WO2015182625A1

WO2015182625A1 - Ras ACTIVITY INHIBITOR AND USE THEREOF

Info

Publication number: WO2015182625A1
Application number: PCT/JP2015/065150
Authority: WO
Inventors: 松田　道行; 勝広齋藤; 真也大石; 直貴小松; 一洋青木; 春杰李
Original assignee: 国立大学法人京都大学
Priority date: 2014-05-26
Filing date: 2015-05-26
Publication date: 2015-12-03
Also published as: JPWO2015182625A1

Abstract

The present invention provides an Ras activity inhibitor which is safe and effective for therapy for cancer, RAS/MAPK syndrome and the like. Specifically provided is an Ras activity inhibitor which contains a compound represented by formula (I), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound. (In the formula, -X- represents a group represented by formula (AA) or a single bond; n represents 0, 1 or 2; R₁ represents a halogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group, and in cases where a plurality of R₁ moieties are present, the R₁ moieties are the same as or different from each other; -Y represents a group represented by formula (BB) or a group represented by -NH-R₂; and R₂ represents an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted arylcarbonyl group, an optionally substituted alkoxycarbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic carbonyl group or an optionally substituted thiocarbonyl group.)

Description

Ras activity inhibitor and use thereof

The present invention relates to a Ras activity inhibitor, a cell growth inhibitor, a cancer and a prophylactic and / or therapeutic agent for RAS / MAPK syndrome and the like comprising dantrolene and a compound having a similar structure.

The pathway for activating ERK MAP kinase from the epidermal growth factor receptor via Sos, Ras, Raf, and MEK is known as an oncogene information transmission system, and abnormalities are observed in many human cancers. Many compounds targeting each molecule have been developed, and epidermal growth factor receptors (eg, trastuzumab, veltuzumab, vanitumumab, gefitinib, erlotinib, lapatinib), Raf (eg, sorafenib), MEK (eg, trametinib) Are already used in human clinical practice (FIG. 1).
On the other hand, Ras is an oncogene that is mutated in about 30% of human cancers. For this reason, many pharmaceutical companies have been searching for compounds that inhibit the activity of Ras protein since the 1980s (see, for example, Patent Documents 1-3), but none have been used clinically. Many drugs that change the localization of Ras have also been tried, but they have almost no specificity and have not been approved as pharmaceuticals due to side effects. In 2013, a drug that inhibits the binding between Ras and a target molecule was reported for the first time (Non-Patent Document 1), but it is completely unknown whether it can be used as a pharmaceutical product.

Also, RAS / MAPK syndrome is known as a genetic disease having a mutation in the oncogene information transmission system (FIG. 1). Although known as the most common genetic disease in the United States, no effective treatment has yet been found.

By the way, the present inventors invented the FRET biosensor for measuring Ras activation for the first time in the world (FIG. 2) (Patent Document 4). Furthermore, a technique for increasing the sensitivity of the FRET biosensor and creating a drug screening system using cultured cells has been devised (Patent Document 5).

Japanese Patent Laid-Open No. 11-012279 Special table 2009-517401 No. 00/004014 International Publication No. 2002/014373 Pamphlet International Publication No. 2012/043477 Pamphlet

As described above, Ras is an oncogene with the most mutations in human cancer, and has been shown to play an extremely important role in cell carcinogenesis. Therefore, if the Ras activity of cancer cells can be inactivated, the growth of cancer cells can be suppressed, and it is considered as one of the most promising target molecules for anticancer agents (FIG. 1). . In addition, it may be a drug discovery target for RAS / MAPK syndrome for which no effective treatment has been found so far. However, despite the fact that many pharmaceutical companies have tried to search for Ras activity inhibitors for more than 30 years, no effective drugs have been found yet.
Therefore, an object of the present invention is to provide a Ras activity inhibitor effective for the treatment of cancer, RAS / MAPK syndrome and the like (FIG. 1).

In order to achieve the above object, the present inventors constructed a drug screening system using cultured cells in which a Ras FRET biosensor is expressed, and used this to screen for compounds that inhibit Ras activity. As a result, it has been found that dantrolene and a compound having a structure similar thereto, which have a muscle relaxant action and are used as a spastic paralysis relieving agent, a malignant syndrome therapeutic agent, inhibit the activity of Ras. Since azumolene, which improved the water solubility while maintaining the anticonvulsant action of dantrolene, did not show the Ras activity inhibitory action, dantrolene exerted Ras by a mechanism different from the muscle relaxation action via the ryanodine receptor. It became clear to inhibit. Furthermore, as a result of examining Ras activity inhibitory activity for various dantrolene analogs, the inventors succeeded in identifying a common basic skeleton important for Ras activity inhibitory action.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
[1] Formula (I):

(Wherein -X- is

A group or a single bond represented by:
n represents 0, 1 or 2;
R ₁ represents a halogen atom, an alkyl group which may have a substituent, or an alkoxy group which may have a substituent;
When there are a plurality of R _{1 s,} they may be the same or different;
-Y is

Or a group represented by —NH—R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; An optional thiocarbonyl group is shown. )
Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, a Ras activity inhibitor.
[2] In the formula (I), -X- is

(Wherein n represents 0 or 1;
R ₁ represents a halogen atom. )
The agent of [1], which is a group represented by:
[3] In the formula (I), -X- is a single bond,
-Y is a group represented by -NH-R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; The agent according to [1], which may be a thiocarbonyl group.
[4] In the formula (I), when Y is a group represented by —NH—R ₂ , R ₂ may have an optionally substituted alkyl group having 1 to 6 carbon atoms, Optionally having a heterocyclic group selected from 1,3,5-triazinyl, phthalazinyl, pyridyl, quinolinyl, thiazolyl and thienopyrimidinyl, optionally having a benzoyl group, having a substituent The agent according to [2] or [3], which may be a phenyl group or an aminothiocarbonyl group.
[5] The compound represented by the formula (I) is dantrolene or any one of the following structural formulas:

The agent of [1], which is a compound represented by:
[6] The agent according to any one of [1] to [5], which is an inhibitor of cell proliferation by signal transduction via Ras.
[7] The agent according to [6], which is a cancer cell growth inhibitor.
[8] The agent according to any one of [1] to [5], which is a prophylactic and / or therapeutic agent for a disease associated with abnormal enhancement of a signal transduction pathway via Ras.
[9] The agent according to [8], wherein the disease is cancer or RAS / MAPK syndrome.
[10] Formula (I):

(Wherein -X- is

Or a group represented by —NH—R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; An optional thiocarbonyl group is shown. )
Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, wherein the method comprises inhibiting Ras activity in a subject.
[11] In the formula (I), -X- is

(Wherein n represents 0 or 1;
R ₁ represents a halogen atom. )
The method of [10] description which is group represented by these.
[12] In the formula (I), -X- is a single bond,
-Y is a group represented by -NH-R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; The method according to [10], wherein the thiocarbonyl group may be used.
[13] In the formula (I), when Y is a group represented by —NH—R ₂ , R ₂ may have an optionally substituted alkyl group having 1 to 6 carbon atoms, Optionally having a heterocyclic group selected from 1,3,5-triazinyl, phthalazinyl, pyridyl, quinolinyl, thiazolyl and thienopyrimidinyl, optionally having a benzoyl group, having a substituent The method according to [11] or [12], which may be a phenyl group or an aminothiocarbonyl group.
[14] The compound represented by the formula (I) is dantrolene or any one of the following structural formulas:

The method of [10] which is a compound represented by these.
[15] The method according to any one of [10] to [14], which is a method of inhibiting cell proliferation by signal transduction via Ras.
[16] The method according to [15], which is a method of inhibiting cancer cell proliferation.
[17] The method according to any one of [10] to [14], which is a method for preventing and / or treating a disease associated with abnormal enhancement of a signal transduction pathway via Ras.
[18] The method according to [17], wherein the disease is cancer or RAS / MAPK syndrome.
[19] Formula (I) for use in inhibiting RAS activity:

(Wherein -X- is

Or a group represented by —NH—R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; An optional thiocarbonyl group is shown. )
Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
[20] In the formula (I), -X- is

(Wherein n represents 0 or 1;
R ₁ represents a halogen atom. )
Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
[21] In the formula (I), -X- is a single bond,
-Y is a group represented by -NH-R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; The compound according to [19], which is an optionally thiocarbonyl group, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
[22] In the formula (I), when Y is a group represented by —NH—R ₂ , R ₂ may have a substituent, an alkyl group having 1 to 6 carbon atoms, Optionally having a heterocyclic group selected from 1,3,5-triazinyl, phthalazinyl, pyridyl, quinolinyl, thiazolyl and thienopyrimidinyl, optionally having a benzoyl group, having a substituent The compound according to [20] or [21], which is an optionally substituted phenyl group or aminothiocarbonyl group, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
[23] The compound represented by the formula (I) is dantrolene or any one of the following structural formulas:

Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
[24] The compound according to any one of [19] to [23], or a pharmaceutically acceptable salt, hydrate, or solvate thereof for use in inhibition of cell proliferation by signal transduction via Ras Or a prodrug.
[25] The compound according to [24] or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof for use in inhibiting cancer cell growth.
[26] The compound according to any one of [19] to [23] or a pharmaceutically acceptable salt thereof for use in the prevention and / or treatment of a disease involving an abnormal increase in a signal transduction pathway mediated by Ras Salt, hydrate, solvate or prodrug.
[27] The compound according to [26], wherein the disease is cancer or RAS / MAPK syndrome, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

The Ras activity inhibitor found by the present invention can block Ras / Raf-mediated signal transduction by inhibiting the binding between Ras and its target molecule, Raf, so that Ras activity is enhanced. It is useful as a therapeutic and prophylactic agent for cancers that are cancerous and for RAS / MAPK syndromes that have genetic abnormalities in RAS / MAPK signaling molecules. Dantrolene has a muscle relaxant action, is widely used as a spastic paralysis ameliorating agent and a malignant syndrome therapeutic agent, and has a large accumulation of safety in long-term administration. Since dantrolene's Ras activity inhibitory action is exerted below the concentration used clinically, it is highly expected as an effective and safe drug.

It is a figure which shows the action point of an oncogene information transmission system and the existing anticancer agent. The epidermal growth factor converts the inactivated GDP-bound Ras to the activated GTP-bound Ras via the Ras guanine nucleotide exchange factor Sos. GTP-bound Ras activates serine threonine kinases Raf, MEK, and ERK, and induces cell proliferation and canceration. It is a figure which shows the screening system of the Ras activity inhibitor using the FRET biosensor used in the Example. It is a figure which shows the Ras activity inhibitory activity of dantrolene sodium salt (DSS) and its analog. It is a schematic diagram of a rapamycin-induced Ras activation system for confirming inhibition of Ras activation in a physiological environment. FIG. 6 shows inhibition of rapamycin-induced Ras activation by dantrolene. It is a figure which shows the structure activity relationship of a dantrolene analog. FIG. 6 shows inhibition of EGF-dependent Ras activation by dantrolene. It is a figure which shows that Ras activity inhibition by dantrolene is nonspecific Ras guanine nucleotide exchange factor. It is a figure which shows the growth inhibitory effect with respect to various human cancer origin cell lines of dantrolene. It is a figure which shows the tumor growth inhibitory effect in the Xenograft model of dantrolene. It is a figure which shows the Ras activation inhibitory effect in a test tube of dantrolene and CX103. It is a figure which shows the cell growth inhibitory effect of CX103. It is a figure which shows the influence of dantrolene (CX001) and CX103 with respect to Rac1 activity.

The present invention provides a Ras activity inhibitor comprising dantrolene or an analog thereof. As used herein, “Ras activity” means an action in which a Ras protein is converted to a GTP-linked form by a reaction catalyzed by a Ras guanine nucleotide exchange factor. The GTP-bound Ras protein binds to a target molecule group including Raf protein. The Ras guanine nucleotide exchange factor may be any of guanine nucleotide exchange factors having a CDC25 homologous region, such as Sos, RasGRP1 / CalDAGII, and RasGRF.

In the present invention, “dantrolene or an analog thereof” refers to the formula (I):

(Wherein -X- is

Or a group represented by —NH—R ₂ ;
R ₂ represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; An optional thiocarbonyl group is shown. )
Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof. In the formula (I), —X— is

N is 0 and Y is the following formula

In the case of the group represented by formula (I), the compound represented by the formula (I) is dantrolene.

The present invention will be described below. The terms used in this specification have meanings commonly used in the art unless otherwise specified.
The terms used in this specification are defined below.

“Halogen atom” includes fluorine atom, chlorine atom, bromine atom and iodine atom. A bromine atom is preferable.

“Alkyl group” means a linear or branched alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, Examples include isopentyl, neopentyl, tert-pentyl, hexyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, octyl, and the like. Among them, an alkyl group having 1 to 6 carbon atoms is preferable, and methyl Groups are more preferred.

“Alkylcarbonyl group” means a carbonyl group substituted with an alkyl group (described above), and examples thereof include acetyloxy, propionyloxy, butyryloxy, isobutyryloxy and the like.

“Alkoxy group” means a linear or branched alkoxy group having 1 to 6 carbon atoms, and specifically includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy. Tert-butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy, neopentyloxy, 2-pentyloxy, 3-pentyloxy, n-hexyloxy, 2-hexyloxy and the like.

“Alkoxycarbonyl group” means a carbonyl group substituted with an alkoxy group (described above), and specifically includes methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert- And butoxycarbonyl.

“Aryl group” means an aryl group having 6 to 10 carbon atoms, and specific examples thereof include phenyl, naphthyl, etc. Among them, a phenyl group is preferred.

“Arylcarbonyl group” means a carbonyl group substituted with an aryl group (described above), and specific examples include benzoyl and naphthoyl.

Unless otherwise specified, the “heterocyclic group” includes an aromatic heterocyclic group and a non-aromatic heterocyclic group.
Examples of the aromatic heterocyclic group include 4 to 7 members (preferably 5 or 6 members) containing 1 to 4 heteroatoms selected from oxygen atoms, sulfur atoms and nitrogen atoms in addition to carbon atoms as ring constituent atoms. And monocyclic aromatic heterocyclic groups and condensed aromatic heterocyclic groups. Examples of the condensed aromatic heterocyclic group include a ring corresponding to the 4- to 7-membered monocyclic aromatic heterocyclic group and a 5- or 6-membered aromatic heterocyclic ring containing 1 or 2 nitrogen atoms. (Eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine) from a ring in which 1 or 2 selected from a 5-membered aromatic heterocycle containing one sulfur atom (eg, thiophene) and a benzene ring are condensed Examples include groups to be derived.
As the “aromatic heterocyclic group”, for example,
Furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3- Thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3- Monocyclic aromatic heterocyclic groups such as triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl;
Benzofuranyl, isobenzofuranyl, benzo [b] thienyl, indolyl, isoindolyl, 1H-indazolyl, benzoimidazolyl, benzoxazolyl, benzo [d] isoxazolyl, benzothiazolyl, benzo [d] isothiazolyl, 1H-benzotriazolyl, quinolyl , Isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthalidinyl, purinyl, pteridinyl, carbazolyl, α-carbolinyl, β-carbolinyl, γ-carbolinyl, acridinyl, phenoxazinyl, phenothiazinyl, phenoxatinyl, trithinyl, , Phenanthrolinyl, indolizinyl, pyrrolo [1,2-b] pyridazinyl, thienopyrimidinyl, pyra B [1,5-a] pyridyl, imidazo [1,2-a] pyridyl, imidazo [1,5-a] pyridyl, imidazo [1,2-a] pyridazinyl, imidazo [1,2-a] pyrimidinyl, Condensed aromatic heterocyclic groups such as 1,2,4-triazolo [4,3-a] pyridyl, 1,2,4-triazolo [4,3-b] pyridazinyl;
Is mentioned.
Examples of the non-aromatic heterocyclic group include 4 to 7 members (preferably 5 or 6 members) containing 1 to 4 heteroatoms selected from oxygen atoms, sulfur atoms and nitrogen atoms in addition to carbon atoms as ring constituent atoms. ) Monocyclic non-aromatic heterocyclic group and condensed non-aromatic heterocyclic group. Examples of the condensed non-aromatic heterocyclic group include a ring corresponding to the 4- to 7-membered monocyclic non-aromatic heterocyclic group, and a 5- or 6-membered aromatic containing 1 or 2 nitrogen atoms. 1 or 2 rings selected from a heterocyclic ring (eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine), a 5-membered aromatic heterocyclic ring containing 1 sulfur atom (eg, thiophene) and a benzene ring Examples thereof include a group derived from a condensed ring and a group obtained by partial saturation of the group.
As the “non-aromatic heterocyclic group”, for example,
Monocyclic and non-aromatic heterocyclic groups such as azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuryl, thiolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, piperidyl, tetrahydropyranyl, dihydropyrimidinyl, morpholinyl, thiomorpholinyl, piperazinyl; Dihydrobenzopyranyl, dihydroquinolyl, isochromenyl, chromenyl (2H-chromenyl, 4H-chromenyl), 1,2,3,4-tetrahydroisoquinolyl, 1,2,3,4-tetrahydroquinolyl, 2,3 -Condensed non-aromatic heterocyclic groups such as dihydrobenzofuranyl, benzo [1,3] dioxolyl;
Is mentioned.

“Heterocycle-carbonyl group” means a carbonyl group substituted with a heterocyclic group (described above).

The above “alkyl group”, “alkoxy group”, “aryl group”, “arylcarbonyl group”, “alkoxycarbonyl group”, “alkylcarbonyl group”, “heterocyclic group”, “heterocyclic-carbonyl group” and “thio” As the substituent that the “carbonyl group” may have,
(1) a halogen atom (for example, fluorine, chlorine, bromine, iodine; preferably chlorine),
(2) a lower alkyl group (for example, a C _1-6 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.),
(3) a cycloalkyl group (for example, a C _3-6 cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.),
(4) a lower alkynyl group (for example, a C _2-6 alkynyl group such as ethynyl, 1-propynyl, propargyl, etc.),
(5) a lower alkenyl group (for example, a C _2-6 alkenyl group such as vinyl, allyl, isopropenyl, butenyl, isobutenyl, etc.),
(6) Aralkyl groups (for example, C _7-12 aralkyl groups such as benzyl, α-methylbenzyl, phenethyl, etc.),
(7) an aryl group (for example, a C _6-10 aryl group such as phenyl or naphthyl, preferably a phenyl group),
(8) Lower alkoxy groups (for example, C _1-6 alkoxy groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.),
(9) aryloxy group (for example, C _6-10 aryloxy group such as phenoxy),
(10) Formyl group or lower alkanoyl group (for example, C _1-6 alkyl-carbonyl group such as acetyl, propionyl, butyryl, isobutyryl, etc.),
(11) arylcarbonyl group (for example, C _6-10 aryl-carbonyl group such as benzoyl, naphthoyl, etc.),
(12) Formyloxy group or lower alkanoyloxy group (for example, C _1-6 alkyl-carbonyloxy group such as acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, etc.),
(13) arylcarbonyloxy groups (for example, C _6-10 aryl-carbonyloxy groups such as benzoyloxy and naphthoyloxy),
(14) a 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.),
(16) an aralkyloxycarbonyl group (for example, a C _7-12 aralkyloxy-carbonyl group such as benzyloxycarbonyl),
(17) a carbamoyl group,
(18) Mono-, di- or tri-halogeno-lower alkyl groups (for example, mono-, di- or tri-halogeno- such as chloromethyl, dichloromethyl, trifluoromethyl, 2,2,2-trifluoroethyl) C _1-6 alkyl group),
(19) an oxo group,
(20) an amidino group,
(21) an imino group,
(22) an amino group,
(23) mono-lower alkylamino groups (for example, mono-C _1-6 alkylamino groups such as methylamino, ethylamino, propylamino, isopropylamino, butylamino, etc.),
(24) Di-lower alkylamino groups (for example, di-C _1-6 alkylamino groups such as dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, N-ethyl-N-methylamino, etc.),
(25) optionally having 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in addition to the carbon atom and one nitrogen atom which may have a substituent; 8-membered nitrogen-containing heterocyclic group (for example, halogen atom, nitro group, cyano group, hydroxy group, optionally halogenated C _1-6 alkyl group, optionally halogenated C _1-6 alkoxy group) 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, Contact Fine halogenated optionally C _1-6 alkyl - carbonyl amino group may have 1 to 5 substituents selected from oxo group and the like, in addition to carbon atoms and one nitrogen atom A 3- to 8-membered nitrogen-containing heterocyclic group which may contain 1 to 3 heteroatoms selected from a nitrogen 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 groups (for example, C _1-3 alkylenedioxy groups such as methylenedioxy and ethylenedioxy),
(27) a hydroxy group,
(28) a nitro group,
(29) a cyano group,
(30) a mercapto group,
(31) a sulfo group,
(32) a sulfino group,
(33) a phosphono group,
(34) a sulfamoyl group,
(35) a sulfonyl group,
(36) Mono-lower alkylsulfamoyl groups (for example, N-methylsulfamoyl, N-ethylsulfamoyl, N-propylsulfamoyl, N-isopropylsulfamoyl, N-butylsulfamoyl, etc.) Mono-C _1-6 alkylsulfamoyl groups),
(37) Di-lower alkylsulfamoyl groups (for example, N, N-dimethylsulfamoyl, N, N-diethylsulfamoyl, N, N-dipropylsulfamoyl, N, N-dibutylsulfamoyl, etc.) Di-C _1-6 alkylsulfamoyl group of
(38) a lower alkylthio group (for example, a C _1-6 alkylthio group such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, tert-butylthio, etc.),
(39) arylthio groups (for example, C _6-10 arylthio groups such as phenylthio and naphthylthio),
(40) a lower alkylsulfinyl group (for example, a C _1-6 alkylsulfinyl group such as methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl, etc.),
(41) arylsulfinyl group (for example, C _6-10 arylsulfinyl group such as phenylsulfinyl, naphthylsulfinyl, etc.),
(42) a lower alkylsulfonyl group (for example, a C _1-6 alkylsulfonyl group such as methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, etc.),
(43) arylsulfonyl group (for example, C _6-10 arylsulfonyl group such as phenylsulfonyl, naphthylsulfonyl, etc.),
(44) a lower alkylcarbonylamino group (for example, a C _1-6 alkylcarbonylamino group such as methylcarbonylamino),
(45) Arylamino group optionally substituted with a halogen atom (eg, chlorine) and / or a lower alkyl group (eg, methyl) (eg, C _6-10 arylamino group such as phenylamino, naphthylamino, etc.) ,
(46) optionally having 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in addition to the carbon atom and one nitrogen atom which may have a substituent; An amino group substituted with an 8-membered nitrogen-containing heterocyclic group (as defined in (25) above) (for example, phenylthiazolylamino)
And a substituent selected from the group consisting of (referred to herein as substituent group A).
These substituents are bonded to each group at 1 to several, preferably 1 to 2, more preferably 1 at the bondable positions. When the number of substituents is 2 or more, they may be the same or different. Further, these substituents may be substituted with any substituent selected from the substituent group A (eg, halogen atom).

In the present specification, a compound represented by the general formula (I) including a novel compound and a known compound may be referred to as a compound (I) or a compound of the present invention.

In general formula (I):
-X- is preferably

Wherein each symbol is as defined above;
n is preferably 0 or 1, more preferably 0;
R ¹ is preferably a halogen atom (eg, bromine);
-Y is preferably the following formula

Or a group represented by —NH—R ₂ ;
R ₂ is preferably an optionally substituted alkyl group having 1 to 6 carbon atoms (eg, methyl), an optionally substituted heterocyclic group (eg, 1,3,5- Triazinyl, phthalazinyl, pyridyl, quinolinyl, thiazolyl, thienopyrimidinyl), optionally substituted arylcarbonyl group (eg, benzoyl), optionally substituted aryl group (eg, phenyl) and amino Thiocarbonyl.

In the general formula (I), -X- is a single bond,
-Y is a group represented by -NH-R ₂ ;
R ₂ may have an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, an alkoxy which may have a substituent A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; An embodiment that may be a thiocarbonyl group is also a preferred embodiment.

More specifically, preferred compounds of the present invention are each of the following Example compounds, more preferably dantrolene (compound number: CX001), compound numbers CX003 to 007, CX101 to 105, CX108, CX112 to 114, CX116 to 118, CX121, CX122, CX125, CX126, CX129, CX130, CX134 and CX135, and more preferably the following compounds.

In one embodiment, among the above compounds, the most preferred compound is CX103.

Compound No. CX103

Examples of the salt of the compound represented by the formula (I) include pharmacologically acceptable salts such as trifluoroacetic acid, acetic acid, lactic acid, succinic acid, maleic acid, tartaric acid, citric acid, and gluconic acid. , Ascorbic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, cinnamic acid, fumaric acid, phosphonic acid, hydrochloric acid, nitric acid, hydrobromic acid, hydroiodic acid, sulfamic acid, sulfuric acid, etc. Acid addition salts; for example, metal salts such as sodium, potassium, magnesium, calcium; for example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, N-methylpyrrolidine, N-methylpiperidine, N-methylmorpholine, etc. Is mentioned.

When compound (I) has an isomer such as an optical isomer, a stereoisomer, a positional isomer, or a rotational isomer, any one isomer or a mixture of isomers is included in compound (I). Is done. For example, when compound (I) has an optical isomer, the optical isomer resolved from the racemate is also encompassed in compound (I). Each of these isomers can be obtained as a single product by a known synthesis method or separation method (concentration, solvent extraction, column chromatography, recrystallization, etc.).

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

Compound (I) may be a solvate (for example, a hydrate) or a non-solvate, and both are encompassed in compound (I).

Compound (I) may be labeled with an isotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, etc.) and the like.

For example, when compound (I) is unstable to acid, it can be provided in the form of an acid-stable prodrug by a method known per se in order to prevent degradation by gastric acid when administered orally.

The compound of the present invention can be synthesized, for example, by the production method described in Snyder® HR, “Jr”, “Davis” CS, “Bickerton® RK,” “Halliday” RP. “J.” Med. “Chem.” 10, 807 (1967). In the formula (I), the same production method can be synthesized regardless of whether X is phenylene or a single bond. It should be noted that those skilled in the art can make changes and modifications as needed. For example, the methods of Baliani A, Bueno GJ, Stewart ML, Yardley V, Brun R, Barrett MP, Gilbert IH. J. Med. Chem. 48, 5570 (2005) can be used as well.

Since the compound of the present invention can inhibit Ras from binding to and activating Raf as a target molecule, suppression of cell proliferation by Ras-mediated signal transduction, for example, Ras activity is enhanced. Of cancer cells in cancers (eg, lung cancer, breast cancer, malignant glioblastoma, glioma, etc., in which EGF receptor mutations or upregulation are observed), and RAS / MAPK signaling signaling molecules (Eg, PTPN11, RAF-1, SOS1, KRas) abnormal RAS / MAPK syndrome (eg, Noonan syndrome, LEOPARD syndrome, Costello syndrome, CFC syndrome, etc.) due to a genetic abnormality of Ras-mediated signaling pathways It is effective for prevention and / or treatment of related diseases.

Since cells having mutations in BRAF, Ras, and EGFR are highly sensitive to the above compound, in one embodiment, the above compound may prevent and / or treat malignant melanoma, pancreatic cancer, colon cancer, lung cancer, and glioblastoma. It is effective for.

The compound (I) of the present invention has low toxicity, and as such or according to a method known per se, a pharmaceutical composition mixed with a pharmacologically acceptable carrier, such as tablets (including sugar-coated tablets and film-coated tablets), powders, As a preparation such as granules, capsules (including soft capsules), orally disintegrating tablets, solutions, injections, suppositories, sustained-release agents, patches, or the like, orally or non-humanly for humans or other mammals It can be safely administered orally (eg, topical, rectal, intravenous, etc.).

The content of compound (I) in the pharmaceutical composition of the present invention is about 0.01% to 100% by weight of the whole composition. The dose varies depending on the type of compound, administration subject, administration route, disease, etc. For example, when dantrolene is orally administered to an adult (60 kg) as an anticancer agent, it is about 10 Up to about 300 mg / day, preferably about 50 to about 150 / day can be administered once a day or divided into 2-3 times. Alternatively, in the case of intravenous injection, about 0.1 to about 5 mg / kg / dose, preferably about 0.5 to about 3 mg / kg / dose is administered to a total amount of about 10 mg / kg, preferably about 7 mg / kg. Can do. For other compounds, a suitable dose can be appropriately selected in consideration of Ras inhibitory activity and drug tolerability using the dose of dantrolene as an index.

Examples of pharmacologically acceptable carriers that may be used in the production of the pharmaceutical composition of the present invention include various organic or inorganic carrier substances that are commonly used as pharmaceutical materials. Examples of the additives include binders, disintegrants, water-soluble polymers, basic inorganic salts; solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, etc. in liquid preparations. Further, if necessary, additives such as ordinary preservatives, antioxidants, colorants, sweeteners, sour agents, foaming agents, and fragrances can be used.

Examples of the “excipient” include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, titanium oxide and the like.
Examples of the “lubricant” include magnesium stearate, sucrose fatty acid ester, polyethylene glycol, talc, stearic acid and the like.

Examples of the “binder” include hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, starch, polyvinylpyrrolidone, gum arabic powder, gelatin, pullulan, low-substituted hydroxypropylcellulose, and the like.

Examples of the “disintegrant” include (1) crospovidone, (2) disintegrants called super disintegrants such as croscarmellose sodium (FMC-Asahi Kasei), carmellose calcium (Gotoku Pharmaceutical), (3) carboxymethyl Examples include starch sodium (eg, Matsutani Chemical Co., Ltd.), (4) low-substituted hydroxypropylcellulose (eg, Shin-Etsu Chemical Co., Ltd.), (5) corn starch and the like. The “crospovidone” is crosslinked with a chemical name of 1-ethenyl-2-pyrrolidinone homopolymer, including those called polyvinylpolypyrrolidone (PVPP) and 1-vinyl-2-pyrrolidinone homopolymer. Specific examples include Kollidon CL (manufactured by BASF), Polyplastidone XL (manufactured by ISP), Polyplaston XL-10 (manufactured by ISP), and Polyplastidone. INF-10 (manufactured by ISP).

Examples of the “water-soluble polymer” include ethanol-soluble water-soluble polymers [for example, cellulose derivatives such as hydroxypropylcellulose (hereinafter sometimes referred to as HPC), polyvinylpyrrolidone, etc.], ethanol-insoluble water-soluble polymers Molecules [for example, hydroxypropylmethylcellulose (hereinafter sometimes referred to as HPMC), cellulose derivatives such as methylcellulose, sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum, etc.] and the like.

Examples of the “basic inorganic salt” include basic inorganic salts of sodium, potassium, magnesium and / or calcium. Preferred is a basic inorganic salt of magnesium and / or calcium. More preferred is a basic inorganic salt of magnesium. Examples of the basic inorganic salt of sodium include sodium carbonate, sodium hydrogen carbonate, disodium hydrogen phosphate and the like. Examples of the basic inorganic salt of potassium include potassium carbonate and potassium hydrogen carbonate. Examples of the basic inorganic salt of magnesium include heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg ₆ Al ₂ ( OH) ₁₆ · CO ₃ · 4H ₂ O] and alumina / magnesium hydroxide, preferably heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide and the like. Examples of the basic inorganic salt of calcium include precipitated calcium carbonate and calcium hydroxide.

Examples of the “solvent” include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
Examples of the “dissolution aid” include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

Examples of the “suspending agent” include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; And hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

Examples of the “isotonic agent” include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.
Examples of the “buffering agent” include buffer solutions of phosphate, acetate, carbonate, citrate, and the like.

Examples of the “soothing agent” include benzyl alcohol and the like.
Examples of the “preservative” include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

Examples of the “antioxidant” include sulfite, ascorbic acid, α-tocopherol and the like.
Examples of the “colorant” include edible pigments such as edible yellow No. 5, edible red No. 2, and edible blue No. 2; edible lake pigments, bengara and the like.

Examples of the “sweetening agent” include saccharin sodium, dipotassium glycyrrhizin, aspartame, stevia, thaumatin and the like.
Examples of the “sour agent” include citric acid (anhydrous citric acid), tartaric acid, malic acid and the like.

Examples of the “foaming agent” include sodium bicarbonate.
The “fragrance” may be a synthetic product or a natural product, and examples thereof include lemon, lime, orange, menthol, and strawberry.

The compound of the present invention is compression-molded according to a method known per se, for example, by adding a carrier such as an excipient, a disintegrant, a binder or a lubricant, and then, if necessary, masking of taste, enteric properties or sustainability. Therefore, it is possible to obtain a preparation for oral administration by coating by a method known per se. In the case of an enteric preparation, an intermediate layer may be provided between the enteric layer and the drug-containing layer by a method known per se for the purpose of separating both layers.

When the compound of the present invention is made into an orally disintegrating tablet, for example, a core containing crystalline cellulose and lactose is coated with the compound of the present invention and, if necessary, a basic inorganic salt, and further coated with a coating layer containing a water-soluble polymer. The resulting composition is coated with a polyethylene glycol-containing enteric coating layer, then coated with a triethyl citrate-containing enteric coating layer, and further coated with a polyethylene glycol-containing enteric coating layer. It can be produced by a method of coating with mannitol to obtain fine particles, mixing the obtained fine particles and additives, and molding.

Examples of the “enteric coating layer” include cellulose acetate phthalate (CAP), hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, methacrylic acid copolymer [for example, Eudragit® L30D-55 (trade name; Rame Co., Ltd.), Kollicoat MAE30DP (trade name; manufactured by BASF Corp.), Polykid PA30 (trade name; manufactured by Sanyo Kasei Co., Ltd.), etc.], carboxymethyl ethyl cellulose, shellac and other water-based enteric polymer bases; Sustained release bases such as coalescent [eg Eudragit NE30D (trade name), Eudragit RL30D (trade name), Eudragit RS30D (trade name), etc.]; water-soluble polymer; triethyl citrate, polyethylene glycol, acetylated monoglyceride De, triacetin, include one or a layer consisting of such a mixture of two or more such plasticizers such as castor oil.

Examples of the “additives” include water-soluble sugar alcohols (eg, sorbitol, mannitol, maltitol, reduced starch saccharified product, xylitol, reduced palatinose, erythritol, etc.), crystalline cellulose (eg, Theolas KG 801, Avicel PH 101) , Avicel PH 102, Avicel PH 301, Avicel PH 、 302, Avicel RC-591 (crystalline cellulose, carmellose sodium), etc., low-substituted hydroxypropylcellulose (eg, LH-22, LH-32, LH-23, LH) -33 (Shin-Etsu Chemical Co., Ltd.) and mixtures thereof, etc.), binders, acidulants, foaming agents, sweeteners, fragrances, lubricants, colorants, stabilizers, excipients, Disintegrants are also used.

The compound of the present invention may be used in combination with other active ingredients. As the concomitant drug, for example, when the compound of the present invention is used as an anticancer agent, various compounds having a preventive and / or therapeutic effect against cancer can be appropriately blended. For example, other active ingredients include alkylating drugs (eg, mustards, nitrosoureas), antimetabolites (eg, folic acid, pyrimidines, purines), antitumor antibiotics (eg, anthracyclines), Hormone analogs (eg, antiestrogens, antiandrogens, LH-RH agonists, progesterone, estradiol), platinum preparations, topoisomerase inhibitors (eg, topoisomerase I inhibitors, topoisomerase II inhibitors), biological preparations (eg, interferon) , Interleukins), molecular targeted drugs (eg, antibodies (eg, trastuzumab, panitumumab), small molecules (gefinitib, erlotinib, sorafenib), retinoin) and the like.

The “other active ingredient” and the compound of the present invention are mixed according to a method known per se, and a single pharmaceutical composition (eg, tablet, powder, granule, capsule (including soft capsule), liquid, injection, suppository) is prepared. Preparations, sustained release preparations, etc.) may be formulated and used together, or each may be formulated separately and administered to the same subject simultaneously or with a time lag.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

Reference Example 1 Construction of Ras Activity Inhibitor Screening System In HeLa cells, plasmid pPBbsr2-Raichu 3706NES (Komatsu, N. et al. (2011) Mol. Biol. Cell) expressing Raichu-Ras wild type lacking a cell membrane translocation signal. 22 (23), 4647-4656; Mochizuki, N. et al. (2001) Nature, 411, 1065-1068) HeLa cells stably expressing Ras FRET biosensor were established. Furthermore, a plasmid pT2Apuro-mSos1 expressing mouse guanine nucleotide exchange factor Sos1 was introduced to express Sos, and cells with increased FRET efficiency were prepared (Raichu-Ras wild type / mSos1 / HeLa cells) (FIG. 2). . On the other hand, a HeLa cell (Raichu-RasS17N cell) expressing a Raichu-RasS17N mutant was similarly prepared and used as an indicator of the maximum inhibitory effect. FRET images were created as previously reported (Komatsu et al., 2011).

Example 1 Identification of a drug that inhibits Ras activity Reduces FRET efficiency in Raichu-Ras wild type / mSos1 / HeLa cells prepared in Reference Example 1 from tens of thousands of library compounds at the Kyoto University One-Stop Drug Discovery Base Drugs were screened.
10 μM drug was administered to Raichu-Ras wild type / mSos1 / HeLa cells and photographed under a fluorescence microscope the next day to measure FRET efficiency. The FRET efficiency was determined by using 440 nm excitation / 530 nm fluorescence intensity / 440 nm excitation / 480 nm fluorescence intensity as an index (Komatsu et al., 2011). The inhibition rate was determined by the following formula.
Inhibition rate = (FRET efficiency in the absence of drug—FRET efficiency in the presence of drug) / (FRET efficiency in the absence of drug—FRET efficiency in S17N cells in the presence of drug)
That is, the drug effect was determined with 100% inhibition as the case where the FRET efficiency of Raichu-RasS17N cells decreased. As a result, dantrolene sodium salt (DSS; hereinafter, also simply referred to as dantrolene) and its analogs (KPTJ10488 (the same compound as CX003), KPTJ11521 (the same compound as CX004), KPTJ19352 (the same compound as CX005)) have improved FRET efficiency. It was found that it was lowered (FIG. 3).

Example 2 Confirmation of Ras Activity Inhibition in Physiological Environment In order to confirm inhibition of Ras activation in a physiological environment, Ras FRET biosensor was expressed on the cell membrane, and rapamycin-induced Sos cell membrane translocation A system was made (FIG. 4). Raipu-Ras wild-type plasmid pPBbsr2-Raichu 3706X with cell membrane translocation signal, vector pMCsbsr-FLAG-FKBP-mSos1-linkercat with FKBP protein expressing only the catalytic region of mouse Sos1 protein and rapamycin binding with cell membrane translocation signal A vector pCX4puro-LDR (Kamioka, Y. et al. (2010) J. Biol. Chem., 285 (43), 33540-33548) expressing the protein was introduced into HeLa cells (FKBP-mSos1 cells). PMCsbsr-FLAG-FKBP-mSos1-linkercat expresses a fusion protein of the Sos1 guanine nucleotide exchange factor catalytic region (CDC25 homologous region) and FKBP. These cells have low FRET efficiency when rapamycin is not added, but when rapamycin is added, Sos1 moves to the cell membrane and activates Ras. Along with this, an increase in FRET efficiency can be observed.
FKBP-mSos1 cells were administered with dantrolene having a final concentration of 10 μM, and the next day, time-lapse observation was performed with a microscope. Ten minutes later, rapamycin was added at a final concentration of 50 nM. As a result, it was found that dantrolene inhibits Ras activation on the cell membrane (FIG. 5). Furthermore, in this reaction, it is known that cell membrane ruffling (wave formation) and lobular extension occur in a Ras-dependent manner, but dantrolene also suppressed this reaction. This indicates that dantrolene inhibited the activation of endogenous Ras.

Example 3 Structure-activity relationship of dantrolene analogues The following compounds having dantrolene and similar structures (CX101 to 135 were purchased from Namiki Shoji) were subjected to the same experiment as in Example 2, and Rasmycin 10 minutes after the addition of rapamycin Activation was measured. DMSO-treated cells were standardized as 100%, and Ras activation inhibitory activity was evaluated in three stages. In addition, IC ₅₀ was determined for drugs that were highly effective. In addition, inhibition of Ras-dependent cell membrane ruffling was also examined, and the inhibitory activity was evaluated in three stages. As a result, dantrolene showed an IC ₅₀ lower than the blood concentration estimated from the doses conventionally used for the treatment of spastic paralysis and generalized rot. In addition, a number of compounds having an IC ₅₀ higher than dantrolene by one order or more and remarkable ruffling inhibitory activity were identified (FIG. 6). On the other hand, it was found that azumolene, which has a muscle relaxing action like dantrolene, is ineffective in inhibiting Ras activity.

Compound No. CX001 (dantrolene sodium salt)

Compound No. CX002 (azumolene)

Compound No. CX003

Compound No. CX004

Compound No. CX005

Compound number CX006

Compound No. CX007

Compound No. CX009

Compound No. CX010

Compound No. CX011

Compound No. CX012

Compound No. CX013

Compound No. CX101

Compound No. CX102

Compound No. CX103

Compound No. CX104

Compound No. CX105

Compound No. CX106

Compound No. CX107

Compound No. CX108

Compound No. CX109

Compound No. CX110

Compound No. CX111

Compound No. CX112

Compound No. CX113

Compound No. CX114

Compound No. CX115

Compound No. CX116

Compound No. CX117

Compound No. CX118

Compound No. CX119

Compound No. CX120

Compound No. CX121

Compound No. CX122

Compound No. CX125

Compound No. CX126

Compound No. CX127

Compound No. CX128

Compound No. CX129

Compound No. CX130

Compound No. CX131

Compound No. CX132

Compound No. CX133

Compound No. CX134

Compound No. CX135

Thus, a common basic skeleton important for the Ras activity inhibitory action represented by the formula (I) was clarified.

Example 4 Inhibition of EGF-Dependent Ras Activation by Dantrolene Next, it was observed whether dantrolene can suppress an epidermal growth factor (EGF) -dependent Ras activation that is a physiological stimulus.
HeLa cells (Raichu-Ras cells) expressing Raichu-Ras wild-type plasmid pBBsr2-Raichu3706X expressing cell membrane translocation signals were administered final concentrations of 10 μM, 20 μM and 50 μM dantrolene, and time-lapse observation was performed 18 hours later. . EGF was added at a final concentration of 10 nM and FRET efficiency was measured. As a result, as shown in FIG. 7, dantrolene significantly suppressed EGF-dependent Ras activation.

Example 5 Specificity of Ras Guanine Nucleotide Exchange Factor Next, in order to examine whether or not the action of dantrolene for inhibiting Ras activity is specific to Sos, the Sos site in FIG. 4 was placed in the catalytic region of RasGRP1 / CalDAGII or RasGRF ( Cells exchanged for the CDC25 homologous region) were prepared. These cells were stimulated with rapamycin and Ras activation was measured. As a result of examining Ras activation in the presence of 10 μM CX001 (dantrolene), it showed almost the same effect as that on Sos (FIG. 8). That is, it was shown that there was almost no specificity with respect to Ras activator.

Example 6 Cell growth inhibitory effect of dantrolene Various human cancer-derived cell lines including cells having mutations such as EGF receptor, KRas, PI3K, BRaf were examined for the growth inhibitory effect of dantrolene. As a result, none of the cells was effective at 10 μM and effective at 50 μM (left side of FIG. 9). The time course of H2170 cells that showed the strongest effect is shown (FIG. 9 right).

Example 7 Xenograft tumors increased inhibition of the model effects human lung squamous carcinoma cell line NCI-H2170 (Her2 / Neu positive, 10 ⁶ cells) were inoculated into nude mice. After palpation of the tumor (Day 0), 20 μM (final concentration) of dantrolene was intraperitoneally administered daily (N = 12). Tumor size was measured blind. The results are shown in FIG. 10 (displayed as mean ± standard error). In the dantrolene-administered group, tumor growth was significantly suppressed compared to the control (PBS-administered) group.

Example 8 Ras Activation Inhibitory Effect in Test Tube Purified 1 μM HRas protein, 0.5 μM Sos protein, and mant-GTP were reacted in 100 μl of buffer solution. The composition of the buffer is as follows. 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM MgCl ₂ , 1 mM DTT, 5% Glycerol, various concentrations of inhibitors (dantrolene, CX103). DMSO was used as a control. The reaction was carried out at 20 ° C., and the amount of Ras bound to mant-GTP was measured with a fluorescence spectrophotometer at excitation light of 355 nm and fluorescence of 448 nm. The results are shown in FIG. Three times of results are shown in an overlapping manner. It can be seen that both dantrolene and CX103 suppress the Sos-dependent GTP exchange reaction.

Example 9 Cell Growth Inhibitory Effect of CX103 The growth inhibitory effect of CX103 was examined on various human cancer-derived cell lines including cells having mutations such as EGF receptor, KRas, PI3K, BRaf and the like. Various cells used were commercially available. As a result, when compared with normal cells (NIH3T3, CV-1, Cos-1), etc., cells with mutations in BRAF, EGFR, Ras (cells with mutations in BRAF such as HT29, Colo205, A375; EGFR such as H4006, H1975) Cells with mutations in Ras; cells with mutations in Ras such as Aspc-1, H358, and HCT116) have lower IC ₅₀ (FIG. 12), indicating that these cells have a stronger effect.

Example 10 Rac1 inhibitory effect It is known that cell membrane ruffling by Sos depends on Rac1. Thus, the influence of DMSO, dantrolene (CX001) and CX103 on Rac1 activity was analyzed using the Sos activation system described in FIG. Rac1 activity was analyzed by a pull-down method. Dantrolene (CX001) or CX103 was administered to FKBP-mSos1 cells, and rapamycin was administered 30 minutes later. After 10 minutes, the cells were solubilized, and GTP-bound Rac1 was quantified by a pull-down method according to a conventional method. As shown in FIG. 13, CX103 significantly suppressed Rac1 activation.

Since the compound of the present invention has a Ras activity inhibitory action, it is useful for suppressing cell growth by Ras-mediated signal transmission, particularly for suppressing growth of cancer cells with enhanced Ras activity. In particular, dantrolene has already been widely used as a muscle contracture ameliorating agent, and its safety in long-term administration has been confirmed. Therefore, it may have clinical applications including combination therapy with other anticancer agents. Is extremely high.
The compounds of the present invention are also promising as therapeutic agents for RAS / MAPK syndrome for which no effective treatment has been found so far.

This application is based on Japanese Patent Application No. 2014-108572 filed on May 26, 2014, the entire contents of which are included in this specification.

Claims

Formula (I):

(Wherein -X- is

A group or a single bond represented by:
n represents 0, 1 or 2;
R 1 represents a halogen atom, an alkyl group which may have a substituent, or an alkoxy group which may have a substituent;
When there are a plurality of R 1 s, they may be the same or different;
-Y is

Or a group represented by —NH—R 2 ;
R 2 represents an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, and an alkoxy which may have a substituent. A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; An optional thiocarbonyl group is shown. )
Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, a Ras activity inhibitor.
In the formula (I), -X- is

(Wherein n represents 0 or 1;
R 1 represents a halogen atom. )
The agent of Claim 1 which is group represented by these.
In the formula (I), -X- is a single bond,
-Y is a group represented by -NH-R 2 ;
R 2 may have an alkyl group which may have a substituent, an aryl group which may have a substituent, an arylcarbonyl group which may have a substituent, an alkoxy which may have a substituent A carbonyl group, an optionally substituted alkylcarbonyl group, an optionally substituted heterocyclic group, an optionally substituted heterocyclic-carbonyl group or a substituted group; The agent according to claim 1, which may be a thiocarbonyl group.
In the formula (I), when Y is a group represented by —NH—R 2 , R 2 may have a substituent, an alkyl group having 1 to 6 carbon atoms, each having a substituent. Or a heterocyclic group selected from 1,3,5-triazinyl, phthalazinyl, pyridyl, quinolinyl, thiazolyl and thienopyrimidinyl, optionally having a benzoyl group, optionally having a substituent The agent according to claim 2 or 3, which is a phenyl group or an aminothiocarbonyl group.
The compound represented by the formula (I) is dantrolene or any one of the following structural formulas:

The agent of Claim 1 which is a compound represented by these.
The agent according to any one of claims 1 to 5, which is an inhibitor of cell proliferation by signal transduction via Ras.
The agent according to claim 6, which is a cancer cell growth inhibitor.
The agent according to any one of claims 1 to 5, which is a prophylactic and / or therapeutic agent for a disease associated with abnormal enhancement of a signal transduction pathway via Ras.
The agent according to claim 8, wherein the disease is cancer or RAS / MAPK syndrome.