CN113045498B - A kind of 1,5-diarylpyrazole derivative, synthesis method and application - Google Patents

Abstract

Translated fromChinese

本发明涉及一种1,5‑二芳基吡唑衍生物及其抗菌应用，本发明在塞来昔布结构基础上对1,5‑二芳基进行结构修饰得到1,5‑二芳基吡唑衍生物，合成得到的目标分子经结构确证后进行抗菌活性体内外研究。本发明的1,5‑二芳基吡唑衍生物具有较好的抗菌活性，可用于细菌的抗感染治疗，为临床上细菌感染提供更多的用药选择。

The invention relates to a 1,5-diarylpyrazole derivative and its antibacterial application. The present invention modifies the structure of the 1,5-diaryl group on the basis of the structure of celecoxib to obtain a 1,5-diaryl group Pyrazole derivatives, the synthesized target molecules were confirmed for their antibacterial activity in vivo and in vitro. The 1,5-diarylpyrazole derivatives of the present invention have good antibacterial activity, can be used for anti-infection treatment of bacteria, and provide more drug options for clinical bacterial infections.

Description

Translated fromChinese

一种1,5-二芳基吡唑衍生物、合成方法及用途A kind of 1,5-diarylpyrazole derivative, synthesis method and application

技术领域technical field

本发明属于药物化学领域，具体涉及一种1,5-二芳基吡唑衍生物、合成方法及其在抗菌中的应用。The invention belongs to the field of medicinal chemistry, and specifically relates to a 1,5-diarylpyrazole derivative, a synthesis method and an antibacterial application thereof.

背景技术Background technique

根据最新的临床数据，病原菌的多药耐药现象呈现快速增长趋势，包括革兰氏阳性菌耐甲氧西林金黄色葡萄球菌(MRSA)，革兰氏阴性菌绿脓杆菌和鲍曼不动杆菌。这是细菌感染世界上一大主要健康威胁，因此临床对于用于治疗多药耐药的病原菌的新型抗生素和替代疗法的需求十分迫切。According to the latest clinical data, the phenomenon of multidrug resistance of pathogenic bacteria shows a rapid increase trend, including Gram-positive bacteria Methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii . Bacterial infections are a major health threat worldwide, and there is an urgent clinical need for new antibiotics and alternative therapies for the treatment of multidrug-resistant pathogens.

尽管科研人员已经做出了很多努力，但是在近二十年间也只有小部分的抗生素进入了临床。新药发现失败的原因是多因素的，一方面受限于化合物穿透膜的渗透性的理解不够完全，也受限于化学多样性等诸多原因。Although researchers have made a lot of efforts, only a small number of antibiotics have entered the clinic in the past two decades. The reasons for the failure of new drug discovery are multifactorial. On the one hand, it is limited by the incomplete understanding of the permeability of compounds through membranes, and is also limited by chemical diversity and many other reasons.

重定位策略提供了抗菌药物发现的一种新策略，该策略可以分为四种类型：1)药物重定位(老药新用)；2)靶标重定位；3)先导化合物重定位和4)基于表型和靶标的重定位。抗菌药物表现出与其他药物显著不同的理化性质，一个抗菌药物常发现是跟多个靶点相结合的，新型抗菌药物的发现用重新定位策略的前3种策略往往不太合适。第四种策略一般是基于表型的筛选，优先考虑抗菌活性而不是筛选特定的酶和受体，通过这种策略，可以发现高质量的先导化合物和并被进一步的开发，因此第四种策略非常适合用于新的抗菌药物的发现，同时也能满足抗菌药物的多靶点需求和紧迫的临床医学需要。The repositioning strategy provides a new strategy for antimicrobial drug discovery, which can be divided into four types: 1) drug repositioning (old drug repurposed); 2) target repositioning; 3) lead compound repositioning and 4) Phenotype and target based retargeting. Antibacterial drugs exhibit significantly different physical and chemical properties from other drugs. An antibacterial drug is often found to be combined with multiple targets. The first three repositioning strategies are often not suitable for the discovery of new antibacterial drugs. The fourth strategy is generally phenotype-based screening, prioritizing antibacterial activity rather than screening specific enzymes and receptors. Through this strategy, high-quality lead compounds can be found and further developed, so the fourth strategy It is very suitable for the discovery of new antibacterial drugs, and can also meet the multi-target requirements of antibacterial drugs and urgent clinical medical needs.

塞来昔布是一个COX-2选择性非甾类抗炎药物，被用于治疗疼痛和炎症。塞来昔布和他的衍生物被发现具有抗菌活性，Chiu等人发现塞来昔布和他们的衍生物表现出抗革兰阴性菌Francisella tularensis的活性，因此，塞来昔布被重定位为局部广谱抗菌药物。塞来昔布的抗菌活性作用机制主要是可以抑制DNA，RNA和蛋白质合成，还可以逆转MRSA的多药耐药性现象。这些结果表明塞来昔布可能是一个抗菌药物发现的目标重定位的较好的选择。塞来昔布的结构可以被修饰，特别是在苯环的5位上连接上大的基团时，可以增强其活性，但是毒性也随之增强。Celecoxib is a COX-2 selective nonsteroidal anti-inflammatory drug used to treat pain and inflammation. Celecoxib and its derivatives were found to have antibacterial activity. Chiu et al. found that Celecoxib and their derivatives showed activity against Gram-negative bacteria Francisella tularensis. Therefore, Celecoxib was repositioned as Topical broad-spectrum antimicrobials. The mechanism of antibacterial activity of celecoxib is mainly to inhibit the synthesis of DNA, RNA and protein, and it can also reverse the phenomenon of multidrug resistance of MRSA. These results suggest that celecoxib may be a better choice for target retargeting in antimicrobial drug discovery. The structure of celecoxib can be modified, especially when a large group is attached to the 5-position of the benzene ring, which can enhance its activity, but its toxicity will also be enhanced.

因此本领域需要发现具有更强抗菌活性的塞来昔布衍生物，为人们提供一种新的、安全有效的用药选择。Therefore, it is necessary to find celecoxib derivatives with stronger antibacterial activity in this field, so as to provide people with a new, safe and effective medication option.

发明内容Contents of the invention

本发明的目的旨在解决上述背景技术中提及的技术问题，提供新型抗菌化合物，具体涉及一种1,5-二芳基吡唑衍生物、合成方法及其在抗菌中的应用。The purpose of the present invention is to solve the technical problems mentioned in the above-mentioned background technology, and to provide novel antibacterial compounds, in particular to a 1,5-diarylpyrazole derivative, its synthesis method and its application in antibacterial.

一方面，本发明提供一种1,5-二芳基吡唑衍生物，其特征在于，用通式结构(I)表示为：In one aspect, the present invention provides a 1,5-diarylpyrazole derivative, characterized in that, it is represented by the general structure (I):

其中R₁取代基为-SO₂NH₂，-NHSO₂NH₂，-CONH₂；Wherein R₁ substituents are -SO₂ NH₂ , -NHSO₂ NH₂ , -CONH₂ ;

R₂、R₃、R₄、R₅、R₆各自独立选自-H，-CH₃，-CH₂CH₃，-F，-Cl，-Br，-I，-OCH₃，-OCH₂CH₃，-NO₂，CN，COOH；R₂ , R₃ , R₄ , R₅ , and R₆ are each independently selected from -H, -CH₃ , -CH₂ CH₃ , -F, -Cl, -Br, -I, -OCH₃ , -OCH_2CH3 ,_-NO2 , CN, COOH;

所述1,5-二芳基吡唑衍生物不是塞来昔布。The 1,5-diarylpyrazole derivative is not celecoxib.

作为本发明的进一步优选方案，所述的1,5-二芳基吡唑衍生物中R₁取代基为-SO₂NH₂；R₂-R₆取代基自独立选自-H，-CH₃，-CH₂CH₃，-F，-Cl，-OCH₃，-OCH₂CH₃。As a further preferred solution of the present invention, the R₁ substituent in the 1,5-diarylpyrazole derivative is -SO₂ NH₂ ; the R₂ -R₆ substituents are independently selected from -H, -CH₃ ,_-CH2CH3 ,_-F ,_-Cl ,_-OCH3 ,_-OCH2CH3 .

作为本发明的进一步优选方案，所述的1,5-二芳基吡唑衍生物的结构为：As a further preferred solution of the present invention, the structure of the 1,5-diarylpyrazole derivative is:

另一方面，本发明提供一种上述结构式(I)所述的1,5-二芳基吡唑衍生物在制备治疗革兰氏阳性菌或革兰氏阴性菌感染的药物中的应用。In another aspect, the present invention provides a use of a 1,5-diarylpyrazole derivative described in the above structural formula (I) in the preparation of a medicament for treating infections caused by Gram-positive or Gram-negative bacteria.

作为本发明的进一步优选方案，所述格革兰氏阳性菌为金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌；革兰氏阴性菌为鲍曼不动杆菌或绿脓杆菌。As a further preferred solution of the present invention, the Gram-positive bacteria are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus; the Gram-negative bacteria are Acinetobacter baumannii or Pseudomonas aeruginosa.

另一方面，本发明提供一种具有治疗革兰氏阴性菌感染的药物组合物，所述药物组合物含有结构式(I)所示的1,5-二芳基吡唑衍生物和多粘菌素B。In another aspect, the present invention provides a pharmaceutical composition for treating Gram-negative bacterial infections, the pharmaceutical composition contains 1,5-diarylpyrazole derivatives represented by structural formula (I) and polymyxa Prime B.

另一方面，本发明提供一种如上式(I)所示的1,5-二芳基吡唑衍生物在制备治疗革兰氏阴性菌感染的药物组合物中的应用，所述药物组合物含有多粘菌素B。In another aspect, the present invention provides an application of a 1,5-diarylpyrazole derivative represented by the above formula (I) in the preparation of a pharmaceutical composition for treating Gram-negative bacterial infections, the pharmaceutical composition Contains polymyxin B.

另一方面，本发明提供一种1,5-二芳基吡唑衍生物的制备方法，其特征在于通过如下制备步骤获得：In another aspect, the present invention provides a method for preparing 1,5-diarylpyrazole derivatives, which is characterized in that it is obtained through the following preparation steps:

其中，R₂-R₆取代基各自独立选自-H，-CH₃，-CH₂CH₃，-F，-Cl，-Br，-I，-OCH₃，-OCH₂CH₃，-NO₂，CN，COOH。Wherein, R₂ -R₆ substituents are each independently selected from -H, -CH₃ , -CH₂ CH₃ , -F, -Cl, -Br, -I, -OCH₃ , -OCH₂ CH₃ , -NO₂ , CN, COOH.

另一方面，本发明提供一种1,5-二芳基吡唑衍生物的制备方法，其特征在于通过如下制备步骤获得：In another aspect, the present invention provides a method for preparing 1,5-diarylpyrazole derivatives, which is characterized in that it is obtained through the following preparation steps:

其中，R₂-R₆取代基各自独立选自-H，-CH₃，-CH₂CH₃，-F，-Cl，-Br，-I，-OCH₃，-OCH₂CH₃，-NO₂，CN，COOH。Wherein, R₂ -R₆ substituents are each independently selected from -H, -CH₃ , -CH₂ CH₃ , -F, -Cl, -Br, -I, -OCH₃ , -OCH₂ CH₃ , -NO₂ , CN, COOH.

本发明的再一个目的是通过使用这类衍生物进行抗耐药菌的感染治疗。本发明的目的是通过如下的技术方案实现的：本发明提供了1,5-二芳基吡唑衍生物的合成方法，采用取代苯乙酮为起始原料，与三氟醋酸乙酯缩合形成苯环取代的1,3-二酮，磺酰胺类化合物是将1,3-二酮与对肼基苯磺酰胺盐酸盐反应生成1,5-二芳基吡唑磺酰胺类衍生物，磺酰脲类衍生物是将1,3-二酮与对硝基苯肼盐酸盐缩合得到1,5-二芳基吡唑母核，再用SnCl₂将硝基还原成胺基，与氯磺酰胺缩合得到1,5-二芳基磺酰脲类衍生物，经分离纯化得到纯品，经核磁、质谱确证其结构，具体的化合物见下表1。Another object of the present invention is to treat infections against drug-resistant bacteria by using such derivatives. The purpose of the present invention is achieved through the following technical scheme: the present invention provides a synthetic method for 1,5-diarylpyrazole derivatives, which uses substituted acetophenone as the starting material and condenses with ethyl trifluoroacetate to form 1,3-diketones substituted by benzene rings, sulfonamide compounds are produced by reacting 1,3-diketones with p-hydrazinobenzenesulfonamide hydrochloride to generate 1,5-diarylpyrazolesulfonamide derivatives, Sulfonylurea derivatives are obtained by condensing 1,3-diketone and p-nitrophenylhydrazine hydrochloride to obtain a 1,5-diarylpyrazole core, and then reducing the nitro group to an amine group with SnCl₂ , and The 1,5-diarylsulfonylurea derivatives were obtained by condensation of chlorosulfonamides. The pure products were obtained after separation and purification, and their structures were confirmed by NMR and mass spectrometry. The specific compounds are shown in Table 1 below.

表1本发明的化合物列表Table 1 List of compounds of the present invention

本发明测定了1,5-二芳基吡唑衍生物在不同种类细菌上的抗菌活性。The invention measures the antibacterial activity of 1,5-diarylpyrazole derivatives on different kinds of bacteria.

本发明公开了一种1,5-二芳基吡唑衍生物的合成及其抗菌应用，本发明在塞来昔布结构基础上对1,5-二芳基分别进行结构修饰得到1,5-二芳基吡唑衍生物，合成得到的目标分子经结构确证后进行抗菌活性体内外研究。本发明的1,5-二芳基吡唑衍生物具有较好的抗菌活性，可用于细菌的抗感染治疗，为临床上细菌感染提供更多的用药选择。The invention discloses the synthesis of a 1,5-diarylpyrazole derivative and its antibacterial application. The invention makes structural modifications to 1,5-diaryl groups on the basis of the structure of celecoxib to obtain 1,5-diaryl pyrazole derivatives. -Diarylpyrazole derivatives, the synthesized target molecules were confirmed for their antibacterial activity in vivo and in vitro. The 1,5-diarylpyrazole derivatives of the present invention have good antibacterial activity, can be used for anti-infection treatment of bacteria, and provide more drug options for clinical bacterial infections.

另外，本发明的1,5-二芳基吡唑衍生物和多粘菌素B组合使用时，抗菌效果显著提高。In addition, when the 1,5-diarylpyrazole derivative of the present invention is used in combination with polymyxin B, the antibacterial effect is significantly improved.

附图说明Description of drawings

图1是塞来昔布及其衍生物对MRSA菌株作用的Time-killing实验结果。Figure 1 shows the results of the Time-killing experiment of celecoxib and its derivatives on MRSA strains.

图2是塞来昔布衍生物小鼠体内抗菌活性测定结果中腹腔注射后小鼠的存活率。Fig. 2 is the survival rate of mice after intraperitoneal injection in the results of in vivo antibacterial activity measurement of celecoxib derivatives in mice.

图3是塞来昔布衍生物小鼠体内抗菌活性测定结果中空白组和实验组小鼠血清中TNF-α和IL-10水平。Fig. 3 shows the levels of TNF-α and IL-10 in the serum of mice in the blank group and the experimental group in the measurement results of the antibacterial activity of celecoxib derivatives in mice.

图4是塞来昔布衍生物体内抗菌活性测定中空白组和实验组的小鼠组织中的代表性组织切片图。Fig. 4 is a diagram of representative tissue sections in mouse tissues of the blank group and the experimental group in the in vivo antibacterial activity measurement of celecoxib derivatives.

图5是塞来昔布衍生物的DNA螺旋酶和拓扑异构酶的抑制凝胶图片。Figure 5 is a gel picture of the inhibition of DNA helicase and topoisomerase by celecoxib derivatives.

具体实施方式Detailed ways

为使本发明的技术方案和有益效果能够更加明显易懂，下面通过列举具体实施例的方式进行详细说明。除非另有定义，本文所使用的技术和科学术语与本申请所属的技术领域中的技术和科学术语的含义相同。In order to make the technical solutions and beneficial effects of the present invention more obvious and comprehensible, the following describes in detail by enumerating specific examples. Unless otherwise defined, technical and scientific terms used herein have the same meanings as technical and scientific terms in the technical field to which this application belongs.

实施例1Example 1

2,6-二甲基塞来昔布,合成步骤如下所示：2,6-Dimethylcelecoxib, the synthesis steps are as follows:

合成过程：取2,6-二甲基苯乙酮1g溶解于30mL甲醇中，配成2,6-二甲基苯乙酮/甲醇溶液。取17.5mL三氟乙酸乙酯溶解于20mL甲醇中，配成三氟乙酸乙酯/甲醇溶液。Synthesis process: Dissolve 1 g of 2,6-dimethylacetophenone in 30 mL of methanol to prepare a 2,6-dimethylacetophenone/methanol solution. Dissolve 17.5 mL of ethyl trifluoroacetate in 20 mL of methanol to form a trifluoroacetate/methanol solution.

取三口瓶将3.5g甲醇钠溶于20mL甲醇中，冰浴降温搅拌，控制温度在4-5℃之间，缓慢滴加三氟乙酸乙酯/甲醇溶液(控制流速为1滴/s)，滴加完成后再滴加2,6-二甲基苯乙酮/甲醇溶液(控制流速为1滴/s)，待到溶液全部滴加完毕后升温至80℃反应，反应期间TLC检测反应进度，点板显示反应结束。旋蒸干燥后，加入200mL去离子水，使用浓盐酸调节pH至6-7之间。使用乙酸乙酯萃取(50ml*3)，收集乙酸乙酯层，用无水硫酸钠干燥。再次旋蒸萃取液，称重得到产物1.074g。Take a three-necked flask and dissolve 3.5g of sodium methoxide in 20mL of methanol, cool and stir in an ice bath, control the temperature between 4-5°C, and slowly add ethyl trifluoroacetate/methanol solution dropwise (control the flow rate to 1 drop/s), After the dropwise addition, add 2,6-dimethylacetophenone/methanol solution dropwise (control the flow rate at 1 drop/s). After the solution is completely added dropwise, the temperature is raised to 80°C for reaction. During the reaction, TLC detects the reaction progress , the dot plate shows the end of the reaction. After rotary evaporation and drying, add 200 mL of deionized water, and use concentrated hydrochloric acid to adjust the pH to between 6-7. It was extracted with ethyl acetate (50ml*3), and the ethyl acetate layer was collected and dried over anhydrous sodium sulfate. The extract was rotary evaporated again, and 1.074 g of the product was obtained by weighing.

在装有回流装置的250ml圆底烧瓶中装入上一步生成物1.074g，用50ml无水乙醇溶解，加入1.8g对肼基苯磺酰胺盐酸盐，加热回流反应12h(过夜反应)，TLC检测(石油醚：乙酸乙酯2:1)反应完全后，停止加热，搅拌冷却至室温。过滤，分别收集滤饼与滤液，使用TLC检测(石油醚：乙酸乙酯2:1)，产品大部分在滤液中。旋干滤液，加入100ml乙酸乙酯溶解，再使用去离子水(50ml*3)洗涤，分层取有机层，加入无水硫酸钠干燥。再次旋蒸萃取液后得产物1.02g。使用乙醇/水重结晶得到产物。结构信息结果如下所示：¹H NMR(DMSO-d₆,500MHz)δ(ppm)7.83(d,J＝8.6Hz,2H),7.42-7.47(m,4H),7.32(d,J＝7.6Hz),7.18(d,2H),7.13(s,1H),2.01(s,6H)；¹³C NMR(DMSO-d₆,500MHz)δ(ppm)144.12,143.54,143.04,142.74,141.34,137.77,133.46,130.40,129.77,128.62,128.45,128.28,127.94,127.27,124.11,113.36,107.65,40.54,40.37,40.21,39.87,39.71,39.54；EI/mass(m/z):calculated395.09,found[M-H]^-394.08。In a 250ml round bottom flask equipped with a reflux device, put 1.074g of the product of the previous step, dissolve it with 50ml of absolute ethanol, add 1.8g of p-hydrazinobenzenesulfonamide hydrochloride, heat and reflux for 12h (reaction overnight), TLC After the detection (petroleum ether: ethyl acetate 2:1) reaction is complete, stop heating, stir and cool to room temperature. Filter, collect the filter cake and filtrate respectively, and use TLC detection (petroleum ether: ethyl acetate 2:1), most of the product is in the filtrate. Spin the filtrate to dryness, add 100ml of ethyl acetate to dissolve, then wash with deionized water (50ml*3), separate the organic layer, add anhydrous sodium sulfate to dry. After rotating the extract again, 1.02 g of the product was obtained. Recrystallization using ethanol/water gave the product. The structural information results are as follows:¹ H NMR (DMSO-d₆ , 500MHz) δ (ppm) 7.83 (d, J = 8.6 Hz, 2H), 7.42-7.47 (m, 4H), 7.32 (d, J = 7.6 Hz),7.18(d,2H),7.13(s,1H),2.01(s,6H);¹³ C NMR(DMSO-d₆ ,500MHz)δ(ppm)144.12,143.54,143.04,142.74,141.34,137.77 , 133.46, 130.40, 129.77, 128.62, 128.45, 128.28, 127.94, 127.27, 124.11, 113.36, 107.65, 40.54, 40.37, 40.21, 39.87, 39.71, 39.54; MH]^- 394.08.

实施例2Example 2

化合物8可以参照实施例1的合成方法，以苯乙酮为原料制备得到，结构信息结果如下所示：¹H NMR(DMSO-d₆,400MHz)δ(ppm)7.91(d,J＝4.0Hz,2H),7.48(d,J＝8.0Hz,2H),7.39(m,3H),7.23(d,2H),6.78(s,1H),4.88(s,2H)；EI/mass(m/z):calculated 367.35,found[M+H]⁺368.0。Compound 8 can be prepared by referring to the synthesis method of Example 1, using acetophenone as a raw material, and the structural information results are as follows:¹ H NMR (DMSO-d₆ , 400MHz) δ (ppm) 7.91 (d, J = 4.0Hz ,2H),7.48(d,J＝8.0Hz,2H),7.39(m,3H),7.23(d,2H),6.78(s,1H),4.88(s,2H); EI/mass(m/ z): calculated 367.35, found [M+H]⁺ 368.0.

实施例3Example 3

化合物9可以参照实施例1的合成方法，以2-甲基苯乙酮为原料制备得到，结构信息结果如下所示：¹H NMR(CDCl₃,400MHz)δ(ppm)7.82(d,J＝8.0Hz,2H),7.35-7.41(m,3H),7.21-7.25(m,3H),6.70(s,1H),4.99-5.08(m,2H),2.00(s,3H)；EI/mass(m/z):calculated381.38,found[M+H]⁺382.06。Compound 9 can be prepared by referring to the synthesis method of Example 1, using 2-methylacetophenone as a raw material, and the structural information results are as follows:¹ H NMR (CDCl₃ , 400 MHz) δ (ppm) 7.82 (d, J = 8.0Hz,2H),7.35-7.41(m,3H),7.21-7.25(m,3H),6.70(s,1H),4.99-5.08(m,2H),2.00(s,3H); (m/z): calculated 381.38, found [M+H]⁺ 382.06.

实施例4Example 4

化合物10可以参照实施例1的合成方法，以3-甲基苯乙酮为原料制备得到，结构信息结果如下所示：¹H NMR(CDCl₃,400MHz)δ(ppm)7.92(d,J＝12Hz,2H),7.48-7.51(m,2H),7.20(d,J＝8.0Hz,2H),7.13(d,J＝8Hz,2H),6.77(s,1H),5.04(d,J＝4Hz,2H),2.40(s,3H)；EI/mass(m/z):calculated 381.37,found[M+H]⁺382.08。Compound 10 can be prepared by referring to the synthesis method of Example 1, using 3-methylacetophenone as a raw material, and the structural information results are as follows:¹ H NMR (CDCl₃ , 400 MHz) δ (ppm) 7.92 (d, J = 12Hz, 2H), 7.48-7.51(m, 2H), 7.20(d, J=8.0Hz, 2H), 7.13(d, J=8Hz, 2H), 6.77(s, 1H), 5.04(d, J= 4Hz, 2H), 2.40(s, 3H); EI/mass(m/z): calculated 381.37, found [M+H]⁺ 382.08.

实施例5Example 5

化合物12可以参照实施例1的合成方法，以2，4-二甲基苯乙酮为原料制备得到，结构信息结果如下所示：¹H NMR(CDCl₃,400MHz)δ(ppm)7.84(d,J＝8.0Hz,2H),7.42(d,J＝8.0Hz,2H),7.05-7.09(m,3H),6.66(s,1H),4.81(s,2H),2.37(s,3H),1.96(s,3H)；EI/mass(m/z):calculated 395.41,found[M+H]⁺396.09。Compound 12 can be prepared by referring to the synthesis method of Example 1, using 2,4-dimethylacetophenone as a raw material, and the structural information results are as follows:¹ H NMR (CDCl₃ , 400MHz) δ (ppm) 7.84 (d ,J=8.0Hz,2H),7.42(d,J=8.0Hz,2H),7.05-7.09(m,3H),6.66(s,1H),4.81(s,2H),2.37(s,3H) , 1.96 (s, 3H); EI/mass (m/z): calculated 395.41, found [M+H]⁺ 396.09.

实施例6Example 6

化合物13可以参照实施例1的合成方法，以2，5-二甲基苯乙酮为原料制备得到，结构信息结果如下所示：¹H NMR(DMSO-d₆,400MHz)δ(ppm)7.81(d,J＝8.8Hz,2H),7.45-7.47(m,4H),7.19(d,J＝7.6Hz,1H),7.16(d,2H),7.12(s,1H),2.25(s,3H),1.91(s,3H)；EI/mass(m/z):calculated 395.3,found[M+H]⁺396.1。Compound 13 can be prepared by referring to the synthesis method of Example 1, using 2,5-dimethylacetophenone as a raw material, and the structural information results are as follows:¹ H NMR (DMSO-d₆ , 400MHz) δ (ppm) 7.81 (d,J=8.8Hz,2H),7.45-7.47(m,4H),7.19(d,J=7.6Hz,1H),7.16(d,2H),7.12(s,1H),2.25(s, 3H), 1.91(s, 3H); EI/mass(m/z): calculated 395.3, found [M+H]⁺ 396.1.

实施例7Example 7

2,4-二甲基磺酰脲类塞来昔布合成步骤如下所示：The synthetic steps of 2,4-dimethylsulfonylurea celecoxib are as follows:

合成过程：将2,4-二甲基苯乙酮2g溶于30mL无水乙醇中。冰浴降温至4-5℃。将5g乙醇钠溶于20mL无水乙醇中。缓慢控温滴加乙醇钠的乙醇溶液，滴加温度控制不超过10℃，滴加完成后，保温搅拌30min，将三氟乙酸乙酯30mL溶于10mL无水乙醇中后加入新的滴液漏斗，控温10℃左右缓慢滴加，滴加完成后，升温至回流反应2h，TLC检测(石油醚：乙酸乙酯5:1)反应未结束，补加1g乙醇钠与3mL三氟乙酸乙酯进三口烧瓶中。2h后反应完成，旋干反应液，加入100mL水，滴加浓盐酸调pH至6-7，乙酸乙酯50mL萃取三次，收集乙酸乙酯层，无水硫酸钠干燥，旋干称重，得到3.5g的1,3-二酮中间体。Synthesis process: Dissolve 2g of 2,4-dimethylacetophenone in 30mL of absolute ethanol. Cool down to 4-5°C in an ice bath. Dissolve 5 g of sodium ethoxide in 20 mL of absolute ethanol. Add the ethanol solution of sodium ethoxide dropwise under temperature control, and the dropping temperature is controlled not to exceed 10°C. After the dropping is completed, keep stirring for 30 minutes, dissolve 30 mL of ethyl trifluoroacetate in 10 mL of absolute ethanol, and add a new dropping funnel , temperature controlled at about 10°C and slowly added dropwise. After the dropwise addition was completed, the temperature was raised to reflux for 2 hours. TLC detection (petroleum ether: ethyl acetate 5:1) the reaction was not completed, and 1g of sodium ethoxide and 3mL of ethyl trifluoroacetate were added Into the three-necked flask. After 2 hours, the reaction was completed, the reaction liquid was spin-dried, 100 mL of water was added, the pH was adjusted to 6-7 by adding concentrated hydrochloric acid dropwise, extracted three times with 50 mL of ethyl acetate, the ethyl acetate layer was collected, dried over anhydrous sodium sulfate, spin-dried and weighed to obtain 3.5 g of the 1,3-diketone intermediate.

在装有回流装置的250mL圆底烧瓶中装入1,3-二酮中间体3.5g，用40mL无水乙醇溶解，加入2.711g对硝基苯肼盐酸盐，加热回流反应4-5h，TLC检测(石油醚：乙酸乙酯5:1)。反应完全后，停止加热，搅拌冷却至室温，过滤，分别收集滤饼和滤液，使用TLC检测滤饼和滤液(石油醚：乙酸乙酯2:1)，产品应该在滤液中，收集滤液旋干，加入乙酸乙酯(50mL)和水(50mL)萃取三次，合并有机相，加入无水硫酸钠干燥后旋干得粗产物，过柱子提纯(展开剂：石油醚)后得到2.7g 3-三氟甲基-1-(4-硝基苯基)-5-(2’,4’-二甲基苯基)吡唑。Put 3.5g of 1,3-diketone intermediate in a 250mL round bottom flask equipped with a reflux device, dissolve it in 40mL of absolute ethanol, add 2.711g of p-nitrophenylhydrazine hydrochloride, and heat to reflux for 4-5h. TLC detection (petroleum ether: ethyl acetate 5:1). After the reaction is complete, stop heating, stir and cool to room temperature, filter, collect the filter cake and filtrate respectively, use TLC to detect the filter cake and filtrate (petroleum ether: ethyl acetate 2:1), the product should be in the filtrate, collect the filtrate and spin dry , adding ethyl acetate (50mL) and water (50mL) to extract three times, combining the organic phases, adding anhydrous sodium sulfate to dry and spinning to obtain a crude product, which was purified by column (developing solvent: petroleum ether) to obtain 2.7g of 3-tri Fluoromethyl-1-(4-nitrophenyl)-5-(2',4'-dimethylphenyl)pyrazole.

将6.75g SnCl₂ 2H₂O在常温下溶解在50mL乙醇中，加入2.7g 3-三氟甲基-1-(4-硝基苯基)-5-(2’,4’-二甲基苯基)吡唑，回流3h后使用TLC检测(石油醚：乙酸乙酯5:1)发现反应未结束，补加1.68g SnCl₂ 2H₂O后1h用TLC检测仍未反应结束继续补加1.68g SnCl₂2H₂O，1h后反应结束，旋干反应液，加入50mL 1mol/L HCl调节pH至2左右，发现溶解度不高，无法全部形成盐酸盐溶清，故加入1mol/L NaOH溶液调节pH至11，形成大量乳白色固体沉淀，加入乙酸乙酯后抽滤，抽滤过后将滤饼转移至烧瓶中，加入乙酸乙酯搅拌洗涤，再进行抽滤，得滤液后使用乙酸乙酯与水萃取三次得有机层，无水硫酸钠干燥，有机层旋蒸得到还原产物2.2g。Dissolve 6.75g of SnCl₂ 2H₂ O in 50mL of ethanol at room temperature, add 2.7g of 3-trifluoromethyl-1-(4-nitrophenyl)-5-(2',4'-dimethyl Phenyl)pyrazole, after refluxing for 3 hours, use TLC detection (petroleum ether: ethyl acetate 5:1) to find that the reaction is not complete, add 1.68g of SnCl₂ 2H₂ O and then use TLC to detect that the reaction has not ended after 1 hour and continue to add 1.68 g SnCl₂ 2H₂ O, the reaction is over after 1h, spin the reaction solution, add 50mL 1mol/L HCl to adjust the pH to about 2, it is found that the solubility is not high, and the hydrochloride cannot be completely dissolved, so add 1mol/L NaOH solution Adjust the pH to 11 to form a large amount of milky white solid precipitates, add ethyl acetate and filter with suction, transfer the filter cake to a flask after suction filtration, add ethyl acetate to stir and wash, and then perform suction filtration to obtain the filtrate and use ethyl acetate and The organic layer was extracted three times with water, dried over anhydrous sodium sulfate, and the organic layer was rotary evaporated to obtain 2.2 g of the reduced product.

将2.2g还原产物溶解在50mL二氯甲烷中，0℃搅拌10min后，滴加含有0.85g氨基磺酰氯的5mL二氯甲烷溶液和1mL三乙胺。滴加完成后，常温反应，TLC检测显示反应完成后。然后用水洗涤，调节pH到中性。有机层使用无水硫酸钠干燥，随后旋蒸浓缩，得到粗品，过柱得到纯净产物。结构信息结果如下所示：¹H NMR(DMSO-d₆,500MHz)δ(ppm)7.10(t,2H),7.03(d,J＝7.65Hz,2H),6.91(t,3H),6.51(d,J＝8.5Hz,2H),5.37(s,2H),2.29(s,3H),2.00(s,3H)；¹³C NMR(DMSO-d₆,500MHz)δ(ppm)149.31,144.14,141.01,140.71,139.19,136.94,131.26,130.99,128.08,126.88,126.63,126.08,123.19,121.06,113.76,106.05,60.18,40.51,40.35,40.18,39.85,39.68,39.51,21.14,19.91,14.45；EI/mass(m/z):calculated410.10,found[M-H]^-409.09。Dissolve 2.2g of the reduced product in 50mL of dichloromethane, stir at 0°C for 10min, then add dropwise a solution of 0.85g of sulfamoyl chloride in 5mL of dichloromethane and 1mL of triethylamine. After the dropwise addition was completed, the reaction was carried out at room temperature, and TLC detection showed that the reaction was completed. Then wash with water and adjust the pH to neutral. The organic layer was dried over anhydrous sodium sulfate, then concentrated by rotary evaporation to obtain a crude product, which was passed through a column to obtain a pure product. The structural information results are shown below:¹ H NMR (DMSO-d₆ , 500 MHz) δ (ppm) 7.10 (t, 2H), 7.03 (d, J = 7.65 Hz, 2H), 6.91 (t, 3H), 6.51 ( d, J=8.5Hz, 2H), 5.37(s, 2H), 2.29(s, 3H), 2.00(s, 3H);¹³ C NMR (DMSO-d₆ , 500MHz) δ(ppm) 149.31, 144.14, 141.01,140.71,139.19,136.94,131.26,130.99,128.08,126.88,126.63,126.08,123.19,121.06,113.76,106.05,60.18,40.51,40.35,40.18,39.85,39.68,39.51,21.14,19.91,14.45；EI/ mass(m/z): calculated 410.10, found [MH]^- 409.09.

实验例1.塞来昔布衍生物对标准菌株及临床菌株的敏感性测定Experimental example 1. Sensitivity determination of celecoxib derivatives to standard strains and clinical strains

菌株及实验用试剂均来自商业化购买，如MH肉汤(CAMHB药敏培养液)购买自上海科马嘉生物有限公司的肉汤培养基；菌株信息：S.aureus,ATCC29213；MRSA,ATCC1026；S.pneumoniae,ATCC49619；E.faecalis,ATCC29212；E.coli,ATCC25922；A.baumannii,临床菌株；P.aeruginosa,ATCC27853；K.pneumoniae,临床菌株。The strains and experimental reagents were purchased commercially, for example, MH broth (CAMHB drug-sensitive culture medium) was purchased from the broth culture medium of Shanghai Kemajia Biological Co., Ltd.; strain information: S.aureus, ATCC29213; MRSA, ATCC1026; S.pneumoniae, ATCC49619; E.faecalis, ATCC29212; E.coli, ATCC25922; A.baumannii, clinical strains; P.aeruginosa, ATCC27853; K.pneumoniae, clinical strains.

测定塞来昔布衍生物对8种不同类型的标准菌株及不少于15株临床耐药菌株(MRSA及A.baumannii)的敏感性，结果以MIC值表示，具体步骤如下：Determination of the sensitivity of celecoxib derivatives to 8 different types of standard strains and no less than 15 strains of clinical drug-resistant strains (MRSA and A.baumannii), the results are expressed as MIC values, and the specific steps are as follows:

1)塞来昔布衍生物药敏板的制备1) Preparation of celecoxib derivative drug-sensitive plate

将倍比稀释后不同浓度的塞来昔布衍生物7-13溶液分别加到无菌的96孔板中，冰冻干燥后密封，-20℃以下保存备用；Add celecoxib derivatives 7-13 solutions of different concentrations after doubling dilution to a sterile 96-well plate, freeze-dry, seal, and store below -20°C for future use;

2)测试菌株2) Test strain

金黄色葡萄球菌ATCC29213，MRSA(标准菌株ATCC1026及不少于20株的临床菌株)，肺炎链球菌ATCC49619，粪肠球菌ATCC29212，大肠埃希菌ATCC25922，鲍曼不动杆菌(对抗菌药物全敏感的临床菌株)，多重耐药鲍曼不动杆菌(不少于20株临床菌株)，铜绿假单胞菌ATCC27853，肺炎克雷伯菌(临床菌株)；Staphylococcus aureus ATCC29213, MRSA (standard strain ATCC1026 and no less than 20 clinical strains), Streptococcus pneumoniae ATCC49619, Enterococcus faecalis ATCC29212, Escherichia coli ATCC25922, Acinetobacter baumannii (all sensitive to antibiotics) clinical strains), multidrug-resistant Acinetobacter baumannii (no less than 20 clinical strains), Pseudomonas aeruginosa ATCC27853, Klebsiella pneumoniae (clinical strains);

3)菌悬液制备3) Preparation of bacterial suspension

将试验菌株活化培养18-24h，用生理盐水直接菌悬液法制备的浓度相当于0.5麦氏比浊标准的菌悬液(浓度约为1.5×10⁸CFU/mL)，再用MH肉汤稀释菌液，使最终菌液浓度为5×10⁵CFU/mL，每孔加150μL稀释菌液于药敏孔中，空白对照只加150μL培养基，阴性对照加菌悬液和培养基的混合物150μL，15分钟内接种完毕，每组试验重复三次。35℃孵育16-24h判断结果。The test strains were activated and cultured for 18-24 hours, and the bacterial suspension with a concentration equivalent to 0.5 McFarland turbidimetric standard (concentration about 1.5×10⁸ CFU/mL) was prepared by the direct bacterial suspension method of physiological saline, and then MH broth Dilute the bacterial solution so that the final concentration of the bacterial solution is 5×10⁵ CFU/mL, add 150 μL of the diluted bacterial solution to the drug-sensitive well, add only 150 μL of medium for the blank control, and add the mixture of bacterial suspension and medium for the negative control 150 μL was inoculated within 15 minutes, and each experiment was repeated three times. Incubate at 35°C for 16-24h to judge the result.

4)结果判断4) Result judgment

以在小孔内完全抑制细菌生长的最低药物浓度为MIC(μg/mL)。当阳性对照孔(即不含抗生素)内细菌明显生长试验才有意义，当在微量肉汤稀释法出现单一的跳孔时，应记录抑制细菌生长的最高药物浓度，以阴性对照孔中细菌接种血平板以判断菌株有无污染。根据CLSI提供的人用抗生素的判定标准(M45-A2)判断结果。MIC (μg/mL) was defined as the minimum drug concentration that completely inhibited bacterial growth in the small well. The test is only meaningful when the bacteria grow significantly in the positive control well (that is, without antibiotics). When a single hole jumps in the micro-broth dilution method, the highest drug concentration that inhibits bacterial growth should be recorded, and the bacteria in the negative control well should be inoculated. Blood plate to determine whether the strain is contaminated. The results were judged according to the judgment standard (M45-A2) of antibiotics for human use provided by CLSI.

5)塞来昔布衍生物的MIC结果5) MIC results of celecoxib derivatives

实验结果表明化合物12(2,4-二甲基塞来昔布)对S.aureus、耐甲氧西林金黄色葡萄球菌(MRSA)、鲍曼不动杆菌及MRSA临床菌株具有较好的抑制作用。The experimental results show that compound 12 (2,4-dimethylcelecoxib) has a good inhibitory effect on S.aureus, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and clinical strains of MRSA .

表2塞来昔布衍生物对革兰氏阳性菌的抗菌活性结果Table 2 The antibacterial activity results of celecoxib derivatives to Gram-positive bacteria

表3塞来昔布衍生物对革兰氏阴性菌的抗菌活性结果Table 3 The antibacterial activity results of celecoxib derivatives to Gram-negative bacteria

表4塞来昔布类似物对临床分离的MRSA耐药菌株的抗菌活性测定结果Table 4 celecoxib analogues to the antibacterial activity determination result of clinically isolated MRSA drug-resistant strains

实验例2.塞来昔布衍生物增强细菌对传统抗菌药物敏感性测定Experimental Example 2. Celecoxib Derivatives Enhance Bacterial Sensitivity to Traditional Antibacterial Drugs

在无菌的96孔板中先后加入亚抑制浓度(1/2MIC)的传统抗生素(包括万古霉素、左氧氟沙星、利奈唑胺、多粘菌素B)溶液与塞来昔布衍生物的倍比稀释梯度溶液(0.125-64μg/mL)，冷冻干燥后置于-20℃保存，再以上述所述步骤加入MRSA，A.baumannii，P.aeruginosa，K.pneumoniae，每个浓度设置三个复孔，并记录实验结果MIC值，结果见表5。Add sub-inhibitory concentration (1/2 MIC) of traditional antibiotics (including vancomycin, levofloxacin, linezolid, polymyxin B) solution and the doubling ratio of celecoxib derivatives in a sterile 96-well plate Dilute the gradient solution (0.125-64μg/mL), freeze-dry and store at -20°C, then add MRSA, A.baumannii, P.aeruginosa, K.pneumoniae according to the above steps, and set up three replicate wells for each concentration , and record the MIC value of the experimental results, the results are shown in Table 5.

结果表明当加入1/2MIC浓度的多粘菌素B时，塞来昔布衍生物对革兰氏阴性菌表现出较好的抑制效应，尤其是化合物12的抑制效应最为明显。The results showed that when 1/2MIC concentration of polymyxin B was added, celecoxib derivatives showed better inhibitory effect on Gram-negative bacteria, especially the inhibitory effect ofcompound 12 was the most obvious.

表5塞来昔布及衍生物与多粘菌素B联用时对G-菌的抗菌活性结果When table 5 celecoxib and its derivatives are used in combination with polymyxin B, the antibacterial activity results against G-bacteria

实验例3.塞来昔布衍生物对细菌的Time-killing实验Experimental example 3. Time-killing experiment of celecoxib derivatives on bacteria

根据塞来昔布衍生物测定的MIC结果，在无菌的96孔板中加入2MIC，4MIC，8MIC浓度的优选化合物溶液，冷冻干燥后置于-20℃保存，再以上述所述步骤加入MRSA菌液，每个浓度设置三个复孔，35℃孵育24h，期间在孵育0h，4h，8h，12h，16h，24h时间点时每孔取样1μL接种至血平板中，培养24h对皿内菌落数进行计数，当24h孔内残余的菌落数小于原来菌落数的0.01％时视为杀菌剂，否则视为抑菌剂，结果见说明书附图1。According to the MIC results of the determination of celecoxib derivatives, add 2MIC, 4MIC, 8MIC concentration of the preferred compound solution in a sterile 96-well plate, freeze-dry and store at -20°C, and then add MRSA according to the above-mentioned steps Bacterial solution, set up three replicate wells for each concentration, incubate at 35°C for 24 hours, during the period of incubation at 0h, 4h, 8h, 12h, 16h, and 24h time points, take 1μL from each well and inoculate it into a blood plate, and cultivate the colonies in the plate for 24h count, and when the number of remaining colonies in the 24h hole is less than 0.01% of the original number of colonies, it is regarded as a bactericide, otherwise it is regarded as a bacteriostatic agent. The results are shown in Figure 1 of the description.

实验例4.对塞来昔布衍生物进行人哺乳细胞的生长抑制测定Experimental Example 4. Determination of Growth Inhibition of Human Mammalian Cells Using Celecoxib Derivatives

1)将细胞消化、计数，配制细胞悬液6.0×10⁴个/mL，96孔细胞培养板中每孔加入100μL细胞悬液；¹ ) Digest and count the cells, prepare a cell suspension of 6.0×104 cells/mL, and add 100 μL of the cell suspension to each well of a 96-well cell culture plate;

2)96孔细胞培养板置于37℃，5％CO₂培养箱中培养24h；2) The 96-well cell culture plate was placed in a 37°C, 5% CO₂ incubator for 24 hours;

3)用培养基稀释药物至所需工作液浓度，每孔加入100μL相应的含药培养基，同时设立阴性对照组；3) Dilute the drug with the medium to the required concentration of the working solution, add 100 μL of the corresponding drug-containing medium to each well, and set up a negative control group at the same time;

4)96孔细胞培养板分别置于37℃，5％CO2培养箱培养48小时；4) 96-well cell culture plates were placed in a 37°C, 5% CO2 incubator for 48 hours;

5)将96孔板进行MTT染色，λ＝490nm，测定OD值；5) Stain the 96-well plate with MTT, λ=490nm, and measure the OD value;

6)每孔加入20μL MTT(5mg/mL)，在培养箱继续培养4小时；6) Add 20 μL of MTT (5 mg/mL) to each well and continue culturing in the incubator for 4 hours;

7)弃上清，每孔加入150μL DMSO，摇床10分钟轻轻地混匀；λ＝490nm，酶标仪读出每孔的OD值，计算抑制率，结果见表6。7) Discard the supernatant, add 150 μL DMSO to each well, shake gently for 10 minutes; λ=490 nm, read the OD value of each well with a microplate reader, and calculate the inhibition rate.

计算各组别抑制率。Calculate the inhibition rate of each group.

表6塞来昔布及衍生物对HepG-2细胞毒生长抑制实验结果Table 6 Celecoxib and its derivatives inhibit the growth of HepG-2 cells

实验结果表明化合物12对真核细胞和原核细菌之间的选择性最好。The experimental results showed thatcompound 12 had the best selectivity between eukaryotic cells and prokaryotic bacteria.

实验例5.塞来昔布及其衍生物体内抗菌活性测定Experimental Example 5. Determination of Antibacterial Activity of Celecoxib and Its Derivatives in Vivo

1)ICR小鼠适应性饲养一周，正常饮水饮食，昼夜光照循环；1) ICR mice were adaptively fed for one week, with normal drinking water and diet, and day and night light cycle;

2)实验前一天将试验菌株活化培养18-24h，将MRSA菌落挑出置于生理盐水中制备浓度约为2.4×10⁹CFU/mL菌悬液；2) The test strain was activated and cultured for 18-24 hours the day before the experiment, and the MRSA colony was picked out and placed in physiological saline to prepare a bacterial suspension with a concentration of about 2.4×10⁹ CFU/mL;

3)购买猪肠胃的粘液素用生理盐水配制浓度为10％(W/V)的溶液，加热搅拌40min，采用碳酸氢钠调节pH至碱性，形成混悬液；3) Prepare a solution of 10% (W/V) concentration of mucin from pig intestines and stomach with physiological saline, heat and stir for 40 minutes, adjust the pH to alkaline with sodium bicarbonate, and form a suspension;

4)将菌悬液和粘液素溶液等体积混合，菌悬液终浓度为1.2×10⁹CFU/mL，粘液素的终浓度为5％；4) Mix the bacterial suspension and the mucin solution in equal volumes, the final concentration of the bacterial suspension is 1.2×10⁹ CFU/mL, and the final concentration of the mucin is 5%;

5)塞来昔布及其衍生物的溶液配置，基于前期发现化合物的溶解性较差，采用乙醇：吐温80：水＝1:1:8的比例混悬药物，阳性药万古霉素水溶性较好，用水溶解，塞来昔布及其衍生物设置浓度为20mg/kg，万古霉素的剂量为30mg/kg；5) The solution configuration of celecoxib and its derivatives, based on the poor solubility of the compounds found in the early stage, adopts the ratio of ethanol: Tween 80: water = 1:1:8 to suspend the drug, and the positive drug vancomycin is dissolved in water Good sex, dissolved in water, the concentration of celecoxib and its derivatives is set at 20mg/kg, and the dosage of vancomycin is 30mg/kg;

5)小鼠称重后对小鼠腹腔注射0.5mL的混合液(菌悬液+粘液素)，感染半小时和八小时后分别腹腔注射药物，观察小鼠48h内的生理状态，体重变化，体温变化，及小鼠的存活率。在感染后2h和24h摘眼球取血，分离血清，测定其炎症因子和化学因子的含量。感染后24h解剖小鼠，分离其组织，用无菌生理盐水漂洗后一半称重后置于无菌生理盐水中匀浆得到组织匀浆液，及时取样接种至血平板中培养24h后进行菌落计数。另一半在生理盐水中漂洗后置于福尔马林中，H&E染色后制作蜡块切片，显微观察组织病理变化，结果见说明书附图2、说明书附图3和说明书附图4。5) After the mice were weighed, 0.5mL of the mixed solution (bacteria suspension + mucin) was injected intraperitoneally into the mice, and the drugs were injected intraperitoneally after half an hour and eight hours after infection, and the physiological state and body weight changes of the mice were observed within 48 hours. Body temperature changes, and the survival rate of mice. At 2h and 24h after infection, the eyeballs were removed to collect blood, the serum was separated, and the contents of inflammatory factors and chemical factors were determined. 24 hours after infection, the mice were dissected, and the tissues were separated. After rinsing with sterile normal saline, half of the tissues were weighed and homogenized in sterile normal saline to obtain a tissue homogenate. Samples were inoculated in a blood plate in time and cultured for 24 hours before colony counting. The other half was rinsed in normal saline and then placed in formalin. After H&E staining, a wax block section was made, and histopathological changes were observed microscopically. The results are shown in Figure 2, Figure 3 and Figure 4 of the specification.

说明书附图2中A为腹腔注射11后的生存率，B为腹腔注射12后的生存率，C为组织收集肝脏，脾脏，肾脏、匀浆、接种后的菌落计数。结果显示：腹腔注射化合物12可以改善MRSA感染小鼠急性腹膜炎模型的生存率。In Figure 2 of the manual, A is the survival rate after intraperitoneal injection of 11, B is the survival rate after intraperitoneal injection of 12, and C is the colony count after tissue collection of liver, spleen, kidney, homogenate, and inoculation. The results showed that intraperitoneal injection ofcompound 12 could improve the survival rate of the acute peritonitis model of MRSA-infected mice.

表7各个实验组的组织病变程度打分结果Table 7 Scoring results of the degree of tissue lesions in each experimental group

采用MRSA标准菌株对ICR小鼠感染形成急性腹膜炎模型。结果用平均值±SD表示(每组6只小鼠)，**p<0.01当与阴性对照组相比较具有显著性差异，#p<0.05和##p<0.01与MRSA感染小鼠相比较具有极其显著性差异，结果显示：模型组的24小时内动物存活率为0，给药组24小时内动物存活率为20％，组织内模型组病变主要为脾脏巨噬细胞增生，吞噬现象增强，噬有大量的细胞核碎片。肾小管上皮细胞重度水肿。药物应用后脾脏和肾脏病变程度明显减轻，化合物12给药组最轻，其次是万古霉素组，化合物11给药组小鼠脾脏、肾脏病变程度也较模型组明显减轻。The standard strain of MRSA was used to infect ICR mice to form an acute peritonitis model. The results are expressed as mean ± SD (6 mice per group), **p<0.01 when compared with the negative control group, there is a significant difference, #p<0.05 and ##p<0.01 compared with MRSA-infected mice There is an extremely significant difference. The results show that: the animal survival rate within 24 hours of the model group is 0, and the animal survival rate of the drug administration group within 24 hours is 20%. The lesion in the model group in the tissue is mainly the proliferation of spleen macrophages, and the phagocytosis is enhanced. , phagocytosis of a large number of nuclear debris. Severe edema of renal tubular epithelial cells. After drug application, the degree of spleen and kidney lesions was significantly reduced, and thecompound 12 administration group was the mildest, followed by the vancomycin group. The degree of spleen and kidney lesions in thecompound 11 administration group was also significantly reduced compared with the model group.

实验例6.塞来昔布及其衍生物的酶抑制测定Experimental Example 6. Enzyme Inhibition Determination of Celecoxib and Its Derivatives

基于前期使用塞来昔布及其衍生物与多种细菌来源的酶进行分子对接，结果表明，塞来昔布及其衍生物与二氢叶酸还原酶、DNA螺旋酶、拓扑异构酶之间存在作用的可能性，因此采用金黄色葡萄球菌二氢叶酸还原酶、DNA螺旋酶以及拓扑异构酶(采用细菌培养，大肠杆菌转染、酶提取)进行体外抑制实验测定，结果如下所示：Based on the previous molecular docking of celecoxib and its derivatives with enzymes from various bacteria, the results showed that the interactions between celecoxib and its derivatives and dihydrofolate reductase, DNA helicase, and topoisomerase There is a possibility of action, so Staphylococcus aureus dihydrofolate reductase, DNA helicase and topoisomerase (using bacterial culture, Escherichia coli transfection, enzyme extraction) were used for in vitro inhibition test determination, the results are as follows:

表8塞来昔布及其衍生物对金黄色葡萄球菌中二氢叶酸还原酶、DNA螺旋酶的抑制活性结果Table 8 Celecoxib and its derivatives are the inhibitory activity results of dihydrofolate reductase and DNA helicase in Staphylococcus aureus

塞来昔布衍生物的DNA螺旋酶和拓扑异构酶的抑制凝胶图片见说明书附图5，结果表明表明化合物12与塞来昔布对二氢叶酸还原酶和DNA螺旋酶均有抑制作用，其中2,4-二甲基塞来昔布对二氢叶酸还原酶的IC₅₀为105.1μg/mL，DNA螺旋酶的抑制IC₅₀为122.8μg/mL。Inhibitory gel pictures of DNA helicase and topoisomerase of celecoxib derivatives are shown in Figure 5 of the instruction manual, and the results show thatcompound 12 and celecoxib have inhibitory effects on both dihydrofolate reductase and DNA helicase , where the IC₅₀ of 2,4-dimethylcelecoxib on dihydrofolate reductase is 105.1 μg/mL, and the IC₅₀ of DNA helicase inhibition is 122.8 μg/mL.

应当理解，以上实施例均为示例性的，不用于包含权利要求所包含的所有可能的实施方式。在不脱离本公开的范围的情况下，还可以在以上实施例的基础上做出各种变形和改变。同样的，也可以对以上实施例的各个技术特征进行任意组合，以形成可能没有被明确描述的本发明的另外的实施例。因此，上述实施例仅表达了本发明的几种实施方式，不对本发明专利的保护范围进行限制。It should be understood that the above embodiments are exemplary and not intended to cover all possible implementations covered by the claims. Various modifications and changes can also be made on the basis of the above embodiments without departing from the scope of the present disclosure. Likewise, various technical features of the above embodiments can also be combined arbitrarily to form other embodiments of the present invention that may not be explicitly described. Therefore, the above-mentioned embodiments only express several implementation modes of the present invention, and do not limit the protection scope of the patent of the present invention.

Claims (2)

1. A pharmaceutical composition for treating gram-negative bacterial infection, comprising a compound of formula 12 or a pharmaceutically acceptable salt thereof, said pharmaceutical composition further comprising polymyxin B;

2. use of a compound according to formula 12 for the preparation of a pharmaceutical composition for the treatment of gram-negative bacterial infections, said pharmaceutical composition comprising polymyxin B, said pharmaceutical composition further comprising a pharmaceutically acceptable salt of a compound according to formula 12;

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