NO881204L - MITOSANDERIVATER. - Google Patents

Description

The present invention relates to mitomycin analogues which contain one or more amidino groups. These compounds are mitomycin C derivatives, in which either the 7-amino group or the carbamido nitrogen atom or both are built into an amidino substituent. These compounds are active antitumor agents in experimental animal tumors.

Nomenclature: The systematic name for mitomycin C is according to "Chemical Abstracts": [laR-(laa,8 B,8aa,8ba) ]-6-amino-8-[ ((aminocarbonyl)oxy)--methyl]-1, 1a,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl-azirino[2',2',3,4]-pyrrolo[1,2-a]-indole-4,7-dione, according to which the azirinopyrroloindole ring system is numbered as follows:

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

Although this system is convenient and appropriate for one

series of simple derivatives such as those bearing N-substituents at the azirino ring nitrogen atom or at the 7- or 9a-positions, it suffers from certain ambiguities and shortcomings for general use. With regard to the compounds according to the present invention, some of which have substituents both on the azirino ring nitrogen atom and on the side chain carbamoyl nitrogen atom, there is no conventional numbering that can separate these positions. In the present description, it has therefore been chosen to refer to the azirino nitrogen atom as N"*"a and the carbamoyl nitrogen atom as N"^ using the mitosan nomenclature system. With regard to the stereochemical configuration of the compounds in question, it is intended that, when they are identified by the basic name "mitosan" or by a structural formula, to identify it as the chemical configuration of mitomycin C.

Mitomycin C is an antibiotic that is produced by fermentation and is already commercially available with the approval of the "Food and Drug Administration" in the therapy of disseminating adenocarcinomas of the stomach or pancreas in post-tested combinations with other approved chemotherapeutic agents and for palliative treatment , when other options have failed ("Mutamycin", Bristol Laboratories, Syracuse,

New York, 13201, Physicians 1 Desk Reference 3 5'.'-:ed. , : 19 81,

pp. 717 and 718). Mitomycin C and its preparation by fermentation are the subject of US-PS 3,660,578 of May 2, 1982 and with priority from earlier applications, including from Japan on April 6, 1957.

The structures of the mitomycins A, B, C and of porfiromycin were first published..by~J.S. Webb et al. of Lederle Laboratories Division American Cyanamid Company, "J. Amer. Chem. Soc.",

84, 3185-3187 (1962). One of the chemical transformations used in this structural study to relate mitomycin A and mitomycin C was the transformation of the former 7 — Sbt — dimethoxymitosane by reaction with ammonia into the latter

7-amino-9α-methoxymitosane. Exploitation of the 7-methoxy group in mitomycin A has proven to be a reaction of considerable interest in the production of antitumor active derivatives of mitomycin C. The following articles and patents all deal with the conversion of mitomycin A to a 7-substituted amino-mitomycin C- derivative with antitumor activity. The purpose of this research work was to produce derivatives that were very active and, in particular, that were less toxic than mitomycin C.

Matsui et al., "The Journal of Antibiotics", XXI, 189-198 (1968).

Kinoshita 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 Annual Meeting of the American Chemical Society,

Las Vegas, Nevada, March 1982, Abstract No. MEDI 72.

The following patents show the preparation of 7-substituted aminomitosane derivatives by reacting mitomycin A, mitomycin B or an N^<a->substituted derivative thereof with a primary or secondary amine: Cosulich et al., US-PS 3,332,944 of 25 July 1967;

Matsui et al., US-PS 3,420,846 of January 7, 1969;

Matsui et al., US-PS 3,450,705 of June 17, 1969;

Matsui et al., US-PS 3,514,452 of May 26, 1970;

Nakano et al., US-PS 4,231,926 of November 4, 1980;

Remers, US-PS 4,268,676 of May 19, 1981.

Mitomycin C derivatives with a substituted amino substituent in the 7-position have also been prepared by direct biosynthesis, i.e.

by adding a variety of primary amines to fermentation substrates and performing the conventional mitomycin fermentation.

(CA. Claridge et al., Abst. of the Annual Meeting of Amer. Soc. for Microbiology 1982. Abstract 028).

Mitomycin C is the primary mitomycin produced by fermentation and is the commercially available form. The current technology for the conversion of mitomycin C to mitomycin A for use in the preparation of semisynthetic substituted amino analogues of mitomycin C, and referred to in the above-mentioned publications, involves the hydrolysis of mitomycin C to the corresponding 7-hydroxy-mitosane, a highly unstable compound, and then methylation of this with diazomethane, which is a very dangerous substance to handle. An attempt to avoid the use of diazomethane for the methylation of 7-0-demethyl-mitomycin A which is produced by the hydrolysis of mitomycin C involves the use of 7-acyl-oxymitosanes (Kyowa Hakko Kogyo KK, Japanese Patent Document No. J5 6073-085 , Farmdoc No. 56227 D/31).

The present invention provides a hitherto unknown group of monoguanidino or mono- and bis-amidino analogues of mitomycin C, where either the 7-amino nitrogen atom or the N^-carbamoy1 nitrogen atom of mitomycin C, or both, is part of an amino substituent, or the 7-aminonitrogen is part of a guanidino group. Corresponding analogues of mitomycin A with the methoxy group in the 7-position and the amidino group in the N^-position are also included. The relevant compounds correspond to the following structural formula:

there • ■ -:- ■ ■ ■

in

A is amino or methoxy7

B is amino and

R 1 is methyl or formyl.

The person skilled in the art will see that tautomeric forms of some of these preceding amidino groups exist. Such shall also be covered by the above stated formulas and by the present invention.

The above-mentioned compounds of formula (I) have antitumor activity in experimental animals. They are also useful as intermediates in the preparation of other compounds with antitumor activity in animals. The present invention provides methods for the preparation of the first-mentioned compounds and their conversion into other valuable compounds

with antitumor activity in laboratory animals, as described below.

The preceding compounds are used as intermediates for^; preparation of other compounds with antitumor activity in animals involves reacting a compound of formula (I), where A or both A and B is the amidino group, with a primary -amine, resulting in cleavage of the N -aminomethylene substituent when the is present, with its conversion to the N^ group present in mitomycin A and mitomycin C.

With certain exceptions, the primary amines also react in the 7-position by removing the amidino group and replacing it with the amino substituent corresponding to the reactant. These procedures are illustrated in the following reaction equations. R denoting the nitrogen substituent of various known and unknown 7-substituted amino-mitomycin C compounds,

as described herein, and of the primary amines, which are capable of displacing the 7-amidino group in the compound of formula (II), are selected from C 1-6 alkyl, except for tert-alkyl, C 1-1 6 alkoxy. C2-alkenyl, C1-1g-alkynyl, C1-1g-haloalkyl, C1-1g-hydroxyalkyl, C4-g-cycloalkyl, aryl or lower aralkyl or lower aralkyl with up to 12 carbon atoms each, or a heteroalicyclic or heteroaromatic group with from 3-8 ring atoms, of which at least two are carbon atoms. R meaning the nitrogen substituent of the primary amines capable of cleaving only the N 2 -amidino substituent is the residue of a very weakly basic aliphatic amine or a strongly hindered alkylamine or aralkylamine. Examples are trifluoroethylamine, benzhydrylamine (ie aminodiphenylmethane) or tert-butylamine.

The compounds of formula (I) are prepared by reacting mitomycin C, 7-hydroxy-9α-methoxymitosane, or mitomycin A or an N"<*>"<a->substituted analog of any of the foregoing, with an amide acetal. The compounds of formula (I), where A, but not B, is the amidino group, can also be prepared by reacting "mitomycin C or an N"<*>"<a->substituted analog thereof with a strong base for formation of an anion at N^, followed by reaction of the anion with a reagent capable of forming the aminomethylene group, such as a halomethyleneiminium salt.

The preferred compounds produced according to the invention are mitomycin C analogues, where the 7-amino group is embedded in a substituted or unsubstituted amidino group.

They have a strong antitumor effect against experimental animal tumors. These compounds are prepared by reacting mitomycin C with a reagent capable of converting the 7-amino group into a 7-amidino group. Preferred reagents for this purpose are the amide acetals which react in good yield and under mild conditions with mitomycin C (Examples 1-5 as well as 18). Another group of amidine-forming reagents are the imidoyl halides (Example 17), the halogen-methyleneiminium salts (Example 15), the 2-halo-1-alkyl-pyridinium halides (Example 16), and the iminoethers or iminothioethers (Examples 13 and 14) which react with the anionic form of mitomycin C, which is formed by deprotonation of its 7-amino group by treatment with a strong base. Conditions for deprotonation of mitomycin C involve treatment of mitomycin C in dimethylformamide solution with approx. 1.5 mole parts sodium hydride at room temperature. Reaction of the thus formed anionic form with one

or several of the above-mentioned reagents preferably use 1-1.5 mole parts thereof in relation to mitomycin C at a temperature from room temperature to approx. -60°C. Aprotic, polar, organic solvents such as dimethylformamide, hexamethylphosphoramide, dimethylsulfoxide or pyridine,

is used as a reaction medium. However, the method is not limited to the formation of anionic mitomycin C in this specific way, as modifications will be realized by the person skilled in the art.

The preferred process for the preparation of the compounds of formula (I), where B or both A and B is the amidino group of the formula

R<2>

R R -N-C=N-, consists in the reaction of mitomycin C or mitomycin A or an N^-substituted derivative of one of these compounds with an amidacetal of the formula

where R2, R3 and R4 have the meaning given above and R^ is lower alkyl or cycloalkyl with up to 6 carbon atoms, or the two R groups are connected as an alkylene chain, which together with the two oxygen atoms and the intermediate carbon atom forms a cyclic structure with 5 or 6 ring-

atoms. The reaction of such amide acetals with primary amines is well known in the art and the person skilled in the art will know how the reaction should take place with mitomycin C, mitomycin A or the N<1a->alkyl derivatives thereof. It is referred to e.g. to US-PS 3,121,084 dated February 11, 1964 and to R.F. Abdullah

et al., "The Chemistry of Formamide Acetals", Tetrahedron, vol. 35, pp. 1720-24 (1979).

It is preferred to carry out the reaction in a liquid, anhydrous reaction medium where the diluent is a liquid which is compatible with the reaction conditions. Preferably, the latter is a lower halogenated, aliphatic hydrocarbon or a lower alkanol or preferably a mixture of these. Chloroform and methanol and mixtures thereof are quite suitable, the reaction takes place at a temperature of 40-65°C for a period of time sufficient for the reaction to complete.

When a large excess (approx. 60 times) of the acetal is used, the predominant product formed is the bis-amidino compound, i.e. the compounds of formula (I), where both A and B form the amido group. The N"^a-formyl derivative is formed in the meantime as a by-product. However, when a limited amount (about 10 times) of the acetal is used, in addition to the bis-amidino compound, a mono-amidino compound is also formed, i.e. a compound of formula (I ), where A is the amino group and B is the amidino group Mixtures of the above-mentioned reaction products are easily separated by chromatography as described in the following examples.

Some commercially available amide acetals that can be used in this process are listed in Table 1, which is taken from the above-mentioned article by Abdulla et al., p. 1685. The compounds of formula (I), where R 2 is cyano, dilave- alkylamino, lower alkoxy or lower alkylthio, are prepared by using the following orthocarbonate derivatives instead of the amide acetals by the aforementioned method.

These reagents are readily available from the following sources; .in ^itiiu. -.oeta i e - The amidino derivatives of formula (I), where A is or

is produced from the anionic form of mitomycin

C or an N^-substituted derivative thereof as described above. Suitable halomethyleneininium salts for use in this method are described in the literature. Representative are those listed by W. Kantlehner in "Advances in Organic Chemistry", vol 9, part 2, Wiley Interscience, 1979, pp. 81 and 82. Table II below is taken from Kantlehner's summary.* When a halomethyleneiminium salt as reactant, as shown in Table II, it is sometimes appropriate to use the corresponding amide as solvent, i.e.

the amide from which the iminium salt was prepared. In cases where the corresponding amides are solid, hexamethylphosphoramide or pyridine can be used. This is illustrated in examples 17 and 18 below.

The imidoyl chlorides derived from N-substituted formamides

are also suitable reactants for this purpose. Their preparation is well known to those skilled in the art, as illustrated in Table III, which is taken from H. Ulrich in "The Chemistry of Imidoyl Halides", Plenum Press, New York, 1968, pp. 74-76. Their reaction with amines to form amidines is also well known, as elucidated by S.R. Sandler and W. Karo in "Organic Chemistry", vol. 12-111, A.T. Blomquist and H. Wasserman, publishers, Academic Press, New York, 1972, p. 227.

The compounds of formula (I), where A is the nitrogen-unsubstituted amidino group, or

is produced

by reacting an amino-protected iminoether with mitomycin C,

an N^-substituted derivative thereof or an N-^a-lower alkyl derivative of one of these in anionic form as described above. The protecting group is then removed in a conventional manner. Isopropyl formidate, where the amino group is protected by the ε-trimethylsilylethoxycarbonyl group, is a suitable reactant (Example 13).

When A is (1-lower alkyl-2(1H)-pyridinylidene)amino or the group of the formula the preparation involves reaction of the anionic form of mitomycin C with cyclic halogerimethyleneiminium salts or imidoyl halides, where R 2 and R 3 of the formula

are connected to form a ring. Suitable reagents for reaction with mitomycin C are 2-chloro-1-methylpyridinium iodide (Example 16), 2-chloro-4,5-dihydro-1-methyl-1(3H)-pyrrolidinium chloride (Table 11), N,N <1->dimethyl-N,N'-trimethylenechloroform-amidimium chloride (Example 28) and other cyclic imidoyl halides derived from 2-azetidinones, 2-pyrrolidinones, 2-piperidinones and 2-azepinones. Again R<7> is now used

or R qi the final product is hydrogen, a: protecting group, as above, in the cyclic halomethyleneiminium salt intermediate.

The compounds of formula (I), where A or both A and B are

an amidino group of the formula

reacts with 5 5 primary amines of the formula R NH, where R is selected from C-L_1g-alkyl, except tert-alkyl, C1-1g-alkenyl, C^.^g"alkynyl, C-^^g-haloalkyl, C1-1g-hydroxyalkyl . incompatible with the reaction conditions is that the amino nitrogen atom is attached to a carbon atom bearing at least one hydrogen atom and less than two aryl groups. An anhydrous, liquid organic compound is used as the reaction medium and any such substance may be used, as long as it is compatible with the reaction conditions and does not participates in the reaction deleteriously. An excess of the primary amine reactant on a molar basis is usually employed. A reaction temperature in the range of about -15°C to +15°C is preferred. The resulting product of this reaction is a 7-substituted amino-9α-methoxymitosane, namely a mitomycin C derivative with a substituent as defined for R 1 on the 7-amino group. Such compounds are known from the prior art to have a significant degree of antitumor activity in experimental animals. Some primary amines, indicated by the formula R^NP^, have been shown to be unable to remove the 7-amidino group by the method described in the previous section. R 1 is alkyl, cycloalkyl, cycloalkylalkyl, aralkyl or heteroalicyclic with from 4-18 carbon atoms, in which the carbon atom carrying the amino group is a tertiary carbon atom, or a secondary carbon atom carrying 2 aryl groups. Certain other weakly basic aliphatic amines, such as trifluoroethylamine, also cannot remove the 7-amidino group. These amines are useful for converting a compound of formula (I), wherein both A and B are the said amidino group, with the formula

to a compound of formula (I), where only A is

the amidino group. Although these amines lack the ability to ff ■ j er ri é: '•' eatLdlliro gr-up peh>!;! 'e r-'' lde!; al l;i ké ve 1 ^ i ••stari d tila cleave the amidino group indicated by B to NH^under formation of the carbamido function which is characteristic of the unsubstituted mitosans. The amine itself can serve as the reaction medium, or one can use a solvent system as indicated in the previous section. This method is preferably carried out1 in the range 20-60°C.

The invention shall be described in more detail in the following examples. Melting points were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. The temperatures are expressed in °C. The nuclear magnetic resonance spectra, NMR, are recorded on a Varian XL100 spectrometer in pyridine-d^, unless otherwise stated. The infrared spectra, IR, were obtained with a Beckman 4240 spectrophotometer and the sample was pressed into a pellet of potassium bromide. The IR numbers are

umaksi cm ^' ne syn-'-:'-9e UV spectra were recorded on a Varian-Cary 219 spectrophotometer.

Thin-layer chromatography, tic, was performed on 0.25 mm precoated silica gel plates using UV light as visualizing agent. •Flash chromatography was performed using Silica Woelm (32-63 µm). The solvent was evaporated under reduced pressure and below 50°C.

Example 1

Connect (V)

7-[..( dimethylamino) methylene ] amino-N1^- ( dimethylamino)methylene-9a-methoxymitosane • " •;':

Compound ( VI) 7 - [ ( dimethylamino)methylene ] amino-N"'"^-( dimethylamino)methylene-N"*"3-formyl-9a-methoxymitosane

To a suspension of 500 mg or 1.50 mM mitomycin C i

25 ml of chloroform was added to a total of 9.6 ml (2.4 ml portions after 0, 18, 21 and 23 hours) of N,N-dimethylformamide-dimethylacetal and the suspension was stirred at approx. 50°C for 41 hours.

Evaporation of the solvent and excess reagent under reduced pressure gave a dark green residue;

tic (methylene chloride/methanol 20:1) showed the absence of mitomycin C and the presence of two new green components (Rf = 0.16 and 0.22). The major component (Rf = 0.16) was isolated by flash chromatography using methylene chloride/methanol 20:1 as eluent and as a green solid (340 mg, 51.5%) which on dissolution in diethyl ether followed by addn. of hexane gave compound (V) as a dark green, amorphous powder.

NMR (pyridine d5,6): 2.18 (s, 3H), 2.70 (bs, 1H), 2.76 (s, 3H), 2.82 (s, 3H), 2.86 (s, 6H), 3.22 (s, 3H), 3.30 (bs, 1H), 3.60 (d, J=12Hz), 4.12 (dd, 1H, J=10, 4 Hz), 4, 43

(d, 1H, J=12 Hz), 4.90 (bs, 1H), 5.10 (t, 1H, J=10 Hz),

5.52 (dd, 1H, J=10, 4 Hz), 7.85.(s, 1H), 8.64 (s, 1H).

IR (KBr) u k<c>m"<1>: 3300, 2930, 1675, 1620, 1545, 1230, 1060.

UV (H2<0>)<*>max'nm: 390 and 244.

Analysis calculated for c2iH28N6°5: C 56'71' H 6'08'N 18.90

Found: C 56.20, H 6.28, N 17.88

The minor component, Rf = 0.22, which was isolated (180 mg, 25.35%) as an amorphous solid by precipitation from diethyl ether and hexane, was identified as compound (VI).

NMR (pyridine d5, s): 2.20 (s, 3H), 2.60-3.00 (3 singlets, 12H), 3.2 (s, 3H), 3.65 (m, 2H), 4 .04 (d, 1H, J=4 Hz),

4.16 (dd, 1H, J=12, 4Hz), 4.60 (d, 1H, J=13 Hz), 4.86 (t, 1H, J=12 Hz), 4.90 (s, 1H ), 5.48 (dd, 1H, J=12, 4 Hz),

7.90 (s, 1H), 8.64 (s, 1H), 9.06 (s, 1H).

IR (KBr) umax/cm -<1>: 2490, 2860, 1698, 1630, 1600, 1540, 1250, 1060.

UV (H20), *max>nm: 390 and 244.

Analysis calculated for C22H28<N>6°6<:>C 55'89'K 5'93'N l7/78

Found: C 55.41, H 5.96, N 16.99.

Solutions of (V) and (VI) in either ethyl acetate or N,N-dimethylformamide-dimethyl acetal showed, on standing at room temperature for more than 10 hours, that compound (VI)

(Rf = 0.22) is converted to compound (V) (Rf = 0.16) forming a solution that was highly enriched in the latter compound.

Examples 2-7 were carried out according to the method according to example 1 with the modifications indicated below for the formation of various additional compounds according to the invention.

Example 2

Compound (VII) 7-[(diisopropylamino)methylene]amino-N<1>^-(diisopropylamino)-methylene-9a-methoxymitosane

A suspension of mitomycin C (200 mg, 0.6 rM) in N,N-diiso-propylformamide-diethyl acetal (3 ml) was heated with stirring at 53°C for 15 hours. The reaction mixture was poured into 50 ml of water and extracted with ethyl acetate (3 x 30 ml). The combined organic extract was dried over Na2SO4 and evaporated to give a dark green syrup, tic (methylene chloride/methanol 10:1) showed a major green component at Rf = 0.43 with impurities (Rf = 0.45-0.50) as moved faster. The major component (VII) was isolated as a dark green solid (156 mg, 46.8%) by two flash chromatography procedures using methylene chloride/methanol 20:1 as the eluent.

NMR (CDCl 3 , 6): 1.10-1.50 (5 singlets, 24H), 1.94 (s,

3H), 2.78 (dd, 1H, J=4, 2 Hz), 3.05 (d, 1H, J=4 Hz),

3.22 (s, 3H), 3.60 (m, 5H), 3.75 (dd, 1H, J=10, 4 Hz),

4.24 (d, 1H, J= 12 Hz), 4.56 (t, 1H, J=10 Hz), 4.88

(dd, 1H, J=10, 4 Hz), 7.83 (s, 1H), 8.67 (s, 1H).

IR (KBr), "max/cm"<1>: 3320, 2990, 2940, 1680, 1630, 1600, 1500, 1235,1060.

UV (MeOH) *max, nm: 24 6 and 3 93.

Analysis calculated for C2gH44<N>g05: C 62.55, H 7.91, N 15.10

Found: C 62.03, H 7.80, N 14.60

Example 3

Connection ( XIV)

7-[(dimethylamino)methylene]amino-N<1>^-(dimethylamino)methylene-ga-me tok sy -Nla-me ty Imitos an

In this example, porfiromycin (N<1a>i methyl-mitomycin C), 130 mg (0.37 mM) was used instead as starting material for reaction with 0.8 ml (1.5 mM) N,N-dimethylformamide dimethyl acetal using 10 ml of chloroform and 2 ml of methanol as reaction solvent and a reaction time of 50 minutes at 50°C. Compound (IX) was obtained as a syrup after evaporation of the reaction solvent and purified by flash chromatography using 20 g of silica gel and methylene chloride/methanol (20:1) as the eluting solvent.

NMR (pyridine d5,6): 2.22 (bs, 4H), 2.28 (s, 3H), 2.70

(d, 1H, J=4 Hz), 2.80 (s, 3H), 2.84 (s, 3H), 2.90 (s, 6H), 3.20 (s, 3H), 3.52 ..{.dd, 1H, J=2, 12 Hz) , 4.10- .(dd, 1H,,-J=4, 11 Hz), 4.38 (d, 1H, J=12 Hz), 4.92 (t, 1H, J=ll Hz), 4.96 (bs, 1H), 5.46 (dd, 1H,J=4, 11 Hz), 7.86 (s, 1H),

8.70 (p, 1H).

Rf = 0.53, thin layer chromatography with 9:1 methylene chloride/methanol.

IR (KBr) umax^cm"<1>: 2930, 1680, 1620, 1545, 1230, 1115.

UV (MeOH) *max, nm: 386 and 243.

Analysis calculated for C22H30<N>6°5<:>C 57.60, H 6.55, N 18.33

Found: C 57.11, H 6.11, N 17.99

This procedure gave compound (XV), 7-amino-N^-dimethylaminomethylene-9a-methoxy-N<1a->methylmitosane, as a by-product in 30% yield, tic Rf = 0.40 (methylene chloride/-

methanol 9:1).

NMR (pyridine d5, 6): 2.02 (s, 3H), 2.16 (dd, 1H, J= 2, 5 Hz), 2.25 (s, 3H), 2.66 (d, 1H, J=5 Hz), 2.76 (s, 3H), 2.86

(s, 3H), 3.18 (s, 3H), 3.51 (dd, 1H, J=2, 12 Hz), 4.08

(dd, 1H, J=4, 10 Hz), 4.50 (d, 1H, J=10 Hz), 4.90 (t,

1H, J=10Hz), 5.05 (bs), 5.43 (dd, 1H, J=4, 10 Hz),

8.70 (p, 1H).

IR (KBr)"max cm"<1>: 3430, 3330, 3270, 2940, 2960, 1690 1625, 1553, 1230, 1125.

UV (MeOH) *max/nm: 358, 244 and 216.

Analysis calculated for C19H25N505: c 56'53'H 6.20, N 17.38

Found: C 54.68, H 6.13, N 16.59

Example 4

Connection ( IX)

9α-Methoxy-7-[(1-piperidinylamino)methylene]amino-N<10->(1-piperidinylmethylene)mitosane 3 ml of N-(diethoxymethyl)piperidine and 200 mg of mitomycin C were allowed to react at 60°C for 2.5 hours in chloroform solution (3 ml). The product was obtained in a yield of 27.6%, tic Rf = 0.20 (methylene chloride/methanol 20:1).

NMR (pyridine d5, 6): 1.38 (bs, 12H), 2.20 (s, 3H), 2.80 (bs, '1H),... 3.24 (s, 3H), 3 , 00-3, 40 (m, 5H), 3 , 40-3 , 80 (m, 5H), 4.13 (dd, 1H, J=10, 4 Hz), 4.45 (d, 1H, J =12 Hz), 4.90 (bs, 2H), 5.12 (t, 1H, J=10 Hz), 5.56 (dd, 1H, J=10, 4 Hz), 7.87 (s, 1H), 8.70 (s, 1H).

IR (KBr) u k f cm"<1>: 3300, 2950, 2870, 1680, 1630, 1610, 1550, 1200, 1070.

UV (H20)^max</>nm: 394 and 246.

Analysis calculated for C27H36<N>605: C 61.79, H 6.87, N 16.02

Found: C 61.01, H 6.85, N 15.34

The N<1α->formyl derivative of the preceding compound, compound (VIII), N<1α-formyl-9a-methoxy-7-[(1-piperidinylamino)-methylene]-amino-N<1>^-(1-piperidinylmethylene) mitosan, was obtained as a major component in 43% yield, tic Rf = 0.25 (methylene chloride/methanol 20:1).

NMR (pyridine d5, 6): 1.38 (bs, 12H), 2.23 (s, 3H), 3.00-3.40 (m, 4H), 3.23 (s, 3H), 3, 40-3.90 (m, 6H), 4.07 (d, 1H, J=4 Hz), 4.18 (dd, 1H, J=ll, 4 Hz), 4.63 (d, 1H), 4.90

(t, 1H, j=ll Hz), 4.94 (bs, 1H), 5.54 (dd, 1H, J=ll, 4 Hz), 7.94 (s, 1H), 8.71 (s , 1H), 9.08 (s, 1H).

IR (KBr)°max</><c>m"<1>: 2490, 2860, 1698, 1630, 1600, 1540, 1250, 1060.

UV J H20) amax, nm: 3 94 and 24 7 .

Analysis calculated for C28H36<N>6°6<:>C 60'08'H 6'52'N 15.21

o r.;! Ugh---. ■ . Found: C 59, 99 , H 6.17, N 15.07

Example 5

Connection ( X)

9a-Methoxy-7-[(1-morpholino)methylene]amino-N"^-(1-morpholino-methylene)mitosane

A stirred suspension of mitomycin C (200 mg, 0.6 mM) in chloroform (10 mL) and N-diethoxymethylmorpholine (4 mL) was heated to ca. 53°C for 42 hours. The reaction mixture was concentrated to syrup under high vacuum. A crude separation by flash chromatography (methylene chloride/methanol 25:1) was carried out to isolate the green colored components from the excess of reagents. The combined green components were dissolved in 20 ml of ethyl acetate and washed with 3 x 20 ml of water. The combined washings were re-extracted with 3 x 15 ml ethyl acetate. All ethyl acetate fractions were combined, dried over Na2SO^ and evaporated to a dark green syrup, tic (methylene chloride/methanol 10:1) showed a distinct green component at Rf = 0.33 with several green impurities (Rf = 0.35-0, 40). Flash chromatography isolated the component at Rf 0.33 (130 mg, 56.8%) as a dark-green, amorphous solid characterized as compound

(X) .

NMR (CDCl 3 , 6): 1.91 (s, 3H), 2.80 (bs, 1H), 3.13 (d, 1H, J= 2 Hz), 3.22 (s, 3H), 3, 30-3.94 (m, 18H), 4.20 (d, 1H, J=12 Hz), 4.40 (bs, 1H), 4.54 (t, 1H,J=10 Hz), 4, 88 (dd, 1H, J=10 Hz, 4 Hz), 7.74 (s, 1H), 8.51 (s, 1H).

IR (KBr)<u>max, cm<_1>: 3300, 2970, 2920, 1680, 1625, 1550, 1235, 1070.

UV (MeOH) xmax/nm: 28 6 and 24 4.

Analysis calculated for<C>25<H>32<N>6°7<:>C 56'78'H 6'06'N 15.90

Found: C 53.07, H 6.03, N 15.37

Example 6

Connection ( XVI)

7-amino-N^-dimethylaminomethylene-9a-methoxymitosane Mitomycin C (200 mg, 0.6 mM) was dissolved in 10 ml of chloroform and 2 ml of methanol, N,N-dimethylformamide dimethyl acetal (0.64

ml, 4.8 mM) was added and the solution was stirred at approx. 50°C for 50 minutes. Thin-layer chromatography (methylene chloride/-

methanol 90:10) showed traces of unreacted mitomycin C (Rf = 0.22) and two new components (Rf = 0.42 and 0.33 respectively). The solution was concentrated under reduced pressure and a syrup was obtained which was flash chromatographed (25 g silica gel) using methylene chloride/methanol in the ratio 20:1 as eluent.

The fastest component (Rf = 0.42) was isolated as a green, amorphous solid (60 mg, 22.5%) and identified as compound (V) on the basis of the NMR spectrum (pyridine d5).

The .blue ..main component. (Rf ■= . 0 , 33 ) was isolated as an amorphous solid (148 mg, 63.3%) and characterized as compound (XVI). An analytical sample was obtained by precipitation from methylene chloride and n-pentane.

NMR (pyridine, d5#6): 2.02 (s, 3H), 2.76 (bs, 4H), 2.86 (s, 3H), 3.21 (s, 3H), 3.28 (d , 1H, J=4 Hz), 3.62 (dd, 1H, J=2, 13 Hz), 3.94 (bs), 4.14 (dd, 1H, J=4, 12 Hz), 4, 56 (d, 1H, J=13 Hz), 5.12 (t, 1H, J=10 Hz), 5.52 (dd,

1H, J=4, 10 Hz).

IR (KBr) umax, cm"<1>: 3430, 3320, 3280, 2930, 1675, 1615, 1650, 1230, 1115.

UV (H20) *max, nm: 364, 244 and 219.

Analysis calculated for C]_8N23N505: C 55, 48, H 5.91, N 17.98 Found: C 54 , 70, H 6.14, N 17.95

Example 7

Connection (XVII)

7,9α-dimethoxy-N<10->dimethylaminomethylenemitosane Mitomycin A (170 mg) was used instead of mitomycin C

in Example 1 and allowed to react with N,N-dimethylformamide-dimethyl acetal (0.6 ml) in chloroform/methanol solution (10:1) at 5.0°C for 1 hour. The desired product was

reached in 48% yield, tic Rf = 0.50 (methylene chloride/methanol 9:1).

NMR (pyridine d5,6): 1.83 (s, 3H), 2.76 (bs, 4H), 2.86

(s, 3H), 3.22 (s, 3H), 3.28 (d, 1H), 3.56 (dd, 1H, J=2,

13 Hz), 4.02 (s, 3H), 4.10 (dd, 1H, J=4, 10 Hz), 4.24

(d, J=13 Hz), 5.10 (t, 1H, J=10 Hz), 5.50 (dd, 1H, J=4, 10 Hz), 8.67 (s, 1H).

IR (KBr) umax, cm"<1>: 3300, 2930, 1675, 1655, 1625, 1500, 1235, 1120.

UV (H20) xmax, nm: 530, 316 and 244.

Analysis calculated for C19H24N406: C 56.39, H 5.94, N 13.85

Found: C 56.51, H 5.92, N 13.71

The N<1a->formyl derivative of compound (XVII) was obtained as compound (XVIII), 7, ga-dimethoxy-N^-dimethylaminomethylene-N -formylmitosane in a yield of 16.5%, tic Rf = 0.61 (methylene chloride /methanol 9:1).

NMR (pyridine d5, «): 1.88 (s, 3H), 2.76 (s, 3H), 2.85 (s, 3H), 3.54 (d, 1H), 3.62 (bs, 1H) , 4.05 (s, 3H), 4.05 (s, 3H), 4.05 (bs, 1H), 4.14 (dd, 1H, J=4, 12 Hz), 4.40 ( d, 1H, J=13 Hz), 4.86 (t, 1H,J=12Hz), 5.42 (dd, 1H, J=4, 12Hz), 8.66 (s, 1H), 9.08 (p, 1H).

Example 8

Connection (XIX)

7-(Dimethylaminomethylene)amino-9α-methoxymitosane To compound (V) (600 mg, 1.35 mM) dissolved in 10 mL of methanol was added aminodiphenylmethane (2.2 mL, 10.8 mM) and the resulting solution was stirred at 54°C for 4 hours. The course of the reaction was measured by tic (methylene chloride/methanol 90:10). At the end of the 4 hours, the starting material (Rf = 0.35) had disappeared and a new main green zone (Rf = 0.29) appeared in its place. The solution was concentrated by reducing

pressure and the resulting syrup flash chromatographed, 25 g silica gel, using methylene chloride/ethanol 20:2

as eluent. Fractions containing the green component (Rf = 0.29) were pooled, dried over Na 2 SO 4 and concentrated. Compound (XIX) was obtained as an amorphous solid (215 mg, 41%).

NMR (pyridine dg, 6): 2.18 (s, 3H), 2.70 (bs, 1H), 2.80

(s, 3H), 2.88 (s, 3H), 3.08 (bs, 1H), 3.24 (s, 3H),

3.56 (bd, 1H, J=12 Hz), 4.00 (dd, 1H), 4.44 (d, 1H, J=12

Hz), 5.06 (t, 1H, J=10 Hz), 5.56 (dd, 1H, J=10, 4 Hz),

7.58 (bs, 2H), .7.88 (s, 1H).

IR (KBr) umax/cm"<1>: 3300-3450, 2960-2910, 1715, 1620, 1535, 1050 .

UV (H20) xmax, nm: 390 and 226.

Analysis calculated for C18H23N5°5<:>C 55'48'H 5>91'N 17.98

Found: C 54.83, H 5.67, N 16.90

When the N<la>formyl derivative, compound (VI), was used as starting material in example 8, but using room temperature for 20 hours as reaction conditions, compound (XIX) was obtained in essentially the same way and with the same yield.

Example 9

Connection ( XX)

7-(Dimethylaminomethylene)amino-9a-methoxy-N 1a -methylmitosane Compound (XIV), (1 g, 2.18 mM) was dissolved in 20 ml of meta-

nol, aminodiphenylmethane (3.5 mL, 17.18 mM) was added and the resulting solution stirred at room temperature for 5

hours and at 40°C for 5 hours. Thin layer chromatography (CH2Cl2/MeOH 90:10) of the reaction mixture showed that almost all the starting material (Rf = 0.55) had been consumed and that a new green main zone (Rf = 0.48) had emerged. Processing

in a similar manner as in Example 8 gave compound (XX) as an amorphous solid in an amount of 350 mg. Further purification occurred by flash chromatography (7g, silica gel)

using CH2Cl3/MeOH (250 mL, 96/4 vol/vol) and precipitation of the resulting solid (Rf = 0.48) from 5 mL methylene chloride and 50 mL hexane to give analytically pure (XX) (314 mg, 35.7%) as a solid.

NMR (CDCl 3 , 6): 1.93 (s, 3H), 2.26 (bs, 1H), 2.26 (s, 3H), 3.06 (s, 3H), 3.08 (bs, 1H ), 3.10 (s, 3H), 3.20 (s, 3H), 3.46 (bd, 1H, J=12, 1Hz), 3.58 (dd, 1H, J=4, 10 Hz) , 4.17 (d, 1H, J=12 Hz), 4.38 (t, 1H, J=10 Hz), 4.68 (m, 2H),

4.76 (dd, 1H, J=4.10 Hz), 7.72 (s, 1H).

IR (KBr) u . , cm"1: 3440, 3350, 3190, 3020, 2940, 2910, IT13.K S 1725, 1630, 1550, 1055.

UV (MeOH)Xmax, nm: 386 and 231

Analysis calculated for C19H25<N>5°5<:>c 56'53'H 6.20, N 17.36

Found: C 53.90, H 5.13, N 15.81

Example 10

Connection (XI)

7-(n-propyl)amino-9a-methoxymitosane

Compound (V) (330 mg, 0.74 mM) was dissolved in 10 ml of anhydrous methanol and 1.0 ml of n-propylamine was added. The reaction mixture was stirred for 6 hours at room temperature and for 16 hours at 0-4°C. The solvent and excess reagent were evaporated under reduced pressure and the residue flash chromatographed using silica gel as absorbent. The obtained by elution with methylene chloride/methanol 30:1

blue component (Rf = 0.40) was re-precipitated from methylene chloride with hexane and compound (XI) was obtained as an amorphous gray powder (125 mg, 44.5%).

NMR (pyridine dg,6): 0.80 (t, 3H), 1.42 (m, 2H), 2.11 (s, 3H), 2.74 (bs, 1H), 3.12 (bs, 1H), 3.22 (s, 3H), 3.36

(q, 2H), 3.60 (d, 1H, J=12 Hz), 3.96 (dd, 1H, J=11Hz,

4 Hz), 4.54 (d, 1H, J=12 Hz), 5.00 (m, 3H), 5.36 (dd,

1H, J=11, 4 Hz), 6.90 (t, 1H).

IR (KBr)<U>max/cm"<1>: 3440, 3300, 2960, 2940, 1715, 1630, 1600, 1550, 1510, 1220, 1060.

UV (H20)Xmax, nm: 372 and 222

Analysis calculated for C18H24<N>4°5<:>C 57'40'H 6.38, N 14.88

Found: C 57.28, H 6.41, N 14.08

Example 11

Compound (XII)

7-(2-Hydroxyethyl)amino-9a-methoxymitosane

Compound (V) (330 mg, 0.74 mM) was dissolved in 5 ml of anhydrous methanol and 2 ml of ethanolamine was added. The reaction mixture was stirred at room temperature for 2 hours and then diluted with 50 ml of water and extracted with 5 x 60 ml of ethyl acetate. The combined ethyl acetate extracts were dried over Na 2 SO 4 and concentrated to a bluish-purple residue which by column chromatography using 10% methanol in methylene chloride and concentrating the combined fractions containing the blue compound gave 105 mg or 37% of compound (XII) as an amorphous solid .

NMR (pyridine dg,6): 2.14 (s, 3H), 2.81 (bs, 1H), 3.18 (d, 1H, J=4 Hz), 3.24 (s, 3H), 3 .65 (dd, 1H, J=2, 12 Hz), 3.70-4.20 (m, 5H), 4.52 (D, 1H,J=13-Hz), 4.96 (t, 1H , J=12 Hz), 7.38 (t, 1H), 7.58 (bs).

IR (KBr) umax, cm"<1>: 3300-3500, 2930, 1710, 1630, 1600, 1540, 1510, 1200, 1055.

UV (<H>2<0>)<*>max, nm: 371 and 221

Analysis calculated for C17H.J.O,: C 53, 92 , H 5.82, N 14.80

Found: C 51.30, H 5.88, N 14.80

Example 12

Compound (XIII)

7-(2-Benzylthioethyl)amino-9a-methoxymitosane

Compound (V) (200 mg, 0.45 mM) was dissolved in 2 ml of methanol, 0.5 ml of S-benzyl-2-aminoethanethiol was added and the solution stirred at room temperature for 16 hours. The residue obtained by evaporation of the solvent under reduced pressure was flash chromatographed (40 g, silica gel) using 6% methanol/methylene chloride in an amount of 400 ml as eluent. The blue component (Rf ca. 0.5 in 10% MeOH/CH2Cl2) was isolated as an amorphous solid in an amount of 65 mg or 29.8%. The compound's spectral data (NMR, IR, UV and mass spectrum) were consistent with the given structure.

Analysis calculated for C24<H>28M4°5<S:>C 59'49'H 5'82, N 11.56

Found: C 59.72, H 5.94, N 11.08

Example 13

Manufacture of

(A) To a solution of isopropylformimidate hydrochloride (1 mmol) in dimethylformamide (DMF), 2 ml, is slowly added diisopropylethylamine (2.1 mmol) at 0°C under a nitrogen atmosphere. To the resulting solution is added dropwise 6-trimethylsilylethyl chloroformate at 0°C. The resulting clear solution is called solution A. (B) A solution of 1 mmol mitomycin C in 5 ml DMF is added to a suspension of 1.5 mmol sodium hydride in 3 ml DMF.

The solution is stirred at room temperature for 20 minutes and cooled to -40 to -50°C before adding solution A

(above). The solution is kept at -40°C for one hour and then allowed to warm to room temperature. After resting at room temperature for approx. 6-18 hours, the reaction mixture was diluted with Cl 2 Cl 2 and filtered. The solid residue obtained after evaporation of the filtrate is chromatographed on silica gel to isolate the amidino-protected title compound. (C) The amidino protecting group of the preceding intermediate is removed by the procedure disclosed by Carpino and Tsao ("J. Chem. Soc. Chem. Comm." 358 (1978)) for obtaining the unsubstituted amidino title compound.

Example 14

Manufacture of

(A) To a solution of 1 mmol of isopropylformimidate hydrochloride in 2 ml of DMF, slowly add 21 mmol of diisopropylethylamine at 0°C under a nitrogen atmosphere. To the resulting solution is added methyl iodide at 0°C. The resulting solution is called solution B. (B) The procedure given in Example 13(B) is repeated using solution B instead of solution. A for

to achieve the title connection.

Example 15

A 0.5 M solution of N,N-dimethylchloromethyleneiminium chloride is prepared by dropwise addition of oxalyl chloride (1.57 g, 12.5 mmol) at 0°C to a solution of DMF (915 mg, 12.5 mmol) in 25 mL CHCl^, followed by stirring at room temperature for 30 minutes. Separately, a solution of mitomycin C (334 mg, 1 mmol) in 5 mL DMF is added to a suspension of NaH (36 mg, 1.5 mmol) in 3 mL DMF. The solution is stirred at room temperature for 20 minutes and cooled to -40 to -50°C, and the above solution of N,N-dimethylchloromethyleneiminium chloride (3 ml, 1.5 mmol) is then added. Additional NaH (18 mg, 0.75 mmol) is added after 10 minutes of stirring at -40°C. The solution was kept at -40°C for one hour and then diluted with CH 2 Cl 2 and filtered. The residue obtained after evaporation of the filtrate is chromatographed by thin-layer chromatography, tic, on silica gel (10% CH3OH-CH2Cl as eluent). Extraction of the major green band gave 78 mg or 43%, calculated on mitomycin C recovered, of an amorphous solid whose NMR spectrum and tlc behavior were identical to those of compound (XIX) prepared in Example 8. Extraction of the purple band gave 150 mg mitomycin C. Example 16 7(1-methyl-2-(H)-pyridinylidene)amino-9a-methoxymitosane

To a mixture of mitomycin C (242 mg, 0.725 mmol) and

To NaH (43.5 mg, 1.81 mmol) was added 4 ml of DMF. After stirring for 15 minutes, 2-chloro-1-methylpyridinium iodide (370 mg, 1.45 mmol) was added at room temperature. The solution was stirred for 1.5 h and then diluted with ethyl acetate (EtOAc) and filtered. The residue obtained after evaporation of the filtrate was chromatographed on silica gel (5% CH^OH-CH2Cl2 as eluent).<v>The minor product in an amount of 12 mg was compound (XIX) (Example 8). The major product in the amount of 75 mg was further purified by silica gel-tlc (10% CH 3 OH-CH 2 Cl 2 ) to obtain 6 mg or 2% of the title compound.

NMR (pyridine dg, &) : 2.11 (s, 3H), 2.76 (bs, 1H), 3.20

(m, 1H), 3.26 (s, 3H), 3.49 (s, 3H), 3.63 (dd, 1H, J=

13, 1 Hz), 4.01 (dd, 1H, J=ll, 4 Hz), 4.51 (d, 1H, J=13Hz),

5.10 (t, 1H, J=10 Hz), 5.43 (dd, 1H, J=10, 4 Hz), 5.99 (dt, 1H, J=9, 2 Hz), 6.09 ( dd, 1H, J=9, 1 Hz), 6.95 (dd, 1H, J=9, 7, 2 Hz), 7.32 (dd, 1H, J= 7, 1 Hz).

Example 17

7-[(methylaminomethylene)amino]-9a-methoxymitosane

Sodium hydride (12 mg, 0.5 mmol) is added under a nitrogen atmosphere to a solution of mitomycin C (167 mg, 0.5 mmol) in 2 ml of hexamethylphosphoramide. To this solution is added N-methylformimidoyl chloride (19 mg, 0.25 mmol, N.H. Bosshard and H. Zollinger, "Heiv. Chim. Acta", 42, 1659 (1959)). The solution is stirred at room temperature for 10 minutes and then NaH (6 mg, 0.25 mmol) and N-methylformimidoyl chloride (9.5 mg, 0.13 mmol) are added. After stirring for 6-12 hours, the solution is diluted with ethyl acetate and filtered. Evaporation of the solvent followed by chromatographic purification of the residue affords the title compound.

Example 18

Connection ( XXI)

9α-Methoxy-7-(1-morpholinomethylene)aminomitosane

To mitomycin C (600 mg, 1.8 mM) suspended in 30 ml of chloroform is added 4-diethoxymethylmorpholine (12.5 ml) and the resulting suspension is heated to 58°C for 48 hours. At the end of the 48 hours, tic (20% MeOH in CH 2 Cl 2 ) showed that the reaction was incomplete. The solution was concentrated under reduced pressure and to the resulting syrup was added 100 ml of water. After stirring for 20 minutes, the dark green solution was extracted with 5 x 50 mL of methylene chloride and the combined extract was dried and concentrated to a syrup. To this syrup in 20 ml of methanol was added 6.5 ml of aminodiphenylmethane and the resulting solution stirred at 30-35°C for 18 hours. Thin layer chromatography (20% MeOH in CH 2 Cl 2 ) showed a major green zone with a smaller, slower purple zone. The solution was concentrated under reduced pressure and the resulting syrup purified by the usual flash chromatography technique to obtain the title compound as a dark green amorphous solid in an amount of 75 mg corresponding to 10%. An analytical sample was obtained by precipitation from a methylene chloride solution with n-hexane.

NMR (pyridine dg,6): 2.16 (s,.3H), 2.76 (dd, 1H, J=5 and 1 Hz), 3.16 (d, 1H,J=5Hz), 3.24 (s, 3H), -3.28-3.80 (m, 10H), 4.02 (dd, 1H,J=10and 4Hz), 4.40 (d, 1H, J=12 Hz), 5, 06 (t, 1H, J=10Hz), ■ 5.46 (dd, 1H, J=10 and 4 Hz), 7.90 (s, 1H).

IR (KBr)"max,<c>m<_1>: 3360, 3280, 2960, 2920, 1720, 1600, 1520, 1230, 1050.

UV (MeOH) *max: 384 and 234

Analysis calculated for C20H25N5°6<:>C 55'64'H 5'80'N 16'23

Found: C 55.07, H 5.55, N 15.88

Example 19

Connection ( XXII)

7-(1-pyrrolidinylmethylene)amino-9a-methoxymitosane

A 0.5 M solution of pyrrolidinylchloromethyleneiminium chloride was prepared by dropwise addition of oxalyl chloride (3.17 g, 25 mmol) at 0°C to a solution of 1-formylpyrrolidine (2.48 g, 25 mmol) in 50 mL of CHCl^, followed by stirring at room temperature for 30 minutes. Sodium hydride (24 mg, 1 mmol) was added separately under a nitrogen atmosphere to a solution of mitomycin C (334 mg, 1 mmol) in 3 mL of 1-formylpyrrolidine.

After stirring for 20 minutes at room temperature, the solution was cooled to -40 to -50°C and the above iminium salt solution (1 ml, 0.5 mmol) was added. To this mixture were added alternately at 10 minute intervals to 12 mg (0.5 mmol) NaH, 0.5 ml (0.25 mmol) of the iminium salt solution, 6 mg (0.25 mmol) NaH, 0.25 ml (0.125 mmol) of the iminium salt solution, and finally 3 mg (0.125 mmol) of NaH

and 0.125 mL (0.063 mmol) of the iminium salt solution. After

stirring for 30 minutes at -30°C, the mixture was warmed to room temperature. It was diluted with ethyl acetate and inorganic salt filtered off. The obtained residue was chromatographed after evaporation of the solvent by thin-layer chromatography on silica gel (10% CH3OH-CH2Cl2). Extraction of the green band gave 120 mg or 15% of the title compound.

NMR (pyridine dg, 6): 1.58 (m, 4H), 2.29 (s, 3H), 2.73 (m, 1H), 3.06-3.50 (m, 8H), 3, 59 (dd, 1H, J=13, 1Hz), 4.03 (dd, 1H, J=10, 4Hz), 4.44 (d, 1H, J=12 Hz), 5.05 (t, 1H, J= 10 Hz), 5.45 (dd, 1H, J=10, 4 Hz), 8.04 (s, 1H).

IR (KBr)"max,<c>m"<1>:3420, 3280, 2960-2870, 1715, 1625, 1560, 1300, 1055.

Example 20

7-[N-methyl-N-(methylamino)methyl]amino-9a-methoxymitosane

The procedure of Example 17 was repeated using 9α-methoxy-7-(N-methylamino)mitosane (Matsui et al., "The Journal of Antibiotics", XXI, 189-198 (1968)) instead of mitomycin C in equivalent molar amount.

Example 21

Connection ( XXIII)

7 - [ 1-( dimethylamino) ethylidene ] amino-N11^- [ 1-( dimethylamino) - ethylidene]-9-methoxymitosane

A suspension of 600 mg (1.79 mM) mitomycin C in 2 ml of methanol was prepared and treated with 3 ml of N,N-dimethylacetamide-dimethyl acetal. The suspension was heated to 75-80°C with stirring for 2 hours. At this stage, tic (CE₂C₂/methanol 10:1) showed that almost all of the mitomycin C was consumed by the reaction. The product appeared as a green zone. The solvent

and volatiles were removed by concentrating the reaction mixture to dryness under reduced pressure,

which gave a syrup which was dissolved in methylene chloride and loaded onto a silica gel column (40 g silica gel), and the column developed with 1% methanol in 200 ml methylene chloride, 2% methanol in 200 ml methylene chloride and 5% methanol in 400 ml methylene chloride. The fractions containing the green zone were combined and concentrated to an amorphous solid weighing 110 mg corresponding to 13%. This material was dissolved in 2 ml of acetone and precipitated from the solution by the addition of hexane. The product was collected by filtration.

Analysis calculated for C23H32<N>6°5<:>C 58'46'H 6'83'N 17.79

Found: C 58.89, H 6.89, N 17.64

UV (MeOH) X max. pp, nm: 235, 364.

IR (KBr) u ITldK S, cm"<1>: 3440, 3295, 2925, 1770, 1660,1620,

1580, 1550, 1300, 1055.

The H NMR spectrum in pyridine is consistent with the structure of the title compound.

Example 22

Connection ( XXIV)

7-[1-(dimethylamino)ethylideneamino1-9a-methoxymitosane

A solution of 100 mg (0.21 mM) of compound (XXIII)

in 2 ml of chloroform was added to 2 ml of aminodiphenylmethane and the solution was heated to approx. 55-60°C for hours.

At this stage, trace amounts of compound (XXIII) remained in the reaction mixture, but it was nevertheless concentrated and the residue chromatographed over neutral alumina using gradient elution from methylene chloride and terminated with methanol/methylene chloride 2.5:1. The green main zone was isolated as an amorphous green solid in an amount of 25 mg corresponding to 29.4%. This material was purified by dissolving in acetone and adding hexane to the acetone solution to precipitate. The product was collected by filtration and dried.

Analysis calculated for C25H32<N>6°5<:>C 56'58'H 6'20'N 17.37 Found: C 55.71, H 6.34, N 15.23

UV (H2<0>)<*>max, nm: 374, 230 (shoulder)

IR (KBr) umax, cm"<1>: 3420, 3350, 3280, 2920, 2710, 1610, 1540, 1300, 1050.

The H NMR spectrum in pyridine is consistent with the structure.

Example 23

Connection (XXV)

7-[(1-methyl-2-pyrrolidinylidene)amino]-N<10->[(1-methyl-2-pyrrolidinylidene)amino]-9a-methoxymitosane

2,2-dimethoxy-1-methylpyrrolidine (H. Eilingsfeld et al., "Angew. Chem.", 72, 836 (1960)), 1.5 g (10.3 mM) and 280

mg of mitomycin C (0.34 mM) in 20 ml of methanol was heated to 55°C for 5 hours. The reaction mixture was tested by thin layer chromatography on an alumina plate using methylene chloride/methanol 97:3 as solvent. tic showed a large green spot representing the product and a small blue spot representing the mitomycin C starting material. The solvent was removed by distillation in vacuo at 40°C and the residue was dissolved in methylene chloride and loaded onto a 4.5 cm column containing 150 g of alumina. Elution with 50 ml of methylene chloride followed by 600 ml of 1% methanol in methylene chloride. Major impurities were removed, but no pure fractions were isolated. The combined eluent was concentrated by distillation at 20°C to an oily residue which apparently contained some 2,2-dimethoxy-1-methylpyrrolidine. This material was chromatographed again on an alumina column (25 g alumina) using 200 ml of methylene chloride followed by 100 ml of 1% methanol in methylene chloride. This resulted in the removal of 2,2-dimethoxy-1-methylpyrrolidine and resulted in a number of fractions containing minor impurities and several pure fractions, which were confirmed by tic (a green spot) and which constituted the desired product in a yield of 53 mg .

Analysis calculated for 5H32<N>6°5•<0>'85H2°:

C 58.66, H 6.64, H 16.42 Found: C 58.63, H 6.46, H 16.50

UV (MeOH) X , nm: 354, 239

max _,

IR (KBr) umax, cm :3300, 3220, 2940, 1660, 1620, 1550, 1290, 1055.

"'"The H-NMR spectrum of pyridine dg is consistent with the structure of the title compound.

Example 24

7-[(1-methyl-2-pyrrolidinylidene)amino]-9a-methoxymitosane

A solution of 80 mg (0.16 mM) of the compound (XXV) and 0.48 ml of n-butylamine in 15 ml of chloroform was heated under reflux for 48 hours. tic (methanol/methylene chloride, 2% on aluminum oxide) gave a large green spot and a small preceding blue spot as well as a small trailing red spot which all corresponded to the starting material. The reaction solution was loaded onto a column containing 50 g of alumina and eluted with 20 ml of 1% methanol in methylene chloride followed by 400 ml of 2% methanol in methylene chloride. The fractions containing a single large green component as shown by tic were combined and concentrated to a residue of the desired product in an amount of 24 mg.

NMR (pyridine dg, 6): 1.72 (q, 2H), 2.04 (s, 3H), 2.16

(q, 2H), 2.72 (bs, 1H), 2.84 (s, 3H), 3.12 (m, 3H),

3.24 (s, 3H), 3.60 (dd, 1H, J=14, 2Hz), 4.00 (dd, 1H, j=12, 6 Hz), 4.40 (d, 1H, J= 14 Hz), 5.04 (t, 1H, J=14Hz),

5.38 (dd, 1H, J=12, 6 Hz), 7.48 (bs, 2H).

Example 25

Connection (XXVI)

7-[(Methoxyamino)methylene]amino-9α-methoxymitosane

A solution of compound (XIX) 660 mg (1.7 mM) in 10 ml of methanol was prepared and 170 mg (2.0 mM) of methoxyamine hydrochloride was added. The solution was stirred at 10°C for 3 hours and at room temperature for 2 hours. tic showed only a trace of unreacted compound (XIX). A black precipitate formed on standing was collected and washed with acetone and the yield of the desired product was 380 mg or 57%.

Analysis calculated for <C>]_7H2i<N>5°6<:>c 52'19'H 5'40',N 17.90

Found: C 51.64, H 5.40, N 17.83

UV (MeOH)<x>max, nm: 376, 242

IR (KBr) umax, cm"<1>: 3440, 3250, 3140, 2920, 1730, 1645, 1615, 1560, 1450, 1320, 1050.

The "*"H NMR spectrum in pyridine d^ is consistent with the structure of the title compound or its tautomer with respect to C-7, i.e.

Example 26

Connection (XXVII)

7-[(benzyloxyamino)methylene]amino-9a-methoxymitosane

A solution of compound (XIX), 100 mg (0.26 mM) in 2 ml of methanol containing 0.5 ml of triethylamine was prepared and thereto was added 400 mg (2.5 mM) of O-benzylhydroxylamine hydrochloride. The reaction was allowed to continue for 2.5 hours at room temperature. tic (Cl^C^/methanol 10:1) showed a large orange-brown zone before the green zone, the latter corresponding to compound (XIX). The reaction mixture was concentrated to a residue which was flash chromatographed over 20 g of silica gel using Cl₂C₂:methanol 20:1 as eluent. The large brown zone constituting the desired product was collected as an amorphous solid in an amount of 80 mg or 65.6%. '

Analysis calculated for<C>23<H>25<N>5°6<:>C 59'10'H 5'35'N 14.97

Found: C 58.43, H 5.48, N 14.62

UV (MeO<H>)<x>max, nm: 376, 245, 209

IR (KBr) u maK, s, cm"<1>: 3460, 3300, 2945, 2920, 1745, 1720, 1570, 1275, 1220, 1060.

The NMR spectrum in pyridine d^ is consistent with the structure of either the title compound or its tautomer at C-7,

i.e.

Unreacted starting material, compound (XIX)-, in an amount of 10 mg, was recovered.

Example 27

Connection ( XXVIII)

7-(1,3-Dimethyl-2-imidazolinylidene)-9a-methoxymitosane

Mitomycin C, 0.34 g (1 mmol) was dissolved in 5 ml of 1,3-dimethyl-2-imidazoline and 0.1 g of sodium hydride (50% in oil, 2.08 mmol) was added at room temperature. The mixture was kept at room temperature for 20 minutes and then cooled in an ice-salt bath (-15°C). The mixture was kept for 10 minutes at this temperature, and then 0.65 g (2 mmol) of 2-chloro-1,3-dimethyl-4,5-dihydro-(3H)-imidazoliminium chloride was added. The whole was kept at -15°C for 1 hour and then diluted with ethyl acetate and chromatographed on an alumina column. The column is diluted with methylene chloride, followed by methylene chloride containing 2% vol/vol methanol. A green colored fraction consisting of the desired product is obtained which is further purified by chromatography on alumina using methylene chloride containing 10% vol/vol methanol, in a yield of 20 mg or 5%.

Analysis calculated for <C>^0<H>26N6°5'1-1/4H2°:

C 53.03, H 6.34, N 18.55 Found: C 52.68, H 6.21, N 18.15

NMR (pyridine-d5,6): 2.32 (s, 3H), 2.47 (s, 3H), 2.59 (s, 3H), 2.74 (m, 1H), 3.03-3 .32 (m, 5H), 3.26 (s, 3H), 3.66 (bd, 1H, J-12 Hz), 4.02 (dd, 1H, J=ll, 4 Hz), 4.75 (d, 1H, J=12 Hz), 5.09 (bt, 1H, J=11 Hz), 5.44 (dd, 1H, J=11,~4 Hz).

IR (KBr): 3400, 3280, 2930, 1700, 1610, 1480, 1330,

1055 cm"<1>.

UV (MeOH, xmax) = 600.375, 252 (sh), 222 nm.

Example 28

Connection (XXIX)

7-[(1,3-dimethyltetrahydropyrimidinylidene)amino]-9a-methoxymitosane

Sodium hydride (50% oil dispersion, 200 mg, 4.2 mmol) was added under nitrogen to a solution of mitomycin C (680 mg, 2 mmol) in 8 mL of 1,3-dimethyl-3,4,5,6-tetrahydro( 1H,3H)-2-pyrimidinone. The mixture is kept for 20 minutes at room temperature and then cooled to 25°C. 2-Chloro-1,3-dimethyl-2,3,4,5-tetrahydro-pyrimidinium chloro, 0.73 g (4 mmol) was then added and the mixture kept at -25°C for 3 hours. It is then diluted with ethyl acetate and 2 ml of methanol. The mixture is loaded without further treatment on a dry chromatographic alumina column and eluted first with methylene chloride and then with 2% vol/vol methanol/methylene chloride, which gives the desired product in an amount of 0.35 g corresponding to 39.5% and with a melting point of 138 -140°C.

Analysis calculated for C2iH27<N>6°5'<H>2°<:>

C 54.65, H 6.33, N 18.21 Found: C 54.78, H 6.18, N 18.21

NMR (pyridine-d5,6): 1.80 (m, 2H), 2.42 (s, 3H), 2.52 (s, 3H), 2.64 (s, 3H), 2.76 (m , 1H), 2.90-3.30 (m, 5H), 3.26 (s, 3H), 3.74 (d, 1H,J=12Hz), 4.05 (dd, 1H, J=ll , 4 Hz), 4.97 (d, 1H, J=12 Hz), 5.09 (t, 1H, J=11.Hz), 5.41 (dd, 1H, J=11, 4Hz).

IR (KBr): 3430, 3280, 2930, 1710, 1570, 1480, 1450, 1350, 1050 cm"<1>.

UV (MeO<H,><A>max) = 635, 377, 264 (sh), 223 nm.

Example 29

Connection ( XXX)

7-(Tetramethyldiaminomethylene)amino-9a-methoxymitosane

Mitomycin C, 425 mg (1.42 mmol) is mixed with a 50% dispersion in oil of 85.3 mg of sodium hydride and 4 ml of dimethylformamide is added. The mixture is stirred at room temperature under an atmosphere of argon for 10 minutes and then cooled

-35°C. Tetramethylchloroformamidium chloride, 289 mg (2.13 mmol) is added and the mixture is allowed to warm to 5°C

within 2 hours. Crushed dry ice is then added to the mixture to cool the reaction and the solvent is removed by distillation under reduced pressure. The residue is chromatographed on a 100 g alumina column using 3% vol/vol methanol in methylene chloride for the elution. This material is further purified by alumina-tlc (5% vol/vol methanol in methylene chloride), giving two fractions of 17 mg and 76 mg. The latter is crystallized from acetone-ether to obtain the desired product in a yield of 12% and with a melting point of 193-195°C.

Analysis calculated for<c>2oH28<N>6°5: C 55.54, H 6'53'N 19.43

Found: C 54.92, H 6.53, N 19.29

NMR (pyridine-d5,6): 2.26 (s, 3H), 2.59 (s, 6H), 2.68 (s, 6H), 2.75 (m, 1H), 3.15 (d , 1H, J=4 Hz), 3.26 (s, 3H), 3.65 (d, 1H, J=12 Hz), 4.00 (dd, 1H, J=ll, 5 Hz), 4, 62

(d, 1H, J=12 Hz), 5.04 (t, 1H, J=11 Hz), 4.38 (dd, 1H, J=11, 5 Hz).

IR (KBr): 3430, 3280, 2920, 1710, 1610, 1495, 1335, 1055 cm"<1.>

UV (MeO<H,><x>max)= 610, 380, 260, 220 nm.

Example 30

Connection (XXXI)

7-(1-piperidinylmethylene)amino-9a-methoxymitosane

A 0.5 M solution of piperidinylchloromethyleneiminium chloride is prepared by dropwise addition of oxalyl chloride (380 mg, 3 mmol) to 6 ml of chloroform containing 0.34 g (3 mmol) of 1-formylpiperidine. Sodium hydride (50% oil dispersion, 96 mg, 2 mmol) is added separately under nitrogen to a solution of mitomycin C (334 mg, 1 mmol) in 3 mL of 1-formylpiperidine. After stirring for 15 minutes at room temperature, the solution is cooled to -25°C and the iminium salt solution prepared above (4 ml, .2 mmol) is added. The reaction mixture is kept at -25°C for 1 hour and cooled by adding dry ice. After adding 1 ml of methanol, the product mixture is absorbed on neutral alumina. This material is placed on an alumina column (30 g). The column is first eluted with methylene chloride and then with 3% vol/vol methanol in methylene chloride, which gives 360 mg or 84% of the title compound with melting point 68-70°C.

Analysis calculated for c2iH25N5°6'1_1/4 H20:

C 55.80, H 6.58, N 15.49 Found: C 55.57, H 6.21, N 15.91 NMR (pyridine-d5, 6): 1.42 (bs, 6H), 2, 19 (s, 3H), 2.72 (m, 1H), 3.06-3.30 (m, 3H), 3.25 (s, 3H), 3.48-3.70 (m, 2H) , 3.57 (d, 1H, J=13 Hz), 4.01 (dd, 1H, J=ll, 4 Hz), 4.43 (d, 1H, J=13Hz), 5.02 (bt, 1H, J=11 Hz), 5.55 (dd, 1H, J=11, 4 Hz), 7.86 (s, 1H).

IR (KBr): 3440, 3350, 3300, 2935, 2835, 1710, 1615, 1520, 1445, 1305, 1250, 1200, 1055 cm"<1>.

UV (MeO<H,><x>max): 590, 389, 362 (sh), 234, 212 (sh) nm.

Example 31

7-hydroxy-N<1>^-dimethylaminomethylene-9a-methoxymitosane

Dimethylformamide-dimethyl acetal (1 ml) is added to a solution of 7-hydroxy-9a-methoxymitosane (20 mg) in methylene chloride (3 ml) and the solution is stirred at approx. 65°C for 30 minutes. The progress of the reaction is followed at tic (10:1 CH2Cl2/MeOH). The product is recovered by concentrating the mixture under reduced pressure and the residue is chromatographed over silica gel, whereby the title compound is obtained.

Activity against P-388 mouse leukemia

Table IV contains the results of laboratory experiments with female CDF^ mice implanted intraperitoneally with a tumor inoculation of 106 ascites cells of P-388 murine leukemia and treated with various doses of either a test compound of formula (I) or mitomycin C. The compounds were administered by intraperitoneal injection. Groups of six mice were used for each dose level and they were treated with a single dose of the compound only on day 1. A group of ten saline-treated control mice was included in each series of experiments. The mitomycin C-treated groups were included as a positive control. A 30 day protocol was used, in which the mean survival time of 1 day was determined 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 6th day. Weight change was taken as a measure of drug toxicity. Mice weighing 20 g each were used and a weight loss of up to 2 g was not considered excessive. The results were determined in terms of %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 as % T/C and the dose that produces this effect is indicated. Values in parentheses are those obtained with mitomycin C as a positive control in the same experiment. A measure of the relative activity of the compounds in question in relation to mitomycin C can therefore be understood.

A minimal effect in terms of % T/C was considered to be 125. The minimal effective dose indicated in the following table is the dose which gives a % T/C of approx. 125. The two values which in each case are stated in the "average weight change" column are respectively the average weight change per mice at the maximum effective dose and the minimum effective dose. Compounds (XIX) and (XX) are of particular interest, as their activity is clearly better than that of mitomycin C, both in terms of maximum effect and mg strength, (comparative dosage sizes for equivalent effects).

They are both compounds of formula (I), where A is the amidino group and B is -NH.-,,, or in other words mitomycin C derivatives, which at N 7 are substituted with an aminomethylene-

group with formula

2 3 4 where R , R and R are as indicated

above.

The relevant bis-amidino compounds of formula (I), where both A and B are the amidino group, are also of considerable interest as active antitumor compounds. Reference is made to the results in the table for the compounds (V), (VI), (VII), (IX), (X) and (XIV), which correspond to this structural requirement.

Table V contains results of antitumor experiments using B16 melanomas grown in mice. BDF-^ mice were used and inoculated intraperitoneally with tumor implant ion.

A 60 day protocol was used. Groups of 10 mice were used for each tested dose and the mean survival time for each group was determined. Control animals were inoculated in the same way as the experimental animals and treated with the injection vehicle without drug and they showed an average survival time of 21 days. The survival time relative to that of the control animals (% 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 defined as the dose that showed 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 intraperitoneally. The average weight change on the indicated day at the maximum effective dose and the minimum effective dose was used as a measure of toxicity. A weight loss of 2 g for a 20 g mouse was not considered excessive.

Compounds (XXX) (Example 29) and Compound (XXIX)

(Example 28) was tested on the B16 mouse melanomas using subcutaneous tumor implantation and intravenous treatment with the drug. The treatment schedule and assessments of survival time (a 40-day protocol was used) were determined as before. The weight change was measured on day 12. The maximum effective dose of compound (XXX) was 1 mg per kg, giving a % T/C of 156 and a weight gain of 1.5 g. Groups of 6 animals were used and 3 animals survived the full 40 day protocol at this dose. The minimum effective dose was 0.25 mg/kg at which dose the 12 day weight change was 1.0 g. For compound (XXIX) the maximum effective dose was 8 mg/kg at a % T/C of 177 and at a weight change of -0.6. The minimum effective dose was 4 mg/kg with a weight gain of +0.8.

In the same experiment, the maximum effective dose of mitomycin C was 3 mg/kg at a %T/C of 195 and a weight change of -0.5. The minimum effective dose of mitomycin C was not determined.

In a short toxicological protocol using groups of 5 male BDF-^ mice per dose administered in a single intraperitoneal dose of compound (XIX) there was no significant decrease in the lymphocyte count at the optimal effective dose of this compound in an amount of 1.6 mg/kg i.p. At this dose, there was no significant increase in blood urea nitrogen (BUN) or serum glutamine phosphotransferase (SGPT), suggesting no adverse effect on renal or hepatic function or suppression of lymphocytic activity.

In view of the unique antitumor activity observed in experimental animal tumors, and the reduced toxicity compared to mitomycin C, the invention encompasses the use of the compounds of formula (I) for the inhibition of tumors in mammals. For this purpose, they are administered systemically to a mammal with a tumor in a substantially non-toxic but antitumor effective dose.

Claims (1)

Mitosane derivatives, characterized in that they have the formula: there A is amino or methoxy, B is amino, and R 1 is methyl or formyl.