NO821220L - NITROSOUR STUFF DERIVATIVES WITH ANTITUMOR ACTIVITY - Google Patents

Description

Title

Nitrosourinous derivatives with antitumor activity.

Related applications

This application is a "continuation-in-part" of

US application 117,940 filed August 14, 1980.

Technical area

The present invention relates to new nitrosourinous substance derivatives and pharmaceutically acceptable salts thereof which are useful due to their antitumor activity and anticoagulant activity. The invention also encompasses pharmaceutical preparations containing these compounds and methods for using them.

State of the art

In the last decade, nitrosourinous substances have gained acceptance as active antitumor agents (T.P. Johnston et al, J. Med. Chem., 14, 600 (1971)). The accepted mode of action appears to involve release of isocyanate in vivo. The compounds most frequently used clinically are 1-cyclohexy1-3-(2-chloroethyl)-3-nitrosourinate (CCNU), 1,3-bis-(2-chloroethyl)-3-nitrosourinate

(BCNU) and MeCCNU '(1-(4-trans-methylcyclohexyl)-3-(2-chloroethyl)-3-nitrosourinous substance which releases in vivo an isocyanate derived from the unitrosated side of the molecule, and an alkylating agent from the other side thereof MeCCNU has been found to have the highest degree of activity against most tumor systems, particularly solid tumor systems.

Numerous investigations are aimed at the metabolic products formed in vivo and in vitro in aqueous media, mainly consisting of 2-chloroethanol, vinyl chloride, acetaldehyde and dichloroethane from the nitrosated side of the molecule. (R.P. Johnston et al,

J. Med. Chem., 18, 634 (1975)). It is also known that the N-nitroso-N-alkyl-ureido part of the molecule alkylates DNA (deoxyribonucleic acid). in vivo and in vitro (Frei et al, Biochem. J., 17 4: 1031 (1978)). It has actually been shown that the carcinogenic effectiveness of such agents as N-methyl-N-nitrosourinous substance coincides with the degree of alkylation of the guanine part in DNA in the target tissue at the oxygen-6 atom.

Alkylation of DNA takes place within 1 hour after administration of the nitrosourin substance, and the half-life for the alkylated products is approx. 24 to 48 hours (D. J. Reed et al, Cancer Res., 35^ : 568 (1975)). The investigation showed that low doses of nitrosourinous substances only represent a small threat as mutagens and are therefore not significantly carcinogenic.

A number of isocyanates have recently been shown to be active inhibitors of transglutaminase (Gross et al, J. Biol. Chem., 250: 7693 (1975)), a calcium-dependent enzyme that can catalyze lysine-glutamine cross-linking in certain proteins which are present on neoplastic cell surfaces. This enzyme (Yancey et al, Ann. N.Y. Acad. Sei., 202: 344 (1972)), and other similar enzymes such as gamma-glutamyl transpeptidase (Novogrodsky et al, Proe. Nati, Acad. Sei. USA, 73 : 2414 (1976); Fiala et' al,

J. Nati. Cancer Inst., 57: 591 (1976); Cameron et al, Cancer Res., .38: 823 (1978)), has been implicated in the unregulated growth of cancer cells and fibrin cross-linking. It is generally recognized that these cross-linked proteins form an extra-cellular coating that makes the cell unrecognizable by the cellular immune system, thereby preventing the normal destruction of the foreign neoplastic tissue. These enzymes are quite specific for glutamine and glutamic acid residues as substrates, and isocyanates similar to these residues have been found to be the most effective inhibitors (Gross et al, J. Biol. Chem., 250: 7693 (1975)) . Other enzymes with similar functions and properties may also be involved, such as other glutamyl cycle enzymes. On this basis, it can be postulated that the more the isocyanate formed by the breakdown of the nitrosourin substance shows specificity towards such enzymes, the lower the required dose, resulting in a reduced risk of carcinogenesis due to the antitumor agent.

The structure of the active site in transglutaminase has been found to contain the pentapeptide sequence —Tyr-Gly-Gln-Cys-Trp— and has the form of a pocket that is 5 x 5 Angstroms in diameter (Folk et al, J. Biol. Chem. , 241: 3238 (1966)). Gamma-glutamyl transpeptidase can be expected to have a somewhat similar structure for the active site.

Toxic side effects such as myelosuppression are

of significant importance in chemotherapy when nitrosourinous substances

are used. Apparently, this effect is not directly related to the alkylating agent which is released in vivo from the nitrosated side of the nitrosourin substance molecule. This is supported by the fact that large doses of the nitrosourinous substances Streptozotocin and Chlorozotocin in relation to other nitrosourinous substances produce a relatively low level of myelosuppression (Schein et al, Cancer, 34: 933 (1974)). Compounds such as CCNU and BCNU exhibit myelosuppression as their most toxic side effects. All four agents release similar alkylating agents in vivo. Streptozotocin and Chlorozotocin have further been shown to liberate the same isocyanate which then undergoes an intramolecular carbamoylation to form a cyclic carbamate (Montgomery et al, Cancer Treat. Rep., 60: 651 (1976)). This intramolecular reaction can therefore be eliminated by direct application of a cyclic carbamate or another carbamate.

Description of the invention

According to the hypothesis on which the invention is based,

a superior inhibitor of transglutaminase and related enzymes will have, based on the above-stated shape and size, hydrophobic moieties directed away from, but in the vicinity of,

the pocket at the active site. The substrate may have groups that promote hydrogen bonding to the tyrosine hydroxyl group at the active site. Other compounds that the present inventor has previously discovered (US Patent Application 68,470) by treating the problem in this manner have proven to be successful antitumor and anticoagulant agents.

According to the present invention, some 1-cycloalkyl-methyl-3-(2-chloroethyl)-3-nitrosourin substances bearing hydroxyl, halogen or other hydrogen-bonding groups in the tertiary position of the cycloalkyl ring are prepared to be investigated as antitumor -agents and anticoagulants. The hypothesis according to the invention is supported by the fact that hydrophobic groups which are bound to the beta or gamma carbon atom in glutamine represent excellent substrates for transglutaminase (Gross et al, J. Biol. Chem., 248: 130 (1973)) and similar enzymes ,

and nitrosourin substances which release isocyanates analogously to these substrates are active antitumor agents (US patent application 68,470). The hydroxyl group bound to tyrosine at the active site repre-

centers an area where hydrogen bonding can occur, and the hydroxylated and halogenated isocyanates that are released from the nitrosourin substances according to the present invention should therefore have a much higher affinity for the active site in transglutaminase and possibly other similar enzymes than alkyl isocyanates that are not substituted. This may explain the effectiveness of such compounds as BCNU and CCNU, since BCNU contains a chlorine group that can form a hydrogen bond with the tyrosine hydroxyl group at the active site, and CCNU and MeCCNU are known to be hydroxylated in the liver to derivatives that can contribute to its binding to the active site in a similar manner. However, this hydroxylation lowers lipid solubility and prevents crossing the blood-brain barrier.

The inhibition mechanism for the isocyanate is via the alkyl thiocarbamate ester formation at the single sulfhydryl group at the active site of the enzyme (Gross et al, J. Biol. Chem., 250: 7693 (1975)):

where R means an alkyl group.

According to the present invention, nitroso-urea derivatives release the following cycloalkyl isocyanates:

where R is a group which can form a hydrogen bond with the tyrosyl group at the active site in the enzyme, preferably a halogen, carboxy or hydroxyl group, or a derivative thereof, and particularly preferably a chlorine or hydroxyl group. The cycloalkyl ring can further be substituted with 1 or more preferably 1 or 2 alkyl groups, preferably methyl groups, or 1 or more preferably 1 or 2 hydroxyl groups.

Later studies have shown that the activity of nitrosourinous substances is markedly increased by means of the 2-chloroethyl or 2-fluoro-ethyl groups present on the nitrosated side of the bond (Montgomery, Cancer Treat, Rep., 60: 651 (1976 ); Johnston et al, J. Med. Chem., 9: 892 (1966); Farmer et al,

J. Med. Chem., 21: 514 (1978)). Since the 2-chloroethyl and 2-fluoroethyl groups also increase solubility, they are the preferred groups according to the invention.

According to the invention, they have new compounds which

possesses the structural criteria necessary to selectively inhibit transglutaminase and similar glutamyl-cycle enzymes, and therefore has potential importance as antitumor agents and as anticoagulants, the following formula:

where n is 4 to 7, hal is chlorine or fluorine, and R is a group capable of forming a hydrogen bond with the tyrosyl group in the active site of the enzyme, preferably a halogen, ;: carboxyl or hydroxyl group, or a derivative thereof,

and more preferably a chlorine or hydroxyl group. The resulting 5- to 8-carbon ring may be substituted with one or more, preferably 1 or 2 lower alkyl groups, preferably methyl groups, to increase selectivity, or certain hydrophilic groups, such as hydroxyl groups, to increase solubility, or other groups which is described extensively in the literature.

Although there is only one carbon atom between the ring and the NH group, the addition of an additional carbon atom should not be considered outside the scope of the present invention, since isocyanates released from these compounds would also conform to the enzyme-inhibiting criteria, since "branching" will actually take place at the gamma carbon atom attached to the isocyanate group.

It is therefore an object of the present invention to provide new compounds which have antitumor activity in humans and other mammals.

It is another object of the invention to provide new compounds which inhibit the activity of transglutaminase and enzymes with similar functions which have similar specifications.

A further object of the invention is to provide new compounds that are effective antitumor agents at low doses, i.e. that they have a lower toxicity than MeCCNU, to minimize harmful mutagenic and/or carcinogenic effects, and to provide compounds with minimal or non-existent myelosuppressive effects.

A further object of the invention is to provide preparations containing the new compounds and methods for using them.

A further object of the invention is to provide new nitrosourin substance derivatives which have anticoagulant effect.

Best embodiment of the invention

Melting points were determined using a Thomas-Hoover capillary melting point apparatus and are uncorrected. Infrared (IR) spectra were obtained using a P.erkin Eimer 397 spectrophotometer, and NMR spectra were taken on a Nicolet 200 MHz instrument using CDCl 3 as solvent and TMS as an internal standard.

Example 1

Neostatin

1- tert- hydroxycyclohexylmethyl- 3-( 2- chloroethyl)- 3- nitrosourin

1-Aminomethyl-1-cyclohexanol hydrochloride (16.6 g, 0.1 mol) was stirred with 13.9 mL (0.1 mol) of triethylamine in 75 mL of anhydrous diethyl ether and cooled to below 5°. 2-Chloroethyl isocyanate (10.5 g, 0.1 mol) was dissolved in 25 mL of ether and added in small portions with stirring while keeping the temperature below 5°. After 2 hours of further stirring, the reaction mixture was filtered, and the residue was washed with two 20 mL portions of ether and

suspended in 20 ml of water, filtered, resuspended in 20 ml of water, filtered again and dried in vacuo over CaCl 2 /KOH. The yield of the urea was 19.5 g (83%) as a white powder, m.p. 109-110°.

An 11.7 g (0.05 mol) amount of the above compound was dissolved in 50 ml of 98% formic acid and cooled to 0°. Sodium nitrite (6.9 g, 0.1 mol) was added portionwise with vigorous stirring while maintaining the temperature below 5°. After stirring for a further hour, 100 ml of water was added. The mixture was extracted with 30 mL of ether, washed with 5% NaHCO 3 , and evaporated to dryness in vacuo to give 13.2 g (82%) of the nitrosourin as an oily solid. IR analysis showed bands at 1710 cm"<1>(C=0) and 1530 cm<-1>(C-N-H). NMR showed a triplet at 4.2 ppm (2H,J=6.5Hz), a triplet at 3.5 ppm (2H, J=6.5 Hz), and a multiplet at 1.6 ppm (12H).The 1-nitroso isomer was also present to the extent of about 30%.

Example 2

(1-chloro-1- cyclohexyl)methyl-3-(2-chloroethyl)-3- nitrosourin

The above compound was prepared using the method of Example 1, replacing the hydroxy starting material with the corresponding chlorine compound. The dividends were equivalent to approx. 33% isomer present.

Other compounds according to the invention, such as methyl- or hydroxyl-substituted cycloalkyl derivatives can be synthesized by the method described in example 1 using a suitably substituted 1-aminomethyl-1-cycloalkanol hydrochloride which can be synthesized by known methods.

Antitumor trial

Antitumor trial data were obtained through

National Cancer Institute, Drug Evaluation Branch, National Institutes of Health, Bethesda, MD. Test data for

MeCCNU was obtained simultaneously for comparison purposes.

L-1210 lymphoid leukemia tumors (10^ cells) were implanted into CDF^ mice according to NIH Protocols. The results given in Table I are single-dose reactions where survival is determined 5 days after i.p. injection of Neostatin (six days after implantation). The compound was injected as a suspension in 10% EtOH, 10% emulphor and 80% saline. Log kill data shows the number of tumor cells that were killed, and survivors after 30 days are designated as "cured". The degree of antitumor activity is expressed as a ratio of treated animals over control animals (T/C) using NCI test assessment values, as specified in individual protocols. Table II contains comparative data for MeCCNU, the most active agent against this tumor system in use at the present time.

Neostatin was found to have an activity that was similar

as good as MeCCNU in the L-1210 system with lower toxicity at higher doses as measured by weight loss. Since L-1210 leukemia responds well to a variety of nitrosourinous substances, the additional, less reactive tumor system (P-1534) was used for further investigation. Using this system, it will be seen that Neostatin is far better than MeCCNU at similar dose amounts.

Corresponding clinical results can be obtained by using the other compounds according to the invention.

The nitrosourin substances according to the invention can be prepared into a form suitable for administration by methods well known in the art. E.g. they may be mixed with pharmaceutically acceptable carriers or diluents such as ethanol, lactose, starch, magnesium stearate, gum tragacanth, gelatin and sodium carboxymethyl cellulose, and the resulting mixture or solution may be prepared by conventional methods into pharmaceutical unit dosage forms such as capsules, tablets, powders, pills , ampoules, suppositories and the like.

The compounds of the invention, such as Neostatin, can be administered orally or parenterally. E.g. the agent can be given intravenously by first dissolving the compound to be administered in 0.5-10 ml of ethanol and adding 50-90% water to this. Further dilution can be made with physiological saline or 5% dextrose (USP), which results in a solution with a slightly acidic pH. Intravenous administration may be continued for a period of up to 2 hours. The nitrosourinous substances can be administered parenterally or orally in doses of approx. 0.5-3 mg/kg.

Other parenteral routes of administration may be employed using any formulation known in the art to permit emulsification, dissolution and suspension of relatively water-insoluble agents or other compounds prior to parenteral administration.

When administered orally in unit dose form, such compounds as Neostatin are active when prepared in a gelatin capsule or tablet in combination with pharmaceutically acceptable binders, fillers or other additives known in the art.

After the invention has been thus described, it will be obvious that it can be varied in many ways. Such variations are not considered to go beyond the scope of the invention, and all such modifications are considered to fall within the scope of the following claims.

Claims (8)

1. Compounds with the formula: and pharmaceutically acceptable salts thereof, wherein Hal is chlorine or fluorine; R is a hydroxy, halogen, carboxylic acid group or derivatives thereof, and n is 4 to 7, where the cycloalkyl group may optionally be substituted with one or more methyl groups or hydroxyl groups. 2. Compound according to claim 1, where n is 4 or 5. 3. Compound according to claim 2, where R is chlorine or a hydroxyl group. 4. Compound according to claim 3 where Hal is Cl. 5. Compound according to claim 1 where n is 5. 6. Compound according to claim 3 where n is 5. 7. Compound according to claim 1 with the formula: 8. Method for the treatment of mammalian and human tumors, comprising administering to a mammal or human.