CN113143924A - Application of thioimidazolidinone medicine in treating COVID-19 diseases - Google Patents
Application of thioimidazolidinone medicine in treating COVID-19 diseases Download PDFInfo
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Abstract
The invention belongs to the technical field of medicines, and particularly relates to an application of a thioimidazolidinone medicine or a pharmaceutically acceptable salt thereof in preparing medicines for treating ACE 2and TMPRSS2 protein disorder-related diseases, in particular to an application in preparing medicines for treating COVID-19 diseases.
Description
Technical Field
The invention belongs to the technical field of antiviral drugs, and particularly relates to an application of a thioimidazolidinone drug in treating COVID-19 diseases.
Background
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a severe respiratory disease caused by 2019 coronavirus disease (COVID-19) which is outbreak in 12 months in 2019. SARS-CoV-2 belongs to the coronavirus type B, and is the same as SARS-CoV and MERS-CoV. The characteristics of SARS-CoV-2 are highly similar to those of SARS-CoV. The key to the infection of human cells by the novel coronavirus is the binding of the coronavirus S protein (Spike Glycoprotein, TMPRSS2) to the ACE2 protein (Angiotensin converting enzyme 2) in humans (Letko, M., Marzi, A. & Munser, V.functional assessment and cell entry and receiver use for SARS-CoV-2and other lines B beta-coronaviruses. Nat. Microbiol.5, 562-569 invasion (2020), by binding to it and inducing infection in humans. The new crown virus uses the S protein to bind to angiotensin converting enzyme 2 (ACE 2 for short) receptor on the Cell, and in turn activates the S protein through TMPRSS2 to facilitate Entry of the new crown virus into the Cell (Hoffmann, M.et. SARS-CoV-2Cell Entry on ACE 2and TMPRSS 2and Is Blocked by a Clinical Proven protein enzyme inhibitor. Cell S0092867402220394 (2020) doi: 10.1016/j.cell.2020.02.052.).
Currently, clinical research work for treating Covid-19 patients in the prior art is completed or is ongoing, including a Hurrill Covid-19 prophylactic vaccine, Covid-19 neutralizing antibody bamlanivimab from Gift, and an antiviral drug remdesivir from Gilidide, which have definite clinical significance, but there is no standard therapeutic means for COVID-19.
This virus is relatively stable at the beginning of the SARS-CoV-2 pandemic, but several variants of this virus have recently been discovered and are spreading rapidly. The viral variants b.1.1.7, b.1.351 and p.1, which were first present in the uk, south africa and brazil, respectively, mutated in the spike protein, some of which mutations occur in the regions targeted by currently used antiviral drugs and vaccines.
In the prior art (cell.2021Apr 29; 184(9):2384-2393.e12.doi:10.1016/j. cell.2021.03.036), the German primate center, Markus Hoffmann and Stefan, the Labrinz Primates institute
A research team leading by Jan Munch, university of Ulm medical center, Germany, found that SARS-CoV-2 variants B.1.351 and P.1 are no longer inhibited by antibodies used in COVID-19 therapy. Furthermore, these variants are less efficiently inhibited by antibodies from convalescent patients and vaccinees. Thus, recovery from COVID-19 and vaccination may only provide incomplete protection against these viral variants.
Therefore, the discovery and development of the current COVID-19 treatment is an urgent need to meet medical needs, especially against the epidemic caused by the SARS-CoV-2 variants, such as variants B.1.351, P.1 and B.1.1.7.
Disclosure of Invention
In order to solve the problems, the invention provides a thioimidazolidinone compound with a structure shown as the following formula (I) or a pharmaceutically acceptable salt thereof, and the thioimidazolidinone compound is used for treating diseases related to the imbalance of ACE 2and TMPRSS2 proteins:
in another aspect, the invention also provides a thioimidazolidinone compound with a structure as shown in the formula (I) or a pharmaceutically acceptable salt thereof, and an application of the thioimidazolidinone compound or the pharmaceutically acceptable salt thereof in preparing medicines for treating diseases related to the imbalance of ACE 2and TMPRSS2 proteins.
In one embodiment, Y in the compound of formula (I) is independently selected from hydrogen, halogen, C1-C3Alkoxy, hydroxy, trifluoromethoxy or cyano, Z is independently selected from trifluoromethyl, halogen, cyano, C optionally substituted with one or more halogens1-C4Alkyl, or C optionally substituted with one or more halogens1-C4An alkoxy group.
In another embodiment, the ACE 2and TMPRSS2 dysregulation is selected from the group consisting of diseases associated with ACE 2and TMPRSS2 protein overexpression.
In another embodiment, the disease associated with overexpression of ACE 2and TMPRSS2 proteins is a coronavirus-induced disease, wherein the coronavirus-induced disease is COVID-19, and more preferably a coronavirus after mutation of SARS-CoV-2; the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 or SARS-CoV-2B.1.526 type variants, preferably SARS-CoV-2P.1, SARS-CoV-2 B.1.1.7.
In another embodiment, the compound of formula (I) wherein Y is F and Z is CF3The structural formula is shown as the formula (I-A):
in another embodiment, the present invention provides the use of the thioimidazolidinone having a structure according to formula (I-A) for treating a COVID-19 disease; the COVID-19 disease is more preferably a disease caused by coronavirus after mutation of SARS-CoV-2, and the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 or SARS-CoV-2B.1.526 type variant, preferably SARS-CoV-2P.1, SARS-CoV-2 B.1.1.7.
In another embodiment, the invention also provides the use of the thioimidazolidinone with a structure of formula (I-A) in the preparation of a medicament for treating a COVID-19 disease; the COVID-19 disease is more preferably a disease caused by coronavirus after mutation of SARS-CoV-2, and the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 or SARS-CoV-2B.1.526 type variant, preferably SARS-CoV-2P.1, SARS-CoV-2 B.1.1.7.
In another embodiment, the thioimidazolidinone having a structure according to formula (I-A) is in the form of a polymorph, pseudopolymorph, amorphous, or hydrate.
In another embodiment, the thioimidazolidinone having a structure according to formula (I-a) is crystalline form a, and the X-ray powder diffraction 2 θ thereof comprises the following peaks: 9.2 plus or minus 0.2, 14.6 plus or minus 0.2, 14.9 plus or minus 0.2, 16.5 plus or minus 0.2, 17.9 plus or minus 0.2, 18.2 plus or minus 0.2, 21.8 plus or minus 0.2, 22.4 plus or minus 0.2 and 23.5 plus or minus 0.2. The specific X-ray powder diffraction pattern can be seen in CN 201510861715.0.
In another embodiment, the medicament is in the form of an oral solid formulation, preferably a tablet, capsule or suspension. In the case that the oral solid preparation is a tablet, the effective dose of the thioimidazolidinone with the structure of formula (I-A) in the tablet is 100mg, and the effective dose of the thioimidazolidinone is 200mg or 300mg taken by a patient every day, and the thioimidazolidinone is taken once a day for 14 to 15 days; for example, 200mg per day for 15 consecutive days, or 300mg per day for 14 consecutive days.
In another embodiment, the present invention provides the use of the thioimidazolidinone compound of formula (I-A) in combination with any one of Apalutamide, Abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119, and SHR3680 for the preparation of a medicament for the treatment of COVID-19.
In another embodiment, the present invention also provides the use of the thioimidazolidinone compound of formula (I-A) in combination with any one of Apalutamide, Abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119, and SHR3680 for the treatment of COVID-19 diseases.
In addition, the invention also provides application of any one single compound of Apalutamide, Abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119 and SHR3680 in preparation of a medicament for treating COVID-19 diseases.
Preferably, in the above use, the thioimidazolidinone compound of the formula (I) or a pharmaceutically acceptable salt thereof is a thioimidazolidinone compound of the formula (I-a) or a pharmaceutically acceptable salt thereof.
In another embodiment, the thioimidazolidinone having a structure according to formula (I-A) is crystalline form A, and the present invention accordingly provides the use of crystalline form A in combination with any one or more of Enzalutamide, Apalutamide, Bicalutamide, Abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119, and SHR3680 for the treatment of COVID-19 diseases; more preferably, the COVID-19 disease is a disease caused by a coronavirus after mutation of SARS-CoV-2, and the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 or SARS-CoV-2B.1.526 type variant, preferably SARS-CoV-2P.1 and SARS-CoV-2 B.1.1.7.
In another embodiment, the thioimidazolidinone having a structure according to formula (I-A) is crystalline form A, and the present invention accordingly provides the use of crystalline form A in combination with any one or more of Enzalutamide, Apalutamide, Bicalutamide, Abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119, and SHR3680 for the preparation of a medicament for the treatment of a COVID-19 disorder; more preferably, the COVID-19 disease is a disease caused by a coronavirus after mutation of SARS-CoV-2, and the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 or SARS-CoV-2B.1.526 type variant, preferably SARS-CoV-2P.1 and SARS-CoV-2 B.1.1.7.
The CAS number of the Enzalutamide is 915087-33-1; apalutamide has a CAS number of 956104-40-8; the CAS number of Bicalutamide is 90357-06-5; the CAS of Abiraterone is 154229-19-3; ODM-201 has CAS number 1297538-32-9; EPI-001 has a CAS number of 227947-06-0; the CAS number of ONC1-13B is 1351185-54-0; HC-1119 has CAS number 1443331-82-5.
EM-5854 structural formula is
SHR3680 has a structural formula of
The applicant finds that the thioimidazolidinone medicament with the structure shown in the formula (I) in the invention, in particular the thioimidazolidinone with the structure shown in the formula (I-A), can obviously reduce the expression levels of ACE 2and TMPRSS2 proteins in Lncap, A549 and RLE-6 TN; and surprisingly found that an unexpected technical effect is achieved in treating COVID-19, which can reduce the hospitalization rate of patients with COVID-19, the use condition of breathing machines and possibly the death rate of the patients, thereby having a remarkable treatment effect.
Especially against the SARS-CoV-2 mutation of type P.1, the compounds of the invention still have effects; aiming at SARS-CoV-2 mutant patients (both male and female patients) of type P.1, the thioimidazolidinone compound with the structure of formula (I-A) in the experimental group of the invention is used as a test drug, all the patients survive (14/14), and no death case occurs; in 16 patients in the control group, 5 cases of death (5/16) are achieved, so that the medicine can effectively reduce the death rate of SARS-CoV-2P.1 mutant patients. In addition, cytological experiments show that the medicine of the present invention may be also effective on SARS-CoV-2B.1.1.7 infection. Pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts disclosed herein can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by the following method: reacting the free acid or base forms of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of both; generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. A list of suitable salts is described in Remington's Pharmaceutical Sciences, 17 th edition, Mack Publishing Company, Inc., of Isatoton, Pa, 1985, p. 1418 and Journal of Pharmaceutical Science,66,2(1977), which are incorporated herein by reference in their entirety.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and other problems or complications commensurate with a reasonable benefit/risk ratio.
Defining: SARS-CoV-2P.1 refers to the strain P.1 according to the "Pango lineages" typing method; other classifications are named the same way.
For information on the "Pango lines" typing method and its viral strains, seehttps://cov- lineages.org/index.html。
Drawings
FIG. 1: influence of Thioimidazolidone of formula (I-A) on expression of ACE 2and TMPRSS2 proteins, KT in FIG. 1 represents Thioimidazolidone of formula (I-A).
FIG. 2: the effect of the thioimidazolidinone with the structure of formula (I-A) for inhibiting SARS-COV-2 infection is shown in the figure.
Detailed Description
The invention may be further understood by the examples, however, it is to be understood that these examples are not limiting of the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the invention as described herein and claimed below.
In the examples, the thioimidazolidinone crystal form having the structure of formula (I-a) is used as crystal form a, and the preparation thereof can be carried out according to the teaching of CN 201510861715.0, unless otherwise specified.
Influence of thioimidazolidinone with structure of formula (I-A) on expression of ACE 2and TMPRSS2 proteins in Lncap, A549 cells
A Western-blot method is used for detecting the degradation effect of the thioimidazolidinone with the structure shown in the formula (I-A) on proteins of ACE 2and TMPRSS2 in a prostate cancer cell strain LnCap and a lung cancer cell A549.
1.1 Experimental materials
| Cells | Source | Culture medium |
| Lncap | Cell bank of Chinese academy of sciences | RPMI 1640+10％FBS |
| A549 | Cell bank of Chinese academy of sciences | RPMI 1640+10％FBS |
1.2 test methods
1.2.1 culture of cells
Collecting LNCaP and A549 cells frozen in liquid nitrogen tank, recovering and culturing cells to growth phase, culturing with CSS culture medium (Charcol Linear bovine serum (FBS) treated with activated carbon), spreading into six-well plate (each well is about 1 × 10)6One).
1.2.2 pharmaceutical formulation
Preparing thioimidazolidinone with the structure of the formula (I-A) by DMSO (dimethyl sulfoxide) to ensure that the storage concentration of the thioimidazolidinone is 10mM, diluting the drugs according to concentration gradient, adding the diluted drugs into a cell culture solution, adding DHT (Double Hydrogen Testosterone) to stimulate cells, acting on the cells for 48 hours, and collecting the cells.
1.2.3 Collection of cells
The cells were collected by centrifugation, the supernatant was washed with PBS (Phosphate Buffer Saline), centrifuged, washed again with PBS, and the PBS was discarded completely.
1.2.4 Collection of proteins
The collected cells were lysed with an appropriate amount of lysate (proteasome inhibitor PMSF was added to the lysate) on ice for 30min, centrifuged at 12000rpm for 20min, and the supernatant was taken.
1.2.5BCA assay for Total protein
1) Preparing standard protein with gradient concentration, and putting 20 mu l of the standard protein into a 96-well plate;
2) dissolving 2 μ l of protein lysate in 18 μ l of PBS, and making a multiple hole for each sample;
3) add 200. mu.l BCA working solution to each well, incubate for 30min at 37 ℃;
4) measuring absorbance at 562 nM;
5) the protein concentration of each sample was calculated and the volume of protein lysate required to be 50. mu.g was calculated.
1.2.6Western-blot detection of protein expression level
1) Denaturation: taking protein lysate, adding a proper amount of 5 × loading buffer, and denaturing at 100 ℃ for 5 min;
2) electrophoresis: performing electrophoresis with 10% prefabricated gel at 200V for 30 min;
3) film transfer: cutting off the concentrated gel, transferring the protein on the gel to a PVDF membrane by a wet transfer method, and performing treatment at 270mA for 2 h;
4) and (3) sealing: placing the PVDF membrane in 5% skimmed milk, and sealing at room temperature for 1 h;
5) a first antibody: cutting the PVDF membrane according to the molecular weight of Mark, respectively placing the PVDF membrane in AR, ACE2, TMPRSS 2and GAPDH antibodies, and incubating overnight in a shaking table at 4 ℃;
6) washing the membrane: placing the PVDF membrane in PBST, and shaking and cleaning the PVDF membrane for 4 times and 6 minutes each time by a shaking table;
7) secondary antibody: placing the PVDF membrane in a corresponding secondary antibody solution, oscillating in a shaking table, and incubating at room temperature for 1-2 h;
8) and (3) detection: detection by ECL.
1.3 results of the experiment
The experimental result shows that the thioimidazolidinone with the structure of formula (I-A) can reduce the expression level of ACE 2and TMPRSS2 proteins in LNCaP and A549; in addition, Dihydrotestosterone (DHT) induced expression of ACE 2and TMPRSS2 proteins in LNCaP and a549 cells, indicating that androgen-AR signaling regulates key proteins for SARS-CoV-2 binding and entry into host cells (figure 1).
Secondly, the clinical effect of the medicine of the thioimidazolidinone with the structure of the formula (I-A) on treating COVID-19
Thioimidazolidone of the structure of formula (I-A) was prepared into a tablet (reference CN 201510861715.0) containing 100mg of the active ingredient as a test drug.
The clinical study was a prospective, interventional, placebo-controlled, double-blind, randomized parallel partition study. 214 eligible patients (of which the experimental group had 114 and 100 placebo controls) were included in this trial.
The following criteria are required for diagnosis and inclusion: 1) male; 2) the age is more than or equal to 50 years old; 3) patients who exhibit Gabrin's disease, androgenetic alopecia, have a Hamilton ■ Nowood rating of three or more levels; 4) the rtPCR test shows positive SARS-CoV-2 in the past 7 days; 5) no hospitalization due to acute respiratory symptoms; 6) patients with appropriate bone marrow, liver and kidney functions; 7) serum creatinine is less than or equal to 1.5xULN or creatinine clearance is more than or equal to 60mL/min (calculated by using Cockcroft-Gault formula); 8) and (3) coagulation: INR is less than or equal to 1.5 multiplied by ULN, APTT is less than or equal to 1.5 multiplied by ULN; 9) written informed consent was obtained prior to any screening procedures.
Group entry exclusion criteria: 1) subjects participated in a study to study COVID-19 drugs; 2) subjects taking any type of antiandrogen, including: androgen-lowering therapy, 5-alpha reductase inhibitors, and the like; 3) patient excipients allergic to study products or similar drugs (or other drugs); 4) subjects with malignancy in the last 5 years, with the exception of basal cell and squamous cell carcinomas where resection is done, completely resecting any type of carcinoma in situ; 5) syndrome of the past 6 months known subjects with severe cardiovascular disease, congenital long QT syndrome, condyloma acuminatum, myocardial infarction, or arterial thrombosis, unstable angina or congestive heart failure are classified as New York Heart Association (NYHA) grade 3 or higher, or ventricular ejection fraction (LVEF) < 50%, QTcF >450 ms; 6) subjects with uncontrolled medical conditions may be compromised in research (e.g., uncontrolled hypertension, hypothyroidism, diabetes); 7) patients enrolled in the experimental drug study within 4 weeks began the study treatment; 8) known Human Immunodeficiency Virus (HIV) diagnosis, hepatitis c, active hepatitis b, treponema pallidum (non-mandatory examination); 9) unwilling or unable to provide informed consent.
The treatment period is as follows: for 30 days.
Therapy, dosage and mode of administration:
1) in the control group, male subjects took daily: 200mcg/kg of ivermectin, q.d, continuously taking the medicine for 5 days; azithromycin 500 mg, q.d, is administered continuously for 5 days.
2) Experimental group, male subjects took daily: 200mcg/kg of ivermectin, q.d, continuously taking the medicine for 5 days; 500 mg of azithromycin per day, q.d, for 5 days; test dose 200mg, q.d, was administered for 15 days.
Clinical evaluation criteria: 1. efficacy, percentage of subjects hospitalized with COVID-19; 2. safety, adverse events occurring during treatment.
The common primary endpoints included the percentage of subjects hospitalized with COVID-19 and the clinical symptoms assessment (using a 7-point scale) within 30 days (control group was administered 5 days after treatment, dose and administration regimen, and the trial statistics were performed on day 6-30, and experimental group was administered 16-30 after the treatment, dose and administration regimen, and the trial statistics were performed on day 30) as follows:
a stage: death; b, stage: receiving invasive mechanical ventilation or Extracorporeal Membrane Oxygenation (ECMO); c, stage: hospitalization using non-invasive ventilation or high flow nasal catheters; d stage: supplementing oxygen for hospitalization; e, level: oxygen is not supplemented during hospitalization; f, stage: hospitalization with limited activity (persistent symptoms) is not present; grade g: no symptoms.
Clinical results: analysis of the results of 114 test group subjects and 100 placebo control group subjects showed that the hospitalization rate of the patients in the test group was 0.8% (only 1 hospitalization after the above treatment in the test group), the percentage of ventilator used was 0%, the percentage of death was 0%, and no adverse events were found. The hospitalization rate of the control patients was 27.0%, the percentage of ventilator use was 9%, and the percentage of death was 2%.
In addition, the percentage rtPCR positive tests from day 0 to day 30 for the experimental and control groups were counted as in table 1:
TABLE 1 percentage rtPCR positivity over time
Starting on day 7, the test group of virus-positive patients was significantly reduced compared to the control group, and this trend continued up to day 30 results. (rtPCR, RT-PCR, is a method in which primers and probes are highly matched with a RNA region specific to the nucleic acid of the novel coronavirus, and the RNA region is judged to be positive or negative according to a CT value defined by the specification of the detection kit used)
In conclusion, the thioimidazolidinone with the structure shown in the formula (I-A) can inhibit the expression of ACE 2and TMPRSS2 so as to block SARS-COV-2 virus from entering host cells, and clinical data also show that the thioimidazolidinone with the structure shown in the formula (I-A) can reduce the hospitalization rate and the use condition of a breathing machine of a patient and can reduce the death rate of the patient, so that a remarkable treatment effect is generated, and the thioimidazolidinone is a clinically urgent treatment means at present.
Thirdly, the clinical effect of the compound of the thioimidazolidinone with the structure of the formula (I-A) on treating SARS-CoV-2P.1 mutant hospitalized patients
Preparing thioimidazolidinone with a structure of formula (I-A) into a tablet (CN 201510861715.0) with the compound active ingredient content of 100mg as a test drug; is used for evaluating the clinical effect of the test drug on SARS-CoV-2P.1 mutant patients (male and female).
Subject enrollment criteria: 1) hospitalized patients diagnosed with COVID-19 symptoms; 2) male and female patients aged 18 or older; 3) the laboratory rtPCR detection is confirmed to be positive for SARS-CoV-2 seven days before random grouping; 4) clinical status of COVID-19: hospitalization, no oxygen supplementation-no longer continuous medical care; hospitalization, no oxygen supplementation-continued medical care (COVID-19 related or otherwise) is required; and c hospitalization, requiring oxygen supplementation; 5) blood coagulation: INR is less than or equal to 1.5 multiplied by ULN, APTT is less than or equal to 1.5 multiplied by ULN; 6) subjects had written informed consent prior to any study procedure 7) subjects consented not to participate in other clinical trial studies of COVID-19.
Group entry exclusion criteria: 1) subjects participated in a study to study COVID-19 drugs; 2) mechanical ventilation is required; 3) the subjects were involved in any kind of anti-androgen therapy: androgen deprivation therapy, 5-alpha reductase inhibitors, and the like; 4) patients allergic to study products or similar drugs (or any excipients); 5) subjects with malignancy in the last 5 years, except for basal and squamous cell carcinomas where resection is done, with total resection of any type of carcinoma in situ; 6) subjects known to have severe cardiovascular disease, congenital long QT, have a past 6 month syndrome, condyloma acuminatum, myocardial infarction, or arterial thrombosis, unstable angina or congestive heart failure classified as New York Heart Association (NYHA) grade 3 or higher, or ventricular ejection fraction (LVEF) < 50%, QTcF >450 ms; 7) subjects with uncontrolled medical conditions may be compromised in research (e.g., uncontrolled hypertension, hypothyroidism, diabetes); 8) confirmed diagnosis is Human Immunodeficiency Virus (HIV) diagnosis, hepatitis c, active hepatitis b, treponema pallidum (mandatory examination); 9) alanine Aminotransferase (ALT) or aspartate Aminotransferase (AST) > 5 times the upper normal limit; 10) estimating glomerular filtration rate (eGFR) <30 ml/min; 11) severe kidney disease requiring dialysis; 12) women with fertility potential are defined as all physiologically competent to become pregnant women, unless they use highly effective contraceptive measures. 13) Sexually active men must use condoms during intercourse while taking the test medications, and should not give birth to children during the 3 months after discontinuation of the medication. Men with vasectomized males also need to use condoms to prevent drug delivery through the semen. 14) The subject may be transferred to another hospital within the next 28 days; 15) the subject is unwilling or unable to provide informed consent.
The treatment period is as follows: day 28, with dosing on days 1 to 14 and no dosing on days 14 to 28 (medical observation only).
Therapy, dosage and mode of administration:
1) control group: the subjects took daily: placebo 300mg once daily for 14 days; placebo was administered for 14 days while being treated in combination with hospital standard therapy.
The placebo replaces its thioimidazolidinone of formula (I-a) structure with microcrystalline cellulose, according to the teaching of the prior CN 201510861715.0, specification 100mg (see table 6 thereof).
2) Experimental groups: the subjects took daily: 300mg of test drug (calculated by the effective dose of the active ingredients) is taken once a day for 14 days; the drug administration was carried out for 14 days while combining the standard hospital therapy.
The clinical evaluation criteria were: the number of deaths of the subjects within 28 days after the administration of the drug was counted, and the results are shown in table 2 (sample data from a part of samples from the NCT04728802 experiment, 30 hospitalized patients with SARS-CoV-2 mutation detected as p.1 type (male/female: 18/12, age between 38.0 and 58.0, average age 44.5 years) were randomly selected, wherein the experimental group was 14 (male/female: 9/5, age between 38.7 and 60.5, average age 47.0 years), and the control group was 16 (male/female: 9/7, age between 37.2 and 51.7, average age 44.5).
Table 2 clinical results:
the clinical experiment result shows that 14 patients in the experimental group survive and die in 30 SARS-CoV-2P.1 mutant hospitalized patients; 5 deaths occurred in 16 of the controls. The results of the experimental group and the control group are very different. Therefore, the compounds of the present invention can effectively treat diseases caused by variant SARS-CoV-2, especially the mutation of P.1 SARS-CoV-2.
Wherein the patients with the strain of the P.1 strain are identified by the following detection method: after a patient is hospitalized, collecting nasopharyngeal swab samples to extract virus RNA, confirming a positive infection sample with ct <26 by RT-PCR, sending the positive infection sample to a sequencing laboratory, confirming a high virus load sample by RT-PCR again, carrying out library construction and second-generation sequencing, carrying out genome assembly after original off-line data is subjected to joint removal, carrying out strain classification on the assembled genome after quality control by using PANGLIN (https:// githu. com/hCoV-2019/PANGOLIN), and confirming a P.1 mutant strain. The detection method can be implemented by referring to the teaching in the prior art (genes (Basel) 2020 Aug; 11(8):949.doi:10.3390/genes 11080949).
Fourthly, the effect of the thioimidazolidinone compound with the structure of the formula (I-A) on SARS-COV-2and SARS-CoV-2B.1.1.7 infected cells
And (3) testing the sample: the compound of formula (I-A) was dissolved in DMSO and stored at-20 ℃.
Test cell lines: LNCaP cells were purchased from American Type Culture Collection (ATCC).
Observation indexes are as follows: AlexaFluor 647 fluorescence intensity under fluorescence microscope.
The evaluation method comprises the following steps: the antiviral effect of the drug is analyzed by detecting the fluorescence intensity under a fluorescence microscope.
The experimental method comprises the following steps:
1) LNCaP cell culture: culturing in RPMI1640 medium containing 10% fetal calf serum in 5% CO2And culturing in a cell culture box at 37 ℃ under saturated humidity. Subculture according to ATCC instructions.
2) Experiment of viral infection
Taking LNCaP cells in logarithmic growth phase at 1 × 104Individual cells were seeded in 384-well plates (Corning, 3603) with cells in 384-well plates at 5% CO2After incubation for 18 hours at 37 ℃ for 24 hours, the compound of formula (I-A) was added, starting at a maximum concentration of 3. mu.M, diluted 2-fold, for a total of 10 concentrations, each of which was repeated in 5 duplicate wells. After 24h, SARS-COV-2 (isolated from: USA-WA1/2020, https:// www.beiresources.org/Catalog/animalviroses/NR-52281. aspx) and SARS-CoV-2B1.1.7 virus were added to LNCaP cells in the BSL3 laboratory for 48 h. 4% paraformaldehyde fixed, permeabilized with 0.03% Triton X-100, and blocked with antibody buffer (1.5% bovine serum albumin, 1% goat serum, 0.0025% Tween-20). Plates were sealed, surface decontaminated and transferred to BSL2 laboratory staining. Staining was performed with SARS-CoV-2 nucleoprotein primary antibody (ProSci Cat No.: ProSci; 35-579,1:2000) followed by anti-mouse IgG: AlexaFluor 647secondary (Invitrogen Cat No.: A21235,1: 1000) and Hoechst 33342(Invitrogen Cat No.: under H3570). Each well has two fluorescence channels (Hoechst-386/23nm, N-protein-650/13nm), and each well captures a total of 9 fields of view. The percentage of infected cells was quantified using CellProfiler. Infected cell regions were first identified in the N protein image by two types of Otsu segmentation. Nuclei are then identified in a similar manner and associated with the infected cell region using the related objects module. If the nucleus is located within the area of infected cells, the infected cells are identified and the percentage of each well is calculated from the infected cells/total cell count. Simultaneously, the vitality of the cells is detected, and the influence of the compound on the vitality (vitality) of the cells is analyzed.
And (3) data analysis:
and observing the strength of fluorescence combination under a fluorescence microscope. Dose-response curves were fitted using a four-parameter logistic model in Graphpad Prism and IC is listed50The value is obtained. Plate-based normalization was performed using 32 infected (0% effect) and 32 uninfected (100% effect) control wells (mean LNCaP infectivity range: 18-25%). Viability was assessed by comparing cell counts in treated wells to the average cell count (100% viability) in 32 uninfected control wells.
Results of the experiment
Applicants evaluated the effect of compounds of formula (I-A) on SARS-CoV-2and SARS-CoV-2B.1.1.7 virus infection of LNcap cells. The SARS-CoV-2 virus nucleocapsid protein is detected and quantified by high content immunofluorescence imaging technique, and the cell nucleus is counted by Hoechst-33342. Using this SARS-CoV-2 bioassay platform, we demonstrate that the compound of formula (I-A) can effectively inhibit SARS-CoV-2and SARS-CoV-2B.1.1.7 virus from infecting LNCaP cells in LNCaP cells, and is dose-dependent. IC that inhibits viral SARS-Cov-2and SARS-Cov-2B.1.1.7 infection50The values were 69nM and 48nM, respectively (FIG. 2), demonstrating that the compounds of formula (I-A) of the present invention have therapeutic effects on SARS-CoV-2B.1.1.7 virus infection.
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (11)
1. Use of a thioimidazolidinone compound having a structure according to formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a disease associated with the dysregulation of ACE 2and TMPRSS2 proteins:
y in the compound of the formula (I) is independently selected from hydrogen, halogen and C1-C3Alkoxy, hydroxy, CF3O or cyano, Z is independently selected from CF3Halogen, cyano, C optionally substituted by one or more halogens1-C4Or C optionally substituted by one or more halogens1-C4Alkoxy group of (2).
2. The use of claim 1, wherein the ACE 2and TMPRSS2 protein dysregulation related disease is selected from ACE 2and TMPRSS2 protein overexpression related diseases.
3. The use of claim 2, wherein the disease associated with overexpression of ACE 2and TMPRSS2 proteins is a disease caused by a coronavirus.
4. Use according to claim 3, wherein in the compound of formula (I) Y is F and Z is CF3The structural formula is shown as the formula (I-A):
5. the use according to claim 4, which is the use of the thioimidazolidinone with a structure represented by formula (I-A) in the preparation of a medicament for treating a COVID-19 disease or the use of the thioimidazolidinone with a structure represented by formula (I-A) in the preparation of a medicament for treating a SARS-CoV-2 mutated coronavirus-caused disease; the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 or SARS-CoV-2B.1.526 type variants, and is selected from SARS-CoV-2P.1 and SARS-CoV-2 B.1.1.7.
6. The use according to claim 5, wherein the thioimidazolidinone having a structure according to formula (I-A) is a polymorph, pseudopolymorph, amorphous, or hydrate.
7. The use according to claim 6, wherein the thioimidazolidinone having a structure according to formula (I-A) is crystalline form A, having an X-ray powder diffraction 2 θ comprising the following peaks: 9.2 plus or minus 0.2, 14.6 plus or minus 0.2, 14.9 plus or minus 0.2, 16.5 plus or minus 0.2, 17.9 plus or minus 0.2, 18.2 plus or minus 0.2, 21.8 plus or minus 0.2, 22.4 plus or minus 0.2 and 23.5 plus or minus 0.2.
8. The use according to any one of claims 1-7, wherein the medicament is in the form of an oral solid formulation which is a tablet, capsule, suspension or pill.
9. The use according to claim 8, wherein the oral solid preparation is a tablet, the thioimidazolidinone having a structure according to formula (I-a) is contained in the tablet in an effective amount of 100mg, and the patient takes the tablet once a day in an effective amount of 200mg or 300mg for 14 to 15 consecutive days.
The use of any one of Apalutamide, Abiraterone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119 and SHR3680 in the preparation of a medicament for treating diseases caused by COVID-19 coronavirus; or in preparing medicine for treating SARS-CoV-2 mutation caused coronavirus diseases, the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 and SARS-CoV-2B.1.526 variant, preferably SARS-CoV-2P.1, SARS-CoV-2 B.1.1.7.
11. Use of a thioimidazolidinone having a structure according to claim 4 and any one or more of Enzalutamide, Apalutamide, Bicalutamide, abirtarone, ODM-201, EPI-001, ONC1-13B, EM-5854, JNJ-63576, TAS-3681, HC-1119, and SHR3680 in combination for the preparation of a medicament for the treatment of a covi-19 disorder; or in preparing medicine for treating SARS-CoV-2 mutation caused coronary virus disease, the SARS-CoV-2 mutation is any one of SARS-CoV-2B.1.351, SARS-CoV-2P.1, SARS-CoV-2B.1.1.7, SARS-CoV-2B.1.617.2, SARS-CoV-2A.23.1, SARS-CoV-2B.1.525, SARS-CoV-2B.1.427, SARS-CoV-2B.1.429 and SARS-CoV-2B.1.526 variant, and is selected from SARS-CoV-2P.1, SARS-CoV-2 B.1.1.7.
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