BENZOTHIOPHENE, THIENOPYRIDINE AND THIENOPYRIMIDINE DERIVATIVES FOR THE MODULATION OF STING

Abstract

A compound of formula (I): wherein: Y is (CH2)n, where n is from 2 to 4; W1 and W11 are independently selected from OH and ORP, where RP is Me or Et.

##STR00001##

Claims

1. A compound of formula I: ##STR00059## wherein: Y is (CH.sub.2).sub.n, where n is from 2 to 4; W.sup.1 and W.sup.11 are independently selected from OH and OR.sup.P, where R.sup.P is Me or Et; A.sup.1 is CR.sup.A or N; A.sup.2 is CR.sup.B or N; A.sup.3 is CR.sup.C or N; A.sup.4 is CR.sup.D or N; where no more than two of A.sup.1, A.sup.2, A.sup.3, and A.sup.4 may be N; one or two of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, OEt, CH.sub.2OH, CH.sub.2OMe and OH; the remainder of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are H; A.sup.11 is CR.sup.AA or N; A.sup.12 is CR.sup.BB or N; A.sup.13 is CR.sup.CC or N; A.sup.14 is CR.sup.DD or N; where no more than two of A.sup.11, A.sup.12, A.sup.13 and A.sup.14 may be N; one or two of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, OEt, CH.sub.2OH, CH.sub.2OMe and OH; the remainder of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD, (if present) are H; R.sup.C1, R.sup.C3 and R.sup.C4 are independently selected from H, Cl, F, Br, Me, OMe, OEt, cyano, CF.sub.3, CH.sub.2OH, CH.sub.2OMe, C.sub.2-4 alkenyl and C.sub.5heterocyclyl; R.sup.C11, R.sup.C13 and R.sup.C14 are independently selected from H, Cl, F, Br, Me, OMe, OEt, cyano, CF.sub.3, CH.sub.2OH, CH.sub.2OMe, C.sub.2-4alkenyl and C.sub.5heterocyclyl.

2. A compound according to claim 1, wherein A.sup.11=A.sup.1, A.sup.12=A.sup.2, A.sup.13=A.sup.3, A.sup.14=A.sup.4, R.sup.C11=R.sup.C1, R.sup.C13=R.sup.C3, R.sup.C14=R.sup.C4, W.sup.11=W.sup.1.

3. A compound according to claim 1 or 2, wherein A.sup.1 is CR.sup.A, A.sup.2 is CR.sup.B, A.sup.3 is CR.sup.C, and A.sup.4 is CR.sup.D.

4. A compound according to any one of claims 1 to 3, wherein A.sup.11 is CR.sup.AA, A.sup.12 is CR.sup.BB, A.sup.13 is CR.sup.CC, and A.sup.14 is CR.sup.DD.

5. A compound according to any one of claims 1 to 3 wherein the compound is selected from formulae IIIb, IIIc, IIId and IIIe: ##STR00060##

6. A compound according to any one of claims 1 to 5, wherein: R.sup.A (if present) is selected from Cl, Br and OMe; R.sup.B (if present) is H; R.sup.C (if present) is H; R.sup.D (if present) is selected from H, F, Br, Me and OMe.

7. A compound according to any one of claims 1 to 4, wherein A.sup.1, A.sup.2, A.sup.3 and A.sup.4 are selected from combinations 1-10: TABLE-US-00010 Combination A.sup.1 A.sup.2 A.sup.3 A.sup.4 1 CCl CH CH CH 2 CCl CH CH CCH.sub.3 3 CCl CH CH CBr 4 CBr CH CH CH 5 CCl CH CH CF 6 CCl CH CH C—OCH.sub.3 7 CBr CH CH CF 8 C—OCH.sub.3 CH CH CH 9 CCF.sub.3 H H H 10 CCH.sub.3 H H H

8. A compound according to any one of claims 1 to 7, wherein: R.sup.AA (if present) is selected from Cl, Br and OMe; R.sup.BB (if present) is H; R.sup.CC (if present) is H; R.sup.DD (if present) is selected from H, F, Br, Me and OMe.

9. A compound according to any one of claims 1 to 4, wherein A.sup.11, A.sup.12, A.sup.13 and A.sup.14 are selected from combinations 1-10: TABLE-US-00011 Combination A.sup.11 A.sup.12 A.sup.13 A.sup.14 1 CCl CH CH CH 2 CCl CH CH CCH.sub.3 3 CCl CH CH CBr 4 CBr CH CH CH 5 CCl CH CH CF 6 CCl CH CH C—OCH.sub.3 7 CBr CH CH CF 8 C—OCH.sub.3 CH CH CH 9 CCF.sub.3 H H H 10 CCH.sub.3 H H H

10. A compound according to any one of claims 1 to 9, wherein R.sup.C11=R.sup.C1, R.sup.C13=R.sup.C3 and R.sup.C14=R.sup.C4.

11. A compound according to claim 10, wherein A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.11, A.sup.12, A.sup.13 and A.sup.14 are selected from combinations 1, 4, 9 and 10: TABLE-US-00012 Combination A.sup.1/A.sup.11 A.sup.2/A.sup.12 A.sup.3/A.sup.13 A.sup.4/A.sup.14 1 CCl CH CH CH 4 CBr CH CH CH 9 CCF.sub.3 H H H 10 CCH.sub.3 H H H

12. A compound according to any one of claims 1 to 10, wherein R.sup.C1, R.sup.C3 and R.sup.C4 are selected from combinations 1-3: TABLE-US-00013 Combination R.sup.C1 R.sup.C3 R.sup.C4 1 H H H 2 H H F 3 OMe H H

13. A compound according to any one of claims 1 to 12, wherein R.sup.C11, R.sup.C13 and R.sup.C14 are selected from combinations 1-3: TABLE-US-00014 Combination R.sup.C11 R.sup.C13 R.sup.C14 1 H H H 2 H H F 3 OMe H H

14. A compound according to any one of claims 1 to 13, wherein Y is (CH.sub.2).sub.n, where n is from 2 to 3.

15. A compound according to any one of claims 1 to 13, wherein Y is (CH.sub.2).sub.2.

16. A compound according to any one of claims 1 to 15, wherein W.sup.1 and W.sup.11 are OH.

17. A compound as defined in any one of claims 1 to 16, for use in a method of therapy.

18. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 16, and a pharmaceutically acceptable excipient.

19. A method of treatment or prevention of a disease ameliorated by the modulation of STING, comprising administering to a patient in need of treatment, a compound as defined in any one of claims 1 to 16, or a pharmaceutical composition according to claim 18.

20. The use of a compound as defined in any one of claims 1 to 16, in the manufacture of a medicament for treating or preventing disease ameliorated by the modulation of STING.

21. A compound as defined in any one of claims 1 to 16, or pharmaceutical composition according to claim 18 for use in the treatment or preventing of disease ameliorated by the modulation of STING.

Description

EXAMPLES

[0370] The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

[0371] Acronyms

[0372] For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), n-butyl (nBu), tert-butyl (tBu), phenyl (Ph), benzyl (Bn), methoxy (MeO), ethoxy (EtO), trimethylsilyl (TMS), and acetyl (Ac).

[0373] For convenience, many chemical compounds are represented using well known abbreviations, including but not limited to, methanol (MeOH), deuterated methanol (MeOD-d.sub.4 or CD.sub.3OD) ethanol (EtOH), isopropanol (i-PrOH), ether or diethyl ether (Et.sub.2O), ethyl acetate (EtOAc), acetic acid (AcOH), acetonitrile (MeCN or ACN), dichloromethane (methylene chloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), deuterated chloroform (CDCl.sub.3), diethylamine (DEA), deuterated dimethylsulfoxide (DMSO-d.sub.6), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl.HCl), meta-chloroperoxybenzoic acid (mCPBA), 1,1′-bis(diphenylphosphino)ferrocene (dppf), tert-butyloxycarbonyl (Boc, BOC), 2-(trimethylsilyl)ethoxymethyl (SEM), triethylamine (Et.sub.3N or TEA), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIPEA or DIEA), 1,1′-bis(diphenylphosphino)ferrocene dichloropalladium (II) (PdCl.sub.2(dppf)), trans-dichlorobis(triphenylphosphine)palladium(II) (PdCl.sub.2(PPh.sub.3).sub.2), tris(dibenzylideneacetone) dipalladium(0) (Pd.sub.2(dba).sub.3), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh.sub.3).sub.4), propylphosphonic anhydride (T3P), hexamethylphosphoramide (HMPA), 1,2-dichloroethane (DCE), chromium(VI) oxide (CrO.sub.3), n-bromosuccinimide (NBS), potassium hydroxide (KOH), benzoyl peroxide (BPO), carbon tetrachloride (CCl.sub.4), petroleum ether (Pet. Ether), potassium carbonate (K.sub.2CO.sub.3), sodium sulfate (Na.sub.2SO.sub.4), lithium diisopropylamine (LDA), azobisisobutyronitrile (AlBN), N-methylmorpholine N-oxide (NMO), benzoyl peroxide (BPO) and 1-hydroxybenzotriazole (HOBt).

[0374] Other abbreviations: thin layer chromatography (TLC), retention time (rt).

[0375] General Experimental Details

[0376] Unless otherwise stated the following generalizations apply. .sup.1H NMR spectra were recorded on a Bruker AVANCE III (400 MHz). The multiplicity of a signal is designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; tt, triplet of triplets; td, triplet of doublets; ddd, doublet of doublet of doublets br, broad; m, multiplet. All observed coupling constants, J, are reported in Hertz. Exchangeable protons are not always observed.

[0377] LCMS data was generated using the conditions described below. Chlorine isotopes are reported as .sup.35Cl, Bromine isotopes are reported as either .sup.79Br or .sup.81Br or both .sup.79Br/.sup.81Br.

[0378] LC-MS Method A (LCMS-A):

[0379] Equipment Information

[0380] LC model: Agilent 1200

[0381] (Pump type: Binary Pump, Detector type: DAD)

[0382] MS model: Agilent G6110A Quadrupole

[0383] Parameters of LCMS

[0384] LC: Column: Xbridge-C18, 2.5 μm, 2.1×30 mm

[0385] Column temperature: 30° C.

[0386] Acquisition of wavelength: 214 nm, 254 nm

[0387] Mobile phase: A: 0.07% HCOOH aqueous solution, B: MeOH

[0388] MS: Ion source: ES+ (or ES−) MS range: 50˜900 m/z

[0389] Fragmentor: 60 Drying gas flow: 10 L/min

[0390] Nebulizer pressure: 35 psi Drying gas temperature: 350° C.

[0391] Vcap: 3.5 kV

[0392] Gradient Table:

TABLE-US-00002 Flow (mL/min) T (min) A (%) B (%) 0.5 0.0 70 30 0.5 0.2 70 30 0.5 1.8 5 95 0.5 2.4 5 95 0.5 2.6 70 30 0.5 3.5 70 30

[0393] Sample Preparation

[0394] The sample was dissolved in methanol, the concentration about 0.11˜1 mg/mL, then filtered through syringe filter with 0.22 μm. (Injection volume: 1˜10 μL)

[0395] LC-MS Method B (LCMS-B):

[0396] Equipment Information

[0397] LC model: Agilent 1200 (Pump type: Binary Pump, Detector type: DAD)

[0398] MS model: Agilent G6110A Quadrupole

[0399] Parameters of LCMS

[0400] LC: Column: Xbridge-C18, 2.5 μm, 2.1×30 mm

[0401] Column temperature: 30° C.

[0402] Acquisition of wavelength: 214 nm, 254 nm

[0403] Mobile phase: A: 0.07% HCOOH aqueous solution, B: MeOH

[0404] MS: Ion source: ES+ (or ES−) MS range: 50˜900 m/z

[0405] Fragmentor: 60 Drying gas flow: 10 L/min

[0406] Nebulizer pressure: 35 psi Drying gas temperature: 350° C.

[0407] Vcap: 3.5 kV

[0408] Gradient Table:

TABLE-US-00003 Flow (mL/min) T (min) A (%) B (%) 0.5 0.0 70 30 0.5 0.3 70 30 0.5 0.6 50 50 0.5 0.9 40 60 0.5 1.2 30 70 0.5 3.2 5 95 0.5 3.5 5 95 0.5 4.0 70 30 0.5 5.0 70 30

[0409] Sample Preparation

[0410] The sample was dissolved in methanol, the concentration about 0.11˜1 mg/mL, then filtered through the syringe filter with 0.22 μm. (Injection volume: 1˜10 μL)

[0411] LC-MS Method C (LCMS-C):

[0412] Equipment Information

[0413] LC model: Waters 2695 alliance (Pumptype: Quaternary Pump, Detector: 2996 Photodiode Array Detector)

[0414] MS model: Micromass ZQ

[0415] Parameters of LCMS

[0416] LC: Column: Xbridge-C18, 3.5 μm, 2.1×50 mm

[0417] Column temperature: 30° C.

[0418] Acquisition of wavelength: 214 nm, 254 nm

[0419] Mobile phase: A: 0.07% HCOOH aqueous solution, B: MeOH

[0420] MS: Ion source: ES+ (or ES−) MS range: 50˜900 m/z

[0421] Capillary: 3 kV Cone: 3 V Extractor: 3 V

[0422] Drying gas flow: 600 L/hr Cone: 50 L/hr

[0423] Desolvation temperature: 300° C.

[0424] Source temperature: 100° C.

[0425] Gradient Table:

TABLE-US-00004 Flow (mL/min) T (min) A (%) B (%) 0.3 0.0 80 20 0.3 0.5 80 20 0.3 0.8 50 50 0.3 1.2 35 65 0.3 2.0 20 80 0.3 4.0 5 95 0.3 5.0 5 95 0.3 5.8 15 85 0.3 6.2 80 20 0.3 8.0 80 20

[0426] Sample Preparation

[0427] The sample was dissolved in methanol, the concentration about 0.11˜1 mg/mL, then filtered through the syringe filter with 0.22 μm. (Injection volume: 1˜10 μL)

[0428] Preparative RP-HPLC:

[0429] Instrument type: Varian 940-LC series;

[0430] Pump type: Quaternary Pump;

[0431] Detector type: Diode Array Detector

[0432] HPLC conditions: Waters Sunfire prep C18 OBD, 5 μm 19×100 mm column, eluting with a gradient of MeOH in water with 0.07% TFA at a flow rate of 15 mL/min. Acquisition wavelength 214 nm, 254 nm.

[0433] Analytical thin-layer chromatography was performed on Merck silica gel 60 F254 aluminium-backed plates which were visualized using fluorescence quenching under UV light or a basic KMnO.sub.4 dip or Ninhydrin dip.

[0434] Preparative thin-layer chromatography (prep TLC) was performed using Tklst (China), grand grade: (HPTLC): 8±2 μm>80%; (TLC): 10-40 μm. Type: GF254. Compounds were visualized by UV (254 nm).

[0435] Column chromatography was performed using Tklst (China), grand grade, 100-200 meshes silica gel.

[0436] Microwave irradiation was achieved using a CEM Explorer SP Microwave Reactor.

[0437] Where necessary, anhydrous solvents were purchased from Sigma-Aldrich or dried using conventional methods. Solutions of inorganic acids or bases were made up as aqueous solutions unless stated otherwise.

[0438] Additional Cartridges used are as follows:

[0439] Phase Separator:

[0440] Manufacturer: Biotage

[0441] Product: ISOLUTE® Phase Separator (3 mL unless otherwise stated)

[0442] SCX and SCX-2 Cartridges:

[0443] Manufacturer: Biotage

[0444] Product: ISOLUTE® SCX 1 g, (6 mL SPE Column unless otherwise stated)

[0445] Manufacturer: Biotage

[0446] Product: ISOLUTE® SCX-2 1 g (6 mL Column)

[0447] Manufacturer: Silicycle

[0448] Product: SCX-2 500 mg or 5 g

[0449] Manufacturer: Agilent

[0450] Product: Bond Elut® SCX 10 g

[0451] Sample Extraction Cartridge:

[0452] Manufacturer: Waters

[0453] Product: Oasis® HLB 35 cc (6 g) LP extraction cartridge

[0454] Solutions of hydrogen chloride, sodium hydroxide, potassium carbonate and sodium bicarbonate are aqueous, unless otherwise stated.

[0455] Intermediate Preparations

(i) 4,4′-(Ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4)

[0456] ##STR00021##

(a) tert-Butyl (2((4-carbamoyl-2-nitrophenyl)amino)ethyl)carbamate (I1)

[0457] A suspension of 4-chloro-3-nitrobenzamide (8.0 g, 39.9 mmol), tert-butyl (2-aminoethyl)carbamate (6.40 g, 39.9 mmol) and Et.sub.3N (8.1 g, 79.8 mmol) in NMP (80 mL) was heated at 150° C. under N.sub.2 overnight. Water (500 mL) was added followed by Pet. Ether/EtOAc (5:1, 300 mL) and the resulting precipitate was collected by filtration to give the title compound I1 (12.0 g, 92%) as a yellow solid. LCMS-B: rt 3.2 min, m/z 347.1 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.65 (s, 1H), 8.44 (t, J=5.8 Hz, 1H), 8.05-7.92 (m, 2H), 7.27 (s, 1H), 7.14 (d, J=9.1 Hz, 1H), 7.04 (t, J=5.8 Hz, 1H), 3.46-3.45 (m, 2H), 3.20-3.19 (m, 2H), 1.35 (s, 9H).

(b) 4((2-Aminoethyl)amino)-3-nitrobenzamide hydrochloride (I2)

[0458] A mixture of tert-butyl (2-((4-carbamoyl-2-nitrophenyl)amino)ethyl)carbamate (I1) (12.0 g, 37.0 mmol) and a 5.5 M HCl in dioxane solution (200 mL) was stirred at room temperature overnight. The solvent was then removed under reduced pressure to give the title compound I2 (8.0 g, 96%) as a yellow solid. LCMS-B: rt 1.2 min, m/z 225.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.65 (d, J=2.1 Hz, 1H), 8.42 (s, 1H), 8.25 (s, 2H), 8.05 (dd, J=9.0, 2.2 Hz, 1H), 7.25 (d, J=9.0 Hz, 1H), 5.62 (br s, 3H), 3.76-3.74 (m, 2H), 3.01-2.97 (m, 2H).

[0459] A suspension of 4-((2-aminoethyl)amino)-3-nitrobenzamide hydrochloride (I2) (7.6 g, 33.9 mmol), 4-chloro-3-nitrobenzamide (6.80 g, 33.9 mmol) and Et.sub.3N (20.6 g, 203 mmol) in NMP (80 mL) was heated at 150° C. under N.sub.2 overnight. Additional 4-((2-aminoethyl)amino)-3-nitrobenzamide hydrochloride (I2) (800 mg, 3.57 mmol) and Et.sub.3N (3.0 g, 29.6 mmol) were added and the mixture was heated at 150° C. under N.sub.2 overnight. The mixture was allowed to cool to room temperature, diluted with Pet. Ether/EtOAc (1:1, v/v, 500 mL) and the resulting precipitate was collected by filtration to give the title compound I3 (12.0 g, 91%) as a yellow solid. LCMS-B: rt 1.3 min, m/z 389.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.62 (s, 4H), 7.99-7.95 (m, 4H), 7.30-7.28 (m, 4H), 3.70-3.64 (m, 4H).

(d) 4,4′-(Ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4)

[0460] A suspension of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-nitrobenzamide) (I3) (12 g, 30.9 mmol), and 10% Pd/C (2.0 g) in DMF (200 mL) was heated at 100° C. under a H.sub.2 atmosphere overnight. The mixture was filtered and the filtrate was concentrated under reduced pressure to give the title compound I4 (9.0 g, 90%) as a black solid. LCMS-B: rt 0.3 min, m/z 329.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.44 (s, 2H), 7.13 (d, J=8.2 Hz, 2H), 7.11 (s, 2H), 6.74 (s, 2H), 6.46 (d, J=8.0 Hz, 2H), 5.05 (s, 2H), 4.58 (s, 4H), 3.41-3.36 (m, 4H).

(ii) 4,4′-(Propane-1,3-diylbis(azanediyl))bis(3-aminobenzamide) (I8)

[0461] ##STR00022##

(a) tert-Butyl (3-((4-carbamoyl-2-nitrophenyl)amino)propyl)carbamate (I5)

[0462] To a solution of 4-chloro-3-nitrobenzamide (2 g, 10 mmol) in NMP (30 mL) was added tert-butyl (3-aminopropyl)carbamate (1.7 g, 9.97 mmol) and Et.sub.3N (2.1 g, 19.9 mmol) and the mixture was heated at 150° C. overnight. Water was added and the resulting mixture was extracted with EtOAc (150 mL×3). The combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=100:1 to 50:1) to give the title compound I5 (2.1 g, 62%) as a yellow solid. LCMS-B: rt 3.42 min, m/z 361.1 [M+Na].sup.+.

(b) 4-((3-Aminopropyl)amino)-3-nitrobenzamide hydrochloride (I6)

[0463] A mixture of tert-butyl (3-((4-carbamoyl-2-nitrophenyl)amino)propyl)carbamate (I5) (2.1 g, 6.2 mmol) and a 6 M solution of HCl in dioxane (40 mL) was stirred at room temperature overnight. The solvent was then removed under reduced pressure to give the title compound I6 (1.5 g, 88%) as a yellow solid. LCMS-B: rt 0.29 min, m/z 239.3 [M+H].sup.+.

[0464] To a mixture of 4-((3-aminopropyl)amino)-3-nitrobenzamide hydrochloride (I6) (2.0 g, 7.31 mmol) in NMP (50 mL) was added 4-chloro-3-nitrobenzamide (1.5 g, 7.31 mmol) and Et.sub.3N (2.2 g, 21.9 mmol) and the mixture was heated at 150° C. overnight. Water was added and the mixture was extracted with EtOAc (300 mL×3). The combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=100:1 to 50:1) to give the title compound I7 (1.3 g, 45%) as a black solid. LCMS-B: rt 2.98 min, m/z 403.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.65 (s, 2H), 8.60-8.48 (m, 2H), 7.99 (s, 4H), 7.29 (s, 2H), 7.12 (d, J=9.1 Hz, 2H), 3.62-3.48 (m, 4H), 2.01-1.88 (m, 2H).

(d) 4,4′-(Propane-1,3-diylbis(azanediyl))bis(3-aminobenzamide) (I8)

[0465] To a solution of 4,4′-(propane-1,3-diylbis(azanediyl))bis(3-nitrobenzamide) (I7) (1 g, 2.5 mmol) in DMF (30 mL) was added 10% Pd/C (100 mg) and the mixture was heated at 90° C. under a H.sub.2 atmosphere overnight. The mixture was filtered and the filtrate was concentrated under reduced pressure to give the title compound I8 (777 mg, 91%) as a black solid. LCMS-B: rt 0.27 min, m/z 343.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.41 (s, 2H), 7.17-6.96 (m, 4H), 6.69 (s, 2H), 6.48-6.29 (m, 2H), 5.22-4.12 (m, 6H), 3.27-3.12 (m, 4H), 1.99-1.87 (m, 2H).

(iii) 4,4′-(Butane-1,4-diylbis(azanediyl))bis(3-aminobenzamide) (I12)

[0466] ##STR00023##

(a) tert-Butyl (4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate (I9)

[0467] To a solution of 4-chloro-3-nitrobenzamide (2.0 g, 10.0 mmol) in NMP (20 mL) was added tert-butyl (4-aminobutyl)carbamate (2.24 g, 10.0 mmol) and Et.sub.3N (2.02 g, 20.0 mmol) and the mixture was heated at 150° C. overnight. The mixture was partitioned between water (100 mL) and EtOAc (100 mL), the layers were separated and the aqueous layer was extracted with EtOAc (100 mL×3). The combined organic layers were washed with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=100:1 to 40:1) to give the title compound I9 (2.0 g, 57%) as a yellow solid. LCMS-B: rt 3.44 min, m/z 375.1 [M+Na].sup.+.

(b) 4-((4-Aminobutyl)amino)-3-nitrobenzamide hydrochloride (I10)

[0468] A mixture of tert-butyl (4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate (I9) (2.27 g, 6.44 mmol) and a 6 M HCl in dioxane solution (30 mL) was stirred at room temperature for 16 h. The solvent was removed under reduced pressure to give the title compound I10 (1.61 g, 100%) as a yellow solid. LCMS-B: rt 3.47 min, m/z 253.1 [M+H].sup.+.

[0469] To a solution of 4-((4-aminobutyl)amino)-3-nitrobenzamide hydrochloride (I10) (1.50 g, 5.95 mmol) in NMP (50 mL) was added 4-chloro-3-nitrobenzamide (1.19 g, 5.95 mmol) and Et.sub.3N (1.81 g, 17.85 mmol) and the mixture was heated at 150° C. for 16 h. The mixture was partitioned between water (200 mL) and EtOAc (200 mL) which resulted in a precipitate forming. The precipitate was collected by filtration to give the title compound I11 (1.22 g, 49%) as a black solid. LCMS-B: rt 3.12 min, m/z 417.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.64 (s, 2H), δ 8.41 (s, 2H), 8.10-7.88 (m, 4H), 7.27 (s, 2H), 7.12 (d, J=8.7 Hz, 2H), 3.55-3.40 (m, 4H), 1.73 (s, 4H).

(d) 4,4′-(Butane-1,4-diylbis(azanediyl))bis(3-aminobenzamide) (I12)

[0470] To a solution of 4,4′-(butane-1,4-diylbis(azanediyl))bis(3-nitrobenzamide) (I11) (1.0 g, 2.40 mmol) in DMF (20 mL) was added 10% Pd/C (100 mg) and the mixture was heated at 90° C. under a H.sub.2 atmosphere for 16 h. The mixture was filtered and the filtrate was concentrated under reduced pressure to give the title compound I12 (842 mg, 98%) as a black solid. LCMS-B: rt 0.32 min, m/z 357.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.41 (s, 2H), 7.16-7.04 (m, 4H), 6.71 (s, 2H), 6.39 (d, J=8.2 Hz, 2H), 4.92 (s, 2H), 4.60 (s, 4H), 3.13 (s, 4H), 1.73 (s, 4H).

(iv) Ethyl 3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I16)

[0471] ##STR00024##

(a) Ethyl 4-hydroxy-3-methylbenzo[b]thiophene-2-carboxylate (I13)

[0472] To a solution of 1-(2-fluoro-6-hydroxyphenyl)ethan-1-one (2.0 g, 3.24 mmol) in DMF (20 mL) was added ethyl 2-mercaptoacetate (2.4 g, 4.86 mmol) and K.sub.2CO.sub.3 (3.6 g, 26.1 mmol) and the mixture was heated at 100° C. under nitrogen overnight. Water (100 mL) was added and the mixture was extracted with EtOAc (100 mL×3). The combined organic extracts were washed with water (100 mL×3), brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=1:0 to 10:1) to give the title compound I13 (1.8 g, 60%) as a grey solid. LCMS-A: rt 2.64 min; m/z 259.0 [M+Na].sup.+.

(b) Ethyl 4-methoxy-3-methylbenzo[b]thiophene-2-carboxylate (I14)

[0473] To a solution of ethyl 4-hydroxy-3-methylbenzo[b]thiophene-2-carboxylate (I13) (350 mg, 1.48 mmol) and methyl iodide (2.1 g, 14.8 mmol) in DMF (8 mL) was added K.sub.2CO.sub.3 (613 mg, 4.44 mmol) and the mixture was stirred at room temperature in a sealed tube overnight. Water was added and the mixture was extracted with EtOAc. The combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=10:1) to give the title compound I14 (380 mg, 100%). LCMS-A: rt 2.65 min; m/z 251.0 [M+H].sup.+.

[0474] To a suspension of ethyl 4-methoxy-3-methylbenzo[b]thiophene-2-carboxylate (I14) (380 mg, 1.48 mmol) and NBS (268 mg, 1.50 mmol) in CCl.sub.4 (20 mL) was added BPO (92 mg, 0.38 mmol) and the mixture was heated at reflux overnight. Water was added and the mixture was extracted with EtOAc. The organic extract was washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=10/1) to give the title compound (360 mg, 76%). LCMS-A: rt 2.73 min; m/z 350.8/352.9 [M+Na].sup.+.

(d) Ethyl 3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I16)

[0475] To a solution of ethyl 3-(bromomethyl)-4-methoxybenzo[b]thiophene-2-carboxylate (I15) (1.0 g, 3.0 mmol) in THF (30 mL) was added NMO (1.41 g, 12.0 mmol) and the mixture was heated at reflux overnight. The mixture was allowed to cool to room temperature, poured into water (50 mL) and extracted with EtOAc (50 mL×3). The combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=50:1 to 10:1) to give the title compound (330 mg, 42%) as a yellow solid. LCMS-B: rt 4.0 min; m/z 265.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.6 (s, 1H), 7.67 (dd, J=8.2, 0.8 Hz, 1H), 7.55 (t, J=8.1 Hz, 1H), 7.05 (d, J=8.0 Hz, 1H), 4.32 (q, J=7.1 Hz, 2H), 3.90 (s, 3H), 1.30 (t, J=7.1 Hz, 3H).

(v) Ethyl 4-chloro-3-formylbenzo[b]thiophene-2-carboxylate (I19)

[0476] ##STR00025##

(a) Ethyl 4-chloro-3-methylbenzo[b]thiophene-2-carboxylate (I17)

[0477] A solution of 1-(2-chloro-6-fluorophenyl)ethanone (25.0 g, 145 mmol), ethyl 2-mercaptoacetate (17.4 g, 145 mmol) and K.sub.2CO.sub.3 (30.0 g, 217 mmol) in DMF (200 mL) was heated at 100° C. overnight. The mixture was allowed to cool to room temperature then poured into water (1.0 L) and EtOAc (500 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (500 mL×3). The combined organic extracts were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by re-crystallization from Pet. Ether/EtOAc to give the title compound I17 (28.0 g, 76%) as a yellow solid. LCMS-A: rt 2.82 min; m/z 255.0 [M+H].sup.+.

(b) Ethyl 3-(bromomethyl)-4-chlorobenzo[b]thiophene-2-carboxylate (I18)

[0478] A suspension of ethyl 4-chloro-3-methylbenzo[b]thiophene-2-carboxylate (I17) (28.0 g, 0.11 mol), NBS (19.6 g, 0.11 mol) and BPO (2.67 g, 0.011 mol) in CCl.sub.4 (200 mL) was heated at 115° C. for 1.5 h. The mixture was allowed to cool to room temperature then concentrated under reduced pressure and the residue was purified by re-crystallization from DCM/Pet. Ether to give the title compound I18 (26.0 g, 71%) as a purple solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.74 (dd, J=8.0, 1.2 Hz, 1H), 7.46 (dd, J=7.6, 0.8 Hz, 1H), 7.40 (t, J=8.0 Hz, 1H), 5.61 (s, 2H), 4.44 (q, J=7.1 Hz 2H), 1.44 (t, J=7.1 Hz, 3H).

[0479] A mixture of ethyl 3-(bromomethyl)-4-chlorobenzo[b]thiophene-2-carboxylate (I18) (10.00 g, 29.97 mmol) and NMO (14.00 g, 119.88 mmol) in THF (250 mL) was heated at reflux for 16 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 50:1) to give the title compound I19 (2.70 g, 34%) as a white solid. LCMS-B: rt 4.16 min, m/z 269.0 [M+H].sup.+, 290.9 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.7 (s, 1H), 8.17 (dd, J=7.6, 1.5 Hz, 1H), 7.66-7.58 (m, 2H), 4.36 (q, J=7.1 Hz, 2H), 1.32 (t, J=7.1 Hz, 3H).

(vi) Ethyl 4-chloro-7-fluoro-3-formylbenzo[b]thiophene-2-carboxylate (I24)

[0480] ##STR00026##

(a) 1-(6-Chloro-2,3-difluorophenyl)ethan-1-ol (I20)

[0481] To a solution of diisopropylamine (4.4 g, 43.9 mmol) in dry THF (100 mL) at −78° C. under nitrogen was added n-BuLi (2.5 M solution in n-hexane, 17.6 mL, 43.9 mmol) dropwise followed by 4-chloro-1,2-difluorobenzene (5.0 g, 33.8 mmol) and the mixture was stirred for 1 h. Acetaldehyde (4.4 g, 101.4 mmol) was added dropwise and the mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was quenched by addition of a saturated aqueous NH.sub.4Cl solution and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=1:0 to 20:1) to give the title compound I20 (3.0 g, 46%) as a yellow oil. LCMS-B: rt 3.39 min; m/z 175.0 [M−OH].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.41-7.33 (m, 1H), 7.31-7.25 (m, 1H), 5.54 (d, J=4.4 Hz, 1H), 5.25-5.16 (m, 1H), 1.45 (d, J=6.7 Hz, 3H).

(b) 1-(6-Chloro-2,3-difluorophenyl)ethan-1-one (I21)

[0482] To a solution of 1-(6-chloro-2,3-difluorophenyl)ethan-1-ol (I20) (3.0 g, 15.6 mmol) in dry DCM (50 mL) was added Dess-Martin periodinane (19.8 g, 46.7 mmol) and the mixture was stirred at room temperature overnight. The mixture was filtered and the filtrate diluted with water and extracted with EtOAc. The combined organic extracts were washed with a saturated aqueous NaHCO.sub.3 solution, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. ether) to give the title compound I21 (2.3 g, 79%) as yellow oil, which was used directly in the next step.

[0483] To a mixture of 1-(6-chloro-2,3-difluorophenyl)ethan-1-one (I21) (1.8 g, 9.3 mmol) and K.sub.2CO.sub.3 (3.9 g, 28.0 mmol) in DMF (20 mL) under nitrogen was added ethyl 2-mercaptoacetate (1.3 g, 11.2 mmol) and the mixture was stirred at room temperature overnight. Water was added and the mixture was extracted with EtOAc. The combined organic extracts were washed with water, brine and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=1:0) to give the title compound I22 (1.5 g, 60%) as a brown solid. LCMS-A: rt 3.54 min; m/z 273.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.55-7.50 (m, 1H), 7.40 (t, J=8.7 Hz, 1H), 4.35 (q, J=7.1 Hz, 2H), 2.97 (s, 3H), 1.34 (t, J=7.1 Hz, 3H).

(d) Ethyl 3-(bromomethyl)-4-chloro-7-fluorobenzo[b]thiophene-2-carboxylate (I23)

[0484] To a solution of ethyl 4-chloro-7-fluoro-3-methylbenzo[b]thiophene-2-carboxylate (I22) (1.5 g, 5.5 mmol) in CCl.sub.4 (50 mL) was added NBS (978 mg, 5.5 mol) and AlBN (451 mg, 2.75 mmol) and the mixture was heated at 80° C. under N.sub.2 overnight. The mixture was diluted with water, extracted with EtOAc and the combined organic extracts were washed with water, brine and concentrated under reduced pressure to give the title compound I23 (1.3 g, 80%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.67-7.61 (m, 1H), 7.50 (t, J=8.7 Hz, 1H), 5.47 (s, 2H), 4.41 (q, J=7.1 Hz, 2H), 1.36 (t, J=7.0 Hz, 3H).

(e) Ethyl 4-chloro-7-fluoro-3-formylbenzo[b]thiophene-2-carboxylate (I24)

[0485] To a solution of ethyl 3-(bromomethyl)-4-chloro-7-fluorobenzo[b]thiophene-2-carboxylate (I23) (1.0 g, 2.8 mmol) in THF (20 mL) under N.sub.2 was added NMO (1.33 g, 11.4 mmol) and the mixture was heated at reflux for 16 h. The mixture was diluted with water, extracted with EtOAc and the combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=1:0 to 100:1) to give the title compound I24 (250 mg, 31%) as a white solid. LCMS-B: rt 4.39 min; m/z 286.9 [M+H].sup.+, 308.9 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.7 (s, 1H), 7.73-7.66 (m, 1H), 7.57 (t, J=8.9 Hz, 1H), 4.38 (q, J=7.1 Hz, 2H), 1.32 (t, J=7.1 Hz, 3H).

(vii) 4,4′-(Ethane-1,2-diylbis(azanediyl))bis(3-amino-2-methoxybenzamide) (I33)

[0486] ##STR00027## ##STR00028##

(a) Methyl 4-fluoro-2-hydroxybenzoate (I25)

[0487] A mixture of 4-fluoro-2-hydroxybenzoic acid (50.0 g, 320.3 mmol) and concentrated H.sub.2SO.sub.4 (40 mL, 672.7 mmol) in MeOH (600 mL) was heated at reflux under N.sub.2 for 16 h. The mixture was poured into water (500 mL) and extracted with EtOAc (500 mL×3). The combined organic extracts were washed with water (500 mL×2), brine (500 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 20:1) to give the title compound (50.0 g, 91%) as a white solid. LCMS-A (ES-API): rt 1.80 min, m/z 171.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.8 (s, 1H), 7.83 (dd, J=8.9, 6.8 Hz, 1H), 6.90-6.70 (m, 2H), 3.88 (s, 3H).

(b) Methyl 4-fluoro-2-hydroxy-3-nitrobenzoate (I26)

[0488] To a solution of methyl 4-fluoro-2-hydroxybenzoate (I25) (50.0 g, 294.0 mmol) in concentrated H.sub.2SO.sub.4 (100 mL) at 0° C. under N.sub.2 was added concentrated HNO.sub.3 (20 mL, 382.1 mmol) dropwise and the mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was slowly poured into water (2.0 L) and extracted with EtOAc (1.5 L×3). The combined organic extracts were washed water (1.5 L×2), brine (1.5 L), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (100% Pet. Ether) to give a mixture of the title compound and an uncharacterised regioisomer (50 g, ˜3.75:1 uncharacterised regioisomer/title compound). The mixture was used in the next step without further purification or characterization. LCMS-A (ES-API): rt 1.57 min (minor) m/z 216.0 [M+H].sup.+ and rt 1.68 min (major) m/z 216.1 [M+H].sup.+.

[0489] A mixture of methyl 4-fluoro-2-hydroxy-3-nitrobenzoate (containing a major, uncharacterised regioisomer) (I26) (50.0 g), CH.sub.3I (99.0 g, 697 mmol) and K.sub.2CO.sub.3 (64.1 g, 465 mml) in DMF (200 mL) was stirred at room temperature under N.sub.2 for 16 h. The mixture was poured into water (1.0 L), extracted with EtOAc (1.0 L×3) and the combined organic extracts were washed water (1.0 L×2), brine (1.0 L), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 40:1) and the fractions containing the minor component were collected to give the title compound (14.0 g, 21% over two steps) as a white solid. LCMS-C (ES-API): rt 3.53 min, m/z 230.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.12 (dd, J=9.1, 6.5 Hz, 1H), 7.50 (t, J=9.0 Hz, 1H), 3.93(s, 3H), 3.91 (s, 3H).

(d) Methyl 4-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-methoxy-3-nitrobenzoate (I28)

[0490] A mixture of methyl 4-fluoro-2-methoxy-3-nitrobenzoate (I27) (6.0 g, 26.2 mmol), tert-butyl (2-aminoethyl) carbamate (4.2 g, 26.2 mmol) and Et.sub.3N (5.3 g, 52.4 mmol) in NMP (100 mL) was heated at 80° C. under N.sub.2 for 16 h. The mixture was poured into water (500 mL), extracted with EtOAc (500 mL×3) and the combined organic extracts were washed with water (500×2 mL), brine (500 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 10:1) to give the title compound (9.5 g, 98%) as a yellow solid. LCMS-C (ES-API): rt 4.43 min, m/z 392.1 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.79 (d, J=9.2 Hz, 1H), 6.96 (t, J=5.5 Hz, 1H), 6.74 (d, J=9.2 Hz, 1H), 6.70 (t, J=5.7 Hz, 1H), 3.79 (s, 6H), 3.27-3.21 (m, 2H), 3.11-3.06 (m, 2H), 1.37 (s, 9H).

(e) Methyl 4-((2-aminoethyl)amino)-2-methoxy-3-nitrobenzoate hydrochloride (I29)

[0491] A mixture of methyl 4-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-methoxy-3-nitrobenzoate (I28) (9.5 g, 25.7 mmol) and a 3 M HCl in dioxane solution (200 mL) was stirred at room temperature under N.sub.2 for 16 h. The mixture was concentrated under reduced pressure to give the title compound (6.0 g, 87%) as a white solid. LCMS-C (ES-API): rt 0.82 min, m/z 270.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.81 (dd, J=9.2, 3.8 Hz, 1H), 6.82-6.71 (m, 2H), 3.79 (s, 3H), 3.75 (s, 3H), 3.31-3.28 (m, 4H).

(f) Dimethyl 4,4′-(ethane-1,2-diylbis(azanediyl))bis(2-methoxy-3-nitrobenzoate) (I30)

[0492] A mixture of methyl 4-((2-aminoethyl)amino)-2-methoxy-3-nitrobenzoate hydrochloride (I29) (5.5 g, 18.0 mmol), methyl 4-fluoro-2-methoxy-3-nitrobenzoate I27 (4.68 g, 20.4 mmol) and Et.sub.3N (6.2 g, 61.3 mmol) in NMP (100 mL) was heated at 80° C. under N.sub.2 for 16 h. The mixture was poured into water (600 mL) and the resulting precipitate was collected by filtration to give the title compound (8.0 g, 82%) as a yellow solid. LCMS-C (ES-API): rt 4.75 min, m/z 477.1 [M−H].sup.−. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.76 (d, J=9.2 Hz, 2H), 6.77-6.74 (m, 4H), 3.78 (s, 6H), 3.77 (s, 6H), 3.40 (br s, 4H).

(g) 4,4′-(Ethane-1,2-diylbis(azanediyl))bis(2-methoxy-3-nitrobenzoic acid) (I31)

[0493] A mixture of dimethyl 4,4′-(ethane-1,2-diylbis(azanediyl))bis(2-methoxy-3-nitrobenzoate) (I30) (4.0 g, 8.4 mmol) and NaOH (2.0 g, 50.2 mmol) in EtOH/water (50 mL/50 mL) was heated at 80° C. for 16 h. Most of the EtOH was removed under reduced pressure and the residue was diluted with water (50 mL) and acidified to pH 4-5 with a 2 M aqueous HCl solution. The resulting precipitate was collected by filtration to give the title compound (3.5 g, 93%) as a yellow solid. LCMS-C (ES-API): rt 3.45 min, m/z 473.1 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.6 (br s, 2H), 7.77 (d, J=9.1 Hz, 2H), 6.74 (d, J=9.3 Hz, 2H), 6.69 (t, J=5.5 Hz, 2H), 3.78 (s, 6H), 3.44 (br s, 4H).

(h) 4,4′-(Ethane-1,2-diylbis(azanediyl))bis(2-methoxy-3-nitrobenzamide) (I32)

[0494] A mixture of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(2-methoxy-3-nitrobenzoic acid) (I31) (3.4 g, 7.6 mmol), NH.sub.4Cl (2.4 g, 45.3 mmol), HOBt (3.1 g, 22.7 mmol), EDCl.HCl (4.3 g, 22.7 mmol) and DIPEA (5.9 g, 45.3 mmol) in DMF (300 mL) was stirred at room temperature under N.sub.2 for 16 h. The mixture was poured into water (600 mL) and the resulting precipitate was collected by filtration to give the title compound (2.4 g, 71%) as a yellow solid. LCMS-C (ES-API): rt 3.02 min, m/z 471.1 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.63 (d, J=9.0 Hz, 2H), 7.42 (br s, 4H), 6.77 (d, J=9.1 Hz, 2H), 6.60 (t, J=4.8 Hz, 2H), 3.77 (s, 6H), 3.39-3.36 (m, 4H).

(i) 4,4′-(Ethane-1,2-diylbis(azanediyl))bis(3-amino-2-methoxybenzamide) (I33)

[0495] A mixture of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(2-methoxy-3-nitrobenzamide) (I32) (1.0 g, 2.3 mmol) and 10% Pd/C (200 mg) in MeOH (150 mL) was stirred at room temperature under a H.sub.2 atmosphere (3 MPa) for 3 days. The mixture was filtered and the filtrate was concentrated under reduced pressure give the title compound (120 mg). The filter cake was washed with DMSO and the filtrate was freeze-dried to give additional title compound (680 mg). The isolated solids were combined to give the title compound (800 mg, 92%) as a brown solid. LCMS-B (ES-API): rt 1.09 min, m/z 388.8 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.39 (br s, 2H), 7.11 (d, J=8.4 Hz, 2H), 7.10 (br s, 2H), 6.37 (d, J=8.5 Hz, 2H), 5.23 (br s, 2H), 4.42 (br s, 4H), 3.66 (s, 6H), 3.35 (br s, 4H).

(viii) Ethyl 3-formylbenzo[b]thiophene-2-carboxylate (I36)

[0496] ##STR00029##

(a) Ethyl 3-methylbenzo[b]thiophene-2-carboxylate (I34)

[0497] A solution of 1-(2-fluorophenyl)ethan-1-one (25.0 g, 181 mmol), ethyl 2-mercaptoacetate (26.1 g, 217 mmol) and K.sub.2CO.sub.3 (75.1 g, 543 mmol) in DMF (250 mL) was heated at 75° C. under N.sub.2 for 16 h. Water was added and the resulting mixture was extracted EtOAc. The combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:0 to 100:1) to give the title compound (38.8 g, 97%) as a light green solid. LCMS-B (ES-API): rt 4.51 min; m/z 221.1 [M+H].sup.+.

(b) Ethyl 3-(bromomethyl)benzo[b]thiophene-2-carboxylate (I35)

[0498] A solution of ethyl 3-methylbenzo[b]thiophene-2-carboxylate (I34) (10.0 g, 45.4 mmol), NBS (8.08 g, 45.4 mmol) and BPO (1.09 g, 4.5 mmol) in CCl.sub.4 (200 mL) was heated at reflux under N.sub.2 for 16 h. Water was added and the resulting mixture was extracted with EtOAc. The combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM) to give title compound (11.8 g, 86%) as a light yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.11-8.08 (m, 2H), 7.61-7.57 (m, 2H), 5.30 (s, 2H), 4.41 (q, J=7.2 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

[0499] A mixture of ethyl 3-(bromomethyl)benzo[b]thiophene-2-carboxylate (I35) (5 g, 16.7 mmol) and NMO (7.83 g, 66.8 mmol) in THF (100 mL) was heated at reflux under N.sub.2 overnight. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 50:1) to give title compound (2.0 g, 50%) as a yellow solid. LCMS-B (ES-API): rt 4.18 min; m/z 235.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.7 (s, 1H), 8.63-8.57 (m, 1H), 8.21-8.15 (m, 1H), 7.67-7.57 (m, 2H), 4.44 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H).

(ix) Ethyl 7-fluoro-3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I40)

[0500] ##STR00030##

(a) 1-(2,3-Difluoro-6-methoxyphenyl)ethan-1-one (I37)

[0501] To a solution of 1,2-difluoro-4-methoxybenzene (10.0 g, 69.4 mmol) in THF (300 mL) at −78° C. was added n-BuLi (2.5 M solution in hexanes, 30.5 mL, 76.3 mmol) dropwise and the mixture was stirred at −78° C. for 40 min. A solution of N-methoxy-N-methylacetamide (11.3 g, 69.4 mmol) in THF (50 mL) was then added dropwise at −78° C. and the mixture was stirred at −78° C. for a further 1 h. The reaction was quenched with a saturated aqueous NH.sub.4Cl solution, diluted with water (300 mL) and extracted with EtOAc (500 mL×3). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=50:1 to 10:1) to give the title compound (2.1 g, 16%) as a yellow oil. LCMS-B (ES-API): rt 3.56 min, m/z 187.0 [M+H].sup.+. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.17-7.10 (m, 1H), 6.63-6.60 (m, 1H), 3.82 (s, 3H), 2.54 (d, J=0.8 Hz, 3H).

(b) Ethyl 7-fluoro-4-methoxy-3-methylbenzo[b]thiophene-2-carboxylate (I38)

[0502] A mixture of 1-(2,3-difluoro-6-methoxyphenyl)ethan-1-one (I37) (1.88 g, 10.1 mmol), ethyl 2-mercaptoacetate (1.21 g, 10.1 mmol) and K.sub.2CO.sub.3 (1.39 g, 10.1 mmol) in DMF (75 mL) was heated at 75° C. under N.sub.2 overnight. The mixture was poured into water, extracted with EtOAc and the combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=500:1 to 100:1) to give the title compound (2.19 g, 81%) as white solid. LCMS-B (ES-API): rt 3.56 min, m/z 291.2 [M+Na].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.34 (t, J=9.0 Hz, 1H), 6.93 (dd, J=8.8, 3.8 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 3.91 (s, 3H), 2.90 (s, 3H). 1.34 (t, J=7.1 Hz, 3H).

[0503] To a solution of ethyl 7-fluoro-4-methoxy-3-methylbenzo[b]thiophene-2-carboxylate (I38) (1.7 g, 6.21 mmol) in CCl.sub.4 (75 mL) was added NBS (1.2 g, 6.69 mmol) and AlBN (500 mg, 3.11 mmol) and the mixture was heated at 80° C. under N.sub.2 overnight. The mixture was diluted with water (70 mL), extracted with EtOAc (70 mL×3) and the combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=500:1 to 150:1) to give the title compound (1.85 g, 84%) as a white solid..sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.53-7.30 (m, 2H), 5.39 (s, 2H), 4.41- 4.36 (m, 2H), 3.97 (s, 3H), 1.37-1.33 (m, 3H).

(d) Ethyl 7-fluoro-3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I40)

[0504] To a solution of ethyl 3-(bromomethyl)-7-fluoro-4-methoxybenzo[b]thiophene-2-carboxylate (I39) (1.8 g, 0.005 mol) in THF (25.0 mL) was added NMO (2.5 g, 0.02 mol) and the mixture was heated at reflux under N.sub.2 overnight. The mixture was diluted with water (60 mL), extracted with EtOAc (60 mL×3) and the combined organic extracts were washed brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=500:1 to 40:1) to give the title compound (810 mg, 54%) as a white solid. LCMS-B (ES-API): rt 3.33 min, m/z 283.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.5 (s, 1H), 7.47 (dd, J=9.5, 8.7 Hz, 1H), 7.05 (dd, J=8.8, 3.6 Hz, 1H), 4.34 (q, J=7.1 Hz, 2H), 3.89 (s, 3H), 1.31 (t, J=7.1 Hz, 3H).

(x) Ethyl 4-cyano-3-formylbenzo[b]thiophene-2-carboxylate (I44)

[0505] ##STR00031##

[0506] Compound I41 is compound I61 of WO 2019/219820.

(a) Ethyl 4-cyano-3-methylbenzo[b]thiophene-2-carboxylate (I42)

[0507] To a solution of ethyl 4-bromo-3-methylbenzo[b]thiophene-2-carboxylate (I41) (1.2 g, 4.01 mmol) in DMF (40 mL) was added CuCN (717 mg, 8.02 mmol) and Pd[PPh.sub.3].sub.4 (463 mg, 0.40 mmol) and the mixture was heated at 120° C. under N.sub.2 overnight. The mixture was filtered and the filtrate was diluted with water and extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 80:1) to give the title compound (600 mg, 60%) as a white solid. LCMS-B (ES-API): rt 4.27 min, m/z 246.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.40 (d, J=8.2 Hz, 1H), 8.02 (d, J=7.3 Hz, 1H), 7.68 (t, J=8.0 Hz, 1H), 4.36 (q, J=7.1 Hz, 2H), 2.99 (s, 3H), 1.34 (t, J=7.1 Hz, 3H).

(b) Ethyl 3-(bromomethyl)-4-cyanobenzo[b]thiophene-2-carboxylate (I43)

[0508] A mixture of ethyl 4-cyano-3-methylbenzo[b]thiophene-2-carboxylate (I42) (450 mg, 1.83 mmol), NBS (392 mg, 2.20 mmol) and BPO (44 mg, 0.18 mmol) in CCl.sub.4 (20 mL) was heated at reflux under N.sub.2 for 16 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 20:1) to give the title compound (360 mg, 60%) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.52 (dd, J=8.3, 0.9 Hz, 1H), 8.14 (dd, J=7.4, 0.9 Hz, 1H), 7.77 (dd, J=8.1, 7.6 Hz, 1H), 5.49 (s, 2H), 4.43 (q, J=7.1 Hz, 2H), 1.37 (t, J=7.1 Hz, 3H).

[0509] A solution of ethyl 3-(bromomethyl)-4-cyanobenzo[b]thiophene-2-carboxylate (I43) (450 mg, 1.39 mmol) and NMO (651 mg, 5.56 mmol) in THF (15 mL) was heated at reflux under N.sub.2 for 16 h. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 50:1) to give the title compound (180 mg, 50%) as a white solid. LCMS-A (ES-API): rt 1.56 min, m/z 260.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.7 (s, 1H), 8.57 (dd, J=8.3, 0.8 Hz, 1H), 8.13 (dd, J=7.4, 0.8 Hz, 1H), 7.78 (t, J=8.0 Hz, 1H), 4.44 (q, J=7.1 Hz, 2H), 1.36 (t, J=7.1 Hz, 3H).

(xi) Ethyl 3-formyl-4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate (I47)

[0510] ##STR00032##

(a) Ethyl 3-methyl-4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate (I45)

[0511] A mixture of 1-(2-fluoro-6-(trifluoromethyl)phenyl)ethan-1-one (2.0 g, 0.01 mol), ethyl 2-mercaptoacetate (1.44 g, 0.012 mol) and K.sub.2CO.sub.3 (4.15 g, 0.03 mol) in DMF (40 mL) was heated at 100° C. under N.sub.2 overnight. The mixture was diluted with water (50 mL), extracted with EtOAc (50 mL×3) and the combined organic extracts were washed with water and brine then concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=120:1 to 100:1) to give the title compound (2.4 g, 86%) as a white solid. LCMS-B (ES-API): rt 4.6 min, m/z 289.6 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.40 (d, J=8.1 Hz, 1H), 7.94 (d, J=7.5 Hz, 1H), 7.68 (t, J=7.8 Hz, 1H), 4.36 (q, J=7.1 Hz, 2H), 2.83 (d, J=2.0 Hz, 3H), 1.34 (t, J=7.1 Hz, 3H).

(b) Ethyl 3-(bromomethyl)-4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate (I46)

[0512] A mixture of ethyl 3-methyl-4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate (I45) (1.2 g, 4.2 mmol), NBS (0.78 g, 4.4 mmol) and AlBN (65.7 mg, 0.4 mmol) in CCl.sub.4 (20 mL) was heated at reflux under N.sub.2 for 3 h. More NBS (0.28 g, 1.6 mmol) was added and the mixture was heated at reflux for a further 2 h. More NBS (0.28 g, 1.6 mmol) was added and the mixture was heated at reflux overnight. The mixture was diluted with water (20 mL), extracted with EtOAc (30 mL×3) and the combined organic extracts were washed with water, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1) to give the title compound (1.17 g, 80%) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.47 (d, J=8.1 Hz, 1H), 8.02 (d, J=7.5 Hz, 1H), 7.74 (t, J=7.9 Hz, 1H), 5.33 (s, 2H), 4.16 (q, J=7.1 Hz, 2H), 1.37 (t, J=7.1 Hz, 3H).

[0513] A solution of ethyl 3-(bromomethyl)-4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate (I46) (1.5 g, 4.1 mmol) and NMO (2.06 g, 17.6 mmol) in THF (20 mL) was heated at reflux under N.sub.2 overnight. Water (40 mL) was added and the mixture was extracted with EtOAc (40 mL×3). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 70:1) to give the title compound (560 mg, 45%) as a white solid. LCMS-B (ES-API): rt 4.20 min, m/z 303.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.5 (d, J=2.1 Hz, 1H), 8.55 (d, J=8.3 Hz, 1H), 8.01 (d, J=7.5 Hz, 1H), 7.80 (t, J=7.9 Hz, 1H), 4.39 (q, J=7.1 Hz, 2H), 1.33 (t, J=7.1 Hz, 3H).

(xii) Ethyl 4-bromo-7-fluoro-3-formylbenzo[b]thiophene-2-carboxylate (I49)

[0514] ##STR00033##

[0515] Compound I48 is compound I52 of WO 2019/219820.

[0516] A solution of ethyl 4-bromo-3-(bromomethyl)-7-fluorobenzo[b]thiophene-2-carboxylate (I48) (676 mg, 1.71 mmol) and NMO (800 mg, 6.84 mmol) in THF (5 mL) was heated at reflux under N.sub.2 for 16 h. The mixture was partitioned between water (30 mL) and EtOAc (30 mL), the layers were separated and the aqueous layer was further extracted with EtOAc (30 mL×3). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 30:1) to give the title compound (243 mg, 43%) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.8 (s, 1H), 7.85 (dd, J=8.4, 4.4 Hz, 1H), 7.52 (t, J=9.2 Hz, 1H), 4.39 (q, J=7.2 Hz, 2H), 1.33 (t, J=7.2 Hz, 3H).

(xiii) Ethyl 4-bromo-3-formylbenzo[b]thiophene-2-carboxylate (I51)

[0517] ##STR00034##

[0518] Compound I50 is compound I62 of WO 2019/219820.

[0519] A solution of ethyl 4-bromo-3-(bromomethyl)benzo[b]thiophene-2-carboxylate (I50) (100 mg, 2.64 mmol) and NMO (124 mg, 10.6 mmol) in THF (20 mL) was heated at reflux under N.sub.2 overnight. The mixture was partitioned between water and EtOAc, the layers were separated and the aqueous layer was further extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=200:1 to 50:1) to give the title compound (290 mg, 35%) as a white solid. LCMS-B (ES-API): rt 3.33 min; m/z 312.9 [M+H].sup.+.

(xiv) Ethyl 3-formyl-4-methylbenzo[b]thiophene-2-carboxylate (I52)

[0520] ##STR00035##

[0521] A mixture of ethyl 4-bromo-3-formylbenzo[b]thiophene-2-carboxylate (I51) (225 mg, 0.78 mmol), 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (361 mg, 2.87 mmol), K.sub.2CO.sub.3(397 mg, 2.87 mmol) and Pd(PPh.sub.3).sub.4 (125 mg, 0.11 mmol) in dioxane (10 mL) was heated at reflux overnight under N.sub.2. Water (50 mL) was added and the mixture was extracted with EtOAc (50 mL×3). The combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered, concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=100:1 to 50:1) to give the title compound (50 mg, 28%) as a white solid. LCMS-B (ES-API): rt 4.26 min, m/z 249.1 [M+H].sup.+.

(xv) Ethyl 4-ethoxy-3-formylbenzo[b]thiophene-2-carboxylate (I54)

[0522] ##STR00036##

[0523] Compound I53 is compound I40 of WO 2019/219820.

[0524] A solution of ethyl 3-(bromomethyl)-4-ethoxybenzo[b]thiophene-2-carboxylate (I53) (2.0 g 6.00 mmol) and NMO (4.7 g, 24.0 mmol) in THF (100 mL) was heated at reflux overnight. The mixture was diluted with water, extracted with EtOAc and the combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether) to give the title compound (600 mg, 37%) as a gray solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.6 (s, 1H), 7.65 (d, J=8.1 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.02 (d, J=7.9 Hz, 1H), 4.32 (q, J=7.1 Hz, 2H), 4.17 (q, J=6.9 Hz, 2H), 1.36 (t, J=6.9 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H).

(xvi) Ethyl 3-formyl-4-methoxythieno[3,2-c]pyridine-2-carboxylate (I56)

[0525] ##STR00037##

[0526] Compound I55 is compound I29 of WO 2019/219820.

[0527] A mixture of ethyl 3-(bromomethyl)-4-methoxythieno[3,2-c]pyridine-2-carboxylate (I55) (1 g, 3.03 mmol) and NMO (2.15 g, 12.2 mmol) in THF (20 mL) was heated at reflux overnight. Water (100 mL) was added and the mixture was extracted with EtOAc (50 mL×3). The combined organic extracts were washed with water (100 mL×2), brine (100 mL×2), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=50:1 to 5:1) to give the title compound (260 mg, 32%) as a yellow solid. LCMS-B (ES-API): rt 3.87 min; m/z 266.0 [M+H].sup.+.

EXAMPLES

3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (1.1 and 1.2)

[0528] ##STR00038##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (A1)

[0529] To a solution of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (20 mg, 0.061 mmol) in EtOH (3 mL) was added ethyl 4-chloro-3-formylbenzo[b]thiophene-2-carboxylate (I19) (33 mg, 0.12 mmol) and AcOH (0.3 mL) and the mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with a saturated aqueous NaHCO.sub.3 solution, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC (Agilent 10 prep-C18, 10 μm, 250×21.2 mm column, eluting with a gradient of ACN in water with 0.1% TFA, at a flow rate of 20 mL/min) to give the title compound A1 (5 mg, 10%) as a yellow solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-B: rt 3.43 min; m/z 825.1/827.1 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD) δ 8.29 (s, 2H), 8.14 (dd, J=8.3, 0.8 Hz, 1H), 8.12-8.05 (m, 2H), 8.04-7.91 (m, 3H), 7.63 (t, J=8.0 Hz, 1H), 7.54-7.48 (m, 2H), 7.23 (d, J=7.2 Hz, 1H), 4.77-4.67 (m, 2H), 4.53-4.40 (m, 2H), 4.17-4.09 (m, 2H), 3.85-3.71 (m, 2H), 1.08 (t, J=7.2 Hz, 3H), 0.91 (t, J=7.2 Hz, 3H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (1.1 and 1.2)

[0530] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (A1) (150 mg, 0.18 mmol) in EtOH (10 mL) was added a solution of NaOH (43 mg, 1.08 mmol) in water (2 mL) and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and the residue was diluted with water, acidified to pH 3-4 with a 1 M aqueous HCl solution and the resulting precipitate was collected by filtration. The solid was purified by prep. HPLC (Agilent 10 prep-C18, 10 μm, 250×21.2 mm column, eluting with a gradient of ACN in water with 0.1% TFA, at a flow rate of 20 mL/min) to give separated atropisomers of the title compound 1.1 (peak 1: 20 mg, 14%) and title compound 1.2 (peak 2: 15 mg, 11%) as yellow solids.

[0531] Peak 1 (1.1) (prep HPLC: rt 13.27 min): LCMS-B: rt 2.81 min; m/z 769.0/771.0 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD) δ 8.30 (d, J=1.2 Hz, 2H), 8.11 (dd, J=8.3, 0.8 Hz, 2H), 8.02 (dd, J=8.7, 1.6 Hz, 2H), 7.87 (d, J=8.8 Hz, 2H), 7.62 (t, J=8.0 Hz, 2H), 7.49 (dd, J=7.7, 0.8 Hz, 2H), 4.70-4.67 (m, 2H), 4.60-4.57 (m, 2H). Peak 2 (1.2) (prep HPLC: rt 15.45 min): LCMS-B: rt 2.94 min; m/z 769.0/771.0 [M+H].sup.+. .sup.1H NMR (400 MHz, MeOD) δ 8.29 (d , J=1.2 Hz, 2H), 8.11 (dd, J=8.8, 0.8 Hz, 2H), 8.02 (d, J=8.4 Hz, 2H), 7.92 (d, J=8.4 Hz, 2H), 7.51 (t, J=8.0 Hz, 2H), 7.22 (d, J=7.6 Hz, 2H), 4.78-4.76 (m, 2H), 4.52-4.50 (m, 2H).

3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chloro-7-fluorobenzo[b]thiophene-2-carboxylic acid) (2.1 and 2.2)

[0532] ##STR00039##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chloro-7-fluorobenzo[b]thiophene-2-carboxylate) (A2)

[0533] To a solution of ethyl 4-chloro-7-fluoro-3-formylbenzo[b]thiophene-2-carboxylate (I24) (250 mg, 0.87 mmol) in DMF (10 mL) was added 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (143 mg, 0.44 mmol) and NaHSO.sub.3(90 mg, 0.87 mmol) and the mixture was heated at 120° C. under N.sub.2 overnight. The mixture was partitioned between EtOAc and water, the layers were separated and the organic layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=10:1 to 1:1 to DCM/MeOH=10:1) to give the title compound A2 (90 mg, 24%) as a grey solid (mixture of atropisomeric diastereomers): .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-B: rt 3.55 min; m/z 861.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.23 (s, 2H), 8.09-7.96 (m, 6H), 7.70-7.58 (m, 2H), 7.50 (t, J=8.9 Hz, 1H), 7.39-7.29 (m, 3H), 4.54-4.32 (m, 4H), 4.08 (q, J=7.1 Hz, 2H), 3.84 (q, J=7.1 Hz, 2H), 0.96 (t, J=7.1 Hz, 3H), 0.85 (t, J=7.1 Hz, 3H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chloro-7-fluorobenzo[b]thiophene-2-carboxylic acid) (2.1 and 2.2)

[0534] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chloro-7-fluorobenzo[b]thiophene-2-carboxylate) (A2) (80 mg, 0.09 mmol) in EtOH/water (3 mL/3 mL) was added NaOH (11 mg, 0.28 mmol) and the mixture was stirred at room temperature overnight. Most of the EtOH was removed under reduced pressure and the aqueous residue was acidified to pH 5 with a 1 M aqueous HCl solution. The resulting precipitate was collected by filtration and purified by preparative HPLC (Varian-940LC, prep-C18, 5 μm, 150×20 mm column, eluting with a gradient of ACN in water with 0.1% formic acid, at a flow rate of 15 mL/min) to give separated atropisomers of the title compound 2.1 (peak 1: 5 mg, 7%) and title compound 2.2 (peak 2: 5 mg, 7%) as white solids.

[0535] Peak 1 (2.1) (HPLC: rt 6.78 min): LCMS-B: rt 2.87 min; m/z 805.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.21 (s, 2H), 8.02 (s, 2H), 7.93 (d, J=8.5 Hz, 2H), 7.85 (br s, 2H), 7.59-7.47 (m, 4H), 7.30 (s, 2H), 4.52-4.41 (m, 2H), 4.41-4.29 (m, 2H).

[0536] Peak 2 (2.2) (HPLC: rt 8.00 min): LCMS-B: rt 2.97 min; m/z 804.9 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.21 (s, 2H), 8.03 (s, 2H), 8.00-7.93 (m, 2H), 7.81 (br s, 2H), 7.44-7.35 (m, 2H), 7.33-7.25 (m, 4H), 4.62-4.53 (m, 2H), 4.31-4.11 (m, 2H).

3,3′-(Butane-1,4-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4 chlorobenzo[b]thiophene-2-carboxylic acid) (3)

[0537] ##STR00040##

(a) Diethyl 3,3′-(butane-1,4-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (A3)

[0538] A mixture of 4,4′-(butane-1,4-diylbis(azanediyl))bis(3-aminobenzamide) (I12) (100 mg, 0.28 mmol), ethyl 4-chloro-3-formylbenzo[b]thiophene-2-carboxylate (I19) (151 mg, 0.56 mmol) and NaHSO.sub.3(59 mg, 0.56 mmol) in DMF (8 mL) was heated at 80° C. under nitrogen for 16 h. The mixture was partitioned between water (50 mL) and EtOAc (50 mL), the layers were separated and the aqueous layer extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=100:1 to 10:1) to give title compound A3 (130 mg, 54%) as a black solid (mixture of atropisomeric diastereomers: analytical HPLC analysis (Agilent 1200 series, Waters Nova-pak, 3.9×150 mm, 4 μm C18 column, eluting with a gradient of MeOH in water with 0.05% TFA, at a flow rate of 0.5 mL/min) showed a 1:1 mixture of components with rt 11.59 min and 11.80 min.). LCMS-B: rt 3.57 min; m/z 853.1/855.1. The mixture was used in the next step without further purification.

(b) 3,3′-(Butane-1,4-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (3)

[0539] A solution of diethyl 3,3′-(butane-1,4-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (A3) (80 mg, 0.09 mmol) and NaOH (23 mg, 0.54 mmol) in EtOH/water (6 mL/2 mL) was stirred at room temperature for 16 h. The solvent was removed under reduced pressure and the residue was diluted with water and acidified to pH 4-5 with a 2 M aqueous HCl solution. The resulting precipitate was collected by filtration and purified by prep HPLC (Agilent 10 Prep-C18, 250×21.2 mm column, eluting with a gradient of ACN in water with 0.1% TFA, at a flow rate of 20 mL/min) to give separated atropisomers of the title compound 3 (peak 1: 5 mg, 6%) and (peak 2: 10 mg, impure) as gray solids.

[0540] Peak 1: (3) (prep HPLC: rt 11.34 min): LCMS-B: rt 2.81 min; m/z 797.0/799.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.17 (s, 4H), 8.01 (s, 2H), 7.84 (s, 2H), 7.66-7.46 (m, 4H), 7.36-7.16 (m, 4H), 4.01-3.75 (m, 4H), 1.56-1.41 (m, 4H).

[0541] Peak 2: (prep HPLC: rt 14.51 min): LCMS-B: rt2.90 min; m/z 797.1/799.1 [M+H].sup.+.

3,3′-(Propane-1,3-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (4.1 and 4.2)

[0542] ##STR00041##

(a) Diethyl 3,3′-(propane-1,3-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (A4)

[0543] To a solution of 4,4′-(propane-1,3-diylbis(azanediyl))bis(3-aminobenzamide) (I8) (200 mg, 0.58 mmol) in DMF (15 mL) was added ethyl 4-chloro-3-formylbenzo[b]thiophene-2-carboxylate (I19) (314 mg, 1.17 mmol) and NaHSO.sub.3 (122 mg, 1.17 mmol) and the mixture was heated at 80° C. under nitrogen overnight. Water was added and the mixture was extracted with EtOAc (100 mL×3). The combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/ EtOAc=10:1 to DCM/MeOH=15:1) to give the title compound A4 (155 mg, 32%) as a gray solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-B: rt 3.48 min; m/z 839.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.28-8.15 (m, 4H), 8.05-7.99 (m, 2H), 7.84-7.75 (m, 2H), 7.65-7.40 (m, 6H), 7.32-7.26 (m, 2H), 4.10-3.94 (m, 8H), 2.30-2.04 (m, 2H), 0.81 (t, J=7.2 Hz, 3H), 0.77 (t, J=7.2 Hz, 3H).

(b) 3,3′-(Propane-1,3-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (4.1 and 4.2)

[0544] To a solution of diethyl 3,3′-(propane-1,3-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (A4) (80 mg, 0.095 mmol) in EtOH/water (4 mL/2 mL) was added NaOH (23 mg, 0.57 mmol) and the mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was diluted with water and acidified to pH 4-5 with a 2 M aqueous HCl solution. The resulting precipitate was collected by filtration and purified by preparative HPLC (Agilent 10 Prep-C18, 250×21.2 mm column, eluting with a gradient of ACN in water with 0.1% TFA, at a flow rate of 20 mL/min) to give separated atropisomers of the title compound 4.1 (peak 1: 5 mg, 6%) and title compound 4.2 (peak 2: 6 mg, 7%) as white solids.

[0545] Peak 1 (4.1) (HPLC: rt 9.10 min): LCMS-B: rt 2.82 min; m/z 783.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.27-8.17 (m, 4H), 8.01 (br s, 2H), 7.78 (d, J=8.5 Hz, 2H), 7.59 (t, J=7.9 Hz, 2H), 7.50-7.40 (m, 4H), 7.31 (br s, 2H), 4.09-4.06 (m, 4H), 2.26-2.03 (m, 2H).

[0546] Peak 2 (4.2) (HPLC: rt 11.50 min): LCMS-B: rt 2.90 min; m/z 783.0 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.24-8.16 (m, 4H), 8.00 (br s, 2H), 7.82 (d, J=8.3 Hz, 2H), 7.64-7.47 (m, 4H), 7.38 (d, J=7.7 Hz, 2H), 7.29 (br s, 2H), 4.18-3.95 (m, 4H), 2.21-2.08 (m, 2H).

3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylic acid) (5.2)

[0547] ##STR00042##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylate) (A5)

[0548] A mixture of ethyl 3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I16) (270 mg, 1.02 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (167 mg, 0.51 mmol) and NaHSO.sub.3 (53 mg, 0.51 mmol) in DMF (25 mL) was heated at 120° C. under nitrogen overnight. The mixture was cooled to room temperature, poured into water (50 mL) and extracted with EtOAc (50 mL). The organic extract was washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=100:1 to 10:1) to give the title compound A5 (112 mg, 13%) as a white solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 3:1 mixture). The material was used in the next step without further purification. LCMS-B: rt 3.45 min; m/z 817.3. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.18 (br s, 2H), 8.10-7.92 (m, 4H), 7.90-7.80 (m, 1H), 7.78-7.68 (m, 2H), 7.66-7.42 (m, 3H), 7.28 (br s, 2H), 6.87 (d, J=7.8 Hz, 1.5 H), 6.58 (d, J=7.6 Hz, 0.5H), 4.59-4.36 (m, 3H), 4.26-4.06 (m, 3H), 4.01-3.91 (m, 1H), 3.67-3.59 (m, 1H), 3.30 (s, 4.5H, coincident with water peak), 3.18 (s, 1.5H), 0.86 (t, J=7.6 Hz, 1.5H), 0.67 (t, J=7.6 Hz, 4.5H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylic acid) (5.2)

[0549] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylate) (A5) (83 mg, 0.1 mmol) in EtOH/water (5:1, 10 mL) was added NaOH (40 mg, 1.0 mmol) and the mixture was stirred at room temperature overnight. The mixture was acidified to pH 6 with a 1 M aqueous HCl solution then concentrated under reduced pressure. The residue purified by preparative HPLC (Agilent PrepStar 218, prep-C18, 10 μm, 250×2.12 mm column, eluting with a gradient of MeOH in water with 0.1% TFA, at a flow rate of 20 mL/min) to give the title compound 5.2 (10 mg, 13%) as a white solid. Only one component was isolated from the reaction mixture of unknown stereochemistry.

[0550] LCMS-B: rt 2.85 min; m/z 761.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.18 (s, 2H), 7.99 (br s, 2H), 7.83 (d, J=8.4 Hz, 2H), 7.70 (d, J=8.0 Hz, 2H), 7.57 (t, J=8.0 Hz, 2H), 7.35 (d, J=8.5 Hz, 2H), 7.27 (br s, 2H), 6.86 (d, J=8.0 Hz, 2H), 4.32-4.15 (m, 4H), 3.36 (s, 6H).

3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylic acid) (5.1 and 5.2)

[0551] ##STR00043##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylate) (A6)

[0552] A solution of ethyl 3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I16) (400 mg, 1.52 mmol), 3,3′-(ethane-1,2-diylbis(azanediyl))bis(4-aminobenzamide) (I4) (250 mg, 0.76 mmol) and NaHSO.sub.3 (78.1 mg, 0.76 mmol) in DMF (15 mL) was heated at 120° C. under N.sub.2 overnight. Water (50 mL) was added and the mixture was extracted with dichloromethane (50 mL×3). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give the title compound (450 mg, 36%) as a brown solid. LCMS-B (ES-API): rt 3.12 min, m/z 817.6 [M+H].sup.+

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylic acid) (5.1 and 5.2)

[0553] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxybenzo[b]thiophene-2-carboxylate) (A6) (430 mg, 0.53 mmol) in EtOH/H.sub.2O (5:1, 10 mL) was added NaOH (212 mg, 5.3 mmol) and the mixture was stirred at room temperature overnight. Most of the EtOH was removed under reduced pressure and the aqueous residue was adjusted to pH 5 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Agilent, BOSTON, 250×2.12 mm, 10 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 20.0 mL/min) to give separated atropisomers of the title compound (5.1) (peak 1: 11 mg, 3%) as a white solid and the title compound (5.2) (peak 2: 22 mg, 5%) as a brown solid.

[0554] Peak 1 (Prep HPLC, rt 7.53 min): (5.1); LCMS-B (ES-API): rt 2.57 min, m/z 761.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.17 (s, 2H), 7.99 (br s, 2H), 7.87 (d, J=8.4 Hz, 2H), 7.63 (d, J=8.4 Hz, 2H), 7.46-7.43 (m, 4H), 7.27 (br s, 2H), 6.61 (d, J=8.0 Hz, 2H), 4.37-4.32 (m, 2H), 4.11-4.09 (m, 2H), 3.18 (s, 6H).

[0555] Peak 2 (Prep HPLC, rt 8.60 min): (5.2); LCMS-B (ES-API): rt 2.51 min, m/z 761.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.17 (s, 2H), 7.98 (br s, 2H), 7.80-7.73 (m, 2H), 7.69 (d, J=8.1 Hz, 2H), 7.54 (t, J=8.1 Hz, 2H), 7.35-7.11 (m, 4H), 6.85 (d, J=8.0 Hz, 2H), 4.23-4.20 (m, 2H), 4.14-4.12 (m, 2H), 3.34 (s, 6H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxythieno[3,2-c]pyridine-2-carboxylate) (6) and 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxythieno[3,2-c]pyridine-2-carboxylic acid) (7)

[0556] ##STR00044##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxythieno[3,2-c]pyridine-2-carboxylate) (6)

[0557] A solution of ethyl 3-formyl-4-methoxythieno[3,2-c]pyridine-2-carboxylate (I56) (162 mg, 0.61 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (100 mg, 0.305 mmol) and NaHSO.sub.3 (64 mg, 0.61 mmol) in DMF (3 mL) was heated at 80° C. overnight. Water (30 mL) was added and the mixture was extracted with EtOAc (30 mL×3). The combined organic extracts were washed with water (30 mL×2), brine (30 mL×2), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC (Varian-940-LC, 150×20 mm, 5 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 15.0 mL/min) to give the title compound (6 mg, 2%) as a white solid. LCMS-B (ES-API): rt 3.08 min; m/z 819.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.20 (s, 4H), 8.02-7.94 (m, 6H), 7.83 (d, J=5.2 Hz, 2H), 7.31 (br s, 2H), 4.54-4.48 (m, 2H), 4.20-4.13 (m, 2H), 3.79-3.73 (m, 2H), 3.65-1.66 (m, 2H), 3.54 (s, 6H), 0.77 (t, J=6.8 Hz, 6H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxythieno[3,2-c]pyridine-2-carboxylic acid) (7)

[0558] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methoxythieno[3,2-c]pyridine-2-carboxylate) (6) (70 mg, 0.086 mmol) in EtOH/H.sub.2O (2.0 mL/0.5 mL) was added NaOH (11 mg, 0.257 mmol) and the mixture was stirred at room temperature overnight. The mixture was diluted with water (5 mL) and most of the EtOH was removed under reduced pressure. The aqueous residue was adjusted to pH 4-5 with 2 M aqueous HCl and the resulting precipitate was collected by filtration and purified by prep-HPLC (Varian-940-LC, 150×20 mm, 5 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 15.0 mL/min) to give the title compound (4 mg, 6%) as a white solid. LCMS-B (ES-API): rt 2.53 min, m/z 761.0 [M−H].sup.−..sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.19 (s, 2H), 8.06 (d, J=5.6 Hz, 2H), 8.01 (br s, 2H), 7.94 (d, J=8.4 Hz, 2H), 7.74-7.66 (m, 4H), 7.28 (br s, 2H), 4.46-4.42 (m, 2H), 4.17-4.15 (m, 2H), 3.32 (s, 6H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(benzo[b]thiophene-2-carboxylate) (8) and 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(benzo[b]thiophene-2-carboxylic acid) (9)

[0559] ##STR00045##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(benzo[b]thiophene-2-carboxylate) (8)

[0560] A solution of ethyl 3-formylbenzo[b]thiophene-2-carboxylate (I36) (0.600 g, 2.57 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (0.423 g, 1.29 mmol) and NaHSO.sub.3 (0.268 g, 2.57 mmol) in DMF (40 mL) was heated at 130° C. under N.sub.2 for 48 h. Water was added and the mixture was extracted with EtOAc. The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=80:1 to 40:1) to give the title compound (440 mg, 46%) as a brown solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-B (ES-API): rt2.95 min; m/z 757.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.23 (s, 1H), 8.17 (d, J=8.2 Hz, 1H), 8.04 (br s, 1H), 7.85-7.79 (m, 2H), 7.72 (d, J=8.2 Hz, 2H), 7.56 (t, J=7.6 Hz, 1H), 7.38-7.30 (m, 4H), 7.24 (t, J=7.6 Hz, 1H), 7.15-7.13 (m, 2H), 7.00-6.97 (m, 2H), 6.13 (d, J=8.2 Hz, 1H), 4.79-4.76 (m, 1H), 4.52-4.49 (m, 1H), 4.31-4.29 (m, 1H), 4.19-3.95 (m, 5H), 0.98 (t, J=7.2 Hz, 3H), 0.91 (t, J=7.2 Hz, 3H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(benzo[b]thiophene-2-carboxylic acid) (9)

[0561] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(benzo[b]thiophene-2-carboxylate) (8) (0.200 g 0.26 mmol) in EtOH/H.sub.2O (20 mL/4 mL) was added NaOH (0.106 g 2.6 mmol) and the mixture was stirred at room temperature overnight. Most of the EtOH was removed under reduced pressure and the aqueous residue was acidified to pH 4-5 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep-HPLC (Shimadzu LC-8A, 150×20 mm, 10 μm column, eluting with a gradient of ACN in water with 0.1% TFA at a flow rate of 15.0 mL/min) to give the title compound (5 mg, 2%) as a white solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-C (ES-API): rt 3.14 min; m/z 701.2 [M+H].sup.+. .sup.1H NMR (400 MHz, 60° C., DMSO-d.sub.6) δ 8.23 (s, 1H), 8.15 (d, J=8.0 Hz, 1H), 7.85-7.82 (m, 3H), 7.73 (dd, J=8.4, 1.7 Hz, 1H), 7.56 (t, J=7.7 Hz, 1H), 7.38-7.36 (m, 3H), 7.23-7.18 (m, 3H), 7.13-7.05 (m, 3H), 6.90 (d, J=8.8 Hz, 1H), 6.28 (d, J=8.4 Hz, 1H), 4.73-4.70 (m, 1H), 4.67-4.34 (m, 2H), 4.17-4.14 (m, 1H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromobenzo[b]thiophene-2-carboxylate) (10) and 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromobenzo[b]thiophene-2-carboxylic acid) (11.1 and 11.2)

[0562] ##STR00046##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromobenzo[b]thiophene-2-carboxylate) (10)

[0563] A solution of ethyl 4-bromo-3-formylbenzo[b]thiophene-2-carboxylate (I51) (0.300 g, 0.96 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (0.157 g, 0.48 mmol) and NaHSO.sub.4 (0.199 g, 1.92 mmol) in DMF (15 mL) was heated at 80° C. under N.sub.2 overnight. Additional NaHSO.sub.4 (0.199 g, 1.92 mmol) was added and the mixture was heated at 100° C. overnight. The mixture was diluted with water, extracted with EtOAc (100 mL×3) and the combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=60:1 to 20:1) to give the title compound (180 mg, 21%) as a white solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-B (ES-API): rt 3.18 min; m/z 912.9, 914.8 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.30 (dd, J=8.0, 0.8 Hz, 1H), 8.25-8.22 (m, 2H), 8.19-8.16 (m, 1H), 8.04-7.95 (m, 5H), 7.92-7.90 (m, 1H), 7.71 (dd, J=8.0, 0.8 Hz, 1H), 7.57 (t, J=8.0 Hz, 1H), 7.49-7.46 (m, 2H), 7.32 (br s, 2H), 4.63-4.57 (m, 1H), 4.50-4.46 (m, 1H), 4.38-4.34 (m, 1H), 4.28-4.21 (m, 1H), 4.05 (q, J=7.2 Hz, 2H), 3.65-3.57 (m, 2H), 0.94 (t, J=7.2 Hz, 3H), 0.78 (t, J=7.2 Hz, 3H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromobenzo[b]thiophene-2-carboxylic acid) (11.1. and 11.2)

[0564] To a solution of 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromobenzo[b]thiophene-2-carboxylate) (10) (150 mg, 0.16 mmol) in EtOH/H.sub.2O (10 mL/2 mL) was added NaOH (39 mg, 0.98 mmol) and the mixture was stirred at room temperature overnight. Most of the EtOH was removed under reduced pressure and the aqueous residue was adjusted to pH 4-5 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Agilent 10 prep-C18, 10 μm, 250×21.2 mm column, eluting with a gradient of ACN in water with 0.1% TFA, at a flow rate of 20 mL/min) to give separated atropisomers of the title compound (11.1) (peak 1: 10 mg, 7%) and the title compound (11.2) (peak 2: 14 mg, 10%) as white solids.

[0565] Peak 1 (Prep HPLC, rt 8.99 min): (11.1); LCMS-B (ES-API): rt 0.56 min, m/z 856.9, 858.9 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.21-8.19 (m, 6H), 7.97 (br s, 2H), 7.64 (d, J=8.0 Hz, 2H), 7.59-7.53 (m, 2H), 7.42-7.39 (m, 2H), 7.20 (br s, 2H), 4.35-4.33 (m, 2H), 4.27-4.24 (m, 2H).

[0566] Peak 2 (Prep HPLC, rt 12.15 min): (11.2); LCMS-B (ES-API): rt 0.67 min, m/z 856.9, 858.9 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.23-8.16 (m, 6H), 8.01 (br s, 2H), 7.89-7.81 (m, 2H), 7.51 (d, J=8.0 Hz, 2H), 7.44-7.42 (m, 2H), 7.25 (br s, 2H), 4.57-4.50 (m, 2H), 4.19-4.07 (m, 2H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(7-fluoro-4-methoxybenzo[b]thiophene-2-carboxylate) (12) and 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(7-fluoro-4-methoxybenzo[b]thiophene-2-carboxylic acid) (13.1 and 13.2)

[0567] ##STR00047##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(7-fluoro-4-methoxybenzo[b]thiophene-2-carboxylate) (12)

[0568] A solution of ethyl 7-fluoro-3-formyl-4-methoxybenzo[b]thiophene-2-carboxylate (I40) (158 mg, 0.56 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (37 mg, 0.28 mmol) and NaHSO.sub.3 (58 mg, 0.56 mmol) in DMF (3 mL) was heated at 100° C. under N.sub.2 overnight. The mixture was poured into water (30 mL), extracted with EtOAc (30 mL×3) and the combined organic extracts were washed brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (Pet. Ether/EtOAc=40:1 to 20:1 and DCM/MeOH=10:1) to give the title compound (80 mg, 16%) as a yellow solid (mixture of atropisomeric diastereomers). LCMS-B (ES-API): rt 1.21 min, m/z 853.1 [M+H].sup.+.

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(7-fluoro-4-methoxybenzo[b]thiophene-2-carboxylic acid) (13.1 and 13.2)

[0569] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(7-fluoro-4-methoxybenzo[b]thiophene-2-carboxylate) (12) (25 mg, 0.03 mmol) in EtOH/H.sub.2O (5 mL/1 mL) was added NaOH (7 mg, 0.18 mmol) and the mixture was stirred at room temperature overnight. Water (5 mL) was added and the mixture was washed with EtOAc (5 mL×3). The aqueous layer was acidified to pH 4-5 with 3 M aqueous HCl and the resulting precipitate was collected by filtration and purified by prep. HPLC (Shimadzu LC-20AP, BOSTON, 250×2.12 mm, 10 μm column, eluting with a gradient of ACN in water with 0.1% TFA at a flow rate of 20.0 mL/min) to give separated atropisomers of the title compound (13.1) (peak 1: 3.5 mg, 15%) and (13.2) (peak 2: 4 mg, 17%) as yellow solids.

[0570] Peak 1 (Prep HPLC, rt 12.70 min): (13.1); LCMS-B (ES-API): rt 0.56 min, m/z 797.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.18 (s, 2H), 8.02 (br s, 2H), 7.97 (d, J=8.6 Hz, 2H), 7.82 (d, J=8.6 Hz, 2H), 7.43 (t, J=9.1 Hz, 2H), 7.29 (br s, 2H), 6.82 (dd, J=8.8, 3.1 Hz, 2H), 4.38-4.34 (m, 2H), 4.22-4.19 (m, 2H), 3.51 (s, 6H).

[0571] Peak 2 (Prep HPLC, rt 13.46 min): (13.2); LCMS-B (ES-API): rt 0.65 min, m/z 797.0 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.19 (s, 2H), 8.06-7.97 (m, 4H), 7.89 (d, J=8.6 Hz, 2H), 7.31-7.25 (m, 4H), 6.55 (dd, J=8.8, 3.3 Hz, 2H), 4.44-4.39 (m, 2H), 4.21-4.16 (m, 2H), 3.19 (s, 6H).

Diethyl 3,3′-(1,1′-(ethane-1,2-diyl)bis(5-carbamoyl-1H-benzo[d]imidazole-2,1-diyl))bis(4-methylbenzo[b]thiophene-2-carboxylate) (14) and 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methylbenzo[b]thiophene-2-carboxylic acid) (15.1 and 15.2)

[0572] ##STR00048##

(a) Diethyl 3,3′-(1,1′-(ethane-1,2-diyl)bis(5-carbamoyl-1H-benzo[d]imidazole-2,1-diyl))bis(4-methylbenzo[b]thiophene-2-carboxylate) (14)

[0573] A solution of ethyl 3-formyl-4-methylbenzo[b]thiophene-2-carboxylate (I52) (50 mg, 0.75 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (23 mg, 0.07 mmol) and NaHSO.sub.3 (14.6 mg, 0.14 mmol) in DMF (2 mL) was heated at 80° C. overnight under N.sub.2. The mixture was diluted with water (10 mL), extracted with EtOAc (10 mL×3) and the combined organic extracts were washed with water, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (DCM/MeOH=20/1) to give the title compound (20 mg, 13%) as a white solid (mixture of atropisomeric diastereomers in ˜2:1 ratio). LCMS-B (ES-API): rt 3.32 min, m/z 785.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.25-8.24 (m, 2H), 8.11-8.04 (m, 3H), 8.03-7.93 (m, 3H), 7.86 (d, J=8.5 Hz, 1.35H), 7.59 (d, J=8.6 Hz, 0.65H), 7.54-7.48 (m, 1.35H), 7.46-7.41 (m, 0.65H), 7.32 (br s, 2H), 7.21 (d, J=7.1 Hz, 1.35H), 7.02 (d, J=7.3 Hz, 0.65H), 4.78-4.69 (m, 1.35H), 4.59-4.54 (m, 0.65H), 4.15-3.96 (m, 4H), 3.51-3.40 (m, obscured by solvent), 1.70 (s, 4H), 1.54 (s, 2H), 0.90 (t, J=7.1 Hz, 2H), 0.73 (t, J=7.1 Hz, 4H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methylbenzo[b]thiophene-2-carboxylic acid) (15.1 and 15.2)

[0574] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-methylbenzo[b]thiophene-2-carboxylate) (14) (140 mg, 0.18 mmol) in EtOH (10 mL) and water (2 mL) was added NaOH (43 mg, 1.10 mmol) and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and the residue was diluted with water (3 mL) and adjusted to pH 4-5 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Varian-940-LC, YMC-C18, 150×20 mm, 5 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 15.0 mL/min) to give separated atropisomers of the title compound (15.1) (peak 1: 9 mg, 7%) and (15.2) (peak 2: 12.7 mg, 10%) as white solids.

[0575] Peak 1 (Prep HPLC, rt 10.85 min): (15.1); LCMS-B (ES-API): rt 2.82 min, m/z 729.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.24 (d, J=1.7 Hz, 2H), 8.02 (br s, 2H), 7.97 (d, J=8.2 Hz, 2H), 7.87 (d, J=8.6 Hz, 2H), 7.46-7.39 (m, 2H), 7.36-7.26 (m, 4H), 7.05 (d, J=7.2 Hz, 2H), 4.57-4.49 (m, 2H), 3.99-3.95 (m, 2H), 1.56 (s, 6H).

[0576] Peak 2 (Prep HPLC, rt 12.01 min): (15.2); LCMS-B (ES-API): rt 2.80 min, m/z 729.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.24 (d, J=1.6 Hz, 2H), 8.07-7.96 (m, 6H), 7.72 (d, J=8.5 Hz, 2H), 7.48 (t, J=7.7 Hz, 2H), 7.28 (br s, 2H), 7.17 (d, J=7.3 Hz, 2H), 4.30-4.21 (m, 4H), 1.66 (s, 6H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-cyanobenzo[b]thiophene-2-carboxylate) (16) and 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-cyanobenzo[b]thiophene-2-carboxylic acid) (17)

[0577] ##STR00049##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-cyanobenzo[b]thiophene-2-carboxylate) (16)

[0578] A solution of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (139 mg, 0.42 mmol), ethyl 4-cyano-3-formylbenzo[b]thiophene-2-carboxylate (I44) (220 mg, 0.84 mmol) and NaHSO.sub.3 (88 mg, 0.84 mmol) in DMF (15 mL) was heated at 120° C. under N.sub.2 for 16 h. The mixture poured into water (80 mL), extracted with EtOAc (80 mL×3) and the combined organic extracts were washed water (80×2 mL), brine (80 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=1:0 to 20:1) to give the title compound (130 mg, 38%) as a brown solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-C (ES-API): rt 3.42 min, m/z 807.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.58 (d, J=8.3 Hz, 1H), 8.54 (d, J=7.8 Hz, 1H), 8.24 (s, 2H), 8.06-8.00 (m, 7H), 7.83-7.72 (m, 3H), 7.34 (br s, 2H), 4.59-4.44 (m, 2H), 4.38-4.25 (m, 2H), 4.09 (q, J=7.0 Hz, 2H), 3.89 (q, J=7.0 Hz, 2H), 0.98 (t, J=7.1 Hz, 3H), 0.88 (t, J=7.1 Hz, 3H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-cyanobenzo[b]thiophene-2-carboxylic acid) (17)

[0579] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-cyanobenzo[b]thiophene-2-carboxylate) (16) (125 mg, 0.15 mmol) in EtOH/water (5 mL/5 mL) was added NaOH (6 mg, 0.90 mmol) and the mixture was stirred at room temperature for 16 h. The mixture was concentrated under reduced pressure and the residue was diluted with water (5 mL) and adjusted to pH 4-5 with 2 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Agilent, BOSTON, 250×2.12 mm, 10 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 20.0 mL/min) to give the title compound as a mixture of atropisomers (1:1 racemate:meso, 15 mg, 12%). LCMS-C (ES-API): rt 2.43 min, m/z 751.1 [M+H].sup.+ and 2.72 min, m/z 751.1 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.48-8.45 (m, 2H), 8.22 (s, 2H), 8.10-7.98 (m, 2H), 7.94-7.55 (m, 7H), 7.40-7.21 (m, 3H), 4.54-4.52 (m, 2H), 4.22-4.10 (m, 2H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate) (18) and 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-(trifluoromethyl)benzo[b]thiophene-2-carboxylic acid) (19.1 and 19.2)

[0580] ##STR00050##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate) (18)

[0581] To a solution of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (197 mg, 0.6 mmol) in DMF (4 mL) was added H.sub.2O (0.1 mL) followed by oxone (369 mg, 0.6 mmol). A solution of ethyl 3-formyl-4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate (I47) (300 mg, 1.0 mmol) in DMF (2 mL) was then added dropwise over 1 h and the mixture was heated at 80° C. for 2 days. Water (20 mL) was added and the resulting precipitate was collected by filtration and triturated with a 1:1 mixture of Pet. Ether/EtOAc to give the title compound (183 mg, 21%) as a black solid (mixture of atropisomeric diastereomers in ˜1.5:1 ratio). LCMS-B (ES-API): rt 3.20 min, m/z 893.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.66-8.64 (m, 1H), 8.52-8.50 (m, 1H), 8.23-8.20 (m, 2H), 8.06-7.98 (m, 5H), 7.96-7.84 (m, 4H), 7.78-7.76 (m, 1H), 7.32 (br s, 2H), 4.52-4.35 (m, 2.4H), 4.14-4.07 (m, 1.6H), 4.05 (q, J=7.1 Hz, 1.6H), 3.54 (q, J=7.1 Hz, 2.4H), 0.90 (t, J=7.1 Hz, 2.4H), 0.74 (t, J=7.1 Hz, 3.6H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-(trifluoromethyl)benzo[b]thiophene-2-carboxylic acid) (19.1 and 19.2)

[0582] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-(trifluoromethyl)benzo[b]thiophene-2-carboxylate) (18) (183 mg, 0.20 mmol) in EtOH/water (8 mL, 10:1) was added NaOH (80 mg, 2.0 mmol) and the mixture was stirred at room temperature overnight. Most of the EtOH was removed under reduced pressure and the aqueous layer was adjusted to pH 5 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Agilent Prep Star 218, 150×20 mm, 5 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 20.0 mL/min) to give separated atropisomers of the title compound (19.1) (peak 1: 5 mg, 3%) and (19.2) (peak 2: 10.8 mg, 7%) as brown solids.

[0583] Peak 1 (Prep HPLC, rt, 10.27 min): (19.1); LCMS-B (ES-API): rt 2.66 min, m/z 837.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.51 (d, J=7.1 Hz, 2H), 8.21 (s, 2H), 8.01 (br s, 2H), 7.94-7.70 (m, 8H), 7.27 (br s, 2H), 4.29-4.19 (m, 4H).

[0584] Peak 2 (Prep HPLC, rt, 14.98 min): (19.2); LCMS-B (ES-API): rt 2.77 min, m/z 837.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.50-8.48 (m, 2H), 8.22 (s, 2H), 8.01 (br s, 2H), 7.93-7.86 (m, 2H), 7.79-7.69 (m, 6H), 7.28 (br s, 2H), 4.43-4.41 (m, 2H), 4.07-4.05 (m, 2H).

3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromo-7-fluorobenzo[b]thiophene-2-carboxylic acid) (20.1 and 20.2)

[0585] ##STR00051##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromo-7-fluorobenzo[b]thiophene-2-carboxylate) (A7)

[0586] A mixture of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (200 mg, 0.61 mmol) and ethyl 4-bromo-7-fluoro-3-formylbenzo[b]thiophene-2-carboxylate (I49) (404 mg, 1.22 mmol) in AcOH/EtOH (10 mL/10 mL) was heated at 50° C. for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc, washed with water and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM/MeOH=20:1) to give the title compound (260 mg, 45%) as a yellow solid. LCMS-B (ES-API): rt 3.61 min; m/z 475.0 [M/2+H].sup.+.

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromo-7-fluorobenzo[b]thiophene-2-carboxylic acid) (20.1 and 20.2)

[0587] To a solution of 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-bromo-7-fluorobenzo[b]thiophene-2-carboxylate) (A7) (260 mg, 0.27 mmol) in EtOH (10 mL) was added a solution of NaOH (65 mg, 1.62 mmol) in water (2 mL) and the mixture was heated at 50° C. overnight. Most of the EtOH was removed under reduced pressure and the aqueous residue was adjusted to pH 3-4 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Agilent 10 prep-C18, 250×21.2 mm, 10 μm column, eluting with a gradient of MeOH in water with 0.1% TFA, at a flow rate of 20 mL/min) to give separated atropisomers of the title compound (20.1) (peak 1:25 mg, 10%) and title compound (20.2) (peak 2: 90 mg, 37%) as yellow solids.

[0588] Peak 1: (Prep HPLC, rt, 8.59 min): (20.1); LCMS-B (ES-API): rt 2.83 min, m/z 892.8, 894.9 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.21 (s, 2H), 8.02 (br s, 2H), 7.87 (d, J=8.4 Hz, 2H), 7.70-7.59 (m, 4H), 7.42 (t, J=8.8 Hz, 2H), 7.27 (br s, 2H), 4.51-4.34 (m, 4H).

[0589] Peak 2: (Prep HPLC, rt, 10.72 min): (20.2); LCMS-B (ES-API): rt 3.02 min, m/z 892.8, 894.8 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.26-7.75 (m, 8H), 7.44-7.32 (m, 6H), 4.51-4.29 (m, 4H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-4-methoxy-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (21) and 3,3′-(ethane-1,2-diylbis(5-carbamoyl-4-methoxy-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (22.1 and 22.2)

[0590] ##STR00052##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-4-methoxy-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (21)

[0591] A mixture of 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-amino-2-methoxybenzamide) (I33) (145 mg, 0.37 mmol), ethyl 4-chloro-3-formylbenzo[b]thiophene-2-carboxylate (I19) (200 mg, 0.74 mmol) and NaHSO.sub.3 (78 mg, 0.74 mmol) in DMF (15 mL) was heated at 120° C. under N.sub.2 for 16 h. The mixture was poured into water (80 mL), extracted with EtOAc (80 mL×3) and the combined organic extracts were washed water (80×2 mL), brine (80 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by prep. TLC (DCM/MeOH=15:1) to give the title compound (130 mg, 39%) as a yellow solid (mixture of atropisomeric diastereomers: .sup.1H NMR showed a 1:1 mixture of racemate:meso). LCMS-C (ES-API): rt 4.53 min, m/z 885.2, 887.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.27-8.25 (m, 1H), 8.16-8.14 (m, 1H), 7.89 (s, 2H), 7.67-7.65 (m, 3H), 7.54-7.43 (s, 6H), 7.29-7.27 (m, 1H), 4.47-4.42 (m, 2H), 4.25-4.23 (m, 10 H), 4.06-4.05 (m, 2H), 0.95 (t, J=6.4 Hz, 3H), 0.79 (t, J=6.8 Hz, 3H)

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-4-methoxy-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylic acid) (22.1 and 22.2)

[0592] To a solution of 3,3′-(ethane-1,2-diylbis(5-carbamoyl-4-methoxy-1H-benzo[d]imidazole-1,2-diyl))bis(4-chlorobenzo[b]thiophene-2-carboxylate) (21) (130 mg, 0.15 mmol) in EtOH/H.sub.2O (8 mL/8 mL) was added NaOH (35 mg, 0.90 mmol) and the mixture was heated at 80° C. for 16 h. The solvent was removed under reduced pressure and the residue was diluted with water (10 mL) and adjusted to pH 4-5 with 2 M aqueous HCl. The resulting precipitate was collected by filtration and purified by prep. HPLC (Agilent, BOSTON, 250×2.12 mm, 10 μm column, eluting with a gradient of ACN in water with 0.1% formic acid at a flow rate of 20.0 mL/min) to give separated atropisomers of the title compound (22.1) (peak 1: 5 mg, 4%) and the title compound (22.2) (peak 2: 10 mg, 8%) as white solids.

[0593] Peak 1 (Prep HPLC, rt 12.08 min): (22.1); LCMS-C (ES-API): rt 3.58 min, m/z 829.1, 831.0 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.17 (d, J=8.1 Hz, 2H), 7.72 (d, J=8.2 Hz, 2H), 7.67 (s, 2H), 7.58 (t, J=7.9 Hz, 2H), 7.46-7.44 (m, 4H), 7.25-6.61 (m, 2H), 4.37-4.28 (m, 2H), 4.29 (s, 6H), 4.18-4.16 (m, 2H).

[0594] Peak 2 (Prep HPLC, rt 14.84 min): (22.2); LCMS-C (ES-API): rt 3.80 min, m/z 829.1, 831.0 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.14 (d, J=7.7 Hz, 2H), 7.91-7.73 (m, 2H), 7.68 (s, 2H), 7.58-7.48 (m, 2H), 7.44 (s, 2H), 7.30 (d, J=7.3 Hz, 2H), 7.19-7.04 (m, 2H), 4.45-4.43 (m, 2H), 4.28 (s, 6H), 4.12-4.01 (m, 2H).

Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-ethoxybenzo[b]thiophene-2-carboxylate) (23) and 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-ethoxybenzo[b]thiophene-2-carboxylic acid) (24)

[0595] ##STR00053##

(a) Diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-ethoxybenzo[b]thiophene-2-carboxylate) (23)

[0596] A solution of ethyl 4-ethoxy-3-formylbenzo[b]thiophene-2-carboxylate (I54) (500 mg, 1.8 mmol), 4,4′-(ethane-1,2-diylbis(azanediyl))bis(3-aminobenzamide) (I4) (300 mg, 0.91 mmol) and NaHSO.sub.3 (175 mg, 1.8 mmol) in DMF (25 mL) was heated at 80° C. overnight. The mixture was diluted with water, extracted with EtOAc and the combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give the title compound (200 mg, 26%) as a pink solid. LCMS-B (ES-API): rt 3.20 min, m/z 845.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.19 (s, 2H), 8.05 (br s, 2H), 7.95 (d, J=8.4 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 7.71 (d, J=8.3 Hz, 2H), 7.57-7.47 (m, 2H), 7.29 (br s, 2H), 6.82 (d, J=8.1 Hz, 2H), 4.55-4.44 (m, 2H), 4.09-4.07 (m, 2H), 3.69-3.57 (m, 4H), 3.51-3.45 (m, 4H), 0.74 (t, J=7.0 Hz, 6H), 0.35 (t, J=7.0 Hz, 6H).

(b) 3,3′-(Ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-ethoxybenzo[b]thiophene-2-carboxylic acid) (24)

[0597] To a solution of diethyl 3,3′-(ethane-1,2-diylbis(5-carbamoyl-1H-benzo[d]imidazole-1,2-diyl))bis(4-ethoxybenzo[b]thiophene-2-carboxylate) (23) (15 mg, 0.018 mmol) in EtOH (8 mL) was added a solution of NaOH (7 mg, 0.18 mmol) in H.sub.2O (1 mL) and the mixture was stirred at room temperature overnight. Most of the EtOH was removed under reduced pressure and the aqueous residue was adjusted to pH 5 with 1 M aqueous HCl. The resulting precipitate was collected by filtration and dried under reduced pressure to give the title compound as a ˜1.8:1 mixture of atropisomers (5 mg, 35%) as a black solid. LCMS-C (ES-API): rt 2.98 min, m/z 789.2 [M+H].sup.+ & 3.07 min, m/z 789.2 [M+H].sup.+. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.13-8.08 (m, 2H), 7.96-7.90 (m, 2H), 7.65-7.48 (m, 4H), 7.34 (t, J=7.8 Hz, 2H), 7.19 (br s, 0.7H), 7.16 (s, 1.3H), 6.74-6.63 (m, 2H), 6.49-6.42 (m, 0.7H), 6.36-6.30 (m, 1.3H), 4.54-4.45 (m, 1H), 4.24-4.11 (m, 2H), 4.06-3.93 (m, 2H), 3.83-3.76 (m, 1H), 3.58-3.50 (m, 2H), 1.08 (t, J=7.3 Hz, 6H).

[0598] Assays

[0599] Protein Production and Purification

[0600] Biophysical experiments were performed with three different recombinant human STING protein variants designated according to allelic nomenclature of Yi et al., (2013). Codon optimized DNA sequences (for expression in Escherichia coli) encoding amino acid residues 149 to 345 (Swiss Prot Q86WV6) of human STING (WT), human STING (HAQ) and human STING (R232H) were synthesized by GenScript USA Inc (Piscataway, N.J., USA). These were ligated into a modified pET43a E. coli expression vector designed to encode N-terminal His tag followed by tobacco etch virus protease (TEV) cleavage site and a STING gene sequence. The resulting protein sequences for the three allelic variants are listed below:

TABLE-US-00005 His-TEV-hSTING(WT) MGHHHHHHGTENLYFQGSE.sup.149KGNFNVAHGLAWSYYIGYLRLILPELQA RIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLP QQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYS QAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFS LSQEVLRHLRQEEKEEVTVGS.sup.345 His-TEV-hSTING(R232H) MGHHHHHHGTENLYFQGSE.sup.149KGNFNVAHGLAWSYYIGYLRLILPELQA RIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLP QQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYS QAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFS LSQEVLRHLRQEEKEEVTVGS.sup.345 His-TEV-hSTING(HAQ) MGHHHHHHGTENLYFQGSE.sup.149KGNFNVAHGLAWSYYIGYLRLILPELQA RIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLP QQTADRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYS QAGFSREDRLEQAKLFCQTLEDILADAPESQNNCRLIAYQEPADDSSFS LSQEVLRHLRQEEKEEVTVGS.sup.345

[0601] To produce recombinant human STING proteins, expression plasmid encoding above-described constructs were transformed into E. coli BL21 DE3 strain and grown with shaking at 37° C. in 2×1 L volumes of Terrific broth (TB) supplemented with 100 μg/ml Ampicillin until OD.sub.600 of 0.8 was reached. Cultures were then cooled to 16° C. and protein expression induced by the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM and the cultures shaken overnight for further 16 hours. Following expression, cell cultures were centrifuged at 5000×g for 20 min and cell pellet stored frozen at −70° C.

[0602] Protein purification was initiated by thawing the cell pellet in Lysis buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM DTT, 2 mM MgCl.sub.2, 10 mM imidazole, 0.5 mg/ml lysozyme, benzonase endonuclease [EMD Millipore], 1 mM PMSF, complete protease inhibitor tablets EDTA-free [Roche]) using a ratio of 7 ml of buffer per 1 g of cells. Cells were further lysed by 3 passes through an ice cooled Avestin C5 cell crusher and then centrifuged at 48,000×g at 4° C. Supernatant (cell lysate) was filtered through a 5 μm filter and loaded onto 5 mL HiTrap IMAC Sepharose FF column (GE Healthcare) pre-equilibrated with IMAC wash buffer 1 (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM DTT, 10 mM imidazole) using Profinia Affinity chromatography purification system (Bio-Rad). The IMAC column was then sequentially washed with IMAC Wash buffer 1 and IMAC Wash buffer 2 (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM DTT, 20 mM Imidazole) and bound His-TEV-hSTING protein eluted with IMAC Elution buffer (25 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM DTT, 250 mM Imidazole). IMAC-eluted protein was further purified by passing through a HiLoad 26/60 Superdex 75 column pre-equilibrated in storage buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM DTT 0.02% [w/v] sodium azide). Finally, hSTING protein was concentrated to ≥2 mg/ml using Amicon Ultra centrifugal filter unit (Utra-15 MWCO 10 kDa), flash-frozen in liquid nitrogen and stored in −70° C. freezer.

[0603] Differential Scanning Fluorimetry (DSF)

[0604] Differential scanning fluorimetry (DSF) is a rapid screening method for identifying low-molecular-weight ligands that bind and, in doing so, stabilize (or sometimes destabilize) purified proteins (Niesen 2007). DSF monitors thermal unfolding of protein in the presence of a fluorescent dye and is typically performed using a real-time PCR instrument. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein that are gradually being exposed during unfolding. The fluorescence of the dye is quenched in aqueous environments, but when the dye associates with hydrophobic sites on unfolded protein, its fluorescence increases. The fluorescence intensity is plotted as a function of the temperature, generating a sigmoidal curve that can be described by a two-state transition. The inflection point of the transition curve (Tm) is calculated using simple equations such as that of Boltzmann.

[0605] Thermal stability of STING protein (with and without bound ligand) was measured using previously described methodology (Seabrook & Newman, 2013), with samples tested in triplicate using 96-well PCR microplate (AB Gene, ABGAB-0600/W). In a final volume of 20 μL, 2 μM protein in 1× HBS buffer (50 mM HEPES, pH 7.4, 150 mM NaCl) was mixed with SYPRO Orange dye (Sigma-Aldrich S5692, final reaction mix dilution 1:1200) and a compound (final concentration at 100 μM). Sealed plates were placed into Bio-Rad CFX96/C1000 thermocycler and FRET scanning mode (λ.sup.excitation of 490 nm and reads at a λ.sup.emission of 570 nm). Melting curves were recorded from 20° C. to 100° C. in 0.5° C. increments every 30 seconds with a read at each increment. Data were analyzed using “Meltdown analysis” protocol described by Rosa (2015). The melting temperature (T.sub.m) obtained for STING protein alone (1% [v/v] DMSO) was subtracted from T.sub.m obtained for protein incubated with ligand to generate ΔT.sub.m values listed in the table below. DSF data was generated for each compound against 3 different STING protein variants—human STING (WT), human STING (HAQ) and human STING (R232H).

TABLE-US-00006 DSF huSTING DSF huSTING DSF huSTING (HAQ) (WT) (R232H) Example ΔTm (° C.) ΔTm (° C.) ΔTm (° C.) 1.1 31.8 19.9 12.1 1.2 0.13 −7.14 −4.3 2.1 32 21 11.8 2.2 22.2 8.85 6.9 3 −0.55 −1.83 −0.15 4.1 2.85 −0.82 −2.0 4.2 4.56 −0.76 −0.91 5.2 30.94 19.17 11.27 5.1 23.59 6.4 7.51 7 6.96 0.6 −0.34 9 6.7 2.36 1.03 11.1 31.05 17.83 10.45 11.2 −1.75 −5.12 −1.64 13.1 31.23 18.17 11.81 13.2 0.89 −1.43 −2.9 15.1 24.64 10.53 6.6 15.2 31.87 20.41 10.23 17 3.27 −2.58 −3.79 19.1 21.36 9.38 8.21 19.2 12.26 2.74 1.85 20.1 31.76 22.71 14.94 20.2 0.44 −4.12 −4.15 21 4.33 3.41 37.65 22.1 45.505 37.67 30.585 22.2 45.31 35.685 28.13 24 3.15 −0.39 0.81 A1 0.36 1.84 18.93

[0606] Surface Plasmon Resonance (SPR)

[0607] Binding interactions of ligands with STING proteins were quantified using Surface Plasmon Resonance (SPR) with a minimally biotinylated STING protein immobilized on a streptavidin chip surface. In this manner highly active STING protein surfaces were obtained that were not compromised by a low pH required for an amine coupling method. Minimal biotinylation of purified huSTING proteins was performed using a previously described methodology (Chhabra 2012). Briefly, approximately 20 nmol of recombinant STING protein in 1× TBS buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM DTT) was mixed with of EZ-Link™ Sulfo-NHS-LC-LC-Biotin (Thermofisher Scientific, cat #21338) at a molar ratio of 1 to 0.6 and incubated on ice for 2 hours. To remove any unreacted biotin reagent, protein/biotin mixture was passed through a Superdex 75 (10/300 GL) column equilibrated with 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM DTT, 5% [v/v] glycerol. A protein peak containing biotinylated huSTING protein was collected and stored in aliquots at −80° C.

[0608] Streptavidin was simultaneously immobilized in all four channels of a CM5 sensor chip docked in a Biacore instrument (either Biacore S200 or Biacore T200, GE Healthcare) as described previously (Zender 2013). Minimally biotinylated STING protein was captured onto a streptavidin coated chip surface at 8° C. in SPR binding buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 2% [v/v] DMSO) by gradually injecting in a single channel at a constant flow-rate of 2 μL/min until desired capture level was achieved, typically 3000 to 7000 RU (1 RU=1 pg/mm.sup.2).

[0609] All binding experiments were performed at 8° C. in SPR binding buffer. To determine binding affinity, compound interaction with immobilized STING protein was analyzed using dose-response experiments. Fresh 10 mM DMSO solutions of compound were diluted directly into SPR binding buffer typically to a concentration of 50 μM and then further diluted 2-fold or 3-fold aiming for either a 5- or 7-point concentration series range. Each ligand concentration series was injected at a constant flow rate of 60 μL/min with a 90 second association and a 180 second dissociation time. These were modified for compounds with longer residence times, so that curves could reach steady-state, or so that compound would be fully dissociated before the subsequent injection. Where appropriate, tighter-binding compounds (roughly K.sub.D<1 uM) were tested using a single-cycle kinetics format (Karlsson 2006), with long association and dissociation times (typically 450 s and 1800 s, respectively).

[0610] Scrubber 2 (www.biologic.com.au) was utilized for data processing, where signals were referenced against the blank surface (streptavidin+D-biotin) and further corrected for DMSO refractive index change and then “double-referenced” using a buffer-blank injection (Papalia 2006). Responses were fitted to either a 1:1 steady-state affinity model or a 1:1 kinetic model (that included a mass-transport component), available within Scrubber.

TABLE-US-00007 SPR (HAQ) SPR (WT) Example K.sub.D (μM) K.sub.D (μM) 1.1 0.0017 0.119 1.2 — — 2.1 0.0074 0.031 2.2 0.124 — 3 5.06 6.3 4.1 35.13 500 4.2 16.61 23 5.1 0.02091 0.692 5.2 0.0014 0.122 7 28 80 9 10.1 11.1 0.001638 0.063 11.2 1.43 13.1 0.004002 0.29995 13.2 2.371 15.1 0.43 15.2 0.004984 0.191 17 38 20.1 0.001652 0.016 24 37 A1 14.9 15.95

[0611] THP-1 Reporter Cell Line Assays

[0612] THP-1 STING Lucia ISG cells (InvivoGen #thpl-isg) were cultured in RPMI-1640 containing 2 mM L-glutamine, 25 mM HEPES, 100 μg/mL Normocin (InvivoGen) and 10% heat-inactivated FBS. Cells were seeded at a density of 7×10.sup.5 cells/mL, maintained at 37° C./5% CO.sub.2, and passaged every 3-4 days. Selection pressure was maintained by the addition of 100 μg/mL Zeocin every second passage.

[0613] Assay conditions: cells were harvested and resuspended at a concentration of 5×10.sup.5 cells/mL in fresh growth media. Of this cell preparation 20 μL per well was dispensed into 384 well cell culture plates (Greiner, #781098X) and incubated at 37° C./5% CO.sub.2 for 2 hours.

[0614] Concentration Response Curves (commonly 11 doses) were prepared by 2.5 fold serial dilution starting from 10mM compound stock solution in DMSO. Compound dilutions were transferred into the cell culture plates using a pintool (0.1 μL transfer), control wells were matched for DMSO. Positive control wells received ML RR-S2 CDA (Med Chem Express HY12885B) in 5 μL of media to a final concentration of 40 μM. All other wells received additional 5 μL of media only to equalize volume. The plates were incubated for 24 h at 37° C./5% CO.sub.2.

[0615] For the detection step, QUANTI-Luc (InvivoGen #rep-qlc1) was prepared according to manufacturer's instruction and 10 μL were added per well to the culture plates. Plates were shaken for ten seconds on an orbital shaker and left in the dark at room temperature for two minutes, before luminescence was read on a Perkin Elmer Envision plate reader.

[0616] Data analysis: Luminescence raw data was normalized % activity relative to the signal of the positive control wells and negative control wells which were not treated with compound. The following formula was used to calculate normalized % activity from raw signals:

% activity=(sample−negative)/(positive−negative)

[0617] The THP-1 STING Lucia ISG cell EC.sub.50 provides an assessment of activity at the human HAQ isoform of STING. For assessing compound activity against the WT human STING isoform, an equivalent method was conducted where the cells used were THP-1-Dual KI-hSTING-R232 (InvivoGen #thpd-r232).

TABLE-US-00008 EC.sub.50 THP-I STING EC.sub.50 THP-1 Dual KI- Example Lucia ISG cells (μM) hSTING-R232 cells (μM) 1.1 0.681 0.271 1.2 14.2 6.33 2.1 1.83 0.518 2.2 8.32 4.30 3 >40 >40 4.1 >40 >40 4.2 >40 18.97 5.1 >40 13.59 5.2 1.66 0.489 6 >40 >40 7 >40 7.523 8 >40 >40 9 >40 3.653 10 19.886 12.666 11.1 0.182 0.172 11.2 5.478 2.828 12 5.082 9.181 13.1 8.306 3.747 13.2 >40 >40 14 >40 >40 15.1 1.541 1.012 15.2 0.272 0.199 16 >40 >40 17 14.927 9.399 18 3.737 >40 19.1 0.189 0.196 19.2 2.60 3.663 20.1 1.576 0.632 20.2 14.672 5.809 21 0.019 0.007 22.1 0.256 0.130 22.2 1.014 0.498 23 >40 >40 24 >40 >40 A1 18.397 11.987 A2 9.528 3.166

[0618] Statements of Invention

[0619] 1. A compound of formula I:

##STR00054##

[0620] wherein:

[0621] Y is (CH.sub.2).sub.n, where n is from 2 to 4;

[0622] W.sup.1 and W.sup.11 are independently selected from OH and OR.sup.P, where R.sup.P is Me or Et;

[0623] A.sup.1 is CR.sup.A or N;

[0624] A.sup.2 is CR.sup.B or N;

[0625] A.sup.3 is CR.sup.C or N;

[0626] A.sup.4 is CR.sup.D or N;

[0627] where no more than two of A.sup.1, A.sup.2, A.sup.3, and A.sup.4 may be N;

[0628] one or two of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, OEt, CH.sub.2OH, CH.sub.2OMe and OH;

[0629] the remainder of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are H;

[0630] A.sup.11 is CR.sup.AA or N;

[0631] A.sup.12 is CR.sup.BB or N;

[0632] A.sup.13 is CR.sup.CC or N;

[0633] A.sup.14 is CR.sup.DD or N;

[0634] where no more than two of A.sup.11, A.sup.12, A.sup.13 and A.sup.14 may be N;

[0635] one or two of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, OEt, CH.sub.2OH, CH.sub.2OMe and OH;

[0636] the remainder of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD, (if present) are H;

[0637] R.sup.C1, R.sup.C3 and R.sup.C4 are independently selected from H, Cl, F, Br, Me, OMe, OEt, cyano, CF.sub.3, CH.sub.2OH, CH.sub.2OMe, C.sub.2-4 alkenyl and C.sub.5heterocyclyl;

[0638] R.sup.C11, R.sup.C13 and R.sup.C14 are independently selected from H, Cl, F, Br, Me, OMe, OEt, cyano, CF.sub.3, CH.sub.2OH, CH.sub.2OMe, C.sub.2-4alkenyl and C.sub.5heterocyclyl.

[0639] 2. A compound according to statement 1, wherein A.sup.11=A.sup.1, A.sup.12=A.sup.2, A.sup.13=A.sup.3, A.sup.14=A.sup.4, R.sup.C11=R.sup.C1, R.sup.C13=R.sup.C3, R.sup.C14=R.sup.C4, W.sup.11=W.sup.1.

[0640] 3. A compound according to statements 1 or 2, wherein A.sup.1 is CR.sup.A.

[0641] 4. A compound according to statements 1 or 2, wherein A.sup.1 is N.

[0642] 5. A compound according to any one of statements 1 to 4, wherein A.sup.2 is CR.sup.B.

[0643] 6. A compound according to any one of statements 1 to 4, wherein A.sup.2 is N.

[0644] 7. A compound according to any one of statements 1 to 6, wherein A.sup.3 is CR.sup.C.

[0645] 8. A compound according to any one of statements 1 to 6, wherein A.sup.3 is N.

[0646] 9. A compound according to any one of statements 1 to 6, wherein A.sup.4 is CR.sup.D.

[0647] 10. A compound according to any one of statements 1 to 6, wherein A.sup.4 is N.

[0648] 11. A compound according to statements 1 or 2, wherein A.sup.1 is CR.sup.A, A.sup.2 is CR.sup.B, A.sup.3 is CR.sup.C, and A.sup.4 is CR.sup.D.

[0649] 12. A compound according to statements 1 or 2, wherein one of A.sup.1, A.sup.2, A.sup.3 and A.sup.4 are N.

[0650] 13. A compound according to statements 1 or 2, wherein two of A.sup.1, A.sup.2, A.sup.3 and A.sup.4 are N.

[0651] 14. A compound according to any one of statements 1 to 13, wherein A.sup.11 is CR.sup.AA.

[0652] 15. A compound according to any one of statements 1 to 13, wherein A.sup.11 is N.

[0653] 16. A compound according to any one of statements 1 to 15, wherein A.sup.12 is CR.sup.BB.

[0654] 17. A compound according to any one of statements 1 to 15, wherein A.sup.12 is N.

[0655] 18. A compound according to any one of statements 1 to 17, wherein A.sup.13 is CIR.sup.CC.

[0656] 19. A compound according to any one of statements 1 to 17, wherein A.sup.13 is N.

[0657] 20. A compound according to any one of statements 1 to 19, wherein A.sup.14 is CR.sup.DD.

[0658] 21. A compound according to any one of statements 1 to 19, wherein A.sup.14 is N.

[0659] 22. A compound according to any one of statements 1 to 13, wherein A.sup.11 is CR.sup.AA, A.sup.12 is CR.sup.BB, A.sup.13 is CR.sup.CC, and A.sup.14 is CR.sup.DD.

[0660] 23. A compound according to any one of statements 1 to 13, wherein one of A.sup.11, A.sup.12, A.sup.13 and A.sup.14 are N.

[0661] 24. A compound according to any one of statements 1 to 13, wherein two of A.sup.11, A.sup.12, A.sup.13 and A.sup.14 are N.

[0662] 25. A compound according to any one of statements 1 to 13, wherein the compound is of formula IIIb:

##STR00055##

[0663] 26. A compound according to any one of statements 1 to 13, wherein the compound is of formula IIIc:

##STR00056##

[0664] 27. A compound according to any one of statements 1 to 13, wherein the compound is of formula IIId:

##STR00057##

[0665] 28. A compound according to any one of statements 1 to 13, wherein the compound is of formula IIIe:

##STR00058##

[0666] 29. A compound according to any one of statements 1 to 28, wherein one or two of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano and OMe, the remainder (if present) are H.

[0667] 30. A compound according to any one of statements 1 to 28, wherein one or two of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl and OMe, the remainder (if present) are H.

[0668] 31. A compound according to any one of statements 1 to 28, wherein one or two of R.sup.A, R.sup.B, R.sup.C and R.sup.D, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3 and OMe, the remainder (if present) are H.

[0669] 32. A compound according to any one of statements 1 to 31, wherein: [0670] R.sup.A (if present) is selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, OEt and CH.sub.2OMe; [0671] R.sup.B (if present) is H; [0672] R.sup.C (if present) is H; [0673] R.sup.D (if present) is selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, CH.sub.2OH and CH.sub.2OMe.

[0674] 33. A compound according to any one of statements 1 to 31, wherein: [0675] R.sup.A (if present) is selected from F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano and OMe; [0676] R.sup.B (if present) is H; [0677] R.sup.C (if present) is H; [0678] R.sup.D (if present) is selected from H, F, Br, Me and OMe.

[0679] 34. A compound according to any one of statements 1 to 31, wherein: [0680] R.sup.A (if present) is selected from Cl, Br and OMe; [0681] R.sup.B (if present) is H; [0682] R.sup.C (if present) is H; [0683] R.sup.D (if present) is selected from H, F, Br, Me and OMe.

[0684] 35. A compound according to statement 11, wherein: [0685] R.sup.A is Cl; [0686] R.sup.B is H; [0687] R.sup.C is H; [0688] R.sup.D is H.

[0689] 36. A compound according to statement 11, wherein: [0690] R.sup.A is Cl; [0691] R.sup.B is H; [0692] R.sup.C is H; [0693] R.sup.D is Me.

[0694] 37. A compound according to statement 11, wherein: [0695] R.sup.A is Cl; [0696] R.sup.B is H; [0697] R.sup.C is H; [0698] R.sup.D is Br.

[0699] 38. A compound according to statement 11, wherein: [0700] R.sup.A is Br; [0701] R.sup.B is H; [0702] R.sup.C is H; [0703] R.sup.D is H.

[0704] 39. A compound according to statement 11, wherein: [0705] R.sup.A is Cl; [0706] R.sup.B is H; [0707] R.sup.C is H; [0708] R.sup.D is F.

[0709] 40. A compound according to statement 11, wherein: [0710] R.sup.A is Cl; [0711] R.sup.B is H; [0712] R.sup.C is H; [0713] R.sup.D is OMe.

[0714] 41. A compound according to statement 11, wherein: [0715] R.sup.A is Br; [0716] R.sup.B is H; [0717] R.sup.C is H; [0718] R.sup.D is F.

[0719] 42. A compound according to statement 11, wherein: [0720] R.sup.A is OMe; [0721] R.sup.B is H; [0722] R.sup.C is H; [0723] R.sup.D is H.

[0724] 43. A compound according to statement 11, wherein: [0725] R.sup.A is CF.sub.3; [0726] R.sup.B is H; [0727] R.sup.C is H; [0728] R.sup.D is H.

[0729] 44. A compound according to statement 11, wherein: [0730] R.sup.A is CH.sub.3; [0731] R.sup.B is H; [0732] R.sup.C is H; [0733] R.sup.D is H.

[0734] 45. A compound according to any one of statements 1 to 44, wherein one or two of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano and OMe, the remainder (if present) are H.

[0735] 46. A compound according to any one of statements 1 to 44, wherein one or two of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl and OMe, the remainder (if present) are H.

[0736] 47. A compound according to any one of statements 1 to 44, wherein one or two of R.sup.AA, R.sup.BB, R.sup.CC and R.sup.DD, (if present) are selected from H, F, Cl, Br, Me, CF.sub.3 and OMe, the remainder (if present) are H.

[0737] 48. A compound according to any one of statements 1 to 44, wherein: [0738] R.sup.AA (if present) is selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, OEt and CH.sub.2OMe; [0739] R.sup.BB (if present) is H; [0740] R.sup.CC (if present) is H; [0741] R.sup.DD (if present) is selected from H, F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano, OMe, CH.sub.2OH and CH.sub.2OMe.

[0742] 49. A compound according to any one of statements 1 to 44, wherein: [0743] R.sup.AA (if present) is selected from F, Cl, Br, Me, CF.sub.3, cyclopropyl, cyano and OMe; [0744] R.sup.BB (if present) is H; [0745] R.sup.CC (if present) is H; [0746] R.sup.DD (if present) is selected from H, F, Br, Me and OMe.

[0747] 50. A compound according to any one of statements 1 to 44, wherein: [0748] R.sup.AA (if present) is selected from Cl, Br and OMe; [0749] R.sup.BB (if present) is H; [0750] R.sup.CC (if present) is H; [0751] R.sup.DD (if present) is selected from H, F, Br, Me and OMe.

[0752] 51. A compound according to statement 22, wherein: [0753] R.sup.AA is Cl; [0754] R.sup.BB is H; [0755] R.sup.CC is H; [0756] R.sup.DD is H.

[0757] 52. A compound according to statement 22, wherein: [0758] R.sup.AA is Cl; [0759] R.sup.BB is H; [0760] R.sup.CC is H; [0761] R.sup.DD is Me.

[0762] 53. A compound according to statement 22, wherein: [0763] R.sup.AA is Cl; [0764] R.sup.BB is H; [0765] R.sup.CC is H; [0766] R.sup.DD is Br.

[0767] 54. A compound according to statement 22, wherein: [0768] R.sup.AA is Br; [0769] R.sup.BB is H; [0770] R.sup.CC is H; [0771] R.sup.DD is H.

[0772] 55. A compound according to statement 22, wherein: [0773] R.sup.AA is Cl; [0774] R.sup.BB is H; [0775] R.sup.CC is H; [0776] R.sup.DD is F.

[0777] 56. A compound according to statement 22, wherein: [0778] R.sup.AA is Cl; [0779] R.sup.BB is H; [0780] R.sup.CC is H; [0781] R.sup.DD is OMe.

[0782] 57. A compound according to statement 22, wherein: [0783] R.sup.AA is Br; [0784] R.sup.BB is H; [0785] R.sup.CC is H; [0786] R.sup.DD is F.

[0787] 58. A compound according to statement 22, wherein: [0788] R.sup.AA is OMe; [0789] R.sup.BB is H; [0790] R.sup.CC is H; [0791] R.sup.DD is H.

[0792] 59. A compound according to statement 11, wherein: [0793] R.sup.A is CF.sub.3; [0794] R.sup.B is H; [0795] R.sup.C is H; [0796] R.sup.D is H.

[0797] 60. A compound according to statement 11, wherein: [0798] R.sup.A is CH.sub.3; [0799] R.sup.B is H; [0800] R.sup.C is H; [0801] R.sup.D is H.

[0802] 61. A compound according to any one of statements 1 to 60, wherein R.sup.C11=R.sup.C1, R.sup.C13=R.sup.C3 and R.sup.C14=R.sup.C4.

[0803] 62. A compound according to any one of statements 1 to 61, wherein R.sup.C1, R.sup.C3 and R.sup.C4 are independently selected from H, Cl, F, Br, Me, OMe, cyano, CF.sub.3 and CH.sub.2OH.

[0804] 63. A compound according to any one of statements 1 to 61, wherein R.sup.C1, R.sup.C3 and R.sup.C4 are independently selected from H, Cl, F, CF.sub.3, OMe and CH.sub.2OH

[0805] 64. A compound according to any one of statements 1 to 61, wherein R.sup.C1, R.sup.C3 and R.sup.C4 are independently selected from H, F and OMe.

[0806] 65. A compound according to any one of statements 1 to 64, wherein two of R.sup.C1, R.sup.C3 and R.sup.C4 are H, and the other is selected from the defined groups, except H.

[0807] 66. A compound according to any one of statements 1 to 64, wherein one of R.sup.C1, R.sup.C3 and R.sup.C4 is H, and the other two are independently selected from the defined groups, except H.

[0808] 67. A compound according to any one of statements 1 to 61, wherein R.sup.C1, R.sup.C3 and R.sup.C4 are H.

[0809] 68. A compound according to any one of statements 1 to 61, wherein: [0810] R.sup.C1 is H; [0811] R.sup.C3 is H; and [0812] R.sup.C4 is F.

[0813] 69. A compound according to any one of statements 1 to 61, wherein: [0814] R.sup.C1 is OMe; [0815] R.sup.C3 is H; and [0816] R.sup.C4 is H.

[0817] 70. A compound according to any one of statements 1 to 69, wherein R.sup.C11, R.sup.C13 and R.sup.C14 are independently selected from H, Cl, F, Br, Me, OMe, cyano, CF.sub.3 and CH.sub.2OH.

[0818] 71. A compound according to any one of statements 1 to 69, wherein R.sup.C11, R.sup.C13 and R.sup.C14 are independently selected from H, Cl, F, CF.sub.3, OMe and CH.sub.2OH.

[0819] 72. A compound according to any one of statements 1 to 69, wherein R.sup.C11, R.sup.C13 and R.sup.C14 are independently selected from H, F and OMe.

[0820] 73. A compound according to any one of statements 1 to 72, wherein two of R.sup.C11, R.sup.C13 and R.sup.C14 are H, and the other is selected from the defined groups, except H.

[0821] 74. A compound according to any one of statements 1 to 72, wherein one of R.sup.C11, R.sup.C13 and R.sup.C14 is H, and the other two are independently selected from the defined groups, except H.

[0822] 75. A compound according to any one of statements 1 to 69, wherein R.sup.C11, R.sup.C13 and R.sup.C14 are H.

[0823] 76. A compound according to any one of statements 1 to 69, wherein: [0824] R.sup.C11 is H; [0825] R.sup.C13 is H; and [0826] R.sup.C14 is F.

[0827] 77. A compound according to any one of statements 1 to 69, wherein: [0828] R.sup.C11 is OMe; [0829] R.sup.C13 is H; and [0830] R.sup.C14 is H.

[0831] 78. A compound according to any one of statements 1 to 77, wherein Y is (CH.sub.2).sub.n, where n is from 2 to 3.

[0832] 79. A compound according to any one of statements 1 to 77, wherein Y is (CH.sub.2).sub.2.

[0833] 80. A compound according to any one of statements 1 to 77, wherein Y is (CH.sub.2).sub.3.

[0834] 81. A compound according to any one of statements 1 to 77, wherein Y is (CH.sub.2).sub.4.

[0835] 82. A compound according to any one of statements 1 to 81, wherein W.sup.1 and W.sup.11 are OH.

[0836] 83. A compound according to any one of statements 1 to 82, wherein W.sup.1 and W.sup.11 are OR.sup.P, where R.sup.P is Me or Et.

[0837] 84. A compound as defined in any one of statements 1 to 83, for use in a method of therapy.

[0838] 85. A pharmaceutical composition comprising a compound as defined in any one of statements 1 to 83, and a pharmaceutically acceptable excipient.

[0839] 86. A method of treatment or prevention of a disease ameliorated by the modulation of STING, comprising administering to a patient in need of treatment, a compound as defined in any one of statements 1 to 83, or a pharmaceutical composition according to statement 85.

[0840] 87. The use of a compound as defined in any one of statements 1 to 83, in the manufacture of a medicament for treating or preventing disease ameliorated by the modulation of STING.

[0841] 89. A compound as defined in any one of statements 1 to 83, or pharmaceutical composition according to statement 85 for use in the treatment or preventing of disease ameliorated by the modulation of STING.

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BENZOTHIOPHENE, THIENOPYRIDINE AND THIENOPYRIMIDINE DERIVATIVES FOR THE MODULATION OF STING

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C07D409/14

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A61P31/00

HUMAN NECESSITIES

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C07D471/14

CHEMISTRY; METALLURGY

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C07D519/00

CHEMISTRY; METALLURGY

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A61P35/00

HUMAN NECESSITIES

International classification

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C07D519/00

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Abstract

Claims

Description