CN113527331B - Nitroimidazole derivative and preparation method and application thereof - Google Patents

Nitroimidazole derivative and preparation method and application thereof Download PDF

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CN113527331B
CN113527331B CN202010307399.3A CN202010307399A CN113527331B CN 113527331 B CN113527331 B CN 113527331B CN 202010307399 A CN202010307399 A CN 202010307399A CN 113527331 B CN113527331 B CN 113527331B
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antitubercular
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CN113527331A (en
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赵传生
向军
刘广才
董吉
宋海峰
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Nanjing Changao Pharmaceutical Science and Technology Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/06—Antibacterial agents for tuberculosis

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Abstract

The invention discloses a nitroimidazole derivative, a preparation method and application thereof. The compounds have the following general formula (I):

Description

Nitroimidazole derivative and preparation method and application thereof
Technical Field
The invention belongs to the fields of pharmacology, medicinal chemistry and pharmacology, and more particularly relates to a novel nitroimidazole compound, a preparation method thereof and application of the compound in treating diseases related to infection caused by tubercle bacillus.
Background
Tuberculosis is caused by infection of mycobacterium tuberculosis (Mycobacterium tuberculosis), one of the oldest diseases of human beings, and until now, tuberculosis still seriously endangers human health. According to WHO statistics, about 1/3 of the world's people are infected with tubercle bacillus, which is an infectious disease (higher than HIV) that causes the greatest number of deaths.
According to the report of global tuberculosis in 2019 issued by World Health Organization (WHO), the number of patients is about 1000 ten thousand in 2018, and the number of patients is stable in recent years. Worldwide, 2018 estimated that the number of HIV-negative tuberculosis deaths was about 120 ten thousand, and that of HIV-positive tuberculosis deaths was about 25.1 ten thousand. Meanwhile, about 48.4 thousands of people are newly developed rifampicin-resistant tuberculosis (RR-TB) in 2018 worldwide, and 78% of these people are multi-drug resistant tuberculosis (MDR-TB). Of the MDR-TB patients, 6.2% were estimated to be broadly resistant to tuberculosis (XDR-TB).
At present, four-medicine combined treatment strategies of rifampicin, isoniazid, ethambutol and pyrazinamide are adopted for the first-line treatment of sensitive tuberculosis, and although the treatment success rate can reach more than 85%, the treatment period is as long as 6 months, and the treatment side effects are large, for example, if the rifampicin and isoniazid are combined, serious hepatotoxicity is possibly caused, and the ethambutol can cause optic nerve damage and the like. Some people cannot be treated normally, and some people develop drug-resistant tuberculosis (rifampicin resistance or multi-drug resistance) due to incomplete treatment or improper treatment. For drug-resistant tuberculosis, the treatment period is longer, the treatment side effect is larger, and the treatment success rate is only about 55%. Drug resistant tuberculosis, especially multi-drug resistant tuberculosis and extensively drug resistant tuberculosis, are the leading causes of death in tuberculosis patients, especially in patients with immunodeficiency, such as patients with both aids and tuberculosis.
WO9701562 discloses a number of nitroimidazoles, representing the compound PA-824 (pretomanid), which have a novel mechanism of action and can be used for the treatment of tuberculosis. In month 8 2019, the FDA announced approval of pretomanid, developed by the non-profit organization global tuberculosis drug development consortium (TB Alliance), for the treatment of specific highly resistant Tuberculosis (TB) patients in combination with bedaquiline (bedaquiline) and linezolid (linezolid). However, PA-824 has low bioavailability due to its low water solubility, requires a complex formulation of tablets for oral administration, and requires further improvement of its antitubercular activity [ bioorg. Med. Chem. Lett,2008,18 (7), 2256-2262 ].
OPC-67683 of Katsukamu pharmaceutical Co., ltd (Otsuka Pharmaceutical Co., ltd. [ J.Med. Chem. ], 2006,49 (26), 7854-7860.] has a mechanism of action similar to that of PA-824, and is used for the treatment of tuberculosis. The compound was approved by the European Union in 5 months 2014 for treatment of adult patients with multidrug resistant pulmonary tuberculosis (MDR-TB). Although the compound has strong activity, the compound has the same problems as PA-824, and has small solubility in water, poor plasma stability and limited pharmacokinetic properties, and needs to be taken 2 times per day. Meanwhile, PA-824 and OPC-67683 have strong inhibition activity on hERG potassium current, and have serious cardiotoxicity problems due to side effects of QT-QTc interval prolongation in clinic. Therefore, there is room for further optimization and perfection of the target drug. The target drug is systematically researched, good results are obtained (patent publication number: CN 105732659A), but when a representative compound is subjected to a toxicological experiment, PK-PD research shows that the representative compound has a capping phenomenon, is difficult to further research, and the in-vivo efficacy of the mouse does not exceed PA-824.
PA-824 and OPC-67683 formulas
In view of the above, there is still an urgent need in the art to develop novel antitubercular drugs. The novel drug should have the following characteristics: is effective for drug-resistant bacteria, especially multi-drug resistant bacteria; can be combined with the currently used first-line antitubercular drugs; has ideal metabolic properties, can be orally administered, and can be done once a day; the safety is superior to the existing medicines.
Disclosure of Invention
The invention aims to provide a novel antituberculosis drug which is effective to drug-resistant bacteria, in particular to multi-drug-resistant bacteria; has ideal metabolic properties, can be orally administered, and can be done once a day; the safety is superior to the existing medicines.
In a first aspect of the present invention there is provided an antitubercular compound which is a compound of formula (I) or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:
in formula (I): m, n represents an integer between 0 and 4;
X is oxygen or NH;
R 1 is selected from hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl, which alkyl, cycloalkyl, or cycloalkylalkyl is unsubstituted or optionally substituted with one to three groups independently selected from halogen, alkyl;
R 2 is selected from hydrogen, alkyl, cycloalkyl, alkoxy, alkylthio, cycloalkoxy, halogen, cyano, or nitro, the alkyl, cycloalkyl, alkoxy, alkylthio, or cycloalkoxy being unsubstituted or substituted with one to three groups independently selected from halogen, alkyl, alkoxy.
In another embodiment, R 1 is hydrogen or C 1-4 alkyl, said C 1-4 alkyl being unsubstituted or optionally substituted with one to three halogens.
In another embodiment, R 2 is C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylthio, halogen, or cyano, the C 1-4 alkyl, C 1-4 alkoxy, or C 1-4 alkylthio is unsubstituted or optionally substituted with one to three halogens.
In another aspect, the pharmaceutically acceptable salt comprises: salts of the compounds of formula (I) with acids; wherein the acid comprises: an inorganic acid, an organic acid or an acidic amino acid; the inorganic acid includes: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid; the organic acid includes: formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, p-toluenesulfonic acid, ethanesulfonic acid or benzenesulfonic acid; the acidic amino acids include: aspartic acid or glutamic acid.
In another preferred example, the compound is a compound of formula 1, a compound of formula 2, a compound of formula 3, a compound of formula 4, a compound of formula 5, a compound of formula 6, a compound of formula 7, a compound of formula 8, a compound of formula 9, a compound of formula 10, a compound of formula 11, a compound of formula 12, a compound of formula 13, a compound of formula 14, a compound of formula 15, a compound of formula 16, a compound of formula 17, a compound of formula 18, a compound of formula 19, a compound of formula 20, a compound of formula 21, a compound of formula 22, a compound of formula 23, a compound of formula 24, a compound of formula 25, a compound of formula 26, a compound of formula 27, a compound of formula 28, a compound of formula 29, a compound of formula 30, a compound of formula 31, a compound of formula 32, or a compound of formula 33:
In a second aspect of the present invention there is provided a method of preparing an antitubercular compound as provided by the present invention as described above, the method comprising the steps of:
(1) The compound with the structure shown as the formula I-5 is reduced to obtain the compound with the structure shown as the formula I-6;
(2) The compound with the structure shown as the formula I-6 is chloridized to obtain the compound with the structure shown as the formula I-7; and
(3) Mixing a compound with a structure shown as a formula I-7 with a compound with a structure shown as a formula I-8, and reacting to obtain the compound with the structure shown as the formula I;
m, n, R 1 and R 2 are as defined above; x is oxygen.
The invention also provides a preparation method of the antitubercular compound, which comprises the following steps: mixing a compound with a structure shown as a formula I-5 with a compound with a structure shown as a formula II-1, and reacting in the presence of a reducing agent to obtain a compound with a structure shown as a formula I;
m, n, R 1 and R 2 are as defined above; x is NH.
In another embodiment, the compound of formula I-5 is obtained by the following steps:
(a) Coupling reaction is carried out on the compound with the structure shown as the formula I-3 and tri-n-butyl vinyl tin, so as to obtain the compound with the structure shown as the formula I-4; and
(B) The double bond of the compound with the structure shown as the formula I-4 is oxidized and cut off to obtain the compound with the structure shown as the formula I-5;
。
in a third aspect of the invention there is provided the use of an antitubercular compound as provided above in the manufacture of a medicament for the treatment of a disease associated with infection by tubercle bacillus.
In another preferred example, the antitubercular compound provided by the invention is used for preparing medicines for treating infectious diseases caused by multi-drug resistant tubercle bacillus.
In a fourth aspect of the invention there is provided a pharmaceutical composition for use in the treatment of a disease associated with an infection by tubercle bacillus comprising a therapeutically effective amount of an antitubercular compound provided by the invention as described above and a pharmaceutically acceptable excipient or carrier.
Accordingly, the present invention provides a novel antitubercular drug effective against drug-resistant bacteria, particularly multi-drug-resistant bacteria; can be combined with the currently used first-line antitubercular drugs; has ideal metabolic properties, can be orally administered, and can be done once a day; the safety is superior to the existing medicines.
Detailed Description
The inventor has conducted extensive researches, and has synthesized and screened a large number of compounds, and found that the compound of the formula (I) has unexpected advantages in terms of pharmacokinetics and pharmacodynamics, has strong inhibition activity on tubercle bacillus, and is effective on drug-resistant bacteria, particularly multi-drug-resistant bacteria. The present invention has been completed on the basis of this finding.
The invention provides a compound shown in a structure of a formula (I), or an optical isomer or a pharmaceutically acceptable salt thereof:
In the formula (I), m, n, X, R 1 and R 2 are as defined above.
Some representative compounds of the present invention and their structural formulas are listed in the following table:
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Unless specifically indicated otherwise, the following terms used in the specification and claims have the following meanings:
"alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 6 carbon atoms. Lower alkyl groups having 1 to 4 carbon atoms are preferred, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl.
"Cycloalkyl" means a 3 to 7 membered all-carbon monocyclic aliphatic hydrocarbon group or ring in which one carbon atom is replaced by a heteroatom such as oxygen, sulfur, etc., wherein one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system. For example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexane, cyclohexadiene, and the like.
"Alkoxy" refers to an alkyl group bonded to the remainder of the molecule through an ether oxygen atom. Representative alkoxy groups are those having 1 to 4 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy groups, particularly alkoxy groups substituted with one or more halogens. Preferred alkoxy groups are selected from OCH 3,OCF3,CHF2O,CF3CH2 O, iPrO, nPrO, iBuO, cPrO, nBuO or tBuO.
"Alkylthio" refers to an alkyl group bonded to the remainder of the molecule through a sulfur atom. Representative alkylthio groups are alkylthio groups having 1 to 4 carbon atoms such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio and tert-butylthio. As used herein, "alkylthio" includes unsubstituted and substituted alkylthio, especially alkylthio substituted with one or more halogens. Preferred alkylthio groups are selected from SCH3, SCF3, CHF2S, CF3CH2S, iPrS, nPrS, iBuS, cPrS, nBuS or tBuS.
"Halogen" means fluorine, chlorine, bromine or iodine.
"Chemical bond" refers to the collective term for strong interaction forces between two or more adjacent atoms (or ions) within a pure molecule or within a crystal.
"Optionally substituted" or "substituted" means that the reference group may be substituted with one or more additional groups independently and independently selected from alkyl, alkoxy, and halogen.
"An integer between 0 and 4" means 0,1,2,3,4; "an integer between 1 and 4" means 1,2,3,4.
The compounds of the invention contain at least 1 asymmetric center and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures, as well as pure or partially pure compounds, are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.
As used herein, "pharmaceutically acceptable salts" means salts which are not particularly limited as long as they are pharmaceutically acceptable, and include inorganic salts and organic salts. Specifically, salts of the compounds of the present invention with acids may be mentioned, and suitable salts-forming acids include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, phosphoric acid, and the like, organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like, and acidic amino acids such as aspartic acid, glutamic acid.
The invention also provides a preparation method of the novel antituberculosis compound or the pharmaceutically acceptable inorganic or organic salt thereof.
The following specifically describes the preparation method of the compound of the general formula (I) of the present invention, but these specific methods do not limit the present invention in any way.
The structural compound of the general formula (I) of the present invention can be produced by a method, however, the conditions of the method, such as reactants, solvents, bases, amounts of compounds used, reaction temperature, time required for the reaction, etc., are not limited to the following explanation. The compounds of the present invention may also optionally be conveniently prepared by combining the various synthetic methods described in this specification or known in the art, such combination being readily apparent to those skilled in the art to which the present invention pertains.
The preparation method of the antituberculous antibacterial compound of the invention comprises the following steps:
Scheme 1: flow when x=o
Firstly, reducing a compound with a structure shown as a formula I-5 to obtain a compound with a structure shown as a formula I-6;
secondly, chloridizing the compound with the structure shown as the formula I-6 to obtain the compound with the structure shown as the formula I-7;
And thirdly, mixing a compound with a structure shown as a formula I-7 with a compound with a structure shown as a formula I-8, and reacting to obtain the compound with the structure shown as the formula I.
The reduction reaction of the first step may be carried out in a suitable solvent, optionally with the use of a suitable reducing agent; such solvents include, but are not limited to, alcohols such as methanol, ethanol, ethers such as tetrahydrofuran, and the like; the reducing agents used include, but are not limited to, sodium borohydride, potassium borohydride, lithium borohydride, and lithium aluminum hydride.
The chlorination reaction in the second step can be optionally performed in a suitable solvent, and a suitable chlorinating agent is used; the solvent includes, but is not limited to, methylene chloride, chloroform, toluene; the chlorinating agent used includes, but is not limited to, thionyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride.
In one embodiment of the present invention, the compound of the structure shown in the formula I-8 in the third step is mixed with a proper solvent, then placed at a low temperature (for example, -20-0 ℃), added with strong base to react for a period of time (for example, 0.5-2 h), and then added with the compound of the structure shown in the formula I-7 to react continuously; the suitable solvent is selected from Dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), tetrahydrofuran; the strong base includes, but is not limited to, sodium hydride, potassium hydride, sodium t-butoxide, potassium t-butoxide.
In one embodiment of the present invention, after the reaction in the third step is completed, the compound having the structure shown in formula i is obtained by separation through conventional treatment; such conventional treatments include, but are not limited to, extraction, washing, drying, concentration, chromatography, and the like.
In one embodiment of the present invention, a compound having the structure of formula I-5 may be obtained from a compound having the structure of formula I-3, and the process may include the steps of:
Firstly, carrying out coupling reaction on a compound with a structure shown as a formula I-3 and tri-n-butyl vinyl tin to obtain a compound with a structure shown as a formula I-4; and
And secondly, oxidizing and cutting the double bond of the compound with the structure shown in the formula I-4 to obtain the compound with the structure shown in the formula I-5.
In one embodiment of the present invention, the catalyst for the coupling reaction of the first step includes, but is not limited to, pd (PPh 3)4、Pd(dppf)2Cl2、Pd2(dba)3), the reaction solvent for the coupling reaction may include, but is not limited to, dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), and the coupling reaction may be carried out at a temperature of 50-120 ℃.
In one embodiment of the present invention, the oxidizing agents that may be used for the oxidative cleavage in the second step include, but are not limited to, sodium periodate, potassium periodate; examples of catalysts include, but are not limited to, potassium osmium, ruthenium trichloride; suitable solvents for the oxidative cleavage include, but are not limited to, mixed solvents of dioxane and water, and suitable temperatures may be in the range of 0-50 ℃.
In one embodiment of the present invention, the process 1 may include the steps of:
Firstly, carrying out coupling reaction on a compound with a structure shown as a formula I-3 and tri-n-butyl vinyl tin to obtain a compound with a structure shown as a formula I-4;
secondly, oxidizing and cutting the double bond of the compound with the structure shown as the formula I-4 to obtain the compound with the structure shown as the formula I-5;
Thirdly, reducing the compound with the structure shown as the formula I-5 to obtain the compound with the structure shown as the formula I-6;
fourthly, chloridizing the compound with the structure shown as the formula I-6 to obtain the compound with the structure shown as the formula I-7;
And fifthly, mixing the compound with the structure shown as the formula I-7 with the compound with the structure shown as the formula I-8, and reacting to obtain the compound with the structure shown as the formula I.
The invention takes 5-bromo-2-chloropyrimidine as a starting material, and can obtain the compound with the structure shown as the formula I-3 in the first step through two ways:
One approach is to dissolve 5-bromo-2-chloropyrimidine and primary amine compounds in solvents (such as, but not limited to, N-butanol, N-propanol, isobutanol), take appropriate bases (such as, but not limited to, N, N-Diisopropylethylamine (DIPEA), triethylamine) as acid-binding agents, and carry out substitution reaction at a certain temperature (such as 80-120 ℃) to obtain compounds with the structure shown as formula I-1; the compound with the structure shown in the formula I-1 is firstly mixed with a proper solvent and then placed at a low temperature (such as-20-0 ℃), then strong alkali is added for reaction for a period of time (such as 0.5-2 h), and then iodides or bromides are added for continuous reaction to obtain the compound with the structure shown in the formula I-3; the suitable solvent is selected from Dimethylformamide (DMF), dimethylacetamide (DMAc), N-methylpyrrolidone (NMP); the strong base includes, but is not limited to, sodium hydride, potassium hydride, LDA, naHMDS, liHMDS.
Another way is to dissolve 5-bromo-2-chloropyrimidine and primary amine compound in solvent (such as but not limited to ethanol, isopropanol, n-butanol, isobutanol) and carry out substitution reaction at a certain temperature (such as 80-100deg.C) to obtain compound with structure shown as formula I-2; the compound with the structure shown in the formula I-2 and halogenated aryl are subjected to coupling reaction under the condition that copper (such as but not limited to cuprous iodide and cuprous bromide) is used as a catalyst and amino acid (such as but not limited to L-proline, dimethyl ethylenediamine and N, N' -dimethyl-1, 2-cyclohexanediamine) is used as a ligand, so that the compound with the structure shown in the formula I-3 can be obtained.
In one embodiment of the present invention, the process 1 can be performed according to the following reaction scheme and the description thereof:
R 1、R2, m, n are as defined for formula (I) herein, wherein R 1 is not equal to hydrogen.
(1) Dissolving a starting material of 5-bromo-2-chloropyrimidine and a primary amine compound in a solvent (such as n-butanol), taking a proper base (such as DIPEA) as an acid binding agent, and carrying out substitution reaction at a certain temperature (such as 80-120 ℃) to obtain an intermediate I-1;
(2) Adding strong base (such as sodium hydride) into intermediate I-1 in proper solvent (such as DMF or DMAc) at low temperature (-20-0deg.C) for reacting for a certain time (such as 0.5-2 h), adding iodo compound or bromo compound, and separating to obtain intermediate I-3;
(3) The starting materials of 5-bromo-2-chloropyrimidine and primary amine compounds are dissolved in a solvent (such as ethanol) and substitution reaction is carried out at a certain temperature (such as 80-100 ℃) to obtain an intermediate I-2. Intermediate I-3 can also be obtained by coupling reaction of intermediate I-2 and halogenated aryl under the condition that copper (such as cuprous iodide) is used as a catalyst and amino acid (such as L-proline) is used as a ligand;
(4) The intermediate I-3 and tri-n-butyl vinyl tin are subjected to coupling reaction under the condition of palladium catalyst (such as Pd (PPh 3)4) and at a proper temperature (such as 50-120 ℃), and conventional column chromatography separation is finished to obtain an intermediate I-4;
(5) The double bond of the intermediate I-4 is oxidized and cut off under the condition that sodium periodate is used as an oxidant and potassium osmium is used as a catalyst and under the condition that a proper solvent (for example, a mixed solvent of dioxane and water) and a proper temperature (for example, 0-50 ℃) are adopted to obtain an aldehyde intermediate I-5;
(6) Intermediate I-5 is reduced in a suitable solvent (e.g., methanol) with a suitable reducing agent (e.g., sodium borohydride) to provide alcohol intermediate I-6;
(7) The intermediate I-6 is chloridized in a proper solvent (such as methylene dichloride) by a proper chloridizing reagent (such as thionyl chloride) to obtain a chloridized intermediate I-7;
(8) Intermediate I-8 is reacted in a suitable solvent (e.g., DMF or DMAc) at low temperature (-20-0deg.C) with the addition of a strong base (e.g., sodium hydride) for a period of time (e.g., 0.5-2 h), followed by the addition of chlorinated intermediate I-7 to continue the reaction. After the reaction is finished, the target compound (I) is obtained through conventional post-treatment separation.
Scheme 2: scheme when x=nh
The compound with the structure shown in the formula I-5 and the compound with the structure shown in the formula II-1 are mixed and reacted in the presence of a reducing agent to obtain the compound with the structure shown in the formula I.
In one embodiment of the invention, the compound with the structure shown in the formula I-5 and the compound with the structure shown in the formula II-1 are reacted for 2-20 hours in the presence of organic base to obtain intermediate imine, and then a reducing agent is added for 1-22 hours to obtain the compound with the structure shown in the formula I. Such organic bases include, but are not limited to, triethylamine, N-Diisopropylethylamine (DIPEA); the reaction solvent for obtaining the intermediate imine comprises, but is not limited to, dichloromethane and dichloroethane; the reducing agent includes, but is not limited to, sodium triacetoxyborohydride, sodium borohydride, sodium cyanoborohydride.
The compound with the structure shown as the formula I-5 can be obtained by the method as described above.
In one embodiment of the present invention, the process 2 can be performed according to the following reaction scheme and the description thereof:
R 1、R2, m, n are as defined for formula (I) herein, wherein R 1 is not equal to hydrogen.
Intermediate I-5 and II-1 are reacted in a solvent (e.g., methylene chloride) in the presence of an organic base (e.g., triethylamine) for a period of time (e.g., 2-20 hours) to produce an intermediate imine, and then a reducing agent (e.g., sodium triacetoxyborohydride) is added to react for a suitable period of time (e.g., 1-22 hours) to give the imine-reduced target compound (I).
In one embodiment of the present invention, there is also provided scheme 3, which proceeds according to the following reaction scheme and its associated description:
(1) Intermediate I-1 is used as a raw material, and intermediate III-1 is synthesized according to the method of document WO 2017/176817.
(2) Intermediate III-1 is reduced in a suitable solvent (e.g. methanol) by a suitable reducing agent (e.g. sodium borohydride) to obtain an alcohol intermediate III-2;
(3) The intermediate III-2 is chloridized in a proper solvent (such as methylene dichloride) by a proper chloridizing reagent (such as thionyl chloride) to obtain a chloridized intermediate III-3;
(4) Intermediate I-8 is reacted in a suitable solvent (e.g., DMF or DMAc) at low temperature (-20-0deg.C) with the addition of a strong base (e.g., sodium hydride) for a period of time (e.g., 0.5-2 h), followed by the addition of chlorinated intermediate III-3 to continue the reaction. After the reaction, the target compound 33 was isolated by a conventional work-up.
The structural general formula involved in the preparation method provided by the invention is shown in the following table:
Wherein m, n, R 1 and R 2 are as defined above.
The invention also provides application of the novel antituberculosis compound, or optical isomer or pharmaceutically acceptable salt thereof in treating diseases related to infection caused by tubercle bacillus.
The compound shown in the general formula (I) has strong anti-mycobacterium tuberculosis effect, and particularly has excellent effect on multi-drug resistant and widely drug resistant mycobacterium tuberculosis.
The compounds of the general formula (I) have better in vitro and in vivo activity, no inhibition on hERG potassium current and better pharmacokinetics property. The compound has important significance for improving the activity of anti-mycobacterium tuberculosis, improving the drug effect, reducing side effects and saving the cost.
In the present invention, the "active ingredient" refers to a compound represented by the general formula (I), and a pharmaceutically acceptable inorganic or organic salt of the compound of the general formula (I). The compounds of the invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures, as well as pure or partially pure compounds, are included within the scope of the invention. The present invention is meant to include all such isomeric forms of these compounds.
In addition, if desired, some of the compounds of the present invention may be prepared by reacting a portion of the compounds with a pharmaceutically acceptable acid in a polar protic solvent, such as methanol, ethanol, isopropanol, to form a pharmaceutically acceptable salt. The pharmaceutically acceptable inorganic or organic acid may be: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid, and the like.
The term "caused by tubercle bacillus", as used herein, refers to caused by tubercle bacillus sensitive to clinical tubercle drugs, tubercle bacillus resistant to clinical drugs and tubercle bacillus resistant to wide spread drugs.
The terms "disease caused by infection with tubercle bacillus" or "tubercle bacillus infectious disease" are used interchangeably, as used herein, all refer to tuberculosis, lymphoid tuberculosis, intestinal tuberculosis, bone tuberculosis, tuberculous pleurisy, tuberculous meningitis, etc.
Because the compound has excellent anti-tubercle bacillus activity, the compound and pharmaceutically acceptable inorganic or organic salts and the pharmaceutical composition containing the compound as main active ingredients can be used for treating diseases related to tubercle bacillus. According to the prior art, the compounds of the present invention are useful in the treatment of tuberculosis and other infectious diseases.
The invention also provides a pharmaceutical composition for treating diseases related to infection caused by tubercle bacillus, which comprises the nitroimidazole compound with effective treatment dose and a pharmaceutically acceptable excipient or carrier.
The pharmaceutical composition of the invention comprises the nitroimidazole compound of the invention in a safe and effective amount range and pharmaceutically acceptable excipients or carriers. Wherein "safe, effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-1000mg of the compound/dose of the invention, preferably 5-500mg of the compound/dose of the invention, more preferably 10-200mg of the compound/dose of the invention.
The compounds of the present invention and pharmaceutically acceptable salts thereof can be formulated into a variety of formulations comprising a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe, effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the specific conditions such as age, illness and treatment course of the subject.
"Pharmaceutically acceptable excipient or carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable excipients or carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulphate, vegetable oils (e.g. soya oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiersWetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water, and the like.
The compounds of the present invention may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.
In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.
The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
When a pharmaceutical composition is used, a safe and effective amount of a compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective dosage, and for a human having a body weight of 60kg, the daily dosage is usually 1 to 1000mg, preferably 10 to 500mg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this specification may be used in combination with any combination of features, provided that the combination of features is not inconsistent and all such combinations are contemplated as falling within the scope of the present specification. The various features disclosed in the specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.
The main advantages of the invention include:
1. The compound has specific effect on mycobacterium tuberculosis and excellent effect on multidrug-resistant and widely drug-resistant mycobacterium tuberculosis.
2. The compounds of the present invention have good pharmacokinetic properties. The compound has important significance for improving the activity of anti-mycobacterium tuberculosis, improving the drug effect, reducing side effects and saving the cost.
3. The compound has no inhibition on hERG potassium current, and has better safety on the cardiovascular system.
4. The compound has excellent bactericidal effect in mice, and remarkably reduces colony count of lungs of infected mice.
The details of the various specific aspects, features and advantages of the above-described compounds, methods, pharmaceutical compositions will be set forth in the following description in order to provide a thorough understanding of the present application. It is to be understood that the detailed description and examples, which follow, describe specific embodiments for reference only. Various changes and modifications to the present application will become apparent to those skilled in the art upon reading the description of the application, and such equivalents are intended to fall within the scope of the application.
The present invention is more specifically explained in the following examples. It should be understood, however, that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Parts and percentages are parts by weight and percentages by weight unless otherwise indicated.
1 H-NMR was recorded on a Varian Mercury 400M or 600M NMR apparatus in all examples, chemical shifts were expressed as delta (ppm); measurement of Ms was performed using a Shimadzu LC-Ms-2020 mass spectrometer. The silica gel for separation is not illustrated as 200-300 meshes, and the ratio of the eluents is volume ratio.
Example 1: (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 1)
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(1) 4-Trifluoromethoxyaniline (26.7 g,0.15 mol), 5-bromo-2-chloropyrimidine (19.34 g,0.1 mmol), DIPEA (25.85 g,0.2 mol) were dissolved in n-butanol (180 mL) and stirred overnight at 120 ℃. TLC (Petroleum ether: ethyl acetate=10:1), after the reaction, the reaction solution was dried by spin-drying, a small amount of methylene chloride was added to dissolve the concentrate, petroleum ether was added dropwise, and the solid was precipitated and filtered to give pale yellow solid 1-1 (13.85 g, yield: 42%).
1H-NMR(400MHz,CDCl3)δ8.44(s,2H),7.66-7.56(m,2H),7.37(s,1H),7.24-7.15(m,2H).ESI-Ms:233.9[M+1]+.
(2) 1-1 (6.68 G,20 mmol) was dissolved in dry DMF (70 mL), naH (1.2 g,30 mmol) was added thereto under argon protection in ice bath, stirred for 30min under ice bath, then methyl iodide (5.68 g,40 mmol) was added thereto, slowly warmed to room temperature and stirred overnight for reaction. LC-Ms monitoring, reaction is completed. Ethyl acetate, water, liquid separation, extraction with ethyl acetate, washing the combined organic phases with saturated brine, drying, and concentration were added to the system, followed by column chromatography (petroleum ether: ethyl acetate=20:1) to give 1-3 (5.46 g, yield: 78.5%) as pale yellow solid.
1H-NMR(400MHz,CDCl3)δ8.33(s,2H),7.36-7.30(m,2H),7.26-7.23(m,2H),3.50(s,3H).ESI-Ms:348.0[M+1]+.
(3) 1-3 (5.22 G,15 mmol) was dissolved in dry DMF (60 mL), tributylvinyltin (7.13 g,22.5 mmol) was added and Pd (PPh 3)4 (0.87 g,0.75 mmol) was protected with argon and reacted overnight at 120℃LC-MS monitoring, reaction was complete.
1H-NMR(400MHz,CDCl3)δ8.39(s,2H),7.36-7.30(m,2H),7.26-7.22(m,2H),6.50(dd,J＝17.2Hz,0.4Hz,1H),5.61(dd,J＝17.2Hz,0.4Hz,1H),5.17(dd,J＝11Hz,0.4Hz,1H),3.53(s,3H).ESI-Ms:296.1[M+1]+.
(4) 1-4 (2.36 G,8.0 mmol) was dissolved in a mixed solution of dioxane/water (30 mL, 1:1), sodium periodate (6.82 g,32 mmol) was added, and K 2OsO4.2H2 O (29 mg,0.08 mmol) was reacted at room temperature. LC-MS monitoring, reaction was complete. Water was added to the system, extraction was performed with ethyl acetate, and the combined organic phases were washed with dilute hydrochloric acid, saturated aqueous sodium bicarbonate, saturated brine in this order, dried, and concentrated to give 1-5 (2.80 g) as a white solid, which was directly fed to the next step without purification. 298.1[ M+1] +.
(5) 1-5 (2.80 G) was dissolved in methanol (30 mL), naBH 4 (0.45 g,12 mmol) was added thereto under an ice bath, and then slowly warmed to post room temperature and reacted for 2 hours. LC-MS monitoring, reaction was complete. Water was added to the system to quench, concentrate, and column chromatography (petroleum ether: ethyl acetate=3:1) gave 1-6 (1.48 g, yield: 62.1%) as a white solid.
1H-NMR(400MHz,CDCl3)δ8.25(s,2H),7.35-7.29(m,2H),7.25-7.22(m,2H),4.48(s,2H),3.49(s,3H).ESI-Ms:300.1[M+1]+.
(6) 1-6 (598 Mg,2.0 mmol) was dissolved in methylene chloride (10 mL), to which was added 5mL of thionyl chloride (5 mL), and reacted at room temperature. LC-MS monitoring, after the reaction was complete, concentrating, adding dichloromethane, steaming SOCl 2 twice, obtaining white solid 1-7 (623 mg) directly to the next step. ESI-Ms 318.1[ M+1] +.
(7) Intermediate I-8 (400 mg,2.14 mmol) was added to dry DMF (5 mL), naH (340 mg,8.5 mmol) was added thereto under ice-bath and argon protection, and the reaction was continued for half an hour under ice-bath, and a solution of intermediate 1-7 (623 mg) in DMF (5 mL) was added, and the reaction was continued for 1 hour after the ice-bath was warmed to room temperature. LC-MS monitored completion of the reaction, ethyl acetate, water, liquid separation, extraction, combined organic phases, washing with saturated brine, drying, concentration, column chromatography (dichloromethane: methanol=20:1) gave compound 1 (191 mg, yield: 20.5%) as a pale yellow solid.
1H NMR(400MHz,CDCl3)δ8.28(s,2H),7.38(s,1H),7.34–7.29(m,2H),7.24-7.21(m,2H),4.63-4.59(m,1H),4.56(d,J＝11.5Hz,1H),4.44(d,J＝11.4Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.6Hz,1H),4.12-4.07(m,2H),3.51(s,3H).ESI-Ms:467.1[M+1]+.
Example 2: (S) -N-ethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 2)
Compound 2 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.20(s,2H),7.33(s,1H),7.19(s,4H),4.55(d,J＝11.8Hz,1H),4.49(d,J＝11.6Hz,1H),4.38(d,J＝11.3Hz,1H),4.29(d,J＝12.2Hz,1H),4.15(dd,J＝13.6,4.8Hz,1H),4.08-4.03(t,J＝8.5Hz,2H),3.95(q,J＝6.9Hz,2H),1.15(t,J＝6.9Hz,3H).ESI-Ms:481.1[M+1]+.
Example 3: (S) -N-propyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 3)
Compound 3 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.19(s,2H),7.33(s,1H),7.21 -7.17(m,4H),4.60–4.51(m,1H),4.48(d,J＝11.4Hz,1H),4.37(d,J＝11.4Hz,1H),4.30(d,J＝12.3Hz,1H),4.15(dd,J＝13.4,4.6Hz,1H),4.08-4.01(m,2H),3.87–3.80(m,2H),1.61-1.56(m,2H),0.85(t,J＝7.4Hz,3H).ESI-Ms:494.2[M+1]+.
Example 4: (S) -N-isopropyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 4)
Compound 4 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.17(s,2H),7.32(s,1H),7.22(d,J＝8.7Hz,2H),7.07(d,J＝8.7Hz,2H),5.12-5.05(m,1H),4.53(d,J＝12.1Hz,1H),4.46(d,J＝11.5Hz,1H),4.36(d,J＝11.5Hz,1H),4.29(d,J＝12.0Hz,1H),4.14(dd,J＝13.5,4.4Hz,1H),4.04(d,J＝10.9Hz,2H),1.08(d,J＝6.7Hz,6H).ESI-Ms:494.2[M+1]+.
Example 5: (S) -N-trifluoroethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 5)
Compound 5 was prepared by the same method as in example 1.
1H NMR(600MHz,CDCl3)δ8.25(s,2H),7.33(s,1H),7.23(d,J＝6.1Hz,4H),4.66-4.60(m,2H),4.57(d,J＝11.6Hz,1H),4.53(d,J＝10.9Hz,1H),4.42(d,J＝10.8Hz,1H),4.31(d,J＝11.6Hz,1H),4.17(d,J＝10.3Hz,1H),4.09(s,2H).ESI-Ms:535.1[M+1]+.
Example 6: (S) -N-cyclopropylmethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 6)
Compound 6 was prepared by the same method as in example 1.
1H NMR(600MHz,CDCl3)δ8.19(s,2H),7.33(s,1H),7.24(d,J＝8.8Hz,2H),7.19(d,J＝8.7Hz,2H),4.57–4.52(m,1H),4.48(d,J＝11.5Hz,1H),4.37(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.15(dd,J＝13.5,4.6Hz,1H),4.05(d,J＝12.4Hz,2H),3.77(d,J＝6.9Hz,2H),1.08–1.03(m,1H),0.39-0.34(m,2H),0.10-0.06(m,2H).ESI-Ms:507.2[M+1]+.
Example 7: (S) -N-cyclopentylmethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 7)
Compound 7 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.20(s,2H),7.35(s,1H),7.24(d,J＝8.8Hz,2H),7.19(d,J＝8.7Hz,2H),4.56–4.52(m,1H),4.45(d,J＝11.8Hz,1H),4.32(d,J＝11.6Hz,1H),4.29(d,J＝12.0Hz,1H),4.13(dd,J＝13.4,4.4Hz,1H),4.06(d,J＝12.2Hz,2H),3.75(d,J＝6.9Hz,2H),1.88-1.72(m,2H),1.52-1.40(m,7H).ESI-Ms:535.2[M+1]+.
Example 8: (S) -N- ((4, 4-Difluoromethylcyclohexyl) methyl) -5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 8)
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Compound 8 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.18(s,2H),7.20(s,1H),7.22(d,J＝8.7Hz,,2H),718(d,J＝8.8Hz,2H),4.58-4.49(m,1H),4.48(d,J＝12.0Hz,1H),4.34(d,J＝12.2Hz,1H),4.0(d,J＝12.3Hz,1H),4.18-4.14(m,1H),4.08(d,J＝12.0Hz,2H),3.65(d,J＝7.0Hz,2H),1.68-1.34(m,8H),1.42-1.37(m,1H).ESI-Ms:585.2[M+1]+.
Example 9: (S) -N-cyclopropyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 9)
(1) Cyclopropylamine (11.1 g,0.195 mol), 5-bromo-2-chloropyrimidine (12.5 g,65 mmol) was dissolved in ethanol (130 mL) and stirred at 80℃for 3h. After cooling, the solid was precipitated and filtered to give 9-2 (5.67 g, yield: 68%) as a white solid, which was directly carried forward without further purification. ESI-Ms 213.9[ M+1] +.
(2) Intermediate 9-2 (4.27 g,0.02 mol), cuI (762 mg,0.04 mol), cs 2CO3 (16.3 g,0.05 mol), L-proline (920 mg,0.008 mol), p-trifluoromethoxy iodobenzene (12.6 g,0.045 mol) were dissolved in DMSO (40 ml), and reacted overnight at 120℃under argon. LC-MS monitoring and finishing the reaction. Ethyl acetate, water, liquid separation, extraction, combination of organic phases, washing with saturated brine, and column chromatography (petroleum ether: ethyl acetate=4:1) was concentrated to give 9-3 (1.5 g, yield 20%) as pale yellow solid.
1H-NMR(400MHz,CDCl3)δ8.32(s,2H),7.34-7.29(m,2H),7.24-7.20(m,2H),3.10-3.05(m,1H),1.06-0.93(m,2H),0.60-0.51(m,2H).ESI-Ms:374.0[M+1]+.
(3) Compound 9 was subsequently produced using intermediate 9-3 as a starting material in the same manner as in example 1.
1H-NMR(400MHz,CDCl3)δ8.32(s,2H),7.38(s,1H),7.24–7.19(m,4H),4.62-4.58(m,1H),4.56(d,J＝11.7Hz,1H),4.46(d,J＝11.6Hz,1H),4.35(d,J＝12.3Hz,1H),4.21(dd,J＝13.4,4.5Hz,1H),4.15–4.08(m,2H),3.17–3.10(m,1H),0.96-0.91(m,2H),0.55–0.47(m,2H).ESI-Ms:492.1[M+1]+.
Example 10: (S) -N-cyclopentyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 10)
Compound 10 was prepared by the same method as in example 9.
Compounds of formula (I) 10：1H-NMR(400MHz,CDCl3)δ8.23(s,2H),7.35(s,1H),7.23(m,-7.18(m,4H),4.60-4.56(m,1H),4.55(d,J＝11.8Hz,1H),4.42(d,J＝12.0Hz,1H),4.30(d,J＝12.1Hz,1H),4.19(dd,J＝13.1,4.7Hz,1H),4.14-4.06(m,2H),3.09-3.04(m,1H),1.82-1.52(m,8H).ESI-Ms:521.2[M+1]+.
Example 11: (S) -N- (3-chloro-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 11)
Compound 11 was produced by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.30(s,2H),7.44(d,J＝2.5Hz,1H),7.39(s,1H),7.32(dd,J＝8.9,1.2Hz,1H),7.25-7.22(m,1H),4.65–4.59(m,1H),4.57(d,J＝11.5Hz,1H),4.46(d,J＝11.5Hz,1H),4.35(d,J＝12.3Hz,1H),4.20(dd,J＝13.3,4.6Hz,1H),4.14–4.08(m,2H),3.51(s,3H).ESI-Ms:501.1[M+1]+.
Example 12: (S) -N- (3-bromo-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 12)
Compound 12 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.30(s,2H),7.59(d,J＝2.1Hz,1H),7.39(s,1H),7.34–7.26(m,2H),4.64–4.59(m,1H),4.57(d,J＝11.4Hz,1H),4.46(d,J＝11.4Hz,1H),4.35(d,J＝12.2Hz,1H),4.21(dd,J＝13.3,4.6Hz,1H),4.15–4.07(m,2H),3.51(s,3H).ESI-Ms:544.0[M+1]+.
Example 13: (S) -4- (methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine) benzonitrile (compound 13)
Compound 13 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.28(s,2H),7.46(d,J＝8.8Hz,2H),7.40(d,J＝8.8Hz,2H),7.38(s,1H),4.64-4.58(m,1H),4.57(d,J＝11.6Hz,1H),4.45(d,J＝11.6Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.6Hz,1H),4.12-4.08(m,2H),3.52(s,3H).ESI-Ms:408.1[M+1]+.
Example 14: (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (3, 4, 5-trifluorophenyl) pyrimidin-2-amine (compound 14)
Compound 14 was produced in the same manner as in example 1.
1H-NMR(400MHz,CDCl3)δ8.28(s,2H),7.38(s,1H),7.23-7.17(m,2H),4.60-4.57(m,1H),4.56(d,J＝11.4Hz,1H),4.43(d,J＝11.4Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.6Hz,1H),4.12-4.06(m,2H),3.50(s,3H).ESI-Ms:437.1[M+1]+.
Example 15: (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) benzyl) pyrimidin-2-amine (compound 15)
Compound 15 was produced in the same manner as in example 1.
1H NMR(600MHz,CDCl3)δ8.22(s,2H),7.34(s,1H),7.19(J＝8.2Hz,2H),7.08(d,J＝8.2Hz,2H),4.82(s,2H),4.58–4.53(m,1H),4.49(d,J＝11.4Hz,1H),4.38(d,J＝11.4Hz,1H),4.31(d,J＝12.2Hz,1H),4.16(dd,J＝13.5,4.6Hz,1H),4.12-4.07(m,2H),3.07(s,3H).ESI-Ms:481.1[M+1]+.
EXAMPLE 16 (S) -N-cyclopropyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) benzyl) pyrimidin-2-amine (compound 16)
Compound 16 was prepared by the same method as in example 1.
1H NMR(600MHz,CDCl3)δ8.27(s,2H),7.34(s,1H),7.16(d,J＝8.6Hz,2H),7.05(d,J＝8.2Hz,2H),4.83(s,2H),4.59–4.54(m,1H),4.51(d,J＝11.4Hz,1H),4.39(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.15(dd,J＝13.5,4.7Hz,1H),4.10–4.04(m,2H),2.73–2.67(m,1H),0.82 -0.79(m,2H),0.63–0.58(m,2H).ESI-Ms:507.2[M+1]+.
Example 17 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenethyl) pyrimidin-2-amine (compound 17)
Compound 17 was prepared by the same method as in example 1.
1H NMR(600MHz,CDCl3)δ8.20(s,2H),7.34(s,1H),7.18(J＝8.6Hz,2H),7.06(d,J＝8.6Hz,2H),4.57–4.53(m,1H),4.48(d,J＝11.4Hz,1H),4.36(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.15(dd,J＝13.5,4.6Hz,1H),4.03-4.07(m,2H),3.52(t,J＝7.2Hz,2H),3.05(s,3H),2.82(t,J＝7.2Hz,2H).ESI-Ms:495.2[M+1]+.
Example 18 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (3- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 18)
Compound 18 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.30(s,2H),7.42–7.36(m,2H),7.25(d,J＝8.0Hz,1H),7.19(s,1H),7.07(d,J＝8.2Hz,1H),4.63–4.59(m,1H),4.57(d,J＝11.5Hz,1H),4.45(d,J＝11.4Hz,1H),4.35(d,J＝12.1Hz,1H),4.20(dd,J＝13.5,4.6Hz,1H),4.14–4.07(m,2H),3.53(d,J＝0.4Hz,3H).ESI-Ms:467.2[M+1]+.
Example 19 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (2- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 19)
Compound 19 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.27(s,2H),7.38(s,1H),7.34(s,4H),4.60–4.52(m,2H),4.44(d,J＝11.6Hz,1H),4.33(d,J＝12.1Hz,1H),4.18(dd,J＝13.4,4.5Hz,1H),4.13–4.05(m,2H),3.44(s,3H).ESI-Ms:467.2[M+1]+.
EXAMPLE 20((S) -N-ethyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (3- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 20)
Compound 20 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.29(s,2H),7.42–7.36(m,2H),7.26(d,J＝8.0Hz,1H),7.19(s,1H),7.06(d,J＝8.2Hz,1H),4.64–4.59(m,1H),4.57(d,J＝11.5Hz,1H),4.45(d,J＝11.4Hz,1H),4.36(d,J＝12.1Hz,1H),4.21(dd,J＝13.5,4.6Hz,1H),4.14–4.07(m,2H),3.93(q,J＝6.9Hz,2H),1.13(t,J＝6.9Hz,3H).ESI-Ms:481.1[M+1]+.
EXAMPLE 21 (S) -N- (4-methoxyphenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 21)
Compound 21 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.24(s,2H),7.33(s,1H),7.23(d,J＝8.4Hz,2H),7.17(d,J＝8.4Hz,2H),4.54-4.43(m,2H),4.42(d,J＝12.1Hz,1H),4.32(d,J＝11.2Hz,1H),4.17(dd,J＝13.5Hz,4.6Hz,1H),4.10 -4.07(m,2H),3.92(s,3H),3.50(s,3H).ESI-Ms:413.1[M+1]+.
EXAMPLE 22 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethyl) phenyl) pyrimidin-2-amine (compound 22)
Compound 22 was prepared by the same method as in example 1.
1H-NMR(400MHz,CDCl3)δ8.29(s,2H),7.43(d,J＝8.6Hz,2H),7.37(s,1H),7.35(d,J＝8.8Hz,2H),4.64-4.55(m,2H),4.56(d,J＝11.4Hz,1H),4.45(d,J＝11.4Hz,1H),4.36(d,J＝12.2Hz,1H),4.20(dd,J＝13.3Hz,4.6Hz,1H),4.13-4.09(m,2H),3.53(s,3H).ESI-Ms:451.1[M+1]+.
Example 23 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethylthio) phenyl) pyrimidin-2-amine (compound 23)
Compound 23 was produced in the same manner as in example 1.
1H-NMR(400MHz,CDCl3)δ8.26(s,2H),7.42(d,J＝8.6Hz,2H),7.38(s,1H),7.29(d,J＝8.8Hz,2H),4.62-4.57(m,1H),4.56(d,J＝11.6Hz,1H),4.44(d,J＝11.6Hz,1H),4.32(d,J＝12.3Hz,1H),4.17(dd,J＝13.3,4.6Hz,1H),4.13-4.08(m,2H),3.51(s,3H).ESI-Ms:483.1[M+1]+.
EXAMPLE 24 (S) -N- (4- (difluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 24)
Compound 24 was produced in the same manner as in example 1.
1H NMR(600MHz,CDCl3)δ8.26(s,2H),7.36-7.33(m,2H),7.28(J＝8.6Hz,2H),7.17(d,J＝8.6Hz,2H),4.57–4.52(m,1H),4.47(d,J＝11.4Hz,1H),4.35(d,J＝11.4Hz,1H),4.30(d,J＝12.2Hz,1H),4.17(dd,J＝13.5,4.6Hz,1H),4.12-4.07(m,2H),3.50(s,3H).ESI-Ms:449.1[M+1]+.
Example 25 (S) -N- (3-fluoro-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 25)
Compound 25 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.30(s,2H),7.39(s,1H),7.29(t,J＝8.7Hz,1H),7.21(dd,J＝11.2,2.4Hz,1H),7.13–7.08(m,1H),4.64–4.59(m,1H),4.57(d,J＝11.4Hz,1H),4.46(d,J＝11.4Hz,1H),4.35(d,J＝12.1Hz,1H),4.21(dd,J＝13.4,4.6Hz,1H),4.14-4.09(m,2H),3.51(s,3H).ESI-Ms:485.1[M+1]+.
EXAMPLE 26 (S) -N- (2-chloro-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 26)
Compound 26 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.28(s,2H),7.38(s,1H),7.36(d,J＝1.8Hz,1H),7.32(d,J＝8.7Hz,1H),7.21–7.16(m,1H),4.63–4.57(m,1H),4.55(d,J＝11.5Hz,1H),4.44(d,J＝11.5Hz,1H),4.35(d,J＝12.2Hz,1H),4.21(dd,J＝13.4,4.5Hz,1H),4.14–4.07(m,2H),3.42(s,3H).ESI-Ms:501.1[M+1]+.
EXAMPLE 27 (S) -N- (2-methyl-4- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 27)
Compound 27 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.29(s,2H),7.40(s,1H),7.19-7.14(m,2H),7.11(d,J＝8.9Hz,1H),4.66–4.59(m,1H),4.56(d,J＝11.4Hz,1H),4.44(d,J＝11.4Hz,1H),4.36(d,J＝12.0Hz,1H),4.21(dd,J＝13.3,4.4Hz,1H),4.15–4.08(m,2H),3.41(s,3H),2.14(s,3H).ESI-Ms:481.1[M+1]+.
EXAMPLE 28 (S) -N- (4-fluoro-3- (trifluoromethoxy) phenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 28)
Compound 28 was prepared by the same method as in example 1.
1H NMR(600MHz,CDCl3)δ8.23(s,2H),7.35(s,1H),7.22(d,J＝5.5Hz,1H),7.17-7.13(m,2H),4.56(d,J＝11.9Hz,1H),4.50(d,J＝11.3Hz,1H),4.41(d,J＝11.4Hz,1H),4.32(d,J＝12.0Hz,1H),4.19(dd,J＝13.0,4.6Hz,1H),4.10(s,2H),3.44(s,3H).ESI-Ms:485.1[M+1]+.
Example 29 (S) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxo) methyl) -N-phenylpyrimidin-2-amine (Compound 29)
Compound 29 was prepared by the same method as in example 1.
1H NMR(400MHz,CDCl3)δ8.26(s,2H),7.35(s,1H),7.33–7.26(m,4H),7.18-7.14(m,1H),4.62-4.57(m,1H),4.56(d,J＝11.5Hz,1H),4.44(d,J＝11.5Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.4,4.6Hz,1H),4.14-4.07(m,2H),3.43(s,3H).ESI-Ms:383.1[M+1]+.
Example 30 (S) -N- (4-cyclopropylphenyl) -N-methyl-5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) pyrimidin-2-amine (compound 30)
Compound 30 was produced in the same manner as in example 1.
1H NMR(400MHz,CDCl3)δ8.26(s,2H),7.36(s,1H),7.32(d,J＝8.6Hz,2H),7.28(d,J＝8.7Hz,2H),4.64-4.59(m,1H),4.55(d,J＝11.5Hz,1H),4.43(d,J＝11.5Hz,1H),4.34(d,J＝12.3Hz,1H),4.19(dd,J＝13.3,4.5Hz,1H),4.13-4.07(m,2H),3.46(s,3H),2.01–1.95(m,1H),1.59-1.52(m,2H),1.16-1.08(m,2H).ESI-Ms:423.2[M+1]+.
Example 31 (S) -N- ((2- (methyl (4- (trifluoromethoxy) phenyl) amino) pyrimidin-5-yl) methyl) -2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-amine (compound 31)
Intermediate 1-5 (148 mg,0.50 mmol), triethylamine (66 mg,0.65 mmol) were dissolved in dichloromethane (10 mL), then added to starting material II-1 (84 mg,0.50 mmol), reacted overnight at room temperature, added NaBH (OAc) 3 (424 mg,2.0 mmol) and reacted overnight at room temperature. Sodium bicarbonate solution was added, the layers separated, the aqueous layer extracted with dichloromethane, the dichloromethane layers combined, washed with saturated sodium chloride solution, dried, and concentrated to give compound 31 (116 mg, yield 52.7%) as pale yellow powder by column chromatography (dichloromethane: methanol=50:1).
1H-NMR(400MHz,CDCl3)δ8.21(s,2H),7.46(s,1H),7.35(d,J＝8.6Hz,2H),7.26(d,J＝8.6Hz,2H),4.51(dd,J＝11.4,2.6Hz,1H),4.43(dd,J＝11.4,5.6Hz,1H),4.23(dd,J＝12.2,4.4Hz,1H),3.98(dd,J＝12.2,4.4Hz,1H),3.81(q,J＝13.4Hz,2H),3.50–3.40(m,1H),3.20(s,3H).ESI-Ms:466.1[M+1]+.
Example 32 (S) -N- ((2- (methyl (4- (trifluoromethoxy) benzyl) amino) pyrimidin-5-yl) methyl) -2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-amine (compound 32)
Compound 32 was prepared by the same method as in example 31.
1H NMR(400MHz,CDCl3)δ8.33(s,2H),7.45(s,1H),7.30(d,J＝8.2Hz,2H),7.20(d,J＝8.2Hz,2H),4.94(s,2H),4.50(dd,J＝11.6,2.6Hz,1H),4.43(dd,J＝11.8,5.5Hz,1H),4.24(dd,J＝12.2,4.3Hz,1H),3.99(dd,J＝12.2,4.4Hz,1H),3.81(q,J＝13.4Hz,2H),3.50–3.41(m,1H),3.19(s,3H).ESI-Ms:480.2[M+1]+.
Example 33 (S) -5- (((2-nitro-6, 7-dihydro-5H-imidazo [2,1-b ] [1,3] oxazin-6-yl) oxy) methyl) -N- (4- (trifluoromethoxy) phenyl) pyrimidin-2-amine (compound 33)
Intermediate III-1 was synthesized in the same manner as in example 1, starting from 1-1, according to the method of document WO2017/176817, and then compound 33 was produced.
1H NMR(600MHz,CDCl3)δ8.32(s,2H),7.57(d,J＝9.0Hz,2H),7.35(s,1H),7.13(d,J＝8.6Hz,2H),4.62–4.58(m,1H),4.56(d,J＝11.4Hz,1H),4.44(d,J＝11.4Hz,1H),4.32(d,J＝12.2Hz,1H),4.18(dd,J＝14.0,4.9Hz,1H),4.12–4.06(m,2H).ESI-Ms:453.1[M+1]+.
Pharmacological examples
Example 34: in vitro efficacy experiment of partial Compounds on Mycobacterium tuberculosis H37Rv Strain
Transferring the tested strain H37Rv into a liquid culture medium, culturing for 2 weeks at 37 ℃, sucking a little of culture bacteria liquid, placing in 4mL of liquid culture medium, adding 10-20 particles of sterile glass beads with the diameter of 2-3mm, oscillating for 20-30S, standing for precipitation l0-20 min, sucking the bacterial suspension supernatant, and regulating turbidimetry to 1 McU with the liquid culture medium, wherein the turbidimetry is equivalent to 1X 10 7 CFU/mL for standby. Each drug was dissolved to 1mg/mL with an appropriate amount of DMSO and filtered through a 0.22 μm filter. And then diluting to the required experimental concentration by using a liquid culture medium. The final concentration of the test drug was set up as follows ：0.0039μg/mL、0.0078μg/mL、0.0156μg/mL、0.03125μg/mL、0.0625μg/mL、0.125μg/mL、0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、4μg/mL, total 11 concentration gradients. Taking 100 mu L of each drug solution, adding the drug solution into a 96-well micro-pore plate, and then adding 100 mu L of bacterial liquid with the concentration of 1mg/mL to ensure that the drug concentration reaches the set final concentration, and culturing at 37 ℃. Three groups of parallel controls are arranged on the same medicine dilution, no medicine is added in the control group, and the inoculation amount is respectively set to be 100%, 10% and 1%. After 14 days of incubation at 37 ℃, the colony growth of each group was observed, and the minimum concentration of the sterile, falling-grown drug group was used as the MIC value of the test compound for the strain. The Minimum Inhibitory Concentration (MIC) of each compound for Mycobacterium tuberculosis was observed and compared to the MIC results of the control drug PA-824. The results are shown in Table 1.
TABLE 1 in vitro Activity of Mycobacterium tuberculosis of H37Rv type-Minimum Inhibitory Concentration (MIC)
As can be seen from Table 1, the compounds of the present invention exhibit significantly better in vitro MIC than the control drug PA-824. For example, the in vitro activity (MIC) of compound 1, compound 13, compound 18, compound 22, compound 23, compound 28, compound 31 and compound 33 was 0.03125 μg/mL, which is 4-fold the in vitro potency of the control drug PA-824.
Example 35: in vitro efficacy experiment of partial Compounds on drug-resistant Mycobacterium tuberculosis Strain
The test strains (1146-14: streptomycin resistant; 4061-15: isoniazid resistant; 3997-7: rifampicin resistant; B2, MDR-TB; B6, B29 and B53, XDR-TB) were isolated clinically from Mycobacterium tuberculosis from Shanghai lung family hospitals. The method comprises the following steps: a. collecting sputum specimens of hospitalized patients in tuberculosis of the hospital of lung th department of Shanghai, performing alkali treatment, inoculating to an improved Roche culture medium, and culturing for 2 weeks; b. drug sensitivity is measured by an absolute concentration method: fresh cultures were scraped from the slant of the medium, turbidded to 1 McO unit (1 mg/mL) with physiological saline, diluted to 10-2mg/mL, 0.1mL was inoculated on the drug sensitive medium, and the results were observed after four weeks. Reference materials: the test procedure of tuberculosis diagnosis laboratory, the basic professional committee of Chinese anti-tuberculosis society, chinese education culture Press, 1 month in 2006) is transferred into a liquid culture medium, cultured for 2 weeks at 37 ℃, a little culture bacteria liquid is absorbed, placed in 4mL of the liquid culture medium, 10-20 particles of sterile glass beads with the diameter of 2-3mm are added, shaking is carried out for 20-30S, resting precipitation l0-20 min, bacterial suspension supernatant is absorbed, and turbidness is adjusted to 1 McUth unit by the liquid culture medium, which is equivalent to 1X 10 7 CFU/mL for standby. Each drug was dissolved to 1mg/mL with an appropriate amount of DMSO and filtered through a 0.22 μm filter. And then the liquid culture is used for dilution to the required experimental concentration. When the final concentration of the tested medicine is set as ：0.0039μg/mL、0.0078μg/mL、0.0156μg/mL、0.03125μg/mL、0.0625μg/mL、0.125μg/mL、0.25μg/mL、0.5μg/mL、1μg/mL、2μg/mL、4μg/mL, concentration gradient detection, 100 mu L of the medicine solution is taken and added into a 96-well micro-pore plate, and then 100 mu L of 1mg/mL of bacterial liquid is added, so that the medicine concentration reaches the set final concentration, and the medicine is cultured at 37 ℃. Three groups of parallel controls are arranged on the same medicine dilution, no medicine is added in the control group, and the inoculation amount is respectively set to be 100%, 10% and 1%. The Mycobacterium tuberculosis of each drug is observed
And simultaneously compared to the MIC results of PA-824. The results are shown in Table 2.
TABLE 2 in vitro Activity against drug resistant Mycobacterium tuberculosis-Minimum Inhibitory Concentration (MIC)
S is streptomycin, H is isoniazid, R is rifampicin B2 is MDR-TB, B53, B29, B6 is XDR-TB.
As can be seen from Table 2, the inventive and control compounds PA-824 exhibited excellent in vitro antibacterial activity against streptomycin-resistant strains, isoniazid-resistant strains, rifampicin-resistant strains, multi-resistant strains (B2) and broadly resistant strains (B53, B29 and B6), and also showed that the inventive compounds had better in vitro activity against various resistant strains than the control drug PA-824. This shows that the compounds of the present invention, like PA-824, are useful in the treatment of diseases caused by drug-resistant tubercle bacillus, especially multi-drug-resistant and broadly drug-resistant tubercle bacillus.
Meanwhile, the compound disclosed by the invention has the same action mechanism as PA-824, is a brand-new action mechanism, and has no cross drug resistance with the existing drug. Thus, the compounds listed in Table 2 were removed, and the other compounds described in the present invention were inhibitory to various drug-resistant strains as long as they had inhibitory activity against H37Rv strain.
Example 36: in vivo pharmacokinetic experiments on partial Compounds
Test compounds were formulated using 0.5% CMC-Na in water as a uniform suspension with a final concentration of 2mg/mL for oral administration. The medicine is orally and parenterally administered, the dosage of single administration is 10mg/kg, the administration volume is 10mL/kg., and 0.15mL of blood sample is taken 15min, 30min, 1h, 2h, 4h, 6h, 10h, 12h and 24h after administration through the retrobulbar venous plexus of the mice.
Preparing a sample with final concentration of 0.5mg/mL for intravenous administration, wherein the solvent for preparing the sample is 5%
Dmso+20% ea+50% peg400+25% saline (physiological saline) in water, single dose 2mg/kg.: blood samples were collected 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 24h after dosing. CD-1 mice, males, 6-8 weeks of week old at the beginning of administration, 20-30g of body weight at the beginning of administration, and marker number were selected. Animal body weight was measured prior to dosing and healthy animals with similar body weights were selected for inclusion in the experiment without randomized group, during which time all animals were free to drink water.
After collection, the plasma is placed in a marked centrifuge tube, rapidly separated at 3500 rpm for 10 minutes and at 4 ℃, and then the plasma is placed below-40 ℃ for preservation and measurement. The drug concentration in plasma was determined by LC-MS/MS method and its drug substitution parameters were calculated.
TABLE 3 pharmacokinetic experiment results in CD-1 mice
As can be seen from the data in Table 3 above, some of the compounds of the present invention have better pharmacokinetic properties after a single oral administration in mice. Table 3 is only representative of some compounds, the remaining compounds may also have excellent pharmacokinetic properties.
These all indicate that the compounds of the invention have good drug properties and are likely to develop into effective tuberculosis therapeutic drugs. In addition, since the in vitro activity of a part of the compounds of the present invention is significantly higher than that of the control drug, it is reasonable to believe that a part of the compounds of the present invention will exhibit excellent in vivo efficacy.
EXAMPLE 37 acute infection model in mice partial Compounds are tested for their in vivo efficacy
BALB/c mice, females, weighing about 20g, were infected with Mycobacterium tuberculosis H37Rv (ATCC STRAIN 27274) by aerosol route using an inhalation exposure system at a dose of about 5000CFU. 5 untreated mice were euthanized on the day of treatment to determine the infection dose. The drug to be tested was formulated as a suspension using 0.5% w/v carboxymethylcellulose (CMC). Before use, the mixture is preserved at 4 ℃. Control mice were treated with only 0.5% cmc.
Mice were grouped, weighed, 5 mice per group, and gastric lavage was started, five days a week, once a day, for four weeks. After 3 days of washout following the last dose, the experimental mice were euthanized, both lungs were aseptically removed, ground, and homogenized in 3ml Hank's Balanced Salt Solution (HBSS). After ten-fold dilution of HBSS solution, colony forming units were counted by incubation on Middlebrook 7H11 agar plates for three weeks. Results are expressed as average LogCFU values for each group of mice.
TABLE 4 in vivo efficacy test in H37Rv acute infected BALB/c female mice
5 Non-dosed mice were euthanized at 24 days post infection due to the explosive infection.
For H37Rv acutely infected BALB/c female mice, none of the three groups PA-824, compound 1 and Compound 2 died after the end of the dosing. As can be seen from table 4, at the end of compound 2 administration, the CFU number per dose group was significantly lower than that of control PA-824, significantly better than that of the control drug, especially at the 30mg/kg dose group, compound 2 had a significant bactericidal effect on tubercle bacillus, CFU was reduced by 0.73 log (compared to 0 days), and the CFU number of PA-824 was increased (compared to 0 days). Compound 1 was reduced by 0.43 log units over the CFU number of PA-824 in the 100mg/kg dose group.
These data indicate that the compounds of the present invention have superior in vivo bactericidal activity relative to PA-824, and in particular, compound 2 has more pronounced advantages at medium doses, i.e. lower doses of the compound can exert better therapeutic effects while reducing side effects.
EXAMPLE 38 test of Compounds for inhibition of hERG Potassium ion channels
HEK-293 cells stably expressing hERG (CREACELLTM, france) were subjected to recording of hERG potassium channel currents using whole cell patch clamp technique at room temperature. The voltage stimulation protocol for whole cell patch clamp recording whole cell hERG potassium current is as follows: the cell membrane voltage was clamped at-80 mV after the whole cell seal was formed. The clamp voltage is divided from-80 mV to-50 mV for 0.5s (used as leakage current detection), then is stepped to 30mV for 2.5s, and then is quickly restored to-50 mV for 4s, so that the tail current of the hERG channel can be excited. Data were collected repeatedly every 10s and the effect of drug on hERG tail current was observed. The leakage current was measured with a stimulus of-50 mV for 0.5 s. Test data were collected by the EPC-10 amplifier (HEKA) and stored in PATCHMASTER (HEKA) software.
Data analysis: the current after each drug concentration was first normalized to the current for the blankThen calculating the inhibition rate/>, corresponding to each drug concentration Average and standard errors were calculated for each concentration and the semi-inhibitory concentration for each compound was calculated using the following equation:
A non-linear fit was made to the dose-dependent effect using the equation above, where C represents drug concentration, IC50 is half-inhibitory concentration, and h represents the hill coefficient. Curve fitting and IC50 calculation was done using the IGOR software.
Sample preparation: 1. preparing a measured sample into a stock solution with corresponding concentration by using DMSO; 2. sequentially diluting the test sample stock solution into diluted solutions of 0.5mM,2mM and 8mM by using DMSO; 3. sequentially diluting the sample diluent with extracellular fluid to prepare 0.5 mu M,2 mu M,8 mu M and 32 mu M liquid, and performing ultrasonic treatment for 20min at all concentrations; 4. the solubility of the sample to be tested was visually and microscopically examined and then tested.
Inhibition of hERG by some compounds of table 5:
Compounds of formula (I) IC50(μM)
Compound 2 >32
PA-824 5.8
Table 5 shows that the compounds of the invention are non-inhibitory to hERG potassium current, suggesting that the compounds of the invention have good safety to the cardiovascular system, which is superior to the control drug (PA-824).
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, which is defined broadly in the appended claims, and any person skilled in the art to which the invention pertains will readily appreciate that many modifications, including those that fall within the metes and bounds of the claims, or equivalence of such metes and bounds thereof.

Claims (14)

1. An antitubercular compound which is a compound of formula (I) or a pharmaceutically acceptable salt thereof:
In formula (I): m represents 0,1 or 2, n represents 1, 2 or 3;
X is oxygen or NH;
R 1 is selected from hydrogen or C 1-4 alkyl;
R 2 is selected from C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylthio, halogen, or cyano, said C 1-4 alkoxy is unsubstituted or optionally substituted with one to three halogens, and said C 1-4 alkyl or C 1-4 alkylthio is optionally substituted with one to three halogens.
2. An antitubercular compound according to claim 1, wherein R 1 is hydrogen, methyl or ethyl; the R 2 is methyl, methoxy, methylthio, fluoro, chloro, or cyano, the methoxy is unsubstituted or optionally substituted with one to three fluoro, and the methyl or methylthio is optionally substituted with one to three fluoro.
3. An antitubercular compound according to claim 1, wherein the pharmaceutically acceptable salt is a salt of a compound of formula (I) with an acid; wherein the acid is inorganic acid, organic acid or acidic amino acid.
4. An antitubercular compound according to claim 3, wherein the mineral acid is hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid.
5. An antitubercular compound according to claim 3, wherein the organic acid is formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, p-toluenesulfonic acid, ethanesulfonic acid, benzenesulfonic acid.
6. An antitubercular compound according to claim 3, wherein the acidic amino acid is aspartic acid, glutamic acid.
7. An antitubercular compound according to any one of claims 1 to 6, wherein the compound is a compound of formula 1, formula 2, formula 11, formula 13, formula 14, formula 17, formula 18, formula 20, formula 21, formula 22, formula 23, formula 24, formula 25, formula 28, formula 31, formula 32, formula 33:
8. a method of preparing an antitubercular compound according to any one of claims 1 to 7, comprising the steps of:
(1) The compound with the structure shown as the formula I-5 is reduced to obtain the compound with the structure shown as the formula I-6; the reducing agent is sodium borohydride, potassium borohydride, lithium borohydride and lithium aluminum hydride;
(2) The compound with the structure shown as the formula I-6 is chloridized to obtain the compound with the structure shown as the formula I-7; the chlorinating reagent used is sulfoxide chloride, phosphorus oxychloride, phosphorus trichloride and phosphorus pentachloride;
(3) The compound with the structure shown in the formula I-8 is firstly mixed with a solvent and then placed at the temperature of minus 20-0 ℃, then strong alkali is added for reaction for 0.5-2 hours, and then the compound with the structure shown in the formula I-7 is added for continuous reaction to obtain the compound with the structure shown in the formula I; the solvent is selected from dimethylformamide, dimethylacetamide, N-methylpyrrolidone or tetrahydrofuran; the strong base is sodium hydride, potassium hydride, sodium tert-butoxide and potassium tert-butoxide;
m, n, R 1 and R 2 are as defined above; x is oxygen.
9. A method of preparing an antitubercular compound according to any one of claims 1 to 7, comprising the steps of: mixing a compound with a structure shown as a formula I-5 with a compound with a structure shown as a formula II-1, and reacting in the presence of a reducing agent to obtain a compound with a structure shown as a formula I; the reducing agent is sodium triacetoxyborohydride, sodium borohydride and sodium cyanoborohydride;
m, n, R 1 and R 2 are as defined above; x is NH.
10. The method of preparing as claimed in claim 9, characterized in that it comprises the steps of: reacting a compound with a structure shown as a formula I-5 with a compound with a structure shown as a formula II-1 in the presence of organic base for 2-20 hours to obtain intermediate imine, and then adding a reducing agent to react for 1-22 hours to obtain the compound with the structure shown as the formula I; the organic base is triethylamine or N, N-diisopropylethylamine; the reaction solvent for obtaining the intermediate imine is dichloromethane and dichloroethane.
11. The process according to any one of claims 8 to 10, wherein the compound of formula i-5 is obtained by:
(a) Coupling reaction is carried out on the compound with the structure shown as the formula I-3 and tri-n-butyl vinyl tin, so as to obtain the compound with the structure shown as the formula I-4; the catalyst of the coupling reaction is Pd (PPh 3)4、Pd(dppf)2Cl2、Pd2(dba)3; the reaction solvent of the coupling reaction is dimethylformamide, dimethylacetamide and N-methylpyrrolidone, and the coupling reaction is carried out at 50-120 ℃;
(b) The double bond of the compound with the structure shown as the formula I-4 is oxidized and cut off to obtain the compound with the structure shown as the formula I-5; the used oxidizing agents are sodium periodate and potassium periodate; the catalyst used is potassium osmium and ruthenium trichloride; the temperature of the oxidation cutting is 0-50 ℃;
12. Use of an antitubercular compound according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of diseases associated with infections caused by tubercle bacillus.
13. The use according to claim 12, wherein the antitubercular compound is used for the preparation of a medicament for the treatment of infectious diseases caused by multidrug-resistant tubercle bacillus.
14. A pharmaceutical composition for the treatment of a disease associated with an infection by mycobacterium tuberculosis, comprising a therapeutically effective amount of an antitubercular compound according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient or carrier.
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CN101341150A (en) * 2005-12-23 2009-01-07 诺瓦提斯公司 Nitroimidazole compounds
CN101965353A (en) * 2008-03-26 2011-02-02 全球结核病药物研发联盟 Covalently bound dicyclo nitro glyoxaline to the substituted benzene oxazolidinone
CN102234287A (en) * 2010-04-26 2011-11-09 上海阳帆医药科技有限公司 Nitro imidazole compound, its preparation method and application
CN102753558A (en) * 2009-07-31 2012-10-24 全球结核病药物研发联盟 Nitroimidazooxazines and their uses in anti-tubercular therapy
CN105732659A (en) * 2014-12-12 2016-07-06 上海阳帆医药科技有限公司 Nitroimidazole compound, preparation method and pharmaceutical application thereof

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Publication number Priority date Publication date Assignee Title
CN101341150A (en) * 2005-12-23 2009-01-07 诺瓦提斯公司 Nitroimidazole compounds
CN101965353A (en) * 2008-03-26 2011-02-02 全球结核病药物研发联盟 Covalently bound dicyclo nitro glyoxaline to the substituted benzene oxazolidinone
CN102753558A (en) * 2009-07-31 2012-10-24 全球结核病药物研发联盟 Nitroimidazooxazines and their uses in anti-tubercular therapy
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CN105732659A (en) * 2014-12-12 2016-07-06 上海阳帆医药科技有限公司 Nitroimidazole compound, preparation method and pharmaceutical application thereof

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