WO2019148575A1 - Novel use of pyridine compound - Google Patents

Abstract

Disclosed is a use of a pyridine compound in the preparation of a medicament for treating mycobacterial infectious diseases. The pyridine compound comprises a pyrazolopyridine compound and an imidazopyridine compound. The structural formula of the pyrazolopyridine compound is represented by formula (I), and the structural formula of the imidazopyridine compound is represented by formula (II). The present inventors have found that pyrazolopyridines and imidazopyridines are effective in the treatment of mycobacterial infectious diseases, especially diseases caused by infection with Mycobacterium ulcerans, Mycobacterium marinum, and Mycobacterium leprae, and said compounds also have the advantages of convenient use, quick effect, low dosage, low toxic side effects, etc.

Description

New use of a pyridine compound Technical field

The present invention relates to a novel use of a pyridine compound, and in particular to the use of a pyridine compound for the preparation of a medicament for treating a mycobacterial infectious disease.

Background technique

Mycobacterium ulcerans can cause severe skin ulcers, necrosis, and even blindness and death. It is often called Buruli Ulcer and is currently in Africa, Australia, Southeast Asia, and South America. The disease was found in various regions such as Japan. The treatment of this disease is mainly through surgical resection of the patient's tissue and then skin grafting, however, the cure rate is only between 16% and 28%, and must be supplemented with medication. The World Health Organization's recommended treatment for this disease is adjuvant therapy with rifampicin and streptomycin for about 8 weeks. The therapy is based on experimental results in previous mouse models. Other therapies (not yet in clinical practice, mainly in 2016), mainly using clarithromycin, betaxazoline instead of streptomycin and rifampicin (or similar drug rifapentine) Need to be treated for about 8 weeks.

Surgical treatment of the disease is costly and has a low cure rate and must be supplemented with medication. Among the standard drug therapies recommended by WHO, streptomycin requires long-term daily injections, which is inconvenient for patients. The route of administration is not completely sterilized due to equipment sterilization, and patient compliance is poor. In addition, rifampicin and streptomycin have large toxic side effects, and long-term administration has poor patient compliance. There are fewer drugs available, and all need to use rifamycin in combination. Therefore, if patients are resistant to this type of drug, the therapeutic effect of all the treatments will be significantly reduced, and the drugs such as rifampicin will be significant. Reducing the efficacy of other disease treatment drugs, such as the AIDS drug Kelizhi, the use of the two will cause Kelizhi's anti-virus to be ineffective. The long-term treatment effect of clarithromycin combined with rifampicin is poor, and it is still necessary to use streptomycin and rifampicin for consolidation treatment later. The new anti-tuberculosis drug betaxazoline has a large side effect. The FDA has added a black box to limit the use. It is often recommended for the treatment of drug-resistant tuberculosis patients when other drugs are not effective. It has not been recommended to treat Brucella necrosis. .

Summary of the invention

Based on this, it is an object of the present invention to provide a use of a pyridine compound for the preparation of a medicament for treating a mycobacterial infectious disease by overcoming the above-mentioned deficiencies of the prior art, the pyridine compound having the following structural 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 stands for Me;

R3 represents CF ₃ , formula (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 Brucella necroticum.

Preferably, the mycobacterium is Mycobacterium marinum or Mycobacterium leprae.

Preferably, the drug is an oral dosage form or an external dosage form.

Preferably, the oral dosage form comprises tablets, capsules, granules and oral liquids; the topical dosage forms include ointments, gels, band-aids and patches.

Another object of the present invention is to provide a medicament for treating a mycobacterial infectious disease, the medicament comprising a pyridine compound and a pharmaceutically acceptable carrier, the pyridine compound having the following structural 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 stands for Me;

R3 represents CF ₃ , formula (a), formula (b), formula (c), formula (d), formula (e) or formula (f):

Preferably, the pyridine compound has the following structural formula:

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

Preferably, the mycobacterial infectious disease is Brucella necrosis, Mycobacterium tuberculosis infectious disease or leprosy.

Preferably, the drug is an oral dosage form or an external dosage form.

More preferably, the oral dosage form comprises tablets, capsules, granules and oral liquids; the topical dosage forms include ointments, gels, band-aids and patches.

The pyridine compound of the present invention comprises a pyrazolopyridine compound and an imidazopyridine compound; the pyrazolopyridine compound has a structural formula of the formula (I), and the imidazopyridine compound has a structural formula As shown in formula (II).

Studies have shown that the core sites of pyrazolopyridines and imidazopyridines have similar therapeutic effects, and the core sites of the pyrazolopyridines and imidazopyridines having the above structural formula are therapeutic. The structure in the formula.

Compared with the prior art, the beneficial effects of the present invention are as follows: the inventors of the present invention are the first to use pyrazolopyridines and imidazopyridines to effectively treat mycobacterial infectious diseases, especially by Brucella necrotic It is a disease caused by Mycobacterium marinum and Mycobacterium leprae infection, and has the advantages of convenient use, quick effect, low dosage and low side effects.

DRAWINGS

Figure 1 is a graph showing the dynamic inhibition of luminescence values of Mycobacterium breve in vitro by COMX and Q203.

Figure 2 is a graph showing the dynamic changes in foot luminescence values of mice infected with Brilliant Nebulous Mycobacterium tuberculosis after COMX treatment.

Figure 3 is a graph showing the results of luminescence detection of the foot tissue of mice infected with Brilliant Nebulous Mycobacterium tuberculosis after COMX treatment.

Figure 4 is a photograph of the foot of a self-illuminating Brucella necrotic Mice mouse infected on day 7 of COMX treatment (5 days after treatment, every other day).

Figure 5 is a graph showing the dynamic changes in the luminescence value of the foot of mice infected with Brilliant Nebulous Mycobacterium tuberculosis after Q203 treatment.

Figure 6 shows the relative bacterial load of the mice infected with M. leprae mice after 4 weeks of treatment with COMX and Q203 (4 weeks after treatment, 4 weeks apart).

Detailed ways

The present invention will be further described with reference to specific embodiments in order to better illustrate the objects, aspects and advantages of the invention.

Example 1

This example investigates the action of the pyrazolopyridines and imidazopyridines of the present invention to inhibit Brucella necroticus in vitro.

(1) Experimental grouping

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

Pyrazolopyridine compound group: The skeleton represented by the formula (I), and the structures of R1, R2 and R3 of each compound are shown in Table 1:

Table 1 Structure of pyrazolopyridines

Compound number	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)

Imidazopyridine compound group: The skeleton represented by the formula (II), and the structures of R1, R2 and R3 of each compound are shown in Table 2:

Table 2 Structure of imidazopyridine compounds

Compound number

R1

R2

R3

16	5-Me	Me	CF 3
17	5-Me	Me	Formula (a)
18	5-Me	Me	Formula (b)
19	5-Me	Me	Formula (c)
20	5-Me	Me	Formula (d)
twenty one	5-Me	Me	Formula (e)
twenty two	5-Me	Me	Formula (f)
twenty three	4-Me	Me	Formula (f)
twenty four	6-Me	Me	Formula (f)
25	7-Me	Me	Formula (f)
26	5-OMe	Me	Formula (f)
27	5-Cl	Me	Formula (f)
28	5-Et	Me	Formula (f)
29	5-i-Pr	Me	Formula (f)
30	5-t-Butyl	Me	Formula (f)

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

(two) experimental steps

1. Cultivation of self-illuminating Brucella necrotic mycobacteria

Self-illuminating Brucella necroticum was added to 7H11 solid medium and cultured at 30 ° C for 58 days; single colonies were picked from the plate to detect relative light units (RLU) using a luminescence detector, and single colonies that confirmed luminescence were connected. 50 mL of 7H9 liquid medium was cultured for about 7 days, resuspended in sterile physiological saline, filtered through a 300-mesh cell sieve, and filtered to form a single existing bacterial suspension for detection experiments.

2, continuous continuous detection of dosing

Take a 96-well plate, add the prepared compound to the first vertical row on the left side, mix the drug with the bacterial solution using a pipetting gun, and then perform a 2-fold gradient dilution from left to right using a lance. Detecting the bactericidal or bacteriostatic effect of the drug: After the fifth day of incubation, the 96-well plate was placed in a microplate reader, and the luminescence values of each group were detected.

(3) Experimental results

Growth inhibition rate of the compound against bacteria = 1 - (detection value of the drug-added group / luminescence value of the solvent control group)%

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

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

Table 3 Experimental results

From the above results, it was found that the pyrazolopyridine and imidazopyridine series compounds have good inhibitory activity against Brucella necroticum. Among them, the pyrazolopyridine compound of No. 11 (i.e., COMX) and the imidazopyridine compound of No. 27 (i.e., Q203) have the best antibacterial activity.

Example 2

This example investigates the effect of COMX and Q203 on inhibiting Brucella necroticus in vitro.

(1) Experimental grouping

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

COMX group: concentration of 1 μg / mL, 0.2 μg / mL, 0.04 μg / mL, 0.008 μg / mL, 0.0016 μg / mL, 0.00032 μg / mL;

Group Q203: concentration 4μg/mL, 0.8μg/mL, 0.16μg/mL, 0.03μg/mL, 0.005μg/mL; 0.001μg/mL

The above-mentioned COMX and Q203 were dissolved in DMSO for use.

(two) experimental steps

1. The culture of self-luminous Brilliant necrotic mycobacteria was carried out in the same manner as in Example 1.

2, continuous continuous detection of dosing

To each EP tube, 198 μL of the bacterial solution and 2 μL of the drug prepared drug were separately added, and the pipetting drug was mixed with the bacterial liquid using a pipetting gun. Detect drug sterilization or bacteriostatic effect: After putting the EP tube into the luminescence detector, press the detection button to record the RLUs of each EP tube, and record the first time as day 0. It was then tested every other day until the 5th day was detected.

(3) Experimental results

From the beginning of the administration, the relative luminescence values of different groups were periodically detected, and the smaller the relative luminescence value at each time point, the more obvious the bacteriostatic effect of the drug, and vice versa.

The test results are shown in Figure 1. The results showed that both COMX group and Q203 can significantly inhibit Brucella necroticus, and the minimum inhibitory concentration of the two is about 0.001 μg/mL, and the antibacterial activity is very good.

Example 3

This example investigates the inhibitory effect of COMX and Q203 on Brucella necroticum in mice.

(1) Animal grouping and administration

Balb/C mice were selected for about 6 weeks, and the infected mice were injected with self-illuminating Brucella necroticus, and the treatment was started 10 days after infection. The administration groups were as follows: 10 mice per group:

Untreated group: give an equal volume of deionized water after infection as a negative control;

Uninfected group: uninfected bacterial group, as a background baseline for the detected luminescence;

Rifampicin + streptomycin group: rifampicin 10mg/kg and subcutaneous injection of streptomycin 150mg/kg;

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

Q203 group: Q203 administered by intragastric administration were 25mg/kg, 6.25mg/kg, 1.6mg/kg;

The perfusion volume of each drug was 0.2 mL/time/time, and it was administered only once a day for 5 consecutive days.

(2) Indicator detection

The in vivo luminescence value of the mice was detected according to the design time point. After the anesthetized mice on the seventh day, the cervical spine was dissected and sacrificed. The tissue of the foot was ground and the tissue luminescence value was measured. The smaller the luminescence value, the better the drug treatment effect, and vice versa. .

(3) Experimental results

1. The results of continuous detection of the luminescence value of the foot of a living mouse are shown in Fig. 2. As can be seen from Fig. 2, COMX as low as 0.4 mg/kg has exhibited significant activity against Mycobacterium breve. Only 0.8mg/kg of COMX was better than 10mg/kg of rifampicin and 150mg/kg of streptomycin, indicating that COMX has good activity against Mycobacterium bryogenes and is in vivo. The effect is faster than the drugs currently used. Two days after discontinuation of the drug, the first-line therapy (RIF10+STR150) mice showed a rebound in in vivo luminescence, while COMX did not rebound above 3.1 mg/kg.

2. The results of luminescence detection of the foot tissue of 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) treatment group can still detect higher luminescence value, and 12.5 mg. The COMX treatment group above /kg has even reached the background value (the same value as the uninfected group), indicating that COMX can significantly shorten the course of treatment.

3. Photographs of the feet of the mice taken on day 7 (5 days after treatment, every other day). The results are shown in Fig. 4. As is apparent from Fig. 4, the feet of the COMX treatment group returned to normal (and The uninfected group has been basically the same), indicating that COMX has a very good therapeutic effect on Brucella necrosis.

4. The inhibitory mechanism of COMX and Q203 on Mycobacterium smegmatis is similar. The experimental results of inhibition of Mycobacterium bryogenes by B203 in mice are shown in Figure 5. Q203 is also very important for Bruley necrosis. Good treatment effect.

Example 4

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

(1) Animal grouping and administration

Female C57BL/6 mice were selected and infected with M. leprae in the foot of the mice. The drug was administered 12 weeks after infection. The drug administration was as follows, 20 mice per group:

Untreated group: give an equal volume of deionized water as a negative control;

Rifampicin group: the amount of intragastric administration is rifampicin 10 mg/kg;

COMX group: COMX was administered and administered by intragastric administration at a dose of 25 mg/kg;

Group Q203: Q203 was administered and administered by intragastric administration at a dose of 25 mg/kg;

The perfusion amount of each drug was 0.2 mL/time/time, and it was administered only once a day for 4 weeks for continuous treatment and 5 days for weekly treatment.

(2) Indicator detection

After the end of treatment in all groups, wait for 4 weeks, the mice were sacrificed by anesthesia for cervical dislocation, and the soft tissue of the infected foot was taken out with scissors. The tissue genome was extracted and purified by quantitative PCR. The upstream detection primer sequence was: 5 '-GCAGTATCGTGTTAGTGAACAGTGCA-3', the downstream detection primer sequence is 5'-CGCTAGAAGGTTGCCGTATGTGC-3'. The amount of Mycobacterium leprae suspension was determined as a standard control, and the amount of bacteria in the tissues of each group of mice was converted according to the fluorescence quantitative PCR result curve.

(3) Experimental results

The test results are shown in Fig. 6. The mouse bacterial load of the pyrazolopyridine compound (represented by COMX) and the imidazopyridine compound (represented by SQ203) was lower than that of the control group and 10 mg/kg. Rifampicin positive control group. Ming COMX and SQ203 have very good therapeutic effects on leprosy.

Example 5

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

(1) Experimental grouping

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

COMX group: concentration of 0.1 μg / mL, 0.01 μg / mL, 0.001 μg / mL;

Group Q203: concentration of 0.1μg/mL, 0.01μg/mL, 0.001μg/mL

The above-mentioned COMX and Q203 were dissolved in DMSO for use.

(two) experimental steps

1. Self-illuminating Mycobacterium marinum. The self-illuminating Mycobacterium marinum was connected to 7H11 solid medium and cultured at 30 ° C for 7 days; single colonies were picked from the plate and the relative light unit (RLU) was detected using a luminescence detector. The single colony that confirmed the luminescence was incubated in 50 mL of 7H9 liquid medium for about 3 days, resuspended in sterile physiological saline, filtered through a 300-mesh cell sieve, and filtered to form a single existing bacterial suspension for detection. experiment.

2, continuous continuous detection of dosing

To each EP tube, 198 μL of the bacterial solution and 2 μL of the drug prepared drug were separately added, and the pipetting drug was mixed with the bacterial liquid using a pipetting gun. After incubation for 24 hours, detect the bactericidal or bacteriostatic effect of the drug: After placing the EP tube into the luminescence detector, wait for 1-2 seconds, wait until the outside light is quenched, and press the detection button to record the RLUs of each EP tube.

(3) Experimental results

The method for calculating the growth inhibition rate and the minimum inhibitory concentration of the compound against the bacteria was the same as in Example 1. Mycobacterium marinum and Brucella necrotica are highly related, and the genomic similarity rate of the two is 99%. The results showed that both COMX group and Q203 could significantly inhibit Mycobacterium marinum, and the minimum inhibitory concentration of both was 0.01 μg/mL, and the antibacterial activity was very good.

It should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention, although the present invention will be described in detail with reference to the preferred embodiments, The technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. Use of a pyridine compound for the preparation of a medicament for treating a mycobacterial infectious disease, characterized in that the pyridine compound has the following structural 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 stands for Me;

   R3 represents CF ₃ , formula (a), formula (b), formula (c), formula (d), formula (e) or formula (f):

   
2. The use according to claim 1, wherein the pyridine compound has the following structural formula:

   
3. The use according to claim 1, characterized in that the mycobacterium is Brucella necroticum.
4. The use according to claim 1, wherein the mycobacterium is Mycobacterium marinum or Mycobacterium leprae.
5. The use according to claim 1, wherein the drug is an oral dosage form or an external dosage form.
6. The use according to claim 5, wherein the oral dosage form comprises tablets, capsules, granules and oral liquid; the external dosage form comprises ointments, gels, band-aids and patches.
7. A medicament for treating a mycobacterial infectious disease, characterized in that the medicament comprises a pyridine compound and a pharmaceutically acceptable carrier, and the pyridine compound has the following structural 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 CF ₃ , formula (a), formula (b), formula (c), formula (d), formula (e) or formula (f):

   
8. The medicament for treating a mycobacterial infectious disease according to claim 7, wherein the pyridine compound has the following structural formula:

   
9. The medicament for treating a mycobacterial infectious disease according to claim 7 or 8, wherein the mycobacterial infectious disease is Brucella necrosis, Mycobacterium tuberculosis infectious disease or leprosy.
10. The medicament for treating a mycobacterial infectious disease according to claim 9, wherein the medicament is an oral dosage form or an external dosage form; preferably, the oral dosage form comprises a tablet, a capsule, a granule, and an oral liquid; The topical dosage forms include ointments, gels, band-aids and patches.

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