Field of invention

[001] The present invention relates to medicine, and in particular to the treatment of viral infections, more particularly to inhibitors of viral mRNA cap methyltransferases (MTases). Even more particularly, the invention relates to adenosylmercaptane derivatives and pharmaceutical compositions thereof and their use as inhibitors for viral mRNA cap methyltransferases.

Background of invention

[002] Coronaviruses have evolved an mRNA capping apparatus to protect their 5 ’-ends with a cap moiety that is indistinguishable from eukaryotic mRNA cap structures (Decroly, et al. Nat Rev. Microbiol. 2011, 10, 51). The capping is performed by the MTases Nspl4 and Nspl6, which modify the N7 of the guanosine cap and the 2’ -OH group of the two subsequent nucleotides of viral mRNA, respectively (Bouvet, M et al. PLoS Pathog. 2010, 6, el 000863). TheN7-methylguanosine (m7G) cap is required for efficient translation of viral proteins, whereas 2'-O-methylation of the first two nucleotides is important for evasion of host immune response (Decroly, E et al. PLoS Pathog. 2011, 7, el 002059; Chen, Y. at al. Proc. Natl. Acad. Sci. USA 2009, 106, 3484)

[003] Studies on SARS-CoV-1 have shown that mutations in the Nspl4 and Nspl6 genes lead to a significantly attenuated virus that is recognized by the innate immune system. (Chen, Y et al. Proc. Natl. Acad. Sci. USA 2009, 106, 3484; Chen, Y. etal. J. Virol. 2013, 87, 6296.; Menachery, V. D. etal. J. Virol. 2014, 88, 4251.)

[004] In addition, the studies with the Nspl4 and Nspl6 SAM analogues have shown that the inhibition of Nspl4 or Nspl6 can be used to treat coronavirus infections (Pugh, C. S. and Borchardt, R T. Biochemistry 1982, 21, 1535) However, despite the efforts made so far, none of the methyltransferase inhibitors have been advanced into clinical development

Summary of the invention

[005] In a first aspect, the invention features a method of treating viral infections in humans or animals, comprising administering to a human or animal in need of a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug, wherein the compound is an inhibitor of viral mRNA cap methyltransferase. [006] In another aspect, the invention features a pharmaceutical composition for treatment of viral infections comprising a therapeutically effective amount of a composition comprising (i) a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug; and (ii) a pharmaceutically acceptable carrier, wherein the compound is an inhibitor of viral mRNA cap methyltransferase.

[007] In another aspect, the invention features the use of a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug, wherein the compound is an inhibitor of viral mRNA cap methyltransferase, in the manufacture of a medicament for treatment or prevention of viral infections.

[008] In another aspect, the invention features a compound or prodrug thereof, or pharmaceutically acceptable salt or ester of said compound or prodrug for use in treating or preventing viral infections, wherein the compound is an inhibitor of viral mRNA cap methyltransferase.

[009] In one embodiment an inhibitor of viral mRNA cap methyltransferase is a compound of Formula I: wherein:

R ¹ , R ² , R ³ R ⁴ are independently H, C _1-6 alkyl, cycloC _3-12 alkyl, cycloC _3-12 alkyl- C _1-6 alkyl, C _{2- 6} alkenyl, C _2-6 alkynyl, aryl, biaryl, arylC _1-6 alkyl, aryl C _2-6 alkenyl, arylC _2-6 alkynyl, heteroaryl, heteroarylC _1-6 alkyl, heteroarylC _2-6 alkenyl, R ⁵ O(CH ₂ ) _n , R ⁵ S(CH ₂ ) _n , R ⁵ OC(=O)(CH ₂ ) _n , R ⁵ N(R ⁶ )C(=O)(CH ₂ ) _n , R ⁵ N(R ⁶ )(CH ₂ ) _n , -F, -Cl, -Br, -I, -CF ₃ , -CH ₂ CF ₃ , -CF ₂ CF ₂ H, -OH, -L-OH,-O-L-OH, -OR ⁵ , -O-L-NH ₂ , -O-L-NHR ⁵ , -O-L-N(R ⁵ )R ⁶ , -L-OR ⁵ ,-O-L-OR ⁵ ,-OCF ₃ , -OCH ₂ CF ₃ , -OCF ₂ CF ₂ H, -L-OR ⁵ ,-O-L-OR ⁵ ,-OCF ₃ , -OCH ₂ CF ₃ , -OCF ₂ CF ₂ H, SR ⁵ , SCF ₃ , CN, -NO ₂ , -NO ₂ , -NH ₂ , -NHR ⁵ , -NR ⁵ 2, -N(R ⁵ )R ⁶ , -L-NH ₂ , -L-NHR ⁵ , -L-NR ⁵ ₂ , -L-N(R ⁵ )R ⁶ ,-NH-L-NH ₂ , -NH-L-NHR ⁵ , -NH-L-N(R ⁵ )R ⁶ , -NH-L-N(R ⁵ )R ⁶ ,-NR ⁵ -L-NH ₂ , -NR ⁵ -L-NHR ⁵ , -NR ⁵ -L-N(R ⁵ )R ⁶ , -NR ⁵ -L-N(R ⁵ )R ⁶ , -N(R ⁵ )R ⁶ , -C(=O)OH, -C(=O)OR ⁵ , -C(=O)NH ₂ , -C(=O)NHR ⁵ , -C(=O)N(R ⁵ )R ⁶ ,

-C(=O)N(R ⁵ )R ⁶ ,-NHC(=O)R ⁵ , -NR ⁵ C(=O)R ⁶ , -NHC(=O)OR ⁵ ,

-NR ⁵ C(=O)OR ⁶ , -OC(=O)NH ₂ , -OC(=O)NHR ⁵ , -OC(=O)N(R ⁵ )R ⁶ ,

-OC(=O) R ⁵ NR ⁶ ,-OC(=O)R ⁵ , -C(=O)R ⁵ ,-NHC(=O)NH ₂ , -NHC(=O)NHR ⁶ ,

-NHC(=O)NR ⁵ ₂ , -NHC(=O)N(R ⁵ )R ⁶ , -NR ⁵ C(=O)NH ₂ , -N(R ⁵ )C(=O)NHR ⁶ ,

-NR ⁵ C(=O)N (R ⁵ )R ⁶

[010] wherein: n is an integer selected from 0 to 1; and optical isomers, pharmaceutically acceptable salts, hydrates, solvates, and polymorphs thereof; wherein when R ¹ , R ² , R ³ , R ⁴ are H; n is not 0, and when R ¹ , R ² , R ⁴ are H; R ³ is NO ₂ ; then n is not 0.

[011] In one embodiment, the treatment is treatment of a disease or disorder that is mediated by a viral mRNA cap methyltransferase or human mRNA cap methyltransferase.

[012] In one embodiment, the treatment is treatment of a disease or disorder that is ameliorated by the inhibition of a viral mRNA cap methyltransferase or human mRNA cap methyltransferase. [013] In one embodiment, the treatment is treatment of a disease or disorder that is treated by a viral mRNA cap methyltransferase or human mRNA cap methyltransferase inhibitor.

[014] In another aspect, the invention features a kit comprising a adenosylmercaptane derivatives described herein, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging.

[015] In another aspect, the invention features compounds obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.

[016] In another aspect, the invention features compounds obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.

[017] In another aspect, the invention features novel intermediates, as described herein, which are suitable for use in the methods of synthesis described herein.

[018] As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention.

Description of invention [019] Viral nonstructural proteins (Nsp) group have been identified as a group of promising biological targets for the development of broad-spectrum antiviral drugs against coronaviruses (Chen, ¥ et al. Proc. Natl. Acad. Sci. USA 2009, 106, 3484; Chen, Y. et al. J. Virol. 2013, 87, 6296.; Menachery, V. D. et al. J. Virol. 2014, 88, 4251, Pugh, C. S. and Borchardt, R. T. Biochemistry 1982, 21, 1535)

[020] When testing the novel adenosylmercaptane derivatives with general formula I for their ability to inhibit Nspl4 and Nspl6 we have unexpectedly discovered, that said derivatives exhibit pronounced inhibitory properties toward said nonstructural proteins, and thus are useful in treatment of viral infections.

[021] According to this invention, the results from nonstructural protein inhibition studies demonstrate that adenosylmercaptane derivatives with general formula I are novel class inhibitors of nonstructural proteins. Several example compounds from the present invention display nanomolar to low micromolecular nonstructural protein inhibitory potency.

Stereochemistry

[022] Many of the chemical structures shown herein indicate one or more specific stereoisomeric configurations. Similarly, many of the chemical structures shown herein are silent in this respect, and do not indicate any stereoisomeric configuration. Similarly, many of the chemical structures shown herein indicate the specific stereoisomeric configurations at one or more positions, but are silent with respect to one or more other positions. Where a chemical structure herein is silent with respect to the stereoisomeric configuration at a position, that structure is intended to depict all possible stereoisomeric configurations at that position, both individually, as if each possible stereoisomeric configuration was individually recited, and also as a mixture (e.g., a racemic mixture) of stereoisomers.

Combinations

[023] Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited. Examples of Specific Embodiments

[024] The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.

[025] The following adenosylmercaptane derivatives with general formula I were prepared as examples of the current invention: General Synthesis

[026] Compounds 1.1-1.4 were prepared according to Scheme 1. Adenosine 2 was first transformed to chloride 3 which was subsequently used for the alkylation of mercaptobenzoic acids.

Scheme 1

Reagents and conditions: (a) i SOCl ₂ , pyridine, MeCN, 0°C to rt; ii NH ₄ OH, MeOH; (b) 3- mercaptobenzoic acid, CS ₂ CO ₃ , DMF, rt.

[027] For the synthesis of 1.5, protected adenosine 4 was used as a starting material. This was first transformed to S-acetyl mercapto derivative 5 which was deacylated and S-alkylated to give intermediate 6. Finally, deprotection of diol provided target compound 1.5.

Scheme 2

Reagents and conditions: (a) DIAD, PPH ₃ , AcSH, THF, 0°C; (b) appropriate RHal, NaOMe, MeOH, -30°C to rt; (c) LiOH, THF, water, rt; (d) HCOOH, water, rt or 50°C.

5'-Chloro-5'-deoxyadenosine 3

[028] To a suspension of adenosine (2) (1.50 g, 5.61 mmol) in dry acetonitrile (6 ml) under an argon atmosphere was added thionyl chloride (1.2 ml, 16.5 mmol) dropwise cooling in the ice bath and keeping the temperature below 4°C. Pyridine (0.9 ml, 11.1 mmol) was added keeping temperature below 4°C and the mixture was stirred in the ice bath for 3 h, then the mixture was allowed to warm to room temperature and stirred 16 h. The precipitate was dissolved by adding water (15 ml) to the reaction mixture. The solution was neutralized to pH 5 by the slow addition of sodium bicarbonate. [029] The precipitate was collected by vacuum filtration, washed with cold water (2x5 ml), and dried in vacuo to give 1.36 g (77%) of the sulfinyladenosine intermediate. A suspension containing 1.36 g of the sulfinyladenosine in MeOH (15 ml) was treated with 1.5 ml of ammonium hydroxide. Precipitate was formed upon stirring at room temperature for 1 h, the precipitate was filtered, washed with cold MeOH/ammonium hydroxide solution (10:1, 2x5 ml), and dried under vacuum to give 1.11 g (69%) of the product 3 as a white solid.

[030] ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.34 (s, 1H), 8.15 (s, 1H), 7.29 (br s, 2H), 5.93 (d, J = 5.6 Hz, 1H), 5.60 (br d, 1H), 5.46 (br d, 1H), 4.75 (q, J = 5.2 Hz, 1H), 4.22 (d, J = 4.5 Hz, 1H), 4.26 - 4.18 (m, 1H), 4.14 - 4.05 (m, 1fH), 3.94 (dd, J = 11.6, 5.0 Hz, 1H), 3.84 (dd, J = 11.6, 6.3 Hz, 1H). Data are consistent with literature values.

[031] Synthesis of products 1.1-1.4, general method A. Exemplified by the synthesis of 3-

((((2S,3S,4R,5R )-5-(6-Amino-9H -purin-9-yl)-3,4-dihydroxytetrahydrofuran-2- yl)methyl)thio)benzoic acid 1.4

[032] A suspension of 5'-chloro-5'-deoxyadenosine 3 (70 mg, 0.25 mmol), 3 -mercaptobenzoic acid (42 mg, 0.27 mmol) and cesium carbonate (0.18 g, 0.55 mmol) in dry DMF (1.2 ml) were stirred in a closed vial under argon atmosphere for 5 h. The solvent was evaporated under reduced pressure. The residue was treated with water (5 ml), cooled in the ice bath and neutralized to pH 5 with 1 M HC1. The precipitate was filtered off, washed with water (2x2 ml), dried in vacuo. The residue was chromatographed on Cl 8 silica on Biotage, eluent acetonitrile in 0.1% HCOOH, gradient 5-80%, and then freeze-dried to give 30 mg (30%) of the product 1.4. as a white solid. HPLC purity 97%.

[033] NMR (400 MHz, DMSO-d ₆ ) δ 8.33 (s, 1H), 8.15 (s, 1H), 7.85 (t, J = 1.6 Hz, 1H), 7.73 (dt, J = 7.8, 1.3 Hz, 1H), 7.59 (ddd, J = 7.8, 1.9, 1.1 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.29 (s, 2H), 5.89 (d, J = 5.9 Hz, 1H), 5.53 (d, J = 6.1 Hz, 1H), 5.41 (d, J = 4.9 Hz, 1H), 4.82 (q, J = 5.6 Hz, 1H), 4.23 - 4.18 (m, 1H), 4.01 (ddd, J = 7.0, 5.9, 3.3 Hz, 1H), 3.53-3.30 (m, 2H, overlapped with water). ¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 166.9, 156.1, 152.7, 149.5, 140.0, 136.8, 132.3, 131.7, 129.4, 128.4, 126.7, 119.3, 87.6, 82.8, 72.8, 72.6, 35.1. HRMS (ESI/TOF-Q) m/z: [M + H] ⁺ calcd for C17H18N5O5S, 404.1029; found, 404.1036.

By the method analogues to method A the following compounds were obtained:

[034] 5 -Acetylthio-5 -deoxy- 2.3 -O-isopropylideneadenosine 5

Diisopropyl azodicarboxylate (0.6 ml, 3.05 mmol) was added drop wise over 5 min to an ice-cold solution of triphenylphosphine (0.80 g, 3.06 mmol) in dry THF (5 ml). After stirring for 30 min in the ice bath a thick suspension formed, 2 ,3 -O-isopropylideneadenosine 4 (0.47 g, 1.53 mmol) was added, and the stirring was continued for 10 min at 0°C. A solution of thioacetic acid (0.22 ml, 3.08 mmol) in dry THF (1 ml) was added dropwise to the resulting yellow suspension and stirring was continued for another 1 h at 0°C, then allowed to warm to room temperature. The solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel on Biotage, eluent EtOAc:EtOH 3: 1 in petroleum ether, gradient 20-80% to give 0.44 g (78%) of the product 5 as a white solid.

[035] NMR (300 MHz, CDCl ₃ ) δ 8.36 (s, 1H), 7.90 (s, 1H), 6.06 (d, J = 2.1 Hz, 1H), 5.72 (br s, 2H), 5.51 (dd, J = 6.4, 2.1 Hz, 1H), 4.97 (dd, J = 6.4, 3.1 Hz, 1H), 4.34 (td, J = 6.9, 3.1 Hz, 1H), 3.29 (dd, J = 13.8, 7.2 Hz, 1H), 3.18 (dd, J = 13.8, 6.6 Hz, 1H), 2.34 (s, 3H), 1.60 (s, 3H), 1.39 (s, 3H).

[036] Synthesis of intermediate 6, general method B. Exemplified bv the synthesis of 3- (((((3aS,4S,6R, 6aR)-6-(6-Amino-9H-purin-9-yl)-2.2-dimethvltetrahvdrofuro[3, 4-d][3,1dioxol-4- yl)methyl)thio)methyl)benzoic acid 6.1.

[037] To a solution of 5 -acetylthio-5 -deoxy-2 ,3 -O-isopropylideneadenosine (5) (0.11 g, 0.30 mmol) and methyl 3-(bromomethyl)benzoate (76 mg, 0.33 mmol) sodium methoxide solution in MeOH (5.4 M, 0.12 ml, 0.65 mmol) was added dropwise under argon atmosphere cooling reaction mixture in the dry ice-acetone bath (bath temperature -30°C). The reaction mixture was stirred at - 30°C for 1 h, then allowed to warm to room temperature and stirred for 3 h. The solvent was evaporated under reduced pressure. The residue was diluted with EtOAc (20 ml), washed with saturated NH4CI solution, water, dried over anhydrous Na ₂ SO ₄ and concentrated.

[038] The residue was purified by chromatography on silica gel on Biotage, eluent EtOAc:EtOH 3: 1 in petroleum ether, gradient 20-60% to give 90 mg of the product with impurities, which was used in the next step without additional purification. This residue was vigorously stirred with LiOH (14 mg, 0.58 mmol) in THF (2 ml) and water (1 ml) mixture at room temperature for 22 h. The reaction mixture was concentrated, diluted with water (3 ml) and acidified with IN HCl to pH 3, white precipitate was extracted with EtOAc (2x15 ml), washed with brine, dried over anhydrous Na ₂ SO ₄ and evaporated. The residue was washed with small volume of MeOH to give 70 mg (50% in 2 steps) of the product 6.1 as white solid.

[039] ¹ H NMR (400 MHz, Methanol-d ₄ ) δ 8.24 (s, 1H), 8.15 (s, 1H), 7.92 (t, J = 1.5 Hz, 1H), 7.83 (dt, J = 7.7, 1.4 Hz, 1H), 7.37 (dt, J = 7.7, 1.6 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 6.12 (d, J = 2.4 Hz, 1H), 5.42 (dd, J = 6.4, 2.4 Hz, 1H), 4.94 (dd, J = 6.4, 3.2 Hz, 1H), 4.27 (td, J = 6.7, 3.2 Hz, 1H), 3.73 (s, 2H), 2.73 (dd, J = 13.8, 6.8 Hz, 1H), 2.66 (dd, J = 13.8, 6.6 Hz, 1H), 1.54 (s, 3H), 1.33 (s, 3H). ¹³ C NMR (100 MHz, Methanol-d ₄ ) δ 169.6, 156.7, 152.9, 150.0, 142.2, 140.3, 134.5, 132.2, 131.1, 129.5, 129.4, 120.6, 115.5, 91.6, 88.2, 85.2, 85.1, 36.8, 34.5, 27.4, 25.5. HRMS (ESI/TOF-Q) m/z: [M + H] ⁺ calcd for C22H24N5O5S, 458.1493; found, 458.1500.

[040] Synthesis of product 1.5, general method C. 3-(((((2S,3S,4R,5R)-5-(6-Amino-9H-purin-

9-yl)-3.4-dihvdroxvtetrahvdrofuran-2-yl)methyl)thio)methy l)benzoic acid 1.5

Acetonide 6.1 (70 mg, 0.15 mmol) was dissolved in ice-cold HCOOH solution in water (50%, 1 ml). The reaction mixture was stirred at 50°C for 20 h. The solvent was evaporated, then the residue was co-evaporated with EtOH to remove HCOOH. The residue was washed with small amount of hot MeOH, then with acetonitrile, dried in vacuo to give 46 mg (72%) of the product 1.5 as white solid. HPLC purity 91%.

[041] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.95 (br zs, 1H), 8.33 (s, 1H), 8.12 (s, 1H), 7.89 (t, J = 1.5 Hz, 1H), 7.80 (dt, J = 7.7, 1.5 Hz, 1H), 7.46 (dt, J = 7.7, 1.5 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.28 (s, 2H), 5.87 (d, J = 5.6 Hz, 1H), 5.48 (d, J = 6.0 Hz, 1H), 5.30 (d, J = 5.1 Hz, 1H), 4.73 (q, J = 5.6 Hz, 1H), 4.18 -4.13 (m, 1H), 4.01 (td, J = 6.4, 3.9 Hz, 1H), 3.82 (s, 2H), 2.84 (dd, J = 13.9, 5.9 Hz, 1H), 2.70 (dd, J = 13.9, 6.9 Hz, 1H). ¹³ CNMR(100MHz, DMSO-d ₆ ) δ 167.2, 156.1, 152.7, 149.4, 140.0, 139.2, 133.3, 131.0, 129.7, 128.6, 127.8, 119.2, 87.6, 83.6, 72.6, 72.5, 35.2, 33.4. HRMS (ESI/TOF-Q) m/z: [M + H] ⁺ calcd for C18H20N5O5S, 418.1180; found, 418.1189.

In vitro Assay

[042] The compounds have been tested for potential antiviral activity in vitro as Nspl4 and Nspl6 inhibitors according to the following process.

[043] Methyltransferase activity was determined with an EPIgeneous Methyltransferase Assay kit by assaying the conversion of SAM to SAH according to the manufacturer’s directions.

[044] The enzymatic reaction was performed in white ProxiPlate-384 Plus with a final volume of 10 ml. The reaction buffer is composed of 40 mM Tris-HCl pH 8.3 (pH 8.0 for nsp 16) and 100mM NaCl (or 10mM KCl only for nspl6), 1 mM DTT, 2mM MgCh, 0.01% Tween20. 4 ml purified recombinant protein Nspl4 at 0.4 mM or Nsp 16 at 3mM final concentration were added in the assay wells, containing previously dispensed inhibitors. The reaction was started with 4 ml of a mix containing both 4 mM GpppG (Jena Bioscience, cat.nr. NU854) or ~5 pM m7GpppA-RNA (for Nsp 16) and SAM at 10 mM final concentrations and incubated at 37°C for 20 min (2h for Nsp 16).

[045] All compounds suspended in 100% DMSO (0.1 % final DMSO) and were tested at 100 μM in duplicate. ICso values were determined for compounds with a higher than 50% inhibitory effect [046] For the detection of released SAH, 2 ml of EPIgeneous detection buffer one was then added in order to stop the reaction. After 10 min of incubation at room temperature, detection reagents were added: 4 ml of a 1/16 dilution of SAH-d2 conjugate in a first place then 4 ml of Anti-SAH- Lumi4-Tb at a 1/100 dilution. HTRF signals were measured using a Hidex Sense (Finland) with an excitation filter at 337 nm and fluorescence wavelength measurements at 620 and 665 nm, an integration delay of 150 ms, and an integration time of 400 ms.

[047] Results were analysed with a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)] *10 ⁴ (HTRF Ratio).

Calculate the mean HTRF Ratio for each sample:

Mean HTRF Ratio = Mean Sample HTRF Ratio - Blank HTRF Ratio, where ‘blank’ is the signal with compound (or DMSO in control sample) and Anti-SAH-Lumi4-Tb.

[048] Percent inhibition calculated using the following the formula for each inhibitor dilution: % Inhibition = 100-(max signal _comp - min signal _comp ) *100 / (max signal control - min signal control), where ‘max signal’ is the signal ratio without protein (negative control) and ‘min signal’ the signal ratio in sample. The ICso value was calculated using the program Graph Pad Prism® 8.0.

[049] Table 1 Biological activity of adenosylmercaptane derivatives