science.sciencemag.org/content/369/6506/eaba6098/suppl/DC1

Supplementary Materials for

An orally available non-nucleotide STING agonist with antitumor activity

Bo-Sheng Pan*, Samanthi A. Perera*†, Jennifer A. Piesvaux*, Jeremy P. Presland*, Gottfried K. Schroeder*†, Jared N. Cumming*, B. Wesley Trotter*†, Michael D. Altman,

Alexei V. Buevich, Brandon Cash, Saso Cemerski, Wonsuk Chang, Yiping Chen, Peter J. Dandliker, Guo Feng, Andrew Haidle, Timothy Henderson, James Jewell,

Ilona Kariv, Ian Knemeyer, Johnny Kopinja, Brian M. Lacey, Jason Laskey, Charles A. Lesburg, Rui Liang, Brian J. Long, Min Lu, Yanhong Ma, Ellen C. Minnihan,

Greg O’Donnell, Ryan Otte, Laura Price, Larissa Rakhilina, Berengere Sauvagnat, Sharad Sharma, Sriram Tyagarajan, Hyun Woo, Daniel F. Wyss, Serena Xu,

David Jonathan Bennett†, George H. Addona†

*These authors contributed equally to this work. †Corresponding author. Email: samanthi_perera@merck.com (S.A.P.); gottfried.schroeder@merck.com

(G.K.S.); wes.trotter@kronosbio.com (B.W.T.); jonathan.bennett@merck.com (D.J.B.); george_addona@merck.com (G.H.A.)

Published 21 August 2020, Science 369, eaba6098 (2020)

DOI: 10.1126/science.aba6098

This PDF file includes:

Materials and Methods Supplementary Text S1 and S2 Figs. S1 to S11 Tables S1 to S6 References

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TABLE OF CONTENTS Page Materials and Methods 3-66

• Preparation of [3H]-2’,3’-cGAMP .......................................................................................3 • Preparation of [3H]-MSA-2..................................................................................................3 • Determination of pKa of MSA-2 ..........................................................................................6 • High throughput phenotypic screen .....................................................................................6 • Methods for accessing cellular activity of test compounds .................................................6 • Assessment of MSA-2 Activation of the STING-TBK1-IRF-3 Pathway

in THP-1 Cells by Western Blot ..........................................................................................8 • Preparation of recombinant full-length human and mouse STING ....................................9 • Protein production for X-ray and NMR ...............................................................................9 • Recombinant STING cytosolic domains for surface plasma resonance experiments .......10 • Competitive [3H]-2’,3’-cGAMP displacement assay ........................................................11 • Preparation of working stock [3H]-MSA-2 for binding studies .........................................11 • Saturation [3H]-MSA-2 binding of hSTING-WT ..............................................................11 • Homologous [3H]-MSA-2 competition assay ....................................................................12 • 1H NMR experiments to estimate the equilibrium constants of MSA-2

and compound 2 dimerization ............................................................................................12 • Surface plasmon resonance experimental methods ...........................................................13 • Automated Ligand Identification System (ALIS)..............................................................14 • Sample Preparation for ALIS Experiments .......................................................................14 • Derivation of equation describing behavior of Model 2 in a homologous

radioligand competition experiments .................................................................................15 • Derivation of equation describing behavior of Model 3 in homologous

radioligand competition experiments .................................................................................16 • Methods for simulating the effect of extracellular pH on intracellular

[free MSA-2] and STING activation .................................................................................19 • Mice and tumor models .....................................................................................................21 • In Vivo Efficacy Drug Treatments .....................................................................................21 • Pharmacokinetic/pharmacodynamics (PK/PD) studies .....................................................21 • In vivo interstitial pH measurement ...................................................................................23 • Immunohistochemistry (IHC) ...........................................................................................23 • Identification of “STING Agonist Fingerprints” by 2D and 1D NMR .............................23 • Crystallography and Surface Area Calculations ................................................................24 • Computational Design of Covalent Dimers .......................................................................26 • Chemical Synthesis ...................................................................................................... 26-66

Supplementary Text S1 to S2 66-67 Figs. S1 to S11 68-81 Tables S1 to S6 82-90 SAFETY STATEMENT Given the highly potent nature of molecules whose preparation is detailed in this section, and their demonstrated ability to induce cytokine elevation in vitro and in vivo, proper procedures for handling of highly potent compounds should be followed at all times in accord with specific institutional policies.

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Materials and Methods Preparation of [3H]-2’,3’-cGAMP

N

N

ON

HO

NH2N

OH

O PO

HOO

PO

OHO

POH

HOO NN

O

N

OH

HN

O

H2N

OH

OPO OH

O

PO

OHOPO

HO

OH

O

OP O

OP

O

OOH

O N

N

N

N

NH2

OH

OHO

N

N

N

NH

O

NH2

OH

T

TT

+T

TT

A buffer solution was prepared by combining aqueous solutions of the following: 1 M Tris buffer pH 7.5 (10 mL), 5 M NaCl (5 mL), 1 M MgCl2 (2.5 mL); and diluting to a final volume of 250 mL with de-ionized water. This buffer solution was used in the reaction below. Herring DNA solution (1.37 mL of a 0.3 mg Herring DNA / 1 mL buffer solution) and cGAMP synthase enzyme solution (72 µL of a 3.1 mg enzyme / 1 mL buffer solution) were mixed in a 4 mL vial. The solution was stirred for approximately 15 minutes at room temperature then transferred to a 50 mL round bottom flask containing [3H]-ATP (25 mCi; specific activity = 29.8 Ci/mmol; obtained from Perkin Elmer) and [3H]-GTP (10.2 mCi; specific activity = 12.2 Ci/mmol; obtained from Perkin Elmer) in 360 µL of buffer solution. The resulting solution was stirred at 37 oC overnight. The reaction mixture was transferred to an Amicon Ultra-15 10K centrifuge tube and centrifuged for 1 hour at 4,000 g. The resulting filtrate was purified on a semi-prep Mono-Q column using the following conditions: Mobile Phase A (0.05 M Tris Cl, pH 8.5) and Mobile Phase B (0.05 M Tris Cl, 0.5 M NaCl, pH 8.5); and the following elution scheme: 100% A for 5 minutes followed by a linear gradient to 50:50 (A:B) over 25 minutes at a flow rate of 3 mL/min. [3H]-cGAMP was detected by UV absorbance at 254 nm. The collected product fractions were combined to give 13.8 mCi of the desired product (specific activity = 36.6 Ci/mmol; 99.8 % radiochemical purity). Preparation of [3H]-MSA-2

S O

OOH

O

O

S O

OO

O

HO

S O

OO

HO

O

S O

OO

O

CbzO

S O

OO

O

O

S O

OO

O

HO S O

OOH

O

OCT3

TMSCHN2 AlCl3, DCM

NH

N1. CT3I, Cs2CO3

,DMF, 20 °C, overnight

2. LiOH, CH3CN,H2O, 20 °C, overnight

DMF, MeOH50

°C, 30 min

97%

carried on as a mixture (77:23)

20 °C, 24 h

CbzCl Et3N, DCM 20 °C, 2 h

60%, 2 stepsMeOH. DCM0

°C, 1 h

98%

yield: 20.14 mCispecific activity: 73.93 mCi/mmol

4-(5-Methoxy-6-(methoxy-t3)benzo[b]thiophen-2-yl)-4-oxobutanoic acid

4

S O

OOH

O

OCT3

Step 1: Methyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SiN+ N

-

SO

O

O

OOH

SO

O

O

OO

TMS-diazomethane (2.0 M in diethyl ether, 5.5 mL, 11 mmol) was added dropwise to a mixture of 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (2.15 g, 7.30 mmol) in dichloromethane (50 mL) and methanol (50 mL) at 0°C°C. The reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched with acetic acid (added dropwise until bubbling ceased). The reaction mixture was concentrated under reduced pressure to afford 2.20 g (7.13 mmol, 98% yield) of crude methyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate, which was used without purification in the next step. LCMS (C15H17O5S) (ES, m/z): 309 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 8.22 (s, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.61 (s, 3H), 3.35 – 3.30 (m, 2H), 2.71 – 2.66 (m, 2H). Proton (1H) NMR spectra for small molecule characterization were collected on a Varian VNMRS 500 MHz instrument at 25°C. Step 2: Methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate and methyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

AlCl

Cl ClSO

O

O

OO

SHO

O

O

OO

SO

HO

O

OO

Aluminum chloride (5.71 g, 42.8 mmol) was added to a mixture of methyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.20 g, 7.13 mmol) in dichloromethane (250 mL) at 20°C. The reaction mixture was stirred at 20°C for 24 hours. The reaction mixture was cooled to 0°C and quenched with water (50 mL, added dropwise via addition funnel). The reaction mixture was then warmed to 20°C and diluted with additional dichloromethane (250 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate/dichloromethane) to afford an inseparable mixture of methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (77%) and methyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (23%) in a combined yield of 1.82 g (6.18 mmol). LCMS (C14H15O5S) (ES, m/z): 295 [M+H]+. Step 3: Methyl 4-(5-(((benzyloxy)carbonyl)oxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate and methyl 4-(6-(((benzyloxy)carbonyl)oxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

5

SHO

O

O

OO

SO

O

O

OO

O O

OO

Cl

N

SO

O

O

OO

O OSO

HO

O

OO

CBZ-Cl (1.06 mL, 7.42 mmol) was added to a mixture of methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (77%) and methyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (23%) (1.82 g, 6.18 mmol) and triethylamine (1.29 mL, 9.28 mmol) in dichloromethane (30 mL) at 0°C. The reaction mixture was then warmed to 20°C and stirred for an additional 2 hours. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (ethyl acetate/hexanes) to afford: Peak 1: Methyl 4-(5-(((benzyloxy)carbonyl)oxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate, 410 mg, 0.957 mmol, 13% yield over two steps. LCMS (C22H21O7S) (ES, m/z): 429 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 8.30 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.48 – 7.38 (m, 5H), 5.30 (s, 2H), 3.87 (s, 3H), 3.61 (s, 3H), 3.40 – 3.33 (m, 2H), 2.69 (t, J = 6.4 Hz, 2H). Peak 2: Methyl 4-(6-(((benzyloxy)carbonyl)oxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate, 1.84 g, 4.29 mmol, 60% yield over two steps. LCMS (C22H21O7S) (ES, m/z): 429 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 8.32 (s, 1H), 8.01 (s, 1H), 7.71 (s, 1H), 7.47 – 7.37 (m, 5H), 5.31 (s, 2H), 3.85 (s, 3H), 3.62 (s, 3H), 3.41 – 3.36 (m, 2H), 2.74 – 2.68 (m, 2H). Step 4: Methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

NH

N

SO

O

O

OO

O OSHO

O

O

OO

1-Methylpiperazine (1.4 mL, 13 mmol) was added to a mixture of methyl 4-(6-(((benzyloxy)carbonyl)oxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (1.84 g, 4.29 mmol) in dimethylformamide (DMF, 5 mL) and methanol (5 mL) at 20°C. The reaction mixture was then heated to 50°C and stirred for an additional 30 minutes. The reaction mixture was cooled to 20°C and partially concentrated under reduced pressure (to remove methanol). The crude mixture was purified by silica gel chromatography (ethyl acetate/dichloromethane) to afford 1.22 g of methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (4.15 mmol, 97% yield). LCMS (C14H15O5S) (ES, m/z): 295 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.18 (s, 1H), 7.47 (s, 1H), 7.32 (s, 1H), 3.86 (s, 3H), 3.61 (s, 3H), 3.33 – 3.29 (m, 2H), 2.71 – 2.66 (m, 2H).

6

S O

OO

O

HO S O

OOH

O

OCT3

Step 5: 4-(5-Methoxy-6-(methoxy-t3)benzo[b]thiophen-2-yl)-4-oxobutanoic acid Methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (1.0 mg, 0.003 mmol) was added to a Tritium reaction vessel, followed by cesium carbonate (1.0 mg, 0.003 mmol), DMF (0.1 mL), and iodomethane, [3H] (0.05 mL, 50 mCi). The vessel was sealed and the solution was stirred overnight at room temperature. This procedure was repeated a second time, and the combined crude product (45 mCi) was purified by HPLC, transferred to a 10 mL flask, and dried under reduced pressure. Acetonitrile (0.5 mL) and aqueous lithium hydroxide (1 M, 0.1 mL) were added and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure and purified by HPLC. The mobile phase was removed under reduced pressure to afford 4-(5-methoxy-6-(methoxy-t3)benzo[b]thiophen-2-yl)-4-oxobutanoic acid, which was dissolved in ethanol (yield: 20.14 mCi, specific activity 73.83 Ci/mmol). LCMS (C14H12T3O5S) (ES, m/z): 301 [M+H]+. Determination of pKa of MSA-2 The pKa of MSA-2 was determined by potentiometric titrations with the Sirius T3 instrument using a double junction electrode. The electrode was standardized from pH 1.8 to 12.2. The KOH titrant (~0.5 M) was standardized against potassium hydrogen phthalate. MSA-2 was dissolved in DMSO to create a 100.0 mg/mL stock solution. The stock solution (20 μl) was diluted in 1.5 mL of MeOH:ionic strength adjusted (ISA) H2O (0.15 M KCl) in a known ratio. The starting pH of the MSA-2 solution was adjusted with 0.5 M KOH. The MSA-2 solution was titrated at 25°C with 0.5 M HCl from about pH 11 to 2 at 25°C in a known concentration of MeOH:ISA H2O, followed by subsequent dilutions to lower concentrations and re-titrations, all in the same vial. Titrations of MSA-2 were performed in replicate. The pKa value in ISA H2O was determined by Yasuda-Shedlovsky extrapolation to 0% MeOH using Sirius T3Refine v.1.1 software. High throughput phenotypic screen The company’s 2.4 million compound library was assayed in a phenotypic screen for the ability to induce secretion of IFN-β in the human monocytic cell line THP-1 (ATCC TIB-202). 30 nL/well of test compound (20 µM final) was transferred to a 1536-well white high base plate (Corning #7386) by acoustic dispensing using the Echo 555 (Labcyte, San Jose, California). Subsequently, 3 µL of 1.3 × 10e6 cells/mL (4K cells) in assay medium (RPMI-Glutamax containing 0.5% (v/v) fetal bovine serum) were added to each well of the cell culture plate. After incubation at 37°C with 5% carbon dioxide for 5 hours, IFN-β levels were quantified by AlphaLISA kit PEAL265F (Perkin Elmer). The AlphaLISA was performed per the manufacturer’s instructions. The AlphaLISA signal was detected using an EnVision Multilabel Reader (PerkinElmer, Waltham, Massachusetts). EC100 was defined as response to 100 µM 2’3’ cGAMP (internal) and EC0 was defined as response to DMSO. Percent activity was defined as %ACT=100*(RLUsample-MedRLUEC0)/(MedEC100-MedEC0) Methods for accessing cellular activity of test compounds The cell-based potency of test compounds was measured by their ability to induce the secretion of IFN-β in the human monocytic cell line THP-1, a STING genetic knockout derivative THP-

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1.STING.KO (STING -/-) cell line and mouse primary macrophages. The THP-1 cell line endogenously expresses the HAQ variant of the STING protein and treatment of THP-1 cells with STING agonists robustly increases the secretion of IFN-β from these cells into the assay medium. The STING gene in THP-1 cells was subjected to deletion using CRISPR technology to yield a cell line devoid of STING expression, the THP-1.STING.KO (STING -/-) cell line. STING agonists also robustly induce IFN-β in mouse primary macrophages. The THP-1 cell-based assays were optimized using a custom Mesoscale Human Interferon-β assay kit to measure the levels of IFN-β in the assay supernatant. The mouse macrophage assays used the mouse IFN-β Verikine ELISA (PBL). Compounds were serially diluted (3-fold) in 100% dimethyl sulfoxide (DMSO) across a 384-well polypropylene source plate from column 3-12 and column 13-22, to yield 10-point titrations for each test compound. Columns 1, 2, 23 and 24 contained either only DMSO or a pharmacologically known control STING agonist cGAMP. For cross titration experiments incorporating more than one compound per well, a second titration was created in a separate plate in the perpendicular orientation. Once titrations were made, 120 nL of the compounds in 384 well plates were transferred by acoustic dispensing to a 384 assay plate to assay activity in cells. THP-1 cells were reconstituted in assay medium (RPMI1640, 1x Non-essential amino acids, 1x Sodium Pyruvate (Life Technologies, Cat. Nos. 61870-036, 11140-050 and 11360-070), supplemented with 0.5% (v/v) FBS (Sigma, Cat. No. F4135) at a concentration of 1.25 × 106 cells/mL. A 40 µL volume of cell suspension was added to each well. After incubation at 37°C with 5% carbon dioxide in a humidified atmosphere for 5 hours, 40 μL assay medium was added to the wells, mixed and the plates centrifuged at 300 x g for 5 min. Human IFN-β levels were measured using a custom 384-well mesoscale kit (Meso Scale Discovery) and followed the manufacturer’s instructions. Briefly, kit plates were washed 3X 30 μL with Dulbecco’s PBS lacking calcium and magnesium (Hyclone, Cat. No. SH30028-03). IFN-β capture antibody was diluted 1:50 (v/v) in Diluent 100, then10 μL was added to each well of the 384-well plate and incubated at room temperature for 2 hours with shaking. Just prior to test sample addition, plates were washed 3X with 100 μL of PBS. Then 25 μL of cell supernatants or standards were added along with 10 μL human IFN-β detection antibody diluted 1:50 (v/v) in Diluent 100. Assay plate(s) were incubated overnight at 4°C with shaking then washed 3X with 30 μL PBS, prior to addition of 30 μL of 2X MSD read buffer. Plates were then read on an MSD Sector Imager 600 (Meso Scale Discovery, Rockland, MD). IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13 and TNF-α levels were measured using V-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale Discovery, Cat. No. K15049D) following the manufacturer’s instructions. Briefly, 40 μL of cell supernatants or standards were added to the Vplex assay plates pre-coated with a mixture of linker and biotinylated antibodies. Samples and standards were incubated in U-plex plates overnight at 4°C while shaking. The next day, plates were washed 3x with 150 μL of PBS-T wash buffer (1x phosphate-buffered saline and 0.01 % Tween-20) before 50 μL of a mixture of custom detection antibodies specific for each cytokine were added. The U-plex plates were then incubated for 2 hours at room temperature while shaking before washing again with 3x 150 μL of PBS-T. Following the last wash, 150 μL of 2x MSD read T buffer was added to each well and plates were read immediately on an MSD Sector Imager 600 (Meso Scale Discovery, Rockland, MD). Mouse bone marrow macrophage cells (Cell Biologics; Cat. C57-6030F) were thawed into pre-warmed assay medium and centrifuged at 300 x g for 5 min at room temperature. After this, cells were re-suspended in assay medium to a density of 750,000 cells/mL. 40 μL of cell suspension

8

was dispensed into 384-well tissue culture treated plates to which 120 nL of compound had been previously dispensed. After incubating for 5 hours at 37°C, 5% CO2 in a humidified atmosphere 40 μL assay medium was added, mixed and the plates centrifuged at 300 x g for 5 min. Mouse IFN-γ, IL-6 and TNF-α cytokine levels were measured using a custom U-plex cytokine profiling kit (Meso Scale Discovery, Rockland, MD) and following the manufacturer’s instructions. Briefly, tissue and plasma samples were prepared as detailed above, and 50 μL of diluted sample or standard were added to U-plex assay plates pre-coated with a mixture of linker and biotinylated antibodies. Samples and standards were incubated in U-plex plates overnight at 4°C while shaking. The next day, plates were washed 3x with 150 μL of PBS-T wash buffer (1x phosphate-buffered saline and 0.01 % Tween-20) before 50 μL of a mixture of custom detection antibodies specific for each cytokine were added. The U-plex plates were then incubated for 2 hours at room temperature while shaking before washing again with 3x 150 μL of PBS-T. Following the last wash, 150 μL of 2x MSD read T buffer was added to each well and plates were read immediately on an MSD Sector Imager 600 (Meso Scale Discovery, Rockland, MD). Mouse IFN-β concentrations were quantified using the VeriKine™ Mouse Interferon Beta ELISA Kit (PBL, Cat. No. 42400) and following the manufacturer’s instructions. Briefly, transfer 35 μL of sample or IFN-β standard (1000-15.6 pg/mL) were added to the assay plate and incubated at room temperature for 2 hours or overnight at 4°C, while shaking. Following this, assay plates were washed three times with 150 μL of wash solution before 100 μl of diluted detection antibody was added to the assay plate. Assay plates were incubated at room temperature for an additional 2 hours while shaking, then were washed three times with 150 μl of wash solution. 1x Horse Radish Peroxidase (HRP) solution was added to each well and the plate was allowed to incubate for 1 hour at room temperature before washing the plate three times with 150 μl of wash solution. Finally, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well for 5 minutes before the reaction was stopped with 100 μl of stop solution. IFN-β levels were quantified by measuring absorbance at 450 nm using a Flexstation 3 plate reader (Molecular Devices, San Jose, CA). In experiments examining the impact of pH on compound activity, the assays were performed as detailed above but using assay medium supplemented with 25 mM HEPES (pH adjusted with either 1 M HCl or 1 N NaOH) immediately prior to filter sterilization and use. Assessment of MSA-2 Activation of the STING-TBK1-IRF-3 Pathway in THP-1 Cells by Western Blot THP-1 cells (1×107 cells/mL) were re-suspended in assay medium (RPMI-1640 medium supplemented with 2% fetal bovine serum, 50 μM β-mercaptoethanol, and 10 mM HEPES) and were treated with 15 or 50 μg/mL cycloheximide for 50 minutes at 37°C. Compound diluted in DMSO was then added to the desired final concentration, and cells were incubated at 37°C with 5% carbon dioxide for the times specified. Following incubation, cells were pelleted, washed, and lysed with 0.4 mL lysis buffer (Thermofisher). Lysates were further disrupted 2x by sonication for 5 seconds each, then heated to 95°C for 3 minutes before 25 µg of each sample was loaded onto a BOLT 4-12% Bis-Tris Plus electrophoresis gel (Thermofisher) and treated for 60 minutes (150V). Sample protein was then transferred to a polyvinylidene difluoride membrane using Lightning Blot (PerkinElmer) and was subsequently blocked with buffer (Tris-buffered saline with 0.05% Tween 20) containing 5% nonfat dry milk for 60 minutes at room temperature. The membranes were bathed in buffer containing primary antibody (anti-STING 1:1,000; anti-pIRF3 1:500; anti-pTBK1 1:1,000; anti-IFR3 1:2,000; anti-TBK1 1:500; anti-beta Actin 1:2,500; anti-

9

GAPDH 1:1,000) (Cell Signaling Technology, Danvers, MA) for 18 hours while shaking at 4°C before washing with buffer. Membranes were then bathed in buffer containing secondary antibody (1:1000 dilution) for 2 hours at 4°C while shaking before washing away unbound antibody. The bound protein was quantified using ECL substrate (Cell Signaling Technologies) for 1 minute, and bands were viewed using a ChemDoc XRS imager (Bio-Rad, Hercules, California). Preparation of recombinant full-length human and mouse STING Full length hSTING-WT, hSTING-HAQ and mouse STING embedded in insect endoplasmic reticulum membrane were generated using a baculovirus expression system. Amino acid sequences of the human STING reference sequence (NP_938023.1), which is also referred to as hSTING-H232, and mouse STING (NP_082537.1) were obtained from the National Center for Biotechnology Information. The hSTING-WT differs from hSTING-H232 at position 232, which is arginine (R) in the former, whereas histidine (H) in the latter. The hSTING-HAQ differs from hSTING-H232 sequence at positions 71, 230, 232 and 293, which are histidine (H), alanine (A), arginine (R) and glutamine (Q) respectively in the former, whereas arginine (R), glycine (G), histidine (H) and arginine (R) in the latter. Synthetic STING genes were synthesized and cloned into baculovirus transfer plasmid pBAC-1 by Genewiz, Inc. Sf21 cells in Sf-900II SFM medium were co-transfected with pBAC-1-STING plasmid and BestBac 2.0, v-cath/chiA Deleted Linearized Baculovirus DNA in the presence of Cellfectin® II Reagent. The transfected cells were incubated at 27°C for 5 to 7 days to produce P0 recombinant virus, which were amplified two rounds to produce P1 and P2 virus. The infected P1 and P2 Sf21 cells (BIIC cells) were harvested and cryopreserved. To generate STING membranes, P1/P2 BIICs were thawed and used to infect fresh Sf21 cells. The infected Sf21 cells were in turn used to infect a large-scale culture of T.ni insect cells. The T.ni cells were allowed to express STING for 48 hours at 27°C and harvested by centrifugation. The cell pellets were re-suspended in ice-cold Lysis buffer (10 mM magnesium chloride, 20 mM potassium chloride, and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5) containing Benzonase and protease inhibitors, homogenized using a Wheaton Dounce glass homogenizer (20 strokes), and passed through an Emulsiflex-C5 microfluidizer (Avestinx) at a pressure of 5000 Psi. Homogenates were subsequently centrifuged at 100,000 × g for 45 minutes at 4°C. The pellets were re-suspended in Wash buffer (25 mM HEPES, pH 7.5, 1 mM magnesium chloride, 20 mM potassium chloride, and 1 M sodium chloride) supplemented with cOmpleteTM EDTA-free Protease Inhibitor Cocktail, homogenized using a Wheaton Dounce glass homogenizer (20 strokes), and centrifuged at 100,000 × g for 45 min at 4°C. The supernatant was discarded, and the wash step was repeated once. The resultant membrane pellets were re-suspended in a buffer containing 20 mM HEPES, pH 7.5, 500 mM sodium chloride, 10% glycerol, and cOmpleteTM EDTA-free Protease Inhibitor Cocktail, aliquoted and stored at -80°C until use. Protein production for X-ray and NMR hSTING-WT and hSTING-HAQ (amino acids 155-341) flanked by an N-terminal 6x His-SUMO-TEV protease cleavage site were cloned into pET47b expression vectors and transformed into Rosetta 2 (DE3) competent cells (EMD Millipore). Initial inoculation occurred overnight in Superbroth (Fisherbrand) at OD600 = 0.1. Expression cultures were then induced at OD600 = 1 with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and then cultured overnight at 16oC. Harvested cells were lysed in buffer (50 mM tris(hydroxymethyl)aminomethane pH 8.0, 300 mM sodium chloride, 10% glycerol, 10 mM imidazole, 1 tablet cOmplete EDTA-free protease

10

inhibitor/50 ml buffer (Roche), 30 kU lysozyme/ml (EMD Millipore) 100 U/ml Benzonase (Sigma-Aldrich), and 5 mM β-mercaptoethanol) by dounce homogenation, followed by two passes through a Emulsiflex C5 microfluidizer (Avestin, Ottawa, Ontario, Canada). The lysate was clarified by centrifugation at 45,000 x g. Clarified lysate was then IMAC-purified through a hitrap Nickel fast flow column (General Electric). The pooled IMAC peak was dialyzed into TEV reaction buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM β-mercaptoethanol) and digested overnight with a His-tagged TEV protease at 4oC. Undigested target protein and TEV protease was then removed by a subtractive IMAC step. The subtractive IMAC flow-through pool was concentrated and loaded onto two S200 16/60 columns (GE Lifesciences, Marlborough, Massachusetts) connected in series and equilibrated with 20 mM Tris pH 7.5, 150 mM sodium chloride, 5 mM dithiothreitol (DTT), and 1 tablet cOmplete EDTA-free protease inhibitor/100 ml (Roche). The gel-filtration peak is then collected and concentrated. Proteins for NMR were generated as described above, except that [15N]-labeled expression media consisting of 1x M9 salts, 0.3 mM calcium chloride, 2 mM magnesium sulfate, 1x trace metal mix (Teknova), 0.15% glucose, and 1g/L [15N] ammonium sulfate containing 0.1x Bioexpress (Cambridge Isotope Laboratories) was used to inoculate overnight cultures instead of Superbroth. The extent of [15N] incorporation was determined by mass spectrometry. Recombinant STING cytosolic domains for surface plasma resonance experiments Synthetic genes encoding the cytosolic domains of hSTING-WT (aa 140-379), hSTING-HAQ (aa 140-379), hSTING-H232 (aa 140-379) and mouse STING (aa 139-378) were designed based on the known amino acid sequence of each STING and the codon usage preference of E. coli. The genes were synthesized and cloned into the bacterial expression plasmid pET47b by Genewiz, Inc. Each resultant recombinant plasmid was designed to direct expression of a STING cytosolic domain with an N-terminal His-TEV-Avi tag with the sequence MAHHHHHHENLYFQSGLNDIFEAQKIEWHE. The recombinant plasmids were used to transform BL21-CodonPlus (DE3)-RIPL competent E. coli cells. A glycerol E. coli stock derived from a single transformed colony was used to inoculate a pre-culture in LB medium, which was allowed to grow at 37°C in the presence of kanamycin and chloramphenicol for 16 hours. A 10-mL aliquot of pre-culture was then used to inoculate 500 mL of Studier Induction ZYP-5052 Media (Teknova) containing kanamycin and chloramphenicol. After growth at 16°C for 48 hours, the cells were harvested by centrifugation and stored at -80°C. Frozen cell pellets were thawed and lysed in Buffer A (50 mM Tris pH 8.0, 300 mM sodium chloride and 20 mM imidazole) supplemented with 1 mg/mL lysozyme, 25 units/mL Benzonase, a cocktail of cOmplete™ EDTA-free Protease Inhibitor Cocktail, and 20 mM beta-mercaptoethanol. The cell lysate was clarified by ultra-centrifugation and the supernatant was loaded onto a column of HIS-Select HF Nickel Affinity Gel (Sigma) pre-equilibrated with Buffer A. The column was washed, sequentially, with 20 column-volumes (CV) of Buffer A, 20 CV of Buffer B (50 mM Tris, pH 8.0, 1000 mM sodium chloride, 20 mM imidazole, and 20 mM beta-mercaptoethanol), and 20 CV of Buffer A. STING protein was then eluted from the column with Buffer C (50 mM Tris, pH 8.0, 300 mM sodium chloride, 300 mM imidazole, and 5 mM beta-mercaptoethanol). Fractions containing purified STING were concentrated using Amicon Ultra-15 Centrifugal Filter with molecular weight cut off of 10 kDa (EMD Millipore). STING protein was biotinylated in an enzymatic reaction using BirA Biotin-Protein Ligase Bulk Reaction Kit (Avidity), and then purified by size exclusion chromatography using a

11

HiLoad 26/600 Sephadex 200pg column (GE Life Sciences) in a buffer containing 50 mM Tris, pH 8.0, 150 mM sodium chloride, 10 % glycerol, and 5 mM dithiothreitol. Competitive [3H]-2’,3’-cGAMP displacement assay To assess the ability of test compounds to inhibit binding of [3H]-2’,3’-cGAMP to hSTING-HAQ, microsomes containing hSTING-HAQ diluted in DPBS were incubated with serially diluted test compounds in a 96-well NUNC deep-well plate for 60 min at 25 ֩◌C. [3H]-cGAMP was then added to each reaction (final concentration: 2 nM). The reactions were incubated at 25 ֩◌C for an additional 60 minutes. The reactions were terminated by filtration using a Microbeta filtermate-96 cell harvester (Perkin Elmer), and filter-bound radioactivity was determined as described in preceding section. Percent inhibition of [3H]-cGAMP binding at each [MSA-2] was calculated using the following equation: percent inhibition = 100 x {1– (bound CPM in the presence of test compounds/bound CPM in the absence of test compounds)}. To determine IC50, the observed relationship between percent inhibition (denoted as Y) and log10 of [test compound] (denoted as X) was fitted with the following equation using GraphPad Prizm 7: Y = 100/(1 + 10�(LogIC50−X)∙Hill Slope�) (Eq. S3) A similar method was used to assess the ability of test compounds to inhibit binding of [3H]-cGAMP to hSTING-WT. The only differences were that the hSTING-WT membranes were incubated simultaneously with both test compounds and [3H]-cGAMP (, and that the reaction contained a final concentration 8 nM [3H]-cGAMP). Preparation of working stock [3H]-MSA-2 for binding studies A stock solution of 15.6 µM [3H]-MSA-2 (specific activity: 73.83 Ci/mmol) prepared in ethanol was sparged with nitrogen gas for 60 minutes at 25°C to remove ethanol. [3H]-MSA-2 was reconstituted in Dulbecco's phosphate-buffered saline (DPBS, ThermoFisher, catalog number: 14190). The concentration of [3H]-MSA-2 in the resulting aqueous solution was calculated from radioactivity in a small aliquot of the solution, determined by scintillation counting and known specific activity of the original [3H]-MSA-2 stock. The [3H]-MSA-2 (“hot”) solution was then diluted to 800 µM with DPBS and was subsequently mixed with 800 µM non-radioactive (“cold”) MSA-2 in DPBS to generate a working ligand solution (final total MSA-2 concentration: 800 µM; molar ratio of cold:hot ligand = 11.5:1). Saturation [3H]-MSA-2 binding of hSTING-WT Frozen insect microsomes containing hSTING-WT were thawed, re-suspended in DPBS, and homogenized using a Wheaton Dounce glass homogenizer (7 strokes). The membranes (final concentration of total protein: 10µg/ml) were incubated with serially diluted working ligand solution in DPBS in a sealed 96-well NUNC deep-well plate for 18 hours at 25 ֩◌C. The binding reactions were terminated by filtration through a 96-well GF/B filter plate on a Mach III Cell Harvester (Tomtec), washed with 20mM cold HEPES buffer, pH 7.3 (ThermoFisher, BP299500). The filters were subsequently dried in a 55 ֩◌C oven before addition of Ultima Gold F scintillation cocktail. Filter-bound radioactivity was then measured using a TopCount NXT (Perkin Elmer). Non-specific binding of [3H]-MSA-2 was determined using 20 µM cGAMP. Specific binding of [3H]-MSA-2 was calculated as total filter-bound radioactivity minus nonspecific binding. The observed relationship between [total MSA-2] (denoted as Y) and specifically binding of [3H]-MSA-2 (denoted as X) were then fit, by nonlinear least squares regression using equation S1 to

12

determine Bmax, Kd and n, or equation S2 to determine Bmax and Kd. Resultant values and standard error for fitting to equation S1 were Bmax = 150 ± 6, n = 1.6 ± 0.2 and Kd = 8 ± 0.8 µM with R2 = 0.99 (n=1).

Y=Bmax·[X]n/(Kdn + Xn) (Eq. S1)

Y=Bmax·[X]/(Kd + [X]) (Eq. S2) Homologous [3H]-MSA-2 competition assay Microsomes expressing hSTING-WT or mSTING were incubated for 16 hours at 25 ֩◌C in DPBS containing serially diluted unlabeled (“cold”) MSA-2 in the presence of a constant concentration of [3H]-MSA-2 (working stock prepared as described above). The binding reactions were terminated by filtration, using a Mach III Cell Harvester (Tomtec), and filter-associated radioactivity was measured as described above. Non-specific binding of [3H]-MSA-2 was determined from reactions containing 100 µM 2’3’-cGAMP. Specifically bound [3H]-MSA-2 was calculated as the total filter-bound CPM minus the nonspecific binding. The following equation was used to fit the observed bell-shaped relationship between specifically bound [3H]-MSA-2 (denoted as Y) and concentration of “cold” MSA-2 (denoted as x):

Y = C ∙ (hot+x)2/(KD+(hot+x)2) ∙ hot ∙ (2 ∙ hot + x)/(x ∙ hot+hot2+x2) (Eq. S4) In equation S4, “hot” denotes the fixed concentration of [3H]-MSA-2 used in the binding reactions. The values of KD and C were determined by non-linear least squares regression using GraphPad Prism 7. Derivation of the above equation can be found in the section below entitled “Derivation of equation describing behavior of Model 3 in homologous radioligand competition experiments”. 1H NMR experiments to estimate the equilibrium constants of MSA-2 and compound 2 dimerization An initial sample of MSA-2 was prepared to a final concentration of 2 mg/mL in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 at pH 7.4 in D2O) + 25 µM TSP (trimethylsilylpropanoic acid, reference standard) and subsequently diluted to a final concentration series of 5612, 2806, 1871, 1122, 224, and 56 µM for analysis. An initial sample of compound 2 was prepared and diluted similarly to a final concentration series of 3384, 1692, 1128, 676, 135, and 34 µM for analysis. Proton (1H) NMR spectra were collected on a Varian VNMRS 600 MHz instrument at 25°C to determine dimerization properties of MSA-2 or compound 2 (see Fig. 5G-H). The dependence of 1H NMR chemical shifts corresponding to monomeric MSA-2 (C) versus total MSA-2 (T) concentration is described by Eq. S5 below, where δobs is the observed chemical shift, δd is the chemical shift of the dimer (determined during the analysis, see below) and δm is the chemical shift of the monomer (assumed to be equal to the chemical shift of the most dilute sample of MSA-2). Eq. S6 (quadratic solution for [monomer] for a monomer:dimer equilibriuma) relates the KD for dimerization to the concentrations of C (free monomer MSA-2) and T (total MSA-2). The value for the equilibrium constant was determined by solving both Eq S5 and S6 simultaneously by iteration of δd and KD parameters (Excel 2010). This procedure was also repeated for compound 2. Graphical results with MSA-2 (green triangle) and compound 2 (inverted orange triangle) are shown in Fig. S6. The resultant simulated curve (solid line) is only shown for MSA-2. The gray dashed line (y = x) represents the expected relationship for a monomeric compound. (Eq. S5) [𝐶𝐶] = [𝑇𝑇] (𝛿𝛿𝑜𝑜𝑜𝑜𝑜𝑜 − 𝛿𝛿𝑑𝑑)

(𝛿𝛿𝑚𝑚 − 𝛿𝛿𝑑𝑑)

13

(Eq. S6) [𝐶𝐶] = −𝐾𝐾𝐷𝐷+ �𝐾𝐾𝐷𝐷

2+8[𝑇𝑇]𝐾𝐾𝐷𝐷

4

According to the relationship for dimerization shown in Eq. S7, the concentration of the monomer (C) is related to the concentration of the dimer (C2) as shown in Eq. S8. The total amount of MSA-2 (T) is described by Eq. S9. Solving for the concentration of the dimer and subsequent substitution into Eq. S8 ultimately yields the quadratic solution for monomer concentration shown in Eq. S6 above (24). (Eq. S7) [𝐶𝐶] + [𝐶𝐶]

𝐾𝐾𝐷𝐷�� [𝐶𝐶2] (Eq. S8) [𝐶𝐶][𝐶𝐶]

[𝐶𝐶2] = 𝐾𝐾𝐷𝐷 (Eq. S9) [𝑇𝑇] = [𝐶𝐶] + 2[𝐶𝐶2] Surface plasmon resonance experimental methods Surface plasmon resonance (SPR) experiments were conducted using a Biacore® T200 system (GE Healthcare). The biotinylated cytosolic domain of each STING variant was diluted to 1-3 μM in Biacore running buffer containing 10 mM HEPES, 150 mM sodium chloride, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.05% v/v polysorbate 20 (Tween 20), and 1 mM dithiothreitol, and captured on the surface of a Series S Sensor Chip SA, according to the manufacturer's instructions, to a final capture level of ~3100 resonance units (RU; same protein for all 3 channels). Reported values are the average of data from each channel (n=3). All experiments were run at 25°C in running buffer containing 3% v/v dimethyl sulfoxide (DMSO) at a flow rate of 50 µL/minute. Compound samples were prepared from DMSO stock solutions (10 or 100 mM). All resultant sensorgrams were solvent corrected and double reference subtracted (blank injection with reference flow cell). The kinetic binding parameters of MSA-2 (294.3 g/mol) for human WT STING (61.4 kDa, dimer), human HAQ STING, human H232 STING and WT mouse STING (61.65 kDa, dimer) were accessed using a single-cycle kinetics method (contact and dissociation times of 420 and 2000 sec, respectively), which entails sequential injections of a series of samples of increasing MSA-2 concentrations (100-6.25 µM, 5 step, 2-fold dilution) without a dissociation step between sample injections (WT mouse data was collected at a top MSA-2 concentration of 50 µM). A single long dissociation phase (~0.5 h), effected by flowing running buffer over the sensor surface, followed the last MSA-2 injection. Initial attempts to fit the data with a one-step kinetic model (Model 1 in Fig. 5C, independent 1:1 binding) were unsuccessful (Fig. S4A,C,E,G). To assess the ability of Model 2 to account for the SPR data, a two-step kinetic binding model (L+R=RL; RL+L=LRL) with four unique microscopic rate constants was created using Biacore Evaluation software. To fit the SPR data according to Model 3, the nominal concentrations of MSA-2 were adjusted to reflect those of the active homodimeric species (see Model 3, Fig. 5C,F and Eq 3) using the experimentally determined KD1 value for homodimerization of 18 mM. This corresponded to a range of 555 – 2 nM for the dilution series. The resultant sensorgrams were subsequently fitted with a 1:1 interaction model to yield association rate constant (on-rate ka, denoted ka2), dissociation constant (off-rate kd, denoted kd2) and equilibrium constant (KD), denoted KD2) values for all four STING variants (Fig. 5F and S4C,E,G). It follows from Model 3 that these fitted rate parameters describe only the second step (KD2 as depicted in the boxed portion of Fig. 5C). The overall equilibrium constant of Model 3 (KD, which has a unit of µM2; see Eq 1 in Fig. 5C) is equal to the product of the equilibrium constant of the dimerization step (KD1) and the equilibrium constant for interaction between MSA-2 homodimeric MSA-2 and STING (KD2). It is also useful to note that

14

the concentration of MSA-2 at 50% STING occupancy is equal to the square root of this overall KD value (Eq 2 in Fig. 5C). Relevant parameters from the SPR analysis are summarized in Table S5. Model 2 results are addressed in Text S1. Interaction of a covalent dimer of an MSA-2 analog, Compound 3 (Fig. 6C, 584.67 g/mol), with human WT STING was also evaluated using a single-cycle kinetics method (contact and dissociation time of 420 and 800 seconds, respectively) in which each Compound 3 concentration of a 5 point, 3-fold dilution series (900-11 nM) was subsequently injected followed by a single dissociation step. The resultant sensorgrams obtained were then fitted with a direct 1:1 interaction model (Biacore® Evaluation Software) to yield ka, kd, and KD values (see Fig. 6E).

Automated Ligand Identification System (ALIS) ALIS is an affinity selection platform that utilizes 2D chromatography coupled to a mass spectrometer. The chromatography phase consists of a size exclusion chromatography (SEC) column (2.1 mm I.D. x 50 mm length, packed with proprietary gel filtration media; Running buffer: 700 mM ammonium acetate, pH 8.0, flow rate (F) = 300 μL/min, 4°C column temperature) which leads into a reverse phase chromatography (RPC) column (Higgins Analytical, Mountain View, CA – Targa C18, 0.5 mm I.D. x 50 mm, 5μm packing material; Mobile Phases: Water: Acetonitrile with 0.2% formic acid, 0-95% B gradient in 2.5 minutes, F = 20 μL/min, 40°C column temperature). The RPC elutes directly into a high resolution/accurate mass Exactive mass spectrometer (ThermoFisher Scientific, San Jose, CA). The ALIS system is comprised of 3 isocratic pumps, 1 binary capillary chromatography pump, 2 variable wavelength detectors, 1 solvent degasser, 1 chilled microplate autosampler (all Agilent Technologies, Wilmington, DE) and a custom valve box with 4 two-position valves. The SEC utilizes two isocratic pumps, one for the running buffer and one for the SEC wash buffer. The first valve on the valve box switches the flow from running buffer to wash buffer after each injection. The second valve (SEC/RPC valve) on the valve box has a 15 µL loop which isolates the first peak detected by the first variable wavelength detector (typically the void volume containing large molecules such as protein and protein/ligand complexes), and transfers it into the binary pump (RPC) flow path. After the SEC flow is disconnected from the protein capture loop, the flow continues through the second variable wavelength detector to confirm the absence of a protein peak and validate the protein capture. The RPC system utilizes the capillary pump, the third valve on the valve box, and an isocratic pump pumping the initial mobile phase of the RPC method. The third valve is a 10 port, 2 position valve that allows for RPC column switching and background re-equilibration of the columns.

Sample Preparation for ALIS Experiments For each experiment, human STING protein (WT or HAQ) was thawed on ice and diluted to 10 μM (2X screening concentration) with ALIS assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2.5% DMSO). A single 12 point, 2-fold serial dilution of either MSA-2 or compound 2 (250 μM high, 2X screening condition) was prepared from 10 mM DMSO stocks to match the ALIS assay buffer. Additional MSA-2 titration experiments conducted at a constant concentration of compound 2 (1 µM final, where no binding was detected alone) were prepared similarly with the addition of compound 2 (2 µM) to each titration well. The protein solution and the samples were then mixed 1:1 and incubated for 30 min (room temperature) in a sealed plate for a final screening concentration of 125-0.06 μM with 5 µM protein (and 1 µM compound 2, when added). Pre-incubated protein/compound samples were then transferred to an autosampler

15

(4°C) and consequently injected in duplicate into the ALIS system and analyzed in either negative or positive ionization modes (for MSA-2 and compound 2, respectively) in a high resolution Exactive mass spectrometer (ThermoFisher Scientific, San Jose, CA). For experiments containing both compounds, the same samples were injected in duplicate for analysis in positive mode and then injected in duplicate for analysis in negative mode. All experiments were repeated as described twice. Data was collected using a processing method in Xcalibur (Version 4.0; ThermoFisher Scientific, San Jose, CA) which recorded both MS intensities and retention times. Each replicate run was subsequently normalized to the highest MS signal observed (for each compound) and then averaged together.

Derivation of equation describing behavior of Model 2 in a homologous radioligand competition experiments The model assumes that 1) each STING dimer contains two identical MSA-2 binding sites; 2) the monomeric MSA-2 molecules are capable of binding to each of the two sites; 3) binding of a MSA-2 molecule to one of the two sites alters the affinity of the other site for MSA-2. Definition of notations R: ligand-free STING dimer, L: free MSA-2 monomer Lc: free unlabeled MSA-2 Lβ: free [3H]-MSA-2. RLL: STING with two bound MSA-2 molecules regardless of their radiolabeling status RL: STING with either of the two sites occupied by a MSA-2 molecule which is either radiolabeled or unlabeled KD1: macroscopic binding constant for MSA-2 binding to a ligand free STING dimer KD2: macroscopic binding constant for MSA-2 binding to a STING dimer with a bound MSA-2 molecule KD1: microscopic binding constant for monomeric MSA-2 binding to a ligand binding site in a ligand free STING dimer KD2: microscopic binding constant for monomeric MSA-2 binding to a ligand binding site in a STING dimer with a bound MSA-2 molecule. It can be shown that Macroscopic KD1 = 𝐾𝐾𝐷𝐷1

2

Macroscopic KD2 = 2𝐾𝐾𝐷𝐷2 Now let’s derive macroscopic relations. [R]𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 = [RLL] + [RL] + [R] [RL] = [R][L]

KD1= [R][L]

𝐾𝐾𝐷𝐷12�

= 2[R][L]𝐾𝐾𝐷𝐷1

[RLL] = [RL][L]KD2

= [RL][L]2𝐾𝐾𝐷𝐷2

=2[R][L]𝐾𝐾𝐷𝐷1

[L]

2𝐾𝐾𝐷𝐷2= [R][L][L]

𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2

[R]𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 = [R][L][L]𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2

+ 2 [R][L]𝐾𝐾𝐷𝐷1

+ [R] From this point we will only use microscopic binding constants, 𝐾𝐾𝐷𝐷1 and 𝐾𝐾𝐷𝐷2.

16

Assuming total MSA-2 concentration = free MSA-2 concentration = [L] = [LC] + [Lβ]

[R]total = [RLL] + [RL] + [R] =[R]([LC] + [Lβ])2

𝐾𝐾𝐷𝐷1 ∙ 𝐾𝐾𝐷𝐷2+ 2

[R]([LC] + �Lβ�)𝐾𝐾𝐷𝐷1

+ [R]

The total concentration of STING with two bound MSA-2 molecules regardless of their radio-labeling status is

[RLL]total =[R]([LC] + [Lβ])2

𝐾𝐾𝐷𝐷1 ∙ 𝐾𝐾𝐷𝐷2

The concentration of STING occupied by one [3H]-MSA-2 and one unlabeled MSA-2 molecule

[RLLβ] = [R]([LC]+[Lβ])2

𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2∗ � 2�Lβ�

[LC]+�L� � [LC]

[LC]+�L�

The concentration of STING occupied by two [3H]-MSA-2 molecules

[RLβLβ] = [R]([LC]+[Lβ])2

𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2∙ �

�Lβ�[LC]+�Lβ�

�2

The total concentration of STING occupied by a single MSA-2 molecule regardless of its radiolabeling status

[RL]total = 2[R]([LC] + �Lβ�)

𝐾𝐾𝐷𝐷1

The concentration of STING occupied by a [3H]-MSA-2 molecule only

�RLβ� = 2[R]�[LC] + �Lβ��

𝐾𝐾𝐷𝐷1(

�Lβ�[LC] + �Lβ�

)

The molar ratio of bound [3H]-MSA-2 to [STING dimers] = [Lβ]bound

[R]total=

2[R]��LC�+�L�

𝐾𝐾𝐷𝐷1�

�Lβ�

�LC�+�L�+

[R]([LC]+[Lβ])2

𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2∗�

2�Lβ�

�LC�+�L�� [LC]

�LC�+�L�+

2[R]([LC]+[Lβ])2

𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2∙�

�Lβ�

�LC�+�L�2

2[R](�LC�+�Lβ�)

𝐾𝐾𝐷𝐷1+

[R]([LC]+[Lβ])2

𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2+[R]

=

2∙𝐾𝐾𝐷𝐷2�Lβ�+2�Lβ�[LC]+2�Lβ�2

2𝐾𝐾𝐷𝐷2�[LC]+�Lβ��+([LC]+�Lβ�)2+𝐾𝐾𝐷𝐷1∙𝐾𝐾𝐷𝐷2 (Eq. S10)

Derivation of equation describing behavior of Model 3 in homologous radioligand competition experiments Model 3 hypothesize that MSA-2 must dimerize in solution before it can bind dimeric STING. The 2-step reaction is depicted by reaction equation S11 where monomeric MSA-2, dimeric MSA-2, ligand-free STING dimer, and STING dimer occupied by MSA-2 are denoted as L, LL, R and RLL respectively. In a homologous competition experiment, binding of [3H]-MSA-2 (denoted as

17

Lβ) to STING is measure in the presence of a constant [Lβ] and various concentrations of unlabeled MSA-2, i.e., L. There are three parallel reactions, as depicted below, occur simultaneously. In reaction equation S11 two molecules of L dimerize to form LL, which then binds R to form RLL; in reaction equation S12 a molecule of L and a molecule of Lβ dimerizes to form LLβ which then binds R to form RLLβ; in reaction equation S13 two Lβ molecules dimerize to yield LβLβ which in turn binds R forming RLβLβ

The validity of the following derivation depends on the validity of the following assumptions 1) The free concentrations of L and Lβ are approximately equal to total concentrations of L and Lβ due to the fact that a) Kd1 is very large, and b) the total concentration of R is much lower than [L] and [Lβ]. 2) In the absence of STING, the approximate total concentration of each of the three types of MSA-2 dimers at equilibrium can be expressed as below,

[LβLβ] ≈ [Lβ]2

Kd1; [LLβ] ≈

[L]∙[Lβ]

Kd1; [LL] ≈ [L]2

Kd1 (Eq. S14, S15, S16)

[LβLβ] [LLβ]

= [Lβ]2

Kd1[L]∙[Lβ]Kd1

= [Lβ][L]

(Eq. S17)

[LβLβ] = [Lβ][L]

∙ [LLβ] (Eq. S18)

Similarly, [LL] [LLβ]

= [L]2

Kd1[L]∙[Lβ]Kd1

= [L]�Lβ�

, thus [LL] = [L]�Lβ�

∙ [LLβ] (Eq. S19)

Therefore the fraction of MSA-2 dimers containing two radioactive molecules in all MSA-2 dimers can be described by equation S19.

[LβLβ][LβLβ]+[LLβ] + [LL]

= [Lβ][L] ∙�LLβ�

(�LLβ�+[Lβ][L] ∙�LLβ�+

[L]�Lβ�

∙ [LLβ] =

[Lβ][L]

�1+[Lβ][L] +

[L]�Lβ�

�=

[Lβ][L]

[L]∙�Lβ�+[Lβ]2+[L]2

[L]∙�Lβ�

= �Lβ�2

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S19’)

Similarly the molar fraction of MSA-2 dimers containing one radioactive molecule and one non-radioactive molecule in all the MSA-2 dimers can be described by equation S20.

�LLβ�

[LβLβ]+[LLβ] + [LL]= �LLβ�

(�LLβ�+[Lβ][L] ∙�LLβ�+

[L]�Lβ�

∙ [LLβ]= 1

�1+[Lβ][L] +

[L]�Lβ�

�

18

= [L]∙[Lβ]

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S20)

It is important to appreciate that Equation S19 and S20 are also applicable to MSA-2 dimers bound to STING. This important concept is captured by Equation S21 and S22.

[LLβ]STING bound

[LβLβ]STING bound+ [LLβ]STING bound+[LL]STING bound≈

[L]∙[Lβ]

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S21)

[LβLβ]STING bound

[LβLβ]STING bound+ [LLβ]STING bound+[LL]STING bound≈ �Lβ�

2

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S22)

3) In the presence of STING, the overall fractional saturation of STING by MSA-2 (radioactive and nonradioactive MSA-2) is defined by the following equation ��RLLβ�+�RLβLβ�+[RLL]�

[R]total≈ (�Lβ�+[L])2

(Kd+(�Lβ�+[L])2) (Eq. S23)

Total concentration of MSA-2-occupied STING is

�RLLβ� + �RLβLβ� + [RLL] ≈ (�Lβ�+[L])2

(Kd+(�Lβ�+[L])2)∙ [R]total (Eq. S24)

From Equation S21 and S22

�LLβ�STING bound[LβLβ]STING−bound+ �LLβ�STING−bound+[LL]STING−bound

≈�RLLβ�

�RLβLβ�+�RLLβ�+[RLL]=

[L]∙[Lβ][L]∙�Lβ�+[Lβ]2+[L]2

(Eq. S25)

[LβLβ]STING bound

[LβLβ]STING−bound+ [LLβ]STING−bound+[LL]STING−bound≈

�RLβLβ�

�RLβLβ�+�RLLβ�+[RLL]=

�Lβ�2

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S26)

From Equation S24 and S25

�RLLβ� = [R]total ∙(�Lβ�+[L])2

(Kd+(�Lβ�+[L])2)∙

[L]∙[Lβ]

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S27)

Using Equation S24 and S26

�RLβLβ� = [R]total ∙(�Lβ�+[L])2

(Kd+(�Lβ�+[L])2)∙ �Lβ�

2

[L]∙�Lβ�+[Lβ]2+[L]2 (Eq. S28)

Define the total concentration of STING bound monomeric 3H-L-493 as [𝐿𝐿𝛽𝛽]𝑆𝑆𝑇𝑇𝑆𝑆𝑆𝑆𝑆𝑆−𝑏𝑏𝑡𝑡𝑏𝑏𝑏𝑏𝑏𝑏, it is easy to see that

[𝐋𝐋𝛃𝛃]𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒𝐒−𝐛𝐛𝐛𝐛𝐛𝐛𝐛𝐛𝐛𝐛 = [𝐑𝐑]𝐭𝐭𝐛𝐛𝐭𝐭𝐭𝐭𝐭𝐭 ∙(�𝐋𝐋𝛃𝛃�+[𝐋𝐋])𝟐𝟐

(𝐊𝐊𝐛𝐛+(�𝐋𝐋𝛃𝛃�+[𝐋𝐋])𝟐𝟐)∙

[𝐋𝐋]∙�𝐋𝐋𝛃𝛃]�+𝟐𝟐�𝐋𝐋𝛃𝛃�𝟐𝟐

[𝐋𝐋]∙�𝐋𝐋𝛃𝛃�+[𝐋𝐋𝛃𝛃]𝟐𝟐+[𝐋𝐋]𝟐𝟐) (Eq. S29)

In a homologous competition experiment, [𝐿𝐿𝛽𝛽]STING−bound is measured in the presence of a constant �𝐿𝐿𝛽𝛽� (for example, 200 nM) and various [L] (for example, 2 nM to 200 µM). We will first plot observed [𝐿𝐿𝛽𝛽]STING−bound (Y) against [L] (x), and then derive Kd by non-linear least square fit of the data with Equation S29 using Prizm. In the equation “hot” denotes the constant �𝐿𝐿𝛽𝛽� and C is related to [𝑅𝑅]𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡.

(Eq. S29’) Y = C ∙ (hot+x)2/(KD+(hot+x)2) ∙ hot ∙ (2 ∙ hot + x)/(x ∙ hot+hot2+x2)

19

Methods for simulating the effect of extracellular pH on intracellular [free MSA-2] and STING activation Scott et al (23) recently derived equations describing steady state distribution of ionizable molecules (both the neutral and ionized forms) in extracellular space and various intracellular compartments. The equation takes into account the physiochemical properties of a drug (permeability, pKa), the physiological properties of the cell (pH, membrane potential, surface area and volume), Fick’s law (diffusion of neutral species), the Nernst-Planck relationship (diffusion of charged species) and the Henderson-Hasselbalch relationship (ionization state). The ratio of [Intracellular free compound] to [Extracellular free compound] of an anionic compound (𝑲𝑲𝑲𝑲𝒖𝒖𝒖𝒖𝒅𝒅𝒅𝒅 ) can be calculated using equation S30 below.

𝑲𝑲𝑲𝑲𝒖𝒖𝒖𝒖𝒅𝒅𝒅𝒅 = 𝒇𝒇𝒖𝒖𝒅𝒅∙𝑪𝑪𝒅𝒅𝒇𝒇𝒖𝒖𝒅𝒅∙𝑪𝑪𝒅𝒅

= (𝝂𝝂𝝂𝝂𝒅𝒅𝑷𝑷𝒊𝒊 +(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅𝑷𝑷𝒏𝒏)𝒆𝒆𝝂𝝂𝝂𝝂𝝂𝝂𝒅𝒅𝑷𝑷𝒊𝒊+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅𝑷𝑷𝒏𝒏

(Eq. S30)

𝑓𝑓𝑓𝑓𝑏𝑏 Unbound fraction on the donor side 𝑓𝑓𝑓𝑓𝑟𝑟 Unbound fraction on the receiver side 𝐶𝐶𝑏𝑏 Total drug concentration on the donor side 𝐶𝐶𝑟𝑟 Total drug concentration on the receiver sider side 𝑃𝑃𝑏𝑏 The intrinsic permeability of the neutral form of the drug 𝑃𝑃𝑖𝑖 The intrinsic permeability of the ionized form of the drug 𝐻𝐻𝑏𝑏 The fraction ionized on the donor side 𝐻𝐻𝑟𝑟 The fraction ionized on the receiver sider 𝐾𝐾𝑏𝑏 The neutral fraction on the donor side 𝐾𝐾𝑟𝑟 The neutral fraction on the receiver sider 𝜈𝜈 = 𝑧𝑧𝑧𝑧ΔΦ

𝑅𝑅𝑇𝑇

z = -1 for a monoacid such as MSA-2 F is Faraday’s constant = 9.65×104 Coulomb/mol The Faraday constant represents the amount of electric charge carried by one mole, or Avogadro's number, of electrons. It is expressed in coulombs per mole (C/mol). Faraday's constant can be derived by dividing the Avogadro constant, or the number of electrons per mole, by the number of electrons per coulomb. The former is equal to approximately 6.02×1023, and the latter is approximately 6.24×1018. Therefore: F = (6.02×1023) / (6.24×1018) = 9.65×104 C/mol The coulomb (symbol: C) is the International System of Units (SI) unit of electric charge. It is the charge (symbol: Q or q) transported by a constant current of one ampere in one second. It is equivalent to the charge of approximately 6.242×1018 (1.036×10−5 mol) protons, and −1 C is equivalent to the charge of approximately 6.242×1018 electrons. ΔΦ is the membrane potential R is gas constant = 8.3144598 J⋅mol−1⋅K−1 (J is short for Joule, a unit of energy) J = C.V (Coulomb.Volt) T is absolute temperature (Kelvin temperature). The normal internal body temperature is 273.15 + 37 = 310.15 K Let’s calculate the value of 𝜈𝜈 at 37oC 𝜈𝜈 = 𝑧𝑧𝑧𝑧ΔΦ

𝑅𝑅𝑇𝑇= −1•96500•–0.040

8.3144598•310= 1.497585 if membrane potential is -40 mV.

𝐾𝐾𝑏𝑏 = [RCOOH][RCOOH]+[RCOO−]

= 10pKa

10pHd+10pKa (𝑝𝑝𝐻𝐻𝑝𝑝 is pH on the donor sider)

20

𝐻𝐻𝑏𝑏 = [RCOO−][RCOOH]+[RCOO−]

= 1 − 𝐾𝐾𝑏𝑏

𝐾𝐾𝑟𝑟 = [RCOOH][RCOOH]+[RCOO−]

= 10pKa

10pHr+10pKa (𝑝𝑝𝐻𝐻𝑝𝑝 is pH on the receiver sider)

𝐻𝐻𝑟𝑟 =[RCOO−]

[RCOOH] + [RCOO−]= 1 − 𝐾𝐾𝑟𝑟

Assuming that the electrically neutral (protonated) MSA-2 molecules are 1000 times more cell permeable than the anionic (deprotonated) MSA-2 molecules, i.e., 𝑃𝑃𝑛𝑛

𝑃𝑃𝑖𝑖 = 1000

𝑲𝑲𝑲𝑲𝒖𝒖𝒖𝒖𝒅𝒅𝒅𝒅 = 𝒇𝒇𝒖𝒖𝒅𝒅∙𝑪𝑪𝒅𝒅𝒇𝒇𝒖𝒖𝒅𝒅∙𝑪𝑪𝒅𝒅

= (𝝂𝝂𝝂𝝂𝒅𝒅𝑷𝑷𝒊𝒊 +(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅𝑷𝑷𝒏𝒏)𝒆𝒆𝝂𝝂𝝂𝝂𝝂𝝂𝒅𝒅𝑷𝑷𝒊𝒊+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅𝑷𝑷𝒏𝒏

= (𝝂𝝂𝝂𝝂𝒅𝒅𝑷𝑷𝒊𝒊 +(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏∙𝑷𝑷𝒊𝒊)𝒆𝒆𝝂𝝂𝝂𝝂𝝂𝝂𝒅𝒅𝑷𝑷𝒊𝒊+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏∙𝑷𝑷𝒊𝒊

= (𝝂𝝂𝝂𝝂𝒅𝒅+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏)𝒆𝒆𝝂𝝂𝝂𝝂𝝂𝝂𝒅𝒅+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏

When extracellular pH = 7.4, intracellular pH =7.2, the membrane potential = -40 mV, for MSA-2 (pKa = 4.78) 𝜈𝜈 = 𝑧𝑧𝑧𝑧ΔΦ

𝑅𝑅𝑇𝑇= −1•96500•–0.040

8.3144598•310= 1.497585

𝐾𝐾𝑏𝑏 = 10pKa

10pHd+10pKa= 104.78

107.4+104.78 =0.002393 𝐻𝐻𝑏𝑏 = 1 − 𝐾𝐾𝑏𝑏 = 0.997607

𝐾𝐾𝑟𝑟 = 10pKa

10pHr+10pKa= 104.78

107.2+104.78 = 0.003787 𝐻𝐻𝑟𝑟 = 1 − 𝐾𝐾𝑟𝑟 = 0.996213 [intracellular free MSA-2]/[extracellular free MSA-2] =

𝑲𝑲𝑲𝑲𝒖𝒖𝒖𝒖𝒅𝒅𝒅𝒅 = (𝝂𝝂𝝂𝝂𝒅𝒅+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏)𝒆𝒆𝝂𝝂𝝂𝝂𝝂𝝂𝒅𝒅+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏

= (1.497585∙0.997607+(2.7181.497585 −1)∙0.002393∙1000)2.7181.497585 ∙1.497585∙ 0.996213+(2.7181.497585 −1)0.003787∙1000

= 0.4946 When extracellular pH = 6.7, intracellular pH =7.2, and the membrane potential = -40 mV, 𝜈𝜈 = 𝑧𝑧𝑧𝑧ΔΦ

𝑅𝑅𝑇𝑇= −1•96500•–0.040

8.3144598•310= 1.497585

𝐾𝐾𝑏𝑏 = 10pKa

10pHd+10pKa= 104.78

106.7+104.78 =0.01188

𝐻𝐻𝑏𝑏 = 1 − 𝐾𝐾𝑏𝑏 = 0.988120182

𝐾𝐾𝑟𝑟 = 10pKa

10pHr+10pKa= 104.78

107.2+104.78 = 0.003787

𝐻𝐻𝑟𝑟 = 1 − 𝐾𝐾𝑟𝑟 = 0.996213 [intracellular free MSA-2]/[extracellular free MSA-2] =

𝑲𝑲𝑲𝑲𝒖𝒖𝒖𝒖𝒅𝒅𝒅𝒅 = (𝝂𝝂𝝂𝝂𝒅𝒅+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏)𝒆𝒆𝝂𝝂𝝂𝝂𝝂𝝂𝒅𝒅+(𝒆𝒆𝝂𝝂−𝟏𝟏)𝑲𝑲𝒅𝒅∙𝟏𝟏𝟏𝟏𝟏𝟏𝟏𝟏

= (1.497585∙0.988120182+(2.7181.497585 −1)∙0.01188∙1000)2.7181.497585 ∙1.497585∙0.996213 +(2.7181.497585 −1)∙0.003787∙1000

= 2.1555

If [extracellular free MSA-2] = 5 µM, [intracellular free MSA-2] would be 0.4946 × 5 µM = 2.473 µM when extracellular pH = 7.4, while [intracellular free MSA-2] would be 2.1555 × 5 µM = 10.78 µM when extracellular pH = 6.7. In Model 3 with overall KD = 64 µM2, the fractional occupancy (activation) of STING in the cell is 𝜃𝜃 = [𝑖𝑖𝑏𝑏𝑡𝑡𝑟𝑟𝑡𝑡𝑖𝑖𝑖𝑖𝑡𝑡𝑡𝑡𝑏𝑏𝑡𝑡𝑡𝑡𝑟𝑟 𝑓𝑓𝑟𝑟𝑖𝑖𝑖𝑖 𝑀𝑀𝑆𝑆𝑀𝑀2]2

K𝐷𝐷+[𝑖𝑖𝑏𝑏𝑡𝑡𝑟𝑟𝑡𝑡𝑖𝑖𝑖𝑖𝑡𝑡𝑡𝑡𝑏𝑏𝑡𝑡𝑡𝑡𝑟𝑟 𝑓𝑓𝑟𝑟𝑖𝑖𝑖𝑖 𝑀𝑀𝑆𝑆𝑀𝑀2]2 (Eq. S31)

𝜃𝜃 = (2.473 𝜇𝜇𝑀𝑀)2

64𝜇𝜇𝑀𝑀2+(2.473 𝜇𝜇𝑀𝑀)2= 0.087223 ≈ 8.7% when extracellular pH = 7.4

𝜃𝜃 = (10.78 𝜇𝜇𝑀𝑀)2

64𝜇𝜇𝑀𝑀2+(10.78 𝜇𝜇𝑀𝑀)2= 0.644856 ≈ 64.5% when extracellular pH = 6.7

21

Mice and tumor models All animal experimental procedures were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Merck & Co., Inc., Kenilworth, NJ, USA, following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Six-to-eight-week-old female C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME) for MC38, B16F10, TC-1 or LL-2 cell inoculation or BALB/c mice were obtained from Taconic Biosciences (Germantown, NY) for CT-26 cell inoculation. Nude NCr mice were obtained from Taconic and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) from Jackson Laboratories for the MC38 immune deficient studies. STINGgt/gt Goldenticket mice (20) were obtained from Jackson Laboratories. Cells were inoculated subcutaneously into the right lower flank with 1 × 106 MC38 colon adenocarcinoma, 2 × 105 B16F10 melanoma, 1 × 105 TC-1 lung cancer, 3 × 105 LL-2 lung carcinoma or CT-26 colon carcinoma cells in 100 μL of serum-free Dulbecco’s modified Eagle’s medium (DMEM). Animals were assigned to treatment arms of 10 mice each when equivalent mean tumor volume reached ~100 mm3 among all treatment groups except for the advance MC38 model when animals were enrolled at ~300 mm3. Length of time to reach desired tumor volume depended on the cell line used and day of enrollment or start of treatment was designated as day 0. Tumor and body weight measurements were performed twice per week using calipers and weigh scale respectively. Tumor volume (V) was calculated with the formula V = (length × width2)/2. Mice were euthanized when tumor volume approached ~2000 mm3, weight loss exceeded 20%, or if tumors ulcerated. For the re-challenge phase of the study, 1 × 106 MC38 tumor cells were inoculated subcutaneously into the left lower flank of mice that achieved CR and had been tumor free for at least 20 days. Ten naïve C57BL/6J mice were used as controls. In Vivo Efficacy Drug Treatments Animals enrolled in MC38 study were dosed with vehicle (PBS) or MSA-2 intratumorally (IT) at 45, 150 or 450 µg, subcutaneously (SC) at 5, 20 or 50 mg/kg or orally (PO) at 20, 80 or 200 mg/kg. IT doses were administered on days 0, 3 and 7 and SC or PO doses were administered on days 0, 5 and 10 except 50 mg/kg SC and 200 mg/kg PO doses which were dosed only on day 0. In the B16F10 model, MSA-2 was SC dosed at 50 mg/kg on days 0 and 4. CT26 tumor-bearing mice were SC treated with MSA-2 at 25 mg/kg on days 0, 11 and 20, or at 50 mg/kg on days 0 and 11, or PO treated at 60 mg/kg on days 0, 7 and 14. In the advanced MC38 model MSA-2 was administered PO at 80 or 160 mg/kg on days 0 and 4. Monoclonal antibody specific for mouse PD-1 (muDX400; MSD) was injected intraperitoneally (IP) every 5 days for a total of 5 treatments at 5 mg/kg for all models except B16F10 and CT26 which were dosed 10 mg/kg. Pharmacokinetic/pharmacodynamics (PK/PD) studies A single administration of MSA-2 was given IT, SC or PO when tumors had reached a volume >100 mm3. Blood was collected at 0.17, 0.5, 1, 2 and 4 hours for pharmacokinetics (PK) and tumor and blood were collected at 2, 4, 10 or 24 hours for pharmacodynamics (PD) time course studies. Vehicle control samples were obtained at the 4 hour time point and used for baseline measurements. Each group consisted of 3 to 9 mice. Tumors were flash-frozen and blood was used immediately for plasma preparation using EDTA-treated tubes (BD Cat# 367841) and frozen for subsequent analysis. Frozen tissue samples in 2 mL eppendorf tubes were thawed on ice and then resuspended 3 volumes of cold NP40 Lysis buffer (Boston Bioproducts, Cat. No. BP-119) supplemented with 1

22

tablet per 10 mL of both Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Cat. No. 11836170001) and PhosSTOP Phosphatase Inhibitor Cocktail (ThermoFisher Cat. No. 04906845001). The tissue samples were then homogenized by the addition of one 5mm steel bead (Qiagen, Cat. No. 69989) per sample, followed by agitation at 4°C using a Qiagen TissueLyser II for 3 minutes using a frequency of 30 Hz. Samples were then centrifuged at 15,000 x g for 15 minutes at 4°C, and supernatant was removed and stored at -80°C in deep-well 96-well plates until further analysis. For both tissue and plasma samples, IFN-β concentrations were quantified using the VeriKine™ Mouse Interferon Beta ELISA Kit (PBL, Cat. No. 42400) and following the manufacturer’s instructions. Briefly, samples were thawed on ice then diluted 1:20 and 1:150 into sample dilution buffer to ensure that cytokines levels were within the linear range of a standard curve. 100 μL of sample or IFN-β standard were added to the assay plate and incubated at room temperature for 2 hours or overnight at 4°C, while shaking. Following this, assay plates were washed three times with 150 µL of wash solution before 100 μl of diluted detection antibody was added to the assay plate. Assay plates were incubated at room temperature for an additional 2 hours while shaking, then were washed three times with 150 μl of wash solution. 1x HRP solution was added to each well and the plate incubated for 1 hour at room temperature before washing the plate three times with 150 μl of wash solution. Finally, 100 μL of TMB substrate solution was added to each well for 5 minutes before the reaction was stopped with 100 μl of stop solution. IFN-β levels were quantified by measuring absorbance at 450 nm using a Flexstation 3 (Molecular Devices, San Jose, CA) plate reader. IL-6 and TNF-α cytokine levels were measured using a custom U-plex cytokine profiling kits (Meso Scale Discovery, Rockland, MD) and following the manufacturer’s instructions. Briefly, tissue and plasma samples were prepared as detailed above, and 50 μL of diluted sample or standard were added to U-plex assay plates pre-coated with a mixture of linker and biotinylated antibodies. Samples and standards were incubated in U-plex plates overnight at 4°C while shaking. The next day, plates were washed 3x with 150 μL of wash buffer (PBS-T; Dulbecco’s PBS (Hyclone, Cat. No. SH30028-03) and 0.01 % (v/v) Tween-20) before 50 μL of a mixture of custom detection antibodies specific for each cytokine were added. The U-plex plates were then incubated for 2 hours at room temperature while shaking before washing again with 3x 150 μL of PBS-T. Following the last wash, 150 μL of 2x MSD read T buffer was added to each well and plates were read immediately on an MSD Sector Imager 6000 (Meso Scale Discovery, Rockland, MD). Samples were quantified using the standard curve for each cytokine, then multiplying by the dilution factor of the sample. Statistical analysis of differences in cytokine levels between treatment groups was performed using an unmatched one-way ANOVA, followed by correction for multiple comparisons using statistical hypothesis testing (Tukey) or Student’s t-test. A liquid chromatography-mass spectroscopy (LC-MS/MS) assay was used to determine the concentration of MSA-2 in blood or plasma samples. Chromatography was performed using a C18 column and detection was carried out using a triple quadrupole tandem mass spectrometer (API5000 from Applied Biosystems) equipped with an electrospray interface. The Analyst 1.6.2 software (Applied Biosystems) was used to control the system and for analyses. Chromatographic data were collected and integrated using the MultiQuant 3.0.1 data analysis program. The raw data files were imported and processed by means of Watson LIMS software for PK calculation.

23

In vivo interstitial pH measurement MC38, CT26 or B16F10 cells were grown as subcutaneous tumors. Once tumors reached 200-500 mm3, the intratumoral pH was measured using a microelectrode (Orion™ 9863BN Micro pH Electrode) as described previously (25-27). Briefly, animals were sedated using isoflurane, and a bevel needle tipped combination electrode was inserted up to 1.3 cm into the center of the tumor and held in place for up to 1 min, until pH readings stabilized. The needle was rotated once in each location, to allow the pH electrode to re-read at the same depth in order to make two independent measurements per location. These values were averaged to report a mean for each animal. A non-tumor location on contralateral side was used for as a reference location for normal skin pH measurement. The animals were euthanized after the pH was measured in the primary tumor and reference location. Statistical analysis of differences in tumor pH to normal skin was performed using pair-matched Student’s t-test. Results are summarized in Fig. 7F. Immunohistochemistry (IHC) LL-2 tumor bearing mice were enrolled into treatment arms in groups of 4 and when tumors reached 100 - 200 mm3. Animals received either vehicle + 5 mg/kg isotype control or 5 mg/kg muDX400 (IP) on day 0 or 40 mg/kg MSA-2 (SC) on days 0 and 5. Tumors were collected on day 7 following initial dose, fixed in 10% Neutral Buffered Formalin (Fisher Scientific) for 24 hours, processed and embedded in paraffin. CD8a (eBiosciences; clone 4SM15) was detected by IHC using TechMate Instrument (Roche Diagnostics) and the MIPE program. Antibodies were incubated for 1 hour in Reagent Manufacturing Buffer (RMB; QualTek Santa Barbara) with Goat Serum, followed by 25 minutes in Goat anti-Rat Secondary Antibody, detected with Avidin-Biotin Complex (ABC; Vector Labs) and visualized with colorimetric chromogen (DAB; GBI Labs). Nuclei were counterstained using hematoxylin (blue stain) to assess cell and tissue morphology. An abundance score was used to evaluate the presence of CD8a within the tumor population. On this scale 0=0 positive lymphocytes, 5=1-9 positive lymphocytes, 15=10-19 positive lymphocytes, 33=20-49 positive lymphocytes, 63=50-75 positive lymphocytes, 88=76-100 positive lymphocytes and 150=>100 positive lymphocytes. All evaluation was completed by reviewing at least 3 representative fields at 20x magnification. Statistical analysis was performed using unpaired Student’s t-test. Results are summarized in Fig. 8F. Identification of “STING Agonist Fingerprints” by 2D and 1D NMR 15N-labeled human STING cytosolic domain constructs (hSTING) were generated as conformation sensing tools to detect closed-form binders of STING. Confidence in the assignment of an agonist conformation for both cGAMP and MSA-2 was enhanced by the distinct differences in resonance shifts upon binding of a non-agonist ligand to the open human STING conformation (28). To derive ‘STING agonistic’ 1D and 2D NMR spectral signatures test compounds were solubilized and titrated in either 100% DMSO-d6 (e.g., MSA-2) or D2O (e.g., 2’3’-cGAMP) up to 2x (cGAMP) or 10x (MSA-2) excess to saturate 50 µM [15N]-labeled hSTING-WT and hSTING-HAQ (amino acids 155-341, containing an extra N-terminal Ser after TEV cleavage) in 20 mM Tris pH 7.5, 150 mM sodium chloride, 5 mM dithiothreitol (DTT), 2% DMSO-d6, 8% D2O. Total sample volume of 165 µl was transferred to 3mm NMR tubes and 2D 1H-15N SOFAST-HMQC (29) and 1D 1H NMR methyl experiments were acquired at 30 °C on an 800 MHz Bruker Ascend Four Channel AVANCE III HD NMR spectrometer, equipped with a TCI 5mm CryoProbe (ATM). The spectra widths were set to 16 ppm in 1H dimension (centered at 4.7 ppm) and 50 ppm in 15N dimension (centered at 117 ppm). The datasets were acquired with 1024 (2D) or 2048 (1D) data

24

points in the 1H dimension and 92 increments in the 15N dimension; typically, data collection completed in ~1 hour and ~90 seconds for each 2D and 1D NMR spectrum, respectively. For the 2D 1H-15N SOFAST-HMQC, the relaxation delay was set to 150 ms, and the H(N) chemical shift offset and H(N) bandwidth were set to 8.75 ppm and 4 ppm, respectively. For the 1D 1H NMR methyl version, the relaxation delay was set to 200 ms, and the H(C) chemical shift offset and H(C) bandwidth were set to -1 ppm and 4.25 ppm, respectively. NMR spectra were processed with Bruker TopSpin 3.5 software and analyzed using NMRViewJ software [One Moon Scientific, Inc.]. Crystallography and Surface Area Calculations STING protein was prepared as previously described (28). To prepare crystals of STING in complex with compounds, 1 µL compound stock (typically 100 mM in DMSO) was added to 25 µL freshly thawed protein for a final compound concentration of 12 mM (MSA-2) or 4 mM (other compounds). DTT (1 M freshly-prepared stock solution in water) was added to the sample to a final concentration of 20 mM. The sample, often containing undissolved compound, was lightly vortexed to mix and then incubated on ice. After 15 minutes, the sample was lightly centrifuged to clarify before dispensing drops. Into a pre-greased VDX plate (Hampton Research, catalog # HR3-172) was dispensed 1 mL reservoir solution containing 20-30% w/v PEG 6000 across the plate, 100 mM Tris pH 8.5, 200 mM NaCl, and 2 mM DTT. 3 µL protein solution was mixed with 1 µL reservoir solution on a siliconized cover slip and sealed atop each reservoir. The plate was incubated at 18°C. Typically, clusters of crystals appeared after 3 days and could be used to streak seed other wells. Single crystals typically appeared one day later and were prepared for data collection by swishing through 2 µL of perfluoropolyether cryo oil (Hampton Research, catalog # HR2-814) immediately before plunging into liquid nitrogen. At either APS 17-ID or SLS PXI-S06SA, 720 frames of 0.25° rotation were collected from a single crystal. Data were scaled, merged, and reduced using autoPROC (30). Structures were solved by molecular replacement using the program MOLREP (31, 32) with a probe constructed from the STING monomer present in PDB entry 4KSY (3). Restraint dictionaries for the compounds were produced using GRADE (33), and compounds were fit manually into difference density using Coot (34). Coordinate refinement was conducted using autoBUSTER (35). Iterative cycles of refinement and analysis were conducted using Coot and MolProbity (36, 37). TLS treatment was applied to the structure with MSA-2 and 4. NCS restraints (38) were used where there were multiple copies of the STING monomer in the asymmetric unit (see Table S6). Data collection, processing, refinement, and validation information is summarized in Table S6. Figures of STING complexes, bound ligands alone (Fig. 6D) and corresponding omit maps (Fig. S8B) were prepared using the PyMOL software (The PyMOL Molecular Graphics System, Schrödinger, LLC). To generate omit maps, structure factors were first calculated by running autoBUSTER (35) in “MapOnly” mode using a model lacking all ligand atoms. Omit maps were then calculated using the program fft and trimmed to within 5 Å of the ligand using the program mapmask (32). All surface area calculations were performed using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC), using the method to calculate buried solvent-accessible surface area (SASA) described in https://pymolwiki.org/index.php/Get_Area. The STING homodimer was constructed by expanding crystallographic symmetry in accordance with REMARK 350 in the PDB file. The STING dimer “apoprotein” was constructed by removal of the bound ligand(s) from the structure of the dimer. Water molecules were excluded from this calculation. For the cGAMP complex, only one cGAMP molecule was included in the protein complex with the ligand, since

25

only one cGAMP molecule may bind at a time. For the MSA-2 complex, both MSA-2 molecules were included since they bind simultaneously. A PyMOL script to calculate these values is included below (text with gray background). The total solvent-accessible surface area buried in the STING dimer by bound ligand(s) is calculated by subtracting the SASA of the complex from the sum of the SASA of the two components – STING dimer apoprotein and the isolated ligand(s). The total surface area buried between two MSA-2 molecules against each other in the absence of protein is calculated in the same manner. Since the MSA-2 dimer is symmetrical, it is possible to determine the buried SASA per MSA-2 molecule by halving the total buried SASA. # PyMOL commands, after creating individual objects of A, B, and AB, using script from https://pymolwiki.org/index.php/Get_Area # * A = apoprotein # * B = ligand(s) extracted ligand binding site # * AB = protein in complex with ligand(s) # Total buried surface area is area(A) + area(B) - area(AB) # load structures. They are called 4ksy and 6ukm. # in the case of 4ksy, note that symmetry expansion produces a second cGAMP molecule atop the first, so we will only consider one. # generate dimer using: symexp 4ksy_s, 4ksy, organic, 3 symexp 6ukm_s, 6ukm, organic, 3 # change chain ID for symmetry mate to make selections easier. alter 4ksy_s01000000, chain='B' alter 6ukm_s01000000, chain='B' # generate various objects for area calculation. You may need to adjust object name for symmetry-mate. Waters are excluded. create 4ksy_dimer, (4ksy or 4ksy_s01000000) and not solvent create 4ksy_lig, 4ksy_dimer and organic and chain A create 4ksy_apo, 4ksy_dimer and not organic create 6ukm_dimer, (6ukm or 6ukm_s01000000) and not solvent create 6ukm_ligs, 6ukm_dimer and organic create 6ukm_apo, 6ukm_dimer and not organic create 6ukm_lig1, 6ukm_ligs and chain L create 6ukm_lig2, 6ukm_ligs and chain B # get hydrogens onto everything (NOTE: must have valid valences on e.g. small organic molecules) h_add # make sure all atoms within an object occlude one another flag ignore, none set dot_density, 4 # solvent-accessible surface set dot_solvent, 1 # get areas get_area 4ksy_apo get_area 4ksy_lig get_area 4ksy_dimer get_area 6ukm_apo get_area 6ukm_ligs get_area 6ukm_dimer get_area 6ukm_lig1 get_area 6ukm_lig2

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In the previously published STING complex with cGAMP (PDB 4KSY), there is a total of 1145 Å2 of solvent-accessible surface area (SASA) buried by cGAMP (see Table S3). This includes buried SASA contributed by the ligand as well as the binding pocket. The SASA of the ligand-binding cavity alone in this cGAMP complex is 386 Å2. In the complex of STING with MSA-2, there is a comparable amount of buried SASA due to binding of the two MSA-2 molecules observed bound to the STING dimer: 1047 Å2. The SASA of the ligand-binding cavity in this MSA-2 complex is 327 Å2. In both cases, the contribution to the total buried SASA is higher for the bound ligand(s) (759 and 720 Å2, respectively) than for the interior of the binding site (386 and 327 Å2, respectively). This difference in areal contribution is likely due to the convexity of the ligand solvent-accessible surface and the complementary concavity of the interior of their binding sites in the STING dimer. Considering the bioactive dimer of MSA-2 molecules alone, an analogous calculation results in burial of 158 Å2 SASA per MSA-2 molecule against the another, excluding the protein. Computational Design of Covalent Dimers To design predicted-optimal linkers between the two MSA-2 monomers, a computational protocol was developed to enumerate thousands of potential core and linker combinations as well as score their ability to replicate the geometry of the MSA-2 dimer in a low-energy conformation. Three isolated cores were considered for linking: 6-methoxybenzothiophene, 5,6-dimethoxybenzothiophene, and 4-fluoro-6-methoxybenzothiophene. Potential dimers were generated through combinatorial enumeration of all possible linkers containing methylene, oxygen, amide, and ester units up to a maximum linear length of 6 atoms connecting any pair of free positions across the cores. Enumerated linkers containing functionality likely to be chemically unstable or undesirable, such as peroxides and acetals, were filtered out. All combinatorial enumeration and filtering was performed using the OEChem Toolkit v2016.01 (OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com), and, in total, 8057 possible linked dimers were considered. To score each linked dimer, first a conformational ensemble with a free-energy ranking was generated using FREEFORM v1.9.0.3 (OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com). Next, the conformational ensemble was aligned to the STING-bound crystallographic geometry of the MSA-2 dimer (isolated benzothiophene cores) using ROCS v3.2.0.4 (OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com). The RefTverskyCombo score was used as the measure of overlay quality to avoid penalizing for the linker. Considering all conformations across all linked dimers, a plot of the free-energy of selecting the conformer from its solution ensemble (conf_dG in the FREEFORM output) versus the RefTverskyCombo overlay score was constructed (Fig. 6A, main text). A linked dimer was prioritized if it had a conformation that simultaneously achieved a high overlay score and a low free energy. SAFETY STATEMENT Given the highly potent nature of molecules whose preparation is detailed in this section, and their demonstrated ability to induce cytokine elevation in vitro and in vivo, proper procedures for handling of highly potent compounds should be followed at all times in accord with specific institutional policies.

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Chemical Synthesis MSA-2: 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

OHO

MeO

MeO OS Step 1:

S O

OH

MeO

MeO

S O

Cl

MeO

MeO(COCl)2, DMF

THF To 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (5.00 g, 21.0 mmol, 1 equiv) in THF

(60 mL) at 0°C under an argon atmosphere was added oxalyl chloride (7.35 mL, 84.0 mmol, 4 equiv) followed by DMF (0.162 mL, 2.10 mmol, 0.1 equiv). The reaction mixture was stirred at 0°C for 1 hour and then warmed to room temperature and stirred for an additional 2 hours. The reaction mixture was then concentrated under reduced pressure and the resulting 5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride was subsequently used without purification. Step 2:

S O

Cl

MeO

MeO

S OMeO

MeO

OO

O

O

ZnBr

THF

SO

OCu

To copper(I) thiophene-2-carboxylate (5.52 g, 29.0 mmol, 1.38 equiv) in THF (30 mL) was

added (3-ethoxy-3-oxopropyl)zinc(II) bromide (0.5 M in THF, 53.7 mL, 26.9 mmol, 1.28 equiv) The reaction mixture was stirred for 20 min at 0°C followed by addition of 5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride (5.39 g, 21.0 mmol, 1.00 equiv) as a solution in THF (100 mL). The resulting reaction mixture was allowed to warm to and stirred at room temperature for 6 h before being poured into saturated aqueous ammonium chloride (150 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water and brine and then concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-40% ethyl acetate / hexanes) to afford ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.89 g, 13% yield over two steps). LCMS (C16H19O5S) (ES, m/z): 323 [M+H]+. 1H NMR (500 MHz, CHCl3-d) δ 7.92 (s, 1H), 7.28 (s, 1H), 7.27 (s, 1H), 4.19 (q, J = 7.1 Hz, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.34 (t, J = 6.8 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).

Step 3:

S OMeO

MeO

OO OH

O

MeO

MeO OS

LiOH

THF, MeOH

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To a solution of ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.47 g, 1.46 mmol, 1.00 equiv) in THF (3 mL) and methanol (3 mL) was added lithium hydroxide (1.0 M in water, 3.00 mL, 3.00 mmol, 2.06 equiv). After the reaction mixture was stirred for 1 hour at room temperature, HCl (1.0 M in water, 3.00 mL, 3.00 mmol, 2.06 equiv) was added. A precipitate formed within 30 min and was collected by filtration to afford 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (0.43 g, 78% yield). LCMS (C14H15O5S) (ES, m/z): 295 [M+H]+. 1H NMR (500 MHz, DMSO- d6) δ 12.20 (br s, 1H), 8.21 (s, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.30 – 3.23 (m, 2H), 2.64 – 2.57 (m, 2H). MSA-1: Diammonium (2R,5S,7R,8R,10R,12aR,14R,15R,15aS)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide

1) TFA·Py, H2O

2) t-BuNH2

DCA, H2O, DCM;

Et3SiH, Py

O

O

HO

N

N N

NH

O

NH

ODMTrO

N

N N

N

NHBz

O OTBS

HP

O

ONH

O

P ON CN

O

O

DMTrO

N

N N

NH

O

NH

HP

O

O

O

O

O

DMTrO

N

N N

NH

O

NH

PO

N

O

CN

Q1Q2 Q3

Q4

O

O

O

N

N N

NH

O

NH

PO OS O

N

N N

N

NHBz

O OTBSPOS

NC

O

O

O

N

N N

NH

O

NH

O

N

N N

N

NHBz

O OTBSPOS

NC

HP

O

ODCA, H2O, DCM;

Et3SiH, Py

PhOP

OPh

O

Cl

O

O

O

N

N N

NH

O

NH

O

N

N N

N

NHBz

O OTBSP

HP

O

ONH

OS

NC

SS

O

1) MeNH2

2) Et3N·3HF

Q5Q6

Q7 MSA-1

t-BuNH3

OO

iPr

O

NH4

1)

2) DDTTDMTrO

HO

1)

2)

O

O

O

N

N N

NH

O

NH2

PO OS O

N

N N

N

NH2

O OHPOS

NH4

NH4

Na

(2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (Q2): To a stirred solution of (2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy) methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Q1, 514 mg, 0.612 mmol) in MeCN/H2O (150/1, 3 mL) at rt was added pyridin-1-ium 2,2,2-trifluoroacetate (142 mg, 0.734 mmol) in one portion. After 1 h, tert-butylamine (3mL) was added and the resulting solution was stirred at rt for 30min. Then, it was concentrated under reduced pressure. The residue

29

was co-evaporated with anhydrous MeCN (2 × 5mL) to give a white foam, which was used for the next step without purification. LCMS (ES, m/z): 704.3 [M + H]+. (2R,3R,5S)-5-(hydroxymethyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (Q3): To a stirred solution of crude Q2 from the previous step in DCM (6 mL) at rt was added water (0.12 mL) and dichloroacetic acid in DCM (6%, 7.2 mL, 5.2 mmol). The color of the solution changed to red immediately. After 30 min, triethylsilane (17.2 mL) was added, and it was stirred for 1.5 h as the red color of the solution disappeared. The reaction was cooled to 0°C and pyridine (0.83 mL, 10 mmol) was added. After 10 min, it was concentrated under reduced pressure. The residue was triturated with MTBE/Hexane (1/1, 8 mL), and the supernatant was decanted. This process was repeated twice. The final residue was dried under reduced pressure over P2O5 for 20 h and was used for the next step without purification. LCMS (ES, m/z): 402.1 [M + H]+. Pyridin-1-ium (2R,3R,5S)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (Q5): To a stirred solution of the crude Q3 (10 g, ~7 mmol) in MeCN (80 mL) under Ar was added activated 4Å molecular sieve (1 g), and the mixture was stirred at rt for 30 min. (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy) tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (Q4, 7.61 g, 7.81 mmol) was co-evaporated with anhydrous MeCN (3 × 30 mL) and then, re-dissolved in MeCN (80 mL). It was dried by adding activated 4Å molecular sieve (1 g) under Ar. After 30 min, it was added to the solution of Q3 dropwise. The mixture was stirred at rt for 30 min. Then, N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)methanimidamide (DDTT, 1.58 g, 7.70 mmol) was added. The resulting mixture was stirred at rt for 1 h and then, concentrated to give a yellow crude solid. It was used for the next reaction step directly. LCMS (ES, m/z): 1318.1 [M - H]-. Ammonium (2R,3R,5S)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-2-(2-isobutyramido-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl phosphonate (Q6): To a stirred solution of crude Q5 from the previous step in DCM (68 ml) was added water (1.4 mL) and dichloroacetic acid in DCM (6%, 82 mL, 59 mmol). The mixture turned to red immediately. After 10 min, Et3SiH (30 mL) was added and the mixture was stirred at rt as it turned to colorless over 1.5 h. Then, the reaction was cooled to 0°C and pyridine (9.5 ml) was added. After 10 min, it was concentrated under reduced pressure. The residue was purified by reverse phase chromatography (C18) eluted with 0 to 31% MeCN in aqueous NH4HCO3 (5 mmol) to give the product (1.35 g, 1.30 mmol, 17.0% yield over two steps) as a light yellow solid. LCMS (ES, m/z): 1018.0 [M + H]+. P-NMR: (162 MHz, CD3OD): δ 68.52-67.7 (m), 2.75-2.54 (m). Sodium (2R,5S,7R,8R,10R,12aR,14R,15R,15aR)-15-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-7-{2-[(2-methylpropanoyl)amino]-6-oxo-1,6-dihydro-9H-purin-9-yl}-14-{6-[(phenylcarbonyl)amino]-9H-purin-9-yl}octahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-10-thiolate 10-oxide 2-sulfide (Q7): A

30

solution of Q6 (116 mg, 0.114 mmol) in anhydrous pyridine (5 ml) was evaporated under reduced pressure. It was repeated twice, and the residue was redissolved in anhydrous pyridine (4.5 ml). To the solution at rt under Ar was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (0.063 g, 0.34 mmol). The resulting mixture was stirred at rt for 1 h. Then, water (0.062 mL) and 3H-benzo[c][1,2]dithiol-3-one (29 mg, 0.17 mmol) were added to the reaction. After 1 h, the reaction mixture was poured into aq NaHCO3 (0.33 M, 18 ml) and stirred for 5 min. EtOAc (15 ml) and Et2O (15 ml) were added to the stirred solution and layers were separated. The aq layer was extracted with EtOAc/Et2O (1/1, 2 × 30 ml). The combined organic layers were dried (MgSO4), concentrated, and purified by prep-TLC (20 cm × 20cm) eluted with 10% MeOH in DCM to give the product (15 mg, 0.015 mmol, 13% yield) as a light yellow solid. LCMS (ES, m/z): 1033.3 [M + H]+. P-NMR: (162 MHz, CD3OD): δ 65.88 (s), 55.68 (s). Diammonium (2R,5S,7R,8R,10R,12aR,14R,15R,15aS)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15-hydroxyoctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-bis(thiolate) 2,10-dioxide (MSA-1): Q7 (15 mg, 0.015 mmol) was dissolved in MeNH2 in EtOH (30%, 1 mL) and it was stirred at rt for 4 h. Then, the solution was concentrated to give a yellow solid. LCMS (ES, m/z): 805.1 [M + H]+. It was dissolved in pyridine/Et3N (5/2, 1 ml) and the solution was evaporated under reduced pressure. It was repeated twice, and the residue was redissolved in pyridine (0.5 ml) under Ar. To the reaction was added triethylamine (0.18 ml, 1.3 mmol) and triethylamine trihydrofluoride (0.053 mL, 0.33 mmol). The mixture was heated at 50°C for 16 h. Then, it was cooled down to rt, concentrated and purified by prep-HPLC (T3 OBD) eluted with 0 to 14% MeCN in aq NH4HCO3 (50 mM) to give the product (2.9 mg, 4.0 µmol, 28 % yield) as a white solid. LCMS (ES, m/z): 689.2 [M - H]-. H-NMR: (400 MHz, D2O): δ 8.26 (s, 1H), 8.18 (s, 1H), 7.76 (s, 1H), 6.10 (s, 1H), 5.69 (s, 1H), 5.68 (s, 1H), 5.16 (m, 1H), 4.56-4.46 (m, 3H), 4.23 (m, 1H), 4.17-3.97 (m, 2H), 2.48 (m, 2H). 31PNMR: (162 MHz, D2O): δ 55.54 (s), 51.75 (s). HRMS (ESI/TOF) m/z: [M + H]+ Calcd for C20H25N10O10P2S2 691.0672; Found 691.0674. Compound 2: 4-(5,6-Dimethoxy-1H-indol-2-yl)-4-oxobutanoic acid

O

OOH

O

OHN

Step 1: tert-Butyl 5,6-dimethoxy-1H-indole-1-carboxylate

O

O

HN O

O

NO

OO O

O

O

O

To a mixture of 5,6-dimethoxy-1H-indole (4.90 g, 27.7 mmol) in DCM (150 mL) was

added triethylamine (7.71 mL, 55.3 mmol), DMAP (1.013 g, 8.30 mmol) and di-tert-butyl dicarbonate (8.67 mL, 37.3 mmol). The reaction mixture was stirred at 15°C for 16 hours. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (eluting 0-20% ethyl acetate/petroleum ether) to afford tert-butyl 5,6-dimethoxy-1H-indole-1-carboxylate (7.0 g, 25 mmol, 91%). 1H NMR (500 MHz, CDCl3): δ 7.78 (br s, 1H), 7.45 (br s, 1H), 7.01 (s, 1H), 6.46 (s, 1H), 3.96 (s, 3H), 3.92 (s, 3H), 1.68 (s, 9H).

31

Step 2: 4-(1-(tert-Butoxycarbonyl)-5,6-dimethoxy-1H-indol-2-yl)-4-oxobutanoic acid

O

OOH

O

ONO

O

O

O

NO

OO O O

tert-Butyllithium (1.3M in THF, 20.3 mL, 26.4 mmol) was added dropwise to a stirred

solution of tert-butyl 5,6-dimethoxy-1H-indole-1-carboxylate (4.60 g, 16.6 mmol) in THF (100 mL) at -78°C under a nitrogen atmosphere. The reaction mixture was stirred at -78°C for one hour, and then a solution of dihydrofuran-2,5-dione (2.76 g, 27.5 mmol) in THF (20 mL) was added dropwise to the mixture at -78°C. The reaction mixture was stirred and slowly warmed to 15°C over a period of 2.5 hours. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (300 mL) and then extracted with ethyl acetate (300 mL). The organic layer was separated and washed with additional saturated aqueous sodium bicarbonate solution (150 mL). The two aqueous layers were combined and acidified by the addition of saturated aqueous citric acid (pH = 5). The mixture was then extracted with ethyl acetate (2 x 300 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford 4-(1-(tert-butoxycarbonyl)-5,6-dimethoxy-1H-indol-2-yl)-4-oxobutanoic acid (5.0 g, 9.3 mmol, 56%). LCMS (C19H24NO7-C4H8) (ES, m/z): 322 [M-tBu+H]+. Step 3: 4-(5,6-Dimethoxy-1H-indol-2-yl)-4-oxobutanoic acid

O

OOH

O

ONO

O

O

OOH

O

OHNHCl

A solution of HCl (1.0M in ethyl acetate, 10 mL, 10 mmol) was added to a stirred solution

of 4-(1-(tert-butoxycarbonyl)-5,6-dimethoxy-1H-indol-2-yl)-4-oxobutanoic acid (1.80 g, 4.77 mmol) in DCM (40 mL). The reaction mixture was stirred at 15°C for 30 minutes. The reaction mixture was concentrated under reduced pressure and the residue was diluted with ethyl acetate (40 mL) and stirred. The mixture was filtered and the isolated solids were dried under in vacuo to afford 4-(5,6-dimethoxy-1H-indol-2-yl)-4-oxobutanoic acid (1.20 g, 4.31 mmol, 90%). LCMS (C14H16NO5) (ES, m/z): 278 [M+H]+. 1H NMR (500 MHz, DMSO-d6): δ 11.48 (s, 1H), 7.40 – 7.10 (br s, 1H), 7.23 (s, 1H), 7.11 (s, 1H), 6.88 (s, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.17 – 3.08 (m, 2H), 2.63 – 2.56 (m, 2H).

Compound 3: 4,4'-((propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S

O

HOO

O S

O

OHO

O

32

Step 1: 5-Bromo-2-fluoro-4-methoxybenzaldehyde

O F

H

OBr

O F

H

O 2-Fluoro-4-methoxybenzaldehyde (9.0g, 58mmol) was added slowly (portion-wise) to a solution of Br2 (6.0mL, 120mmol) in MeOH (40mL) at 0°C. The reaction mixture was stirred at 0°C for 2h. A solution of NaHSO3 (24.3g, 234mmol) in H2O (300mL) was added slowly to the reaction mixture at 0°C. The resulting suspension was then stirred for 30 min at 0°C. The reaction mixture was filtered, and the filtrate was washed with additional H2O (3x25mL). The filtrate was then dried under reduced pressure to afford 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 58.4 mmol, 78%). The product was used without purification. 1H NMR (500MHz, DMSO-d6): δ 10.02 (s, 1H), 7.98 (d, J = 7.5Hz, 1H), 7.26 (d, J = 13.0Hz, 1H), 3.97 (s, 3H). Step 2: tert-Butyl 5-bromo-6-mehoxybenzo[b]thiophene-2-carboxylate

O F

H

OBr

O

Br

S O

O

K2CO3 (19.0g, 137mmol) was added slowly (portion-wise) to a solution of 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7g, 45.8mmol) and tert-butyl 2-mercaptoacetate (6.65mL, 45.8mmol) in DMF (50mL) at 20°C under Ar atmosphere. The reaction mixture was stirred and heated to 100°C for 16h. The reaction mixture was then cooled to RT and diluted with Et2O (1000mL). The mixture was then washed with H2O (500mL, then 2x250mL), and the combined aq layers were extracted with Et2O (2x200mL). The organic layers were then combined and washed with brine (50mL). The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure to afford tert-butyl 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylate (15.45 g, 45.0 mmol, 98%). The product was used without purification. 1H NMR (500MHz, DMSO-d6): δ 8.26 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 3.92 (s, 3H), 1.55 (s, 9H). Step 3: 5-Bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid

O

Br

S O

O

O

Br

S O

OH HCl (56mL, 4.0M in 1,4-dioxane, 230mmol) was added to a solution of tert-butyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (15.5g, 45.0mmol) in DCM (200mL) at 20°C. The reaction mixture was stirred at 20°C for 3days. The reaction mixture was then diluted by the dropwise addition of Hex (500mL). The resulting suspension was stirred for an additional 2h post-addition at RT. The reaction mixture was filtered, and the collected material was washed with Hex (2x50mL) and dried under reduced pressure to afford 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylic acid (12.5 g, 43.4 mmol, 96%), which was used without purification. 1H NMR (500MHz, DMSO-d6): δ 13.42 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.80 (s, 1H), 3.93 (s, 3H). Step 4: 5-Bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride

O

Br

S O

OH

O

Br

S O

Cl DMF (0.049mL, 0.63mmol) was added slowly (dropwise) to a solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (6.0g, 21mmol) and (COCl)2 (5.5mL, 63mmol) in THF (100mL) at 0°C under Ar atmosphere. The reaction mixture was stirred at 0°C for 2h and then allowed to warm to RT. The reaction mixture was stirred for 18h at RT. The reaction mixture

33

was then concentrated under reduced pressure to afford 5-bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride (6.39 g, 20.9 mmol, 100%). The product was used without purification. Step 5: Ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

O

Br

S O

Cl

O

Br

S O

OO

A solution of (3-ethoxy-3-oxopropyl)zinc(II) bromide (13.8mL, 0.50M in THF, 6.9mmol) was added to an oven-dried flask containing ((thiophene-2-carbonyl)oxy)copper (1.31g, 6.87mmol) under Ar at 0°C. The reaction mixture was stirred for 20 min at 0°C under Ar. An Ar-degassed solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride (1.52g, 4.98mmol) in THF (25.0mL) was then added via cannula to the reaction mixture at 0°C; the resulting suspension was allowed to warm to RT and was stirred for an additional 3h. The reaction mixture was cooled to 0°C and quenched with sat aq NH4Cl (50mL). The mixture was allowed to warm to RT and stirred for an additional 10min. The mixture was filtered, and the filtrate was diluted with EtOAc (500mL) and brine (50mL). The organic layer was separated, washed with additional brine (25mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc in DCM) to afford ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (1.14 g, 6.59 mmol, 62%). LCMS (C15H16BrO4S) (ES, m/z): 371, 373 [M+H]+. 1H NMR (500MHz, DMSO-d6): δ 8.27 (s, 1H), 8.26 (s, 1H), 7.81 (s, 1H), 4.07-4.02 (m, 2H), 3.94 (s, 3H), 3.35-3.25 (m, 2H), 2.68-2.64 (m, 2H), 1.20-1.14 (m, 3H). Step 6: Ethyl 4-(5-allyl-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

Br

O

OO

S OO

OO

To a vial containing ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (5.0g, 13mmol), tetrakis(triphenylphosphine)palladium(0) (1.6g, 1.3mmol), and dioxane (15mL), was added allyltri-n-butyltin (5.4mL, 18mmol). The reaction was heated to 90°C for 18h. Upon cooling to RT, the mixture was diluted with DCM, filtered through CELITE and added to flask containing aq KF (0.5M, 200mL). The mixture stirred, and the organic layer was then separated, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (0-->30% EtOAc gradient in hexanes) to afford (2.46g,7.4 mmol, 55%) of ethyl 4-(5-allyl-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate. LCMS (C18H21O4S) (ES, m/z): 333 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.23 (s, 1H), 7.69 (s, 1H), 7.58 (s, 1H), 5.96 (dq, J = 15.9, 6.6 Hz, 1H), 5.04 (d, J = 4.5 Hz, 1H), 5.02 (s, 1H), 4.01 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.37 (d, J = 6.3 Hz, 2H), 3.27 (dd, J = 11.0, 4.3 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 1.13 (t, J = 7.1 Hz, 3H). Step 7: Ethyl 4-(6-methoxy-5-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)benzo[b]thiophen-2-yl)-4-oxobutanoate

S OO

OO

S OO

OO

BO O

34

To a mixture of 1,4-bis(diphenylphosphino)butane (0.45g, 1.1mmol), chloro(1,5-cyclooctadiene)iridium(i) dimer (0.35g, 0.53mmol), ethyl 4-(5-allyl-6-methoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (3.5g, 11mmol), and THF (20 mL) was added pinacolborane (1.0M in THF, 15.8mL, 15.8mmol). The reaction was stirred at RT for 4 hours. The solvent was then removed under reduced pressure, and the residue was purified by silica gel column chromatography (0-->20% EtOAc gradient in hexanes) to afford ethyl 4-(6-methoxy-5-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)benzo[b]thiophen-2-yl)-4-oxobutanoate (1.57g, 3.4 mmol, 65%). 1H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.66 (s, 1H), 7.53 (s, 1H), 4.02 (q, J = 7.0 Hz, 2H), 3.84 (s, 3H), 3.27 (t, J = 6.2 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 2.58 (t, J = 7.4 Hz, 2H), 1.58 (p, J = 7.4 Hz, 2H), 1.16-1.11 (m, 15H), 0.67 (t, J = 7.6 Hz, 2H). Step 8: 4,4'-(5,5'-(propane-1,3-diyl)bis(6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S OO

OO

BO O

S O

Br

O

OO

S

O

HOO

O S

O

OHO

O

To a mixture of ethyl 4-(6-methoxy-5-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)propyl)benzo[b]thiophen-2-yl)-4-oxobutanoate (69.1 mg, 0.15 mmol), ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (84 mg, 0.225 mmol), PdCl2(dppf)-CH2Cl2 (24.50 mg, 0.030 mmol) and cesium carbonate (195 mg, 0.600 mmol) was added dioxane (0.8 mL) and water (0.2 mL). The reaction was heated at 100 oC overnight. The reaction mixture was then filtered, washed with dioxane and the solvent was removed under reduced pressure. The residue was then purified via prep-HPLC (ACN/H2O with 0.1% NH4OH) to afford 4,4'-(5,5'-(propane-1,3-diyl)bis(6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid) (5.2 mg 0.009 mmol, 5.75%). LCMS (C29H28O8S2) (ES, m/z): 566 [M-H]-. 1H NMR (600 MHz, DMSO-d6) δ 8.11 (s, 2H), 7.68 (s, 2H), 7.50 (s, 2H), 3.82 (s, 6H), 3.16 – 3.09 (m, 4H), 2.66 (t, J = 6.6 Hz, 4H), 2.40 (s, 4H), 1.93 – 1.83 (m, 2H). HRMS (ES) calculated for C28H25FO10S2: 569.1304 [M+H]+, found 569.1287. Compound 4: 4,4'-((propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S

OOH

O

O

O

OS

OHO

O

Step 1: 5-Bromo-2-fluoro-4-methoxybenzaldehyde

O F

H

OBr

O F

H

O

35

2-Fluoro-4-methoxybenzaldehyde (9.0g, 58mmol) was added slowly (portion-wise) to a solution of Br2 (6.0mL, 120mmol) in MeOH (40mL) at 0°C. The reaction mixture was stirred at 0°C for 2h. A solution of NaHSO3 (24.3g, 234mmol) in H2O (300mL) was added slowly to the reaction mixture at 0°C. The resulting suspension was then stirred for 30 min at 0°C. The reaction mixture was filtered, and the filtrate was washed with additional H2O (3x25mL). The filtrate was then dried under reduced pressure to afford 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 58.4 mmol, 78%). The product was used without purification. 1H NMR (500MHz, DMSO-d6): δ 10.02 (s, 1H), 7.98 (d, J = 7.5Hz, 1H), 7.26 (d, J = 13.0Hz, 1H), 3.97 (s, 3H). Step 2: tert-Butyl 5-bromo-6-mehoxybenzo[b]thiophene-2-carboxylate

O F

H

OBr

O

Br

S O

O

K2CO3 (19.0g, 137mmol) was added slowly (portion-wise) to a solution of 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7g, 45.8mmol) and tert-butyl 2-mercaptoacetate (6.65mL, 45.8mmol) in DMF (50mL) at 20°C under Ar atmosphere. The reaction mixture was stirred and heated to 100°C for 16h. The reaction mixture was then cooled to RT and diluted with Et2O (1000mL). The mixture was then washed with H2O (500mL, then 2x250mL), and the combined aq layers were extracted with Et2O (2x200mL). The organic layers were then combined and washed with brine (50mL). The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure to afford tert-butyl 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylate (15.45 g, 45.0 mmol, 98%). The product was used without purification. 1H NMR (500MHz, DMSO-d6): δ 8.26 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 3.92 (s, 3H), 1.55 (s, 9H). Step 3: 5-Bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid

O

Br

S O

O

O

Br

S O

OH HCl (56mL, 4.0M in 1,4-dioxane, 230mmol) was added to a solution of tert-butyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (15.5g, 45.0mmol) in DCM (200mL) at 20°C. The reaction mixture was stirred at 20°C for 3days. The reaction mixture was then diluted by the dropwise addition of Hex (500mL). The resulting suspension was stirred for an additional 2h post-addition at RT. The reaction mixture was filtered, and the collected material was washed with Hex (2x50mL) and dried under reduced pressure to afford 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylic acid (12.5 g, 43.4 mmol, 96%), which was used without purification. 1H NMR (500MHz, DMSO-d6): δ 13.42 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.80 (s, 1H), 3.93 (s, 3H). Step 4: 5-Bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride

O

Br

S O

OH

O

Br

S O

Cl DMF (0.049mL, 0.63mmol) was added slowly (dropwise) to a solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (6.0g, 21mmol) and (COCl)2 (5.5mL, 63mmol) in THF (100mL) at 0°C under Ar atmosphere. The reaction mixture was stirred at 0°C for 2h and then allowed to warm to RT. The reaction mixture was stirred for 18h at RT. The reaction mixture was then concentrated under reduced pressure to afford 5-bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride (6.39 g, 20.9 mmol, 100%). The product was used without purification. Step 5: Ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

36

O

Br

S O

Cl

O

Br

S O

OO

A solution of (3-ethoxy-3-oxopropyl)zinc(II) bromide (13.8mL, 0.50M in THF, 6.9mmol) was added to an oven-dried flask containing ((thiophene-2-carbonyl)oxy)copper (1.31g, 6.87mmol) under Ar at 0°C. The reaction mixture was stirred for 20 min at 0°C under Ar. An Ar-degassed solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride (1.52g, 4.98mmol) in THF (25.0mL) was then added via cannula to the reaction mixture at 0°C; the resulting suspension was allowed to warm to RT and was stirred for an additional 3h. The reaction mixture was cooled to 0°C and quenched with sat aq NH4Cl (50mL). The mixture was allowed to warm to RT and stirred for an additional 10min. The mixture was filtered, and the filtrate was diluted with EtOAc (500mL) and brine (50mL). The organic layer was separated, washed with additional brine (25mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc in DCM) to afford ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (6.62 g, 16.6 mmol, 84%). LCMS (C15H16BrO4S) (ES, m/z): 371, 373 [M+H]+. 1H NMR (500MHz, DMSO-d6): δ 8.27 (s, 1H), 8.26 (s, 1H), 7.81 (s, 1H), 4.07-4.02 (m, 2H), 3.94 (s, 3H), 3.35-3.25 (m, 2H), 2.68-2.64 (m, 2H), 1.20-1.14 (m, 3H). Step 6: Ethyl 4-(6-methoxy-5-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)benzo[b]thiophen-2-yl)-4-oxobutanoate

S O

Br

O

OO

S OO

OO

OO

To a mixture of ethyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (13g, 35mmol), and CPhos Pd G4 (1.4g, 1.7mmol) was added (3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)zinc(II) bromide (0.50M in THF, 100 mL, 50mmol) at once. The reaction was heated to 40°C for 2 hours. The mixture was then allowed to cool to RT and filtered through CELITE. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (0-->30% EtOAc gradient in hexanes) to afford ethyl 4-(6-methoxy-5-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)benzo[b]thiophen-2-yl)-4-oxobutanoate (11.4g, 26.3 mmol, 75% ). LCMS (C23H31O6S) (ES, m/z): 435 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.71 (s, 1H), 7.55 (s, 1H), 4.50 (s, 1H), 4.02 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 3.70 (t, J = 8.1 Hz, 1H), 3.65-3.58 (m, 1H), 3.40-3.34 (m, 1H), 3.33-3.29 (m, 3H), 2.73-2.59 (m, 4H), 1.79 (p, J = 6.7 Hz, 2H), 1.69 (d, J = 8.7 Hz, 1H), 1.58 (t, J = 7.9 Hz, 1H), 1.48-1.34 (m, 4H), 1.14 (t, J = 7.1 Hz, 3H). Step 7: Ethyl 4-(5-(3-bromopropyl)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S OO

OO

OO

S OO

OO

Br

To a mixture of 4-(6-methoxy-5-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)benzo [b]thiophen-2-yl)-4-oxobutanoate (6.2g, 14mmol) and DCM (100 mL) at 0°C was added triphenylphosphine dibromide (9.03 g, 21.4mmol) portion-wise. The mixture was allowed to warm to RT and then stirred for 1 hour. The mixture was then quenched with water and diluted

37

with DCM. The organic layer was separated, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (0-->30% EtOAc gradient in hexanes) to afford ethyl 4-(5-(3-bromopropyl)-6-methoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (5.8g, 13.9 mmol, 98%). LCMS (C18H22BrO4S) (ES, m/z): 413, 415 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.23 (s, 1H), 7.73 (s, 1H), 7.58 (s, 1H), 4.02 (q, J = 7.0 Hz, 2H), 3.86 (s, 3H), 3.50 (t, J = 6.5 Hz, 2H), 3.27 (d, J = 6.4 Hz, 2H), 2.75 (t, J = 7.3 Hz, 2H), 2.63 (t, J = 6.2 Hz, 2H), 2.07 (p, J = 6.7 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H). Step 8: 4-(5-(3-((2-(3-carboxypropanoyl)-6-methoxybenzo[b]thiophen-5-yl)oxy)propyl)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S O

HO

O

OO

S O

O

O

OOH

S

O

HOO

O

S OO

OO

Br

To a mixture of ethyl 4-(5-(3-bromopropyl)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (83 mg, 0.2 mmol), tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (78 mg, 0.240 mmol), and K2CO3 (27.6 mg, 0.200 mmol) was added acetonitrile (1 mL). The reaction was heated at 65 oC overnight. The reaction mixture was then diluted with 4mL of ACN and filtered. The solvent was then removed under reduced pressure and the residue diluted with DCM (1 mL) and TFA (1 mL) and stirred at room temperature for 2 hours. The solvent was again then removed under reduced pressure. The resulting residue was again diluted with THF (2 mL), MeOH (0.5 mL), Water (1.000 mL) and LiOH (47.9 mg, 2.000 mmol) was then added. The reaction was stirred at rt for 2 hours. The reaction mixture was quenched with 0.400 mL of AcOH and the solvent was removed under reduced pressure. The resulting residue was then purified via prep-HPLC (ACN/H2O with 0.1% TFA) to afford of 4-(5-(3-((2-(3-carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)propyl)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (12.7 mg, 0.021 mmol, 20.5%). LCMS (C29H28O9S2) (ES, m/z): 585 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.24 (s, 1H), 8.16 (s, 1H), 7.79 (s, 1H), 7.62 (d, J = 6.3 Hz, 2H), 7.46 (s, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.95-388 (m, 6H), 3.3-3.24 (m, 4H), 2.86 (t, J = 7.4 Hz, 2H), 2.63-2.57 (m, 4H), 2.15-2.07 m, 2H). HRMS (ES) calculated for C29H28O9S2: 585.1253 [M+H]+, found 585.1256. Compound 5: 4,4'-((ethane-1,2-diylbis(oxy))bis(6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S

OO

O

OOH

O

OSO

HOO

Step 1: 5-Bromo-2-fluoro-4-methoxybenzaldehyde

O F

H

OBr

O F

H

O

38

2-Fluoro-4-methoxybenzaldehyde (9.0 g, 58 mmol) was added slowly (portion-wise) to a solution of bromine (6.0 mL, 120 mmol) in methanol (40mL) at 0°C. The reaction mixture was stirred at 0°C for 2 hours. A solution of sodium bisulfite (24.3 g, 234 mmol) in water (300 mL) was added slowly to the reaction mixture at 0°C. The resulting suspension was then stirred for 30 minutes at 0°C. The reaction mixture was filtered, and the filtrate was washed with additional water (3 x 25 mL). The filtrate was then dried under reduced pressure to afford 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 58.4 mmol, 78%). The product was used without purification in the subsequent step. 1H NMR (500MHz, DMSO-d6): δ 10.02 (s, 1H), 7.98 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 13.0 Hz, 1H), 3.97 (s, 3H). Step 2: tert-Butyl 5-bromo-6-mehoxybenzo[b]thiophene-2-carboxylate

O F

H

OBr

O

Br

S O

O

Potassium carbonate (19.0 g, 137 mmol) was added slowly (portion-wise) to a solution of 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 45.8 mmol) and tert-butyl 2-mercaptoacetate (6.65 mL, 45.8 mmol) in DMF (50 mL) at 20°C under argon. The reaction mixture was stirred and heated to 100°C for 16 hours. The reaction mixture was then cooled to room temperature and diluted with diethyl ether (1000 mL). The mixture was then washed with water (500 mL, then 2 x 250 mL), and the combined aqueous layers were extracted with diethyl ether (2 x 200 mL). The organic layers were then combined and washed with saturated aqueous sodium chloride (50 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford tert-butyl 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylate (15.45 g, 45.0 mmol, 98%). The product was used without purification in the subsequent step. 1H NMR (500MHz, DMSO-d6): δ 8.26 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 3.92 (s, 3H), 1.55 (s, 9H). Step 3: 5-Bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid

O

Br

S O

O

O

Br

S O

OH HCl (4.0M in 1,4-dioxane, 56 mL, 230 mmol) was added to a solution of tert-butyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (15.5 g, 45.0 mmol) in dichloromethane (200 mL) at 20°C. The reaction mixture was stirred at 20°C for 3 days. The reaction mixture was then diluted by the dropwise addition of hexanes (500mL). The resulting suspension was stirred for an additional 2 hours post-addition at room temperature. The reaction mixture was filtered, and the collected material was washed with hexanes (2 x 50 mL) and dried under reduced pressure to afford 5-bromo-6-methoxybenzo[b] thiophene-2-carboxylic acid (12.5 g, 43.4 mmol, 96%), which was used without purification. 1H NMR (500MHz, DMSO-d6): δ 13.42 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.80 (s, 1H), 3.93 (s, 3H). Step 4: 5-Bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride

O

Br

S O

OH

O

Br

S O

Cl DMF (0.049 mL, 0.63 mmol) was added slowly (dropwise) to a solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (6.0 g, 21 mmol) and oxalyl chloride (5.5 mL, 63 mmol) in THF (100 mL) at 0°C under argon. The reaction mixture was stirred at 0°C for 2 hours and then warmed to room temperature. The reaction mixture was stirred for 18 hours at room temperature. The reaction mixture was then concentrated under reduced pressure to afford

39

5-bromo-6-methoxybenzo[b] thiophene-2-carbonyl chloride (6.39 g, 20.9 mmol, 100%). The product was used without purification in the subsequent step. Step 5: tert-Butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

ClBr

O

O

O

ZnBr

S O

Br

O

OO

CPhos Pd G4

A mixture of CPhos Pd G4 (0.161 g, 0.196 mmol) and 5-bromo-6-methoxybenzo[b]

thiophene-2-carbonyl chloride (6.00 g, 19.6 mmol) was degassed with argon and then diluted with THF (50 mL). The reaction mixture was cooled to 0℃ and then a solution of (3-(tert-butoxy)-3-oxopropyl)zinc(II) bromide (0.50M in THF, 50 ml, 25 mmol) was added dropwise over 30 minutes. The reaction mixture was removed from the ice bath and stirred for 2 hours at room temperature. The reaction mixture was quenched at 0℃ with saturated aqueous ammonium chloride and then diluted with dichloromethane. The reaction mixture was filtered through Celite and the organic layer was separated. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / hexanes) to afford tert-butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (6.62 g, 16.6 mmol, 84%). LCMS (C17H20BrO4S – C4H9) (ES, m/z): 343, 345 [M+H-tBu]+. 1H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 1H), 8.20 (s, 1H), 7.75 (s, 1H), 3.90 (s, 3H), 3.21 (t, J = 6.2 Hz, 2H), 2.54 (t, J = 6.2 Hz, 2H), 1.32 (s, 9H). Step 6: tert-Butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

Br

O

OO

S O

HO

O

OO

NOH

RockPhos Pd G3

To a mixture of RockPhos Pd G3 (0.105 g, 0.125 mmol), benzaldoxime (3.03 g, 25.0 mmol), tert-butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (5.0 g, 13 mmol), and cesium carbonate (12.2 g, 37.6 mmol) was added DMF (40 mL). The reaction mixture was heated to 80°C for 18 hours. The reaction mixture was cooled to room temperature and then poured into a flask containing aqueous hydrochloric acid (0.5M in water, 100 mL, 50 mmol). The resulting mixture was extracted with dichloromethane. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexanes) to afford tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.1 g, 6.2 mmol, 50%). LCMS (C17H20O5SNa) (ES, m/z): 359 [M+Na]+. 1H NMR (600 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.12 (s, 1H), 7.49 (s, 1H), 7.27 (s, 1H), 3.83 (s, 3H), 3.18 (t, J = 6.2 Hz, 2H), 2.52 (t, J = 6.2 Hz, 2H), 1.33 (s, 9H). Step 7: Tert-butyl 4-(5-(2-bromoethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SO

HO

O

OO

Cs2CO3

BrBr SO

O

O

OO

Br

To a mixture of tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.065 g, 0.2 mmol), Cs2CO3 (0.326 g, 1.000 mmol), and Acetonitrile (2 mL) was added 1,2-dibromoethane (1 mL, 9.85 mmol) and the reaction was heated at 65 oC for 2 hours. The mixture was then filtered, washed with THF and diluted with hexanes. The solvent was

40

removed under reduced pressure to yield tert-butyl 4-(5-(2-bromoethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate. The compound was used crude without further purification. LCMS (C22H28O6S) (ES, m/z): 433, 445 [M+H]+. Step 8: 4-(5-(2-((2-(3-carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S

O

O

O

OO

Br

S

O

O

HO

OO

Cs2CO3 S

OO

O

OOH

O

OSO

HOO

To a mixture of tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.081 g, 0.24 mmol), crude tert-butyl 4-(5-(2-bromoethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.089 g, 0.24 mmol) and Cs2CO3 (0.091 g, 0.279 mmol) was added acetonitrile (1 mL). The reaction mixture was then heated at 85 oC for 1 hour. The reaction was then diluted with THF, filtered, and the solvent was removed under reduced pressure. The resulting residue was then dissolved in DCM (1 mL) and TFA (1 mL) and stirred at room temperature for 2 hours and the solvent was removed under reduced pressure. The residue was then purified via prep-HPLC (ACN/H2O with 0.1% TFA) to afford 4,4'-((ethane-1,2-diylbis(oxy))bis(6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid) (79.6mg, 0.136 mmol, 69%). LCMS (C28H26O10S2) (ES, m/z): 587 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 2H), 7.63 (d, J = 17.1 Hz, 4H), 4.44 (s, 4H), 3.88 (s, 6H), 3.30 – 3.26 (m, 4H), 2.61 (t, J = 6.3 Hz, 4H). HRMS (ES) calculated for C28H26O10S2: 587.1045 [M+H]+, found 587.1041 Compound 6: 4-(5-(2-((2-(3-carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S

OO

O

OOH

O

OSO

HOO

F

Step 1: 5-Bromo-2-fluoro-4-methoxybenzaldehyde O F

H

OBr

O F

H

O 2-Fluoro-4-methoxybenzaldehyde (9.0 g, 58 mmol) was added slowly (portion-wise) to a solution of bromine (6.0 mL, 120 mmol) in methanol (40mL) at 0°C. The reaction mixture was stirred at 0°C for 2 hours. A solution of sodium bisulfite (24.3 g, 234 mmol) in water (300 mL) was added slowly to the reaction mixture at 0°C. The resulting suspension was then stirred for 30 minutes at 0°C. The reaction mixture was filtered, and the filtrate was washed with additional water (3 x 25 mL). The filtrate was then dried under reduced pressure to afford 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 58.4 mmol, 78%). The product was used without purification in the subsequent step. 1H NMR (500MHz, DMSO-d6): δ 10.02 (s, 1H), 7.98 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 13.0 Hz, 1H), 3.97 (s, 3H). Step 2: tert-Butyl 5-bromo-6-mehoxybenzo[b]thiophene-2-carboxylate

O F

H

OBr

O

Br

S O

O

41

Potassium carbonate (19.0 g, 137 mmol) was added slowly (portion-wise) to a solution of 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 45.8 mmol) and tert-butyl 2-mercaptoacetate (6.65 mL, 45.8 mmol) in DMF (50 mL) at 20°C under argon. The reaction mixture was stirred and heated to 100°C for 16 hours. The reaction mixture was then cooled to room temperature and diluted with diethyl ether (1000 mL). The mixture was then washed with water (500 mL, then 2 x 250 mL), and the combined aqueous layers were extracted with diethyl ether (2 x 200 mL). The organic layers were then combined and washed with saturated aqueous sodium chloride (50 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford tert-butyl 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylate (15.45 g, 45.0 mmol, 98%). The product was used without purification in the subsequent step. 1H NMR (500MHz, DMSO-d6): δ 8.26 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 3.92 (s, 3H), 1.55 (s, 9H). Step 3: 5-Bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid

O

Br

S O

O

O

Br

S O

OH HCl (4.0M in 1,4-dioxane, 56 mL, 230 mmol) was added to a solution of tert-butyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (15.5 g, 45.0 mmol) in dichloromethane (200 mL) at 20°C. The reaction mixture was stirred at 20°C for 3 days. The reaction mixture was then diluted by the dropwise addition of hexanes (500mL). The resulting suspension was stirred for an additional 2 hours post-addition at room temperature. The reaction mixture was filtered, and the collected material was washed with hexanes (2 x 50 mL) and dried under reduced pressure to afford 5-bromo-6-methoxybenzo[b] thiophene-2-carboxylic acid (12.5 g, 43.4 mmol, 96%), which was used without purification. 1H NMR (500MHz, DMSO-d6): δ 13.42 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.80 (s, 1H), 3.93 (s, 3H). Step 4: 5-Bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride

O

Br

S O

OH

O

Br

S O

Cl DMF (0.049 mL, 0.63 mmol) was added slowly (dropwise) to a solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (6.0 g, 21 mmol) and oxalyl chloride (5.5 mL, 63 mmol) in THF (100 mL) at 0°C under argon. The reaction mixture was stirred at 0°C for 2 hours and then warmed to room temperature. The reaction mixture was stirred for 18 hours at room temperature. The reaction mixture was then concentrated under reduced pressure to afford 5-bromo-6-methoxybenzo[b] thiophene-2-carbonyl chloride(6.39 g, 20.9 mmol, 100%). The product was used without purification in the subsequent step. Step 5: tert-Butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

ClBr

O

O

O

ZnBr

S O

Br

O

OO

CPhos Pd G4

A mixture of CPhos Pd G4 (0.161 g, 0.196 mmol) and 5-bromo-6-methoxybenzo[b]

thiophene-2-carbonyl chloride (6.00 g, 19.6 mmol) was degassed with argon and then diluted with THF (50 mL). The reaction mixture was cooled to 0℃ and then a solution of (3-(tert-butoxy)-3-oxopropyl)zinc(II) bromide (0.50M in THF, 50 ml, 25 mmol) was added dropwise over 30 minutes. The reaction mixture was removed from the ice bath and stirred for 2 hours at room temperature. The reaction mixture was quenched at 0℃ with saturated aqueous ammonium

42

chloride and then diluted with dichloromethane. The reaction mixture was filtered through Celite and the organic layer was separated. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / hexanes) to afford tert-butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (6.62 g, 16.6 mmol, 84%). LCMS (C17H20BrO4S – C4H9) (ES, m/z): 343, 345 [M+H-tBu]+. 1H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 1H), 8.20 (s, 1H), 7.75 (s, 1H), 3.90 (s, 3H), 3.21 (t, J = 6.2 Hz, 2H), 2.54 (t, J = 6.2 Hz, 2H), 1.32 (s, 9H). Step 6: tert-Butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

Br

O

OO

S O

HO

O

OO

NOH

RockPhos Pd G3

To a mixture of RockPhos Pd G3 (0.105 g, 0.125 mmol), benzaldoxime (3.03 g, 25.0 mmol), tert-butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (5.0 g, 13 mmol), and cesium carbonate (12.2 g, 37.6 mmol) was added DMF (40 mL). The reaction mixture was heated to 80°C for 18 hours. The reaction mixture was cooled to room temperature and then poured into a flask containing aqueous hydrochloric acid (0.5M in water, 100 mL, 50 mmol). The resulting mixture was extracted with dichloromethane. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexanes) to afford tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.1 g, 6.2 mmol, 50%). LCMS (C17H20O5SNa) (ES, m/z): 359 [M+Na]+. 1H NMR (600 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.12 (s, 1H), 7.49 (s, 1H), 7.27 (s, 1H), 3.83 (s, 3H), 3.18 (t, J = 6.2 Hz, 2H), 2.52 (t, J = 6.2 Hz, 2H), 1.33 (s, 9H). Step 7: Tert-butyl 4-(5-(2-bromoethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SO

HO

O

OO

Cs2CO3

BrBr SO

O

O

OO

Br

To a mixture of tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.065 g, 0.2 mmol), Cs2CO3 (0.326 g, 1.000 mmol), and Acetonitrile (2 mL) was added 1,2-dibromoethane (1 mL, 9.85 mmol) and the reaction was heated at 65 oC for 2 hours. The mixture was then filtered, washed with THF and diluted with hexanes. The solvent was removed under reduced pressure to yield tert-butyl 4-(5-(2-bromoethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate. The compound was used without further purification. LCMS (C19H23BrO5S) (ES, m/z): 443, 445 [M+H]+. Step 8: 4-(5-(2-((2-(3-carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S

O

O

O

OO

Br

S

O

O

HOF

OO

Cs2CO3 S

OO

O

OOH

O

OSO

HOO

F

To a mixture of ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.078 g, 0.24 mmol), tert-butyl 4-(5-(2-bromoethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.089 g, 0.24 mmol) and K2CO3 (0.060 g, 0.24 mmol) was added acetonitrile (1 mL). The reaction mixture was then heated at 65 oC for 1 hour.

43

The reaction was then diluted with THF, filtered, and the solvent was removed under reduced pressure. The resulting residue was then dissolved in DCM (1 mL) and TFA (1 mL) and stirred at room temperature for 2 hours and the solvent was removed under reduced pressure. THF (1 mL), methanol (0.2 mL), water (0.5 mL), and LiOH (0.013 g, 0.559 mmol) was added to the residue and the reaction was again stirred for 2 hours. The reaction mixture was quenched with AcOH and the solvent was again removed under reduced pressure. The residue was then purified via prep-HPLC (ACN/H2O with 0.1% TFA) to afford 4-(5-(2-((2-(3-carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)ethoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (17.3 mg, 0.029 mmol, 14.3%). LCMS (C28H25FO10S2) (ES, m/z): 605 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.18 (s, 1H), 7.58 (m, 3H), 7.52 (s, 1H), 4.48 – 4.44 (m, 2H), 4.35 – 4.30 (m, 2H), 3.89 (s, 3H), 3.76 (s, 3H), 3.34-3.32 (m, 4H), 2.59 (q, J = 6.1 Hz, 4H). HRMS (ES) calculated for C28H25FO10S2: 605.0951 [M+H]+, found 605.0946. Compound 7: 4,4'-((propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S

OO

O

OOH

O

OSO

HOO

F

F

Step 1: Methyl 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylate S

O

O O

O

S

O

O O

OF

SelectFluor

1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (SELECTFLUOR™, 77 mg, 0.22 mmol) was added to a mixture of methyl 5,6-dimethoxybenzo[b] thiophene-2-carboxylate (50 mg, 0.20 mmol) in acetonitrile (1 mL) at room temperature. The resulting mixture was stirred at 45°C for 15 hours. The mixture was cooled to room temperature, diluted with saturated aqueous sodium bicarbonate (10 mL), and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, ethyl acetate / petroleum ether) to afford methyl 4-fluoro-5,6-dimethoxybenzo[b] thiophene-2-carboxylate (11 mg, 0.041 mmol, 21%). LCMS (C12H11FO4SNa) (ES, m/z): 293 [M+Na]+. 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.08 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H). Step 2: 4-Fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid

S

O

O O

O

S

O

O O

OH

F F

LiOH

LiOH·water (71 mg, 1.7 mmol) was added portion-wise to a mixture of methyl 4-fluoro-5, 6-dimethoxybenzo[b]thiophene-2-carboxylate (46 mg, 0.17 mmol) in THF (3 mL), methanol (1 mL), and water (1 mL) at room temperature. The reaction mixture was stirred for 15 hours. The mixture was adjusted to pH=5 with 1N HCl and extracted with ethyl acetate (3 x 10mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by

44

prep-HPLC (acetonitrile / water with 0.1% TFA) to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (23 mg, 0.09 mmol, 53%). LCMS (C11H10FO4S) (ES, m/z): 257 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 8.12 (s, 1H), 7.09 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H). Step 3: 4-Fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride

S OO

O OH

S OO

O Cl

F F

Cl

OCl

O

To a stirred solution of 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (150 mg, 0.60 mmol) in anhydrous THF (5 mL) was added oxalyl chloride (0.21 mL, 2.4 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 1 hour and then at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride, which was used in the subsequent step without purification. Step 4: Ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

OO

O

O

S OO

O ClF F

ZnBr

O

O

A suspension of copper(I) thiophene-2-carboxylate (125 mg, 0.65 mmol) was sparged with nitrogen for 5 minutes and then cooled to 0°C. A solution of (3-ethoxy-3-oxopropyl)zinc(II) bromide (0.5M in THF, 17.7mL, 9 mmol) was added under nitrogen at 0°C, and the reaction mixture was stirred for 20 minutes at 0°C. A nitrogen-sparged solution of 4-fluoro-5,6-dimethoxybenzo [b]thiophene-2-carbonyl chloride (130 mg, 0.47 mmol) in THF (3 mL) was then added at 0°C. The resulting suspension was warmed to room temperature and stirred for 8 hours. The reaction mixture was then poured into saturated aqueous ammonium chloride (20 mL) and stirred. The mixture was extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with water and brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / hexanes) to afford ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (3.85 g, 11.3 mmol, 69%). LCMS (C16H18FO5S) (ES, m/z): 341 [M+H]+. 1H NMR (500 MHz, CDCl3) δ 8.02 (d, J = 0.7 Hz, 1H), 7.10 (t, J = 1.0 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 4.05-3.97 (m, 6H), 3.36 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H), 1.29 (t, J= 7.2 Hz, 3H). Step 5: Ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S OO

OF O

O

S OO

HOF O

O

AlCl3

To a mixture of ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (3.6 g, 11 mmol) in dichloromethane (50 mL) was added aluminum chloride (5.64 g, 42.3 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with water (50 mL, added slowly via addition funnel) followed by HCl (1N,

45

50mL, added slowly via addition funnel). The reaction mixture was then diluted with 20% isopropanol in dichloromethane. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / dichloromethane) to afford ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.15 g, 6.59 mmol, 62%). LCMS (C15H16FO5S) (ES, m/z): 327 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.25 (s, 1H), 7.47 (s, 1H), 4.06 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 3.39-3.34 (m, 2H), 2.68-2.63 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). Step 6: Ethyl 4-(5-(2-bromoethoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SO

HOF

O

OO

Cs2CO3

BrBr SO

OF

O

OO

Br

To a mixture of ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate(0.065 g, 0.2 mmol), Cs2CO3 (0.326 g, 1.000 mmol), and Acetonitrile (2 mL) was added 1,2-dibromoethane (1 mL, 9.85 mmol) and the reaction was heated at 65 oC for 2 hours. The mixture was then filtered, washed with THF and diluted with hexanes. The solvent was removed under reduced pressure to yield ethyl 4-(5-(2-bromoethoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate. The crude compound was used without further purification. LCMS (C17H18BrFO5S) (ES, m/z): 433, 435 [M+H]+. Step 7: 4,4'-((ethane-1,2-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S

O

O

OF

OO

Br

S

O

O

HOF

OO

Cs2CO3 S

OO

O

OOH

O

OSO

HOO

F

F To a mixture of ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.024 g, 0.073 mmol), ethyl 4-(5-(2-bromoethoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.025 g, 0.056 mmol) and Cs2CO3 (0.091 g, 0.279 mmol) was added acetonitrile (1 mL). The reaction mixture was then heated at 65 oC for 1 hour. The reaction was then diluted with THF, filtered, and the solvent was removed under reduced pressure. The resulting residue was then dissolved in THF (1 mL), methanol (0.2 mL), water (0.5 mL), and LiOH (0.013 g, 0.559 mmol) was added and the reaction stirred for 2 hours. The reaction mixture was quenched with AcOH and the solvent was again removed under reduced pressure. The residue was then purified via prep-HPLC (ACN/H2O with 0.1% TFA) to afford 4,4'-((ethane-1,2-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid) (6 mg, 0.009 mmol, 9.6%). LCMS (C28H22F2O10S2) (ES, m/z): 623 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.29 (s, 2H), 7.56 (s, 2H), 4.38 (s, 4H), 3.9 (s, 6H), 3.34-3.32 (m, 4H), 2.60 (t, J = 6.3 Hz, 4H). HRMS (ES) calculated for C28H22F2O10S2: 623.0857 [M+H]+, found 623.0858. Compound 8: 4,4'-((propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S O

O

O

OOH

O

OSO

HOO

F F

46

Step 1: Methyl 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylate S

O

O O

O

S

O

O O

OF

SelectFluor

1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (SELECTFLUOR™, 77 mg, 0.22 mmol) was added to a mixture of methyl 5,6-dimethoxybenzo[b] thiophene-2-carboxylate (50 mg, 0.20 mmol) in acetonitrile (1 mL) at room temperature. The resulting mixture was stirred at 45°C for 15 hours. The mixture was cooled to room temperature, diluted with saturated aqueous sodium bicarbonate (10 mL), and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, ethyl acetate / petroleum ether) to afford methyl 4-fluoro-5,6-dimethoxybenzo[b] thiophene-2-carboxylate (11 mg, 0.041 mmol, 21%). LCMS (C12H11FO4SNa) (ES, m/z): 293 [M+Na]+. 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.08 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H). Step 2: 4-Fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid

S

O

O O

O

S

O

O O

OH

F F

LiOH

LiOH·water (71 mg, 1.7 mmol) was added portion-wise to a mixture of methyl 4-fluoro-5, 6-dimethoxybenzo[b]thiophene-2-carboxylate (46 mg, 0.17 mmol) in THF (3 mL), methanol (1 mL), and water (1 mL) at room temperature. The reaction mixture was stirred for 15 hours. The mixture was adjusted to pH=5 with 1N HCl and extracted with ethyl acetate (3 x 10mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (acetonitrile / water with 0.1% TFA) to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (23 mg, 0.09 mmol, 53%). LCMS (C11H10FO4S) (ES, m/z): 257 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 8.12 (s, 1H), 7.09 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H). Step 3: 4-Fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride

S OO

O OH

S OO

O Cl

F F

Cl

OCl

O

To a stirred solution of 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (150 mg, 0.60 mmol) in anhydrous THF (5 mL) was added oxalyl chloride (0.21 mL, 2.4 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 1 hour and then at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride, which was used in the subsequent step without purification. Step 4: Ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

47

S O

OO

O

O

S OO

O ClF F

ZnBr

O

O

A suspension of copper(I) thiophene-2-carboxylate (125 mg, 0.65 mmol) was sparged with nitrogen for 5 minutes and then cooled to 0°C. A solution of (3-ethoxy-3-oxopropyl)zinc(II) bromide (0.5M in THF, 17.7mL, 9 mmol) was added under nitrogen at 0°C, and the reaction mixture was stirred for 20 minutes at 0°C. A nitrogen-sparged solution of 4-fluoro-5,6-dimethoxybenzo [b]thiophene-2-carbonyl chloride (130 mg, 0.47 mmol) in THF (3 mL) was then added at 0°C. The resulting suspension was warmed to room temperature and stirred for 8 hours. The reaction mixture was then poured into saturated aqueous ammonium chloride (20 mL) and stirred. The mixture was extracted with ethyl acetate (2 x 20 mL). The combined organic layers were washed with water and brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / hexanes) to afford ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (3.85 g, 11.3 mmol, 69%). LCMS (C16H18FO5S) (ES, m/z): 341 [M+H]+. 1H NMR (500 MHz, CDCl3) δ 8.02 (d, J = 0.7 Hz, 1H), 7.10 (t, J = 1.0 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 4.05-3.97 (m, 6H), 3.36 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H), 1.29 (t, J= 7.2 Hz, 3H). Step 5: Ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S OO

OF O

O

S OO

HOF O

O

AlCl3

To a mixture of ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (3.6 g, 11 mmol) in dichloromethane (50 mL) was added aluminum chloride (5.64 g, 42.3 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with water (50 mL, added slowly via addition funnel) followed by HCl (1N, 50mL, added slowly via addition funnel). The reaction mixture was then diluted with 20% isopropanol in dichloromethane. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / dichloromethane) to afford ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.15 g, 6.59 mmol, 62%). LCMS (C15H16FO5S) (ES, m/z): 327 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.25 (s, 1H), 7.47 (s, 1H), 4.06 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 3.39-3.34 (m, 2H), 2.68-2.63 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). Step 6: Ethyl 4-(5-(3-bromopropoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SO

HOF

O

OO

Cs2CO3

Br Br SO

OF

O

OO

Br

To a mixture of ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate(0.065 g, 0.2 mmol), Cs2CO3 (0.326 g, 1.000 mmol), and Acetonitrile (2 mL) was added 1,3-dibromopropane (1 mL, 9.85 mmol) and the reaction was heated at 65 oC for 2 hours. The mixture was then filtered, washed with THF and diluted with hexanes. The solvent was removed under reduced pressure to yield ethyl 4-(5-(3-bromopropoxy)-4-fluoro-6-

48

methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate. The compound was used without further purification. LCMS (C23H30O6S) (ES, m/z): 447, 449 [M+H]+. Step 7: 4,4'-(5,5'-(propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid)

S

O

O

OF

OO

S

O

O

HOF

OO

Br S O

O

O

OOH

O

OSO

HOO

F F

Cs2CO3

To a mixture of ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.024 g, 0.073 mmol), ethyl 4-(5-(3-bromopropoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.025 g, 0.056 mmol) and Cs2CO3 (0.091 g, 0.279 mmol) was added acetonitrile (1 mL). The reaction mixture was then heated at 65 oC for 1 hour. The reaction was then diluted with THF, filtered, and the solvent was removed under reduced pressure. The resulting residue was then dissolved in THF (1 mL), methanol (0.2 mL), water (0.5 mL), and LiOH (0.013 g, 0.559 mmol) was added and the reaction stirred for 2 hours. The reaction mixture was quenched with AcOH and the solvent was again removed under reduced pressure. The residue was then purified via prep-HPLC (ACN/H2O with 0.1% TFA) to afford 21.4 mg (0.036 mmol, 65% yield) of ( 4,4'-(5,5'-(propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(4-oxobutanoic acid). LCMS (C29H26F2O10S2) (ES, m/z): 637 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.26 (s, 2H), 7.55 (s, 2H), 4.28 (q, J = 7.1, 6.6 Hz, 4H), 3.99 – 3.80 (m, 6H), 3.34-3.32 (m, 4H), 2.60 (q, J = 6.4 Hz, 4H), 2.09 (dt, J = 11.5, 5.8 Hz, 2H). HRMS (ES) calculated for C29H26F2O10S2: 637.1013 [M+H]+, found 637.1008. Compound 9: 4-(5-(3-((2-(3-carboxypropanoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

SO

O

O

OHO

OS

O

HOO

O

Step 1: 5-Bromo-2-fluoro-4-methoxybenzaldehyde

O F

H

OBr

O F

H

O 2-Fluoro-4-methoxybenzaldehyde (9.0 g, 58 mmol) was added slowly (portion-wise) to a solution of bromine (6.0 mL, 120 mmol) in methanol (40mL) at 0°C. The reaction mixture was stirred at 0°C for 2 hours. A solution of sodium bisulfite (24.3 g, 234 mmol) in water (300 mL) was added slowly to the reaction mixture at 0°C. The resulting suspension was then stirred for 30 minutes at 0°C. The reaction mixture was filtered, and the filtrate was washed with additional water (3 x 25 mL). The filtrate was then dried under reduced pressure to afford 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 58.4 mmol, 78%). The product was used without purification in the subsequent step. 1H NMR (500MHz, DMSO-d6): δ 10.02 (s, 1H), 7.98 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 13.0 Hz, 1H), 3.97 (s, 3H). Step 2: tert-Butyl 5-bromo-6-mehoxybenzo[b]thiophene-2-carboxylate

O F

H

OBr

O

Br

S O

O

49

Potassium carbonate (19.0 g, 137 mmol) was added slowly (portion-wise) to a solution of 5-bromo-2-fluoro-4-methoxybenzaldehyde (10.7 g, 45.8 mmol) and tert-butyl 2-mercaptoacetate (6.65 mL, 45.8 mmol) in DMF (50 mL) at 20°C under argon. The reaction mixture was stirred and heated to 100°C for 16 hours. The reaction mixture was then cooled to room temperature and diluted with diethyl ether (1000 mL). The mixture was then washed with water (500 mL, then 2 x 250 mL), and the combined aqueous layers were extracted with diethyl ether (2 x 200 mL). The organic layers were then combined and washed with saturated aqueous sodium chloride (50 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford tert-butyl 5-bromo-6-methoxybenzo [b]thiophene-2-carboxylate (15.45 g, 45.0 mmol, 98%). The product was used without purification in the subsequent step. 1H NMR (500MHz, DMSO-d6): δ 8.26 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 3.92 (s, 3H), 1.55 (s, 9H). Step 3: 5-Bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid

O

Br

S O

O

O

Br

S O

OH HCl (4.0M in 1,4-dioxane, 56 mL, 230 mmol) was added to a solution of tert-butyl 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (15.5 g, 45.0 mmol) in dichloromethane (200 mL) at 20°C. The reaction mixture was stirred at 20°C for 3 days. The reaction mixture was then diluted by the dropwise addition of hexanes (500mL). The resulting suspension was stirred for an additional 2 hours post-addition at room temperature. The reaction mixture was filtered, and the collected material was washed with hexanes (2 x 50 mL) and dried under reduced pressure to afford 5-bromo-6-methoxybenzo[b] thiophene-2-carboxylic acid (12.5 g, 43.4 mmol, 96%), which was used without purification. 1H NMR (500MHz, DMSO-d6): δ 13.42 (s, 1H), 8.26 (s, 1H), 7.98 (s, 1H), 7.80 (s, 1H), 3.93 (s, 3H). Step 4: 5-Bromo-6-methoxybenzo[b]thiophene-2-carbonyl chloride

O

Br

S O

OH

O

Br

S O

Cl DMF (0.049 mL, 0.63 mmol) was added slowly (dropwise) to a solution of 5-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (6.0 g, 21 mmol) and oxalyl chloride (5.5 mL, 63 mmol) in THF (100 mL) at 0°C under argon. The reaction mixture was stirred at 0°C for 2 hours and then warmed to room temperature. The reaction mixture was stirred for 18 hours at room temperature. The reaction mixture was then concentrated under reduced pressure to afford 5-bromo-6-methoxybenzo[b] thiophene-2-carbonyl chloride (6.39 g, 20.9 mmol, 100%). The product was used without purification in the subsequent step. Step 5: tert-Butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

ClBr

O

O

O

ZnBr

S O

Br

O

OO

CPhos Pd G4

A mixture of CPhos Pd G4 (0.161 g, 0.196 mmol) and 5-bromo-6-methoxybenzo[b]

thiophene-2-carbonyl chloride (6.00 g, 19.6 mmol) was degassed with argon and then diluted with THF (50 mL). The reaction mixture was cooled to 0℃ and then a solution of (3-(tert-butoxy)-3-oxopropyl)zinc(II) bromide (0.50M in THF, 50 ml, 25 mmol) was added dropwise over 30 minutes. The reaction mixture was removed from the ice bath and stirred for 2 hours at room temperature. The reaction mixture was quenched at 0℃ with saturated aqueous ammonium

50

chloride and then diluted with dichloromethane. The reaction mixture was filtered through Celite and the organic layer was separated. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / hexanes) to afford tert-butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (6.62 g, 16.6 mmol, 84%). LCMS (C17H20BrO4S – C4H9) (ES, m/z): 343, 345 [M+H-tBu]+. 1H NMR (600 MHz, DMSO-d6) δ 8.21 (s, 1H), 8.20 (s, 1H), 7.75 (s, 1H), 3.90 (s, 3H), 3.21 (t, J = 6.2 Hz, 2H), 2.54 (t, J = 6.2 Hz, 2H), 1.32 (s, 9H). Step 6: tert-Butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

Br

O

OO

S O

HO

O

OO

NOH

RockPhos Pd G3

To a mixture of RockPhos Pd G3 (0.105 g, 0.125 mmol), benzaldoxime (3.03 g, 25.0 mmol), tert-butyl 4-(5-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (5.0 g, 13 mmol), and cesium carbonate (12.2 g, 37.6 mmol) was added DMF (40 mL). The reaction mixture was heated to 80°C for 18 hours. The reaction mixture was cooled to room temperature and then poured into a flask containing aqueous hydrochloric acid (0.5M in water, 100 mL, 50 mmol). The resulting mixture was extracted with dichloromethane. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexanes) to afford tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.1 g, 6.2 mmol, 50%). LCMS (C17H20O5SNa) (ES, m/z): 359 [M+Na]+. 1H NMR (600 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.12 (s, 1H), 7.49 (s, 1H), 7.27 (s, 1H), 3.83 (s, 3H), 3.18 (t, J = 6.2 Hz, 2H), 2.52 (t, J = 6.2 Hz, 2H), 1.33 (s, 9H). Step 7: tert-Butyl 4-(6-methoxy-5-(3-((5-methoxy-2-(4-methoxy-4-oxobutanoyl)benzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

SO

O

O

OO

SO

HO

O

OO

S O

OO

OO

O S O

OO

O

Br

A mixture of methyl 4-(6-(3-bromopropoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-

oxobutanoate (35 mg, 0.084 mmol), tert-butyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (28 mg, 0.084 mmol), and potassium carbonate (47 mg, 0.34 mmol) was degassed with argon. DMF (1.0 mL) was added to the mixture, and the reaction mixture was stirred and heated at 40°C for 18 hours. The reaction mixture was cooled to room temperature and was used in the subsequent step without workup or purification. LCMS (C34H38O10S2Na) (ES, m/z): 693 [M+Na]+. Step 8: 4-(5-(3-((2-(3-Carboxypropanoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S O

OO

OO

O S O

OO

O

SO

O

O

OHO

OS

O

HOO

ONaOH

Sodium hydroxide (1.0M in water, 0.60 mL, 0.60 mmol) was added to a mixture of tert-butyl 4-(6-methoxy-5-(3-((5-methoxy-2-(4-methoxy-4-oxobutanoyl)benzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (57 mg, 0.085 mmol) in DMSO (2.0 mL).

51

The reaction mixture was then stirred at room temperature for 30 minutes. The reaction mixture was quenched with TFA (0.079 mL, 1.0 mmol) and filtered. The filtrate was purified by reverse phase HPLC (acetonitrile in water, with 0.1% TFA modifier) to afford 4-(5-(3-((2-(3-carboxypropanoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (3.4 mg, 5.7 µmol, 6.7% yield over two steps). LCMS (C29H29O10S2) (ES, m/z): 601 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 12.19 (s, 2H), 8.20 (s, 1H), 8.17 (s, 1H), 7.67 (s, 1H), 7.61 (s, 1H), 7.55 (s, 1H), 7.50 (s, 1H), 4.28 (t, J = 6.1 Hz, 2H), 4.22 (t, J = 6.2 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.29 – 3.24 (m, 4H), 2.63 – 2.58 (m, 4H), 2.30 (q, J = 6.1 Hz, 2H). HRMS (ES) calculated for C29H29O10S2: 601.1202 [M+H]+, found 601.1198.

Compound 10: 4-(6-(3-((2-(3-Carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

SO

O

O

OHO

OS

O

HOO

OF

Step 1: Methyl 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylate

S

O

O O

O

S

O

O O

OF

SelectFluor

1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (SELECTFLUOR™, 77 mg, 0.22 mmol) was added to a mixture of methyl 5,6-dimethoxybenzo[b] thiophene-2-carboxylate (50 mg, 0.20 mmol) in acetonitrile (1 mL) at room temperature. The resulting mixture was stirred at 45°C for 15 hours. The mixture was cooled to room temperature, diluted with saturated aqueous sodium bicarbonate (10 mL), and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, ethyl acetate / petroleum ether) to afford methyl 4-fluoro-5,6-dimethoxybenzo[b] thiophene-2-carboxylate (11 mg, 0.041 mmol, 21%). LCMS (C12H11FO4SNa) (ES, m/z): 293 [M+Na]+. 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.08 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H). Step 2: 4-Fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid

S

O

O O

O

S

O

O O

OH

F F

LiOH

LiOH·water (71 mg, 1.7 mmol) was added portion-wise to a mixture of methyl 4-fluoro-5, 6-dimethoxybenzo[b]thiophene-2-carboxylate (46 mg, 0.17 mmol) in THF (3 mL), methanol (1 mL), and water (1 mL) at room temperature. The reaction mixture was stirred for 15 hours. The mixture was adjusted to pH=5 with 1N HCl and extracted with ethyl acetate (3 x 10mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (acetonitrile / water with 0.1% TFA) to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (23 mg, 0.09 mmol, 53%). LCMS (C11H10FO4S)

52

(ES, m/z): 257 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 8.12 (s, 1H), 7.09 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H). Step 3: 4-Fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride

S OO

O OH

S OO

O Cl

F F

Cl

OCl

O

To a stirred solution of 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (150 mg, 0.60 mmol) and DMF (4.6 µL, 0.060 mmol) in anhydrous THF (5 mL) was added oxalyl chloride (0.21 mL, 2.4 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 1 hour and then at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride, which was used in the subsequent step without purification. Step 4: Ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S O

OO

O

O

S OO

O ClF F

ZnBr

O

O

THF was added to a mixture of 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride (4.50 g, 16.4 mmol) and CPhos Pd G4 (0.269 g, 0.328 mmol) under an argon atmosphere. A solution of (3-ethoxy-3-oxopropyl)zinc(II) bromide (50 mL, 0.50M in THF, 25 mmol) was then added to the reaction mixture and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was quenched with saturated aqueous ammonium chloride and then diluted with DCM. The organic layer was separated and washed with saturated aqueous sodium bicarbonate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissovled in 20 mL of DCM, and 150 mL of hexanes was added slowly. The precipiated solids were filtered and washed with hexanes to yield ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (3.85 g, 11.3 mmol, 69%). LCMS (C16H18FO5S) (ES, m/z): 341 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 8.33 (s, 1H), 7.58 (s, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 3.86 (s, 3H), 3.40 – 3.35 (m, 2H), 2.70 – 2.64 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). Step 5: Ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S OO

OF O

O

S OO

HOF O

O

AlCl3

To a mixture of ethyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (3.6 g, 11 mmol) in dichloromethane (50 mL) was added aluminum chloride (5.64 g, 42.3 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with water (50 mL, added slowly via addition funnel) followed by HCl (1N, 50mL, added slowly via addition funnel). The reaction mixture was then diluted with 20% isopropanol in dichloromethane. The organic layer was separated, dried over sodium sulfate,

53

filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / dichloromethane) to afford ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.15 g, 6.59 mmol, 62%). LCMS (C15H16FO5S) (ES, m/z): 327 [M+H]+. 1H NMR (600 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.25 (s, 1H), 7.47 (s, 1H), 4.06 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 3.39-3.34 (m, 2H), 2.68-2.63 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). Step 6: Ethyl 4-(4-fluoro-6-methoxy-5-(3-((5-methoxy-2-(4-methoxy-4-oxobutanoyl)benzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate

SO

O

O

OO

SO

HOF

O

OO

Br

K2CO3

SO

O

O

OO

OS

O

OO

OF

A mixture of methyl 4-(6-(3-bromopropoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (35 mg, 0.084 mmol), ethyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (28 mg, 0.084 mmol), and potassium carbonate (47 mg, 0.34 mmol) was degassed with argon. DMF (1.0 mL) was added to the mixture, and the reaction mixture was stirred and heated at 40°C for 18 hours. The reaction mixture was cooled to room temperature and was used in the subsequent step without workup or purification. LCMS (C32H34FO10S2) (ES, m/z): 661 [M+H]+. Step 7: 4-(6-(3-((2-(3-Carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

SO

O

O

OO

OS

O

OO

OF

SO

O

O

OHO

OS

O

HOO

OF

NaOH

Sodium hydroxide (1.0M in water, 0.59 mL, 0.59 mmol) was added to a solution of ethyl 4-(4-fluoro-6-methoxy-5-(3-((5-methoxy-2-(4-methoxy-4-oxobutanoyl)benzo[b]thiophen-6-yl)oxy)propoxy)benzo[b]thiophen-2-yl)-4-oxobutanoate (crude mixture from previous step, 56 mg, 0.085 mmol) in DMSO (2.0 mL). The reaction mixture was stirred at room temperature for 5 minutes. The reaction mixture was quenched with TFA (0.078 mL, 1.0 mmol) and filtered. The filtrate was purified by reverse phase HPLC (acetonitrile / water, with 0.1% TFA modifier) to afford 4-(6-(3-((2-(3-carboxypropanoyl)-4-fluoro-6-methoxybenzo[b]thiophen-5-yl)oxy)propoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (16 mg, 0.026 mmol, 30% yield over two steps). LCMS (C29H28FO10S2) (ES, m/z): 619 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 12.20 (s, 2H), 8.29 (s, 1H), 8.20 (s, 1H), 7.64 (s, 1H), 7.56 (s, 1H), 7.47 (s, 1H), 4.30 (t, J = 6.2 Hz, 2H), 4.24 (q, J = 5.8 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 3.33 – 3.29 (m, 2H), 3.29 – 3.24 (m, 2H), 2.60 (q, J = 7.4 Hz, 4H), 2.20 (q, J = 6.1 Hz, 2H). HRMS (ES) calculated for C29H28FO10S2: 619.1108 [M+H]+, found 619.1118. Compound 11: 4,4'-((propane-1,3-diylbis(oxy))bis(5-methoxybenzo[b]thiophene-6,2-diyl))bis(4-oxobutanoic acid)

SO

O

O

OHO

O

SO

HOO

O

54

Step 1: Methyl-5,6-dimethoxybenzo[b]thiophene-2-carboxylate

F

OO

O

S

O

OHSO

OMe

K2CO3

O

O

To a stirred solution of 2-fluoro-4,5-dimethoxybenzaldehyde (18.7 g, 102 mmol) in DMF (600 mL) was added methyl 2-mercaptoacetate (11.9 g, 112 mmol) and potassium carbonate (42.1 g, 305 mmol). The resulting mixture was then heated at 60°C for 15 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was diluted with water (500 mL) and extracted with dichloromethane (600 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford methyl 5,6-dimethoxybenzo[b]thiophene-2-carboxylate (23.3 g, 83 mmol, 90%). LCMS (C12H13O4S) (ES, m/z): 253 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.03 (s, 1H), 7.60 (s, 1H), 7.49 (s, 1H), 3.85 (s, 6H), 3.82 (s, 3H). Step 2: 5,6-Dimethoxybenzo[b]thiophene-2-carboxylic acid

S

O

OKOH

O

OH

S

O

O O

O To a suspension of methyl 5,6-dimethoxybenzo[b]thiophene-2-carboxylate (23 g, 91 mmol) in methanol (200 mL), THF (200 mL) and water (200 mL) was added KOH (51 g, 910 mmol). The resulting suspension was heated to 60°C for 30 minutes. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. Water (600 mL) was added to the resulting residue, and then citric acid was added to the solution to adjust to pH 6. The precipitated material was collected via filtration to afford 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (17.5 g, 66.1 mmol, 90%). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H), 7.58 (s, 1H), 7.48 (s, 1H), 3.85 (s, 3H), 3.82 (s, 3H). Step 3: 5,6-Dimethoxybenzo[b]thiophene-2-carbonyl chloride

S

OH

O S

Cl

O

O

OO

O

(COCl)2

DMF, cat To a stirring solution of 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (5.0 g, 21 mmol) in THF (200 mL) at 0°C under argon was added (COCl)2 (5.5 mL, 63 mmol) followed by DMF (0.1 mL, 1.3 mmol). The reaction mixture was stirred at 0°C for 1 hour and then warmed to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the resulting 5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride was used in the next step without purification. 1H NMR (600 MHz, CH3CN-d3): δ 8.25 (s, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 3.92 (s, 3H), 3.88 (s, 3H). Step 4: Ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

S

O

O

Cl

O S

O

O O

OO

O

O

BrZn

A flask containing copper (I) chloride (416 mg, 4.20 mmol) was flushed with nitrogen. (3-Ethoxy-3-oxopropyl) zinc (II) bromide (0.50M in THF, 16.8 mL, 8.4 mmol) was added and the resulting mixture was stirred at room temperature for 30 minutes. A suspension of 5,6-dimethoxybenzo[b]thiophene-2-carbonyl chloride (1.1 g, 4.2 mmol) in THF (8.4 mL) was

55

added and the mixture was stirred for 45 minutes at room temperature. The reaction mixture was quenched by the slow addition of aqueous saturated ammonium chloride (10 mL) while stirring vigorously. After 5 minutes aqueous saturated sodium bicarbonate (30 mL) was added and stirring was continued for an additional 5 minutes. The reaction mixture was extracted with ethyl acetate (2 x 50 mL). The organic layers were combined, washed with water (20 mL) and brine (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate in hexanes) to afford ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (900 mg, 2.8 mmol, 67%). LCMS (C16H19O5S) (ES, m/z): 323 [M+H]+. Step 5: 4-(5,6-Dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S

O

O O

OO

S

O

O O

OHO

LiOH

Lithium hydroxide (1.0M in water, 3.0 mL, 3.0 mmol) was added to a mixture of ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.47 g, 1.46 mmol) in THF (3 mL) and methanol (3 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then neutralized with hydrochloric acid (1.0M in water, 3.0 mL, 3.0 mmol). The resulting suspension was stirred for 30 minutes, and the solids were filtered and dried under vacuum to afford 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (333 mg, 1.13 mmol, 78%). LCMS (C14H15O5S) (ES, m/z): 295 [M+H]+. Step 6: Methyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SiN+ N

-

SO

O

O

OOH

SO

O

O

OO

TMS-diazomethane (2.0M in diethyl ether, 5.5 mL, 11 mmol) was added dropwise to a

mixture of 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (2.15 g, 7.30 mmol) in dichloromethane (50 mL) and methanol (50 mL) at 0°C. The reaction mixture was stirred at 0°C for 1 hour. The reaction mixture was quenched with acetic acid (added dropwise until bubbling ceased). The reaction mixture was concentrated under reduced pressure to afford methyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate, which was used without purification in the next step (2.20 g, 7.13 mmol, 98%). LCMS (C15H17O5S) (ES, m/z): 309 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 8.22 (s, 1H), 7.61 (s, 1H), 7.49 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.61 (s, 3H), 3.35 – 3.30 (m, 2H), 2.71 – 2.66 (m, 2H). Step 7: Methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate and methyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

AlCl

Cl ClSO

O

O

OO

SHO

O

O

OO

SO

HO

O

OO

Aluminum chloride (5.71 g, 42.8 mmol) was added to a mixture of methyl 4-(5,6-

dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (2.20 g, 7.13 mmol) in dichloromethane (250 mL) at room temperature. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was cooled to 0°C and quenched with water (50 mL, added dropwise via addition funnel). The reaction mixture was then warmed to room temperature and diluted with additional

56

dichloromethane (250 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate/dichloromethane) to afford an inseparable mixture of methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (77%) and methyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (23%) (mixture of regioisomers: 1.82 g, 6.19 mmol, 77%). LCMS (C14H15O5S) (ES, m/z): 295 [M+H]+. Step 8: Methyl 4-(5-(((benzyloxy)carbonyl)oxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate and methyl 4-(6-(((benzyloxy)carbonyl)oxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SHO

O

O

OO

SO

O

O

OO

O O

OO

Cl

N

SO

O

O

OO

O OSO

HO

O

OO

CBZ-Cl (1.06 mL, 7.42 mmol) was added to a mixture of methyl 4-(6-hydroxy-5-

methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (77%) and methyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (23%) (1.82 g, 6.18 mmol) and triethylamine (1.29 mL, 9.28 mmol) in dichloromethane (30 mL) at 0°C. The reaction mixture was then warmed to room temperature and stirred for an additional 2 hours. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (ethyl acetate / hexanes) to afford:

Peak 1: methyl 4-(5-(((benzyloxy)carbonyl)oxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (410 mg, 0.96 mmol, 15%). LCMS (C22H21O7S) (ES, m/z): 429 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 8.30 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.48 – 7.38 (m, 5H), 5.30 (s, 2H), 3.87 (s, 3H), 3.61 (s, 3H), 3.40 – 3.33 (m, 2H), 2.69 (t, J = 6.4 Hz, 2H).

Peak 2: methyl 4-(6-(((benzyloxy)carbonyl)oxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (1.84 g, 4.29 mmol, 69%). LCMS (C22H21O7S) (ES, m/z): 429 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 8.32 (s, 1H), 8.01 (s, 1H), 7.71 (s, 1H), 7.47 – 7.37 (m, 5H), 5.31 (s, 2H), 3.85 (s, 3H), 3.62 (s, 3H), 3.41 – 3.36 (m, 2H), 2.74 – 2.68 (m, 2H). Step 9: Methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

NH

N

SO

O

O

OO

O OSHO

O

O

OO

1-Methylpiperazine (1.4 mL, 13 mmol) was added to a mixture of methyl 4-(6-

(((benzyloxy)carbonyl)oxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (1.84 g, 4.29 mmol) in DMF (5 mL) and methanol (5 mL) at room temperature. The reaction mixture was then heated to 50°C and stirred for an additional 30 minutes. The reaction mixture was cooled to room temperature and partially concentrated under reduced pressure (to remove methanol). The crude

57

mixture was purified by silica gel chromatography (ethyl acetate / dichloromethane) to afford methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (1.22 g, 4.15 mmol, 97%). LCMS (C14H15O5S) (ES, m/z): 295 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.18 (s, 1H), 7.47 (s, 1H), 7.32 (s, 1H), 3.86 (s, 3H), 3.61 (s, 3H), 3.33 – 3.29 (m, 2H), 2.71 – 2.66 (m, 2H). Step 10: Methyl 4-(6-(3-bromopropoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate

SHO

O

O

OO

SO

O

O

OO

BrBr Br

K2CO3

1,3-Dibromopropane (0.17 mL, 1.7 mmol) was added to a mixture of methyl 4-(6-hydroxy-

5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (50 mg, 0.17 mmol) and potassium carbonate (70 mg, 0.51 mmol) in DMF (0.5 mL) at room temperature. The reaction mixture was then heated to 50°C and stirred for 1 hour. The reaction mixture was cooled to room temperature, diluted with dichloromethane (1 mL), and filtered to remove inorganic salts. The filtrate was directly purified by silica gel chromatography (ethyl acetate / dichloromethane) to afford methyl 4-(6-(3-bromopropoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (58 mg, 0.14 mmol, 82%). LCMS (C17H20BrO5S) (ES, m/z): 415, 417 [M+H]+. Step 11: Dimethyl 4,4'-((propane-1,3-diylbis(oxy))bis(5-methoxybenzo[b]thiophene-6,2-diyl))bis(4-oxobutanoate)

SO

O

O

OO

SHO

O

O

OO

SO

O

O

OO

O

SO

OO

OBr

K2CO3 A mixture of methyl 4-(6-(3-bromopropoxy)-5-methoxybenzo[b]thiophen-2-yl)-4-

oxobutanoate (35 mg, 0.084 mmol), methyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (25 mg, 0.084 mmol), and potassium carbonate (47 mg, 0.34 mmol) was degassed with argon. DMF (1.0 mL) was added to the mixture, and the reaction mixture was stirred and heated at 40°C for 18 hours. The reaction mixture was cooled to room temperature and was used in the subsequent step without workup or purification. LCMS (C31H33O10S2) (ES, m/z): 629 [M+H]+. Step 12: 4,4'-((Propane-1,3-diylbis(oxy))bis(5-methoxybenzo[b]thiophene-6,2-diyl))bis(4-oxobutanoic acid)

SO

O

O

OO

O

SO

OO

O SO

O

O

OHO

O

SO

HOO

ONaOH

Sodium hydroxide (1.0M in water, 0.59 mL, 0.59 mmol) was added to a mixture of

dimethyl 4,4'-(6,6'-(propane-1,3-diylbis(oxy))bis(5-methoxybenzo[b]thiophene-6,2-diyl))bis(4-oxobutanoate) (crude from previous step, 53 mg, 0.084 mmol) in DMSO (2.0 mL). The reaction mixture was stirred at room temperature for 5 minutes. The reaction mixture was quenched with TFA (0.078 mL, 1.0 mmol) and filtered. The filtrate was purified by reverse phase HPLC (acetonitrile / water, with 0.1% TFA) to afford 4,4'-(6,6'-(propane-1,3-diylbis(oxy))bis(5-methoxybenzo[b]thiophene-6,2-diyl))bis(4-oxobutanoic acid) (3.1 mg, 5.2 µmol, 6.1% yield over two steps). LCMS (C29H29O10S2) (ES, m/z): 601 [M+H]+. 1H NMR (499 MHz, DMSO-d6) δ 12.18 (s, 2H), 8.20 (s, 2H), 7.67 (s, 2H), 7.49 (s, 2H), 4.26 (t, J = 6.2 Hz, 4H), 3.85 (s, 6H), 3.29 –

58

3.24 (m, 4H), 2.64 – 2.58 (m, 4H), 2.30 (p, J = 6.0 Hz, 2H). HRMS (ES) calculated for C29H29O10S2: 601.1202 [M+H]+, found 601.1201. Compound 12: 4-(4-(3-(2-(3-carboxypropanoyl)-5-methoxybenzo[b]thiophen-6-yl)propyl)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

S

OMeO

OHOO

HOO

OMe

S

Br

FO

Br

FO

H

O

To a mixture of 1-bromo-3-fluoro-5-methoxybenzene (7.5g, 37mmol) in THF (120mL) at -78°C was added LDA (2.0M in THF, 22mL, 44mmol), and the mixture was allowed to stir for 30 min at -78°C. After 30 min, DMF (3.4mL, 44mmol) was added dropwise, and the mixture was then allowed to stir for 30 min. The mixture was then quenched with water, warmed to RT, and then EtOAc was added. The layers were separated, and the water layer was extracted with EtOAc two more times. The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product residue was purified by silica gel chromatography to afford 2-bromo-6-fluoro-4-methoxybenzaldehyde. The product was contaminated with 4-bromo-2-fluoro-6-methoxybenzaldehyde. This mixture was taken on without further purification. LCMS (C8H7BrFO2) (ES, m/z): 233, 235 [M+H]+.

Br

FO

H

O Br

O S

O

O To a mixture of 2-bromo-6-fluoro-4-methoxybenzaldehyde (2.5g, 11mmol) in DMSO (54mL) was added TEA (3.0mL, 21mmol). After 10 min, methyl thioglycolate (3.1mL, 32mmol) was added, and the mixture was allowed to stir for 30 min at RT. After 30 min, the mixture was heated to 60°C for 1 h. Upon cooling to RT, the mixture was diluted with sat aq NaHCO3 and EtOAc. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product residue was purified by silica gel column chromatography (0→15% EtOAc gradient in Hex) to afford methyl 4-bromo-6-methoxybenzo[b] thiophene-2-carboxylate. LCMS (C11H10BrO3S) (ES, m/z): 301, 303 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 7.93 (s, 1H), 7.73 (s, 1H), 7.44–7.37 (m, 1H), 3.89 (s, 3H), 3.87 (s, 3H).

O

SMeO O

BrO

SMeO OH

Br

To a mixture of methyl 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (1.7g, 5.5mmol) in THF (14mL), MeOH (7.0mL), and water (7.0mL) was added LiOH (0.66g, 28mmol), and the mixture was heated to 40°C for 2 h. After 2 h, the mixture was allowed to cool to RT. The mixture was quenched with aq HCl (2.0M in water, 14mL, 28mmol). The mixture was filtered, and the residue was washed with EtOAc. The residue was then dried under vacuum and used without further purification (1.4g, 4.9mmol, 89% yield). LCMS (C10H8BrO3S) (ES, m/z):

59

287, 289 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 13.57 (s, 1H), 7.86 (s, 1H), 7.71 (s, 1H), 7.38 (d, J=1.7 Hz, 1H), 3.86 (s, 3H).

S

O

OHMeO SMeO

BrBrDBUDMA

To a microwave vial containing 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (1.84 g, 6.41 mmol) was added DMA (18 ml). DBU (2.415 ml, 16.02 mmol) was added and the mixture was irradiated in the microwave at 200°C for 2 hours. After 2 hours, the mixture was allowed to cool to room temperature and then diluted with ethyl acetate and saturated aquesous sodium bicarbonate. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated. The resulting residue was purified by silica gel column chromatography to afford 4-bromo-6-methoxybenzo[b]thiophene (1.1g, 4.4mmol, 69% yield). LCMS (C9H8BrOS) (ES, m/z): 243, 245 [M+H]+. 1H NMR (500 MHz, Chloroform-d) δ 7.37 (d, J = 5.5 Hz, 1H), 7.32 (d, J = 5.5 Hz, 1H), 7.31 – 7.29 (m, 1H), 7.23 (d, J = 1.7 Hz, 1H), 3.88 (s, 3H).

SMeO

Br

SMeO

BrO O

OH

OO O

AlCl3 To a mixture of 4-bromo-6-methoxybenzo[b]thiophene (1.1g, 4.4mmol) in dichloromethane (44mL) at 0°C was added succinic anhydride (0.57g, 5.7mmol) and then aluminum chloride (1.2g, 8.8mmol). The mixture was allowed to stir at room temperature for one hour. After one hour, the mixture was diluted with ethyl acetate and aqueous saturated sodium bicarbonate. Solids crashed out of solution. Aqueous HCl (1.0N) was added to a pH ~3 and some of the solids went into solution. The mixture was filtered. The organic layer was then separated, dried over magnesium sulfate and filtered. Silica gel (10 grams) was added and the mixture was concentrated under reduced pressure. The mixture was put under vacuum overnight and then column chromatography was used for purification (40G column, 0-50% ethyl acetate gradient in hexanes) to afford 4-(4-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (0.99g, 1.6mmol, 55% yield). LCMS (C13H12BrO4S) (ES, m/z): 343, 345 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 12.19 (s, 1H), 8.15 (s, 1H), 7.70 (s, 1H), 7.41 – 7.38 (m, 1H), 3.87 (s, 3H), 2.60 (t, J = 6.1 Hz, 2H).

SMeO

BrO O

OHSMeO

BrO O

OTMS-diazomethane

To a mixture of 4-(4-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (99mg, 2.9mmol) in methanol (29mL) was added TMS-Diazomethane (2.0M in diethyl ether, 4.3mL, 8.6mmol) slowly. After 30 minutes, more TMS-Diazomethane (2.0M in diethyl ether, 4.3mL, 8.6mmol) was added slowly. After 1 hour, the mixture was slowly quenched with acetic acid until the mixture stopped bubbling. The mixture was then concentrated and the resulting residue was purified by silica gel column chromatography. The fractions containing product were pooled and then purified by SFC (biphenyl column (15% methanol with 0.25% DMEA in CO2) to afford methyl 4-(4-bromo-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.77g, 2.2mmol, 75% yield). LCMS (C14H14BrO4S) (ES, m/z): 357, 359 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.70 (s, 1H), 7.40 (d, J = 1.7 Hz, 1H), 3.87 (s, 3H), 3.61 (s, 3H), 3.41 (t, J = 6.3 Hz, 2H), 2.68 (t, J = 6.2 Hz, 2H).

60

S O

OO

Br

O

Br F

O

To a stirred solution of 4-bromo-2-fluoro-5-methoxybenzaldehyde (5.00g, 21.5mmol) in DMF (100mL) was added methyl 2-mercaptoacetate (2.51g, 23.6mmol) and K2CO3 (8.90g, 64.4mmol). The reaction mixture was degassed with N2 3 times. The resulting mixture was then stirred at RT for 15h. EtOAc (500mL) and H2O (1200mL) were added to the reaction mixture. The organic layer was separated and washed with sat aq NaCl (2x200mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EtOAc in PE) to give methyl 6-bromo-5-methoxybenzo [b]thiophene-2-carboxylate. LCMS (C11H10BrO3S) (ES, m/z): 301, 303 [M+H]+. 1H NMR (400MHz, CDCl3): δ=8.01 (s, 1H), 7.93 (s, 1H), 7.26 (s, 1H), 3.96 (s, 3H), 3.94 (s, 3H).

S O

OO

Br S O

OHO

Br

To a suspension of methyl 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylate (1.45g, 4.81mmol) in MeOH (20mL), THF (20mL), and H2O (20mL) was added NaOH (1.93g, 48.1mmol). The resulting suspension was heated to 50°C for 0.5h. The reaction mixture was concentrated under reduced pressure to remove the solvent. H2O (200mL) was added to the residue, and citric acid was added to adjust the solution to pH=6. The remaining aq suspension was extracted with EtOAc (3x50mL). The combined organic layers were washed with sat aq NaCl (100mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid, which was used without further purification. 1H NMR (400MHz, DMSO-d6): δ=13.52 (br s, 1H), 8.35 (s, 1H), 8.01 (s, 1H), 7.65 (s, 1H), 3.90 (s, 3H).

S O

ClO

BrS O

OHO

Br

To a stirred solution of 6-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid (0.80g, 2.8mmol) in anhydrous THF (6.0mL) was added (COCl)2 (1.1g, 8.4mmol) dropwise at 0°C. The mixture was then heated at 75°C for 15h and then cooled to RT. The solvent was removed under reduced pressure to give the crude 6-bromo-5-methoxybenzo[b]thiophene-2-carbonyl chloride, which was used without further purification.

S

MeO

Br O

Cl

S

MeO

Br O

OO

To a round bottom flask was added CuCl (0.24g, 2.4 mmol). The flask was evacuated and then opened to N2. This was repeated three times. THF (4.0mL) was added and the mixture was cooled to 0°C. A mixture of (3-(tert-butoxy)-3-oxopropyl)zinc(II) bromide (0.50M in THF, 9.6mL, 4.8mmol) was added dropwise at 0°C over 10 min. The resulting mixture was allowed to stir for 30 min. 6-bromo-5-methoxybenzo[b]thiophene-2-carbonyl chloride (0.73 g, 2.4mmol) was added. The mixture was removed from the ice bath and allowed to warm to RT. The mixture was stirred for 2 h. The mixture was then cooled to 0°C, and concentrated NH4OH (4.5mL) was added. To the resulting suspension was added water (240mL) and MeOH (60mL). The mixture was stirred for 5 min and sonicated in a bath sonicator. The mixture was then diluted with EtOAc, and the organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product residue was purified by silica gel chromatography to

61

afford tert-butyl 4-(6-bromo-5-methoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (0.16g, 0.40mmol, 17% yield). LCMS (C17H19BrO4SNa) (ES, m/z): 421, 423 [M+Na]+.

S O

OO

S O

OO

Br

MeO

MeO OSi

To a flask containing tert-butyl 4-(6-bromo-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.16g, 0.40mmol) and THF (2.0mL) was added [(2-dicyclohexylphosphino-2′,6′-bis(N,N-dimethylamino) -1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)] palladium(II) methane sulfonate (C-Phos Pd G3, 16mg, 0.020mmol). The flask was evacuated and backfilled 3 times with N2. (3-((tert-butyldimethylsilyl)oxy)propyl)zinc(II) bromide (0.50M in THF, 2.4mL, 1.2mmol) was added, and the mixture was allowed to stir at RT for 2.5h. The mixture was then quenched with a mixture of EtOAc and 10% aqueous sodium citrate. The organic layer was separated, washed with sat aq NaCl, dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography to afford tert-butyl 4-(6-(3-((tert-butyldimethylsilyl)oxy)propyl)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.11g, 0.23mmol, 58% yield). LCMS (C26H41O5SSi-C4H8) (ES, m/z): 437 [M-C4H8]+.

S O

OO

MeO

OSi S O

OO

MeO

HO

To a flask containing tert-butyl 4-(6-(3-((tert-butyldimethylsilyl)oxy)propyl)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.11g, 0.23 mmol) was added MeOH (1.5mL), water (1.5mL) and HOAc (1.5mL). The mixture was allowed to stir for 4 h. The mixture was diluted with EtOAc and then washed with water (3x50mL). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography to afford tert-butyl 4-(6-(3-hydroxypropyl)-5-methoxy-benzo[b]thiophen-2-yl)-4-oxobutanoate (74mg, 0.20mmol, 85% yield). LCMS (C20H27O5S - C4H8) (ES, m/z): 323 [M-C4H8]+.

S

OMeO

HO

OO

S

OMeO

Br

OO

To a mixture of tert-butyl 4-(6-(3-hydroxypropyl)-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (74mg, 0.20mmol) and triphenylphosphine (82mg, 0.31mmol) in THF (1.0mL) at 0°C was added NBS (52mg, 0.29mmol). After 15 min at 0°C, the mixture was quenched with sat aq NH4Cl and diluted with EtOAc. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography to afford tert-butyl 4-(6-(3-bromopropyl)-5-methoxybenzo [b]thiophen-2-yl)-4-oxobutanoate (72mg, 0.16mmol, 83% yield). LCMS (C20H26BrO4S-C4H8) (ES, m/z): 385, 387 [M-C4H8].

62

S

OMeO

Br

OO SMeO

BrO O

OS

OMeO

OO

S

MeO

OO

O

To a vial was added tert-butyl 4-(6-(3-bromopropyl)-5-methoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (72mg, 0.16mmol), methyl 4-(4-bromo-6-methoxybenzo[b] thiophen-2-yl)-4-oxobutanoate (58mg, 0.16mmol), NaI (12mg, 0.082mmol), nickel(II) bromide ethylene glycol dimethyl ether complex (15mg, 0.049mmol), Mn (36mg, 0.65mmol) and 4,4’-dimethoxy-2,2’-bipyridine (11mg, 0.049mmol). To the vial was added DMPU (1.6mL) followed by the addition of 5% v/v solutions in DMPU of pyridine (130µl, 0.082mmol) and TMS-Cl (130µl, 0.049mmol). The vial was degassed with Ar for 5 min. The mixture was heated to 90°C for 1 h. After 1 h, the mixture was allowed to cool to RT and then diluted with EtOAc and water. The organic layer was separated, dried over MgSO4, filtered and concentrated under reduced pressure. The resulting mixture was used without further purification or characterization.

S

OMeO

OO

S

MeO

OO

O

S

OMeO

OHO

S

MeO

OO

OH

To a mixture of tert-butyl 4-(5-methoxy-6-(3-(6-methoxy-2-(4-methoxy-4-oxobutanoyl)benzo[b]thiophen-4-yl)propyl)benzo[b]thiophen-2-yl)-4-oxobutanoate (100mg, 0.16mmol) and MeOH (1.6mL) was added NaOH (5M in water, 0.65mL, 3.3mmol), and the mixture was heated to 50°C for 1 h. Upon cooling to RT, the mixture was purified by prep-HPLC (ACN/H2O with 0.1% TFA) to afford 4-(4-(3-(2-(3-carboxypropanoyl)-5-methoxybenzo [b]thiophen-6-yl)propyl)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (6.2mg, 11µmol, 7% yield). HRMS (ESI) m/z: [M+H]+ Calcd for C29H29O8S2 569.1304; Found 569.1290. 1H NMR (500 MHz, DMSO-d6) δ 12.23 (s, 2H), 8.26 (d, J = 10.3 Hz, 2H), 7.81 (d, J = 9.8 Hz, 1H), 7.50 (s, 1H), 7.44 (s, 1H), 6.93 (s, 1H), 3.85 – 3.82 (m, 6H), 3.31 – 3.23 (m, 4H), 3.03 (t, J = 7.3 Hz, 2H), 2.82 – 2.77 (m, 2H), 2.63 – 2.57 (m, 4H), 2.04 – 1.98 (m, 2H). mono-13: 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoic acid

O

O

S OOH

O

F

63

S

O

O

O

OS

O

O

O

O

F

N

N

F

Cl

BF4

BF4

1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (SELECTFLUOR™, 77mg, 0.22mmol) was added to a mixture of methyl 5,6-dimethoxybenzo[b]thiophene-2-carboxylate (50mg, 0.20mmol) in acetonitrile (1.0mL) at rt. The resulting mixture was heated to 45°C for 15h. The mixture was cooled to rt, diluted with sat aq NaHCO3 (10mL), and extracted with EtOAc (3x10mL). The combined organic layers were washed with brine (10mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, EtOAc in PE) to give methyl 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylate. LCMS (C12H12FO4S) (ES, m/z): 293 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.61 (s, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H).

S

O

O

O

O S

O

O

O

OH

F F

LiOH·H2O

LiOH·H2O (71mg, 1.7mmol) was added portionwise to a mixture of methyl 4-fluoro-5, 6-dimethoxybenzo[b]thiophene-2-carboxylate (46mg, 0.17mmol) in THF (3.0mL), MeOH (1.0mL), and H2O (1.0mL) at rt. The mixture was stirred for 15h. After 15h, the mixture was adjusted to pH=5 with 1N HCl and extracted with EtOAc (3x10ml). The combined organic layers were washed with brine (10mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (ACN/H2O with 0.1% TFA) to give 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (4.7g, 18mmol, 99% yield). LCMS (C11H9FO4S) (ES, m/z): 257 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 7.93 (s, 1H), 7.59 (s, 1H), 3.92 (s, 3H), 3.86 (s, 3H).

S

O

O

O

OH S

O

O

F F

CuQuinoline

To a flask containing 4-fluoro-5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (10.4g, 40.7mmol) and quinoline (65.0mL) was added copper powder (4.40g, 69.1mmol). The resulting mixture was heated to 190°C for 2 hours. After 2 hours, the mixture was allowed to cool to room temperature. The mixture was then diluted with ethyl acetate (300mL). The mixture was washed with aqueous hydrochloric acid (2.0M in water, 1.20L, 2.40mmol) followed by brine (50mL). The mixture was then dried over magnesium sulfate, filtered and concentrated under reduced pressure to afford the crude product as a black oil. The crude oil was purified by silica gel column chromatography (330G Snap column, 100% hexanes). The fractions containing product were combined, concentrated and repurified by silica gel column chromatography (330G Snap column, 100% hexanes) to afford 4-fluoro-5,6-dimethoxybenzo[b]thiophene (7.18g, 33.8mmol, 83% yield) as a white solid. LCMS (C10H10FO2S) (ES, m/z): 213 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 7.64 (d, J = 5.5 Hz, 1H), 7.54 (s, 1H), 7.37 (d, J = 5.5 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 3H).

64

O

O

S O

O

S OOH

OOO O

F FAlCl3 To a flask containing 3,3-dimethyldihydrofuran-2,5-dione (2.7mL, 24mmol) and 4-fluoro-5,6-dimethoxybenzo[b]thiophene (2.5g, 12mmol) in dichloromethane (59mL) at 0°C was added aluminum chloride (2.0g, 15mmol). The mixture was allowed to stir at 0°C for 2 hours and then allowed to warm to ambient temperature. The mixture was stirred at ambient temperature for 16 hours. After 16 hours, the mixture was quenched slowly with aqueous hydrochloric acid (2.0M in water, 24mL, 48mmol) and then diluted with ethyl acetate. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting mixture was purified by silica gel column chromatography to afford 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoic acid (1.4g, 4.1mmol, 35% yield) as a white solid. HRMS (ESI) m/z: [M+H]+ Calcd for C16H18FO5S 341.0859; Found 341.0876. 1H NMR (500 MHz, DMSO-d6) δ 12.07 (s, 1H), 8.30 (s, 1H), 7.58 (s, 1H), 3.93 (s, 3H), 3.86 (s, 3H), 3.37 (s, 2H), 1.23 (s, 6H). Compound 13: 4,4'-((propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(2,2-dimethyl-4-oxobutanoic acid)

OS

HO

O

O

O S O

OHO

OOFF

S

O OOH

O

S

O OO

O

O O

F F

To a mixture of 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoic acid (2.3g, 6.8mmol) in DMF (45mL) was added K2CO3 (2.3g, 17mmol). After 10 minutes, CH3I (2.1mL, 34mmol) was added, and the mixture was stirred for 18 h at RT. The mixture was then diluted with water and Et2O. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The mixture was then purified by silica gel chromatography to afford methyl 4-(4-fluoro-5,6-dimethoxybenzo[b] thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (1.1g, 3.1mmol, 46% yield). LCMS (C17H20FO5S) (ES, m/z): 355 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.59 (s, 1H), 3.92 (s, 3H), 3.85 (s, 3H), 3.57 (s, 3H), 3.44 (s, 2H), 1.23 (s, 6H).

S

O

O

OF

OO

S

O

O

HOF

OO

To a mixture of methyl 4-(4-fluoro-5,6-dimethoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (1.1g, 3.1mmol) and DCM (20mL) was added AlCl3 (1.7g, 12mmol). The reaction mixture was stirred at RT for 18 h. The reaction mixture was poured into a flask containing ice and 1N HCl and stirred for 5 min. EtOAc was then added. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The mixture was

65

then purified by silica gel chromatography to afford methyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (0.67g, 2.0mmol, 63% yield). LCMS (C16H18FO5S) (ES, m/z): 341 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.24 (s, 1H), 7.47 (s, 1H), 3.91 (s, 3H), 3.57 (s, 3H), 3.43 (s, 2H), 1.23 (s, 6H).

O

HO

S O

OO

O

O

S O

OO

Br ClCl

F FK2CO3

A mixture of methyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (0.20g, 0.59mmol), 1-bromo-3-chloropropane (0.17mL, 1.8mmol), and potassium carbonate (0.33g, 2.4mmol) was degassed with argon. Acetonitrile (2.5 mL) was added to the mixture, and the reaction mixture was stirred and heated to 45°C for 18 hours. After 18 hours, the mixture was allowed to cool to room temperature. The mixture was diluted with dichloromethane (10mL) and the mixture was filtered. The filtrate was concentrated under reduced pressure and the crude product residue was purified by silica gel chromatography to afford methyl 4-(5-(3-chloropropoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (0.21g, 0.50mmol, 85% yield). LCMS (C19H23ClFO5S) (ES, m/z): 417 [M+H]+. 1H NMR (500 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.59 (s, 1H), 4.15 (t, J = 5.8 Hz, 2H), 3.92 (s, 3H), 3.85 (t, J = 6.4 Hz, 2H), 3.56 (s, 3H), 3.43 (s, 2H), 2.12 (p, J = 6.1 Hz, 2H), 1.23 (s, 6H).

O S O

OO

OCl

O S O

OO

HOF

OS

O

O

O

O S O

OO

OOFF F

K2CO3

A mixture of methyl 4-(4-fluoro-5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (41mg, 0.12mmol), methyl 4-(5-(3-chloropropoxy)-4-fluoro-6-methoxybenzo[b]thiophen-2-yl)-2,2-dimethyl-4-oxobutanoate (50mg, 0.12mmol), and potassium carbonate (66mg, 0.48mmol) was degassed with argon. DMF (0.50mL) was added to the mixture, and the reaction mixture was stirred and heated to 50°C for 2 hours. After 2 hours, the heated was increased to 70°C and heated for 2 hours. After 2 hours, the reaction mixture was cooled to ambient temperature and diluted with ethyl acetate and water. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude mixture was taken on to the next step without further purification. LCMS (C35H39F2O10S2) (ES, m/z): 721 [M+H]+.

OS

O

O

O

O S O

OO

OOFF

OS

HO

O

O

O S O

OHO

OOFF

NaOH

To a mixture of dimethyl 4,4'-(5,5'-(propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(2,2-dimethyl-4-oxobutanoate) (96mg, 0.13mmol) in methanol (1.3mL) was added aqueous sodium hydroxide (0.75mL, 3.8mmol) and the mixture was heated to 50°C for 1.5 hours. After 1.5 hours, the mixture was cooled to room temperature and then acidified to a ph~3 with aqueous hydrochloric acid (2.7mL, 2.7mmol). The mixture was diluted with ethyl acetate and water. The organic layer was separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude residue was dissolved in DMSO and purified by RP-HPLC [C18 column, water (0.1% TFA)-CH3CN] to afford 4,4'-((propane-1,3-diylbis(oxy))bis(4-fluoro-6-methoxybenzo[b]thiophene-5,2-diyl))bis(2,2-dimethyl-4-oxobutanoic acid) (28mg, 0.040mmol, 30% yield). HRMS (ESI) m/z: [M+H]+ Calcd

66

for C33H35F2O10S2 693.1639; Found 693.1654. 1H NMR (500 MHz, DMSO-d6) δ 12.08 (s, 2H), 8.25 (s, 2H), 7.55 (s, 2H), 4.28 (t, J = 6.1 Hz, 4H), 3.87 (s, 6H), 3.37 (s, 4H), 2.14 – 2.05 (m, 2H), 1.24 (s, 12H).

Supplementary Text Text S1 Fitting the WT human STING surface plasmon resonance data using the two-step kinetic model based on Model 2 (see Methods, Fig. S4B) yielded individual rate constants with significant experimental variance not present in the raw data (note equivalent replicates in Fig. S4B), suggesting that these parameters are not well constrained. This was additionally reflected in a resultant fit that exhibited heterogeneity clearly not present in the actual sensorgram, particularly at low MSA-2 concentrations (black triangles, Fig. S4B). This was principally a reflection of the exceedingly rapid off-rate predicted for kd1 coupled with a very slow on-rate for ka2, ultimately yielding equilibrium constants that differed by more than 1,000-fold. Within the context of Model 2, it is also difficult to rationalize these predicted equilibrium constant values in physical terms, for if the initial binding event is truly described by a weak, rapid equilibrium (KD1 > 100 µM) then binding of the second molecule is essentially reduced to that of a three body collision in solution. In light of these results, the more complex Model 2 was not considered further. Text S2 MSA-2 titrations with both human STING isoforms (WT/HAQ) were fit by non-linear regression to a 4-parameter curve with variable slope, yielding values for the EC50 and Hill coefficient (see Fig. S7A-B). Overall values (and rank order) were comparable to those determined by other biophysical approaches (see Fig. 5). Compound 2 appears to bind human STING significantly more weakly (Fig. S7C-D) and fails to saturate at the highest tested concentration (125 µM). This is consistent with the inability of compound 2 (at 20 µM) to inhibit binding of radiolabeled cGAMP to STING and in keeping with a correspondingly lower concentration of bioactive dimer (Fig. 5H, S6). As seen with MSA-2, compound 2 is observed bound at lower compound concentrations to hSTING-HAQ than hSTING-WT (ALIS). But as indicated, no binding signal was observed at a compound 2 concentration of 1 µM with either protein isoform. This concentration was subsequently selected for combination studies with MSA-2 (Fig. S7E and Fig. 5I). The data sets for MSA-2 and compound 2 overlaid in Fig. S7E (and Fig. 5I) are normalized independently, reflecting intrinsic differences in their respective ionization potentials (and the necessity of analysis in different MS modes). Therefore, their absolute relative amounts cannot be inferred from this experiment. However, consistent with solution heterodimer formation, in all cases both species are only observed bound to human STING simultaneously. As shown in Fig. S7 and 5I, bound compound 2 (orange) increased concurrently with bound MSA-2 (green) as [MSA-2] increased until bound compound 2 reached a plateau; further increases of [MSA-2] caused concentration-dependent decline of bound compound 2 accompanied by progressive increases in bound MSA-2. In addition, the presence of even (relatively) low amounts of MSA-2 renders the binding of compound 2 observable at a significantly lower concentration (as denoted by the orange brackets) than in its absence. Moreover, as described in the main text, the observed trends for both bound molecules are consistent with Model 3. Thus, on the basis of this model, addition of MSA-2 effectively ‘rescues’ the apparent weak activity of compound 2, due to the formation of a solution heterodimer (MSA-2:2) which is more favorable than the formation of a compound 2 homodimer (Fig. 5H).

67

It may also be worth noting that the result of this experiment in some ways mirrors that shown in Fig. 5E, with the MSA-2:2 heterodimer replacing the mixed radioactive/unlabeled MSA-2 homodimer (an experimental ‘heterodimer’), yielding a similar trend but with simultaneous observation of the previously ‘silent’ binding event (bound MSA-2 homodimer). Lastly, the combination MSA-2:2 experiment conducted in THP-1 cells expressing hSTING-HAQ shown in Fig. 5J may be compared with the biochemical (ALIS) results shown in Fig. S7E for hSTING-HAQ. Although significantly higher concentrations of compound 2 were used in the cell based assay (10-160 µM vs 1 µM), in both cases addition of compound 2 at a concentration insufficient to elicit response alone caused a measurable and significant increase in response when combined with MSA-2. These observations can be rationalized by heterodimer formation consistent with Model 3.

68

Fig. S1. Biochemical evidence for MSA-2 activation of STING pathway in THP-1 cells. (A) Interferon-β production in THP-1 (○) or STING-/- THP-1 (●) cells in response to MSA-2 titrations (6 hr incubation, n=3). (B) Western blot of STING-TBK1-IRF3 pathway proteins from THP-1 cells (endogenously expressing hSTING-HAQ) following MSA-2 (30 µM) treatment for increasing periods of time. A relatively rapid increase in phosphorylated TBK1, followed by an increase in phosphorylated IRF3, was observed as shown. Western blotting was not performed for STING-/- THP-1 cells due to lack of response in (A).

69

Fig. S2. Effect of MSA-2 on body weight and plasma and tumor cytokine levels in MC38 tumor bearing WT and STING knockout (KO) mice, including comparative assessment of tumor volume and body weight. (A-C) Percent body weight change of mice treated with up to three injections of vehicle or MSA-2 intratumorally (A), subcutaneously (B) or orally (C). All groups were dosed on days indicated by black arrows except the 50 mg/kg SC dose which was dosed only on day 0. Data are mean ± standard error and mean from 10 animals per group. (D) IFN-β and (E) TNF-α levels in plasma (left) and tumor (right) over a 24-hour period post treatment with MSA-2 via IT, SC or PO routes. Data are mean ± standard deviation from 3-5 animals per group. It may be worth noting that, in general, cytokine levels exhibited somewhat faster decay rates in plasma than in tumor. (F-H) Effects of MSA-2 (indicated SC regimens) on tumor growth

70

kinetics (F), body weight (G) and tumor/plasma IFN-β levels at 4 hours post dose (H) in MC38 tumor bearing WT C57BL6 and STINGgt/gt Goldenticket (STING null phenotype) mice (20). Arrows indicate dosing frequency. Data are shown as mean ± standard error of the mean (SEM) from 10 animals per group for F-G and mean ± standard deviation (SD) from 4 animals per group for H.

71

Fig. S3. Effects of systemic (SC) and IT delivery of cyclic dinucleotide MSA-1 versus oral dosing (PO) of MSA-2 in MC38 tumor bearing mice. (A) Effects of indicated SC (red) and IT (teal) regimens of MSA-1 (×) on tumor growth volume (dosing frequency marked, ▲). Same experiments as shown in Fig. 3D-F; vehicle and single oral dose of MSA-2 (yellow) from Fig. 3F included for reference, others omitted for clarity. (B) Percent body weight change of mice following treatment with MSA-1 via SC (red) and IT (teal) delivery (n=10). Same experiments as shown in Fig. S2A-C; vehicle and single oral dose of MSA-2 (yellow) included from Fig. S2C for reference, others omitted for clarity. IL-6 levels in tumor (C) and plasma (D) in mice over 24 hours (n=3-5) following single SC (red) and IT (teal) doses of MSA-1 (×). Same experiment(s) as shown in Fig. 3I and 3J, respectively; single oral dose of MSA-2 (yellow) included for reference, others omitted for clarity. Statistical significance determined by one-way ANOVA. Data points in B,E represent mean ± SEM and C-D represent mean ± SD.

72

Fig. S4. Real time kinetics of MSA-2 binding to multiple STING isoforms in representative SPR experiments (green lines) fitted (black lines) with the models shown in Fig. 5C (see text,

73

methods). Relevant kinetic parameters shown represent n=1 (Models 1 and 2) or n=3 (Model 3, Table S5) data. Extended upper insets correspond to Model 3 (see Fig. 5F). (A) Human WT data (see Fig. 5F) fit to Model 1, note determined on-rate (ka, red) is outside the measurable limits of the instrument and the systematic deviations of the fit from the data. This theoretical ka value is also significantly lower than the lowest on-rate previously reported for a one-step binding reaction. It may also be worth noting that micromolar concentrations of MSA-2 were required to elicit detectable binding to cytosolic domain of hSTING-WT, but paradoxically, bound MSA-2 exhibited a slow off rate (t1/2 = 1.3 h) which is usually a characteristic of high affinity ligands. (B) Fit of human WT data (Fig. 5F, S4A) to Model 2 (see methods and Text S1). Replicate data (green) from two different channels are overlaid (note only slight bifurcation of the raw data signals at 100 µM). Specific deviations of the fit from the data are also annotated (black triangles). (C) Failed fit of human HAQ data to Model 1. (D) Successful fit of human HAQ data to Model 3 (see text, methods). (E) Failed fit of human H232 data to Model 1. (F) Successful fit of human H232 data to Model 3. (G) Failed fit of mouse WT data to Model 1. Note that top [MSA-2] is only 50 µM. (H) Successful fit of mouse WT data to Model 3.

74

Fig. S5. Simulated behavior of MSA-2 in saturation binding and homologous radioligand competition experiments, respectively, according to Model 2 (A-B) and Model 3 (C-D). The behavior of Model 2 and Model 3 in homologous competition experiments (B,D) were simulated using Eq. S10 and Eq. S29 with a fixed radioligand ([Lβ] = 0.2 µM) and the assumed equilibrium constants indicated in the figure. Simulated Scatchard plots were generated using the same equations with [Lc] (A) or [L] (C) set to zero.

75

Fig. S6. Plot of total concentration of either MSA-2 (green triangles) or compound 2 (orange inverted triangles) versus the corresponding concentration of the monomeric species (C), determined as described in methods. Solid line represents the best fit to the experimental data by iterative optimization (see methods) to estimate the equilibrium constant for MSA-2 homodimerization (KD1). The dashed line denotes the expected behavior of a purely monomeric compound.

76

Fig. S7. Results of ALIS (Automated Ligand Identification System) binding experiments probing the interaction of MSA-2 alone (green triangles) with hSTING-HAQ (A) and hSTING-WT (B) in negative MS mode. The solid line represents a non-linear regression fit to a 4-parameter curve with a variable slope. Values for the fitted parameters are shown in the figure inset. ALIS results for compound 2 alone (inverted orange triangles) with hSTING-HAQ and hSTING-WT (positive MS mode) are shown in (C) and (D), respectively. No binding was observed in the areas indicated by the orange brackets. (E) Results of ALIS assay titration of MSA-2 at a constant concentration of compound 2 (1 µM) with hSTING-HAQ. MS signals were normalized for each compound independently (each from their respective modes). The orange bracket denotes additional concentrations of MSA-2 sufficient to yield observable simultaneously bound MSA-2:2. See Text S2 for additional information.

77

Fig. S8. Additional data relevant to Fig. 6. (A) Continuation of chart from Fig. 6C. Values (n=2, except where noted) are reported as mean±SD. an=1. (B) Individual omit maps (dark blue mesh, contoured at 3 σ) for MSA-2 and each ligand in Fig. 6D. Ligands are depicted as sticks with green carbon atoms. In cases where crystallographic or non-crystallographic symmetry would place a second copy of a ligand atop or near the first, the second ligand is depicted with yellow carbon atoms. Any hydrogen atoms included in the model have been removed for clarity.

78

Fig. S9. Additional simulations regarding MSA-2 and measured cytokine levels in mouse tissues following MSA-2 treatment relating to Fig. 7. (A) Simulated amount of membrane permeable (neutral, solid line) and anionic (dashed line) MSA-2 (pKa = 4.78) as a function of pH. Note increase in percent of MSA-2 monomers in the neutral form with decreasing pH. Vertical lines and symbols (legend) are colored as results in Fig. 7. IL-6 (B) and TNFα (C) levels in various tissues of MC38 tumor bearing C57BL6 mice 4 hr after indicated dose of MSA-2 (SC or PO administration). Data points represent mean ± SD (n=5). Statistical significance determined by one-way ANOVA (Tukey’s multiple comparisons test vs. tumor).

79

Fig. S10A-D.

80

Fig. S10E-F.

Fig. S10. Anti-PD-1 antibody treatment combined with systemically dosed MSA-2 elicits tumor growth inhibition in a panel of syngeneic mouse tumor models. Individual tumor growth curves of (A) B16F10 (light green box), (B) LL-2 (gray box) (C) CT26 (dark yellow box) and (D) advanced MC38 (brown box) syngeneic tumor-bearing mice treated with vehicle, muDX400 (PD-1 mAb; injected intraperitoneally (IP), MSA-2 (SC or PO) or muDX400 combined with MSA-2 at indicated doses and dose intervals. Mean (±SEM) tumor volumes of (E) B16F10, (F) LL-2 (G) CT26 and (H) advanced MC38 syngeneic mouse tumors treated with indicated doses. Days of dosing are indicated by arrowheads (A-D) or triangles (E-F); blue for MSA-2 (SC), orange for MSA-2 (PO) and green for muDX400. Control IP injections are isotype controls (mouse IgG) matched to dose levels of muDX400 as shown (see Methods). *P<0.05, **P<0.01, ****P<0.0001 (one-way ANOVA; Tukey post-test) with n=10 mice per group.

81

Fig. S11. IL-6 induction (n=4) (A) and body weight loss (n=10) (B) in T-cell deficient NCr Nude, NSG, and normal C57BL mice following vehicle or MSA-2 (SC) administration. Days of dosing are indicated by solid triangles (black).

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Table S1. Activity of MSA-2 (10 µM) in an off-target assay panel. Assay Name Species % Inhibition Cholinesterase, Acetyl, ACES human -5 Cyclooxygenase COX-1 human -11 Cyclooxygenase COX-2 human -22 Monoamine Oxidase MAO-A human 6 Monoamine Oxidase MAO-B human -1 Peptidase, CASP8 (Caspase 8) human 3 Peptidase, CASP9 (Caspase 9) human 2 Peptidase, CTSG (Cathepsin G) human 7 Phosphodiesterase PDE3 human 11 Protein Serine/Threonine Kinase, MAPK14 (p38α) human 10 Protein Serine/Threonine Kinase, MAPK3 (ERK1) human -11 Protein Serine/Threonine Kinase, MARK3 human 29 Protein Serine/Threonine Kinase, PKC, Non-Selective rat -2 Protein Serine/Threonine Kinase, PRKACA (PKA) human 9 Protein Tyrosine Kinase, EGF Receptor human -7 Protein Tyrosine Kinase, Insulin Receptor human -27 Tyrosine Hydroxylase rat -9 Adenosine A1 human -14 Adenosine A2A human -3 Adrenergic α1A rat 4 Adrenergic α1B rat -6 Adrenergic α1D human 13 Adrenergic α2A human 19 Adrenergic α2B human -10 Adrenergic α2C human -5 Adrenergic β1 human 0 Adrenergic β2 human -5 Adrenergic β3 human -5 Androgen (Testosterone) human 14 Angiotensin AT1 human -2 Atrial Natriuretic Factor (ANF) guinea pig 6 Bradykinin B1 human 2 Cannabinoid CB1 human 7 Cannabinoid CB2 human 1 Chemokine CCR1 human -8 Chemokine CXCR2 (IL-8RB) human -3 Chemokine CXCR4 human -5 Cholecystokinin CCK1 (CCKA) human 0 Cholecystokinin CCK2 (CCKB) human -21 Corticotropin Releasing Factor CRF1 human -1 CysLT2 (LTC4) human 1

83

Dopamine D1 human -1 Dopamine D2S human -2 Endothelin ETA human 7 Endothelin ETB human -5 Estrogen ERα human 3 GABAA, Chloride Channel, TBOB rat 7 GABAA, Ro-15-1788, Hippocampus rat -15 GABAB, Non-Selective rat 8 Glucocorticoid human 11 Glutamate, Metabotropic, mGlu5 human -4 Glutamate, NMDA, Agonism rat 6 Glutamate, NMDA, Glycine rat -16 Glutamate, NMDA, Phencyclidine rat -9 Glutamate, NMDA, Polyamine rat 2 Glycine, Strychnine-Sensitive rat -6 Histamine H1 human -2 Histamine H2 human -8 Histamine H3 human 2 Histamine H4 human 16 IP (PGI2) human -3 Melanin-Concentrating Hormone MCH1 (SLC1) human 12 Melanocortin MC1 human 5 Melanocortin MC3 human -2 Melanocortin MC4 human -6 Melanocortin MC5 human -1 Muscarinic M1 human -7 Muscarinic M2 human -2 Muscarinic M3 human 0 Muscarinic M4 human 9 Muscarinic M5 human -8 Neuropeptide Y Y1 human 1 Neuropeptide Y Y2 human 9 Neurotensin NT1 human 5 Nicotinic Acetylcholine human -2 Opiate κ(OP2, KOP) human 1 Opiate μ(OP3, MOP) human -2 Orphanin ORL1 human 0 Platelet Activating Factor (PAF) human 5 Potassium Channel [KATP] hamster 0 PPARγ human 11 Progesterone PR-B human 0 Prostanoid EP1 human 0 Prostanoid EP3 human -19

84

Prostanoid FP human -5 Purinergic P2Y rat 7 Retinoid X Receptor RXRα human 14 Serotonin (5-Hydroxytryptamine) 5-HT1A human -5 Serotonin (5-Hydroxytryptamine) 5-HT1B rat 9 Serotonin (5-Hydroxytryptamine) 5-HT2A human -11 Serotonin (5-Hydroxytryptamine) 5-HT2B, [3H]Mesulergine human -1 Serotonin (5-Hydroxytryptamine) 5-HT2C human -12 Serotonin (5-Hydroxytryptamine) 5-HT3 human -2 Serotonin (5-Hydroxytryptamine) 5-HT6 human -4 Somatostatin sst2 human -12 Tachykinin NK1 human 20 Tachykinin NK2 human 4 Thyrotropin Releasing Hormone (TRH) rat 8 Transporter, Adenosine guinea pig 4 Transporter, Choline rat -15 Transporter, Dopamine (DAT) human -15 Transporter, GABA rat 2 Transporter, Norepinephrine (NET) human 15 Transporter, Serotonin (5-Hydroxytryptamine) (SERT) human 7 Vasopressin V1A human 0 Vasopressin V1B human -14 Vasopressin V2 human 11

Assay Name Species % Peroxisome Proliferator Activated Receptor PPARα (Agonism) human

0

Peroxisome Proliferator Activated Receptor PPARα (Antagonism) human

0

Counter-screening (n=1) was conducted for MSA-2 (10 uM) at Eurofins Panlabs Taiwan, Ltd. Assay methods were adapted from the scientific literature and were run with integrated reference standards. A significant hit was defined as ≥50% inhibition. Substantial negative inhibition can be caused by physical properties or insolubility of compound in final reaction conditions.

85

Table S2. Pharmacokinetics of MSA-2 in mouse.

Route Dose Cmax (µM) AUC (µM·hr)

MSA-2

IT 45 µg 3.6 1.355

150 µg 11.7 4.296 450 µg 42.6 15.22

SC 5 mg/kg 10.98 2.987

20 mg/kg 51.1 12.78 50 mg/kg 139.8 44.31

PO 20 mg/kg 21.7 7.62 80 mg/kg 60.9 28.76

200 mg/kg 291.7 249.6

86

Table S3. Summary of total buried Solvent-Accessible Surface Area (SASA, 1.4 Å probe radius) calculations for crystallographic STING complexes. Apo denotes ligand-free.

4KSY (cGAMP complex) SASA (Å2) apoprotein 16493 single ligand 759 dimer 16107 total buried area 1145 apo - complex 386

6UKM (MSA-2 complex) SASA (Å2) apo 15821 dimer of ligands 720 dimer 15494 ligand 1 518 ligand 2 518 total buried area 1047 apo - complex 327 buried area between ligands 316 same, but per-ligand 158

87

Table S4. Equilibrium constants of MSA-2 binding to full-length hSTING-WT and mouse STING.

Protein KD (µM2)a KD2 (nM)b [MSA-2]50 (µM)c

hSTING-WT 158.3 ± 13.0 (n = 3) 8.8 12.6

mSTING-WT 30.2 ± 4.2 (n = 3) 1.7 5.5

a- KD is overall equilibrium constant of the two-step reactions as defined by Model 3. KD was determined experimentally by homologous radioligand competition experiments as described in methods. b- KD2 is the equilibrium constant of the interaction between dimeric MSA-2 and dimeric STING. KD2 was calculated from KD using equation KD2 = KD/KD1 (KD1 = 18 mM). c- [MSA-2]50 is the concentration of MSA-2 required to achieve 50% STING occupancy. [MSA-2]50 was calculated from KD using equation [MSA-2]50 =�𝐾𝐾𝐃𝐃.

88

Table S5. Kinetic rate parameters of MSA-2 with various STING isoforms determined by SPR using Model 3.

Ligand ka2 (M-1s-1) a,b t1/2 (min) c KD2 (nM) d overall KD (µM2) e

[MSA-2] for 50% STING occupancy (µM) f

hSTING-H232 3.3 ± 0.2 × 104 0.57 ± 0.02 610 ± 50 11000 105 hSTING-WT 4.88 ± 0.02 × 104 78 ± 2 3.0 ± 0.1 54 7.3

hSTING-HAQ 6.11 ± 0.02 × 104 266 ± 46 0.7 ± 0.1 12.6 3.5 mSTING-WT 2.69 ± 0.02 × 106 11.6 ± 0.1 0.370 ± 0.004 6.7 2.6

a - The fitted association rate constant (on-rate) for the interaction of dimeric MSA-2 and STING. b - All parameters (n=3) determined based on an equilibrium constant (KD1) of 18 mM for the dimerization of MSA-2 in solution, as measured by NMR. c - Calculated as ln(2)/kd2. d - Equilibrium constant for the interaction of dimeric MSA-2 and STING (kd2/ka2). e - Product of the equilibrium constants for each step (KD1 × KD2). e - The square root of the overall KD is equal to the [MSA-2] at 50% STING occupancy based on Model 3. See text & Fig. 5C (gray box).

89

Table S6. Crystallographic data collection, processing, and refinement information Compound MSA-2 3 9 10 11 12 6 4 Type Monomer Dimer Dimer Dimer Dimer Dimer Dimer Dimer Linkage n/a C5-CCC-C5 C5-OCCCO-C6 C5-OCCCO-C6 C6-OCCCO-C6 C4-CCC-C6 C5-OCCO-C5 C5-OCCC-C5 Other modifications C4-F C4-F PDB code 6UKM 6UKU 6UKV 6UKW 6UKX 6UKY 6UKZ 6UL0 Data collection and processing

Beamline APS 17-ID SLS PXI-X06SA

APS 17-ID APS 17-ID APS 17-ID APS 17-ID SLS PXI-X06SA SLS PXI-X06SA

Wavelength (Å) 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 Space group C2 C2 P21 P21 P21 P41212 P21 C2 Cell parameters a, b, c (Å) 98.6, 61.2, 36.2 87.0, 73.2, 37.1 36.5, 109.4, 59.1 36.4, 109.5, 59.3 36.5, 109.3, 59.0 110.8, 110.8, 35.8 36.4, 109.1, 59.0 98.4, 61.5, 36.3 α, β, γ (°) 90, 103.7, 90 90, 102.3, 90 90, 95.7, 90 90, 95.8, 90 90, 95.5, 90 90, 90, 90 90, 95.4, 90 90, 104.6, 90 Diffraction limits (Å) (Highest resolution shell)*

55–1.738 (1.744–1.738)

42–1.682 (1.711–1.682)

59–1.831 (1.862–1.831)

59–1.974 (2.008–1.974)

59–1.933 (1.966–1.933)

55–1.950 (1.956–1.950)

36–1.522 (1.527–1.522)

48–1.762 (1.891–1.762)**

No. of observed reflections

64537 (642) 79100 (863) 138533 (6852) 100463 (5214) 116868 (5988) 209330 (2254) 237229 (2382) 55448 (766)

No. of unique reflections 19712 (189) 23503 (245) 40497 (1998) 31056 (1584) 34271 (1703) 16545 (169) 67964 (677) 16734 (837) Multiplicity 3.3 (3.4) 3.4 (3.5) 3.4 (3.4) 3.2 (3.3) 3.4 (3.5) 12.7 (13.3) 3.5 (3.5) 3.3 (2.1) Average I/σ(I) 12.1 (2.4) 19.5 (2.1) 13.9 (2.1) 8.7 (3.9) 12.0 (2.0) 13.3 (2.0) 20.2 (2.0) 12.2 (1.0) Completeness (%) 99.7 (94.5) 91.1 (99.2) 99.7 (99.9) 95.5 (97.7) 99.7 (99.8) 98.1 (100.0) 96.9 (99.7) 89.3 (41.6) Rmerge (%) 6.1 (56.9) 2.6 (48.9) 5.2 (52.8) 12.0 (83.4) 5.8 (56.7) 12.6 (135) 2.7 (52.8) 5.5 (74.3) Rmeas (%) 7.3 (67.9) 3.1 (57.9) 6.1 (62.8) 14.3 (98.6) 6.9 (66.9) 13.5 (142) 3.1 (62.5) 6.5 (120) Rpim (%) 3.9 (36.7) 1.7 (30.5) 3.3 (33.5) 7.7 (52.1) 3.7 (35.2) 3.7 (38.3) 1.7 (33.0) 3.5 (68.8) CC1/2 0.997 (0.742) 1.000 (0.891) 0.999 (0.741) 0.985 (0.549) 0.999 (0.763) 0.998 (0.595) 1.000 (0.754) 0.999 (0.435) Overall Wilson B (Å2) 30.2 34.0 27.8 28.9 30.8 33.8 25.7 26.9

Refinement Resolution used in refinement (Å)

48–1.74 19–1.68 27–1.83 25–1.97 59–1.93 55–1.95 22–1.52 48–1.76

No. of reflections 19658 23485 40481 30979 34271 16537 67946 16732 No. of reflections in Rfree set

1002 1190 2012 1540 1700 874 3388 821

Rwork (%) 17.9 20.3 18.9 21.0 18.9 20.1 19.9 21.2 Rfree (%) 19.2 22.1 20.8 24.1 21.7 22.5 21.3 24.2 RMSDs Bonds (Å) 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.010 Angles (°) 1.01 0.99 0.096 0.096 1.00 1.03 1.01 1.12

90

No. of STING monomers per asymmetric unit

1 1 2 2 2 1 2 1

Average B (Å2) / No. of non-H atoms Protein 38.5 / 1448 42.3 / 1497 32.7 / 3125 31.3 / 2814 36.0 / 2845 37.6 / 1404 31.1 / 2878 38.3 / 1442 Waters 43.3 / 111 47.9 / 124 40.1 / 289 35.4 / 232 45.4 / 303 46.3 / 136 41.9 / 364 37.6 / 76 Ligand 21.6 / 20 25.4 / 39 17.4 / 82 16.6 / 84 21.3 / 41 20.8 / 39 16.9 / 82 17.9 / 40

Model validation Ramachandran analysis (%) Favored 97.1 97.7 97.4 97.6 96.8 97.6 98.0 96.0 Outliers 0.6 0.0 0.0 0.0 0.3 0.0 0.0 0.0 MolProbity (after automatically adding missing hydrogens at electron-cloud positions) MolProbity score / percentile

1.42 / 95th 1.33 / 97th 1.26 / 99th 1.31 / 99th 1.68 / 89th 1.72 / 88th 1.39 / 92nd 1.41 / 96th

Clashscore / percentile 2.07 / 99th 2.32 / 99th 3.5 / 98th 2.82 / 99th 3.34 / 99th 3.19 / 99th 3.27 / 98th 2.41 / 99th

* Values in parentheses define the boundaries of the highest-resolution shell. Subsequent parenthetical values reflect calculations for reflections within that shell. ** Data processed using the STARANISO methodology (39)

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