WO2015168979A1 - Derivatives of naftifine hydrochloride, and preparation method and use thereof - Google Patents

Abstract

Disclosed in the present invention are derivatives of naftifine hydrochloride, and a preparation method and use thereof. The derivatives of naftifine hydrochloride of the present invention have a structure as shown by formula (I), wherein Ar has the definition as described in the description and claims. By inhibiting the expression and/or function of key catalyzing enzyme CrtN in a golden yellow pigment synthesis pathway and powerfully inhibiting the synthesis of golden yellow pigments, the virulence of bacteria can thus be lowered. The key catalyzing enzyme CrtN in the golden yellow pigment synthesis pathway can be used as a drug target; and compounds capable of inhibiting the expression and/or function of the catalyzing enzyme CrtN can be used for preparing anti-bacterial drugs. The naftifine hydrochloride and the derivatives thereof in the present invention can be used as inhibitors for the catalyzing enzyme CrtN to powerfully inhibit the synthesis of golden yellow pigments, so that the virulence of Staphylococcus aureus can be lowered, and same can thus be used for preparing the anti-bacterial drug, and especially a drug for resisting Staphylococcus aureus infections.

Description

 Derivative of naftifine hydrochloride, preparation method and use thereof

 Technical field

 The invention relates to the fields of pharmacology, medicinal chemistry and pharmacotherapy, and more particularly to naftifine hydrochloride and its derivatives, preparation method, new antibacterial use, and a target CrtN for exerting antibacterial action.

 Background technique

 Staphylococcus aureus 03⁄4^ /0«3⁄4^^ awre^) is an important pathogen that seriously endangers human health. As a representative of Gram-positive bacteria, it is the most common pathogen causing human suppurative infection, which can directly lead to localized pus infection, pneumonia, pseudomembranous colitis, pericarditis, etc., and even systemic infections such as sepsis and sepsis.

 With the development of life sciences and medicine, it has been found that pathogens including Staphylococcus aureus are pathogenic because they help colonization, adhesion, cytotoxicity of bacteria by producing a variety of Virulence factors. Immune evasion and the like allow the bacteria to successfully carry out the infection.

 Anti-virulence drugs are becoming a hot spot for new antibacterial infections because of the emergence and spread of various drug-resistant bacteria. At present, anti-bacterial virulence drugs mainly play through five ways: (1) to inhibit the expression of toxins in target bacteria; (2) to block quorum sensing between bacteria; (3) to inhibit the secretion and transmission of toxins; (4) resistance Break all links of bacterial adhesion; (5) inhibit bacterial immune escape. Any of the drugs having one of the above five effects can reduce the pathogenicity of bacteria and effectively prevent and treat various infectious diseases.

 In 2005, Professor Victor Nizet of the University of California, San Diego (UCSD) discovered that Staphyloxanthin of Staphylococcus aureus has the ability to help Staphylococcus aureus escape the active oxygen produced by the innate immune system, which determines the pathogenicity of bacteria. A key factor in ability. Professor Eric Oldfield of the University of Illinois at Urbana-Champaign has successfully discovered that a known cholesterol synthesis inhibitor BPH-652 can inhibit the formation of golden yellow pigment in Staphylococcus aureus, thereby reducing the pathogenicity of Staphylococcus aureus in mice. . There are also some reports that golden yellow pigments can increase the resistance of bacteria to oleic acid. In the subcutaneous infection model of mice, the abscess area caused by the mutant strain that can not produce pigment is significantly reduced compared with the wild type strain, suggesting that the pigment can improve bacteria. The ability to resist oxidation increases the virulence of the bacteria. These existing studies have initially demonstrated that inhibition of the synthesis of the virulence factor golden yellow pigment of S. aureus is a new and effective antibacterial strategy.

 China is one of the countries with the most serious abuse of antibiotics in the world. The resulting problem of bacterial resistance is particularly acute. Some bacteria isolated from clinical isolates have the highest resistance to certain antibiotics in the world. In the face of severe bacterial antibiotic resistance, we urgently need to find new antibacterial targets and new antibacterial drugs.

 Therefore, research and development of antibacterial drugs for anti-gold yellow pigment synthesis has important practical and scientific value. Summary of the invention

 It is an object of the present invention to provide a process for the preparation and novel use of naftifine hydrochloride and its derivatives.

 Another object of the present invention is to provide a novel structure of a naftifine hydrochloride derivative.

In addition, the invention also provides the use of a catalytic enzyme CtrN inhibitor and a method of reducing bacterial pathogenicity or toxicity. In a first aspect of the invention, there is provided a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the preparation of an antibacterial agent,

 Wherein Ar is a C6-C10 aryl group, a C1-C6 fluorenyl substituted C6-C10 aryl group.

 In another preferred embodiment, the pharmaceutically acceptable salt is a hydrochloride salt.

 In another preferred embodiment, the antibacterial agent is a drug against Staphylococcus aureus.

In a second aspect of the invention, there is provided a use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a catalytic enzyme CrtN inhibitor, or for the preparation of inhibition,

 Wherein Ar is a C6-C10 aryl group, a C1-C6 fluorenyl substituted C6-C10 aryl group.

 In another preferred embodiment, the pharmaceutically acceptable salt is a hydrochloride salt.

 In another preferred embodiment, Ar is a phenyl group, a naphthyl group, or a C1-C6 fluorenyl substituted phenyl group.

式中，

1^为。2-。6垸基， R₂为氢； In a third aspect of the invention, there is provided a salt of the formula,

In the formula,

1^ is. 2-. 6 fluorenyl, R ₂ is hydrogen;

Or, R ₂ together with an adjacent carbon atom form a C6-C10 aryl group.

In another preferred embodiment, it is. 3-. 5 fluorenyl, R ₂ is hydrogen;

Or R ₂ together with an adjacent carbon atom form a benzene ring.

 In another preferred embodiment, the pharmaceutically acceptable salt of the compound of formula I is a hydrochloride salt selected from the group consisting of

 In a fourth aspect of the invention, there is provided a process for the preparation of a compound of formula I or a pharmaceutically acceptable salt thereof, the process comprising the steps of:

 II ΠΙ j

(a) reacting a compound of formula II with a compound of formula III to form a compound of formula I; and optionally

(b) a step of producing a hydrochloride salt of a compound of formula I from a compound of formula I,

 In each formula, Ar is a C6-C10 aryl group, a C1-C6 fluorenyl substituted C6-C10 aryl group.

 According to a fifth aspect of the invention, there is provided an antibacterial pharmaceutical composition comprising a compound which reduces the activity of a catalytic enzyme CrtN; and a pharmaceutically acceptable carrier.

 In another preferred embodiment, the antibacterial means controlling bacterial pathogenicity.

 In another preferred embodiment, the compound which reduces the activity of the catalytic enzyme CrtN means a compound which can directly bind to the catalytic enzyme CrtN, thereby inhibiting the activity of the catalytic enzyme CrtN.

 In another preferred embodiment, the compound which reduces the activity of the catalytic enzyme CrtN refers to a compound which binds to certain sequences of the bacteria themselves, thereby causing a decrease in the expression of the catalytic enzyme CrtN.

In another preferred embodiment, the reduced catalytic I compound or a pharmaceutically acceptable salt thereof,

Wherein Ri, R _{2 are} independently hydrogen, C1-C6 fluorenyl;

Or, R ₂ together with an adjacent carbon atom form a C6-C10 aryl group.

 In another preferred embodiment, the antibacterial pharmaceutical composition is a pharmaceutical composition against Staphylococcus aureus.

 According to a sixth aspect of the invention, a pharmaceutical composition for inhibiting the catalytic enzyme CrtN or inhibiting the synthesis of golden yellow pigment, comprising a compound of formula I or a pharmaceutically acceptable salt thereof;

a pharmaceutically acceptable carrier,

Wherein Ri, R _{2 are} independently hydrogen, C1-C6 fluorenyl;

Or Ri, R ₂ together with adjacent carbon atoms form a C6-C10 aryl group.

 In a seventh aspect of the invention, there is provided a use of a catalytic enzyme CrtN inhibitor for the preparation of a pharmaceutical composition or an antibacterial composition for reducing the pathogenicity of bacteria.

 According to an eighth aspect of the invention, a method for reducing bacterial pathogenicity or toxicity, comprising the steps of:

 The bacteria are contacted with an inhibitor of the catalytic enzyme CrtN to reduce bacterial pathogenicity or toxicity.

 In another preferred embodiment, the method is a non-therapeutic method.

 In another preferred embodiment, the method is a therapeutic method.

 In another preferred embodiment, the contacting results in a decrease in the expression and/or activity of the catalytic enzyme CrtN in the bacteria.

In another preferred embodiment, the catalytic enzyme CrtN inhibitor comprises: a compound of formula I or a pharmaceutically acceptable salt thereof, An antisense nucleic acid or miRNA, an antibody against CrtN, or a combination thereof that inhibits expression of CrtN.

 According to a ninth aspect of the present invention, there is provided an attenuated bacterial strain, wherein a decrease or loss of activity of a catalytic enzyme CrtN in the bacterial strain causes a decrease in toxicity of the bacteria.

 In another preferred embodiment, the decrease or loss of CrtN activity is achieved by administration of a CrtN inhibitor.

 In another preferred embodiment, the decrease or loss of CrtN activity is achieved by gene interference or knockout.

 In another preferred embodiment, the attenuated bacterial strain is an attenuated S. aureus strain.

 In a tenth aspect of the invention, the use of the bacterial strain of the ninth aspect is provided for screening for a compound which reduces the pathogenicity and/or toxicity of S. aureus.

 In an eleventh aspect of the invention, there is provided a method of screening for a compound which reduces pathogenicity and/or toxicity of a bacterium, comprising the steps of:

 (a) providing a compound to be tested, and determining whether the compound to be tested interacts with the catalytic enzyme CrtN, and selecting a catalytic enzyme CrtN inhibitor, wherein if the test compound lowers the catalytic enzyme CrtN activity, or causes a catalytic enzyme A decrease in CrtN expression indicates that the compound to be tested is a catalytic enzyme CrtN inhibitor;

 (b) In the experimental group, the catalytic enzyme CrtN inhibitor selected in the previous step was contacted with bacteria to determine the pathogenicity and/or toxicity of the bacteria, and compared with the control group to select bacteria-reducing bacteria. Pathogenic and/or toxic compounds.

 In another preferred embodiment, the other experimental conditions are the same as those of the experimental group except that the control group is not in contact with the catalytic enzyme CrtN inhibitor.

 According to a twelfth aspect of the invention, there is provided a compound which reduces the pathogenicity and/or toxicity of bacteria, which compound is selected by the method described in the eleventh aspect.

 In a thirteenth aspect of the invention, there is provided a method of inhibiting the catalytic enzyme CrtN or inhibiting the synthesis of golden yellow pigment, administering a safe and effective amount of a compound of formula I to a subject or to the environment.

 In another preferred embodiment, the desired subject includes a human or a non-human mammal, preferably a human, a mouse or a rat.

 In a fourteenth aspect of the invention, a method of anti-S. aureus is provided, wherein a safe and effective amount of a compound of formula I is administered to a subject in need thereof or a bacteriostatically effective amount of a compound of formula I is administered to the environment.

 In another preferred embodiment, the desired subject comprises cells cultured in vitro, human or non-human mammals, preferably human, mouse or rat.

 In a fifteenth aspect of the invention, an antibacterial method, wherein a safe and effective amount of a compound of formula I is administered to a subject in need thereof. In another preferred embodiment, the desired subject comprises cells cultured in vitro, human or non-human mammals, preferably human, mouse or rat.

 According to a sixteenth aspect of the present invention, a method for treating a bacterial infection, comprising the steps of: administering a safe and effective amount of a drug or a pharmaceutical composition to a subject infected with a bacterium, wherein

 The drug is a compound which reduces the activity of the catalytic enzyme CrtN;

 The pharmaceutical composition includes a compound which reduces the activity of the catalytic enzyme CrtN, and a pharmaceutically acceptable carrier. In another preferred embodiment, the drug or pharmaceutical composition is contacted with bacteria for a period of time to reduce the pathogenicity and/or toxicity of the bacteria.

In another preferred embodiment, the desired subject includes a human or non-human mammal, preferably a human, mouse or rat. In another preferred embodiment, the manner of administration to the subject is not particularly limited, and includes, but is not limited to, oral administration, injection, Inhalation, topical use.

 In the present invention, "safe and effective amount" means that the amount of the active ingredient (the compound of formula I) is sufficient to significantly improve the condition without causing serious side effects.

 In the present invention, the catalytic enzyme CrtN refers to a protein having homology to the catalytic enzyme CrtN from the dehydrosqualene desaturase of the golden yellow pigment synthesis pathway in S. aureus. In another preferred embodiment, the protein having homology to the catalytic enzyme CrtN refers to a protein having a certain similarity (homology greater than 30%) to the S. aureus catalytic enzyme CrtN. In another preferred embodiment, proteins having a sequence homology greater than 30% are generally considered to be advanced from the same ancestor. In another preferred embodiment, the catalytic enzyme CrtN may also refer to a protein having the same or similar function as the S. aureus CrtN protein.

 In the present invention, the dehydrosqualene desaturase in the S. aureus golden yellow pigment synthesis pathway

(dehydrosqualene desaturase), capable of continuously catalyzing a three-step dehydrogenation reaction to introduce three double bonds in the 4,4'-diapo phytoene, which in turn form 4,4'-didehexamethylene 4,4'-diapophytofluene, 4,4'-dipo- _Z eta-carotene and 4,4'-didecoa Oxidoreductase of 4,4'-diaponeurosporene. Among them, 4,4'-dideoxysporine (4,4'-diaponeurosporene) is the main C _3Q carotenoid in staphylococci, which is golden yellow and can be further modified to form the main gold in staphylococci. Yellow pigment (staphyloxanthin). The enzyme requires FAD as a cofactor to participate in the reaction, and its catalytic properties are as follows: 15-cis-4,4'-didehydro phytoene (15-cis-4,4'-diapophytoene) + 4 FAD = all-trans-4,4'-bias lycopene (all-trans-4,4'-diapolycopene) + 4 FADH2.

 In the present invention, the bacterium comprises a Gram-positive bacterium, and in another preferred embodiment, the bacterium comprises Staphylococcus aureus. The bacteria also include other bacteria that produce carotenoids by the catalytic enzyme CrtN and its homologous protein.

 The present inventors have found for the first time to inhibit the synthesis of golden yellow pigment by inhibiting the expression and/or function of the key catalytic enzyme CrtN in the golden yellow pigment synthesis pathway, thereby reducing the pathogenicity of bacteria. The key catalytic enzyme CrtN in the golden pigment synthesis pathway can be used as a drug target, and compounds capable of inhibiting the expression and/or function of the catalytic enzyme CrtN can be used for the preparation of antibacterial drugs.

The present invention finds for the first time that naftifine hydrochloride and its derivatives can strongly inhibit the synthesis of Staphylococcus aureus golden yellow pigment by inhibiting CrtN, a key catalytic enzyme in the golden yellow pigment synthesis pathway. In the mouse subcutaneous and systemic infection models, the pathogenicity of the crtN gene mutant was found to be significantly reduced by 500-5000 times, confirming that CrtN is a new drug target against S. aureus virulence. In animal infection experiments, naftifine hydrochloride (Compound 1) was found to significantly reduce the colonization of S. aureus (Newman) in the kidney, heart, and liver of mice, and to reduce drug-resistant Staphylococcus aureus Mu50 (A) Colonization of oxycillin and vancomycin in the heart and liver of mice, reducing the colonization of drug-resistant Staphylococcus aureus USA400 (methicillin-resistant) in the heart of mice and in the kidney . In response to a lethal dose of S. aureus (Newman, USA400) infection, naftifine hydrochloride (Compound 1) significantly prolonged the survival of mice with similar efficacy to vancomycin. The above results indicate that the four compounds of the present invention can be developed into antibacterial drugs for new uses. In vivo studies have confirmed that naftifine hydrochloride and its derivatives have clear efficacy against methicillin-sensitive Staphylococcus aureus (Newman) and methicillin-resistant Staphylococcus aureus (Mu50, USA400). In the future, further design and development of new anti-bacterial infection drugs laid the structural and theoretical basis. The naftifine hydrochloride and its derivative of the invention can be used as an inhibitor of the catalytic enzyme CrtN, and can strongly inhibit the synthesis of golden yellow pigment, thereby reducing the pathogenicity of Staphylococcus aureus, and can be used for preparing antibacterial drugs, especially Preparation of a drug against Staphylococcus aureus infection. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, it will not be repeated here. DRAWINGS

 Fig. 1 is a diagram showing the synthesis of a yellow pigment in the compound 1 of the present invention. The concentration of the compound 1 from left to right is 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ, 0 μΜ.

 Fig. 2 is a diagram showing the synthesis of a yellow pigment in the compound 2 of the present invention, and the concentration of the compound 2 from left to right is 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ, respectively.

 Figure 3 is a diagram showing the synthesis of a yellow pigment in the compound 3 of the present invention, and the concentration of the compound 3 from left to right is 50 μM in this order.

10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ.

 Fig. 4 is a diagram showing the synthesis of a yellow pigment in the compound 4 of the present invention, and the concentration of the compound 4 from left to right is 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ, respectively.

 Fig. 5 is a graph showing the dose-effect relationship of Compound 1 for inhibiting the synthesis of golden yellow pigment.

 Fig. 6 is a graph showing the dose-effect relationship of Compound 2 for inhibiting the synthesis of golden yellow pigment.

 Figure 7 is a graph showing the dose-effect relationship of Compound 3 inhibiting the synthesis of golden yellow pigment.

 Figure 8 is a graph showing the dose-effect relationship of Compound 4 for inhibiting the synthesis of golden yellow pigment.

 Figure 9 is a HPLC analysis of the intermediate metabolites of the golden yellow pigment synthesis pathway.

 Figure 10 is a HPLC analysis of metabolites overexpressing crtM in E. coli, E. coli, and overexpressing crtMN in E. coli.

 Figure 11 is a diagram showing the inhibition of the synthesis of golden pigments, wherein (A) from left to right: crtN overexpressing strain, wild type strain, ispA overexpressing strain; (B) from left to right: crtN overexpressing strain + compound 1 (The final concentration is 10 μΜ, 1 μΜ,

0 μΜ), and wild-type strain; (C) skin to right: ispA overexpressing strain + compound 1 (final concentration is 10 μΜ,

1 μΜ, 0 μΜ), as well as wild-type strains.

 Figure 12 is a graph showing the results of Coomassie blue staining of CrtN protein (size: 56.7 Kd) by thermolysin. M: marker protein (Marker); a: CrtN control (no compound 1 and thermolysin); b: compound l: CrtN=672: l (m/m); c: compound l: CrtN=336: l (m/m); d: Compound l: CrtN = 168: 1 (m/m); e: Compound l: CrtN=84: l (m/m); f: Compound l: CrtN=42: l (m /m); g: CrtN control (; no compound 1, plus thermolysin).

 Figure 13 shows a significant reduction in the subcutaneous virulence of S. aureus caused by inactivation of the crtN gene.

 Figure 14 is a pharmacodynamic diagram of Compound 1 against Staphylococcus aureus Newman in a systemic mouse infection experiment.

 Figure 15 shows a significant decrease in the virulence of the S. aureus system caused by the inactivation of the crtN gene.

 Figure 16 is a graph showing the pharmacodynamics of Compound 1 against drug-resistant Staphylococcus aureus Mu50 in a system-infected mouse experiment.

 Figure 17 is a graph showing the survival time of Compound 1 prolonged infection with S. aureus Newman in a system-infected mouse experiment.

 Figure 18 is a pharmacodynamic diagram of Compound 1 against S. aureus USA400 in a systemic mouse infection experiment.

Figure 19 is a graph showing the survival time of Compound 1 prolonged infection with S. aureus USA400 in a system-infected mouse experiment. Figure 20 is a graph showing the pharmacodynamics of Compound 2 against drug-resistant Staphylococcus aureus Mu50 in a system-infected mouse experiment. detailed description

 The inventors of the present application have extensively and intensively studied, and for the first time, unexpectedly discovered that naftifine hydrochloride and its derivatives can inhibit the synthesis of golden yellow pigment by inhibiting the key catalytic enzyme CrtN, thereby reducing the pathogenicity of Staphylococcus aureus, and can be used for Preparation of antibacterial drugs, especially for the preparation of anti-S. aureus drugs. On the basis of this, the present invention has been completed.

 the term

 C1-C6 thiol refers to a straight or branched fluorenyl group having 1 to 6 carbon atoms, such as hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. Butyl or tert-butyl and the like.

 The C6-C10 aryl group means an aromatic ring group having 6 to 10 carbon atoms such as a phenyl group, a naphthyl group or the like.

 Naftifine hydrochloride

 The chemical name is (£)-N-methyl (naphthalene-1-yl), and the structure is as follows:

 Naftifine hydrochloride was marketed in Germany, Austria, Malaysia and Singapore in 1985 under the trade name ExoderiL as a representative of allylamine ergosterol synthesis inhibitors. Naftifine is a highly effective, low toxicity topical antifungal. medicine. There have been no reports of anti-bacterial use of naftifine hydrochloride.

 a compound of formula I or a pharmaceutically acceptable salt thereof

The compound of formula I in the present invention means having

 Wherein Ar is a C6-C10 aryl group, a C1-C6 fluorenyl substituted C6-C10 aryl group.

其中， R₂独立地为 C1-C6垸基或氢； 或者 、 R₂与相邻的碳原子共同形成 C6-C10芳基。 在另一优选例中， 为。2-。6垸基， R₂为氢； 或 Ri、 R₂与相邻的碳原子共同形成 C6-C10芳基。 在另一优选例中， 为。3-。5垸基， R₂ 为氢； 或 Ri、 R₂与相邻的碳原子共同形成苯环。 In another preferred embodiment, the pharmaceutically acceptable salt is a hydrochloride salt. In another preferred embodiment, Ar is a phenyl group substituted with a phenyl group, a naphthyl group, or a C1-C6 fluorenyl group. In another preferred embodiment, Ar is a naphthyl group, and a C2-C6 fluorenyl group is substituted in another preferred embodiment, Ar

Wherein R _{2 is} independently C1-C6 fluorenyl or hydrogen; or R ₂ together with an adjacent carbon atom forms a C6-C10 aryl group. In another preferred embodiment, it is. 2-. 6 fluorenyl, R ₂ is hydrogen; or Ri, R ₂ together with adjacent carbon atoms form a C6-C10 aryl group. In another preferred embodiment, it is. 3-. 5 fluorenyl, R ₂ is hydrogen; or Ri, R ₂ together with adjacent carbon atoms form a benzene ring.

In another preferred embodiment, it is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and R ₂ is hydrogen.

In another preferred embodiment, the hydrochloride salt of the compound of formula I is selected from the group consisting of:

 Preparation

 A method of preparing a compound of formula I of the present invention or a pharmaceutically acceptable salt thereof, comprising the steps of:

 II ΠΙ

(a) reacting a compound of formula II with a compound of formula III to form a compound of formula I; and optionally

(b) a step of producing a hydrochloride salt of a compound of formula I from a compound of formula I,

Formulas, Ar is a C6-C10 aryl group, C1-C6 alkyl with the substituted C6-C10 aryl group _t

其中， R₂独立地为 C1-C6垸基或氢； 或者 、 R₂与相邻的碳原子共同形成 C6-C10芳基。 In another preferred embodiment, Ar, C1-C6 fluorenyl substituted phenyl. In another preferred example,

Wherein R _{2 is} independently C1-C6 fluorenyl or hydrogen; or R ₂ together with an adjacent carbon atom forms a C6-C10 aryl group.

In another preferred embodiment, it is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and R ₂ is hydrogen.

In another preferred embodiment, a compound of formula II is employed:

 II

 Reaction of 1-(chloromethyl)naphthalene with methylamine to give a compound of formula II: N-methyl-naphthalene-1-methylamine c

In another preferred embodiment, the compound of formula III is synthesized using the following steps: Ar,, Br

V IV HI

 (i) a compound of formula V is subjected to a reduction reaction to give a compound of formula IV;

 (ii) The compound of formula IV is subjected to a halogenation reaction to give a compound of formula III.

 In another preferred embodiment, the preparation method of the four compounds of naftifine hydrochloride (1) and derivatives thereof (2), (3), and (4) includes the following

盐酸萘替芬（1)和衍生物（2)、 （3)、 （4)

Naftifine hydrochloride (1) and derivatives (2), (3), (4)

 Wherein Ar is phenyl (1), 4-tolyl (2), 4-tert-butylphenyl (3) and naphthalene-2 (4).

 1) An aqueous solution of methylamine is dissolved in tetrahydrofuran, and then a solution of 1-(chloromethyl)naphthalene in tetrahydrofuran is slowly added dropwise to the reaction system, and reacted at 20 to 30 ° C for 10 20 hours. After completion of the reaction, concentration and column chromatography gave the intermediate N-methyl-naphthalene-1-methylamine (intermediate 11).

 2) E)-3-Ar-Acrolein CV) is dissolved in methanol, and sodium borohydride is added in portions under ice bath, and reacted at 20 to 30 ° C for 10 30 minutes. After concentration, water was added, and the residue was evaporated, evaporated, evaporated, evaporated

 3) Dissolve Intermediate IV in anhydrous ether, nitrogen-protected ice bath, add phosphorus tribromide, and react at 20~30 °C for 10~20 hours. After the reaction was completed, the reaction mixture was poured into EtOAc EtOAc (EtOAc) Ar-3-bromo-propene (intermediate 111).

 4) Add intermediate II, intermediate III, potassium carbonate to N,N-dimethylformamide, and react at 20~30 °C for 10~20 hours. After the completion of the reaction, water was added to the reaction mixture, and the mixture was extracted with EtOAc EtOAc (EtOAc)EtOAc. 1-yl)-3-Ar-prop-2-en-1-amine (1).

5) Add (£)-N-methyl-N-(naphthalen-1-yl;)-3-Ar-prop-2-en-1-amine I to ethyl acetate, and introduce homemade hydrogen chloride gas. 1 to 5 minutes, distillate the solvent under reduced pressure, and add 1/100 petroleum ether/ethyl acetate mixed solvent to the residue to precipitate The solid, suction filtration and washing afforded compound (£)-N-methyl(naphthalen-1-yl)-3-Ar-prop-2-en-1-amine hydrochloride (1-4). application

 The compound of the formula I of the present invention can be used for the preparation of an antibacterial agent; for the preparation of a catalytic enzyme CrtN inhibitor; or for the preparation of a medicament for inhibiting the synthesis of S. aureus golden yellow pigment.

 The antibacterial agent is a drug against Staphylococcus aureus, and the compound of the formula I of the present invention can reduce the pathogenicity of Staphylococcus aureus.

 The antibacterial agent is a drug against Staphylococcus aureus infection.

 The S. aureus is resistant to methicillin-sensitive Staphylococcus aureus (Newman) or methicillin-resistant Staphylococcus aureus (Mu50, USA400).

 In another preferred embodiment, the synthesis is resistant to S. aureus by inhibiting the synthesis of S. aureus golden yellow pigment. Pharmaceutical composition

 The present invention provides an antibacterial pharmaceutical composition, a pharmaceutical composition for inhibiting the catalytic enzyme CrtN or a pharmaceutical composition for inhibiting the synthesis of Staphylococcus aureus golden pigment.

 The pharmaceutical composition of the present invention comprises the compound of the formula I or a pharmaceutically acceptable salt thereof as an active ingredient; and a pharmaceutically acceptable carrier.

 Usually, the pharmaceutical composition contains from 1 to 2000 mg of the active ingredient per agent, more preferably from 10 to 200 mg of the active ingredient per agent. Preferably, the "one dose" is a tablet.

 "Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By "compatibility" it is meant herein that the components of the composition are compatible with the active ingredients of the present invention and with respect to each other without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid). , magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), moist Wet agents (such as sodium decyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

 In another preferred embodiment, the compound of the formula I of the present invention can form a complex with a macromolecular compound or a polymer by non-bonding. In another preferred embodiment, the compound of the formula I of the present invention can be linked as a small molecule to a macromolecular compound or a polymer by a chemical bond. The macromolecular compound may be a biological macromolecule such as a polysaccharide, a protein, a nucleic acid, a polypeptide or the like.

 The administration form of the active ingredient or the pharmaceutical composition of the present invention is not particularly limited, and representative administration forms include, but are not limited to, oral, intratumoral, rectal, gastrointestinal (intravenous, intramuscular or subcutaneous) and the like.

 Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) moisturizing An agent, for example, glycerin; (d) a disintegrant, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; f) absorption accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc , calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, Or a mixture thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.

 The solid dosage forms can also be prepared with coatings and shell materials, such as casings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials.

 Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances. In addition to these inert diluents, the compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.

 In addition to the active ingredient, the suspension may contain a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan ester, microcrystalline cellulose, aluminum methoxide and agar or a mixture of these and the like.

 Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and sterile powder for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

 The compounds of the invention may be administered alone or in combination with other therapeutic agents.

 When a pharmaceutical composition is used, a safe and effective amount of a compound of the invention is applied to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight, The dose to be administered is usually from 1 to 2000 mg, preferably from 20 to 500 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

 The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, may be arbitrarily combined. All of the features disclosed in this specification can be used in combination with any of the compositions, and the various features disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Therefore, the features disclosed are only general examples of equal or similar features, unless otherwise stated.

 The invention is advantageous in that:

 (1) The present invention provides a naftifine hydrochloride derivative having a novel structure.

 (2) The present invention provides a preparation method of naftifine hydrochloride and a derivative thereof, which is simple and efficient.

 (3) The present invention has found for the first time a new use of naftifine hydrochloride and its derivatives, which can be used for the preparation of antibacterial drugs, particularly for the preparation of anti-S. aureus drugs.

 The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.

将 10毫升 20%甲胺的水溶液溶于 20毫升四氢呋喃中， 然后向反应体系中缓慢滴加 3 克 1- (氯甲基)萘的 10毫升四氢呋喃溶液， 室温反应过夜。 反应结束后， 浓缩， 柱层析得到 标题化合物 (中间体 11)， 2.2克黄色油状物， 收率 75%。 Example 1 N-Methyl-naphthalene-1-methylamine (Intermediate II)

10 ml of a 20% aqueous solution of methylamine was dissolved in 20 ml of tetrahydrofuran, and then a solution of 3 g of 1-(chloromethyl)naphthalene in 10 ml of tetrahydrofuran was slowly added dropwise to the reaction system, and the mixture was allowed to react at room temperature overnight. After completion of the reaction, the title compound was obtained from mjjjjjjj

 1H-NMR (400 MHz, acetone) 8.23 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.48 (ddd, J = 22.4, 14.0, 7.1 Hz, 4H), 4.17 (s, 2H), 2.46 (s, 3H).

Example 2 Preparation of (E)-3-(4-methylphenyl)-propane-2.

 100 mg of (£)-3-(4-methylphenyl)-propenal was dissolved in 10 ml of methanol, and 26 mg of sodium borohydride was added portionwise in an ice bath, and reacted at room temperature for 15 minutes. After concentrating, the residue was evaporated. Directly cast the next reaction.

Example 3 Preparation of (E)-l-(4-methylphenyl)-3-bromo I-1)

 370 mg of the intermediate IV-1 was dissolved in 20 ml of anhydrous diethyl ether, and under ice-protected ice bath, 85 μl of phosphorus tribromide was added and allowed to react at room temperature overnight. After the reaction, the reaction mixture was poured into EtOAc EtOAc EtOAc. The yield was 85%.

1H-NMR (400 MHz, CDC1 ₃ ) δ 7.27 (t, J = 7.4 Hz, 2H), 7.13 (d, J = 7.4 Hz, 2H), 6.61 (d, J = 15.6 Hz, 1H), 6.34 (dt , J = 15.6, 7.8 Hz, 1H), 4.16 (d, J = 7.7 Hz, 2H), 2.34 (s, 3H).

 Example 4

(E)-N-methyl-AL (naphthalene small group)_ ₃ _( ₄ _tolyl)-prop-2-en-1-amine (compound 1-1) and (E)-N-methyl ( Naphthalen-1-yl) _ ₃ _( ₄ _ A

 100 mg of the intermediate II, 105 mg of the intermediate 111-1, 83 mg of potassium carbonate were added to 10 ml of N,N-dimethylformamide and allowed to react at room temperature overnight. After the reaction, water was added to the reaction mixture, and the mixture was evaporated. Color oil, yield 73%.

 For purification, compound 1-1 was dissolved in 1 ml of ethyl acetate, and hydrogen chloride gas was introduced for 1 minute to prepare a hydrochloride salt, and the solvent was evaporated to dryness. Solvent, white hydrochloride solid was precipitated, suction filtered, and washed to give Compound 2. iH-MR is its hydrochloride form data.

 1H-NMR (400 MHz, MeOD) δ 8.20 (d, J = 8.6 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.04 (d, J

= 7.6 Hz, 1H), 7.78 (d, J = 6.3 Hz, 1H), 7.73-7.59 (m, 3H), 7.43 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H ), 6.95 (d, J = 15.8 Hz, 1H), 6.37 (dt, J = 15.5, 7.5 Hz, 1H), 5.08 (d, J = 13.6 Hz, 1H), 4.74 (d, J = 13.6 Hz, 1H ), 4.10 (ddd, J = 36.9, 13.3, 7.8 Hz, 2H), 2.87 (s, 3H), 2.37 (s, 3H).; MS (ESI) m/z 302.0 [M+H]+. Example 5

 Preparation of (E)-N-methyl-N-(naphthalen-1-yl)-3-(4-tert-salt (3)

 In addition to replacing (3-(4-methylphenyl)-propenal with (£ 3-(4-tert-butylphenyl)-propenal, the remaining starting materials, reagents and preparation methods are the same as in Examples 1-4, 113 mg of white solid hydrochloride (Compound 3) was obtained in a yield of 79%.

 1H- MR (400 MHz, MeOD) δ 8.20 (d, J = 8.5 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 6.2 Hz, 1H), 7.74-7.59 (m, 3H), 7.52-7.42 (m, 4H), 6.96 (d, J = 15.8 Hz, 1H), 6.39 (dt, J = 15.4, 7.5 Hz, 1H ), 5.08 (d, J = 13.6 Hz, 1H), 4.74 (d, J = 13.5 Hz, 1H), 4.11 (ddd, J = 36.6, 13.3, 7.8 Hz, 2H), 2.88 (s, 3H), 1.35 (s, 9H).; MS (ESI) m/z 344.1 [M+H]+.

 Example 6

 Preparation of (E)-N-methyl-N-(naphthalen-1-yl)-3-(naphthalene-)

 In addition to replacing (^)-3-(4-methylphenyl)-propenal with (£)-3-(naphthalen-2-yl)-propenal, the remaining starting materials, reagents and preparation methods are the same as in Example 1. -4, 109 mg of the title compound m.

 H-NMR (400 MHz, MeOD) δ 8.24 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.3 Hz, 1H), 8.03 (d, J

= 8.1 Hz, 1H), 7.96-7.85 (m, 4H), 7.82 (d, J = 6.9 Hz, 1H), 7.76 (dd, J = 8.6, 1.3 Hz, 1H), 7.71 (dd, J = 1 1.1 , 4.1 Hz, 1H), 7.64 (t, J = 7.6 Hz, 2H), 7.58-7.44 (m, 2H), 7.15 (d, J = 15.8 Hz, 1H), 6.67-6.40 (m, 1H), 5.12 (d, J = 13.6 Hz, 1H), 4.80 (d, J = 13.6 Hz, 1H), 4.19 (ddd, J = 35.3, 13.2, 7.6 Hz, 2H), 2.92 (s, 3H).; MS (ESI ) m/z 338.0 [M+H]+.

 Example 7 Preparation of (E)-N-methyl-N-(naphthalen-1-yl) acid salt (1)

 Except that (£)-3-(4-methylphenyl)-propenal was replaced by (£)-3-phenyl-propenal, the remaining starting materials, reagents and preparation methods were the same as those in Examples 1-4, and 122 were obtained. Mg white solid hydrochloride the title compound, yield 80%.

1H-NMR (400 MHz, CDC1 ₃ ) δ 8.29 (d, J = 8.3 Hz, 1H), 7.81 (t, J = 8.5 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.57-7.34 (m, 6H), 7.29 (t, J = 7.5 Hz, 2H), 7.19 (dd, J = 13.1, 6.0 Hz, 1H), 6.56 (d, J = 15.9 Hz, 1H), 6.45-6.28 (m, 1H), 3.92 (s, 2H), 3.26 (d, J = 6.6 Hz, 2H), 2.26 (s, 3H).; MS (ESI) m/z 288.0 [M+H]+.

 Example 8 Compound 1-Compound 4 Initial screening experiment for inhibiting the activity of golden yellow pigment synthesis

Experimental strain: Freshly activated Staphylococcus aureus Newman wild strain (^top/^/ococc^ aureus subsp. aureus str. Newman, see "Duthie, ES, and LL Lorenz. 1952. Staphylococcal coagulase: Mode of action and antigenicity. J. Gen. Microbiol. 6:95-107") and its homologous crtN insertion mutant (no golden yellow pigment synthesis) (see Lan, LF, Cheng, A., Dunman, PM, Missiakas, D., He, C. Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus. J. Bacteriol. 2010, 192(12): 3068.).

 Experimental medium: Tryptone Soy broth (TSB), British Oxid product, prepared with distilled water, 121 ° C, sterilized for 15 minutes, ready for use.

 Primary screening test method:

 (1) Preparation of compound: The compound of the present invention was dissolved in dimethyl sulfoxide (DMSO) to prepare a mother liquor having a concentration of 10 mM. Dilute 100 μm of mother liquor with 400 μl of DMSO to a concentration of 2 mM. Mix well and take 250 μ (2 mM) of the solution and continue to add an equal amount of DMSO for 2 fold until the solution concentration is 0.0625 mM.

 (2) Culture of the strain: A Newman strain monoclonal was picked from the TSA plate to a test tube containing 4 mL of a sterile TSB medium, and cultured at 37 ° C, 250 rpm for 12 hours, and used.

 (3) Compound 1-Compound 4 Initial screening for inhibition of the synthesis of golden pigment in S. aureus: A sterile test tube was taken, and freshly sterilized TSB medium (3980 μί) was added to each tube. Subsequently, 20 prepared compound solutions having a concentration of 10 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM were separately added to the test tube to make the final concentration of the compound of the present invention 50 μΜ, 10, respectively. μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ. At the same time, 20 DMSO solution (final concentration 0.5%) was added to another tube as a negative control without compound. To each tube, add 40 broth for 12 hours (inoculation amount: medium = 1: 100), and after culturing at 37 ° C, 250 rpm for 24 hours, remove the bacterial solution 1.5 mL, centrifuge 14000 g 2 After a minute, the supernatant was removed and it was observed whether the synthetic golden yellow pigment had a significant decrease compared to the negative control after the addition of a specific concentration of the compound of the present invention.

 Compound 1-compound 4 inhibits the activity of gold pigment synthesis. The preliminary screening results are shown in Figures 1-4. 1 is the synthesis diagram of the compound 1 of the present invention for inhibiting the golden yellow pigment, and the concentration of the compound 1 from left to right is 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ, 0 μΜ; 2 is a diagram showing the synthesis of the inhibitory yellow pigment of the compound 2 of the present invention, and the concentration of the compound 2 from left to right is 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ, respectively; Compound 3 inhibited the synthesis of golden yellow pigment, and the concentration of compound 3 from left to right was 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ, respectively; The pigment synthesis map, the concentration of compound 4 from left to right was 50 μΜ, 10 μΜ, 5 μΜ, 2.5 μΜ, 1.25 μΜ, 0.625 μΜ, 0.3125 μΜ.

 The results showed that the addition of the compound 1 of the present invention to the medium at a final concentration of 50 μM, 10 μM and 5 μM completely inhibited the synthesis of the golden yellow pigment at a concentration of 2.5 μM and below, and the golden yellow pigment. The synthesis increased as the concentration of Compound 1 decreased, indicating that Compound 1 has a significant concentration dependence on the synthesis of golden yellow pigment. Compound 2, Compound 3 and Compound 4 were modified and modified on the basis of Compound 1, and the ability to inhibit the synthesis of golden pigment was significantly increased. Among them, compound 2 and compound 4 can completely inhibit the production of pigment at the lowest concentration of this experiment (0.3125 μΜ); compound 3 at the lowest concentration of this experiment (0.3125 μΜ), the synthetic golden yellow pigment has the same concentration of compound 1 A significant decrease indicates that Compound 2, Compound 3 and Compound 4 are significantly more active than Compound 1.

Example 9 IC _5e assay for inhibition of gold yellow pigment synthesis activity by Compound 1 - Compound 4 Selection of compound concentration: Based on the preliminary screening results, the ability of each compound to inhibit the synthesis of golden yellow pigment was determined. For compounds with strong activity, such as strong inhibition of pigment production at the lowest concentration of the primary screening, the experiment can be continued in a similar manner to the primary screening until the compound is substantially unable to inhibit the production of golden yellow pigment. According to the experimental results, 11 different concentration gradients were designed for each compound, and the ability to inhibit pigment synthesis basically included 0% to 100%.

 Culture of strain: Pick Newman strain and crtN mutant monoclonal from TSA plate to 4 mL sterile

The TSB medium was cultured in a test tube at 37 ° C, 250 rpm for 12 hours, and was used.

 1^ Determination: Take sterile tubes and add 3980 μί of freshly sterilized TSB medium to each tube. Subsequently, 20 μL of the formulated 11 concentration gradients of the compound of the invention were separately added to the test tube. At the same time, 20 μl of DMSO solution (final concentration 0.5%) was added to the other two tubes as a control without compound. To each of the two tubes to which the 20 ^ DMSO solution was added, 40 μL of Newman (negative control, no compound addition) and crtN mutant (positive control, no pigment production) were added for 12 hours. The remaining tubes of the compound were added to 40 tubes of Newman strain for 12 hours. All tubes were incubated at 37 ° C, 250 rpm for 12 hours, then switched to 30 ° C, and continued to incubate at 250 rpm for 36 hours to increase pigment accumulation. After the completion of the culture, take 3 mL of the bacterial solution in a 2 mL EP tube, centrifuge at 14000 g for 2 minutes, remove the supernatant, wash twice with PBS buffer (1 mL each time), add 300 methanol solution, and mix the mixture. The mixture was heated and heated in a 55 ° C water bath for 3 minutes to extract the pigment. After centrifugation at 14,000 g for 2 minutes, the methanol extract was pipetted into a 1.5 mL EP tube, and an equal amount of methanol solution was added thereto, and the extraction was repeated twice, and the three extracted pigments were combined. Using the methanol extract in the crtN mutant as a blank control, the absorbance values of each sample at 450 nm were measured, and the absorbance values of the compound-free negative control were determined.

 The relative levels of pigment synthesis at each concentration of the compounds of the invention = A450 nm (sample) / A450 nm (negative control) * 100%.

The molar concentration of the compound is plotted on the abscissa, and the relative level of pigment synthesis is plotted as the ordinate (ie A450 (sample) / A450 (negative control) * 100%). Inhibitor concentration - synthetic pigment level is performed in Graphpad prism 5.0 software. The curve fit of (log(inhibitor) vs response), and the IC _{50 of the} compound inhibiting pigment synthesis was calculated by the software based on the fitting result.

Table 1 Inhibitory activity data of compound (1-4) on golden yellow pigment synthesis (IC _5Q , nM)

IC ₅₀ (n

 Compound number

 M)

 1 731.6

 2 44.7

 3 173.7

 4 51.1

The dose-effect relationship curve of Compound 1 - Compound 4 for inhibiting the synthesis of golden yellow pigment is shown in Figures 5-8. According to the measurement results, the inhibitor concentration-pigment synthesis curve was fitted, and the correlation coefficients (R ² ) were 0.9939, 0.9854, 0.9844 and 0.9889, respectively, indicating that the concentration of the inhibitor has a high correlation with the level of pigment synthesis. From the experimental results, the four compounds have strong activity in inhibiting the synthesis of golden pigment, and the 95% value is used as the confidence interval. The IC ₅₀ ranges are: compound 1: 558.6 nM - 958.2 nM _; : 30.33 nM - 65.73 nM; Compound 3: 110.7 nM - 272.4 nM; Compound 4: 35.16 nM - 74.23 nM.

The activity data is shown in Table 1. According to the experimental results, the reference half effective inhibition concentration IC _5Q given by the software is 731.6 nM, 44.7 nM, 173.7 nM and 51.1 nM. The IC _{5Q of} Compound 1 was 16.4, 4.2 and 14.2 times that of Compound 2, Compound 3 and Compound 4, respectively.

Example 10 demonstrates that Compound 1 of the present invention inhibits the synthesis of golden pigment by inhibiting the extraction of the functional gene of the cr N gene and its intermediate metabolites: overnight culture of wild-type strain (Newman) crtM gene mutant strain (crtM, see Lan, LF, Cheng, A., Dunman, PM, Missiakas, D., He, C. Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus. J. Bacteriol. 2010, 192(12):3068.) crtN gene mutation Strains (crtN~, see Lan, LF, Cheng, A., Dunman, PM, Missiakas, D., He, C. Golden Pigment Production and Virulence Gene Expression Are Affected by Metabolisms in Staphylococcus aureus. J. Bacteriol. 2010, 192 (12): 3068.), the complementary strain of crtN gene mutant (crtNVcrtN) was transferred to 50 ml of fresh sterile TSB or compound 1 (10 μΜ) in a ratio of 1:100 (bacterial solution: medium). After incubation for 24 hours at 37 ° C, 250 rpm in TSB medium, the cells were collected by centrifugation at 8000 g for 4 min and washed twice with PBS buffer. Add 20 ml of acetone to the cells, vortex to extract the pigment and its intermediates, and then add 10 ml of hexamidine and 10 ml of NaCl (10%, mass/volume) solution to the extract and shake vigorously to remove The oil component in the extract is extracted, and then the ruthenium layer containing the pigment and its intermediate product is collected, and 10 ml of hexamidine is further added, and the extraction process is repeated once. The two extracts of hexane were combined and dried with anhydrous MgS0 ₄ . The sample was concentrated on a rotary evaporator and then dissolved in a solvent of 500 μΐ of pure acetonitrile-isopropyl alcohol (85:15) to dissolve the sample with 0.45 μm. The membrane was filtered to remove impurities. Detection by HPLC.

The construction method of the complementary cell ( _C rtN-/crtN) of the crtN gene mutant used was as follows: First, the construction of the complementary plasmid pYJ335: :crtN (for the detailed construction process, see Example 12). The difference is that the material is verified by sequencing, and after determining the abasic deletion and mutation, it needs to be transferred to the crtN gene mutant to obtain the complementary strain of crtN gene mutant (crtN7pYJ335: :crtN).

 HPLC conditions: column: Spherisorb ODS2 column (250 * 4.6 mm; 5 μιη particle size; Waters); mobile phase: acetonitrile-isopropanol (85: 15, volume/volume); flow rate: l ml/min; Sample size: 50μ1; liquid chromatograph: Agilent 1260 infinity C with diode array detector), detection wavelength: 286 nm.

 The metabolites of the golden pigment synthesis pathway in five samples were analyzed by HPLC (24-hour analysis results). The five samples were: wild type strain (Newman), crtM gene mutant strain (crtM), crtN gene mutant strain (crtN crtN gene mutant complement strain (crtNVcrtN) and compound 1 (10 μΜ) grown under culture conditions. Pigment intermediate metabolite of wild type strain (Newman+1).

The results showed (Fig. 9) that the HPLC profile of the gold-yellow pigment synthesis pathway metabolite in the wild-type strain grown under the culture condition containing Compound 1 (10 μΜ) was very similar to the HPLC profile of the crtN gene mutant (crtN"). Mutation and addition of Compound 1 resulted in the appearance of metabolites (detection wavelength 286 nm) with retention times of 10.8 minutes and 1 1.4 minutes, respectively. Functional complementation (crtN7 _crt N) experiments further confirmed that these two metabolites are involved in the function of crtN It is speculated that these two metabolites may be the catalytic products of CrtM, and the function of CrtN is inhibited and accumulated in large amounts.

 Example 11

 To verify that the compound of the present invention 1 inhibits the synthesis of golden yellow pigment by inhibiting the function of cr N gene

The crtM and crtMN were overexpressed in E. coli (.co/), and the metabolite changes were analyzed by HPLC. The protein expression vector pet28a and E. coli £.co/CDE3 were purchased from Invitrogen. Pet28a: :crtM/E.coli(OE3), pet28a:: crtMM£.co/ (DE3) The build process is as follows:

 Construction of the plasmid:

 According to the genomic sequence of the wild type strain Newman in NCBI, primers were designed to clone the crtM gene and the crtMN gene into the pet28a vector, respectively, to construct pet28a: :crtM and pet28a::crtMV.

 The primers (synthesized by Shanghai Jierui Bioengineering Co., Ltd.) are as follows:

 Pet28a-crt -F(Bam HI): CGCGGATCC ATGAC A ATGATGGAT ATGAATTTT AA(5 ' -3 ' ); pet28a-crt -R(Xho I):CCGCTCGAGCTATATTCTATGATATTTACTATTT(5'-3');

 Pet28a-crt Chef -F(Bam HI): CGCGGATCCATGACAATGATGGATATGAATTTTAA (5'-

3');

 Pet28a-crt kitchen -R(Xho I): CCGCTCGAGTT AT ACGCCCCGCTC AAT ATCTTTA (5 ' -3 '). The underlined portions are the cleavage sites of Bam HI and Xho I, respectively.

Construction and validation of the plasmid: Using the Newman genomic DNA as a template and pet28a-crtM-F/R and pet28a-crtMV-F/R as primers, the crtM and crtMN gene fragments were amplified by PCR. The PCR reaction was carried out according to the PrimeSTAR® HS DNA Polymerase instruction (; Bio-Engineering (Dalian) Co., Ltd.). The specifically amplified gene fragment was confirmed by 1% agarose gel electrophoresis, and the PCR product was recovered using a PCR product purification kit (Omega). They were taken after 2 _μ β PCR product was recovered (crtM and crtMV) and 2 g pet28a vector, each double digestion reaction Bam HI and Xho I at 50 μΐ system, the reaction conditions according to the conditions recommended by New England biolabs company. After the digestion reaction, the pet28a vector was selected for 0.8% agarose gel electrophoresis, and the uncleared pet28a vector was used as a control to confirm the successful completion of the digestion. The digested PCR product and the pet28a vector were purified by a kit (Omega), and the purified PCR product and the pet28a vector were mixed in a molar ratio (8:1 to 10:1) to a total volume of ΙΟμΙ. Contains T4 DNA ligase), ligated overnight at 16 °C. The ligation product and the pet28a empty vector were separately transformed into .co/(DE3) competent cells by thermal shock at 42 °C, and after resuscitation, applied to LB solid plate containing kanamycin (50μ _§ /ιη1). Above, and cultured overnight in a 37 °C incubator to obtain a single pet28a/£.co/ (DE3), pet28a: :crtM/E.coli (OE3), pet28a::crtMM£.co/ (DE3) clone. Pick up the monoclonal antibody produced on kanamycin (50 g/ml) LB solid plate for streak culture to amplify the amount of bacteria, and then pick a small number of cells for PCR colony verification. The primers used are primers used in plasmid construction. . The clones with positive PCR results were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing confirmation.

Extraction of pigments and their intermediate metabolites: overnight cultured pet28a/E.con{DET), pet28a: :crtM/E.coli(OE3), pet28a: :crtMN/E.co/ (DE3) by 1:100 ( The ratio of the bacterial solution: medium was transferred to 50 ml of fresh sterile LB + kanamycin kanamycin (final concentration: 50 g / ml) medium, and cultured at 37 ° C, 250 rpm for 24 hours, 8000 g The cells were collected by centrifugation at 4 min and washed twice with PBS buffer. Add 20 ml of acetone to the cells, vortex to extract the pigment and its intermediates, and then add 10 ml of hexamidine and 10 ml of NaCl (10%, mass/volume) solution to the extract and shake vigorously to remove The oil component in the extract is extracted, and then the ruthenium layer containing the pigment and its intermediate product is collected, and 10 ml of hexamidine is further added, and the extraction process is repeated once. Combine the two extracts of hexane and dry them with anhydrous MgS0 ₄ . Concentrate the sample on a rotary evaporator and add 500 μM of chromatographically pure acetonitrile-isopropyl alcohol (85:15) to dissolve the sample and use 0.45 μm. The membrane was filtered to remove impurities. Detection by HPLC. HPLC experimental conditions: Column: Spherisorb ODS2 column (250 * 4.6 mm; 5 μιη particle size; Waters); mobile phase: acetonitrile-isopropanol (85: 15, volume / volume;); flow rate: 1 ml / min; Injection volume: 50μ1; liquid Phase Chromatograph: Agilent 1260 infinity C with diode array detector), detection wavelength: 286 nm, 440 nm.

 The results (Fig. 10) showed that metabolites with retention times of 10.7 minutes and 11.3 minutes were apparent in metabolites of E. coli overexpressing crtM, and the two metabolites predicted in Example 10 may be synthetic products of CrtM. Consistent. In the metabolites of E. coli overexpressing crtMV, these two metabolites with retention times of 10.7 minutes and 11.3 minutes were significantly reduced compared to E. coli overexpressing crtM. However, a metabolite with a retention time of 6.2 minutes (detection wavelength of 440 nm) has appeared sharply. These results suggest that in E. coli overexpressing crtMN, the product of CrtM (retention time 10.7 minutes and 11.3 minutes, detection wavelength 286 nm) was further synthesized by CrtN to a retention time of 6.2 minutes (detection wavelength 440 nm). product.

 Example 12

 To verify that the compound of the present invention 1 inhibits the synthesis of golden yellow pigment by inhibiting the function of cr N gene

 Analysis of overexpression of crtN in S. aureus attenuates the inhibitory effect of Compound 1 on the production of golden yellow pigment.

 (1) Construction of cr gene overexpressing strain and ispA gene overexpressing strain:

 According to the genomic sequence of the wild-type strain Newman in NCBI, the primers were designed to clone the crtN gene and the ispA gene into the Escherichia coli-Staphylococcus aureus shuttle vector pYJ335 (see Ji, Y., A. Marra, M. Rosenberg, and G. Woodnutt. 1999. Regulated anti sense RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection. J. Bacteriol. 181 :6585-6590.), construct pYJ335 : :crtN and pYJ335::

 The primers (synthesized by Shanghai Jierui Bioengineering Co., Ltd.) are as follows:

 pYJ335- -F: AAAGAAGAAGCTGAGGATGTAAAAA (5 ' -3 ');

 pYJ335 -ispA -R: TTGCTTTT AGTGATCCCTGCT A(5 '-3');

 pYJ335 -crtN-F: T AAAT ATC AT AGAAT AT AGGTGGTTG (5 '-3');

 pYJ335-crtN-R: CCCTTAT ACTTTTCTC AC ATCT (5 ' -3 ').

pYJ335: :crtM£.co/ (DH5a)的单克隆。 挑 取含羧苄青霉素 (100μ_§/ιη1)的 LB 固体平板上生出的单克隆进行划线培养以扩增菌体量，而 后挑取少量菌体进行 PCR菌落验证，所用引物为 pYJ335载体通用引物: pYJ335-universal-F: C AAT AC AATGT AGGCTGCTCT AC AC(5 ' -3 ') 与 质 粒 构 建 时 所 用 的 反 向 弓 I 物 (pYJ335-/^-R或 pYJ335-crtN-R)， 以确保基因连接的方向正确。 将 PCR验证结果为阳性 的克隆送上海美吉生物医药科技有限公司进行测序确证。 Plasmid construction and validation: Using the Newman genomic DNA as a template, pYJ335-3⁄44-F/R and pYJ335-crtN-F/R were used as primers, and the ispA and crtN gene fragments were amplified by PCR. The PCR reaction was carried out according to the PrimeSTAR® HS DNA Polymerase instruction (Bao Bioengineering (Dalian) Co., Ltd.). The specifically amplified gene fragment was confirmed by 1% agarose gel electrophoresis, and the PCR product was recovered using a PCR product purification kit (Omega). The 2 μ _§ pYJ335 vector was used to carry out the single-digestion reaction of EcoR V in a 50 μΐ system. The reaction conditions were as recommended by New England biolabs. After the digestion reaction, the pYJ335 vector was selected for 0.8% agarose gel electrophoresis, and the pYJ335 vector which was not digested was used as a control to confirm the successful completion of the digestion. The PCR product and the digested pYJ335 vector were purified by a kit (Omega), and the purified PCR product and the pYJ335 carrier molar ratio (8:1 10:1) were added to the total volume of the ΙΟμΙ linkage system (including Τ4 DNA ligase), ligated overnight at 16 °C. At 42 ° C using the heat shock method to connect the product together PYJ335 empty vector were transformed into £. _C o / (DH5a) competent cells, after recovery, applied containing carbenicillin (100μ _§ / ιη1) of solid LB Plate and incubate overnight in a 37 °C incubator to obtain pYJ335/£.co//(DH5a) pYJ335:

pYJ335: A monoclonal clone of :crtM£.co/ (DH5a). The monoclonal clones on LB solid plates containing carbenicillin (100μ _§ /ιη1) were picked and streaked to amplify the amount of cells, and then a small number of cells were picked for PCR colony verification. The primers used were pYJ335 vector universal primers. : pYJ335-universal-F: C AAT AC AATGT AGGCTGCTCT AC AC (5 ' -3 ') and the reverse arch I used in the construction of the plasmid (pYJ335-/^-R or pYJ335-crtN-R) to ensure that the direction of gene connection is correct. The clones with positive PCR results were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing confirmation.

 The constructed clone was transformed into the competent form of E. coli DH5a by heat shock, and the monoclonal clone (100 g/ml) on the carbenicillin-resistant plate was picked and cultured overnight. The plasmid was extracted and verified by sequencing. After base deletion and mutation, electroporation into S. aureus Newman, finally obtained crtN gene overexpression strain (Newman/p YJ335:: crtN) and 'ψΑ gene overexpressing strain (Newman/p YJ335 :: ispA), The p YJ335 empty vector was electroporated into Newman strain (Newman/p YJ335) as a negative control.

(2) Culture of the strain and observation of the pigment: Newman strain (containing pYJ335 empty vector), crtN gene overexpressing strain and ispA gene overexpressing strain monoclonal were picked from TSA plate to 4 mL sterile TSB medium (including In a test tube with a final concentration of 10 μ _§ /ιη1 of erythromycin and chloramphenicol, cultured at 37 ° C, 250 rpm for 12 hours, and set aside.

(3) Take 7 sterile tubes, and add freshly sterilized TSB medium (containing erythromycin and chloramphenicol at a final concentration of 10 μ _§ /ιη1) to 3980 μί. Subsequently, 20 prepared compound solutions having a concentration of 2 mM and 0.2 mM were separately added to the test tubes to give the present compounds a final concentration of 10 μM and ΙμΜ (two tubes each). At the same time, 20 mL of a DMSO solution (final concentration of 0.5%) was added to the other three tubes to give a compound concentration of 0 μM. Add 40 μL of the crtN gene overexpressing broth and ispA gene overexpressing broth (inoculation amount: medium = 1: 100) (three each) to the test tubes of different concentrations of compounds, and Newman's solution containing pYJ335 empty vector was added to one tube. After incubating all the tubes at 37 ° C, 250 rpm for 24 hours, 1.5 mL of the bacterial solution was taken out, centrifuged at 14,000 g for 2 minutes, and then the supernatant was removed to observe the ability of the strain to synthesize a golden yellow pigment after adding a specific concentration of the compound of the present invention. Whether there is a significant change compared to the control.

 As shown in Figure 11, Compound 1 (10 μM) did not effectively inhibit the production of golden yellow pigment in crtN overexpressing strains. However, at the same concentration, Compound 1 (10 μΜ) can effectively inhibit pA (an enzyme encoding the enzyme responsible for the synthesis of golden pigments upstream of CrtN - geranyl transferase;) overexpression of the golden yellow pigment in the strain The production. It was further confirmed that the compound 1 inhibits the production of golden yellow pigment by inhibiting the activity of the CrtN protein.

 Example 13

 To verify that the compound of the present invention 1 inhibits the synthesis of golden yellow pigment by inhibiting the function of cr N gene

 CrtN pure protein can be obtained from pet28a: :crtN/£.co/ (DE3) by nickel column affinity chromatography and desalting purification. (1) Construction and cultivation of pet28a:: crtM£.co/ (DE3):

 According to the genomic sequence of the wild type strain Newman in NCBI, primers were designed to clone the CrtN gene into the protein expression vector pet28a to construct pet28a: :crtN, and pet28a::crtM/£.co/ (DE3) and pet28a in Reference Example 11: :crtMV7£.co/ (DE3) Construction of experimental steps and conditions and verification of pet28a: :crtN construction.

 The primers (synthesized by Shanghai Jierui Bioengineering Co., Ltd.) are as follows:

 pet28a-crtN-F(Bam HI): CGCGGATCCATGAAGATTGCAGTAATTGGTGCAG;

 pet28a-crtN-R (Xho I): CCGCTCGAGTTATACGCCCCGCTCAATATCTTTA, underlined are the cleavage sites of Bam HI and Xho I, respectively.

The constructed clone was transformed into E. coli DH5a competent state by heat shock, and a single clone of kanamycin-resistant plate (50 g/ml) was picked and cultured overnight, and the plasmid was extracted. The material was verified by sequencing. After abasic deletion and mutation, it was transformed into the protein expression strain £.co/i(DE3) to obtain lj pet28a: '.crtNI E. co//(DE3). Pick pet28a: :crtM£.co/ CDE3;) Monoclonal to 10ml LB medium (with kanamycin at a final concentration of 50 g/ml;), 37 . C, 250 rpm overnight culture, transfer to 1L LB medium (containing the same concentration of kanamycin), culture at 37 ° C for about 3 hours to OD of about 0.5, the temperature is reduced to 16 ° C, add different After propyl-β-D-thiogalactopyranoside (IPTG, final concentration of 1 mM) was induced to express protein for 16 hours, the protein-containing cells were collected by centrifugation.

 (2) Purification of CrtN protein:

 Buffer buffer used:

 Buffer A: 50 mM Tris-HCl, 200 mM NaCl, 2 mM DTT, 50 mM imidazole, pH 8.0

 Buffer B: 50 mM Tris-HCl, 200 mM NaCl, 500 mM imidazole, 2 mM DTT, pH 8.0

 Lysis buffer: buffer A+5% glycerol

 The following operations were performed at 4 ° C or on ice. The collected 1L cells were added with 30 ml of lysis buffer, crushed on a sonicator for 30 minutes, 25000 g, centrifuged for 25 minutes, and the supernatant was taken. The supernatant containing the CrtN protein was pumped to a pre-packed nickel affinity column (5*5 ml, HistrapTM HP, GE) using a peristaltic pump. The completed nickel column was connected to an AKTA protein purification system for purification. Purification conditions: bufferA balance 5-6 column volumes, linear gradient elution (100% buffer A, 0% buffer B to 0% buffer A, 100% buffer B), flow rate 3ml / min, total elution time is about 45 minutes, detection wavelength 280 nm. 2 ml of the eluted CrtN protein was collected and directly subjected to desalting, desalting column (100*5 ml, Histrap desalting, GE), and the desalting buffer was buffer A without imidazole. Purification conditions: Desalting buffer balance After 5-6 column volumes, desalting purification was carried out at a flow rate of 1.5 ml/min and a detection wavelength of 280 nm. The protein after the desalting purification is used for the enzyme digestion reaction.

 The conditions of the digestion reaction were as follows: Reaction buffer: citric acid-sodium citrate (50 mM), sodium chloride (150 mM), dithiothreitol (2.0 mM), pH 5.5. The protein CrtN concentration was 0.337 mg/ml, which was about 5.95 μΜ. Thermolysin: CrtN = 1:600 (m/m), digested for 30 minutes at room temperature.

 Figure 12 is a graph showing the results of Coomassie blue staining of CrtN protein (size: 56.7 Kd) digested with thermolysin. Wherein, M: protein Marker; a: CrtN control (no compound 1 and thermolysin); b: compound l: CrtN = 672: l (m/m); c: compound 1: CrtN = 336: l (m/m d: Compound l: CrtN = 168: 1 (m/m); e: Compound l: CrtN = 84: l (m/m); f: Compound l: CrtN = 42: l (m/m); g: CrtN control (no compound 1, plus thermolysin). The final concentrations of Compound 1 in b~g were: 4 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0 mM. The results of restriction endonuclease analysis of CrtN pure protein showed that: Compound 1 can protect CrtN protein from degradation by thermophilic protease thermolysin, indicating that compound 1 binds to CrtN and protects the cleavage site in CrtN. Point, thus directly demonstrating the interaction of Compound 1 with the CrtN protein.

 Compound 2, Compound 3, and Compound 4 were verified by the methods of Examples 10-13. The results showed that Compound 2, Compound 3, and Compound 4 also inhibited the synthesis of golden yellow pigment by inhibiting the function of the crtN gene.

 Example 14 Mutation of the cr N gene results in a significant decrease in the pathogenicity of S. aureus

 Experimental SPF mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. under sterile conditions. In the subcutaneous infection experiment in mice, female CD-I mice of 10-14 weeks old were randomly divided into two groups of 10 animals each. All mice were cut off with hair clippers on both sides of the back two days before infection.

 The overnight cultured Staphylococcus aureus (Newman) strain and its crtN mutant were transferred to fresh sterile tryptone Soy broth (TSB) and cultured for 3 hours to exponential growth phase. After washing twice with PBS buffer, it was suspended in PBS for use.

The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg), and then injected subcutaneously on both sides of the hair removal. Lxl O ⁸ CFU Newman strain and its crtN mutant strain were used to infect mice. Five days after infection, the mice were sacrificed by inhalation of CO ₂ . The area of the mouse subcutaneous abscess was removed and homogenized uniformly in 1 mL of sterile PBS buffer (containing 0.01% triton X-100). The homogenate was serially diluted and 10 μL of the diluted droplets were taken onto a TSA plate to measure the bacterial CFU count. The number of bacteria colonized under the skin was quantified by bacterial CFU counts in the homogenate.

 In the mouse subcutaneous infection model, the pathogenicity of the crtN gene-mutated S. aureus strain decreased significantly (Fig.

13). Wild-type Staphylococcus aureus (Newman) causes subcutaneous abscesses, while mutants of the crtN gene lose their ability to cause subcutaneous abscesses (Fig. 13A). The experimental results of the bacterial count showed that the viability of the bacteria in the mice decreased by about 1000 times due to the mutation of the crtN gene (Fig. 13B). These results indicate that CrtN, a key catalytic enzyme in the S. aureus golden yellow pigment synthesis pathway, can be used as an antibacterial target.

 Example 15 Compound 1 (naphtholidine hydrochloride) against Staphylococcus aureus

The experimental SPF mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. under sterile conditions. The overnight cultured Staphylococcus aureus (Newman) strain was transferred to fresh sterile tryptone Soy broth (TSB) and cultured for 3 hours to exponential growth phase. After washing twice with PBS buffer, it was suspended in PBS for use. In a systemic mouse infection experiment, 6-8 week old female BALB/c mice were randomized into two groups of 15 each. All mice by intraperitoneal injection of sodium pentobarbital (80mg / kg) anesthesia, and then injected about retroorbital 1.5><10 ⁶ CFU of strain Newman infected mice. For the compound 1 (naftotifen hydrochloride) treatment group, the mice were intraperitoneally injected with 16 mg/kg of Compound 1 each time, the first time 12 hours before the bacterial infection, and 8 times within 4 days after infection (2 times a day, 9 times in total). After the experiment, the mice were inhaled. 0 ₂ was executed. The heart, kidney and liver of the mice were removed and uniformly disrupted in 1 mL of sterile PBS buffer (containing 0.01% triton X-100). The crushed solution was serially diluted, and 10 dilutions of the droplets were taken onto the TSA plate to measure the bacterial CFU count. The degree of colonization of bacteria in different organs is quantified by bacterial CFU counts in the disrupted fluid of different organs. The results of the experiment (Fig. 14) showed that Compound 1 (naphtholidine hydrochloride) significantly reduced the colonization of S. aureus (Newman) in the kidney, heart, and liver of mice, showing potent anti-S. aureus Drug effect.

 Example 16 Mutation of the cr N gene results in a significant decrease in the pathogenicity of S. aureus

All experimental methods and materials as in Example 15, except that the strain Newman 1.5 X 10 ⁶ CFU of strain Newman was respectively replaced by 3 xl 0 ⁶ CFU and 3 xl 0 ⁶ CFU of the mutant strain crtN, each group of mice a further 15 Only 12 were reduced, and neither group of mice were given compound treatment.

 In the animal infection system experiment, the pathogenicity of the crtN gene-mutated S. aureus strain to mice was significantly reduced. Compared to the wild-type strain (Newman), the number of bacteria isolated from the kidney and heart of mice infected with the crtN mutant strain was significantly less than that of the wild-type strain under the same dose of S. aureus (Fig. 15A). Visual observation of the degree of bacterial colonization in the kidneys of mice revealed that a large number of wild-type strains (Newman) were colonized on mouse kidneys (Fig. 15B), whereas no obvious renal colonization was observed in the crtN mutant strain (Fig. 15C). ). These results indicate that CrtN, a key catalytic enzyme in the golden yellow pigment synthesis pathway of Staphylococcus aureus, is a key virulence factor affecting bacterial pathogenicity and can be used as an antibacterial target.

 Example 17 Compound 1 (naphtholidine hydrochloride) against Staphylococcus aureus

All experimental methods and materials as in Example 15, except replacing strain Newman 1.5 xl 0 ⁶ CFU into

1.5 X 10 ⁷ CFU of Mu50 strain (methicillin-resistant, vancomycin-resistant, purchased from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), another The number of mice in the group was reduced from 15 to 13. The experimental results (Fig. 16A) indicate that Compound 1 (naphtholidine hydrochloride) significantly reduced the colonization of drug-resistant Staphylococcus aureus Mu50 in the mouse heart and in the liver. Visual observation of the degree of bacterial colonization in the liver of mice revealed that a large amount of drug-resistant Staphylococcus aureus Mu50 was colonized on the liver of mice without Compound 1 (Fig. 16B), whereas mouse liver supplemented with Compound 1 No obvious bacterial colonization was observed on (Figure 16C). It shows that Compound 1 has potent anti-resistance to Staphylococcus aureus.

 Example 18 Compound 1 (naphtholidine hydrochloride) against Staphylococcus aureus

All the experimental methods and materials similar to Example 15, except replacing strain Newman 1.5 x l0 ⁶ CFU of strain Newman to 1.5 X 10 ⁷ CFU's. For the compound 1 (naftotifen hydrochloride) treatment group, the mice were intraperitoneally injected with 16 mg/kg of Compound 1 each time, the first time 12 hours before the bacterial infection, and 12 times within 6 days after the infection (2 times a day, A total of 13 times). For the vancomycin hydrochloride (positive control) treatment group, the mice were intraperitoneally injected with 25 mg/kg vancomycin hydrochloride for the first time, 12 hours before the bacterial infection, and 12 times within 6 days after the infection (2 times a day). , a total of 13 times). For the negative control group (no compound treatment group;), the mice were intraperitoneally injected with 100 μΐ sterile saline each time, the first time 12 hours before the bacterial infection, and 12 times within 6 days after the infection (2 times a day, total 13 times). The number of deaths of the mice after infection was recorded daily, and the end of the day was recorded on the 12th day, and the survival curve was drawn within 12 days.

The results (FIG. 17) shows that, strain Newman 1.5 x l0 ⁷ CFU of lethal dose in mice without drug group, all mice died within 1 infection. For the lethal dose of Staphylococcus aureus (Newman) infection, although the efficacy was slightly worse than the positive control vancomycin hydrochloride (protection rate = 100% in 12 days), Compound 1 (naftotifen hydrochloride) significantly prolonged the mice Survival time (protection rate >70% in 12 days). It is indicated that Compound 1 has potent anti-S. aureus efficacy.

 Compound 2, Compound 3, and Compound 4 were examined by the methods of Example 15, Example 17, and Example 18. The results showed that Compound 2, Compound 3, and Compound 4 also showed potent anti-S. aureus drugs. effect.

 Example 19 Compound 1 (naphtholidine hydrochloride) against Staphylococcus aureus

All experimental methods and materials as in Example 15, except replacing strain Newman of 1.5 X 10 ⁶ CFU to 2.5 x l0 ⁷ CFU of strain USA400 (methicillin-resistant, available from the American Library resistant Staphylococcus aureus ( Network on Antimicrobial Resistance in Staphylococcus aureus, NARSA).

 The experimental results (Figure 18) demonstrate that Compound 1 (naphtholidine hydrochloride) significantly reduces drug-resistant Staphylococcus aureus

Colonization of USA400 in the heart of mice, as well as in the kidneys. The colony count showed that Compound 1 reduced the number of Staphylococcus aureus colonized in the mouse heart by about 10 times. At the same time, Compound 1 reduced the number of Staphylococcus aureus colonized in the mouse kidney by about 100 times. It is shown that Compound 1 has potent anti-resistant S. aureus efficacy.

 Example 20

All the experimental methods and materials similar to Example 15, except replacing strain Newman 1.5 x l0 ⁶ CFU into 3 x USA400 strains of l0 ⁸ CFU. For the compound 1 (naphtholidine hydrochloride) treatment group, the mice were intraperitoneally injected with 32 mg/kg of Compound 1 each time, the first time 12 hours before the bacterial infection, and 12 times within 6 days after infection (2 times a day, 13 times in total ;). For the negative control group (no compound treatment group;), the mice were intraperitoneally injected with 200 μΐ sterile saline each time, the first time 12 hours before bacterial infection, and 12 times within 6 days after infection (2 times a day, total 13 times). The number of deaths of the mice after infection was recorded daily, and the end of the day was recorded, and the survival curve was plotted within 7 days.

The experimental results (Fig. 19) showed that the 3×10 ⁸ CFU USA400 strain was a lethal dose, and in the unmedicated group, all mice died within 6 days of infection. Infection against a lethal dose of Staphylococcus aureus (USA400), compound 1 (naphtholidine hydrochloride) significantly prolonged the survival time of mice (protection rate = 40% in 7 days) with a very significant difference (P < 0.001). It is indicated that Compound 1 has potent anti-S. aureus efficacy.

 Example 21 Compound 2 ((E)-N-methyl(naphthalen-1-yl)-3-(4-methylphenyl)-prop-2-en-1-amine hydrochloride) against Staphylococcus aureus

 All experimental methods and materials were the same as in Example 17.

 The experimental results (Fig. 20) show that the compound 2 ((E)-N-methyl(naphthalen-1-yl)-3-(4-methylphenyl)-prop-2-en-1-amine hydrochloride) can be Significantly reduced colonization of drug-resistant Staphylococcus aureus Mu50 in mouse liver. Visual observation of the degree of bacterial colonization in the liver of mice showed that a large amount of drug-resistant Staphylococcus aureus Mu50 was colonized on the liver of mice without Compound 2, but was not observed on the liver of Compound 2 plus Compound 2. Significant bacterial colonization, ie, Compound 2 and Compound 1 have similar activity against S. aureus infection. Colony counts showed that Compound 2 reduced the number of S. aureus colonized in the liver of the mice by a factor of about 1000, indicating that Compound 2 also has potent anti-resistance to S. aureus.

 Compound 3 and Compound 4 were examined by the method of Example 21, and it was revealed that Compound 3 and Compound 4 also showed potent anti-S. aureus efficacy.

 As can be seen from Examples 8-21, the four compounds of the present invention, naftifine hydrochloride (1) and its derivatives (2), (3), (4) can strongly inhibit Staphylococcus aureus golden yellow in vitro. The synthesis of pigments, which inhibits the synthesis of golden pigments, inhibits the key catalytic enzyme CrtN in the golden yellow pigment synthesis pathway. In the subcutaneous and systemic infection models of mice, the pathogenicity of the mutant of crtN gene was found to be significantly reduced by 500,000 times, confirming that CrtN is a new drug target against S. aureus virulence. In animal infection experiments, naftifine hydrochloride (Compound 1) was found to significantly reduce the colonization of S. aureus (Newman) in the kidney, heart, and liver of mice, and to reduce drug-resistant Staphylococcus aureus Mu50 (A) Colonization of oxycillin and vancomycin in the heart and liver of mice, reducing the colonization of drug-resistant Staphylococcus aureus USA400 (methicillin-resistant) in the heart of mice and in the kidney . In response to a lethal dose of Staphylococcus aureus (Newman, USA400), naftifine hydrochloride (Compound 1) significantly prolonged the survival of mice with similar efficacy to vancomycin. The above results indicate that the four compounds of the present invention can be developed into new-purpose antibacterial drugs.

 The (£)-N-methyl(naphthalen-1-yl)-3-Ar-prop-2-en-1-amine hydrochloride (1-4) compound of the invention has a simple molecular structure and a simple preparation process. The production cost is low, the antibacterial mechanism in vivo and in vitro is clear, and the drug effect is remarkable. Therefore, it is expected to develop not only a novel single-medication antibacterial drug, but also an antibacterial drug which is combined with an existing antibiotic.

 All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request 1 - A compound of formula I or a compound thereof pharmaceutically characterized in that it is used for the preparation of antibacterial drugs,

In the formula, Ar is a C6-C10 aryl group or a C6-C10 aryl group substituted by a C1-C6 alkyl group. 2. The application according to claim 1, wherein the antibacterial drug is an anti-Staphylococcus aureus drug. 3. Application of a compound of formula I or a pharmaceutically acceptable salt thereof, characterized in that it is used to prepare a catalytic enzyme CrtN inhibitor; or to prepare a golden pigment that inhibits

In the formula, Ar is a C6-C10 aryl group or a C6-C10 aryl group substituted by a C1-C6 alkyl group. 4. The application according to any one of claims 1 to 3, characterized in that Ar is phenyl, naphthyl, or C1-C6 alkyl-substituted phenyl. 5. A compound of formula I or a pharmaceutically acceptable compound thereof

In the formula, Ar

for. 2-. 6 alkyl, R ₂ is hydrogen; Or Ri, R ₂ and adjacent carbon atoms together form a C6-C10 aryl group. 6. The compound of formula I according to claim 5, characterized in that, is. 3 5 alkyl group, R ₂ is hydrogen; or R ₂ and adjacent carbon atoms form a benzene ring. 7. The compound of formula I according to claim 5, wherein the pharmaceutically acceptable salt of the compound of formula I is selected from the group consisting of:

8. A method for preparing a compound of formula I or a pharmaceutically acceptable salt thereof, characterized in that the method includes the following steps: (a) reacting a compound of formula II with a compound of formula III to form a compound of formula I; and optionally (b) the step of producing the hydrochloride salt of the compound of formula I from the compound of formula I,

In each formula, Ar is a C6-C10 aryl group or a C6-C10 aryl group substituted by a C1-C6 alkyl group. 9. An antibacterial pharmaceutical composition, characterized in that the composition contains a compound that reduces the activity of the catalytic enzyme CrtN; and a pharmaceutically acceptable carrier. 10. A pharmaceutical composition that inhibits the catalytic enzyme CrtN or inhibits the synthesis of golden pigment, characterized in that it contains a compound of formula I or a pharmaceutically acceptable salt thereof; with,

In the formula, Ri and R ₂ are independently hydrogen and C1-C6 alkyl; Or, R ₂ and adjacent carbon atoms together form a C6-C10 aryl group. 11. The use of a catalytic enzyme CrtN inhibitor, which is characterized in that it is used to prepare pharmaceutical compositions or antibacterial compositions that reduce the pathogenic ability of bacteria. 12. A method for reducing bacterial pathogenicity or toxicity, characterized by comprising the steps: Exposure of bacteria to inhibitors of the catalytic enzyme CrtN reduces bacterial pathogenicity or virulence. 13. An attenuated bacterial strain, characterized in that the decrease or loss of the activity of the catalytic enzyme CrtN in the bacterial strain results in a decrease in the toxicity of the bacteria. 14. Use of the bacterial strain according to claim 13, characterized in that the bacterial strain is used for screening compounds that reduce the pathogenicity and/or toxicity of Staphylococcus aureus. 15. A method for screening compounds that reduce the pathogenicity and/or toxicity of bacteria, characterized by comprising the steps of: (a) providing a compound to be tested, and determining whether the compound to be tested interacts with the catalytic enzyme CrtN function, and select inhibitors of catalytic enzyme CrtN, where if the test compound reduces the activity of catalytic enzyme CrtN, or causes a decrease in the expression of catalytic enzyme CrtN, it indicates that the compound to be tested is an inhibitor of catalytic enzyme CrtN; (b) In the experimental group, contact the catalytic enzyme CrtN inhibitor selected in the previous step with the bacteria, determine the pathogenicity and/or toxicity of the bacteria, and compare it with the control group to screen out the inhibitors that reduce the bacterial pathogenicity and/or toxicity. Pathogenic and/or toxic compounds. 16. A compound that reduces the pathogenicity and/or toxicity of bacteria, characterized in that the compound is screened out by the method described in claim 15.