JP2001233894A - Novel terpenoids and medicines utilizing the biological activity of these compounds - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel physiologically active substance that has high inhibitory action against DNA synthetase group, particularly against the reverse transcriptase originating from DNA synthetases α and β, and HIV. SOLUTION: The objective novel terpenoids 1 and 2 have the chemical structures represented by the following formula. These terpenoids manifest high inhibitory action against the reverse transcriptase originating from DNA synthetase α, DNA synthetase β and HIV.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel terpenoid.

[0002]

2. Description of the Related Art DNA synthases of higher organisms are fundamentally involved in cell division, differentiation and the like in living organisms, and are essential elements for maintaining homeostasis of organisms. DNA synthases possessed by higher organisms include DNA synthase α, β, γ,
Six types, δ, ε and ζ, are known, and the characteristics of each enzymatic reaction have been clarified in the last 10 years mainly from in vitro proof.

For example, DNA synthases α and δ are
It is said that DNA synthase γ is involved in NA replication, DNA synthase γ is involved in mitochondrial DNA replication, and DNA synthase ζ is involved in repair. It has also been suggested that the DNA synthases β and ε are also involved in DNA repair, but these enzymes are not capable of recombining DNA chains.
It is also said to be an enzyme deeply involved in n). Here, “recombination” refers to recombination between DNAs in one cell, and is different from “recombination” in genetic engineering in which DNA encoding a specific protein is incorporated into E. coli or the like.

[0004] In order to study the physiological activities of these DNA synthases, substances that selectively inhibit each enzyme are useful. For example, as a compound that selectively inhibits DNA synthase β, dideoxy TTP is known (Tersa et al., Annual Review of Biochemistry 1).
991 60 513-552). However, dideoxy TTP
Is highly toxic in vivo, and no outstanding physiological activity has been observed.

On the other hand, among the enzymes of the DNA synthase group, HI
As is well known, V-derived reverse transcriptase is an enzyme that plays a central role in HIV infection. The ability to inhibit HIV-derived reverse transcriptase to produce a therapeutic effect on HIV has also been demonstrated with commercially available clinical drugs such as azidothymidine (edited by Nippon Pharmaceutical Information Center, Pharmaceutical Co., Ltd.) Published by Hosha, "Medical Drugs, Japan Pharmaceutical Collection 1996", p. 599). However, it is difficult to cope with easily mutated HIV only with existing drugs such as azidothymidine, and development of new inhibitors against HIV-derived reverse transcriptase is desired.

[0006] Many DNA synthetase inhibitors, including dideoxy TTP and azidothymidine, have a D-structure.
An analog of NA that exhibits competitive inhibition in DNA synthesis reactions. Antagonistic inhibitors have good inhibition rates, but have the disadvantage that they are also taken up by various other DNA-metabolizing enzymes and can contribute to side effects on living organisms.

[0007]

DISCLOSURE OF THE INVENTION The present invention is directed to the DNA synthases, particularly the DNA synthases α and β and HI
An object of the present invention is to provide a novel physiologically active substance which non-antagonistically exerts a high inhibitory effect on V-derived reverse transcriptase and a drug using the activity.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that specific terpenoids extracted from basidiomycetes have strong inhibitory activities against DNA synthases α and β and HIV-derived reverse transcriptase. Heading, the present invention has been completed. These terpenoids are new substances.

That is, the present invention provides (1) novel terpenoids 1 and 2 having the following structural formulas:

Embedded image

(2) a DNA synthase α inhibitor comprising at least one of terpenoids 1 and 2 according to (1) as an active ingredient; and (3) at least one of terpenoids 1 and 2 according to (1). DNA synthase β as an active ingredient
Inhibitor, and (4) Terpenoid 1 according to (1)
And an HIV-derived reverse transcriptase inhibitor comprising at least one of the following as an active ingredient:

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The terpenoid 1 of the present invention (hereinafter also referred to as "lucidic acid O") and the terpenoid 2
(Hereinafter, also referred to as “lucidic acid lactone”) will be described in detail.

The lucidic acid O and lucidic acid lactone of the present invention can be obtained from the basidiomycete, Ganod
erma lucidum) can be extracted as follows.

The fruit body of Ganoderma lucidum is ground in a small amount of acetone and then extracted with acetone. Subsequently, acetone is distilled off under reduced pressure to obtain a brown wax-like concentrate. This concentrate is mixed with ethyl acetate / distilled water =
Partitioning was performed under 1/1 (v / v) and pH 7 conditions to obtain an ethyl acetate layer, and the ethyl acetate was distilled off under reduced pressure. This fraction,
The mixture was subjected to silica gel column chromatography, and ethyl acetate: methanol: distilled water = 50: 1: 0.1 (v / v /
Elute with the mixture of v). Among the obtained fractions, DN
Fractions A and B that inhibit the activity of A synthase, for example, DNA synthase β, are collected and concentrated under reduced pressure, respectively.

The fraction B obtained later is subjected to silica gel column chromatography, and eluted with isopropyl alcohol: methanol: distilled water = 5: 1: 0.1 (v / v / v) to obtain the lucidene of the present invention. An acid O is obtained.

On the other hand, the fraction A obtained above was subjected to silica gel column chromatography, and chloroform: methanol: distilled water = 5: 1: 0.1 (v / v / v).
By eluting with the mixture of v), the lucidic acid lactone of the present invention is obtained.

[0016] Mannentake used as a raw material is commercially available, and can be obtained, for example, from Japan Microbial Chemistry Co., Ltd.

The process for producing lucidic acid O and lucidic acid lactone of the present invention is not limited to the above-mentioned extraction method, but includes an extraction solvent, column chromatography,
The elution solvent and other conditions can be appropriately changed.

The lucidic acid O and lucidic acid lactone of the present invention can be obtained by using the DNA synthase α as shown in the following Examples.
And strong inhibitory activity against β and HIV-derived reverse transcriptase. The present invention relates to a medicine utilizing these inhibitory activities, specifically, a DNA synthetase α inhibitor, a DNA synthase β inhibitor and an HIV-derived DNA inhibitor containing at least one of lucidic acid O and lucidic acid lactone as an active ingredient. Also provided are reverse transcriptase inhibitors.

The lucidic acid O and lucidic acid lactone of the present invention can be administered, for example, orally or parenterally. Lucidic acid O and lucidic acid lactone of the present invention,
Pharmaceutical preparations can be prepared by combining with appropriate pharmaceutically acceptable excipients or diluents depending on the administration route.

The dosage forms suitable for oral administration include those in the form of solid, semi-solid, liquid or gas, and specifically, tablets, capsules, powders, granules, solutions, suspensions and the like. Suspending agents, syrups, elixirs and the like can be exemplified, but not limited thereto.

In order to formulate the lucidic acid O and lucidic acid lactone of the present invention into tablets, capsules, powders, granules, solutions, suspensions and the like, using a method known per se, The lucidic acid O and lucidic acid lactone of the present invention are mixed with a binder, a tablet disintegrant, a lubricant and the like, and further, if necessary, a diluent, a buffer, a wetting agent, a preservative,
It can be performed by mixing with a flavoring agent or the like. For example, the binder includes crystalline cellulose, a cellulose derivative, corn starch, gelatin, and the like, the tablet disintegrant includes corn starch, potato starch, sodium carboxymethyl cellulose, and the like, and the lubricant includes talc, magnesium stearate, and the like. And further, conventionally used additives such as lactose, mannitol and the like can be used.

The lucidic acid O and lucidic acid lactone of the present invention can be used in the form of a liquid or a fine powder together with a gas or liquid propellant or, if necessary, a known auxiliary such as a wetting agent. Along with the agent, it may be filled in a non-pressurized container such as an aerosol container and a nebulizer and administered in the form of an aerosol or an inhalant. As a propellant,
A pressurized gas such as dichlorofluoromethane, propane, or nitrogen can be used.

When lucidic acid O and lucidic acid lactone of the present invention are administered parenterally, they can be administered, for example, by rectal administration and injection.

For rectal administration, it can be administered, for example, as a suppository. Suppositories can be prepared by the known methods of lucidic acid O and lactone lactone, in a manner known per se.
Cacao butter, which melts at body temperature but solidifies at room temperature,
It can be formulated by mixing with excipients such as carbon wax and polyethylene glycol and molding.

For administration by injection, it can be administered subcutaneously, intradermally, intravenously, intramuscularly, or the like. These injectable preparations may be prepared, in a manner known per se, by converting the lucidic acid O and the lactone lactone of the invention into aqueous or non-aqueous aqueous solutions such as vegetable oils, synthetic fatty acid glycerides, esters of higher fatty acids, propylene glycol. It can be dissolved, suspended or emulsified in a solvent and further formulated as necessary with conventionally used additives such as a solubilizer, an osmotic pressure regulator, an emulsifier, a stabilizer and a preservative.

In order to form the lucidic acid O and lucidic acid lactone of the present invention in the form of a solution, suspension, syrup, elixir or the like, a pharmaceutically acceptable solution such as sterile water for injection or a normal saline solution. Solvents can be used.

The lucidic acid O and lucidic acid lactone of the present invention can be used as a pharmaceutical preparation in combination with a pharmaceutically acceptable compound having another activity.

DNA synthases α and β containing lucidic acid O and lucidic acid lactone of the present invention as active ingredients
In addition, the HIV-derived reverse transcriptase inhibitor can be appropriately set and adjusted according to the administration form, administration route, degree and stage of the disease to be treated, and the like.

For example, when administered orally, 1 to 100 mg / kg body weight of lucidic acid O or lucidic acid lactone is used.
/ Day, when administered as an injection, lucidic acid O or lucidic acid lactone is 1 to 50 mg / kg body weight / day, when rectally administered, lucidic acid O or lucidic acid lactone is 1 to 5
It can be set to 0 mg / kg body weight / day, but is not limited thereto.

When lucidic acid O and lucidic acid lactone of the present invention are used as a medicament, the stereoisomers, polymorphs, salts and solvates of these compounds may be used as long as the activity of the medicament is not lost. Is included as an active substance.

[0031]

EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1 The fruit body of Ganoderma lucidum was purchased from Nippon Microbial Chemistry Co., Ltd. The purchased Ganoderma lucidum fruit body was collected in Gunma Prefecture and
& Forest Products Res. Inst. (FFPRI)
It has been confirmed to belong to erma lucidum.

[0033] The fruit body of Mannentake (150 g) was ground in a small amount of acetone, and then extracted with 5 liters of acetone (hereinafter, liter is also referred to as "L") for 3 days at room temperature. Subsequently, acetone was distilled off under reduced pressure to obtain a brown wax-like concentrate. This concentrate was partitioned under conditions of ethyl acetate / distilled water = 1/1 (v / v) and pH 7, to obtain an ethyl acetate layer, and ethyl acetate was distilled off under reduced pressure.

This fraction was subjected to silica gel column chromatography (Wakogel C-200, 5.0 × 50c).
m), and ethyl acetate: methanol: distilled water = 50:
The mixture was eluted at a flow rate of 3 mL / min using a mixture of 1: 0.1 (v / v / v), and 20 mL was collected per fraction.

The activity of each fraction to inhibit DNA synthase β was measured by a method according to the method described in Measurement Example 3 below, and each fraction was subjected to two large fractions having an activity (fractions A and B). ) And concentrated under reduced pressure.

The fraction B (fraction No.
105-134, after concentration under reduced pressure: 0.2 g), and further subjected to silica gel column chromatography (Wakogel C-3).
00, 2.0 × 50 cm), and isopropyl alcohol: methanol: distilled water = 5: 1: 0.1 (v / v /
Purification by elution in v) gave lucidic acid O of the present invention.

Fraction A (fraction No. 5) eluted earlier by chromatography on Wakogel C-200
4 to 60, after concentration under reduced pressure: 0.6 g) was subjected to silica gel column chromatography (Wakogel C-300, 2.0).
× 30 cm), chloroform: methanol:
It was eluted with a mixed solution of distilled water = 5: 1: 0.1 (v / v / v) and purified. The fraction previously eluted by this chromatography (fraction Nos. 35 to 41, 1
6.4 mg) to give the lactone lactone of the present invention. The fraction eluted later (fraction N
o. 49-58), it was confirmed that another terpenoid (Celebiterol, Biol. Pharm. Bull. 1999, 22, 111-116) was eluted.

Various physical properties of the lucidic acid O and lucidic acid lactone obtained in Example 1 were measured.

<Measurement Example 1-1> The optical rotation of lucidic acid O (amorphous solid, white powder) obtained in Example 1 was measured.
Optical rotation [α] _D ²³ = + 71 ° (c = 0.2, MeOH)
I got

<Measurement Example 1-2> The lucidic acid O obtained in Example 1 was subjected to high-resolution high-speed atom bombardment mass spectrometry (HR-FA).
BMS).

From the obtained spectrum, HR-FABM
S m / z 475.2712 (MH) was obtained.
This result indicated that the molecular formula of this substance was C ₂₇ H ₄₀ O ₇ .

<Measurement Example 1-3> Lucidic acid O obtained in Example 1 was subjected to the following thin layer chromatography.
Rf value: 0.49 was obtained.

Adsorbent: silica gel plate (Kieselgel 6)
0F ₂₅₄ Merck Co.) Developing solvent: isopropanol: methanol: water = 5:
1: 0.5 <Measurement Example 1-4> The ultraviolet absorption characteristics of lucidic acid O obtained in Example 1 were measured, and λmax (MeOH) nm
(Ε): 253 (7490) was obtained.

<Measurement Example 1-5> ¹ H-NMR spectrum of lucidic acid O obtained in Example 1 was measured using CD ₃ OD as a solvent and TMS as an internal standard.

The following data was obtained from the obtained spectrum.

¹ H NMR (600 MHz in CD ₃ OD): δ 4.96 and
4.95 (1H, br.s, H-19), 4.81 (lH, dd, J = 9.8,
7.3Hz, H-16), 4.62 (lH, dd, J = 9.8, 7.3Hz, H-7),
3.60 (1H, dd, J = 11.7, 4.9Hz, H-3α), 3.52 and 3.31
(1H, d, J = 11.2Hz, H-24), 2.91 (1H, d, J = 15, l
Hz, H-12α), 2.88 (1H, dd, J = 9.3, 9.3Hz, H-14), 2.6
9 (lH, ddd, J = 13.7, 2.9, 2.9Hz, H-1β), 2.44 (2H,
m, H-21), 2.34 (lH, m, H-15β), 2.26 (lH, d, J = 1
5.1 Hz, H-12β), 2.21 and 2.31 (1H, m, H-2 respectively)
0), 2.03 (lH, br.dd, J = 12.7, 7.3 Hz, H-60α), 1.
70 (lH, m, H-15α), 1.69 (1H, m, H-2β), 1.62 (lH,
m, H-2α), 1.57 (lH, ddd, J = 12.7, 12.7, 9.8Hz, H-
6β), 1.39 (lH, br.d, J = 12.7Hz, H-5), 1.31 (3H,
s, H-27), 1.27 (3H, s, H-25), 0.94 (1H, m, H-1
α), 0.79 (3H, s, H-26), 0.72 (3H, s, H-23).

<Measurement Example 1-6> The ¹³ C-NMR spectrum of lucidic acid O obtained in Example 1 was measured using CD ₃ OD as a solvent and TMS as an internal standard.

The following data was obtained from the obtained spectrum.

¹³ C NMR (125 MHz in CD ₃ 0D): δ 202.05
(C-1l), 177.40 (C-22), 161.32 (C-8), 148.35 (C-1
8), 142.89 (C-9), 111.34 (C-19), 73.63 (C-16), 72.
41 (C-3), 69.75 (C-7), 66.16 (C-24), 54.95 (C-17),
52.16 (C-12), 49.3 (C-14), 48.5 (C-13), 43.40 (C-
4), 42,67 (C-5), 39.58 (C-10), 35.52 (C-1), 33.69
(C-15), 34.32 (C-21), 33.55 (C-20), 27.97 (C-2), 1
2.91 (C-23), 28.52 (C-6), 20.18 (C-25), 20.13 (C-2
7), 19.37 (C-26).

<Measurement Example 2-1> The optical rotation of the lactone lucidate (amorphous solid, white powder) obtained in Example 1 was measured, and [α] _D ²⁴ = + 13 ° (C = 0.1 ° C.) , MeOH)
I got

<Measurement Example 2-2> The lucidic acid lactone obtained in Example 1 was subjected to high-resolution high-speed atom bombardment mass spectrometry (HR-FABMS).

From the obtained spectrum, HR-FABM
S m / z 477.2810 (M + H) was obtained, indicating that the molecular formula of this material is C ₂₇ H ₄₀ O ₇ .

<Measurement Example 2-3> The lucidic acid lactone obtained in Example 1 was subjected to the following thin layer chromatography to obtain an Rf value of 0.40.

Adsorbent: silica gel plate (Kieselgel 6)
0F ₂₅₄ Merck) Development solvent: chloroform: methanol: water = 5: 1:
0.5.

<Measurement Example 2-4> The ultraviolet absorption characteristics of the lactone lucidene obtained in Example 1 were measured, and λ max (M
MeOH) nm (ε): 255 (6050) was obtained.

[0056] <Example 2-5 Measurement> For Rushiden acid lactone obtained in Example 1, CDCl _3, using TMS as an internal standard as a solvent, was analyzed by ¹ H-NMR spectrum.

The following data was obtained from the obtained spectrum.

¹ H NMR (600 MHz in CDCl ₃ ): δ 4.87 (1H,
dd, J = 9.3, 7.3 Hz, H-16), 4.54 (lH, dd, 9.8, 7.3
Hz, H-7), 3.58 (2H, s, H-19), 3.15 (lH, dd, J = 11.
7, 4.4Hz, H-3), 2.94 (1H, d, J = 15.1 Hz, H-12α),
2.74 (lH, ddd, J = 18.1, 11.2, 8.8 Hz, H-21), 2.73
(1H, d, J = 15.1 Hz, H-1β), 2.64 (1H, dd, J = 11.0,
7.8 Hz, H-14), 2.55 (lH, ddd, J = 18.l, l0.7, 3.4
Hz, H-21), 2.39 (lH, d.J = 15.1 Hz, H-12β), 2.34
(lH, ddd, J = 13.2, l0.7, 8.8Hz, H-20). 2.20 (lH, dd
d, J = 13.2, 11.2, 3.4Hz, H-20), 2.20 (lH, ddd, J =
14.2, 9.3, 7.8 Hz, H-15β), 2.12 (lH, ddd, J = 12.7,
7.3, 1.0 Hz, H-6α), 1.77 (lH, ddd, J = 14.2, 11.0,
7.3 Hz, H-15), 1.65 (lH, m, H-2α), 1.60 (1H, dd,
12.7, 11.7, 9.8Hz, H-6β), 1.58 (1H, m, H-2β),
1.28 (3H, s, H-27), 1.24 (3H, s, H-25), 1.01 (3H,
s, H-24), 0.98 (3H, s, H-26), 0.96 (lH, m, H-1α),
0.94 (lH, dd, 11.7, 1.0Hz, H-5), 0.83 (3H, s, H-2
3).

<Measurement Example 2-6> The ¹³ C-NMR spectrum of the lactone lucidate obtained in Example 1 was measured using CD ₃ OD as a solvent and TMS as an internal standard.

The following data was obtained from the obtained spectrum.

[0061] ^{13 C NMR (125 MHz in CD} 3 0D): δ 201.49
(C-11), 160.93 (C-8), 143.02 (C-9), 93.41 (C-18),
79.55 (C-22), 78.91 (C-3), 72.77 (C-16), 70.01 (C-
7), 68.37 (C-19), 53.09 (C-12), 50.43 (C-5), 55.05
(C-17), 47.95 (C-13), 47.55 (C-14), 39.76 (C-10),
39.68 (C-4), 35.90 (C-1), 30.82 (C-21), 32.07 (C-1
5), 29.02 (C-6), 28.35 (C-2), 16.35 (C-23), 28.68
(C-24), 27.30 (C-20), 20.62 (C-27), 19.64 (C-25).

<Measurement Example 3> The lucidate O obtained in Example 1 was tested for its inhibitory activity against DNA synthases α and β and HIV-derived reverse transcriptase.

The DNA synthase α used was a sample extracted and purified from bovine thymus by a conventional method. As the DNA synthase β, a sample prepared by incorporating the relevant gene from a rat into Escherichia coli by an ordinary gene recombination method and producing the same was used. HIV-derived reverse transcriptase is commercially available (Worthington Biochemical)
Was used.

For the measurement of the inhibitory effect on these DNA synthases, a general DNA synthase reaction system (edited by the Biochemical Society of Japan, Shinsei Chemistry Laboratory Course 2, Nucleic Acid IV, Tokyo Chemical Dojin, pp. 63-66) Was used. That is, DMSO
Lucidic acid dissolved in water, labeled with a radioisotope [ ³
0.05 U (final concentration) of each enzyme was incubated at 37 ° C. for 60 minutes with a reaction mixture containing [H] -dTTP and DNA template molecules. After the reaction, only [ ³ H] -dTTP incorporated into the DNA template molecule is measured by a liquid scintillation counter, and the value is defined as the amount of synthesized DNA.

The inhibition rate was determined using (a) the synthetic D
The amount of NA and (b) the amount of synthetic DNA in the presence of lucidic acid prepared in Example 1 were evaluated as {(ab) / a} × 100 = inhibition rate (%).

FIG. 1 shows the obtained results.

From the results shown in FIG. 1, the lucidic acid O
DNA synthases α and β and HIV-derived reverse transcriptase
Inhibition is concentration dependent, 50% inhibitory concentration
(IC ₅₀) Are 35 μM, 72 μM and 67 μM, respectively.
M.

<Measurement Example 4> The lucidic acid lactone obtained in Example 1 was subjected to DN treatment in the same manner as in Measurement Example 3 above.
The inhibitory activities of A synthase α, DNA synthase β and HIV-derived reverse transcriptase were tested.

FIG. 2 shows the obtained results.

From the results shown in FIG. 2, the inhibition of lucidic acid lactone against DNA synthase α and β and HIV-derived reverse transcriptase is concentration-dependent, and the 50% inhibitory concentration (IC ₅₀ ) 42 μM, 99 μM and 6
9 μM.

[Brief description of the drawings]

FIG. 1 is a graph showing the enzyme inhibitory activity of lucidic acid O of the present invention.

FIG. 2 is a graph showing the enzyme inhibitory activity of lucidic acid lactone of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI theme coat ゛ (reference) C07J 21/00 C07J 21/00 F term (reference) 4C086 AA01 AA02 AA03 DA10 DA11 ZC20 ZC55 4C091 AA01 BB20 CC01 DD01 EE04 FF02 FF06 GG02 GG13 HH01 JJ03 KK12 LL03 LL04 LL06 LL09 NN01 PA02 PA05 PA13 PB04 QQ01

Claims (4)

[Claims] 1. New terpenoids 1 and 2 having the structural formula shown below. Embedded image 2. The terpenoids 1 and 2 according to claim 1.
A DNA synthase α inhibitor comprising at least one of the following as an active ingredient: 3. The terpenoids 1 and 2 according to claim 1.
A DNA synthase β inhibitor comprising at least one of the following as an active ingredient: 4. The terpenoids 1 and 2 according to claim 1.
An HIV-derived reverse transcriptase inhibitor comprising at least one of the following as an active ingredient: