WO2018043567A1 - Compound for removing human pluripotent stem cells - Google Patents

Abstract

The purpose of the present invention is to discover a cytotoxic anticancer drug that serves as a common substrate for both ABCB1 and ABCG2 which are suppressed in human pluripotent stem cells, and to provide a method for efficiently removing undifferentiated cells using said drug. Specifically, the present invention pertains to: a compound represented by formula (I); synthesis intermediates thereof; a method in which said compound is used to remove undifferentiated cells in a cell population in which the differentiation of stem cells has been induced; and a drug that selectively damages cells that express ABCB1 and ABCG2 transporters, the drug containing said compound as an active ingredient.

Description

Compounds that remove human pluripotent stem cells

The present invention relates to a compound for removing undifferentiated cells remaining after induction of differentiation of human pluripotent stem cells such as iPS cells and ES cells, and a method for using the same.

Human induced pluripotent stem cells (hiPSCs) are considered to be a very valuable resource for both basic research and regenerative treatment. However, there are many limitations on its clinical application, for example, the risk that undifferentiated cells become tumors during transplantation has been pointed out (Non-patent Documents 1 and 2). For the safety of stem cell therapy, selective removal of fully differentiated or undifferentiated cells is required. Several methods for selective removal have already been reported: cell selection and precipitation using antibodies against stem cell surface antigens (3, 4); cytotoxicity specific for human pluripotent stem cells A non-patent document 5 (non-patent document 5); modified cell culture conditions (non-patent documents 6 and 7); and a lectin-toxin fusion protein specific to human pluripotent stem cells (non-patent document 8). Several chemical methods have also been shown to be effective, such as a small molecule inhibitor of stearoyl CoA desaturase (SCD1) (Non-patent Document 9) and a small molecule inhibitor of survivin (Non-patent Document 10). Despite these promising studies, none of these removal methods has reached the clinical application stage of regenerative medicine. Development of new chemicals with different removal mechanisms is sought to ensure complete removal of undifferentiated cells that are prone to tumors, either alone or by complementing other methods.

The present inventors previously reported that Kyoto probe 1 (KP-1), which is a fluorescent molecule, selectively labels hiPSCs (Non-patent Document 11, Patent Document 1). Mechanistic studies have shown that the selection is primarily due to the different expression patterns of the ATP binding cassette (ABC) transporter and the transporter selectivity of KP-1 in human pluripotent stem cells. Two ABC transporters, ABCB1 (MDR1) and ABCG2 (BCRP), which cause KP-1 efflux, are suppressed in human pluripotent stem cells.

International Publication No. WO2013 / 05051

ABC transporters play an important role in drug resistance mechanisms in cancer treatment (K. Ueda et al., Proceedings of the National Academy of Sciences of United States of America (1987) 84: 3004-300.C.P. Cole et al., Science (1992) 258: 1650-1654; J. Fletcher et al., Nat Rev Cancer (2010) 10: 147-156). A wide range of cytotoxic anticancer agents have been reported as substrates for ABC transporters. However, previous studies included conflicting results regarding their transporter selectivity. Therefore, the present inventors have first aimed to develop a cytotoxic anticancer agent that is a common substrate for both ABCB1 and ABCG2 that are suppressed in human pluripotent stem cells.

First, we analyzed the effects of 13 cytotoxic anticancer agents on four cell lines in order to find a common substrate for both ABCB1 and ABCG2 among the existing anticancer agents. The transporter selectivity was compared. KB3-1 cells do not express ABC transporters at detectable levels. KB3-1-derived cells (KB / ABCB1 (Y. Taguchi et al., Biochemistry (1997) 36: 8883-8889), KB / ABCC1 (K. Nagata et al., J Biol Chem (2000) 275: 17626-17630), and KB / ABCG2 (N. Hirata et al., Cell Rep (2014) 6: 1165-1174.) Overexpresses ABCB1, ABCC1, and ABCG2, respectively. Most drugs showed at least one ABC transporter-mediated resistance.

For example, camptothecin and etoposide were substrates for both ABCB1 and ABCC1. These results are the results of previous studies by the present inventors (N. Hirata et al., Cell Rep (2014) 6: 1165-1174) and reported results (JJ Strouse et al., Anal Biochem (2013) 437). : 77-87). However, a common substrate for both ABCB1 and ABCG2 could not be found among the compounds tested. This result means that it is very difficult to find a common substrate for both ABCB1 and ABCG2 even though ABCB1 and ABCC1 share the substrate and ABCC1 and ABCG2 share the substrate. It was.

Substances described in WO2013 / 103501 (hereinafter collectively referred to as “KP-1 etc.”) exhibit excellent selectivity for human pluripotent stem cells, but KP-1 etc. have cytotoxicity. Because human pluripotent stem cells cannot be removed. Therefore, the present inventors bound KP-1 and the like to a cytotoxic substance, so that the resulting complex has characteristics as a common substrate for both ABCB1 and ABCG2 such as KP-1, We believe that selective and relatively safe substances can be found if both cytotoxicity of cytotoxic anticancer drugs can be maintained, and various cytotoxic anticancer drugs such as KP-1 Were examined by covalent bonding. First, the inventors bound five anticancer agents including combretastatin A-4 (CA4), which is a cytotoxic anticancer agent, and KP-1 etc., and used them as a substrate for a transporter. Specificity and cytotoxic activity were analyzed. As a result, it was found that the substrate specificity to ABCG2 such as KP-1 was lost by binding to these cytotoxic anticancer agents. Therefore, an object of the present invention is to find a compound that, when combined with KP-1 or the like, gives a complex of a cytotoxic anticancer agent that becomes a common substrate for both ABCB1 and ABCG2.

The present inventors create a substance that is a common substrate for both ABCB1 and ABCG2 and has cytotoxicity by binding a cytotoxic anticancer agent that is a substrate of ABCG2 to KP-1 and the like. We have found that this is possible and have completed the present invention.

Therefore, the present invention relates to a complex in which a cytotoxic anticancer agent that is a substrate for ABCG2 is bound to KP-1 or the like. In particular, the complex is a common substrate for both ABCB1 and ABCG2 and is a substance having cytotoxicity. More specifically, the present invention relates to:
(1) Compound represented by the following formula (I)

[Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
R ² and R ³ are the same or different and are each a hydrogen atom or a C1-6 alkyl group,
X is an oxygen atom, a sulfur atom, or NR ⁷ ;
Here, R ⁷ is a hydrogen atom or a C1-6 alkyl group,
L represents a linker moiety represented by — (CH ₂ ) _k (NH) _m (CO) _n (CH ₂ ) _p —,
Here, k represents an integer of 3 to 7, m is 0 or 1, n is 0 or 1, p is 0 or 1,
D represents ABCG2 selective cytotoxic substance;
Here, ABCG2 selective cytotoxic agent, SN38, Mitoxantrone, Irinotecan, 9-Aminocamptothecin, Doxorubicin, Daunorubicin, Epirubicin, Idarubicinol, Flavopiridol, CI1033, BBR3390, Methotrexate, Prazocin, Indolocarbazole, a topoisomerase I inhibitor NB- 506 and J-107088, Zidovudine (AZT), and lamivudine].
(2) The compound according to (1), represented by the following formula (II)

[Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
L represents a linker moiety represented by — (CH ₂ ) _k (NH) _m (CO) _n (CH ₂ ) _p —,
Here, k represents an integer of 3 to 7, m is 0 or 1, n is 0 or 1, p is 0 or 1,
D represents ABCG2 selective cytotoxic substance;
Here, ABCG2 selective cytotoxic agent, SN38, Mitoxantrone, Irinotecan, 9-Aminocamptothecin, Doxorubicin, Daunorubicin, Epirubicin, Idarubicinol, Flavopiridol, CI1033, BBR3390, Methotrexate, Prazocin, Indolocarbazole, a topoisomerase I inhibitor NB- 506 and J-107088, Zidovudine (AZT), and lamivudine].
(3) The compound according to (1) or (2), wherein R ¹ is a halogen atom or a —OP (O) (OR ⁵ ) (OR ⁶ ) group (R ⁵ and R ⁶ are (1) or Follow the definition in (2)).
(4) L represents a linker moiety represented by — (CH ₂ ) _k (NH) (CO) (CH ₂ ) —, wherein k represents an integer of 3 to 5, (1) to ( 3. The compound according to any one of 3).
(5) The compound according to any one of (1) to (4), wherein D is an ABCG2-selective cytotoxic substance that is a topoisomerase inhibitor.
(6) The compound according to any one of (1) to (5), wherein D is SN38 or Mitoxantrone.
(7) A compound represented by any one of the following formulas (in the formula, a terminal not explicitly showing an atom represents CH ₃ ).

(8) Compound represented by the following formula (Ia) or (Ib)

[Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
R ² and R ³ are the same or different and are each a hydrogen atom or a C1-6 alkyl group,
X is an oxygen atom, a sulfur atom, or NR ⁷ ;
Here, R ⁷ is a hydrogen atom or a C1-6 alkyl group,
k represents an integer of 3 to 7.]
(9) Compound represented by the following formula (IIa) or (IIb)

[Wherein R1 represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. Yes,
Here, R5 and R6 are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R5 and R6 are not present,
k represents an integer of 3 to 7.]
(10) The compound according to (8) or (9), wherein R ¹ is a halogen atom or a —OP (O) (OR ⁵ ) (OR ⁶ ) group.
(11) The compound according to any one of (8) to (10), wherein k represents an integer of 3 to 5.
(12) The method according to any one of (1) to (7), comprising reacting the compound according to any one of (8) to (11) with an ABCG2-selective cytotoxic substance. Wherein ABCG2-selective cytotoxic substances are SN38, Mitoxantrone, Irinotecan, 9-Aminocamptothecin, Doxorubicin, Daunorubicin, Epirubicin, Idrubicinol, Boldinol NB-506 and J-107088, Zidovudine (AZT), and lami, inhibitors of topoisomerase I Is a material selected from udine, method.
(13) Contacting the compound according to any one of (1) to (7) with a cell population in which differentiation of stem cells has been induced for a period of time during which undifferentiated cells die, and remaining (1) to (7) A method for removing undifferentiated cells remaining after induction of differentiation of stem cells, comprising removing the compound according to any one of the above by washing.
(14) The removal method according to (13), wherein the stem cell is an iPS cell.
(15) A drug that selectively damages cells in which the expression of ABCB1 and ABCG2 transporters is suppressed, comprising the compound according to any one of (1) to (7) as an active ingredient.
(16) The drug according to (15), wherein the cells in which expression of ABCB1 and ABCG2 transporter is suppressed are undifferentiated cells.
(17) A kit for selectively damaging a cell in which expression of ABCB1 and ABCG2 transporter is suppressed, comprising the compound according to any one of (1) to (7).

In this specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and preferably a fluorine atom.

In the present specification, the “C1-6 alkyl group” means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, Examples include i-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, pentyl group, isopentyl group, 2,3-dimethylpropyl group, hexyl group, and cyclohexyl group. , C1-5 alkyl group, more preferably methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, pentyl group, An isopentyl group or a 2,3-dimethylpropyl group. More preferred is a C1-3 alkyl group, for example, a methyl group, an ethyl group, an n-propyl group, and an i-propyl group, and a methyl group or an ethyl group is most preferred.

In the present specification, the “C1-6 alkoxy group” means a group ((C1-6 alkyl group) -O— group) bonded to the C1-6 alkyl group through an oxygen atom. The base part may be linear or branched. The C1-6 alkoxy group means that the alkyl group moiety has 1 to 6 carbon atoms. Examples of the alkoxy group include methoxy group, ethoxy group, 1-propyloxy group, 2-propyloxy group, 2-methyl-1-propyloxy group, 2-methyl-2-propyloxy group, and 2,2-dimethyl group. -1-propyloxy group, 1-butyloxy group, 2-butyloxy group, 2-methyl-1-butyloxy group, 3-methyl-1-butyloxy group, 2-methyl-2-butyloxy group, 3-methyl-2- Butyloxy, 1-pentyloxy, 2-pentyloxy, 3-pentyloxy, 2-methyl-1-pentyloxy, 3-methyl-1-pentyloxy, 2-methyl-2-pentyloxy , 3-methyl-2-pentyloxy group, 1-hexyloxy group, 2-hexyloxy group, 3-hexyloxy group and the like. The C1-6 alkoxy group is preferably a C1-5 alkoxy group, and more preferably a methoxy group, ethoxy group, n-propyloxy group, i-propyloxy group, n-butyloxy group, sec-butyloxy group, t -Butyloxy group, isobutyloxy group, pentyloxy group, isopentyloxy group, and 2,3-dimethylpropyloxy group, and more preferably a C1-3 alkoxy group (methoxy group, ethoxy group, and propyloxy group) And more preferably a methoxy group or an ethoxy group.

In this specification, “phenyloxy group” means a group (Ph—O— group) bonded to a phenyl group via an oxygen atom.

In the present specification, as the “—OP (O) (OR ⁵ ) (OR ⁶ ) group”, R ⁵ and R ⁶ are preferably the same, and more preferably —OP (O) (OCH ₂ CH ₃ ) (OCH ₂ CH ₃ ) group, —OP (O) (OCH ₂ Ph) (OCH ₂ Ph) group, or “—OPO ₃ ^2- group”.

In the compound of the present invention, R ¹ is preferably a hydrogen atom, a halogen atom, a C1-6 alkyl group, a hydroxyl group or a —OP (O) (OR ⁵ ) (OR ⁶ ) group (where R ⁵ and R ⁶ are Are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ do not exist), more preferably a hydrogen atom, a fluorine atom, A chlorine atom, a propyl group, and a hydroxyl group, and most preferably a fluorine atom. When R ¹ is a phosphate group, uptake into cells can be promoted by utilizing alkaline phosphatase by utilizing the negative charge of the phosphate group. The substitution position for R1 is preferably the para position.

Substitution of R ² has little influence on transporter selectivity and may be any group of a hydrogen atom and a C1-6 alkyl group, preferably a hydrogen atom, a methyl group, or an ethyl group, Preferably it is a hydrogen atom.

Substitution of R ³ has a low influence on transporter selectivity and may be any group of a hydrogen atom and a C1-6 alkyl group, preferably a hydrogen atom, a methyl group, or an ethyl group, Preferably it is a hydrogen atom.

X is an oxygen atom, a sulfur atom, or NR ⁷ (where R ⁷ is a hydrogen atom or a C1-6 alkyl group), preferably an oxygen atom or a sulfur atom, and most preferably an oxygen atom. .

R ¹ to R ³ and X are preferably a combination in which R ¹ is a fluorine atom, R ² and R ³ are hydrogen atoms, and X is an oxygen atom.

In the present specification, L represents a linker that binds a KP-1 equivalent moiety and an ABCG2 selective cytotoxic substance moiety, and — (CH ₂ ) _k (NH) _m (CO) _n (CH ₂ ) _p − Here, k represents an integer of 3 to 7, preferably 3 to 6, more preferably 3 to 5 or 3 to 4, and most preferably 3. m is 0 or 1, preferably 1. n is 0 or 1, preferably 1. p is 0 or 1, preferably 1. The combination of k, m, n, and p is preferably 3 to 5, 1, 1, 1, and more preferably 3, 1, 1, 1, respectively.

In the present specification, “ABCG2 selective cytotoxic substance” represented by D means a substance that is a selective substrate of ABCG2 and has cytotoxicity. Specifically, SN38, Mitoxantrone , Irinotecan, 9-aminocamptothecin, cited Doxorubicin, Daunorubicin, Epirubicin, Idarubicinol, Flavopiridol, CI1033, BBR3390, Methotrexate, Prazocin, Indolocarbazole, NB-506 and J-107088, Zidovudine a topoisomerase I inhibitor (AZT), and lamivudine (L. Doyle et al., Oncoge e (2003) 22: 7340-7358). As the ABCG2 selective cytotoxic substance, SN38 and Mitoxantrone are preferable.

The binding site on the side of the ABCG2 selective cytotoxic substance is not particularly limited as long as it is a site that does not easily lose the ABCG2 selectivity and cytotoxicity of the ABCG2 selective cytotoxic substance. In view of the common technical knowledge in, these compounds have hydroxyl group, acyl group, acetyl group, formyl group, benzoyl group, carboxyl group, amide group, imide group, cyano group, sulfonic acid group, urea group, isonitrile group ( -NC), allene groups (> C = C = C <), ketene groups (-C = C = O), diimide groups (-N = C = N-), isocyanate groups (-N = C = O), Isothiocyanate group (—N═C═S), carbonyl group, amino group, imino group, cyano group, azo group, azide group, thiol group, sulfo group, nitro group, ether bond, ester bond, amino group Binding, and can be attached using functional groups such as urethane bond.

The present invention also relates to a drug that selectively damages cells in which the expression of ABCB1 and ABCG2 transporters is suppressed, containing the compound of the present invention as an active ingredient. Throughout this specification, whether a cell is suppressed in expression of ABCB1 and ABCG2 transporters is determined by confirming the expression of ABCB1 and ABCG2 transporter genes in the test cells at the mRNA or protein level. can do. Alternatively, transport of ABCB1 and ABCG2 transporter-specific marker molecules to the outside of the cells can be confirmed, and cells that do not transport can be determined as the expression of ABCB1 and ABCG2 transporters being suppressed. Preferably, the cell in which the expression of ABCB1 and ABCG2 transporter is suppressed is an undifferentiated cell. Throughout this specification, “expression of ABCB1 and / or ABCG2 transporter is suppressed” does not require that ABCB1 and / or ABCG2 transporter is not expressed, and is the same environment. It includes a low expression level of ABCB1 and / or ABCG2 transporter compared to other cells present in the medium (eg, in the same culture medium). For example, the expression level of ABCB1 and / or ABCG2 transporter is 25% or less, 20% or less, 15% or less, 10% or less compared to other cells present in the same environment (for example, in the same culture medium) , 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less. In addition, throughout the present specification, “selectively causing damage” does not need to mean that no damage is given to cells other than the target cell that causes damage, and the purpose is compared with other cells. Including more strongly damaging cells. As an example, selectively damaging may mean selectively killing, i.e., it can kill the desired cell, but not the other cells. May be. In addition, the selectivity of the present invention may be brought about not only by the transporter but also by the toxicity mechanism (for example, topoisomerase inhibitory activity, etc.) of the drug to be bound.

A kit for selectively damaging cells in which the expression of ABCB1 and ABCG2 transporters containing the compound of the present invention is suppressed includes a container and instructions for encapsulating the compound in addition to the compound. You can leave.

Since the compound represented by the formula (I) or the formula (II) of the present invention can selectively damage a cell in which the expression of ABCB1 and ABCG2 transporter is suppressed, the expression of these transporters This is useful for removing undifferentiated cells in which the suppression is suppressed. In particular, since the compound represented by the formula (I) or the formula (II) of the present invention is a fluorescent substance, the remaining state after removal of undifferentiated cells can be detected by using fluorescence, so that it is reliable. Clearance evaluation is possible. In addition, the compound represented by the formula (Ia), (Ib), (IIa) or (IIb) of the present invention is used for synthesizing the compound represented by the formula (I) or the formula (II) of the present invention. Useful as a synthetic intermediate.

It is a figure which shows the outline | summary of the structure activity relationship of KP-1. The numerical values indicate the fluorescence intensities obtained by calculating from ImageJ fluorescence images of KB3-1 cells, KB / ABCB1 cells (ABCB1), KB / ABCC1 cells (ABCC1), and KB / ABCG2 cells (ABCG2), respectively. The selectivity of the complex 16 is shown. (A) The chemical structure of the composite 16 is shown. (B) It is a table | surface which shows IC50 value with respect to KB3-1 cell, KB / ABCB1 cell, KB / ABCC1 cell, and KB / ABCG2 cell of CA4, compound 14 (14), and composite 16 (Conjugate 16). (C) Fluorescent microscopic images of KB3-1 cells, KB / ABCB1 cells, KB / ABCC1 cells, and KB / ABCG2 cells treated with complex 16 (1 μM). The scale bar represents 200 μm. The selectivity of the complex 17 is shown. (A) The chemical structure of the complex 17 is shown. (B) A table showing IC50 values of SN38 and complex 17 (Conjugate 17) for KB3-1 cells, KB / ABCB1 cells, KB / ABCC1 cells, and KB / ABCG2 cells (n = 2). (C) KB3-1 cells, KB / ABCB1 cells, KB / ABCC1 cells, and KB / ABCG2 treated with complex 17 (1 μM) in the presence or absence of CsA (10 μM) or Ko143 (10 μM) It is a photograph of the fluorescence microscope image of a cell. The scale bar represents 200 μm. The selectivity of the complex 18 is shown. (A) The chemical structure of the complex 18 is shown. (B) A table showing IC50 values of mitoxantrone and complex 17 (Conjugate 17) for KB3-1 cells, KB / ABCB1 cells, KB / ABCC1 cells, and KB / ABCG2 cells (n = 2). (C) KB3-1 cells, KB / ABCB1 cells, KB / ABCC1 cells, and KB / ABCG2 treated with complex 18 (1 μM) in the presence or absence of CsA (10 μM) or Ko143 (10 μM) It is a photograph of the fluorescence microscope image of a cell. The scale bar represents 200 μm. FIG. 3 is a photograph in which partially differentiated hiPSCs colonies are labeled with KP-1 (1 μM), complex 17 (1 μM), or complex 18 (1 μM). The central part of the colony represents differentiated cells. The scale bar represents 200 μm. It is a photograph showing the effect of complex 17 (5 μM), complex 18 (5 μM), or control DMSO (0.1%) on alkaline phosphatase activity of iPS colonies and partially differentiated iPSCs. . Cells were treated with the complex for 72 hours before alkaline phosphatase colorimetric staining. It is a graph showing the result of the qPCR analysis of nanog in the iPSCs partially differentiated which were processed with the complex 17, the complex 18, or DMSO. Partially differentiated hiPSCs were prepared by culturing hiPSCs with all-trans retinoic acid (0.5 μM) for 48 hours, and then treated with each compound (5 μM) for 72 hours. The expression level of nanog was normalized by gadph. The vertical axis shows the percentage (%) relative to GAPDH, and the horizontal axis shows each processed compound. It is a graph showing the result of the qPCR analysis of sox2 in the partially differentiated iPSCs treated with the complex 17, the complex 18, or DMSO. Partially differentiated hiPSCs were prepared by culturing hiPSCs with all-trans retinoic acid (0.5 μM) for 48 hours, and then treated with each compound (5 μM) for 72 hours. The expression level of sox2 was normalized by gadph. The vertical axis shows the percentage (%) relative to GAPDH, and the horizontal axis shows each processed compound. FIG. 10 is a graph showing the results of oct3 / 4 qPCR analysis in partially differentiated iPSCs treated with Complex 17, Complex 18, or DMSO. Partially differentiated hiPSCs were prepared by culturing hiPSCs with all-trans retinoic acid (0.5 μM) for 48 hours, and then treated with each compound (5 μM) for 72 hours. The expression level of oct3 / 4 was normalized by gadph. The vertical axis shows the percentage (%) relative to GAPDH, and the horizontal axis shows each processed compound. It is a graph showing the viability of hiPSCs (201B7 and 253G1) and human primary cells treated with Complex 17 for 72 hours. Dark gray circles represent iPS201B7, dark gray squares represent iPS253G1, light gray squares represent adrenal microvascular cells, light gray circles represent astrocytes, diamonds represent brain microvascular cells, downward Triangles represent prostate epithelial cells, and upward triangles represent hepatocytes. The vertical axis represents the survival rate (%), and the horizontal axis represents the logarithmic value (Log) of the concentration (μM) of the complex 17. It is a graph showing the survival rate of the human primary cell processed by the complex 17 in the presence or absence (black circle) of cyclosporine (black square) or Ko143 (black triangle). Viability was determined by a WST-8-based colorimetric assay (Cell Counting kit-8, Dojindo). Absorbance at 450 nm was measured. Transporter inhibitors, cyclosporin A (10 μM) or Ko143 (10 μM) were added 1 hour prior to incubation with complex 17. The vertical axis represents the survival rate (%), and the horizontal axis represents the logarithmic value (Log) of the concentration (μM) of the complex 17. It is a graph showing the selectivity of SN38 and the complex 17. hiPS or human primary somatic cells (adrenal microvasculature, astrocytes, brain microvascular cells, prostate epithelial cells, and hepatocytes) were treated with SN38 (0.1 or 1 μM) or complex 17 (1 or 10 μM) for 72 hours. did. (A) Comparison of SN38 (0.1 μM) and complex 17 (1 μM) when the viability level of hiPSCs is ˜20%. (B) Comparison of SN38 (1 μM) with Complex 17 (10 μM) when the viability level of hiPSCs is ˜5%. In both cases, viability was determined by a WST-8 based colorimetric assay. It was revealed that Complex 17 showed higher selectivity than SN38. The vertical axis represents the survival rate (%), and the horizontal axis represents the results of iPS201B7 cells, adrenal microvascular cells, astrocytes, brain microvessels, prostate epithelial cells, and hepatocytes in order from the left. It is a photograph showing inhibition by SN38 and complex 17. Supercoiled DNA premixed with SN38 (0, 1, or 10 μM) or complex 17 (0, 1 or 10 μM) was incubated with recombinant human topoisomerase I at 37 ° C. for 30 minutes. “Super Coiled” represents supercoiled DNA, and “TOPO I” represents human recombinant topoisomerase I. The reaction mixture was loaded on a 1% agarose gel. After electrophoresis, the gel was stained with ethidium bromide in TAE buffer and photographed under UV illumination. 5 is a graph showing the removal of the complex 17. Partially differentiated hiPSCs were prepared by incubating with all-trans retinoic acid (0.5 μM) for 48 hours and then treated with complex 17 (5 μM) for 72 hours. After the cells were treated with complex 17 for 3 days, the medium was collected. The cells were then washed 3 times with 2 mL of fresh ES medium and the washed medium was collected. Media samples were centrifuged and analyzed for fluorescence intensity (excitation at 528 nm). The vertical axis of the graph indicates the fluorescence intensity, and the horizontal axis indicates the wavelength (nm).

(Method for producing the compound of the present invention)
The compound of the present invention synthesizes an intermediate derived from KP-1 etc. in consideration of the synthesis of compounds 1 to 13 in the Examples of the present application, and then an intermediate derived from an ABCG2 selective cytotoxic substance as necessary. And finally, an intermediate such as KP-1 and an ABCG2-selective cytotoxic substance or an intermediate derived therefrom are reacted with each other.

For example, an intermediate derived from KP-1 etc. of the present invention can be synthesized by the following steps.

[Wherein R ¹ , R ² , R ³ and X follow the above definition. L ′ may be the same as or a part of the linker L]

The reaction between an intermediate such as KP-1 and an ABCG2-selective cytotoxic substance is appropriately performed in the field of organic chemistry depending on the type of the functional group of the terminal group of L 'and the ABCG2-selective cytotoxic substance. Can be achieved by bonding by a well-known method. In addition, if necessary, a group that easily binds to the terminal group of L ′ is introduced into ABCG2-selective cytotoxic substance in advance to synthesize an intermediate derived from ABCG2-selective cytotoxic substance, and then KP It may be reacted with an intermediate such as -1.

(Undifferentiated cell removal method)
In one embodiment, the present invention differentiates stem cells comprising contacting a cell population in which differentiation of stem cells has been induced with the compound of the present invention for a time during which undifferentiated cells die, and removing the remaining compound by washing. The present invention relates to a method for removing undifferentiated cells remaining after induction.

In the present specification, “stem cell” means a cell having pluripotency and self-renewal ability. In this specification, “pluripotency” is synonymous with pluripotency, and means a state of a cell that can differentiate into cells of a plurality of lineages by differentiation. In the present specification, pluripotency refers to a state that can be differentiated into all types of cells constituting a living body (totipotency), and a state that can be differentiated into all types of cells other than extraembryonic tissues (differentiation). Pluripotency), a state that can differentiate into cells belonging to some cell lineages (multipotency), and a state that can differentiate into one type of cell (unipotency) Including. Therefore, “stem cells” in the present specification include stem cells, ES cells, iPS cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, or germline stem cells. Preferably, the “stem cell” in the present specification is a cell having pluripotency, more preferably an embryonic stem cell (ES cell) and an induced pluripotent stem cell (iPS cell). Whether a cell is a stem cell is determined by, for example, a cell that forms an embryoid body in an in vitro culture system, or a cell that differentiates into a desired cell after culturing under differentiation-inducing conditions (differentiation treatment). It can be confirmed as a stem cell. Or, whether or not it is a stem cell is determined by using a living body to transplant to an immunodeficient mouse, a cell that forms a teratoma, a cell that forms a chimeric embryo by injection into a blastocyst, or a living tissue It can be confirmed that cells proliferating by transplantation or injection into ascites are stem cells. Alternatively, whether the cells are stem cells is determined by determining whether the cells are positive for alkaline phosphatase staining, SSEA3 staining, SSEA4 staining, TRA-1-60 staining, and / or TRA-1-81 staining; oct3 / 4, nanog, sox2, clipto , Dax1, eras, fgf4, esg1, rex1, zfp296, utf1, gdf3, all4, tbx3, tcf3, dnmt3l, and / or dnmt3b gene; miR-290 and / or miR-302 are expressed It can also be confirmed that the cell is a stem cell. Alternatively, whether or not a certain cell is a stem cell can be determined as a cell having a high expression level of telomerase reverse transcriptase or survivin, for example.

Preferably, the stem cell has pluripotency capable of differentiating into all cells existing in a living body, and also has proliferative ability. Examples of the pluripotent stem cells include embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (“ntES cells”), germ stem cells (“GS cells”), embryonic germ cells ("EG cells"), and induced pluripotent stem (iPS) cells, but are not limited to these. Examples of preferred pluripotent stem cells include ES cells, ntES cells, and iPS cells.

In this specification, “differentiation” refers to a phenomenon in which daughter cells having specific functional or morphological characteristics are generated by the division of pluripotent cells. In the present specification, “differentiation treatment” and “differentiation induction treatment” are synonymous and mean treatment for inducing stem cells into differentiated cells. It has been reported that cell differentiation is induced by various methods. Pluripotent cells can be differentiated by differentiation-inducing treatment using a differentiation-inducing substance or the like according to the type of cells to be differentiated. Many differentiation induction methods are already known to those skilled in the art. For example, differentiation induction methods for neural stem cells described in JP-A No. 2002-291469, pancreatic stem-like cells described in JP-A No. 2004-121165 are described. Examples thereof include differentiation induction methods and differentiation induction methods for hematopoietic cells described in JP-T-2003-505006. Furthermore, examples of the differentiation induction method by the formation of embryoid bodies include the method described in JP-T-2003-523766. “Differentiated cells” refers to daughter cells that have a specific functional or morphological characteristic resulting from differentiation. Differentiated cells are usually stable, their ability to proliferate is low, and differentiation into other types of cells occurs only exceptionally.

“Undifferentiated cell” means an undifferentiated cell, an undifferentiated cell in the middle of differentiation, or a cell that is incompletely differentiated. In the present specification, unless otherwise understood in particular, “undifferentiated cell” means a cell that has not been differentiated even though it has undergone differentiation induction treatment. The undifferentiated cells in the present specification do not necessarily need to have the above-mentioned properties of stem cells completely, and have a higher (self) proliferative capacity than differentiated cells (for example, the proliferation rate is twice or more, (3 times or more, 5 times or more, 10 times or more) means a cell. Whether or not a cell is an undifferentiated cell can be determined, for example, as a cell in which a marker indicating undifferentiation such as c-Myc is activated, or a cell having a high expression level of telomerase reverse transcriptase . In the method for removing undifferentiated cells of the present invention, the expression of ABCB1 and / or ABCG2 transporter is suppressed in undifferentiated cells.

The time for which the compound of the present invention is brought into contact with the cell population in which the differentiation of stem cells is induced is not particularly limited as long as the undifferentiated cells are killed. For example, 24 to 120 hours, 36 to 96 hours, or 72 hours. The contact of the compound with the cell population can be performed by allowing the compound of the present invention to coexist under normal cell culture conditions.

After the undifferentiated cells have been killed, the remaining compound can be easily removed by washing with a medium or PBS. Washing is preferably performed twice or more. Further, the amount of the removed compound can be measured by measuring the fluorescence intensity using the medium or PBS used for washing as a sample. Alternatively, the residual compound amount can be measured by directly measuring the fluorescence of the collected differentiated cells. Desirably, washing is performed until the fluorescence intensity in the sample and / or differentiated cells of the medium and PBS used for washing is comparable to the background.

Hereinafter, the present invention will be described in more detail with reference to examples, but this does not limit the scope of the present invention. References cited throughout the present specification are incorporated herein by reference in their entirety.

(Materials and methods)
The chemical reagents were purchased from Sigma Aldrich Japan Co., Ltd., Wako Pure Chemical Industries, Ltd. and Tokyo Chemical Industry (TCI) and used as they were. The solvent used for chemical synthesis was dried before use. High performance liquid chromatography was performed using Shimadzu LC-2010C and Hitachi HPLC system (L-7260 autosampler, L-7150 pump, D-7600 interface, L-7410 UV detector). Mass spectra were recorded in ESI mode using Shimadzu LCMS-2010. High resolution mass spectra of the three complexes were obtained using JEOL JMS LG-2000 in FAB mode. Solution 1H-NMR spectra were collected on a JEOL JNM-ECP 300 MHz or JEOL JNM-ECA 600 MHz spectrometer. The fluorescence spectrum was recorded using an LS55 fluorescence spectrometer (Perkin Elmer).

(Cell culture)
KB3-1 and KB3-1-derived cells (KB / ABCB1 stably expressing ABCB1 (Y. Taguchi et al., Biochemistry (1997) 36: 8883-8889), KB / ABCC1 stably expressing ABCC1 (K. Nagata et al., J Biol Chem (2000) 275: 17626-17630) and KB / ABCG2 stably expressing ABCG2 (N. Hirata et al., Cell Rep (2014) 6: 1165-1174.)), 10 Cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 1% heat-inactivated fetal bovine serum (Equitech-Bio, Kerrville, TX) and 1% antibiotic (Sigma Aldrich, St. Louis, MO). SNL feeder cells containing 4.5% of 7% heat inactivated fetal bovine serum (Equitech Bio, Kerrville, TX), 1% L-glutamine (200 mM, Gibco), and 1% antibiotics / L glucose-containing Dulbecco's modified Eagle medium (DMEM High Glucose, Nacalai Tesque, Japan). Human primary cells were purchased from Cell Systems, in MSCM (bronchial epithelial cells), BEGM (adrenal microvascular cells), or CSC (astrocytes, brain microvascular cells, prostate epithelial cells, and hepatocytes) In culture. For hiPSCs, gelatin pre-coated dishes seeded with SNL feeder cells and ES primate medium (Reprocell) containing 4 ng / mL bFGF (Reprocell) were used for regular maintenance. All cells were maintained in a humidified incubator at 37 ° C. and 5% CO ₂ .

(Preparation of partially differentiated hiPSCs)
After hiPSCs were subcultured for 24 hours, 0.5 μM all-trans retinoic acid was added and the cells were further incubated for 48 or 72 hours. Subsequently, partially differentiated hiPSCs were treated with the test compound for 72 hours.

(Cell-based assay)
Test cells were seeded in a 96-well plate at 5000 cells per well. 24 hours after sowing, test compounds were added at various concentrations. After 24 or 72 hours incubation, cells were washed with PBS. Viability (%) was determined by a WST-8 based colorimetric assay (Cell Counting Kit-8, Dojindo). Absorbance at 450 nm was measured and IC50 was calculated based on dose response values. The survival rate (%) was determined with the value when 5000 cells were seeded as 100. For experiments with transporter inhibitors, cyclosporin A (10 μM) or Ko143 (10 μM) was added 1 hour prior to incubation with test compounds.

(HiPSC-colony formation assay)
Human ESCs or hiPSCs were seeded in 6-well plates, allowed to grow for 72 hours, and then treated with the test compound for 72 hours. The cells were fixed with 4% paraformaldehyde for 1 hour, washed twice with PBS, and stained with 1% crystal violet solution (Sigma Aldrich) for 15 minutes. The plates were then washed 3-5 times with PBS and distilled water and allowed to air dry before taking images. For quantification, 1% SDS solution was added to each well and incubated for 15 minutes. The absorbance at 570 nm of the eluate was measured.

(Fluorescence microscope imaging of KB3-1 cell line and hiPSC colony)
KB3-1 and a KB3-1-derived cell line were seeded on a 96-well plate (Nunc 165305, Thermo Fisher Scientific) at 5000 cells per well. Test compounds (1 μM or 10 μM) were added 24 hours after cell seeding and the cells were incubated at 37 ° C. for 2 hours. The treated cells were washed with PBS and observed using a confocal microscope with a laser excitation wavelength of 405,488 and 561 nm in DMEM containing 10% FBS (Cell Voyager 1000, Yokogawa Electric Corporation). Quantitative analysis was performed by measuring fluorescence intensity and analyzing using NIH ImageJ, version 1.30. hiPSCs (clone # 201B7) were seeded on SNL feeder cells in a μ-dish (height 35 mm, ibidi). After 6 days of incubation, donut-like colonies of iPS cells were obtained. Colonies were incubated with KP-1 (1 μM), complex 17 (1 μM), or complex 18 (10 μM) at 37 ° C. for 2 hours. Treated cells were washed with PBS and observed in ES primate medium containing 4 ng / mL bFGF. The fluorescence imaging was taken using a confocal microscope (Cell Voyager 1000, Yokogawa Electric Corporation) with laser excitation wavelengths of 405 and 488 and 561 nm.

(Tubulin polymerization assay)
Tubulin polymerization assay kit was purchased from Cytoskeleton (BK006P) and assayed according to manufacturer's instructions. The final concentration of each component was PIPES (pH 6.9) 80 mM, MgCl _{2 2} mM, EGTA 0.5 mM, glycerol 10.2%, GTP 1 mM, and tubulin 3 mg / mL. The reaction system was pre-warmed to 37 ° C. without tubulin and then the reaction was initiated by the addition of tubulin. Absorption at 340 nm was measured using SpectraMax M5 (Molecular Devices) immediately after the addition of tubulin, and thereafter for 60 minutes at 1 minute intervals. CA4 and taxol were used as positive controls and molecule 14 and DMSO alone were used as negative controls.

(Alkaline phosphatase assay)
The alkaline phosphatase activity of hiPSCs was measured using an alkaline phosphatase substrate kit (SK-5300, Vector (registered trademark) blue). hiPSC colonies and partially differentiated hiPSCs were prepared as described above. Cells were then treated with SK-5300, incubated for 1 hour at room temperature, washed and finally maintained in PBS buffer.

(Quantitative polymerase chain reaction (QPCR))
Partially differentiated hiPSCs were prepared by incubating with all-trans retinoic acid (0.5 μM) for 48 hours and then treated with test compounds for 72 hours. Total mRNA was isolated using ISOGEN (Nippon Gene Co., Ltd.), and cDNA was synthesized using PrimeScript (Takara Shuzo Co., Ltd.). QPCR was performed on a 7500 Fast real-time PCR system (Applied Biosystems) using a high-speed SYBR (registered trademark) Green master mix. The primer sequences are shown in Table 1 below.

(Topoisomerase I inhibition assay)
The inhibitory activity of complex 17 topoisomerase I was tested by the supercoiled DNA relaxation assay. Recombinant human topoisomerase I (wild type protein, TopoGEN) pre-mixed with supercoiled DNA (TopoGEN) with complex 17 (0, 1, or 10 μM) or SN38 (0, 1, or 10 μM) as a control ) At 37 ° C. for 30 minutes. The reaction was terminated with stop buffer (TopoGEN) and the reaction mixture was loaded onto a 1% agarose gel. After electrophoresis, the gel was stained with ethidium bromide in TAE buffer and photographed under UV illumination.

(Removal of complex 17)
Partially differentiated hiPSCs were prepared by incubating with all-trans retinoic acid (0.5 μM) for 48 hours and then treated with complex 17 (5 μM) for 72 hours. Three days after cell treatment with Complex 17, the medium was collected. The cells were washed 3 times with 2 ml of fresh ES medium and the washed medium was collected. The obtained culture medium sample was centrifuged and analyzed for fluorescence intensity (excitation wavelength: 528 nm).

(Synthesis of compounds 1 to 15)

Example 1 Synthesis of Compounds 1-8

Compound 1-8 was synthesized according to the following method. A suspension of 3-aminophenol (3.0 equivalents) and substituted benzaldehyde (1.0 equivalents) in methanesulfonic acid was stirred at 130 ° C. for 24 hours. The reaction mixture was then cooled to ambient temperature, diluted with ice water, adjusted to about pH 6 with K ₂ CO ₃ and filtered. The precipitate was washed with water, dissolved in methanol and concentrated under reduced pressure. The residue was purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0).

Compound 1. Purple oil. Yield: 0.5%. 1H-NMR (300 MHz, CD3OD) δ 7.72-7.64 (m, 1H), 7.44-7.39 (m, 1H), 7.29-7.24 (m, 4H), 6 .86 (dd, J = 9.1, 2.2 Hz, 2H), 6.82 (d, J = 2.2 Hz, 2H); ESIMS m / z = 305 [M] +

Compound 2. Purple oil. Yield: 0.6%. 1H-NMR (300 MHz, CD3OD) δ 7.76-7.69 (m, 1H), 7.51-7.40 (m, 3H), 7.23 (dd, J = 6.3, 1. 1 Hz, 2H), 6.87 (dd, J = 9.1, 2.2 Hz, 2H), 6.83 (d, J = 2.2 Hz, 2H); ESIMS m / z = 305 [M ] +

Compound 3. Purple oil. Yield: 1.8%. 1H-NMR (300 MHz, CD3OD) δ 7.68 (dd, J = 5.5, 1.9 Hz, 2H), 7.46 (dd, J = 6.5, 2.1 Hz, 2H), 7.28 (d, J = 9.1 Hz, 2H), 6.87 (dd, J = 9.1, 2.2 Hz, 2H), 6.82 (d, J = 9.1 Hz, 2H) ); ESIMS m / z = 321 [M] +

Compound 4. Purple oil. Yield: 1.5%. 1H-NMR (300 MHz, CD3OD) δ 7.84 (dd, J = 6.6, 1.9 Hz, 2H), 7.39 (dd, J = 6.6, 1.9 Hz, 2H), 7.28 (d, J = 9.1 Hz, 2H), 6.86 (dd, J = 9.1, 2.2 Hz, 2H), 6.81 (d, J = 2.2 Hz, 2H) ); ESIMS m / z = 365 [M] +

Compound 5. Purple oil. Yield: 1.3%. 1H-NMR (300 MHz, CD3OD) δ 7.67-7.64 (m, 3H), 7.47-7.44 (m, 2H), 7.29 (d, J = 9.1 Hz, 2H) ), 6.86 (dd, J = 9.1, 2.2 Hz, 2H), 6.82 (d, J = 1.9 Hz, 2H); ESIMS m / z = 287 [M] +

Compound 6. Purple oil. Yield: 0.7%. 1H-NMR (300 MHz, CD3OD) δ 7.49 (d, J = 8.3 Hz, 2H), 7.38 (dd, J = 8.3, 1.9 Hz, 2H), 7.29 ( d, J = 9.1 Hz, 2H), 6.85 (dd, J = 9.1, 1.9 Hz, 2H), 6.82 (d, J = 2.2 Hz, 2H), 2. 76 (t, J = 7.7 Hz, 2H), 1.82-1.70 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H); ESIMS m / z = 329 [ M] +

Compound 7. Purple oil. Yield: 0.2%. 1H-NMR (300 MHz, CD3OD) δ 7.44 (d, J = 9.1 Hz, 2H), 7.31 (dd, J = 6.6, 2.2 Hz, 2H), 7.05 ( dd, J = 6.6, 1.9 Hz, 2H), 6.86 (dd, J = 9.1, 1.9 Hz, 2H), 6.80 (d, J = 2.2 Hz, 2H) ); ESIMS m / z = 303 [M] +

Compound 8. Purple oil. Yield: 0.2%. 1H-NMR (300 MHz, CD3OD) δ 7.42 (d, J = 8.7 Hz, 2H), 7.31 (dd, J = 6.6, 1.9 Hz, 2H), 7.05 ( dd, J = 6.6, 1.9 Hz, 2H), 6.85 (dd, J = 9.1, 2.2 Hz, 2H), 6.78 (d, J = 2.2 Hz, 2H) ); ESIMS m / z = 303 [M] +

Example 2 Synthesis of Compounds 9 and 10

A suspension of 5-fluoro-1,3-isobenzofurandione (498 mg, 3 mmol) and 3-aminophenol (982 mg, 9 mmol) in methanesulfonic acid (9 mL) was added at 130 ° C. For 24 hours. After cooling to ambient temperature, methanol (25 mL) was added to the reaction and refluxed overnight. The reaction mixture was then cooled to ambient temperature, diluted with ice water, adjusted to about pH 6 with K ₂ CO ₃ and filtered. The precipitate was washed with water, dissolved in methanol, and concentrated under reduced pressure. The residue was purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0), 0.9 mg of compound 9 and purple 0.8 mg of compound 10 was obtained as an oil.

Compound 9. Purple oil. Yield: 0.8%. 1H-NMR (300 MHz, CD3OD) δ 8.06 (dd, J = 9.6, 2.1 Hz, 1H), 7.66 (ddd, J = 8.1, 8.1, 2.8 Hz) , 1H), 7.49 (dd, J = 8.9, 5.5 Hz, 1H), 7.22 (d, J = 8.9 Hz, 2H), 7.15 (d, J = 2. 1 Hz, 1H), 6.99 (dd, J = 9.0, 2.0 Hz, 1H), 6.96 (dd, J = 9.6, 2.0 Hz, 1H), 6.92 ( d, J = 2.0 Hz, 2H), 3.65 (s, 3H); ESIMS m / z = 363 [M] +

Compound 10. Purple oil. Yield: 0.7%. 1H-NMR (300 MHz, CD3OD) δ 8.40 (dd, J = 8.9, 5.5 Hz, 1H), 7.57 (ddd, J = 8.3, 8.3, 2.8 Hz) , 1H), 7.32 (dd, J = 8.3, 2.8 Hz, 1H), 7.23 (d, J = 9.6 Hz, 2H), 7.16 (d, J = 2. 1 Hz, 1H), 6.99 (dd, J = 9.6, 2.1 Hz, 1H), 6.97 (dd, J = 9.6, 2.1 Hz, 1H), 6.92 ( d, J = 2.1 Hz, 1H), 3.62 (s, 3H); ESIMS m / z = 363 [M] +

Example 3 Synthesis of Compound 11

A suspension of resorcinol (1.32 g, 12 mmol) and 4-fluorobenzaldehyde (422 μL, 4 mmol) in methanesulfonic acid (12 mL) was stirred at 130 ° C. for 24 hours. The reaction mixture was then cooled to ambient temperature, diluted with ice water, adjusted to about pH 6 with K ₂ CO ₃ and filtered. The precipitate was washed with water, dissolved in methanol and concentrated under reduced pressure. The residue was purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0) to give 3 mg of compound 11 as a purple oil. Obtained. Yield: 0.1%.

1H-NMR (300 MHz, CD3OD) δ 7.70-7.61 (m, 4H); 7.51-7.48 (m, 2H), 7.24-7.15 (m, 4H); ESIMS m / z = 307 [M + H] +

Example 4 Synthesis of Compound 12

A suspension of 3- (methylamino) phenol (411 mg, 3.3 mmol) and 4-fluorobenzaldehyde (176 μL, 1.67 mmol) in methanesulfonic acid (11 mL) was stirred at 130 ° C. for 24 hours. The reaction mixture was then cooled to ambient temperature, diluted with ice water, adjusted to about pH 6 with K ₂ CO _{3 and} filtered. The precipitate was washed with water, dissolved in methanol, and concentrated under reduced pressure. The residue was purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0) to give 23 mg of compound 12 as a purple oil. Obtained. Yield: 3.1%

1H-NMR (300 MHz, CD3OD) δ 7.48-7.36 (m, 2H); 7.21 (d, J = 9.1 Hz, 2H), 7.04-6.90 (m, 2H) ), 6.83 (dd, J = 9.3, 1.7 Hz, 2H), 6.69 (s, 2H), 2.99 (s, 6H); ESIMS m / z = 333 [M] +

Example 5 Synthesis of Compound 13

3-((3-aminopropyl) amino) phenol hydrobromide (2.5 g, 10 mmol) (Firmino, ADG; Goncalves, MS Tetrahedron Lett. 2012, 53, 4946 .), 3-aminophenol (1.1 g, 10 mmol), and 4-fluorobenzaldehyde (710 μL, 6.7 mmol) in methanesulfonic acid (25 mL) were stirred at 130 ° C. for 24 hours. The reaction mixture was then cooled to ambient temperature, diluted with ice water, adjusted to about pH 6 with K ₂ CO ₃ and filtered. The precipitate was washed with water, dissolved in methanol, and concentrated under reduced pressure. The residue was purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0) to give 150 mg of compound 13 as a purple oil. Obtained. Yield: 4.7%.

1H-NMR (300 MHz, CD3OD) δ 7.53-7.48 (m, 2H); 7.45-7.39 (m, 2H), 7.31 (dd, J = 9.1, 2. 2 Hz, 2H), 6.93-6.90 (m, 2H), 6.86 (dd, J = 10.4, 2.2 Hz, 2H), 3.51 (t, J = 7.1) Hz, 2H), 3.09 (t, J = 7.7 Hz, 2H), 2.12-2.00 (m, 2H); ESIMS m / z = 362 [M] +

Example 6 Synthesis of Compound 14

To a solution of compound 13 (20 mg, 42 μmol) in dry DMF (600 μL), 2-methoxyacetic acid (3.8 mg, 42 μmol), HATU (19 mg, 54 μmol), HOBT (7 mg, 54 μmol), and DIPEA (20 mg, 155 μmol) were added. Added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was diluted with methanol and purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0). 8.8 mg of compound 14 was obtained as an oil. Yield: 38.3%.

1H-NMR (300 MHz, CD3OD) δ 7.52-7.48 (m, 2H); 7.44-7.38 (m, 2H), 7.29 (dd, J = 9.1, 1. 1 Hz, 2H), 6.88-6.82 (m, 4H), 3.89 (s, 2H), 3.44-3.33 (m, 7H), 1.87-1.97 (m , 2H); ESIMS m / z = 434 [M] +

Example 7 Synthesis of Compound 15

Compound 15 was synthesized by the method already reported (Hirata, N. et al., M. Cell Rep (2014) 6, 1165.). Purple oil. Yield: 2.4%.

1H-NMR (600 MHz, acetic acid-d4) δ 7.51 (dd, J = 8.9, 5.5 Hz, 2H), 7.40 (dd, J = 8.9, 8.9 Hz, 2H), 7.31 (d, J = 8.9 Hz, 2H), 6.98-6.93 (m, 4H), 3.40 (m, 2H), 3.04 (m, 2H), 1.74-1.67 (m, 2H), 1.44-1.32 (m, 14H); ESIMS m / z = 460 [M] +

(Synthesis of complexes 16-18)

Example 8 Synthesis of Complex 16

To a solution of compound 13 (18 mg, 37 μmol) in dry DMF (600 μl), C16-1 (Kamal, A .; Mallareddy, A .; Janaki Ramaiah, M .; Pushpavalli, SN; Suresh, P .; Kishor, Raty, NS; Ghosh, S .; Addlagata, A .; Pal-Bhadra, M. Eur. J. Med. Chem. 2012, 56, 166.) (14 mg 37 μmol), HATU (16 mg, 41 μmol), HOBT (6 mg, 41 μmol), and DIPEA (16 mg, 124 μmol) were added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was diluted with methanol and purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0). 14 mg of complex 16 was obtained as an oil. Yield: 44.4%.

1H-NMR (300 MHz, CD3OD) δ 7.50-7.37 (m, 4H), 7.27 (d, J = 9.1 Hz, 1H), 7.24-7.15 (m, 1H) 6.92 (s, 2H), 6.89-6.82 (m, 3H), 6.78 (dd, J = 8.2, 2.2 Hz, 2H), 6.50-6.43. (M, 4H), 4.35 (s, 2H), 3.83 (s, 3H), 3.69 (s, 3H), 3.59 (s, 6H), 3.41-3.34 ( m, 4H), 1.94-1.89 (m, 2H); ESIMS m / z = 718 [M] +; HRMS (FAB) calcd for [M] + m / z = 718.923, found 718. 2946.

Example 9 Synthesis of Complex 17

A suspension of SN38 (30 mg, 76 μmol), tert-butylchloroacetic acid (46 mg, 307 mmol), and DIPEA (65 mg, 504 μmol) in DMF (1.5 mL) was stirred at 70 ° C. for 48 hours. The solvent was removed in vacuo and the residue was purified by flash column (DCM: methanol = 20: 1) to give 30 mg of C17-1 as a yellow solid. Trifluoroacetic acid (500 μl) was added dropwise to a solution of C17-1 (11 mg, 22 μmol) in DCM (500 μL). The reaction mixture was stirred at room temperature overnight then diluted with methanol and purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0), 5.5 mg of C17-2 was obtained as a yellow solid. 2-step yield: 43.3%.

1H-NMR (300 MHz, CD3OD) δ 7.9 (d, J = 9.1 Hz, 1H), 7.54 (s, 1H), 7.49 (dd, J = 9.4, 2.8) Hz, 1H), 7.34 (d, J = 2.5 Hz, 1H), 5.55 (d, J = 16 Hz, 1H), 5.35 (d, J = 16 Hz, 1H), 5 .16 (s, 2H), 4.86 (s, 2H), 3.14 (q, J = 7.7 Hz, 2H), 1.94 (q, J = 7.1 Hz, 2H), 1 .36 (t, J = 7.4 Hz, 3H), 1.00 (t, J = 7.4 Hz, 3H); ESIMS m / z = 451 [M + H] +

To a solution of compound 13 (10 mg, 21 μmol) in dry DMF (600 μl), C17-2 (12 mg, 21 μmol), HATU (8.8 mg, 23 μmol), HOBT (4 mg, 23 μmol), and DIPEA (13.5 mg, 105 μmol) Was added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was diluted with methanol and purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0). 5 mg of complex 17 was obtained as an oil. Yield: 26.2%.

1H-NMR (300 MHz, CD3OD) δ 8.03 (d, J = 9.1 Hz, 1H), 7.57 (d, J = 9.1 Hz, 2H), 7.38 (brs, 4H) , 7.28 (s, 2H), 7.09 (d, J = 9.3 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.55 (d, J = 1.7 Hz, 2H), 5.42 (brs, 1H), 5.26 (brs, 1H), 5.16-5.05 (m, 2H), 4.83 (s, 2H), 3. 64 (brs, 2H), 3.13-2.85 (m, 6H), 1.93 (brs, 4H), 1.38 (t, J = 7.4 Hz, 3H), 0.89 (brs) , 3H); ESIMS m / z = 794 [M] +; HRMS (FAB) calcd for [M] + m / z = 794.2985, fou d 794.2962.

Example 10 Synthesis of Complex 18

To a solution of compound 13 (30 mg, 63 μmol) in dry DMF (600 μL), chloroacetic acid (6 mg, 63 μmol), HATU (26.3 mg, 69 μmol), HOBT (9.4 mg, 71 μmol), and DIPEA (26.7 mg, 207 μmol). ) Was added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was diluted with methanol and purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0). 12 mg of C18-1 was obtained as an oil. Yield: 34.6%.

1H-NMR (300 MHz, CD3OD) δ 7.56-7.48 (m, 2H), 7.44-7.38 (m, 2H), 7.29 (dd, J = 9.1, 2. 5 Hz, 2H), 6.89-6.82 (m, 4H), 4.06 (s, 2H), 3.49-3.32 (m, 4H), 1.95-1.91 (m , 2H); ESIMS m / z = 438 [M] +

A suspension of C18-1 (12 mg, 22 μmol), mitoxantrone dihydrochloride (22.8 mg, 44 μmol), and DIPEA (56.4 mg, 440 μmol) in DMF (300 μL) was stirred at 40 ° C. for 24 hours. The reaction mixture was then cooled to ambient temperature, diluted with methanol and purified by HPLC (Inertsil ODS-3, methanol: 0.1% (v / v), TFA-H ₂ O = 0: 1 to 1: 0), 9.9 mg of complex 18 was obtained as a dark purple oil. Yield: 46.9%.

1H-NMR (300 MHz, CD3OD) δ 7.48-7.35 (m, 6H), 7.16-6.94 (m, 4H), 6.74-6.66 (m, 2H), 6 .49 (s, 1H), 6.42 (s, 1H), 4.13 (s, 2H), 3.97 (s, 2H), 3.92 (brt, J = 4.7 Hz, 2H) 3.82-3.79 (m, 6H), 3.72 (brs, 2H), 3.53 (brs, 2H), 3.47 (brs, 2H), 3.38 (brs, 2H), 1.93 (brs, 2H); ESIMS m / z = 846 [M] +; HRMS (FAB) calcd for [M] + m / z = 846.33621, found 846.3611.

Example 11 Comparison of Transporter Selectivity of Cytotoxic Anticancer Agents As cytotoxic anticancer agents, SN38, Camptothecin, 9-Aminocamptothecin, 9-nitrocamptothecin, 7-ethylcamptothecin, Doxorubicinine, Mitoxanthropnectin C, 5-FUdR, Dactinomycin, Taxol, and Vinblastin were used. The above (cell-based assay) was performed using KB3-1 and KB3-1-derived cells (KB / ABCB1, KB / ABCC1, and KB / ABCG2 cells), and IC50 values of each compound were determined (n ≧ 2) .

The results are shown in Table 2. KB3-1 represents a control without drug resistance. None of the cells showed resistance to 7-ethylcamptothecin and Mitomycin C. For Doxorubicin, Dactinomycin, Taxol, and Vinblastin, KB / ABCB1 showed resistance. SN38, 9-Aminocamptothecin, 9-nitrocamptothecin, and 5-FUdR were resistant to KB / ABCG2. Camptothecin and Etoposide were resistant to KB / ABCB1 and KB / ABCC1. Mitoxantrone was resistant to KB / ABCC1 and KB / ABCG2. No compound was found that B / ABCB1 and KB / ABCG2 are resistant to.

(Example 12) Structure-activity relationship of KP-1 In order to examine the sites capable of binding in KP-1, the structure-activity relationship was examined. First, 15 kinds of KP-1 derivatives having different substituents on the benzene “head” and xanthone “arm” of KP-1 were synthesized according to the above-described method (compounds 1 to 15), and their fluorescence spectra (λ _ex and λ _em ) was measured. Subsequently, the selectivity of compounds 1 to 15 for ABC transporter was evaluated by staining patterns of four types of cell lines (KB3-1, KB / ABCB1, KB / ABCC1, and KB / ABCG2).

The fluorescence spectra of compounds 1 to 15 are shown in Table 3. In addition, the selectivity of compounds 1 to 15 for ABC transporter is shown in FIG. The KP-1 derivatives excluding Compound 9 and Compound 11 clearly stained the parent KB3-1 cells and hardly stained the KB / ABCB1 cells. This indicates that the modification of benzene “head” and xanthone “arm” does not impair the selectivity of KP-1 for ABCB1. The selectivity for ABCG2 was more sensitive to structural transformation compared to ABCB1, and was particularly affected by substituents on the benzene “head”. Compound 1 and Compound 2 did not show ABCG2-mediated efflux such as KP-1 and showed that the para substituent was important for ABCG2 selectivity (Cl) (compound 3), By substituting the fluorine atom (F) with a bromine atom (Br) (compound 4), a hydrogen atom (H) (compound 5), a propyl group (compound 6), or a hydroxyl group (compound 7), the selectivity for ABCG2 is improved. Decreased to various degrees. Modification of the amine “arm” had a lesser effect on selectivity. Similarly, KP-1, Compound 12, and Compound 14 showed excellent selectivity for both ABCB1 and ABCG2, and low selectivity for ABCC1. Compound 14 was selected for binding with cytotoxic anticancer agents.

(Example 13) Cytotoxicity and ABC transporter selectivity of complex 16 A conjugate (complex 16) of compound 14 and combretastatin A-4 (CA4), which is a cytotoxic anticancer agent, Whether or not the ABC transporter selectivity of KP-1 was maintained was examined by binding compound 14 and CA4. This naturally occurring tubulin inhibitor is highly effective against cell lines that highly express ABCB1 and ABCG2, indicating that CA4 is not a substrate for either ABCB1 or ABCG2. (LM Greene et al., J Pharmacol Exp Ther (2010) 335: 302-313). The structure of compound 16 is shown in FIG. 2A. In order to confirm the ability of Compound 16 to inhibit tubulin polymerization, a tubulin polymerization assay was performed. In addition, in order to examine the ABC transporter selectivity of the complex 16, two types of cell-based assays, a cell viability assay and a fluorescence imaging assay, were performed simultaneously.

In the tubulin polymerization assay, CA4 inhibited tubulin polymerization, whereas compound 14 did not show detectable inhibition of tubulin polymerization at concentrations up to 30 μM. In contrast to compound 14, complex 16 inhibited tubulin polymerization.

In the cell viability assay, complex 16 showed significantly superior cytotoxicity than compound 14. This suggests that the toxicity of complex 16 is due to the CA4 moiety. Although the complex 16 maintained the property as a substrate for ABCB1, it could not maintain the selectivity for ABCG2 (FIG. 2B). The results of the fluorescence imaging assay were consistent with the results of the cell viability assay (FIG. 2C). Complex 16 stained KB / ABCG2 cells, but was excreted from KB / ABCB1 cells. From these results, it was suggested that the complex of compound 14 and CA4 maintains selectivity for ABCB1, but does not maintain selectivity for ABCG2.

Similarly, a cell viability assay and a fluorescence imaging assay were performed by synthesizing a complex of four types of cytotoxic anticancer agents other than CA4 and compound 14, and the result was the same as that of complex 16. .

Example 14 Cytotoxicity and ABC Transporter Selectivity of Complex 17 ABCG2 is generally thought to have a narrower efflux substrate profile than ABCB1 (JJ Strouse et al., Anal Biochem (2013)). 437: 77-87). By using an ABCG2 selective cytotoxic agent instead of CA4 in Example 13, it was examined whether the resulting complex would be a substrate for both ABCB1 and ABCG2. Several cytotoxic anticancer agents have been reported as substrates for ABCG2 (L. Doyle et al., Oncogene (2003) 22: 7340-7358). The anticancer agent SN38 having a long use calendar was selected from these, and the complex 17 was synthesized (FIG. 3A). To examine the ABC transporter selectivity of Complex 17, two cell-based assays, a cell viability assay and a fluorescence imaging assay, were performed simultaneously.

In the cell viability assay, as previously reported, KB / ABCG2 cells showed resistance to SN38, but SN38 showed strong cytotoxicity in KB / ABCB1 and KB / ABCC1 cells. Complex 17 was slightly less cytotoxic than SN38, but resistance was confirmed in both KB / ABCB1 and KB / ABCG2 cells (FIG. 3B).

As with KP-1, the fluorescence intensity of complex 17 was significantly lower in KB / ABCB1 cells and KB / ABCG2 cells than in KB3-1 cells. Fluorescence was restored by treatment with a selective inhibitor of ABCB1 (cyclosporin A, 10 μM) or a selective inhibitor of ABCG2 (Ko143, 10 μM) (FIG. 3C). These results indicate that complex 17 is an excellent cytotoxic substrate for both ABCB1 and ABCG2.

Example 15: Cytotoxicity and ABC transporter selectivity of complex 18 Further verify that the combination of Compound 14 and ABCG2 selective cytotoxic agent can be a cytotoxic substrate for both ABCB1 and ABCG2 Therefore, Compound 14 (FIG. 5A) was synthesized by combining compound 14 with another ABCG2-selective cytotoxic agent mitoxantrone. To examine the ABC transporter selectivity of Complex 18, two cell-based assays, a cell viability assay and a fluorescence imaging assay, were performed simultaneously.

As a result of cell viability and fluorescence image analysis, it was shown that mitoxantrone has excellent selectivity for ABCC1 and ABCG2, but low selectivity for ABCB1. In contrast, complex 18 was a substrate for both ABCB1 and ABCG2, but not ABCC1 (FIG. 4B, C).

(Example 16) Undifferentiated cell labeling ability and removal ability of complex 17 and complex 18 It was confirmed whether complex 17 and complex 18 can label and eliminate hiPSCs in the cell mixture. When hiPSCs overgrow, cells form donut-shaped colonies. The central part of the colony represents differentiated cells (N. Hirata et al., Cell Rep (2014) 6: 1165-1174). First, hiPSCs in which donut-shaped colonies were formed were treated with the complex 17 (1 μM) or the complex 18 (10 μM), and observed immediately afterwards with a confocal microscope.

Next, hiPSCs or hiPSCs partially differentiated on SNL feeder cells were cultured for 72 hours in the presence of complex 17 (5 μM) or complex 18 (5 μM). Undifferentiated hiPSCs were detected by colorimetric staining for alkaline phosphatase (ALP) activity which is a pluripotency marker (MD O'Connor et al., Stem Cells (2008) 26: 1109-1116). In order to confirm the removal of undifferentiated cells, three types of representative pluripotency markers (J. Cai et al., Stem Cells (2006) 24: 516-) in cells cultured in the presence of each complex or DMSO control. 530; M. Stadtfeld et al., Genes Dev (2010) 24: 2239-2263) was measured by quantitative polymerase chain reaction (qPCR). The relative expression levels of nanog, sox2, and oct3 / 4 were normalized by the housekeeping gene GAPDH.

Similar to KP-1, Complex 17 and Complex 18 fluorescently labeled undifferentiated cells more strongly than differentiated cells (FIG. 5A). In addition, ALP-positive cells were removed by culturing in the presence of complex 17 or complex 18, but ALP-negative cells were hardly affected (FIG. 5B). hiPSCs were removed from the cell mixture. The cell mixture treated with DMSO alone expressed relatively high nanog (0.7%), oct3 / 4 (6.7%), and sox2 (0.4%). Culture in the presence of Complex 17 or Complex 18 reduces nanog expression level to 0.1%, oct3 / 4 expression level to 0.8%, and sox2 expression level to 0.03% (FIGS. 5C, 5D, 5E), consistent with selective removal of pluripotent cells.

(Example 17) The ability of complex 17 to remove differentiated cells and undifferentiated cells The effect of complex 17 on five different human primary cells compared to two kinds of hiPSC clones, 201B7 and 253G1, was compared. In addition, in order to examine whether SN38 itself is selective for hiPSCs, the effects of SN38 alone and complex 17 on various human primary cells and hiPSCs were compared.

Hepatocytes (metabolic function), prostate epithelium (genital organs), brain microvascular cells (barrier function), astrocytes (central nervous system), and adrenal microvascular cells (secretory function) are resistant to complex 17 ( IC50> 10 μM), but the two hiPSC cell lines reacted with high sensitivity. (201B7: IC50 = 0.2 μM; 253G1: IC50 = 0.4 μM) (FIG. 5F). Addition of cyclosporine (ABCB1 selective inhibitor) or Ko143 (ABCG2 selective inhibitor) increased the cytotoxicity of complex 17 against somatic cells that showed resistance (FIG. 6). Addition of a transporter inhibitor to the medium restored the cytotoxicity of complex 17 to human primary somatic cells, but this cytotoxicity was not complete. This suggests that Complex 17 exhibits selective cytotoxicity by a mechanism other than ABC transporter-mediated excretion. It is known to express high levels of ABC transporters (N. Hirata et al., Cell Rep (2014) 6: 1165-1174), hepatocytes, prostate epithelial cells, and brain microvascular cells are either cyclosporine or Ko143. The resistance was maintained even in the presence of (Fig. 6).

SN38 showed mild selectivity for hiPSCs, but not as much as complex 17 (FIG. 7). An SN38 dose of 1 μM almost completely eliminated iPS cells (viability <5%) and more than 70% of human primary cells. In contrast, complex 17 showed a weaker effect on human primary cells than SN38 even at 10 μM. These results suggest that SN38 is originally selective for hiPSCs, but the selectivity is enhanced by binding to KP-1.

(Example 18) TopoI inhibitory activity of complex 17 The selectivity inherently of SN38 may be derived from its mechanism of action. (C.P. Garcia et al., Stem Cell Res (2014) 12: 400-414; O. Momcilovic, PLoS One (2010) 5: e13410) SN38 exhibits antiproliferative activity by inhibiting poisomerase I (TopoI). (BL Staker et al., Proc Natl Acad Sci USA (2002) 99: 15387-15392). High growth rates are considered important for PSCs pluripotency and self-renewal (S. Ruiz et al., Curr Biol (2011) 21: 45-52). In contrast, differentiated cells usually show a low growth rate, which may possibly confer some resistance to TopoI inhibitors. To examine whether Complex 17 inhibits TopoI, an in vitro biochemical assay was performed using recombinant human topoisomerase I.

The results are shown in FIG. SN38 inhibited TopoI at 10 μM. However, Complex 17 inhibited TopoI more strongly.

(Example 19) TopoI inhibitory activity of complex 17 After the undifferentiated cells that are likely to cause tumors are removed by the complex, it is necessary to remove the complex from the cell sample before transplantation. Using the fluorescence characteristics, the remaining amount of the complex 17 was monitored.

The culture medium containing 5 μM complex 17 showed strong fluorescence at 548 nm. After simple washing with fresh medium, the fluorescence intensity decreased significantly to about 2%. Furthermore, a simple wash again reduced the fluorescence to the background level (medium only). The fluorescence of conjugate 17 or other KP-1-drug conjugates will be advantageous for future applications by ensuring clearance of cytotoxic reagents in the resulting cell sample.

Claims (17)

1. Compound represented by the following formula (I)
   

   

   
   [Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
   Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
   R ² and R ³ are the same or different and are each a hydrogen atom or a C1-6 alkyl group,
   X is an oxygen atom, a sulfur atom, or NR ⁷ ;
   Here, R ⁷ is a hydrogen atom or a C1-6 alkyl group,
   L represents a linker moiety represented by — (CH ₂ ) _k (NH) _m (CO) _n (CH ₂ ) _p —,
   Here, k represents an integer of 3 to 7, m is 0 or 1, n is 0 or 1, p is 0 or 1,
   D represents ABCG2 selective cytotoxic substance;
   Here, ABCG2 selective cytotoxic agent, SN38, Mitoxantrone, Irinotecan, 9-Aminocamptothecin, Doxorubicin, Daunorubicin, Epirubicin, Idarubicinol, Flavopiridol, CI1033, BBR3390, Methotrexate, Prazocin, Indolocarbazole, a topoisomerase I inhibitor NB- 506 and J-107088, Zidovudine (AZT), and lamivudine].
2. The compound of Claim 1 represented by following formula (II)
   

   

   
   [Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
   Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
   L represents a linker moiety represented by — (CH ₂ ) _k (NH) _m (CO) _n (CH ₂ ) _p —,
   Here, k represents an integer of 3 to 7, m is 0 or 1, n is 0 or 1, p is 0 or 1,
   D represents ABCG2 selective cytotoxic substance;
   Here, ABCG2 selective cytotoxic agent, SN38, Mitoxantrone, Irinotecan, 9-Aminocamptothecin, Doxorubicin, Daunorubicin, Epirubicin, Idarubicinol, Flavopiridol, CI1033, BBR3390, Methotrexate, Prazocin, Indolocarbazole, a topoisomerase I inhibitor NB- 506 and J-107088, Zidovudine (AZT), and lamivudine].
3. The compound according to claim 1 or 2, wherein R ¹ is a halogen atom or a -OP (O) (OR ⁵ ) (OR ⁶ ) group.
4. L represents a linker moiety represented by-(CH ₂ ) _k (NH) (CO) (CH ₂ )-, wherein k represents an integer of 3 to 5. The compound according to any one of the above.
5. The compound according to any one of claims 1 to 4, wherein D is an ABCG2-selective cytotoxic substance that is a topoisomerase inhibitor.
6. 6. The compound according to any one of claims 1 to 5, wherein D is SN38 or Mitoxantrone.
7. A compound represented by any one of the following formulae.
   

   

   
   

   
8. Compound represented by the following formula (Ia) or (Ib)
   

   

   
   

   

   
   [Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
   Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
   R ² and R ³ are the same or different and are each a hydrogen atom or a C1-6 alkyl group,
   X is an oxygen atom, a sulfur atom, or NR ⁷ ;
   Here, R ⁷ is a hydrogen atom or a C1-6 alkyl group,
   k represents an integer of 3 to 7.]
9. The compound according to claim 8, which is represented by the following formula (IIa) or (IIb):
   

   

   
   

   

   
   [Wherein R ¹ represents a hydrogen atom, a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, a phenyl group, a phenyloxy group, a hydroxyl group, or an —OP (O) (OR ⁵ ) (OR ⁶ ) group. And
   Here, R ⁵ and R ⁶ are the same or different and each represents a C1-6 alkyl group which may be substituted with a phenyl group, or R ⁵ and R ⁶ are not present,
   k represents an integer of 3 to 7.]
10. The compound according to claim 8 or 9, wherein R ¹ is a halogen atom or a -OP (O) (OR ⁵ ) (OR ⁶ ) group.
11. The compound according to any one of claims 8 to 10, wherein k represents an integer of 3 to 5.
12. A compound according to any one of claims 1 to 7, comprising reacting the compound according to any one of claims 8 to 11 with an ABCG2-selective cytotoxic substance. a synthetic method, wherein, ABCG2 selective cytotoxic substance, SN38, Mitoxantrone, Irinotecan, 9-aminocamptothecin, Doxorubicin, Daunorubicin, Epirubicin, Idarubicinol, Flavopiridol, CI1033, BBR3390, Methotrexate, Prazocin, Indolocarbazole, topoisomerase I Inhibitors NB-506 and J-107088, Zidovudine (AZT), and la It is a material selected from Ivudine, method.
13. The cell population induced to differentiate stem cells is contacted with the compound according to any one of claims 1 to 7 for a period of time during which undifferentiated cells die, and any one of claims 1 to 7 which remains. A method for removing undifferentiated cells remaining after differentiation induction of stem cells, comprising removing the compound according to item 1 by washing.
14. The removal method according to claim 13, wherein the stem cells are iPS cells.
15. A drug which selectively damages cells in which the expression of ABCB1 and ABCG2 transporters is suppressed, comprising the compound according to any one of claims 1 to 7 as an active ingredient.
16. The drug according to claim 15, wherein the cells in which expression of ABCB1 and ABCG2 transporter is suppressed are undifferentiated cells.
17. A kit for selectively damaging a cell in which expression of ABCB1 and ABCG2 transporter is suppressed, comprising the compound according to any one of claims 1 to 7.

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