阅读说明：本技术 异白叶藤碱类似物、从诺氟沙星到异白叶藤碱类似物的制备方法和应用 (Isoleucinine analogue, preparation method and application of isoleucinine analogue from norfloxacin to isoleucinine analogue ) 是由 黄帅 楚红英 许俊丽 路长远 晋秋芝 张堋梁 胡国强 于 2021-10-26 设计创作，主要内容包括：本发明提出了一种异白叶藤碱类似物、从诺氟沙星到异白叶藤碱类似物的制备方法和应用,用以解决如何以白叶藤类生物碱为先导物,以原子经济策略设计新型结构的吲哚并喹啉类抗结核药物的技术问题。本发明选择诺氟沙星制备异白叶藤碱类似物,实现了由氟喹诺酮结构到吲哚并喹啉骨架的有效化学构建,扩展了异白叶藤碱的结构修饰新途径,达到了氟喹诺酮药物与天然吲哚并喹啉类生物碱的优势结构的互补。体外抗结核活性测试结果表明,化合物对测试结核菌株有较好的生长抑制活性,部分化合物的活性与对照异烟肼相当,且兼有抗耐药性和较低的细胞毒性,可作为全新结构的抗结核药物进一步开发制备。(The invention provides an isoleucinine analogue, a preparation method and application of the isoleucinine analogue from norfloxacin to the isoleucinine analogue, which are used for solving the technical problem of how to design an indoloquinoline antituberculosis drug with a novel structure by taking a solanum dulcamara alkaloid as a precursor and adopting an atom economic strategy. The invention selects norfloxacin to prepare the isoleucin analogue, realizes effective chemical construction from a fluoroquinolone structure to an indoloquinoline skeleton, expands a new way of structural modification of isoleucin, and achieves the complementation of the advantageous structures of the fluoroquinolone medicament and natural indoloquinoline alkaloid. The in vitro antituberculosis activity test result shows that the compound has better growth inhibition activity on a tested tuberculosis strain, the activity of part of the compound is equivalent to that of a control isoniazid, and the compound has the advantages of drug resistance and lower cytotoxicity, and can be further developed and prepared as an antituberculosis drug with a brand new structure.)

1. An isoalburnine analogue is characterized in that the chemical structural general formula is shown as formula I:

the substituent R in the formula I can be independently hydrogen atom-H, methoxyl-OCH₃methyl-CH₃Fluorine atom-F, chlorine atom-Cl or xanthylamino-SO₂NH₂。

2. The isoalburnine analogue of claim 1, wherein the isoalburnine analogue has the chemical formula:

3. a preparation method of an analog from norfloxacin to isoleucinine is characterized by comprising the following steps:

s1, carrying out reduction decarboxylation reaction on norfloxacin shown in a formula II serving as a raw material and potassium borohydride to obtain 2, 3-dihydroquinolin-4-one shown in a formula III;

s2, preparing the cryptolepine alkaloid with the mother nucleus structure characteristic of the indoloquinoline by the Fischer indole synthesis method by using the 2, 3-dihydroquinoline-4-ketone and the phenylhydrazine obtained in the step S1;

s3, carrying out post-treatment on the Sinomenine alkaloid with the mother nucleus structure characteristic of the indoloquinoline obtained in the step S2 to obtain the isoSinomenine analogue as claimed in claim 1 or 2.

4. The process for preparing an analog of norfloxacin to isbehcerine according to claim 3, wherein the step S1 specifically comprises the steps of:

y1, mixing norfloxacin and a solvent to prepare a suspension, slowly adding potassium borohydride into the suspension for several times under stirring at normal temperature, heating the mixed reactant in a water bath, and stirring and refluxing to react until the norfloxacin disappears to obtain a mixed solution;

y2, placing the mixed solution obtained in the step Y1 to room temperature, and evaporating the solvent in the mixed solution by using an evaporator to obtain a residue;

y3, adding the remainder obtained in the step Y2 into deionized water, mixing, adjusting the pH to be approximately equal to 2 by using concentrated hydrochloric acid, adding activated carbon for decoloring, adjusting the pH to be approximately equal to 10 by using sodium hydroxide with the mass concentration of 30%, and standing to separate out a solid;

y4, and recrystallizing the solid precipitated in the step Y3 by hot water and n-hexane in sequence to obtain the 2, 3-dihydroquinolin-4-one.

5. The process for preparing an analog of norfloxacin to isbehcerine according to claim 3, wherein the step S2 specifically comprises the steps of:

t1, dissolving the 2, 3-dihydroquinolin-4-one obtained in the step S1 in absolute ethyl alcohol, adding phenylhydrazines, and stirring at normal temperature until the 2, 3-dihydroquinolin-4-one disappears to obtain a mixed solution;

t2, adding a cyclization catalyst into the mixed solution obtained in the step T1 dropwise, carrying out heating reflux reaction, standing to room temperature, and filtering to collect the generated albophylline alkaloid with the mother nucleus structure characteristics of indoloquinoline.

6. The process for preparing an analog of norfloxacin to isbehcerine according to claim 3, wherein the step S3 specifically comprises the steps of:

z1, dissolving the cryptolepine alkaloid with the mother nucleus structure characteristics of the indoloquinoline obtained in the step S3 in deionized water, adding activated carbon for reflux and decoloration, adding concentrated ammonia water to adjust the pH to be approximately equal to 10, and filtering to collect the generated solid;

and Z2, recrystallizing the solid obtained in the step T3 by using an absolute ethyl alcohol-ethyl acetate mixed solvent to obtain the light yellow crystal isoalbutine analogue.

7. The preparation method of norfloxacin-to-isoleucamine analogues according to claim 4, wherein the solvent in step Y1 is absolute methanol, absolute ethanol or 95% ethanol, and the molar ratio of norfloxacin to potassium borohydride is 1 (1-3).

8. The process according to claim 5, wherein the molar ratio of 2, 3-dihydroquinolin-4-one to phenylhydrazines in step T1 is 1 (1-2), the phenylhydrazines are phenylhydrazine, p-methylphenylhydrazine, m-methylphenylhydrazine, o-methylphenylhydrazine, p-fluorophenylhydrazine, m-fluorophenylhydrazine or m-fluorosulfonylaminobenzhydrazide, the cyclization catalyst in step T2 is concentrated hydrochloric acid, concentrated sulfuric acid, phosphoric acid, polyphosphoric acid, glacial acetic acid or trifluoroacetic acid, and the reflux reaction time is 10-24 h.

9. Use of the isoalbuterol analogue according to any one of claims 1, 2, 4 to 8 in the manufacture of an anti-tuberculosis medicament.

10. Use of isoalburnine analogues according to claim 9 for the preparation of anti-tubercular drugs, wherein said isoalburnine analogues are used for the preparation of drugs inhibiting mycobacterium tuberculosis, said mycobacterium tuberculosis being H₃₇Ra or H₃₇Rv。

Technical Field

The invention relates to the technical field of medicinal chemistry related to organic synthesis and new medicament research and development, in particular to an isoalbutine analogue, and also relates to a preparation method of the isoalbutine analogue from norfloxacin and an application of the isoalbutine analogue in preparation of antituberculosis medicaments.

Background

Tuberculosis is a chronic infectious disease with high morbidity caused by tubercle bacillus, and is an urgent public health and social problem facing the world due to the lack of effective therapeutic drugs. Meanwhile, the tubercle bacillus is easy to generate drug resistance to the existing drugs, especially the generation of multi-drug resistance, and provides new challenges for developing anti-tuberculosis drugs, so that no novel compound is available for treating tuberculosis for more than half a century since the rifampicin anti-tuberculosis drugs are found. Therefore, the development of antituberculosis drugs is a complicated intellectual innovation engineering with high time consumption and high investment, and is receiving attention. Wherein, the effective components of natural medicines are used as leads, and the structure of the natural medicines is optimized and modified, which is the most economic and effective strategy for finding new medicines. In the research and development of various natural active ingredients, the cryptolepine alkaloids with indoloquinoline as the structural characteristic skeleton, such as cryptolepine (A), isocryptolepine (B), neocryptolepine (C) and the like,

the structure is unique, and the anti-plasmodium falciparum and anti-tumor biological activities are better, so that the research interest is aroused, but the reports on the anti-tuberculosis activity are few. However, the source of the solanum lyratum alkaloid is difficult, and the bioavailability is low due to the poor water solubility, so that the clinical application is limited. Therefore, how to design the indoloquinoline antituberculosis drugs with novel structures by using the solanum glaucescens alkaloids as a precursor and an atom economic strategy is very important. On one hand, the fluoroquinolone-based medicine is not only a clinically important antibacterial medicine, but also a clinical second-line antituberculosis medicine, for example, the advantageous structures of the solanum lyratum alkaloid and the fluoroquinolone are combined to play the advantages of respective pharmacophores, and a novel indoloquinoline antituberculosis medicine is possibly designed and found; on the other hand, by introducing effective substituent groups in fluoroquinolone medicine molecules, the pharmacodynamics and pharmacokinetic properties of the fluoroquinolone medicine molecules are further improved so as to overcome the defects of the conventional alkaloid and promote the development of the pharmacy of novel indoloquinoline antituberculosis medicines.

Disclosure of Invention

The invention provides an isoleucinine analogue, and also relates to a preparation method of the isoleucinine analogue from norfloxacin, and application of the isoleucinine analogue in preparing antituberculosis drugs, aiming at the technical problem of how to design an indoloquinoline antituberculosis drug with a novel structure by taking a leucinia alkaloid as a precursor and adopting an atom economic strategy. The invention uses the commercially available fluoroquinolone drug norfloxacin (II) as a raw material, and the norfloxacin (II) is reduced and deacidified to quinolinone (6-fluoro-1-ethyl-7-piperazine-1-yl-2, 3-dihydro-quinoline-4 (1H) -ketone, III), and then the quinolinone and phenylhydrazine are successfully synthesized into the isoalbuterol analogue by a Fischer indole synthesis method; isoalbuterone (B) in the albuterone alkaloids is selected as a precursor, indolo [3,2-c ] quinoline is used as a dominant skeleton, hydrophilic basic piperazinyl in a fluoroquinolone medicine structure is introduced to increase water solubility and improve bioavailability, and introduction of fluorine atoms can increase permeability of medicine molecules to improve bioactivity.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

an isoalburnine analogue has a chemical structural general formula shown in formula I:

the substituent R in the formula I can be independently hydrogen atom-H, methoxy-OCH₃methyl-CH₃Fluorine atom-F, chlorine atom-Cl or xanthylamino-SO₂NH₂。

Preferably, the chemical structural formula of the isoalburnine analogue is:

a preparation method of norfloxacin-isoleucinine analogues comprises the following steps:

s1, carrying out reduction decarboxylation reaction on norfloxacin shown in a formula II serving as a raw material and potassium borohydride to obtain 2, 3-dihydroquinolin-4-one shown in a formula III;

s2, preparing the cryptolepine alkaloid with the mother nucleus structure characteristic of the indoloquinoline by the Fischer indole synthesis method by using the 2, 3-dihydroquinoline-4-ketone and the phenylhydrazine obtained in the step S1;

s3, carrying out post-treatment on the Sinomenine alkaloid with the mother nucleus structure characteristic of the indoloquinoline obtained in the step S2 to obtain the isoSinomenine analogue as the claim 1 or 2.

Preferably, 6-fluoro-1-ethyl-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one, III, of formula III, is prepared by a reductive decarboxylation reaction of a commercially available norfloxacin of formula II starting with a commercially available analytical pure chemical reagent, sodium borohydride, by a similar procedure to that described in the references to preparation of 6-fluoro-1-ethyl-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one, III, of formula III (Kondo H, Sakamoto F, et al, study on precursors, 7.Synthesis and antimicrobial activity of 3-formalquinolone derivatives, J Med Chem,1988,31(1): 221-.

As a further improvement, the potassium borohydride which is cheap, difficult to absorb moisture and mild in reaction is used for replacing sodium borohydride in the literature.

Step S1 specifically includes the following steps:

y1, mixing norfloxacin and a solvent to prepare a suspension, slowly adding potassium borohydride into the suspension for several times under stirring at normal temperature, heating the mixed reactant in a water bath, and stirring and refluxing to react until the norfloxacin disappears to obtain a mixed solution;

y2, placing the mixed solution obtained in the step Y1 to room temperature, and evaporating the solvent in the mixed solution by using an evaporator to obtain a residue;

y3, adding the remainder obtained in the step Y2 into deionized water, mixing, adjusting the pH to be approximately equal to 2 by using concentrated hydrochloric acid, adding activated carbon for decoloring, adjusting the pH to be approximately equal to 10 by using sodium hydroxide with the mass concentration of 30%, and standing to separate out a solid;

y4, and recrystallizing the solid precipitated in the step Y3 by hot water and n-hexane in sequence to obtain the 2, 3-dihydroquinolin-4-one.

Preferably, step S2 specifically includes the following steps:

t1, dissolving the 2, 3-dihydroquinolin-4-one obtained in the step S1 in absolute ethyl alcohol, adding phenylhydrazines, and stirring at normal temperature until the 2, 3-dihydroquinolin-4-one disappears to obtain a mixed solution;

t2, adding a cyclization catalyst into the mixed solution obtained in the step T1 dropwise, carrying out heating reflux reaction, standing to room temperature, and filtering to collect the generated albophylline alkaloid with the mother nucleus structure characteristics of indoloquinoline.

Preferably, step S3 specifically includes the following steps:

z1, dissolving the cryptolepine alkaloid with the mother nucleus structure characteristics of the indoloquinoline obtained in the step S3 in deionized water, adding activated carbon for reflux and decoloration, adding concentrated ammonia water to adjust the pH to be approximately equal to 10, and filtering to collect the generated solid;

and Z2, recrystallizing the solid obtained in the step T3 by using an absolute ethyl alcohol-ethyl acetate mixed solvent to obtain the light yellow crystal isoalbutine analogue.

Preferably, the solvent in the step Y1 is absolute methanol, absolute ethanol or 95% ethanol, and the molar ratio of norfloxacin to potassium borohydride is 1 (1-3).

Preferably, the molar ratio of the 2, 3-dihydroquinolin-4-one to the phenylhydrazines in the step T1 is 1 (1-2), the phenylhydrazines are phenylhydrazine, p-methylphenylhydrazine, m-methylphenylhydrazine, o-methylphenylhydrazine, p-fluorophenylhydrazine, m-fluorophenylhydrazine or m-fluorosulfonylaminophenylhydrazine, the cyclization catalyst in the step T2 is concentrated hydrochloric acid, concentrated sulfuric acid, phosphoric acid, polyphosphoric acid, glacial acetic acid or trifluoroacetic acid, and the reflux reaction time is 10-24 h.

An application of isohederin analogue in preparing antituberculosis drugs is provided.

Preferably, the isoalbuterone analogue is used for preparing medicine for inhibiting mycobacterium tuberculosis, wherein the mycobacterium tuberculosis is H₃₇Ra or H₃₇Rv。

The invention has the beneficial effects that:

1. the isoleucinine analogue of the invention reserves the predominant skeleton of isoleucinine-indoloquinoline mother nucleus, and simultaneously has the characteristic structure of fluoroquinolone medicaments, namely quinoline ring, especially as the effective modifying group of fluoroquinolone medicaments, and the hydrophilic basic piperazinyl is used as the modifying group of the indoloquinoline mother nucleus, thereby not only effectively improving the water solubility of the isoleucinine and improving the bioavailability, being beneficial to the development of drug properties, but also increasing the medicament osmosis effect by introducing fluorine atoms, and realizing the effects of enhancing the efficiency and reducing toxicity and resisting drug resistance.

2. The indoloquinoline parent nucleus and the quinoline ring in the isoleucinine analogue realize the complementation and the activity superposition of different structural pharmacophores, and the in vitro antitubercular activity test result in an experimental example shows that the compound has better growth inhibition activity on a test tuberculosis strain, the activity of part of the compound is equivalent to that of a control isoniazid, and the compound has the advantages of drug resistance, lower cytotoxicity, excellent in vitro inhibition activity on the growth of tubercle bacillus, and can be further developed as a novel antitubercular medicament with the structural characteristics of indoloquinoline.

3. According to the preparation method, norfloxacin is used for preparing 2, 3-dihydroquinoline-4-ketone through reduction decarboxylation reaction and potassium borohydride, then 2, 3-dihydroquinoline-4-ketone and phenylhydrazine are used for preparing the isoalbuterol analogue through a Fischer indole synthesis method, effective chemical construction from a fluoroquinolone structure to an indoloquinoline skeleton is achieved, a new approach of structural modification of isoalbuterol is expanded, and complementation of the advantageous structures of a fluoroquinolone medicine and natural indoloquinoline alkaloids is achieved.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

An isoalburnine analogue has a chemical structural general formula shown in formula I:

in this example, where the substituent R in formula I is H, the chemical structure of 2-fluoro-3-piperazin-1-yl-5-ethyl-5H-indolo [3,2-c ] quinoline is:

this example uses a preparation method from norfloxacin to isoleucinine analogue for preparing the isoleucinine analogue (I-1), which specifically includes the following steps:

s1, carrying out reduction decarboxylation reaction on norfloxacin shown in a formula II serving as a raw material and potassium borohydride to obtain 2, 3-dihydroquinolin-4-one shown in a formula III.

Specifically, the method comprises the following steps: y1, mixing norfloxacin and a solvent to prepare a suspension, slowly adding potassium borohydride into the suspension for several times under stirring at normal temperature, heating the mixed reactant in a water bath, and stirring and refluxing to react until the norfloxacin disappears to obtain a mixed solution; y2, placing the mixed solution obtained in the step Y1 to room temperature, and evaporating the solvent in the mixed solution by using an evaporator to obtain a residue; y3, adding the remainder obtained in the step Y2 into deionized water, mixing, adjusting the pH to be approximately equal to 2 by using concentrated hydrochloric acid, adding activated carbon for decoloring, adjusting the pH to be approximately equal to 10 by using sodium hydroxide with the mass concentration of 30%, and standing to separate out a solid; y4, and recrystallizing the solid precipitated in the step Y3 by hot water and n-hexane in sequence to obtain the 2, 3-dihydroquinolin-4-one.

In this embodiment, the method specifically includes: suspending 18.0g (55.0mmol) of norfloxacin II in 500mL of anhydrous methanol, slowly adding 7.4g (138.0mmol) of potassium borohydride in portions under stirring at normal temperature, heating the mixed reactant in a water bath, and stirring and refluxing to react until the raw material II disappears. The mixture was left at room temperature, and the solvent was evaporated under reduced pressure by a rotary evaporator. And adding 500mL of deionized water into the residue, adjusting the pH value to be approximately equal to 2.0 by using concentrated hydrochloric acid, adding a proper amount of activated carbon for decolorization, adjusting the pH value of the filtrate to be approximately equal to 10.0 by using a sodium hydroxide solution with the mass concentration of 30%, and standing to separate out a solid. And recrystallizing the crude product by using hot water and n-hexane in sequence to obtain the 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one shown in the formula III, wherein the yield is 64.7 percent and the m.p.58-60 ℃.¹H NMR(400MHz,CD₃Cl) δ: 7.51(1H, d,5-H), 6.17(1H, d,8-H), 3.05-3.36 (8H, piperazine-H), 3.42-3.48 (4H, m, 2-H and 1-CH)₂) 2.25 to 2.64(3H, m, 3-H and NH), 1.22(3H, t, CH)₃)；MS(m/z)：278[M+H]⁺Calculating (C)₁₅H₂₀FN₃O)：277.34。

S2, preparing the cryptolepine alkaloid with the mother nucleus structure characteristics of the indoloquinoline by the Fischer indole synthesis method by using the 2, 3-dihydroquinoline-4-ketone and the phenylhydrazine obtained in the step S1. Specifically, the method comprises the following steps: t1, dissolving the 2, 3-dihydroquinolin-4-one obtained in the step S1 in a solvent, adding a phenylhydrazine, and stirring at normal temperature until the 2, 3-dihydroquinolin-4-one disappears to obtain a mixed solution; t2, adding a cyclization catalyst into the mixed solution obtained in the step T1 dropwise, carrying out heating reflux reaction, standing to room temperature, and filtering to collect the generated albophylline alkaloid with the mother nucleus structure characteristics of indoloquinoline.

In this embodiment, the method specifically includes: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, 0.50g (4.6mmol) of phenylhydrazine was added, and the reaction was stirred at room temperature for 20 hours, whereby a large amount of precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 12h and allowed to stand overnight. The resulting solids were collected by filtration. The solid is the bynine alkaloid with the mother nucleus structure characteristic of the indoloquinoline.

S3, carrying out post-treatment on the Sinomenine alkaloid with the mother nucleus structure characteristic of the indoloquinoline obtained in the step S2 to obtain the isoSinomenine analogue as claimed in claim 1 or 2. Specifically, the method comprises the following steps: z1, dissolving the cryptolepine alkaloid with the mother nucleus structure characteristics of the indoloquinoline obtained in the step S3 in deionized water, adding activated carbon for reflux and decoloration, adding concentrated ammonia water to adjust the pH to be approximately equal to 10, and filtering to collect the generated solid;

and Z2, recrystallizing the solid obtained in the step T3 by using an absolute ethyl alcohol-ethyl acetate mixed solvent to obtain the light yellow crystal isoalbutine analogue.

In this embodiment, the method specifically includes: dissolving the solid with 50mL of deionized water, adding a proper amount of active carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. And (3) collecting the generated solid by filtration, drying, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 5:1), so as to obtain a light yellow crystal target compound, namely the formula I-1, wherein the yield is 52.4%, and m.p.232-234 ℃.¹H NMR(400MHz,CD₃Cl)δ：1.42(3H,t，CH₃) 3.12 to 3.45(8H, m, piperazine-H), 3.62(1H, br, NH), 4.54(2H, q, CH)₂) 7.17-8.12 (6H, m, 1-H, 4-H and Ph-H), 8.72(1H, s, 6-H); MS (m/z): 349[ M + H]⁺Calculating (C)₂₁H₂₁FN₄)： 348.43。

Example 2

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is methoxy, the chemical structure of isofraxinine analog, i.e. 2-fluoro-5-ethyl-8-methoxy-3-piperazin-1-yl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-2), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, 0.62g (4.5mmol) of p-methoxyphenylhydrazine was added thereto, and the reaction was stirred at room temperature overnight with the formation of a significant precipitate. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. The resulting solid was collected by filtration, dried, and recrystallized from an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 5:1) to give a pale yellow crystalline target compound of formula I-2, yield 53.6%, m.p.233 to 235 ℃.¹H NMR(400MHz, CD₃Cl)δ：1.45(3H,t，CH₃) 3.16-3.47 (8H, m, piperazine-H), 3.68(1H, br, NH), 3.86(3H, s, OCH)₃)，4.62(2H，q，CH₂) 7.45-8.23 (5H, m, 1-H, 4-H and Ph-H), 8.86(1H, s, 6-H); MS (m/z): 376[ M + H]⁺Calculating (C)₂₂H₂₃FN₄O)：378.45。

It is worth noting that in this example, the process for preparing 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one is essentially identical to that of example 1, except that in this example, the molar ratio of norfloxacin to potassium borohydride is set to 1: 2, anhydrous methanol used in example 1 as a solvent was replaced with anhydrous ethanol as a solvent.

Example 3

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is methoxy, the chemical structure of isofraxinine analog, i.e. 2-fluoro-5-ethyl-3-piperazin-1-yl-9-methoxy-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-3), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one is dissolved in 15mL of anhydrous ethanol, 0.83g (6.0mmol) of m-methoxyphenylhydrazine is added, and the reaction is stirred at normal temperature for 24H, with the formation of a significant precipitate. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 12h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. And (3) collecting the generated solid by filtration, drying, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 6:1) to obtain a light yellow crystal target compound, namely the formula I-3, wherein the yield is 42.5%, and m.p.234-236 ℃.¹H NMR(400MHz, CD₃Cl)δ：1.44(3H,t，CH₃) 3.18 to 3.46(8H, m, piperazine-H), 3.70(1H, br, NH), 3.88(3H, s, OCH)₃)，4.64(2H，q，CH₂) 7.53-8.25 (5H, m, 1-H, 4-H and Ph-H), 8.87(1H, s, 6-H); MS (m/z): 376[ M + H]⁺Calculating (C)₂₂H₂₃FN₄O)：378.45。

It is worth noting that in this example, the process for preparing 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one is essentially identical to that of example 1, except that in this example, the molar ratio of norfloxacin to potassium borohydride is set to 1: 1, the absolute methanol used in example 1 as solvent was replaced with 95% ethanol as solvent.

Example 4

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is methoxy, the chemical structure of isofraxinine analog, i.e. 2-fluoro-5-ethyl-3-piperazin-1-yl-10-methoxy-5H-indolo [3,2-c ] quinoline, is as follows:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-4), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, 0.72g (5.0mmol) of o-methoxyphenylhydrazine was added, and the reaction was stirred at room temperature for 24 hours, whereupon a precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 12h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. And (3) filtering and collecting the generated solid, drying, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 3:1) to obtain a light yellow crystal target compound, namely the formula I-4, wherein the yield is 40.2%, and m.p. is 226-228 ℃.¹H NMR(400MHz,CD₃Cl) δ：1.45(3H,t，CH₃) 3.23-3.47 (8H, m, piperazine-H), 3.65(1H, br, NH), 3.86(3H, s, OCH)₃)，4.62(2H，q，CH₂) 7.55-8.26 (5H, m, 1-H, 4-H and Ph-H), 8.86(1H, s, 6-H); MS (m/z): 376[ M + H]⁺Calculating (C)₂₂H₂₃FN₄O)：378.45。

It is worth noting that in this example, the process for preparing 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one is essentially identical to that of example 1, except that in this example, the molar ratio of norfloxacin to potassium borohydride is set to 1: 2, the absolute methanol used in example 1 as solvent was replaced with 95% ethanol as solvent.

Example 5

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is methyl, the chemical structure of isofraxinine analog, i.e., 2-fluoro-3-piperazin-1-yl-5-ethyl-8-methyl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-5), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one represented by formula III was dissolved in 15mL of anhydrous ethanol, and 0.70g (5.7mmol) of p-tolylhydrazine was added thereto, and the reaction was stirred at room temperature overnight to form a precipitate. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 24h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. And (3) filtering and collecting the generated solid, drying, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 3:1) to obtain a light yellow crystal target compound, namely the formula I-5, wherein the yield is 36.4%, and m.p. is 232-234 ℃.¹H NMR(400MHz, CD₃Cl)δ：1.41(3H,t，CH₃)，2.27(3H，s，Ph-CH₃) 3.05 to 3.37(8H, m, piperazine-H), 3.62(1H, br, NH), 4.57(2H, q, CH)₂) 7.42-8.18 (5H, m, 1-H, 4-H and Ph-H), 8.86(1H, s, 6-H); MS (m/z): 363[ M + H]⁺Calculating (C)₂₂H₂₃FN₄)：362.45。

It is worth noting that in this example, the process for preparing 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one is essentially identical to that of example 1, except that in this example, the molar ratio of norfloxacin to potassium borohydride is set to 1: 3, anhydrous methanol used in example 1 as a solvent was replaced with anhydrous ethanol as a solvent.

Example 6

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is F atom, the chemical structure of isofraxinine analog, i.e. 2, 8-difluoro-3-piperazin-1-yl-5-ethyl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-6), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, 0.60g (4.8mmol) of p-fluorophenylhydrazine was added thereto, and the reaction was stirred at room temperature for 24 hours, whereby a large amount of precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 20h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. The resulting solid was collected by filtration, dried, and recrystallized from an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 6:1) to obtain a pale yellow crystalline target compound of formula I-6, yield 52.4%, m.p.241 to 243 ℃.¹H NMR(400MHz,CD₃Cl) δ：1.52(3H,t，CH₃) 3.31 to 3.52(8H, m, piperazine-H), 3.72(1H, br, NH), 4.70(2H, q, CH)₂) 7.62-8.26 (5H, m, 1-H, 4-H and Ph-H), 8.90(1H, s, 6-H); MS (m/z): 367[ M + H]⁺Calculating (C)₂₁H₂₀F₂N₄)：366.42。

Example 7

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is F atom, the chemical structure of isofraxinine analog, i.e. 2, 9-difluoro-3-piperazin-1-yl-5-ethyl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-7), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, 0.66g (5.2mmol) of m-fluorophenylhydrazine was added thereto, and the reaction was stirred at room temperature overnight with the formation of a significant precipitate. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. The resulting solid was collected by filtration, dried, and recrystallized from an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 5:1) to give a pale yellow crystalline target compound of formula I-7, yield 42.6%, m.p.230 to 232 ℃.¹H NMR(400MHz,CD₃Cl) δ：1.48(3H,t，CH₃) 3.33-3.62 (8H, m, piperazine-H), 3.73(1H, br, NH), 4.72(2H, q, CH)₂) 7.58-8.25 (5H, m, 1-H, 4-H and Ph-H), 8.91(1H, s, 6-H); MS (m/z): 367[ M + H]⁺Calculating (C)₂₁H₂₀F₂N₄)：366.42。

Example 8

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is chlorine atom, the chemical structure of isofraxinine analog, i.e. 2-fluoro-8-chloro-3-piperazin-1-yl-5-ethyl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-8), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous0.74g (5.2mmol) of p-chlorophenylhydrazine was added to ethanol, and the mixture was stirred at room temperature for 24 hours to react, whereby a large amount of precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 24h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. And (3) collecting the generated solid by filtration, drying, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 5:1) to obtain a light yellow crystal target compound, namely the formula I-8, wherein the yield is 41.5%, and m.p.232-234 ℃.¹H NMR(400MHz,CD₃Cl) δ：1.43(3H,t，CH₃) 3.27-3.60 (8H, m, piperazine-H), 3.68(1H, br, NH), 4.70(2H, q, CH)₂) 7.47-8.24 (5H, m, 1-H, 4-H and Ph-H), 8.89(1H, s, 6-H); MS (m/z): 383 (Cl)³⁵)[M+H]⁺，385(Cl³⁷)[M+H]⁺Calculating (C)₂₁H₂₀ClFN₄)：382.87。

Example 9

An isofraxinine analog, which is different from example 1 in that the substituent R in formula I is chlorine atom, the chemical structure of isofraxinine analog, i.e. 2-fluoro-9-chloro-3-piperazin-1-yl-5-ethyl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-9), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one is dissolved in 15mL of anhydrous ethanol, 0.70g (5.0mmol) of m-chlorophenylhydrazine is added, and the reaction is stirred at normal temperature for 24 hours, so that a remarkable precipitate is generated. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. Filtering to collect the solid, drying, and mixing with anhydrous alcohol-ethyl acetate (v is 6:1) to obtain a light yellow crystal target compound, namely the formula I-9, wherein the yield is 41.3%, and m.p.231-233 ℃.¹H NMR(400MHz,CD₃Cl) δ：1.48(3H,t，CH₃) 3.30-3.67 (8H, m, piperazine-H), 3.72(1H, br, NH), 4.73(2H, q, CH)₂) 7.50-8.26 (5H, m, 1-H, 4-H and Ph-H), 8.90(1H, s, 6-H); MS (m/z): 383 (Cl)³⁵)[M+H]⁺，385(Cl³⁷)[M+H]⁺Calculating (C)₂₁H₂₀ClFN₄)：382.87。

Example 10

An isoleucinine analog, which is different from example 1 in that the substituent R in formula I is a sulfonamide group, the chemical structure of isoleucinine analog, i.e., 2-fluoro-8-sulfonamide-3-piperazin-1-yl-5-ethyl-5H-indolo [3,2-c ] quinoline, is:

this example uses a preparation method from norfloxacin to isoleucinine analogs for preparing the isoleucinine analog (I-10), which specifically includes the following steps: 1.0g (3.6mmol) of 1-ethyl-6-fluoro-7-piperazin-1-yl-2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, and 0.67g (3.6mmol) of p-chlorosulfonylaminophenyl hydrazine was added thereto, and the reaction was stirred at room temperature overnight, whereby a large amount of precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 24h and allowed to stand overnight. And (3) filtering and collecting the generated solid, dissolving the solid in 50mL of deionized water, adding a proper amount of activated carbon, and refluxing and decoloring for 1 h. Hot filtration, pH of filtrate adjusted with ammonia about 10.0. And (3) collecting the generated solid by filtration, drying, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 5:1), so as to obtain a light yellow crystal target compound, namely the formula I-10, wherein the yield is 53.6%, and m.p.244-246 ℃.¹H NMR(400MHz,CD₃Cl)δ：：1.57(3H,t，CH₃) 3.37-3.78 (8H, m, piperazine-H), 3.75(1H, br, NH), 4.82(2H, q, CH)₂) 7.32 to 8.56(7H, m, 1-H, 4-H, Ph-H and NH)₂)，9.08(1H，s，6-H)；MS(m/z)：428[M+H]⁺Calculating (C)₂₁H₂₂FN₅O₂S)：427.50。

The invention also provides application of the isoalbutine analogue in preparing anti-tuberculosis drugs, in particular to application of the isoalbutine analogue in preparing drugs for inhibiting mycobacterium tuberculosis, wherein the mycobacterium tuberculosis is H₃₇Ra or H₃₇Rv, the following experimental examples of the use of isospergualin analogs in the preparation of anti-tubercular drugs are described in detail.

Examples of the experiments

First, the in vitro anti-tubercle bacillus activity assay of the isoalbugine analogs provided in examples 1 to 10

1. Experimental reagent

Positive control substances Isoniazide (INH) and norfloxacin (norfloxacin) were purchased from the institute for food and drug, hannan province; 7H9 liquid culture medium was purchased from Difco, USA. Under the aseptic condition, a positive control and the samples of the examples I-1 to I-10 are prepared into a 4mg/mL solution by using dimethyl sulfoxide (DMSO), the solution is filtered by a 0.22 mu m filter membrane after being dissolved sufficiently by ultrasound, and the filtrate is used as a stock solution and is stored at-20 ℃ for standby (when in use, the concentration of the DMSO in the culture solution is less than 0.5 percent in order to avoid the influence of the DMSO on the experimental result).

2. Tuberculosis strain

The experimental tuberculosis strains are respectively a mycobacterium tuberculosis standard strain H₃₇Ra(ATCC25177)、 H₃₇The clinical separated drug-resistant mycobacterium tuberculosis of Rv (ATCC27294) and 3 strains are respectively numbered as H6, H7 and H10, and are provided by the Henan province disease prevention and control center and provide the measurement of experimental data. Wherein, H6 and H7 are multi-drug resistant strains for isoniazid, rifampicin, ethambutol, streptomycin and ofloxacin, and H10 is a drug resistant strain for isoniazid and rifampicin.

3. Experimental methods

1) Preparation of a strain suspension: taking out the to-be-detected tubercle bacillus cultured for 2-3 weeks, inoculating into a sterilized small bottle, uniformly mixing to form milk, diluting with normal saline, preparing the bacterial liquid into 1mg/mL bacterial liquid by turbidimetric reaction with a NO.1 McLee standard turbidimetric tube, and diluting with normal saline to 1 × 10⁵The CFU is ready for use.

2) To a 96-well plate, 200. mu.L of a test compound solution of an appropriate concentration was added (test compound was diluted to 200. mu.g/mL with sterile 7H9 broth), and then test compound was further diluted as necessary (fold-by-fold dilution to 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.097, 0.048, 0.024, 0.012. mu.g/mL), and drug-free control wells were set.

3) Adding the diluted bacteria solution into all detection wells and drug-free facing wells, placing the plates in a constant temperature incubator, each plate being at 37 deg.C and 5% CO₂Cultured under the conditions for 21 days. The lowest concentration at which no bacterial strain grows is observed by naked eyes through a microscope at 40 times is the Minimum Inhibitory Concentration (MIC) of the drug. Meanwhile, isoniazid and norfloxacin are used as positive controls, and DMSO and a culture solution without any compound are used as negative controls. Each data was measured in triplicate and averaged, and the results are shown in Table 1.

TABLE 1 in vitro antitubercular Activity (MIC) of test samples

The results in Table 1 show that, of the compounds provided in examples 1 to 10, examples 1, 4, 6, 7 and 10 are H₃₇Ra and H₃₇The MIC values of two standard strains of the Rv mycobacterium tuberculosis are lower than that of the control norfloxacin, especially the activities of the two standard strains of the Rv mycobacterium tuberculosis are equivalent to that of isoniazid, and the two standard strains of the Rv mycobacterium tuberculosis show better in-vitro anti-tubercle bacillus activity. Meanwhile, the MIC values of the compounds provided in examples 1-10 to 3 clinically isolated drug-resistant strains H6, H7 and H10 are far lower than those of control norfloxacin or isoniazid, and the compounds show better anti-drug resistance activity.

Second, in vitro cytotoxicity assay of examples 1 to 10

1. Experimental reagent

Positive control substances Isoniazide (INH) and norfloxacin were purchased from the institute of food and drug inspection in henna; normal cells were VerO, a Vero cell line, purchased from Shanghai Tong Seiki Seisakusho Biotech, Ltd. RPMI, Trypsin (TRGPSIN) and fetal calf serum were purchased from Hangzhou ilex bioengineering materials, Inc.; brominating- (4,5) -dimethyl-2-thiazole-2, 5-diphenyl tetrazole (MTT, AMRESCO split charging); sodium Dodecyl Sulfate (SDS), sodium dihydrogen phosphate purchased from Tianjin department Europe chemical reagent development center; ethylenediaminetetraacetic acid disodium salt (EDTA) and dimethyl sulfoxide (DMSO) were purchased from Tianjin Deen chemical Co., Ltd.

2. Preparation of test solution

Under the aseptic condition, the positive reference substance and the test substances of the examples I-1 to I-10 are prepared into 1.0 multiplied by 10 by dimethyl sulfoxide (DMSO)^-4mol·L^-112 stock solutions with concentration, and then diluting the stock solutions with 5 concentration gradients (0.1, 1.0, 5.0, 10.0, 50.0 μmol. L) by using 10% calf serum RPMI-1640 culture solution^-1) The working solution is fully dissolved by ultrasonic waves and then filtered by a filter membrane of 0.22 mu m, and the filtrate is taken as a test solution and is stored at the temperature of minus 20 ℃ for standby.

3. Experimental method (MTT method)

Taking VERO African green monkey kidney cell strain in logarithmic growth phase, inoculating 6000 cells per well into 96-well plate, adding working solution with 5 concentration gradients of the above 12 samples, adding 5 g.L per well after 48 hr^–1mu.L of MTT (thiazole blue) solution was added, and after further culturing for 4 hours, 100. mu.L of a 10% by mass Sodium Dodecyl Sulfate (SDS) solution was added. After 24 hours of incubation, the absorbance (OD) was measured at a wavelength of 570nm using a microplate reader. The inhibition rate of cell proliferation is calculated according to the formula:

inhibition rate [ (1-experimental OD value)/control OD value ] × 100%

Then, linear regression is carried out on the VERO cell inhibition rate corresponding to each concentration according to the pair value of each concentration of each sample to obtain a dose-effect equation, and the half Inhibition Concentration (IC) of the sample to the experimental VERO cell is calculated from the obtained dose-effect equation₅₀) (ii) a Each data was measured in triplicate and averaged, the results are shown in Table 2.

TABLE 2 in vitro VERO cytotoxicity assay for test samples(IC₅₀)

Table 2 results show that the compounds provided in examples 1-10 have half the growth Inhibitory Concentration (IC) on VERO cells₅₀) The positive control norfloxacin is equivalent, shows a lower cytotoxic effect, and simultaneously, the IC of the compound provided in examples 1-10₅₀IC higher than positive isoniazid₅₀The values indicate that the cytotoxicity of the compounds provided in examples 1-10 is lower than that of the positive control isoniazid.

In summary, the compounds provided in embodiments 1 to 10 have not only better anti-tubercle bacillus activity but also potential anti-drug resistance activity in vitro, and show lower cytotoxic effect. Based on the new drug research rule, the norfloxacin-isoalbutinine analogue is expected to develop an anti-tuberculosis drug with high efficiency and low toxicity.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.