TW201118100A - A novel antibacterial agent targeting on Wa1R(YycF) response regulator - Google Patents

Abstract

Provided is an antibacterial agent effective for Gram-positive bacteria using a compound of formula (I) or pharmaceutical acceptable salt or solvate or N-oxide thereof. [wherein: R1 represents a 6- to 10-membered aryl substituted by C1-6 haloalkyl, R2 and R3 each independently represents hydrogen, C1-6 alkyl, C3-6 cycloalkyl or C1-6 alkyl substituted by 6- to 10-membered aryl, X represents oxygen or sulfur.]

Description

201118100 VI. Description of the invention: [Technical field to which the invention pertains] * The invention relates to a novel toxoflavin derivative and an antibacterial agent containing the same. [Prior Art] Many antibacterial agents have been used as therapeutic agents for bacterial infections in the past. Most of the antibacterial agents known to date act by inhibiting bacterial nucleic acid synthesis, protein synthesis, glycan synthesis, and the like. Since the target sites of these targets are mainly for the purpose of inhibiting the metabolic synthesis pathway, it is easy for these antimicrobial agents to develop resistant bacteria, especially in recent years, there are multiple resistant bacteria that are resistant to a plurality of antibiotics. problem. In recent years, it has become a problem. One of the heavy resistant bacteria, methici 11 in resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are all Gram-positive bacteria. On the other hand, the two-component signal transduction system (TCS) of bacteria is known to play an important role in drug resistance and etiology (Non-Patent Document 1). WalK/WalR TCS is a two-component message transmission system unique to gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus. The two-component signaling system consists of the sensor protein WalK (also known as YycG) on the cell membrane surface and the regulatory protein WalR (also known as YycF) in the cell. When the induced protein is stimulated from the outside, 'self-phosphorylation by adding a phosphate group to its His site produces histidine kinase (HK) activity, and this acid group is transferred to a regulatory protein' thereby regulating the protein and the target gene 4 322420 201118100 Combined with the regulation of gene expression S. It is known that in the two-component tfl transmission, the dimerization of the regulatory protein is required, and the first is not (Non-Patent Document 2). The process of screening for the antibacterial agent which is effective for the multi-drug resistant bacteria, the present inventors and the like disclose a screening method for the two component custody system of the main signal transduction mechanism of the bacterial cell (Patent Documents 1 to 3). [Patent Document] [Patent Document] Japanese Patent Laid-Open Publication No. JP-A No. 2004-256. Non-Patent Document] [Non-Patent Document 1] Utsumi R ed. (2008), Bacterial Signal Transduction · Networks and Drug Targets [Non-Patent Document 2] Gao R, Mack TR, Stock AM (2007), Trends Biochem Sci 32: 225 - 234 SUMMARY OF THE INVENTION (Problems to be Solved by the Invention) With the advent of multiple resistant bacteria such as MRSA and VRE, it is still desired to develop an antibacterial agent which is effective and novel for Gram-positive bacteria. (Means for Solving the Problem) The present inventors focused on the two-component control system of the main signal transduction mechanism of the bacterial cell, and the results of careful research, in the two-component regulation system of the Gram-positive bacteria, particularly the inhibition on the WalR The message transmission route 5 322420 201118100 was successfully developed for the Gram-positive bacteria by screening. The antibacterial agent acting on WalR is the first disclosed by the present invention. > The antibacterial agent of the present invention exerts an effect by inhibiting the expression of a gene involved in cell wall synthesis (Fig. 1). The compound represented by the following formula (I) is contained. That is, the present invention provides, in the first aspect, a compound represented by the formula (1) or a pharmaceutically acceptable salt or solvate thereof (N-〇xide),

Ri N

R2 (I) [in the formula

Ri represents an aryl group of a 6 to 1 member ring substituted by an alkyl group, and h and R3 each independently represent hydrogen, a Cl 6 alkyl group, a CM cycloalkyl group or a Cm alkane substituted with an aryl group of 6 to 10 membered rings. Base, X represents oxygen or sulfur]. In a second aspect of the invention, as a preferred compound of the first aspect compound, a compound of the formula (1) or a pharmaceutically acceptable salt or solvate thereof is provided. 322420 6 201118100

(i) [wherein R represents an aryl group of a 6 to i member ring substituted with a g-6 alkyl group, and L and R3 each independently represent hydrogen or a Cl 6 alkyl group, and X represents oxygen or sulfur]. Further, in a third aspect of the present invention, a compound which is particularly preferred as the compound of the first and second aspects is a compound or a pharmaceutically acceptable salt or solvate thereof, which is the above formula (丨)in,

Ri is a phenyl group substituted with a trifluoromethyl group at the 4-position, 1 is hydrogen or a methyl group or an ethyl group, R3 is a fluorenyl group, and X is an oxygen group. Further, in the fourth aspect of the present invention, as a specific compound contained in the first aspect compound of the present invention, a compound selected from the group consisting of pharmaceutically acceptable salts or solvates thereof, or a pharmaceutically acceptable salt or solvate thereof is provided. Or N_oxide 1,6--fluorenyl-3-[4-(trifluoromethyl)phenyl]pyrimidine[5, 4_e] [1,2,4]triter-5,7-di _, 1-phenylhydrazino-6-methyl-3-[4-(trifluoromethyl)phenyl], and [5,4_e][l,2,4]dioxin-5,7_dioxin , 1-cyclohexyl-6-mercapto-3-[4-(trifluoromethyl)phenyl]pyrimidin 322420 7 201118100 .[5, 4-e][l,2,4]triphthyl-5, 7-diketone, b-ethyl-6-methyl+[4-(trifluoromethyl)phenyl] mouth bite and [5,4-6][1,2,4]trin-5,7-dione And 6-methyl-3-[4-(difluoromethyl)phenyl]_8H-battered and *[1,2,4]trin-5,7-dione. The present invention is in a fifth aspect, and as a particularly preferred compound contained in the above second or second compound, a compound selected from the group consisting of pharmaceutically acceptable salts or solvates thereof is provided. 6-Dimethyl-3-[4-(trimethylmethyl)phenyl]-bited and [5,4 e] [1,2,4] tri-n--5,7-dione, 1-ethyl -6-methyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4-e][ 1,2, 4]三卩井-5, 7-dinet and 6 methyl 3 [4-(Difluoromethyl)phenyl] intimate and [5,4_e] [1,2,4] tri-n--5, 7-dione. In a sixth aspect, the present invention provides an antibacterial agent comprising any one of the compounds described above as an active ingredient.

Further, in the seventh aspect of the present invention, there is provided an anti-killing agent which comprises MRSA or VRE as an active ingredient, and comprises any of the compounds described above as an active ingredient. Further, the present invention provides a WalR reaction modulating agent, which comprises any of the compounds described above as an active ingredient. As an active ingredient of the antibacterial agent of the sixth and seventh aspects of the present invention and the WalR reaction regulator inhibitor of the eighth aspect, preferred compounds are the compounds provided in the above second aspect, and more preferably the above compounds are 8 322420 201118100 Compounds provided in the three-state sample, particularly preferred compounds are those provided in the fifth aspect above. The term "alkyl" as used herein, when used as part of a radical or radical, refers to a hydrocarbon chain having a straight or branched chain of the specified number of carbon atoms. For example, C16 alkyl means a hydrocarbon chain having a straight or branched chain of one or more and six or less carbon atoms. Examples of the alkyl group include mercapto (Me), ethyl (Et), n-propyl, isopropyl, n-hexyl and isohexyl groups, but are not limited thereto. The term "cycloalkyl" as used herein, when used as part of a group or group, refers to a non-aromatic, saturated, cyclic hydrocarbon having a specified number of carbon atoms. Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group, but are not limited thereto. The term "aryl" as used herein means at least one ring of aromatic, C6-10 monocyclic or bicyclic hydrocarbon ring. Examples of such a group include a phenyl group and a naphthyl group. The above aryl group may also be substituted with a Ci-6 haloalkyl group. The term "alkyl" as used herein refers to an alkane having a specified number of carbon atoms, substituted with one or more halo substituents selected from the group consisting of fluorine, chlorine, bromine and iodine, suitably substituted with from 1 to 6 substituents. base. The haloalkyl group is preferably a trifluoromethyl group. The compound represented by the formula (I) of the present invention may, for example, be 1,6-dimercapto-3-[4-(trifluoromethyl)phenyl]pyrimido[5,44][1,2,4 Tris-5- 7-diketone, 1-phenylhydrazin-6-mercapto-3-[4-(trifluoromethyl)phenyl]pyrimido[5,44][1,2,4] Three tillage-5,7-dione, 1-cyclohexyl-6-methyl

B 9 322420 201118100 3 [4~(Difluoromethyl)phenyl]pyrimido[5,4_6][1,2,4]three tillage-5,7-one, 1-ethyl-6-methyl -3-[4-(trifluoromethyl)phenyl]pyrimido[5' 4-e][l,2' 4]three tillage-5, 7-di-marine and 6-methyl-3-[4_ (Trifluoromethyl)phenyl]Rutho[5,4_6][1,2,4]triterpene _5,7-dioxime and the like. The compound of the present invention can also be crystallized or recrystallized from a solvent such as an aqueous or organic solvent. In this case τ, a solvate can be formed. Such solvates are included within the scope of the invention. Further, the phrase "the compound represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof or ruthenium-oxide" is intended to include the compound of the formula (1) and the formula (I), respectively. An oxide, a pharmaceutically acceptable salt of a compound of formula (1), a solvate of formula (I) or a pharmaceutically acceptable combination thereof. Therefore, as a non-limiting example used in the present specification, the compound of the formula (G) or a pharmaceutically acceptable salt or solvate thereof may further comprise a compound of the formula (I) which is a solvate. A pharmaceutically acceptable salt. Further, in the present invention, the Ν-oxide is a compound in which ν at any position of the formula (I) becomes a ruthenium oxide, and a compound which becomes a ruthenium-oxide at the 4-position is preferable. Specific compounds in the present invention include the compounds described in the examples and the pharmaceutically acceptable rhodium-oxides, salts and solvates thereof. The pharmaceutically acceptable salt of the above compound of the formula (I) comprises an acid addition salt or a quaternary ammonium salt, such as a mineral acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, or acetic acid or fumaric acid. a salt of an organic acid such as succinic acid, maleic acid, citric acid, benzoic acid, ρ-p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid or tartaric acid. Further, the compound of the formula (I) can also be prepared as a cerium-oxide. The invention extends to all such derivatives. 10 S 322420 201118100 . Further, when the compound of the formula (containing one or more asymmetric carbon atoms) can also exist in the form of a racemic form and an optically active form, the compound of the formula (I) of the present invention comprises All of these isomers and mixtures thereof. The compounds of formula (I) of the present invention can be made by Scheme I shown below.

Scheme I Add 6 gas-3-alkyl (aryl) uracil or 6-chloro-3-alkyl (aryl), 2, IL uridine 'pyridine (a) to alkyl (aryl) hydrazine and three In the ethanol mixture of alkali such as ethylamine, the mixture was stirred under heating and then cooled to room temperature. The brewing organism was added to the reaction solution, and reacted at room temperature to obtain a knee (^) (hydraZ〇ne) ^ This was unrefined and dissolved in THF, and hydrochloric acid was added under cooling, followed by addition. Sodium nitrite was stirred at room temperature to give the formula (abbreviated as 11 322420 201118100 罅 and its 4-N oxide body).

U Refer to Nagamatsu T, Yamasaki tHirota T, Yamato Μ, Kido Υ, Shibata Μ, Yoneda F (1993) Chem Pharm Bull (Tokyo) 41 : 362-368 for this procedure. (Effect of the invention)

The compound of the formula (I) of the present invention can inhibit the WalK/WalR system, particularly the waiR reaction regulator, of the two-component regulatory system unique to Gram-positive bacteria. Thereby, the compound of the present invention is useful as an effective antibacterial agent peculiar to Gram-positive bacteria. [Embodiment] The present invention provides, in the first to fifth aspects, a compound represented by the formula (1), or a salt, a solvate or an N-oxide which is acceptable on a sauce. Related compounds are used as antibacterial agents and, in addition, for the development of antibacterial agents. In the formula (1) / in the chelate compound 'particularly the second, third and fifth aspect of the compound' by inhibiting the GalK/WalR TCS in the Gram-positive bacteria, in particular the Wa丄R response regulation is shown to Gram The antibacterial activity of positive bacteria. As far as the observation of n(4) is concerned, the WeiR reaction regulator is not focused, and therefore the antibacterial agent of the present invention has a novel antibacterial activity which has a completely different mechanism of action from the past. Therefore, the present invention provides, in the sixth and seventh aspects, a compound of the antibacterial agent (1), or a pharmaceutically acceptable salt, a solution or a batter oxide thereof as an active ingredient. As effective antibacterial agents, the compounds of the second, third and fifth aspects are particularly preferred. Furthermore, the antibacterial agent MRSA or VRE of the present invention is a galore yang 12 322420

S 201118100 • Sexual bacteria have a unique role. When the compound of the formula (1) is used as an antibacterial agent, the compound of the formula (I) or a salt thereof can be usually used as an oral or parenteral preparation. As the preparation for oral administration, various solid preparations and liquid preparations such as a key, a capsule, a granule, a powder, a preparation, and a liquid can be used. As the parenteral administration, a solution such as a solution, a suspension, or an emulsion, or a freeze-dried or powdery preparation which is dissolved or suspended during use can be used. Further, a non-oral preparation such as a transdermal preparation or a suppository such as an ointment or a patch may be used. These preparations may also contain a general carrier, an additive or the like in a pharmaceutical preparation, and may be produced by a general method. The dosage of the preparation of the pharmaceutical preparation containing the compound of the formula (I) may be appropriately determined depending on the sex, age, weight, severity of the symptoms, etc. of the patient, however, when the dosage is administered orally, The compound of the formula (1) is administered in an amount of from 0.1 to 1000 mg per liter, preferably from 1 to 10 mg, and from 0.0001 to 1 mg when administered orally, from 1 to 1 〇mg is better. According to an eighth aspect of the present invention, there is provided a WaiR reaction modifier inhibitor comprising the compound of the formula (I), or a pharmaceutically acceptable salt, solvate or N-oxide thereof as an active ingredient. As the active ingredient of the relevant WalR reaction regulator inhibitor, preferred are the compounds of the second, third and fifth aspects. As another aspect, the present invention provides a sterilizing lotion, a fat elixir or the like, and comprises a compound or a salt, a solvate or an N-oxide represented by the formula (I) as an active ingredient. The present invention will be described in more detail and specifically as shown in the following examples, but the 322420 13 201118100 embodiment is not intended to limit the inventors. [Example 1] Example 1-1 Production of 1,6-diamidyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4-e] [1,2, 4] Plowing-5, 7-diketone (Compound 1)

Add 6-chloro-3-indolyl urination (300 mg, 1.86 mmol) to a solution of hydrazine (103 mg, 2.23 mmol), triethylamine (0.40 mL, 2.86 mmol) in ethanol (4 mL). After stirring at 80 ° C for 12 hours, it was cooled to room temperature. A solution of 4-trifluoromethylbenzaldehyde (270 mg, 2.23 min) in ethanol (2 mL) was added and stirred at room temperature for 2 hr. The ethanol solvent of the obtained mixture was distilled off under reduced pressure to give a yellow solid as a crude product. THF (6 mL) and 2N-hydrochloric acid (3 mL) were added to the obtained yellow solid and cooled to 0 °C. An aqueous solution (1 mL) of sodium nitrite (153 mg, 2.23 mmol) was added dropwise at 0 ° C, and the mixture was stirred at room temperature for 2 hr. The organic phase was dried over anhydrous sodium sulfate (MgSO4). An N-oxide derivative (138 mg, 0.39 mmol) as a title compound (yield: 21%) was obtained. 4 NMR (500MHz, CDC13) 5 3. 53 (s, 3H), 4. 26 (s, 3H), 7. 79 (d, 2H, J = 8. 2Hz), 8. 44 (d, 2H, J = 8.2 Hz); 13C NMR (125 MHz, CDC13) (5 29.2, 43. 7, 126.0, 127.6, s 14 322420 201118100 133. 2, 135. 145. 4, 149. 0, 151.8, 154 2, 158. 4, 161 IR (neat) 1715, 1620, 1210 cm1; melting point 257 to 260 ° C Example 1 - 2 Production of 1-ethyl-6-methyl-3-[4-(trifluoromethyl) Phenyl]pyrimido[5,4_6][1,2,4]triterpenoid_5,7-di-class (compound 2)

Add 6-chloro-3-methyluracil 1 (300 mg, L86nm〇l) to a solution of acetamidine-grass salt (337 mg, 2.25 mmol) and triethylamine (〇.80mIj, 5.73mmol) in ethanol (4mL). ), at 80. (: After stirring for 12 hours, it was cooled to room temperature. A solution of 4-trifluoromethylbenzaldehyde (388 mg, 2.23 mmol) in ethanol (2 mL) was added and stirred at room temperature for 2 hr. The crude product of the knee 3c was obtained as a yellow solid. THF (6 mL) and 2N-hydrochloric acid (3 mL) were added to the obtained yellow solid, and then cooled to rc. The sodium nitrite (153 mg, 2.23_n) was dropped. After the aqueous solution (10)) was dropped for 2 hours at room temperature, it was extracted with acetic acid. After the separation was dried on anhydrous sodium sulfate, the solvent was removed, and the residue was purified by chromatography. The toxanthin derivative & 228 mg '〇.65 mmol) (yield 35% from i) was obtained as a yellow solid. Further, a flavonoid-N-oxide derivative 5c (61 mg, 0.16 mmol) was obtained as a yellow solid 322420 15 201118100 (yield 9% from 1). *H NMR (300MHz, CDCh) δ 1. 65(t, 3Η, J=7. 2Hz), 3. 55(s, 3H), 4. 74(q, 2H, J=7. 2Hz), 7. 80(d, 2H, J=8. 2Hz), 8. 45(d, 2H, J= 8.2Hz); 13C NMR(75MHz, CDCh) δ 13. 1, 29. 2, 51. 0, 126. 0 , 126. 1, 127. 6, 133. 5, 135. 3, 145. 5, 148. 6, 152. 0, 154. 4, 158. 5: Example 1 - 3 Manufacture of 6-methyl-3- [4-(Trifluoromethyl)phenyl]-8H-pyrimido[5,4-e] [1,2,4]triterp-5,7-dione (Compound 3)

A solution of the toxic flavin derivative 4 (50 mg, 0.15 mmol) in DMF (2 mL) was stirred at 130 ° C for one hour. The solvent was distilled off in vacuo, and the residue was purified eluted eluted eluted eluted eluted elution elution !H NMR (300MHz, CDCh) 5 3. 19(s, 3H), 7. 82(d, 2H, J=8. 2Hz) 8. 72(d, 2H, J=8. 2Hz), 9. 18 -9. 24(b, 1H): Example 4 Preparation of 1-phenylhydrazino-6-mercapto-3-[4-(trifluoromethyl)phenyl]pyrimidine and 16 1 322420 201118100 [5, 4 -e][1,2, 4]triterpenic-5,7-dioxime (compound 4)

2a 3a 4a(TF0001) 5a in the scorpion scorpion (匕61^711^(1犷321116)-hydrochloride (356111 palace,

2. Add 24-chloro-3-methylurea l (300 mg, 1.86 mmol) to a solution of 24 mmol), triethylamine (〇·80 mL, 5.73 mmol) in ethanol (4 mL), at 80 °C After mixing for 12 hours, it was cooled to room temperature. A solution of 4-trimethylmethyl benzoic acid _ (388 mg ' 2 · 23 mmol) in ethanol (2 mL) was added and the mixture was stirred at room temperature for 2 hr. The crude product of 3a. THF (^u and 2N-hydrochloric acid (3 mL)' was added to the obtained yellow solid and cooled to hydrazine. (:: An aqueous solution (1 mL) of sodium nitrite (153 mg ' 2 · 23 mmol) was added dropwise at 〇 ° c, in the room After stirring for 2 hours, the mixture was extracted with ethyl acetate. The separated organic phase was dried over anhydrous sodium sulfate, and then the solvent was evaporated, and the residue was purified by silica gel column chromatography to obtain a yellow solid lutein. Derivative 4a (176 mg, 0.42 nim) (yield from 1% to 23%). In addition, a flavonoid oxidizing organism 5a (56 mg, 0.13 mmol) was obtained as a yellow solid (yield from 7%). ' !H NMR (300MHz, CDCl3) (5 3. 29(s, 3H), 5. 60(s, 2H) 7 25-7. 38(m, 3H), 7. 57(d,2H, J= 8. 3 Hz), 7. 75 (d, 2H' J = 8 2 Hz) 7.90 (d, 2H, J = 8.2 Hz); ' ' 17 322420 s 201118100 13C NMR (75MHz, CDC13) 5 28. 1,60. 2, 125. 3, 126. 1,126. 4, 127. 1, 127. 7, 128. 6, 134. 2, 135. 3, 141. 1, 145. 7, 148. 5, 15 1. 9 , 155· 1,158. 3 : Example 1-5 Production of 1-cyclohexyl-6-mercapto-3-[4-(trifluoromethyl)phenyl]pyrimido[5, 4-e] [1 , 2, 4] trimorph-5,7-dione (compound 5)

1 2b 3b 4b(TF-0002) 5b To a solution of cyclohexane-hydrochloride (339 mg, 2.25 mmol), triethylamine (0. 80 mL, 5.73 mmol) in ethanol (4 mL) 3-methyluracil 1 (300 mg, 1.86 mmol) was stirred at 80 ° C for 12 hours and then cooled to room temperature. A solution of 4-trifluoromethylbenzialdehyde (388 mg, 2.23 mmol) in ethanol (2 mL) was added and stirred at room temperature for 2 hr. The ethanol solvent of the obtained mixture was evaporated under reduced pressure to give a crude product of yt. After adding THF (6 mL) and 2N-hydrochloric acid (3 mL) to the obtained yellow solid, the mixture was cooled to 0 °C. An aqueous solution (1 mL) of sodium nitrite (153 mg, 2.23 mmol) was added dropwise at 0 ° C, and stirred at room temperature for 2 hr. After the separated organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel column chromatography to afford to obtain a yellow solid as a flavin derivative. 18 18 322420 201118100 4b (90 mg, 0·22 mmol) (12% yield from 1). Further, a lutein-N-oxide derivative 5b (54 mg, 0.13 mmol) having a yellow solid (yield 7% from 1) was obtained. ]H NMR (300MHz, CDCI3) δ 1. 48-2. 24(m, 10H), 3. 48(s, 3H) 4. 25-4. 35(m, 1H), 7. 76(d, 2H , J=8. 2Hz), 8. 21(d, 2H, J=8. 2 Hz); , 3C NMR (75MHz, CDCh) δ 22. 3, 27. 2, 28. 7, 29. 4, 52 5, 126. 1, 126. 3, 127. 5, 134. 2, 135. 5, 146. 1, 148. 7, 151. 8, 15 4. 6, 158. 3 : [Example 2] Confirmation WalR Reaction Modulator Inhibitory Activity of the Compound of the Invention The WalR response modifier inhibitory activity of the compound of the present invention was confirmed by the screening system schematically shown in Fig. 2. Without the Wa 1 r response modifier inhibitor, since WalR forms a dimer and inhibits the expression of egfp, fluorescence is not observed. If a WalR reaction regulator inhibitor is present, egfp will be observed, and fluorescence can be observed. Further, in the screening system of Fig. 2, if the rib portion is replaced with a gene encoding a reaction regulator other than WalR, each reaction regulator inhibitor can be selected. The operation operation is performed based on the report using GFP described in Japanese Patent Laid-Open Publication No. Hei. No. 6-56787 (which is based on the method of analyzing the immersion (10). The plastid pFIGG1 (black square in Fig. 4) and pFI (10) 3 are included. JM109 of pFI014 (white circle in Figure 4), pArcA (black triangle in Figure 4) or p〇MpR (black rectangle in Figure 4) was cultured at 37t in LB containing ampicillin 19 322420 201118100 (ampici 11 in) The culture medium 1 #1 was added to a 96-microplate containing 200/1 LB-amp medium and 1 to 1 test compound, and the culture was incubated at 37 ° C for 20 hours. In lac 1420 ARVOsx (PerkinElmer), the optical density (OD595) at 595 nm was measured by the Model 3550 microplate detection system (Bi〇-Rad) by the excitation wavelength of 485 nm and the emission wavelength of 535 nm. Light intensity minus

01) 595値 is used for standardization. Further, PFI014 includes a gene encoding a DNA binding site of IclR of Escherichia coli (shown as N100 in Fig. 2) and a dimer formation site of WalR (shown as HD in Fig. 2). If the test compound is inhibited

WalR activity ' Because of the inability of WalR-ΝΙΟΟ to bind to the IclR promoter region, EGFp behaves to produce fluorescence. On the other hand, if the test compound does not inhibit WalR activity, WalR-N100 binds to the IclR promoter region and inhibits the expression of EGFP. Further, as a control group, the system used includes a gene encoding a DNA binding site of IclR and a dimer formation site of IC1R. Results As a result of the compound i of the compound of the present invention, as shown in Fig. 4, gamma (4) 14, gg - #, , and temple, fluorescence was observed. The compound 2, 3, 1R of the present invention has a specific inhibitory ability. Similarly, for the compound of the present invention, the compounds 6 and 7 were compared (the compounds 6 and 7 are commercially available, and the compounds shown in Table u are indicated as HD sheds). Matter and 3 also showed unique inhibition of WalR. 322420 20 201118100 [’Table 1]

[Example 3]

S 21 322420 201118100 Effect of the compound of the present invention on the performance of yoeB In order to confirm the effect of the compound of the present invention on the Wa 1 K/Wa 1R line, 'the use of B. subtilis has the effect of yoeB gene by inhibiting WalK/WalR The NBS280 strain composed of Py〇eB-bgaB was evaluated. A strain of NBS242 having a composition of PrpoB-bgaB was used as a comparison. ^ - Analysis of galactose 'except that the sample was cultured at 55 ° C, by (Nanam i ya H, Ohashi Y, Asai K, Moriya S, Ogasawara N, Fujita M, Sadaie Y, Kawamura F (1998) Mol Microbiol 29: 505-513) The method implemented in s. The specific activity is represented by M i 11 er Uni ts. As shown in Fig. 5, treatment with the compound i as the compound of the present invention showed an increase in the performance of yoeB in the NBS280 strain, and no change in the expression of rpoB in the NBS242 strain. This result shows that the compound 1 of the present invention regards WalR as a target' and thereby destroys B. subt i 1 i s and S. aureus. [Example 4] It was confirmed that the effect of the compound of the present invention on the expression of the gene of MRSA is such that the effect of the compound of the present invention on the expression of the gene of MRSA is more pronounced 'the genes for the synthesis of S· aureus N315 cell wall areaA, lytM, and ssaA performs S1 nuclease analysis. The gene rpsJ, which is not the target of waiR, was also used as a control.

The whole RNA S. aureus N315 strain was prepared in an LB medium at 3 (TC was aerated and propagated in the state of 0 D coffee, and the test compound was added thereto at 0, 50, 100 or 20 〇eg/ml. , culturing for 15 minutes. Centrifuge the cells ' and immediately suspend them in RNA-later (Ambion). These s 22 322420 201118100 homogenize with TE saturated phenol, kill by ethanol. RNase- After the decomposition of free DNase-I (Takara), the whole rnA is extracted by cumbersome, precipitated by ethanol, and dissolved in RNase-free water. S1 Nuclease Analysis

32P was produced by the method described in (Yamamoto K, Ishihama A (2006) Biosci Biotechnol Biochem 70: 1688-1695) using the gene DNA of S. aureus N315 and the primer shown in Fig. 6 (SEQ ID NO: 1 to 8). Identify DNA probes and perform SI nuclease assays. The 32P-labeled probe was cooled in a hybrid buffer (80% formaldehyde, 0.4 M NaCl, 20 Mm HEPES (pH 6.4)) and 100/zg of total RNA simultaneously at 75 ° C for 10 minutes. After incubation to 37 ° C for one night, it was decomposed with S1 nuclease (Takara). The decomposed DNA was precipitated with ethanol and dissolved in a furfural dye solution, and analyzed by electrophoresis of a 6% polypropylene guanamine gel containing 8 M urea. As a result, as shown in Fig. 7, the rpsj as a control showed no change in the expression of the compound 1 gene as a compound of the present invention, and the genes related to cell wall synthesis areaA, lytM, and ssaA, which are compounds of the present invention. 1 The concentration rises and the performance is suppressed. This phenomenon indicates that the compound of the present invention initially inhibits the expression of genes associated with cell wall synthesis, thereby eliminating gram-positive bacteria. [Example 5] Determination of antibacterial activity of the compound of the present invention The minimum growth inhibitory concentration (MIC) of the compound of the present invention was used s 23 322420 201118100

Mueller-Hinton agar medium (Difco) was determined by sequential cold-dilution method (Igarashi M, Sawa R'Kinoshita N, Hashizume H, Nakagawa N, Homma Y, Nishimura Y, Akamatsu Y (2008) J Antibiot (Tokyo) 61: 387-393). The final concentration that completely inhibits proliferation is taken as the MIC. The MICs of the compounds 1 to 5 of the present invention are shown in Table i (MIC measurement column), and the MICs of the compounds 1 to 3 of the present invention are shown in Table 2. In Table 2, the unit is v g/mi. [Table 2] Vg/ml, 96-microporous disk ^ 167 strain of grass bacterium, Rhizoctonia solani N315 strain, Escherichia coli W3110 strain Compound 1

3. 13TiW Compound 2 L 95 15. 62 Compound 3 15. 62 62, 5 [Example 6] Observation of the antibacterial action of the compound of the present invention The compound of the present invention shows inhibition of the WalR reaction regulator. The WalK/WalR TCS is a cell division of bacteria, which is predicted to cause the bacteria inhibited to become filamentous and agglutinous. In order to confirm this prediction, the appearance of B subtilis 168 and Saureus N315 was observed by a common light-focus laser microscope in the state where Compound 1 of the present invention was added at 12.5 / zg / ml. The compound 1 to which the present invention was not added was observed as a control. Operation The rapidly growing cells were transferred to a 2% agaric sugar pad containing the culture solution, and stained by SYNP Green (membrane) and SYBR Green (DNA) for microscopic observation. The image was obtained by 〇lympusFV3〇〇 conjugated focal laser microscope 24 322420 201118100 (xlOOO). As a result, in the case of the compound 1 in which the compound of the present invention was not added, the cells were divided and propagated, and the compound 1 to which the compound of the present invention was added was inhibited from cell division, and filamentous or agglutinated (Fig. 8). [Example 7] Inhibition of phosphoryl transfer (WalR) and autophosphorylation (WaijQ experiment, WalR protein (1 in μ) was reacted with phase-diluted compounds 1 to 4, comparative compounds 6 and 7 of the present invention at 37 ° C After 60 minutes, the KalK protein (0.25//M) phosphorylated with (r-32P) ATP was added, and the phosphoryl transfer reaction was carried out for 15 minutes at 37 ° C. After the reaction was completed, SDS-PAGE was performed by means of an image analyzer. The phosphorylation inhibition of the WalR protein caused by Compounds 1 to 4, 6 and 7 was measured as the intensity of RI, and the analysis software was used to calculate the 50% inhibitory concentration. Results The results using Compounds 1 to 4, 6 and 7 are shown in Table 1. (Expressed as IC5.) The results of using Compounds 1 to 3 are shown in Table 3. Compounds 1 to 3 have a particularly strong phosphorylation inhibitory effect on the target protein WalR. [Table 3]

25 322420 # M,Bsub Compound 1 201118100 ; This month provides the compound of formula (1), and contains these as the 'reported aging' anti- _, can be considered for medicine, music: agricultural music, lotion, biological In the field of membrane inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the mechanism of action of the compound of the present invention. Fig. 2 is a schematic view showing the screening method for the presence or absence of the inhibitory effect of Wam in the compound of the present invention. Fig. 3 is a chart showing the strains and plasmids used in the examples of the present invention. Fig. 4 is a view showing the results of screening of a compound which is a compound of the present invention. The vertical axis represents the fluorescence intensity, and the horizontal axis represents the amount of the compound 1 added. Contained in Escherichia coli JM109, white circles indicate pfI〇14 plastids, black circles indicate pF 1001 plastids, black triangles indicate pArcA plastids, and black rectangles indicate pOmpR plastids. Figure 5 is a graph showing the effect of y〇e]B on the compound 1 of the present invention. Indicates the growth of the strain (black mark) and the _galactosidase activity (white mark). Circles, triangles, and rectangles respectively indicate the amount of addition of Compound 1 (0.78 " g/m 〇 · 39 / z g / ml, 〇). Fig. 6 is a view showing a map of the plastids used in the S1 nuclease analysis. Fig. 7 is a view showing the result of supplying the compound 1 as the quinone of the present invention in the S1 nuclease analysis. e AG band is a fragment of the Maxam-G i 1 bert reaction. The bands 1 to 4 are respectively added as The compound 1 of the compound of the present invention is lanthanum, 5 〇, 1 〇〇, 200 /zg/ml. Fig. 8 is a view showing the state of the compound 1 which was not added as a compound of the invention of 26 322420 201118100 by a conjugated-focus laser microscope (control group) and the addition of B. subti 1 is 168 of 12. 5 // g/ml and S. The result graph of aureus N315. [Main component symbol description] None

S 27 322420 201118100 Sequence Listing. <110> School of Kinki, University of Kinki < 120> A novel antibacterial agent based on the reaction regulator WalR (YycF), <130> 558414 <150> JP2009-230384 <151> 2009-10-02 <160> 8 <210> 1 <211> 32 <212> DNA <213> Artificial sequence <220〉

<223> Designed oligonucleotide primer for amplifying Staphylococcus aureus isaA <400〉 1 gggttgattg gattctaaag ggcacatatt tc 32 <210〉 2 <211> 29 <212> DNA <213> Artificial sequence <220> 〉 Designed oligonucleotide primer for amplifying Staphylococcus aureus isaA <400〉 2 gcgtaacctg ttacacctaa tgccactgc 29 <210> 3 <211> 25 <212> DNA <213> Artificial sequence <220〉

&lt;223&gt; Designed oligonucleotide primer for amplifying Staphylococcus aureus lytM &lt;400> 3 gcgattgaga atggaatttg gttga 25 &lt;210> 4 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213> Artificial sequence &lt;220&gt;&lt;223&gt; Designed oligonucleotide primer for amplifying Staphylococcus aureus lytM &lt;400&gt; 4 gcgccattgt aaatgtagcg aagccc 26 &lt;210> 5 &lt;211&gt; 26 &lt;212> DNA &lt;213> Artificial sequence &lt;220> &lt;223&gt; Designed oligonucleotide primer for Amplifying Staphylococcus aureus ssaA &lt;400> 5 catagaaact tgatcgttcg tgtcga 26 &lt;210> 6 &lt;211&gt; 30 <212> DNA <213> Artificial sequence &lt;220&gt;&lt;223&gt; Designed oligonucleotide primer for amplifying Staphylococcus aureus ssaA <400 〉 6 1 322420 201118100 -----〇—〇- tgcgatagta gctgtagcga όϋ * &lt;210&gt; 7 &lt;211&gt; 29 &lt;212&gt; DNA • &lt;213> Artificial Sequence &lt;220〉

- &lt;223&gt; Designed oligonucleotide primer for amplifying Staphylococcus aureus rpsJ &lt;400〉 7 catattcatt caccaccgtt cttatgact 29 &lt;210〉 8 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213> Artificial sequence &lt;220〉 &lt;223&gt Designed oligonucleotide primer for amplifying Staphylococcus aureus rpsj &lt;400&gt; 8 gattgatcaa tcacgcggtg atcataagct 30 2 322420 s

Claims (1)

201118100 ' VII. Patent application scope: 'L A compound represented by the formula (丨) or a pharmaceutically acceptable salt or solvate thereof or N-oxide R3, N, N -Ri N R2 (I) Wherein Ri represents an aryl group of a 6 to 10 membered ring substituted with a Ci-e halogen group, and R2 and R3 each independently represent hydrogen, a Ci-6 alkyl group, a Cw cycloalkyl group, or a 6 to 1 member ring. An aryl group substituted with c, -6 alkyl, and X represents oxygen or sulfur. 2. A compound according to claim 1 or a pharmaceutically acceptable salt or solvate thereof or N-oxide, a compound of the formula (1) or a pharmaceutically acceptable salt or solvent thereof Compound, 〇R3&gt; Ri R2 (I) [wherein R1 represents an aryl group of a 6 to 10 membered ring substituted with a C1-6i alkyl group, and h and R3 each independently represent hydrogen or a C1-6 alkyl group, 322420 1 201118100 X means oxygen or sulfur]. 3. The compound of claim 2 or a pharmaceutically acceptable salt or solvate or N-oxide thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein μ Ri is a phenyl group substituted with a trifluoromethyl group at the 4-position, hydrazine is hydrogen or a methyl group or an ethyl group, R3 is a methyl group, and X is oxygen. 4. A compound according to claim 1 or a pharmaceutically acceptable salt or solvate or N-oxide thereof, which is selected from the group consisting of or a pharmaceutically acceptable compound thereof Salt or solvate or 1'6-dimethyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4_e] [1,2,4]trim-5,7- Diketone, m-methyl-6-methyl-3-[4-(tris[5,4-e][l,2,4]tris-5,7-di 1'cyclohexyl-6-oxime 3-[4-(trifluoromethyl)phenyl]pyridin[5,4-6][1,2,4]trin-5,7-dione, ethyl-6-methyl +[4-(Trifluoromethyl)phenyl](tetra) and [5,4e] [1,2, 4]三卩井-5, 7-dione and methyl-3-[4-(difluoromethyl) Base) Stupid base 8H, bite and [5, 4 e] [1, 2, 4] three tillage-5,7-dione. For example, the compound described in the first paragraph to the scope of the patent application or the pharmaceutically acceptable salt or compound or N-oxide thereof is selected from the group consisting of 2 a 322420 201118100 a compound or a pharmaceutically acceptable salt or solvate thereof, 1,6-dimethyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4_e] [1,2,4二卩井-5,7-二嗣, Buethyl-6-methyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5, 4-e] [1,2,4] Plowing -5,7-dione and 6-methyl-3-[4-(trifluoromethyl)phenyl]-8H-pyrimido[5,4-e] [1,2, 4] 5, 7-Diketone 6. An antibacterial agent is a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate or N-oxide thereof. As an active ingredient. 7. The antibacterial agent as described in claim 6 is targeted at MRSA or VRE. 8. A reaction regulator (reSp0nse reguiat〇r) inhibitor, which comprises a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt or solvate thereof An oxide is used as an active ingredient. 3 322420