JP2001039969A - New compounds with multidrug resistance overcoming effects - Google Patents

Abstract

(57) [Summary] [Object] To obtain a novel compound having an action of overcoming multidrug resistance and effective for overcoming multidrug resistance which is a problem in anticancer drug treatment. Kind Code: A1 Chromomycinone-5-C-glycoside, which is a compound represented by the formula 1, a pharmaceutically acceptable salt thereof, a method for producing the same, and a drug-resistance overcoming agent containing this compound as an active ingredient. Embedded image (In the formula, R represents a structure in which the hydroxyl group at position 1 of the sugar is removed.)

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound, chromomycinone-5-C-, which has a function of overcoming multidrug resistance and is effective for overcoming multidrug resistance which is a problem in anticancer drug treatment. The present invention relates to a drug resistance overcoming agent containing a glycoside and a pharmaceutically acceptable salt thereof as an active ingredient.

[0002]

2. Description of the Related Art In recent years in Japan, cancer has become one of the most socially ill diseases, accounting for about one-quarter of the Japanese deaths. Currently, treatments for cancer include surgery, radiation therapy, and chemotherapy.
These methods alone have not yielded a sufficient therapeutic effect.

[0003] In cancer chemotherapy, it is a serious problem that cancer cells show resistance to drugs used, that is, the development of drug resistance.

[0004] One of the major causes of drug resistance is P
-Glycoproteins. P-glycoprotein is a protein present in the cell membrane and acts as a pump for discharging a drug out of a cell, and has a function of reducing the drug concentration in the cell. It is known that in some cancer cells, the MDR1 gene encoding this P-glycoprotein is highly expressed, and the amount of P-glycoprotein on the cell membrane is increased. In such cancer cells, the intracellular drug concentration is reduced and becomes lower than the effective concentration,
This leads to resistance to the drug. In addition, P-glycoprotein has low substrate specificity, and in cancer cells in which MDR1 is highly expressed, not only the previously used drug but also other drugs are simultaneously acquired, and chemotherapy is acquired. It is often impossible. Such a phenomenon is called multidrug resistance. Drugs that cause cancer cells to become resistant due to multidrug resistance include adriamycin, vinblastine, vincristine, actinomycin D, colchicine, and the like.
Drugs with different chemical structures and mechanisms of action (Molecular mechanism of drug resistance, edited by Takashi Tsuruo, pp10-19, 1994, Yodosha,
Tokyo). Since these are typical drugs frequently used in chemotherapy, the seriousness of the multidrug resistance problem can be understood.

[0005]

In order to solve these problems, a search for a substance that inhibits the function of P-glycoprotein has been conducted.
Development is being attempted. For example, calcium antagonists such as verapamil, cyclosporin A, FK506
It has been reported that immunosuppressants such as P-glycoprotein inhibit the function of P-glycoprotein. However, when these are administered for the purpose of releasing drug resistance, their inherent effects of calcium antagonism and immunosuppression appear as strong side effects and are not suitable for actual administration. Also, SDZPSC833 which is a cyclosporin inducer and M which is a quinoline derivative
Although S-209 and the like are also being developed, they have not yet been applied to clinical applications due to problems with efficacy and toxicity (Molecular mechanism of drug resistance, edited by Takashi Tsuruo, pp20-39, 1994)
Year, Youtosha, Tokyo).

[0006] In recent years, as the analysis of cell functions at the genetic level has progressed, the development of drugs that inhibit the expression of harmful proteins themselves, rather than inhibiting the function of harmful proteins already expressed on cells, has been increasing. It is thriving. For example, Japanese Patent No. 214500 discloses that quercetin, one of the plant components, suppresses an increase in the expression of P-glycoprotein caused by arsenic acid stimulation. Kim et al. Also report that quercetin increases the sensitivity of multidrug-resistant cancer cells to vinblastine and vincrestin (Kim et al., Exp. Mol. Med., 30, 87-92 (1998)). .
However, in Patent No. 214500, the expression of P-glycoprotein is forcibly increased by stimulating the cells with arsenite, and the same effect as that in the resistant cancer cells expressed in the normal human body is observed. The question remains. Furthermore, quercetin is a flavonoid having no sugar in its structure, and is a substance which is hardly dissolved in water and therefore difficult to handle in a pharmaceutical preparation. Furthermore, it is unknown whether or not the concentration of these in vitro experimental results can provide a sufficient effect on humans, such as the clinical summary.

[0007] In addition, humans have various genes, and the properties of cancer cells caused by genetic abnormalities also vary widely. In fact, effective drugs differ greatly from one cancer cell to another, and there are many cancer types for which no effective drug has yet been found, such as certain solid cancers. Clinically, it can be said that even showing better effects on some cancers than conventional drugs is sufficient.

[0008] That is, when a new substance that effectively suppresses the expression of the MDR1 gene and reduces the amount of P-glycoprotein is developed, its industrial utility is immense.

[0009]

DISCLOSURE OF THE INVENTION The present invention provides a chromomycinone-5-C-glycoside having a drug resistance overcoming action, a pharmaceutically acceptable salt thereof, a process for producing the same, and a process for producing the same. Provide an agent for overcoming drug resistance.

As a result of intensive studies, the present inventors have completed a method for introducing a dideoxy sugar, a kind of sugar, into the naphthalene ring portion of chromomycinone, which is known as an aglycone of aureolic acids, and further obtained therefrom. The present inventors have found that a novel chromomycinone-5-C-glycoside suppresses the expression of the MDR1 gene, and completed the present invention.

Accordingly, the present invention provides a chromomycinone-5-C-glycoside having a drug resistance overcoming action, a pharmaceutically acceptable salt thereof, a method for producing the same, and a drug resistance overcoming agent containing the same. provide. The present invention is a compound represented by formula 1, chromomycinone-5-C-glycoside, and a pharmaceutically acceptable salt thereof.

[0012]

Embedded image (In the formula, R represents a structure in which the hydroxyl group at position 1 of the sugar has been removed.
Here, the sugar refers to hexose, pentose, deoxysugar, dideoxysugar, aminosugar and the like. Preferably, R in formula 1 is orolith (formula 2) or oribose (formula 3).

[0013]

Embedded image

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Chromomycinone-5 of the present invention
C-glycosides can be obtained by mildly heating aureolic acids having chromomycinone as a mother nucleus, such as mitramycin and chromomycin, in the presence of methanol and an acid.

The aureolic acids used as raw materials are Streptic
solvent extraction from actinomycetes such as omyces tanashiensis,
It can be isolated using conventional means such as chromatography. It can also be obtained by ordinary chemical synthesis.

The chromomycinone-5-C-glycoside of the present invention has a unique chemical structure (C-glycoside) in which the carbon at position 1 of the sugar is directly bonded to the naphthalene ring portion of the aglycone chromomycinone. Body structure). In nature,
Most glycosides are O-glycosides in which aglycone and sugar are bonded via an oxygen atom, C-glycosides are vitexin,
It is an extremely rare compound species such as sapolenatin and orientin that have been discovered only a few types so far.
Little is known about its biosynthesis. In addition, all of the C-glycosides known so far use flavonoids as aglycones, and those using chromomycinone as aglycones are reported for the first time by the product of the present invention.

In recent years, it has been found that C-glycoside compounds have various physiological activities different from O-glycoside compounds, and attention has been paid to C-glycoside compounds. In the present invention, an extremely simple method for synthesizing chromomycinone-5-C-glycoside, which has not been known so far, is also presented. Law is likely to be valuable.

The chromomycinone-5-C-glycoside of the present invention is characterized in that the aureolic acids as the starting materials are
It is obtained by mixing with an acid in a solvent and heating gently. The stoichiometric ratio at this time is not particularly limited, but the reaction can be carried out with 0.1 equivalent or more of methanol and 1 equivalent or more of acid with respect to 1 equivalent of aureolic acid. The acid is not particularly limited, and inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as trifluoroacetic acid and acetic acid can be used. Examples of the solvent include water, lower alcohols such as ethanol and propanol, acetone, ethyl acetate, chloroform, and TH.
F, DMSO and the like can be used, and a mixture of two or more of them can be used. The presence of methanol is essential in the reaction system.

The heating temperature is not particularly limited.
The temperature is preferably about 100 ° C., and more preferably 30 to 40 ° C. The heating time is not particularly limited, but is preferably 15 minutes or more, preferably about 1 to 4 hours.

The obtained chromomycinone-5-C-glycoside can be purified by a conventional method, for example, reverse phase chromatography, ion exchange chromatography or the like, to give a pure product or a salt thereof.

When the chromomycinone-5-C-glycoside is used as a pharmaceutically acceptable salt, a metal or cation as a salt may be added to an alcohol solution of the compound in addition to a method using an ion exchange resin. There is also a method of dissolving the volatile organic compound. As the ion-exchange resin, a general cation-exchange resin can be used without any particular limitation, and ethanol is suitable as the alcohol, but other solvents do not cause any problem. The pharmaceutically acceptable salts mentioned herein include sodium salts, potassium salts, magnesium salts, aluminum salts, ammonium salts, zinc salts, iron salts, copper salts, cobalt salts, nickel salts, spermine salts, spermidine salts, and taurine. And the like.

When the compound of the present invention is used as an agent for overcoming drug resistance, it can be administered in an appropriate dosage form such as a tablet, capsule, soft capsule, powder, injection or patch. In various formulations with these dosage forms,
Excipients, solubilizers, binders, stabilizers, flavors and the like can be used.

Specific examples of adjuvants that can be mixed with tablets, capsules and the like are as follows. Binders such as tragacanth gum, gum arabic, corn starch or gelatin, excipients such as microcrystalline cellulose, disintegrants such as constarch, fully gelled starch, alginic acid, lubricants such as magnesium stearate, sucrose A sweetener such as lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry, and, when the unit use form is a capsule, containing a liquid carrier such as a fatty oil in addition to the above type of material. Can be. Various other materials may be present as coatings or to otherwise alter the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.

The sterile composition for injection is water for injection, sesame oil,
By dissolving or suspending the active substance in excipients, such as naturally occurring vegetable oils, such as coconut oil, peanut oil, cottonseed oil, and the like, can be formulated according to conventional pharmaceutical practice. Buffers, preservatives, antioxidants and the like can be mixed as necessary.

The medicament of the present invention can be used for intravenous injection, subcutaneous injection,
Various injections such as intramuscular injection or oral administration, can be carried out by various methods such as transdermal administration, particularly preferably oral administration and administration by injection, the dosage is generally oral administration 5 to 900m a day
g, In the case of intravenous administration, it is preferably 1 to 500 mg per day, and may be administered several times a day. The dose generally varies depending on the type and symptoms of the disease caused by drug resistance, the method of administration, and the like, and the dose can be administered outside the above range.

[0026]

The present invention is not limited to the following examples. Example 1 Synthesis of Chromomycinone-5-C-Olibose Form and Chromomycinone-5-C-Oliose Form Mitramycin 110 mg purified from Streptomyces sp.
l, add 10 ml of methanol, and stir at 35 ° C in a water bath.
Left for 1 hour. After the reaction, the reaction solution was dried under reduced pressure, and the dried product was subjected to reverse phase HPLC under the following conditions to measure the absorbance (FIG. 1). The peaks a and b shown in FIG. 1 were collected and freeze-dried. Chromomycinone-5-C-oliose (8 mg) was obtained from peak a, and chromomycinone-5 was obtained from peak b.
The -C-olivoses (14 mg) were each obtained as a yellow powder. HPLC conditions: instrument, Shimadzu LC-8A system; Column, Shimadzu Shim-Pack ODS 20mmI.D. × 25cm; mobile phase, (A solution: B
Liquid) = linear gradient from (70:30) to (30:70) for 40 minutes Liquid A: 10% aqueous acetonitrile containing 0.1% hydrochloric acid, liquid B: 60% aqueous acetonitrile containing 0.1% hydrochloric acid; flow rate, 10 ml / min: detection, UV 220 nm.

Analysis data of chromomycinone-5-C-oliose compound (peak a) mp .: 121-124 ° C. (dec.) HR-FAB-MS (+): M + 1 = 551.2136
(C ₂₇ H ₃₅ O ₁₂ , calc. 551.2128) [α] ²² _D : +91.3 (c = 0.1,
(MeOH) IR (KBr, cm ^-1 ): 1063, 1094, 1188, 1265, 162
9, 3396.UV: λmax (nm) (logε), 420 (3.92), 326 (3.81), 28
2 (4.58), 233 (4.40), λmin (nm) (logε), 358 (3.5
6), 318 (3.77), 251 (4.14), 219 (4.31) ¹ H-NMR [position] (in CD ₃ OD, δppm): [2] 4.48 (1H,
d, J = 11.3Hz), [3] 2.81 (1H, m), [4] 2.74 (1H, m),
3.01 (1H, m), [10] 6.96 (1H, s), [1 '] 4.91 (1H,
m), [3 '] 4.24 (1H, d, J = 2.7Hz), [4'] 4.28 (1H, d
q, J = 2.7, 6.4Hz), [5 '] 1.28 (3H, d, J = 6.4Hz), [O-
Me1 '] 3.48 (3H, s), [6'] 2.16 (3H, s), [C-1] 5.3
1 (1H, dd, J = 1.9, 11.7Hz), [C-2] 2.16 (1H, m), 1.8
2 (1H, m), [C-3] 3.82 (1H, m), [C-4] 3.15 (1H, dd,
J = 9.1, 9.1Hz), [C-5] 3.59 (1H, dq, J = 6.4, 9.1Hz),
[C-6] 1.45 (3H, d, J = 6.4Hz). ¹³ C-NMR [position] (in CD ₃ OD, δppm): [1] 204.2
(s), [2] 72.7 (d), [3] 43.9 (d), [4] 27.8 (t), [5]
109.8 (s), [6] 159.8 (s), [7] 110.8 (s), [8] 156.0
(s), [9] 164.4 (s), [10] 111.4 (d), [4a] 136.6
(s), [8a] 106.4 (s), [9a] 107.0 (s), [10a] 134.9
(s), [1 '] 82.8 (d), [2'] 212.4 (s), [3 '] 79.1
(d), [4 '] 68.3 (d), [5'] 18.7 (q), [OMe1 '] 3.48
(3H, s), [6 '] 7.1 (q), [C-1] 74.7 (d), [C-2] 32.8
(t), [C-3] 69.3 (d), [C-4] 70.2 (d), [C-5] 75.6
(d), [C-6] 16.8 (q).

Instrumental analysis data of chromomycinone-5-C-olibose compound (peak b) mp: 127-131 ° C. (dec.), HR-FAB-MS (+): M + 1 = 551.2141
(C ₂₇ H ₃₅ O ₁₂ , calc. 551.2128) [α] ²² _D : +92.0 (c = 0.
1, MeOH), IR (KBr, cm ^-1 ): 1070, 1186, 1261, 1628,
3396.UV: λmax (nm) (logε), 419 (3.94), 324 (3.76), 28
2 (4.59), 233 (4.40), λmin (nm) (logε), 353 (3.4
6), 317 (3.72), 252 (4.13), 219 (4.31). ¹ H-NMR [position] (in CD ₃ OD, δppm): [2] 4.48 (1H,
d, J = 11.2Hz), [3] 2.77 (1H, m), [4] 2.69 (1H, m),
3.02 (1H, m), [10] 6.93 (1H, s), [1 '] 4.91 (1H,
m), [3 '] 4.24 (1H, d, J = 2.7Hz), [4'] 4.28 (1H, d
q, J = 2.7, 6.4Hz), [5 '] 1.28 (3H, d, J = 6.4Hz), [O-
Me1 '] 3.48 (3H, s), [6'] 2.15 (3H, s), [C-1] 5.5
0 (1H, dd, J = 2.0, 12.0Hz), [C-2] 2.12 (1H, m), 1.8
1 (1H, m), [C-3] 3.82 (1H, m), [C-4] 3.15 (1H, dd,
J = 9.1, 9.3Hz), [C-5] 3.59 (1H, dq, J = 6.4, 9.1Hz),
[C-6] 1.45 (3H, d, J = 6.4Hz). ¹³ C-NMR [position] (in CD ₃ OD, δppm): [1] 204.2
(s), [2] 72.7 (d), [3] 43.9 (d), [4] 27.8 (t), [5]
108.6 (s), [6] 159.4 (s), [7] 110.8 (s), [8] 156.0
(s), [9] 164.6 (s), [10] 111.4 (d), [4a] 136.8
(s), [8a] 106.6 (s), [9a] 107.1 (s), [10a] 134.9
(s), [1 '] 82.8 (d), [2'] 212.3 (s), [3 '] 79.1
(d), [4 '] 68.3 (d), [5'] 18.6 (q), [OMe1 '] 3.48
(3H, s), [6 '] 7.1 (q), [C-1] 75.8 (d), [C-2] 39.0
(t), [C-3] 71.8 (d), [C-4] 77.7 (d), [C-5] 77.6
(d), [C-6] 17.6 (q).

Example 2 (Preparation of Chromomycinone-5-C-oliose Form Potassium Salt) 5 mg of chromomycinone-5-C-oliose form was dissolved in 1 ml of ethyl alcohol, and 0.5 mg of metal potassium was added. . After leaving at room temperature for 30 minutes, the reaction solution was filtered, and ethyl alcohol was distilled off under reduced pressure to give chromomycinone-5.
5.5 mg of -C-olibose potassium salt was obtained.

Example 3 (Preparation of Chromomycinone-5-C-oliose Form Sodium Salt) Chromomycinone-5-C-oliose Form Potassium Salt 5m
g was dissolved in 1 ml of distilled water and passed through 2.5 ml of a Na-type cation exchange resin (Dowex 50W-X8 manufactured by Dow Chemical Company). The passing solution was concentrated to dryness under reduced pressure to obtain 6 mg of chromomycinone-5-C-oliose sodium salt.

Example 4 (Tablets, Capsules) Chromomycinone-5-C-oliose Form 0.2 Lactose 7.5 Magnesium Stearate 2.3 Total 10.0 The above parts by weight (g) were uniformly used. To make tablets and capsules according to a conventional method. Chromomycinone-5
Similarly, tablets and capsules were prepared for the C-olivoses and their potassium and sodium salts.

Example 5 (Powder, Granule) Chromomycinone-5-C-oliose 0.5 Starch 4.5 Lactose 5.0 Total 10.0 The above parts by weight (g) are uniformly mixed. Then, powders and granules were prepared according to a conventional method. Chromomycinone-5-C-
The olivoses and their potassium and sodium salts were similarly powdered and granulated.

Example 6 (Injection) Chromomycinone-5-C-oliose 0.1 Surfactant 0.9 Physiological saline 9.0 Total 10.0 Heat the above parts by weight (g) It was mixed and sterilized to give an injection. In addition, the chromomycinone-5-C-olibose derivative and its potassium salt and sodium salt were similarly used as injections.

[0034]Example 7(MDR1 expression inhibitory effect) In RPMI1640 medium containing 2 μM adriamycin
Cultured human small cell lung cancer cells SBC-3 / ADM
The obtained chromomycinone-5-C-olibose form and
Lomomycinone-5-C-oliose form is 0.1-0.6 mg /
ml, and in the same medium at 37 ° C in an atmosphere containing 5% carbon dioxide.
Cultured for 48 hours. After culturing, collect the cells and follow the routine
Was extracted. MDR1 and β as a control by RT-PCR
_Two−m (β _Two-Microglobulin) after amplifying the RNA
After electrophoresis, the expression levels of MDR1 were compared. The result
As shown in FIG. In FIG. 2, lane 1 is a control (no sample added).
Lane 2 and 3 are chromomycinone-5-C-O
When adding 0.1 mg / ml and 0.3 mg / ml ribose, respectively,
And chromomycinone-5-C-oliose body
Of MDR1 (0.1 mg / ml and 0.6 mg / ml, respectively)
Stage) and β_Two-M (lower) expression level by antibody staining
Is shown.

Chromomycinone-5-C-olivose and chromomycinone-5-C-oliose are MD
Although the expression of R1 was suppressed, the expression of the control β ₂ -m was not suppressed. That is, the expression of MDR1 was selectively and nontoxicly suppressed. That is, it was revealed that the present compound has an activity of releasing multidrug resistance caused by the MDR1 gene.

As described above, the chromomycinone of the present invention
5-C-glycoside and its salt have low cytotoxicity, suppress the expression of the MDR1 gene in multidrug-resistant cancer cells, and have an action of overcoming multidrug resistance. Therefore, the chromomycinone-5-C-glycoside of the present invention and its salt are useful as an agent for overcoming multidrug resistance.

[Brief description of the drawings]

FIG. 1 shows Chromomycinone-5-C-
Oribose (a) and chromomycinone-5-C-
Reverse phase HPLC chromatogram of the purified oriose (b).

FIG. 2 is an electrophoretic diagram showing the MDR1 expression-suppressing effect of chromomycinone-5-C-olivose and chromomycinone-5-C-oliose in cells overexpressing P-glycoprotein in Example 7.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mika Nakao 1-1-21 Midori, Moriya-cho, Kitasoma-gun, Ibaraki Asahi Breweries Co., Ltd. 1-1-21 Asahi Breweries, Ltd. Food and Drug Laboratories (72) Inventor Kazunori Ochiai 1-2-5-6 Shinmachi, Setagaya-ku, Tokyo (72) Inventor Seiji Isonishi 3-5-7, Narita Nishi, Suginami-ku, Tokyo (72) Inventor Aiko Okamoto 4-5-2 Igusa, Suginami-ku, Tokyo F-term (reference) 4C062 AA18 4C086 AA01 AA03 AA04 BA07 MA01 MA04 NA14 ZB26 4H039 CA61 CD10

Claims (4)

[Claims] 1. A chromomycinone-5-C-glycoside which is a compound represented by the formula 1, and a pharmaceutically acceptable salt thereof. Embedded image (In the formula, R represents a structure in which the hydroxyl group at position 1 of the sugar is removed.) 2. The chromomycinone-5 according to claim 1, wherein R in the formula 1 is oliose (formula 2) or oliboose (formula 3).
-C-glycosides and pharmaceutically acceptable salts thereof. Embedded image 3. A chromomycinone-5-C-glycoside characterized by heating an aureolic acid having chromomycinone as a mother nucleus in the presence of methanol and an acid, and a pharmaceutically acceptable pharmaceutically acceptable salt thereof. Method for producing salt. 4. An agent for overcoming multidrug resistance comprising, as an active ingredient, chromomycinone-5-C-glycoside and a pharmaceutically acceptable salt thereof.