TITLE OF THE INVENTION
HITOMYCIN DERIVATIVES HAVING REDUCED BONE MARROW TOXICITY.
PROCESSES FOR THEIR PREPARATION, AND THE USES THEREOF
FIELD OF THE INVENTION:
The invention is in the field of pharmaceutical agents and the uses thereof.
BACKGROUND OF THE INVENTION
The mitomycins are a family of compounds having the following general formula (I):
Mitomycins A, B and C are related to one another as set forth in Table 1 below, the designations X, Y and Z being those of formula I.
TABLE 1
Mitomycin: X Y
A -OCH3 -OCH3 -H
B -OCH3 -OH -CH3
C -NH2 -OCH3 -H
Mitomycins are derived from mitosane compounds having the following skeleton (II):
The mitosanes are formed during the cultivation of the microorganism Streptomyces caespitosus in a liquid nutrient medium under artificially controlled conditions. After separating the resulting mycellium, the various mitomycins may be isolated from the latter by active carbon or preferably non-ion exchange resin adsorption, organic solvent extraction or chromatography on alumina, as disclosed in U.S. Patent No.3,660,578 to Hata et al.
Although the nitosanes are excellent antibiotics, they have limited utility due to their toxicity to human blood (see U.S. Patent No. 3,450,705 to Matsui et al.). The relatively highly toxin nature of the compounds has prompted search for derivatives of mitomycin to increase the antibiotic activity and to decrease toxicity.
For example, Matsui et al., U.S. Patent No. 3,450,705, disclose mitomycin compounds substituted at the 7-position with amino, lower alkylamino, phenylamino, or pyridyl, and substituted at the la position with haloalkanoyl, halobenzoyl, nitrobenzoyl, alkenoyl, acetyl glycyl, sorboyl, or acetyl methionyl.
Matsui et al., U.S. Patent No. 3,558,651, disclose mitosane derivatives comprising la-acyl-7-acyloxy-9a-methoxy compounds.
Certain mitomycins and mitomycin derivatives also possess antitumor activity. Oboshi et al., Gann 58:315-321 (1967); Usubuchi et al., Gann 58:307-313 (1967); Matsui et al., J. Antibiotics XXI.:189-198 (1968); Japanese Patent No. 6806627 to Matsui et al. (Chemical Abstracts 69:86986k (1968)); and Cheng et al., J. Med. Chem. 20:767-770 (1977).
While mitomycin C is active against a relatively broad spectrum of experimental tumors, its toxicity and myelosuppressive effects limit its use in clinical practice (Mitomycin C: Current Status and New Developments. Carter et al. (eds.), Academic Press, New York (1979)). In preclinical and clinical studies, mitomycin C has shown activity against a variety of murine and human neoplasms, but has also shown severe, delayed bone marrow toxicity. Goldin, A., et al., NCI -EORTC Symposium on Mitomycin C. Brussels, Belgium (1981).
In other studies, a combination of 5-fluorouracil, adriamycin and mitomycin C was found to be effective for the treatment of patients with advanced gastric and colorectal cancer. This regimen incorporated mitomycin C administration in a single dose schedule every two months, to decrease the treatment-limiting delayed myelosuppressive effects of the compound. Schein, P.S., et al., Mitomvcin C: Current Status and New Developments, pp. 133-143, Carter et al. (eds.), Academic Press, New York (1979).
Numerous synthetic derivatives of mitomycin C have been prepared in the hope of obtaining compounds with improved therapeutic properties. These derivatives include substitution on the aziridine ring, carbamoyl, or acyl group substitution on the hydroxymethyl side chain,
and replacement of the 7- substituent in the quinone ring with other functional groups, especially substituted amines. However, as disclosed by Remers, U.S. Patent No. 4,268,676, none of these analogs have emerged as a clinical agent, with the possible exception of the 7-hydroxy analog of the mitomycin C, which has been involved in a recent study in Japan. This analog is asserted to be less leukopenic than mitomycin C, but is also less potent. Also disclosed by Remers, suora. are totally synthetic mitomycin analogs of the mitosane type (Mott et al., J. Med. Chem. 21:493 (1978)), prepared mainly for their antibacterial activity.
Kinoshita, S., et al., J. Med. Chem. 14:103-112 (1971), disclose several derivatives of mitomycin substituted in the la, 7, and 9a positions. In particular, compounds substituted at the la position with sulfonyl, ortho-substituted benzoyl, and acyl derivatives were reported.
Iyengar, B.S., et al., J. Med. Chem. £4:975-981 (1981), disclose a series of 31 mitomycin C and porfiromycin analogues with various substituents at the 7- and la-positions. The most active substituents at the 7-position included aziridine, 2-methylaziridine, propargylamine, furfuryl amine, methyl glycinate and 3-aminopyridine.
Iyengar, B., et al., J. Med. Chem. 26:16-20 (1983), disclose a series of 7-substituted mitomycin C and porfiromycin derivatives and the screening thereof in standard antitumor systems. The authors report that the 7- position controls the reduction of the quinone ring, thus suggesting that it would be possible to alter the substitution of the 7- position to gain selectivity between normal cells and certain cancer cells.
Iyengar, B.S., et al., J. Med: Chem. 26:1453-1457 (1983), disclose 20 mitomycin C analogues substituted with secondary amines at the 7-position. Eleven of these analogues were more active than mitomycin C against P388 murine leukemia and two of these eleven were significantly less leukopenic. The authors report that no quantitative correlation between antitumor activity and physiochemical
properties of the analogues was evident, although the relative ease of quinone reduction may be related to activity.
Iyengar, B.S., et al., J. Med. Chem. 29:1864-1868 (1986), disclose the preparation of 7-substituted amino 1,2-aziridinomitosenes. The authors reported that a Methyl group on the aziridine nitrogen gave Increased potency. The 7-amino mitosene derivatives which were difficult to reduce to hydroquinones were essentially Inactive.
Sami, S., et al., J. Med. Chem. 27:701-708 (1984), disclose a series of 30 N7-phenyl-substituted Mitomycin C analogs. Two of the compounds having pyrazolyl or aminopyridyl substituents at the 7-position were disclosed as clearly superior to mitomycin C in activity against P388 murine leukemia.
Sami, T., et al., J. Med. Chem. 22:247-250 (1979), also disclose N-(2-chloroethyl)-N-nitrosocarbamoyl derivatives of glycosylamines, including three disaccharide derivatives which exhibited strong antitumor activity against leukemia 1210 in mice. In addition, glucopyranose derivatives of N-nitrosoureas possess immunogenic and marrow-sparing properties. Anderson et al., Cancer Research 35:761-765 (1975); Panasci et al., J. Clin. Invest. 64:1103-1111 (1979).
In U.S. Patent No. 4,720,543, compounds having the following general formula (III) are disclosed:
where
R1 is selected from the group consisting of NH2, C1-C4 alkoxy and a glycosyl residue; and
R2 is selected from hydrogen, C1-C4 alkyl, and a glycosyl residue, with the proviso that either R1 or R2, but not both, contain a glycosyl group.
The compounds represented by formula III have excellent anti- πeoplastic activity and at the same time possess reduced bone marrow toxicity and lower overall toxicity.
Despite the above-listed mitomycin derivatives, a need continues to exist for improved mitomycin derivatives having good anti-neo- plastic properties and low bone marrow and overall toxicity.
SUMMARY OF THE INVENTION
The invention relates to a mitomycin derivative having the following general formula (IV):
wherein
n is 0 or 1;
Y is selected from the group consisting of glucopyranosyl, galactopyranosyl, mannopyranosyl, xylopyranosyl, cellobiosyl, lactosyl, glucofuranosyl, maltosyl, and 2-amino-1,3- cyclohexanediol, or a hydroxyl-protected peracetate derivative thereof;
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted by phenyl, hydroxyphenyl, Indolyl, mercapto, C1-C4 alkylthio, hydroxy, carboxy, amino, guanidino, imidazole or carbamyl; or
R and R1 together form a five or six membered nitrogen containing ring.
The Invention also relates to a mitomycin derivative having the following general formula (V):
wherein n, R, R1 and Y are as defined above and R2 is NH2- or CH3O-.
The invention also relates to a mitomycin derivative having the following structural formula (VI):
wherein
R2 is as defined above; and
R3 is a 2-(3-cyano-4-morpholinyl)-2-deoxypyranosyl saccharide or a 2-(4-morpholinyl)-2-deoxypyranosyl saccharide.
The Invention also relates to a process for preparing a mitomycin derivative having the formula (IV)
wherein
n is 1;
Y is selected from the group consisting of glucopyranosyl, galactopyranosyl, mannopyranosyl, xylopyranosyl, cellobiosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclohexanediol-2-yl, or a hydroxyl -protected peracetate derivative thereof;
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted by phenyl, hydroxyphenyl, indolyl, mercapto, C1-C4 alkylthio, hydroxy,. carboxy, amino, guanidino, imidazole or carbamyl; or
R and R1 together form a five or six membered nitrogen containing ring;
comprising:
(a) condensing an N-protected amino acid with an alcohol in the presence of a dehydration reagent to give an activated ester,
(b) condensing the activated ester obtained in step (a) with an amino compound to give a protected amino acid-amino compound conjugate,
(c) removing the protecting group of the protected amino acid-amino compound conjugate obtained in step (b) to give an amino acid-amino compound conjugate, and
(d) condensing the amino acid-amino compound conjugate obtained in step (c) with mitomycin A to give the mitomycin derivative.
The invention also relates to a process for the preparation of a mitomycin derivative having the formula (VI):
wherein
R2 is NH2- or CH3O-; and
R3 is a 2-(3-cyano-4-morpholinyl)-2-deoxy saccharide;
comprising
(a) condensing bis(acetaldehyde-2-yl) ether with a 2-amino-2-deoxy saccharide in the presence of a salt of cyanoborohydride to give a 2-deoxy-2-(3-cyano-4-morpholinyl) saccharide and a 2-deoxy-4-morpholinyl saccharide;
(b) separation of the 2-deoxy-2-(3-cyano-4-morpholinyl) saccharide from the 2-deoxy-4-morpholinyl saccharide obtained in step
(a);
(c) reaction of the 2-deoxy-2-(3-cyano-4-morpholinyl) saccharide obtained in step (b) with an acetyl halide to give a 2-deoxy 1-halo-2-(3-cyano-4-morpholinyl) peracetyl saccharide;
(d) treatment of the 2-deoxy-1-halo-2-(3-cyano-4-morpholinyl) peracetyl saccharide obtained in step (c) with silver thiocyanate to give a saccharide-1-thiocyanate;
(e) reaction of the saccharide-1-thiocyanate obtained in step (d) with mitomycin C or mitomycin A to give a mitomycin C- or mitomycin A-saccharide peracetate carbothioamide; and
(f) hydrolysis of the acetate groups of the mitomycin-C-saccharide peracetate obtained in step (e) to give the mitomycin derivative.
The invention also relates to a process for the preparation of a mitomycin derivative having the following formula (VI)
wherein
R2 is NH2- or CH3O-; and
R3 is a (4-morpholinyl)-2-deoxy saccharide;
comprising
(a) condensation of bis(acetaldehyde-2-yl) ether with a 2-amino-2-deoxy saccharide in the presence of a salt of cyanoborohydride to give a 2-deoxy-2-(3-cyano-4-morpholinyl) saccharide and a 2-deoxy-2-(4-morpholinyl) saccharide;
(b) separation of said 2-deoxy-2-(4-morpholinyl) saccharide from said 2-deoxy-2-(3-cyano-4-morphplinyl) saccharide obtained in step (a);
(c) reaction of the 2-deoxy-2-(4-morpholinyl) saccharide obtained in step (b) with an acetyl halide to give a 2-deoxy-1-halo-2- (4-morpholinyl) peracetyl saccharide;
(d) treatment of the 2-deoxy-1-halo-2-(4-morpholinyl) peracetyl saccharide obtained in step (c) with silver thiocyanate to give a saccharide-1-thiocyanate;
(e) reaction of the saccharide-1-thiocyanate obtained in step (d) with mitomycin A or C to give a mitomycin A- or C-saccharide percetate carbothioamide; and
(f) hydrolysis of the acetate groups of the mitomycin-C-saccharide peracetate obtained in step (e) to give the mitomycin derivative.
The invention also relates to a process for the preparation of a mitomycin derivative having the following formula (V)
wherein
n is 0 or 1;
Y is selected from the group consisting of glucopyranosyl, galactopyranosyl, mannopyranosyl, xylopyranosyl, cellobiosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclohexanediol-2-yl;
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted by phenyl, hydroxyphenyl, indolyl, mercapto, C1-C4 alkylthio, hydroxy, carboxy, amino, guanidino, imidazole or carbamyl; or
R and R1 together form a five or six membered nitrogen containing ring;
R2 is NH2-;
comprising:
(a) condensation of mitomycin C with succinic anhydride under basic conditions to give mitomycin C-la-succinic acid ester;
(b) condensation of the mitomycin C-la-succinic acid ester obtained in step (a) with a compound of the Formula (VII).
wherein R, R1 and n are defined above and Yp is a hydroxyl-protected saccharide selected from the group consisting of the hydroxyl-protected derivatives of glucopyranosyl, galactopyranosyl, mannopyranosyl, xylopyranosyl, cellobiosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclohexamediol-2-yl;
(c) removal of the hydroxyl protecting groups to give the mitomycin derivative.
The invention also relates to a process for the preparation of a mitomycin derivative having the following formula (V):
wherein
n is 0 or 1;
Y is selected from the group consisting of glucopyranosyl, galactopyranosyl, mannopyranosyl, xylopyranosyl, cellobiosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclohexanediol-2-yl;
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted by phenyl, hydroxyphenyl, indolyl, mercapto, C1-C4 alkylthio, hydroxy, carboxy, amino, guanidino, imidazole or carbamyl; or
R and R1 together form a five or six membered nitrogen containing ring;
R2 is CH3O-;
comprising:
(a) condensation of mitomycin A with succinic anhydride under basic conditions to give mitomycin A-la-succinic acid ester;
(b) condensation of the mitomycin A-la-succinic acid ester obtained in step (a) with a compound of the Formula (VII)
wherein R, R1 and n are as defined above and Yp is a hydroxyl-protected saccharide selected from the group consisting of the hydroxyl-protected derivatives of glucopyranosyl, galactopyranosyl, mannopyranosyl, xylopyranosyl, cellobiosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclohexanediol-2-yl;
(c) removal of the hydroxyl protecting groups to give the mitomycin derivative.
The invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of the mitomycin derivatives of the invention together with a pharmaceutically acceptable carrier.
The invention also relates to methods for the treatment of bacterial infections comprising administering the pharmaceutical compositions of the invention to an animal.
The invention also relates to methods for the treatment of cancer by suppressing growth of cancer cells susceptible to growth suppres
sion comprising administering the pharmaceutical compositions of the invention to an animal.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The synthetic preparation of the mitomycin derivatives of the invention have, as their starting point, mitomycin C. Mitomycin C may be prepared according to the methods generally disclosed in Cheng et al., J. Med. Chem.20:767-770 (1977). Alternatively, mitomycin C can be obtained from mitomycin A by treatment of mitomycin A with a methanolic-ammonia solution as described by Matsui, M., et al., J. Antibiotics XXI:189 (1968).
The mitomycin derivatives of Formula IV, wherein n=0 (X), may be obtained by displacement of the methoxy group of mitomycin A (VIII) with the amino group of an amino compound, for example, glucosamine (Y-NH2; (IX)) under basic conditions in a polar organic solvent to give the N7-substituted mitomycin derivative (X) (see Scheme 1 below).
Amino compounds (Y-NH2) which may be substituted at the 7-position include, but are not limited to glucosamine, galactosamine, mannosamine, xylosamine, cellobiosamine, maltosamine and 2-amino-1,3- cyclohexanediol and the hydroxyl -protected peracetate derivatives thereof. Preferably, the saccharide comprising the group "Y" is substituted at the 2-position with the amino group. Polar organic solvents which may be used in the practice of the invention include methanol, ethanol, propanol, dimethylsulfoxide, and dimethylformamide. Suitable bases for providing the basic conditions of the reaction include alkylamines such as C1-C3 trialkyl amines, diisopropylethyl-amine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and dimethylamino-pyridine (DMAP). In general, mitomycin A and the amino derivative are present in a 1:1 molar ratio, although excess amino derivative may be present. Sufficient base is present in the reaction mixture to insure that the reaction remains basic throughout.
Preferred mitomycin derivatives having Formula X include N7-(2- deoxyglucopyranosyl)mitomycin C, N7-(2-deoxygalactopyranosyl)mitomycin C, N7-(tetraacetyl-2-deoxyglucopyranosyl)mitomycin C, and N7-(tetra-acetyl-2-deoxygalactopyranosyl)mitomycin C.
Scheme I
The amino acid linked mitomycin derivatives of formula IV, wherein n=1, may be prepared (Scheme II) from mitomycin A by condensation of the N-protected amino acid, for example, the N-benzyloxycarbonyl derivative (XI), with an alcohol such as N-hydroxysuccinamide, which is capable of generating an activated ester, and a dehydrating reagent to give the activated ester (XII). Dehydrating reagents which may be used in this process include, but are not limited to dicyclohexylcarbodiimide (DCC) and diethylazodicarboxyl ate (DEAD) and triphenylphosphine. Treatment of the activated ester (XII) with any of the above-listed amino compounds (IX) gives the protected amino acid-amino compound conjugate (XIII). Removal of the protecting group, for example, by hydrogenolysis of the N-benzyloxycarbonyl group, gives the free amino derivative (XIV). Compound (XIV) may then be condensed with mitomycin A (VIII) by displacement of -OCH3 as described above to give the amino acid linked mitomycin derivative (IV).
Preferred mitomycin derivatives having Formula (IV), wherein n=1, R1 = H and R = H include N7-[[[(2-deoxy-2-glucopyranosyl)amino] carbonyl]methyl] mitomycin C and N7-[[[(tetraacetyl-2-deoxy-2-glucopyranosyl)aminojcarbonyl]methyl]mitomycin C.
Scheme II
Mitomycin derivatives having Formula (V), wherein R2 is NH2 and n is 0 (Formula (XV), below), may be prepared (Scheme III) by condensation of mitomycin C (XVI) with succinic anhydride to give the amide (XVII) which May then be condensed with the hydroxyl-protected amino derivative YP-NH2 (XVIII) using any of the above-listed dehydrating reagents followed by deprotectlon to give (XV). Protecting groups for the amino derivative Include, but are not limited to, C2-C4 acyl esters.
Preferred mitomycin derivatives having Formula (XV) include N1- [[2-[[(2-deoxy-2-glucopyranosyl)amino]carbonyl]ethyl]carbonyl] mitomycin C.
Scheme III
Mitomycin derivatives having Formula (V), wherein R2 is -OCH3 and n is 0 (Formula (XIX), below), may be prepared (Scheme IV) by treatment of mitomycin C (XVI) with sodium methoxide in absolute methanol to give mitomycin A (VIII) followed by condensation with succinic anhydride to give the mitomycin A-la-succinic add ester (XX). Condensation of the carboxylic acid group of (XX) with the hydroxyl- protected amino derivative YP-NH2 (XVIII), as described above, followed by deprotection gives (XIX).
Preferred mitomycin derivatives having Formula XVIII include N1- [[2-[[(2-deoxy-2-glucopyranosyl)amino]carbonyl]ethyl]carbonyl] mitomycin A.
Scheme IV
The mitomycin derivatives of Formula VI may be prepared according to the sequence depicted in Scheme V. Treatment of 3,4-dihydroxy-tetrahydrofuran (XXI) with aqueous sodium periodate in a polar organic solvent gives bis(acetaldehyde-2-yl) ether (XXII) which may be condensed with a 2-amino-2-deoxy-saccharide (XXIII) in the presence of a salt of cyanoborohydride to give a mixture of 2-deoxy-2-(3-cyano-4-morpholino) saccharide ((XXIVa), Q = -CN), and 2-deoxy-4-morpholinyl saccharide ((XXIVb), Q = -H) which may be separated, for example, by column chromatography. Salts of cyanoborohydride may include any of the alkali metal salts of cyanoborohydride, preferably sodium cyanoborohydride. Treatment of the saccharide derivative (XXIVa) with an acetyl halide gives the 2-deoxy-1-halo-(4-morpholinyl) peracetyl saccharide which may be reacted with silver thiocyanate to give a 1- thiocyanate saccharide (XXV). Condensation of the thiocyanate (XXV) with mitomycin C (XVI) gives the mitomycin C-saccharide peracetate carbothioamide (XXVI). Deacylation of (XXVI), for example, with methanolic ammonia, gives (VI) (Q = -CN or H).
Preferred mitomycin derivatives having Formula VI include 2- (3- cyano-4-morpholinyl) 2-deoxyglucopyranosyl mitomycin-la-carbothioamide and 2-(3-cyano-4-morpholinyl)-2-deoxygalactopyanosylmitomycin-la-carbothioamide.
Scheme V
The compounds of the invention may be present as pharmaceutically acceptable salts. Among the preferred anionic counter ions are those of the halides (derived from hydrohalic acids), such as chloride, bromide, or fluoride. Other anions include sulfonate, or p-toluene-sulfonate.
As an antibiotic, the compounds of the present invention are useful against all microorganisms susceptible to the anti-bacterial action of the parent compounds, these microorganisms including, but not limited to, Pseudomonas. Staohylococcus. Sarcinia. Diolococcus. Streptococcus. Corvnebacterium. Hemophilus. Escherichia. Klebsiella. Proteus. Salmonella. Shioella. Brucella. Mycobacterium. Nocardia. Saccharomyces. Candida. Penicillium. and Asperqillus. Specific microorganism treatable with the compounds of the present invention include Pseudomonas aeruqinosa. Staphylococcus aureus. Staphylococcus albus. Staphylococcus citreus. Sarcina lutea. Diolococcus pneumoniae. Streptococcus hemolyticus. Streptococcus lactis. Corynebacterium diphtheriae. Hemophilus pertussis. Escherichia coli. Klebsiella pneumoniae. Proteus vuloaris. Salmonella tvohosa. Salmonella paratyphi. Shioella dvsenteriae. Brucella abortus. Brucella megatherium. Brucella mycoides. Brucella anthracius. Mvcobacterium ATCC 607. Mycobacterium avium. Mycobacterium ohlei. Nocardia asteroides. Saccharomyces cervisiae. Candida albicans. Penicillium qlacum, and Asperqillus niger.
The mitomycin derivatives of the present invention are useful in vitro as antiseptics, i.e. for disinfecting. The compounds are also useful topically and Internally as therapeutic agents in combating pathogenic bacteria, e.g. in cases of staphylodermatitis, bacterial pneumoniae, leptopserosis, rickettsiosis, salmonellosis, and the like.
Typically, for topical application, the mitomycins of this Invention are applied in compositions having concentrations in the range of 0.01 to 1000 ug/ml.
As antineoplastic agents, the compounds of the present invention are useful in treating a variety of cancers, including, but not
limited to, those cancers susceptible to cell growth suppression b the parent compounds. Treatment of cancers with the parent compounds are described in the following references:
Driscoll, J.S. et al., Cancer Chemotherapy Rep. 4:1 (1974).
Kojima, R., et al., Cancer Chemotherapy Rep. 3:111 (1972).
Sugiura, K., Cancer Res. 19:438 (1959).
Oboshi, S., et al., Gjnn 52:315 (1967).
Sugiura, K., Cancer Chemotherapy Rep. 13:51 (1961).
Venditti, J.M., et al., Advances in Cancer Chemotherapy, pp. 201- 209 (1978) Editors; H. Umezawa et al., Japan Soc. Press, Tokyo/Univ. Park Press, Baltimore.
Usubuchi, I., et al., Gann 58:307 (1967).
Typical cancers treated by the mitomycin derivatives of this invention include, but are not limited to gastric and pancreatic neoplasms (Schein, P.S. et al., in Mitomycin C: Current Status and New Developments, pp. 133-143, Carter et al . Eds., Academic Press, New York (1979)). Other cancers that may be treated using the compounds of the invention include lung, breast, anal, colorectal, head and neck, and melanoma.
The compounds of the invention are also active against the following tumor systems: Leukemia L-1210, Leukemia P388, P1534 leukemia, Friend Virus Leukemia, Leukemia L4946, Mecca lymphosarcoma, Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, Sarcoma 180 (ascites), Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CD8F, Mammary Carcinoma, Colon 38, Carcinoma 1025, Ehrlich carcinoma (ascites & solid), Krubs 2 carcinoma (ascites), Bashford carcinoma 63, Adenocarcinoma E 0771, B16 Melanoma, Hardin-Passey melanoma, Giloma. 26, Miyona adenocarcinoma, Walker carcinosarcoma 256, Flexner-Jobling carcinoma, Jensen sarcoma, Iglesias sarcoma, Iglesias ovarian tumor, Murphy-Stum lymphosarcoma, Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.
The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
Compositions within the scope of this invention include all compositions wherein the mitomycin derivative is contained in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is with the skill of the art. Typical dosage forms contain 10 to 300 μmole/kg animal of the mitomycin derivative, or an equivalent amount of the pharmaceutically acceptable salt thereof.
In addition to the pharmacologically active compounds, the new pharmaceutical preparations may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 25 to 75 percent of active compound(s), together with the excipient.
The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing
the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tri-calcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, steric acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
Other pharmaceutical preparations which can be used orally Include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active
compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be added.
Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a. suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.
EXAMPLES
Example 1: Preparation of Mitomycin A
Mitomycin C (50 mg, 0.15 mmol) was dissolved in 3 ml of a solution of 50% methanol and 50% 0.1 N NaOH and stirred at room temperature for 18 hrs. After completion of the reaction (TLC, CHCl3:MeOH, 10:1), the reaction mixture was quenched with dry ice to neutralize sodium hydroxide. The mixture was then freeze-dried in vacuo. and the mitosane compound was removed with methanol. The methanol solution was concentrated in vacuo to dryness, and the residue was redissolved in a minimum amount of methanol and then precipitated with ether to give 20 mg of a red-purplish powder. This was dissolved in 15 ml of ethyl acetate and cooled to 5ºC, treated with diazomethane (etherial solution of diazomethane was prepared according to the procedure of Arndt, Pro. Synthesis. Collective Volume II, pp. 165-167), and stirred for 20 minutes (TLC, CHCl3:MeOH, 10:1). After completion of the reaction, the solvent was first removed under water aspirator reduced pressure and then dried in vacuo. The residue was recrystallized from ether to give 18 mg of reddish needles, m.p. 159-160'C. TLC (EtOAc:acetone, 1:1) gave one spot Rf = 0.91, and (acetone:benzene, 4:1) one spot Rf = 0.48. UV (methanol) 216 and 358 mu. NMR (acetone-d6, middle peak of acetone at 2.10), 5, 5.94 (br, 2H); 4.76 (dd, 1H); 4.38 (t, 1H); 4.07 (s, 3H) ; 3.96 (d, 1H); 3.54 (dd, 1H); 3.41 (d, 1H); 3.35 (d, 1H); 3.25 (s, 3H); 2.99-2.264 (mmm); 2.87 (s); 1.640 (s, 3H).
Example 2: Preparation of N7-(2-deoxvolucopyranosyl)mitomycin C
To a solution of mitomycin A (10 mg, 0.028 mmol) in absolute methanol was added a methanolic solution of glucosamine'HCl (70 mg, 0.325 mmol) and diisopropylethylamine (100 ul). This mixture was stirred under N2 atmosphere at room temperature until the reaction was
complete by TLC (EtOAc: acetone, 1:1), at which time (10 hrs.) the solution had changed color from reddish to dark purple. The solution was concentrated by evaporation with a N2 stream and chromatographed on a preparative silica plate eluted with acetone-ethyl acetate (1:1). The purple band remaining close to the origin was scraped off and eluted with Methanol. Further purification by HPLC (C18 reversed phase, semi-preparative column, methanol: 0.1N phosphate buffer, 1:1) gave a purple powder, NMR (D2O) δ 5.32 (d, 1H, saccharide anomeric H); 3.85 (s, 3H, 9a-OCH3); and the disappearance of singlet at 4.09 (Matsui, M., et al., J. Antibiot. 21:189 (1968); Cheng, L., and Remers, W.A., J. Med. Chem. 20:767 (1977); Vyas, D.M., et al., J. Org. Chem. 51:4307 (1986)).
Example 3: Preparation of N-(2,6-Dihydroxycyclohexyl)qlycinamide
To a solution of N-benzyloxycarbonylglycine (3 g, 14.3 mmol) in dioxane was added N-hydroxysuccinimide (1.65 g, 14.3 mmol) and N,N-dicyclohexylcarbodiimide (2.96 g, 14.3 mmol) with cooling. The reaction mixture was stirred at 0-5ºC for one hour and allowed to stand under refrigeration overnight. The urea precipitate was removed by suction filtration and the filtrate was concentrated in vacuo to dryness. The yellowish residue was recrystallized from ethyl acetateether to give an 84% yield of the glycine activated ester mp. 112-114ºC. NMR (CHCl3).
The above-prepared activated ester of glycine (25 g, 0.008 mol) was dissolved in 15 ml of dry DMF (dimethyl formamide), chilled to below 5ºC, and 2-amino-1,3-cyclohexanediol (2.18 g, 0.016 mol) in DMF was added drop-wise with stirring under N2 atmosphere. After completion of the reaction (TLC, CHCl3:MeOH, 10:1), DMF was removed under reduced pressure and the resulting solid residue was crystallized from ethyl acetate to give white crystals in 84% yield, m.p. 170-172ºC. NMR (D2O): δ 7.45 (s, 5H, aromatic H); 5.20 (s, 2H, benzylic-CH2); 3.95 (s, 2H, -CO-CH2-NH2); 3.6 (t, 1H, C1H of cyclohexane ring); 3.45
(m, 2H, C2H and C6H of cyclohexane ring); 2.0, 1.8 and 1.35 (m,m,m, 2 to 1 to 3H; C3, C4 and C5 hydrogens of cyclohexane). The product comprises the N-protected benzyloxycarbonyl derivative of N-(2,6- dihyroxycyclohexyl)glycinamide.
N-protected benzyloxycarbonyl N-(2,6-dihydroxycyclohexyl) glycinamide (3g, 0.093 mol) was dissolved in 100 ml of absolute ethanol with a molar equivalent of 10% HCl. Hydrogenolysis with 5% Pd/C at 30 psi, removal of the catalyst over celite, and subsequent evaporation of solvents in vacuo yielded a pale brownish solid which was triturated with ether and recrystallized from ethyl acetate and ether, m.p. 207-210ºC. NMR (D2O) δ, 3.65 (t, 3H, C1H of cyclohexane ring); 3.55 (m, 2H, C2H and CeH of cyclohexane); 3.4 (s, 2H, -CO-CH2- NH2); 2.05, 1.80, and 1.38 (m,m,m, 2 to 1 to 3H, hydrogens of C3, C4 and C5 of cyclohexane).
Example 4.
Animal Studies
The compound N7-(2-deoxyglucopyranosyl)mitomycin C, prepared according to Example 2, was evaluated for both murine P388 leukemia antitumor activity and toxicity to bone marrow in normal mice.
A. Determination of murine antitumor activity
The murine P388 leukemia system, maintained intraperitoneally in female DBA/2 mice, was used to evaluate antitumor activity. This tumor was selected because of Its known sensitivity to the parent compound, mitomycin C (Driscoll et al., Cancer Chemotherapy Reports 4:1 (1974)). N7-(2-deoxyglucopyranosyl)mitomycin C was dissolved in sterile water (at 4ºC) immediately prior to administration. Mitomycin C was dissolved in ethanol, and the resultant solution was adjusted to 5% ethanol, 95% sterile water.
Each compound was administered intraperitoneally to groups of CD2F1 male mice on Day 1 after intraperitoneal implantation of 1 × 106 P388 leukemia cells. The P388 antileukemic activity of the test compound was assessed by mean survival days and percentage increased life span (ILS). The % ILS was calculated as follows:
%ILS - (T-C)/C × 100;
where T 1s the mean survival days of the treated Mice and C is the mean survival days of the untreated mice.
P388 antitumor activity for N7-(2-deoxyglucopyranosyl)mitomycin C, in comparison with the parent mitomycin C, is summarized in Table 2:.
Table 2
Antitumor Activity Against P388 Leukemia
Drug Dose (mg/kg) %ILS Mean Survival (days)
N7-(2- 5a 42% 14.2
deoxygl ucopyranosyl)
mitomycin C 13.5a 61% 16.1
Mitomycin C 4.5b 81% 18.1
Controlc 10.0 aLD0 dose
bApproximate LD10 dose
cTreated with drug vehicle
B. Determination of the effects of N7-(2-deoxyglucopyranosyl) - mitomycin C on the hematopoietic svstem in mice
Measurement of peripheral leukocyte (WBC) count was performed using a 20-ul sample of retro-orbital sinus blood obtained from normal CD2F1 male mice on Day 3 following i.p. administration of 13.5 mg/kg of N7-(2-deoxyglucopyranosyl)mitomycin C or 4.5 mg/kg of mitomycin C.
Blood samples obtained were diluted in 9.98 ml of Isoton (a neutral, isotonic buffer solution) and counted in a Coulter counter after lysis with Zapoglobin (an enzyme solution which lyses red blood cells, but not white blood cells). WBC counts are expressed as a percentage of values from control mice receiving drug vehicle only. The results are summarized in the following table:
Table 3
In vivo Murine WBC Depression
WBC Count on Day 3 Drug Dose (as percent of control)
N7-(deoxy13.5 mg/kg 94%
glucopyranosyl)
mitomycin C
Mitomycin C 4.5 mg/kg 56-66%
In summary, these in vivo studies demonstrate that N7-(2-deoxyglucopyranosyl)mitomycin C has significant activity against the murine P388 tumor system, at doses producing no significant bone marrow toxicity, as determined by depression of peripheral leukocyte (WBC) count.
Example 5: Antibacterial Activity
N7-(2-deoxyglucopyranosyl)mitomycin C was evaluated for activity against Gram-negative bacteria, in a comparative study with the parent mitomycin C. Minimum Inhibition concentration (M.I.C.) against a Gram-negative strain of bacteria (HB101) was estimated by the dilution method, with graded concentrations of drug added to agar at 37-40ºC. N7-(2-deoxyglucopyranosyl)mitomycin C was dissolved in 50% sterile water-50% ethanol at 4ºC, and mitomycin C was dissolved in ethanol. The agar, containing drug, quickly solidified at room temperature, and
the bacteria were plated immediately. After 24 hours at 37ºC, the agar plates were observed for inhibition of bacterial growth. The results are summarized in Table 4.
Table 4
M.I.C.
Compound Gram-negative Bacteria
N7-(2-deoxyglucopyranosyl) 1.66-3.3 mcg/ml
mitomycin C
Mitomycin C 0.3 -0.5 mcg/ml
Having now fully described this invention, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof.