WO2013183656A1

WO2013183656A1 - Conjugate of g-protein coupled receptor binding ligand and nucleic acid molecule

Info

Publication number: WO2013183656A1
Application number: PCT/JP2013/065512
Authority: WO
Inventors: 龍永田; 雅則戸邊; 拓士中川; 慶介柿口
Original assignee: 大日本住友製薬株式会社
Priority date: 2012-06-04
Filing date: 2013-06-04
Publication date: 2013-12-12

Abstract

Provided is a transporter for efficiently incorporating nucleic acid medicine into diseased cells. More specifically, a new chemical compound (conjugate) of a nucleic acid molecule and the transporter is provided. The conjugate includes a linker between a G-protein coupled receptor binding ligand and a nucleic acid molecule. The linker includes triazolylene, and a polyalkylene glycol chain which may be substituted, or an alkylene chain which may be substituted (the alkylene chain may have one or more divalent groups inserted so as to interrupt the chain, or at the end thereof, selected from the group consisting of -O-, -S-, -NH-, =N-, -N=, -SO₂-, -C(=O)-, (C6-C10) arylene, (C2-C9) heteroarylene, (C3-C8) cycloalkylene and (C3-C7) heterocyclene).

Description

G protein-coupled receptor binding ligand and nucleic acid molecule conjugate

The present invention relates to a conjugate of a G protein coupled receptor (GPCR) ligand and a nucleic acid molecule. In particular, the present invention provides a novel compound containing a conjugate of a GPCR ligand and a nucleic acid molecule, and a pharmaceutical composition containing the novel compound as an active ingredient. Furthermore, the present invention provides methods for making these compounds and methods for introducing nucleic acid molecules into cells using these compounds for the treatment of various diseases associated with cells with GPCRs. .

Nucleic acid drugs use nucleic acid molecules such as antisense oligonucleotides, siRNAs, and micro RNAs (miRNAs) as drugs, and are attracting attention as next-generation drugs next to low-molecular drugs and antibody drugs.
An antisense oligonucleotide is an oligonucleotide having a structure complementary to a partial sequence region of mRNA encoding a target protein. Antisense oligonucleotides can inhibit translation into proteins by binding to the corresponding mRNA, and in addition, the activation of RNase H degrades the corresponding mRNA and suppresses mRNA expression. Potential control of gene expression using oligonucleotides and application to therapy is expected (Non-patent Document 1).
siRNA is a double-stranded RNA that is converted into a single strand in the process of RNA interference, and then incorporated into RISC (RNA-induced silencing complex) to target RNA that has at least one highly complementary binding site. Recognize sequence-specifically. As a result, the target RNA is cleaved / degraded by the nuclease in RISC. Therefore, siRNA is expected as a new type of pharmaceutical that can suppress the expression of genes that cause diseases (Non-patent Document 2).
miRNA is single-stranded RNA, and is incorporated into RISC in the process of RNA interference like siRNA, and shows specific gene expression suppression. miRNA has been reported to be associated with various diseases including cancer, such as involvement in suppression or promotion of cancer, and the possibility of treatment using miRNA is expected (Non-patent Documents 3 and 4). .
An antisense oligonucleotide having a complementary structure to a partial sequence region of miRNA has been reported to inhibit the function of endogenous miRNA upon intravenous administration to mice, and an antisense oligonucleotide targeting miRNA is a nucleic acid. Expected to be a pharmaceutical product (Non-Patent Document 5).
The therapeutic methods using nucleic acid molecules as described above are considered to provide solutions to diseases that have been considered difficult so far, and more disease-related gene sequences are being identified. Clinical trials of therapeutic methods using nucleic acid molecules for are currently underway. On the other hand, in spite of the usefulness of such nucleic acid molecules as therapeutic agents, pharmacology has solved problems such as stability in serum, delivery to appropriate organs or cells, and uptake into cells. There is still a need for nucleic acid molecules with properties.

Techniques for improving the uptake of nucleic acid molecules into cells include protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, and transformation through calcium phosphate. However, in many of these techniques, the types of cells enabling uptake into cells and the conditions for achieving uptake are limited (Non-Patent Document 6).
Unlike these intracellular uptake techniques, endocytosis via receptors expressed on cell membranes is known as one of the methods for taking up nucleic acid molecules into cells (Non-patent Document 7). A specific ligand for a membrane receptor expressed on the cell membrane is conjugated to the nucleic acid molecule. In this conjugate, after the specific ligand moiety binds to the receptor, the membrane receptor is activated and taken up into the cell by endocytosis. This intracellular uptake mechanism involves the migration of the conjugate bound to the membrane receptor through the invagination of the cell membrane structure to the inside of the region surrounded by the membrane. So far, several intracellular uptake of nucleic acid molecules by receptor-mediated endocytosis has been reported. For example, it has been reported that a sugar chain receptor or a folate receptor is selected as a receptor, and as a result of conjugating a specific ligand to the nucleic acid molecule, incorporation of the nucleic acid molecule into the cell has been achieved. (Patent Documents 1 and 2).
However, the conjugates reported so far are limited to liver diseases because sugar chain receptors are highly expressed in the liver, and folate receptors are limited to cancer because they are expressed in cancer cells. , Delivering nucleic acid molecules for limited diseases. Accordingly, development of a technique for incorporating intracellular nucleic acid molecules with great versatility is strongly desired.
GPCRs are a member of the cell membrane receptor superfamily. These receptors are biologically important and various GPCRs are involved in many diseases. For example, hypertension, myocardial infarction, arrhythmia, atherosclerosis, renal failure, diabetes, asthma, chronic obstructive pulmonary disease, rhinitis, inflammatory disease, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, pain, depression It is involved in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, multiple sclerosis, and cancer, but it is also considered to be involved in other than these diseases (Non-patent Documents 8 and 9).
Most GPCRs are stimulated by binding to GPCR-specific agonists, then phosphorylated by GPCR kinase, undergo endocytosis with GPCR-specific agonists through binding of arrestins, and are taken up into cells (Non-Patent Documents 10 and 11).

WO2009 / 082606 WO2009 / 073809

The present invention provides a transporter for effectively incorporating a nucleic acid drug into a diseased cell, more specifically, a novel compound (conjugate) of a nucleic acid molecule and a transporter (hereinafter referred to as “ It may be abbreviated as “the compound of the present invention”).

In order to solve the above problems, the present inventors have focused on GPCRs that are specifically expressed in diseases. Therefore, when a compound in which a compound capable of becoming a GPCR ligand and a nucleic acid molecule are bound with a linker is produced and allowed to act on a cell, the target nucleic acid molecule is introduced into the target cell, and the present invention is achieved. It came to be completed. It was also confirmed that the nucleic acid molecule portion in the compound functions. Furthermore, it was confirmed that GPCR agonists function effectively in GPCR ligands.

That is, the present invention is as follows.
[1] A conjugate comprising a linker between a G protein-coupled receptor binding ligand (hereinafter sometimes referred to as “GPCR ligand”) and a nucleic acid molecule, wherein the linker is substituted with triazolylene and An optionally substituted polyalkylene glycol chain or an optionally substituted alkylene chain (wherein the alkylene chain is one or more —O—, —S—, —NH—, ═N—, —N═, — ₂ selected from the group consisting of SO _2- , -C (= O)-, (C6-C10) arylene, (C1-C9) heteroarylene, (C3-C8) cycloalkylene and (C3-C7) heterocyclylene. Which may be blocked with a valent group or inserted at the end.
[2] The linker is represented by the following formula (I):

[Where:
^* Represents a binding site with a GPCR ligand;
^** represents a binding site with a nucleic acid molecule,
L is from a phosphate group, a phosphorothioate group, a boranophosphate group, a phosphoroselenate group, a boranophosphate ester group, a hydrogen phosphonate group, a phosphoramidate group, an alkylphosphonate group, an arylphosphonate group, and a phosphotriester group. Represents a group selected from the group consisting of, and is covalently bonded to a pentose group of a nucleic acid molecule,
R ¹ is — (CH ₂ ) _n1 —, — (CH ₂ ) _n2 —O— (CH ₂ ) _n3 —, — (CH ₂ CH ₂ O) _n4 —CH ₂ CH ₂ —, —CH ₂ CH ₂ — S—S—CH ₂ CH ₂ —C (O) —NH— (CH ₂ ) ₆ — and —CH ₂ CH ₂ —O—C (O) —NH— (CH ₂ ) ₆ — are selected from the group consisting of Divalent groups (wherein — (CH ₂ ) _n1 —, — (CH ₂ ) _n2 —O— (CH ₂ ) _n3 — and — (CH ₂ CH ₂ O) _n4 —CH ₂ CH ₂ — are substitutable. Each independently at 1 to 3 (C1-C6) alkyl group optionally substituted with 1 to 3 hydroxyl groups and 1 to 5 substituents selected from the group consisting of hydroxyl groups. even if _{_{_{well, -CH 2 CH 2 -S-S}}} -CH 2 CH 2 -C (O) - H- _(CH _{2) 6} - is substituted at possible ^{^{positions, R 30 R 31 N-C}} (O) - , and ^R 32 OC (O) - identical or selected from the group consisting of a different one to three substituents In which R ³⁰ , R ³¹ and R ³² each independently represents a hydrogen atom or a (C1-C6) alkyl group), n1, n2, n3 and n4 each independently represents an integer of 3 to 20,
R ² represents — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 —, —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n8 —, —C (O) —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n9 —, —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n10 —C (O) —, —C (O) —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n11 —NH—C (O) —, —C (O) — (CH ₂ ) _n12 —, —O— (CH ₂ ) _n13 —, — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— _(CH _{2) n16} - and _{_{- (CH 2) n17 -NH-}} C (O) - (CH 2 CH 2 O) n18 -CH 2 CH 2 -NH-C (O) - (CH 2) n19 - consisting A divalent group selected from the group (wherein the divalent groups are each independently a substitutable position, 1 to 3 hydroxyl groups (C1-C6) optionally substituted with 1 to 3 hydroxyl groups, optionally substituted with the same or different 1 to 10 substituents selected from the group consisting of hydroxyl groups, n5, n6, n7, n8, n9, n10, n11, n12, n13, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 0 to 20,
Q ¹ is represented by the formula (IIa):

(Wherein, ^{R 3} is (represents C1-C6) alkyl or (C3-C8) cycloalkyl group, ^{R 4} is a hydrogen atom, (C1-C6) alkyl or (C3-C8) cycloalkyl group R ³ and R ⁴ may be bonded to each other to form a (C3-C8) member ring, and W ¹ represents —NH—, —O—, —NH—C (O) — (CH ₂ ) _m1 — and —NH—C (O) — (CH ₂ ) _m2 —O— represents a divalent group selected from the group consisting of m1 and m2, each independently representing an integer of 1 to 20 ), Formula (IIb):

(Wherein R ⁵ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W ² represents — (CH ₂ ) _m3 —, — (CH ₂ ) _m4 —NH — And — (CH ₂ ) _m5 —O— represents a divalent group selected from the group consisting of m3, m4 and m5 each independently represents an integer of 1 to 20), Formula (IIc):

(Wherein R ⁶ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W ³ represents — (CH ₂ ) _m6 —, — (CH ₂ ) _m7 —NH _-And- (CH ₂ ) _m8- represents a divalent group selected from the group consisting of -O-, and m6, m7 and m8 each independently represents an integer of 1 to 20), formula (IId):

(Wherein R ^{5 ′} represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W ⁴ represents — (CH ₂ ) _m9 —, — (CH ₂ ) _m10 — NH— and — (CH ₂ ) _m11 —O— represents a divalent group selected from the group consisting of m9, m10 and m11 each independently represents an integer of 1 to 20, and formula (IIe) :

(Wherein R ^{6 ′} represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W ⁵ represents — (CH ₂ ) _m12 —, — (CH ₂ ) _m13 — NH— and — (CH ₂ ) _m14 —O— represents a divalent group selected from the group consisting of m12, m13 and m14 each independently represents an integer of 1 to 20), formula (IIf) :

_Wherein W ⁶ is a divalent group selected from the group consisting of — (CH ₂ ) _m15 — and —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _m16 — (the divalent group is a substitutable position) Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m15 and m16 are Each independently represents an integer of 1 to 10, and W ⁷ represents a single bond or —C (O) —NH— (CH ₂ ) _m17 —, —NH—C (O) — (CH ₂ ) _m18 A divalent group selected from the group consisting of — and —O— (CH ₂ ) _m19 — (the divalent group is each independently a —C (O) —NH— (C1-C6) at a substitutable position. ) Alkyl and —NH—C (O) — (C1-C6) alkyl M17, m18 and m19 each independently represents an integer of 1 to 20), which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of Formula (IIg):

(Wherein, ^{W 8} is, - _(CH _{2) m20} - and _{_{_{_{-CH 2 CH 2 - (OCH 2}}}} CH 2) m21 - divalent group (the bivalent group selected from the group consisting of the substitutable position Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m20 and m21 Each independently represents an integer of 1 to 20, R ⁷ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and R ⁸ and R ⁹ are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R ⁷ and R ⁸ may be bonded to each other to form a (C3-C8) member ring), and IIh):

_Wherein W ⁹ is a divalent group selected from the group consisting of — (CH ₂ ) _m22 — and —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _m23 — (the divalent group is a substitutable position) Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, and may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m22 and m23 are Each independently represents an integer of 1 to 20, R ¹⁰ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and R ¹¹ and R ¹² are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R ¹⁰ and R ¹¹ may be bonded to each other to form a (C3-C8) member ring)
Represents a divalent group selected from the group consisting of
a represents an integer of 0 or 1,
Q ² represents formula (IIIa):

Or formula (IIIb):

(Wherein p1, p2, p3 and p4 each independently represents an integer of 1 to 20, and (CH ₂ ) _p1 and (CH ₂ ) _p3 represent one or more —O—, —C Selected from the group consisting of (═O) —, —C (═O) —NH—, —C (═O) —N (Me) —, —C (═O) —O— and —SO ₂ —NH—. Which may be blocked or inserted at the end with a divalent group)
A ¹ represents (C3-C8) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, (C1-C9) heteroarylene, —O—. (C1-C9) heteroarylene, (C1-C9) heteroarylene-O-, (C3-C7) heterocyclylene, formula (IVa):

Formula (IVb):

Formula (IVc):

And formula (IVd):

And (C3-C8) cycloalkylene may be substituted with 1 to 10 identical or different (C1-C6) alkyl groups, and (C6-C10) arylene. , -O- (C6-C10) arylene, (C6-C10) arylene-O-, (C1-C9) heteroarylene, -O- (C1-C9) heteroarylene, (C1-C9) heteroarylene-O- , (C3-C7) heterocyclylene, the arylene, heteroarylene and heterocyclylene moieties in formula (IVa), formula (IVb), formula (IVc) and formula (IVd) are halogen atoms, (C1-C6) alkyl Group, (C1-C6) alkoxy group, (C1-C6) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group And it may be substituted with 1-6 substituents selected from the group consisting of a trifluoromethoxy group,
b represents an integer of 0 or 1. ]
The conjugate according to [1] above, represented by:
[3] The linker is represented by the following formula (I):

[Where:
^* Represents a binding site with a GPCR ligand;
^** represents a binding site with a nucleic acid molecule,
L is from a phosphate group, a phosphorothioate group, a boranophosphate group, a phosphoroselenate group, a boranophosphate ester group, a hydrogen phosphonate group, a phosphoramidate group, an alkylphosphonate group, an arylphosphonate group, and a phosphotriester group. Represents a group selected from the group consisting of, and is covalently bonded to a pentose group of a nucleic acid molecule,
R ¹ is — (CH ₂ ) _n1 —, — (CH ₂ ) _n2 —O— (CH ₂ ) _n3 —, — (CH ₂ CH
_A divalent group selected from the group consisting of ₂ O) _n4 —CH ₂ CH ₂ — and —CH ₂ CH ₂ —O—C (O) —NH— (CH ₂ ) ₆ —, where — (CH ₂ ) _n1 —, — (CH ₂ ) _n2 —O— (CH ₂ ) _n3 — and — (CH ₂ CH ₂ O) _n4 —CH ₂ CH ₂ — are each independently a substitutable position of 1 to 3 N1 and n2 (which may be substituted with the same or different 1 to 5 substituents selected from the group consisting of (C1-C6) alkyl groups and hydroxyl groups). , N3 and n4 each independently represents an integer of 3 to 20,
R ² represents — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 —, —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n8 —, —C (O) —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n9 —, —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n10 —C (O) —, —C (O) —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _n11 —NH—C (O) —, —C (O) — (CH ₂ ) _n12 —, —O— (CH ₂ ) _n13 — and — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— A divalent group selected from the group consisting of (CH ₂ ) _n16 — (wherein the divalent group may be independently substituted with 1 to 3 hydroxyl groups at substitutable positions; C1-C6) the same or different 1 selected from the group consisting of alkyl groups and hydroxyl groups N5, n6, n7, n8, n9, n10, n11, n12, n13, n14, n15 and n16 each independently represents 0 to 20 Represents an integer,
Q ¹ is represented by the formula (IIa):

Or formula (IIIb):

(Wherein p1, p2, p3 and p4 each independently represents an integer of 1 to 20, and (CH ₂ ) _p1 and (CH ₂ ) _p3 represent one or more —O—, —C Blocked with a divalent group selected from the group consisting of (═O) —, —C (═O) —NH—, —C (═O) —N (Me) — and —C (═O) —O—. Which may be inserted at the end)
A ¹ represents (C3-C8) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, (C1-C9) heteroarylene, —O—. (C1-C9) heteroarylene, (C1-C9) heteroarylene-O-, (C3-C7) heterocyclylene, formula (IVa):

Formula (IVb):

Formula (IVc):

And formula (IVd):

And (C3-C8) cycloalkylene may be substituted with 1 to 10 identical or different (C1-C6) alkyl groups, and (C6-C10) arylene. , -O- (C6-C10) arylene, (C6-C10) arylene-O-, (C1-C9) heteroarylene, -O- (C1-C9) heteroarylene, (C1-C9) heteroarylene-O- , (C3-C7) heterocyclylene, the arylene, heteroarylene and heterocyclylene moieties in formula (IVa), formula (IVb), formula (IVc) and formula (IVd) are halogen atoms, (C1-C6) alkyl Group, (C1-C6) alkoxy group, (C1-C6) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group And it may be substituted with 1-6 substituents selected from the group consisting of a trifluoromethoxy group,
b represents an integer of 0 or 1. ]
The conjugate according to [1] above, represented by:
[4] L is the formula (V):

(Wherein, Z ¹ and Z ² each independently represents an oxygen atom or a sulfur atom) The conjugate according to any one of [2] and [3] above.
[5] The conjugate according to any one of [1] to [4], wherein the nucleic acid molecule is a single-stranded or double-stranded nucleic acid molecule.
[6] The conjugate according to any one of [1] to [5], wherein the nucleic acid molecule is a nucleic acid molecule having 7 to 100 bases.
[7] The conjugate according to any one of [1] to [5] above, wherein the nucleic acid molecule is a nucleic acid molecule having 15 to 100 bases.
[8] The conjugate according to any one of [1] to [7], wherein the nucleic acid molecule is a nucleic acid molecule that interacts with mRNA or a nucleic acid molecule that induces RNA interference.
[9] The conjugate according to any one of [1] to [8] above, wherein the nucleic acid molecule is selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, and antagonistMir.
[10] The conjugate according to any one of [1] to [9] above, wherein the GPCR ligand is a non-peptide ligand structure that binds to GPCR.
[11] The conjugate according to any one of [1] to [10], wherein the GPCR ligand has a partial structure of a GPCR agonist.
[12] The conjugate according to any one of [1] to [11] above, wherein the GPCR ligand is a non-peptide ligand structure that binds to a β ₂ receptor or a 5-HT ₄ receptor.
[13] The GPCR ligand is of formula (VIa):

(Wherein B ¹ represents —CH ₂ OH, —NHCHO or — (CH ₂ ) ₂ OH), formula (VIb):

Formula (VIc):

(Wherein B ² represents a hydrogen atom, methyl or ethyl), formula (VId):

Formula (VIe):

Or formula (VIf):

The conjugate according to any one of [1] to [12], which is a structure represented by:
[14] The GPCR ligand is of formula (VIa):

Formula (VIc):

(Wherein B ² represents a hydrogen atom, methyl or ethyl), or formula (VId):

The conjugate according to any one of [1] to [12], which is a structure represented by:
[15] The GPCR ligand is of formula (VIIa):

Formula (VIIb):

(Wherein B ³ represents a hydrogen atom or methoxy), formula (VIIc):

(Wherein B ⁴ represents methyl or ethyl, and B ⁷ represents a methylene or oxygen atom), formula (VIId):

Or formula (VIIe):

The conjugate according to any one of [1] to [12], which is a structure represented by:
[16] The GPCR ligand is of formula (VIIa):

Formula (VIIb):

(Wherein B ³ represents a hydrogen atom or methoxy), formula (VIIc):

(Wherein B ⁴ represents methyl or ethyl), formula (VIId):

Or formula (VIIe):

The conjugate according to any one of [1] to [12], which is a structure represented by:

[17] A pharmaceutical composition comprising the conjugate according to any one of [1] to [16] as an active ingredient.
[18] Formula (1-1):

(Wherein R ² , A ¹ , p1, b and * are as defined in the above [2] or [3]) and the formula (1-2):

(Wherein R ¹ , Q ¹ , L, p2, a and ** have the same meanings as the above [2] or [3]), and the reaction is carried out in the presence of a catalytic amount of a copper salt. Including
Formula (I-1):

(Each symbol in a formula is synonymous with said [2] or [3].) The manufacturing method of the conjugate as described in said [2] or [3].
[19] Formula (2-1):

(Wherein R ² , A ¹ , p3, b and * are as defined above in [2] or [3]), and a compound represented by formula (2-2):

(Wherein R ¹ , Q ¹ , L, p4, a, and ** are synonymous with the above [2] or [3]), the reaction is carried out in the presence of a catalytic amount of a copper salt. Including
Formula (I-2):

(Each symbol in a formula is synonymous with said [2] or [3].) The manufacturing method of the conjugate as described in said [2] or [3].

According to the present invention, a novel compound comprising a conjugate of a GPCR ligand and a nucleic acid molecule that has an ability to efficiently incorporate into cells by binding to the GPCR is provided.

Hereinafter, the present invention will be described in more detail.

(Definition)

In the present specification, “G protein-coupled receptor binding ligand (hereinafter sometimes referred to as“ GPCR ligand ”)” means a compound that acts on GPCR. GPCR (G protein coupled receptor) refers to a group of receptors that couple with G protein and signal when GPCR ligand is bound. Preferred GPCR ligands in the present invention include non-peptide small molecule agonists.
Specifically, GPCR ligands include non-peptide ligand structures for GPCRs that are known to be internalized by agonists. Preferred examples include non-peptidic ligand structures that bind to β ₂ receptor or 5-HT ₄ receptor, and more preferably formula (VIa):

Formula (VIc):

(Wherein B ² represents a hydrogen atom, methyl or ethyl), formula (VId):

Or formula (VIf):

A non-peptidic ligand structure that binds to the β ₂ receptor represented by formula (VIIa):

Formula (VIIb):

(Wherein B ³ represents a hydrogen atom or methoxy), formula (VIIc):

Or formula (VIIe):

And a non-peptidic ligand structure that binds to the 5-HT ₄ receptor represented by the formula (VIa), (VIb), (VIe) or (VIf) described above is particularly preferred. Non-peptidic ligand structures that bind to the β ₂ receptor, and non-peptidic ligand structures that bind to the 5-HT ₄ receptor represented by formula (VIIa), formula (VIIb), or formula (VIIc). And most preferred are non-peptide ligand structures that bind to the β ₂ receptor represented by the formula (VIa), formula (VIb) or formula (VIf) described above.

As another aspect, more preferably, the formula (VIa):

Formula (VIc):

(Wherein B ² represents a hydrogen atom, methyl or ethyl), or formula (VId):

Formula (VIIb):

(Wherein B ³ represents a hydrogen atom or methoxy), formula (VIIc):

(Wherein B ⁴ represents methyl or ethyl), formula (VIId):

Or formula (VIIe):

And a non-peptidic ligand structure that binds to the 5-HT ₄ receptor represented by the formula (VIa), the formula (VIb), the formula (VIc) or the formula (VId) described above is particularly preferable. Non-peptidic ligand structures that bind to the β ₂ receptor.

In the present specification, the “nucleic acid molecule” means a molecule (oligonucleotide) obtained by polymerizing a nucleotide structure composed of a nucleobase, a pentose group and a phosphate group as a basic unit. This includes modified nucleic acid molecules, and any of the nucleobases, pentose groups or phosphate groups may be modified. The nucleic acid molecule may be DNA, RNA, or a chimera (a nucleic acid containing DNA and RNA in a single-stranded nucleic acid), and may be single-stranded or double-stranded. Examples of the length of the nucleic acid molecule include about 7 to 100 bases, preferably about 15 to 100 bases. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, a double-stranded chimera, an RNA / DNA hybrid, an RNA / chimeric hybrid, a DNA / chimeric hybrid, or a chimeric / chimeric hybrid. Specific examples of nucleic acid molecules include nucleic acid molecules that interact with mRNA (eg, antisense oligonucleotides, antigomir (or antiMir) or exon skip nucleic acid molecules), or nucleic acid molecules that induce RNA interference (eg, siRNA). , MiRNA or shRNA). Specific examples include antisense oligonucleotides, siRNA, miRNA, antigoMir (or antiMir) and the like.

Examples of the “antisense oligonucleotide” include a nucleic acid molecule of about 7 to 100 bases having a direct or indirect interaction with a partial sequence region of mRNA encoding a target protein, preferably 7 to 30 bases. A nucleic acid molecule of about 15 to 20 bases is more preferable.
In another embodiment, the “antisense oligonucleotide” includes a nucleic acid molecule of about 15 to 100 bases having a complementary structure to the partial sequence region of mRNA encoding the target protein, preferably 15 to 30 bases. A nucleic acid molecule of about 15 to 20 bases is more preferable.

“SiRNA” includes a double-stranded nucleic acid molecule (eg, RNA) of about 15 to 100 bases involved in RNA interference that suppresses gene expression specifically for the target mRNA sequence, and preferably 15 to 30. A double-stranded nucleic acid molecule having about a base is mentioned, and a double-stranded nucleic acid molecule having about 21 to 23 bases is more preferable.

“MiRNA” includes a single-stranded nucleic acid molecule (eg, RNA) of about 15 to 100 bases having a function of regulating the expression of other genes, preferably a single-stranded nucleic acid molecule of about 15 to 30 bases. A nucleic acid molecule, and more preferably a single-stranded nucleic acid molecule of about 21 to 25 bases. Further, the miRNA may be pri-miRNA or pre-miRNA which is a precursor of miRNA.

“AntagMir (or antiMir)” includes a single-stranded nucleic acid molecule (eg, RNA) of about 7 to 100 bases having a complementary structure to the partial sequence region of miRNA, and preferably about 7 to 30 bases. A single-stranded nucleic acid molecule is mentioned, More preferably, a single-stranded nucleic acid molecule of about 21 to 25 bases is mentioned.
In another embodiment, “antagoMir (or antiMir)” includes a single-stranded nucleic acid molecule (eg, RNA) of about 15 to 100 bases having a complementary structure to a partial sequence region of miRNA, preferably 15 Examples thereof include single-stranded nucleic acid molecules of about 30 to 30 bases, and more preferably single-stranded nucleic acid molecules of about 21 to 25 bases.

The “complementary structure” may not be a structure that maintains perfect complementarity, but may be a structure that partially maintains complementarity.

The nucleic acid molecule may have 5 bases or less at the 5 'or 3' end, preferably 2 bases, and have an additional base that does not form a base pair. The additional base may be DNA or RNA. Such additional base sequences include, for example, ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, tttt- Examples of the sequence include 3 ′ and uuu-3 ′, but are not limited thereto.

“Nucleic acid base” includes, for example, a purine base or a pyrimidine base. Specific examples thereof include adenine, guanine, thymine, cytosine, uracil and the like.

“Modified nucleobases” include, for example, modified purine bases and modified pyrimidine bases. Specific examples of the modified purine base include hypoxanthine, xanthine, isoguanine, 2-position substituted adenine derivative and 2-position substituted purine derivative, 2-position substituted with amino group, alkylamino, mercapto group, alkylthio group, etc. 2-substituted alkyladenine derivatives substituted with alkyl groups such as methyl groups and 2-position alkyl-substituted hypoxanthine derivatives, 2-position modified guanine derivatives modified with 2-position nitrogen with alkyl groups such as methyl groups, 6-positions such as methyl 6-position alkyl-substituted purine derivatives and 2-position amino 6-position alkyl-substituted purine derivatives modified with an alkyl group, 6-position modified adenine derivatives in which the 6-position nitrogen is modified with an alkyl group such as a methyl group, and the 6-position oxygen with an alkyl such as a methyl group 6-modified guanine derivative and 6-position modified hypoxanthine derivative modified with a group, 2-position nitrogen 2-position modified 6-position modified guanine derivative in which 6-position oxygen is modified with alkyl group such as methyl group, 7-position modified guanine derivative modified in position 7 with methyl group, 8-position alkyl group, halogen, amino group, mercapto And 8-position substituted adenine derivatives and 8-position substituted guanine derivatives substituted with a group, alkylthio group, hydroxyl group and the like. Furthermore, modified purine bases include, for example, deaza derivatives such as 7-deazaadenine, and indole derivatives such as 5-nitroindole.

Specific examples of the modified pyrimidine base include, for example, 2-thiocytosine derivatives and 2-thiouracil derivatives, 3-position alkyl-substituted uracil derivatives substituted with an alkyl group such as a methyl group, 3-position alkyl-substituted cytosine derivatives, 4-thiouracil Derivatives: 4-position modified cytosine derivative in which 4-position nitrogen is modified with a substituent such as acetyl group, 5-position is halogen, trifluoromethyl group, alkyl group, amino group, alkylamino group, alkylaminomethyl group, arylamino group, 5-substituted uracil derivatives, 5-substituted 2-thiouracil derivatives, 5-substituted cytosine derivatives, 5-substituted pyrimidine derivatives, 5-uracil modified with substituents such as alkoxy, propynyl, methoxycarbonylmethyl, and acetic acid oxy groups Derivatives (pseudouracil derivatives), etc. . Further modified pyrimidine bases include, for example, dihydrouracil derivatives, 3-deaza-5-aza-cytosine derivatives, 6-azauracil derivatives, 6-azacytosine derivatives, 6-azothymine derivatives, 6-azapyrimidine derivatives, triazole derivatives, 3-azole derivatives, Also included are pyrrole derivatives such as nitropyrrole, 2-pyridinone derivatives and the like. Other modified nucleobases include J. et al. Org. Chem. , 2011, 76, 7295, Angew. Chem. Int. Ed. , 1991, 30, 613.

Examples of the “pentose group” include a ribose group and a 2-deoxyribose group.

Examples of the “modified pentose group” include modification and conversion of the hydroxyl group at the 2-position of the ribose group, that is, modification of the 2-position of the 2-deoxyribose group. Specific examples of the substituent are selected from the group consisting of a hydrogen atom; a hydroxyl group; a halogen atom; a cyano group, an amino group optionally substituted with a (C1-C6) alkyl group, and a (C1-C6) alkoxy group. (C1-C6) alkoxy group optionally substituted by one or two or more groups which are the same or different; cyano group; azido group; mercapto group; (C1-C6) thioalkoxy group; (C1-C6) An amino group optionally substituted with an alkyl group; (C1-C6) an aminooxy group optionally substituted with an alkyl group; an aminooxy group; a (C1-C6) alkyl group; a (C1-C6) alkenyl group; (C1-C6) alkynyl group; (C1-C6) alkylcarbonylamino group and the like. Preferred examples of the substituent at the 2-position include a hydrogen atom, a hydroxyl group, a fluorine atom, a (C1-C6) alkoxy group, a 2-cyanoethoxy group, and a 2-methoxyethoxy group. In addition, a locked nucleic acid (LNA) in which a hydroxyl group at the 2-position of the ribose group, an amino group, a mercapto group, and the like are bonded to the carbon at the 4-position of the ribose group by a methylene bridge, a hydroxyl group at the 2-position of the ribose group, A locked nucleic acid (ENA) in which an amino group, a mercapto group, etc. are bonded to the carbon at the 4-position of the ribose group by an ethylene bridge, the 2-position of the ribose group and the 4-position of the ribose group are an ether group, an amino group, and a methylene group Also included herein are bridged nucleic acids (BNA) cross-linked by one or two or more identical or different substituents selected from the group consisting of groups. Further, a nucleic acid in which the pentose group is substituted with a morpholinyl group (morpholino nucleic acid (PMO)), a nucleic acid substituted with a cyclobutyl group, a nucleic acid substituted with a pyrrolidinyl group, or a 4′-thionucleic acid substituted with a tetrahydrothiophene group A nucleic acid (peptide nucleic acid (PNA)) in which a pentose group and a phosphate group are substituted with units such as N- (2-aminoethyl) glycine, and a chain structure in which the pentose group is opened Also included herein are substituted nucleic acids (unlocked nucleic acids (UNA)). As an example of LNA, Chem. Rev. 2007, 107, 4672, as an example of BNA, for example, Nucleic Acids Res. , 2009, 37, 1225 and Bioorg. Med. Chem. 2008, 16, 9230, and examples of PMO include J. Pharm. Sci. 2002, 91, 1009, and examples of PNA include FASEB J., et al. 2000, 14, 1041, and examples of UNA include, for example, Bioorg. Med. Chem. , 1995, 3, 19, and J.A. Am. Chem. Soc. 2003, 125, 654.

Examples of the “modified phosphate group” include phosphorothioate group, phosphorodithioate group, boranophosphate group, phosphoroselenate group, boranophosphate ester group, hydrogen phosphonate group, phosphoramidate group, alkylphosphonate group Arylphosphonate groups and phosphotriester groups. Also included herein are compounds in which the linking oxygen of the phosphate group is replaced with sulfur (bridged phosphorothioate), nitrogen (bridged phosphoramidate) or carbon (bridged methylenephosphonate).

The linker in the present invention is not particularly limited as long as it is a part connecting the GPCR ligand and the nucleic acid molecule and has a structure having biocompatibility.
Preferably, it contains triazolylene and an optionally substituted polyalkylene glycol chain or an optionally substituted alkylene chain, and the alkylene chain further comprises one or more —O—, —S—, —NH. -, = N-, -N =, -SO _2- , -C (= O)-, arylene, heteroarylene, cycloalkylene, heterocyclylene, blocked or substituted at the end with a divalent group selected from the group consisting of It has a structure that may be present, and more preferably has a structure represented by the above formula (I).

The “conjugate” which is the compound of the present invention means a compound in which the GPCR ligand and the nucleic acid molecule are bound, and specifically includes the linker between them.

In the present specification, the “polyalkylene glycol chain” means a polyether chain having a structure in which alkylene glycol is polymerized. Specific examples thereof include a polyethylene glycol chain and a polypropylene glycol chain.

In the present specification, the “alkylene chain” means a linear alkylene chain, preferably a linear alkylene chain having 1 to 20 carbon atoms. Specific examples thereof include methylene chain, ethylene chain, propylene chain, butylene chain, pentylene chain, hexylene chain, heptylene chain, octylene chain, nonylene chain, decanylene chain, dodecanylene chain, tetradecanylene chain, hexadecanylene chain, icosylene chain, and the like. Can be mentioned.

Examples of the substituent in the “optionally substituted polyalkylene glycol chain and optionally substituted alkylene chain” include a (C1-C6) alkyl group or hydroxyl group optionally substituted with 1 to 3 hydroxyl groups. Can be mentioned. Preferably, a (C1-C6) alkyl group is used.

In the present specification, “may be blocked by a divalent group or inserted at the end” means a structure in which a divalent group is inserted at any position or at the end of a polyalkylene glycol chain or an alkylene chain. .

In this specification, “L is covalently bonded to the pentose group of a nucleic acid molecule” means, for example, the formula (VIIIa):

(Wherein B ⁵ represents a nucleobase, Y ¹ represents a substituent at the 2-position of the ribose group, and N ¹ represents a single-stranded or double-stranded oligonucleotide) or formula (VIIIb) :

(Wherein B ⁶ represents a nucleobase, Y ² represents a substituent at the 2-position of the ribose group, and N ² represents a single-stranded or double-stranded oligonucleotide). Is bound to the sugar moiety of the nucleic acid molecule.

In the present specification, “(C1-C6) alkyl” means a linear or branched alkyl group having 1 to 6 carbon atoms. Specific examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl and the like.

In the present specification, “(C3-C8) cycloalkyl” means a saturated monocyclic cycloalkyl having 3 to 8 carbon atoms. Specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

In the present specification, “(C1-C6) alkoxy” means straight or branched alkoxy having 1 to 6 carbon atoms. Specific examples thereof include methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy and the like.
In addition, “(C1-C6) alkoxy” in “(C1-C6) alkoxycarbonyl” has the same meaning.

In the present specification, “(C3-C8) cycloalkylene” means a divalent saturated monocyclic cycloalkylene having 3 to 8 carbon atoms. Specific examples thereof include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene and the like.

In the present specification, “(C6-C10) arylene” means a bivalent monocyclic or bicyclic aromatic ring having 6 to 10 carbon atoms. Specific examples thereof include phenylene, 1-naphthylene, 2-naphthylene and the like. Here, when “(C6-C10) arylene” is a bicyclic aromatic ring, one in which one ring is saturated or partially saturated is also included. Specific examples thereof include 1,2,3,4-tetrahydronaphthylene or 2,3-dihydro-1H-indenylene.

In this specification, “(C1-C9) heteroarylene” is a monocyclic or bicyclic ring having 1 to 9 carbon atoms and containing 1 to 4 nitrogen atoms, oxygen atoms and / or sulfur atoms. A 5- to 10-membered divalent aromatic heterocyclic ring. Specific examples thereof include pyridylene, pyridazylene, pyrazinylene, pyrimidinylene, triazinylene, pyrrolylene, pyrazolylene, triazolylene, tetrazolylene, imidazolylene, furylene, thienylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, thiadiazolylene, oxadiazolylene, etc. Monocyclic 5- to 7-membered divalent aromatic heterocycles containing one nitrogen atom, oxygen atom and / or sulfur atom, indoleylene, indazolylene, quinolylene, isoquinolylene, chromenylene, benzofurylene, benzothienylene, benzoxazolylene 1 to 3 nitrogen atoms, oxygen atoms such as benzothiazolylene, benzisoxazolylene, benzisothiazolylene, benzotriazolylene or benzimidazolylene Beauty / or divalent aromatic heterocyclic 9-10 membered ring 2 ring containing a sulfur atom. Preferred heteroarylenes include pyridylene, pyridazylene, pyrazinylene, pyrimidinylene, pyrrolylene, pyrazolylene, furylene, thienylene, indolenylene, indazolylene, quinolylene, isoquinolylene.

In this specification, “(C3-C7) heterocyclylene” is a monocyclic 5 having 3 to 7 carbon atoms and containing 1 to 3 nitrogen atoms, oxygen atoms and / or sulfur atoms. Means a ˜8 membered divalent heterocyclo ring; Specific examples thereof include two monocyclic five-membered rings containing 1 to 3 nitrogen atoms, oxygen atoms and / or sulfur atoms such as tetrahydrofuranylene, pyrrolidinylene, pyrazolidinylene, imidazolidinylene, thiazolidinylene or oxazolidinylene. A monovalent 6-membered divalent ring containing 1 to 3 nitrogen, oxygen and / or sulfur atoms such as a valent heterocyclo ring, piperidinylene, morpholinylene, thiomorpholinylene, piperazinylene, tetrahydropyranylene or dioxanylene And a monocyclic 7-membered divalent heterocyclo ring containing 1 to 3 nitrogen atoms, oxygen atoms and / or sulfur atoms such as azepanylene, homopiperazinylene or oxepanylene. Preferable heterocyclylene includes pyrrolidinylene, piperidinylene and the like.

The two bonding positions in “cycloalkylene”, “arylene”, “heteroarylene” and “heterocyclylene” may be anywhere as long as possible.

In the present specification, “triazolylene” means a monocyclic 5-membered divalent heterocyclo ring containing three nitrogen atoms, and specific examples thereof include 1,2,3-triazolylene or 1,2 , 4-triazolylene. Preferred triazolylene includes 1,2,3-triazolylene.

In the present specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
In the present specification, the “(C3-C8) membered ring” means a divalent saturated monocyclic cycloalkane having 3 to 8 carbon atoms. Specific examples thereof include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and the like.

(Compound of the present invention)
The compound of the present invention is a conjugate comprising a linker between a GPCR ligand and a nucleic acid molecule, wherein the linker is triazolylene and an optionally substituted polyalkylene glycol chain or an optionally substituted alkylene chain. (Wherein the alkylene chain is one or more —O—, —S—, —NH—, ═N—, —N═, —SO ₂ —, —C (═O) —, (C6-C10 And a divalent group selected from the group consisting of arylene, (C1-C9) heteroarylene, (C3-C8) cycloalkylene and (C3-C7) heterocyclylene. It is the above conjugate. Specifically, the linker is represented by the following formula (I):

[Each symbol in the formula is as defined above. ]
It is a conjugate represented by these.

Hereinafter, each group of the compound of the present invention will be described.

R ¹ is preferably — (CH ₂ ) _n1 —, —CH ₂ CH ₂ —SS—CH ₂ CH ₂ —C (O) —NH— (CH ₂ ) ₆ — and —CH ₂ CH ₂ A divalent group selected from the group consisting of —O—C (O) —NH— (CH ₂ ) ₆ — (wherein n1 represents an integer of 3 to 10, and — (CH ₂ ) _n1 — is substitutable. And may be substituted with the same or different 1 to 5 substituents selected from the group consisting of (C1-C4) alkyl group, hydroxyl group and hydroxymethyl group, and —CH ₂ CH ₂ —SS —CH ₂ CH ₂ —C (O) —NH— (CH ₂ ) ₆ — is a substitutable position from the group consisting of R ³⁰ R ³¹ N—C (O) — and R ³² OC (O) —. It may be substituted with 1 to 3 identical or different substituents selected. In ^this, R ^{30, R 31} and ^{R 32} each independently represent a hydrogen atom or a (C1-C6) alkyl group.) Can be mentioned.
In another embodiment, R ¹ is preferably a divalent group selected from the group consisting of — (CH ₂ ) _n1 — and —CH ₂ CH ₂ —O—C (O) —NH— (CH ₂ ) ₆ —. A group (wherein n1 represents an integer of 3 to 10, and — (CH ₂ ) _n1 — represents a substitutable position selected from the group consisting of a (C1-C4) alkyl group, a hydroxyl group and a hydroxymethyl group. Or may be substituted with 1 to 5 different substituents).
More preferred is a divalent group represented by — (CH ₂ ) _n1 — (where n1 represents an integer of 3 to 10).

R ² is preferably — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 —, — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— ( CH ₂₎ _n16 - and _{_{- (CH 2) n17 -NH-}} C (O) - (CH 2 CH 2 O) n18 -CH 2 CH 2 -NH-C (O) - (CH 2) n19 - group consisting of (Wherein n5, n6, n7, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 0 to 20, and the divalent group can be substituted) Each independently at 1 to 3 (C1-C6) alkyl group optionally substituted with 1 to 3 hydroxyl groups and 1 to 10 substituents selected from the group consisting of hydroxyl groups. May be included) . More preferably, — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 —, — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— (CH ₂ ) _n16 Two selected from the group consisting of — and — (CH ₂ ) _n17 —NH—C (O) — (CH ₂ CH ₂ O) _n18 —CH ₂ CH ₂ —NH—C (O) — (CH ₂ ) _n19 — A valent group (here, n5, n6, n7, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 0 to 20, and the divalent group is a substitutable position; Each independently may be substituted with 1 to 3 (C1-C4) alkyl groups).
In another embodiment, R ² is preferably — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 — and — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ). _a divalent group selected from the group consisting of _n15 —O— (CH ₂ ) _n16 — (wherein n5, n6, n7, n14, n15 and n16 each independently represents an integer of 0 to 12, The divalent group is independently substituted with 1 to 5 identical or different substituents selected from the group consisting of a (C1-C4) alkyl group, a hydroxyl group and a hydroxymethyl group at substitutable positions. May be included). More preferably, — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 — and — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— (CH ₂ ) _n16 A divalent group selected from the group consisting of (wherein n5, n6, n7, n14, n15 and n16 each independently represents an integer of 0 to 12, and the divalent group is a substitutable position) And each independently may be substituted with 1 to 3 (C1-C4) alkyl groups).

R ³ is preferably a (C1-C6) alkyl group. R ⁴ is preferably a hydrogen atom or a (C1-C6) alkyl group. Alternatively, preferably, a (C3-C8) member ring formed by combining R ³ and R ⁴ with each other can be mentioned.

R ⁵ , R ^{5 ′} , R ⁶ , R ^{6 ′} , R ⁹ and R ¹² are preferably each independently a hydrogen atom or a (C1-C6) alkyl group.

R ⁷ and R ¹⁰ are preferably each independently a hydrogen atom or a (C1-C6) alkyl group. R ⁸ and R ¹¹ are preferably each independently a (C1-C6) alkyl group.

Q ¹ is preferably a divalent group selected from the group consisting of the formula (IIa), formula (IIb), formula (IIc), formula (IIe) and formula (IIf) described above.
More preferably, a divalent group selected from the group consisting of the formula (IIb), the formula (IIc) and the formula (IIe) described above can be used, and more preferably the formula (IIc) and the formula (IIe) described above. A divalent group selected from the group consisting of
In another embodiment, Q ¹ preferably includes a divalent group selected from the group consisting of the formula (IIa), formula (IIb), formula (IIc) and formula (IIf) described above.
More preferably, a divalent group selected from the group consisting of the formula (IIb) and the formula (IIc) described above is used.

As Q ² , (CH ₂ ) _p1 and (CH ₂ ) _p3 described above may preferably be blocked or inserted at one end with one or more —O— or —SO ₂ —NH—. Examples thereof include a divalent group selected from the group consisting of the above-described formula (IIIa) and formula (IIIb).
In another aspect, as Q ² , preferably, (CH ₂ ) _p1 and (CH ₂ ) _p3 described above may be blocked or inserted at one end with one or more —O—. And a divalent group selected from the group consisting of formula (IIIa) and formula (IIIb).

A ¹ is preferably (C3-C7) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, and (C3-C7) heterogene. Cyclylene, a divalent group selected from the group consisting of formula (IVa), formula (IVb), formula (IVc) and formula (IVd) as described above (wherein (C3-C7) cycloalkylene and (C3-C7) ) Heterocyclylene may be substituted with 1 to 4 identical or different (C1-C4) alkyl groups, (C6-C10) arylene, —O— (C6-C10) arylene and (C6-C10) The arylene moiety in) arylene-O— is a halogen atom, a (C1-C4) alkyl group, a (C1-C4) alkoxy group, or a (C1-C4) alkoxy group. And may be substituted with 1 to 4 substituents selected from the group consisting of a sulfonyl group, a carboxyl group, a cyano group, a hydroxyl group, a trifluoromethyl group, and a trifluoromethoxy group.
More preferably, (C6-C10) arylene, -O- (C6-C10) arylene and (C6-C10) arylene-O-, the above-described formula (IVa), formula (IVb), formula (IVc) and formula A divalent group selected from the group consisting of (IVd) (wherein (C6-C10) arylene, -O- (C6-C10) arylene and (C6-C10) arylene-O-, the arylene moiety is a halogen atom, 1 to 4 selected from the group consisting of (C1-C4) alkyl group, (C1-C4) alkoxy group, (C1-C4) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group and trifluoromethoxy group. Optionally substituted with 4 substituents). Particularly preferably, a divalent group selected from the group consisting of (C6-C10) arylene (preferably phenylene), —O— (C6-C10) arylene (preferably —O-phenylene) and the above-described formula (IVc). In the groups (wherein (C6-C10) arylene and -O- (C6-C10) arylene are halogen atoms, (C1-C4) alkyl groups, (C1-C4) alkoxy groups, (C1-C4)) And may be substituted with 1 to 4 substituents selected from the group consisting of an alkoxycarbonyl group, a carboxyl group, a cyano group, a hydroxyl group, a trifluoromethyl group, and a trifluoromethoxy group.

Examples of W ¹ include —NH—C (O) — (CH ₂ ) _m1 —, —NH—C (O) — (CH ₂ ) _m2 —O—, —NH—, or —O—.

W ² , W ³ , W ⁴ and W ⁵ are preferably each independently, — (CH ₂ ) _m3 —, — (CH ₂ ) _m6 —, — (CH ₂ ) _m9 — or — (CH ₂ ) _M12- .

W ⁶ , W ⁸ and W ⁹ are preferably each independently, — (CH ₂ ) _m15 —, — (CH ₂ ) _m20 — or — (CH ₂ ) _m22 — (wherein the divalent group Each independently may be substituted at the substitutable position with the same or different 1 to 10 substituents selected from the group consisting of (C1-C6) alkyl groups, hydroxyl groups and amino groups. Can be mentioned.

W ⁷ is preferably a single bond or a divalent group selected from the group consisting of —C (O) —NH— (CH ₂ ) _m17 — and —O— (CH ₂ ) _m19 — (wherein The divalent group is independently selected from the group consisting of C (O) —NH— (C1-C6) alkyl group and NH—C (O) — (C1-C6) alkyl at substitutable positions. And may be substituted with 1 to 10 substituents which may be the same or different.

Z ¹ and Z ² are preferably oxygen atoms.

N1 to n4 are each independently and preferably an integer of 3 to 10, and n5 to n16 are each independently and preferably an integer of 0 to 10.

M1 to m23 are each independently preferably an integer of 1 to 10.

P1 to p4 are preferably each independently an integer of 1 to 10.

A is 0 or 1, and b is preferably 1.

One aspect of the present invention is a conjugate in which R ¹ , R ² , Q ¹ , Q ² , A ¹ , L, a and b and the GPCR ligand in formula (I) respectively represent:
R ¹ is — (CH ₂ ) _n1 — (where n1 represents an integer of 3 to 10) or —CH ₂ CH ₂ —O—C (O) —NH— (CH ₂ ) ₆ —. Represents a divalent group;
R ² represents — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 — or — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— (CH ₂ ) _n16 -(Wherein n5, n6, n7, n14, n15 and n16 each independently represents an integer of 0 to 10);
Q ¹ is represented by formula (IIa):

(Wherein, ^{R 3} is (represents C1-C6) alkyl or (C3-C8) cycloalkyl group, ^{R 4} is a hydrogen atom, (C1-C6) alkyl or (C3-C8) cycloalkyl group R ³ and R ⁴ may be bonded to each other to form a (C3-C8) member ring, and W ¹ represents —NH—, —O—, —NH—C (O) — (CH ₂ ) _m1 — and —NH—C (O) — (CH ₂ ) _m2 —O— represents a divalent group selected from the group consisting of m1 and m2, each independently representing an integer of 1 to 20 ), Formula (IIb ′):

(Wherein R ⁵ represents a hydrogen atom or a (C1-C4) alkyl group, W ² represents a divalent group represented by — (CH ₂ ) _m3 —, and m3 represents an integer of 1 to 10. And formula (IIc ′):

(Wherein R ⁶ represents a hydrogen atom or a (C1-C4) alkyl group, W ³ represents a divalent group represented by — (CH ₂ ) _m6 —, and m6 represents an integer of 1 to 10. And formula (IIe):

(Wherein R ^{6 ′} represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W ⁵ represents — (CH ₂ ) _m12 —, — (CH ₂ ) _m13 — NH— and — (CH ₂ ) _m14 —O— represents a divalent group selected from the group consisting of m12, m13 and m14 each independently represents an integer of 1 to 20) or formula (IIf) :

_Wherein W ⁶ is a divalent group selected from the group consisting of — (CH ₂ ) _m15 — and —CH ₂ CH ₂ — (OCH ₂ CH ₂ ) _m16 — (the divalent group is a substitutable position) Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m15 and m16 are Each independently represents an integer of 1 to 10, and W ⁷ represents a single bond or —C (O) —NH— (CH ₂ ) _m17 —, —NH—C (O) — (CH ₂ ) _m18 A divalent group selected from the group consisting of — and —O— (CH ₂ ) _m19 — (the divalent group is each independently a —C (O) —NH— (C1-C6) at a substitutable position. ) Alkyl and —NH—C (O) — (C1-C6) alkyl M17, m18 and m19 each independently represents an integer of 1 to 20), which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of Represents a divalent group represented;
Q ² represents a divalent group represented by the above formula (IIIa) or (IIIb);
A ¹ is (C6-C10) arylene, —O— (C6-C10) arylene and (C6-C10) arylene-O—, the above-mentioned formula (IVa), formula (IVb), formula (IVc) and formula A divalent group selected from the group consisting of (IVd) (wherein (C6-C10) arylene, -O- (C6-C10) arylene and (C6-C10) arylene-O-, the arylene moiety is a halogen atom, 1 to 4 selected from the group consisting of (C1-C4) alkyl group, (C1-C4) alkoxy group, (C1-C4) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group and trifluoromethoxy group. And optionally substituted with 4 substituents;
L represents the above-described formula (V) (wherein Z ¹ and Z ² each independently represents an oxygen atom or a sulfur atom);
a represents 0 or 1;
b represents 0 or 1;
The GPCR ligand represents formula (VIa), formula (VIb), formula (VIc) or formula (VId) as described above.
Another aspect of the invention is a conjugate in which R ¹ , R ² , Q ² , A ¹ , L, a and b and the GPCR ligand in formula (I) each represent:
R ¹ is — (CH ₂ ) _n1 — (where n1 represents an integer of 3 to 10) or —CH ₂ CH ₂ —O—C (O) —NH— (CH ₂ ) ₆ —. Represents a divalent group;
R ² represents — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 — or — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— (CH ₂ ) _n16 -In which n5, n6, n7, n14, n15 and n16 each independently represents an integer of 3 to 10;
Q ² represents a divalent group represented by the above formula (IIIa) or (IIIb);
A ¹ is a divalent group selected from the group consisting of phenylene, —O-phenylene, phenylene-O—, formula (IVa), formula (IVb), formula (IVc) and formula (IVd) described above (herein , Phenylene, —O-phenylene and (phenylene in —O— include a halogen atom, a (C1-C4) alkyl group, a (C1-C4) alkoxy group, a (C1-C4) alkoxycarbonyl group, a carboxyl group, a cyano group, And optionally substituted with 1 to 4 substituents selected from the group consisting of a group, a hydroxyl group, a trifluoromethyl group, and a trifluoromethoxy group;
L represents the above-described formula (V) (wherein Z ¹ and Z ² each independently represents an oxygen atom or a sulfur atom);
a represents 0;
b represents 1;
The GPCR ligand represents formula (VIa) as described above.
Yet another aspect of the invention is the conjugate in which R ¹ , R ² , Q ² , A ¹ , L, a and b and the GPCR ligand in formula (I) respectively represent:
R ¹ is — (CH ₂ ) _n1 —, —CH ₂ CH ₂ —S—S—CH ₂ CH ₂ —C (O) —NH— (CH ₂ ) ₆ — or —CH ₂ CH ₂ —O—C A divalent group which is (O) —NH— (CH ₂ ) ₆ — (where n1 represents an integer of 3 to 10, and —CH ₂ CH ₂ —S—S—CH ₂ CH ₂ —C (O) —NH— (CH ₂ ) ₆ — is a substitutable position and 1 to 3 R ³⁰ R ³¹ N—C (O) — (wherein R ³⁰ , R ³¹ and R ³² are each independently Represents a hydrogen atom or a (C1-C6) alkyl group), and may be substituted);
R ² represents — (CH ₂ ) _n5 —, — (CH ₂ ) _n6 —O— (CH ₂ ) _n7 —, — (CH ₂ ) _n14 — (OCH ₂ CH ₂ ) _n15 —O— (CH ₂ ) _n16 — Or — (CH ₂ ) _n17 —NH—C (O) — (CH ₂ CH ₂ O) _n18 —CH ₂ CH ₂ —NH—C (O) — (CH ₂ ) _n19 — (where n5, n6 N7, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 3 to 10).
Q ¹ represents a divalent group of formula (IIb), formula (IIc) or formula (IIe) as described above;
Q ² represents the above-described formula (IIIa) or (IIIb) (wherein p1, p2, p3 and p4 each independently represents an integer of 1 to 20, and (CH ₂ ) _p1 and ( CH ₂ ) _p3 represents a divalent group that may be blocked or inserted at the end with a divalent group selected from the group consisting of one or more —O— and —SO ₂ —NH—;
A ¹ is a divalent group selected from the group consisting of phenylene, —O-phenylene, phenylene-O— and the above-described formula (IVc) (wherein phenylene, —O-phenylene and phenylene in (phenylene-O—) The moiety consists of a halogen atom, (C1-C4) alkyl group, (C1-C4) alkoxy group, (C1-C4) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group and trifluoromethoxy group. Which may be substituted with 1 to 4 substituents selected from the group;
L represents the above-described formula (V) (wherein Z ¹ and Z ² represent an oxygen atom);
a represents 0 or 1;
b represents 0 or 1;
The GPCR ligand represents a structure represented by the above formula (VIa), formula (VIb), formula (VIe), formula (VIf), formula (VIIa), formula (VIIb) or formula (VIIc).
Each of the above compound groups limited by one or a combination of any of these exemplifications is also an embodiment of a preferred compound of the present invention.

In the present specification, the number of substituents in the group defined as “optionally substituted” is not particularly limited as long as it is substitutable unless otherwise defined, and is one or more. In addition, unless otherwise specified, the description of each group also applies when the group is a part of another group or a substituent.

The compounds of the present invention may form pharmaceutically acceptable salts, such as salts with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid; formic acid, Salts with organic carboxylic acids such as acetic acid, fumaric acid, maleic acid, oxalic acid, citric acid, malic acid, tartaric acid, aspartic acid, glutamic acid; methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, hydroxybenzenesulfonic acid , Salts with sulfonic acids such as dihydroxybenzene sulfonic acid; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salt; triethylamine salt, pyridine salt, picoline salt, Ethanolamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine and Salts and the like.

The compound of the present invention and a pharmaceutically acceptable salt thereof may be a hydrate or a solvate such as an ethanolate, and these hydrates and / or solvates are also compounds according to the present invention. And pharmaceutically acceptable salts thereof.

When the compound of the present invention has a stereoisomer, a tautomer and / or an optical isomer, the compound of the present invention includes a mixture of these isomers and an isolated isomer.

The compound of the present invention and a pharmaceutically acceptable salt thereof may be a crystal, and may be a single crystal form or a crystal form mixture.

Compounds labeled with isotopes (eg, ² H, ³ H, ¹⁴ C) and the like are also encompassed in the compounds of the present invention. Further, compound (I) may be a deuterium converter.

(Method for producing the compound of the present invention)
The production method of the compound of the present invention is not particularly limited. For example, the compound of the present invention in which the linker is represented by the formula (I) can be produced by the following production methods 1 and 2 and the like.

The raw material compound can be produced by the production method shown below and the like, and can be easily obtained as a commercial product unless otherwise specified, or can be produced according to a method known per se or a method analogous thereto.

Further, the compounds of the present invention whose linker is other than the formula (I) can be produced in the same manner using known methods.

In addition, the compound obtained at each step in the following reaction formula can be used in the next reaction as a reaction solution or as a crude product. Moreover, the compound used by the following manufacturing method may form a salt in the range which does not interfere with reaction. Alternatively, the compound can be isolated from the reaction mixture according to a conventional method, and can be easily purified by usual separation means such as recrystallization, distillation, chromatography and the like.

The compound of the present invention or a pharmaceutically acceptable salt thereof is a novel compound, and can be produced, for example, by the method described below, the examples described later and a method analogous thereto.

[Production Method 1]
In the formula (I), Q ² represents the following formula (IIIa):

[Compound of the following formula (I-1)] can be produced, for example, according to the following method.

(Wherein R ¹ , R ² , Q ¹ , A ¹ , L, p ¹ , p 2, a, b, ^*, and ^** have the same meanings as [2] or [3] above).

[Step 1]
Compound (I-1) is produced by reacting compound (1-1) with compound (1-2). The reaction can be carried out according to the reaction conditions of a conventional Huisgen azide-alkene cyclization reaction using a catalytic amount of a copper salt (Lallana et al., Angew. Chem. Int. Ed. (2011) 50: 8794-8804). . Specifically, examples of the copper salt include divalent copper salts such as copper sulfate, and monovalent copper salts such as copper bromide, copper iodide, and copper trifluoromethylsulfonate. Additives and ligands may be used. Examples of the additive include reducing agents such as sodium ascorbate and tris (2-carboxyethyl) phosphine (TCEP). Examples of the ligand include tris (benzyltriazolylmethyl) amine (TBTA), Examples include tris (hydroxypropyltriazolylmethyl) amine (THPTA), bathophenanthroline disulfonate (BPDS), and the like. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents. (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, etc.), ketone solvent (acetone, etc.), water (including buffer solution), alcohol solvent (methanol, ethanol, t-butanol, etc.), or these The mixed solvent is mentioned. The reaction temperature can usually be selected from the range of −20 to 100 ° C., preferably 0 ° C. to room temperature.

[Production Method 2]
In the formula (I), Q ² represents the following formula (IIIb):

[Compound of the following formula (I-2)] can be produced, for example, according to the following method.

(Wherein R ¹ , R ² , Q ¹ , A ¹ , L, p3, p4, a, b, ^*, and ^** have the same meanings as [2] or [3] above).

[Step 1]
Compound (I-2) is produced from compound (2-1) and compound (2-2) by the same method as in Step 1 described in Production Method 1.

[Production method of (1-2)]
The starting compound (1-2) used in Production Method 1 can be produced, for example, according to the following method.

(Wherein R ¹ , Q ¹ , L, p2, a and ^** have the same ^{meanings as} the above [2] or [3], and X ¹ is, for example, a chlorine atom, a bromine atom, alkoxycarbonyloxy, phenoxy, Activating group or hydroxyl group of a carboxylic acid derivative such as p-nitrophenoxy or succinimidyloxy)

[Step 1]
Compound (1-2) is synthesized by reacting compound (3-1) with compound (3-2). When X ¹ is an activating group of a carboxylic acid such as chlorine atom, bromine atom, alkoxycarbonyloxy, phenoxy, p-nitrophenoxy or succinimidyloxy, for example, the reaction can be carried out in the presence or absence of a base. Do it. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). DMSO, NMP, acetonitrile, etc.), ketone solvents (acetone, etc.), water (including buffer solution), alcohol solvents (methanol, ethanol, etc.), or mixed solvents thereof. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- Diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably potassium carbonate, diisopropylethylamine, triethylamine. Can be mentioned. The amount of the base used is usually selected from the range of 1 to 5 equivalents relative to compound (3-2). The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably in the range of −78 to 50 ° C.

When X ¹ is a hydroxyl group, the reaction is performed in the presence of a condensing agent. Examples of the condensing agent include carbodiimide (dicyclohexylcarbodiimide, 1-ethyl-3- (dimethylaminopropyl) carbodiimide, etc.), phosphonium salt (BOP, etc.), guanidinium salt (HBTU, etc.), etc. Agents (1-hydroxybenzotriazole, etc.) and bases (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4] .3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, acetonitrile, etc.), ketone solvents (acetone, etc.), or mixed solvents thereof. The reaction temperature is usually selected from the range of 0 to 100 ° C., preferably 0 to 50 ° C.

In the case where X ¹ is a hydroxyl group, if necessary, the hydroxyl group is converted into an activating group such as a chlorine atom, a bromine atom, alkoxycarbonyloxy or succinimidyloxy, and then compound (3-2) Compound (1-2) is produced by reacting with.

When X ¹ is converted from a hydroxyl group to a chlorine atom or a bromine atom, for example, thionyl chloride, oxalyl chloride, phosphorus trichloride, phosphorus pentachloride, phosphorus oxychloride, dichlorotriphenylphosphorane, carbon tetrachloride-triphenylphosphine, odor Thionyl chloride, dibromotriphenylphosphorane and the like are used. Examples of the solvent include an aprotic solvent (such as toluene), a halogen-based solvent (such as methylene chloride and chloroform), or a mixed solvent thereof. Moreover, you may use additives, such as DMF, as needed. The reaction temperature can usually be selected from the range of −78 to 100 ° C.

When X ¹ is converted from a hydroxyl group to alkoxycarbonyloxy, it is carried out by reacting, for example, an alkyl halide formate such as ethyl chloroformate, isopropyl chloroformate, or isobutyl chloroformate in the presence of a base. The amount of the halogenated alkyl formate used is usually selected from the range of 1 to 5 equivalents relative to the carboxylic acid. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), or a mixed solvent thereof. Is mentioned. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- And diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like, preferably N-methylmorpholine, triethylamine, diisopropyl Ethylamine is mentioned. The amount of the base used is usually selected from the range of 1 to 10 equivalents relative to the carboxylic acid. The reaction temperature can usually be selected from the range of −78 to 100 ° C., preferably in the range of −78 ° C. to room temperature.

X ¹ is converted from a hydroxyl group to succinimidyloxy by reacting N-hydroxysuccinimide in the presence of a condensing agent. Examples of the condensing agent include carbodiimide (dicyclohexylcarbodiimide, 1-ethyl-3- (dimethylaminopropyl) carbodiimide, etc.), phosphonium salt (BOP, etc.), guanidinium salt (HBTU, etc.), etc. Agents (1-hydroxybenzotriazole, etc.) and bases (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4] .3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like. The amount of N-hydroxysuccinimide and condensing agent used is usually selected from the range of 1 to 5 equivalents relative to the carboxylic acid. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, acetonitrile, etc.), ketone solvents (acetone, etc.), or mixed solvents thereof. The reaction temperature is usually selected from the range of 0 to 100 ° C., preferably 0 to 50 ° C.

The compound (3-2) is synthesized according to an ordinary nucleic acid synthesis method such as a phosphoramidite method using amidite reagent (Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, New York (2000)).

[Production method of (2-2)]
The starting compound (2-2) used in Production Method 2 can be produced, for example, according to the following method.

(In the formula, R ¹ , Q ¹ , L, p4, a and ^** are as defined in [2] or [3] above, P ¹¹ is a protecting group for carboxylic acid, and X ² is a chlorine atom. , Bromine atom, iodine atom, methanesulfonyloxy group (hereinafter sometimes referred to as “OMs”), p-toluenesulfonyloxy group (hereinafter sometimes referred to as “OTs”), trifluoromethanesulfonyloxy group (Hereinafter, it may be referred to as “OTf”.) Or a hydroxyl group.)

[Step 1]
Compound (4-2) is synthesized by reacting compound (4-1) with an azidation reagent in the presence or absence of a base.

When X ² is a chlorine atom, bromine atom, iodine atom, OMs, OTs or OTf, examples of the azide reagent include metal azide compounds such as sodium azide, organic azide compounds such as trimethylsilyl azide, tetrabutylammonium azide And quaternary ammonium azide compounds. The amount of the azidation reagent used is usually selected from the range of 1 to 10 equivalents relative to the compound (4-1). Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, HMPT, etc.), Water, alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof can be used. If necessary, for example, a phase transfer catalyst such as tetrabutylammonium chloride may be added as an additive. The reaction temperature can usually be selected from the range of 0 ° C. to 150 ° C., and preferably in the range of room temperature to 100 ° C.

When X ² is a hydroxyl group, examples of the azidation reagent include diphenyl phosphate azide, and the amount used is usually selected from the range of 1 to 10 equivalents relative to compound (4-1). Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide, DMSO). , NMP, acetonitrile, HMPT, etc.), ketone solvents (acetone, etc.) or mixed solvents thereof. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), organic bases ( N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, 1 , 4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably 1,4-diazabicyclo [5.4.0] undec-7-ene. The amount of the base used is usually selected from the range of 1 to 10 equivalents relative to compound (4-1). The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably 0 to 100 ° C.

Also, when ^{X 2} is a hydroxyl group, once, it is also possible to convert the ^{X 2} chlorine atom, a bromine atom, an iodine atom, OMs, the OTs or OTf.

When X ² is converted from a hydroxyl group to a chlorine atom, a bromine atom or an iodine atom, it is carried out by reacting a halogenating agent in the presence or absence of an acid. Examples of the halogenating agent include chlorinating agents such as thionyl chloride, oxalyl chloride, phosphorus trichloride, phosphorus pentachloride, phosphorus oxychloride, carbon tetrachloride-triphenylphosphine, N-chlorosuccinimide-triphenylphosphine, Brominating agents such as thionyl, hydrobromic acid, phosphorus tribromide, carbon tetrabromide-triphenylphosphine, N-bromosuccinimide-triphenylphosphine, bromine-triphenylphosphine, hydroiodic acid, potassium iodide, And an iodinating agent such as iodine-triphenylphosphine. Examples of the acid used include hydrochloric acid, sulfuric acid, phosphoric acid and the like. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents. (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, HMPT, etc.), water, or a mixed solvent thereof may be mentioned, and no solvent may be used. Moreover, you may add a base as needed. Examples of the base used include organic bases (imidazole, N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like). The reaction temperature can usually be selected from the range of −78 to 100 ° C., preferably 0 to 100 ° C.

When X ² is converted from a hydroxyl group to OMs, OTs, or OTf, the reaction is performed by reacting a sulfonylating agent in the presence of a base. Examples of the sulfonylating agent include methanesulfonyl chloride, tosyl chloride, trifluorosulfonic acid anhydride, and the like. The amount of the sulfonylating agent used is usually selected from the range of 1 to 5 equivalents relative to the compound wherein X ² is a hydroxyl group. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), ketone solvents (acetone, etc.) or a mixed solvent thereof. It is done. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), organic bases ( N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, 1 , 4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably N-methylmorpholine, triethylamine, diisopropylethylamine. The amount of the base used is usually selected from the range of 1 to 10 equivalents relative to compound (4-1). The reaction temperature can usually be selected from the range of −78 to 100 ° C., preferably in the range of −78 ° C. to room temperature.

[Step 2]
For example, by using the general deprotection conditions for protecting groups of carboxylic acids shown in the literature (Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.)), compound (4-2) can be used. Compound (4-3) is produced.

[Step 3]
Compound (2-2) is produced from compound (4-3) and compound (3-2) by the same method as in the case where X ^{1 in} Step 1 of the production method described in (1-2) is a hydroxyl group. Is done. Specifically, a method using a condensing agent or a hydroxyl group of the carboxylic acid of the compound (4-3) is converted into an activating group such as a chlorine atom, a bromine atom, alkoxycarbonyloxy or succinimidyloxy. Thereafter, compound (2-2) is produced by a method of reacting with compound (3-2).

[Production method of (3-1a)]
In compound (3-1), Q ¹ is

The compound of the following formula (3-1a) in which a is 1 can be produced, for example, according to the following method.

(Wherein R ³ , R ⁴ and p2 have the same meanings as [2] above, W ¹¹ has the same meaning as W ^{1 in} [2] above, and P ¹² represents a hydrogen atom or a general amino group or hydroxyl group. X ³ is a protecting group, for example, a chlorine atom, a bromine atom, an activation group of a carboxylic acid such as alkoxycarbonyloxy or succinimidyloxy, or a hydroxyl group, and X ⁴ is phenoxy, p-nitrophenoxy or Activating group such as alkoxy.)

[Step 1]
Compound (5-3) is produced from compound (5-1) and compound (5-2) by the same method as in Step 1 described in the production method for (1-2).

[Step 2]
If P ¹² is a protecting group, for example, the literature (Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.)) By using the general deprotection conditions shown ^in, once the ^{P 12,} Convert to hydrogen atom. Subsequently, in the presence of a base, for example, an aryl halide formate such as phenyl chloroformate or chloroformate (p-nitrophenyl), an alkyl formate such as ethyl chloroformate, isopropyl chloroformate or isobutyl chloroformate is reacted. To produce compound (3-1a). The amount of aryl halide formate or alkyl halide formate to be used is usually selected from the range of 1 to 5 equivalents relative to compound (5-3). Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), or a mixed solvent thereof. Is mentioned. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- And diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like, preferably N-methylmorpholine, triethylamine, diisopropyl Ethylamine is mentioned. The amount of the base used is usually selected from the range of 1 to 10 equivalents relative to the carboxylic acid. The reaction temperature can usually be selected from the range of −78 to 100 ° C., preferably in the range of −78 ° C. to room temperature.

[Production method of (3-1b)]
In compound (3-1), Q ¹ represents the following formula (IIb):

Or formula (IIc):

The compound of the following formula (3-1b) in which a is 1 can be produced, for example, according to the following method.

(Wherein, p2 has the same meaning as the above-mentioned ^{[2], R 21} has the same meaning as ^{R 5} or ^{R 6} of the above ^{[2], W 21} has the same definition as ^{W 2} or ^{W 3} of the [2] , X ¹ is synonymous with the description of the production method of (1-2), X ³ is synonymous with the description of the production method of (3-1a), and R ²² is a carboxylic acid or a protected carboxylic acid, a hydrogen atom Or a protective group for a hydroxyl group or an amine, and X ⁵ is a chlorine atom, a bromine atom, an iodine atom, OMs, OTs, OTf, or a hydroxyl group.

[Step 1]
Compound (6-1) is produced from compound (5-2) and hydrazine by the same method as in Step 1 described in the production method of (1-2).

[Step 2]
Compound (6-4) is synthesized by reacting compound (6-2) with compound (6-3). When X ⁵ is, for example, a chlorine atom, a bromine atom, an iodine atom, OMs, OTs, or OTf, the reaction is performed in the presence of a base. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, NMP, acetonitrile, etc.), ketone solvents (acetone, etc.) or a mixed solvent thereof. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- Diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably cesium carbonate, potassium carbonate, hydrogenated Sodium is mentioned. The amount of the base used is usually selected from the range of 1 to 5 equivalents relative to compound (6-2). The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably in the range of room temperature to 100 ° C.
When X ⁵ is a hydroxyl group, an azo compound such as diethyl azodicarboxylate or diisopropyl azodicarboxylate and a phosphorus reagent such as triphenylphosphine are used according to the Mitsunobu method (eg Synthesis, 1 (1981)). By using it, the compound (6-4) can be produced. Examples of the reaction solvent include an inert solvent such as THF, and examples of the reaction temperature include a range of 0 ° C. to the boiling point of the solvent.

[Step 3]
Compound (6-5) is synthesized by reacting compound (6-4) with compound (6-1) in the presence or absence of an acid. Examples of the acid used include acetic acid, hydrochloric acid, sulfuric acid, p-toluenesulfonic acid and the like, and preferably acetic acid. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). DMSO, NMP, acetonitrile, HMPT, etc.), water (including a buffer solution), alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof. The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably 0 to 100 ° C.

[Step 4]
When R ²² is a protecting group, for example, by using general deprotection conditions shown in the literature (Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.)), R ²² is once Convert to carboxylic acid or hydrogen atom. Subsequently, W ²¹ is — (CH ₂ ) _m3 — or — (CH ₂ ) _m6 — (wherein m3 and m6 each independently represents an integer of 1 to 20), and R ²² is In the case of a carboxylic acid, the compound (6-5) is converted to the compound (3-1b) by converting the hydroxyl group by the method described in Step 1 of the production of the raw material compound (1-2) as necessary. can do. W ²¹ is — (CH ₂ ) _m4 —NH—, — (CH ₂ ) _m5 —O—, — (CH ₂ ) _m7 —NH— or — (CH ₂ ) _m8 —O— (where m4, m5 and m7 each independently represents an integer of 1 to 20, and when R ²² is a hydrogen atom, the compound (3-1) is produced by the same method as in Step 2 of the production method of (3-1a). 6-5) is converted to compound (3-1b).

[Production method of (3-1c)]
In compound (3-1), Q ¹ represents the following formula (IId):

Or formula (IIe):

The compound of the following formula (3-1c) in which a is 1 can be produced, for example, according to the following method.

(Wherein, p2 has the same meaning as the above-mentioned ^{[2], R 23} has the same meaning as R ^{5 'or} R ^6' of the ^{[2], W 22} is synonymous with ^{W 4} or ^{W 5} of [2] X ¹ has the same meaning as described in the production method of (1-2), R ²⁴ is a carboxylic acid or a protected carboxylic acid, a hydrogen atom, a hydroxyl group or an amine protecting group, and X ⁶ is Chlorine atom, bromine atom, iodine atom, OMs, OTs or OTf.)

[Step 1]
Compound (7-1) is synthesized by reacting compound (6-4) ′ with hydroxylamine. The amount of hydroxylamine to be used is usually selected from the range of 1 to 10 equivalents relative to compound (6-4) ′. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, HMPT, etc.), Water, alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof can be used. If necessary, for example, sodium acetate may be added as an additive. The reaction temperature can usually be selected from the range of 0 ° C. to 150 ° C., and preferably in the range of room temperature to 100 ° C.

[Step 2]
Compound (7-3) is synthesized by reacting compound (7-1) with compound (7-2) in the presence of a base. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, NMP, acetonitrile, etc.), ketone solvents (acetone, etc.) or a mixed solvent thereof. Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- Diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably cesium carbonate, potassium carbonate, hydrogenated Sodium is mentioned. The amount of the base to be used is usually selected from the range of 1 to 5 equivalents relative to compound (7-1). The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably in the range of room temperature to 100 ° C.

[Step 3]
Compound (3-1c) is produced from compound (7-3) by the same method as in Step 4 described in the production method for (3-1b).

[Production method of (3-1d)]
In compound (3-1), Q ¹ represents the following formula (IIf):

The compound of the following formula (3-1d) in which a is 1 can be produced, for example, according to the following method.

(In the formula, R ¹ , W ⁶ , W ⁷ , p2, L and ^** have the same ^{meanings as} the above [2] or [3].)

[Step 1]
Compound (8-3) is synthesized by reacting compound (8-1) with compound (8-2) in the presence or absence of an acid. Examples of the acid used include acetic acid, hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, boron trifluoride diethyl ether complex and the like, and preferably acetic acid. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). DMSO, NMP, acetonitrile, HMPT, etc.), ketone solvents (acetone, etc.), water (including buffer solution), alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof. The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably 0 to 50 ° C.

[Step 2]
Compound (8-4) is produced from compound (8-3) and compound (3-2) in the same manner as in the case where X ^{1 in} Step 1 of the production method of (1-2) is a hydroxyl group. Is done. Specifically, the method using a condensing agent or the hydroxyl group of the carboxylic acid of the compound (8-3) is converted into an activating group such as a chlorine atom, a bromine atom, alkoxycarbonyloxy or succinimidyloxy. Thereafter, compound (8-4) is produced by a method of reacting with compound (3-2).

[Step 3]
Compound (1-3d) is produced by reacting compound (8-4) with compound (8-5). Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, acetonitrile, etc.), water ( Buffer solution), alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof. The reaction temperature can usually be selected from the range of 0 to 100 ° C., preferably in the range of room temperature to 80 ° C.

[Production Method 4]
In the formula (I), R ² represents —C (CH ₃ ) ₂ —CH ₂ —NH—C (O) — (CH ₂ CH ₂ O) ₄ —CH ₂ CH ₂ —NH—C (O) — ( A compound that is CH ₂ ) ₅ — is synthesized by the methods of published patent (Yamazaki et al., WO2010 / 053115), Reference Example 35 and Example 34.

[Production Method 5]
In the formula (I), a compound wherein R ¹ is —CH ₂ CH ₂ —SS—CH ₂ CH ₂ —C (O) —NH— (CH ₂ ) ₆ — [in the formula (9-3) below Compound] can be produced, for example, according to the following method.

(In the formula, R ² , Q ¹ , Q ² , A ¹ , L, a, b, ^*, and ^** are synonymous with the above [2] or [3].)

[Step 1]
Compound (9-3) is produced by reacting compound (9-1), which can be synthesized by a known method, with compound (9-2). The reaction is synthesized by reacting compound (9-1) with compound (9-2) in the presence or absence of a buffer solution. Examples of the buffer solution used include borate buffer solution, phosphate buffer solution, acetate buffer solution, citrate buffer solution, and preferably borate buffer solution. Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). DMSO, NMP, acetonitrile, HMPT, etc.), ketone solvents (acetone, etc.), water (including buffer solution), alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof. The reaction temperature can usually be selected from the range of −78 to 150 ° C., preferably 0 ° C. to room temperature (see Reference Example 37, Reference Example 53, and Example 44).

Other raw material compounds of the compound of the present invention are known compounds or can be synthesized from known compounds by combining known synthesis methods.

When the compound of the present invention or an intermediate thereof has a functional group such as an amino group, a carboxy group, a hydroxyl group, or an oxo group, a protection or deprotection technique can be used as necessary. Suitable protecting groups, protecting methods, and deprotecting methods are described in detail in the aforementioned Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.) and the like.

The compound of the present invention or an intermediate for producing the compound can be purified by methods known to those skilled in the art. For example, it can be purified by column chromatography (for example, silica gel column chromatography or ion exchange column chromatography) or recrystallization. For example, as the recrystallization solvent, alcohol solvents such as methanol, ethanol or 2-propanol, ether solvents such as diethyl ether, ester solvents such as ethyl acetate, aromatic hydrocarbon solvents such as benzene or toluene, acetone, etc. A ketone solvent such as hexane, a hydrocarbon solvent such as hexane, an aprotic solvent such as dimethylformamide or acetonitrile, water, or a mixed solvent thereof can be used. As other purification methods, the methods described in Experimental Chemistry Course (edited by the Chemical Society of Japan, Maruzen) Vol. 1 etc. can be used.

The administration route of the compound of the present invention is roughly classified into oral administration and parenteral administration. The dose of the compound varies depending on the type of compound, administration form, administration method, patient symptom / age, etc., but is usually 0.005 to 150 mg / kg / day, preferably 0.05 to 20 mg / kg / day. And can be administered in one or several divided doses.

The compound of the present invention is usually administered as a pharmaceutical in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier. Specific examples include oral preparations such as tablets and capsules, ointments, intravesical injections and the like. External preparations such as liquid preparations for external use, patches, inhalants, nasal drops, injections such as intradermal injections, subcutaneous injections or intracavitary injections such as intraperitoneal and intraarticular cavity. These pharmaceutical compositions are prepared according to conventional methods.

As the pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the compound according to the present invention is used. Specific examples of pharmaceutical carriers used for tablet and capsule production include lactose, corn starch, sucrose, mannitol, calcium sulfate, excipients such as crystalline cellulose, croscarmellose sodium, modified starch, carmellose calcium, Crospovidone, disintegrants such as low substituted hydroxypropylcellulose, binders such as methylcellulose, gelatin, gum arabic, ethylcellulose, hydroxypropylcellulose, povidone, light anhydrous silicic acid, magnesium stearate, talc, sucrose fatty acid ester And lubricants such as hydrogenated oil. The tablet may be coated by a well-known method using a coating agent such as carnauba wax, hydroxypropylmethylcellulose, macrogol, cellulose acetate phthalate, hydroxypropylmethylcellulose acetate phthalate, sucrose, titanium oxide, sorbitan fatty acid ester, calcium phosphate.

Specific examples of the base of the patch include polymer bases such as polyvinyl pyrrolidone, polyisobutylene, vinyl acetate copolymer, acrylic copolymer, glycerin, propylene glycol, polyethylene glycol, triethyl citrate, citric acid Plasticizers such as acetyltriethyl, diethyl phthalate, diethyl sebacate, dibutyl sebacate, and acetylated monoglycerides. The dermal absorption enhancer may be any pharmacologically acceptable one, for example, alcohols such as ethanol and diethylene glycol; polar solvents such as dodecylpyrrolidone; urea; ethyl laurate; azone; olive oil Etc.

If necessary, inorganic fillers such as kaolin, bentonite, zinc oxide, titanium oxide; agarose, carrageenan, alginic acid or salts thereof, tragacanth, acacia gum, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, carboxyvinyl polymer, gelatin, corn starch Viscosity modifiers such as polymers such as xanthan gum, dextrin and polyvinyl alcohol; anti-aging agents; pH regulators; humectants such as glycerin and propylene glycol may be added. Further, a surfactant may be added. Examples thereof include fatty acid alkali salts such as potassium laurate, potassium palmitate and potassium myristate; sodium lauryl sulfate, sodium cetyl sulfate, castor oil sulfate (funnel oil) Ionic surfactants such as sulfate esters such as sorbitan stearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate (so-called Span); polysorbate 20, polysorbate 40, polysorbate 60, Polysorbate 65, polysorbate 80, polysorbate 85, polyoxyethylene sorbitan fatty acid ester (so-called Tween); polyoxyethylene hydrogenated castor oil (so-called HCO); polyoxyethylene lauryl ester Polyoxyethylene alkyl ethers such as tellurium, polyoxyethylene cetyl ether and polyoxyethylene oleyl ether; polyethylene glycol fatty acid esters such as polyethylene glycol monolaurate and polyethylene glycol monostearate; nonionic surface activity such as poloxamer (so-called pluronic) Agents. Further, lecithin (including purified phospholipids isolated from lecithin such as phosphatidylcholine and phosphatidylserine) or derivatives thereof including hydrogenated products thereof may be added.

An inhalant can be produced by converting the compound of the present invention into a powder or liquid form and blending it into an inhalation spray or carrier and filling the inhalation container such as a metered dose inhaler or a dry powder inhaler. it can. It may be a spray, an aerosol, or a spray. As the inhalable propellant, conventionally known ones can be widely used. For example, Freon-11, Freon-12, Freon-21, Freon-22, Freon-113, Freon-114, Freon-123, Freon- 142c, CFC-134a, CFC-227, CFC-318, CFCs such as 1,1,1,2-tetrafluoroethane, CFC alternatives such as HFA-227 and HFA-134a, propane, isobutane, butane, etc. Examples thereof include hydrocarbon gas, diethyl ether, nitrogen gas, carbon dioxide gas and the like. As the carrier, conventionally known ones can be widely used, and examples thereof include sugars, sugar alcohols, amino acids and the like.

For inhalation solutions, preservatives (benzalkonium chloride, parabens, etc.), coloring agents, buffering agents (sodium phosphate, sodium acetate, etc.), isotonic agents (sodium chloride, concentrated glycerin, etc.), It is prepared by appropriately selecting a sticking agent (such as carboxyvinyl polymer), an antiseptic (such as benzalkonium chloride, paraben), an absorption accelerator, and the like as necessary.

In the case of powders for inhalation, lubricants (stearic acid and its salts, etc.), binders (starch, dextrin, etc.), excipients (lactose, cellulose, etc.), coloring agents, absorption enhancers, etc. are required. According to the selection, it is prepared as appropriate.

The nasal drops can take various forms such as a drop type, a coating type, and a spray type. In the case of a spray type, manual pump type nasal drops with a mechanism for ejecting liquid by manually moving the pump attached to the container, compressed gas (air or oxygen, nitrogen, carbonic acid, or mixed gas), etc. In addition, an aerosol type nasal spray having a mechanism in which a liquid agent is automatically ejected by moving a valve attached to the container and filling the container with the propellant is automatically included.

For injection, the compound of the present invention is dissolved in distilled water for injection and, if necessary, a solution to which a solubilizing agent, buffer, pH adjuster, tonicity agent, soothing agent, preservative and the like are added. May be prepared by suspending the compound in distilled water for injection or vegetable oil, and in this case, a base, a suspending agent, a thickening agent, etc. may be added as necessary. Can do. Moreover, the form which melt | dissolves a powder or a lyophilized product at the time of use may be sufficient, and an excipient | filler etc. can be added as needed.

The content of the compound according to the present invention in the pharmaceutical composition varies depending on the dosage form, but is usually 0.0025 to 20% by weight in the total composition. These pharmaceutical compositions may also contain other therapeutically effective substances. In the combined use, the administration timing of the compound of the present invention and the concomitant drug is not particularly limited, and the compounding ratio of the compound of the present invention and the concomitant drug can be appropriately set depending on the administration subject, administration method, disease, combination and the like. For example, the content of the concomitant drug in the concomitant drug of the present invention varies depending on the form of the preparation, but is usually 0.0025 to 20% by weight in the total composition.

Hereinafter, the present invention will be described more specifically with reference to reference examples, examples, and test examples, but these examples do not limit the present invention. Compound identification includes elemental analysis values, mass spectrum, high performance liquid chromatography / mass spectrometer (LCMS), time-of-flight mass spectrometer (TOF-MS), IR spectrum, NMR spectrum, high performance liquid chromatography (HPLC), etc. It went by.
In order to simplify the description of the specification, the following abbreviations may be used in the examples and tables in the examples. Symbols used for nucleic acid sequences include 3′- for the 3 ′ end, 5′- for the 5 ′ end, A, G, C, U, and T for adenine, guanine, cytosine, uracil, and thymine, respectively, as nucleobases. The ribonucleotides a, g, c and t are deoxyribonucleotides having adenine, guanine, cytosine and thymine as nucleobases, respectively. A (M), G (M), C (M), and U (M) represent 2′-O-methylated adenine, guanine, cytosine, and uracil, respectively, and A (F), G (F ), C (F) and U (F) mean ribonucleotides having a nucleobase of adenine, guanine, cytosine and uracil fluorinated at the 2 ′ position, respectively. Furthermore, ^ means a phosphorothioate group.
“Room temperature” in the following examples usually indicates about 10 ° C. to about 35 ° C. The ratio shown in the mixed solvent is a volume ratio unless otherwise specified. Unless otherwise indicated, “%” indicates “% by weight”.

Reference example 1
4-pentynoic acid succinimidyl ester

Under a nitrogen atmosphere, dicyclohexylcarbodiimide (17.6 g) was added to a dioxane solution (100 mL) of 4-pentynoic acid (8.00 g) and N-hydroxysuccinimide (9.85 g), and the mixture was stirred at room temperature for 24 hours. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 70 / 30-30 / 70) to give the title compound as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 2.82-2.88 (m, 6H), 2.57-2.62 (m, 2H), 2.03 (t, J = 2.7 Hz, 1H).

Reference example 2
5-hexynoic acid succinimidyl ester

The reaction and treatment were conducted in the same manner as described in Reference Example 1 to give the title compound from 5-hexynoic acid.

Reference example 3
10-Undecynoic acid succinimidyl ester

The title compound was obtained from 10-undecylic acid by reacting and treating in the same manner as described in Reference Example 1.

Reference example 4
5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one

(1) 5-Bromoacetyl-2-hydroxybenzaldehyde Under a nitrogen atmosphere, a methylene chloride suspension (556 mL) of bromoacetic acid bromide (500 g) was added to a methylene chloride suspension (3990 mL) of aluminum chloride (1.10 kg) at room temperature. It was dripped at. After completion of the dropwise addition, the temperature was raised to 30 ° C. and stirred at the same temperature for 1 hour. To this mixture, a methylene chloride solution (551 mL) of salicylaldehyde (248 g) was added dropwise and heated to reflux for 36 hours. After cooling the mixture, it was slowly poured into ice water (10.6 L), and the organic layer was separated. The aqueous layer was extracted with chloroform, and the combined organic layer was washed with water and saturated brine in this order. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was washed with a mixed solution of hexane / chloroform = 1: 1 to obtain the title compound (165 g) as a light brown solid.

(2) 2-Bromo-1- [4-hydroxy-3- (hydroxymethyl) phenyl] ethanone A solution of 3-bromoacetyl-2-hydroxybenzaldehyde (165 g) obtained above in acetic acid (3384 mL) under a nitrogen atmosphere To the mixture, sodium borohydride (25.7 g) was added in 8 portions while cooling to 10-20 ° C. The mixture was warmed to room temperature and stirred for 1 hour. The mixture was concentrated under reduced pressure, water was added to the residue, and neutralized with saturated sodium hydrogen carbonate solution. The mixture was extracted twice with ethyl acetate, and the combined organic layer was washed with water and then saturated brine. After drying over anhydrous magnesium sulfate, concentration under reduced pressure gave the title compound (157 g) as a brown solid.

(3) 2-bromo-1- [2,2-dimethyl-4H-1,3-benzodioxin-6-yl) ethanone 2-bromo-1- [4-hydroxy-] obtained above under a nitrogen atmosphere To a methylene chloride suspension (3160 mL) of 3- (hydroxymethyl) phenyl] ethanone (155 g) and p-toluenesulfonic acid monohydrate (1.2 g), 2,2-dimethoxypropane (78.8 g) was added. Methylene chloride solution (1220 mL) was added dropwise over 30 minutes, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was washed with saturated sodium bicarbonate solution (1 L), and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 4/1) to give the title compound (73.5 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.82 (dd, J = 8.7, 2.1 Hz, 1H), 7.70 (d, J = 2.1 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 4.90 (s, 2H), 4.38 (s, 2H), 1.57 (s, 6H).
MS (ESI) m / z 285 (M + H) ⁺ .

(4) Di-tert-butyl [2- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxoethyl] imide dicarbonate A suspension of bromo-1- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) ethanone (73.5 g) and di-tert-butyl iminocarboxylate (55.9 g) in acetonitrile ( (756 mL) was added cesium carbonate (83.8 g), and the mixture was stirred at room temperature for 24 hours. Water (1260 mL) was added to the reaction mixture, and the mixture was extracted with diethyl ether (1260 mL). The organic layer was washed with saturated brine (630 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 5/1) to give the title compound (86.9 g) as a white solid.

(5) tert-butyl [2- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxoethyl] carbamate di-tert-butyl obtained above under nitrogen atmosphere 2- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxoethyl] imide dicarbonate (86.9 g) in methylene chloride solution (938 mL) and trifluoroacetic acid (19.3 mL) ) And stirred at room temperature for 4 hours. To the reaction mixture was added 0.5N aqueous sodium hydroxide solution (750 mL), and the layers were separated. The organic layer was washed with water (938 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 4/1) to give the title compound (53.0 g) as a white solid.

(6) tert-butyl [2- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-hydroxyethyl] carbamate tert-butyl [2] obtained above under nitrogen atmosphere Boron hydride while cooling to −20 ° C. in a methanol solution (984 mL) of (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxoethyl] carbamate (50.4 g) Sodium (11.8 g) was added. After stirring at −20 ° C. for 2 hours, water (394 mL) was added, and methanol was distilled off under reduced pressure. The residue was extracted with ethyl acetate (1270 mL), and the aqueous layer was further extracted with ethyl acetate (830 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the title compound (49.8 g) as a white solid.

(7) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one 55% hydrogenation cooled to 0-5 ° C. under nitrogen atmosphere To a DMF suspension (152 mL) of sodium (8.79 g), tert-butyl [2- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-] obtained above was added. Hydroxyethyl] carbamate (49.8 g) in DMF (455 mL) was added dropwise over 97 minutes. After completion of dropping, the mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled to 0 ° C., diluted with water (205 mL), and neutralized with 1N aqueous hydrochloric acid (201 mL). Further, water (205 mL) and ethyl acetate (774 mL) were added for liquid separation. The organic layer was washed with saturated brine (194 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was crystallized by adding a mixed solution of diethyl ether / hexane to give the title compound (29.7 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.16 (dd, J = 8.4, 2.0 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 5.54 (t, J = 8.1 Hz, 1H), 5.38 (s, 1H), 4.85 (s, 2H), 3.93 (t, J = 8.6 Hz, 1H), 3.56-3.53 (m, 1H), 1.55 (s , 6H).
MS (ESI) m / z 272 (M + Na) ⁺ .

Reference Example 5
(4- {4-[(6-Bromohexyl) oxy] butyl} phenyl) methanol

(1) tert-Butyl 4-bromobenzoate p-Bromobenzoyl chloride (50.8 g) was added to a dehydrated THF solution (240 mL) of tert-butoxypotassium (31.2 g) cooled to −40 to −30 ° C. in a nitrogen atmosphere. A dehydrated THF solution (240 mL) was added dropwise over 3 hours. The reaction mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was diluted with tert-butyl methyl ether, and washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 19/1) to give the title compound (55.4 g) as a pale yellow oil.

(2) tert-butyl 4- (4-hydroxybut-1-in-1-yl) benzoate Under a nitrogen atmosphere, tert-butyl 4-bromobenzoate (55.4 g) obtained above and 3-butyne-1 -To a solution of ol (19.6 g) in triethylamine (450 mL) were added bistriphenylphosphine palladium dichloride (7.71 g) and copper iodide (832 mg), and the mixture was stirred at room temperature for 18 hours. The residue obtained by concentrating the reaction mixture under reduced pressure was diluted with chloroform and washed with a 1N aqueous hydrochloric acid solution and saturated brine in this order. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 4/1) to give the title compound (49.0 g) as a light brown oil.

(3) tert-butyl 4- (4-hydroxybutyl) benzoate Ethanol solution (2500 mL) of tert-butyl 4- (4-hydroxybut-1-in-1-yl) benzoate (49.0 g) obtained above ) Was added with 10% palladium / carbon (16.0 g, 50% wet) and stirred for 24 hours in a hydrogen atmosphere. The reaction mixture was filtered through celite and concentrated under reduced pressure to give the title compound (47.2 g) as a pale yellow oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.90 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.3 Hz, 2H), 3.71-3.58 (m, 2H), 2.76-2.51 (m , 2H), 1.80-1.51 (m, 13H).

(4) tert-butyl 4- {4-[(6-bromohexyl) oxy] butyl} benzoate tert-butyl 4- (4-hydroxybutyl) benzoate (46.0 g) obtained above and 1,6- To a mixture of dibromohexane (135 g), tetrabutylammonium hydrogen sulfate (189 mg) and 50% aqueous sodium hydroxide solution (276 mL) were added and stirred for 5 hours. Tetrabutylammonium hydrogen sulfate (189 mg) was added to the reaction mixture, and the mixture was further stirred for 43 hours. The reaction mixture was extracted with tert-butyl methyl ether. The organic layer was washed with water and saturated brine in this order, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / tert-butyl methyl ether = 20/1) to give the title compound (55.3 g) as a pale yellow oil.

(5) 4- {4-[(6-Bromohexyl) oxy] butyl} benzoic acid tert-butyl 4- {4-[(6-bromohexyl) oxy] butyl} benzoate (55.3 g) obtained above ) Was added to trifluoroacetic acid (300 mL) and stirred for 15 hours. The reaction mixture was concentrated under reduced pressure, 1,4-dioxane (500 mL) was added, and the operation of concentration under reduced pressure was repeated three times to obtain the title compound (45.8 g) as a white solid.

(6) (4- {4-[(6-Bromohexyl) oxy] butyl} phenyl) methanol The above obtained 4- {4-[(6-bromohexyl) cooled to 5-8 ° C. under nitrogen atmosphere. Borane-THF (145 mL, 1.06 M THF solution) was added to a dehydrated THF solution (730 mL) of) oxy] butyl} benzoic acid (45.8 g). The reaction mixture was warmed to room temperature and stirred for 4 hours. Saturated aqueous ammonium chloride solution (730 mL) was slowly added to the reaction mixture, and the layers were separated. The aqueous layer was extracted with ethyl acetate (500 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 5/1) to give the title compound (41.1 g) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.28 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 4.66 (d, J = 5.3 Hz, 2H), 3.49- 3.34 (m, 6H), 2.63 (t, J = 7.3 Hz, 2H), 1.92-1.80 (m, 2H), 1.75-1.52 (m, 6H), 1.51-1.30 (m, 4H).
MS (ESI) m / z 365 (M + Na) ⁺ .

Reference Example 6
({4-[(5-Bromopentyl) oxy] benzyl} oxy) (tert-butyl) dimethylsilane

(1) tert-butyl [(4-{[tert-butyl (dimethyl) silyl] oxy} benzyl) oxy] dimethylsilane Imidazole was added to a DMF solution (580 mL) of p-hydroxybenzyl alcohol (23.0 g) under a nitrogen atmosphere. (50.4 g) and tert-butyldimethylsilyl chloride (69.8 g) were added, and the mixture was heated with stirring at 50 ° C. for 4 hours. The reaction mixture was cooled to room temperature, water (2300 mL) was added, and the aqueous layer was extracted 3 times with tert-butyl methyl ether. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product of the title compound (74.9 g) as a yellow oil.

(2) 4-({[tert-Butyl (dimethyl) silyl] oxy} methyl) phenol In a nitrogen atmosphere, tert-butyl [((15.6 g)] was added to an ethanol solution (185 mL) of potassium hydroxide (15.6 g). 4-([tert-Butyl (dimethyl) silyl] oxy} benzyl) oxy] dimethylsilane crude product (74.9 g) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and 3N aqueous hydrochloric acid and saturated aqueous ammonium chloride were added to the resulting residue to adjust to pH = 5. After adding tert-butyl methyl ether to the reaction mixture and extracting three times, the combined organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The dried organic layer was concentrated under reduced pressure, and the obtained residue was purified by silica gel chromatography (hexane / ethyl acetate = 10/1) to give the title compound (43.4 g) as a yellow oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.19 (d, J = 8.3 Hz, 2H), 6.79 (d, J = 8.3 Hz, 2H), 4.66 (s, 2H), 0.93 (s, 9H), 0.09 (s, 6H).

(3) (4- {4-[(6-Bromohexyl) oxy] butyl} phenyl) methanol 4-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenol obtained above under nitrogen atmosphere (43.4 g), 1,5-dibromopentane (63.4 g), potassium carbonate (33.0 g) and tetrabutylammonium iodide (10.2 g) in an acetone suspension (305 mL) were heated to reflux for 2 hours. . The reaction mixture was cooled to room temperature and then concentrated under reduced pressure. To the residue are added ethyl acetate and water, and a saturated aqueous ammonium chloride solution is added to adjust the pH to 7-8, followed by extraction twice with ethyl acetate. The combined organic layer is washed with saturated brine, and anhydrous sodium sulfate. And dried. The residue obtained by concentration of the dried organic layer under reduced pressure was purified by silica gel chromatography (hexane / ethyl acetate = 20/1) to give the title compound (34.0 g) as a white solid.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.18-7.10 (m, 2H), 6.83-6.71 (m, 2H), 4.58 (s, 2H), 3.92-3.82 (m, 2H), 3.41-3.30 ( m, 2H), 1.92-1.67 (m, 4H), 1.62-1.43 (m, 2H), 0.84 (s, 9H), 0.00 (s, 6H).

Reference Example 7
4- {2-[(6- {4- [4- (azidomethyl) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) phenol

(1) 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- (6- {4- [4- (hydroxymethyl) phenyl] butoxy} hexyl) -1, 3-Oxazolidin-2-one

To a DMF suspension (100 mL) of 60% sodium hydride (1.54 g) cooled in ice under a nitrogen atmosphere, 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl)- A DMF solution (30 mL) of 1,3-oxazolidine-2-one (Reference Example 4, 8.0 g) was added, and the mixture was warmed to room temperature and stirred for 30 minutes. The reaction mixture was ice-cooled, a DMF solution (30 mL) of (4- {4-[(6-bromohexyl) oxy] butyl} phenyl) methanol (Reference Example 5, 16.5 g) was added, and the temperature was raised to room temperature. And stirred for 14 hours. Toluene (300 mL) and water (150 mL) were added to the reaction mixture, and the layers were separated. The organic layer was washed with water and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 1/1) to give the title compound (13.4 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.28 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 7.12 (dd, J = 8.4, 2.2 Hz, 1H), 7.00 (d, J = 2.2 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.39 (t, J = 8.1 Hz, 1H), 4.84 (s, 2H), 4.65 (d, J = 5.1 Hz, 2H), 3.84 (t, J = 8.7 Hz, 1H), 3.48-3.17 (m, 7H), 2.63 (t, J = 7.3 Hz, 2H), 1.75-1.49 (m, 8H), 1.54 (s , 6H), 1.44-1.28 (m, 4H).
MS (ESI) m / z 534 (M + Na) ⁺ .

(2) 3- (6- {4- [4- (azidomethyl) phenyl] butoxy} hexyl) -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3 -Oxazolidin-2-one

Under a nitrogen atmosphere, the 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- (6- {4- [4- (hydroxymethyl) phenyl] butoxy obtained above was obtained. } Hexyl) -1,3-oxazolidine-2-one (1.0 g) and triethylamine (407 μL) in THF (30 mL) were added dropwise methanesulfonyl chloride (181 μL) under ice cooling. The reaction mixture was warmed to room temperature and stirred for 2 hours. Ethyl acetate and saturated aqueous sodium hydrogen carbonate solution were added to the reaction mixture, and the phases were separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Sodium azide (191 mg) was added to a DMF (6.5 mL) solution of the obtained residue, and the mixture was stirred at 80 ° C. for 3 hours. After the reaction mixture was cooled to room temperature, ethyl acetate and water were added and the layers were separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane / ethyl acetate = 1/1) to give the title compound (942 mg) as a white solid. Got as.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.25-7.16 (m, 4H), 7.12 (dd, J = 8.4, 2.0 Hz, 1H), 7.00 (d, J = 2.0 Hz, 1H), 6.84 (d , J = 8.4 Hz, 1H), 5.39 (t, J = 8.1 Hz, 1H), 4.84 (s, 2H), 4.30 (s, 2H), 3.85 (t, J = 8.7 Hz, 1H), 3.47-3.18 (m, 7H), 2.64 (t, J = 7.4 Hz, 2H), 1.73-1.50 (m, 8H), 1.54 (s, 6H), 1.46-1.29 (m, 4H).
MS (ESI) m / z 559 (M + Na) ⁺ .

(3) 4- {2-[(6- {4- [4- (azidomethyl) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) phenol

3- (6- {4- [4- (Azidomethyl) phenyl] butoxy} hexyl) -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1 obtained above , 3-Oxazolidin-2-one (942 mg) in THF (8.8 mL) was added potassium trimethylsilanoate (1.13 g), and the mixture was heated to reflux for 1 hour. The reaction mixture was cooled to room temperature, ethyl acetate and water were added, and the layers were separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Acetic acid (6.0 mL) and water (2.0 mL) were added to a compound obtained by purifying the obtained residue by silica gel chromatography (chloroform / methanol = 10/1), and the mixture was stirred at 80 ° C. for 1 hour. The reaction mixture was purified by silica gel chromatography (amino silica gel, chloroform / methanol = 10/1) to obtain the title compound (942 mg) as a pale yellow solid.
¹ H-NMR (300MHz, d ₆ -DMSO) δ: 7.31-7.14 (m, 5H), 6.96 (dd, J = 8.2, 2.0 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 5.00 (brs, 2H), 4.52-4.41 (m, 3H), 4.37 (s, 2H), 3.40-3.25 (m, 4H), 2.63-2.43 (m, 6H), 1.64-1.31 (m, 8H), 1.31 -1.19 (m, 4H).
MS (ESI) m / z 471 (M + H) ⁺ .

Reference Example 8
4- [2-({5- [4- (Azidomethyl) phenoxy] pentyl} amino) -1-hydroxyethyl] -2- (hydroxymethyl) phenol

(1) 3- {5- [4-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenoxy] pentyl} -5- (2,2-dimethyl-4H-1,3-benzodioxin-6 -Yl) -1,3-oxazolidine-2-one

({4-[(5-bromopentyl) oxy] benzyl} oxy) (tert-butyl) dimethylsilane (Reference Example 6, 34.0 g) and 5- (2,2-dimethyl-4H-1,3- Using benzodioxin-6-yl) -1,3-oxazolidine-2-one (Reference Example 4, 19.7 g), the reaction and treatment were conducted in the same manner as described in Reference Example 7- (1). Compound (38.3 g) was obtained as a colorless oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.18-7.10 (m, 2H), 7.08-7.00 (m, 1H), 6.92 (s, 1H), 6.80-6.71 (m, 3H), 5.37-5.27 ( m, 1H), 4.75 (s, 2H), 4.58 (s, 2H), 3.91-3.73 (m, 3H), 3.38-3.13 (m, 3H), 1.80-1.37 (m, 12H), 0.84 (s, 9H), 0.00 (s, 6H).

(2) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine-2 -on

Under a nitrogen atmosphere, the 3- {5- [4-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenoxy] pentyl} -5- (2,2-dimethyl-4H-1, 1M tetra-n-butylammonium fluoride / THF solution (69.0 mL) was added dropwise to a THF solution (383 mL) of 3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (38.3 g). . The reaction mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. Ethyl acetate (778 mL) and water (156 mL) were added to the obtained residue for liquid separation. The organic layer was washed with saturated brine (156 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1/2) to give the title compound (27.9 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.28 (d, J = 9.0 Hz, 2H), 7.11 (dd, J = 8.4, 2.2 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.86 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 8.4 Hz, 1H), 5.40 (t, J = 8.4 Hz, 1H), 4.82 (s, 2H), 4.61 (d, J = 5.7 Hz, 2H), 3.96 (t, J = 6.2 Hz, 2H), 3.86 (t, J = 8.4 Hz, 1H), 3.49-3.23 (m, 3H), 1.90-1.73 (m, 2H), 1.69-1.48 (m, 4H), 1.54 (s, 6H).
MS (ESI) m / z 464 (M + Na) ⁺ .

(3) 3- {5- [4- (azidomethyl) phenoxy] pentyl} -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2- on

5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine obtained above The reaction was treated with -2-one (441 mg) in the same manner as described in Reference Example 7- (2) to give the title compound (383 mg) as a colorless oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.23 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H), 6.89-6.83 (m, 3H ), 5.40 (t, J = 8.0 Hz, 1H), 4.84 (s, 1H), 4.26 (s, 2H), 3.96 (t, J = 8.0 Hz, 2H), 3.87 (t, J = 8.0 Hz, 1H ), 3.44-3.26 (m, 3H), 1.87-1.08 (m, 2H), 1.67-1.62 (m, 4H), 1.56-1.51 (m, 2H), 1.54 (s, 6H).
MS (ESI) m / z 489 (M + Na) ⁺ .

(4) 4- [2-({5- [4- (Azidomethyl) phenoxy] pentyl} amino) -1-hydroxyethyl] -2- (hydroxymethyl) phenol

3- {5- [4- (azidomethyl) phenoxy] pentyl} -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine- obtained above The title compound (75 mg) was obtained as a white amorphous product by reacting and treating in a similar manner to that described in Reference Example 7- (3) using 2-one (206 mg).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.23 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.89 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 8.0 Hz, 1H), 4.77 (s, 2H), 4.55 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 4.26 (s, 2H), 3.96 ( t, J = 8.0 Hz, 2H), 3.26 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 2.71-2.58 (m, 3H), 1.83-1.76 (m, 2H), 1.60-1.46 (m , 4H).
MS (ESI) m / z 401 (M + H) ⁺ .

Reference Example 9
4- (2-{[5- (4-{[4- (2-azidoethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol

(1) tert-butyl [4- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl) phenoxy] dimethylsilane under nitrogen atmosphere, 2- (4-hydroxyphenyl) ethanol (23.0 g) and imidazole To a DMF solution (250 mL) of (68.0 g), tert-butyldimethylsilyl chloride (70.1 g) was added and stirred at room temperature for 3 hours. Water and ethyl acetate were added to the reaction mixture and the phases were separated. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated. The obtained residue was purified by silica gel chromatography (elution solution was hexane / ethyl acetate) to give the title compound (61.1 g) as a colorless oil.

(2) 4- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl) phenol Under nitrogen atmosphere, tert-butyl [4- (2-{[tert-butyl (dimethyl) silyl] obtained above ] To a THF solution (800 mL) of [oxy} ethyl) phenoxy] dimethylsilane (61.1 g), a 1M tetra n-butylammonium fluoride / THF solution (166 mL) was added dropwise under ice cooling. The reaction mixture was stirred in an ice bath for 20 minutes, saturated aqueous ammonium chloride solution (500 mL) was added, and the mixture was extracted with tert-butyl methyl ether. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1/2) to give the title compound (24.8 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.09 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 3.77 (t, J = 7.1 Hz, 2H), 2.76 ( t, J = 7.1 Hz, 2H), 0.89 (s, 9H), 0.00 (s, 6H).

(3) 3- [5- (4-{[4- (2-{[tert-Butyl (dimethyl) silyl] oxy} ethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl- 4H-1,3-benzodioxin-6-yl) -1,3-oxazolidin-2-one 4- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl obtained above under nitrogen atmosphere ) Phenol (1.26 g), 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1, To a solution of 3-oxazolidine-2-one (Reference Example 8- (2), 2.0 g) and triphenylphosphine (1.54 g) in THF (23 mL) under ice-cooling, diisopropyl azodicarboxylate (1.23 mL) Was added dropwise and stirred for 2 hours . Water and ethyl acetate were added to the reaction solution and the layers were separated. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography (hexane / ethyl acetate = 2/1) to give the title compound (1.88 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ 7.33 (d, J = 8.6 Hz, 2H), 7.16-7.07 (m, 3H), 7.00 (d, J = 2.0 Hz, 1H), 6.93-6.79 (m, 5H), 5.40 (t, J = 8.1 Hz, 1H), 4.96 (s, 2H), 4.84 (s, 2H), 3.96 (t, J = 6.3 Hz, 2H), 3.87 (t, J = 8.7 Hz, 1H), 3.76 (t, J = 7.2 Hz, 2H), 3.47-3.22 (m, 3H), 2.76 (t, J = 7.2 Hz, 2H), 1.90-1.74 (m, 2H), 1.71-1.46 (m , 4H), 1.54 (s, 6H), 0.87 (s, 9H), -0.01 (s, 6H).
MS (ESI) m / z 698 (M + Na) ⁺ .

(4) 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[4- (2-hydroxyethyl) phenoxy] methyl} phenoxy) Pentyl] -1,3-oxazolidine-2-one 3- [5- (4-{[4- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl) phenoxy] methyl} obtained above Phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (1.88 g) was used to give Reference Example 8- The reaction and treatment were conducted in a manner similar to that described in (2) to give the title compound (1.64 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.34 (d, J = 8.4 Hz, 2H), 7.19-7.09 (m, 3H), 7.00 (d, J = 1.8 Hz, 1H), 6.98-6.79 (m , 5H), 5.40 (t, J = 8.2 Hz, 1H), 4.97 (s, 2H), 4.84 (s, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.90-3.77 (m, 3H) , 3.48-3.22 (m, 3H), 2.81 (t, J = 6.6 Hz, 2H), 1.90-1.74 (m, 2H), 1.72-1.46 (m, 4H), 1.54 (s, 6H).
MS (ESI) m / z 584 (M + Na) ⁺ .

(5) 3- [5- (4-{[4- (2-Azidoethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl ) -1,3-Oxazolidin-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[4] obtained above -(2-Hydroxyethyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidine-2-one (1.64 g) was used in the same manner as described in Reference Example 7- (2). Treatment gave the title compound (1.49 g) as a white solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.34 (d, J = 8.8 Hz, 2H), 7.17-7.09 (m, 3H), 7.00 (d, J = 1.8 Hz, 1H), 6.96-6.81 (m , 5H), 5.41 (t, J = 8.1 Hz, 1H), 4.96 (s, 2H), 4.84 (s, 2H), 3.97 (t, J = 6.3 Hz, 2H), 3.87 (t, J = 8.7 Hz , 1H), 3.52-3.22 (m, 5H), 2.84 (t, J = 7.2 Hz, 2H), 1.90-1.77 (m, 2H), 1.71-1.47 (m, 4H), 1.54 (s, 6H).
MS (ESI) m / z 609 (M + Na) ⁺ .

(6) 4- (2-{[5- (4-{[4- (2-azidoethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol The resulting 3- [5- (4-{[4- (2-azidoethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl ) -1,3-oxazolidine-2-one (1.49 g) was reacted and treated in the same manner as described in Reference Example 7- (3) to give the title compound (370 mg) as a white solid Obtained.
¹ H-NMR (300MHz, d ₆ -DMSO) δ: 7.33 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 2.0 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.98 (dd, J = 8.0, 2.0 Hz, 1H), 6.91 (d, J = 8.4 Hz, 4H), 6.68 (d, J = 8.0 Hz, 1H), 4.96 (s, 2H), 4.52 (t, J = 6.2 Hz, 1H), 4.45 (s, 2H), 3.94 (t, J = 6.4 Hz, 2H), 3.49 (d, J = 7.1 Hz, 2H), 2.76 (d, J = 7.1 Hz, 2H), 2.69-2.55 (m, 4H), 1.79-1.63 (m, 2H), 1.57-1.35 (m, 4H).
MS (ESI) m / z 521 (M + H) ⁺ .

Reference Example 10
4- (2-{[5- (4-{[3- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol

(1) 3-({[tert-Butyl (dimethyl) silyl] oxy} methyl) phenol Using 3- (hydroxymethyl) phenol (10.0 g), Reference Examples 9- (1) and 9- (2) The title compound (11.5 g) was obtained as a colorless oil by reaction and treatment in the same manner as described in 1.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.19 (t, J = 7.9 Hz, 1H), 6.91-6.80 (m, 2H), 6.70 (dd, J = 7.9, 2.7 Hz, 1H), 4.70 (s , 2H), 0.94 (s, 9H), 0.10 (s, 6H).

(2) 3- [5- (4-{[3-({[tert-Butyl (dimethyl) silyl] oxy} methyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H- 1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one 3-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenol (1.78 g) obtained above and , 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine-2-one (Reference Example 8- (2), 3.0 g) was used for the reaction and treatment in the same manner as described in Reference Example 9- (3) to give the title compound (3.58 g) as a white solid. It was.

(3) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[3- (hydroxymethyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidine-2-one 3- [5- (4-{[3-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenoxy] methyl} phenoxy) pentyl] obtained above -5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (3.58 g) was used in Reference Example 8- (2). Reaction and treatment in the same manner as described gave the title compound (1.46 g) as a white amorphous.

(4) 3- [5- (4-{[3- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl)- 1,3-oxazolidine-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[3- ( Hydroxymethyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidin-2-one (1.46 g) was reacted and treated in the same manner as described in Reference Example 7- (2) to give the title The compound (1.29 g) was obtained as a colorless oil.

(5) 4- (2-{[5- (4-{[3- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol obtained above 3- [5- (4-{[3- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1, The title compound (97 mg) was obtained as a white solid by reacting and treating in a similar manner as described in Reference Example 7- (3) using 3-oxazolidine-2-one (343 mg).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.34 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 8.0 Hz, 1H), 7.12 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 6.98 (s, 1H), 6.94-6.89 (m, 5H), 6.81 (d, J = 8.0 Hz, 1H), 4.99 (s, 2H), 4.78 (s, 2H), 4.56 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 4.30 (s, 2H), 3.96 (t, J = 8.0 Hz, 2H), 2.78 (dd, J = 12.0 Hz, J = 4.0 Hz, 1H), 2.71- 2.59 (m, 3H), 1.82-1.75 (m, 2H), 1.59-1.45 (m, 4H).
MS (ESI) m / z 507 (M + H) ⁺ .

Reference Example 11
4- (2-{[5- (4-{[4- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol

(1) 4- (3-{[tert-butyl (dimethyl) silyl] oxy} propyl) phenol Using 4- (3-hydroxypropyl) phenol (10.5 g), Reference Examples 9- (1) and 9 Reaction and treatment were conducted in the same manner as described in (2) to give the title compound (11.5 g) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.05 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz, 2H), 4.61 (s, 1H), 3.62 (t, J = 6.3 Hz, 2H), 2.60 (t, J = 7.8 Hz, 2H), 1.86-1.72 (m, 2H), 0.90 (s, 9H), 0.05 (s, 6H).
MS (ESI) m / z 267 (M + H) ⁺ .

(2) 3- [5- (4-{[4- (3-{[tert-Butyl (dimethyl) silyl] oxy} propyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl- 4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one 4- (3-{[tert-butyl (dimethyl) silyl] oxy} propyl) phenol (2 .06g) and 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine Using -2-one (Reference Example 8- (2), 3.0 g) in the same manner as described in Reference Example 9- (3), the title compound (4.5 g) Obtained as a solid.

(3) 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[4- (3-hydroxypropyl) phenoxy] methyl} phenoxy) Pentyl] -1,3-oxazolidine-2-one 3- [5- (4-{[4- (3-{[tert-butyl (dimethyl) silyl] oxy} propyl) phenoxy] methyl} obtained above Phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (4.5 g) was used to give Reference Example 8- The reaction and treatment were conducted in a manner similar to that described in (2) to give the title compound (1.9 g) as a white solid.

(4) 3- [5- (4-{[4- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6- Yl) -1,3-oxazolidine-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[ 4- (3-Hydroxypropyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidine-2-one (1.9 g) was used in the same manner as described in Reference Example 7- (2). To give the title compound (1.07 g) as a white solid.

(5) 4- (2-{[5- (4-{[4- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol 3- [5- (4-{[4- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6 obtained in -Il) -1,3-oxazolidine-2-one (360 mg) was used for the reaction and treatment in the same manner as described in Reference Example 7- (3) to give the title compound (132 mg) as a white amorphous product. It was.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.30 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.95-6.76 (m, 6H), 6.72 (d, J = 8.0 Hz, 1H), 4.91 (s, 2H), 4.78-4.71 (m, 1H), 4.52 (s, 2H), 3.88 (t, J = 8.0 Hz, 2H), 3.25 (t, J = 8.0 Hz , 2H), 2.90-2.76 (m, 4H), 2.68-2.54 (m, 2H), 1.89-1.82 (m, 2H), 1.78-1.64 (m, 4H), 1.50-1.41 (m, 2H).
MS (ESI) m / z 535 (M + H) ⁺ .

Reference Example 12
4- (2-{[5- (4-{[3- (2-azidoethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol

(1) 3- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl) phenol Using 3- (2-hydroxyethyl) phenol (17.5 g), Reference Examples 9- (1) and 9 Reaction and treatment in the same manner as described in (2) to give the title compound (31.2 g) as a pale yellow oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.16 (t, J = 7.9 Hz, 1H), 6.79 (d, J = 7.3 Hz, 1H), 6.76-6.64 (m, 2H), 4.67 (s, 1H ), 3.81 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H), 0.89 (s, 9H), 0.01 (s, 6H).

(2) 3- [5- (4-{[3- (2-{[tert-Butyl (dimethyl) silyl] oxy} ethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl- 4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one 3- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl) phenol (2 18 g) and 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine Using -2-one (Reference Example 8- (2), 2.95 g) in the same manner as in Reference Example 9- (3), the title compound (4.31 g) was obtained as a white solution. Obtained as a solid.

(3) 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[3- (2-hydroxyethyl) phenoxy] methyl} phenoxy) Pentyl] -1,3-oxazolidine-2-one 3- [5- (4-{[3- (2-{[tert-butyl (dimethyl) silyl] oxy} ethyl) phenoxy] methyl} obtained above Phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidin-2-one (4.31 g) was used to give Reference Example 8- The reaction and treatment were conducted in a manner similar to that described in (2) to give the title compound (1.75 g) as a white solid.

(4) 3- [5- (4-{[3- (2-Azidoethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl ) -1,3-oxazolidine-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[3 Using-(2-hydroxyethyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidine-2-one (1.75 g), the reaction was carried out in the same manner as described in Reference Example 7- (2). Treatment gave the title compound (1.16 g) as a white solid.

(5) 4- (2-{[5- (4-{[3- (2-azidoethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol The resulting 3- [5- (4-{[3- (2-azidoethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl ) -1,3-oxazolidine-2-one (352 mg) was used in the same manner as in the method described in Reference Example 7- (3) to give the title compound (47 mg) as a colorless oil. .
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.34 (d, J = 8.0 Hz, 2H), 7.23 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.02 ( s, 1H), 6.90 (d, J = 8.0 Hz, 2H), 6.87-6.81 (m, 4H), 4.97 (s, 2H), 4.81 (s, 2H), 4.61 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 3.96 (t, J = 8.0 Hz, 2H), 3.50 (t, J = 8.0 Hz, 2H), 2.88-2.81 (m, 3H), 2.73-2.64 (m, 3H), 1.83 -1.76 (m, 2H), 1.61-1.48 (m, 4H).
MS (ESI) m / z 521 (M + H) ⁺ .

Reference Example 13
4- (2-{[5- (4-{[4- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol

(1) 3- [5- (4-{[4-({[tert-Butyl (dimethyl) silyl] oxy} methyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H- 1,3-Benzodioxin-6-yl) -1,3-oxazolidin-2-one 4-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenol (Reference Example 6- (2), 78 g) and 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine- Using 2-one (Reference Example 8- (2), 3.0 g) in the same manner as in Reference Example 9- (3), the title compound (4.03 g) was obtained as a white solid. Obtained as a thing.

(2) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[4- (hydroxymethyl) phenoxy] methyl} phenoxy) pentyl] 1,3-Oxazolidin-2-one 3- [5- (4-{[4-({[tert-butyl (dimethyl) silyl] oxy} methyl) phenoxy] methyl} phenoxy) pentyl] obtained above Using 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (4.03 g), Reference Example 8- (2) Reaction and treatment in the same manner as described gave the title compound (1.68 g) as a white solid.

(3) 3- [5- (4-{[4- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl)- 1,3-oxazolidine-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[4- ( Hydroxymethyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidin-2-one (1.68 g) was reacted and treated in the same manner as described in Reference Example 7- (2) to give the title The compound (1.17 g) was obtained as a white solid.

(4) 4- (2-{[5- (4-{[4- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol obtained above 3- [5- (4-{[4- (azidomethyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1, The title compound (82 mg) was obtained as a white amorphous substance by reacting and treating in a similar manner as described in Reference Example 7- (3) using 3-oxazolidine-2-one (343 mg).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.34 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.00 ( s, 1H), 6.97 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.0Hz, 2H), 6.83 (d, J = 8.0 Hz, 1H), 4.98 (s, 2H), 4.80 ( s, 2H), 4.57 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.27 (s, 2H), 3.96 (t, J = 8.0 Hz, 2H), 2.80 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 2.72-2.59 (m, 3H), 1.83-1.75 (m, 2H), 1.59-1.47 (m, 4H).
MS (ESI) m / z 507 (M + H) ⁺ .

Reference Example 14
4- (2-{[5- (4-{[3- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol

(1) 3- (3-{[tert-butyl (dimethyl) silyl] oxy} propyl) phenol By using 3- (3-hydroxypropyl) phenol (10.0 g), Reference Examples 9- (1) and 9 Reaction and treatment in the same manner as described in (2) gave the title compound (12.0 g) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.14 (t, J = 7.6 Hz, 1H), 6.77 (d, J = 7.5 Hz, 1H), 6.70-6.61 (m, 2H), 4.65 (s, 1H ), 3.63 (t, J = 6.3 Hz, 2H), 2.63 (t, J = 7.8 Hz, 2H), 1.91-1.76 (m, 2H), 0.91 (s, 9H), 0.05 (s, 6H).
MS (ESI) m / z 267 (M + H) ⁺ .

(2) 3- [5- (4-{[3- (3-{[tert-Butyl (dimethyl) silyl] oxy} propyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl- 4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one 3- (3-{[tert-butyl (dimethyl) silyl] oxy} propyl) phenol (1 88g) and 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- {5- [4- (hydroxymethyl) phenoxy] pentyl} -1,3-oxazolidine Using -2-one (Reference Example 8- (2), 3.0 g) in the same manner as in Reference Example 9- (3), the title compound (3.84 g) was obtained as a white solution. Obtained as a solid.

(3) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[3- (3-hydroxypropyl) phenoxy] methyl} phenoxy) Pentyl] -1,3-oxazolidine-2-one 3- [5- (4-{[3- (3-{[tert-butyl (dimethyl) silyl] oxy} propyl) phenoxy] methyl} obtained above Phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (3.84 g) was used to give Reference Example 8- The reaction and treatment were conducted in a manner similar to that described in (2) to give the title compound (2.04 g) as a white solid.

(4) 3- [5- (4-{[3- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6- Yl) -1,3-oxazolidine-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [5- (4-{[ 3- (3-Hydroxypropyl) phenoxy] methyl} phenoxy) pentyl] -1,3-oxazolidin-2-one (2.04 g) was reacted in the same manner as described in Reference Example 7- (2) To give the title compound (1.76 g) as a colorless oil.

(5) 4- (2-{[5- (4-{[3- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] amino} -1-hydroxyethyl) -2- (hydroxymethyl) phenol 3- [5- (4-{[3- (3-azidopropyl) phenoxy] methyl} phenoxy) pentyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6 obtained in -Ill) -1,3-oxazolidine-2-one (360 mg) was used for the reaction and treatment in the same manner as described in Reference Example 7- (3) to give the title compound (128 mg) as a colorless oil. Obtained.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.33 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.96 ( s, 1H), 6.89 (d, J = 8.0 Hz, 2H), 6.84-6.77 (m, 4H), 4.96 (s, 2H), 4.70 (s, 2H), 4.59 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 3.94 (t, J = 8.0 Hz, 2H), 3.27 (t, J = 8.0 Hz, 2H), 2.80-2.58 (m, 6H), 1.95-1.86 (m, 2H), 1.81 -1.73 (m, 2H), 1.61-1.52 (m, 2H), 1.52-1.44 (m, 2H).
MS (ESI) m / z 535 (M + H) ⁺ .

Reference Example 15
Nucleic acid sequence: 3′-ttCGUGUCCUCUCUUCUCGUA-5 ′ (SEQ ID NO: 1)
(1) Synthesis of oligonucleotides All oligonucleotides were synthesized by an AKTA oligopilot synthesizer. Commercially available aminated nonporous polystyrene solid support (Primer Support 200 dT Synth GE Healthcare), RNA phosphoramidite with standard protecting group (5'-O-dimethoxytrityl-N ⁶ -benzoyl-2'- Triisopropylsilyloxymethyl-adenosine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-N ⁴ -acetyl-2′-triisopropylsilyloxymethyl-cytidine -3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N ² -isobutyryl-2'-triisopropylsilyloxymethyl-guanosine-3'-ON , N'-Diisopropyl-2-cyanoethyl phosphoroa And 5'-O-dimethoxytrityl-2'-triisopropylsilyloxymethyl-uridine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite (Sigma Aldrich)), and standard A DNA phosphoramidite (5′-O-dimethoxytrityl-thymidine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite manufactured by Sigma-Aldrich) having an appropriate protecting group was used. All phosphoramidites were used at a concentration of 0.1M in acetonitrile. A 15 minute ligation / reuse time was used for RNA and a 5 minute ligation / reuse time was used for DNA. As the phosphoramidite activator, 5-benzylmercaptotetrazole (0.25M, manufactured by Wako Pure Chemical Industries, Ltd.) was used, and iodine / water / pyridine was used for the oxidation of phosphorous acid to phosphoric acid.
(2) Deprotection I (Cleavage from the carrier)
The polystyrene carrier (1 g) obtained in (1) was allowed to act on an equal amount mixture (15 mL) of 40% aqueous methylamine solution and 33% methylamine ethanol solution at 45 ° C. for 13 hours. The oligonucleotide was cleaved from the support while deprotecting the base moiety. The mixture was filtered and the polystyrene support was washed twice with ethanol / water (1: 1 v / v, 40 mL). The filtrate was distilled off on a rotary evaporator (roto-vap) and dried.
(3) Deprotection II (removal of 2′-triisopropylsilyloxymethyl (2′-TOM) group)
The dry residue obtained in (2) is suspended in a deprotection solution, that is, a 1M tetra-n-butylammonium fluoride / tetrahydrofuran solution (0.25 mL / synthetic oligonucleotide 1 μmol), and is suspended at 50 ° C. for 10 minutes, then Heat at 14 ° C. for 14 hours. The reaction was quenched with 1M Tris-HCl buffer (pH 7.5) in the same volume as the deprotection solution and stored in a freezer until used for the next purification step.
(4) Purification by HPLC The crude oligonucleotide obtained in (3) was purified by reversed-phase ion exchange HPLC using a Source 15 RPC gel column to obtain a fraction containing the 5′-dimethoxytrityl form of the title compound. . After removing the dimethoxytrityl group using 0.6% trifluoroacetic acid, the fraction containing the full-length oligonucleotide was purified by reverse phase ion exchange HPLC. After desalting, the title compound was obtained by lyophilization.

Reference Example 16

Synthesized from corresponding raw materials in the same manner as in Reference Example 15. However, as the polystyrene solid carrier, an aminated nonporous polystyrene solid carrier (Custom Primer Support C6 amino 80 manufactured by GE Healthcare) was used.

Reference Example 17

Synthesized from corresponding raw materials in the same manner as in Reference Example 15. However, as the 5 ′ terminal aminated phosphoramidite, 1-N- [2 ′-(monomethoxytrityl) aminoethoxycarbonyl] amino-6-hexyl-6-ON, N′-diisopropyl-2-cyanoethylphospho Loamidite (manufactured by Sigma Aldrich) was used.

Reference Example 18

2 mM aqueous solution (2.2 mL) of the compound of Reference Example 16 (30.8 mg) and 4-pentynoic acid succinimidyl ester (Reference Example 1) under sodium carbonate buffer (pH 9.0, final concentration 0.1 M) ( 1.72 mg) of dimethyl sulfoxide / acetonitrile mixed solution (4/1, 1.76 mL) was mixed and stirred at room temperature for 1 hour. The reaction solution was purified by reverse phase ion exchange HPLC on a Source 15 RPC gel column. As the mobile phase, acetonitrile / 0.1 M triethylamine acetate buffer (pH 7.0) (5/95 to 90/10) was used. Fractions containing the title compound were collected, desalted and lyophilized to give the title compound (22.7 mg).

Reference Example 19

In the same manner as in Reference Example 18, the title compound (7.0 mg) was obtained from the compound of Reference Example 16 (19.6 mg) and 5-hexynoic acid succinimidyl ester (Reference Example 2).
MALDI-TOF / MS: m / z = 7089.79 (MH) ^- (theoretical 7090.51).

Reference Example 20

In the same manner as in Reference Example 18, the title compound (25.4 mg) was obtained from the compound of Reference Example 16 (30 mg) and 10-undecic acid succinimidyl ester (Reference Example 3).
MALDI-TOF / MS: m / z = 7157.90 (MH) ^- (theoretical 7716.64).

Reference Example 21

In the same manner as in Reference Example 18, the title compound (3.6 mg) was obtained from the compound of Reference Example 17 (7.5 mg) and 4-pentynoic acid succinimidyl ester (Reference Example 1).
MALDI-TOF / MS: m / z = 7161.49 (MH) ^- (theoretical 7716.43).

Reference Example 22

In the same manner as in Reference Example 18, the title compound (3.6 mg) was obtained from the compound of Reference Example 17 (7.5 mg) and 5-hexynoic acid succinimidyl ester (Reference Example 2).
MALDI-TOF / MS: m / z = 7175.15 (MH) ^- (theoretical value 7174.46).

Reference Example 23
2-propynyl-4-pentynoic acid succinimidyl ester

Using 2-propynyl-4-pentynoic acid (300 mg) synthesized by the method described in the literature (Journal of the Chemical Society, Perkin Transactions 1, 1986, 7, 1215-24) and the method described in Reference Example 1 The same reaction and treatment were performed to obtain the title compound (354 mg) as a colorless solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 3.10 (m, 1H), 2.82 (s, 4H), 2.79-2.76 (m, 4H), 2.09 (t, J = 2.7 Hz, 2H).

Reference Example 24
N ′-[(1E) -1- (4- {4-[(2,5-dioxopyrrolidin-1-yl) oxy] -4-oxobutoxy} phenyl) ethylidene] pent-4-inhydrazide

(1) 4- (4-{(1E) -1- [2- (pent-4-inoyl) hydrazinylidene] ethyl} phenoxy) butyric acid literature (Journal of the Chemical Society, Perkin Transactions 1, 1987, 11, 2511- Acetic acid (1.94 mL) was added to a methanol solution (19.4 mL) of pent-4-inhydrazide (580 mg) and 4- (4-acetylphenoxy) butyric acid synthesized by the method described in 16) at 45 ° C. Stir for 5 hours. The reaction mixture was cooled to room temperature, and the precipitated solid was washed with methanol to give the title compound (1.18 g) as a colorless solid.
¹ H-NMR (400MHz, DMSO) δ: 10.4 (s, 0.5H), 10.3 (s, 0.5H), 7.73-7.65 (m, 2H), 6.95-6.90 (m, 2H), 4.04-3.99 (m , 2H), 2.88-2.74 (m, 2H), 2.46-2.34 (m, 5H), 2.23-2.15 (m, 3H), 2.00-1.91 (m, 2H).

(2) N ′-[(1E) -1- (4- {4-[(2,5-dioxopyrrolidin-1-yl) oxy] -4-oxobutoxy} phenyl) ethylidene] pent-4-yne Hydrazide 4- (4-{(1E) -1- [2- (pent-4-inoyl) hydrazinylidene] ethyl} phenoxy) butyric acid (1.18 g) obtained above and N-hydroxysuccinimide (518 mg) The title compound (1.20 g) was obtained as a colorless solid by reaction and treatment in the same manner as described in Reference Example 1.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 8.44 (brs, 1H), 7.69 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 4.12-4.05 (m, 2H ), 3.06-3.00 (m, 2H), 2.91-2.77 (m, 6H), 2.68-2.58 (m, 2H), 2.30-2.21 (m, 2H), 2.17 (s, 3H), 2.02-1.98 (m , 1H).

Reference Example 25
N ′-[(E)-(4- {4-[(2,5-dioxopyrrolidin-1-yl) oxy] -4-oxobutoxy} phenyl) methylidene] pent-4-inhydrazide

(1) 4- (4-{(E)-[2- (pent-4-inoyl) hydrazinylidene] methyl} phenoxy) butyric acid Pent synthesized by the method described in the literature (Tetrahedron, 2003, 59, 6121-6130) Using 4--4-hydrazide (452 mg) and 4- (4-formylphenoxy) butyric acid (700 mg), the reaction and treatment were conducted in the same manner as in Reference Example 24- (1) to give the title compound (885 mg). Obtained as a colorless solid.
¹ H-NMR (400MHz, DMSO) δ: 11.3 (s, 0.5H), 11.2 (s, 0.5H), 8.09 (s, 0.5H), 7.92 (s, 0.5H), 7.62-7.57 (m, 2H ), 7.00-6.97 (m, 2H), 4.04-4.00 (m, 2H), 2.82-2.77 (m, 2H), 2.51-2.37 (m, 6H), 1.96-1.93 (m, 2H).

(2) N ′-[(E)-(4- {4-[(2,5-dioxopyrrolidin-1-yl) oxy] -4-oxobutoxy} phenyl) methylidene] pent-4-inhydrazide Using 4- (4-{(E)-[2- (pent-4-inoyl) hydrazinylidene] methyl} phenoxy) butyric acid (885 mg) obtained in the same manner as in the method described in Reference Example 1, Treatment gave the title compound (844 mg) as a colorless solid.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 8.62 (brs, 1H), 7.65 (s, 1H), 7.59 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.15-4.06 (m, 2H), 3.06-2.99 (m, 2H), 2.93-2.80 (m, 7H), 2.66-2.58 (m, 2H), 2.29-2.20 (m, 2H), 2.00 (s, 1H ).

Reference Example 26
N ′-[1- (3- {4-[(2,5-dioxopyrrolidin-1-yl) oxy] -4-oxobutoxy} phenyl) ethylidene] pent-4-inhydrazine

(1) 4- (3- {1- [2- (pent-4-inoyl) hydrazinylidene] ethyl} phenoxy) butyric acid in literature (Journal of the Chemical Society, Perkin Transactions 1, 1987), 11, 2511-16) Using pent-4-inhydrazide (580 mg) and ethyl 4- (3-acetylphenoxy) butanoate (970 mg) synthesized by the method described in the above, the same reaction and treatment as in the method described in Reference Example 24- (1) To give the title compound (1.08 g) as a colorless solid.
¹ H-NMR (400MHz, DMSO) δ: 10.5 (s, 0.5H), 10.4 (s, 0.5H), 7.40-7.22 (m, 3H), 6.98-6.94 (m, 1H), 4.09-3.95 (m , 2H), 2.92-2.74 (m, 3H), 2.60-2.2.34 (m, 4H), 2.31-2.18 (m, 3H), 2.02-1.90 (m, 2H).

(2) N ′-[1- (3- {4-[(2,5-dioxopyrrolidin-1-yl) oxy] -4-oxobutoxy} phenyl) ethylidene] pent-4-inhydrazine obtained above Using the obtained 4- (3- {1- [2- (pent-4-inoyl) hydrazinylidene] ethyl} phenoxy) butyric acid (1.08 g), the reaction and treatment were conducted in the same manner as described in Reference Example 1. To give the title compound (903 mg) as a colorless solid.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.33-7.28 (m, 3H), 6.97-6.93 (m, 1H), 4.14-4.08 (m, 2H), 3.11-3.02 (m, 2H), 2.90- 2.79 (m, 6H), 2.67-2.60 (m, 2H), 2.28-2.21 (m, 2H), 2.19 (s, 3H), 2.00-1.98 (m, 1H).

Reference Example 27
1-{[4- (4-{(E)-[(prop-2-yn-1-yloxy) imino] methyl} phenoxy) butanoyl] oxy} pyrrolidine-2,5-dione

(1) Ethyl 4- {4-[(E)-(hydroxyimino) methyl] phenoxy} butanoate Ethyl 4- (4-formylphenoxy) butanoate (5.0 g) in ethanol solution (30 mL) was added hydroxylamine hydrochloride. (1.77 g) and sodium acetate (2.09 g) were added, and the mixture was stirred at 70 ° C. for 3 hours. After the reaction mixture was cooled to room temperature, ethyl acetate and water were added and the layers were separated. The organic layer is washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue is purified by silica gel chromatography (hexane / ethyl acetate) to give the title compound (3.71 g) as a colorless oil. It was.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 8.06 (s, 1H), 7.48 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.16-3.99 (m, 4H ), 2.50 (t, J = 5.8 Hz, 2H), 2.12-2.08 (m, 2H), 1.24 (t, J = 7.5 Hz, 3H).

(2) Ethyl 4- (4-{(E)-[(prop-2-yn-1-yloxy) imino] methyl} phenoxy) butanoate Ethyl 4- {4-[(E)-( Propargyl bromide (515 μL) and cesium carbonate (2.90 g) were added to a DMF solution (30 mL) of hydroxyimino) methyl] phenoxy} butanoate (1.5 g), and the mixture was stirred at 60 ° C. for 2 hours. After the reaction mixture was cooled to room temperature, ethyl acetate and water were added and the layers were separated. The organic layer is washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue is purified by silica gel chromatography (hexane / ethyl acetate) to give the title compound (1.28 g) as a colorless oil. It was.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 8.05 (s, 1H), 7.51 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 4.72 (s, 2H), 4.13 (q, J = 7.6 Hz, 2H), 4.01 (t, J = 6.0 Hz, 2H), 2.52-2.47 (m, 2H), 2.12-2.02 (m, 2H), 1.24 (t, J = 7.6 Hz , 3H).

(3) 4- (4-{(E)-[(prop-2-yn-1-yloxy) imino] methyl} phenoxy) butyric acid Ethyl 4- (4-{(E)-[( To a mixed solution of prop-2-in-1-yloxy) imino] methyl} phenoxy) butanoate (1.0 g) in THF (18 mL) and methanol (6 mL) was added an aqueous solution (6 mL) of lithium hydroxide (290 mg). And stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in water and neutralized with 1N hydrochloric acid. The precipitated crystals were collected by filtration to give the title compound (717 mg) as a colorless solid.
¹ H-NMR (300MHz, DMSO) δ: 12.2 (brs, 1H), 8.21 (s, 1H), 7.55 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 4.71 (d, J = 1.8 Hz, 2H), 4.01 (d, J = 6.0 Hz, 2H), 3.48 (t, J = 1.8 Hz, 1H), 2.37 (t, J = 6.7 Hz, 1H), 1.98-1.89 (m, 2H).

(4) 1-{[4- (4-{(E)-[(prop-2-yn-1-yloxy) imino] methyl} phenoxy) butanoyl] oxy} pyrrolidine-2,5-dione obtained above 4- (4-{(E)-[(prop-2-yn-1-yloxy) imino] methyl} phenoxy) butyric acid (245 mg) was used for the same reaction and treatment as in the method described in Reference Example 1. To give the title compound (305 mg) as a colorless solid.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 8.05 (s, 1H), 7.52 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 4.73 (d, J = 1.7 Hz, 2H), 4.06 (t, J = 6.3 Hz, 2H), 2.86-2.83 (m, 7H), 2.48 (t, J = 1.7 Hz, 1H), 2.38-2.15 (m, 2H).

Reference Example 28
tert-butyl (1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-3-yl) methylcarbamate

(1) {4- [4- (6-Azidohexyloxy) butyl] phenyl} methanol (4- {4-[(6-bromohexyl) oxy] butyl} phenyl) methanol (3 0.0 g) in DMF (20 mL) was added sodium azide (684 mg), and the mixture was stirred at 80 ° C. for 5 hours. Water and ethyl acetate were added to the reaction mixture, and the organic layer was separated. The organic layer was washed with water and saturated brine in that order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (2.66 g) as a pale yellow oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.26 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 4.63 (s, 2H), 3.41-3.21 (m, 6H ), 2.64-2.59 (m, 2H), 1.64-1.24 (m, 12H).

(2) 4- {4-[(6-Azidohexyloxy] butyl} benzaldehyde 4- {4-[(6-azidohexyloxy) butyl] phenyl} methanol (2.66 g) obtained above in methylene chloride To the solution (20 mL) were added N-methyl-morpholine N-oxide (1.22 g), tetrapropylammonium perruthenate (152 mg) and molecular sieves 4A (800 mg), and the mixture was stirred at room temperature for 5 hours under a nitrogen atmosphere. After filtration of the mixture, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (2.26 g) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 9.95 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 3.42-3.21 (m, 6H ), 2.72-2.67 (m, 2H), 1.71-1.35 (m, 12H).
MS EM 303.19 found 304.4.

(3) tert-butyl (1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-3-yl) methylcarbamate 4- {4-[(6-azidohexyl) obtained above Add tert-butyl piperidin-3-ylmethylcarbamate (259 mg), magnesium sulfate (291 mg) and triethylamine (0.1 mL) to a methanol solution (3 mL) of oxy] butyl} benzaldehyde (368 mg) and stir at room temperature for 7 hours. Sodium tetrahydroborate (229 mg) was added to the reaction mixture, and the mixture was stirred at room temperature for 15 hours, saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by chromatography (hexane / ethyl acetate) gave the title compound (292 mg) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.18 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 4.61 (brs, 1H), 3.47-3.21 (m, 10H ), 3.0-2.57 (m, 6H), 1.96-0.99 (m, 26H).
MS EM501.37 Measured 502.5

Reference Example 29
tert-Butyl (1- (4- (4- (6-Azidohexyloxy) butyl) benzyl) morpholin-2-yl) methylcarbamate

To a THF solution (3 mL) of 4- {4-[(6-azidohexyloxy] butyl} benzaldehyde (368 mg) obtained in Reference Example 28- (2), tert-butyl morpholin-2-ylmethylcarbamate (259 mg) Acetic acid (0.14 mL) and sodium triacetoxyborohydride (513 mg) were added, and the mixture was stirred at room temperature for 15 hours, saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (228 mg) as a colorless oil.
MS EM503.35 found 504.5

Reference Example 30
tert-butyl 1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-4-yl) carbamate

To a methanol solution (5 mL) of 4- {4-[(6-azidohexyloxy] butyl} benzaldehyde (808 mg) obtained in Reference Example 28- (2), tert-butyl piperidin-4-ylcarbamate (533 mg), Magnesium sulfate (642 mg) and triethylamine (0.22 mL) were added, and the mixture was stirred at room temperature for 7 hours, sodium tetrahydroborate (503 mg) was added to the reaction mixture, and the mixture was stirred for 15 hours at room temperature. An aqueous sodium solution was added, and the mixture was extracted with ethyl acetate.The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate). The title compound (641 mg) was obtained as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.18 (d, J = 8.0Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 4.39 (brs, 1H), 3.42-3.21 (m, 9H ), 2.79-2.57 (m, 4H), 2.08-1.21 (m, 27H).
MS EM 487.35 found 488.6

Reference Example 31
4- {2-[(6- {4- [3- (azidomethyl) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) phenol

(1) tert-butyl 3- (4-hydroxybut-1-in-1-yl) benzoate Using tert-butyl 3-iodobenzoate (31.13 g) and 3-butyn-1-ol (9.33 g) The title compound (23.7 g) was obtained as a white solid by reaction and treatment in the same manner as described in Reference Example 5- (2).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 8.01 (s, 1H), 7.95-7.92 (m, 1H), 7.56-7.51 (m, 1H), 7.39-7.33 (m, 1H), 3.88-3.79 ( m, 2H), 2.75-2.65 (m, 2H), 1.59 (s, 9H).

(2) tert-butyl 3- (4-hydroxybutyl) benzoate The tert-butyl 3- (4-hydroxybut-1-in-1-yl) benzoate (27.6 g) obtained above was used as a reference. The reaction and treatment were conducted in a manner similar to that described in Example 5- (3), and the title compound (27.4 g) was obtained as a colorless solid.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.82-7.98 (m, 2H), 7.39-7.25 (m, 2H), 3.68-3.65 (m, 2H), 2.70-2.63 (m, 2H), 1.77- 1.64 (m, 2H), 1.61-1.57 (m, 11H).

(3) tert-butyl 3- {4-[(6-bromohexyl) oxy] butyl} benzoate Using the tert-butyl 3- (4-hydroxybutyl) benzoate (27.4 g) obtained above, The reaction and treatment were conducted in a manner similar to that described in Example 5- (4), and the title compound (33.1 g) was obtained as a colorless solid.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.83-7.75 (m, 2H), 7.38-7.23 (m, 2H), 3.42-3.39 (m, 6H), 2.70-2.61 (m, 2H), 1.91- 1.82 (m, 2H), 1.78-1.49 (m, 15H), 1.45-1.30 (m, 4H).

(4) 3- {4-[(6 bromohexyl) oxy] butyl} benzoic acid tert-butyl 3- {4-[(6-bromohexyl) oxy] butyl} benzoate obtained above (33.0 g) The title compound (28.7 g) was obtained as a colorless solid by reaction and treatment in the same manner as in Reference Example 5- (5).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.94-7.90 (m, 2H), 7.43-7.31 (m, 2H), 3.43-3.35 (m, 6H), 2.72-2.63 (m, 2H), 1.85- 1.32 (m, 12H).

(5) (3- {4-[(6-Bromohexyl) oxy] butyl} phenyl) methanol 3- {4-[(6 bromohexyl) oxy] butyl} benzoic acid obtained above (28.7 g) The title compound (23.6 g) was obtained as a colorless solid by reaction and treatment in the same manner as described in Reference Example 5- (6).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.30-7.26 (m, 1H), 7.20-7.16 (m, 2H), 7.13-7.09 (m, 1H), 4.67 (s, 2H), 3.43-3.38 ( m, 6H), 2.66-2.62 (m, 2H), 1.92-1.83 (m, 2H), 1.73-1.52 (m, 6H), 1.50-1.28 (m, 4H).

(6) 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- (6- {4- [3- (hydroxymethyl) phenyl] butoxy} hexyl) -1, 3-Oxazolidin-2-one Using the compound of Reference Example 4 (2.67 g) and (3- {4-[(6-bromohexyl) oxy] butyl} phenyl) methanol (3.72 g) obtained above. The title compound (4.39 g) was obtained as a white solid by reaction and treatment in the same manner as described in Reference Example 7- (1).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.30-7.08 (m, 5H), 6.99 (s, 1H), 6.85-6.82 (m, 1H), 5.45-5.37 (m, 1H), 4.84 (s, 2H), 4.73-4.64 (m, 2H), 3.90-3.80 (m, 1H), 3.49-3.20 (m, 7H), 2.70-2.61 (m, 2H), 1.94-1.88 (m, 1H), 1.72- 1.45 (m, 14H), 1.43-1.29 (m, 4H).

(7) 3- (6- {4- [3- (azidomethyl) phenyl] butoxy} hexyl) -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3 -Oxazolidin-2-one 4- {2-[(6- {4- [3- (azidomethyl) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) obtained above The title compound (3.78 g) was obtained as a colorless solid by reacting and treating with phenol (4.39 g) in the same manner as described in Reference Example 7- (2).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.33-7.25 (m, 3H), 7.18-7.09 (m, 2H), 7.02 (s 1H), 6.86-6.81 (m, 1H), 5.43-5.35 (m , 1H), 4.84 (s, 2H), 4.31 (s, 2H), 3.89-3.81 (m, 1H), 3.45-3.18 (m, 7H), 2.68-2.61 (m, 2H), 1.74-1.49 (m , 14H), 1.42-1.30 (m, 4H).

(8) 2-[(6- {4- [3- (azidomethyl) phenyl] butoxy} hexyl) amino] -1- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) ethanol 3- (6- {4- [3- (azidomethyl) phenyl] butoxy} hexyl) -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1 obtained above , 3-Oxazolidin-2-one (2.0 g) was reacted and treated in the same manner as described in Reference Example 7- (3) to give the title compound (281 mg) as a white solid.
MS (ESI) m / z 472 (M + H) ⁺ .

Reference Example 32
4- {2-[(6- {4- [4- (2-azidoethoxy) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) phenol

(1) 3- [6- (but-3-in-1-yloxy) hexyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine -2-one 1-bromo-6- (but-3-in-1-yloxy) hexane (33.0 g) and the compound of Reference Example 4 (22.3 g) were used to give Reference Example 7- (1). Reaction and treatment in the same manner as described gave the title compound (33.6 g) as a white solid.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.13-7.11 (m, 1H), 7.00 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 5.41-5.38 (m , 1H), 4.84 (s, 2H), 3.86 (t, J = 8.9 Hz, 1H), 3.55 (t, J = 7.0 Hz, 2H), 3.47-3.27 (m, 5H), 2.48-2.44 (m, 2H), 1.99-1.98 (m, 1H), 1.58-1.54 (m, 10H), 1.38-1.36 (m, 4H).

(2) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- [6-({4- [4- (2-hydroxyethoxy) phenyl] but-3- In-1-yl} oxy) hexyl] -1,3-oxazolidine-2-one 3- [6- (but-3-in-1-yloxy) hexyl] -5- (2,2 -Dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidin-2-one (4.20 g), 2- (3-bromophenoxy) ethanol (3.17 g), triethylamine (89 mL) ) In acetonitrile (49 mL), and cuprous iodide (200 mg) and bistriphenylphosphine palladium (II) dichloride (444 mg) were added, followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to give the title compound (4.81 g) as a brown oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.22-7.16 (m, 1H), 7.13-7.09 (m, 1H), 7.02-6.97 (m, 2H), 6.94 (s, 1H), 6.86-6.80 ( m, 2H), 5.41-5.34 (m, 1H), 4.84 (s, 2H), 4.08-4.02 (m, 2H), 4.00-3.91 (m, 2H), 3.86-3.80 (m, 1H), 3.65- 3.58 (m, 2H), 3.51-3.45 (m, 2H), 3.40-3.16 (m, 3H), 2.70-2.64 (m, 2H), 2.13-2.07 (m, 1H), 1.66-1.49 (m, 9H ), 1.48-1.29 (m, 4H).

(3) 5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -3- (6- {4- [3- (2-hydroxyethoxy) phenyl] butoxy} hexyl)- 1,3-oxazolidine-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- [6-{{4- [4- ( 2-hydroxyethoxy) phenyl] but-3-yn-1-yl} oxy) hexyl] -1,3-oxazolidin-2-one (4.81 g) in ethanol (66 mL) and ethyl acetate (13 mL) To the mixture, platinum dioxide (681 mg) was added and the atmosphere was replaced with hydrogen, followed by stirring at room temperature for 2.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (3.66 g) as a colorless oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.22-7.16 (m, 1H), 7.11-7.07 (m, 1H), 7.00 (s, 1H), 6.88-6.70 (m, 4H), 5.42-5.35 ( m, 1H), 4.84 (s, 2H), 4.10-4.03 (m, 2H), 3.98-3.92 (m, 2H), 3.88-3.80 (m, 1H), 3.45-3.19 (m, 7H), 2.65- 2.57 (m, 2H), 2.15-2.05 (m, 1H), 1.72-1.45 (m, 13H), 1.43-1.28 (m, 4H).

(3) 3- (6- {4- [4- (2-azidoethoxy) phenyl] butoxy} hexyl) -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl)- 1,3-Oxazolidin-2-one 5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -3- (6- {4- [3- (2 -Hydroxyethoxy) phenyl] butoxy} hexyl) -1,3-oxazolidin-2-one (1.91 g) was reacted and treated in the same manner as described in Reference Example 7- (2) to give the title compound. (3.25 g) was obtained as a colorless oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.21-7.14 (m, 1H), 7.12-7.08 (m, 1H), 6.99 (s, 1H), 6.83-6.77 (m, 2H), 6.77-6.59 ( m, 2H), 5.42-5.35 (m, 1H), 4.84 (s, 2H), 4.16-4.10 (m, 2H), 3.86-3.81 (m, 1H), 3.60-3.55 (m, 2H), 3.44- 3.20 (m, 6H), 2.64-2.57 (m, 2H), 1.71-1.47 (m, 14H), 1.41-1.28 (m, 4H).

(4) 4- {2-[(6- {4- [4- (2-azidoethoxy) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) phenol obtained above 3- (6- {4- [4- (2-azidoethoxy) phenyl] butoxy} hexyl) -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1, The title compound (428 mg) was obtained as a colorless oil by reacting and treating in a similar manner to the method described in Reference Example 7- (3) using 3-oxazolidine-2-one (1.91 g).
MS (ESI) m / z 501 (M + H) ⁺ .

Reference Example 33
N- (2-azidoethyl) -3- (4-{[6-({2-hydroxy-2- [4-hydroxy-3- (hydroxymethyl) phenyl] ethyl} amino) hexyl] oxy} butyl) benzenesulfone Amide

(1) 3- [4-({6- [5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxo-1,3-oxazolidine-3-yl] Hexyl} oxy) but-1-in-1-yl] -N- (2-hydroxyethyl) benzenesulfonamide 3- [6- (but-3-in-1) obtained in Reference Example 28- (1) -Iroxy) hexyl] -5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -1,3-oxazolidine-2-one (7.61 g) and 3-bromo-N- (2-Hydroxyethyl) benzenesulfonamide (5.58 g) was used for the reaction and treatment in the same manner as described in Reference Example 7- (1) to give the title compound (8.92 g) as a colorless oil. Obtained.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.89 (s, 1H), 7.79-7.73 (m, 1H), 7.58-7.55 (m, 1H), 7.46-7.43 (m, 1H), 7.15-7.10 ( m, 1H), 7.02 (s, 1H), 6.85-6.83 (m, 1H), 5.49-5.41 (m, 1H), 5.31 (brs, 1H), 4.85 (s, 2H), 3.85-3.90 (m, 1H), 3.70-3.67 (m, 2H), 3.65-3.58 (m, 2H), 3.55-3.47 (m, 2H), 3.46-3.40 (m, 1H), 3.39-3.20 (m, 2H), 3.12- 3.08 (m, 2H), 2.71-2.67 (m, 2H), 1.60-1.30 (m, 13H).

(2) 3- [4-({6- [5- (2,2-Dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxo-1,3-oxazolidine-3-yl] Hexyl} oxy) butyl] -N- (2-hydroxyethyl) benzenesulfonamide 3- [4-({6- [5- (2,2-dimethyl-4H-1,3-benzodioxin) obtained above] -6-yl) -2-oxo-1,3-oxazolidine-3-yl] hexyl} oxy) but-1-in-1-yl] -N- (2-hydroxyethyl) benzenesulfonamide (8.92 g The title compound (8.95 g) was obtained as a colorless oil by reaction and treatment in the same manner as described in Reference Example 32- (2).
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.70-7.68 (m, 2H), 7.41-7.39 (m, 2H), 7.15-7.11 (m, 1H), 7.01 (s, 1H), 6.85-6.81 ( m, 1H), 5.43-5.39 (m, 1H), 4.85 (s, 2H), 3.91-3.85 (m, 1H), 3.75-3.70 (m, 2H), 3.63-3.61 (m, 2H), 3.43- 3.23 (m, 6H), 3.11-3.08 (m, 2H), 2.73-2.69 (m, 2H), 1.73-1.65 (m, 2H), 1.61-1.25 (m, 15H).

(3) N- (2-azidoethyl) -3- [4-({6- [5- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-oxo-1, 3-Oxazolidin-3-yl] hexyl} oxy) butyl] benzenesulfonamide 3- [4-({6- [5- (2,2-dimethyl-4H-1,3-benzodioxin-) obtained above 6-yl) -2-oxo-1,3-oxazolidin-3-yl] hexyl} oxy) butyl] -N- (2-hydroxyethyl) benzenesulfonamide (8.15 g) in dichloromethane (16 mL) Pyridine (3.34 mL) and paratoluenesulfonyl chloride (7.71 g) were added at 0 ° C., and the mixture was warmed to room temperature and stirred for 27 hours and a half. Water was added to the reaction mixture, and the target product was extracted with chloroform. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Sodium azide (1.75 g) was added to a dimethyl sulfoxide solution (135 mL) of the obtained residue, and the mixture was stirred at 35 ° C. for 12 hours. Water was added to the reaction mixture, and the target product was extracted with ethyl acetate. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (7.51 g) as a colorless oil.
¹ H-NMR (400MHz, CDCl ₃ ) δ: 7.71-7.68 (m, 2H), 7.42-7.40 (m, 2H), 7.14-7.10 (m, 1H), 6.84-6.81 (m, 1H), 6.85- 6.82 (m, 1H), 5.41-5.36 (m, 1H), 5.02-4.97 (m, 1H), 4.84 (s, 2H), 3.46-3.21 (m, 8H), 3.15-3.08 (m, 2H), 2.79-2.72 (m, 2H), 1.75-1.65 (m, 2H), 1.73-1.22 (m, 13H), 1.43-1.28 (m, 4H).

(4) N- (2-azidoethyl) -3- {4-[(6-{[2- (2,2-dimethyl-4H-1,3-benzodioxin-6-yl) -2-hydroxyethyl] Amino} hexyl) oxy] butyl} benzenesulfonamide N- (2-azidoethyl) -3- [4-({6- [5- (2,2-dimethyl-4H-1,3-benzo) obtained above] Dioxin-6-yl) -2-oxo-1,3-oxazolidine-3-yl] hexyl} oxy) butyl] benzenesulfonamide (2.30 g) and the method described in Reference Example 7- (3) The reaction and treatment were conducted in a similar manner to give the title compound (408 mg) as a colorless solid.
MS (ESI) m / z 565 (M + H) ⁺ .

Reference Example 34
2-[(6- {4- [4- (azidomethyl) phenyl] butoxy} hexyl) amino] -1- (2-chlorophenyl) ethanol

(1) 5- (2-chlorophenyl) -1,3-oxazolidine-2-one To a solution of 2-amino-1- (2-chlorophenyl) ethanol hydrochloride (300 mg) in dichloromethane (10 mL) at 0 ° C. with triethylamine ( 536 μL) and triphosgene (259 mg) were added, and the mixture was warmed to room temperature and stirred for 2 hours. Water was added to the reaction mixture, and the target product was extracted with methylene chloride. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (301 mg) as a colorless oil.
MS EM 197.02 found 198.0

(2) 5- (2-chlorophenyl) -3- (6- {4- [4- (hydroxymethyl) phenyl] butoxy} hexyl) -1,3-oxazolidine-2-one 5- (5) obtained above 2-Chlorophenyl) -1,3-oxazolidine-2-one (301 mg) was used for the reaction and treatment in the same manner as described in Reference Example 7- (1) to give the title compound (657 mg) as a colorless solid Got as.
MS EM459.22 Measured 460.7

(3) 3- (6- {4- [4- (Azidomethyl) phenyl] butoxy} hexyl) -5- (2-chlorophenyl) -1,3-oxazolidine-2-one 5- (2 -Chlorophenyl) -3- (6- {4- [4- (hydroxymethyl) phenyl] butoxy} hexyl) -1,3-oxazolidine-2-one (657 mg) was added to Reference Example 7- (2). Reaction and treatment in the same manner as described gave the title compound (447 mg) as a colorless solid.
MS EM484.22 Measured 485.9

(4) 2-[(6- {4- [4- (azidomethyl) phenyl] butoxy} hexyl) amino] -1- (2-chlorophenyl) ethanol 3- (6- {4- [4] obtained above Using-(azidomethyl) phenyl] butoxy} hexyl) -5- (2-chlorophenyl) -1,3-oxazolidine-2-one (447 mg), the reaction was carried out in the same manner as described in Reference Example 7- (3). To give the title compound (73 mg) as a colorless oil.
MS EM 458.24 Found 459.8

Reference Example 35
22-azido-N- (2-{[3- (9H-carbazole-4-yloxy) -2-hydroxypropyl] amino} -2-methylpropyl) -17-oxo-4,7,10,13-tetraoxa -16-azadocosan-1-amide

(1) tert-butyl (2-{[3- (9H-carbazol-4-yloxy) -2-hydroxypropyl] amino} -2-methylpropyl) carbamate 4-glycidyloxycarbazole (976 mg) and (2-amino -2-Methylpropyl) -carbamic acid tert-butyl ester (852 mg) was added to 1-butanol (2 mL), and the mixture was heated to reflux at 90 ° C. for 5 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (1.26 g) as a colorless oil.
HRMS-ESI (m / z): [M + H] + calcd.for C24H33N3O4, 428.2544; found, 428.2541.

(2) 1-[(1-Amino-2-methylpropan-2-yl) amino] -3- (9H-carbazol-4-yloxy) propan-2-ol dihydrochloride obtained in (1) above The compound (1.12 g) was dissolved in chloroform (30 mL), 4N hydrogen chloride ethyl acetate solution (5 mL) was added, and the mixture was stirred at room temperature for 6 hr. The reaction mixture was concentrated under reduced pressure, chloroform (30 mL) was added, and the mixture was concentrated again under reduced pressure to give the title compound (840 mg) as a colorless oil.
HRMS-ESI (m / z): [M + H] + calcd.for C19H25N3O2, 328.2020; found, 328.2018.

(3) tert-butyl [22- (9H-carbazol-4-yloxy) -21-hydroxy-18,18-dimethyl-15-oxo-3,6,9,12-tetraoxa-16,19-diazadocos-1 -Yl] carbamate DMF (3 mL) solution of the compound obtained in (2) above (548 mg) and 15-tert-butyloxycarbonylamino-4,7,10,13-tetraoxa-pentadecanoic acid (500 mg) HATU (572 mg) and diisopropylethylamine (107 μL) were added thereto, and the mixture was stirred at 4 ° C. for 1 hour in a nitrogen atmosphere. Chloroform (100 mL) was added to the reaction mixture, and the organic layer was washed with water (50 mL) and saturated brine (50 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Purification by silica gel column chromatography gave the title compound (359 mg) as a colorless oil.
HRMS-ESI (m / z): [M + H] + calcd.for C35H54N4O9, 675.3964; found, 675.3960.

(4) 1-amino-N- (2-{[3- (9H-carbazole-4-yloxy) -2-hydroxypropyl] amino} -2-methylpropyl) -3,6,9,12-tetraoxa Pentadecane-15-amide trihydrochloride To a solution of the compound (302 mg) obtained in (3) above in chloroform (30 mL) was added 4N hydrogen chloride ethyl acetate solution (5 mL), and the mixture was stirred at room temperature for 6 hours. The solvent was concentrated under reduced pressure, chloroform (30 mL) was added, and the mixture was concentrated again under reduced pressure to give the title compound (298 mg) as a colorless solid.
HRMS-ESI (m / z): [M + H] + calcd.for C30H46N4O7, 575.3439; found, 575.3440.

(5) 22-azido-N- (2-{[3- (9H-carbazole-4-yloxy) -2-hydroxypropyl] amino} -2-methylpropyl) -17-oxo-4,7,10, 13-tetraoxa-16-azadocosan-1-amide Triethylamine was added to a mixed solution of the compound obtained in (4) (54 mg) in THF (1.4 mL), DMF (0.2 mL) and methylene chloride (1.0 mL). 1-[(6-azido-1-oxohexyl) oxy] -2,5- synthesized according to the method of (11.6 mL) and literature (Vasilyeva et al., Bioorganic & Medicinal Chemistry, (2013) 21: 703-711). Pyrrolidinedione (21 mg) was added and stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography (0.1% TFA acetonitrile solution / 0.1% TFA water) to give the title compound (32 mg) as a colorless oil.
MS (ESI) m / z 714 (M + H) ⁺ .

Reference Example 36
N- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl) -1- (2-methoxyphenyl) propan-2-amine

To a solution of 2-methoxyphenylacetone (100 mg) and 11-azido-3,6,9-trioxaundecan-1-amine (146 mg) in tetrahydrofuran (5 mL) was added acetic acid (70 μL) and sodium triacetoxyborohydride (258 mg). ) And stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (101 mg) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.19-7.09 (s, 2H), 6.88-6.81 (m, 2H), 3.79 (s, 3H), 3.66-3.53 (m, 12H), 3.38-3.34 ( m, 2H), 2.94-2.75 (m, 4H), 2.55-2.49 (m, 1H), 1.01 (d, J = 6.0 Hz, 3H).

Reference Example 37
N ^2- (3- {1- [4- (4-{[6-({2-hydroxy-2- [4-hydroxy-3- (hydroxymethyl) phenyl] ethyl} amino) hexyl] oxy} butyl) Benzyl] -1H-1,2,3-triazol-4-yl} propanoyl) -N-methylcysteine amide

(1) S-para-tolylpent-4-inthioate To a solution of 4-pentinoic acid (1.2 g) and 4-methylbenzenethiol (1.5 g) in methylene chloride (40 mL) at room temperature with dicyclohexylcarbodiimide (2. 8 g) was added and stirred for 12 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (2.00 g) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.29-7.19 (m, 4H), 2.86 (t, J = 9.0 Hz, 2H), 2.57-2.52 (m, 2H), 2.36 (s, 3H), 2.00 -1.98 (m, 1H).

(2) S- (4-Methylphenyl) 3- {1- [4- (4-{[6-({2-hydroxy-2- [4-hydroxy-3- (hydroxymethyl) phenyl] ethyl} amino) ) Hexyl] oxy} butyl) benzyl] -1H-1,2,3-triazol-4-yl} propanethioate S-para-tolylpent-4-inthioate (160 mg) obtained above and 4- {2 -[(6- {4- [4- (azidomethyl) phenyl] butoxy} hexyl) amino] -1-hydroxyethyl} -2- (hydroxymethyl) phenol (342 mg) in THF (4 mL) and ethanol (4 mL) Add 0.533M sodium ascorbate aqueous solution (850 μL) and 0.263M copper sulfate pentahydrate aqueous solution (850 μL) at room temperature, and stir for 12 hours. It was. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase column chromatography (acetonitrile / water) to give the title compound (214 mg) as a colorless oil.
MS EM674.35 found 675.6

(3) N ^2- (3- {1- [4- (4-{[6-({2-hydroxy-2- [4-hydroxy-3- (hydroxymethyl) phenyl] ethyl} amino) hexyl] oxy } Butyl) benzyl] -1H-1,2,3-triazol-4-yl} propanoyl) -N-methylcysteine amide obtained above S- (4-methylphenyl) 3- {1- [4- ( 4-{[6-({2-hydroxy-2- [4-hydroxy-3- (hydroxymethyl) phenyl] ethyl} amino) hexyl] oxy} butyl) benzyl] -1H-1,2,3-triazole- 4-yl} propanethioate (214 mg) and literature (Journal of the American Chemical Society, 1982, 104, 15, 4283-4285) To a mixed solution of 2-amino-3-mercapto-N-methylpropanamide hydrochloride (70 mg) synthesized in accordance with the method of guanidine buffer (10 mL) and acetonitrile (5 mL) at room temperature with tris (2-carboxyethyl) phosphine hydrochloride Salt (380 mg) was added and stirred for 12 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase column chromatography (0.1% TFA acetonitrile solution / 0.1% TFA water) to give the title compound (84 mg) as a colorless oil.
MS EM 684.37 found 685.8

Reference Example 38
4-Amino-N-((1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-3-yl) methyl) -5-chloro-2-ethoxybenzamide

To a solution of tert-butyl (1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-3-yl) methylcarbamate (292 mg) obtained in Reference Example 28 in dioxane (3 mL) was added 4N A hydrogen chloride dioxane solution (5 mL) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and methylene chloride (5 mL) was added to the resulting residue. Further, triethylamine (0.16 mL), 4-amino-5-chloro-2-ethoxybenzoic acid (125 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (111 mg) and 1-hydroxybenzotriazole ( 72 mg) and stirred at room temperature for 15 hours. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (251 mg) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 8.08 (s, 1H), 7.78 (m, 1H), 7.17 (d, J = 7.9Hz, 2H), 7.07 (d, J = 7.9 Hz, 2H), 6.23 (s, 1H), 4.33 (s, 2H), 4.06-3.97 (m, 2H), 3.21-3.1 (m, 11H), 2.83-2.55 (m, 4H), 2.02-1.02 (m, 21H).
MS EM 598.34 found 600.5

Reference Example 39
4-Amino-N-((1- (4- (4- (6-azidohexyloxy) butyl) benzyl) morpholin-2-yl) methyl) -5-chloro-2-ethoxybenzamide

To a solution of tert-butyl (1- (4- (4- (6-azidohexyloxy) butyl) benzyl) morpholin-2-yl) methylcarbamate (228 mg) obtained in Reference Example 29 in dioxane (3 mL) was added 4N A hydrogen chloride dioxane solution (5 mL) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and methylene chloride (5 mL) was added to the resulting residue. Further, triethylamine (0.13 mL), 4-amino-5-chloro-2-ethoxybenzoic acid (98 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (87 mg) and 1-hydroxybenzotriazole ( 61 mg) was added and stirred at room temperature for 15 hours. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (251 mg) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 8.19 (brs, 1H), 8.08 (s, 1H), 7.20-7.09 (m, 4H), 6.24 (s, 1H), 4.33 (s, 2H), 4.04 (q, J = 7.0 Hz, 2H), 3.86-3.21 (m, 15H), 2.78-2.57 (m, 5H), 2.15-1.97 (m, 2H), 1.62-1.34 (m, 12H).
MS EM600.32 found 602.1

Reference Example 40
4-Amino-N- (1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-4-yl) -5-chloro-2,3-dihydrobenzofuran-7-benzamide

To a solution of tert-butyl 1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-4-yl) carbamate (250 mg) obtained in Reference Example 30 in dioxane (3 mL) was added 4N hydrogen chloride. A dioxane solution (5 mL) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and methylene chloride (5 mL) was added to the resulting residue. In addition, triethylamine (0.19 mL), 4-amino-5-chloro-2,3-dihydrobenzofuran-7-carboxylic acid (156 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (99 mg) and 1-Hydroxybenzotriazole (140 mg) was added and stirred at room temperature for 15 hours. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (273 mg) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 7.83 (s, 1H), 7.24 (m, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.11 (d, J = 8.1 Hz, 2H), 4.76-4.70 (m, 2H), 4.13 (s, 2H), 3.96 (m, 1H), 3.45-3.34 (m, 6H), 3.25-2.57 (m, 8H), 2.18-1.93 (m, 4H), 1.72-1.33 (m, 14H).
MS EM 582.31 found 583.5

Reference Example 41
4-Amino-N- (1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-4-yl) -5-chloro-2-methoxybenzamide

To a solution of tert-butyl 1- (4- (4- (6-azidohexyloxy) butyl) benzyl) piperidin-4-yl) carbamate (250 mg) obtained in Reference Example 30 in dioxane (3 mL) was added 4N hydrogen chloride. A dioxane solution (5 mL) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and methylene chloride (5 mL) was added to the resulting residue. Further, triethylamine (0.19 mL), 4-amino-5-chloro-2-methoxybenzoic acid (148 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (99 mg) and 1-hydroxybenzotriazole ( 140 mg) was added, and the mixture was stirred at room temperature for 15 hours. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (273 mg) as a colorless oil.
¹ H-NMR (300MHz, CDCl ₃ ) δ: 8.07 (s, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.20 (d, J = 8.1 Hz, 2H), 7.11 (d, J = 8.1 Hz, 2H), 6.25 (s, 1H), 4.34 (s, 2H), 3.99 (m, 1H), 3.83 (s, 3H), 3.46-3.21 (m, 10H), 2.74-2.57 (m, 4H) , 2.21-1.95 (m, 4H), 1.66-1.33 (m, 12H).
MS EM570.15, measured 572.2

Reference Examples 42-44, 46, 47
In the same manner as in Reference Example 16, RNA phosphoramidite, namely 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-triisopropylsilyloxymethyladenosine-3′-ON, N′-diisopropyl -2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-N ⁴ -acetyl-2′-triisopropylsilyloxymethylcytidine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-N ² -isobutyryl-2′-triisopropylsilyloxymethylguanosine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl- 2'-Triisopropylsilyloxymethyluridine-3'-ON, N'-diisopropyl Lopyl-2-cyanoethyl phosphoramidite (manufactured by Sigma Aldrich), 2′-O-methyl phosphoramidite, ie, 5′-O-dimethoxytrityl-N ⁶ -benzoyl-2′-methoxyadenosine-3′-O -N, N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N ⁴ -acetyl-2'-methoxycytidine-3'-ON, N'-diisopropyl-2-cyanoethylphospho Loamidite, 5′-O-dimethoxytrityl-N ² -isobutyryl-2′-methoxyguanosine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-2 '-Methoxythymidine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'O-dimethoxytrityl-2'-methoxyuridine-3'-ON,N'-diisopropyl-2-cyanoethyl phosphoramidite (Chemgene), 2'-F-phosphoramidite, ie 5'-O -Dimethoxytrityl-N ⁴ -acetyl-2'-fluorocytidine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, and 5'-O-dimethoxytrityl-2'-fluorouridine-3 '-O-N, N'-diisopropyl-2-cyanoethyl phosphoramidite (manufactured by Sigma-Aldrich), and DNA phosphoramidite, 5'-O-dimethoxytrityl-N ⁶ -benzoyladenosine-3'-O- N, N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl--N ⁴ - acetyl Cytidine -3'-O-N, N'- diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl -N ² - isobutyryl guanosine -3'-O-N, N'- diisopropyl -2 Using the corresponding starting compounds from -cyanoethyl phosphoramidite and 5'-O-dimethoxytritylthymidine-3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite (manufactured by Sigma-Aldrich) Formula (XX):

The compounds shown in Table 1 shown below were obtained. However, for the conversion of phosphorous acid to a phosphorothioate group, 3-((N, N-dimethylaminomethylidene) amino) -3H-1,2,4-dithiazole-5-thione (DDTT) / pyridine / acetonitrile The solution was used.

Reference Example 48-52
In the same manner as in Reference Example 18, using the corresponding starting material compound, the following formula (XXI):

The compounds shown in Table 2 shown below were obtained.

Reference Example 53

Under a borate buffer (pH 8.0, final concentration 0.5 M), a 1.0 mM aqueous solution (180 μL) of the compound of Reference Example 16 and 10.0 mM dimethyl sulfoxide / N-succinimidyl 3- (2-pyridyldithio) propionate / Acetonitrile mixed solution (4/1, 70 μL) was mixed and stirred at room temperature for 4 hours. The reaction solution was ultrafiltered and concentrated by centrifugation to give the title compound (2.0 mg).
MALDI-TOF / MS: m / z = 7194.30 (theoretical value 7194.68)

Reference Examples 54, 56 and 58
In the same manner as in Reference Example 53, using the corresponding starting material compound, the following formula (XXII):

The compounds shown in Table 3 shown below were obtained.

Reference Example 59-65
The oligonucleotides shown in Table 4 were obtained in the same manner as in Reference Example 15 using the corresponding raw material compounds.

Reference Example 66

(1) Synthesis of oligonucleotides Synthesis was performed from corresponding raw materials in the same manner as in Reference Example 15. However, as the polystyrene solid carrier, a commercially available aminated solid phase carrier (manufactured by Phthalamido Amino C6 lcaa CPG Chem Genes), and 1′-O-dimethoxytritylhexyl disulfide, 1′-2-cyanoethyl- N, N-diisopropyl phosphoramidite (Glen Research) was used for oligonucleotide synthesis. All phosphoramidites were used at a concentration of 0.1M in acetonitrile. It took 10 minutes for RNA, 3 minutes for DNA, and 15 minutes for 5 ′ end modification. As the phosphoramidite activator, 5-benzylmercaptotetrazole (0.25M, manufactured by Wako Pure Chemical Industries, Ltd.) was used, and iodine / water / pyridine was used for the oxidation of phosphorous acid to phosphoric acid.

(2) Deprotection I (Cleavage from the carrier)
The CPG carrier obtained above was transferred to a glass bottle. Oligonucleotide was removed from the carrier while simultaneously deprotecting the base and the phosphate group at 25 ° C. for 3 hours using 1.0 mL of a mixed solution of 28% aqueous ammonia and 40% aqueous methylamine with respect to 1 μmol of the carrier. Disconnected. Thereafter, the solvent was distilled off with an ultrafiltration device and dried with a concentration centrifuge (manufactured by IWAKI).

(3) Deprotection II (removal of 2′-triisopropylsilyloxymethyl (2′-TOM) group)
The dry residue obtained in (2) was suspended in a 1M tetra-n-butylammonium fluoride / tetrahydrofuran solution (0.25 mL / synthetic oligonucleotide 1 μmol) and heated at 65 ° C. for 3 hours. The reaction was quenched with 1M Tris-HCl buffer (pH 7.5) in the same amount as the deprotection solution, ethanol precipitated, and dried.

(4) Removal of disulfide group The dry residue obtained in (3) is dissolved in 1M Tris-HCl buffer (pH 7.5, 0.5 mL / synthetic oligonucleotide 1 μmol), 0.12 M dithiothreitol solution and 0 . The same amount of a mixed solution of 5M phosphate buffer (pH 8.0) solution was added and reacted at 25 ° C. for 6 hours. The reaction product was evaporated by ultrafiltration and stored in a freezer.

(5) Purification by HPLC The crude oligonucleotide obtained in (4) was purified by reverse phase HPLC using a C18 reverse phase column (X-bridge Waters). The buffer was 100.0 mM hexafluoroisopropanol and 8.0 mM triethylamine mixed aqueous solution (pH 8.0) (buffer A) and methanol (buffer B). Fractions containing the full length oligonucleotide were collected, ultrafiltered and lyophilized to give the title compound.
MALDI-TOF / MS: m / z = 719.70 (theoretical molecular weight 7193.59)

Reference Example 67

A 1.0 mM deionized RNase-free aqueous solution (1.9 mL) of the compound of Reference Example 66 and 4.9 mL of 0.5 M borate buffer (pH 8.0) were mixed. A 10.0 mM solution of the compound of Reference Example 3 (dimethyl sulfoxide / acetonitrile = 4/1, 0.7 mL) was added, and the mixture was stirred at 25 ° C. for 4 hours. The reaction solution was added to a centrifugal ultrafiltration device, and centrifugal concentration was performed at 20 ° C. and 4000 rpm. 10 mL of deionized RNase-free water was added to the top, and centrifugal concentration was performed again to obtain the title compound (7.5 mg).
MALDI-TOF / MS: m / z = 7354.45 (theoretical value 7357.84)

Reference Example 68

134 μL of 6.0 mM deionized RNase-free aqueous solution of the compound obtained in Reference Example 67 and 134 μL of 0.5 M borate buffer (pH 8.0) were mixed. 18.0 mM Alexa Fluor (registered trademark) 594 C5 maleimide solution (dimethyl sulfoxide / acetonitrile = 4/1, 134 μL) was added to the stirred solution, and the mixture was stirred at 20 ° C. for 12 hours. The reaction solution was added to a centrifugal ultrafiltration device, and centrifugal concentration was performed at 20 ° C. and 4000 rpm. 10 mL of deionized RNase-free water was added to the top, and centrifugal concentration was performed again to obtain the title compound (4.0 mg).
MALDI-TOF / MS: m / z = 8245.08 (theoretical value 8243.82)

Reference Example 69

(1) Click reaction Copper sulfate was dissolved in 2 mM aqueous solution, L-ascorbic acid was dissolved in 10 mM aqueous solution, and the compound of Reference Example 68 was dissolved in deionized RNase-free water so as to be a 0.47 mM aqueous solution. Further, a 10 mM solution of the compound of Reference Example 18 and a 3.5 mM solution of tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (hereinafter abbreviated as TBTA). So that DMF: acetonitrile (4: 1) was used. The prepared solution was 2 mM copper sulfate aqueous solution (888 μL), 3.5 mM TBTA solution (1332 μL), 10 mM ascorbic acid aqueous solution (888 μL), 0.1 M phosphate buffer (pH = 7.0, 666 μL) and the compound of Reference Example 62 Aqueous solution (0.47 mM, 560 μL) was mixed in order. An aqueous solution (10 mM, 888 μL) of the compound of Reference Example 18 was added to the well mixed solution, and the reaction was performed at 25 ° C. for 2 hours. After the reaction was completed, HPLC analysis was performed using a C18 reverse phase column (X-bridge Nippon Waters) to confirm the reaction.

(2) HPLC purification The reaction solution obtained in (1) was purified by reverse phase ion exchange HPLC using an X-bridge gel column (Nihon Waters). As the mobile phase, acetonitrile / 0.1 M triethylamine acetate buffer (pH 7.0) (5/95 to 90/10) was used. After desalting, freeze-dried to obtain a click reaction product.
MALDI-TOF / MS: m / z = 8714.03 (MH) ^- (theoretical 8714.42).

(3) Annealing The click results obtained in (2) and the compound of Reference Example 15 were dissolved in RNase-free water so as to be 200 μM, and equal amounts were mixed. The mixture was prepared to be a 10 mM Tris-HCl buffer (pH 8.0) solution of 20 mM sodium chloride at a final concentration. The solution was heated at 70 ° C. for 10 minutes, and then the temperature was decreased by 1 ° C. per minute to 30 ° C. for annealing. Annealing was confirmed by 19% non-denaturing polyacrylamide gel electrophoresis to obtain the title compound.

Reference Example 70

In the same manner as in Reference Example 18, the title compound was obtained from the compound of Reference Example 46.
MALDI-TOF / MS: m / z = 7085.40 (M−H) ⁻ (theoretical value 7085.48).

Reference Example 71

In the same manner as in Reference Example 18, the title compound was obtained from the compound of Reference Example 43.
MALDI-TOF / MS: m / z = 6651.71 (M−H) ⁻ (theoretical value 6649.47).

Reference Example 72

In the same manner as in Reference Example 16, the title compound (6.9 mg) was obtained from the corresponding starting material compound.
MALDI-TOF / MS: m / z = 68887.74 (M−H) ⁻ (theoretical value 6883.16).

Reference Example 73

In the same manner as in Reference Example 53, the title compound (2.6 mg) was obtained from the compound of Reference Example 72.
MALDI-TOF / MS: m / z = 6972.00 [M-pyridylthio]-(theoretical value 7085.03).

Example 1

(1) Click reaction The compound of Reference Example 18 (3.5 mg) was prepared as an aqueous solution and used in the reaction. Each of the reaction solutions had a final concentration of 0.2 mM copper sulfate, 0.7 M tris- (benzyltriazolemethyl) amine, 1.33 mM ascorbic acid, 10 mM phosphate buffer (pH 7.0), 0.14 mM Reference Example 18 And 1.33 mM 4- (2- (5- (4-((3- (azidomethyl) phenoxy) methyl) phenoxy) pentylamino) -1-hydroxyethyl) -2- (hydroxymethyl) phenol (Reference Example 10) ) And stirred at 25 ° C. for 3 hours.
(2) HPLC purification The reaction solution obtained in (1) was purified by reverse phase ion exchange HPLC using an X-bridge gel column (Nihon Waters). As the mobile phase, acetonitrile / 0.1 M triethylamine acetate buffer (pH 7.0) (5/95 to 90/10) was used. After desalting, the product was freeze-dried to obtain a click reaction product (1.8 mg).
MALDI-TOF / MS: m / z = 7579.68 (MH) ^- (theoretical value 7584.27).
(3) Annealing The click results obtained in (2) and the compound of Reference Example 15 were dissolved in RNase-free water so as to be 200 μM, and equal amounts were mixed. The mixture was prepared to be a 10 mM Tris-HCl buffer (pH 8.0) solution of 20 mM sodium chloride at a final concentration. The solution was heated at 70 ° C. for 10 minutes, and then the temperature was decreased by 1 ° C. per minute to 30 ° C. for annealing. Annealing was confirmed by 19% non-denaturing polyacrylamide gel electrophoresis, and the compound of Example 1 was obtained.

Example 2-17
In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXIII):

The compounds shown in Tables 5-1 and 5-2 were obtained.

Example 18-21
In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXIV):

The compounds shown in Table 6 shown below were obtained.

Examples 22-34
In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXV):

The compounds shown in Table 7 shown below were obtained.

Examples 35-41
In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXVI):

The compounds shown in Table 8 shown below were obtained.

Examples 42-43
In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXVII):

The compounds shown in Table 9 shown below were obtained.

Example 44

(1) The compound of Reference Example 53 was dissolved in deionized RNase-free water so as to be 0.50 mM. A DMF solution (70 μL) of the compound of Reference Example 37 prepared to 10 mM was added to a mixed solution of 560 μL of this aqueous solution and 770 μL of 0.5 M borate buffer (pH 8.0), and the mixture was stirred at 25 ° C. for 1 hour.

(2) HPLC purification The reaction solution was purified by reverse phase HPLC using a C18 reverse phase column. As the buffer, 100 mM hexafluoroisopropanol and 8 mM triethylamine mixed aqueous solution (pH 8.0) (buffer A) and methanol (buffer B) were used. Fractions containing the modified oligonucleotide were collected and subjected to ultrafiltration and lyophilization to obtain a disulfide (1.2 mg).

(3) Annealing The title compound was obtained by annealing the disulfide obtained in (2) and the compound of Reference Example 15 in the same manner as in Example 1.

Examples 45-49
In the same manner as in Example 44, using the corresponding starting material compound, the following formula (XXVIII):

The compounds shown in Table 10 shown below were obtained.

Example 50

In the same manner as in Example 44, the title compound (1.4 mg) was obtained from the compounds of Reference Example 37 and Reference Example 73. It was obtained as a single-chain derivative without annealing.
MALDI-TOF / MS: m / z = 7655.51 (theoretical value 7658.75).

The pharmacological activity of the compound of the present invention was confirmed by the following test.
Test Example 1: cAMP Assay of β ₂ Receptor Expressing HEK293 Cells HEK293 cell line was seeded at 6 × well plate at 3 × 10 ⁵ cells / 2 mL / well and cultured overnight. As the medium, antibiotic-free 10% FBS / DMEM was used. The beta ₂ receptor was inserted into the mammalian expression vector such as pcDNA3.1 and pCI, were transfected with LipofectamineLTX (overnight culture at 37 ° C.). The cells were detached with trypsin, suspended in 2 mL of FBS (−) / DMEM, and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed, and the cells were suspended in 1 × 10 ⁶ cells / mL with assay buffer (0.5 mM IBMX / 0.1% BSA / DMEM). To 384-well Low Volume Black Round Bottom PS NBS Microplate (Corning, Cat # 3676), 5 μL / well of the evaluation compound prepared to twice the final concentration was added, and 5 μL / well of the cell suspension was further added. After standing at 37 ° C. for 0.5 hour, the cAMP concentration produced by the cells was quantified using cAMP HTRF Kit (Cisbio, Cat # 62AM6PEJ). The measurement of homogeneous time-resolved fluorescence (HTRF) was performed with RUBYstar (Ex337 nm, Em665 nm / Em620 nm).
The evaluation results of the test compound are shown in Table 11. Emax values of cAMP concentration increase to a maximum a beta ₂ agonist Salmeterol is 100%, and expressed in percentage of the maximum cAMP concentration increase to each compound on it. The EC ₅₀ value was calculated as the concentration of the compound that resulted in a cAMP increase of 50% of the Emax value of each compound. The EC ₅₀ value of Salmeterol was 0.1 nM. The compounds of the invention showed an increase in cAMP in beta ₂ receptor expressing HEK293 cells.

Test Example 2: Receptor internalization assay 6-well plate using the beta ₂ receptor expressing HEK293 cells with GFP2 crowded seeded HEK293 cell lines ^{3x10 5 cells / 2 mL / well} , and cultured overnight. As the medium, antibiotic-free 10% FBS (0.22 μm-filtered) / DMEM was used. those that combines GFP2 the beta ₂ receptor is a mammalian expression vector pcDNA3.1 and pCI or various viral expression vector pAxcwit2, pDON-5 DNA, pLVSIN -CMV Neo, inserted like pFastBacMam, virus created Infected (37 ° C., 4-6 hours). Cells are detached with trypsin, seeded at 2 × 10 ⁴ cells / 0.1 mL / well in a Poly-D-Lysine-coated 96-well plate (Black / Clear Bottom) (BD, BIOCOAT, Cat # 356640), and cultured overnight. did. An evaluation compound having a concentration 11 times the final concentration was added at 10 μL / well and allowed to stand at 37 ° C. for 5 hours. A 4% paraformaldehyde solution containing Hoechst 33342 (1 μg / mL) was added at 0.1 mL / well, and the mixture was allowed to stand at room temperature for 1 hour. The supernatant was discarded, and 0.2 mL / well of D-PBS (−) was added. Nuclei and β ₂ -GFP2 images were captured with an imaging cytometer IN Cell Analyzer 1000. The measurement conditions are as shown in Table 12. The amount of internalized β ₂ -GFP2 per nucleus was calculated.

The evaluation results of the test compound are shown in Table 13. Emax value, the concentration of a beta ₂ agonist Salmeterol is inherent to the maximum as 100%, was expressed at a ratio of concentrations indicating the maximum internalization of each compound thereto. The EC ₅₀ value was calculated as the concentration of the compound that resulted in internalization of 50% of the Emax value of each compound. The EC ₅₀ value of Salmeterol was 1.7 nM. The compounds of the present invention showed internalization in beta ₂ receptor expressing HEK293 cells with GFP2.

Test Example 3: Knockdown experiments method HEK293 cell line beta ₂ receptor expression plasmid using beta ₂ receptor expressing HEK293 cells are transfected with LipofectamineLTX (overnight culture at 37 ° C.). Cells are detached with trypsin and suspended in 10% FBS / DMEM. Cells are seeded on a 12-well plate at 1.5 × 10 ⁵ cells / 2 mL / well and cultured for 6 hours. Add compound and incubate for 24 hours. The culture fluid of the cultured cells is removed, and total RNA is prepared using the QuickGene-800 automatic nucleic acid extraction system (FUJIFILM) and RNA cultured cell kit S (FUJIFILM) according to the instructions. The total RNA is used as a template, and a reverse transcriptase (RT) reaction is carried out according to the instructions of the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For the PCR reaction, Power SYBR Green PCR Master Mix (Applied Biosystems) is used, and quantitative PCR is performed by the Applied Biosystems 7900HT real-time PCR system according to the instructions. Specifically, 5 μL of the RT-reacted solution, 0.5 μL of the following 20 μM Forward Primer, 0.5 μL of the following 20 μM Reverse Primer, 6.5 μL of DW and 12.5 μL of the Power SYBR solution are mixed, Step 1: 50 ° C., 2 min, Step 2: 95 ° C., 10 min, Step 3: 95 ° C., 15 sec, 60 ° C., 1 min for 40 cycles, Step 4: 95 ° C., 15 sec, Step 5: 60 ° C., 15 sec, Step 6: 95 ° C., 15 sec. PCR primers are peptidylprolylomerase B (cyclophilin B) (hereinafter referred to as PPIB), PPIB Forward Primer: 5'-gctagataggcaagcatgtgtgtgtt-3 ', PPIBrevert' Ribosomal protein, large, P0 (hereinafter referred to as RPLP0) is used as an internal standard gene, and PCR primers are RPLP0 Forward Primer: 5′-aggtgtcgacatggcatcatcact-3 ′, RPLP0 Reverse Primet 5 To do.

Test Example 4: Intracellular Uptake Assay Using Fluorescent Labeled Compound HEK293 cell line was seeded at 6 × well plate at 3 × 10 ⁵ cells / 2 mL / well and cultured overnight at 37 ° C. in a 5% CO ₂ incubator. As the medium, antibiotic-free 10% FBS / DMEM was used. Human beta ₂ receptor was inserted into the mammalian expression vector, such as PTran3.1, were transfected with LipofectamineLTX (overnight culture at 37 ° C.). Cells were detached with trypsin, suspended in 10% FBS / DMEM, and then 3 × 10 ⁵ cells / 0.1 mL in Poly-D-Lysine-coated Glass Bottom Culture Dishes (MatTek, Cat # P35GC-1.5-10-C). / Well and seeded overnight. The culture supernatant was removed, GDX-117 was added to a final concentration of 1 μM, and the mixture was cultured at 37 ° C. for a predetermined time. After adding Hoechst 34580 (final concentration 2.5 μg / mL, 10-20 minutes, room temperature), the culture supernatant was removed and immobilized with 4% paraformaldehyde (room temperature, 20 minutes). After 0.3% TritonX-100 / PBS treatment (room temperature, 3 minutes), immunostaining was performed with a β2 receptor-specific antibody, and the nucleus and the compound of Reference Example 69 were analyzed with a confocal laser microscope system (LEICA DMIRE2). An image of β ₂ receptor was captured to confirm the uptake of the compound into cells.

Test Example 5: cAMP assay of 5-HT ₄ receptor-expressing HEK293 cells HEK293 cell line was seeded on a 6-well plate at 1.5 × 10 ⁵ cells / 2 mL / well and cultured overnight. As the medium, antibiotic-free 10% FBS / DMEM was used. 5-HT ₄ receptor was inserted into the pcDNA3.1 mammalian expression vector, were transfected with Trans-IT LT1 reagent (cultured overnight at 37 ° C.). The cells were detached with trypsin, suspended in 2 mL of FBS (−) / DMEM, and centrifuged at 1000 rpm for 2 minutes. The supernatant was removed, and the cells were suspended in 5 × 10 ⁵ cells / mL with assay buffer (0.5 mM IBMX / 0.1% BSA / DMEM). 384-Well Low Volume Black Round PS PS NBS Microplate (Corning, Cat # 3676) was added 5 μL / well of the compound of Example 30 prepared to twice the final concentration, and 5 μL / well of cell suspension was further added. did. After standing at 37 ° C. for 0.5 hour, the cAMP concentration produced by the cells was quantified using cAMP HTRF Kit (Cisbio, Cat # 62AM6PEJ). The measurement of homogeneous time-resolved fluorescence (HTRF) was performed with RUBYstar (Ex337 nm, Em665 nm / Em620 nm). When the cAMP concentration at which 5-HT was maximized was 100%, the proportion of cAMP concentration increased by 30 μM of the compound of Example 30 was 52%.

The novel compound comprising a conjugate of an antisense oligonucleotide, siRNA, and miRNA represented by a miRNA and a ligand that binds to GPCR in the present invention has an efficient intracellular uptake ability via GPCR, The functional expression of oligonucleotides can be shown and contributes greatly to the industrial fields such as research and medical fields.

This application is based on Japanese Patent Application No. 2012-126788 filed in Japan, the contents of which are incorporated in full herein.

Claims

A conjugate comprising a linker between a G protein-coupled receptor binding ligand (hereinafter sometimes referred to as “GPCR ligand”) and a nucleic acid molecule, wherein the linker may be triazorylene and substituted. A polyalkylene glycol chain or an optionally substituted alkylene chain (wherein the alkylene chain is one or more —O—, —S—, —NH—, ═N—, —N═, —SO 2 —) A divalent group selected from the group consisting of: -C (= O)-, (C6-C10) arylene, (C1-C9) heteroarylene, (C3-C8) cycloalkylene and (C3-C7) heterocyclylene. The conjugate as described above, which may be blocked or inserted at the end.
The linker is represented by the following formula (I):

[Where:
* Represents a binding site with a GPCR ligand;
** represents a binding site with a nucleic acid molecule,
L is from a phosphate group, a phosphorothioate group, a boranophosphate group, a phosphoroselenate group, a boranophosphate ester group, a hydrogen phosphonate group, a phosphoramidate group, an alkylphosphonate group, an arylphosphonate group, and a phosphotriester group. Represents a group selected from the group consisting of, and is covalently bonded to a pentose group of a nucleic acid molecule,
R 1 is — (CH 2 ) n1 —, — (CH 2 ) n2 —O— (CH 2 ) n3 —, — (CH 2 CH 2 O) n4 —CH 2 CH 2 —, —CH 2 CH 2 — S—S—CH 2 CH 2 —C (O) —NH— (CH 2 ) 6 — and —CH 2 CH 2 —O—C (O) —NH— (CH 2 ) 6 — are selected from the group consisting of Divalent groups (wherein — (CH 2 ) n1 —, — (CH 2 ) n2 —O— (CH 2 ) n3 — and — (CH 2 CH 2 O) n4 —CH 2 CH 2 — are substitutable. Each independently at 1 to 3 (C1-C6) alkyl group optionally substituted with 1 to 3 hydroxyl groups and 1 to 5 substituents selected from the group consisting of hydroxyl groups. even if well, -CH 2 CH 2 -S-S -CH 2 CH 2 -C (O) - H- (CH 2) 6 - is substituted at possible positions, R 30 R 31 N-C (O) - , and R 32 OC (O) - identical or selected from the group consisting of a different one to three substituents R 30 , R 31 and R 32 each independently represents a hydrogen atom or a (C1-C6) alkyl group), and n1, n2, n3 and n4 Each independently represents an integer from 3 to 20,
R 2 represents — (CH 2 ) n5 —, — (CH 2 ) n6 —O— (CH 2 ) n7 —, —CH 2 CH 2 — (OCH 2 CH 2 ) n8 —, —C (O) —CH 2 CH 2 — (OCH 2 CH 2 ) n9 —, —CH 2 CH 2 — (OCH 2 CH 2 ) n10 —C (O) —, —C (O) —CH 2 CH 2 — (OCH 2 CH 2 ) n11 —NH—C (O) —, —C (O) — (CH 2 ) n12 —, —O— (CH 2 ) n13 —, — (CH 2 ) n14 — (OCH 2 CH 2 ) n15 —O— (CH 2) n16 - and - (CH 2) n17 -NH- C (O) - (CH 2 CH 2 O) n18 -CH 2 CH 2 -NH-C (O) - (CH 2) n19 - consisting A divalent group selected from the group (wherein the divalent groups are each independently a substitutable position, (C1-C6) which may be substituted with 1 to 3 hydroxyl groups and may be substituted with 1 to 10 identical or different substituents selected from the group consisting of alkyl groups and hydroxyl groups. , N5, n6, n7, n8, n9, n10, n11, n12, n13, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 0 to 20,
Q 1 is represented by the formula (IIa):

(Wherein, R 3 is (represents C1-C6) alkyl or (C3-C8) cycloalkyl group, R 4 is a hydrogen atom, (C1-C6) alkyl or (C3-C8) cycloalkyl group R 3 and R 4 may be bonded to each other to form a (C3-C8) member ring, and W 1 represents —NH—, —O—, —NH—C (O) — (CH 2 ) m1 — and —NH—C (O) — (CH 2 ) m2 —O— represents a divalent group selected from the group consisting of m1 and m2, each independently representing an integer of 1 to 20 ), Formula (IIb):

(Wherein R 5 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W 2 represents — (CH 2 ) m3 —, — (CH 2 ) m4 —NH — And — (CH 2 ) m5 —O— represents a divalent group selected from the group consisting of m3, m4 and m5 each independently represents an integer of 1 to 20), Formula (IIc):

(Wherein R 6 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W 3 represents — (CH 2 ) m6 —, — (CH 2 ) m7 —NH -And- (CH 2 ) m8- represents a divalent group selected from the group consisting of -O-, and m6, m7 and m8 each independently represents an integer of 1 to 20), formula (IId):

(Wherein R 5 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W 4 represents — (CH 2 ) m9 —, — (CH 2 ) m10 — NH— and — (CH 2 ) m11 —O— represents a divalent group selected from the group consisting of m9, m10 and m11 each independently represents an integer of 1 to 20, and formula (IIe) :

(Wherein R 6 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and W 5 represents — (CH 2 ) m12 —, — (CH 2 ) m13 — NH— and — (CH 2 ) m14 —O— represents a divalent group selected from the group consisting of m12, m13 and m14 each independently represents an integer of 1 to 20), formula (IIf) :

Wherein W 6 is a divalent group selected from the group consisting of — (CH 2 ) m15 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m16 — (the divalent group is a substitutable position) Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m15 and m16 are Each independently represents an integer of 1 to 10, and W 7 represents a single bond or —C (O) —NH— (CH 2 ) m17 —, —NH—C (O) — (CH 2 ) m18 A divalent group selected from the group consisting of — and —O— (CH 2 ) m19 — (the divalent group is each independently a —C (O) —NH— (C1-C6) at a substitutable position. ) Alkyl and —NH—C (O) — (C1-C6) alkyl M17, m18 and m19 each independently represents an integer of 1 to 20), which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of Formula (IIg):

(Wherein, W 8 is, - (CH 2) m20 - and -CH 2 CH 2 - (OCH 2 CH 2) m21 - divalent group (the bivalent group selected from the group consisting of the substitutable position Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m20 and m21 Each independently represents an integer of 1 to 20, R 7 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and R 8 and R 9 are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R 7 and R 8 may be bonded to each other to form a (C3-C8) member ring), and IIh):

Wherein W 9 is a divalent group selected from the group consisting of — (CH 2 ) m22 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m23 — (the divalent group is a substitutable position) Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, and may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m22 and m23 are Each independently represents an integer of 1 to 20, R 10 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and R 11 and R 12 are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R 10 and R 11 may be bonded to each other to form a (C3-C8) member ring)
Represents a divalent group selected from the group consisting of
a represents an integer of 0 or 1,
Q 2 represents formula (IIIa):

Or formula (IIIb):

(Wherein p1, p2, p3 and p4 each independently represents an integer of 1 to 20, and (CH 2 ) p1 and (CH 2 ) p3 represent one or more —O—, —C Selected from the group consisting of (═O) —, —C (═O) —NH—, —C (═O) —N (Me) —, —C (═O) —O— and —SO 2 —NH—. Which may be blocked or inserted at the end with a divalent group)
A 1 represents (C3-C8) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, (C1-C9) heteroarylene, —O—. (C1-C9) heteroarylene, (C1-C9) heteroarylene-O-, (C3-C7) heterocyclylene, formula (IVa):

Formula (IVb):

Formula (IVc):

And formula (IVd):

And (C3-C8) cycloalkylene may be substituted with 1 to 10 identical or different (C1-C6) alkyl groups, and (C6-C10) arylene. , -O- (C6-C10) arylene, (C6-C10) arylene-O-, (C1-C9) heteroarylene, -O- (C1-C9) heteroarylene, (C1-C9) heteroarylene-O- , (C3-C7) heterocyclylene, the arylene, heteroarylene and heterocyclylene moieties in formula (IVa), formula (IVb), formula (IVc) and formula (IVd) are halogen atoms, (C1-C6) alkyl Group, (C1-C6) alkoxy group, (C1-C6) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group And may be substituted with 1-6 substituents selected from the group consisting of a trifluoromethoxy group, b represents an integer of 0 or 1. ] The conjugate of Claim 1 represented by these.
L is the formula (V):

The conjugate according to claim 2, wherein Z 1 and Z 2 each independently represents an oxygen atom or a sulfur atom.
The conjugate according to any one of claims 1 to 3, wherein the nucleic acid molecule is a single-stranded or double-stranded nucleic acid molecule.
The conjugate according to any one of claims 1 to 4, wherein the nucleic acid molecule is a nucleic acid molecule having 7 to 100 bases.
The conjugate according to any one of claims 1 to 5, wherein the nucleic acid molecule is a nucleic acid molecule that interacts with mRNA or a nucleic acid molecule that induces RNA interference.
The conjugate according to any one of claims 1 to 6, wherein the nucleic acid molecule is selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, and tagoMir.
The conjugate according to any one of claims 1 to 7, wherein the GPCR ligand has a non-peptide ligand structure that binds to GPCR.
The conjugate according to any one of claims 1 to 8, wherein the GPCR ligand has a partial structure of a GPCR agonist.
GPCR ligands, beta 2 receptor or 5-HT 4 conjugate according to claims 1, which is a non-peptide ligands structure that binds to a receptor to any one of 9.
The GPCR ligand is of formula (VIa):

(In the formula, B 1 represents —CH 2 OH, —NHCHO, or — (CH 2 ) 2 OH.)
Formula (VIb):

Formula (VIc):

(In the formula, B 2 represents a hydrogen atom, methyl or ethyl.)
Formula (VId):

Formula (VIe):

Or formula (VIf):

The conjugate according to any one of claims 1 to 10, which has a structure represented by:
The GPCR ligand is of formula (VIIa):

Formula (VIIb):

(In the formula, B 3 represents a hydrogen atom or methoxy.)
Formula (VIIc):

(Wherein B 4 represents methyl or ethyl, and B 7 represents a methylene or oxygen atom.)
Formula (VIId):

Or formula (VIIe):

The conjugate according to any one of claims 1 to 10, which has a structure represented by:
A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 12 as an active ingredient.