KR100484014B1 - How to treat malignant tumors with thyroxine analogs that do not have hormonal activity - Google Patents

Abstract

The present invention provides a method of treating cancer, particularly malignant tumors, with thyroxine analogs lacking hormonal activity. Thyroxine analogs are administered to diseased mammals in an effective amount capable of inhibiting the growth of malignant tumors and treating cancer. Particularly suitable thyroxine analogs are characterized by their ability to interfere with the initial rate of microtubule protein incorporation by more than 35% in vitro.

Description

How to treat malignant tumors with thyroxine analogs with hormonal activity

Related Applications

This application is a continuation of US application file 08 / 655,267 filed June 4, 1996, which is incorporated by reference.

Technical field

The present invention relates to the field of cancer treatment. More specifically, the present invention utilizes thyroxine analogs that do not have hormonal activity, particularly methyl 3,5-diaido-4- (4'-meth) as a potent, selective, non-toxic anti-tumor agent. Oxyphenoxy) benzoate.

The growth of malignant cancers poses a serious challenge to modern medicine because of their unique properties. These features include uncontrollable cell proliferation, uncontrollable growth of malignant tissues, penetration into local and remote tissues, poor differentiation, no detectable symptoms, and most seriously effective treatment and It includes becoming unable to prevent it.

Cancer can occur in tissues of any organ, regardless of age. Although the cause of cancer has not been clearly identified, it is genetically susceptible, or mechanisms such as chromosomal cutting disease, viruses, environmental factors, and immunological diseases appear to be involved in both growth and modification of malignant tumors.

Currently, chemotherapy to prevent renal tissue formation uses several groups of drugs, including alkylates, purine antagonists, and anti-tumor antibiotics. Alkylating agents alkylate cellular proteins and nucleic acids to prevent cell proliferation, disrupt cell metabolism and eventually kill cells. Common alkylating agents include nitrogen mustard, cyclophosphamide and chlorambutyl. Toxicity in the treatment with alkylates includes an increased risk of developing nausea, vomiting, alopecia, hemorrhagic cystitis, pulmonary fibrosis and acute leukemia.

Purines, pyrimidines and folate antagonists are characteristic of cellular processes and phases, in order to promote anti-tumor effects, such agents require cells to be on the DNA process of replication and replication. Purine antagonists, such as 6-mercaptopurine or 6-thioguanidine, initially interfere with purine synthesis and interconversion of purines. Pyrimidine antagonists such as cytarabine, 5-fluorouracil or flosuridine interfere with deoxycytidylated kinase and DNA polymerase to inhibit DNA synthesis.

Folate antagonists, such as methotrexate, bind firmly to the intracellular enzyme dihydrofolate reductase, which kills cells by preventing them from synthesizing pyrimidine. Toxicity associated with these compounds includes alopecia, myelosuppression, vomiting, nausea, cerebellar ataxia, and the like.

Plant alkaloids such as vancristine, vinblastine, or grapephytotoxin etoposide and teniposide generally interfere with mitosis, DNA synthesis and RNA dependent protein synthesis. The toxicity of these drugs is similar to those described above and includes myopathy, myelosuppression, peripheral neuropathy, vomiting, nausea and hair loss, and the like.

Anti-tumor antibiotics such as doxorubicin, daunorrubicin, and actinomycin act to insert into DNA, interfering with cell proliferation, interfering with the synthesis of DNA-dependent RNA, and inhibiting DNA polymerase Do it. Bleomycin cleaves DNA, and mitomycin acts as a substance that interferes with DNA synthesis by bifunctional alkylation. The toxicity of these antibiotics is quite high, including necrosis, myelosuppression, hypersensitivity, loss of appetite, dose-dependent cardiac toxicity and pulmonary fibrosis.

Other compounds used to treat cancer with chemotherapy include inorganic ions such as cisplatin, biological response modifiers such as interferon, enzymes and hormones. All these materials carry toxic side effects as described above.

Thus, it would be of great benefit to provide a safe and non-toxic chemotherapy composition that can effectively inhibit and inhibit tumor cell proliferation and renal tissue growth. It is also of great benefit to provide safe, effective, non-toxic chemotherapeutic agents that are easy to administer.

Accordingly, it is an object of the present invention to identify safe, nontoxic, orally administrable organic compounds capable of inhibiting malignancies in mammals and to use these compounds for cancer treatment.

Summary of the Invention

The present invention utilizes thyroxine analogs without hormone activity, in particular methyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate ("DIME") to inhibit the growth of malignant tumors, Related to treating. The method is generally administered to mammals with an amount of thyroxine effective to inhibit malignant tumor growth or to treat cancer. In general, thyroxine analogs are characterized by no significant hormonal activity.

In particular, the present invention relates to a method of treating malignant tumors in a mammal, the method comprising administering an amount of thyroxine analog to a mammal having a tumor sufficient to inhibit the growth of the malignant tumor, wherein the thyroxine analog is in vitro Can interfere with the initial coalescence rate of microtubule protein at about 35% or more, preferably 70%, most preferably at least 90%, in vitro do.

In an embodiment of the present invention, the thyroxine analogs that can be used in the methods of the present invention are compounds of Formula 1 and pharmaceutically acceptable salts thereof;

[Formula 1]

X = 0, S, CH ₂ , carboxyl or absent;

Y = 0 or S;

R ₁ = methyl or ethyl;

R ₂ , R ₃ , R ₄ R ₅ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy and halogen;

R ₆ , R ₇ , R ₈ , R ₉ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, ( C ₁ -C ₄ ) alkoxy, halogen, NO ₂ , NH ₂ .

In another embodiment, thyroxine analogs that may be used in the methods of the present invention include the following compounds of Formula 2 and their pharmaceutically acceptable salts;

[Formula 2]

X = 0, S, CH ₂ , carboxyl or absent;

Y = 0 or S;

R ₁ = methyl or ethyl;

R ₂ , R ₃ , R ₄ R ₅ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy and halogen;

R ₆ , R ₇ , R ₈ , R ₉ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, ( C ₁ -C ₄ ) alkoxy, halogen, NO ₂ , NH ₂ .

In an embodiment of the invention, the thyroxine analog is methyl 3,5-diaido-4- (4'-methoxyfe

Xoxy) benzoate ("DIME").

Figure 1 illustrates the effect on cell morphology after culturing E-ras 20 cells with 4μM DIME for 18-24 hours. Panel A is not drug treated (baseline); Panel B is drug treated; Panel C was also drug treated. Panels A and B are 150x magnified; Panel C is 300x magnified.

2 is a graph illustrating the loss of tumorigenic capacity in DIME-treated E-ras cell transformed bovine endothelial cells.

3 is a graph illustrating oral bioavailability of serum half-life (t½) and DIME in rats.

In FIG. 4, 10 ⁵ or 10 ⁶ E-ras 20 cells per inoculation were pretreated with DIME (100 M for 4 days) to show tumorigenic effects on tumor formation.

5 shows the effect of DIME concentration on colony formation rate by MDA-MB-231 cells.

Figure 6 shows induction of DNA cleavage at 0.0 μ M (a), 2.0 μ M (b), 5 μ M (c), 10 μ M (d). E-ras cells (2 × 10 ⁴ cells / cm 2) were treated with DIME for 18 hours prior to TUNEL testing.

Figure 7 shows TLC of DIME (D) and TLC of its carboxylic acid (A) degradation product by A-549 (lung cancer) cell extract (corresponding to 2 × 10 ⁶ cells). In the experiments shown in lines 1 and 2, the esterase activity of the extract occurs during 4 hours of incubation with 1 μM DIME. Line 2 illustrates the esterase interference by 125 μM BNPP. Lines 3 and 4 describe the same experiment as lines 1 and 2 where single cell extracts were prepared from native cells pre-cultured with 1 μM DIME, then washed and extracted. This experiment explains that DIME does not induce esterases.

8 shows that BNPP increases DIME interference in A-59 cell growth.

9 graphically illustrates the effect of DIME on MDA-MB231 cells. The initial inoculation density was 0.05x 10 ⁶ / 2㎠.

10 shows flow cytometry analysis performed on MDA-MB 231 human mammary sarcoma cell nucleus, indicating that the G2 content of DNA accumulated in the nucleus 18 hours after exposure to the drug, suggesting that phase M was blocked. .

FIG. 11 is an image explaining that mitosis is delayed by 13 hours and then becomes 6 progeny cells due to irregular division. Frame 1, 0 hr.:0 min .; Frame 2, 10:55; Frame 3, 24:20; Frame 4, 24: 40; Frame 5, 24:55; Frame 6, 25:10; Frame 7, 26: 35; Frame 8, 28:20.

12 shows the effect of DIME on retention time on M of MDA-MB_231 cells.

Figure 13 shows the quantitative analysis of abnormal cell division induced by DIME.

Figure 14 shows the effect of DIME on the rate of entry into the M phase of MDA-MB-231 cells.

Figure 15 (a, b, c) shows a hybrid of chromosome 19 DNA in DIME-treated MDA-MB-231 cells. FIG. 16A is a reference, FIG. 16B is a DNA stained image, and FIG. 16C shows a hybrid of chromosome 19 treated with 1 μM DIME for 5 days (mid mitosis).

Figure 16 (a, b, c) shows the effect of DIME treatment in the mitotic spindle of MDA-MB-231 cells. Panel A is baseline (no drug); Panel B was treated with 1 μM DIME for 18 hours; Panel C was treated with 1 μM DIME for 5 days.

Figure 17 is an optical test of the microtube copolymer (MTP polymerization). The concentration of GTP is 1 μM (right curve) and the effect of 4 μM DIME is explained in the left curve. Other conditions are the same as described in Table 13.

18 shows the effect of increasing the concentration of DIME in MTP polymerization. The concentration of GTP is 1 mM and the other conditions are the same as shown in Table 13.

FIG. 19 shows the Michaelis-Menten analysis of the effect of 1 μ M DIME on the initial linear rate of MTP polymerization as a function of GTP concentration (0, no drug). Other conditions are the same as those in Table 13.

Definition of terms used here

" Alkyl " refers to a saturated branched, straight chain or cyclic hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, osbutyl, cyclobutyl, tert-butyl, pentyl, hexyl and the like.

" Alkenyl " refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond. The radical refers to the cis or trans shape for the double bond. Common alkenyl groups include ethenyl, propenyl, isopropenyl, cyclopropenyl, butenyl, isobutenyl, cyclobutenyl, tert-butenyl, pentenyl, hexenyl and the like.

" Alkynyl " refers to an unsaturated branched, straight chain or cyclic hydrocarbon having at least one carbon-carbon triple bond. Common alkynyl groups include ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, and the like.

" Alkoxy " is an -OR radical, where R refers to alkyl, alkenyl or alkynyl as described above.

" Halogen " refers to fluorine , chlorine, bromine and iodo substituents.

" Mammal " refers to an animal or a person.

" Pharmaceutically acceptable salts " are biologically effective and have free base properties, inorganic acids such as hydrogen chloride, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. It refers to a salt of a compound obtained by reaction with. Pharmaceutically acceptable salts include alkali metal salts such as sodium, potassium, alkaline tomium and ammonium salts, and the like.

" Pharmacophore " refers to an important tertiary arrangement (or electron density distribution) of molecular parts or fragments that can be recognized by a receptor (Burger's Medicinal Chemistry and Drug Delivry Vol. I. Principles and Practice 619, 5th Edition, John Wiley & Sons, New York).

A " therapeutically effective amount " refers to an amount effective to inhibit the growth of malignant tumors or to reduce symptoms associated with cancer disease.

Description of Specific Embodiments

The present invention relates to a method for treating malignant tumors and cancers in mammals with thyroxine analogs characterized by no significant hormonal activity. Suitably, the present invention is based on the surprising finding that certain thyroxine analogs that do not exhibit hormonal activity serve as potent, selective, non-toxic growth inhibitors in malignant tumor growth. Preferred thyroxine analogs may be DIME as mentioned herein.

Thyroxine is an amino acid of the thyroid gland (Merck Index, 1989, 9348: 1483) and thyroxine analogs are well known in the art. According to the literature, thyroid hormones, especially thyroxine T3 and T4, have two distinct biological functions; One acts on cell metabolism and the other on cell differentiation and development (Jorgensen, 1978, "Thyroid Hormones and Analogues II. Structure-Activity Relationships," In: Hormonal Proteins and Peptides, Vol, VI, pp, 107 -204, CH Li, ed., Academic Press, NY). For example, thyroxine inhibits the thyroid from taking iodine (Money et al, 1959, "The Effect of Various Thyroxine Analogues on Suppression of Iodine-131 Uptake by the Rat Thyroid," Endocrinology 64: 123-125); Tadpoles induce cell differentiation when studied through metamorphosis (Money et al., 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana Pipiens Tadpoles," Endocrinology 63: 20-28). In addition, thyroxine and certain thyroxine analogs inhibit the growth of pituitary gland tumors of non-malignant mice (Kumaoka et al., 1960, "The Effect of Thyroxine Analogues on a Transplantable Mouse Pituitary Tumor," Endocrinology 66: 32- 38; Grinberg et al., 1962, "Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of Various Thyroxine Analogs on Growth and Secretion," Cancer Research 22: 835-841). Essential structures required by thyroxine and thyroxine analogs to induce metabolic stimulation and cell differentiation are not identical (Jorgensen, 1978, "Thyroid Hormones and Analogues II.Structure-Activity Relationships," In: Hormonal Proteins and Peptides, Vol. VI. , p. 150, CH Li, ed., Academic Press, NY). For example, Money et al., Found no correlation between inhibiting thyroid iodine acceptance and tadpole metamorphosis (Money et al., 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on"). the Metamorphosis of Rana Pipiens Tadpoles, "Endocrinology 63: 20-28).

Based on these findings, it was conceived that one could alter or elicit cellular responses not identified by certain thyroxine analogs that do not have the action (metabolic or differentiation) exhibited by thyroxine T3 and T4.

compound

Thyroxine analogs that may be used in the methods of the invention include the following compounds and their pharmaceutically acceptable salts;

X = 0, S, CH ₂ , carboxyl or absent;

Y = 0 or S;

R ₁ = methyl or ethyl;

R ₂ , R ₃ , R ₄ R ₅ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy and halogen;

R ₆ , R ₇ , R ₈ , R ₉ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, ( C ₁ -C ₄ ) alkoxy, halogen, NO ₂ , NH ₂ .

In suitable embodiments, the thyroxine analogs that can be used in the methods of the invention include the following compounds and pharmaceutically acceptable salts thereof;

X = 0, S, CH ₂ , carboxyl or absent;

Y = 0 or S;

R ₁ = methyl or ethyl;

R ₂ , R ₃ , R ₄ R ₅ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, (C ₁ -C ₄ ) alkoxy and halogen;

R ₆ , R ₇ , R ₈ , R ₉ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxy, ( C ₁ -C ₄ ) alkoxy, halogen, NO ₂ , NH ₂ .

In certain embodiments of the invention, the thyroxine analog is methyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate ("DIME").

Thyroxine analogues such as DIME are described in the literature. However, unlike thyroxine, DIME has not been reported to have metabolic or cellular differentiation activity (as observed by tadpole metamorphosis) (Money et al., 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana Pipiens Tadpoles, "Endocrinology 63: 20-28; Stasilli et al., 1959," Antigoitrogenic and Calorigenic Activities of Thyroxine Analogues in Rats, "Endocrinology 64: 62-82). For example, the entry of iodine into rat thyroid has been shown to interfere with DIME by 15% when compared to thyroxine (Money et al., 1959, "The Effect of Various Thyroxine Analogues on Suppression of Iodine-"). 131 Uptake by the Rat Thyroid, "Endocrinology 64: 123-125). In addition, DIME has been shown to have no interfering activity against the growth of non-malignant pituitary adenoma (Kumaoka et al., 1960, "The Effect of Thyroxine Analogues on a Transplantable Mouse Pituitary Tumor," Endocrinology 66: 32-38; Grinberg et. al., 1962, "Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of Various Thyroxine Analogs on Growth and Secretion," Cancer Research 22: 835-841). No malignant cells have been studied.

In particular, certain thyroxine analogs with no hormonal activity, in particular DIME, inhibit the growth of many malignant cells (see Table 3), as well as induce tumor cell apoptosis progressed by micronucleation. I found that. Inhibiting such cellular activity and killing cells is sensitive to structure. Testing with 13 DIME analogs and verbs showed that even with minor changes in the methyl ester and 4'-methoxyoxy substituents, the molecule was completely inactive. DIME was very active in cytology and in vivo, while the 4'-propoxy and ethyl ester verbs were completely inactive. Thus, DIME has significant chemotherapy potential as a structure that defines a specific arrangement of molecules or inhibits certain cellular activities or has lethal activity.

Without being bound by theory, the most likely mode of action of thyroxine analogs described herein is to disrupt cellular processes and induce apoptosis.

Eukaryotic progression through cell division is largely regulated by cyclin-dependent protein kinase activity. The best studied is the transition from G2 to M phase, which is regulated by the cdc2 kinase complexed with cyclin B (Dunphy, 1994, Trends Cell. Biol. 4: 202-207). Phosphorylation is required for cdc2 kinase activity, which is regulated by protein phosphorase 2A (Wera & Hennings, 1995, Biochem. J. 311: 17-29).

In addition, the thyroxine analogs described herein were found to specifically activate protein phosphatase 2A in vitro and in vivo. In vivo, activation of protein phosphatase 2A is consistent with inhibition of cdc2 kinase and dephosphorylation of MAP kinase and topoisomerase II, which inactivates both enzymes. DIME does not metabolize nor interfere with the biosynthetic pathways of DNA, RNA and proteins. Thus, the most likely mode of action is to disrupt cellular processes and induce apoptosis through the dephosphorylation of these key regulatory proteins. Thus, the interfering action of cdc2 kinase at the same time as activating phosphatase 2A is an important and powerful therapeutic target for treating cancer.

Changes in the ester and 4'-position appear to have a significant impact on the effects of DIME, although thyroxine analogs useful in inhibiting malignant tumor growth and treating cancer are not limited to DIME. For example, 4'-ethoxy verbs showed about 25-30% maximal cytotoxic activity in human cancer cells as compared to DIME (Example 4). DIME can also predict that activity can be substituted on aromatic ring positions or oxygen linkages without significant effect.

It is known that the aromatic ring of thyroxine is not coplanar (Jorgensen, 1978, "Thyroid Hormones and Analogues II.Structure-Activity Relationship," In: Hormonal Proteins and Peptides, Vol. VI, pp. 107-204, CH Li, ed., Academic Press, NY). It is also known that ring positions on all of the aromatic rings in thyroxine can be substituted with various substituents, such as alkyl, halogen, nitrogen, and amino groups (ibid) with various hormonal activities. In addition, the oxygen linked to the ring may be absent or may be substituted with other groups or atoms that are not limited to aromatic groups in the same plane, for example with methylene groups, carboxyl groups or sulfur, and with hormonal activity (ibid) It is not affected. Thus, it can be predicted that similar substitutions to DIME will not affect the significant loss of anticancer activity. Importantly, the 2'-goat analogs of DIME have up to 25% inhibitory activity on human cancer cell growth when compared to DIME (Example 5). Because of the close correlation between in vitro and in vivo effects (Examples 2-7), compounds effective in the methods of the present invention can be conveniently identified through in vitro screening tests. Such a test can screen the ability of certain compounds to activate protein phosphatase 2A as described in Examples 2-3. In general, compounds useful in the methods of the present invention are those that increase protein phosphatase 2A activity by about two to three factors when measured according to the tests described in Examples 2 or 3.

Such a test can screen for the ability of certain compounds to interfere with malignant tumor cell growth or to remove malignant cells' tumorigenic capacity in vitro or in vivo as described in Examples 4-6. In general, the active compounds that can be used in the methods of the present invention have a concentration of compound that kills 50% of the cell culture when measured as described in Example 4 (I ₅₀ (compared to the reference culture). ) Is in the range of 0.5 μm to 5.0 μm.

As is known to those skilled in the art, many different malignant tumor cells and cell lines are used to screen for activity, such as HL-60, HT-144, E-ras-20, DU-145, MDA-168, MCF-7, 855- Including but not limited to 2. Of course, it will be appreciated by those skilled in the art that other in vitro and in vivo assays can be used to screen anti-tumor and anti-cancer activity that can be used to identify effective thyroxine analogs useful in the present invention.

The chemical formula mentioned herein may represent automerism or shape isomerism. Although the structural drawings herein show only one of the possible tautomers or shape isomers, it will be appreciated that the present invention includes isomers that exhibit similar biological or pharmacological activity to the DIME described herein.

In addition to the aforementioned compounds and their pharmaceutically acceptable salts, the present invention may utilize dissolved or undissolved forms (hydrated forms) of the compounds.

The compound mentioned above can be prepared by the arbitrary process which can be used for preparing a chemical compound. Appropriate processes are described in representative examples. Essential starting materials can be obtained by standard processes in organic chemistry.

cancer

The thyroxine analogs detailed herein are useful for treating a variety of cancers. Such cancers include carcinomas such as throat, colon, rectum, pancreas, stomach, liver, lungs, breast, skin, prostate, ovary, brain, urethra and bladder cancer; Leukemia, lymphoma; Glioma; Retinoblastoma; Includes but is not limited to sarcoma.

Pharmaceutical Compositions and Routes of Administration

The thyroxine analogs available in the present invention can be administered to a patient on their own or in a pharmaceutical composition in combination with a pharmaceutically acceptable salt or therapeutically effective amount of a pharmaceutically acceptable carrier or excipient. Is an amount that eliminates the symptoms associated with cancer disease or inhibits the growth of malignant tumor cells.

Route of administration

The thyroxine analogs and pharmaceutical compositions described herein can be administered via a variety of routes. Suitable routes of administration include, for example, oral, rectal, transmucosal or visceral administration; Extra-intestinal injections including muscle, subcutaneous, intramedullary; Intravesical, direct intraventricular, venous, peritoneal, nasal or ocular injections.

Alternatively, the compound may be administered locally, rather than systemically, for example by direct injection of the compound into a solid tumor or in a depot or delayed release formulation.

The compounds may also be administered in a targeted drug transport system such as liposomes coated with tumor-specific antibodies. Liposomes target tumors, and only tumors selectively receive liposomes.

In suitable embodiments, the thyroxine analogs and pharmaceutical compositions detailed herein are administered orally.

Composition / Formulation

The pharmaceutical compositions described herein can be prepared in a known manner, for example, by conventional mixing, dissolving, granulating, dragee, powdering, emulsifying, encapsulating, encapsulating or And freeze drying.

Pharmaceutical compositions that can be used in the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers consisting of excipients and auxiliaries having the ability to treat the active compounds in pharmaceutically acceptable formulations. . Proper formulation is dependent upon the route of administration chosen. For injection, the materials of the invention are made in physiologically applicable buffers such as aqueous solutions, suitably Hank's solution, Ringer's solution or physiological salt buffer. For transmucosal administration, penetrants that can penetrate the barrier properly are used in the formulation. Such penetrants are well known in the art.

For oral administration, the compounds can be used in combination with pharmaceutically acceptable carriers known in the art. Such carriers are used to make the compounds into formulations that can be administered to the oral cavity of a patient to be treated, such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Pharmaceutical compositions used for oral administration can be made into tablets or dragees, if desired, after mixing the solid excipients with the compounds, optionally grinding the resulting mixture, treating the particle mixture, and adding the appropriate adjuvant. . Suitable excipients include, in particular, fillers such as sugars, including lactose, sucrose, mannitol, sorbitol; Cellulose materials such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragatan gum, methyl cellulose, hydroxypropylmethyl-cellulose, carboxymethylcellulose sodium or polyvinylpyrrolidone (PVP). If desired, disintegrating agents such as crosslinked polyvinyl pyrrolidone, agar or salts thereof such as alginic acid or sodium alginate can be added.

Properly coat the Party. To this end, concentrated sugar solutions are used, which may optionally include gum arabic, active, polyvinylpyrrolidone, carboful gel, polyethylene glycol, titanium dioxide, lacquer solutions, suitable organic solvents or solvent mixtures, and the like. . A dye or the like may be added to tablets or dragees to distinguish or identify complexes with different amounts of active compound.

Pharmaceutical compositions available orally include push-fit capsules made of gelatin and plasticizers such as glycerol or sorbitol and sealed capsules made of gelatin. Push-fit capsules may include the active ingredient in admixture with a filler such as lactose, a binder such as starch and optionally a mixture such as a stabilizer. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids such as fatty acid oils, liquid paraffin or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration are in a dosage form suitable for the mode of administration.

For buccal administration, the compositions can be made into tablets or lozenges prepared in a conventional manner.

For administration by inhalation, compounds usable in the present invention may be provided by aerosol spray from a pressurized pack or nebulizer, wherein dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, Propellants such as carbon dioxide or other suitable gas may be used together. In the case of a pressurized aerosol, the dosage unit can be determined using a valve to provide a measured amount. Gelatin capsules or medicines for use in an inhaler or pulverizer are prepared to contain a compound powder mixture and a suitable powder such as lactose or starch. The compounds may be prepared for extragranular administration by injection and may be administered, for example, by pill administration or continuous infusion. Injectable formulations may be presented in unit dosage form, eg, in multidose containers, with ampoules or preservatives added. The composition may be provided in suspension, solution, emulsion, or in an oil or water soluble carrier, and may include formulations such as suspensions, stabilizers or dispersants.

Pharmaceutical formulations for extra enteral administration include aqueous solutions of the active compounds in forms that are soluble in water. Suspensions of the active compounds can also be made as appropriate oil injection suspensions. Suitable lipophilic solvents or carriers include fatty acid oils such as sesame oil or synthetic oleic esters such as ethyl oleate or triglycerides and liposomes. Aqueous injection suspensions include substances such as carboxymethylcellulose sodium, sorbitol or dextran to increase the viscosity of the suspension. Optionally, the suspension may contain a solution that increases the solubility of the compound or a suitable stabilizer to produce a very high concentration solution.

Alternatively, the active ingredient may be in powder form that can be reconstituted with a suitable carrier such as sterile pyrogen-free water before use.

The compounds may also be prepared in rectal compositions such as suppositories or retention intestines, including conventional suppository bases such as cocoa butter or other glycerides.

In addition to the compositions described above, the compounds may also be made into depot formulations. Compositions that act for a long time can also be administered as explants (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be made of suitable polymers or hydrophobic materials (eg emulsions in acceptable oils) or ion exchange resins or insoluble derivatives such as insoluble salts.

Suitable pharmaceutical carriers for the hydrophobic compounds of the present invention are cosolvent systems consisting of benzyl alcohol, nonpolar surfactants, organic polymers mixed with water and aqueous phases. The cosolvent system may be a VPD cosolvent system. VPD consists of 3% (w / v) benzyl alcohol, 8% (w / v) nonpolar surfactant polysorbate 80, 65% (w / v) polyethylene glycol 300, which is filled with absolute alcohol. The VDP cosolvent system (VPD: 5W) consists of VDP diluted 1: 1 with 5% (w / v) dextrose / water. Such a cosolvent system dissolves hydrophobic compounds well, and itself is very low in systemic administration. Originally, the proportion of the cosolvent system can vary widely without altering its solubility and toxicity characteristics. In addition, the nature of the cosolvent system is very diverse. For example, other low toxicity nonpolar surfactants may be used in place of polysorbate 80; Aliquots of polyethylene glycol can also vary in size; Other biocompatible polymers may be substituted for polyethylene glycol, for example polyvinyl pyrrolidone and the like; Other sugars or polysaccharides may be used instead of dextrose.

Or other delivery systems for hydrophobic pharmaceutical compounds. Liposomes or emulsions are known as carriers or carriers that carry hydrophobic drugs. Certain organic solvents, such as dimethylsulfoxide, can be used but they are toxic. In addition, the compounds may be delivered using delayed-release systems such as solid hydrophobic polymeric semipermeable matrices containing the therapeutic agent. Various forms of delayed release materials are known and are well known to those skilled in the art. Delayed-release capsules release the compounds for weeks or up to 100 days, depending on their chemical characteristics. Pharmaceutical compositions also consist of a carrier or excipient in a suitable solid or gel state. Examples of such carriers or excipients include, but are not limited to, polymers such as calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polyethylene glycols.

Other formulations suitable for administering the thyroxine analogs detailed above are known in the art and can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition.

Effective dosage

Suitable pharmaceutical compositions for use in the present invention include therapeutically effective amounts of the active ingredient compounds. Determination of the effective amount can be made by those skilled in the art, and in particular, the determination can be made based on the above-described details. In any compound that may be used in the methods of the invention, the therapeutically effective dose may be determined first from cell culture assays. For example, in order to obtain a range of circulatory concentrations, the dose is determined in an animal model, which can be measured in cell culture, either I ₅₀ (which refers to the concentration of test compound required to kill 50% of the cell culture) or I ₁₀₀ (which is Concentration of test compound required to kill 100% of the cell culture). This information can be used to more accurately determine the useful dose in humans. Initial doses can be made by comparing the effects of known anticancer agents such as vincristine with the effects of thyroxine analogs in cell culture assays. In such a method, the initial dose can be obtained by multiplying the ratio of the effective amount obtained in the cell culture of the thyroxine analog by the effective amount of the known anticancer agent by the ratio of the effective amount of the known anticancer agent. For example, if the thyroxine analog is twice as effective in cell culture assays as vincristine (eg, I ₅₀ ^DIME equals ½ times I ₅₀ ^vincristine in the same assay), the initial dose of thyroxine analog is known vincristine. It will be ½ of the dose. Using these initial guidelines, one skilled in the art will be able to determine effective dosages in humans. For example, administration of 250 mg / kg once daily for 5 days a week for 32 days showed significant reduction in the growth of breast cancer xenografts (MDA-MB-231) in nude rats. (Example 7.3). According to the study, DIME has a half-life (t½) in serum of about 2-2.5 hours and can use 87% of the dose administered (Example 7.2). One skilled in the art can optimize the dosages that can be administered to humans based on this data.

Dosages and intervals of administration are adjusted to the individual to maintain plasma levels of the active compound sufficient to maintain the therapeutic effect. The dosage of a typical patient orally administered will be about 50-2000 mg / kg / day, typically 250-1000 mg / kg / day, preferably 500-700 mg / kg / day, most preferably about 350-550mg / kg / day. Preferably, therapeutically effective plasma levels can be obtained by administering multiple doses daily.

In the case of topical administration and selective reception, the effective local concentration of the drug is independent of the plasma concentration. Those skilled in the art will be able to optimize therapeutically effective topical doses without experimentation.

Of course, the dosage of the compound to be administered depends on the patient to be treated, depending on the patient's weight, the severity of the disease, the mode of administration and the judgment of the attending physician.

Chemotherapy can be repeated intermittently while the tumor is detected or while the tumor is not detected. In addition, due to its superficial non-toxicity (discussed below), the treatment may be given alone or in combination with other anticancer agents such as AZT, anti-inflammatory, antibiotics, corticosteroids, vitamins and the like.

Possible co-effects between the thyroxine analogs described above and other drugs can be predicted here. It is also possible to predict possible coordination effects between many thyroxine analogs.

toxicity

Toxicity and therapeutic effects of the thyroxine analogs detailed herein can be determined by standard pharmacological processes in cell cultures or experimental animals such as LD ₅₀ (a dose that kills 50% of the population) and ED ₅₀ ( ₅₀ of the population). To a therapeutically effective dose). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio LD ₅₀ and ED ₅₀ . Compounds with a high therapeutic index are preferred. Data from these cell culture tests and animal tests can be used to determine the nontoxic dose range available to humans. Doses of such compounds are preferably in the range of circulatory concentrations including ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Depending on the patient's condition, the personal physician may select the correct formulation, route of administration, and dosage (Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1).

One of the advantages of using the thyroxine analogs described herein in the treatment of cancer is the lack of toxicity. For example, if a dose of 1 g / kg is administered for 12-15 days, it can be found that there is no sore effect in nude rats (Example 7.1). The iv plasma half-life (t _1/2 ) of DIME is about 2-2,5 hours, with repeated daily administration of the thyroxine analogs described herein, the pain will disappear.

The present invention is described by the following examples, which are intended to illustrate the present invention, but are not intended to limit the present invention.

Example 1: Compound Synthesis

Fourteen thyroxine analogs were synthesized, purified and characterized. The structure and physical data of each synthesized compound are shown in Table 1 below.

1.1 Methyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate (Compound 1)

Methyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate is described in Borrows et al., 1949 "The synthesis of Thyroxine and Related Substances. Part I. The Preparation of Tyrosine and some of its Derivates, and a New Route to Tyroxine "J. Chem. Soc. 1949 (Supp. Issue No. 1). Prepare according to the method described above in S185-S190 and recrystallize from 95% ethanol. Melting Point: 153-l55 ℃

Mass spectrum: FAB, m / z (relative intensity): 510 (M ⁺ , 100), 479 (4.5), 384 (4.5). High resolution data for M + peak: C ₁₅ H ₁₂ I ₂ O ₄ requires 509.882513; Calcd 509.882960 (deviation = -0.9 ppm).

¹ H NMR spectrum in DMSO-d ₆ (δ (ppm) values relative to TMS): 3.719 (3H, singlet), 3.876 (3H, singlet), 6.693 (2H, doublet, J = 9.45Hz, plus fine-splitting) , 6.845 (2H, doublet, H = 9.36 Hz, plus fine-splitting), 8.390 (2H, singlet).

1.2 Methyl 3,5-dinitro-4- (4'-ethoxyphenoxy) benzoate (Compound 2)

In a 50 mL flask at room temperature, 4-ethoxy-phenol (Aldrich) (1492 mg, 10.8 mmoles) is stirred with 2.0 M water soluble KOH (5.50 mL) to make 4-ethoxy-phenolate potassium. Methyl 4-chloro-3,5-dinitrobenzoate (Ullmann, 1909, Annalen der Chemie 366: 92-93; commercial source: Spectrum Chemical Company, Gardena, CA; 2606 mg, 10.0 mmoles) was added and the mixture was heated Reflux for 1 hour, cool in an ice bath, and precipitate rubber components in the product. Cooling Aqueous 1.0 M KOH (20 mL) is added and cooling continuously allows the product to solidify. The yellow-orange solid is crushed, collected in a suction filter, washed with water and dried. The material (3.08 g) was recrystallized in hot 95% ethanol (50 mL) to give 2.56 g (70.6% yield) of methyl 3,5-dinitro-4- (4'-ethoxyphenoxy) benzoate. . Melting point; 101-103 ℃

Mass spectrum (EI): M + in high-resolution: C ₁₆ H ₁₄ N ₂ O ₈ requires 362.075016; Found, 362.074793 (deviation = 0.6 ppm)

1.2.2 Methyl 3,5-diaido-4- (4'-ethoxyphenoxy) benzoate

A portion of methyl 3,5-dinitro-4- (4'-methoxyphenoxy) benzoate (724.4 mg, 2.00 mmoles) was added to 10% palladium in a Parr Model 4561 mini reactor filled with H ₂ (43 psi) atmosphere. Dissolve in glacial acetic acid (50 mL) mixed with a carbon catalyst (Aldrich) (200 mg) and stir rapidly at room temperature until the reaction stops and the pressure drops (6 min, final 16 psi). The mixture is filtered directly through a celite bed to remove the catalyst and the acetic acid solvent is removed on a rotary evaporator to give a brown oil residue which represents an unrefined 3,5-diamine derivative. The crude diamine was dissolved in glacial acetic acid (6.0 mL) and added dropwise for 3 minutes with sodium nitrate solution (345 mg, 5 mmoles) in concentrated sulfuric acid (3.5 mL) cooled with stirred ice for tetrazothia reaction. After stirring for 30 minutes at ice bath temperature, the viscous mixture is pipetted directly into potassium iodine (3.0 g) solution in distilled water (2.5 mL) which is rapidly stirring at room temperature. The dark mixture is stirred for 30 minutes and finally heated to 70 ° C. for 5 minutes. The mixture is taken up in ethyl acetate (100 mL) and water (50 mL) is added. The two phase mixture is transferred to a separate funnel and the product is extracted with ethyl acetate. The organic (ethyl acetate) layer is washed with two additions (50 mL each) and dried over anhydrous sodium sulfate. Subsequent evaporation of the ethyl acetate removes a dark tar residue.

This unpurified product is dissolved in acetone (8 ml) and purified on the prepared TLC plate 5 (Whatman, silica gel, 1000 μm layer, 20 cm × 20 cm, using fluorescent indicators together). The plate is developed with n-hexane: ethyl acetate: acetic acid (3: 1: 0.8 v / v / v). Product bands visible in UV light (R _f = 0.84) were collected from each plate and eluted from silica gel (in a sintered glass funnel) with ethyl acetate (3 x 50 mL). Removal of ethyl acetate yields off-white crystals that crystallize from 95% ethanol (10 mL). Yield; 275 mg of two white crystals (26% based on dinitro precursor 2mmoles). Melting Point: 123-l25 ℃

Mass spectrum: EI, m / z (relative intensity): 524 (M +, 100), 496 (16.7), 310 (9.1), 242 (6.1), 211 (7.6), 155 (6.1). High resolution data for M + peak: C ₁₆ H ₁₄ I ₂ O ₄ calcd., 523.898163; Found, 523.898737 (deviation = -1.1 ppm).

¹ H NMR spectrum in DMSO-d ₆ (δ (ppm) values relative to TMS): 1.303 (3H, triplet, J = 6.94 Hz), 3.877 (3H, singlet), 3.971 (2H, quartet, J = 6.95 Hz) , 6.678 (2H, doublet, J = 8.98 Hz, plus fine-splitting), 6.879 (2H, doublet, J = 9.06 Hz, Plus fine-splitting), 8.398 (2H, singlet).

1.3. Methyl 3,5-diaido-4- (4'-n-propoxyphenoxy) benzoate (Compound 3)

Prepare methyl 3,5-diaido-4- (4'-n-propoxyphenoxy) benzoate (Compound 3) according to Example 1.2. Dinitro precursors were synthesized by treatment of aqueous 4-n-propoxy-phenolate (prepared from commercially available 4-n-propoxy-phenol) with aqueous methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product is reduced to H ₂ / Pd (C) to form a diamine derivative, followed by tetrazo reaction with NaNO ₂ / H ₂ SO ₄ , and potassium iodide to convert to the diido product (Sandmeyer reaction). . Purification is by crystallization with TLC.

1.4. Methyl 3,5-diaido-4- (4'-n-butoxyphenoxy) benzoate (Compound 4)

Methyl 3,5-diaido-4- (4'-n-butoxyphenoxy) benzoate (Compound 4) was prepared as detailed in Example 1.2. Dinitro precursors were synthesized by treatment of aqueous potassium 4-n-butoxy-phenolate (prepared from commercially available 4-n-butoxy-phenol) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product is reduced to H ₂ / Pd (C) to form a diamine derivative, followed by tetrazo reaction with NaNO ₂ / H ₂ SO ₄ , and potassium iodide to convert to the diido product (Sandmeyer reaction). . Purification is by crystallization with TLC.

1.5 ethyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate (compound 5)

Ethyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate (Compound 5) is 3,5-diaido-4- (4'methoxyphenoxy) benzoyl chloride (Borrows et al. , 1949, J. Chem. Soc. 1949: S185-S190). Thus, in a 10 ml flask, 3,5-diaido-4 (4'-methoxyphenoxy) benzoic acid (99.2 mg, 0.200 mmol) was added to 3,5-diaido-4- (4'-methoxyphenoxy To benzoyl chloride. After removing the excess thionyl chloride under vacuum, anhydrous ethanol (5.0 mL) is added with stirring and the mixture is heated to 70 ° C. for 5 minutes. Excess ethanol is removed, the dry residue can be dissolved in hot 95% ethanol (4.0 mL) and the product ester crystallized in a refrigerator (3 ° C.). Yield; 55.8 mg (53%) melting point of pale yellow crystals; 96-98 ℃

Mass spectrum (EI): high resolution data for M + peak: C ₁₆ H ₁₄ I ₂ O ₄ , calculated 523.898163; Found, 523.898202 (deviation = -0.1 ppm)

¹ H NMR spectrum in DMSO-d ₆ (δ (ppm) values relative to TMS): 1.336 (3H, triplet, J = 7.19 Hz), 3.717 (3H, singlet), 4.336 (2H, quartet, J = 7.06Hz) , 6.695 (2H, doublet, J = 9.34 Hz, plus fine-splitting), 6.895 (2H, doublet, J = 9.20, plus fine-splitting), 8.389 (2H, singlet).

1.6 3,5-diaido-4- (4'-methoxyphenoxy) benzoic acid (compound 6)

3,5-Diaido-4- (4'-methoxyphenoxy) benzoic acid (Compound 6) is described in Borrows et al., 1949, J. Chem. Soc. 1949: synthesized according to the method described in S185-S190.

1.7 3,5 diaido-4- (4'-methoxyphenoxy) benzaamide (Compound 7)

3,5-Diado-4- (4'-methoxyphenoxy) benzaamide (Compound 7) is synthesized by amidating Compound 1. In a 125 mL flask, methyl 3,5-diaido-4- (4'-methoxyphenoxy) benzoate (Compound 1) (100 mg, 0.196 mmol) is dissolved in anhydrous methanol (60 mL). After standing for 1 hour in the closed flask, the ammonia gas treatment was repeated (5 minutes), and the mixture was left in the closed flask for 48 hours. Methanol / ammonia is removed by fire before evaporation, and the dry residue is dissolved in methanol: water (7: 3 v / v) (30 mL) and crystals are formed in a refrigerator (3 ° C.). Yield: 58.3 mg (60% yield) of pale yellow crystals, melting point 207-209 ° C.

Mass spectrum (FAB): high resolution data for M + peak: C ₁₄ H ₁₁ I ₂ NO ₃ , calculated 494.882847; Experimental value, 494,881880 (deviation = 2.0 ppm)

¹ H NMR spectrum in DMSO-d ₆ (δ (ppm) values relative to TMS): 3.716 (3H, singlet), 6.682 (2H, doublet, J = 8.93Hz), 6.895 (2H, doublet, J = 8.99Hz) , 7.528 (1H, singlet), 8.113 (1H, singlet), 8.402 (2H, singlet).

1.8 3,5-Diado-4- (4'-methoxyphenoxy) -N-methyl benzamide (Compound 8)

3,5-Diado-4- (4'-methoxyphenoxy) -N-methyl benzamide (Compound 8) is a 3,5-diaido-4- (4'methoxyphenoxy) benzoyl chloride ( See Example 1.5). Hydrochloric acid is reacted with excess methylamine in tetrahydrofuran at room temperature (1 hour), filtered to remove the methylamine-hydrochloride precipitate, the solvent is vaporized, and the product is crystallized from 95% ethanol.

1.9 3,5-Diado-4- (4'-methoxyphenoxy) -N, N-dimethyl benzamide (Compound 9)

3,5-Diado-4- (4'-methoxyphenoxy) -N, N-dimethyl benzamide (Compound 9) is 3,5-diaido-4- (4'methoxyphenoxy) benzoyl Synthesis using chloride (see Example 1.5). Hydrochloric acid is reacted with excess methylamine in tetrahydrofuran at room temperature (1 hour), filtered to remove the methylamine-hydrochloride precipitate, the solvent is vaporized, and the product is crystallized from 95% ethanol.

1.10 Methyl 3,5-diaido-4- (4'-hydroxyphenoxy) benzoate (Compound 10)

Methyl 3,5-diaido-4- (4'-hydroxyphenoxy) benzoate (Compound 10) was prepared as described above in Example 1.2. Dinitro precursors were synthesized by treating aqueous potassium phenolate solution (prepared from commercial phenols) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product is reduced to H ₂ / Pd (C) to form a diamine derivative, followed by tetrazo reaction with NaNO ₂ / H ₂ SO ₄ , and potassium iodide to convert to the diido product (Sandmeyer reaction). ). Purification is by crystallization with TLC.

1.10 Methyl 3,5-diaido-4- (4'-hydroxyphenoxy) benzoate (Compound 10)

Methyl 3,5-diaido-4- (4'-hydroxyphenoxy) benzoate (Compound 10) was prepared as described above in Example 1.2. Dinitro precursors are described in Borrows et al., 1949, "The Synthesis of Thyroxine Related Substances. Part II. The Preparation of Dinitrodiphenyl Ethers" J. Chem. Soc. 1949 (Supp. Issue No. 1): Hydroquinone in pyridine solution can be prepared by reacting 4-chloro-3,5-dinitrobenzoate according to the method described in S190-S199.

1.11 Methyl 3,5-diaido-4-phenoxybenzoate (Compound 11)

Methyl 3,5-diaido-4- wasteoxybenzoate (Compound 11) was prepared as described above in Example 1.2. Dinitro precursors were synthesized by treating aqueous potassium phenolate solution (prepared from commercial phenols) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product is reduced to H ₂ / Pd (C) to form a diamine derivative, followed by tetrazo reaction with NaNO ₂ / H ₂ SO ₄ , and potassium iodide to convert to the diido product (Sandmeyer reaction). ). Purification is by crystallization with TLC.

1.12 Methyl 3,5-diaido-4- (4'-idodophenoxy) benzoate (Compound 12)

Methyl 3,5-diaido-4- (4'-idenophenoxy) benzoate (Compound 12) was prepared as described above in Example 1.2. Since the iodo substituent on the dinitro precursor is itself unstable to H ₂ / Pd (C), the iodine-dinitro precursor is reduced to iodo-diamine, which is iron in acetic acid / 95% ethanol. Gemmill et al., 1956, "3-Iodo-, 3,3'-Diiodo- and 3,3'-Diiodo-5-bromothyroxine," J. Am. Chem. Soc. 78: 2434-2436 ). Iodine diamine is tetra-harmonized, which is converted to triiodine product using the Sandmeyer reaction. After purification by TLC, the product can be crystallized from ethanol (mp 139 ° C to 141 ° C).

Mass spectrum (EI). High resolution analytical data for M ⁺ peak: C ₁₄ H ₉ O ₃ I ₃ , calculated 605.768600; Theoretical, 605.767839 (deviation = 1.3 ppm)

¹ H NMR spectrum in DMSO-d ₆ (δ (ppm) values relative to TMS): 3.879 (3H, singlet), 6.628 (2H, doublet, J = 8.97Hz plus fine-splitting), 7.670 (2H, doublet, J = 9.12 Hz plus fine-splitting), 8.396 (2H, singlet).

1.13 Methyl 3,5-diaido-4- (3'-methoxyphenoxy) benzoate (Compound 13)

Methyl 3,5-diaido-4- (3'-methoxyphenoxy) benzoate (Compound 13) was synthesized according to the method of Example 1.2. Dinitro precursors were synthesized by treating 3-methoxy phenolate potassium aqueous solution (prepared from commercial 3-methoxyphenol) with methyl 4-chloro-3,5-dinitrobenzoate. The dinitro product is reduced to H ₂ / Pd (C) to form a diamine derivative, followed by tetrazo reaction with NaNO ₂ / H ₂ SO ₄ , and potassium iodide to convert to the diido product (Sandmeyer reaction). ). Purification is by crystallization with TLC.

1.14 Methyl 3,5-diaido-4- (2'-chloro-4'-methoxyphenoxy) benzoate (Compound 14)

Methyl 3,5-diaido-4- (2'-chloro-4'-methoxyphenoxy) benzoate (Compound 14) can be synthesized by the general method described in Example 1.2, except that dinitro It is carried out by another method for the recirculation of the precursor.

1.14.1 Methyl 3,5-dinitro-4- (2'-chloro-4'-methoxyphenoxy) benzoate

The dinitro precursors were prepared with 2-chloro-4-methoxyphenol with methyl 4-chloro-3,5-dinitrobenzoate and 2-chloro-4-methoxyphenolate potassium as detailed in Example 1.2.1. Aldrich Chemical Co., Milwaukee, WI) is prepared by the reaction. Methyl 3,5-dinitro-4- (2'-chloro-4'-methoxyphenoxy) benzoate product (66% yield) from ethanol is crystallized to give orange crystals. Melting Point: 116-119 ℃

Mass spectrum (EI): M ⁺ in high resolution: C ₁₅ H ₁₁ ClN ₂ O ₆ , calculated 382.020393; Found, 382.020187 (deviation = 0.5 ppm)

1.14.2 Methyl 3,5-diaido-4- (2'-chloro-4'-methoxyphenoxy) benzoate

Since the 2'-chloro substituent in the dinitro precursor is unstable for reduction by H ₂ / Pd (C), the precursor was converted from acetic acid / 95% ethanol to iron powder and 2'-chloro diamine, similar to Example 1.12. Reduce. Thus, in a 250 ml flask, 3,5-dinitro-4- (2'-chloro-4-methoxyphenoxy) benzoate (765.5 mg, 2.00 mmol) was dissolved in glacial acetic acid (35 ml) and 95% ethanol (35 ML), the solution is heated to 70 ° C. and iron powder (2.00 g) is added. The mixture is vigorously stirred in a heating bath (70 ° C.). After 3 minutes, the mixture is brownish. Stirring is continued at 70 ° C. for 35 minutes. The mixture is then transferred to a separate funnel, water (250 mL) and ethyl acetate (250 mL) are added, the product is extracted with an ethyl acetate layer and the ethyl acetate phase is separated from the aqueous phase (3 hours). The extract is dried over anhydrous Na ₂ S0 ₄ , filtered and ethyl acetate is removed using a rotary evaporator to yield the 3,5-diamino product which solidifies.

The resulting diamino product was directly dissolved in biacetic acid (6.0 mL) as described in Example 1.2, subjected to tetrazo reaction via Sandmeyer reaction, and methyl 3,5-diaido-4- (2'-chloro -4'-methoxyphenoxy) benzoate. Purification by TLC (R _f = 0.70) as in Example 1.2 and the product crystallized from 95% ethanol (250.8 mg off-white crystals, 23% yield). Melting point; 132-134 ℃

Mass spectrum: EI, m / z (relative intensity): 546 (34), 545 (16), 544 (M ⁺ , 100), 418 (6), 382 (6). High resolution analytical data for M ⁺ peak: C ₁₅ H ₁₁ ClI ₂ O ₄ , calculated 543,843541; Found, 543.843424 (deviation = 0.2 ppm)

¹ H NMR spectrum in DMSO-D ₆ (δ (ppm) values relative to TMS): 3.747 (3H, singlet), 3.881 (3H, singlet), 6.328 (H, doublet, J = 8.97 Hz), 6.780 (1H, doublet of doublets, J = 9.10Hz and J = 2.95Hz), 7.195 (1H, doublet, J = 3.02Hz), 8.400 (2H, singlet)

1.15 Other Compounds

The additional thyroxine analogs detailed herein can be synthesized from the appropriate starting materials using the synthesis described above, which will be apparent to those skilled in organic chemistry. For further guidance, see Borrows et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine," J. Chem. Soc. 1949 (Supp. Issue No. 1): S185-S190; Borrows et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part II Preparation of Dinitrophenyl Ethers," J. Chem. Soc. 1949 (Supp. Issue No. 1): S190-S199; Clayton et al., 1951, "The Synthesis of Thyroxine and Related Substances. Part VIII. The Preparation of Some Halogeno- and Nitro-diphenyl Ethers," J. Chem. Soc. 1951: 2467-2473; Gemmill et al., 1956, "3-Iodo-, 3,3'-Diiodo- and 3,3'-Diiodo-5-bromothyroxine," J. Am. Chem. Soc. 78: 2434-2436; Meltzer et al., 1957, "Thyroxine Analogs," J. Org. Chem. 22: 1577-1581; Crowder et al., 1958, "Bisbenzylisoquinolines. Part II. The Synthesis of 5- (2-Aminoethyl) -4'-carboxy-2,3-dimethoxydiphenyl Ether," J. Chem. Soc. 1958: 2142-2149; Jorgensen, 1978, "Thyroid Hormones and Analogues, I. Synthesis, Physical Properties and Theoretical Calculations" In: Hormonal Proteins and Peptides Vol. VI, pp. 57-105, C. H. Li, Ed., Academic Press, NY (and references cited therein), and Jorgensen, 1978, "Thyroid Hormones and Anslogues, II.Structure-Activity Relationships," In: Hormonal Proteins and Peptides, Vol. VI, pp. 107-204, C. H. Li, Ed., Academic Press, NY (and references cited therein).

Example 2 Activity of Purified Protein Phosphorase 2A

Compounds 1 and 3 (identified in Table 1) were tested for protein phosphatase 2A activity.

2.1 ³² Preparing P-labeled Histone H1 Substrate

Histone H1 is phosphorylated with protein kinase C (Upstate Biotechnology, Inc., Lake Placid, NY) at 100 μl according to standard protocols. Alternatively, histone H1 is phosphorylated with p34cdc2 kinase purified from Mytilus Edulis embryos rapidly increased in stages 4-8 after separation by P ¹³ -Suc agarose technology (Smythe and Newport, 1992, "Coupling of Mitosis to the Completiom of S Phase in Xenopus Occurs via Modulation of the Tyrosine Kinase that Phosphorylates p34cdc2," Cell 68: 787-797).

2.2 Phosphorase Test

125 ng purified protein phosphatase 2A (Upstate Biotechnology, Inc., Lake Placid, NY; Usui et al., 1983, J. Biol. Chem, 258: 10455-10463) was buffered (20 mM MOPS or Tris, pH 7.5, 1 mM MgCl ₂ , 60 μΜ β-mercaptoethanol), pre-incubated for 10 minutes at 23 ° C. with thyroxine analogs (50 μΜ). The total reaction volume is 20 μl. ³² P-labeled histone H1 substrate (10 μg, 10 ⁵ cpm) is added and the dephosphorylation is allowed to proceed at 23 ° C. for 5 minutes and then the reaction is terminated by addition of 24 μl Laemmli buffer. The same reaction involving 125 ng untreated protein phosphatase 2A was conducted as a reference.

Phosphorylated and diphosphorylated histone H1 was separated by gel electrophoresis (12% SDS-PAGE), and the band containing phosphorylated histone H1 was cut out and examined for ³² P activity through a scintillation counter. .

2.3 Results

The 5 minute dephosphorylation reaction rate represents the initial rate of dephosphorylation reaction (V _init ). V _init for compounds 1 and 3 are shown in Table 2 below.

From these results, it can be seen that preincubation of protein phosphatase 2A with DIME (Compound 1) for a short time (10 minutes) makes V _init of histone H1 dephosphorylation reaction more than twice. The 4′-propoxy verb did not activate protein phosphatase 2A. These data show that even minor changes at the ends of the DIME Parmacopores structures (such as methoxy and methyl ester groups) have a significant effect on activity. These data correlate significantly with the protein phosphatase 2A activity observed in the in vitro malignant cell assay (Example 3) and closely related to the in vivo anti-tumor effect as observed in mice (Examples 4 and 6). Indicates a correlation.

Example 3 Activation of Protein Phosphorase 2A in Tumor Cell Cultures

According to the protocol (Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a Rac-Transformed Bovine Endothelial Cell Line by Treatment with 5-Iodo-6-Amino-1,2-Benzopyrone (INH) ₂ BP), “International J. of Oncology 8: 239-252), compound 1-13 was tested for protein phosphatase 2A activity in malignant tumor cells in vitro. The results for activation by DIME (Compound 1) are shown in Table 3 below. All other compounds were ineffective.

In these results, 50 μM DIME activated protein phosphatase 2A at least twice as much in both E-ras transformed bovine endothelial cells and DU-145 cells.

Example 4 Lethal Activity in Human Cancer Cells

In vitro mortality of compounds 1-13 was tested in 7 human cancer cell lines. DIME (Compound 1) is the most active and ethoxy derivative (Compound 2) has 25-30% maximum activity.

4.1 Experimental Protocol

Seven human cancer cell lines are obtained from the American Tissue Culture Collection (Rockille, MD) and maintained in the recommended growth medium. Cells are seeded in wells (2 cm 2) with 2 × 10 ⁴ cells / cm 2. Varying concentrations of compounds 1-13 participate in the medium at inoculation time.

Cultures are incubated at 37 ° C. (5% CO ₂ atmosphere) for 72 hours. After incubation, the cells are separated with trypsin and the number is measured using a yeast cytometer.

4.2 Results

DIME (Compound 1) has the highest activity, and ethoxy analog (Compound 2) has 25-30% maximum activity. All other analogs tested were not active at all.

Experimental results for DIME (Compound 1) are shown in Table 4 below. I ₁₀₀ represents the concentration when no viable remaining cells are present; I ₅₀ represents the concentration when 50% live cells remain when compared to baseline.

Example 5 Interruption of Tumor Cell Growth

Compounds 1 and 14 were tested for growth inhibition of MDA-MD-231 cancer cells. Compound 14 showed about 25% interference activity when compared to DIME (Compound 1).

5.1 Experimental Process

MDA-MD-231 human cancer cells were obtained from the American Tissue Culture Collection (Rockville, MD) and maintained in the recommended growth medium. Cells are grown at 37 ° C. for 3 days in the presence of various concentrations of compounds 1 and 14.

5.2 Results

The experimental results are shown in Table 5 below.

DIME 2'chloro analog (Compound 14) exhibited about 25% inhibitory activity when compared to DIME (Compound 1).

Example 6 Loss of Tumor Formability of E-ras Transformed Bovine Endothelial Cells

Morphological action of DIME (Compound 1) was tested in E-ras modified bovine endothelial cells with very high tumorigenesis.

6.1 Experimental Protocol

E-ras transformed bovine endothelial cells (Bauer et al, 1996, Intl. J. Oncology 8: 239-252) are exposed to 102 M DIME for 3 days. DIME-treated cells (10 ⁵ or 10 ⁶ cells / 100 μl) were injected subcutaneously in nude mice and tumor progression was observed for 25 days.

6.2 Results

As can be seen in FIG. 1, exposure of E-ras transformed endothelial cells to 10 μM DIME for 3 days induces significant micronucleation, consistent with the loss of tumorigenic capacity. As illustrated in FIG. 2, animals exposed to untreated cells showed tumor growth (stomach curve) and died by tumor around 25 days. Cells exposed to 10 μM DIME prior to injection almost completely lost tumorigenicity (below curve). Tumors did not appear after 3 months without in vivo drug treatment.

Example 7: in vivo experiment

The following examples illustrate the in vivo effects and non-toxicity, bioavailability, serum half-life (t _1/2 ) of DIME in treating human breast cancer grafts in mice.

7.1 Toxicity

Ten nude rats were administered orally (1.0 g / kg, 0.1 ml corn oil) with ¹⁴ C-labeled DIME (Compound 1) daily. There was no adverse effect in rats during the entire treatment period.

7.2 Serum Half-Life (t _1/2 ) And bioavailability

Rats are orally administered 126 mg / kg ¹⁴ C-labeled DIME (Compound 1). After administration, the blood sampling frequency is 15 minutes, 30 minutes, 1, 2, 4, 6, 8, 24 hours. A certain amount of blood (50 μl) was examined using a liquid scintillation counter and the data is expressed in μg per ml. Blood level data were analyzed by the RSTRIP method (Micromath, Salt Lake City, UT).

A similar rat population was administered intravenously with 24.5 mg / kg ¹⁴ C-labeled DIME and blood sampling times were 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 8 hours.

7.2.1 Results

3 illustrates blood serum levels of ¹⁴ C-labeled DIME (mg-eq./ml). The area under the blood concentration-time curve is 665.28 μg-hr. / Ml for oral administration and 156 μg-hr / ml for intravenous administration (shown in squares). Bioavailability of orally administered DIME can be seen to be about 83% calculated from these data using standard ratio × dose method. The DIME half life (t _1/2 ) is about 2-2.5.

7.3. in vivo effect

The ability of human tumors to grow as xenografts in thymus-free mice (such as nude mice) in studying biological responsiveness to human tumor therapy can be a useful model in vivo. Because of the first successful xenograft of human tumors in nude mice (Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scnad. 77: 758-760), many different human tumor cell lines (eg, breast, lung, genitourinary, Stomach, head and neck, glioblastoma, bone and malignant melanoma) were successfully implanted and grown in nude mice. Human breast tumor cell lines including MCF-7, ZR75-1, MDA-MB-231 were made with subcutaneous grafts in nude mice (Warri et al., 1991, Intl. J. Cancer 49: 616-23; Ozzello & Sordat) , 1980, "Behaviour of Tumors Produced by Transplantation of Human Mammary Cell Lines in Athymic Nude Mice," Eur. J. Cancer 16: 553-559; Osbourne et al., 1985, Cancer Res. 45: 584-590: Siebert et al., 1983, Cancer Res. 43: 2223-2239).

These experiments explain the inhibition of MDA-MB-231 xenografts in nude mice.

7.3.1 Experimental Protocol

MDA-MD-231 (human breast cancer) is obtained from the American Type Culture Collection (Rockville, MD) and maintained in the recommended growth medium. Each 20 nude mice were inoculated subcutaneously with MDA-MB-231 (10 ⁶ cells / 100 μl). DIME is administered to a group of 10 animals in the gastrointestinal tract (250 mg / kg, 10 ml / kg corn oil) once a day, 5 days a week, for a total of 32 days. Another group of 10 rats (baseline) are administered as carriers twice a week following the same dosage procedure. Using Vernier caliper, tumors are measured twice a week and the average tumor volume is determined at each time point. Using the unpair two-tailed t-test, the results were compared between groups and analyzed by the variable analysis.

7.3.2 Results

The mean tumor mass at 14, 21, 28 and 32 days after inoculation for the treated and untreated mice is shown in Table 5.

These data were effective in significantly reducing malignant tumor growth even under unoptimized DIME treatment.

7.4 In vivo effect

The other thyroxine analogs detailed herein were tested as described above. According to these tests, the analog is expected to have activity.

Example 8 Formulation

The following examples illustrate, but are not limited to, formulations for administering thyroxine analogs of the invention to mammals, particularly human patients. Although methods for formulating DIME have been described, it will be appreciated that any of the thyroxine analogs presented herein may be formulated as provided in the following examples.

8.1 Tablet Formulation

Each formulation containing 60 mg of active ingredient is made as follows;

DIME 60mg

Starch 45mg

Microcrystalline Cellulose 45mg

Carboxymethyl Starch Sodium 4.5mg

Talc 1mg

4 mg polyvinylpyrrolidone (10% in water)

Magnesium Stearate 0.5mg

                                         150 mg

The active ingredients starch and cellulose are passed through 45 mesh (U.S. sieve) and mixed thoroughly. The polyvinylpyrrolidone solution mixes with the resulting powder, which passes through 14 mesh (U.S. sieve). The particles are dried at 50-60 ° C. and passed through 18 mesh (U.S. sieve). Carboxymethyl starch sodium, magnesium stearate, talc are previously passed through 60 mesh (U, S. sieve, added to the particles, and the mixture is compressed with a tablet machine to make tablets of 150 mg each.

A tablet is prepared from the ingredients shown in Table 1 by wet granulation by compression.

8.2 Gelatin Capsules

Hard gelatin capsules are prepared using the following ingredients;

DIME 250mg / capsule

Dry starch 200mg / capsule

Magnesium Stearate 10mg / capsule

The ingredients are mixed and filled into 460 mg hard gelatin capsules.

8.3 Aerosol Solutions

The aerosol solution is prepared to contain the following components;

DIME 0.25% (w / w)

Ethanol 29.75% (w / w)

Propellant 22 (chlorodifluoromethane) 77.00% (w / w)

The active compound is mixed with ethanol, the mixture is added to a portion of the propellant 22, cooled to -30 ° C and transferred to the filling device. The required amount is supplied to a stainless steel container and diluted with the remaining propellant. The valve unit is fixed to the container.

8.4 suppositories

Suppositories each containing 225 mg of the active ingredient are prepared as follows;

DIME 225 mg

2,000mg saturated fatty acid glyceride

The active ingredient is passed through Mesh 60 (U.S. Sieve) and suspended in molten saturated fatty acid glycerides using the minimum heat required. The mixture is then placed in a 2 gram weak mold and allowed to cool.

8.5 Suspension

A suspension containing 50 mg of drug per 5 ml dose is made as follows;

DIME 50 mg

Carboxymethyl Cellulose Sodium 50mg

Syrup 1.25ml

0.10 ml benzoic acid solution

Spices q.v.

Color developer q.v.

Adjust to 5 ml with purified water

The active ingredient is passed through 45 mesh (U.S. sieve) and mixed with carboxymethyl cellulose sodium and syrup to make a soft dough. The benzoic acid solution, fragrance and some color developer are diluted with some water, added and stirred. Then add enough water to get the desired volume.

Example 9

Substitution of the H atom at position 5 of 6-amino1,2-benzofurene with iodine significantly enhances the parent compound's pADPRT inhibitory, anti-HIV and anti-tumor activity (Cole, et al. 1991). "Inhibition of HIV-1 IIIb Replication in AA-2 Cells in Culture by Two Ligands of Poly (ADP-Ribose) Polymerase: 6-Amino-1, 2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone `` Biochem Biophys. Res. Commun. 180: 504-514; Bauer et al., 1996, "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-Transformed Bovine Endothelial Cell Line by Treatment with 5-iodo-6 -amino-1,2-benzopyrone (INH ₂ BP) "Intl. J. Oncol. 8: 239-252). Increasing the number of iodine substituents on a particular aromatic molecule raises the question whether the pharmacological properties of these molecules can be altered. To address this question, we decided to analyze the cellular action of known diaido-compounds, such as thyroid hormone analogs.

It has been found that the metabolic or metabolic effects of thyroid hormone analogs depend on their chemical structure (Jorgensen, E. 1978, "Thyroid Hormones and Analogs. II.Structure-Activity Relationships: In Hormonal Protein and Peptides" Vol VI, Li CH (ed.) Academic Press, New York, pp. 108-203). Hormonally inactive methyl-3,5-diaido-4- (4'-methoxyphenoxy) benzoate (DIME) was first synthesized in 1949 (Borrows, et al. 1949, "The Synthesis of thyroxine and Related Substances.Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine ", J. Chem. Soc Suppl Issue No. 1: S185-S190), (Money et al. 1958, "The Effect of Change in Chemical Structure of Some Thyroxine Analogues on the Metamorphosis of Rana pipiens Tadpoles", Endocrinology 63: 20-28; Stasili et al., 1959, "Antigoitrogenic and Calorigenic Activities of Thyroxine Analogues in Rats ", Endocrinology 64: 62-82; Money et al., 1959," The Effect of Various Thyroxine Analogues on Suppression of ¹³¹ I Uptake by the Rat Thyroid ", Endocrinology 64: 123-125; Kumaoka et al., 1960, "The Effect of Thyroxine Analogues on a Transplantable Mouse Pituitary Tumor", Endocrinology 66: 32-38; Gr inberg et al., 1962, "Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of Various Thyroxine Analogs on Growth and Secretion", Cancer Res. 22: 835-841). Initial work by the inventors has shown that DIME can be a potent tumor killing agent in cell culture and in vivo (Kun et al., 1996, "Induction of Thmor Apotosis by methyl-3,5-diioao-4-). (4'-methoxyphenoxy) benzoate (DIME) ", Abstract No. 102, Int. J. Oncol. 9: supplement 829; Zhen, et al., 1997," Induction of Metaphase Block and Endoreduplication in Human Cancer Cells by 3, 5-diiodo-4 (4'-methoxyphenoxy) benzoate (DIME) .Abs., Abstract, Amer.Assoc.Cancer Res, Symposium on Cell Signaling and Cancer Synthesis, and DIME and Structural Tests of Verb and Analogs Difference Only in Side Chain Substitutions And US patent application 08 / 655,267 filed June 4, 1996 (named "Method of Treating Maligant Tumors with Thyroxine Analogs having No Significant Hormonal Activity"), which is characteristic of tumor lethal activity of DIME. The structure is shown.

The following work compares the structure and action of DIME and its 17 analogs and describes the tumor lethal activity of DIME itself at the cellular level. Drug metabolism and cell acceptance testing using DIME explain why it is not toxic in animals in vivo. Cell analysis and biochemical mechanisms of the way DIME works are the objectives of the next study.

Substituted phenols for the synthesis of compounds 1-4 and 10-18 in Table 1 were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). Methyl-4-chloro-3,5-dinitrobenzoate (Ullmann F, 1909, "die 4-chlor-3,5-dinitrobenzoesaure" Annalen der Chemie 36: 92-93) is 4-chloro-3,5- Prepared from dinitrobenzoic acid (Aldrich).

9.1 General Synthesis

Each substituted phenol (potassium potassium) reacts with methyl-4-chloro-3,5-dinitrobenzoate to give methyl-3,5-dinitro-4- (substituted phenoxy) benzoate And then reduced to the corresponding 3,5-diamine and converted to the targeted 3,5-diaido compound by Sandmeyer reaction (Kun et al., 1996, "Induction of Tumor Apotosis by methyl-3 , 5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME) ", Abstract No. 102, Int. J. Oncol. 9: supplement 829). In general, reduction is carried out by a hydration reaction with a catalyst. When R ₁ or R ₃ is a halogen atom (compounds 12 and 14), reduction is performed with iron powder in acetic acid / ethanol to prevent halogen from being removed ( Kun et al., 1996, "Induction of Tumor Apotosis by Methyl-3,5-Diiodo-4- (4'-Methoxyphenoxy) Benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: supplement 829 ). Purification generally consists of TLC chromatography and crystallization. In the case of compounds in which R ₂ is other than methoxy in Table 7 (compound 5-9), an additional synthetic reaction is used. Compound 6 is prepared by base hydrolysis of Compound 1 (Borrows, et al. 1949, "The Synthesis of Thyroxine and Related Substances, Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine" , J. Chem. Soc. Suppl.Issue No. 1: S185-S190). Compounds 5, 8, and 9 are hydrochloric acid of 6 (Borrows, et al., 1949, "The Synthesis of Thyroxine and Related Substances. Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine", J Chem. Soc. Suppl. Issue No. 1: S185-S190) and prepared by reaction with anhydrous ethanol, methylamine and dimethylamine, respectively (Kun et al., 1996, "Induction of Tumor Apotosis by methyl-3, 5-diiodo-4- (4'-methoxyphenoxy) Benzoate (DIME) ", Abstract No. 102, Int. J. Oncol. 9: supplement 829. Compound 7 is prepared by reaction of Compound 1 with ammonia in anhydrous methanol. All compounds were characterized by melting point and high resolution mass spectrum except for the known carboxylic acid 6. The ¹ H NMR spectrum was measured at compound 1-14 and showed satisfactory results in all cases.

9.2 Synthesis of Compound 1

Compound 1 (DIME) is described in Borrows, et al., 1949, "The Synthesis of thyroxine and Related Substances. Part I. The Preparation of Tyrosine and Some of its Derivatives, and a New Route to Thyroxine", J. Chem. Soc. Suppl. Issue No. 1: S185-S190, mp 153-155 ° C. was synthesized as described above. Basic spectral measurements that have not been reported previously for this compound are as follows; UV absorption spectrum in ethanol; τ max (ε): 289 nm (4.20 × 10 ³ ), 232 nm (3.08 × 10 ⁴ ), 213 nm (2.48 × 10 ⁴ ). Mass spectrum: FAB, m / z (relative intensity): 510 (M ⁺ , 100), 479 (4.5), 384 (4.5). High resolution analytical data for M ⁺ peak: C ₁₅ H ₁₂ I ₂ O ₄ , calculated 509.882513; Experimental, 509.882960 (deviation = -0.9 ppm) ¹ H NMR spectrum in DMSO-d6 (δ (ppm) values relative to TMS): 3.719 (3H, singlet), 3.876 (3H, singlet), 6.693 (2H, doublet, J = 9.45Hz, plus fine-splitting), 6.845 (2H, doublet, H = 9.36Hz, plus fine-splitting), 8.390 (2H, singlet)

9.3 Synthesis of Compound 7

Compound 7 [3,5-diaido-4- (4'methoxyphenoxy) benzaamide] was subjected to ammonia bubbles in a solution of Compound 1 (100 mg, 0.196 mmole) in anhydrous methanol (60 mL) at room temperature for 5 minutes. You can get it. After standing for 1 hour in a stoppered flask, the mixture is again treated with ammonia and then closed for 48 hours and left. Methanol / ammonia is removed by rotary evaporator, and the dry residue is dissolved in warm methanol: water (7: 3 v / v) (30 mL) and crystals are made in a refrigerator (3 ° C.). Yield; 58.3 mg (60%) light yellow crystals. Melting point; 207-209 ° C. mass spectrum (FAB): high resolution data for M ⁺ peak; C ₁₄ H ₁₁ I ₂ NO ₃ , calculated 494.882847; Found, 494.881880 (deviation = 2.0 ppm). ¹ H NMR spectrum in DMSO-d6 (δ (ppm) values relative to TMS): 3.716 (3H, singlet), 6.682 (2H, doublet, J = 8.93 Hz, plus fine-splitting), 6.895 (2H, doublet, J = 8.99 Hz, Plus fine-splitting), 7.528 (1H, singlet), 8.113 (1H, singlet), 8.402 (2H singlet).

9.4 Cell Culture

E-ras 20 cells are described in Bauer et al., 1996, "Moldification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP) "Intl. J. Oncol. 8: 239-252; culture as described above; HT-144 (melanoma), DU-145 (prostate cancer); HeLa (cervical cancer); HL 60 (progenitor cell leukemia); MDA-MB-231 (breast cancer); SK-Br-3 (breast cancer); T47D (mammary carcinoma); A559 (Lung Cancer) is obtained from the American Type Culture Collection (Rockville, MD) and cultured in the medium described above. The effect of DIME was tested in culture with an initial cell density of 2 × 10 ⁴ cells / cm 2, and the effect of DIME on cell growth by directly counting the number of cells after treatment with trypsin on hemocytometer for 72 hours after drug addition. (Eigen cells identified by trypan blue extrusion) were compared.

9.5 Tumorogenesis of E-ras 20 Cells

Tumor-forming ability of E-ras 20 cells in nude mice without thymus was examined as previously described (Bauer et al., 1996, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP) "Intl. J. Oncol. 8: 239-252). In vivo, the tumor-inhibiting effect of DIME was examined in tumor-free rats inoculated with 10 ⁶ MDA-MB-231 cells. Around 10-14 days, when tumors appear subcutaneously, DIME treatment consisting of po administration of DIME suspension (once per day) is initiated and lasts 28-32 days as reported (Kun et al., 1996). , "Induction of tumor apotosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: supplement 829).

9.6 Tests to Quantify Colony Formation

Assays to quantify colony formation are described in Vidair, et al., 1986 "Evaluation of a Role for Intracellular Na ⁺ , K ⁺ , Ca2 ⁺ , and Mg ²⁺ in hyperthermic cell killing" Radiation Res. This was done as reported in 105: 187-200.

9.7. DIME-Sepharose Affinity Column

EAH-Sepharose (Pharmacia, Piscataway, NJ, USA) (2.0 g wet) was washed with water and solvent exchanged with 60% DMF (aq). The wet cake was resuspended with 60% DMF (0.1 mL) containing a carboxylic acid derivative of Compound (Compound 6) (60 mg) and a 10-fold excess of N, N ¹ -dicyclohexycarbodiimide (Sigma). The suspension is gently rotated at room temperature for 16 hours. Sepharose beads are collected in a sintered glass filter, washed successively with DMF, dioxane, DMF, water-soluble DMF (66%, 50%, 33%), and finally with water. Based on the UV absorption spectrum of the substituted beads, the content of DIME-groups ranges from 1-2 μmol per ml of wet cake.

9.8 [ ¹⁴ C] -DIME Metabolism

(a) Mouse tissue homogenate (brain, kidney, kidney) consisting of 0.5 g of tissue in 1.0 ml of homogenization buffer (50 mM Tris, pH 7.4, 400 mM NaCl, 10 mM MgCl ₂ , 1 mM EDTA, 0.5% NP-40, 0.5 mM PMSF) Liver, lung) each was combined with 1.0 ml MES buffer (100 mM pH 6.5) containing 20 μM [ ¹⁴ C] -DIME (10.55 mCi / mmol) and the mixture was incubated at 37 ° C. for 4 hours. Ethyl acetate (1.5 mL), ammonium sulfate (900 mg), 60% perchloric acid (160 μL) is then added successively to each tube and vortex after addition. After standing for 10 minutes, phase separation is performed using a benchtop centrifuge. The upper layer (ethyl acetate) is removed and the lower aqueous phase and intermediate are extracted with ethyl acetate (1.5 mL). The ethyl acetate extract (complexed) contains at least 90% of the total cpm (ca. 4 × 10 ⁵ cpm) in the original culture. The extract was evaporated and dried using N ² , and each residue taken with ethyl acetate (100 μl) and one drop (10 μl) was analyzed for analytical silica gel TLC plate (Whatman PE SIL G / UV flexible plate, thickness of 250 μm, 10 cm × 20 cm) and develop with 3: 1: 0.8 v: v: v n-hexane. Assay criteria, including [ ¹⁴ C] -DIME, can be seen on the plate under UV light. (b) metabolism of [ ¹⁴ C] -DIME in cells in culture; Cells (2-5 × 10 ⁶ in each test) are incubated with [ ¹⁴ C] -DIME, homogenized and tested for metabolites as in (a).

9.9 Intracellular Concentration of DIME

9.6㎠ well (3㎖ medium) exposes the ^{[14 C] -DIME (10.55 mCi} / mmol) for 24 hours in monolayer culture and then to be washed seven times with medium containing 1㎖ DIME untested label. Cells are dissolved in 1 ml 4% Na ₂ CO ₃ and 0.2 M NaOH and the radioactivity is measured using a scintillation counter. Cell volume is measured by haematocrit and counts cell numbers in the side wells treated with unlabeled DIME.

9.10 Test for DNA Cleavage

DNA cleavage assays indicative of apoptosis were performed via terminal deoxy nucleotidyl transferase dUTP nick end labeling (TUNEL), as specified in the test kit (Boehringer Mannheim, Indianapolis, IN, USA, Cat. No. 168-4817). Conduct.

9.11 results

Carboxyl-esterase inhibitor bis [p-nitrophenyl] phosphate (BNPP) (Heymann, et al., 1968, "Inhibition of phenacetin and acetanilide-induced methemoglobinemia in the rat by the carboxyl esterase inhibitor of bis [p- nitrophenyl] phosphate ", Biochem. Pharmacol. 18: 801-811), was purchased from Sigma.

The structural specificity of DIME and its verbs and analogs can be assessed by examining Tables 7 and 8. We prepared 17 new DIME structural analogs and compared them with the antitumor activity of 10 of the compounds (Table 8) to draw some conclusions. The biological test chosen was to measure the ability of E-ras cells to inhibit the oncogenicity of the drug against in vivo oncogenic in nude mice (Bauer et al., 1996, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ₂ BP) "Intl. J. Oncol. 8: 239-252). E-ras 20 cells (10 ⁵ or 10 ⁶ ) were incubated with 10 μM DIME for 4 days, then 10 ⁵ and 10 ⁶ cells were inoculated subcutaneously in nude rats (5 per test) and directly Tumor formation was quantified in a volumetric manner (cf. 2). In the case of DIME itself (FIG. 4), tumor formation was completely discarded. The same tumorigenicity suppression test was conducted using nine DIME analogs, taking the effect of DIME as 100% (comparing tumor size on day 25), and calculating the antitumor effect as a percentage of DIME activity. Obviously, E-ras 20 cells were less sensitive to DIME than human tumors, so this result was used only as a comparison of DIME analogs. The results summarized in Table 8 demonstrate that the substituents on R ₁ and R ₂ determine antitumor effects. This effect is a surprising discovery that, particularly compared to _{R 1 CH 3 O, EtO,} n-Pro, nBuO in R _1, the side chain parameters, not to be bonded to a center structure "similar to the thyroxine" is assigned to specific pharmacologically It was.

This conclusion was validated by preliminary protein binding studies using DIME-Sepharose affinity columns. In this affinity column, DIME covalently binds to the matrix at R ₂ , and thus, inferred from the results of Table 8, it can be seen that DIME derivatives have significantly reduced tumor mortality than DIME (Kun et al., US Patent Application.Serial No. 08 / 655,267, filed June 4, 1996, "Method of treating malignant tumors with thyroxine analogs having no significant hormonal activity"). Protein was bound to the affinity column by osmosis of the cell extract through this column; One of the proteins absorbed was identified as tubulin. The previously reported centricon method was used to characterize protein binding of DIME (Bauer et al., 1996, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5- iodo-6-amino-1,2-benzopyrone (INH ₂ BP) "Intl. J. Oncol. 8: 239-252). From the Scatchard plot, the K _D values of the proteins in the cell extracts were obtained from 10 ⁹ to 10 ¹⁰ , indicating that the DIME is fractionally associated with various proteins. Based on the results shown in the DIME-Sepharose Affinity column, we believe that the cause of this binding is in the "center" structure of DIME.

Although the relative activity of DIME analogs can be deduced from Table 8, current experiments focus on the cytotoxic action of DIME itself and define an experimental system suitable for more detailed analysis of DIME analogs. Extending DIME's ability to inhibit tumorigenicity (FIG. 1) in vivo, 70-85% of tumors were inhibited by administering 0.25 to 1.0 g / kg daily for 20-25 days with DIME in nude mice (Kun et al., 1996, "Induction of tumor apotosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: supplement 829). At such large doses there was no noticeable toxicity for the possible reasons described below. The 0.25g / kg and 1.0g / kg DIMEs do not appear to have a dose-response relationship because they have the same antitumor effect. This hypothesis can be explained by the selective acceptance of drugs in tumor cells in vivo as discussed.

In the current study, the focus is on microscopic methods of analyzing cell death. As described in FIG. 5, colony forming ability of MDA-MB-231 cells is significantly reduced by DIME in the 1-2 μM range for 10 days. When DIME is removed at the end of 8 hours of incubation with cells, it prevents cells from dying and the morphological changes induced by the drug are completely reversed. Cells replated for up to 8 hours after pre-incubation with 1-10μ M DIME can be found to resume normal cell growth and replication if washed for DIME free. After eight hours of drug treatment, an important event feature creates an irreversible pathway to induce cell death and the reason for this is yet to be identified, which is for further study purposes. However, one of the causes of the ultimate cell death induced by DIME is caused by the breakdown of DNA according to the drug concentration, as illustrated in FIG.

A surprising property of DIME is the selectivity of tumors in vivo. We performed drug metabolism and DIME acceptance testing of cells, which need further study. As described above, culturing cells in culture with ¹⁴ DIME (Bauer et al., 1996, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5 -iodo-6-amino-1,2-benzopyrone (INH ₂ BP) "Intl. J. Oncol. 8: 239-252) The cells receive the drug, which is the same concentration of extracellular extracellularly added during the culture. PBS containing inactive DIME will not be washed away from the cells. Drug acceptance rates are significantly higher in cells (MDA-MP-231), which are directly killed by DIME when compared to cells that are sensitive to DIME (Table 9). We developed a transformed phenotype of normal renal cells, CV-1 cells, which lost contact inhibition and had the ability to multiply rapidly (12 hours of doubling time compared to 54 hours of untransformed phenotype). . As can be seen in Table 9, tumor-forming transformed cells generally receive DIME faster than untransformed cells (CV-1) or cells that are less susceptible to death by DIME (E-ras 20). . Stable cell lines become permanently isolated, thus becoming strictly physiologically inoperable cells, and our lack of drug results and the superficial lack of DIME toxicity in vivo have caused normal or minor DIME in normal animals. Say you can't accept it. On the other hand, organ homogenates of normal rats actively metabolize DIME as described in Table 10 (de-esterification). The maximum rate of "DIME-esterase" activity occurs in brain homogenates.

The correlation between drug metabolism and tumor lethal action of DIME is described in experiments with human lung tumor cells (A-549). As can be seen in Figure 4, A-549 cells cleave ester bonds (R ₂ ) in DIME, and bis [p-nitrophenyl] phosphate interferes with esterase activity (Heymann, et al., 1968, "Inhibition of Phenacetin and Acetanilide-Induced Methemoglobinemia in the Rat by the Carboxyl Esterase Inhibitor of bis [p-Nitrophenyl] Phosphate", Biochem. Pharmacol. 18: 801-811), as can be seen in FIG. -549 cells cause more effective cell death by DIME. Table 11 shows a comparison of the action of DIME that interferes with cell growth in various cancer cells expressed at the DIME concentration at which 50% of the cells ceased growth for 3 days. Figure 6 shows the time-dependent action of DIME at 0.5, 1.0, 2.0μ M final concentration in MDA-MB-231 cells.

DIME is a unique tumor lethal molecule, unless it has the toxicity observed macroscopically in vivo, except for tumor cells growing in native animals. From drug acceptance tests, cells sensitive to cell death by DIME are most likely to accept drugs. Replication of non-tumor cells in culture is variously hampered by DIME (data not shown), but the drug appears to be nontoxic in vivo. Thus, cell permeability to DIME in cell culture appears to be different from cell function in vivo. The cause for this apparent cell specificity may be due to the difference between the cell-growth in animal tissues and the part-yet-uncovered-cell membrane properties of the cells growing in culture, but further research is needed. Differences in permeability to DIME between cells grown in these cell cultures (both tumor cells and non-tumor cells) and cells acting in vivo are not maintained for tumor cells growing in vivo (Kun et al. , 1996, "Induction of tumor apoptosis by methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: supplement 829), The reason is that they die from the supply of DIME in vivo (Kun et al., 1996, "Induction of tumor apoptosis by methyl-3,5-diiodo-4- (4'l-methoxyphenoxy) benzoate (DIME)", Abstract No. 102, Int. J. Oncol. 9: supplement 829). Since the amount of DIME given in vivo (0.25-1.0 g / kg) is significantly higher than the extracellular tumor lethal concentration (0.5-2.0 μM) obtained in cell culture, tumor cells in vivo have been administered DIMEI Tumor cells that only accept a small portion of and grow in vivo have the same sensitivity to DIME as in cell culture, ie tumor cells in culture and exhibit similar drug in vivo. In vivo tumor cell membranes resembling tumor cells growing in cell culture are a new trend in tumor cells and require further analysis. In addition to in vivo selectivity for DIME permeability in tumors, tumor cells differ from normal cells where detectable DIME-esterase activity is different, except for A-549 cells (lung cancer).

In studies of these cells (FIGS. 4 and 5), it was surprising that DIME itself, not the metabolite produced by esterase, was a tumor lethal molecule. As can be seen from Table 8, significant anti-tumor effect of certain DIME analogs in E-ras 20 cells when the methyl-ester group (R ₂ ) of DIME is substituted with a longer chain ethyl-ester group or amide group You can see that it still holds. Although only DIME was analyzed in more detail in this study, it is also noteworthy that DIME analogs with a variety of "activity" could have cellular and in vivo effects, which could be further used in chemotherapy. In the case of iodine atoms in DIME and its analogs, these large atoms are undoubtedly sandwiched between two benzene rings, which at the same time are the major determinants of cell penetration.

Example 10 Action of DIME on Morphology of Cells and Nuclei

The following provides evidence for a broader concept of using tyrosine analogs to treat a variety of cancers. We report that DIME is a potent substance that induces cell death in cell cultures and tumor cells in vivo. Selective tumor lethal action has been explained by selective penetration of the drug into tumor cells in vivo (Mendeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl-3,5-Diiodo-4- (4). '-Methoxyphenoxy) Benzoate (DIME), "Intl. J. Oncol., In press). As described herein, exposure of 1 to 4 DIME to tumor cells results in cytological modification and mitotic blockade is associated with several biochemical sites of DIME. The important thing is to first define how DIME acts on the cells by cytometry before analyzing the site at the biochemical level. One embodiment relates to the action of DIME on cell and nuclear morphology and to the action of DIME in progression through mitosis.

10.1 Microscopic View

Mendeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl-3,5-Diiodo-4- (4'-Methoxyphenoxy) Benzoate (DIME)," for morphology of E-ras cells under a microscope. " Intl. J. Oncol., In press; Bauer et al., 1996, reported in "Modification of Growth Related Enzymatic Pathways and Apparent Loss of Tumorigenicity of a ras-Transformed Bovine Endothelial Cell Line by Treatment With 5-iodo-6-amino-1,2-benzopyrone (INHBP) There is a bar.

10.2 Preparation for viewing M phase

MDA-MB-231 cells were obtained from ATCC (Rockville, MD, USA) and the cells were cultured at 37 ° C. in T-75 flasks containing minimal essential medium-alpha supplemented with 10% FCS. As a reference, 0.1 μg / ml colceemia is added to the culture for 4 hours and cells are branched, without receiving DIME, and for 4 hours prior to collecting cells blocked in mid-term. The effects of DIME and colsemide are indistinguishable from blocking cellular processes on M, and therefore no colsemide is added when testing the effects of DIME on medium-term spreads. Cells (10 ⁷ ) were separated from T-75 trypsinized for 5 minutes with 0.25% trypsin, resuspended, stored in 10ml 75mM KCl for 10 minutes at 37 ° C and then precipitated again and fixed in 10ml 75mM KCl The methanol-acetic acid is changed four times in succession. One drop of the final cell suspension (100 [mu] l) is dropped onto a ethanol wiped slide and air dried.

10.3 in situ hybrid reactions

Human chromosome specific probes are produced by ONCOR (Gaithersburg, MD, USA). Pinkel et al., 1986, "Cytogentic Analysis Using Quantitative High-Sensitivity Fluorescence Hybridization," Proc. Natl. Acad. Sci. A hybrid reaction was carried out by modifying the method described in USA 83: 2934-2938. Cells on the slides were treated with pepsin (20 μg / ml 0.01 N HCI) at 37 ° C. for 10 minutes, then dehydrated with 70%, 85%, 100% ethanol, DNA soaked in 70% formamide, denatured, Treat with 2X standard citrate sodium (IX SSC is 0.5M NaCl, 0.015M citrate sodium, pH 7.0) at 70 ° C. for 2 minutes and dehydrate in ethanol as described above. The total 10 μL volume hybrid mixture consists of 50% formamide, 2 × SSC, 10% dextran sulfate, 0.5 μg herring sperm DNA, 1-5 μg proteinase K-treated human placental DNA. Herring sperm DNA and human placental DNA are previously sonicated with 200-600 bp fragments, ˜40 ng of degosigenylated probe DNA (denatured at 70 ° C. for 5 min) is added and incubated at 37 ° C. for 1 hour. The mixture was placed on a slide containing fixed cells, covered with a cover, and incubated at 37 ° C. for 2-3 days. After the hybridization was completed, the slides were washed three times (3 × 5 min) with 50% formamide, 2 × SSC, pH 7,0, and PN buffer (0.1M NaH ₂ PO ₄ and 0.1M HaH at 45 ° C.). Wash twice with ₂ PO ₄ mixture), 5 μg / ml antidigoxigenin FITC, 2 μg / ml rabbit anti-sheep Boehringer Mannheim / PNM buffer (5% with 0.02% azide sodium) Non-fat dry oil, centrifuged to remove solids, 20 minutes at room temperature, 0.4 μ MD API (4,6-diamino-2) in antifade solution (Vector labs, Burlingame, CA, USA) DNA was stained with -phenylindole). The slides were observed with a Zeiss fluorescence microscope (Chroma Technology, Brattleboro, VT, USA) with a multi-band pass filter to determine the number of FISH signals in each nucleus.

10.4 Time-Lapse Video Microscopy

Cells in sealed T-25 tissue culture flasks are placed in a temperature-controlled culture chamber and placed in an inverted microscope. The chamber is isolated from the attention room light. Microscopic light is provided every 12 minutes for 5 seconds, during which time the image is captured by an imaging system provided by Compix, Inc. (Mars, PA, USA). The sequence is analyzed by software from the same company.

10.5 Flow cytometry

Cells exposed to various other treatments are grown to join, treated with trypsin, and washed with phosphate buffer salt (PBS). For nuclear analysis, cells are fixed in Vindelov cell citric acid buffer (sucrose 250 mM, trisodium citrate, 2H ₂ O 40 mM, DMSO 50 mL / 1000 mL solution, pH 7.6). Normal human lymphocytes are separated from the blood and used as a reference. Each cell sample and reference count the number of hemocytometers and adjust the cell concentration to 2 × 10 ⁶ cells / ml. 20,000 cells from each cell line were washed twice with a total volume of 2 ml, treated with 200 µg / ml RNAase at 37 ° C. for 30 minutes, and 10 µg / ml prodium iodide for 45 minutes. Coloring is done. Flow cytometry analysis was performed using a FACScan benchtop flow cytometer (Becton-Dickinson, San Jose, Calif., USA) equipped with an air-cooled argon laser that can be adjusted to 488 nm. For analysis and record keeping, 20,000 cases were collected in a 6-variable list method. Dual identification can be obtained by 1024 data channel analysis with two optical scan parameters (front and side), propidium iodide fluorescence measured at 575/26 nm and above 620 nm, fluorescence pulse width and area. The starting value is set in the case of positive iodide propidium on channel 100 and above. Small debris is removed, and large clamps and cell duplications are performed after obtaining values.

10.6 Immunocytochemistry

Cells are fixed in methanol at −20 ° C. for 5 minutes. The primary antibody is murine monoclonal anti-beta tubulin (Amersham) and the secondary antibody is an antibody obtained from goat conjugated to floresin isothiocyanate obtained from Cappel (Durham, NC, USA). The DNA of the nucleus is stained with 0.05 µg / ml 4'-6-diimidono-2-phenylindole (DAPI) for 10 minutes. Stained cells can be seen through Zeiss 40 × PlanONeofluar or Nikon 60 × PlanApo objectives. In order to classify cells on mitosis, it is difficult to distinguish the electricity from the middle phase, so the preceding and the middle periods fall into one category.

10.7 results

The effect on the cell morphology after incubation with E-ras 20 cells for 18-24 hours in 4μM DIME is illustrated in FIG. 10. Tumor cells expand, especially when magnified 300 times, many micronuclei appear. The cause of these cytological changes should be further studied. In flow cytometry analysis (FIG. 11) performed on MDA-MB 231 human breast carcinoma, when exposed to the drug for 18 hours, nuclei having a G2 content of DNA accumulate, indicating that phase M has stopped.

Following the above observations, we decided to immediately examine the dynamics of mitosis in cells exposed to DIME. Cells were cultured in medium containing 1 μM DIME and subjected to time-lapse video microscopy as mentioned in Materials and Methods. 12 shows that mitosis is delayed by 13 hours, then irregular division occurs and divides into 6 progeny cells. In contrast, it can be seen that the reference cell had a mitosis delayed by about 0.5 hours (see FIG. 13) and always divided into exactly two progeny cells (data not shown).

Time-lapse video microscopy allows the monitoring of the progeny of progeny cells due to the aforementioned irregular cell divisions. Twenty percent of the mitosis seen in the presence of 1 μM DIME produces fused progeny cells (Table .12), which results in large nuclei with multiple nuclei as shown in FIG. 10. Reference cells do not exhibit such cell division after fusion.

Cell division occurring in the presence of 1 μM was examined to quantitatively assess the number of progeny cells due to each mitosis (FIG. 14). The number of progeny cells ranged from one to six per mitosis. In contrast, the reference cell always divides into two progeny cells (33/33). Thus, as mentioned above, prolonged mitosis induced by the drug is delayed, resulting in cell division in a very abnormal manner.

As can be seen in Figure 15, it shows the rate of mitosis between cells cultured in 1μ M DIME and reference cells. You can see that the curves are nearly identical, which shows that DIME does not affect the rate at which cells move between phases. In contrast, it can be seen that the mean time of each cell spent in mitosis increased about 20 times or more when induced by the drug (FIG. 13). Rounded cells blocked by DIME in mitosis can be identified by aggregated large chromatin (FIG. 16) and abnormal mitotic spindles (FIG. 17).

Chromosomal analysis by hybridization of the intermediate nuclei was performed with probes specific for chromosomes 1, 2, 7, 11, and 19. All of these gave similar results, and therefore only the case of chromosome 19 is described. Representative results are shown for chromosome 19 in a, b, c of FIG. 16. In all cases MDA-MB-231 (human breast cancer) cells were used. 16A shows a hybrid reaction on chromosome 19. Exposure to 1 μM DIME for 18 hours had no appreciable effect, and FIGS. 16A and 16B show reference and drug treated cells. FIG. 16B shows DNA staining (DAPI), which is consistent with chromatin-coloured DNA while 16a shows chromosome 19 staining. However, exposure of cells to drug (1 μM DIME) for 5 days results in significant accumulation of medium-term chromatin in some cells (about 40 chromosome-19 signals, 100 chromosomes or more) (FIG. 16C). There is no evidence that the chromosome is destroyed, but ultimately cells are killed through drug treatment (endeleyev et al., 1997, "Structural Specificity and Tumoricidal Action of Methyl-3,5-Diiodo-4- (4'-Methoxyphenoxy ) Benzoate (DIME), "Intl. J. Oncol 10: 689-695).

17 shows the mitotic spindle structure after 18 hours of exposure to 1 μM DIME. 17A shows tubulin-colored mitotic spindles in MDA-MB-231 cancer cells not treated with drugs. Figure 17b illustrates the change in tubulin distribution that appeared to be quite odd after exposure to the drug for 18 hours. This multicentroid structure was exacerbated after being exposed to 1 μM DIME for 5 days. The appearance of tubulin in a multicentral distribution in cells is described in Mitchison et al., 1984, "Microtubule Assembly Nucleated by Isolated Centrosomes," by adding a centroid in vitro to the nucleus of tubulin. It was shown that aggregation was similar to what happened. However, we could not detect the number of abnormal centrosomes by immunofluorescence (data not shown). Thus, after 18 hours of treatment with 1 μM DIME, multicenter nucleation of tubulin appears to be independent of centrosomes. This feature did not predict whether 1 μ M DIME induced or inhibited tubulin depolymerization.

In preliminary studies (data not shown), we used immunocytologic techniques to study the behavior of proteins associated with specific spindles. Cdc-2 kinase has not been associated with spindles without 1 μM DIME, but is demonstrated to induce cdc-2-kinase- spindle coupling after 18 hours of cell exposure to drug. Drug treatment does not alter the pattern of pigmentation by antiserum against the centroid protein percentrin because only two foci appear. Cyclin B-microtubule- spindle associations also remain unchanged. However, the abnormal tissue of the spindle central axis induced by the drug is still bound to cyclin B. The protein phosphatase 2A (pp2a)-spindle association is not affected by drug treatment (1 μM, 18 h), as is the case with percentrin or cyclin B.

Such studies are based on cell biological research. Preliminary experiments also tested the possible role of phosphatase in protein- spindle spindle association. Exposure to extracellularly added 100 nM ocdanoic acid for 18 hours removes only cdc-2 kinase and pp2a- spindle associations, which is associated with protein phosphatase (data not shown).

These results seem to distinguish between the initial effects of 1 μM DIME within 18-24 hours after drug exposure and the results seen in the number of chromosomes that occur after 5 days of drug treatment. However, what happens later simply reflects a series of cellular responses initiated by the binding of DIME at specific cell sites. What happens early is a clear cellular change, especially cells with large multinucleated aggregates. The formation of the micronucleus nucleus is known to occur mainly after radiation damage, because the out-of-center chromosome fragments fail to migrate to the poles during the late period, without the presence of both centriole and microtubule spindle attachments. (Bedford, JS, 1991, "Sublethal Damage, Potentially Lethal Damage, and Chromosomal Aberration in Mammalian Cells Exposed to Ionizing Radiation," J. Radiation Oncol. Biol. Phys 21: 1457-1469). Time-lapse studies have shown that vinblastine sulfate also induces multinucleated cells that block intermediate phases, and Kirshan, 1968, "Time Lapse and Ultrastructure Studies on the Reversal of Mitotic Arrest Induces by Vinblastine Sulfate in Earl's L Cells. , "J. Natl. Cancer Institute 41: 581-595), again looking at the results (FIG. 10), vinca alkaloids show serious toxic effects in animals, while DIME does not show toxic effects. Overexpression of protein phosphatase 2A (pp2a) induces multinucleation (Wera et al., 1995, "Deregulation of Transitional Control of the 65 kDa Regulatory Subunit (PR65 Alpha) of Protein Phosphatase 2A leads to Multinucleated Cells," J. Biol Chem. 270: 21374-2138l), which suggests that the mechanism of development of such complex processes (multi-core aggregation) is probably associated with a variety of enzymatic activities. These results suggest that the activity of pp2a in DIME cells (l.c.1) is due to the direct action of DIME on this enzyme. The involvement of various cellular enzymes in the way DIME works may be the subject of separate biochemical studies.

One of the best observable cellular phenomena induced by DIME is the blockade of M phase (FIGS. 11 and 13), which is very similar to the action of colchemid or especially vinca alkaloids instead of DIME. The effect (5 days) behind the DIME described by DNA-fluorescence hybridization with probes for chromosomes 1, 2, 7, 11, and 19 results in the accumulation of chromosomes because cell division fails. DIME does not directly affect DNA. Cleavage in double chain-DNA (Mendeleyev et al., 1997, "Structural specificity and tumoricidal action of methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)" Int. J. Oncology 10: 689 -695) are the result of the subsequent interaction of DIME with cancer cells, mostly poly-ADP- in DIME-treated cells, through indirect activation of poly (ADP-ribose) glycohydrolase (footnote I in ref 1). This reflects the up-control of DNA endonucleases that release ribosylation. Endonuclease activation does not only induce predetermined cell death by DIME because it is known to induce apoptosis due to various molecular mechanisms (Wertz et al., 1996, "Diverse Molecular Provocation of Programmed Cell Death"). , "TIBS 21: 359-364). The occurrence of abnormal mitotic spindles in DIME-treated cells indicates the initiation of DIME cellular activity in the tubulin system, which is known to play an important role in division (Murray et al., 1993, "The Cell Cycle: An Introduction., "Oxford University Press, New York.

Because DIME is a "non-hormone-active" thyroid hormone analog, metabolic precursors (or metabolites) of thyroid hormones play an important role in maintaining physiological differentiation, acting as "modulatory" correction regulators. Doubts arise. This is the basis for the study of thyroid hormone metabolites with tumor death.

Example 11 Influence on Tubulin Polymerization

The following experiments show that DIME, a thyroid hormone analog without hormone activity, interferes with the GTP-dependent polymerization of MTP at concentrations of 1 to 5 μM (determined by optical testing). This disturbance mainly depends on the GTP concentration. Under the linear rate of MTP polymerization, the quantitative correlation between DIME and GTP concentrations follows the Michaelis-Menten kinetics, and the inhibition is expressed as "mixed", where k _m and U _max of GTF change simultaneously. DIME chemical analogs interfere with MTP polymerization in parallel with their ability to inhibit tumor formation in vivo. The MTP site is one of the early cellular response sites of DIME.

Exposure of human breast cancer cells (MDA-MB-231) to 1 μM DIME induces abnormal spindle structure for 18 hours of drug treatment, thus presuming DIME-microtubule-protein (MTP) interactions to the initial drug Zhen, et al., 1997, "Cellular Analysis of the mode of action of methyl-3-5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME) on tumor cells, Intl. J. Oncol) .Aberrant spindle structure appears to be the result of DIME-MTP interactions or the effects of DIME and components of microtubule tissue-centered or DIME interactions with undefined systems. The quantitative analysis was not appropriate for measuring the initial rate, so we adopted the neurotubule in vitro coalescence system as a model for the quantitative analysis of the interaction between MTP and DIME. (Gaskin , et al., 1974, "Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules", J. Mol. Biol. 89: 737-758; and Kirschner, et al., 1974, "Microtubules from mammalian brain: some properties of their depolymerization products and a propose mechanism of assembly and disassembly ", Proc. Natl. Acad. Sci. USA 71: 1159-1163; this system is suitable for kinetic assay of MTP assembly in vitro. The process consists of initiation, proliferation and termination steps (Gaskin, et al., 1974, "Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules", J. Mol. Biol. 89: 737-758). Under defined conditions, the growth rate is linear enough to be subjected to epidemiological analysis, and thus the concentration of DIME and GTP can be assessed. As can be seen, interference of MTP coalescence by DIME occurs at the same range of drug concentrations that are necessary to prevent tumor formation in vivo or to inhibit cell proliferation or eventually to kill cells (Mendeleyev, et al., 1997. "Structural specificity and tumoricidal action of methyl-3,5-diiodo-4- (4'-methoxyphenoxy) benzoate (DIME)" Int. J. Oncol., 10: 689-695 and Table 8) Thus, DIME-MTP interactions are probably considered to be a component of many cellular mechanisms of DIME.

11.1 Isolation of Microtubule Protein (MTP)

Optical testing of MTP preparation and polymerization is carried out according to published methods (Gaskin, et al., 1974, "Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules", J. Mol. Biol. 89: 737- 758; Tiwari, et al., 1993, "A pH and temperature-dependent cycling method that doubles the yield of microtubule protein", Anal. Biochem. 215: 96-103). Bovine or rabbit brain is homogenized with ice-cold oriental buffer containing 100 mM Pipes / K ⁺ (pH 7.4), 4 mM EGTA, 1 mM MgCl ₂ , 0.5 mM DTT, 0.1 mM PMSF and 39,000 g for 1 hour at 4 ° C. Centrifuge at. DMSO (final concentration 8%) and GTP (final concentration 1 mM) are added to the supernatant and incubated at 37 ° C for 30 minutes. The microtubules are pelleted by centrifugation at 100,000 g at 37 ° C. for 30 minutes, which are resuspended in ice cooled PEM buffer (100 mM Pipes / K + (pH 6.9), 1 mM EGTA, 1 mM MgCl ₂ ). The heated polymerization and cold depolymerization were repeated one or more times, and cold resuspended monomer MTP (8-10 mg / ml protein) was used for the optical test for polymerization kinetics. The same MTP material is obtained from rabbit or cerebellum.

The optical test configuration was described according to the drawings and the legend of FIG. 12. The polymerization was initiated by the addition of 100 μl MTP solution (which is equivalent to 0.8-1.0 mg protein), and the initial linear velocity was found to increase the absorbance at 350 nm and a cubic holder with automatic temperature control at 37 ° C. Record on a Perkin-Elmer 552 dual beam spectrophotometer (see FIG. 1).

11.2 Results and Discussion

18 illustrates the accuracy of the optical inspection of the MTP polymerization reaction. Experimental conditions are the same as in the legend of Table 7, except that the GTP concentration is 1 mM (right curve) and the DIME is 4 μM (left curve). Obviously, the rate of MTP polymerase proceeds in a linear fashion, so the conditions for the maximum rate of polymerization are described in Berne, B, 1974, "Interpretation of light scattering from long rods", J. Mol. Biol. 89: 755-758. Therefore, by varying the concentrations of drugs and active substances and comparing linear rates, a quantitative correlation can be determined between [DIME] and [GTP]. Gradually increasing the concentration of DIME at 1 mM GTP concentration interferes with MTP polymerization (FIG. 18).

As can be seen in FIG. 20, 1 μ M DIME is quantitatively related to [GTP] and double interplot shows “mixed” inhibition (Dixon, et al., 1964 Enzymes, pp. 234-237, Acad. Press, Inc., New York). Both V _max and k _m change, with 50% k _m GTP changing from 6.7 μ M to 14 μ M, while V _max decreases by nearly 50%. Based on the Briggs-Haldane equation (cf.7), the simplest interpretation of mixed inhibition is that k ₂ , the decomposition of [ES], into E and P (product) is indirectly affected and therefore both k _m and V _max Transform. The exact characteristics of k ₂ are not known at present, but in order to colonize it, we need to analyze the reaction products of GTP hydrolysis, which we know work elsewhere. Similar results can be obtained with allosteric modifications (Dixon, et al., 1964 Enzymes, pp. 234-237, Acad. Press, Inc, New York). The purpose of this experiment was to compare the effects of some of its analogs with DIME in MTP polymerization (see Table 12) and to correlate the results with cytopathological processes (inhibition of tumorigenicity in vivo).

There is a clear correlation between the chemical structure of seven DIMEs and their analogs in MTP polymerization (Table 13) and their inhibitory capacity in tumor formation in vivo (compare Table 8 or Mendeleyev et al.,). Substitution of the substituents of R ₁ with EtO and n-BuO in CH ₃ O gradually decreases the inhibition of the MTP polymerization, thus reducing the tumorigenic effect (compare Table 8 or Mendeleyev et al.,). On the other hand, replacing R ₂ with a methyl ester in the carboxylic acid completely eliminated the inhibitory effect on the MTP polymerization and reduced the tumorigenic activity by half when E-ras 20 was treated (Table 8, FIG. 5 or Mendeleyev et al.,). It is possible that such quantitative differences result in a specific change in cell type.

The test system in Table 13 consists of PEM buffer (containing 8% DMSO), GTP (final concentration 1 mM), drug (add 3 μl, final concentration 10 μM) or solvent basis and 0.6 mg MTP (60 μl). . The microtubule union at 37 ° C. is monitored at λ = 350 nm and the initial rate is calculated at mA ₃₅₀ / min. The reference is the initial speed is 180mA ₃₅₀ / min. The value is the average of two tests repeated.

Interruption of the MTP polymerization has very complex cellular effects. In cytolysis, such interference interferes with the contractile forces of tubulin, preventing the formation of cleavage grooves essential for cell division (Burton, et al., 1997, "Traction forces of cytokinesis measured with optically modified elastic substrate", Nature 385: 450-454). Disruption of MTP polymerization by DIME seems to be related to the biochemical site of the drug. In comparison to that of Mendeleyev et al., DIME directly activates pp2-ase, and therefore needs to coordinate this effect with mitosis-related phenomena induced by DIME. For example, according to a recent report (Kawabe, et al., 1997, "HOXII interacts with protein phosphatase pp2a and pp1 and disrupts G2 / M cell cycle check point" Nature 385: 454-458), pp2-ase is G2 / Regulating M transition, pp2-ase is also a potent oncogen, which interferes with promoting oncogenation. Activation of pp-2ase by DIME may antagonize oncogenogenesis.

Based on this experiment, thyroxine analogs such as DIME appear to be able to block mitosis in cancer cells. The present invention provides for the rapid selection of such compounds using these techniques, cell sorting, using chromosomal blots or other DNA analysis in cells.

The foregoing specification describes the invention in detail so that one skilled in the art can practice it. Various changes may be made in practicing the invention and those skilled in the art of pharmacy or other related fields will recognize that they are within the scope of the following claims.

Claims (8)

In the pharmaceutical composition for treating malignant tumors having a thyroxine analog of Formula 1 as an active ingredient and sensitive to a thyroxine analog, the thyroxine analog can suppress the growth of a malignant tumor without hormonal activity. A pharmaceutical composition, characterized in that it can interfere with the initial rate of microtubule protein coalescence in vitro to 35% or more. X = 0, S, CH ₂ , carboxy or absent; Y = 0 or S; R ₁ = methyl or ethyl; R ₂ , R ₃ , R ₄ R ₅ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy and halogen; R ₆ , R ₇ , R ₈ , R ₉ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenal, (C ₁ -C ₄ ) alkynyl, hydroxyl, ( C ₁ -C ₄ ) alkoxy, halogen, NO ₂ , NH ₂ . The pharmaceutical composition of claim 1, wherein the thyroxine analog has the formula: or a pharmaceutically acceptable salt thereof. X = 0, S, CH ₂ , carboxy or absent; Y = 0 or S; R ₁ = methyl or ethyl; R ₂ , R ₃ , R ₄ R ₅ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) alkoxy and halogen; R ₇ and R ₈ are each independently H, (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkenyl, (C ₁ -C ₄ ) alkynyl, hydroxyl, (C ₁ -C ₄ ) Alkoxy, halogen, NO ₂ , NH ₂ . The pharmaceutical composition of claim 1, wherein the thyroxine analog is methyl 3,5-diaido-4- (4′-methoxyphenoxy) benzoate. The pharmaceutical composition of claim 1, wherein the thyroxine analog is effective for regressing malignant tumors. The pharmaceutical composition of claim 1, wherein the malignant tumor is selected from carcinoma, sarcoma. The pharmaceutical composition of claim 1, wherein the thyroxine analog is administered orally. A pharmaceutical composition for treating cancer, comprising the thyroxine analog of Formula 1 as an active ingredient. X = 0, S, CH ₂ , carboxy or absent; Y = 0 or S; R ₁ = methyl or ethyl; _{R 2, R 3, R 4} , R 5 are each independently H, (C ₁ -C ₄₎ alkyl, (C ₁ - C ₄₎ alkenyl, (C ₁ - C ₄₎ alkynyl, hydroxyl, ( C ₁ -C ₄ ) alkoxy and halogen; _{R 6, R 7, R 8} , R 9 are each independently H, (C ₁ -C ₄₎ alkyl, (C ₁ - C ₄₎ alkenyl, (C ₁ - C ₄₎ alkynyl, hydroxyl, ( C ₁ -C ₄ ) alkoxy, halogen, NO ₂ , NH ₂ . 8. The pharmaceutical composition of claim 7, wherein the cancer is selected from carcinoma, sarcoma.