DK169478B1 - 7-oxomitosan derivatives, process for their preparation and pharmaceutical compositions containing them - Google Patents

Description

in DK 169478 B1

The present invention relates to novel mitomycin analogs containing a di-sulfide group (class 260, subclass 326.24) and processes for their preparation and pharmaceutical compositions containing them. Mitomycin A is an antibiotic the use of which is known and its 7-O-substituted mitosan analogs thereof can be used in a similar manner.

Nomenclature - The systematic name for mitomycin A according to Chemical Abstracts based on a recent review (Shirhata et al., J. Am. Chem.

Soc., 105, 7199 (1983)) are: 10 [a1 S- (la8,8 / 3,8aa, 8b)) - 8 - [((aminocarbonyl) oxy) methyl] -6,8a-dimethoxy-1 , 1a, 3,8,8a, 8b-hexahydro-5-methyl-arizino [2 ', 3', 3,4] -pyrrolo- [1,2-a] indole-4,7-dione, according to which the name of the azirinopyrroloindole system is numbered as follows: 15 I. 8 6 iT ^ \ 8a V ^ 8b T> 1 4 '2 ^' 20

Chemical Abstracts

A nomenclature-trivial system that has found widespread use in the mitomycin literature identifies the preceding ring system comprising several of the characteristic substituents of the mitomycins as mitosan.

10 O CH-OCONH, 30 ϋ \

L

J> ° 3 2 35

Mi tosan DK 169478 B1 2

Acc. this system is mitomycin A 7,9a-dimethoxymitosan and mitomycin C is 7-amino-9a-methoxymitosan. As to the stereochemical configuration of the products of the invention, it is intended that when identified by the common name "mitosan" or by a structural formula, they should be identified by the same stereochemical configuration as that of mitomycin A or C.

O CH2OCONH2 R »^ 1, Mitomycin A R '= R'j = 0CH3 10 _ Mitomycin C R' = NH ,,, R '^ OCH, o' V ·" "'' '

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 gastric or pancreatic adenocarcinoma in tested combinations with other approved chemotherapeutic agents and for palliative therapy when other modalities have failed ("Mutamycin®", Bristol Laboratories, Syracuse, New York 13221,

Physicians' Desk Reference, 37th Edition, 1983, pp. 747 and 748). Mitomycin 20 C and its preparation by fermentation are disclosed in U.S. Patent No. 3,660,578, issued May 2, 1972, which requires priority from prior applications including an application filed in Japan on April 6, 1957.

The structure of mitomycins A, B, C and porfiromycin was first published by J.S. Webb et al., Lederle Laboratories Division American Cyanamid Company, J. Am. Chem. Soc., 84, 3185-3187 (1962). One of the chemical transformations used in this structural study to compare mitomycin A with mitomycin C was the conversion of the former, 7,9a-dimethoxymitosan, by reaction with ammonia 30 to the latter, 7-amino-9a-methoxymitosan. Replacement of the 7-methoxy group of mitomycin A has been shown to be a reaction of considerable interest in the preparation of active antitumor derivatives of mitomycin C. Recently, it has been found that the stereochemical configurations of 1-, 1-, 8- and the 8b substitutions are as shown above in connection with the nomenclature of Chemical Abstracts (Shirhata et al., J. Am. Chem.

Soc., 105, 7199-7200 (1983)). The previous literature refers to the enantiomer.

The following articles and patents deal with i.a. converting DK 169478 B1 3 of mitomycin A into a 7-substituted aminomitomycin C derivative with antitumor activity. The purpose of this research was to produce derivatives that were more active and especially less toxic than mitomycin C:

Matsui et al., J. Antibiotics, XXI, 189-198 (1969), Konishita et al., J. Med. Chem., 14, 103-109 (1971),

Iyengar et al., J. Med. Chem., 24, 975-981 (1981),

Iyengar, Sami, Remers and Bradner, Abstracts of Papers, 183.

Annual Meeting of The American Chemical Society, Las Vegas, Nevada, March 1982, Abstract No. MEDI 72, 10 Cosulich et al., U.S. Patent No. 3,332,944, issued July 25, 1967,

Matsui et al., U.S. Patent No. 3,420,846, issued January 7, 1969,

Matsui et al., U.S. Patent No. 3,450,705, issued June 17, 1969,

Matsui et al., U.S. Patent No. 3,514,452, issued March 26, 1970,

Nakano et al., U.S. Patent No. 4,231,916, issued November 4, 1980, 20 Remers, U.S. Patent No. 4,268,676, issued May 19, 1981.

The following patent applications relate to the preparation of 7-substituted aminomitomycin C derivatives, wherein the substituent comprises a disulfide bond.

Kono et al., European Patent Application No. 116,208 (1984), Vyas et al., DE Publication No. 3,413,489 (1984).

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

Mitomycin C is the most important mitomycin produced by fermentation and is the commercially available form. Current technology for converting mitomycin C to mitomycin A suffers from a number of shortcomings. Hydrolysis of mitomycin C to the corresponding 7-hydroxy-9a-methoxy-mitosan and subsequent methylation of this substance requires diazomethane, a substance whose handling on a production scale is very risky and the 7-hydroxy intermediate is very unstable (Matsui et al., J. Antibiotics, XXI, 189-198 (1968)). An attempt to avoid these difficulties involves the use of 7-acyloxymitosans (Kyowa Hakko Kogyo DK 169478 B1 4 KK Japanese Patent Application J5 6073-085, Farmdoc No. 56227 D / 31). Alcoholysis of mitomycin A as described by Urakawa et al., J. Antibiotics, 23, 804-809 (1980) is limited solely to the preparation of specific 7-alkoxy structure types due to the availability and reactivity of the alcoholic starting materials.

The present invention relates to a group of mitomycin A analogs with an organic dithio substituent incorporated into the alkoxy group at the 7-position. These compounds are represented by the following general formula (I) wherein R is C1-6 alkanoylethyl, C1-6 alkanoyloxyethyl, C1-6 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 by one or two substituents from nitro, methoxy, amino and carboxy, or a pharmaceutically acceptable salt thereof, pyridyl optionally substituted with nitro, or a glutathione derived residue of the formula nh2 ho2cchch2ch2conh 30 hcch2-ho2cch2nhco 35 or a pharmaceutically acceptable salt thereof, R1 is hydrogen or lower alkyl and Alk is C 1-6 alkylene.

The compounds of the invention act as inhibitors of experimental tumors in animals. In particular, the compounds listed in Examples 2 and 3-16 DK 169478 B1 are novel substances. They are used in a similar way to mitomycin C. Depending on their toxicity, the dosages used are adjusted according to the toxicity of mitomycin C. In cases where the new compound is less toxic, a higher dose is used.

In a further aspect of the invention, there is provided a novel process for the preparation of mitosanes of formula (I). This novel process comprises reacting at least one equivalent of a triazene of formula (VI) 10

Ar-N = N-NH-Alk-SS-R (VI) wherein R and Alk have the meaning set forth in claim 1 and Ar is the organic residue of a diazotable aromatic amine, with an equivalent of a mitosan of formula ( IV)

ISLAND

Wherein R * is as defined in Claim 1, under reaction conditions 25 in an inert organic solvent at a temperature of about 10 DEG. 0 ° to approx. 60 ° until a sufficient amount of the product of formula (I) is obtained and when R in the compound of formula (I) is nitrosubstituted pyridyl, it is optionally converted by reaction in an inert solvent at a temperature of 0 ° C to 60 ° C, optionally in the presence of at least 30 one equivalent of a strong base, with a thiol of the formula R'SH, wherein R 'has the meaning given above for R other than nitrosubstituted pyridyl and, if desired, is converted to a obtained compound of formula (I) into a pharmaceutically acceptable salt thereof.

The above-mentioned derivatives according to the invention, wherein R is nitrosubstituted pyridyl, have the formula (Ib) DK 169478 B1 / NO2 * p

A lt X

('' Vss-Alkr — o, Λ \ / ΎΓ \ AND, \ = ζΐ / || V · 3 Ib 5 CH 3 - ^ \ X ^ n ^ J-r

The di sulfide mitosans of formula (Ib) are prepared according to the triazene process described herein. More particularly, the mitosane of formula 10 (Ib) wherein Alk is ethyl one and R * is hydrogen, described in Example 2 and in U.S. Patent Application No. 646,888, filed September 4, 1984. The process of the invention is illustrated below in Scheme 2nd

Scheme 2

O

o 11 ° ch2ocnh2

_ - ^ -r1 Ar-N = N-NH-Al k-SS-R

20 ^ 3 π \

WE

TV

wherein R, R * and Alk have the meanings set forth above and Ar is the organic residue of a diazotizable aromatic amine.

The aryl triazenes of formula (VI) used can be prepared according to the general methods described by E.H. White et al., In Org. Syn., Vol. 48, 1968, pp. 102-105 and Tetrahedron Letters, No. 21, 1961, p.

761 except that the alkyl amines used therein are replaced by amino disulfides of formula (XII) R-SS-A1k-NHg (XII) wherein R and Alk have the meaning given above.

Amino disulfides of formula (XII) are known compounds and can be prepared by various methods. For example, they can be prepared by reacting the appropriate thiol RSH with a Bunte salt of formula DK 169478 B1 7 NH2Alk-SSO3Na (XV) or with a sulfenylthiocarbonate of formula 5 NH2Alk-SSCOCH3 (XVI) according to that of Klayman et al ., J. Org. Chem., 29, 3737-3738 (1964).

The di sulfide starting material of formula (XII) is preferably prepared by reacting the appropriate thiol with a sulfenyl thiocarbonate of formula (XVI). This is the method of S.J. Brois et al., J. Am.

Chem. Soc., 92, 7629-7631 (1970).

As a preferred embodiment of the process of the invention, the following variant is provided for the preparation of disulfide mitons of formula (Ia) and CHOO Inh7 20 R2-SS-CH, CHO0 in TK1 3a CH3 S ^ NO0> 25 2 wherein R has the same meaning as R except for 3-nitro-2-pyridyl.

For the preparation of the di-sulfide mitosans of formula (Ia), it is preferred to use the 9α-methoxy-7- [2- (3-nitro-2-pyridyldithio) -ethoxy] mitosane of formula (XVII) in a thiol exchange process with an o 30 appropriate organic thiol of formula R SH as shown in Scheme 3.

The reason for the formation of the di sulphides of formula (Ia) is the stability of the by-product, namely 3-nitro-2-mercaptopyridine, which exists only as the thione (XVIII).

8 UK ΒΊ

Scheme 3 / πΓ2 X C ^ ° ^ ®2 W 2 2 Y | ^ \ Λ + r2sh _ ^

XVII

10 2,? CH-oInH- R -SS-CH.CH-O ^^^^ Å ^ Χΐί02

15 in χν ^ + (X

ch3'an ^ X ^ * n · ^ jh XVIII- 20 la

Alternatively, if mitosans of formula (I) are desired in which Alk is other than ethyl one, such as trimethylene or propyl one, the appropriate triazine of formula (VI) is used in the process of di-sulfide mitosanes shown in Scheme 2 of formula (Ib)

N ° 2 O

/ r \ 1 <= 2 ^ 30 \ = / 1 ifX? 113 \ 3 ^ "r1 wherein Alk and R * have the meanings set forth above.

In the examples, two general synthesis methods for the preparation of lipophilic as well as hydrophilic m tosans of formula (Ia) are described. The general process A is used to prepare either lipophilic or moderately soluble di sulfides of formula (Ia), whereas the general process B is used for water-soluble disulfides of formula (Ia), which are preferably isolated as sodium salts or as zwitterionic forms. Preferably, at least one equiv-2-lent mercaptan R SH is used per equivalent of the mitosis of formula (XVII), and the reaction may be carried out in the presence of approx. 1 equivalent base per equivalent 2 mercaptan R SH. Preferred bases are the tertiary amines, e.g. triethylamine, N-methylmorpholine, N-methylpiperidine, pyridine, 2.6-1 utidine and the inorganic bases, e.g. sodium bicarbonate, potassium carbonate, potassium bicarbonate and the like. Suitable inert solvents for the reaction of starting materials of formula (XVII) and RH are the lower alcannols, lower alcanoic acid-lower alkyl esters, lower aliphatic ketones, cyclic aliphatic ethers, lower polyhalogenated aliphatic hydrocarbons and water. The organic solvents contain up to 8 carbo atoms, but solvents boiling at temperatures of less than 100 ° C are preferred. Particularly preferred solvents are methylene chloride, methanol, acetone, water and mixtures thereof. The reaction may be carried out at the reflux temperature of the reaction mixture or at up to approx.

60 ° C. It is preferred to carry out the reaction at room temperature or below, e.g. in the range of 0 to 25 ° C.

The above reaction conditions and precautions can generally be used to prepare other disulfide mitosans of formulas (Ia) and (Ib) according to the general process depicted in Scheme 3.

Compounds of the invention are considered to possess antibacterial activity against gram-positive and gram-negative microorganisms in a manner similar to that observed in 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 murine leukemia 30 Table 1 contains the results of laboratory experiments with CDF

* C

having been implanted intraperitoneally with a tumor graft in the form of 10 ascites cells of P-388 mouse leukemia and treated with different doses of either a test compound of formula (I) or with mitomycin C. The compounds were administered by intraperitoneal injection. Groups of 35 6 mice were used for each dosing amount and treated with a single dose of the compound the day after inoculation. A group of 10 saline-treated control mice were included in each experimental series. The mitomycin C treated groups were included as a positive control. A 30 day DK 169478 B1 protocol was recorded over the average 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 is taken as a measure of the drug's toxicity. Five mice weighing 20 g were used and a weight loss of up to approx. 2 g was not considered unusually large. Results were determined in terms of% T / C, which is the ratio of the average survival time of the treated group to the average survival time of the saline-treated control group multiplied by 100. The saline-treated 10 control animals usually died within 9 days. The "maximum effect" in the following table is expressed in% T / C and the dose giving this effect is given. The values in parentheses are those with mitomycin C as the positive control values obtained by sarane experiments. Thus, the relationship between the relative activity of the substances listed and mitomycin C 15 can be assessed. The minimum effect expressed in% T / C was considered to be 125.

The minimum effective dose reported in the following table is the dose giving a% T / C of approx. 125. In each case, the 2 values indicated in the column, referred to as "average weight change", respectively. the average weight change per mouse at the maximum effective dose 20 and at the minimum effective dose.

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Activity against B16 melanoma

Table 2 contains results of antitumor tests using B16 melanomas grown in mice. BDF 2 mice were used which were inoculated subcutaneously with the tumor implant. A 60-day protocol was kept. Groups 5 of 10 mice were used for each dosage amount tested and the average survival time for each group was determined. Control animals grafted in the same way as the test animals and treated with a non-drug injection method showed an average survival time of 24 days. The survival time versus the survival time of the control animals (% 10 T / C) was used as a measure of efficacy and the maximum effective dose and minimum effective dose for each test compound were determined. The minimum effective dose was determined as the dose exhibiting a% T / C value of 125. For each dose level, the test animals were treated with the test compound on days 1, 5 and 9 by intravenous route.

15

Table 2 B16 melanoma

Compound in Maximum effect Minimum Average 1 12 20 Example No.% T / C dose effective dose weight change 10 167 (112) 3 1.6 (3) 3 <0.4 -0.9; +1.4> 214 (145) 2.4 (3) <1.6 -2.4; -1.9 25 2 110 (112) 3.2 (3) 3.2 +0.5 +0.5 8 152 (145) 1.6 (3) <1.6 -0.6 -0.6 30 1. mg / kg body weight 2. average number of g / day for each maximum and minimally effective dose 3. values in parentheses have been tested for mitomycin C in the same course 35 Considering the antitumor activity observed in experimental animal tumors, the compounds of the invention may be is used to inhibit mammalian tumors. For this purpose, these are systematically administered to a mammal suffering from a tumor at a substantially non-toxic dose with antitumor effect in the DK 169478 B1 13.

The compounds of the invention are primarily intended for use in injection form in a similar manner and for the same purpose as mitomycin C. Slightly larger or smaller doses may be used depending on the particular tumor sensitivity. They can be readily distributed as dry pharmaceutical compositions containing diluents, buffers, stabilizers, solubilizers and ingredients contributing to pharmaceutical usefulness. These compositions are then constituted with an injectable liquid medium made to order immediately prior to use. Suitable injectable fluids include water, isotonic saline and the like.

In the following methods and examples, all temperatures are given in ° C and the melting points are uncorrected. The proton nuclear magnetic resonance spectra (½ NMR) were recorded on a "Varian XL100", "Joel FX-15 90Q" or "Bruker WM 360" spectrometer in either pyridine-d_g or D2O as shown. When pyridine-dg is used as the solvent, the pyridine resonance at 5 = 8.57 is used as an internal reference, whereas the TSP is used as an internal reference in the case of DgO as the solvent. Chemical shifts are reported in 5 units and coupling constants in Hertz.

20 Split patterns are denoted as follows: s, singlet, d, doublet, t, triplet, q, quartet, m, multiplet, bs, wide signal, dd, doublet of doublet, dt, doublet of triplet. Infrared spectra were determined on either a "Beckman Model 4240" spectrometer or a "Nicolet 5DX FT" IR spectrometer and are given in reciprocal centimeters. Ultraviolet (UV) spectra were determined either on a "Cary Model 290" spectrometer or a "Hewlitt Packard 8450A" spectrometer equipped with a multi-diode detector. Thin-layer chromatography (TLC) 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 / tm) and the 30 solvents indicated. All solvent evaporation was carried out under reduced pressure at below 40 ° C.

The 1-alkyl-3-aryl triazenes form a class of reagents known to be used for reaction with carboxylic acids to form the corresponding lower alkyl esters. 1-Methyl-3- (4-methylphenyl) triazene 35 can be prepared as follows: DK 169478 B1 14

Process 1 7-Hydroxy-9a-methoxymitosane. Mitomycin C (2.2 g, 6.6 mmol) was dissolved in 140 ml of 0.1N methanol in NaOH (50%) and the reaction mixture was stirred at room temperature for 30 hours. The solution was then adjusted to ca. pH 5 4.0 with 1N HCl and extracted with ethyl acetate (4 x 500 ml). The combined ethyl acetate extracts were dried (Na 2 SO 4) and evaporated under reduced pressure at ca. 30 ° to approx. 35 ° C to give a solid residue which, after dissolving in ether and treating with excess hexane, gave a purple-colored precipitate. The precipitate was collected and dried to give the title compound as a fine purple powder (1.4 g, 63%).

1 H NMR (pyridine-d 6, 6): 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).

15

Example 1 1- [2- (3-Nitro-2-pyridyldithio) ethyl] -3- (4-methylphenyl) triazene

A solution of 4-methylphenyldiazonium chloride is prepared as described in E.H. White et al., Org. Vision. Vol. 48, 1968, pages 102-195 out of 20 from p-toluidine, and adjusted to pH 6.8-7.2 at 0 ° C also as described in this process. In this way, a solution containing 21.15 mmol of diazonium salt in 45 ml of solution was prepared and placed in a separatory funnel connected to a 250 ml 3-necked, round-bottom flask containing 5.34 g (20.0 mmol) of 2- (3-nitro-2-pyridyldi-thio) ethylamine, 7 g of sodium carbonate and 150 ml of dioxane which had been added to the flask in this order. 6 ml of saturated aqueous sodium carbonate solution and 10 g of ice were added to the flask. The flask was cooled in an ice bath and the contents stirred mechanically. From the separatory funnel, the diazonium salt solution was then added dropwise over 1 hour. After addition 30 was added, the reaction mixture was allowed to warm to room temperature and then extracted with three 400 ml portions of ether. Drying and evaporation of the extracts gave the desired product, 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). 35 xane: 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 TLC) were combined and evaporated to give 2.5 g of the title compound.

DK 169478 B1. 15

Example 2 9a-Methoxy-7-β- (3-nitro-2-pyridyldi thio) ethoxylmyl tosane (20) 7-Hydroxy-9a-methoxymitosan, 580 mg (1.73 mmol) is placed in a round bottom flask and dissolve in 60 ml of methylene chloride. The triazine from Example 1, ca. 2.5 g (5.7 mmol) 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 reaction was monitored by silica TLC using methylene chloride: methanol (9: 1). The reaction mixture was kept at room temperature for an additional 26 hours and then worked up by column chromatography on a column 8.4 mm in diameter, 304.8 mm long and packed with alumina. The solvents used sequentially for development and elution were 200 ml aliquots respectively. 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 title compound, 470 mg.

Analysis for ^ 22 ^ * 23 ^ 5 ^ 8 ^ 2:

Calculated: C, 45.65; H, 4.09; N, 11.82 (corrected for 0.5 mole% CH 2 Cl 2): Found: C, 45.74; H, 4.14; N, 11.61.

IR (KBr), i / max, cm -1: 3440-3200, 3060, 2930, 1720, 1570, 1510, 1395, 1335, 1210, 1055.

1 H NMR (pyridine-d 6, S): 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 procedures of Examples 1 and 2 to other <? - (3-nitro-2-pyridyldithio) alkylamines of 2-6 carbon atoms in the alkyl group, mitosan derivatives of the following formula can be prepared.

30 o N o CH 2 OCNH 2 + - SS- (CH 2) n -O N 2 O 2 - 1 / OCH 3 = n = 2-6 1 R = H, or C 1-6 alkyl DK 169478 B1 16

Examples 3-13 7-Alkoxydithio-9α-methoxymitosanes 3-13 in subsequent Table 3 were prepared according to the general methods A or B as described below and shown in Table 3. The physical data for mitosan compounds 3-5 16 are reported in the following the following Table 4.

Process A

To a deoxygenated solution of mitosan of Example 2 (-0.1 mol) in acetone (3-5 ml) is added with stirring, under argon, triethylamine (-1.1 equivalents) followed by dropwise or portion addition of a mercaptan3 ( 1 equivalent) in acetone (1-2 ml). For most reactions, the course of reaction is monitored by silica gel thin layer chromatography (10% CH₂OH in CH₂Cl₂) - The end of the reaction is marked by a disappearance of the spot corresponding to the starting material and the appearance of the product stain. At this point, the reaction mixture is concentrated under reduced pressure (at -30 ° C) and the residue is chromatographed on a slurry packed neutral "Woelm" alumina column (6.4 x 25.4 mm) with 2-5% CH 2 OH in CH 2 Cl 2. This process separates the desired mito-san product from the pyridylthione by-product retained by the column.

The product thus eluted using 2-5% CH₂OH in CHgCl₂ is further carefully purified by flash silica gel chromatography using 5-7% CH₂OH in CH₂Cl₂ as eluent. The headband corresponding to the product is isolated and the amorphous 7-alkoxydithio-9a-methoxy-mitosan is characterized.

a) In those cases where the starting mercaptan is impure,> 1 equivalent of thiol is required.

B) In those cases where the starting mitosane of Example 20 and the product exhibit very close R 2 values by TLC, high pressure liquid chromatography (HPLC) monitoring (n / tBondapak C18 "column) is used.

Method B

To a solution of mitosan of Example 2 (-0.1 mmol) in 2-5% acetone 3 in methanol (10 mL) is added a saturated aqueous NaHCO 3 solution (6 drops) followed by addition of mercaptan (1 equivalent) in DK 169478 B1 17 methanol (1 ml). The reaction is monitored by TLC (silica gel, 10% CH 2 OH in CH 2 Cl 2). After completion of the reaction, the reaction mixture is diluted with water (15 ml) and concentrated to ca. 10 ml under reduced pressure at approx. 30 ° C. The resulting solution is chromatographed on a reverse phase C-18-5 column with stepwise gradient elution (100% H 2 O to 80% CH 2 OH in H 2 O). The product, eluted as a red larger band, is collected and concentrated to give the 7-alkoxydithio-9a-methoxymitosane as an amorphous solid. If further purification is required, repeat the above chromatography step.

A) Methylene chloride may also be used, but acetone is preferred.

b) In the case where 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 product retained on the column.

DK 169478 B1 18

Table 3 7-Alkoxydithio-9α-methoxymi tosans (j> π CH-OCNH- O T 2 2 ® A or 'Vj \ -PCH_ RSH + 2 - ^ (i li

2 N P) JH

Eks.nr. Thiol (RSH) Method Product 10

ISLAND

to 3 ethyl-2-me reap to- A R ^ Cl- ^ CH ^ QCCH ^ acetate

4. 3-mercapto-1,2 A R = -CH 2 CH (OH) CH 2 OH

propanediol 5 3-mercaptopropion- BR = -CH2CH «COO Na" 1 "acid 6 cysteine BR = -CH2CH (NH3 +) COO ~ 7 thiophenol AR = 8 p-ni trobenzene thiol AR = -y xV-NO2 g = 10 p-aminobenzenthiol AR = - / γ-NH? \ zz / DK 169478 B1 19

Table 3 (continued)

Eks.nr. Thiol (RSH) Process Product 5 COO ~ Na + / Vp

11 2-mercaptobenzoo- B R = -M

acid \ _ / 12 2-nitro-4-mercapto- BR = ^ -02 benzoic acid - \ COO ~ Na + 13 4-mercaptopyridine AR = - ^ / Vj N \ 14 2-mercaptomethyl-1-AR = -C ^ | methylimidazole N 2 [ch 3 · NHO I 2 _ + NHCOCH-CH-CHCOO Na I 2 2

Glutathione B R = -CH_CH

21 -f.

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Claims (34)

HCCH2-H2O2CH2NHCO or a pharmaceutically acceptable salt thereof, R R is hydrogen or lower alkyl and Alk is C C_g alkylene. 2. A derivative according to claim 1, characterized in that Alk is ethyl and R 2 is hydrogen. 3. A derivative according to claims 1-2, characterized in that R is acetylaminoethyl. 4. A derivative according to claims 1-2, characterized in that R is acetyloxyethyl. DK 169478 Pg 29 5. A derivative according to claims 1-2, characterized in that R is 1,2-di hydroxy-prop-3-yl. 6. A derivative according to claims 1-2, characterized in that R is carboxyethyl 5 or a non-toxic pharmaceutically acceptable salt thereof. 7. A derivative according to claims 1-2, characterized in that R is 1-carboxyaminoethyl or a non-toxic, pharmaceutically acceptable salt thereof. A derivative according to claims 1-2, characterized in that R is 1-methylimidazol-2-yl-methyl. 9. A derivative according to claims 1-2, characterized in that R is dimethylaminoethyl. 15 10. A derivative according to claims 1-2, characterized in that R is phenyl. The derivative of claims 1-2, characterized in that R is 4-nitro-phenyl. 20 12. A derivative according to claims 1-2, characterized in that R is 4-methoxy-phenyl. 13. A derivative according to claims 1-2, characterized in that R is 4-aminophenyl. A derivative according to claims 1-2, characterized in that R is 2-carboxy-phenyl or a non-toxic, pharmaceutically acceptable salt thereof. The derivative of claims 1-2, characterized in that R is 4-nitro-3-carboxyphenyl or a non-toxic, pharmaceutically acceptable salt thereof. The derivative of claims 1-2, characterized in that R is 4-pyridyl. 35 DK 169478 B1 30 17. A derivative according to claims 1-2, characterized in that it has the formula? H2 O ho2cchch2ch2conh O ch2ocnh2 5 CHCHOSSCH_CH "0 1 I 2 λ λ \ ØCH0 ho2cck2nhco 3" 'T 10 or a non-toxic, pharmaceutically acceptable salt thereof. 18. A derivative according to claims 1-2, characterized in that it has the formula (Ib) _, NO2 O / r \ lv 1 f2c £ m2 ('/ -SS-Alk-O. Jk \ / tT ir \ OCH, VZZN7 Ib '3 Ib 20 ° wherein R and Al kg are as defined in claim 1. 19. A derivative according to claims 1-2, characterized in that it has the formula 25 -F ° 2 O ch2ocnh2 \> ECH, XVII 30 \ - N 3 3 o A pharmaceutical composition, characterized in that it comprises a derivative according to claim 1 in connection with a pharmaceutically acceptable solvent, diluent, adjuvant or carrier. DK 169478 B1 31 A process for preparing a derivative according to claim 1, characterized in that at least one equivalent of a triazene of formula (VI) is reacted. Ar-N = N-NH-Alk-SS-R (VI) wherein R and Alk have the meaning set forth in claim 1, and Ar is the organic residue of a diazotable aromatic amine, with an equivalent of a mitosane of formula ( IV) 10. i? CH 2 OCNH 2 / OCH 3 wherein R * is as defined in claim 1, under reaction conditions in an inert organic solvent at a temperature of about 0 ° to approx. 60 ° until a sufficient amount of the product of formula (I) is obtained and when R in the compound of formula (I) is nitrosubstituted pyridyl, it is optionally converted by reaction in an inert solvent at a temperature of 0 ° C to 60 ° C. ° C, optionally in the presence of at least one equivalent of a strong base, with a thiol of the formula R'SH, wherein R 'has the meaning given above for R except nitrosubstituted pyridyl and if desired is converted where possible; an obtained compound of formula (I) for a pharmaceutically acceptable salt thereof. 22. A process according to claim 21, characterized in that 1- (2- (3-nitro-2-pyridyldithio) ethyl] - 3- (4-methylphenyl) triazene is used as the triazene of formula (VI). A process according to claim 21, characterized in that Alk is ethyl and R * is hydrogen. The process according to claim 21, characterized in that an equivalent base is used in the reaction. DK 169478 Pg 32 25. A process according to claims 21-24, characterized in that a lower alkanol, a lower alkanoic acid-lower alkyl ester, a lower aliphatic ketone, a cyclic al in fatty ether or a lower polyhalogenated aliphatic hydrocarbon having up to 8 carbon atoms or water as 5 reaction medium. The process of claim 25, characterized in that a reaction inert organic liquid solvent is used as the reaction medium for the tosan. 10 27. A process according to claim 25, characterized in that a lower alkanol, a lower one-alkyl alkanoic acid ester, a lower-lower alkyl ether, a lower polyhalogenated aliphatic hydrocarbon or a cyclic aliphatic ether having up to 8 carbon atoms are used as the reaction medium. 15 28. A process according to claim 25, characterized in that methylene chloride, methanol, diethyl ether, ethyl acetate or a mixture of two or more thereof is used as the reaction medium. Process according to claim 25, characterized in that the reaction temperature is from 0 ° to 60 ° C. The process of claim 25, characterized in that the reaction temperature is from 0 ° to 25 ° C. 25 Bristol-Myers Squibb Company at TH. OSTENFELD PATENT BUREAU A / S 30 i_ Peter Engl ^ ' 35 Copenhagen, 23 September 1994