WO2018221479A1 - Novel fluorescent substance for transporter function analysis - Google Patents

Abstract

The purpose of the present invention is to provide a fluorescent substrate for a transporter, the fluorescent substrate maintaining high transport performance while being unlikely to be quenched and exhibiting strong fluorescent intensity. The present invention relates to a compound represented by formula (I). Formula (I): A-L-F [In the formula: A denotes cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, α-muricholic acid, β-muricholic acid, ω-muricholic acid, hyocholic acid, hyodeoxycholic acid, 7-oxo-deoxycholic acid, 7-oxo-lithocholic acid, 1-oxo-lithocholic acid, 2-oxo-lithocholic acid, or a derivative of these; L denotes a linker; and F denotes Tokyo Green (TG), Tokyo Magenta (TM), hydroxycoumarin (HC), or a derivative of these.]

Description

New transporter functional analysis phosphor

The present invention relates to a fluorescent substrate used for functional analysis of a transporter.

In drug discovery, it is important to evaluate transporters responsible for transporting developed drugs into and out of cells in order to evaluate the kinetic properties, efficacy (drug efficacy) and safety of drugs. Following the EMA), the US Food and Drug Administration (FDA) issued guidance on transporter studies for developed drugs from the perspective of drug interactions. Transporters are important membrane transport proteins responsible for the uptake and excretion of various substances in cells, and govern the transport of drugs into and out of cells. Recent developments in post-genomic research have identified many transporters, and their functions and types of substrates have become clear. Based on such a background, development of a drug discovery screening system capable of more quickly and simply evaluating transporter transport ability for drugs is desired.

Currently, in screening systems using transporter substrates, methods using a substrate labeled with a radioisotope (RI) and methods for measuring a non-RI-labeled substrate by LC / MS / MS are the mainstream. These methods are excellent in that the transportability of the transporter can be quantified without changing the specificity of the substrate. However, due to the complexity of the measurement operation and the need for an expensive measurement device, a technique that replaces the conventional method is eagerly desired. As described above, although the transporter substrate is labeled with a fluorescent dye (fluorescent substrate), it is positioned as a tool for next-generation drug discovery screening systems because of its superiority in measurement procedures and simplification of measurement equipment. However, at present, fluorescently labeled substrates themselves are limited, and no promising drug discovery screening system has been established.

In addition, a fluorescent substrate in which chenodeoxycholic acid (CDCA) is labeled with nitrobenzooxadiazole (NBD) has been produced and reported (Non-Patent Document 1), but there are problems such as easy quenching and weak fluorescence. In addition, when used in high-throughput screening (HTS) or the like, a high S / N ratio is required, but no fluorescent substrate having an S / N ratio that can be used in HTS has been reported so far. .

Therefore, an object of the present invention is to provide a fluorescent substrate that is difficult to quench and has high fluorescence intensity while maintaining the high transport ability of the transporter substrate.

As a result of evaluating the transport activity of combinations of various fluorescent substances and substrates, the present inventors have found that a probe exhibiting a high S / N ratio can be created by imparting water solubility to the fluorescent substrate. In addition, a fluorescent substrate in which various bile acids and Tokyo Green (TG), Tokyo Magenta (TM), or hydroxycoumarin (HC), or derivatives thereof are bonded via a linker is quenched while maintaining high transport ability. It has been found that it becomes a fluorescent substrate of a transporter that is difficult and has high fluorescence intensity. For example, the S / N ratio at the time of saturation of CDCA-NBD uptake (after 10 minutes), that is, (uptake by transporter expressing cells) / (uptake by transporter non-expressing cells (mock cells)) In contrast to 2.5, TG is 5.7 and HC is 3.6, indicating an excellent S / N ratio, so that a fluorescent substrate that can be used in HTS has been successfully provided.

The present invention has been made on the basis of such findings. Therefore, the present invention relates to a compound represented by the following formula (I):
ALF (I)
[Wherein, A is cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, α-mulicholic acid, β-mulicholic acid, ω-mulicholic acid, hyocholic acid, hyodeoxycholic acid 7-oxo-deoxycholic acid, 7-oxo-lithocholic acid, 1-oxo-lithocholic acid, or 2-oxo-lithocholic acid, or derivatives thereof,
L represents a linker, and
F represents a monovalent group of a compound represented by the following formula:

[In the above formula, R ¹ , R ³ , R ⁴ , R ⁶ , R ⁹ , and R ¹¹ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonic acid group, a salt of a sulfonic acid group, C1˜ Represents a 6 alkyl group or a C1-6 alkenyl group,
R ² represents an oxygen atom or an optionally substituted nitrogen atom,
R ⁵ represents a hydroxyl group or an optionally substituted nitrogen atom,
R ⁷ represents an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, a carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
n represents a natural number from 0 to 4,
R ⁸ and R ¹² are each independently a hydroxyl group, a halogen atom, an amino group, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, or a C1-6 alkoxycarbonyl group. , A carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
R ¹⁰ represents a hydroxyl group or an optionally substituted nitrogen atom, and
X represents an oxygen atom or an optionally substituted silicon atom;
Here, the nitrogen atom which may be substituted and the substituent of the silicon atom which may be substituted are the same or different, and each may be a C1-6 alkyl group which may be substituted. The substituents of the C1-6 alkyl group which may be the same or different are each a halogen atom, a carboxylic acid, a carboxylic acid salt, a sulfonic acid, or a sulfonic acid salt.

The group represented by A in the compound represented by the formula (I) in the present specification represents a monovalent group of a compound taken up by OATP1B1 and / or OATP1B3, specifically, cholic acid, chenodeoxycholic acid, Deoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, α-mulicholic acid, β-mulicholic acid, ω-mulicholic acid, hyocholic acid, hyodeoxycholic acid, 7-oxo-deoxycholic acid, 7-oxo- It means a monovalent group of lithocholic acid, 1-oxo-lithocholic acid, and 2-oxo-lithocholic acid (hereinafter referred to as “cholic acid and the like”) or derivatives thereof. A “derivative” such as cholic acid is a compound in which an arbitrary part of a group is substituted with another group or an arbitrary group is introduced while maintaining a basic steroid skeleton in cholic acid or the like. Meaning a compound that maintains the ability to be taken up by transporting polypeptide 1B1 (OATP1B1) and / or OATP1B3. The linking group for cholic acid or the like or a derivative thereof to bind to L may be at any position of cholic acid or the like or a derivative thereof, but is preferably bonded at a carboxylic acid residue moiety. The bonding mode can be appropriately selected according to the type of the linking group. For example, when the linking group is a carboxylic acid, the bonding mode can be bonded by an ester bond. In the present specification, the “ester bond” includes an amide bond as well as an ester bond.

In this specification, “organic anion transporting polypeptide 1B1 (OATP1B1)” is also known as SLCO1B1. “Organic anion transporting polypeptide 1B3 (OATP1B3)” is also known as SLCO1B3. OATP1B1 and OATP1B3 are both a single carrier transporter (SLC) transporter that is specifically expressed on the hepatocyte basement membrane side and acts on the incorporation of many statins into hepatocytes. Orally administered statins are selectively taken up by hepatocytes via OATP1B1 and inhibit cholesterol biosynthesis.

Whether a derivative such as cholic acid is taken up by OATP1B1 and / or OATP1B3 can be examined by the following method. Cells that expressed OATP1B1, OATP1B3, and mock cells were seeded at about 2 × 10 ⁵ cells / well in 24-well plates that were appropriately coated, and after 72 hours of growth, the cells were washed with a buffer solution. After one to several washes, buffer is added to the cells and preincubated for 10 minutes. Add a buffer containing a test cholic acid derivative conjugated with a fluorescent label, and after incubation for a certain period of time, remove the buffer and add an ice-cold buffer containing 1% bovine serum albumin (BSA). Stop the capture. The cells are further washed several times with an ice-cold buffer solution, dried, and then the fluorescence of a measurement sample obtained by lysing the cells by adding Lysis Buffer is measured. When the fluorescence of the measurement sample is confirmed, it is determined that a derivative such as cholic acid is taken up by OATP1B1 and / or OATP1B3.

In one embodiment, A is a group represented by the following formula:

[In the above formula, R ¹³ represents a hydrogen atom or a hydroxyl group,
R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, respectively, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom.] .

In the present specification, the group represented by F in the compound represented by formula (I) represents a fluorescent substance, more specifically, a monovalent group of the compound represented by the following formula:

[In the above formula, R ¹ , R ³ , R ⁴ , R ⁶ , R ⁹ , and R ¹¹ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonic acid group, a salt of a sulfonic acid group, or C1. Represents 6 alkyl groups,
R ² represents an oxygen atom or an optionally substituted nitrogen atom,
R ⁵ represents a hydroxyl group or an optionally substituted nitrogen atom,
R ⁷ represents an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, a carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
n represents a natural number from 0 to 4,
R ⁸ and R ¹² are each independently a hydroxyl group, a halogen atom, an amino group, a C1-6 alkyl group, a C2-6 alkenyl group, a C2-6 alkynyl group, a C1-6 alkoxy group, or a C1-6 alkoxycarbonyl group. , A carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
R ¹⁰ represents a hydroxyl group or an optionally substituted nitrogen atom, and
X represents an oxygen atom or an optionally substituted silicon atom;
Here, the nitrogen atom which may be substituted and the substituent of the silicon atom which may be substituted are the same or different, and each may be a C1-6 alkyl group which may be substituted. The substituents of the C1-6 alkyl group which may be the same or different are each a halogen atom, a carboxylic acid, a carboxylic acid salt, a sulfonic acid, or a sulfonic acid salt.

In the present specification, F in the compound represented by the formula (I) is preferably TokyoGreen (registered trademark) represented by the following formula that emits green fluorescence.

[Wherein n represents a natural number of 0 to 4, and R ¹⁷ independently represents a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, a hydroxyl group, a carboxy group, Group, a sulfonic acid group, a C1-6 alkoxycarbonyl group, a halogen atom, or an amino group, and R ¹⁸ represents a C1-6 alkyl group or a C1-6 alkoxy group. ]

As Tokyo Green (registered trademark), the following compounds are preferable.

[Wherein R ¹⁷ represents a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, a hydroxyl group, a carboxy group, a sulfonic acid group, a C1-6 alkoxycarbonyl group, a halogen atom. Or an amino group, and R ¹⁸ represents a C1-6 alkyl group or a C1-6 alkoxy group. ]

In the above, R ¹⁷ is preferably a C1-4 alkyl group or a C1-4 alkoxy group, and more preferably a methyl group or a methoxy group. R ¹⁸ is preferably a C1-4 alkyl group or a C1-4 alkoxy group, and more preferably a methyl group or a methoxy group.

Moreover, as F in the compound represented by the formula (I) in this specification, TokyoMagenta represented by the following formula is preferable.

[In the formula, n represents a natural number of 0 to 4, and each R ¹⁹ independently represents a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, a hydroxyl group, a carboxy group, It represents group, a sulfonic acid group, C1 ~ 6 alkoxycarbonyl group, a halogen atom, or an amino ^{group, R 20} represents a C1 ~ 6 alkyl group or a C1 ~ 6 alkoxy group. ]

As TokyoMagenta, the following compounds are preferable.

[Wherein R ¹⁹ is a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, a hydroxyl group, a carboxy group, a sulfonic acid group, a C1-6 alkoxycarbonyl group, a halogen atom. Or an amino group, and R ²⁰ represents a C1-6 alkyl group or a C1-6 alkoxy group. ]

In the above, R ¹⁹ is preferably a C1-4 alkyl group or a C1-4 alkoxy group, and more preferably a methyl group or a methoxy group. R ²⁰ is preferably a C1-4 alkyl group or a C1-4 alkoxy group, and more preferably a methyl group or a methoxy group.

Further, as the F in the compound represented by the formula (I) in the present specification, hydroxycoumarin represented by the following formula is preferable.

The compound of the present invention is preferably a compound represented by the following formula (V) or formula (VI).

[In the above formula, R ¹ , R ³ , R ⁴ , R ⁶ , R ⁹ , and R ¹¹ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonic acid group, a salt of a sulfonic acid group, C1˜ Represents a 6 alkyl group or a C1-6 alkenyl group,
R ² represents an oxygen atom or an optionally substituted nitrogen atom,
R ⁵ represents a hydroxyl group or an optionally substituted nitrogen atom,
R ⁷ represents an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, a carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
n represents a natural number from 0 to 4,
R ⁸ and R ¹² are each independently a hydroxyl group, a halogen atom, an amino group, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, or a C1-6 alkoxycarbonyl group. , A carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
R ¹⁰ represents a hydroxyl group or an optionally substituted nitrogen atom, and
X represents an oxygen atom or an optionally substituted silicon atom;
R ¹³ represents a hydrogen atom or a hydroxyl group,
R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, respectively, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom,
Here, the nitrogen atom which may be substituted and the substituent of the silicon atom which may be substituted are the same or different, and each may be a C1-6 alkyl group which may be substituted. The substituents of the C1-6 alkyl group which may be the same or different are each a halogen atom, a carboxylic acid, a carboxylic acid salt, a sulfonic acid, or a sulfonic acid salt.

In the compound, R ¹ is preferably a hydrogen atom or a halogen atom.
R ² is preferably an oxygen atom.
R ³ is preferably a hydrogen atom or a halogen atom.
R ⁴ is preferably a hydroxyl group or a nitrogen atom which may be substituted with a C1-6 alkyl group, and more preferably a hydroxyl group or a C1-4 alkyl group. It is a good nitrogen atom, more preferably a hydroxyl group or a nitrogen atom which may be substituted with a methyl group.
R ⁵ is preferably a hydrogen atom or a halogen atom.
R ⁶ is preferably a hydroxyl group or an optionally substituted nitrogen atom.
R ⁷ is preferably a hydroxyl group or an optionally substituted nitrogen atom.
X is preferably an oxygen atom or a silicon atom optionally substituted by 1 to 2 C1-6 alkyl groups, more preferably an oxygen atom or 2 C1-6 A silicon atom substituted with a 4 alkyl group, more preferably an oxygen atom, or a silicon atom substituted with two methyl groups.
R ⁹ is preferably a hydrogen atom.
R ¹⁰ is preferably a hydroxyl group or a di (C1-6 alkyl) amino group (preferably a dimethylamino group or a diethylamino group).
R ¹¹ is preferably a hydrogen atom.
R ¹³ is preferably a hydrogen atom or a hydroxyl group.
R ¹⁴ and R ¹⁵ are preferably a hydrogen atom, a hydroxyl group or an oxygen atom. The solid and dotted lines represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, respectively. When R ¹⁴ or R ^{15 to} be bonded is an oxygen atom, it represents a double bond.

Therefore, in one example, the compound of the present invention is a compound represented by the formula (V),
R ¹ represents a hydrogen atom or a halogen atom,
R ² represents an oxygen atom or an optionally substituted nitrogen atom,
R ³ represents a hydrogen atom or a halogen atom,
R ⁴ represents a hydroxyl group or an optionally substituted nitrogen atom,
R ⁵ represents a hydrogen atom or a halogen atom,
R ⁶ represents a hydroxyl group or an optionally substituted nitrogen atom,
R ⁷ represents a methyl group or a methoxy group, and
X represents an oxygen atom or an optionally substituted silicon atom;
Here, the optionally substituted nitrogen atom and the optionally substituted silicon atom substituent are the same or different and each is a C1-6 alkyl group.
R ¹³ represents a hydrogen atom or a hydroxyl group,
R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom, respectively. It is.

In another embodiment, the present invention is an intermediate compound represented by the following formulas (VII) to (IX). These compounds can be used as intermediates for synthesizing the compounds of the present invention represented by the formula (I).

[Wherein, definitions of X, R ¹ to R ⁷ , R ⁹ to R ¹¹ , and R ¹³ to R ¹⁵ , and lines represented by solid lines and dotted lines are the same as described above. ]

In the present specification, the group represented by L in the compounds represented by formulas (I), (V), (VI), (VII), (VIII), and (IX) represents a linker. The linker is a group that binds cholic acid or the like or a derivative thereof to the fluorescent dye compound represented by F in formula (I). The linker may be any group as long as the compound of the present invention can maintain the high transport ability of the transporter substrate represented by A and can maintain the fluorescence intensity of the fluorescent substance represented by F. There may be. Preferably, the linker moiety is a group that can be bonded to A and F at both ends by an ester bond, an amide bond, or a sulfonic acid amide bond, respectively. For example, L has a linear or branched C2-14 alkylene chain which may be substituted. Examples of the substituent when the alkylene chain of L is substituted include a hydroxyl group, amino group, halogen atom, oxo group, C1-4 alkoxy group, C1-4 alkoxycarbonyl group, C1-4 alkoxycarbonyloxy group, C1˜ A 4-alkylaminocarboxy group, a carboxylic acid group, or a —CONH— (CH ₂ ) ₂ —SO ₃ H group can be mentioned. When the alkylene chain of L is substituted, the number of substituents may be 1 to 4, 1 to 3, 1 to 2, or 1. Preferably L is —NH— (CH ₂ ) _p — (CHR ²¹ ) _q — (CH ₂ ) _r —NHCO— (the CO group is a group derived from R ⁸ or R ¹² ), or —NH— (CH _{2 P} − (CHR ²¹ ) _q — (CH ₂ ) _r —NHSO ₂ — (wherein the SO ₂ group is a group derived from R ⁸ or R ¹² ) [wherein, p and r are respectively , The same or different, a natural number of 2 to 10,
q is 0 or 1, and
R ²¹ is a carboxylic acid group or a —CONH— (CH ₂ ) ₂ —SO ₃ H group].

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. A C1-4 alkyl group, more preferably a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group, a t-butyl group, and an isobutyl 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 most preferred is a methyl group or an ethyl group.

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 via 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 a methoxy group, an ethoxy group, a 1-propyloxy group, a 2-propyloxy group, a 2-methyl-1-propyloxy group, a 2-methyl-2-propyloxy group, and 2,2-dimethyl. -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 group, 1-pentyloxy group, 2-pentyloxy group, 3-pentyloxy group, 2-methyl-1-pentyloxy group, 3-methyl-1-pentyloxy group, 2-methyl-2-pentyloxy group 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 C1-4 alkoxy group, more preferably a C1-3 alkoxy group, still more preferably a methoxy group or an ethoxy group.

In this specification, the “C1-6 alkoxycarbonyl group” means a group ((C1-6 alkyl group) -C (═O) bonded to the C1-6 alkoxy group via an oxo group (> C═O). ) -Group), and the alkyl group moiety may be linear or branched. The C1-6 alkoxycarbonyl group means that the alkyl group moiety has 1 to 6 carbon atoms. Examples of the alkoxy group include a methoxycarbonyl group, an ethoxycarbonyl group, a 1-propyloxycarbonyl group, a 2-propyloxycarbonyl group, a 2-methyl-1-propyloxycarbonyl group, and a 2-methyl-2-propyloxycarbonyl group. 2,2-dimethyl-1-propyloxycarbonyl group, 1-butyloxycarbonyl group, 2-butyloxycarbonyl group, 2-methyl-1-butyloxycarbonyl group, 3-methyl-1-butyloxycarbonyl group, 2-methyl-2-butyloxycarbonyl group, 3-methyl-2-butyloxycarbonyl group, 1-pentyloxycarbonyl group, 2-pentyloxycarbonyl group, 3-pentyloxycarbonyl group, 2-methyl-1-pentyl Oxycarbonyl group, 3-methyl-1-penty Examples include oxycarbonyl group, 2-methyl-2-pentyloxycarbonyl group, 3-methyl-2-pentyloxycarbonyl group, 1-hexyloxycarbonyl group, 2-hexyloxycarbonyl group, and 3-hexyloxycarbonyl group. . The C1-6 alkoxycarbonyl group is preferably a C1-4 alkoxycarbonyl group, more preferably a C1-3 alkoxycarbonyl group, and still more preferably a methoxycarbonyl group or an ethoxycarbonyl group.

The “C2-6 alkenyl group” is a carbon atom formed by removing one hydrogen atom from any carbon atom of a linear or branched unsaturated hydrocarbon having one or more carbon-carbon double bonds. It means a monovalent group having 2 to 6 numbers. Examples of the C2-6 alkenyl group include a vinyl group, propenyl group, isopropenyl group, 1-butenyl group, 2-butenyl group, 3-butenyl group, 1-methyl-1-propenyl group, 2-methyl-1- Propenyl group, 1-methyl-2-propenyl group, 2-methyl-2-propenyl group, 1-methylidene-1-propane group, 1-pentenyl group, 1-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1-methyl-1-butenyl group, 1-methyl-2-butenyl group, 1-methyl-3-butenyl group, 1-methylidenebutyl group, 2-methyl-1-butenyl group, 2-methyl-2-butenyl group, 2-methyl-3-butenyl group, 2-methylidenebutyl group, 3-methyl-1-butenyl group, 3-methyl-2-butenyl group, 3-methyl-3-butenyl group, 1-ethyl-1 Propenyl group, 1-ethyl-2-propenyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, 5-hexenyl group, 1-methyl-1-pentenyl group, 1-methyl- 2-pentenyl group, 1-methyl-3-pentenyl group, 1-methyl-4-pentenyl group, 1-methylidenepentyl group, 2-methyl-1-pentenyl group, 2-methyl-2-pentenyl group, 2- Methyl-3-pentenyl group, 2-methyl-4-pentenyl group, 2-methylidenepentyl group, 3-methyl-1-pentenyl group, 3-methyl-2-pentenyl group, 3-methyl-3-pentenyl group, 3-methyl-4-pentenyl group, 3-methylidenepentyl group, 4-methyl-1-pentenyl group, 4-methyl-2-pentenyl group, 4-methyl-3-pentenyl group, 4 Methyl-4-pentenyl group, 1-ethyl-1-butenyl group, 1-ethyl-2-butenyl group, 1-ethyl-3-butenyl group, 2-ethyl-1-butenyl group, 2-ethyl-2-butenyl group Group, 2-ethyl-3-butenyl group, 1- (1-methylethyl) -1-propenyl group, 1- (1-methylethyl) -2-propenyl group, 1-ethyl-2-methyl-1-propenyl And a 1-ethyl-2-methyl-2-propenyl group. A C2-4 alkenyl group is preferable, and a vinyl group is more preferable.

The “C2-6 alkynyl group” is a carbon atom obtained by removing one hydrogen atom from any carbon atom of a linear or branched unsaturated hydrocarbon having one or more carbon-carbon triple bonds. It means a monovalent group having 2 to 6 numbers. Examples of the C2-6 alkynyl group include ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 2-butynyl group, 3-butynyl group, pentynyl group, hexynyl group, phenylethynyl group and the like. it can. A C2-4 alkynyl group is preferable, and an ethynyl group is more preferable.

“Halogen atom” means a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom, a chlorine atom and a bromine atom, more preferably a fluorine atom or a chlorine atom. .

In the present specification, the term “salt” in a salt of a sulfonic acid group and a salt of a carboxy group means a salt formed by combining the compound of the present invention with an inorganic or organic base at the sulfonic acid group or carboxy group. . Examples of the salt include alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium; ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris (hydroxymethyl) aminomethane, N, N -Salts of amines such as bis (hydroxyethyl) piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, L-glucamine; or bases such as lysine, δ-hydroxylysine, arginine A salt with a functional amino acid can be formed.

In another aspect, the present invention relates to a transporter function measurement kit comprising the compound. Transporters include OATP1B1 and / or OATP1B3. In addition to the compound, the kit of the present invention may comprise a container for storing the compound, an instruction manual, and a package for storing the components of the kit such as a paper box or a plastic case.

The fluorescent substrate of the present invention can provide a fluorescent substrate for a transporter that can be put to practical use because it maintains high transporter transportability and is not easily quenched and has high fluorescence intensity.

₂ is a graph showing the time dependency of uptake of CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, and CDCA-Lys-Et ₂ AC in OATP1B1 and OATP1B3-expressing HEK293 cells. The concentration of each fluorescent substrate was CDCA-Lys-TG 0.025 μM (A), CDCA-Lys-HC 0.1 μM (B), CDCA-Lys-DCTM 0.01 μM (C), CDCA-Lys-Et ₂ AC 0. Set to 1 μM (D). Data represent mean ± standard error (n = 3). The vertical axis of each graph represents the amount of uptake per 1 mg of protein (uptake) (pmol / mg protein), and the horizontal axis represents the elapsed time (minutes). A white circle represents a cell into which an empty vector has been introduced (control), a black triangle represents a cell into which OATP1B1 has been introduced, and a black square represents a cell into which OATP1B3 has been introduced. 2 is a graph showing the concentration dependency of CDCA-Lys-TG uptake in OATP1B1-expressing HEK293 cells. The data show the mean ± standard error (n = 3), the vertical axis represents the uptake after 30 seconds per 1 mg protein (pmol / mg protein / 30 sec), and the horizontal axis represents the concentration (μM) of the fluorescent substrate. (Same in FIGS. 2B-D and FIGS. 3A-D). The vertical axis of the small graph represents the uptake amount after 30 seconds per mg of protein (pmol / mg protein / 30 sec), and the horizontal axis represents the uptake amount after 30 seconds per mg of protein divided by the concentration of the fluorescent substrate. Value (ml / mg protein / 30 sec) (same in FIGS. 2B to 3D and FIGS. 3A to 3D). 2 is a graph showing the concentration dependence of CDCA-Lys-HC uptake in OATP1B1 and OATP1B3 expressing HEK293 cells. It is a graph showing the concentration dependence of CDCA-Lys-DCTM uptake in OATP1B1-expressing HEK293 cells. It is a graph showing the concentration dependence of uptake of CDCA-Lys-Et ₂ AC in OATP1B1-expressing HEK293 cells. 2 is a graph showing the concentration dependency of CDCA-Lys-TG uptake in OATP1B3-expressing HEK293 cells. 2 is a graph showing the concentration dependency of CDCA-Lys-HC uptake in OATP1B3-expressing HEK293 cells. It is a graph showing the concentration dependence of CDCA-Lys-DCTM uptake in OATP1B3-expressing HEK293 cells. It is a graph showing the concentration dependence of the uptake of CDCA-Lys-Et ₂ AC in OATP1B3-expressing HEK293 cells. OATP1B1 and OATP1B3 expressing HEK293 cells CDCA-Lys-TG, CDCA- Lys-HC, CDCA-Lys-DCTM, cyclosporine A against _CDCA-Lys-Et 2 AC _{uptake, rifampicin, T 4, T 3} , pravastatin, BSP, E It is a graph showing the inhibitory effect by ₃ S, PAH (control), and CDCA. Data are shown as mean ± standard error (n = 3-6). The vertical axis represents the uptake ratio (% of control) of each fluorescent substrate, and the horizontal axis represents the type of fluorescent substrate. Black bars represent OATP1B1-expressing HEK293 cells and white bars represent OATP1B3-expressing HEK293 cells. It is a graph showing the result of the time-dependent test of CDCA-Lys-TM, CDCA-Lys-DFTG, and CDCA-Lys-SiR uptake in OATP1B1 and OATP1B3-expressing HEK293 cells. The vertical axis of each graph represents the amount of uptake per mg of protein (uptake) (pmol / mg protein), the horizontal axis represents the elapsed time (minutes), and the white circle represents the cells into which the empty vector was introduced (control). The black triangle represents a cell into which OATP1B1 has been introduced, and the black square represents a cell into which OATP1B3 has been introduced (the same applies to FIGS. 5B and C and FIG. 6). It is a graph showing the result of the time-dependent test of CDCA-C2-DCTM, CDCA-C5-DCTM, and CA-Lys-DCTM uptake in OATP1B1 and OATP1B3-expressing HEK293 cells. It is a graph showing the result of the time-dependent test of CDCA-Tauro-nor-HC and CA-Tauro-nor-TG uptake in OATP1B1 and OATP1B3-expressing HEK293 cells. It is a graph showing the result of the time-dependent test of CA-Lys-DCTM, CDCA-Lys-TM, and CDCA-Lys-DFTG uptake in OATP1B1 and OATP1B3-expressing HEK293 cells.

1. Production Method The compound of the present invention can be obtained by binding cholic acid or the like or a derivative thereof and a fluorescent dye F through a linker while referring to the Examples of the present application. For example, it can be obtained by attaching a linker to cholic acid or the like or a derivative thereof, and then attaching a fluorescent dye to the remaining end of the linker. Alternatively, it can be obtained by binding a linker to a fluorescent dye and then binding cholic acid or the like or a derivative thereof to the remaining end of the linker.

Cholic acid or the like or derivatives thereof can be synthesized by methods described in the literature, or can be obtained as commercially available reagents. Moreover, the coupling | bonding of cholic acid etc. or those derivatives, and a linker can be performed according to the method of literature description, such as making a linker amide bond to the carboxy group of cholic acid.

Fluorescent dye F is disclosed in WO 2012/099218 and Yasuteru Urano et al. am. Chem. soc (2005) 127: 4888-4894 and the like. The binding between the fluorescent dye and the linker is carried out, for example, by introducing an N-succinimide group into the carboxy group of the fluorescent dye using N-hydroxysuccinimide (NHS), and then this N-succinimide fluorescent dye and cholic acid or the like It can be obtained by binding a derivative-bound linker.

2. Screening Method Since the fluorescent substrate of the present invention is transported by OATP1B1 and / or OATP1B3, it can be used for screening a substance transported by OATP1B1 and / or OATP1B3. Therefore, in one aspect, the present invention is a method for determining whether a test substance is transported by OATP1B1 and / or OATP1B3, the method comprising the following steps:
Contacting the test substance and the compound of the present invention with an OATP1B1 and / or OATP1B3-expressing cell culture,
Removing the test substance and the compound of the present invention that have not been taken up into the cells,
A step of measuring the fluorescence intensity of the compound of the present invention incorporated into the cell, and the obtained fluorescence intensity is contacted with the OATP1B1 and / or OATP1B3 expressing cell in the absence of the test substance in the same manner; And a step of determining that the test substance is transported by OATP1B1 and / or OATP1B3 when it is smaller than the fluorescence intensity of the compound of the present invention incorporated therein.

The step of bringing the test substance and the compound of the present invention into contact with an OATP1B1 and / or OATP1B3 expressing cell culture comprises culturing OATP1B1 and / or OATP1B3 expressing cells on a dish, This can be done by replacing with a buffer containing the compound. The test substance and the compound of the present invention that have not been taken up into the cells can be removed, for example, by removing the buffer solution and then adding a buffer solution containing BSA. The fluorescence intensity of the compound of the present invention incorporated into the cells is measured by measuring the fluorescence of the cell lysate obtained by drying the cells and then lysing the cells by adding a cell lysis buffer. be able to. The fluorescence can be measured with a commercially available measuring instrument by selecting an excitation wavelength and a fluorescence wavelength suitable for the fluorescent substance that binds to the compound of the present invention used (see Table 1).

The determination of whether or not the test substance is transported by OATP1B1 and / or OATP1B3 is based on the control in which only the compound of the present invention is contacted with the OATP1B1 and / or OATP1B3 expressing cell culture without contacting the test substance. This is done by comparing with the obtained fluorescence intensity. If the fluorescence intensity is comparable to the control, it can be determined that it is not transported by OATP1B1 and / or OATP1B3, and if the fluorescence intensity is lower than the control, it can be determined that it is transported by OATP1B1 and / or OATP1B3. The fluorescence intensity of the control is such that only the compound of the present invention is brought into contact with OATP1B1 and / or OATP1B3 expressing cell culture, the compound of the present invention that has not been taken up into the cells is removed, and It is obtained by measuring the fluorescence intensity of the compound.

Alternatively, the screening method of the present invention can be performed by quenching using a quencher without removing the test substance and the compound of the present invention. That is, in one aspect, the present invention is a method for determining whether a test substance is transported by OATP1B1 and / or OATP1B3, the method comprising the following steps:
Contacting the test substance and the compound of the present invention with an OATP1B1 and / or OATP1B3-expressing cell culture,
Adding a quencher to the cell culture to quench the compounds of the invention that have not been taken up into the cells;
A step of measuring the fluorescence intensity of the compound of the present invention incorporated into the cell, and the obtained fluorescence intensity is contacted with the OATP1B1 and / or OATP1B3 expressing cell in the absence of the test substance in the same manner; And a step of determining that the test substance is transported by OATP1B1 and / or OATP1B3 when it is smaller than the fluorescence intensity of the compound of the present invention incorporated therein.

The compound used as a quencher is not particularly limited as long as it can quench the compound of the present invention and is not taken up by cells, and can be selected according to the fluorescent dye to be used. For example, brilliant black (4-acetylamino-5-hydroxy-6- [7-sodiosulfo-4- [4- (sodiosulfo) phenylazo] -1-naphthylazo] naphthalene-1,7-disulfonic acid disodium as a quencher CAS number 2519-30-4) can be used.

In the screening method of the present invention, the fluorescence intensity per unit weight of the protein can also be used as the fluorescence intensity. Therefore, the present invention is a method for determining whether or not a test substance is transported by OATP1B1 and / or OATP1B3, the method comprising the following steps:
Contacting the test substance and the compound of the present invention with an OATP1B1 and / or OATP1B3-expressing cell culture,
A step of removing the test substance and the compound of the present invention that has not been taken up into the cells, or a quencher is added to the cell culture to quench the compound of the present invention that has not been taken up into the cells. Step to make,
Measuring the fluorescence intensity of the compound of the present invention incorporated into the cells,
The step of measuring the protein concentration in the cell lysate, and the obtained (fluorescence intensity / protein concentration) are contacted with OATP1B1 and / or OATP1B3 expressing cells in the same manner in the absence of the test substance. It is related to the step of determining that the test substance is transported by OATP1B1 and / or OATP1B3 when it is small compared with (fluorescence intensity / protein concentration) of the compound of the present invention incorporated in the inside.

Protein concentration can be measured by Bradfold or the like. For example, it can be performed by measuring the fluorescence of the cell lysate obtained by drying the cells into which the fluorescent substrate has been incorporated and then adding NaOH or the like to lyse the cells. The protein concentration can be determined by creating a calibration curve using BSA as a standard substance and the absorbance obtained in the sample as the concentration on the calibration curve.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. It should be noted that all documents cited throughout this application are incorporated herein by reference in their entirety.

Example 1 Synthesis of Compounds 1 to 10 In order to evaluate transporter transport ability, the following compounds 1-10 were synthesized.

(1) Synthesis of fluorescent substrate First, the following compounds 11-17 were synthesized. Compounds 11-14 and 17 were synthesized with reference to literature.

Compounds 15 and 16 were synthesized by the following method.
(A) Synthesis of Compound 15

TM-COOH (43.0 mg, 0.111 mmol) and NHS (25.5 mg, 0.222 mmol) in a 50 mL eggplant flask were dissolved in methylene chloride (3.00 mL). DIC (43.0 microliters, 0.278 mmol) was added there, and it stirred at room temperature for 2.5 hours. The reaction solution was concentrated and roughly purified by silica gel column chromatography. The obtained crude product of compound 15 was used in the next reaction as it was.

(B) Synthesis of Compound 16

DCTM-COOH (30.9 mg, 0.0676 mmol) and NHS (9.33 mg, 0.0811 mmol) in a 30 mL eggplant flask were dissolved in methylene chloride (1.50 mL). DIC (12.5 microliters, 0.0811 mmol) was added there, and it stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by silica gel column chromatography to obtain 18.4 mg (0.0332 mmol) of Compound 16 in a yield of 49%.

1H NMR (acetone-d6,400MHz)
δ 8.14 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 6.85 (d, J = 9. 3Hz, 2H), 6.72-6.63 (m, 2H), 3.00 (s, 4H), 2.20 (s, 3H), 0.88 (s, 6H)
MS (ESI)
calcd. for [M−H] ⁻ 552.04, found 552.03

(2) Synthesis of chenodeoxycholic acid derivative Next, chenodeoxycholic acid derivatives 18, 20, and 21 required for coupling with compound 11-17 and cholic acid derivative 19 were synthesized with reference to literature.

(3) Synthesis of Compounds 1 to 10 Compound 1-10 was synthesized according to the route described below.

c) -1. Synthesis of compound 1

Compound 11 (19.9 mg, 0.0657 mmol) and compound 18 (46.9 mg, 0.0739 mmol) in a 20 mL eggplant flask were dissolved in DMF (1.00 mL). Et ₃ N (31.0 μL, 0.224 mmol) was added thereto, and the mixture was stirred at room temperature for 16 hours. 0.1N HCl was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated, and purified by silica gel column chromatography to obtain Compound 1 (14.4 mg, 0.0203 mmol) in a yield of 31%.

1H NMR (CD ₃ OD, 400 MHz)
δ 8.63 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 6.70 (dd, J = 2.2, 8.8 Hz, 1H), 6.52 (d, J = 2.2 Hz 1H), 4.28 (m, 1H), 3.75 (s, 1H), 3.45-3.33 (m, 3H), 2.38-2.05 (m, 3H) 1.98-0.85 (m, 35H), 0.63 (s, 3H)
MS (ESI)
calcd. ^{for [M-2H] 2- 353.19} , found 353.17

c) -2. Synthesis of compound 2

Compound 12 (26.7 mg, 0.0746 mmol) and compound 18 (41.9 mg, 0.0660 mmol) were dissolved in DMF (1.30 mL). Et ₃ N (28.0 μL, 0.202 mmol) was added thereto, and the mixture was stirred at room temperature for 2 hours. 0.1N HCl was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated, and purified by silica gel column chromatography to obtain 27.5 mg (0.0360 mmol) of Compound 2 in a yield of 55%.

1H NMR (CD ₃ OD, 400 MHz)
δ 9.05 (m, 1H), 8.61 (s, 1H), 7.55 (d, J = 8.9 Hz, 1H), 6.82 (d, J = 8.9 Hz, 1H), 6 .55 (s, 1H), 4.37 (m, 1H), 3.74 (s, 1H), 3.53 (q, J = 7.1 Hz, 4H), 3.45-3.33 (m , 3H), 2.35-2.07 (m, 3H), 2.02-0.80 (m, 41H), 0.61 (s, 3H)
MS (ESI)
calcd. for [M−H] ⁻ 762.47, found 762.43

c) -3. Synthesis of compound 3

Compound 13 (10.0 mg, 0.0226 mmol) and compound 18 (12.7 mg, 0.0200 mmol) in a 20 mL eggplant flask were dissolved in DMF (0.600 mL). Et ₃ N (9.00 μL, 0.0650 mmol) was added thereto, and the mixture was stirred at room temperature for 24 hours. 0.1N HCl was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated and purified by silica gel column chromatography to obtain 7.10 mg (0.00836 mmol) of Compound 3 in a yield of 42%.

1H NMR (CD ₃ OD, 400 MHz)
δ 7.93 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.04 (d, J = 9. 2Hz, 2H), 6.81-6.68 (m, 4H), 4.37 (m, 1H), 3.77 (s, 1H), 3.45 (m, 1H), 3.21 (m , 2H), 2.35-2.10 (m, 6H), 2.00-0.85 (m, 35H), 0.67 (s, 3H)
MS (ESI)
calcd. ^{for [M-2H] 2- 423.22} , found 423.20

c) -4. Synthesis of compound 4

Compound 14 (5.00 mg, 0.0104 mmol) and compound 18 (7.03 mg, 0.0135 mmol) in a 30 mL eggplant flask were dissolved in methylene chloride (4.50 mL) and DMF (1.00 mL). IPr ₂ NEt (10.0 μL, 0.0575 mmol) was added thereto, and the mixture was stirred at room temperature for 20 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated, and purified by silica gel column chromatography to obtain 4.50 mg (0.00508 mmol) of Compound 4 in a yield of 49%.

1H NMR (CD ₃ OD, 400 MHz)
δ 7.94 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 6.68 (d, J = 7. 4Hz, 2H), 6.58 (dd, J = 1.3, 11.4Hz, 2H), 4.31 (m, 1H), 3.76 (s, 1H), 3.44 (m, 2H) 3.38-3.31 (m, 1H), 2.37-2.10 (m, 6H), 1.99-0.85 (m, 35H), 0.66 (s, 3H)

c) -5. Synthesis of compound 5

Crude compound 15 (14.5 mg) and compound 18 (7.71 mg, 0.0148 mmol) in a 30 mL eggplant flask were dissolved in methylene chloride (3.00 mL). There _iPr 2 NEt _{(7.72μL, 0.0444mmol)} and stirred at room temperature for 20 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated, and purified by silica gel column chromatography to obtain 4.30 mg (0.00483 mmol) of compound 5 in a yield of 33%.

1H NMR (CD ₃ OD, 400 MHz)
δ 7.85 (s, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.04 (d, J = 2. 2 Hz, 2H), 6.88 (d, J = 9.5 Hz, 2H), 6.44 (m, 2H), 4.31 (m, 1H), 3.76 (s, 1H), 3.43 (M, 2H), 3.37-3.32 (m, 1H), 2.37-2.10 (m, 6H), 2.00-0.85 (m, 35H), 0.66 (s , 3H), 0.51 (d, J = 5.6 Hz, 6H)

c) -6. Synthesis of Compound 6

Compound 16 (4.00 mg, 0.00722 mmol) and compound 18 (20.8 mg, 0.0327 mmol) in a 20 mL eggplant flask were dissolved in DMF (1.00 mL). Et ₃ N (14.0 μL, 0.101 mmol) was added thereto, and the mixture was stirred at room temperature for 16 hours. 0.1N HCl was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated and purified by silica gel column chromatography to obtain 3.20 mg (0.00333 mmol) of Compound 6 in a yield of 46%.

1H NMR (CD ₃ OD, 400 MHz)
δ 7.81 (d, J = 6.8 Hz, 1H), 7.75 (s, 1H), 7.70 (d, J = 6.8 Hz, 1H), 6.73 (d, J = 9. 6Hz, 2H),
6.42 (d, J = 9.6 Hz, 2H), 4.19 (m, 1H), 3.68 (s, 1H), 3.43-3.25 (m, 3H), 2.25 0.65 (m, 50H)
MS (ESI)
calcd. for [M-2H] 2- 478.20, found 478.20

e) Synthesis of Compound 7

Compound 16 (5.00 mg, 0.00903 mmol) and compound 20 (9.22 mg, 0.0212 mmol) in a 50 mL eggplant flask were dissolved in methylene chloride (3.00 mL). IPr ₂ NEt (20.0 μL, 0.115 mmol) was added thereto, and the mixture was stirred at room temperature for 5 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated, and purified by silica gel column chromatography to obtain 4.50 mg (0.00515 mmol) of Compound 7 in a yield of 57%.

1H NMR (CD ₃ OD, 400 MHz)
δ 8.13 (m, 1H), 7.85 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 6 .84 (dd, J = 1.7, 9.3 Hz, 2H), 6.55 (d, J = 9.3 Hz, 2H), 3.77-3.69 (m, 2H), 3.55 ( m, 2H), 3.49-3.42 (m, 2H), 3.26-3.20 (m, 1H), 2.32-2.09 (m, 6H), 2.01-0. 85 (m, 35H), 0.65 (s, 3H)

f) Synthesis of Compound 8

Compound 16 (8.92 mg, 0.0161 mmol) and compound 21 (9.30 mg, 0.0195 mmol) in a 50 mL eggplant flask were dissolved in methylene chloride (3.00 mL). IPr ₂ NEt (17.0 μL, 0.0975 mmol) was added thereto, and the mixture was stirred at room temperature for 10 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated and purified by silica gel column chromatography to obtain 10.0 mg (0.0109 mmol) of Compound 8 in a yield of 68%.

1H NMR (CD ₃ OD, 400 MHz)
δ 8.58 (m, 1H), 7.98 (m, 1H), 7.85 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 9.5 Hz, 2H), 6.57 (d, J = 9.5 Hz, 2H), 3.78 (s, 1H), 3.48. −3.33 (m, 3H), 3.26−3.12 (m, 2H), 2.32-2.18 (m, 2H), 2.14−2.04 (m, 4H), 2 .00-0.86 (m, 41H), 0.66 (s, 3H)

c) -9. Synthesis of compound 9

Compound 17 (5.00 mg, 0.00765 mmol) and compound 18 (6.98 mg, 0.0134 mmol) in a 30 mL eggplant flask were dissolved in methylene chloride (3.00 mL). IPr ₂ NEt (6.99 μL, 0.0402 mmol) was added thereto, and the mixture was stirred at room temperature for 20 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated and purified by silica gel column chromatography to obtain 3.60 mg (0.00340 mmol) of Compound 9 in a yield of 44%.

1H NMR (CD ₃ OD, 400 MHz)
δ 7.88 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 2.8 Hz, 2H), 7.24 (d, J = 7. 9 Hz, 1H), 7.04 (d, J = 9.6 Hz, 2H), 6.78 (dd, J = 2.8, 9.6 Hz, 2H), 4.40 (m, 1H), 3. 76 (s, 1H), 3.50-3.43 (m, 2H), 3.39-3.30 (m, 13H), 2.37-2.09 (m, 6H), 2.05- 0.89 (m, 35H), 0.67 (s, 3H), 0.61 (d, J = 5.9 Hz, 6H)

(D) Synthesis of Compound 10

Compound 16 (8.92 mg, 0.0161 mmol) and compound 19 (9.40 mg, 0.0175 mmol) in a 50 mL eggplant flask were dissolved in methylene chloride (3.00 mL). IPr ₂ NEt (17.4 μL, 0.100 mmol) was added thereto, and the mixture was stirred at room temperature for 10 hours. A saturated aqueous ammonium chloride solution was added to the reaction solution, and the organic layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried, concentrated and purified by silica gel column chromatography to obtain Compound 10 (4.3 mg, 0.00441 mmol) in a yield of 27%.

1H NMR (CD ₃ OD, 400 MHz)
δ 7.82 (s, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 6.77 (dd, J = 0. 80, 9.3 Hz, 2H), 6.39 (d, J = 9.3 Hz, 2H), 4.29 (m, 1H), 3.93 (s, 1H), 3.77 (m, 1H) 3.45-3.33 (m, 3H), 2.37-2.10 (m, 6H), 2.03-0.83 (m, 39H), 0.68 (s, 3H)

(Example 2) Evaluation of uptake by OATP1B1 and OATP1B3 expressing cells (1) Culturing of OATP1B1 and OATP1B3 expressing HEK293 cells OATP1B1 and OATP1B3 stably expressing HEK293 cells have already been established (Yamaguchi et al., 200, Cancer, et al. 163-169; Yamaguchi et al., Biol. Pharm. Bull. 2011; 34; 389-395). As a control, HEK293 cells (mock cells) into which an empty vector was introduced were used. OATP1B1 / HEK293, OATP1B3 / HEK293 cells and mock cells were used in a medium containing 10% FBS and G-418 (0.5 mg / mL) in Dulbecco's modified Eagle's medium in a 5% CO ₂ environment. Cultured at 37 ° C. These cells were maintained by subculture using a plastic dish every 3 or 4 days.

(2) Fluorescent substrate As fluorescent substrates, CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, CDCA-Lys-Et ₂ AC, CDCA-Lys-TM, CDCA-Lys-DFTG, CDCA-Lys -SiR, CDCA-C2-DCTM, CDCA-C5-DCTM, CDCA-Lys-DCTM, CA-Tauro-nor-HC, and CA-Tauro-nor-TG were used. Table 1 shows the measurement excitation wavelength, (Excitation wavelength) (nm), and fluorescence wavelength (nm) of each fluorescent substrate.

(3) Uptake test method OATP1B1 / HEK293, OATP1B3 / HEK293 cells and mock cells (Vec / HEK293) were seeded at about 200,000 cells / well on poly-L-lysine coated 24-well plates and grown for 72 hours. . The medium was changed 24 hours before the uptake experiment. The cells were mixed with Krebs-Henseleit (KH) buffer (118 mM NaCl, 23.8 mM NaHCO ₃ , 4.83 mM KCl, 0.96 mM KH ₂ PO ₄ , 1.20 mM MgSO ₄ , 12.5 mM N- (2-hydroxyethyl) piperazine- After washing once with N′-2-ethanesulfonic acid (HEPES), 5.0 mM D-glucose, and 1.53 mM CaCl ₂ , pH 7.4), KH buffer was added to the cells, and preincubation was performed for 10 minutes.

Incorporation was started by replacing with KH buffer containing each fluorescently labeled compound (or each fluorescently labeled compound and inhibitor). The uptake reaction was stopped by removing the incubation buffer after the specified time of incubation and adding ice-cooled KH buffer containing 1% of bovine serum albumin (BSA). The cells were further washed twice with ice-cooled KH buffer, dried, and lysed with Lysis Buffer added to obtain a fluorescence measurement sample. A microplate reader Infinite 200 PRO (Tecan Japan, Kanagawa, Japan) was used for fluorescence measurement. Wash with ice-cooled KH buffer twice, dry, add 0.5N NaOH and dissolve it as a protein quantification sample, and determine protein concentration by Bradford method using bovine serum albumin (BSA) as standard did.

(4) Protein quantification method The protein quantification sample performed protein quantification by Bradford method. To each well of 96 well plate, 5 μL of a protein quantification sample and 200 μL of 5-fold diluted Bio-Rad Protein Assay Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA, USA) were added, and after 5 minutes, the absorbance at an absorption wavelength of 595 nm was measured. It was measured. A calibration curve was prepared using BSA as the standard protein.

(5) Statistical analysis The experimental results are shown as mean ± standard error (mean ± SE). The transporter-specific uptake was calculated by subtracting the transporter non-specific uptake by mock cells (Vec / HEK cells) from the uptake of OATP1B1 / HEK cells and OATP1B3 / HEK cells. Michaelis-Menten wave and Eadie Hofste Plot were created using data analysis software Kaleida Graph (HuLinks Inc, Tokyo, Japan). In the inhibition test, the amount of transporter-specific uptake in the absence of an inhibitor was taken as 100%, and Uptake% of Control was calculated. The test of significant difference between the Control group and the inhibitor (or PAH) group was performed by Dannett test using statistical analysis software JMP Pro 12 (SAS Institute Inc., North Carolina, USA) (* p <0.05). ).

(6) Time dependency test The time dependency of CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, and CDCA-Lys-Et ₂ AC uptake into OATP1B1 and OATP1B3 expressing HEK293 cells was tested. (N = 3). The results are shown in FIG. Concentration of each fluorescent substrate was CDCA-Lys-TG 0.025 μM (A), CDCA-Lys-HC 0.1 μM (B), CDCA-Lys-DCTM 0.01 μM (C), CDCA-Lys-Et ₂ AC 0. Set to 1 μM (D). For all four fluorescent substrates, a significant increase in uptake in OATP1B1 and OATP1B3 expressing cells was observed compared to mock cells. Data represent mean ± standard error.

(7) Concentration dependency test The concentration dependency of uptake of CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, and CDCA-Lys-Et ₂ AC in OATP1B1 and OATP1B3-expressing HEK293 cells was tested twice. (N = 3). The respective results are shown in FIGS. 2A to 2D and FIGS. 3A to 3D. Uptake of the four fluorescent substrates increased in a concentration-dependent manner, and saturation was observed in the high concentration range. From the results of the time-dependent test described above, the uptake time of all four fluorescent substrates was 30 seconds at which the initial rate of uptake through OATP1B1 and OATP1B3 can be evaluated. Data are shown as mean ± standard error. Further, the apparent Michaelis constant (Km value) was calculated from Eadie Hofste Plot to calculate the kinetic parameter. The results are shown in Table 2 below.

(8) Inhibitory effect of typical substrate / typical inhibitor on CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, CDCA-Lys-Et ₂ AC uptake of HEA 293 cells expressing OATP1B1 and OATP1B3 CDCA-Lys-TG 0.02 μM (A), CDCA-Lys-HC 0.1 μM (B), CDCA-Lys-DCTM 0.01 μM (C), CDCA-Lys-Et ₂ AC 0.1 μM (D) Cyclosporine A, rifampicin, T ₄ , T ₃ , pravastatin, BSP, and E ₃ S as typical substrates and inhibitors of OATP1B1 and OATP1B3, CDCA, which is an unlabeled bile acid, as a negative substance, As control With PAH, it was assessed inhibitory effect on uptake via OATP1B1 and OATP1B3. The results are shown in FIG. Data are shown as mean ± standard error (n = 3-6). A decrease in uptake by OATP1B1 and OATP1B3 typical substrates and inhibitors was observed for all four fluorescent substrates. It became clear that these inhibition sensitivities show the same tendency as known transport substrates. On the other hand, when PAH was added, the uptake amount was almost the same as that of the control.

(9) Calculation of inhibition constant (K _i value) of OATP1B1 and OATP1B3 typical substrate / typical inhibitor using CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, CDCA-Lys-Et ₂ AC Each fluorescent substrate concentration was set to CDCA-Lys-TG 0.025 μM, CDCA-Lys-HC 0.1 μM, CDCA-Lys-DCTM 0.01 μM, CDCA-Lys-Et ₂ AC 0.1 μM, and OATP1B1 and OATP1B3 typical Inhibitory constants for the inhibitors Cyclosporine A, Rifampicin, Ritonavir, Erythromycin, Verapamil, and Probenecid were calculated. Inhibition constants of each inhibitor in transport through OATP1B1 are shown in Table 3, and inhibition constants of each inhibitor in transport through OATP1B3 are shown in Table 4 (in the table, [1] Izumi et al., Drug Metab Dispos. 2013 Oct; 41 (10): 1859-66; [2] Matsushima et al., Drug Metab Dispos. 2008 Apr; 36 (4): 663-9; [3] Tahara et al., Drug Metas 34: 74 -747, 2006). The inhibition constant in the transport via OATP1B1 and OATP1B3 shows that the value of most inhibitors is 0.1 when compared with the case where a fluorescent substrate is used and the case where Estradiol-17β-glucuronide or Fexofenadine which are known substrates are used. Correlation within -10 times was shown. This suggests that CDCA-Lys-TG, CDCA-Lys-HC, CDCA-Lys-DCTM, and CDCA-Lys-Et ₂ AC exhibit inhibitory sensitivities comparable to known substrates of OATP1B1 and OATP1B3.

(10) CDCA-Lys-TM, CDCA-Lys-DFTG, CDCA-Lys-SiR, CDCA-C2-DCTM, CDCA-C5-DCTM, CA-Lys-DCTM, CA-Tauro- in OATP1B1 and OATP1B3-expressing HEK293 cells Time-dependent test of uptake of nor-HC and CA-Tauro-nor-TG The uptake experiment was conducted with each fluorescent substrate concentration set to 0.1 μM. The results are shown in FIGS. All fluorescent substrates showed a significant increase in uptake in OATP1B1 and OATP1B3 expressing cells compared to mock cells. Data are shown as mean ± standard error (n = 3).

(11) Time-dependent test of CDCA-Lys-TM, CDCA-Lys-DFTG, and CA-Lys-DCTM uptake in OATP1B1 and OATP1B3-expressing HEK293 cells The uptake experiment was conducted with each fluorescent substrate concentration set to 0.005 μM . The results are shown in FIG. All fluorescent substrates showed a significant increase in uptake in OATP1B1 and OATP1B3 expressing cells compared to mock cells. Data are shown as mean ± standard error (n = 3).

Claims (10)

1. Compound represented by the following formula (I):
   ALF (I)
   [Wherein, A is cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursocholic acid, ursodeoxycholic acid, α-mulicholic acid, β-mulicholic acid, ω-mulicholic acid, hyocholic acid, hyodeoxycholic acid 7-oxo-deoxycholic acid, 7-oxo-lithocholic acid, 1-oxo-lithocholic acid, or 2-oxo-lithocholic acid, or derivatives thereof,
   L represents a linker, and
   F represents a monovalent group of a compound represented by the following formula:
   

   

   [In the above formula, R ¹ , R ³ , R ⁴ , R ⁶ , R ⁹ , and R ¹¹ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonic acid group, a salt of a sulfonic acid group, Represents a 6 alkyl group or a C1-6 alkenyl group,
   R ² represents an oxygen atom or an optionally substituted nitrogen atom,
   R ⁵ represents a hydroxyl group or an optionally substituted nitrogen atom,
   R ⁷ represents an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, a carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
   n represents a natural number from 0 to 4,
   R ⁸ and R ¹² are each independently a hydroxyl group, a halogen atom, an amino group, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, or a C1-6 alkoxycarbonyl group. , A carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
   R ¹⁰ represents a hydroxyl group or an optionally substituted nitrogen atom, and
   X represents an oxygen atom or an optionally substituted silicon atom;
   Here, the nitrogen atom which may be substituted and the substituent of the silicon atom which may be substituted are the same or different, and each may be a C1-6 alkyl group which may be substituted. The substituents of the C1-6 alkyl group which may be the same or different are each a halogen atom, a carboxylic acid, a carboxylic acid salt, a sulfonic acid, or a sulfonic acid salt.
2. The compound according to claim 1, wherein A is represented by the following formula (IV):
   

   

   [In the above formula, R ¹³ represents a hydrogen atom or a hydroxyl group,
   R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
   A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, respectively, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom.] .
3. The compound according to claim 1, which is represented by the following formula (V) or formula (VI):
   

   

   

   [In the above formula, R ¹ , R ³ , R ⁴ , R ⁶ , R ⁹ , and R ¹¹ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonic acid group, a salt of a sulfonic acid group, Represents a 6 alkyl group or a C1-6 alkenyl group,
   R ² represents an oxygen atom or an optionally substituted nitrogen atom,
   R ⁵ represents a hydroxyl group or an optionally substituted nitrogen atom,
   R ⁷ represents an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, a carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
   n represents a natural number from 0 to 4,
   R ⁸ and R ¹² are each independently a hydroxyl group, a halogen atom, an amino group, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, or a C1-6 alkoxycarbonyl group. , A carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
   R ¹⁰ represents a hydroxyl group or an optionally substituted nitrogen atom, and
   X represents an oxygen atom or an optionally substituted silicon atom;
   R ¹³ represents a hydrogen atom or a hydroxyl group,
   R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
   A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, respectively, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom,
   Here, the nitrogen atom which may be substituted and the substituent of the silicon atom which may be substituted are the same or different, and each may be a C1-6 alkyl group which may be substituted. The substituents of the C1-6 alkyl group which may be the same or different are each a halogen atom, a carboxylic acid, a carboxylic acid salt, a sulfonic acid, or a sulfonic acid salt.
4. A compound represented by the formula (V) according to claim 3,
   R ¹ represents a hydrogen atom or a halogen atom,
   R ² represents an oxygen atom or an optionally substituted nitrogen atom,
   R ³ represents a hydrogen atom or a halogen atom,
   R ⁴ represents a hydroxyl group or an optionally substituted nitrogen atom,
   R ⁵ represents a hydrogen atom or a halogen atom,
   R ⁶ represents a hydroxyl group or an optionally substituted nitrogen atom,
   R7 represents a methyl group or a methoxy group, and
   X represents an oxygen atom or an optionally substituted silicon atom;
   Here, the optionally substituted nitrogen atom and the optionally substituted silicon atom substituent are the same or different and each is a C1-6 alkyl group.
   R ¹³ represents a hydrogen atom or a hydroxyl group,
   R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
   A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom, respectively. .
5. L is linked to A and F by an ester bond, an amide bond, or a sulfonamide bond, and has a linear or branched C2-14 alkylene chain which may be substituted. 5. The compound according to any one of 4.
6. 6. A compound compound according to claim 5, wherein L is
   —NH— (CH ₂ ) _p — (CHR ²¹ ) _q — (CH ₂ ) _r —NHCO—, or —NH— (CH ₂ ) _p — (CHR ²¹ ) _q — (CH ₂ ) _r —NHSO ₂ —
   A compound represented by the formula: wherein p and r are the same or different and are each a natural number of 2 to 10;
   q is 0 or 1, and
   R ²¹ is a carboxylic acid group or a —CONH— (CH ₂ ) ₂ —SO ₃ H group].
7. A compound represented by any one of the following formulas (VII) to (IX):
   

   

   [In the above formula, R ¹ , R ³ , R ⁴ , R ⁶ , R ⁹ , and R ¹¹ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonic acid group, a salt of a sulfonic acid group, C1˜ Represents a 6 alkyl group or a C1-6 alkenyl group,
   R ² represents an oxygen atom or an optionally substituted nitrogen atom,
   R ⁵ represents a hydroxyl group or an optionally substituted nitrogen atom,
   R ⁷ represents an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, a carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
   n represents a natural number from 0 to 4,
   R ⁸ and R ¹² are each independently a hydroxyl group, a halogen atom, an amino group, a C1-6 alkyl group, a C1-6 alkenyl group, a C1-6 alkynyl group, a C1-6 alkoxy group, or a C1-6 alkoxycarbonyl group. , A carboxy group, a salt of a carboxy group, a sulfonic acid group, or a salt of a sulfonic acid group,
   R ¹⁰ represents a hydroxyl group or an optionally substituted nitrogen atom, and
   X represents an oxygen atom or an optionally substituted silicon atom;
   R ¹³ represents a hydrogen atom or a hydroxyl group,
   R ¹⁴ and R ¹⁵ represent a hydrogen atom, a hydroxyl group or an oxygen atom,
   A solid line and a dotted line represent a single bond when R ¹⁴ or R ^{15 to} be bonded is a hydrogen atom or a hydroxyl group, respectively, and a double bond when R ¹⁴ or R ^{15 to} be bonded is an oxygen atom,
   Here, the nitrogen atom which may be substituted and the substituent of the silicon atom which may be substituted are the same or different, and each may be a C1-6 alkyl group which may be substituted. The substituents of the C1-6 alkyl group which may be the same or different are each a halogen atom, a carboxylic acid, a carboxylic acid salt, a sulfonic acid, or a sulfonic acid salt.
8. A transporter function measurement kit comprising the compound according to any one of claims 1 to 6.
9. A method for determining whether a test substance is transported by OATP1B1 and / or OATP1B3, the method comprising the following steps:
   Contacting the test substance and the compound according to any one of claims 1 to 6 with OATP1B1 and / or OATP1B3-expressing cells;
   A step of removing the test substance and the compound according to any one of claims 1 to 6 that have not been taken up into the cell, or adding a quencher to the cell culture, Quenching the compound according to any one of claims 1 to 6, which is not incorporated in
   The step of measuring the fluorescence intensity of the compound according to any one of claims 1 to 6 incorporated into cells, and the obtained fluorescence intensity in the absence of the test substance. The compound according to any one of claims 1 to 6, wherein the compound according to any one of claims 6 to 6 is taken into a cell contacted with an OATP1B1 and / or OATP1B3 expressing cell in the same manner. A step of determining that the test substance is transported by OATP1B1 and / or OATP1B3 when it is smaller than the fluorescence intensity.
10. A method for determining whether a test substance is transported by OATP1B1 and / or OATP1B3, the method comprising the following steps:
    Contacting the test substance and the compound according to any one of claims 1 to 6 with an OATP1B1 and / or OATP1B3-expressing cell culture,
    A step of removing the test substance and the compound according to any one of claims 1 to 6 that have not been taken up into the cell, or adding a quencher to the cell culture, Quenching the compound according to any one of claims 1 to 6, which is not incorporated in
    The step of measuring the fluorescence intensity of the compound according to any one of claims 1 to 6 incorporated into cells, and the obtained fluorescence intensity in the absence of the test substance. The compound according to any one of claims 1 to 6, wherein the compound according to any one of claims 6 to 6 is taken into a cell contacted with an OATP1B1 and / or OATP1B3 expressing cell in the same manner. A step of determining that the test substance is transported by OATP1B1 and / or OATP1B3 when it is smaller than the fluorescence intensity.

Images