JPH06206893A - New disaccharide derivative - Google Patents
New disaccharide derivativeInfo
- Publication number
- JPH06206893A JPH06206893A JP5222449A JP22244993A JPH06206893A JP H06206893 A JPH06206893 A JP H06206893A JP 5222449 A JP5222449 A JP 5222449A JP 22244993 A JP22244993 A JP 22244993A JP H06206893 A JPH06206893 A JP H06206893A
- Authority
- JP
- Japan
- Prior art keywords
- activity
- compound
- reaction
- lipid
- present
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Saccharide Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Compounds Of Unknown Constitution (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【産業の利用分野】本発明は、多様な生物活性を有し、
かつ致死毒性および発熱原性が非常に弱く低毒性である
という特性を有する新規なジサッカライド誘導体および
その塩に関するものである。The present invention has various biological activities,
The present invention also relates to a novel disaccharide derivative and a salt thereof, which have the characteristics that they are extremely weak in lethal toxicity and pyrogenicity and have low toxicity.
【0002】[0002]
【従来の技術】各種グラム陰性菌の細胞壁外膜に含まれ
るリポポリサッカライド(LPS)は、リピドAと呼ば
れる糖脂質に各種の糖類が結合したものであり、以前か
ら内毒素の主成分であることが知られている。LPSは
生体の各種免疫機能促進活性を有することが知られてお
り、その主たる活性発現部位はリピドAにあることが明
らかにされており免疫調整作用および抗腫瘍作用の他、
多様な生物活性を有することが認められている。2. Description of the Related Art Lipopolysaccharide (LPS) contained in the outer wall of cell walls of various Gram-negative bacteria is a glycolipid called lipid A in which various sugars are bound, and has been a main component of endotoxin for a long time. It is known. LPS is known to have various immune function-promoting activities in the living body, and it has been clarified that its main activity expression site is in lipid A. In addition to the immunomodulatory action and antitumor action,
It is recognized to have diverse biological activities.
【0003】リピドAの化学構造は大腸菌を初め各種グ
ラム陰性菌において明らかにされてきた("Structure o
f the lipopolysaccharide from an E. Coli Heptose-l
essMutant", Marcha R. R., Jiunn-yann T., Israel
B., and H. Gobind Khorana,The Journal of Biologic
al Chemistry, vol. 254, No.13 p5906-5917(1979))。
その中で大腸菌由来リピドAについては完全に化学合成
がなされ、各種誘導体も化学合成されている。その結果
一部合成されたリピドA誘導体については腫瘍壊死因子
(TNF)誘導作用およびマイトジェン活性において大
腸菌由来リピドAと同等または同等以上であることが認
められている(特開昭59−48497号)。The chemical structure of lipid A has been elucidated in various Gram-negative bacteria including E. coli ("Structure o").
f the lipopolysaccharide from an E. Coli Heptose-l
essMutant ", Marcha RR, Jiunn-yann T., Israel
B., and H. Gobind Khorana, The Journal of Biologic
al Chemistry, vol. 254, No. 13 p5906-5917 (1979)).
Among them, E. coli-derived lipid A is completely chemically synthesized, and various derivatives are also chemically synthesized. As a result, the partially synthesized lipid A derivative was found to have the tumor necrosis factor (TNF) -inducing action and mitogenic activity equivalent to or higher than that of E. coli-derived lipid A (JP-A-59-48497). .
【0004】しかしながら大腸菌由来リピドAおよびリ
ピドA誘導体については発熱原性、壊死毒性活性等の好
ましくない作用が認められることから、さらに広範囲に
誘導体の合成研究が行われた(特開昭61−22758
6号)。またリピドA様活性を有する単糖構造を基本骨
格とするものを初め種々の置換基による修飾、置換基導
入部位の検討が詳細に行われ各種類縁体も合成され、生
物活性、免疫活性および毒性について調べられたが
(「リピドA類似体の生物活性」,小川裕示,木曽真,
長谷川明,代謝,第26巻,No.5,p15〜27,1
989、「合成リピドAとその誘導体」,本間遜,免疫
薬理,第8巻,No.4,1990,p25〜32)、3
位,3′位および4′位の水酸基が遊離であるような化
合物に言及した例はなく、未だ医薬品として実用化しう
るものは開発されていない。However, since E. coli-derived lipid A and lipid A derivatives have unfavorable actions such as pyrogenicity and necrotoxic activity, the synthetic studies of the derivatives have been conducted more extensively (JP-A-61-2758).
No. 6). In addition, modification with various substituents including those having a monosaccharide structure having lipid A-like activity as a basic skeleton and study of the site of introduction of the substituents were carried out in detail, and various types of analogs were also synthesized, resulting in biological activity, immunological activity and toxicity. ("Bioactivity of Lipid A analogues", Yuji Ogawa, Makoto Kiso,
Akira Hasegawa, Metabolism , Volume 26, No. 5, p15-27, 1
989, "Synthetic lipid A and its derivatives", Haruka Honma, Immune
Pharmacology , Volume 8, No. 4, 1990, p25-32), 3
There is no reference to a compound in which the hydroxyl groups at the 3'-position, 3'-position and 4'-position are free, and a compound that can be put to practical use as a pharmaceutical has not been developed yet.
【0005】[0005]
【発明が解決しようとする課題】したがって、上記の理
由からリピドA類似体のうちでより低毒性でより強い有
用活性を有する化合物が強く望まれている。Therefore, among the lipid A analogs, a compound having a lower toxicity and a stronger useful activity is strongly desired for the above reasons.
【0006】本発明は、強いマイトジェン活性、アジュ
バント活性、非特異的感染防御活性、抗ウイルス活性、
免疫賦活作用等の種々の有用な生物活性を有し、かつ発
熱原性および致死毒性等の有害作用がほとんどなく、特
に医薬品等として有用である新規なジサッカライド誘導
体を提供するものである。The present invention has strong mitogenic activity, adjuvant activity, nonspecific infection protection activity, antiviral activity,
The present invention provides a novel disaccharide derivative which has various useful biological activities such as immunostimulatory activity and has almost no harmful effects such as pyrogenicity and lethal toxicity and which is particularly useful as a drug or the like.
【0007】[0007]
【課題を解決するための手段】本発明者らは、ヒトの口
腔内に常在しており、現在歯周病の原因と考えられてい
る細菌の一つであるPorphyromanas(Bacteroides) ging
ivalisの細胞壁外膜中に含まれるLPSがマイトジェン
活性等を有し、同時に致死毒性および発熱原性が非常に
弱いことを見いだした。さらに、LPS中の活性発現部
位を調製後精製し、構造解析を行い、鋭意研究の結果、
本発明の活性化合物はグルコサミンβ(1−6)ジサッ
カライドの構造の1位にリン酸基がエステル結合をした
ものを基本骨格とし、2位のアミノ基に3-hydroxy-15-m
ethylhexadecanoic acidがアミド結合し、2′位のアミ
ノ基に3-hexadecanoyloxy-15-methylhexadecanoic acid
がアミド結合していることを見いだした。本発明の化合
物の構造は、従来のリピドA誘導体と大きく異なり4′
位にリン酸基を持たず、3位および3′位の水酸基が遊
離のままである点に特徴がある。したがって本発明の化
合物は式;Means for Solving the Problems The present inventors have found that Porphyromanas (Bacteroides) ging, which is one of the bacteria that is resident in the human oral cavity and is currently considered to be the cause of periodontal disease.
It was found that LPS contained in the outer wall of ivalis cell has mitogenic activity and at the same time has very weak lethal toxicity and pyrogenicity. Furthermore, after the active expression site in LPS was prepared and purified, the structure was analyzed and the results of earnest research showed that
The active compound of the present invention has, as a basic skeleton, a structure in which a phosphoric acid group is ester-bonded at the 1-position of the structure of glucosamine β (1-6) disaccharide, and 3-hydroxy-15-m at the 2-position amino group.
Ethylhexadecanoic acid is amide-bonded and 3-hexadecanoyloxy-15-methylhexadecanoic acid is added to the amino group at the 2'position.
Was found to have an amide bond. The structure of the compound of the present invention is significantly different from that of the conventional lipid A derivative.
It is characterized in that it has no phosphate group at the position and the hydroxyl groups at the 3 and 3'positions remain free. Thus the compounds of the present invention have the formula:
【化1】 で表されるような構造を有することが推定される。また
本願化合物は強いマイトジェン活性、アジュバント活
性、多クローン性B細胞活性化(非特異的感染防御)活
性、ナチュラルキラー活性等多様な生物活性を有する一
方、従来のリピドAおよびリピドA誘導体においてみら
れたマイクロファージからの腫瘍壊死因子(TNF)や
IL−1等のいわゆる炎症性サイトカインの産生を誘導
する活性が殆ど見られないことを見いだした。したがっ
て従来、リピドAおよびリピドA誘導体において問題と
なっていた致死毒性および発熱原性等の有害作用を示す
ことなく、免疫賦活剤として有用である。さらには大腸
菌のリピドAが誘導するIL−1の産生を抑え、さらに
IL−1レセプターアンタゴニスト(IL−1ra)の
産生を誘導することを見いだしたが、これらの活性は、
強い非特異的感染防御活性を有することと併せて、大腸
菌等グラム陰性菌の感染によって引き起こされる病態、
特に敗血症、あるいは敗血症性ショックの予防・治療用
剤として有用である。また、IL−1の産生を抑え、さ
らにIL−1レセプターアンタゴニスト(IL−1r
a)の産生を誘導することは、IL−1自身の異常産生
状態になっていると考えられるような病態、例えば慢性
関節リューマチ等の治療用剤としても有用である。さら
に、ナチュラルキラー細胞を活性化し、抗腫瘍活性を有
することを見いだした上、抗ウイルス活性を有すること
も明らかになった。抗腫瘍活性からは、抗腫瘍剤として
の有用性が考えられ、抗ウイルス活性と併せて強い非特
異的感染防御活性を有することからは、抗ウイルス剤と
しての有用性がある。このような特性から、本願発明化
合物である新規なジサッカライド誘導体またはその塩
は、医薬用担体および/または希釈剤と配合してなる医
薬組成物として特に有用であると期待される。[Chemical 1] It is presumed that it has a structure represented by. In addition, the compound of the present invention has various biological activities such as strong mitogenic activity, adjuvant activity, polyclonal B cell activation (non-specific infection protection) activity, natural killer activity, etc., while it is found in conventional lipid A and lipid A derivatives. It was found that the activity of inducing the production of so-called inflammatory cytokines such as tumor necrosis factor (TNF) and IL-1 from microphages was hardly observed. Therefore, it is useful as an immunostimulant without exhibiting harmful effects such as lethal toxicity and pyrogenicity, which have heretofore been problems for lipid A and lipid A derivatives. Furthermore, they have found that E. coli lipid A-induced IL-1 production is suppressed, and further that an IL-1 receptor antagonist (IL-1ra) production is induced.
In addition to having strong non-specific defense against infection, pathological conditions caused by infection with Gram-negative bacteria such as Escherichia coli,
Particularly, it is useful as a prophylactic / therapeutic agent for sepsis or septic shock. In addition, IL-1 production is suppressed, and further, an IL-1 receptor antagonist (IL-1r
Inducing the production of a) is also useful as a therapeutic agent for pathological conditions that are considered to be in the abnormal production state of IL-1 itself, such as rheumatoid arthritis. Furthermore, it was found that it activates natural killer cells and has antitumor activity, and that it also has antiviral activity. It is considered to be useful as an antitumor agent from the viewpoint of antitumor activity, and useful as an antiviral agent since it has a strong nonspecific infection protective activity in addition to the antiviral activity. From such characteristics, it is expected that the novel disaccharide derivative or the salt thereof which is the compound of the present invention will be particularly useful as a pharmaceutical composition prepared by mixing with a pharmaceutical carrier and / or diluent.
【0008】なお、本化合物には種々の立体異性体が存
在し得るが、単離されたこれらの異性体および異性体混
合物は、いずれも本発明の技術的思想に包含される。The compound may have various stereoisomers, and the isolated isomers and isomer mixtures are all included in the technical idea of the present invention.
【0009】本願発明の化合物は微生物源より調製し、
精製して使用することができる。The compounds of the present invention are prepared from microbial sources,
It can be purified before use.
【0010】さらに種々の化学合成法により製造するこ
とも可能である。Further, it can be produced by various chemical synthesis methods.
【0011】以下に各製造法による具体的な製造例を示
す。Specific manufacturing examples by each manufacturing method are shown below.
【0012】(1)微生物を用いた製造例 本願化合物を微生物により製造する場合には、前記〔化
1〕により示される本願発明の化合物を生産することが
できる微生物であればいずれも使用できるが、例えばPo
rphyromanas(Bacteroides) gingivalis(ATCCカタ
ログ番号33277)等を使用することができる。(1) Production Example Using Microorganism When the compound of the present invention is produced by a microorganism, any microorganism can be used as long as it can produce the compound of the present invention represented by the above-mentioned [Chemical formula 1]. , For example Po
rphyromanas (Bacteroides) gingivalis (ATCC catalog number 33277) and the like can be used.
【0013】培養に使用される培地は液状または固状で
よいが、通常は液状培地による嫌気静置培養が便利であ
る。培地は本願発明化合物生産菌が生育して本願発明化
合物を生産するものであればどのようなものでもよい。
培養温度、培養期間、培養の液性等の条件は、本願発明
化合物の生産量が最大となるように適宜選択、調節され
得るが、好ましくは嫌気的条件下に25℃〜40℃、好
ましくは37℃にて、12〜36時間、好ましくは26
時間培養され、その培地のpHは6.0〜8.0、好ま
しくは7.3に維持される。The medium used for culture may be liquid or solid, but anaerobic static culture using a liquid medium is usually convenient. The medium may be any medium as long as the compound-producing compound of the present invention grows to produce the compound of the present invention.
Conditions such as culture temperature, culture period, and liquidity of culture can be appropriately selected and adjusted so that the production amount of the compound of the present invention is maximized, but preferably 25 ° C to 40 ° C under anaerobic conditions, preferably At 37 ° C, 12 to 36 hours, preferably 26
After culturing for a time, the pH of the medium is maintained at 6.0 to 8.0, preferably 7.3.
【0014】上記条件で培養することにより本発明化合
物が生産、蓄積される。培養物を濾過または遠心分離し
て菌体を回収し、目的物の分離、精製を行う。菌体から
のLPSの分離、精製には化合物の化学的特性に基づく
種々の手段を用いることができる。例えば熱フェノール
−水抽出法にて抽出し、各種酵素処理後に遠心して精
製、溶媒分画、各種樹脂を用いたカラム法を使用し、こ
れらを適当に組み合わせることによりLPSを分離、精
製することができる。By culturing under the above conditions, the compound of the present invention is produced and accumulated. The culture is filtered or centrifuged to collect the bacterial cells, and the desired product is separated and purified. Various means based on the chemical characteristics of the compound can be used for the isolation and purification of LPS from the bacterial cells. For example, it is possible to separate and purify LPS by extracting with a hot phenol-water extraction method, purifying by centrifugation after treatment with various enzymes, solvent fractionation, and a column method using various resins, and appropriately combining these. it can.
【0015】次に精製を繰り返したLPSまたは粗LP
Sを弱酸加水分解する。弱酸加水分解は、本願発明化合
物が遊離する方法であればどのような方法でもよく、好
ましくは0.05〜0.2規定の酢酸を用い、90℃〜
110℃、2〜3時間の反応でよい。反応物から目的物
の分離、精製には本願発明化合物の化学的特性に基づく
種々の手段が採用できる。すなわち、溶媒分画法、各種
樹脂を使用したカラム法等が用いられ、これらを適当に
組み合わせることにより本願発明に係わる物質を分離、
精製することができる。Next, LPS or crude LP which has been repeatedly purified
S is hydrolyzed with a weak acid. The weak acid hydrolysis may be carried out by any method as long as the compound of the present invention is liberated, preferably using 0.05 to 0.2 N acetic acid and 90 ° C to
The reaction may be 110 ° C. for 2 to 3 hours. Various means based on the chemical characteristics of the compound of the present invention can be adopted for the separation and purification of the desired product from the reaction product. That is, a solvent fractionation method, a column method using various resins, and the like are used, and by appropriately combining these, a substance according to the present invention can be separated,
It can be purified.
【0016】(2)化学合成法を用いた製造例 適当な保護基で適当な位置を保護したN−グルコサミン
誘導体を配糖体結合形成反応によってジサッカライド誘
導体とし、これを脂肪酸によるN−アシル化、還元末端
1位のリン酸化後、脱保護する。または所望の脂肪酸に
よってN−アシル化された保護N−グルコサミン誘導体
を配糖体結合形成反応によってジサッカライド誘導体と
し、これをリン酸化後、脱保護基を行うことによっても
合成することができる。(2) Production Example Using Chemical Synthesis Method An N-glucosamine derivative in which an appropriate position is protected by an appropriate protecting group is converted into a disaccharide derivative by a glycoside bond forming reaction, which is N-acylated with a fatty acid. , Phosphorylation of the reducing terminal 1-position is followed by deprotection. Alternatively, a protected N-glucosamine derivative N-acylated with a desired fatty acid may be converted to a disaccharide derivative by a glycoside bond-forming reaction, which may be phosphorylated and then deprotected to perform deprotection.
【0017】上記の説明にしたがって得られた本発明化
合物は、リン酸基部分で塩を形成しうる化合物であり、
公知の方法により容易に塩に変換することができる。そ
のような塩の例としては、例えばナトリウムまたはカリ
ウムのようなアルカリ金属の塩、カルシウム、マグネシ
ウム等のアルカリ土類金属の塩、アンモニウム塩および
薬学的に許容されるアミン塩、等が含まれる。非毒性ア
ミン類の塩としては、テトラメチルアンモニウムのよう
なテトラアルキルアンモニウムの塩、およびメチルアミ
ン塩、トリエチルアミン塩、シクロペンチルアミン塩、
ベンジルアミン塩、ピリジン塩、ピペリジン塩、ジエタ
ノールアミン塩、リジン塩、アルギニン塩のような有機
アミン塩等が例として挙げられる。このようにして得ら
れた本発明化合物であるジサッカライド誘導体またはそ
の塩は、治療または予防の目的で用いる医薬組成物とし
て通常全身的あるいは局所的に経口または非経口で投与
される。投与量としては、年齢、体重、症状、投与方法
等により異なるが、通常成人ひとり当たり1回につき
0.01mg〜100mgの範囲で1日1回から数回、経口
あるいは非経口で投与される。勿論投与量は種々の条件
で異なるので、上記投与量より少ない範囲で充分効果の
ある場合もあるし、逆にこれらの範囲を超えて投与する
必要のある場合もある。本発明による経口投与のための
固形医薬組成物には、錠剤、散剤、顆粒剤等が含まれ
る。このような固形組成物においては、少なくともひと
つの不活性な希釈剤、例えば乳糖、ブトウ糖、微結晶セ
ルロース、デンプン、ポリビニルピロリドン、メタケイ
酸アルミン酸マグネシウム等と混合される。組成物は、
さらに不活性な希釈剤以外の添加剤、例えば、ステアリ
ン酸マグネシウムのような潤滑剤や、繊維素グルコン酸
カルシウムのような崩壊剤を含有していてもよい。錠剤
または丸剤は、必要により白糖、ゼラチン、ヒドロキシ
プロピルセルロース等の胃溶解性あるいは腸溶解性物質
のフィルムで被覆してもよいし、また2以上の層で被覆
してもよい。さらにゼラチンのように吸収されうる物質
のカプセルであってもよい。経口投与のための液体医薬
組成物は、薬学的に許容される乳濁剤、溶液剤、懸濁
剤、シロップ剤等を含み、一般的に用いられる不活性な
希釈剤としては、例えば精製水、エタノール等を含む。
不活性な希釈剤以外に、補助剤として湿潤剤、懸濁剤
等、さらに甘味剤、風味剤、芳香剤、防腐剤等を含有し
ていてもよい。また、経口投与のための組成物には、常
法により処方されるスプレー剤も含まれる。本発明によ
る非経口投与のための注射剤組成物としては、無菌の水
性または非水性の溶液剤、懸濁剤、乳濁剤等が含まれ
る。水性の溶液剤、懸濁剤としては、例えば注射用蒸留
水および生理食塩水が含まれる。非水性の溶液剤、懸濁
剤としては、例えばプロピレングリコール、ポリエチレ
ングリコール、オリーブ油等の植物油、エタノール等の
アルコール類、ポリソルベート80(登録商標)等があ
る。このような組成物は、さらに防腐剤、湿潤剤、乳化
剤、分散剤のような補助剤を含んでもよい。これらは、
特定の濾過や、殺菌剤の配合または照射によって無菌化
される。これらはまた、無菌の固体組成物を製造し、使
用前に無菌水または無菌の注射用溶媒に溶解して使用す
ることもできる。非経口投与のための組成物としては、
このほか公知の方法により処方される外用液剤、軟膏の
ような塗布剤、および座剤、ペッサリー等も含まれる。The compound of the present invention obtained according to the above explanation is a compound capable of forming a salt at the phosphate group moiety,
It can be easily converted into a salt by a known method. Examples of such salts include salts of alkali metals such as sodium or potassium, salts of alkaline earth metals such as calcium and magnesium, ammonium salts and pharmaceutically acceptable amine salts. As salts of non-toxic amines, tetraalkylammonium salts such as tetramethylammonium, and methylamine salts, triethylamine salts, cyclopentylamine salts,
Examples thereof include benzylamine salt, pyridine salt, piperidine salt, diethanolamine salt, lysine salt, and organic amine salt such as arginine salt. The thus-obtained compound of the present invention, which is a disaccharide derivative or a salt thereof, is generally orally or parenterally administered systemically or locally as a pharmaceutical composition used for the purpose of treatment or prevention. The dose varies depending on the age, body weight, symptoms, administration method, etc., but is usually in the range of 0.01 mg to 100 mg per adult, once to several times a day, orally or parenterally. Of course, since the dose varies depending on various conditions, there may be a case where the dose is less than the above dose and the effect is sufficient, and conversely, it may be necessary to administer beyond the range. Solid pharmaceutical compositions for oral administration according to the present invention include tablets, powders, granules and the like. Such solid compositions are mixed with at least one inert diluent such as lactose, butter sugar, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminometasilicate and the like. The composition is
Further, it may contain additives other than an inert diluent, for example, a lubricant such as magnesium stearate and a disintegrating agent such as calcium fibrin gluconate. If necessary, the tablets or pills may be coated with a film of a gastric-soluble or enteric-soluble substance such as sucrose, gelatin, hydroxypropylcellulose, or may be coated with two or more layers. Further, it may be a capsule made of an absorbable substance such as gelatin. Liquid pharmaceutical compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and the like, and commonly used inert diluents include, for example, purified water. , Ethanol, etc. are included.
In addition to the inactive diluent, a wetting agent, a suspending agent, etc., and a sweetening agent, a flavoring agent, an aromatic agent, a preservative, etc. may be contained as an auxiliary agent. The composition for oral administration also includes sprays formulated by a conventional method. The injectable composition for parenteral administration according to the present invention includes sterile aqueous or non-aqueous solutions, suspensions, emulsions and the like. Examples of the aqueous solution and suspension include distilled water for injection and physiological saline. Examples of non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and Polysorbate 80 (registered trademark). Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. They are,
It is sterilized by specific filtration, blending of a bactericide or irradiation. They can also be used by preparing a sterile solid composition and dissolving it in sterile water or a sterile solvent for injection before use. As a composition for parenteral administration,
In addition, liquid formulations for external use prescribed by known methods, coating agents such as ointments, suppositories, pessaries and the like are also included.
【0018】また本願化合物は強いマイトジェン活性、
アジュバント活性、多クローン性B細胞活性化(非特異
的感染防御)活性、ナチュラルキラー活性等多様な生物
活性を有する一方、従来のリピドAおよびリピドA誘導
体においてみられたマイクロファージからの腫瘍壊死因
子(TNF)やIL−1等のいわゆる炎症性サイトカイ
ンの産生を誘導する活性が殆ど見られないことを見いだ
した。したがって従来、リピドAおよびリピドA誘導体
において問題となっていた致死毒性および発熱原性等の
有害作用を示すことなく、免疫賦活剤として有用であ
る。さらには大腸菌のリピドAが誘導するIL−1の産
生を抑え、さらにIL−1レセプターアンタゴニスト
(IL−1ra)の産生を誘導することを見いだした
が、これらの活性は、強い非特異的感染防御活性を有す
ることと併せて、大腸菌等グラム陰性菌の感染によって
引き起こされる病態、特に敗血症、あるいは敗血症性シ
ョックの予防・治療用剤として有用である。また、IL
−1の産生を抑え、さらにIL−1レセプターアンタゴ
ニスト(IL−1ra)の産生を誘導することは、IL
−1自身の異常産生状態になっていると考えられるよう
な病態、例えば慢性関節リューマチ等の治療用剤として
も有用である。さらに、ナチュラルキラー細胞を活性化
し、抗腫瘍活性を有することを見いだした上、抗ウイル
ス活性を有することも明らかになった。抗腫瘍活性から
は、抗腫瘍剤としての有用性が考えられ、抗ウイルス活
性と併せて強い非特異的感染防御活性を有することから
は、抗ウイルス剤としての有用性がある。The compound of the present invention has a strong mitogenic activity,
Tumor necrosis factor from microphages found in conventional lipid A and lipid A derivatives, while having various biological activities such as adjuvant activity, polyclonal B cell activation (non-specific infection protection) activity, and natural killer activity It was found that there is almost no activity that induces the production of so-called inflammatory cytokines such as (TNF) and IL-1. Therefore, it is useful as an immunostimulant without exhibiting harmful effects such as lethal toxicity and pyrogenicity, which have heretofore been problems for lipid A and lipid A derivatives. Furthermore, they found that it suppressed the lipid A-induced IL-1 production of Escherichia coli, and further induced the production of an IL-1 receptor antagonist (IL-1ra). In addition to having activity, it is useful as a prophylactic / therapeutic agent for pathological conditions caused by infection with Gram-negative bacteria such as Escherichia coli, particularly sepsis or septic shock. Also, IL
Suppressing the production of IL-1 and further inducing the production of IL-1 receptor antagonist (IL-1ra)
-1 It is also useful as a therapeutic agent for pathological conditions such as rheumatoid arthritis that is considered to be in the abnormal production state of -1 itself. Furthermore, it was found that it activates natural killer cells and has antitumor activity, and that it also has antiviral activity. It is considered to be useful as an antitumor agent from the viewpoint of antitumor activity, and useful as an antiviral agent since it has a strong nonspecific infection protective activity in addition to the antiviral activity.
【0019】このような特性から、本願発明化合物であ
る新規なジサッカライド誘導体またはその塩は、医薬用
担体および/または希釈剤と配合してなる医薬組成物と
して特に有用であると期待される。From such characteristics, it is expected that the novel disaccharide derivative or the salt thereof which is the compound of the present invention is particularly useful as a pharmaceutical composition prepared by mixing with a pharmaceutical carrier and / or a diluent.
【0020】以下、本願発明に関する化合物について実
施例に基づき詳細に説明するが、本発明を以下に記載す
る特別な態様に限定することを意図するものではない。Hereinafter, the compounds relating to the present invention will be described in detail based on examples, but the present invention is not intended to be limited to the specific embodiments described below.
【0021】[0021]
実施例1微生物源からの製造 Porphyromanas(Bacteroides) gingivalisを160リッ
トルのGAMブイヨン(日水製薬株式会社製)培地(p
H7.3)で嫌気的に37℃で26時間培養した。培養
完了後培養液の遠心を行い菌体を回収し、凍結乾燥によ
り乾燥菌体100gを得た。この乾燥菌体を熱フェノー
ル−水抽出法にて抽出処理して粗LPSを得た。すなわ
ち100gの乾燥菌体に3.5リットルの蒸留水を加え
68℃に加熱し、別に68℃に加熱した90%フェノー
ルを加え68℃にて20分間撹拌し、氷冷後遠心して水
層を分取後、再度3.5リットルの蒸留水を加え抽出操
作を繰り返した。得られた水層を合わせ、蒸留水に対し
て十分透析後、濃縮し凍結乾燥を行い粗抽出物12.4
7gを得た。Example 1 Production from microbial source Porphyromanas (Bacteroides) gingivalis was added with 160 liters of GAM broth (manufactured by Nissui Pharmaceutical Co., Ltd.) medium (p
H7.3) was anaerobically cultured at 37 ° C. for 26 hours. After the completion of the culture, the culture solution was centrifuged to collect the cells, and freeze-dried to obtain 100 g of dried cells. The dried cells were subjected to extraction treatment with a hot phenol-water extraction method to obtain crude LPS. That is, 3.5 liters of distilled water was added to 100 g of dried cells and heated to 68 ° C., 90% phenol heated to 68 ° C. was added, and the mixture was stirred at 68 ° C. for 20 minutes, ice-cooled and centrifuged to form an aqueous layer. After the collection, 3.5 liters of distilled water was added again and the extraction operation was repeated. The obtained aqueous layers were combined, sufficiently dialyzed against distilled water, concentrated and freeze-dried to obtain a crude extract 12.4.
7 g was obtained.
【0022】粗抽出物10gを1リットルの蒸留水に懸
濁し超遠心沈渣をヌクレアーゼP1(ヤマサ醤油株式会
社製)およびプロナーゼ(カルビオケミカル製;米国)
酵素処理を二度づつ繰り返し、蒸留水で超遠心沈渣の洗
浄を二度繰り返し、凍結乾燥を行い粗LPS画分250
mgを得た。粗LPS画分250mgを50mMトリス塩酸緩
衝液(pH7.4)に懸濁後、セファロース4Bカラム
クロクトグラフィー(内径1.5cm、長さ90cm)に供
し、排除体積画分を分取後、エタノール沈殿を行い、蒸
留水で二度洗浄後凍結乾燥を行った。LPS画分110
mgを得た。LPS画分を0.1規定の酢酸で105℃、
2.5時間弱酸加水分解を行い、反応物を遠心後、遠心
沈渣を得た。この画分をシリカゲルカラムクロマトグラ
フィー(クロロホルム/メタノール/水/トリエチルア
ミン=30/12/1.5/0.1)で精製を行い、
4.5mgの本発明化合物を得た。10 g of the crude extract was suspended in 1 liter of distilled water, and the ultracentrifugal precipitate was suspended with nuclease P1 (Yamasa Shoyu Co., Ltd.) and Pronase (Calbio Chemical; USA).
The enzyme treatment was repeated twice, the ultracentrifugal precipitate was washed twice with distilled water, and freeze-dried to obtain a crude LPS fraction 250.
to obtain mg. After suspending 250 mg of the crude LPS fraction in 50 mM Tris-HCl buffer (pH 7.4), it was subjected to Sepharose 4B column chromatography (internal diameter 1.5 cm, length 90 cm), the excluded volume fraction was collected, and ethanol was collected. Precipitation was performed, washed twice with distilled water, and then freeze-dried. LPS fraction 110
to obtain mg. The LPS fraction was treated with 0.1 N acetic acid at 105 ° C,
After performing a weak acid hydrolysis for 2.5 hours and centrifuging the reaction product, a centrifugal precipitate was obtained. This fraction was purified by silica gel column chromatography (chloroform / methanol / water / triethylamine = 30/12 / 1.5 / 0.1),
4.5 mg of the compound of the invention was obtained.
【0023】実施例2構造解析 上記、実施例1で得られた化合物の物理化学的性質を検
討したところ、以下の結果が得られた。Example 2 Structural Analysis When the physicochemical properties of the compound obtained in Example 1 were examined, the following results were obtained.
【0024】(1)糖分析および脂肪酸分析; ア.グルコサミンβ(1−6)ジサッカライド構造の1
位にリン酸基がエステル結合したものを基本骨格とし、 イ.2位のアミノ基に3-hydroxy-15-methylhexadecanoi
c acidがアミド結合し、 ウ.2′位のアミノ基に3-hexadecanoyloxy-15-methylh
exadecanoic acidがアミド結合し、 エ.4′位にリン酸基を持たず、 オ.3位,3′位および4′位の水酸基が遊離のままで
ある。(1) Sugar analysis and fatty acid analysis; Glucosamine β (1-6) disaccharide structure 1
The basic skeleton has a phosphoric acid group ester-bonded at position b. 3-hydroxy-15-methylhexadecanoi on the 2-position amino group
c acid forms an amide bond, and c. 3-hexadecanoyloxy-15-methylh at the 2'position amino group
Exadecanoic acid is amide-bonded, and d. It does not have a phosphate group at the 4'position. The hydroxyl groups at the 3 ', 3'and 4'positions remain free.
【0025】(2)分子式;C62H119 O17N2 P (3)呈色反応;硫酸反応に陽性、ディットマー・レス
ター反応に陽性、ニンヒドリン反応に陰性、TTC反応
に陰性である。(2) Molecular formula: C 62 H 119 O 17 N 2 P (3) Color reaction: positive for sulfuric acid reaction, positive for Dittmer-Lester reaction, negative for ninhydrin reaction, negative for TTC reaction.
【0026】(4)物質の色、形状;白色粉末 (5)質量スペクトル;ネガティブFAB−MS−M
S、M/Z 1193(M−H),937(M−2H−C15H31
COO) (6)NMRスペクトル; 1H−NMR(300MHz,CD
Cl3 +MeOD+D2O)δ:0.81(12H,d)、0.8
2(3H,t)、1.1-1.6(72H,m,CH2 ,CH)、2.2
-2.5 (6H,m,CO−CH2 )、3.1-4.3(13H,
m)、4.46(1H,d)、5.15(1H,m)、5.45(1H,
m) 以上の結果より、本発明化合物の構造は〔化1〕で表さ
れるものと推定された。(4) Color and shape of substance; white powder (5) Mass spectrum; Negative FAB-MS-M
S, M / Z 1193 (M -H), 937 (M-2H-C 15 H 31
COO) (6) NMR spectrum; 1 H-NMR (300 MHz, CD
Cl 3 + MeOD + D 2 O) δ: 0.81 (12H, d), 0.8
2 (3H, t), 1.1-1.6 (72H, m, CH 2 , CH), 2.2
-2.5 (6H, m, CO- CH 2), 3.1-4.3 (13H,
m), 4.46 (1H, d), 5.15 (1H, m), 5.45 (1H,
m) From the above results, the structure of the compound of the present invention was presumed to be represented by [Chemical Formula 1].
【0027】実施例3活性測定 次に本願発明の化合物について試験した生理活性の結果
を示す。Example 3 Activity Measurement Next, the results of physiological activity tested for the compounds of the present invention are shown.
【0028】(1)マイトジェン活性 マイトジェン活性は、例えば単離されたマウスリンパ系
培養細胞中に取り込まれた 3H−チミジンの量を測定す
ることにより行った。すなわちBALB/cマウスの脾
臓を擂り潰し、5×105 個/well(200μl)に調
製した脾細胞に所定濃度の本願発明化合物、あるいは比
較化合物を添加、または培地のみを添加して培養した。
培養終了6時間前に37kBq/well(10μl)の 3H−
チミジンを加え、培養終了後細胞に取り込まれた 3H−
チミジンの量(放射線量)を定量した。結果は下式で示
すStimulation Index で表した。(1) Mitogenic activity Mitogenic activity was measured by measuring the amount of 3 H-thymidine incorporated into the isolated mouse lymphoid cultured cells, for example. That is, the spleens of BALB / c mice were crushed, and 5 × 10 5 cells / well (200 μl) prepared spleen cells were added with a predetermined concentration of the compound of the present invention or a comparative compound, or the medium alone was cultured.
6 hours before the end of culture, 37 kBq / well (10 μl) of 3 H-
Thymidine was added, and 3 H- was taken up by cells after the culture was completed.
The amount of thymidine (radiation dose) was quantified. The results are expressed by the Stimulation Index shown below.
【0029】Stimulation Index=化合物の添加群(cp
m)/培地のみ添加群(cpm) 本発明化合物は、表1に示されるようにマイトジェン活
性を有することを示した。比較化合物としては、合成リ
ピドAである506を用いた。506は、6−O−〔2
−デオキシ−2−(3−ドテカノイルオキシテトラデカ
ノイルアミノ)−3−O−(3−テトラデカノイルオキ
シテトラデカノイル)−4−O−ホスホノ−β−D−グ
ルコピラノシル〕−2−デオキシ−2−(3−ヒドロキ
シテトラデカノイルアミノ)−3−O−テトラデカノイ
ル−1−O−ホスホノ−α−D−グルコピラノースであ
る。Stimulation Index = Compound addition group (cp
m) / medium only addition group (cpm) As shown in Table 1, the compounds of the present invention were shown to have mitogenic activity. As a comparative compound, synthetic lipid A 506 was used. 506 is 6-O- [2
-Deoxy-2- (3-dotecanoyloxytetradecanoylamino) -3-O- (3-tetradecanoyloxytetradecanoyl) -4-O-phosphono-β-D-glucopyranosyl] -2-deoxy 2- (3-hydroxytetradecanoylamino) -3-O-tetradecanoyl-1-O-phosphono-α-D-glucopyranose.
【0030】[0030]
【表1】 [Table 1]
【0031】(2)NK(ナチュラルキラー)活性 本試験は以下の方法により検討した。すなわち、BAL
B/cマウスを用い100μgの本発明化合物、あるい
は比較化合物を0日目および7日目に静脈内に注射し、
14日目に脾細胞を採取した。調製した脾細胞(1×1
06 個/0.1ml)に51Crで標識した標的細胞(Molon
eyウイルス誘発リンパ種YAC−1:2×104 個/
0.1ml)を加え、4時間培養した。なお、最小遊離対
照群には脾細胞を含まない培養液のみを加え、最大遊離
対照群には0.1N NaOHを加えた。培養終了後、
培養上清0.1mlを採取し、標的細胞が障害を受けるこ
とにより遊離した51Crの量(放射線量)を測定した。
活性は各価を以下の式に代入し、NK Activity(%)を求
めた。(2) NK (Natural Killer) Activity This test was examined by the following method. That is, BAL
B / c mice were used to inject 100 μg of the compound of the present invention or a comparative compound intravenously on days 0 and 7,
Splenocytes were collected on the 14th day. Prepared splenocytes (1 x 1
Target cells (Molon) labeled with 51 Cr in 0 6 cells / 0.1 ml)
ey virus-induced lymphoma YAC-1: 2 × 10 4 cells /
0.1 ml) was added and the cells were cultured for 4 hours. It should be noted that only a culture solution containing no splenocytes was added to the minimum free control group, and 0.1N NaOH was added to the maximum free control group. After culturing,
0.1 ml of the culture supernatant was collected, and the amount of 51 Cr released by the damage to the target cells (radiation dose) was measured.
For the activity, NK Activity (%) was calculated by substituting each value into the following formula.
【0032】NK Activity(%)={(実験群−最小遊離対
照群)(cpm)/(最大遊離対照群−最小遊離対照群)(cp
m)}×100 本発明化合物は、上式で算出した結果50.1のNK Act
ivity(%)という値を示し、合成リピドAである506
と同等の活性を有することが明らかとなった。 (3)抗腫瘍活性 本試験はメチルコラントレン誘発線維肉腫(Meth
A)に対する増殖抑制活性により検討した。すなわち、
BALB/cマウスより取り出した脾細胞より付着細胞
(マクロファージ)を採取した。調製したマクロファー
ジ(2×105個/0.1ml)と標的細胞(Meth
A:2×104 個/0.1ml)に所定濃度の本発明化合
物あるいは比較化合物を加え、18時間培養した後、1
4.8kBq/well(10μl)の 3H−チミジンを加えて
さらに6時間培養した。培養終了後細胞に取り込まれた
3H−チミジンの量(放射線量)を定量した。結果は得
られた値を次式に代入して 3H−チミジンの取り込み抑
制率を計算し、マクロファージの腫瘍細胞増殖抑制活性
として表した。 Cytostatic Activity(%)={1−(マクロファージおよ
び標的細胞−マクロファージのみ)(cpm)/標的細胞のみ
(cpm)}×100 本発明化合物は表2で示されるような値を示し、合成リ
ピドAである506とほぼ同等の腫瘍増殖抑制活性を示
した。NK Activity (%) = {(experimental group-minimum release control group) (cpm) / (maximum release control group-minimum release control group) (cp
m)} × 100 The compound of the present invention has a NK Act of 50.1 as calculated by the above formula.
506, which is a synthetic lipid A showing the value of ivity (%)
It was found to have the same activity as. (3) Antitumor activity The present study was conducted in accordance with methylcholanthrene-induced fibrosarcoma (Meth
It was examined by the growth inhibitory activity against A). That is,
Adherent cells (macrophages) were collected from splenocytes taken out from BALB / c mice. Prepared macrophages (2 × 10 5 cells / 0.1 ml) and target cells (Meth
A: 2 × 10 4 cells / 0.1 ml) was added with the compound of the present invention or the comparative compound at a predetermined concentration and incubated for 18 hours, then 1
4.8 kBq / well (10 μl) of 3 H-thymidine was added, and the mixture was further cultured for 6 hours. Incorporated into cells after culture
The amount of 3 H-thymidine (radiation dose) was quantified. The obtained values were substituted into the following formula to calculate the 3 H-thymidine uptake inhibition rate, which was expressed as the tumor cell growth inhibitory activity of macrophages. Cytostatic Activity (%) = {1- (macrophages and target cells-macrophages only) (cpm) / target cells only
(cpm)} × 100 The compound of the present invention showed the values as shown in Table 2 and showed a tumor growth inhibitory activity almost equal to that of synthetic lipid A 506.
【0034】[0034]
【表2】 [Table 2]
【0035】(4)アジュバント活性 本試験は1群6匹の雄性BALB/cマウスを用い、0
日目と28日目に牛血清アルブミン(BSA)100μ
gに本発明化合物あるいは比較化合物100μgを添加
あるいは無添加にて、フロイントの不完全アジュバント
(FIA)を用いて油中水型乳剤として皮下注射し、追
加免疫後5日目に血清中に産生した抗BSAIgG抗体
量をELISA法により測定した。結果は、次式で示す
Stimulation Index で表した。(4) Adjuvant activity In this test, 6 male BALB / c mice per group were used.
Bovine serum albumin (BSA) 100μ on days 28 and
100 μg of the compound of the present invention or 100 g of the comparative compound was subcutaneously injected as a water-in-oil emulsion using Freund's incomplete adjuvant (FIA), and produced in serum 5 days after the booster immunization. The amount of anti-BSA IgG antibody was measured by the ELISA method. The result is shown by the following formula.
It is represented by the Stimulation Index.
【0036】Stimulation Index =FIAに化合物とB
SAの添加群(μg/ml)/FIAにBSAのみの添加群
(μg/ml) 本発明化合物は、表3に示されるように合成リピドAで
ある506より強いアジュバント活性を有することが明
らかとなった。Stimulation Index = FIA compound and B
SA addition group (μg / ml) / FIA only addition group (μg / ml) As shown in Table 3, it was revealed that the compound of the present invention has a stronger adjuvant activity than synthetic lipid A 506. became.
【0037】[0037]
【表3】 [Table 3]
【0038】(5)抗ウイルス活性 本試験は、水疱性口内炎ウイルス(VSV;Vesic
ular stomatitis virus)のマウ
ス線維芽細胞株L929に対する作用の抑制を指標にし
た。すなわち、各ウェルにL929細胞を4×104 個
/0.1mlに入れ24時間培養後、本発明化合物あるい
は比較化合物(1mg/ml)の希釈系列より各希釈試料液
を0.1mlずつ加えさらに24時間培養した。培養上清
除去後、100TCID50/0.1mlに調製したVSV
を加え24時間培養後、培養液を除去し、5%ホルムア
ルデヒド溶液により20分間固定し、続いて0.5%ク
リスタルバイオレット染色液にて20分間染色した。水
洗後乾燥し、吸光度600nmで測定を行った。活性はL
929細胞が50%生存する試料液の元の試料濃度(1
mg/ml)に対する希釈倍数の逆数で表した。本発明化合
物は表4に示すように合成リピドAである506より強
い抗ウイルス活性を有することが明らかとなった。(5) Antiviral activity In this test, vesicular stomatitis virus (VSV; Vesic) was used.
The inhibition of the action of ultra statitis virus) on mouse fibroblast cell line L929 was used as an index. That is, L929 cells were placed in 4 × 10 4 cells / 0.1 ml in each well, cultured for 24 hours, and then 0.1 ml of each diluted sample solution was added from the dilution series of the compound of the present invention or the comparative compound (1 mg / ml). It was cultured for 24 hours. After removing the culture supernatant, VSV adjusted to 100 TCID 50 /0.1 ml
After culturing for 24 hours, the culture solution was removed, fixed with 5% formaldehyde solution for 20 minutes, and then stained with 0.5% crystal violet staining solution for 20 minutes. After washing with water and drying, the absorbance was measured at 600 nm. Activity is L
The original sample concentration of the sample solution in which 929 cells survive 50% (1
It was expressed as the reciprocal of the dilution factor for mg / ml). As shown in Table 4, it was revealed that the compound of the present invention has a stronger antiviral activity than that of synthetic lipid A 506.
【0039】[0039]
【表4】 [Table 4]
【0040】(6)多クローン性B細胞活性化活性 本試験はエリスポット(Enzyme-Linked Immunospot; EL
ISPOT)法を用いて検討した。BALB/cマウスの脾細
胞(2.5×106 個)に所定量の本発明化合物あるい
は比較化合物の添加あるいは無添加の条件下で5%の牛
胎児血清(FBS)を含むRPMI1640培地にて、
37℃、72時間培養した。洗浄後、抗体産生細胞はE
LISPOT法にて測定した。(6) Polyclonal B cell activating activity This test was carried out using Elispot (Enzyme-Linked Immunospot; EL
It was examined using the (ISPOT) method. In the RPMI1640 medium containing 5% fetal bovine serum (FBS) under the condition that the spleen cells (2.5 × 10 6 cells) of BALB / c mouse were added with or without a predetermined amount of the compound of the present invention or the comparative compound. ,
It was cultured at 37 ° C. for 72 hours. After washing, the antibody-producing cells are E
It was measured by the LISPOT method.
【0041】すなわち、ヤギ抗マウスイムノグロブリン
をコートし、5%FBS処理したプレートの各ウェルに
上述した脾細胞を加え4時間培養した。細胞を洗浄除去
後、プレートをビオチン標識したヤギ抗マウスμ鎖特異
抗血清と25℃で一晩反応し、生理緩衝食塩水(PB
S)で洗浄後、ペルオキシダーゼ標識ストレプトアビジ
ンにて処理した。活性は抗体産生細胞により生じるスポ
ット数(細胞数)を実体顕微鏡下で計測し、供試化合物
添加の細胞数に対する供試化合物無添加の細胞数をコン
トロールしたときのStimulation Index にて表した。な
お、供試化合物の用量は、細胞2.5×106 個当たり
のμgで表した。本発明化合物は、表5に示されるよう
に合成リピドAである506と同等もしくはそれ以上の
多クローン性B細胞活性化活性を有することが明らかと
なり、従って強い非特異的感染防御活性を有するものと
考えられる。That is, the above-mentioned splenocytes were added to each well of a plate coated with goat anti-mouse immunoglobulin and treated with 5% FBS, and cultured for 4 hours. After washing and removing the cells, the plate was reacted with a biotin-labeled goat anti-mouse μ chain-specific antiserum at 25 ° C. overnight to obtain physiological buffered saline (PB).
After washing with S), it was treated with peroxidase-labeled streptavidin. The activity was represented by a Stimulation Index when the number of spots (cell number) generated by antibody-producing cells was measured under a stereoscopic microscope and the number of cells without test compound to the number of cells with test compound was controlled. The dose of the test compound was expressed in μg per 2.5 × 10 6 cells. As shown in Table 5, the compounds of the present invention were found to have a polyclonal B cell activating activity equal to or higher than that of synthetic lipid A 506, and therefore, a compound having a strong nonspecific infection protective activity. it is conceivable that.
【0042】[0042]
【表5】 [Table 5]
【0043】(7)サイトカイン産生誘導活性 サイトカイン産生誘導活性試験は、TNF−α測定用E
LISAシステム(アマーシャム・ジャパン株式会社
製)およびIL−1β測定用ELISAシステム(大塚
製薬株式会社製)を用いて検討した。すなわち、ヒト末
梢血単球(5×105 個)に所定濃度の本発明化合物あ
るいは比較化合物を加え、24時間培養後、培養上清中
のサイトカイン量をELISA法により定量した。(7) Cytokine production inducing activity The cytokine production inducing activity test was carried out using E for TNF-α measurement.
The examination was performed using a LISA system (manufactured by Amersham Japan Co., Ltd.) and an IL-1β measurement ELISA system (manufactured by Otsuka Pharmaceutical Co., Ltd.). That is, a predetermined concentration of the compound of the present invention or a comparative compound was added to human peripheral blood monocytes (5 × 10 5 cells), and after culturing for 24 hours, the amount of cytokine in the culture supernatant was quantified by the ELISA method.
【0044】その結果、ヒト末梢血単球からのTNF−
αやIL−1βの産生を誘導する活性は、本発明化合物
には殆ど認められなかった。さらに、合成リピドAであ
る化合物506あるいは大腸菌由来のLPSと本発明化
合物(50倍量)を同時に加えて培養することによっ
て、化合物506あるいは大腸菌由来のLPSによって
誘導されるIL−1βの産生を抑制することが明らかと
なった。さらに、IL−1レセプターアンタゴニスト
(IL−1ra)測定用ELISAシステム(R&D社
製)により、本発明化合物はヒト末梢血単球の培養上清
中にIL−lraを合成リピドAである化合物506よ
り多量に産生することも明らかとなった。As a result, TNF- from human peripheral blood monocytes was detected.
Almost no activity of inducing α or IL-1β production was observed in the compound of the present invention. Furthermore, the production of IL-1β induced by LPS derived from compound 506 or Escherichia coli is suppressed by simultaneously adding and culturing the compound 506 or LPS derived from Escherichia coli, which is a synthetic lipid A, and the compound of the present invention (50-fold amount). It became clear to do. Further, the compound of the present invention was analyzed by an ELISA system (manufactured by R & D) for measuring IL-1 receptor antagonist (IL-1ra) to obtain IL-lra in the culture supernatant of human peripheral blood monocytes from compound 506 which is a synthetic lipid A. It was also revealed that a large amount was produced.
【0045】(8)ガラクトサミン負荷致死毒性 ガラクトサミン負荷致死毒性は以下の実験系を用いて求
めた。(8) Lethal toxicity of galactosamine loading The lethal toxicity of galactosamine loading was determined using the following experimental system.
【0046】C57BLマウス(雄性、8週齢)に、1
6mgのD−ガラクトサミン/HClを腹腔内投与し、そ
の直後に本願発明化合物を静脈注射し、24時間後に状
態観察を行った。To C57BL mice (male, 8 weeks old), 1
6 mg of D-galactosamine / HCl was intraperitoneally administered, immediately thereafter, the compound of the present invention was intravenously injected, and the condition was observed 24 hours later.
【0047】本願発明化合物は、以下の表6に示される
ような活性を示し、低毒性であることが明らかになっ
た。The compounds of the present invention exhibited the activities shown in Table 6 below, and were found to have low toxicity.
【0048】[0048]
【表6】 [Table 6]
【0049】(9)その他の毒性局所シュワルツマン反応 本試験は以下の方法により検討した。即ち雄のウサギの
皮内に0.2mlの生理食塩水で所定濃度に調製した供試
化合物を注射し、24時間後、100μg/ml/kgのサ
ルモネラミネソタ9700LPS−W(ディフコ社製)
を静脈より惹起注射して4時間後の皮内の出血反応を観
察した。その結果、本発明化合物は100μg/siteで
出血反応は見られなかった。(9) Other Toxicity Local Schwarzman Reaction This test was examined by the following method. That is, a test compound prepared at a predetermined concentration with 0.2 ml of physiological saline was intradermally injected into a male rabbit, and 24 hours later, 100 μg / ml / kg of Salmonella Minnesota 9700LPS-W (manufactured by Difco).
Was intradermally injected 4 hours after the injection was observed. As a result, no bleeding reaction was observed with the compound of the present invention at 100 μg / site.
【0050】発熱原性試験 本試験はウサギを用い、供試化合物を5ml/kgの生理食
塩水に所定濃度に調製したものを静脈注射し、直腸温度
の測定を実施した。注射後4時間の間に、0.6℃以上
の体温上昇が認められたものについて発熱作用ありと判
定した。その結果、合成リピドAである506は0.0
1μgで発熱作用が認められるのに対して、本発明化合
物は10μg/kgの用量においても発熱作用が認められ
なかった。Pyrogenicity test In this test, rabbits were used, and 5 ml / kg of the test compound prepared to a predetermined concentration was intravenously injected, and the rectal temperature was measured. Those in which a body temperature increase of 0.6 ° C. or more was observed within 4 hours after the injection were judged to have a fever effect. As a result, synthetic lipid A 506 is 0.0
While the pyrogenic effect was observed at 1 μg, the compound of the present invention showed no pyrogenic effect even at a dose of 10 μg / kg.
【0051】リムルス試験 本試験はエンドトキシン測定試薬であるプレゲル(生化
学工業株式会社製)を使用して検定した。即ち日本産カ
ブトガニのライセートより調製した凍結乾燥品を用い
て、ゲル形成能の有無を判定した。その結果、本発明化
合物の最小有効量は、合成リピドAである506の10
00倍であり、非常に低毒性であった。 Limulus test This test was performed using Pregel (manufactured by Seikagaku Corporation) which is an endotoxin measuring reagent. That is, the presence or absence of gel-forming ability was determined using a freeze-dried product prepared from Japanese horseshoe crab lysate. As a result, the minimum effective dose of the compound of the present invention is 10 of 506 of synthetic lipid A.
It was 00 times and had very low toxicity.
【0052】本願発明化合物は、リムルス活性、局所シ
ュワルツマン反応および発熱性等の諸試験において低毒
性であった。The compound of the present invention had low toxicity in various tests such as Limulus activity, local Schwarzman reaction and pyrogenicity.
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成5年12月7日[Submission date] December 7, 1993
【手続補正1】[Procedure Amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0002[Name of item to be corrected] 0002
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0002】[0002]
【従来の技術】各種グラム陰性菌の細胞壁外膜に含まれ
るリポポリサッカライド(LPS)は、リピドAと呼ば
れる糖脂質に各種の糖類が結合したものであり、以前か
ら内毒素の主成分であることが知られている。LPSは
生体の各種免疫機能促進活性を有することが知られてお
り、その主たる活性発現部位はリピドAにあることが明
らかにされており免疫調節作用および抗腫瘍作用の他、
多様な生物活性を有することが認められている。2. Description of the Related Art Lipopolysaccharide (LPS) contained in the outer wall of cell walls of various Gram-negative bacteria is a glycolipid called lipid A in which various sugars are bound, and has been a main component of endotoxin for a long time. It is known. LPS is known to have a variety of immune function promotion activity of the living body, other its principal active expression site are immune regulatory effects and anti-tumor effect is shown to be in the lipid A,
It is recognized to have diverse biological activities.
【手続補正2】[Procedure Amendment 2]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0007[Correction target item name] 0007
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0007】[0007]
【課題を解決するための手段】本発明者らは、ヒトの口
腔内に常在しており、現在歯周病の原因と考えられてい
る細菌の一つであるPorphyromonas(Bacteroides) gingi
valisの細胞壁外膜中に含まれるLPSがマイトジェン
活性等を有し、同時に致死毒性および発熱原性が非常に
弱いことを見いだした。さらに、LPS中の活性発現部
位を調製後精製し、構造解析を行い、鋭意研究の結果、
本発明の活性化合物はグルコサミンβ(1−6)ジサッ
カライドの構造の1位にリン酸基がエステル結合をした
ものを基本骨格とし、2位のアミノ基に3-hydroxy-15-m
ethylhexadecanoic acidがアミド結合し、2′位のアミ
ノ基に3-hexadecanoyloxy-15-methylhexadecanoic acid
がアミド結合していることを見いだした。本発明の化合
物の構造は、従来のリピドA誘導体と大きく異なり4′
位にリン酸基を持たず、3位および3′位の水酸基が遊
離のままである点に特徴がある。したがって本発明の化
合物は式;[Means for Solving the Problems] The present inventors have found that Porphyromonas (Bacteroides) gingi, which is one of the bacteria that is resident in the human oral cavity and is currently considered to be the cause of periodontal disease.
It was found that LPS contained in the cell wall outer membrane of valis has mitogenic activity and the like, and at the same time, its lethal toxicity and pyrogenicity are very weak. Furthermore, after the active expression site in LPS was prepared and purified, the structure was analyzed and the results of earnest research showed that
The active compound of the present invention has, as a basic skeleton, a structure in which a phosphoric acid group is ester-bonded at the 1-position of the structure of glucosamine β (1-6) disaccharide, and 3-hydroxy-15-m at the 2-position amino group.
Ethylhexadecanoic acid is amide-bonded and 3-hexadecanoyloxy-15-methylhexadecanoic acid is added to the amino group at the 2'position.
Was found to have an amide bond. The structure of the compound of the present invention is significantly different from that of the conventional lipid A derivative.
It is characterized in that it has no phosphate group at the position and the hydroxyl groups at the 3 and 3'positions remain free. Thus the compounds of the present invention have the formula:
【化1】 で表されるような構造を有することが推定される。また
本願化合物は強いマイトジェン活性、アジュバント活
性、多クローン性B細胞活性化(非特異的感染防御)活
性、ナチュラルキラー活性等多様な生物活性を有する一
方、従来のリピドAおよびリピドA誘導体においてみら
れたマクロファージからの腫瘍壊死因子(TNF)やI
L−1等のいわゆる炎症性サイトカインの産生を誘導す
る活性が殆ど見られないことを見いだした。したがって
従来、リピドAおよびリピドA誘導体において問題とな
っていた致死毒性および発熱原性等の有害作用を示すこ
となく、免疫賦活剤として有用である。さらには大腸菌
のリピドAが誘導するIL−1の産生を抑え、さらにI
L−1レセプターアンタゴニスト(IL−1ra)の産
生を誘導することを見いだしたが、これらの活性は、強
い非特異的感染防御活性を有することと併せて、大腸菌
等グラム陰性菌の感染によって引き起こされる病態、特
に敗血症、あるいは敗血症性ショックの予防・治療用剤
として有用である。また、IL−1の産生を抑え、さら
にIL−1レセプターアンタゴニスト(IL−1ra)
の産生を誘導することは、IL−1自身の異常産生状態
になっていると考えられるような病態、例えば慢性関節
リューマチ等の治療用剤としても有用である。さらに、
ナチュラルキラー細胞を活性化し、抗腫瘍活性を有する
ことを見いだした上、抗ウイルス活性を有することも明
らかになった。抗腫瘍活性からは、抗腫瘍剤としての有
用性が考えられ、抗ウイルス活性と併せて強い非特異的
感染防御活性を有することからは、抗ウイルス剤として
の有用性がある。このような特性から、本願発明化合物
である新規なジサッカライド誘導体またはその塩は、医
薬用担体および/または希釈剤と配合してなる医薬組成
物として特に有用であると期待される。[Chemical 1] It is presumed that it has a structure represented by. In addition, the compound of the present invention has various biological activities such as strong mitogenic activity, adjuvant activity, polyclonal B cell activation (non-specific infection protection) activity, natural killer activity, etc., while it is found in conventional lipid A and lipid A derivatives. tumor necrosis factor from macrophages was (TNF) and I
It was found that there is almost no activity that induces the production of so-called inflammatory cytokines such as L-1. Therefore, it is useful as an immunostimulant without exhibiting harmful effects such as lethal toxicity and pyrogenicity, which have heretofore been problems for lipid A and lipid A derivatives. Furthermore, it suppresses the production of IL-1 induced by lipid A of Escherichia coli.
It was found that it induces the production of L-1 receptor antagonist (IL-1ra), and these activities, together with having a strong non-specific protective activity against infection, are caused by infection with Gram-negative bacteria such as Escherichia coli. It is useful as a prophylactic / therapeutic agent for pathological conditions, particularly sepsis or septic shock. It also suppresses the production of IL-1 and further suppresses IL-1 receptor antagonist (IL-1ra).
Inducing the production of is also useful as a therapeutic agent for pathological conditions that are considered to be in the abnormal production state of IL-1 itself, such as rheumatoid arthritis. further,
In addition to activating natural killer cells and having antitumor activity, it was revealed that they also have antiviral activity. It is considered to be useful as an antitumor agent from the viewpoint of antitumor activity, and useful as an antiviral agent since it has a strong nonspecific infection protective activity in addition to the antiviral activity. From such characteristics, it is expected that the novel disaccharide derivative or the salt thereof which is the compound of the present invention will be particularly useful as a pharmaceutical composition prepared by mixing with a pharmaceutical carrier and / or diluent.
【手続補正3】[Procedure 3]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0012[Correction target item name] 0012
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0012】(1)微生物を用いた製造例 本願化合物を微生物により製造する場合には、前記〔化
1〕により示される本願発明の化合物を生産することが
できる微生物であればいずれも使用できるが、例えばPo
rphyromonas(Bacteroides) gingivalis(ATCCカタ
ログ番号33277)等を使用することができる。(1) Production Example Using Microorganism When the compound of the present invention is produced by a microorganism, any microorganism can be used as long as it can produce the compound of the present invention represented by the above-mentioned [Chemical formula 1]. , For example Po
rphyromonas (Bacteroides) gingivalis (ATCC catalog number 33277) and the like can be used.
【手続補正4】[Procedure amendment 4]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0018[Correction target item name] 0018
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0018】また本願化合物は強いマイトジェン活性、
アジュバント活性、多クローン性B細胞活性化(非特異
的感染防御)活性、ナチュラルキラー活性等多様な生物
活性を有する一方、従来のリピドAおよびリピドA誘導
体においてみられたマクロファージからの腫瘍壊死因子
(TNF)やIL−1等のいわゆる炎症性サイトカイン
の産生を誘導する活性が殆ど見られないことを見いだし
た。したがって従来、リピドAおよびリピドA誘導体に
おいて問題となっていた致死毒性および発熱原性等の有
害作用を示すことなく、免疫賦活剤として有用である。
さらには大腸菌のリピドAが誘導するIL−1の産生を
抑え、さらにIL−1レセプターアンタゴニスト(IL
−1ra)の産生を誘導することを見いだしたが、これ
らの活性は、強い非特異的感染防御活性を有することと
併せて、大腸菌等グラム陰性菌の感染によって引き起こ
される病態、特に敗血症、あるいは敗血症性ショックの
予防・治療用剤として有用である。また、IL−1の産
生を抑え、さらにIL−1レセプターアンタゴニスト
(IL−1ra)の産生を誘導することは、IL−1自
身の異常産生状態になっていると考えられるような病
態、例えば慢性関節リューマチ等の治療用剤としても有
用である。さらに、ナチュラルキラー細胞を活性化し、
抗腫瘍活性を有することを見いだした上、抗ウイルス活
性を有することも明らかになった。抗腫瘍活性からは、
抗腫瘍剤としての有用性が考えられ、抗ウイルス活性と
併せて強い非特異的感染防御活性を有することからは、
抗ウイルス剤としての有用性がある。The compound of the present invention has a strong mitogenic activity,
Adjuvant activity, polyclonal B cell activation (non-specific defense against infection) activity, while having a natural killer activity such diverse biological activities, tumor necrosis factor from macrophages seen in conventional lipid A and lipid A derivative It was found that there is almost no activity that induces the production of so-called inflammatory cytokines such as (TNF) and IL-1. Therefore, it is useful as an immunostimulant without exhibiting harmful effects such as lethal toxicity and pyrogenicity, which have heretofore been problems for lipid A and lipid A derivatives.
Furthermore, it suppresses the production of IL-1 induced by lipid A of E. coli, and further suppresses IL-1 receptor antagonist (IL
-1ra), it was found that these activities, in addition to having a strong non-specific protective activity against infection, are associated with pathological conditions caused by infection with Gram-negative bacteria such as Escherichia coli, particularly sepsis or sepsis. It is useful as a prophylactic / therapeutic agent for sexual shock. In addition, suppressing the production of IL-1 and further inducing the production of IL-1 receptor antagonist (IL-1ra) is a pathological condition that is considered to be in the abnormal production state of IL-1 itself, for example, a chronic condition. It is also useful as a therapeutic agent for rheumatoid arthritis and the like. In addition, activates natural killer cells,
It was found that it has antitumor activity and also has antiviral activity. From the antitumor activity,
It is considered to be useful as an antitumor agent, and since it has a strong non-specific defense against infection in addition to its antiviral activity,
It has utility as an antiviral agent.
【手続補正5】[Procedure Amendment 5]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0021[Correction target item name] 0021
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0021】[0021]
【実施例】 実施例1微生物源からの製造 Porphyromonas(Bacteroides) gingivalisを160リッ
トルのGAMブイヨン(日水製薬株式会社製)培地(p
H7.3)で嫌気的に37℃で26時間培養した。培養
完了後培養液の遠心を行い菌体を回収し、凍結乾燥によ
り乾燥菌体100gを得た。この乾燥菌体を熱フェノー
ル−水抽出法にて抽出処理して粗LPSを得た。すなわ
ち100gの乾燥菌体に3.5リットルの蒸留水を加え
68℃に加熱し、別に68℃に加熱した90%フェノー
ルを加え68℃にて20分間撹拌し、氷冷後遠心して水
層を分取後、再度3.5リットルの蒸留水を加え抽出操
作を繰り返した。得られた水層を合わせ、蒸留水に対し
て十分透析後、濃縮し凍結乾燥を行い粗抽出物12.4
7gを得た。Example 1 Production from Microbial Source Porphyromonas (Bacteroides) gingivalis 160 g of GAM broth (manufactured by Nissui Pharmaceutical Co., Ltd.) medium (p
H7.3) was anaerobically cultured at 37 ° C. for 26 hours. After the completion of the culture, the culture solution was centrifuged to collect the cells, and freeze-dried to obtain 100 g of dried cells. The dried cells were subjected to extraction treatment with a hot phenol-water extraction method to obtain crude LPS. That is, 3.5 liters of distilled water was added to 100 g of dried cells and heated to 68 ° C., 90% phenol heated to 68 ° C. was added, and the mixture was stirred at 68 ° C. for 20 minutes, ice-cooled and centrifuged to form an aqueous layer. After the collection, 3.5 liters of distilled water was added again and the extraction operation was repeated. The obtained aqueous layers were combined, sufficiently dialyzed against distilled water, concentrated and freeze-dried to obtain a crude extract 12.4.
7 g was obtained.
【手続補正6】[Procedure correction 6]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0030[Name of item to be corrected] 0030
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0030】[0030]
【表1】 [Table 1]
【手続補正7】[Procedure Amendment 7]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0031[Correction target item name] 0031
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0031】(2)NK(ナチュラルキラー)活性 本試験は以下の方法により検討した。すなわち、BAL
B/cマウスを用い100μgの本発明化合物、あるい
は比較化合物を0日目および7日目に静脈内に注射し、
14日目に脾細胞を採取した。調製した脾細胞(1×1
06 個/0.1ml)に51Crで標識した標的細胞(Molon
eyウイルス誘発リンパ腫YAC−1:2×104 個/
0.1ml)を加え、4時間培養した。なお、最小遊離対
照群には脾細胞を含まない培養液のみを加え、最大遊離
対照群には0.1N NaOHを加えた。培養終了後、
培養上清0.1mlを採取し、標的細胞が障害を受けるこ
とにより遊離した51Crの量(放射線量)を測定した。
活性は各価を以下の式に代入し、NK Activity(%)を求
めた。(2) NK (Natural Killer) Activity This test was examined by the following method. That is, BAL
B / c mice were used to inject 100 μg of the compound of the present invention or a comparative compound intravenously on days 0 and 7,
Splenocytes were collected on the 14th day. Prepared splenocytes (1 x 1
Target cells (Molon) labeled with 51 Cr in 0 6 cells / 0.1 ml)
ey virus-induced lymphoma YAC-1: 2 × 10 4 cells /
0.1 ml) was added and the cells were cultured for 4 hours. It should be noted that only a culture solution containing no splenocytes was added to the minimum free control group, and 0.1N NaOH was added to the maximum free control group. After culturing,
0.1 ml of the culture supernatant was collected, and the amount of 51 Cr released by the damage to the target cells (radiation dose) was measured.
For the activity, NK Activity (%) was calculated by substituting each value into the following formula.
【手続補正8】[Procedure Amendment 8]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0034[Correction target item name] 0034
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0034】[0034]
【表2】 [Table 2]
【手続補正9】[Procedure Amendment 9]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0049[Correction target item name] 0049
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0049】(9)その他の毒性局所シュワルツマン反応 本試験は以下の方法により検討した。即ち雄のウサギの
皮内に0.2mlの生理食塩水で所定濃度に調製した供試
化合物を注射し、24時間後、100μg/ml/kgの S
almonella minnesota9700LPS−W(ディフコ社
製)を静脈より惹起注射して4時間後の皮内の出血反応
を観察した。その結果、本発明化合物は100μg/si
teで出血反応は見られなかった。(9) Other Toxicity Local Schwarzman Reaction This test was examined by the following method. That is, a test compound prepared at a predetermined concentration with 0.2 ml of physiological saline was injected into the skin of a male rabbit, and 24 hours later, 100 μg / ml / kg of S
Almonella minnesota 9700LPS-W (manufactured by Difco) was intravenously induced and injected, and the intradermal bleeding reaction was observed 4 hours later. As a result, the compound of the present invention was 100 μg / si
No hemorrhagic reaction was seen on te.
フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 // C12P 19/26 7432−4B (C12P 19/26 C12R 1:01) Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location // C12P 19/26 7432-4B (C12P 19/26 C12R 1:01)
Claims (3)
性、ディットマー・レスター反応に陽性、ニンヒドリン
反応に陰性、TTC反応に陰性であって、 (2)NMRスペクトルとして、 1H−NMR(300MHz,
CDCl3 +MeOD+D2 O)δ:0.81(12H,
d)、0.82(3H,t)、1.1-1.6(72H,m,CH2,C
H)、2.2-2.5 (6H,m,CO−CH2 )、3.1-4.3(13
H,m)、4.46(1H,d)、5.15(1H,m)、5.45(1
H,m)の物性値をもち、 (3)質量スペクトルとして、ネガティブFAB−MS
−MS、M/Z 1193(M−H)、937(M−2H−C15
H31COO)の物性値を持つ、新規ジサッカライド誘導
体、またはその塩。(1) The color reaction is positive for a sulfuric acid reaction, positive for a Dittmer-Lester reaction, negative for a ninhydrin reaction, and negative for a TTC reaction. (2) As an NMR spectrum, 1 H-NMR ( 300MHz,
CDCl 3 + MeOD + D 2 O) δ: 0.81 (12H,
d), 0.82 (3H, t), 1.1-1.6 (72H, m, CH 2 , C
H), 2.2-2.5 (6H, m , CO-CH 2), 3.1-4.3 (13
H, m), 4.46 (1H, d), 5.15 (1H, m), 5.45 (1
H, m), and (3) as a mass spectrum, negative FAB-MS
-MS, M / Z 1193 (M -H), 937 (M-2H-C 15
H 31 COO), a novel disaccharide derivative or a salt thereof.
カライド構造の1位にリン酸基がエステル結合したもの
を基本骨格とし、 (2)2位のアミノ基に3-hydroxy-15-methylhexadecan
oic acidがアミド結合し、 (3)2′位のアミノ基に3-hexadecanoyl-15-methylhe
xadecanoic acid がアミド結合し、 (4)4′位にリン酸基を持たず、 (5)3位,3′位が遊離のままである、新規ジサッカ
ライド誘導体またはその塩。2. A basic skeleton having (1) a glucosamine β (1-6) disaccharide structure in which a phosphate group is ester-bonded at the 1-position, and (2) 3-hydroxy-15- at the 2-position amino group. methylhexadecan
(3) 3-hexadecanoyl-15-methylhe is attached to the amino group at the 2'position by amide bond of oic acid.
A novel disaccharide derivative or salt thereof in which xadecanoic acid is amide-bonded, (4) does not have a phosphate group at the 4'position, and (5) remains free at the 3'and 3'positions.
その薬学的に許容される塩を、医薬用担体および/また
は希釈剤と配合してなる医薬組成物。3. A pharmaceutical composition comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutical carrier and / or diluent.
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP5222449A JPH06206893A (en) | 1992-09-07 | 1993-09-07 | New disaccharide derivative |
EP94909291A EP0668289A4 (en) | 1993-09-07 | 1994-03-09 | Novel disaccharide derivative. |
PCT/JP1994/000376 WO1995007285A1 (en) | 1993-09-07 | 1994-03-09 | Novel disaccharide derivative |
US08/433,354 US5654289A (en) | 1993-09-07 | 1994-03-09 | Disaccharide derivative |
AU62196/94A AU679970B2 (en) | 1993-09-07 | 1994-03-09 | Novel disaccharide derivative |
CA002148824A CA2148824A1 (en) | 1993-09-07 | 1994-03-09 | Novel disaccharide derivative |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP23788692 | 1992-09-07 | ||
JP4-237886 | 1992-09-07 | ||
JP5222449A JPH06206893A (en) | 1992-09-07 | 1993-09-07 | New disaccharide derivative |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH06206893A true JPH06206893A (en) | 1994-07-26 |
Family
ID=26524891
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP5222449A Pending JPH06206893A (en) | 1992-09-07 | 1993-09-07 | New disaccharide derivative |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH06206893A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09505071A (en) * | 1993-11-17 | 1997-05-20 | ラボラトワル・オーエム・ソシエテ・アノニム | Glucosamine disaccharides, processes for their production, pharmaceutical compositions containing them and their use |
JP2003518086A (en) * | 1999-12-22 | 2003-06-03 | オーエム ファルマ | Novel acyl-dipeptide-like compounds with additional functionalized side chains |
JP2018070652A (en) * | 2012-04-12 | 2018-05-10 | アバンティ・ポーラ・リピッド・インコーポレイテッド | Disaccharide synthetic lipid compounds and uses thereof |
JP2021504463A (en) * | 2017-11-23 | 2021-02-15 | ルスロ ビーブイビーエー | Method for preparing gelatin hydrolyzate with low endotoxin content |
-
1993
- 1993-09-07 JP JP5222449A patent/JPH06206893A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH09505071A (en) * | 1993-11-17 | 1997-05-20 | ラボラトワル・オーエム・ソシエテ・アノニム | Glucosamine disaccharides, processes for their production, pharmaceutical compositions containing them and their use |
JP2003518086A (en) * | 1999-12-22 | 2003-06-03 | オーエム ファルマ | Novel acyl-dipeptide-like compounds with additional functionalized side chains |
JP4902924B2 (en) * | 1999-12-22 | 2012-03-21 | オーエム ファルマ | Novel acyl-dipeptide-like compounds with additional functionalized side chains |
JP2018070652A (en) * | 2012-04-12 | 2018-05-10 | アバンティ・ポーラ・リピッド・インコーポレイテッド | Disaccharide synthetic lipid compounds and uses thereof |
JP2021504463A (en) * | 2017-11-23 | 2021-02-15 | ルスロ ビーブイビーエー | Method for preparing gelatin hydrolyzate with low endotoxin content |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DE2655500C2 (en) | ||
EP0466838B1 (en) | Novel lipid a derivatives and uses therefor | |
US4446128A (en) | Antigen derivatives and processes for their preparation | |
US5952313A (en) | LPS antagonists and methods of making and using the same | |
FI75172C (en) | Process for the preparation of therapeutically useful N-glycosylated e-carboxylic acid amide derivatives. | |
EP0038750B1 (en) | Immunologically active dipeptidyl 4-0-, 6-0-acyl-2-amino-2-deoxy-d-glucose derivatives and methods for their preparation | |
US4315913A (en) | Immunologically active dipeptidyl 2-amino-1,2-dideoxy-D-glucose derivatives and methods of preparation | |
EP0003833B2 (en) | Antigen derivatives, process for their preparation, pharmaceutical compositions containing them | |
US4310514A (en) | Immunologically active dipeptidyl 5-0,6-0-acyl-2-amino-2-deoxy-D-glucofuranose derivatives and methods of preparation | |
US4868155A (en) | Dipeptidyl 4-0-,6-0-acyl-2-amino-2-deoxy-D-glucose compositions and methods of use in AIDS-immunocompromised human hosts | |
US5041427A (en) | Lipid A derivatives | |
US4501693A (en) | Method of preparing immunostimulant proteoglycans which induce production of interferon, proteoglycans obtained and pharmaceutical compositions containing them | |
JPH06206893A (en) | New disaccharide derivative | |
DK170170B1 (en) | Alpha-D-Glucopyranose Derivatives, Method of Preparation and Pharmaceutical Composition Containing | |
EP0668289A1 (en) | Novel disaccharide derivative | |
US4868157A (en) | Dipeptidyl 2-amino-1,2-dideoxy-D-glucose derivatives as host resistance enhancers in AIDS-immunocompromised hosts and methods of use | |
US4866035A (en) | Dipeptidyl saccharides as host resistance enhancers in AIDS-immuno-compromised hosts and methods of use | |
AU679970B2 (en) | Novel disaccharide derivative | |
US4391800A (en) | Immunologically active peptidyl disaccharides and methods of preparation | |
US9005622B2 (en) | Cell wall components of enterococci and uses thereof | |
EP0118364B1 (en) | Immunostimulatory dipeptidyl d-glucose derivatives and methods of preparation | |
JP2514070B2 (en) | Novel glucopyranose derivative | |
US4866036A (en) | Dipeptidyl 5-0,6-0-acyl-2-amino-2-deoxy-D-glucofuranose compositions and methods of use in aids-immunocompromised human hosts | |
JPS63179885A (en) | Novel glucopyranose derivative, production thereof and medicine containing said derivative as active ingredient | |
WO2018155051A1 (en) | Lipid a |