AT393125B - Cytorhodin S derivatives, process for their preparation and medicinal products containing them - Google Patents

Abstract

Cytorhodin S derivatives, in particular cytorhodin S- immunoglobulin complexes, are described. Cytorhodin S itself has a very high anticancer activity, but the complexes have a more selective action on cancer cells and are more useful as anticancer agents. To prepare cytorhodin S-immunoglobulin complexes, the oxo group on the saccharide chain of cytorhodin S is reduced with an alkali metal cyanoborohydride in the presence of an ammonium salt or of a compound with an amino group to an amino group or substituted amino group. The cytorhodin S derivatives prepared in this way are then linked by means of a bifunctional crosslinker, a condensing agent or a spacer to immunoglobulin. Novel intermediate derivatives are also useful as anticancer agents.

Description

AT 393 125 B

The present invention relates to cytorhodin S derivatives, in particular to cytorhodin S immunoglobulin complexes.

The cytorhodin S derivatives of the invention are selective for cancer cells and are useful as an anti-cancer agent.

It has been reported that cytorhodin S is produced by acid treatment of a crude cytorhodin mixture, which is produced by cultivating Streptomyces Y-11472 (deposit no. DSM-2658) and has anti-cancer effects (Japanese Patent Application Sho 6048994). Although cytorhodin S itself has very high anti-cancer effects, it is nevertheless desirable to develop agents which act selectively on cancer cells and have high bioavailability.

As a result of extensive studies on the development of cytorhodin S derivatives that have superior selective specificity against cancer cells, we have been able to obtain cytorhodin S immunoglobulin complexes that retain the anti-cancer effects of cytorhodin S itself. The present invention has been accomplished based on the above finding.

According to the invention, cytorhodin S derivatives, processes for their preparation and an anti-cancer agent containing them are available as follows 1) A cytorhodin S derivative which is represented by the general formula (I)

3 CH

OH

N-CH

0 3 O OH

CH 3 CH 3 X © is represented, wherein X is a group of the formula -NH2. -NH-R ^ NHj, -NH-R ^ COOH, -NH-R ^ -SH,

or -NH-R3-C-NH (IgG) 1 / n II

O -2-

AT 393 125 B, where R ^ is a divalent hydrocarbon group which can be substituted by any substituent, is a divalent hydrocarbon group, is a divalent organic group, IgG is an immunoglobulin residue, and n is an integer from 1 to 15, and salts thereof. 2) A cytorhodin S derivative according to item 1) above, wherein R ^ is R * or a group of the formula

stands, wherein R4 represents a divalent hydrocarbon group. 3) A cytorhodin S derivative according to item 2) above, wherein R ^ is a propylene group and R4 is a methylene group. 4) A cytorhodin S derivative according to any one of items 1) to 3) above, where n is 5 to 12. 5) a cytorhodin S derivative according to any one of items 1) to 4) above, wherein the IgG is a residue of the antibody against carcinoembiyonic antigen (CEA) 6) a method for producing a cytorhodin S derivative of the general formula (I), wherein X is a group of the formula -NH2, -NH-R ^ NHo,

O

-NH-C-R2-N II O

O or

O

-NH-R1 -NH-C-R2 -N

O -NH-R3 - C'NH-tl sG) χ / α

Is O, where R1 is a divalent hydrocarbon group which can be substituted by any substituent, R ^ is a divalent hydrocarbon group, R ^ is a divalent organic radical, IgG is an immunoglobulin radical, and n is an integer from 1 to 15, -3-

AT 393 125 B which comprises: reducing a Cy torhodin S of the formula (Π)

with an alkali metal cyanoborohydride in the presence of an ammonium salt or a compound represented by NI ^ -R ^ Ni ^, where R * has the same meaning as defined above, to obtain a cytorhodin S derivative, wherein X in formula (I) above - Is NH2 or -NH-R1-NH2, if necessary reacting the product with a maleimide compound of the formula (IQ)

m

wherein R ^ has the same meaning as defined above, to obtain a cytorhodin S derivative CD, wherein X

O

-NH-C- 0 -NH-R1 -4-

AT393 125 B and, if necessary, reacting the product with a thiol derivative of immunoglobulin of the formula (IV) HS-R4-C-NH (IgG) 1 / n. (IV)

II

O

wherein R4 is a divalent hydrocarbon group, IgG has the same meaning as defined above, and n 'is an integer from 1 to 20, to obtain a cytorhodin S derivative (I), wherein X is -NH-R1 2-C-NH (IgG) 1 / n

II

O 7) A method for producing a cytorhodin S derivative according to item 6) above, wherein R2

is. 8) A process for the preparation of a cytorhodin S derivative of the general formula (I), in which X denotes a group -NH-R ^ -SH or -NH-R2-C-NH (IgG) y / jj, in which R * a divalent hydrocarbon group,

O which may be substituted by any substituent means R2 means a divalent organic group, IgG means an immunoglobulin residue, and n means an integer from 1-15 which includes; reducing a cytorhodin S of formula (II) mentioned above with an alkali metal cyanoborohydride in

Presence of a compound represented by bffl ^ -R ^ -SH, where R * has the same meaning as defined above, to obtain a cytorhodin S derivative (I), wherein X in the above-mentioned formula (I) -ΝΉ-R * -SH and, if necessary, reacting the product with a maleimide derivative of immunoglobulin of the formula (V)

-C-NH-tl gG) II O 1 / n '(V) -5- 1 2

wherein R and IgG have the same meanings as defined above, and n * is an integer from 1-20, to obtain a cytorhodin S derivative (I), wherein X

AT 393 125 B -NH-R3-C-NHflgG) 1 / n

II is 0. 9) A method for producing a cytorhodin 5 derivative according to item 8) above, wherein R3 0

is. 10) A process for the preparation of a cytorhodin S derivative of the general formula (I), in which X denotes an -NH * R * -COOH or -NH-R3-C-NH (IgG) j ^ group, in which R1 a divalent

O

Means hydrocarbon group, which can be substituted by any substituent, R3 means a divalent organic group, IgG means an immunoglobulin residue, and n means an integer from 1-15, which includes reducing cytorhodin S of formula (II) above an alkali metal cyanoborohydride in the presence of a compound represented by N ^ -R ^ -COOH, in which R * has the same meaning as defined above and, if necessary, reacting the product with an immunoglobulin of the formula (VI) NH2agG) 1 / nm in which IgG is the same Meaning as defined above, and n is an integer from 1-15, to obtain a cytorhodin S derivative (I), wherein X in the formula (0 -NH-R3-C-NH (IgG) 1 / n

II

O is. 11) A method for producing a cytorhodin S derivative according to item 10) above, wherein R3 is R *. 12) An anti-cancer agent containing a cytorhodin S derivative represented by the above formula 0).

Brief description of the drawing

1 shows the mass spectrum of cytorhodin S-aminoderivaL

Hg. 2 shows elution patterns of the cytorhodin S immunoglobulin complex and of cytorhodin S in column chromatography.

3 shows the UV spectrum of the cytorhodin S immunoglobulin complex.

Fig. 4 graphically shows the cell growth inhibitory activities of cytorhodin S and the cytorhodin S-rabbit-immunoglobulin complex against L1210 cells.

Figure 5 graphically shows cell growth inhibitory efficacy of cytorhodin S, cytorhodin S-MA 204-Imm uncomplex, cytorhodin S-rabbit immunoglobulin complex, and control against Colo 205 cells.

Figure 6 graphically shows the cell growth inhibitory efficacy of the cytorhodin S immunoglobulin complex and the aclacinomycin immunoglobulin complex against L1210 cells.

Detailed description of the invention

As preferred examples of R * in the formula (I) given above, groups are mentioned which are composed of known a-amino acids, β ·, γ. and ε-amino acids and ß-, γ- and ε-thiolamines. -6-

AT 393 125 B

As preferred examples of R in the above formula (I), lower alkylene groups such as methylene, ethylene and propylene, phenylene groups such as p-phenylene and m-phenylene, C 1 -C 4 cycloalkylene groups such as cyclohexylene, phenylenyl lower alkyl groups such as phenylenylmethyl, and C ^ -Cj-Cycloalkenyl-lower alkyl groups, such as cyclohexylenylmethyl, mentioned. Groups which are represented by the formulas are mentioned as preferred examples

O

be represented.

As preferred examples of lower alkylene groups such as methylene, ethylene and propylene, phenylene groups such as p-phenylene and m-phenylene groups, C ^ -C ^ cycloalkylene groups such as cyclohexylene, phenylenyl-lower alkyl groups such as phenylenylmethyl, and C ^ -Cy -Cycloalkylenyl-lower alkyl groups, such as cyclohexylenylmethyl, mentioned. The immunoglobulin residue represented by IgG means an immunoglobulin residue in which n amino groups are removed from an immunoglobulin. IgG in formula (I) includes one in which some of the thiol groups (-NH-C-R ^ -SH) introduced into the immunoglobulin remain.

This is because not all of the thiol groups in the thiol derivative of immunoglobulin (IV) are reacted with the cytorhodin S derivative, and some of the ’lhiol groups remain unreacted.

An antibody that specifically binds to cancer cells is preferred as the immunoglobuliniest. 1 / n indicates the number of immunoglobulin molecules per molecule of cytorhodin S, which is 1/1 to 1/15. This means that 1 to 15 molecules of the cytorhodin S are bound to one molecule of the immunoglobulin.

A compound represented by formula (I), wherein X -NH2, -NH-R ^ NH ^ -NHR1 -COOH, -NHR ^ SH,

O

or R2 - N -NH-R-c-

O -7-

AT 393 125 B is an intermediate compound for the production of immunoglobulin complexes which of course maintains anti-cancer effects of Cytorhodin S and, as it is, can be an anti-cancer agent.

In general, when a complex is formed between an anti-cancer agent and immunoglobulin, the anti-cancer agent is bound to the immunoglobulin by means of a bifunctional crosslinking agent, a condensing agent or a spacer. The above objective is achieved by combining an anti-cancer agent with a bifunctional crosslinking agent, a condensing agent or a spacer via a functional group (such as an amino, carboxyl or thiol group) of the former. However, cytorhodin S has no such functional groups and should necessarily be chemically modified to a

10 Heating up a derivative containing amino, carboxyl or thiol groups In order to obtain such derivatives of cytorhodin S without losing its anti-cancer effects, the oxo group on the saccharide chain can be converted into an amino group.

General procedures for reducing the oxo group include the use of lithium aluminum hydride (L1AIH4), sodium borohydride (NaBH ^) or the like. The reagent that can be used to selectively reduce the oxo group on the IS saccharide chain without reducing the oxo group on the anthracycline ring is sodium cyanoborohydride ( NaBCNHg) or lithium cyanoborohydride (LiBCNHß). Will be one

If reducing agents are used in the presence of an ammonium salt or an organic compound containing amino group (s), the oxo group on the saccharide chain is selectively reduced to the amino group, the derivative of cytorhodin S being formed. In fact, cytorhodin S of formula (Π) 20

25 30 35 40 45 into a cytorhodin S derivative (I), in which X is -NHj or -NH-R ^ -N ^, -NH-R ^ -COOH or -NHR ^ SH, by reduction with an alkali metal cyanoborohydride in the presence of an ammonium salt or an organic compound containing an amino group represented by NHj-R ^ -N ^, Ni ^ -R ^ -COOH or NHj-R ^ -SH

Examples of the ammonium salt include ammonium acetate, ammonium propionate and 50 ammonium chloride. Known α-amino acids such as lysine and cysteine, β-, γ- and ε-amino acids such as γ-aminobutyric acid, amino acid esters such as the ethyl ester of glycine and thiolamines such as aminoethanethiol are examples of the amino compound (s) containing organic compound and aminothiophenol mentioned. Examples of the alkali metal cyanoborohydride include lithium (or sodium) cyanoborohydride.

The above reaction is conveniently accomplished by dissolving cytorhodin S and an ammonium salt or an organic compound containing 55 amino group (s) in a water-miscible organic solvent (such as methanol or ethanol) or in any one containing no primary and secondary amines , buffer solution adjusted to pH 8 (such as a phosphate or borate buffer or 2,4,6-trimethylpyridine-hydrochloric acid buffer) and addition of an alkali metal cyanoborohydride to the solution at room temperature or with ice cooling -8-

AT 393 125 B

The product contained in the reaction mixture can be obtained by conventional means. For example, it is obtained by adjusting the reaction mixture to pH 8, extraction with a suitable organic solvent, such as dichloromethane, and distilling off the solvent from the extract, or by means of preparative HPLC.

The amino derivative of cytoihodin S thus obtained, in which X is -NH2 or -NH-R ^ N ^, is then reacted with a maleimide compound of the above formula (ΠΙ) to form a cytorhodin S derivative of the above formula (I), in which X 0

and O 'λ

-NH-R1-NH-C-R2-N II O is implemented. N-hydroxysuccinimide is formed as a by-product.

Examples of the maleimide compound (ΙΠ) are mentioned: Ng-maleimidobutyiyloxy-succinimide [GMBS, R2: (CH ^ ß], succinimidyl-4- (N-maleimidtMethyl) -cyclohexanecarboxylate (R2 :—( > —CH2-), m -

Maleimidobenzoyl-N-hydroxysuccinimide ester (R2: -) and Suecinimidyl 4- (p-maleimidophenyl) butyrate (R2: (CH2) 3- £ ^ -).

The above reaction can be conveniently carried out by treating a cytorhodin S-amino derivative with a maleimide compound in a suitable solvent (e.g. dichloromethane, dichloroethane, chloroform, pyridine, triethylamine, etc.) at room temperature or under ice-cooling.

The cytorhodin S derivatives thus obtained, into which a maleimide group has been introduced, are suitable for undergoing an addition reaction with any of the thio group-containing proteins or any of the thiol group-containing compounds. The double bond of the maleimide group takes part in the electrophilic addition reaction with the thiol group. Therefore, as described below, the addition reaction between the maleimide group and the thiol group introduced into the immunoglobulin enables a complex to be formed.

In general, the immunoglobulin used to prepare the complex is preferably a monoclonal or polyclonal antibody against the membrane protein specifically formed in cancer cells. It is also desirable in clinical use to use the antibody from which the Fc portion has been removed if an antibody of non-human origin is used. Taking into account the effectiveness in the addition of cytorhodin S, however, the entire antibody is preferably used.

A particularly preferred immunoglobulin is a monoclonal antibody against cancer-specific protein, such as carcinoembryonic antigen (CEA) or α-fetoprotein (AFP). The monoclonal antibody against carcinoembryonic antigen is produced by a conventional method in which mice are treated with the antigen, the spleen is isolated from the mice, the spleen cells are fused with HAT-sensitive myeloma cells according to the polyethylene glycol method, and the hybridoma using HAT Medium is selected as the selection medium and transplanted into mice.

Before the reaction of immunoglobulin with cytorhodin S, which has a maleimide group to produce a complex, a thiol group is introduced into the immunoglobulin. In order to introduce the thiol group, the immunoglobulin is treated with an N-succinimide compound of the general formula (VII) -9-

AT 393 125 B

wherein has the same meaning as defined above, and a thiol protecting group, e.g. B. means an acyl group such as acetyl, or a 2-pyridylthio group, to obtain a thiol derivative of immunoglobulin with the above-mentioned formula (IV).

As a preferred example of the N-succinimide compound (VII), N-succinimidyl-S-acetylthioacetate (ch3-c-s-ch2-c-o-n I! IIo o

V, SATA) mentioned. SATA becomes 0 with the amino group in the immunoglobulin to form an amide bond -NH-C-CHj-S-C-CHj

II II

O O implemented in which the thiol group is protected by an acetyl group, and N-hydroxysuccinimide is formed as a by-product. The deprotection of the thiol group is e.g. B. with hydroxylamine at pH 7.5 to convert the former into the reactive group -NH-C-CH2-SH.

II

O

The thiol group is suitable to be oxidized under certain conditions with the result that immunoglobulin polymers are formed. It is therefore desirable to cleave the acetyl protecting group at 25 ° C for about 1 hour and immediately before the reaction with a cytorhodin S derivative having the maleimide group.

Depending on the amount of SATA used, the thiol group is introduced in a proportion of 1-20, preferably 5-20, per molecule of immunoglobulin. The introduction does not result in a loss of antibody activity of the immunoglobulin.

The use of N-succinimidyl-3- (2-pyridyldithio) propionate

O CH2-CH2-C-0-N, QSS- as a thiol introducing reagent instead of SATA and the use of dithiothretol as a deprotecting agent, together with the amino group in the immunoglobulin, leads to the formation of the group -NH-C- CI ^ CHj-SH.

II

O

The immunoglobulin (IV) which has a thiol group and is formed in this way is reacted with the maleimide-containing cytorhodin S to produce the immunoglobulin complex according to the invention.

The above reaction can be conveniently carried out by mixing a solution of the maleimide group-containing cytorhodin S derivative in a water-miscible organic solvent (e.g. methanol, ethanol, dimethylformamide) with an aqueous solution of the thiol group-containing immunoglobulin at pH 7.5.

Cytorhodin S derivatives with an introduced thiol group in which X is -NH-R ^ -SH are capable of forming a -10- with any protein or any compound with an introduced maleimide group.

AT 393 125 B

Enter addition reaction. The double bond of the maleimide group is reacted with the thiol group by an electrophilic addition reaction. Therefore, as described below, by adding the thiol group to the maleimide group, a complex can be formed in the immunoglobulin with the maleimide group introduced.

The immunoglobulin to be used to prepare the complex is as described above. S Before the reaction of immunoglobulin with an introduced thiol group containing cytorhodin S maleimide groups are introduced into the immunoglobulin. In order to introduce the maleimide groups, immunoglobulin is mixed with a maleimide compound of the formula (wird)

Λ wherein R has the same meaning as defined above, with the formation of a maleimide derivative of 20 immunoglobulin with the formula (V) given above. Preferred examples of the maleimide compound (III) are Ν-γ-maleimidobutyryloxysuccinimide and others as mentioned above.

The maleimide compound (ΙΠ) forms an amide bond through a nucleophilic substitution reaction with amino groups in the immunoglobulin, a maleimide derivative of immunoglobulin of the formula (V)

25 30 and, as a by-product, N-hydroxysuccinimide arise. 35 Depending on the amount of the maleimide compound (ΠΙ), the maleimide group is introduced in a proportion of 1-20, and preferably 5-20, per molecule of the immunoglobulin. However, the introduction does not lead to a loss of effectiveness of the immunoglobulin as an antibody.

The immunoglobulin complexes of the invention are prepared by reacting the introduced maleimide group-containing immunoglobulin (V) with the cytorhodin S having an introduced thiol group. The reaction is conveniently carried out by mixing a pH 7.5 solution of the cytorhodin S having an introduced thiol group in one with Water-miscible solvent (e.g. methanol, ethanol, dimethylformamide or the like) or a buffer solution of pH 7.5 (the same as that used to dissolve immunoglobulin) with an aqueous solution of the immunoglobulin with maleimide group introduced. 45 The cytorhodin S derivatives in which X is -NH-R ^ -COOH are suitable for binding with amino groups of a protein or a compound with a peptide bond with the aid of a corresponding condensing agent. The peptide bond is formed by nucleophilic reaction of an active ester of the carboxyl group which has been formed by the condensing agent with the amino group in the protein or the compound 50. Therefore, a complex can be formed by introducing a cytorhodin S derivative via a peptide bond with the into the amino group (side chain, like lysine residue), which was originally contained in the immunoglobulin, newly introduced carboxyl group.

The cytorhodin S derivatives in which X is -NH-R ^ -COOH are converted into an active ester by a condensing agent to form a complex. To produce the active ester, the 55 cytorhodin S derivative in which X in the above formula (I) -NH-R ^ -COOH is reacted with a carbodiimide.

As preferred examples of the carbodiimide, dicyclohexylcarbodiimide, 1,3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, diethylcaibodiimide, diphenylcarbodiimide, dibenzylcarbodiimide, cianamide and the like. The reaction is carried out by dissolving a solution of the cytorhodin S derivative with the 60 carbodiimide in a water-miscible organic solvent (e.g. methanol, ethanol, -11-

AT 393 125 B

Dimethylformamide or the like) or in a buffer solution at pH 7.5 (the same as that used to dissolve immunoglobulin) at room temperature for 1 hour, after which the reaction mixture to an aqueous solution of the immunoglobulin at pH 7.5 is added. bi Depending on the amount of the cytorhodin S derivative used for the reaction, the cytorhodin S derivative is bound in a proportion of 1-20, and preferably 5-20, per molecule of the immunoglobulin. However, the binding does not lead to a loss of the effectiveness of immunoglobulin as an antibody.

The binding ratio of cytorhodin S to immunoglobulin in the complex according to the invention is 1-15: 1, i.e. H. 1-15 molecules of cytorhodin S are bound per molecule of immunoglobulin.

Such a high proportion of the anti-cancer component is effective in producing high anti-cancer activities.

The clinical dose for the cytorhodin S derivative of the invention can be varied depending on the route of administration and is in the range of 1 to 20 mg per day in adults in terms of cytorhodin S.

The dose can be given intravenously, intraperitoneally or rectally. In the case of intravenous administration, an intravenous drip can be used in addition to the usual intravenous injection.

Pharmaceutical preparations which contain the cytorhodin S derivative (Γ) are produced by customary processes using customary carriers and additives

The injectable preparation can e.g. B. be a powdered preparation for injection purposes. It is by adding one or more appropriate excipients, such as. B. mannitol, sucrose, lactose, maltose, glucose and fructose, to the cytorhodine S derivative (Ί), dissolving the mixture in water and dividing the solution into vials or ampoules and then lyophilizing and sealing

The rectal preparation can be made from a lipophilic base, such as the semi-synthetic base, which is prepared by mixing cocoa oil or a fatty acid triglyceride with a fatty acid monoglyceride or a fatty acid diglyceride in various proportions, or a hydrophilic base, such as a polyethylene glycol or a glycerogelatin, while heating the mixture to a solution and adding the solution to a homogeneous mixture and forming the preparation in a mold.

Examples of the invention are given below.

example 1

10 mg of cytorhodin S were dissolved in 2 ml of methanol. A methanol solution (concentration 10 mg / ml) of 5.8 mg of ammonium acetate, which corresponds to 10 molar equivalents of cytorhodin S, and a methanol solution (concentration 10 mg / ml) of 1 were added to the solution , 4 mg of sodium cyanoborohydride, which corresponds to 3 molar equivalents, was added. The mixture was reacted for 4 hours at 25 ° C. The reaction mixture was poured into 20 ml of cooled, 0.5% strength aqueous acetic acid, and the mixture was mixed with 2 portions of 5 ml Extracted dichloromethane to remove unreacted cytorhodin S. The resulting aqueous acetic acid solution was adjusted to pH 8 with saturated aqueous sodium bicarbonate solution. The aqueous solution was extracted with 3 portions to 20 ml dichloromethane, an amino derivative being kept. The dichloromethane solution was dried over anhydrous sodium sulfate and the dichloromethane was removed by vacuum distillation. 4.8 mg of an amino derivative of cytorhodin S (hereinafter referred to as aminocytorhodin S) were obtained. The product was analyzed for purity by high pressure liquid chromatography. The conditions were as follows Column SSC-Aquasil PE-2.4.6 mm x 250 mm (Senshu Kagaku); Solvent 2.0% triethylammonium phosphate (pH 3), 75%, and acetonitrile, 25%; Retention time cytorhodin S (25.0 min), aminocytorhodin S (12.9 min). In addition, the mass number for the cytorhodin S derivative obtained by HPLC was measured by means of an FAB / MS spectrometer (Nihon Denshi JMS-DX300), a mass spectrum of 942 (M + H *) being obtained, which was calculated using the calculated mass number was identical.

A specific absorption at 495 nm was observed in the absorption spectrum of the derivative, which originates from the anthracycline ring of the cytorhodin S. This derivative shows the same spectrum as the starting cytorhodin S, from which it follows that the anti-cancer effects in the derivative have not been lost.

Example ^

4.8 mg of the aminocytorhodin S prepared in Example 1 were dissolved in 2 ml of dichloromethane. A dichloromethane solution (concentration 10 mg / ml) of 3.8 mg Ν-γ-maleimidobutyryloxysuccinimide (manufactured by Behring Diagnostics, hereinafter referred to as GMBS) was added to the solution, which corresponds to 5 mol equivalents of aminocytorhodin S. The mixture was reacted at 25 ° C for 2 hours. The reaction mixture was purified by preparative thin layer chromatography, whereby a GMBS derivative of cytorhodin S was obtained. The conditions for thin layer chromatography are as follows: thin layer plate, 2 mm silica gel 60F-254 (manufactured by Merck); Development solvent, methanol: dichloromethane = 1: 4; Rf value of the GMBS derivative (0.82), the aminocytorhodin S (0.29): The GMBS derivative thus produced was extracted from the thin-layer plate, the extract was dried over anhydrous sodium sulfate and the solvent was removed by -12-

AT 393 125 B

Vacuum distillation removed. 2.7 mg of the GMBS derivative of cytorhodin S were obtained. Furthermore, the purity of the GMBS derivative was analyzed by HPLC, and the purification was carried out. The column and the condition used in the analysis were as follows: column, SSC-Aquasil PE-2.4.6 mm x 250 mm (Senshu Kagaku); Solvent, acetonitrile in 2.0% triethylammonium phosphate (TEAP, pH 3.0) was added over a 30 min period using a linear gradient of 0% - 30% and the state of 30% acetonitrile was maintained for an additional 30 min; Flow rate 1 ml / min; Retention time 36 min for cytorhodin S and 38 min and 38.6 min for the GMBS derivative. The column and the conditions used for the purification were as follows: column, Senshu Pak NP-318-4252.10 mm x 250 mm (Senshu Kagaku); Solvent, acetonitrile in 2.0% TEAP (pH 3.0) was added over a 20 min period in a linear gradient of 10% - 30% and the state of 30% acetonitrile was maintained for an additional 15 min. In the same way, a change was made over a period of 5 minutes with a gradient of 30% - 100%; Flow rate 4 ml / min; Retention times 28 min and 28.5 min for the GMBS derivative. The eluate from the HPLC was applied to a SEP-PAK (Cjg) cartridge (manufactured by Nihon Waters) to adsorb the cytorhodin S derivative, followed by washing with 0.5% aqueous acetic acid and 0.5 % acetic acid-methanol solution was eluted. The eluate was collected and dried under reduced pressure, holding about 3.2 mg of the GMBS derivative. The purity of the product was checked again under the same conditions as described above by HPLC. In addition, the mass number of said cytorhodin S derivative obtained by HPLC was measured by an FAB / MS spectrometer (manufactured by Nihon Denshi, JMS-DX300), a mass spectrum of 1215 (M + H4) was obtained, which was confirmed by the »calculated mass number and was the mass number of a complex with thioglycerin, which had been used in the measurement of the mass spectrum. The specific absorption of the anthracycline ring around 495 nm was also well maintained in the absorption spectrum of the derivative.

In addition, the introduction of the maleimide group was also confirmed by the reaction with aminoethanethiol, in which the positions d spots and peaks in thin-layer chromatography and in d HPLC were changed. The column and HPLC conditions used in d »analysis are the same as described above. Retention time: 19.5 min and 20.5 min for the aminocytorhodin S, and 23 min and 23.5 min for the GMBS-D'ivat-Aminoethanthk > l adduct (A comparison was made using a mixture of the starting aminocytorhodin S with aminoethanethiol, produced under the same conditions as with the GMBS derivative. It was found that the aminocytorhodin S was not influenced at all by the incorporation of the thiol, as far as the peaks of the HPLC are concerned.)

BsispisU.

In 1 ml of 50 mM phosphate buffer solution (pH 7.5) containing 1 mM EDTA, 18 mg of rabbit immunoglobulin (manufactured by Sigma) was dissolved. To the solution was added 10 µΐ 100 mM dimethylformamide solution of N-succinimidyl-S-acetylthioacetate (manufactured by Behring Diagnostics, hereinafter referred to as S ATA), and the mixture was reacted at 25 ° C for 20 minutes. To the reaction mixture was added 50 μl of 1M Tris hydrochloride solution (pH 7.8) and the mixture was allowed to stand at 25 ° C. for 5 minutes. The reaction mixture was then fractionated on a PD-10 column (manufactured by Pharmacia), which had been brought into equilibrium with 50 mM phosphate buffer solution (pH 7.5) containing EDTA. An S ATA derivative of immunoglobulin was obtained. 0.5 mM aqueous hydroxylamine solution (pH 7.5) containing 0.25 mM EDTA, which corresponds to 50 equivalents of SATA, was added to the S_ATA derivative. The mixture was reacted for 1 hour at 25 ° C for deacylation. To the above aqueous solution containing immunoglobulin with introduced SH group, a solution of the GMBS derivative which was 10 times equivalent to the SH group in 20 μΐ dimethylformamide was added, and the mixture was stirred at 5 for 12 hours ° C implemented. After the reaction was completed, the reaction mixture was fractionated on a 20 mm x 400 mm Sephadex G-25 column (manufactured by Pharmacia), which was equilibrated with 50 mM phosphate buffer solution (pH 7.5) containing 1 mM EDTA had been brought. A fraction corresponding to protein 160,000 was isolated. The eluate was monitored at 280 nm and 495 nm in order to identify protein and cytorhodin S fractions.

The cytorhodin S-immunoglobulin complex thus obtained was desalted by dialysis and lyophilized to give 20 mg of a product. The amount of bound cytorhodin S per molecule of the immunoglobulin was calculated from the absorption specific for cytorhodin S at 495 nm and from the dry weight of the complex, it being found that 5 molecules of cytorhodin S were bound per molecule of the immunoglobulin. In addition, 12 molecules of cytorhodin S could be bound by increasing the amount of SATA.

3 shows the UV spectrum of the complex, in which approximately 12 molecules of cytorhodin S are bound per molecule of the immunoglobulin. The UV spectra of the complex show ") that as the proportion of bound cytorhodin S" increases, the absorption maximum for cytorhodin S increases -13-

AT 393 125 B

Example 4

A methanol solution of 2.6 mg of y-aminobutyric acid (concentration 1 mg / ml), which corresponds to 6 moles per mole of cytorhodin S, was added to a solution of 5 mg of cytorhodin S in 1 ml of methanol. After adjusting the pH to 8 with triethylamine, 1.4 mg of sodium cyanoborohydride, corresponding to 3 molar equivalents, was added, and the mixture was reacted at 25 ° C for 24 hours. The reaction mixture thus formed was checked for the presence or absence of the product by thin layer chromatography, whereupon an analysis for purity by means of HPLC and a purification were carried out. The Rf values in thin layer chromatography under the same conditions as in Example 2 were 0.57 for cytorhodin S and 0.28 for the γ-aminobutyrate derivative. The retention times in the HPLC analysis under the same conditions as in Example 2 were 36 min for Cytorhodin S and 29 min for the γ-aminobutyrate derivative. Purification by HPLC under the same conditions as in Example 2 gave about 14 mg of the γ-aminobutyrate derivative. In addition, measurements of the mass number of the derivative mentioned using an FAB / MS spectrometer (JMS-DX300) gave a mass spectrum of 1028 (M + H *), which was identical to the mass number as calculated.

In addition, the specific absorption around 495 nm for the anthracycline ring of cytorhodin S was well maintained in the absorption spectrum of the derivative.

Example 5

A DMF solution of 515 pg dicyclohexylcarbodiimide (concentration 50 mg / ml), which corresponds to an equimolar amount, based on the cytorhodin S derivative, was added to a solution of 24 mg of cytorhodin syaminobutyiate derivative in DMF (concentration 50 mg / ml) , added. The mixture was reacted at room temperature for 1 hour.

To a solution of 20 mg of rabbit immunoglobulin (manufactured by Sigma) in 1 ml of 50 mM phosphate buffer solution (pH 7.5) was added the DMF solution of the cytorhodin S derivative prepared above. The mixture was reacted at room temperature for 30 minutes. The cytorhodin S-y-aminobutyrate derivative corresponds to 20 molar equivalents, based on the rabbit immunoglobulin. Then, IM Tris-hydrochloric acid buffer solution (pH 7.8) was added rapidly, and the mixture was stirred and allowed to stand for 10 minutes. The resulting mass was applied to a PD-10 column (manufactured by Pharmacia), which had previously been equilibrated with 50 mM phosphate buffer solution, for separation and purification.

Example 6

To a solution of 5 mg of cytorhodin S in 1 ml of methanol were added an 1M aqueous acetic acid solution of 3.3 mg of lysine (concentration 10 mg / ml), corresponding to 6 mol equivalents, based on the cytorhodin S, and 1.4 mg of sodium cyanoborohydride, corresponding to 3 mol. Equivalents added. The mixture was reacted for 24 hours at 25 ° C. The reaction mixture thus obtained, after checking for the presence or absence of product, was analyzed for purity by HPLC and then purified. The Rf value for the lysine derivative was 0.20 when the thin layer chromatography was carried out under the same conditions as in Example 2.

The retention time for the lysine derivative in the HPLC analysis under the same conditions as in Example 2 was 27 min and 28 min. Purification by HPLC under the same conditions as in Example 2 gave about 2.6 mg of the lysine derivative. In addition, the mass number of the derivative mentioned was measured using a FAB / MS spectrometer (JMS-DX300), a mass spectrum of 1071 (M + H *) being obtained, which was identical to the calculated mass number.

In addition, a specific absorption around 495 nm for the anthracycline ring of cytorhodin S was well retained in the absorption spectrum of the derivative.

Example 7

To a solution of 5 mg of cytorhodin S in 1 ml of methanol was added a methanol solution of 1.7 mg of aminoethanethiol (concentration 1 mg / ml), corresponding to 6 molar equivalents, based on the cytorhodin S, and 1.4 mg of sodium cyanoborohydride, corresponding to 3 Mol equivalents added. The mixture was reacted at 25 ° C for 24 hours. The reaction mixture thus obtained was checked for purity by HPLC and then purified by HPLC for the presence or absence of the product. The Rf value for the aminoethanethiol derivative in thin layer chromatography was under the same condition «! as in Example 2 0.25. The retention time in the HPLC analysis under the same conditions as in Example 2 was 28 min for the aminoethanethiol derivative. Purification by HPLC under the same conditions as in Example 2 gave about 1.6 mg of the -14-

AT 393 125 B

Aminoethanethiol derivative. In addition, the mass number of the derivative mentioned was measured by means of an FAB / MS spectrometer (JMS-DX300), a mass spectrum of 1002 (M + H *) being obtained which was identical to the calculated mass number.

In addition, a specific absorption around 495 nm for the anthracycline ring of cytorhodin S was well maintained in the absorption spectrum of the derivative.

On the other hand, the introduction of the -SH group was additionally confirmed by the reaction with N-ethylmaleimide, the position of the spots and peaks changing in thin-layer chromatography and HPLC. (The changes observed for the aminoethanethiol derivative were not observed for cytorhodin S and aminocytarhodin S)

Example 8

A methanol solution of 2.7 mg cysteine (concentration 1 mg / ml), corresponding to 6 mol equivalents, based on cytorhodin S, and 1.4 mg sodium cyanoborohydride, corresponding to 3 mol, were added to a solution of 5 mg cytorhodin S in 1 ml methanol Equivalents, added. The mixture was reacted for 24 hours at 25 ° C. The reaction mixture thus obtained was analyzed for purity by HPLC after checking for the presence or absence of the product by thin-layer chromatography and then purified. The Rf value for the cysteine derivative at thin layer chromatography under the same conditions as in Example 2 was 0.35. The retention time for the cysteine derivative in the HPLC analysis under the same conditions as in Example 2 was 32 min. Purification by HPLC under the same conditions as in Example 2 gave about 1.8 mg of the cysteine derivative. In addition, the mass number of the above derivative was measured by an FAB / MS spectrometer (manufactured by Nihon Denshi), whereby a mass spectrum of 1046 (M + H +) was obtained, which was identical to the calculated mass number.

In addition, the introduction of the -SH group was confirmed in the same manner as in Example 7 by reaction with N-ethylmaleimide, whereby the positions of the spots and peaks (thin layer chromatography and HPLC) changed.

EsisäsLS

To a solution of 11 mg of rabbit immunoglobulin (manufactured by Sigma) in 1 ml of 50 mM phosphate buffer solution (pH 7.5) containing 1 mM EDTA, a DMF solution of 191 μg Ν-γ-maleimidobutyryloxysuccinimide (manufactured by from Behring Diagnostics, hereinafter referred to as GMBS) (concentration 50 mg / ml). The mixture was reacted at 25 ° C for 2 hours. To the reaction solution was added 50 μl of 1M Tris-hydrochloric acid buffer solution (pH 7.8), and the mixture was allowed to stand at 25 ° C. for 5 minutes. The reaction mixture was then fractionated on a PD-10 column (manufactured by Pharmacia), which had previously been equilibrated with 50 mM phosphate buffer solution (pH 7.5) containing 1 mM EDTA, using a rabbit immunoglobulin of the maleimide-introduced type. The inserted SH group cytorhodin S derivative as generated in Example 7 above was reacted with the maleimide type rabbit immunoglobulin.

A solution of 1.5 mg of the cytorhodin S-aminoethanediol derivative in DMF (concentration 100 mg), which corresponds to 2 molar equivalents based on the maleimide group introduced into the rabbit immunoglobulin, was introduced into an aqueous solution of the rabbit immunoglobulin type with the introduced one Maleimide as prepared above was added. The mixture was reacted at 5 ° C for 12 hours

BeigpifiUD

To a solution of 5 mg of cytorhodin S in 1 ml of methanol were added a methanol solution of 3.2 mg of glycine ethyl ester (concentration 1 mg / ml), corresponding to 6 molar equivalents, based on the cytorhodin S, and 1.4 mg of sodium cyanoborohydride, corresponding to 3 Molar equivalents added The mixture was reacted at 25 ° C for 24 hours. The reaction mixture thus obtained was checked for the presence or absence of the product for purity by HPLC and then purified by thin-layer chromatography. The RF value for the glycine ethyl ester derivative under the same conditions as in Example 2 was 034. The retention time was 034. The retention time for the glycine ethyl ester derivative in the HPLC analysis carried out under the same conditions as in Example 2 was 32 min and 33 min. Purification by HPLC under the same conditions as in Example 2 gave about 1.4 mg of the glycine ethyl ester derivative. In addition, the mass number of the derivative mentioned was measured by means of a FAB / MS spectrometer (JMS-DX300), a mass spectrum of 1028 (M + H +) being obtained, which is identical to the calculated mass number.

A specific absorption around 495 nm for the -15-

AT 393 125 B

Anthracycline ring of Cytoihodin S well maintained. ghostly

To female BALB / C mice 7-10 weeks old, 25 pg of purified human CEA per mouse was administered intraperitoneally together with complete Freund's adjuvant. After 10 weeks, a solution of 25 pg CEA in physiological saline was administered intravenously. On the third day after administration, the spleen was isolated and the spleen cells were fused with non-productive type X63-Ag 8,653 myeloma cells. Cell fusion and subsequent cultivation and goning were fundamentally carried out according to the method of Oi and co-workers (Oi VT & Herzberg, LA, Selected methods in Cellular Immunology, BB Mishell & SM Shiigi, ed., P. 351, Freeman & Co ., San Francisco 1980). The ratio of the spleen cells to the myeloma cells was 10: 1. The mixture was centrifuged for a short time to obtain a bead. 0.5 ml of 42.5% polyethylene glycol (MW 2,000, containing 15% dimethyl sulfoxide) was added dropwise to the bead and the mixture was slowly stirred at 37 ° C. for 2 minutes. The resulting mixture was mixed with RPMI medium in a ratio of 20 : 1 diluted After being centrifuged, it was suspended in RPMI-1640 medium containing bovine embryonic serum. The suspension was distributed on a 96-well microtiter plate in a quantity of 5 × 10 4 cells each. After incubation overnight at 37 ° C., a drop of HAT medium (100 μΜ hypoxanthine, 4 × 10 ** μΜ aminopterin, 1.6 × 10 ′ ^ μΜ thymidine) was added

The selection of hybridomas using HAT medium was carried out on days 2, 3, 5, 7, 10 and 13 by exchanging half of the medium in each hole with fresh HAT medium. The anti-CEA activity of the supernatant from the culture was tested by RIA or £ IA. The cells which were found to be active in the test were transferred to a larger culture system or cloned using a restrictive dilution method. The fused cells thus obtained were found in the peritoneum was transplanted from mice of the same strain that had previously been treated with 0.5 ml of tetramethylpentadecane. One to two weeks after the transplant, several nd ascites were obtained in which homogeneous, monoclonal antibodies were contained in a concentration of 5-10 mg / ml.

A cytorhodin S complex with the monoclonal antibody against CEA (MA 204) thus obtained was prepared in the same manner as in Example 3

Testing Cvtorhodin S Immunoglobulin Comolex for Anti-Cancer Effectiveness

200 μl of tumor cell line L1210 (5x10 * cells / ml), which had been grown in RPMI1640-10% fetal calf serum (1 x 10 * cells / hole), were distributed in a 96-well microtiter plate. 10 μl of cytorhodin S (CYT) and cytorhodine S immunoglobulin complex [CYT rabbit - (= Rab) IgG] were added to the cell culture. The mixture was incubated at 37 ° C. for 18 hours. Then 25 μl of tritiated thymidine were added at a concentration of 0.5 µΟ / well and the mixture was incubated at 37 ° C for 6 hours. Then, the cells were separated by a cell collector on a filter, dried, and the amount of the labeled thymidine incorporated into the cell was measured by a candle scintillation counter for comparison of the DNA synthesis activity of the cell. The results are shown in Ed. 4. FIG. 4 shows that the anti-cancer activity of CYT is approximately retained in the CYT rabbit IgG.

In the same way, the effect of the cytorhodin S derivative (cytorhodin S-anti-CEA-monoclonal-antibody complex) was tested on Colo 205, which is a CEA-producing cell. The results are shown in FIG. 5. The amounts of those used Test materials were as follows

Samples: drug / antibody, ratio CYT 0.2 pg / ml CYT-MA 204 * 0.2 pg ** / 6.3 pg / ml; 2: 1 CYT-Rab IgG 0.2 µ l * / 12.5 pg / ml; 2: 1 MA204 * 0.5 pg / ml cell Colo 205.5 x 10 * / hole * MA 204 is anti-CEA-Monoclonal »antibody. ** indicates CYT activity equivalent.

Because MA 204 has a higher selection specificity than Rab IgG, the higher cell growth inhibitory activity of CYT-MA 204 over that of CYT-Rab IgG, shown in Fig. 5, indicates that the cytorhodin S-immunoglobulin complex of the invention is selective for cancer cells acts -16-

Claims (12)

AT 393 125 B Comparison with Aclacinomvcin-Immunoglobulin Comolex For comparison purposes, an immunoglobulin complex with Aclacinomycin A which is structurally similar to cytorhodin S, prepared in the same manner as in Example 3. While cytorhodin S was added in an amount of 5-12 molecules per molecule of the antibody, aclacinomycin was added in an amount as small as 2 molecules per molecule of the antibody. The DNA synthesis inhibitory activity of the two antibody complexes in L1210 was determined from the amount of tritiated thymidine that was incorporated into the cells. The results are shown in Fig. 6. Figure 6 shows that the activity of the cytorhodin S complex against the cells was higher than that of the aclacinomycin complex. PATENT CLAIMS 1. Cytorhodin S derivative, characterized by the general formula (I) 5 AT 393 125 B wherein X is a group of the formula -nh2, -NH-R ^ NH ^ -NH-R ^ COOH, -NH-R ^ SH, I nh-c-r2-n. 10 0 -NH-R1-NH-CR * -N or 15 0 -NH-R3-C-NH (IgG) 1 / n 20 O darslelli, where R * is a divalent hydrocarbon gnome, which can be substituted by any substituent , R ^ is a divalent hydrocarbon gnoc, R3 means a divalent organic group, IgG means an immunoglobulin residue, and n means an integer from 1 to 15, and a salt thereof. 25 30 35 2. Cytorhodin S derivative according to claim 1, characterized in that R3 for R * or a group of the formula 40 45 stands, wherein R ^ means a divalent hydrocarbon group. 3. Cytorhodin S derivative according to claim 2, characterized in that R ^ is a propyl group and R ^ is a methyl group. 4. Cytorhodin S derivative according to any one of claims 1 to 3, characterized in that n is 5 to 12. -18- AT393 125 B 5. cytorhodin S derivative according to any one of claims 1 to 4, characterized in that the IgG is a residue of the antibody against carcinoembryonic antigen (CEA) 6. Process for the preparation of a cytorhodin S derivative of the general formula (I) wherein X is a group of the formula -NH2. -NH-R ^ NHj, -NH-R ^ COOH, -NH-R ^ SH, O -NH-C-R2-N II O -nh-r1-nh-cr II 0 -NH-R3-C-NH (IgG) 1 / n II 0, where R * is a divalent hydrocarbon group which can be substituted by any substituent, R ^ a divalent Hydrocarbon group means R3 means a divalent organic residue, IgG means an immunoglobulin residue, and n means an integer from 1 to 15, characterized in that a cytorhodin S of formula (II) -19- 5 10 15 20 AT 393 125 B 25 30 with an alkali metal cyanoborohydride in the presence of an ammonium salt or a compound represented by NH2-R ^ -NH2, where R1 has the same meaning as defined above, is reduced to obtain a cytorhodin S derivative, wherein X in the formula (I ) -NH2 or -NH-RI-NH2, and if necessary this product with a maleimide compound of the formula (BQ 35 40 (ΠΙ). ο in which R * has the same meaning as defined above, is implemented to obtain a cytorhodin S derivative (I) in which X 45 0 50 -NH-C-R2 -N j) or 0 Ύ 0 O II 55 -NH -R1-NH-CR * 11 O -N 60 II 0 -20- AT 393 125 B, and further, if necessary, this product with a thiol derivative of immunoglobulin of the formula (IV) HS-R4-C-NH (IgG ) 1 / n. (IV) II O wherein R4 is a divalent hydrocarbon group, IgG has the same meaning as defined above, and n 'is an integer from 1 to 20, is reacted to obtain a cytorhodin S derivative (I), wherein X is -NH -R3-C-NH (IgG) 1 / n II O 7. A method for producing a cytorhodin S derivative according to claim 6, characterized in that R3 is. 8. Process for the preparation of a cytorhodin S derivative of the formula (0 -21- AT 393 125 B in which X denotes a group -NH-R ^ -SH or -N-R3-C-NH (IgG) j, in which R ^ is a divalent II 0 hydrocarbon group which can be substituted by any substituent, R3 denotes a divalent organic group, IgG denotes an immunoglobulin unit, and n denotes an integer from 1 to 15, characterized in that a cytorhodin S of the formula (II) with an alkali metal cyanoborohydride in the presence of a compound represented by NH2-R1-SH, where r1 has the same meaning as defined above, is reduced to obtain a cytorhodin S-derivative (I) > wherein X in the above-mentioned formula (I) is -NH-R ^ -SH, and if necessary this product with a maleimide derivative of immunoglobulin of the formula (V) N-R2 -C-ΝΗτΙ * G > II O 1 / n '(V). wherein R3 and IgG have the same meanings as defined above, and n 'is an integer from 1 to 20, is reacted to obtain a cytorhodin S derivative (I), wherein X is -NH-R3-C-NH (IgG) 1 / n is O. 9. A method for producing a cytorhodin S derivative according to claim 8, characterized in that R3 -22- AT393 125 B Ο 10. Process for the preparation of a cytorhodin S derivative of the formula (I) where X is a -NH-R ^ -COOH- or -NH-R ^ -C-NH (IgG) yn group, in which R1 is a divalent O hydrocarbon group which can be substituted by any substituent, a divalent, organic Group means IgG means an immunoglobulin residue, and n means an integer from 1 to 15, characterized in that cytorhodin S of the formula (II) (Π) -23- AT 393 125 B is reduced with an alkali metal cyanoborohydride in the presence of a compound represented by NH2-R1-COOH, which has the same meaning as defined above, to obtain a cyctorhodin S derivative, wherein X in the above formula ( I) -NH-R ^ COOH and, if necessary, this product with an immunoglobulin of the formula (VI) NIW ^ l / n (VI) wherein IgG has the same meaning as defined above, and n is an integer from 1 to 15 , is implemented to obtain a cytorhodin S derivative (I), wherein X in the formula (I) -NH-R3-C-NH (IgG) 1 / n II O. 11. A method for producing a cytorhodin-S derivative according to claim 10, characterized in that R3 is R1. 12. Anti-cancer agent containing a cytorhodin-S derivative, which is represented by the general formula (I) CO is represented, where X is a group of the formula -nh2, -NH-R ^ NH ^ -NH-R ^ COOH, -NH-R ^ SH, O O-NH-R1 or -24- AT 393 125 B -NH-R3-C-NHagG) 1 / n II o 5, where R1 is a divalent hydrocarbon group which can be substituted by any substituent Ί 3, R is a divalent hydrocarbon group, RJ means a divalent organic group, and n is an integer from 1 to 15 means 10 to 4 sheets of drawings -25-