WO2016037573A1 - 作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物、制备方法及应用 - Google Patents

作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物、制备方法及应用 Download PDF

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WO2016037573A1
WO2016037573A1 PCT/CN2015/089286 CN2015089286W WO2016037573A1 WO 2016037573 A1 WO2016037573 A1 WO 2016037573A1 CN 2015089286 W CN2015089286 W CN 2015089286W WO 2016037573 A1 WO2016037573 A1 WO 2016037573A1
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hydrogen
derivative according
difluoro
derivative
aminobenzamide
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French (fr)
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黄国贤
陈德恒
陈建辉
陈声
吕学乔
鄞荣耀
邱家琪
叶康坚
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Hong Kong Polytechnic University HKPU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A61K31/431—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems containing further heterocyclic rings, e.g. ticarcillin, azlocillin, oxacillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
    • C07C237/30—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having the nitrogen atom of the carboxamide group bound to hydrogen atoms or to acyclic carbon atoms

Definitions

  • the invention belongs to the technical field of medicine, and relates to a 3-aminobenzamide derivative, a preparation method thereof and application thereof in preparing a ⁇ -lactam auxiliary antibiotic, and the derivative can be used for treating methicillin-resistant Staphylococcus infection.
  • Staphylococcus aureus is the most common bacterial pathogen in developed countries. Severe bacterial infections caused by such pathogens, especially multidrug-resistant Staphylococcus aureus (MRSA), have caused a large number of patient deaths worldwide. Currently, more than two-thirds of the strains of Staphylococcus aureus are resistant to methicillin (ie, second-generation ⁇ -lactam antibiotics). It is worrying that clinical strains have recently emerged that are resistant to vancomycin, the ultimate drug for the treatment of MRSA. In 2005, approximately 95,000 people in the United States were infected with MRSA, nearly 19,000 of them died.
  • MRSA multidrug-resistant Staphylococcus aureus
  • CA-MRSA community-infected multidrug-resistant Staphylococcus aureus
  • the technical problem to be solved by the present invention is to provide a 3-aminobenzamide derivative for use as a ⁇ -lactam adjuvant antibiotic in view of the lack of prior art defects in the efficient treatment of MRSA-infected drugs.
  • a further problem to be solved by the present invention is to provide a process for preparing a 3-aminobenzamide derivative which is simple and efficient in process.
  • Another problem to be solved by the present invention is to provide an application of the above derivative in the preparation of a ⁇ -lactam adjuvant antibiotic.
  • a 3-aminobenzamide derivative as a ⁇ -lactam auxiliary antibiotic which is a compound having the following chemical formula (I):
  • R 1 , R 2 and R 3 are each independently hydrogen, fluorine or bromine;
  • R 4 is hydrogen, methyl, ethyl or fluorobenzyl
  • R 5 is C 4 -C 10 alkyl, alkoxyalkyl, C 4 -C 10 alkenyl, fluorine a benzyl or alkoxy substituted benzyl or acyl group
  • each of R 1 , R 2 , R 3 and R 4 is hydrogen and R 5 is an octyl group.
  • the derivative is 3-(octylamino)benzamide.
  • each of R 1 , R 2 , R 3 and R 4 is hydrogen and R 5 is a fluorenyl group.
  • the derivative is 3-(decylamino)benzamide.
  • each of R 1 , R 2 and R 3 is hydrogen, R 4 is a methyl group, and R 5 is an octyl group.
  • the derivative is 3-(methyl(octyl)amino)benzamide.
  • each of R 1 , R 2 and R 3 is hydrogen, R 4 is a methyl group, and R 5 is a fluorenyl group.
  • the derivative is 3-(methyl(indenyl)amino)benzamide.
  • each of R 1 and R 3 is hydrogen, R 2 is F, R 4 is hydrogen, and R 5 is a fluorenyl group.
  • the derivative is 2-fluoro-5-(nonylamino)benzamide.
  • each of R 1 and R 3 is hydrogen
  • R 2 and R 4 are each F
  • R 5 is a fluorenyl group.
  • the derivative is 2,4-difluoro-5-(nonylamino)benzamide.
  • step (b) When the product obtained in the step (a) is 3-(decylamino)benzamide or 3-(octylamino)benzamide, it is separately combined with dimethyl sulfate, potassium carbonate, and acetonitrile. The mixture was stirred uniformly and heated to reflux for 12 hours, and then purified to give a product.
  • the above preparation method is as follows:
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is an octyl group.
  • the derivative is 2,6-difluoro-3-(octylamino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is a fluorenyl group.
  • the derivative is 2,6-difluoro-3-(decylamino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is a heptyl group.
  • the derivative is 2,6-difluoro-3-(heptylamino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 3,7-dimethyl-2,6-octane Alkenyl.
  • the derivative is (E)-3-((3,7-dimethyl-2,6-octadienyl)amino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 2-nonenyl.
  • the derivative is (Z)-2,6-difluoro-3-(2-nonenylamino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is (4'-butoxy)butyl.
  • the derivative is 3-((4'-butoxy)butylamino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is a fluorenyl group.
  • the derivative is 3-(nonylamino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F
  • R 3 is bromine
  • R 4 is hydrogen
  • R 5 is a fluorenyl group.
  • the derivative is 4-bromo-2,6-difluoro-3-(decylamino)benzamide.
  • R 1 and R 2 are F
  • R 3 is hydrogen
  • R 4 is a methyl group
  • R 5 is an octyl group.
  • the derivative is 2,6-difluoro-3-(methyl(octyl)amino)benzamide.
  • R 1 and R 2 are F
  • R 3 is hydrogen
  • R 4 is an ethane group
  • R 5 is an octyl group.
  • the derivative is 3-(ethane(octyl)amino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F
  • R 3 is hydrogen
  • R 4 is a methyl group
  • R 5 is a fluorenyl group.
  • the derivative is 2,6-difluoro-3-(methyl(indenyl)amino)benzamide.
  • step (c) further alkylation of the 2,6-difluoro-3-(octylamino)benzamide obtained from the step (a) using methyl iodide or ethyl bromide, and uniformly stirring with potassium carbonate and acetonitrile After refluxing for 14 hours, it is purified to obtain 2,6-difluoro-3-(methyl(octyl)amino)benzamide or 3-(ethane(octyl)amino)-2,6-difluorobenzamide. ;
  • the 3-aminobenzamide derivative preferably the R 1, R 2 is F; R 3 and R 4 are each independently hydrogen, R 5 is nonyl amide group.
  • the derivative is 2,6-difluoro-3-nonyl amido benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is pyrrolidine.
  • the derivative is 2,6-difluoro-3-(pyrrolidin-1-amino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is piperidine.
  • the derivative is 2,6-difluoro-3-(piperidin-1-amino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 3', 4'-difluorobenzyl.
  • the derivative is 3-(3',4'-difluorobenzylamino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; R 3 is hydrogen, R 4 is 3', 4'-difluorobenzyl, and R 5 is 3', 4' - difluorobenzyl.
  • the derivative is 3-(bis(3',4'-difluorobenzyl)amino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 2',4'-difluorobenzyl.
  • the derivative is 3-(2',4'-difluorobenzylamino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; R 3 is hydrogen, and each of R 4 and R 5 is 2',4'-difluorobenzyl.
  • the derivative is 3-(bis(2',4'-difluorobenzyl)amino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 2',6'-difluorobenzyl.
  • the derivative is 3-(2',6'-difluorobenzylamino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; R 3 is hydrogen, and each of R 4 and R 5 is 2',6'-difluorobenzyl.
  • the derivative is 3-(bis(2',6'-difluorobenzyl)amino)-2,6-difluorobenzamide.
  • a technical solution of the third preparation method a preparation method of the above derivative, comprising the following steps:
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 3'-butoxybenzyl.
  • the derivative is 3-((3'-butoxybenzyl)amino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 3'-(sec-butoxy)benzyl.
  • the derivative is 3-((3'-(sec-butoxy)benzyl)amino)-2,6-difluorobenzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 3'-(pentyloxy)benzyl.
  • the derivative is 2,6-difluoro-3-((3'-(pentyloxy)benzyl)amino)benzamide.
  • R 1 and R 2 are F; each of R 3 and R 4 is hydrogen, and R 5 is 3'-((4"-(trifluoromethyl).
  • Benzyl)oxy)benzyl is 2,6-difluoro-3-((3'-((4"-(trifluoromethyl)benzyl)oxy)benzyl)amino)phenyl) Amide.
  • the preparation method of the above derivative includes the following steps:
  • the benzaldehyde is 3-butoxybenzaldehyde, 3-(pentyloxy)benzaldehyde , 3-(sec-butoxy)benzaldehyde or 3-((4'-(trifluoromethyl)benzyl)oxy)benzaldehyde.
  • the above preparation method is as follows:
  • a further disclosed technical solution of the present invention is the use of the above derivative in the preparation of a ⁇ -lactam adjuvant antibiotic.
  • the ratio of the amount of the derivative to the body weight of the human body is 50 mg/kg body weight, and the biocompatible of the derivative after oral administration is 12%.
  • FtsZ temperature sensitive mutant Z
  • GTP guanosine triphosphatase
  • FtsZ plays such an important role in bacterial cell division, it is considered to be a highly regarded drug target in the development of new antibacterial drugs.
  • Several high definition X-ray crystal structures are decomposed into several FtsZ homologues, presenting an important stable tissue as shown in Figure 1a, which consists of two separate foldable domains.
  • the N-terminal domain forms a GTP binding site
  • the C-terminal domain includes an elastic ring (T7 loop).
  • the two domains are connected to each other by a long central spiral 7 (H7).
  • the present invention discloses a novel compound (I) derived from 3-aminobenzamide (Fig. 1b, formula 4), which has the lowest minimum inhibitory effect against Staphylococcus aureus in an antibacterial sensitivity test. Concentration (MIC), which has low toxicity to mouse fibroblasts in cytotoxicity tests. And the present invention further bioassays the two derivatives having strong antibacterial activity, for example, Light scattering detection, GTPase activity analysis, cell morphology analysis, microscopic fluorescence analysis. All results clearly indicate that the two derivatives interact with the cell division protein FtsZ.
  • the present invention also demonstrates that such derivatives exhibit high levels of synthetic antimicrobial activity against clinically isolated, community-infected Staphylococcus aureus (USA300) and other MRSA strains after synthesis with clinically applied beta-lactam antibiotics.
  • the oral dose of F332 is 50 mg/kg body weight, and the oral biocompatibility is about 12%, which supports its use in the preparation of ⁇ -lactam-assisted antibiotics.
  • the oxyxicillin-infected drug that is, clinically, the compound of the present invention can be used as a ⁇ -lactam-assisted antibiotic to treat methicillin-resistant Staphylococcus infection by an oral route.
  • Figure 1a is the crystal structure of Staphylococcus aureus FtsZ
  • Figure 1b is the chemical structure of PC190723, its derivatives (Formula 1, 2, 3) and the basic structure of 3-aminobenzamide (Formula 4);
  • Figure 5 is a fourth preparation method disclosed in the embodiment of the present invention.
  • FIG. 6 is an electron micrograph of Bacillus subtilis 168 cells grown in a dimethyl sulfoxide solution for 4 hours according to an embodiment of the present invention
  • Figure 7 is an electron micrograph of Bacillus subtilis 168 cells grown in a 1.56 ⁇ M F332 solution for 4 hours;
  • Figure 8 is an electron micrograph of Bacillus subtilis 168 cells grown in a 0.78 ⁇ M F361 solution for 4 hours;
  • Figure 9 is an SDS-page gel diagram of Staphylococcus aureus FtsZ (trajectories 4-8);
  • Figure 10a is a structural diagram of the expression plasmid of the Bacillus subtilis FtsZ-eGFP fusion protein
  • Figure 10b is a fluorescence micrograph of B. subtilis 168 transformed with the expression plasmid of FtsZ-eGFP fusion protein and cultured for 4 hours without any of the derivatives of the present invention
  • Figure 10c is a phase contrast microscope image
  • Fig. 10d is a fluorescence micrograph of B. subtilis 168 transformed with an expression plasmid of FtsZ-eGFP fusion protein and cultured at 3 ⁇ M F332 for 4 hours
  • Fig. 10e is a phase contrast micrograph.
  • Figure 11 is a graph showing the effect of F332 on the polymerization of FtsZ in Staphylococcus aureus FtsZ (5 ⁇ M);
  • Figure 12 is a graph showing the effect of F361 on the polymerization of FtsZ in Staphylococcus aureus FtsZ (5 ⁇ M).
  • Figure 13 is the effect of F332 on GTPase activity of S. aureus FtsZ.
  • Figure 14 is the effect of F361 on the GTPase activity of S. aureus FtsZ.
  • Figure 15 is an electron micrograph of a FtsZ polymer.
  • Figure 16 is an electron micrograph of a FtsZ polymer.
  • Figure 17 is the pharmacokinetic parameters of F332.
  • the present invention divides the preparation of 3-aminobenzamide derivatives into four cases according to different raw materials and products, and lists the following four preparation methods:
  • the two compounds are further alkylated to a tertiary amine to produce F413 and F414.
  • F342 and F345 can be synthesized separately by mixing with bromodecane in acetonitrile to meet the basic conditions of reflux temperature.
  • the starting material A is 3-aminobenzamide, 2-fluoro-5-aminobenzamide or 2,4-difluoro-5-aminobenzamide
  • starting material B is bromodecane or bromooctane
  • step (b) When the product obtained in the step (a) is 3-(decylamino)benzamide or 3-(octylamino)benzamide, it is separately combined with dimethyl sulfate, potassium carbonate, and acetonitrile. Stir well and heat to reflux for 12 hours to give the product.
  • the second preparation method synthesis of compounds F332, F333, F334, F350, F361, F369, F370, F371, F391, F409 and F410 (see the corresponding numbered compounds in Figure 3), as in the following Examples 7-18:
  • Figure 3 shows the synthetic scheme of the compounds F332, F333, F334, F350, F361, F369, F370, F371, F391, F409 and F410 of the present invention.
  • 2,6-difluoro-3-aminobenzamide utilizes commercially available 2,6-difluoro-3-nitrobenzoic acid ( 2,6-difluoro-3-nitrobenzonic acid) is carried out in two steps.
  • 2,6-difluoro-3-nitrobenzoic acid and thionyl chloride are dissolved in an aqueous ammonia solution to produce 2,6-difluoro-3-nitrobenzamide (2,6-difluoro).
  • -3-nitrobenzamide which is then further converted to 2,6-difluoro-3-aminobenzamide by the incorporation of concentrated hydrochloric acid using stannous chloride (tin(II) chloride).
  • 2,6-difluoro-3-aminobenzamide is mixed with various alkyl halides, such as bromodecane, bromooctane, bromodecane, 1-bromo-4-butoxybutane 1-bromo-4-butoxybutane, geranyl bromide, (Z)-1-bromo-2-nonene [trans-1-bromonon-2-ene], bromoheptane ( 1-bromoheptane), which was mixed in acetonitrile (ACN) for 4 hours to synthesize F332, F333, F334, F350, F369, F370, F391.
  • alkyl halides such as bromodecane, bromooctane, bromodecane, 1-bromo-4-butoxybutane 1-bromo-4-butoxybutane, geranyl bromide, (Z)-1-bromo-2-nonene [trans-1-bromonon-2-ene], bromoh
  • 2,6-Difluoro-3-nitrobenzoic acid 44 g, 216 mmol
  • thionyl chloride 100 mL
  • 2,6-Difluoro-3-nitrobenzonic acid chloride which was used in the next step without further purification.
  • 2,6-Difluoro-3-nitrobenzenetoluoyl chloride was dropwise added to a uniformly stirred 30% aqueous ammonia solution (300 mL) at a temperature of 0 °C.
  • Figure 4 shows the chemical synthesis pathways of the compounds F357, F358, and F362 to F368 of the present invention.
  • Compound F368 was prepared by dissolving 2,6-difluoro-3-aminobenzamide and nonanoyl chloride at 0 ° C in pyridine and dichloromethane. (dichloromethane) 4 hours, as shown in Figure 4.
  • F357 and F358 are synthesized by 2,6-difluoro-3-aminobenzamide, 1,4-bromopentane, 1, 5-Dibromobutane (1,4-dibromobutane) was added to potassium carbonate in acetonitrile and stirred uniformly for 4 hours.
  • F362, F363, F364, F365, F366, F367 were also synthesized from 2,6-difluoro-3-aminobenzamide by 3,4-difluorobromobenzyl (3,4-difluorobenzyl bromide), 2,4-difluorobenzyl bromide, 2,6-difluorobenzyl bromide, potassium carbonate, in acetonitrile Heat to reflux for 4 hours.
  • Example 21 3-((3',4'-difluorobenzyl)amino)-2,6-difluorobenzamide (F362) and 3-(bis(3',4'-difluoro) Benzyl)amino)-2,6-difluorobenzamide (F363)
  • Example 22 3-((2',4'-difluorobenzyl)amino)-2,6-difluorobenzamide (F364) and 3-(bis(2',4'-difluoro) Benzyl)amino)-2,6-difluorobenzamide (F365)
  • F364 (0.29 g, 42%) and F365 (0.21 g, 21%) were obtained by 2,6-difluoro-3-aminobenzamide (0.40 g, 2.3, respectively) Methyl) and 2,4-difluorobenzyl bromide (0.60 g, 2.9 mmol), acetonitrile (20 mL) and potassium carbonate K 2 CO 3 (0.42 g, 3.0 mmol) And F363's preparation process.
  • Example 23 3-((2',6'-difluorobenzyl)amino)-2,6-difluorobenzamide (F366) and 3-(bis(2',6'-difluoro) Benzyl)amino)-2,6-difluorobenzamide (F367)
  • F366 (0.29 g, 42%) and F367 (0.21 g, 21%) were obtained by using 2,6-difluoro-3-aminobenzamide (0.40 g, 2.3 mmol) and 2,6-difluorobenzyl bromide, respectively.
  • (2,6-difluorobenzyl bromide) (0.60 g, 2.9 mmol)
  • acetonitrile (20 mL
  • potassium carbonate K 2 CO 3 (0.42 g, 3.0 mmol
  • the fourth preparation method synthesis of compounds F373, F376, F377 and F378 (specifically corresponding to Figure 5 The same numbered compounds), as in Examples 25-28:
  • Figure 5 shows the synthetic route of the compounds F373, F376, F377 and F378 of the present invention. These compounds are obtained by reductive amination of 2,6-difluoro-3-aminobenzamide with the corresponding benzaldehyde, and the yield is high.
  • the specific experimental procedure is shown in Figure 5: (a) various aldehydes, catalyst (cat.) p-toluenesulfonic acid (pTsOH), methanol (MeOH), room temperature (rt), 2 hours, sodium cyanoborohydride (NaBH 3 CN), 12 hours.
  • This compound (0.13 g, 38%) was obtained by using 2,6-difluoro-3-aminobenzamide (0.17 g, 1.0 mmol), 3-(pentyloxy)benzaldehyde (3-(pentyloxy)benzaldehyde) (0.19 g, 1.0 mmol)], p-toluenesulfonic acid monohydrate (0.02 g, 0.11 mmol), methanol (10 mL) and sodium cyanoborohydride (0.63 g, 10 mmol) After mixing, it was obtained according to the above-mentioned F373 preparation procedure.
  • This compound (0.12 g, 36%) was obtained by using 2,6-difluoro-3-aminobenzamide (0.17 g, 1.0 mmol), 3-(s). 3-(sec-butoxy)benzaldehyde (0.17 g, 1.0 mmol), p-toluenesulfonic acid monohydrate (0.02 g, 0.11 mmol), methanol (10 mL) and cyanide Sodium cyanoborohydride (0.63 g, 10 mmol) was mixed and obtained according to the procedure described above for F373.
  • This compound (0.16 g, 37%) was obtained by using 2,6-difluoro-3-aminobenzamide (0.17 g, 1.0 mmol), 3-((4-(trifluoromethyl)benzyl)oxy) 3-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde) (0.28 g, 1.0 mmol), p-toluenesulfonic acid monohydrate (0.02 g, 0.11 mmol), methanol ( 10 mL) and sodium cyanoborohydride (0.63 g, 10 mmol) were mixed and obtained according to the procedure of the above F373.
  • the strains used in the microbial sensitivity test were Escherichia coli, Staphylococcus aureus 29213 and Bacillus subtilis 168.
  • Escherichia coli and Staphylococcus aureus 29213 were obtained through the laboratory of the Hong Kong Polytechnic University, and Bacillus subtilis 168 was purchased from the American Standard Library.
  • the mammalian cell line L929 mouse peritoneal fibroblast
  • LCC6 a native breast cancer cell
  • LCC6MDR P-gp high expression breast cancer cell line
  • Nutrient Agar, tryptone and yeast extract were purchased from Oxoid Limited (Niping, Ontario, Canada).
  • LB liquid medium (Luria-Bertani) was obtained by dissolving 2 g of tryptone, 1 g of yeast extract and 2 g of sodium chloride in 200 ml of deionized water.
  • Müller-Hinton liquid medium (MHB) for microbial susceptibility testing, positive ion regulated Müller-Hinton liquid medium (MHB) and trypticase soy medium (TSB) were purchased from BD Corporation of the United States (New Jersey, USA) ).
  • Bacterial growth and cytotoxicity were measured using a microplate reader (Bio-Rad Laboratories, Model 680). Use Olympus Bacillus subtilis cells were observed by a biological imaging navigation microscope.
  • MIC minimum inhibitory concentration
  • the OD 600 value was measured using a microplate reader, and the percentage of inhibitory concentration was calculated. If the MIC value is equal to or higher than 90%, the compound should be determined to be active, meaning that the compound inhibits bacterial growth by more than 90%. Table 1 summarizes the MICs for all synthetic compounds.
  • the cell lines used in the cytotoxicity test were obtained from the inventory of the Polytechnic University.
  • mouse fibroblasts (L929), a native breast cancer cell line (LCC6), and a P-gp-high expression breast cancer cell line (LCC6MDR) were mixed with various concentrations of compounds at 96-
  • the final volume of the microtiter plate reached 100 ⁇ L.
  • the cells were cultured for 3 days at 37 °C. Determining the value of a half-maximal inhibition of a compound (IC 50) is obtained by CellTiter 96AQueous One Solution Cell Proliferation Assay Kit (Promega).
  • F361 extended the cell length of B. subtilis by a factor of six, from 3.5 ⁇ 0.7 ⁇ m to 21.1 ⁇ 1.8 ⁇ m.
  • the study found that F332 and F361 can produce Bacillus subtilis 168 cells at a sublethal concentration to produce a filamentous morphology.
  • the results show that both compounds can inhibit bacterial growth by blocking cytoplasmic cell division proteins.
  • FtsZ-eGFP fusion protein expression plasmid was constructed as follows (the plasmid structure is shown in Figure 10a):
  • Primer pairs were designed based on the DNA sequence of plasmid pGK12 of a Gram-positive bacterium (G+):
  • the amplified PCR fragment contains the chloramphenicol resistance gene, the G+ origin of replication, and most of the pGK12 sequence.
  • a PCR fragment of about 1,000 base pairs was amplified, containing the origin of replication of pBR322 and the T7 promoter.
  • the two PCR fragments were digested with restriction endonucleases XbaI and BglII, ligated and transformed into E. coli BL21 (DE3), and screened on chloramphenicol plates to obtain plasmid pFZ1.0.
  • Amplification of the PCR fragment approximately 3,800 base pairs in length, removed excess pGK12 portion.
  • a PCR fragment of about 1,200 base pairs was amplified, containing the lacA gene and its lacI promoter.
  • the two PCR fragments were digested with restriction endonucleases XhoI and EcoRI, ligated and transformed into E. coli BL21 (DE3), and screened on chloramphenicol plates to obtain plasmid pFZ1.1.
  • Primer pairs were designed based on the DNA sequence of pFZ1.1:
  • PCR fragment was amplified, approximately 5,000 base pairs in length, and digested with restriction enzymes EcoRI and BamHI. Primer pairs were designed based on the DNA sequence of G+ vector pHT43 (MoBiTec):
  • a PCR fragment of about 450 base pairs was amplified, containing the groE promoter and the lacO sequence, and digested with the restriction enzymes EcoRI and BglII. The two fragments were ligated and transformed into E. coli BL21 (DE3), and screened on chloramphenicol plates to obtain plasmid pFZ2.0.
  • Primer pairs were designed based on the FtsZ gene sequence of Bacillus subtilis 168:
  • the PCR fragment was amplified and was approximately 1,200 base pairs in length.
  • primer pairs were designed based on the DNA sequence of pEGFP:
  • the eGFP gene fragment of about 700 base pairs was amplified, and the two fragments were ligated by the overlap extension PCR method, and then digested with the endonucleases EcoRI and BamHI and ligated to the corresponding inscribed sites of pFZ2.0.
  • the cells were transformed into E. coli BL21 (DE3) and screened on chloramphenicol plates to obtain plasmid pFZ-FtsZ-eGFP.
  • the extracted plasmid was transformed into Bacillus subtilis 168, grown in LB medium containing chloramphenicol, and the expression of FtsZ-eGFP gene was observed by adding different concentrations of lactose or IPTG.
  • the B. subtilis shown in Figures 10b and 10c was cultured without F332, and a strong green fluorescence appeared in the middle part of the B. subtilis cell of Figure 10b under a fluorescence microscope, indicating that the expression plasmid of the FtsZ-eGFP fusion protein was localized and Z-ring integrated into the middle of the cell to form a cell division.
  • the Bacillus subtilis shown in Fig. 10d and 10e was cultured in the case of F332, and under the fluorescence microscope, green fluorescence was observed in various parts of the Bacillus subtilis cell in Fig. 10d, and the cells were grown, reflecting the expression plasmid of the FtsZ-eGFP fusion protein.
  • the location in the cell is affected by F332. Therefore, the expression of this protein is not limited to the location where cell division is performed.
  • E. coli BL21 cells carrying the Staphylococcus aureus ftsz gene were obtained from the laboratory of the University of Technology.
  • the Escherichia coli strain extracted from the bacterial library was cultured in 5 mL of LB medium and 100 ⁇ g/mL ampicillin for 14-16 hours at a temperature of 37 °C.
  • the overnight cultured cell culture medium (2 mL) was transferred to 200 mL of 2X TY medium containing 100 ⁇ g/mL ampicillin, and the cells were grown at 37 ° C until the optical absorbance (OD 600 ) reached 0.8.
  • Protein expression was induced 4 hours after the addition of 0.4 mM isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG).
  • Bacterial cells were aggregated under a centrifugal force of 9000 rpm for 20 minutes at a temperature of 4 °C. The cell pellet was then resuspended in 20 mL of starting buffer (0.02 M sodium phosphate, 0.5 M sodium chloride, pH 7.4). The cells were homogenized by sonication, increasing by 50%, pulsed for 30 seconds, and pulsed for 30 seconds, for 5 cycles. The insoluble cell debris was removed under a 1.5 hour 13,000 rpm centrifugal separation force at a temperature of 4 °C. The float layer was collected and filtered through a 0.22 ⁇ m sterile filter.
  • the FtsZ protein in the floating layer was purified by Fast Protein Liquid Chromatography (FPLC).
  • FPLC Fast Protein Liquid Chromatography
  • a 5 mL HiTrap chelate layer was activated with a nickel (II) sulfate solution to capture the S. aureus FtsZ protein. It is then equilibrated with the starting buffer.
  • a soluble protein solution comprising the protein of interest is passed through the chelate layer along with the starting buffer.
  • the target protein (Peak A) was then eluted with an elution buffer (0.02 M sodium phosphate, 0.5 M sodium chloride, 0.5 M isopyrazole, pH 7.4). Collection and analysis were performed by SDS-PAGE (Fig. 9), followed by dialysis against 50 mM MOPS buffer at a storage temperature of -80 °C.
  • the light scattering test can determine the extent of FtsZ polymerization by measuring the 90° light scattering signal using a fluorescence spectrometer (Perkin-Elmer, USA). The excitation and emission wavelengths were set to 600 nm and the slit width was 5 nm. Staphylococcus aureus FtsZ (12 ⁇ M) was incubated for 10 minutes under continuous dilution in 50 mM MOPS buffer (pH 6.8) at a temperature of 25 °C. The control sample included only 1% dimethyl sulfoxide. KCl (final concentration: 50 mM) and MgCl 2 (final concentration: 10 mM) were then added to the mixture, and cultured at 37 ° C for 8 minutes to establish a baseline.
  • KCl final concentration: 50 mM
  • MgCl 2 final concentration: 10 mM
  • the final concentration of 1 mM GTP was then added and the light scattering signal was measured for 1,500 seconds.
  • the calculation of the relevant light scattering signal in the test device is based on a comparative test. Three tests were performed for each compound concentration. Two compounds, F332 and F361, were selected for testing, and the results are shown in Figures 11 and 12, respectively. Both compounds inhibit light scattering of FtsZ polymerization. The reduced light scattering signal shows that both compounds reduce the polymeric nature of the FtsZ fibrils in a dose dependent manner.
  • the GTPase activity of S. aureus FtsZ was determined by a 96-well miniplate, and the concentration of released inorganic phosphate during GTP hydrolysis was tested using the CytoPhosTM phosphate assay Biochem KitTM (Cytoskeleton, USA). .
  • Recombinant Staphylococcus aureus FtsZ (7.5 ⁇ M) was incubated for 10 minutes under continuous dilution in 50 mM MOPS buffer (pH 6.5) at a temperature of 25 °C.
  • the control sample included only 1% dimethyl sulfoxide.
  • KCl final concentration: 200 mM
  • MgCl 2 final concentration: 5 mM
  • GTP final concentration: 500 mM
  • the insoluble polymer was removed by centrifugation at 13,500 rpm for 1 minute at room temperature.
  • 80 ⁇ L of the floating layer was transferred to a well of a 96-well microplate and incubated with 120 ⁇ L of Cytophos reagent for 10 minutes at room temperature.
  • the inorganic phosphate in the mixture was determined by measuring the absorbance at 650 nm using a 96-well microplate reader (BioRad 680).
  • test results of the samples are based on comparative tests. Three tests were performed for each compound concentration. Two compounds, F332 and F361, were selected for testing, and the results are shown in Figures 13 and 14, respectively. As shown in Figures 13 and 14, no significant changes in GTPase activity of S. aureus FtsZ were observed after the addition of the two compounds. Based on the results of light scattering tests and GTPase activity tests, the two compounds are likely to have the ability to inhibit the polymerization of FtsZ without affecting GTPase activity.
  • TEM provides a direct observation of the morphology of FtsZ polymerization.
  • Two compounds, F332 and F361 were selected for testing.
  • Staphylococcus aureus FtsZ (12 ⁇ M) was cultured in various concentrations of compounds and incubated in 50 mM MOPS buffer (pH 6.5) for 10 minutes at a temperature of 25 °C.
  • the control sample included only 1% dimethyl sulfoxide.
  • KCl final concentration: 50 mM
  • MgCl 2 final concentration: 5 mM
  • GTP final concentration: 1 mM
  • the fibrils obtained after the addition of the inhibitor were compared with the relatively short linear single-chain FtsZ fibrils produced by the polymerization of 100 ⁇ M F332 or F361. This phenomenon coincides with the light scattering test because both F332 and F361 compounds exhibit inhibition of FtsZ polymerization in a dose-dependent manner.
  • the strains used in the present invention are clinically isolated MRSA strains USA300#757 and #2960, both obtained from the laboratory of the Polytechnic University.
  • the previously used Müller-Hinton Liquid Medium (MHB) dilution method is used to determine the Grading Inhibitory Concentration Index (FICI) 23 .
  • 12 rounds of serial dilutions were performed on PC190723, and F332 and F361 were prepared using 96-well microplates at a concentration ranging from 512 ⁇ g/mL to 0.25 ⁇ g/mL.
  • a series of double dilutions of ampicillin, cloxacillin and cefuroxime were performed using the same procedure.
  • each microplate plate contains 100 ⁇ L of MHB medium supplemented with compound alone, supplemented with ⁇ -lactams alone, or supplemented with a mixture of compound and ⁇ -lactam.
  • Staphylococcus aureus cultured overnight in Müller-Hinton liquid medium was diluted in saline to an OD 595 value of 0.08-0.1, added to each microplate well, and cultured at 37 ° C for 20 hours. All tests were repeated once to visually judge the degree of inhibition of bacterial growth.
  • FICI (MIC of compound and ⁇ -lactam mixture / MIC of compound) + (MIC of MIC/ ⁇ -lactam of compound and ⁇ -lactam mixture) .
  • Table 3 The results are shown in Table 3.
  • F332 was administered with the strains in Table 4 in combination with five antibiotics, respectively.
  • the different FICI values are shown in Table 4:
  • the UPLC-MS/MS system includes Waters Ultra High Performance Liquid Chromatography (Acquity Waters UPLC) coupled to a mass spectrometer (Micromass type Quattro Ultima) using positive ion mode electrospray ionization.
  • the ACQUITY UPLC BEH C18 1.7 ⁇ M (2.1 x 50 mm) layer was chromatographed. (Chromatographic Analysis)
  • the mobile phase included methanol + 0.1% formic acid (solvent B) and Milli-Q ultrapure water + 0.1% formic acid (dissolved A).
  • the multiple reaction monitoring mode was set to monitor F332[M+H] + at 299 > 142 m/z.
  • Collision energy, injection cone voltage, source temperature, desolvation temperature, and capillary voltage were 25, 30, 150 ° C, 350 ° C, and 3 Kv, respectively.
  • the cone gas volume and the de-dissolved gas volume were 150 L/Hr and 600 L/Hr, respectively.
  • mice Male Sprague–Dawley (SD) mice (body weight 250-280 g) were obtained from the Animal Facilities Center of the Hong Kong Polytechnic University. Animals were housed under specially controlled temperature and humidity conditions, maintained for 12 hours of normal light, and fed standard amounts of food and water. The Hong Kong Polytechnic University Animal Subject Ethics Committee agrees Animal experiment agreement. The right jugular vein puncture was performed the day before the experiment. Animals were fasted the night before and water could be fed during the experiment.
  • F332 was prepared with 5% castor oil, Cremophor EL, 5% ethanol, and 90% saline at a concentration of 2 mg/ml. F332 was prepared on the day of use and used for animal research within half an hour.
  • F332 was administered by oral feeding and intravenous (IV).
  • Blood samples (approximately 500 ⁇ L) were administered intravenously by penetrating heparin tubes (20 u nits) in the jugular vein and 5, 10, 30, 45, 60, 120, 240 and 420 minutes after use.
  • plasma samples were taken at 2, 10, 30, 45, 60, 120, 240, 480 and 600 minutes and collected for 10 minutes under a centrifugal force of 16,100 G. All plasma samples were spiked with 3 times the volume of methanol.
  • the floating layer was filtered through a syringe filter prior to UPLC-MS/MS analysis.
  • Pharmacokinetic parameters in plasma were analyzed by non-segmented pharmacokinetic data by Pharmacokinetics Solutions 2.0 software (manufactured by Summit Research Services, Montrose, USA).
  • the determined pharmacokinetic parameters include maximum plasma concentration (Cmax), maximum plasma concentration time (Tmax), mean residence time (MRT), absorption/distribution half-life (t1/2 ⁇ ), elimination half-life (t1/2 ⁇ ), distribution Capacity (Vd), total clearance (CL), and area under the concentration curve (AUC). The results are shown in Figure 17 and Table 5.
  • the preparation method of the present invention is a novel derivative derived from 3-aminobenzamide, for various strains such as (Gram-positive and Gram-negative), cancer.
  • Cells (LCC6 and LCC6MDR) and mouse peritoneal fibroblasts (L929) have cytotoxic/strong antibacterial activity; have low toxicity to mouse fibroblasts; interact with cell division protein FtsZ; After synthesis of ⁇ -lactam antibiotics, it showed high synthetic antibacterial activity against clinically isolated and community-infected Staphylococcus aureus (USA300) and other MRSA strains, and the inhibitory concentration index was as low as 0.01; pharmacodynamic studies showed that One of the derivatives, F332, is orally administered at a dose of 50 mg/kg of body weight and has an oral biocompatibility of about 12%; the derivative of the present invention is used in the preparation of a medicament for treating methicillin-resistant Staphylococcus infection. It can be used clinically as a

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Abstract

一种作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物、制备方法及应用，衍生物为化学式（I）的化合物。该衍生物能作为β-内酰胺辅助抗生素，用于治疗耐甲氧西林葡萄球菌感染。

Description

作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物、制备方法及应用 技术领域
本发明属于医药技术领域，涉及一种3-氨基苯甲酰胺衍生物、制备方法及在制备β-内酰胺辅助抗生素中的应用，该衍生物可以用于治疗耐甲氧西林葡萄球菌感染。
背景技术
金黄葡萄球菌是世界发达国家最普遍的细菌性病原体。这类病原体导致的严重的细菌感染，尤其是多药耐药金黄葡萄球菌(MRSA)已经在世界上造成大量病人死亡。目前，金黄葡萄球菌中有超过三分之二的菌种对甲氧西林(即第二代β-内酰胺抗生素)具有耐药性。令人担忧的是，近来已经出现了对治疗MRSA的终极药物盐酸万古霉素(vancomycin)具有耐药性的临床菌株。2005年，在美国约有95,000人感染MRSA，其中接近19,000死亡。在香港，MRSA也成为最重要的一种病原体，尤其是社区感染的多药耐药金黄葡萄球菌(CA-MRSA)。所通报的CA-MRSA感染病例从2007年的82例到2013年的988例一直在迅速飙升，七年内大约增加了12倍。在这些通报的各类传染病例中，CA-MRSA是香港最严重的四种传染病之一。更为重要的是，根据香港特别行政区卫生署卫生防护中心2007年发布的报告称，两名病人因感染CA-MRSA死亡。鉴于MRSA的出现和临床上用以治疗的抗生素种类有限，迫切需要开发新药品治疗MRSA感染。
发明内容
本发明要解决的技术问题在于，针对现有技术中缺少高效治疗MRSA感染药物的缺陷，提供一种用于作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物。
本发明进一步要解决的问题就是提供一种工艺简单、高效的3-氨基苯甲酰胺衍生物的制备方法。
本发明另外要解决的问题就是提供一种上述衍生物在制备一种β-内酰胺辅助抗生素中的应用。
本发明解决其技术问题所采用的技术方案是：一种作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物，为具以下化学式(I)的化合物:
Figure PCTCN2015089286-appb-000001
其中，R1、R2和R3各自分别为氢、氟或溴；
R4与R5不同时，R4为氢、甲基、乙基或氟代苄基，R5为C4-C10的烷基、烷氧基烷基、C4-C10的烯基、氟代苄基、烷氧基取代的苄基或酰基；
R4和R5相同时，R4和R5都为CH2-(CH2)n-CH2，n＝2或3。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2、R3和R4各自分别为氢，R5为辛基。所述衍生物为3-(辛基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2、R3和R4各自分别为氢，R5为壬基。所述衍生物为3-(壬基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2和R3各自分别为氢，R4为甲基，R5为辛基。所述衍生物为3-(甲基(辛基)氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2和R3各自分别为氢，R4为甲基，R5为壬基。所述衍生物为3-(甲基(壬基)氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1和R3各自分别为氢，R2为F，R4为氢，R5为壬基。所述衍生物为2-氟-5-(壬基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1和R3各自分别为氢，R2和R4各自为F，R5为壬基。所述衍生物为2,4-二氟-5-(壬基氨基)苯甲酰胺。
第一种制备方法的技术方案：上述衍生物的制备方法，包括以下步骤：
(a)将原料A与原料B、碳酸钾和乙腈均匀搅拌形成反应混合液，加热回流反应4小时，再精制后得到产物；原料A为3-氨基苯甲酰胺、2-氟-5-氨基苯甲酰胺或2,4-二氟-5-氨基苯甲酰胺；原料B为溴代壬烷或溴代辛烷；
(b)在步骤(a)得到的产物为3-(壬基氨基)苯甲酰胺或3-(辛基氨基)苯甲酰胺时，再将其分别与硫酸二甲酯、碳酸钾、和乙腈均匀搅拌，并加热回流12小时，再精制后得到产物。上述制备方法如下所示：
Figure PCTCN2015089286-appb-000002
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为辛基。所述衍生物为2,6-二氟-3-(辛基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为壬基。所述衍生物为2,6-二氟-3-(壬基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为庚基。所述衍生物为2,6-二氟-3-(庚基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为3,7-二甲基-2,6-辛二烯基。所述衍生物为(E)-3-((3,7-二甲基-2,6-辛二烯)氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为2-壬烯基。所述衍生物为(Z)-2,6-二氟-3-(2-壬烯基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为(4’-丁氧基)丁基。所述衍生物为3-((4’-丁氧基)丁基氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为癸基。所述衍生物为3-(癸氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F，R3为溴，R4为氢，R5为壬基。所述衍生物为4-溴-2,6-二氟-3-(壬基氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F，R3为氢，R4为甲基，R5为辛基。所述衍生物为2,6-二氟-3-(甲基(辛基)氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F，R3为氢，R4为乙烷基，R5为辛基。所述衍生物为3-(乙烷基(辛基)氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F，R3为氢，R4为甲基，R5为壬基。所述衍生物为2,6-二氟-3-(甲基(壬基)氨基)苯甲酰胺。
第二种制备方法的技术方案：上述衍生物的制备方法，包括以下步骤：
(a)将原材料2,6-二氟-3-氨基苯甲酰胺与溴代烷烃、碳酸钾及乙腈均匀搅拌 形成反应混合液，并加热回流4小时，再精制后得到产物；所述溴代烷烃为溴代壬烷、溴代辛烷、溴代癸烷、溴化香叶醇、(Z)-1-溴-2-壬烯或溴代庚烷；
(b)将步骤(a)反应得到的2,6-二氟-3-(壬基氨基)苯甲酰胺进行溴化处理并在室温下搅拌12小时，精制得到4-溴-2,6-二氟-3-(壬基氨基)苯甲酰胺；
(c)使用碘甲烷或溴乙烷对从步骤(a)得到的2,6-二氟-3-(辛基氨基)苯甲酰胺，进一步烷基化处理，并与碳酸钾和乙腈均匀搅拌回流14小时，精制得到2,6-二氟-3-(甲基(辛基)氨基)苯甲酰胺或3-(乙烷基(辛基)氨基)-2,6-二氟苯甲酰胺；
(d)使用硫酸二甲酯对从步骤(a)得到的2,6-二氟-3-(壬基胺基)苯甲酰胺进行烷基化处理，并与碳酸钾和乙腈均匀搅拌回流12小时，精制得到2,6-二氟-3-(甲基(壬基)氨基)苯甲酰胺。上述制备方法如下所示：
Figure PCTCN2015089286-appb-000003
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为壬酰胺基。所述衍生物为2,6-二氟-3-壬酰胺基苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为吡咯烷。所述衍生物为2,6-二氟-3-(吡咯烷-1-氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为哌啶。所述衍生物为2,6-二氟-3-(哌啶-1-氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为3’,4’-二氟苄基。所述衍生物为3-(3’,4’-二氟苄氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3为氢，R4为3’,4’-二氟苄基，R5为3’,4’-二氟苄基。所述衍生物为3-(双(3’,4’-二氟苄基)氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为2’,4’-二氟苄基。所述衍生物为3-(2’,4’-二氟苄氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3为氢，R4、R5各自分别为2’,4’-二氟苄基。所述衍生物为3-(双(2’,4’-二氟苄基)氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为2’,6’-二氟苄基。所述衍生物为3-(2’,6’-二氟苄氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3为氢，R4、R5各自分别为2’,6’-二氟苄基。所述衍生物为3-(双(2’,6’-二氟苄基)氨基)-2,6-二氟苯甲酰胺。
第三种制备方法的技术方案：上述衍生物的制备方法，包括以下步骤：
(a)将原材料2,6-二氟-3-氨基苯甲酰胺和壬酰氯在0℃下溶入吡啶和二氯甲烷反应4小时，精制得到2,6-二氟-3-壬基氨基苯甲酰胺；
(b)将原材料2,6-二氟-3-氨基苯甲酰胺与1,4-二溴丁烷或1,5-二溴代戊烷分别加入碳酸钾在乙腈中均匀搅拌4小时反应，精制分别得到2,6-二氟-3-(吡咯 烷-1-氨基)苯甲酰胺和2,6-二氟-3-(哌啶-1-氨基)苯甲酰胺；
(c)将原材料2,6-二氟-3-氨基苯甲酰胺与原料C以及碳酸钾，在乙腈中加热回流4小时，精制分别得到3-(3’,4’-二氟苄氨基)-2,6-二氟苯甲酰胺或3-(双(3’,4’-二氟苄基)氨基)-2,6-二氟苯甲酰胺、3-(2’,4’-二氟苄氨基)-2,6-二氟苯甲酰胺或3-(双(2’,4’-二氟苄基)氨基)-2,6-二氟苯甲酰胺、以及3-(2’,6’-二氟苄氨基)-2,6-二氟苯甲酰胺或3-(双(2’,6’-二氟苄基)氨基)-2,6-二氟苯甲酰胺；原料C为3,4-二氟溴苄、2,4-二氟溴苄或2,6-二氟溴苄。上述制备方法如下所示：
Figure PCTCN2015089286-appb-000004
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为3’-丁氧基苄基。所述衍生物为3-((3’-丁氧基苄基)氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别 为氢，R5为3’-(仲丁氧基)苄基。所述衍生物为3-((3’-(仲丁氧基)苄基)氨基)-2,6-二氟苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为3’-(戊氧基)苄基。所述衍生物为2,6-二氟-3-((3’-(戊氧基)苄基)氨基)苯甲酰胺。
所述3-氨基苯甲酰胺衍生物中，优选所述R1、R2为F；R3和R4各自分别为氢，R5为3’-((4”-(三氟甲基)苄基)氧)苄基。所述衍生物为2,6-二氟-3-((3’-((4”-(三氟甲基)苄基)氧)苄基)氨基)苯甲酰胺。
第四种制备方法的技术方案：上述衍生物的制备方法，包括以下步骤：
(a)将原材料2,6-二氟-3-氨基苯甲酰胺分别与相应的苯甲醛以及催化剂对甲苯磺酸(pTsOH)和甲醇进行还原胺化，在室温下混合液搅拌2小时；
(b)然后分部分加入氰基硼氢化钠，再混合搅拌12小时，反应精制分别得到上述的衍生物；所述苯甲醛为3-丁氧基苯甲醛、3-(戊氧基)苯甲醛、3-(仲丁氧基)苯甲醛或3-((4’-(三氟甲基)苄基)氧)苯甲醛。上述制备方法如下所示：
Figure PCTCN2015089286-appb-000005
本发明另外公开的技术方案为：上述衍生物在制备一种β-内酰胺辅助抗生素中的应用。
所述衍生物的用量与人体体重的比例为50毫克/千克体重，衍生物在口服后的生物兼容性为12％。
近来，细菌细胞分裂机制在学术界和医疗产业引起了极大关注，旨在探寻新型抗菌药物的药物靶标。在目前这些已知选项中，长丝的温度敏感突变体Z(FtsZ)是最有希望和最具特色的一种。FtsZ是一种鸟苷三磷酸酶(guanosine triphosphatase)，在多种细菌中具有高度保守性。为使细菌进行细胞分裂，FtsZ单体首先定位于细胞中部，以鸟苷三磷酸酶(GTP)的独立方式与直线单链原纤维头尾相连进行聚合。单链FtsZ原纤维之间的侧部接触首先形成FtsZ束，最终形成Z环。随后引入其它下行蛋白质形成细胞膜和细胞壁，从而完成细胞分裂过程。正因为FtsZ在细菌细胞分裂过程中发挥了如此重要的作用，它在开发新型抗菌药物中被认为是一种备受关注的药物靶标。几种高清晰度的X光晶体机构分解为若干FtsZ同系物，呈现出一种如图1a所示的重要的稳定组织，该组织由两个独立的可折叠域组成。N-终端域形成三磷酸鸟苷结合位点(GTP binding site)，C-终端域包括弹性环(T7环)。两域通过一条长的中心螺旋线7(H7)相互连接。据已有报告称，多种FtsZ互动化合物与三磷酸鸟苷结合位点或T7环绑定连接，一些化合物具有强大的抗菌活性，在微摩尔级水平上达到最低抑菌浓度(MIC)。
本发明公开一种衍生自3-氨基苯甲酰胺(3-aminobenzamide)(图1b，式4)的新型化合物(I)，该化合物在抗菌敏感性试验中对金黄葡萄球菌具有最低的最小抑菌浓度(MIC)，在细胞毒性测试中对小鼠成纤维细胞具有低毒性。并且本发明进一步对这两种具有强大的抗菌活性的衍生物进行生物测定，例如 光散射检测、GTPase活性分析、细胞形态学分析、显微镜下荧光分析。所有结果均清楚表明这两种衍生物与细胞分裂蛋白FtsZ有相互作用。本发明也证明该类衍生物与临床应用的β-内酰胺抗生素合成后表现出对临床分离而社区感染的金黄葡萄球菌(USA300)和其它MRSA菌株具有高度的合成抗菌活性。在药代动力学研究结果中，F332的口服用量为50毫克/千克体重，口服的生物兼容性约为12％，从而支持其用于β-内酰胺辅助抗生素制备上，通过口服途径治疗耐甲氧西林葡萄球菌感染药物上，即在临床上，本发明化合物可用作β-内酰胺辅助抗生素通过口服途径治疗耐甲氧西林葡萄球菌感染。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1a是金黄葡萄球菌FtsZ的晶体结构；
图1b是PC190723的化学结构式、其衍生物(式1，2，3)以及3-氨基苯甲酰胺(式4)的基本结构；
图2是本发明实施例公开的第一种制备方法；
图3是本发明实施例公开的第二种制备方法；
图4是本发明实施例公开的第三种制备方法；
图5是本发明实施例公开的第四种制备方法；
图6是本发明实施例枯草杆菌168细胞在二甲亚砜溶液中生长4小时电镜图；
图7是枯草杆菌168细胞在1.56μM F332溶液中生长4小时电镜图；
图8是枯草杆菌168细胞在0.78μM F361溶液中生长4小时电镜图；
图9是金黄葡萄球菌FtsZ(轨迹4-8)的SDS-page凝胶图；
图10a是枯草杆菌FtsZ-eGFP融合蛋白的表达质粒结构图；
图10b是枯草杆菌168经FtsZ-eGFP融合蛋白的表达质粒转化后并在没有任何本发明的衍生物影响的情况下培养4小时后荧光显微镜图；
图10c是相差显微镜图；
图10d是枯草杆菌168经FtsZ-eGFP融合蛋白的表达质粒转化后并在3μM F332下培养4小时后荧光显微镜图，图10e是相差显微镜图。
图11是F332在金黄葡萄球菌FtsZ(5μM)中对FtsZ聚合的影响趋势图；
图12是F361在金黄葡萄球菌FtsZ(5μM)中对FtsZ聚合的影响趋势图。
图13是F332对金黄葡萄球菌FtsZ的GTPase活性的影响。
图14是F361对金黄葡萄球菌FtsZ的GTPase活性的影响。
图15是FtsZ聚合物的电子显微图。
图16是FtsZ聚合物的电子显微图。
图17是F332的药代动力学参数。
具体实施方式
为了对本发明的技术特征、目的和效果有更加清楚的理解，现对照附图详细说明本发明的具体实施方式。
本发明根据不同的原料和产物将3-氨基苯甲酰胺衍生物的制备分为四种情况，分别列出如下四种制备方法：
第一种制备方法：合成化合物F411，F412，F413，F414，F342及F345 (见附图2对应标号的化合物)，这些化合物对应以下实施例1-6。其合成步骤如图2所示。
利用市场上可买到的3-氨基苯甲酰胺与溴代壬烷(1-bromooctane)或溴代 辛烷(1-bromooctane)在乙腈(ACN)中混合，满足回流温度的基本条件可以分别合成F411和F412。
通过使用硫酸二甲酯(dimethyl sulphate)并满足基本条件，这两种化合物被进一步烷基化为三级胺(tertiary amine)，从而产生F413和F414。
同样，利用市售成品2-氟-5-氨基苯甲酰胺(2-fluoro-5-aminobenzamide)或2,4-二氟-5-氨基苯甲酰胺(2,4-difluoro-5-aminobenzamide)与溴代壬烷在乙腈中混合，满足回流温度的基本条件可以分别合成F342和F345。
实验程序如图2所示，具体包括以下步骤：
(a)将原料A与原料B、碳酸钾和乙腈均匀搅拌形成反应混合液，加热回流反应4小时得到产物；原料A为3-氨基苯甲酰胺、2-氟-5-氨基苯甲酰胺或2,4-二氟-5-氨基苯甲酰胺；原料B为溴代壬烷或溴代辛烷；
(b)在步骤(a)得到的产物为3-(壬基氨基)苯甲酰胺或3-(辛基氨基)苯甲酰胺时，再将其分别与硫酸二甲酯、碳酸钾、和乙腈均匀搅拌，并加热回流12小时，得到产物。
实施例1：3-(壬基氨基)苯甲酰胺(F411)
将3-氨基苯甲酰胺(0.20g,1.4mmol)和溴代壬烷(0.32g,1.5mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.23g,1.6mmol)中。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，该反应混合液将通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.15g)的产率为39％：1H NMR(400MHz，氯仿-d)(t,J＝7.83Hz,1H),7.11(t,J＝1.96Hz,1H),7.03(d,J＝7.34Hz,1H),6.75(dd,J＝2.20,7.58Hz,1H),6.15(br.s.,1H),5.99(br.s.,1H),3.15(t,J＝7.09Hz,2H),1.63(quin,J＝7.21Hz,2H),1.25-1.45(m,12H),0.84-0.95(m,3H)；13C NMR (101MHz,氯仿-d)170.2,148.8,134.4,129.3,116.1,115.3,111.6,43.9,31.9,29.6,29.4,29.3,27.1,22.7,14.1；LRMS(ESI)m/z 263(M++H,100),285(M++Na,8)；HRMS(ESI)C16H27N2O(M++H)计算值263.2123，实验值263.2112。
实施例2：3-(辛基氨基)苯甲酰胺(F412)
将3-氨基苯甲酰胺(0.20g,1.4mmol)和溴代辛烷(0.29g,1.5mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.23g,1.6mmol)中。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，该反应混合液将通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.18g)的产率为47％：1H NMR(400MHz，氯仿-d)7.23(t,J＝7.83Hz,1H),7.07-7.18(m,1H),6.97-7.07(m,1H),6.75(dd,J＝2.20,7.58Hz,1H),6.15(br.s.,1H),5.98(br.s.,1H),3.15(t,J＝7.09Hz,2H),1.64(quin,J＝7.21Hz,2H),1.22-1.47(m,10H),0.91(t,J＝6.85Hz,3H)；13C NMR(101MHz,氯仿-d)170.1,148.8,134.4,129.3,116.1,115.3,111.6,43.9,31.8,29.4,29.4,29.3,27.1,22.7,14.1；LRMS(ESI)m/z 249(M++H,100),271(M++Na,10)；HRMS(ESI)C15H25N2O(M++H)计算值249.1967，实验值249.1957。
实施例3：3-(甲基(壬基)氨基)苯甲酰胺(F413)
将3-(壬基氨基)苯甲酰胺(0.09g,0.3mmol)和硫酸二甲酯(dimethyl sulphate)(0.06g,0.5mmol)溶液在乙腈(10mL)中均匀搅拌，加入碳酸钾K2CO3(0.06g,0.4mmol)中。将该反应混合液加热回流12小时，在薄层色谱法(TLC)显示原料完全消失后，用乙酸乙酯(ethyl acetate)(20mL)对该反应混合液进行稀释， 使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.04g)的产率为42％：1H NMR(400MHz，氯仿-d)7.23-7.32(m,1H),7.21(s,1H),6.99(d,J＝7.34Hz,1H),6.84(dd,J＝2.45,8.31Hz,1H),6.15(br.s.,1H),5.95(br.s.,1H),3.30-3.41(m,2H),2.96(s,3H),1.53-1.64(m,2H),1.22-1.37(m,12H),0.83-0.95(m,3H)；13C NMR(101MHz,氯仿-d)170.5,149.5,134.3,129.2,115.3,113.8,111.1,52.7,38.4,31.9,29.6,29.5,29.3,27.1,26.7,22.7,14.1；LRMS(ESI)m/z 277(M++H,100),299(M++Na,7)；HRMS(ESI)C17H29N2O(M++H)计算值277.2280，实验值277.2271。
实施例4：3-(甲基(辛基)氨基)苯甲酰胺(F414)
将3-(辛基氨基)苯甲酰胺(0.08g,0.3mmol)和硫酸二甲酯(dimethyl sulfate)(0.05g,0.4mmol)溶液在乙腈(10mL)中均匀搅拌，加入碳酸钾K2CO3(0.06g,0.4mmol)中。将该反应混合液加热回流12小时。在薄层色谱法(TLC)显示原料完全消失后，用乙酸乙酯(ethyl acetate)(20mL)对该反应混合液进行稀释，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.04g)的产率为47％：1H NMR(400MHz，氯仿-d)7.23-7.32(m,1H),7.21(s,1H),6.99(d,J＝7.82Hz,1H),6.84(dd,J＝2.45,8.31Hz,1H),6.14(br.s.,1H),5.97(br.s.,1H),3.29-3.41(m,2H),2.98(s,3H),1.52-1.66(m,2H),1.23-1.39(m,10H),0.85-0.96(m,3H)；13C NMR(101MHz,氯仿-d)170.5,149.5,134.3,129.2,115.3,113.8,111.1,52.7,38.4,31.8,29.5,29.3,27.2,26.7,22.7,14.1；LRMS(ESI)m/z 263(M++H,100),285(M++Na,10)；HRMS(ESI)C16H27N2O(M++H)计算值263.2123，实验值263.2113。
实施例5：2-氟-5-(壬基氨基)苯甲酰胺(F342)
将2-氟-5-氨基苯甲酰胺[2-fluoro-5-aminobenzamide](0.20g,1.3mmol)和溴代壬烷(0.30g,1.4mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.25g,1.8mmol)中。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.11g)的产率为30％：1H NMR(400MHz，氯仿-d)7.23-7.34(m,1H),6.94(d,J＝8.80Hz,1H),6.76(s,1H),6.62-6.71(m,1H),6.28(br.s.,1H),3.70(br.s.,1H),3.11(t,J＝7.09Hz,2H),1.61(quin,J＝7.09Hz,2H),1.22-1.44(m,12H),0.89(t,J＝6.60Hz,3H)；13C NMR(101MHz,氯仿-d)165.6,154.8,152.5,145.4,145.4,120.2,120.0,117.5,117.4,116.7,116.4,114.3,44.4,31.9,29.5,29.4,29.3,27.1,22.7,14.1；LRMS(ESI)m/z281(M++H,100),303(M++Na,50)；HRMS(ESI)C16H26N2OF(M++H)计算值281.2029，实验值281.2033。
实施例6：2,4-二氟-5-(壬基氨基)苯甲酰胺(F345)：
将2,4-二氟-5-氨基苯甲酰胺(2,4-difluoro-5-aminobenzamide)(0.20g,1.1mmol)和溴代壬烷(0.28g,1.4mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.23g,1.7mmol)中。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原材料完全消失后，使其通过一小段硅凝胶层。将得到的滤液在对比压力下进行蒸发脱水，使用硅凝胶进行快速柱层析。所得化合物(0.09g)的产出率为26％：1H NMR(400MHz，氯仿-d)7.54-7.34(m,1H),6.75-6.92(m,1H),6.60-6.75(m,1H),6.23(br.s.,1H),3.79(br.s.,1H),3.45-3.09(m,2H),1.56-1.71(m,2H),1.19-1.44(m,14H),0.80-0.96(m,3H)；LRMS (ESI)m/z 299(M++H,100),321(M++Na,85)；13C NMR(101MHz,氯仿-d)164.9,162.7,154.1,153.5,151.5,151.1,134.3,134.1,131.7,131.6,118.6,115.9,113.4,113.3,104.0,103.9,103.6,103.6,103.3,77.4,77.2,77.1,76.7,43.8,40.1,31.9,29.5,29.5,29.4,29.4,29.3,29.3,29.2,27.0,27.0,22.7,14.1；LRMS(ESI)m/z 299(M++H,100),321(M++Na,85)；HRMS(ESI)C16H25N2OF2(M++H)计算值299.1935，实验值299.1939。
第二种制备方法：合成化合物F332,F333,F334,F350,F361,F369,F370,F371, F391,F409和F410(见附图3对应标号的化合物)，具体如下实施例7-18：
图3示出本发明化合物F332,F333,F334,F350,F361,F369,F370,F371,F391,F409和F410的合成流程。
2,6-二氟-3-氨基苯甲酰胺(2,6-Difluoro-3-aminobenzamide)的大规模合成是利用市场上可买到的2,6-二氟-3-硝基苯甲酸(2,6-difluoro-3-nitrobenzonic acid)，分为两个步骤进行。
首先，将2,6-二氟-3-硝基苯甲酸和氯化亚砜(thionyl chloride)溶入氨水溶液产生2,6-二氟-3-硝基苯甲酰胺(2,6-difluoro-3-nitrobenzamide),随后通过使用氯化亚锡(tin(II)chloride)溶入浓盐酸进一步转化为2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)。
然后将2,6-二氟-3-氨基苯甲酰胺与各种烷基卤化物混合,例如溴代壬烷,溴代辛烷，溴代癸烷,1-溴-4-丁氧基丁烷(1-bromo-4-butoxybutane),溴化香叶酯(geranyl bromide),(Z)-1-溴-2-壬烯[trans-1-bromonon-2-ene]，溴代庚烷(1-bromoheptane),满足基本条件在乙腈(ACN)中混合4小时，分别合成F332,F333,F334,F350,F369,F370,F391。
对F332进行溴化处理产生F371。
使用硫酸二甲酯(dimethyl sulphate)或溴乙烷(bromoethane)对F332和F333进行进一步烷化处理产生F409,F410和F361。
具体实验程序如图3所示：(a)各种溴代烷烃,碳酸钾,乙腈,回流,4小时；(b)Br2,DCM,室温,12小时；(c)碘甲烷(methyl iodide)或溴乙烷(bromoethane),碳酸钾,乙腈，回流，14小时；(d)硫酸二甲酯,碳酸钾,乙腈，回流，12小时。
实施例7：2,6-二氟-3-氨基苯甲酰胺
将2,6-二氟-3-硝基苯甲酸(44g,216mmol)和氯化亚砜(thionyl chloride，100mL)加热回流2小时。通过蒸发除去多余的氯化亚砜，产生2,6-二氟-3-硝基苯甲酰氯(2,6-difluoro-3-nitrobenzonic acid chloride),在下一步骤的使用中无需进一步提纯。将2,6-二氟-3-硝基苯甲苯甲酰氯物逐滴滴入搅拌均匀的30％氨水溶液(300mL)，温度为0℃。之后，将形成的沉淀物进行过滤和收集，得到2,6-二氟-3-硝基苯甲酰胺(40g,91％),在下一步骤的使用中无需进一步提纯。将均匀搅拌的氯化亚锡(80g,421mmol)在0℃溶入浓盐酸(200mL)，分部分加入2,6-二氟-3-硝基苯甲酰胺。随后，将反应混合液在室温下搅拌12小时。通过向反应混合液中加入多余的氢氧化钾溶液(potassium hydroxide solution)使化学反应淬灭，直至在0℃时pH值大于12。用乙酸乙酯(200mL x4)萃取水溶液。用无水硫酸镁(MgSO4)将混合有机层进行干燥、过滤並蒸除溶剂，产生2,6-二氟-3-氨基苯甲酰胺(18g,53％):1H NMR(400MHz,丙酮)7.36(br.s.,1H),7.14(br.s.,1H),6.87(dt,J＝5.38,9.29Hz,1H),6.77(t,J＝9.05Hz,1H),4.67(br.s.,2H)；13C NMR(101MHz,丙酮)162.3,151.7,151.7, 149.3,148.2,148.1,145.8,145.7,133.0,132.9,132.8,132.8,116.4,116.4,116.3,116.3,115.5,115.3,115.3,115.1,111.0,110.9,110.7,110.7。
实施例8：2,6-二氟-3-(壬基氨基)苯甲酰胺(F332)
将2,6-二氟-3-氨基苯甲酰胺(0.74g,4.3mmol)和溴代壬烷(1.20g,5.8mmol)溶液在乙腈(50mL)中均匀搅拌，加入碳酸钾K2CO3(1.20g,8.7mmol)和碘化钠NaI(0.08g，催化量)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.49g)的产率为38％：1H NMR(400MHz，氯仿-d)6.77-6.94(m,1H),6.69(dt,J＝5.38,9.29Hz,1H),6.12(br.s.,1H),6.05(br.s.,1H),3.82(br.s.,1H),3.12(t,J＝7.09Hz,2H),1.58-1.73(m,2H),1.23-1.46(m,12H),0.90(t,J＝6.85Hz,3H)；13C NMR(101MHz,氯仿-d)162.8,151.9,149.5,149.2,146.8,134.2,134.1,113.1,113.0,111.4,111.4,111.2,111.2,43.9,31.9,29.5,29.4,29.3,29.2,27.0,22.7,14.1；LRMS(ESI)m/z 299(M++H,97),321(M++Na,100)；HRMS(ESI)C16H24N2OF2Na(M++Na)计算值321.1754，实验值321.1756。
实施例9：2,6-二氟-3-(辛基氨基)苯甲酰胺(F333)
将2,6-二氟-3-氨基苯甲酰胺(0.40g，2.3mmol)和溴代辛烷(0.45g,2.3mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.40g,2.9mmol)和碘化钠NaI(0.04g，催化量)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.26g)的产率为39％：1H NMR(400MHz,氯 仿-d)6.83(t,J＝9.29Hz,1H),6.68(dt,J＝5.38,9.05Hz,1H),6.36(br.s.,1H),6.09(br.s.,1H),3.81(br.s.,1H),3.06-3.17(m,2H),1.59-1.69(m,2H),1.23-1.43(m,10H),0.90(t,J＝6.60Hz,3H)；13C NMR(101MHz,氯仿-d)163.0,151.9,149.5,149.5,149.2,149.1,146.7,134.2,134.2,134.1,134.1,113.1,113.1,113.0,113.0,112.5,111.4,111.4,111.2,111.2,43.9,31.9,31.8,29.4,29.3,29.2,27.0,22.6,14.1；LRMS(ESI)m/z 285(M++H,100),307(M++Na,20)；HRMS(ESI)C15H23N2OF2(M++H)计算值285.1778，实验值285.1773。
实施例10：3-(癸氨基)-2,6-二氟苯甲酰胺(F334)
将2,6-二氟-3-氨基苯甲酰胺(0.40g，2.3mmol)和溴代癸烷(0.56g，2.5mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.40g，2.9mmol)和碘化钠NaI(0.04g，催化量)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.27g)的产率为37％：1H NMR(400MHz,氯仿-d)6.77-6.89(m,1H),6.68(dt,J＝5.14,9.17Hz,1H),6.29(br.s.,1H),6.10(br.s.,1H),3.75-3.89(m,1H),3.12(q,J＝6.52Hz,2H),1.59-1.68(m,2H),1.21-1.49(m,14H),0.90(t,J＝6.36Hz,3H)；13C NMR(101MHz,氯仿-d)163.3,162.9,134.2,134.1,113.1,113.0,113.0,112.9,111.4,111.4,111.2,111.1,57.9,43.9,42.3,31.9,31.9,29.6,29.6,29.6,29.5,29.4,29.3,29.3,27.7,27.3,27.0,22.7,14.1；LRMS(ESI)m/z 313(M++H,28),335(M++Na,95)；HRMS(ESI)C17H26N2OF2Na(M++Na)计算值335.1911，实验值335.1923。
实施例11：3-((4’-丁氧基)丁基氨基)-2,6-二氟苯甲酰胺(F350):
将2,6-二氟-3-氨基苯甲酰胺(0.17g，1.0mmol)和1-溴-4-丁氧基丁烷(1-bromo-4-butoxybutane)(0.21g，1.0mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.15g，1.1mmol)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.05g)的产率为17％:1H NMR(400MHz,氯仿-d)6.75-6.93(m,1H),6.68(dt,J＝5.38,9.29Hz,1H),6.32(br.s.,1H),6.09(br.s.,1H),3.95(br.s.,1H),3.44(td,J＝6.17,12.59Hz,4H),3.16(br.s.,2H),1.65-1.76(m,4H),1.53-1.61(m,2H),1.32-1.44(m,2H),0.93(t,J＝7.34Hz,3H)；13C NMR(101MHz,氯仿-d)163.0,151.9,146.7,134.1,113.1,113.0,113.0,112.9,111.4,111.4,111.1,70.8,70.3,43.7,31.8,27.2,26.2,19.4,13.9；LRMS(ESI)m/z 301(M++H,40)；HRMS(ESI)C15H23N2O2F2(M++H)计算值301.1728，实验值301.1716。
实施例12：2,6-二氟-3-(甲基(壬基)氨基)苯甲酰胺(F361):
将2,6-二氟-3-(壬基氨基)苯甲酰胺(2,6-difluoro-3-(nonylamino)benzamide)(0.15g，0.5mmol)和硫酸二甲酯(0.15g，1.2mmol)溶液在丙酮(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.15g，1.1mmol)。将该反应混合液加热回流14小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.03g)的产率为19％:1H NMR(400MHz,氯仿-d)6.90-7.02(m,1H),6.79-6.90(m,1H),6.39(br.s.,1H),6.05(br.s.,1H),3.00-3.08(m,2H),2.79(s,3H),1.53(br.s.,2H),1.27(br.s.,12H),0.89(t,J＝6.60Hz,3H)；13C NMR(101MHz,氯仿-d)163.0,154.9,153.5,152.4,137.6,121.2,121.1,116.9,111.2,111.2,111.0,55.6,55.5,40.0,40.0, 31.9,29.6,29.5,29.3,27.2,27.0,22.7,14.1；LRMS(ESI)m/z 313(M++H,100)；HRMS(ESI)C17H27N2OF2(M++H)计算值313.2091，实验值313.2083。
实施例13：(E)-3-((3,7-二甲基-2,6-辛二烯)氨基)-2,6-二氟苯甲酰胺(F369)
将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.34g,2.0mmol)和溴化香叶醇(geranyl bromide)(0.42g,2.0mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.29g，2.1mmol)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.29g)的产率为48％:1H NMR(400MHz,氯仿-d)6.83(dt,J＝1.47,9.29Hz,1H),6.68(dt,J＝5.38,9.05Hz,1H),6.48(br.s.,1H),6.11(br.s.,1H),5.30(t,J＝6.11Hz,1H),5.03-5.13(m,1H),3.83(br.s.,1H),3.72(d,J＝6.85Hz,2H),2.02-2.16(m,4H),1.66-1.75(m,6H),1.62(s,3H)；13C NMR(101MHz,氯仿-d)163.1,152.1,149.7,146.8,146.8,139.9,134.1,133.9,131.8,123.8,120.7,113.4,113.4,113.3,113.3,111.4,111.2,111.1,41.8,39.5,26.3,25.7,17.7,16.4；LRMS(ESI)m/z 309(M++H,100),321(M++Na,6)；HRMS(ESI)C17H23N2OF2(M++H)计算值309.1778，实验值309.1779。
实施例14：(Z)-2,6-二氟-3-(2-壬烯基氨基)苯甲酰胺(F370)
将2,6-二氟-3-氨基苯甲酰胺(0.17g，1.0mmol)和(Z)-1-溴代壬烷-2-烯[(Z)-1-bromonon-2-ene](0.21g,1.0mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.15g，1.1mmol)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸 除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.13g)的产率为44％:1H NMR(400MHz,氯仿-d)6.84(dt,J＝1.47,9.29Hz,1H),6.70(dt,J＝5.38,9.29Hz,1H),6.28(br.s.,1H),6.08(br.s.,1H),5.52-5.61(m,1H),5.32-5.43(m,1H),3.88(br.s.,1H),3.09-3.18(m,2H),2.40(q,J＝6.85Hz,2H),2.06(q,J＝7.17Hz,2H),1.24-1.41(m,6H),0.90(t,J＝6.85Hz,3H)；13C NMR(101MHz,氯仿-d)162.9,159.0,152.0,149.6,149.2,133.9,133.4,125.4,113.3,113.2,113.2,113.1,111.4,111.2,111.2,43.4,31.5,29.3,27.3,27.2,27.0,22.5,14.0,14.0；LRMS(ESI)m/z 297(M++H,100),319(M++Na,35)；HRMS(ESI)C16H23N2OF2(M++H)计算值297.1778，实验值297.1768。
实施例15：4-溴-2,6-二氟-3-(壬基氨基)苯甲酰胺(F371)
将2,6-二氟-3-(壬基氨基)苯甲酰胺(2,6-difluoro-3-(nonylamino)benzamide)(0.3g，1.0mmol)和二氯甲烷(dichloromethane)(20mL)溶液中加入多余的液溴(1mL)，在室温下搅拌12小时。在薄层色谱法(TLC)显示原料完全消失后，将该反应混合液倒进装有饱和硫代硫酸钠溶液(sodium thiosulfate solution)(30mL)的分液漏斗中，用乙酸乙酯(ethyl acetate)(20mL x 3)萃取。用硫酸镁(MgSO4)将混合有机层进行干燥、过滤並蒸除溶剂，对得到的粗制品进行快速柱层析，所得化合物(0.28g)的产率为74％:1H NMR(400MHz,氯仿-d)7.11(dd,J＝1.96,8.80Hz,1H),6.76(br.s.,1H),6.19(br.s.,1H),3.74(br.s.,1H),3.28(t,J＝6.11Hz,2H),1.52-1.61(m,2H),1.24-1.38(m,12H),0.83-0.92(m,3H)；13C NMR(101MHz,氯仿-d)162.4,152.9,152.8,150.9,150.9,150.4,150.3,148.3,133.0,133.0,132.9,132.9,115.8,115.7,115.5,115.5,114.9,114.9,114.8,114.7,113.9,113.7,113.5,47.3,47.2,31.9,31.6,30.7,29.5, 29.3,29.2,26.8,22.7,14.2,14.1；LRMS(ESI)m/z 377(M++H,96),399(M++Na,16)；HRMS(ESI)C16H24N2OF2Br(M++H)计算值377.1040，实验值377.1049。
实施例16：2,6-二氟-3-(庚基氨基)苯甲酰胺(F391):
将2,6-二氟-3-氨基苯甲酰胺(0.70g，4.1mmol)和溴代庚烷(1-bromoheptane)(0.80g,4.4mmol)溶液在乙腈(50mL)中均匀搅拌，加入碳酸钾K2CO3(0.60g,4.4mmol)和NaI(0.08g，催化量)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.40g)的产率为36％:1H NMR(400MHz,氯仿-d)6.85(dt,J＝1.96,9.29Hz,1H),6.69(dt,J＝5.38,9.29Hz,1H),6.14(br.s.,1H),6.05(br.s.,1H),3.12(t,J＝7.09Hz,2H),1.59-1.69(m,2H),1.27-1.45(m,8H),0.86-0.96(m,3H)；13C NMR(101MHz,氯仿-d)166.4,154.2,149.6,145.8,140.8,134.2,113.1,111.5,111.2,105.2,43.9,31.8,29.3,29.1,27.0,22.6,14.1；LRMS(ESI)m/z 271(M++H,100),293(M++Na,60)；HRMS(ESI)C14H21N2OF2(M++H)计算值271.1622，实验值271.1612。
实施例17：2,6-二氟-3-(甲基(辛基)氨基)苯甲酰胺(F409)
将2,6-二氟-3-(辛基氨基)苯甲酰胺(2,6-difluoro-3-(octylamino)benzamide)(0.12g,0.4mmol)和碘甲烷(methyl iodide)(0.30g,2.1mmol)溶液在乙腈(10mL)中均匀搅拌，加入碳酸钾K2CO3(0.30g,2.1mmol)。将该反应混合液加热回流14小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小 段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.03g)的产率为24％:1H NMR(400MHz,氯仿-d)6.95(dt,J＝5.62,9.17Hz,1H),6.80-6.90(m,1H),6.63(br.s.,1H),6.11(br.s.,1H),2.99-3.07(m,2H),2.79(s,3H),1.47-1.58(m,2H),1.22-1.35(m,10H),0.89(t,J＝6.85Hz,3H)；13C NMR(101MHz,氯仿-d)163.2,154.9,154.9,153.6,152.4,151.0,137.5,121.2,121.1,121.1,114.1,113.9,111.2,111.2,111.0,111.0,55.6,55.5,40.0,31.8,29.5,29.3,27.2,27.0,22.6,14.1；LRMS(ESI)m/z 299(M++H,100),321(M++Na,26)；HRMS(ESI)C16H25N2OF2(M++H)计算值299.1935，实验值299.1928。
实施例18：3-(乙烷基(辛基)氨基)-2,6-二氟苯甲酰胺(F410):
将2,6-二氟-3-(辛基氨基)苯甲酰胺(2,6-difluoro-3-(octylamino)benzamide)(0.12g,0.4mmol)和溴代乙烷(bromoethane)(0.30g,2.7mmol)溶液在乙腈(10mL)中均匀搅拌，加入碳酸钾K2CO3(0.30g,2.1mmol)。将该反应混合液加热回流14小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.04g)的产率为30％:1H NMR(400MHz,氯仿-d)6.95(dd,J＝5.62,9.05Hz,2H),6.76-6.86(m,1H),6.20(br.s.,1H),3.12(q,J＝7.01Hz,2H),3.03(t,J＝7.58Hz,2H),1.43(br.s.,2H),1.24(br.s.,10H),1.02(t,J＝7.09Hz,3H),0.86(t,J＝6.60Hz,3H)；13C NMR(101MHz,氯仿-d)163.4,155.3,154.7,152.8,152.8,152.1,135.5,123.6,123.5,114.3,114.1,113.9,111.1,111.0,110.8,110.8,52.3,52.3,47.2,47.2,31.8,29.4,29.2,27.3,27.1,22.6,14.0,12.4；LRMS(ESI)m/z 313(M++H,100),335(M++Na,16)；HRMS(ESI)C17H27N2OF2(M++H)计算值313.2091，实验值313.2097。
第三种制备方法：合成化合物F357,F358,以及F362到F368(见附图 4对应标号的化合物)，具体如实施例19-24：
图4示出本发明化合物F357,F358,以及F362至F368的化学合成途径。化合物F368是通过将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)和壬酰氯(nonanoyl chloride)在0℃下溶入吡啶(pyridine)和二氯甲烷(dichloromethane)4小时，如图4所示。
F357和F358通过2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)合成,将1,4-二溴代戊烷(1,5-bromopentane)、1,5-二溴丁烷(1,4-dibromobutane)分别加入碳酸钾在乙腈中均匀搅拌4小时。F362,F363,F364,F365,F366,F367也由2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)合成,分別通过与3,4-二氟溴苄(3,4-difluorobenzyl bromide)、2,4-二氟溴苄(2,4-difluorobenzyl bromide)、2,6-二氟溴苄(2,6-difluorobenzyl bromide)一起加入碳酸钾，在乙腈中加热回流4小时。
具体实验程序如图4所示：(a)壬酰氯(nonanoyl chloride)，Py/DCM,0℃,4小时；(b)1,5-二溴戊烷(1,5-dibromopentane)或1,4-二溴丁烷(1,4-dibromobutane),碳酸钾,乙腈，回流，4小时；(c)各种二氟溴苄(difluorobenzyl bromides),碳酸钾,乙腈,回流,4小时。
实施例19：2,6-二氟-3-(吡咯烷-1-氨基)苯甲酰胺(F357)
将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.17g,1.0mmol)和1,4-二溴丁烷(1,4-dibromobutane)(0.23g,1.1mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.18g,1.3mmol)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶 层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.11g)的产率为46％:1H NMR(400MHz,氯仿-d)6.82(t,J＝8.80Hz,1H),6.69(dt,J＝5.38,9.29Hz,1H),6.13(br.s.,1H),5.99(br.s.,1H),3.30-3.41(m,4H),1.97(td,J＝3.48,6.24Hz,4H)；13C NMR(101MHz,氯仿-d)163.5,156.9,155.9,152.4,150.9,138.2,121.6,116.5,116.3,111.3,50.0,50.0,25.1；LRMS(ESI)m/z227(M++H,100),249(M++Na,57)；HRMS(ESI)C11H13N2OF2(M++H)计算值227.0996，实验值227.1005。
实施例20：2,6-二氟-3-(哌啶-1-氨基)苯甲酰胺(F358)
将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.17g,1.0mmol)和1,5-二溴丁烷(1,5-dibromopentane)(0.25g,1.1mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.18g,1.3mmol)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。所得化合物(0.10g)的产率为38％:1H NMR(400MHz,氯仿-d)7.00(dt,J＝5.87,9.05Hz,1H),6.80-6.94(m,1H),6.36(br.s.,1H),6.08(br.s.,1H),2.91-3.02(m,4H),1.69-1.80(m,4H),1.49-1.64(m,2H)；13C NMR(101MHz,氯仿-d)162.9,153.2,151.6,138.4,121.1,121.1,114.0,111.3,111.2,52.5,52.4,26.1,24.0；LRMS(ESI)m/z 241(M++H,100),263(M++Na,30)；HRMS(ESI)C12H15N2OF2(M++H)计算值241.1152，实验值241.1149。
实施例21：3-((3’,4’-二氟苄)氨基)-2,6-二氟苯甲酰胺(F362)和3-(双(3’,4’-二氟 苄)氨基)-2,6-二氟苯甲酰胺(F363)
将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.40g,2.3mmol)和3,4-二氟溴苄(3,4-difluorobenzyl bromide)(0.60g,2.9mmol)溶液在乙腈(20mL)中均匀搅拌，加入碳酸钾K2CO3(0.42g,3.0mmol)。将该反应混合液加热回流4小时。在薄层色谱法(TLC)显示原料完全消失后，使其通过一小段硅凝胶层。减压蒸除溶剂，使用硅凝胶进行快速柱层析。化合物F362(0.25g)从F363中提取,产率为36％，F363(0.19g)产率为19％。
F362:1H NMR(400MHz,氯仿-d)7.04-7.22(m,3H),6.77(t,J＝9.29Hz,1H),6.55(dt,J＝5.14,9.17Hz,1H),6.50(br.s.,1H),6.17(br.s.,1H),4.44(br.s.,1H),4.34(d,J＝5.87Hz,2H)；13C NMR(101MHz,氯仿-d)162.8,162.6,152.4,151.9,150.8,150.1,146.8,135.6,135.5,133.2,133.1,122.9,122.8,122.8,122.8,117.6,117.5,116.0,115.8,113.5,113.4,113.4,113.4,111.5,111.4,111.3,111.2,46.9；LRMS(ESI)m/z 299(M++H,100),321(M++Na,20)；HRMS(ESI)C14H11N2OF4(M++H)计算值299.0808，实验值299.0794；
F363:1H NMR(400MHz,氯仿-d)6.94-7.15(m,7H),6.88(dd,J＝3.18,9.05Hz,1H),6.68-6.78(m,1H),6.39(br.s.,1H),4.15(s,4H)；13C NMR(101MHz,氯仿-d)163.0,156.2,154.9,153.6,152.5,151.6,151.5,150.9,150.7,149.2,149.0,148.4,148.3,134.6,134.6,134.6,134.5,124.8,124.5,124.2,124.2,124.1,124.1,117.7,117.6,117.3,117.1,117.0,116.9,116.7,114.7,114.5,114.3,111.4,111.4,111.2,111.1,55.6；LRMS(ESI)m/z 425(M++H,100),447(M++Na,28)；HRMS(ESI)计算值C21H15N2OF6(M++H)425.1089，实验值425.1082。
实施例22：3-((2’,4’-二氟苄)氨基)-2,6-二氟苯甲酰胺(F364)和3-(双(2’,4’-二氟 苄)氨基)-2,6-二氟苯甲酰胺(F365)
F364(0.29g,42％)和F365(0.21g,21％)是通过分别将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.40g,2.3mmol)和2,4-二氟溴苄(2,4-difluorobenzyl bromide)(0.60g,2.9mmol),加入乙腈(20mL)和碳酸钾K2CO3(0.42g,3.0mmol)后按照上述F362和F363的制配程序所得。
F364:1H NMR(400MHz,氯仿-d)7.25-7.36(m,1H),6.74-6.90(m,3H),6.64(dt,J＝5.38,9.05Hz,2H),6.20(br.s.,1H),4.38(s,3H)；13C NMR(101MHz,氯仿-d)163.7,163.5,163.0,162.6,162.1,161.9,161.2,161.1,159.5,152.4,150.1,150.0,149.3,133.1,133.1,133.0,133.0,130.0,130.0,129.9,129.9,121.3,121.3,121.2,121.1,113.5,113.4,113.4,113.3,112.9,112.7,111.6,111.5,111.5,111.4,111.4,111.3,111.3,111.2,104.3,104.0,103.8,41.1；LRMS(ESI)m/z299(M++H,100),321(M++Na,20)；HRMS(ESI)calcd for C14H11N2OF4(M++H)299.0808，实验值299.0793。
F365:1H NMR(400MHz,氯仿-d)7.25-7.38(m,2H),6.90(dt,J＝5.87,9.05Hz,1H),6.72-6.85(m,5H),6.56(br.s.,1H),6.08(br.s.,1H),4.27(s,4H)；13C NMR(101MHz,氯仿-d)163.5,163.4,162.6,162.2,162.1,161.1,161.0,159.8,159.7,134.5,131.4,131.3,131.3,131.2,124.9,120.3,120.3,120.2,114.0,111.5,111.4,111.3,111.2,111.2,104.0,103.7,103.4,49.3；LRMS(ESI)m/z 425(M++H,100),447(M++Na,25)；HRMS(ESI)calcd for C21H15N2OF6(M++H)425.1089，实验值425.1080。
实施例23：3-((2’,6’-二氟苄)氨基)-2,6-二氟苯甲酰胺(F366)和3-(双(2’,6’-二氟 苄)氨基)-2,6-二氟苯甲酰胺(F367)
F366(0.29g,42％)和F367(0.21g,21％)是通过分别将2,6-二氟-3-氨基苯甲酰胺(0.40g,2.3mmol)和2,6-二氟溴苄(2,6-difluorobenzyl bromide)(0.60g,2.9mmol),加入乙腈(20mL)和碳酸钾K2CO3(0.42g,3.0mmol)后按照上述F362和F363的制配程序所得。
F366:1H NMR(400MHz,氯仿-d)7.22-7.31(m,1H),6.78-6.95(m,4H),6.38(br.s.,1H),6.12(br.s.,1H),4.45(d,J＝6.36Hz,2H),4.34(br.s.,1H)；13C NMR(101MHz,氯仿-d)162.8,162.5,160.4,160.3,152.6,150.1,149.6,132.9,129.8,129.7,129.6,114.2,114.0,113.8,111.7,111.6,111.5,111.5,111.5,111.4,111.3,111.3,35.8,35.7；LRMS(ESI)m/z 299(M++H,100),321(M++Na,30)；HRMS(ESI)C14H11N2OF4(M++H)计算值299.0808，实验值299.0800。
F367:1H NMR(400MHz,氯仿-d)7.10-7.20(m,2H),7.08(br.s.,1H),6.96(dt,J＝6.11,8.93Hz,1H),6.77(t,J＝7.82Hz,4H),6.65(t,J＝9.05Hz,1H),6.19(br.s.,1H),4.38(s,4H)；13C NMR(101MHz,氯仿-d)163.1,163.0,160.6,160.5,157.4,157.3,154.9,154.8,133.3,133.3,133.2,133.2,129.5,129.4,129.3,127.8,114.1,113.9,113.8,113.7,113.6,113.4,111.3,111.2,111.1,111.0,110.8,110.8,44.8；LRMS(ESI)m/z 425(M++H,100),447(M++Na,40)；HRMS(ESI)C21H15N2OF6(M++H)计算值425.1089，实验值425.1082。
实施例24：2,6-二氟-3-壬酰胺基苯甲酰胺(F368)
将2,6-二氟-3-氨基苯甲酰胺(0.17g,1.0mmol)、二氯甲烷(DCM)(5mL)、吡啶(5mL)在0℃下均匀混合搅拌，逐滴滴入壬酰氯(nonanoyl chloride)(0.23g,1.3mmol)，将该反应混合液在0℃下混合搅拌4小时。将该反应混合液倒进装有1M HCl(50mL)的分液漏斗中，使化学反应淬灭，用二氯甲烷(DCM) (20mL x 3)萃取。用碳酸氢钠(NaHCO3)对该混合有机层进行清洗，用硫酸镁(MgSO4)进行干燥、过滤並减压蒸除溶剂，得到反应物粗品，进行快速柱层析，所得化合物(0.11g)的产率为36％:1H NMR(400MHz,丙酮-d6)8.95(br.s.,1H),8.16(dt,J＝6.36,8.80Hz,1H),7.51(br.s.,1H),7.19(br.s.,1H),7.02(dt,J＝1.71,8.93Hz,1H),2.47(t,J＝7.58Hz,2H),1.70(quin,J＝7.34Hz,2H),1.25-1.43(m,10H),0.81-0.98(m,3H)；13C NMR(101MHz,丙酮-d6)171.7,161.2,124.3,123.6,110.9,110.7,110.6,36.3,31.7,25.3,22.4,19.1,18.5,13.4；LRMS(ESI)m/z 313(M++H,100)；HRMS(ESI)C16H22N2O2F6(M++H)计算值313.1728，实验值313.1716。
第四种制备方法：合成化合物F373,F376,F377和F378(具体对应附图5中 相同标号的化合物)，具体如实施例25-28：
图5示出本发明化合物F373,F376,F377和F378的合成途径。这些化合物是通过2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)与相应的苯甲醛(benzaldehyde)进行还原胺化反应所得，产率较高。具体实验程序如图5所示：(a)各类醛，催化剂(cat.)对甲苯磺酸(pTsOH)，甲醇(MeOH)，室温(r.t.)，2小时,氰基硼氢化钠(NaBH3CN)，12小时。
实施例25：3-((3’-丁氧基苄基)氨基)-2,6-二氟苯甲酰胺(F373)
将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.17g,1.0mmol)和3-丁氧基苯甲醛(3-butoxybenzaldehyde)(0.17g,1.0mmol)在0℃下与甲醇(10mL)均匀搅拌，加入对甲苯磺酸一水合物(p-toluenesulfonic acid monohydrate)(0.02g,0.11mmol),将该反应混合液搅拌2小时。之后，将 氰基硼氢化钠(sodium cyanoborohydride)(0.63g,10.0mmol)分部分加入该反应混合物，然后再混合搅拌12小时。将该反应混合液倒进装有50mL水的分液漏斗中，使化学反应淬灭，用乙酸乙酯(ethyl acetate)(20mL x 3)萃取。用硫酸镁(MgSO4)在对比压力下进行干燥、过滤並减压蒸除溶剂，得到反应粗品，进行快速柱层析，所得化合物(0.15g)的产率为45％:1H NMR(400MHz,氯仿-d)7.26(t,J＝7.82Hz,1H),6.86-6.97(m,2H),6.73-6.86(m,2H),6.63(dt,J＝5.38,9.05Hz,1H),6.56(br.s.,1H),6.16(br.s.,1H),4.27-4.40(m,3H),3.96(t,J＝6.36Hz,2H),1.71-1.81(m,2H),1.44-1.57(m,2H),0.99(t,J＝7.34Hz,3H)；13C NMR(101MHz,氯仿-d)163.1,159.6,152.2,149.2,146.7,140.0,133.7,133.5,129.8,119.2,113.5,113.5,113.4,113.4,111.4,111.4,111.2,111.2,67.7,47.9,31.3,19.2,13.9；LRMS(ESI)m/z 335(M++H,60),357(M++Na,50)；HRMS(ESI)C18H21N2O2F2(M++H)计算值335.1571，实验值335.1568。
实施例26：2,6-二氟-3-((3’-(戊氧基)苄基)氨基)苯甲酰胺(F376)
该化合物(0.13g,38％)是通过将2,6-二氟-3-氨基苯甲酰胺(0.17g,1.0mmol)、3-(戊氧基)苯甲醛(3-(pentyloxy)benzaldehyde)(0.19g,1.0mmol)],对甲苯磺酸一水合物(p-toluenesulfonic acid monohydrate)(0.02g,0.11mmol)、甲醇(10mL)和氰基硼氢化钠(sodium cyanoborohydride)(0.63g,10mmol)混合后按照上述F373的制配程序所得。1H NMR(400MHz,丙酮-d6)7.37(br.s.,1H),7.24(t,J＝7.82Hz,1H),7.10(br.s.,1H),6.93-7.01(m,2H),6.72-6.85(m,2H),6.65(dt,J＝5.38,9.29Hz,1H),5.54(br.s.,1H),4.42(d,J＝5.87Hz,2H),3.92-4.02(m,2H),1.71-1.82(m,2H),1.34-1.49(m,4H),0.87-0.97(m,3H)；13C NMR(101MHz,Acetone)159.6,141.3,129.4,119.1,113.3,112.7,112.2, 110.4,67.5,46.9,22.2,13.4；LRMS(ESI)m/z 349(M++H,100),371(M++Na,50)；HRMS(ESI)calcd for C19H23N2O2F2(M++H)349.1728，实验值349.1739.
实施例27：3-((3’-(仲丁氧基)苄基)氨基)-2,6-二氟苯甲酰胺(F377)
该化合物(0.12g,36％)是通过将2,6-二氟-3-氨基苯甲酰胺(2,6-difluoro-3-aminobenzamide)(0.17g,1.0mmol)、3-(仲丁氧基)苯甲醛(3-(sec-butoxy)benzaldehyde)(0.17g,1.0mmol)、对甲苯磺酸一水合物(p-toluenesulfonic acid monohydrate)(0.02g,0.11mmol)、甲醇(10mL)和氰基硼氢化钠(sodium cyanoborohydride)(0.63g,10mmol)混合后按照上述F373的制配程序所得。1H NMR(400MHz,氯仿-d)7.24(t,J＝8.07Hz,1H),6.85-6.93(m,2H),6.71-6.85(m,3H),6.61(dt,J＝5.14,9.17Hz,1H),6.27(br.s.,1H),4.38(br.s.,1H),4.27-4.34(m,3H),1.68-1.79(m,1H),1.57-1.67(m,1H),1.25-1.31(m,3H),0.97(t,J＝7.58Hz,3H)；13C NMR(101MHz,氯仿-d)163.3,158.7,152.2,149.8,149.7,149.2,149.1,146.7,146.6,140.1,133.7,133.6,133.6,133.5,129.8,119.1,114.8,114.6,113.5,113.4,113.4,113.3,112.9,112.8,112.6,111.4,111.4,111.2,111.1,75.0,47.9,29.2,19.2,9.8；LRMS(ESI)m/z 335(M++H,40),357(M++Na,40)；HRMS(ESI)C18H21N2O2F2(M++H)计算值335.1571，实验值335.1580。
实施例28：2,6-二氟-3-((3’-((4”-(三氟甲基)苄基)氧)苄基)氨基)苯甲酰胺(F378)
该化合物(0.16g,37％)是通过将2,6-二氟-3-氨基苯甲酰胺(0.17g,1.0mmol)、3-((4-(三氟甲基)苄基)氧)苯甲醛(3-((4-(trifluoromethyl)benzyl)oxy)benzaldehyde)(0.28g,1.0mmol)、对甲苯磺酸一水合物(p-toluenesulfonic  acid monohydrate)(0.02g,0.11mmol)、甲醇(10mL)和氰基硼氢化钠(sodium cyanoborohydride)(0.63g,10mmol)混合后按照上述F373的制配程序所得。1H NMR(400MHz,氯仿-d)7.66(d,J＝8.31Hz,2H),7.56(d,J＝7.82Hz,2H),7.29(d,J＝6.85Hz,1H),6.94-7.03(m,2H),6.88-6.93(m,1H),6.72-6.83(m,1H),6.62(td,J＝4.59,8.93Hz,1H),6.25(br.s.,1H),6.09(br.s.,1H),5.13(s,2H),4.36(s,3H)；13C NMR(101MHz,氯仿-d)158.9,141.0,140.3,130.0,127.4,125.6,125.5,125.5,120.0,113.7,113.6,111.4,69.1,47.8；LRMS(ESI)m/z 437(M++H,56),459(M++Na,50)；HRMS(ESI)C22H18N2O2F5(M++H)计算值437.1288，实验值437.1292。
本发明的衍生物进行如下生物测试：
(A)菌株和细胞株
微生物敏感性试验中使用的菌株是大肠杆菌，金黄葡萄球菌29213和枯草杆菌168。其中，大肠杆菌和金黄葡萄球菌29213是通过香港理工大学的实验室获得，枯草杆菌168购自美国标准菌库。哺乳动物细胞株L929(小鼠腹膜成纤维细胞)、LCC6(一种原株乳腺癌细胞)、LCC6MDR(P-gp高表达乳腺癌细胞株)是从理工大学的库存中获得。营养琼脂(Nutrient Agar)、胰蛋白胨(tryptone)和酵母提取物购自Oxoid Limited公司(加拿大安大略省尼平)。LB液体培养基(Luria-Bertani)通过将2g胰蛋白胨、1g酵母提取物和2g氯化钠溶入200ml去离子水杀菌后获得。用于微生物敏感性试验的Müller-Hinton液体培养基(MHB)、正离子调节Müller-Hinton液体培养基(MHB)和胰酶解酪蛋白大豆培养基(TSB)购自美国BD公司(美国新泽西州)。需用酶标仪(Bio-Rad Laboratories公司680型)测量细菌生长和细胞毒性。使用奥林巴斯
Figure PCTCN2015089286-appb-000006
生物成像导航显微镜观察枯草杆菌细胞。
(B)抗菌敏感性试验
每种化合物的最小抑菌浓度(MIC)是依据美国国家临床实验室标准化委员会(CLSI)标准通过微量液体稀释法测定的。所有化合物在二甲基亚砜(DMSO)溶解，以双倍稀释。将TSB琼脂板的单菌落菌株溶入5mL MHB或CA-MHB。菌细胞在37℃下进行培养，直至生长细胞的最适密度(OD600)达到1.0。然后将其稀释到最终浓度约为5x 105CFU/mL，在96-微量滴定板(日本易威奇公司)双倍稀释。化合物3和PC190723用作阳性对照。在37℃下培养18小时后,使用酶标仪测量OD600值，计算抑菌浓度百分比。如果MIC值等于或高于90％，该化合物应测定为活性，是指该化合物造成的抑制细菌生长比例高于90％。表1对所有合成化合物的MIC进行了概述。
表1.每种化合物对不同菌株的MIC值
Figure PCTCN2015089286-appb-000007
Figure PCTCN2015089286-appb-000008
(C)细胞毒性试验
细胞毒性试验中使用的细胞株是从理工大学的库存中取得的。在本试验中，将小鼠成纤维细胞(L929)、一种原株乳腺癌细胞线(LCC6)和P-gp-高表达乳腺癌细胞株(LCC6MDR)与各种浓度化合物混合，在96-微量滴定板的最终容积达到100μL。细胞在37℃下培养3天。化合物的最大半数抑制值(IC50)的确定是通过CellTiter 96AQueous单溶液细胞增殖检测试剂盒(普洛麦格)。3-(4,5-二甲基噻唑-2-氧基)-5-(3-羧基-甲氧基苯基)-2-(4-磺苯基)-2H-四唑(3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)(2mg/mL,MTS,普洛麦格)和吩嗪硫酸甲酯(phenzaine methosulfate)(0.92mg/mL PMS,西格玛奥德里奇)以20:1的比例混合。将MTS/PMS混合液(10μL)加入每个井洞，在滴定板上培养2小时，温度为37℃。然后用酶标仪(Bio-Rad)测试490nm吸光度。IC50值通过使用GraphPad(Prism 4.0)绘图软件进行计算。每种化合物的选择性指标通过L929毒性等级 和金黄葡萄球菌29213的MIC值的比率计算。如果选择性指标大于10，化合物定义为潜在抗生素。F332和F361被选作测试三种哺乳动物细胞株。结果如表2所示。
表2.F332和F361对不同哺乳动物细胞株的IC50值。
Figure PCTCN2015089286-appb-000009
(D)对枯草杆菌细胞形态的影响
作为最有潜力的两种化合物，选择测试F332和F361对枯草杆菌168细胞形态的影响。使用两种化合物对枯草杆菌进行对数期培育测试，使终浓度达到MIC半数值。使用1％DMSO溶液进行控制。结果显示，两种化合物能使枯草杆菌细胞产生丝状形态。如图6所示，枯草杆菌细胞的平均长度为3.5±0.7μm。如图7所示，F332使枯草杆菌细胞长度延长三倍。F332使枯草杆菌细胞的平均长度从3.5±0.7μm延长至11.7±4.5μm。另一方面，如图8所示，F361使枯草杆菌细胞长度延长六倍，从3.5±0.7μm延长至21.1±1.8μm。研究发现，F332和F361能使枯草杆菌168细胞在亚致死浓度下产生丝状形态。结果显示，两种化合物都可以通过阻塞胞质细胞分裂蛋白质从而抑制细菌繁殖。
(E)荧光显微镜分折
通过使用绿荧光蛋白标识的细胞分裂蛋白FtsZ对F332在枯草杆菌中所产 生的成丝作用在荧光显微镜下进行定位分折以。一种FtsZ-eGFP融合蛋白表达质粒的建构如下(质粒结构示于图10a)：
根据一个革兰氏阳性菌(G+)的质粒pGK12的DNA序列，设计引物对：
序列1 
Figure PCTCN2015089286-appb-000010
序列2 
Figure PCTCN2015089286-appb-000011
扩增PCR片断，长约4,000个碱基对，内含氯霉素抗性基因、G+复制原点和大部分pGK12序列。
为了引入革兰氏阴性菌(G-)的复制原点及使氯霉素抗性基因能在大肠杆菌有效表达，根据G-质粒pRSET-A(Invitrogen公司)的DNA序列，设计引物对：序列3 
Figure PCTCN2015089286-appb-000012
序列4 
Figure PCTCN2015089286-appb-000013
扩增长约1,000个碱基对的PCR片断，内含pBR322的复制原点和T7启动子。两个PCR片断经限制性内切酶XbaI和BglII消化后连接并转化到大肠杆菌BL21(DE3)，在氯霉素平板上进行筛选，获得质粒pFZ1.0。
通过引入乳糖操纵子机制，可有效地控制枯草杆菌FtsZ基因的过量表达所引起宿主的致死效应。根据pFZ1.0的DNA序列，设计引物对：
序列5 
Figure PCTCN2015089286-appb-000014
序列6 
Figure PCTCN2015089286-appb-000015
扩增PCR片断，长约3,800个碱基对，去除多余的pGK12部分。
根据pMAL-c2(NEB公司)的DNA序列，设计引物对：
序列7 
Figure PCTCN2015089286-appb-000016
序列8 
Figure PCTCN2015089286-appb-000017
扩增长约1,200个碱基对的PCR片断，内含lacA基因及其lacI启动子。 两个PCR片断经限制性内切酶XhoI和EcoRI消化后连接并转化到大肠杆菌BL21(DE3)，在氯霉素平板上进行筛选，获得质粒pFZ1.1。
根据pFZ1.1的DNA序列，设计引物对：
序列9 
Figure PCTCN2015089286-appb-000018
序列10 
Figure PCTCN2015089286-appb-000019
扩增PCR片断，长约5,000个碱基对，用限制性内切酶EcoRI和BamHI进行消化。再根据G+载体pHT43(MoBiTec公司)的DNA序列，设计引物对：
序列11 
Figure PCTCN2015089286-appb-000020
序列12 
Figure PCTCN2015089286-appb-000021
Figure PCTCN2015089286-appb-000022
扩增长约450个碱基对的PCR片断，内含groE启动子和lacO序列，用限制性内切酶EcoRI和BglII进行消化。两个片断连接后转化到大肠杆菌BL21(DE3)，在氯霉素平板上进行筛选，获得质粒pFZ2.0。
根据枯草杆菌168的FtsZ基因序列，设计引物对：
序列13 
Figure PCTCN2015089286-appb-000023
序列14 
Figure PCTCN2015089286-appb-000024
扩增PCR片断，长约1,200个碱基对。
此外根据pEGFP的DNA序列，设计引物对：
序列15 
Figure PCTCN2015089286-appb-000025
序列16 
Figure PCTCN2015089286-appb-000026
扩增长约700个碱基对的eGFP基因片断，通过重叠延伸PCR方法连接两个片断，再用内切酶EcoRI和BamHI消化并连接到pFZ2.0的对应内切位点 上，转化到大肠杆菌BL21(DE3)，在氯霉素平板上进行筛选，获得质粒pFZ-FtsZ-eGFP。提取质粒转化到枯草杆菌168，在含氯霉素的LB培养液内生长，加入不同浓度的乳糖或IPTG观察FtsZ-eGFP基因的表达。
图10b和10c所示的枯草杆菌是在没有F332的情况下培养，在荧光显微镜下图10b的枯草杆菌细胞中间部分出现了较强的绿色荧光，表示FtsZ-eGFP融合蛋白的表达质粒被定位并整合于细胞中间以形成细胞分裂的Z环(Z-ring)。
图10d和10e所示的枯草杆菌是在F332的情况下培养，在荧光显微镜下图10d的枯草杆菌细胞多个部分出现了绿色荧光，并使细胞增长，反映FtsZ-eGFP融合蛋白的表达质粒在细胞中的定位被F332影响。因此，该蛋白的表达不只于在进行细胞分裂的位置。
(F)金黄葡萄球菌FtsZ蛋白质(S.aureus FtsZ protein)的表达和提纯
携带金黄葡萄球菌ftsz基因的大肠杆菌BL21细胞是从理工大学实验室菌库中获得的。将菌库中提取的大肠杆菌菌株在5mL LB培养液和100μg/mL氨苄青霉素(ampicillin)中培养14-16小时，温度为37℃。隔夜培养的细胞培养液(2mL)转至200mL的2X TY培养液中，其中含100μg/mL氨苄青霉素，使细胞在37℃下生长，直至光学吸收率(OD600)达到0.8。蛋白质表达在添加0.4mM异丙基-β-D-1-硫代半乳糖苷(isopropyl-β-D-1-thiogalactopyranoside)(IPTG)后再过4小时被诱发。细菌细胞在20分钟9000rpm离心分离力作用下得以聚集，温度为4℃。然后细胞团块在20mL起始缓冲液(0.02M磷酸钠,0.5M氯化钠,pH 7.4)中再次悬浮。通过声波降解法将细胞均质化，增幅为50％，30秒带脉冲，30秒无脉冲，进行 5个循环。不能溶解的细胞残骸在1.5小时13,000rpm离心分离力作用下排除，温度为4℃。浮层通过0.22μm无菌过滤器进行收集和过滤。
浮层中的FtsZ蛋白质通过快速蛋白质液相层析法(Fast Protein Liquid Chromatography(FPLC))进行提纯。在FPLC中，用硫酸镍(II)溶液激活5mL HiTrap螯合层，捕获金黄葡萄球菌FtsZ蛋白质。然后用起始缓冲液使其平衡。包含目标蛋白质的可溶性蛋白质溶液和起始缓冲液一起通过该螯合层。然后用洗提缓冲液(0.02M磷酸钠,0.5M氯化钠,0.5M异吡唑,pH 7.4)对目标蛋白质(Peak A)进行洗提。通过SDS-PAGE法(图9)进行收集和分析,然后用50mM MOPS缓冲液渗析，储存温度为-80℃。
(G)光散射试验
光散射试验可以确定FtsZ聚合的程度，通过使用荧光光谱仪(Perkin-Elmer,USA)测量90°光散射信号。激发和发射波长设定为600nm，狭缝宽度为5nm。金黄葡萄球菌FtsZ(12μM)在50mM MOPS缓冲液(pH 6.8)中连续稀释条件下培养10分钟，温度为25℃。对照样品只包括1％的二甲基亚砜。然后将KCl(终浓度:50mM)和MgCl2(终浓度:10mM)加入混合液中，在37℃下培养8分钟，建立一个基线。然后加入终浓度1mM GTP，对光散射信号测量1,500秒。试验装置中的相关光散射信号的计算是参照对比试验。对每种化合物浓度分别进行三次试验。选择F332和F361这两种化合物进行测试，结果分别在图11和图12显示。两种化合物都能抑制FtsZ聚合的光散射。减少的光散射信号显示，两种化合物都能在剂量依赖性方式下减少FtsZ原纤维的聚合物质。
(H)GTP酶(GTPase)活性测试
金黄葡萄球菌FtsZ的GTPase活性是通过96孔微型板块确定，使用CytoPhosTM磷酸盐荧光检测试剂盒(CytoPhosTM phosphate assay Biochem KitTM)(Cytoskeleton,USA)，对GTP水解过程中的释放的无机磷酸盐浓度进行测试。重组的金黄葡萄球菌FtsZ(7.5μM)在50mM MOPS缓冲液(pH 6.5)中连续稀释条件下培养10分钟，温度为25℃。对照样品只包括1％的二甲亚砜。然后将KCl(终浓度:200mM)和MgCl2(终浓度:5mM)加入混合液中，加入GTP(终浓度:500mM)，在37℃下培养30分钟。不能溶解的聚合物在室温下经过1分钟13,500rpm离心分离力排除。80μL浮层转至96孔微型板块的一孔中，用120μL Cytophos试剂在室温下培养10分钟。混合液中的无机磷酸盐通过96孔微型板块读写器(BioRad 680)测试650nm的吸收率确定。样品的测试结果是参照对比试验。对每种化合物浓度分别进行三次试验。选择F332和F361这两种化合物进行测试，结果分别在图13和图14显示。如图13及14所示，加入两种化合物后，没有观察到金黄葡萄球菌FtsZ的GTPase活性有显著变化。基于光散射试验结果和GTPase活性测试，两种化合物很可能有能力在不影响GTPase活性前提下抑制FtsZ的聚合作用。
(I)透射电子显微镜法(TEM)
TEM提供了一种对FtsZ聚合形态的直接观察法。选择F332和F361这两种化合物进行测试。金黄葡萄球菌FtsZ(12μM)在不同浓度的化合物中培养，在50mM MOPS缓冲液(pH 6.5)中培养10分钟，温度为25℃。对照样品只包括1％的二甲亚砜。然后将KCl(终浓度:50mM)和MgCl2(终浓度:5mM)加入混合液中，加入GTP(终浓度:1mM)，在37℃下培养15分钟。在辉光 放电涂碳铜载网(400网格)上加入10μL反应混合液，在25℃下培养10分钟。在铜载网上加入10μL 0.5％磷钨酸(PTA)将蛋白质染色。对铜载网进行通宵风干。用透射式电子显微镜(JEOL型JEM 2010，工作电压200kV，配置Gutan MSC 794CCD摄像机)观察FtsZ聚合形态。F332和F361的电子显微图分别见图15和图16。FtsZ原纤维形成的宽大厚束网可以通过添加抑制剂进行观察。比较添加抑制剂后得到的原纤维和加入100μM F332或F361聚合产生的相对短小的直线单链FtsZ原纤维。这一现象与光散射试验不谋而合，因为F332和F361这两种化合物都表现出在剂量依赖性方式下抑制FtsZ聚合作用。
(J)确定分级抑制浓度指数(FICI)
本发明使用的菌株是在临床上分离的MRSA菌株USA300#757和#2960，都是从理工大学的实验室获得的。之前所述Müller-Hinton液体培养基(MHB)稀释法采用的即是确定分级抑制浓度指数(FICI)23。对PC190723进行12轮连续的双倍稀释,用96孔微型板块制配F332和F361，浓度从512μg/mL变为0.25μg/mL。用同样的程序进行对氨苄青霉素、氯唑西林(cloxacillin)和头孢呋辛(cefuroxime)进行一系列的双倍稀释。最后，将化合物与β-内酰胺(β-lactams)按照1:1比例进行混合，再依照上述程序进行双倍稀释。每个微型板块孔洞包含100μL MHB培养基，单独用化合物增补，单独用β-内酰胺(β-lactams)增补，或用化合物和β-内酰胺混合液增补。将Müller-Hinton液体培养基中过夜培养的金黄葡萄球菌在盐水中稀释至OD595值为0.08-0.1，添加进每个微型板块孔洞，在37℃下培养20小时。所有测试都重复做一次，通过视觉判断对细菌生长的抑制程度。化合物和β-内酰胺的FICI计算公式如 下：FICI＝(化合物和β-内酰胺混合液的MIC/化合物的MIC)+(化合物和β-内酰胺混合液的MIC/β-内酰胺的MIC)。结果见表3。
表3.PC190723,F332和F361对两种MRSA菌株(USA300#757；USA300#2960)的FICI值：
Figure PCTCN2015089286-appb-000027
为了进一步证明可应用在不同菌株上，其中，F332分别与五种抗生素联合施用在表4中的菌株，不同的FICI值示于表4：
表4 F332与以下抗生素联合施用对不同菌株的FICI值
Figure PCTCN2015089286-appb-000028
Figure PCTCN2015089286-appb-000029
#ATCC菌株
(J)药代动力学参数研究及其在药物制备在的应用
化合物F332需要进一步进行体内药代动力学研究。UPLC-MS/MS系统包括沃特世公司超高压液相色谱(Acquity Waters UPLC)，与质谱计(Micromass型Quattro Ultima)相连，使用正离子模式电喷雾电离法。对ACQUITY UPLC BEH C18 1.7μM(2.1x 50mm)层进行层析分离。(色谱分析)流动相包括甲醇+0.1％甲酸(溶剂B)和Milli-Q超纯水+0.1％甲酸(溶A)。设置多重反应监测模式(MRM)对F332[M+H]+在299>142m/z情况下进行监测。碰撞能量、进样锥电压、源温度、退溶温度和毛细管电压分别为25,30,150℃,350℃和3Kv。锥孔气量和退溶气量分别为150L/Hr和600L/Hr。
雄性Sprague–Dawley(SD)小鼠(体重250-280g)从香港理工大学动物设施中心获得。将动物在特别控制的温度和湿度环境下饲养，保持12小时正常光照，喂养标准量的食物和水。香港理工大学动物主体伦理小组委员会认同 动物实验协定。在进行实验的前一天进行右颈静脉穿刺术。头天晚上对动物禁食，在实验过程中可以喂水。F332是用5％蓖麻油聚氧乙烯醚(CremophorEL)、5％乙醇、90％盐水在2mg/ml浓度下现配的。F332在使用当天制配，在半小时内用于动物研究。在当前研究中，F332的使用是通过口部喂食和静脉注射(IV)。血样(大约500μL)通过在颈静脉穿插肝素管(20u nits)，在使用后5,10,30,45,60,120,240和420分钟后进行静脉注射研究。对于口部喂食研究，则在2,10,30,45,60,120,240,480和600分钟采集血浆样品，在16,100G离心分离力作用下采集10分钟。所有的血浆样品需添加3倍容量的甲醇进行蛋白质沉淀。在进行UPLC-MS/MS分析前通过注射器式滤器对浮层进行过滤。血浆中的药动学参数通过Pharmacokinetics Solutions 2.0软件(美国蒙特罗斯Summit Research Services公司生产)进行非分割性药物动力学数据分析。所确定的药动学参数包括最大血浆浓度(Cmax)、达到最大血浆浓度时间(Tmax)、平均滞留时间(MRT)、吸收/分布半衰期(t1/2α),消除半衰期(t1/2β)、分布容量(Vd)、总清除率(CL)以及浓度曲线下面积(AUC)。结果如图17和表5所示。
表5体内药代动力学参数
Figure PCTCN2015089286-appb-000030
Figure PCTCN2015089286-appb-000031
通过上述测试结果可以看出：本发明的制备方法从3-氨基苯甲酰胺(3-aminobenzamide)衍生出的新型衍生物，对多种菌株例如(革兰氏阳性和革兰氏阴性)、癌细胞(LCC6和LCC6MDR)以及小鼠腹膜成纤维细胞(L929)具细胞毒性/强大的抗菌活性；对小鼠成纤维细胞具有低毒性；其与细胞分裂蛋白FtsZ起相互作用；其与临床应用的β-内酰胺抗生素合成后表现出对临床分离而社区感染的金黄葡萄球菌(USA300)和其它MRSA菌株具有高度的合成抗菌活性，抑菌浓度指数更低至0.01；药效动力学研究结果显示，其中一种名为F332的衍生物，其口服用量为50毫克/千克体重，口服的生物兼容性约为12％；本发明的衍生物应用于治疗耐甲氧西林葡萄球菌感染的药物制备中，可在临床上用作β-内酰胺辅助抗生素通过口服途径治疗耐甲氧西林葡萄球菌感染。

Claims (37)

  1. 一种作为β-内酰胺辅助抗生素的3-氨基苯甲酰胺衍生物，其特征在于，为具以下化学式(I)的化合物:
    Figure PCTCN2015089286-appb-100001
    其中，R1、R2和R3各自分别为氢、氟或溴；
    R4与R5不同时，R4为氢、甲基、乙基或氟代苄基，R5为C4-C10的烷基、烷氧基烷基、C4-C10的烯基、氟代苄基、烷氧基取代的苄基或酰基；
    R4和R5相同时，R4和R5都为CH2-(CH2)n-CH2，n＝2或3。
  2. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2、R3和R4各自分别为氢，R5为辛基。
  3. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2、R3和R4各自分别为氢，R5为壬基。
  4. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2和R3各自分别为氢，R4为甲基，R5为辛基。
  5. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2和R3各自分别为氢，R4为甲基，R5为壬基。
  6. 根据权利要求1所述的衍生物，其特征在于，所述R1和R3各自分别为氢，R2为F，R4为氢，R5为壬基。
  7. 根据权利要求1所述的衍生物，其特征在于，所述R1和R3各自分别为氢，R2和R4各自为F，R5为壬基。
  8. 一种权利要求1-7任一项的所述衍生物的制备方法，其特征在于，包括以下步骤：
    (a)将原料A与原料B、碳酸钾和乙腈均匀搅拌形成反应混合液，加热回流反应4小时，再精制后得到产物；所述原料A为3-氨基苯甲酰胺、2-氟-5-氨基苯甲酰胺或2,4-二氟-5-氨基苯甲酰胺；所述原料B为溴代壬烷或溴代辛烷；
    (b)在步骤(a)得到的产物为3-(壬基氨基)苯甲酰胺或3-(辛基氨基)苯甲酰胺时，再将其分别与硫酸二甲酯、碳酸钾、和乙腈均匀搅拌，并加热回流12小时，再精制后得到产物。
  9. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为辛基。
  10. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为壬基。
  11. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为庚基。
  12. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为3,7-二甲基-2,6-辛二烯基。
  13. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为2-壬烯基。
  14. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为(4’-丁氧基)丁基。
  15. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为癸基。
  16. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F，R3为溴，R4为氢，R5为壬基。
  17. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F，R3 为氢，R4为甲基，R5为辛基。
  18. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F，R3为氢，R4为乙烷基，R5为辛基。
  19. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F，R3为氢，R4为甲基，R5为壬基。
  20. 一种权利要求1或9-19中任一项的所述衍生物的制备方法，其特征在于，包括以下步骤：
    (a)将原材料2,6-二氟-3-氨基苯甲酰胺与溴代烷烃、碳酸钾及乙腈均匀搅拌形成反应混合液，并加热回流4小时，再精制后得到产物；所述溴代烷烃为溴代壬烷、溴代辛烷、溴代癸烷、溴化香叶醇、(Z)-1-溴-2-壬烯或溴代庚烷；
    (b)将步骤(a)反应得到的2,6-二氟-3-(壬基氨基)苯甲酰胺进行溴化处理并在室温下搅拌12小时，精制得到4-溴-2,6-二氟-3-(壬基氨基)苯甲酰胺；
    (c)使用碘甲烷或溴乙烷对从步骤(a)得到的2,6-二氟-3-(辛基氨基)苯甲酰胺，进一步烷基化处理，并与碳酸钾和乙腈均匀搅拌回流14小时，精制得到2,6-二氟-3-(甲基(辛基)氨基)苯甲酰胺或3-(乙烷基(辛基)氨基)-2,6-二氟苯甲酰胺；
    (d)使用硫酸二甲酯对从步骤(a)得到的2,6-二氟-3-(壬基胺基)苯甲酰胺进行烷基化处理，并与碳酸钾和乙腈均匀搅拌回流12小时，精制得到2,6-二氟-3-(甲基(壬基)氨基)苯甲酰胺。
  21. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为壬酰胺基。
  22. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为吡咯烷。
  23. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为哌啶。
  24. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为3’,4’-二氟苄基。
  25. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3为氢，R4为3’,4’-二氟苄基，R5为3’,4’-二氟苄基。
  26. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为2’,4’-二氟苄基。
  27. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3为氢，R4、R5各自分别为2’,4’-二氟苄基。
  28. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为2’,6’-二氟苄基。
  29. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3为氢，R4、R5各自分别为2’,6’-二氟苄基。
  30. 一种权利要求1或21-29中任一项的所述衍生物的制备方法，其特征在于，包括以下步骤：
    (a)将原材料2,6-二氟-3-氨基苯甲酰胺和壬酰氯在0℃下溶入吡啶和二氯甲烷反应4小时，精制得到2,6-二氟-3-壬基氨基苯甲酰胺；
    (b)将原材料2,6-二氟-3-氨基苯甲酰胺与1,4-二溴丁烷或1,5-二溴代戊烷分别加入碳酸钾在乙腈中均匀搅拌4小时反应，精制分别得到2,6-二氟-3-(吡咯烷-1-氨基)苯甲酰胺和2,6-二氟-3-(哌啶-1-氨基)苯甲酰胺；
    (c)将原材料2,6-二氟-3-氨基苯甲酰胺与原料C以及碳酸钾，在乙腈中加热回流4小时，精制分别得到3-(3’,4’-二氟苄氨基)-2,6-二氟苯甲酰胺或3-(双(3’,4’-二氟苄基)氨基)-2,6-二氟苯甲酰胺、3-(2’,4’-二氟苄氨基)-2,6-二氟苯甲酰胺或3-(双(2’,4’-二氟苄基)氨基)-2,6-二氟苯甲酰胺、以及3-(2’,6’-二氟苄氨基)-2,6-二氟苯甲酰胺或3-(双(2’,6’-二氟苄基)氨基)-2,6-二氟苯甲酰胺；所述原料C为3,4-二氟溴苄、2,4-二氟溴苄或2,6-二氟溴苄。
  31. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3 和R4各自分别为氢，R5为3’-丁氧基苄基。
  32. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为3’-(仲丁氧基)苄基。
  33. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为3’-(戊氧基)苄基。
  34. 根据权利要求1所述的衍生物，其特征在于，所述R1、R2为F；R3和R4各自分别为氢，R5为3’-((4”-(三氟甲基)苄基)氧)苄基。
  35. 一种权利要求1或31-34中任一项的所述衍生物的制备方法，其特征在于，包括以下步骤：
    (a)将原材料2,6-二氟-3-氨基苯甲酰胺分别与相应的苯甲醛以及催化剂对甲苯磺酸(pTsOH)和甲醇进行还原胺化，在室温下混合液搅拌2小时；
    (b)然后分部分加入氰基硼氢化钠，再混合搅拌12小时，反应精制分别得到权利要求31到34任一项所述的衍生物；所述苯甲醛为3-丁氧基苯甲醛、3-(戊氧基)苯甲醛、3-(仲丁氧基)苯甲醛或3-((4’-(三氟甲基)苄基)氧)苯甲醛。
  36. 权利要求1、10或19中所述的衍生物在制备β-内酰胺辅助抗生素中的应用。
  37. 根据权利要求36所述的应用，其特征在于，所述衍生物的用量与人体体重的比例为50毫克/千克体重，所述衍生物在口服后的生物兼容性为12％。
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