NO169441B - PROCEDURE FOR THE PREPARATION OF 7-OXOMITOSANDER DERIVATIVES - Google Patents

Description

The present invention relates to a new method for the production of hitherto unknown 7-oxomitosan compounds which contain a disulfide group. These compounds are mitomycin A analogues where the 7-alkoxy group has an organic substituent comprising a disulphide group.

Nomenclature: The systematic name for mitomycin A according to Chemical Abstracts based on a recent revision (Shirhata et al., J. Am. Chem Soc, Vol. 105, 1983, p. 7199) is: [laS-(lap,8p,8a ~,8bp)]-8-[((aminocarbonyl)oxy)methyl]-6,8a-dimethoxy-1-la,3,8,8a,8b-hexahydro-5-methyl-arizino [2' , 3', 3, 4] -pyrrolo [1, 2- a.] indole-4, 7-dione.

According to this name, the azirinopyrroloindole ring system is numbered as follows:

Chemical Abstracts

A nomenclature trivial system that has found widespread use in the mitomycin literature designates the above ring system, which contains several of the characteristic

substituents of the mitomycins, such as mitosan.

Mitosan

According to this system, mitomycin A is 7,9α-dimethoxymitosane, and mitomycin C is 7-amino-9α-methoxymitosane. As regards the stereochemical configuration of the compounds according to the invention, it is intended that when they are indicated by the generic name "mitosan" or by a structural formula, they should be identified with the same stereochemical configuration as that of mitomycin A or C.

Mitomycin C is an antibiotic produced by fermentation and currently sold with the approval of the "Food and Drug Administration" for the treatment of disseminated adenocarcinoma of the stomach or pancreas in proven combinations with other approved chemotherapeutic agents and for palliative treatment when other mortalities have failed ("Mutamycin", Physicians Desk Reference, 37th ed., 1983, pp. 747 and 748). Mitomycin C and its preparation by fermentation are known from US patent 3,660,578.

The structure of mitomycins A, B and C and porfiromycin was first published by J.S. Webb et al., J. Am. Chem. Soc, Vol. 84, 1962, pp. 3185-3187. One of the chemical transformations used in this structural investigation to compare mitomycin A with mitomycin C was the transformation of the former, 7,9a-dimethoxymitosane, by reaction with ammonia, into the latter, 7-amino-9a-methoxymitosane. Replacement of the 7-methoxy group in mitomycin A has proven to be a reaction of considerable interest in the preparation of active anti-tumor derivatives of mitomycin A. It has recently been shown that the stereochemical configurations in 1-, 1a-, 8a - and the 8b positions are as shown above in connection with the nomenclature according to Chemical Abstracts (Shirhata et al., J. Am. Chem. Soc, Vol. 105, 1983, p. 7199-7200). Previous literature refers to the enantiomer.

The following articles and patent documents concern, among other things, conversion of mitomycin A into a 7-substituted aminomitomycin C derivative with antitumor activity. The purpose of this research was the preparation of derivatives which were more active and especially less toxic than mitomycin C: Matsui et al., J. Antibiotics, Vol. XXI, 1969, p. 189-198,

Konishita et al., J. Med. Chem, Vol. 14, 1971, pp. 103-109,

Iyengar et al., J. Med. Chem., Vol. 24, 1981, pp. 975-981.

Iyengar, Sami, Remers and Bradner, Abstracts of Papers, 1983. Annual Meeting of The American Chemical Society, Las Vegas, Nevada, March 1982, Abstract No. MEDI 72,

US Patents 3,332,944, 3,420,846, 3,450,705, 3,514,452, 4,231,916 and 4,268,676.

European patent application 116,208 and GB patent application 2,140,799 relate to the production of 7-substituted aminomitomycin C derivatives where the substituent contains a disulfide bond.

7-Alkoxy-substituted mitosans, which are structurally related to mitomycin A, are described in an article by Urakawa et al., J. Antibiotics, Vol 23, 1980, p. 804-809 as valuable antibiotics with activity against experimental tumors in animals .

Mitomycin A is the most important mitomycin produced by fermentation, and is the commercially available form. Known technology for converting mitomycin C to mitomycin A has a number of shortcomings. Hydrolysis of mitomycin C to the corresponding 7-hydroxy-9a-methoxymitosane and subsequent methylation of this compound requires diazomethane, a compound whose handling on a production scale is very risky, and the 7-hydroxy intermediate is very unstable (Matsui et al., J. Antibiotics, Vol. XXI, 1968, pp. 189-198). An attempt to avoid these difficulties includes the use of 7-acyloxymitosanes (Japanese Patent Document J. 5 6073-085, Farmdoc No. 56227 D/31). Alcoholysis of mitomycin A as described by Urakawa et al., J. Antibiotics, Vol. 23, 1980, p. 804-809 is limited to the exclusive production of specific 7-alkosy structure types because of the availability and reactivity of the alcoholic starting materials.

The above-mentioned methylation of 7-hydroxy-9a-methoxymitosane with diazomethane is the only method known to date for the selective alkylation of the 7-hydroxy position, as other alkyl reagents tried so far react with the residue in the la-N position and with C-10 -the carbamoyl residue or a combination of these. As mentioned above, the diazomethane method is not harmless. From the technique, e.g. Ukraine. Crimea. Zhur., Vol 21, 1985 p. 469-498 (Chemical Abstracts, Vol 50, 1956 5549) it is also known that triazene reagents react with imides, whereby the person skilled in the art will expect that triazene reagents will react with the C-10 carbamoyl residue on similarly to the alkylating agent dimethylformamide-dimethylacetal, which selectively alkylates said C-10-carbamoyl residue.

With the method according to the invention, it now surprisingly turns out that it is possible to selectively alkylate the 7-hydroxy function in 7-hydroxymitosane using a triazene reagent.

The present invention relates to a method for producing a group of mitomycin A analogues with an organic dithiosubstituent incorporated in the alkoxy group in the 7-silence. These compounds are represented by the following general formula where R is <C>1-7-alkanoyl-ethyl, C1-7-alkanoyloxyethyl, C1-7-alkanoylaminoethyl, 1,2-dihydroxyprop-3-yl, carboxyethyl or a pharmaceutical acceptable salt thereof, carboxyaminoethyl or a pharmaceutically acceptable salt thereof, N-methylimidazolylmethyl, di-C 6 -alkylaminoethyl, phenyl, which is unsubstituted or optionally substituted with one or two substituents selected from nitro, methoxy, amino, carboxy or a pharmaceutically acceptable salt thereof, pyridyl, which is optionally substituted with nitro, or a residue derived from glutathione of the formula

or a pharmaceutically acceptable salt thereof,

R<1> is hydrogen, and Alk2 is ethylene.

The method is characterized by at least one equivalent of a triazene with the formula (V)

where

R and Alk2 have the meaning given above, and Ar is the organic residue of a diazotizable aromatic amine, is reacted with an equivalent of a mitosan with the formula (IV) where R<*> has the meaning given above, under reaction conditions in an inert organic solvent at a temperature from 0°C to 60°C, until a sufficient amount of the compound of formula (I) is obtained, and when R in the compound of formula (I) is nitro-substituted pyridyl, this is optionally converted by reaction in an inert solvent at a temperature of from 0°C to 60°C, optionally in the presence of at least one equivalent of a strong base, with a thiol of the formula RSH, where R has the above meaning except for nitro-substituted pyridyl, and if desired, where it is possible, compounds are converted to a pharmaceutically acceptable salt, thereof.

The compounds produced according to the invention act as inhibitors of experimental tumors in animals. In particular, the compounds given in examples 2-16 and 17 are hitherto unknown substances. They are used in a similar way to mitomycin C. Depending on their toxicity, the dosages used are set in relation to the toxicity of mitomycin C. In cases where the new compound is less toxic, a higher dose is used.

The triazene reactants of formula (V) used can be prepared according to the general methods described by E.H. White et al., in Org. Syn., Vol 48, 1968, p. 102-105 and in Tetrahedron Letters, no. 21, 1961, p. 761, except that the alkylamines used in these general methods are replaced by aminodisulfides with the formula:

where R and Alk2 have the above meaning.

As a preferred embodiment, an alternative method according to the invention is provided for the production of disulfide mitosans with the formula (Ia)

where R 2 is an organic group, i.e. the structural component of an organic thiol with the formula R^SH, which is alternatively described by R^Alk^, or R<4>, where R^, R<4> and Alk1 have the meanings given above. '

For the preparation of the disulphide mitosans with the formula (Ia), it is preferred to use the 9a-methoxy-7-2-(-3-nitro-2-pyridyl-thio)ethoxy mitosan in a thiol replacement process with a suitable organic thiol of the formula R^SH. The reason for the formation of the disulfides with the formula (Ia) is the stability of the by-product, namely 3-nitro-2-mercaptopyridine, which only exists in thione form.

If it is alternatively desired to produce mitosans with the formula (I) where Alk2 has a different meaning than ethylene, such as trimethylene or propylene, the appropriate triase with the formula (V) is used. In the process in question for the production of disulfide mitosans with the formula (Ib)

where Alk2 and R have the meanings given above.

Two general methods for the production of both lipophilic and hydrophilic mitosans with the formula (Ia) are described here. The general method A is used for the production of either lipophilic or moderately soluble disulfides with the formula (Ia), while the general method B is used for water-soluble disulfides with the formula (Ia), which are preferably isolated as sodium salts or as zwitterionic forms. Preferably, at least one equivalent of mercaptan R^SH is used per equivalent mitosan with the formula (XVII), and the reaction can be carried out in the presence of approx. 1 equivalent base per equivalent mercaptan R<2>SH. Preferred bases are the tertiary amines, e.g. triethylamine, N-methylmorpholine, N-methylpiperidine, pyridine, 2,6-lutidine and the inorganic bases, e.g. sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate etc. Suitable inert solvents for reacting starting materials of the formula (XVII) with R<2>SH are the lower alkanols, lower alkanoic acids-lower alkyl esters, lower aliphatic ketones, cyclic aliphatic ethers, lower polyhalogenated aliphatic hydrocarbons and water. The organic solvents contain up to 8 carbon atoms, but solvents which boil at temperatures below 100°C are preferred. Particularly preferred solvents are methylene chloride, methanol, acetone, water and mixtures thereof. The reaction can be carried out at the reflux temperatures of the reaction mixture or at up to 60°C. It is preferred to carry out the reaction at room temperature or lower, e.g. in the range 0-25°C.

The compounds according to the invention are considered to have antibacterial activity against gram-positive and gram-negative microorganisms in a similar manner to that observed for the naturally occurring mitomycins, and are thus potentially valuable as therapeutic agents for the treatment of bacterial infections in humans and animals.

Activity against P-388 mouse leukemia

Table I contains the results of laboratory experiments with CDF^ mice, which have been intraperitoneally implanted with a tumor inoculum in the form of 10^ ascites cells of P-388 mouse leukemia and treated with different doses of either a test compound of the formula (I) or (II), or with mitomycin C. The compounds were administered by intraperitoneal injection. Groups of 6 mice were used for each dose and were treated with a single dose of the compound days after inoculation. A group of 10 saline-treated control mice was included in each series of experiments. The groups treated with mitomycin C were included as a positive control. A 30-day record was kept of the mean survival time expressed in days for each group of mice, and the number of survivors at the end of the 30-day period was noted.

The mice were weighed before treatment and again on the sixth day. The weight change was taken as a measure of the drug's toxicity. Mice weighing 20 g were used, and a weight loss of up to 2 g was not considered unusually large. The results were specifically expressed in % T/C, which is the ratio of the mean survival time of the treated group to the mean survival time of the saline-treated control group multiplied by 100. The saline-treated control animals usually died within 9 days. The "maximum effect" in the following table is expressed in % T/C, and the dose producing this effect is indicated. The values in parentheses are those with mitomycin C as the positive control values achieved in the same trial. Thereby, the relationship between the relative activity of the listed substances and mitomycin C can be assessed. The minimal effect expressed in % T/C was considered to be 125. The minimal effective dose, which is indicated in the following table, is the dose which gives % T/C ? to approx. 125. The two values entered in each case in the "average weight change" column are the average weight change per mice at the maximum effective dose and at the minimum effective dose, respectively.

Activity against B16 melanoma

Table 2 contains results of antitumor tests using Bl6 melanomas grown in mice. BDF^ mice were used, which were grafted subcutaneously with the tumor implant. A 60-day protocol was kept. Groups of 10 mice were used for each dosage amount tested, and the mean survival time for each group was determined. Control animals, which were inoculated in the same way as the test animals and treated with the injection vehicle without drug, had an average survival time of 24 days. The survival time relative to the survival time of the control animals (% T/C) was used as a measure of efficacy, and the maximum effective dose and the minimum effective dose for each test compound were determined. The minimal effective dose was determined as the dose that had a % T/C value of 125. For each dose level, the test animals were treated intravenously with the test compound on days 1, 5 and 9.

In view of the observed anti-tumor activity in experimental animal tumors, the compounds according to the invention can be used to inhibit tumors in mammals. To this end, these are systematically administered to a mammal suffering from a tumor in a largely non-toxic dose with an anti-tumor effect.

The compounds according to the invention are primarily intended for use in injection form in a similar way and with the same purpose as mitomycin C. Slightly larger or smaller doses can be used depending on the particular tumor sensitivity. They can be easily distributed as dry pharmaceutical preparations containing diluents, buffers, stabilizers, solubilizers and ingredients that contribute to pharmaceutical applicability. These preparations are then prepared with an injectable liquid medium, which is made to order, immediately before use. Suitable injectable fluids include water, isotonic saline and the like.

In the following methods and examples, all melting points are uncorrected. The proton nuclear magnetic resonance spectra (<1>H NMR) were recorded in a "Varian XL100", "Joel FX-90Q" or "Bruker WM 360" spectrometer, either in pyridine-d,. or D20 as indicated. When pyridine-dj. is used as solvent, the pyridine resonance at 6=8.57 is used as an internal reference, while on the other hand TSP is used as an internal reference with D20 as solvent. Chemical shifts are given in £ units, and coupling constants in Hertz. Splitting patterns are indicated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad signal, dd = doublet of doublet, dt = doublet of triplet. IR spectra were determined in either a "Beckman Model 4240" spectrometer or a "Nicolet 5DX FT" IR spectrometer and are given in reciprocal centimeters. UV spectra were determined either in a "Cary Model 290" spectrometer or a "Hewlitt Packard 84 50A" spectrometer equipped with a multi-diode detector. Thin layer chromatography was performed on 0.25 mm "Analtech silica gel GF" plates. Flash chromatography was performed with either "Woelm" neutral alumina (DCC grade) or "Woelm" silica gel (32-63°) and the solvents listed. All solvent evaporations were carried out at reduced pressure and at below 40°C.

The 1-alkyl-3-aryltriazenes constitute a class of reactants, which are known to be able to be used for reaction with carboxylic acids to form the corresponding lower alkyl esters. 1-methyl-3-(4-methylphenyl)triazene can be prepared in the following way:

Procedure 1

7-Hydroxv- 9a- Methoxvmitosan. 2.2 g (6.6 mmol) mitomycin

C was dissolved in 140 mL of 0.1N methanolic NaOH (50%), and the reaction mixture was stirred at room temperature for 30 hours. The solution was then set to approx. pH 4.0 with IN HCl and extracted with 4 x 500 ml ethyl acetate. The combined ethyl acetate extracts were dried with Na 2 SO 4 and evaporated under reduced pressure at 30-35°C to give a solid residue, which after dissolution in ether and treatment with excess hexane gave a purple precipitate. The precipitate was collected and dried,

whereby the title compound was obtained as a fine purple-

colored powder (1.4 g, 63%).

<1>H NMR (pridine-d5, ): 2.05 (s, 3H), 2.14 (bs, 1H), 2.74

(bs, 1H) , 3.13 (d, 1H), 3.24 (s, 3H) , 3.56 (d, 1H) , 4.00 (dd,

1H) , 4.37 (d, 1H) , 5.05 (t, 1H) , 5.40 (dd, 1H) , 5.90 (bs, 2H) . Example 1

1-\ 2 -( 3- nitro- 2- pvridvldithio) ethyl! - 3(4-methylphenyl)triazene

A solution of 4-methylphenyldiazonium chloride was prepared

as described in E.H. White et al., Org. Syn., Vol 48, 1968, P. 102-195 from p-toluidine and adjusted to pH 6.8-7.2 at 0PC, likewise as described in this method. A solution containing 21.15 mmol of diazonium salt in 45 ml of solution was then

when prepared and placed in a separatory funnel, which was connected to a 250 mL three-necked round-bottomed flask containing 5.34 g (20.0

mmol) of 2-(3-nitro-2-pyridyldithio)ethylamine, 7 g of sodium carbonate and 150 ml of dioxane, which had been introduced into the flask in this order. 6 ml of saturated aqueous sodium carbonate solution and 10 g of ice were introduced into the flask. The flask was cooled in an ice bath and the contents stirred mechanically. From the separation tract, the diazonium salt solution was then added dropwise over the course of one hour.

After the addition was complete, the reaction mixture was allowed to reach room temperature

erature and was then extracted with 3 x 4.00 ml portions of ether. Drying and evaporation of the extracts gave the desired compound, which was purified by chromatography using an alumina packed column, 2.54 cm in diameter and 25.4 cm

long, using hexane: methylene chloride = 4:1, hexane: methylene chloride = 3:2, hexane: methylene chloride = 1:4 and finally methylene chloride containing 1% methanol to develop and elute the column. The appropriate fractions (identified by thin layer chromatography) were combined and evaporated to give 2.5 g of the title compound.

Example 2

9a-methoxy-7-f2-(3-nitro-2-pyridylthio)ethoxysvlmitosane (20)

580 mg (1.73 mmol) of 7-hydroxy-9α-methoxymitosane was placed in a round bottom flask and dissolved in 60 ml of methylene chloride. About. 2.5 g (5.7 mmol) of the triazene from Example 1 was added to the solution in the flask, and the mixture was stirred at 5 C for 14 hours, and then at room temperature for 8 hours. The course of the reaction was monitored by silica thin layer chromatography using methylene chloride:methanol = 9:1. The reaction mixture was kept at room temperature for a further 26 hours and then worked up by column chromatography in a column which was 8.4 mm in diameter, approx. 305 mm long and packed with aluminum oxide. The solvents, which were used in order for development and elution, were respectively 200 ml portions of methylene chloride, 0.5% methanol in methylene chloride, 1.0% methanol in methylene chloride, 1.5% methanol in methylene chloride, 2% methanol in methylene chloride and 4% methanol in methylene chloride. The appropriate fractions were combined and evaporated to give 470 mg of the title compound.

Analysis: calculated for C22<H>23<N>5°8<S>2: C: 45'65; H: 4'9; N: 11, 82

corrected for 0.5 mol% CH2Cl2:

found: C: 45.74; H: 4.14; N: 11.61.

IR (KBr), vmax, cm"1: 3440-3200, 3060, 2960, 2930, 1720, 1570, 1510, 1395, 1335, 1210, 1055.

<1>H NMR (pyridine-d5, ): 1.81 (s, 3H), 2.00 (bs, 1H), 2.61 (bs, 1H) , 2.98 (bs, 1H) , 3, 08, (s, 3H) , 3.20 (m, 2H) , 3.39 (d, 1H), 3.83 (dd, 1H) , 4.07, (d, 1H), 4.59-4 , 89) (m, 3H) 5.21 (dd, 1H), 7.16 (dd, 1H), 8.31 (dd, 1H), 8.71 (dd, 1H). By adapting the methods from examples 11 and 20 to other -(3-nitro-2-pyridylthio)alkylamines with 2-6 carbon atoms in the alkyl group, mitosans with the following formula can be prepared:

n = 2-6

R 1 = H, or C 1-6 alkyl

Example 3- 16

The 7-alkoxydithio-9a-methoxymitosanes .3-16' in the following table 3 were prepared according to the general methods A or B as described below and shown in table 3. The physical data for the mitosan compounds 3-16 are given in the following table 4.

Procedure A

To a deoxygenated solution of the mitosan from example 2 (approx. 0.1 mmol) in 3-5 ml of acetone is added while stirring, under argon, approx. 1.1 equivalents of triethylamine, followed by dropwise or portionwise addition of one equivalent of mercaptan in 1-2 ml of acetone. In most reactions, the course of the reaction is monitored by silica gel thin-layer chromatography (10% CH^OH in CH2CL2). Completion of the reaction is marked by the disappearance of the spot corresponding to the starting material and the appearance of the spot corresponding to the desired compound. At this point the reaction mixture is concentrated under reduced pressure (at about 30°C) and the residue is chromatographed on a flash-packed neutral "Woelm" alumina column (6.4 x 25.4 cm) with 2-5% CH 3 OH in CH 2 Cl 2 . In this way, the desired mitosan compound is separated from the pyridyl ion by-product, which is retained by the column. The compound thereby eluted using 2-5% CH 2 OH in CH 2 Cl 2 is further carefully purified by flash silica gel chromatography using 5-7% CH 2 OH in CH 2 Cl 2 as eluent. The main band, corresponding to compound, is isolated, and the amorphous 7-alkoxydithio-

9a-Methoxymitosane is characterized.

Procedure B

To a solution of the mitosan from example 2 (approx. 0.1 mmol) in 2-5% acetone<3>^ in 10 ml of methanol is added approx. 6 drops of a saturated aqueous NaHCO^ solution b), followed by 1 equivalent of mercaptan in 1 ml of methanol<0>^. The course of the reaction is monitored by thin-layer chromatography (silica gel, 10% CH^OH in CH2C12). After completion of the reaction, the reaction mixture is diluted with 15 ml of water and concentrated to approx. 10 ml with reduced pressure at approx. 30°C. The resulting solution is chromatographed on a reversed phase C-18 column with stepwise gradient elution (from 100% ci)H 2 O to 80% CH^OH in H 2 O). The compound, which elutes as a major red band, is collected and concentrated, whereby the 7-alkoxydi-thio-9a-methoxymitosane is obtained as an amorphous solid. If further purification is necessary, the chromatography step described above is repeated. a) Methylene chloride can also be used, but acetone is preferred. b) When the mercaptan is L-cysteine, this base is not used.

c) Water is used if the starting thiol is water soluble.

d) Elution with water separates the yellow pyridylthione by-product from the compound, which is retained in the column.

Example 17

A solution of 2.4 equivalents of the triazene in CH 2 Cl 2 :methanol = 4:1 was added to a solution of 7-hydroxy-9a-methoxymitosane in CH 2 Cl 2 :methanol = 4:1. The reaction mixture was stirred at room temperature, and the course of the reaction was monitored by thin-layer chromatography (10% MeOH in CH2C12). The reaction mixture was chromatographed on "Woelm" alumina after the reaction was considered complete on the basis of thin layer chromatography, and the 7-Alkoxy-9a-methoxymitosane was obtained as an amorphous solid. The compound produced is listed as in Table 5.

Claims (1)

Process for the preparation of a 7-oxomitosan compound else with the general formula (I) where R is C^_^-alkanoyl-ethyl 1, C^_^-alkanoyloxyethyl, C^_7-alkanoylaminoethyl, 1,2-dihydroxy-prop-3-yl, carboxyethyl or a pharmaceutically acceptable salt thereof, carboxyaminoethyl or a pharmaceutically acceptable salt thereof, N-methylimidazolylmethyl, di-C 1-6 -alkylaminoethyl, phenyl, which is unsubstituted or optionally substituted with one or two substituents selected from nitro, methoxy, amino, carboxy or a pharmaceutically acceptable salt thereof, pyridyl, which is optionally substituted with nitro, or a glutathione-derived residue with forme len or a pharmaceutically acceptable salt thereof, R"*" is hydrogen, and Alk2 is ethylene, characterized in that at least one equivalent of a triazene of the formula (V) where R and Alk2 have the meaning stated above, and Ar is the organic residue of a diazotizable aromatic amine, is reacted with an equivalent of a mitosan with the formula (IV) where R"<*>" has the above meaning, under reaction conditions in an inert organic solvent at a temperature from 0°C to 60°C, until a sufficient amount of the compound of formula (I) is obtained, and when R in the compound of formula (I) is nitro-substituted pyridyl, this is optionally converted by reacting in an inert solvent at a temperature from 0°C to 60°C, optionally in the presence of at least 1 equivalent of a strong base, with a thiol of the formula RSH, where R has the above meaning except for nitro-substituted pyridyl, and if desired, where possible, compounds are converted to a pharmaceutically acceptable salt, thereof.