JP4022360B2 - New bioactive substance - Google Patents

Description

(2). モルティエレラ・アルピナ・エムイーアール・エフ2368(Mortierella alpina Mer-f2368, FERM P-17653)を栄養培地中で培養し、その培養液から(1)に記載の化合物を採取する事を特徴とする、(1)に記載の化合物の製造方法、
に関する。
【０００５】
【発明の実施の形態】
本発明について、１．生産菌のスクリーニング法、２．分離された生産菌の性状、３．生産菌の培養法、４．活性物質の精製法、５．活性物質の抗真菌剤としての利用法の順に詳細に説明する。
【０００６】
１．生産菌のスクリーニング法
哺乳類細胞、好ましくは真菌が接着する細胞である腸管上皮細胞を培養し、適当な方法、例えばエタノールにより固定する。そこへ被検サンプルと、適当な時間インキュベートしたカンジダ・アルビカンスを接種し、一定時間培養後バッファーで洗浄して、寒天培地、例えばサブロー・デキストロース寒天培地（Difco）を重層する。30℃一晩培養後、CFUをカウントし、付着率を計算する。
【０００７】
２．分離された生産菌の性状
本発明化合物の精製原料として、モルティエレラ属の菌種いずれも使用可能であると期待されるが、本発明の代表的な菌株として、神奈川県で採取された土壌より分離された菌株で、本発明者らがエムイーアール・エフ2368(Mer-f2368)と番号を付した菌株が挙げられる。このエムイーアール・エフ2368菌株の菌学的性状は次の通りであった。
培養は25℃で行った。培地はポテトデキストロース寒天培地を用いた。
本菌株の栄養菌糸はよく発達し、気生菌糸を形成した。気生菌糸より生じる胞子嚢柄は分枝せず、長さ80μmほどで、先端に向け細くなる傾向があった。胞子嚢は直径15μmの亜球形を成し、胞子嚢膜は平滑で溶菌により胞子を放出した。
胞子は無色の単細胞で平滑を呈し、楕円形から腎臓型を示し、大きさは2.5〜4×1.5〜2μmであった。
以上の形態的特性より本菌はモルティエレラ・アルピナ(Mortierella alpina)種であると同定した。本発明者らは、本菌をモルティエレラ・アルピナ・エムイーアール・エフ2368(Mortierella alpina Mer-f2368)として工業技術院生命工学工業技術研究所に FERM P-17653の番号で寄託している。
【０００８】
３．生産菌の培養法
上記微生物の培養方法は、通常は液体培養による振盪培養法、通気攪拌培養法などの好気的条件下で行なうのが好適である。
培養に用いられる培地としては、モルティエレラ属に属する微生物が利用できる栄養源を含有する培地であればよく、各種の合成培地、半合成培地、天然培地などいずれも用いることができる。培地組成としては炭素源としてのグルコース、シュークロース、フルクトース、グリセリン、デキストリン、澱粉、糖蜜、コーン・スティープ・リカー、有機酸などを単独または組み合わせて用い得る。窒素源としてはファーマメデイァ、ペプトン、肉エキス、酵母エキス、大豆粉、カゼイン、アミノ酸、尿素などの有機窒素源、硝酸ナトリウム、硫酸アンモニウムなどの無機窒素源を単独または組み合わせて用い得る。
ナトリウム塩、カリウム塩、マグネシウム塩、燐酸塩、その他の重金属塩なども必要に応じて添加使用され得る。なお、培養中発泡の著しいときは公知の各種消泡剤を適宜培地中に添加することもできるが、その添加は目的物質の生産に悪影響をあたえないものとする必要がある。
培地のpHは微生物の至適pH範囲、通常中性付近とするのが望ましい。培養温度は、微生物が良好に生育する温度、通常20〜40℃、特に好ましくは25℃付近に保つのがよい。培養時間は液体培養の場合、1〜14日間程度とされる。
上述した各種の培養条件は、使用微生物の種類や特性、外部条件などに応じて適宜変更でき、またそれぞれに応じて上記範囲から最適条件を選択、調節される。
【０００９】
４．活性物質の精製法
培養終了後、培養液から化合物EM-f2368を採取するためには、一般に微生物代謝産物をその培養液から単離するために用いられる分離、精製の方法が利用できる。例えば、ブタノール、酢酸エチル、クロロホルム等を用いた有機溶媒抽出、各種のイオン交換クロマトグラフィー、セファデックスLH-20等を用いたゲル濾過クロマトグラフィー、活性炭、シリカゲル等による吸着クロマトグラフィー、もしくは薄層クロマトグラフィーによる吸脱着処理、あるいは逆相カラム等を用いた高速液体クロマトグラフィー等の公知のあらゆる方法がこれにあたる。また、ここに示した方法に特に限定されるのもではない。
これらの方法を単独あるいは任意の順序に組み合わせ、また反復して用いることにより、化合物EM-f2368を単離、そして採取することができる。
【００１０】
５．活性物質の抗真菌剤としての利用法
単離した化合物は真菌の付着阻害活性を有していた。従って該化合物は、第一に真菌感染症のリスクファクターの高い患者さんへの予防投与、第二に難治性真菌感染症に対して既存の抗真菌剤との併用使用が想定される。
該化合物を各種疾患治療・予防剤として投与する場合、錠剤、散剤、顆粒剤、カプセル剤、シロップ剤などとして経口的に投与してもよいし、また噴霧剤、坐剤、注射剤、外用剤、点滴剤として非経口的に投与してもよい。投与量は症状の程度、年齢、肝疾患の種類などにより著しく異なるが、通常成人１日当たり約 1mg〜100mg を１日１〜数回にわけて投与する。
製剤化の際は通常の製剤担体を用い、常法により製造する。すなわち、経口用固形製剤を調製する場合は、主薬に賦形剤、更に必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤などを加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤などとする。これらの錠剤、顆粒剤には糖衣、ゼラチン衣、その他必要により適宜コーティングすることは勿論差し支えない。
注射剤を調製する場合には、主薬に必要によりpH調整剤、緩衝剤、安定化剤、可溶化剤などを添加し、常法により皮下、筋肉内、静脈内用注射剤とする。
【００１１】
【発明の効果】
本発明により、真菌の付着を抑制し病原性を減弱させることが可能となった。
【００１２】
【実施例】
以下の実施例により本発明をさらに詳細に説明するが、本発明はこれらに限定されるものではない。
【００１３】
［実施例１］精製方法
モルティエレラ・アルピナ・エムイーアール・エフ2368(Mortierella alpina Mer-f2368)株の斜面培地（ポテト・デキストロース寒天培地）から１白金耳を20 mlの種培地（馬鈴薯デンプン2％、グルコース1％、大豆粉2％、リン酸第一カリウム0.1％、硫酸マグネシウム７水和物0.05％、pH 無調整）を入れた250ml容の三角フラスコに接種し、25℃で３日間回転攪拌機上で培養して種培養液を得た。
この種培養液1mlを上記と同じ組成の培地60mlを含む500ml容の三角フラスコに接種して、25℃で96時間回転攪拌機上で培養を行なった。
培養終了後、得られた培養液3Lを3Lのn-ブタノールで抽出した。得られたn-ブタノール抽出物を、減圧下で濃縮し粗抽出物3.0gを得た。
この粗抽出物を少量のジメチルスルホキシドに溶解し、ODSカラムクロマトグラフィー（YMC-GEL ODS-AM 120-S50（ワイエムシィ社製)、内径50mm、長さ500mm）に付した。30％アセトニトリルで洗浄し、続いて、50％アセトニトリルで溶出した。溶出液は高速液体クロマトグラフで分析し、 EM-f2368（カラム：J’sphere ODS-H80、内径4.6mm、長さ75mm、ワイエムシィ社製、移動層：アセトニトリル−0.01％TFA＝30:70、流速：1ml/min、検出：210nmにおける紫外吸収、保持時間；7.6 min）を含む溶出液を集め、 減圧下濃縮乾固してEM-f2368粗抽出物を得た。
得られた粗抽出物を少量のジメチルスルホキシドに溶解し、分取高速液体クロマトグラフィー（カラム：J’sphere ODS-H80、内径20mm、長さ250mm、ワイエムシィ社製、移動層：アセトニトリル−0.01％TFA＝30:70、流速：10ml/min、検出：210nmにおける紫外吸収、保持時間；30.0min）に付した。この分取をくりかえし、 EM-f2368を含む溶出液をそれぞれ併せ、減圧下濃縮乾固してEM-f2368の純粋な白色粉末を10.0mg得た。
【００１４】
［実施例２］EM-f2368の理化学的性質
１．色および性状：白色粉末
２．分子式：C₄₃H₆₀N₁₀O₈
３．分子量：844
４．紫外部吸収スペクトル：
λ_max(MeOH, nm(ε))：280 (1400)
５．赤外部吸収スペクトル：
（KBr, cm^-1）3260, 1680, 1540, 1440, 1400, 1210, 1140, 840, 800, 730
６．¹H核磁気共鳴スペクトル
（600MHz, DMSO-d₆）, δppm:
10.73(1H,bs),9.11(1H,s),8.43(1H,bs),7.97(1H,d,7.4),7.91(1H,bs),7.86(1H,d,8.5),7.72(1H,bs),7.52(1H,d,7.4),7.30(1H,d,8.2),7.26(2H,dd,7.4,6.6),7.23(2H,d,7.4),7.18(1H,dd,6.6,6.6),7.13(1H,bs),7.03(1H,dd,8.2,7.4),6.94(1H,dd,7.4,7.4),6.54(1H,bs),6.43(1H,bs),4.62(1H,m),4.34(1H,bs),4.24(1H,m),4.20(1H,m),4.11(1H,m),3.21(1H,dd,15.2,5.0),3.15(1H,m),3.04(1H,m),2.99(1H,m),2.77(1H,m),1.91(1H,m),1.84(3H,s),1.64(1H,m),1.53(3H,d,6.9),1.50(1H,m),1.40(2H,m),1.22(1H,m),1.14(2H,m),0.81(3H,d,6.5),0.80(3H,d,6.5),0.73(3H,d,6.3),0.70(3H,d,6.3)
７．溶解性：
可溶： ジメチルスルホキシド
不溶：ヘキサン、酢酸エチル
８．高速液体クロマトグラフィー分析条件：
カラム：J’sphere ODS-H80、内径4.6mm、長さ75mm（ワイエムシィ社製）
溶媒：アセトニトリル−0.01％TFA＝30:70
流速：1ml/min
検出：210nmにおける紫外部吸収
保持時間：7.6min
【００１５】
［実施例３］f2368の真菌付着阻害活性
6穴マルチウェルプレートの各穴に10％牛胎児血清および2mMグルタミンを含むD-MEM培地（日水製薬）で1×10⁵個/mlに調整したIEC-18細胞を3mlずつ分注した。該プレートを炭酸ガスインキュベータ内で37℃、3日間培養後、培養上清を除去し、エタノール固定した。各濃度のf2368含有サブロー・デキストロース液体培地で30℃、48時間培養したカンジダ・アルビカンスを4×10²個/mlに調整し、該プレートの各穴に1ml接種した。30℃、1時間培養後、培養上清を除去し、PBSで洗浄後、サブロー・デキストロース寒天培地（Difco）を2ml重層した。30℃、一夜培養後、CFUをカウントし、付着率を算出した。その結果を図１に示す。
f2368は、カンジダ・アルビカンスのIEC-18細胞への付着を阻害する活性を示した。
【図面の簡単な説明】
【図１】 f2368の濃度とカンジダ付着率との関係を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a drug that inhibits fungal adhesion and inhibits fungi from exerting pathogenicity.
[0002]
[Prior art]
In recent years, countermeasures against opportunistic infections have become increasingly important due to an increase in the number of patients and elderly people whose immune functions have been reduced by advanced chemotherapy and the like. Visceral fungal infections caused by Candida, Aspergillus, Cryptococcus, etc. are a part of these opportunistic infections, the incidence is increasing year by year, and despite being a direct cause of death, there are few effective therapeutic agents.
Conventional antifungal treatments have been centered on the strategy of chemically modifying known skeletons to develop new compounds, but there is a problem of resistant bacteria, and the development of new drugs based on new mechanisms is eagerly desired.
As one promising approach, attention has been paid to attempts to prevent pathogenicity from being exerted by inhibiting the attachment of pathogens to the host. Recent studies have also reported multiple associations between adhesion and infection (Janet F et al., Science, 283: 1535-1538 (1999), Ed T. Buurman et al., Proc. Natl. Acad. Sci. USA, 95: 7670-7675 (1998), Cheryl A. Gale et al., Science, 279: 1355-1358 (1998)).
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a therapeutic agent for fungal infection by isolating a novel substance that inhibits fungal cell adhesion and inhibits fungal pathogenicity.
[0004]
[Means for Solving the Problems]
In view of the above situation, the present inventors conducted screening screening for substances that inhibit fungal cell attachment using a microorganism culture solution as a raw material. As a result, it has been found that substances that inhibit the adhesion of fungi to cells are produced in the culture solution of microorganisms belonging to the genus Mortierella. As a result of isolation and structure determination of this active substance, it was found to be a novel active substance.
That is, the present invention
(1). A compound represented by formula (I).
[Chemical 2]

(2) . Mortierella alpina Mer-f2368, FERM P-17653) is cultured in a nutrient medium, and the compound described in (1) is collected from the culture solution. A method for producing the compound according to (1),
About.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
About the present invention: 1. Screening method for production bacteria 2. Properties of the isolated production bacteria; 3. Culture method of production bacteria 4. Purification method of active substance It demonstrates in detail in order of the utilization method as an antifungal agent of an active substance.
[0006]
1. Screening method of producing bacteria Intestinal epithelial cells, which are cells to which mammalian cells adhere, preferably fungi adhere, are cultured and fixed by an appropriate method, for example, ethanol. A test sample and Candida albicans incubated for an appropriate time are inoculated there, and after incubation for a certain period of time, washed with a buffer and overlaid with an agar medium, for example, Sabouraud dextrose agar medium (Difco). After incubation at 30 ° C overnight, count CFU and calculate the adhesion rate.
[0007]
2. Properties of the isolated production bacteria As a purification raw material of the compound of the present invention, it is expected that any species of the genus Mortierella can be used, but as representative strains of the present invention, from soil collected in Kanagawa Prefecture Among the isolated strains, the strains numbered by the present inventors as M-F2368 may be mentioned. The mycological properties of this MRF 2368 strain were as follows.
The culture was performed at 25 ° C. As the medium, potato dextrose agar medium was used.
The vegetative mycelium of this strain developed well and formed aerial hyphae. The spore sac produced from aerial hyphae was not branched, was about 80 μm long, and tended to narrow toward the tip. The spore sac was submicron with a diameter of 15 μm, and the spore sac was smooth and released spores by lysis.
The spores were colorless single cells that were smooth, oval to kidney-shaped, and the size was 2.5-4 × 1.5-2 μm.
Based on the above morphological characteristics, the bacterium was identified as Mortierella alpina. The present inventors have deposited this bacterium as Mortierella alpina Mer-f2368 at the Institute of Biotechnology, Institute of Industrial Science under the number FERM P-17653.
[0008]
3. Culture method of producing microorganism The above-mentioned microorganism culture method is usually preferably carried out under aerobic conditions such as a shaking culture method by liquid culture and an aeration stirring culture method.
The medium used for the culture may be a medium containing a nutrient source that can be used by microorganisms belonging to the genus Mortierella, and any of various synthetic media, semi-synthetic media, natural media, and the like can be used. As the medium composition, glucose as a carbon source, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, organic acid and the like may be used alone or in combination. As the nitrogen source, organic nitrogen sources such as pharmame media, peptone, meat extract, yeast extract, soybean flour, casein, amino acid, urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can be used alone or in combination.
Sodium salts, potassium salts, magnesium salts, phosphates, other heavy metal salts, and the like may be added and used as necessary. It should be noted that when foaming is significant during culture, various known antifoaming agents can be added to the medium as appropriate, but the addition should not adversely affect the production of the target substance.
The pH of the medium is preferably in the optimum pH range of the microorganism, usually near neutral. The culture temperature is preferably maintained at a temperature at which microorganisms grow well, usually 20 to 40 ° C, particularly preferably around 25 ° C. In the case of liquid culture, the culture time is about 1 to 14 days.
The various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms used, external conditions, and the like, and optimum conditions are selected and adjusted from the above ranges according to each.
[0009]
4). Purification method of active substance In order to collect the compound EM-f2368 from the culture solution after completion of the culture, separation and purification methods generally used for isolating microbial metabolites from the culture solution can be used. For example, organic solvent extraction using butanol, ethyl acetate, chloroform, etc., various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20, etc., adsorption chromatography using activated carbon, silica gel, etc., or thin layer chromatography Any known method such as adsorption / desorption treatment by chromatography or high performance liquid chromatography using a reverse phase column or the like corresponds to this. Moreover, it is not specifically limited to the method shown here.
Compound EM-f2368 can be isolated and collected by using these methods alone, in any order, or repeatedly.
[0010]
5). Utilization of active substance as antifungal agent The isolated compound had fungal adhesion inhibitory activity. Therefore, it is envisaged that the compound is first used for preventive administration to patients with a high risk factor of fungal infection, and secondly used in combination with an existing antifungal agent for intractable fungal infection.
When the compound is administered as a therapeutic or prophylactic agent for various diseases, it may be administered orally as tablets, powders, granules, capsules, syrups, etc., and sprays, suppositories, injections, and external preparations. Alternatively, it may be administered parenterally as an infusion. The dose varies significantly depending on the degree of symptom, age, type of liver disease, etc., but about 1 mg to 100 mg per adult is usually administered once to several times a day.
In formulating, it is produced by a conventional method using an ordinary pharmaceutical carrier. That is, when preparing an oral solid preparation, after adding an excipient to the main ingredient, and further adding a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, etc., if necessary, a tablet by a conventional method, Coated tablets, granules, powders, capsules, etc. Of course, these tablets and granules may be appropriately coated with sugar coating, gelatin coating, etc. if necessary.
When preparing an injection, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. are added to the main drug as necessary, and an injection for subcutaneous, intramuscular or intravenous use is prepared by a conventional method.
[0011]
【The invention's effect】
According to the present invention, it has become possible to suppress fungal adhesion and attenuate pathogenicity.
[0012]
【Example】
The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.
[0013]
[Example 1] Purification method From a slant medium (potato dextrose agar medium) of Mortierella alpina Mer-f2368 strain, 1 platinum ear is added to 20 ml of seed medium (potato starch 2% Inoculated into a 250 ml Erlenmeyer flask containing 1% glucose, 2% soybean powder, 0.1% potassium phosphate, 0.05% magnesium sulfate heptahydrate, pH unadjusted), and a rotary stirrer at 25 ° C for 3 days The seed culture solution was obtained by culturing above.
1 ml of this seed culture was inoculated into a 500 ml Erlenmeyer flask containing 60 ml of the medium having the same composition as described above, and cultured on a rotary stirrer at 25 ° C. for 96 hours.
After completion of the culture, 3 L of the obtained culture broth was extracted with 3 L of n-butanol. The obtained n-butanol extract was concentrated under reduced pressure to obtain 3.0 g of a crude extract.
This crude extract was dissolved in a small amount of dimethyl sulfoxide and subjected to ODS column chromatography (YMC-GEL ODS-AM 120-S50 (manufactured by YMC), inner diameter 50 mm, length 500 mm). Wash with 30% acetonitrile followed by elution with 50% acetonitrile. The eluate was analyzed by high performance liquid chromatography. EM-f2368 (column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75 mm, manufactured by YMC, moving bed: acetonitrile-0.01% TFA = 30:70, flow rate : 1 ml / min, detection: ultraviolet absorption at 210 nm, retention time; 7.6 min) was collected, and concentrated to dryness under reduced pressure to obtain a crude extract of EM-f2368.
The obtained crude extract was dissolved in a small amount of dimethyl sulfoxide, and preparative high performance liquid chromatography (column: J'sphere ODS-H80, inner diameter 20 mm, length 250 mm, manufactured by YMC Co., Ltd., moving bed: acetonitrile-0.01% TFA) = 30:70, flow rate: 10 ml / min, detection: UV absorption at 210 nm, retention time: 30.0 min). This fractionation was repeated, and the eluates containing EM-f2368 were combined and concentrated to dryness under reduced pressure to obtain 10.0 mg of pure white powder of EM-f2368.
[0014]
[Example 2] Physicochemical properties of EM-f2368 Color and properties: white powder Molecular formula: C ₄₃ H ₆₀ N ₁₀ O ₈
3. Molecular weight: 844
4). Ultraviolet absorption spectrum:
λ _max (MeOH, nm (ε)): 280 (1400)
5). Red external absorption spectrum:
(KBr, cm ^-1 ) 3260, 1680, 1540, 1440, 1400, 1210, 1140, 840, 800, 730
6). ¹ H nuclear magnetic resonance spectrum _{(600MHz, DMSO-d 6)} , δppm:
10.73 (1H, bs), 9.11 (1H, s), 8.43 (1H, bs), 7.97 (1H, d, 7.4), 7.91 (1H, bs), 7.86 (1H, d, 8.5), 7.72 (1H, bs), 7.52 (1H, d, 7.4), 7.30 (1H, d, 8.2), 7.26 (2H, dd, 7.4, 6.6), 7.23 (2H, d, 7.4), 7.18 (1H, dd, 6.6, 6.6) ), 7.13 (1H, bs), 7.03 (1H, dd, 8.2,7.4), 6.94 (1H, dd, 7.4,7.4), 6.54 (1H, bs), 6.43 (1H, bs), 4.62 (1H, m ), 4.34 (1H, bs), 4.24 (1H, m), 4.20 (1H, m), 4.11 (1H, m), 3.21 (1H, dd, 15.2,5.0), 3.15 (1H, m), 3.04 ( 1H, m), 2.99 (1H, m), 2.77 (1H, m), 1.91 (1H, m), 1.84 (3H, s), 1.64 (1H, m), 1.53 (3H, d, 6.9), 1.50 (1H, m), 1.40 (2H, m), 1.22 (1H, m), 1.14 (2H, m), 0.81 (3H, d, 6.5), 0.80 (3H, d, 6.5), 0.73 (3H, d , 6.3), 0.70 (3H, d, 6.3)
7). Solubility:
Soluble: Dimethyl sulfoxide Insoluble: Hexane, ethyl acetate High-performance liquid chromatography analysis conditions:
Column: J'sphere ODS-H80, inner diameter 4.6mm, length 75mm (manufactured by YMC)
Solvent: acetonitrile-0.01% TFA = 30: 70
Flow rate: 1ml / min
Detection: UV absorption retention time at 210 nm: 7.6 min
[0015]
[Example 3] Fungal adhesion inhibitory activity of f2368
3 ml of IEC-18 cells adjusted to 1 × 10 ⁵ cells / ml with D-MEM medium (Nissui Pharmaceutical) containing 10% fetal bovine serum and 2 mM glutamine were dispensed into each well of a 6-well multiwell plate. After culturing the plate at 37 ° C. for 3 days in a carbon dioxide incubator, the culture supernatant was removed and ethanol fixed. Candida albicans cultivated for 48 hours at 30 ° C. in a Sabouraud dextrose liquid medium containing f2368 at each concentration was adjusted to 4 × 10 ² cells / ml, and 1 ml was inoculated into each well of the plate. After culturing at 30 ° C. for 1 hour, the culture supernatant was removed, washed with PBS, and overlaid with 2 ml of Sabouraud dextrose agar medium (Difco). After overnight culture at 30 ° C., CFU was counted and the adhesion rate was calculated. The result is shown in FIG.
f2368 showed the activity of inhibiting Candida albicans adhesion to IEC-18 cells.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between f2368 concentration and Candida deposition rate.

Claims (2)

A compound represented by formula (I).

  Mortierella alpina. Mer-f2368, FERM P-17653 is cultured in a nutrient medium, and the compound according to claim 1 is collected from the culture solution. The manufacturing method of the compound of Claim 1.

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