JP2013082657A - New compound exhibiting production inhibitory capacity of yellow pigment produced by mrsa and usage of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound which suppresses biosynthesis of a yellow pigment produced by an MRSA (Methicillin-Resistant Staphylococus aureus) and becomes prevention or therapeutic agent of MRSA infectious disease.SOLUTION: Citridone A1 substance and citridone A2 substance are compounds respectively represented by formula [I] and formula [II].

Description

  The present invention is a novel aromatic compound that is expected to be useful for the prevention and treatment of MRSA infection by specifically inhibiting the production of yellow pigment produced by Staphylococcus aureus (MRSA) showing methicillin resistance. The present invention relates to derivative substances and methods for producing them.

  Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (hereinafter abbreviated as MRSA) are widespread worldwide, and have been confirmed as community-acquired infections, which is a major social problem. In addition, there have been many reports of deaths due to multidrug-resistant bacteria (such as Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter) that many antibiotics do not work recently, and there is a concern that there will be no antibiotics in the future. The background of this problem of resistant bacteria is the abuse of antibacterial drugs, but other than this, there is an increasing number of patients with reduced immunity and an aging society, and bacterial infections will continue to increase in the future. It is expected to increase.

  In general, antibiotics such as glycopeptide vancomycin and aminoglycoside arbekacin are used for the treatment of MRSA infection. In addition, combination therapy of β-lactam antibiotics or β-lactam antibiotics with other antibiotics having different action points has been performed (Non-patent Document 1). As for vancomycin and arbekacin, resistant bacteria to these drugs have already appeared. In order to cope with such a situation, exploratory research for new anti-MRSA drugs is currently in progress.

  In 2006, a research team from Merck reported a new antibiotic, platensimycin, which is also effective against MRSA from Streptomyces platensis isolated from the soil in South Africa (non- Patent Document 2). However, the development of a drug that acts directly on such bacteria to sterilize, that is, the conventional antibiotic concept, will eventually induce the drug resistance of the bacteria, and as a result, "wealth of drugs and bacteria" will begin again is expected.

  Therefore, an approach to an anti-infective drug based on a completely new concept that does not directly show antibacterial activity to MRSA is required. Staphylococcus aureus is known to produce yellow pigment to protect itself from human immunity after infecting the human body. The main yellow pigment is staphyloxanthin. It has already been reported in experiments at the gene level that this dye is essential for establishing infection with MRSA by escaping from the bactericidal oxidative action by neutrophils etc. of the host (Non-patent Document 3). In other words, if the production of the yellow pigment, particularly staphyloxanthin, is inhibited, infection by MRSA can be prevented.

  Therefore, the compound that inhibits the production of the yellow pigment invalidates not only Staphylococcus aureus but also MRSA, the defense mechanism of the bacteria to escape the bactericidal oxidation action by the host neutrophils and the like. Is expected. That is, such a compound is expected to reduce the infectivity to the host without affecting the growth of MRSA and to have a low possibility of emergence of new resistant bacteria. At the same time, since the compound that inhibits the production of the yellow pigment is not related to the drug resistance mechanism possessed by MRSA, it is expected to bring about the same effect on various drug-resistant bacteria.

Yumi Hasegawa et al., Science of Drug Administration, p. 264-273, 1998. Wang J. et al., Nature, 441, p. 358-361, 2006. Liu G.Y. et al., J. Exp. Med., 202, p. 209-215, 2005.

  The present invention provides a substance that attenuates the infectivity of MRSA, in particular, suppresses the biosynthesis of the yellow pigment produced by MRSA, thereby inhibiting the antibactericidal oxidative action exhibited by MRSA, thereby preventing infection of the host. It is an object of the present invention to provide a novel compound that can be a safe and effective pharmaceutical with little possibility of emergence of new resistant bacteria, and to provide a method for producing the compound.

  As a result of searching for a substance having an activity of inhibiting the production of a yellow pigment produced by MRSA (hereinafter also simply referred to as a yellow pigment) for metabolites produced by microorganisms, the present inventors have found that citridon A [(3R) -2,3α, 8aα-trimethyl-7-phenyl-3aα, 8a-dihydro-3H-8-oxa-5-azacyclopenta [a] inden-4 (5H) -one], Two newly synthesized substances were found to have the desired inhibitory activity, and these substances were named as citridon A1 substance and citridon A2 substance, respectively.

  The present invention completed based on this finding is a citridon A1 substance which is a compound represented by the following formula [I] and a citridon A2 substance which is a compound represented by the following formula [II].

  The present invention also provides a staphyloxanthin biosynthesis inhibitor containing citridon A1 substance as an active ingredient, a pharmaceutical composition for infection prevention or treatment of MRSA infection, and a staphyloxanthine biosynthesis inhibitor containing citridon A2 substance as an active ingredient. And a pharmaceutical composition for preventing or treating MRSA infection.

  ADVANTAGE OF THE INVENTION According to this invention, the novel substance citridon A1 substance and the citridon A2 substance which have the biosynthesis inhibitory effect of staphyloxanthin and the infection prevention effect of MRSA infection are provided. By administering a pharmaceutical composition containing these substances as active ingredients to an inpatient who is at risk of nosocomial infection by MRSA, for example, nosocomial infection of MRSA can be prevented. The compounds are also useful for the treatment of MRSA infections.

  Since the citridon A1 substance and the citridon A2 substance according to the present invention prevent infection of the host by suppressing the production of yellow pigment produced by MRSA, it is safe and effective with little possibility of appearance of new resistant bacteria. Become a medicine.

1 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of the substance Citridone A1 according to the present invention. 2 shows a proton nuclear magnetic resonance spectrum (in deuterated chloroform) of the citridone A2 substance according to the present invention.

  The citridone A1 substance and the citridone A2 substance of the present invention were prepared in the same manner as in Examples 1 and 2 with reference to the synthesis procedure of citridone A described in Organic Letters, 2011, 13, 1158-1161 (T. Miyagawa et al.). It was synthesized by the method described in 1.

The physicochemical properties of the obtained citridon A1 substance and citridon A2 substance of the present invention will be described below.
The physicochemical properties of the citridon A1 substance are as follows.
(1) Property: Oily (2) Molecular formula: C ₁₈ H ₁₇ NO ₂
HRESI-MS (m / z) [M + Na] ⁺ calculated value 302.1157, actual value 302.1143
(3) Molecular weight: 279
(4) Infrared absorption spectrum: An infrared absorption spectrum measured by the potassium bromide tablet method shows a characteristic absorption maximum at νmax 2926, 1739, 1654, 1369, 1227 cm ^{−1 and the} like.
(6) Optical rotation: [α] D27.6 -129.6 ° (c = 0.1, chloroform)
(7) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) measured by Varian 400M nuclear magnetic resonance spectrometer in deuterated dimethyl sulfoxide (Figure 1) and carbon chemical shift (ppm) Are as follows:
¹ H-NMR (400 Hz, CDCl ₃ ) δ 7.54-7.50 (3H), 7.43-7.36 (1H), 7.34-7.28 (2H), 5.95 (1H), 5.73 (1H), 3.25 (1H), 3.14- 3.12 (1H), 1.69 (3H), 1.29 (3H);
¹³ C-NMR (100 MHz, CDCl ₃ ) δ 164.7, 163.1, 141.4, 134.5, 133.2, 131.1, 128.5, 127.6, 127.4, 127.3, 104.8, 55.5, 46.3, 26.0, 21.6.

  As a result of examining various physicochemical properties and spectrum data of the above-mentioned citridon A1 substance in detail, it was determined that the citridon A1 substance has a chemical structure represented by the following formula [I]. This compound can be denoted as demethylcitridone A.

The physicochemical properties of the citridon A2 substance are as follows.
(1) Property: white solid substance (2) Molecular formula: C ₁₉ H ₁₉ NO ₂
HRESI-MS (m / z) [M + Na] ⁺ calculated value 294.1494, actual value 294.1485
(3) Molecular weight: 293
(4) Infrared absorption spectrum: The infrared absorption spectrum measured by the potassium bromide tablet method shows a characteristic absorption maximum at νmax 2965, 1652, 1600, 1454, 1431 cm ^{−1 and the} like.
(5) Optical rotation: [α] D27.6 -107.6 ° (c = 1, chloroform)
(6) Proton and carbon nuclear magnetic resonance spectra: Hydrogen chemical shift (ppm) measured by Varian 400M nuclear magnetic resonance spectrometer in deuterated dimethyl sulfoxide (Figure 2) and carbon chemical shift (ppm) Are as follows:
¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.54-7.51 (3H), 7.41-7.37 (2H), 7.32-7.28 (1H), 5.40 (1H), 3.28 (1H), 2.90 (1H), 1.73 ( 3H), 1.67 (3H), 1.30 (3H);
¹³ C-NMR (100 MHz, CDCl ₃ ) δ 165.4, 162.6, 150.6, 134.3, 133.5, 128.7, 127.7, 127.4, 126.4, 111.8, 104.3, 56.7, 49.2, 26.4, 20.4, 14.9.

  As a result of detailed examination of various physicochemical properties and spectrum data of the above-mentioned citridon A2 substance, it was determined that the citridon A2 substance has a chemical structure represented by the following formula [II].

  The citridone A1 substance and citridone A2 substance of the present invention have a biosynthesis inhibitory activity of a yellow pigment (staphyloxanthine) produced by MRSA, as shown in the test examples described later. Therefore, these substances are useful as inhibitors of staphyloxanthin biosynthesis, and further can suppress the infection and progression of MRSA, and thus are useful in pharmaceutical compositions for preventing or treating MRSA infections.

  The citridone A1 substance and the citridone A2 substance of the present invention can be used in medicine each alone or in combination. Formulation may be performed by a conventional method. For example, the substance of the present invention is used as an active ingredient, and conventional carriers and excipients, if necessary, binders, disintegrants, lubricants, buffers, suspending agents, stabilizers, pH regulators, colorants A pharmaceutical composition to which a flavoring agent, a fragrance, etc. are added can be formulated in the form of a solution, suspension, tablet, granule, powder, capsule or the like. The route of administration is not particularly specified. Both oral administration and parenteral administration such as injection and infusion are possible.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the examples are not intended to limit the present invention and are merely illustrative.

  This example illustrates the synthesis of the citridone A1 substance. Citridon A1 substance can be synthesized according to the following reaction pathway. In the following, compound 1 is the target compound citridon A1.

Under a nitrogen atmosphere, t-BuOK (4.2 mg, 0.038 mmol) was added to a DMF (250 μL) solution of compound 3 (9 mg, 0.013 mmol), and reacted at 100 ° C. for 1 hour. Water was added to stop the reaction, and the reaction solution was extracted with CH ₂ Cl ₂ three times. The combined organic layers are dried over Na ₂ SO ₄ and concentrated, and the resulting residue (crude material) is purified by column chromatography (silica gel 500 mg, CH ₂ Cl ₂ : MeOH = 50: 1). As a result, Citridon A1 substance (Compound 1) (2.1 mg, 57%) was obtained.

Various physicochemical properties and spectrum data of the obtained citridone A1 substance are as described above.
Compound 3 of the starting material can be synthesized from (+) pulegone and transcinnamic acid as described in Organic Letters, 2011, 13, 1158-1161 (T. Miyagawa et al.).

  This example illustrates the synthesis of the citridon A2 material. Citridon A2 substance can be synthesized according to the following reaction pathway. In the following, compound 2 is the target compound citridon A2, and the meanings of the symbols are as follows.

TBDPS: tert-butyldiphenylsilyl group (t-BuPh ₂ Si-);
TBAF: tetrabutylammonium fluoride;
DBU: Diazabicycloundecene;
AIBN: Azobisisobutyronitrile.

Synthesis of Compound 5 I ₂ (84.6 mg, 0.669 mmol) was added to a solution of Compound 4 (148 mg, 0.268 mmol) in CH ₂ Cl ₂ (5.4 mL) under a nitrogen atmosphere and reacted for 2.5 hours. The reaction was stopped by adding an aqueous Na ₂ S ₂ O ₃ solution, and the reaction solution was extracted with CH ₂ Cl ₂ three times. The combined organic layers were dried over Na ₂ SO ₄ and concentrated, and the resulting residue (crude material) was purified by column chromatography (silica gel 8.60 g, CH ₂ Cl ₂ : MeOH = 100: 1). Compound 5 (115 mg, 63%) was obtained as an oily substance.

¹ H-NMR (400 Hz, CDCl ₃ ) δ 7.68-7.66 (2H), 7.60-7.57 (2H), 7.48-7.31 (12H), 4.64 (1H), 3.88 (1H), 3.73 (1H), 3.21 ( 1H), 2.35-2.26 (1H), 1.68 (3H), 1.65-1.54 (1H), 1.26 (3H), 0.96 (9H);
HRMS (ESI) [M + Na] ⁺ calcd for C ₃₅ H ₃₈ INNaO ₃ Si 698.1563, found 698.1547.

Compound 4 as the starting material can be synthesized from (+) pulegone and trans-cinnamic acid as described in Organic Letters, 2011, 13, 1158-1161 (T. Miyagawa et al.).
Synthesis of Compound 6 Under a nitrogen atmosphere, TBAF (1.0 M THF solution) (0.47 mL, 0.47 mmol) was added dropwise to a solution of Compound 5 (320 mg, 0.474 mmol) in THF (4.7 mL). For 90 hours. The reaction was stopped by adding H ₂ O, and the reaction solution was extracted with AcOEt three times. The combined organic layers were dried over Na ₂ SO ₄ and concentrated, and the resulting residue was purified by column chromatography (silica gel 4.30 g, CH ₂ Cl ₂ : MeOH = 60: 1) to give a tan oil. Compound 6 (159 mg, 77%) was obtained.

¹ H-NMR (300 MHz, CD ₃ CD) δ 7.41-7.30 (5H), 7.28-7.22 (1H), 4.43 (1H), 3.74 (1H), 3.70 (1H), 3.17 (1H), 2.05-1.94 (1H), 1.66-1.60 (1H), 1.63 (3H), 1.35 (3H);
HRMS (ESI) [M + H] ⁺ calcd for C ₁₉ H ₂₁ INO ₃ 438.0566, found 438.0567.

Synthesis of Compound 8 Under a nitrogen atmosphere, pyridine (3.6 mL) and DMSO (3.6 mL) were added to a CH ₂ Cl ₂ (3.6 mL) solution of Compound 6 (159 mg, 364 μmol), and Dess-Martin periodinane (DMP, 309 mg, 728 μmol) was added, and the resulting mixture was stirred at room temperature for 30 minutes. The reaction was stopped by adding a saturated aqueous Na ₂ S ₂ O ₃ solution, and then a saturated aqueous NaHCO ₃ solution was added, and then the reaction solution was extracted with AcOEt three times. The combined organic layers were dried over Na ₂ SO ₄ and concentrated to obtain compound 7 as a crude product. This compound 7 was used in the next step without purification.

Under a nitrogen atmosphere, DBU (163 μL, 1.09 mmol) was added dropwise to a solution of compound 7 in CH ₂ Cl ₂ (7.3 mL) at 0 ° C., and the mixture was stirred at room temperature for 15 minutes. H ₂ O was added to stop the reaction, and the reaction solution was extracted with CH ₂ Cl ₂ three times. The combined organic layers were dried over Na ₂ SO ₄ and concentrated, and the resulting residue was purified by column chromatography (silica gel 4.30 g, CH ₂ Cl ₂ : MeOH = 50: 1). Compound 8 (72.7 mg, 2 steps 65%) was obtained.

¹ H-NMR (300 MHz, CDCl ₃ ) δ 9.83 (1H), 8.06 (1H), 7.43-7.31 (5H), 6.69 (1H), 3.41 (1H), 3.24 (1H), 1.81 (3H), 1.31 (3H);
HRMS (ESI) [M + H] ⁺ calcd for C ₁₉ H ₁₈ NO ₃ 308.1287, found 308.1292.

Under nitrogen atmosphere compound 9, compound 8 (72.7 mg, 237μmol) of CH ₂ Cl ₂ (2.4 mL) was added ZnI ₂ (2.6 mg, 7.11μmol) and _{Me 3 SiSSCH 2 CH 2 SSiMe 3} (79μL, 308 mol ) Was added and stirred at room temperature for 14 hours. Additional ZnI ₂ (2.6 mg, 7.11 μmol) and Me ₃ SiSSCH ₂ CH ₂ SSiMe ₃ (79 μL, 308 μmol) were added to the reaction mixture, and the mixture was further stirred for 4 hours, and then saturated aqueous Na ₂ S ₂ O ₃ solution and saturated NaHCO ₃ solution. _{3 The} aqueous solution was added to stop the reaction, and the reaction solution was extracted with CH ₂ Cl ₂ three times. The combined organic layers were dried over Na ₂ SO ₄ and concentrated, and the resulting residue was purified by column chromatography (silica gel 2.15 g, CH ₂ Cl ₂ : MeOH = 100: 1) to give a tan oily compound 9 (86.9 mg, 83%) was obtained.

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.54-7.49 (3H), 7.42-7.26 (3H), 5.86 (1H), 5.06 (1H), 3.34 (1H), 3.32-3.26 (1H), 3.23- 3.05 (4H), 1.67 (3H), 1.38 (3H);
HRMS (ESI) [M + Na] ⁺ calcd for C ₂₁ H ₂₁ NNaO ₂ S ₂ 406.0911, found 406.0907.

Synthesis of citridon A2 substance (compound 2) Under a nitrogen atmosphere, Bu ₃ SnH (240 μL, 907 μmol) and AIBN (1.5 mg, 9.08 μmol) were added to a solution of compound 9 (86.9 mg, 22.7 μmol) in benzene (2.3 mL). The mixture was stirred at 80 ° C. for 23 hours. Additional Bu ₃ SnH (240 μL, 907 μmol) and AIBN (1.5 mg, 9.08 μmol) were added, and the mixture was further stirred for 2.5 hours. The reaction solution was returned to room temperature, hexane was added, and the mixture was extracted 3 times with MeOH. The obtained MeOH layers were combined and concentrated, and purified by column chromatography (silica gel 2.15 g, CH ₂ Cl ₂ : MeOH = 100: 1) to obtain citridon A2 substance (32.1 mg, 49%). . Various physicochemical properties and spectrum data of the obtained citridon A2 substance are as described above.

(Test Example 1)
MRSA has been reported to exhibit resistance to the host immune system by producing a yellow pigment (staphyloxanthin) having antioxidant activity (Patent Document 3). Therefore, the yellow pigment (staphyloxanthine) production inhibitory activity was evaluated for the citridon A1 substance and the citridon A2 substance of the present invention.

  MRSA K-24 strain, a clinical isolate, was inoculated into 10 mL of Mueller Hinton Broth (BD) using a platinum loop and cultured at 37 ° C. for 18 hours. Then, 5 mL of MHB was added to 1 mL of the culture solution, and the mixture adjusted to 0.5 McFarland was used as the MRSA bacterial solution.

Next, TYB medium (Marshall J H. et al. J. Bacteriol., 147, p.900-913, 1982) (Bacto Tryptone (BD) 1.7%, yeast extract (DIFCO) 1.0%, NaCl (Wako) 0.5 %, K ₂ HPO ₄ (Wako) 0.25%, Bacto agar (DIFCO) 1.5%) and sterilized at 121 ° C. for 15 minutes. Glycerol monoacetate (Wako) solution sterilized with a sterilizing filter (Minisart 0.20μm, Sartorius Stedim) was added to TYB medium to a final concentration of 1.5%, and 25 ml of this medium was spread on a No. 2 petri dish (Eiken). . After confirming that the agar medium had hardened, the MRSA bacterial solution prepared earlier was applied to the agar surface using a sterile cotton swab and cultured at 37 ° C. for 4 hours. Thereafter, a paper disk having a diameter of 6 mm containing 50 μg of the test compound was placed almost at the center on the agar medium and cultured at 37 ° C. for 68 hours. The activity was evaluated by measuring the diameter of a white circle formed around the paper disk (the growth circle of MRSA that could no longer produce a yellow pigment).

  As a result, both the citridon A1 substance and the citridon A2 substance formed a 13 mm white circle. From these results, the citridon A1 substance and the citridon A2 substance according to the present invention do not exhibit antibacterial activity and inhibit the biosynthesis of yellow pigment (staphyloxanthin), which is useful for preventing infection of MRSA to the host. It is believed that there is.

  The method for evaluating the yellow pigment production inhibitory activity produced by MRSA used here is newly developed by the present inventors. In this evaluation method, it has been confirmed that only the target yellow pigment inhibitory activity can be selectively evaluated without causing the antibacterial activity of an antibacterial agent such as vancomycin to appear. That is, when the paver disc contains a compound having yellow pigment inhibitory activity, a white circle is formed, but even if a paper disc containing an antibacterial agent is placed on the agar medium, a white circle is formed around it. A circle does not appear.

  In addition, when the staphyloxanthin biosynthesis inhibitory activity was investigated about the citridon A by the same evaluation method, it was confirmed that it shows substantially the same activity as the citridon A1 substance and the citridon A2 substance. Therefore, citridon A is also useful for the prevention and treatment of MRSA infections.

Claims (6)

Citridone A1 substance which is a compound represented by the following formula [I].

Citridon A2 substance which is a compound represented by the following formula [II].

  A staphyloxanthine biosynthesis inhibitor comprising the citridon A1 substance or the citridon A2 substance according to claim 1 or 2 as an active ingredient.   A pharmaceutical composition for preventing or treating infection of MRSA infection comprising the citridon A1 substance or citridon A2 substance according to claim 1 or 2 as an active ingredient.   Staphyloxanthin biosynthesis inhibitor containing citridon A as an active ingredient.   A pharmaceutical composition for preventing or treating infection of MRSA infection comprising citridon A as an active ingredient.

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