JPWO2018016523A1 - New radiation protective agent - Google Patents

Abstract

The present invention relates to a radiation protective agent comprising a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient (wherein, ring A represents a fused ring, R 1 represents a hydrogen atom or 1 carbon atom) A radiation protective agent is provided, which comprises as an active ingredient 6 to 6 alkyl groups), in particular the following compounds: The radioprotective agent of the present invention reduces cell damage and cell death resulting from radiation exposure and has a protective effect on immune related cells.

Description

  The present invention relates to a radiation protective agent comprising a compound having an action capable of reducing radiation damage after radiation exposure, ie, a radiation protective action. More particularly, it relates to a radioprotectant that reduces cell damage and cell death resulting from radiation exposure and has a protective effect on immune related cells.

Agents that can reduce radiation damage after radiation exposure are called radiation protective agents. Significant radiation exposure primarily causes damage in 1) the hematopoietic system, 2) the digestive system, and 3) the cardiovascular / central nervous system. Radioprotective agents are also being investigated as agents to aid in the radiation treatment of cancer.
Conventional radioprotectants are considered to exhibit protective effects such as reduction of radicals and active oxygen generated by indirect action of radiation. For example, aminothiol derivatives such as cysteamine (mercaptoethylamine) and WR-2721 (S-2- (3-aminopropylamino) ethyl dihydrogen phosphorothioate; amiphostin) are representative thereof. However, all currently known drugs have not been put to practical use because they have strong side effects such as reduced judgment and agility and vomiting. Furthermore, existing radioprotectants need to be administered just before exposure to be effective, and accidental accidents can also occur, so development of radioprotectants that exert effects even after exposure It is desired.

Lysophosphatidic acid (LPA) is a lipid mediator produced during tissue injury and has seven transmembrane receptors coupled to various G proteins (G _{q / 11/14} , G _12/13 , G _{i / o} ) It is known to act on the body (LPA ₁ , LPA ₂ , LPA ₃ , LPA ₄ , LPA ₅ , LPA ₆ ) and as a trophic factor for various cells including nerve and glial cells. In recent years, it has been reported that LPA ₂ receptor agonists are effective as a radioprotectant (Patent Document 1 and Non-patent Document 1), but they have not yet been put into practical use in terms of effects, safety and the like.

Japanese Patent Publication No. 2010-510238

Patil, R. et al., Chemistry & Biology 22, 206-216, 2015

  An object of the present invention is to provide a radioprotectant that reduces cell damage and cell death resulting from radiation exposure and has a protective effect on immune-related cells.

The present inventors focused on LPA ₂ receptor agonists and conducted various studies on their radioprotective effects. As a result, LPA ₂ receptor agonists with certain specific structures have a strong inhibitory effect on radiation-induced apoptosis (especially intestinal cells), and additionally T cells and B cells after irradiation It was also found to have a protective effect on immune cells. Furthermore, it has been confirmed in a mouse individual (in vivo) that these compounds have an excellent survival effect after irradiation, and the present invention has been completed.

That is, the present invention provides the following.
[1] A radioprotector containing a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

(Wherein, ring A represents a fused ring, R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
[2] The agent according to [1] above, wherein ring A is a nitrogen-containing fused ring.
[3] The agent according to the above-mentioned [2], wherein ring A is indole or quinoline.
[4] The agent according to the above-mentioned [1], wherein ring A is naphthalene.
[5] The agent according to the above-mentioned [1], wherein ring A is tetrahydrobenzothiophene.
[6] The agent according to any one of the above [1] to [5], wherein R ₁ is a methyl group.
[7] The agent according to the above [1], wherein the compound represented by the formula (I) is selected from the following compound group.

[8] The agent according to the above [1], wherein the compound represented by the formula (I) is selected from the following compound group.

[9] The agent according to the above [1], wherein the compound represented by the formula (I) is the following compound.

[10] The agent according to any one of the above [1] to [9], which has a protective effect on immune cells after irradiation.
[11] The agent according to any one of the above [1] to [10], which has a protective effect on intestinal cells after irradiation.
[12] The agent of the above-mentioned [10] or [11], wherein the irradiation is radiation treatment of cancer.
[13] The agent according to any one of the above-mentioned [1] to [12], which is a preventive and / or therapeutic agent for radiation injury.
[14] The agent according to the above-mentioned [13], wherein the radiation injury is a side effect during radiation treatment of cancer.
[15] A preventive and / or therapeutic agent for radiation injury, which comprises the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

(Wherein, ring A represents a fused ring, R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
[16] The prophylactic and / or therapeutic agent according to the above-mentioned [15], wherein the radiation damage is a side effect during radiation treatment of cancer.
[17] A method for the prophylaxis and / or treatment of radiation disorders, which comprises administering an effective amount of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof.

(Wherein, ring A represents a fused ring, R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
[18] The method for prophylaxis and / or treatment according to the above-mentioned [17], wherein the radiation injury is a side effect during radiation treatment of cancer.
[19] A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof for use in the prophylaxis and / or treatment of radiation disorders.

(Wherein, ring A represents a fused ring, R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
[20] The compound of the above-mentioned [19] or a pharmaceutically acceptable salt thereof, wherein the radiation damage is a side effect during radiation treatment of cancer.
Hereinafter, the compound represented by formula (I) (also referred to as compound (I)) and a pharmaceutically acceptable salt thereof may be generically referred to as a compound of the present invention.

  The compound of the present invention suppresses the apoptosis of stem cells caused by irradiation and as a result exerts an immunocytoprotective effect on small intestinal villi tissue or T cells. Furthermore, such a protective effect is exhibited even after irradiation. Therefore, the compound of the present invention is useful as an unprecedented radioprotective agent effective at a low concentration and having a protective effect on immune cells, and as a preventive and / or therapeutic agent for so-called radiation injury caused by irradiation. is there.

It is the graph which showed the apoptosis suppression effect of this invention compound (compound 1 and compound 2) to the intestinal-cultured cell. Compound 1 was also examined for enantiomers (R form and S form). The percentage of apoptotic cells induced by radiation was determined. It is a figure which shows the result of having investigated the survival effect of this invention compound (compound 1) at the time of irradiation to a mouse | mouth individual. The upper row shows mouse individual survival rate, and the lower row shows mouse individual weight transition. The compound of the present invention (N = 11) or vehicle (N = 22) was intraperitoneally administered 30 minutes before and after γ-ray irradiation. p <0.01 It is a figure which shows the result of having investigated the survival effect of this invention compound (compound 2) at the time of irradiation to a mouse | mouth individual. It is a figure which shows the result of having investigated the survival effect of this invention compound at the time of irradiation to a mouse | mouth individual. The compounds of the present invention or vehicle were intraperitoneally administered after gamma irradiation (0.5, 1, 3, 6 hours later). It is a figure which shows the result of having investigated the small intestine protective effect of this invention compound at the time of irradiation. The upper row is an image obtained by observing villi of small intestine under a microscope, the lower left figure is a graph showing the result of examining the villi area of the small intestine, and the lower figure is a graph showing the result of examining the villi length of the small intestine. . FIG. 6 shows that the small intestine protective effect of the compound of the present invention upon irradiation is almost completely abolished in LPA ₂ gene-deficient mice (LPA ₂ KO). It is a graph which shows the result of having investigated the small intestinal tract crypt cell maintenance effect of this invention compound at the time of irradiation. The number of crypt structures containing Ki67 positive cells was measured using Ki67, which is a marker for dividing cells, as an index. It is a graph which shows the result of having investigated the protective effect of the small intestine immune cell of this invention compound at the time of irradiation. The percentage of T cells and B cells in small intestinal villi tissue was measured. The expression of each marker was examined 9 days after γ irradiation using CD3 as a marker for T cells and B220 as a marker for B cells. It is a graph which shows the protective effect of this invention compound (compound 1) in the spleen and bone marrow of this invention compound at the time of irradiation. The protective effect on the spleen was evaluated by measuring the CD3 positive area and the protective effect on the bone marrow by measuring the number of Ki67 positive cells. It is a graph which shows the result of having investigated the radioprotective effect of this invention compound (compound 1) with respect to SAS cell which is 1 type of an oral squamous cell carcinoma cell line. The upper row shows that the expression of the LPA ₂ receptor is low in SAS cells. It has been shown that the compounds of the present invention do not exert a radioprotective effect on SAS cells. It is a graph which shows the result of having investigated the inhibitory effect of this invention compound in the apoptosis induced by radiation.

Hereinafter, the present invention will be described. The terms used herein have meanings commonly used in the art unless otherwise stated.
The terms used in the present specification are defined below.

As used herein, "fused ring" refers to a cyclic structure in which two or more rings are linked by two or more atoms.
The fused ring is preferably a fused ring containing one monocyclic aromatic ring or monocyclic heteroaromatic ring, more preferably a monocyclic aromatic ring, heteroaromatic ring, aliphatic ring and heteroaliphatic ring A fused ring consisting of two rings selected from the group consisting of
As a monocyclic aromatic ring, benzene is mentioned, for example. As a monocyclic heteroaromatic ring, for example, 1 to 4 hetero atoms selected from an oxygen atom, a sulfur atom (the sulfur atom may be oxidized) and a nitrogen atom in addition to a carbon atom as a ring constituting atom Examples thereof include 5- or 6-membered heteroaromatic rings. Preferable specific examples of the 5-membered heteroaromatic ring include, for example, furan, thiophene, pyrrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, isoxazole, oxadiazole (eg, 1,2,4-oxadiazole, etc.) , 1,3,4-oxadiazole), thiadiazole (eg, 1,2,4-thiadiazole, 1,3,4-thiadiazole), triazole (eg, 1,2,3-triazole, 1,3,4) -Triazole) etc. are mentioned. Preferred specific examples of the 6-membered heteroaromatic ring include, for example, pyridine, pyrimidine, pyridazine, pyrazine, triazine (eg, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-) Triazine etc.).
Examples of monocyclic aliphatic rings include cyclopentane, cyclohexane, cycloheptane, cyclooctane and the like. As the hetero aliphatic ring, those obtained by hydrogenating the hetero aromatic ring can be mentioned.
Specific examples of the fused ring include naphthalene, indole, benzofuran, benzothiophene, tetrahydrobenzothiophene, isobenzofuran, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, indolizine, purine, pteridine and the like. Not limited to these.

The fused ring may optionally have a substituent at a substitutable position. As the substituent which the fused ring may have,
(1) halogen atom (for example, fluorine, chlorine, bromine, iodine),
(2) Lower alkyl group (eg, C _1-6 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl etc.),
(3) cycloalkyl group (for example, C _3-6 cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl etc.),
(4) Lower alkynyl groups (eg, C _2-6 alkynyl groups such as ethynyl, 1-propynyl, propargyl, etc.),
(5) Lower alkenyl groups (eg, C _2-6 alkenyl groups such as vinyl, allyl, isopropenyl, butenyl, isobutenyl etc.),
(6) Aralkyl groups (eg, C _7-12 aralkyl groups such as benzyl, α-methylbenzyl, phenethyl etc., etc.),
(7) aryl groups (eg, C _6-10 aryl groups such as phenyl and naphthyl);
(8) Lower alkoxy group (for example, C _1-6 alkoxy group such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like),
(9) aryloxy group (eg, C _6-10 aryloxy group such as phenoxy etc.),
(10) formyl group or lower alkanoyl group (eg, C _1-6 alkyl-carbonyl group such as acetyl, propionyl, butyryl, isobutyryl etc.),
(11) arylcarbonyl group (eg, C _6-10 aryl-carbonyl group such as benzoyl, naphthoyl, etc.),
(12) formyloxy group or lower alkanoyloxy group (eg, C _1-6 alkyl-carbonyloxy group such as acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, etc.),
(13) arylcarbonyloxy group (eg, C _6-10 aryl-carbonyloxy group such as benzoyloxy, naphthoyloxy, etc.),
(14) Carboxyl group,
(15) Lower alkoxycarbonyl group (for example, C _1-6 alkoxy-carbonyl group such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl etc., etc.),
(16) aralkyloxycarbonyl group (eg, C _7-12 aralkyloxy-carbonyl group such as benzyloxycarbonyl etc., etc.),
(17) carbamoyl group,
(18) Mono-, di- or tri-halogeno-lower alkyl group (for example, mono-, di- or tri-halogeno such as chloromethyl, dichloromethyl, trifluoromethyl, 2,2,2-trifluoroethyl etc. C _1-6 alkyl group etc.),
(19) Oxo group,
(20) amidino group,
(21) Imino group,
(22) amino group,
(23) mono-lower alkylamino group (eg, mono-C _1-6 alkylamino group such as methylamino, ethylamino, propylamino, isopropylamino, butylamino etc.),
(24) Di-lower alkylamino group (eg, di-C _1-6 alkylamino group such as dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, N-ethyl-N-methylamino, etc.),
(25) 3 to 3 which may have a substituent and may contain 1 to 3 hetero atoms selected from nitrogen atom, oxygen atom and sulfur atom in addition to carbon atom and 1 nitrogen atom 8-membered nitrogen-containing Hajime Tamaki (e.g., halogen atom, nitro group, cyano group, hydroxy group, an optionally halogenated C _1-6 alkyl group, a C _1-6 alkoxy group which may be halogenated , Amino group, mono-C _1-6 alkylamino group, di-C _1-6 alkylamino group, carboxyl group, C _1-6 alkyl-carbonyl group, C _1-6 alkoxy-carbonyl group, carbamoyl group, mono- C _1-6 alkyl - carbamoyl group, a di _{-C 1-6} alkyl - carbamoyl _{group, C 6-10} aryl - carbamoyl _{group, C 6-10} aryl _{group, C 6-10} aryloxy group and, Halogenated which may be C _1-6 alkyl - carbonyl amino group may have 1 to 5 substituents selected from oxo group, a nitrogen in addition to carbon atoms and one nitrogen atom 3 to 8 membered nitrogen-containing heterocyclic group which may contain 1 to 3 hetero atoms selected from an atom, an oxygen atom and a sulfur atom; for example, aziridinyl, azetidinyl, pyrrolidinyl, pyridyl, pyrrolinyl, pyrrolyl, imidazolyl, Pyrazolyl, imidazolidinyl, piperidyl, oxadiazolyl, isoxazolyl, morpholinyl, dihydropyridyl, tetrahydropyridyl, piperazinyl, N-methylpiperazinyl, N-ethylpiperazinyl etc.),
(26) alkylenedioxy group (eg, methylene dioxy, C _1-3 alkylenedioxy group such as ethylenedioxy, etc.),
(27) hydroxy group,
(28) nitro group,
(29) cyano group,
(30) mercapto group,
(31) sulfo group,
(32) Sulfino group,
(33) phosphono group,
(34) sulfamoyl group,
(35) Mono-lower alkylsulfamoyl group (eg, N-methylsulfamoyl, N-ethylsulfamoyl, N-propylsulfamoyl, N-isopropylsulfamoyl, N-butylsulfamoyl, etc. Mono-C _1-6 alkylsulfamoyl group etc.),
(36) Di-lower alkylsulfamoyl group (eg, N, N-dimethylsulfamoyl, N, N-diethylsulfamoyl, N, N-dipropylsulfamoyl, N, N-dibutylsulfamoyl, etc. Di-C _1-6 alkylsulfamoyl group etc.),
(37) Lower alkylthio group (eg, C _1-6 alkylthio group such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, tert-butylthio etc., etc.),
(38) arylthio groups (eg, C _6-10 arylthio groups such as phenylthio, naphthylthio and the like),
(39) Lower alkylsulfinyl groups (eg, C _1-6 alkylsulfinyl groups such as methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl and the like),
(40) arylsulfinyl groups (eg, C _6-10 arylsulfinyl groups such as phenylsulfinyl, naphthylsulfinyl and the like),
(41) a lower alkylsulfonyl group (eg, a C _1-6 alkylsulfonyl group such as methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl and the like),
(42) arylsulfonyl group (for example, C _6-10 arylsulfonyl group such as phenylsulfonyl, naphthylsulfonyl etc., etc.),
(43) Substituents selected from the group consisting of lower alkylcarbonylamino groups (for example, C _1-6 alkylcarbonylamino groups such as methylcarbonylamino etc.) and the like (herein referred to as substituent group A) Be

  In the present specification, the "C1-C6 alkyl group" means a linear or branched C1-C6 alkyl group, and specifically, methyl, ethyl, n-propyl, isopropyl , N-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl and the like.

  The compound (I) will be described below.

In formula (I), ring A represents a fused ring.
The fused ring in ring A is preferably a nitrogen-containing heterocyclic ring, more preferably indole or quinoline. As the fused ring in ring A, naphthalene and tetrahydrobenzothiophene are also preferred.
In formula (I), R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, preferably an alkyl group having 1 to 6 carbon atoms.
The alkyl group having 1 to 6 carbon atoms in R ₁ is preferably methyl.

  Among the compounds (I), preferred is a compound represented by the following formula.

More preferably, it is a compound represented by the following formula.

  Particularly preferred is a compound represented by the following formula.

  As a pharmaceutically acceptable salt of compound (I), for example, trifluoroacetic acid, acetic acid, lactic acid, succinic acid, maleic acid, maleic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, benzoic acid, methanesulfonic acid, p- Acid addition salts with acids such as toluenesulfonic acid, cinnamic acid, fumaric acid, phosphonic acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, sulfuric acid, etc .; eg sodium, potassium, magnesium, calcium And metal salts such as, for example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, N-methylpyrrolidine, N-methylpiperidine, N-methylmorpholine, and the like.

  When compound (I) has one or more isomers such as optical isomers, stereoisomers, positional isomers and rotational isomers, either one isomer or a mixture of isomers is also included in compound (I). Be done. For example, when compound (I) has an optical isomer, an optical isomer resolved from a racemate is also encompassed in compound (I). Each of these isomers can be obtained as a single product by a synthesis method known per se, a separation method (concentration, solvent extraction, column chromatography, recrystallization etc.). In addition, structural isomers such as tautomers and geometric isomers exist in Compound (I), and such isomers are also within the scope of the present invention.

  Compound (I) may be in a crystalline or amorphous form. When Compound (I) is a crystal, Compound (I) is encompassed whether the crystal form is single or a mixture of crystal forms. The crystals can be produced by crystallization using a crystallization method known per se.

  Compound (I) may be a solvate (eg, hydrate etc.) or a non-solvate, both of which are encompassed in compound (I).

The compounds of the present invention have a remarkable inhibitory effect on radiation-induced apoptosis, and further have a protective effect on immune cells after irradiation, in particular, immune cells in small intestinal villi and spleen. Due to the excellent radioprotective activity of the compound of the present invention, the compound of the present invention is preferably a mammal (eg, human, monkey, cat, pig, horse, cow, mouse, rat, guinea pig, dog, rabbit, etc., preferably) For humans, it is useful as a radioprotectant and as a preventive and / or therapeutic agent for radiation damage caused by radiation exposure.
Radiation in the present invention includes alpha rays, beta rays, gamma rays emitted from radioactive materials, artificially created X rays, proton rays, carbon rays, neutral rays, and electron rays, and radiation damage in the present invention Is a disorder associated with such radiation exposure and radiation therapy, and does not include exposure (natural exposure) from naturally occurring radiation sources. For example, acute and / or late radiation damage due to general radiation exposure due to nuclear accident or nuclear explosion, or local radiation exposure due to radiation for medical purposes such as cancer treatment or radiation exposure accident etc. Acute and / or late radiation damage may be included. Among these, the compound of the present invention is preferably used for protection of local or systemic acute radiation damage, and particularly preferably for protection of hematopoietic / immune system damage.
Moreover, the protection in the present invention includes prevention and / or treatment of such radiation exposure and disorders (side effects) involved in radiation treatment of cancer, and is preferably used for treatment.
Side effects of radiation therapy for cancer include acute and late disorders. Acute disorders include skin disorders, mucosal disorders such as the oral cavity, gastrointestinal tract disorders such as nausea, vomiting and diarrhea, pharyngeal edema, dyspnea, hair loss, increased intracranial pressure and the like. Late-onset disorders include cataract, brain necrosis, necrosis of teeth or jaw bone, esophageal stricture, interstitial pneumonia / pulmonary fibrosis, heart failure, ileus, intestinal hemorrhage, cystitis or secondarily induced cancer etc. It can be mentioned. Since the compound of the present invention is excellent in the protective effect of immune cells and intestinal cells, it is effective in reducing the side effects during radiation treatment of cancer.
Damage from radiation exposure also follows the side effects of radiation treatment for cancer, but may present with more severe symptoms.

  The radioprotective agent of the present invention or the preventive and / or therapeutic agent for radiation damage is within 60 minutes, preferably within 30 minutes, more preferably within 15 minutes, still more preferably within 10 minutes, before or after radiation exposure. Preferably, administration is initiated within 5 minutes. Further, additional administration after one day after radiation exposure is also preferable from the viewpoint of life extension effect and improvement of survival rate.

  The dose of the radioprotective agent or preventive and / or therapeutic agent for radiation damage of the present invention depends on conditions such as type of compound to be administered, purpose of protection (prevention or treatment) of radiation damage, age and condition of the patient, etc. Although it can be selected as appropriate, for example, the concentration of the compound of the present invention or a pharmaceutically acceptable salt thereof in vivo may be 0.5 to 20 μM, preferably 1 to 15 μM, more preferably 5 to 10 μM immediately after administration. Do so as to The dose per single dose is 5 to 50 mg, more preferably 20 to 40 mg, and particularly preferably 25 to 30 mg as compound (I) per kg of body weight.

  The compound of the present invention is mixed with a pharmaceutically acceptable carrier, and solid preparations such as tablets, capsules, granules and powders; liquid preparations such as syrups and injections; patchs, ointments, plasters and the like It can be formulated suitably as a percutaneous absorption agent; inhalation agent; suppository.

  As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials can be used. Specifically, it includes an excipient in a solid preparation, a lubricant, a binder, a disintegrant, a solvent in a liquid preparation, a solubilizing agent, a suspending agent, a tonicity agent, a buffer, a soothing agent, etc. can do. Moreover, formulation additives such as preservatives, antioxidants, coloring agents, and sweeteners can also be used, if necessary.

  Examples of excipients include lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, licorice powder, mannitol, sodium hydrogen carbonate, calcium phosphate, calcium sulfate and the like.

  Examples of lubricants include magnesium stearate, stearic acid, calcium stearate, purified talc, colloidal silica and the like.

  Examples of the binder include crystalline cellulose, sucrose, mannitol, dextrin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone and the like.

  Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like.

  Preferred examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.

  Preferred examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Examples of suspending agents include, for example, surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, etc .; polyvinyl alcohol, polyvinyl pyrrolidone, Carboxymethylcellulose sodium, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and the like can be mentioned.

  Preferred examples of the tonicity agent include sodium chloride, glycerin, D-mannitol and the like.

Preferred examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
Preferred examples of the soothing agent include benzyl alcohol and the like.

Preferred examples of preservatives include, for example, p-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferred examples of the antioxidant include sulfites, ascorbic acid and the like.
Preferred examples of the colorant include, for example, tar dyes, caramel, ferric oxide, titanium oxide, riboflavins and the like.
Preferred examples of sweetening agents include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, simple syrup and the like.

Compound (I), an isomer, a solvate and pharmaceutically acceptable salts thereof are commercially available or utilize characteristics based on the basic skeleton or the type of substituent, Various known (previously reported) synthetic methods can be applied, and further, if desired, they can be appropriately modified and manufactured. Such modifications include alkylation, acylation, amination, imination, halogenation, reduction, oxidation and the like, and any reaction or method generally used in the art is used. At that time, depending on the type of functional group, if it is effective in production technology to replace the functional group with a suitable protecting group (group that can be easily converted to the functional group) at the raw material or intermediate stage. There is. The chemical nature of the protecting group, the method of its introduction, and its removal are detailed, for example, in T. Greene and P. Wuts “Protective Groups in Organic Synthesis” (3 ^rd ed.), John Wiley & Sons NY (1999) ing.

  Unless otherwise stated, starting compounds are readily commercially available or can be provided by known compound library resources, and based on the information, methods known per se or these are It can manufacture according to an equivalent method.

Moreover, in each reaction of each reaction and raw material compound synthesis, a generally known solvent may be used during the reaction.
Commonly known solvents include, for example, ethers such as tetrahydrofuran, diethyl ether, 1,2-dimethylethane, 1,4-dioxane, etc., esters such as ethyl acetate and butyl acetate, and aromatic carbonization such as benzene and toluene Hydrogen, an aromatic heterocyclic compound such as pyridine or lutidine, an amide such as N, N-dimethylformamide or N-methylpyrrolidone, a halide such as chloroform or methylene chloride, methanol, ethanol, 2-propanol, 2,2 Alcohols such as dimethylethanol, hydrocarbon compounds such as hexane, heptane and petroleum ether, carboxylic acids such as formic acid and acetic acid, and water.
In addition, as a solvent used in the reaction, a single solvent may be used, or two to six solvents may be mixed and used.
The reaction may be carried out, for example, in the presence of an amine such as triethylamine, N, N-diisopropylamine, pyridine, N-methylmorpholine, or a base such as sodium hydroxide or potassium carbonate.
The reaction may be carried out in the presence of an acid such as hydrochloric acid, sulfuric acid or acetic acid.

  The present invention will be described in more detail by way of the following examples, which do not limit the scope of the present invention. Also, reagents and materials to be used are commercially available unless otherwise specified.

Synthesis example 1

2-quinolinecarboxaldehyde (471 mg) was dissolved in trifluoroethanol, 2-pipecoline (297 mg) was added, and stirred at 60 ° C. for 15 minutes. The temperature was lowered to 40 ° C. and sodium borohydride (114 mg) was added and stirred for 1 hour. The reaction mixture was diluted with saturated aqueous ammonium chloride solution (40 mL) and extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. The solution is evaporated under reduced pressure, and the residue is purified by column chromatography (developing solvent: acetic acid ethyl ester: methanol = 10: 1) to give 2-((2-methylpiperidin-1-yl) methyl) quinoline as brownish brown Obtained as a liquid product (65 mg, 9% yield).
¹ H NMR (500 MHz, CDCl ₃ ) δ 1.18 (d, J = 6.1 Hz, 3 H), 1.31-1. 45 (m, 2 H), 1.50-1. 56 (m, 2 H), 1.61-1. 71 (m, 2 H) , 2.17-2.22 (m, 1H), 2.46- 2.49 (m, 1H), 2.77 (dt, J = 3.7, 11.8 Hz, 1 H), 3.59 (d, J = 14.4 Hz, 1 H), 4.27 (d, J = 14.4 Hz, 1 H), 7.48-7.52 (m, 1 H), 7.67-7.72 (m, 2 H), 7. 79 (d, J = 8.1 Hz, 1 H), 8.06 (d, J = 8.3 Hz, 1 H), 8.10 (d, J = 8.6 Hz, 1 H).

Synthesis example 2

(1) 2-quinolinecarboxaldehyde (1.0 g) was dissolved in methanol, sodium borohydride (292 mg) was added under ice-cooling, and the mixture was stirred for 1 hour. The reaction mixture was evaporated under reduced pressure, and the residue was extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. The solution was evaporated under reduced pressure to give a white solid (1.0 g). The obtained white solid (1.0 g) was dissolved in 10 mL of dichloromethane, thionyl chloride (500 μL) was added, and the mixture was stirred at room temperature for 10 hours. After completion of the reaction, the reaction solution was diluted with aqueous potassium carbonate solution (10 mL) and extracted with dichloromethane. The extract was washed successively with water and saturated brine and dried over magnesium sulfate. The solution is evaporated under reduced pressure, and the residue is purified by column chromatography (developing solvent: ethyl acetate: hexane = 1: 2) to give 2- (chloromethyl) quinoline as a white solid (816 mg, 73% yield) Got as.
¹ H NMR (500 MHz, CDCl ₃ ) δ 4.85 (s, 2 H), 7.56-7.59 (m, 1 H), 7.63 (d, J = 8.6 Hz, 1 H), 7.73-7. 76 (m, 1 H), 7.84 (d, J = 8.1 Hz, 1 H), 8.08 (d, J = 8.6 Hz, 1 H), 8.21 (d, J = 8.6 Hz, 1 H).
(2) Dissolve 2- (chloromethyl) quinoline (300 mg) obtained in the above (1) in acetone, add 2-pipecoline (200 mg) and potassium carbonate (464 mg), and stir at room temperature for 20 hours did. The reaction mixture was diluted with water and extracted with ethyl acetate. The extract was washed successively with water and saturated brine, and dried over magnesium sulfate. The solution is evaporated under reduced pressure, and the residue is purified by column chromatography (developing solvent: ethyl acetate: methanol = 10: 1) to give 2-((2-methylpiperidin-1-yl) methyl) as a brownish liquid. Obtained 365 mg (90%) of quinoline.

Production Example Acquisition of a Compound Having the Basic Structure of the Compound of the Present Invention The 9600 compounds contained in the Core Library of the University of Tokyo Drug Discovery Open Innovation Center were screened for LPA ₂ receptor agonist activity by Gq calcium assay, and excellent LPA ₂ receptor The compound having agonist activity was obtained and its basic structure was found. Subsequently, optimization of compound synthesis was performed based on the basic structure (see the above-mentioned Synthesis Examples 1 and 2 for Compound 1) to obtain the following compounds (Compounds 1 to 4). The obtained compound was examined for its radiation protective effect.

(※ Gq calcium assay)
Cells stably expressing the LPA ₂ receptor were used to investigate their ability to induce LPA-induced calcium transients (EC ₅₀ ). The specific test method is as follows.
Cells in which human LPA ₂ receptor was forcibly expressed by Lipofectamine 2000 (Thermo Fisher Scientific) against B103 cells are cultured for 24 hours. Then, the cells were seeded in a 384 well plate so as to be 10000 cells / well, and 8 μM QUEST FLUO-8H, 1 mg / ml Amaranth, 0.1% BSA were added and incubation was performed for 30 minutes. Furthermore, after adding a test compound adjusted to an appropriate concentration, the fluorescence intensity from the green fluorescent calcium indicator QUEST FLUO-8H was measured using Functional Drug Screening System / μCell (Hamamatsu Photonics). This calcium elevation was taken as the activation intensity of the LPA ₂ receptor by the compound.
Similarly, cells stably expressing the LPA ₁ receptor, LPA ₃ receptor, LPA ₄ receptor or LPA ₅ receptor are prepared, and these cells are used to induce an LPA-induced calcium transient. The ability (EC ₅₀ ) was examined.
Agonist activity for each LPA receptor measured for Compounds 1 and 2 is shown in Table 1.

Example 1: Apoptosis inhibitory effect of the compound of the present invention in cultured intestinal epithelial cells-1
Intestinal epithelial cells (IEC 6 cells) cultured in DMEM medium supplemented with 5% fetal bovine serum and insulin (10 μg / ml) were used for the experiment. The cells were irradiated with 9 gray γ-rays ([ ¹³⁷ Cs]) using a γ-ray irradiation apparatus (Pony Industrial PS-3100SB) to induce apoptosis.
Using Compounds 1 to 4 obtained in Production Examples, the proportion of cells in which apoptosis was induced was examined to evaluate its suppressive effect. Specifically, the proportion of apoptotic cells was evaluated as follows.
Anti-phosphorylated histone H2AX antibody (BioLegend 613401) against histone H2AX which is known to be distributed throughout the nucleus only in apoptotic cells after fixing IEC6 cells with 4% paraformaldehyde 24 hours after γ-irradiation The percentage of apoptotic cells was determined by fluorescence immunostaining using.
The results for Compound 1 and Compound 2 are shown in FIG.
By using this method, significant inhibitory effects on apoptosis were observed for both compound 1 and compound 2 of the present invention.
Moreover, as a result of examining also about the enantiomer of compound 1, as compared with S form, compound 1 of R form has strong agonist activity compared with S form, and R form compound 1 is lower than S form compound 1 in low concentration conditions. It was confirmed that a stronger anti-apoptotic effect was shown (FIG. 1).

Example 2: Life-preserving effect of the compound of the present invention upon irradiation 9 Gray γ-rays ([ ¹³⁷ Cs]) were irradiated to the whole body of a mouse individual. The compound of the present invention (compound 1) (30 mg / kg) or vehicle was intraperitoneally administered 30 minutes before and after γ-ray irradiation, and the relationship between the presence or absence of administration and the survival rate of the mouse and weight transition was examined. Specifically, it investigated as follows.
Eight-week-old C57BL / 6J male mice were whole-body irradiated with 9 gray γ-rays using a γ-ray irradiation apparatus (Pony Industrial PS-3100SB) under anesthesia. Thereafter, breeding of the mice was continued with a positive pressure clean rack, and the survival time after γ-irradiation of each mouse was measured.
The results are shown in FIG. A significant survival effect and a weight loss suppression effect were confirmed in a population in which the compound of the present invention was intraperitoneally administered to mice 30 minutes before and after γ irradiation.
Similarly, a significant survival benefit was also confirmed in a population in which the compound of the present invention (compound 2) was intraperitoneally administered 30 minutes before and after γ-irradiation (FIG. 3).
Furthermore, the timing of administration of the compound of the present invention (compound 1) and the life prolongation effect were examined. Administration was performed only after irradiation, and the compounds of the present invention were administered 0.5, 1, 3 and 6 hours after irradiation. The results are shown in FIG. Even after administration only after irradiation, a significant survival benefit was obtained, and in particular, administration after 0.5 to 1 hour had a significant survival benefit.

Example 3: The small intestine protective action of the compound of the present invention at the time of radiation irradiation The compound of the present invention (compound 1) (30 mg / kg) or vehicle was intraperitoneally administered 30 minutes before and after γ-ray irradiation, with and without administration and after γ-ray irradiation The relationship with the protective effect of intestinal tract was examined. Specifically, after irradiating the whole body of a mouse individual with 9 gray gamma rays ([ ¹³⁷ Cs]), the small intestine was excised immediately after euthanasia treatment by cervical dislocation on the 9th, and 4% paraformaldehyde was used. Fixing was performed at 4 ° C. for 16 hours. Thereafter, a small intestinal tissue paraffin section of 5 μm in thickness was prepared using a paraffin block making apparatus (Milestone) and a paraffin section making apparatus (Thermo Fisher). Hematoxylin and eosin staining was performed on the small intestine tissue paraffin sections, and imaging of the small intestine tissue was performed using an all-in-one fluorescence microscopic BIOREVO (KEYENCE). The area and length of villi in the small intestine were measured using the image analysis software ImageJ (NIH) on the photographed image.
The results are shown in FIG. At 72 hours after γ-irradiation, the average area of individual villi in the small intestine is larger and the average length is longer in the compound-administered group of the present invention compared to the vehicle-administered control group compared with the vehicle-administered control group It was close. From this, it can be seen that the compound of the present invention alleviates damage to intestinal tissue caused by radiation.
The radioprotective effect of Compound 1 in the small intestine was almost completely abolished in LPA ₂ gene-deficient mice (LPA2KO). A typical example is shown in FIG. This result was also reproducible in another LPA ₂ gene-deficient mouse. The LPA ₂ gene deficient mouse was provided by Dr. Jerold Chun (Scripps Institute, La jolla, CA).

Example 4: Intestinal crypt dividing cell maintenance effect of the compound of the present invention at the time of irradiation The villus tissue of the small intestine maintains the villus structure by supplying new cells from the cryptosystem including the dividing cell immediately below it. It has been reported that this cryptogenic cell is damaged by irradiation.
Therefore, the compound of the present invention (compound 1) (30 mg / kg) or vehicle was intraperitoneally administered 30 minutes before and after γ-ray irradiation to a mouse individual to be irradiated with 12 gray γ-rays ([ ¹³⁷ Cs]). The relationship between the presence or absence of administration and the state of cryptogenic dividing cells in the small intestine after gamma irradiation was examined. The effect of the compound of the present invention on maintenance of crypt structure containing Ki67 positive cells was examined 12 hours and 72 hours after γ-irradiation, using Ki67 as a marker for dividing cells as an index. Specifically, quantitative analysis of Ki67 positive cells was performed on the small intestine tissue paraffin sections prepared by the above method by fluorescence immunostaining using an anti-Ki67 antibody (Abcam ab15580).
The results are shown in FIG. The γ-irradiation confirmed a decrease in the number of crypt structures containing Ki67 positive cells. On the other hand, the decrease was suppressed in the compound administration group of the present invention. From this, it is suggested that the compound of the present invention protects division cell death by radiation.

Example 5: Immunocytoprotective effect of the compound of the present invention at the time of irradiation 9 gray γ-rays ([ ¹³⁷ Cs]) were irradiated to the whole body of a mouse individual. The compound of the present invention (compound 1) (30 mg / kg) or vehicle was intraperitoneally administered 30 minutes before and after γ-ray irradiation, and the relationship between the presence or absence of the administration and the influence on immune cells after γ-ray irradiation was examined. Specifically, gamma rays were irradiated to 8-week-old C57BL / 6J male mice in the same manner as in the preparation of small intestine paraffin sections in Example 3, and spleens were removed immediately after euthanasia treatment was performed on the 9th day after irradiation. And postfixation with 4% paraformaldehyde was carried out at 4 ° C. for 16 hours. Quantitative analysis of T cells and B cells by fluorescence immunostaining using anti-CD3 antibody (Abcam ab16669) as a T cell marker and anti-B220 antibody (BioLegend 103201) as a B cell marker for small intestine and spleen tissue paraffin sections Did.
The results of examining the proportion of T cells and B cells in small intestinal villus tissue are shown in FIG. Although γ-irradiation reduces immune cells, administration of the compound of the present invention suppresses the decrease and confirms proliferation of immune cells. In particular, the effect was remarkable in T cells.
The protective effect of the compound of the present invention (Compound 1) was examined on T cells in the spleen, which is a secondary lymphatic tissue supplying these T cells and B cells, and proliferation cells in the bone marrow, which is a primary lymphatic tissue. The results are shown in FIG.
First, in the spleen 9 days after 9 gray γ-irradiation, Compound 1 administration protected the decrease in the number of T cell marker CD3 positive cells in comparison with the vehicle group. Moreover, this protective effect was not exhibited in the group where Compound 1 was administered to LPA2KO irradiated with γ-ray, suggesting that Compound 1 exerts a protective effect through the LPA ₂ receptor. Furthermore, in the bone marrow 9 days after 9 gray γ-irradiation, the reduction in the number of Ki67-positive cells, which is a dividing cell marker, due to irradiation was protected by administration of Compound 1.
Furthermore, the protection in the present invention includes the prevention and / or treatment of a disorder (side effect) involved in radiation treatment of cancer. LPA ₂ Compound 1 is a receptor agonist is assumed not to exert a protective effect on cells not expressing LPA ₂ receptor, thus applied to the time radiotherapy for tumors lower expression of LPA ₂ receptor It is believed that Gene expression analysis identified low expression of the LPA ₂ receptor in SAS cells, which is a type of oral squamous cell carcinoma cell line (FIG. 10, upper row). Twenty days after the transplantation of the SAS cells into the left foot, mice were irradiated with 24 gray γ-rays, and the site of subsequent transplantation was observed. The results are shown in the lower part of FIG.
The inhibitory effect equivalent to that of the vehicle group was also confirmed in the group of mice to which Compound 1 was administered with respect to the growth of cancer cells by γ-irradiation. This indicates that Compound 1 does not protect cancer cell lines from radiation, and it is a side effect reducing agent during radiation therapy that Compound 1 protects normal cells in the bone marrow etc. but not target cancer cells. Suggest that can be

Example 6: Apoptosis inhibitory effect of the compound of the present invention in cultured intestinal epithelial cells-2
The apoptosis inhibitory effect of the compound of the present invention (compound 1) was examined in the same manner as in Example 1. In addition, DBIBB (2- [4- (1,3-dioxo-1H, 3H-benzoisoquinolin-2-yl) butylsulfamoyl] benzoic acid) and GRI 977143 (2), which are known LPA ₂ receptor agonists The apoptosis inhibitory effect of-((3- (1,3-dioxo-1H-benzo [de] isoquinolin-2 (3H) -yl) propyl) benzoic acid) was also examined.

The results are shown in FIG. While the compound of the present invention showed a significant apoptosis inhibitory effect at a dose of 1.0 μM, the existing LPA ₂ receptor agonists DBIBB and GRI977143 required higher doses. From this, it can be seen that the compound of the present invention is superior to the existing LPA ₂ receptor agonists in radioprotective action.

  The compound of the present invention suppresses the apoptosis of stem cells caused by irradiation and as a result exerts an immunocytoprotective effect on small intestinal villi tissue or T cells. Furthermore, such a protective effect is exhibited even after irradiation. Therefore, the compound of the present invention is useful as a novel radioprotective agent which is effective at low concentration and has a protective effect on immune cells, and a preventive and / or therapeutic agent for radiation injury caused by irradiation.

  This application is based on patent application No. 2016-142646 filed in Japan (filing date: July 20, 2016), the contents of which are incorporated in full herein.

Claims (16)

A radioprotector comprising the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
(Wherein, ring A represents a fused ring, R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)   The agent according to claim 1, wherein ring A is a nitrogen-containing fused ring.   The agent according to claim 2, wherein ring A is indole or quinoline.   The agent according to claim 1, wherein ring A is naphthalene.   The agent according to claim 1, wherein ring A is tetrahydrobenzothiophene. The agent according to any one of claims 1 to 5, wherein R ₁ is a methyl group. The agent according to claim 1, wherein the compound represented by the formula (I) is selected from the following compound group.
The agent according to claim 1, wherein the compound represented by the formula (I) is selected from the following compound group.
The agent according to claim 1, wherein the compound represented by the formula (I) is the following compound.
  The agent according to any one of claims 1 to 9, which has a protective effect on immune cells after irradiation.   The agent according to any one of claims 1 to 10, which has a protective effect on intestinal cells after irradiation.   The agent according to claim 10 or 11, wherein the irradiation is by radiation treatment of cancer.   The agent according to any one of claims 1 to 12, which is a preventive and / or therapeutic agent for radiation injury.   The agent according to claim 13, wherein the radiation injury is a side effect of radiation treatment of cancer. A preventive and / or therapeutic agent for radiation injury comprising the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
(Wherein, ring A represents a fused ring, R ₁ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)   The prophylactic and / or therapeutic agent according to claim 15, wherein the radiation damage is a side effect during radiation treatment of cancer.