JPH0348617A - Antiandrogen agent - Google Patents

Abstract

PURPOSE:To obtain an antiandrogen agent, containing a 5-hydroxyflavonoid derivative and effectively applicable to respective symptoms, such as prostatomegaly, cancerous tissue and alopecia capitis totalis, etc. CONSTITUTION:An antiandrogen agent containing at least one compound selected from compounds expressed by formula I (R is OH or H), i.e., pinobanksin in which R is OH, pinocembrin in which R is H and 3,7- dimethylquercetin expressed by formula II as an active ingredient. The aforementioned compound can be obtained by extraction and separation thereof from flowers and buds of poplars with ethanol and has higher inhibiting ability against testosterone-5alpha reductase activity than that of a poplar extract.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to anti-androgens, and more particularly to testosterone-5α-reductase activity inhibitors.

[Prior Art] The mechanism of hormonal action expression in androgen target organs is currently believed to be due to selective uptake of testosterone, reduction of testosterone-5α, and gene activation by the 5α-dehydrotestosterone receptor complex.

In this case, it is known that testosterone exhibits hormonal effects only after it is reduced by 5α-reductase to produce 5α-dihydrotestosterone (hereinafter referred to as 5α-DHT).

Therefore, it is expected to have the potential to treat various symptoms such as prostatic hypertrophy and cancerous tissue that are said to be androgen-dependent, as well as frontal hair loss due to androgen excess, and to inhibit or suppress the hormone action in androgen target organs. There is a demand for the development of various drugs.

Furthermore, poplar plant extract is commercially available as a hair growth agent, but its mechanism of action is not clear.

[Problems to be Solved by the Invention] Under the current situation, the present invention aims to provide an anti-androgen agent that has an effect of inhibiting or suppressing the hormone action in androgen target organs, and in particular, it aims to clarify the hair growth effect of poplar plant extract. The object of the present invention is to provide an anti-androgen agent that inhibits testosterone-5α-reductase activity and suppresses the production of 5α-DHT.

(Means for Solving the Problems) According to the present invention, a 5-hydroxyflavonoid derivative containing at least one of the compounds represented by the following formulas [1], [iii] and [iii], and containing testosterone-5α- An anti-androgen agent characterized by inhibiting reductase activity is provided.

The present invention will be explained in more detail below.

The anti-androgen agent of the present invention has the above formula [1], [il
1 and [ijl, both of which contain one type of 5-hydroxyflavonoid derivative.

Specifically, the compound [11 pinobankcin, the compound [
ii ] 3,7-dimethylquercetin or the compound iii ] pinosemprin.

These 5-=hydroxyflavonoid derivatives can be produced by known synthetic methods. It can also be obtained by extraction and separation from an ethanol extract of poplar flowers and buds (hereinafter simply referred to as poplar extract).

Poplar extract contains 5α-DHT and 4-testosterone.
The anti-androgen compound' of the present invention exhibits 11% inhibition of the enzymatic conversion reaction to androstene-3,7-sion, but the antiandrogen compound' of the present invention has a higher testosterone
Inhibitory ability against 5α reductase activity (hereinafter simply referred to as T-5α
Reductase inhibitory ability. ).

[Example] Below, the above-mentioned compounds [il,
An example of a method for separating [111 and [iii] from a poplar extract will be described. However, the present invention is not limited to this example.

The reagents and analytical instruments used in the examples are as follows.

(1) 50% ethanol poplar extract: manufactured by Mitsubishi Kasei (
2) 4-"C Testosterone (52 mci/mmol) manufactured by New England Nuclear Corporation (3) Non-cariogenic steroid (testosterone, 5α-D)
HT, 4-androstene-3,17-dione, and estradiol (special grade reagents): Tokyo Chemical Industry Co., Ltd. (4) High performance liquid chromatography HPLC-1 column packing material; Nova-PAK cll Rad
ial-PAK mobile phase; A) ammonium acetate (10
nM, pH 4,0) B) Acetonitrile B/A; 0χ-100%, linear gradient ++P
LC-2 column packing material; Nova-PAK C10Radial
-PAK mobile liquid phase; 40χ acetonitrile/ammonium acetate (10mM, p) 14.0) (5) Thin layer chromatography PTLC4: ShiIJ force gel/1zCH30)! -C
HCIIPTLC-2: C'J Power Gel/2% C
HsOH-CHChPTLC-3: Silica gel 15χ
C1l:108-CHCI:1 Silica gel: Silica gel 60Fzs manufactured by Merck & Co. (6) Analytical equipment ■Mass spectrometry JEOL ■i1. rEoLJMS-0300 mass spectrometer IH-NMR and 3C-NMR JEOL JEOL FX-270 (270MHz and 67.8-Hz) Melting point measurement Yanagimoto Microscope Hot Stage manufactured by Yanagimoto Seisakusho Photometric measurement Old P-360 polarimeter manufactured by JASCO Corporation - Separation from poplar extract - Figure 1 shows the extraction and fractionation process from poplar extract and the T-5α reductase inhibition ability (IA and (1)

) measurement results are shown.

Note that IA is a value calculated using formulas (1) and (2) described later.

In Figure 1, 50% ethanol poplar extract 42.
70 g was extracted and fractionated with ethyl acetate and water to obtain 33.10 g of ethyl acetate extract. This ethyl acetate extract showed 5% lAl. Furthermore, 2 of this ethyl acetate extract
mg was taken and subjected to high performance liquid chromatography (hereinafter referred to as HPL).
Let it be C. ) was preliminarily divided into 8 fractions 1-1 to 1-8, and the IA of each fraction was measured. FIG. 1 shows this process diagram and results.

Fractions 1-3.1-4 and 1-5 were 22%, respectively;
It showed an IA of 21% and 44%. As a result of HPLC, it was found that fractions 1-3 and 1-4 mainly consisted of a single compound, and fraction 1-5 contained two compounds.

Based on the above preliminary HPLC results, the ethyl acetate extract was fractionated by silica gel column chromatography and further purified by silica gel thin layer chromatography (referred to as PTLC). A diagram of this fractionation process is shown in FIG.

In FIG. 2, 590 mg of the ethyl acetate extract was fractionated by silica gel column chromatography using chloroform/methanol as a developing agent, and fractionated into fractions 2-1 to 2-6. Among these, large capacity 2-1, 2-2, and 2-
Each fraction of 5 was further purified by PTLC,
Compounds [iv], [mountain] and [il and (ii l
I got it. However, for fraction 2-5, PTLCO
After that, the compound [111 was obtained by high performance liquid chromatography (1 (PLC-2)). In addition, the compound [ivl] obtained from fraction 2-1 had a low solubility in the solvent during the enzymatic conversion reaction. It had a low ability to inhibit T-5α reductase.

Melting point, mass spectrometry, IH-NM for each compound obtained
R and "C-NMR spectra were measured to identify the compound.

In the 'H-NMR and 'C-NMR spectra, the chemical shift (δ) is expressed in ppm from the internal standard tetramethylsilane (TMS).

In addition, in the signals in the spectrum, S indicates a single signal, d indicates a double signal, t indicates a triple signal, q indicates a quadruple signal, m indicates a multiple signal, and dd indicates a double signal.

The results of these analytical measurements were as follows.

[About compounds (il)]

Colorless plate crystals (chloroform recrystallization), melting point 175-
177°C1 [α12: +14.3° (c = 0°5, methanol) H-NMR (measured in DMSO-d6) δ: 4.6
1 (LH, d, J=11.2Hz, Fl-3), 5.1
8 (18, d, J・11.2 fiz, ll-2), 5
．． 88 (IH, d, J=2.0Hz, +(-6), 5.
92 (IH.

d, J=2.0Hz, It-8), 7.30-7.
50 (311, m, H-3', fl-4',
Htl-5), 7.52 (28, d, J=6.111
z, old 2")l-6')"C-NMR (D gate 5o-d6
) δ near 1.5 (d, C-3), 82.8 (d
, C-2), 95. Hd, C-8), 96.0d, C
-6),IOo,3(s,C-10),128.0
(d, C-2C-6'), 128.1 (d, c-3"
, C-5'), 128.6 (d, C-4') 137.
2 (s, C-1'), 162.4 (s, C-9)
, 163.3 (s, C-5), 167.0 (s, C-
7), 197.4 (s, C-4) Mass spectrometry: m/
z = 272 (M”), 243, 153, 120.
91 From the above measurement results, it was confirmed that the compound [il was pinobankcin.

[About compound [ii]]

Yellow needles (methanol recrystallization), melting point 242-24
3°C' H-NMR (measured in DMSO-d6) δ: 3
．． 80 (3Ls, 7-OClh), 3.86 (3t],
s, 3-OCI(z), 6°37(18,d, J=
2.0Hz, tl-6), 6.71 (LH, d, J=
2.0Hz, 118), 6.91 (IH, d, J=8
．． 0Hz, H-5'), 7.49(111,dd
, J=8.0.2.0)1z, t(-6'), 7.
59(IH, d, J=2.0)lz,)l-2')3C
-NMR (measured in DMsO-d&) δ: 56. O
(q, 7-OCHi) , 59.6 (q, 7-OCt1
3), 92.1 (a, c8), 97.6 (d, C
-6), 105.1 (s, C-10), 115.4
(d.

C-2'), 115.6 (d, c-5'), L20.
4 (d, C-6'), 120.6 (s, C-1'),
137.8 (s, C-3), 145.3 (s, C-3'
) 149.0 (s, C-4°), 155.9 (s,
C-2), 156.2 (s, C-5), 160.9
(s, C-9) , 165.0 (s, C-7) , 1
77.9 (s, C-4) Mass spectrometry: m/z =3
30 (M”), 301,287° From the above measurement results,
Compound [ii) was confirmed to be 3,7-synthylquercetin.

[About compound 1] Colorless fine needle crystals (chloroform recrystallization), melting point 20
1-202°C1[α]”: 46.0° (C-0
, 9, acetone)' H-NMR (oMso-d, measured in) δ: 2
．． 78(1)1. dd, J=17. ], 3.0Hz, H
-3α), 3.24 (18°dd, J=12.5,1
7.1Hz, H-3β), 5.58 (18, dd.

J=3.0, 12.5Hz, )I-2), 5.92(
1)1. d, J=2.0Hz, H-6)5.95(11
1, d, J=2.0tlz, ll-8), 7.30-7
．． 50(3t(, m, It-3'.

)1-4', H-5'), 7.52 (2H, d, J=6
．． 6Hz, H-2°, H-6')1''C-N M
R (measured in DMSO-66) δ242.1 (t
C-3), 78.3(d, C-2), '15
．． 0 (d, C-8), 95.9 (d, C-6),
101.8 (s, C-10), 126.5 (d, C-
2'C-6'), 12L5 (d, C-3', C-4°
, C-5'), 128.6 (d.

C-4'), 138.6 (s, C-1"), 162.
7 (s, C-9), 163.2 (s, C-5), 1
66.5 (s, C-7), 195.7 (s, C-
4) Mass spectrometry: m/z = 256 (M"), l'
19,152,124° From the above measurement results, compound [1
iil was confirmed to be pinosemprin.

Testosterone - 5α reductase inhibitory ability - [Maedate 1IJ
i! Liquid method] Wistar normal adult male rat (200-220g)
After cutting the neck, immediately remove the prostate gland and
TRl5 at low temperature 50 (mM), pH 7,4
Si? Night (NaC 161mM, KCl 1.5mM, M
gCl2 ・6Hz01mM, 2+Chin7miF'1
5mM, fumaric acid 1mM. ).

Thereafter, the prostate was taken out from the TRIS buffer, wiped off with a filter paper, weighed, cut into small pieces with scissors, and homogenized using a homogenizer while cooling with ice water after adding 4 times the amount of TRl5 buffer. It became.

Further, the mixture was centrifuged at 11,000 rpm for 3 minutes to separate the supernatant, and the separated pellet was further washed with 1.4 times TRl5 loose fluid and centrifuged again in the same manner. The supernatants obtained in both cases were combined and centrifuged again, and the supernatant obtained here was used as prostatic fluid in the enzyme conversion reaction test.

[Enzyme conversion reaction test] For enzyme conversion reaction! A test tube was prepared in which 4-I4C1 testosterone (0.2 μCi, 3.8 nmol) and unlabeled testosterone (2.9 nmol+) were dissolved in ethanol and injected.

Estradiol as a positive indicator and each extract or fraction dissolved in methanol were added to each of the test tubes prepared above, and then the solvent was evaporated to dryness under a nitrogen stream.

Each residue was dissolved in 50 μl methanol and further 2 d
of TRl5 buffer and 1 mg in 1 mfl of TRl5 buffer.
Dissolved coenzyme nicotinamide adenine dinucleotide phosphate (referred to as NADPI) tetrasodium salt was added.

This mixture was preincubated at 37°C for 5 minutes, the previously prepared prostatic fluid 1+d was added, and the mixture was subsequently incubated at 37°C for 30 minutes in a shaking water bath. The reaction was terminated by the addition of 5-ethyl acetate, followed by shaking at 25 Orpm for 20 minutes and centrifugation at 3000 rpm for 4 minutes.

Thereafter, the ethyl acetate phase and the aqueous phase were separated, and the aqueous phase portion was further extracted twice with 4 mfl of ethyl acetate. Combine with the ethyl acetate phase and add 10ml of saturated aqueous sodium bicarbonate solution!
After washing with 10 ml of a 50% sodium chloride aqueous solution.

The ethyl acetate phase was then dried over 2 g of anhydrous sodium sulfate overnight, filtered, and then evaporated to dryness under reduced pressure at 45°C.

To each of the obtained dry extracts, 200 μg of testosterone, 5α-DHT and 4-androstene-3,17-sion were added as markers, and each was subjected to thin layer chromatography (adsorption layer Nijiri gel/developing agent: Ct(Ch/
CzHsOCzlls=85/15).

The adsorbed layer containing each steroid compound was extracted with dichloromethane at 30 Orpm for 10 minutes, and centrifuged at 3000 rpm for 3 minutes. 1 ml of each methanol extract
Add guasoL2 to 10mft of
loka) Manufacturing method scintillation counter LSC7
Radioactivity was measured at 0.00.

From the measurement of I40, the T-5α reductase inhibitory ability of each sample was calculated using the following formula.

Table 1: T-5α reductase inhibition ability (I
The results of A) are shown in Table 1.

(Hereinafter, blank space) As is clear from these results, compounds [1], [ii
l and [iii I are higher T-5 than poplar extract.
In particular, compound i+j) has an ability to inhibit T-5α reductase comparable to that of estradiol.

Therefore, the compound [il
, (iii and [iii]) are known to inhibit T-5α reductase activity and act as anti-androgens, and since frontal hair loss is thought to be caused by androgen excess, poplar extract It is also found that it has a hair growth effect.

〔Effect of the invention〕

The present invention provides the above formulas [il, 1111 and [ii
A compound containing at least one compound represented by 1, which acts effectively as an anti-androgen agent that inhibits or suppresses hormone action in androgen target organs, and is effective against prostatic hypertrophy, cancer tissue, and the forehead. It can be effectively applied to various symptoms such as hair loss.

[Brief explanation of drawings]

Figure 1 is a diagram showing the fractionation process for fractionating components from a poplar extract containing the compounds constituting the anti-androgen agent of the present invention, and Figure 2 shows the content of the ethyl acetate extract in the poplar extract. It is a diagram of the fractionation process of components.

Claims (1)

[Claims] (1) A 5-hydroxyflavonoid derivative with the following formula [
An anti-androgen agent characterized by containing at least one of the compounds represented by [i], [ii] and [iii] and inhibiting testosterone-5α-reductase activity. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, R in the formula is [i]R=OH, or [iii]
Represents an atom or functional group consisting of R=H. ) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼