FI80700C - FOERFARANDE FOER FRAMSTAELLNING AV TERAPEUTISKT ANVAENDBARA MITOMYCINANALOGER. - Google Patents

Description

1 80700

A method for preparing therapeutically useful mitomycin analogs

The invention relates to the preparation of novel mitomycin analogs containing a disulfide group. These compounds are analogs of mitomycin A in which the 7-alkoxy group has an organic substituent which introduces a disulfide group into the compound. Mitomycin A is an antibiotic that has been shown to be useful, and its 7-O-substituted mitosan-10 analogs have similar utility.

Nomenclature - A systematic, recent correction of Chemical Abstracts [Shirhata et al., J. Am. Chem. Soc., 105, 7199 (1983)] is [laS- (lap, 8β, 8ao, 8bp)] - 8 - (((aminocarbonyl) ox-15si) methyl] -6,8a-dimethoxy-1, 1α, 2,8,8a, 8b-Hexahydro-5-methylazirino- [2 ', 3', 3,4] pyrrolo [1,2-a] indole-4-7-dione, according to which the azirinopyrroloindole ring system is numbered as follows: 20 7 8 25

The trivial nomenclature widely used in the mitomycin literature names the ring system described above, which is associated with several substituents characteristic of mitomycins, as mitosan.

30 10 CH2OCONH2 1 3 CH3y ^ NflS> \ o 4 2 80700 According to this system, mitomycin A is 7,9a-dimethoxymitosane and mitomycin C is 7-amino-9a-methoxymitosan. With respect to the stereochemical configuration of the products of this invention, when identified by the base portion of the name "mitosan" or structural formula, it is meant that they have the same stereochemical configuration as mitomycin A or C.

10 q Cl ^ OCONl · ^ Mitomycin A R = R ^ = OCH ^ R I R-, Mitomycin C R = NH ~, - \ R ^ och CHXx) s ^ 3 o N \ 15

Mitomycin C is an antibiotic produced by fermentation and is currently marketed as approved by the Food and Drug Administration for the treatment of gastric or pancreatic diffuse adenocarcinoma in combination with other approved chemotherapeutic agents and for palliative treatment when other therapies have failed (Bramir Laboratories, Syracuse, New York 13221, Physicians' Desk Reference, 37th ed., 1983, pp. 747 and 748). Mitomycin C and its preparation by fermentation are the subject of U.S. Patent 3,660,578, issued on May 2.5. 1972 and has been requested to take precedence over previous applications, e.g. On the basis of an application lodged in Japan on 6 April 1957.

30 J.S. Webb et al. Lederle Laboratories Division Ame Rican Cyanamid Company, J. Am. Chem. Soc., 84, 3185 -3187, (1962) first published the structures of mitomycins A, B and C and profiromycin. One of the chemical conversion reactions used to determine mitomycin A and mitomycin C in this structural study was 3,80700 was the conversion of the former, 7,9a-dimethoxymitosane, by reaction with ammonia to the latter, 7-amino-9a-methoxymitosane. Substitution of the 7-methoxy group of mitomycin A has proven to be a remarkably interesting reaction in the preparation of mitomycin C derivatives having antitumor activity. It has recently been shown that the stereochemical configuration of positions 1, 1a, 8a and 8b is as described above with reference to the nomenclature of Chemical Abstracts [Shirata et al., J. 10 Am. Chem. Soc., 105, 7199-7200 (1983)]. The previous literature talks about the enantiomer.

The following articles and patents discuss, inter alia, the conversion of mitomycin A to a 7- (substituted amino) mitomycin C derivative having antitumor activity. The purpose of this study was to prepare derivatives that were more potent and, in particular, less toxic than mitomycin C; Matsui et al., J., Antibiotic, XXI, 189-198 (1968); Konishita et al., J. Med. Chem. 14, 103-109 (1971); 20 Iyengar et al., J. Med. Chem., 24, 975-981 (1981);

Iyengar, Sami, Remers, and Bradner, Abstracts of Papers, 183rd Annual Meeting of the American Chemical Society, Las Vegas, Nevada, March 1982, Abstract No. MEDI 72; Consulich et al., U.S. Patent No. 3,332,944, issued July 25, 1967;

Matsui et al., U.S. Patent No. 3,420,836, issued January 7, 1969;

Matsui et al., U.S. Patent No. 3,450,705, issued June 17, 1969; 30 Matsui et al., U.S. Patent No. 3,514,452, issued May 26, 1970;

Nakano et al., U.S. Patent No. 4,231,936, issued November 4, 1980;

Remes, U.S. Patent No. 4,268,676, issued May 19, 1981.

The following patent applications relate to the preparation of 7,80700 7- (substituted amino) mitomycin C derivatives in which the substituent introduces a disulfide bond into the compound:

Kono et al., EPO Patent Application No. 116,208 (1984); 5 Vyas et al., GB Patent Application No. 2,140,799 (1984).

7-Alkoxy-substituted mitosans with a structure close to mitomycin Aita are described as useful antibiotics with activity in animal tumor experiments, Urakawa et al., J. Antibio-10 ties, 23, 804-809 (1980).

Mitomycin C is the major mitomycin produced by fermentation and is a commercially available form. The technique currently used to convert mitomycin C to mitomycin A is hampered by numerous shortcomings. Hydrolysis of mitomycin C to the corresponding 7-hydroxy-9α-methoxynitosan and methylation of this material requires diazomethane, which is a very hazardous substance to be processed on a production scale, and the 7-hydroxy intermediate is very unstable [Matsui et al., J. Antibiotics, XXI; 189 - 198 20 (1968)]. One method that seeks to avoid these difficulties uses 7-acyloxymitosans (Kyowa Hakko Kogyo KK JP Patent No. J5 6073-085, Farmdoc No. 56227 D / 31). Urakawan et al [J. Antibiotics, 23, 804-809 (1980)], the alcoholysis of mitomycin A is limited to the availability and reactivity of alcohols used as starting materials for the preparation of certain types of 7-alkoxy structures.

The invention relates to a process for the preparation of therapeutically useful mitomycin analogues of the formula (II) or (III) and their non-toxic pharmaceutically acceptable salts.

II

5 80700 o n CH2OCNH2

5 R3-Alk, -SS- (CH2) ~ -0 v. A

"\" 3 “3 ·" <»> ° o CH2OCNH2 r4-ss- ^, och3 15 CH3'7X | ^ \ ^^ Xn'h (III) 20 in which formulas Alkx is ethylene1 or methylene, R3 is acetyloxy , 1,2-dihydroxyethyl, carboxy, 1-carboxyaminomethyl or 1-methylimidazol-2-yl, NH, NHCOCH 2 CH 2 CHCOO'Na * or dimethylamino, 1 2 3 4 5 6 7 8 9 10 11

-CH

2 3 CONHCH2 COO- Na * 4 R4 is phenyl, 4-nitrophenyl, 4-methoxyphenyl, 4-aminophenyl, 2-carboxyphenyl, 4-nitro-3-carboxyphenyl, 4-5 pyridyl, 2- (3-nitropyridyl) .

The compounds of this invention are inhibitors of experimental tumors in animals 7. In particular, the compounds shown herein as compounds of Examples 17, 20 and 21-34 are novel substances. They are used in the same way as mitomycin C. The dosages used are adjusted according to their toxicity compared to 6 80700 mitomycin C toxicity. In cases where the toxicity of the new compound is lower, higher doses are used.

Mitosanes of formulas II and III are prepared according to the invention in such a way that at least one equivalent of triazene of formula (V) or (VI)

Ar-N = N-NH- (CH2) 2 -SS-Alkj -R3 Ar-N = N-NH- (CH2) 2 -SS-R4 (V) (VI) 10 is reacted with one equivalent of mitosan having formula (IV)

O

O CH2OCNH2

HO II W

NH (IV) 0 W "20 wherein R 3, R 4 and AlkL are as defined above and Ar is an organic residue of a diazotizable aromatic amine, under suitable reaction conditions in an inert organic solvent at a temperature of about 0 to 60 ° C, provided a sufficient amount a compound of formula (II) or (III) is formed, or b) at least one equivalent of a thiol of formula R 3 Alk: SH or formula R 4 SH is reacted with one equivalent of mitosan of formula (Ib) 30 / NO 2 ° / -ζ O CH20iNH2 ('^ ^ CH ^ -O OCH, (Ib) ch3 il k

Wherein R 3, R 4 and Alkx are as defined above, optionally in the presence of at least one base equivalent, in an inert solvent at a temperature of about 0 to 60 ° C until a sufficient amount of compound 5 of formula (II) or (III) is formed. .

The term non-toxic, "pharmaceutically acceptable salt" is intended to include salts of the compounds of formula II or III with any non-toxic pharmaceutically acceptable acid or base. Such acids are well known and include hydrogen chloride, hydrogen bromide, sulfur, sulfamine, phosphorus, nitrogen, maleic, fumaric, succinic, oxalic, benthic, methanesulfonic, wine , citric, camphorsulfonic and levulinic acids, etc. Such bases are also well known and include, for example, non-toxic metal salts such as sodium, potassium, calcium, and magnesium salts, ammonium salts, and non- toxic amines, e.g., trialkylamines, procaine, dibenzylamine, pyridine, N-methylmorpholine, N-methylpiperidine, and the like.

Salts of 20 amines. The salts are prepared by methods known in the art.

In the process of the invention, a mitosan of formula IV is reacted with a triazene of formula V or VI as shown in Scheme 2.

25 Scheme 2 0 0 CH.OCNH »HO Π fc 2 2 yVV0CH3

30 CH3aVx ^]> - H

Ar-N = N-NH- (CH2) 2-SS-R 3, Alk 1

+ —---> II or III

35 or Ar-N = N-NH- (CH2) 2-SS-R4

VI

8 80700 wherein R 3, R 4 and Alk 1 are as defined above and Ar is an organic residue of a di-azotatable aromatic amine.

Aryltriazenes of formula V or VI can be prepared by conventional methods starting from aminodisulphides of formula XII

R2 SS- (CH2) 2 NH2 XII

10 which may alternatively be represented by formulas XIII and XIV

R3 -Alkj -SS- (CH2) 2NH2 and R4-SS- (CH2) 2NH2 XIII XIV

15

Aminodisulfides of formula XIli or XIV are known compounds and can be prepared by a variety of methods. They can be prepared, for example, by reacting a suitable thiol of formula R3AklxSH or R4 SH with a Bunte salt of formula

NH2 (CH2) 2 SS03Na XV

or sulfenylthiocarbonate of formula

25 O

II

NH2 (CH2) 2 SSCOCHj XVI

Klayman et al., [J. Org. Chem., 29, 3737-3738 (1964)] have prepared the following compounds by the Bunte salt method: 2-aminoethyl-n-butyl disulfide; 2-aminoethyl-n-heksyylidisulfidi; 2-aminoethyl-n-oktyylidisulfidi; 2-aminoetyylifenyylidisulfidi; 35 2-Aminoethylbenzyl disulfide.

Il 9 80700

Methanol has been found to be the preferred reaction solvent for the reaction between the Bunte salt and the thiol. When using this solvent, reaction temperatures of 9 to -10 ° C have been found to be preferred. When other solvents were used, higher temperatures were necessary. The main disadvantage of this method is the formation of symmetrical disulfides as a by-product, presumably as a result of the disproportionation of the desired mixed disulfide.

The mixed disulfides used as starting materials of formulas XIII and XIV are preferably prepared by the reaction of a suitable thiol with a sulfenyl thiocarbonate of formula XVI. This is the method described in S.J. Brois et al., J. Am. Chem. Soc., 92, 7629-7631 (1970). Typically, this method of preparation involves the addition of a thiol to a methanol solution of an aminoalkyl sulfenyl thiocarbonate of formula XVI, followed by allowing the reaction to proceed at a temperature in the range of 0-25 ° C. The reaction times vary practically from the instantaneous reaction to several hours, depending on the thiol used in each case. The progress of the reaction can be monitored by measuring the presence of unreacted thiol in the reaction vessel. If the reaction is slow, a catalytic amount of triethylamine can be added to accelerate it.

1- (Substituted disulfine-25-di) -3-arylthiazenes of formula V or VI are prepared by reacting aminodisulfides of formula XII with aryldiazonium salts by conventional methods. As the source of the aryl portion of the 1,3-disubstituted triazene, any arylamine having 6 to 12 carbon atoms and easily forming a diazonium salt can be used. Examples of disulfide triazenes prepared in this manner and used in accordance with this invention include 1 - [- (2-acetamidoethyldithio) ethyl] -3- (4-methylphenyl) triazene and 1- [2- (3-nitro-2-pyridyldithio) ethyl] - 3- (4-methylphenyl) triazine. The following compounds are also examples of formula V.

Suitable triazenes for use as starting materials in this invention according to 10 80700 or VI: 1- [2- (3-nitro-2-pyridyldithio) ethyl] -3- (4-chlorophenyl) triazene, 5- [2- (3-nitro -2-pyridyldithio) propyl] -3- (4-methylphenyl) triazene, 1- [2- (2-pyridylthio) ethyl] -3- (4-methylphenyl) triazene, 1- [2- (phenyldithio) ) ethyl] -3- (4-methylphenyl) triazene, 1- [2- (butyldithio) ethyl] -3- (4-methylphenyl) triazene, 1- [2- (4-methoxyphenyldithio) ethyl] -3- ( 4-methylphenyl) triazene, 1- [2- (4-nitrophenyldithio) ethyl] -3- (4-methylphenyl) triazene, 1- {2- [2-benzoylaminoethyl) dithio] ethyl} -3- (4 -methyl phenyl) triazene, 1- [2- (4-chloro-2-naphthyldithio) ethyl] -3- (4-methylphenyl) triazene, 1- {2 - [(2-phenoxyethyl) dithio] ethyl} - 3- (4-methylphenyl) -20 triazene.

One preferred embodiment of the present invention is an alternative process for the preparation of disulfide mitosanes of formula Ia 25 0 2 I? CH_0CNH_ R “ss_CH2CH2 °> v ^ / JJsvs ^ _ ^ Jl ^ 2 M ΗΓ \ s ° CH, V * 3 30 γ \ ^ y wherein R2 is an organic group, namely a moiety of an organic thiol according to R2SH, which may alternatively be described as a group R3Alkx or R4, wherein R3, R4 and Alkx are

II

11 80700 as defined above.

For the preparation of disulfide mitotanes of formula Ia, it is preferred to use 9a-methoxy-7- [2- (3-nitro-2-pyridylthio) ethoxy] mitosan of formula XVII in a thiol exchange process with an organic thiol of formula R2 SH as described in Scheme 3. The formation of disulfides of the formula Ia is based on the stability of the by-product, namely 3-nitro-2-mercaptopyridine, which is only a thione of the formula XVIII.

10 Scheme 3 _po2 j) r. Λ o ch2ocnh2 '-N Nfj + r2sh -> 15

XVII

20 R2 A ftoLj R - ^ ävVA ^ o * ° 2 I ιΓχί! 3 * l 25

XVIII

Ia Described herein are two general synthetic methods for preparing both lipophilic and hydrophilic mitosans of formula Ia. General method A is used to prepare either lipophilic or moderately soluble disulfides of formula Ia, while general method B is used to prepare water-soluble disulfides of formula Ia, which are preferably isolated as sodium salts or in the zwitterionic form. Preferably, at least one equivalent of mercaptan of formula R2SH is used per equivalent of Mitoan of formula XVII and the reaction can be carried out in the presence of about one base equivalent of mercaptan of formula R2 R2 SH per equivalent.

Preferred bases include tertiary amines, e.g., triethylamine, N-methylmorpholine, N-methylpiperidine, pyridine, 2,6-lutidine, and inorganic bases, e.g., sodium bicarbonate, potassium carbonate, potassium bi-10 carbonate, etc. Suitable solvents for inert acids, etc. for the reaction of the starting materials according to are lower alkanols, lower alkyl esters of lower alkanoic acids, lower aliphatic ketones, cyclic aliphatic ethers, lower polyhalogenated aliphatic carbons and water. Organic solvents contain up to 8 carbon atoms but those having a boiling point below 100 ° C are preferred. Individual preferred solvents include methylene chloride, methanol, acetone, water, and mixtures thereof. The reaction may be carried out at the reflux temperature of the reaction mixture or at a maximum of about 60 ° C. It is recommended that the reaction be carried out at room temperature or below, for example in the range of 0 to 25 ° C.

The above reaction conditions and preconditions 25 are applicable to the preparation of other mitosans of formula Ia according to the general procedure shown in Scheme 3.

With respect to compounds representing thiols of formula R 3 Alkx SH or R 4 SH which can be converted by reaction with Bunte salt XV or sulfenylthiocarbonate XVI to intermediates of formula XIII or XIV, which in turn are converted to the products of this invention as described above, the invention The processes according to the invention are limited only by the use of thiols which contain primary alkylamines as terminal groups, 13,80700 which could lead to the formation of product mixtures, and by the use of heteroaromatic thiols which do not react with the compounds of the formula Ia.

The utility of the compounds of the invention in antineoplastic therapeutic methods is apparent from the results of in vivo screening experiments in which the compounds were administered at various doses to mice first induced with P-388 leukemia or B16 melanoma.

The compounds of this invention are believed to have antibacterial activity against gram-positive and gram-negative microorganisms in the same manner as natural mitomycins and are thus potentially useful as therapeutic agents in the treatment of bacterial infections in humans and animals.

Table I shows the results of laboratory experiments performed on CDFj mice transplanted intraperitoneally with a tumor implant of 106 ascites cells of P-388 murine leukemia treated with different doses of either test compound II or III or mitomycin C. . The compounds were administered by intraperitoneal injection. Groups of six mice were used for each dose and treated with a single dose of compound the day after tumor implantation. Ten control mice treated with saline were included in each set of experiments. Mitomycin C-treated groups were included in the experiments as positive controls. For 30 days, a protocol was kept to determine the mean survival time for each group of 30 mice and to record the number of surviving mice at the end of the 30-day period. Mice were weighed before treatment and again on the sixth day. The weight change was marked as drug toxicity. Mice weighing 20 g each and up to about 2 grams weighed 14,80700; the reduction was not considered excessive. Results were expressed as% 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 5,100. Control animals treated with saline generally died within nine days. The "maximum effect" in the following table is given as% T / C and the dose at which this effect is reached is given. Values in parentheses are those obtained with mitomycin C used as a positive control in the same experiment. Thus, the relative activity of the present substances relative to mitomycin C can be approximated. The minimum potency expressed as% T / C was considered to be 125. The effective minimum dose given in the following table is the dose that gives a% T / C of about 125. In each case, the two values given in the column "mean weight change" are the mean weight change per mouse at effective maximum dose and effective The minimum dose.

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18 80700

Table II shows the results of antitumor efficacy experiments using mouse-grown B16 melanoma. BDF1 mice were used and implanted subcutaneously with a tumor graft. A 60-5 day protocol was used. Groups of ten mice were used for each dose studied and the mean survival time was determined for each group. Control animals inoculated with the tumor as well as test animals and treated with drug-free injection vehicle had a mean survival time of 24 days. The ratio of survival versus survival time (% T / C) was used as a measure of efficacy and the maximum effective dose and the effective minimum dose were determined for each test compound. The effective minimum dose of Te-15 was defined as the dose that gave a% T / C of 125. At each dose, experimental animals were treated intravenously with test compound on days 1, 5, and 9.

20

Table II

Compound Maximum potency ^ Effective minimum- Average e.g. No.% T / C Dose dose_ weight change 28,167 (112) 1 2 3 1.6 (3) 3 0.4 -0.9; +1.4 25> 214 (145) 2.4 (3) 1.6 -2.4; -1.9 20 110 (112) 3.2 (3) 3.2 +0.5; +0.5 26 152 (145) 1.6 (3) 1.6 -0.6; -0.6 35 The values in mg / kg body weight obtained in animal experiments in mg / kg body weight 30 2. The average in grams / day for each maximum 2 and minimum dose 3 are the values obtained for mitomycin C in a parallel experiment.

Based on the results of 80,80700, the compounds of the invention can be used to prevent tumors in mammals. For this purpose, they are administered systemically in a substantially non-toxic, antitumor-effective dose to a mammal having a tumor.

The compounds of this invention are intended to be administered primarily by injection, in much the same manner and for some of the same purposes as mitomycin C. Somewhat higher or lower doses may be used depending on the sensitivity of the tumor in question. They are easily dispensed as dry pharmaceutical compositions containing diluents, buffers, stabilizers, solubilizers, and ingredients to aid in providing a pharmaceutically acceptable product. These compositions are then reconstituted with an injectable liquid medium just prior to use. Suitable injectable liquids include water, isotonic saline, and the like.

In the following methods and examples, all temperatures are given in degrees Celsius and melting points are uncorrected. Nuclear magnetic resonance (1 H NMR) spectra were determined on a Varian XL100, Joel FX-900 or Bruker WM 360 spectrometer in either pyridine-d5 or D20 as indicated. When pyridine-d5 is used as the solvent, the resonance of pyridine at 6-5.57 is used as an internal reference, while TSP is used as an internal comparison when D 2 O is used as a solvent. Chemical shifts are given in δ-units and coupling constants in Hertz. The distribution types are denoted as follows: s singlet, d doublet, t triplet, g quartet, m multiplet, bs wide singlet, dd double doublet dt double triplet. Infrared spectra were determined on either a Bekman Model 4240 spectrometer or a Nicolet 35 5DX-IR spectrometer and are reported in units of 20 80,700 cm-1. Ultraviolet (UV) spectra were determined on either a Cary Model 290 spectrometer or a Hewlitt Packard 8450A spectrometer equipped with a multidiod table! 1-detector for. Thin layer chromatography (TLC) was performed on 0.25 mm Analtech silica gel GF plates. Flash chromatography was performed on either Woelm-neutral alumina (DCC grade) and Woelm-silica gel (32-63) and the indicated solvents. All evaporations were performed under reduced pressure and below 40 ° C.

10'-Alkyl-3-aryltriazenes are a group of reagents known to be useful in the reaction to form the corresponding lower alkyl esters of carboxylic acids. 1-Methyl-3- (4-methylphenyl) triazene can be prepared as follows: Method 1 E. H. White et al., Org. Syn., 48, 102-195 (1968).

l-methyl-3-p-tolyylitrlatseeni. p-Toluidine (50.2 g, 0.47 mol) is added to a 2-liter flask equipped with a 200 mL dropping funnel and a powerful stirrer, and the flask is immersed in an ice-salt bath n.

-10 ° C. A solution of 46.8 g (0.55 mol) of potassium nitrite in 150 ml of water is placed in a dropping funnel and a mixture of 250 g of crushed ice and 140 ml of concentrated hydrochloric acid is added to p-toluidine with stirring. The potassium nitrite solution is added slowly while stirring for 1 to 2 hours until a positive starch potassium iodide test is obtained (Note 1) and the mixture is stirred for a further hour to ensure that all the toluidine reacts.

The pH of the 30-p-toluenediazonium chloride solution is then adjusted to 6.8-7.2 at 0 ° with cold, concentrated aqueous sodium carbonate solution, whereupon the solution turns red to orange and a small amount of red material precipitates. Transfer the cold, neutral solution to a Ti-35 dropping funnel and add slowly, while stirring vigorously, a mixture of 150 g of sodium carbonate, 300 ml of a 30 to 35% aqueous solution of methylamine (Note 2) and 100 g of crushed ice in 3 l. in the bottle. The reaction mixture is kept at about -10 ° C for the addition of ai-5, which takes about 45 minutes (Note 3). The solution is extracted with three 1 liter portions of ether. The ether extracts are dried over anhydrous sodium sulfate and evaporated on a rotary evaporator at room temperature to give 65 g of crude 1-methyl-3-p-tolyltriazene (Note 4). This is placed in a water-cooled sublimation apparatus and the triazene is sublimed at 50 ° C (1 mm); 43.3 g (0.29 mol, 62%) of a yellow crystalline sublimate are obtained, melting point 77-80 ° C (Note 5). The sublimed material can be recrystallized from hexane to give the triazene as white needles, m.p. 80.5 - 81.5. More simply, it is dissolved in a minimum amount of ether and the solution is diluted 2-fold with a volume of hexane and cooled to 0 ° C to give smooth plates that are slightly yellowish; mp. 79-81 °. The yield of pure triazene-20 is 33-37 g (47-53%) (Note 6).

1. Individual experiments with starch-potassium iodide paper should be performed within 1-2 minutes after the addition of potassium nitrile is stopped.

2. A 40% aqueous solution of methylamine may be used instead.

3. The reaction is complete when a drop of solution no longer gives a red color with the solution formed by β-naphthol in aqueous sodium carbonate solution.

4. The major impurity is 1,5-di-p-tolyl-3-methyl-1,4-30 pentaazadiene (m.p. 148 ° C). This can be removed by fractional crystallization, but it is easier to sublime the triazene from the reaction mixture.

5. The sublimed material contains traces of 1,3-di-p-tolyltriazene as evidenced by thin layer chromatography. Recrystallization gives pure 1-methyl-3-p-tolyl-22,80700 triazene.

6. This procedure works well only for water-soluble amines. Method 2 below is more suitable for the preparation of triacene from water-insoluble amines.

Method 2 E.H.White el ai., Tetrahedron Letters No. 21, pp. 761 (1961).

l-n-butyl-3-p-kloorlfenyylltrlatseeni. A solution of p-chlorobenzenediazonium hexafluorophosphate (recrystallized from acetone-methanol) (2.87 g, 10.1 mol) in dimethylformamide (which did not contain dimethylamine) was added slowly while stirring n-butylamine (0.73 g, 10 .0 mol), a mixture of powdered sodium carbonate (15 g) and dimethylformamide (30 ml), which was stirred and kept at -5 ° C. The diazonium salt solution can be used at room temperature, but a purer product is usually obtained if the diazonium salt solution is prepared and added from a separatory funnel maintained at about -50 ° C. The solution was warmed to 0 ° C and stirred until a negative result was obtained in an experiment with 2-naphthol (usually only a few minutes). Ether was added, the mixture was filtered and the filtrate was washed thoroughly with water and dried, (at this point the triazene can be isolated and recrystallized from pentane at time temperatures). Method 3 7-Hydroxy-9α-methoxymitosane. Mitomycin C (2.2 g, 6.6 mmol) was dissolved in 140 mL of 0.1 N methanolic NaOH (50%) and the reaction mixture was stirred at room temperature for 30 hours. The pH of the solution was then adjusted to about 30 with HCl and extracted with ethyl acetate, the combined ethyl acetate extracts dried (Na 2 SO 4) and evaporated under reduced pressure at about 30-35 ° C to give a solid residue which was dissolved in ether and treated. purple with excess hexane to give a precipitate. The precipitate was collected and dried over air to give the title compound as a fine purple powder (1.4 g, 63%).

1 H NMR (pyridine-d 5, δ): 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, 2 H).

Method 4

Mitomycin A. 100 mg (0.30 mmol) of 7-hydroxy-9α-methoxymitosane and 100 mg (0.67 mmol) of 3-methyl-1-p-tolyl-10-litriazene were dissolved in 2 ml of methylene chloride and 10% of any diethyl ether. After gently refluxing for 8 hours, the solution was stirred at room temperature for 18 hours. TCL (methylene chloride / methanol, 90:10) showed a deep purple spot at Rf = 0.36 and traces of impurities at Rf = 0.41. The reaction mixture was evaporated to dryness and chromatographed on Woelmi neutral alumina using methylene chloride and methylene chloride / methanol (30: 1) as eluents. Fractions containing a component with an Rf of 0.36 were combined and evaporated to dryness. Combination of the dry residue from methylene chloride and hexane gave the title compound as a fine amorphous purple powder (25 mg, 24%), m.p. 161 ° C. Analysis for C 6 H 19 N 3 O 6: 25 calculated C 54.96; H 5.44; N 12.02 found C 53.96; H 5.37; N 11.99.

IR (KBr), ν max, cm -1: 3400, 3300, 2950, 1700, 1630, 1575, 1200, 1060.

1 H NMR (pyridine-d 5,): 1.82 (s, 3H), 2.74 (dd, 1H), δ 3.12 (d, 1H), 3.24 (s, 3H), 3.54 (dd, 1H), 3.96 (dd, 1H), 4.02 (s, 3H), 4.22 (d, 1H), 4.84 (bs, 2H), 5.02 (t, 1H) , 5.38 (dd, 1 H).

The yield of Method 4 increases to 63% when methylene chloride is used as the reaction solvent and room temperature is observed for 24 hours.

24 80700

Method 5 A 250 mL one-necked round bottom flask was charged with solid Na 2 CO 3, a 35% aqueous solution of the amine (amount the same as in Method 1), and ice and sus-5 suspension were stirred at -5 ° C (ice-salt bath). To this suspension was added dropwise a cold suspension of p-chlorobenzene diazonium hexafluorophosphate (Aldrich Chemical Co.) in ice, water and Na 2 CO 3 (pH of the solution about 7). When the addition was complete, the reaction mixture was extracted with diethyl ether. The combined diethyl ether extracts were washed with water, dried (Na 2 SO 4) and concentrated. The yellowish solid residue was purified by column chromatography on Woelm alumina using hexane / methylene chloride (1: 1) as eluent (* Η NMR determined).

15 Examples 1-10

The triazenes 1-7 shown in Table III below were prepared according to General Method 1 described above, which exemplified the triazene of Example 1. The triazenes were purified by column chromatography on Woelm alumina.

Triazenes 8-10 of Table III were prepared according to General Method 5 above.

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Example 11 1- [2- (3-Nitro-pyridyldithio) ethyl] -3- (4-methyl-phenyl) -triazene A solution of 4-methylphenyldiazonium chloride is prepared as described in Method 1 from p-toluidine and adjusted to pH 6.8. - 7.2 at 0 ° C as described in said method.

In this way, a solution containing 21.15 mmol of diazonium salt in 45 ml of solution was prepared and placed in a dropping funnel connected to a 250 ml round-bottomed three-necked 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 added to the flask at this stage. To the flask were added 6 ml of saturated aqueous sodium carbonate solution and 10 g of ice.

The flask was cooled in an ice bath and its contents were stirred mechanically. Diazonium saline was then added dropwise within one hour of the dropping funnel. After complete addition, the reaction mixture was allowed to warm to room temperature and then extracted with three 400 mL portions of ether. After the extracts were dried and evaporated, the desired product was obtained, which was purified by chromatography on an alumina packed column, 25.4 mm in diameter and 254 mm in length, to develop a column and eluted with hexane / methylene chloride (4: 1), hexane / methylene chloride (3 : 2), hexane / methylene chloride (1: 4) and finally methylene chloride containing 1% methanol. The appropriate fractions (detected by TLC) were combined and evaporated to give 2.5 g of the title compound.

Examples 12 to 19

General procedure for the preparation of 7-alkoxy-9a-methoxymitosans (12-19)

A solution of triazene (2.4 equivalents) in 35 Cl 2 Cl 2 / methanol (4: 1) was added to a solution of 7-hydroxy-9α-methoxy-28,80700 mitosan (prepared in Method 3) in CH 2 Cl 2 / methanol (4: 1). 1). The reaction mixture was stirred at room temperature and the reaction was monitored by thin layer chromatography (TLC) (10% MeOH in Cl 2 Cl 2).

The 7-alkoxy-9α-methoxymitosane product appears as a dark purple spot on TLC. The reaction mixture is chromatographed on Woelm alumina when the reaction is considered complete by TLC to give 7-alkoxy-9α-methoxymitosane as an amorphous solid.

The products prepared are identified as Examples 12 to 19 in Table IV.

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Example 20 9a-Methoxy-7-Z2- (3-nitro-2-pyridylthio) ethoxy.7 Mitosane (20) 7-Hydroxy-9a-methoxymitosane was measured in 580 mg of a 5 (1.37 mmol) round bottom flask and dissolved in 60 mL. of methylene chloride. The triazene of Example 11 was added to a solution of 2.5 g (5.7 mmol) 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 another 26 hours and then further subjected to column chromatography on a column with a diameter of 8.5 mm and a length of 305 mm packed with alumina.

The solvent cycles used to develop and elute the column were 200 ml aliquots of solvents: 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 the title compound, 470 mg.

Analysis for C 22 H 23 N 5 O 8 S 2 (corrected for 0.5 mol% CH 2 Cl 2): calculated C 45.65; H 4.09; N 11.82 Found C 45.74; H 4.14; N 11.61.

IR (KBr), νmax, cm-3440-3200, 3060, 2930, 1720, 1570, 1510, 1395, 1335, 1210, 1055.

1 H NMR (pyridine> d 5, δ): 1181 (s, 3H), 2.00 (bs, 1H), 2t61 (bs, 1H), 2.98 (bs, 1H), 3.08 (s, 3H), 3f 2 O (m, 2H), 3.39 (d, 1H), 3r83 (dd, 1H), 4.07 (d, 1H), 4.59-4.89 (m, 3H), 35 5.21 (dd, 1H), 7.16 (dd, 1H), 8.31 (dd, 1H), 8r 71 (dd, 1H).

Il 33 80700

By applying the methods of Examples 11 and 20 to other (3-nitro-2-pyridyldithio) alkylamines having 2 to 6 carbon atoms in the alkyl group, mitosane derivatives of the following formula can be prepared.

5 o O CH-OCNH, Π '"M" SS- (CH-) -O, J Λ 2 2 n' 0CH2 10 CH3'y 'n = 2-6 R = H or alkyl 15

Examples 21-34 The 7-alkoxy-dithio 9-a methoxymitosans 21-34 mentioned in Table V below were prepared according to the general method A or B shown in Table V below. For mitosan compounds 20 to 34, the physical constants are shown in Table VI, which also follows.

Method A

To a solution of the mitosan (--0.1 mmol) of Example 20 in deoxygenated acetone (3-5 mL) is added, with stirring and under argon, triethylamine (1,11.1 eq) followed by dropwise or portionwise addition of mercaptan (^1 mL). equivalent) in acetone (h) (1-2 ml). In the case of most reactions, the progress of the reaction is monitored by silica gel thin layer chromatography. 30 by graph (10% CH 3 OH in CH 2 Cl 2). The completion of the reaction is indicated by the disappearance of the spot corresponding to the starting material and the appearance of the product spot.

At this point, the reaction mixture is concentrated under reduced pressure (3030 ° C) and the residue is chromatographed on a neutral Wielm alumina column (8.3 mm x 254 mm) packed using a slurry in a mixture of 2 to 5% CH 2 OH CH 2 Cl 2. :in. This procedure separates the desired mitosane product from the by-product pyridylthione compound which remains on the column.

The product thus eluted with a mixture of 2 to 5% CH 2 OH in CH 2 Cl 2 is further thoroughly purified by flash silica gel chromatography using a mixture of 5 to 7% CH 2 OH in CH 2 Cl 2 as eluent. The main line corresponding to the product is isolated and the amorphous 7-alkoxydithio-9a-methoxymitosan is characterized.

(a) In cases where the starting mercaptan is impure, more than 1 equivalent of thiol is required.

15 (b) In the case where the starting material of Example 20

Saab and the product have very close R 1 values by RLC, high pressure liquid chromatography (HPLC) is used to monitor the reaction (μ (Bondapak-C 6 g column). Method B

To a solution of the mitosan (0,10.1 mmol) of Example 20 in a mixture of 2 to 5% acetonyl in methanol (10 mL) was added saturated aqueous NaHCO 3 (6 drops) followed by mercaptan ( c) (1 equivalent) in methanol (1 ml). The progress of the reaction is monitored by TLC (silica gel, 10% CH 2 OH in CH 2 Cl 2). When the reaction is complete, the reaction mixture is diluted with water (15 mL) and concentrated to about 10 mL under reduced pressure at about 30 ° C. The resulting solution is chromatographed on a reverse phase: C-18 column using a gradual gradient of eluent (from 100% H 2 O to 80% CH 2 OH in H 2 O). The product, which elutes as the richest red band, is recovered and concentrated to give 7-alkoxydithio-9a-methoxymitosane as an amorphous solid. If further purification 35 is required, repeat the chromatography step above.

li 35 80700 a) Methylene chloride can also be used, but acetone is preferred.

b) If the mercaptan is L-cysteine, this base is not used.

5 c) Water is used if the starting ethiol is water soluble, d) Elution with water separates the yellow pyridylthione formed as a by-product from the product which remains on the column.

36 80700

Table V

7-alkoxydithio-9a-methoxymitosanes

If o CH2OCNH2. . RSS ^ - \ vOCH, A or 3

RSH + 20 -> JU

B CH3 n LFu

Example Thiol

Well. (RSH) Method Product

O

II

21 ethyl-2-mercapto-A R = -CH2CH2OCCH3 acetate

22 3-mercapto-1,2-R = -CH2CH (OH) CH2OH

propanediol 1 2 3 4 5 6 3-mercaptopropioni- BR = -CH2CH2COO Na +: acid 2 cysteine BR = -CH2CH (NH3 +) COO_ 3 thiophenol AR = ^ 4 p-nitrobenzenethiol AR = ^ -NO2 5 p-methoxybenzene AR = ^^ -OCH ^ 6 p-aminobenzenethiol AR = ^ ^ ~ NH2 37 80 700

Table V (continued)

Example Triol

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/OF

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Claims (3)

46 80700 A process for the preparation of therapeutically useful mitomycin analogs of formula (II) or (III) and non-toxic pharmaceutically acceptable salts thereof, O q ch2ocnh2 10 R -Alk, -SS- (CH „) _-0 Jl Jf CH3 ui) 15 2 o CH2OCNH2 r4-ss- <ch,), - ov IL J rrv ^ CH3 (111) wherein Alkx is ethylene or methylene, R3 is acetyloxy, 1,2-dihydroxyethyl, carboxy, 1-carboxy-aminomethyl or 1-methylimidazol-2-yl, group NH2 NHCOCH2 CH2 (LhCOO-Na + or dimethylamino, -CH CONHCH 2 COO * Na * 30 R 4 is phenyl, 4-nitrophenyl, 4-methoxyphenyl, 4-aminophenyl, 2-carboxyphenyl, 4-nitro-3-carboxyphenyl, 4-pyridyl, 2- (3- nitropyridyl), characterized in that a) at least one equivalent of triazene of formula (V) or (VI) II 47 80700 Ar-N = N-NH- (CH2) 2 -SS-Alk1 -R3 Ar-N = N-NH- (CH2) 2 -SS-R4 (V) (VI is reacted with one equivalent of mitosan, 5 of formula (IV) O o ch2ocnh2 jdch3 3 o \ J ^ ”H (IV) in which formulas R3, R4 and Alkx are as defined above and Ar is an organic residue of a diazotizable aromatic amine, under suitable reaction conditions in an inert organic solvent at a temperature of about 0 to 60 ° C until a sufficient amount of the compound of formula (II) or (III) or b) at least one equivalent of a thiol of formula R3AlkjSH or formula R4 SH is reacted with one equivalent of mitosan of formula (Ib) / NO2 2 25 / T \ N CH2 ° = NH2 V7s5 '(CH2> In the formulas R 3, R 4 and Alk 2 are as defined above, optionally in the presence of at least one base equivalent, in an inert solvent at a temperature of about 0 to 60 ° C until a sufficient amount of the formula Compound 48 (8) or (III) 48 8 0 7 0 0 is formed. Process according to Claim 1, characterized in that a compound of the formula VH 2 O ho 2cchch 2ch 2cohh O CH.OCJJH, CHCH «SSCH-CH-O I 2 is prepared 10 H02CCH2NHCO3 N (N 3 N 2 -H 15 or a non-toxic pharmaceutically acceptable salt thereof. Process according to Claim 1, characterized in that a compound of the formula 20 is prepared. chXh V X) -SS-CH, CH, -Ox Λ 25 '—N j I fevil) II 49 80700