JP2688182B2 - Novel differentiation-inducing aid used for cancer differentiation therapy and chemoprevention method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to cancer differentiation therapy and chemoprevention method, and more particularly to a novel differentiation-inducing auxiliary agent that acts on an abnormal methylation enzyme of cancer cells.

[0002]

Abbreviations used here are: AdoHcy: s-adenosylhomocysteine AdoMet: s-adenosylmethionine Met: methionine adenosyltransferase MT: methyltransferase NBT: nitroblue tetrazolium RA: retinoic acid SAHH: s- Adenosyl homocysteine hydrolase. [Differentiation therapy for cancer] If a large number of differentiation inducer receptors are present in the target cancer cells and responds to them, it is considered that differentiation therapy that is effective and has few side effects is an ideal cancer therapy. For example, the therapeutic effects of interferon (Reference 1) for hairy cell leukemia and retinoic acid (References 2, 3) for acute promyelocytic leukemia have been recognized.
However, since differentiation therapy is still in the development stage and its application is limited, there are many problems to be solved. The current situation is that interferon is effective in few cancers and the palliative period of retinoic acid therapy is short. Moreover, the mechanism of differentiation therapy is not completely understood. [Role of methyltransferases in cell division and differentiation] Expression of genes involved in cell differentiation function is essential for the differentiation process. In stem cells, these genes are in a repressed state. Methylation of DNA molecules is being reported to play an important role in some gene repression mechanisms (references 4-6). This methylation acts on cytosine residues in the DNA molecule and converts it into 5 mC, so DN
Only the CG sequence in the A molecule will be changed.
To recover from this change, cells should be split twice and DN
Since demethylation by A replication is required, it will be affected by gene expression.

Among the in-vivo methylation reactions, DNA methylation is the most important one because it affects gene expression and is therefore involved in cell growth and differentiation processes. However, methylation of rRNA is also involved in it.
Most rRNA methylation reactions act on 2'-o-ribose and play a crucial role in protecting the rRNA sequence during ribosome biosynthesis (reference 10). rRNA is
First, we synthesize a large 45S precursor, which is divided into two subunits in the nucleus. All methylated nucleotides are housed in the rRNA sequence, thereby
The action of cell degrading enzymes can be avoided. Therefore,
Ribosome biosynthesis is largely dependent on methylation of rRNA precursors.

Since the proteins used for DNA synthesis are required for chromosome replication, it is necessary to construct and place ribosomes before entering the S phase. Administration of actinomycin D, an inhibitor of rRNA production, to the production of ribosomes in cells stimulated to grow (reference document 11), or to give a temperature-sensitive mutant strain an inappropriate temperature shift (reference document 1).
When selectively suppressed by 2), DNA synthesis is interrupted and the cell cycle is stopped in the G1 phase. From these experiments, it was found that ribosome production is essential for cell proliferation. [Involvement of abnormal methylation enzyme in malignant growth] The fact that the methylation reaction of nucleic acid is often involved in gene expression and ribosome production suggests the importance of the role of the methylation enzyme. The methylating enzyme is MAT-MT-SAH
It is a complex composed of three H enzymes. There are various MTs, but each is an enzyme with extremely high specificity,
Has different functions. However, the same MAT and SAHH
When combined with these enzymes, these enzymes form a family with a common expression regulation mechanism. Methylases are generally regulated by certain activators, which are steroids in steroid hormone target tissues (13). If this activator is not present,
The complex consisting of the three enzymes collapses, and each enzyme is immediately destroyed by the endogenous proteolytic enzyme. This enzyme complex is stable and appears to be the unit of action of methylating enzymes. Normal methylating enzymes are said to depend entirely on exogenous activators. Cells other than the steroid hormone target cells produce a methylating enzyme activator by stimulation of growth.

The methylating enzyme in cancer cells is abnormal, and a protein factor peculiar to cancer has a toe-type bond with the enzyme. These abnormal enzymes were discovered by Hi et al. (Refs. 14-16) and they pointed out that the methylating enzyme isozymes in cancer cells are kinetic abnormal. Km values for normal isozymes referred sur over L, to the MAT ^L and SAHH ^L, respectively are 3μM methionine and 0.35μM adenosine. In contrast, the Km values of sur-LT, a cancer cell methylating enzyme isozyme, are 20 μM methionine (for MAT ^LT ) and 2.2 μM adenosine (for SAHH ^LT ). The presence of MAT isozymes in this abnormal cancer cell was reported by Surfrin and Lombarini (Reference 17) and Kapple.
r et al. (reference 18). This oncoprotein factor not only enhances the activity and kinetic properties of the enzyme, but also alters its stability and regulation. This factor acts like an activator of a methylating enzyme and can stabilize the cancer cell while maintaining the activity of the methylating enzyme. Cancer cells obtain a sufficient methylation function by producing this endogenous protein factor without depending on an exogenous activator, and a functional RNA required for cell growth.
Production and maintenance of malignant phenotypes can ensure replication of DNA methylation patterns. Inappropriate methyltransferases are therefore the most important challenges facing cancer.

This unusual cancer MAT ^LT is present in primary cultures of rat hepatoma cells and transplanted cells thereof (reference document 15) (reference document 15), and melanoma, sarcoma, lymphoma and colon, lung from human, Adenocarcinomas of the breast, liver, ovary, uterus and rhinitis head were also found in xenografted cells in athymic nude mice (reference 19). The same abnormal phenomenon was observed in the cancer tissue cells obtained by surgery and the HL-60 leukemia cultured cells. The level of MAT ^{LT in} cancer tissues was high, and a good correlation was also obtained between the growth rate of xenografted human cancer cells and the level of abnormal enzymes (Reference 19).
This abnormal enzyme is not found in regenerating liver, dying tissue, bone marrow, and small intestinal mucosa cells with a high proliferation rate, and is an abnormal phenomenon peculiar to cancer cells. This cancer-specific aberrant methylation phenomenon can be used for a therapeutic method. By binding to a cancer-specific protein factor, the methylation enzyme has an abnormal response to the regulatory factor. Our research results are
It was pointed out that cancer RNA methylating enzyme responds well to suppressors such as oligonucletide and DNA intercalating agent, but does not respond much to stimulators such as ATP and polyphosphate (references 10 and 20). It is clear that this abnormal phenomenon of cancer is a target for its treatment, as normal methylases are the opposite. [Aberrant Methyl Enzymes Become Selective Targets in Cancer Differentiation Therapy] As described above, it is a basic cause of cancer that cancer cells cannot enter into the differentiation process due to an abnormality in methylation enzymes. It is a phenomenon. If these abnormal enzymes are removed, it is expected that cancer cells can be introduced into the differentiation process. Our rudimentary research results also support this assumption. surely,
When the abnormal enzyme present in the Novicoff's ascites hepatoma cells was removed (reference document 16), the synthesis of macromolecules was later inhibited and cell proliferation was also stopped (reference document 21). Poly (I)
(C) seems to act indirectly because it cannot enter cells. Poly (I) (C) has been shown to induce oligoisoadenylate synthases such as interferon (reference 22). The product of this derived enzyme, oligoisoadenylate, is a trinucleotide, which is an inhibitory agent that removes the aberrant methylating enzyme. Prior to that, we discovered that cancer rRNA methylases had oligonucleotides (especially trinucleotides).
It was reported that it was sensitive to the inhibitory effect of the drug (reference document 10).
Oligonucleotides are products of differentiating cells, and complex oligopeptide and natural substances such as organic acids have similar functions (References 23 and 24). These natural substances are
It has a selective antitumor effect and was named antineoplaston by Burzynski (reference 25). These antineoplastons are low molecular weight metabolites,
In the living body, it is reabsorbed by the kidneys and can return to the blood circulation without being partially excreted. Antineoplastons in the body are then held there above a certain level and help control cancers or phenomena. Takashi et al. Call this natural chemical defense mechanism chemical monitoring (reference document 26).

The purified active ingredient of antineoplaston induces terminal differentiation of HL-60 cells (reference document 2).
3), it has high activity of inhibiting colony formation of HBL-100 cells (reference document 24). These active ingredients are dye-conjugated peptides and organic acids. The chemical properties are quite different, but the biological activities are similar. When tested on purified MAT isozymes, these active ingredients show activity in inhibiting and normalizing abnormal aberrant cancer isozymes. The active ingredient of antineoplaston inhibits the cancer-specific protein factor of MAT ^LT and suppresses its effect on the methyltransferase. This anti-cancer mechanism is to act on the demethylation of DNA and rRNA precursors and modify the abnormal methylating enzyme (reference document 9). As a result, the cancer cells are differentiated, and normal cells are not affected because they do not have this protein factor.

Enzymatically, it causes terminal differentiation,
It is crucial to normalize aberrant methyltransferases.
The results of examination of intracellular pools of AdoMet and AdoHcy strongly support the above conclusions. De La Rosa et al. (Ref. 27) pointed out that the AdoMet pool thirds in cancer cells are relatively large, which is due to the fact that cancer MA
This is because the Km value of T isozyme is high. Chiba et al. (Reference document 27) also reported that if HL-60 cells were terminally differentiated,
We have reported that the pool size of AdoMet and AdoHcy shrinks, which is evidence that the cancer methylating enzyme is normalized and its high Km value becomes low. From various perspectives,
It is suggested that the cause of cancer cells not going to terminal stage differentiation is an abnormality of methylase. [Natural Chemical Surveillance and Canceration Phenomena] Anti-neoplaston, which suppresses the development of cancer, exists in the human body in the same way that humans cause disease in the presence of the immune system.
Humans also get cancer. The cause is that the surveillance system collapses. Our findings indicate that cancer patients may be unable to maintain a chemical surveillance system due to excessive excretion of low molecular weight substances (Ref. 26,
29), which results from the excretion of low molecular weight endogenous antineoplastons. The growth of cancer cells is to further enhance the loss of endogenous antineoplastons,
As a result, this chemical surveillance system is almost absent in end-stage cancer patients. So, besides the methylation enzyme abnormalities, disruption of this chemical surveillance system is another important etiology of cancer. A treatment method that suppresses both of these is likely to be a method with a high success rate, and in fact, antineoplastone purified from urine was pointed out to be a very effective anticancer agent (References 23 and 29). The effect of this antineoplaston therapy, which suppresses excessive excretion of peptides while normalizing abnormal enzymes, is clear (Reference 30).

A possible cause for cancer patients to excrete large amounts of low molecular weight metabolites is inflammation. Due to inflammation, macrophages release cactectin, which causes cachexia that excretes a large amount of metabolites, increased catabolism such as lipid migration, and anorexia (references 31-33). Since the onset of acute inflammation is short, it has little relationship with cancer, but chronic inflammation seems to have a large contribution to cancer. For example, AIDS and B
The carcinogenic rate of hepatitis C patients is high. Cancer cells contribute to chronic inflammation due to abnormal gene expression. Every time a cancer cell grows, cachexia becomes worse. Controlling cachexia is beneficial in treating cancer, but it is completely ignored by traditional cancer therapies. We reported that cytotoxic agents cause excessive excretion of peptides (ref. 26), suggesting that cytotoxic agents disrupt the chemical surveillance system. When this formulation is administered for a long period of time, the cell resistance is completely lost. If cancer cells survive the cytotoxic drug therapy, the growth of these cells becomes uncontrolled. In other words, cytotoxic drug therapy aims to kill cancer cells completely,
That itself is very difficult. Antineoplaston therapy, on the other hand, is the rapid regeneration of chemical surveillance systems to protect the body with a natural defense mechanism. Killing all cancer cells is not necessary.

Phenylacetylglutamine, a component of antineoplaston formulations, can restore the overexcretion of peptides commonly found in cancer patients. Although this compound does not act on cultured cancer cells, it treats early cancer (reference document 26) and prevents chemical canceration (reference document 34-).
36) is effective. The curative and preventive effects of this cancer appear to be due to the restoration and protection of the chemical surveillance system. The fact that cultured cells are more likely to cause artificial canceration than living organisms means that the chemical monitoring system of living organisms must be destroyed first. Inflammatory agents such as proton oil act as promoters of the carcinogenic phenomenon, while drugs such as phenylacetylglutamine protect the chemical surveillance system and prevent the carcinogenic phenomenon. That is, the use of an accurate chemical monitoring system is useful for treating or preventing cancer. [Role of differentiation-inducing adjuvant in differentiation therapy] Differentiation-inducing agents are directly (antineoplaston, etc.) or indirect (retinoic acid, interferon and poly (I) (C) (polyinosinic acid: cytidylic acid)) Various methylating enzymes can be normalized (references 9, 16, 39). Indirect inducers have limited applications because they rely entirely on the presence of the receptor. Recently, Taku et al. Reported a series of compounds that have no differentiation-inducing activity, but greatly enhance the activity of inducers (Reference 40). These compounds are called differentiation-inducing auxiliary agents, and most of them are inhibitors of the constituent enzymes of the methylase complex. For example, competitive inhibitors of MAT. Butyric acid is a compound that has the best effect as a differentiation-inducing auxiliary agent among MAT competitive inhibitors, and phenylacetic acid also has that activity (Reference 40). Differentiation induction inhibitors belonging to this group have relatively low toxicity (Reference 41-
43). From a therapeutic point of view, these compounds are particularly useful in expanding the therapeutic range of indirect differentiation inducers, and the low cost of the compounds themselves can reduce the financial burden on patients. . In some special cases, the differentiation-inducing adjunct alone is also effective. Since the brain is shielded by the blood-brain barrier, endogenous antineoplastons in the brain are less likely to be washed away than in other compartments. Therefore, for brain cancer, especially for astrocytoma, the effect appears only by administering the differentiation inducer. Not all cytotoxic agents work on astrocytomas, so
Phenylacetic acid is the only therapeutic agent. Therefore, the differentiation-inducing auxiliary agent is considered to be useful for treating early-stage cancer diseases and preventing cancer.

[0011]

The present invention shows a differentiation-inducing auxiliary agent that can contribute to cancer differentiation therapy and chemoprevention, and systematically researched it. We sought to synthesize compounds with better efficacy and utility by chemically modifying them while selecting effective differentiation-inducing adjuvants. Compounds that are excellent differentiation-inducing aids are methyl and ethylphenylacetamide, phenylacetic acid ethyl ester, 2,4-dichlorophenylacetic acid and indoleacetic acid. Methyl and ethylphenylacetamide and phenyl are competitive inhibitors of MAT. There are ethyl acetate, 2,4-dichlorophenylacetic acid and indoleacetic acid.

The present invention provides a pharmaceutical composition effective in the differentiation therapy and prevention of cancer, particularly brain cancer and prostate cancer, which comprises:
The pharmaceutical composition has a single composition or a mixed composition selected from methyl and ethyl phenylacetamide, phenylacetic acid ethyl ester, 2,4-dichlorophenylacetic acid and indoleacetic acid. This pharmaceutical composition has a dosage form suitable for oral, parenteral or topical administration.

In this pharmaceutical composition, a differentiation inducer used for cancer differentiation therapy may be added as an active ingredient.

[0014]

[Examples] Even if it is called a differentiation-inducing aid, it not only assists the effect of the differentiation-inducing agent, but is indispensable in the differentiation therapy of cancer. The differentiation of the cells induced by the addition of the differentiation-inducing agent is complete as compared with the result of the differentiation-inducing agent alone (reference document 40). From the results of our study, it is rare that the number of NBT + cells is higher than 85% with the inducing agent alone, and there are few uninduced cells, but they are always present. The presence of this uninduced cell causes a short remission phase of RA therapy (ref. 44). The differentiation process is complete D
It is a long process because it requires two mitotic processes including NA demethylation to take place (references 45-47).
In the process, if the cells are damaged and DNA synthesis cannot be completed, the cells may return to a cancerous state by repairing DNA. In that case, the symptoms reappear. In cancer cells, a unique protein factor maintains a complex structure of methylase and stabilizes it, so that the level of methylase is high. Once the effect of this protein factor is inhibited by antineoplaston or its analogues induced by the differentiation inducer, the methylase complex structure collapses and the constituent enzymes are released. Some free methyltransferases act as nucleases (ie,
Latent nuclease), damages cells (Reference 10), and stops the differentiation process. The differentiation-inducing auxiliary agent has the effect of controlling damage from the latent nuclease and completing the differentiation process. That is, since the differentiation-inducing auxiliary agent promotes the differentiation process and suppresses the recurrence of symptoms, it is essential for differentiation therapy. 1. Synthesis of Phenylacetic Acid Derivatives Phenylacetic acid is a therapeutic agent for astrocytomas (Ref. 30,
43), it has already been reported that it is a differentiation induction auxiliary agent (reference document 40). However, the drug itself has an unpleasant odor, and the minimum concentration as an auxiliary agent reaches several mM, which is not satisfactory. Focusing on improving its activity and odor, we discovered and synthesized the following derivatives. 1-1. Synthesis of N-Substituted Phenylacetamide The N-substituted phenylacetamides here are already listed in reference 51, but Scheme 1 shows how we synthesize it.

[0015]

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Here, R is a lower alkyl group. Scheme 1 Phenylacetyl chloride is replaced with methylamine or ethylamine.
After the reaction with benzene with benzene solution, the product was subjected to column chromatography.
It was generated by matography and recrystallization method. Example I-1-1: Phenylacetamide (3-1) 15 g (0.1 mol) of phenylacetyl chloride was added to 100%.
Dissolve in ml of benzine and stir at 20-30 ° C.
Amonia gas was introduced into the benzine solution. After the reaction
The solid obtained by filtration is collected, washed with water and dried.
Was. Wash the filtrate in a separatory funnel with water and dry with anhydrous magnesium sulfate.
It was dried under vacuum and concentrated under reduced pressure. Residue obtained by concentration
Silica gel column chromatography
Chromatograph and elute with chloroflume. Remove the eluate
Recrystallize with roloflum to give 11 g of compound 3-1 as white crystals.
Crystals were obtained (yield 80%). mp .: 153-155 ° C. FIR (KBr) νmax: 3300, 3120 (NH), 1670
(C = O) cm^-1;¹H-NMR (CDCl _Three): 3.58 (s, 2H, -CH₎, 5.60 (br,
2H, -NH_Two), 7.24-7.30 (m, 5H, -C₆H_Five); MS, m / z: 135 (M +);
 Elemental analysis value: C₈H₉NO, Calculated: C: 71.09, H: 6.71, N:
10.36, Actual: C: 71.15, H: 6.93, N: 10.26. Example I-1-2: N-methylphenylacetamide (3
-2) Add 15 g (0.1 mol) of phenylacetyl chloride to 100
Dissolve in ml of benzine and stir at 20-30 ° C.
Methylamine was introduced into the benzine solution. Compound 3
Purify by the same procedure of -1, and use N-methylphenylacetoacetate
Light yellow crystals of amide were obtained (12 g, yield 80%). mp .: 56-59 ° C. FIR (KBr) νmax: 3340 (NH), 1630 (C = O) cm
^-1;¹H-NMR (CDCl_Three) d: 2.75 (s, 3H, -CH_Three), 3.59 (s, 2H, -CH
_Two ), 5.59 (br, 1H,-NH), 7.28-7.31 (m, 5H, -C₆H_Five); MS,
 m / z: 149 (M⁺); Elemental analysis value: C₉H₁₁NO, calculated: C: 70.0
4, H: 8.08, N: 10.21, Actual value: C: 70.18, H: 8.05,
N: 10.07. Example I-1-3: N-ethylphenylacetamide (3
-3) Add 15 g (0.1 mol) of phenylacetyl chloride to 100
Dissolve in ml of benzine and stir at 20-30 ° C.
25 g of ethylamine (0.5 mol) was dissolved in benzine
It was introduced into the liquid. The reaction product is washed with water, and anhydrous sulfuric acid is added.
After drying with nedium, it was concentrated under reduced pressure. Compound residue
Purify by column chromatography by the same procedure of 3-1.
Yellow crystals of N-ethylphenylacetamide were obtained (1
4 g, yield 85%). mp .: 72-74 ° C. FIR (KBr) νmax: 3360 (NH), 1630 (C = O) cm
^-1;¹H-NMR (CDCl_Three) d: 1.02 (t, 3H, -CH_Three), 3.25 (m, 2H, -N-
CH_Two ), 3.50 (s, 2H, -CH_Two-CO-), 7.20-7.33 (m, 5H, -C ₆H_Five);
 MS, m / z: 163 (M⁺); Elemental analysis value: C_TenH₁₃NO, calculated:
C: 73.59, H: 8.03, N: 8.58, Actual value: C: 73.67, H: 8.0
0, N: 8.41. I-2. Synthesis of Alkyl Phenyl Acetate Esters of phenylacetic acid are described in References 52, 5
3 is shown in Scheme 2 in the present invention.
Synthesized by the method.

[0017]

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Where R is lower alkyl. Scheme 2 Phenylacetic acid was esterified by reacting with alcohol in the presence of Lewis acid. Example I-2-1: Methylphenylacetic acid ester (5-
1) 13 g (0.1 mol) of phenylacetic acid was dissolved in 500 ml of anhydrous methanol, and while introducing hydrogen chloride gas, 3
Refluxed for hours. The ester residue obtained by concentration under reduced pressure was dissolved in benzine, and washed successively with water and a 10% aqueous sodium hydroxide solution. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, eluting with chloroflume. A yellow liquid of methylphenylacetic acid ester purified by concentration under reduced pressure was obtained (6 g, yield 40%). bp .: 218 ° C. IR (KBr) νmax: 1700 (C = O) cm ^-1 ; ¹ H-NMR (CDC
l ₃ ) d: 3.62 (s, 2H, -CH ₂ ), 3.70 (s, 3H, -CH ₃ ), 7.30-7.32
_{(m, 5H, -C 6 H} 5); MS, m / z: 150 (M +). Example I-2-2: Emethylphenylacetic acid ester (5-
2) 13 g (0.1 mol) of phenylacetic acid was dissolved in 500 ml of absolute ethanol and reacted and purified by the same procedure as Example I-2-2 to obtain a yellow liquid of ethylphenylacetic acid ester (6.5 g). , Yield 40%). bp .: 227 ° C. IR (KBr) νmax: 1700 (C = O) cm ^-1 ; ¹ H-NMR (CDC
l ₃ ) d: 1.24 (s, 3H, -CH ₃ ), 3.61 (s, 2H, -CH ₂ -CO-), 4.15 (s,
2H, -OCH _2- ), 7.29-7.32 (m, 5H, -C ₆ H ₅ ); MS, m / z: 164
(M ⁺ ). I-3.2,4-dichlorophenylacetic acid, indole-3-acetic acid, and other other differentiation-inducing aids we have found so far were purchased from Sigma Chemistry.

Compounds such as all-trans-retinoic acid, phenylacetic acid, phenylacetyl chloride, butyric acid and hexanoic acid used in the experiments were purchased from Merck. II. Measurement of activity of differentiation-inducing auxiliary agent The activity of the differentiation-inducing auxiliary agent was measured by the procedure developed by Hi et al. (Reference 40). This method was based on measuring the induced differentiation of HL-60 cells with the NBT assay. HL-
60 cells were subcultured at a starting concentration of 1.5 × 10 ⁵ cells / ml. Several sets of five flasks having RA concentrations of 0 to 0.125 μM were prepared, and 10 ml of the culture solution was added to each flask. Since RA was added as a methanol solution, it did not affect the growth and differentiation of HL-60 cells, so the methanol concentration was kept below 2%. One set among them was used as a control, and a differentiation induction auxiliary agent was added to the other set, and its effect was observed. After 96 hours, the number of cells was counted and NBT assay was performed by the method of Reference 40. Control NBT + cells to which neither an inducer nor an adjuvant was added were 4% or less in all, and NBT in a group to which only the differentiation aid was added
+ Cells were always below 10%. The experimental value was the value obtained by subtracting the control value. The ED ₅₀ is the effective concentration of the inducer that results in 50% NBT + cells, which is the NB vs. the concentration of inducer in the presence or absence of adjuvant.
It is the value obtained from the plot of the percentage of T + cells. FIG.
As shown in, ED ₅₀ of RA is a 0.12, ED ₅₀ in the presence of iodide ethidium as 1μM and 2μM adjuvants, respectively 0.056μM and 0.
It became 03 μM. The extinction index of an adjuvant is the ED ₅₀ obtained in the presence of the adjuvant divided by that obtained in the absence. The lower the value, the better the effect as an adjuvant.

The biological activity of the differentiation-inducing auxiliary agent is closely related to its chemical structure. As shown in Fig. 2, butyric acid is MAT
In contrast to being the most effective of the competitive inhibitors, acetic acid has no activity. If a phenyl group is introduced into acetic acid, it tends to have higher activity. Compared to butyric acid,
Hexanoic acid is much less active. Butyric acid, which is highly active in vitro, has an unpleasant odor and is apt to be rapidly metabolized in the body, and is not suitable as a therapeutic drug. Phenylacetate has a slow bio-metabolism but is not highly active and has the drawback of giving an unpleasant odor. The following compounds were selected as excellent differentiation-inducing aids through a series of systematic considerations. II-1. N-Methylphenylacetamide N-methylphenylacetamide was dissolved in methanol and the activity was measured. As shown in Fig. 3, the extinction index is 0.5.
The concentration required to be 0.65 mM. In contrast, phenylacetic acid requires 4 mM.
No effect was observed on the growth of HL-60 cells at 2 mM or less,
Has no unpleasant odor. The only drawback is that it is insoluble in water, which makes it unsuitable for parenteral formulations, but is optimal for oral formulations. II-2. Ethylphenylacetamide Chemistry and biological properties are very similar to methylphenylacetamide, but the activity is rather low. As shown in FIG. 4, when the extinction index is 0.5, the concentration is 0.87 mM.
It is. II-3. Ethylphenylacetic acid ester A yellow liquid with an aroma, which was dissolved in methanol and its activity as an auxiliary agent was measured. As shown in FIG. 5, it is a highly active compound. 10 times more active than phenylacetic acid
To reach the extinction index of 0.5
M concentration is required. Since it is easily hydrolyzed in an acidic state, further enteric coating is required for oral administration as a soft gel agent. II-4.2,4-Dichlorophenylacetic acid It is a colorless crystal having an aroma. The activity was measured as the sodium salt. As shown in FIG. 6, the concentration at which the extinction index reaches 0.5 is 0.3 mM, indicating high activity. Since it is a chloro substitution derivative, its toxicity needs to be examined before it can be used clinically. However, it is judged to be a drug suitable for short-term treatment in patients with terminal cancer. II-5. Indole Acetic Acid By replacing the phenol group with an indole group as shown in FIG. 7, the activity is enhanced and the concentration required to reach the extinction index of 0.5 is only 0.25 mM. Very active auxiliaries themselves are often excellent inducers. As shown in FIG. 8, it is not surprising that it has an activity as an inducer at 0.5 mM or more.

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[Brief description of the drawings]

FIG. 1 shows the enhancing effect of ethidium iodide on RA-induced terminal differentiation, and the concentration of ethidium iodide was 0.0 μM (triangle in the figure) and 1.0 μM, respectively.
(Black circle in the figure) and 2.0 μM (white circle in the figure).

FIG. 2 is a diagram showing the effect of acetic acid, butyric acid, hexanoic acid and phenylacetic acid as differentiation-inducing aids.

FIG. 3 is a graph showing the activity of methylphenylacetamide as a differentiation-inducing auxiliary agent and the cell-inhibitory effect thereof.

FIG. 4 is a graph showing the activity of ethylphenylacetamide as a differentiation-inducing auxiliary agent and the cell-inhibitory effect thereof.

FIG. 5 is a graph showing the activity of phenylacetic acid ethyl ester as a differentiation-inducing auxiliary agent and the cell-inhibitory effect thereof.

FIG. 6 is a graph showing the activity of 2,4-dichlorophenylacetic acid as a differentiation-inducing aid and the cell-inhibitory effect thereof.

FIG. 7 is a graph showing the activity of indoleacetic acid as a differentiation-inducing aid and the cell-inhibitory effect thereof.

FIG. 8 is a graph showing the inducing effect of indole acetate on terminal differentiation.

Claims (4)

(57) [Claims] 1. Methylphenylacetamide, ethylphenylacetamide, phenylacetic acid ethyl ester, which are auxiliary agents for differentiation induction used for differentiation therapy and prevention of cancer.
A novel differentiation-inducing aid used for cancer differentiation therapy and chemoprevention, which has a single composition or a mixed composition selected from 4-dichlorophenylacetic acid and indoleacetic acid. 2. A novel differentiation-inducing auxiliary agent for use in the cancer differentiation therapy and chemoprevention method, which is a pharmaceutical composition according to claim 1, having a dosage form suitable for oral, parenteral or topical administration. 3. A novel differentiation-inducing auxiliary agent for use in the cancer differentiation therapy and chemoprevention method, which is the pharmaceutical composition according to claim 1, further comprising a differentiation inducer used in cancer differentiation therapy as an active ingredient. 4. A novel differentiation-inducing auxiliary agent for use in the cancer differentiation therapy and chemoprevention method, which is the pharmaceutical composition according to claim 1, wherein the cancer represents brain cancer and prostate cancer.