CN108159049B

CN108159049B - New application of pyridine compound

Info

Publication number: CN108159049B
Application number: CN201810106538.9A
Authority: CN
Inventors: 张天宇; 刘洋
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2018-02-01
Filing date: 2018-02-01
Publication date: 2021-01-05
Anticipated expiration: 2038-02-01
Also published as: WO2019148575A1; CN108159049A

Abstract

The invention discloses an application of pyridine compounds in preparing a medicament for treating mycobacterial infectious diseases, wherein the pyridine compounds comprise pyrazolopyridine compounds and imidazopyridine compounds; the structural general formula of the pyrazolopyridine compound is shown as a formula (I), and the structural general formula of the imidazopyridine compound is shown as a formula (II). The inventor of the application discovers for the first time that pyrazolopyridine compounds and imidazopyridine compounds can effectively treat mycobacterial infectious diseases, particularly diseases caused by infection of Mycobacterium bruxides (Mycobacterium ulcerans), Mycobacterium marinum (Mycobacterium marinum) and Mycobacterium leprae), and the pyrazolopyridine compounds and the imidazopyridine compounds have the advantages of convenience in use, quickness in taking effect, low use dosage, low toxic and side effects and the like.

Description

New application of pyridine compound

Technical Field

The invention relates to a new application of a pyridine compound, in particular to an application of the pyridine compound in preparing a medicament for treating mycobacterial infectious diseases.

Background

Brucella necrotizing Mycobacterium (Mycobacterium ulcerans) can cause severe skin ulceration, necrosis, and even blindness and death, most known as Brucella necrotizing (Buruli Ulcer), and is currently found in several areas of Africa, Australia, southeast Asia, and south America, Japan. The treatment method for the disease is mainly to perform skin grafting after patient tissues are removed through surgical operation, however, the cure rate is only 16% to 28%, and the treatment method needs to be assisted by medicine. The recommended treatment for this disease by the world health organization is the combined treatment of rifampicin and streptomycin for about 8 weeks as an adjuvant therapy. This therapy is based on experimental results in previous mouse models. Other therapies (which have not been clinically developed at present and have been completed in 2016) mainly involve the use of clarithromycin, bedaquiline instead of streptomycin in combination with rifampin (or the like, rifapentin) for about 8 weeks.

The cost of surgery for treating the disease is high, the cure rate is low, and the adjuvant drug therapy is necessary. In the standard drug therapy recommended by the world health organization, streptomycin needs to be injected and administered every day for a long period, which is inconvenient for patients, and the administration route has high risk of incomplete infection due to equipment disinfection and poor patient compliance. In addition, rifampicin and streptomycin have large toxic and side effects, and the patient compliance is poor after long-term administration. The existing available medicines are few and medicines of rifamycins are required to be used together, so if a patient is resistant to the medicines, the treatment effect of all the treatments can be obviously reduced, and medicines of rifampicin can obviously reduce the drug effect of other disease treatment medicines, such as AIDS medicine Gakeli, and the use of the medicines of rifampicin and Gakeli can cause the antiviral effect of Gakeli. The long-term treatment of clarithromycin in combination with rifampicin is less effective and there is still a need for later consolidation therapy with streptomycin and rifampicin. The novel antituberculosis drug Bedaquin has large toxic and side effects, FDA has added a black frame to limit the use, and the treatment for drug-resistant tuberculosis patients is usually recommended only under the condition that other drugs are ineffective, and the treatment for brucelli necrosis is not recommended.

Disclosure of Invention

Based on the above, the present invention aims to overcome the defects of the prior art and provide the use of pyridine compounds in the preparation of drugs for treating mycobacterial infectious diseases, wherein the pyridine compounds have the following structural general formula:

wherein R1 represents 5-Me, 4-Me, 6-Me, 7-Me, 5-OMe, 5-Cl, 5-Et, 5-i-Pr or 5-t-Butyl;

r2 represents Me;

r3 represents CF₃Formula (a), formula (b), formula (c), formula (d), formula (e) or formula (f):

preferably, the pyridine compound has the following structural formula:

preferably, the mycobacterium is mycobacterium bruelii necrosis.

Preferably, the mycobacterium is mycobacterium marinum or mycobacterium leprae.

Preferably, the medicament is in an oral dosage form or an external dosage form.

Preferably, the oral dosage forms include tablets, capsules, granules and oral liquids; the external preparation comprises ointment, gel, band-aid and medicinal patch.

Another object of the present invention is to provide a drug for treating mycobacterial infectious diseases, which comprises a pyridine compound and a pharmaceutically acceptable carrier, wherein the pyridine compound has the following structural formula:

r2 represents Me;

preferably, the pyridine compound has the following structural formula:

the compound shown in the formula (III) is abbreviated as COMX in the invention; the compound represented by the formula (IV) is abbreviated as Q203.

Preferably, the mycobacterial infectious disease is brucellosis, mycobacterium marinum infectious disease or leprosy.

More preferably, the oral dosage forms include tablets, capsules, granules, and oral liquids; the external preparation comprises ointment, gel, band-aid and medicinal patch.

The pyridine compounds comprise pyrazolopyridine compounds and imidazopyridine compounds; the structural general formula of the pyrazolopyridine compound is shown as a formula (I), and the structural general formula of the imidazopyridine compound is shown as a formula (II).

Researches show that the core parts of the pyrazolopyridines and the imidazopyridines which have the structural general formulas have similar structures, and the core parts of the pyrazolopyridines and the imidazopyridines which have the structural general formulas and have the therapeutic effects are the structures in the general formulas.

Compared with the prior art, the invention has the beneficial effects that: the invention discloses a pyrazolopyridine compound and an imidazopyridine compound for the first time, which can effectively treat mycobacterial infectious diseases, particularly diseases caused by infection of mycobacterium brunelli, mycobacterium marinum and mycobacterium leprae, and have the advantages of convenience in use, quick response, low use dose, low toxic and side effects and the like.

Drawings

FIG. 1 is a graph of the dynamic inhibition of luminescence of Mycobacterium brucelly necrosis in vitro by COMX and Q203.

FIG. 2 is a graph showing the results of dynamic changes in foot luminescence values of mice infected with Mycobacterium brucelly necrotizing bacterium after COMX treatment.

FIG. 3 is a graph showing the results of luminescence detection of foot tissues of mice infected with self-luminous Mycobacterium brucelly after COMX treatment.

FIG. 4 is a photograph of the feet of mice infected with self-illuminating Mycobacterium brucei necrotizing strain on day 7 of COMX treatment (5 days of treatment, after one day).

FIG. 5 is a graph showing the results of dynamic changes in foot luminescence values of mice infected with Mycobacterium brucellosis after Q203 treatment.

FIG. 6 is the relative bacterial load calculated by RT-PCR assays for foot tissue from mice infected with Mycobacterium leprae 4 weeks after COMX and Q203 treatment (4 weeks after treatment, 4 weeks apart).

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

Example 1

This example studies the effect of pyrazolopyridines and imidazopyridines of the present invention in inhibiting mycobacterium bruelii in vitro.

(one) Experimental groups

Solvent group: an equal volume of drug solvent was given as a control;

pyrazolopyridine compound group: the structures of R1, R2 and R3 of the respective compounds are shown in Table 1 with a skeleton represented by formula (I):

TABLE 1 structural formula of pyrazolopyridines

Compound numbering	R1	R2	R3
				1	5-Me	Me	CF ₃
2	5-Me	Me	Formula (a)
				3	5-Me	Me	Formula (b)
4	5-Me	Me	Formula (c)
				5	5-Me	Me	Formula (d)
6	5-Me	Me	Formula (e)
				7	5-Me	Me	Formula (f)
8	4-Me	Me	Formula (f)
				9	6-Me	Me	Formula (f)
10	7-Me	Me	Formula (f)
				11	5-OMe	Me	Formula (f)
12	5-Cl	Me	Formula (f)
				13	5-Et	Me	Formula (f)
14	5-i-Pr	Me	Formula (f)
				15	5-t-Butyl	Me	Formula (f)

Imidazopyridines group: the structures of R1, R2, and R3 of the respective compounds are shown in table 2 with a skeleton represented by formula (ii):

TABLE 2 structural formulas of imidazopyridines

The pyrazolopyridines and imidazopyridines used above are dissolved in DMSO for use.

(II) Experimental procedure

1. Culture of self-luminous Bruli necrosis mycobacteria

Inoculating the self-luminous brucella necrosis mycobacteria into a 7H11 solid culture medium, and culturing for 58 days at 30 ℃; a single colony is picked from a plate, a Relative Light Unit (RLU) is detected by using a luminescence detector, the single colony confirmed to emit light is inoculated into 50mL of 7H9 liquid culture medium for about 7 days, and is filtered by a 300-mesh cell screen after being resuspended by sterile normal saline, and the single colony is nearly formed into a single bacterial suspension for detection experiments.

2. Dosing co-incubation continuous detection

And (3) taking a 96-well plate, adding the prepared compound into the first vertical row on the left, uniformly blowing the medicine and the bacterial liquid by using a pipette gun, and then performing gradient dilution by 2 times from left to right by using the pipette gun. Detecting the sterilization or bacteriostatic effect of the medicament: after incubation for 5 days, the 96-well plate was placed in an microplate reader, and the luminescence values of each group were measured.

(III) results of the experiment

The inhibition rate of the compound on the growth of bacteria was 1- (test value of drug-added group/luminescence value of solvent control group)%

The minimum inhibitory concentration is the lowest concentration at which the inhibition rate of the compound is higher than 90%.

The detection results of the minimum inhibitory concentration of the pyrazolopyridine compound group and the imidazopyridine compound group are shown in table 3:

TABLE 3 results of the experiment

From the above results, it is clear that pyrazolopyridine and imidazopyridine compounds have a good inhibitory activity against Mycobacterium bruxides. Of these, pyrazolopyridine compound No. 11 (i.e., COMX) and imidazopyridine compound No. 27 (i.e., Q203) are the most superior in bacteriostatic activity.

Example 2

This example investigates the effect of COMX and Q203 in inhibiting mycobacterium bruelii necrosis in vitro.

(one) Experimental groups

Solvent group: an equal volume of drug solvent was given as a control;

group COMX: the concentration is 1 mug/mL, 0.2 mug/mL, 0.04 mug/mL, 0.008 mug/mL, 0.0016 mug/mL, 0.00032 mug/mL;

group Q203: the concentration is 4. mu.g/mL, 0.8. mu.g/mL, 0.16. mu.g/mL, 0.03. mu.g/mL, 0.005. mu.g/mL; 0.001. mu.g/mL

COMX and Q203 used above were dissolved in DMSO until use.

(II) Experimental procedure

1. The procedure of the cultivation of self-luminescent Mycobacterium brucelly necrosis was the same as in example 1.

2. Dosing co-incubation continuous detection

198 mul of bacteria solution and 2 mul of prepared drug were added to each EP tube, and the pipetting gun was used to mix the pipetting drug with the bacteria solution. Detecting the sterilization or bacteriostatic effect of the medicament: after the EP tube is put into the luminescence detector, the detection key is pressed, the RLUs of each EP tube are recorded, and the first detection is recorded as day 0. The assay was then run every 1 day until day 5.

(III) results of the experiment

From the beginning of the administration, the relative luminescence values of different groups are detected regularly, and the smaller the relative luminescence value at each time point is, the more obvious the bacteriostatic effect of the medicine is, and vice versa.

The detection results are shown in FIG. 1, and the results show that: the COMX group and the Q203 group can obviously inhibit the Brucella necrosis mycobacteria, the minimum inhibitory concentration of the two groups is about 0.001 mu g/mL, and the antibacterial activity is very good.

Example 3

This example investigates the inhibitory effect of COMX and Q203 on mycobacterium bruelii necrosis in mice.

(one) animal grouping and administration

Selecting Balb/C mice about 6 weeks, injecting self-luminous Brucella necrosis mycobacteria to the rear feet of the mice, and starting administration treatment after 10 days of infection, wherein the administration groups are as follows, and each group comprises 10 mice:

untrained group: after infection, an equal volume of deionized water was given as a negative control;

uninfected group: uninfected bacterial groups as background baseline for detected luminescence;

rifampin + streptomycin group: the administration of rifampicin 10mg/kg by intragastric administration and the subcutaneous injection of streptomycin 150 mg/kg;

group COMX: the administration amount of COMX by intragastric administration is 50mg/kg, 25mg/kg, 12.5mg/kg, 6.25mg/kg, 3.2mg/kg, 1.6mg/kg, 0.8mg/kg and 0.4mg/kg respectively;

group Q203: the administration dosage of Q203 by intragastric administration is respectively 25mg/kg, 6.25mg/kg and 1.6 mg/kg;

the intragastric administration volume of each medicine is 0.2 mL/time, and the medicine is administered once a day for 5 days continuously.

(II) index detection

Detecting the luminous value of the mouse living body according to the design time point, carrying out death by a cervical dislocation method after anesthetizing the mouse on the seventh day, taking foot tissues for grinding, and determining the luminous value of the tissues, wherein the smaller the luminous value is, the better the medicine treatment effect is, otherwise, the poor the medicine treatment effect is.

(III) results of the experiment

1. The results of continuous measurement of the luminescence values of the feet of the living mice are shown in FIG. 2, and it can be seen from FIG. 2 that COMX shows significant activity against Mycobacterium bruelii as low as 0.4 mg/kg. The combined use of only 0.8mg/kg of COMX with rifampicin at 10mg/kg and streptomycin at 150mg/kg shows that COMX has excellent anti-Brucella necrosis mycobacteria activity and has faster effect in vivo than the current commonly used drugs. After stopping taking the medicine for 2 days, the first-line therapy (RIF10+ STR150) mice have rebound luminous value in living bodies, but the COMX has not rebound above 3.1 mg/kg.

2. The results of the foot tissue luminescence test of the infected mice are shown in FIG. 3. As can be seen from FIG. 3, after 5 days of treatment, the standard first line therapy (RIF10+ STR150) treated group still can detect a higher luminescence value, while the COMX treated group of 12.5mg/kg or more has even reached the background value (same as the value of the uninfected group), which indicates that the COMX can significantly shorten the treatment period.

3. The results of the photographs taken of the mouse feet at day 7 (5 days of treatment, after one day) are shown in fig. 4, and it is evident from fig. 4 that the feet of the COMX-treated group were substantially normal (which is substantially identical to that of the unfected group), indicating that COMX had a very good therapeutic effect on bruise necrosis.

4. The inhibition mechanism of COMX and Q203 on Brucella necrosis mycobacteria is similar, the inhibition experiment result of Q203 on Brucella necrosis mycobacteria in mice is shown in figure 5, and Q203 also has a very good treatment effect on Brucella necrosis.

Example 4

This example investigates the inhibitory effect of COMX and Q203 on mycobacterium leprae in mice.

(one) animal grouping and administration

Female C57BL/6 mice were selected, mice were injected in their feet with mycobacterium leprae infection and treatment was initiated 12 weeks after infection, with the following groups of 20 mice per group:

untrained group: giving equal volume of deionized water as negative control;

rifampin group: the administration amount of the gavage is 10mg/kg of rifampicin;

group COMX: COMX was administered by gavage at a dose of 25 mg/kg;

group Q203: q203 is administrated, and the stomach is administrated, and the dosage is 25 mg/kg;

the gavage amount of each drug is 0.2 mL/time/dose, and each drug is administered once a day for 4 weeks and 5 days a week.

(II) index detection

After all groups of treatment are finished, waiting for 4 weeks, carrying out anesthesia cervical dislocation to kill the mouse, carefully taking out the mouse by using scissors to infect soft tissues at feet, extracting tissue genome, carrying out fluorescent quantitative PCR detection after purification, wherein the upstream detection primer sequence is as follows: 5'-GCAGTATCGTGTTAGTGAACAGTGCA-3', and the sequence of the downstream detection primer is 5'-CGCTAGAAGGTTGCCGTATGTGC-3'. And (3) converting the bacterial load in the tissue of each group of mice according to a fluorescent quantitative PCR result curve by taking the determined content of the mycobacterium leprae suspension as a standard control.

(III) results of the experiment

As shown in FIG. 6, the mouse load of the treated groups of pyrazolopyridine compound (represented by COMX) and imidazopyridine compound (represented by SQ 203) was lower than that of the control group and that of the rifampin-positive control group at 10 mg/kg. The Ming COMX and SQ203 have very good treatment effect on the leprosy.

Example 5

This example investigates the inhibitory effect of COMX and Q203 on mycobacterium marinum in vitro.

(one) Experimental groups

Solvent group: an equal volume of drug solvent was given as a control;

group COMX: the concentration is 0.1 mug/mL, 0.01 mug/mL and 0.001 mug/mL;

group Q203: the concentrations were 0.1. mu.g/mL, 0.01. mu.g/mL, 0.001. mu.g/mL

COMX and Q203 used above were dissolved in DMSO until use.

(II) Experimental procedure

1. Culturing self-luminous mycobacterium marinum, namely inoculating the self-luminous mycobacterium marinum into a 7H11 solid culture medium, and culturing for 7 days at 30 ℃; a single colony is picked from a plate, a Relative Light Unit (RLU) is detected by using a luminescence detector, the single colony confirmed to emit light is inoculated into 50mL of 7H9 liquid culture medium for about 3 days, and is filtered by a 300-mesh cell screen after being resuspended by sterile normal saline, and the single colony is nearly formed into a single bacterial suspension for detection experiments.

2. Dosing co-incubation continuous detection

198 mul of bacteria solution and 2 mul of prepared drug were added to each EP tube, and the pipetting gun was used to mix the pipetting drug with the bacteria solution. After incubation for 24 hours, the bactericidal or bacteriostatic effect of the drug is detected: and (3) after the EP tube is placed in a luminescence detector, waiting for 1-2 seconds until external light is quenched, pressing a detection key, and recording the RLUs of each EP tube.

(III) results of the experiment

The growth inhibition rate and the minimum inhibitory concentration of the compound on bacteria were calculated in the same manner as in example 1. The affinity of the mycobacterium marinum and the mycobacterium bruuli necrosis is high, and the genome similarity rate of the mycobacterium marinum and the mycobacterium bruuli necrosis reaches 99%. The results show that: the COMX group and the Q203 group can obviously inhibit mycobacterium marinum, the minimum inhibitory concentration of the COMX group and the Q203 group is about 0.01 mu g/mL, and the antibacterial activity is very good.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of the pyridine compound in preparing the medicine for treating infectious diseases of the Brucella necrosis mycobacteria or the leprosy mycobacteria is characterized in that the pyridine compound has the following structural general formula:

(I) or

（Ⅱ）；

In the formula (I), R1 represents 5-Me, 6-Me, 7-Me, 5-OMe, 5-Cl, 5-Et, 5-i-Pr or 5-t-Butyl; and R2 represents Me; and R3 represents CF₃Formula (a), formula (b), formula (c), formula (d), formula (e) or formula (f);

in formula (II), R1 represents 5-Me, 5-OMe, 5-Cl, 5-Et, 5-i-Pr or 5-t-Butyl; and R2 represents Me; and R3 represents CF₃Formula (a), formula (b), formula (c), formula (d), formula (e) or formula (f);

、

、

、

、

、

。

2. use according to claim 1, characterized in that the pyridine compound has the following formula:

(III) or

（Ⅳ）。

3. The use according to claim 1, wherein the medicament is in an oral or topical dosage form.

4. The use according to claim 3, wherein the oral dosage forms include tablets, capsules, granules and oral liquids; the external preparation comprises ointment, gel and patch.