Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
  • C07C251/32—Oximes
  • C07C251/34—Oximes with oxygen atoms of oxyimino groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C255/00—Carboxylic acid nitriles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
  • C07C259/12—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. N-hydroxyamidines
  • C07C259/14—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. N-hydroxyamidines having carbon atoms of hydroxamidine groups bound to hydrogen atoms or to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
  • C07C259/12—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. N-hydroxyamidines
  • C07C259/18—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. N-hydroxyamidines having carbon atoms of hydroxamidine groups bound to carbon atoms of six-membered aromatic rings

Definitions

• hydroxybenzohydroxamic acids are known to be inhibitors of ribonucleotide reductase according to the Elford, Wampler and Van't Riet group—see Cancer Res., 22, 589 (1979), Abstracts, Proc. Am. Assoc. Cancer Res., 18, 177 (1977), 19, 63 (1978), 20 , 149 (1979), 22, 18 (1981), 23, 202 (1982), Va. J. Sci., 29, 81 (1978), papers, J. Pharm. Sci., 69, 856 (1980), New Approaches to the Design of Antineoplastic Agents-Bardos and Kalman, eds.
• This invention provides compounds of the formula:
• n 2-5, m is 0 or 1
• R' is NOH or NH
• R is NH 2 or NHOH when R' is NOH
• R is NH 2 or O-C 1-3 alkyl when R' is NH
• R" is H or OH
• Preferred compounds according to formula I are those in which at least two of the hydroxyls in the phenyl ring are vicinal.
• An even more preferred group of compounds are those in which the vicinal hydroxyls are at C-3 and C-4 of the phenyl ring.
• Representative compounds according to formula I above include: 2,3-dihydroxybenzohydroxyamidoxime 3 ,4-dihydroxybenzbhydroxyamidoxime methyl 2,3, 4-trihydroxybenzamidate isopropyl 3, 5-dihydroxybenzamidate ethyl 3,4, 5-trihydroxybenzamidate ethyl 3,4-dihydroxybenzamidate
• Benzamidoximes and phenylacetamidoximes according to I above, where R' is NOH and R is NH 2 , can be prepared reacting a nitrile of the formula
• this nitrile may be a transitory intermediate formed by reacting an aldehyde of the formula
• n 2-5 with a mixture of an alkali metal cyanide and hydroxylamine hydrochloride in aqueous solution.
• the mandelonitrile (a cyanohydrin) forms initially but then reacts at once with hydroxylamine to give the desired mandelamidoxime.
• amidates (I where R is O-C 1-3 alkyl and R' is NH) can be prepared by reacting the above nitrile (II) with a lower alkanol (C 1-3 alkylOH) to which has been added an acid such as gaseous HC1.
• the reaction medium here should be non-aqueous, and Lewis acids other than HC1 can be employed.
• Nitriles useful in the above two synthetic procedures are readily available by processes set forth in the art. 2,3,4-Trihydroxybenzonitrile, the three tetrahydroxybenzonitriles and pentahydroxybenzonitrile are, however, new and their synthesis will be set forth in detail below.
• n, m and R' ' have the same significance as before by reaction with hydroxylamine hydrochloride.
• the hydroxy-substituted benzamidines, phenylacetamidines and mandelamidines can be prepared from the corresponding amidate by reaction with ethanolic ammonia.
• the following more detailed examples illustrate the preparation of the compounds of this invention. Other equally useful methods for their preparation will readily suggest themselves to those skilled in the art.
• Ethyl 3,4,5-trihydroxybenzamidate can be prepared by neutralization of the hydrochloride salt, extraction of the ester into ether and removal of the ether by evaporation.
• the suspension was poured into a mixture of ice and 120 ml. of 12N aqueous hydrochloric acid. The volume was raised to about 900 ml. with water. This mixture was then heated. Complete dissolution occurred at about 95 °C at which point, 700 mg. of thioacetamide were added and heating continued until black copper sulfide had formed. The mixture was filtered through carbon.
• the corresponding methyl ester was prepared by heating 3,4 ,5-trimethoxysalicylic acid monohydrate in methanol containing 2% sulfuric acid, as set forth in the above reference.
• 2,3,5,6-tetrahydroxybenzonitrile can be prepared from 2,3,5-trimethoxybenzoic acid (with 2,3,4,5-tetrahydroxybenzonitrile as a contaminant).
• 2,3,4,6-tetrahydroxybenzonitrile can be prepared from 2,3,4-trimethoxybenzoic acid.
• pentahydroxybenzamidate can be prepared from the corresponding pentahydroxybenzonitrile by reaction with ethanol in the presence of anhydrous HC1.
• the compound is converted to the hydrochloride salt and isolated as such according to the procedure of Example 3.
• Compounds represented by formula I above have the ability to inhibit ribonucleotide reductase, an enzyme involved in the reductive conversion of ribonucleotides to deoxyribonucleotides. This enzymatic reaction is a rate controlling step in the biosynthetic pathway leading to DNA and cell replication.
• the ribonucleotide reductase level is closely correlated with cellular replication.
• the compounds of this invention which are potent ribonucleotide reductase inhibitors, are also capable of prolonging the life of mice carrying transplanted tumors since replication of tumor cells is equally inhibited.
• administration of a compound of this invention coming within the scope of formula I above prolongs the life of mice inoculated with L1210 leukemia, a tumor not ordinarily susceptible to chemotherapy.
• the compounds have shown activity against P388 leukemia and B16 melanoma.
• Table 1 gives ribonucleotide reductase data for representative compounds of formula I.
• column 1 gives the substitution pattern in the benzene ring
• column 2 the (CHR'') group
• column 3 the group
• column 4 the ID 50 (inhibitory dose in micromolar concentration which inhibits ribonucleotide reductase by 50%) in ⁇ moles
• ribonucleotide reductase is partially purified from HeLa cells or Ehrlich ascites cells by a procedure similar to that set forth by Elford et al. J. Biol. Chem., 245, 5228 (1970).
• the activity of the enzyme was measured by a slightly modified assay procedure originally developed by Reichard et. al. id, 236, 1150 (1969). This procedure measures the conversion of CDP to dCDP.
• L-1210 leukemia was maintained by weekly passage of 10 L-1210 cells intraperitoneally into DBA/2 mice. Diluted ascitic fluid, 0.1 ml. (10 5 cells), was administered ip to female B6D 2 F 1 mice weighing about 20 g. Drugs were administered ip 24 hours after tumor transplantation and injections were continued daily for a total of eight days. A group of control mice receiving only the injection medium were maintained.
• Table 2 which follows gives the antitumor activity against L-1210 leukemia for certain compounds of this invention. In the table, column 1 gives the name of the compound, column 2 the dose in mg./kg., and column 3 the percent increase in survival time over controls at each dose level.
• the drugs were also used to treat melanotic melanoma B 16 .
• the protocol for testing against this tumor is as follows:
• a tumor homogenate prepared by homogenizing 1 g. of tumor with 10 ml. of cold balanced solution is implanted ip in groups of 10 B 6 C 3 F 1 mice.
• the drug is administered daily for a total of nine days starting one day after tumor inoculation.
• the results are expressed as mean survival time of treated group versus control group (T/C) as a percent. Table 4 which follows gives the results of this test.
• a compound of this invention was also tested on the solid colon tumor model, colon 38.
• the tumor was implanted subcutaneously and the drug injected intraperitoneally twice a day on day two (seven hours apart) and twice on day nine.
• the median tumor weight estimated from tumor diameter is the parameter of tumor inhibition and is measured on day 20.
• the median tumor weight of treated versus control T/C percent. Table 5 which follows gives the results of this test.
• CDF 1 mice were injected ip with 10 cells.
• the drug was administered in the first day after tumor inoculation and continued daily for five treatments.
• the antineoplastic activity of the compounds of this invention is due in part to their ability to scavenge free-radicals.
• the compounds of this invention are cancer protective agents and this utility also may be a manifestation of their free-radical scavenging capability.
• Free-radical scavenging agents may also be useful in detoxifying mammals in whom an excess of free radicals is a cause and/or result of the toxicity.
• Other possible uses of free radical scavengers are to inhibit protaglandin transformation, leucotriene interconversion and lipogenesis, to act as inflammaoty modulators and as food additive or preservatives to prevent lipid oxidation.
• the free radical scavenging ability of the compounds of the invention was determined by measuring the destruction of the stable free radical, diphenylpicrylhydrazyl, in the presence of the test compound in a manner similar to that reported by Venker and Herzmann, Naturwiss, 47, 133-134 (1960). The absorbance at 518 nM of a 100 ⁇ M solution of diphenylpicryhydrazyl free radical in acetone was monitored in a Gilford spectrophotometer. The test compound was added at a final concentration of 25 ⁇ M and the rate of reduction of absorption at 518 nM was observed. Table 7 below gives the free radical scavenging abilities of the compounds of this invention expressed as the initial rate of decrease in optical density units/min.
• the method for the generation of the tyrosine free radical and its destruction by a compound of this invention was accomplished by pulse radiolysis experiments at Brunei University, Oxbridge England on the Brunel 4 MeV linear accelerator using a 1.5 cm optical cell and a 200 ns pulse delivering a dose of approximately 1 krad as measured by thiocyanate dosimetry. All solutions were purged with nitrogen using a syringe-deaerating technique prior to use. The detailed description of the methodology can be found in R.L. Willson, Chemistry and Industry, 183-193 (1977).
• tyrosine radical sodium azide was utilized as an intermediate between the pulse radiolysis generated free electron and tyrosine. Hydroxyurea was used as a reference point for the ability of these compounds to scavange tyrosine free radical since hydroxyurea inhibition of ribonucleotide reductase has been attributed to its ability to scavenge the free radical of the active mammalian ribonucleotide reductase.—See Akerblom et al (loc. cit). See also I.K. Larsen et al., Eur. J. Biochem,
• a rate constant of 1.9 x 10 6 M -1 S -1 was determined for hydroxyurea and 4.5 x 10 8 M -1 S -1 for 3,4-dihydroxybenzamidoxime hydrochloride.
• 3, 4-dihydroxybenzamidoxime hydrochloride was 100+ times a faster scavenger of tyrosine free radical than hydroxyurea.
• Table 8 which follows gives anti-tumor data vs L-1210 and P-388 leukemia for these novel nitriles.
• the compounds of this invention are administered parenterally to mammals suffering from a neoplastic disease, preferably using a water soluble salt of the drug.
• Intravenous injection of an isotonic salt solution of the drug salt is the preferred route of administration.

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• 1983
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