CN113816958A - Isoleucinine analogue prepared from pefloxacin as raw material, and preparation method and application thereof - Google Patents
Isoleucinine analogue prepared from pefloxacin as raw material, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an analogue from pefloxacin to isoleucamine, a preparation method and application thereof, and the characteristic chemical structure is shown as the following formula I:
the substituent R in the formula I can be independently-H-OCH or-OCH3-F, -Cl or-SO2NH2. The isoleucinine analogue takes pefloxacin as a raw material, realizes effective chemical construction from a fluoroquinolone structure to an indoloquinoline skeleton, expands a new way of structural modification of isoleucinine, achieves the complementation of the advantageous structures of fluoroquinolone medicines and natural indoloquinoline alkaloids, further increases the anti-tubercle bacillus activity and the anti-drug resistance of the compounds, reduces the toxicity to normal cells, and can be further developed as an anti-tubercle medicine with a brand new structureAnd (4) sending.
Description
Technical Field
The invention belongs to the technical field of medicinal chemistry related to organic synthesis and new medicament research and development, and particularly relates to an isoalbutine analogue, a method for preparing the isoalbutine analogue by taking pefloxacin as a raw material, and application of the isoalbutine analogue in preparing 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 aloperine alkaloids taking indoloquinoline as a structural characteristic skeleton, such as cryptolepine (A), isocoryphylline (B), neocryptolepine (C) and the like, are found to arouse research interest due to unique structures and better various biological activities such as anti-plasmodium, anti-tumor and the like, but reports on the anti-tuberculosis activity of the alophylline alkaloids 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. In view of this, isohederin B in the hederamine alkaloids is selected as a precursor, indolo [3,2-c ] quinoline is used as an advantageous 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.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an isoalbuterol analogue with the function and efficacy of resisting tubercle bacillus, which is obtained by using fluoroquinolone drug pefloxacin as a raw material, reducing and deacidifying the fluoroquinolone drug pefloxacin to quinolinone (6-fluoro-1-ethyl-7- (4-methylpiperazine-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one), and then successfully constructing the quinolinone and phenylhydrazine by a Fischer indole synthesis method.
The invention also provides a method for preparing the isoleucamine analogue by taking pefloxacin as a raw material, and also provides application of the isoleucamine analogue. The isoleucinine analogue realizes effective chemical construction from a fluoroquinolone structure to an indoloquinoline skeleton, expands a new way of structural modification of isoleucinine, achieves the complementation of the advantageous structures of fluoroquinolone medicaments and natural indoloquinoline alkaloids, further increases the anti-tubercle bacillus activity and the anti-drug resistance of the compounds, reduces the toxicity to normal cells, and can be further developed as an anti-tubercle medicament with a brand new structure.
In order to achieve the purpose, the invention adopts the following technical scheme:
an isoalburnine analogue, whose chemical structure general formula is shown in formula I:
the substituent R in the formula I can be independently hydrogen atom-H, methoxy-OCH3methyl-CH 3, fluorine atom-F. Chlorine atom-Cl or xanthylamino-SO2NH2The compound is a compound with the following specific structure:
the invention provides a method for preparing the isoleucamine analogue by taking pefloxacin as a raw material, which comprises the following specific steps: pefloxacin shown in a formula II is used as a raw material, the pefloxacin and potassium borohydride are subjected to reduction deacidification reaction in the presence of a solvent to prepare a compound 1-ethyl-6-fluoro-7- (4-methylpiperazine-1-yl) -2, 3-dihydro-quinoline-4 (1H) -ketone shown in a formula III, then the compound and a phenylhydrazine compound are subjected to Fischer indole synthesis to construct an albuterone alkaloid with an indoloquinoline parent nucleus structure characteristic, and the isoalbuterone analogue is prepared through post-treatment;
in the method for preparing the isoquercitine analogue, the molar ratio of the pefloxacin to the reducing agent potassium borohydride is 1.0: 1.0-3.0, and the molar ratio of the 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one to the phenylhydrazine compound is 1.0: 1.0-2.0. As a further improvement, the solvent can be at least one of absolute methanol, absolute ethanol or 95% ethanol, and the absolute methanol is preferred. The phenylhydrazine compounds can be phenylhydrazine, p-methoxyphenylhydrazine, m-methoxyphenylhydrazine, o-methoxyphenylhydrazine, p-methylphenylhydrazine, p-fluorophenylhydrazine, m-fluorophenylhydrazine, p-chlorophenylhydrazine, m-chlorophenylhydrazine, p-sulfonamido phenylhydrazine, etc.
The invention also provides application of the isoalburnine analogue in preparing anti-tuberculosis drugs. Furthermore, the application of the compound can be used for preparing the drug for inhibiting the mycobacterium tuberculosis. The Mycobacterium tuberculosis is H37Ra or H37Rv, and the like.
The preparation of the isoleucamine analogue is prepared by taking the commercially obtained pefloxacin shown in the formula II as a raw material, and the specific preparation steps are as follows:
1) the preparation method of the 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinoline-4 (1H) -ketone shown in the formula III is disclosed in the literature (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, potassium borohydride which is not easy to absorb moisture and is low in price is used for replacing sodium borohydride in the literature, and the preparation operation process comprises the following steps:
suspending 18.0g (55.0mmol) of pefloxacin II in 500mL of solvent anhydrous methanol, gradually adding 7.4g (138.0mmol) of potassium borohydride while stirring at normal temperature, heating the mixed reactant in a water bath, and stirring and refluxing to react until the raw material II disappears (TLC detection). The mixture was left at room temperature, and the solvent was evaporated under reduced pressure by a rotary evaporator. Adding 500mL of deionized water into the residue, adjusting the pH value to be about 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 about 10.0 by using a sodium hydroxide solution with the mass concentration of 30%, and standing to separate out a solid. Recrystallizing the crude product by using boiling water and n-hexane in sequence to obtain the 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinoline-4 (1H) -ketone shown in the formula III, wherein the yield is 64.7 percent and the m.p.113-115 ℃.
2) 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -ketone shown in formula III and an analytically pure chemical reagent phenylhydrazine compound are subjected to a Fischer indole synthesis reaction in absolute ethyl alcohol under the catalysis of acid, and after the reaction is completed, a target compound shown in formula I is obtained by treatment:
wherein, the substituent R in the formula I can be independently hydrogen atom-H, methoxy-OCH3methyl-CH3Fluorine atom-F, chlorine atom-Cl or xanthylamino-SO2NH2。
The general synthetic preparation procedure for the target compound of formula i is: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one represented by the formula III was dissolved in 15mL of anhydrous ethanol, and a phenylhydrazine compound (3.3mmol) was added thereto, and the reaction mixture was stirred at room temperature until the raw material III disappeared (TLC detection). And (3) dropwise adding 0.5mL of cyclization catalyst, carrying out reflux reaction on the mixed reactants for 10-24 h, standing at room temperature, and filtering to collect the generated hydrochloride solid crude product shown in the formula I. Dissolving the solid crude hydrochloride of the formula I in 50mL of deionized water, adding a proper amount of activated carbon, refluxing and decoloring for 1.0h, and adjusting the pH of the filtrate to about 10.0 by using concentrated ammonia water. Filtering and collecting the generated solid, and recrystallizing by using an absolute ethyl alcohol-ethyl acetate mixed solvent to obtain a light yellow crystalline target compound shown as the formula I.
As a further improvement, the amount (mol) ratio of the 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one shown in the formula III to the phenylhydrazine compound substance is 1: 1.0-2.0. The cyclization catalyst is at least one of concentrated hydrochloric acid, concentrated sulfuric acid, phosphoric acid, polyphosphoric acid, glacial acetic acid or trifluoroacetic acid, and preferably concentrated hydrochloric acid is used as the catalyst.
Compared with the prior art, the invention has the following beneficial effects:
the isoleucinine analogue reserves the predominant skeleton of isoleucinine-indoloquinoline mother nucleus, and simultaneously has the characteristic structure of fluoroquinolone medicaments, namely a quinoline ring, particularly as an effective modifying group of the fluoroquinolone medicaments, and the hydrophilic basic piperazinyl of the isoleucinine analogue is used as a modifying group of the indoloquinoline mother nucleus, so that the water solubility of the isoleucinine alkaloid can be effectively improved, the bioavailability is improved, the development of drug properties is facilitated, and the introduction of an F atom can increase the permeation of the medicaments, thereby realizing the effects of enhancing the effect, reducing the toxicity and resisting the drug resistance. The isoalburnine analogue realizes the complementation and activity superposition of pharmacophores with different structures, has excellent in-vitro tubercle bacillus growth inhibition activity, and can be further developed as a novel antitubercular medicament with structural characteristics of indoloquinoline.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
In the following examples, the compound 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one of the formula III used was prepared by the following procedure:
suspending 18.0g (55.0mmol) of pefloxacin II in 500mL of solvent anhydrous methanol, gradually adding 7.4g (138.0mmol) of potassium borohydride while stirring at normal temperature, heating the mixed reactant in a water bath, and stirring and refluxing to react until the raw material II disappears (TLC detection). The mixture was left at room temperature, and the solvent was evaporated under reduced pressure by a rotary evaporator. Adding 500mL of deionized water into the residue, adjusting the pH value to be about 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 about 10.0 by using a sodium hydroxide solution with the mass concentration of 30%, and standing to separate out a solid. Recrystallizing the crude product by using boiling water and n-hexane in sequence to obtain the 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinoline-4 (1H) -ketone shown in the formula III, wherein the yield is 64.7 percent and the m.p.113-115 ℃.1H NMR(400MHz,CD3Cl) δ: 7.54(1H, d,5-H), 6.23(1H, d,8-H), 3.06-3.65 (8H, piperazine-H), 2.42-2.68 (6H, m, 2-H, 3-H and 1-CH)2)，2.28(3H，s，N-CH3)，1.23(3H，t，CH3)；MS(m/z)：292[M+H]+Calculating (C)16H22FN3O)：291.37。
Example 1
2-fluoro-3- (4-methylpiperazin-1-yl) -5-ethyl-5H-indolo [3,2-c ] quinoline (I-1) having the chemical formula:
namely, R in the formula I is an H atom.
The preparation method of the compound (I-1) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of absolute ethanol, 0.50g (4.6mmol) of phenylhydrazine was added thereto, and the reaction was stirred at room temperature for 20 hours (TLC was observed to disappear from the starting material III), whereby a large amount of precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added as a cyclization catalyst, and the reaction mixture was refluxed for 12 hours and allowed to stand overnight (12 hours, the same below). The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an absolute ethanol-ethyl acetate mixed solvent (v: v ═ 5:1) to give a pale yellow crystalline target compound of formula I-1, yield 60.5%, m.p.228 to 230 ℃.1H NMR(400MHz,CD3Cl)δ：1.36(3H,t，CH3)，2.32(3H，s，N-CH3) 3.05 to 3.56(8H, m, piperazine-H), 4.42(2H, q, CH)2) 7.26-8.14 (6H, m, 1-H, 4-H and Ph-H), 8.87(1H, s, 6-H); MS (m/z): 363[ M + H]+Calculating (C)22H23FN4)：362.45。
Example 2
2-fluoro-5-ethyl-8-methoxy-3- (4-methylpiperazin-1-yl) -5H-indolo [3,2-c ] quinoline (I-2) having the chemical formula:
namely, R in the formula I is methoxyl.
The preparation method of the compound (I-2) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of absolute ethanol, 0.62g (4.5mmol) of p-methoxyphenylhydrazine was added thereto, and the reaction was stirred at room temperature overnight (TLC was observed to disappear from the starting material III), and a significant precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ 5:1) to give a pale yellow crystalline target compound of formula I-2, yield 51.4%, m.p.235 to 237 ℃.1H NMR(400MHz,CD3Cl)δ：1.42(3H,t，CH3)，2.34(3H，s，N-CH3) 3.07 to 3.65(8H, m, piperazine-H), 3.87(3H, s, OCH)3)，4.46(2H，q，CH2) 7.52-8.18 (5H, m, 1-H, 4-H and Ph-H), 8.88(1H, s, 6-H); MS (m/z): 393[ M + H ] in]+Calculating (C)23H25FN4O)：392.48。
Example 3
2-fluoro-5-ethyl-3- (4-methylpiperazin-1-yl) -9-methoxy-5H-indolo [3,2-c ] quinoline (I-3) having the chemical formula:
namely, R in the formula I is methoxyl.
The preparation method of the compound (I-3) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one is dissolved in 15mL of absolute ethyl alcohol, 0.83g (6.0mmol) of m-methoxyphenylhydrazine is added, the reaction is stirred at normal temperature for 24H (TLC is used for observing the disappearance of the raw material III), and obvious precipitate is generated. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 12h and allowed to stand overnight. The resulting solid was collected by filtration, dissolved in 50mL of deionized water and addedAppropriate amount of active carbon, and refluxing and decoloring for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an absolute ethanol-ethyl acetate mixed solvent (v: v ═ 6:1) to give a pale yellow crystalline target compound of formula I-3, yield 55.0%, m.p.232 to 234 ℃.1H NMR(400MHz,CD3Cl)δ：1.42(3H,t，CH3)，2.32(3H，s，N-CH3) 3.11 to 3.68(8H, m, piperazine-H), 3.88(3H, s, OCH)3)，4.52(2H，q，CH2) 7.56-8.25 (5H, m, 1-H, 4-H and Ph-H), 8.92(1H, s, 6-H); MS (m/z): 393[ M + H ] in]+Calculating (C)23H25FN4O)：392.48。
Example 4
2-fluoro-5-ethyl-3- (4-methylpiperazin-1-yl) -10-methoxy-5H-indolo [3,2-c ] quinoline (I-4) having the chemical formula:
namely, R in the formula I is methoxyl.
The preparation method of the compound (I-4) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of absolute ethanol, 0.72g (5.0mmol) of o-methoxyphenylhydrazine was added thereto, and the reaction was stirred at room temperature for 24 hours (TLC was observed to disappear from the starting material III), whereby a precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The generated solid is collected by filtration, dried and recrystallized by an absolute ethyl alcohol-ethyl acetate mixed solvent (v: v ═ 3:1), and a light yellow crystal target compound, namely the formula I-4 is obtained, the yield is 44.7%, and m.p.226-228 ℃.1H NMR(400MHz,CD3Cl)δ：1.45(3H,t，CH3)，2.34(3H，s，N-CH3) 3.16 to 3.68(8H, m, piperazine-H), 3.87(3H, s, OCH)3)，4.56(2H，q，CH2) 7.62-8.27 (5H, m, 1-H, 4-H and Ph-H), 8.91(1H, s, 6-H); MS (m/z)：393[M+H]+Calculating (C)23H25FN4O)：392.48。
Example 5
2-fluoro-3- (4-methylpiperazin-1-yl) -5-ethyl-8-methyl-5H-indolo [3,2-c ] quinoline (I-5) having the chemical formula:
namely, R in the formula I is methyl.
The preparation method of the compound (I-5) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one represented by the formula III was dissolved in 15mL of anhydrous ethanol, 0.70g (5.7mmol) of p-tolylhydrazine was added thereto, and the reaction was stirred at room temperature overnight (TLC was observed to cause disappearance of the raw material III), resulting in formation of a precipitate. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 15h and allowed to stand overnight. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ 3:1) to give a pale yellow crystalline target compound of formula I-5, yield 42.6%, m.p.225 to 227 ℃.1H NMR(400MHz,CD3Cl)δ：1.42(3H,t，CH3)，2.25,2.32(6H，s，Ph-CH3And N-CH3) 3.06-3.62 (8H, m, piperazine-H), 4.52(2H, q, CH)2) 7.26-8.02 (5H, m, 1-H, 4-H and Ph-H), 8.86(1H, s, 6-H); MS (m/z): 377[ M + H]+Calculating (C)23H25FN4)：376.48。
Example 6
2, 8-difluoro-3- (4-methylpiperazin-1-yl) -5-ethyl-5H-indolo [3,2-c ] quinoline (I-6) having the chemical formula:
namely, R in the formula I is an F atom.
The preparation method of the compound (I-6) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-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 (TLC was observed to disappear from the starting material III), 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 solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ 7:1) to give a pale yellow crystalline target compound of formula I-6, yield 58.3%, m.p.231 to 233 ℃.1H NMR(400MHz,CD3Cl)δ：1.45(3H,t，CH3)，2.34(3H，s，N-CH3) 3.15 to 3.68(8H, m, piperazine-H), 4.62(2H, q, CH)2) 7.63-8.25 (5H, m, 1-H, 4-H and Ph-H), 8.93(1H, s, 6-H); MS (m/z): 381[ M + H ]]+Calculating (C)22H22F2N4)：380.44。
Example 7
2, 9-difluoro-3- (4-methylpiperazin-1-yl) -5-ethyl-5H-indolo [3,2-c ] quinoline (I-7) having the chemical formula:
the preparation method of the compound (I-7) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, and 0.66g (5.2mmol) of m-fluorophenylhydrazine was added thereto, and the reaction was stirred at room temperature overnight (TLC was observed to disappear from the starting material III), and a significant precipitate was formed. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and dissolved in an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ v)5:1) recrystallizing to obtain a light yellow crystal target compound shown as a formula I-7, wherein the yield is 42.6%, and m.p.230-232 ℃.1H NMR(400MHz,CD3Cl)δ：1.44(3H,t，CH3)，2.36(3H，s，N-CH3) 3.11 to 3.65(8H, m, piperazine-H), 4.58(2H, q, CH)2) 7.57-8.23 (5H, m, 1-H, 4-H and Ph-H), 8.90(1H, s, 6-H); MS (m/z): 381[ M + H ]]+Calculating (C)22H22F2N4)：380.44。
Example 8
2-fluoro-8-chloro-3- (4-methylpiperazin-1-yl) -5-ethyl-5H-indolo [3,2-c ] quinoline (I-8) having the chemical formula:
namely, R in the formula I is chlorine atom.
The preparation method of the compound (I-8) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one was dissolved in 15mL of anhydrous ethanol, 0.74g (5.2mmol) of p-chlorophenylhydrazine was added thereto, and the reaction was stirred at room temperature for 24 hours (TLC was observed to have disappeared the starting material III), 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. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ 5:1) to give a pale yellow crystalline target compound of formula I-8, yield 52.6%, m.p.227 to 229 ℃.1H NMR(400MHz,CD3Cl)δ：1.45(3H,t，CH3)，2.33(3H，s，N-CH3) 3.13 to 3.65(8H, m, piperazine-H), 4.61(2H, q, CH)2) 7.55-8.17 (5H, m, 1-H, 4-H and Ph-H), 8.91(1H, s, 6-H); MS (m/z): 397 (Cl)35)[M+H]+，399(Cl37)[M+H]+Calculating (C)22H22ClFN4)：396.90。
Example 9
2-fluoro-9-chloro-3- (4-methylpiperazin-1-yl) -5-ethyl-5H-indolo [3,2-c ] quinoline (I-9) having the chemical formula:
namely, R in the formula I is chlorine atom.
The preparation method of the compound (I-9) comprises the following steps: 1.0g (3.4mmol) of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one is dissolved in 15mL of absolute ethyl alcohol, 0.70g (5.0mmol) of m-chlorophenylhydrazine is added, and the reaction is stirred at normal temperature for 24 hours (TLC is used for observing the disappearance of the raw material III), so that obvious precipitate is generated. Concentrated hydrochloric acid (0.50mL) was added and the reaction was mixed and refluxed for 16h and allowed to stand overnight. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ 6:1) to give a pale yellow crystalline target compound of formula I-9, yield 45.7%, m.p.223 to 225 ℃.1H NMR(400MHz,CD3Cl)δ：1.46(3H,t，CH3)，2.34(3H，s，N-CH3) 3.15 to 3.67(8H, m, piperazine-H), 4.62(2H, q, CH)2) 7.53-8.26 (5H, m, 1-H, 4-H and Ph-H), 8.90(1H, s, 6-H); MS (m/z): 397 (Cl)35)[M+H]+，399(Cl37)[M+H]+Calculating (C)22H22ClFN4)：396.90。
Example 10
2-fluoro-8-sulfonamido-3- (4-methylpiperazin-1-yl) -5-ethyl-5H-indolo [3,2-c ] quinoline (I-10) having the chemical structure:
namely, R in the formula I is sulfonamide.
The preparation method of the compound (I-10) comprises the following steps: taking 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro1.0g (3.4mmol) of (E) -quinolin-4 (1H) -one was dissolved in 15mL of absolute ethanol, 0.67g (3.6mmol) of p-sulfonamidophenylhydrazide was added thereto, and the reaction was stirred at room temperature overnight (disappearance of starting material III was observed by TLC), and 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. The resulting solid was collected by filtration, dissolved in 50mL of deionized water, added with an appropriate amount of activated carbon, and decolorized under reflux for 1 h. Filtering while the solution is hot, and adjusting the pH of the filtrate to be approximately equal to 10.0 by using ammonia water. The resulting solid was collected by filtration, dried, and recrystallized from an anhydrous ethanol-ethyl acetate mixed solvent (v: v ═ 5:1) to give a pale yellow crystalline target compound of formula I-10, yield 50.2%, m.p.240 to 242 ℃.1H NMR(400MHz,CD3Cl)δ：1.55(3H,t，CH3)，2.38(3H，s，N-CH3) 3.35 to 3.76(8H, m, piperazine-H), 4.66(2H, q, CH)2) 7.26 to 8.52(7H, m, 1-H, 4-H, Ph-H and NH)2)，9.06(1H，s，6-H)；MS(m/z)：442[M+H]+Calculating (C)22H24FN5O2S)：441.53。
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)、H37Rv (ATCC27294) and 3 clinically isolated drug-resistant Mycobacterium tuberculosis (numbered H6, H7 and H10 in the application) are provided by the Henan province disease prevention and control center and provide experimental data for determination. Wherein, H6H7 is a multi-drug resistant strain 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 culture 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 the 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 control 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 andH37the 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 on 3 clinically isolated drug-resistant strains H6, H7 and H10 are 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 1 to 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 10% by mass of RPMI-1640 culture solution of fetal calf serum to have 5 concentration gradients (0.1, 1.0, 5.0, 10.0, 50.0. mu. mol. L)-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–110 μ L of MTT (thiazole blue) solution, and after further culturing for 4 hours, 100 μ L of twelve substances with a concentration of 10% by mass were addedSodium alkyl sulfate (SDS) solution. 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 (IC) of test samples50)
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 isoalburnine analogue is expected to develop an anti-tuberculosis drug with high efficiency and low toxicity.
Claims (6)
1. An isoalburnine analogue, which is a typical compound of the following structure:
2. a method for preparing the isoleucamine analogue of claim 1 by using pefloxacin as a raw material, which is characterized by comprising the following specific steps: pefloxacin shown in a formula II is used as a raw material, and is subjected to reduction deacidification reaction with potassium borohydride to prepare a compound 1-ethyl-6-fluoro-7- (4-methylpiperazine-1-yl) -2, 3-dihydro-quinoline-4 (1H) -ketone shown in a formula III, then the compound and a phenylhydrazine compound are subjected to Fischer indole synthesis to construct an albinine alkaloid with the mother-nucleus structural characteristics of indoloquinoline, and the isoalbinine analogue is prepared through post-treatment;
3. the method for preparing the isoquercitine analogue as claimed in claim 2, wherein the molar ratio of pefloxacin to potassium borohydride is 1.0: 1.0-3.0, and the molar ratio of 1-ethyl-6-fluoro-7- (4-methylpiperazin-1-yl) -2, 3-dihydro-quinolin-4 (1H) -one to phenylhydrazine compound is 1.0: 1.0-2.0.
4. Use of the isoalburnine analogue of claim 1 in the preparation of an anti-tuberculosis medicament.
5. The use of isoalburnine analogues according to claim 4 for the preparation of anti-tubercular drugs, characterized by the use for the preparation of drugs inhibiting mycobacterium tuberculosis.
6. The use of isoalburnine analogues for the preparation of anti-tuberculosis drugs, according to claim 5, wherein said Mycobacterium tuberculosis is H37Ra or H37Rv。
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