CN115260162A - 3-Hydroxy-4-pyridone-ciprofloxacin conjugate and its preparation method and use - Google Patents

Abstract

本发明还同时提供了上述3‑羟基‑4‑吡啶酮—环丙沙星耦合物在制备治疗细菌引起的疾病的药物中的应用。所述细菌为环丙沙星耐药鲍曼不动杆菌(ABA)、环丙沙星耐药肺炎克雷伯菌(KP)。The invention belongs to the technical field of medicine, and in particular relates to a 3-hydroxy-4-pyridone-ciprofloxacin conjugate and a preparation method and application thereof. The invention discloses a 3-hydroxy-4-pyridone-ciprofloxacin coupling compound, and the general structural formula I is:

The present invention also provides the application of the above-mentioned 3-hydroxy-4-pyridone-ciprofloxacin conjugate in the preparation of a medicine for treating diseases caused by bacteria. The bacteria are ciprofloxacin-resistant Acinetobacter baumannii (ABA) and ciprofloxacin-resistant Klebsiella pneumoniae (KP).

Description

3-Hydroxy-4-pyridone-ciprofloxacin coupled substance and its preparation method and use

technical field

The invention belongs to the technical field of medicine, and in particular relates to a 3-hydroxy-4-pyridone-ciprofloxacin coupling product and its preparation method and application.

Background technique

Antimicrobial resistance is a serious threat to human health. As a frequently used antibacterial drug in clinical practice, the problem of bacterial resistance to quinolones is particularly serious, and its resistance mechanisms are generally believed to include: target enzyme gene mutation, decreased membrane permeability, active efflux system, and plasmid-mediated resistance. medicine. Among them, decreased cell membrane permeability is more important because it enhances the activity of other drug resistance mechanisms and can reduce the final drug concentration of the target. The phenomenon of bacterial resistance to antibiotics has sounded the alarm to the medical community and the pharmaceutical industry. In addition to the correct use of existing effective drugs, new antibacterial drugs need to be developed.

As a fluoroquinolone, ciprofloxacin has broad-spectrum antibacterial activity, excellent pharmacokinetic properties and less side effects. However, the problem of clinical resistance to ciprofloxacin is relatively serious at present, such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii, and the drug resistance rate reaches 30% to 90%. . How to improve its anti-drug-resistant bacteria activity and avoid the problem of drug resistance has become an urgent research problem.

Iron is an important nutrient element necessary for microorganisms. The free iron ion concentration in blood and tissues is only 10 ^-24 mol/L, and the iron ion concentration required for bacterial growth is at least 10 ^-6 mol/L. Microorganisms have evolved an iron transport system composed of siderophore and homologous membrane receptor proteins with extremely high specific affinity for Fe ³⁺ (binding constant up to 10 ^-52 M ~ 10 ^-20 M).

Since the bacteria’s recognition and transport of the complex formed by siderophore and Fe ³⁺ is a specific and efficient active process, the siderophore and antibiotics are combined to form a coupling through the connecting chain, and the negative bacteria’s own iron transport system is used. When bacteria actively transport siderophore, it is an effective way to overcome the drug resistance caused by the decrease of outer membrane permeability of negative bacteria and the production of drug efflux pumps.

To this end, researchers have developed some siderophore-antibacterial drug conjugates. BAL30072 is a 1,3-dihydroxy-4-pyridone-monocyclic β-lactam conjugate reported by Baselia, which is stable to multiple β-lactamases and resistant to multiple drug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii has good antibacterial activity. Cefiderocol (Cefiderocol), which uses catechol as siderophore, developed by Shionogi Company in Japan, has strong activity against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. It was approved by the United States in November 2019. FDA-approved for the treatment of urinary tract infections.

Contents of the invention

The technical problem to be solved in the present invention is to provide a 3-hydroxyl-4-pyridone-ciprofloxacin coupling and its preparation method and application, the 3-hydroxyl-4-pyridone-ciprofloxacin coupling can Solve the drug resistance problem of ciprofloxacin.

In order to solve the above-mentioned technical problems, the present invention provides a 3-hydroxyl-4-pyridone-ciprofloxacin coupling, the general structural formula I is as follows:

where X is

As an improvement of the 3-hydroxyl-4-pyridone-ciprofloxacin coupling of the present invention, it is the general formula II or compound I-4:

General formula Ⅱ:

Compound I-4:

The general formula II is specifically any of the following: compound I-1, compound I-2, compound I-3;

Compound I-1

Compound I-2:

Compound I-3:

The present invention also provides tautomers, optical isomers or pharmaceutically acceptable salts of the above-mentioned 3-hydroxy-4-pyridone-ciprofloxacin coupled product.

The present invention also provides the preparation method of above-mentioned 3-hydroxyl-4-pyridone-ciprofloxacin coupling substance simultaneously:

One, the preparation method of general formula II (being compound I-1, I-2, I-3) is to comprise the following steps:

(1.1) compound V and compound VI are reacted under reflux for 12±1 hours with an aprotic polar solvent as a solvent under inorganic base conditions to obtain compound VII;

Compound V:

Compound VI:

Compound VII:

式中的R₃为羟基保护基；所述X为

所述R₂为羧基保护基； _The R1 is

R in the _formula is a hydroxyl protecting group; the X is

The R ₂ is a carboxyl protecting group;

(1.2) Compound VII is deprotected in a mixed solvent under the catalysis of hydrogen and palladium/carbon to obtain the compound described in the general formula II (ie compound I-1～compound I-3);

Two, the preparation method of compound I-4 is to comprise the following steps:

(2.1) Compound VIII is reacted with a condensing agent at room temperature for 12±1 hours under the condition of an organic base, using an aprotic polar solvent as a solvent, to obtain Compound IX;

Compound VIII:

Compound IX:

In the above reaction formula, X is

_R1 is

R ₂ is a leaving group, the leaving group is:

(2.2) Compound IX and ciprofloxacin were reacted at room temperature for 12±1 hours in an aprotic polar solvent under organic base conditions to obtain compound I-4.

As the improvement of the preparation method of 3-hydroxyl-4-pyridone-ciprofloxacin coupling substance of the present invention:

R ₃ is a hydroxyl protecting group, and the protecting group is benzyl and benzhydryl;

R ₂ is a carboxyl protecting group, and the protecting group is benzyl or benzhydryl.

As the improvement of the preparation method of 3-hydroxyl-4-pyridone-ciprofloxacin coupling substance of the present invention:

In the described step (1.1), the inorganic base is potassium carbonate, sodium carbonate, potassium hydroxide; the aprotic polar solvent is acetonitrile, dimethylformamide.

In the step (1.2): the mixed solvent can be methylene chloride/ethanol, methylene chloride/tetrahydrofuran;

In the step (2.1): the organic base is triethylamine, N,N-diisopropylethylamine; the condensing agent is N,N'-succinimide carbonate, 1,1'-carbonyldiimidazole; The aprotic polar solvent is dichloromethane, tetrahydrofuran;

In the step (2.2): the organic base may be triethylamine, N,N-diisopropylethylamine; the aprotic polar solvent may be methylene chloride or tetrahydrofuran.

The present invention also provides the application of the above-mentioned 3-hydroxy-4-pyridone-ciprofloxacin coupling in the preparation of medicines for treating diseases caused by bacteria.

As an improvement in the application of the present invention: the bacteria are Gram-negative bacteria. The details are as follows: the bacteria are ciprofloxacin-resistant Acinetobacter baumannii (ABA) and ciprofloxacin-resistant Klebsiella pneumoniae (KP).

In the present invention:

(I-1), (I-2) and (I-3) compounds of the present invention can be prepared according to the method of reaction formula (1):

Reaction formula (1)

In above-mentioned reaction formula (1), X is

其中R₃为羟基保护基，所述保护基例如可以选自：苄基、二苯甲基等； _R1 is

Wherein _R3 is a hydroxyl protecting group, and the protecting group can be selected from, for example, benzyl, benzhydryl, etc.;

R ₂ is a carboxyl protecting group, and the protecting group can be selected from, for example, benzyl, benzhydryl, etc.;

(a) compound V and compound VI are reacted under reflux for 12±1 hours with an aprotic polar solvent as a solvent under inorganic base conditions to obtain compound VII;

The inorganic base may be, for example, potassium carbonate, sodium carbonate, and potassium hydroxide; the aprotic polar solvent may, for example, be acetonitrile or dimethylformamide.

(b) Compound VII is deprotected in a mixed solvent under the catalysis of hydrogen and palladium/carbon to obtain compounds I-1-3. Described mixed solvent can be methylene chloride/ethanol, methylene chloride/tetrahydrofuran;

The compound (I-4) of the present invention can be prepared according to the method of Reaction Formula (2).

Reaction formula (2)

In the above reaction formula, X is

_R1 is

等；R ₂ is a leaving group, which can be selected from, for example:

Wait;

(a) Compound VIII is reacted with a condensing agent at room temperature for 12±1 hours under the condition of an organic base, using an aprotic polar solvent as a solvent, to obtain Compound IX;

The organic base can be triethylamine, N,N-diisopropylethylamine; the condensing agent can be N,N'-succinimide carbonate, 1,1'-carbonyldiimidazole; The aprotic polar solvent can be, for example, dichloromethane and tetrahydrofuran.

(b) IX and ciprofloxacin were reacted at room temperature for 12 hours to obtain compound I-4 under the condition of an organic base, using an aprotic polar solvent as a solvent;

The organic base may be triethylamine, N,N-diisopropylethylamine; the aprotic polar solvent may be methylene chloride or tetrahydrofuran.

The usage of the present invention can refer to 1,3-dihydroxy-4-pyridone-monocyclic β-lactam coupling.

In the present invention, the general formula (I) links the piperazine N of the ciprofloxacin molecule with the siderophore 3-hydroxyl-4-pyridone, wherein the 3-hydroxyl-4-pyridone can be used as the siderophore with Fe ^{3 +} forms a complex, which is recognized by the homologous receptors on the cell membrane as an iron donor, and then passes through the outer membrane of the cell in the form of active transport, thereby overcoming the low membrane permeability and active efflux of drug-resistant bacteria. drug resistance.

As a class of bidentate iron carriers, 3-hydroxy-4-pyridone has good selectivity and affinity for Fe ³⁺ ; at the same time, 3-hydroxy-4-pyridone-iron complex has good in vivo stability and safety. Aiming at the problem of drug resistance of Gram-negative bacteria, the present invention combines 3-hydroxy-4-pyridone siderophores with ciprofloxacin antibiotics to form a "Trojan horse" coupling, so that ciprofloxacin can be mediated by bacterial siderophores The iron transport pathway is actively transported into the cell to play an antibacterial effect and solve the problem of resistance of Gram-negative bacteria to ciprofloxacin.

In summary, the present invention utilizes the "Trojan horse" strategy to design couplings, that is, utilizes the specific recognition of siderophores by bacteria, and connects 3-hydroxy-4-pyridone and ciprofloxacin through a linking chain to form siderophores—antibacterial drugs The coupler enables Gram-negative bacteria to specifically recognize siderophores and at the same time actively transports antibacterial drugs into the bacteria to exert antibacterial effects, thereby overcoming the drug resistance of negative bacteria.

Detailed ways

The present invention will be further described below in conjunction with the examples, but these examples are by no means any limitation to the present invention. In all embodiments, ¹ H-NMR is measured with Bruker 500MHz NMR instrument of the public experimental platform of School of Pharmacy, Zhejiang University, chemical shifts are expressed in δ (ppm), and TMS is an internal standard; high-resolution mass spectra are determined by Agilent1290-HPLC-6224 Determination by combined instrument; melting point was determined by Büchi B-540 melting point apparatus.

The synthesis of compound VI can refer to literature (J.Med.Chem.2013, 56, 2690-2694); the raw materials ciprofloxacin and maltol (II) were purchased from Shanghai Beide Pharmaceutical Technology Co., Ltd.

Synthesis of target compounds

Example 1: 1-cyclopropyl-6-fluoro-7-(4-(2-(2-(3-hydroxy-2-methyl-4-pyridonyl)ethyl)amino)-2-carbonyl Preparation of ethyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-1)

Step 1: Preparation of Benzyl Maltol (III)

Maltol (II, 50.4g, 0.40mol) was dissolved in a mixed solvent of 80mL ethanol and 80mL water, and sodium hydroxide (17.6g, 0.44mol) and benzyl chloride (46.8g, 0.37mol) were added successively under stirring, React at 60°C overnight (ie, about 12 hours). After TLC detected that the benzyl chloride reaction was complete, the ethanol was removed by rotary evaporation, and the remaining mixed solution was extracted twice with 80 mL of dichloromethane. It was dried over sodium sulfate and concentrated by rotary evaporation to obtain the crude product, which was recrystallized from PE and EA (1:1, v/v) to obtain brown solid III (62 g, 79%).

Melting point: 55-57°C; HRMS (ESI): m/z theoretical value is C ₁₃ H ₁₃ O ₃ [M+H] ⁺ : 217.0865, measured value is 217.0863.

Step 2: Preparation of 1-(2-aminoethyl)-3-benzyloxy-2-methylpyridin-4-one (IV-1)

Dissolve III (4.32g, 20mmol), ethylenediamine (1.26g, 21mmol) and sodium hydroxide (0.72g, 18mmol) in a mixed solvent of 20mL water and 20mL ethanol, and react at 80°C for 1.5 hours. After TLC detects that the III reaction is complete, the reaction solution is rotary evaporated to remove ethanol, and then extracted three times with 50 mL of dichloromethane, the organic layer is combined, washed with 30 mL of water and saturated brine successively, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, The crude product was purified by silica gel column chromatography (dichloromethane:methanol:triethylamine=10:1:0.1, v/v/v, flow rate was 3mL/min) to obtain brown oil IV-1 (2.37g, 45 %).

HRMS(ESI): m/z theoretical value is C ₁₅ H ₁₉ N ₂ O ₂ [M+H] ⁺ : 259.1447, found value is 259.1447.

Step 3: Preparation of N-(2-(3-(benzyloxy)-2-methyl-4-pyridonyl)ethyl)-2-chloroacetamide (V-1)

Dissolve IV-1 (738.7mg, 2.86mmol) and triethylamine (347.1mg, 3.43mmol) in 20mL of dichloromethane, put it in an ice bath, and add chloroacetyl chloride (451.6mg, 4.00 mmol), keep the temperature below 5°C during the dropwise addition, remove the ice bath after the dropwise addition, and slowly rise to room temperature (the heating rate is about 1.5°C/min), from the beginning of the dropwise addition to the complete reaction of IV-1 for 2 hours . 20 mL of water was added to quench the reaction, the organic layer was washed successively with 20 mL of water and saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a crude product, which was then purified by silica gel column chromatography (dichloromethane:methanol=40:1- 10:1, v/v, with a flow rate of 3 mL/min) to obtain V-1 (802 mg, 84%) as a yellow oil.

HRMS(ESI): m/z theoretical value is C ₁₇ H ₂ ClN ₂ O ₃ [M+H] ⁺ : 335.1162, measured value is 335.1159.

Step 4: 7-(4-(2-(3-(Benzyloxy)-2-methyl-4-carbonylpyridine-ethyl)amino)-2-carbonylethyl)piperazin-1-yl)- Preparation of 1-cyclopropyl-6-fluoro-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid benzyl ester (VII-1)

Dissolve V-1 (67.0mg, 0.2mmol), VI (105.4mg, 0.25mmol) in 10mL of acetonitrile, add potassium carbonate (69.1mg, 0.5mmol), and reflux for 12 hours. After the complete reaction of raw material V-1 was detected by TLC, the reaction solution was cooled to room temperature, suction filtered, the filtrate was rotary evaporated to remove acetonitrile, and 50 mL of ethyl acetate was added to the residue to dissolve, followed by washing with 20 mL of water and saturated brine. Dry over sodium sulfate, and concentrate by rotary evaporation to obtain a crude product, which is then purified by silica gel column chromatography (dichloromethane:methanol=50:1, v/v, flow rate 3mL/min) to obtain brown oil VII-1 (53mg ,37%).

HRMS(ESI): m/z theoretical value is C ₄₁ H ₄₃ FN ₅ O ₆ [M+H] ⁺ : 720.3197, measured value is 720.3196.

Step 5: 1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-hydroxy-2-methyl-4-pyridinonyl)ethyl)amino)-2-carbonylethyl Preparation of base) piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-1)

Dissolve VII-1 (50.4mg, 0.07mmol) in a mixed solvent of 10mL of dichloromethane and 10mL of ethanol, add 5mg of 10% palladium/carbon by mass fraction, first vacuumize, and then use a hydrogen bag to inject hydrogen, repeat three times , so that the reaction flask is filled with hydrogen. , reacted at room temperature for 3 hours (there was still excess hydrogen in the reaction bottle at this time). After the completion of the reaction of VII-1 was detected by TLC, the reaction solution was filtered with diatomaceous earth, and the obtained filtrate was washed with 20 mL of ethanol three times, then concentrated by rotary evaporation to obtain a crude product, and then beaten with 10 mL of ether three times to obtain a gray solid I-1 (38 mg, 95%).

Melting point 158～160℃; ¹ H NMR (500MHz, DMSO-d ₆ ) δ8.66(s, 1H), 8.10(s, 1H), 7.89(d, J=13.0Hz, 1H), 7.54–7.51(m ,2H),6.13(d,J＝7.0Hz,1H),4.05(s,2H),3.88–3.85(m,1H),3.38–3.32(m,6H),3.02–3.00(m,2H), 2.64–2.60(m,4H),2.31(s,3H),1.36–1.35(m,2H),1.25–1.23(m,2H); ¹³ C NMR(125MHz,DMSO-d ₆ )δ176.77,170.21,169.49 ,166.40,154.45(d,J=248.0Hz),148.43,145.84,145.63(d,J=9.0Hz),139.64,138.46,129.25,114.04,111.51(d,J=24.0Hz),110.94,107.19,106.70 , 61.35, 52.90, 52.23, 49.76, 38.89, 36.35, 11.94, 8.06; the theoretical value of HRMS (ESI) was C ₂₇ H ₃₁ FN ₅ O ₆ [M+H] ⁺ = 540.2258, and the measured value was 540.2255.

Example 2: (R)-1-cyclopropyl-6-fluoro-7-(4-(2-(2-(3-hydroxyl-2-methyl-4-carbonylpyridin-1-yl)-3 Preparation of -phenylpropoxy)-2-carbonylethyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-2)

Step 1: Preparation of Benzyl Maltol (III)

See Example 1, Step 1.

Step 2: Preparation of (R)-3-(benzyloxy)-1-(1-hydroxy-3-phenylpropan-2-yl)-2-methylpyridin-4-one (IV-2)

Brown oil IV-2 (2.60g ,65%).

That is, specifically: change the ethylenediamine in Step 2) of Example 1 into (R)-2-amino-3-phenylpropanol (4.54g, 30mmol), and the rest are the same as in Step 2) of Example 1 to prepare A brown oily substance IV-2 was obtained.

HRMS (ESI): m/z theoretical value is C ₂₂ H ₂₄ NO ₃ [M+H] ⁺ : 350.1756, measured value is 350.1752.

Step 3: (R)-2-(3-(Benzyloxy)-2-methyl-4-carbonylpyridin-1-yl)-3-phenylpropyl 2-chloroacetate (V-2) preparation of

Compound IV-2 (1.0 g, 2.86 mmol) and chloroacetyl chloride (451.6 mg, 4.00 mmol) were prepared in a similar manner to step 3 in Example 1 to obtain brown oil V-2 (2.76 g, 65%).

That is, specifically: change IV-1 in step 3) of Example 1 to compound IV-2 (1.0 g, 2.86 mmol), and the rest is the same as step 3) of Example 1, brown oil IV-2.

HRMS(ESI): Theoretical m/z value is C ₂₄ H ₂₅ ClNO ₄ [M+H] ⁺ : 426.1472, found value is 426.1475.

Step 4: (R)-7-(4-(2-(2-(3-(Benzyloxy)-2-methyl-4-carbonylpyridin-1-yl)-3-phenylpropoxy) Preparation of -2-carbonylethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid benzyl ester (VII-2)

Compound V-2 (85.2mg, 0.2mmol) and VI (105.4mg, 0.25mmol) were prepared in a similar manner to step 4 in Example 1 to obtain brown oil VII-2 (66mg, 41%).

That is, specifically: change V-1 in Step 4) of Example 1 to compound V-2 (85.2 mg, 0.2 mmol), and the rest is the same as Step 4) of Example 1 to obtain brown oil VII-2.

HRMS(ESI): m/z theoretical value is C ₄₈ H ₄₈ FN ₄ O ₇ [M+H] ⁺ : 811.3507, measured value is 811.3510.

Step 5: (R)-1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-hydroxy-2-methyl-4-carbonylpyridin-1-yl)-3- Preparation of phenylpropoxy)-carbonyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-2)

Compound VII-2 (56.8mg, 0.07mmol) was prepared in a similar manner to step 5 in Example 1 to obtain gray solid I-2 (41mg, 92%).

That is, specifically: change VII-1 in step 5) of Example 1 to compound VII-2 (56.8 mg, 0.07 mmol); the rest is the same as step 5) of Example 1 to obtain gray solid I-2.

Melting point 134～136℃; ¹ H NMR (500MHz, DMSO-d ₆ ) δ8.67(s, 1H), 7.85(d, J=13.0Hz, 1H), 7.56(d, J=7.0Hz, 1H), 7.51–7.47(m,2H),7.39–7.35(m,3H),7.34–7.32(m,1H),6.16(d,J=8.0Hz,1H),4.84(d,J=11.0Hz,1H) ,4.78(d,J＝11.0Hz,2H),4.56–4.52(m,1H),4.37–4.34(m,1H),3.60–3.58(m,1H),3.17–3.06(m,6H),2.63 –2.59(m,4H),1.88(s,3H),1.25–1.19(m,2H),1.15–1.04(m,2H); HRMS(ESI) m/z theoretical value is C ₃₄ H ₃₆ FN ₄ O ₇ [M+H] ⁺ = 631.2568, the measured value is 631.2569.

Example 3: (S)-1-cyclopropyl-6-fluoro-7-(4-(2-(2-(3-hydroxyl-2-methyl-4-carbonylpyridin-1-yl)-3 Preparation of -methylbutoxy)-2-carbonylethyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-3)

Step 1: Preparation of Benzyl Maltol (III)

See Example 1, step 1.

Step 2: Preparation of (S)-3-(benzyloxy)-1-(1-hydroxy-3-methylbutan-2-yl)-2-methylpyridin-4-one (IV-3)

Compound III (4.32g, 20mmol) and (S)-2-amino-3-methylbutanol (3.10g, 30mmol) were prepared in a similar manner to step 2 in Example 1 to obtain brown oil IV-3 (1.99 g, 55%).

That is, specifically: change the ethylenediamine in Step 2) of Example 1 into (S)-2-amino-3-methylbutanol (3.10g, 30mmol), and the rest is the same as Step 2) of Example 1, IV-3 was obtained as a brown oil.

HRMS(ESI): m/z theoretical value is C ₁₈ H ₂₄ NO ₃ [M+H] ⁺ : 302.1756, measured value is 302.1756

Step 3: (S)-2-(3-(Benzyloxy)-2-methyl-4-carbonylpyridin-1-yl)-3-methylbutyl 2-chloroacetate (V-3) preparation of

Compound IV-3 (862.0 mg, 2.86 mmol) and chloroacetyl chloride (451.6 mg, 4.00 mmol) were prepared in a similar manner to Step 3 in Example 1 to obtain brown oil V-3 (1.85 g, 56%).

That is, specifically: change IV-1 in step 3) of Example 1 to compound IV-3 (862.0 mg, 2.86 mmol), and the rest is the same as step 3) of Example 1, brown oil V-3.

HRMS(ESI): The theoretical value of m/z is C ₂₀ H ₂₅ ClNO ₄ [M+H] ⁺ : 378.1472, and the measured value is 378.1474

Step 4: (S)-7-(4-(2-(2-(3-(Benzyloxy)-2-methyl-4-carbonylpyridin-1-yl)-3-methylbutoxy) Preparation of -2-carbonylethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid benzyl ester (VII-3)

Compound V-3 (75.6mg, 0.2mmol) and VI (105.4mg, 0.25mmol) were prepared in a similar manner to step 4 in Example 1 to obtain brown oil VII-3 (67mg, 44%).

That is, specifically: change V-1 in Step 4) of Example 1 to compound V-3 (75.6 mg, 0.2 mmol), and the rest is the same as Step 4) of Example 1 to obtain brown oil VII-3.

HRMS(ESI): m/z theoretical value is C ₄₄ H ₄₈ FN ₄ O ₇ [M+H] ⁺ : 763.3507, measured value is 763.3506

Step 5: (S)-1-Cyclopropyl-6-fluoro-7-(4-(2-(2-(3-hydroxy-2-methyl-4-carbonylpyridin-1-yl)-3- Preparation of methylbutoxy)-2-carbonylethyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-3)

Compound VII-3 (53.4mg, 0.07mmol) was prepared in a similar manner to step 5 in Example 1 to obtain brown solid I-3 (34mg, 84%).

That is, specifically: change VII-1 in step 5) of Example 1 to compound VII-3 (53.4 mg, 0.07 mmol), and the rest is the same as step 5) of Example 1 to obtain brown solid I-3.

Melting point 143～144℃; ¹ H NMR (500MHz, DMSO-d ₆ ) δ8.67(s, 1H), 7.89(d, J=13.0Hz, 1H), 7.73(d, J=7.5Hz, 1H), 7.55(d,J=7.0Hz,1H),6.21(d,J=7.5Hz,1H),4.53–4.50(m,1H),4.45–4.41(m,2H),3.88–3.83(m,1H) ,3.29–3.17(m,6H),2.58–2.54(m,4H),2.32(s,3H),1.36–1.31(m,2H),1.26–1.22(m,1H),1.21–1.14(m, 2H),1.09–1.03(m,3H),0.70–0.67(m,3H); ¹³ C NMR(125MHz,DMSO-d ₆ )δ171.75,171.59,169.55,164.56,153.56(d,J＝245.0Hz), 148.37, 144.37, 143.82(d, J=10.0Hz), 138.02, 137.49, 136.69, 135.34, 121.92, 111.73(d, J=23.0Hz), 108.82, 106.28, 71.73, 65.22, 63.987, 51.624, 4.9. 29.94, 19.23, 18.79, 7.49; HRMS (ESI) m/z theoretical value of C ₃₀ H ₃₆ FN ₄ O ₇ [M+H] ⁺ = 583.2568, found 583.2565.

Example 4: (S)-1-cyclopropyl-6-fluoro-7-(4-((2-(3-hydroxyl-2-methyl-4-carbonylpyridin-1-yl)-3-benzene Preparation of propoxy)carbonyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-4)

Step 1: Preparation of (S)-3-hydroxyl-1-(1-hydroxyl-3-phenyl-2-propyl)-2-methylpyridin-4-one hydrochloride (VIII) Maltol ( II, 189.2mg, 1.5mmol), (R)-2-amino-3-phenylpropanol (251.0mg, 1.66mmol) and boric acid (92.7mg, 1.5mmol) were added to 50mL of water and heated to reflux. After the maltol reaction was detected by TLC, the reaction solution was cooled to room temperature, adjusted to pH 8.5 with 40% sodium hydroxide solution, extracted twice with 100 mL of dichloromethane, and the organic layer was dried with anhydrous sodium sulfate, concentrated by rotary evaporation, The residue was adjusted to pH=1 by adding 6M hydrochloric acid solution, heated to dissolve at 40°C, then cooled slowly to an ice bath, and stirred until solid precipitated. Suction filtration, the filter cake was washed with 10 mL of water, dried, and recrystallized from n-butanol and acetonitrile (3:1, v/v) to obtain yellow solid VIII (154 mg, 61%).

Melting point 186-187°C; HRMS(ESI): m/z theoretical value is C ₈ H ₁₂ NO ₃ [M+H] ⁺ : 169.0739, measured value is 169.0736.

Step 2: (S)-2,5-Dioxypyrrolidin-1-yl (2-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)-3-phenylpropyl ) Preparation of carbonate (IX)

Dissolve VIII (346.7mg, 1mmol) in 30mL tetrahydrofuran, add triethylamine (556.6mg, 5.5mmol) and N,N'-succinimide carbonate (384.3mg, 1.5mmol), stir at room temperature for 2 hours, TLC After the completion of the reaction of VIII was detected, it was concentrated by rotary evaporation, and the crude product of IX was directly sent to the next step.

Step 3: (S)-1-cyclopropyl-6-fluoro-7-(4-((2-(3-hydroxy-2-methyl-4-carbonylpyridin-1-yl)-3-phenylpropane Preparation of oxy)carbonyl)piperazin-1-yl)-4-carbonyl-1,4-dihydroquinoline-3-carboxylic acid (I-4)

Add 10ml of dichloromethane to the crude product of IX to dissolve, then add ciprofloxacin (397.7mg, 1.2mmol) and triethylamine (404.8mg, 4mmol), stir at room temperature for two hours (at this moment, TLC detects that IX disappears), and add The reaction was quenched with 20 mL of water. Liquid separation, the aqueous layer was extracted twice with 50 mL of dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol=30:1, flow rate was 3ml/ min), a pink solid (400 mg, 65%) was obtained.

Melting point 136-138°C; 1H NMR (500MHz, DMSO-d ₆ ) δ8.66(s, 1H), 7.96(d, J=7.5Hz, 1H), 7.89(d, J=13.0Hz, 1H), 7.52 (d,J=7.5Hz,1H),7.28–7.22(m,2H),7.20–7.17(m,3H),6.21(d,J=7.5Hz,1H),4.86–4.82(m,1H), 4.54–4.45(m,1H),4.42–4.38(m,1H),3.87–3.84(m,1H),3.51–3.47(m,4H),3.27–3.06(m,6H),2.01(s,3H ),1.35–1.30(m,2H),1.20–1.15(m,2H); 13C NMR(125MHz,DMSO-d ₆ )δ176.78,169.34,166.40,154.41(d,J＝249.0Hz),154.17,148.47, 145.38(d,J=10.0Hz),145.04,139.55,137.05,134.42,130.05,129.44,128.94,127.27,119.43(d,J=8.0Hz),111.72,111.47(d,J=24.0Hz),107.32, 107.20, 66.52, 60.22, 43.62, 43.59, 36.56, 36.36, 12.03, 8.05; HRMS (ESI) m/z theoretical value is C ₃₃ H ₃₄ FN ₄ O ₇ [M+H] ⁺ = 617.2412, measured value is 617.2410.

Test example 1, in vitro anti-drug-resistant negative bacteria activity experiment of compound

1.1 Test strains

The clinical isolates selected for the in vitro antibacterial activity test are: ciprofloxacin-resistant Acinetobacter baumannii (ABA), ciprofloxacin-resistant Klebsiella pneumoniae (KP), see: Yongkang Carbon-tolerant Drug-resistant phenotype and molecular typing of penem-like Klebsiella pneumoniae. Zhejiang Medical Sciences 2022, 44(10), 1063-1066; Molecular typing of multidrug-resistant Acinetobacter baumannii in Yongkang, Zhejiang Province. Chinese Journal of Preventive Medicine 2015, 16(5), 379-383.

Source of strains: The strains used in this experiment are clinically isolated pathogenic bacteria collected in Zhejiang in May 2021. Before the experiment, each strain of bacteria was purified by drawing a single colony on an agar plate, and the freshly cultured cells at 37°C overnight were properly diluted for the experiment.

1.2 In vitro antibacterial test method

The microbroth dilution method recommended by the American Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Test Operating Procedures was used to measure each test sample, and the tested strains were tested in normal MHB medium and iron-deficient MHB medium Measure the MIC value.

1.3 Experimental steps

1.3.1 Measure MIC value in normal culture medium

1.3.1.1 MHB liquid medium configuration

Weigh 11g of MHB dry powder into 500ml of ultrapure water, stir to dissolve, put it into a high-pressure steam sterilizer (121°C, 20min), take it out after the sterilization is completed, and place it in a constant temperature incubator at 37°C after cooling.

1.3.1.2 Preparation of drug stock solution

Weigh 3 μmol of the target compound or positive control drug and dissolve it in 3 ml DMSO to prepare a 1 mM compound solution. Add 2 mL of Tris-HCl buffer solution at pH 8.0, add 1 mL of FeCl3 solution (1 mM), shake well, and configure drug stock solution (0.5 mM).

1.3.1.3 Bacterial suspension preparation

The strains frozen at -70°C were taken out, inoculated on the blood agar plate (BA) in different regions, and cultivated in a 35°C incubator for 16-24h, and waited for a single colony to grow for later use. Before the experiment, a single colony was picked with a sterile inoculation loop and placed in 0.45% normal saline, and adjusted to a concentration of 0.5 McFarland turbidity (1.0×10 ⁸ CFU/ml).

1.3.1.4 Microbroth dilution drug susceptibility test

Draw 512 μL of drug stock solution, dilute to 1 mL with MHB culture medium, and make broth working solution with a concentration of 256 μM. in a clean bench,

① Add 200 μl of diluted drug solution to the first well of the 96-well plate, add 100 μL of MHB broth to the second to tenth wells, add 200 μL of bacterial solution to the eleventh well as a negative control for bacterial solution, and add 200 μL of MHB to the twelfth well Broth served as a positive control.

② Use an eight-channel pipette to draw 100 μL of antibacterial drugs from the first column to the second column, mix well, then draw 100 μL from the second column to the third column, and mix well. In this two-fold serial dilution method until the tenth column, aspirate 100 μL and discard.

③Then add 100 μL of bacterial suspension to the first to tenth columns respectively. At this time, the final concentrations of the compounds in the first to tenth columns are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μM .

④ Mark the 96-well plate and place it in a 35°C incubator for 16-24 hours to observe the results. The negative control wells of the bacterial solution showed turbidity, the positive control wells showed clearness, and the quality control was within the specified range. The lowest concentration at which no bacterial growth was observed by the naked eye was the minimum inhibitory concentration (Minimal Inhibitory Concentration, MIC) of the compound.

1.3.2 Measuring MIC value in iron-deficient medium

By adding 2,2'-bipyridyl to normal medium, this compound can chelate iron in the medium. In an iron-deficient environment, bacteria secrete a large amount of siderophore to compete with 2,2'-bipyridine for iron in the medium, and the unique iron uptake pathway of bacteria is stimulated, which is conducive to the antibacterial effect of the siderophore-antibacterial drug conjugate. In addition, in humans and animals, iron is complexed by various proteins, resulting in an extremely low concentration of free iron that can be used by bacteria. The iron-deficiency medium added with 2,2'-bipyridine is just the right solution to the iron-deficiency environment in vivo. Simulated, iron-deficient medium conditions can more effectively reflect the antibacterial effects of compounds.

1.3.2.1 MHB iron-deficiency medium configuration

Weigh 11g of MHB dry powder into 500ml of ultrapure water, stir to dissolve, put it into a high-pressure steam sterilizer (121°C, 20min), take it out after the sterilization is completed, and place it in a constant temperature incubator at 37°C after cooling.

1.3.2.2 Preparation of Drug Stock Solution

Weigh 3 μmol of the target compound or positive control drug and dissolve it in 3 ml DMSO to prepare a 1 mM compound solution. Add 2 mL of Tris-HCl buffer solution at pH 8.0, add 1 mL of FeCl3 solution (1 mM), shake well, and configure drug stock solution (0.5 mM).

1.3.2.3 Bacterial suspension preparation

The strains frozen at -70°C were taken out, inoculated on the blood agar plate (BA) in different regions, and cultivated in a 35°C incubator for 16-24h, and waited for a single colony to grow for later use. Before the experiment, a single colony was picked with a sterile inoculation loop and placed in 0.45% normal saline, and adjusted to a concentration of 0.5 McFarland turbidity (1.0×10 ⁸ CFU/ml).

1.3.2.4 Broth microdilution drug susceptibility test

Draw 512 μL of drug stock solution, dilute to 1 mL with MHB culture medium, and make broth working solution with a concentration of 256 μM. in a clean bench,

① Add 200 μl of diluted drug solution to the first well of the 96-well plate, add 100 μL of iron-deficient MHB broth to the second to tenth wells, add 200 μL of bacterial solution to the eleventh well as a negative control for bacterial solution, and add 200 μL of iron-deficient MHB broth was used as a positive control.

② Use an eight-channel pipette to draw 100 μL of antibacterial drugs from the first column to the second column, mix well, then draw 100 μL from the second column to the third column, and mix well. In this two-fold serial dilution method until the tenth column, aspirate 100 μL and discard.

③Then add 100 μL of bacterial suspension to the first to tenth columns respectively. At this time, the final concentrations of the compounds in the first to tenth columns are 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μM .

④ Mark the 96-well plate and place it in a 35°C incubator for 16-24 hours to observe the results. The negative control wells of the bacterial solution showed turbidity, the positive control wells showed clearness, and the quality control was within the specified range. The lowest concentration at which no bacterial growth was observed by the naked eye was the minimum inhibitory concentration (Minimal Inhibitory Concentration, MIC) of the compound.

1.4 The in vitro anti-drug-resistant negative bacteria test results of the compound

Table 1 MIC values of compounds against drug-resistant negative bacteria (unit: μM)

The data in the table shows that under normal conditions, the compound of the present invention has moderate antibacterial activity against drug-resistant Klebsiella pneumoniae and Acinetobacter baumannii, which is equivalent to or slightly weaker than ciprofloxacin. The iron-deficiency medium simulates the iron-deficiency environment of in vivo infection, and can more effectively reflect the antibacterial activity of the compound. Under iron-deficiency conditions, the antibacterial activity of compound I-1 against drug-resistant Klebsiella pneumoniae was double that of ciprofloxacin, and compounds I-2, I-3 and I-4 were effective against drug-resistant Acinetobacter baumannii Its antibacterial activity is double that of ciprofloxacin.

In summary, the compound of the present invention has a novel chemical structure, and has improved antibacterial activity against ciprofloxacin-resistant bacteria under iron-deficiency conditions, and is expected to be developed as a drug for treating infectious diseases caused by drug-resistant Gram-negative bacteria.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (10)

1.3-hydroxyl-4-pyridone-ciprofloxacin coupled substance is characterized in that the general structural formula I is as follows:

where X is

2. The 3-hydroxy-4-pyridone-ciprofloxacin coupling according to claim 1, characterized in that it is the general formula II or compound I-4: General formula Ⅱ:

Compound I-4:

3. The 3-hydroxyl-4-pyridone-ciprofloxacin coupling according to claim 2, characterized in that the general formula II is any of the following: compound I-1, compound I-2, compound I-3 ; Compound I-1

Compound I-2:

Compound I-3:

4. The tautomers, optical isomers or pharmaceutically acceptable salts of the 3-hydroxy-4-pyridone-ciprofloxacin coupling as claimed in any one of claims 1-3. 5. The preparation method of 3-hydroxyl-4-pyridone-ciprofloxacin coupling substance, is characterized in that: One, the preparation method of general formula II is to comprise the following steps: (1.1) compound V and compound VI are reacted under reflux for 12±1 hours with an aprotic polar solvent as a solvent under inorganic base conditions to obtain compound VII; Compound V:

Compound VI:

Compound VII:

_The R1 is

R in the _formula is a hydroxyl protecting group; the X is

The R ₂ is a carboxyl protecting group; (1.2) Compound VII is deprotected in a mixed solvent under the catalysis of hydrogen and palladium/carbon to obtain the compound described in the general formula II; Two, the preparation method of compound I-4 is to comprise the following steps: (2.1) Compound VIII is reacted with a condensing agent at room temperature for 12±1 hours under the condition of an organic base, using an aprotic polar solvent as a solvent, to obtain Compound IX; Compound VIII:

Compound IX:

_R1 is

R ₂ is a leaving group, the leaving group is:

(2.2) Compound IX and ciprofloxacin were reacted at room temperature for 12±1 hours in an aprotic polar solvent under organic base conditions to obtain compound I-4. 6. the preparation method of 3-hydroxyl-4-pyridone-ciprofloxacin coupling thing according to claim 5, is characterized in that: R ₃ is a hydroxyl protecting group, and the protecting group is benzyl and benzhydryl; R ₂ is a carboxyl protecting group, and the protecting group is benzyl or benzhydryl. 7. according to the preparation method of the 3-hydroxyl-4-pyridone-ciprofloxacin coupling thing described in claim 5 or 6, it is characterized in that: In described step (1.1), inorganic base is potassium carbonate, sodium carbonate, potassium hydroxide; Aprotic polar solvent is acetonitrile, dimethylformamide; In the step (1.2): the mixed solvent is methylene chloride/ethanol, methylene chloride/tetrahydrofuran; In the step (2.1): the organic base is triethylamine, N,N-diisopropylethylamine; the condensing agent is N,N'-succinimide carbonate, 1,1'-carbonyldiimidazole; The aprotic polar solvent is dichloromethane, tetrahydrofuran; In the step (2.2): the organic base is triethylamine and N,N-diisopropylethylamine; the aprotic polar solvent is dichloromethane and tetrahydrofuran. 8. The use of the 3-hydroxy-4-pyridone-ciprofloxacin conjugate as claimed in any one of claims 1 to 3 in the preparation of medicines for treating diseases caused by bacteria. 9. The application according to claim 8, characterized in that: the bacteria are Gram-negative bacteria. 10. The application according to claim 8 or 9, characterized in that: the bacteria are ciprofloxacin-resistant Acinetobacter baumannii (ABA), ciprofloxacin-resistant Klebsiella pneumoniae (KP) .