HRP940452A2 - Derivatives, substituted for the methylamino nitrogen - Google Patents

Description

The proposed invention relates to staurosporine derivatives substituted on the methylamine nitrogen, especially to those of the general formula

[Stau]-N(CH3)-R (I)

where [Stau] represents the rest of the partial formula

[image]

and R represents hydrocarbyl R° or acyl Ac, which preferably contain at most 30 carbon atoms, as well as on salts of compounds of formula I which can form salts.

The invention also relates to the process for preparing the compounds defined above, to pharmaceutical preparations containing these compounds, and to their preparation, further to the medical use of these compounds and preparations, as well as to the appropriate therapeutic method.

Staurosporin of the formula [Stau]-NH-CH3 (II) (meaning of the residue [Stau] see above), as a basic compound in the sense of the invention, was isolated as early as 1977 from the cultures of Strptotomyces staurosporeus AWAYA, TAKAHASHI and OMURA, sp. new. AM 2282, prim. Omura, S.; Iwai, Y.; Hirano, A.; Nakagawa, A.; Awaya, J.; Tsuchiya, H.; Takahashi, Y. and Masuma, R.. J. Antibiot. 30, 275-281 (1977), and tested for antimicrobial effectiveness. At the same time, they also determined that the compound is effective against microorganisms from yeast species and fungi (MIC approximately 3 to 25 mcg/ml), while as a hydrochloride it has an LD50 of 6.6 mg/kg (mouse, intraperitoneally). An extensive search has recently shown that the compound has a strong inhibitory effect on protein kinase C (from rat brain) (cf. Tamaoki, T.; Nomoto, H.; Takahashi, I.; Kato, Y.; Morimoto, M. and Tomita, F.. Biochem. in Biophys. Research Commun. 135 (No. 2), 397-402 (1986).

Protein kinase, dependent on phospholipids and calcium, appears in cells in several forms, and participates in its various basic processes, such as signal transmission, proliferation and differentiation, as well as the release of hormones and neurotransmitters. Activation of these enzymes takes place, as is known, either by hydrolysis of phospholipids of the cell membrane, through receptors, or by direct interaction with certain active substances, which promote tumors. The sensitivity of the cell against receptor-mediated signal transduction can be influenced by changing the activity of protein kinase C (as a signal transmitter). Compounds, which can selectively change the activity of protein kinase C, can be used as active substances that suppress tumors and inflammation, modulate immunity, because they act antibacterially, and are very interesting as agents against atherosclerosis and diseases of the cardiovascular system and central nervous system.

In fact, staurosporine has a strong inhibitory effect on proteokinase C (see above), so it also strongly inhibits other protein kinases and therefore lacks the selectivity required for therapeutic use. Surprisingly, it has now been shown that the removal of the hydrogen in the methylamino group of staurosporine by substitution causes such N-substituted derivatives to retain the selective inhibitory activity of staurosporine against protein kinase C, but are significantly less effective against other peotein kinases. Due to this significantly increased selectivity, the compounds according to the invention also exhibit the aforementioned suitability for therapeutic use in the above-mentioned indication areas, primarily for influencing cell proliferation.

To determine the inhibitory effect on protein kinase C, protein kinase C is first isolated from the brain of guinea pigs and purified in the manner described in T. Uchida and C.R. Filburn in J. Biol. Chem. 259, 12311-4 (1984). Determining the inhibitory effect of compounds of formula I on protein kinase is done based on the methodology of D. Fabro et al. Arch. Biochem. Biophys. 239, 102-111 (1985). Significant inhibition of protein kinase C occurred above a concentration of approximately 0.01 µ/l.

In accordance with this, the compounds of formula I and their pharmaceutically usable salts can be used, for example, as drugs, especially for suppressing tumors and inflammation, modeling immunization, against bacteria, and also as agents against arteriosclerosis, diseases of the cardiovascular system and the central nervous system. A further object of the proposed invention is the use of the compounds in the sense of the invention for the preparation of medicines, for example for the above-mentioned use, for therapeutic and prophylactic treatment of the human and also the animal body. This also includes the commercial formulation of active substances.

Hydrocarbyl (hydrocarbon residue) R° is an acyclic (aliphatic), carbocyclic or carbocyclic-acyclic hydrocarbon residue, which has a total of preferably no more than 30, especially no more than 18 carbon atoms, and can be saturated or unsaturated, unsubstituted or substituted. It can also contain two or more carbon atoms instead of one, as well as the same or different heteroatoms, such as oxygen, sulfur and nitrogen, in the acyclic and/or cyclic part; in the last example it is designated as a heterocyclic residue (heterocyclyl residue) or as a heterocyclic-acyclic residue.

Unsaturated residues are those that contain one or more, especially conjugated and/or isolated multiple bonds (double and/or triple bonds). The term cyclic residues also includes aromatic residues, for example, those where at least one six-membered carbocyclic or five-membered to eight-membered heterocyclic ring contains the maximum number of non-cumulative double bonds. Carbocyclic residues, where at least one ring occurs as a six-membered, aromatic ring (ie, a benzene ring), are designated as aryl residues.

Unless otherwise stated, the organic residues in the proposed representation marked with "lower" contain no more than 7, preferably no more than 4 carbon atoms.

An acyclic, unsubstituted hydrocarbon residue is particularly a straight or branched lower alkyl, lower alkenyl, lower alkadienyl or lower alkynyl residue. Lower alkyl is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, furthermore also n-pentyl, isopentyl, n-hexyl, isohexyl and n-heptyl; lower alkenyl is, for example, allyl, propenyl, isopropenyl, 2- or 3-methallyl and 2- or 3-butenyl; lower alkadienyl is eg 1-penta-2,4-dienyl; lower alkynyl is, for example, propargyl or 2-butynyl. In the corresponding unsaturated residues, the double bond is localized towards the free valence, especially in a higher position than the α-position.

The carbocyclic hydrocarbon radical is in particular a mono-, bi- or polycyclic cycloalkyl, cycloalkenyl or cycloalkadienyl radical, or a corresponding aryl radical. Primarily, these are residues with a maximum of 14, especially 12, carbon atoms in the ring and 3- to 8-, preferably 5- to 7-, primarily six-membered rings, whereby they can carry one or more, e.g. two acyclic residues, e.g. mentioned above, and especially lower alkyl residues, or further carbocyclic residues. Carbocyclic-acyclic residues are those, where the acyclic residue, especially the one with no more than 7, preferably no more than 4 carbon atoms, and as above all methyl, ethyl and vinyl, carry one or more carbocyclic, in the given case aromatic residues of the above definition. In particular, cycloalkyl-lower alkyl and aryl-lower alkyl residues can be mentioned, as well as their ring and/or chain unsaturated analogues, which bear a ring at the end C-atom of the chain.

Cycloalkyl has primarily 3 up to and including 10 carbon atoms and is, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, as well as bicyclo-(2,2,2)-octyl, 2-bicyclo-(2 , 2, 1) -heptyl) and adamantyl, which can also be substituted with one, two or more, for example, lower alkyl residues, especially methyl residues; cycloalkenyl is, for example, one of the already mentioned monocyclic cycloalkyl residues, which carries a double bond in position 1, 2 or 3. Cycloalkyl-lower-alkyl or -lower-alkenyl is, for example, -methyl, -1- or -2-ethyl, - 1- or -2-vinyl, -1-, -2- or -3-propyl, i.e. -allyl substituted with one of the above-mentioned cycloalkyl residues, with preference being given to those substituted at the end of the linear chain.

The aryl residue is primarily phenyl, further naphthyl, such as 1- or 2-naphthyl, biphenyl, such as 4-biphenylyl, further also anthryl, fluorophenyl and azulenyl, as well as their aromatic analogues with one or more saturated rings. These are primarily aryl-lower-alkyl and lower-alkenyl residues, for example phenyl-lower-alkyl or phenyl-lower-alkenyl with a terminal phenyl residue, such as for example benzyl, phenethyl, 1-. 2- or 3-phenylpropyl, diphenylmethyl (benzhydryl), trityl and cinnamyl, furthermore also 1- or 2-naphthylmethyl. Under aryl radicals, which bear acyclic radicals as lower alkyl, mention may be made in particular of o-, m- and p-tolyl and xylyl radicals with differently substituted methyl radicals.

Heterocyclic residues, including heterocyclic-acyclic residues, are particularly monocyclic, and also bi or polycyclic, aza-, thia-, oxa-, thiaza-, oxaza-, diaza-, triaza- or tetraazacyclic residues of aromatic character, as well as corresponding partially or primarily fully saturated heterocyclic residues of that type, wherein residues of that type, in a given case, e.g. as the carbocyclic or aryl residues mentioned above, may carry further acyclic, carbocyclic or heterocyclic residues, and/or they may be mono-, di- or polysubstituted with functional groups. The acyclic part in heterocyclic-acyclic residues has, for example, the meaning given for the corresponding carbocyclic-acyclic residues. If the heterocyclyl is found as a direct substituent R° on the methylamino group of staurosporine, its free valence must come from one of its C-atoms. First of all, these are unsubstituted or substituted monocyclic residues with a nitrogen, oxygen or sulfur atom, such as 2-aziridinyl, and especially aromatic residues of this type such as pyryl, e.g. 2-pyryl or 3-pyryl, pyridyl, e.g. 2- , 3- or 4-pyridyl, further thienyl, eg 2- or 3-thienyl, or furyl, eg 2-furyl; analogous bicyclic residues with a nitrogen, oxygen or sulfur atom are, for example, indolyl, as 2- or 3-indolyl, quinolyl as 2- or 4-quinolyl, isoquinolyl as 3- or 5-isoquinolyl, benzofuryl as 2-benzofuryl, chromenyl as 3 -chromenyl or benzothienyl as 2- or 3-benzothienyl; these are primarily monocyclic or bicyclic residues with more heteroatoms, e.g. imidazolyl as 2-imidazolyl, pyrimidinyl as 2- or 4-pyrimidinyl, oxazolyl as 2-oxazolyl, isoxazolyl as 3-isoxazolyl, or thiazolyl as 2-thiazolyl, or benzimidazolyl as 2-benzimidazolyl, benzoxazolyl as 2-benzoxazolyl, or quinazolyl as 2-quinazolyl. Suitable partially or fully saturated analog residues, such as 2-tetrahydrofuryl, 2- or 3-pyrrolidyl, 2-, or 3- or 4-piperidyl, and then also 2- or 3-morpholinyl, 2- or 3-thiomorpholinyl, 2-piperazinyl and N,N'-bis-lower-alkyl-2piperazinyl residues. These residues may also carry one or more acyclic, carbocyclic or heterocyclic residues, especially those listed above. Heterocyclic-acyclic residues are particularly derived from acyclic residues with a maximum of 7, preferably a maximum of 4 carbon atoms, e.g. between those listed above, and may carry one, two or more heterocyclic residues, e.g. those listed above, whereby the ring may be connected to a chain also via one of its nitrogen atoms.

As we have already mentioned, hydrocarbonyl (including heterocyclyl) can be substituted with one, two or more identical or different substituents (functional groups); the following substituents come into consideration in particular: free, etherified and esterified hydroxyl groups; mercapto as also lower alkylthio and optionally substituted phenylthio groups; halogen atoms such as chlorine and fluorine, and also bromine and iodine; oxo groups, which are in the form of formyl (ie aldehyde) and keto groups, and also as corresponding acetals or ketals; azido and nitro groups; primary, secondary and especially tertiary amino groups, with the usual protective groups of primary or secondary amino groups, acylamino groups and diacylamino groups, as well as, in a given case, functionally changed sulfo groups such as sulfoamoyl, or sulfo groups that occur in the form of salts. However, all these functional groups must not be located on the C-atom from which the free valency emerges, and preferably they are separated from it by two or more carbon atoms. The hydrocarbyl residue can also carry free and functionally converted carboxyl groups, as esterified carboxyl groups that appear in the form of salts, in a given case carbamoyl, ureido or guanidine groups, which carry, in a given case, one or two carbon atoms, and cyano groups.

The etherified hydroxyl group, which appears as a substituent in the hydrocarbyl, is, for example, a lower alkoxy group, such as a methoxy, ethoxy, propoxy, isopropoxy, butoxy and tert.butoxy group, which may also be substituted. Thus, it can be an alkoxy group substituted with halogen atoms, e.g. singly, doubly or multiply, especially in position 2, such as in a 2,2,2-trichloroethoxy, 2-chloroethoxy or 2-iodethoxy residue, or with a hydroxy, or lower alkoxy residues, in this case preferably single, especially in position 2, as in the 2-methoxyethoxy residue. A form with a particular advantage of etherified hydroxyl groups appears in oxaalkyl residues, where in one, preferably linear, alkyl, one or more carbon atoms are replaced by oxygen atoms, which are preferably separated from each other by more (primarily two) carbon atoms, so to form the group - (-O-CH2CH2-)n-, where n is 1 to 14, whereby this group is repeated multiple times in the given case. Furthermore, such etherified hydroxyl groups are also in the given case substituted phenoxy and phenyl-lower-alkoxy residues, such as primarily benzyloxy, benzylhydryloxy and triphenylmethoxy (trityloxy), as well as heterocyclyloxy residues, such as especially 2-tetrahydropyranyloxy. The methylenedioxy and ethylenedioxy groups can be highlighted as special etherified groups, where the former usually bridges two adjacent carbon atoms, especially in aryl residues, and the latter is attached to one and the same carbon atom and is considered a protective group for oxo. Etherified groups in this context also include silylated hydroxyl groups, such as those appearing, for example, in tri-lower-alkylsilyloxy, such as trimethylsilyloxy and dimethyl-tert.butylsilyloxy, or phenyl-di-lower-alkyl-silyloxy, or lower alkyl-diphenylsilyloxy.

The esterified hydroxyl group, which appears as a substituent in the hydrocarbyl, further carries an acyl residue Ac as defined below, especially one with up to 12 carbon atoms, or is lactanized with a carboxyl group, which is also present in the hydrocarbyl.

An esterified carboxyl group that appears as a substituent in hydrocarbyl is one in which the hydrogen atom is replaced by one of the hydrocarbon residues defined above, preferably with a lower alkyl or phenyl-lower alkyl residue; as an example of an esterified carboxyl group, we can mention, for example, a lower alkoxycarbonyl or, in a given case, a phenyl-lower-alkoxycarbonyl substituted in the phenyl part, especially a methoxy, ethoxy, tert.butoxy and benzyloxycarbonyl group, and also a lactonized carboxyl group.

The primary amino group -NH2 as a hydrocarbonyl substituent can also appear in a protected form, as an acylamino group of the formula -NH-Ac° corresponding to that group, where Ac° has the meaning given below. The secondary amino group instead of one of the two hydrogen atoms carries a hydrocarbyl residue, preferably unsubstituted, such as one of the above-mentioned, especially lower alkyl, and it can also appear in a protected form, as an acylamino group derived from it with the below-mentioned, monovalent acyl residue Acº .

The acyl residue Acº, which is used as an amino protecting group, is preferably derived from a semi-derivative of carbonic acid and it is preferably, in a given case, especially lower alkyloxycarbonyl or aryl-lower-alkyloxycarbonyl substituted with lower alkyl, lower alkoxy, nitro and/or halogen , such as rnetoxycarbonyl, ethoxycarbonyl, tert.butoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-iodethoxycarbonyl, benzyloxycarbonyl, 2-phenyl-2-propoxycarbonyl, 2-p-tolyl-2-propoxycarbonyl, 2-(p-biphenylyl )-2-propoxycarbonyl or 9-fluorenylmethoxycarbonyl.

The tertiary amino group appearing as a substituent in hydroxycarbyl carries two different or preferably the same hydrocarbyl residues (including heterocyclic residues), such as the above-mentioned unsubstituted hydrocarbyl residues, especially lower alkyl.

In particular, the amino group is that of the formula R1-N-R2, where R1 and R2 mean, independently of each other, hydrogen, unsubstituted acyclic C1-C7-hydrocarbyl (such as C1-C4-alkyl or C1-C4-alkenyl) or monocyclic, in a given case with C1-C4-alkyl, C1-C4-alkoxy, halogen and/or nitro-substituted aryl, aralkyl or aralkenyl with up to 10 carbon atoms, wherein the carbon-containing residues are linked by a carbon-carbon bond or by oxygen, sulfur or in a given case with a nitrogen atom substituted with a hydrocarbyl. In such a case, together with the nitrogen atom, the amino group forms a nitrogen-containing heterocyclic ring. As examples of free amino groups, the following can be mentioned with advantage: di-lower-alkylamino such as dimethylamino, diethylamino, pyrrolidino, piperidino, morpholino, thiomorpholino and piperazino or 4-methylpiperazino, in a given case especially diphenylamino and dibenzylamino substituted in the phenyl part, e.g. .with lower alkyl, lower alkoxy, halogen and/or nitro; then, of the protected ones, especially lower alkoxycarbonylamino such as tert.butoxycarbonylamino, phenyl-lower-alkoxycarbonylamino such as 4-methoxybenzyloxycarbonylamino as well as 9-fluorenylmethoxycarbonylamino.

If not stated otherwise, the aromatic, carbocyclic and heterocyclic hydrocarbyl residues mentioned above and below can be substituted singly or multiply, as doubly or triply, especially with alkyl with 1 to 4 carbon atoms, alkoxyl with 1 to 4 carbon atoms, halogen, nitro, with trifluoromethyl, further with carboxyl, alkoxycarbonyl, with 1 to 4 carbon atoms, methylenedioxyl and/or with cyano. The above and below, the insufficient data for the substituents, can be considered as those with an advantage.

Preferably compounds of formula I in the sense of the invention are, for example, those, where R represents hydroxycarbonyl R° preferably with the following meaning of acyclic hydroxycarbyl: C1-C20-alkyl, C2-C20-hydroxyalkyl, whose hydroxyl group is located in any position, except position 1 , and preferably in position 2, cyano-(C1-C20)-alkyl, whose cyano group is preferably in position 1 or ω, or carboxy-(C1-C20)-alkyl, whose carboxyl group is preferably in position 1 or ω, and in the given case also in salt form or as C1-C4-alkylester (C1-C4-Alkoxycarbonyl) or benzylester (benzyloxycarbonyl), as well as C3-C20-alkenyl, whose free valence is not located on the same carbon atom as double bond, wherein all said residues, with the exception of those with a C3-C5-alkyl basic structure, have a linear (unbranched) alkyl chain; furthermore, also linear (mono-, di- to hexa)-oxalalkyl with 4 to 20 ring members, where one or more carbon atoms are numbered from C-3, linear C4-C20-alkyl substituted with oxygen atoms, which are mutually separated by at least two carbon atoms, and are preferably located in positions 3, 6, 9, 12, 15 and 18.

Primarily compounds of formula I in terms of the invention are also such, where R represents hydrocarbyl R° preferably with the following meanings of carbocyclic or heterocyclic, as well as carbocyclic-acyclic or heterocyclic-acyclic hydrocarbyl: bicyclic or preferably monocyclic aryl, primarily phenyl, further naphthyl , which can carry one or more of the following substituents: halogen atoms, especially fluorine, chlorine and bromine, C1-C4-alkyl radicals, especially methyl, C1-C4- alkyl groups, especially methoxy, methylenedioxy, nitro groups and/or carboxyl groups, which can be free, in the form of salts or as

C1-C4-alkylesters, especially methoxycarbonyl or ethoxycarbonyl. First of all, aryl residues do not carry more than two substituents, especially those of the same nature, or however one alone; and above all they are unsubstituted. As a heterocyclic hydrocarbyl (heterocyclyl), we primarily consider, for example, one that is analogous to the above-mentioned aryl residue, and instead of one or two carbon atoms each time contains one heteroatom, especially nitrogen, such as pyridyl or quinolyl, i.e. quinazolyl, whereby is a free valence localized on the carbon atom, and can also be appropriately substituted. Primarily carbocyclic-acyclic and heterocyclic-acyclic hydrocarbyl residues are those, where C1-C3-alkyl carries 2 or 3, and preferably only one of the above-defined cyclic residues, preferably unsubstituted, where all are preferably located on one carbon atom, preferably on the last; the greatest preference is given to unsubstituted benzyl.

First of all, the compounds of formula I are those, where Rº represents C1-C7-alkyl, especially C1-C4-alkyl, hydroxy-C2-C18-alkyl, especially hydroxy-C2-C14-alkyl, cyano-C1-C7-alkyl, especially cyano -C1-C4-alkyl, carboxy-C1-C7-alkyl, especially carboxy-C1-C4-alkyl, C1-C7-alkoxy-carbonyl-C1-C7-alkyl, especially C1-C4-alkoxy-carbonyl-C1-C4 -alkyl, benzyloxy-carbonyl-C1-C7-alkyl, especially benzyloxy-carbonyl-C1-C4-alkyl, C3-C7-alkenyl, phenyl, naphthyl, pyridyl, quinolyl, or quinazolyl, or phenyl-C1-C7-alkyl, especially phenyl-C1-C3-alkyl, wherein all aromatic residues can also be further substituted with alkyl with 1 to 7 carbon atoms, especially with alkyl with 1 to 4 carbon atoms, alkoxyl with 1 to 7 carbon atoms, and especially with 1 up to 4 carbon atoms, by halogen, nitro, trifluoromethyl, further by carboxyl, C1-C4-alkoxycarbonyl, methylenedioxyl and/or cyano, whereby the hydroxyl group is located in an appropriately substituted alkyl residue, especially in position 2, and cyano-, carboxy-, Alkoxy-carbonyl, Benzy oxy-carbonyl or phenyl group in a suitably substituted alkyl residue, especially in position 1 or ω.

Of particular preference are those compounds of formula I, where Rº is C1-C4-alkyl, such as methyl or ethyl, hydroxy-C2-C14-alkyl, such as 2-hydroxy-propyl, -hexyl, -decyl or -tetradecyl, cyano-C1 -C4-alkyl, as 2-cyano-ethyl, carboxy-C1-C4-alkyl, as carboxymethyl, C1-C4-alkoxycarbonyl-C1-C4-alkyl, as methoxycarbonyl-methyl or -ethyl, C3-C7-alkenyl, as allyl, or phenyl, wherein the hydroxyl group is found in a suitably substituted alkyl, preferably in position 2, and the cyano-, carboxy-, or alkylcarbonyl group is particularly in position 1 or ω.

The acyl residue Ac is derived from, in a given case, a functionally modified carboxylic acid, an organic sulfonic acid or, in a given case, an esterified phosphoric acid, such as pyro- or orthophosphoric acid, and preferably has at most 30 carbon atoms.

Acyl, derived from, in a given case, a functionally changed carboxylic acid, which we designate as Ac1, is particularly that of the partial formula Z-C (=W) -, where W can represent oxygen, sulfur or amino, and Z is hydrogen, hydrocarbyl R°, hydrocarbyloxy R°O, an amino group, especially that of the formula [image] or when W represents oxygen or sulfur and also chlorine. The meanings of R°, R1 and R2 correspond to the above, general and prominent, with the latter also representing the generally preferred choice here.

An acyl derived from an organic sulfonic acid, denoted Ac2, is particularly that of the partial formula R°-SO2-, where the hydrocarbyl R° represents the above-mentioned, generally emphasized meanings, the latter being also here also the generally preferred choice .

The acyl derived from, in the given case, esterified phosphoric acid, designated as Ac3, is particularly that of the partial formula

[image]

where R1 and R2 independently have the above, general and distinguished meanings. R1 and R2 preferably have the same meaning.

Acyl residues Ac1 are preferably carboxylic acid acyl residues, which are indicated with the partial formula R°b-CO-, where R°b represents either hydrogen (and with it forms a formyl residue), or however has one of the above-mentioned general and hydrocarbyl residues R °, which is given the meaning with preference, and are derived with it, in a given case, from a substituted acyclic, carbocyclic, carbocyclic-acyclic, heterocyclic or heterocyclic-acyclic monocarboxylic acid. First of all, the hydrocarbyl in such an acyl is, for example, C1-C19-alkyl, primarily C1-C7- or C1-C4-alkyl, especially such, which has a linear chain with more than 5 carbon atoms, and which can also carry the following substituents: a carboxyl group, which in a given case also comes in the form of a salt or as a cyano group or as a C1-C4-alkylester (C1-C4-alkoxycarbonyl group) and which is preferably located in the ω position, an amino group with the above-defined formula [image] preferably one where R 1 and R 2 are hydrogen and then preferably located in position 1, or one or more halogen atoms, especially fluorine or chlorine, located preferably in the vicinity of the carbonyl group. The next acyl is preferably bicyclic or especially monocyclic aroyl, above all benzoyl, which can also carry one or more of the following substituents: halogen atoms, especially chlorine or fluorine atoms, nitro groups, C1-C4-alkyl radicals, especially methyl, hydroxyl groups and etherified hydroxyl groups, especially C1-C4-Alkoxy such as methoxy, phenoxy or methylenedioxy, as well as carboxyl groups, which can also occur in salt form or as a cyano group or C1-C4-alkylester (C1-C4-Alkoxy-carbonyl). Aroyl residues primarily carry a maximum of 2, and primarily only one such substituent. Analogous heteroaroyl residues are also preferred, especially those derived from pyridine, furan, thiophene and imidazole, and their analogues with a condensed benzene ring (such as quinoline, isoquinoline, benzofuran and benzimidazole), and in a given case they are also substituted, as which is stated above. Primarily acyl residues of this type are also derived from monocyclic aryl-alkenyl, for example the corresponding aryl-C2-C5-alkenyl such as benzyl and styryl (ie phenacetyl and cinnamoyl), and can also be substituted in the above-mentioned manner. Acyl residues of this type form the corresponding acylamides with the basic structure of staurosporine, whereby those with the above-mentioned values for Ac1 are particularly advantageous. For example, staurosporine amides derived from the following carboxylic acids can be mentioned: aliphatic monocarboxylic acids with a maximum of 20 carbon atoms as lower alkanecarboxylic acids, e.g. propionic, butyric, isobutyric, valeric, isovaleric, caproic, trimethylacetic, enanthic and diethylacetic acids, primarily acetic, as well as lauric, myristic, palmitic and stearic acids, as well as oleic, elaidic, linoleic and linolenic acids, and also corresponding halogenated lower alkanecarboxylic acids such as chloroacetic, trifluoro- or trichloroacetic, bromoacetic or α-bromosovaleric acid, carbocyclic or carbocyclic-acyclic monocarboxylic acids, for example cyclopropane, cyclopenta and cyclohexane-carboxylic acids, or cyclopentane- or cyclohexaneacetic or -propionic acids; aromatic carbocyclic carboxylic acids, eg benzoic acid, which may be mono- or poly-substituted, as mentioned above; aryl- or aryloxy-lower alkanecarboxylic acids and their chain-unsaturated analogues, e.g. in the given case, as stated above for benzoic acid, substituted phenylacetic or phenoxy-acetic acids, phenylpropionic acids and cinnamic acids; further heterocyclic acids, e.g. furan-2-carboxylic, 5-tert.butyl-furan-2-carboxylic, thiophene-2-carboxylic, nicotinic or isonicotinic acid, 4-pyridinepropionic acid, and in the given case, pyrrole-2- or -3-carboxylic acids substituted with lower alkyl residues; also of corresponding α-amino acids, especially in natural α-amino acids from the L-type, e.g. glycine, phenylglycine, alanine, phenylalanine, proline, leucine, serine, valine, tyrosine, arginine, histidine and asparagine, primarily in N-protected forms, i.e. in those in which the amino group is substituted with a conventional, for example, one of the above-mentioned protecting groups for amino; furthermore also dicarboxylic acids such as oxalic, malonic, mono- or di-lower alkylmalonic acids, succinic, glutaric, adipic, erucic acids, maleic acids, with halogens such as fluorine, chlorine or bromine and/or lower alkyl, hydroxyl, lower alkoxy and nitro in a given case substituted, phthalo-isoquinolinic or phenyl-succinic acid, and also glutamic and aspartic acid, both last mentioned acids coming preferably with protected amino groups. As we have already mentioned, the second carboxyl group can be present not only in free forms, but also in functionally modified forms, for example as a C1-C4-alkylester or as a salt, preferably as a physiologically transferable salt, with a base component that can form a salt . Thus, metal or ammonium salts come into consideration in the first place as salts with alkali or alkaline earth metals, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or similar organic amines.

Furthermore, acyl Ac1, which is preferred, is derived from a carbonic acid monoester and is indicated by the partial formula R°-O-CO-. Then this acyl with the basic structure of staurosporine forms the corresponding N-disubstituted urethanes. Among the hydrocarbyl residues R°, of particular advantage in these derivatives, the following can be mentioned, for example: acyclic hydrocarbyl, especially C1-C20-alkyl, preferably linear, which can be substituted with a carboxyl group, preferably in functionally modified forms, such as a salt, cyano or C1-C4-alkylester, located preferably in the ω-position, or an analogous linear (mono- to hexa-)-oxa-alkyl with 4 to 20 members in the chain, especially with such, which we denoted above as s a special advantage. Regarding the meaning of R°, substituted phenyl and benzyl radicals are also preferred in the given case, for example those we mentioned above as those with advantage.

A further preferred acyl Ac1 is derived from a carbonic acid amide (or also a thiocarbonic acid) and is indicated by the formula

[image]

where R1 and R2 have the meanings given above, and W represents sulfur and especially oxygen. Then, this acyl residue with the basic structure of staurosporine forms the corresponding ureas, i.e. thioureas.

Among the compounds according to the invention, which are preferred, and which carry this acyl, those where W represents oxygen, one of the residues R1 and R2 is hydrogen, and the other represents C1-C7-alkyl, which can be substituted with hydroxyl , mercapto, methylthio, phenyl, p-hydroxyphenyl, p-methoxyphenyl, 2-indolyl, 2-imidazolyl and above all with carboxyl (in free form or in a functionally modified form as C1-C4-Alkoxycarbonyl, carbamoyl or amido) whereby one of them, it is located primarily in position 1, and primarily corresponds to a radical, whose free valence stands in place of an amino group in a common amino acid such as β-alanine, -aminobutyric acid or norvaline, and especially one of the L-type α-amino acids, which in appears in nature as an integral part of a peptide, or its antipode. Acyl compounds of the last mentioned type can also be highlighted, where W represents sulfur, one of the residues R1 and R2 is hydrogen, and the other is C1-C7-alkyl or especially C1-C7-alkenyl, where the free valency originates from another atom carbon as a double bond, as allyl.

The compounds according to the invention of the formula I, where R represents chloroformyl or thiochloroformyl, can also be highlighted, which are distinguished in particular as suitable intermediates for the preparation of modified acyl esters of carbonic acid.

The acyl residue Ac2 is derived from an acyclic, carbocyclic or heterocyclic, further also carbocyclic-acyclic or heterocyclic-acyclic sulfonic acid and corresponds to the aforementioned partial formula R°-SO2-, where R° represents hydrocarbyl with the above-mentioned general, and especially the meanings given advantage. Among the compounds in the sense of the invention, which carry the residue Ac2, those where R° represents C1-C7-alkyl, especially linear, bicyclic or especially monocyclic aryl such as especially phenyl, which can be substituted analogously, as we mentioned above for the highlighted aroyls, can be highlighted leftovers. Analogously structured bicyclic and monocyclic, aromatic heterocyclic residues, where one or two of the carbon atoms are replaced by heteroatoms, such as pyrimidyl, eg 2- or 4-pyrimidyl, quinolyl or isoquinolyl, can also be highlighted. Also, heterocyclyl residues can bear substituents, especially those highlighted for aroyl (wherein, for example, a hydroxyl derivative with a tautomeric attraction of the double bond is equal to a dihydro-oxo-derivative).

The acyl residue Ac3, derived from phosphoric acid, is, for example, derived from pyrophosphoric acid, and primarily orthophosphoric acid, which can also appear in a functionally altered form, for example as a salt, hydrocarbyl ester or amide. Among the compounds of formula I in terms of the invention, where R represents Ac3, those in which Ac3 corresponds to the partial formula can be highlighted in particular

[image]

where R1 and R2 have the above-mentioned general and prominent meanings, and they are, preferably both, equal, and represent hydrogen or unsubstituted alkyl with 1 to 7 carbon atoms, especially linear alkyl, primarily methyl or ethyl, or however in the given case, phenyl substituted in particular with alkyl with 1 to 4 carbon atoms, alkyl with 1 to 4 carbon atoms, halogen and/or nitro.

Of particular preference are those compounds of formula I where R is acyl of the partial formula Z-C(=W)-, where W represents oxygen, then sulfur, and Z is alkyl with 1 to 7 carbon atoms, which can be substituted with halogen, carboxy or C1 -C4-Alkoxy-carbonyl.

Of particular preference are those compounds of formula I, where R is acyl with the partial formula Z-C(=W)-, where W represents oxygen, then sulfur, and Z is phenyl, then pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, which is unsubstituted or substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, chalogen, nitro, trifluoromethyl, carboxyl, C 1 -C 4 -alkoxycarbonyl, methylenedioxyl and/or cyano.

Of particular advantage are those compounds of formula I, where R is acyl of the partial formula Rb°-CO-, where Rb° represents alkyl with 1 to 7 carbon atoms, especially alkyl with 1 to 4 carbon atoms such as methyl or tert.butyl, which can be substituted also with halogen, such as fluorine or chlorine, carboxy or C1-C4-alkoxycarbonyl such as methoxycarbonyl, such as CF3- or CCl3-methyl, 2-carboxy- or 2-methoxycarbonylethyl.

Particular preference is given to such compounds of the formula I, where R is acyl of the partial formula Rbº-CO-, where Rbº represents phenyl, which can be unsubstituted or further substituted with alkyl with 1 to 4 carbon atoms, alkoxyl with 1 to 4 carbon atoms, halogen as fluorine or chlorine, nitro, trifluoromethyl, carboxyl, or C1-C4-alkoxy-carbonyl.

Particular preference is given to such compounds of formula I, where R is acyl of the partial formula R°-SO2-, where R° represents alkyl with 1 to 7 carbon atoms, especially alkyl with 1 to 4 carbon atoms.

Particular preference is given to such compounds of formula I, where R is acyl of the partial formula R°-SO2-, where R° represents phenyl, further pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, which is unsubstituted or substituted with C1 -C4-alkyl, C1-C4-alkoxy, halogen, nitro, trifluoromethyl, carboxyl, C4-C4-alkoxycarbonyl, methylenedioxyl and/or cyano.

Particular preference is given to such compounds of formula I, where R is acyl of the partial formula R°-SO2-, where R° represents phenyl, or phenyl or isoquinolyl substituted with alkyl with 1 to 4 carbon atoms or halogen, such as 5-isoquinolyl.

Particular preference is given to such compounds of formula I, where R is acyl of the partial formula R°-O-CO-, where R° is alkyl with 1 to 7 carbon atoms, especially alkyl with 1 to 4 carbon atoms.

Particular preference is given to such compounds of formula I, where R is acyl of the partial formula R°-O-CO-, where R° is phenyl, further pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, which is always unsubstituted or substituted with alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halogen, nitro, trifluoromethyl, carboxyl, C 1 -C 4 -alkoxycarbonyl, methylenedioxyl and/or cyano.

Particular preference is given to the compound of formula I, where R is acyl of the partial formula R°-O-CO-, where R° is unsubstituted phenyl.

Particular preference is given to those compounds of formula I, where R is acyl of the partial formula

[image]

where W represents sulfur or especially oxygen, R1 is hydrogen, and R2 represents C1-C7-alkyl, especially C1-C4-alkyl, C3-C7-alkenyl or phenyl, further pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, which may be unsubstituted or substituted with alkyl with 1 to 4 carbon atoms, alkoxy with 1 to 4 carbon atoms, halogen, nitro, trifluoromethyl, carboxyl, C 1 -C 4 -alkoxycarbonyl, methylenedioxyl and/or cyano.

Compounds of formula I, where R is derived from an α-amino acid, especially in the naturally occurring form of an L-type α-amino acid, are of particular advantage.

Such compounds of the formula I, where R is derived from an α-amino acid, selected from glycine, phenylglycine, alanine, phenylalanine, proline, leucine, serine, valine, tyrosine, arginine, histidine and asparagine, are particularly preferred.

Particular preference is given to such compounds of formula I, where R is derived from an α-amino acid selected from glycine, alanine, phenylalanine, serine, arginine and histidine.

Depending on their nature, if they have groups that can form salts, the compounds of the invention can also appear in the form of salts, especially pharmaceutically usable salts, i.e. physiologically transferable salts. Pharmaceutically inappropriate salts can also be used for isolation or purification. For therapeutic use, however, only pharmaceutically usable salts are considered, which are preferred.

Thus, for example, compounds appear with free acid groups, such as a free sulfo, phosphoryl or carboxyl group, especially with the one found in the acyl residue Ac, in the form of a salt, primarily as a physiologically transferable salt, with a base component that can form a salt . First of all, metal or ammonium salts come into consideration as salts of alkali and alkaline earth metals, e.g. sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or similar organic amines, especially tertiary monoamines and heterocyclic bases, e.g. triethylamine, tri-(2-hydroxyethyl)-amine, N-ethylpiperidine or N,N'-dimethylpiperazine. When such an acid group is present in the hydrocarbyl residue R°, it can also form the basic structures of staurosporine with the amine nitrogen, or also an internal salt with another amino group present in the given case.

Compounds in the sense of the invention of a basic character can also appear as addition salts, especially as acid addition salts with inorganic or organic acids, and also as quaternary salts. Thus, compounds of formula I, which carry a basic group as an amino group as a substituent in the Ac residue, can form acid addition salts with common acids. In particular, the addition salts of the formula can be highlighted

[image]

where [Stau] and R° have the meanings given in the introduction, R°q represents hydrogen or unsubstituted, preferably linear C1-C4-alkyl, especially ethyl or above all methyl, or however benzyl, and X- is an anion of an inorganic or organic acid, or of carboxyl, which appears in the residue R°, wherein the physiologically transferable salts are advantageous. The quaternary salts of formula IA primarily mean those where the first carbon atom in the hydroxycarbyl R° is methylene.

For the formation of the anion X-, the following common acids are suitable, for example, among others: hydrohalic acids, for example hydrochloric and hydrobromic acids, sulfuric, phosphoric, nitric or perchloric acids, or aliphatic, alicyclic, aromatic or heterocyclic carboxylic or sulfonic acids, such as formic, acetic, propionic, amber, glycolic, milky, malic, tartaric, citric, fumaric, maleic, hydroxymaleic, oxalic, pyruvate, phenyloctene, benzoic, p-aminobenzoic, anthranilic, p-hydroxybenzoic, salicylic, p-aminosalicylic, embonic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, ethylenedisulfonic, halobenzenesulfonic, toluenesulfonic and naphthalenesulfonic acid or sulfanilic acid, furthermore methionine, tryptophan, lysine or arginine, as well as ascorbic acid. In quaternary salts, as X- are primarily anions of strong inorganic acids, as hydrohalic acids, especially bromides and iodides, or anions of organic sulfonic acids, such as methanesulfonates (mesylates), p-toluenesulfonates (tosylates), p-bromobenzenesulfonates (brosylates) and p- nitrobenzenesulfonates.

Compounds of the formula I or IA described in the examples are of particular advantage.

The compounds of the formula I according to the invention and their salts are prepared such that staurosporine of the formula [Stau]-NH-CH3 (II), where [Stau] has the above meaning, or its acid addition salt

a) is chemically converted with a reagent of the formula

R-Y (III)

where R has the above meaning, and Y represents an activated hydroxyl group, capable of reaction, or an additional single bond, the other end of which replaces the hydrogen in the R residue, or

b) for the preparation of compounds of formula I, where R represents the remainder of the partial formula

H-R°a-,

where R°a represents a divalent hydrocarbyl residue of aliphatic character, which corresponds to the structure of R° (i.e. one in which the functionalized carbon atom is connected by single bonds to the neighboring carbon and/or hydrogen atom), is chemically converted with the carbonyl reagent of the formula

R°a=O (IV)

where R°a has the above meaning,

and at the same time or then it is chemically converted with a reducing agent, and if desired, the obtained compound of formula I is converted into another compound of formula I and/or the compound of formula I obtained in free form is converted into its salt, and/or the compound of formula I, obtained as salt, it is translated into its free form or some other salt.

The chemical transformation of staurosporine in terms of the invention with reagent type a), i.e. that of formula III, takes place under known process conditions, which are generally common in organic chemistry for amine substitution, usually at temperatures between the freezing point and the boiling point of the reaction mixture, as in the temperature range from about -10 to about +160°C, especially from about +20 to about +50°C, under atmospheric or elevated pressure, in a heterogeneous phase (as a suspension) with stirring or shaking, or however especially in a homogeneous liquid phase, as in an excess of liquid reagent, or especially in the presence of solvents, especially organic solvents, and in a given case, in the presence of inorganic or organic acid binding agents. Suitable solvents are, for example, aprotic organic solvents of low polarity, such as aliphatic and aromatic hydrocarbons such as pentane, hexane, heptane and cyclohexane, i.e. benzene, toluene and xylenes, as well as halogenated, chlorinated, aliphatic hydrocarbons such as chloroform and dichloromethane, and especially polar aprotic solvents such as aliphatic and cyclic ethers, e.g. diethylether, 1,2-dimethoxyethane and diisopropylether, i.e. dioxane and tetrahydrofuran, lower aliphatic esters and amides, such as ethyl acetate, i.e. formamide, acetamide, N,N-dimethylacetamide and dimethylformamide, as well as acetonitrile , dimethylsulfoxide and hexamethylphosphortriamide; under certain conditions water can also be used as a solvent, and e.g. methanol, ethanol, an organic solvent such as a lower alkanol, e.g. ethanol, isopropyl alcohol and tert.butyl alcohol, and also glycol- or diglycol monoether, e.g. 2-methoxy- ethanol. In this case, it is often appropriate to speed up the reaction with elevated pressure, i.e. partly in closed vessels, to raise the boiling point and reaction temperature. Solvents can also be used in purposeful combinations, for example to increase the solubility of components.

In principle, any basic compounds can be used as an acid binding agent, such as organic bases containing nitrogen, for example tertiary amines of the type triethylamine, ethyldiisopropylamine, N,N-dimethylaniline, N-ethylpiperidine or N,N'-dimethylpiperazine or aromatic heterocyclic bases such as pyridine, collidine, quinoline or 4-dimethylaminopyridine, and on the other hand also inorganic compounds that react with bases, especially hydroxides, carbonates and hydrogencarbonates of alkali metals, and also salts of carboxylic acids such as sodium or potassium acetate.

Finally, this role can also be taken over by neutral nitrogen-containing compounds, which often simultaneously represent also suitable solvents, for example amides of carboxylic acids, especially amides of lower aliphatic carboxylic acids, as mentioned above, and cyclic amides, such as N-methylpyrrolidone, and also amides carbonic acid derivatives, such as urethanes and urea.

Although the exchange reaction always takes place according to the same principle and the chemical conversion takes place according to the same basic scheme, for an optimal result in practical implementation it is necessary to take into account the properties of the reaction components, in the first place of each reaction agent of formula III.

In the sense of the above definition, the residue R can be hydrocarbyl R° with the above general and highlighted meanings. In this case, Y represents primarily an esterified hydroxyl group capable of reaction (as a reactive activated hydroxyl group of the special, above-mentioned form), i.e. one that is esterified with a strong inorganic acid such as hydrohalic acid (e.g. hydrochloric, hydrobromic and hydroiodic acid), mineral acids that contain oxygen, such as phosphoric acid and especially sulfuric acid, or strong organic acids such as aliphatic or aromatic sulfonic acids (e.g. methane- and ethane- or benzene-, p-toluene-, p-nitrobenzene- and p- chlorobenzenesulfonic acid). If R° has an aliphatic character, i.e. if its free valence comes from the carbon atom that is bound to the neighboring C or H atom, with only single bonds, it can be freely chosen from the esterified hydroxyl groups, mentioned above as an example; if, however, R° has an aromatic character, i.e. if its free valence comes from a carbon atom that is an integral part of an aromatic carbocyclic or heterocyclic ring, preference is given to hydrohalic acid esters, especially bromides and iodides.

The reagent of formula III, where Y represents an additional single bond to the hydrocarbyl residue R° (with replacement of one of its hydrogens), is nor. an alkene, especially one whose double bond is structurally special, as in 2-methylpropene, or additionally activated by substitution, as especially in acrylonitrile. The definition for Y also includes a single bond, the other end of which is not directly connected to the carbon atom of the hydrocarbyl residue R°, but to a heteroatom, which appears as a substituent, such as with oxygen (ie the oxygen of the hydroxyl group), or with nitrogen (amio group) whereby it replaces the hydrogen of that group); reagents of the formula R°Y containing an α-epoxide (oxidized) or α-imine (azilidated) group are particularly preferred and are used as a suitable source of residues R° with a 2-hydroxy- or 2-aminoalkyl group. The chemical conversion of these reagents takes place primarily in the presence of lower alkanes at an elevated temperature, for example at approximately +100 to approximately 150°C and in a given case (to increase the boiling point of the reaction mixture) under elevated pressure, or in a basic environment and especially with an excess of reagents.

In the sense of the introductory definition, the residue R can represent acyl Ac and therefore represents the basis of the acylating agent of the formula AcY, in which both Ac and Y have the indicated general and corresponding meanings, as indicated for Ac1, Ac2 and Ac3. Y primarily represents halogen, especially chlorine, bromine and iodine.

In acylating agents derived from the above-defined acyl residue Ac1 carboxylic acid, Y can represent, for example, a hydroxyl group activated so that it is capable of reaction. This is how it occurs already in the free carboxyl group of a carboxylic acid of the formula R°-COOH, if due to the particularity of the structure it has satisfactory reactivity, as in trifluoroacetic acid and above all in formic acid, and especially if it has been previously activated by the action of an activating reagent, e.g. carbodiimide, such as dicyclohexylcarbodiimide or di-(2-imidazolyl)-carbodiimide and other analogous compounds, and in the given case, in the presence of auxiliary substances, which form active esters, such as substituted phenols and especially N-hydrocyamine compounds of the 1-hydroxy-benzotriazole type , N-hydroxyphthalimide and N-hydroxymaleinimide or -succinimide.

An activated hydroxyl group, suitable for acyl residues of all types, e.g. for Ac1, Ac2 and Ac3, is a reactive hydroxyl group, esterified with strong acids, as defined above in connection with hydrocarbyl R°, which forms a mixed acid anhydride with the acyl residue. Among them, mixed anhydrides of hydrohalic acids, especially with hydrobromic and above all hydrochloric acid, i.e. acid bromides or acid chlorides, for example those with the formulas

[image]

where Hal represents bromine and preferably chlorine, and Z, W, R°, R1 and R2 have the above meaning; whereby phosgene and thiophosgene can be mentioned as a special embodiment.

In the case of acyl residues Ac1 of carboxylic acids (including acyl residues of functionally changed carbonic acid) is a reactive, esterified hydroxyl group that can also be esterified or with the residue of another carboxylic acid, especially stronger carboxylic acids such as formic, chloroacetic or especially trifluoroacetic acids, and the base for a mixed anhydride, or however it can be esterified with the same acyl residue, forming a symmetrical carboxylic acid anhydride of the formula Ac1-O-Ac1, especially that of the formula

R°-CO-O-CO-R° or R°-O-CO-O-CO-O-R° (or its sulfur analog).

When acylating with the above-mentioned acylating agents, it is done primarily in the presence of an acid binding agent, as mentioned above, which is used above all in an equivalent amount or a small excess (which normally does not exceed two equivalents).

Acylating agents of the formula AcY, where Y represents an additional bond to the Ac residue, are derived especially from the acyl residues of Ac1 carboxylic acids, especially those that carry hydrogen on the adjacent atom to the carboxyl group, i.e. on the adjacent carbon or nitrogen atom; belong to the category of ketenes or isocyanates and correspond to the formulas

R°a=O=O or R1-N=C=O,

where R°a has the above-defined meaning of divalent hydrocarbyl of aliphatic character, which corresponds to the residue R°, R1 and has the above-mentioned general and specific corresponding meanings, with the exception of hydrogen. An analogous sulfur-containing acylating agent may also be mentioned, i.e. the isocyanate of the formula

R1-N=C=S

where R 1 has the above general and corresponding meanings, except for hydrogen. Acylation with agents of this type can take place, depending on their nature, also without acid binding agents, with the exclusion of moisture and/or protic solvents, which is recommended.

Version b) of the process is generally known as "reductive alkylation" and is often used. Among the carbonyl reagents of the formula R°a=O (IV) defined above are primarily aldehydes, and also ketones, including cyclic ketones, in which the carbonyl group is part of an alicyclic ring; preferably these reagents are derived from unsubstituted hydrocarbyl residues, or however at least bear substituents which are resistant against reduction. As reductants, complex metal hydrides such as aluminum and especially boron hydrides with alkali metals come into consideration, e.g. lithium-aluminum hydride, potassium borohydride, lithium borohydride and above all sodium borohydride, as well as their derivatives, where one or more hydrogen atoms are replaced with alkoxy residues or with cyano, for example methoxy sodium borohydride, tri-(tert.butoxy)-lithium borohydride or di-(2-methoxyethoxy)-disodium lithium hydride, i.e. sodium cyanoborohydride, and also diborane. These reductants are primarily added only in the second stage of alkylation, i.e. after the initial addition of the carbonyl reagent. - Furthermore, elemental hydrogen is often used as a reducing agent, which is used under the usual conditions of catalytic hydrogenation, at temperatures approximately +20 to approximately +100°C, and if necessary under overpressure up to approximately 147 bar, simultaneously with the carbonyl component. Raney's nickel and also palladium or platinum are usually used as a catalyst, preferably on an inert support such as calcium carbonate, barium sulfate or aluminum oxide. In a further variant of reductive alkylation, formic acid is used as a reductant, which is particularly suitable for methylation with formaldehyde.

In terms of the invention, the obtained compound of formula I can be conveniently converted into another compound of formula I; accordingly, the functional group, which appears especially in the residue R, is converted into another, e.g. functionally changed, especially protected, hydroxyl, carboxyl or amino group, is translated into their free forms, or the chlorine atom capable of reaction (as in the chloroformyl residue) replace with the residue R°-O- or R1N(-R2)-. - The release of a functionally changed group is, for example, the conversion of an esterified carboxyl group into a free carboxyl group, which can generally be carried out by usual hydrolysis, primarily by the action of bases (especially hydroxides, carbonates or hydrogencarbonates of alkali metals), or also with suitable esters, such as esters of tertiary alcohols (e.g. tert.butylalcohol), by acidolysis, e.g. using hydrogen fluoride or trifluoroacetic acid. Esters with benzyl alcohols can also be cleaved by conventional hydrolysis. Since esterification is one of the most common methods of protecting carboxyl groups, the above conversion represents a simultaneously effective method of removing carboxyl protecting groups.

The cleaving groups and methods used for pre-protection of hydroxyl groups are also generally known, e.g. from peptide synthesis. Hydroxyl groups are particularly protected in the form of esters with carboxylic acids, such as with lower alkanoic acids or monoesters of carboxylic acids (e.g., on the one hand, thormates or acetates or on the other hand, tert.butoxy - or benzyloxy-carbonates), or however in the form of ethers, especially those with tertiary alcohols (e.g. tert.butyl alcohol), or also in the form of acetals (e.g. especially as 2-tetrahydropyranyl ether). The first protecting groups are removed analogously to the esterified carbonyl groups; both last ones are primarily removed by acidolysis.

The protective groups, used for the preliminary protection of primary and secondary amino groups, correspond to those that have been exhaustively investigated in the synthesis of peptides, and the amino-protecting groups mentioned in the introduction are the most widely used - their separation, which is generally based on their specific nature, takes place under generally known conditions of hydrolysis (especially basic hydrolysis), or acidolysis, or hydrogenolysis.

First of all, the general conditions for the usual separation of functionally converted groups are chosen so that neither the bond between the residue R and the methylamine group of staurosporine, nor its basic structure, is touched; since these structural features are generally characterized by good stability, the usual reaction conditions can be applied without special safety measures.

If desired, the subsequent conversion of the chlorine atom capable of reacting according to the invention takes place in particular by converting the chloroformyl group (Cl-CO-) into a hydrocarbyloxycarbonyl group (R°-O-CO-) or an aminocarbonyl-(carbamoyl)-group [R1- N(-R2)-CO-]. This conversion takes place under known conditions, so that N-chloroformylstaurosporine is chemically converted with an alcohol of the formula R°-OH, i.e. with an amine (including ammonia) of the formula R1-NH-R2, preferably in the presence of an acid binding agent, as organic base (eg one of the above, tertiary amines). The general reaction conditions are analogous to those described in detail above for chemical conversions with reagents having an esterified reactive hydroxyl group (especially for acid chlorides).

If desired, feasible salt formation and release of the basic forms of the compound of formula I from its salts takes place in a generally known, conventional manner. - Thus, the acyl derivatives of formula I bearing carboxyl are converted into corresponding salts with bases, primarily salts of alkali metals, by treatment with a suitable base, especially with a compound that reacts alkaline, such as hydroxide, carbonate or bicarbonate; salts can be converted into free compounds by acidification, e.g. with inorganic acids, especially hydrohalic acids. - Final substances, which react basicly, for example tertiary or quaternary amines of the formula I or IA, can be converted into their salts with acids, for example by treatment with an acid, suitable for the formation of salts, such as one of the above. Conversely, treatment with basic reactive agents, such as inorganic hydroxides, carbonates and bicarbonates, or organic bases and ion exchangers, releases the basic basic form of the tertiary amine of formula I.

Suitable compounds in terms of the proposed invention can also form internal salts, e.g. by usual acid-base filtration up to the point, i.e. up to the isoelectric point, or e.g. by treatment with a quaternizing agent, which corresponds to the residue R°q, such as with an ester of a hydroxyl compound capable of reaction with a strong acid, such as hydrohalic acid, sulfuric acid or a strong organic sulfonic acid, of quaternary ammonium salts of formula IA.

These and other salts of new compounds, such as, for example, picrates, can also be used to purify the obtained compounds, with the fact that the free compounds are converted into salts, and then they are separated and the free compounds are obtained again from the salt. Due to the close connection between the compounds in their free form and their salt forms, in what has been said above and in the following, the free compounds are meaningfully and purposefully understood in the given case as well as the corresponding salts (including quaternary salts).

Certain carbonyl functions can, for example, be converted into the corresponding thio form by means of suitable reagents, which cause the exchange of O with S. Thus, for example, by chemical conversion with Lawesson's reagent [2,4-bis-(4-methoxy-phenyl)-2,4-dithioxo-1,3,2,4-dithiadiphosphetane] in compounds of formula I and their salts, which have as carbonyl function, for example carboxamide, ketone and lactone groups, the oxygen atom can be replaced with a sulfur atom.

The invention also relates to those embodiments of the process, according to which the starting point is from compounds obtained in any stage of the process as intermediates, and insufficient stages are carried out, or the starting substance is used in the form of a derivative, e.g. a salt, or it is formed under reaction conditions.

According to the process in terms of the proposed invention, known starting substances or available by known methods are used, primarily those that lead to the compounds described in the introduction as particularly valuable.

Considering the pharmacological properties of the new compounds described above, the proposed invention also includes the use of the effective substances themselves in terms of the invention, in a given case together with auxiliary substances, or in combination with other active substances, e.g. antibiotics or chemotherapeutics, as means for the treatment of diseases, in of which - as we described above - cell growth was mentioned, both prophylactically and curatively. When used as medicine, active substances in the sense of the invention are administered in prophylactically or curatively effective amounts, primarily in the form of pharmaceutical compositions, together with usual pharmaceutical carriers or auxiliary substances. At the same time, warm-blooded animals with a body weight of approximately 70 kg, depending on the species, body weight, age and individual condition, as well as on the method of administration and especially depending on the picture of the disease in question, are given daily doses of approximately 1 to 1000 mg, which in acute cases they may also exceed. A meaningful invention also includes a suitable method of medical care.

The invention further relates to pharmaceutical compositions containing compounds within the meaning of the proposed invention as active substances, and to methods for preparing these substances.

In the case of pharmaceutical compositions in the sense of the invention, these are, for example, those intended for enteral as well as peroral or rectal administration, as well as for parenteral administration to warm-blooded creatures. Suitable dosage units for oral administration, eg dragees, tablets or capsules, contain preferably 5 to 500 mg, especially approximately 10 to 100 mg, of the active substance together with pharmaceutically usable carriers or excipients.

Suitable carriers are especially fillers such as sugars, e.g. lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate, furthermore binders, such as starch adhesives (using e.g. corn, wheat, rice or potato starch), gelatin, tragacanth, methylcellulose and/or optionally disintegrants, such as the above-mentioned types of starch, further carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid or its salt such as sodium alginate. Auxiliary agents are, first of all, flow control agents and lubricants, for example flint, talc, stearic acid or its salt such as magnesium or calcium stearate and/or polyethylene glycol. The dragee cores can be equipped with suitable coatings, which in the given case are resistant against gastric juice, using, among other things, concentrated sugar solutions, which in the given case may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycols and/or titanium dioxide , or solutions of lacquers in suitable organic solvents or solvent mixtures, or for the preparation of coatings, resistant against gastric juice, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropyl-methylcellulose phthalate. Dyes or pigments can be added to tablets or dragee coatings, for example to identify or mark different doses of the active substance.

Further pharmaceutical preparations for oral use are plug-in capsules and gelatin, and also soft, closed capsules made of gelatin and emollients such as glycerin or sorbitol. Plug-in capsules can contain the active substance in the form of granules, for example in a mixture with fillers such as lactose, binders such as starch and/or sliding agents such as talc or magnesium stearate, and in the given case stabilizers.

In soft capsules, the active substance is preferably dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols, whereby stabilizers can also be added.

As pharmaceutical preparations for reactive use, for example, suppositories, which consist of a combination of an active substance with a base mass for suppositories, come into consideration. Natural or synthetic glycerides, paraffinic hydrocarbons, polyethylene glycols or higher alkanols are suitable as a base material for suppositories. Furthermore, gelatin rectal capsules containing a combination of the active substance with the base mass can also be used; liquid triglycerides, polyethylene glycol or paraffinic hydrocarbons can be considered as the base mass.

For parenteral use, aqueous solutions of water-soluble forms of active substances, e.g. water-soluble salts or aqueous injectable suspensions, which contain substances to increase viscosity, e.g. sodium carboxymethylcellulose, sorbitol and/or dextran, are suitable for parenteral use, and in a given case stabilizers. In this case, the active substance can appear together with auxiliary substances, also in the form of a lyophilizate, and before parenteral administration it can be converted into a solution by the addition of suitable solvents.

The pharmaceutical compositions in terms of the proposed invention can be prepared in a manner known per se, for example by means of the usual mixing, granulating, drizzling, dissolving or lyophilizing procedures. In this way, pharmaceutical preparations for oral use can be obtained with the fact that the active substance is combined with solid carriers, the resulting mixture is granulated in the given case and the mixture, i.e. granulate, if desired or necessary, after the addition of suitable substances, is processed into tablets or dragee cores .

The following examples illustrate the invention described above, but in no way limit its scope. Temperatures are given in degrees Celsius.

The nomenclature of the product is derived from the complete structure of staurosporine [Stau]-NH-CH3

[image]

wherein the substituent marked with N is located on the nitrogen of the methylamino group.

Example 1

N-methoxycarbonylmethyl-staurosporine

To a mixture of 233 mg (0.5 mmol) of staurosporine, 0.1 ml (0.59 mmol) of N,N-diisopropylethylamine and 2 ml of dimethylformamide, 0.056 ml (0.6 mmol) of bromooctene methyl ester is added at room temperature. acid. The reaction mixture is stirred in a closed flask for 48 hours at room temperature; with the addition of 1 ml of water, the product is precipitated and then recrystallized from methanol. Tal. ~ 210°C (decomposition, above 170°C brown coloring).

[image]

Example 2

n-carboxymethyl-staurosporine

269 mg (0.5 mmol) of N-methoxycarbonylmethyl-staurosporine (Example 1) was boiled in 15 ml of methanol and 0.3 ml of 2 N sodium hydroxide solution for 18 hours under reflux. After cooling to room temperature, it is neutralized with 0.1 ml of acetic acid and the product is precipitated by adding 15 ml of water. Tal. above 230°C (decomposition, above approximately 220°C brown colouration).

Example 3

N-(1-methoxycarbonylethyl)-staurosporine

To a mixture of 233 mg (0.5 mmol) of staurosporine, 0.12 ml (0.71 mmol) of N,N-diisopropylethylamine and 2 ml of dimethylformamide, 0.085 ml (0.75 mmol) of methyl ester α is added at room temperature. - bromopropionic acid. The reaction mixture is stirred in a closed flask for 20 hours at room temperature. After adding a further 0.044 ml (0.038 mmol) of α-bromopropionic acid methyl ester, it is heated for another 20 hours at 80°C. After cooling to room temperature, the product is precipitated by adding 2 ml of water. The crude product is purified by chromatography on silica gel (eluent: methylene chloride/ethanol 9:1). Tal. ~ 150°C (decomposition).

Example 4

N-benzyl-staurosporine

To a mixture of 116.5 mg (0.25 mmol) of staurosporine, 0.06 ml (0.35 mmol) of N,N-diisopropylethylamine and 1 ml of dimethylformamide was added at room temperature 0.048 ml (0.38 mmol) of benzyl bromide and the reaction mixture is stirred in a closed flask for 6 hours at room temperature. The product is precipitated by adding 1 ml of water, filtered and recrystallized from methanol. Tal. ~ 170°C (decomposition).

Example 5

N-allyl-staurosporine

To a mixture of 116.5 mg (0.25 mmol) of staurosporine, 0.06 ml (0.35 mmol) of N,N-diisopropyl-ethylamine and 1 ml of dimethyl-formamide is added at room temperature 0.032 ml (0.38 mmol) of allyl bromide and the reaction mixture was stirred in a closed flask for 6 hours at room temperature. The product is precipitated by adding 1 ml of water and filtered. Purification is carried out by chromatography on silica gel with methylene chloride/ethanol 9:1 as eluent. Tal. 160°C (decomposition).

Example 6

N,N-dimethyl-staurosporine-iodide (N-methyl-staurosporine-iodo-methylate)

To a mixture of 233 mg (0.5 mmol) of staurosporine, 0.12 ml (0.71 mmol) of N,N-diisopropylethylamine and 2 ml of dimethylformamide, 0.046 ml (0.75 mmol) of methyl iodide is added at room temperature and the reaction mixture is stirred in a closed flask at room temperature. After about 1 hour, a precipitate forms. After the addition of a further 0.023 ml (0.038 mmol) of methyl iodide, it is stirred for another 4 hours at room temperature and filtered after the addition of 2 ml of water; the solid product is poured over with warm methanol and after cooling it is again filtered and dried. Tal. 260°C (decomposition).

Example 7

N-ethyl-staurosporine

At room temperature, 0.029 ml (0.38 mmol) of ethyl- of iodide and the reaction mixture is stirred in a closed flask for 24 hours at room temperature. The product is precipitated by adding 2 ml of water and filtered. Tal. 170°C (decomposition).

Example 8

N,N-ethyl-methyl-staurosporine-iodide (N-ethyl-staurosporine-iodo-methylate)

To a mixture of 115 mg (0.2 mmol) of N-ethyl-staurosporine (Example 7) and 2 ml of dimethylformamide, 0.018 ml (0.3 mmol) of methyl iodide was added at room temperature. After 16 hours at room temperature and 16 hours at 50°C, a further 0.018 ml (0.3 mmol) of methyl iodide is added and the mixture is stirred for another 5 hours at 80°C. The crude product is obtained by precipitation with 2 ml of water and recrystallized from dimethylformamide/chloroform. Tal. 265°C (decomposition).

Example 9

N-(2-hydroxyhexyl)-staurosporine

A suspension of 116.5 mg (0.25 mmol) of staurosporine and 0.054 ml (0.45 mmol) of 1-hexenoxide in 3.5 ml of absolute ethanol is heated for 36 hours in a bomb tube at 110°C. The cooled reaction mixture is diluted with water and extracted with methylene chloride. The organic phase is dried over magnesium sulfate, evaporated and chromatographed on silica gel (eluent methylene chloride/ethanol 9:1); recrystallization from ether/petroleum ether yields a product with a melting point of 110°C (decomposition).

Example 10

N-(2-hydroxyeradecyl)-staurosporine

A suspension of 116.5 mg (0.25 mmol) of staurosporine and 0.075 ml (0.30 mmol) of 1-tetradecenoxide in 3.5 ml of absolute ethanol is heated for 68 hours in a bomb tube at 110°C. The cooled reaction mixture is diluted with water and extracted with methylene chloride. The organic phase is dried over magnesium sulfate, evaporated and chromatographed on silica gel (eluent methylene chloride/ethanol 9:1). Recrystallization from ether/petroleum ether yields a product with a melting point of 120°C (decomposition).

Example 11

N-(2-hydroxydecyl)-staurosporine

A suspension of 116.5 mg (0.25 mmol) of staurosporine and 0.055 ml (0.30 mmol) of 1-decene oxide in 3.5 ml of absolute ethanol is heated for 43 hours in a bomb tube at 110ºC. The cooled reaction mixture is diluted with water and extracted with methylene chloride. The organic phase is dried over magnesium sulfate, evaporated and chromatographed on silica gel (eluent methylene chloride/ethanol 9:1). Recrystallization from ether/petroleum ether yields a product with a melting point of 140°C.

Example 12

N-(2-cyanoethyl)-staurosporine

A suspension of 116.5 mg (0.25 mmol) of staurosporine in 2.5 ml (38 mmol) of acrylonitrile is heated for 70 hours in a bomb tube at 140°C. After cooling, the reaction mixture is diluted, evaporated and chromatographed on silica gel (eluent methylene chloride/ethanol 9:1). Recrystallization from ether/petroleum ether yields a product with a melting point of ~ 210°C.

Example 13

N-(2-acetyl)-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 0.03 ml (0.3 mmol) of acetic anhydride was added at room temperature and it is stirred for 2 hours in a closed flask. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The product is recrystallized from chloroform/methanol; melting point 240°C.

Example 14

N-(3-carboxypropionyl)-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine in 0.065 ml (38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 40 mg (0.4 mmol) of succinic anhydride is added at room temperature and stirred. 28 hours in a closed flask. The reaction mixture is diluted with chloroform, washed with 0.1 N hydrochloric acid solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride/ethanol 9:1); melting point 140ºC.

Example 15

N-(5-isoquinolylsulfonyl)-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.118 ml (69 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 105 mg (0.4 mmol) of 5-isoquinolylsulfonyl chloride was added at room temperature and stirred. 29 hours in a closed flask. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The product is recrystallized from chloroform/methanol; melting point 240°C (with decomposition).

Example 16

N-methylsulfonyl-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (38 mmol) of N,N-diisopropyl-ethylamine in 2 ml of chloroform, 0.023 ml (0.38 mmol) of methanesulfochloride was added at room temperature and stirred for 24 hours. in a closed flask. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, with 0.1 N hydrochloric acid solution and saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride/ethanol 9:1); melting point 230°C.

Example 17

N-(p-tosyl)-staurosporine

At room temperature, 57 mg (0.3 mmol) of p-toluene- sulfochloride and stirred for 68 hours in a closed flask. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride/ethanol 9:1); melting point 245ºC.

Example 18

N-benzoyl-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (0.38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 0.035 ml (0.3 mmol) of benzoyl chloride was added at room temperature and mixed. 10 minutes. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride/ethanol 30:1); melting point 235 to 247°C with brown coloring.

Example 19

N-trifluoroacetyl-staurosporine

To a solution of 233 mg (0.5 mmol) of staurosporine and 0.13 ml (0.6 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 0.5 ml (3.57 mmol) of trifluoroacetic anhydride is added at room temperature acid and mixed for 15 minutes. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride/ethanol 20:1); melting point above 220°C.

Example 20

N-phenoxycarbonyl-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (0.38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 0.035 ml (0.28 mmol) of phenylester of chloroformic acid was added at room temperature and it is stirred for 30 minutes in a closed flask. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The residue is cleaned with hot methanol and after cooling it is filtered off and dried. Melting point above 210°C (decomposition).

Example 21

N-methoxycarbonyl-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (0.38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform, 0.025 ml (0.32 mmol) of chloroformic acid methyl ester was added at room temperature and stir for 1 hour in a closed flask. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated. The crude product is recrystallized from methanol; melting point above 220°C (with decomposition).

Example 22

N-allylaminothiocarbonyl-staurosporine (N-allylthiocarbamoyl-staurosporine)

To a solution of 116.5 mg (0.25 mmol) of staurosporine in 2.5 ml of chloroform, 0.029 ml (0.3 mmol) of allyl isothiocyanate was added at room temperature and stirred for 12 hours in a closed flask. The reaction mixture is evaporated and the crude product is recrystallized from methanol; melting point 220°C (with decomposition).

Example 23

N-methylaminothiocarbonyl-staurosporine (N-methylthiocarbamoyl-staurosporine)

To a solution of 116.5 mg (0.25 mmol) of staurosporine in 2.5 ml of chloroform, 0.022 ml (0.3 mmol) of methylisothiocyanate was added at room temperature and stirred for 12 hours in a closed flask at room temperature. The reaction mixture is evaporated and the crude product is recrystallized from chloroform/methanol; melting point 235 to 238ºC.

Example 24

N-phenylcarbamonyl-staurosporine

0.033 ml (0.3 mmol) of phenylisocyanate was added to a solution of 126.5 mg (0.25 mmol) of staurosporine in 2.5 ml of chloroform and stirred for 15 minutes at room temperature. The reaction mixture is evaporated and the crude product is recrystallized from chloroform/methanol; melting point 225 to 229°C (brown colouration).

Example 25

N-trichloroacetyl-staurosporine

0.04 ml (0.35 mmol) of trichloroacetyl chloride is added to a solution of 116.5 mg (0.25 mmol) of staurosporine in 0.1 ml (0.58 mmol) of N,N-diisopropyl-ethylamine in 1 ml of chloroform at room temperature and stir for 1 hour. The reaction mixture is diluted with chloroform, washed with sodium bicarbonate solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent ethyl acetate); IR: 1682 (strong); FAB-MS: 661.

Example 26

N-(3-chlorobenzoyl)-staurosporine

At room temperature, 0.038 ml (0.38 mmol) of 3-chlorobenzoyl chloride and stir for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 240°C (decomposition).

Example 27

N-(2-chlorobenzoyl)-staurosporine

At room temperature, 0.038 ml (0.38 mmol) of 2-chlorobenzoyl chloride and stir for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 255ºC (decomposition).

Example 28

N-(3-nitrobenzoyl)-staurosporine

At room temperature, 55.5 mg (0.30 mmol) of 3- of nitrobenzoyl chloride and stirred for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 230°C (decomposition).

Example 29

N-(4-methoxybenzoyl)-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine and 0.065 ml (0.38 mmol) of N,N-diisopropylethylamine in 2 ml of chloroform is added at room temperature 0.083 ml (0.38 mmol) of 58% solution of 4-methoxybenzoyl chloride in toluene and stirred for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 220°C (decomposition).

Example 30

N-(4-fluorobenzoyl)-staurosporine

At room temperature, 0.036 ml (0.30 mmol) of 4-fluorobenzoyl chloride and stir for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 225ºC (decomposition).

Example 31

N-(4-chlorobenzoyl)-staurosporine

At room temperature, 0.077 ml (0.6 mmol) of 4-chlorobenzoyl chloride and stir for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 220ºC (decomposition).

Example 32

N-(3-fluorobenzoyl)-staurosporine

At room temperature, 0.072 ml (0.6 mmol) of 3-fluorobenzoyl chloride and stir for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 2 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 240°C (decomposition).

Example 33

N-(4-nitrobenzoyl)-staurosporine

At room temperature, 0.11 ml (0.3 mmol) of 4- of nitro-benzoyl chloride and stirred for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 255ºC.

Example 34

N-(4-methoxycarbonylbenzoyl)-staurosporine

To a solution of 466 mg (1 mmol) of staurosporine and 0.26 ml (1.52 mmol) of N,N-diisopropylethylamine in 8 ml of chloroform, 237 mg (1.2 mmol) of 4-methoxycarbonylbenzoyl chloride are added at room temperature and mixed. 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated.

The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point 240ºC (decomposition).

Example 35

N-thiobenzoyl-staurosporine

A mixture of 180 mg (0.31 mmol) of N-benzoyl-staurosporine (Example 18) and 132 mg (0.326 mmol) of Lawesson's reagent (Fluka AG) in 2 ml of toluene was stirred for 48 hours at room temperature. For processing, it is diluted with methylene chloride, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent ethyl ester acetic acid): FD-MS: 586; H-NMR (300 MHz in CDCl3): 2.99 s (3H); 2.62 s (3H); 2.56 s (3H).

Example 36

N-tert.butoxycarbonyl-staurosporine

To a solution of 116.5 mg (0.25 mmol) of staurosporine in 2 ml of tetrahydrofuran, a solution of 65 mg (0.297 mmol) of di-tert.butyl-dicarbonate in 1 ml of tetrahydrofuran was added at room temperature and stirred for 9 hours. The reaction mixture is evaporated and chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point ~160°C.

Example 37

Na-salt of N-(4-carboxybenzoyl)-staurosporine

A mixture of 314 mg (0.5 mmol) of N-(4-methoxy-carbonylbenzoyl)-staurosporine (Example 34), 10 ml of methanol and 0.32 ml of 2 N sodium hydroxide solution was heated under reflux for 24 hours. After cooling, it is diluted with 10 ml of water and neutralized with 0.1 ml of acetic acid; in doing so, the title compound is precipitated as an acid (melting point 275°C). To prepare the sodium salt, the acid is suspended in 10 ml of methanol and 1 equivalent (5 ml) of 0.1 N sodium hydroxide solution is added. The resulting solution is evaporated and the residue is recrystallized from methanol/ether; FAB-MS: 637 (M+M) + ; 659 (M+Na)+.

Example 38

N-(3,5-dinitrobenzoyl)-staurosporine

At room temperature, 138 mg (0.6 mmol) of 3,5- of dinitrobenzoyl chloride and stirred for 1 hour. The reaction mixture is diluted with chloroform and washed with sodium bicarbonate solution, 1 N hydrochloric acid and saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The crude product is chromatographed on silica gel (eluent methylene chloride-ethanol 95:5); melting point ~240°C (decomposition).

Example 39

N-[(tert.butoxycarbonylamino)-acetyl]-staurosporine

264 mg (1.5 mmol) of BOC-glycine (Fluka AG) and 340 mg (1.65 mmol) of dicyclohexylcarbodiimide are added to 699 mg (1.5 mmol) of staurosporine in 40 ml of dry chloroform and stirred for 1 hour. The reaction mixture is then diluted with chloroform and washed with sodium bicarbonate solution and saturated sodium chloride solution, dried over magnesium sulfate and evaporated. The residue is poured over with some methylene chloride and filtered (removal of dicyclohexylurea). The filtrate is evaporated and dried. Melting point 190°C.

Example 40

N-(2-aminoacetyl)-staurosporine

To a solution of 187 mg (0.3 mmol) of N-[(tert.butoxycarbonylamino)-acetyl]-staurosporine (Example 39) in 1 ml of ethyl acetic acid is added at room temperature 1 ml of an unsaturated solution of hydrochloric acid in ethyl acetic acid. A precipitate immediately forms. The suspension is stirred for another 10 hours, the product is filtered off and washed with acetic acid ethyl ester; melting point 280°C (decomposition).

Example 41

N-(2-hydroxy-propyl)-staurosporine

A mixture of 23.3 mg (50 μmol) of staurosporine) in 1 ml of dioxane, 0.5 ml (0.05 M) of borate buffer (pH 10.0) and 100 μl (1.5 mmol) of propylene oxide was stirred for 13 days. The mixture is extracted twice with dichloromethane. The organic phase is dried over sodium sulfate and the solvent is removed in vacuo. The reaction product was purified by semi-preparative HPLC (Lichrosorb Si 60.5 μm, 8 x 250 mm), using dichloromethane/2-propanol (98:2, vol./vol.) saturated with water at a flow rate of 5 ml. /min and a detector with 295 nm. 20 injections are made. The retention time of the product is 15.4 minutes. The structure was confirmed by E1-MS and 1H-NMR (360 Hz).

Example 42

N-phenyl-staurosporine

A solution of 2.4 mg (51 μmol) of staurosporine) in 0.5 ml of dioxane and 50 μl of a 1 N solution of phenyldiazonium chloride (Organikum, 13th ed., Deutscher Verlag der Wissenschaften, Berlin, 1974, p. 583) is mixed for 1 hour and 1 ml of 1 N sodium bicarbonate, 50 ml of dichloromethane and 5 ml of methanol are added to the mixture. The organic phase was dried over sodium sulfate and the solvent was removed and the product was purified by semi-preparative HPLC under the conditions described in Example 41. The retention time of the product was 5.1 minutes. The structure was confirmed by E1-MS and 1H-NMR (360 Hz).

Example 43

In an analogous way, as described in the above implementation examples, can be prepared:

N-alanyl-staurosporine,

N-arginyl-staurosporine,

N-phenylalanyl-staurosporine,

N-histidyl-staurosporine,

N-seryl-staurosporine.

Example 44

Tablets containing 20 mg of the active substance, eg N-methoxy-carbonylmethyl-staurosporine, are prepared with the following composition and in the usual way:

Composition:

active substance 20 mg

wheat starch 60 mg

milk sugar 50 mg

colloidal silica 5 mg

talc 9 mg

magnesium stearate 1 mg

145 mg

Preparation:

The active substance is mixed with a part of wheat starch, with milk sugar and colloidal flint and the mixture is pushed through a sieve. A further part of the cornstarch is slurried with five times the amount of water in a water bath and the powder mass is kneaded with this glue until a slightly plastic mass is obtained.

The plastic mass is pushed through a sieve approximately 3 mm wide, dried and the resulting dry granulate is again pushed through the sieve. Then the remaining wheat starch, talc and magnesium stearate are mixed in and the mixture is pressed into 145 mg tablets with a dividing comma.

Example 45

Tablets containing 1 mg of active substance, for example N-methoxy-carbonylmethyl-staurosporine, are prepared with the following composition and in the usual way:

Composition:

active substance 1 mg

wheat starch 60 mg

milk sugar 50 mg

colloidal silica 5 mg

talc 9 mg

magnesium stearate 1 mg

126 mg

Preparation:

The active substance is mixed with a part of wheat starch, with milk sugar and colloidal flint and the mixture is pushed through a sieve. A further part of the cornstarch is slurried with five times the amount of water in a water bath and the powder mass is kneaded with this glue until a slightly plastic mass is obtained.

The plastic mass is pushed through a sieve approximately 3 mm wide, dried and the resulting dry granulate is again pushed through the sieve. Then the remaining wheat starch, talc and magnesium stearate are mixed in and the mixture is pressed into 126 mg tablets with a dividing comma.

Example 46

Capsules containing 10 mg of the active substance, eg N-methoxy-carbonylmethyl-staurosporine, are prepared in the usual way as follows:

Composition:

active substance 2500 mg

talc 200 mg

colloidal silica 50 mg

Preparation:

The active substance is thoroughly mixed with talc and colloidal flint, the mixture is pushed through a sieve about 0.5 mm wide, and then 11 mg of this mass are filled into solid gelatin capsules of a suitable size.

Example 47

Instead of the compound described in examples 44 to 46, pharmaceutical preparations can be prepared that contain one of the compounds described in examples 1 to 43 as an active substance.

Claims (32)

1. Some N-substituted staurosporine derivative of the general formula [Stau]-N(CH3)-R (I) indicated that [Stau] represents the rest of the partial formula [image] and R is some acyclic, carbocyclic or carbocyclic-acyclic hydrocarbon radical R° which has a total of at most 30 carbon atoms and can be saturated or unsaturated, and unsubstituted or substituted, in which radicals can be present at the position of one, two or more carbon atoms, identical or different heteroatoms selected from oxygen, sulfur and nitrogen; or some acyl radical Ac having a maximum of 30 carbon atoms; or a salt of a compound of formula I which has salt-forming properties. 2. The compound of formula I according to claim 1, characterized in that R represents a C1-C20 alkyl radical, a C1-C20 hydroxyalkyl radical whose hydroxyl group is in any position other than position 1, a cyano-[C1-C20]alkyl radical, a carboxy-[ C1-C20]alkyl radical whose carboxyl group can also exist in the form of a salt or in the form of a C1-C4alkyl ester or benzyl ester, or a C1-C20alkenyl radical whose free valence is not on the same carbon atom on which the double bond is located. 3. The compound according to claim 1, indicated by the fact that R represents a bicyclic or monocyclic radical or an analogous heterocyclic radical with one or two heteroatoms, wherein the free valence is located on a carbon atom, or R represents one of those radicals to which one or more of the following substituents: halogen atoms, C1-C4 alkyl radicals, C1-C4 alkoxy groups, methylenedioxy, nitro groups and/or carboxy groups which may be present in free form or as salts or in the form of C1-C4 alkyl ester groups. 4. The compound according to claim 1, characterized in that R° represents C1-C7alkyl, hydroxy-C2-C18alkyl, cyano-C1-C7alkyl, carboxy-C1-C7alkyl, C1-C7alkoxycarbonyl-C1-C7alkyl, benzyloxycarbonyl-C1-C7alkyl, C3-C7alkenyl, phenyl, naphthyl, pyridyl, quinolyl, quinazolyl or phenyl-C1-C7alkyl, wherein it is possible for said aromatic radicals to also be substituted with C1-C7alkyl, C1-C7alkoxy, halogen, nitro, trifluoromethyl, carboxy, C1- C4-Alkoxycarbonyl, methylenedioxy and/or cyano, or their salt, if groups capable of forming salts are present. 5. A compound according to claim 1, characterized in that R° represents C1-C4alkyl, hydroxy-C2-C14alkyl, cyano-C1-C4alkyl, carboxy-C1-C4alkyl, C1-C4alkoxycarbonyl-C1-C4alkyl, C3-C7alkenyl or phenyl , or their salt, if groups capable of forming salts are present. 6. A compound according to claim 1, characterized in that R represents an acyl radical having a maximum of 30 carbon atoms, of the partial formula Z-C(=W)- in which W represents oxygen, sulfur or imino, and Z represents hydrogen; hydrocarbyl or hydrocarbyloxy, where hydrocarbyl is an acyclic, carbocyclic or carbocyclic-acyclic hydrocarbon radical that has a total of at most 18 carbon atoms and can be saturated or unsaturated and substituted or unsubstituted, in which the radical can be present in the place of one, two or more carbon atoms , the same or different heteroatoms selected from oxygen, sulfur and nitrogen; or some amino group; or if W represents oxygen or sulfur, Z can also represent chlorine, or their salts if groups capable of forming salts are present. 7. The compound according to claim 6, indicated by the fact that R represents an acyl radical of the partial formula Z-C(=W)- in which W represents oxygen or sulfur, and Z represents C1-C7alkyl, which can also be substituted by halogen, carboxy or C1-C4alkyloxycarbonyl , phenyl, pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, each of which is unsubstituted or substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, nitro, trifluoromethyl, carboxy, C 1 -C 4 alkoxycarbonyl, methylenedioxy and /or cyano; or their salt if groups that can form salts are present. 8. The compound according to claim 1, indicated by the fact that R represents an acyl radical, derived from an organic sulfonic acid, of the partial formula R°-SO2- in which R° represents an acyclic, carbocyclic or carbocyclic-acyclic hydrocarbon radical that has a total of at most 30 carbon atoms of atoms and can be saturated or unsaturated and unsubstituted or substituted, in which radicals can be present in the position of one, two or more carbon atoms, the same or different heteroatoms selected from oxygen, sulfur and nitrogen, or their salt if there are groups that can form salts. 9. The compound according to claim 1, characterized in that R represents an acyl radical, derived from an optionally esterified phosphoric acid of the partial formula [image] in which each of R1 and R2 independently represent hydrogen, an unsubstituted C1-C7 alkyl radical or a phenyl radical that is unsubstituted or substituted with C1-C4 alkyl, C1-C4 alkoxy, halogen and/or nitro, or their salt if groups are present that can form salts. 10. The compound according to claim 1, indicated by the fact that R represents an acyl radical of the partial formula R°b-O- in which R° represents hydrogen, a C1-C19 alkyl group that has a straight chain with more than 5 carbon atoms, or such an alkyl group substituted with amino, halogen and/or carboxy in the form of a free acid, salt, cyano group or C1-C4 alkyl ester, or their salt if groups capable of forming salts are present. 11. The compound according to claim 1, indicated by the fact that R represents an acyl radical of the partial formula R°b-CO- in which R°b represents a C1-C7alkyl radical that can be substituted by halogen, carboxy or C1-C4alkoxycarbonyl, or represents phenyl which may be unsubstituted or substituted with C1-C4 alkyl, C1-C4 alkoxy, halogen, nitro, trifluoromethyl. carboxyl or C1-C4alkoxycarbonyl, or their salt if groups capable of forming salts are present. 12. The compound according to claim 1, characterized in that R represents a bicyclic or monocyclic aroyl radical, which can also carry one or more of the following substituents: halogen atoms of the nitro group, C1-C4 alkyl radicals, hydroxyl groups, C1-C4 alkoxy, phenoxy, methylenedioxy and / or carboxy groups (the latter also in the form of salts or in the form of cyano groups or C1-C4 alkyl ester groups), or some analogous heteroaroyl radical derived from pyridine, furan, thiophene or imidazole or from their analogues with condensed benzo rings, or their salt if groups that can form salts are present. 13. The compound according to claim 1, characterized in that R represents an acyl radical of the partial formula R°-SO2- in which R° represents C1-C7alkyl, phenyl, pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, each of which is unsubstituted or substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, nitro, trifluoromethyl, carboxy or C 1 -C 4 alkoxycarbonyl, methylenedioxy and/or cyano, or a salt thereof if salt-forming groups are present. 14. The compound according to claim 1, characterized in that R represents an acyl radical derived from a carboxylic acid monoester of the partial formula R°-O-CO- in which R° represents a C1-C20-alkyl radical that can be unsubstituted or substituted by carboxy group (also in the form of a salt, cyano group or C1-C4-alkyl ester group), or some analogous linear (mono-to hexa-)-oxaalkyl radical with 4 to 20 chain members, or alternatively some unsubstituted or substituted phenyl or benzyl radical . 15. Compound according to claim 1, characterized in that R represents an acyl radical of the partial formula R°-O-CO- in which R° represents a C1-C7alkyl, phenyl, pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, each of which is unsubstituted or substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, nitro, trifluoromethyl, carboxy, C 1 -C 4 alkoxycarbonyl, methylenedioxy and/or cyano, or a salt thereof if salt-forming groups are present. 16. A compound according to claim 1, characterized in that R represents a carboxylic acid amide of the formula [image] in which W represents oxygen, one of the radicals R1 and R2 is hydrogen and the other is a C1-C7alkyl radical which can be unsubstituted or substituted with hydroxy, mercapto, methylthio, phenyl, p-hydroxyphenyl, p-methoxyphenyl, 2-indolyl, 2-imidazolyl and/or with carboxy (in free form or in the form of a C1-C4 alkoxycarbonyl, carbamoyl or amidino group), one of which is in position 1, or their salt if groups capable of forming salts are present. 17. A compound according to claim 1, characterized in that R represents an acyl radical of the partial formula [image] wherein W is sulfur or oxygen, R1 is hydrogen and R2 is C1-C7alkyl, C3-C7-alkenyl, phenyl, pyridyl, furyl, thienyl, imidazolyl, quinolyl, isoquinolyl, benzofuranyl or benzimidazolyl, each of which is unsubstituted or substituted with C1-C4alkyl, C1-C4alkyl, halogen, nitro, trifluoromethyl, carboxy, C1-C4 alkoxycarbonyl, methylenedioxy and/or cyano, or their salt if groups capable of forming salts are present. 18. Compound according to claim 1, characterized in that R is derived from some α-amino acid selected from glycine, phenylglycine, alanine, phenylalanine, proline, leucine, serine, valine, tyrosine, arginine, histidine and asparagine, or their salts. 19. A compound, indicated by the fact that it is selected from the group consisting of: N-(3-carboxypropionyl)-staurosporine, N-benzoyl-staurosporine, N-trifluoroacetyl-staurosporine, N-methylaminothiocarbonyl-staurosporine and N-phenylcarbamoyl-staurosporine. 20. A compound, indicated by the fact that it is selected from the group consisting of: N-(3-nitrobenzoyl)-staurosporine, N-(3-fluorobenzoyl)-staurosporine, N-tert-butoxycarbonyl-staurosporine, sodium salt of N-(4-carboxybenzoyl)-staurosporine, N-(3,5-dinitrobenzoyl)-staurosporine, N-[(tert-butoxycarbonylamino)-acetyl]-staurosporine and N-(2-aminoacetyl)-staurosporine. 21. Compound according to claim 1, characterized in that it is an addition salt of the formula [image] wherein [Stau] and Rº have the meanings set forth in any of claims 1 to 7, R°b represents hydrogen or an unsubstituted C1-C4 alkyl or benzyl radical, and X- is an anion of an inorganic or organic acid or a carboxy group present in to the radical Rº. 22. The compound according to claim 1, characterized in that it is N-carboxymethylstaurosporine or its pharmaceutically acceptable salt. 23. The compound according to claim 1, characterized in that it is N-ethylstaurosporine or its pharmaceutically acceptable salt. 24. The compound according to claim 1, characterized in that it is N-benzoylstaurosporine. 25. The compound according to claim 1, characterized in that it is N-trifluoroacetyl-staurosporine. 26. The compound according to claim 1, characterized in that it is N-(phenylcarbamoyl)-staurosporine. 27. The compound according to claim 1, characterized in that it is N-glycylstaurosporine or its pharmaceutically acceptable salt. 28. The compound according to claim 1, characterized in that it is N-alanylstaurosporine or its pharmaceutically acceptable salt. 29. A compound according to any one of claims 1 to 28, indicated to be used as a protein kinase C inhibitor. 30. A process for the preparation of a compound of formula 1 or its salt, indicated by the fact that it includes the reaction of staurosporine of the formula [Stau]-NH-CH3 where [Stau] has the meaning described in claim 1, or its acid addition salt, or a) with the reagent of the formula R-Y (III) in which R has the meaning specified in claim 1 and Y is a reactively activated hydroxyl group, or an additional single bond whose other end replaces a hydrogen atom in the radical R, or b) for the preparation of the compound of the formula I in which R represents a radical of the partial formula H-R°a where R°a is a divalent aliphatic radical corresponding to the general structure of the acyclic hydrocarbon radical R° defined in claim 1, with a carbonyl reagent of the formula R°a=O (IV ) in which R°a has the above-mentioned meaning, and simultaneously or consecutively with a reductive reagent, and as desired, the conversion of the resultant compound of formula I into various compounds of formula I and/or the conversion of the compound of formula I obtained in free form into its salt and/or conversion of the compound of formula I obtained in the form of a salt into its free form or into another salt. 31. Pharmaceutical preparation, characterized in that it contains as an active ingredient at least one of the compounds defined in any of claims 1 to 28 and one or more pharmaceutically acceptable additives. 32. Application of the compound according to any one of claims 1 to 28, characterized in that it serves for the preparation of pharmaceutical preparations for the preventive and curative treatment of diseases in which the inhibition of protein kinase C is important.