CN114478486B - Three-molecule conjugate taking para-aminosalicylic acid as parent nucleus, intermediate, preparation method and application - Google Patents

Three-molecule conjugate taking para-aminosalicylic acid as parent nucleus, intermediate, preparation method and application Download PDF

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CN114478486B
CN114478486B CN202210106754.XA CN202210106754A CN114478486B CN 114478486 B CN114478486 B CN 114478486B CN 202210106754 A CN202210106754 A CN 202210106754A CN 114478486 B CN114478486 B CN 114478486B
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aminosalicylic acid
para
conjugate
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molecule conjugate
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CN114478486A (en
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范莉
杨大成
任艳会
谢建平
许峻旗
代乐平
毛丹
杨茜
周成合
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Southwest University
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
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Abstract

The invention discloses a three-molecule conjugate taking p-aminosalicylic acid (PAS) as a mother nucleus, which is shown in a formula I and is formed by conjugation of three structural units of PAS, isonicotinic acid and fluoroquinolone; through an anti-human pathogenic bacteria activity test, the three-molecule conjugate synthesized by the invention has a certain antibacterial activity, particularly has a plurality of molecules with high antibacterial activity on mycobacterium tuberculosis, salmonella, pseudomonas aeruginosa and staphylococcus aureus, and meanwhile, the safety of the molecules is verified through a hemolytic test, so that the three-molecule conjugate has the potential of further developing antibacterial drugs, particularly antituberculosis drugs.

Description

Three-molecule conjugate taking para-aminosalicylic acid as parent nucleus, intermediate, preparation method and application
Technical Field
The invention belongs to the technical field of medicine synthesis, and relates to a three-molecule conjugate taking p-aminosalicylic acid (p-aminosalicylic acid, PAS) as a parent nucleus, an intermediate, a preparation method and pharmaceutical application.
Background
Tuberculosis (TB) is a chronic lethal infectious disease caused by mycobacterium Tuberculosis (Mycobacterium Tuberculosis, MTB). Tuberculosis is the leading cause of death for a single infectious disease, ranked higher than aids, before coronavirus (covd-19) is pandemic. To date, TB remains a global public health problem.
World health organization (World health organization, WHO) classifies antitubercular drugs into first-line and second-line antitubercular therapeutic drugs according to their effects and magnitude of side effects. The first-line TB drugs Streptomycin (SM), isoniazid (INH, H), pyrazinamide (PZA, Z), rifampicin (RIF, R) and Ethambutol (Ethambutol, EMB, E) have obvious curative effects and fewer side effects, can kill tubercle bacillus in the rapid propagation period and the slow propagation period simultaneously, and are the first-choice varieties for TB treatment. Two-line TB has a large variety of drugs, quinolones (FQ S ) Macrolides, phenothiazines, aminoglycosides, oxazolidinones, cycloserine, PAS are representative thereof, but have relatively more side effects and relatively poor therapeutic effects. Because of the specificity of TB, treatment of sensitive TB always employs a combination regimen of multiple simultaneous doses, with approximately 85% of TB patients employing a 2hrze+4hr 6 month regimen comprising rifampicin, generally enabling therapeutic success. Although the treatment of sensitive TB has achieved tremendous success, drug-resistant tuberculosis (DR-resistant Tuberculosis, DR-TB), including multi-Drug resistant tuberculosis and widespread Drug resistant tuberculosis, remains worldwideThe most serious challenge. The multi-drug resistance and high drug resistance rate are the main characteristics of patients with the pulmonary tuberculosis, and even if the combined treatment mode of the multi-drug recommended by WHO is adopted, the clinical problems of long treatment period, drug resistance and the like still exist, and one of the reasons is that the compliance of the patients is poor, and the drug resistance is further generated.
Disclosure of Invention
The design strategy of the multi-target medicine is a new idea for solving the problem of complex diseases, especially multi-target diseases. The pharmacophore connection method can introduce medicines with multiple action targets into a single molecule, so as to realize the design of the medicines with multiple targets.
The coupling of two or more antitubercular molecules into a single molecule may have the same therapeutic effect and may reduce the single dose, so that the problem of poor compliance of patients can be improved compared with the combined treatment scheme of WHO recommends simultaneous administration of multiple drugs, and further the generation of drug resistance is reduced, which is a valuable drug study subject.
INH is a prodrug that is activated by the catalase-peroxidase (KatG) of MTB by passive transport into the MTB to form isonicotinyl radicals, which are then reacted with NAD + Binding, to form an IN-NAD adduct, inhibits enoyl-acyl carrier protein reductase (InhA), interferes with mycolic acid synthesis, and disrupts bacterial cell walls.
The antibacterial agent FQs has good treatment effect on sensitive TB and DR-TB, and the curative effect of the antibacterial agent combined with other anti-TB drugs is more remarkable than that of the antibacterial agent singly used. FQs drug targets DNA helicases, inhibits the MTB replication cycle, leading to cell death.
PAS is suitable for pulmonary and extrapulmonary tuberculosis caused by Mycobacterium tuberculosis, and must be combined with other antitubercular drugs. Can delay the generation of drug resistance of tubercle bacillus to SM or INH when being used together. PAS has three functional groups of carboxyl, hydroxyl and amino, can be used as a linker (linker) of a multi-target drug, can be used as an antituberculosis drug, takes the linker as a parent nucleus design molecule, can conjugate three drug fragments into the same molecule (3-in-1), can reduce the molecular weight of a target molecule, can possibly improve the patentability of the whole molecule under the advantage of reducing the synthesis difficulty, and has potential research prospect.
In view of the above, the present invention aims to use PAS as a parent nucleus, derive PAS into corresponding carboxylic acid ester, leave amino and hydroxyl to be connected with isonicotinic acid (INA) and FQs, design and synthesize a class of three-molecule conjugates, and test the antibacterial activity of the three-molecule conjugates.
As an attempt, a target molecular pattern was designed by selecting a hydroxyl-linked FQs, amino-linked INA (see Mode of target molecule in FIG. 1), in which Linker 1 Linker linking carboxyl group of INA and amino group of PAS 2 Linking the hydroxyl group of PAS with the amine group of FQs. The Linker selection is an important part of the pharmacophore splicing method. Suitable linker must possess the function of linking two or more fragments while meeting the requirements of as small a molecular weight as possible, moderate molecular rigidity and flexibility, non-toxic metabolic fragments, etc. Based on the assumption that molecules are nontoxic, simple in structure, capable of connecting hydroxyl groups and amino groups and the like, the invention selects halogenated acyl chloride as a Linker 1 And Linker 2 According to which the Target molecule (see Target molecule in FIG. 1) is designed. The invention further designs a Linker based on the requirement of the minimum molecular weight 1 Is acetyl, linker 2 Target molecules TM1 and TM2 (see FIG. 1) which are propionyl groups. Through synthetic route design and reaction condition exploration, 16 novel molecules which are not reported yet are successfully synthesized, and through anti-human pathogenic bacteria activity test, the molecules have certain antibacterial activity, particularly a plurality of molecules with high antibacterial activity on mycobacterium tuberculosis, salmonella, pseudomonas aeruginosa and staphylococcus aureus exist, and meanwhile, the safety of the molecules is verified through hemolytic test. Therefore, the invention finally provides the following technical scheme:
1. a three-molecule conjugate with para-aminosalicylic acid as a parent nucleus represented by formula I:
in the formula I, R is methyl or ethyl;
x is cyclopropyl, ethyl or 4-halogeno phenyl;
y is-represents the linker to the carbonyl group; />Represents a linker to an aromatic ring;
z is N or C-R 1 ；R 1 Hydrogen, halogen or methoxy.
Further, X is cyclopropyl, ethyl or 4-fluorophenyl; r is R 1 Hydrogen, fluorine, chlorine or methoxy.
Further, the three-molecule conjugate with para-aminosalicylic acid as a parent nucleus shown in the formula I is any one of the following compounds:
further, the three-molecule conjugate with para-aminosalicylic acid as a parent nucleus shown in the formula I is any one of the following compounds: TM1a, TM1b, TM1f, TM1h, TM2a, TM2b, TM2c, TM2h.
2. An intermediate of formula II or a pharmaceutically acceptable salt thereof, for use in preparing a trimolecular conjugate of formula I having para-aminosalicylic acid as a parent core, or a pharmaceutically acceptable salt thereof:
in formula II, R is methyl or ethyl.
3. The preparation method of the three-molecule conjugate or the pharmaceutically acceptable salt thereof taking para-aminosalicylic acid as a mother nucleus shown in the formula I comprises the following steps:
a. PAS and alcohol ROH are heated to react under the catalysis of concentrated sulfuric acid at 60-70 ℃ to prepare an intermediate IM1;
b. the intermediate IM1 and chloroacetyl chloride are stirred in methylene Dichloride (DCM) in an ice bath for reaction in the presence of sodium bicarbonate to prepare an intermediate IM2;
c. the intermediate IM3 is prepared by the oil bath stirring reaction of the intermediate IM2 and INA in dimethyl sulfoxide (DMSO) at 60 ℃ in the presence of triethylamine;
d. FQs and 3-chloropropionyl chloride are stirred in DCM in the presence of sodium bicarbonate in an ice bath for reaction to prepare an intermediate IM4;
e. the three-molecule conjugate taking para-aminosalicylic acid as a parent nucleus shown in the formula I is prepared by carrying out oil bath stirring reaction on the intermediates IM3 and IM4 in N, N-Dimethylformamide (DMF) at 60 ℃ in the presence of potassium carbonate and a catalyst NaI;
in the alcohols ROH, intermediates IM1, IM2 and IM3, R has the meaning given in formula I;
FQs and intermediate IM4, X, Y and Z have the definition given in formula I.
4. The application of the three-molecule conjugate with the para-aminosalicylic acid as the mother nucleus shown in the formula I or the pharmaceutically acceptable salt thereof in the preparation of antibacterial drugs.
Further, the antibacterial agent is an agent against one or more of mycobacterium tuberculosis, staphylococcus aureus, micrococcus luteus, escherichia coli, acinetobacter baumannii, salmonella and pseudomonas aeruginosa.
5. The application of the intermediate shown in the formula II or the pharmaceutically acceptable salt thereof in preparing antibacterial drugs.
Further, the antibacterial agent is an agent against one or more of mycobacterium tuberculosis, staphylococcus aureus, micrococcus luteus, escherichia coli, acinetobacter baumannii, salmonella and pseudomonas aeruginosa.
The term "pharmaceutically acceptable salt" in the present invention may be an acidic salt or a basic salt, such as an inorganic acid salt, an organic acid salt, an inorganic base salt or an organic base salt, unless otherwise specified. The term "halogen" refers to F, cl, br and I.
The invention has the beneficial effects that: the invention adopts the principle of splicing the medicine fragments, splices PAS, INA and FQs into a single molecule through a linker, and designs and synthesizes a three-molecule conjugate taking PAS as a parent nucleus. Through an anti-human pathogenic bacteria activity test, the molecules have certain antibacterial activity, particularly a plurality of molecules with high antibacterial activity on mycobacterium tuberculosis, salmonella, pseudomonas aeruginosa and staphylococcus aureus, and meanwhile, the safety of the molecules is verified through a hemolytic test, so that the anti-human pathogenic bacteria antibacterial agent has the potential of further developing antibacterial agents, particularly anti-tuberculosis agents.
Drawings
FIG. 1 is a schematic diagram of a target molecule TM1/TM2.
FIG. 2 is a synthetic route diagram of the target molecule TM1/TM2.
FIG. 3 shows the results of a hemolysis test of the target molecule TM1a/TM2 a.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below.
Main chemical reagent and instrument
PAS (Shanghai taitan Co., ltd., 98%); INA, concentrated sulfuric acid, methanol, ethanol, sodium bicarbonate, and potassium carbonate (Chongqing Chuan chemical Co., ltd.); clinafloxacin (CLX), ciprofloxacin (CIP), sarafloxacin (SAR), enoxacin (ENX), balofloxacin (BAL), gatifloxacin (GAT) (zheng, keltai biochemical technology limited, > 95%); norfloxacin (NOR), lomefloxacin (lomefloxane) (AR, shanghai dary fine chemical company, inc.); chloroacetyl chloride, 3-chloropropionyl chloride (Chongqing titanium new chemical Co., ltd.); the remaining reagents were commercially available chemically pure or analytically pure products, which were used without purification.
Nuclear magnetic resonance apparatus (Bruker, ADVANCE III) TM 600MHz, TMS as internal standard); high resolution mass spectrometer (QTOF-MS, bruker Impact II, bremen, germany); melting point tester (X-6, beijing Fukai instruments Co., ltd.).
1 Synthesis of target molecule TM
a. Synthesis of intermediate IM1a/IM1b
Taking PAS and methanol (MeOH) for example for preparing IM1a, the reaction conditions (catalyst type and amount, reaction temperature, etc.) of this step were explored (see Table 1).
TABLE 1 synthetic reaction condition search experiment of intermediate IM1a
Through exploration, the optimal conditions of the reaction in the step are as follows: concentrated sulfuric acid is used as a catalyst, the molar ratio of PAS to concentrated sulfuric acid is 1:2, and reflux reaction is carried out at 60-70 ℃.
PAS and methanol/ethanol (EtOH) are heated to react under the catalysis of concentrated sulfuric acid according to the optimal reaction conditions to obtain the intermediate IM1a/IM1b. The results of the synthesis experiments are shown in Table 2.
Operation example: PAS 1.53g (10 mmol) and methanol/ethanol 10mL are added into a 100mL round bottom flask, the mixture is stirred at room temperature, concentrated sulfuric acid 1.06mL (20 mmol) is slowly added dropwise, the mixture is subjected to oil bath reflux reaction, thin Layer Chromatography (TLC) is monitored until the reaction is finished, ice bath cooling is performed, a saturated sodium carbonate solution is used for adjusting pH to be 8-9, a large amount of solid is separated out, suction filtration is performed, a filter cake is washed by saturated NaCl solution, and the filter cake is dried, so that an intermediate IM1a/IM1b, an off-white solid, m.p. is respectively 100.3-104.0 ℃ and 113.2-113.9 ℃.
TABLE 2 results of synthetic experiments on intermediates IM1a/IM1b
b. Synthesis of intermediate IM2a/IM2b
The intermediate IM1a/IM1b and chloroacetyl chloride were reacted in DCM with ice-bath stirring in the presence of sodium bicarbonate (acid binding agent) to give intermediate IM2a/IM2b. The results of the synthesis experiments are shown in Table 3.
Operation example: in a 100mL reaction flask, IM1a/IM1b (10 mmol), 10mL DCM were added, stirred at room temperature, naHCO added 3 (30 mmol,2.522 g), continuously stirring, cooling in an ice bath, dropwise adding chloroacetyl chloride (12 mmol,1 mL), continuously stirring in an ice bath for reaction, monitoring the reaction to be finished by TLC, removing DCM by screwing, adding 20mL of water, filtering, washing a filter cake with saturated NaCl solution (15 mL multiplied by 3) and water (15 mL multiplied by 3) in sequence, and drying in vacuum to obtain an intermediate IM2a/IM2b as a white solid, wherein m.p. is 180.7-181.5 ℃ and 189.9-190.7 ℃ respectively.
TABLE 3 results of Synthesis experiments of intermediates IM2a/IM2b
c. Synthesis of intermediate IM3a/IM3b
Taking the reaction of IM2a and INA to prepare IM3a as an example, the reaction conditions (type of base, solvent, reaction temperature) of this step were examined (see Table 4).
TABLE 4 synthetic reaction condition exploration experiment of intermediate IM3a
Through exploration, the optimal conditions of the reaction in the step are as follows: with triethylamine (Et) 3 N) is alkali, DMSO is solvent, and the reaction is carried out at 60 ℃.
According to the optimal reaction conditions, intermediates IM2a/IM2b and INA are reacted in Et 3 And (3) in the presence of N, carrying out oil bath stirring reaction at 60 ℃ in DMSO to obtain an intermediate IM3a/IM3b. The results of the synthesis experiments are shown in Table 5.
Operation example: into a 100mL reaction flask, IM2a/IM2b (5 mmol) and 5mL DMSO were added, and the mixture was stirred at room temperature and Et was added 3 N (7.5 mmol), continuing stirring for 0.5h, adding INA (7.5 mmol,0.931 g), stirring at 60deg.C for reaction, monitoring the reaction to end by TLC, adding water, precipitating solid, suction filtering, washing filter cake with saturated NaCl solution (15 mL×3) and water (5 mL×3) in sequence, vacuum drying to obtain crude product, and purifying by column chromatography (PE: EA=5:1-1:1, v/v) to obtain intermediate IM3a/IM3b, wherein m.p. is 207.3-208.0deg.C, 186.5-187.3 deg.C respectively.
TABLE 5 results of Synthesis experiments of intermediate IM3a/IM3b
d. Synthesis of intermediates IM4 a-IM 4h
FQs (CLX/NOR/CIP/SAR/ENX/BAL/LOM/GAT) and 3-chloropropionyl chloride were reacted in DCM with ice-bath stirring in the presence of sodium bicarbonate (base) to give intermediate IM4. The results of the synthesis experiments are shown in Table 6.
Operation example: adding FQs (10 mmol) and DCM (20 mL) into a 100mL round bottom flask, stirring uniformly at room temperature, adding finely ground sodium bicarbonate (40 mmol), continuing stirring uniformly, moving to a 0 ℃ ice salt bath, slowly dropwise adding a 3-chloropropionyl chloride (25 mmol) DCM (2.5 mL) solution, after about 1h dropwise addition, continuing ice bath stirring reaction, monitoring the reaction until the reaction is finished by TLC, adding 20mL of saturated NaCl solution, regulating pH=2-3 by 2N HCl, standing for layering, separating liquid, collecting an organic phase, drying for removing water, rotating to remove a solvent to obtain a solid crude product, adding Ethyl Acetate (EA) 15mL, stirring for 30min, adding Petroleum Ether (PE) 40mL for dispersion, standing, suction filtering, monitoring the solid by TLC, repeating for a plurality of times if a mixed point is found, and finally drying in vacuum to obtain an intermediate IM4; when FQs is BAL and GAT, the oily crude product is obtained, 20mL DCM is added for recrystallization, yellow solid is separated out, and the pure product is obtained by suction filtration and drying.
TABLE 6 results of Synthesis experiments of intermediates IM4 a-IM 4h
e. Synthesis of target molecule TM1/TM2
Taking the reaction of IM3a and IM4a to prepare TM1a as an example, the reaction conditions (types of solvents and bases, reaction temperature, etc.) of this step were examined (see Table 7).
TABLE 7 synthetic reaction conditions exploration experiment of target molecule TM1a
Note that: * Catalytic amounts of NaI are added to the reaction.
Through exploration, the optimal conditions of the reaction in the step are as follows: in K 2 CO 3 Is alkali, DMF is solvent, the reaction is carried out at 60 ℃, and the feeding mole ratio is controlled to be IM3:IM4:K 2 CO 3 ＝1:1.2:1.5。
The intermediates IM3a/IM3b and IM4 were reacted at K according to the optimum reaction conditions described above 2 CO 3 Stirring and reacting in DMF at 60 ℃ in the presence of NaI (catalyst) to obtain the target molecule TM1/TM2. The results of the synthesis experiments are shown in Table 8.
Operation example: into a 100mL reaction flask, IM3a/IM3b (1 mmol) and 2mL DMF were added, and the mixture was stirred at room temperature and K was added 2 CO 3 (1.5 mmol,0.207 g), stirring for 0.5 hr, adding IM4 (1.2 mmol) and NaI (0.01 mmol), stirring for 0.5 hr, oil bath stirring at 60deg.C, TLC monitoring to the end of the reaction, adding ice-cold saturated NaCl solution, adjusting pH to 3-4 with 2N HCl solution, separating out solid, refrigerating, vacuum filtering, washing the filter cake with saturated NaCl solution (10 mL×1), ice water (15 mL×2), vacuum drying to obtain crude product, column chromatography (DCM: CH) 3 Oh=150:1 to 20:1, v/v) and part of the compound was purified by thin layer chromatography (DCM: CH 3 OH=50:1, v/v) to obtain the target compound TM1/TM2.
TABLE 8 results of Synthesis experiments of target molecules TM1/TM2
The structural formula and structural characterization data of the intermediate IM3a/IM3b are as follows:
IM3a white solid, m.p.:207.3-208.0 ℃. 1 H NMR(600MHz,DMSO-d6)δ10.61(s,1H),8.71(d,J＝5.6Hz,2H),7.74(d,J＝5.8Hz,2H),7.68(d,J＝8.5Hz,1H),7.54(s,1H),7.19(d,J＝8.3Hz,1H),5.02(s,2H),3.87(s,3H).
IM3b white solid, m.p. 186.5-187.3 ℃. 1 H NMR(600MHz,DMSO-d6)δ10.41(s,1H),8.77(d,J＝5.7Hz,2H),7.81(d,J＝5.8Hz,2H),7.76(d,J＝8.7Hz,1H),7.41(d,J＝1.5Hz,1H),7.12(d,J＝1.6Hz,1H),5.01(s,2H),4.35(q,J＝7.1Hz,2H),1.33(t,J＝12.7,5.6Hz,3H).
The structural formula and structural characterization data of the target molecule TM1/TM2 are as follows:
TM1a white solid, m.p. 228.3-229.2 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.51(s,1H,),10.61(s,1H),10.55(s,1H),8.88–8.81(m,2H),7.94(d,J＝11.7Hz,1H),7.91(d,J＝5.6Hz,2H),7.75(d,J＝8.7Hz,1H),7.35(d,J＝1.1Hz,1H),7.09(d,J＝7.4Hz,1H),5.73(dd,J＝10.5,1.8Hz,2H),5.01(s,2H),4.41(d,J＝3.3Hz,1H),4.02(dt,J＝20.6,10.4Hz,1H),3.87(s,3H),3.76(s,3H),3.35(s,4H),2.08(d,J＝7.4Hz,1H),1.20(dd,J＝13.1,6.9Hz,3H),1.01(s,2H). 13 C NMRδ176.59,172.45,169.15,166.09,165.56,165.14,164.89,161.81,156.91,155.24,153.12,151.28,145.14,143.85,143.76,138.31,136.80,128.60,127.98,123.13,120.30,110.98,108.41,108.17,106.85,64.14,61.59,60.21,55.27,46.40,41.99,30.47,21.44,19.10,14.45,11.29.HR MS calcd for C 36 H 33 ClFN 5 O 10 ,[M+H] + :750.1973,found:750.1972.
TM1b, white solid, m.p. 216.2-217.0 ℃. 1 H NMR(600MHz,DMSO-d6)δ11.13(s,1H,H-22),10.64(s,1H),10.57(s,1H),9.97(s,1H),8.86(d,J＝3.8Hz,2H),7.92(d,J＝4.2Hz,2H),7.76(d,J＝8.7Hz,1H),7.72(d,J＝8.1Hz,1H),7.37(s,1H),7.11(d,J＝8.6Hz,1H),5.68(s,2H),5.00(d,J＝28.9Hz,2H),4.23(t,J＝6.4Hz,2H),4.03(d,J＝5.7Hz,1H),3.88(s,3H),3.37(s,9H),1.65(t,J＝7.3Hz,3H). 13 C NMRδ176.58,172.15,169.39,166.06,165.50,165.08,163.69,161.63,153.15,148.88,146.24,145.44,145.16,143.86,143.04,138.33,131.34,128.66,127.93,120.32,111.12,111.02,108.38,108.19,106.89,64.21,62.49,52.67,51.10,46.38,42.00,35.77,30.49,19.11,11.31.HR MS calcd for C 35 H 34 FN 5 O 10 ,[M+Na] + :7 726.2182,found:726.2164.
TM1c white solidBody, m.p. 234.2-235.1 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.19(s,1H,H-23),10.62(s,3H,H-5),10.57(s,2H,H-19),8.85(d,J＝4.9Hz,2H,H-9and H-10),8.66(s,1H,H-17),7.91(d,J＝5.4Hz,2H,H-7and H-8),7.75(d,J＝8.7Hz,1H,H-2),7.57(d,J＝6.9Hz,1H,H-3),7.35(s,1H,H-18),7.09(d,J＝8.6Hz,1H,H-4),5.01(s,2H,H-12),4.23(s,4H,H-6and H-11),3.87(s,3H,H-1),3.81(t,J＝16.1Hz,2H,H-14),3.42(s,4H,H-13and H-15),3.36(s,3H,H-16and H-20),1.33(d,J＝6.4Hz,1H,H-21),1.21(d,J＝30.7Hz,2H,H-22),0.88–0.79(m,1H,H-21). 13 C NMRδ176.57,172.14,169.14,168.53,166.04,165.49,165.08,163.68,161.81,155.24,153.13,148.87,146.24,145.44,145.16,143.03,138.31,131.24,130.94,128.64,127.93,110.98,108.38,108.18,106.85,65.47,64.19,62.48,61.57,61.52,55.32,41.99,35.77,30.49,14.46,11.31.HR MS calcd for C 36 H 34 FN 5 O 10 ,M+H] + :716.2362,found:716.2359,[M+Na] + :738.2182,found:738.2179.
TM1d: white solid, m.p.:207.3-208.0 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.11(s,1H),10.63(s,1H),10.57(s,1H),8.85(d,J＝5.3Hz,2H),8.64(s,1H),7.99(d,J＝13.0Hz,2H),7.91(d,J＝5.4Hz,2H),7.83–7.70(m,2H),7.53(t,J＝8.4Hz,1H),7.35(s,1H),7.09(d,J＝8.6Hz,2H),5.74(s,2H),5.01(s,2H),3.87(s,3H),3.74–3.54(m,2H),3.41(d,J＝24.9Hz,8H). 13 C NMRδ176.61,172.49,169.15,166.11,165.60,165.16,164.90,161.79,156.90,155.23,153.13,151.27,145.13,143.85,143.76,138.31,136.80,131.92,131.27,129.09,128.95,128.57,128.04,123.51,123.14,120.29,111.11,110.98,108.42,108.14,106.85,64.13,61.60,55.26,41.99,30.46,21.44,19.09,14.45.HR MS calcd for C 39 H 33 F 2 N 5 O 10 ,[M+H] + :770.2268,found:770.2269.
TM1e: white solid, m.p.:213.6-214.5 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.30(s,1H),10.62(s,1H),10.57(s,1H),8.96(s,1H),8.85(d,J＝4.4Hz,2H),8.07(d,J＝13.3Hz,1H),7.91(d,J＝4.4Hz,2H),7.74(d,J＝8.7Hz,1H),7.35(s,1H),5.01(s,2H),4.56–4.42(m,2H),3.87(s,3H),3.77(d,J＝35.3Hz,3H),3.43(s,10H),1.40(t,J＝6.8Hz,3H). 13 C NMRδ176.60,172.16,169.14,166.06,165.54,165.10,163.69,161.80,155.24,153.15,148.88,146.24,145.44,145.15,143.01,131.26,129.10,128.62,127.98,111.26,110.99,108.40,108.17,106.86,65.48,64.19,62.46,61.58,42.00,35.76,30.47,19.10,14.46,11.30.HR MS calcd for C 34 H 33 FN 6 O 10 ,[M+H] + :705.2314,found:705.2315.
TM1f yellow solid, m.p. 297.9-298.8 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.94(s,1H),10.62(s,1H),8.86(d,J＝4.5Hz,1H),8.69(s,2H),7.91(d,J＝4.5Hz,2H),7.74(d,J＝11.9Hz,4H),4.56(s,2H),4.17(s,3H),4.03(dd,J＝14.3,7.2Hz,2H),3.87(s,3H),3.79(s,4H),3.14(dd,J＝28.2,10.4Hz,3H),3.00(s,3H),2.92(d,J＝14.52,2H),2.82(m,2H),1.90(Hz,2H),1.22–1.14(m,2H). 13 C NMRδ176.74,169.71,169.39,166.05,162.76,161.63,155.35,151.32,150.76,146.43,145.17,139.74,139.66,136.81,134.49,131.35,131.04,129.69,127.62,123.12,121.41,121.35,111.00,107.03,106.86,64.16,63.23,63.19,60.20,52.67,51.00,41.28,36.61,30.45,27.29,25.79,14.53,9.49,9.27.HR MS calcd for C 39 H 40 FN 5 O 11 ,[M+Na] + :796.2601,found:796.2608.
TM1g pale yellow solid, m.p. 209.5-210.3 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.08(s,1H),10.61(s,1H),9.95(s,1H9),8.93(d,J＝8.6Hz,2H),8.64(s,1H),7.72(d,J＝8.7Hz,1H),7.87(d,J＝8.7,2H),7.79(s,1H),7.53(t,J＝8.6,1.7Hz,1H),5.72(d,J＝10.7Hz,2H),4.59(d,J＝3.2Hz,8H),3.87(s,3H),3.51(d,J＝9.7Hz,2H),3.36(d,J＝9.6Hz,3H),1.45(t,J＝6.7Hz,6H). 13 C NMRδ176.61,172.18,169.39,166.08,165.56,163.69,161.62,153.16,148.88,146.24,145.44,145.14,143.01,138.33,131.36,128.62,128.00,111.29,111.12,111.03,110.97,108.40,108.16,106.99,106.90,65.49,64.20,62.45,52.68,42.00,41.05,35.75,30.47,19.10,13.95,11.30.HR MS calcd for C 36 H 35 F 2 N 5 O 10 ,[M+H] + :736.2425,found:736.2437.
TM1h yellow solid, m.p. 225.6-226.3 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.93(s,1H),10.63(s,1H),10.57(s,1H),8.85(d,J＝4.3Hz,2H),8.72(s,1H),7.91(d,J＝4.3Hz,2H),7.76(d,J＝6.2Hz,1H),7.35(s,1H),7.09(d,J＝8.5Hz,1H),5.73(d,J＝13.6Hz,4H),5.01(s,2H),4.21(dd,J＝24.5,17.9Hz,1H),3.87(s,3H),3.72(s,2H),3.46(s,4H),3.36(s,3H),3.20(d,J＝17.6Hz,1H),1.28(d,J＝45.5Hz,3H),1.13(s,1H),1.03(s,1H),0.95–0.89(m,1H),0.87–0.80(m,1H). 13 C NMRδ176.74,169.42,166.09,166.07,161.64,156.83,155.17,151.28,150.95,148.86,146.23,145.42,145.15,140.01,139.94,136.79,134.56,131.33,127.88,123.12,111.03,108.37,107.18,107.14,106.90,65.48,64.18,64.14,64.07,62.44,55.27,52.66,50.95,41.08,30.47,19.10,13.93,9.45.HR MS calcd for C 38 H 38 FN 5 O 11 ,[M+Na] + :782.2444,found:782.2427.
TM2a yellow solid, m.p. 236.3-237.1 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.48(s,1H),10.69(s,1H),10.54(s,1H),8.84(d,J＝3.1Hz,2H),8.82(s,1H),7.90(d,J＝2.5Hz,2H),7.73(d,J＝8.6Hz,1H),7.33(s,1H),7.08(d,J＝8.3Hz,1H),5.01(s,2H),4.34(q,J＝6.9Hz,2H),3.74(s,2H),3.42(s,8H),3.32(s,3H),1.32(t,J＝6.9Hz,3H),1.20(d,J＝5.7Hz,2H),1.00(s,1H),0.87(dd,J＝44.5,6.3Hz,1H). 13 C NMRδ176.59,169.15,166.08,165.54,164.89,161.81,156.91,155.25,153.11,151.28,145.14,143.85,143.76,138.31,136.80,131.25,128.61,127.96,123.12,120.31,111.11,110.98,108.41,108.18,106.86,65.48,64.14,61.58,55.27,51.63,51.08,46.36,41.98,31.38,30.47,14.45,11.29.HR MS calcd for C 37 H 35 ClFN 5 O 10 ,[M+Na] + :786.1949,found:786.1936.
TM2b yellow solid, m.p. 201.6-201.3 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.25(s,1H),10.70(s,1H),10.56(s,1H),8.95(s,1H),8.86(d,J＝5.3Hz,2H),7.91(d,J＝5.7Hz,2H),7.76(d,J＝8.7Hz,1H),7.35(s,1H),7.19(d,J＝6.2Hz,1H),7.09(d,J＝8.6Hz,1H),5.02(s,2H),4.59(dd,J＝13.7,6.7Hz,1H),4.34(q,J＝7.0Hz,2H),3.89–3.64(m,4H),3.35(s,8H),2.92(dd,J＝14.6,8.2Hz,1H),1.42(t,J＝7.0Hz,3H),1.33(t,J＝7.0Hz,3H). 13 C NMRδ176.74,169.39,166.07,164.88,162.78,161.62,156.98,155.33,151.31,150.76,146.41,145.45,145.17,139.75,139.67,136.80,134.48,131.35,131.04,123.13,121.38,121.32,111.00,108.36,107.01,64.15,63.22,52.68,51.00,41.28,36.59,36.24,31.24,30.45,25.78,14.52.HR MS calcd for C 36 H 36 FN 5 O 10 ,[M+Na] + :740.2338,found:740.2331.
TM2c white solid, m.p. 235.8-236.6 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.18(s,1H),10.70(s,1H),10.56(s,1H),8.86(d,J＝4.4Hz,2H),8.66(s,1H),7.91(d,J＝5.4Hz,2H),7.75(d,J＝8.7Hz,1H),7.57(d,J＝7.1Hz,1H),7.35(s,1H),7.09(d,J＝8.4Hz,1H),5.01(s,2H),4.34(q,J＝6.9Hz,2H),3.80(d,J＝20.1Hz,4H),3.37(s,13H),1.33(t,J＝6.9Hz,3H). 13 C NMRδ176.74,169.42,166.11,166.07,164.89,163.68,161.63,155.16,151.27,150.94,148.86,146.23,145.42,145.13,142.99,140.01,136.79,134.55,131.33,127.92,123.13,111.02,108.36,107.16,106.98,106.89,65.48,64.17,64.13,64.07,62.43,55.27,52.67,41.09,30.46,19.09,13.93,9.44.HR MS calcd for C 37 H 36 FN 5 O 10 ,[M+Na] + :752.2338,found:752.2333.
TM2d yellow solid, m.p.:212.1-213.0 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.09(s,1H),10.68(s,1H),9.95(s,1H),8.65(s,1H),8.01(d,J＝13.0Hz,2H),7.79(t,J＝6.0Hz,2H),7.54(t,J＝8.1Hz,2H),7.15(s,1H),6.80(dd,J＝16.6,10.5Hz,4H),6.41(d,J＝7.2Hz,1H),5.69(d,J＝10.8Hz,1H),4.34(dd,J＝13.7,6.7Hz,2H),4.02(t,J＝6.3Hz,2H),3.78(t,J＝6.3Hz,2H),3.65(m,8H),1.33(t,J＝6.9Hz,3H). 13 C NMRδ176.75,172.19,169.78,169.26,169.14,166.18,166.12,164.90,161.78,151.28,150.75,146.38,145.38,145.13,139.74,137.66,136.80,134.98,134.46,131.29,130.99,128.50,123.14,121.36,121.30,111.27,110.99,108.43,108.10,106.99,106.85,64.13,63.21,63.17,61.61,41.26,36.57,30.44,25.75,14.45.HR MS calcd for C 40 H 35 F 2 N 5 O 10 ,[M+H] + :784.2425,found:784.2419.
TM2e, white solid, m.p. 215.7-216.5 ℃. 1 H NMR(600MHz,DMSO-d6)δ15.28(s,1H),10.70(s,1H),10.55(s,1H),8.97(s,1H,H-18),8.86(d,J＝3.6Hz,2H),8.08(d,J＝13.3Hz,1H),7.91(d,J＝3.5Hz,2H),7.75(d,J＝8.6Hz,1H),7.35(s,1H),5.01(s,2H),4.50(d,J＝6.6Hz,2H),4.34(q,J＝6.9Hz,2H),4.23(t,J＝6.8Hz,2H),4.02(t,J＝6.8Hz,2H),3.96–3.63(m,8H),1.40(t,J＝6.6Hz,3H),1.33(t,J＝6.8Hz,3H). 13 C NMRδ176.73,169.16,166.12,166.06,164.88,163.67,161.80,151.22,150.89,148.85,146.22,145.41,145.11,142.97,140.00,139.92,134.52,131.22,127.93,123.14,110.98,108.37,107.13,106.85,65.48,64.87,62.40,61.60,61.54,55.24,41.08,30.45,19.08,14.42,9.43.HR MS calcd for C 35 H 35 FN 6 O 10 ,M+Na] + :741.2291,found:741.2297.
TM2f yellow solid, m.p. 208.0-208.8 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.95(s,1H),10.70(s,1H,H-6),10.56(s,1H),8.86(d,J＝4.7Hz,2H,H-10and H-11),8.70(s,1H,H-20),7.91(d,J＝4.6Hz,2H,H-8and H-9),7.76(d,J＝8.5Hz,1H),7.35(s,1H),7.09(d,J＝8.5Hz,1H),5.01(s,2H),4.34(dd,J＝14.1,7.0Hz,2H),4.17(t,J＝6.8Hz,2H),4.02(s,3H),3.79(d,J＝6.5Hz,3H),3.39(s,3H),3.26(s,2H),3.20–3.06(m,1H),3.04–2.69(m,2H),2.16(dd,J＝29.1,8.7Hz,1H),1.78–1.91(m,3H),1.33(t,J＝7.0Hz,3H),1.26–0.94(m,4H).. 13 C NMRδ176.79,172.17,169.13,166.11,164.89,162.65,161.80,151.33,150.80,146.40,145.17,139.75,136.81,134.50,131.31,130.99,128.52,123.13,121.43,111.28,110.99,108.44,107.88,107.05,106.86,64.16,63.23,63.19,62.47,61.58,51.00,50.01,43.18,41.28,34.44,30.45,30.30,25.78,14.48,9.26.HR MS calcd for C 40 H 42 FN 5 O 11 ,M+H] + :788.2938,found:788.2953.
TM2g white solid, m.p.:223.9-224.8 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.88(s,1H),10.71(s,1H),10.57(s,1H),8.92(s,1H),8.85(d,J＝4.6Hz,2H),7.91(d,J＝4.6Hz,2H),7.87(d,J＝11.6Hz,1H),7.75(d,J＝8.7Hz,1H),7.34(s,1H),5.74(t,J＝18.2Hz,7H),5.01(s,2H,H-7),4.58(s,2H,H-12),4.34(q,J＝13.9,6.9Hz,2H),4.02(t,J＝6.9Hz,4H),1.45(t,J＝6.6Hz,3H),1.33(t,J＝7.0Hz,6H). 13 C NMRδ176.74,172.20,169.42,166.12,166.08,164.89,163.68,161.63,155.14,151.28,150.95,148.88,146.23,145.43,145.13,142.97,139.94,136.77,134.55,131.34,123.13,111.00,108.34,107.13,106.86,65.49,64.17,64.08,62.42,55.28,52.68,51.00,41.09,30.46,19.10,13.94,9.44.HR MS calcd for C 37 H 37 F 2 N 5 O 10 ,[M+H] + :750.2581,found:750.2578.
TM2h yellow solid, m.p.:233.2-234.0 ℃. 1 H NMR(600MHz,DMSO-d6)δ14.92(s,1H),10.71(s,1H),10.57(s,1H),8.85(d,J＝5.6Hz,2H),8.71(s,1H),7.91(d,J＝5.6Hz,2H),7.75(d,J＝9.1Hz,2H),7.35(s,1H),5.74(s,4H),5.01(s,2H),4.34(dd,J＝14.1,7.0Hz,2H),4.16(s,1H),3.72(s,3H,),3.35(d,J＝12.1Hz,7H),1.33(t,J＝7.1Hz,6H,),1.08(m,4H). 13 C NMRδ176.72,172.15,169.16,166.08,166.05,163.67,161.82,156.82,151.26,150.91,148.85,146.23,145.42,145.14,143.00,140.00,139.92,134.53,131.23,127.87,123.12,111.24,110.98,108.37,106.85,65.47,64.17,64.05,62.44,61.58,61.53,55.26,50.97,41.08,30.47,19.09,14.43,13.92,9.44.HR MS calcd for C 39 H 40 FN 5 O 11 ,[M+H] + :774.2781,found:774.2780.
2 target molecule TM1/TM2 anti-human pathogenic bacteria Activity test
2.1 anti-Mycobacterium tuberculosis Activity
Mycobacterium smegmatis is an experimental model for studying Mycobacterium tuberculosis and other pathogenic mycobacteria. The present invention determines the Minimum Inhibitory Concentration (MIC) of the target molecule TM1/TM2 against Mycobacterium smegmatis Mycolicibacterium smegmatis (strain ATCC 700084/mc (2) 155). The test results are shown in Table 9.
TABLE 9 test results of anti-Mycobacterium smegmatis Activity of target molecules TM1/TM2
As shown in Table 9, for the wild type Mycobacterium smegmatis tested, the MIC of 8 FQs drugs is 0.0011-0.024 mu mol/mL, and 7 out of 16 target molecules have MIC less than or equal to 0.0054 mu mol/mL, and the wild type Mycobacterium smegmatis has strong antibacterial activity; wherein the MIC (about 0.0051. Mu. Mol/mL) of TM1f, TM1h and TM2h are equivalent to that of the positive control drug NOR, SAR, ENX (0.0051-0.0061. Mu. Mol/mL); the bacteriostatic activity of TM2b (0.0043. Mu. Mol/mL) was 5.6 times that of CIP (0.024. Mu. Mol/mL); the bacteriostatic activity of TM1a (0.0013. Mu. Mol/mL) was comparable to that of CLX (0.0011. Mu. Mol/mL), 4 times that of SAR (0.0051. Mu. Mol/mL) and 18 times that of CIP (0.0236. Mu. Mol/mL); TM2a (0.00063. Mu. Mol/mL) showed the strongest activity, 1.7 times that of CLX, 8.1-9.7 times that of NOR, SAR, ENX, and 37 times that of CIP. The above results indicate that the hybridization of PAS, INA and FQ can give compounds having high inhibitory activity against Mycobacterium smegmatis.
Analysis of the structure-activity relationship revealed that the PAS linked INA and the derivative of CLX (TM 1a/TM2 a) was more active than all FQs, showing the strongest anti-Mycobacterium smegmatis activity; the inhibitory activity of the ethyl esterification derivative TM2 (0.00063-0.14. Mu. Mol/mL) against Mycobacterium smegmatis was stronger than that of the methyl esterification derivative TM1 (0.0013-0.14. Mu. Mol/mL).
2.2 Activity against other pathogenic bacteria
To examine the antimicrobial spectrum of the target molecules, the MIC of the target molecules TM1/TM2 for Staphylococcus aureus (Staphyloccocus aureus) ATCC25129, staphylococcus aureus ATCC14125, micrococcus luteus (Micrococcus luteus), escherichia coli ATCC25922, acinetobacter baumannii (Acinetobacter baumannii) ATCC19606, salmonella (Salmonella Enteritidis) ATCC13076, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC27853 were further tested according to the National Committee for Clinical Laboratory Standardization (NCCLS) recommended microdilution method. The test results are shown in Table 10.
TABLE 10 results of Activity of target molecules TM1/TM2 against other pathogenic bacteria (MIC, μg/mL)
Note that: * The units of partial MIC values in the columns are (. Mu.g/mL)/(. Mu. Mol/mL).
As can be seen from Table 10, overall, the antibacterial activity of the target molecule TM1/TM2 is stronger than that of the parent PAS and the intermediate IM3a/IM3b; the antibacterial activity of PAS methyl ester is larger than that of PAS ethyl ester.
For E.coli, the inhibitory activity of the target molecule is generally weaker than FQs but stronger than PAS, where 11 molecules have MIC of 8-64 μg/mL,2 molecules have MIC of 16 μg/mL, and TM1a has a MIC as low as 8 μg/mL; in general, the inhibitory activity TM1> TM2.
For salmonella, overall, inhibitory activity TM1> TM2; MIC of 6 target molecules is less than or equal to 32 mug/mL, and is stronger than positive control medicines NOR (64 mug/mL) and PAS (> 256 mug/mL); in particular, TM1a and TM1b had MICs of 0.00267 and 0.00284. Mu. Mol/mL, respectively, which were stronger than the 8 FQs (0.00514-0.200. Mu. Mol/mL) tested.
For Acinetobacter baumannii, out of 8 FQs, 5 had MICs of 256 μg/mL and 2 had MICs of 128 μg/mL; of the 16 target molecules, 13 had MICs of 256. Mu.g/mL and 3 had MICs of 128. Mu.g/mL. Overall, both FQs and target molecules have poor inhibitory activity.
MIC for Pseudomonas aeruginosa, 8 FQs was 0.0052-0.100. Mu. Mol/mL; all target molecules were more potent than NOR (0.100 μmol/mL); MIC values of TM1c, TM2d and TM2h are 8 mug/mL and are stronger than BAL; MICs of TM1h and TM2a were as low as 4 μg/mL, and the inhibitory activity was comparable to or stronger than that of FQs tested; the MICs of TM1a and TM1b were as low as 2. Mu.g/mL, with greater inhibitory activity than those tested FQs.
The inhibitory activities of 8 micrococcus luteus FQs are greatly different, and the MIC is 0.00109-0.802 mu mol/mL; the MIC of 16 target molecules is 0.00262-0.0870 mu mol/mL, and the inhibition activity difference is relatively small; the MIC of TM1a and TM2a with the CLX structural unit reached 0.00267 and 0.00262. Mu. Mol/mL, respectively, with inhibition activity 300 times that of NOR (0.802. Mu. Mol/mL) and 2 times that of GAT (0.00536. Mu. Mol/mL).
The overall activity of the target molecules is weaker than FQs for staphylococcus aureus ATCC25129, and MIC is 2-64 mug/mL, wherein MIC of 12 target molecules is less than or equal to 16 mug/mL. Notably, MICs for TM1a and TM2h were as low as 0.00267 and 0.00258. Mu. Mol/mL, with inhibition activity being 38 times that of NOR (0.100. Mu. Mol/mL) and 2 times that of GAT (0.00536. Mu. Mol/mL). MIC to staphylococcus aureus ATCC14125,8 FQs is 2-4 mug/mL, MIC to 16 target molecules is 0.125-32 mug/mL, and overall activity TM1> TM2; wherein the MIC of 6 target molecules is 0.000167-0.00284 mu mol/mL, which is stronger than all FQs; in particular, MICs of TM1a and TM1h were as low as 0.000167 and 0.000329. Mu. Mol/mL, respectively, much stronger than all FQs (0.00536-0.0125. Mu. Mol/mL), and worthy of subsequent intensive investigation.
Hemolysis test of 3 target molecule TM1a/TM2a
Hemolysis refers to the phenomenon of rupture and lysis of Red Blood Cells (RBCs). To examine the safety of the highly active target molecule TM1a/TM2a, a hemolytic test was performed. Human erythrocytes separated from fresh blood of healthy people are resuspended by normal saline to obtain erythrocyte suspension, and after incubation with test compounds with different concentrations for 4 hours at 37 ℃, the suspension is centrifuged, the supernatant is taken to measure absorbance, the hemolysis rate is calculated, and the normal saline is used as a negative control, and a normal saline solution of 0.1% Triton X-100 is used as a positive control. The results are shown in FIG. 3.
As can be seen from FIG. 3, the hemolysis ratio of TM1a/TM2a increases with increasing compound concentration, and the hemolysis ratio of TM1a is 1.32% -14.40% and the hemolysis ratio of TM2a is 0.0015% -8.20% at a test concentration of 1-64 μg/mL; when the test concentrations are 8 mug/mL and 32 mug/mL respectively, the hemolysis rates of TM1a and TM2a are 3.73% and 3.19% respectively, the hemolysis rates are lower than 5%, and according to the international standard, TM1a and TM2a have relative safety at the corresponding concentrations; at the same concentration, the hemolysis ratio of TM2a is lower than that of TM1a, i.e., the hemolysis ratio of the PAS ethyl ester derivative is lower than that of the PAS methyl ester derivative, which may be related to the longer the alkyl chain of the PAS carboxyl esterification, the greater its fat solubility.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (6)

1. A three-molecule conjugate with para-aminosalicylic acid as a parent nucleus represented by formula I:
in the formula I, R is methyl or ethyl;
x is cyclopropyl, ethyl or 4-halogeno phenyl;
y is-represents the linker to the carbonyl group; />Representation and representationIs connected with the connecting end of the connecting rod;
z is N or C-R 1 ；R 1 Hydrogen, halogen or methoxy.
2. The trimolecular conjugate or pharmaceutically acceptable salt thereof comprising p-aminosalicylic acid as a parent core according to claim 1, wherein the trimolecular conjugate is characterized byIn the following steps: x is cyclopropyl, ethyl or 4-fluorophenyl; r is R 1 Hydrogen, fluorine, chlorine or methoxy.
3. A three-molecule conjugate using para-aminosalicylic acid as a parent core or a pharmaceutically acceptable salt thereof, characterized in that: a three-molecule conjugate with para-aminosalicylic acid as a parent core is any one of the following compounds:
4. a trimolecular conjugate or pharmaceutically acceptable salt thereof comprising para-aminosalicylic acid as a parent core according to claim 3, wherein: a three-molecule conjugate with para-aminosalicylic acid as a parent core is any one of the following compounds: TM1a, TM1b, TM1f, TM1h, TM2a, TM2b, TM2c, TM2h.
5. A method of preparing a trimolecular conjugate having para-aminosalicylic acid as a parent nucleus or a pharmaceutically acceptable salt thereof according to claim 1, comprising the steps of:
a. heating p-aminosalicylic acid (PAS) and alcohol ROH under the catalysis of concentrated sulfuric acid to perform a reaction to obtain an intermediate IM1;
b. the intermediate IM1 and chloroacetyl chloride are stirred and reacted in methylene dichloride in an ice bath in the presence of sodium bicarbonate to prepare an intermediate IM2;
c. the intermediate IM2 and isonicotinic acid, INA, are stirred and reacted in dimethyl sulfoxide at 60 ℃ in an oil bath in the presence of triethylamine to prepare an intermediate IM3;
d. stirring fluoroquinolone FQs and 3-chloropropionyl chloride in methylene dichloride in an ice bath in the presence of sodium bicarbonate to react to prepare an intermediate IM4;
e. the three-molecule conjugate taking para-aminosalicylic acid as a parent nucleus in the formula I is prepared by carrying out oil bath stirring reaction on intermediate IM3 and IM4 in N, N-dimethylformamide at 60 ℃ in the presence of potassium carbonate and a catalyst NaI;
in the alcohols ROH, intermediates IM1, IM2 and IM3, R has the meaning given in formula I in claim 1;
FQs and intermediate IM4, X, Y and Z have the meanings given in formula I in claim 1.
6. Use of the trimolecular conjugate with para-aminosalicylic acid as a parent nucleus or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for the preparation of an antibacterial drug against one or more of mycobacterium tuberculosis, staphylococcus aureus, micrococcus luteus, escherichia coli, acinetobacter baumannii, salmonella and pseudomonas aeruginosa.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005058888A2 (en) * 2003-12-18 2005-06-30 Morphochem Aktiengesellschaft für kombinatorische Chemie Oxazolidinone-quinolone hybrid antibiotics
CN112159354A (en) * 2020-09-25 2021-01-01 西南大学 Fluoroquinolone derivative of p-aminosalicylic acid and intermediate, preparation method and application thereof
CN112159355A (en) * 2020-09-25 2021-01-01 西南大学 Fluoroquinolone p-aminosalicylate derivative and intermediate, preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005058888A2 (en) * 2003-12-18 2005-06-30 Morphochem Aktiengesellschaft für kombinatorische Chemie Oxazolidinone-quinolone hybrid antibiotics
CN112159354A (en) * 2020-09-25 2021-01-01 西南大学 Fluoroquinolone derivative of p-aminosalicylic acid and intermediate, preparation method and application thereof
CN112159355A (en) * 2020-09-25 2021-01-01 西南大学 Fluoroquinolone p-aminosalicylate derivative and intermediate, preparation method and application thereof

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