WO2020105701A1

WO2020105701A1 - Novel polymer-active drug conjugate and use thereof

Info

Publication number: WO2020105701A1
Application number: PCT/JP2019/045604
Authority: WO
Inventors: 滋充武田; 知紘三池; 一弥小竹; 俊輔 ▲高▼嶋
Original assignee: 京都薬品工業株式会社
Priority date: 2018-11-22
Filing date: 2019-11-21
Publication date: 2020-05-28

Abstract

The present invention addresses the problem of providing a polymer-anticancer agent-nitric monoxide-releasing compound conjugate that, once administered intravitally, circulates continuously through the whole body and moves to a tumor tissue and accumulates there more efficiently than conventional polymer-anticancer agent conjugates, and that remains there for an extended period of time to release the anticancer agent in the low-pH environment around the tumor, thereby demonstrating excellent antitumor activity with fewer adverse effects. The present invention can provide a polymer compound, or a pharmaceutically-accepted salt thereof, that demonstrates excellent anti-cancer activity with few adverse effect, and that comprises: a constituent unit (1) represented by formula (1) [symbols in the formula are as defined in the specification]; a constituent unit (2) represented by formula (2) [symbols in the formula are as defined in the specification]; and a constituent unit (3) derived from N-(2-hydroxypropyl) methacrylamide.

Description

Novel polymer-active drug-conjugate and its use

The present invention relates to a novel polymer compound obtained by conjugating N- (2-hydroxypropyl) methacrylamide (HPMA) with two components, an anthracycline anticancer agent and a nitric oxide-releasing compound. The present invention also relates to an anticancer agent, a preventive or therapeutic agent for cancer metastasis and / or cancer recurrence, which comprises the above novel polymer compound or a pharmaceutically acceptable salt thereof. Furthermore, the present invention relates to a pharmaceutical composition containing the polymer compound or a pharmaceutically acceptable salt thereof.

Many low-molecular-weight anticancer drugs used in cancer chemotherapy are metabolized or excreted relatively quickly, and therefore, their concentration may not reach a sufficient level in tumor tissue after administration, or they may not be produced for a sufficient period of time necessary for treatment. It may not be able to maximize the therapeutic effect because it does not stay in the body. In addition, many low-molecular-weight anticancer agents are distributed not only in tumor tissues but also in normal tissues, and the occurrence of side effects due to this is a problem. Therefore, if the anticancer agent can be accumulated in cancer cells and selectively act on it, it is possible to provide an excellent anticancer agent having a high therapeutic effect and reduced side effects. On the other hand, in cancer tissues, the barrier of neovascularization is incomplete, and in normal tissues, macromolecules that do not penetrate into the tissues migrate and / or accumulate, and the lymphatic system is immature, and thus the accumulated macromolecules are difficult to remove from the tissues. A phenomenon of staying for a long time (EPR effect) is known. Utilizing this EPR effect, attempts have been made to develop a drug delivery system (DDS) that efficiently delivers and / or distributes an anticancer drug to a tumor tissue that is a target site. Examples of such DDS agents include liposome-encapsulated preparations (Patent Documents 1 to 3), polymer conjugate preparations (Patent Document 4), and derivatives that produce conjugates with proteins in vivo (Patent Documents 5 to 9). Are known. Among these, only the liposome preparation is clinically used, but it is also reported from the clinical study meta-analysis that the liposome preparation does not always show an excellent anticancer effect due to the EPR effect. (Non-patent document 1). Studies on the EPR effect and anticancer effects of high molecular weight DDS preparations, which are expected to have the EPR effect, have been conducted mainly using experimental animal models such as mice transplanted with cancer cells. It is known that the density of blood vessels in tissues and the volume of interstitium are significantly different from those of transplanted tumors in animals because they spontaneously occur and proliferate and grow over a long period. In fact, some high molecular weight DDS anti-cancer agents have shown effects in animal models, but in clinical trials, sufficient effects were not observed and they have not been launched (Non-patent Documents 2 and 3).

HPMA copolymers are biocompatible polymers and have been widely used as DDS carriers for tumor tissues (Non-patent Documents 4 and 5). It has been reported that a high molecular weight DDS in which one or two low molecular weight anti-cancer agents are conjugated to HPMA accumulates in tumor tissues in various experimental animal models of cancer by the EPR effect and exhibits an excellent anti-cancer effect. As such low molecular weight anticancer agents, anthracyclines and platinum compounds which are cytotoxic anticancer agents, and gemcitabine which is a metabolism inhibitor are mainly used. Further, as a specific example of a polymer in which two kinds of anticancer agents are conjugated, a combination of an anthracycline anticancer agent and an aromatase inhibitor (Non-Patent Document 6) or gemcitabine (Non-Patent Document 7) has been reported. .. By conjugating a plurality of active ingredients to the same carrier, it is expected that each active ingredient will be simultaneously released in the tumor tissue to exert a synergistic effect. However, compared with the experimental animal model, human tumor tissue is considered to have a low EPR effect (Non-patent Document 3), and therefore, high molecular weight DDS conjugated with a plurality of anticancer agents also migrates / accumulates into human tumor tissue. Possibility of retention is low. Further, the sensitivity of a plurality of anti-cancer agents to cancer cells differs depending on the cancer type, and the toxicity also differs for each anti-cancer agent, so that it is necessary to optimize the conjugate amount ratio of each active ingredient drug in the polymer.
On the other hand, it has been attempted to enhance the EPR effect of a polymer DDS anticancer drug by using it in combination with other drugs or treatment such as ultrasonic irradiation to expect its effectiveness in human tumor tissues. For example, a combination of a pressor agent such as angiotensin for delivering a drug against high interstitial pressure in human tumor tissue (Non-Patent Document 8), and a combination of nitric oxide releasing agent for increasing blood flow in tumor tissue. (Non-patent document 9), relaxation of a blood vessel barrier by microbubbles and ultrasonic waves, and the like have been reported (non-patent document 10). However, there is no report of an EPR effect-enhancing polymer DDS anticancer agent in which HPMA is conjugated with a low molecular weight anticancer agent and a pressor or a nitric oxide-releasing compound.

International Publication No. 1998/16201 International Publication No. 2005/011633 International Publication No. 2014/050509 Japanese Patent No. 4718117 US Patent No. 8642555 Japanese Patent No. 4617296 Japanese Patent No. 5392965 US Patent No. 8153581 International Publication No. 2016/205738

An object of the present invention is to further conjugate a nitric oxide-releasing compound to a DDS drug in which a biocompatible polymer is conjugated with a low molecular weight anticancer drug, so that the DDS drug is more easily transferred to a tumor tissue. And a high polymer concentration in tumor tissue, an excellent antitumor effect, and a reduced side effect. Furthermore, it is to provide a therapeutic agent for solid cancer containing the polymer compound.

Most of the drugs currently in clinical use are low molecular weight compounds, usually show high clearance, and are widely distributed in various organs. Therefore, the drug may not reach the site of action sufficiently, or may not exist at the site of action for a sufficient period of time and disappear, resulting in the expected therapeutic effect not being obtained, and it may also be distributed and act on normal tissues. May cause adverse side effects. Particularly, the side effect due to the distribution of the drug in normal tissues is particularly remarkable in the anticancer drug having high cytotoxicity. On the other hand, it is known that the polymer compound accumulates in the tumor tissue and stays longer than the normal tissue due to the EPR effect. However, it is considered that the EPR effect is low in human tumor tissue, and the accumulation property, retention property, and anticancer action of the polymer DDS anticancer agent are low.
On the other hand, nitric oxide or nitric oxide-releasing compounds are known to enhance the antitumor effect of polymeric DDS anticancer agents. Also, many solid tumors are known to exhibit a low pH environment compared to normal cells. Focusing on these phenomena, an anthracycline anticancer agent and a nitric oxide-releasing compound are conjugated to methacryloylated HPMA having a terminal ε-amino acid unit having a hydrazide structure at a terminal via a pH-sensitive hydrazone bond. As a result of synthesizing the polymer compound thus prepared and examining its antitumor activity, it was found that the present polymer compound or a pharmaceutically acceptable salt thereof solves this problem, and has completed the present invention.

That is, the present invention is
[1] Methacryloylated α-amino acid, ε-amino acid or hydrazone-derived constituent unit of oligopeptide having an anthracycline anticancer agent residue at the terminal, and methacryloylated having a nitric oxide-releasing compound residue at the terminal Further, a polymer compound or a pharmaceutically acceptable salt thereof, comprising a structural unit derived from hydrazone of α-amino acid, ε-amino acid or oligopeptide and a structural unit derived from N- (2-hydroxypropyl) methacrylamide ..
[2] Formula (1):

[In the formula,
B represents an anthracycline anticancer drug residue. ]
The structural unit (1) represented by the formula (2):

[In the formula,
R ¹ represents a hydrogen atom, an optionally substituted C _1-6 alkyl group or an optionally substituted C _6-14 aryl group;
A represents an arylene group or a divalent heterocyclic group, each of which may be further substituted;
D is a single bond, -CH = CH-, -O-, -S-, -N (R ² )-, ^* -CON (R ² )- ^** or ^* -SO ₂ N (R ² )- ^{*. *} (Wherein R ² represents a hydrogen atom or an optionally substituted C _1-6 alkyl group; ^* represents a bonding position with A; and ^** represents a bonding position with E) .); And E represents a C _1-6 alkylene group which may be further substituted. ] The polymer compound containing the structural unit (2) represented by the following formula and the structural unit (3) derived from N- (2-hydroxypropyl) methacrylamide (hereinafter referred to as "the compound (I) of the present invention" or "the present invention"). Or a pharmaceutically acceptable salt thereof.
[3] The polymer compound according to the above [2] or a pharmaceutically acceptable salt thereof, further comprising a structural unit (4) derived from N- (5-hydrazinocarbonylpentyl) methacrylamide.
[4] In the above [2] or [3], R ¹ of the structural unit (2) is a hydrogen atom, and A is a phenylene group, a biphenylylene group or an indolinylene group which may be further substituted. The polymer compound described above or a pharmaceutically acceptable salt thereof.
[5] D of the structural unit (2) is a single bond, -O-, -S-, ^* -CONH- ^** or ^* -SO ₂ NH- ^** (where ^* and ^** in the formula are 2], and the pharmaceutically acceptable salt thereof, according to any one of the above [2] to [4].
[6] The structural unit (2), -ADDE-ONO _2, has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to the above [2] or [3], which is
[7] The structural unit (2), -ADDE-ONO _2, has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to the above [2] or [3], which is
[8] The structural unit (2), -ADDE-ONO _2, has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to the above [2] or [3], which is
[9] The structural unit (2), -ADE-ONO _2, has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to the above [2] or [3], which is
[10] The polymer compound or a pharmaceutically acceptable salt thereof according to any one of the above [2] to [9], wherein B of the structural unit (1) is an epirubicin residue.
[11] The polymer compound or a pharmaceutically acceptable salt thereof according to any one of the above [3] to [10], which comprises the structural units (1), (2), (3) and (4).
[12] Containing 1 to 40% by weight of anthracycline anticancer drug residues and 0.5 to 20% by weight of nitric oxide-releasing compound residues, based on 100% by weight of the polymer compound or a pharmaceutically acceptable salt thereof. The polymer compound or a pharmaceutically acceptable salt thereof according to any one of [1] to [11] above.
[13] The polymer compound or a pharmaceutically acceptable salt thereof according to any one of the above [1] to [12], which has a weight average molecular weight of 10,000 to 300,000.
[14] A pharmaceutical composition comprising the polymer compound according to any one of [1] to [13] or a pharmaceutically acceptable salt thereof as an active ingredient.
[15] An anticancer agent containing the polymer compound or the pharmaceutically acceptable salt thereof according to any one of [1] to [13] as an active ingredient.
[16] A prophylactic or therapeutic agent for cancer, cancer metastasis, and / or cancer recurrence, which comprises the polymer compound or the pharmaceutically acceptable salt thereof according to any one of [1] to [13] above as an active ingredient.

The compound (I) or a pharmaceutically acceptable salt thereof of the present invention is a highly safe compound that is retained in vivo, continuously retained, and efficiently converted to an active drug after reaching tumor tissue. It is useful as an anti-cancer agent, a preventive or therapeutic agent for cancer metastasis and / or cancer recurrence.

The definitions of terms and symbols used in this specification are explained below.

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

In the present specification, the “C _1-6 alkyl group” means a linear or branched, monovalent saturated hydrocarbon group having 1 to 6 carbon atoms. Examples of the “C _1-6 alkyl group” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 4-methylpentyl, hexyl and the like. Be done.

In the present specification, a “C _2-6 alkenyl group” is a linear or branched monovalent hydrocarbon group having at least one carbon-carbon double bond and having 2 to 6 carbon atoms. Means Examples of the “C _2-6 alkenyl group” include ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2- Examples thereof include butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl and 5-hexenyl.

In the present specification, the “C _2-6 alkynyl group” is a linear or branched monovalent hydrocarbon group having at least one carbon-carbon triple bond and having 2 to 6 carbon atoms. means. Examples of the “C _2-6 alkynyl group” include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 3-methyl-2-butynyl, 1-pentynyl, 2- Examples include pentynyl, 3-pentynyl, 4-pentynyl, 4-methyl-1-pentynyl and the like.

In the present specification, the “C _3-8 cycloalkyl group” means a monovalent group derived from a saturated hydrocarbon ring having 3-8 carbon atoms. Further, the C _3-8 cycloalkyl may be crosslinked. Examples of the “C _3-6 cycloalkyl group” include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo [1,1,1] pentane and the like. Of these, a “C _3-6 cycloalkyl group” is preferable.

In the present specification, the “C _1-6 alkoxy group” means a group in which the above-mentioned “C _1-6 alkyl group” is bonded to an oxygen atom, that is, a straight chain or branched chain alkoxy group having 1 to 6 carbon atoms. To do. Examples of the “C _1-6 alkoxy group” include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, 1-ethylpropyl. Oxy, hexyloxy and the like can be mentioned.

In the present specification, the “C _1-6 alkylene group” means a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms. Examples of the “C _1-6 alkylene group” include methylene, ethylene, propylene, trimethylene, 1,3-butylene, 1,4-butylene, pentylene, hexylene and the like.

In the present specification, the “aryl group” means a monocyclic or polycyclic (fused) hydrocarbon group exhibiting aromaticity, and specifically, for example, phenyl, 1-naphthyl, 2-naphthyl. And C _6-22 aryl groups such as biphenylyl, terphenyl, diphenylnaphthyl, 2-anthryl and phenanthryl. Of these, a C _6-14 aryl group is preferable. Moreover, the "aryl group" may be partially hydrogenated. The position to be hydrogenated is not particularly limited. Examples of the partially hydrogenated aryl group include tetrahydronaphthyl, indanyl and the like.

In the present specification, examples of the “C _6-14 aryl group” include phenyl, 1-naphthyl, 2-naphthyl, biphenylyl and the like, and phenyl is particularly preferable.

In the present specification, the “aryl C _1-6 alkyl group” means a group in which a C _1-6 alkyl group is substituted with an aryl group, and the carbon number range is not particularly limited, but preferably C _6-14 It is an aryl C _1-6 alkyl group.

In the present specification, the “C _6-14 aryl C _1-6 alkyl group” is a group in which the “C _6-14 aryl group” is substituted on the “C _1-6 alkyl group”, for example, benzyl, 1- Phenylethyl, 2-phenylethyl, (naphthyl-1-yl) methyl, (naphthyl-2-yl) methyl, 1- (naphthyl-1-yl) ethyl, 1- (naphthyl-2-yl) ethyl, 2- Examples thereof include (naphthyl-1-yl) ethyl, 2- (naphthyl-2-yl) ethyl, biphenylylmethyl and the like.

In the present specification, the “arylene group” means a divalent aromatic hydrocarbon group derived from the above “aryl” group. Examples of the “arylene group” include 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 4,4′-biphenylylene, 2,2′-biphenylylene, 3,3′-biphenylylene, 4 , 3′-biphenylylene and the like are preferable, and C _6-14 arylene is preferable, and 1,3-phenylene, 1,4-phenylene and 4,3′-biphenylylene are particularly preferable.

In the present specification, examples of the “heterocyclic group” include an aromatic heterocyclic group and a non-aromatic heterocyclic group.

Here, the aromatic heterocyclic group is, for example, a 4- to 7-membered (preferably 5 or 7-membered) containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom and nitrogen atom in addition to carbon atoms as ring-constituting atoms. 6-membered) monocyclic aromatic heterocyclic groups and condensed aromatic heterocyclic groups. Examples of the condensed aromatic heterocyclic group include a ring corresponding to the 4- to 7-membered monocyclic aromatic heterocyclic group and a 5- or 6-membered aromatic heterocyclic ring containing 1 or 2 nitrogen atoms. (Eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine), a 5-membered aromatic heterocycle containing one sulfur atom (eg, thiophene) and a ring formed by fusion of 1 or 2 selected from a benzene ring. Examples include groups that are derived.

Preferable examples of the aromatic heterocyclic group include furyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl and triazinyl. Cyclic aromatic heterocyclic group; quinolyl, isoquinolyl, quinazolyl, quinoxalyl, benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzimidazolyl, benzpyrazolyl, benzotriazolyl, indolyl, indazolyl, pyrrolopyrazinyl And fused aromatic heterocyclic groups such as imidazopyridyl, thienopyridyl, imidazopyrazinyl, pyrazolopyridyl, pyrazolothienyl, pyrazolotriazinyl, and pyridopyridyl.

As another preferable example of the aromatic heterocyclic group, two or more aromatic heterocyclic groups or a group in which an aromatic heterocyclic group and an aryl group are linked (eg, bipyridyl, terpyridyl, pyridylphenyl, pyrimidinylphenyl, etc. ) And the like.

The non-aromatic heterocyclic group includes, for example, 3 to 7-membered (preferably 4 to 7-membered) containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom and nitrogen atom in addition to carbon atoms as ring-constituting atoms. , And more preferably 5 or 6-membered) monocyclic non-aromatic heterocyclic groups and condensed non-aromatic heterocyclic groups. Examples of the fused non-aromatic heterocyclic group include a ring corresponding to these 3- to 7-membered monocyclic non-aromatic heterocyclic groups and 5- or 6-membered aromatic groups containing 1 or 2 nitrogen atoms. 1 or 2 rings selected from a heterocycle (eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine), a 5-membered aromatic heterocycle containing one sulfur atom (eg, thiophene) and a benzene ring Examples thereof include a group derived from a condensed ring, a group obtained by partial saturation of the group, and the like.

Preferable examples of the non-aromatic heterocyclic group include azetidinyl, pyrrolidinyl, piperidyl, morpholinyl (eg, morpholino), thiomorpholinyl, piperazinyl, hexamethyleneiminyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, oxazolinyl, thiazolinyl, imidazolyl, diazolinyl, oxazolinyl, and thiazolinyl. A monocyclic non-aromatic heterocyclic group such as dihydrooxadiazolyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrothiopyranyl, tetrahydrofuryl, pyrazolidinyl, pyrazolinyl, tetrahydropyrimidinyl, dihydrotriazolyl, tetrahydrotriazolyl; Indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzodioxinyl, dihydrobenzodioxepinyl, tetrahydrobenzofuranyl, chromenil, dihydrochromenil, dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, tetrahydroisoquinolyl And condensed non-aromatic heterocyclic groups such as dihydrophthalazinyl and hexahydrofuropyrrolyl.

In the present specification, the “divalent heterocyclic group” means a divalent group derived from the above “heterocycle” group. Preferable examples of the "divalent heterocyclic group" include, for example, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, thiadiazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrimidinyl and the like. Formula Aromatic heterocyclic group; indolyl, isoindolyl, indazolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, quinolyl, isoquinolyl, benzoxazinyl, benzothiazinyl, furo [2,3-b] pyridyl , Thieno [2,3-b] pyridyl, naphthyridinyl, imidazopyridyl, oxazolopyridyl, thiazolopyridyl, quinolyl, carbazolyl, dibenzothiophenyl, etc., fused aromatic heterocyclic groups; indolinyl, etc. A heterocyclic group obtained by partial saturation; a group in which two or more aromatic heterocyclic groups such as bipyridyl and terpyridyl are linked; a group in which an aromatic heterocyclic group such as pyridylphenyl and pyrimidinylphenyl and an aryl group are linked And a divalent group. Of these, 1,6-indolinylene is preferable.

In the present specification, the “residue of anthracycline anticancer drug” means a residue obtained by removing the carbonyl oxygen atom from the 13th position of the anthracycline anticancer drug having a carbonyl group at the 13th position. Examples of the "anthracyclin anticancer agent" include doxorubicin, daunorubicin, epirubicin, amrubicin, pirarubicin, idarubicin, etc., and preferably epirubicin.

In the present specification, examples of the “nitric oxide-releasing compound” include nitrates, nitrites, S-nitrosothiols, NONOates, sydnonimines and furoxanes, and the like, preferably nitrates And a compound having a formyl group or a carbonyl group at the terminal which can form a hydrazone bond with a hydrazide residue.

In the present specification, the “nitric oxide-releasing compound residue” means a residue obtained by removing a carbonyl oxygen atom from the formyl group or carbonyl group of the “nitric oxide-releasing compound”.

The term “optionally substituted” means that the compound has 1 to 5 (preferably 1 to 3) substituents at non-substituted or substitutable positions, and each substituent is the same. Or may be different.

Examples of the “optionally substituted” substituent include a halogen atom, a hydroxy group, a carboxy group, a cyano group, a nitro group, an amino group, a sulfo group, a phospho group, a diC _1-6 alkylphosphate group, C _1-6 alkyl group, C _1-6 alkoxy group, C _3-8 cycloalkyl group, C _2-6 alkenyl group, C _2-6 alkynyl group, aryl group, aryl C _1-6 alkyl group, heterocyclic group Etc., each of which may be further substituted with a carboxy group, an amino group, or a hydroxy group.

In the present specification, the “pharmaceutically acceptable salt” means a salt that can be used as a medicine. When the compound of the present invention has an acidic group or a basic group, it can be converted to a basic salt or an acidic salt by reacting with a base or an acid, and therefore the salt is shown.
Examples of the pharmaceutically acceptable "basic salt" of compound (I) of the present invention include, for example, alkali metal salts such as sodium salt, potassium salt and lithium salt; alkaline earth metal salts such as magnesium salt and calcium salt; Organic base salts such as N-methylmorpholine salt, triethylamine salt, tributylamine salt, diisopropylethylamine salt, dicyclohexylamine salt, N-methylpiperidine salt, pyridine salt, 4-pyrrolidinopyridine salt, picoline salt; glycine salt, lysine salt , Amino acid salts such as arginine salt, ornithine salt, glutamate and aspartate, and the like, and alkali metal salts are preferable.

Examples of the pharmaceutically acceptable “acid salt” of the compound (I) of the present invention include, for example, hydrohalides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide. Inorganic acid salts such as nitrates, perchlorates, sulfates and phosphates; lower alkane sulfonates such as methane sulfonate, trifluoromethane sulfonate and ethane sulfonate, benzene sulfonate, p-toluene Organic acid salts such as aryl sulfonates such as sulfonates, acetates, malates, fumarates, succinates, citrates, ascorbates, tartrates, oxalates, maleates; glycine Examples thereof include salts, lysine salts, arginine salts, ornithine salts, glutamate salts, amino acid salts such as aspartate salts, and the like, and hydrohalide salts (particularly hydrochloride salts) are preferable.

As used herein, the term “prevention” refers to, for example, a patient who has not developed the disease or symptom expected to be at high risk of developing the disease or a symptom, or has developed the disease, due to some factor related to the disease or symptom. Administration of a drug containing the compound (I) of the present invention to a patient having no symptom, or treatment of the illness or symptom, and then treatment of the illness or symptom, to a patient who is concerned about recurrence of the illness or symptom, Administering a medicament comprising I).
"Treatment" is the cure of a disease or condition.

In the present specification, “α-amino acid” means an amino acid in which an amino group is also bonded to a carbon (α carbon) to which a carboxyl group is bonded.

In the present specification, “ε-amino acid” means an amino acid in which a carboxyl group and an amino group are bound via 5 carbon atoms.

In the present specification, the “oligopeptide” means a peptide consisting of 2 to 20 amino acids, and examples thereof include dipeptide, tripeptide, tetrapeptide, pentapeptide and the like.

As used herein, the term “methacryloylated α-amino acid, ε-amino acid or hydrazone-derived constituent unit of an oligopeptide having an anthracycline-based anticancer drug residue at the terminal” refers to methacrylic acid-derived carboxy constituting a polymer. The group is bound to the α-amino acid, ε-amino acid or the amino group terminal of the oligopeptide by a peptide bond, and further the carboxy terminal of the amino acid or oligopeptide is linked to the hydrazine (H ₂ N-NH ₂ ) by a peptide bond. Then, it means a constitutional unit in which the hydrazine terminal amino group (hydrazide residue) and the 13-position carbonyl group of the anthracycline anticancer agent form a hydrazone by dehydration condensation. As a preferable embodiment of the constitutional unit, for example, a constitutional unit (1) represented by the formula (1) described later can be mentioned.

In the present specification, “methacryloylated α-amino acid, ε-amino acid or hydrazone-derived constituent unit of oligopeptide having a nitric oxide-releasing compound residue at the terminal” is derived from methacrylic acid constituting a polymer. The carboxy group of α-amino acid, ε-amino acid or the amino group terminal of the oligopeptide is bonded by a peptide bond, and the carboxy terminal of the amino acid or oligopeptide and hydrazine (H ₂ N-NH ₂ ) are bonded by a peptide bond. After binding, it means a structural unit in which the amino group (hydrazide residue) at the hydrazine terminal and the formyl group or carbonyl group of the nitric oxide-releasing compound form a hydrazone by dehydration condensation. Preferable embodiments of the structural unit include, for example, a structural unit (2) represented by the formula (2) described below.

In the present specification, “methacryloylated α-amino acid, ε-amino acid or hydrazide-derived constituent unit of oligopeptide” means a methacrylic acid-derived carboxy group constituting a polymer, an α-amino acid and an ε-amino acid. Or, it means a constitutional unit having a hydrazide residue in which the amino group terminal of the oligopeptide is linked by a peptide bond, and further the carboxy terminal of the amino acid or oligopeptide and hydrazine (H ₂ N-NH ₂ ) are linked by a peptide bond. .. Preferable embodiments of the structural unit include, for example, a structural unit (4) represented by the formula (4) described below.

In the present specification, the “structural unit derived from N- (2-hydroxypropyl) methacrylamide” means the formula (3a):

Formula (3) formed by polymerizing N- (2-hydroxypropyl) methacrylamide (hereinafter also referred to as “HPMA”) represented by

Means a structural unit (3) represented by

In the present specification, the “structural unit derived from N- (5-hydrazinocarbonylpentyl) methacrylamide” is represented by the formula (4a):

Formula (4) formed by polymerizing N- (5-hydrazinocarbonylpentyl) methacrylamide (hereinafter, also referred to as “Ma-ah-NHNH ₂ ”) represented by the carbon-carbon double bond:

Means a structural unit (4) represented by

In the present specification, the “structural unit” means a partial unit structure constituting a polymer compound. The constituent unit may be present in the compound (I) of the present invention a plurality of times consecutively, or a plurality of the constituent units may be present in the compound (I) of the present invention separately and independently. Good.

In the present specification, the “polymerization initiator” is not particularly limited, but a compound that generates a radical species by heating is preferable, and specifically, for example, 2,2′-azobisisobutyro Nitrile (AIBN), 2,2'-azobis (2-methylbutyronitrile), 2,2'-azobis (2,4-dimethylvaleronitrile), 1,1'-azobis (1-cyclohexanecarbonitrile), Azo compounds such as 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile) and 2- (carbamoylazo) isobutyronitrile, dibenzoyl peroxide, dilauroyl peroxide, distearoyl peroxide 1,1-di (tert-butylperoxy) -2-methylcyclohexane, 1,1-di (tert-hexylperoxy) -3,3,5-trimethylcyclohexane, 1,1-di (tert-hexylperoxy) Cyclohexane, 1,1-di (tert-butylperoxy) cyclohexane, di-tert-hexyl peroxide, tert-butylcumyl peroxide, di-tert-butyl peroxide, 1,1,3,3-tetramethylbutylperoxy Examples thereof include peroxides such as -2-ethylhexanoate, tert-hexylperoxy-2-ethylhexanoate, tert-butylperoxy-2-ethylhexanoate and tert-butylperoxyisopropylmonocarbonate. Of these, AIBN is particularly preferable. The addition amount of the polymerization initiator is not particularly limited, and those skilled in the art can select an appropriate amount.

(Compound (I) of the present invention)
The compound (I) of the present invention has the formula (1):

[In the formula,
R ¹ represents a hydrogen atom, an optionally substituted C _1-6 alkyl group or an optionally substituted C _6-14 aryl group;
A represents an arylene group or a divalent heterocyclic group, each of which may be further substituted;
D is a single bond, -CH = CH-, -O-, -S-, -N (R ² )-, ^* -CON (R ² )- ^** or ^* -SO ₂ N (R ² )- ^{*. *} (Wherein R ² represents a hydrogen atom or an optionally substituted C _1-6 alkyl group; ^* represents a bonding position with A; and ^** represents a bonding position with E) .); And E represents a C _1-6 alkylene group which may be further substituted. ] A polymer compound containing the structural unit (2) represented by the above formula and the structural unit (3) derived from N- (2-hydroxypropyl) methacrylamide, or a pharmaceutically acceptable salt thereof.

Hereinafter, each group in the formula (1) and the formula (2) which is each constitutional unit of the compound (I) of the present invention will be described.

B indicates an anthracycline anticancer drug residue.

B is preferably a doxorubicin residue, a daunorubicin residue, an epirubicin residue, an amrubicin residue, a pirarubicin residue, or an idarubicin residue, and particularly preferably an epirubicin residue.

R ¹ represents a hydrogen atom or an optionally substituted C _1-6 alkyl group.

R ¹ is preferably a hydrogen atom or a C _1-4 alkyl group, more preferably a hydrogen atom or a methyl group, and particularly preferably a hydrogen atom.

A represents an arylene group or a divalent heterocyclic group, each of which may be further substituted.

A is preferably a C _6-14 arylene group which may be further substituted, or a heterocyclic group obtained by partial saturation of a divalent fused aromatic heterocyclic group, and more preferably, 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 4,4′-biphenylylene, 2,2′-biphenylylene, 3,3′-biphenylylene, 4,3 ′, which may be substituted -Biphenylylene or indolinylene, particularly preferably 1,3-phenylene, 1,4-phenylene, 4,3'-biphenylylene or 1,6-indolinylene, each of which may be further substituted.

D is a single bond, -CH = CH-, -O-, -S-, -N (R ² )-, ^* -CON (R ² )- ^** or ^* -SO ₂ N (R ² )- ^{*. *} (Wherein R ² represents a hydrogen atom or an optionally substituted C _1-6 alkyl group; ^* represents a bonding position with A; and ^** represents a bonding position with E) .) Is shown.

D is preferably a single bond, -O-, -S-, ^* -CON (R ² )- ^** or ^* -SO ₂ N (R ² )- ^** (each symbol in the formula is as defined above). Synonymous with each other), and more preferably a single bond, -O-, ^* -CONH- ^** or ^* -SO ₂ NH- ^** (each symbol in the formula has the same meaning as described above). is there.

E represents a C _1-6 alkylene group which may be further substituted

E is preferably a C _1-3 alkylene group which may be further substituted, more preferably a 1,2-propylene group, a methylene group or an ethylene group which may be further substituted by an ONO ₂ group. Is.

The following are preferable as the structural unit (1) of the compound (I) of the present invention.

[Structural unit (1A)]
Structural unit (1) in which B is a doxorubicin residue, a daunorubicin residue, an epirubicin residue, an amrubicin residue, a pirarubicin residue, or an idarubicin residue.

[Structural unit (1B)]
Building block (1), wherein B is an epirubicin residue.

The following are preferable as the structural unit (2) of the compound (I) of the present invention.

[Structural unit (2A)]
R ¹ is a hydrogen atom or a C _1-4 alkyl group;
A is a C _6-14 arylene group, which may be further substituted, or a heterocyclic group obtained by partial saturation of a divalent fused aromatic heterocyclic group;
D is a single bond, -O-, -S-, ^* -CON (R ² )- ^** or ^* -SO ₂ N (R ² )- ^** (wherein R ² is a hydrogen atom or a substituted C _1-6 alkyl group which may be present; ^* indicates a bonding position with A; and ^** indicates a bonding position with E); and E is further substituted. Structural unit (2) which is also a C _1-3 alkylene group.

[Structural unit (2B)]
R ¹ is a hydrogen atom or a methyl group;
1,2-phenylene, 1,3-phenylene, 1,4-phenylene, 4,4′-biphenylylene, 2,2′-biphenylylene, 3,3′-biphenylylene, each of which A may be further substituted , 4,3′-biphenylylene, or indolinylene;
D is a single ^bond, -O-, * -CONH- in ^** or ^* -SO ₂ NH- ^** ^{(wherein, *} indicates the bonding position to A; and ^** is the bonding position of the E , And E is a 1,2-propylene group, a methylene group, or an ethylene group which may be further substituted with an ONO ₂ group, the structural unit (2).

[Structural unit (2C)]
R ¹ is a hydrogen atom;
A is 1,3-phenylene, 1,4-phenylene, 4,3′-biphenylylene, or 1,6-indolinylene, each of which may be further substituted;
D is a single ^bond, -O-, * -CONH- in ^** or ^* -SO ₂ NH- ^** ^{(wherein, *} indicates the bonding position to A; and ^** is the bonding position of the E , And E is a 1,2-propylene group, a methylene group, or an ethylene group which may be further substituted with an ONO ₂ group, the structural unit (2).

[Structural unit (2D)]
The group -ADDE-ONO _{2 in the} above formula (2) has the following formula:

The structural unit (2), which is, can be mentioned as another preferable embodiment.

The compound (I) of the present invention is preferably a copolymer composed of the structural unit (1), the structural unit (2), the structural unit (3), and the structural unit (4), and more preferably the structural unit. A copolymer composed of (1A), structural unit (2A), structural unit (3), and structural unit (4), and more preferably structural unit (1B), structural unit (2B), structural unit (3). ) And a structural unit (4), particularly preferably a structural unit (1B), a structural unit (2C), a structural unit (3), and a structural unit (4). is there. The compound (I) of the present invention is preferably a copolymer composed of the structural unit (1B), the structural unit (2D), the structural unit (3), and the structural unit (4).

The compound (I) of the present invention contains 1 to 40% by weight, preferably 5 to 20% by weight, of the anthracycline anticancer drug residue in the structural unit (1) based on the total amount.

The compound (I) of the present invention contains 0.5 to 20% by weight, preferably 5 to 15% by weight, of the nitric oxide-releasing compound residue in the structural unit (2) with respect to the total amount. To do.

The degree of anticancer activity and side effects of the compound (I) of the present invention can be adjusted by adjusting the amount ratio of each raw material monomer used at the time of its synthesis and the amounts of the anthracycline anticancer agent and nitric oxide-releasing compound used. Etc. can be adjusted freely.

The compound (I) of the present invention may contain a structural unit other than the structural units (1) to (4) as long as the anticancer activity thereof is not impaired. The structural units other than the structural units (1) to (4) are not particularly limited as long as they do not impair the anticancer activity.

The content of the anthracycline anticancer drug residue contained in the structural unit (1) and the content of the nitric oxide-releasing compound residue contained in the structural unit (2) in the compound (I) of the present invention are The released anthracycline anticancer agent and nitric oxide-releasing compound can be determined by measurement using a known measuring device according to a known method. The content of the structural unit (4) having a hydrazide residue can be measured by a known measuring device according to a known method (Masayoshi Tachizawa, Analytical Chemistry 11, 1055-1059 (1965)). is there.

The weight average molecular weight of the compound (I) of the present invention is 10 to 300 kDa (10,000 to 300,000), preferably 10 to 100 kDa (10,000 to 100,000).

The weight average molecular weight and polydispersity of the compound (I) of the present invention can be determined by a method known per se (for example, PJ Wyatt, Anal. Chim. Acta 272, 1-40 (1993); X.Jiang et al., Poly. Chem., 5, 4915-4925 (2014)), column for size exclusion column chromatography, multi-angle static light scattering measurement device (miniDAWN TREOS II, Wyatt Technology) and differential refractometer (Optilab T-rEX, Wyatt Technology) connected high performance liquid chromatograph (HPLC) and analysis software (ASTRA 7.1.2, Wyatt Technology). In addition, the particle size is measured according to a method known per se (JIS Z8826: 2005 particle analysis-photon correlation method; I.C. Carina, Polymer 68, 41-46 (2015)), and a dynamic light scattering measurement device (nano Partica SZ-100, HORIBA, Ltd.).

(Method for producing compound (I) of the present invention)
As a method for producing the compound (I) of the present invention, the case where B is an epirubicin hydrochloride residue is shown below as an example, and the same applies when B in the above formula (1) is another anthracycline anticancer drug residue. Can be manufactured. Further, the method for producing the compound (I) of the present invention is not limited to the following method.
In addition, the raw material compound in each step can be obtained as a commercially available product and used as it is, or can be produced according to a method known per se or a method analogous thereto, unless a specific production method is mentioned. It is possible. In addition, the intermediate produced in the following production method may be isolated and purified by a conventional separation method such as precipitation method, column chromatography, recrystallization, or may be used in the next step without isolation. ..
Furthermore, when a structural unit other than the structural units (1) to (4) is included, coexistence of a monomer component corresponding to the structural unit other than the structural units (1) to (4) in the first step described later. The corresponding compound (I) of the present invention can be produced by copolymerizing under the following.

The compound (I) of the present invention is a copolymer synthesized from (1) N- (2-hydroxypropyl) methacrylamide (HPMA) and N- (5-hydrazinocarbonylpentyl) methacrylamide (Ma-ah-NHNH ₂ ). And a step of conjugating an epirubicin residue to the copolymer via a hydrazone bond by a reaction of a hydrazide group in the copolymer with a carbonyl group of epirubicin at the 13-position (second step), and Or a hydrazide group remaining in the conjugate, a formyl group or a carbonyl group of the nitric oxide-releasing compound is further conjugated via a hydrazone bond (3rd step), or (2) After the hydrazide group in the copolymer obtained in the step is first conjugated via the formyl group or carbonyl group of the nitric oxide-releasing compound (second step), it remains in the obtained conjugate. To a carbonyl group at the 13-position of epirubicin by a reaction with a hydrazide group to form an epirubicin residue via a hydrazone bond (3 ′ step), or (3) in the 1st step. It can be produced by any method of producing via a step (second '' step) of simultaneously conjugating an epirubicin residue and a nitric oxide-releasing compound residue to a hydrazide group in the obtained copolymer. .. In addition, the compound (I) of the present invention can be purified by a conventional separation method such as a precipitation method or column chromatography.

(Step 1) Production of HPMA-Ma-ah-NHNH ₂ Copolymer In this step, HPMA (Compound (3a)) and Ma-ah-NHNH ₂ (Compound (4a)) are co-existed in the presence of a polymerization initiator in a random copolymer. Polymerize and formula (3):

The structural unit (3) represented by and the formula (4):

Is a step of producing a copolymer (compound (5)) consisting of the structural unit (4) represented by. Compounds (3a) and (4a) can be produced by a method known per se (J. Control. Release., 64, 63-79 (2000), International Publication No. 2007/028347) or a method analogous thereto. ..

The amount of compound (3a) to be used is generally 1 to 100 molar equivalents, preferably 4 to 20 molar equivalents, relative to compound (4a).
Examples of the polymerization initiator include the above-mentioned polymerization initiators, but AIBN can be preferably used. The amount of the polymerization initiator used is usually 0.01 to 1 molar equivalent based on compound (4a).

Examples of the inert solvent used include alcohols such as methanol, ethanol, propanol, 2-propanol and butanol; aromatic hydrocarbons such as benzene, toluene and xylene; diethyl ether, diisopropyl ether, tetrahydrofuran, 1, Ethers such as 4-dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide Halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, fluorobenzene, trichloromethylbenzene, trifluoromethylbenzene; acetonitrile; or a mixture thereof. Etc. Alcohols are preferred as the solvent.

The reaction temperature varies depending on the raw material compound or the solvent used, but it is usually from room temperature to the reflux temperature of the reaction mixture, and preferably from 60 ° C. to the reflux temperature of the reaction mixture.

The reaction time varies depending on the raw material compound, the solvent used or the reaction temperature, but is usually 30 minutes to 96 hours, and preferably 30 minutes to 24 hours.

The weight average molecular weight of the copolymer (compound (5)) obtained in this step can be adjusted by changing the ratio of methanol-ethyl acetate in the precipitation method using methanol-ethyl acetate.

(Second Step) Production of Polymer-Epirubicin-Conjugate In this step, the end of the structural unit (4) in the copolymer (compound (5)) produced in the first step in the presence of an acid in an inert solvent is used. To the hydrazide group of the following formula (6):

Epirubicin hydrochloride represented by the formula (compound (6)) is condensed to give formula (1B):

Is a step of producing a polymer-epirubicin-conjugate (compound (7)) comprising the structural unit (1B) represented by, the structural unit (3), and the structural unit (4).

As the acid, for example, organic acids such as acetic acid, oxalic acid, maleic acid, citric acid, tartaric acid, malic acid and succinic acid can be used.
The amount of acid used is usually 1 to 200 molar equivalents, preferably 1 to 50 molar equivalents, relative to compound (6).

Examples of the inert solvent used include alcohols such as methanol, ethanol, propanol, 2-propanol and butanol; aromatic hydrocarbons such as benzene, toluene and xylene; diethyl ether, diisopropyl ether, tetrahydrofuran, 1, Ethers such as 4-dioxane and 1,2-dimethoxyethane; amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide Halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, fluorobenzene, trichloromethylbenzene, trifluoromethylbenzene; acetonitrile; water; dimethyl sulfoxide Or a mixture of these and the like. Alcohols are preferred as the solvent.

The reaction temperature varies depending on the type of acid used or the solvent used, but is usually 0 ° C. to the reflux temperature of the reaction mixture, and preferably room temperature to the reflux temperature of the reaction mixture.

The reaction time varies depending on the type of acid used, the solvent used, or the reaction temperature, but is usually 30 minutes to 96 hours, and preferably 30 minutes to 24 hours.

(Third Step) Polymer-Epirubicin-Nitric Oxide-Releasing Compound-Production of Conjugate In this step, the polymer-epirubicin-prepared in the second step in the presence or absence of an acid in an inert solvent is used. The hydrazide group at the terminal of the structural unit (4) remaining in the conjugate (compound (7)) has the formula (8):

[In the formula, R ¹ , A, D and E have the same meanings as described above. ]
A nitric oxide-releasing compound (compound (8)) represented by

[In the formula, R ¹ , A, D and E have the same meanings as described above. ]
Represented by the structural unit (2), the structural unit (1B), the structural unit (3), and the structural unit (4): epirubicin-nitric oxide-releasing compound-conjugate polymer (compound of the present invention This is a step of manufacturing (I)).

The amount of the compound (8) used is usually 0.1 to 100 molar equivalents, preferably 0.5 to 10 molar equivalents, relative to the amount of the epirubicin residue bound to the compound (7).

As the acid, for example, organic acids such as acetic acid, oxalic acid, maleic acid, citric acid, tartaric acid, malic acid and succinic acid can be used.
The amount of the acid used is usually 0 to 200 molar equivalents, preferably 1 to 50 molar equivalents, relative to compound (8).

The reaction temperature varies depending on the raw material compound or the solvent used, but is usually 0 ° C. to the reflux temperature of the reaction mixture, and preferably room temperature to the reflux temperature of the reaction mixture.

The reaction time varies depending on the raw material compound, the solvent used, or the reaction temperature, but is usually 30 minutes to 96 hours, and preferably 30 minutes to 24 hours.

(Step 2 ′) Production of Polymer—Nitric Oxide-Releasing Compound—Conjugate In this step, the copolymer produced in the above-mentioned step 1 in the presence or absence of an acid (compound (5 )) In the terminal hydrazide group of the structural unit (4) is represented by the formula (8):

[In the formula, R ¹ , A, D and E have the same meanings as described above. ]
Is a step of producing a polymer-a nitric oxide-releasing compound-conjugate (compound (9)) comprising the structural unit (2) represented by, the structural unit (3), and the structural unit (4).

As the acid, an organic acid such as acetic acid, oxalic acid, maleic acid, citric acid, tartaric acid, malic acid or succinic acid can be used as in the third step.
The amount of the acid used is usually 0 to 200 molar equivalents, preferably 1 to 50 molar equivalents, relative to compound (8).

(3 'step) Preparation of polymer-epirubicin-nitric oxide-releasing compound-conjugate In this step, the polymer-epirubicin-nitric oxide-releasing compound prepared in the 2'step in the presence of an acid in an inert solvent is used. Epirubicin hydrochloride (compound (6)) is condensed with the terminal hydrazide group of the structural unit (4) remaining in the compound-conjugate (compound (9)) to give the compound of formula (1B):

For producing a polymer-epirubicin-nitric oxide-releasing compound-conjugate comprising the structural unit (1B) represented by the formula (1B), the structural unit (2), the structural unit (3), and the structural unit (4) Is.

The amount of the compound (6) used is usually 0.1 to 100 molar equivalents, preferably 0.5 to 10 molar equivalents, relative to the amount of the nitric oxide-releasing compound bound to the compound (9).

As the acid, as in the second step, for example, an organic acid such as acetic acid, oxalic acid, maleic acid, citric acid, tartaric acid, malic acid, succinic acid can be used.
The amount of acid used is usually 1 to 200 molar equivalents, preferably 1 to 50 molar equivalents, relative to compound (6).

(Second step) Production of polymer-epirubicin-nitric oxide-releasing compound-conjugate (compound (I) of the present invention) This step is carried out in the presence of an acid in an inert solvent in the first step. In the hydrazide group at the terminal of the structural unit (4) in the produced copolymer (compound (5)), epirubicin hydrochloride (compound (6)) and formula (8):

[In the formula, R ¹ , A, D and E have the same meanings as described above. ]
By condensing a nitric oxide-releasing compound (compound (8)) represented by the formula (1B):

The structural unit (1B) represented by the formula (2):

[In the formula, R ¹ , A, D and E have the same meanings as described above. ]
Is a step of producing a polymer-epirubicin-nitric oxide-releasing compound-conjugate comprising the structural unit (2) represented by, the structural unit (3), and the structural unit (4).

The amount of compound (6) and compound (8) used is usually 0.1 to 100 molar equivalents, preferably 0.5 to 10 molar equivalents, relative to compound (5).

As the acid, organic acids such as acetic acid, oxalic acid, maleic acid, citric acid, tartaric acid, malic acid and succinic acid can be used as in the above.
The amount of acid used is usually 1 to 200 molar equivalents, preferably 1 to 50 molar equivalents, relative to compound (6) or compound (8).

Examples of the inert solvent used include alcohols such as methanol, ethanol, propanol, 2-propanol and butanol; aromatic hydrocarbons such as benzene, toluene and xylene; diethyl ether, diisopropyl ether, tetrahydrofuran, 1 , 4-dioxane, 1,2-dimethoxyethane and other ethers; N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, hexamethylphosphorotriamide and the like Amides; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, fluorobenzene, trichloromethylbenzene, trifluoromethylbenzene; acetonitrile; water; dimethyl Examples thereof include sulfoxide; or a mixture thereof. Alcohols are preferred as the solvent.

Structural unit (1) containing an anthracycline anticancer drug residue in compound (I) of the present invention (in the above example, structural unit (1B) containing an epirubicin residue) and a structural unit containing a nitric oxide-releasing compound residue The proportion of the unit (2) can be adjusted by appropriately changing the amounts of the compound (6) and the compound (8) used and the reaction conditions in each of the above steps to obtain a desired ratio of polymer-compound (6) -compound (8)-. The conjugate can be manufactured.

The compound (I) of the present invention or a pharmaceutically acceptable salt thereof may be a geometric isomer such as cis isomer or trans isomer, a tautomer or an optical isomer such as d isomer or l isomer depending on the kind and combination of substituents. Although various isomers such as isomers may exist, the compound (I) of the present invention includes all isomers, stereoisomers and any ratio of these isomers and stereoisomer mixtures unless otherwise specified. It also includes.

The cancer to which the compound (I) of the present invention is applied is, for example, colon cancer (eg, colon cancer, rectal cancer, anal cancer, familial colon cancer, hereditary non-polyposis colon cancer, gastrointestinal stromal tumor), Lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (eg, pancreatic ductal cancer, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (eg, papillary cancer) Adenocarcinoma, mucinous adenocarcinoma, adenosquamous cell carcinoma, duodenal cancer, small intestine cancer, breast cancer (eg, invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (eg, epithelial ovarian cancer) , Extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low-grade tumor), testicular tumor, prostate cancer (eg, hormone-dependent prostate cancer, hormone-independent prostate cancer, castration-resistant prostate cancer), liver Cancer (eg, hepatocellular carcinoma, primary liver cancer, extrahepatic cholangiocarcinoma), thyroid cancer (eg, medullary thyroid carcinoma), kidney cancer (eg, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (Eg, endometrial cancer, cervical cancer, endometrial cancer, uterine sarcoma), gestational choriocarcinoma, brain tumor (eg, medulloblastoma, glioma, pineal astrocytoma, pilocytic) Astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (eg, basal cell tumor, malignant melanoma), sarcoma (eg, rhabdomyosarcoma, Leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma, malignant bone tumor, bladder cancer, hematological cancer (eg, multiple myeloma, leukemia (eg, acute myelogenous leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disease) ), Cancers of unknown primary origin and the like.
Among them, the compound (I) of the present invention is used for breast cancer, pancreatic cancer, bladder cancer, prostate cancer, esophageal cancer, gastric cancer, uterine cancer, ovarian cancer, brain tumor, colon cancer (eg, colon cancer, rectal cancer), blood cancer ( For example, it is effective against at least one cancer selected from the group consisting of acute myelogenous leukemia, multiple myeloma), liver cancer (eg, hepatocellular carcinoma), skin cancer, lung cancer and thyroid cancer.

When the compound (I) of the present invention or a pharmaceutically acceptable salt thereof is used as an anticancer drug, it can be used in combination with other drugs, for example, existing anticancer drugs, as long as the drug efficacy is not impaired. At this time, the timing of administration is not limited, and these may be administered to the administration subject at the same time or at different times. The dose can be appropriately selected based on the dose clinically used. The compounding ratio of the compound of the present invention or a pharmaceutically acceptable salt thereof and the concomitant drug can be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination and the like.

Examples of existing anticancer agents include chemotherapeutic agents, hormone therapeutic agents, immunotherapeutic agents, molecular targeting agents, immune checkpoint inhibitors (anti-PD-1 antibody, anti-PD-L1 antibody) and the like.
As the "chemotherapeutic agent", for example, an alkylating agent, an antimetabolite, an anticancer antibiotic, a plant-derived anticancer agent, etc. are used.
Examples of the “alkylating agent” include nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melamine. Phalan, dacarbazine, ranimustine, bendamustine, procarbazine, estramustine phosphate sodium, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, etogluside, carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin, altretamine hydrochloride, ambromustine, ambastine, ambromustine. Pydium, photemustine, predonimustine, pumitepa, ribomustine, temozolomide, threosulfan, trofosfamide, dinostatin stimalamer, adzelesin, systemustin, viserecin, and DDS preparations thereof are used.

Examples of the “antimetabolite” include mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, pemetrexed, enocitabine, cytarabine, cytarabine ocfosphate, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, doxifluridine, carmofur, gallocitabine, emitefur, capecitabine), aminopterin, Neruzarabin, leucovorin calcium, tabloid, Butoshin, Folli Nate calcium, levo Folli Nate calcium, cladribine, clofarabine, nelarabine, emitefur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin Statins, pyritrexime, idoxyuridine, mitoguazone, thiazofurin, and DDS preparations thereof are used.

Examples of the "anti-cancer antibiotic" include actinomycin D, actinomycin C, mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, peomycin, peplomycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride. , Neocarzinostatin, misramycin, zarcomycin, carcinophylline, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride, and their DDS preparations are used.

As the “plant-derived anticancer agent”, for example, etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, vinorelbine, and DDS preparations thereof are used.

Examples of the “hormone therapeutic agent” include phosfestrol, diethylstilbestrol, chlorotrianisene, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone, mepartricin, Raloxifene, olmeroxifene, levormeroxifene, anti-estrogens (eg, tamoxifen citrate, toremifene citrate), pill preparations, mepithiostane, testrolactone, aminoglutethiimide, LH-RH agonists (eg, goserelin acetate, buserelin acetate) , Leuprorelin), droloxifene, epithiostanol, ethinyl estradiol sulfonate, aromatase inhibitors (eg, fadrozole hydrochloride, anastrozole, letrozole, exemestane, borozole, formestane), antiandrogens (eg, flutamide, Bicalutamide, nilutamide), 5α-reductase inhibitors (eg, finasteride, epristeride), corticosteroids (eg, dexamethasone, prednisolone, betamethasone, triamcinolone), androgen synthesis inhibitors (eg, abiraterone), retinoids and retinoid metabolism Drugs that delay the onset (eg, Liarozole) and the like are used.

The “immunotherapeutic agent” includes biological response modifiers (eg, picibanil, krestin, schizophyllan, lentinan, ubenimex, interferon, interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum, levamisole, polysaccharide K, procodazole, anti-CTLA4 antibody) and the like are used.

The "molecular target drug", for example, tositumomab, ibritumomab, alemtuzumab, axitinib, bevacizumab, AFATINIB, Oshimeruchinibu, bortezomib, bosutinib, carfilzomib, cetuximab, dasatinib, denosumab, edrecolomab, erlotinib, everolimus, VISMODEGIB, gefitinib, gemtuzumab Ozogamicin, imatinib, ipilimumab, lapatinib, lenalidomide, nilotinib, nimotuzumab, olaparib, panitumumab, pazopanib, pertuzumab, rituximab, ofatumunibumab, mogamulizumab, brentuximab, glutinumab, brentuximab, glutinumab, brentuximab. Thalidomide, trastuzumab, trastuzumab emtansine, tretinoin, bandetanib, vorinostat, cabozantinib, trametinib, dabrafenib, crizotinib, alectinib, seritinib, rivesolib, rukisolib, rivisobrib, riboxibreb, riboxiculib, riboxiculib, riboxiculib, riboxiculib, riboxiculib, riboxiculib, riboxiclib, riboxibreb, riboxiculib, riboxibreb, riboxibreb, riboxibreb, riboxibreb, riboxibreb, riboxibreb, riboxibreb, riboxibreb, riboxibreb, riboxibreb.

As the “immunity checkpoint inhibitor”, for example, ipilimumab, tremelimumab, nivolumab, pembrolizumab, avelumab, atezolizumab, durvalumab, etc. are used.

Since the compound (I) of the present invention is a polymer obtained by simultaneously conjugating an anticancer drug and a nitric oxide-releasing compound, it enhances the EPR effect and thus migrates to a tumor tissue as compared with a conventional HPMA-conjugated DDS anticancer drug. It is expected that the anti-cancer effect will be improved and a superior anti-cancer effect will be exhibited. Therefore, it is useful as an anticancer agent, a preventive or therapeutic agent for cancer metastasis and / or cancer recurrence.

The present invention relates to the following synthesis of a nitric oxide-releasing compound (compound (8)), weight average molecular weight of polymer-active drug-conjugate, content of epirubicin and nitric oxide-releasing compound, and particle size. Measurement of physical properties, synthesis of HPMA-Ma-ah-NHNH ₂ copolymer (compound (5)), synthesis of polymer-epirubicin-conjugate (compound (7)), polymer-epirubicin-nitric oxide-releasing compound-conjugate The synthesis of the gates (Examples 1 to 20) and the measurement of their anti-cancer activity (Test Examples 1 and 2) will be explained in detail without being limited thereto and without departing from the scope of the present invention. You may change in the range. The reagents and raw material compounds used are commercially available, unless otherwise specified.
"Room temperature" in the following synthesis examples, measurement examples, examples, and test examples usually indicates about 10 ° C to about 35 ° C. The ratios shown in the mixed solvent are volume ratios unless otherwise specified. Unless otherwise specified,% means% by weight.

The nitric oxide-releasing compound (compound (8)) was produced by the methods shown in Synthesis Examples 1 to 22 below.

Synthesis example 1:
(1) 2-nitrooxymethylbenzaldehyde

2-Bromomethylbenzonitrile (5.00 g, 25.5 mmol) was dissolved in toluene (50 mL), and a solution of 1.0 M diisobutylaluminum hydride in toluene (39 mL, 39 mmol) was added dropwise over 5 minutes under ice-cooling. Stir for 1 hour. 100 mL of 1.0 M hydrochloric acid was added to the reaction solution, the insoluble material was filtered off with Celite, and washed with 100 mL of ethyl acetate three times. The two layers of the filtrate were separated, and the organic layer was washed successively with 1.0 M hydrochloric acid (100 mL) and saturated brine (100 mL) and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain a mixture of a black oily substance and a solid substance.
The obtained residue is dissolved in 50 mL of acetonitrile, 6.51 g (38.3 mmol) of silver nitrate (I) is added, and after stirring for 3.5 hours at room temperature, 2.17 g (12.8 mmol) of silver nitrate (I) is added, and at room temperature. It was stirred for another 2 hours. The insoluble matter was filtered off through Celite, washed with 100 mL of ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give 1.45 g (2 steps, yield 31%) of a yellow oily substance of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 5.97 (2H, s), 7.60-7.77 (3H, m), 8.02 (1H, d, J = 7.3 Hz), 10.19 (1H, s).

Synthesis example 2:
(2) 4-nitrooxymethylbenzaldehyde

4-Bromomethylbenzonitrile (5.00 g, 25.5 mmol) was dissolved in toluene (50 mL), and a solution of 1.0 M diisobutylaluminum hydride in toluene (39 mL, 39 mmol) was added dropwise over 7 minutes under ice-cooling. It was stirred for 30 minutes. 100 mL of 1.0 M hydrochloric acid was added to the reaction solution, the insoluble matter was filtered off with Celite, and then washed with 100 mL of ethyl acetate. The two layers of the filtrate were separated, and the aqueous layer was extracted with 50 mL of ethyl acetate. The organic layers were combined, washed successively with 1.0 M hydrochloric acid and 50 mL of saturated saline, and then dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain 4.19 g of white powder.
4.19 g of the obtained white powder was dissolved in acetonitrile, 5.36 g (31.6 mmol) of silver nitrate (I) was added, and the mixture was stirred at room temperature for 1 hour. The insoluble matter was filtered off with Celite, washed with 100 mL of ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 3, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 3.34 g (2 steps, yield 72%) of a white powder of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 5.50 (2H, s), 7.54-7.59 (2H, m), 7.90-7.95 (2H, m), 10.04 (1H, s).

Synthesis example 3:
(3) 3-nitrooxymethylbenzaldehyde

Dissolve 6.53 g (33.3 mmol) of 3-bromomethylbenzonitrile in 67 mL of toluene, and under ice cooling, add 50 mL (50 mmol) of a 1.0 M solution of diisobutylaluminum hydride in toluene over 8 minutes, and at the same temperature. Stir for 3.5 hours. To the reaction solution was added 150 mL of 1.0 M hydrochloric acid, the mixture was stirred at room temperature for 30 minutes, the insoluble material was filtered off with Celite, and washed with 100 mL of ethyl acetate. The two layers of the filtrate were separated, and the organic layer was washed successively with 100 mL of 1.0 M hydrochloric acid and 100 mL of saturated saline and then dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain a mixture of a pale yellow oily substance and a solid substance.
The obtained residue was suspended in 67 mL of acetonitrile, 11.3 g (66.6 mmol) of silver nitrate (I) was added, and the mixture was stirred at room temperature for 1.5 hours. The insoluble matter was filtered off through Celite, washed with 100 mL of ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 3: 17, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 4.60 g (2 steps, yield 76%) of a pale yellow oily substance of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 5.68 (2H, s), 7.67 (1H, t, J = 7.7 Hz), 7.81 (1H, d, J = 7.7 Hz), 7.92-8.02 ( 2H, m), 10.04 (1H, s).

Synthesis example 4:
(4a) 4- (2-bromoethyl) phenylmethanol

Dissolve 10.0 g (43.7 mmol) of 4-bromoethylbenzoic acid in 250 mL of tetrahydrofuran, and under ice cooling, add 100 mL (90 mmol) of a 0.90 M borane-tetrahydrofuran complex solution in tetrahydrofuran over 10 minutes, and add 2 at room temperature. Stir for hours. 500 mL of saturated aqueous ammonium chloride was added to the reaction solution, extracted with 200 mL of ethyl acetate, and the organic layer was washed successively with saturated aqueous ammonium chloride, water and saturated saline, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain 9.49 g (quantitative) of the title compound as a white powder.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.10 (2H, t, J = 7.1 Hz), 3.70 (2H, t, J = 7.1 Hz), 4.46 (2H, d, J = 5.6 Hz) , 5.10 (1H, t, J = 5.6 Hz), 7.18-7.27 (4H, m).

(4b) 4- (2-Bromoethyl) benzaldehyde

4- (2-Bromoethyl) phenylmethanol 5.00 g (23.0 mmol) prepared in Synthesis Example 4 (4a) was dissolved in 150 mL of methylene chloride, pyridinium chlorochromate (5.95 g, 27.6 mmol) was added, and the mixture was stirred at room temperature for 4 hours. Stir for hours. The insoluble matter was filtered off with Celite, and the solvent of the filtrate was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 3, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (4.39 g, yield 90%) as a white powder.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.24 (2H, t, J = 7.1 Hz), 3.79 (2H, t, J = 7.1 Hz), 7.49-7.54 (2H, m), 7.83- 7.88 (2H, m), 9.98 (1H, s).

(4) 4- (2-Nitrooxyethyl) benzaldehyde

4- (2-Bromoethyl) benzaldehyde 4.39 g (20.6 mmol) produced in Synthesis Example 4 (4b) was dissolved in acetonitrile 41 mL, silver nitrate (I) 5.25 g (30.9 mmol) was added, and the mixture was allowed to stand at room temperature for 15.5 hours. The mixture was stirred at 50 ° C for 23.5 hours. After cooling, the insoluble matter was filtered off with Celite, washed with 100 mL of ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 3: 17, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 1.81 g (yield 45%) of a pale yellow oily substance of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.13 (2H, t, J = 6.4 Hz), 4.81 (2H, t, J = 6.4 Hz), 7.50-7.57 (2H, m), 7.83- 7.90 (2H, m), 9.98 (1H, s).

Synthesis example 5:
(5) 4- (3-nitrooxypropyl) benzaldehyde

Dissolve 7.63 mL (50.2 mmol) of 3-bromopropylbenzene in 100 mL of methylene chloride, add 12.0 mL (110 mmol) of titanium (IV) chloride under ice cooling, and stir at the same temperature for 50 minutes, then dichloromethylmethyl. Ether 4.92 mL (55.2 mmol) was added, and the mixture was stirred at room temperature for 35 minutes. The reaction solution was poured into ice water, the two layers were separated, and the organic layer was washed successively with 150 mL of saturated aqueous sodium hydrogen carbonate and 150 mL of saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain a mixture of yellow solid and oil.
The obtained residue was dissolved in 100 mL of acetonitrile, 12.8 g (75.4 mmol) of silver nitrate (I) was added, and the mixture was stirred at room temperature for 14 hours and at 50 ° C for 24 hours. After cooling, the insoluble matter was filtered off with Celite, washed with 100 mL of ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). After evaporating the solvent of the target fraction under reduced pressure, the obtained residue was purified again by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (3.51 g, two steps, yield 33%) as a pale-yellow oil.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 1.97-2.08 (2H, m), 2.78 (2H, t, J = 7.7 Hz), 4.54 (2H, t, J = 6.5 Hz), 7.45- 7.50 (2H, m), 7.82-7.87 (2H, m), 9.97 (1H, s).

Synthesis example 6:
(6a) 2-nitrooxyethylamine nitrate

To 60 mL of methylene chloride under ice cooling, 9.0 mL of fuming nitric acid was added dropwise over 2 minutes, and after stirring for 10 minutes at the same temperature, 2.9 mL (48.2 mmol) of ethanolamine was added dropwise over 1 minute and at the same temperature. Stir for 50 minutes. Acetic anhydride 6.0 mL (63.5 mmol) was added dropwise at the same temperature over 1 minute, stirred at room temperature for 40 minutes, cooled again with ice, and then 20 mL of ethanol was added for 2 minutes and 60 mL of ethyl acetate was added for 5 minutes. After dropping, the mixture was stirred at room temperature for 1 hour. The insoluble matter was collected by filtration, washed with 20 mL of ethyl acetate, and then dried under reduced pressure at room temperature for 40 minutes to obtain 4.91 g of white powder (crude product) containing the title compound.

(6) 4-Formyl-N- (2-nitrooxyethyl) benzamide

4-Formylbenzoic acid 1.00 g (6.66 mmol) was dissolved in N, N-dimethylformamide 20 mL, 1,1'-carbonyldiimidazole 1.62 g (9.99 mmol) was added, and the mixture was stirred at room temperature for 1 hour, then synthesized. White powder (1.69 g) containing 2-nitrooxyethylamine nitrate prepared in Example 6 (6a) and N, N-diisopropylethylamine (1.8 mL, 10 mmol) were added, and the mixture was stirred at room temperature for 18 hours. 100 mL of water was added to the reaction solution, and the mixture was extracted twice with 70 mL of ethyl acetate. The organic layers were combined, washed with 50 mL of water and 50 mL of saturated saline, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 3: 7 → 7: 3, V / V). The solvent of the target fraction was evaporated under reduced pressure, and the obtained residue was purified again by column chromatography (ethyl acetate: n-hexane, 3: 7 → 7: 3, V / V). After evaporating the solvent of the target fraction under reduced pressure, 0.48 g of the obtained residue was left to stand overnight in a freezer to solidify. 10 mL of a mixture of tert-butyl methyl ether-n-hexane (1:10) was added to the solid, and the insoluble material was collected by filtration, and then a mixture of tert-butyl methyl ether-n-hexane (1:10) 5 It was washed with mL. The obtained powder was dried under reduced pressure at room temperature for 1 hour to obtain 419 mg (yield 26%) of a white powder of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.61-3.68 (2H, m), 4.65-4.70 (2H, m), 7.97-8.06 (4H, m), 8.89-8.97 (1H, m) , 10.08 (1H, s).

Synthesis example 7:
(7a) 3,5-bis (bromomethyl) phenylmethanol

Dissolve 5.26 g (31.3 mmol) of 1,3,5-benzenetrimethanol in 157 mL of acetonitrile, add 22.5 g (68.9 mmol) of carbon tetrabromide under ice cooling, and then add 18.1 g (68.9 mmol) of triphenylphosphine. ) Was added over 3 minutes, and the mixture was stirred at room temperature for 2 hours. The solvent of the reaction solution was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (4.74 g, yield 52%) as a white solid.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.75 (1H, t, J = 5.8 Hz), 4.47 (4H, s), 4.70 (2H, d, J = 5.8 Hz), 7.34 (3H, s) .

(7b) 3,5-bis (bromomethyl) benzaldehyde

3,5-bis (bromomethyl) phenylmethanol (4.74 g, 16.1 mmol) produced in Synthesis Example 7 (7a) was dissolved in 81 mL of methylene chloride, and manganese dioxide (14.0 g, 161 mmol) was added, and the mixture was allowed to stand at room temperature for 15 hours. It was stirred. The insoluble matter of the reaction solution was filtered off with Celite, the solvent of the filtrate was evaporated under reduced pressure, and the obtained residue was subjected to column chromatography (ethyl acetate: n-hexane, 0: 1 → 3: 17, V / V). ). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (2.57 g, yield 55%) as a white solid.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 4.81 (4H, s), 7.85 (1H, s), 7.93 (2H, s), 10.00 (1H, s).

(7) 3,5-bis (nitrooxymethyl) benzaldehyde

2.57 g (8.80 mmol) of 3,5-bis (bromomethyl) benzaldehyde produced in Synthesis Example 7 (7b) was dissolved in 18 mL of acetonitrile, and 4.48 g (26.4 mmol) of silver nitrate (I) was added, and the mixture was stirred at room temperature for 70 minutes. It was stirred. The insoluble material was filtered off with Celite, washed with 100 mL of ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (2.11 g, yield 94%) as a colorless oil.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 5.71 (4H, s), 7.90 (1H, s), 8.05 (2H, s), 10.05 (1H, s)

Synthesis Example 8:
(8a) 3-formylbenzoic acid

Triphenylphosphine 1.27 g (4.83 mmol) and iodine 1.23 g (4.83 mmol) were suspended in toluene 16 mL and stirred at room temperature for 10 minutes, and then 3-iodobenzoic acid 1.00 g (4.03 mmol) and palladium acetate 27 mg ( 0.12 mmol) and triethylamine 3.4 mL (24 mmol) were added, and the mixture was stirred at 80 ° C. for 4 hours in a sealed tube. After allowing to cool, 30 mL of 1.0 M hydrochloric acid was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried (Na ₂ SO ₄ ) and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (chloroform: methanol, 1: 0 → 9: 1, V / V), and the solvent of the target fraction was evaporated under reduced pressure to remove the title compound and triphenylphosphine oxide. 700 mg of a pale yellow oily substance was obtained as a mixture.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 7.74 (1H, t, J = 7.6 Hz), 8.14 (1H, d, J = 7.6 Hz), 8.24 (1H, d, J = 7.6 Hz) , 8.44 (1H, s), 10.09 (1H, s).

(8) 3-Formyl-N- (2-nitrooxyethyl) benzamide

700 mg of a pale yellow oil containing 3-formylbenzoic acid prepared in Synthesis Example 8 (8a) was dissolved in 3 mL of methylene chloride, and 0.14 mL (1.6 mmol) of oxalyl chloride and 1 drop of N, N-dimethylformamide were added. The mixture was stirred at room temperature for 1.5 hours. The solvent of the reaction solution was distilled off under reduced pressure to obtain a pale yellow oily substance.
The obtained oil was dissolved in 3 mL of methylene chloride, and under ice cooling, 267 mg of white powder containing 2-nitrooxyethylamine nitrate produced in Synthesis Example 6 (6a) and 0.24 mL (3.2 mmol) of pyridine were added, The mixture was stirred at the same temperature for 1 hour. 1.0 M hydrochloric acid was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 1, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (46 mg) as a colorless oil.
The same reaction and treatment were carried out using 520 mg (1.56 mmol) of a slightly yellow powder containing 3-formylbenzoic acid produced in Synthesis Example 8 (8a) to obtain the pale yellow oil of the title compound. The pale yellow oil obtained and the colorless oil obtained above (46 mg) were combined and purified three times by column chromatography (ethyl acetate: n-hexane, 1: 4 → 2: 3, V / V). .. The solvent of the target fraction was distilled off under reduced pressure, 3 mL of tert-butyl methyl ether was added to the obtained residue, the mixture was irradiated with ultrasonic waves, and the insoluble material was collected by filtration to give 83 mg of the white powder of the title compound (2 steps, Yield 13%) was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.83-3.90 (2H, m), 4.69 (2H, t, J = 5.1 Hz), 6.63-6.73 (1H, br), 7.65 (1H, t, J = 7.6 Hz), 8.01-8.12 (2H, m), 8.27 (1H, s), 10.07 (1H, s).

Synthesis Example 9:
(9a) Methyl 2,3'-dimethylbiphenylyl-4-carboxylate

Dissolve 4.97 g (18.0 mmol) of methyl 4-iodo-3-methylbenzoate and 2.93 g (21.6 mmol) of 3-methylphenylboronic acid in 100 mL of 1,4-dioxane and add tetrakistriphenylphosphine palladium 1.04 g (0.90 (2.0 mmol) and 2.0 M aqueous potassium carbonate solution (27 mL, 54 mmol) were added, and the mixture was stirred at 100 ° C. for 5 hours under a nitrogen atmosphere. After cooling, ethyl acetate was added, and the mixture was washed successively with saturated aqueous ammonium chloride solution and saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (toluene: n-hexane, 1: 1, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (4.12 g, yield 95%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 2.31 (3H, s), 2.41 (3H, s), 3.93 (3H, s), 7.08-7.14 (2H, m), 7.18 (1H, d, J = 7.3 Hz), 7.24-7.35 (2H, m), 7.88 (1H, d, J = 7.8 Hz), 7.94 (1H, s).

(9b) Methyl 2,3'-bis (bromomethyl) biphenylyl-4-carboxylate

Methyl 2,3'-dimethylbiphenylyl-4-carboxylate 4.12g (17.1 mmol) produced in Synthesis Example 9 (9a) was dissolved in 100 mL of carbon tetrachloride, and N-bromosuccinimide 6.26g (35.1 mmol) and Azoisobutyronitrile 0.14 g (0.86 mmol) was added, and the mixture was heated under reflux for 8 hours. After cooling, the insoluble matter was filtered off, washed with ethyl acetate, and the solvent of the filtrate was evaporated under reduced pressure. The obtained residue was purified by column chromatography (toluene: n-hexane, 1: 1, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 4.40 g (crude product) of a white powder containing the title compound.

(9c) 2,3′-bis (bromomethyl) biphenylyl-4-ylmethanol

1.40 g of a white powder containing methyl 2,3'-bis (bromomethyl) biphenylyl-4-carboxylate produced in Synthesis Example 9 (9b) was dissolved in 15 mL of methylene chloride, and 1.0 M diisobutylaluminum hydride was prepared under ice cooling. Toluene solution 10.6 mL (11 mmol) was added dropwise, and the mixture was stirred at the same temperature for 1 hour. 30 mL of 1.0 M hydrochloric acid was added to the reaction solution, extracted twice with 20 mL of methylene chloride, the organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 4 → 1: 3, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 1.12 g (crude product) of a white powder containing the title compound.

(9d) 2,3’-Bis (bromomethyl) biphenylyl-4-carbaldehyde

1.12 g of white powder containing 2,3′-bis (bromomethyl) biphenylyl-4-ylmethanol produced in Synthesis Example 9 (9c) was dissolved in 15 mL of methylene chloride, and dimethylsulfoxide 1.08 mL (15.2 mmol) under ice cooling. ), N, N-diisopropylethylamine 2.65 mL (15.2 mmol) and sulfur trioxide-pyridine complex 1.21 g (7.58 mmol) were added, and the mixture was stirred at the same temperature for 0.5 hr. After adding 8 mL of 1.0 M hydrochloric acid to the reaction solution and extracting twice with 20 mL of methylene chloride, the organic layers were combined, washed successively with 1.0 M hydrochloric acid and water, dried (Na ₂ SO ₄ ) and the solvent was depressurized. I distill off. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 1: 4, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 692 mg (crude product) of a colorless oily substance containing the title compound.

(9) 2,3'-Bis (nitrooxymethyl) biphenylyl-4-carbaldehyde

A colorless oil 692 mg containing 2,3'-bis (bromomethyl) biphenylyl-4-carbaldehyde prepared in Synthesis Example 9 (9d) was dissolved in 10 mL of acetonitrile, and silver nitrate (I) 958 mg (5.64 mmol) was added. In addition, the mixture was stirred at room temperature for 1 hour. The insoluble matter was filtered off with Celite, washed with acetonitrile, and the solvent of the filter wash solution was distilled off under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). The solvent of the target fraction was distilled off under reduced pressure, 10 mL of n-hexane was added to the obtained residue, the solvent was distilled off under reduced pressure, and 421.1 mg of the colorless oil of the title compound (4 steps, yield 23%) Got
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 5.58 (2H, s), 5.65 (2H, s), 7.43-7.51 (1H, m), 7.53 (1H, s), 7.55-7.68 (3H , m), 8.04 (1H, d, J = 7.8 Hz), 8.14 (1H, s), 10.09 (1H, s).

Synthesis example 10:
(10a) 2-nitrooxy-1-nitrooxymethylethylamine nitrate

5.1 mL of fuming nitric acid was added dropwise to 35 mL of methylene chloride under ice cooling over 3 minutes, and the mixture was stirred at the same temperature for 10 minutes, and then 2-amino-1,3-propanediol 2.50 g (27.4 mmol) was added over 2 minutes. The mixture was added and stirred at the same temperature for 50 minutes. Acetic anhydride 7.8 mL (83 mmol) was added dropwise at the same temperature over 2 minutes, and the mixture was stirred at room temperature for 40 minutes and ice-cooled again. After dropping, the mixture was stirred at room temperature for 30 minutes. The insoluble matter was collected by filtration, washed twice with 10 mL of ethyl acetate, and then dried under reduced pressure at room temperature for 1 hour to obtain 4.19 g (yield 63%) of a white powder of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.99-4.07 (1H, m), 4.67-4.75 (2H, m), 4.79-4.86 (2H, m), 8.40-8.56 (3H, br) .

(10) 4-Formyl-N- (2-nitrooxy-1-nitrooxymethylethyl) benzamide

4-Formylbenzoic acid 492 mg (3.28 mmol) was suspended in methylene chloride 12 mL, and 2-nitrooxy-1-nitrooxymethylethylamine nitrate 800 mg (3.28 mmol) and 1-ethyl produced in Synthesis Example 10 (10a) were prepared. -3- (3-Dimethylaminopropyl) carbodiimide hydrochloride 942 mg (4.92 mmol) and N, N-diisopropylethylamine 0.56 mL (3.3 mmol) were added, and the mixture was stirred at room temperature for 15 minutes. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with 5% citric acid water, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 3, V / V). The solvent of the target fraction was distilled off under reduced pressure, 10 mL of tert-butyl methyl ether was added to the obtained residue, the mixture was irradiated with ultrasonic waves, and the insoluble matter was collected by filtration to give 413 mg of a white powder of the title compound (yield 40 %) Was obtained.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 4.63-4.85 (5H, m), 8.03 (4H, s), 8.94 (1H, d, J = 6.6 Hz), 10.09 (1H, s).

Synthesis Example 11:
(11a) 1- (2-bromoethyl) indoline-6-ylmethanol

3.33 g (22.3 mmol) of 6-hydroxymethylindoline was suspended in 21.8 mL of dibromoethane and 10.8 mL of triethylamine, and the mixture was stirred at 90 ° C for 40 minutes. After allowing to cool, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified by column chromatography (chloroform: methanol, 1: 0 → 9: 1, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 3.16 g (yield 55%) of a pale brown solid of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 2.99 (2H, t, J = 8.3 Hz), 3.42-3.56 (6H, m), 4.60 (2H, d, J = 4.4 Hz), 6.49 (1H, s), 6.64 (1H, d, J = 7.3 Hz), 7.04 (1H, d, J = 7.3 Hz).

(11b) 1- (2-Bromoethyl) indoline-6-carbaldehyde

1- (2-bromoethyl) indoline-6-ylmethanol 3.16 g (12.3 mmol) produced in Synthesis Example 11 (11a) was dissolved in 129 mL of methylene chloride, and under ice cooling, dimethyl sulfoxide 8.24 mL (116 mmol), 10.0 mL (58.2 mmol) of N, N-diisopropylethylamine and 9.26 g (58.2 mmol) of sulfur trioxide-pyridine complex were added, and the mixture was stirred at the same temperature for 25 minutes. 100 mL of water and 100 mL of ethyl acetate were added to the reaction solution, and the mixture was concentrated under reduced pressure. After distilling methylene chloride off, the two layers were separated. The aqueous layer was extracted twice with 100 mL of ethyl acetate, the organic layers were combined, washed twice with 100 mL of water and once with saturated brine, dried (Na ₂ SO ₄ ) and the solvent was removed under reduced pressure. Distilled off. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 2: 3, + 3% chloroform, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 2.91 g (yield 93%) of a yellow oily substance of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.09 (2H, t, J = 8.3 Hz), 3.48-3.61 (6H, m), 6.90 (1H, s), 7.15 (1H, d, J = 7.3 Hz), 7.20 (1H, d, J = 7.3 Hz), 9.86 (1H, s).

(11) 1- (2-nitrooxyethyl) indoline-6-carbaldehyde

1- (2-Bromoethyl) indoline-6-carbaldehyde 2.91 g (11.5 mmol) produced in Synthesis Example 11 (11b) was dissolved in 23 mL of acetonitrile, and silver nitrate (I) 2.94 g (17.3 mmol) was added thereto, and the mixture was stirred at room temperature. It was stirred for 137 minutes. 100 mL of ethyl acetate was added to the reaction solution, and the mixture was stirred for 10 minutes, then the insoluble matter was filtered off with Celite, and washed twice with 50 mL of ethyl acetate. The solvent of the filter wash was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 2: 3, + 3% chloroform, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 2.49 g of a blackish brown oily matter. 2.49 g of the obtained blackish brown oily matter was purified again by column chromatography (ethyl acetate: n-hexane, 1: 9 → 2: 3, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (2.33 g, yield 86%) as a dark brown oil.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ (ppm); 3.01 (2H, t, J = 8.3 Hz), 3.47-3.57 (4H, m), 4.75 (2H, t, J = 5.1 Hz) , 6.93 (1H, s), 7.18 (1H, d, J = 7.3 Hz), 7.25 (1H, d, J = 7.3 Hz), 9.83 (1H, s).

Synthesis Example 12:
(12a) 3- (2-bromoethoxy) benzaldehyde

2.44 g (20.0 mmol) of 3-hydroxybenzaldehyde was dissolved in 25 mL of acetonitrile, 10 mL (0.12 mol) of dibromoethane and 6.90 g (49.9 mmol) of potassium carbonate were added, and the mixture was heated under reflux for 5 hours. After allowing to cool, ethyl acetate and 150 mL of water were added, the two layers were separated, and the aqueous layer was extracted twice with 100 mL of ethyl acetate. The organic layers were combined, washed successively with 150 mL of water and 150 mL of saturated brine, dried (Na ₂ SO ₄ ), the solvent was evaporated under reduced pressure, and the obtained residue was subjected to column chromatography (ethyl acetate: n -Hexane, 0: 1 → 1: 1, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 3.60 g (yield 76%) of a pale yellow oily substance of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.67 (2H, t, J = 6.1 Hz), 4.36 (2H, t, J = 6.1 Hz), 7.18-7.23 (1H, m), 7.37-7.40 ( 1H, m), 7.43-7.51 (2H, m), 9.97 (1H, s).

(12) 3- (2-Nitrooxyethoxy) benzaldehyde

3- (2-Bromoethoxy) benzaldehyde 3.60 g (15.7 mmol) produced in Synthesis Example 12 (12a) was dissolved in 30 mL of acetonitrile, and silver nitrate (I) 3.86 g (22.7 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. The mixture was stirred at 60 ° C for 18 hours. After allowing to cool, 50 mL of ethyl acetate was added to the reaction solution, and after stirring for 20 minutes, the insoluble material was filtered off with Celite and washed twice with 100 mL of ethyl acetate. The solvent of the filtrate was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 1, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (2.44 g, yield 76%) as a pale-yellow oil.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 4.38-4.44 (2H, m), 4.88-4.94 (2H, m), 7.28-7.35 (1H, m), 7.44-7.48 (1H, m) , 7.52-7.57 (2H, m), 9.98 (1H, s).

Synthesis Example 13:
(13a) 3- (2-bromoethylsulfanyl) -N-methoxy-N-methylbenzamide

3-Mercaptobenzoic acid 500 mg (3.24 mmol) was dissolved in tetrahydrofuran 10 mL, dibromoethane 2.79 mL (32.4 mmol) and triethylamine 0.54 mL (3.9 mmol) were added, and the mixture was stirred at room temperature for 16 hours. 5% aqueous citric acid was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and a white powder 1.17 g was obtained. 1.17 g of the obtained white powder was suspended in 10 mL of methylene chloride, N, O-dimethylhydroxylamine hydrochloride 320 mg (3.28 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 785 mg. (4.10 mmol) and triethylamine 0.45 mL (3.3 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Water was added to the reaction solution, the mixture was extracted twice with ethyl acetate, the organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 3: 7, V / V). The solvent of the target fraction was evaporated under reduced pressure to give 730 mg (2 steps, yield 74%) of the title compound as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.29-3.39 (5H, m), 3.42-3.50 (2H, m), 3.56 (3H, s), 7.36 (1H, t, J = 7.6 Hz), 7.47 (1H, d, J = 7.6 Hz), 7.55 (1H, d, J = 7.6 Hz), 7.69 (1H, s).

(13b) 3- (2-Bromoethylsulfanyl) benzaldehyde

730 mg (2.40 mmol) of 3- (2-bromoethylsulfanyl) -N-methoxy-N-methylbenzamide produced in Synthesis Example 13 (13a) was dissolved in 8 mL of tetrahydrofuran, and 1.0 M diisobutylaluminum hydride was cooled with ice. 2.9 mL (2.9 mmol) of the toluene solution of was added dropwise, and the mixture was stirred at the same temperature for 1 hour. 1.0 M Hydrochloric acid was added to the reaction mixture, the mixture was extracted twice with ethyl acetate, the organic layers were combined, washed with 50 mL of saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 9, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (430 mg, yield 73%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.33-3.41 (2H, m), 3.44-3.52 (2H, m), 7.50 (1H, t, J = 7.6 Hz), 7.62 (1H, d, J = 7.6 Hz), 7.74 (1H, d, J = 7.6 Hz), 7.87 (1H, s), 9.99 (1H, s).

(13) 3- (2-Nitrooxyethylsulfanyl) benzaldehyde

430 mg (1.75 mmol) of 3- (2-bromoethylsulfanyl) benzaldehyde produced in Synthesis Example 13 (13b) was dissolved in 6 mL of acetonitrile, 894 mg (5.26 mmol) of silver nitrate (I) was added, and the mixture was stirred at room temperature for 1 hour. Stir for hours. The insoluble matter was filtered off with Celite, washed with ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 9, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (324 mg, yield 81%) as a colorless oil.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.45 (2H, t, J = 6.6 Hz), 4.69 (2H, t, J = 6.6 Hz), 7.57 (1H, t, J = 7.8 Hz) , 7.71-7.78 (2H, m), 7.91 (1H, s), 9.99 (1H, s).

Synthesis Example 14:
(14a) 3-methylaminobenzyl alcohol

40 mL of formic acid was added to 3.00 g (24.4 mmol) of 3-aminobenzyl alcohol, and the mixture was heated under reflux for 2 hours. The reaction mixture was concentrated under reduced pressure, ethyl acetate was added to the obtained residue to dilute it, and the mixture was washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain 4.63 g of a brown oily substance.
The obtained brown oily substance 4.63 g was dissolved in N, N-dimethylformamide 50 mL, cesium carbonate 11.9 g (36.5 mmol) and methyl iodide 1.8 mL (29 mmol) were added, and the mixture was stirred at the same temperature for 18.5 hours. .. Water was added to the reaction solution, the mixture was extracted twice with ethyl acetate, the organic layers were combined, washed successively with water and saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain 2.75 g of a yellow oily substance.
Methanol 14 mL and 5.0 M sodium hydroxide aqueous solution 13.8 mL (69 mmol) were added to the obtained yellow oily substance 2.75 g, and it heated and refluxed for 4 hours. The reaction solution was allowed to cool, 30 mL of saturated brine was added, and the mixture was extracted 3 times with chloroform. The organic layers were combined, dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure to give a brown oil of the title compound (1.84 g, 3 steps, yield 55%).
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.55-1.65 (1H, br), 2.85 (3H, s), 3.63-3.85 (1H, br), 4.60-4.65 (2H, br), 6.52-6.57 (1H, m), 6.61-6.65 (1H, m), 6.66-6.72 (1H, m), 7.17 (1H, t, J = 7.6 Hz).

(14b) {3- [Methyl (2-bromoethyl) amino] phenyl} methanol

To 920 mg (6.70 mmol) of 3-methylaminobenzyl alcohol produced in Synthesis Example 14 (14a), 6.7 mL of 1,2-dibromoethane and 3.35 mL of triethylamine were added, and the mixture was stirred at 90 ° C for 1.5 hours. After allowing to cool, the reaction solution was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 3, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (1.29 g, yield 57%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.55-1.65 (1H, br), 3.02 (3H, s), 3.46 (2H, t, J = 7.6 Hz), 3.75 (2H, t, J = 7.6 Hz), 4.65 (2H, s), 6.61-6.67 (1H, m), 6.70-6.76 (2H, m), 7.20-7.26 (1H, m).

(14c) 3- [Methyl (2-bromoethyl) amino] benzaldehyde

{3- [Methyl (2-bromoethyl) amino] phenyl} methanol (1.29 g, 5.20 mmol) produced in Synthesis Example 14 (14b) was dissolved in 26 mL of methylene chloride, and 2.35 mL (31.7 mmol) of dimethyl sulfoxide under ice cooling. ), N, N-diisopropylethylamine 2.74 mL (15.9 mmol) and sulfur trioxide-pyridine complex 2.52 g (15.9 mmol) were added, and the mixture was stirred at the same temperature for 0.5 hr. The reaction solution was washed with water, dried (Na ₂ SO ₄ ) and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (1.10 g, yield 87%) as a yellow oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.08 (3H, s), 3.48 (2H, t, J = 7.3 Hz), 3.80 (2H, t, J = 7.3 Hz), 6.94-7.00 (1H, m), 7.16-7.27 (2H, m), 7.40 (1H, t, J = 7.8 Hz), 9.95 (1H, s).

(14) 3- [Methyl (2-nitrooxyethyl) amino] benzaldehyde

1.10 g (4.54 mmol) of 3- [methyl (2-bromoethyl) amino] benzaldehyde produced in Synthesis Example 14 (14c) was dissolved in 9 mL of acetonitrile, and 1.16 g (6.82 mmol) of silver nitrate (I) was added, and the mixture was warmed to room temperature. And stirred for 14 hours. Ethyl acetate was added to the reaction solution, the insoluble matter was filtered off with Celite, washed with ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 3: 7, V / V), and the solvent of the target fraction was evaporated under reduced pressure to give the title compound as a yellow oil. 921 mg (yield 90%) of the product was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.07 (3H, s), 3.76 (2H, t, J = 5.6 Hz), 4.64 (2H, t, J = 5.6 Hz), 6.95-7.02 (1H, m), 7.16-7.28 (2H, m), 7.42 (1H, t, J = 7.8 Hz), 9.95 (1H, s).

Synthesis Example 15:
(15a) N- (2-bromoethyl) -4-formylbenzenesulfonamide

4-Chlorosulfonylbenzonitrile (2.44 g, 11.9 mmol) was suspended in methylene chloride (30 mL), and triethylamine (3.5 mL, 25 mmol) and bromoethylamine hydrobromide (2.00 g, 9.92 mmol) were added under ice-cooling. Stir at temperature for 1 hour. Water was added to the reaction solution, extracted twice with chloroform, the organic layers were combined, dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure to give a pale yellow powder. 3.70 g was obtained.
3.70 g of the obtained pale yellow powder was suspended in 40 mL of methylene chloride, 22 mL (22 mmol) of a 1.0 M solution of diisobutylaluminum hydride in toluene was slowly added while cooling with ice, and the mixture was stirred at room temperature for 5 hours. To the reaction solution, 11 mL (11 mmol) of a 1.0 M solution of diisobutylaluminum hydride in toluene was slowly added, and after stirring at room temperature for 1.5 hours, 20 mL of 2.0 M hydrochloric acid was slowly added under ice cooling and extracted twice with chloroform. .. The organic layers were combined and dried (Na ₂ SO ₄ ), then the solvent was evaporated under reduced pressure, and the obtained residue was subjected to column chromatography (ethyl acetate: n-hexane, 1: 4 → 2: 3, V / V). Purified in. The solvent of the target fraction was evaporated under reduced pressure to obtain 1.22 g (2 steps, yield 42%) of the title compound as white powder.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.40-3.49 (4H, m), 5.00-5.14 (1H, br), 8.05 (4H, s), 10.11 (1H, s).

(15) 4-Formyl-N- (2-nitrooxyethyl) benzenesulfonamide

N- (2-bromoethyl) -4-formylbenzenesulfonamide 600 mg (2.05 mmol) produced in Synthesis Example 15 (15a) was dissolved in acetonitrile 4 mL, and silver nitrate (I) 523 mg (3.08 mmol) was added, After stirring for 11.5 hours at room temperature, 523 mg (3.08 mmol) of silver nitrate (I) was added, stirred for 2 hours at 60 ° C., 523 mg (3.08 mmol) of silver nitrate (I) was added again, and at the same temperature for 0.5 hours. It was stirred. The reaction solution was allowed to cool, then the insoluble material was filtered off with Celite, washed with ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 3: 7 → 1: 1, V / V). The solvent of the target fraction was distilled off under reduced pressure, 4 mL of tert-butyl methyl ether was added to the obtained residue, the mixture was irradiated with ultrasonic waves, and the insoluble matter was collected by filtration to give 427 mg of a white powder of the title compound (yield 76 %) Was obtained.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 3.15-3.23 (2H, m), 4.49 (2H, t, J = 5.1 Hz), 7.97-8.03 (2H, m), 8.09-8.14 (2H , m), 8.22-8.29 (1H, br), 10.10 (1H, s).

Synthesis example 16:
(16) 4'-formyl-N- (2-nitrooxyethyl) biphenylyl-4-carboxamide

4-Formylphenylboronic acid 2.00 g (13.3 mmol) and 4-bromobenzoic acid 1.79 g (8.90 mmol) were suspended in 1,4-dioxane 30 mL, and 2.0 M potassium carbonate aqueous solution 13 mL (26 mmol) and tetrakistri were added. Phenylphosphine palladium (514 mg, 0.445 mmol) was added, and the mixture was stirred at 100 ° C for 2 hours. After allowing to cool, 30 mL of 2.0 M hydrochloric acid was added to the reaction solution, the mixture was stirred at room temperature for 5 minutes, 30 mL of ethyl acetate was added, and the mixture was further stirred for 5 minutes. The insoluble matter was collected by filtration and washed twice with 10 mL of ethyl acetate to obtain 2.98 g of gray powder.
415 mg of the obtained gray powder was suspended in 6 mL of methylene chloride, and 465 mg of white powder containing 2-nitrooxyethylamine nitrate produced in Synthesis Example 6 (6a), 1-ethyl-3- (3-dimethylaminopropyl). ) Carbodiimide hydrochloride 527 mg (2.75 mmol) and triethylamine 0.38 mL (2.8 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Water was added to the reaction solution, extracted twice with chloroform, the organic layers were combined, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (chloroform: methanol, 1: 0 → 19: 1, V / V). The solvent of the target fraction was evaporated under reduced pressure, 10 mL of tert-butyl methyl ether-n-hexane (2: 1) was added to the obtained residue, and the mixture was irradiated with ultrasonic waves, and the insoluble material was collected by filtration to give the title compound As a result, 133 mg of white powder of (2 steps, yield 23%) was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.82-3.92 (2H, m), 4.69 (2H, t, J = 4.9 Hz), 6.44-6.54 (1H, br), 7.69-7.81 (4H, m ), 7.85-7.91 (2H, m), 7.95-8.01 (2H, m), 10.07 (1H, s).

Synthesis Example 17:
(17a) 3 '-(2-bromoethoxy) biphenylyl-4-carbaldehyde

4-Bromobenzaldehyde (1.00 g (5.40 mmol)) and 3-hydroxyphenylboronic acid (894 mg (6.48 mmol)) were suspended in 1,4-dioxane (20 mL), and 2.0 M potassium carbonate solution (8.1 mL (16 mmol)) and tetrakistriphenylphosphine were suspended. Palladium (312 mg, 0.270 mmol) was added, and the mixture was stirred at 100 ° C for 2 hr. After allowing to cool, water and ethyl acetate were added to the reaction solution, and the insoluble material was filtered off with Celite. The mixture was extracted twice with ethyl acetate, the organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 1, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 1.22 g of a pale yellow oily substance.
The obtained pale yellow oily substance 1.22 g was suspended in acetonitrile 5.4 mL, potassium carbonate 1.87 g (13.5 mmol) and dibromoethane 4.65 mL (54.0 mmol) were added, and the mixture was heated under reflux for 14 hr. After allowing to cool, water was added to the reaction solution, extracted twice with chloroform, the organic layers were combined and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (1.23 g, two steps, yield 75%) as a pale-yellow oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.68 (2H, t, J = 6.4 Hz), 4.37 (2H, t, J = 6.4 Hz), 6.93-7.00 (1H, m), 7.16-7.20 ( 1H, m), 7.22-7.30 (1H, m), 7.40 (1H, t, J = 7.8 Hz), 7.71-7.77 (2H, m), 7.92-7.98 (2H, m), 10.06 (1H, s)

(17) 3 '-(2-nitrooxyethoxy) biphenylyl-4-carbaldehyde

1.23 g (4.03 mmol) of 3 '-(2-bromoethoxy) biphenylyl-4-carbaldehyde produced in Synthesis Example 17 (17a) was dissolved in 20 mL of acetonitrile, and 1.81 g (10.7 mmol) of silver nitrate (I) was added. After stirring at room temperature for 95 minutes, 1.81 g (10.7 mmol) of silver (I) nitrate was added, and the mixture was stirred at 60 ° C for 17 hours. The insoluble matter was filtered off with Celite, washed with ethyl acetate, and the solvent of the filter wash solution was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 3: 7, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (1.01 g, yield 99%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 4.31-4.36 (2H, m), 4.83-4.89 (2H, m), 6.92-6.98 (1H, m), 7.14-7.19 (1H, m), 7.22 -7.30 (1H, m), 7.41 (1H, t, J = 8.0 Hz), 7.70-7.77 (2H, m), 7.92-7.99 (2H, m), 10.06 (1H, s).

Synthesis Example 18:
(18a) 1- (3-bromopropyl) indoline-6-ylmethanol

1.44-g (9.65 mmol) of 6-hydroxymethylindoline was suspended in 10.7 mL (105 mmol) of dibromopropane and 5.1 mL (37 mmol) of triethylamine, and stirred at 90 ° C for 25 minutes. After allowing to cool, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified by column chromatography (chloroform: methanol, 1: 0 → 19: 1, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (1.01 g, yield 39%) as a pale-yellow oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 2.15 (2H, quintet, J = 6.3 Hz), 2.96 (2H, t, J = 8.3 Hz), 3.25 (2H, t, J = 6.3 Hz), 3.37 (2H, t, J = 8.3 Hz), 3.53 (2H, t, J = 6.3 Hz), 4.60 (2H, d, J = 5.9 Hz), 6.53 (1H, s), 6.64 (1H, d, J = 7.3 Hz), 7.04 (1H, d, J = 7.3 Hz).

(18b) 1- (3-Bromopropyl) indoline-6-carbaldehyde

1.01 g (3.74 mmol) of 1- (3-bromopropyl) indoline-6-ylmethanol produced in Synthesis Example 18 (18a) was dissolved in 25 mL of methylene chloride, and 1.6 mL (22.4 mmol) of dimethyl sulfoxide was cooled under ice cooling. , N, N-diisopropylethylamine 1.93 mL (11.2 mmol) and sulfur trioxide-pyridine complex 1.79 g (11.2 mmol) were added, and the mixture was stirred at the same temperature for 25 minutes. Water and ethyl acetate were added to the reaction solution, the mixture was concentrated under reduced pressure, methylene chloride was distilled off, and the two layers were separated. The aqueous layer was extracted twice with ethyl acetate, the organic layers were combined, washed twice with water and once with saturated brine, dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 870 mg (yield 87%) of a yellow oily substance of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 2.17 (2H, quintet, J = 6.8 Hz), 3.05 (2H, t, J = 8.6 Hz), 3.31 (2H, t, J = 6.8 Hz), 3.47 (2H, t, J = 8.6 Hz), 3.52 (2H, t, J = 6.8 Hz), 6.93 (1H, s), 7.14 (1H, d, J = 7.3 Hz), 7.19 (1H, d, J = 7.3 Hz), 9.87 (1H, s).

(18) 1- (3-Nitrooxypropyl) indoline-6-carbaldehyde

870 mg (3.24 mmol) of 1- (3-bromopropyl) indoline-6-carbaldehyde produced in Synthesis Example 18 (18b) was dissolved in 11 mL of acetonitrile, and 1.65 g (9.73 mmol) of silver nitrate (I) was added, The mixture was stirred at room temperature for 30 minutes and at 60 ° C for 30 minutes. The insoluble matter was filtered off with Celite and washed twice with 50 mL of ethyl acetate. The solvent of the filter wash solution was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 639 mg (yield 79%) of a dark green oily substance of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 1.90-2.02 (2H, m), 2.93-3.03 (2H, m), 3.16-3.47 (4H, m), 4.56-4.65 (2H, m) , 6.88 (1H, s), 7.12-7.28 (2H, m), 9.83 (1H, s).

Synthesis Example 19:
(19a) 2- (3-hydroxymethylphenoxy) malonate diethyl

3-Hydroxybenzyl alcohol 5.00 g (40.3 mmol) was dissolved in acetone 100 mL, potassium carbonate 8.35 g (60.4 mmol) and diethyl bromomalonate 10.2 mL (60.6 mmol) were added, and the mixture was heated under reflux for 3 hours. After allowing to cool, 120 mL of 2.0 M hydrochloric acid was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 6.27 g (yield 55%) of a yellow oily substance of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.31 (6H, t, J = 7.1 Hz), 1.68-1.78 (1H, br), 4.31 (4H, q, J = 7.1 Hz), 4.67 (2H, d, J = 4.2 Hz), 5.22 (1H, s), 6.84-6.90 (1H, dd, J = 8.3, 2.2 Hz), 6.97-7.07 (2H, m), 7.27-7.31 (1H, m).

(19b) 2- (3-tert-Butyldimethylsilyloxymethylphenoxy) diethyl malonate

Diethyl 2- (3-hydroxymethylphenoxy) malonate 6.27 g (22.2 mmol) produced in Synthesis Example 19 (19a) was dissolved in N, N-dimethylformamide 60 mL, and imidazole 1.81 g (26.6 mmol) and tert- Butyldimethylsilyl chloride (3.68 g, 24.4 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate, the organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (6.27 g, yield 87%) as a yellow oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 0.10 (6H, s), 0.94 (9H, s), 1.30 (6H, t, J = 7.1 Hz), 4.26-4.36 (4H, m), 4.70 ( 2H, s), 5.20 (1H, s), 6.80-6.86 (1H, m), 6.94-7.01 (2H, m), 7.20-7.25 (1H, m).

(19c) 2- (3-tert-Butyldimethylsilyloxymethylphenoxy) propane-1,3-diol

Lithium aluminum hydride 1.83 g (48.2 mmol) was suspended in 70 mL of diethyl ether, and diethyl 2- (3-tert-butyldimethylsilyloxymethylphenoxy) malonate produced in Synthesis Example 19 (19b) 7.65 g (19.3 mmol). 10 mL of diethyl ether solution was added dropwise over 2 minutes, and the mixture was stirred at room temperature for 1 hour. Water (2 mL), 5.0 M aqueous sodium hydroxide solution (2 mL) and water (6 mL) were sequentially added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. The insoluble matter was filtered off with Celite, the solvent of the filtrate was evaporated under reduced pressure, and the obtained residue was subjected to column chromatography (ethyl acetate: n-hexane, 1: 4 → 7: 3, V / V). Purified. The solvent of the target fraction was evaporated under reduced pressure to give the title compound (4.08 g, yield 68%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 0.11 (6H, s), 0.95 (9H, s), 1.98 (2H, t, J = 6.1 Hz), 3.85-3.98 (4H, m), 4.44 ( 1H, quintet, J = 4.6 Hz), 4.71 (2H, s), 6.82-6.89 (1H, m), 6.90-6.95 (1H, m), 6.95-7.00 (1H, m), 7.20-7.25 (1H, m)

(19d) 1- (2-Bromo-1-bromomethylethoxy) -3-bromomethylbenzene

2- (3-tert-butyldimethylsilyloxymethylphenoxy) propane-1,3-diol 4.08 g (13.1 mmol) produced in Synthesis Example 19 (19c) was dissolved in 70 mL of methylene chloride, and triphenyl was dissolved under ice cooling. Phosphine (1.20 g, 4.57 mmol) and carbon tetrabromide (1.52 g, 4.57 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The solvent of the reaction solution was evaporated under reduced pressure, diethyl ether was added to the residue, the insoluble material was filtered off, and the solvent of the filtrate was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 1: 4, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (1.70 g, yield 30%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.69 (4H, d, J = 5.1 Hz), 4.45 (2H, s), 4.59 (1H, quintet, J = 5.1 Hz), 6.85-6.94 (1H, m), 6.95-7.03 (1H, m), 7.04-7.11 (1H, m), 7.22-7.33 (1H, m).

(19e) 3- (2-Bromo-1-bromomethylethoxy) benzaldehyde

1- (2-bromo-1-bromomethylethoxy) -3-bromomethylbenzene 1.70 g (4.39 mmol) produced in Synthesis Example 19 (19d) was dissolved in dimethyl sulfoxide, and sodium hydrogencarbonate 738 mg (8.78 mmol) Was added and the mixture was stirred at 80 ° C. for 1 hour. After allowing to cool, water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 1: 9, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (320 mg, yield 23%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.71 (4H, d, J = 5.1 Hz), 4.69 (1H, quintet, J = 5.1 Hz), 7.25-7.30 (1H, m), 7.44-7.58 ( 3H, m), 9.98 (1H, s).

(19) 3- (2-Nitrooxy-1-nitrooxymethylethoxy) benzaldehyde

3- (2-bromo-1-bromomethylethoxy) benzaldehyde 320 mg (0.994 mmol) produced in Synthesis Example 19 (19e) was dissolved in acetonitrile 3 mL, and silver nitrate (I) 507 mg (3.00 mmol) was added, The mixture was stirred at room temperature for 1 hour, 60 ° C. for 24 hours, and room temperature for 20 hours. To the reaction solution, 1.01 g (5.95 mmol) of silver nitrate (I) and 3 mL of acetonitrile were added, and after stirring at 80 ° C for 4 hours, 507 mg (3.00 mmol) of silver nitrate (I) was added and 3.5 at 80 ° C. Stir for hours. After cooling, the insoluble matter was filtered off with Celite, and the solvent of the filtrate was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 1: 9, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound as a colorless oil (238 mg, yield 76%).
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 4.84 (2H, dd, J = 12.2, 5.8 Hz), 4.94 (2H, dd, J = 12.2, 3.4 Hz), 5.20-5.28 (1H, m ), 7.35-7.42 (1H, m), 7.53-7.62 (3H, m), 9.98 (1H, s).

Synthesis Example 20:
(20a) (E) -3- (3- [1,3] dioxolan-2-ylphenyl) methyl acrylate

3-Bromobenzaldehyde 4.00 g (21.6 mmol) was dissolved in toluene 80 mL, ethylene glycol 6.03 mL (108 mmol) and p-toluenesulfonic acid monohydrate 186 mg (0.978 mmol) were added, and the mixture was heated under reflux for 4 hours. .. After allowing to cool, the reaction solution was washed once with 80 mL of saturated aqueous sodium hydrogen carbonate and 80 mL of water and twice with 80 mL of saturated saline solution, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure at 50-60 ° C to obtain 5.48 g of a colorless oily substance (crude product).
The obtained colorless oil (5.48 g) was dissolved in N, N-dimethylformamide (86 mL), and methyl acrylate (2.52 mL, 28.1 mmol), sodium bicarbonate (3.63 g, 43.2 mmol) and tri (o-tolyl) phosphine, 263 mg (0.864) were dissolved. mmol) and dichlorobis (triphenylphosphine) palladium (II) 303 mg (0.432 mmol) were sequentially added, and after nitrogen substitution, the mixture was stirred at 120 ° C. for 2 hours and at 150 ° C. for 1.5 hours. After allowing to cool, 150 mL of toluene and 150 mL of water were added to the reaction solution, the two layers were separated, and the aqueous layer was extracted twice with 50 mL of ethyl acetate. The organic layers were combined, washed successively with 100 mL of water three times and 100 mL of saturated saline once, then dried (Na ₂ SO ₄ ), and the solvent was distilled off under reduced pressure. It was purified by chromatography (ethyl acetate: n-hexane, 1: 19 → 1: 3, V / V). The solvent of the target fraction was evaporated under reduced pressure to obtain 4.52 g (2 steps, yield 89%) of a pale yellow solid of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 3.81 (3H, s), 4.01-4.18 (4H, m), 5.82 (1H, s), 6.47 (1H, d, J = 16.1 Hz), 7.37- 7.44 (1H, m), 7.46-7.58 (2H, m), 7.65 (1H, s), 7.70 (1H, d, J = 16.1 Hz).

(20b) (E) -3- (3-Hydroxypropen-1-yl) benzaldehyde

4.52 g (19.3 mmol) of methyl (E) -3- (3- [1,3] dioxolan-2-ylphenyl) acrylate produced in Synthetic Example 20 (20a) was suspended in 39 mL of toluene and cooled under ice-cooling. , 1.0 M diisobutylaluminum hydride-toluene solution 48.3 mL (48 mmol) was added dropwise over 17 minutes, and the mixture was stirred at the same temperature for 30 minutes, and then 1.0 M diisobutylaluminum hydride-toluene solution 20.0 mL (20 mmol) was added to 6 mL. The mixture was added dropwise over minutes and stirred at the same temperature for 30 minutes. 10 mL of ethyl acetate was added dropwise to the reaction solution, 100 mL of saturated aqueous sodium potassium tartrate solution was slowly added, and the mixture was stirred at room temperature for 13 hours. After separating the two layers, the aqueous layer was extracted twice with 100 mL of ethyl acetate, the organic layers were combined, washed successively with 100 mL of water and 100 mL of saturated brine, dried (Na ₂ SO ₄ ) and the solvent. Was distilled off under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 3: 2, V / V), and the solvent of the target fraction was evaporated under reduced pressure to give a yellow oil (2.36 g) Got
The obtained yellow oil (2.36 g) was dissolved in tetrahydrofuran (28.5 mL) and water (28.5 mL), p-toluenesulfonic acid monohydrate (217 mg, 1.14 mmol) was added, and the mixture was heated under reflux for 45 min. After allowing to cool, 50 mL of saturated aqueous sodium hydrogen carbonate was added to the reaction solution, the two layers were separated, and the aqueous layer was extracted twice with 50 mL of ethyl acetate. The organic layers were combined, washed successively with 50 mL of water twice and 100 mL of saturated brine once, then dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure to give a pale yellow oil containing the title compound. The product (1.76 g, crude product) was obtained.

(20c) (E) -3- (3-Chloropropen-1-yl) benzaldehyde

1.44 g of (E) -3- (3-hydroxypropen-1-yl) benzaldehyde produced in Synthesis Example 20 (20b) was dissolved in 27 mL of tetrahydrofuran, and 1.88 g (44.4 mmol) of lithium chloride and 1.36 mL of triethylamine (9.77). mmol) and methanesulfonyl chloride 0.76 mL (9.8 mmol) were added, and the mixture was stirred at room temperature for 65 minutes. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 3: 17, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound (890 mg, 3 steps, yield 29%) as a colorless oil.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 4.27 (2H, d, J = 7.1 Hz), 6.43 (1H, dt, J = 15.6, 7.1 Hz), 6.72 (1H, d, J = 15.6 Hz) , 7.51 (1H, t, J = 7.6 Hz), 7.65 (1H, d, J = 7.6 Hz), 7.78 (1H, d, J = 7.6 Hz), 7.90 (1H, s), 10.02 (1H, s) .

(20) (E) -3- (3-Nitrooxypropen-1-yl) benzaldehyde

890 mg (4.93 mmol) of (E) -3- (3-chloropropen-1-yl) benzaldehyde produced in Synthesis Example 20 (20c) was dissolved in 10 mL of acetonitrile, and silver nitrate (I) (I) 1.26 g ( 7.39 mmol) was added and the mixture was stirred at 80 ° C. for 20 minutes. After cooling, the insoluble matter was filtered off with Celite, and the solvent of the filtrate was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 3: 17, V / V). The solvent of the target fraction was evaporated under reduced pressure to give the title compound as a colorless oil (980 mg, yield 96%).
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 5.24 (2H, d, J = 6.6 Hz), 6.55 (1H, dt, J = 15.8, 6.6 Hz), 6.97 (1H, d, J = 15.8) Hz), 7.61 (1H, t, J = 7.6 Hz), 7.82-7.90 (2H, m), 8.05 (1H, s), 10.02 (1H, s).

Synthesis example 21:
(21a) Methyl 3-bromo-5-hydroxymethylbenzoate

Dissolve 5.00 g (18.3 mmol) of dimethyl 5-bromoisophthalate in 18 mL of tetrahydrofuran, add 762 mg (20.1 mmol) of sodium borohydride under ice cooling, stir at the same temperature for 1 minute, and then with 1.8 mL of methanol. A mixed solvent of tetrahydrofuran 6.9 mL was added, and the mixture was further stirred at the same temperature for 30 minutes. Water and 1.0 M hydrochloric acid were added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed successively with water and saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, the resulting residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 2: 3, V / V), and the solvent of the target fraction was distilled off under reduced pressure. Then, 3.24 g (yield 72%) of a colorless oily substance of the title compound was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.90 (1H, t, J = 5.9 Hz), 3.93 (3H, s), 4.74 (2H, d, J = 5.8 Hz), 7.73 (1H, s), 7.95 (1H, s), 8.08 (1H , s).

(21b) Methyl 3-hydroxymethyl-5-vinylbenzoate

Methyl 3-bromo-5-hydroxymethylbenzoate 3.24 g (13.2 mmol) produced in Synthesis Example 21 (21a) was dissolved in 1,4-dioxane 45 mL, potassium vinyltrifluoroborate 3.54 g (26.4 mmol), [1,1'-Bis (diphenylphosphino) ferrocene] palladium (II) dichloride Methylene chloride adduct 1.08 g (1.32 mmol) and triethylamine 5.5 mL (40 mmol) were added, and under nitrogen atmosphere, at 90 ° C for 13 hours. It was stirred. After allowing to cool, the insoluble matter was filtered off with Celite, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 1, V / V), and the solvent of the target fraction was evaporated under reduced pressure to give the title compound as a light brown color. An oily substance 2.32 g (yield 91%) was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.81 (1H, t, J = 5.9 Hz), 3.93 (3H, s), 4.76 (2H, d, J = 5.9 Hz), 5.34 (1H, d, J = 11.0 Hz), 5.85 (1H, d , J = 17.6 Hz), 6.75 (1H, dd, J = 17.6, 11.0 Hz), 7.62 (1H, s), 7.92 (1H, s), 8.00 (1H, s).

(21c) 3-tert-Butyldimethylsilyloxymethyl-5-vinylmethyl benzoate

2.32 g (12.1 mmol) of methyl 3-hydroxymethyl-5-vinylbenzoate prepared in Synthesis Example 21 (21b) was dissolved in 40 mL of N, N-dimethylformamide, and 1.23 g (18.1 mmol) of imidazole and tert-butyl were dissolved. 2.18 g (14.5 mmol) of dimethylsilyl chloride was added, and the mixture was stirred at room temperature for 50 minutes. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed successively with water and saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 4, V / V), and the solvent of the target fraction was distilled off under reduced pressure. Then, 3.53 g (yield 95%) of a pale yellow oily substance of the title compound was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 0.11 (6H, s), 0.96 (9H, s), 3.92 (3H, s), 4.77 (2H, s), 5.32 (1H, d, J = 11.0 Hz), 5.82 (1H, d, J = 17.6 Hz), 6.75 (1H, dd, J = 17.6, 11.0 Hz), 7.59 (1H, s), 7.86 (1H, s), 7.95 (1H, s).

(21d) 3-tert-Butyldimethylsilyloxymethyl-5-formyl benzoate methyl

Dissolve 1.26 g (4.11 mmol) of methyl 3-tert-butyldimethylsilyloxymethyl-5-vinylbenzoate produced in Synthesis Example 21 (21c) in 40 mL of 1,4-dioxane-water (3: 1), 2,6-lutidine 0.96 mL (8.2 mmol), microencapsulated osmium (VIII) oxide (purity: 10%) 300 mg (0.12 mmol) and sodium periodate 3.52 g (16.4 mmol) were added, and the mixture was allowed to stand at room temperature. Stir for hours. 5% Aqueous sodium thiosulfate was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 1: 9, V / V), and the solvent of the target fraction was distilled off under reduced pressure. Then, 980 mg (yield 77%) of a white powder of the title compound was obtained.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 0.13 (6H, s), 0.96 (9H, s), 3.96 (3H, s), 4.84 (2H, s), 8.06 (1H, s), 8.25 (1H, s), 8.41 (1H, s) , 10.07 (1H, s).

(21e) 3-Formyl-5-hydroxymethylbenzoic acid

1.82 g (5.90 mmol) of methyl 3-tert-butyldimethylsilyloxymethyl-5-formylbenzoate produced in Synthesis Example 21 (21d) was dissolved in 20 mL of methanol, and the mixture was cooled with ice to give a 1.0 M aqueous lithium hydroxide solution 7.1. mL (7.1 mmol) was added, and the mixture was stirred at the same temperature for 75 minutes and at room temperature for 5 minutes, then 6 mL of tetrahydrofuran was added, and the mixture was stirred at the same temperature for 1 hour. 7.1 mL (7.1 mmol) of 1.0 M aqueous lithium hydroxide solution was added to the reaction solution, and the mixture was further stirred for 1 hour, 1.0 M hydrochloric acid was added to the reaction solution under ice cooling, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure to give 1.68 g of a white powder.
1.68 g of the obtained white powder was dissolved in 15 mL of tetrahydrofuran, 15 mL of 6.0 M hydrochloric acid was added, and the mixture was stirred at room temperature for 30 minutes. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, tert-butyl methyl ether-n-hexane (1: 4) 25 mL was added to the obtained residue, and the insoluble material was collected by filtration after sonication. Obtained 899 mg (2 steps, yield 85%).
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 4.66 (2H, s), 5.46-5.57 (1H, br), 8.07 (1H, s), 8.20 (1H, s), 8.32 (1H, s), 10.09 (1H, s), 13.20-13.50 ( 1H, br).

(21) 3-formyl-5-nitrooxymethylbenzoic acid

3-Formyl-5-hydroxymethylbenzoic acid 300 mg (1.67 mmol) produced in Synthesis Example 21 (21e) was dissolved in tetrahydrofuran 6 mL, and fuming nitric acid (purity: 94%) was added to acetic anhydride 1.5 mL under ice cooling. A solution prepared by adding 0.18 mL (4.0 mmol) to the mixture was added dropwise at room temperature over 3 minutes, and the mixture was stirred at the same temperature for 1.5 hours. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, n-hexane (4 mL) was added to the obtained residue, and the insoluble material was collected by filtration to give the title compound (307 mg, yield 82%) as a pale-yellow powder.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 5.75 (2H, s), 8.23 (1H, s), 8.34 (1H, s), 8.47 (1H, s), 10.10 (1H, s).

Synthesis Example 22:
(22a) 2,3,5,6-tetramethylphenol

Dissolve 13.0 mL (162 mmol) of diiodomethane in 250 mL of toluene, and add 121 mL (0.12 mol) of a 1.0 M diethylzinc-toluene solution over 10 minutes while cooling with ice, and then add 2,3,5-trimethylphenol 11.0. A solution of g (80.7 mmol) in 100 mL of toluene was added over 2 minutes, the mixture was stirred at the same temperature for 5 minutes, and then heated under reflux for 3 hours. After cooling with ice, saturated aqueous sodium hydrogen carbonate was added to the reaction solution, and the two layers were separated. The aqueous layer was extracted twice with ethyl acetate, the organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 19 → 1: 9, V / V). The solvent of the target fraction was evaporated under reduced pressure to give 981 mg (crude product) of a yellow powder containing the title compound.

(22b) 3-Hydroxy-2,4,5-trimethylbenzaldehyde

After dissolving 3.76 g (8.49 mmol) of lead (IV) acetate in 20 mL of chloroform, 981 mg of yellow powder containing 2,3,5,6-tetramethylphenol prepared in Synthesis Example 22 (22a) under ice cooling was added. Chloroform 10 mL suspension was added, and the mixture was stirred at room temperature for 45 minutes. Saturated aqueous sodium hydrogen carbonate was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain 1.35 g of a yellow oily substance.
The obtained yellow oil (1.35 g) was dissolved in dimethyl sulfoxide (10 mL), sodium bicarbonate (686 mg) (8.16 mmol) was added at room temperature, and the mixture was stirred at 80 ° C for 13.5 hr. After allowing to cool, water was added to the reaction solution and the mixture was extracted 3 times with ethyl acetate. The organic layers were combined, washed with water and saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 1: 4, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 350 mg (3 steps, yield 2.7%) of a yellow powder of the title compound.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 2.17 (3H, s), 2.24 (3H, s), 2.44 (3H, s), 7.17 (1H, s), 8.37-8.62 (1H, br ), 10.10 (1H, s).

(22c) 3-Hydroxymethyl-2,5,6-trimethylphenol

3-hydroxy-2,4,5-trimethylbenzaldehyde 310 mg (1.89 mmol) produced in Synthesis Example 22 (22b) was dissolved in ethanol 6 mL, sodium borohydride 79 mg (2.1 mmol) was added, and the mixture was allowed to come to room temperature. And stirred for 45 minutes. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate, the organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 9 → 2: 3, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 270 mg (yield 86%) of a pale yellow powder of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.43 (1H, t, J = 5.6 Hz), 2.17 (3H, s), 2.24 (3H, s), 2.26 (3H, s), 4.63 (2H, d, J = 5.6 Hz), 4.71 (1H, s), 6.76 (1H, s).

(22d) 3-Hydroxy-5-hydroxymethyl-2,4-dimethylbenzaldehyde

After dissolving 383 mg (0.865 mmol) of lead (IV) acetate in 2 mL of chloroform, 3-hydroxymethyl-2,5,6-trimethylphenol 100 mg (0.602 mmol) of chloroform 1 produced in Synthesis Example 22 (22c) was dissolved in chloroform 1 A mL solution was added, and the mixture was stirred at room temperature for 1 hour. Saturated aqueous sodium hydrogen carbonate was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was distilled off under reduced pressure to obtain 0.16 g of a yellow oily substance.
0.16 g of the obtained yellow oil was dissolved in 1 mL of dimethyl sulfoxide, 70 mg (0.83 mmol) of sodium bicarbonate was added at room temperature, and the mixture was stirred at 80 ° C for 12 hours. After allowing to cool, ethyl acetate was added to the reaction solution, washed with water and saturated saline, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 1: 4 → 3: 2, V / V). The solvent of the target fraction was evaporated under reduced pressure to give 18 mg (2 steps, yield 17%) of a yellow powder of the title compound.
The same operation was carried out using 430 mg (2.86 mmol) of 3-hydroxymethyl-2,5,6-trimethylphenol to obtain 130 mg (2 steps, yield 25%) of a yellow powder of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 1.67 (1H, t, J = 5.6 Hz), 2.33 (3H, s), 2.58 (3H, s), 4.75 (2H, d, J = 5.6 Hz) , 5.01 (1H, s), 7.44 (1H, s), 10.18 (1H, s).

(22e) 5-Bromomethyl-3-hydroxy-2,4-dimethylbenzaldehyde

3-hydroxy-5-hydroxymethyl-2,4-dimethylbenzaldehyde 18 mg (0.099 mmol) produced in Synthesis Example 22 (22d) was suspended in methylene chloride 1 mL, and triphenylphosphine 39 mg (0.15 mmol) under ice cooling. ) And N-bromosuccinimide (27 mg, 0.15 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, dried (Na ₂ SO ₄ ), and the solvent was evaporated under reduced pressure.
3-hydroxy-5-hydroxymethyl-2,4-dimethylbenzaldehyde 130 mg (0.505 mmol) produced in Synthesis Example 22 (22d) was suspended in methylene chloride 5 mL, and triphenylphosphine 199 mg (0.757 mmol) was added under ice cooling. ) And N-bromosuccinimide (135 mg, 0.757 mmol) were added, and the mixture was stirred at the same temperature for 5 minutes. Water was added to the reaction solution and the mixture was extracted twice with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure, and the obtained residue was combined with the above residue and purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 3: 7, V / V). The solvent of the target fraction was distilled off under reduced pressure to obtain 92 mg (yield 63%) of a yellow powder of the title compound.
¹ H-NMR (CDCl ₃ , 400 MHz) δ; 2.36 (3H, s), 2.58 (3H, s), 4.54 (2H, s), 5.01 (1H, s), 7.40 (1H, s), 10.15 ( 1H, s).

(22) 3-Hydroxy-5-nitrooxymethyl-2,4-dimethylbenzaldehyde

5-bromomethyl-3-hydroxy-2,4-dimethylbenzaldehyde 92 mg (0.38 mmol) produced in Synthesis Example 22 (22e) was dissolved in acetonitrile 0.80 mL, silver nitrate (I) 96 mg (0.57 mmol) was added, The mixture was stirred at room temperature for 2 hours. Ethyl acetate was added to the reaction solution, the insoluble material was filtered off with Celite, and the solvent of the filtrate was evaporated under reduced pressure. The obtained residue was purified by column chromatography (ethyl acetate: n-hexane, 0: 1 → 3: 7, V / V), and the solvent of the target fraction was evaporated under reduced pressure. To the obtained residue was added tert-butyl methyl ether-n-hexane (1: 3) 3 mL, and the insoluble material was collected by filtration to give the title compound (24 mg, yield 28%) as a white powder.
¹ H-NMR (DMSO-d ₆ , 400 MHz) δ; 2.27 (3H, s), 2.50 (3H, s), 5.64 (2H, s), 7.44 (1H, s), 8.85-8.94 (1H, br ), 10.17 (1H, s).

Various physical properties of various polymer compounds (polymer-active drug conjugates) obtained in the following synthesis examples and examples were analyzed by the methods shown in the following measurement examples 1 to 5.

Measurement Example 1: Measurement of weight average molecular weight and polydispersity About 10 mg of a sample was precisely weighed, and a mobile phase was added to make exactly 5 mL to prepare a sample solution. 50 μL of the sample solution was injected into a high-performance liquid chromatograph (HPLC) connected with a multi-angle static light scatterometer (TREOSII, Wyatt Technology) and a differential refractometer (Optilab T-rEX, Wyatt Technology) as detectors, and the size was injected. Exclusion chromatography was performed. The refractive index concentration increment was measured from the differential refractometer and the light scattering intensity was measured from the static light scatterometer, and the weight average molecular weight and polydispersity were measured from these measured values using analysis software (ASTRA 7.1.2, Wyatt Technology). I asked for the degree.
HPLC conditions: column TSKgel α-4000, 7.8 × 300 mm (Tosoh), column temperature 40 ° C., mobile phase methanol / 30 mM phosphate buffer (pH 7.8) mixed solution (2: 1), flow rate 0.5 mL / min.

Measurement Example 2: Content of constitutional unit (4) having a hydrazide residue in HPMA-Ma-ah-NHNH ₂ copolymer (compound (5)) About 0.1 g of a sample was precisely weighed and an ammonium thiocyanate reagent and a copper ammine reagent were measured. After exactly adding 3 mL in order, water was added to make exactly 25 mL. After centrifuging this solution, 20 mL of the supernatant was accurately weighed, and 20 mL of 1 M ammonium chloride reagent solution and 80 mg of murexide indicator were added. The point at which the green color of the sample solution disappeared was used as the end point and titration was performed with a 20 mM EDTA solution to quantify copper (II) ions. The amount of the structural unit (4) having a hydrazide residue was calculated from the amount of copper (II) ions consumed by the hydrazide group, which was obtained by subtracting the titration amount of the sample solution from the titration amount of the blank test solution.

Measurement Example 3: Measurement of particle size To a glass tube with a lid, 3 mL of Dulbecco's phosphate buffered saline (pH 7.4, Nacalai Tesque, Kyoto, Japan) and about 3 mg of a sample were added in order, and the mixture was gently inverted and dispersed. It was filtered with a membrane filter (DISMIC 13HP020AN). Excluding the first 1.5 mL or more, the following filtrate 1 mL or more was used as a sample solution, and the particle size distribution was measured by a dynamic light scattering device (DLS) (nano Partica SZ-100, Horiba Ltd.). The analysis software used SZ-100 for Windows (Horiba).
DLS measurement conditions: measurement mode nano-analysis mode, data acquisition time 120 seconds, number of measurements 10 times, light receiving position 173 °, gate time 320 nsec, ND filter 100% T.

Measurement Example 4: Measurement of bound epirubicin content The hydrazone bond of the epirubicin residue in each polymer compound was cleaved under acidic conditions, and the total amount of released epirubicin was measured to obtain the bound epirubicin content. However, since a trace amount of unbound epirubicin may be mixed in the polymer compound, the unbound epirubicin content was measured, and the bound epirubicin content was determined by correcting by subtracting the unbound epirubicin content from the total epirubicin content. ..
(1) Total epirubicin content
About 5 mg of the sample was precisely weighed and water was added to make exactly 5 mL. Accurately measuring 1.5 mL of this solution, 0.6 mL of 1 M hydrochloric acid and 4.5 mL of acetonitrile were added, and the mixture was allowed to stand at 20 to 25 ° C for 1.5 hours. To this solution, 6 mL of 0.1 M disodium hydrogen phosphate solution, 5 mL of methanol, and 10 mM phosphate buffer solution (pH 7.0) were added to make exactly 25 mL to prepare a sample solution. Separately, 5 mg of epirubicin hydrochloride was precisely weighed, and methanol was added to make exactly 50 mL to prepare a standard stock solution. Accurately add 1 mL, 3 mL, and 5 mL of the standard stock solution to 15 mL of the placebo stock solution (described later) .Methanol or acetonitrile for the solution containing 1 mL of the standard stock solution, and methanol for the solution containing 3 mL of the standard stock solution. Alternatively, after adding 2 mL of acetonitrile, water was added to each to make exactly 25 mL, which was used as a standard sample solution for a calibration curve. 10 μL of each of the sample solution and the standard sample solution for the calibration curve was injected into HPLC, and the total epirubicin content was calculated from the calibration curve prepared from the standard sample solution for the calibration curve and the peak area of the sample solution.
Placebo stock solution: HPMA-Ma-ah-NHNH ₂ copolymer approx. 30 mg in 30 mL water, 12 mL 1 M hydrochloric acid, 90 mL acetonitrile, 120 mL 0.1 M disodium hydrogen phosphate solution and 10 mM phosphate buffer (pH 7 .0) was added to make exactly 300 mL.
HPLC conditions: Detector UV-visible spectrophotometer (measurement wavelength 496 nm), column Triart DIOL-HILIC, 4.6 × 250 mm, 5 μm (YMC), column temperature 25 ° C, mobile phase acetonitrile / 10 mM phosphate buffer (PH 7.0) mixed solution (10: 3), flow rate 1.0 mL / min.
(2) Unbound epirubicin content About 10 mg of the sample was precisely weighed, and methanol was added to make exactly 5 mL to prepare a sample solution. Separately, 8 mL of the standard stock solution of epirubicin hydrochloride of Measurement Example 4 (1) was accurately measured, and methanol was added to make exactly 20 mL. 1 mL, 4 mL, and 7.5 mL of this solution were accurately weighed, and methanol was added to each to make exactly 10 mL, which was used as a standard solution for a calibration curve. 20 μL of each of the sample solution and the standard solution for the calibration curve was injected into HPLC, and the unbound epirubicin content was calculated from the calibration curve prepared from the standard solution for the calibration curve and the peak area of the sample solution. The HPLC conditions were in accordance with those of Measurement Example 4 (1).

Measurement Example 5: Measurement of bound nitric oxide-releasing compound content The total amount of nitric oxide-releasing compounds released by cleaving the hydrazone bond of nitric oxide-releasing compound residues in various polymer compounds under acidic conditions Was measured as the content of bound nitric oxide-releasing compound. However, since a small amount of unbound nitric oxide-releasing compound may be mixed in the polymer compound, the content of unbound nitric oxide-releasing compound is measured, and the amount of unbound nitric oxide-releasing compound is calculated from the total content of unbound nitric oxide-releasing compound. The bound nitric oxide-releasing compound content was determined by subtracting and correcting the nitric oxide-releasing compound content.
(1) Total nitric oxide-releasing compound content A sample solution was prepared according to Measurement Example 4. Separately, about 25 μg / mL of a nitric oxide-releasing compound in methanol or acetonitrile was prepared and used as a standard stock solution. Accurately add 1 mL and 3 mL of the standard stock solution to 15 mL of the placebo stock solution of Measurement Example 4 (1), and add 1 mL of the standard stock solution to a solution containing 4 mL of methanol or acetonitrile and 3 mL of the standard stock solution. After adding 2 mL of methanol or acetonitrile, water was added to each to make exactly 25 mL, which was used as a standard sample solution for a calibration curve. 20 μL of each of the sample solution and the standard sample solution for calibration curve was injected into HPLC, and the total nitric oxide-releasing compound content was calculated from the calibration curve prepared from the standard sample solution for calibration curve and the peak area of the sample solution.
HPLC conditions: Detector UV-visible spectrophotometer (measurement wavelength 254 nm), column Cosmosil 5C ₁₈ -ARII, Cosmosil 5C ₁₈ -MSII or Cosmosil PBr, both 4.6 × 150 mm, 5 μm (Nacalai Tesque) Column temperature 25 ° C, mobile phase appropriately selected according to nitric oxide-releasing compound, mobile phase gradient of acetonitrile or methanol and 10 mM phosphate buffer (pH 7.0), flow rate 1.0 mL / min.
(2) Content of unbound nitric oxide-releasing compound A sample solution was prepared according to Measurement Example 4 (2). Separately, prepare solutions of about 0.4 μg / mL, 4 μg / mL, and 8 μg / mL nitric oxide-releasing compound in methanol or water / acetonitrile (1: 1) mixture as a solvent, and use them as standard solutions for calibration curves. did. 20 μL of each of the sample solution and the standard solution for the calibration curve was injected into HPLC, and the content of unbound nitric oxide-releasing compound was calculated from the calibration curve prepared from the standard solution for the calibration curve and the peak area of the sample solution. The HPLC conditions were in accordance with those of Measurement Example 5 (1).

Synthesis Examples 23-1 to 23-4: Synthesis of HPMA-Ma-ah-NHNH ₂ Copolymer (Compound (5))

Synthesis Example 23-1
In a 100 mL eggplant flask, N- (2-hydroxypropyl) methacrylamide (HPMA) (compound (3a)) 12.0 g (84.0 mmol), N- (5-hydrazinocarbonylpentyl) methacrylamide (Ma-ah-NHNH ₂ ) (Compound (4a)) 1.49 g (7.00 mmol), 2,2'-azobis (isobutyronitrile) (AIBN) 575 mg (3.50 mmol) and 42 mL of methanol were added and dissolved, and nitrogen was added for 10 minutes. I bubbled. The reaction vessel was immersed in an oil bath heated to 80 ° C., heated under reflux for 8 hours, added with 12 mL of methanol to the reaction solution, and allowed to cool. 600 mL of ethyl acetate was added to a 1 L eggplant flask, the above reaction solution was added dropwise over 7 minutes while stirring the solution, and the mixture was washed twice with 4 mL of methanol and then stirred at room temperature for 0.5 hour. The precipitate was collected by filtration, washed twice with 50 mL of ethyl acetate, and dried at room temperature under reduced pressure to give 13.37 g of a crude product of the compound of Synthesis Example 23-1 (Mw: 24.8 kDa, polydispersity: 2.07) was obtained as a white powder. 10.6 g (Mw: 24.8 kDa, polydispersity: 2.07) of white powder of the obtained crude product was dissolved in 106 mL of methanol, and 175 mL of ethyl acetate was added dropwise over 25 minutes under ice cooling, and then at room temperature. Stir for 1.5 hours. After separating the precipitate by filtration, 249 mL of ethyl acetate was added to the filtrate, and the mixture was stirred at room temperature for 21 hours. The precipitate was collected by filtration, washed twice with 30 mL of ethyl acetate: methanol (4: 1), and dried under reduced pressure to give a white powder of the compound of Synthesis Example 23-1 (Compound (5-1)) (6.57). g (Mw: 21.2 kDa, polydispersity: 1.62, content of structural unit (4) 8.3% by weight) was obtained.

Synthesis Example 23-2
In a 500 mL eggplant flask, N- (2-hydroxypropyl) methacrylamide (HPMA) (compound (3a)) 60.1 g (420 mmol), N- (5-hydrazinocarbonylpentyl) methacrylamide (Ma-ah-NHNH ₂ ) (Compound (4a)) 7.46 g (35.0 mmol), 2,2'-azobis (isobutyronitrile) (AIBN) 1.44 g (8.75 mmol) and 210 mL of methanol were added and dissolved, and nitrogen was added for 10 minutes. I bubbled. The reaction vessel was immersed in an oil bath heated to 85 ° C., heated under reflux for 22 hours, 70 mL of methanol was added to the reaction solution, and the mixture was allowed to cool. 3000 mL of ethyl acetate was added to a 5 L eggplant flask, the reaction solution was added dropwise over 12 minutes while stirring the solution, and the mixture was washed twice with 20 mL of methanol and then stirred at room temperature for 0.5 hours. The precipitate was collected by filtration, washed twice with 250 mL of ethyl acetate, and dried under reduced pressure at room temperature to give 67.1 g of a crude product of the compound of Synthesis Example 23-2 (Mw: 35.3 kDa, polydispersity: 2.05) was obtained as a white powder.
67.1 g (Mw: 35.3 kDa, polydispersity: 2.05) of white powder of the obtained crude product was dissolved in 670 mL of methanol, 1000 mL of ethyl acetate was added dropwise over 15 minutes, and after stirring at room temperature for 1.5 hours. , 44 mL of methanol were added, and the mixture was further stirred for 22.5 hours. The precipitate was filtered off, 1142 mL of ethyl acetate was added to the filtrate, and the mixture was stirred at room temperature for 22.5 hours. The precipitate was collected by filtration, washed twice with 75 mL of ethyl acetate: methanol (3: 1), and dried under reduced pressure to give 42.8 white powder of the compound of Synthesis Example 23-2 (Compound (5-2)). g (Mw: 32.7 kDa, polydispersity: 1.72, content of structural unit (4) 8.4% by weight) was obtained.

Synthesis Example 23-3
In a 500 mL eggplant flask, N- (2-hydroxypropyl) methacrylamide (HPMA) (compound (3a)) 60.1 g (420 mmol), N- (5-hydrazinocarbonylpentyl) methacrylamide (Ma-ah-NHNH ₂ ) (Compound (4a)) 7.46 g (35.0 mmol), 2,2'-azobis (isobutyronitrile) (AIBN) 1.44 g (8.75 mmol) and ethanol 175 mL were added and dissolved, and nitrogen was added for 10 minutes. I bubbled. The reaction vessel was immersed in an oil bath heated to 98 ° C, heated under reflux for 8 hours, 140 mL of ethanol was added to the reaction solution, and the mixture was allowed to cool. 3000 mL of ethyl acetate was added to a 5 L eggplant flask, the above reaction solution was added dropwise over 12 minutes while stirring the solution, and the mixture was washed twice with 20 mL of ethanol, and then stirred at room temperature for 0.5 hours. The precipitate was collected by filtration, washed twice with 250 mL of ethyl acetate, and dried under reduced pressure at room temperature to give 70.25 g of a crude product of the compound of Synthesis Example 23-3 (Mw: 26.5 kDa, polydispersity: 1.85) was obtained as a white powder.
White powder 70.25 g (Mw: 26.5 kDa, polydispersity: 1.85) of the obtained crude product and 700 mL of methanol were added to dissolve the white powder, and 2.8 L of ethyl acetate was added over 24 minutes while stirring. After that, the mixture was stirred at room temperature for 4 hours. The precipitate was collected by filtration, washed twice with 75 mL of ethyl acetate: methanol (4: 1), and dried under reduced pressure to give a white powder of the compound of Synthesis Example 23-3 (Compound (5-3)) 60 g (Mw: 28.2 kDa, polydispersity: 1.92, content of structural unit (4) 8.3% by weight) was obtained.

Synthesis Example 23-4
In a 200 mL eggplant-shaped flask, N- (2-hydroxypropyl) methacrylamide (HPMA) (Compound (3a)) 18.5 g (126 mmol), N- (5-hydrazinocarbonylpentyl) methacrylamide (Ma-ah-NHNH) ₂ ) (Compound (4a)) 2.24 g (10.5 mmol), 2,2'-azobis (isobutyronitrile) (AIBN) 172 g (1.05 mmol) and 63 mL of ethanol were added and dissolved, and nitrogen was added for 10 minutes. I bubbled. The reaction vessel was immersed in an oil bath heated to 98 ° C, heated under reflux for 22 hours, 15 mL of ethanol was added to the reaction solution, and the mixture was allowed to cool. 900 mL of ethyl acetate was added to a 2 L round-bottomed flask, the reaction solution was added dropwise over 8 minutes while stirring the solution, and the mixture was washed twice with 6 mL of ethanol and then stirred at room temperature for 0.5 hour. The precipitate was collected by filtration, washed twice with 75 mL of ethyl acetate, and dried under reduced pressure at room temperature to give 19.4 g of a crude product of the compound of Synthesis Example 23-4 (Mw: 47.9 kDa, polydispersity: 1.82) was obtained as a white powder.
White powder 19.4 g (Mw: 47.9 kDa, polydispersity: 1.82) of the obtained crude product was dissolved in 200 mL of methanol, and 270 mL of ethyl acetate was added dropwise over 15 minutes, followed by stirring at room temperature for 4 hours. . After separating the precipitate by filtration, 530 mL of ethyl acetate was added to the filtrate, and the mixture was stirred at room temperature for 2 hours. The precipitate was collected by filtration, washed twice with 30 mL of ethyl acetate: methanol (4: 1), and dried under reduced pressure to give a white powder of the compound of Synthesis Example 23-4 (Compound (5-4)) 14.7 g (Mw: 40.4 kDa, polydispersity: 1.76, content of structural unit (4) was 8.9% by weight) was obtained.

Synthesis Example 24: Synthesis of Polymer-Epirubicin-Conjugate (Compound (7))

Synthesis Examples 24-1 to 24-3: Synthesis of polymer-epirubicin-conjugate having a bound epirubicin content of about 11 to 12% The HPMA-Ma-ah-NHNH2 copolymer (compound (5-3 )) 20.0 g (M _w 28.2 kDa) was dissolved in 200 mL of methanol, 2.77 g (4.78 mmol) of epirubicin hydrochloride (Compound (6)) was added, and then 10.9 mL (191 mmol) of acetic acid was added, and the mixture was protected from light. The mixture was stirred at room temperature for 15 hours. Ethyl acetate (800 mL) was added dropwise to the reaction solution over 37 minutes, and the mixture was stirred at room temperature for 2 hours under light shielding, and the insoluble material was collected by filtration. The obtained powder was washed twice with 200 mL of ethyl acetate-methanol (4: 1), then dissolved again in 200 mL of methanol, and 800 mL of ethyl acetate was added dropwise over 32 minutes. Stir for hours. The insoluble material was collected by filtration, the obtained powder was washed twice with 200 mL of ethyl acetate-methanol (4: 1), and then dried under reduced pressure at 50 ° C. for 15 hours to give Synthesis Example 24-2. 21.2 g ( _Mw 40.3 kDa, polydispersity 1.35, bound epirubicin content 11.1 wt%) of a pale orange powder of the compound (Compound (7-2)) was obtained.
Using the HPMA-Ma-ah-NHNH ₂ copolymers (Compound (5-1) and Compound (5-4)) produced in Synthesis Examples 23-1 and 23-4, the reaction and purification were performed under the same conditions as above. This gave the compound of Synthesis Example 24-1 (Compound (7-1)) and the compound of Synthesis Example 24-3 (Compound (7-3)). Table 1 shows the analytical values of Synthesis Example 24-1 to Synthesis Example 24-3 (Compound (7-1) to Compound (7-3)).

Synthesis Example 24-4: Synthesis of polymer-epirubicin-conjugate having a bound epirubicin content of about 6% HPMA-Ma-ah-NHNH2 copolymer (Compound (5-2)) 3.00 g (M _w 32.7 kDa) was dissolved in 30 mL of methanol, and after adding 208 mg (0.359 mmol) of epirubicin hydrochloride, 1.64 mL (28.7 mmol) of acetic acid was added, and the mixture was stirred at room temperature for 15 hours in the dark. 120 mL of ethyl acetate was added dropwise to the reaction solution over 11 minutes, and the mixture was stirred at room temperature for 2 hours under light shielding, and the insoluble material was collected by filtration. The obtained powder was washed twice with 30 mL of ethyl acetate-methanol (4: 1), dissolved again in 30 mL of methanol, and 120 mL of ethyl acetate was added dropwise over 15 minutes. Stir for hours. The insoluble material was collected by filtration, and the obtained powder was washed twice with 30 mL of ethyl acetate-methanol (4: 1) and then dried under reduced pressure at 50 ° C. for 15 hours to give the compound of Synthesis Example 24-4 ( As a result, 2.86 g ( _Mw 39.7 kDa, polydispersity 1.40, bound epirubicin content 6.32% by weight) of pale orange powder of the compound (7-4) was obtained.

Examples 1-18: Synthesis of Polymer-Epirubicin-Nitric Oxide Releasing Compound-Conjugate (Compound of the Invention)

The polymer-epirubicin-conjugate (compound (7-2)) (500 mg) prepared in Synthesis Example 24-2 was dissolved in methanol (4.5 mL), and acetic acid (50 μL, 0.87 mmol) was prepared according to the method of Synthesis Example 3-. A solution of nitrooxymethylbenzaldehyde (16.5 mg, 91.1 μmol) in 0.2 mL of methanol was added, and the mixture was washed 3 times with 0.1 mL of methanol, and the mixture was stirred at room temperature for 1 hour in the dark. 50 mL of ethyl acetate was added dropwise to the reaction solution over 9 minutes, and the mixture was stirred at room temperature for 1 hour under light shielding, and the insoluble material was collected by filtration. The obtained powder was washed twice with 5 mL of ethyl acetate and dried under reduced pressure at room temperature for 15 hours to give 452.6 mg (M _w 42.4 kDa, polydispersity 1.45, binding) of a pale orange powder of the compound of Example 1. An epirubicin content of 10.9% by weight, a bound nitric oxide-releasing compound content of 2.99% by weight, a bound nitric oxide-releasing compound / bound epirubicin (mol ratio) 0.83, and an average particle size of 18.8 nm) were obtained.
Various nitric oxide-releasing compounds synthesized by the methods of Synthesis Examples 4 to 10, 12 to 19, 21 and 22 were condensed with polymer-epirubicin-conjugate (compound (7-2)) by the same method as above. Then, the polymer of the present invention-epirubicin-nitric oxide-releasing compound-conjugate (Examples 2 to 18) was synthesized. Table 2 shows the weight average molecular weight, polydispersity, bound epirubicin content (wt%), bound nitric oxide liberate compound content (wt%) of each of the synthesized polymers-epirubicin-nitric oxide-releasing compound-conjugate. The bound nitric oxide-releasing compound / bound epirubicin (mol ratio) and the average particle size are shown.

Further, as shown in Example 19 below, a fixed amount of nitric oxide was released to the polymer-epirubicin-conjugate (compound (7-4)) synthesized in Synthesis Example 24-4 and having different bound epirubicin contents. It is possible to synthesize a polymer-epirubicin-nitric oxide-releasing compound-conjugate in which the ratio of the content of bound epirubicin to the content of bound nitric oxide-releasing compound is changed by binding the functional compound.

Example 19
600 mg of the polymer-epirubicin-conjugate (compound (7-4)) produced in Synthesis Example 24-4 was dissolved in 5 mL of methanol, and 60 μL (1.1 mmol) of acetic acid was added, and then, produced in Synthesis Example 3. A solution of 3-nitrooxymethylbenzaldehyde (33.0 mg, 0.182 mmol) in 1 mL of methanol was added, and the mixture was stirred at room temperature for 1 hour under light shielding. 60 mL of ethyl acetate was added dropwise to the reaction solution over 8 minutes, and the mixture was stirred at room temperature for 1 hour under light shielding, and the insoluble material was collected by filtration. The obtained powder was washed twice with 6 mL of ethyl acetate and dried under reduced pressure at room temperature for 17 hours to give 640.9 mg (M _w 42.7 kDa, polydispersity 1.43, bound epirubicin content) of the title compound as a pale orange powder. %, 5.17% by weight of bound nitric oxide-releasing compound, 5.77% of bound nitric oxide-releasing compound / bound epirubicin (mol ratio), and an average particle diameter of 12.2 nm).

Also, using the polymer-epirubicin-conjugate (Compound (7-4)) prepared in Synthesis Example 24-4 and 4-formyl-N- (2-nitrooxyethyl) benzamide prepared in Synthesis Example 6, A polymer-epirubicin-nitric oxide-releasing compound-conjugate was obtained in the same manner as in Example 19 (Example 20).
In Table 3, the weight average molecular weight, polydispersity, bound epirubicin content, bound nitric oxide free compound content, and bound monoxide of the polymer-epirubicin-nitric oxide releasing compound-conjugate synthesized in Examples 19 and 20 are shown. The nitrogen-releasing compound / bound epirubicin (mol ratio) and the average particle size are shown.

Test Example 1: Antitumor effect of polymer-epirubicin-nitric oxide-releasing compound-conjugate (Example 1) in B16-F0 tumor-bearing mice (method)
Seven-week-old male C57BL / 6N mice (Japan SLC, Shizuoka, Japan) were subcutaneously implanted with B16-F0 cells (2 × 10 ⁵ cells / body) derived from mouse malignant melanoma under anesthesia. Ten days after transplantation, the polymer-epirubicin-conjugate (Compound (7-2)) and the polymer-epirubicin-nitric oxide-releasing compound-conjugate (Example 1) produced in Synthesis Example 24-2 were respectively prepared. The solution was dissolved in phosphate buffered saline (PBS) and epirubicin equivalent to 10 mg / kg of each solution was intravenously administered once by tail vein. Incidentally, PBS alone was intravenously administered to the control group. The number of cases in each group was 20. Tumor diameters (major axis and minor axis) at the transplant site were periodically measured for 14 days after administration, and the tumor volume was determined by the following formula. Furthermore, 14 days after the administration, whole blood was collected from the heart under deep anesthesia and euthanized, the tumor was excised, and the wet weight was measured.
Tumor volume (mm ³ ) = (major axis × minor axis ² ) / 2
(result)
The tumor volume of the polymer-epirubicin-conjugate (compound (7-2)) administration group was 61.1 to 77.4% of the tumor volume of the control group during the administration period, and the tumor weight 14 days after administration was 69.1%. It was That is, the tumor volume and tumor weight were significantly reduced. The tumor volume and tumor weight of the polymer (epirubicin-nitric oxide-releasing compound-conjugate (Example 1)) were calculated with the tumor volume and tumor weight of the compound (7-2) administration group set to 100%. The tumor volume during the administration period of Example 1 remained at 76.3 to 89.3% of the tumor volume of the compound (7-2) administration group, and the tumor weight 14 days after administration was the compound (7-2) administration group. 78.5% of the tumor volume. That is, Example 1 significantly reduced the tumor volume and weight as compared with Compound (7-2).

Test Example 2: Antitumor effect of polymer-epirubicin-nitric oxide-releasing compound-conjugate (Example 1) in C26 tumor-bearing mice (method)
Seven-week-old male BALB / c mice (Japan SLC, Shizuoka, Japan) were subcutaneously transplanted with mouse colon cancer-derived C26 cells (4 × 10 ⁶ cells / body) under anesthesia. The polymer-epirubicin-conjugate (Compound (7-2)) and the polymer-epirubicin-nitric oxide-releasing compound-conjugate (Example 1) prepared in Synthesis Example 24-2 were prepared 17 days after the transplantation, respectively. The solution was dissolved in PBS, and 10 mg / kg of epirubicin of each solution was administered by single intravenous injection into the tail vein. Incidentally, PBS alone was intravenously administered to the control group. The number of cases in each group was 10. Tumor diameters (major axis and minor axis) at the transplant site were periodically measured for 14 days after administration, and the tumor volume was determined by the following formula. Furthermore, 14 days after the administration, whole blood was collected from the heart under deep anesthesia and euthanized, the tumor was excised, and the wet weight was measured.
Tumor volume (mm ³ ) = (major axis × minor axis ² ) / 2
(result)
The tumor volume of the polymer-epirubicin-conjugate (compound (7-2)) administration group was 66.6-97.9% of the control group tumor volume during the administration period, and the tumor weight 14 days after administration was 68.0%. there were. That is, it significantly reduced tumor volume and tumor weight. The tumor volume and tumor weight of the polymer (epirubicin-nitric oxide-releasing compound-conjugate (Example 1)) were calculated with the tumor volume and tumor weight of the compound (7-2) administration group set to 100%. The tumor volume during the administration period of Example 1 was 68.5 to 90.5% of the tumor volume of the compound (7-2) administration group, and the tumor weight 14 days after administration was the compound (7-2) administration group. 75.3% of the tumor volume. That is, Example 1 significantly reduced the tumor volume and weight as compared with Compound (7-2).

The compound (I) or a pharmaceutically acceptable salt thereof of the present invention is a highly safe compound that is retained in vivo, continuously retained, and efficiently converted to an active drug after reaching tumor tissue. .. In addition, the compound (I) of the present invention is a polymer in which an anticancer drug residue and a nitric oxide-releasing compound are simultaneously conjugated, has an enhanced EPR effect, and is more efficient in tumor tissues than conventional polymer DDS anticancer drugs. It is useful as an anti-cancer agent, a prophylactic or therapeutic agent for cancer metastasis and / or cancer recurrence, because it can be transferred to and accumulated in E. coli and exert an excellent effect.

This application is based on Japanese Patent Application No. 2018-219678, the contents of which are incorporated in full herein.

Claims

Methacryloylated α-amino acid, ε-amino acid or hydrazone-derived constituent unit of oligopeptide having an anthracycline anticancer drug residue at the terminal, and methacryloylated α having a nitric oxide-releasing compound residue at the terminal -A polymer compound or a pharmaceutically acceptable salt thereof, comprising a structural unit derived from an hydrazone of an amino acid, an ε-amino acid or an oligopeptide, and a structural unit derived from N- (2-hydroxypropyl) methacrylamide.
Formula (1):

[In the formula,
B represents an anthracycline anticancer drug residue. ]
The structural unit (1) represented by the formula (2):

[In the formula,
R 1 represents a hydrogen atom, an optionally substituted C 1-6 alkyl group or an optionally substituted C 6-14 aryl group;
A represents an arylene group or a divalent heterocyclic group, each of which may be further substituted;
D is a single bond, -CH = CH-, -O-, -S-, -N (R 2 )-, * -CON (R 2 )- ** or * -SO 2 N (R 2 )- *. * (Wherein R 2 represents a hydrogen atom or an optionally substituted C 1-6 alkyl group; * represents a bonding position with A; and ** represents a bonding position with E) .); And E represents a C 1-6 alkylene group which may be further substituted. ]
A polymer compound comprising the structural unit (2) represented by: and a structural unit (3) derived from N- (2-hydroxypropyl) methacrylamide, or a pharmaceutically acceptable salt thereof.
The polymer compound according to claim 2, which further comprises a structural unit (4) derived from N- (5-hydrazinocarbonylpentyl) methacrylamide, or a pharmaceutically acceptable salt thereof.
The polymer compound according to claim 2 or 3, wherein R 1 of the structural unit (2) is a hydrogen atom, and A is a phenylene group, a biphenylylene group or an indolinylene group, which may be further substituted, respectively. Pharmaceutically acceptable salts.
D of the structural unit (2) is a single bond, -O-, -S-, * -CONH- ** or * -SO 2 NH- ** (wherein * and ** are as defined in claim 2) The polymer compound or the pharmaceutically acceptable salt thereof according to any one of claims 2 to 4, wherein
In the structural unit (2), -ADE-ONO 2 has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to claim 2 or 3, wherein
In the structural unit (2), -ADE-ONO 2 has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to claim 2 or 3, wherein
In the structural unit (2), -ADE-ONO 2 has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to claim 2 or 3, wherein
In the structural unit (2), -ADE-ONO 2 has the following formula:

The polymer compound or a pharmaceutically acceptable salt thereof according to claim 2 or 3, wherein
The polymer compound or a pharmaceutically acceptable salt thereof according to any one of claims 2 to 9, wherein B in the structural unit (1) is an epirubicin residue.
The polymer compound or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 10, which comprises the structural units (1), (2), (3) and (4).
An amount of 1 to 40% by weight of an anthracycline anticancer drug residue and 0.5 to 20% by weight of a nitric oxide-releasing compound residue based on 100% by weight of a polymer compound or a pharmaceutically acceptable salt thereof. The polymer compound or a pharmaceutically acceptable salt thereof according to any one of 1 to 11.
The polymer compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, which has a weight average molecular weight of 10,000 to 300,000.
A pharmaceutical composition comprising the polymer compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof as an active ingredient.
An anticancer agent comprising the polymer compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof as an active ingredient.
A preventive or therapeutic agent for cancer, cancer metastasis and / or cancer recurrence, which comprises the polymer compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof as an active ingredient.