Chemical compoundsid50000167790940 p. doc 2025-05-07 filing no.:19

Abstract

Compounds of formula (I) and pharmaceutically acceptable salts thereof, methods of preparing them, formulations comprising them, and their use in treating coronavirus infections are described.

Description

[0001] CHEMICAL COMPOUNDS

[0002] FIELD

[0003] The present specification relates to chemical compounds, specifically chemical compounds that treat coronavirus infections. The present specification further relates to methods of preparing the chemical compounds, formulations comprising them, and their use in therapy.

[0004] BACKGROUND

[0005] Coronaviruses are a group of RNA viruses that cause disease, particularly respiratory tract infections, in mammals and birds. Coronaviruses represent a serious threat to both human and non-human mammalian health.1Seven Coronaviridae are known to infect humans, four causing mild upper-respiratory tract illness: HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 and three pathogenic coronaviridae, SARS-CoV-1 , MERS-CoV and SARS-CoV-2 responsible for the Sudden Acute Respiratory Syndrome (SARS), the Middle Eastern Respiratory Syndrome (MERS) and Coronavirus Disease 2019 (COVID-19) responsible for significant mortality and morbidity.2SARS- CoV-2 spread rapidly around the world infecting nearly 500 million people and causing more than 6 million mortalities by April 2022. New cases of COVID-19 infection are on the rise and are still increasing rapidly.

[0006] There are also coronaviruses that infect a variety of animal species3both agriculturally important species such as pigs: transmissible gastroenteritis virus of swine (TGEV), porcine epidemic diarrhoea virus (PEDV), swine acute diarrhea syndrome (SADS-CoV), and porcine deltacoronavirus (PDCoV); cattle: bovine coronavirus (BCoV); and egg laying and meat producing birds: infectious bronchitis virus (IBV); as well as diseases of companion animals such as feline infectious peritonitis (FIP) in cats and the canine coronaviruses in dogs (CCoV-l, CCoV-ll and CRCoV). The transmission of coronaviridae between species is highly precedented with all of the coronaviridae currently infecting human having come from zoonotic host reservoirs in bat or rodents either directly or having had an intermediate mammalian host.

[0007] The replication cycle of all coronaviridae requires the transcribed polypeptide to be cleaved into the protein components of the viral capsid.4This is affected by the viral proteases Mpro (also known as 3CL pro), and a second protease PLpro. Inhibition of the coronaviridae proteases has been shown to be a viable method for inhibition of viral replication.

[0008] The first Mpro inhibitor used to treat mammals was the covalent peptidomimetic GC376 to treat FIP5. The agents showing clinical effectiveness in humans against the SARS-C0V2 virus are nirmatrelvir,6 7ibuzatrelvir8 9and ensitrelvir.10 11Nirmatrelvir and ibuzatrelvir are peptidomimetic covalent inhibitors of the SARS-CoV-2 Mpro, whereas ensitrelvir is a non-peptidomimetic compound. Other covalent Mpro inhibitors of SARS-CoV-2 include: RAY1216,12simnotrelvir,13pomotrelvir,14EDP-235,15 / 16and WPV-0117however these all are either focussed on SARS-CoV-2 or combined SARS-CoV-1 and SARS-CoV-2 activity.

[0009] A large number of coronavirus inhibitors have been described, predominantly derived from knowledge of the peptide sequence of the substrates. It is desirable to have multiple inhibitors available to treat current coronavirus derived infections as this gives the opportunity to use agents in combination, to combat resistant strains of virus which evolve as agents are used clinically, and to have the potential for use when new viruses spill over from their zoonotic hosts.

[0010] The compounds described herein are active against coronaviruses, in particular the highly pathogenic human coronaviridae SARS-CoV-2 and MERS-CoV, and are structurally distinct from established agents.

[0011] SUMMARY

[0012] This specification describes, in part, a compound of formula (I): wherein:

[0013] R1is selected from 1-methyl-1 ,2,3-triazol-4-yl, 3-methyl-isoxazole-5-yl, pyran-4-yl and

[0014] 4-methyl-pyrimidin-6-yl; and

[0015] R2is selected from hydrogen and methyl; or a pharmaceutically acceptable salt thereof.

[0016] This specification also describes, in part a pharmaceutical composition which comprises a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as described herein, in association with a pharmaceutically-acceptable diluent or carrier.

[0017] This specification also describes, in part a compound of formula (I) or a pharmaceutically acceptable salt thereof for use as a medicament. This specification also describes, in part a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0018] This specification also describes, in part the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of coronavirus infection.

[0019] This specification also describes, in part a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of a SARS-CoV-2 and / or MERS-CoV infection.

[0020] This specification also describes, in part a method of treating COVID-19 and / or Middle East Respiratory Syndrome in a human, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] Many embodiments of the invention are detailed throughout the specification and will be apparent to a reader skilled in the art. The invention is not to be interpreted as being limited to any of the recited embodiments.

[0023] “A” (or “an”) means “at least one”. In any embodiment where “a” is used to denote a given material or element, “a” may mean one.

[0024] “Comprising” means that a given material or element may contain other materials or elements. In any embodiment where “comprising” is mentioned the given material or element may be formed of at least 10% w / w, at least 20% w / w, at least 30% w / w, or at least 40% w / w of the material or element. In any embodiment where “comprising” is mentioned, “comprising” may also mean “consisting of” (or “consists of”) or “consisting essentially of” (or “consists essentially of”) a given material or element.

[0025] “Consisting of” or “consists of” means that a given material or element is formed entirely of the material or element. In any embodiment where “consisting of” or “consists of” is mentioned, the given material or element may be formed of 100% w / w of the material or element.

[0026] “Consisting essentially of” or “consists essentially of” means that a given material or element consists almost entirely of that material or element. In any embodiment where “consisting essentially of” or “consists essentially of” is mentioned the given material or element may be formed of at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w or at least 99% w / w of the material or element.

[0027] In any embodiment where “is” or “may be” is used to define a material or element, “is” or “may be” may mean the material or element “consists of” or “consists essentially of” the material or element. Claims are embodiments.

[0028] Embodiments may be combined.

[0029] Salts, chiral compounds and solvates

[0030] A suitable pharmaceutically acceptable salt of a compound of the invention is, for example, an acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic, citric, methansulfonic acid, fumaric acid or maleic acid.

[0031] Compounds of the formula (I) have chiral centres, and it is to be understood that the invention encompasses all such optical, diastereoisomers and geometric isomers with coronavirus activity. The invention further relates to any and all tautomeric forms of the compounds of the formula (I) with coronavirus activity.

[0032] References to a particular atom refer to that atom in any isotopic form unless otherwise stated. For example, ‘H’ or ‘hydrogen’ may be in any isotopic form, including1H,2H (deuterium), and3H (tritium); ‘C’ or ‘carbon’ may be in any isotopic form, including12C,13C, and14C; and ‘O’ or ‘oxygen’ may be in any isotopic form, including16O and18O; and the like.

[0033] It is also to be understood that certain compounds of the formula (I) can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms with coronavirus activity.

[0034] Specific Embodiments

[0035] In one embodiment the stereo centre marked is in the S configuration. In one embodiment the stereo centre marked R configuration.

[0036] In one embodiment the stereo centre marked #: R configuration.

[0037] In one embodiment the stereo centre marked #: S configuration.

[0038] In one embodiment R1is selected from 1 -methyl-1 ,2,3-triazol-4-yl.

[0039] In one embodiment R1is selected from 3-methyl-isoxazole-5-yl.

[0040] In one embodiment R1is selected from pyran-4-yl. In one embodiment R1is selected from 4-methyl-pyrimidin-6-yl.

[0041] In one embodiment R2is selected from hydrogen.

[0042] In one embodiment R2is selected from methyl.

[0043] In one embodiment the compound of formula (I) is selected from:

[0044] (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1-methyltriazol-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0045] (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0046] (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0047] (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or

[0048] (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or a pharmaceutically acceptable salt thereof.

[0049] In one embodiment the compound of formula (I) is selected from:

[0050] (4R,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1 -methyltriazol-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0051] (4R,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0052] (4R,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0053] (4R,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or

[0054] (4R,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or a pharmaceutically acceptable salt thereof.

[0055] In one embodiment the compound of formula (I) is selected from:

[0056] (4S,4'R)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1 -methyltriazol-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0057] (4S,4'R)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0058] (4S,4'R)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0059] (4S,4'R)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or (4S,4'R)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or a pharmaceutically acceptable salt thereof.

[0060] In one embodiment the compound of formula (I) is selected from:

[0061] (4R,4'R)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1 -methyltriazol-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0062] (4R,4'R)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0063] (4R,4'R)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;

[0064] (4R,4'R)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or

[0065] (4R,4'R)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; or a pharmaceutically acceptable salt thereof.

[0066] In one embodiment the compound of formula (I) is (4S,4'S)-6-chloro-4'-

[0067] [(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1-methyltriazol-4-yl)methyl]spiro[3H-isoquinoline- 4,3'-pyrrolidine]-1 ,2'-dione: or a pharmaceutically acceptable salt thereof.

[0068] In one embodiment the compound of formula (I) is (4S,4'S)-6-chloro-4'-

[0069] [(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5-yl)methyl]spiro[3H-isoquinoline- 4,3'-pyrrolidine]-1 ,2'-dione: or a pharmaceutically acceptable salt thereof. In one embodiment the compound of formula (I) is(4S,4'S)-6-chloro-4'-

[0070] [(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4-ylmethyl)]spiro[3H-isoquinoline-4,3'- pyrrolid i ne]- 1 ,2'-dione: or a pharmaceutically acceptable salt thereof.

[0071] In one embodiment the compound of formula (I) is (4S,4'S)-6-chloro-4'-

[0072] [(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4-yl)methyl]spiro[3H-isoquinoline- 4,3'-pyrrolidine]-1 ,2'-dione: or a pharmaceutically acceptable salt thereof.

[0073] In one embodiment the compound of formula (I) is (4S,4'S)-6-chloro-4'-

[0074] [(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4-ylmethyl)]spiro[3H-isoquinoline-

[0075] 4,3'-pyrrolidine]-1 ,2'-dione: or a pharmaceutically acceptable salt thereof.

[0076] General procedure for the preparation of compounds of the invention

[0077] It will be appreciated that certain of the various ring substituents in the compounds of the present invention may be introduced by standard aromatic substitution reactions or generated by conventional functional group modifications either prior to or immediately following the processes mentioned above, and as such are included in the process aspect of the invention. Such reactions and modifications include, for example, introduction of a halogen by means of an electrophilic aromatic substitution reaction or nitration followed by diazonium salt preparation and treatment with a copper halide. The reagents and reaction conditions for such procedures are well known in the chemical art.

[0078] It will also be appreciated that in some of the reactions mentioned herein it may be necessary / desirable to protect any sensitive groups in the compounds. The instances where protection is necessary or desirable and suitable methods for protection are known to those skilled in the art. Conventional protecting groups may be used in accordance with standard practice (for illustration see T.W. Green, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991 ). Thus, if reactants include groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein.

[0079] A suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl ort-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions forthe above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a t-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.

[0080] A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions forthe above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide.

[0081] Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon. A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.

[0082] The protecting groups may be removed at any convenient stage in the synthesis using conventionaltechniques well known in the chemical art.

[0083] Doses and routes of administration

[0084] According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in association with a pharmaceutically-acceptable diluent or carrier.

[0085] The composition may be in a form suitable for oral administration, for example as a tablet or capsule, liquid formulation, oral thin film, lollipop, mouth wash rinse, or pre-rinse; for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) as a sterile solution, suspension or emulsion; for topical administration as an ointment or cream; or for rectal administration as a suppository.

[0086] In general the above compositions may be prepared in a conventional manner using conventional excipients.

[0087] The compound of formula (I) will normally be administered to a warm-blooded animal at a unit dose within the range 0.1 -100 mg / kg, and this normally provides an effective dose. Preferably a daily dose in the range of 0.1 -100 mg / kg is employed. However the daily dose will necessarily be varied depending upon the host treated, the particular route of administration, and the severity of the illness being treated.

[0088] Accordingly the optimum dosage may be determined by the practitioner, for example a medical practitioner, who is treating any particular patient.

[0089] Uses

[0090] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use as a medicament.

[0091] In one embodiment there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein as a medicament.

[0092] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in therapy. As used herein, the terms “treatment” and “treat” refer to preventing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In one embodiment, treatment may be conducted before infection has occurred. In one embodiment, treatment may be conducted in subjects exposed to infection. In one embodiment, treatment may be conducted in subjects at risk of infection. In one embodiment, treatment may be conducted after one or more symptoms have developed. In other embodiments, treatment may be conducted in the absence of symptoms. For example, treatment may be conducted to a susceptible individual prior to the onset of symptoms (e.g. in light of a history of symptoms and / or in light of genetic or other susceptibility factors and / or indicative diagnostic tests). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0093] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of an infection.

[0094] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of a viral infection.

[0095] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of coronavirus infection.

[0096] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of a SARS-CoV-2 and / or MERS- CoV infection.

[0097] In one embodiment there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of COVID-19 and / or Middle East Respiratory Syndrome.

[0098] COVID-19 has a number of different symptoms including, but not limited to: fever, cough, tiredness, loss of taste or smell, shortness of breath or difficulty breathing, muscle aches, chills, sore throat, runny nose, headache, chest pain, conjunctivitis, nausea, vomiting, diarrhoea, multisystem inflammatory syndrome, and / or rash. The severity of COVID-19 symptoms can range from very mild / asymptomatic to severe. COVID-19 symptoms can persist for more than four weeks after diagnosis (post- or long- COVID).

[0099] In one embodiment there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for the manufacture of a medicament for the treatment of an infection.

[0100] In one embodiment there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for the manufacture of a medicament for the treatment of a viral infection. In one embodiment there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for the manufacture of a medicament for the treatment of coronavirus infection.

[0101] In one embodiment there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for the manufacture of a medicament for the treatment of a SARS-CoV-2 and / or MERS-CoV infection.

[0102] In one embodiment there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for the manufacture of a medicament for the treatment of COVID-19 and / or Middle East Respiratory Syndrome.

[0103] Pharmaceutical compositions

[0104] In one embodiment there is provided a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0105] In one embodiment there is provided a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of an infection.

[0106] In one embodiment there is provided a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of a viral infection.

[0107] In one embodiment there is provided a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of coronavirus infection.

[0108] In one embodiment there is provided a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of a SARS-CoV-2 and / or MERS-CoV infection.

[0109] In one embodiment there is provided a pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in the treatment of COVID-19 and / or Middle East Respiratory Syndrome.

[0110] Methods of treatment

[0111] In one embodiment there is provided a method of treating an infection in a warm-blooded animal, such as man, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein. In one embodiment there is provided a method of treating a viral infection in a warmblooded animal, such as man, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein.

[0112] In one embodiment there is provided a method of treating coronavirus infection in a warmblooded animal, such as man, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein.

[0113] In one embodiment there is provided a method of treating a SARS-CoV-2 and / or MERS-CoV infection in a warm-blooded animal, such as man, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein.

[0114] In one embodiment there is provided a method of treating COVID-19 and / or Middle East Respiratory Syndrome in a human, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein.

[0115] Kits

[0116] In one embodiment there is provided a kit comprising: a) a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein; b) container means for containing said compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0117] In one embodiment there is provided a kit comprising: a) a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein; b) container means for containing said compound of formula (I) or a pharmaceutically acceptable salt thereof; and optionally c) instructions for use.

[0118] In one embodiment there is provided a kit comprising: a) a compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein; b) container means for containing said compound of formula (I) or a pharmaceutically acceptable salt thereof; c) a coronavirus diagnostic test or testing kit; and optionally d) instructions for use. Combination Therapy

[0119] The compound of formula (I) or a pharmaceutically acceptable salt thereof as described herein may be applied as a sole therapy or may involve, in addition to the compound of the invention.

[0120] Such conjoint treatment may include another antiviral medication; or a medication demonstrated to enhance the response of a human patient; or a medication that might provide support to organs being damaged by viral infection.

[0121] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.

[0122] In the above other use, pharmaceutical composition, method, and combination features, the alternative and preferred embodiments of the compounds of the invention described herein also apply.

[0123] EXAMPLES

[0124] General Experimental

[0125] General methods

[0126] All reactions were carried out using commercial materials and reagents supplied by Piramal without further purification unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) on silica gel plates (Kiselgel 60 F254, Merck) and / or ultra-performance liquid chromatography (UPLC). Visualization of the spots on TLC plates was achieved by UV light and / or by staining the TLC plates in potassium permanganate and charring with a heat gun. All products were characterized by1H NMR and UPLC. Chemical shifts are expressed in parts per million values (ppm) and are designated as s (singlet); br. s (broad singlet); d (doublet); t (triplet); q (quartet); quint (quintet) or m (multiplet). Flash column chromatography was performed on silica gel 60 using a Biotage Isolera Four apparatus using Silicycle Siliasep columns (pre-packed with spherical silica 25 pm) and / or Teledyne Redisep gold cartridge and reagent grade n-heptane and ethyl acetate, as eluent for normal phase chromatographic purification.

[0127] Analytical methods

[0128] NMR Instrument details

[0129] Bruker Avance III HD 400 MHz, JEOL ECX 400 mHz with 30H5X / FG (inverse probe,1H only) or 40TH5AT / FG2 (standard probe) probe UPLC2-MS Acid methods

[0130] Waters Acquity UPLC H-Class system

[0131] Waters Acquity Quaternary pump (Flow 0.8 mL / min)

[0132] Waters Acquity Autosampler

[0133] Waters Acquity QDA

[0134] Waters Acquity PDA (Total plot 210 - 350 nm)

[0135] Solvents: A) water B) acetonitrile D) 2% formic acid in water, the gradients run with 5% D throughout, with only A and B changing percentage.

[0136] Wash solvents:

[0137] Wash solvent = 50:50 MeCN: Water

[0138] Seal wash = 90:10 Water: MeOH

[0139] Purge solvent = 90:10 MeCN: Water

[0140] Column: CSH C18 1 .7 pm 2.1 x 50 mm or BEH C8 1 .7 pm 2.1 x 50 mm

[0141] UPLC2-MS (Long Acid 2 to 95%)

[0142] Run Time: 4.60 min

[0143] Solvents A) water B) acetonitrile D) 2% formic acid in water: the gradient runs with 5% D throughout.

[0144] Gradient: 2-95% B with A and 5% D in 4.00 min, hold at 95% B 5% D to 4.60 min @ 0.8 mL / min, 40 °C

[0145] Waters UPLC H-Class system (UPLC3) instrument details:

[0146] Waters Acquity Sample manager, Quaternary pump, sample manager FTN, PDA detector, SQD (mass spectrometer)

[0147] Flow rate = 0.8 ml / min

[0148] Column temperature = 40 °C

[0149] Total plot = 215 -350 nm

[0150] Solvents: A) Water B) MeOH C) MeCN D1 ) 2% formic acid in water D2) 2% ammonia in water D3 to

[0151] D6) Buffer lines

[0152] Wash solvents:

[0153] Wash solvent = 50:50 MeCN: Water

[0154] Seal wash = 90:10 Water: MeOH

[0155] Purge solvent = 90:10 MeCN:Water

[0156] Buffers:

[0157] 10 mM ammonium formate pH3 10mM ammonium formate pH9 10mM ammonium acetate pH10 10mM ammonium bicarbonate

[0158] 10mM ammonium acetate

[0159] Column: CSH C182.1x50 mm, 1 .7 um

[0160] 2to95 C with A 5% D1 or D2

[0161] 2% MeCN with 5% D1 / D2 and 93% Water to 95% MeCN, 5% D1 / D2 in 4.50 min. Hold at 95% MeCN to 5.00 min.

[0162] UPLC5 Base methods

[0163] Waters Acquity UPLC H-Class system

[0164] Waters Acquity Quaternary pump (Flow 0.8 mL / min)

[0165] Waters Acquity Auto sampler

[0166] Waters Acquity PDA (Total plot 210 - 350 nm)

[0167] 3100 MS

[0168] Waters Acquity ELSD

[0169] Column Temp: 40 °C

[0170] Solvents: A) Water C) Acetonitrile D) 2% ammonia in water, the gradients run with 5% D (from D2 valve) throughout, with only A and C changing percentage. For Base methods D line uses 2% ammonia. (Ammonia was made up from 35% w / w aqueous ammonia solution, 10 mL into 500 mL, and actual % ammonia in D was 0.7% w / w)

[0171] Wash solvents:

[0172] Wash solvent = 50:50 MeCN: Water

[0173] Seal wash = 90:10 Water: MeOH

[0174] Purge solvent = 90:10 MeCN: Water

[0175] Column: BEH C182.5 pm 2.1 x 50 mm or BEH C82.5 pm 2.1 x 50 mm

[0176] UPLC5 (Long Base 50 to 95%) BEH C18 and C8

[0177] Run Time: 5.00 min

[0178] Solvents A) water C) acetonitrile D) 2% ammonia in water: the gradient runs with 5% D throughout. Gradient: 50-95% C with A and 5% D in 4.50 min, hold at 95% C 5% D to 5.00 min @ 0.8 mL / min, 40 °C

[0179] UPLC6-MS Base methods

[0180] Waters Acquity UPLC H-Class system

[0181] Waters Acquity Quaternary pump (Flow 0.8 mL / min)

[0182] Waters Acquity Autosampler

[0183] Waters Acquity QDA

[0184] Waters Acquity PDA (Total plot 210 - 350 nm) Solvents: A) water B) acetonitrile C) 2% ammonia in water, the gradients run with 5% C throughout, with only A and B changing percentage. (Ammonia was made up from 35% w / w aqueous ammonia solution, 10mL into 500 mL, and actual % ammonia in C was 0.7% w / w)

[0185] Wash solvents:

[0186] Wash solvent = 50:50 MeCN: Water

[0187] Seal wash = 90:10 Water: MeOH

[0188] Purge solvent = 90:10 MeCN:Water

[0189] Column: BEH C182.5 m 2.1 x 50 mm or BEH C8 1 .7 pm 2.1 x 50 mm

[0190] UPLC6-MS (Long Base 2 to 95%) BEH C18 and C8

[0191] Run Time: 4.60 min

[0192] Solvents A) water B) acetonitrile C) 2% ammonia in water: the gradient runs with 5% C throughout. Gradient: 2-95% B with A and 5% C in 4.00 min, hold at 95% B 5% C to 4.60 min @ 0.8 mL / min, 40 °C

[0193] Intermediate 1: tert-butyl (5S)-5-[[tert-butyl(dimethyl)silyl]oxymethyl]-2,2-dioxo- oxathiazolidine-3-carboxylate Boc

[0194] Prepared in 4 steps from 2(S)-3-aminopropane-1 ,2-diol by methods in the literature.18 19

[0195] Intermediate 2: methyl 4-chloro-2-(2-methoxy-2-oxo-ethyl)benzoate

[0196] Reaction was performed in 6 x 1 kg scale.

[0197] To a solution of 1-bromo-2-(bromomethyl)-4-chloro-benzene (1 .00 kg, 3.52 mol) in MeOH (7 L) were added [1 ,1 '-Bis(diphenylphosphino)ferrocene]dichloropalladium(ll) (172 g, 211 mmol) and DIPEA (1 .05 kg, 8.09 mol, 1 .41 L). Argon was purged through the vessel for 10 mins and then CO (217 g, 7.74 mol, 274 mL) was charged to 30 bar. The reaction mixture was heated incrementally to 95 °C for 5 h. Once uptake has stopped, the vessels were cooled to 30 °C and slowly vented. The reaction mixture was concentrated until a gelatinous wet solid was observed. EtOAc (5 L) was added, and the resulting precipitate was filtered. The filter cake was washed with EtOAc (2 x4 L), and the filtrate passed through a silica plug (1 .5 kg) before concentrating to dryness to give crude 2 as a brown solid.

[0198] All crude material from the 6 reactions were combined and then purified by recrystallization with MeOH (14 L) and water (3.5 L) to afford methyl 4-chloro-2-(2-methoxy-2-oxo-ethyl)benzoate 2 (2072 g, 7.52 mol, 71 .8 % yield, 88.07% purity) as a black solid. MF: CnHnO4Cl MW: 242.66. CJJPLC6-MS: (BEH-C18 Long Base 2 to 95%) Rt = 2.01 min (88.07%), does not ionise.1H NMR (400 MHz, CDCl3) 5 [ppm] = 7.96 (d, J = 8.4 Hz, 1 H), 7.34 (dd, J = 2.1 , 8.4 Hz, 1 H), 7.26 (s, 1 H), 3.98 (s, 2H), 3.86 (s, 3H), 3.70 (s, 3H).

[0199] Intermediate 3: 4-Chloro-2-(1-methoxycarbonylvinyl)benzoic acid

[0200] To a solution of methyl 4-chloro-2-(2-methoxy-2-oxo-ethyl)benzoate 2 (1 .68 kg, 4.85 mol, 70% purity) in toluene (25 L) were added paraformaldeyde (321 g, 10.7 mol), TBAI (179 g, 485 mmol) and potassium carbonate - 325 Mesh (2.00 kg, 14.5 mol). The reaction mixture was stirred at RT under an argon atmosphere for 18 h and then was heated to 80 °C for 5 h. The reaction mixture was cooled to RT and then water (12.5 L) was added. The layers were separated, and the aqueous layer extracted with EtOAc (2 x 6.5 L). The initial organic layers were discarded, and the aqueous layer acidified to pH = 2 with 4M HCl (11 L) before extracting with EtOAc (3 x 6.5 L). The combined organic layers were washed with brine (6.5 L) and dried over MgSO4before filtering and concentrating to dryness to give 4-chloro-2-(1-methoxycarbonylvinyl)benzoic acid 3 (890 g, 91 J~l% purity, 76% yield) as a brown solid. Taken onto next step with no further purification. MF: CnH9O4Cl MW: 240.64. UPLC2-MS: (CSH-C18 Long Acid 2 to 95%) Rt = 1 .77 min (90.13%), MS (ESIneg): m / z= [M- H]-238.9 / 240.9 Cl isotope pattern - Ring opened product; Rt = 1 .82 min (1 .64%), MS (ESIneg): m / z= [M-H]- 238.9 / 240.8 Cl isotope pattern. Ring closed product.1H NMR (400 MHz, CDCl3): 5 [ppm] = 8.05 (d, J = 8.4 Hz, 1 H) 7.45 (dd, J = 8.4, 2.1 Hz, 1 H) 7.33 (d, J = 2.1 Hz, 1 H) 6.46 (d, J = 0.8 Hz, 1 H) 5.78 (d, J = 0.8 Hz, 1 H) 3.74 (s, 3H). Intermediate 4: Methyl 6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-1-oxo-3,4- dihydroisoquinoline-4-carboxylate

[0201] To a solution of 4-chloro-2-(1 -methoxycarbonylvinyl)benzoic acid 3 (790 g, 3.02 mol) in 1 ,4- dioxane (12 L) were added triethylamine (664 g, 6.56 mol, 915 mL) and (2,4- dimethoxyphenyl)methanamine (825 g, 4.93 mol) and the reaction mixture was stirred at 65 °C for 4 h. More (2,4-dimethoxyphenyl)methanamine (55.0 g, 329 mmol) was added and the reaction mixture heated to 65 °C for a further 2 h. The reaction mixture was cooled down to RT and then concentrated to dryness. The residue was dissolved in MeOH (10 L) and then 4 M HCl in dioxane (4 M, 3.44 mol, 860 mL) was added slowly. The mixture was stirred at RT for 18 h to give a suspension. The precipitate was filtered, and the filter cake rinsed with ice-cold MeOH to obtain first crop of product. The filtrate was cooled to -40 °C and stirred for 1 h to give a suspension. The precipitate was filtered, and the filter cake rinsed with ice-cold MeOH to obtain second crop of product. Both crops were combined and dried to give methyl 6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-1 -oxo- 3,4-dihydroisoquinoline-4-carboxylate rac-4 (863 g, crude, 97.51 % purity, 73% yield) as an off- white solid. Taken onto next step with no further purification. MF: C20H20O5NCI MW: 389.83. UPLC2- MS: (CSH-C18 Long Acid 2 to 95%) Rt = 2.27 min (97.51 %), MS (ESIpos): m / z=[M+H]+390.1 / 392.1 Cl isotope pattern observed.1H NMR (400 MHz, CDCl3): 5 [ppm] = 8.06 - 8.12 (m, 1 H) 7.40 (dd, J = 8.4, 2.0 Hz, 1 H) 7.24 - 7.30 (m, 2H) 6.42 - 6.49 (m, 2H) 4.65 - 4.81 (m, 2H) 3.95 (dd, J = 12.3, 3.4 Hz, 1 H) 3.83 (s, 3H) 3.81 (s, 3H) 3.70 - 3.79 (m, 2H) 3.60 (s, 3H).

[0202] Intermediate 5: methyl 4-[(2R)-1-[(tert-butoxycarbonyl)amino]-3-[(tert- butyldimethylsilyl)oxy]propan-2-yl]-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-1-oxo-3H- isoquinoline-4-carboxylate

[0203] A solution of methyl (4S)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-1 -oxo-3,4- dihydroisoquinoline-4-carboxylate rac-4 (863 g, 2.21 mol) in A / ,A / -dimethylformamide (9490 mL) was degassed with argon overnight. The solution was cooled to -20 °C and then sodium hydride 60% dispersion in mineral oil (93.0 g, 2.33 mol, 60% purity) was added portion wise over 40 mins. The reaction mixture was stirred at -20 °C for 2h. A solution of tert-butyl (5S)-5-[[tert- butyl(dimethyl)silyl]oxymethyl]-2,2-dioxo-oxathiazolidine-3-carboxylate 1 (975 g, 2.65 mol) in N,N- dimethylformamide (3400 mL) was degassed with argon and then added to the main reaction mixture dropwise over 40 mins. The reaction mixture was stirred at -20 °C for 30 mins and then warmed to RT. 2 M HCl (4400 mL) was added dropwise over 30 min and then stirred at RT for 1 h. The reaction mixture was added to MTBE (4400 mL) and the layers separated. To the aqueous / DMF layer was added water (4400 mL) slowly and then extracted with MTBE (2 x 4400 mL). The combined organic layers were washed with brine (8800 mL x 2) and then dried over MgSO4before filtering and concentrating to dryness to give methyl (4SR)-4-[(1R)-1 -[(tert-butoxycarbonylamino)methyl]-2-[tert- butyl(dimethyl)silyl]oxy-ethyl]-6-chloro-2-[(2,4-dimethoxyphenyl) methyl] -1 -oxo-3H-isoquinoline- 4-carboxylate (RS,R)-5 (1 .55 kg, crude) as an orange glass. Taken onto next step with no further purification or further analysis (1H NMR and UPLC data not written up due to mixture of products).

[0204] Intermediate 6: rel-(4'S)-4,-(Aminomethyl)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]spiro[3H- isoquinoline-4,3'-tetrahydrofuran]-1,2'-dione hydrochloride

[0205] To a solution of methyl (4SR)-4-[(1 R)-1-[(tert-butoxycarbonylamino)methyl]-2-[tert- butyl(dimethyl)silyl]oxy-ethyl]-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-1 -oxo-3H-isoquinoline-4- carboxylate (RS,R)-5 (1 .22 kg, 1 .40 mol) in MeOH (6100 mL) was added acetyl chloride (673 g, 8.57 mol, 520 mL) dropwise at -2 °C over 1 h. The reaction mixture was stirred at 0 °C for 30 mins, and then warmed to RT over 18 h. The reaction mixture was concentrated to dryness to afford rel-(4'S)- 4'-(aminomethyl)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl] spiro[3H-isoquinoline-4,3'- tetrahydrofuran]-1 ,2'-dione (RS,R)-6.HCl (900 g, crude, 78.19% purity) as an off-white solid. Taken onto next step with no further purification orfurther analysis (1H NMR data not written up due to mixture of diastereomers). MF: C22H24O5N2CI2 MW: 467.34. UPLC2-MS: (CSH-C18 Long Acid 2 to 95%) Minor Diastereomer: Rt = 1 .20 min (31 .28%), MS (ESIpos): m / z=[M+H]+ 431 .3, 433.3 Cl isotope pattern. Major Diastereomer: Rt = 1 .25 min (46.91%), MS (ESIpos): m / z=[M+H]+ 431 .3, 433.3 Cl isotope pattern (as the free base). Intermediate 7: rel-(4'R)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'-(hydroxymethyl)-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1,2'-dione

[0206] To a solution of rel-(4'S)-4'-(aminomethyl)-6-chloro-2-[(2,4- dimethoxyphenyl)methyl]spiro[3H-isoquinoline-4,3'-tetrahydrofuran]-1 ,2'-dione (RS,R)-6.HCl (650 g, 1.09 mol) in acetonitrile (13 L) were added 4-bromoisoquinoline (238 g, 1.14 mol), potassium carbonate, anhydrous powder 325 mesh (451 g, 3.26 mol), copper (I) Iodide (228 g, 1 .20 mol) and A / ,A / '-dimethylethylenediamine (144 g, 1 .63 mol, 176 mL). The reaction mixture was stirred at 90 °C for 24 h. The reaction mixture was cooled down to RT and then filtered through dicalite (1 kg). The filtrate was concentrated to about half the volume and then EtOAc (6500 mL) and 20% aqueous ammonia (6500 mL) were added. The layers were separated, and the organic layer was washed with 20% aqueous ammonia (6500 mL). The aqueous layerwas back-extracted with EtOAc (2 x 3250 mL) and the combined organic layers were dried over MgSO4before filtering and concentrating to dryness to give rel-(4'R)-6-chloro-2-[(2,4-dimethoxy phenyl)methyl]-4'- (hydroxymethyl)-l '-(4-isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (RS,R)-7 (625 g, crude) as a dark-red foamy solid. MF: C3iH28O5N3Cl MW: 558.02. CJJPLC2-MS: (CSH-C18 Long Acid 2 to 95%) Diastereomer 1 : Rt = 1 .90 min (25.75%), MS (ESIpos): m / z=[M+H]+558.30. 560.3 - Cl isotope pattern. Diastereomer 2: Rt = 1 .92 min (26.89%), MS (ESIpos): m / z=[M+H]+ 558.30, 560.3 Cl isotope pattern.

[0207] Note: Purification was done in 3 batches.

[0208] Batch 1 : Rel-(4'R)-6-chloro-2-[(2,4-dimethoxy phenyl)methyl]-4'-(hydroxymethyl)-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (RS,R)-7 (200 g, crude) was purified by Normal Phase Column Chromatography (Biotage Isolera LS, 1 .6 kg SiliCycle cartridge; eluent 100% DCM for 0.5 CV then 0-100% MeCN in DCM over 7 CV then 100% MeCN for 2 CV).

[0209] Batch 2: Rel-(4'R)-6-chloro-2-[(2,4-dimethoxy phenyl)methyl]-4'-(hydroxymethyl)-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (RS,R)-7 (200 g, crude) was purified by Normal Phase Column Chromatography (Biotage Isolera LS, 1 .6 kg SiliCycle cartridge; eluent 100% DCM for 0.5 CV then 0-100% MeCN in DCM over 7 CV then 100% MeCN for 2 CV).

[0210] Batch 3: Rel-(4'R)-6-chloro-2-[(2,4-dimethoxy phenyl)methyl]-4'-(hydroxymethyl)-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (RS,R)-7 (225 g, crude) was purified by Normal Phase Column Chromatography (Biotage Isolera LS, 1 .6 kg SiliCycle cartridge; eluent 100% DCM for 0.5 CV then 0-100% MeCN in DCM over 7 CV then 100% MeCN for 2 CV).

[0211] Fractions containing product from all 3 purifications were combined and concentrated to dryness to afford product (4SR,4'R)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'-(hydroxymethyl)-1 '-(4- isoquinolyl)spiro[3H-iso quinoline-4,3'-pyrrolidine]-1 ,2'-dione (RS,R)-7 (234 g, 386 mmol, 96.8% yield, 92.15% purity) as a light-orange solid.

[0212] MF: C3iH28O5N3Cl MW: 558.02. C_UPLC2-MS: (CSH-C18 Long Acid 2 to 95%) Diastereomer 1 : Rt = 1 .89 min (45.29%), MS (ESIpos): m / z =[M+H]+ 558.3. Diastereomer 2 : Rt = 1 .91 min (46.86%), MS (ESIpos): m / z=[M+H]+ 558.3.

[0213] Resolution of Intermediate 7: (4S,4,R)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'- (hydroxymethyl)-1'-(4-isoquinolyl)spiro[3H-isoquinoUne-4,3,-pyrrolidine]-1,2,-dione

[0214] Rel-(4'R)-6-chloro-2-[(2,4-dimethoxy phenyl)methyl]-4'-(hydroxymethyl)-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (RS,R)-7 (85 g) was dissolved to 100 mg / mL in MeOH and was then purified by HPLC (Lux iC5 column 30mm x 250mm, 5um, MeOH (0.2% v / v NH3), isocratic conditions). Combined fractions of each diastereomer were then evaporated to near dryness using a rotary evaporator, transferred into final vessels with MeOH:DCM which was removed under a stream of compressed air at 35°C before being stored in a vacuum oven at 35°C and 5 mbar until constant weight to afford (4S,4'R)-6-chloro-2-[(2,4- dimethoxyphenyl)methyl]-4'-(hydroxymethyl)-1 '-(4-isoquinolyl)spiro[3H-isoquinoline-4,3'- pyrrolidine]-1 ,2'-dione (S,R)-7 (35.52 g, 99.68% pure, 45.20% yield) and (4R,4'R)-6-chloro-2-[(2,4- dimethoxyphenyl)methyl]-4'-(hydroxymethyl)-1 '-(4-isoquinolyl)spiro[3H-isoquinoline-4,3'- pyrrolid i ne]- 1 ,2'-dione (R,R)-7 (34.43 g, 99.70% pure, 43.82% yield) as light brown solids. Data obtained for the desired diastereomer (S,R)-7: MF: C31H28N3O5CI MW: 558.02. S_UPLC3-MS (PM-CSH-C18, 4.5 min long acidic, 5 to 95%): Rt = 1 .85 min (99.4%), MS (ESIpos): m / z = [M+H]+ 558.2 & 560.1 [Cl isotopes].1H NMR (400 MHz, Methanol-d4): 5 [ppm] = 9.27 (s, 1 H), 8.49 (s, 1 H), 8.19 (d, J = 8.5 Hz, 1 H), 8.06 (d, J = 8.5 Hz, 1 H), 7.90-7.83 (m, 2H), 7.76-7.72 (m, 1 H), 7.49 (dd, J = 8.5, 1 .8 Hz, 1 H), 7.44 (d, J = 1 .8 Hz, 1 H), 7.24 (d, J = 8.5 Hz, 1 H), 6.56 (d, J = 2.4 Hz, 1 H), 6.46 (dd, J = 8.2, 2.1 Hz, 1 H), 5.01 (d, J = 14.5 Hz, 1 H), 4.56 (d, J = 14.5 Hz, 1 H), 4.11 -4.02 (m, 2H), 3.88-3.82 (tm,

[0215] 2H), 3.84 (s, 3H), 3.75 (s, 3H), 3.75-3.68 (m, 2H), 2.97-2.90 (m, 1 H).

[0216] Intermediate 8: 4-(Chloromethyl)-1 -methyl-triazole hydrochloride

[0217] Under argon, a solution of (1 -methyltriazol-4-yl)methanol, 17, CAS [77177-21-0] (1 .50 g, 12.6 mmol) in DCM (15 mL) was cooled to 0 °C. Thionyl chloride (2.24 g, 18.9 mmol, 1 .38 mL) was added dropwise followed by N,N-dimethylformamide (18.9 mg, 258 pmol, 20.0 pL) and the reaction mixture was stirred at RT for 18 h. The reaction mixture was concentrated to dryness to give 4- (chloromethyl)-l -methyl-triazole hydrochloride, 18.HCI (2.18 g, quant.) as a peach solid. MF: C4H7N3C12 MW: 168.02. CJJPLC6-MS: (BEH-C18 Short Base 2 to 20%) Rt = 0.64 min (100%), MS (ESIpos): m / z=[M+H]+ 131 .94. 1 H NMR (400 MHz, Methanol-d4): 5 [ppm] = 8.18 (s, 1 H), 4.74 (s, 2H), 4.13 (s, 3H).

[0218] Intermediate 9: 4-(Chloromethyl)-1 -methyl-triazole

[0219] A solution of 4-(chloromethyl)-1 -methyl-triazole hydrochloride, 8.HCI (1 .08 g, 6.36 mmol) in MeOH (10 mL) was cooled in an ice bath. Triethylamine (966 mg, 9.55 mmol, 1 .33 mL) was added dropwise and the reaction mixture stirred for 20 minutes. The solution was concentrated to dryness. To the residue was added methyl tert-butyl ether (10 mL) and the suspension was sonicated for 5 minutes. The methyl tert-butyl ether was decanted from the solid and filtered through cotton wool. This process was repeated two further times. The combined methyl tert-butyl ether filtrates were concentrated to dryness to give 4-(chloromethyl)-1 -methyl-triazole, 9 (820 mg, 6.09 mmol, 95.7% yield, 97.65% purity) as a brown oil. MF: C4H6CIN3 MW: 131 .56. C JJPLC5-MS: (BEH-C18 Short Base 2 to 20%) Rt = 0.61 min (97.65%), MS (ESIpos): m / z=[M+H]+ 131 .98. 1 H NMR (400 MHz, Methanol-d4): 5 [ppm] = 7.99 (s, 1 H), 4.74 (s, 2H), 4.11 (s, 3H). Intermediate 10: [(3'R,4S)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-1,-(4-isoquinolyl)-1,5'- dioxo-spiro[3H-isoquinoline-4,4,-pyrrolidine]-3,-yl]methyl methanesulfonate

[0220] To a solution of (4S,4'R)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'-(hydroxymethyl)-1 (4-isoquinolyl)spiro [3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (S,R)-7 (30.0 g, 53.8 mmol) in DCM (360 mL) was added triethylamine (13.6 g, 134 mmol, 18.7 mL) and methanesulfonyl chloride (6.77 g, 59.1 mmol, 4.59 mL) dropwise over 5 min. at -5 °C. The reaction mixture was stirred at RT under a N2atmosphere for 0.5 h. The reaction mixture was washed with ammonium chloride (2 x 150 mL) then sodium bicarbonate (2 x 150 mL). The organic layers were dried over MgSO4before filtering and concentrating to dryness to give [(3'R,4S)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]- 1 '-(4-isoquinolyl)-1 ,5'-dioxo-spiro[3H-isoquinoline-4,4'-pyrrolidine]-3'-yl]methyl methanesulfonate (S,R)-10 (35.5 g, 96.9 % yield, 93.3% purity) as a pale tan foam. MF: C32H30CIN3O7S MW: 636.11 . UPLC5-MS: (Base BEH-C18 Long 5 to 95%) Rt= 2.17 min (94.6%), MS (ESIpos): m / z=[M+H]+636.2 / 638.1 [Cl isotopes].1H NMR (400 MHz, CDCl3): 5 [ppm] = 9.30 (s, 1 H), 8.52 (s, 1 H), 8.19 (d, J = 8.5 Hz, 1 H), 8.12-8.10 (m, 1 H), 7.87-7.83 (m, 1 H), 7.75-7.71 (m, 2H), 7.47 (dd, J = 8.5, 2.4 Hz, 1 H), 7.42 (d, J = 7.9 Hz, 1 H), 7.29 (d, J = 1 .8 Hz, 1 H), 6.53-6.49 (m, 2H), 4.87-4.71 (m, 2H), 4.29-4.17 (m, 2H), 4.11-4.01 (m, 2H), 3.91 -3.86 (m, 5H), 3.81-3.77 (m, 6H), 3.06-2.97 (m, 1 H).

[0221] Intermediate 11: (4S,4,S)-6-chloro-2-(2,4-dimethoxybenzyl)-4,-((ethylamino)methyl)-1'- (isoquinolin-4-yl)-2,3-dihydro-1H-spiro[isoquinoUne-4,3'-pyrrolidine]-1,2,-dione

[0222] [(3'R,4S)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-1 '-(4-isoquinolyl)-1 ,5'-dioxo-spiro[3H- isoquinoline-4,4'-pyrrolidine]-3'-yl]methyl methanesulfonate, (S,R)-10 (35.5 g, 52.1 mmol), methanol (284 mL) and ethylamine (70% in water) (335 g, 5.21 mol, 335 mL) were combined in a pressure vessel and heated at 70°C for 24 h. The mixture was evaporated under reduced pressure and dissolved in EtOAc (500 mL), washed with 10% aqueous sodium bicarbonate (200 mL), Water (200 mL), dried (MgSO4), filtered and evaporated under reduced pressure to give an off-white foam (31 .2 g). A 10 g portion was purified by flash column chromatography, eluting with 95:5:0.5 (dichloromethane:MeOH:cNH3), to give (4S,4'S)-6-chloro-2-(2,4-dimethoxybenzyl)-4'- ((ethylamino)methyl)-1 '-(isoquinolin-4-yl)-2,3-dihydro-1 H-spiro[isoquinoline-4,3'-pyrrolidine]-1 ,2'- dione, (S,S)-11 (7.39 g, 12.63 mmol, 79.5% yield, 98.7% purity) as a cream coloured foam. MF: C33H33N4O4CI MW: 585.09. SJJPLC3-MS (PM-BEH-C18, 4.5 min long basic, 5 to 95%): Rt = 2.18 min (98.7%), MS (ESIpos): m / z = [M+H]+ 585.2 & 587.2 [Ci isotopes]. 1 H-NMR (396 MHz, METHANOL- D4) 5 9.28 (s, 1 H), 8.47 (s, 1 H), 8.22 (d, J = 7.9 Hz, 1 H), 8.08 (d, J = 8.5 Hz, 1 H), 7.92-7.88 (m, 1 H), (m, 2H), 7.49 (td, J = 9.4, 1 .8 Hz, 2H), 7.30 (d, J = 8.5 Hz, 1 H), 6.59 (d, J = 2.4 Hz, 1 H), 6.48 (dd, J = 8.2, 2.1 Hz, 1 H), 4.70-4.82 (2H), 4.11 -4.04 (m, 2H), 3.88 (s, 3H), 3.88-3.79 (m, 2H), 3.75 (s, 3H), 3.10-3.01 (m, 1 H), 2.64 (dd, J = 11 .8, 3.9 Hz, 1 H), 2.51 (t, J = 11 .2 Hz, 1 H), 2.43-2.35 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H).

[0223] Intermediate 12: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4-isoquinolyl)-2,3-dihydro-1 H- spiro[isoquinoUne-4,3'-pyrrolidine]-1,2,-dione:

[0224] (4S,4'S)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'-(ethylaminomethyl)-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione, (S,S)-11 (7.39 g, 12.6 mmol) was dissolved in trifluoroacetic acid (74.5 g, 653 mmol, 50.0 mL) and heated under an atmosphere of nitrogen at ~50°C for 90 minutes then left overnight at room temperature. The volatiles were removed under reduced pressure. The residue was evaporated from a mixture of MeOH (20 mL) and 4 M HCl in dioxane (30 mL) and the residue stirred rapidly with MeOH (50 mL) and filtered to remove the solids, washing with MeOH (2 x 25 mL) and the filtrate evaporated under reduced pressure. The resultant gum was stirred with water (50 mL) and basified with solid potassium carbonate (10 g). The aqueous was decanted away and the solids dissolved in dichloromethane:MeOH:cNH3(90:10:1 , 75 mL) and evaporated under reduced pressure. The residue was dissolved in dichloromethane (50 mL), dried (K2CO3), filtered and evaporated. The residue was purified by flash column chromatography, eluting with 92.5:7.5:0.75 (dichloromethane:MeOH:cNH3) through 90:10:1 to afford a gum. This was dissolved in EtOAc (50 mL) and partially evaporated to give a gum that solidified on standing to give(4S,4'S)-6-chloro-4'- [(ethylamino)methyl]-1 '-(4-isoquinolyl)-2,3-di ydro-1 H-spiro[isoquinoline-4,3'-pyrrolidine]-1 ,2'- dione, 12 (4.725 g, 10.86 mmol, 86% yield, 98.3% purity) as a white solid. MF: C24H23N4O2CI MW: 434.92. S_UPLC3-MS (PM-BEH-C18, 4.5 min long basic, 5 to 95%): Rt= 1 .63 min (98.3% by UV-254 nM), MS (ESIpos): m / z = [M+H]+435.2 & 437.1 [Ci isotopes].1H-NMR (396 MHz, METHANOL-D4) 5 9.29 (s, 1 H), 8.57 (s, 1 H), 8.23 (d, J = 8.5 Hz, 1 H), 8.04 (d, J = 8.5 Hz, 1 H), 7.95-7.90 (m, 2H), 7.82- 7.77 (m, 1 H), 7.51 (td, J = 8.8, 2.0 Hz, 2H), 4.14 (dd, J = 10.3, 7.9 Hz, 1 H), 4.04-3.97 (m, 2H), 3.84 (d, J = 12.7 Hz, 1 H), 3.15-3.07 (m, 1 H), 2.98 (t, J = 10.9 Hz, 1 H), 2.89 (dd, J = 12.1 , 4.2 Hz, 1 H), 2.64-2.52 (m, 2H), 1 .04 (t, J = 7.3 Hz, 3H) NH protons not seen in MeOD.

[0225] Intermediate 13: (4S,4,S)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'- [(isopropylamino)methyl]-1'-(4-isoquinolyl) spiro[3H-isoquinoUne-4,3'-pyrroUdine]-1,2'-dione

[0226] In an autoclave, a solution of [(3'R,4S)-6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-1 '-(4- isoquinolyl)-1 ,5'-dioxo-spiro[3H-isoquinoline-4,4'-pyrrolidine]-3'-yl]methyl methanesulfonate, (R,S)-10 (25.2 g, 35.2 mmol) in propan-2-amine (173 g, 2.92 mol, 250 mL) was heated at 70 °C for 40 h. The reaction mixture was cooled and concentrated under vacuum. The residue was taken up into ethyl acetate (300 mL) and washed with saturated sodium hydrogen carbonate solution (300 mL). The organic layer was separated, dried over MgSO4, filtered and concentrated to give (4S,4'S)- 6-chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)spiro[3H- isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione, (S,S)-13 (22.2 g, quant). Taken onto next step with no further purification. MF: C34H35N4O4Cl MW: 599.12. CJJPLC5-MS: (BEH-C18 Long Base 2 to 95%) Rt= 2.39 min (90.06%), MS (ESIpos): m / z=[M+H]+599.31 .1H NMR (400 MHz, CDCU): 5 [ppm] = 9.28 (s, 1 H), 8.53 (s, 1 H), 8.19 (d, J = 8.4 Hz, 1 H), 8.10 (d, J = 8.2 Hz, 1 H), 7.84 - 7.79 (m, 2H), 7.73 - 7.69 (m, 1 H), 7.44 (dd, J = 8.4, 2.0 Hz, 1 H), 7.37 (d, J = 8.3 Hz, 1 H), 7.30 (d, J = 2.0 Hz, 1 H), 6.54 - 6.45 (m, 2H), 5.03 (d, J = 14.3 Hz, 1 H), 4.63 (d, J = 14.3 Hz, 1 H), 4.16-4.07 (m, 1 H), 3.99 (d, J = 13.3 Hz, 1 H), 3.88 (s, 3H), 3.87 - 3.76 (m, 6H), 2.86 - 2.80 (m, 1 H), 2.70 - 2.54 (m, 3H), 0.95 (dd, J = 6.2, 3.4 Hz, 6H). Intermediate 14: (4S,4'S)-6-Chloro-4,-[(isopropylamino)methyl]-1,-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1,2'-dione

[0227] (4S,4'S)-6-Chloro-2-[(2,4-dimethoxyphenyl)methyl]-4'-[(isopropylamino)methyl]-1 '-(4- isoquinolyl)spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione, (S,S)-13 (7.40 g, 11.1 mmol) was dissolved in 33% hydrogen bromide in acetic acid (55.1 g, 225 mmol, 37.0 mL) and mixture heated to 50 °C and stirred for 1 h, then allowed to stand at RT for 16 h. Water (50 mL) was added and the mixture was filtered through celite to remove the precipitate. The filtrate was basified with solid sodium hydroxide and then extracted with EtOAc (3 x 100 mL). The organic layers were combined and dried with Na2SO4. The solution was filtered, and then concentrated to dryness to give (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)spiro[2,3-dihydroisoquinoline-4,3'- pyrrolidine]-1 ,2'-dione, (S,S)-14 (4.92 g, 10.1 mmol, 90.7 % yield, 92.09% purity) as a brown solid. MF: C25H25N4CIO2 MW: 448.94. C_UPLC6-MS (BEH-C18, Long Base, 2 to 95%): Rt = 1 .73 min (92.09%), MS (ESIpos): m / z = [M+H]+ 449.14. 1 H NMR (400 MHz, CDC13) 59.29 (s, 1 H), 8.58 (s, 1 H), 8.18 - 8.08 (m, 2H), 7.89 -7.79 (m, 2H), 7.78 - 7.66 (m, 1 H), 7.47 (dd, J = 8.3, 2.0 Hz, 1 H), 7.36 (d, J = 1 .8 Hz, 1 H), 6.12 (br d, J = 4.8 Hz, 1 H), 4.20 - 4.07 (m, 2H), 3.95 - 3.84 (m, 2H), 3.13 - 3.06 (m, 1 H), 3.01 - 2.92 (m, 1 H), 2.81 - 2.68 (m, 2H), 1 .02 (dd, J = 6.2, 4.2 Hz, 6H). Amine NH not observed.

[0228] Preparation of Example 1: Intermediate 14: (4S,4,S)-6-Chloro-4,-[(isopropylamino)methyl]-1'- (4-isoquinolyl)spiro[2,3-dihydroisoquinoline-4,3'-pyrrolidine]-1,2'-dione to Example 1(4S,4'S)- 6-chloro-4'-[(isopropylamino)methyl]-1'-(4-isoquinolyl)-2-[(1-methyltriazol-4- yl)methyl]spiro[3H-isoquinoUne-4,3'-pyrrolidine]-1,2,-dione

[0229] A suspension of (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione, 14 (2.47 g, 5.03 mmol) in acetonitrile (22 mL) and N,N-dimethylformamide (5 mL) was cooled to 0 °C. Potassium t-butoxide (845 mg, 7.53 mmol) was added portion wise and the reaction mixture warmed to RT and stirred for 30 minutes. The solution was cooled to 0 °C and a solution of 4-(chloromethyl)-1 -methyl-triazole, intermediate 9, (813 mg, 6.03 mmol) in acetonitrile (7.5 mL) was added dropwise. The resulting solution was warmed to RT and stirred overnight. The reaction mixture was combined with crude reaction mixtures from three reactions carried out with (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione, 14 (1 .75 g, 3.56 mmol), (2.37 g, 4.83 mmol), (2.28 g, 4.64 mmol). In total (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione, 14 (8.87 g, 18.06 mmol) was used.

[0230] The crude reaction mixtures were combined and quenched by addition of water (100 mL). The aqueous layer was extracted with EtOAc (3 x 150 mL), and the combined organic layers washed with brine (2 x 150 mL), dried over MgSO4, filtered and concentrated. To the crude residue was added EtOAc (75 mL) and the mixture was sonicated for 2 minutes. Heptane (5 mL) was added, and the mixture was sonicated for 2 minutes. Further EtOAc (10 mL) was added, and the mixture was sonicated for 2 minutes. The resulting solid was collected by filtration to give a cream solid (5.26 g, 96.78% purity). This solid was recrystallised from acetonitrile and the resulting solid was collected by filtration, washed with ice-cold acetonitrile (5 mL) and dried under vacuum to give a cream solid, batch 1 (2.63 g, 98.46% purity).

[0231] The EtOAc filtrate was concentrated, the residue was taken up in dichloromethane (20 mL) and washed with water (2 x 20 mL) then brine (2 x 40 mL). The organic phase was dried over sodium sulfate, filtered and concentrated. The residue was purified by normal phase chromatography (Biotage Isolera, 80 g Siliasep cartridge; eluent 50-75% acetone in EtOAc [column equilibrated with 1 :1 acetone : EtOAc with 5% triethylamine]). Appropriate fractions were concentrated, and the resulting material was recrystallised from acetonitrile. The resulting solid was collected by filtration, washed with ice-cold acetonitrile (10 mL) and dried under vacuum to give a cream solid, batch 2 (1.05 g, 99.19% purity).

[0232] The acetonitrile filtrate was concentrated and recrystallised from n-propanol. The solid was then recrystallised from acetonitrile twice more to give a cream solid, batch 3 (557 mg, 99.01 % purity).

[0233] Batch 1 (2.63 g), batch 2 (1 .05 g) and batch 3 (557 mg) were combined and EtOAc (50 mL) added. The slurry was heated at refluxfor 30 minutes then cooled to RT and concentrated to dryness. The solid was then dried in the vacuum oven at 45 °C for 18 h to give (4S,4'S)-6-chloro-4'- [(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1-methyltriazol-4-yl)methyl]spiro[3H-isoquinoline- 4,3'-pyrrolidine]-1 ,2'-dione, Example 1 as an off white solid (4.08 g, 7.36 mmol, 58.6 % yield, 98.27% purity). MF: C29H30N7O2CI MW: 544.05. CJJPLC6-MS: (BEH-C18 Long Base 2 to 95%) Rt = 1 .88 min (98.27%), MS (ESIpos): m / z=[M+H]+ 544.12. 1 H NMR (400 MHz, DMSO-d6): 5 [ppm] = 9.37 (s, 1 H), 8.61 (s, 1 H), 8.26 (d, J = 8.2 Hz, 1 H), 8.02 - 8.00 (m, 2H), 7.93 - 7.87 (m, 2H), 7.79 (ddd, J = 8.0, 6.3, 1 .7 Hz, 1 H), 7.56 (dd, J = 8.3, 2.0 Hz, 1 H), 7.49 (d, J = 2.0 Hz, 1 H), 5.06 (d, J = 15.0 Hz, 1 H), 4.58 (d, J = 14.9 Hz, 1 H), 4.23 (d, J = 13.2 Hz, 1 H), 4.01 - 3.88 (m, 6H), 3.06 - 2.98 (m, 1 H), 2.66 - 2.61 (m, 2H), 2.57-2.50 (m, 1 H), 1 .41 (br s, 1 H), 0.88-0.85 (m, 6H).

[0234] Preparation of Example 2: Intermediate 12: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4- isoquinolyl)-2,3-dihydro-1H-spiro[isoquinoUne-4,3'-pyrroUdine]-1,2'-dione to Example 2: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoUne-4,3'-pyrrolidine]-1,2,-dione

[0235] (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2,3-dihydro-1 H-spiro[isoquinoline-4,3'- pyrrolidine]-1 ,2'-dione 12 (4.43 g, 9.31 mmol) was dissolved in DMF (50 mL) under an atmosphere of nitrogen. While cooling in an ice / water bath, potassium t-butoxide (1 .57 g, 14.0 mmol) was added in one portion and after stirring for 15 minutes, 5-(bromomethyl)-3-methyl-isoxazole (1 .97 g, 11 .2 mmol) in DMF (5 mL) was added dropwise. After 5 minutes, the reaction was quenched with 2M HCl solution (10 mL) and evaporated under reduced pressure. The residue was stirred rapidly with saturated sodium carbonate (10 mL), water (20 mL) and EtOAc (50 mL). The organic layer was separated, the aqueous extracted again with EtOAc (20 mL), organics combined, dried (MgSO4), filtered and evaporated under reduced pressure. The crude material was purified by flash column chromatography, eluting with 97.5:2.5:2.5 dichloromethane:MeOH: cNH3through 95:5:0.5 to give an off-white solid (4.85 g, 98.7% purity). This was combined with other crude batches obtained from reactions using ((4S,4'S)-6-Chloro-4'-(ethylaminomethyl)-1 '-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione 12 (200 mg, 0.42 mmol), (4S,4'S)-6-Chloro-4'- (ethylaminomethyl)-l '-(4-isoquinolyl)spiro[2,3-dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione 12 (2.79 g, 5.86 mmol) and ((4S,4'S)-6-Chloro-4'-(ethylaminomethyl)-1 '-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione 12 (200 mg, 0.42 mmol) and recrystallised from MiBKto give (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione Example 2, (5.64 g, 9.30 mmol, 73.1 % yield, 100% purity) as a white solid. MF: CzgHzsNsOaCl MW: 530.02. SJJPLC3-MS (PM-BEH-C18, 4.5 min long basic, 5 to 95%): Rt= 1 .89 min (100% by UV-254 nM, and 220 nM and TAC), MS (ESIpos): m / z = [M+H]+530.1 & 532.1 [Cl isotopes].1H-NMR (396 MHz, METHANOL-D4) 59.28 (s, 1 H), 8.52 (s, 1 H), 8.22 (d, J = 8.5 Hz, 1 H), 8.08 (d, J = 8.5 Hz, 1 H), 7.89 (dd, J = 13.0, 7.6 Hz, 2H), 7.79-7.75 (m, 1 H), 7.53 (td, J = 8.2, 2.0 Hz, 2H), 6.28 (s, 1 H), 5.04 (d, J = 16.3 Hz, 1 H), 4.76 (d, J = 15.7 Hz, 1 H), 4.22 (d, J = 13.3 Hz, 1 H), 4.12-3.97 (m, 3H), 3.25-3.18 (m, 1 H), 2.78-2.67 (m, 2H), 2.52 (qd, J = 12.9, 7.3 Hz, 2H), 2.17 (s, 3H), 1 .01 (t, J = 7.3 Hz, 3H) NH proton not seen in MeOD.

[0236] Preparation of Example 3: Intermediate 12: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4- isoquinolyl)-2,3-dihydro-1H-spiro[isoquinoUne-4,3'-pyrroUdine]-1,2'-dione to Example 3: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoUne-4,3'-pyrrolidine]-1,2,-dione

[0237] NaH (129 mg, 3,22 mmol, 60% dispersion in mineral oil) was added to a solution of (4S,4'S)- 6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2,3-dihydro-1 H-spiro[isoquinoline-4,3'- pyrrolidine]-1 ,2'-dione 12 (0.70 g, 1 .61 mmol) in DMF (7 mL) with cooling under ice-water bath. After 30 min of stirring 4-(iodomethyl)tetrahydropyran (400 mg, 1 .77 mmol) was added. The resulting mixture was stirred at room temperature for 18 hr. The reaction mixture was diluted with NH4Cl aq. (10 mL) and extracted with EtOAc (3*15 mL). The combined organic layers were washed with brine (2*20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to flash column chromatography (SiO2, gradient ACN - MeOH) to afford partially purified product (200 mg, 78% LCMS). This material was subjected to HPLC (0-1 - 7 min., 20-20-50% water- ACN, +0.1 % vol. of 25% aq. NH3, flow: 60 mL / min, column: XBridge OBD C18 100x30 mm, 5 pm) to afford (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2- [(tetrahydropyran-4-ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione Example 3 (56.0 mg, 105 pmol, 3.26% yield, 100% purity) as a white solid. MF: C30H33N4O3CI MW: 532.22. LCMS(ESI): [M+H]+m / z: calcd 533.28; found 533.2. 1 H NMR (600 MHz, DMSO-d6) 50.91 (t, 3H), 1 .12 - 1 .21 (m, 1 H), 1 .24 - 1 .32 (m, 1 H), 1 .46 - 1 .66 (m, 3H), 1 .96 - 2.05 (m, 1 H), 2.34 - 2.51 (m, 2H), 2.66- 2.71 (m, 2H), 3.07 (dd, 1 H), 3.12 - 3.19 (m, 2H), 3.22 - 3.27 (m, 1 H), 3.74- 3.85 (m, 3H), 3.89 (d, 1 H), 3.95 (t, 1 H), 4.02 (t, 1 H), 4.07 (d, 1 H), 7.51 - 7.54 (m, 2H), 7.77 (t, 1 H), 7.89 (t, 1 H), 7.96 (t, 2H), 8.24 (d, 1 H), 8.65 (s, 1 H), 9.35 (s, 1 H). Preparation of Example 4: Intermediate 12: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4- isoquinolyl)-2,3-dihydro-1H-spiro[isoquinoUne-4,3'-pyrroUdine]-1,2'-dione to Example 4: (4S,4'S)-6-chloro-4,-[(ethylamino)methyl]-1,-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoUne-4,3'-pyrrolidine]-1,2,-dione t-BuOK (120 mg, 1.10 mmol) was added to a solution of (4S,4'S)-6-chloro-4'- [(ethylamino)methyl]-1 '-(4-isoquinolyl)-2,3-dihydro-1 H-spiro[isoquinoline-4,3'-pyrrolidine]-1 ,2'- dione 12 (120 mg, 276 pmol) in DMF (2.0 mL) under argon atmosphere at -10 °C. The resulting mixture was stirred at this temperature for 30 min. Then, 4-(chloromethyl)-6-methyl-pyrimidine (75 mg, 524 pmol) was added to the mixture. The resulting mixture was allowed to warm to room temperature and stirred for 18 hr. LCMS showed 60% conversion. The reaction mixture was cooled to -10 °C and another portion of t-BuOK (59 mg, 524 pmol) was added to the mixture. The resulting mixture was stirred for 30 min. Then, 4-(chloromethyl)-6-methyl-pyrimidine (39.3 mg, 276 pmol) was added to the mixture. The resulting mixture was allowed to warm to room temperature and stirred for 18 hr. LCMS showed full conversion. The reaction mixture was quenched by addition of saturated aqueous solution of NH4Cl (1 mL) and subjected to HPLC (0-1 -7 min., 20-20-50% water - ACN, +0.1 % vol. of 25% aq. NH3, flow: 60 mL / min, column: XBridge OBD C18 100><30 mm, 5 pm) to afford (4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione Example 4 (96.0 mg, 174 pmol, 63.0% yield) as a yellow solid. MF: C3oH29N602Cl MW: 540.20. LCMS(ESI): [M+H]+m / z: calcd 541 .25; found 541 .2. 1 H NMR (500 MHz, CD3OD) 5 1 .07 (t, 3H), 2.30 (s, 3H), 2.55 - 2.65 (m, 2H), 2.85 - 2.90 (m, 2H), 3.32 - 3.40 (m, 1 H), 4.02 - 4.15 (m, 2H), 4.26 (dd, 2H), 4.56 (d, 1 H), 5.23 (d, 1 H), 7.42 (s, 1 H), 7.56 (dd, 1 H), 7.64 (d, 1 H), 7.75 - 7.81 (m, 2H), 7.85 (d, 2H), 8.10 (d, 1 H), 8.23 (d, 1 H), 8.49 (s, 1 H), 8.94 (s, 1 H), 9.30 (s, 1 H). Preparation of Example 5: Intermediate 14: (4S,4,S)-6-Chloro-4,-[(isopropylamino)methyl]-1'- (4-isoquinolyl)spiro[2,3-dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione to Example 5: (4S,4'S)-6-chloro-4,-[(isopropylamino)methyl]-1,-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoUne-4,3'-pyrrolidine]-1,2,-dione

[0238] (4S,4'S)-6-Chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)spiro[2,3- dihydroisoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (0.12 g, 267 pmol) was dissolved in DMF (2 mL). The resulting solution was cooled to 0 °C and then sodium hydride (44.91 mg, 1 .87 mmol, 60% dispersion in mineral oil) was added thereto. The reaction mixture was allowed to warm up to ambient temperature and stirred for 20 min. To the obtained mixture 4- (iodomethyl)tetrahydropyran (423 mg, 1 .87 mmol) was added in small portions at ambient temperature during 2 days until aliquots showed full conversion by LCMS. The reaction mixture was poured into 20 mL of saturated aqueous ammonium chloride solution. The resulting mixture was extracted with EtOAc (30 mL), the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected HPLC (0-1 -6 min., 22-22-60% water- ACN, +0.1 % vol. of 25% aq. NH3, flow: 30 mL / min, column: XBridge BEH C18 100><19 mm, 5 pm) to afford (4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran- 4-ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione (76.1 mg, 139 pmol, 52.0% yield) as a white solid. MF: C3iH35N4O3Cl MW: 546.24. LCMS(ESI): [M+H]+m / z: calcd 547.30; found 547.2.1H NMR (500 MHz, CD3OD) 5 1 .12 (d, 6H), 1.31 - 1 .49 (m, 2H), 1 .58 - 1 .72 (m, 2H), 2.05 - 2.15 (m, 1 H), 2.71 - 2.78 (m, 1 H), 2.80 -2.90 (m, 2H), 3.17 (dd, 1 H), 3.20 - 3.31 (m, 2H), 3.37 - 3.45 (m, 1 H), 3.85 - 3.91 (m, 1 H), 3.92 - 3.97 (m, 1 H), 3.97 -4.18 (m, 5H), 7.52 - 7.58 (m, 2H), 7.81 (t, 1 H), 7.93 - 7.99 (m, 2H), 8.06 (d, 1 H), 8.25 (d, 1 H), 8.58 (s, 1 H), 9.31 (s, 1 H).

[0239] Biological Assays

[0240] SAR-CoV-2 MPro Enzyme inhibition IC50 determination

[0241] The inhibition of the SAR-CoV-2 MPro Enzyme was measured by the method of Mesecar et al.20: The fluorescence of the cleaved FAM peptide (excitation 480 nm / emission 520 nm) was measured using a fluorescence intensity protocol on a Pherastar FX (BMG). The assay reaction buffer contained 20 mM HEPES (pH 7.3), 50 mM NaCl, 10% Glycerol, 1 mM TCEP, 0.01 % Tween-20. Compounds were pre-incubated with 5 nM SARS-CoV-23CL protease for 15 minutes at 23 °C. Enzyme reactions were initiated with the addition of 375 nM [5-FAM]-AVLQSGFR-[Lys(Dabcyl)-K- amide substrate and allowed to proceed for 30 minutes at 23 °C.

[0242] Percent inhibition or activity was calculated based on control wells containing no compound (0% inhibition / 100% activity). IC5o values were generated using a 4-parameter hill slope fit model using Vault software (CDD).

[0243] MERS-CoV MPro Enzyme inhibition IC50 determination

[0244] The inhibition of the MERS-CoV-2 MPro Enzyme was measured by an analogous method to that of Mesecar et al.20The fluorescence of the cleaved FAM peptide (excitation 480 nm / emission 520 nm) is measured using a fluorescence intensity protocol on a Pherastar FX (BMG).The assay reaction buffer contained 20 mM HEPES (pH 7.3), 50 mM NaCl, 1 mM TCEP, 0.1 mg / ml BSA and 0.01% Triton x-100.

[0245] Compounds were pre-incubated with 50 nM MERS 3CL protease for 15 minutes at 23° C. Enzyme reactions were initiated with the addition of 550 nM (5-FAM)-GVLQSGLV-K(Dabcyl)-K-NH2 substrate and allowed to proceed for 60 minutes at 23 °C.

[0246] Percent inhibition or activity was calculated based on control wells containing no compound (0% inhibition / 100% activity). IC5o values were generated using a 4-parameter hill slope fit model using Vault software (CDD).

[0247] SARS-CoV-2 Hela ACE2 EC50 determination and MERS-CoV Vero TMPRSS2 EC50 determination

[0248] Four thousand HeLa-ACE2 or Vero-TMPRSS2 cells (BPS Bioscience) were seeded into 96- well plates in DMEM (10% FBS) and incubated for 24 hours at 37°C, 5% CO2. Two hours before infection, the medium was replaced with 100 pL of DMEM (2% FBS) containing the compound of interest at concentrations 50% greater than those indicated, including a DMSO control. Plates were then transferred into the BSL3 facility and an MOI of 0.25 of SARS-CoV-2 or MERS-CoV was added in 50 pL of DMEM (2% FBS), bringingthe final compound concentration to those indicated. Plates were then incubated for 24 hours at 37 °C. After infection, supernatants were removed and cells were fixed with 4% formaldehyde for 24 hours prior to being removed from the BSL3 facility. The cells were then immunostained for the viral N protein (an inhouse mAb 1C7, provided by Dr. Andrew Duty, (Icahn School of Medicine at Mount Sinai), with a DAPI counterstain. Infected cells (488 nm) and total cells (DAPI) were quantified using the Cytation 1 (Biotek) imaging cytometer. Infectivity was measured by the accumulation of viral N protein (fluorescence accumulation). Percent infection was quantified as ((Infected cells / Total cells) - Background) *100 and the DMSO control was then set to 100% infection for analysis. Data was fit using nonlinear regression and IC50s for each experiment were determined using GraphPad Prism version 10.0.0 (San Diego, CA). Cytotoxicity was also performed using the MTT assay (Roche), accordingto the manufacturer’s instructions. Cytotoxicity was performed in uninfected cells with same compound dilutions and concurrent with viral replication assay. All assays were performed in biologically independent triplicates.

[0249] Assessment of anti-viral spectrum of action

[0250] The spectrum of potential coronavirus inhibition was assessed by the use of a cellular fluorescent reporter assay to assess Mpro activity from multiple coronaviridae according to the method of Leonard et al21Activity against the following coronaviridae MPro were assessed: alpha- coronaviridae: Lucheng-19, CDPHE15, HKU8, CHB25, HKU33, 229E, NL63; beta coronaviridae: HKU1 , OC43; gamma-coronaviridae IBV, SW1 ; delta-coronaviridae: HKU11 , PDCoV, HKU19.

[0251] All cells were treated with 2 zM of a P-glycoprotein efflux inhibitor to limit cellular efflux. Reported significance of signal values when treated with 10 zM of drug normalized to DMSO control where a * indicates a p-value s 0.05 in multiple Mann-Whitney U test, with ns indicating p>0.05.

[0252] Human liver microsome clearance determination

[0253] Microsomal incubations were carried out in 96-well plates in 5 aliquots of 30 pL each (one for each time point). Liver microsomal incubation medium comprised of phosphate buffer (100 mM, pH 7.4), MgCl2(3.3 mM), NADPH (3 mM), glucose-6-phosphate (5.3 mM), glucose-6- phosphate dehydrogenase (0.67 units / ml) with 0.42 mg of liver microsomal protein per ml. Human Liver Microsomes were pooled, mixed gender (XenoTech, H0630 / lot #2110263). In the control reactions, the NADPH-cofactor system was substituted with phosphate buffer. Test compounds (2 pM, final solvent concentration 1 .6 %) were incubated with microsomes at 37 °C, shaking at 100 rpm. Five time points over 40 minutes were analysed. The reactions were stopped by adding 5 volumes of acetonitrile with internal standard to incubation aliquots, followed by protein sedimentation by centrifuging at 5500 rpm for 5 minutes. Each reaction was performed in duplicates. Supernatants were analysed using the HPLC system coupled with a tandem mass spectrometer.

[0254] The elimination constant (ket), half-life (ti / 2), and intrinsic clearance (Clint) were determined in a plot of In(AUC) versus time, using linear regression analysis. In order to indicate the quality of the linear regression analysis, the R2 (determination coefficient) values are provided. In some cases, the last time point is excluded from the calculations to ensure acceptable logarithmic linearity of decay.

[0255] Biological data

[0256] Antiviral spectrum Assessment References

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Claims

ClaimsWhat is claimed is:

1. A compound of formula (I):wherein:R1is selected from 1-methyl-1 ,2,3-triazol-4-yl, 3-methyl-isoxazole-5-yl, pyran-4-yl and 4-methyl-pyrimidin-6-yl; andR2is selected from hydrogen and methyl; or a pharmaceutically acceptable salt thereof.

2. A compound of formula (I) as claimed in claim 1 , or a pharmaceutically acceptable salt thereof, wherein the stereo centre markedis in the S configuration.

3. A compound of formula (I) as claimed in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the stereo centre marked #:is in the S configuration.

4. A compound of formula (I) as claimed in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R1is selected from1 -methyl- 1 ,2,3-triazol-4-yl.

5. A compound of formula (I) as claimed in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1is selected from 3-methyl-isoxazole-5-yl.

6. A compound of formula (I) as claimed in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1is selected from pyran-4-yl.

7. A compound of formula (I) as claimed in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1is selected from 4-methyl-pyrimidin-6-yl.

8. A compound of formula (I) as claimed in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R2is selected from hydrogen.

9. A compound of formula (I) as claimed in any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein R2is selected from methyl.

10. A compound of formula (I) as claimed in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, selected from:(4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(1-methyltriazol-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;(4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(3-methylisoxazol-5- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;(4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione;(4S,4'S)-6-chloro-4'-[(ethylamino)methyl]-1 '-(4-isoquinolyl)-2-[(6-methylpyrimidin-4- yl)methyl]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione; and(4S,4'S)-6-chloro-4'-[(isopropylamino)methyl]-1 '-(4-isoquinolyl)-2-[(tetrahydropyran-4- ylmethyl)]spiro[3H-isoquinoline-4,3'-pyrrolidine]-1 ,2'-dione.

11. A compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-10 for use as a medicament.

12. A pharmaceutical composition which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-10 and a pharmaceutically acceptable diluent or carrier.

13. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 -10 for the manufacture of a medicament for the treatment of coronavirus infection.

14. A compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-10 for use in the treatment of a SARS-CoV-2 and / or MERS-CoV infection.

15. A method of treating COVID-19 and / or Middle East Respiratory Syndrome in a human, which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 -10.

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