WO2015037706A1 - 多能性幹細胞の心筋分化を促進する化合物 - Google Patents
多能性幹細胞の心筋分化を促進する化合物 Download PDFInfo
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- WO2015037706A1 WO2015037706A1 PCT/JP2014/074233 JP2014074233W WO2015037706A1 WO 2015037706 A1 WO2015037706 A1 WO 2015037706A1 JP 2014074233 W JP2014074233 W JP 2014074233W WO 2015037706 A1 WO2015037706 A1 WO 2015037706A1
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- 0 CCc(ccc(CCCC*C(C(C1)=Nc(ccc(I)c2)c2C1=*)N)c1)c1[U]C Chemical compound CCc(ccc(CCCC*C(C(C1)=Nc(ccc(I)c2)c2C1=*)N)c1)c1[U]C 0.000 description 2
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- C07D277/62—Benzothiazoles
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- the present invention relates to a compound that promotes myocardial differentiation of pluripotent stem cells and use thereof.
- Heart disease is the world's leading cause of death, and heart transplantation is currently the only treatment for patients with severe heart failure, but heart transplantation has a problem of lack of donors.
- transplantation of cardiomyocytes derived from pluripotent stem cells (iPS / ES cells and the like) is considered promising, and immediate realization is desired.
- the present inventors have developed a compound having a myocardial differentiation promoting activity of pluripotent stem cells (Patent Document 1, Non-Patent Document 1).
- the novel compound KY02111 has a very strong effect compared with known myocardial differentiation promoting compounds and proteins.
- KY02111 is very useful as a myocardial differentiation promoting agent for pluripotent stem cells, but in order to obtain a sufficient myocardial differentiation promoting effect, it must be added to the culture solution at a relatively high concentration. From the viewpoint of further cost reduction and safety improvement, a compound that exhibits an effect of promoting myocardial differentiation at a lower concentration is desirable.
- An object of the present invention is to provide a compound that enables myocardial differentiation of pluripotent stem cells with higher efficiency and lower cost than KY02111.
- the present inventors synthesize compounds in which the Cl group of KY02111 is substituted with another halogen group, or compounds in which the carbon number (n) of the carbon chain is changed, and compare the myocardial differentiation promoting effects of these compounds. As a result, a compound showing an effect of promoting myocardial differentiation at an extremely lower concentration than KY02111 was found, and the present invention was completed.
- the present invention provides the following. 1. Formula (II) [Wherein m is 1 to 4, R 16 and R 17 are each independently selected from a methoxy group, an ethoxy group, and a propoxy group] Or a salt thereof. 2. 2. The compound according to 1 or a salt thereof, wherein R 16 is a methoxy group. 3. 3. The compound or a salt thereof according to 2 above, wherein R 17 is a methoxy group or a propoxy group. 4). 4. The compound or salt thereof according to any one of 1 to 3, wherein m is 1 to 3. 5. A compound having one of the following formulas or a salt thereof: 6).
- An agent for promoting myocardial differentiation of pluripotent stem cells comprising the compound or salt thereof according to any one of 1 to 5 above. 7).
- a kit for promoting myocardial differentiation of pluripotent stem cells comprising the compound or salt thereof according to any one of 1 to 5 above. 10.
- a method for inducing differentiation of pluripotent stem cells into cardiomyocytes comprising culturing the pluripotent stem cells in a medium containing the compound or salt thereof according to any one of 1 to 5 above. 11. Or 11. The method according to 10 above, comprising culturing pluripotent stem cells in a medium containing 12 12. The method according to 10 or 11, wherein the medium contains the compound at 0.4 to 2 ⁇ M. 13.
- a method for producing cardiomyocytes from pluripotent stem cells comprising culturing pluripotent stem cells in a medium containing the compound or salt thereof according to any one of 1 to 5 above. 14 Or 14. The method according to 13, comprising culturing pluripotent stem cells in a medium containing 15. 15. The method according to 13 or 14 above, wherein the medium contains the compound at 0.4 to 2 ⁇ M.
- cardiomyocytes can be produced from pluripotent stem cells with higher efficiency and lower cost.
- B Serum-free medium (20 ⁇ M).
- C Serum-free medium (3 ⁇ M).
- R 1 to R 5 each independently represent a hydrogen atom; a halogen atom; a hydroxyl group; a straight or branched alkoxy group having 1 to 5 carbon atoms; a straight chain having 1 to 5 carbon atoms that is unsubstituted or substituted with a halogen atom.
- R 12 and R 13 are each independently a straight chain of 1 to 5 carbon atoms substituted with a hydrogen atom, an oxygen atom, or an unsubstituted or halogen atom, or Where two adjacent R 1 —R 5 together form —O—CH 2 —O— or —O— (CH 2 ) 2 —O—.
- R 6 to R 9 each independently represent a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; and a 1 to 5 carbon atom substituted with a group —C (O) A.
- a straight-chain or branched alkoxy group (A is a saturated or unsaturated 5- or 6-membered ring which is unsubstituted or substituted with a straight-chain or branched alkyl group having 1 to 5 carbon atoms, and the ring is a nitrogen atom, an oxygen atom, and 1 or it may contain two atoms) selected from sulfur atom independently, a linear or branched alkyl group unsubstituted or carbon atoms substituted with a halogen atom 1-5; or a group -NR 12 R 13 (R 12 and R 13 each independently represents a hydrogen atom, an oxygen atom, or a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom), Although in two adjacent of R 6 -R 9 together O-CH 2 -O- or -O- (CH 2) may form a 2 -O-, R 10 to R 11 are each independently a hydrogen atom
- KY02111 is a compound having the following formula.
- the compound of the present invention Formula (II) [Wherein m is 1 to 4, R 16 and R 17 are each independently selected from a methoxy group, an ethoxy group, and a propoxy group] It is a compound which has this.
- R 17 is a methoxy group, an ethoxy group, or a propoxy group, and R 16 is a methoxy group. In a further preferred embodiment, R 17 is a methoxy group or a propoxy group, and R 16 is a methoxy group.
- m is 1 to 3, more preferably 2 or 3.
- the compound of the invention is selected from:
- Examples of the salt of the compound of the present invention include sodium salt, potassium salt, calcium salt, magnesium salt and ammonium salt.
- the compound of the present invention can be synthesized according to the method described in the examples.
- the term “pluripotent stem cell” means pluripotency capable of differentiating into all cells constituting an adult and self-replication capable of maintaining the pluripotency even after cell division. It means a cell having the ability.
- the “pluripotent stem cell” includes embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells).
- the species of “pluripotent stem cells” is not particularly limited, but is preferably a mammal, more preferably a rodent or a primate.
- the present invention is suitable for monkey or human pluripotent stem cells, particularly ES cells or iPS cells.
- ES cells are pluripotent stem cells derived from early embryos, and can be established from the inner cell mass of blastocysts or epiblasts of early embryos after implantation.
- ES cells include humans (Thomson J. A. et al., Science 282: 1145-1147 (1998), Biochem Biophys Res Commun. 345 (3), 926-32 (2006); primates such as rhesus monkeys and marmosets (Thomson J. A. et al., Proc. Natl. Acad. Sci. USA 92: 7844-7848 (1995); Thomson J. A. et al., Biol. Reprod.
- EG cells are pluripotent stem cells derived from primordial germ cells, such as human EG cells (Shamblott, et al., Proc. Natl. Acad. Sci USA 95: 13726-13731 (1998)) which forms part of the specification).
- iPS cell means a pluripotent stem cell derived from a cell other than a pluripotent stem cell such as a somatic cell or a tissue stem cell.
- Methods for producing iPS cells include, for example, WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, Cell Stem Cell 3 (5): 568-574 (2008), Cell Stem Cell 4 (5 ): 381-384 (2009), Nature 454: 646-650 (2008), Cell 136 (3): 411-419 (2009), Nature Biotechnology 26: 1269-1275 (2008), Cell Stem Cell 3: 475- 479 (2008), Nature Cell Biology 11: 197-203 (2009), Cell 133 (2): 250-264 (2008), Cell 131 (5): 861-72 (2007), Science 318 (5858): 1917 -20 (2007), which are hereby incorporated by reference.
- cells produced by any method are included in the “i
- the final concentration of the compound of the present invention in the myocardial differentiation medium of pluripotent stem cells is 0.1 to 20 ⁇ M, preferably 0.1 to 10 ⁇ M, more preferably 0.4 to 2 ⁇ M, still more preferably 0.4 to 1 ⁇ M.
- the myocardial differentiation medium is not particularly limited as long as it is a composition generally used for myocardial differentiation of pluripotent stem cells.
- myocardial differentiation medium based on IMDM medium (the following are used in the examples) ), DMEM-based myocardial differentiation medium (DMEM / F12 medium (Sigma) 200 ml, fetal bovine serum (GIBCO) 50 ml, MEM non-essential amino acid solution (Sigma) 2.5 ml, penicillin-streptomycin (GIBCO) 2.5 ml, 200 mM L-glutamine 2.5 ml, 2-mercaptoethanol) or StemPro-34SFM (GIBCO) + BMP4 (10 ng / ml) is exemplified.
- DMEM-based myocardial differentiation medium DMEM / F12 medium (Sigma) 200 ml, fetal bovine serum (GIBCO) 50 ml, MEM non-essential amino acid solution (Sigma) 2.5 ml, penicillin-streptomycin (GIBCO) 2.5 ml, 200 mM L-glutamine 2.5 ml, 2-
- the compound of the present invention can be generally used in a culture method suitable for myocardial differentiation of pluripotent stem cells.
- Examples of the culture method include an adhesion culture method, a suspension culture method, and a suspension culture method.
- the timing and period of culturing the pluripotent stem cells in the medium containing the compound of the present invention is the type of pluripotent stem cells used, the composition of the myocardial differentiation medium, etc. Can be changed as appropriate.
- the 2nd to 14th days of the myocardial differentiation culture 2 days or more (specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days), preferably 3 to 10 days, more preferably 4 to 10 days
- the culture may be performed for 1 day, more preferably 4 to 8 days, and even more preferably 4 to 6 days.
- the compound of the present invention may be used together with another myocardial differentiation promoting factor such as nitrobin, cytokine (combination of bFGF, BMP4, VEGF, DKK1 and activin A), Wnt signal inhibitor and the like.
- Wnt signal inhibitor means a substance that inhibits the Wnt signal pathway, and includes, for example, compounds such as IWP2, XAV939, and IWR1, and proteins such as IGFBP4 and Dkk1.
- the “myocardial differentiation promoting factor” in the present invention includes all substances having a myocardial differentiation promoting effect.
- the compound of the present invention comprises (1) a step of culturing pluripotent stem cells in a medium containing one or more Wnt signal activators, and (2) a cell obtained in step (1) with the compound of the present invention. It can be used in a method comprising the step of culturing in a medium containing.
- Wnt signal activator means a substance that activates the Wnt signal pathway.
- Wnt signal activators include GSK3 ⁇ inhibitors such as BIO and CHIR99021. In the above method, two or more, for example 2, 3, or 4 types of Wnt signal activators may be used in combination.
- the medium preferably contains albumin.
- albumin examples include bovine serum albumin and human serum albumin.
- a culture dish coated with gelatin or laminin for example, human laminin 211) can be used.
- pluripotent stem cell cardiomyocyte differentiation When a serum-free medium containing albumin is used, serum, cytokines, supporting cells, and other proteins other than albumin and components derived from a species different from the pluripotent stem cell used (ie, heterogeneous components) are not present Under pluripotent stem cell cardiomyocyte differentiation can be induced.
- Step (2) may be started immediately after the end of step (1), or may be started after a certain period from the end of step (1).
- the Wnt signal activator and the compound of the present invention may be added to the early stage and the middle stage of myocardial differentiation of pluripotent stem cells, respectively.
- the initial stage of myocardial differentiation of pluripotent stem cells means the stage of induction of differentiation from pluripotent stem cells to mesoderm, where expression of the mesoderm marker gene occurs, and the mid-stage is from mesoderm It means the differentiation induction period to the myocardium.
- mesoderm marker genes include T, MIXL1, NODAL and the like.
- 2 or 3 days from the beginning of myocardial differentiation culture that is, 0-2 days or 0-3 days of myocardial differentiation culture Step (1) is carried out for 1 day, and then 2 days or more of the period until the 14th day of myocardial differentiation culture (specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) Or 12 days), preferably 3 to 10 days, more preferably 4 to 10 days, still more preferably 4 to 8 days, and even more preferably 4 to 6 days.
- step (2) 4 to 6 days out of the 10th day of the myocardial differentiation culture, for example, 3rd to 9th day, 3 to 8th day, 3 to 7th day, 4 to 10th of the myocardial differentiation culture It is preferably carried out on the 4th to 9th day or the 4th to 8th day.
- the concentration of the Wnt signal activator is not particularly limited. When BIO or CHIR99021 is used as the Wnt signal activator, it may be used at a final concentration of 100 nM to 100 ⁇ M, preferably 1 ⁇ M to 10 ⁇ M.
- the compound of the present invention may be added so that the final concentration is 0.1 to 20 ⁇ M, preferably 0.1 to 10 ⁇ M, more preferably 0.4 to 2 ⁇ M, and still more preferably 0.4 to 1 ⁇ M.
- the compound of the present invention can be used for the production of cardiomyocytes. Differentiation into cardiomyocytes can be confirmed by, for example, the number of beating cardiomyocytes, expression of myocardial markers, expression of ion channels, response to electrophysiological stimulation, and the like.
- Myocardial markers include ⁇ MHC, ⁇ MHC, cTnT, ⁇ -actinin, and NKX2.5.
- Examples of the ion channel include HCN4, Nav1.5, Cav1.2, Cav3.2, HERG1b, and KCNQ1.
- the cardiomyocytes obtained by the method of the present invention can be used for in vitro drug safety tests or as transplanted cardiomyocytes for heart disease and the like.
- the present invention also provides a pluripotent stem cell myocardial differentiation promoting agent and myocardial differentiation promoting kit comprising the compound of the present invention or a salt thereof.
- the kit of the present invention may contain another myocardial differentiation promoting factor in addition to the compound of the present invention.
- the compound of the present invention and another myocardial differentiation promoting factor may be stored in separate containers or in the same container.
- the present invention also includes a method for inducing differentiation of pluripotent stem cells into cardiomyocytes, comprising culturing the pluripotent stem cells in a medium containing the compound of the present invention or a salt thereof, and the compound of the present invention or a salt thereof.
- a method for producing cardiomyocytes from pluripotent stem cells comprising culturing pluripotent stem cells in a medium containing the same. The method of the present invention can be carried out according to the explanation of the method of using the compound of the present invention.
- the present invention also provides use of the compound of the present invention or a salt thereof for the manufacture of a myocardial differentiation promoting agent or myocardial differentiation promoting kit of pluripotent stem cells.
- GFP green fluorescent protein
- IMDM medium (Sigma) 200 ml, fetal calf serum (GIBCO) 50 ml, MEM 4 to 8 days after initiation of differentiation induction by culturing in 2.5 ml of a non-essential amino acid solution (Sigma), 2.5 ml of penicillin-streptomycin (GIBCO), 2.5 ml of 200 mM L-glutamine, 2 ⁇ l of 2-mercaptoethanol Compound was added. On the 10th day, the amount of GFP fluorescence was analyzed with the Metamorph imaging system.
- SO2031 (KY02-I) is about 5.2 times, 2 times, and 1.6 times at 0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M, and 30 ⁇ M, respectively, compared with SO087 in which the R 7 group is a Br group. 1.1 times the myocardial differentiation effect was obtained. From these results, it became clear that the iodine-substituted compound has the highest effect particularly at a low concentration.
- the medium components were IMDM (Sigma) (1% MEM non-essential amino acid solution (Sigma), 1% penicillin-streptomycin (Gibco), 2 mM L-glutamine (Sigma), 0.5 mM L-carnitine (Sigma ), 0.001% 2-mercaptoethanol (Gibco), and 0.4% human serum albumin (Sigma)).
- the culture plate was subjected to myocardial differentiation in a suspension culture system using an ultra-low adhesion culture dish (Corning). During the first 2 days, 3 ⁇ M of CHIR99021 as a Wnt activator was added, and 2 ⁇ M of SO2031 (KY02-I) was added from day 3 to day 8 after culture.
- Myocardial differentiation efficiency was determined by calculating the percentage of cardiomyocytes by flow cytometry using an antibody of cardiac troponin T (cTnT), which is a cardiac muscle specific marker molecule.
- cTnT cardiac troponin T
- the proportion of cardiomyocytes increased from 3.7% to 56.8% by the addition of SO2031 (KY02-I), and human ES cells (KhES-3 strain)
- the percentage of cardiomyocytes increased from 17.9% to 77.9% by the addition of SO2031 (KY02-I).
- the myocardial differentiation is efficiently performed at a relatively low concentration of iodine-substituted SO2031 (KY02-I) and without stimulation of cytokines or growth factors. It turned out to promote.
- the solution (Sigma) 2.5 ml, penicillin-streptomycin (GIBCO) 2.5 ml, 200 mM L-glutamine 2.5 ml, 2-mercaptoethanol 2 ⁇ l) was dissolved at 3 ⁇ M or 20 ⁇ M and observed 24 hours later (FIG.
- reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution.
- the extract was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was recrystallized from ethanol to obtain 167 mg of 2- (2- (3,4-dimethoxyphenyl) acetamido) -6-iodobenzothiazole in a yield of 50%.
- reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution.
- the extract was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was recrystallized from ethanol to obtain 83 mg of 2- (3- (3,4-dimethoxyphenyl) propanamide) -6-iodobenzothiazole in a yield of 48%.
- reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution.
- the extract was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was recrystallized from ethanol to obtain 131 mg of 2- (4- (3,4-dimethoxyphenyl) butanamide) -6-iodobenzothiazole in a yield of 30%.
- reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution.
- the extract was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was recrystallized from ethanol to obtain 217 mg of 2- (3- (3-methoxy-4-propoxyphenyl) propanamide) -6-iodobenzothiazole in a yield of 60%.
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Abstract
Description
1.式(II)
R16およびR17は、それぞれ独立してメトキシ基、エトキシ基、およびプロポキシ基から選択される]
を有する化合物またはその塩。
2.R16がメトキシ基である、前記1記載の化合物またはその塩。
3.R17がメトキシ基またはプロポキシ基である、前記2記載の化合物またはその塩。
4.mが1~3である、前記1~3のいずれかに記載の化合物またはその塩。
5.以下のいずれかの式を有する化合物またはその塩。
7.
8.前記化合物の最終濃度が0.4~2μMとなるよう多能性幹細胞の心筋分化培地に添加される、前記6または7に記載の多能性幹細胞の心筋分化促進剤。
9.前記1~5のいずれかに記載の化合物またはその塩を含む、多能性幹細胞の心筋分化促進用キット。
10.前記1~5のいずれかに記載の化合物またはその塩を含む培地中で多能性幹細胞を培養することを含む、多能性幹細胞を心筋細胞に分化誘導する方法。
11.
12.前記培地が前記化合物を0.4~2μMで含む、前記10または11に記載の方法。
13.前記1~5のいずれかに記載の化合物またはその塩を含む培地中で多能性幹細胞を培養することを含む、多能性幹細胞から心筋細胞を製造する方法。
14.
15.前記培地が前記化合物を0.4~2μMで含む、前記13または14に記載の方法。
式(I):
R1-R5は、各々独立して、水素原子;ハロゲン原子;水酸基;炭素数1~5の直鎖又は分岐アルコキシ基;非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基;又は基-NR12R13(R12及びR13は、各々独立して、水素原子、酸素原子、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖または分岐アルキル基である)である、ここでR1-R5のうち隣接する2つが一緒になって-O-CH2-O-または-O-(CH2)2-O-を形成していてもよい、
R6-R9は、各々独立して、水素原子;ハロゲン原子;水酸基;炭素数1~5の直鎖又は分岐アルコキシ基;基-C(O)Aで置換された炭素数1~5の直鎖又は分岐アルコキシ基(Aは、非置換又は炭素数1~5の直鎖または分岐アルキル基で置換された飽和または不飽和5または6員環であり、該環は窒素原子、酸素原子、及び硫黄原子から独立に選択される1または2個の原子を含んでいてもよい);非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基;又は基-NR12R13(R12及びR13は、各々独立して、水素原子、酸素原子、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖または分岐アルキル基である)である、ここでR6-R9のうち隣接する2つが一緒になって-O-CH2-O-または-O-(CH2)2-O-を形成していてもよい、
R10-R11は、各々独立して、水素原子;又は炭素数1~5の直鎖又は分岐アルキル基である、
Xは、-CR14(R14は、水素原子、ハロゲン原子、水酸基、炭素数1~5の直鎖又は分岐アルコキシ基、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基である);酸素原子;硫黄原子;セレン原子;又は基-NR15(R15は、水素原子、炭素数1~5の直鎖又は分岐アルキル基、又は炭素数1~5の直鎖又は分岐アシル基である)である、および
nは、0から6の整数である]。
式(I)のR7基に相当するKY02111のCl基を他のハロゲン基等に置換した化合物を合成し、既報のとおり(Cell Reports, Volume 2, Issue 5, 1448-1460, 25 October 2012、国際出願公開第2012/026491号公報;参照により本明細書の一部をなす)、サルES細胞において濃度依存的に心筋分化効果を確認した。具体的には、心筋分化マーカーであるα-MHC遺伝子のプロモーターの制御下で緑色蛍光タンパク質(GFP)を発現するベクターをサルES細胞(カニクイザルCMK6.4株)に導入し、6ウェルプレート(旭硝子/ 5816-006 :Ezview カルチャープレート)に4.0×105細胞/ウェルにて播種し、IMDM培地を基本とした心筋分化培地(IMDM培地(Sigma)200ml、ウシ胎児血清(GIBCO)50ml、MEM 非必須アミノ酸溶液(Sigma)2.5ml、ペニシリン-ストレプトマイシン(GIBCO)2.5ml、200mM L-グルタミン 2.5ml、2-メルカプトエタノール 2μl)にて培養して、分化誘導開始後4~8日に化合物を添加した。10日目にGFP蛍光量をMetamorph イメージングシステムで解析した。
上記1の解析の結果、R7基にH<F<CH3<Cl<Br<Iの順に心筋分化効果が高くなるという構造活性相関が導かれた(図2)。特に、ヨウ素置換体であるSO2031(以下、KY02-Iとも記載する)は、R7基がCl基であるKY02111と比較して、0.1μM、1μM、10μM、30μMにおいて、それぞれ約16倍、6.3倍、2.1倍、1.5倍の心筋分化効果が得られた。また、SO2031(KY02-I)は、R7基がBr基であるSO087と比較しても、0.1μM、1μM、10μM、30μMにおいて、それぞれ約5.2倍、2倍、1.6倍、1.1倍の心筋分化効果が得られた。これらの結果から、特に低濃度ではヨウ素置換体の化合物が最も高い効果を持つことが明らかとなった。
KY02-Iの炭素鎖の炭素数(n)を変えたものを合成し、心筋分化効果を確認した(図3)。その結果、炭素鎖n=0のSO3030(KY00-I)についてはほとんど活性を示さなかったが、炭素鎖n=1のSO3031(KY01-I)、n=2のSO2031(KY02-I)、n=3のSO3042(KY03-I)に関しては、KY02111よりも高い心筋分化促進効果が見られた。また、0.1μMの低濃度においては、炭素鎖n=1のSO3031(KY01-I)よりも、炭素鎖n=2とn=3のSO2031(KY02-I)とSO3042(KY03-I)の方が高い効果(それぞれ2.7倍、2.8倍)を持つことが分かった。さらに、10μMと30μMの高濃度においては、SO2031(KY02-I)よりSO3042(KY03-I)の方が僅かに高い分化効果(約1.3倍)を持つ傾向が見られた。
炭素鎖n=2のSO2031(KY02-I)と炭素鎖n=3のSO3042(KY03-I)の心筋分化促進効果を、図3に示す結果とは別に、再度濃度依存的に確認した(図4)。その結果、0.1μM、0.4μM、2μMにおいては2つの化合物の分化促進効果に差がなかったが、比較的高濃度の10μMではS3042の方が僅かに効果が高かった(約1.1倍)。図3の結果と合わせると、高濃度ではn=3のSO3042(KY03-I)の方が僅かに効果が高いと考えられた。
式(I)の一般式のR2基およびR3基(KY02111のジメトキシ基)を置換した化合物を合成し、心筋分化効果を確認した(図5)。その結果、ジメトキシ基構造を失った化合物は、R2のメトキシ基をプロポキシ基に置換したSO2077を除き、いずれも分化促進効果が低くなった。SO2077は、ジメトキシ基を有するSO2031(KY02-I)と同等の活性が見られた。
ヒトES細胞(KhES-3株)およびiPS細胞(IMR90-1株)を用いて、SO2031(KY02-I)の心筋分化促進効果を確認した(図6)。心筋分化誘導は、既報の方法に従って行った(Cell Reports, Volume 2, Issue 5, 1448-1460, 25 October 2012、参照により本明細書の一部をなす)。具体的には、培地成分は、IMDM (Sigma)(1% MEM 非必須アミノ酸溶液 (Sigma)、1% ペニシリン-ストレプトマイシン (Gibco)、2 mM L-グルタミン (Sigma)、0.5 mM L-カルニチン (Sigma)、0.001% 2-メルカプトエタノール (Gibco)、および0.4% ヒト血清アルブミン (Sigma)含有)を用いた。培養プレートは超低接着培養ディッシュ (Corning)を用いて、浮遊培養系にて心筋分化を行った。最初の2日間はWnt活性剤であるCHIR99021を3μM添加し、培養後3日目から8日目までSO2031(KY02-I)を2μM添加した。心筋分化効率は、心筋特異的マーカー分子である心筋トロポニンT(cTnT)の抗体を用いてフローサイトメトリーにより心筋細胞の割合を計算することで求めた。その結果、ヒトiPS細胞(IMR90-1株)においては、SO2031(KY02-I)添加により心筋細胞の割合が3.7%から56.8%まで増加し、ヒトES細胞(KhES-3株)においては、SO2031(KY02-I)添加により心筋細胞の割合が17.9%から77.9%まで増加した。このことから、サルES細胞だけではなくヒトES/iPS細胞においても、ヨウ素置換体であるSO2031(KY02-I)が比較的低濃度で、かつサイトカインや成長因子の刺激なしでも心筋分化を効率的に促進することが分かった。
SO2031(KY02-I)を20%血清含有培地(IMDM培地(Sigma)200ml、ウシ胎児血清(GIBCO)50ml、MEM 非必須アミノ酸溶液(Sigma)2.5ml、ペニシリン-ストレプトマイシン(GIBCO)2.5ml、200mM L-グルタミン 2.5ml、2-メルカプトエタノール 2μl)に20μMで、または血清不含培地(IMDM培地(Sigma)200ml、MEM非必須アミノ酸溶液(Sigma)2.5ml、ペニシリン-ストレプトマイシン(GIBCO)2.5ml、200mM L-グルタミン 2.5ml、2-メルカプトエタノール 2μl)に3μMまたは20μMで溶解させ、24時間後に観察した(図7)。いずれの培地でも大きな結晶の析出は観察されず(図7A~C)、また、血清不含培地でも、十分な心筋分化促進効果が得られる3μMでは、結晶の析出はほとんど観察されなかった(図7C)。結晶が析出しないことにより、培地中の濃度が析出により下がることがなく安定に維持される。また、析出した結晶が細胞に傷害を与える可能性がなく、さらに細胞内に残存した結晶が移植適用時に持ち越されてホストに毒性を与えたりする危険性がない。
SO3031(KY01-I)
1H NMR (DMSO-d6): δ 12.61 (s, 1H), 8.37 (s, 1H), 7.73-7.69 (m ,1H), 7.54 (d, J = 8.0 Hz, 1H), 6.97-6.84 (m, 3H), 3.75-3.72 (m, 8H).
MS (ESI) Found; 455 [M+H]+
1H NMR (DMSO-d6): δ 12.42 (s, 1H), 8.37 (s, 1H), 7.72-7.69 (m, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.85-6.83 (m, 2H), 6.75-6.72 (m, 1H), 3.71 (s, 3H), 3.69 (s, 3H), 2.90-2.76 (m, 4H).
MS (ESI) Found; 469 [M+H]+
1H NMR (DMSO-d6): δ 12.37 (s, 1H), 8.37 (s, 1H), 7.72-7.69 (m, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.86-6.79 (m, 2H), 6.70 (d, J = 8.0 Hz, 1H), 3.73 (s, 3H), 3.70 (s, 3H), 2.58-2.48 (m, 4H), 1.96-1.86 (m, 2H).
MS (ESI) Found; 483 [M+H]+
1H NMR (DMSO-d6): δ 12.42 (s, 1H), 8.38-8.37 (m, 1H), 7.72-7.69 (m, 1H), 7.54-7.51 (m, 1H), 6.85-6.82 (m, 2H), 6.72 (d, J = 8.0 Hz, 1H), 3.86-3.82 (m, 2H), 3.72 (s, 3H), 2.87-2.78 (m, 4H), 1.72-1.65 (m, 2H), 094 (t, J = 7.3 Hz, 3H).
MS (ESI) Found; 497 [M+H]+
Claims (15)
- R16がメトキシ基である、請求項1記載の化合物またはその塩。
- R17がメトキシ基またはプロポキシ基である、請求項2記載の化合物またはその塩。
- mが1~3である、請求項1~3のいずれかに記載の化合物またはその塩。
- 請求項1~5のいずれかに記載の化合物またはその塩を含む、多能性幹細胞の心筋分化促進剤。
- 前記化合物の最終濃度が0.4~2μMとなるよう多能性幹細胞の心筋分化培地に添加される、請求項6または7に記載の多能性幹細胞の心筋分化促進剤。
- 請求項1~5のいずれかに記載の化合物またはその塩を含む、多能性幹細胞の心筋分化促進用キット。
- 請求項1~5のいずれかに記載の化合物またはその塩を含む培地中で多能性幹細胞を培養することを含む、多能性幹細胞を心筋細胞に分化誘導する方法。
- 前記培地が前記化合物を0.4~2μMで含む、請求項10または11に記載の方法。
- 請求項1~5のいずれかに記載の化合物またはその塩を含む培地中で多能性幹細胞を培養することを含む、多能性幹細胞から心筋細胞を製造する方法。
- 前記培地が前記化合物を0.4~2μMで含む、請求項13または14に記載の方法。
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