JP2579730B2 - Peptide-lipid derivatives and liposomes - Google Patents

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【０００１】[0001]

【産業上の利用分野】本発明は、Ａｒｇ−Ｇｌｙ−Ａｓ
ｐからなる配列を有するペプチドに、脂質が直接にまた
はリンカーを介して結合していることを特徴とするペプ
チド−脂質誘導体、及びこの誘導体を有するリポソーム
に関する。The present invention relates to Arg-Gly-As
The present invention relates to a peptide-lipid derivative characterized in that a lipid is bound to a peptide having a sequence consisting of p directly or via a linker, and a liposome having this derivative.

【０００２】[0002]

【従来の技術】Ａｒｇ−Ｇｌｙ−Ａｓｐ配列（ＲＧＤ配
列）は、細胞表面に存在する接着分子であるインテグリ
ンをリセプターとするフィブロネクチン等のリガンド中
に存在し、インテグリンの一部はこのＲＧＤ配列を認識
してこれと結合することが知られている（Pierschbache
r, M.D., et al., Nature, 309, 30〜33 (1984) ）。2. Description of the Related Art An Arg-Gly-Asp sequence (RGD sequence) is present in a ligand such as fibronectin having an integrin, which is an adhesion molecule on the cell surface, as a receptor, and a part of the integrin recognizes this RGD sequence. Is known to combine with this (Pierschbache
r, MD, et al., Nature, 309 , 30-33 (1984)).

【０００３】このことより、外から与えられたＲＧＤ配
列を含むペプチド（ＲＧＤペプチド）はフィブロネクチ
ンと競合して例えば癌細胞に結合し、その結果、フィブ
ロネクチンによる細胞間接着等を阻害すると考えられ
る。実際、ＲＧＤ配列が癌細胞の転移を抑制することが
観察されている（Humphries M.J., et al., Science,2
33, 467 (1986)）。[0003] From this, it is considered that a peptide containing an RGD sequence provided from outside (RGD peptide) competes with fibronectin and binds to, for example, cancer cells, and as a result, inhibits intercellular adhesion and the like by fibronectin. In fact, it has been observed that the RGD sequence suppresses metastasis of cancer cells (Humphries MJ, et al., Science, 2
33 , 467 (1986)).

【０００４】[0004]

【発明が解決しようとする課題】そこで本発明者は、フ
ィブロネクチンと拮抗的にインテグリンに結合し、か
つ、ＲＧＤペプチドより、より強く癌細胞表面インテグ
リンに結合する物質を発見できれば、より強く癌細胞の
転移を抑制できると考えた。即ち、本発明は癌細胞の転
移抑制物質を提供しようとするものである。Therefore, if the present inventors can find a substance that binds to integrin in a competitive manner with fibronectin and that binds more strongly to the integrin on the cancer cell surface than the RGD peptide, the present inventors will find that the stronger the cancer cells are, We thought that metastasis could be suppressed. That is, the present invention aims to provide a substance that inhibits metastasis of cancer cells.

【０００５】[0005]

【課題を解決するための手段】本発明者は、リポソーム
表面にＲＧＤペプチドを多数存在せしめれば、そのリポ
ソーム表面の複数のＲＧＤ配列に癌細胞が結合すること
により、ＲＧＤペプチド単独よりもより強く癌細胞に結
合するのではないかと考え、表面にＲＧＤペプチドを有
するリポソームを得ようとした。Means for Solving the Problems The present inventors have proposed that if a large number of RGD peptides are present on the surface of a liposome, the cancer cells will bind to a plurality of RGD sequences on the surface of the liposome, whereby the RGD peptide will be stronger than the RGD peptide alone. We thought that it might bind to cancer cells, and tried to obtain liposomes having an RGD peptide on the surface.

【０００６】そこで、ＲＧＤペプチドの脂質誘導体（Ｒ
ＧＤペプチド−脂質誘導体）を用いてリポソームを調製
したところ、表面にＲＧＤペプチドを有するリポソーム
を調製することができた。Accordingly, lipid derivatives of the RGD peptide (R
When a liposome was prepared using (GD peptide-lipid derivative), a liposome having an RGD peptide on the surface could be prepared.

【０００７】すなわち、本発明は、ＲＧＤペプチドに脂
質が直接にまたはリンカーを介して結合していることを
特徴とするペプチド−脂質誘導体、及びこのようなペプ
チド−脂質誘導体を有することを特徴とするリポソーム
に関する。That is, the present invention is characterized in that a peptide-lipid derivative is characterized in that a lipid is bonded to an RGD peptide directly or via a linker, and that such a peptide-lipid derivative is provided. Related to liposomes.

【０００８】以下、本発明を逐次詳細に説明する。Hereinafter, the present invention will be described in detail.

【０００９】先ず、本発明のペプチド−脂質誘導体は新
規物質であるので、その製造法とともにこれを説明す
る。First, since the peptide-lipid derivative of the present invention is a novel substance, it will be described together with its production method.

【００１０】ＲＧＤペプチドは、先述のようにＲＧＤ配
列を含むペプチドの略称であって、ＲＧＤ配列を含むペ
プチドであれば全てこれに包含される。従って、ＲＧＤ
ペプチドは、ＲＧＤ配列のみからなるペプチドでもよ
く、また、ＲＧＤ配列とそのＣ−末端またはＮ−末端に
結合したアミノ酸またはペプチドとからなるペプチドで
もよく、更にはＲＧＤ配列がペプチド鎖のＣ−末端又は
Ｎ−末端以外の場所にあっても良い。[0010] The RGD peptide is an abbreviation of a peptide containing an RGD sequence as described above, and any peptide containing an RGD sequence is included in this. Therefore, RGD
The peptide may be a peptide consisting only of the RGD sequence, or may be a peptide consisting of the RGD sequence and an amino acid or peptide bound to its C-terminus or N-terminus. It may be at a place other than the N-terminal.

【００１１】ＲＧＤペプチドの全体のアミノ酸の重合度
は、２０以下とするのが合成上便利である。The degree of polymerization of the entire amino acid of the RGD peptide is conveniently 20 or less in terms of synthesis.

【００１２】ＲＧＤペプチドに結合させてペプチド−脂
質誘導体を得るために用いられる脂質としては、コレス
テロール及び炭素原子数８〜１８のアルキル基が好適で
ある。脂質がアルキル基であるときは、直鎖アルキル基
であっても、分枝状アルキル基であってもよい。更に、
２個以上の直鎖アルキル基が、グルタミン酸、エチレン
ジアミン等の２個以上の官能基を有するリンカーに接続
して分枝状となっていてもよい。[0012] Cholesterol and an alkyl group having 8 to 18 carbon atoms are suitable as lipids used for obtaining peptide-lipid derivatives by binding to the RGD peptide. When the lipid is an alkyl group, it may be a linear alkyl group or a branched alkyl group. Furthermore,
Two or more linear alkyl groups may be branched by connecting to a linker having two or more functional groups such as glutamic acid and ethylenediamine.

【００１３】ＲＧＤペプチドに脂質を結合させる方法と
しては、例えば、ペプチドのアミノ基またはカルボキシ
ル基に、これらＲＧＤペプチドの官能基と共有結合を形
成しうる官能基を有するようにしたコレステロール誘導
体またはアルキル基を反応せしめて、ＲＧＤペプチドに
脂質を直接結合させる方法を挙げることができる。ま
た、エタノールアミン、γ−アミノ酪酸、コハク酸等の
リンカーを介して、ＲＧＤペプチドに脂質を導入しても
良い。As a method for binding a lipid to an RGD peptide, for example, a cholesterol derivative or an alkyl group having a functional group capable of forming a covalent bond with a functional group of the RGD peptide at an amino group or a carboxyl group of the peptide. To directly bind a lipid to the RGD peptide. In addition, a lipid may be introduced into the RGD peptide via a linker such as ethanolamine, γ-aminobutyric acid, and succinic acid.

【００１４】これを詳述するに、例えばコレステロール
をＲＧＤペプチドのＮ−末端に導入するには、コレステ
ロールをその水酸基を利用してカルボニル誘導体とし
て、ウレタン結合またはウレア結合によりＮ−末端に導
入することができる。また、このコレステロールのカル
ボニル誘導体はリジンのアミノ基の一方と反応せしめた
後、もう一方のアミノ基を利用してＲＧＤペプチドのＣ
−末端に連結することもできる。更に、Ｃ−末端への導
入については、コレステロールの水酸基を利用して直接
にＣ−末端に繋げることもできる。In detail, for example, in order to introduce cholesterol to the N-terminus of an RGD peptide, cholesterol is introduced into the N-terminus by a urethane bond or a urea bond as a carbonyl derivative utilizing its hydroxyl group. Can be. The carbonyl derivative of cholesterol reacts with one of the amino groups of lysine and then utilizes the other amino group to react with the CGD of the RGD peptide.
-It can also be linked to the terminus. Furthermore, for introduction into the C-terminal, it is also possible to directly link to the C-terminal using the hydroxyl group of cholesterol.

【００１５】また、アルキル基を脂質として用いる場合
には、長鎖脂肪酸のカルボキシル基とＲＧＤペプチドの
末端アミノ基またはリジンのε−アミノ基とを反応せし
めて結合せしめることができる。また、ＲＧＤペプチド
をその末端カルボキシル基またはグルタミン酸のγ−カ
ルボキシル基を介してアルキル基と繋ぐには、カルボキ
シル基と反応しうる例えば水酸基、アミノ基等の官能基
を導入したアルキル基とこれらカルボキシル基とを反応
せしめればよい。ＲＧＤペプチドとアルキル脂質との間
に重合度２〜６のポリエチレングリコールをスペーサー
として導入した方がよい場合がある。When an alkyl group is used as a lipid, a carboxyl group of a long-chain fatty acid can be reacted with a terminal amino group of the RGD peptide or an ε-amino group of lysine to bond them. In order to connect the RGD peptide to an alkyl group via its terminal carboxyl group or the γ-carboxyl group of glutamic acid, an alkyl group capable of reacting with a carboxyl group, for example, an alkyl group into which a functional group such as a hydroxyl group or an amino group has been introduced, and these carboxyl groups May be reacted. In some cases, it is better to introduce polyethylene glycol having a degree of polymerization of 2 to 6 as a spacer between the RGD peptide and the alkyl lipid.

【００１６】上記のように、予め調製したＲＧＤペプチ
ドにアルキル基を導入することもできるが、固相法また
は液相法にてアミノ酸を順次繋げてゆく段階でアルキル
基を導入してもよい。例えば、コレステロールの活性エ
ステルに液相法にてカルボキシル基を保護したアミノ酸
を順次繋げてもよいし、または、アミノ酸のカルボキシ
ル基を固相担体に固定し、これにアミノ基を保護したア
ミノ酸を順次繋げてもよい。As described above, an alkyl group can be introduced into a previously prepared RGD peptide. Alternatively, an alkyl group may be introduced at a stage where amino acids are successively connected by a solid phase method or a liquid phase method. For example, an amino acid having a carboxyl group protected by a liquid phase method may be successively linked to an active ester of cholesterol, or the carboxyl group of an amino acid may be immobilized on a solid support, and an amino acid having an amino group protected thereon may be sequentially added thereto. May be connected.

【００１７】次に、本発明のリポソームについて説明す
る。Next, the liposome of the present invention will be described.

【００１８】上述のようにして得た本発明のペプチド−
脂質誘導体を用いてリポソームを調製するには、特別の
制限はなく、それ自体公知の方法に準ずることができ
る。The peptide of the present invention obtained as described above
There are no particular restrictions on preparing liposomes using lipid derivatives, and any method known per se can be used.

【００１９】本発明のリポソームは、上述した本発明の
物質であるＲＧＤペプチド−脂質誘導体を配合して得ら
れるリポソームであって、該物質の特定の性質を専ら利
用する物である。The liposome of the present invention is a liposome obtained by blending the above-described RGD peptide-lipid derivative, which is the substance of the present invention, and exclusively utilizes the specific properties of the substance.

【００２０】このリポソームを調製するには、ホスファ
チジルコリン、スフィンゴミエリン、ホスファチジルエ
タノールアミン等の脂質やジアルキル型合成界面活性剤
等の膜成分物質と本発明の物質とを予め混合し、これを
公知の方法（Ann. Rev. Biophys. Bioeng., 9, 467 (1
980)）に従いリポソームの水分散液を調製する。かかる
リポソームは、膜安定化剤としてコレステロール等のス
テロール類、ジアルキルリン酸、ステアリルアミン等の
荷電物質およびトコフェロール等の酸化防止剤を含んで
いてもよい。To prepare these liposomes, lipids such as phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine, and membrane component substances such as a dialkyl-type synthetic surfactant are mixed in advance with a substance of the present invention, and the mixture is prepared by a known method. (Ann. Rev. Biophys. Bioeng., 9 , 467 (1
980)) to prepare an aqueous dispersion of liposomes. Such liposomes may contain sterols such as cholesterol, charged substances such as dialkylphosphoric acid and stearylamine, and antioxidants such as tocopherol as membrane stabilizers.

【００２１】[0021]

【実施例】以下実施例により本発明を更に説明する。The present invention will be further described with reference to the following examples.

【００２２】実施例１（ＲＧＤペプチド−脂質誘導体の
合成（その１）） 本実施例においては、脂質（アンカー）がアルキル基で
あり、スペーサーとして重合度が３のポリエチレングリ
コールを有するＲＧＤペプチド−脂質誘導体を合成し
た。Example 1 (Synthesis of RGD peptide-lipid derivative (No. 1)) In this example, the RGD peptide-lipid having a lipid (anchor) as an alkyl group and having a polyethylene glycol having a polymerization degree of 3 as a spacer Derivatives were synthesized.

【００２３】すなわち、目的化合物を、スペーサー・ア
ンカー部（下記スキームＩａにおけるＨＯ−Ｒ）からア
ミノ酸残基を１個ずつ伸長させ、得られた保護体をトリ
フルオロ酢酸（ＴＦＡ）または弗化水素（ＨＦ）によっ
て脱保護する方法で合成した。基本的には古典的なペプ
チド合成法（液相法）によるものである。合成の概略
は、下記スキームＩａ〜Ｉｄの通りである。That is, the target compound is extended one amino acid residue at a time from the spacer-anchor portion (HO-R in the following scheme Ia), and the resulting protected form is converted to trifluoroacetic acid (TFA) or hydrogen fluoride ( HF). Basically, it is based on a classical peptide synthesis method (liquid phase method). The outline of the synthesis is as shown in the following schemes Ia to Id.

【００２４】[0024]

【化１】 Embedded image

【００２５】[0025]

【化２】 Embedded image

【００２６】[0026]

【化３】 Embedded image

【００２７】[0027]

【化４】 Embedded image

【００２８】（ａ）化合物１−１の合成 トリエチレングリコールモノｎ−オクタデシルエーテル
１１．０２３ｇ、フタル酸無水物４．２３９ｇ及びトリ
フェニルホスフィン８．６１６ｇをテトラヒドロフラン
（ＴＨＦ）１５０ｍｌに溶かし、氷冷下撹拌した。ここ
にジエチルアゾジカルボキシレート５．１７ｍｌを加
え、そのまま２２時間撹拌した。溶媒を減圧下留去し、
残渣をシリカゲルカラムクロマトグラフィーで精製し
（酢酸エチル：ｎ−ヘキサン ４：１）、無色非晶質物
１３．６８４ｇを得た。(A) Synthesis of Compound 1-1 11.023 g of triethylene glycol mono n-octadecyl ether, 4.239 g of phthalic anhydride and 8.616 g of triphenylphosphine were dissolved in 150 ml of tetrahydrofuran (THF), and cooled under ice-cooling. Stirred. 5.17 ml of diethyl azodicarboxylate was added thereto, and the mixture was stirred for 22 hours. The solvent is distilled off under reduced pressure,
The residue was purified by silica gel column chromatography (ethyl acetate: n-hexane 4: 1) to obtain 13.684 g of a colorless amorphous substance.

【００２９】ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H)
，1.25(br s, 3OH) ， 1.53-1.56(m, 2H) ，3.41(t, 2
H, J=6.8Hz)，3.50-3.52(m, 2H)，3.57-3.62(m, 4H)，
3.64-3.66(m, 2H)，3.74(t, 2H, J=5.9Hz)，3.90(t, 2
H, J=5.9Hz)，7.70-7.72(m, 2H)，7.84-7.85(m, 2H)。NMR (δ, CDCl ₃ ): 0.88 (t, 3H)
, 1.25 (br s, 3OH), 1.53-1.56 (m, 2H), 3.41 (t, 2
H, J = 6.8Hz), 3.50-3.52 (m, 2H), 3.57-3.62 (m, 4H),
3.64-3.66 (m, 2H), 3.74 (t, 2H, J = 5.9Hz), 3.90 (t, 2H
H, J = 5.9Hz), 7.70-7.72 (m, 2H), 7.84-7.85 (m, 2H).

【００３０】（ｂ）化合物１−２の合成 化合物１−１、１３．６８４ｇにエタノール１３０ｍｌ
及びヒドラジン一水和物２．５０ｍｌを加えて加熱還流
下で４．５時間撹拌した。放冷した後氷冷し、析出した
結晶を濾去した。溶媒を減圧下留去し、残渣をクロロホ
ルム−２Ｎ水酸化ナトリウム間に分配させ、有機層を分
離した。水層をクロロホルム抽出し、抽出液を合わせて
飽和食塩水で洗い、硫酸マグネシウム上乾燥させた。溶
媒を減圧下留去し、無色非晶質物１０．０７３ｇを得
た。(B) Synthesis of Compound 1-2 Compound 13.1-1.
And hydrazine monohydrate (2.50 ml) were added, and the mixture was stirred under reflux for 4.5 hours. After allowing to cool, the mixture was cooled with ice, and the precipitated crystals were removed by filtration. The solvent was distilled off under reduced pressure, the residue was partitioned between chloroform-2N sodium hydroxide, and the organic layer was separated. The aqueous layer was extracted with chloroform, the extracts were combined, washed with saturated saline, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain 10.073 g of a colorless amorphous substance.

【００３１】ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H,
J=7.0Hz)，1.25(br s, 3OH) ，1.55-1.60(m, 2H)，2.86
(t, 2H, J=5.2Hz)，3.45(t, 2H, J=6.7Hz)，3.51(t, 2
H, J=5.2Hz)，3.58-3.60(m, 2H)，3.63-3.68(m, 6H)。NMR (δ, CDCl ₃ ): 0.88 (t, 3H,
J = 7.0Hz), 1.25 (br s, 3OH), 1.55-1.60 (m, 2H), 2.86
(t, 2H, J = 5.2Hz), 3.45 (t, 2H, J = 6.7Hz), 3.51 (t, 2H
H, J = 5.2Hz), 3.58-3.60 (m, 2H), 3.63-3.68 (m, 6H).

【００３２】（ｃ）化合物１−３の合成 Ｎ−ベンジルオキシカルボニルプロリン２．５４９ｇ、
化合物１−２、３．７７４ｇ及び１−ヒドロキシベンゾ
トリアゾール１．５２４ｇをＮ，Ｎ−ジメチルホルムア
ミド（ＤＭＦ）５０ｍｌとＴＨＦ３０ｍｌとの混合物に
溶かし、氷冷下撹拌した。ここにＮ，Ｎ′−ジシクロヘ
キシルカルボジイミド（ＤＣＣ）２．０３６ｇを加え、
そのまま２３時間撹拌した。生じた沈殿を濾去し、溶媒
を減圧下留去し、残渣を酢酸エチルに溶かし、１０％ク
エン酸、１０％炭酸ナトリウム及び飽和食塩水でこの順
に洗い、硫酸マグネシウム上乾燥させ、溶媒を減圧下留
去した。残渣をシリカゲルカラムクロマトグラフィーで
精製し（塩化メチレン→塩化メチレン：メタノール ７
０：１）、無色粘稠の油状物を４．７３１ｇ得た。(C) Synthesis of compound 1-3 2.549 g of N-benzyloxycarbonylproline,
3.774 g of compound 1-2 and 1.524 g of 1-hydroxybenzotriazole were dissolved in a mixture of 50 ml of N, N-dimethylformamide (DMF) and 30 ml of THF, and the mixture was stirred under ice-cooling. 2.036 g of N, N'-dicyclohexylcarbodiimide (DCC) was added thereto,
The mixture was stirred for 23 hours as it was. The resulting precipitate was removed by filtration, the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with 10% citric acid, 10% sodium carbonate and saturated saline in this order, dried over magnesium sulfate, and concentrated under reduced pressure. Distilled off under. The residue is purified by silica gel column chromatography (methylene chloride → methylene chloride: methanol 7).
0: 1), 4.731 g of a colorless viscous oil was obtained.

【００３３】［α］_D ²²＝−１７．８°（ｃ＝１．０
８，ＥｔＯＨ）． ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H, J=7.0Hz)，1.
25(br s, 3OH) ，1.55-1.60(m, 4H)，1.84-2.01(br d,
2H) ，2.06-2.32(br d, 2H) ，3.38-3.66(m, 12H) ，3.
43(t, 2H, J=6.8Hz)，4.32(br s, 1H)，5.11(d, 1H, J=
12.5Hz) ，5.18(d, 1H, J=12.5Hz) ，6.46(br s, 0.47
H) ，6.90(br s, 0.53H) ，7.36(br s, 5H)。[Α] _D ²² = −17.8 ° (c = 1.0
8, EtOH). NMR (δ, CDCl ₃ ): 0.88 (t, 3H, J = 7.0 Hz), 1.
25 (br s, 3OH), 1.55-1.60 (m, 4H), 1.84-2.01 (br d,
2H), 2.06-2.32 (br d, 2H), 3.38-3.66 (m, 12H), 3.
43 (t, 2H, J = 6.8Hz), 4.32 (br s, 1H), 5.11 (d, 1H, J =
12.5Hz), 5.18 (d, 1H, J = 12.5Hz), 6.46 (brs, 0.47
H), 6.90 (brs, 0.53H), 7.36 (brs, 5H).

【００３４】（ｄ）化合物１−４の合成 化合物１−３、４．６４０ｇにメタノール８０ｍｌを加
えて溶かした。ここに触媒として１０％Ｐｄ−Ｃ（ｄｒ
ｙ）０．２０ｇを加え、常圧水素雰囲気下で２時間撹拌
した。触媒を濾去し、溶媒を減圧下留去して無色非晶質
物３．６３２ｇを得た。(D) Synthesis of compound 1-4 80 ml of methanol was added to 4.640 g of compound 1-3 and dissolved. Here, 10% Pd-C (dr
y) 0.20 g was added, and the mixture was stirred under a normal pressure hydrogen atmosphere for 2 hours. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 3.632 g of a colorless amorphous substance.

【００３５】ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H,
J=6.8Hz)，1.25(br s, 3OH) ，1.55-1.60(m, 2H)，1.64
-1.76(m, 2H)，1.84-1.94(m, 2H)，2.09-2.16(m, 1H)，
2.88-2.93(m, 1H)，2.97-3.02(m, 1H)，3.38-3.50(m, 4
H)，3.55-3.59(m, 4H)，3.61-3.67(m, 6H)，3.72(dd, 1
H, J=5.5Hz, 9.0Hz)，7.84(br s, 1H)。NMR (δ, CDCl ₃ ): 0.88 (t, 3H,
J = 6.8Hz), 1.25 (br s, 3OH), 1.55-1.60 (m, 2H), 1.64
-1.76 (m, 2H), 1.84-1.94 (m, 2H), 2.09-2.16 (m, 1H),
2.88-2.93 (m, 1H), 2.97-3.02 (m, 1H), 3.38-3.50 (m, 4
H), 3.55-3.59 (m, 4H), 3.61-3.67 (m, 6H), 3.72 (dd, 1
H, J = 5.5Hz, 9.0Hz), 7.84 (brs, 1H).

【００３６】（ｅ）化合物１−５の合成 Ｎ−ベンジルオキシカルボニルＯ−ｔ−ブチルセリン
２．３５４ｇ、化合物１−４、３．６１４ｇ及び１−ヒ
ドロキシベンゾトリアゾール１．１７５ｇをＤＭＦ２５
ｍｌに溶かし、氷冷下撹拌した。ここにＤＣＣ１．７１
９ｇを加え、そのまま１１時間撹拌し、生じた沈殿を濾
去し、溶媒を減圧下留去した。残渣を酢酸エチルに溶か
し、１０％クエン酸、１０％炭酸ナトリウム及び飽和食
塩水でこの順に洗い、硫酸マグネシウム上乾燥させ、溶
媒を減圧下留去した。残渣をシリカゲルカラムクロマト
グラフィーで精製し（塩化メチレン→塩化メチレン：メ
タノール １００：１〜７０：１）、淡黄色粘稠の油状
物４．７３１ｇを得た。(E) Synthesis of compound 1-5 2.354 g of N-benzyloxycarbonyl Ot-butylserine, 3.614 g of compounds 1-4 and 1.175 g of 1-hydroxybenzotriazole were added to DMF25.
The mixture was dissolved in ml and stirred under ice cooling. Here DCC 1.71
9 g was added, and the mixture was stirred as it was for 11 hours. The resulting precipitate was removed by filtration, and the solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate, washed with 10% citric acid, 10% sodium carbonate and saturated saline in this order, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (methylene chloride → methylene chloride: methanol 100: 1 to 70: 1) to obtain 4.731 g of a pale yellow viscous oil.

【００３７】［α］_D ²²＝−２５．３°（ｃ＝１．１
７，ＥｔＯＨ）． ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H, J=6.8Hz)，1.
18(s, 9H) ，1.25(brs, 3OH) ，1.54-1.60(m, 2H)，1.9
9(m, 3HH)，2.26(m, 1H) ，3.27-3.33(m, 1H)，3.43-3.
63(m, 15H) ，3.68-3.71(m, 1H)，3.65-3.80(m, 1H)，
4.58(br d, 1H)，4.65(br q, 1H)，5.10(s, 2H) ，5.63
(d, 1H, J=7.5Hz)，6.98(br s)，7.31-7.36(br s, 5H)
。[Α] _D ²² = −25.3 ° (c = 1.1
7, EtOH). NMR (δ, CDCl ₃ ): 0.88 (t, 3H, J = 6.8 Hz), 1.
18 (s, 9H), 1.25 (brs, 3OH), 1.54-1.60 (m, 2H), 1.9
9 (m, 3HH), 2.26 (m, 1H), 3.27-3.33 (m, 1H), 3.43-3.
63 (m, 15H), 3.68-3.71 (m, 1H), 3.65-3.80 (m, 1H),
4.58 (br d, 1H), 4.65 (br q, 1H), 5.10 (s, 2H), 5.63
(d, 1H, J = 7.5Hz), 6.98 (br s), 7.31-7.36 (br s, 5H)
.

【００３８】（ｆ）化合物１−６の合成 化合物１−５、４．９５０ｇにメタノール８０ｍｌを加
えて溶かした。ここに１０％Ｐｄ−Ｃ（ｄｒｙ）０．２
０４ｇを加え、常圧水素雰囲気下で２時間撹拌した。触
媒を濾去し、溶媒を減圧下留去して無色粘稠の油状物
３．８５５ｇを得た。(F) Synthesis of Compound 1-6 80 ml of methanol was added to 4.950 g of Compounds 1-5 and dissolved. Here, 10% Pd-C (dry) 0.2
After adding 04 g, the mixture was stirred for 2 hours under a normal pressure hydrogen atmosphere. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 3.855 g of a colorless viscous oil.

【００３９】ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H,
J=6.8Hz)，1.12(s, 9H) ，1.25(brs, 3OH) ，1.55-1.60
(m, 2H)，1.86-2.18(m, 3H)，2.23-2.32(m, 1H)，3.29-
3.38(m, 1H)，3.42-3.65(m, 15H) ，3.73-3.82(m, 2
H)，4.58(dd, 1H, J=2.8Hz, 8.3Hz)，4.80(dd, 1H, J=
2.3Hz, 8.8Hz)，7.02(br s, 1H)。NMR (δ, CDCl ₃ ): 0.88 (t, 3H,
J = 6.8Hz), 1.12 (s, 9H), 1.25 (brs, 3OH), 1.55-1.60
(m, 2H), 1.86-2.18 (m, 3H), 2.23-2.32 (m, 1H), 3.29-
3.38 (m, 1H), 3.42-3.65 (m, 15H), 3.73-3.82 (m, 2
H), 4.58 (dd, 1H, J = 2.8Hz, 8.3Hz), 4.80 (dd, 1H, J =
2.3Hz, 8.8Hz), 7.02 (brs, 1H).

【００４０】（ｇ）化合物１−７の合成 Ｎ−ベンジルオキシカルボニルβ−ｔ−ブチルアスパラ
ギン酸一水和物２．２５５ｇにベンゼンおよびＴＨＦを
加えて溶かし、溶媒を減圧下留去した。残渣に再びベン
ゼンを加えて溶かし、溶媒を減圧下留去した。ここに化
合物１−６、３．８５５ｇ及び１−ヒドロキシベンゾト
リアゾール０．９７４ｇをＤＭＦ３０ｍｌに溶かしたも
のを加え、氷冷下撹拌した。ここにＤＣＣ１．４２５ｇ
を加え、そのまま１２．５時間撹拌した。生じた沈殿を
濾去し、溶媒を減圧下留去し、残渣を酢酸エチルに溶か
し、１０％クエン酸、１０％炭酸ナトリウム及び飽和食
塩水でこの順に洗い、硫酸マグネシウム上乾燥させ、溶
媒を減圧留去した。残渣をシリカゲルカラムクロマトグ
ラフィーで精製し（塩化メチレン→塩化メチレン：メタ
ノール １００：１〜５０：１）、無色粘稠の油状物
５．０５６ｇを得た。(G) Synthesis of Compound 1-7 Benzene and THF were added to 2.255 g of N-benzyloxycarbonyl β-t-butylaspartic acid monohydrate to dissolve, and the solvent was distilled off under reduced pressure. Benzene was added again to the residue to dissolve it, and the solvent was distilled off under reduced pressure. A solution prepared by dissolving Compound 1-6, 3.855 g and 1-hydroxybenzotriazole in 0.97 g of DMF was added thereto, followed by stirring under ice-cooling. Here, DCC 1.425g
Was added, and the mixture was stirred as it was for 12.5 hours. The resulting precipitate was removed by filtration, the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with 10% citric acid, 10% sodium carbonate and saturated saline in this order, dried over magnesium sulfate, and concentrated under reduced pressure. Distilled off. The residue was purified by silica gel column chromatography (methylene chloride → methylene chloride: methanol 100: 1 to 50: 1) to obtain 5.056 g of a colorless viscous oil.

【００４１】［α］_D ²²＝−２９．０°（ｃ＝１．０
４，ＥｔＯＨ）． ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H, J=7.0Hz)，1.
17(s, 9H) ，1.25(brs, 3OH) ，1.43(s, 9H) ，1.54-1.
57(m, 2H)，1.94-2.04(m, 3H)，2.24-2.29(m,1H)，2.60
(dd, 2H, J=5.8Hz, 16.8Hz) ，2.94(dd, 1H, J=5.3Hz,
16.8Hz) ，3.28-3.37(m, 1H)，3.41-3.67(m, 16H) ，3.
86-3.89(m, 1H)，4.52-4.58(m, 2H)，4.79-4.83(m, 1
H)，5.11(d, 1H, J=12.0Hz) ，5.15(d, 1H, J=12.0Hz)
，5.91(d, 1H, J=9.0Hz)，6.96(t, 1H, J=5.5Hz)，7.1
8(d, 1H, J=9.0Hz)，7.31-7.38(m,5H)。[Α] _D ²² = −29.0 ° (c = 1.0
4, EtOH). NMR (δ, CDCl ₃ ): 0.88 (t, 3H, J = 7.0 Hz), 1.
17 (s, 9H), 1.25 (brs, 3OH), 1.43 (s, 9H), 1.54-1.
57 (m, 2H), 1.94-2.04 (m, 3H), 2.24-2.29 (m, 1H), 2.60
(dd, 2H, J = 5.8Hz, 16.8Hz), 2.94 (dd, 1H, J = 5.3Hz,
16.8Hz), 3.28-3.37 (m, 1H), 3.41-3.67 (m, 16H), 3.
86-3.89 (m, 1H), 4.52-4.58 (m, 2H), 4.79-4.83 (m, 1
H), 5.11 (d, 1H, J = 12.0Hz), 5.15 (d, 1H, J = 12.0Hz)
, 5.91 (d, 1H, J = 9.0Hz), 6.96 (t, 1H, J = 5.5Hz), 7.1
8 (d, 1H, J = 9.0Hz), 7.31-7.38 (m, 5H).

【００４２】（ｈ）化合物１−８の合成 化合物１−７、４．９３６ｇにメタノール１００ｍｌを
加えて溶かした。ここに１０％Ｐｄ−Ｃ（ｄｒｙ）０．
２１０ｇを加え、常圧水素雰囲気下で２時間撹拌した。
触媒を濾去し、溶媒を減圧下に留去して無色粘稠の油状
物４．０６０ｇを得た。(H) Synthesis of Compound 1-8 To 4.936 g of Compound 1-7, 100 ml of methanol was added and dissolved. Here, 10% Pd-C (dry) 0.
210 g was added, and the mixture was stirred under a normal pressure hydrogen atmosphere for 2 hours.
The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 4.060 g of a colorless viscous oil.

【００４３】ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H,
J=7.0Hz)，1.18(s, 9H) ，1.25(brs, 3OH) ，1.45(s, 9
H) ，1.54-1.60(m, 2H)，1.87-2.04(m, 3H)，2.26-2.30
(m,1H)，2.52(dd, 2H, J=8.5Hz, 16.5Hz) ，2.81(dd, 1
H, J=3.5Hz, 16.5Hz) ，3.24-3.36(m, 1H)，3.42-3.68
(m, 16H) ，3.86-3.90(m, 1H)，4.45-4.50(m, 0.2H)，
4.57-4.59(br d, 0.8H) ，4.82-4.87(m, 2H)，7.00(br
d, 1H)，7.86(d, 0.2H, J=6.0Hz)，8.01(d, 0.8H, J=8.
0Hz)。NMR (δ, CDCl ₃ ): 0.88 (t, 3H,
J = 7.0Hz), 1.18 (s, 9H), 1.25 (brs, 3OH), 1.45 (s, 9H)
H), 1.54-1.60 (m, 2H), 1.87-2.04 (m, 3H), 2.26-2.30
(m, 1H), 2.52 (dd, 2H, J = 8.5Hz, 16.5Hz), 2.81 (dd, 1
H, J = 3.5Hz, 16.5Hz), 3.24-3.36 (m, 1H), 3.42-3.68
(m, 16H), 3.86-3.90 (m, 1H), 4.45-4.50 (m, 0.2H),
4.57-4.59 (br d, 0.8H), 4.82-4.87 (m, 2H), 7.00 (br
d, 1H), 7.86 (d, 0.2H, J = 6.0Hz), 8.01 (d, 0.8H, J = 8.
0Hz).

【００４４】（ｉ）化合物１−９の合成 Ｎ−ベンジルオキシカルボニルグリシン１．２５１ｇ、
化合物１−８、４．０５３ｇ及び１−ヒドロキシベンゾ
トリアゾール０．８７６ｇをＤＭＦ２５ｍｌに溶かし、
氷冷下撹拌した。ここにＤＣＣ１．２８６ｇを加え、そ
のまま１３．５時間撹拌した。生じた沈殿を濾去し、溶
媒を減圧下留去し、残渣を酢酸エチルに溶かし、１０％
クエン酸、１０％炭酸ナトリウム及び飽和食塩水でこの
順に洗い、硫酸マグネシウム上乾燥させ、溶媒を減圧留
去した。残渣をシリカゲルカラムクロマトグラフィーで
精製し（塩化メチレン→塩化メチレン：メタノール１０
０：１〜３０：１）、無色粘稠の油状物４．６１１ｇを
得た。(I) Synthesis of compound 1-9 1.251 g of N-benzyloxycarbonylglycine,
Compounds 1-8, 4.053 g and 1-hydroxybenzotriazole 0.876 g were dissolved in DMF 25 ml,
The mixture was stirred under ice cooling. To this, 1.286 g of DCC was added, and the mixture was stirred for 13.5 hours. The resulting precipitate was removed by filtration, the solvent was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate.
The extract was washed with citric acid, 10% sodium carbonate and saturated saline in this order, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue is purified by silica gel column chromatography (methylene chloride → methylene chloride: methanol 10).
0: 1 to 30: 1) to obtain 4.611 g of a colorless viscous oil.

【００４５】［α］_D ²²＝−３０．３°（ｃ＝１．０
１，ＥｔＯＨ）． ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H, J=6.8Hz)，1.
17(s, 9H) ，1.25(brs, 3OH) ，1.44(s, 9H) ，1.53-1.
59(m, 2H)，1.87-2.04(m, 3H)，2.22-2.29(m,1H)，2.54
-2.64(m, 2H)，2.83-2.96(m, 1H)，3.25-3.32(m, 1H)，
3.43-3.65(m,16H) ，3.84-3.99(m, 3H),4.35-4.40(m,
0.2H) ，4.54-4.56(br d, 0.8H) ，4.77-4.81(br q, 2
H) ，5.11-5.17(br q, 2H) ，5.65(br s, 0.8H)，5.79
(br s, 0.2H)，6.97(br t, 1H)，7.18-7.25(m, 2H)，7.
30-7.38(m, 5H)。[Α] _D ²² = -30.3 ° (c = 1.0
1, EtOH). NMR (δ, CDCl ₃ ): 0.88 (t, 3H, J = 6.8 Hz), 1.
17 (s, 9H), 1.25 (brs, 3OH), 1.44 (s, 9H), 1.53-1.
59 (m, 2H), 1.87-2.04 (m, 3H), 2.22-2.29 (m, 1H), 2.54
-2.64 (m, 2H), 2.83-2.96 (m, 1H), 3.25-3.32 (m, 1H),
3.43-3.65 (m, 16H), 3.84--3.99 (m, 3H), 4.35-4.40 (m,
0.2H), 4.54-4.56 (br d, 0.8H), 4.77-4.81 (br q, 2
H), 5.11-5.17 (br q, 2H), 5.65 (br s, 0.8H), 5.79
(br s, 0.2H), 6.97 (br t, 1H), 7.18-7.25 (m, 2H), 7.
30-7.38 (m, 5H).

【００４６】（ｊ）化合物１−１０の合成 化合物１−９、４．４９３ｇにメタノール１２０ｍｌを
加えて溶かした。ここに１０％Ｐｄ−Ｃ（ｄｒｙ）０．
２１０ｇを加え、常圧水素雰囲気下で２時間撹拌した。
触媒を濾去し、溶媒を減圧下留去して無色粘稠の油状物
３．９５１ｇを得た。(J) Synthesis of compound 1-10 120 ml of methanol was added to 4.493 g of compound 1-9 and dissolved. Here, 10% Pd-C (dry) 0.
210 g was added, and the mixture was stirred under a normal pressure hydrogen atmosphere for 2 hours.
The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 3.951 g of a colorless viscous oil.

【００４７】（ｋ）化合物１−１１の合成 Ｎ−ベンジルオキシカルボニルＮ^G −４−メトキシ−
２，３，５−トリメチルベンゼルスルホニルアルギニン
シクロヘキシルアミン塩１９．９５ｇを酢酸エチル３
５０ｍｌに懸濁させた。ここに０．１Ｎ硫酸２００ｍｌ
を加え、激しく撹拌し、結晶が溶解したら有機層を分離
し、水層を酢酸エチル抽出した。抽出液は合わせて飽和
食塩水で洗い、硫酸マグネシウム上乾燥させ、溶媒を減
圧下留去して無色非晶質物１８．２７ｇを得た。これを
メタノール３００ｍｌに溶かし、ここに１０％Ｐｄ−Ｃ
（ｄｒｙ）１．５００ｇを加え、常圧水素雰囲気下で３
時間撹拌した。触媒を濾去し、溶媒を減圧下留去して無
色粘稠の油状物１０．３１ｇを得た。(K) Synthesis of compound 1-11 N-benzyloxycarbonyl ^NG -4-methoxy-
19.95 g of 2,3,5-trimethylbenzenesulfonylarginine cyclohexylamine salt was added to ethyl acetate 3
It was suspended in 50 ml. 200 ml of 0.1N sulfuric acid
Was added and stirred vigorously. When the crystals were dissolved, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The extracts were combined, washed with a saturated saline solution, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 18.27 g of a colorless amorphous substance. This was dissolved in 300 ml of methanol, and 10% Pd-C
(Dry) 1.500 g, and added under normal pressure hydrogen atmosphere.
Stirred for hours. The catalyst was removed by filtration, and the solvent was distilled off under reduced pressure to obtain 10.31 g of a colorless viscous oil.

【００４８】（ｌ）化合物１−１２の合成 化合物１−１１、１０．３１ｇに蒸留水２０ｍｌ及びト
リエチルアミン（ＴＥＡ）７．４４ｍｌを加えて溶かし
た。これを氷冷下撹拌し、ここにｔ−ブチルＳ−４，６
−ジメチルピリミジン−２−イル−チオカーボネート
（Ｂｏｃ−Ｓ）７．０５ｇを１，４−ジオキサンに溶か
して加えた。(L) Synthesis of compound 1-12 To 10.31 g of compound 1-11, 20 ml of distilled water and 7.44 ml of triethylamine (TEA) were added and dissolved. This was stirred under ice cooling, and t-butyl S-4,6 was added.
-Dimethylpyrimidin-2-yl-thiocarbonate (Boc-S) (7.05 g) dissolved in 1,4-dioxane was added.

【００４９】室温で１４時間撹拌し、蒸留水で全量が約
５００ｍｌとなるよう希釈し、酢酸エチルで２回洗浄し
た。氷冷し、１０% クエン酸でｐＨ＝３として酢酸エチ
ルで抽出した。有機層を氷冷した１Ｎ塩酸（３回）及び
飽和食塩水で洗い、硫酸マグネシウム上乾燥させ、溶媒
を減圧下留去して無色非晶質物１３．２４ｇを得た。The mixture was stirred at room temperature for 14 hours, diluted with distilled water to a total volume of about 500 ml, and washed twice with ethyl acetate. The mixture was cooled on ice, adjusted to pH = 3 with 10% citric acid, and extracted with ethyl acetate. The organic layer was washed with ice-cooled 1N hydrochloric acid (3 times) and saturated brine, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 13.24 g of a colorless amorphous substance.

【００５０】ＮＭＲ（δ，ＣＤＣｌ₃ ）：1.43(s, 9H)
，1.63(br s, 2H)，1.74(br s, 1H)，1.87(br s, 1
H)，2.12(s, 3H) ，2.57(s, 3H) ，2.66(s, 3H) ，3.23
(br s, 2H)，3.83(s, 3H) ，4.25(br s, 1H)，5.61(br
d, 1H)，6.2-6.5(br s, 3H) ，6.53(s, 1H) 。NMR (δ, CDCl ₃ ): 1.43 (s, 9H)
, 1.63 (br s, 2H), 1.74 (br s, 1H), 1.87 (br s, 1H)
H), 2.12 (s, 3H), 2.57 (s, 3H), 2.66 (s, 3H), 3.23
(br s, 2H), 3.83 (s, 3H), 4.25 (br s, 1H), 5.61 (br
d, 1H), 6.2-6.5 (br s, 3H), 6.53 (s, 1H).

【００５１】（ｍ）化合物１−１３の合成 化合物１−１２、０．６９０ｇ、化合物１−１０、１．
０２８ｇ及び１−ヒドロキシベンゾトリアゾール０．２
０８ｇをＤＭＦ１０ｍｌに溶かし、氷冷下撹拌した。こ
こにＤＣＣ０．３０５ｇを加え、そのまま１４時間撹拌
した。生じた沈殿を濾去し、溶媒を減圧下留去し、残渣
を酢酸エチルに溶かし、１０％クエン酸、１０％炭酸ナ
トリウム及び飽和食塩水でこの順に洗い、硫酸マグネシ
ウム上乾燥させ、溶媒を減圧下留去した。残渣をシリカ
ゲルカラムクロマトグラフィーで精製し（塩化メチレ
ン：メタノール １００：１〜３０：１）、無色非晶質
物１．２３４ｇを得た。(M) Synthesis of compound 1-13 0.690 g of compound 1-12, compound 1-10,
028 g and 1-hydroxybenzotriazole 0.2
08 g was dissolved in DMF10 ml and stirred under ice cooling. 0.305 g of DCC was added thereto, and the mixture was stirred as it was for 14 hours. The resulting precipitate was removed by filtration, the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with 10% citric acid, 10% sodium carbonate and saturated saline in this order, dried over magnesium sulfate, and concentrated under reduced pressure. Distilled off under. The residue was purified by silica gel column chromatography (methylene chloride: methanol 100: 1 to 30: 1) to obtain 1.234 g of a colorless amorphous substance.

【００５２】［α］_D ²²＝−２４．３°（ｃ＝１．０
８，ＥｔＯＨ）． ＦＡＢ−ＭＳ：［Ｍ＋Ｈ］⁺ ；ｍ／ｚ＝１３３８． ＮＭＲ（δ，ＣＤＣｌ₃ ）：0.88(t, 3H, J=6.8Hz)，1.
15(s, 9H) ，1.25(brs,30H)，1.43(s, 9H) ，1.44(s, 9
H) ，1.52-1.67(m, 4H)，1.67-1.79(m, 1H)，1.82-2.25
(m, 4H)，2.12(s, 3H) ，2.63(s, 3H) ，2.60-2.69(m,
1H)，2.70(s 3H)，2.82-2.96(br q, 1H) ，3.22-3.28
(m, 3H)，3.43-3.65(m, 17H) ，3.82(s,3H) ，3.81-3.8
8(m, 3H)，3.98-4.05(br q, 1H) ，4.20-4.28(br s, 1
H) ，4.35-4.42(m, 0.2H)，4.43-4.47(br d, 0.8H) ，
4.73-4.78(br s, 2H) ，5.78(br d,1H)，6.35(br s, 1
H)，6.39(br s 2H) ，6.52(s, 1H) ，6.93(br s, 1H)，
7.23-7.32(m, 1H)，7.39-7.48(m, 2H)。[Α] _D ²² = −24.3 ° (c = 1.0
8, EtOH). FAB-MS: [M + H] ⁺ ; m / z = 1338. NMR (δ, CDCl ₃ ): 0.88 (t, 3H, J = 6.8 Hz), 1.
15 (s, 9H), 1.25 (brs, 30H), 1.43 (s, 9H), 1.44 (s, 9H)
H), 1.52-1.67 (m, 4H), 1.67-1.79 (m, 1H), 1.82-2.25
(m, 4H), 2.12 (s, 3H), 2.63 (s, 3H), 2.60-2.69 (m,
1H), 2.70 (s 3H), 2.82.2.96 (br q, 1H), 3.22-3.28
(m, 3H), 3.43-3.65 (m, 17H), 3.82 (s, 3H), 3.81-3.8
8 (m, 3H), 3.98-4.05 (br q, 1H), 4.20-4.28 (br s, 1
H), 4.35-4.42 (m, 0.2H), 4.43-4.47 (br d, 0.8H),
4.73-4.78 (br s, 2H), 5.78 (br d, 1H), 6.35 (br s, 1
H), 6.39 (br s 2H), 6.52 (s, 1H), 6.93 (br s, 1H),
7.23-7.32 (m, 1H), 7.39-7.48 (m, 2H).

【００５３】（ｎ）化合物１−１４の合成 化合物１−１３、０．４９５８ｇにアニソール１ｍｌを
加え、氷冷した。ここに氷冷したＴＦＡ１０ｍｌを加
え、そのまま６時間撹拌し、さらに室温で１８．５時間
撹拌した。溶媒を減圧下に留去し、残渣にジエチルエー
テル及びｎ−ヘキサンを加えて遠心分離（３０００ｒｐ
ｍ、１０分）した。上清を除き、沈殿物を高速液体クロ
マトグラフィー（ＨＰＬＣ）で精製した。分析条件及び
分取条件は下記の通りである。(N) Synthesis of Compound 1-14 To 0.4958 g of Compound 1-13, 1 ml of anisole was added, and the mixture was cooled with ice. 10 ml of ice-cooled TFA was added thereto, and the mixture was stirred as it was for 6 hours, and further stirred at room temperature for 18.5 hours. The solvent was distilled off under reduced pressure, and diethyl ether and n-hexane were added to the residue, followed by centrifugation (3000 rpm).
m, 10 minutes). The supernatant was removed, and the precipitate was purified by high performance liquid chromatography (HPLC). The analysis conditions and preparative conditions are as follows.

【００５４】[0054]

【表１】 [Table 1]

【００５５】得られたフラクション中のアセトニトリル
を減圧留去し、さらに２回凍結乾燥して目的化合物を無
色非晶質物として０．２１１４ｇを得た。Acetonitrile in the obtained fraction was distilled off under reduced pressure, and further freeze-dried twice to obtain 0.2114 g of the target compound as a colorless amorphous substance.

【００５６】［α］_D ²¹＝−２７．７°（ｃ＝１．０
５，ＣＨＣｌ₃ −ＭｅＯＨ−Ｈ₂ Ｏ １０：１０：
３）． ＦＡＢ−ＭＳ：［Ｍ＋Ｈ］⁺ ；ｍ／ｚ＝９１４． アミノ酸分析：Ａｓｐ，１．００；Ｓｅｒ，０．９３；
Ｐｒｏ，１．０１；Ｇｌｙ，０．９９；Ａｒｇ，１．０
１。[Α] _D ²¹ = −27.7 ° (c = 1.0
_{5, CHCl 3 -MeOH-H 2} O 10:10:
3). FAB-MS: [M + H] ⁺ ; m / z = 914. Amino acid analysis: Asp, 1.00; Ser, 0.93;
Pro, 1.01; Gly, 0.99; Arg, 1.0
One.

【００５７】実施例２（ＲＧＤペプチド−脂質誘導体の
合成（その２）） 本実施例においては、脂質がアルキル基であるがスペー
サーを挿入していない下記ＲＧＤペプチド−脂質誘導体
を合成した。Example 2 (Synthesis of RGD peptide-lipid derivative (No. 2)) In this example, the following RGD peptide-lipid derivative in which the lipid was an alkyl group but did not have a spacer inserted was synthesized.

【００５８】[0058]

【化５】 Embedded image

【００５９】すなわち、目的化合物はＢｏｃ法（Boc st
rategy）に基いて固相法によりペプチド鎖部分を合成し
た。使用した固相合成機は、アプライド・バイオシステ
ム社の「モデル４３０Ａ」である。Ｎ−末端のアシル化
体については、固相法で合成したレジンのＮ−末端をフ
リーとした後、活性エステル法により行なった。脱保護
はＨＦにより行ない、得られた粗生成物はＨＰＬＣによ
り精製した。なお、ＨＦ脱保護装置としては、ペプチド
研究所の「HF-Reaction Apparatus Type IV 」を使用し
た。以下、詳述する。That is, the target compound is prepared by the Boc method (Boc st
The peptide chain was synthesized by the solid phase method based on the rategy). The solid phase synthesizer used was "Model 430A" from Applied Biosystems. The N-terminal acylated product was prepared by the active ester method after the N-terminal of the resin synthesized by the solid phase method was made free. Deprotection was performed by HF, and the obtained crude product was purified by HPLC. As the HF deprotection device, "HF-Reaction Apparatus Type IV" from Peptide Research Institute was used. The details will be described below.

【００６０】固相合成機により下記のレジンを合成し
た。合成は０．５mmolスケールで行ない、１．２００ｇ
のレジンを得た。The following resin was synthesized by a solid phase synthesizer. The synthesis was performed on a 0.5 mmol scale, and 1.200 g
Resin was obtained.

【００６１】[0061]

【化６】 Embedded image

【００６２】こうして得たレジン全量に５０％（Ｖ／
Ｖ）ＴＦＡ−ＣＨ₂ Ｃｌ₂ ４０ｍｌを加え、開放して１
分間、さらに栓をして１５分間撹拌し、レジンを濾取し
た。ＣＨ₂ Ｃｌ₂ 、ＭｅＯＨ及びＣＨ₂ Ｃｌ₂ でこの順
で２回ずつ洗い、減圧乾燥した。回収したレジンにＤＭ
Ｆを加えて窒素をバブルさせた後、ＤＭＦを濾去した
（３回）。次いで、１０％ジイソプロピルエチルアミン
（ＤＩＥＡ）−ＤＭＦを加え、１分間窒素をバブルさせ
た後、ＤＩＥＡ−ＤＭＦを濾去した（２回）。さらにＤ
ＭＦ及びＣＨ₂ Ｃｌ₂ でこの順に２回ずつ洗い、減圧乾
燥した。得られたレジンをＤＭＦ４ｍｌとＣＨ₂ Ｃｌ₂
１０ｍｌとの混合物に懸濁させ、パルミチン酸スクシン
イミドエステル(palmitoyl-OSu) １．７６８ｇをＣＨ₂
Ｃｌ₂ １０ｍｌに溶かして加え、室温で５日間撹拌し
た。レジンを濾取し、ＣＨ₂ Ｃｌ₂ 、ＭｅＯＨ及びＣＨ
₂ Ｃｌ₂ でこの順で２回ずつ洗い、減圧乾燥し、レジン
０．９３９ｇを得た。The total amount of the resin thus obtained was 50% (V /
_{V) TFA-CH 2 Cl 2} 40ml was added, open to 1
The mixture was further stoppered and stirred for 15 minutes, and the resin was collected by filtration. The mixture was washed twice with CH ₂ Cl ₂ , MeOH and CH ₂ Cl ₂ in this order, and dried under reduced pressure. Add DM to the recovered resin
After adding F and bubbling nitrogen through, DMF was filtered off (three times). Then, 10% diisopropylethylamine (DIEA) -DMF was added, nitrogen was bubbled for 1 minute, and then DIEA-DMF was filtered off (twice). And D
The resultant was washed twice with MF and CH ₂ Cl ₂ in this order, and dried under reduced pressure. The obtained resin was mixed with 4 ml of DMF and CH ₂ Cl ₂
Suspended in a mixture with 10 ml, and 1.768 g of succinimide palmitate (palmitoyl-OSu) was added to CH _2.
It was dissolved in 10 ml of Cl ₂ and added, followed by stirring at room temperature for 5 days. The resin was filtered off, CH ₂ Cl ₂ , MeOH and CH
This was washed twice with ₂ Cl ₂ in this order, and dried under reduced pressure to obtain 0.939 g of a resin.

【００６３】ここにアニソール９４０μｌを加え、ＨＦ
約３０ｍｌで脱保護した（０℃、２時間）。ｎ−ヘキサ
ンを加えて上清を捨て（２回）、蒸留水に溶かしてオル
ガノ社製イオン交換樹脂「Amberlite IRA-93ZU」（Ａｃ
ＯＨ型）を加えてｐＨ＝４とし、樹脂を濾去した。濾液
を凍結乾燥し、残渣をＨＰＬＣにより下記条件で精製し
た。To this, 940 μl of anisole was added, and HF was added.
Deprotected in about 30 ml (0 ° C., 2 hours). The supernatant was discarded by adding n-hexane (twice), dissolved in distilled water, and ion-exchange resin "Amberlite IRA-93ZU" (Ac)
(OH type) to pH = 4 and the resin was filtered off. The filtrate was lyophilized and the residue was purified by HPLC under the following conditions.

【００６４】[0064]

【表２】 [Table 2]

【００６５】なお、このようにして合成したＲＧＤペプ
チド−脂質誘導体(palmitoyl-(RGDSP)₂ - OH・2HCl) に
ついて、質量分析計「ＪＥＯＬ ＨＸ−１００」及び旋
光度計「Parkin-Elmer 430」を用いて質量スペクトル及
び比旋光度をそれぞれ測定した結果は次の通りであっ
た。For the RGD peptide-lipid derivative (palmitoyl- (RGDSP) ₂ -OH.2HCl) thus synthesized, a mass spectrometer “JEOL HX-100” and a polarimeter “Parkin-Elmer 430” were used. The results of the measurement of the mass spectrum and the specific rotation measured by using the same were as follows.

【００６６】ＦＡＢ−ＭＳ；［Ｍ＋Ｈ］⁺ ，ｍ／Ｚ：１
２８２． ［α］_D ¹⁶＝−６６．０°（ｃ＝１．０１，ＣＨＣｌ₃
−ＭｅＯＨ−Ｈ₂ Ｏ １０：１０：３）。FAB-MS; [M + H] ⁺ , m / Z: 1
282. [Α] _D ¹⁶ = −66.0 ° (c = 1.01, CHCl _{3
-MeOH-H 2 O 10: 10} : 3).

【００６７】実施例３（ＲＧＤペプチド−脂質誘導体の
合成（その３）） 本実施例においては、コレステロール脂質がカルボニル
基を介してＲＧＤペプチドと結合した形のＲＧＤペプチ
ド−脂質誘導体（Ｃｈｏｌ−ＣＯ−ＲＧＤペプチド、こ
こにＣｈｏｌはコレステリル残基を表わす。）をいくつ
か合成した。Example 3 (Synthesis of RGD Peptide-Lipid Derivative (Part 3)) In this example, an RGD peptide-lipid derivative (Chol-CO- Several RGD peptides, where Chol represents a cholesteryl residue, were synthesized.

【００６８】Ｃｈｏｌ−ＣＯ−ＲＧＤペプチドの合成の
概略は、次の通りである。The outline of the synthesis of the Chol-CO-RGD peptide is as follows.

【００６９】（１）ペプチドの合成 自動化ポリペプチド合成機「Ｍｏｄｅｌ ４３０Ａ」
（アプライド・バイオシステムズ社製）を用いて、同社
の標準操作手順（ｔ−Ｂｏｃ法、０．５mmolスケール）
に準拠して固相合成した。固相からのペプチドの切断
は、トリフルオロメタンスルホン酸（ＴＦＭＳＡ）／ト
リフルオロ酢酸（ＴＦＡ）を用いる同社標準操作手順に
従い、得られた粗ペプチド（ＲＧＤペプチド）はメタノ
ールまたはエタノールに溶解後ジエチルエーテル（エー
テル）により粉末として、次の３−コレステリルカルボ
ニル化反応（Ｃｈｏｌ−ＣＯ化）に供した。(1) Synthesis of Peptide Automated polypeptide synthesizer “Model 430A”
(Applied Biosystems) using the company's standard operating procedure (t-Boc method, 0.5 mmol scale)
Solid phase synthesis was performed according to Cleavage of the peptide from the solid phase was carried out according to the company's standard operating procedure using trifluoromethanesulfonic acid (TFMSA) / trifluoroacetic acid (TFA). The obtained crude peptide (RGD peptide) was dissolved in methanol or ethanol, and then dissolved in diethyl ether (RGD). Ether) to give a powder in the next 3-cholesterylcarbonylation reaction (Chol-CO conversion).

【００７０】（２）ペプチドＮ−末端のＣｈｏｌ−ＣＯ
化 上記粗ペプチドとその１．０〜１．５倍モルの活性エス
テルＮ−［（３−コレステリル）カルボニルオキシ］ス
クシンイミド（Ｃｈｏｌ−ＣＯ−ＯＳｕ）を、過剰量の
炭酸水素ナトリウム存在下、クロロホルム、メタノール
及び蒸留水の８：１０：３の混合溶媒中にて室温で撹拌
した。(2) Chol-CO at N-terminal of peptide
The above crude peptide and its active ester N-[(3-cholesteryl) carbonyloxy] succinimide (Chol-CO-OSu) at 1.0 to 1.5 times the molar amount thereof were converted to chloroform, in the presence of an excess amount of sodium hydrogen carbonate, The mixture was stirred at room temperature in a mixed solvent of 8: 10: 3 of methanol and distilled water.

【００７１】この反応の過程は下記の高速液体クロマト
グラフィー（ＨＰＬＣ）にてチェックした。簡潔に記す
と、反応液約５０μｌを小試験管に採取し、ドライアー
にて加熱、送風して内容物の溶媒を留去してアメ状と
し、これにＨＰＬＣグラジェント初期溶離液を約１００
μｌ加えて溶解した。その一部（２０〜３０μｌ）を定
性分析用のＨＰＬＣシステムに注入し、分析した。The process of this reaction was checked by the following high performance liquid chromatography (HPLC). Briefly, about 50 μl of the reaction solution was collected in a small test tube, heated and blasted with a dryer to evaporate the solvent of the content to form a candy, to which about 100 μl of the HPLC gradient initial eluate was added.
μl was added to dissolve. An aliquot (20-30 μl) was injected into a HPLC system for qualitative analysis and analyzed.

【００７２】（３）ＨＰＬＣによる定性分析および分取
精製 「Model 150A Separation System」（アプライド・バイ
オシステムズ社製）を用い、２１４ｎｍの紫外部吸収に
て検出した。(3) Qualitative analysis by HPLC and preparative purification Purification was detected by ultraviolet absorption at 214 nm using "Model 150A Separation System" (manufactured by Applied Biosystems).

【００７３】（定性分析）Ｃ−８タイプの逆相系カラム
（「Aquapore RP-300 」、ポアサイズ 30 nm、 7μm 球
状オクチルシリル化シリカゲル、4.6 mmφ×220 mm、ア
プライド・バイオシステムズ社製）を用い、流速1.0 ml
/min、0.1% TFAを含むアセトニトリル水溶液のアセトニ
トリル濃度を１０分間に１０％から８０％まで直線的に
増加させ、さらに引続き８０％アセトニトリル水溶液に
て１０分間溶出するグラジエントプログラムを適用し
た。(Qualitative analysis) Using a C-8 type reverse phase column (“Aquapore RP-300”, pore size 30 nm, 7 μm spherical octylsilylated silica gel, 4.6 mmφ × 220 mm, manufactured by Applied Biosystems) , Flow rate 1.0 ml
The acetonitrile concentration of the acetonitrile aqueous solution containing 0.1% TFA / min was linearly increased from 10% to 80% in 10 minutes, and then a gradient program was applied which was eluted with the 80% acetonitrile aqueous solution for 10 minutes.

【００７４】（分取、精製）Ｃ−８タイプの逆相カラム
（「Aquapore Prep-10」、ポアサイズ 30 nm、20μm 球
状オクチルシリル化シリカゲル、10mmφ×25mm、アプラ
イド・バイオシステムズ社製）を用いた。Chol-CO-RGD
ペプチドの精製は、０．０５または０．１％ＴＦＡを含
むアセトニトリルのリニアグラジェント（１５分間に１
０→９０％、流速６．０ｍｌ／ｍｉｎ）により行なっ
た。(Preparation and Purification) A C-8 type reverse phase column (“Aquapore Prep-10”, pore size 30 nm, 20 μm spherical octylsilylated silica gel, 10 mmφ × 25 mm, manufactured by Applied Biosystems) was used. . Chol-CO-RGD
Purification of the peptide was performed using a linear gradient of acetonitrile containing 0.05 or 0.1% TFA (1 per 15 minutes).
0 → 90%, flow rate 6.0 ml / min).

【００７５】（４） ¹Ｈ−ＮＭＲ測定方法 各試料は４〜５ｍｇ／ｍｌの濃度の溶液に調製し、これ
にＴＦＡを１滴加えて試料溶液とした。この溶液を「Va
rian VXR-500S 」（500 MHz 、Varian社製）にて測定し
た。(4) ¹ H-NMR Measurement Method Each sample was prepared as a solution having a concentration of 4 to 5 mg / ml, and one drop of TFA was added thereto to prepare a sample solution. This solution is called "Va
rian VXR-500S "(500 MHz, Varian).

【００７６】（５）Ｈ−（ＲＧＤＳＰ）₂ −ＯＨの調製 実施例２におけると同様にして合成したレジン、すなわ
ち官能基が保護された（Ａｒｇ−Ｇｌｙ−Ａｓｐ−Ｓｅ
ｒ−Ｐｒｏ）₂ が結合している固相２．４１ｇ（０．９
２mmol）を１００ｍｌナス形コルベンに入れ（なるべく
器壁に付着しないように）、さらにチオアニソール２．
５ｍｌおよびエタンジチオール１．２ｍｌをそれぞれ加
え、室温で１０分間放置した。これにＴＦＡ２０ｍｌを
氷冷下に加え、室温で１０分間撹拌した。これを再び冷
却し、ＴＦＭＳＡ３ｇ滴加し、室温で４５分間撹拌し
た。この反応液にエーテルを氷冷下加えてその全量を約
１００ｍｌとし、固相および白色析出物をグラスフィル
ター（Ｇ３）にて濾取した。(5) Preparation of H- (RGDSP) ₂ —OH A resin synthesized in the same manner as in Example 2, that is, a resin whose functional group was protected (Arg-Gly-Asp-Se)
2.41 g (0.9%) of the solid phase to which r-Pro) ₂ is bound
2 mmol) in a 100 ml eggplant-shaped corben (preferably not to adhere to the vessel wall).
5 ml and 1.2 ml of ethanedithiol were added, respectively, and left at room temperature for 10 minutes. To this, 20 ml of TFA was added under ice cooling, and the mixture was stirred at room temperature for 10 minutes. It was cooled again, 3 g of TFMSA was added dropwise and stirred at room temperature for 45 minutes. Ether was added to this reaction solution under ice-cooling to make the total amount about 100 ml, and the solid phase and white precipitate were collected by a glass filter (G3).

【００７７】これをエーテルにて数回洗浄し、さらに吸
引にてエーテルをできるかぎり除去した。エーテル５０
０ｍｌを入れたビーカーの液面上にこのグラスフィルタ
ーを装着し、ビーカー内を撹拌しながらグラスフィルタ
ー中にＴＦＡ５ｍｌを加えて可溶物を溶出させ、エーテ
ル中に白色析出物を得た。この操作を当該析出物が得ら
れなくなるまで繰返した。得られた白色析出物をグラス
フィルター（Ｇ４）にて濾取し、エタノールにて可溶物
を溶出させた。この溶出液を約５ｍｌまで減圧下濃縮
し、これをエーテル３００ｍｌ中に撹拌しながら滴加し
た。得られた白色析出物をグラスフィルター（Ｇ４）に
て濾取し、エーテルにて数回洗浄した。この析出物を、
エーテルで湿った状態でサンプル瓶中に入れ、減圧下乾
燥した。収量１．１１ｇ。This was washed several times with ether, and the ether was removed by suction as much as possible. Ether 50
The glass filter was mounted on the liquid surface of a beaker containing 0 ml, and 5 ml of TFA was added to the glass filter while stirring the inside of the beaker to elute a soluble substance, thereby obtaining a white precipitate in ether. This operation was repeated until the precipitate could not be obtained. The obtained white precipitate was collected by filtration with a glass filter (G4), and the soluble matter was eluted with ethanol. The eluate was concentrated under reduced pressure to about 5 ml, and added dropwise to 300 ml of ether while stirring. The obtained white precipitate was collected by filtration with a glass filter (G4) and washed several times with ether. This precipitate is
The sample was put in a sample bottle while being moist with ether, and dried under reduced pressure. Yield 1.11 g.

【００７８】その他のＲＧＤペプチドについても、上記
同様の処理（アプライド・バイオシステムズ社標準操作
手順）にて固相より切断した。Other RGD peptides were also cleaved from the solid phase by the same treatment as above (standard procedure of Applied Biosystems).

【００７９】以下、本発明のＲＧＤペプチド−脂質誘導
体の具体的合成例を掲げる。Hereinafter, specific synthesis examples of the RGD peptide-lipid derivative of the present invention will be described.

【００８０】（ａ）Ｃｈｏｌ−ＣＯ−ＧＲＧＤＳＰ−Ｏ
Ｈの合成（Ｈ−ＧＲＧＤＳＰ−ＯＨのＣｈｏｌ−ＣＯ
化）：Ｈ−ＧＲＧＤＳＰ−ＯＨ（Ｇｌｙ−Ａｒｇ−Ｇｌ
ｙ−Ａｓｐ−Ｓｅｒ−Ｐｒｏ）０．５９ｇ（１．０mmo
l）を脱イオン水８ｍｌおよびメタノール２８ｍｌの混
合液に溶解させ、過剰量の炭酸水素ナトリウムを加え
た。この溶液にＣｈｏｌ−ＣＯ−ＯＳｕ０．６０ｇ
（１．１mmol）およびクロロホルム２４ｍｌそれぞれ加
えて室温にて撹拌した。３時間後、ＨＰＬＣにてＨ−Ｇ
ＲＧＤＳＰ−ＯＨの消失を確認した。(A) Chol-CO-GRGDSP-O
Synthesis of H (Chol-CO of H-GRGDDSP-OH)
): H-GRGDSP-OH (Gly-Arg-Gl
y-Asp-Ser-Pro) 0.59 g (1.0 mmo
l) was dissolved in a mixture of 8 ml of deionized water and 28 ml of methanol, and excess sodium hydrogen carbonate was added. 0.60 g of Chol-CO-OSu was added to this solution.
(1.1 mmol) and 24 ml of chloroform, respectively, and the mixture was stirred at room temperature. After 3 hours, HG by HPLC
The disappearance of RGDSP-OH was confirmed.

【００８１】４．５時間後に反応液を減圧下濃縮し、エ
タノールを加えてさらに数回濃縮を繰返した後、これに
クロロホルムを加え、減圧下濃縮して乾固させた。得ら
れた残渣にエーテル約５０ｍｌ加えて固化させた。得ら
れた白色の不溶物を濾取し、１０％メタノール水溶液３
０ｍｌに溶解し、この液を一旦１Ｎ水酸化ナトリウム水
溶液にてｐＨ約１０とし、引き続き１Ｎ塩酸にてｐＨ４
とすると白色析出物が得られた。これにメタノール１０
０ｍｌを加えると白色析出物はさらに増加し、これを静
置すると同析出物はコルベンの内壁に付着した。この液
を捨ててメタノール５０ｍｌを加えて得られた析出物を
固化させ、白色粉末を濾取して減圧下乾燥した。この一
部を１０％メタノール水溶液に溶解させて、分取用ＨＰ
ＬＣにて精製した。回収した溶出液は減圧濃縮し、エタ
ノールを加えて再度約５ｍｌにまで減圧濃縮し、メタノ
ール約２０ｍｌおよび１Ｎ水酸化ナトリウム水溶液を加
えてｐＨ５〜６として静置した。得られた白色析出物を
濾取し、減圧乾燥した。収量は３４１ｍｇ。After 4.5 hours, the reaction solution was concentrated under reduced pressure, ethanol was added thereto, and the mixture was further concentrated several times. Then, chloroform was added thereto, and the mixture was concentrated under reduced pressure to dryness. About 50 ml of ether was added to the obtained residue to solidify. The obtained white insolubles were collected by filtration, and 10% aqueous methanol solution 3
0 ml, and this solution is adjusted to pH about 10 with 1N aqueous sodium hydroxide solution, and then adjusted to pH 4 with 1N hydrochloric acid.
Then, a white precipitate was obtained. Add methanol 10
When 0 ml was added, the amount of the white precipitate further increased. When the white precipitate was allowed to stand, the precipitate adhered to the inner wall of the Kolben. This liquid was discarded, and the resulting precipitate was solidified by adding 50 ml of methanol, and the white powder was collected by filtration and dried under reduced pressure. A part of this was dissolved in a 10% aqueous methanol solution to prepare a preparative HP
Purified by LC. The collected eluate was concentrated under reduced pressure, ethanol was added thereto, and the mixture was concentrated again to about 5 ml under reduced pressure. The obtained white precipitate was collected by filtration and dried under reduced pressure. The yield is 341 mg.

【００８２】¹Ｈ−ＮＭＲ（ＣＤ₃ ＯＤ）：δ 5.37(1H,
broad d), 4.7-4.9(2H,m), 4.3-4.5(3H,m), 3.6-3.9(8
H,m), 3.20(1H,t), 2.80(1H,dd), 2.74(1H,dd), 2.32(2
H,d),2.25(1H,m), 2.1-1.8(9H,m), 1.8-0.8(36H,m), 0.
71(3H,s) 。 ¹ H-NMR (CD ₃ OD): δ 5.37 (1H,
broad d), 4.7-4.9 (2H, m), 4.3-4.5 (3H, m), 3.6-3.9 (8
H, m), 3.20 (1H, t), 2.80 (1H, dd), 2.74 (1H, dd), 2.32 (2
H, d), 2.25 (1H, m), 2.1-1.8 (9H, m), 1.8-0.8 (36H, m), 0.
71 (3H, s).

【００８３】（ｂ）Ｃｈｏｌ−ＣＯ−（ＲＧＤＳＰ）₂
−ＯＨの合成（Ｈ−（ＲＧＤＳＰ）₂−ＯＨのＣｈｏｌ
−ＣＯ化）：Ｈ−（ＲＧＤＳＰ）₂ −ＯＨ（Ａｒｇ−Ｇ
ｌｙ−Ａｓｐ−Ｓｅｒ−Ｐｒｏ）１．００ｇ（０．９６
mmol）を脱イオン水６ｍｌおよびメタノール１０ｍｌの
混合液に溶解させ、泡が出なくなるまで炭酸水素ナトリ
ウムを加えてさらにこれを１００ｍｇ追加した。これに
メタノール１０ｍｌを追加して、Ｃｈｏｌ−ＣＯ−ＯＳ
ｕ ３７０ｍｇ（０．７０mmol）およびクロロホルム１
５ｍｌをそれぞれ加えて室温にて撹拌した。５時間後、
薄層クロマトグラフィー（ＴＬＣ）にてＣｈｏｌ−ＣＯ
−ＯＳｕの消失を確認した。(B) Chol-CO- (RGDSP) ₂
Synthesis of -OH (Chol of H- (RGDSP) ₂ -OH
-CO): H- (RGDSP) _2- OH (Arg-G
ly-Asp-Ser-Pro) 1.00 g (0.96
mmol) was dissolved in a mixture of 6 ml of deionized water and 10 ml of methanol, and sodium hydrogencarbonate was added until no more bubbles were formed. To this, 10 ml of methanol was added, and Chol-CO-OS was added.
u 370 mg (0.70 mmol) and chloroform 1
5 ml each was added and stirred at room temperature. After 5 hours,
Chol-CO by thin layer chromatography (TLC)
-The disappearance of OSu was confirmed.

【００８４】反応液を減圧下濃縮し、エタノールを加え
てさらに数回濃縮を繰返した後これにクロロホルムを加
え、減圧下濃縮して乾固させた。得られた残渣にエーテ
ル約５０ｍｌ加えて固化させた。得られた淡黄色の不溶
物を濾取し、１０％メタノール水溶液３０ｍｌに溶解さ
せ、この液を１Ｎ塩酸にてｐＨ５とすると白色析出物が
得られた。これにメタノール４０ｍｌを加えると白色析
出物はさらに増加し、これを静置すると同析出物はコル
ベンの内壁に付着した。この液を捨ててメタノール３０
ｍｌを加えて得られた析出物を固化させ、ほとんど粘性
を示さない白色粉末を濾取して減圧下乾燥した。この一
部を１０％メタノール水溶液に溶解させ、分取用ＨＰＬ
Ｃにて精製した。回収した溶出液は減圧濃縮し、エタノ
ールを加えて再度約５ｍｌにまで減圧濃縮して、メタノ
ール約２０ｍｌおよび１Ｎ水酸化ナトリウム水溶液を加
えてｐＨ５〜６として静置した。得られた白色析出物を
濾取し、減圧乾燥した。収量は７７６ｍｇ。The reaction solution was concentrated under reduced pressure, ethanol was added thereto, and the mixture was further concentrated several times. Then, chloroform was added thereto, and the mixture was concentrated under reduced pressure to dryness. About 50 ml of ether was added to the obtained residue to solidify. The obtained pale yellow insoluble matter was collected by filtration, dissolved in 30 ml of a 10% aqueous methanol solution, and the solution was adjusted to pH 5 with 1N hydrochloric acid to obtain a white precipitate. When 40 ml of methanol was added thereto, the amount of white precipitate further increased. When the precipitate was allowed to stand, the precipitate adhered to the inner wall of the Kolben. Discard this liquid and remove methanol 30
The resulting precipitate was solidified by adding ml, and a white powder having almost no viscosity was collected by filtration and dried under reduced pressure. A part of this was dissolved in a 10% methanol aqueous solution, and the preparative HPL was
Purified by C. The collected eluate was concentrated under reduced pressure, ethanol was added thereto, and the mixture was concentrated again under reduced pressure to about 5 ml. The obtained white precipitate was collected by filtration and dried under reduced pressure. The yield is 776 mg.

【００８５】¹Ｈ−ＮＭＲ（ＣＤ₃ ＯＤ）：δ 5.37(1H,
broad d), 5.0-4.7(m), 4.4-4.5(3H,m), 4.33(1H,m),
4.07(1H,m), 3.9-3.7(12H,m), 3.20(4H,m), 2.9-2.7(4
H,m), 2.4-2.2(4H,m), 2.1-1.8(13H,m), 1.8-0.87(39H,
m), 0.71(3H,s) 。 ¹ H-NMR (CD ₃ OD): δ 5.37 (1H,
broad d), 5.0-4.7 (m), 4.4-4.5 (3H, m), 4.33 (1H, m),
4.07 (1H, m), 3.9-3.7 (12H, m), 3.20 (4H, m), 2.9-2.7 (4
H, m), 2.4-2.2 (4H, m), 2.1-1.8 (13H, m), 1.8-0.87 (39H,
m), 0.71 (3H, s).

【００８６】（ｃ）Ｃｈｏｌ−ＣＯ−（ＲＧＤＳＰ）₂
−ＮＨ₂ の合成（Ｈ−（ＲＧＤＳＰ）₂ −ＮＨ₂ のＣｈ
ｏｌ−ＣＯ化）：Ｈ−（ＲＧＤＳＰ）₂ −ＮＨ₂ （Ａｒ
ｇ−Ｇｌｙ−Ａｓｐ−Ｓｅｒ−Ｐｒｏ−ＮＨ₂ ）０．８
０ｇ（０．７７mmol）を脱イオン水１０ｍｌおよびメタ
ノール３０ｍｌの混合液に溶解させ、トリエチルアミン
を加えてｐＨ約９とした。この溶液にＣｈｏｌ−ＣＯ−
ＯＳｕ ０．５０ｇ（０．９５mmol）およびクロロホル
ム２０ｍｌをそれぞれ加えて室温にて撹拌した。６時間
後、ＨＰＬＣにてＨ−（ＲＧＤＳＰ）₂ −ＮＨ₂ の消失
を確認した。(C) Chol-CO- (RGDSP) ₂
Synthesis of —NH ₂ (Ch of H- (RGDSP) ₂ —NH ₂
ol-CO _{reduction): H- (RGDSP) 2 -NH} 2 (Ar
g-Gly-Asp-Ser- Pro-NH 2) 0.8
0 g (0.77 mmol) was dissolved in a mixture of 10 ml of deionized water and 30 ml of methanol, and the pH was adjusted to about 9 by adding triethylamine. Chol-CO-
0.50 g (0.95 mmol) of OSu and 20 ml of chloroform were added, respectively, and the mixture was stirred at room temperature. After 6 hours, disappearance of H- (RGDSP) ₂ -NH ₂ was confirmed by HPLC.

【００８７】反応液を減圧下濃縮し、エタノールを加え
てさらに数回濃縮を繰返した後、これにクロロホルムを
加え、減圧下濃縮して乾固させた。得られた残渣にエー
テル約５０ｍｌ加えて固化させた。得られた白色の不溶
物を濾取し、１０％メタノール水溶液３０ｍｌに溶解
し、この液を１Ｎ塩酸にてｐＨ５とすると白色析出物が
得られた。これにメタノール４０ｍｌを加えると白色析
出物はさらに増加し、これを静置すると同析出物はコル
ベンの内壁に付着した。この液を捨ててメタノール２０
ｍｌを加えて得られた析出物を固化させ、白色粉末を濾
取して減圧下乾燥した。この一部を１０％メタノール水
溶液に溶解させて分取用ＨＰＬＣにて精製した。回収し
た溶出液は減圧濃縮し、エタノールを加えて再度約５ｍ
ｌにまで減圧濃縮し、メタノール約２０ｍｌおよび１Ｎ
水酸化ナトリウム水溶液を加えてｐＨ５〜６として静置
した。得られた白色析出物を濾取し、減圧乾燥した。収
量は２０４ｍｇ。The reaction solution was concentrated under reduced pressure, and ethanol was added thereto. After concentration was repeated several times, chloroform was added thereto, and the mixture was concentrated under reduced pressure to dryness. About 50 ml of ether was added to the obtained residue to solidify. The resulting white insolubles were collected by filtration, dissolved in 30 ml of a 10% aqueous methanol solution, and the solution was adjusted to pH 5 with 1N hydrochloric acid to obtain a white precipitate. When 40 ml of methanol was added thereto, the amount of white precipitate further increased. When the precipitate was allowed to stand, the precipitate adhered to the inner wall of the Kolben. Discard this liquid and add methanol 20
The resulting precipitate was solidified by adding ml, and the white powder was collected by filtration and dried under reduced pressure. This part was dissolved in a 10% aqueous methanol solution and purified by preparative HPLC. The collected eluate was concentrated under reduced pressure, and ethanol was added thereto.
and concentrated under reduced pressure to about 20 ml of methanol and 1N
An aqueous solution of sodium hydroxide was added to adjust the pH to 5 to 6, and the mixture was allowed to stand. The obtained white precipitate was collected by filtration and dried under reduced pressure. Yield 204 mg.

【００８８】¹Ｈ−ＮＭＲ（ＣＤ₃ ＯＤ）：δ 5.39(1H,
broad d), 4.8(4H,m), 4.4-4.5(3H,m), 4.35(1H,m), 4.
08(1H,m), 3.9-3.7(12H,m), 3.21(4H,m), 2.89(2H,m),
2.76(4H,m), 2.4-2.2(4H,m), 2.2-1.8(13H,m), 1.8-0.8
6(39H,m), 0.71(3H,s) 。 ¹ H-NMR (CD ₃ OD): δ 5.39 (1H,
broad d), 4.8 (4H, m), 4.4-4.5 (3H, m), 4.35 (1H, m), 4.
08 (1H, m), 3.9-3.7 (12H, m), 3.21 (4H, m), 2.89 (2H, m),
2.76 (4H, m), 2.4-2.2 (4H, m), 2.2-1.8 (13H, m), 1.8-0.8
6 (39H, m), 0.71 (3H, s).

【００８９】（ｄ）Ｃｈｏｌ−ＣＯ−（ＲＧＤ）₃ −Ｏ
Ｈの合成（Ｈ−（ＲＧＤ）₃ −ＯＨのＣｈｏｌ−ＣＯ
化）：Ｈ−（ＲＧＤ）₃ −ＯＨ１．００ｇ（１．０mmo
l）を脱イオン水５ｍｌおよびメタノール５ｍｌの混合
液に溶解させ、過剰量の炭酸水素ナトリウムを加えた。
この溶液にＣｈｏｌ−ＣＯ−ＯＳｕ ０．４０ｇ（０．
７６mmol）およびクロロホルム１０ｍｌをそれぞれ加え
て室温にて撹拌した。６時間後、ＨＰＬＣにてＣｈｏｌ
−ＣＯ−ＯＳｕの消失を確認した。(D) Chol-CO- (RGD) ₃ -O
Synthesis of H (H- (RGD) _3- OH Chol-CO
): 1.00 g of H- (RGD) _3- OH (1.0 mmol)
l) was dissolved in a mixture of 5 ml of deionized water and 5 ml of methanol, and an excess amount of sodium hydrogen carbonate was added.
0.40 g of Chol-CO-OSu (0.
76 mmol) and 10 ml of chloroform were added, and the mixture was stirred at room temperature. After 6 hours, HPLC
The disappearance of -CO-OSu was confirmed.

【００９０】反応液を減圧下濃縮し、エタノールを加え
てさらに数回濃縮を繰返した後、これにクロロホルムを
加え、減圧下濃縮してアメ状とした。得られた残渣にエ
ーテル約５０ｍｌ加えて固化させた。得られた白色の不
溶物を濾取し、１０％メタノール水溶液３０ｍｌに溶解
し、この液を一旦１Ｎ水酸化ナトリウム水溶液にてｐＨ
約１０とし、引き続き１Ｎ塩酸にてｐＨ５とすると白色
析出物が得られた。これにメタノール３０ｍｌを加える
と白色析出物はさらに増加し、これを静置すると同析出
物はコルベンの内壁に付着した。この液を捨ててメタノ
ール２０ｍｌを加えて得られた析出物を固化させ、白色
粉末を濾取して減圧下乾燥した。この一部を１０％メタ
ノール水溶液に溶解して、分取用ＨＰＬＣにて精製し
た。回収した溶出液は減圧濃縮し、エタノールを加えて
再度約５ｍｌにまで減圧濃縮し、メタノール約２０ｍｌ
および１Ｎ水酸化ナトリウム水溶液を加えてｐＨ５〜６
として静置した。得られた白色析出物を濾取し、減圧乾
燥した。収量は７３１ｍｇ。The reaction solution was concentrated under reduced pressure, ethanol was added thereto, and the mixture was further concentrated several times. Then, chloroform was added thereto, and the mixture was concentrated under reduced pressure to give a candy-like state. About 50 ml of ether was added to the obtained residue to solidify. The resulting white insolubles were collected by filtration, dissolved in 30 ml of a 10% aqueous methanol solution, and the solution was once adjusted to pH with a 1N aqueous sodium hydroxide solution.
When the pH was adjusted to about 10 and then to pH 5 with 1N hydrochloric acid, a white precipitate was obtained. When 30 ml of methanol was added thereto, the amount of white precipitate further increased, and when this was allowed to stand, the precipitate adhered to the inner wall of the Kolben. This liquid was discarded, and the obtained precipitate was solidified by adding 20 ml of methanol. The white powder was collected by filtration and dried under reduced pressure. This part was dissolved in a 10% aqueous methanol solution and purified by preparative HPLC. The collected eluate was concentrated under reduced pressure, ethanol was added thereto, and the mixture was again concentrated under reduced pressure to about 5 ml.
And 1N aqueous sodium hydroxide solution to add pH 5-6
As it was. The obtained white precipitate was collected by filtration and dried under reduced pressure. The yield is 731 mg.

【００９１】¹Ｈ−ＮＭＲ（ＣＤ₃ ＯＤ）：δ 5.37(1H,
broad d), 4.75(2H,m), 4.4-4.2(3H,m), 4.12(1H,t),
4.0-3.8(6H,m), 3.19(6H,m), 2.9-2.7(6H,m), 2.31(2H,
broad d), 2.1-0.8(51H,m), 0.71(3H,s)。 ¹ H-NMR (CD ₃ OD): δ 5.37 (1H,
broad d), 4.75 (2H, m), 4.4-4.2 (3H, m), 4.12 (1H, t),
4.0-3.8 (6H, m), 3.19 (6H, m), 2.9-2.7 (6H, m), 2.31 (2H, m
broad d), 2.1-0.8 (51H, m), 0.71 (3H, s).

【００９２】（ｅ）Ｃｈｏｌ−ＣＯ−ＧＧＧＲＧＤＳＰ
−ＯＨの合成（Ｈ−ＧＧＧＲＧＤＳＰ−ＯＨのＣｈｏｌ
−ＣＯ化）：Ｈ−ＧＧＧＲＧＤＳＰ−ＯＨ（Ｇｌｙ−Ｇ
ｌｙ−Ｇｌｙ−Ａｒｇ−Ｇｌｙ−Ａｓｐ−Ｓｅｒ−Ｐｒ
ｏ）０．８５ｇ（１．２１mmol）を脱イオン水５ｍｌお
よびメタノール５ｍｌの混合液に溶解させ、過剰量の炭
酸水素ナトリウムを加えた。この溶液にＣｈｏｌ−ＣＯ
−ＯＳｕ ０．４２ｇ（０．８０mmol）およびクロロホ
ルム１０ｍｌをそれぞれ加えて室温にて撹拌した。６時
間後、ＨＰＬＣにてＣｈｏｌ−ＣＯ−ＯＳｕの消失を確
認した。(E) Chol-CO-GGGRGDDSP
Synthesis of -OH (Chol of H-GGGRGDDSP-OH
-CO conversion): H-GGGRGDDSP-OH (Gly-G
ly-Gly-Arg-Gly-Asp-Ser-Pr
o) 0.85 g (1.21 mmol) was dissolved in a mixture of 5 ml of deionized water and 5 ml of methanol, and an excess amount of sodium hydrogen carbonate was added. Chol-CO was added to this solution.
0.42 g (0.80 mmol) of -OSu and 10 ml of chloroform were added thereto, and the mixture was stirred at room temperature. After 6 hours, disappearance of Chol-CO-OSu was confirmed by HPLC.

【００９３】反応液を減圧下濃縮し、エタノールを加え
てさらに数回濃縮を繰返した後、これにクロロホルムを
加え、減圧下濃縮してアメ状とした。得られた残渣にエ
ーテル約５０ｍｌ加えて固化させた。得られた白色の不
溶物を濾取し、１０％メタノール水溶液３０ｍｌに溶解
し、この液を一旦１Ｎ水酸化ナトリウム水溶液にて約ｐ
Ｈ１０とし、引き続き１Ｎ塩酸にてｐＨ５とすると白色
析出物が得られた。これにメタノール３０ｍｌを加える
と白色析出物はさらに増加し、これを静置すると同析出
物はコルベンの内壁に付着した。この液を捨ててメタノ
ール２０ｍｌを加えて得られた析出物を固化させ、白色
粉末を濾取して減圧下乾燥した。この一部を１０％メタ
ノール水溶液に溶解し、分取用ＨＰＬＣにて精製した。
回収した溶出液は減圧濃縮し、エタノールを加えて再度
約５ｍｌにまで減圧濃縮し、メタノール約２０ｍｌおよ
び１Ｎ水酸化ナトリウム水溶液を加えてｐＨ５〜６とし
て静置した。得られた白色析出物を濾取し、減圧乾燥し
た。収量は４２３ｍｇ。The reaction solution was concentrated under reduced pressure, and ethanol was added thereto. After concentration was repeated several times, chloroform was added thereto, and the mixture was concentrated under reduced pressure to give a candy-like state. About 50 ml of ether was added to the obtained residue to solidify. The obtained white insoluble matter was collected by filtration, dissolved in 30 ml of a 10% aqueous methanol solution, and this solution was temporarily dissolved in a 1N aqueous sodium hydroxide solution to about p.
When the pH was changed to H10 and then to pH 5 with 1N hydrochloric acid, a white precipitate was obtained. When 30 ml of methanol was added thereto, the amount of white precipitate further increased, and when this was allowed to stand, the precipitate adhered to the inner wall of the Kolben. This liquid was discarded, and the obtained precipitate was solidified by adding 20 ml of methanol. The white powder was collected by filtration and dried under reduced pressure. A part of this was dissolved in a 10% aqueous methanol solution and purified by preparative HPLC.
The collected eluate was concentrated under reduced pressure, ethanol was added thereto, and the mixture was concentrated again to about 5 ml under reduced pressure. The obtained white precipitate was collected by filtration and dried under reduced pressure. The yield is 423 mg.

【００９４】¹Ｈ−ＮＭＲ（ＣＤ₃ ＯＤ）：δ 5.37(1H,
broad d), 4.8-4.7(2H,m), 4.0-3.7(12H,m), 3.19(2H,
t), 2.9-2.7(2H,m), 2.32(2H,d), 2.26(1H,m), 2.1-1,7
(9H,m), 1.7-1.8(36H,m), 0.71(3H,s)。 ¹ H-NMR (CD ₃ OD): δ 5.37 (1H,
broad d), 4.8-4.7 (2H, m), 4.0-3.7 (12H, m), 3.19 (2H, m
t), 2.9-2.7 (2H, m), 2.32 (2H, d), 2.26 (1H, m), 2.1-1,7
(9H, m), 1.7-1.8 (36H, m), 0.71 (3H, s).

【００９５】実施例４（リポソームの調製、細胞接着阻
害実験） 本実施例の実験は、Pierschbacher, M.D., et al., Nat
ure, 309, 30(1984)に記載の方法に準じて行なった。す
なわち、エライザ（ＥＬＩＳＡ）用９６穴マイクロプレ
ートをフィブロネクチンでコーディングし、ウシ血清ア
ルブミン（ＢＳＡ）でブロッキングした。検体を分注
し、トリプシン処理にて調製したマウス肺癌細胞株３Ｌ
Ｌ（Saiki,I., et al., Br. J. Cancer,59, 194(1989)
参照）を４×１０⁴ 個加え、３７℃で４０分間インキュ
ベートした。非接着細胞を除去した後、接着細胞に 3−
［4,5-Dimethylthiazol-2-yl］−2,5-diphenyltetrazol
iumbromide （ＭＴＴ）を添加して発色させ、吸光度か
ら細胞接着率を算出した。Example 4 (Preparation of liposome, cell adhesion inhibition experiment) The experiment of this example was carried out by Pierschbacher, MD, et al., Nat.
ure, 309 , 30 (1984). That is, a 96-well microplate for ELISA was coded with fibronectin and blocked with bovine serum albumin (BSA). A mouse lung cancer cell line 3 L prepared by dispensing a sample and treating with trypsin
L (Saiki, I., et al., Br. J. Cancer, 59 , 194 (1989)
See) 4 × 10 ⁴ cells were added and incubated at 37 ° C. 40 min. After removing non-adherent cells, 3-
[4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazol
iumbromide (MTT) was added for color development, and the cell adhesion was calculated from the absorbance.

【００９６】なお、検体としては、本発明のペプチド修
飾リポソーム及びコントロールとしての遊離のペプチド
（ＲＧＤペプチドそのもの）の計２点を使用した。As the specimen, a total of two points were used: the peptide-modified liposome of the present invention and a free peptide (RGD peptide itself) as a control.

【００９７】ここに、本発明のペプチド修飾リポソーム
は、Ｌ−α−ジパルミトイルフォスファチジルコリン
（ＤＰＰＣ）、コレステロール（ＣＨＯＬ）、ジセチル
リン酸（ＤＣＰ）及び本発明の物質である実施例２の方
法で合成されたＲＧＤペプチド−脂質誘導体（脂質（ア
ンカー）がアルキル基）がそれぞれ５８．７２mg、３
０．９４mg、４．３８mg及び１０．９５mgの脂質組成を
有する。製造法としては、これらを５０mlの遠沈管にと
り、クロロホルムおよびメタノールの混液（容積比１：
１）に溶かし、次に、窒素ガス気流中で有機溶媒を除去
して遠沈管のガラス壁にリピッドフィルムを生成させ
た。一晩デシケータ内で減圧乾燥後、これに予め約４５
℃に加温したリン酸緩衝化生理食塩水（ＰＢＳ）８mlを
加えて約５０℃に保温しながら振盪し、更に軽く超音波
処理してリポソームの懸濁液を調製した。これを６０℃
に加温し、０．２μｍ、０．１μｍ、０．０８μｍの孔
径を有するポリカーボネート製メンブランフィルターを
順に通過させ、粒径約０．１μｍのリポソームの懸濁液
を調製した。Here, the peptide-modified liposome of the present invention comprises L-α-dipalmitoyl phosphatidylcholine (DPPC), cholesterol (CHOL), dicetyl phosphate (DCP) and the substance of the present invention in the method of Example 2. 58.72 mg of RGD peptide-lipid derivative (lipid (anchor) is an alkyl group) synthesized in
It has a lipid composition of 0.94 mg, 4.38 mg and 10.95 mg. As a production method, these were placed in a 50 ml centrifuge tube, and mixed with chloroform and methanol (volume ratio 1: 1).
The organic solvent was removed in a stream of nitrogen gas to form a lipid film on the glass wall of the centrifuge tube. After drying under reduced pressure overnight in a desiccator, add about 45
8 ml of phosphate-buffered saline (PBS) heated to 0 ° C. was added, and the mixture was shaken while keeping the temperature at about 50 ° C., and further sonicated slightly to prepare a liposome suspension. 60 ℃
Then, the mixture was passed through a polycarbonate membrane filter having pore diameters of 0.2 μm, 0.1 μm, and 0.08 μm in this order to prepare a liposome suspension having a particle size of about 0.1 μm.

【００９８】一方、遊離のペプチドは、実施例３の
（５）の方法で合成されたＲＧＤＳＰＲＧＤＳＰで、リ
ン酸緩衝化生理食塩水（ＰＢＳ）の溶液にして供試し
た。On the other hand, the free peptide was used as a solution in phosphate-buffered saline (PBS) using RGDSPRGDSP synthesized by the method of (3) in Example 3.

【００９９】結果を図１に示す。この図より、本発明の
ペプチド修飾リポソームのほうがより低濃度で３ＬＬ細
胞の、フィブロネクチンへの接着阻害効果を示すことが
判る。なお、図１において、横軸のペプチド濃度は、本
発明のペプチド修飾リポソームについては本発明のＲＧ
Ｄペプチド−脂質誘導体としての濃度である。The results are shown in FIG. From this figure, it can be seen that the peptide-modified liposome of the present invention exhibits a lower concentration of 3LL cell adhesion-inhibiting effect on fibronectin. In FIG. 1, the peptide concentration on the horizontal axis indicates the RG of the present invention for the peptide-modified liposome of the present invention.
It is the concentration as D peptide-lipid derivative.

【０１００】実施例５（リポソームの調製） 本発明のペプチド修飾リポソームを次のようにして調製
した。すなわち、ＤＰＰＣ、ＣＨＯＬ、ＤＣＰ及び本発
明の物質であるＲＧＤペプチド−脂質誘導体（実施例３
の（ｂ）の方法で合成されたもの、脂質（アンカー）は
コレステリル基）をそれぞれ３６．７ｍｇ、１９．４ｍ
ｇ、２．７５ｍｇ及び１４．５ｍｇ（１０：１０：１：
２の割合（モル比））秤取し、これらを５０ｍｌ遠沈管
にとり、これにクロロホルム及びメタノールの１：１混
液を加えて溶解した。次に、４０〜５０℃の水浴中で窒
素ガスを用いて溶媒を除去してリピッドフィルムを調製
した。一晩デシケータ内に保存後、これにリン酸緩衝生
理食塩水（ＰＢＳ）５ｍｌを加え、４０〜５０℃でボル
テックス・ソニケーションを行ない、エクストルーダを
用いて０．２μｍ、０．１μｍ及び０．０８μｍのフィ
ルターで逐次濾過し、リポソームを調製した。粒子径１
６１．２ｎｍ、ゼータ電位−２８．１８ｍＶ。Example 5 (Preparation of liposome) The peptide-modified liposome of the present invention was prepared as follows. That is, DPPC, CHOL, DCP and the RGD peptide-lipid derivative as the substance of the present invention (Example 3)
(36.7 mg, 19.4 m) of lipids (anchors being cholesteryl groups) synthesized by the method of (b).
g, 2.75 mg and 14.5 mg (10: 10: 1:
2 (molar ratio) were weighed, placed in a 50 ml centrifuge tube, and dissolved in a 1: 1 mixture of chloroform and methanol. Next, the solvent was removed using a nitrogen gas in a water bath at 40 to 50 ° C. to prepare a lipid film. After storing in a desiccator overnight, 5 ml of phosphate buffered saline (PBS) was added thereto, and vortex sonication was performed at 40 to 50 ° C., and 0.2 μm, 0.1 μm and 0.08 μm were obtained using an extruder. The mixture was sequentially filtered through a filter of No. 1 to prepare a liposome. Particle size 1
61.2 nm, zeta potential -28.18 mV.

【０１０１】一方、コントロールのリポソームを次のよ
うにして調製した。すなわち、本発明の物質であるＲＧ
Ｄペプチド−脂質誘導体は全く使用せずにＤＰＰＣ、Ｃ
ＨＯＬ及びＤＣＰをそれぞれ上記と同量を秤取し、以下
全く同様にしてリポソームを調製した。粒子径１５０．
８ｎｍ、ゼータ電位−１７．９５ｍＶ。On the other hand, control liposomes were prepared as follows. That is, the substance of the present invention, RG
DPPC, C without any D peptide-lipid derivatives
HOL and DCP were weighed in the same amounts as above, and liposomes were prepared in exactly the same manner. Particle size 150.
8 nm, zeta potential -17.95 mV.

【０１０２】[0102]

【発明の効果】本発明により、リポソームに含有せしめ
られたときに癌細胞の転移を有効に抑制する物質が容易
に提供されるところとなった。According to the present invention, a substance that effectively suppresses the metastasis of cancer cells when contained in a liposome can be easily provided.

【図面の簡単な説明】[Brief description of the drawings]

【図１】実施例４における実験結果を示す。FIG. 1 shows the experimental results in Example 4.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 金子 英雄 神奈川県横浜市南区中村町１−１−25 (72)発明者 田中 勲 茨城県つくば市二の宮２−５−16 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hideo Kaneko 1-1-25 Nakamuracho, Minami-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Isao Tanaka 2-5-16 Ninomiya, Tsukuba-shi, Ibaraki

Claims (3)

(57)【特許請求の範囲】(57) [Claims] 【請求項１】 下記の一般式で示されるペプチド−脂質
誘導体。 【化１】Ｒ¹ −ＣＯＮＨ−Ｘ−ＣＯ−Ｙ但し、Ｒ ¹ は、水素原子、炭素原子数８〜１８のアルキ
ル基又はコレステロール残基であり、Ｘは、 Ａｒｇ−Ｇ
ｌｙ−Ａｓｐからなる配列を含むペプチドのアミノ基及
びカルボキシル基を除く基であり、Ｙは、Ｒ ¹ が炭素原
子数８〜１８のアルキル基又はコレステロール残基であ
るときは水酸基であり、Ｒ ¹ が水素原子であるときは基 【化２】−ＮＨ−（ＣＨ₂ ＣＨ₂ Ｏ）_n −Ｒ² である。但し、Ｒ ² は、炭素原子数８〜１８のアルキル
基又はコレステロール残基であり、ｎは、１〜４の整数
である。 1. A peptide-lipid derivative represented by the following general formula . R ¹ -CONH-X-CO-Y wherein R ¹ is a hydrogen atom, an alkyl having 8 to 18 carbon atoms.
X is Arg-G
Amino group of peptide containing a sequence consisting of ly-Asp
A group except the fine carboxyl group, Y, R ¹ is a carbon source
An alkyl group having 8 to 18 children or a cholesterol residue
When R ¹ is a hydrogen atom, it is a group : —NH— (CH ₂ CH ₂ O) _n —R ² . However, R ² is an alkyl having 8 to 18 carbon atoms.
Group or cholesterol residue, n is an integer of 1-4
It is. 【請求項２】 Ｒ ¹ 又はＲ ² が炭素原子数１８のアルキ
ル基である請求項１記載のペプチド−脂質誘導体。2. The peptide-lipid derivative according to claim 1, wherein R ¹ or R ² is an alkyl group having 18 carbon atoms. 【請求項３】 請求項１記載のペプチド−脂質誘導体を
有することを特徴とするリポソーム。3. A liposome comprising the peptide-lipid derivative according to claim 1.