CN117255680A - Formulations for oral administration containing triazine derivatives - Google Patents

Formulations for oral administration containing triazine derivatives Download PDF

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Publication number
CN117255680A
CN117255680A CN202280007992.3A CN202280007992A CN117255680A CN 117255680 A CN117255680 A CN 117255680A CN 202280007992 A CN202280007992 A CN 202280007992A CN 117255680 A CN117255680 A CN 117255680A
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Prior art keywords
formula
compound
vii
acid
compound represented
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Inventor
五味真人
堀内健佑
森本甫享
高垣恵介
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Shionogi and Co Ltd
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Shionogi and Co Ltd
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Priority claimed from PCT/JP2022/043092 external-priority patent/WO2023027198A1/en
Publication of CN117255680A publication Critical patent/CN117255680A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/30—Only oxygen atoms
    • C07D251/32—Cyanuric acid; Isocyanuric acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/13—Dicarboxylic acids
    • C07C57/15—Fumaric acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Abstract

The present invention provides an orally administered preparation containing a triazine derivative having a virus proliferation inhibitory effect.

Description

Formulations for oral administration containing triazine derivatives
Technical Field
The present invention relates to an orally administered preparation containing a triazine derivative. More specifically, the present invention relates to an orally administered preparation containing a triazine derivative having coronavirus 3CL protease inhibitory activity, a pharmaceutically acceptable salt thereof, or a complex thereof as an active ingredient.
Background
Coronaviruses belonging to the subfamily Coronaviridae, the order Coronaviridae, have a genome size of about 30 kilobases and are the largest class of single-stranded positive strand RNA viruses among the known RNA viruses. Coronaviruses are classified into 4 kinds of alpha, beta, gamma and delta coronaviruses, and as coronaviruses infected with humans, 2 kinds of alpha coronaviruses (HCoV-229E, HCoV-NL 63) and 5 kinds of beta coronaviruses (HCoV-HKU 1, HCoV-OC43, SARS-CoV, MERS-CoV, SARS-CoV-2) are known in total of 7 kinds. Of these, 4 (HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC 43) are causative agents of the common cold, the remainder being novel coronavirus (SARS-CoV-2), and Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV) causing severe pneumonia.
The number of infection with novel coronavirus infection (covd-19) confirmed at time 21 of 9 of 2022 was 6.1 million or more, and the number of deaths was 650 ten thousand or more (non-patent document 1). As a main infection route of SARS-CoV-2, droplet infection, contact infection and aerosol infection were reported, and SARS-CoV-2 was confirmed to float in the air for about 3 hours together with aerosol, and maintain infectivity (non-patent document 2). The incubation period is about 2 to 14 days, and is typically cold-like symptoms such as fever (87.9%), dry cough (67.7%), tiredness (38.1%), and sputum (33.4%), etc. (non-patent document 3). In severe cases, acute respiratory distress syndrome and respiratory failure due to acute lung injury, interstitial pneumonia, etc. are caused. In addition, multiple organ failure such as renal failure and liver failure has been reported.
In japan, according to drug repositioning of existing drugs, adefovir as an antiviral agent, dexamethasone as an anti-inflammatory agent, and baratinib as a rheumatoid arthritis agent were approved as therapeutic agents against covd-19, and tolizumab as an anti-IL-6 receptor antibody was additionally approved at month 1 of 2022. In addition, in month 7 of 2021, ronnapsave (Casirivimab/imdevimamab) was approved as antibody cocktail therapy, in month 9 of 2021, sotrovmamab was approved in the special case, and in month 12 of 2021, mo Pila vir (molnupiravir) was approved in the special case. There is no adequate evidence for the effectiveness and safety of these agents. Thus, it is urgent to develop a therapeutic agent against covd-19.
Coronaviruses synthesize 2 polyproteins if they infect cells. The 2 multimeric proteins include 2 proteases and replication complexes for producing viral genomes. Proteases play an indispensable role in order to cleave multimeric proteins synthesized by viruses and to make the respective proteins function. Among the 2 proteases, 3CL protease (main protease) is almost responsible for cleavage of polyprotein (non-patent document 4).
The end of the Phase1b assay (NCT 04535167) of Lufotrelvir (PF-07304814), a prodrug of PF-00835231, by the company Pfizer was disclosed on ClinicalTrials. Gov as a 3CL protease-targeted COVID-19 therapeutic agent, month 2021. Furthermore, the company Pfizer published a Phase1 test for the new therapeutic agent PF-07321332 for coronavirus infection, month 2021. The structural formulae of PF-00835231, lufotrelvir and PF-07321332 are shown below, and differ from the chemical structures of the compounds of the present invention (non-patent documents 5, 12 and 13, and patent documents 6 and 7).
PF-00835231：
[ chemical formula 1 ]
Lufotrelvir(PF-07304814)：
[ chemical formula 2 ]
PF-07321332：
[ chemical 3 ]
Further, in 2021, 7 months, a Phase2/3 assay (NCT 04960202) was disclosed on ClinicalTrials. Gov which began the combined use of PF-07321332 and ritonavir with a patient of COVID-19 who had a high risk factor. Furthermore, on the homepage of Pfizer corporation, month 11 of 2021, PAXLOVID (TM) (PF-07321332; ritonavir) was reported to reduce the risk of hospitalization or death by 89% in high-risk patients of adults compared with placebo (non-patent document 14). Further, in 2021, at 12, PAXLOVID (TM) was approved for emergency use in the united states, and in 2022, at 2, 10, PAXLOVID (registered trademark) packaging was approved in japanese special cases.
Non-patent documents 5 to 8 disclose compounds having 3CL protease inhibitory activity, but none of the documents describes or suggests compounds, production methods and synthetic intermediates related to the present invention.
Patent documents 1 to 4 and 8 to 12 disclose that the compound has P2X 3 And/or P2X 2/3 Triazine derivatives and uracil derivatives which are receptor antagonistic, however, in either document, no inhibition of 3CL protease activity is aimed atSex and antiviral effects are described or suggested. In addition, the production method and the synthetic intermediate according to the present invention are not described or suggested.
Non-patent documents 9 to 11 disclose triazine derivatives having antitumor effects, but neither document describes the coronavirus 3CL protease inhibitory activity and antiviral effects, nor does it describe or suggest the compounds, production methods, and synthetic intermediates associated with the present invention.
Although patent document 5 discloses triazine derivatives having a glycylglycine receptor modulating effect, neither document describes or suggests 3CL protease inhibitory activity or antiviral effect. In addition, the production method and the synthetic intermediate according to the present invention are not described or suggested.
Prior art literature
Patent literature
Patent document 1: international publication No. 2012/020749
Patent document 2: international publication No. 2013/089212
Patent document 3: international publication No. 2010/092966
Patent document 4: international publication No. 2014/200078
Patent document 5: international publication No. 2012/009258
Patent document 6: international publication No. 2021/205298
Patent document 7: international publication No. 2021/250648
Patent document 8: chinese patent application publication No. 113620888 specification
Patent document 9: chinese patent application publication No. 113666914 specification
Patent document 10: chinese patent application publication No. 113735838 specification
Patent document 11: chinese patent application publication No. 113773300 specification
Patent document 12: chinese patent application publication No. 113801097 specification
Non-patent literature
Non-patent document 1: "covd-19 Dashboard by the Center for Systems Science and Engineering at Johns Hopkins University", [ online ], johns Hopkins University, [2022, 3, 14 search ], internet < URL: https:// corenavir. Jhu. Edu/map. Html >
Non-patent document 2: the NEW ENGLAND JOURNAL of MEDICINE (2020), volume 382, pages 1564-1567
Non-patent document 3: "Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (covd-19)", [ online ], 28, WHO, [2021, 8, 2) search ], internet < URL: https:// www.who.int/docs/default-source/corenaviuse/wh neighbor-joint-mission-o-covid-19-final-report. Pdf >
Non-patent document 4: science (2003), 300 rolls, pages 1763-1767
Non-patent document 5: "A comparative analysis of SARS-CoV-2antivirals characterizes 3CLpro inhibitor PF-00835231as a potential new treatment for COVID-19", journal of Virology, 2021, 4/26/2/15/2022/1 Session "), internet < URL: https:// journ als. Asm. Org/doi/10.1128/jvi.01819-20> < doi:10.1128/JVI.01819-20 ]
Non-patent document 6: cell Research (2020), volume 30, pages 678-692
Non-patent document 7: science (2020), volume 368, pages 409-412
Non-patent document 8: ACS Central Science (2021), 7, 3, 467-475 pages
Non-patent document 9: cancer Treatment Reviews (1984), 11 rolls, support 1, pages 99 to 110
Non-patent document 10: contributions to Oncology (1984), 18, pages 221-234
Non-patent document 11: arzneimittel-Forschung (1984), volume 11, no. 6, pages 663 to 668
Non-patent document 12:261st Am Chem Soc (ACS) Natl Meet 2021-04-05/2021-04-16. Virtual, N/A. Abst 243
Non-patent document 13: science (2021), volume 374, pages 1586 to 1593
Non-patent document 14: "Pfizer's Novel COVID-19Oral Antiviral Treatment Candidate Reduced Risk Of Hospitalization Or Death By 89%In Interim Analysis Of Phase 2/3EPIC-HR Study", [ online ], 5 th 11 th 2021, pfizer Press Release, [2022, 2, 15 th inspection line ], internet < URL: https:// www.pfizer.com/news/press-release/press-release-tail/pfizer-novel-covid-19-oral-anti-therapeutic-treatment-truck >
Non-patent document 15: AIMECS 2021 (AFMC International Medicinal Chemistry Symposium 2021), online seminar, 2021, 11, 29, 12, 2
Non-patent document 16: bioRxiv preprint doi: https: org/10.1101/2022.01.26.477782, "Discovery of S-217622, aNon-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19"
Non-patent document 17: J.Med.chem. (2022), volume 65, pages 6499-6512, "Discovery of S-217622,a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19"
Disclosure of Invention
Problems to be solved by the invention
The present invention provides a process for producing a triazine derivative having coronavirus 3CL protease inhibitory activity, a pharmaceutically acceptable salt thereof, or a complex thereof.
The present invention also provides an orally administered preparation containing a triazine derivative exhibiting coronavirus 3CL protease inhibitory activity, a pharmaceutically acceptable salt thereof, or a complex thereof as an effective ingredient.
Means for solving the problems
The present invention relates to the following.
(1) A process for producing a compound represented by the formula (III) or a salt thereof, which comprises reacting a compound represented by the formula (I) or a salt thereof with a compound represented by the formula (II) or a salt thereof in the presence of an acid
[ chemical formula 4 ]
(wherein R is 1 Is a substituted or unsubstituted C1-C4 alkyl group, R 2 Each independently is halogen, cyano or methyl, n is an integer from 1 to 5)
[ chemical 5 ]
(wherein R is 3 Each independently is a substituted or unsubstituted C1-C4 alkyl group, m is an integer from 0 to 5)
[ 6 ] A method for producing a polypeptide
(2) The production method according to the above (1), wherein the acid is trifluoroacetic acid.
(3) The production method according to the above (1) or (2), wherein the compound represented by the formula (III) is represented by the formula (III-1):
[ chemical 7 ]
(4) A process for producing a compound represented by the formula (VI) or a salt thereof or a solvate thereof, characterized by reacting a compound represented by the formula (IV) or a salt thereof with a compound represented by the formula (V) or a salt thereof in the presence of an acid
[ chemical formula 8 ]
(wherein R is 4 In the case of a substituted or unsubstituted aromatic heterocyclic group, or a substituted or unsubstituted aromatic carbon ring group, p is 0 or 1, and the other symbols have the same meaning as in (1)
[ chemical formula 9 ]
(wherein R is 5 Each independently is halogen, or substituted or unsubstituted alkyl, q is an integer from 0 to 5)
[ chemical formula 10 ]
(the labels in the formula are as defined above).
(5) The production method according to the above (4), wherein the acid is acetic acid.
(6) The production method according to the above (4) or (5), wherein the compound represented by the formula (VI) is represented by the formula (VII):
[ chemical formula 11 ]
(7) A method for producing a compound represented by the formula (VII), or a salt thereof or a solvate thereof, which comprises: a step of obtaining a compound represented by the formula (III) or a salt thereof by the production method according to any one of the above (1) to (3)
[ chemical formula 12 ]
[ chemical formula 13 ]
(8) A process for producing a fumaric acid co-crystal form I of a compound represented by the formula (VII), characterized by crystallizing a compound represented by the formula (VII) or a salt thereof in the presence of fumaric acid, acetone and water
[ chemical formula 14 ]
(9) The production method according to the above (8), characterized in that the compound represented by the formula (VII) or a salt thereof obtained by using the production method according to any one of the above (1) to (7) is crystallized
[ 15 ] A method of producing a polypeptide
(10) The production method according to the above (8) or (9), wherein the crystallization temperature is 40 to 60℃and the crystallization time is 120 minutes or more.
(11) Formula (VIII):
[ 16 ] the preparation method
A compound as shown, or a salt thereof.
(12) Formula (IX):
[ chemical formula 17 ]
A compound as shown, or a salt thereof.
(13) Formula (X):
[ chemical formula 18 ]
A compound as shown, or a salt thereof.
(14) Formula (XI):
[ chemical formula 19 ]
A compound as shown, or a salt thereof.
(15) Formula (VII):
[ chemical formula 20 ]
Toluene compounds of the compounds shown.
(16) Fumaric acid cocrystal form of a compound of formula (VII) which is substantially free of the free form of the compound of formula (VII)
[ chemical formula 21 ]
(17) The following formula:
[ chemical formula 22 ]
Methanesulfonic acid salts of the compounds shown.
(18) The following formula:
[ chemical formula 23 ]
Methanesulfonic acid salts of the compounds shown.
(19) The following formula:
[ chemical 24 ]
A compound as shown, or a salt thereof.
(20) The following formula:
[ chemical 25 ]
A compound as shown, or a salt thereof.
(21) The salt according to item (20) above, which is represented by the formula:
[ chemical 26 ]
1, 8-diazabicyclo [5.4.0] -7-undecene salts of the compounds shown.
(22) An orally administered formulation (pharmaceutical composition) comprising the formula (VII):
[ chemical formula 27 ]
The compound, pharmaceutically acceptable salt or complex thereof is shown as an active ingredient.
(23) The preparation (pharmaceutical composition) according to the above (22), wherein the active ingredient is a complex of a compound represented by the formula (VII), which is a complex comprising fumaric acid.
(24) The preparation (pharmaceutical composition) according to the above (23), wherein the complex is a co-crystal of the compound represented by the formula (VII) and fumaric acid in a molar ratio of 1:1.
(25) The preparation (pharmaceutical composition) according to any one of the above (22) to (24), wherein a polymer is contained in the preparation.
(26) The preparation (pharmaceutical composition) according to the above (25), wherein the polymer is at least 1 selected from the group consisting of cellulose-based polymers, acrylic-based polymers and vinyl-based polymers.
(27) The preparation (pharmaceutical composition) according to any one of the above (22) to (26), wherein the active ingredient is a compound obtained by the method according to any one of the above (4) to (10), a pharmaceutically acceptable salt thereof or a complex thereof.
(28) The preparation (pharmaceutical composition) according to any one of the above (22) to (27), which is used for the treatment and/or prevention of coronavirus infection.
(29) The preparation (pharmaceutical composition) according to the above (28), wherein the coronavirus infection is a novel coronavirus infection (COVID-19).
(30) The preparation according to the above (22), which contains 125.0mg of the compound represented by the formula (VII) as an active ingredient.
(31) The preparation according to the above (23), wherein the preparation comprises 152.3mg of the compound represented by the formula (VII) and fumaric acid in a molar ratio of 1: 1.
(32) The preparation according to the above (22), which contains 250.0mg of the compound represented by the formula (VII) as an active ingredient.
(33) The preparation according to the above (23), wherein the preparation comprises 304.6mg of the compound represented by the formula (VII) and fumaric acid in a molar ratio of 1: 1.
(34) The preparation according to the above (22), which contains 25.0mg of the compound represented by the formula (VII) as an active ingredient.
(35) The preparation according to the above (23), wherein the preparation comprises 30.46mg of the compound represented by the formula (VII) and fumaric acid in a molar ratio of 1: 1.
(36) The preparation according to any one of the above (22) to (35), which is used for children and adults over 12 years of age.
(37) The preparation according to any one of the above (22) to (35), which is used for children over 6 years old and less than 12 years old.
Effects of the invention
The compound produced by the production method according to the present invention has an inhibitory activity against coronavirus 3CL protease, and is useful as a therapeutic and/or prophylactic agent for coronavirus infection.
The compound produced by the production method according to the present invention is useful as a pharmaceutical raw material.
Further, a pharmaceutical composition containing fumaric acid co-crystals of the compound (I-0005) produced by the production method according to the present invention is very useful as a therapeutic agent for novel coronavirus infection (COVID-19). The production method according to the present invention is a method capable of producing the compound according to the present invention in high yield.
The orally administered preparation (pharmaceutical composition) according to the present invention has an inhibitory activity against coronavirus 3CL protease and is useful as a therapeutic and/or prophylactic agent for coronavirus infection.
Drawings
Fig. 1 shows a powder X-ray diffraction pattern of fumaric acid co-crystal form I (form I) of the compound represented by formula (VII) in example a. The horizontal axis represents 2θ (°), and the vertical axis represents intensity (count).
Fig. 2 shows a peak list of the powder X-ray analysis pattern of fig. 1.
Fig. 3 shows the structure in an asymmetric unit of fumaric acid co-crystal form I (form I) of the compound shown by formula (VII).
Fig. 4 shows the DSC analysis result of fumaric acid co-crystal form I (form I) of the compound shown in formula (VII) showing the powder X-ray analysis pattern of fig. 1. The horizontal axis represents temperature (. Degree. C.) and the vertical axis represents heat (W/g).
Fig. 5 shows TG/DTA analysis results of fumaric acid co-crystal form I (form I) of the compound shown in formula (VII) showing the powder X-ray analysis pattern of fig. 1. The vertical axis represents heat (. Mu.V) or weight change (%), and the horizontal axis represents temperature (. Degree. C.). Cel in the figure refers to degrees Celsius (C.).
Fig. 6 shows DVS analysis results of fumaric acid co-crystal form I (crystal form I) of the compound shown in formula (VII) showing the powder X-ray analysis pattern of fig. 1. No substantial weight change was observed even with varying humidity, and the crystals were stable to moisture.
FIG. 7 shows the HPLC measurement result of Compound I-005 obtained in step 4 of example 1 a. The pa% (peak area) of the compound represented by the formula (VII) (Compound I-005) was about 95pa%.
FIG. 8 shows the HPLC determination result of Compound I-005 obtained in step 4'. The pa% of the compound represented by the formula (VII) (Compound I-005) is about 99pa%.
Fig. 9 shows a powder X-ray diffraction pattern of fumaric acid co-crystal form I (crystal form I) of the compound represented by formula (VII) in example b. The horizontal axis represents 2θ (°), and the vertical axis represents intensity (count).
Fig. 10 shows a peak table of the powder X-ray analysis pattern of fig. 9.
Fig. 11 shows a structural diagram in an asymmetric unit of fumaric acid co-crystal form I (form I) of the compound shown by formula (VII).
FIG. 12 shows the results of HPLC of an undried crystal of the compound represented by formula (VII) obtained in step 5-1 of example 1 b. The pa% of the compound represented by the formula (VII) is about 99pa%.
Fig. 13 shows the analysis results of fig. 12 excluding the toluene-derived peak (rt=about 9.8 min). The pa% of the compound represented by the formula (VII) is about 99.7pa%, and each impurity is about 0.1pa% or less.
FIG. 14 shows DSC analysis results of fumaric co-crystal form I (form I) of the compound represented by formula (VII) obtained in step 5-2 of example 1 b.
FIG. 15 shows the TG/DTA analysis result of the fumaric acid co-crystal form I (form I) of the compound represented by the formula (VII) obtained in step 5-2 of example 1 b. The weight reduction to 150 ℃ was 0.28%.
Fig. 16 shows the results of HPLC of fumaric acid co-crystal form I (form I) of the compound represented by formula (VII) obtained in step 5-2 of example 1 b.
FIG. 17 shows the particle size distribution of fumaric acid co-crystal form I (form I) of the compound represented by formula (VII) obtained in step 5-2 of example 1 b. D50 was 25.35. Mu.m, and D90 was 73.56. Mu.m.
Fig. 18 shows the DVS analysis result of the fumaric acid co-crystal form I (form I) of the compound represented by formula (VII) obtained in step 5-2 of example 1 b. No substantial weight change was observed even with varying humidity, and the crystals were stable to moisture.
Fig. 19 shows a comparison of powder X-ray analysis patterns before and after DVS measurement of fig. 18. The crystal forms did not change before and after DVS measurement, and were stable.
Fig. 20 shows a powder X-ray diffraction pattern of a toluene compound of the compound represented by formula (VII). The horizontal axis represents 2θ (°), and the vertical axis represents intensity (count).
Fig. 21 shows the elution behavior of example 6. The horizontal axis represents time (minutes) and the vertical axis represents dissolution rate (%).
Fig. 22 shows the particle size distribution of the active ingredient (fumaric acid co-crystal I-shaped crystal of the compound represented by formula (VII)) used in the formulations of examples 6A and 6B.
Fig. 23 shows particle size distributions of the active ingredient (fumaric acid co-crystal I-shaped crystals of the compound represented by formula (VII)) used in the formulations of examples 6C and 6D.
FIG. 24 shows the change from baseline in viral titer of SARS-CoV-2 at Phase 2a Part. The vertical axis represents the change in the baseline of viral titer (log 10 (TCID 50 /mL)), the horizontal axis shows the evaluation point.
Fig. 25 shows the time until the virus titer was initially confirmed to be negative. The vertical axis represents the ratio (unit:%) of those with negative SARS-CoV-2 virus titer. The horizontal axis shows the time (units: hours) from the start of the treatment.
FIG. 26 shows the change from baseline in the 12 symptom aggregate score for COVID-19 at each time point. The vertical axis represents the change from the baseline of the 12 symptom aggregate score for covd-19. The horizontal axis represents the evaluation time point.
Fig. 27 shows the elution behavior of example 10. The horizontal axis represents time (minutes), and the vertical axis represents the dissolution rate (%) after the content correction.
Detailed Description
Hereinafter, the meaning of each term used in the present specification is explained. Unless otherwise indicated, all terms used herein are intended to have the same meaning as defined above.
The term "consisting of … …" means having only constituent elements.
The terms "comprising" and "including" are not limited to the constituent elements, and do not exclude the elements not described.
Furthermore, throughout this specification, the expression in the singular should be understood to include the plural form thereof unless specifically stated otherwise. Accordingly, the singular forms (e.g., "a," "an," "the," etc., in the case of english) are to be construed to include the plural forms as well, unless specifically stated otherwise.
Further, the terms used in the present specification should be understood to be used in the meaning commonly used in the above-mentioned field, unless otherwise specified. Accordingly, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs without additional definition. In case of conflict, the present specification, including definitions, will control.
"halogen" is meant to include fluorine, chlorine, bromine, and iodine atoms. Fluorine atoms and chlorine atoms are particularly preferred.
"alkyl" means a straight-chain or branched hydrocarbon group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and still more preferably 1 to 4 carbon atoms. Examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, n-decyl and the like.
Preferable examples of the "alkyl" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and n-pentyl. Further preferable examples include methyl, ethyl, n-propyl, isopropyl and tert-butyl.
"C1-C4 alkyl" means a straight-chain or branched hydrocarbon group having 1 to 4 carbon atoms. Examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like.
"aromatic carbon cyclic group" means a monocyclic or more than 2-ring cyclic aromatic hydrocarbon group. Examples thereof include phenyl, naphthyl, anthryl and phenanthryl. Examples of the 6-membered aromatic carbon ring group include phenyl groups. Examples of the 10-membered aromatic carbon ring group include a naphthyl group and the like. Examples of the 14-membered aromatic carbon ring group include an anthracene group and a phenanthrene group.
As a preferable mode of the "aromatic carbon ring group", a phenyl group is exemplified.
"aromatic carbocycle" refers to a ring derived from the "aromatic carbocyclyl" described above.
"aromatic heterocyclic group" means a monocyclic or 2-ring or more aromatic ring group having 1 or more heteroatoms in the ring, optionally selected from the same or different heteroatoms in O, S and N.
The 2-or more-ring aromatic heterocyclic group also includes a group obtained by fusing a ring in the above "aromatic carbon ring group" to a monocyclic or 2-or more-ring aromatic heterocyclic group, and the bonding position may be present in any ring.
The monocyclic aromatic heterocyclic group is preferably 5 to 8-membered, more preferably 5-membered or 6-membered. Examples of the 5-membered aromatic heterocyclic group include a pyrrolyl group, a pyrimidinyl group, a pyrazolyl group, a triazolyl group, a tetrazolyl group, a furanyl group, a thienyl group, an isoxazolyl group, an oxazolyl group, an oxadiazolyl group, an isothiazolyl group, a thiazolyl group, and a thiadiazolyl group. Examples of the 6-membered aromatic heterocyclic group include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.
The aromatic heterocyclic group having 2 rings is preferably 8 to 10 membered, more preferably 9 or 10 membered. Examples thereof include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzopyrimidinyl, benzisoxazolyl, benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, benzotriazole group, imidazopyridinyl, triazolopyridinyl, imidazothiazolyl, pyrazolopyridazinyl, oxazolopyridinyl, thiazolopyridinyl and the like. Examples of the 9-membered aromatic heterocyclic group include indolyl, isoindolyl, indazolyl, indolizinyl, purinyl, benzopyrimidinyl, benzisoxazolyl, benzoxazolyl, benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazole, benzofuranyl, imidazopyridinyl, triazolopyridinyl, oxazolopyridinyl, and thiazolopyridinyl. Examples of the 10-membered aromatic heterocyclic group include a quinolinyl group, an isoquinolinyl group, a cinnolinyl group, a phthalazinyl group, a quinazolinyl group, a naphthyridinyl group, a quinoxalinyl group, a pteridinyl group, and a pyrazolopyridazinyl group.
The aromatic heterocyclic group having 3 or more rings is preferably 13 to 15 members. Examples thereof include carbazolyl, acridinyl, oxaanthracyl, phenothiazinyl, phenoxathiazinyl, phenoxazinyl, dibenzofuranyl and the like.
As a preferred mode of the "aromatic heterocyclic group", there may be mentioned a triazole group.
"aromatic heterocycle" refers to a ring derived from the "aromatic heterocyclic group" described above.
Examples of the substituent for the "substituted alkyl" include the following substituent group A. The carbon atom at any position may be bonded to 1 or more groups selected from the substituent group a described below.
Substituent group a: halogen, cyano and nitro.
As the substituent for "substituted C1-C4 alkyl", the following substituent group B may be mentioned. The carbon atom at any position may be bonded to 1 or more groups selected from the substituent group B described below.
Substituent group B: halogen, cyano and nitro.
Examples of the substituents on the ring of the "aromatic carbocycle" and "aromatic heterocycle" such as the "substituted aromatic carbocycle group" and the "substituted aromatic heterocycle group" include the following substituent group C. An atom at an arbitrary position on the ring may be bonded to 1 or more groups selected from the substituent group C described below.
Substituent group C: halogen, cyano, nitro and alkyl.
The compounds of formula (VI) and formula (VII) are not limited to a particular isomer, including all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereomers, optical isomers, rotamers, etc.), racemates, or mixtures thereof.
[ chemical 28 ]
For example, the compound represented by the formula (VII) and the compound I-005 include tautomers as described below.
[ chemical 29 ]
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Substantially free of formula (VII):
[ chemical formula 30 ]
The fumaric acid co-crystal form I of the compound represented by the formula (VII) in which the free form of the compound represented by the formula (VII) is not detected by a measuring instrument such as powder X-ray diffraction measurement or the like.
Further, one or more hydrogen, carbon and/or other atoms in the compounds represented by the formula (I), the formula (II), the formula (III), the formula (IV), the formula (V), the formula (VI), the formula (VII), the formula (VIII), the formula (IX), the formula (X), and the formula (XI) (hereinafter referred to as the formula (VII) and the like) may be replaced with isotopes of hydrogen, carbon and/or other atoms, respectively. Examples of such isotopes are, respectively, e.g 2 H、 3 H、 11 C、 13 C、 14 C、 15 N、 18 O、 17 O、 31 P、 32 P、 35 S、 18 F、 123 I and 36 like Cl, it includes hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine. The compounds represented by the formula (VII) and the like also include compounds substituted with such isotopes. The compounds substituted with the isotopes are also useful as pharmaceuticals, including all radiolabeled compounds represented by formula (VII) and the like. In addition, "radiolabeling methods" for making the "radiolabels" are also included in the present invention, which are useful as tools for pharmacokinetic studies, research and/or diagnostics in binding assays.
Furthermore, the crystal of the present invention may be a deuterium transducer. The crystals of the invention may be isotopically substituted (e.g. 3 H、 14 C、 35 S、 125 I), etc.).
Radiolabeled bodies of compounds represented by the formula (VII) and the like can be prepared by methods well known in the art. For example, tritium-labeled compounds represented by the formula (VII) and the like can be produced by introducing tritium into a specific compound represented by the formula (VII) and the like by dehalogenation using a tritium catalyst. The method comprises reacting a precursor of a compound represented by formula (VII) or the like substituted with an appropriate halogen with tritium gas in the presence of an appropriate catalyst, such as Pd/C, in the presence or absence of a base. Other suitable methods for preparing tritium-labeled compounds can be found in "Isotopes in the Physical and Biomedical Sciences, vol.1, labeled Compounds (Part a), chapter 6 (1987)". 14 The C-labelled compound may be prepared by using a compound having the following properties 14 C carbon raw material.
In the preparation of the present invention, pharmaceutically acceptable salts of the compounds represented by the formula (VII) and the like can be used. Examples of pharmaceutically acceptable salts of the compounds represented by the formula (VII) and the like include salts of the compounds represented by the formula (VII) and alkali metals (e.g., lithium, sodium, potassium and the like), alkaline earth metals (e.g., calcium, barium and the like), magnesium, transition metals (e.g., zinc, iron and the like), ammonia, organic bases (e.g., trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, picoline, quinoline and the like), and amino acids, or salts of inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid and the like), and organic acids (e.g., formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, succinic acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid and the like). These salts can be formed by a generally performed method.
As pharmaceutically acceptable salts of the compounds of formula (VII), for example, consisting of the compounds of formula (VII) and a counter molecule or counter ion, any number of counter molecules or counter ions may be included. The pharmaceutically acceptable salt of the compound represented by formula (VII) refers to a substance formed by proton movement between the compound and a counter molecule or counter atom via an ionic bond.
In the preparation of the present invention, a complex of a compound represented by the formula (VII) or a pharmaceutically acceptable salt thereof can be used. The compound represented by the formula (VII) or a pharmaceutically acceptable salt thereof sometimes forms solvates (e.g., hydrates, etc.), co-crystals, and/or inclusion compounds, which are described as "complexes" in the present specification.
As used herein, the term "solvate" means that a compound represented by the formula (VII) or the like can be coordinated to any number of solvent molecules (e.g., water molecules or the like), for example. When a compound represented by the formula (VII) or the like or a pharmaceutically acceptable salt thereof is left in the atmosphere, water is absorbed, and water may be adsorbed, or a hydrate may be formed.
Examples of the solvent molecule include acetonitrile, chlorobenzene, chloroform, cyclohexane, 1, 2-dichloroethylene, methylene chloride, 1, 2-dimethoxyethane, N-dimethylacetamide and N, N-dimethylformamide, 1, 4-dioxane, 2-ethoxyethanol, ethylene glycol, formamide, hexane, methanol, 2-methoxyethanol, methyl butyl ketone, methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine, sulfolane, tetrahydronaphthalene, toluene, 1, 2-trichloroethylene, xylene, acetic acid, anisole, 1-butanol, 2-butanol, N-butyl acetate, tert-butylmethyl ether, cumene, dimethyl sulfoxide, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, tetrahydrofuran, water (i.e. hydrate), ethanol, acetone, 1-diethoxypropane, 1-dimethoxymethane, 2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyl tetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid, preferably acetic acid, anisole, 1-butanol, 2-butanol, N-butyl acetate, tert-butyl methyl ether, cumene, dimethyl sulfoxide, ethyl acetate, diethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, tetrahydrofuran, water (i.e. hydrate) ethanol, acetone, 1-diethoxypropane, 1-dimethoxymethane, 2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone methyl tetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid, more preferably, water (i.e., hydrate), ethanol, acetone, 1-diethoxypropane, 1-dimethoxymethane, 2-dimethoxypropane, isooctane, isopropyl ether, methyl isopropyl ketone, methyl tetrahydrofuran, petroleum ether, trichloroacetic acid and trifluoroacetic acid, and the like.
As used herein, "co-crystal" refers to a regular arrangement of the counter-molecules within the same lattice, and may include any number of counter-molecules. In addition, co-crystals refer to chemical interactions between compounds and counter molecules that are non-covalent and nonionic via hydrogen bonds, van der waals forces, and the like.
For example, as a co-crystal of the compound represented by formula (VII), which is composed of the compound represented by formula (VII) and a counter molecule, any number of counter molecules may be contained. Preferably consisting of a compound of formula (VII) and fumaric acid, any number of fumaric acids may be included. Further preferred are co-crystals of the compound of formula (VII) and fumaric acid in a molar ratio of 1:1.
The co-crystals are distinguished from salts in that the compound remains essentially charge-free or neutral.
The co-crystals are distinguished from hydrates or solvates in that the counter-molecule is not in water or solvent.
The compounds represented by the formula (I), the formula (II), the formula (III), the formula (IV), the formula (V), the formula (VI), the formula (VII), the formula (VIII), the formula (IX), the formula (X), and the formula (XI) or salts thereof of the present invention sometimes form solvates (e.g., hydrates, etc.), co-crystals, and/or polymorphs, and the present invention also includes such various solvates, co-crystals, and polymorphs. The "solvate" may coordinate a compound of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), and formula (XI) with any number of solvent molecules (e.g., water molecules, etc.). In addition, a polymorph is sometimes formed by recrystallizing a compound represented by formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), and formula (XI) or a salt thereof.
As used herein, "crystal" refers to a solid having a three-dimensional regular arrangement of atoms, ions, molecules, etc. that is formed, and is distinguished from an amorphous solid that does not have such a regular internal structure. The crystals of the present invention may be single crystals, twin crystals, polycrystal, etc.
Further, in the "crystal", there are sometimes "polymorphs" having the same composition and different arrangements in the crystal, including these are called "crystalline states".
In addition, the compounds of formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), and formula (XI) may also be converted into these salts or these pharmaceutically acceptable solvates. The crystals of the present invention may be any of these salts, hydrates, solvates, and polymorphs, and even a mixture of two or more kinds is intended to be included in the scope of the present invention.
The crystallinity and crystallinity can be determined by a variety of techniques including, for example, powder X-ray diffraction measurement, raman spectroscopy, infrared absorption spectrometry, moisture absorption and desorption measurement, differential scanning calorimetric measurement, dissolution profile.
In addition, a "polymorph" may be formed by recrystallizing a compound represented by the formula (VII) or the like, a pharmaceutically acceptable salt thereof, or a complex thereof.
In the preparation of the present invention, various salts, complexes (hydrates, solvates, co-crystals, inclusion compounds) and polymorphs thereof can be used, and a mixture of two or more of them can be used.
(powder X-ray diffraction (XRPD))
Powder X-ray diffraction (XRPD) is one of the most sensitive assays for measuring the crystalline state and crystallinity of solids. When the crystal is irradiated with X-rays, the X-rays are reflected by crystal planes and interfere with each other, thereby showing diffraction lines of existing order corresponding to the period of the structure. On the other hand, amorphous solids generally have no repetition period of order in their structure, and therefore do not cause diffraction phenomena, showing a characteristic broad XRPD pattern (also referred to as a halo pattern).
The crystalline state of the compound represented by the formula (VII) and the like can be identified by a powder X-ray diffraction pattern and a characteristic diffraction peak. The crystalline state of the compound represented by the formula (VII) and the like can be distinguished from other crystalline states by the presence of a characteristic diffraction peak.
The characteristic diffraction peak used in this specification is a peak selected by the diffraction pattern observed. The characteristic diffraction peaks are preferably about 10, more preferably about 5, and even more preferably about 3 selected from the diffraction patterns.
A peak identified in this crystal but not identified in other crystals is a preferable characteristic peak in the identification of the crystal, compared with the intensity of the peak, in the differentiation of a plurality of crystals. If the characteristic peaks are one or two, the crystals can be labeled with the characteristic. Comparing the graphs obtained by the measurement, it can be said that the powder X-ray diffraction patterns are substantially uniform if these characteristic peaks are uniform.
In general, since the diffraction angle (2θ) in powder X-ray diffraction may be within a range of ±0.2°, it is to be understood that the value of the diffraction angle in powder X-ray diffraction also includes a value within a range of approximately ±0.2°. Therefore, the present invention includes not only crystals in which diffraction angles of peaks in powder X-ray diffraction are completely uniform, but also crystals in which diffraction angles of peaks are uniform within an error of about ±0.2°.
The intensities of peaks shown in the following tables and figures are generally known to vary depending on various factors, such as the effect of selective orientation of crystals with respect to X-ray beams, the influence of coarse particles, the purity of the substance analyzed, or the crystallinity of the sample. Further, the peak position may be shifted based on the variation in the sample height. Further, if measured using different wavelengths, different displacements are obtained according to the bragg equation (nλ=2dsinθ), but additional XRPD patterns obtained by using such additional wavelengths are also included within the scope of the present invention.
(analysis of Single Crystal Structure)
In one method of identifying a crystal, parameters of crystallography, further atomic coordinates (values indicating spatial positional relationships of atoms) and a three-dimensional structure model in the crystal can be obtained. Reference was made to "guidelines for X-ray structural analysis" skirt chinese house release (1983), stout & Jensen, X-Ray Structure Determination: a Practical Guide, macmillanco., new York (1968), etc. Single crystal structure analysis is useful for identifying the structure of crystals of the complex, salt, optical isomer, tautomer, and geometric isomer of the present invention.
The compounds represented by the formula (VII) and the like have coronavirus 3CL protease inhibitory activity and are therefore useful as therapeutic and/or prophylactic agents for diseases in which coronavirus 3CL protease participates. In the present invention, "therapeutic agent and/or prophylactic agent" also includes symptom-improving agent. The disease in which coronavirus 3CL protease participates may be a viral infection, and preferably a coronavirus infection.
As one embodiment, coronaviruses that infect humans are exemplified. As coronaviruses which infect humans, there may be mentioned HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and/or SARS-CoV-2.
As one embodiment, the coronavirus may be an α coronavirus and/or a β coronavirus, more preferably a β coronavirus, and still more preferably a b coronavirus (sarbeovirus).
As one embodiment, examples of the alpha coronavirus include HCoV-229E and HCoV-NL63. HCoV-229E may be particularly preferred.
As one embodiment, the beta coronavirus may be HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV, and/or SARS-CoV-2. It is preferably HCoV-OC43 or SARS-CoV-2, and particularly preferably SARS-CoV-2.
As one embodiment, as the beta coronavirus, there may be mentioned the beta coronavirus A lineage (beta-coronavirus lineage A), the beta coronavirus B lineage (beta-coronavirus lineage B), and the beta coronavirus C lineage (beta-coronavirus lineage C). More preferred are the beta coronavirus A lineage (beta-coronavirus lineage A), and the beta coronavirus B lineage (beta-coronavirus lineage B), particularly preferred is the beta coronavirus B lineage (beta-coronavirus lineage B).
As the beta coronavirus A lineage (. Beta. -coronavirus lineage A), there may be mentioned, for example, HCoV-HKU1 and HCoV-OC43, preferably, HCoV-OC43. As the beta coronavirus B lineage (. Beta. -coronavirus lineage B), there may be mentioned, for example, SARS-CoV and SARS-CoV-2, preferably SARS-CoV-2. As the beta coronavirus C-lineage (. Beta. -coronavirus lineage B), MERS-CoV is preferred.
As one embodiment, the coronavirus may be HCoV-229E, HCoV-OC43, and/or SARS-CoV-2, and SARS-CoV-2 is particularly preferred.
Examples of the coronavirus infection include infection caused by HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV, and/or SARS-CoV-2. It is preferable that the infection is caused by HCoV-229E, HCoV-OC43 and/or SARS-CoV-2, and particularly preferable that the infection is caused by SARS-CoV-2.
The coronavirus infection is particularly preferably a novel coronavirus infection (COVID-19).
The following describes a manufacturing method according to the present invention.
Step 1 Process for producing Compound of formula (III)
[ chemical 31 ]
The labels in the formula are as defined above.
The present step is a method for producing a compound represented by formula (III), which is characterized by reacting a compound represented by formula (I) with a compound represented by formula (II) in the presence of an acid.
The compound represented by the formula (II) can be used in an amount of usually 1.0 to 5.0 equivalents, for example, 1.0 to 3.0 equivalents, relative to the compound represented by the formula (I).
The solvent is not particularly limited as long as it is a solvent that efficiently performs the above steps, and an acid may be used as the solvent. Examples thereof include acids, toluene, methylene chloride, and dichloroethane, and they may be used alone or in combination. Preferably, an acid is exemplified.
Examples of the acid include a protic acid and a lewis acid, and preferably trifluoroacetic acid.
The amount of the acid to be used is usually 1.0 equivalent to a large excess, for example, 5.0 equivalents to a large excess, relative to the compound represented by the formula (I).
The reaction temperature is not particularly limited, and may be generally carried out at about 0℃to about 50℃and preferably at room temperature to 40 ℃.
The reaction time is not particularly limited, but is 0.1 to 12 hours, preferably 0.1 to 5 hours.
Step 2 Process for producing Compound of formula (VI)
[ chemical formula 32 ]
The labels in the formula are as defined above.
The present step is a method for producing a compound represented by formula (VI), which is characterized by reacting a compound represented by formula (IV) with a compound represented by formula (V) in the presence of an acid.
The compound represented by the formula (V) can be used in an amount of usually 1.0 to 5.0 equivalents, for example, 1.0 to 1.5 equivalents, relative to the compound represented by the formula (IV).
The solvent is not particularly limited as long as it is a solvent that efficiently performs the above steps, and an acid may be used as the solvent. Examples thereof include toluene, t-butanol, t-amyl alcohol, etc., and may be used alone or in combination. Preferably, toluene is exemplified.
Examples of the acid include acetic acid and 2, 2-dimethylbutyric acid. Preferably, acetic acid is exemplified.
The amount of the acid to be used is usually 1.0 to 10 equivalents, for example 3.0 to 10 equivalents, based on the compound represented by the formula (IV).
The reaction temperature is not particularly limited and may be usually carried out at room temperature to about 150℃or under microwave irradiation, preferably at 50 to 150℃or under microwave irradiation.
The reaction time is not particularly limited, but is 0.1 to 12 hours, preferably 3 to 10 hours.
Step 3 Process for producing fumaric acid Co-crystal form I of Compound of formula (VII)
The present step is a method for producing a fumaric acid co-crystal form I of a compound represented by the formula (VII), which is characterized by crystallizing a compound represented by the formula (VII) in the presence of fumaric acid, acetone and water.
The fumaric acid may be used in an amount of usually 1.0 to 3.0 equivalents, for example, 1.0 to 1.5 equivalents, relative to the compound represented by the formula (VII).
The crystallization temperature is not particularly limited, and may be carried out at a temperature of usually 40 to 80 ℃, preferably 40 to 60 ℃.
The crystallization time is not particularly limited, but is usually 1 hour or more, preferably 2 hours or more, and more preferably 2 to 12 hours.
The ratio of acetone to water may be preferably 85:15 to 50:50, as long as the acetone and water are present.
The compound represented by the formula (VII), a pharmaceutically acceptable salt thereof or a complex thereof (hereinafter referred to as a compound represented by the formula (VII)), and a compound produced by the production method of the present invention (a compound represented by the formula (VII)), have coronavirus 3CL protease inhibitory activity, and are therefore useful as a therapeutic and/or prophylactic agent for viral infections.
Further, the compound produced by the production method according to the present invention has pharmaceutical usefulness, and preferably has any one or more of the following excellent characteristics.
a) The inhibition effect on CYP enzymes (e.g., CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, etc.) is weak.
b) Shows good pharmacokinetics with high bioavailability, moderate clearance rate and the like.
c) High metabolic stability.
d) The concentration range of the CYP enzyme (e.g., CYP3A 4) under the measurement conditions described in the specification does not show irreversible inhibition.
e) Has no mutagenicity.
f) The risk of cardiovascular system is low.
g) Showing high solubility.
h) The protein non-binding rate (fu value) was high.
i) Has high selectivity of coronavirus 3CL protease.
j) Has high coronavirus proliferation inhibiting activity. For example, when Human Serum (HS) or Human Serum Albumin (HSA) is added, the coronavirus growth inhibitory activity is high.
Examples of the coronavirus proliferation inhibitor include EC in the CPE inhibition effect confirmation test (SARS-CoV-2) described later 50 10. Mu.M or less, preferably 1. Mu.M or less, more preferably 100nM or less.
The salt, crystal, complex and co-crystal of the compound according to the present invention are useful as a medicine, and preferably have any one or more of the following excellent characteristics.
bb) shows high bioavailability, moderate clearance, high AUC, high maximum blood concentration, etc., good pharmacokinetics.
gg) shows high solubility, high chemical stability, low hygroscopicity.
The pharmaceutical composition containing the compound represented by the formula (VII) and the like (for example, the compound produced by the production method of the present invention) may be administered by any of oral and parenteral methods. Examples of the parenteral administration include transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, transmucosal, inhalation, nasal, eye drop, ear drop, and intravaginal administration.
In the case of oral administration, the pharmaceutical composition may be formulated into any one of commonly used dosage forms such as solid preparations for oral administration (for example, tablets, powders, granules, capsules, pills, films, etc.), solutions for oral administration (for example, suspensions, emulsions, elixirs, syrups, soda water (Limonade) preparations, alcoholic solutions, aromatic water solutions, extracts, decoctions, tinctures, etc.), and the like, according to a conventional method. The tablet can be sugar-coated tablet, film-coated tablet, water-soluble coated tablet, sustained-release tablet, spindle tablet, sublingual tablet, buccal tablet, chewable tablet or orally disintegrating tablet, the powder and granule can be dry syrup, and the capsule can be soft capsule, microcapsule or sustained-release capsule.
In the case of non-oral administration, the composition may be suitably administered in any form commonly used, such as injection, drop, external preparation (for example, eye drop, nose drop, ear drop, aerosol, inhalation, lotion, injection, coating, gargle, enema, paste, plaster, gel, cream, patch, cataplasm, external powder, suppository, etc.). The injection can be O/W, W/O, O/W/O, W/O/W emulsion.
The pharmaceutical composition can be prepared by mixing an effective amount of a compound represented by the formula (VII) (for example, a compound produced by the production method of the present invention) with various pharmaceutical additives such as excipients, binders, disintegrants, lubricants, etc. suitable for the dosage form thereof, if necessary. Further, the pharmaceutical composition can be formulated into a pharmaceutical composition for children, the elderly, critically ill patients or for surgery by appropriately changing the effective amount, dosage form and/or various pharmaceutical additives of the compound of the present invention. For example, the pharmaceutical composition for children may be administered to a newborn (less than 4 weeks after birth), a lactating infant (4 weeks after birth to less than 1 year old), a young child (more than 1 year old and less than 7 years old), a child (more than 7 years old and less than 15 years old), or a patient of 15 years to 18 years old. For example, the pharmaceutical composition for the elderly may be administered to patients over 65 years old.
The amount of the compound represented by the formula (VII) and the like (for example, the compound produced by the production method according to the present invention) (for example, the pharmaceutical composition comprising fumaric acid co-crystal form I of the compound represented by the formula (VII)) to be administered is desirably set in consideration of the age, weight, type, degree, administration route and the like of the patient, and in the case of oral administration, is usually in the range of 0.05 to 200 mg/kg/day, preferably 0.1 to 100 mg/kg/day. The non-oral administration is significantly different depending on the route of administration, and is usually in the range of 0.005 to 200 mg/kg/day, preferably 0.01 to 100 mg/kg/day. It is administered 1 day 1 to several times.
The compound represented by the formula (VII) (for example, a compound produced by the production method of the present invention)) can be used in combination with, for example, another novel therapeutic agent for coronavirus infection (covd-19) (including an approved agent and an agent under development or developed in the future) (hereinafter referred to as a combination agent) for the purpose of enhancing the action of the compound or reducing the amount of the compound to be administered. In this case, the administration period of the compound of the present invention and the pharmaceutical agent used in combination is not limited, and they may be administered to the administration subject simultaneously or with a time difference. The compound of the present invention and the pharmaceutical composition for combined use may be administered in the form of 2 or more preparations containing the respective active ingredients, or may be administered in the form of a single preparation containing these active ingredients.
The administration amount of the combination agents may be appropriately selected from clinically used dosages as a basis. The ratio of the compound of the present invention to the pharmaceutical composition for combination may be appropriately selected according to the administration subject, the administration route, the disease, the symptom, the combination, and the like. For example, in the case of administration to a human, the pharmaceutical composition may be used in an amount of 0.01 to 100 parts by weight based on 1 part by weight of the compound of the present invention.
The compound represented by the formula (VII) can be formulated by mixing an effective amount of an excipient, a binder, a disintegrant, a lubricant and other various pharmaceutical additives suitable for the formulation thereof, if necessary.
The preparation of the invention is orally taken. In the case of oral administration, the pharmaceutical composition may be formulated into any of commonly used dosage forms such as solid preparations for oral administration (for example, tablets, powders, granules, dry syrups, capsules, pills, films, etc.), solutions for oral administration (for example, suspensions, emulsions, elixirs, syrups, soda (Limonade) solutions, alcoholic solutions, aromatic water agents, extracts, decoctions, tinctures, etc.), and the like, according to a conventional method. The tablet can be sugar-coated tablet, film-coated tablet, water-soluble coated tablet, sustained-release tablet, spindle tablet, sublingual tablet, buccal tablet, chewable tablet or orally disintegrating tablet, the powder and granule can be dry syrup, and the capsule can be soft capsule, microcapsule or sustained-release capsule. The solid preparation or suspension for oral administration is preferable, the solid preparation for oral administration is more preferable, and the tablet and the granule are particularly preferable.
The tablet may be any shape, and specifically, may be formed into a tablet having a shape of a circle, an ellipse, a sphere, a rod, or a doughnut. The sheet may be a laminate sheet, a core sheet, or the like, and is preferably a single-layer sheet having a simple manufacturing method. Further, marks such as symbols and characters for improving the recognition may be marked, and dividing lines may be marked.
Further, the preparation of the present invention can be used as a preparation for children, the elderly, critically ill patients or for surgery by appropriately changing the effective amount of the compound represented by the formula (VII), the dosage form and/or various pharmaceutical additives. For example, the pharmaceutical composition for children may be administered to a newborn (less than 4 weeks after birth), a lactating infant (4 weeks after birth to less than 1 year old), a young child (more than 1 year old and less than 7 years old), a child (more than 7 years old and less than 15 years old), or a patient of 15 years to 18 years old. For example, the preparation for aged people can be administered to a patient over 65 years old.
The administration amount of the preparation of the present invention (for example, a preparation containing fumaric acid co-crystal form I of the compound represented by the formula (VII)) is desirably set in consideration of the age, weight, type, extent of disease, administration route and the like of the patient, and in the case of oral administration, is usually in the range of 0.05 to 200 mg/kg/day, preferably 0.1 to 100 mg/kg/day. The non-oral administration is significantly different depending on the route of administration, and is usually in the range of 0.005 to 200 mg/kg/day, preferably 0.01 to 100 mg/kg/day. It is administered 1 day 1 to several times.
The weight of the compound represented by the formula (VII) and the like contained in the preparation of the present invention is not particularly limited as long as the compound is easily taken by a patient and can be produced, and is 1 to 450mg, preferably 5 to 350mg, more preferably 25 to 250mg.
Specifically, the average weight of the compound represented by the formula (VII) contained in 1 tablet, 1 capsule or 1 is 25mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg or 250mg.
In this case, 25mg means a range of 22.5 to 27.5mg, preferably 23.7 to 26.3mg, 50mg means a range of 45.0 to 55.0mg, preferably 47.5 to 52.5mg, 75mg means a range of 67.5 to 82.5mg, preferably 71.2 to 78.8mg, 100mg means a range of 90.0 to 110.0mg, preferably 95.0 to 105.0mg, 125mg means a range of 112.5 to 137.5mg, preferably 118.7 to 131.3mg, 150mg means a range of 135.0 to 165.0mg, preferably 142.5 to 157.5mg, 175mg means a range of 157.5 to 192.5mg, preferably 166.2 to 183.8mg, 200mg means a range of 180.0 to 220.0, preferably 190.0 to 190.0, preferably 225.25 mg, preferably 38 mg to 29.0 mg, preferably 29.5 to 29.5 mg, and preferably 29.5 to 29.0 mg.
The compound represented by the formula (VII) and the like may be used in combination with, for example, other novel therapeutic agents for coronavirus infection (covd-19) (including approved agents and agents under development or later) for the purpose of enhancing the action of the compound or reducing the amount of the compound to be administered (hereinafter referred to as "combination agents"). In this case, the administration timing of the compound represented by the formula (VII) and the pharmaceutical agent to be used in combination is not limited, and they may be administered to the administration subject at the same time or with a time difference. Further, the compound represented by the formula (VII) and the pharmaceutical composition may be administered in the form of 2 or more preparations containing the respective active ingredients, or may be administered in the form of a single preparation containing these active ingredients.
The administration amount of the combination agents may be appropriately selected from clinically used dosages as a basis. The mixing ratio of the compound represented by the formula (VII) and the pharmaceutical composition for use in combination may be appropriately selected according to the administration subject, the administration route, the disease, the symptom, the combination, and the like of the subject. For example, when the composition is administered to a human, the composition may be used in an amount of 0.01 to 100 parts by weight based on 1 part by weight of the compound represented by the formula (VII) and the like.
The preparation of the present invention may contain a polymer, and as the polymer, a polymer which is collected in the japanese pharmacopoeia, the standards of pharmaceuticals other than the japanese pharmacopoeia, the standards of pharmaceutical additives, the food additive specifications, and the like can be used.
The preparation containing the compound represented by the formula (VII), a pharmaceutically acceptable salt thereof or a complex thereof, and a polymer can improve the solubility of the compound represented by the formula (VII) in the preparation.
Examples of the polymer include cellulose polymers, acrylic polymers, vinyl polymers, and polysaccharides.
Examples of the cellulose-based polymer include hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose, butyrate, methylcellulose (MC), methyl hydroxyethyl cellulose, carboxymethyl ethylcellulose, crystalline cellulose, microcrystalline cellulose, crystalline sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, powdered cellulose, low-substitution hydroxypropyl cellulose, fumaric acid, stearic acid, polyvinyl acetal diethylaminoacetate, hydroxypropyl methylcellulose, and the like.
Examples of the acrylic polymer include aminoalkyl acrylate copolymer E, polyvinyl acetal diethylaminoacetate, ethyl acrylate-methyl methacrylate copolymer dispersion, aminoalkyl methacrylate copolymer, methacrylic acid copolymer, 2-methyl-5-vinylpyridine methacrylate-methacrylic acid copolymer, dried methacrylic acid copolymer, dimethylaminoethyl methacrylate-methyl methacrylate copolymer, and the like.
Examples of the vinyl polymer include polyvinylpyrrolidone, polyvinyl alcohol-methyl methacrylate-acrylic acid copolymer, crospovidone, carboxyvinyl polymer, polyvinyl acetal diethylaminoacetate, and polyvinyl alcohol copolymer.
Examples of the polysaccharide include pullulan and the like.
The cellulose-based polymer, acrylic-based polymer and/or vinyl-based polymer are preferable, the cellulose-based polymer is more preferable, and hydroxypropyl methylcellulose (hydroxypropyl methylcellulose) is further preferable.
In another embodiment, the present invention is preferably an orally administered tablet containing the compound represented by the formula (VII), a pharmaceutically acceptable salt thereof or a complex thereof as an active ingredient, and a cellulose polymer.
In the formulation of the present invention, 1 or more additives selected from the group consisting of excipients, binders, disintegrants and lubricants may be used.
In the formulation of the present invention, an excipient (and sometimes a filler) may be used. As the excipient, an excipient which is collected in japanese pharmacopoeia, a standard of pharmaceuticals outside the japanese pharmacopoeia, a standard of pharmaceutical additives, a food additive official gazette, or the like can be used. Examples of the excipient include sugar derivatives, starch derivatives, cellulose derivatives, inorganic excipients, β -cyclodextrin, magnesium stearate, calcium stearate, sucrose fatty acid esters, crospovidone, soybean lecithin, tragacanth powder, acacia, dextran, pullulan and the like.
Sugar derivatives include saccharides and sugar alcohols, examples of which include lactose, white sugar, glucose, fructose, and sucrose, and examples of sugar alcohols include mannitol, sorbitol, erythritol, xylitol, powdered maltose syrup, and maltitol.
Examples of the starch derivative include starch, potato starch, corn starch (corn starch), rice starch, partially alpha-starch, porous starch, sodium carboxymethyl starch, hydroxypropyl starch, sodium carboxymethyl starch with a low substitution degree, and the like.
Examples of the cellulose derivative include crystalline cellulose, powdered cellulose, sodium carboxymethyl cellulose, croscarmellose sodium, carboxymethyl cellulose calcium, carboxymethyl ethyl cellulose, and low-substitution hydroxypropyl cellulose.
Examples of the inorganic excipient include silicate derivatives, phosphates, carbonates, sulfates, magnesium oxide, titanium oxide, calcium lactate, synthetic hydrotalcite, talc, kaolin, dried aluminum hydroxide, magnesium oxide, bentonite, and the like.
Examples of silicate derivatives include silica such as hydrous silica and light anhydrous silicic acid, magnesium metasilicate aluminate, synthetic aluminum silicate, and calcium silicate.
Examples of the phosphate include anhydrous calcium hydrogen phosphate, monocalcium phosphate, calcium hydrogen phosphate, sodium hydrogen phosphate, dipotassium phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, and sodium dihydrogen phosphate.
Examples of the carbonate include precipitated calcium carbonate, magnesium carbonate, and the like. Examples of the sulfate include calcium sulfate.
These excipients may be used in a mixture of 2 or more kinds in a proper ratio.
The excipient in the formulation of the present invention is preferably mannitol and/or croscarmellose sodium.
In the preparation of the present invention, a binder may be used, and a binder contained in the japanese pharmacopoeia, the standards for pharmaceuticals outside the japanese pharmacopoeia, the standards for pharmaceutical additives, the food additive specifications, and the like may be used. For example, as the binder, a cellulose-based binder, a starch-based binder, a vinyl-based binder, polyether, acacia powder, alginic acid, sodium alginate, sucrose, gelatin, dextrin, pullulan, tragacanth powder, xanthan gum, pectin, sodium polyacrylate, agar, phellodendron powder, guar gum, light anhydrous silicic acid, hydrogenated oil, and the like can be given.
Examples of the cellulose-based binder include carboxymethyl cellulose (carboxymethyl cellulose, CMC), sodium carboxymethyl cellulose (sodium carboxymethyl cellulose), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (hydroxypropyl methylcellulose) (HPMC), methyl Cellulose (MC), crystalline cellulose, microcrystalline cellulose, ethyl cellulose, crystalline cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, powdered cellulose, and low-substitution hydroxypropyl cellulose.
Examples of the starch-based binder include starch, gelatinized starch, partially gelatinized starch, potato starch, wheat starch, rice starch, porous starch, corn starch, hydroxypropyl starch, sodium starch glycolate (sodium carboxymethyl starch), and the like.
Examples of the vinyl-based binder include polyvinyl alcohol (PVA), polyvinylpyrrolidone (povidone) (PVP), carboxyvinyl polymer, and copovidone.
Examples of the polyether include polyethylene glycol (polyethylene glycol) 200, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 1540, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 20000, glycerol, polyoxyethylene [105] polyoxypropylene [5] glycol, and propylene glycol.
These binders may be used in a mixture of 2 or more kinds in a proper ratio.
The binder in the formulation of the invention is preferably hydroxypropyl cellulose (HPC).
In the preparation of the present invention, a disintegrating agent can be used, and a disintegrating agent which is collected in the japanese pharmacopoeia, the standards for pharmaceuticals other than the japanese pharmacopoeia, the standards for pharmaceutical additives, the food additive specifications, and the like can be used. Examples of the disintegrating agent include cellulose-based disintegrating agents, starch-based disintegrating agents, vinyl-based disintegrating agents, and magnesium metasilicate aluminate.
Examples of the cellulose-based disintegrants include carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, low-substitution hydroxypropyl cellulose, croscarmellose sodium (Ac-Di-Sol), crystalline cellulose, and powdered cellulose.
Examples of the starch-based disintegrants include partially alpha-starch, potato starch, corn starch, hydroxypropyl starch, sodium carboxymethyl starch with low substitution degree, sodium starch glycolate, alpha-starch, and the like.
Examples of the vinyl-based disintegrants include crospovidone and polyvinyl alcohol.
These disintegrants may be used in a mixture of 2 or more kinds in a proper ratio.
Among the disintegrants, a swelling type disintegrator having a very large swelling ratio is known as a "super-disintegrator". Examples of the super-disintegrants include calcium carboxymethyl cellulose, low-substitution hydroxypropyl cellulose, croscarmellose sodium, crospovidone, sodium starch glycolate, and the like.
These super disintegrants may be used in a mixture of 2 or more kinds in a proper ratio. Furthermore, a disintegrant and a superdisintegrant may be combined.
The disintegrant in the formulation of the present invention is preferably croscarmellose sodium.
The preparation of the present invention may contain a lubricant, and a lubricant contained in the japanese pharmacopoeia, the standards of pharmaceuticals outside the japanese pharmacopoeia, the standards of pharmaceutical additives, the food additive specifications, or the like may be used. Examples of the lubricant include stearic acid and a metal salt of stearic acid, an inorganic lubricant, a hydrophobic lubricant, a hydrophilic lubricant, sodium stearyl fumarate, and the like.
Examples of the stearic acid and the metal salt of stearic acid include magnesium stearate, calcium stearate, stearic acid, stearyl alcohol, and polyethylene glycol stearate 40.
Examples of the inorganic lubricants include talc, light anhydrous silicic acid, hydrous silicon dioxide, magnesium carbonate, precipitated calcium carbonate, dried aluminum hydroxide gel, magnesium metasilicate aluminate, magnesium silicate, synthetic aluminum silicate, magnesium oxide, and magnesium sulfate.
Examples of the hydrophobic lubricant include cocoa butter, carnauba wax, glycerol fatty acid ester, hydrogenated oil, white beeswax, hydrogenated soybean oil, beeswax, cetyl alcohol, and sodium laurate.
Examples of the hydrophilic lubricant include sucrose fatty acid ester and polyethylene glycol (polyethylene glycol).
These lubricants may be used in a mixture of 2 or more kinds in a proper ratio.
The binder in the formulation of the present invention is preferably magnesium stearate.
The preparation of the present invention may contain a fluidizing agent, and a fluidizing agent contained in the japanese pharmacopoeia, the standards for pharmaceuticals other than the japanese pharmacopoeia, the standards for pharmaceutical additives, the food additive specifications, or the like may be used. Examples of the fluidizing agent include silica, stearic acid and metal salts thereof, crystalline cellulose, synthetic aluminum silicate, titanium oxide, heavy anhydrous silicic acid, magnesium aluminum hydroxide, calcium phosphate, talc, corn starch, magnesium metasilicate aluminate, granules of calcium hydrogen phosphate, calcium silicate, anhydrous calcium hydrogen phosphate, and synthetic hydrotalcite.
Examples of the silica include hydrous silica and light anhydrous silicic acid. Examples of the stearic acid and the metal salt thereof include stearic acid, calcium stearate, magnesium stearate, and the like.
These fluidizing agents may be used in a mixture of 2 or more kinds in a proper ratio.
The binder in the formulation of the invention is preferably crystalline cellulose.
In the preparation of the present invention, the method for producing the granule is not particularly limited, and specifically, a method of mixing the active ingredient, the disintegrant, the excipient and other additives to produce a mixed powder and granulating the mixed powder; the wet granulation method, the dry granulation method and the melt granulation method in which water, water containing a binder and a solvent are added to granulate, and compression molding is preferable.
In the preparation of the present invention, the method for producing tablets is not particularly limited, and specifically, the above-mentioned method is a tabletting method in which granules are produced, a disintegrant and a lubricant are mixed with the granules, and the mixed granules are compressed with a tablet press.
The preparation of the present invention may be coated with a coating layer after the production of the above-mentioned granules and tablets. When forming the coating layer on the granules, a fluidized bed granulation coater, a fluidized bed rotary coater, or the like can be used. When forming a coating layer on a tablet, a pan coater, a vent coater, or the like may be used. In the coating machine, the coating liquid is sprayed on the granules and the tablets while the granules and the tablets are flowing, and the granules and the tablets are dried to form a coating layer.
Example (example)
The present invention will be described in further detail below with reference to examples, reference examples, and test examples, but the present invention is not limited thereto.
In addition, abbreviations used in the present specification represent the following meanings.
Boc: t-Butoxycarbonyl group
CDI: carbonyl diimidazoles
DBU:1, 8-diazabicyclo [5.4.0] -7-undecene
DIEA: n, N-diisopropylethylamine
DMA: n, N-dimethylacetamide
DMF: n, N-dimethylformamide
DMSO: dimethyl sulfoxide
DTT: dithiothreitol
EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
EDT:1, 2-ethane dithiol
EDTA: ethylenediamine tetraacetic acid
FBS: fetal bovine serum
HOBT: 1-hydroxybenzotriazoles
LHMDS: lithium bis (trimethylsilyl) amide
MEM: minimum necessary medium for eagle
NMP: n-methylpyrrolidone
Pd(OAc) 2 : palladium acetate
TFA: trifluoroacetic acid
TMSCL: chlorotrimethylsilane
Xantphos:4,5 '-bis (diphenylphosphino) -9,9' -dimethylxanthene
mM：mmol/L
μM：μmol/L
nM：nmol/L
Example a
(method for identifying Compound)
NMR analyses obtained in the examples were carried out at 400MHz using DMSO-d 6 、CDCl 3 、Methanol-d 4 And (5) measuring. Furthermore, showWhen NMR data is obtained, all peaks to be measured may not be described.
RT represents LC/MS: retention time in liquid chromatography/mass spectrometry was measured under the following conditions.
(measurement condition 1')
Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm i.d.2.1X105 mm) (Waters)
Flow rate: 0.8 mL/min
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is an acetonitrile solution containing 0.1% formic acid
Gradient: after a linear gradient of 5% -100% solvent [ B ] was performed at 3.5 minutes, 100% solvent [ B ] was maintained for 0.5 minutes.
(measurement condition 2')
Column: shim-pack XR-ODS (2.2 μm, i.d. 3.0x50mm) (Shimadzu)
Flow rate: 1.6 mL/min
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is an acetonitrile solution containing 0.1% formic acid
Gradient: a linear gradient of 10% to 100% solvent [ B ] was performed at 3 minutes, maintaining 100% solvent [ B ] for 0.5 minutes.
In the specification, the term MS (m/z) refers to a value observed by mass spectrometry.
(conditions for HPLC measurement)
Column: xselect CSH Fluoro-Phenyl (3.5 μm i.d.4.6X1150 mm) (Waters)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 255nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is acetonitrile for liquid chromatography
Gradient: maintaining a linear gradient of 20% to 37% solvent [ B ] for 2 minutes, a linear gradient of 37% to 50% solvent [ B ] for 10 minutes, and a linear gradient of 50% to 95% solvent [ B ] for 2 minutes.
Flow rate: 1.0 mL/min
Injection amount: 10 mu L
(measurement of powder X-ray diffraction Pattern)
The powder X-ray diffraction measurement of the crystals obtained in each example was performed according to the powder X-ray diffraction measurement method described in the general test method of japanese pharmacopoeia. The measurement conditions are shown below.
(apparatus)
SmartLab manufactured by the Locker corporation
(method of operation)
The measuring method comprises the following steps: reflection method
The wavelength is used: cuK alpha line
Tube current: 200mA
Tube voltage: 45kV
Sample plate: aluminum (Al)
Incidence angle of X-rays: 2.5 degree
Sampling width: 0.02 degree
A detector: hyPix-3000 (two-dimensional detection mode)
(measurement of Differential Scanning Calorimeter (DSC))
DSC measurements of the crystals obtained in the examples were performed. The sample was weighed in an aluminum crucible at about 3mg and measured flat. The measurement conditions are shown below. Measurement by Differential Scanning Calorimeter (DSC) may cause errors in the range of ±2℃.
The device comprises: TA Instrument Q1000/TA Instrument
Measuring temperature range: 0-295 DEG C
Heating rate: 10 ℃/min
Atmosphere: n (N) 2 50 mL/min
(measurement of TG/DTA data)
About 3mg of the crystal obtained in example was weighed, filled in an aluminum crucible, and measured in an open system. The measurement conditions are as follows.
The device comprises: high-temperature-resistant TG/DTA STA7200RV
Measuring temperature range: room temperature-350 DEG C
Heating rate: 10 ℃/min
(determination of moisture adsorption and desorption isotherms)
The sample was weighed in a sample crucible to about 15 to 25mg, and the measurement was performed. The measurement conditions are shown below.
The device comprises: surface Measurement Systems DVS Adventure manufactured by Corp
Measurement points: 0% from 5% to 95% RH, and 95% RH from 5% to 0%
Temperature: 25 DEG C
(determination and analysis method for analysis of Single Crystal Structure)
The measurement conditions and the analysis method of the single crystal structure analysis are shown below.
(apparatus)
XtaLAB P200 MM007 manufactured by the Locker corporation
(measurement conditions)
Measuring temperature: 25 DEG C
The wavelength is used: cuK alpha line
Software: crysalisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018)
(data processing)
Software: crysalisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018)
The data were subjected to lorentz and polarization correction and absorption correction.
(analysis of Crystal Structure)
Phase determination was performed using the direct procedure shellxt (shellpaint, g.m., 2015), and finishing was performed using shellxl (shellpaint, g.m., 2015) to implement the full-matrix least squares method. The temperature factors of the non-hydrogen atoms are all refined with anisotropy. The hydrogen atoms were introduced by calculation as a coding atom using the default parameters of ShellXL. All hydrogen atoms were refined with isotropic parameters.
The mapping was performed using PLATON (Spek, 1991)/ORTEP (Johnson, 1976).
Example 1a
Synthesis of Compound (I-005)
[ chemical formula 33 ]
Step 1 Synthesis of Compound 18
Compound 4a (926 mg, 4.04 mmol), acetonitrile (7.41 mL), potassium carbonate (726 mg, 5.25 mmol) and 2,4, 5-trifluorobenzyl bromide (1000 mg, 4.44 mmol) were mixed. The reaction solution was stirred at 80℃for 40 minutes, left to cool, and then diluted with ethyl acetate. After insoluble matter was filtered off, the filtrate was concentrated to give a crude product (1.51 g, 4.04mmol, yield: quantitative) of compound 18.
LC/MS (ESI): m/z=374, rt=2.54 min, LC/MS measurement condition 1'
Step 2 Synthesis of Compound 19
Compound 18 (1.51 g, 4.04 mmol) and TFA (3.02 mL) were mixed. The reaction solution was stirred at room temperature for 4 hours and allowed to stand overnight. TFA was distilled off under reduced pressure, toluene was added to the residue, and the residue was azeotroped. Isopropyl ether was added to the residue, which was suspended and collected by filtration to give compound 19 (1.22 g, 3.84mmol, yield 95%).
LC/MS (ESI): m/z=318, rt=1.68 min, LC/MS measurement condition 1'
Step 3 Synthesis of Compound 20
Compound 19 (200 mg, 0.63 mmol), DMF (1.8 mL), potassium carbonate (261 mg, 1.89 mmol) and 3- (chloromethyl) -1-methyl-1H-1, 2, 4-triazole hydrochloride (159 mg, 0.946 mmol) were mixed. The reaction solution was stirred at 60℃for 2 hours, and a saturated aqueous ammonium chloride solution was added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with saturated brine. The organic layer was dried over magnesium sulfate, filtered and concentrated. The residue was suspended in a mixed solvent of isopropyl ether, hexane, ethyl acetate and chloroform and filtered. The residue was mixed with DMF (1.8 mL), potassium carbonate (261 mg, 1.89 mmol) and 3- (chloromethyl) -1-methyl-1H-1, 2, 4-triazole hydrochloride (159 mg, 0.946 mmol). The reaction solution was stirred at 60℃for 6 hours, and a saturated aqueous ammonium chloride solution was added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with saturated brine. The organic layer was dried over magnesium sulfate, filtered and concentrated. The residue was suspended in a mixed solvent of isopropyl ether, hexane, ethyl acetate and chloroform, and the suspension was filtered to obtain compound 20 (116 mg, 0.281mmol, yield 45%).
LC/MS (ESI): m/z=413, rt=1.84 min, LC/MS measurement condition 1'
Step 4 Synthesis of Compound (I-005)
Compound 20 (115 mg, 0.279 mmol), THF (2.30 mL) and 6-chloro-2-methyl-2H-indazol-5-amine (60.8 mg, 0.335 mmol) were mixed. LHMDS (558. Mu.L, 0.558 mmol) was added dropwise to the reaction solution at 0 ℃. The reaction solution was stirred at 0℃for 2.5 hours and at room temperature for 40 minutes, and a saturated aqueous ammonium chloride solution was added. The organic layer was extracted with chloroform and concentrated. The residue was purified by silica gel column chromatography (chloroform/methanol) to give compound (I-005) (61.8 mg, 0.116mmol, yield 42%). The HPLC measurement result of the obtained compound I-005 is shown in FIG. 7.
1 H-NMR(CDCl 3 )δ：7.96(s,1H),7.82(d,J＝2.5Hz,2H),7.48(br s,1H),7.45-7.37(m,1H),7.08(s,1H),6.97-6.88(m,1H),5.35(s,2H),5.17(s,2H),4.21(s,3H),3.89(s,3H).
LC/MS (ESI): m/z=532, rt=1.70 min, LC/MS measurement condition 1'
Example 2a
To 1170mg of the compound (I-005) were added 278mg (1.1 eq) of fumaric acid and 5.85mL of ethyl acetate, and the mixture was stirred at room temperature for 45 minutes. The solid was collected by filtration and dried, whereby fumaric acid co-crystal form I crystals (1369.4 mg, 94.6%) of the compound represented by the formula (VII) were obtained.
The results of the analysis of the single crystal structure of the fumaric acid co-crystal form I of the compound represented by the formula (VII) are shown below.
R1 (I > 2.00s (I)) was 0.0470, and no loss or misplacement of electron density was confirmed by final difference Fourier.
The crystallographic data are shown in table 1.
[ Table 1 ]
Here, the volume refers to the unit cell volume, and Z refers to the number of molecules in the unit cell.
The structure in the asymmetric unit of the fumaric acid co-crystal form I of the compound of formula (VII) is shown in FIG. 3.
In the asymmetric unit, 1 molecule of each of the compound represented by formula (VII) and fumaric acid exists. No ionic chemical interactions were confirmed, and a 1:1 molar ratio of co-crystals was confirmed.
The bond distance of N10-C9 is aboutThe bond distance of N16-C9 is about +.>From this bond distance, the compound represented by formula (VII) of fumaric acid co-crystal I form was identified as an imino structure:
[ chemical 34 ]
Further, the results of powder X-ray diffraction of fumaric acid co-crystal I-shaped crystals of the compound represented by formula (VII) are shown.
In fig. 1, a powder X-ray diffraction pattern of fumaric acid co-crystal form I (form I) of the compound represented by formula (VII) is shown. The peak table of the powder X-ray diffraction pattern of fig. 1 is shown in fig. 2.
Powder X-ray diffraction pattern, diffraction angle (2θ): peaks were confirmed at 7.8±0.2°, 9.5±0.2°, 10.1±0.2°, 10.9±0.2°, 13.8±0.2°, 14.7±0.2°, 18.6±0.2°, 22.6±0.2°, 23.5±0.2° and 24.6±0.2°.
In the powder X-ray diffraction peak, diffraction angle (2θ): peaks of 9.5.+ -. 0.2 °, 10.9.+ -. 0.2 °, 18.6.+ -. 0.2 °, 23.5.+ -. 0.2 ° and 24.6.+ -. 0.2 ° are particularly characteristic as fumaric acid co-crystal form I crystals of the compound represented by formula (VII).
The DSC analysis result of fumaric co-crystal I-shaped crystals of the compound of formula (VII) showing the powder X-ray analysis pattern of FIG. 1 is shown in FIG. 4. The onset temperature of the endothermic peak shows about 272 ℃.
The TG/DTA analysis result of fumaric acid co-crystal form I (form I) of the compound shown in formula (VII) showing the powder X-ray analysis pattern of fig. 1 is shown in fig. 5.
DVS analysis results of fumaric acid co-crystal form I (form I) of the compound shown in formula (VII) showing the powder X-ray analysis pattern of fig. 1 are shown in fig. 6.
The synthesis of the compound (I-005) represented by the formula (VII) may be performed as follows.
Step 4'
LHMDS (1M in THF;1.46 mL; 1.46 mmol) was added dropwise to compound 20 (300 mg, 0.727 mmol), 6-chloro-2-methyl-2H-indazol-5-amine (172 mg, 0.946 mmol) in THF (6 mL) at 0deg.C. The reaction mixture was stirred at 0℃for 2.5 hours and at room temperature for 40 minutes, and a saturated aqueous ammonium chloride solution was added thereto. The aqueous layer was extracted with EtOAc. The organic layer was washed with saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CHCl 3/MeOH, gradient 0-20% MeOH). The resulting solid was solidified from acetone/H2O to give compound I-005 (95.3 mg, yield 25%, brown solid). The HPLC measurement result of the obtained compound I-005 is shown in FIG. 8 (about 99 pa%).
In addition, abbreviations used in the present specification represent the following meanings.
BINAP:2,2 '-bis (diphenylphosphino) -1,1' -binaphthyl
CPME: cyclopentyl methyl ether
CbzCl: benzoic acid phenylmethyl ester
DME: dimethyl ether
MEK: methyl ethyl ketone
Example b
(method for identifying Compound)
NMR analyses obtained in each of examples and reference examples were carried out at 400MHz using DMSO-d 6 、CDCl 3 And (5) measuring. In addition, in the case of NMR data, all peaks to be measured may not be described.
"RT" or "hold time" refers to the expression LC/MS: retention time in liquid chromatography/mass spectrometry or liquid chromatography (HPLC) was measured under the following conditions.
The term MS (m/z) refers to a value observed by mass spectrometry.
(measurement condition 1)
Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm i.d.2.1X105 mm) (Waters)
Flow rate: 0.8 mL/min
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is an acetonitrile solution containing 0.1% formic acid
Gradient: after a linear gradient of 5% -100% solvent [ B ] was performed at 3.5 minutes, 100% solvent [ B ] was maintained for 0.5 minutes.
(measurement condition 2)
Column: ACQUITY UPLC (registered trademark) BEH C18 (1.7 μm i.d.2.1X105 mm) (Waters)
Flow rate: 0.8 mL/min
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 10mM ammonium carbonate, [ B ] is an acetonitrile solution containing 0.1% formic acid
Gradient: after a linear gradient of 5% -100% solvent [ B ] was performed at 3.5 minutes, 100% solvent [ B ] was maintained for 0.5 minutes.
(measurement condition 4)
Column: xselect CSH C18 (3.5 μm i.d.4.6X150 mm) (Waters)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is acetonitrile for liquid chromatography
Gradient: after a linear gradient of 5% -95% solvent [ B ] was performed at 17 minutes, 95% solvent [ B ] was maintained for 3 minutes.
Flow rate: 1.0 mL/min
Injection amount: 5 mu L
(measurement condition 5)
Column: xselect CSH C18 (3.5 μm i.d.4.6X150 mm) (Waters)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is acetonitrile for liquid chromatography
Gradient: after a linear gradient of 5% -95% solvent [ B ] was performed at 17 minutes, 95% solvent [ B ] was maintained for 3 minutes.
Flow rate: 1.0 mL/min
Injection amount: 10 mu L
(measurement condition 7)
Column: xselect CSH Fluoro-Phenyl (3.5 μm i.d.4.6X1150 mm) (Waters)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 255nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is acetonitrile for liquid chromatography
Gradient: maintaining the solvent [ B ] at 20% for 6 minutes, performing a linear gradient of the solvent [ B ] at 20% -42% for 21 minutes, performing a linear gradient of the solvent [ B ] at 42% -50% for 4 minutes, and performing a linear gradient of the solvent [ B ] at 50% -95% for 3 minutes.
Flow rate: 1.0 mL/min
Injection amount: 10 mu L
(measurement condition 8)
Column: xselect CSH Fluoro-Phenyl (3.5 μm i.d.4.6X1150 mm) (Waters)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 255nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is acetonitrile for liquid chromatography
Gradient: maintaining a linear gradient of 20% to 37% solvent [ B ] for 2 minutes, a linear gradient of 37% to 50% solvent [ B ] for 10 minutes, and a linear gradient of 50% to 95% solvent [ B ] for 2 minutes.
Flow rate: 1.0 mL/min
Injection amount: 10 mu L
(measurement condition 9)
Column: YMC Jsphere ODS-H80 (4 μm i.d.4.6x250 mm)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 250nm
Mobile phase: [A] is an aqueous solution containing 0.2% trifluoroacetic acid, [ B ] is methanol for liquid chromatography
Gradient: after a linear gradient of 10% -70% solvent [ B ] at 6 minutes, a linear gradient of 70% solvent [ B ] at 3 minutes, followed by a linear gradient of 70% -90% at 3 minutes, followed by a linear gradient of 90% solvent [ B ] at 5 minutes, followed by a linear gradient of 90% -95% at 1 minute, followed by a linear gradient of 95% solvent [ B ] at 5 minutes
Flow rate: 1.0 mL/min
Injection amount: 5 mu L
(measurement condition 10)
Column: xselect CSH C18 (3.5 μm i.d.4.6X105 mm)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 254nm
Mobile phase: [A] is an aqueous solution containing 0.1% formic acid, [ B ] is acetonitrile for liquid chromatography
Gradient: after a linear gradient of 5% -95% solvent [ B ] was performed at 17 minutes, 95% solvent [ B ] was maintained for 3 minutes.
Flow rate: 1.0 mL/min
Injection amount: 10 mu L
(measurement condition 11)
Column: XBIdge C18 (3.5 μm i.d.4.6X105 mm)
Column temperature: constant temperature around 40 DEG C
UV detection wavelength: 210nm of
Mobile phase: [A] is an aqueous solution containing 10mM ammonia, [ B ] is methanol for liquid chromatography
Gradient: a linear gradient of 5% solvent [ B ] was maintained at 5 minutes, 5% to 38% solvent [ B ] was performed at 10 minutes, and 38% to 95% solvent [ B ] was performed at 5 minutes.
Flow rate: 0.8 mL/min
Injection amount: 10 mu L
(measurement condition 12)
Column: c18 column
Column temperature: 40 DEG C
UV detection wavelength: 255nm
Mobile phase: [A] is an aqueous solution containing 10mM ammonia formate, [ B ] is methanol for liquid chromatography
The gradient slowly increases the mixing ratio of mobile phase B from 9:1 to 1:9, measured at 28 minutes.
Flow rate: 0.3 mL/min
Injection amount: 4 mu L
(measurement of powder X-ray diffraction Pattern)
The powder X-ray diffraction measurement of the crystals obtained in each example was performed according to the powder X-ray diffraction measurement method described in the general test method of japanese pharmacopoeia. The measurement conditions are shown below. (apparatus)
MiniFlex600 manufactured by the Fecky corporation
(method of operation)
A detector: high-speed one-dimensional detector (D/TecUltra 2)
The type of light source: cu pipe ball
The wavelength is used: cuK alpha line
Tube current: 15mA
Tube voltage: 40Kv
Sample plate: non-reflection sample plate
(determination and analysis method for analysis of Single Crystal Structure)
The measurement conditions and the analysis method of the single crystal structure analysis are shown below.
(apparatus)
XtaLAB P200 MM007 manufactured by the Locker corporation
(measurement conditions)
Measuring temperature: 25 DEG C
The wavelength is used: cuK alpha line
Software: crysalisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018)
(data processing)
Software: crysalisPro 1.171.39.46e (Rigaku Oxford Diffraction, 2018)
The data were subjected to lorentz and polarization correction and absorption correction.
(analysis of Crystal Structure)
Phase determination was performed using the direct procedure shellxt (shellpaint, g.m., 2015), and finishing was performed using shellxl (shellpaint, g.m., 2015) to implement the full-matrix least squares method. The temperature factors of the non-hydrogen atoms are all refined with anisotropy. The hydrogen atoms were introduced by calculation as a coding atom using the default parameters of ShellXL. All hydrogen atoms were refined with isotropic parameters.
The mapping was performed using PLATON (Spek, 1991)/ORTEP (Johnson, 1976).
(measurement of Differential Scanning Calorimeter (DSC))
DSC measurements of the crystals obtained in the examples were performed. The sample was weighed in an SUS crucible at about 2mg, and measured by flattening. The measurement conditions are shown below. Measurement by Differential Scanning Calorimeter (DSC) may cause errors in the range of ±2℃.
The device comprises: DSC 3+
Measuring temperature range: 30-300 DEG C
Heating rate: 10 ℃/min
Atmosphere: n2 40 mL/min
(measurement of TG/DTA data)
About 3mg of the crystal obtained in example was weighed, filled in an aluminum crucible, and measured in an open system. The measurement conditions are as follows.
The device comprises: hitachi-tek, and method for producing the same, using the same, include the steps of Hitachi-tek, tg/DTA7200
Measuring temperature range: 30-350 DEG C
Heating rate: 10 ℃/min
(determination of moisture adsorption and desorption isotherms)
The sample was weighed in a sample crucible to about 15 to 25mg, and the measurement was performed. The measurement conditions are shown below.
The device comprises: surface Measurement Systems DVS Adventure manufactured by Corp
Measurement points: 0% from 5% to 95% RH, and 95% RH from 5% to 0%
Temperature: 25 DEG C
The comparison of the powder X-ray analysis patterns before and after DVS measurement was measured by the following method.
(apparatus)
SmartLab manufactured by the Locker corporation
(method of operation)
The measuring method comprises the following steps: reflection method
The wavelength is used: cuK alpha line
Tube current: 200mA
Tube voltage: 45kV
Sample plate: aluminum (Al)
Incidence angle of X-rays: 2.5 degree
Sampling width: 0.02 degree
A detector: hyPix-3000 (two-dimensional detection mode)
(determination of particle size distribution)
The measurement conditions are as follows.
The manufacturer: fuel tap and fuel cell system
The device comprises: laser diffraction type HELOS & RODOS particle size distribution measuring device
The range is as follows: r4
Dispersion pressure: 3bar
Triggering conditions: the measured concentration is less than or equal to 1% or 10s actual time after 2s
Example 1b
Synthesis of fumaric acid Co-Crystal form I of Compound of formula (VII)
[ 35 ]
Step 1: synthesis of Compound 3
Compound 1 (35.0 kg, 238.8mol, hydrochloride), N-dimethylacetamide (273L), 1, 8-diazabicyclo [ 5.4.0 ] -7-undecene (87.2 kg, 573.1 mol) and compound 2 (26.0 kg, 262.7 mol) were mixed and stirred at 25 ℃ for 10 minutes. N, N' -carbonyldiimidazole (50.3 kg, 310.4 mol) and N, N-dimethylacetamide (7L) were stirred at 50℃for 90 minutes. Methanol (18.4 kg, 573.1 mol) was added to the reaction solution, cooled to 25℃and the pH was adjusted to 2.5 with 10% sulfuric acid. The slurry was cooled to 5℃and the solid was collected by filtration, washed with 20% methanol water and dried, whereby compound 3 (38.12 kg, 162.0mol, yield: 67.9%) was obtained.
HPLC (uv=254 nm): rt=8.9 min, HPLC assay condition 4
Step 2: synthesis of Compound 5
Compound 3 (34.5 kg, 146.7 mol), acetonitrile (345L), diisopropylethylamine (26.5 kg, 205.4 mol) and compound 4 (39.6 kg, 176.0 mol) were mixed and stirred at 60 ℃ for 300 minutes. The reaction solution was cooled to 25℃and water (172.5L) was added. The slurry was cooled to 0℃and the solid was collected by filtration, washed with 66% acetonitrile water and dried, whereby compound 5 (46.10 kg, 121.5mol, yield: 82.9%) was obtained.
HPLC (uv=254 nm): rt=14.7 min, HPLC assay condition 4
Step 3: synthesis of Compound 7
Compound 5 (29.0 kg, 76.4 mol), trifluoroacetic acid (72.5L) and compound 6 (16.5 kg, 152.9 mol) were mixed and stirred at 35℃for 180 minutes. The reaction solution was cooled, ethyl acetate (348L) was added thereto, and the mixture was washed with a 38% aqueous tripotassium phosphate solution, 2.3% saline solution and water. The ethyl acetate solution was concentrated to 203L and heptane (261L) was added. The slurry was cooled to 0℃and the solid was collected by filtration, washed with a mixed solvent of ethyl acetate and heptane, and then dried, whereby compound 7 (23.60 kg, 65.0mol, yield: 85.0%) was obtained.
HPLC (uv=254 nm): rt=12.5 min, HPLC assay condition 5
Step 4: synthesis of Compound 9
Compound 7 (23.3 kg, 64.1 mol), compound 8 (14.0 kg, 83.4mol, hydrochloride), potassium iodide (6.4 kg, 38.5 mol), cesium carbonate (31.3 kg, 96.2 mol) and N, N-dimethylacetamide (139.8L) were mixed and stirred at 40 ℃ for 360 minutes. The reaction solution was cooled to 25℃and acetic acid (34.6 kg, 577.2 mol) was added. Insoluble matter was collected by filtration, and acetonitrile (93.2L) and water (326.2L) were added to the filtrate. The slurry was cooled to 0℃and the solid was collected by filtration, washed with a 20% acetonitrile aqueous solution and dried, whereby compound 9 (20.35 kg, 44.4mol, yield: 69.2%) was obtained.
HPLC (uv=255 nm): rt=25.1 min, HPLC assay condition 7
The same procedure was carried out 2 times for steps 1 to 4.
Step 5-1: synthesis of fumaric acid Co-Crystal form I of Compound of formula (VII)
Compound 9 (39.0 kg, 80.7 mol), compound 10 (16.2 kg, 84.8 mol), acetic acid (30.7 kg, 484.3 mol) and toluene (234L) obtained in steps 1 to 4 were mixed and stirred at 100℃for 360 minutes. Toluene (390L) was added and the resulting slurry was cooled to 25 ℃. The solid was collected by filtration and washed with acetone to give an undried crystal of the compound represented by formula (VII) (the result of measurement of the undried crystal obtained here by HPLC measurement condition 8 is shown in fig. 12 and 13. Rt=9.8 min peak is toluene).
Step 5-2: to half the amount of the obtained undried crystal of the compound represented by the formula (VII) were added acetone (613.5L) and water (109.2L), and the mixture was dissolved at 50 ℃. The obtained solution was treated with activated carbon, and acetone (150.2L) and water (5.9L) were added to the treated solution, followed by concentration to 702L. The temperature of the concentrate was adjusted to 50℃and fumaric acid (4.6 kg, 72.6 mol), acetone (150.2L) and water (5.9L) were added to concentrate to 464L. Acetone (78L) was added to the concentrated solution, concentrated to 265L, and acetone (19.5L) was added. The slurry temperature was adjusted to 55℃and stirred for 120 minutes or more. The slurry was cooled to 0 ℃, and the solid was collected by filtration, washed with acetone and dried (the results of DSC, TG/DTA, HPLC, particle size distribution, and DVS measurement of the dried crystals obtained herein are shown in FIGS. 14 to 18.HPLC as measured by measurement condition 12. FIG. 19 shows a comparison of powder X-ray analysis patterns before and after the DVS measurement).
The same procedure was repeated for the remaining half amount, to thereby obtain fumaric acid co-crystal form I (41.68 kg, 64.3mol, yield: 75.6%) of the compound represented by the formula (VII).
HPLC (uv=255 nm): rt=12.8 min, HPLC assay condition 8
Example 2b
Synthesis of a toluene Compound of the Compound represented by the formula (VII)
Step 1
Compound 9 (150 mg,0.327 mmol) and compound 10 (65.4 mg,0.360 mmol) were mixed with toluene (1.5 mL) and acetic acid (0.87 mL,3.27 mmol) and stirred at 100 ℃ for 9 hours. After cooling to room temperature, heptane (1.5 mL, 10V) was added and filtered, and the resulting crystals were washed 3 times with heptane (0.7 mL). Drying under reduced pressure gave crystals of the compound represented by formula (VII) (168 mg, yield 87%). The resulting crystals contained 0.5 to 0.6 molecular equivalent of toluene as a solvate, and were dried under reduced pressure in the usual operation range without removing toluene. It was confirmed that a toluene compound of the compound represented by the formula (VII) was obtained with good quality.
1H-NMR(400MHz,CDCl 3 )δppm：7.93(s,1H),7.70(d,J＝2.57Hz,2H),7.62(brs,1H),7.35-7,45(m,1H),7.07(m,1H),6.92-6.97(td,J＝9.63,6.42,1H),5.34(s,2H),5.14(s,2H),4.20(s,3H),3.87(s,2H).
At 7.14 to 7.27ppm and 2.35ppm, peaks corresponding to 0.5 to 0.6 molecules of toluene were confirmed.
Reference example 1 Synthesis of Compound S-4
[ chemical formula 36 ]
Step 1: synthesis of Compound S-2
Compound S-1 (5.50 kg, 29.5 mol), acetonitrile (21.7 kg) and glacial acetic acid (115.00 kg) were mixed and cooled to 5 ℃. 17% sodium nitrite aqueous solution (13.03 kg) was added thereto, followed by stirring for 1 hour, heating to 25℃and stirring for 1.5 hours. Insoluble materials were collected by filtration, and washed with acetonitrile (21.7 kg) and tetrahydrofuran (49.0 kg). To the collected filtrate was added water (460L). The slurry was cooled to 0℃and the solid was collected by filtration, washed with water and dried to thereby obtain Compound S-2 (3.75 kg, 19.0mol, yield: 64.4%).
LC/MS (ESI): m/z=196 (M-H), rt=11.8 min, LC/MS assay condition 4
Step 2: synthesis of Compound S-3
Compound S-2 (3.25 kg, 16.4 mol) and ethyl acetate (58.7 kg) were mixed, and trimethyloxonium tetrafluoroborate (2.09 kg, 14.1 mol) was added thereto, followed by stirring at 25℃for 7 hours. To the reaction solution, a mixture of ethyl acetate (29.5 kg) and methanol (10.3 kg) was added. The mixture was added to a 5% aqueous sodium carbonate solution (66.3 kg), and the organic layer and the aqueous layer were separated. The organic layer was washed 2 times with 5% aqueous sodium chloride (65.8 kg), treated with activated carbon, and concentrated to 42kg. Tetrahydrofuran (87.0 kg) was added thereto, the mixture was concentrated to 23kg, the above operation was repeated 2 times, tetrahydrofuran (87.0 kg) was further added thereto, the mixture was concentrated to 18.9kg, and the temperature was raised to 33 ℃. To the mixture was added heptane (47.0 kg). The slurry was cooled to 0℃and the solid was collected by filtration, washed with a mixture of tetrahydrofuran and heptane, and dried, whereby Compound S-3 (1.68 kg, 7.9mol, yield: 48.2%) was obtained.
LC/MS(ESI)：m/z＝212(M+H)、253(M+CH 3 Cn+h) rt=12.4 min, LC/MS assay condition 4
Step 3: synthesis of Compound S-4
Compound S-3 (1040 g, 4.9 mol), 10% palladium on carbon (PE, aqueous) (523 g, 0.25 mol) and ethyl acetate (8.99 kg) were mixed, and hydrazine monohydrate (504 g, 10.1 mol) was added thereto, followed by stirring at 35℃for 3 hours. 10% palladium on carbon was collected by filtration, and 10% palladium on carbon was washed with water (1560 g) and ethyl acetate (9.00 kg). To the collected filtrate, 2mol/L hydrochloric acid (750 g) was added, and the mixture was separated into an organic layer and an aqueous layer. The resulting aqueous layer was extracted with ethyl acetate (4.69 kg). The organic layers were combined, treated with activated carbon and concentrated to 11.09kg. To the concentrate was added 4mol/L of a hydrogen chloride/ethyl acetate solution (1124 g). The solid was collected by filtration, washed with ethyl acetate and dried, whereby Compound S-4 (0.84 kg, 3.9mol, yield: 78.5%) was obtained.
LC/MS(ESI)：m/z＝182(M+H)、223(M+CH 3 CN+H)RT＝6.6min、
LC/MS measurement condition 4
Chlorine concentration (ion chromatography): 16.74%
Reference example 2 Synthesis of Compound A-3
[ FORMS 37 ]
Step 1: synthesis of dichloromethane solution of Compound A-2
Compound A-1 (9.2 kg,65.1 mol) and tetrahydrofuran (64L) were mixed and cooled to 0℃to prepare a slurry. Wherein, while keeping the internal temperature below 8 ℃, it took 60 minutes to drop a Red-AL/tetrahydrofuran solution obtained by mixing sodium bis (2-methoxy) aluminum hydride (Red-AL)/toluene solution (65 wt%) (26.4 kg,84.9 mol) and tetrahydrofuran (28L). Thereafter, stirring was carried out at 0℃to 5℃for 30 minutes. To the reaction solution, acetone (4.9 kg,84.3 mol) was added dropwise over 30 minutes, and the temperature was raised to 25 ℃. In another reactor, a slurry obtained by mixing potassium sodium tartrate/4 hydrate (46 kg,163 mol) and tetrahydrofuran (138L) was prepared, and a reaction solution obtained by quenching the reaction solution with acetone for the previous Red-AL reduction was added dropwise over 30 minutes (the internal temperature in this process reached around 40 ℃). After stirring continuously at 40℃for 2 hours, it was cooled to 25 ℃. Water (2.5 kg) was added thereto and stirred, followed by suction filtration, and the resulting filtrate was concentrated under reduced pressure to 35kg (28L). The filtrate (28L) 3 was aliquoted, toluene (2.6 kg) was added to the first portion, the mixture was concentrated under reduced pressure, the above operation was repeated 8 times, and then the mixture was finally concentrated to dryness. To the concentrated, dried product was added methylene chloride (13.5 kg) to prepare a methylene chloride solution of product A-2. The same procedure was also carried out for the second and third portions, to prepare an A-2/dichloromethane solution (yield: 74.8%) composed of A-2 (5.53 kg) and dichloromethane (42.7 kg).
1H-NMR(400MHz,CDCl3,30℃)δppm：8.00(s,1H),4.74(s,2H),3.90(s,3H).
Step 2: synthesis of Compound 8
To the A-2/methylene chloride solution produced in step 1 (49.8 kg of a methylene chloride solution containing 5.53kg of A-2 (48.8 mol)) was added methylene chloride (44L), and the temperature was adjusted to 25 ℃. It took 30 minutes to drop a mixed solution of thionyl chloride (7.8 kg,65.5 mol) and methylene chloride (27L), and the mixture was washed with methylene chloride (8.2L) to prepare a washing solution, which was then stirred at room temperature for 7 hours. In addition, a 20% aqueous sodium acetate solution (179 kg) was prepared from sodium acetate (36.2 kg,436 mol) and water in the course of a water course (143L). 20% sodium acetate (119 kg) was added dropwise to the previous reaction solution. The pH at the end of the addition was around 4.6. The organic layer obtained in this operation was washed with a 10% aqueous sodium chloride solution prepared from sodium chloride (5.5 kg) and tap water (49L), and the aqueous layer was extracted with dichloromethane (55L) as well. After the combined organic layers (dichloromethane solution) were concentrated to 33L, ethyl acetate (27.5L) was added and concentrated. After concentrating to 33L, ethyl acetate (47.5L) was additionally added, and the mixture was concentrated at normal pressure at a jacket temperature of 60℃to filter out the inorganic salt formed. To the filtrate was added a hydrochloric acid/ethyl acetate solution (4 mol/L,12.6 kg), and the mixture was subjected to hydrochloride formation, stirred at 25℃for 30 minutes, and then cooled to around 5 ℃. After crystallization was completed with stirring for 30 minutes, the resulting crystallized slurry was filtered, washed with cooled ethyl acetate (55L), and dried under reduced pressure to give compound 8 (5.25 kg) (pale yellow powder, yield: 64.8%).
1H-NMR(400MHz,DMSO-D6,30℃)δppm：8.54(s,1H),4.70(s,2H),3.86(s,3H).
Reference example 3 Synthesis of Compound 10
[ chemical 38 ]
Step 1: synthesis of Compound B-2
Compound B-1 (79.1 kg,499 mol) was added in portions (internal temperature kept at 0℃to 5 ℃) to 98% sulfuric acid (395.7L) cooled at 0℃to 5℃under a nitrogen atmosphere. Potassium nitrate (55.5 kg) was held at an internal temperature of 0℃to 12℃and was fed in portions 15 times (at intervals of 20 minutes or more). Stirring at 0-5 deg.c for 5 hr. The reaction mixture was slowly poured into water (791L) cooled to 0℃to 5℃while maintaining the internal temperature at 0℃to 5℃and thoroughly washed with 98% sulfuric acid (39.6L), followed by stirring at 0℃for 5 hours. The slurry was filtered through a centrifuge and washed with water (791L). The crude solid obtained was suspended in water (791L), stirred at 20℃to 30℃for 30 minutes, and then the solid was filtered, washed 3 times with water (791L), and dried under reduced pressure to give Compound B-2 (99.61 kg).
1H-NMR(400MHz,CDCl3)δppm：10.31(s,1H),8.46(d,J＝6.60Hz,1H),7.47(d,J＝9.17Hz,1H).
HPLC (uv=250 nm): rt=10.9 min, HPLC assay conditions 9
Step 2: synthesis of Compound S-2
Ethanol (697L), water (697L) and hydrazine 1 hydrate (73.5 kg,1468 mol) were mixed and heated to 45 ℃. Therein, it took 60 minutes to drop a mixed solution of compound B-2 (99.6 kg,489 mol) and ethanol (299L), and stirring was further carried out at 45 to 50 ℃ for 9 hours at 8 hours. While maintaining the internal temperature at 40℃to 50℃it took 30 minutes or more to drop an aqueous solution prepared from potassium hydrogencarbonate (53.9 kg,538 mol) and water (1295L). Cooling to 0-5 deg.c, stirring for 1 hr and filtering. Water (1335L) and ethanol (657L) were mixed and the previous solids were washed using aqueous ethanol cooled to 0℃to 5 ℃. Drying under reduced pressure gave Compound S-2 (83.25 kg) (yield: 86.9%).
1H-NMR(400MHz,DMSO-d6)δppm：13.56-13.98(m,1H),8.67(s,1H),8.37(d,J＝0.98Hz,1H),7.92(d,J＝0.61,1H).
HPLC (uv=250 nm): rt=10.4 min, HPLC assay conditions 9
Step 3: synthesis of Compound S-3
Compound S-2 (84 kg,430 mol) and ethyl acetate (1596L) were mixed and stirred at 20℃to 30 ℃. Trimethyloxonium tetrafluoroborate (77.6 kg,525 mol) was added in portions, ethyl acetate (84L) was added, and stirring was performed at 25℃for 6 hours. It took 2 hours to quench the excess trimethyloxonium tetrafluoroborate by dropping a mixed solution of methanol (252L) and ethyl acetate (420L) into the previous reaction solution. To an aqueous sodium carbonate solution obtained by mixing sodium carbonate (84 kg) with water (1596L), the reaction solution after the previous quenching was added dropwise over 1 hour, and ethyl acetate (420L) and methanol (84L) were added. The organic layer obtained by the liquid separation operation was washed with saturated brine (1680 kg) 2 times, and the obtained organic layer was subjected to an activated carbon filtration treatment. Thereafter, the organic layer was concentrated under reduced pressure, and then tetrahydrofuran (2520L) was introduced thereinto, followed by further concentration under reduced pressure. After the additional inflow of tetrahydrofuran and the concentration under reduced pressure were performed again, heptane (2139L) was added dropwise, and after cooling to-5℃to 5℃the mixture was stirred at around 0℃for 1 hour, and then crystallized and matured. The crystallization slurry was filtered and washed with a cooled mixed solution of tetrahydrofuran (224L) and heptane (912L). Drying under reduced pressure gave Compound S-3 (65.73 kg) (yield: 74.1%).
1H-NMR(400MHz,CDCl3)δppm：8.31(s,1H),8.13(s,1H),7.81(s,1H),4.27(s,3H).
HPLC (uv=254 nm): rt=10.3 min, HPLC assay condition 10
Step 4: synthesis of Compound 10
Compound S-3 (65.7 kg,310 mol) and ethyl acetate (657L) were mixed, stirred at room temperature, cooled to around 10℃and purged with nitrogen. 5% platinum-carbon (57.7 kg,53% moisture content) was added. After the hydrogen substitution, the internal temperature was adjusted to around 25℃and stirring was performed for 4 hours. After confirming the disappearance of the starting material, nitrogen substitution was performed, and a filtration operation was performed to remove the platinum-carbon catalyst. After the liquid separation operation, the organic layer was concentrated, and heptane was added dropwise to the ethyl acetate solution to form a crystallization slurry. After filtration and washing with heptane/ethyl acetate, drying under reduced pressure was performed to obtain compound 10 (37.24 kg) (yield: 66.2%).
1H-NMR(400MHz,CDCl3)δppm：7.70(s,1H),7.64(s,1H),6.89(s,1H),4.15(s,3H).
HPLC (uv=254 nm): rt=4.8 min, HPLC assay condition 10
Reference example 4 Synthesis of Compound 9
[ chemical formula 39 ]
Step 1: synthesis of Compound C-2
Compound C-1 (10.00 g, 48.0mmol, mesylate), N' -carbonyldiimidazole (8.18 g, 50.4 mmol), acetonitrile (60.00 mL), and diisopropylethylamine (6.83 g, 52.8 mmol) were mixed and stirred at 10℃for 60 minutes. In the reaction mixture, compound 1 (8.09 g, 55.2mmol, hydrochloride) and diisopropylethylamine (7.14 g, 55.2 mmol) were mixed and stirred at 50℃for 210 minutes. The reaction solution was cooled and concentrated to 45g. 2-propanol (100 mL) was added, and after concentrating to 60g, 2-propanol (100 mL) was added. The slurry was cooled to 0℃and the solid was collected by filtration, washed with 2-propanol and dried, whereby Compound C-2 (10.48 g, 42.2mmol, yield: 88%) was obtained.
HPLC (uv=210 nm): rt=14.5 min, HPLC assay condition 11
Step 2: synthesis of Compound C-3
Compound C-2 (8.00 g, 32.2 mmol), N' -carbonyldiimidazole (6.79 g, 41.9 mmol), tetrahydrofuran (80.0 mL), and 1, 8-diazabicyclo [5.4, 0] -7-undecene (5.40 g,35.4 mmol) were mixed and stirred at 25℃for 120 minutes. Tetrahydrofuran (80.0 mL) was added dropwise, and the reaction solution was cooled to 0℃to form a crystallized slurry. The solid was collected by filtration, washed with tetrahydrofuran and then dried under heating, whereby crystals of Compound C-3 (12.6 g, 29.6mmol, 1, 8-diazabicyclo [5.4.0] -7-undecene salt, yield: 92%) were obtained.
HPLC (uv=210 nm): rt=1.9 min, HPLC assay condition 11
Step 3: synthesis of Compound 9
Compound C-3 (1.00 g, 2.3mmol, 1, 8-diazabicyclo [5.4.0] -7-undecene salt), N-dimethylacetamide (5.0 mL) and compound 4 (579.2 mg, 2.6 mmol) were mixed and stirred at 70℃for 300 minutes. The reaction mixture was cooled, acetonitrile (10 mL) was added thereto, and the mixture was concentrated to 9.4g, and the above operation was repeated 2 times. To the concentrated solution were added compound 6 (461 mg, 4.7 mmol) and diisopropylethylamine (456 mg, 3.5 mmol), and the mixture was stirred at 60℃for 160 minutes. The reaction solution was cooled to 25℃and acetic acid (703 mg, 11.7 mmol), water (8.0 mL) and seed crystals were added thereto, and the resulting crystallization slurry was cooled to 0 ℃. To the slurry was added water (12.0 mL), and the solid was collected by filtration, washed with a 20% acetonitrile aqueous solution, and then dried, whereby compound 9 (0.86 g, 1.9mmol, yield: 79.5%) was obtained.
HPLC (uv=255 nm): rt=14.5 min, HPLC assay condition 8
Reference example 5 Synthesis of Compound S-3
[ 40 ]
Step 1: synthesis of Compound S-3
In the same manner as in step 1 of reference example 3, compound B-2 was obtained. Then, compound B-2 (30 g, 147 mmol) and NMP (120 mL) were mixed, boc-carboxylate (56 g, 383 mmol) was added under ice-cooling, and stirring was performed at room temperature for 30 minutes. Diisopropylethylamine (38.6 mL, 221 mmol) was added to the reaction mixture, and the mixture was stirred at 90℃for 20 hours. The reaction mixture was set at 80℃and water (240 mL) was added thereto, followed by cooling to room temperature, and insoluble matters precipitated were removed by filtration. The resulting solid was washed 3 times with a mixture of NMP/water=1/2 (15 mL) and 3 times further with water (30 mL). The resulting solid was suspended in isopropyl acetate (60 mL), heptane (240 mL), and after stirring at room temperature, washed 3 times with isopropyl acetate/heptane=1/4 (30 mL), thereby obtaining compound D-3. The resulting solid was suspended in isopropyl acetate (100 mL). The resulting suspension was added to a mixture of methanesulfonic acid (96 mL, 1474 mmol) and isopropyl acetate (100 mL) at 55deg.C, thoroughly washed with isopropyl acetate (60 mL), and stirred at that temperature for 25 minutes. Water (240 mL), aqueous sodium hydroxide (239 mL, 1916 mmol) and isopropyl acetate (150 mL) were added to the reaction solution under ice-cooling, and stirred at 40 ℃. To the resulting reaction solution was added isopropyl acetate (150 mL). The resulting organic layer was washed 3 times with water (90 mL) and concentrated to 45g. Isopropyl acetate (12 g) and heptane (210 mL) were added, the resulting insoluble matter was collected by filtration, and the solid was washed 3 times with isopropyl acetate/heptane=1/7 (30 mL) and dried, whereby compound S-3 (25.3 g, 120mmol, yield: 81.1%) was obtained.
1H-NMR(400MHz,CDCl3)δppm：8.34(s,1H),8.13(s,1H),7.84(s,1H),4.28(s,3H).
Reference example 6 Synthesis of Compound 10
[ chemical formula 41 ]
Step 1: synthesis of Compound T-2, T-3
Compound T-1 (40 g, 182 mmol), concentrated sulfuric acid (200 mL, 3677 mmol) and 69% nitric acid (23.3 g, 255 mmol) were mixed under ice-cooling, stirred for 3 hours under ice-cooling to room temperature, and then allowed to stand overnight. The mixture was poured into ice water 520mL, and methylene chloride (200 mL) was added thereto to separate the liquid. The resulting dichloromethane solution was washed 2 times with 5% aqueous sodium bicarbonate (400 mL) and concentrated to dryness. Methanol (120 mL) was added to the obtained solid, and the mixture was concentrated until the content reached 116g. After methanol was added to the resulting slurry until the content reached 333g, water (240 mL) was added, the resulting insoluble matter was collected by filtration, and the solid was washed with methanol/water=1/1 (200 mL) and dried, whereby a mixture of compound T-2/T-3=1/2.78 (30.67 g, yield: 58.5%) was obtained.
LC/MS (ESI): as m/z, MS detection was not performed, RT=2.04 min, LC/MS measurement condition 1
Step 2: synthesis of Compound T-4
2-propanol (1.4 mL) was added to a mixture (200 mg) of T-2/T-3=1/2.78 under a nitrogen flow, the temperature was raised to 60℃and triethylamine (0.289 mL, 2.07 mmol) was added thereto and stirring was carried out for 1.5 hours. Thereafter, a solution of methylamine hydrochloride (94 mg, 1.39 mmol) in water (0.4 mL) was added to the reaction solution at 60℃and stirred for 3 hours. To the resulting reaction solution was added water (8 mL) at 60℃and the mixture was cooled to room temperature, followed by stirring for 30 minutes. Insoluble matter precipitated was collected by filtration, and the obtained solid was washed with water (5 mL) and dried, whereby Compound T-4 (194 mg, 0.699mmol, yield: 100%) was obtained.
LC/MS (ESI): m/z=277 (m+h), rt=2.46 min, LC/MS assay condition 2
Step 3: synthesis of Compound T-5
Compound T-4 (500 mg,1.80 mmol) and 2-propanol (2.5 mL) were mixed with tributylphosphine (802 mg, 3.96 mmol) under a nitrogen stream and stirred at 80℃for 1.5 hours. After cooling to room temperature, the mixture was subjected to solvent substitution with toluene (3 mL) 3 times, and the mixture was concentrated until the concentration was 5g. Thereafter, the reaction mixture was ice-cooled to 4℃and 4mol/L hydrochloric acid-ethyl acetate solution (1.5 mL) was added thereto, followed by stirring for 20 minutes. The resulting crystallized slurry was collected by filtration, washed with toluene (2.5 mL), and dried, whereby a solid was obtained. To a mixture of water (4.3 mL) and sodium hydrogencarbonate (0.192 g, 2.29 mmol), the obtained solid was gradually added in small amounts, the pH was adjusted to 7 to 8, and the mixture was stirred for 30 minutes to obtain a crystallization slurry. The extract was collected by filtration, washed with water (8.6 mL), and dried, whereby Compound T-5 (351 mg, 1.43mmol, yield: 79.4%) was obtained.
LC/MS (ESI): m/z=245 (m+h), rt=1.90 min, LC/MS assay condition 1
Step 4: synthesis of Compound 10
Compound T-5 (2.015 g, 8.21 mmol), DME (20 mL), sodium T-butoxide (1.104 g, 11.49 mmol), benzophenone imine (1.645 mL, 9.80 mmol), BINAP (0.153 g, 0.246 mmol), and palladium diacetoxy (0.036 g, 0.160 mmol) were mixed under a nitrogen atmosphere, stirred at 80℃for 9 hours, and allowed to stand overnight. To the resulting suspension was added ethanol (10 mL) and cooled to 5 ℃. To the suspension was gradually added a small amount of 30% sulfuric acid (20 mL) and stirred at room temperature overnight. To the resulting reaction solution were added ethyl acetate (40 mL) and water (20 mL), and the mixture was subjected to a liquid separation operation. The resulting aqueous layer was washed with ethyl acetate (10 mL) and the organic layer was washed with 10% sulfuric acid (10 mL). The aqueous layers were combined, ice-cooled, and neutralized to pH 8 with 48% aqueous sodium hydroxide. Ethyl acetate (20 mL) was added thereto, and precipitated sodium sulfate was collected by filtration to conduct a liquid separation operation. To the resulting aqueous layer was added ethyl acetate (20 mL), and the mixture was separated. The resulting organic layers were combined, concentrated, and further solvent replaced with ethyl acetate (10 mL) 4 times repeatedly. Heptane (12 mL) was added and the resulting crystallization slurry was stirred under ice-cooling for 1 hour. The extract was collected by filtration, washed with ethyl acetate/heptane=1/3 (6 mL), and dried, whereby compound 10 (1.18 g, 6.5mmol, yield: 79.2%) was obtained.
LC/MS (ESI): m/z=182 (m+h), rt=0.88 min, LC/MS assay condition 1
Reference example 7 Synthesis of Compound U-4
[ chemical 42 ]
Step 1: synthesis of Compound U-2
Compound U-1 (2.09 g, 22.6mmol, hydrochloride) and CPME (12.04 g) were mixed with water (7 g), and a solution obtained by dissolving potassium carbonate (4.25 g, 30.8 mmol) in water (7 g) was slowly added so that the temperature of the reaction solution reached 20 to 30 ℃. The resulting mixed solution was vigorously stirred, and CbzCl (3.50 g, 20.5 mmol) was slowly added so that the temperature of the reaction solution reached 20 to 30℃and stirred at room temperature for 1 hour. The resulting solution was subjected to a liquid separation operation, and the organic layer was washed with water (14 g) and then concentrated. CPME (15.05 g) was added to the residue, and the mixture was further concentrated until the residue became 10.5g. The resulting solution was warmed to 45℃and the temperature was maintained while taking 30 minutes to add heptane (9.58 g), after which stirring was further carried out for 30 minutes. After heptane (19.15 g) was added, the resulting crystallization slurry was stirred under ice-cooling for 30 minutes. The obtained solid was washed with a mixture of CPME-heptane (3 g-9.58 g) and dried, whereby Compound U-2 (3.41 g, 17.93mmol, yield: 86%) was obtained.
HPLC (uv=254 nm): rt=9.51 min, HPLC assay condition 5
Step 2: synthesis of Compound U-3
To compound U-2 (8.00 g, 42.1 mmol) was added methanol (31.66 g), and after cooling to 0℃a 28% methanol solution of sodium methoxide (2.43 g, 12.6 mmol) was added thereto, followed by stirring at that temperature for 4 hours. To the resulting solution, a solution of N-methylformazide (3.74 g, 50.5 mmol) dissolved in methanol (19 g) was added at 0 to 5℃and acetic acid (2.53 g, 42.1 mmol) was further added at this temperature, followed by stirring at 0℃for 2 hours. The resulting solution was warmed to 60℃and stirred at that temperature for 4 hours. After the reaction mixture was concentrated to 32g, ethyl acetate (57.73 g) and 5% aqueous sodium hydrogencarbonate solution (67.53 g) were added. The resulting mixed solution was stirred for 10 minutes, and a liquid separation operation was performed. For the resulting aqueous layer, extraction was also performed with ethyl acetate (57.73 g). The combined organic layers were concentrated to 40g. MEK (64.4 g) was added, further concentrated to 40g, and the above operation was repeated 2 times. To the resulting concentrate was added a solution of methanesulfonic acid (4.04 g, 42.0 mmol) dissolved in MEK (32.2 g) at 20 to 30℃and stirred at room temperature for 30 minutes. The precipitated crystal slurry was collected by filtration, and the obtained solid was washed with MEK (25.76 g) and dried, whereby Compound U-3 (10.1 g, 29.5mmol, methanesulfonate, yield: 70%) was obtained.
HPLC (uv=254 nm): rt=7.90 min, HPLC assay condition 5
Step 3: synthesis of Compound C-1
Compound U-3 (10 g, 29.2mol, methanesulfonate) and methanol (79.15 g) were mixed and stirred at room temperature, followed by nitrogen substitution. Palladium-on-carbon (palladium 10%) (0.5 g, 5 wt%) was added, and after hydrogen substitution, stirring was performed at room temperature for 7 hours. After the nitrogen substitution, the palladium-carbon catalyst was removed by a celite (registered trademark) filtration operation. The resulting filtrate was concentrated to 50g. MEK (40.25 g) was added, concentrated to 40g, and the above procedure was repeated 2 times. The resulting crystallized slurry was collected by filtration, washed with MEK (25.76 g), and dried, whereby compound C-1 (5.3 g, 25.5mmol, methanesulfonate, yield: 87%) was obtained.
HPLC (uv=254 nm): rt=2.75 min, HPLC assay condition 11
The results of the analysis of the single crystal structure of the fumaric acid co-crystal form I of the compound represented by the formula (VII) are shown below.
R1 (I > 2.00s (I)) was 0.0470, and no positive or erroneous placement of electron density was confirmed by final difference Fourier.
The crystallographic data are shown in table 2.
[ Table 2 ]
Here, the volume refers to the unit cell volume, and Z refers to the number of molecules in the unit cell.
The atomic coordinates of the non-hydrogen atom are shown in tables 3 to 4. Here, U (eq) refers to the equivalent isotropic temperature factor.
[ Table 3 ]
Atoms x y z U(eq)
Cl36 8115.3(9) 8341.6(8) 5010.7(5) 79.9(3)
F32 8958.5(19) 7981.3(17) 307.5(9) 78.5(5)
O35 7267(2) 5961.4(16) 1399.9(10) 56.3(5)
O34 5322(3) 4254.8(16) 4098.2(11) 63.3(5)
O38 3536(2) 9367.5(19) 8936.3(12) 64.2(5)
N12 6506(2) 7056.8(18) 2611.0(12) 44.2(5)
F33 13870(2) 7642(2) 1402.1(13) 100.3(7)
N16 5475(2) 6174.4(18) 3988.1(12) 48.2(5)
N14 6120(3) 5115.3(18) 2713.0(12) 47.3(5)
N9 2815(3) 8924(2) 7397.8(13) 55.4(6)
N10 5772(3) 8146(2) 3856.1(13) 55.1(6)
N1 1276(3) 8864(2) 7324.6(14) 60.2(6)
F31 12197(3) 7751(3) 3084.6(13) 124.9(9)
N23 3644(3) 4434(2) 1818.7(15) 64.5(6)
N20 3122(3) 4249(2) 1061.4(15) 64.9(6)
C11 6673(3) 6043(2) 2193.8(15) 44.7(6)
C9 5879(3) 7178(2) 3527.6(15) 44.2(6)
C10 5619(3) 5119(2) 3639.3(15) 48.4(6)
N22 5784(3) 3621(2) 814.1(15) 67.8(7)
O39 6151(3) 8893(3) 8285.8(15) 109.2(10)
C12 6985(3) 8068(2) 2049.4(15) 47.2(6)
C20 5248(3) 4044(2) 1633.9(16) 50.7(6)
C7 5022(3) 8298(2) 4770.9(15) 50.8(6)
C4 3693(3) 8762(2) 6554.3(16) 49.4(6)
C13 8823(3) 7976(2) 1872.6(16) 48.8(6)
C5 5385(3) 8700(2) 6267.8(17) 56.4(7)
C19 6380(3) 4009(2) 2279.5(17) 54.5(7)
C14 9741(3) 7934(2) 1013.2(16) 54.7(7)
C3 2685(3) 8593(2) 5965.2(17) 54.4(7)
C6 6015(3) 8469(2) 5392.0(16) 54.3(7)
C23 5121(4) 9287(3) 8898.3(18) 62.1(7)
O41 1842(3) 4874(3) 3529.1(18) 119.8(10)
C8 3370(3) 8376(2) 5054.8(17) 57.4(7)
C24 5542(3) 9730(3) 9679.7(17) 61.9(7)
C18 9684(4) 7917(3) 2570.7(18) 67.1(8)
C15 11431(3) 7827(3) 831.3(19) 67.8(8)
C16 12217(3) 7760(3) 1541(2) 67.9(8)
C2 1134(4) 8667(3) 6497.1(18) 67.4(8)
[ Table 4 ]
Atoms x y z U(eq)
C17 11360(4) 7806(3) 2405(2) 75.0(9)
C21 4400(4) 3767(3) 485.7(19) 70.6(8)
O43 -464(4) 4618(4) 3203.2(19) 154.2(15)
C1 9(4) 8943(3) 8139(2) 81.7(10)
C26 307(4) 4766(4) 3745(2) 93.6(12)
C25 -384(4) 4909(4) 4700(2) 92.1(11)
C22 1397(4) 4562(4) 963(3) 102.7(13)
Next, the atomic coordinates of the hydrogen atom are shown in Table 5. Here, U (iso) refers to an isotropic temperature factor. In addition, the numbers of hydrogen atoms in table 5 are associated with the numbers of bonded non-hydrogen atoms.
[ Table 5 ]
Atoms x y z U(iso)
H38 3370.9 9206.88 8452.86 96
H16 5092.25 6215.55 4554.71 58
H12A 6452.59 8783.45 2347.49 57
H12B 6603.63 8119.01 1479.7 57
H5 6053.99 8811.71 6658.45 68
H19A 6229.72 3381.57 2741.61 65
H19B 7509.94 3824.57 1962.58 65
H41 2202.36 4700.41 3007.94 180
H8 2702.01 8287.11 4656.27 69
H24 6652.83 9619.42 9719.44 74
H18 9115.24 7953.15 3160.4 81
H15 12010.7 7800.84 243.55 81
H2 176.44 8593.16 6310.6 81
H21 4344.44 3553.57 -79.51 85
H1A 260.69 8258.79 8539.89 122
H1B -1049.48 8985.26 7978.29 122
H1C -14.15 9635.57 8433.78 122
H25 -1486.76 4863.66 4886.06 110
H22A 719.4 4375.73 1521.91 154
H22B 1225.91 4127.33 499 154
H22C 1105.77 5390.24 801.98 154
Further, the interatomic bond distances (units: angstrom) are shown in Table 6.
[ Table 6 ]
Fumaric acid cocrystal form I of the compound of formula (VII) in an asymmetric unit, 1 molecule of the compound of formula (VII) is present. The structure in the asymmetric unit of fumaric acid co-crystal form I of the compound of formula (VII) is shown in FIG. 11.
The numbers of the non-hydrogen atoms in tables 3 to 4 and 6 correspond to the numbers shown in fig. 11.
As shown in Table 6, the bond distance of N10-C9 shows aboutThe bond distance of N16-C9 shows about +.>
The bond distance of N10-C9 (about) A bond distance shorter than N16-C9 (about +.>) The compound of formula (VII) in fumaric acid co-crystal form I was therefore identified as an imino structure:
[ chemical formula 43 ]
That is, even in the same compound, there are cases where an imino structure is adopted and cases where an amino structure is adopted depending on crystallization conditions or the like, and even in the case of forming a salt or a complex, there are cases where an imino structure is adopted and cases where an amino structure is adopted depending on the kind of a counter molecule of the salt or the complex, and even in the case of the same counter molecule, there are cases where an imino structure is adopted and cases where an amino structure is adopted depending on crystallization conditions or the like. In addition, there are also mixtures of a compound taking an imino structure, a salt thereof or a complex thereof with a compound taking an amino structure, a salt thereof or a complex thereof.
The results of powder X-ray diffraction of fumaric acid co-crystal form I of the compound represented by formula (VII) obtained by the same production method as in step 5-2 of example 1b are shown.
Powder X-ray diffraction pattern, diffraction angle (2θ): peaks were confirmed at 7.7±0.2°, 9.5±0.2°, 10.0±0.2°, 10.9±0.2°, 13.8±0.2°, 14.6±0.2°, 18.6±0.2°, 22.6±0.2°, 23.4±0.2° and 24.6±0.2°.
The powder X-ray diffraction pattern of fumaric acid co-crystal form I (form I) of the compound represented by formula (VII) is shown in fig. 9. The horizontal axis represents 2θ (°), and the vertical axis represents intensity (count).
The peak table in the powder X-ray diffraction pattern of fig. 9 is shown in fig. 10.
Further, the result of powder X-ray diffraction of the toluene compound of the compound represented by formula (VII) is shown.
In the powder X-ray diffraction pattern, at a diffraction angle (2θ): peaks were confirmed at 7.4±0.2°, 8.1±0.2°, 13.7±0.2°, 15.1±0.2°, 16.3±0.2°, 19.3±0.2°, 21.4±0.2°, 22.6±0.2°, 24.6±0.2°, 26.6±0.2°, 27.8±0.2° and 29.5±0.2°.
The powder X-ray diffraction pattern of the toluene compound of the compound represented by the formula (VII) shows fig. 20. The horizontal axis represents 2θ (°), and the vertical axis represents intensity (count).
The molecular structure (amino body/imino matrix) was not identified for the toluene compound of the compound represented by the formula (VII).
Hereinafter, biological test examples of the compounds of the present invention are described.
The compound represented by the formula (VII) of the present invention has a coronavirus 3CL protease inhibitory action, as long as the coronavirus 3CL protease is inhibited.
Specifically, in the evaluation method described below, the IC50 is preferably 50. Mu.M or less, more preferably 1. Mu.M or less, and still more preferably 100nM or less.
Test example 1: cytopathic effect (CPE) inhibition assay Using human TMPRSS 2-expressing Vero E6 cells (Vero E6/TMPRSS2 cells)
< operation flow >)
Dilution and dispensing of the test sample
Test samples were diluted with DMSO to a suitable concentration in advance, and after 2-5-fold stepwise dilution series were prepared, they were dispensed into 384-well plates.
Dilution and dispensing of cells and SARS-CoV-2
VeroE6/TMPRSS2 cells (JCRB 1819, 5X 10) 3 Cell/well) and SARS-CoV-2 (100 TCID 50 Well) in a medium (MEM, 2% FBS, penicillin-streptomycin), and after dispensing into a well to which a test sample was added, CO was used 2 The incubator was cultured for 3 days.
Measurement of the dispensing and luminescence Signal of CellTiter-Glo (registered trademark) 2.0
After the plate cultured for 3 days was returned to room temperature, cellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed with a plate mixer. After a certain time, the luminescence signal (Lum) was measured with a plate reader.
< calculation of measured item values >
50% concentration of inhibition of death of SARS-CoV-2 infected cells (EC 50 ) Calculation of
When x is a logarithmic value of the compound concentration and y is a% efficacy, an inhibition curve is fitted by using the following Logistic regression equation, and a value of x substituted into y=50 (%) is calculated as EC 50 。
y＝min+(max-min)/{1+(X50/x)^Hill}
% potency = { (sample-virus control)/(cell control-virus control) } 100%
Cell control: mean of Lum of cell control wells
Virus control: average of Lum of virus control wells
min: y-axis lower limit, max: y-axis upper limit, X50: the x-coordinate of the inflection point, hill: slope of curve at midpoint of min and max
The compounds of the invention are tested essentially as described above. The results are shown below.
Compound I-005:0.328 mu M
Test example 2: inhibition Activity assay for SARS-CoV-2 3CL protease
< Material >
Commercially available recombinant SARS-CoV-2 3CL protease
Commercially available substrate peptides
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2(SEQ ID NO:1)
Internal standard peptide
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Gln(SEQ ID NO:2)
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu (13C6,15N) -Gln can be synthesized by reference (Athereon, E.; sheppard, R.C., "In Solid Phase Peptide Synthesis, APractical Approach", IRL Press at Oxford University Pres, 1989. And Bioorg. Med. Chem., volume 5, no. 9, 1997, pages 1883-1891, etc.). An example is shown below.
H-Lys-Thr-Ser-Ala-Val-Leu (13C6,15N) -Glu (resin) -OαOtBu (Lys side chain protected by Boc, thr side chain protected by tert-butyl, ser side chain protected by tert-butyl, C-terminal OH of Glu protected by tert-butyl, carboxylic acid of Glu side chain condensed with resin) was synthesized by Fmoc solid phase synthesis using Rink amide resin. N-terminal Dabcyl group repair 4-dimethylaminoazobenzene-4' -carboxylic acid (Dabcyl-OH) was condensed on the resin using EDC/HOBT. Final deprotection, and cleavage from the resin were performed by treatment with TFA/edt=95:5. Thereafter, purification was performed by reverse phase HPLC.
·RapidFire Cartridge C4 typeA
< operation flow >)
Preparation of assay buffer
In this test, an assay buffer consisting of 20mM Tris-HCl, 100mM sodium chloride, 1mM EDTA, 10mM DTT, 0.01% BSA was used. For IC 50 As a compound having a value of 10nM or less, an assay buffer composed of 20mM Tris-HCl, 1mM EDTA, 10mM DTT, and 0.01% BSA was used.
Dilution and dispensing of the test sample
Test samples were diluted with DMSO to a suitable concentration in advance, and after 2-5-fold stepwise dilution series were prepared, they were dispensed into 384-well plates.
Enzyme addition to substrate, enzyme reaction
To the prepared compound plate, 8. Mu.M of a substrate and 6 or 0.6nM of an enzyme solution were added, and incubation was performed at room temperature for 3 to 5 hours. Thereafter, a reaction stop solution (0.067. Mu.M internal standard, 0.1% formic acid, 10 or 25% acetonitrile) was added to stop the enzyme reaction.
Determination of the reaction products
The plates at the end of the reaction were measured using a Rapid FireSystem 360 and a mass spectrometer (Agilent, 6550iFunnel Q-TOF), or Rapid Fire System 365 and a mass spectrometer (Agilent, 6495CTriple Quadrupole). As mobile phases for the measurement, a solution A (75% isopropanol, 15% acetonitrile, 5mM ammonium formate) and a solution B (0.01% trifluoroacetic acid, 0.09% formic acid) were used.
The reaction Product detected by the mass spectrometer was recorded as a Product area (Product area) value calculated using RapidFire Integrator or a program capable of equivalent analysis. The internal standard of simultaneous detection was also calculated and recorded as an internal standard area (Internal Standard area) value.
< calculation of measured item values >
Calculation of P/IS
The area (area) value obtained in the signed item IS calculated by the following equation, and P/IS calculated.
P/is=product area value/internal standard area value
50% SARS-CoV-2 3CL protease inhibition concentration (IC 50 ) Calculation of
When x is a logarithmic value of the compound concentration and y is% inhibition, an inhibition curve is fitted by using the following Logistic regression equation, and the value of x substituted into y=50 (%) is calculated as IC 50 。
y＝min+(max-min)/{1+(X50/x)^Hill}
% inhibition = {1- (sample-control (-))/control (+) -control (-)) } 100
Control (-): average of P/IS of enzyme inhibition conditioned wells
Control (+): mean P/IS of DMSO control wells
min: y-axis lower limit, max: y-axis upper limit, X50: the x-coordinate of the inflection point, hill: slope of curve at midpoint of min and max
The compounds of the invention are tested essentially as described above. The results are shown below.
Compound I-005: 0.010. Mu.M
Test examples 1-2: cytopathic effect (CPE) inhibition assay Using human TMPRSS 2-expressing Vero E6 cells (Vero E6/TMPRSS2 cells)
< operation flow >)
Dilution and dispensing of the test sample
Test samples were diluted in DMSO to a suitable concentration in advance, and after 3-fold stepwise dilution series were prepared, they were dispensed into 96-well plates.
Dilution and dispensing of cells and SARS-CoV-2
VeroE6/TMPRSS2 cells (JCRB 1819, 1.5X10 cells/well) and SARS-CoV-2hCoV-19/Japan/TY/WK-521/2020, hCoV-19/Japan/QK002/2020, hCoV-19/Japan/QHN/2020, hCoV-19/Japan/QHN002/2020, hCoV-19/Japan/TY7-501/2021、hCoV-19/Japan/TY7-503/2021、hCoV-19/Japan/TY8-612/2021、hCoV-19/Japan/TY11-927-P1/2021(30-1000TCID 50 Well) in a medium (MEM, 2% FBS, penicillin-streptomycin), and after dispensing into a well to which a test sample was added, CO was used 2 The incubator was cultured for 3 days.
Measurement of the dispensing and luminescence Signal of CellTiter-Glo (registered trademark) 2.0
After the plate cultured for 3 days was returned to room temperature, cellTiter-Glo (registered trademark) 2.0 was dispensed into each well and mixed with a plate mixer. After a certain time, the luminescence signal (Lum) was measured with a plate reader.
< calculation of measured item values >
50% concentration of inhibition of death of SARS-CoV-2 infected cells (EC 50 ) Calculation of
When x is a logarithmic value of the compound concentration and y is a% efficacy, an inhibition curve is fitted by using the following Logistic regression equation, and a value of x substituted into y=50 (%) is calculated as EC 50 。
y＝min+(max-min)/{1+(X50/x)^Hill}
% potency = { (sample-virus control)/(cell control-virus control) } 100%
Cell control: mean of Lum of cell control wells
Virus control: average of Lum of virus control wells
min: y-axis lower limit, max: y-axis upper limit, X50: the x-coordinate of the inflection point, hill: slope of curve at midpoint of min and max
The compounds of the invention are tested essentially as described above. The results are shown below.
(SARS-CoV-2hCoV-19/Japan/TY/WK-521/2020)
Fumaric acid co-crystal form I of the compound of formula (VII): 0.37 mu M
Test example 2-2: inhibition Activity assay for SARS-CoV-2 3CL protease
< Material >
Commercially available recombinant SARS-CoV-2 3CL protease
Commercially available substrate peptides
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu(Edans)-NH2(SEQ ID NO:1)
Internal standard peptide
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu(13C6,15N)-Gln(SEQ ID NO:2)
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu (13C6,15N) -Gln can be synthesized by reference (Athereon, E.; sheppard, R.C., "In Solid Phase Peptide Synthesis, APractical Approach", IRL Press at Oxford University Pres, 1989. And Bioorg.Med. Chem., volume 5, no. 9, 1997, pages 1883-1891, etc.). An example is shown below.
H-Lys-Thr-Ser-Ala-Val-Leu (13C6,15N) -Glu (resin) -OαOtBu (Lys side chain protected by Boc, thr side chain protected by tert-butyl, ser side chain protected by tert-butyl, C-terminal OH of Glu protected by tert-butyl, carboxylic acid of Glu side chain condensed with resin) was synthesized by Fmoc solid phase synthesis using Rink amide resin. N-terminal Dabcyl group repair 4-dimethylaminoazobenzene-4' -carboxylic acid (Dabcyl-OH) was condensed on the resin using EDC/HOBT. Final deprotection, and cleavage from the resin were performed by treatment with TFA/edt=95:5. Thereafter, purification was performed by reverse phase HPLC.
·RapidFire Cartridge C4 typeA
< operation flow >)
Preparation of assay buffer
In this test, an assay buffer consisting of 20mM Tris-HCl, 1mM EDTA, 10mM DTT, 0.01% BSA was used.
Dilution and dispensing of the test sample
Test samples were diluted in DMSO to a suitable concentration in advance, and after 3-fold stepwise dilution series were prepared, they were dispensed into 384-well plates.
Enzyme addition to substrate, enzyme reaction
In the prepared compound plate, 8. Mu.M substrate, and 6nM enzyme solution were added and incubation was performed at room temperature for 3 hours. Thereafter, a reaction stop solution (0.072. Mu.M internal standard, 0.1% formic acid, 10% acetonitrile) was added to stop the enzyme reaction.
Determination of the reaction products
The plates at the end of the reaction were measured using a Rapid fire System 360 and a mass spectrometer (Agilent, 6550iFunnel Q-TOF). As mobile phases for the measurement, a solution A (75% isopropanol, 15% acetonitrile, 5mM ammonium formate) and a solution B (0.01% trifluoroacetic acid, 0.09% formic acid) were used.
The reaction Product detected by the mass spectrometer was noted as a Product area (Product area) value calculated using RapidFire Integrator. The internal standard of simultaneous detection was also calculated and recorded as an internal standard area (Internal Standard area) value.
< calculation of measured item values >
Calculation of P/IS
The area (area) value obtained in the signed item IS calculated by the following equation, and P/IS calculated.
P/is=product area value/internal standard area value
50% SARS-CoV-2 3CL protease inhibition concentration (IC 50 ) Calculation of
When x is a logarithmic value of the compound concentration and y is% inhibition, an inhibition curve is fitted by using the following Logistic regression equation, and the value of x substituted into y=50 (%) is calculated as IC 50 。
y＝min+(max-min)/{1+(X50/x)＾Hill}
% inhibition = {1- (sample-control (-))/control (+) -control (-)). 100 × control (-)
Control (-): average P/IS ratio in wells without SARS-CoV-2 3CL protease and test substance
Control (+): average P/IS ratio in wells containing SARS-CoV-2 3CL protease and no test substance
min: y-axis lower limit, max: y-axis upper limit, X50: the x-coordinate of the inflection point, hill: slope of curve at midpoint of min and max
The compounds of the invention are tested essentially as described above. The results are shown below.
Fumaric acid co-crystal form I of the compound of formula (VII): 0.0132 mu M
Example 5
The fumaric acid co-crystal I-shaped crystals of the compound represented by the formula (VII) were weighed so that the amount of the compound represented by the formula (VII) became about 1.8 (w/v)%, and dispersed in an aqueous solution in which 0.3 (w/v)% of the polymer was dissolved.
As the additive, aminoalkyl methacrylate copolymer E (manufactured by Evonik corporation), hydroxypropyl cellulose (manufactured by japan sojoda corporation), hydroxypropyl methylcellulose (manufactured by siegesbeck chemical industry corporation), polyvinyl alcohol (manufactured by Merck corporation), polyvinyl pyrrolidone (manufactured by BASF corporation), polyvinyl alcohol-methyl methacrylate-acrylic acid copolymer (manufactured by daily new chemical industry corporation) were used.
[ Table 7 ]
Test example 3: solubility evaluation
1mL of each of the dispersions of example 5 was added to 14mL of fasted simulated intestinal fluid (FaSSIF), and stirred with a constant temperature stirrer at 37℃and 400rpm for 1 hour. After stirring for 1 hour, the sample was filtered through a 0.45 μm filter, and the concentration of the compound represented by formula (VII) in the filtrate was measured by liquid chromatography.
(assay)
Detector: ultraviolet absorbance photometer (measuring wavelength 222 nm)
Column: ACQUITY UPLC BEH C18 2.1X150 mm, 1.7 μm
Column temperature: a certain temperature around 40 DEG C
Mobile phase a:0.1M ammonium formate solution, mobile phase B: acetonitrile
Liquid feed of mobile phase: the mixing ratio of mobile phase a and mobile phase B was changed to 7:3 or the concentration gradient was controlled as in the table below.
[ Table 8 ]
Flow rate: about 0.6mL/min
Injection amount: 1 mu L or 1.5 mu L
Sample coolant temperature: about 10 DEG C
Auto injector wash: water/methanol mixed solution (7:3)
Area measurement range: sample solution was injected 4 minutes or 7.5 minutes after injection
Calculation of the amount of Compound of formula (VII)
The amount (%) of the compound represented by formula (VII) =atii/Σat×100
ATII: peak area of the compound represented by formula (VII) of the sample solution
Σat: total of peak areas of sample solutions (excluding blank and system peaks)
(results)
The results of the solubility test of the compound represented by the formula (VII) are shown in the following table. As a result, the solubility of the compound represented by formula (VII) is greatly improved by adding a polymer.
[ Table 9 ]
The fumaric acid co-crystal form I crystals of the compound of formula (VII) used in the present experiment had a D50 of 3.44. Mu.m, and a D90 of 8.33. Mu.m.
Example 6
(method for producing tablets)
Tablets containing the fumaric acid co-crystal I-shaped crystals of the compound represented by formula (VII) were produced.
In the table below, the formulation of an average of 1 tablet is shown. Fumaric acid co-crystal form I crystals of the compound represented by the formula (VII), D-mannitol (manufactured by Roquette Co., ltd.), croscarmellose sodium (manufactured by DuPont Co., ltd.), hydroxypropyl cellulose (manufactured by Cabot Co., ltd.), light anhydrous silicic acid (manufactured by Cabot Co., ltd.) and magnesium stearate (manufactured by Mallinckrodt Co., ltd.) were sieved and mixed by a 30-mesh sieve, and then granulated.
Granules obtained by granulation and sizing, crystalline cellulose (manufactured by Asahi chemical Co., ltd.) and magnesium stearate (manufactured by Mallinckrodt Co., ltd.), or light anhydrous silicic acid (manufactured by Cabot Co., ltd.) were added and mixed, and then compressed and molded into tablets having a diameter of 9.0mm by a static compressor or a rotary tablet press, to obtain tablets having the following composition.
[ Table 10 ]
Test example 4: dissolution test of tablets
(dissolution test method)
The elution test was carried out by revising Japanese pharmacopoeia elution test method No. 2 (elution test No. 1 liquid containing a surfactant, paddle method, paddle rotation speed: 50rpm, result: average value of 2 tablets).
(experimental results)
The results of the dissolution test of examples 6A, 6B, 6C and 6D are shown in FIG. 21. As a result, in any of the examples, quick dissolution was exhibited.
The particle size distribution of the active ingredient (fumaric acid co-crystal I-shaped crystal of the compound represented by formula (VII)) used in the formulations of examples 6A and 6B used in test example 4 is shown in fig. 22. 10% of the particles had a particle size of 0.69. Mu.m, 50% had a particle size of 4.00. Mu.m, and 90% had a particle size of 10.80. Mu.m.
The particle size distribution of the active ingredient (fumaric acid co-crystal I-shaped crystal of the compound represented by formula (VII)) used in the formulations of examples 6C and 6D used in test example 4 is shown in fig. 23. 10% of the particles had a particle size of 0.67. Mu.m, 50% of the particles had a particle size of 3.63. Mu.m, and 90% of the particles had a particle size of 10.98. Mu.m.
The measurement conditions of the particle size distribution are shown below.
The manufacturer: fuel tap and fuel cell system
The device comprises: laser diffraction type HELOS & RODOS particle size distribution measuring device
The range is as follows: r2
Dispersion pressure: 3bar
Triggering conditions: the measured concentration is less than or equal to 1% or 10s actual time after 2s
Test example 5: influence of the stabilizer in the formulation in the stability test over time
The results of confirming the stability with time of the same batch of the formulation of example 6A as used in test example 4 are shown.
a. Stability test method
The formulation of example 6A was either 1) stored for two weeks at 60 ℃ with the brown glass vial closed, or 2) stored for one month at 40 ℃ with the brown glass vial open, at 75% relative humidity. Thereafter, the amount of the related substance of the compound represented by the formula (VII) in the preparation of the present invention was measured.
(method for preparing sample solution)
(measurement method)
The amount of the related compound represented by the formula (VII) in the preparation of the present invention was measured by liquid chromatography by the following method and conditions.
Detector: ultraviolet absorbance photometer (measurement wavelength: 222 nm)
Column: c18 column
Column temperature: a certain temperature around 40 DEG C
Mobile phase a:0.01mol/L ammonium formate solution, mobile phase B: acetonitrile
The mixing ratio of mobile phase A and mobile phase B was slowly increased from 9:1 to 1:9, and the measurement was performed at 32 minutes.
Flow rate: 0.6 mL/min
The amount of the related substance was calculated by taking the total of peak areas of chromatograms of the HPLC chart as 100%, and the ratio (%) with respect to the amount thereof was calculated.
b. Results
The results of the stability test are shown in the following table. The total relevant matter amount of the preparation of example 6A was stable after 1) storage for two weeks at 60 ℃ in a brown glass bottle closed state or 2) storage for one month at 40 ℃ and 75% relative humidity in a brown glass bottle open state without increasing.
[ Table 11 ]
Total related substances (%)
Initiation 0.36
After two weeks of storage at 60 DEG C 0.34
40 ℃ and 75% RH, and after one month of storage 0.35
According to the above, the preparation of the invention has high stability to humidity and temperature.
Example 7
Test example 6 clinical trial (Ph 2 a)
The evaluation of the effectiveness and safety of the test agent (active ingredient: fumaric acid co-crystal form I of the compound represented by formula (VII)) when administered orally repeatedly to SARS-CoV-2 infected persons having only mild/moderate and no/mild symptoms was carried out by a double-blind comparative test based on random distribution using a placebo as a comparative subject.
The main evaluation item of Phase 2a Part was common to both mild/moderate and asymptomatic SARS-CoV-2 infected individuals, and was the change in the viral titer of SARS-CoV-2 at each time point from the baseline, confirming the antiviral effect of the test agent.
The mild/moderate SARS-CoV-2 infected patient was selected to meet all of the criteria described below.
(a) Male or female patients over 12 years old and less than 70 years old.
(b) SARS-CoV-2 was diagnosed positive within 120 hours prior to registration.
(c) The time from onset of COVID-19 to registration was less than 120 hours.
(d) Any one of the following symptoms (12 symptoms of COVID-19) caused by COVID-19 at the time of registration has symptoms of 1 item or more (except symptoms existing before the onset of COVID-19) of moderate degree or more (COVID-19 symptom score: 2).
Systemic symptoms: tiredness, muscle or body pain, headache, aversion to cold, fever
Respiratory symptoms: nasal water or nasal obstruction, sore throat, cough and asthma
Digestive symptoms: nausea, vomiting and diarrhea
Asymptomatic SARS-CoV-2 infected individuals select patients who meet all of the criteria described below.
(a) Male or female patients over 12 years old and less than 70 years old.
(b) SARS-CoV-2 was diagnosed positive within 120 hours prior to registration.
(c) Asymptomatic: within 2 weeks prior to registration, the following symptoms of covd-19 (except for those present prior to SARS-CoV-2 infection) were not confirmed.
Systemic symptoms: tiredness, muscle or body pain, headache, aversion to cold, fever, abnormal taste, abnormal smell
Respiratory symptoms: nasal water or nasal obstruction, sore throat, cough and asthma
Digestive symptoms: nausea, vomiting and diarrhea
Asymptomatic/only mild symptoms: within 2 weeks before random assignment, none of the following symptoms (12 symptoms of COVID-19) due to COVID-19 had symptoms of moderate (COVID-19 symptom score: 2) or more (except for symptoms existing before the onset of COVID-19).
Systemic symptoms: burnout sensation (tiredness), muscle pain or body pain, headache, aversion to cold/sweating, fever or fever
Respiratory symptoms: nasal water or nasal obstruction, sore throat, cough and asthma (dyspnea)
Digestive symptoms: nausea, vomiting and diarrhea
Method for administering clinical trial drug
(i) Test agent
250mg tablet: the tablet contains fumaric acid co-crystal I-shaped crystals of the compound represented by the formula (VII), and 250mg of the compound represented by the formula (VII) is contained. The 250mg tablet was prepared by 2-fold mixing of the same compositions as in example 6C.
125mg tablet: the tablet contains fumaric acid co-crystal I-shaped crystals of the compound represented by the formula (VII), and 125mg of the compound represented by the formula (VII) is contained. The 125mg tablet was a tablet having the same composition as in example 6C.
(ii) Placebo
placebo-D tablets: is a tablet indistinguishable from the above 250mg tablet in appearance, and contains no fumaric acid co-crystal I-shaped crystals of the compound represented by the formula (VII).
placebo-B tablets: is a tablet indistinguishable from the above 125mg tablet in appearance, and contains no fumaric acid cocrystal I-shaped crystal of the compound represented by the formula (VII).
Administration amount and administration method
In Phase 2a Part, subjects judged eligible as light/moderate or asymptomatic SARS-CoV-2 infected subjects were randomly assigned to either of 375/125mg group of test agent, 750/250mg group of test agent, and placebo group at a 1:1:1 ratio.
Clinical trial for each given group
375/125mg group
125mg tablets and placebo-D tablets were each administered on day 1, and 1 125mg tablet and placebo-D tablet were each administered every 1 day on days 2 to 5.
750/250mg group
250mg of each of the tablets and placebo-B tablets were administered on day 1, and 1 tablet of each of the 250mg tablets and placebo-B tablets was administered every 1 day on days 2 to 5.
Placebo group
placebo-D and placebo-B tablets were each administered on day 1 and 1 placebo-D and placebo-B tablet was each administered every 1 day on days 2-5.
The "day 1" is given to the first day, and the "2 nd to 5 th days" is given from the first day to the second day to 5 th days.
Main evaluation item of effectiveness (Phase 2a Part)
The main item for evaluating the effectiveness of Phase 2a Part is common to both mild/moderate and asymptomatic SARS-CoV-2 infected individuals, and is the amount of change from the baseline in the viral titer of SARS-CoV-2 at each time point. Defined as the absolute change from baseline in observed SARS-CoV-2 virus titer at each time point.
Analysis of Main evaluation items (Phase 2a Part)
For each of the mild/moderate SARS-CoV-2 infectious agent population, asymptomatic SARS-CoV-2 infectious agent population, and a combination thereof, a summary statistic of the amount of change from the baseline of the SARS-CoV-2 viral titer at each time point was calculated for the mITT population. Further, for the combined population of mild/moderate and asymptomatic SARS-CoV-2 infectors, the van eleten test was applied to significantly level 5% viral titers against SARS-CoV-2 at each time point on both sides, comparing each dose group of the test drug with the placebo group. Among the layers used in the van Elteren test, the mild/moderate SARS-CoV-2 infectious agent population, and the asymptomatic SARS-CoV-2 infectious agent population were used.
Results of the Main evaluation project
(1) Variation from baseline of SARS-CoV-2 viral titer at each time point (Phase 2a Part)
In Phase 2a Part, 69 cases were randomized, 22 cases were assigned to 375/125mg group (1 case was not given), 23 cases were assigned to 750/250mg group, and 24 cases were assigned to placebo group. Of 69 cases, the number of cases positive for the baseline RT-PCR was 44, and the number of cases in which the baseline virus titer was detected was 40. The 40 cases consisted of 14 cases in 375/125mg group, 13 cases in 750/250mg group, and 13 cases in placebo group. The number of these groups and the content thereof were calculated based on the RT-PCR measurement results and the virus titer measurement results which were obtained from the time of 2022, 1, 17 days.
As a final result of Phase 2a Part, the number of cases positive for the baseline RT-PCR was 47 out of 69, and the number of cases in which the virus titer at the baseline was detected was 43. The 43 cases consisted of 15 cases in 375/125mg group, 14 cases in 750/250mg group, and 14 cases in placebo group.
The daily follow-up for admission prescribed in the clinical trial protocol indicates the correspondence with the administration Day (Day) and the allowable width as follows. Op V refers to optional follow-up, indicating optional day of admission.
[ Table 12 ]
The mean value of each group of changes in the baseline of SARS-CoV-2 virus titer was shifted by Modified intention-to-treatment (all subjects who were randomized and positive for both baseline RT-PCR and virus titer) as shown in FIG. 24. The analysis included both mild/moderate SARS-CoV-2 infected persons and asymptomatic SARS-CoV-2 infected persons in the analysis, indicating only follow-up on the necessary hospital admission days. In addition, the virus titer was below the lower detection limit (0.8 log 10 (TCID 50 Per mL)), the viral titer value is 0.8log 10 (TCID 50 /mL). At the time points of visit 3 (day 4 after start of dosing), visit 2 (day 2 after start of dosing) and visit 3 (day 4 after start of dosing) in 375/125mg group, virus titers were statistically significantly reduced at significant levels of 0.05 compared to placebo group. Among the RT-PCR measurement results and the virus titer measurement results which can be obtained at the time of day 17 of 2022, there was a tendency that the virus titer was decreased compared with that of the placebo group by the time of day 1 of 2022, after the follow-up 3 (day 4 after the start of administration) in 375/125mg group, and after the follow-up 2 (day 2 after the start of administration) in 750/250mg group.
The proportion of positive patients with respect to virus titer at each time point is shown below.
At time point 4, the ratio of positive with virus titer above 0.8 in the placebo group was 80% reduced in the 750/250mg group and 63% reduced in the 375/125mg group. At time point 6, the ratio of positive with virus titer above 0.8 in the placebo group was reduced by 54% in 750/250mg group and 100% in 375/125mg group.
At the time points of day 4 and 6, a low tendency was found for the proportion of patients positive for viral titres compared to placebo in both the 750/250mg group and 375/125mg group. Thus, it is suggested that patients who discharge viruses having infectivity can be rapidly reduced by taking the pharmaceutical composition of the present invention.
Results of the secondary evaluation items
(1) Time until virus titer was initially confirmed negative (Phase 2a Part)
The time until the virus titer of SARS-CoV-2 was initially confirmed to be negative is shown in Table 13 and FIG. 25 below.
Of 69 cases of Phase 2a Part, 15 cases were shown in 375/125mg group, 13 cases were shown in 750/250mg group, and 14 cases were shown in placebo group.
[ Table 13 ]
In the stratified log rank test, only the subject layer (mild/moderate, asymptomatic/mild) was recorded as a stratification factor
As shown in table 13, in 375/125mg group, no significant difference was observed for the time until the virus titer was negative for the initial confirmation (p= 0.0159) as short as 49.8 hours in the center value compared to placebo group. In the 750/250mg group, no significant difference was observed for the time until the virus titer negative was initially confirmed (p=0.0205) as short as 48.4 hours in the central value compared to the placebo group.
In addition, as shown in fig. 25, the time until the virus titer of 50% of patients in the placebo group reached negative was approximately 4.6 days after the start of the treatment. In contrast, in the 750/250mg group and 375/125mg group, the time until the virus titer of 50% of patients reached negative was approximately 2.6 days from the start of the treatment. Therefore, the time until the virus titer of 50% of patients was confirmed was shortened by about 2 days.
(2) The amount of change from baseline in the 12 symptom aggregate score of the COVID-19 at each time point (Phase 2a Part)
The change from baseline in the 12 symptom aggregate score for covd-19 at each time point is shown in figure 6.
Of 69 cases of Phase 2a Part, 13 cases in 375/125mg, 12 cases in 750/250mg, and 14 cases in placebo were shown for mild/moderate subjects.
As shown in FIG. 26, there was a tendency that the total score of 12 symptoms of COVID-19 was improved numerically in the 375/125mg group and 750/250mg group compared to the placebo group at all time points after day 2 (after 1 administration).
(3) Severe inhibition effect (Phase 2a Part)
The proportion of subjects whose score first worsened to 3 or more at any time point after the start of administration (order scale of 8-stage classification of clinical severity, table 14) in 69 cases of Phase 2a Part for subjects with mild/moderate symptoms is shown in table 15.
[ Table 14 ]
8-point sequence scale Score of
No symptoms were confirmed 0
Symptomatic but not affecting daily life 1
Symptomatic, affecting daily life 2
Requiring hospitalization or rehabilitation based thereon 3
Needs hospitalization or nursing (less than 5L/min) 4
Subjects in need of hospitalization or rehabilitation based thereon and in need of maintenance (above 5L/min) 5
Subjects in need of hospitalization or rehabilitation based thereon, and artificial respiratory tract 6
Death of 7
[ Table 15 ]
375/125mg group 750/250mg group Placebo group
Deterioration rate 0.0％ 0.0％ 14.3％
Number of deterioration cases/number of analysis target cases 0/13 0/12 2/14
As shown in Table 15, in the 375/125mg group and the 750/250mg group, cases were not confirmed in which the order scale was first changed to 3 or more after the start of administration.
Frequency of discovery of adverse events
In Phase 2a Part, there are no mortality, critical adverse events, and adverse events that lead to discontinuation of administration.
The preparation of the invention can be used for children and adults over 12 years old.
Example 8
(method for producing tablets)
Tablets containing the fumaric acid co-crystal I-shaped crystals of the compound represented by formula (VII) were produced.
In the table below, the formulation of an average of 1 tablet is shown. Fumaric acid co-crystal form I crystals of the compound represented by the formula (VII), D-mannitol (manufactured by Roquette Co., ltd.), croscarmellose sodium (manufactured by DuPont Co., ltd.), hydroxypropyl cellulose (manufactured by Cabot Co., ltd.), light anhydrous silicic acid (manufactured by Cabot Co., ltd.) and magnesium stearate (manufactured by Mallinckrodt Co., ltd.) were sieved and mixed by a 30-mesh sieve, and then granulated.
Granules obtained by granulation and sizing, crystalline cellulose (manufactured by Asahi chemical Co., ltd.) and magnesium stearate (manufactured by Mallinckrodt Co., ltd.), or light anhydrous silicic acid (manufactured by Cabot Co., ltd.) were added and mixed, and then compressed and molded into tablets having the following composition by a static compressor or a rotary tablet press to have a length of 15.4mm X a short diameter of 8 mm.
[ Table 16 ]
Test example 7: dissolution test of tablets
(dissolution test method)
For examples 8A, 8B, 8C and 8D, the elution test was conducted by revising Japanese pharmacopoeia elution test method No. 2 (elution test liquid No. 1 containing a surfactant, paddle method, paddle rotation speed: 50rpm, result: average of 2 tablets) at 18.
(experimental results)
In either example, rapid dissolution is shown.
Example 9
(method for producing tablets)
Tablets containing the fumaric acid co-crystal I-shaped crystals of the compound represented by formula (VII) were produced.
In the table below, the formulation of an average of 1 tablet is shown. Fumaric acid co-crystal form I crystals of the compound represented by the formula (VII), D-mannitol (manufactured by Roquette Co., ltd.), croscarmellose sodium (manufactured by DuPont Co., ltd.), hydroxypropyl cellulose (manufactured by Cabot Co., ltd.), light anhydrous silicic acid (manufactured by Cabot Co., ltd.) and magnesium stearate (manufactured by Mallinckrodt Co., ltd.) were sieved and mixed by a 30-mesh sieve, and then granulated.
Granules obtained by granulation and sizing, crystalline cellulose (manufactured by Asahi chemical Co., ltd.) and magnesium stearate (manufactured by Mallinckrodt Co., ltd.), or light anhydrous silicic acid (manufactured by Cabot Co., ltd.) were added and mixed, and then compressed and molded into tablets having a diameter of 5mm or 5.5mm by a static compressor or a rotary tablet press, to obtain tablets having the following composition.
[ Table 17 ]
Test example 8: dissolution test of tablets
(dissolution test method)
For 2 formulations in example 9, the elution test was carried out by the 18 th revised Japanese pharmacopoeia elution test method No. 2 (elution test No. 1 liquid containing a surfactant, paddle method, paddle rotation speed: 50rpm, result: average of 2 tablets).
(experimental results)
In either example, rapid dissolution is shown.
The preparation of the invention can be used for children and adults over 12 years old.
Example 10
(method for producing granules)
Granules containing fumaric acid co-crystal I-shaped crystals of the compound of formula (VII) are produced.
In the table below, the formulation of the granules on average 1 part is shown. Fumaric acid co-crystal form I crystals of the compound represented by the formula (VII), D-mannitol (manufactured by Roquette Co., ltd.), hydrogenated maltose starch syrup (maltitol, manufactured by Roquette Co., ltd.), croscarmellose sodium (manufactured by DuPont Co., ltd.), hydroxypropyl cellulose (manufactured by Japanese Cauda Co., ltd.), light anhydrous silicic acid (manufactured by Cabot Co., ltd.), magnesium stearate (manufactured by Mallinckrodt Co., ltd.) and sucralose (manufactured by Sanrong source ffi Co., ltd.) were sifted and mixed by a 30-mesh sieve, and granulated.
Granules having the following composition were obtained by adding and mixing granulated and sized granules, light anhydrous silicic acid (manufactured by Cabot corporation) and sucralose (manufactured by Sanrong source ffi corporation).
[ Table 18 ]
/>
Test example 9: dissolution test of granules
(dissolution test method)
For the 2 formulations in example 10, the dissolution test was conducted by the 18 th revised Japanese pharmacopoeia dissolution test method, method 2 (dissolution test liquid 1 containing a surfactant, paddle method, paddle rotation speed: 50rpm, result: average value of 2 granules).
(experimental results)
The results of the elution test after the content correction are shown in FIG. 27, and the results thereof show rapid elution in any of the examples.
Test example 10: stability over time test of granules
For 2 formulations in example 10, results confirming stability over time are shown.
a. Stability test method
The formulation of example 10 was either 1) stored in a DUMA bottle (plastic container, GERRESHEIMER) at 40 ℃ for three weeks at 75% relative humidity, or 2) stored in a DUMA bottle with added silica gel at 40 ℃ for three weeks at 75% relative humidity, or 3) stored in a DUMA bottle with added silica gel at 40 ℃ for one month at 75% relative humidity, or 4) stored in a DUMA bottle with added silica gel at 40 ℃ for one month at 75% relative humidity. Thereafter, the amount of the related substance of the compound represented by the formula (VII) in the preparation of the present invention was measured.
(method for preparing sample solution)
(measurement method)
The amount of the related compound represented by the formula (VII) in the preparation of the present invention was measured by liquid chromatography by the following method and conditions.
Detector: ultraviolet absorbance photometer (measurement wavelength: 222 nm)
Column: c18 column
Column temperature: a certain temperature around 40 DEG C
Mobile phase a:0.01mol/L ammonium formate solution, mobile phase B: acetonitrile
The mixing ratio of mobile phase A and mobile phase B was slowly increased from 9:1 to 1:9, and the measurement was performed at 32 minutes.
Flow rate: 0.3 mL/min
The amount of the related substance was calculated by taking the total of peak areas of chromatograms of the HPLC chart as 100%, and the ratio (%) with respect to the amount thereof was calculated.
b. Results
The results of the stability test are shown in tables 19 and 20 below. The total relevant amount of the formulation of example 10 was not increased under either condition and was stable.
[ Table 19 ]
[ Table 20 ]
According to the above, the preparation of the invention has high stability to humidity and temperature.
The formulation examples shown below are merely illustrative and are not intended to limit the scope of the invention in any way.
The compounds of the invention may be administered as pharmaceutical compositions by any of the conventional routes, in particular enterally, e.g. orally, e.g. in the form of tablets, granules or capsules, or parenterally, e.g. in the form of injection solutions or suspensions, topically, e.g. in the form of lotions, gels, ointments or creams, or in the form of nasal or suppository forms. The pharmaceutical compositions of the compounds of the present invention, comprising the compounds in free form or in pharmaceutically acceptable salt form, together with at least 1 pharmaceutically acceptable carrier or diluent, can be manufactured by conventional methods using mixing, granulating or coating methods. For example, as the composition for oral administration, tablets, granules, capsules containing an excipient, a disintegrant, a binder, a lubricant, etc., an active ingredient, etc., may be used. The injectable composition may be formulated into a solution or suspension, or may be sterilized, and may further contain a preservative, a stabilizer, a buffer, or the like.
Formulation example 1 suspension
To a raw material of the compound represented by the formula (VII), for example, water for injection is added to prepare a suspension.
Formulation example 2 tablet
To the raw material of the compound represented by the formula (VII), for example, D-mannitol or magnesium stearate is added as an additive to prepare a tablet.
Preparation example 3 granule
To the raw material of the compound represented by the formula (VII), for example, D-mannitol and magnesium stearate are added as additives to prepare granules.
Industrial applicability
The compound produced by the production method according to the present invention has an inhibitory effect on coronavirus 3CL protease, and is considered to be useful as a therapeutic and/or prophylactic agent for a disease or a condition in which coronavirus 3CL protease participates. The novel synthetic intermediates or salts thereof according to the present invention and the production method according to the present invention are useful for the production of pharmaceuticals.
The agent of the present invention has an inhibitory effect on coronavirus 3CL protease and is considered to be useful as a therapeutic and/or prophylactic agent for a disease or a state in which coronavirus 3CL protease participates.

Claims (5)

1. A formulation for oral administration comprising formula (VII):
[ chemical formula 1 ]
The compound, pharmaceutically acceptable salt or complex thereof is shown as an active ingredient.
2. The preparation according to claim 1, wherein the active ingredient is a complex of a compound represented by formula (VII), which is a complex containing fumaric acid.
3. The formulation according to claim 2, wherein the complex is a co-crystal of the compound of formula (VII) and fumaric acid in a molar ratio of 1:1.
4. The preparation according to any one of claims 1 to 3, wherein a polymer is contained in the preparation.
5. The preparation according to claim 4, wherein the polymer is 1 or more selected from the group consisting of cellulose-based polymers, acrylic-based polymers and vinyl-based polymers.
CN202280007992.3A 2021-11-24 2022-11-22 Formulations for oral administration containing triazine derivatives Pending CN117255680A (en)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
JP2021-189932 2021-11-24
JP2021-191635 2021-11-26
JP2022-006725 2022-01-19
JP2022-012386 2022-01-28
JP2022-017132 2022-02-07
JP2022-027629 2022-02-25
JP2022-046304 2022-03-23
JP2022-142767 2022-09-08
JP2022142767 2022-09-08
PCT/JP2022/043092 WO2023027198A1 (en) 2021-11-24 2022-11-22 Preparation for oral administration containing triazine derivative

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