WO2024130411A1 - Inhibiteurs de protéase et leurs méthodes d'utilisation - Google Patents
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- WO2024130411A1 WO2024130411A1 PCT/CA2023/051717 CA2023051717W WO2024130411A1 WO 2024130411 A1 WO2024130411 A1 WO 2024130411A1 CA 2023051717 W CA2023051717 W CA 2023051717W WO 2024130411 A1 WO2024130411 A1 WO 2024130411A1
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
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- 210000003712 lysosome Anatomy 0.000 description 1
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- 238000004949 mass spectrometry Methods 0.000 description 1
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- UPSFMJHZUCSEHU-JYGUBCOQSA-N n-[(2s,3r,4r,5s,6r)-2-[(2r,3s,4r,5r,6s)-5-acetamido-4-hydroxy-2-(hydroxymethyl)-6-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@H](OC=2C=C3OC(=O)C=C(C)C3=CC=2)O[C@@H]1CO UPSFMJHZUCSEHU-JYGUBCOQSA-N 0.000 description 1
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Definitions
- Some embodiments relate to protease inhibitors and methods of their use. Some embodiments relate to inhibitors of coronavirus proteases and their methods to treat coronavirus. Some embodiments relate to inhibitors of the SARS-CoV-2 main protease (M pro ) and methods of their use, for example to treat COVID-19. Some embodiments relate to inhibitors of cathepsin L and methods of their use, for example to treat COVID-19.
- COVID- 19 pandemic illustrated the ease with which an unchecked pathogen can interfere with the normal activities of human society. A great deal of effort was expended to try to quickly identify and approve therapies and vaccines that could help to check the spread of the pandemic by treating or limiting the spread of the SARS-CoV-2 virus that causes COVID-19.
- the SARS-CoV-2 M pro protease represents an attractive target for COVID-19 therapeutics.
- the protease is essential for the maturation and replication of the SARS-CoV- 2 virus, and its protease substrate binding pocket is highly similar across multiple different coronaviruses.
- therapeutic agents targeting the M pro protease may potentially be effective across multiple variants of the virus.
- cathepsin L is implicated in entry of the SARS- CoV-2 virus into cells by activating the viral spike protein in the endosome or lysosome, and some studies have shown that inhibitors of cathepsin L can block or decrease the entry of SARS-CoV-2 into cells without being toxic to the mammalian host.
- inhibitors of cathepsin L and in particular compounds active as inhibitors of both SARS-CoV-2 M pro and cathepsin L, may have utility as therapeutic agents in the treatment of SARS-CoV-2.
- inhibitors of SARS-CoV-2 M pro may also have inhibitory effects against M pro from other coronavirus types that are known to infect humans, including for example other beta-coronaviruses including SARS-CoV-1 , MERS-CoV, HCoV- OC43 and HCoV-HKU1 , and alpha-coronaviruses including HCoV-229E and HCoV-NL63 (see e.g. Owen et al., 2021 ; Unoh et al, 2022).
- n 1 or 2.
- a pharmaceutical composition or an oral dosage form containing such a compound is provided.
- a method of inhibiting a main protease (M pro ) of a coronavirus is provided in which the M pro is exposed to a compound, pharmaceutically acceptable salt, pharmaceutical composition or oral dosage form as described herein.
- the method further includes inhibiting cathepsin L.
- the compound, pharmaceutically acceptable salt, pharmaceutical composition or oral dosage form is administered to a mammalian subject.
- the coronavirus is a beta-coronavirus or an alpha-coronavirus, including for example the alpha-coronavirus being HCoV-229E or HCoV-NL63; the beta-coronavirus being SARS- CoV, MERS-CoV, HCoV-OC43, or HCoV-HKU1 ; or the SARS-CoV being SARS-CoV-1 or SARS-CoV-2.
- the compound, pharmaceutical composition or oral dosage form is administered together with a second antiviral, an immune modulator, or a monoclonal antibody treatment.
- the second antiviral can be remdesivir, molnupiravir, ritonavir, nirmatrelvir or ritonavir/nirmatrelvir;
- the immune modulator can be baricitinib; or
- the monoclonal antibody treatment can be bebtelovimab, tixagevimab, cilgavimab, bamlanivimab, etesevimab, casirivimab, imdevimab, or sotrovimab.
- Kits containing the compound, pharmaceutical composition or oral dosage form can be provided together with instructions for carrying out the methods described herein.
- the compound can be used in a method of manufacturing a medicament for use in treatment of a coronavirus infection in a mammalian subject.
- FIG. 1 is a ribbon diagram illustrating three-dimensional structure based on a crystal structure that was obtained for SARS-CoV-2 M pro with an exemplary protease inhibitor bound thereto.
- FIG. 2 shows a partial detail view showing some of the binding interactions of the exemplary protease inhibitor within the substrate binding pocket of the SARS-CoV-2 M pro shown in the ribbon diagram of FIG. 1.
- the inventors have now discovered novel compounds that are useful as inhibitors of SARS-CoV-2 M pro and correspondingly are potentially useful as therapeutic agents for the treatment of infections caused by SARS-CoV-2. Further, since the substrate binding pocket of M pro is highly conserved across different strains of coronavirus and other compounds that are active as inhibitors of SARS-CoV-2 M pro have also been demonstrated to possess activity against the M pro of other coronaviruses, such compounds are potentially useful in the treatment of multiple strains of coronavirus, including for example other betacoronavirus strains including SARS-CoV-1 , CoV MERS, MERS-CoV, HCoV-OC43, HCoV- HKU1 , or the like and alpha-coronavirus strains including HCoV-229E, HCoV-NL63, or the like (see e.g. Owen et al., 2021 ; Unoh et al., 2022).
- EnkiTM Generative Al a powerful generative artificial intelligence platform developed by Variational Al Inc. (Vancouver, Canada). EnkiTM Generative Al allows predictive generation of de novo molecules with properties optimized for pharmaceutical efficacy, safety and synthesizability.
- treat and “treatment” encompass prophylactic or preventative treatment, as well as treatment that reduces or eliminates infection by a pathogen. Treatment includes any steps taken to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms associated with an infection.
- n 1 or 2.
- a compound that has one of the following structures:
- the compounds as described herein can be prepared according to any desired synthetic scheme. While exemplary synthetic schemes are provided herein with reference to the disclosed examples, in other embodiments, any desired synthetic route can be used to obtain the disclosed compounds. Further, in other embodiments, the disclosed compounds may exist as prodrugs, pharmaceutically acceptable salts, in racemic form or in enantiomeric form, or the like, and all such forms are encompassed within the scope of various embodiments of the disclosed compounds.
- the compounds disclosed herein are used to inhibit a protease, including the SARS-CoV-2 M pro or the M pro from another strain of coronavirus, for example M pro from other beta-coronavirus strains including SARS-CoV-1 , CoV MERS, MERS-CoV, HCoV-OC43, HCoV-HKU1 , or the like, or M pro from an alpha-coronavirus strain including HCoV-229E, HCoV-NL63, or the like.
- the compounds disclosed herein are further used to inhibit cathepsin L.
- the compounds disclosed herein are used as antiviral agents, e.g.
- coronavirus including beta-coronavirus strains including SARS-CoV-2, SARS-CoV-1 , CoV MERS, MERS-CoV, HCoV-OC43, HCoV-HKU1 , or the like, or alpha-coronavirus strains including HCoV-229E, HCoV-NL63, or the like.
- the compounds disclosed herein are used to treat a coronavirus infection in a mammalian subject.
- the compounds can be formulated for administration to the mammalian subject in a suitable manner now known or later developed for delivering therapeutic agents, including by being formulated into a suitable pharmaceutical composition.
- the compound can be administered to the mammalian subject using any suitable dosage regime or dosage regimen.
- the compounds disclosed herein are administered alone to treat a coronavirus infection in a mammalian subject.
- the compounds disclosed herein are administered together with one or more additional coronavirus therapeutics.
- the one or more additional coronavirus therapeutics are antiviral agents, for example, remdesivir, molnupiravir, ritonavir, nirmatrelvir, ritonavir/nirmatrelvir (PaxlovidTM) or the like; immune modulators, for example baricitinib or the like; or monoclonal antibody treatments for coronavirus, for example bebtelovimab, tixagevimab, cilgavimab, bamlanivimab, etesevimab, casirivimab, imdevimab, sotrovimab, or the like; or other anti-coronavirus therapeutics now known or later developed.
- antiviral agents for example, remdesivir, molnupiravir, ritonavir, nirmatrelvir, ritonavir/nirmatrelvir (PaxlovidTM
- the co-administration of the compounds as disclosed herein together with one or more coronavirus therapeutics may include administering the combination of active agents together, whether at the same time and/or formulated together into a single pharmaceutical composition, or at different times, e.g. through separate modes of administration or through sequential administration (e.g. via the administration of a course of one of the active agents followed in time by the administration of a course of the second one of the active agents).
- tablets and dosage forms containing the compounds disclosed herein are provided.
- the tablets or dosage forms contain a dose of the active ingredient(s) that is suitable for administration according to a predetermined administration schedule, e.g. once daily, twice daily, three times daily or four times daily.
- the tablets and dosage forms can further contain suitable carriers or excipients for administration, including those suitable for administration in oral form, e.g. a tablet or syrup, although other modes of administration (e.g. any form of parenteral administration or the like) can be used if desired.
- the mammalian subject can be a human, monkey, cat, dog, sheep, rabbit, horse, cow, pig, goat, mouse, guinea pig, or the like. In some embodiments, the mammalian subject is a human.
- kits are provided, the kit containing a pharmaceutical formulation containing a compound as disclosed herein and instructions for the administration or use of the pharmaceutical formulation, for example according to one of the methods disclosed herein. In some embodiments, the kits are contained within suitable packaging.
- Amine (1) (1 ,1 -Dimethylethyl 1- (aminomethyl)-3,4-dihydro-2(1 H)-isoquinolinecarboxylate, CAS# 1207175-15-2) (262 mg, 1 mmol) was added to the mixture and the reaction mixture was stirred at 50 °C for 1 h. The solvent was removed under reduced pressure and the residue was purified by preparative chromatography to give 288 mg (69%) of the intermediate. The intermediate compound was deprotected with MsOH (methanesulfonic acid) (288 mg, 3 mmol) in acetonitrile (3 mL) at room temperature.
- MsOH methanesulfonic acid
- GDI 198 mg, 1.22 mmol
- 2-(1 H-indol-5-yl)acetic acid 201 mg, 1.15 mmol
- acetonitrile 3 mL
- a solution of methanesulfonic acid salt from the previous step and N-methylmorpholine 0.6 mL, 5.45 mmol
- the solution was concentrated under reduced pressure and the residue was dissolved in dichloromethane (10 mL).
- Synthetic Scheme (B) Compound (102) was synthesized following Synthetic Scheme (B). Briefly, GDI (1 ,1'-Carbonyldiimidazole) (198 mg, 1.22 mmol) was added to a stirred solution of 5- chloronicotinic acid (180 mg, 1.14 mmol) in acetonitrile (3 mL). The mixture was stirred at 50 °C for 30 min.
- Amine (1) (1 ,1 -Dimethylethyl 1-(aminomethyl)-3,4-dihydro-2(1 H)- isoquinolinecarboxylate, CAS# 1207175-15-2) (260 mg, 0.99 mmol) was added to the mixture and the reaction mixture was stirred at 50 °C for 1 h. The solvent was removed under reduced pressure and the residue was purified by preparative chromatography to give 380 mg (95%) of the intermediate. The intermediate compound was deprotected with MsOH (methanesulfonic acid) (0.25 mL) in acetonitrile (3 mL) at room temperature.
- MsOH methanesulfonic acid
- GDI (252 mg, 1.56 mmol) was added to a stirred solution of 2-(1 H-indol-5-yl)acetic acid (280 mg, 1.6 mmol) in acetonitrile (3 mL) and the mixture was stirred at 50 °C for 30 min.
- a solution of methanesulfonic acid salt from the previous step and N-methylmorpholine (0.6 mL, 5.45 mmol) was added to the mixture and the reaction mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and the residue was dissolved in dichloromethane (10 mL).
- Amine (2) (1 ,1 -Dimethylethyl 1-(2- aminoethyl)-3,4-dihydro-2(1 H)-isoquinolinecarboxylate, CAS# 222022-64-2) (276 mg, 1 mmol) was added to the mixture and the reaction mixture was stirred at 50 °C for 1 h. The solvent was removed under reduced pressure and the residue was purified by preparative chromatography to give 337 mg (78%) of the intermediate. The intermediate compound was deprotected with MsOH (methanesulfonic acid) (288 mg, 3 mmol) in acetonitrile (3 mL) at room temperature.
- MsOH methanesulfonic acid
- CDI (198 mg, 1.22 mmol) was added to a stirred solution of (thiophene-2- carbonyl)glycine (213 mg, 1.15 mmol) in acetonitrile (3 mL) and the mixture was stirred at 50 °C for 30 min.
- Compound (105) was synthesized following General Synthetic Scheme (E). Briefly, Reagent (3) (1 eq.), Reagent (4) (1.1 eq.), HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) (1.1 eq.), and DIPEA (N,N-Diisopropylethylamine) (2.5 eq.) were mixed in DMSO (dimethyl sulfoxide) (approximately 0.7 ml per 100 mg of product). The mixture was sealed and stirred at ambient temperature for 16 hours.
- DMSO dimethyl sulfoxide
- cleavage cocktail trifluoroacetic acid, triisopropylsilane, water (93:5:2; v/v) approximately 0.7 ml per 100 mg of product
- the mixture was stirred at ambient temperature for 6 hours.
- the solvent was evaporated under reduced pressure, and to the residue DMSO (appr. 0.7 ml per 100 mg of product), DIPEA (5.7 eq.), Reagent (5) (1.2 eq.) and HATU (1.2 eq.) were added.
- the mixture was sealed and stirred at ambient temperature for 16 hours.
- the solution was filtered, analyzed by LCMS, and transferred for HPLC purification.
- N- ⁇ [2-(2-oxo-1 , 2,3, 4-tetrahydroquinazoline-6-carbonyl)-1 , 2,3,4- tetrahydroisoquinolin-1-yl]methyl ⁇ isoquinoline-4-carboxamide (105) was obtained by General Synthetic Scheme (E) using 54 mg (0.206 mmol) of tert-butyl l-(aminomethyl)- 1 ,2,3,4-tetrahydroisoquinoline-2-carboxylate as Reagent (3), 39 mg (0.225 mmol) of isoquinoline-4-carboxylic acid as Reagent (4), 46.9 mg (0.244 mmol) of 2-oxo-1 , 2,3,4- tetrahydroquinazoline-6-carboxylic acid as Reagent (5), 85.1 mg (0.224 mmol) of HATU, 65.7 mg (0.508 mmol) of DIPEA, 149.9 mg (1.16
- Mass spectrometry was carried out using an Agilent LCWISD system with DAD ⁇ ELSD.
- LCMS procedure 1 Column: InfinityLab Poroshell 120 SB-C18 4.6x30mm 2.7 Micron with Guard: UHPLC Guard 3PK InfinityLab Poroshell 120 SB-C18 4.6x5mm 2.7 Micron.
- Mobile phases A - Deionized water: Formic acid (99.9:0.1 %).
- Gradient from A - 99%, B - 1 % to A - 1 %, B - 99%.
- HPLC procedure Column: Chromatorex 18 SMB 100-5T 100A, 5 pm, 19 mm x 100mm with SiliaSphere C18 100A 5pm 100 A, 19mm x 10 mm. Detection: DAD - DAD1A 215 nm, DAD1 B 254 nm. MSD - single quadrupole, AP-ESI. Mobile phases: A - Deionized water (100%). B - HPLC-grade MeOH (100%).
- Fluorogenic enzymatic assays to evaluate the inhibition of the SARS-CoV-2 M pro protease by the tested compounds were carried out using the quenched fluorogenic substrate ⁇ DABCYL ⁇ -Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-(EDANS)- NH2 (SEQ ID NO:1) as described in Tietjen et al., 2021. Briefly, the peptide substrate exhibits low fluorescence prior to cleavage, because the EDANS moiety is quenched by the DABCYL moiety.
- Assays to evaluate the inhibition of cathepsin L contained 25 pM cathepsin L (RD systems: 952-CY-010), 5 uM Z-LR-AMC fluorogenic peptide substrate, 100 nL of test compound in 100% DMSO, in a total of 10 uL of 20 mM KPO4, pH 6.0, 150 mM NaCI, 0.005% Tween20, 5 mM DTT in black low volume 384-well plates.
- the production of AMC (7-amino-4-methylcoumarin) was followed at 5 min intervals at 355 nm excitation, 460 nm emission in an Envision microplate reader (PerkinElmer). Reaction rates were determined by linear regression of the resulting progress curves.
- Rates were normalized to % inhibition, where 0% is equal to the rate in the presence of enzyme, and 100% is equal to the rate in the absence of enzyme.
- test compound For select test compounds, the fluorogenic enzymatic assay measuring inhibition of SARS-CoV-2 M pro was followed by a subsequent chromatographic purification by HPLC to remove any potential fluorescence interference from the test compound.
- Assays contained test compound, 20 nM M pro , 10 uM substrate (DABCYL-KTSAVLQSGFRKME-EDANS, SEQ ID NO:1), 1 uL test compound in 100% DMSO, in a final volume of 100 uL of assay buffer (10 mM HEPES, pH 7.4, 150 mM NaCI, 5 mM DTT, 0.005% Tween20).
- compounds (101), (102) and (103) had IC50 values against the SARS-CoV-2 M pro of 6.4 pM, 6.0 pM, and 4.5 pM respectively, confirming the accuracy of the fluorogenic assays for which the determined IC50 values were 5.9 pM, 6.1 pM and 4.8 pM, respectively.
- M pro The Gene for SARS-COV2 main protease (M pro ) (corresponding to amino acid residues 1-306 of nsp5, SEQ ID NO:2) was synthesised by codon optimization for Escherichia coli (E. coli) expression.
- the M pro synthesised gene was sub-cloned into a pET based vector (pl NX) with the enzymes Ncol and Xhol to generate plNX4 recombinant vector.
- the SUMO (ULP1) cleavage site and 8xHis-tag (SUMO-8HisTag) were added to the N-terminus in the recombinant gene as a cleavable purification and solubility enhancing tag.
- the recombinant plasmid plNX4 encoding M pro 1-306 was transformed into E. coli BL21 (DE3) cells for protein expression.
- E. coli BL21 (DE3) cells in a volume of 200 ml of SOC (Super Optimal broth with Catabolite repression) medium and kanamycin was inoculated with plNX4 and incubated overnight (o/n) at 37 °C at 250 rpm.
- the cells were inoculated into 3 liters of auto induction medium (AIM) and incubated at 37 °C, 150 rpm until the culture reached an OD600nm of 0.6. Then, for auto-induction the temperature in the incubator was decreased to 16 °C to let the cells to grow overnight with constant shaking of 150 rpm.
- Cells were harvested by centrifugation at 4500 rpm 15 min at 4 °C.
- the pellet was resuspended in lysis buffer containing 50 mM TRIS, pH 8, 500 mM NaCI, 5% glycerol, 1 mM PMSF (phenylmethylsulfonyl fluoride), 5 mM beta-mercaptoethanol, antiprotease, lysozyme, DNAse and 5mM MgC .
- Cell suspension was sonicated 6 min in cycles of 10 sec on/ 30 sec off at 25% intensity. After sonication, cell debris was eliminated by centrifugation at 45 000 x g, 1 hour at 4 °C and imidazole was added to a final concentration of 20 mM. Then, the supernatant was clarified by filtration using a 0.45 pm filter.
- Protein was purified at 4 °C by IMAC (immobilized metal-affinity chromatography) using a 5 mL HisTrap column previously equilibrated with buffer 50 mM TRIS, pH8, 500 mM NaCI, 5% glycerol, 1 mM PMSF, 20 mM imidazole. Protein elution was achieved by a lineal gradient of 500 mM Imidazole. Eluted peak was collected and transferred into a dialysis bag and ULP1 (Ubiquitin-like-specific protease 1) was added at a ratio of 1pg Enzyme/100 pg of protein.
- IMAC immobilized metal-affinity chromatography
- Tag removal and buffer exchange was performed in one step by dialysis in buffer 20 mM TRIS, pH8, 200 mM NaCI, 5% Glycerol, 0.5 mM TCEP (tris(2- carboxyethyl)phosphine) overnight at 4 °C.
- Tag free protein was purified by reverse IMAC in a 5 ml HisTrap column. The removal of SUMO-8His-tag was confirmed by SDS-PAGE. M pro tag free protein was finally purified in a size exclusion column HiLoad sdx 200. The quality of the protein obtained was confirmed by SDS-PAGE. After protein purification a total of 273 mg of protein was obtained. Protein was concentrated to 13.4 mg/ml and aliquoted in 200 pl fractions, then fast frozen with liquid N2 and finally stored at -80 °C.
- M pro at initial concentration of 13.4 mg/ml was diluted with buffer 20 mM HEPES, pH 7.5, 200 mM NaCI, 5% Glycerol, 0.5 mM TCEP to concentrations between 5 mg/ml and 10 mg/ml.
- protein was centrifugated at 10 000 x g for 10 min. Initially the commercial crystallization screening JCSG and PACT were used to guide the search.
- M pro was crystalized by sitting drop vapor diffusion method at 4 °C. The best apo crystal hits were reproduced and used to prepare micro-seeds stocks to grow bigger crystals.
- X-ray data were collected on CMCF BM beamline at the Canadian Light Source (CLS). Data integration and scaling were performed using XDS (X-ray Detector Software). The structure was determined by molecular replacement with the CCP4i2 package using the Apo structure previously modeled from the RCSB PDB: 6wtm in MOLREP as a search model.
- COOT Crystalographic Object-Oriented Toolkit
- the aromatic nitrogen of the isoquinoline ring acts as an H-bond acceptor, while the imidazole NH of histidine serves as an H-bond donor.
- This hydrogen bond is frequently observed in M pro protein-ligand complexes and is a common feature in both fully developed molecules and fragments.
- the carbonyl attached to the isoquinoline ring of compound (103) makes a hydrogen bond to the backbone NH of G143. Further, compound (103) forms an additional hydrogen bond through another carbonyl group which extends from the tetrahydro-isoquinoline ring and interacts with the backbone -NH of E166.
- the phenyl group of the heterocyclic tetrahydro-isoquinoline ring participates in a T-shaped TT-TT stacking interaction with H41's imidazole ring, an integral component of the catalytic dyad with C145.
- the thiophene ring of compound (103) is flanked by the side chains of S46, M49, and Q189.
- the carbonyl attached to the thiophene engages in a slightly elongated and weaker hydrogen bond, measuring approximately 3.17 A, with the side chain amide of N142.
- Table 2 Data for crystal structure of M pro with compound (103).
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
L'invention concerne des inhibiteurs de la protéase Mpro du SARS-CoV-2 et des méthodes d'utilisation de ceux-ci dans le traitement d'une infection à coronavirus chez un sujet. Les inhibiteurs peuvent avoir la structure générale (I).
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