Substituted hydantoin compounds, methods for preparation thereof and use thereof in the treatment and / or prevention of a coronavirus disease

Abstract

The disclosure concerns substituted hydantoin compounds of Formula I, methods for preparation thereof and uses thereof. In particular, the disclosure concerns use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment and / or prevention of a disease involving a coronavirus.

Description

[0001] SUBSTITUTED HYDANTOIN COMPOUNDS, METHODS FOR PREPARATION THEREOF AND USE THEREOF IN THE TREATMENT AND / OR PREVENTION OF A CORONAVIRUS DISEASE

[0002] Technical field

[0003] The present disclosure concerns substituted hydantoin compounds, methods for preparation thereof and uses thereof. More specifically, the present disclosure concerns substituted 7-(l / 7-pyrrolo[2,3-c]pyridin-4yl)-5,7-diazaspiro[3.4] octane-6, 8-diones, methods for preparation thereof and use thereof in the treatment and / or prevention of a disease or disorder involving a corona virus, in particular in the treatment and / or prevention of COVID-19.

[0004] Background

[0005] Coronavirus disease 2019, abbreviated COVID-19, is a contagious disease caused by severe acute respiratory syndrome coronavirus 2, abbreviated SARS-CoV-2. The SARS-CoV-2 virus has caused the greatest health crisis of this generation and COVID- 19 has already led to >6 million deaths worldwide, as of 6 January 2023 (reported by WHO). Despite promising vaccination efforts, antiviral drugs will likely be crucial to control future outbreaks of coronaviruses. SARS-CoV-2 will continue to circulate and will likely be a major threat to our society as it is the third deadly coronavirus in recent history. Antiviral agents are needed to treat patients that have been infected, as well as be given prophylactically to patients who run a high risk of being infected.

[0006] Among the proteins encoded by the SARS-CoV-2 genome, the chymotrypsin-like main protease (which is commonly abbreviated to MPro) has emerged as a promising drug target. Inhibition of this enzyme blocks processing of polypeptides produced by translation of the viral RNA, which is essential for viral replication. After the SARS-CoV- 1 (also referred to as SARS-CoV) outbreak in 2002, inhibitors of MProwere identified and several of these were recently confirmed active against the highly homologous SARS-CoV-2 protease.

[0007] J. Am. Chem. Soc. 2022, 144, 2905-2929 discloses the exploration of virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. The resulting lead compound, namely compound 19 (2-(2-chlorophenyl)-7- (isoquinolin-4-yl)-5,7-diazaspiro[3.4]octane-6, 8-dione), is a noncovalent main protease inhibitor. The scaffold of compound 19 is stated to be promising for the development of an antiviral drug targeting SARS-Co-2. \N0 2022 / 229458 discloses substituted 5, 7-diazaspiro[3.4]octane-6, 8-diones, methods of preparation thereof as well as use thereof as inhibitors of MProin the treatment and / or prevention of a disease or disorder caused by a corona virus, in particular COVID-19.

[0008] J. Med. Chem. 2022, vol. 65, pages 6454-6495 discloses the development of azaindolebased frameworks as potential antiviral agents and their future perspectives. It is stated that modulating the position and isosteric replacement of the nitrogen atom of Al analogs notably influences the intrinsic physicochemical properties of lead compounds.

[0009] The further investigation and development of substituted 5,7-diazaspiro[3.4]octane- 6, 8-diones has revealed a need for further inhibitors of Mpro. In particular, there is a need for further inhibitors of Mproexhibiting favourable pharmaceutical properties such as satisfactory solubility and / or metabolic stability.

[0010] Summary

[0011] It is an object of the present disclosure to provide an inhibitor of Mproexhibiting favourable pharmaceutical properties such as satisfactory solubility and / or metabolic stability. Further, it is an object of the present disclosure to provide aspects and / or advantages not provided by hitherto known technique.

[0012] The present disclosure provides a compound of Formula I: or a pharmaceutically acceptable salt thereof, wherein

[0013] R is selected from the group consisting of H, Ci-Csalkyl substituted with 0, 1, 2 or 3

[0014] F, and cyclopropyl; and

[0015] X is selected from the group consisting of F, Cl, Br and I. The present disclosure also provides a pharmaceutical composition comprising the compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable excipient, carrier and / or diluent.

[0016] Further, the present disclosure provides a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use as a medicament in therapy.

[0017] The present disclosure also provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described herein for use in the treatment and / or prevention of a disease or disorder involving a corona virus.

[0018] The present disclosure also provides a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described herein for the manufacture of a medicament for use in the treatment and / or prevention of a disease or disorder involving a corona virus.

[0019] The present disclosure also provides a method for the treatment and / or prevention of a disease or disorder involving a corona virus, said method comprising administering to a mammal, such as a human or an animal, in need thereof, an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described herein.

[0020] Description

[0021] The present disclosure provides a compound of Formula I:

[0022] Formula I or a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of H, Ci-Csalkyl substituted with 0, 1, 2 or 3

[0023] F, and cyclopropyl; and

[0024] X is selected from the group consisting of F, Cl, Br and I.

[0025] The term "Ci-Csalkyl" denotes a straight or branched, saturated alkyl group of one to three carbon atoms. Examples of "Ci-Csalkyl" include, but are not limited to, methyl, ethyl, vinyl, allyl, n-propyl and isopropyl. The C1-C3 alkyl group may be substituted with 0, 1, 2 or 3 F. For instance, the Ci group may be CF3, i.e. trifluoromethyl,

[0026] For example, R may be selected from the group consisting of methyl, ethyl, n-propyl or isopropyl. For instance, R may be ethyl. In a further example, R may be ethyl.

[0027] The R substituent of the compound of formula I may be orientated cis or trans with respect to the hydantoin across the cyclobutane moiety. For example, when the R substituent is orientated cis with respect to the hydantoin nitrogen adjacent to the spiro carbon there is provided a compound of Formula la:

[0028] Formula la

[0029] In a further example, when the R substituent is orientated trans with respect to the hydantoin nitrogen adjacent to the spiro carbon there is provided a compound of Formula lb:

[0030] Formula lb

[0031] It will be appreciated that the compound of Formula la and the compound of Formula lb are diastereoisomers of one another. The compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be provided as a single stereoisomer or as a mixture of stereoisomers. The stereoisomer may be a diastereomer of Formula la or Formula lb. The mixture of stereoisomers may be a mixture of the diastereoisomers of Formula la and Formula lb.

[0032] There is provided a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, wherein X is in the ortho, meta or para position on the phenyl ring. For instance, X may be in the ortho position on the phenyl ring. Alternatively, X may be in the meta position on the phenyl ring.

[0033] It will be appreciated that the substituents X and R of a compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, may have the following values.

[0034] X may be Cl or F. For instance, X may be Cl. In a further example, X may be F.

[0035] R may be H, methyl or ethyl. For instance, R may be H. In a further example, R may be methyl. In still a further example, R may be ethyl.

[0036] Surprisingly, the compounds of Formula I as described herein have been found to exhibit favourable pharmaceutical properties such as good metabolic stability (i.e., being substantially metabolically stable). Additionally, the compounds of Formula I as described herein have been found to exhibit good aqueous solubility (i.e., being substantially soluble) or acceptable aqueous solubility. Such advantages may provide for corresponding useful properties in practice. For example, the compounds of the present disclosure may be more efficacious than, be longer acting than, be more easily absorbed than, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties than compounds known in the prior art.

[0037] The present disclosure provides a compound which is one or more of the following: 2-(2-chlorophenyl)-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7-diazaspiro[3.4]octane-6,8- dione, 2-(2-chlorophenyl)-2-methyl-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspirof 3.4] octane-6, 8-dione, 2-(2-fluorophenyl)-2-methyl-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, 2-(3-fluorophenyl)-2-methyl-7-(lH-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, 2-ethyl-2-(3-fluorophenyl)-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, or a pharmaceutically acceptable salt thereof. Each of these compounds, or a pharmaceutically acceptable salt thereof, may be provided as a single stereoisomer such as a diastereoisomer as described herein.

[0038] The present disclosure also provides the compound 2-methyl-2-phenyl-7-(l / 7- py rrolo[2, 3-c] pyrid in -4-yl)-5,7-diazaspiro[3.4] octane-6, 8-dione, or a pharmaceutically acceptable salt thereof.

[0039] The present disclosure also provides a pharmaceutical composition comprising the compound of Formula I as described herein, or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable excipient, carrier and / or diluent.

[0040] Further, there is provided a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use as a medicament in therapy.

[0041] The pharmaceutical composition described herein may be an oral pharmaceutical composition. For example, the oral pharmaceutical composition may be provided as a liquid such as a syrup. Alternatively, the oral pharmaceutical composition may be provided as a solid such as a tablet, capsule or lozenge.

[0042] The compound of Formula I, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition may be provided in a therapeutically effective amount, i.e., an amount allowing for achieving a desired therapeutic effect. For example, the therapeutically effective amount may be adjusted to provide curing and / or relief of symptoms.

[0043] The present disclosure also provides a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use in the treatment and / or prevention of a disease or disorder involving a coronavirus. Further, there is provided a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for the manufacture of a medicament for use in the treatment and / or prevention of a disease or disorder involving a coronavirus

[0044] The present disclosure also provides a method for the treatment and / or prevention of a disease or disorder involving a coronavirus, said method comprising administering to a mammal, such as a human or an animal, in need thereof, an effective amount of a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.

[0045] It will be appreciated that the coronavirus described herein may be the SARS coronavirus 2, which is commonly denominated as SARS-CoV-2 and the cause of coronavirus disease 2019 (COVID-19). Further, the coronavirus described herein may be the SARS coronavirus 1, which is commonly denominated as SARS-CoV-1 and the cause of Severe Acute Respiratory Syndrome (SARS). Furthermore, the coronavirus described herein may be the MERS coronavirus, commonly denominated as MERS- CoV and the cause of Middle East Respiratory Syndrome (MERS).

[0046] Accordingly, there is provided a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use in the treatment and / or prevention of a disease or disorder selected from the group consisting of coronavirus disease 2019 (COVID-19), Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS).

[0047] The present disclosure further provides a compound of Formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein for use in the treatment and / or prevention of COVID-19.

[0048] Stereoisomers

[0049] The compounds of the present disclosure may exist as stereoisomers such as diasteroisomers and a mixture thereof. For example, the compound of Formula I may be provided as a cis diastereoisomer of Formula la, a trans diastereoisomer of Formula lb or a mixture thereof.

[0050] Further, it will be appreciated that the stereoisomers of the compounds described herein may be separated from each other using techniques known in the art, such as chromatography. It will be appreciated that the compounds described herein may be provided in a diasteromeric excess of 90% or more, such as 95% or more, such as 99% or more. As used herein, a diastereomeric excess (abbreviated de) intends the percentage of one diastereomer of the compound of Formula I, or a pharmaceutically acceptable salt thereof, minus the percentage of another diastereomer, or a pharmaceutically acceptable salt thereof. For example, if a mixture contains 98 mole% of one diastereomer and 2 mole percent of another diastereomer the diasteromeric excess is 96%.

[0051] Salts

[0052] The compounds of the present disclosure may be provided as a pharmaceutically acceptable salt. A suitable pharmaceutically acceptable salt of a compound of the present disclosure may be, for example, an acid addition salt.

[0053] Examples of pharmaceutically acceptable salts include, without limitation, non-toxic inorganic and organic acid addition salts such as hydrochloride, hydrobromide, borate, nitrate, perchlorate, phosphate, sulphate, formate, acetate, aconate, ascorbate, benzenesulphonate, benzoate, cinnamate, citrate, embonate, enantate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulphonate, naphthalene-2-sulphonate, phthalate, propionate, salicylate, sorbate, stearate, succinate, tartrate, toluene-p-sulphonate, and any combination thereof. Such salts may be formed by procedures well known and described in the art.

[0054] It will be appreciated that proton transfer may occur between the compound of Formula I described herein and the acid which together form the salt. The proton transfer may take place to a varying extent.

[0055] Solvates or hydrates

[0056] It will be appreciated that the compound of Formula I described herein may be provided as a solvate or as a solvate of a pharmaceutically acceptable salt of the compound of Formula I. It is to be understood that the present disclosure encompasses all such solvates or hydrates.

[0057] Co-crvstals

[0058] In a salt, proton transfer may occur between the active pharmaceutical ingredient and the counter ion of the salt. However, in some cases there is no or only partial proton transfer and the solid is therefore not a true salt. It is accepted that the proton transfer is in fact a continuum, and can change with temperature, and therefore the point at which a salt is better described as a "co-crystal" may be subjective. The term "cocrystal" as used herein refers to multicomponent system in which there exists a host molecule or molecules (active pharmaceutical ingredient) and a guest (or co-former) molecule or molecules. The guest or co-former molecule is defined as existing as a solid at room temperature in order to distinguish the co-crystal from solvates. However, a co-crystal may itself form solvates. In a co-crystal there is generally predominance for interaction through non-ionic forces, such as hydrogen bonding. It will be appreciated that all co-crystals are included within the scope of the compounds described herein.

[0059] Polymorphs

[0060] The compounds of the present disclosure may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. Thus, it is to be understood that all polymorphs, such as mixtures of different polymorphs, are included within the scope of the claimed compounds.

[0061] Isotopicallv labelled compounds

[0062] The compounds of the present disclosure may contain an atomic isotope at one or more of the atoms that constitute said compounds, i.e., said compound may be labelled with an isotope. For example, the compound of Formula I may be labelled with one or more isotopes, such as for example tritium (3H), deuterium (2H) or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure.

[0063] Prodruos

[0064] Compounds of the present disclosure may be administered in the form of a prodrug. A prodrug is a compound, which may have little or no pharmacological activity itself, but when such compound is administered into or onto the body of a subject such as a patient, it is converted into a compound of Formula I. It will be appreciated that the subject such as a patient may be a human or an animal.

[0065] Combinations

[0066] The compounds of the present disclosure may be combined with other pharmaceutical drugs such as other antiviral drugs and / or metabolism blocking drugs. Methods of preparation

[0067] The compounds of the present disclosure may be prepared as described in this document or as described in WO 2022 / 229458. For example, the compounds may be prepared as described in Scheme 1 herein. Substituted styrenes such as compounds of Formula II in the Scheme 1 may be used as starting materials and may be prepared as described in the literature or in published patent documents such as described in WO 2022 / 229458.

[0068] As shown in Scheme 1, reaction of a compound of Formula II with dimethylacetamide (DMA), trifluoromethanesulfonic anhydride (Tf?O) and 2,4,6-trimethylpyridine in 1,2- dichloroethane (DCE) may provide a compound of Formula III. Heating in a microwave (MW) reactor a mixture of a compound of Formula III with potassium cyanide (KCN) and ammonium carbonate ((NH^COs) in ethanol (EtOH) and water may provide a compound of Formula IV. N-arylation of compounds of Formula IV may be achieved by heating in a MW reactor a mixture of a compound of Formula IV, copper oxide (CuO?) and 4-bromo-lH-pyrrolo[2,3-c]pyridine in dimethylformamide (DMF) and may provide compounds of Formula I.

[0069] It will be appreciated that the substituents R and X in Schemes 1 may be as described herein for the compound of Formula I. The invention will be further described by reference to the following examples, which are not intended to limit the scope of the invention.

[0070] Examples

[0071] In this document, unless otherwise stated, the drawing and naming of the chemical compounds have been made using the software package ChemDraw Ultra 12.0. If the drawing and naming are inconsistent, the drawing of the chemical structure shall be considered to be correct.

[0072] The following abbreviations are used throughout this document.

[0073] Abbreviations

[0074] C nt Intrinsic clearance

[0075] DCE 1,2-dichloroethane

[0076] DMA dimethylacetamide

[0077] FRET Fluorescence Resonance Energy Transfer

[0078] G gravitational force equivalent h hour(s)

[0079] HLM human liver microsomes

[0080] HPLC high-performance liquid chromatography

[0081] HRMS high resolution mass spectrometry

[0082] KD equilibrium dissociation constant kDa kilodalton

[0083] L litre(s)

[0084] LC-MS liquid chromatography-mass spectrometry

[0085] M molar

[0086] MERS Middle East Respiratory Syndrome mg milligram(s)

[0087] MHz megahertz min minute(s) mL millilitre(s) mM millimolar

[0088] MPromain protease

[0089] MW Microwave irradiation nd not determined nm nanometre(s) nM nanomolar

[0090] NMR nuclear magnetic resonance MWCO molecular weight cut-off

[0091] ODeoo optical density at 600 nm

[0092] RFU relative fluorescence units rpm revolutions per minute

[0093] RT room temperature s second(s)

[0094] SARS Severe Acute Respiratory Syndrome

[0095] SEC size-exclusion chromatography

[0096] Tf?O trifluoromethanesulfonic anhydride

[0097] TLC thin-layer chromatography

[0098] T ris tris(hydroxymethyl)aminomethane

[0099] Tween polysorbate pg microgram(s) pL microlitre(s) pm micrometre(s) pM micromolar

[0100] Chemistry

[0101] General procedure for synthesis of cvclobutanones of Formula III

[0102] Trifluoromethanesulfonic anhydride (1.5 equiv.) was added dropwise to a solution of DMA (1.2 equiv.) in DCE (1 M) under stirring at 0 °C. The addition was accompanied by white solid precipitation and the mixture was stirred at the same temperature for 30 min. A mixture of a styrene of Formula II (1 equiv.) and 2,4,6-trimethylpyridine (1.5 equiv.) in DCE (0.25 M) was added dropwise and the reaction mixture was left to reflux for 14 h. The reaction mixture was then cooled to RT, treated with water and refluxed for a further 8 h. The mixture was cooled to RT and the organic phase was separated and extracted using dichloromethane (DCM). Combined organic layers were subsequently washed with water and brine, dried over sodium sulfate (Na2SC>4) and concentrated in vacuo. The crude mixture was purified by flash chromatography using 1-10 % ethyl acetate (EtOAc) in pentane to afford the target compound of Formula III as a colourless oil.

[0103] General procedure for synthesis of hydantoin analogues of Formula IV

[0104] KCN (1.3 equiv.) and (NF ^COs (5.0 equiv.) were dissolved in water and added to a microwave vial. A cyclobutanone of Formula III (1.0 equiv.) in EtOH (1.5 mL / mmol) was then added to the solution and the vial was subsequently sealed, subjected to stirring and irradiated in a microwave reactor at 100 °C for 10 min. The progress of the reaction was monitored by TLC. Once the reaction was complete, the reaction mixture was chilled in an ice bath. Most of the EtOH was removed under pressure and saturated sodium bicarbonate (NaHCOs) was added to the reaction mixture. The organic phase was then separated and extracted using EtOAc. Combined organic layers were subsequently washed with water and brine, dried over Na?SO4 and concentrated in vacuo. The crude mixture was purified by flash chromatography using 0-10 % methanol (MeOH) in DCM to afford a white solid product of the target compound of Formula IV as a mixture of isomers.

[0105] General procedure for synthesis of substituted hydantoin compounds of Formula I

[0106] A microwave vial was charged with a hydantoin analogue of Formula IV (1 equiv.), CU2O (0.7 equiv.) and 4-bromo-lH-pyrrolo[2,3-c]pyridine (1.5 equiv.). The vial was sealed and purged with N2 before adding anhydrous DMF. The reaction mixture was then stirred at 165 °C for 12 h. LC-MS was used to monitor the reaction. Once the reaction was complete, the suspension was cooled to RT and filtered through a pad of celite (washed with EtOAc). The filtrate was then concentrated in vacuo and dissolved back into dimethyl sulfoxide (DMSO). The crude reaction mixture was purified using HPLC with 20-60 % acetonitrile (ACN) in water for 30 min to isolate the target compound as a mixture of isomers or as isolated isomers.

[0107] NMR spectra were recorded on a 400, 500 or 600 MHz spectrometer at 298K.

[0108] The comparative examples were prepared as described in Scheme 1 herein and / or as described in WO 2022 / 229458.

[0109] The compounds in Tables 1 and 2 were prepared as a stereoisomeric mixture which was separated into two stereoisomers denominated Isomer 1 and Isomer 2, respectively. The absolute stereochemistry was not determined and the chemical structure in Table 1 is therefore depicted in the same way as for the stereoisomeric mixture.

[0110] Table 1

[0111] Table 2 Biological Assays

[0112] Enzyme activity assay

[0113] The SARS-CoV-2 MProactivity of the tested example compounds was assessed by detection of hydrolysis of a quenched Fluorescence Resonance Energy Transfer (FRET) substrate, as described in the NCATS (National Center for Advancing Translational Sciences) protocol for their SARS-CoV-2 3CL Protease Enzyme Assay (3CL enzymatic activity assay described at National Institutes of Health (NIH), NCATS OpenData portal).6The FRET substrate used in the assay (DABCYL-Lys-HCoV-SARS Replicase Polyprotein 1 ab (3235-3246)-Glu-EDANS trifluoroacetate salt, >95% pure) was custom synthesised and obtained from Bachem AG, Switzerland.

[0114] Expression and purification of SARS-CoV-2 MProprotease

[0115] The MProused for the catalytic activity assay was obtained from the protein Science Facility (PSF, Karolinska Institutet, Stockholm, Sweden) and prepared as described below.

[0116] MProwas produced adopting a published construct (Chernykh, A. et al., Eur. J. Org. Chem., 6466 (2015)) used for the expression of SARS-CoV MProprotease containing nucleotide sequences corresponding to residues S1-Q306 (Chinese isolate, NCBI accession number VP 009725301). Using this construct, the produced MProis flanked by an N-terminal GST (glutathione S-transferase) tag followed by a MProrecognition sequence for auto proteolysis, and a C-terminal 6xHis-tag preceded by a HRV 3C protease recognition sequence.

[0117] Except for some minor adjustments, the expression and purification of MProwas performed according to the procedure described in Xue, X. et al., J. Mol. Biol., 366, 965 (2007). The vector (pGEX-6P-l) containing the coding sequence of the MProwas transformed into E. coll BL21 (DE3)-T1 R competent cells. L-Broth media (Formedium, Norfolk, UK) supplemented with carbenicillin (100 pg / mL) was inoculated with fresh transformants and grown at 37 °C until an ODeoo of 1.5 was reached. The starter culture was then used to inoculate the main culture in Auto Induction Media (AIM) Terrific Broth base with trace elements (Formedium, Norfolk, UK) supplemented with 1 % glycerol and carbenicillin (100 pg / mL). The cultures were grown at 37 °C until an ODeoo of 2 was reached and the protein expression was continued overnight at 18 °C for 13.5 h. Cells were thereafter harvested by centrifugation (10 min at 4500 x g, 4 °C), resuspended in an Immobilized Metal Chelate Affinity Chromatography (IMAC) lysis buffer (50 mM Tris, 300 mM NaCI, pH 8.0) supplemented with Benzonase nuclease (10 pL / 1.5 L culture, 250 U / pL, E1014, Merck, Darmstadt, Germany), and disrupted by sonication (4 s / 4 s 3 min, 80 % amplitude, Sonics Vibracell-VCX750, Sonics & Materials Inc., Newtown, CT, USA). Lysates were centrifuged at 25 49,000 x g for 20 min at 4 °C. The supernatants were filtered (Corning bottle-top vacuum filter, 0.45 pm, Corning, NY, USA) and imidazole was added to a final concentration of 10 mM before loading onto an IMAC HisTrap HP 5 mL column (Cytiva, Little Chalfont, UK), mounted on an AKTA Xpress FPLC system (Cytiva, Little Chalfont, UK). The column was washed with wash buffer (50 mM Tris, 300 mM NaCI, 25 mM imidazole, pH 8.0) and the bound protein was eluted with elution buffer (50 mM Tris, 300 mM NaCI, 500 mM imidazole, pH 8.0). The protein was further purified by size exclusion chromatography (SEC) using a HiLoad 16 / 60 Superdex 200 preparative grade column (Cytiva, Little Chalfont, UK) preequilibrated with a gel filtration buffer (50 mM Tris, 300 mM NaCI, pH 8.0). To remove the His-tag, the protein containing fractions were pooled and treated with HRV 3C protease (1 pg / 500 pg target protein, SAE0045, Merck, Darmstadt, Germany) overnight at 4 °C in a gel filtration buffer supplemented with 0.5 mM TCEP and 0.5 mM OTT. For the FRET assay the protein was treated with HRV 3C protease directly after the IMAC purification step and the buffer was at the same time exchanged by dialysis (dialysis buffer 50 mM Tris, 300 mM NaCI, 0.5 mM TCEP and 0.5 mM OTT, pH 8.0) with a dialysis cassette (Slide-A-Lyzer Dialysis Cassette, 10K molecular weight cut-off (MWCO), 3 mL, Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 °C. The cleaved MProsamples were subsequently purified by reverse IMAC purification using a HisTrap 1 mL column (Cytiva, Little Chalfont, UK). A wash buffer (50 mM Tris, 300 mM NaCI, 25 mM imidazole, pH 8.0) was used and the flow through was collected. The reverse IMAC purification was followed by a second SEC step using a HiLoad 16 / 60 Superdex 200 preparative grade column (Cytiva, Little Chalfont, UK) preequilibrated with a gel filtration buffer (50 mM Tris, 300 mM NaCI, pH 8.0). Fractions containing the target protein were examined by SDS PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), pooled together, and concentrated with Vivaspin® 20 mL centrifugal concentrators (10 kDa MWCO, Sartorius, Goettingen, Germany) at 4,000 x g, 4 °C. The protein was then flash frozen in liquid nitrogen and stored at -80 °C.

[0118] SARS-CoV-2 MProprotease (MPro) catalytic activity assay

[0119] All test compounds were dissolved in 100% DMSO (Merck KGaA, Darmstadt, Germany) to give 10 mM stocks and transferred to ECHO LDV source plates (Labcyte, Inc, Ca, 20 USA).

[0120] The SARS-CoV-2 MProprotease (MPro) enzyme assay was performed in 20 mM Tris, 50 mM NaCI and 0.1 mM EDTA (Merck KGaA, Darmstadt, Germany), pH 7.5 at RT. The compounds were transferred using an Echo 550 non-contact dispenser (Labcyte, Inc., USA) to a Corning 3575 non-binding 384 well assay plate. MPro(75 nM final concentration) was added to the assay plate using a 16-channel pipette (Integra ViaFlo, BergmanLabora AB, Sweden) and shaken for 15 min at 1500 rpm in an Eppendorf Mixmate shaker. After a pulse centrifugation, a stock solution of the quenched fluorogenic substrate (5 mM in DMSO) was added to the assay plate to give a final concentration of 10 pM, thus contributing 0.2 % DMSO in the final assay, using a Labcyte ECHO 550 non-contact dispenser. After 10 min incubation and a pulse centrifugation, fluorescence was measured in a PerkinElmer Envision plate reader at ambient temperature using kinetic mode, with excitation at 340 nm and emission at 490 nm. Activity was calculated as percent of control activity at each data point (100*(RFU sample - RFU Blank control) / (RFU DMSO control - RFU Blank control)). Non-linear fit of 11-point dose response curves (log(inhibitor) vs. response - Variable slope (four parameters) and ICso calculations were performed using GraphPad Prism version 9.1.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

[0121] Compounds were screened at three concentrations, namely 50 pM, 15 pM and 5 pM, and hits were re-tested in an 11-point concentration series (1 :3 dilutions, starting concentration 50 pM). The dose-response curve was generated using the liquid handler Echo 550 non-contact dispensing from 10 mM compound stocks.

[0122] Tables 3A and 3B show ICso values of the compounds as described herein.

[0123] Table 3A Table 3B

[0124] The IC50 values of the example and comparative compounds of the present disclosure show that the compounds are selective inhibitors of the chymotrypsin-like main protease, MPro. It was observed that the example compounds of the present disclosure had IC50 values that were comparable or lower to those of the comparative examples. It was observed that the IC50 value of 123 nM of the compound of Example IB was lower than the IC50 value of 283 of the Comparative example 6 (CE6).

[0125] Surface plasmon resonance (SPR) biosensor assays

[0126] The direct interaction between the example compounds and MProwas confirmed, and the affinities determined using surface plasmon resonance (SPR) biosensor analysis. Production of Avi -tagged SARS-CoV-2 MProprotease

[0127] Avi-tagged MProwas used for the SPR biosensor assays. The expression vector and method for production was essentially as described in Ref. 6 with some minor modifications. The C-terminal Avi-tag replaces the His-tag, giving the final construct GST-3C-MPro_3C-AviTag inserted between BamHI and Xhol in vector pGEX-P-1. The GST-3C part is autocatalytic removed by MProupon expression. The volume of expression cultures was gradually increased in three steps over 8 h from 1 mL to 100 mL LB, i.e. Luria Broth, supplemented with 100 pg / mL ampicillin (Sigma) and 25 pg / mL chloramphenicol (Sigma). 10 mL of the starter culture was used to inoculate 1 L of auto induction medium (Formedium, Hunstanton, Norfolk, UK) supplemented with 10 mL of glycerol and 100 pg / mL ampicillin and 25 pg / mL chloramphenicol. The cultures were grown at 37 °C, 220 rpm for 5 h then switched to 18 °C, 220 rpm for 10-12 h. The cells were harvested by centrifugation and stored at -80 °C. Cells were resuspended in 50 mM Tris pH 8, 300 mM NaCI, 0.03 pg / ml Benzonase (Merck). The cells were lysed by sonication for 5 min on ice, using 15 s on / 15 s off pulses. The lysate was clarified by centrifugation at 50,000 x g. The supernatant was then poured into a 50 mL tube and Streptavidin Mutein matrix (Roche Diagnostics), prepared according to the manufacturer protocol, was added. Binding was allowed for 1 h at 4 °C. The mixture was then transferred to a disposable column for washing and elution using gravity flow. Washing was performed using 50 mM Tris pH 8, 300 mM NaCI, and elution was performed using 10 mM and 50 mM biotin in the same buffer. Relevant fractions were pooled and concentrated using a 10 kDa MWCO centrifugal filter device. Excess biotin was removed either by PDlO-chromatography (GEHC / Cytiva, Uppsala, Sweden) and / or dialysis.

[0128] Interaction analysis

[0129] The SPR experiments were performed using a Biacore S200 instrument and CM5 biosensor chips (Cytiva, Uppsala, Sweden) at 25 °C. Streptavidin (Sigma) was immobilized by amine coupling. The CM5 chip surface was activated by an injection of a 1: 1 mixture of N-(3-dimethylaminopropyl)N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (Cytiva, Uppsala, Sweden) for 7 min at a flow rate of 10 pL / min. Streptavidin (Sigma) was diluted to 250 pg / mL in sodium acetate buffer (pH 5.0) and injected over the activated surface at a flow rate of 2 pL / min for 10 min. The surface was then deactivated by the injection of 1 M ethanolamine (Cytiva, Uppsala, Sweden) for 7 min. Subsequently, the biosensor chip was conditioned with four pulse injections of 1 M NaCI / 50 mM NaOH solution. MProwas diluted to 100 pg / mL in 1.02 x running buffer (50 mM TrisHCI, pH 7.5, 0.05% Tween-20) and injected at a flow rate of 2 pL / min, reaching a typical immobilisation level of 8000-9000 RU.

[0130] After immobilisation, compounds were injected over the surface using a 10-point concentration series, at a flow rate of 30 pL / min in 50 mM TrisHCI, pH 7.5, 0.05 % Tween-20. An association phase was monitored for 60 s and a dissociation phase for 120 s. Sensorgrams were double referenced by subtracting the signals from a reference surface and the signal from one blank injection. A solvent correction accounting for 2 % DMSO was performed. The data were analysed using Biacore S200 Evaluation Software, v. 3.1 (Cytiva, Uppsala, Sweden). For determination of equilibrium dissociation constants (KD), an equation corresponding to a reversible, one-step, 1 : 1 interaction model was fitted by nonlinear regression analysis to report points taken at the end of the injection, representing steady-state signals. Tables 4A and 4B show the equilibrium dissociation constants (KD) determined for steady-state analysis of the compounds as described herein.

[0131] Table 4A Table 4B The SPR biosensor analysis showed that the interactions between the compounds and MProare well described by a reversible, one-step, 1 : 1 interaction, in accordance with their mode of action as reversible active site binding competitive inhibitors. Kinetic solubility measurements of examples compounds

[0132] The kinetic solubility was measured by utilizing test compound from 10 mM DMSO stock solution, and which was measured at a final compound concentration of 100 pM and 1% DMSO. Test compound was added to 100 mM potassium phosphate buffer and incubated at 37°C for at least 20 hours in a heater-shaker. After incubation, the samples were centrifuged at 3000 x g at 37°C for 30 min to pellet insoluble material and an aliquot of the supernatant was taken for analysis. After dilution of the sample, the concentration of dissolved compound was quantified by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS / MS). The kinetic solubility was found to be as shown in Tables 5A and 5B, respectively.

[0133] Table 5A Table 5B

[0134] It was observed that: the compounds of Examples 1, 1A and IB had higher kinetic solubility that the compound of comparative Example 1, the compounds of Examples 1, 1A and IB had a kinetic solubility that was similar to that of the compound of comparative Example 6, the compound of Example 2A had higher kinetic solubility than the compound of comparative Example 2A, and the compounds of Example 2A, 3A, 4A, and 5A had higher kinetic solubilities than all the comparative Examples.

[0135] Thus, the azaindole moiety of the claimed compounds allows for an increase of the solubility such as the kinetic solubility or at least not substantially negatively impact the kinetic solubility.

[0136] The compound of Example 2A was observed to have a higher solubility than the compound of Example 1A. It was concluded that the substituent on the spiro carbon increased the kinetic solubility.

[0137] The compound of Example 6A was observed to have a lower kinetic solubility than the compounds of Examples 2A, 3A, 3B, 4A and 5A. It was concluded that a substituent on the phenyl ring increased the kinetic solubility.

[0138] It was also observed that increasing the number of nitrogen atoms in the heterocycle as in comparative Examples 3, 4, 5 and 6 resulted in compounds with a kinetic solubility of similar magnitude as the compounds of Examples 1A and IB or a very low kinetic solubility.

[0139] Thermodynamic solubility measurements

[0140] The thermodynamic solubility was determined by utilizing solid form of a test compound. Solid test compound (~2-3 mg) was weighed in a glass HPLC vial and 100 mM KPO4-buffer, pH 7.4 was added to give a theoretical max concentration if everything is dissolved of ~5-6 mg / mL. The vial was incubated at 900 rpm, 37 °C in a rotational shaker for 24h. After the incubation an aliquot (200 pl) was transferred to a glass insert and centrifuged at 10 OOOxg, 37 °C for 20 min to separate any solid material from the solution. The supernatant was transferred to a new HPLC vial and analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS / MS).

[0141] The thermodynamic solubility was found to be as shown in Tables 5A and 5B, respectively.

[0142] Table 6A Table 6B

[0143] It was observed from Tables 6A and 6B that the tested example compounds had significantly higher thermodynamic solubility values than the comparative examples.

[0144] Human liver microsomal (HLM) metabolic stability measurements

[0145] The human liver microsomal metabolic stability was determined in 0.5 mg / mL human liver microsomes at a compound concentration of 1 pM in 100 mM KPO4 buffer pH 7.4 in a total incubation volume of 500 pl. The reaction was initiated by addition of 1 mM NADPH. At various incubation times, i.e. at 0, 5, 10, 20, 40 and 60 min, a sample was withdrawn from the incubation and the reaction was terminated by addition of cold acetonitrile with warfarin as an internal standard. The amount of parent compound remaining was analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS / MS).

[0146] The human liver microsomal (HLM) metabolic stability was found to be as shown in Tables 7A and 7B, respectively.

[0147] Table 7A Table 7B It was observed that all tested example compounds had higher or similar metabolic stability in human microsomes , i.e. lower intrinsic clearance (Clint) and longer half-life (ti / 2), than all comparative examples but comparative Example 5. Thus, the presence of the azaindole moiety improved the metabolic stability. Comparative Example 5 was found to have a high metabolic stability, but as indicated above it had a very low kinetic solubility. Conclusions

[0148] As shown by the results of the enzyme and surface plasmon resonance biosensor assays and solubility and metabolic stability measurements, the compounds of Formula I are inhibitors of the chymotrypsin-like main protease, MPro. Moreover, the compounds of Formula I show satisfactory solubility. Further, the compounds of Formula I show satisfactory metabolic stability.

[0149] References

[0150] 1. Luttens, A, et al. Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses. J. Am. Chem. Soc. 144, 2905 (2022). 2. Chernykh, A, et al. Synthesis and Physicochemical Properties of 3-

[0151] Fluorocyclobutylamines. European Journal of Organic Chemistry, 6466 (2015).

[0152] 3. Xue, X., et al. Production of Authentic SARS-CoV MPro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction. Journal of Molecular Biology 366, 965 (2007). 4. WO 2022 / 229458 Al

[0153] 5. Open Data Portal. National Center for Advancing Translational Sciences. SARS- CoV-2 3CL Protease Enzyme Assay protocol, downloaded at https: / / opendata.ncats.nih.gov / covidl9 / assay7a id =9 (accessed 2023-02-27)

[0154] 6. J. Med. Chem. 2022, vol. 65, pages 6454-6495

Claims

Claims1. A compound of Formula I:Formula I or a pharmaceutically acceptable salt thereof, whereinR is selected from the group consisting of H, Ci-Csalkyl substituted with 0, 1,2 or 3 F, and cyclopropyl; andX is selected from the group consisting of F, Cl, Br and I.

2. The compound of Formula I according to claim 1, which is a compound ofFormula la or Formula lb:or a pharmaceutically acceptable salt thereof.

3. The compound of Formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is in the ortho, meta or para position on the phenyl ring.

4. The compound of Formula I according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein X is in the ortho or meta position on the phenyl ring.

5. The compound of Formula I, or a pharmaceutically acceptable salt thereof, according to any one of the preceding claims, wherein X is Cl or F.

6. The compound of Formula I, or a pharmaceutically acceptable salt thereof, according to any one of the preceding claims, wherein R is H.

7. The compound of Formula I, or a pharmaceutically acceptable salt thereof, according to any one of claims 1-5, wherein R is methyl.

8. The compound of Formula I, or a pharmaceutically acceptable salt thereof, according to any one of claims 1-5, wherein R is ethyl.

9. The compound according to any one of the preceding claims, which is one or more of the following: 2-(2-chlorophenyl)-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, 2-(2-chlorophenyl)-2-methyl-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, 2-(2-fluorophenyl)-2-methyl-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, 2-(3-fluorophenyl)-2-methyl-7-(lH-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, 2-ethyl-2-(3-fluorophenyl)-7-(l / 7-pyrrolo[2,3-c] pyridin-4-yl)-5,7- diazaspiro[3.4] octane-6, 8-dione, or a pharmaceutically acceptable salt thereof.

10. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, which is provided as a single stereoisomer or as a mixture of stereoisomers.

11. A pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, as defined in any one of the preceding claims in admixture with a pharmaceutically acceptable excipient, carrier and / or diluent.

12. The compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 11for use as a medicament in therapy.

13. The compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 11 for use in the treatment and / or prevention of a disease or disorder involving a corona virus.

14. The compound or pharmaceutical composition for use according to claim 13, wherein the disease or disorder is selected from the group consisting of COVID-19, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS).

15. The compound or pharmaceutical composition for use according to claim 13 or 14, wherein the disease or disorder is COVID-19.

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