fungi. Protoberberine derivatives act on the transition step of lanosterol to ergosterol by 7- reduction enzyme, exhibiting antifungal activity by inhibiting enzyme involved in the synthesis of chitin, major component of the cell wall, as well as having effect on formation of the cell membrane. However, the mechanism by which these antifungal agents inhibit physiological activity, has not yet been known. The object of the present invention is to provide the usage of protoberberine derivatives as an inhibitor of specific mitogen-activated protein kinase (MAPK) , more specifically, an inhibitor of specific Wisl and Spcl kinase in the MAPK singal transduction pathway in Saccharomyces pombe, since the mechanisms by which environmental changes are recognized, extracellular stimuli are transmitted to the cell, as well as the cell shape is maintained, are well understood in S . pombe Disclosure of invention Protoberberine derivatives of the present invention have the following chemical formula (I) (I) wherein R^R 2 and R 3 may be the same or different, and represent C 1 -C 5 alkoxy, R 4 represents hydrogen or a group having the following chemical formula (II) and A " represents inorganic acid ion, organic acid ion or halide: (II) wherein Z 1 , Z 2 , Z 3 , Z 4 and Z 5 may be the same or different, independently of one another represent hydrogen, halogen, C1-C5 alkyl, trifluoromethyl, phenyl, substituted phenyl, nitro, C 1 -C 4 alkoxy, trifluoromethoxy, hydroxy, t-butyl dimethylsilyloxy, phenoxy, vinyl or methoxycarboxyl groups . Among the compounds having the above chemical formula (I), the compound wherein R 1 and R 2 are ethylenedioxy (-O-CH 2 -O-) , R 3 is methoxy, and R 4 is 3,4- dimethylbenzyl; the compound wherein R 1 and R 2 are methylenedioxy (-O-CH 2 -O-) , R 3 is methoxy, and R 4 is 4-(t- butyl) benzyl; the compound wherein R 1 , R 2 and R 3 are methoxy, and R 4 is 4-isopropylbenzyl; the compound wherein R 1 and R 2 are methylenedioxy (-0-CH 2 -0-) , R 3 is methoxy, and R 4 is 4- ( -butyldimethylsilyloxy) benzyl; the compound wherein R 1 , R 2 and R 3 are methoxy, and R 4 is 4-(t-butyl dimethylsilyloxy) benzyl; the compound wherein R 1 , R 2 and R 3 are methoxy, and R 4 is 4-phenylbenzyl; or the compound wherein R 1 , R 2 and R 3 are propoxy, and R 4 is hydrogen, are more desirable for the inhibitor of mitogen-activated protein kinase in the present invention. Protoberberine derivatives which inhibit mitogen- activated protein kinase of the present invention, 13- benzylberberine iodide, 13- (4-chlorobenzyl) berberine iodide, 13- (3-bromobenzyl) berberine iodide, 13- (2,3- dichlorobenzyl) berberine chloride, 13- (2-chloro-6-fluoro benzyl) berberine chloride, 13- (4-fluoro-2-trifluoro methylbenzyl) berberine chloride, 13- (2,3, 4, 5, 6- pentafluoro benzyl) berberine chloride, 13- (2, 3,5,6- tetrafluoro-4-tri fluoromethylbenzyl) berberine chloride, 13- (4-methylbenzyl) berberine chloride, 13- (2,4- dimethylbenzyl) berberine chloride, 13- (4-methyl-3- nitrobenzyl) berberine chloride, 13- (trifluoromethylbenzyl) berberine chloride, 13- (4- methoxybenzyl) berberine chloride, 13- (4-trifluoromethoxy benzyl) berberine chloride, 13- (4-methoxycarbonylbenzyl) berberine chloride, 13- (4-t-butylbenzyl) berberine chloride, 13- (4-i-propylbenzyl) berberine chloride, 13- (4-(t-butyl dimethylsilyloxy) benzyl) berberine chloride, 13- (4-t-butylbenzyl)palmatine iodide, 13- (4- (t- butyldimethyl silyloxy) benzyl) palmatine chloride, 2, 3, 9, 10-tetrapropoxy protoberberine iodide, which is described in detail in the specification of the Korean patent application No. 258,849 and U.S. patent No. 6,008,356. In the present invention, Shizosaccharomyces pombe was cultured and minimum concentrations of protoberberines for its inhibition, was determined. Then, the knock-out mutants in three MAP kinase cascades described above were treated with protoberberine derivatives and their effects on the growth of each mutant were measured. The results show that protoberberine derivatives have an effect on knock-out mutant of wisl and spcl . Also, it was studied whether protoberberine derivatives act on whole transduction pathway wherein Wisl MAPKK and Spcl MAPK are involved or only on Wisl MAPKK and Spcl MAPK. From the result that protoberberine derivatives do not act on knock-out mutants of the genes for MAPKKK that function upstream of wisl and spcl , winl and wis4 r it was found that these protoberberine derivatives selectively inhibit the activity of Wisl and Spcl kinase. Brief description of the drawings Fig.l illustrates three signal transduction pathways wherein mitogen-activated protein kinases are involved in Schizosaccharomyces pombe in the present invention. Fig.2 represents the photograph of electroporesis showing inhibition of the activity of Wisl kinase by protoberberine derivatives of the present invention. Fig.3 represents the photograph of electroporesis showing inhibition of the activity of Spcl kinase by protoberberine derivatives of the present invention. Best mode for carrying out the invention The usage of protoberberine derivatives as the inhibitor of MAPK in the present invention will be in more detail illustrated by the following examples and attached figures. But it should be understood that the present invention is not limited to these examples in any manner. Example 1 Measurement of minimum inhibitory concentration (MIC) of protoberberine derivatives on Schizosaccharomyces pombe KGY246 cells, wild type Saccharomyces pombe, were cultured in rich media YE Aϋ (yeast extract, peptone, malt extract, dextrose, agar: pH 6.2) containing 0.75mg/L adenine and uracil to exponential phase. To determine minimum inhibitory concentration of each protoproberberine derivative, cells in exponential phase were transferred to YE A0 medium containing variable concentrations of protoberberine derivatives and cell number was measured every 3 hour. Thereafter, the minimal concentration on which cell number was maintained constantly was determined as minimum inhibitory concentration. The minimum inhibitory concentrations of protoberberine derivatives 13-(4-t- butylbenzyl) berberine chloride, 13- (4- (t- butyldimetylsilyloxy) benzyl) berberine chloride and 2, 3, 9, 10-tetrapropoxyprotoberberine iodide of the present invention were 20ug/mL, 7ug/mL and 20ug/mL respectively. To minimize inhibition by solvent itself, 10% ethanol was used as a solvent of protoberberine derivatives. The processes -of cell culture and measurement of cell number described above were repeated using 13-benzylberberine iodide, 13- (4- chlorobenzyl) berberine iodide, 13- (3- bromobenzyl) berberine iodide, 13- (2, 3-dichlorobenzyl) berberine chloride, 13- (2--chloro-6- fluorobenzyl) berberine chloride, 13- (4-fluoro-2- trifluoromethylbenzyl) berberine chloride, 13- (2, 3, 4, 5, 6- pentafluorobenzyl) berberine chloride, 13- (2, 3,5,6- tetrafluoro-4-trifluoromethylbenzyl) berberine chloride, 13- (4-methylbenzyl) berberine chloride, 13- (2,4- di ethylbenzyl) berberine chloride, 13- (4-methyl-3- nitrobenzyl) berberine chloride, 13- (3-trifluoromethyl benzyl) erberine chloride, 13- (4-methoxybenzyl) berberine chloride, 13- (4-trifluoromethoxybenzyl) berberine chloride, 13- (4-methoxycarbonylbenzyl) berberine chloride, 13- (4-i-propylbenzyl) berberine chloride. These compounds also had the minimum concentration of inhibition at the concentration of 18-22 ug/mL. Example 2 Effects of protoberberine derivatives on various knock- out mutants of Schizosaccharomyces pombe To investigate the effect of protoberberine derivatives on the growth of cells, the cells of each strain were cultured in rich medium YE Aϋ to exponential phase (5X10 7 cells/mL) . These cells were transferred to YE A0 medium and cultured for 12 hours. Then cell number was measured every 3 hours. Specificity of protoberberine derivatives on cell growth was proved through comparison with the result obtained by treatment with 3ug/mL miconazole of which inhibiting effect of yeast cell growth has been known. The strains of Schizosaccharomyces pombe used in the present invention are indicated in table 1. Table 1 The growth of wild type Schizosaccharomyces pombe was inhibited by protoberberine derivatives as indicated in table 2. The knock-out mutants in the MAPK pathways were treated with protoberberine derivatives of the present invention and the results were indicated in tables in below. As indicated in table 5, the knock-out mutant of wisl encoding mitogen-activated protein kinase kinase (MAPKK) was not inhibited by 13- (4-t-butylbenzyl) berberine chloride, 13- (4- ( -butyldimethylsilyloxy) benzyl) berberine chloride and 2,3,9,10- tetrapropoxyprotoberberine iodide. As indicated in table 6, the knock-out mutant of spcl encoding Spcl mitogen- activated protein kinase (MAPK) which is phosphorylated and thereby activated by Wisl mitogen-activated protein kinase kinase (MAPKK) was not inhibited at all by 13- (4- (t-butyldimethylsilyloxy) benzyl) berberine chloride and partially inhibited by 13- (4-t-butylbenzyl) berberine chlroride whereas completely inhibited by 2,3,9,10- tetrapropoxy protoberberineiodide . That is, 13- (4-t- butylbenzyl) berberine chloride, 13- (4- (t- butyldimethylsilyloxy) benzyl) berberine chloride and 2, 3, 9, 10-tetrapropoxyprotoberberine iodide inhibit the activity of Wisl kinase and 2, 3, 9, 10-tetrapropoxy protoberberine iodide inhibit the activity of Spcl kinase. The effects of each compound 13-benzylberberine iodide, 13- (4-chlorobenzyl) berberine iodide, 13- (3- bromobenzyl) berberine iodide, 13- (2, 3-dichlorobenzyl) berberine chloride, 13- (2-chloro-6- fluorobenzyl) berberine chloride, 13- (4-fluoro-2- trifluoro methylbenzyl) berberine chloride, 13- (2, 3, 4, 5, 6-pentafluoro benzyl) berberine chloride, 13- (2,3,5, 6-tetrafluoro-4-trifluoromethylbenzyl) berberine chloride, 13- (4-methylbenzyl) berberine chloride, 13- (2, 4-dimethylbenzyl) berberine chloride, 13- (4-meth " yl-3- nitrobenzyl) berberine chloride, 13- (trifluoromethylbenzyl) berberine chloride, 13- (4- methoxybenzyl) berberine chloride, 13- (4-trifluoro methoxybenzyl) berberine chloride, 13- (4-methoxycarbonyl benzyl) berberine chloride, 13-(4-i- propylbenzyl) berberine chloride on each knock-out. mutant were measured. As a result, these compounds also inhibited the activity of Wisl kinase. Table 2 Effects of a protoberberine derivative 13- (4- (t- butyldimethylsilyloxy) benzyl) berberine chloride on several kinase mutants. Table 3 Effects of protoberberine derivatives on the knock-out mutant of byr2 in Ras-Byr2 pathway Table 4 Effects of protoberberine derivatives on the knock-out mutant of spml in Mhkl-Spml pathway Table 5 Effects of protoberberine derivatives on the knock-out mutant of wisl Table 6 Effects of protoberberine derivatives on the knock-out mutant of spcl Example 3 Effects of protoberberine derivatives on the knock-out mutant of winl and wis4 The knock-out mutant of winl and wis4 was cultured and treated with 13- (4-t-butylbenzyl) berberine chloride, 13- (4- ( -butyldimethylsilyloxy) benzyl) berberine chloride or 2, 3, 9, 10-tetrapropoxyprotoberberine iodide of the compounds 13-benzylberberine iodide, 13-(4-chloro benzyl) berberine iodide, 13- (3-bromobenzyl) berberine iodide, 13- (2, 3-dichlorobenzyl) berberine chloride, 13- (2-chloro-6-fluorobenzyl) berberine chloride, 13- (4- fluoro-2-trifluoromethylbenzyl) berberine chloride, 13- (2, 3, 4, 5, 6-pentafluorobenzyl) berberine chloride, 13- (2,3,5, 6-tetra fluoro-4-trifluoromethylbenzyl) berberine chloride, 13- (4-methylbenzyl) berberine chloride, 13- (2, 4-dimethylbenzyl) berberine chloride, 13- ( 4-methyl-3- nitrobenzyl) berberine chloride, 13- (3- trifluoromethylbenzyl) berberine chloride, 13- (4- methoxybenzyl) berberine chloride, 13- (4-trifluoro methoxybenzyl) berberine chloride, 13- (4-methoxycarbonyl benzyl) berberine chloride, 13- (4-t-butylbenzyl) berberine chloride, 13- (4-i-propylbenzyl) berberine chloride, 13- (4- (t-butyldimethylsilyloxy) benzyl) berberine chloride, 13- (4-t-butylbenzyl) palmatine iodide, ' 13- (4- (t- butyldimethyl silyloxy) benzyl) palmatine chloride and 2, 3, 9, 10-tetra propoxyprotoberberine iodide. As indicated in table 7 and 8, the knock-out mutant of winl and wis4 encoding the two upstream kinases which phosphorylate wisl and spcl were completely inhibited by protoberberine derivatives as in wild type, which indicates that these protoberberine derivatives have effect on specific Wisl and Spcl kinases and on the not whole Wisl-Spcl mitogen-activated protein kinase signal transduction pathway. Table 7 Effect of protoberberine derivatives on the knock-out mutant of winl Table 8 Effect of protoberberine derivatives on the knock-out mutant of wis4 Example 4 Induction of expression and purification of Wisl and Spcl kinases The genes wisl and spcl were cloned into the pRepl vector containing nmtl promoter of which expression was inhibited by thiamin in the medium and able to tag GST. The KGY246 cells, wild type Schizosaccharomyces pombe cells were transformed with this plasmid. The expression of Wisl and Spcl kinase tagged with GST was induced by the cultivation of the transformed cells in MM Aϋ medium containing 0.4mM thiamin followed by cultivation to exponential phase (5X10 6 cells) for about 20 hours in lOOmL of MM AU medium in which thiamin was withdrawn. After osmotic shock (0.6M KC1) and oxidative stress were given to the cells expressing Wisl and Spcl tagged with GST for 25 minutes, cells were recovered at 4°C, washed with ice-cold PBS/NaF (50mM) /NaN 3 (ImM) buffer and HB buffer (25mM HEPES,pH 7.5, 60mM β -qlycerophosphate, 15mM EGTA, o.l mM Na vanadate, 0.1% triton and hydrolyzed using glass bead and bead beater purchased from Sigma. The supernatant of hydrolyzed cells was incubated with lOOmM NaCl for salting-out to remove unnecessary proteins. After the supernatant was incubated with 0.045g/ml GST beads for 1.5 hours, proteins not bound to GST beads were removed. Purified Wisl and Spcl kinases were separated on 7% SDS-PAGE and the degree of purification was confirmed by western blotting using GST antibody. Example 5 The activity of Wisl and Spcl kinase and the effect of protoberberine derivatives on kinase activity Before Wisl and Spcl kinases purified in example 4 were analyzed, they were pre-incubated in analyzing buffer of kinase (50mM Tris-Cl pH7.4, 20mM MgCl 2 , 5mM EGTA, 2mM Na vanadate, ImM DTT) . The kinase reaction was performed at 30 ° C for 30 minutes in the presence of lmg/ml myelin basic protein (MBP) , ImM ATP and 20uCi of [γ- 32p]ATP and then terminated by adding SDS sample buffer and heating at 90 ° C for 5 minutes. The reaction mixtures were separated on SDS-polyacrylamide gel electrophoresis. After fixation with 10% acetic acid : 10% methanol and drying, the gel was exposed on X-ray film. When Wisl is activated by stimulus, it also activates autophosphorylation. Therefore, the degree of Wisl autophosphorylation was also measured. As represented in Fig 2, Wisl, mitogen-activated protein kinase (MAPK) kinase is directly inhibited by 13- (4- ( t-butyldimethylsilyloxy) benzylberberine chloride and 13- (4-t-butylbenzyl) berberine chloride. In additon, the processes of incubation, kinase reaction and measurement of the degree of effect on autophosphorylation were repeated using 13-benzylberberine iodide, 13- (4- chlorobenzyl) erberine iodide, 13- (3- bromobenzyl) berberine iodide, 13-(2,3-di chlorobenzyl) berberine chloride, 13- (2-chloro-6-fluoro benzyl) berberine chloride, 13- (4-fluoro-2- trifluoromethyl benzyl) berberine chloride, 13- (2, 3, 4, 5, 6-pentafluorobenzyl berberine chloride, 13- (2, 3, 5, 6-tetrafluoro-4-trifluoro methylbenzyl)berberine chloride, 13- (4-methylbenzyl) berberine chloride, 13- (2, 4-dimethylbenzyl) berberine chloride, 13- (4-methyl-3- nitrobenzyl) berberine chloride, 13- (3- trifluoromethylbenzyl) berberine chloride, 13- (4- methoxybenzyl) berberine chloride, 13- (4-trifluoromethoxy benzyl) berberine chloride, 13- (4-methoxycarbonylbenzyl) berberine chloride, 13- (4-i-propylbenzyl) berberine chloride. The activity of Wisl kinase was directly inhibited by these compounds. As represented in Fig 3, the Spcl, mitogen-activated protein kinase, was only directly inhibited by 2, 3, 9, 10-tetrapropoxy protoberberine iodide when myelin basic protein was used as a substrate. Industrial applicability The present invention provides protoberberine derivatives which inhibit specifically the activity of Wisl and Spcl kinase in the mitogen-activated protein kinase signal transduction pathway, and protoberberine derivatives of the present invention can be used as a reagent for the study of intracellular signal transduction pathways. Also, it can be used for the development of pharmaceutical product which regulates the activity of mitogen-activated protein kinase such as an inhibitor of SAPK(stress activated protein kinase) /JNK(c-Jun N-terminal kinase) kinase or anticancer drug, since the mitogen-activated protein kinase signal transduction system in Schizosaccharomyces pombe is highly homologous to the SAPK/JNK signaling pathway which transmits environmental stimuli such as cytokines or DNA damages in mammalian cells.

Claims: What is claimed is: 1. An inhibitor for mitogen-activated protein kinase, of protoberberine derivative having the following chemical formula (I) (I) wherein R X ,R 2 and R 3 may be the same or different, and represent C 1 -C 5 alkoxy, R 4 represents hydrogen or a group having the following chemical formula (II), and A " represents inorganic acid ion, organic acid ion or halide: (ID wherein Z X ,Z 2 ,Z 3 ,Z 4 and Z 5 may be the same or different, independently of one another represent hydrogen, halogen, C1-C5 alkyl, trifluoromethyl, phenyl, substituted phenyl, nitro, Cι-C alkoxy, trifluoromethoxy, hydroxy, t- butyldimethylsilyloxy, phenoxy, vinyl or methoxycarboxyl groups. 2. The inhibitor for mitogen-activated protein kinase, protoberberine derivative according to claim 1, wherein R 1 -R 2 is methylenedioxy (-0-CH 2 -0-) , R 3 is methoxy, R 4 is 4- ( -butyl) benzyl and A ~ is chloride. 3. The inhibitor for mitogen-activated protein kinase, of protoberberine derivative according to claim 1, wherein R 1 -R 2 is methylenedioxy (-0-CH 2 -0-) , R 3 is methoxy, R 4 is 4- ( -butyldimethylsilyloxy) benzyl and A " is chloride . 4. The inhibitor for mitogen-activated protein kinase, of protoberberine derivative according to claim 1, wherein R 1 -R 3 is propoxy, R 4 is hydrogen and A " is iodide.

Inventors: JANG MYOUNG-JIN, JWA MI-RI, KIM JUNG-HO, PAIK YOUNG-KI, SONG KI-WON, JANG, MYOUNG-JIN, JWA, MI-RI, KIM, JUNG-HO, PAIK, YOUNG-KI, SONG, KI-WON
Applicants: HANWHA CHEMICAL CORP, JANG MYOUNG JIN, JWA MI RI, KIM JUNG HO, PAIK YOUNG KI, SONG KI WON, HANWHA CHEMICAL CORPORATION, JANG, MYOUNG-JIN, JWA, MI-RI, KIM, JUNG-HO, PAIK, YOUNG-KI, SONG, KI-WON
Application year: 2001
Main classification: A61K31
IPC codes: A61K31, A61P31, A61P35, A61P43, C07D455