Negamycin derivatives

Abstract

[Problem] To provide a novel compound that has read-through activity and is useful as an agent for preventing or treating nonsense-mutation genetic disorders. [Solution] One embodiment of the present invention provides a compound represented by formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof (with the various substituents in formula (1) being as defined in the description).

Description

[Technical Field]

[0001] The present invention relates to negamycin derivatives and uses thereof. [Background technology]

[0002] Genetic diseases are caused by genetic abnormalities due to various factors, resulting in impaired vital functions. Nonsense mutations, which account for 10–30% of genetic diseases, result in the creation of premature termination codons (PTCs) within structural genes, suppressing the expression of functional full-length proteins and resulting in various genetic disorders. It is estimated that there are over 2,500 types of nonsense mutation-related genetic diseases. While hormone replacement therapy is exceptionally effective in cases of hormone deficiency due to genetic abnormalities, there are currently no direct treatments for genetic diseases. Currently, treatments rely on symptomatic treatments to alleviate symptoms, and no cures have yet been discovered. Gene therapy is one of the most promising treatments, but it has not yet reached a stage where it can be fully tolerated in clinical settings.

[0003] Nagashima palmoplantar keratoderma (NPPK), a type of congenital palmoplantar keratoderma, is known as a nonsense mutation-mediated genetic disease. It is an autosomal recessive, nonsyndromic, diffuse palmoplantar keratoderma characterized by erythema and hyperkeratosis that extends not only to the palms and soles but also to the dorsum of the hands and feet, wrists, ankles, Achilles tendons, elbows, and knees. Loss-of-function mutations in the SERPINB7 gene, which encodes a serine protease inhibitor, are thought to be the cause of this disease. More than 90% of patients with this disease have been reported to carry a UGA stop codon due to the nonsense mutation c.796C>T (p.Arg266Ter) (Br. J. Dermatol., 171: pp. 847-853 (2014)).

[0004] The use of compounds with read-through activity for the treatment of nonsense mutation-related genetic diseases has been reported. When specific compounds are administered to patients lacking a specific protein due to a premature stop codon (PTC) caused by a nonsense mutation, the compound acts on the ribosome, causing the ribosome to read past the stop codon and continue translation. This phenomenon is called "read-through." As a result of read-through, wild-type normal protein is synthesized, thereby treating the disease. An example of a compound with such read-through activity is the aminoglycoside antibiotic gentamicin. It has been reported that administration of gentamicin to patients with Duchenne muscular dystrophy (DMD) results in the accumulation of dystrophin protein (see Acta. Myol., 22: pp. 15-21 (2003)). It has also been reported that local administration of gentamicin to the airway epithelium of patients with cystic fibrosis can normalize typical electrophysiological abnormalities (see N. Engl. J. Med., 349: pp. 1433-1441 (2003)). However, like other aminoglycoside antibiotics, gentamicin exhibits significant nephrotoxicity and ototoxicity, making it unsuitable for this treatment, which requires long-term administration. Furthermore, considering side effects, it is necessary to separate the pharmacological activity of the compound from its antibacterial activity.

[0005] Negamycin is a dipeptide antibiotic isolated and identified from actinomycetes in 1970 (see J. Antibiot., 23: pp. 170-171 (1970)). Derivatives of this compound have been reported to have read-through activity. For example, it has been reported that a compound in which the configuration at the 3rd position of the amino acid constituting the left wing of the negamycin structure is inverted and the 5th position does not contain a hydroxyl group has read-through activity equivalent to or greater than that of negamycin and is useful for treating diseases caused by nonsense mutations (see JP 2013-136570 A and ChemMedChem, 9: pp. 2233-2237 (2014)). Furthermore, some literature has described that dipeptide antibiotics such as negamycin have read-through activity and are useful for treating diseases caused by nonsense mutations (see WO 2002 / 102361).

[0006] On the other hand, deoxynegamycin derivatives having a cyclopropane ring in the molecule have been disclosed with their antibacterial activity (see Bioorganic & Medicinal Chemistry Letters, 14: pp. 3103-3107 (2004)). However, there is no disclosure or suggestion that the derivatives disclosed therein have read-through activity. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide novel compounds having read-through activity that are useful as agents for the prevention and treatment of nonsense mutation-related genetic diseases. [Means for solving the problem]

[0008] As a result of extensive research aimed at solving the above problems, the present inventors have found that negamycin derivatives having a specific structure containing a cyclopropane ring, represented by the following formula (1), or pharmaceutically acceptable salts thereof, or solvates thereof, have read-through activity. Because the compounds of the present invention are compounds with read-through activity, they may have an effect of improving nonsense mutation-related genetic diseases (e.g., Nagashima palmoplantar keratosis, etc.). DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention relates to a compound having read-through activity represented by the following formula (1), or a pharmaceutically acceptable salt thereof, or a solvate thereof, and to a pharmaceutical composition containing any of them as an active ingredient. According to the present invention, a novel compound having read-through activity that is useful as an agent for the prevention and treatment of nonsense mutation genetic diseases is provided.

[0010] [ka]

[0011] [The definition of each substituent in formula (1) is the same as that in embodiment [1] described later.] [Aspects of the present invention] More specifically, the present invention includes the following aspects. [1] A first aspect of the present invention is a compound represented by the following formula (1):

[0012] [ka]

[0013] [In formula (1), n represents an integer of 0, 1, or 2; R 1 and R 2 are each independently a hydrogen atom or C 1~3 is an alkyl group; R 3represents a hydrogen atom, the following partial structural formula (S-1) [wherein the formula does not include the part to the right of the dashed line]:

[0014] [ka]

[0015] (In the partial structural formula (S-1), m represents an integer of 0, 1, or 2; R 4 is a hydrogen atom, C 1~10 Alkyl group, C 2~10 Alkenyl group, C 2~10 Alkynyl group, C 4~10 Alkyldienyl group and C 6~10 a group selected from the group consisting of aryl groups; R 4 C in 1~10 Alkyl group, C 2~10 Alkenyl group, C 2~10 Alkynyl groups and C 4~10 Each —CH— group in the alkyldienyl group may be substituted with 1 to 3 —O— groups (ether groups); R 4 C in 1~10 Alkyl group, C 2~10 Alkenyl group, C 2~10 Alkynyl group, C 4~10 Alkyldienyl group and C 6~10 The aryl group contains halogen atoms, hydroxyl groups, C 1~6 Alkoxy group, NR A R B Group (NR A R B R in the group A and R B are each independently a hydrogen atom or C 1~3 alkyl group), C 1~6 Alkylsulfonyl group, C 3~8 Cycloalkyl groups, C 6~10 Aryl group, C 7~16 an aralkyl group, a 3- to 8-membered non-aromatic heterocyclic group, a 5- to 6-membered heteroaryl group (the above C 1~6 Alkoxy group, C 3~8 Cycloalkyl groups, C6~10 Aryl group, C 7~16 The arylalkyl group, the 3- to 8-membered non-aromatic heterocyclic group, or the 5- to 6-membered heteroaryl group may be substituted with a hydroxyl group, a halogen atom, or a C 1~6 Alkyl group, C 3~8 Cycloalkyl group or C 6~10 optionally substituted with 1 to 3 aryl groups), and C 2~7 alkanoylamino groups) Or the following partial structural formula (S-2) [wherein the formula does not include the right side of the dashed line]:

[0016] [ka]

[0017] (In partial structural formula (S-2), R 8 is C 2~7 alkanoylamino group) and R 5 is a hydrogen atom or C 1~3 is an alkyl group; R 6 is a hydrogen atom or C 1~3 is an alkyl group; R 7 is a hydrogen atom, C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 2~10 Alkenyl group, C 6~10 Aryl group, or C 7~16 R is an alkyl group; 7 C in 6~10 Aryl group or C 7~16 Aryl alkyl groups contain halogen atoms, C 1~6 Alkyl group or C 1~6 optionally substituted with 1 to 3 alkoxy groups] or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0018] Hereinafter, each group in the above formula (1) of the present invention and in more specific embodiments of formula (1) described below will be specifically explained.

[0019] In the description of the compounds of the present invention, for example, "C 1~6 " indicates that the number of constituent carbon atoms is 1 to 6, and unless otherwise specified, represents the total number of carbon atoms in the straight-chain or branched-chain group.

[0020] In this specification, unless otherwise specified, "C 1~10 Examples of the "alkyl group" include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 1,2-dimethylpropyl group, a hexyl group, an isohexyl group, and the like. 1~10 Among alkyl groups, those with 1 to 3 carbon atoms are called "C 1~3 Examples of alkyl groups include methyl, ethyl, propyl, and isopropyl groups.

[0021] In this specification, unless otherwise specified, "C 2~10 Examples of the "alkenyl group" include a vinyl group, a propenyl group, a methylpropenyl group, a butenyl group, and a methylbutenyl group.

[0022] In this specification, unless otherwise specified, "C 2~10 Examples of the "alkynyl group" include an ethynyl group, a 1-propynyl group, a 2-propynyl group, a 2-butynyl group, a pentynyl group, and a hexynyl group.

[0023] In this specification, unless otherwise specified, "C 4~10 The "alkyldienyl group" includes, for example, a 1,3-butadienyl group.

[0024] In this specification, unless otherwise specified, "C 6~10The term "aryl group" refers to an aromatic hydrocarbon group having 6 to 10 carbon atoms, and examples thereof include phenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, and 1,2,3,4-tetrahydronaphthyl groups.

[0025] Unless otherwise specified, in this specification, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0026] In this specification, unless otherwise specified, "C 1~6 The alkoxy group is defined as "-OC 1~6 It means an "alkyl group" and includes, for example, groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, or hexyloxy.

[0027] In this specification, unless otherwise specified, "NR A R B The "amino group" refers to the two hydrogen atoms on the nitrogen atom of the "amino group" connected by -R A 、 -R B In this specification, unless otherwise specified, R A and R B are each independently a hydrogen atom or C 1~3 The alkyl group is "NR A R B Examples of the "group" include an amino group, a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, a diethylamino group, and an n-propylamino group.

[0028] In this specification, unless otherwise specified, "C 1~6 "Alkylsulfonyl group" means "-SO2-C 1~6 It means an "alkyl group," and examples thereof include a methanesulfonyl group, an ethanesulfonyl group, a propanesulfonyl group, an isopropanesulfonyl group, a butanesulfonyl group, an isobutanesulfonyl group, a sec-butanesulfonyl group, and a t-butanesulfonyl group.

[0029] In this specification, unless otherwise specified, "C 3~8 The term "cycloalkyl group" refers to a monocyclic or polycyclic saturated hydrocarbon ring group having 3 to 8 carbon atoms, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.

[0030] In this specification, unless otherwise specified, "C 7~16 "Aralkyl" is a group with 7 to 16 carbon atoms. 6~10 C substituted with aryl groups 1~6 It means an "alkyl group," and examples thereof include a benzyl group (phenylmethyl group) and a phenethyl group (phenylethyl group).

[0031] In this specification, unless otherwise specified, the term "3- to 8-membered non-aromatic heterocyclic group" refers to a monovalent group obtained by removing any hydrogen atom from a 3- to 8-membered saturated or unsaturated heterocycle containing 1 to 4 heteroatoms selected from oxygen atoms, sulfur atoms, and nitrogen atoms, and examples thereof include an aziridinyl group, an azetidinyl group, a pyrrolidinyl group, a piperidinyl group, an azepan-1-yl group, an azacyclooctyl group, a pyrazolidinyl group, an imidazoline group, and the like. Examples of such groups include an oxazolidinyl group, an oxazolidinyl group, a thiazolidinyl group, an isoxazolidinyl group, a morpholino group, a thiomorpholino group, a 1,2-oxazinyl group, a 1,3-oxazinyl group, a 1,3-thiazinyl group, a piperazinyl group, a tetrahydropyridazinyl group, a hexahydropyridazinyl group, a tetrahydropyrimidinyl group, a hexahydropyrimidinyl group, a 1,4-thiazepanyl group, and an isothiazolidinyl group.

[0032] Unless otherwise specified, in this specification, the term "5- to 6-membered heteroaryl group" refers to a monovalent group obtained by removing any hydrogen atom from a 5- to 6-membered heteroaryl ring containing 1 to 4 heteroatoms selected from nitrogen, sulfur, and oxygen atoms, and examples thereof include pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, 1,2,4-triazolyl, oxazolyl, thiazolyl, isoxazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidyl, and triazyl groups. All positional isomers of these aromatic heterocyclic groups are also contemplated.

[0033] In this specification, unless otherwise specified, "C 2~7 An "alkanoylamino group" is a group in which one hydrogen atom on the nitrogen atom of the amino group is "C 2~7 "C" refers to a group substituted with an "alkanoyl group," and examples thereof include an acetylamino group, an ethylcarbonylamino group, an n-pentylcarbonylamino group, and the like. 2~7 An alkanoyl group is defined as "-(O=)CC 1~6 It means an "alkyl group," and examples thereof include an acetyl group, an ethylcarbonyl group, or an n-pentylcarbonyl group.

[0034] In this specification, unless otherwise specified, "halogenated C 1~6 The "C alkyl group" refers to the "C 1~6 "Alkyl" means a group optionally substituted with several, preferably 1 to 5, halogen atoms, such as fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 1,1,2,2-tetrafluoroethyl, or pentafluoroethyl.

[0035] [1-1] In the formula (1) of the above aspect [1], n is preferably an integer of 1 or 2; more preferably an integer of 1.

[0036] [1-2] In the formula (1) of the above aspect [1], R 1 and R 2 is preferably R 1 and R2 At least one of R 1 and R 2 are both hydrogen atoms.

[0037] [1-3] In the formula (1) of the above aspect [1], R 3 is preferably a hydrogen atom or the following partial structural formula (S-1) [wherein the formula does not include the right side of the dashed line]:

[0038] [ka]

[0039] [In partial structural formula (S-1), m is an integer of 0 or 1, and R 4 is a hydrogen atom, C 1~10 Alkyl group, C 1~6 Alkoxy-substituted C 1~10 Alkyl group, C 2~10 Alkenyl group, C 2~10 Alkynyl groups and C 4~10 alkyldienyl groups] and more preferably, a hydrogen atom or the partial structural formula (S-1) [in formula (S-1), m is an integer of 0, and R 4 is a hydrogen atom, C 1~10 Alkyl group or C 1~6 Alkoxy-substituted C 1~10 alkyl group]; more preferably, a hydrogen atom or the partial structural formula (S-1) [in formula (S-1), m is an integer of 0, and R 4 is a group selected from the group consisting of an isopropyl group, an n-octyl group, and a methoxy n-hexyl group.

[0040] [1-4] In the formula (1) of the above aspect [1], R 3 is preferably a hydrogen atom or the following partial structural formula (S-1-A) [wherein the formula does not include the right side of the dashed line]:

[0041] [ka]

[0042] [In partial structural formula (S-1-A), R 4A is R in the above aspect [1] 4 is the same definition as is.

[0043] [1-4-1] In the formula (1) of the above aspect [1], R 3 is preferably a hydrogen atom or a partial structural formula (S-1-A) [in formula (S-1-A), R 4A is a hydrogen atom, C 1~10 Alkyl group, C 1~6 Alkoxy-substituted C 1~10 Alkyl group, C 2~10 Alkenyl group, C 2~10 Alkynyl groups and C 4~10 alkyldienyl groups]; more preferably, a hydrogen atom or a group represented by partial structural formula (S-1-A) [in formula (S-1-A), R 4A is a hydrogen atom, C 1~10 Alkyl group or C 1~6 Alkoxy-substituted C 1~10 is an alkyl group]; more preferably, a hydrogen atom or a group represented by partial structural formula (S-1-A) [in formula (S-1-A), R 4A is a group selected from the group consisting of an isopropyl group, an n-octyl group, and a methoxy n-hexyl group.

[0044] [1-5] In the formula (1) of the above aspect [1], R 5 is preferably a hydrogen atom or a methyl group; more preferably a hydrogen atom.

[0045] [1-6] In the formula (1) of the above aspect [1], R 6 is preferably a hydrogen atom or a methyl group; more preferably a methyl group.

[0046] [1-7] In the formula (1) of the above aspect [1], R 7 is preferably a hydrogen atom, C1~6 Alkyl group, C 6~10 Aryl group, or C 7~16 Aryl alkyl group (the above C 6~10 Aryl group or C 7~16 Aryl alkyl groups contain halogen atoms, C 1~6 Alkyl group or C 1~6 and is preferably a hydrogen atom, C 1~6 Alkyl group or C 7~16 Aryl alkyl group (the above C 7~16 The aralkyl group is optionally substituted with 1 to 3 halogen atoms; more preferably, it is a hydrogen atom, a methyl group, a benzyl group, a 2-bromobenzyl group, or a 2-chlorobenzyl group.

[0047] One of the characteristics of the compound represented by formula (1) of the above aspect [1] is that it has a cyclopropane ring in the molecule.

[0048] [1-8] In the formula (1) of the above embodiment [1], the steric configuration of the cyclopropane ring can be any of the following four configurations (a) to (d).

[0049] (a) A form in which the group bonded to the carbon atom at position 1 (upper right in formula (1)) in the cyclopropane ring and the group bonded to the carbon atom at position 2 (upper left in formula (1)) are in a cis configuration, and when n = 0, the configuration of the carbon atom at position 2 in the cyclopropane ring is R configuration (a form represented by the following partial structural formula (CP-C-DW)); (b) A form in which the group bonded to the carbon atom at position 1 (upper right in formula (1)) in the cyclopropane ring and the group bonded to the carbon atom at position 2 (upper left in formula (1)) are in a cis configuration, and when n = 0, the configuration of the carbon atom at position 2 in the cyclopropane ring is S configuration (a form represented by the following partial structural formula (CP-C-UP)); (c) A form in which the group bonded to the carbon atom at position 1 (upper right in formula (1)) in the cyclopropane ring and the group bonded to the carbon atom at position 2 (upper left in formula (1)) are in a trans configuration, and when n = 0, the configuration of the carbon atom at position 2 in the cyclopropane ring is R configuration (a form represented by the following partial structural formula (CP-T-DW)); (d) A form in which the group bonded to the carbon atom at position 1 (upper right in formula (1)) in the cyclopropane ring and the group bonded to the carbon atom at position 2 (upper left in formula (1)) are in a trans configuration, and when n = 0, the configuration of the carbon atom at position 2 in the cyclopropane ring is S configuration (a form represented by the following partial structural formula (CP-T-UP)).

[0050] [ka]

[0051] [1-8-1] In the above-mentioned embodiment [1-8], the configuration of the cyclopropane ring is preferably (a) (the above partial structural formula (CP-C-DW)) or (b) (the above partial structural formula (CP-C-UP)) described in the above-mentioned embodiment [1-8]; more preferably (a) (the above partial structural formula (CP-C-DW)).

[0052] [1-9] In the formula (1) of the above aspect [1], preferably, R 3 is a hydrogen atom or a partial structural formula (S-1-A):

[0053] [ka]

[0054] [In formula (S-1-A), R 4A is a hydrogen atom, C 1~10 Alkyl group, C 1~6 Alkoxy-substituted C 1~10 Alkyl group, C 2~10 Alkenyl group, C 2~10 Alkynyl groups and C 4~10alkyldienyl groups; R 7 is a hydrogen atom, C 1~6 Alkyl group, C 6~10 Aryl group, or C 7~16 Aryl alkyl group (the above C 6~10 Aryl group or C 7~16 Aryl alkyl groups contain halogen atoms, C 1~6 Alkyl group or C 1~6 The configuration of the cyclopropane ring is the partial structural formula (CP-C-DW) or the partial structural formula (CP-C-UP) described in the above embodiment [1-8-1].

[0055] [1-9-1] In the formula (1) of the above aspect [1], R 3 is a hydrogen atom or a partial structural formula (S-1-A) [in formula (S-1-A), R 4A is a hydrogen atom, C 1~10 Alkyl group or C 1~6 Alkoxy-substituted C 1~10 is an alkyl group; R 7 is a hydrogen atom, C 1~6 Alkyl group or C 7~16 Aryl alkyl group (the above C 7~16 The aralkyl group may be substituted with 1 to 3 halogen atoms; and the configuration of the cyclopropane ring is the partial structural formula (CP-C-DW) described in the above embodiment [1-8-1].

[0056] [1-9-2] In the formula (1) of the above aspect [1], R 3 is a hydrogen atom or a partial structural formula (S-1-A) [in formula (S-1-A), R 4A is a group selected from the group consisting of isopropyl, n-octyl, methoxy n-hexyl, 7-aminoheptyl, 4-phenyl n-butyl, phenylmethoxy, morpholino n-butyl, and 1-octenyl; R 7is a hydrogen atom, a methyl group, a benzyl group, a 2-bromobenzyl group, or a 2-chlorobenzyl group; the configuration of the cyclopropane ring is the partial structural formula (CP-C-DW) described in the above embodiment [1-8-1].

[0057] As described above, by appropriately combining each of the aspects [1-1] to [1-9-2] of the present invention and their preferred aspects, as well as the definitions of the substituents, it is possible to arbitrarily form a preferred aspect of the compound represented by the formula (1) of the aspect [1].

[0058] Substituent R 3 Formula (S-1) in (wherein the formula does not include the right side of the dashed line):

[0059] [ka]

[0060] [m and R in formula (S-1)] 4 is the same as defined in the above aspect [1]. Unless otherwise specified, the formula (S-1) in this specification includes the isomers represented by the formula (S-1-a) or the formula (S-1-b).

[0061] [ka]

[0062] Similarly, the substituent R 3 Formula (S-2) [the right side of the dashed line is not included]:

[0063] [ka]

[0064] [R in formula (S-1)] 8 is the same as defined in the above aspect [1]. Unless otherwise specified, in this specification, formula (S-2) is meant to include the isomers represented by formula (S-2-a) or formula (S-2-b).

[0065] [ka]

[0066] When the compound or intermediate compound of the present invention has geometric isomers, configurational isomers, stereoisomers, conformational isomers, etc., they can be isolated by known means.

[0067] When the compound of the present invention or an intermediate compound is an optically active compound, it can be separated into a (+) or (-) form from the corresponding racemic form by a conventional optical resolution method.

[0068] When the compound or intermediate compound of the present invention has optical isomers, stereoisomers, positional isomers, rotational isomers, and tautomers, each isomer can be obtained as a single compound by a synthesis method or a separation method known per se. Examples of the separation method include optical resolution methods such as fractional recrystallization, diastereomeric method, and chiral column method. Each resolution method will be described in detail below.

[0069] Fractional recrystallization: This method involves ionically bonding an optical resolution agent to a racemate to obtain crystalline diastereomers, then separating the crystalline diastereomers by fractional recrystallization, and optionally removing the optical resolution agent to obtain an optically pure compound. Examples of optical resolution agents include (+)-mandelic acid, (-)-mandelic acid, (+)-tartaric acid, (-)-tartaric acid, (+)-1-phenethylamine, (-)-1-phenethylamine, cinchonine, (-)-cinchonidine, and brucine.

[0070] Diastereomeric method: A mixture of racemates is covalently bound to an optical resolution agent to obtain a mixture of diastereomers, which is then separated into optically pure diastereomers by conventional separation means (e.g., fractional recrystallization, silica gel column chromatography, HPLC, etc.), and then optically pure optical isomers are obtained through a step of removing the optical resolution agent by a chemical reaction (e.g., hydrolysis reaction).

[0071] For example, when the compound of the present invention or an intermediate compound has a hydroxyl group or an amino group (primary or secondary), the compound can be condensed with an optically active organic acid (e.g., α-methoxy-α-(trifluoromethyl)phenylacetic acid, (-)-menthoxyacetic acid, etc.) to give diastereomers of esters or amides, respectively. Furthermore, when the compound of the present invention has a carboxy group, the compound can be condensed with an optically active amine or an optically active alcohol to give diastereomers of amides or esters, respectively. The diastereomers obtained by the condensation reaction are separated and subjected to hydrolysis with an acid or base, whereby they are converted to optically pure optical isomers of the original compound.

[0072] Chiral column method: This is a method of direct optical resolution by subjecting a racemate or its salt to chromatography using a chiral column (a column for separating optical isomers).

[0073] For example, in the case of high performance liquid chromatography (HPLC), a mixture of optical isomers can be added to a chiral column (e.g., the CHIRAL series manufactured by Daicel Corporation) and developed with an elution solvent (a single solvent such as water, various buffers (e.g., phosphate buffer), and organic solvents (e.g., ethanol, methanol, isopropanol, acetonitrile, trifluoroacetic acid, diethylamine, etc.), or a mixture thereof), to separate the optical isomers. Furthermore, in the case of gas chromatography, a chiral column (e.g., CP-Chirasil-DeX CB (manufactured by GL Sciences)) can be used to separate the optical isomers. Furthermore, in the case of supercritical fluid chromatography (SFC), a mixture of optical isomers can be added to a chiral column (e.g., the CHIRAL series manufactured by Daicel Corporation) and developed with an elution solvent such as carbon dioxide and an appropriate organic solvent (e.g., methanol, ethanol, isopropanol, trifluoroacetic acid, diethylamine, etc.) to separate the optical isomers.

[0074] When the compound or intermediate compound of the present invention is optically active, it can be synthesized by asymmetric synthesis, which selectively synthesizes one of the optical isomers. Optically active compounds can be synthesized by (1) asymmetric synthesis, which involves enantioselectively reacting a racemic compound to produce an optically active compound, or (2) diastereoselective synthesis from naturally occurring optically active compounds (such as sugars and amino acids).

[0075] [2] In the second aspect of the present invention, in the compounds of formula (1) of aspect [1], preferred compounds are the compounds listed below (compounds TCP-301 to TCP-310, compounds TCP-315 to TCP-319, compounds TCP-320 to TCP-323, compounds TCP-325 to TCP-330, compound TCP-335, compounds TCP-338 to TCP-344, compound TCP-346, compound TCP-348, and compound TCP-359), or pharmaceutically acceptable salts or solvates thereof. Of the above compounds, the structures of compounds TCP-321 and subsequent compounds are those represented by R in formula (1). 3Therefore, among the structures shown below, the structures of compounds TCP-321 and subsequent compounds are the same as those of compound TCP-320 except for the structure of R in formula (1). 3 The structure of

[0076] [ka]

[0077] [ka]

[0078] The pharmaceutically acceptable salt is not particularly limited, but examples thereof include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.

[0079] Suitable examples of metal salts include alkali metal salts such as lithium salts, sodium salts, potassium salts, and cesium salts; alkaline earth metal salts such as calcium salts, magnesium salts, and barium salts; and aluminum salts (including, for example, monosalts, disodium salts, and dipotassium salts).

[0080] Suitable examples of salts with organic bases include salts with methylamine, ethylamine, t-butylamine, t-octylamine, diethylamine, trimethylamine, triethylamine, cyclohexylamine, dicyclohexylamine, dibenzylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, morpholine, pyridine, picoline, lysine, arginine, ornithine, ethylenediamine, N-methylglucamine, glucosamine, phenylglycine alkyl ester, guanidine, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, and N,N'-dibenzylethylenediamine.

[0081] Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, and the like.

[0082] Suitable examples of salts with organic acids include salts with aliphatic monocarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, butyric acid, valeric acid, enanthic acid, capric acid, myristic acid, palmitic acid, stearic acid, lactic acid, sorbic acid, and mandelic acid; salts with aliphatic dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, and tartaric acid; salts with aliphatic tricarboxylic acids such as citric acid; salts with aromatic monocarboxylic acids such as benzoic acid and salicylic acid; salts with aromatic dicarboxylic acids such as phthalic acid; salts with organic carboxylic acids such as cinnamic acid, glycolic acid, pyruvic acid, oxylic acid, salicylic acid, and N-acetylcysteine; salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; and acid addition salts with acidic amino acids such as aspartic acid and glutamic acid.

[0083] Suitable examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc., and suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc.

[0084] The salts can be obtained by a conventional method, for example, by mixing the compound represented by formula (1) with a solution containing an appropriate amount of acid or base to form the desired salt, and then separating and filtering the salt or by distilling off the mixed solvent. In addition, the pharmaceutically acceptable salt of the compound represented by formula (1) can form a solvate.

[0085] Solvate refers to a molecular complex formed between an active compound and one or more pharmaceutically acceptable solvents. Pharmaceutically acceptable solvents include water, ethanol, isopropanol, ethyl acetate, acetic acid, ethanolamine, etc. When the solvent molecule is water, it is specifically called a "hydrate."

[0086] The description of the compound represented by formula (1) of the present invention includes the description of its salts, solvates, and solvates of its salts.

[0087] The compound of formula (1) according to the present invention can also be used as a prodrug. 7 A prodrug can be formed at the moiety represented by the formula: (I) (II). For example, in the case of an ester, which is one form of prodrug, the water solubility of the compound is reduced and the lipid solubility is increased. A compound with increased lipid solubility not only promotes absorption into the body, but is also converted into an active form while being absorbed by esterases present in the digestive tract, etc., thereby increasing enteral absorption, or reaches the site where it is to act in the body, where it is converted into a carboxyl group by esterases present in organs and tissues, and can act at the site. As a result, the bioavailability is ultimately increased. The compounds according to the present invention include compounds in such prodrug forms as well as compounds in non-prodrug forms.

[0088] The compound represented by formula (1) (negamycin derivative) can be produced by taking into consideration the examples described below and the common general technical knowledge at the time of filing of this application. For example, the compound can be produced by referring to the above-mentioned patent and non-patent documents as well as previously reported documents on the total synthesis of negamycin.

[0089] Examples of total synthesis of negamycin include (1) Wang et al., J. Am. Chem. Soc., 104, pp. 6465-6466, 1982; (2) Iida et al., J. Am. Chem. Soc., 108, pp. 4647-4648, 1986; (3) Davies et al., Tetrahedron: Asymmetry, 7, pp. 1919-1922, 1996; and (4) Williams et al., J. Org. Chem., 67, pp. 6361-6365, 2002.

[0090] Here, for example, in formula (1), R 3 The compound (the following formula (1)-A) in which is a hydrogen atom can be produced by the following production method.

[0091] [ka]

[0092] (In the above formula, P 1 means a protecting group for an amino group such as tert-butoxycarbonyl (Boc), and the substituent R 1 ~R 7 is the same as that of formula (1) in the above embodiment.) The amino group of the compound of formula (SM-1) (the compound of formula (SM-1) can be produced according to the method described in the Examples below) is converted into a protecting group (P 1 The compound of formula (IM-A1) obtained by protecting with a protecting group P of formula (IM-A2) is subjected to an amidation reaction with the compound of formula (SM-2) using a dehydration condensation agent such as 1-hydroxybenzotriazole (HOBt) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) to obtain the compound of formula (IM-2A). 1 The above-mentioned predetermined compound can be obtained by deprotecting the group R in formula (SM-1) and formula (SM-2). 1 ~R 7 Depending on the type of R, it may be desirable to use a protecting group during the reaction. For example, 1 and R 2 In the case of a compound in which both are hydrogen atoms, the amino groups substituted by these are 1 It is preferable to protect the group with a group similar to that of R 7 In the synthesis of a compound in which is a hydrogen atom, it is preferable to protect the carboxyl group as a tert-butyl ester, methyl ester, ethyl ester, benzyl ester or the like at the stage of the compound of formula (SM-2).

[0093] In formula (1), R 3The compound represented by partial structural formula (S-1) can be produced by subjecting the compound represented by formula (1)-A above and a carboxylic acid compound corresponding to partial structural formula (S-1) to an amidation reaction similar to that described above. In this case, too, it is desirable to protect the free amino group in partial structural formula (S-1) during the reaction.

[0094] In formula (1), R 1 and R 2 is a hydrogen atom, and R 3 The compound represented by partial structural formula (S-1) (the following formula (1)-B) can also be produced by the following production method.

[0095] [ka]

[0096] (In the above formula, P 2 means a protecting group for an amino group such as Boc, and the substituent R 4 ~R 7 is the same as that of formula (1) in the above embodiment.) First, R 3 The compound of formula (IM-1B) is obtained by using a compound of formula (SM-3) in which a tert-butylsulfonyl (Bus) group, which is a protecting group for an amino group, has been introduced at the position corresponding to the formula (the compound of formula (SM-3) can be produced in accordance with the method described in the Examples below) and a compound of formula (SM-4) as starting materials and carrying out a condensation reaction in the same manner as in the production method of the compound of formula (IM-A2).

[0097] Next, the compound of formula (IM-1B) is subjected to acidic conditions to deprotect the Bus group, and then the compound of formula (SM-5) is used in a condensation reaction in the same manner as above to obtain the compound of formula (IM-2B). Then, the azide (-N3) group of the compound of formula (IM-2B) is reduced by catalytic reduction or the like to remove the protecting group P 2 The above-mentioned predetermined compound can be obtained by deprotecting the compound.

[0098] In the above production methods, by appropriately selecting the configuration of the cyclopropane ring in the starting material, a compound having the same configuration as that of the final product can be obtained.

[0099] In the method for producing the compound represented by formula (1)-A or formula (1)-B, the protecting group P 1 and P 2 The protection or deprotection of the protecting groups used during the reaction, as well as the protection or deprotection of the protecting groups used during the reaction, can be carried out according to a method known in the literature, for example, the method described in "Protective Groups in Organic Synthesis 4th Edition," 4th Edition, 2007, John Wiley & Sons, Greene et al.

[0100] Furthermore, the compound represented by formula (1) (negamycin derivative) may be synthesized by a liquid phase method, as described in the Examples below, or by a solid phase method using a carrier such as resin beads.

[0101] The target compound thus produced can be isolated by known separation and purification means such as extraction, partitioning, column chromatography, etc.

[0102] <<Use (Pharmaceutical composition for preventing and / or treating diseases caused by nonsense mutations)>> In another aspect, the present invention provides a pharmaceutical composition containing at least one of the above-mentioned compounds or a pharmaceutically acceptable salt (or solvate) thereof as an active ingredient, as a use of the above-mentioned compounds. The pharmaceutical composition may contain two or more of the above-mentioned compounds as active ingredients. In particular, the compound of formula (1) according to the present invention acts on ribosomes in a situation where a premature termination codon (PTC) is generated by a nonsense mutation, preventing the production of a specific protein. The ribosomes read through the premature termination codon generated by the nonsense mutation and translate the protein, resulting in the production of a wild-type normal protein. In the present invention, a wild-type normal protein refers to a protein consisting of an amino acid sequence encoded by a normal wild-type gene that does not contain a mutation.

[0103] The term "wild-type normal protein" also encompasses proteins consisting of an amino acid sequence substantially identical to the amino acid sequence described above. Here, "substantially identical protein" refers to, for example, a protein consisting of an amino acid sequence encoded by a normal wild-type gene, in which one or more amino acids have been deleted, substituted, or added, and which has the same function or activity as the protein encoded by the normal wild-type gene. Here, "one or more" preferably refers to 1 to 5, 4, 3, or 2. Furthermore, the term "substantially identical protein" refers to a protein that has 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 96, 97, 98, or 99% or more sequence identity with the amino acid sequence encoded by the normal wild-type gene, as calculated using BLAST or similar (e.g., default, i.e., initial setting parameters), and has the same function or activity as the protein encoded by the normal wild-type gene.

[0104] [3] A third aspect of the present invention is a pharmaceutical composition comprising, as an active ingredient, at least one of the compounds according to any one of aspects [1] or [2], or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0105] [4] A fourth aspect of the present invention is a pharmaceutical composition for preventing and / or treating a disease caused by a nonsense mutation, characterized in that it contains, as an active ingredient, an amount effective for preventing and / or treating the disease of at least one of the compound according to any one of aspects [1] or [2], or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0106] [5] A fifth aspect of the present invention is a preventive and / or therapeutic agent for a disease caused by a nonsense mutation, characterized by containing, as an active ingredient, at least one of the compounds according to any one of aspects [1] or [2] above, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0107] [6] A sixth aspect of the present invention is a method for preventing and / or treating a disease caused by a nonsense mutation, the method comprising administering at least one of the compounds according to any one of aspects [1] or [2], or a pharmaceutically acceptable salt thereof, or a solvate thereof, to a subject in need of prevention and / or treatment of the disease.

[0108] [7] A seventh aspect of the present invention is use of a compound according to any one of aspects [1] or [2], or a pharmaceutically acceptable salt thereof, or a solvate thereof, for the manufacture of a medicament for use in the prevention and / or treatment of a disease caused by a nonsense mutation.

[0109] [8] An eighth aspect of the present invention is a compound according to any one of aspects [1] or [2], or a pharmaceutically acceptable salt thereof, or a solvate thereof, for use in preventing and / or treating a disease caused by a nonsense mutation.

[0110] [9] A ninth aspect of the present invention is a readthrough agent for a premature stop codon formed by a nonsense mutation, characterized by containing, as an active ingredient, at least one of the compounds according to any one of aspects [1] or [2], or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0111] "Nonsense mutation-induced diseases" refers to diseases caused by a point mutation in a gene that results in a premature termination codon (PTC) in the middle of the gene, thereby suppressing the expression of a protein with normal function. Furthermore, nonsense mutation-induced diseases also include diseases caused by the inhibition of protein expression due to the degradation of mRNA containing the premature termination codon.

[0112] Diseases caused by nonsense mutations include Nagashima palmoplantar keratoderma, muscular dystrophy, infantile neuronal ceroid lipofuscinosis, multiple sclerosis, Alzheimer's disease, Tay-Sachs disease, Parkinson's disease, hemophilia, von Willebrand disease, ataxia-telangiectasia, beta-thalassemia, osteogenesis imperfecta, neurofibromatosis, lysosomal storage disorders, Hurler disease, familial hypercholesterolemia, tuberous sclerosis, cystic fibrosis, retinitis pigmentosa, hereditary ATTR amyloidosis, gigantism, dwarfism, Niemann-Pick disease, Marfan syndrome, epidermolysis bullosa, xeroderma pigmentosum, congenital ichthyosis, pseudoxanthoma elasticum, oculocutaneous albinism, atopic dermatitis, and color vision disorders.

[0113] Of the above diseases, the compound represented by formula (1) of the present invention can be preferably used for a disease selected from the group consisting of Nagashima palmoplantar keratosis, muscular dystrophy, cystic fibrosis, epidermolysis bullosa, xeroderma pigmentosum, congenital ichthyosis, pseudoxanthoma elasticum, oculocutaneous albinism, and atopic dermatitis; more preferably, it can be used for Duchenne muscular dystrophy or Becker muscular dystrophy, among muscular dystrophies, and Nagashima palmoplantar keratosis; even more preferably, it can be used for Duchenne muscular dystrophy or Nagashima palmoplantar keratosis; and most preferably, it can be used for Nagashima palmoplantar keratosis.

[0114] The administration route of the pharmaceutical composition of the present invention is not limited, and can be oral, subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, nasal, buccal, transmucosal, or other routes. The dosage form is also not limited. For example, for oral administration, it can be tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, spirits, syrups, extracts, or elixirs. For parenteral administration, it can be, for example, injections such as subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections; transdermal or patch preparations, ointments, or lotions; sublingual preparations and buccal patches for buccal administration; and aerosol preparations for nasal administration. For example, for the treatment of muscular dystrophy, the pharmaceutical composition of the present invention can be administered as an injection (e.g., an intramuscular injection for direct administration into the muscle). For the treatment of Nagashima palmoplantar keratosis, the pharmaceutical composition of the present invention can be administered as an ointment or lotion. The pharmaceutical composition of the present invention may be in a sustained or slow release dosage form.

[0115] The pharmaceutical composition of the present invention may contain various pharmaceutically acceptable ingredients, such as excipients, disintegrants, diluents, lubricants, flavoring agents, coloring agents, sweeteners, corrigents, suspending agents, wetting agents, emulsifiers, dispersing agents, adjuvants, preservatives, buffers, binders, stabilizers, coating agents, etc., and may contain a plurality of these.

[0116] The dosage of the pharmaceutical composition of the present invention is not limited, and is appropriately selected depending on the efficacy of the contained ingredients, the dosage form, the administration route, the type of disease, the weight, age, condition, and other characteristics of the patient to be administered, or the judgment of a physician, etc. For example, it is in the range of about 0.01 μg to about 100 mg per kg of patient weight, preferably about 0.1 μg to about 1 mg. The dosage can be administered once a day or in divided doses, or may be administered intermittently once every few days or weeks.

[0117] The read-through activity of the compound of the present invention can be measured, for example, by constructing a vector containing (i) a promoter, (ii) a first translation initiation codon and a first reporter gene located downstream of the promoter, (iii) a second reporter gene located downstream of the first reporter gene, (iv) a sequence located between the first and second reporter genes and containing a premature stop codon (PTC) derived from a gene causing a disease caused by nonsense mutation, and (v) a translation stop codon located downstream of the second reporter gene, and then introducing the vector into a host cell or an animal to produce a transgenic animal. Specifically, a compound to be evaluated (test compound) is administered to the host cell or transgenic animal, and the ratio of the expression level of the second reporter gene to the expression level of the first reporter gene is compared between the presence and absence of the test compound. The higher the expression level of the second reporter gene, the greater the read-through activity. In this case, for example, a CMV promoter, a β-actin promoter, etc. can be used as the promoter. Furthermore, for example, a β-galactosidase gene, a luciferase gene, a GFP (Green Fluorescent Protein) gene, a CAT (Chroramphenicol Acetyl Transferase) gene, etc. can be used as the first and second reporter genes. Furthermore, as a sequence containing a premature stop codon derived from a gene causing a disease due to a nonsense mutation, a sequence containing a stop codon (UAA, UAG, or UGA) in the reading frame can be used. For example, a sequence containing a premature stop codon of the dystrophin gene, which is a causative gene for muscular dystrophy, or a sequence containing a premature stop codon of the SERPINB7 gene, which is a causative gene for Nagashima palmoplantar keratosis, etc. can be used. [Example]

[0118] Examples will be given below to explain the present invention in more detail, but these examples are merely implementations and do not limit the present invention, and may be modified within the scope of the present invention.

[0119] Nuclear magnetic resonance spectroscopy (NMR) was measured using JEOL JNM-EX-270, JEOL JNM-AL-400, JEOL JMM-ECA-500, and Bruker AVANCE-III (400 MHz).

[0120] Mass spectrometry (HRMS (ESI) m / z) was measured on a Waters Xevo G2-S QTof, a Waters MICRO MASS LCT-premier, a JEOL JMS-700TZ for (FAB), and a JEOL JMS-100GCv for (EI).

[0121] 1 In the H-NMR data, the NMR signal patterns are: s, d, doublet, t, triplet, q, quartet, m, multiplet, br, broad, brs, broad singlet, brd, broad doublet, J, coupling constant, Hz, hertz, CDCl3, deuterated chloroform, DO, heavy water, and DMSO-d6, dimethyl sulfoxide. 1 In H-NMR data, protons of hydroxyl groups (OH), amino groups (NH2), carboxyl groups (COOH), etc. are broadband and cannot be confirmed, so they are not recorded in the data.

[0122] In the HRMS (ESI) m / z data, M is the molecular weight, [M+H] + [M+Na] + means the molecular ion peak.

[0123] Compounds TCP-301-310 and compounds 315-317 were purified by HPLC under the following conditions (instrument name: Waters 600 HPLC System, column: YMC-Pack ODS-AM 250x20mm Column (TCP-301-304, 307) and Waters SunFire C18 5μm 4.6x150mm Column (TCP-305-306, 308-310, 315-317), mobile phase: 0.1% trifluoroacetic acid (TFA) in ultrapure water and 0.1% TFA in acetonitrile, flow rate: 5mL / min, measurement temperature: room temperature, measurement wavelength: UV 222nm). The purified products were obtained as TFA salts by this procedure.

[0124] Compound Examples (Example 1) to (Example 4) [1. Synthesis of TCP-301 to TCP-304] Example A: Synthesis of cyclopropane intermediates (compounds 3 and 4)

[0125] [ka]

[0126] <Step 1> Synthesis of (3R,4S)-3,4-dimethyl-3-(phenylsulfonyl)dihydrofuran-2(3H)-one (Compound IM-A1) Compound IM-A1 was synthesized from (R)-epichlorohydrin according to the method described in the literature (Kazuta, Y. et al. J. Org. Chem., 2002, 67, pp. 1669-1677).

[0127] <Step 2> Synthesis of (1R,2S)-2-(t-butyldiphenylsilyloxy)methyl-1-(N-methoxy-N-methyl)carbamoyl-1-phenylsulfonylcyclopropane (Compound 2) N,O-Dimethylhydroxylamine hydrochloride (4.49 g, 46.0 mmol) was added to dichloromethane (CHCl) (50 mL) to form a suspension. Triethylamine (EtN) (6.22 mL, 45.0 mmol) was added at 0°C, and the mixture was stirred at room temperature for 30 minutes. The resulting precipitate was filtered off. The resulting filtrate was slowly added to a CHCl solution (80 mL) of compound IM-A1 (2.86 g, 12.0 mmol) obtained in (Example A) <Step 1> and aluminum chloride (AlCl) (3.20 g, 24.0 mmol) at 0°C, and the mixture was stirred at room temperature for 8 hours. 1 M aqueous hydrochloric acid was added, and the mixture was partitioned between CHCl and 1 M aqueous hydrochloric acid. The organic layer was washed with saturated aqueous NaHCO and saturated brine, then dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in dimethylformamide (DMF) (100 mL), followed by the sequential addition of imidazole (3.27 g, 48.0 mmol) and tert-butyldiphenylchlorosilane (TBDPSCl) (6.24 mL, 24.0 mmol), and the mixture was stirred at room temperature for 12 hours. Methanol (MeOH) was added to the reaction mixture, and the solvent was evaporated under reduced pressure. The resulting residue was partitioned between ethyl acetate (AcOEt) and HO. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 90:10 → 75:25) to obtain compound 2 (6.05 g) as a colorless amorphous solid.

[0128] <Step 3> Synthesis of (1S,2R)-cis-2-(t-butyldiphenylsilyloxymethyl)cyclopropane-1-(N-methoxy-N-methyl)carboxamide (Compound 3) and (1R,2R)-trans-2-(t-butyldiphenylsilyloxymethyl)cyclopropane-1-(N-methoxy-N-methyl)carboxamide (Compound 4) Compound 2 (5.38 g, 10.0 mmol) obtained in Example A, Step 2, was dissolved in MeOH (80 mL). Magnesium powder (7.44 g) was added at room temperature, and the mixture was stirred at 55°C for 1.5 hours. Ice-cooled 0.5 M aqueous hydrochloric acid was added to the reaction mixture, followed by partitioning between ethyl acetate and 1 M aqueous hydrochloric acid. The organic layer was washed with saturated aqueous NaHCO3 and saturated brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (15-20% AcOEt in hexane) to give compound 3 (cis isomer) (1.31 g) as a white solid and compound 4 (trans isomer) (2.58 g) as a pale yellow oil. The instrumental data for compound 3 were consistent with those reported in the literature (Watanabe, M. et al., J. Med. Chem., 2006, 49, pp. 5587-5596).

[0129] Example B: Synthesis of cyclopropane intermediate (compound 10)

[0130] [ka]

[0131] <Step 1> Synthesis of (1S,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropanecarbaldehyde (Compound 5) Compound 5 was synthesized from Compound 3 obtained in Example A according to the method described in the literature (Watanabe, M. et al., J. Med. Chem., 2006, 49, pp. 5587-5596).

[0132] <Step 2> Synthesis of (S,E)-N-(((1S,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)methylene)-2-methylpropane-2-sulfinamide (Compound 6) Compound 6 was synthesized from Compound 5 obtained in (Example B) <Step 1> according to the method described in the literature (Watanabe, M. et al., Bioorg. Med. Chem., 2011, 19, pp. 5984-5988).

[0133] <Step 3> Synthesis of (R)-benzyl 3-((1S,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 7) Under an argon atmosphere, potassium hexamethyldisilazide (KHMDS) (0.6 M toluene solution, 70 mL, 42 mmol) was added to tetrahydrofuran (THF) (120 mL) at −78°C. A THF solution (20 mL) of benzyl acetate (5.7 mL, 40 mmol) was slowly added dropwise at −78°C, and the mixture was stirred at the same temperature for 10 minutes. A THF solution (55 mL) of compound 6 (11.2 g, 25.4 mmol) obtained in (Example B) <Step 2> was added at −78°C and stirred for 30 minutes. After that, saturated aqueous ammonium chloride solution was added, and the mixture was warmed to room temperature and stirred for 20 minutes. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated aqueous ammonium chloride and saturated brine and dried over sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1 → 1:1) to obtain compound 7 (12.8 g) as a colorless oil as a single diastereomer.

[0134] <Step 4> Synthesis of (R)-benzyl 3-((S)-1,1-dimethylethylsulfinamido)-3-((1S,2R)-2-(hydroxymethyl)cyclopropyl)propanoate (Compound 8) Compound 7 (305 mg, 0.515 mmol) obtained in (Example B) <Step 3> was dissolved in THF (4.5 mL) and stirred under ice-cooling. Tetrabutylammonium fluoride (TBAF) (1 M THF solution, 0.772 mL, 0.772 mmol) was added and stirred under ice-cooling for 30 minutes, followed by stirring at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:ethyl acetate=1:2) to obtain compound 8 (173 mg) as a colorless oil.

[0135] <Step 5> Synthesis of (R)-benzyl 3-((1S,2R)-2-(azidomethyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 9) Under an argon atmosphere, dichloromethane (CHCl) (2.0 mL) was added to compound 8 (72 mg, 0.20 mmol) obtained in (Example B) <Step 4> and stirred under ice-cooling. Triethylamine (0.068 mL, 0.49 mmol) and methanesulfonyl chloride (0.019 mL, 0.24 mmol) were added sequentially and stirred under ice-cooling for 30 minutes. The reaction mixture was partitioned with ethyl acetate and water. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in DMF (2 mL) under an argon atmosphere, sodium azide (20 mg, 0.31 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was partitioned with ethyl acetate and saturated aqueous NaHCO, the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to give compound 9 (66 mg) as a colorless oil.

[0136] <Step 6> Synthesis of (R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoic acid (Compound 10) (Example B) Compound 9 (22 mg, 0.058 mmol) obtained in <Step 5> was added with a 4 M solution of hydrochloric acid in ethyl acetate (0.5 mL, 2.0 mmol) and stirred for 1 hour. The solvent was removed by evaporation under reduced pressure, and the resulting residue was dissolved in methanol (0.6 mL). Palladium on carbon (Pd / C) (6 mg) was added, and the mixture was stirred under a hydrogen atmosphere for 30 minutes. The reaction mixture was filtered through Celite, and the solvent was removed by evaporation under reduced pressure. The resulting residue was dissolved in methanol (0.6 mL). Triethylamine (0.036 mL, 0.26 mmol) and di-tert-butyl dicarbonate (BocO) (0.040 mL, 0.17 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction mixture was partitioned with ethyl acetate and 1 M hydrochloric acid. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to give compound 10 (14 mg) as a colorless oil.

[0137] Example C: Synthesis of cyclopropane intermediate (compound 16)

[0138] [ka]

[0139] <Step 1> Synthesis of (1R,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropanecarbaldehyde (Compound 11) Under an argon atmosphere, diisobutylaluminum hydride (0.95 M hexane solution, 2.6 mL, 2.47 mmol) was added to a solution of compound 4 (937 mg, 2.36 mmol) obtained in Example A in CHCl (15 mL) at −78°C, and the mixture was stirred at the same temperature for 2 hours. After quenching with a small amount of methanol, saturated brine was added and the mixture was stirred at room temperature for 2 hours. The mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 9:1) to obtain compound 11 (781 mg, 98%) as a colorless solid. The instrumental data for compound 11 were consistent with those reported in the literature (Kazuta, Y. et al., J. Org. Chem., 2002, 67, pp. 1669-1677).

[0140] <Step 2> Synthesis of (S,E)-N-(((1R,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)methylene)-2-methylpropane-2-sulfinamide (Compound 12) Compound 12 was synthesized from compound 11 obtained in (Example C) <Step 1> according to the literature (Watanabe, M. et al., Bioorg. Med. Chem, 2011, 19, pp. 5984-5988).

[0141] <Step 3> Synthesis of (R)-benzyl 3-((1R,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 13) In the same manner as in obtaining Compound 7, Compound 13 (548 mg) was obtained as a yellow-brown oily substance from Compound 12 (883 mg, 2.00 mmol) obtained in (Example C) <Step 2>.

[0142] <Step 4> Synthesis of (R)-benzyl 3-((S)-1,1-dimethylethylsulfinamido)-3-((1R,2R)-2-(hydroxymethyl)cyclopropyl)propanoate (Compound 14) In the same manner as in obtaining Compound 8, Compound 14 (271 mg) was obtained as a colorless oily substance from Compound 13 (452 ​​mg, 0.764 mmol) obtained in (Example C) <Step 3>.

[0143] <Step 5> Synthesis of (R)-benzyl 3-((1R,2R)-2-(azidomethyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 15) Compound 15 (207 mg) was obtained as a yellow-brown oily substance from compound 14 (232 mg, 0.656 mmol) obtained in (Example C) <Step 4> in the same manner as in the synthesis of compound 9 in (Example B).

[0144] <Step 6> Synthesis of (R)-3-((tert-butoxycarbonyl)amino)-3-((1R,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoic acid (Compound 16) Compound 16 (113 mg) was obtained as a colorless amorphous solid from compound 15 (167 mg, 0.441 mmol) obtained in (Example C) <Step 5> in the same manner as in the synthesis of compound 10 in (Example B).

[0145] Example D: Synthesis of cyclopropane intermediates (ent-3 and ent-4)

[0146] [ka]

[0147] <Step 1> Synthesis of (1S,5R)-1-(phenylsulfonyl)-3-oxabicyclo[3.1.0]hexan-2-one (compound ent-1) Compound ent-1 was synthesized from (S)-epichlorohydrin according to the method described in the literature (Kazuta, Y. et al., J. Org. Chem., 2002, 67, pp. 1669-1677).

[0148] <Step 2> Synthesis of (1S,2R)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-N-methoxy-N-methyl-1-(phenylsulfonyl)cyclopropanecarboxamide (compound ent-2) Compound ent-2 was synthesized from compound ent-1 obtained in (Example D) <Step 1> in the same manner as in the synthesis of compound 2 in (Example A).

[0149] <Step 3> Synthesis of (1R,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-N-methoxy-N-methylcyclopropanecarboxamide (ent-3) and (1S,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-N-methoxy-N-methylcyclopropanecarboxamide (compound ent-4) Compounds ent-3 and ent-4 were synthesized from compound ent-2 in the same manner as in the synthesis of compounds 3 and 4 in Example A, and in the same manner as in Example D, <Step 2>.

[0150] Example E: Synthesis of cyclopropane intermediate (compound 21)

[0151] [ka]

[0152] <Step 1> Synthesis of (1R,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropanecarbaldehyde (compound ent-5) Compound ent-5 was synthesized from compound ent-3 obtained in Example D according to the method described in the literature (Watanabe, M. et al., J. Med. Chem., 2006, 49, pp. 5587-5596).

[0153] <Step 2> Synthesis of (S,E)-N-(((1R,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)methylene)-2-methylpropane-2-sulfinamide (Compound 17) Compound 17 was synthesized from compound ent-5 obtained in (Example E) <Step 1> according to the method described in the literature (Yoshida, K. et al., Org. Lett., 2008, 10, pp. 3571-3574).

[0154] <Step 3> Synthesis of (R)-benzyl 3-((1R,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 18) Compound 18 (830 mg) was obtained as a yellow oily substance from compound 17 (883 mg, 2.00 mmol) obtained in (Example E) <Step 2> in the same manner as in the synthesis of compound 7 in (Example B).

[0155] <Step 4> Synthesis of (R)-benzyl 3-((S)-1,1-dimethylethylsulfinamido)-3-((1R,2S)-2-(hydroxymethyl)cyclopropyl)propanoate (Compound 19) Compound 19 (408 mg) was obtained as a colorless oil from compound 18 (722 mg, 1.22 mmol) obtained in (Example E) <Step 3> in the same manner as in the synthesis of compound 8 in (Example B).

[0156] <Step 5> Synthesis of (R)-benzyl 3-((1R,2S)-2-(azidomethyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 20) Compound 20 (161 mg) was obtained as a yellow oily substance from compound 19 (235 mg, 0.664 mmol) obtained in (Example E) <Step 4> in the same manner as in the synthesis of compound 9 in (Example B).

[0157] <Step 6> Synthesis of (R)-3-((tert-butoxycarbonyl)amino)-3-((1R,2S)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoic acid (Compound 21) Compound 21 (84 mg) was obtained as a white amorphous solid from compound 20 (137 mg, 0.36 mmol) obtained in <Step 5> of (Example E) in the same manner as in the synthesis of compound 10 of (Example B).

[0158] Example F: Synthesis of cyclopropane intermediate (compound 26)

[0159] [ka]

[0160] <Step 1> Synthesis of (1S,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropanecarbaldehyde (compound ent-11) Compound ent-11 was synthesized from compound ent-4 obtained in (Example D) in the same manner as in the synthesis of compound 11 in (Example C). The instrumental data of compound ent-11 were consistent with those in the literature (Kazuta, Y. et al., J. Org. Chem., 2002, 67, pp. 1669-1677).

[0161] <Step 2> Synthesis of (S,E)-N-(((1S,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)methylene)-2-methylpropane-2-sulfinamide (Compound 22) Compound 22 was synthesized from compound ent-11 obtained in (Example F) <Step 1> according to the method described in the literature (Yoshida, K. et al., Org. Lett., 2008, 10, pp. 3571-3574).

[0162] <Step 3> Synthesis of (R)-benzyl 3-((1S,2S)-2-(((tert-butyldiphenylsilyl)oxy)methyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 23) Compound 23 (448 mg) was obtained as a yellow-brown oily substance from compound 22 (595 mg, 1.35 mmol) obtained in <Step 2> of (Example F) in the same manner as in the synthesis of compound 7 of (Example B).

[0163] <Step 4> Synthesis of (R)-benzyl 3-((S)-1,1-dimethylethylsulfinamido)-3-((1S,2S)-2-(hydroxymethyl)cyclopropyl)propanoate (Compound 24) Compound 24 (142 mg) was obtained as a colorless oil from compound 23 (393 mg, 0.664 mmol) obtained in <Step 3> of (Example F) in the same manner as in the synthesis of compound 8 of (Example B).

[0164] <Step 5> Synthesis of (R)-benzyl 3-((1S,2S)-2-(azidomethyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoate (Compound 25) Compound 25 (111 mg) was obtained as a yellow-brown oily substance from compound 24 (130 mg, 0.368 mmol) obtained in <Step 4> of (Example F) in the same manner as in the synthesis of compound 9 of (Example B).

[0165] <Step 6> Synthesis of (R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2S)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoic acid (Compound 26) Compound 26 (84 mg) was obtained as a colorless amorphous solid from compound 25 (137 mg, 0.362 mmol) in the same manner as in the synthesis of compound 10 in (Example B) and in the same manner as in the synthesis of 10 obtained in (Example F) <Step 5>.

[0166] The following shows schemes for synthesizing compound TCP-301 from compound 16 (Example 1), compound TCP-302 from compound 21 (Example 2), compound TCP-303 from compound 26 (Example 3), and compound TCP-304 from compound 10 (Example 4).

[0167] [ka]

[0168] Example 1: Synthesis of 2-(2-((R)-3-amino-3-((1R,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-301) <Step 1> Synthesis of tert-butyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1R,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 27)

[0169] [ka]

[0170] To a DMF solution (2 mL) of compound 16 (32.5 mg, 90.7 μmol) obtained in Example C, HNN(Me)CHCO t-Bu·p-toluenesulfonate (60.2 mg, 0.181 μmol) and HOBt·HO (27.7 mg, 0.181 μmol) were added at room temperature. EtN (25.1 μL, 0.181 μmol) and EDC·HCl (34.7 mg, 0.181 μmol) were added sequentially under ice cooling, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with 10% aqueous citric acid, saturated aqueous sodium bicarbonate, water, and saturated brine, and dried over NaSO. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol=100:1) to obtain Compound 27 as a colorless solid (39.8 mg).

[0171] <Step 2> Synthesis of 2-(2-((R)-3-amino-3-((1R,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-301)

[0172] [ka]

[0173] To compound 27 (22.4 mg, 44.7 μmol) obtained in (Example 1) <Step 1>, 4 M hydrochloric acid / dioxane (3 mL) was added under ice cooling, and the mixture was stirred at room temperature for 1 hour. The reaction solution was evaporated under reduced pressure, and the resulting residue was purified by HPLC to obtain compound TCP-301 (13.1 mg) as a white solid.

[0174] Example 2: Synthesis of 2-(2-((R)-3-amino-3-((1R,2S)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-302) <Step 1> Synthesis of tert-butyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1R,2S)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 28)

[0175] [ka]

[0176] Compound 21 (33.3 mg, 92.9 μmol) obtained in (Example E) <Step 6> was used in the same manner as in the synthesis of Compound 27 in (Example 1) <Step 1> to obtain Compound 28 as a colorless solid (33.7 mg).

[0177] <Step 2> Synthesis of 2-(2-((R)-3-amino-3-((1R,2S)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-302)

[0178] [ka]

[0179] Compound 28 (19.3 mg, 38.9 μmol) obtained in (Example 2) <Step 1> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-302 as a white solid (9.68 mg).

[0180] Example 3: Synthesis of 2-(2-((R)-3-amino-3-((1S,2S)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-303) <Step 1> Synthesis of tert-butyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2S)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 29)

[0181] [ka]

[0182] Compound 26 (45.0 mg, 0.126 mmol) was used in the same manner as in the synthesis of Compound 27 in (Example 1) <Step 1> to give Compound 29 as a colorless solid (42.8 mg).

[0183] <Step 2> Synthesis of 2-(2-((R)-3-amino-3-((1S,2S)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-303)

[0184] [ka]

[0185] Compound 29 (19.1 mg, 38.2 μmol) obtained in (Example 3) <Step 1> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-303 as a white solid (13.9 mg).

[0186] Example 4 Synthesis of 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-304) <Step 1> Synthesis of tert-butyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 30)

[0187] [ka]

[0188] Compound 10 (38.0 mg, 0.106 mmol) was used in the same manner as in the synthesis of Compound 27 in (Example 1) <Step 1> to obtain Compound 30 as a colorless solid (43.1 mg).

[0189] <Step 2> Synthesis of 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-304)

[0190] [ka]

[0191] Compound 30 (16.3 mg, 32.6 μmol) obtained in (Example 4) <Step 1> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-304 as a white solid (7.80 mg).

[0192] (Example 5) to (Example 9), (Example 14) to (Example 15) [2. Synthesis of Compounds TCP-305, TCP-306, and TCP-315 to TCP-319]

[0193] [ka]

[0194] Here, R in the above scheme 3P and R 3 The chemical structures of the above and the protected amino acid derivatives used in synthesizing compounds 33a to 33g from compound 32 are shown in Tables 1-1 and 1-2 below.

[0195] [Table 1-1]

[0196] [Table 1-2]

[0197] Example G: Synthesis of (R)-3-((1S,2R)-2-(azidomethyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoic acid (Compound 31) To a THF solution (8.8 mL) of compound 9 (332 mg, 0.876 mmol) obtained in Example B, 1 M aqueous lithium hydroxide solution (8.8 mL, 8.8 mmol) was added at room temperature and stirred at the same temperature for 1 hour. The reaction mixture was partitioned with ethyl acetate and 1 M HCl. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was then evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform:methanol = 99:1) to obtain compound 31 (221 mg) as a colorless amorphous solid.

[0198] Example 5 Synthesis of 2-(2-((R)-3-((S)-2-amino-4-methylpentanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-305) <Step 1> Synthesis of benzyl 2-(2-((R)-3-((1S,2R)-2-(azidomethyl)cyclopropyl)-3-((S)-1,1-dimethylethylsulfinamido)propanoyl)-1-methylhydrazinyl)acetate (Compound 32)

[0199] [ka]

[0200] To a DMF solution (3 mL) of compound 31 (134 mg, 0.465 mmol) obtained in Example G, HNN(Me)CHCOBn (180 mg, 0.929 mmol) and HOBt·HO (142 mg, 0.929 mmol) were added at room temperature. EtN (129 μL, 0.929 mmol) and EDC·HCl (178 mg, 0.929 mmol) were added sequentially under ice-cooling, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with 10% aqueous citric acid, saturated aqueous sodium bicarbonate, water, and saturated brine, and dried over sodium sulfate (NaSO). After filtration, the mother liquor was evaporated under reduced pressure. The resulting residue was purified by silica gel chromatography (chloroform:methanol = 100:1) to give compound 32 (132 mg) as a colorless oil.

[0201] <Step 2> Synthesis of (7R,10S)-benzyl 7-((1S,2R)-2-(azidomethyl)cyclopropyl)-10-isobutyl-3,14,14-trimethyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate (Compound 33a)

[0202] [ka]

[0203] To compound 32 (34.9 mg, 75.1 μmol) obtained in Step 1 of Example 5, 4 M hydrochloric acid / dioxane solution (2 mL) was added under ice cooling, and the mixture was stirred at room temperature for 1 hour. The reaction solution was evaporated under reduced pressure, and the resulting residue was used in the next reaction without purification. The residue was dissolved in DMF (3 mL), and protected amino acid derivatives Boc-L-Leu-OH·HO (48.3 mg, 0.194 mmol) and HOBt·HO (29.7 mg, 0.194 mmol) were added at room temperature. EtN (40.9 μL, 0.291 mmol) and EDC·HCl (37.2 mg, 0.194 mmol) were added sequentially under ice cooling, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the resulting residue was dissolved in ethyl acetate. The organic layer was washed with 10% aqueous citric acid, saturated aqueous sodium bicarbonate, water, and saturated brine, and then dried over Na2SO4. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol=100:1) to give compound 33a (39.2 mg) as a white solid.

[0204] <Step 3> Synthesis of 2-(2-((R)-3-((S)-2-amino-4-methylpentanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-305)

[0205] [ka]

[0206] Under an argon atmosphere, 10% Pd / C (2.9 mg) was added to a methanol solution (2 mL) of compound 33a (29.7 mg, 51.8 μmol) obtained in (Example 5) <Step 2>, and the mixture was purged with hydrogen and stirred at room temperature overnight. The reaction solution was filtered through Celite, and the solvent was evaporated under reduced pressure. The resulting residue was used in the next reaction without purification. To the residue was added a 4 M hydrochloric acid / dioxane solution (2 mL) under ice-cooling and stirring, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and the resulting residue was purified by HPLC to give TCP-305 (2.63 mg) as a white solid.

[0207] Example 6: Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((S)-2-aminoundecanamido)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-306) <Step 1> Synthesis of (7R,10S)-benzyl 7-((1S,2R)-2-(azidomethyl)cyclopropyl)-3,14,14-trimethyl-10-nonyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate (Compound 33b)

[0208] [ka]

[0209] Compound 33b was obtained as a white solid (47.4 mg) in the same manner as in the synthesis of Compound 33a in (Example 5, Step 2), except that 41.4 mg (89.1 μmol) of Compound 32 obtained in (Example 5, Step 1) and (S)-2-((tert-butoxycarbonyl)amino)undecanoic acid (69.3 mg, 0.230 mmol) were used as a protected amino acid derivative.

[0210] <Step 2> Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((S)-2-aminoundecanamido)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-306)

[0211] [ka]

[0212] Using compound 33b (42.9 mg, 66.6 μmol) obtained in (Example 6) <Step 1>, TCP-306 was obtained as a white solid (31.3 mg) in the same manner as in the synthesis of TCP-305 in (Example 5) <Step 3>.

[0213] Example 7 Synthesis of 2-(2-((R)-3-((R)-2-amino-4-methylpentanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-315) <Step 1> Synthesis of (7R,10R)-benzyl 7-((1S,2R)-2-(azidomethyl)cyclopropyl)-10-isobutyl-3,14,14-trimethyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate (Compound 33c)

[0214] [ka]

[0215] Compound 33c was obtained as a white solid (46.2 mg) in the same manner as in the synthesis of Compound 33a in (Example 5, Step 2), using 41.9 mg (90.2 μmol) of Compound 32 obtained in (Example 5, Step 1), and Boc-D-Leu-OH·HO (58.0 mg, 0.233 mmol) and HOBt·HO (29.7 mg, 0.233 mmol) as protected amino acid derivatives.

[0216] <Step 2> Synthesis of 2-(2-((R)-3-((R)-2-amino-4-methylpentanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-315)

[0217] [ka]

[0218] Using compound 33c (1 mg, 55.3 μmol) obtained in (Example 7) <Step 1>, TCP-315 was obtained as a white solid (12.6 mg) in the same manner as in the synthesis of TCP-305 in (Example 5) <Step 3>.

[0219] Example 8: Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((R)-2-aminoundecanamido)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-316) <Step 1> Synthesis of (7R,10R)-benzyl 7-((1S,2R)-2-(azidomethyl)cyclopropyl)-3,14,14-trimethyl-10-nonyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate (Compound 33d)

[0220] [ka]

[0221] Compound 33d was obtained as a white solid (59.4 mg) in the same manner as in the synthesis of Compound 33a in (Example 5, Step 2) using 42.9 mg (92.3 μmol) of Compound 32 obtained in (Example 5, Step 1) and (R)-2-((tert-butoxycarbonyl)amino)undecanoic acid (71.8 mg, 0.238 mmol) as a protected amino acid derivative.

[0222] <Step 2> Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((R)-2-aminoundecanamido)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-316)

[0223] [ka]

[0224] Using compound 33d (33.8 mg, 52.5 μmol) obtained in (Example 8) <Step 1>, TCP-316 was obtained as a white solid (13.1 mg) in the same manner as in the synthesis of TCP-305 in (Example 5) <Step 3>.

[0225] Example 9: Synthesis of 2-(2-((R)-3-((S)-2-amino-9-methoxynonanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-317) <Step 1> Synthesis of (7R,10S)-benzyl 7-((1S,2R)-2-(azidomethyl)cyclopropyl)-10-(7-methoxyheptyl)-3,14,14-trimethyl-5,9,12-trioxo-13-oxa-3,4,8,11-tetraazapentadecan-1-oate (Compound 33e)

[0226] [ka]

[0227] Compound 33e was obtained as a colorless solid (53.4 mg) in the same manner as in the synthesis of Compound 33a in (Example 5, Step 2), using 81.0 mg (0.174 mmol) of Compound 32 obtained in (Example 5, Step 1) and (S)-2-((tert-butoxycarbonyl)amino)-9-methoxynonanoic acid (63.5 mg, 0.209 mmol) as a protected amino acid derivative.

[0228] <Step 2> Synthesis of 2-(2-((R)-3-((S)-2-amino-9-methoxynonanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (compound TCP-317)

[0229] [ka]

[0230] Using compound 33e (33.8 mg, 52.3 μmol) obtained in (Example 9) <Step 1>, TCP-317 was obtained as a white solid (2.69 mg) in the same manner as in the synthesis of TCP-305 in (Example 5) <Step 3>.

[0231] Example 14 Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((S)-2,10-diaminodecanamido)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-318) <Step 1> Synthesis of (7R,10S)-benzyl 7-((1S,2R)-2-(azidomethyl)cyclopropyl)-10-((tert-butoxycarbonyl)amino)-3,22,22-trimethyl-5,9,20-trioxo-21-oxa-3,4,8,19-tetraazatricosane-1-oate (Compound 33f)

[0232] [ka]

[0233] (Example 5) Compound 33f was obtained as a brown oily substance (113 mg) using compound 32 (81.5 mg, 0.176 mmol) obtained in <Step 1> and (S)-2,10-bis((tert-butoxycarbonyl)amino)decanoic acid (84.7 mg, 0.211 mmol) in the same manner as in the synthesis of compound 33a.

[0234] <Step 2> Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((S)-2,10-diaminodecanamido)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-318)

[0235] [ka]

[0236] (Example 14) Using compound 33f (66.3 mg, 89.1 mol) obtained in <Step 1>, TCP-318 was obtained as a white solid (0.9 mg) in the same manner as in the synthesis of TCP-305.

[0237] Example 15: Synthesis of (10S,13R)-10-amino-13-((1S,2R)-2-(aminomethyl)cyclopropyl)-17-methyl-11,15-dioxo-2,5,8-trioxa-12,16,17-triazanonadecan-19-oic acid (TCP-319) <Step 1> Synthesis of (10S,13R)-benzyl 13-((1S,2R)-2-(azidomethyl)cyclopropyl)-10-((tert-butoxycarbonyl)amino)-17-methyl-11,15-dioxo-2,5,8-trioxa-12,16,17-triazanonadecan-19-oate (Compound 33g)

[0238] [ka]

[0239] (Example 5) Using compound 32 (67.8 mg, 0.146 mmol) obtained in <Step 1> and (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(2-methoxyethoxy)ethoxy)propanoic acid (53.8 mg, 0.175 mmol), compound 33g was obtained as a white solid (46.1 mg) in the same manner as in the synthesis of compound 33g.

[0240] <Step 2> Synthesis of (10S,13R)-10-amino-13-((1S,2R)-2-(aminomethyl)cyclopropyl)-17-methyl-11,15-dioxo-2,5,8-trioxa-12,16,17-triazanonadecan-19-oic acid (TCP-319)

[0241] [ka]

[0242] (Example 15) Using the compound 33g (24.4 mg, 37.5 μmol) obtained in <Step 1>, TCP-319 was obtained as a white solid (11.1 mg) in the same manner as in the synthesis of TCP-305.

[0243] (Example 10) to (Example 11) [3. Synthesis of Compound TCP-307 and Compound TCP-308]

[0244] [ka]

[0245] Example 10: Synthesis of methyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (compound TCP-307) <Step 1> Synthesis of methyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 34a)

[0246] [ka]

[0247] Compound 10 (31.7 mg, 88.4 μmol) obtained in Example B was used, and HNN(Me)CHCOCH (20.9 mg, 177 μmol) was used instead of HNN(Me)CHCO t-Bu·p-toluenesulfonate, in the same manner as in the synthesis of Compound 27 in Example 1, Step 1, to obtain Compound 34a as a colorless oil (23.3 mg).

[0248] <Step 2> Synthesis of methyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (compound TCP-307)

[0249] [ka]

[0250] Compound 34a (20.2 mg, 44.1 μmol) obtained in (Example 10) <Step 1> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-307 as a white solid (16.2 mg).

[0251] Example 11: Synthesis of benzyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (compound TCP-308) <Step 1> Synthesis of benzyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 34b)

[0252] [ka]

[0253] Compound 34b was obtained as a colorless oil (512 mg) in the same manner as in the synthesis of Compound 27 in Step 1 of Example 1, except that Compound 10 obtained in Example B (425 mg, 1.19 mmol) was used and HNN(Me)CHCOBn (231 mg, 1.79 mmol) was used instead of HNN(Me)CHCOt-Bu·p-toluenesulfonate. <Step 2> Synthesis of benzyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (compound TCP-308)

[0254] [ka]

[0255] Compound 34b (33.2 mg, 62.1 μmol) obtained in (Example 11) <Step 1> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-308 as a white solid (20.0 mg).

[0256] (Example 12) to (Example 13) [4. Synthesis of Compound TCP-309 and Compound TCP-310]

[0257] [ka]

[0258] Example 12 Synthesis of 2-bromobenzyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound TCP-309) <Step 1> Synthesis of 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (Compound 35)

[0259] [ka]

[0260] Under an argon atmosphere, 10% Pd / C (30.3 mg) was added to a methanol solution (5.6 mL) of compound 34b (303 mg, 0.567 mmol) obtained in Example 11, and the mixture was stirred at room temperature for 5 hours after hydrogen substitution. The reaction solution was filtered through Celite, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform:methanol = 80:1) to give compound 35 (122 mg) as a colorless solid.

[0261] <Step 2> Synthesis of 2-bromobenzyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 36a)

[0262] [ka]

[0263] To a DMF solution (2 mL) of compound 35 (60.2 mg, 0.135 mmol) obtained in (Example 12) <Step 1>, o-bromobenzyl alcohol (30.4 mg, 0.162 mmol) was added at room temperature. Under ice-cooling, N,N-dimethyl-4-aminopyridine (DMAP, 1.65 mg, 13.5 μmol) and N,N'-dicyclohexylcarbodiimide (DCC, 30.7 mg, 0.149 mmol) were added sequentially, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the resulting residue was diluted with chloroform and filtered. The organic layer was washed with water and saturated brine and dried over Na2SO4. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol = 100:1) to give compound 36a (50.4 mg) as a white solid.

[0264] <Step 3> Synthesis of 2-bromobenzyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (compound TCP-309)

[0265] [ka]

[0266] Compound 36a (28.6 mg, 46.6 μmol) obtained in (Example 12) <Step 2> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-309 as a white solid (13.3 mg).

[0267] (Example 13) Synthesis of 3-chlorobenzyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound TCP-310) <Step 1> Synthesis of 3-chlorobenzyl 2-(2-((R)-3-((tert-butoxycarbonyl)amino)-3-((1S,2R)-2-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (Compound 36b)

[0268] [ka]

[0269] Compound 35 (52.3 mg, 0.118 mmol) obtained in (Example 12) <Step 1> was used, and m-chlorobenzyl alcohol (20.1 mg, 0.141 mmol) was used instead of o-bromobenzyl alcohol, and the procedure was similar to the synthesis method for compound 36a in (Example 12) <Step 2> to give compound 36b (57.6 mg).

[0270] <Step 2> Synthesis of 3-chlorobenzyl 2-(2-((R)-3-amino-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetate (compound TCP-310)

[0271] [ka]

[0272] (Example 13) Compound 36b (24.0 mg, 42.2 μmol) obtained in <Step 1> was used in the same manner as in the synthesis of compound TCP-301 in (Example 1) <Step 2> to obtain compound TCP-310 as a white solid (8.39 mg). (Example 16) ~ (Example 34) [3. Synthesis of compounds TCP-320~TCP-323, compounds TCP-325~TCP-330, compound TCP-335, compounds TCP-338~TCP-TCP-344, compound TCP-346, compound TCP-348, compound TCP-359]

[0273] [ka]

[0274] Example H: Synthesis of (R)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((1S,2R)-2-(azidomethyl)cyclopropyl)propanoic acid (Compound 37)

[0275] [ka]

[0276] Compound 31 was synthesized by hydrolysis of the benzyl ester using compound 9 (1.13 g, 2.98 mmol) obtained in Example B. Compound 31 was used in the next reaction without purification. 4 M HCl / dioxane (6 mL) was added to the resulting residue under ice-cooling and stirring, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated, and the resulting residue was used in the next reaction without purification. Sodium bicarbonate (1.00 g, 11.9 mmol) and Fmoc-OSu (2.01 g, 5.96 mmol) were added to a dioxane / HO solution (1:1, 15 mL) of the residue at room temperature, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over NaSO. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform) to give compound 37 (1.07 g) as a colorless solid.

[0277] Example I: Synthesis of Resin 1 Resin 1 was synthesized using Wang resin (0.88 mmol / g, 1 eq), Fmoc-NHN(Me)CH2CO2H (4 eq), triphenylphosphine (PPh3, 4 eq), and diisopropyl azodicarboxylate (DIAD, 4 eq) according to the method described in Bioorganic & Medicinal Chemistry Letters, 13 (2003) 2413-2418. The substitution amount (mmol / g) of the synthesized resin 1 was calculated by measuring the absorption of N-(9-fluorenylmethyl)piperidine at 301 nm (substitution amount: 0.194-0.427 mmol / g) according to "Experimental Chemistry Lectures, Vol. 16, Synthesis of Organic Compounds IV: Carboxylic Acids, Amino Acids, and Peptides, 5th Edition."

[0278] Example J: Synthesis of Resin 2 To the resin 1 (1 eq) obtained in Example I, a 20% piperidine / DMF solution was added at room temperature, and the mixture was stirred at the same temperature for 20 minutes to remove the Fmoc group. After washing the resin with DMF, HATU (3 eq) and DIPEA (3 eq) were added to a DMF solution of compound 37 (3 eq) obtained in Example H at room temperature, and the mixture was stirred at the same temperature for 1 minute 30 seconds. This mixed solution was added to the resin, and the mixture was stirred at room temperature for 1 hour 30 minutes. The resulting resin was washed with DMF, methanol, and diethyl ether, and then dried.

[0279] Example K: Synthesis of Resin 3 A 20% piperidine / DMF solution was added to the resin 2 obtained in Example J at room temperature and stirred at the same temperature for 20 minutes to remove the Fmoc group. After washing the resin with DMF, a protected amino acid derivative or acyl derivative was introduced onto the resin by the following condensation method: Condensation method i): Protected amino acid derivative, HOBt·H2O (3 eq), DIPCI (3 eq) and DMF were added to the resin at room temperature and stirred for 1 hour and 30 minutes; Condensation method ii): Protected amino acid derivative, HOAt (5 eq), HATU (5 eq), DIPEA (5 eq) and DMF were added at room temperature and stirred for 30 minutes; Condensation method iii): HATU (5 eq) and DIPEA (5 eq) were added to a DMF solution of the acyl derivative at room temperature, and the mixture was stirred at the same temperature for 1 minute and 30 seconds. This mixture was then added to the resin and stirred at room temperature for 1 hour. Condensation method iv): HOAt (3 eq), HATU (3 eq), and DIPEA (3 eq) were added to a DMF solution of a protected amino acid derivative at room temperature, and the mixture was stirred at the same temperature for 1 minute and 30 seconds. This mixture was then added to the resin and stirred at room temperature for 1 hour. Resin 3 obtained under the above condensation conditions was washed with DMF.

[0280] Resin 3 was then subjected to the following reduction procedure to convert the azide groups on the resin to amino groups: Reduction method i): After washing the resin with THF, PPh3 (3 eq), THF (375 μL) and water (125 μL) were added and stirred at room temperature for 20-24 hours; Reduction method ii): After washing the resin with CH3CN, CH3CN (375 μL), DTT (2.2 eq), DIPEA (1.1 eq), and water (125 μL) were added, and the mixture was stirred at room temperature for 1 hour to overnight.

[0281] The resin obtained by the reduction method described above was washed with DMF, methanol, and Et2O and dried. TFA:triisopropylsilane:HO=95:2.5:2.5 was added to the dried resin and stirred at room temperature for 3 hours. The reaction solution was evaporated by blowing nitrogen, and cold Et2O was added to the resulting residue. The precipitated crude product was centrifuged. The supernatant was discarded, and the crude product was washed with cold Et2O and then dried. The crude product was purified by HPLC to obtain the desired compound.

[0282] Example 16: Synthesis of 2-(2-((R)-3-((S)-2-amino-7-phenylheptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-320)

[0283] [ka]

[0284] Using Resin 2 (100 mg, 43.2 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-7-phenylheptanoic acid (41.6 mg, 0.129 mmol) was introduced by condensation method i). Subsequently, reduction method iii), de-resination, and HPLC purification were carried out to obtain TCP-320 as a white solid (5.06 mg).

[0285] Example 17 Synthesis of 2-(2-((R)-3-((S,E)-2-amino-7-phenylhept-4-enamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-321)

[0286] [ka]

[0287] Using resin 2 (176 mg, 34.1 μmol) obtained in Example J, (S,E)-2-((tert-butoxycarbonyl)amino)-7-phenylhept-4-enoic acid (32.6 mg, 0.102 mmol) was introduced by condensation method i). Subsequently, reduction method i), resin removal, and HPLC purification were carried out to obtain TCP-321 as a white solid (2.38 mg).

[0288] Example 18 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(4-(benzyloxy)phenyl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-322)

[0289] [ka]

[0290] Using resin 2 (176 mg, 20.4 μmol) obtained in Example J, Boc-Ser(Bn)-OH (18.1 mg, 61.2 μmol) was introduced by condensation method i), followed by reduction method i), de-resination, and HPLC purification to obtain TCP-322 as a white solid (1.61 mg).

[0291] Example 19: Synthesis of 2-(2-((R)-3-((S)-2-amino-7-morpholinoheptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-323)

[0292] [ka]

[0293] Using resin 2 (162 mg, 31.4 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-7-morpholinoheptanoic acid (31.1 mg, 94.2 μmol) was introduced by condensation method iv). Subsequently, reduction method i), resin removal, and HPLC purification were carried out to obtain TCP-323 as a white solid (4.12 mg).

[0294] Example 20 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(4-butylpiperazin-1-yl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-325)

[0295] [ka]

[0296] Using resin 2 (120 mg, 25.4 μmol) obtained in (Example J), ​​(S)-2-((tert-butoxycarbonyl)amino)-3-(4-butylpiperazin-1-yl)propanoic acid (25.1 mg, 76.2 μmol) was introduced by condensation method i). Then, reduction method i), resin removal, and HPLC purification were carried out to obtain TCP-325 as a white solid (4.28 mg).

[0297] Example 21 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(1-pentyl-1H-1,2,3-triazol-4-yl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-326)

[0298] [ka]

[0299] Using resin 2 (100 mg, 43.2 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-3-(1-pentyl-1H-1,2,3-triazol-4-yl)propanoic acid (19.8 mg, 0.129 mmol) was introduced by condensation method i). Then, reduction method ii), resin removal, and HPLC purification were carried out to obtain TCP-326 as a white solid (4.47 mg).

[0300] Example 22: Synthesis of 2-(2-((R)-3-((S)-2-amino-6-butylamidohexanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-327)

[0301] [ka]

[0302] Using resin 2 (128 mg, 27.1 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-6-butylamidohexanoic acid (25.7 mg, 81.3 μmol) was introduced by condensation method i). Subsequently, reduction method i), resin removal, and HPLC purification were carried out to obtain TCP-327 as a white solid (6.88 mg).

[0303] Example 23 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-heptanamidopropanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-328)

[0304] [ka]

[0305] Using resin 2 (98.1 mg, 37.3 μmol) obtained in Example J, Boc-Dap(Fmoc)-OH (32.6 mg, 0.109 mmol) was introduced by condensation method i). A 20% piperidine / DMF solution was added to the obtained resin at room temperature and stirred at the same temperature for 20 minutes. After washing the resin with DMF, heptanoic acid (26.5 μL, 0.187 mmol) was introduced to the resin by condensation method iii). Subsequently, reduction method i), de-resination, and HPLC purification were performed to obtain TCP-328 as a white solid (9.14 mg).

[0306] Example 24 Synthesis of 2-(2-((R)-3-((S)-3-amino-2-heptanamidopropanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-329)

[0307] [ka]

[0308] Using resin 2 (96.3 mg, 36.6 μmol) obtained in Example J, Fmoc-Dap(Boc)-OH (46.9 mg, 0.110 mmol) was introduced by condensation method i). A 20% piperidine / DMF solution was added to the obtained resin at room temperature, and the mixture was stirred at the same temperature for 20 minutes. After washing the resin with DMF, heptanoic acid (15.6 μL, 0.110 mmol) was introduced to the resin by condensation method i). Subsequently, reduction method i), de-resination, and HPLC purification were performed to obtain TCP-329 as a white solid (9.80 mg).

[0309] Example 25: Synthesis of 2-(2-((R)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)-3-((S,E)-2-aminoundec-4-enamido)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-330)

[0310] [ka]

[0311] Using resin 2 (95.5 mg, 36.3 μmol) obtained in Example J, (S,E)-2-((tert-butoxycarbonyl)amino)undec-4-enoic acid (32.6 mg, 0.109 mmol) was introduced by condensation method i). Subsequently, reduction method i), de-resination, and HPLC purification were carried out to obtain TCP-330 as a white solid (4.85 mg).

[0312] Example 26: Synthesis of 2-(2-((R)-3-((S)-2-amino-9-hydroxynonanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-335)

[0313] [ka]

[0314] Resin 2 (174 mg, 33.8 μmol) obtained in Example J was used to introduce (S)-2-((tert-butoxycarbonyl)amino)-9-((tert-butyldimethylsilyl)oxy)nonanoic acid (40.8 mg, 0.101 mmol) by condensation method i). The azide group was then converted to an amino group by reduction method i). The resulting resin was washed with THF, and then THF (400 μL) and TBAF (1.0 M in THF, 3 eq) were added, followed by stirring at room temperature for 2 hours. The resulting resin was washed with DMF, methanol, and EtO, dried, and then de-resined to obtain a crude product. The crude product was dissolved in a CHCN:water mixed solvent (1:1 (volume ratio), 1 mL), and saturated aqueous sodium bicarbonate was added dropwise at room temperature to adjust the pH to 8. After stirring at the same temperature for 1 hour, 1 M aqueous HCl was added dropwise to the reaction solution to adjust the pH to 3-4. This reaction solution was purified by HPLC to give TCP-335 as a white solid (2.69 mg).

[0315] Example 27 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(1-benzyl-1H-1,2,3-triazol-4-yl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-338)

[0316] [ka]

[0317] Using the resin 2 (100 mg, 43.2 μmol) obtained in (Example J), ​​(S)-3-(1-benzyl-1H-1,2,3-triazol-4-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (44.8 mg, 0.129 mmol) was introduced by condensation method i). Then, reduction method iii), resin removal, and HPLC purification were carried out to obtain TCP-338 as a white solid (12.4 mg).

[0318] Example 28 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(4-propoxyphenyl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-339)

[0319] [ka]

[0320] Using resin 2 (108 mg, 41.0 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-3-(4-propylphenyl)propanoic acid (39.8 mg, 0.123 mmol) was introduced by condensation method i). Subsequently, reduction method i), resin removal, and HPLC purification were carried out to obtain TCP-339 as a white solid (9.81 mg).

[0321] Example 29 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(4-(cyclopropylmethoxy)phenyl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-340)

[0322] [ka]

[0323] Using resin 2 (91.9 mg, 34.9 μmol) obtained in (Example J), ​​(S)-2-((tert-butoxycarbonyl)amino)-3-(4-(cyclopropylmethoxy)phenyl)propanoic acid (35.1 mg, 0.105 mmol) was introduced by condensation method i). Subsequently, reduction method i), de-resination, and HPLC purification were carried out to obtain TCP-340 as a white solid (1.10 mg).

[0324] Example 30 Synthesis of 2-(2-((R)-3-((S)-2-amino-3-(4-(benzyloxy)phenyl)propanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-341)

[0325] [ka]

[0326] Using resin 2 (95.9 mg, 36.4 μmol) obtained in (Example J), ​​(S)-3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (40.6 mg, 0.109 μmol) was introduced by condensation method i). Subsequently, reduction method i), resin removal, and HPLC purification were carried out to obtain TCP-341 as a white solid (4.36 mg).

[0327] Example 31 Synthesis of 2-(2-((R)-3-((S)-2-amino-7-(piperidin-1-yl)heptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-342)

[0328] [ka]

[0329] Using the resin 2 (91.6 mg, 39.3 μmol) obtained in (Example J), ​​(S)-2-((tert-butoxycarbonyl)amino)-7-(piperidin-1-yl)heptanoic acid (38.7 mg, 0.118 mmol) was introduced by condensation method iv). Then, reduction method iii), resin removal, and HPLC purification were carried out to obtain TCP-342 as a white solid (6.71 mg).

[0330] Example 32 Synthesis of 2-(2-((R)-3-((S)-2-amino-7-(piperazin-1-yl)heptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-343)

[0331] [ka]

[0332] Using resin 2 (90.8 mg, 39.2 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-7-(4-(tert-butoxycarbonyl)piperazin-1-yl)heptanoic acid (48.5 mg, 0.113 mmol) was introduced by condensation method iv). Subsequently, reduction method iii), resin removal, and HPLC purification were carried out to obtain TCP-343 as a white solid (4.66 mg).

[0333] Example 33 Synthesis of 2-(2-((R)-3-((S)-2-amino-7-thiomorpholinoheptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-344)

[0334] [ka]

[0335] Using the resin 2 (103 mg, 44.7 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-7-thiomorpholinoheptanoic acid (46.4 mg, 0.134 mmol) was introduced by condensation method iv). Subsequently, reduction method iii), de-resination, and HPLC purification were carried out to obtain TCP-344 as a white solid (6.72 mg).

[0336] Example 34 Synthesis of 2-(2-((R)-3-((S)-2-amino-7-(pyrrolidin-1-yl)heptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-346)

[0337] [ka]

[0338] Using the resin 2 (90.4 mg, 39.0 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-7-(pyrrolidin-1-yl)heptanoic acid (36.8 mg, 0.117 mmol) was introduced by condensation method iv). Then, reduction method iii), de-resination, and HPLC purification were carried out to obtain TCP-346 as a white solid (2.75 mg).

[0339] Example 35 Synthesis of 2-(2-((R)-3-((S)-2-amino-7-(1H-imidazol-1-yl)heptanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-348)

[0340] [ka]

[0341] Using resin 2 (92.6 mg, 40.0 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-7-(1H-imidazol-1-yl)heptanoic acid (37.3 mg, 0.120 mmol) was introduced by condensation method iv). Then, reduction method iii), de-resination, and HPLC purification were carried out to obtain TCP-348 as a white solid (8.58 mg).

[0342] Example 36 Synthesis of 2-(2-((R)-3-((S)-2-amino-6-cyclohexylhexanamido)-3-((1S,2R)-2-(aminomethyl)cyclopropyl)propanoyl)-1-methylhydrazinyl)acetic acid (TCP-359)

[0343] [ka]

[0344] Using resin 2 (92.7 mg, 40.0 μmol) obtained in Example J, (S)-2-((tert-butoxycarbonyl)amino)-6-cyclohexylhexanoic acid (37.6 mg, 0.120 mmol) was introduced by condensation method i). Subsequently, reduction method iii), resin removal, and HPLC purification were carried out to obtain TCP-359 as a white solid (5.04 mg).

[0345] The compounds synthesized in the above (Example A) to (Example H) and (Example 1) to (Example 36) 1 H-NMR data, 13 C-NMR data, HRMS (ESI) m / z data, and optical rotation ([α] D ) The data are presented in the following tables (Table 2-1, Table 2-2, Table 3-1, Table 3-2, Table 3-3, Table 3-4, Table 3-5, Table 4, Table 5, Table 6-1, Table 6-2, and Table 7).

[0346] [Table 2-1]

[0347] [Table 2-2]

[0348] [Table 3-1]

[0349] [Table 3-2]

[0350] [Table 3-3]

[0351] [Table 3-4]

[0352] [Table 3-5]

[0353] [Table 4]

[0354] [Table 5]

[0355] [Table 6-1]

[0356] [Table 6-2]

[0357] [Table 7]

[0358] <Evaluation of DMD read-through activity using cell lines> In this example, the read-through activity of each compound on dystrophin gene translation in a cell line was evaluated using a dual reporter assay with a vector in which a dystrophin gene containing a nonsense mutation that causes Duchenne muscular dystrophy (DMD) was inserted between the β-galactosidase gene and the firefly luciferase gene.

[0359] [1. Cell culture] COS-7 cells were cultured in a cell culture dish (Falcon, 100 × 20 mm) at 37°C in 5% CO. D-MEM (high glucose, L-glutamine, and phenol red) containing 10% FBS was used as the culture medium.

[0360] [2. Evaluation of read-through activity] The "Reporter Lysis Buffer 5x," "Assay 2x buffer," and "1M Na2CO3" used in the following experiments, as well as the standard used to create the calibration curve, are contained in the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer (Promega).

[0361] 2-1. Addition of compounds to cells COS-7 cells (8.0 × 10 cells) were cultured in a 96-well clear flat-bottom cell culture surface-treated polystyrene microplate (Corning). 3 The culture medium (cells / well) was seeded in 100 μL aliquots and incubated at 37° C. for 12 to 15 hours.

[0362] On the other hand, we prepared a dual reporter vector in which a dystrophin gene containing a nonsense mutation (premature stop codon (PTC)) responsible for DMD was inserted between the upstream β-galactosidase gene and the downstream firefly luciferase gene (see Shiozuka et al., J. Biochem., 2010, 147, 463-470). This nonsense mutation results in a single-nucleotide substitution at the tyrosine-encoding codon (UAC) that converts to a stop codon (UAG). This stop codon acts as a premature stop codon (PTC), preventing translation of codons following the PTC (including the firefly luciferase gene). Therefore, the read-through activity of each compound can be assessed using the firefly luciferase activity relative to the β-galactosidase activity as an index.

[0363] Here, 2 μg of the dual reporter vector prepared above and 4 μL of FuGENE® HD Transfection Reagent (Promega) were prepared, and a total volume of 100 μL of DNA solution was prepared using OPTI-MEM® I (Reduced Serum Medium 1x) (Invitrogen). Next, 4 μL of this DNA solution was added to each well in liquid and incubated at 37°C for 10 to 11 hours.

[0364] After incubation, the culture medium was completely aspirated, and the compound to be evaluated was adjusted to a concentration of 200 μM in the culture medium and added in 200 μL aliquots per well. The same procedure was also performed for systems where the compound concentration was changed to 100 μM or 50 μM. After compound addition, the cells were incubated at 37°C for 48 hours.

[0365] After incubation, the culture medium in the wells was aspirated and washed twice with PBS. Next, Reporter Lysis Buffer 5x was diluted with ultrapure water to make Reporter Lysis Buffer 1x, and 100 μL of this solution was added in successive aliquots and allowed to stand at room temperature for 15 minutes. The contents of each well were then collected, transferred to a Nunc microwell plate (V-bottom) (Thermo Fisher Scientific), and centrifuged (1800 rpm, 15 minutes). 85 μL of the resulting lysate was transferred to each well of a 96-well ultra-low attachment flat-bottom plate (Corning).

[0366] [2-2. Evaluation of β-galactosidase activity] To a 96-well flat-bottom ultra-low attachment plate (Corning), 30 μL of Reporter Lysis Buffer 1X was added per well in successive aliquots, followed by 20 μL of the lysate collected in 2-1 above, for a total volume of 50 μL. Separately, standards required for creating a calibration curve were added at 0, 1, 2, 3, 4, and 5 mU according to the protocol for the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer (Promega). 50 μL of Assay 2X buffer was added to each well and incubated at room temperature for 20 minutes. Next, 150 μL of 1M Na2CO3 (reaction stop solution) was added to each well, and the absorbance intensity (414 nm, reference 0 nm) was immediately measured using a MULTISKAN FC absorbance microplate reader (Thermo Fisher Scientific). A calibration curve was prepared from the absorbance of the standard, and the absorbance was converted into mU according to this calibration curve to obtain the value of β-galactosidase activity.

[0367] [2-3. Evaluation of luciferase activity] 50 μL of the lysate collected in 2-1 above was added to each well of a 96-well white plate (manufactured by Corning Incorporated), and 100 μL of PicaGene® (manufactured by Toyo Ink Co., Ltd.) was added, followed by incubation at room temperature for 15 minutes. After incubation, the luminescence intensity was measured using a luminescence microplate reader, LUMINOSKAN ASCENT (manufactured by Thermo Fisher Scientific).

[0368] [2-4. Calculation of read-through activity] The luciferase activity measured in 2-3 above was divided by the β-galactosidase activity measured in 2-2 above to calculate the read-through activity. The ratio of the read-through activity to the value calculated in the same manner for a control (culture medium) in which the same experiment was performed without adding the compound to be evaluated was calculated, and this was used as the read-through activity. The results are shown in Table 8 below.

[0369] <Evaluation of CF read-through activity using cell lines> In this example, the read-through activity of each compound on the translation of the CFTR gene in a cell line was evaluated by a dual reporter assay using a vector in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing a nonsense mutation (W1282X) that causes cystic fibrosis (CF) was inserted between the Renilla luciferase gene and the firefly luciferase gene.

[0370] [1. Cell culture] The COS-7 cells cultured in the above evaluation of DMD read-through activity were used.

[0371] [2. Evaluation of read-through activity] The "Passive Lysis Buffer, 5x," "LARII Buffer," and "Stop & Glo Buffer" used in the following experiments are contained in the Dual luciferase reporter assay system (Promega).

[0372] 2-1. Addition of compounds to cells COS-7 cells were seeded at 8,000 cells / well in a 96-well bottomed plate (Costar) and cultured for 12–15 hours. Two micrograms of a reporter gene (a construct consisting of a Renilla luciferase gene ligated with a firefly luciferase gene, with a PTC-containing sequence inserted at the junction) and 6 μL of FuGENE® HD Transfection Reagent (Roche) were diluted with OPTI-MEM® I (Reduced Serum Medium 1X, Invitrogen) to a total volume of 100 μL. 4 μL of this solution was added per well and cultured at 37°C for 10–12 hours. The medium was removed, and 200 μL of a test compound solution adjusted to 200 μM was added. A control plate was also cultured with 200 μL of medium. After 46–48 hours of culture, the medium in the wells was removed and washed twice with PBS. Next, 100 μL of 1x Passive Lysis Buffer, prepared by diluting 5x Passive Lysis Buffer with ultrapure water, was added to each well to lyse the cell membrane. The contents of each well were collected, transferred to a Nunc microwell plate (V-bottom, Thermo Fisher Scientific), and centrifuged (1800 rpm, 15 minutes). 85 μL of this lysate was transferred to a Nunc low-binding plate 96-well plate (flat-bottom, clear, Thermo Fisher Scientific).

[0373] [2-2. Evaluation of dual luciferase activity] 5μL of the collected lysate was added to a Corning 96-well white plate (costar® 3912), 50μL of LARII Buffer was added to each well, and the luminescence intensity was immediately measured using a Luminoskan luminescence microplate reader (Thermo Fisher Scientific). After the measurement, 50μL of Stop & Glo Buffer was added to each well of the same plate, and the luminescence intensity was immediately measured using a Luminoskan luminescence microplate reader (Thermo Fisher Scientific).

[0374] [2-3. Calculation of read-through activity] The firefly luciferase activity measured in 2-2 above was divided by the Renilla luciferase activity to calculate the read-through activity. The ratio of the read-through activity to the value calculated in the same manner for a control (culture medium) in which the same experiment was performed without adding the test compound was calculated, and this value was used as the read-through activity. The results are shown in Table 8 below.

[0375] <Evaluation of NPPK read-through activity using a cell-free system> In this experimental example, the read-through activity of each compound on the translation of the SERPINB7 gene in a cell-free system was evaluated by a dual reporter assay using a vector in which the SERPINB7 gene containing a nonsense mutation (c.796C>T) that causes Nagashima palmoplantar keratosis (NPPK) was inserted between the β-galactosidase gene and the firefly luciferase gene.

[0376] [1. Addition of compounds to cell-free evaluation system] HeLa cell lysate, accessory protein solution, and reaction mixture from a cell-free protein synthesis kit (Thermo Fisher Scientific, 1-Step Human Couples IVT Kit, 88882) were mixed according to the kit's instructions. Next, 10 volumes of the cell-free protein synthesis kit mixture were mixed with 1 volume of dual reporter vector solution (125 ng / µL) to prepare a premix solution. The dual reporter vector was prepared by inserting the SERPINB7 gene, containing a nonsense mutation (premature stop codon (PTC)) responsible for NPPK, between the upstream β-galactosidase gene and the downstream firefly luciferase gene, using the same method as described above.

[0377] Next, 1.5 μL of an aqueous solution of the compound to be evaluated and 11.5 μL of the premix solution prepared above were added to a PCR tube (0.2 mL, RNase / DNase-free, manufactured by Trefflab) and mixed. The mixture was then incubated at 30°C for 90 minutes to carry out a protein synthesis reaction. Note that the solution of the compound to be evaluated was diluted by mixing with the premix solution, so it was prepared at a concentration 8.33 times higher than the concentration to be evaluated. As a control, a premix solution to which the same amount of water had been added instead of the compound was used.

[0378] 2. Evaluation of β-galactosidase activity The protein synthesis reaction solution incubated as described above was diluted 3000-fold with the Reporter Lysis Buffer 1X prepared as described above. For this dilution procedure, 1 μL of the reaction solution was first mixed with 199 μL of Reporter Lysis Buffer 1X to prepare a 200-fold diluted solution. Next, 10 μL of this 200-fold diluted solution was mixed with 140 μL of Reporter Lysis Buffer 1 1X to dilute the solution 15-fold, preparing a 3000-fold diluted solution. Separately, as a standard enzyme solution required for preparing a calibration curve, the β-galactosidase enzyme (1 U / μL) contained in the β-Galactosidase Enzyme Assay with Reporter Lysis Buffer kit (Promega) was serially diluted with Reporter Lysis Buffer 1x to prepare concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 μU / μL.

[0379] Next, 50 μL of the diluted protein synthesis reaction mixture and serially diluted standard enzyme solutions were added to each well of a 96-well plate (Thermo Fisher Scientific). Then, 50 μL of Assay 2x Buffer was added to each well and incubated at 37°C for 30 minutes. After incubation, 150 μL of 1M Na2CO3 solution (reaction stop solution) was added to each well, and the absorbance (420 nm, reference 0 nm) was immediately measured using a Multiskan 60 (Thermo Fisher Scientific). A calibration curve was created from the absorbance of the standard enzymes, and enzyme activity (mU / μL) was calculated from the calibration curve and the absorbance of the protein synthesis reaction mixture.

[0380] 3. Evaluation of luciferase activity 20 μL of Reporter Lysis Buffer 1× (Promega) and 1 μL of the protein synthesis reaction solution incubated above were added to a 96-well white plate (COSTAR, 3917) and mixed. Next, 50 μL of Luciferase Assay System solution (Promega, E1500) was added, and the luminescence intensity was immediately measured using a PerkinElmer 2030 Multilabel Reader ARVO™ X5.

[0381] 4. Calculation of read-through activity The luciferase activity measured in 3 above was divided by the β-galactosidase activity measured in 2 above to calculate the read-through activity. The ratio of the read-through activity to the value calculated in the same manner for a control (solvent group) in which the same experiment was performed without adding the compound to be evaluated was calculated, and this was taken as the read-through activity. The results are shown in Table 8 below.

[0382] <Evaluation of NPPK read-through activity using cell lines> In this example, a cell line was used to evaluate the read-through activity of each compound on the translation of the SERPINB7 gene, which contains a nonsense mutation (c.796C>T) that causes Nagashima palmoplantar keratosis (NPPK).

[0383] [1. Cell culture] AD293 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS).

[0384] [2. Evaluation of read-through activity] In this experimental example, AD293 cells transfected with a SERPINB7 gene having a nonsense mutation were treated with an evaluation compound, and the expression level of normal SERPINB7 protein was measured by Western blotting to evaluate read-through activity.

[0385] 2-1. Introduction of vector DNA into cells To analyze SERPINB7 protein containing a nonsense mutation (c796.C>T), we constructed a p3xFLAG-SERPINB7 c.796C>T vector DNA expressing a mutant SERPINB7 protein containing a FLAG tag sequence at the N-terminus. To evaluate transfection efficiency, we simultaneously transfected cells with a firefly luciferase-expressing vector DNA. To transfect cultured cells, 600 ng of p3xFLAG-SERPINB7 c.796C>T vector DNA and 600 ng of firefly luciferase expression vector DNA were added to 30 μL of Opti-MEM 1 Reduced-Serum Medium (Thermo Fisher Scientific) per well of a 24-well plate, and then mixed with 2.4 μL of P3000 Reagent (Thermo Fisher Scientific) to prepare a transfection solution (Solution 1). Next, a solution was prepared by mixing 30 μL of Opti-MEM 1 Reduced-Serum Medium and 3.6 μL of Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) in a separate container (Solution 2). After mixing Solution 1 and Solution 2, the mixture was left to stand at room temperature for 5 minutes or more, and this was designated Solution 3. A cell suspension was prepared from cultured AD293 cells, and the cell concentration was adjusted to 2 × 10 5 The cell suspension was diluted to cells / mL. 600 μL of this cell suspension was prepared per well of a 24-well plate, and Solution 3 was added and seeded into each well. The cells were then cultured at 37°C under conditions of 95% air (volume) and 5% CO2 (volume).

[0386] [2-2. Treatment with evaluation compound] One day after the vector DNA was introduced into the cells, the test compound was added to the cell culture medium. At this time, the test compound was dissolved in distilled water and then diluted to a predetermined concentration with DMEM medium before use. Similar experiments were also carried out using the comparative compounds described below. Among these comparative compounds, gentamicin and G418 were diluted to a predetermined concentration with DMEM medium before use. After the addition of the compound, the cells were cultured for 2 days, and then cell lysates were collected by the following method.

[0387] [2-3. Recovery of cell lysate] The medium was removed from each well, and 100 μL of sample buffer prepared from NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) and NuPAGE Sample Reducing Agent (Thermo Fisher Scientific) was added to each well. After sonication using an Ultrasonic Liquid Processor Q125 (Qsonica), the cells were centrifuged at 14,000 rpm at 4°C for 10 minutes, and the supernatant was used as the cell lysate.

[0388] [2-4. Western blotting analysis] The cell lysate prepared above was inserted into each well of 4-12% NuPAGE Bis-Tris Mini Gels (Thermo Fisher Scientific) and subjected to electrophoresis. After electrophoresis, the gel was immersed in ultrapure water and then in 20% ethanol solution for approximately 5 minutes. Using the iBlot2 Dry Blotting System (Thermo Fisher Scientific), the gel was transferred to an iBlot2 PVDF Regular Stacks membrane (Thermo Fisher Scientific). The gel was immersed in 5% skim milk solution for approximately 1 hour at room temperature for blocking, followed by washing with Tris-Buffered Saline (TBS). The primary antibody was then incubated at room temperature for 1 hour. The primary antibody used to detect SERPINB7 protein containing a FLAG tag sequence at its N-terminus was an anti-FLAG M2 antibody (F3165, Sigma-Aldrich), and the primary antibody used to detect firefly luciferase was an anti-firefly luciferase antibody (ab16466, Abcam). The primary antibody was diluted 1000-fold with Can Get Signal Immunoreaction Enhance Solution 1 (Toyobo Co., Ltd.). After treatment with the primary antibody, the sample was washed with a solution of TBS supplemented with Tween 20 (Sigma-Aldrich) to a concentration of 0.5 w / v% (hereinafter referred to as "TBST"). Subsequently, the secondary antibody was incubated at room temperature for approximately 1 hour, followed by washing with TBST. The secondary antibody used was Peroxidase-Affini Pure goat anti-mouse IgG antibody (115-035-003, manufactured by Jackson), which was diluted 2000-fold with Can Get Signal Immunoreaction Enhance Solution 1 and used in the reaction.

[0389] [2-5. Calculation of read-through activity] After reacting with the secondary antibody in 2-4 above, the membrane was treated with ECL Western Blotting Detection Reagents (GE Healthcare), and signals were detected using an ImageQuant LAS4000 (Fujifilm Corporation). The full-length SERPINB7 protein signal and β-actin signal detected during this process were then quantified using an ImageQuant TL (GE Healthcare), and the former divided by the latter was used to calculate the read-through activity. The results are shown in Table 8 below.

[0390] [Table 8]

[0391] The activity notation in Table 8 is as follows: In Table 8, "ND" means not determined: Cell-based assays bGal-PTC(DMD)-Luc and hRluc-PTC(CFW1282X)-Fluc: (+)_1<activity value ≤ 10; (++)_10<activity value; Cell-based assay SERPINB7 (c.796C>T): (+)_0<activity value≦1; (++)_1<activity value; Cell-free assay for SERPINB7 (c.796C>T): (+)_1 <活性値≦2; (++)_2< / 活性値≦2;

Claims

1. The following formula (2): 【Chemistry 1】 [In formula (2), n represents 1; R １ and R ２ is a hydrogen atom; R ３ represents a hydrogen atom, the following partial structural formula (S-1) [wherein the formula does not include the part to the right of the dashed line]: 【Chemistry 2】 (In partial structural formula (S-1), m represents an integer of 0, 1 or 2; R ４ is C 1-10 alkyl, C 2-10 alkenyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, CH ３ CH ２ C 1-10 alkyl substituted with CONH—, C 2-10 alkenyl substituted with phenyl, —(OCH ２ CH ２ ) ２ -OCH ３ , —O—CH ２ -phenyl, triazole substituted with a benzyl group, or phenyl substituted with a cyclopropylmethyloxy group; Or the following partial structural formula (S-2) [wherein the formula does not include the right side of the dashed line]: 【Transformation 3】 (In partial structural formula (S-2), R ８ is C ６ an alkanoylamino group; R ５ is a hydrogen atom or C １～３ is an alkyl group; R ６ is a methyl group; R ７ is a hydrogen atom, C １～６ Alkyl groups, halogenated C １～６ Alkyl group, C ２～１０ Alkenyl group, C ６～１０ an aryl group, or C ７～１６ is an aralkyl group; R ７ C in ６～１０ Aryl group or C ７～１６ The aralkyl group may be a halogen atom, a C １～６ Alkyl group or C １～６ and optionally substituted with 1 to 3 alkoxy groups. or a pharmaceutically acceptable salt thereof, or a solvate thereof.

2. The R ３ is a hydrogen atom or the partial structural formula (S-1), or a pharmaceutically acceptable salt thereof, or a solvate thereof.

3. The R ３ is a hydrogen atom or the partial structural formula (S-1), The R ３ The compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein when the formula is the partial structural formula (S-1), m is 0.

4. The R ３ is a hydrogen atom or the partial structural formula (S-1), The R ３ is the partial structural formula (S-1), m is 0, and R ４ is C 1-10 alkyl, C 2-10 alkenyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, CH ３ CH ２ The compound according to claim 3, which is C 1-10 alkyl substituted with CONH-, C 2-10 alkenyl substituted with phenyl, or phenyl substituted with a cyclopropylmethyloxy group, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

5. The R ３ is the partial structural formula (S-1), and m is 0; The R ４ is C 1-10 alkyl, C 2-10 alkenyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, CH ３ CH ２ The compound according to claim 4, which is C 1-10 alkyl substituted with CONH- or C 2-10 alkenyl substituted with phenyl, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

6. The R ３ is the partial structural formula (S-1), and m is 0; The R ４ is C 1-10 alkyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, or CH ３ CH ２ 6. The compound according to claim 5, which is C 1-10 alkyl substituted with CONH-, or a pharmaceutically acceptable salt or solvate thereof.

7. A compound represented by the following formula TCP-304, TCP-306, TCP-330 or TCP-341, or a pharmaceutically acceptable salt thereof, or a solvate thereof: 【Chemistry 4】

8. The compound according to claim 7, represented by TCP-306 or TCP-330, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

9. A pharmaceutical composition comprising at least one of the compounds according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient.

10. A pharmaceutical composition for preventing and / or treating a disease caused by a nonsense mutation, comprising, as an active ingredient, an amount effective for preventing and / or treating the disease of at least one of the compounds according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

11. The following formula (2): 【Transformation 5】 [In formula (2), n represents 1; R １ and R ２ is a hydrogen atom; R ３ represents a hydrogen atom, the following partial structural formula (S-1) [wherein the formula does not include the part to the right of the dashed line]: 【Transformation 6】 (In partial structural formula (S-1), m represents an integer of 0, 1 or 2; R ４ is C 1-10 alkyl, C 2-10 alkenyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, CH ３ CH ２ C 1-10 alkyl substituted with CONH—, C 2-10 alkenyl substituted with phenyl, —(OCH ２ CH ２ ) ２ -OCH ３ , —O—CH ２ -phenyl, triazole substituted with a benzyl group, or phenyl substituted with a cyclopropylmethyloxy group; Or the following partial structural formula (S-2) [wherein the formula does not include the right side of the dashed line]: 【Transformation 7】 (In partial structural formula (S-2), R ８ is C ６ an alkanoylamino group; R ５ is a hydrogen atom or C １～３ is an alkyl group; R ６ is a methyl group; R ７ is a hydrogen atom, C １～６ Alkyl groups, halogenated C １～６ Alkyl group, C ２～１０ Alkenyl group, C ６～１０ an aryl group, or C ７～１６ is an aralkyl group; R ７ C in ６～１０ Aryl group or C ７～１６ The aralkyl group may be a halogen atom, a C １～６ Alkyl group or C １～６ and optionally substituted with 1 to 3 alkoxy groups. A readthrough agent for a premature stop codon formed by a nonsense mutation, comprising as an active ingredient at least one of a compound represented by the formula:

12. The R ３ is a hydrogen atom or the partial structural formula (S-1).

13. The R ３ is a hydrogen atom or the partial structural formula (S-1), The R ３ The lead-through agent according to claim 12, wherein when the partial structural formula (S-1) is

14. The R ３ is a hydrogen atom or the partial structural formula (S-1), The R ３ is the partial structural formula (S-1), m is 0, and R ４ is C 1-10 alkyl, C 2-10 alkenyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, CH ３ CH ２ The read-through agent according to claim 13, which is a C 1-10 alkyl substituted with CONH-, a C 2-10 alkenyl substituted with phenyl, or a phenyl substituted with a cyclopropylmethyloxy group.

15. The R ３ is the partial structural formula (S-1), and m is 0; The R ４ is C 1-10 alkyl, C 2-10 alkenyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, CH ３ CH ２ The read-through agent according to claim 14, which is a C 1-10 alkyl substituted with CONH- or a C 2-10 alkenyl substituted with phenyl.

16. The R ３ is the partial structural formula (S-1), and m is 0; The R ４ is C 1-10 alkyl, C 1-10 alkyl substituted with an amino group, C 1-10 alkyl substituted with a methoxy group, C 1-10 alkyl substituted with a hydroxyl group, C 1-10 alkyl substituted with a phenyl, C 1-10 alkyl substituted with a 3- to 8-membered non-aromatic heterocyclic group or a 5- to 6-membered heteroaryl group, or CH ３ CH ２ The read-through agent of claim 15, which is a C 1-10 alkyl substituted with CONH-.

17. A readthrough agent for premature termination codons formed by nonsense mutations, characterized by containing as an active ingredient a compound represented by TCP-304, TCP-306, TCP-330 or TCP-341: 【Transformation 8】

18. The read-through agent according to claim 17, wherein the compound is represented by TCP-306 or TCP-330.

Images