WO2013137302A1 - リジンオリゴマー誘導体及びそれからなる軟骨組織マーカー - Google Patents
リジンオリゴマー誘導体及びそれからなる軟骨組織マーカー Download PDFInfo
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- WO2013137302A1 WO2013137302A1 PCT/JP2013/056974 JP2013056974W WO2013137302A1 WO 2013137302 A1 WO2013137302 A1 WO 2013137302A1 JP 2013056974 W JP2013056974 W JP 2013056974W WO 2013137302 A1 WO2013137302 A1 WO 2013137302A1
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- Prior art keywords
- mmol
- cartilage
- group
- ethyl acetate
- lysine
- Prior art date
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- 210000000845 cartilage Anatomy 0.000 title claims abstract description 82
- 239000004472 Lysine Substances 0.000 title claims abstract description 57
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 239000003550 marker Substances 0.000 title claims abstract description 17
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 19
- 125000003277 amino group Chemical group 0.000 claims abstract description 12
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 4
- 235000018977 lysine Nutrition 0.000 claims description 58
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 11
- 229910052740 iodine Inorganic materials 0.000 claims description 8
- 150000002669 lysines Chemical class 0.000 claims description 8
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- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 4
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
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- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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Definitions
- the present invention relates to a lysine oligomer derivative.
- the present invention also relates to a cartilage tissue marker comprising a lysine oligomer derivative having a property of specifically binding to a cartilage matrix.
- the cartilage tissue is a support tissue composed of a chondrocyte and a substrate surrounding the chondrocyte that exists in order to relieve friction generated between bones and absorb an impact in a joint such as a knee joint.
- OA osteoarthritis
- the total number of osteoarthritis patients in Japan is about 8 million, and the number is expected to further increase as the population ages.
- X-ray photography cannot directly detect the degree of damage or degeneration that cartilage actually suffers, and therefore cannot be quantified, and it is difficult to detect damage to articular cartilage in the absence of advanced symptoms. It is.
- joint fluid testing which is another method, the state of articular cartilage can be grasped using physiological or biochemical changes as indices, but the physical state such as thickness and deformation of articular cartilage is known. It is powerless.
- a method for directly diagnosing articular cartilage there is a method using an arthroscope.
- a fluorescence imaging apparatus that performs optical projection tomography (OPT) of living tissue ex vivo using fluorescent molecules.
- OPT optical projection tomography
- a fluorescent laser-stained biological tissue is irradiated with a pulse laser as excitation light, and photons generated from the irradiated part of the biological tissue are amplified and detected by a photomultiplier tube for each individual pulse irradiation.
- an arbitrary cross-sectional image of the target tissue and an image of the entire tissue are created. can do.
- an in vivo fluorescent imaging system that can detect and image the position of a fluorescent labeling substance in the body of a small animal such as a living rat or mouse has been recently developed and is commercially available (for example, manufactured by GE HEALTHCARE). “EXplore Optix”). According to this, a fluorescent label that specifically accumulates in a target tissue can be administered to an animal, and its three-dimensional distribution can be measured over time and imaged.
- Patent Document 5 a cartilage marker utilizing the fact that a polyarginine peptide (a) or polylysine peptide (b) having a structure represented by the following formula specifically binds to cartilage tissue.
- These polypeptides are those in which the ⁇ -amino group and carboxyl group of arginine or lysine are linked by a peptide bond, as shown in the following formula.
- the compound described in Patent Document 5 is a cartilage tissue marker in which a fluorophore or an X-ray absorbing group is bonded to the N-terminus or C-terminus.
- Patent Document 6 describes ⁇ -polylysine in which an amino group is protected with a urethane bond.
- ⁇ -polylysine is a product in which the ⁇ -amino group and carboxyl group of lysine are linked by a peptide bond.
- Patent Document 6 describes the use of such ⁇ -polylysine for toiletries, cosmetics, feed additives, medicines, agricultural chemicals, food additives, electronic materials, and the like.
- ⁇ -polylysine as a marker for living tissue, and there is no description about introducing a fluorophore or X-ray contrast group into ⁇ -polylysine.
- An object of the present invention is to provide a lysine oligomer derivative that specifically binds to cartilage tissue. Moreover, it is providing the cartilage tissue marker which consists of such a lysine oligomer derivative. And it is providing the reagent (composition) for visualization of the cartilage tissue which combined the fluorophore and the group for X-ray contrast.
- lysine oligomers which are oligopeptides in which the ⁇ -amino group and carboxyl group of lysine are linked by peptide bonds, can serve as cartilage tissue markers. I found it.
- the present invention was completed by successfully visualizing cartilage by binding a group capable of generating or absorbing electromagnetic waves to the lysine oligomer.
- the present invention is a group in which an ⁇ -amino group and a carboxyl group of lysine are linked by a peptide bond, and an electromagnetic wave can be generated or absorbed in the C-terminal carboxyl group, the N-terminal amino group and / or the ⁇ -amino group.
- the group capable of generating or absorbing the electromagnetic wave is preferably a fluorophore or a group for X-ray contrast
- the group capable of generating or absorbing the electromagnetic wave is a group for X-ray contrast containing an iodine atom. More preferably.
- a preferred embodiment of the present invention is a cartilage tissue marker comprising such a lysine oligomer derivative.
- an oligopeptide in which a plurality of lysines are linked by a peptide bond between an ⁇ -amino group and a carboxyl group has a property of specifically accumulating on a cartilage matrix. Therefore, the lysine oligomer derivative of the present invention in which a group capable of generating or absorbing electromagnetic waves is bound to such an oligopeptide is useful as a cartilage tissue marker.
- cartilage tissue marker of the present invention the construction of a cartilage tissue visualization system using a fluorescent imaging device, an X-ray imaging device, or the like in a laboratory animal is achieved.
- this system the basic mass of cartilage can be evaluated over time and quantitatively. Therefore, for example, it is useful for detecting a decrease in cartilage matrix in rheumatoid arthritis, osteoarthritis, or trauma, and an increase in cartilage matrix in cartilage tumors. It is also useful for follow-up after treatment of cartilage diseases. It is also useful as a means for evaluating the state of cartilage in the development of therapeutic agents for cartilage diseases.
- FIG. 2 is a stained image of cartilage when using compounds A to C.
- FIG. It is a stained image of cartilage when using compounds 26-29. 2 is an X-ray CT imaging photograph of cartilage when Compound D is used.
- the lysine oligomer derivative of the present invention is obtained by linking ⁇ -amino group and carboxyl group of lysine with a peptide bond.
- a major feature is that the ⁇ -amino group and the carboxyl group are not linked by a peptide bond as in a general-purpose polylysine, but the ⁇ -amino group and the carboxyl group are linked by a peptide bond.
- the lysine oligomer derivative can specifically bind to cartilage tissue.
- the number of lysines constituting the lysine oligomer derivative of the present invention is not particularly limited, but preferably 3 to 12 lysines are linked.
- the number of lysines is less than 3, there is a possibility that the ability to bind to cartilage tissue is reduced.
- the number of lysines exceeds 12, synthesis may be difficult, and the molecular weight may become too large to make it difficult to diffuse smoothly into the tissue.
- the lysine oligomer derivative of the present invention is obtained by binding a group capable of generating or absorbing electromagnetic waves to a C-terminal carboxyl group, an N-terminal amino group and / or an ⁇ -amino group. By attaching such a group, the cartilage tissue can be visualized.
- n is an integer of 1 to 10. That is, 3 to 12 lysine units constituting the oligomer are contained.
- W is a group capable of generating or absorbing electromagnetic waves, and is preferably a fluorophore or an X-ray contrast group.
- R is independently a group capable of generating or absorbing an amino group or electromagnetic wave. Therefore, all R may be an amino group, a part may be an amino group and a part may be a group capable of generating or absorbing electromagnetic waves, or all may be a group capable of generating or absorbing electromagnetic waves. May be.
- R is preferably a fluorophore or an X-ray contrast group.
- the “group capable of generating or absorbing electromagnetic waves” can generate or absorb electromagnetic waves (radio waves, infrared rays, visible rays, ultraviolet rays, X-rays, gamma rays, etc.).
- electromagnetic waves radio waves, infrared rays, visible rays, ultraviolet rays, X-rays, gamma rays, etc.
- a group capable of absorbing electromagnetic waves for example, a group for X-ray contrast is exemplified, and imaging can be performed by a decrease in electromagnetic waves irradiated from the outside.
- a group that generates electromagnetic waves for example, a fluorophore is exemplified, and imaging is possible by absorbing higher energy and generating fluorescence.
- an iodine-containing group introduced into a lysine oligomer in order to recognize an anionic side chain such as a sulfo group of chondroitin sulfate forming cartilage tissue contains a cationic group such as a guanidino group or an amino group. It is also desirable.
- the fluorophore may be derived from a fluorescent substance (fluorescent dye or the like) having no strong affinity for other tissues around the cartilage. Therefore, calcein that stains bone and Hoechst that stains DNA of cell nuclei should be avoided, but other fluorescent dyes known to those skilled in the art may be appropriately selected.
- fluorescent dyes among such fluorescent substances include benzofurazan dyes such as NBD, rhodamine dyes (eg, rhodamine, carboxy-X-rhodamine, carboxyrhodamine, tetraethylrhodamine, In addition to tetramethylrhodamine, rhodamine red, rhodamine green, etc., fluorescein dyes (eg, fluorescein, carboxynaphthofluorescein, tetrachlorofluorescein, tetrabromosulfone fluorescein), cyanine dyes (eg, , Cy7, Cy5.5, Cy5, Cy3.5, Cy3 Other Cy dyes: GE Healthcare), Alexa Fluors (eg, Alexa Fluor 790, Alexa Fluor 750, Alexa Fluor 700, Alexa Fluor 680, Alexa Fluor 647, Alexa Fluor 633, Alexa Fluor 594
- Qdot a nanocrystal phosphor utilizing quantum dot photon emission (a cadmium mixed with hundreds to thousands of semiconductor materials such as selenium or tellurium, zinc sulfide) And a polymer coated with a biopolymer and then coated with a biopolymer: INVITROGEN].
- the chemical synthesis method of the lysine oligomer derivative of the present invention is not particularly limited. For example, it can be carried out according to the method described in Biopolymers, 1980, 19, 219-229, or according to a method appropriately modified in accordance with this method. Although illustrated below, this invention is not limited to these.
- lysine oligomer For example, the ⁇ -amino group is protected with a Z group (benzyloxycarbonyl group), the ⁇ -amino group is a Boc group (tert-butoxycarbonyl group), and the carboxyl group is a methyl ester.
- a protected lysine derivative as a raw material to form a peptide using a condensing agent that enables peptide bond formation
- a lysine oligomer in which an ⁇ -amino group and a carboxyl group are linked by a peptide bond can be obtained. it can.
- the carboxylic acid body of a lysine oligomer can be obtained by deprotecting the obtained carboxylic acid ester by saponification using lithium hydroxide etc.
- the cationic X-ray contrast agent used here is an aminobenzoic acid ester as a raw material, reductive aminoalkylation to the amino group, and further through guanidinolation of the terminal amino group, in the presence of silver sulfate, methanol It can be obtained by reacting with iodine.
- the lysine oligomer derivative of the present invention may be a pharmaceutically acceptable salt.
- the present invention includes those isomers, and also includes solvates, hydrates, and crystals of various shapes. Is included.
- pharmaceutically acceptable salts include general pharmacologically and pharmaceutically acceptable salts, particularly acid addition salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, Mention may be made of inorganic acid salts such as salts, hydrogen carbonates and perchlorates.
- a lysine oligomer in which the ⁇ -amino group and carboxyl group of lysine are linked by a peptide bond specifically binds to cartilage tissue, and thus can function as a marker for cartilage tissue. Therefore, the lysine oligomer derivative of the present invention into which a group capable of generating or absorbing electromagnetic waves, such as a fluorophore or an X-ray contrast group, is introduced by a method such as a fluorescence microscope, optical projection tomography, X-ray imaging, It can be used as a reagent for visualizing cartilage tissue that can be easily detected.
- a group capable of generating or absorbing electromagnetic waves such as a fluorophore or an X-ray contrast group
- the cartilage tissue marker of the present invention By using the cartilage tissue marker of the present invention as a composition obtained by dissolving in a sterilized aqueous medium (especially water, physiological saline, buffered physiological saline, etc.), it can be used as a reagent for visualizing cartilage tissue. Can do.
- a sterilized aqueous medium especially water, physiological saline, buffered physiological saline, etc.
- the concentration in the composition may be appropriate.
- an appropriate amount of 0.01 mM to 1 mM can be administered, for example, into the joint cavity, but is not limited thereto.
- this lysine oligomer can be used as a carrier for targeting cartilage and delivering drugs to it. That is, a drug in which a drug component used for cartilage treatment is bound to a lysine oligomer linked by a peptide bond between an ⁇ -amino group and a carboxyl group is administered (for example, into a joint cavity). As a result, the drug component can be efficiently accumulated in the cartilage.
- a group capable of generating or absorbing electromagnetic waves may be bound, but it is not necessary to have such a group.
- the lysine oligomer linked by ⁇ bond of the present invention specifically binds to cartilage tissue, a group capable of generating or absorbing electromagnetic waves, such as a fluorophore or a group for X-ray contrast, is bound thereto. This makes it possible to visualize the cartilage tissue and is useful as a means for evaluating the state of cartilage in the development of a therapeutic agent for cartilage diseases.
- the lysine oligomer is also useful as a means for selectively delivering a therapeutic agent to cartilage.
- Sections were thawed at room temperature for 10 seconds, immersed in 100% ethanol for about 30 seconds, stored in 4% paraformaldehyde (PFA) for a while, washed with tap water, and photographed with a fluorescence microscope.
- a GFP filter was used as the fluorescent filter. Since the lysine peptide was labeled with the fluorescent agent NBD, its signal was detected.
- FIG. 1 shows stained images of cartilage when the following three types A to C are used as lysine oligomer derivatives.
- the solvent of the lysine oligomer derivative solution at this time was a physiological saline containing 25% ethanol.
- FIG. 1 is an enlarged photograph of the articular cartilage portion of the sagittal section of the distal femur knee joint.
- a broken line indicates a tide mark that is an interface between the cartilage and the calcified cartilage layer.
- a fluorescent signal (green in the color photograph) is recognized from the surface layer of the cartilage layer to the tide mark, and it can be seen that the lysine oligomer derivative is not bound to the calcified cartilage layer.
- Compound A and B show staining of cartilage tissue, but Compound C limits the staining to surface tissue.
- FIG. 2 shows stained images of cartilage when the following four types are used as lysine oligomer derivatives.
- the solvent of the lysine oligomer derivative solution at this time was physiological saline.
- FIG. 2 is an enlarged photograph of the articular cartilage portion of the sagittal section of the distal femur knee joint. A fluorescent signal is observed from the surface layer of the cartilage layer to the tide mark, and it can be seen that the lysine oligomer derivative does not enter the calcified cartilage layer.
- staining of cartilage tissue was observed. However, staining of compound 26 containing only one lysine unit was negligible.
- Each cartilage fragment sample was imaged and reconstructed with a 10.7 ⁇ m isotropic voxel using a micro CT analysis system (SkyScan 1174, SkyScan, Aartselaar, Belgium).
- the electronic data was further examined using volumetric reconstruction software (NRecon, CTAn, CTvol / CTVox, SkyScan) to obtain a CT image.
- FIG. 3 The obtained X-ray CT image is shown in FIG.
- “K ⁇ 5-TIB” is a sample immersed in an aqueous solution containing compound 48
- “PBS” is a sample immersed only in a phosphate buffer.
- the mesh-like portion of the cartilage captured image is a calcified layer, and the cartilage layer is located above it.
- the cartilage layer was not shown, whereas in the case of the sample immersed in the compound D solution, the cartilage layer was imaged white. Since the density is sufficiently observed, knowledge can be obtained not only about the thickness of the cartilage layer but also about the concentration of chondroitin sulfate contained therein.
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Abstract
Description
例えば、ε-アミノ基をZ基(ベンジルオキシカルボニル基)などで保護し、かつα-アミノ基をBoc基(tert-ブトキシカルボニル基)で、さらにカルボキシル基をメチルエステルなどの保護したリジン誘導体を原料に、ペプチド結合形成を可能にする縮合剤を用いてペプチド形成することなどにより、ε-アミノ基とカルボキシル基とがペプチド結合で連結されたリジンオリゴマーを得ることができる。そして、得られたカルボン酸エステルを水酸化リチウム等を用いたけん化により脱保護することで、リジンオリゴマーのカルボン酸体を得ることができる。
得られたカルボン酸体に、蛍光団やX線造影用の基などを導入し、Boc基を酸を用いて脱保護することで、電磁波を発生又は吸収し得る基が結合されてなるリジンオリゴマー誘導体を得ることができる。
(1)記載の脱Bocリジンオリゴマーに対し、3-ヨードもしくは3,5-ジヨード-4-(2-グアノジノエチルアミノ)安息香酸、3-ヨードもしくは3,5-ジヨード-4-(2-アミノエチルアミノ)安息香酸、2,3,5-トリヨード安息香酸などの含ヨウ素安息香酸誘導体、もしくはこれらの含ヨウ素安息香酸誘導体とジアミンとがペプチド結合してなるアミノ基含有アミドなどのX線造影剤を、一般的なペプチド合成用縮合剤の存在下で反応させ、X線撮像可能なリジンオリゴマー誘導体を得ることが出来る。また、ここで用いられるカチオン性X線造影剤は、アミノ安息香酸エステルを原料に、そのアミノ基への還元的アミノアルキル化、さらに、末端アミノ基のグアニジノ化を経て、硫酸銀存在下、メタノール中、ヨウ素と反応させることで得ることが出来る。
Nα-(tert-ブトキシカルボニル)-L-リジン(1)(985mg、4mmol)を無水1,4-ジオキサン(3mL)、水(3mL)に溶解し、トリエチルアミン(615μL、4.4mmol)を加え、氷冷下、二炭酸ジ-tert-ブチル(960mg、4.4mmol)を滴下し、18時間撹拌した。TLCプレート(酢酸エチル:n-ヘキサン=5:1)で反応終了を確認し、反応液を0.5規定塩酸水溶液(80mL)にあけ、酢酸エチル(3×40mL)で抽出し、有機層を飽和食塩水(80mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン=1:1→2:1)を行い、白色固体の中間体2(1.29g、93%)を得た。
1H-NMR(300MHz,CDCl3)δ: 5.21(br s, 1H), 4.62(br s, 1H), 4.28(br s, 1H), 3.14(d, 2H, J = 6.0Hz), 1.88(br s, 1H), 1.76(br s, 1H), 1.45-1.23(m, 22H), 0.92-0.88(m, 1H)。
Ar雰囲気下、Nα-(tert-ブトキシカルボニル)-Nε-(ベンジルオキシカルボニル)-L-リジン(3)(810mg、2.1mmol)を無水DMF(2mL)、無水メタノール(1mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(390mg、2.6mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(490mg、2.6mmol)の順に加え、氷冷し、2時間撹拌した。TLC(酢酸エチルのみ)で反応終了を確認し、反応液を水にあけ、酢酸エチル(3×70mL)で抽出し、有機層を飽和炭酸水素ナトリウム水溶液(70mL)、水(70mL)、飽和食塩水(70mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン = 1:4→1:2)を行い、無色油状の中間体4(839mg、99%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 7.36-7.33(m, 5H), 5.09(s, 2H), 5.06(s, 1H), 4.81(s, 1H), 3.73(s, 3H), 3.20(q, 2H, J = 6.5 Hz), 1.77-1.67(m, 2H), 1.53-1.51(m, 2H), 1.42-1.37(m, 11H)。
中間体4(839mg、2.1mmol)をメタノール(15mL)に溶解し、Pd/C(触媒量)を加え、水素雰囲気下、室温で40分撹拌した。TLCプレート(酢酸エチル:n-ヘキサン = 2:1)で反応終了を確認し、反応液をセライト濾過し、減圧下で溶媒留去後、無色油状の中間体5(563mg、q.y.)を得、そのまま次の反応に用いた。
Ar雰囲気下、中間体5(560mg、2.2mmol)、Nα-(tert-ブトキシカルボニル)-Nε-(ベンジルオキシカルボニル)-L-リジン(837mg、2.2mmol)(3)を無水DMF(10mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(337mg、2.2mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(423mg、2.2mmol)の順に加え、氷冷し、5.5時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 3:1)で反応終了を確認し、反応液を水にあけ、酢酸エチル(3×70mL)で抽出し、有機層を飽和炭酸水素ナトリウム水溶液(70mL)、水(70mL)、飽和食塩水(70mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、白色固体の中間体6(596mg、44%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 7.36(br s, 5H), 6.18(br s, 1H), 5.19(br s, 2H), 5.10(s, 2H), 4.90(br s, 1H), 4.25(br s, 1H), 4.00(br s, 1H), 3.73(s, 3H), 3.21(br s, 4H), 1.83(br s, 2H), 1.44(d, 18H, J = 3.0Hz)。
中間体6(790mg、1.3mmol)をTHF:水=3:2(32mL)に溶解し、水酸化リチウム一水和物(161mg、3.8mmol)を加え、室温で2時間撹拌した。TLC(酢酸エチル)で反応終了を確認し、反応液を飽和塩化アンモニウム水溶液(50mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を飽和食塩水(2×50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、無色粘性の中間体7(840mg、q.y.)を得、そのまま次の反応に用いた。
Rf = 0.48 (CH2Cl2 : MeOH = 1 : 2); 1H-NMR(300 MHz, CDCl3)δ: 7.34(br s, 5H), 5.14(br s, 1H), 5.09(s, 2H), 4.16-3.84(m, 3H), 3.18(br s, 3H), 1.97-1.90(m, 1H), 1.77(br s, 2H), 1.63-1.21(m, 5H), 1.51(s, 4H), 1.44-1.40(m, 18H)。
中間体6(595mg、1.0mmol)をメタノール(13mL)に溶解し、Pd/C(触媒量)を加え、水素雰囲気下、室温で7.5時間撹拌した。TLCプレート(酢酸エチル)で反応終了を確認し、反応液をセライト濾過し、減圧下で溶媒留去後、白色固体の中間体8(469mg、q.y.)を得、そのまま次の反応に用いた。
Ar雰囲気下、中間体8(376mg、0.77mmol)、Nα-(tert-ブトキシカルボニル)-Nε-(ベンジルオキシカルボニル)-L-リジン(3)(293mg、0.77mmol)を無水DMF(5mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(141mg、0.92mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(176mg、0.92mmol)の順に加え、氷冷し、4時間撹拌した。TLC(酢酸エチルのみ)で反応終了を確認し、反応液を飽和炭酸水素ナトリウム水溶液(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を飽和食塩水(100mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン =1:3)を行い、白色泡状固体の中間体9(504mg、77%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 7.35(br s, 5H), 6.47(br s, 2H), 5.37-5.05(m, 6H), 4.23(br s, 1H), 4.00(br s, 2H), 3.73(s, 3H), 3.21(t, J = 6.5 Hz, 6H), 1.80(br s, 3H), 1.71-1.24(m, 13H), 1.43(d, J = 3.5 Hz, 27H)。
Ar雰囲気下、中間体8(255mg、0.52mmol)、中間体7(319mg、0.52mmol)を無水DMF(5mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(96mg、0.62mmol)、1-(3-ジメチルアミノプロピル) -3-エチルカルボジイミド塩酸塩(119mg、0.62mmol)の順に加え、氷冷し、2時間撹拌した。TLCプレート(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を水(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を水(100mL)、飽和食塩水(100mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン = 9:1)を行い、白色固体の中間体10(464mg、83%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 7.42-7.29(m, 5H), 6.59(br s, 1H), 6.50(br s, 1H), 5.61-5.17(m, 4H), 5.10(s, 2H), 4.24(br s, 1H), 4.00(br s, 3H), 3.73(s, 3H), 3.32(br s, 2H), 3.27-3.17(m, 6H), 1.79-1.35(m, 24H), 1.45-1.41(m, 36H)。
中間体9(504mg、0.6mmol)をメタノール(5mL)に溶解し、Pd/C(触媒量)を加え、水素雰囲気下、室温で1.5時間撹拌した。TLCプレート(酢酸エチル)で反応終了を確認し、反応液をセライト濾過し、減圧下で溶媒留去後、白色泡状固体の中間体11(421mg、99%)を得、そのまま次の反応に用いた。
1H-NMR(300 MHz, CDCl3)δ: 6.57(br s, 1H), 6.50(br s, 1H), 5.44(br s, 1H), 5.26(br s, 2H), 4.23(br s, 1H), 4.07-3.93(m, 2H), 3.73(s, 3H), 3.24(d, J = 5.0 Hz, 4H), 2.71(t, J = 6.5 Hz, 2H), 1.81(br s, 3H), 1.65(br s, 3H), 1.59-1.46(m, 7H), 1.44-1.43(m, 27H), 1.40-1.39(m, 7H)。
中間体10(446mg、0.41mmol)をメタノール(5mL)に溶解し、Pd/C(触媒量)を加え、水素雰囲気下、室温で0.5時間撹拌した。TLCプレート(酢酸エチル)で反応終了を確認し、反応液をセライト濾過し、減圧下で溶媒留去後、白色泡状固体の中間体12(392mg、95%)を得、そのまま次の反応に用いた。
1H-NMR(300 MHz, CDCl3)δ: 6.97(br s, 1H), 5.60-5.49(m, 3H), 4.22(br s, 1H), 4.16-3.98(m, 3H), 3.73(s, 3H), 3.23(br s, 5H), 2.74(br s, 2H), 1.82(br s, 20H), 1.70-1.22(m, 7H), 1.44-1.43(m, 36H)。
Ar雰囲気下、中間体8(469mg、1.0mmol)、中間体2(360mg、1.0mmol)を無水DMF(8mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(176mg、1.2mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(220mg、1.2mmol)の順に加え、氷冷し、13時間撹拌した。TLCプレート(酢酸エチル)で反応終了を確認し、反応液を水にあけ、酢酸エチル(3×90mL)で抽出し、有機層を水(2×90mL)、飽和食塩水(90mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン =2:1→1:0)を行い、黄色固体の中間体13(166mg、21%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 6.45-6.37(m, 2H), 5.27(br s, 1H), 4.71(br s, 1H), 4.25(br s, 1H), 4.90(br s, 1H), 4.25(br s, 1H), 4.01(br s, 2H), 3.74(s, 3H), 3.25(br s, 3H), 3.12(br s, 2H), 1.80(br s, 3H), 1.69-1.64(m, 4H), 1.45(s, 36H)。
Ar雰囲気下、中間体11(143mg、0.2mmol)、中間体2(69mg、0.2mmol)を無水DMF(3mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(37mg、0.24mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(46mg、0.24mmol)の順に加え、氷冷し、14時間撹拌した。TLCプレート(酢酸エチル)で反応終了を確認し、反応液を水(50mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を水(70mL)、飽和食塩水(70mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:メタノール = 19:1)を行い、白色固体の中間体14(118mg、56%)を得た。
Rf = 0.57 (CH2Cl2 : MeOH = 9 : 1); 1H-NMR(300 MHz, CDCl3)δ: 5.47(br s, 1H), 4.24(br s, 1H), 4.00(br s, 2H), 3.31-3.17(m, 7H), 1.81(br s, 4H), 1.61-1.43(m, 14H), 1.43(d, J = 3.5 Hz, 45H)。
Ar雰囲気下、中間体12(189mg、0.20mmol)、中間体2(69.3mg、0.20mmol)を無水DMF(3mL)に溶解し、1-ヒドロキシベンゾトリアゾール一水和物(36.8mg、0.24mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(46.0mg、0.24mmol)の順に加え、氷冷し、20時間撹拌した。TLCプレート(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を飽和塩化アンモニウム水溶液(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を水(100mL)、飽和食塩水(100mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル)を行い、白色固体の中間体15(235mg、92%)を得た。
Rf = 0.51 (CH2Cl2 : MeOH = 9 : 1); 1H-NMR(300 MHz, CDCl3)δ: 7.14(s, 1H), 6.79(s, 1H), 6.58(s, 1H), 5.75(br s, 1H), 5.51(br s, 2H), 5.41(br s, 1H), 4.89(br s, 1H), 4.21(br s, 3H), 4.01(br s, 2H), 3.41(br s, 3H), 3.22(br s, 3H), 3.11(d, J = 6.0 Hz), 1.77-0.90(m, 35H), 1.43(t, J = 2.0 Hz, 36H), 1.42(s, 18H)。
中間体13(90mg、0.11mmol)をTHF:水=3:1(5.5mL)に溶解し、水酸化リチウム一水和物(28mg、0.66mmol)を加え、室温で1時間撹拌した。TLC(酢酸エチル)で反応終了を確認し、反応液を飽和塩化アンモニウム水溶液(20mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を 飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、無色粘性の中間体16(83mg、94%)を得、そのまま次の反応に用いた。
1H-NMR(300 MHz, CDCl3)δ: 5.45(br s, 2H), 4.84(br s, 1H), 4.23-3.84(m, 3H), 3.17(br s, 5H), 1.94-0.19(m, 14H), 1.80(br s, 2H), 1.43(s, 36H). FAB-MS m/e:804[M+H]+
中間体14(180mg、0.17mmol)をTHF:水=3:2(1mL)に溶解し、水酸化リチウム一水和物(43mg、1.0mmol)を加え、室温で2時間撹拌した。TLC(酢酸エチル)で反応終了を確認し、反応液を0.5規定塩酸水溶液(40mL)にあけ、酢酸エチル(3×40mL)で抽出し、有機層を水(2×40mL)、飽和食塩水(40mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、白色固体の中間体17(160mg、92%)を得、そのまま次の反応に用いた。
Rf = 0.49 (CH2Cl2 : MeOH = 9 : 1); 1H-NMR(300 MHz, CDCl3)δ: 5.39(br s, 2H), 4.05(br s, 2H), 3.10(d, J = 5.0 Hz, 2H), 1.80(br s, 5H), 1.56(br s, 7H), 1.44-1.24(m, 9H), 1.44(d, J = 4.0 Hz, 45H)。
中間体15(190mg、0.15mmol)をTHF:水=3:2(18mL)に溶解し、水酸化リチウム一水和物(89mg、2.1mmol)を加え、室温で2時間撹拌した。TLC(酢酸エチル)で反応終了を確認し、反応液を飽和塩化アンモニウム水溶液(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を飽和食塩水(2×50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、無色粘性の中間体18(210mg、q.y.)を得、そのまま次の反応に用いた。
Rf = 0.48 (CH2Cl2 : MeOH = 1 : 2); 1H-NMR(300 MHz, CDCl3)δ: 5.61-5.50(m, 2H), 4.36-3.68(m, 6H), 3.48(d, J = 5.0 Hz, 2H), 3.09(br s, 6H), 2.26-0.86(m, 29H), 1.43(br s, 54H)。
Ar雰囲気下、4-クロロ-7-ニトロベンゾフラザン(NBD-Cl:19)(99.4mg、0.5mmol)を無水DMF(3mL)に溶解し、トリエチルアミン(69μL、0.5mmol)、N-(tert-ブトキシカルボニル)-1,2-ジアミノエタン(87μL、0.6mmol)を加え、室温で2時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:1)で反応終了を確認し、反応液を飽和塩化アンモニウム水溶液(20mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、茶緑色油状の中間体20(171mg、q.y.)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.51(d, 1H, J = 9.0 Hz), 7.70(br s, 1H), 6.17(d, 1H, J = 9.0 Hz), 5.08(br s, 1H), 3.61(br s, 4H), 1.47(s, 9H)。
Ar雰囲気下、中間体20(90mg、0.28mmol)を4.0規定塩化水素1,4-ジオキサン溶液(2mL)に溶解し、遮光し、5.5時間撹拌した。TLCプレート(塩化メチレン:メタノール = 29:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、赤褐色固体の中間体21(68mg、94%)を得た。
1H-NMR(300 MHz, CD3OD)δ: 8.56(d, 1H, J = 9.0 Hz), 6.46(d, 1H, J = 9.0 Hz), 3.87(t, 2H, J = 6.0 Hz), 3.34(br s, 2H)。
Ar雰囲気下、中間体2(100mg、0.3mmol)を無水DMFに溶解し、氷冷下、1-ヒドロキシベンゾトリアゾール一水和物(55mg、0.36mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(69mg、0.36mmol)、中間体21(78mg、0.30mmol)の順に加え、遮光し、室温に戻し2時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を0.5M 塩酸水溶液(40mL)にあけ、酢酸エチル(3×40mL)で抽出し、有機層を水(2×40mL)、飽和食塩水(40mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(n-ヘキサン:酢酸エチル=1:1→1:5)を行い、黄色固体の中間体22(110mg、67%)を得た。
1H-NMR(300 MHz, CD3OD)δ: 6.43(d, J = 9.0 Hz, 1H), 3.90(br s, 1H), 3.66(br s, 2H), 3.55(br s, 2H), 2.97(t, J = 5.5 Hz, 2H), 1.63(br s, 1H), 3.66(br s, J = 2.0 Hz, 18H).
Ar雰囲気下、中間体16(73.9mg、0.092mmol)を無水DMFに溶解し、トリエチルアミン(26μL、0.184mmol)を加え、氷冷下、1-ヒドロキシベンゾトリアゾール一水和物(16.8mg、0.110mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(21.1mg、0.110mmol)、中間体21(23.9mg、0.092mmol)の順に加え、遮光し、室温に戻し20時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を飽和炭酸水素ナトリウム水溶液(30mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を飽和塩化アンモニウム水溶液(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:n-ヘキサン:酢酸エチル:メタノール = 9:5:4:1)を行い、橙色泡状固体の中間体23(74.8mg、81%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.51(d, 1H, J = 8.5 Hz), 8.12(br s, 1H), 7.66(br s, 1H), 6.65(br s, 1H), 6.44(br s, 1H), 6.21(d, 1H, J = 8.5 Hz), 5.60(br s, 1H), 5.48(br s, 2H), 4.75(br s, 1H), 4.00(br s, 2H), 3.67(br s, 3H), 3.26(br s, 1H), 1.82(br s, 3H), 1.43(s, 36H)。
Ar雰囲気下、中間体17(119mg、0.12mmol)、中間体21(31mg、0.12mmol)を無水DMF(3mL)に溶解し、トリエチルアミン(33μL、0.24mmol)を加え、氷冷下、1-ヒドロキシベンゾトリアゾール一水和物(21mg、0.14mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(27mg、0.14mmol)の順に加え、遮光し、室温に戻し8時間撹拌した。TLC(塩化メチレン:メタノール = 19:1)で反応終了を確認し、反応液を飽和炭酸水素ナトリウム水溶液(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を飽和塩化アンモニウム水溶液(50mL)、水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:メタノール=29:1→9:1)を行い、橙色泡状固体の中間体24(113.4mg、79%)を得た。
Rf = 0.55 (CH2Cl2: MeOH = 9 : 1); 1H-NMR(300 MHz, CDCl3)δ: 8.49(d, J = 8.5 Hz, 1H), 8.13(br s, 1H), 6.90(br s, 1H), 6.49(br s, 1H), 6.19(d, J = 8.5 Hz, 1H), 5.78(br s, 1H), 5.41(br s, 1H), 4.76(br s, 1H), 4.23-4.02(m, 1H), 3.67(br s, 3H), 3.48(br s, 3H), 3.10(br s, 3H), 1.79(q, J = 12.5 Hz, 45H), 1.71-1.17 (m, 32H), 1.41(br s, 2H) ; [α]D +14.4o (c = 1.0083, MeOH)。
Ar雰囲気下、中間体18(168mg、0.13mmol)、中間体21(34.6mg、0.13mmol)を無水DMF(3mL)に溶解し、トリエチルアミン(36μL、0.26mmol)を加え、氷冷下、1-ヒドロキシベンゾトリアゾール一水和物(24.5mg、0.16mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(30.7mg、0.16mmol)の順に加え、遮光し、室温に戻し17.5時間撹拌した。TLC(塩化メチレン:メタノール = 19:1)で反応終了を確認し、反応液を飽和炭酸水素ナトリウム水溶液(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を飽和塩化アンモニウム水溶液(50mL)、水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:メタノール = 29:1→9:1)を行い、橙色泡状固体の中間体25(77.4mg、41%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.49(d, J = 8.5 Hz, 1H), 8.22(br s, 1H), 6.19(d, J = 8.5 Hz, 1H), 4.81(br s, 1H), 4.05(br s, 5H), 3.68(br s, 4H), 3.49(br s, 4H), 3.10(br s, 6H), 1.83-1.11(m, 28H), 1.42(t, J = 5.0 Hz, 54H); [α]D +21.7o (c = 1.0108, MeOH)。
Ar雰囲気下、中間体22(83mg、0.15mmol)を無水メタノール(2mL)、4.0規定塩酸1,4-ジオキサン溶液(2mL)に溶解し、遮光し、2時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、赤色固体の目的化合物26(Kε1-NBD)(25mg、40%)を得た。
m.p. 151.4-153.9℃; 1H-NMR(300 MHz, CD3OD)δ: 8.55(d, J = 9.0 Hz, 1H), 6.49(d, J = 9.0 Hz, 1H), 3.87(t, J = 6.0 Hz, 1H), 3.68-3.58(m, 4H), 2.91(t, J = 7.5 Hz, 2H), 1.89-1.78(m, 2H), 1.71-1.61(m, 2H), 1.50-1.41(m, 2H); 13C-NMR(300 MHz, CD3OD)δ: 170.7, 146.5, 145.8, 145.3, 138.4, 123.3, 100.3, 54.3, 52.1, 40.3, 39.0, 32.0, 28.0, 23.0; Absmax/Emmax(PBS): 475 nm/539 nm; [α]23 D = +21.1 cm3 g-1 dm-1 (c = 1.0 g cm-3 in MeOH); Elemental Anal. calcd (%) for C14H23Cl2N7O4・1/2H2O・1/2CH3OH: C 38.76, H 5.83, N 21.82, found: C 38.46, H 5.50, N 21.59; MALDI-TOFMS m/z: 352 [M+H] +.
Ar雰囲気下、中間体23(71.5mg、0.071mmol)を無水メタノール(1mL)、4.0規定塩酸1,4-ジオキサン溶液(1mL)に溶解し、遮光し、5.5時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、橙色固体の目的化合物27(化合物A)(Kε3-NBD)(46.3mg、87%)を得た。
Mp 183.3-184.7℃; 1H-NMR(300 MHz,CD3OD)δ: 8.56(d, 1H, J = 9.0 Hz), 6.51(d, 1H, J = 9.0 Hz), 3.94-3.77(m, 6H), 3.54-3.49(m, 2H), 3.24-3.10(m, 3H), 2.97(t, 2H, J = 7.75 Hz), 1.96-1.81(m, 5H), 1.76-1.68(m, 4H), 1.65-1.60(m, 2H), 1.55-1.38(m, 9H)。MALDI-TOF/MS; 608.319 [M+H](化合物27:C26H45N11O6= 607.36)
Ar雰囲気下、中間体24(102.8mg、0.083mmol)を無水メタノール(2mL)、4.0規定塩酸1,4-ジオキサン溶液(2mL)に溶解し、遮光し、2時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、橙色固体の目的化合物28(Kε4-NBD)(66.2mg、87%)を得た。
1H-NMR(300 MHz, CD3OD)δ: 8.56(d, J = 9.0 Hz, 1H), 6.50(d, J = 9.0 Hz, 1H), 3.87-3.78(m, 7H), 3.55-3.52(m, 1H), 3.21-3.10(m, 5H), 2.96(t, J = 7.5 Hz, 2H), 1.94-1.37(m, 26H); Absmax/Emmax(PBS): 476 nm/540 nm; Anal. calcd for C32H62Cl5N13O7・3H2O: C 39.53, H 7.05, N 18.73, found: C 39.57, H 6.82, N 18.58。
Ar雰囲気下、中間体25(76.4mg、0.052mmol)を無水メタノール(2mL)、4.0規定塩酸1,4-ジオキサン溶液(2mL)に溶解し、遮光し、1時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、橙色固体の目的化合物29(Kε5-NBD)(41.4mg、73%)を得た。
1H-NMR(300 MHz, CD3OD)δ: 8.56(d, J = 9.0 Hz, 1H), 6.51(d, J = 9.0 Hz, 1H), 4.00-3.68 (m, 8H), 3.57-3.48(m, 1H), 3.34-3.07(m, 7H), 2.97(t, J = 7.5 Hz, 2H), 1.92-1.82(m, 8H), 1.79-1.68(m, 4H), 1.61(t, J = 6.5 Hz, 7H), 1.46(br s, 12H); Absmax/Emmax(PBS): 476 nm/539 nm; Anal. calcd for C38H69N15O8・4H2O・CH3OH: C 39.46, H 7.39, N 17.70, found: C 39.44, H 7.00, N 17.55。
無水メタノール(60mL)に、活性化したモレキュラーシーブス4Aと4-アミノ安息香酸エチル(30)(1.7g、10mmol)、酢酸(0.63mL、11mmol)、N-tert-ブトキシカルボニル-2-アミノアセトアルデヒド(1.9g、12mmol)を加え、Ar雰囲気下室温で29時間撹拌した。TLC(酢酸エチル:n-ヘキサン:塩化メチレン = 1:2:3)で反応終了を確認した後、シアノ水素化ほう素ナトリウム(0.6g、10mmol)を加え、室温で14時間撹拌した。TLC(酢酸エチル:n-ヘキサン:塩化メチレン = 1:2:3)で反応終了を確認後、反応液に水(10mL)を加え、酢酸エチルでセライト濾過し、濾液を2規定塩酸水溶液(50mL)、飽和炭酸水素ナトリウム水溶液(100mL)、飽和食塩水(100mL)で洗浄し、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン:塩化メチレン = 1:5:4)を行い、白色固体の中間体31(2.38g、77%)を得た。
1H-NMR(300 MHz,CDCl3)δ: 7.88(d, 2H, J = 9.0 Hz), 6.56(d, 2H, J = 9.0 Hz), 4.81(s, 1H), 4.68(s, 1H), 4.32(q, 2H, J = 7.0 Hz), 3.40(t, 2H, J = 6.0 Hz), 3.31(br s, 2H), 1.46(s, 9H), 1.36(t, 3H, J = 7.0 Hz)。
中間体31(806mg、2.6mmol)、ヨウ素(1,32g、5.2mmol)、硫酸銀(1.78g、5.7mmol)を無水メタノール(10mL)に溶解し、遮光、Ar雰囲気下室温で1.5時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:3)で反応終了を確認した後、反応液に飽和チオ硫酸ナトリウム水溶液を過剰量加え、酢酸エチルでセライト濾過した。濾液を水(50mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン=1:7)を行い、白色固体の中間体32(1.14g、78%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.41(s, 2H), 4.84(br s, 1H), 4.35(q, 2H, J = 7.1 Hz), 4.07(br s, 1H), 3.41(br s, 4H), 1.45(s, 9H), 1.38(t, 3H, J = 7.1 Hz)。
中間体32(562mg、1.0mmol)を無水塩化メチレン(5mL)に溶解し、トリフルオロ酢酸(2mL)を加え、遮光、室温下1.5時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:1)で反応終了を確認し、反応液を水(10mL)にあけ、飽和炭酸水素ナトリウム水溶液(50mL)を加え、酢酸エチル(3×50mL)で抽出し、有機層を水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、黄色粘性の中間体33(364mg、79%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.41(s, 2H), 4.35(q, 2H, J = 7.1 Hz), 3.40(br s, 2H), 3.04(br s, 2H), 2.05(br s, 2H), 1.38(t, 3H, J = 7.1 Hz)。
中間体32(560mg、1.0mmol)をTHF(3.0mL)、水(1.0mL)に溶解し、水酸化リチウム一水和物(62.9mg、1.5mmol)を加え、40℃で13時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:3)で反応終了を確認し、反応液に0.1規定塩酸水溶液(20mL)を加え、析出した固体をろ取し、水で洗浄し、白色固体の中間体34(498mg、94%)を得た。
1H-NMR(300 MHz, CD3OD)δ: 8.38(s, 2H), 6.72(br s, 1H), 3.41(t, 2H, J = 6.0 Hz), 1.43(s, 9H)。
グアニジン塩酸塩(35)(1.45g、15mmol)を無水1,4-ジオキサン(25mL)に溶解し、氷冷下、4.5規定水酸化ナトリウム水溶液(12.5mL)を滴下した後、二炭酸ジ-tert-ブチル(7.2mL、32mmol)を滴下し、24時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:4)で反応終了を確認し、反応液 を水(50mL)にあけ、酢酸エチル(3×70mL)で抽出し、有機層を水(2×50mL)、飽和食塩水(2×50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン:塩化メチレン = 1:6:1)を行い、白色固体の中間体36(1.93g、49%)を得た。
1H-NMR(300 MHz, DMSO-d6)δ: 10.45(br s, 1H), 8.48(br s, 2H), 1.40(s, 18H)。
中間体36(1.93g、7.4mmol)、トリエチルアミン(2.1mL、14.8mmol)をAr雰囲気下、無水塩化メチレン(15mL)に溶かし、-78℃に冷却し、トリフルオロメタンスルホン酸無水物(1.9mL、11.6mmol)を10分かけて滴下し、室温にて20時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:4)で反応終了を確認し、反応液 を飽和塩化アンモニウム水溶液(100mL)にあけ、塩化メチレン(3×50mL)で抽出し、有機層を水(100mL)、飽和食塩水(100mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン = 1:10)を行い、白色固体の中間体37(786mg、27%)を得た。
1H-NMR(300 MHz, DMSO-d6)δ: 11.09(br s, 2H), 1.45(s, 18H)。
Ar雰囲気下、中間体37(364mg、0.8mmol)、中間体33(318mg、0.8mmol)を無水塩化メチレン(5mL)に溶解し、トリエチルアミン(0.44mL、3.2mmol)を加え、室温で16.5時間撹拌した。TLCプレート(酢酸エチル:n-ヘキサン = 1:2)で反応終了を確認し、反応液を飽和塩化アンモニウム水溶液(20mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(酢酸エチル:n-ヘキサン:塩化メチレン = 2:75:50)を行い、白色泡状固体の中間体38(524mg、94%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 11.49(s, 1H), 8.63(br s, 1H), 8.41(s, 2H), 4.34 (q, 2H, J = 7.25 Hz), 3.72(q, 2H, J = 5.75 Hz), 3,54(q, 2H, J = 5.75 Hz), 1.51(s, 9H), 1.49(s, 9H), 1.38(t, 3H, J = 7.25 Hz)。
中間体38(518mg、0.7mmol)をTHF(6.0mL)、水(2.0mL)に溶解し、水酸化リチウム一水和物(111mg、2.6mmol)を加え、40℃で23時間撹拌した。TLC(酢酸エチル:n-ヘキサン = 1:2)で反応終了を確認し、反応液を0.1規定塩酸水溶液(20mL)にあけ、酢酸エチル(3×50mL)で抽出し、有機層を水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、白色泡状固体の中間体39(503mg、q.y.)を得た。
1H-NMR(300 MHz, CDCl3)δ: 11.49(br s, 1H), 8.62(br s, 1H), 8.45(s, 2H), 3.72(q, 2H, J = 5.75 Hz), 3.60(t, 2H, J = 5.75 Hz), 1.51(s, 9H), 1.49(s, 9H)。 FAB-MS m/e:675[M+H]+
Ar雰囲気下、遮光し、化合物27(化合物A)(17.4mg、0.023mmol)、中間体39(74.5mg、0.110mmol)を無水DMF(3mL)に溶解し、トリエチルアミン(29μL、0.207mmol)を加え、氷冷下、HOBt・H2O(18.3mg、0.119mmol)、EDCI・HCl(22.3mg、0.116mmol)の順に加え、室温に戻し27.5時間撹拌した。TLCプレート(酢酸エチル:塩化メチレン = 9:1)で反応終了を確認し、反応液を水(20mL)にあけ、塩化メチレン(3×30mL)で抽出し、有機層を 飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:メタノール = 9:1)を行い、褐色粘性の中間体40(16.3mg、22%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 11.50(s, 4H), 8.46(d, 1H, J = 9.0 Hz), 8.26(s, 2H), 8.21(s, 4H), 8.15(s, 2H), 6.11(d, 1H, J = 9.0 Hz), 4.53(br s, 3H), 3.99(br s, 3H), 3.67-3.63(m, 7H), 3.44(br s, 10H), 1.49(s, 80H)。
Ar雰囲気下、遮光し、化合物27(化合物A)(13.0mg、0.017mmol)、中間体34(51.0mg、0.096mmol)を無水DMF(3mL)に溶解し、トリエチルアミン(21μL、0.153mmol)を加え、氷冷下、1-ヒドロキシベンゾトリアゾール一水和物(13.5mg、0.088mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(16.3mg、0.085mmol)の順に加え、室温に戻し20時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を飽和炭酸水素ナトリウム水溶液(30mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を飽和塩化アンモニウム水溶液(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:n-ヘキサン:酢酸エチル:メタノール = 9:5:4:1)を行い、橙色泡状固体の中間体41(74.8mg、81%)を得た。
1H-NMR(300 MHz,CD3OD)δ: 8.42(d, 1H, J = 9.0 Hz), 8.42-8.20(m, 8H), 8.16(br s, 1H), 8.10(br s, 1H), 6.71(br s, 3H), 6.33(d, 1H, J = 9.0 Hz), 4.43-4.35(m, 3H), 3.63(br s, 3H), 3.56-3.53(m, 3H), 1.78(br s, 6H), 1.43(s, 36H)。
Ar雰囲気下、中間体40(15.7mg、4.1μmol)を無水メタノール(0.5mL)、無水塩化メチレン(0.5mL)、4.0規定塩酸1,4-ジオキサン(1.0mL)に溶解し、遮光し、11.5時間撹拌した。TLCプレート(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、橙色固体の目的化合物42(化合物B)(4.2mg、40%)を得た。
Mp 189.4-191.5℃; 1H-NMR(300 MHz, CD3OD)δ: 8.53-8.47(m, 6H), 8.36-8.31(m, 7H), 8.25-8.13(m, 10H), 6.33(d, 1H, J = 9.0 Hz), 4.36(br s, 3H), 3.66-3.62(m, 4H), 3.48-3.40(m, 25H), 1.85-1.76(m, 9H), 1.65-1.43(m, 21H); MALDI-TOF/MS m/e:2431.913[M+H](化合物42: C66H85I8N27O10=2430.93)
Ar雰囲気下、中間体41(3.9mg、1.5μmol)を無水メタノール(0.5mL)、4.0規定塩酸1,4-ジオキサン溶液(0.5mL)に溶解し、遮光し、10.5時間撹拌した。TLCプレート(塩化メチレン:メタノール = 19:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、橙色固体の化合物43(化合物C)(2.5mg、69%)を得た。
Mp 192.9-195.7℃; 1H-NMR(300 MHz, CD3OD)δ: 8.43-8.23(m, 9H), 6.33(d, 1H, J = 9.0 Hz), 4.38(br s, 4H), 3.66(br s, 7H), 1.79(br s, 8H), 1.49(br s, 18H);MALDI-TOF/MS; 2301.939 [M+HCl+H](化合物43: C62H77I8N19O10= 2262.85)
Ar雰囲気下、2,3,5一トリヨード安息香酸(44)(100mg、0.2mmol)、N-(tert-ブトキシカルボニル) -1,2-ジアミノエタン(32μL、0.2mmol)を無水DMF(3mL)に溶解し、氷冷し、1-ヒドロキシベンゾトリアゾール一水和物(37mg、0.24mmol)、1-(3-ジメチルアミノプロピル) -3-エチルカルボジイミド塩酸塩(46mg、0.24mmol)の順に加え、室温で13,5時間撹拌した。TLCプレート(n-ヘキサン:酢酸エチル = 1:1)で反応終了を確認し、反応液を水(30mL)にあけ、酢酸エチル(3×30mL)で抽出し、有機層を飽和炭酸水素ナトリウム水溶液(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、n-ヘキサン/酢酸エチルで再結晶を行い、白色固体の中間体45(106mg、82%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.22(d, J = 2.0 Hz, 1H), 7.53(d, J = 2.0 Hz, 1H), 6.50(br s, 1H), 4.91(br s, 1H), 3.53(q, J = 5.4 Hz, 2H), 3.40(br s, 2H), 1.44(s, 9H).
Ar雰囲気下、中間体45(415mg、0.65mmol)を無水メタノール(10mL)、4.0規定塩酸1,4-ジオキサン溶液(6mL)に溶解し、2時間撹拌した。TLC(n-ヘキサン:酢酸エチル = 1:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、白色固体の中間体46(326.2mg、87%)を得た。
1H-NMR(300 MHz, CD3OD)δ: 8.34(d, J = 2.0 Hz, 1H), 7.71(d, J = 2.0 Hz, 1H), 3.60(t, J = 6.5 Hz, 2H), 3.17 (t, J = 6.5 Hz, 2H); MALDI-TOFMS m/z: 543 [M+H]+.
Ar雰囲気下、中間体18(289mg、0.23mmol)、中間体46(133mg、0.23mmol)を無水DMF(8mL)に溶解し、氷冷下、1-ヒドロキシベンゾトリアゾール一水和物(43mg、0.28mmol)、1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド塩酸塩(54mg、0.28mmol)の順に加え、室温に戻し2時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を水(70mL)にあけ、酢酸エチル(3×70mL)で抽出し、有機層を水(50mL)、飽和食塩水(50mL)で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下で溶媒留去後、フラッシュカラムクロマトグラフィー(塩化メチレン:メタノール = 19:1)を行い、白色固体の中間体47(158mg、39%)を得た。
1H-NMR(300 MHz, CDCl3)δ: 8.30(d, J = 2.0 Hz, 1H), 7.63(d, J = 2.0 Hz, 1H), 3.94(br s, 5H), 3.44(br s, 3H), 3.20-3.07(m, 10H), 3.02(d, J = 6.5 Hz, 2H), 1.71(br s, 5H), 1.51(d, J = 6.5 Hz, 21H), 1.44-1.42 (m, 54H).
Ar雰囲気下、中間体47(100mg、0.056mmol)を無水メタノール(2mL)、4.0規定塩酸1,4-ジオキサン溶液(1mL)に溶解し、1時間撹拌した。TLC(塩化メチレン:メタノール = 9:1)で反応終了を確認し、反応液を風乾し、減圧下で溶媒留去後、メタノール/酢酸エチルで再結晶を行い、白色固体の目的化合物48(化合物D)(74mg、95%)を得た。
m.p. 208.0-209.5℃; 1H-NMR(300 MHz, CD3OD)δ: 8.32(d, J = 2.0 Hz, 1H), 7.66(d, J = 2.0 Hz, 1H), 3.96-3.85(m, 5H), 3.59-3.47(m, 1H), 3.24(br s, 6H), 2.97(t, J = 7.5 Hz, 2H), 1.87(br s, 10H), 1.74(br s, 2H), 1.60(d, J = 6.5 Hz, 9H), 1.48(br s, 11H); 13C-NMR(300 MHz, CD3OD)δ: 171.5, 170.6, 170.2, 170.1, 148.7, 147.9, 136.3, 113.2, 107.7, 97.2, 95.0, 61.6, 54.5, 54.3, 50.0, 40.4, 40.1, 32.3, 32.2, 29.8, 28.0, 23,4, 23.1, 20.9, 14.5.; [α]21 D =+19.9 cm3 g-1 dm-1 (c = 1.0 g cm-3 in MeOH); Elemental Anal. calcd (%) for C39H75Cl6I3N12O6・3/2H2O: C 32.79, H 5.50, N 11.77, found: C 33.01, H 5.52, N 11.41; MALDI-TOFMS m/z: 1183 [M+H]+.
正常なC57B6/J系マウス(8週齢、雌)2匹を用意し、エーテルにて深麻酔殺後大腿骨遠位端を得、これをリジンオリゴマー誘導体の0.1mM溶液100μLに室温1時間浸漬した。1時間後、緩衝生理食塩水にて2回洗浄した。これを4%CMCコンパウンド内に包埋し、液体窒素とヘキサンで凍結して、硬組織切片用のタングステンカーバイド製ブレードTC-65(Leica製)とCryofilm (Leica製)を用いて、脱灰せずにクライオスタットで7μmの厚さに薄切した。切片を室温に10秒置いて解凍し、100%エタノールに約30秒間浸漬し、4%パラホルムアルデヒド(PFA)内にしばらく保存した後、水道水で洗浄し、蛍光顕微鏡撮影を行った。蛍光フィルターとしては、GFPフィルターを用いた。リジンペプチドは蛍光剤NBDで標識されているので、そのシグナルが検出された。
A:化合物27
B:化合物42
C:化合物43
Kε1-NBD:化合物26
Kε3-NBD:化合物27
Kε4-NBD:化合物28
Kε5-NBD:化合物29
農協にて購入した豚膝関節軟骨片を、カミソリを用いて、約1.0×0.5×0.2cmの大きさにカットした。この軟骨片を化合物D(化合物48:Kε5-TIB)の200mMのリン酸緩衝液およびリン酸緩衝液のみに30分間浸漬した。30分後、いずれの軟骨片もリン酸緩衝液ですすぎ、洗浄した後、キムワイプで余分な水分を拭き取った。いずれの軟骨片サンプルもマイクロCT分析システム(SkyScan 1174,SkyScan,Aartselaar,Belgium)を用いて、10.7μm等方ボクセルにて、撮像、画像再構築を行った。その電子データを容積測定再構築ソフトウェア(NRecon,CTAn,CTvol・CTVox,SkyScan)を用いてさらに精査し、CT撮像画像を得た。
Claims (8)
- リジンのε-アミノ基とカルボキシル基とがペプチド結合で連結されてなり、C末端カルボキシル基、N末端アミノ基及び/又はα-アミノ基に、電磁波を発生又は吸収し得る基が結合されてなることを特徴とする、リジンオリゴマー誘導体。
- 3~12個のリジンが連結されてなる請求項1に記載のリジンオリゴマー誘導体。
- 前記電磁波を発生又は吸収し得る基が、蛍光団又はX線造影用の基である請求項1又は2に記載のリジンオリゴマー誘導体。
- 前記電磁波を発生又は吸収し得る基が、ヨウ素原子を含むX線造影用の基である請求項3に記載のリジンオリゴマー誘導体。
- 請求項1に記載のリジンオリゴマー誘導体からなる軟骨組織マーカー。
- 3~12個のリジンが連結されてなる請求項5に記載の軟骨組織マーカー。
- 前記電磁波を発生又は吸収し得る基が、蛍光団又はX線造影用の基である請求項5又は6に記載の軟骨組織マーカー。
- 前記電磁波を発生又は吸収し得る基が、ヨウ素原子を含むX線造影用の基である請求項7に記載の軟骨組織マーカー。
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US14/383,976 US9724434B2 (en) | 2012-03-13 | 2013-03-13 | Lysine oligomer derivative and cartilage tissue marker made thereof |
JP2014504955A JP6112427B2 (ja) | 2012-03-13 | 2013-03-13 | リジンオリゴマー誘導体及びそれからなる軟骨組織マーカー |
EP13761422.8A EP2826785B1 (en) | 2012-03-13 | 2013-03-13 | Lysine oligomer derivative and cartilage tissue marker made thereof |
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EP (1) | EP2826785B1 (ja) |
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WO2015087839A1 (ja) | 2013-12-13 | 2015-06-18 | Mmipマネジメント株式会社 | 関節軟骨イメージング組成物 |
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CN114845741A (zh) * | 2019-12-04 | 2022-08-02 | 苏黎世大学 | 用于肾中血管和管状结构的3d离体成像的造影剂 |
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US20150080551A1 (en) | 2015-03-19 |
JPWO2013137302A1 (ja) | 2015-08-03 |
EP2826785A4 (en) | 2015-12-02 |
JP6112427B2 (ja) | 2017-04-12 |
EP2826785B1 (en) | 2017-08-09 |
US9724434B2 (en) | 2017-08-08 |
EP2826785A1 (en) | 2015-01-21 |
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