A.; Massa, A.; Perfetto, B.; Donnarumma, G.; Schiraldi, C.; Tufano,
M. A.; de Rosa, M.; Filosa, R.; Hay, A. Antiviral Res. 2013, 99, 125–
135.
8.2, 7.7 Hz, 1H, H13), 6.83 - 6.81 (m, 1H, H14), 6.79 (ddd, J = 7.7, 1.8,
0.9, 1H, H10), 6.70 (dd, J = 8.2, 1.8, 0.9 Hz, 1H, H12), 4.70 (s, 2H,
SCH2), 4.26 (q, J = 7.1 Hz, 2H, CO2CH2CH3), 3.64 (s, 3H, NCH3),
1.39 (t, J = 7.1 Hz, 3H, CO2CH2CH3). δC (150 MHz, MeOD) 166.9,
158.9, 150.6, 145.5, 136.4, 133.6, 131.0, 130.7, 128.0, 124.9, 120.5,
116.0, 114.8, 107.9, 104.8, 60.8, 30.5, 30.4, 14.8. Rf: 0.45 (1% MeOH
in CH2Cl2). HRMS (ESI-TOF): C19H18BrNO4S ([M+H]+) requires
436.0213, found 436.0215
11. Balzarini, J.; Ruchko, E.; Zakharova, E.; Kameneva, I.; Nawrozkij, M.
Chem. Hetero. Comp. 2014, 50, 489–495.
12. Zotova, S. A.; Korneeva, T. M.; Shvedov, V. I.; Fadeeva, N. I.;
Leneva, I. A.; Fedyakina, I. T.; Khristova, M. L.; Nikolaeva, I. S.;
Peters, V. V.; Guskova, T. A. Pharm. Chem. J. 1995, 29, 57–59.
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Ciliberto, G. Bioorg. Med. Chem. 2010, 18, 6143–6148.
15. Scuotto, M.; Abdelnabi, R.; Collarile, S.; Schiraldi, C.; Delang, L.;
Massa, A.; Ferla, S.; Brancale, A.; Leyssen, P.; Neyts, J.; Filosa, R.
Bioorg. Med. Chem. 2017, 25, 327–337.
22. Ethyl
6-bromo-4-((dimethylamino)methyl)-5-hydroxy-2-(((3-
hydroxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate
11. Meta-hydroxy Indole 8 (30.0 mg, 0.069 mmol, 1.0 eq.) and
N,N,N’,N’-tetramethyldiaminomethane (47.0 µL, 0.344 mmol, 5.0 eq.)
were dissolved in CH2Cl2 (30 mL). The reaction was heated to reflux
for 3.5 h before removing the solvent in vacuo to yield the title
product as a pale yellow solid (34 mg, 99%). NMR: δH (500 MHz,
CDCl3) 7.47 (s, 1H, H7), 7.12 (t, J = 7.9 Hz, 1H, H13), 6.90 (d, J = 7.9
Hz, 1H, H14), 6.90 (d, J = 7.9 Hz, 1H, H12), 6.66 (s, 1H, H10), 4.41 (s,
2H, CH2NMe2), 4.34 (s, 2H, CH2SPh), 4.15 (q, J = 7.1 Hz, 2H,
CO2CH2CH3), 3.60 (s, 3H, NCH3), 2.55 (s, 6H, CH2N(CH3)2), 1.33 –
1.21 (m, 3H, CO2CH2CH3). δC (150 MHz, CDCl3) 165.9, 156.7,
150.9, 142.6, 135.1, 132.2, 131.0, 130.0, 128.9, 124.6, 124.3, 119.3,
115.5, 113.4, 108.6, 106.3, 60.8, 58.7, 44.0, 30.4, 29.9, 14.3. Rf: 0.15
(10% MeOH in CH2Cl2). HRMS (ESI-TOF): C22H25BrN2O4S
([M+H]+) requires 493.0791, found 493.0792.
16. Nasser, Z. H.; Swaminathan, K.; Müller, P.; Downard, K. M. Antiviral
Res. 2013, 100, 399–406.
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2009, 81, 132–140.
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206–214.
19. General experimental. Unless otherwise indicated, all reagents were
obtained from chemical suppliers with no further purification. Sodium
bicarbonate refers to a saturated solution of sodium hydrogen
carbonate in water. All water used was either distilled using a
Millipore MilliQ® water purifier with Q-Gard® 2 column and 0.22
µM filter from Millipore or used directly from a bottle of HPLC-grade
water. All reactions were carried out in closed systems under Argon.
NMR spectra were recorded using a Bruker AVIII HD-600, DRX-
500, AVIII-400 and DPX-400 spectrometer (600 MHz, 500 MHz, 400
MHz and 400 MHz, respectively) and all samples were dissolved in
deuterated chloroform unless otherwise stated. Offline data processing
was carried out using the MestreNova software. Chemical shifts (δ)
are given in ppm units relative to tetramethylsilane and coupling
constants (J) are measured in Hertz. Proton (1H) NMR multiplicities
are shown as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (double doublet), dt (double triplet), dq (doublet of
quartets), dt (doublet of triplets), tt (triplet of triplets), br s (broad
singlet), br d (broad doublet). HRMS refers to high resolution mass
spectrometry. Electrospray ionization (ESI) accurate mass was
determined using a ThermoFinnigan LTQ Ion Trap. Flash column
chromatography was carried out using silica gel with particle size <60
µm and reverse phase column chromatography was carried out using
silica gel 60 silanized (53-200 µm). Thin layer chromatography (TLC)
was performed on aluminium backed Sigma-Aldrich TLC plates with
F254 fluorescent indicator. Developed plates were air dried and
analysed under a UV light or by staining with the appropriate
indicator.
23. Ekiert, D. C.; Kashyap, A. K.; Steel, J.; Rubrum, A.; Bhabha, G.;
Khayat, R.; Lee, J. H.; Dillon, M. A.; O'Neil, R. E.; Faynboym, A. M.;
Horowitz, M.; Horowitz, L.; Ward, A. B.; Palese, P.; Webby, R.;
Lerner, R. A.; Bhatt, R. R.; Wilson, I. A. Nature 2012, 489, 526–532.
24. Wu, N. C.; Xie, J.; Zheng, T.; Nycholat, C. M.; Grande, G.; Paulson, J.
C.; Lerner, R. A.; Wilson, I. A. Cell Host Microbe 2017, 21, 742–748.
25. Biolayer interferometry data and analysis. The Kd for ligand
binding to influenza HA was determined by BLI using an Octet Red
instrument (ForteBio). Biotinylated HAs, purified as described
previously,23 were used for these measurements. HAs at ~10–
50ꢀµgꢀml−1 in 1× kinetics buffer (1ꢀ×ꢀPBS, pH 7.4, 2% DMSO and
0.002% Tween 20) were loaded onto streptavidin-coated biosensors
and incubated with varying concentrations of small molecule in
solution. All binding data were collected at 30ꢀ°C. The experiments
comprised five steps: (1) baseline acquisition (60ꢀs); (2) HA loading
onto sensor (1800ꢀs); (3) second baseline acquisition (120ꢀs); (4)
association of small molecule for the measurement of kon (180ꢀs); and
(5) dissociation of small molecule for the measurement of koff (180ꢀs).
Baseline and dissociation steps were carried out in buffer only. The
ratio of kon to koff determines the Kd reported here and is subtracted
from a reference well to remove the effects of non-specific binding, as
previously described.24 The R2 for the model fitting and the standard
error of mean (SEM) for the Kd values reported in Table 1 were
computed by Octet Data Analysis software version 9.0 (ForteBio).
20. Synthesis of Arbidol analogues: Ethyl 5-acetoxy-6-bromo-2-(((3-
hydroxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate
8a. 3-hydroxythiophenol (117 µL, 1.15 mmol, 1.0 eq.) was added to a
solution of sodium carbonate (367 mg, 3.46 mmol, 3.0 eq.) and bromo
indole 2 (500 mg, 1.15 mmol, 1.0 eq.) in dry ethyl acetate (10 mL).
The reaction was heated to 100 °C and stirred for 5 h before cooling,
filtering and removing the solvent in vacuo. The compound was
purified by column chromatography (40% EtOAc in Hexanes) to
produce the title product as a pale yellow solid (240 mg, 44%). NMR:
δH (500 MHz, CDCl3) 7.85 (s, 1H, H7), 7.56 (s, 1H, H4), 7.12 (t, J =
7.9 Hz, 1H, H13), 6.95 – 6.90 (m, 1H, H14), 6.78 (s, 1H, H10), 6.75-
6.71 (m, 1H, H12), 4.69 (s, 2H, SCH2), 4.30 (q, J = 7.4 Hz, 3H,
CO2CH2CH3), 3.66 (s, 3H, NCH3), 2.40 (s, 3H, COCH3), 1.38 (t, J =
7.4 Hz, 3H, CO2CH2CH3). δC (150 MHz, CDCl3) 169.8, 165.0, 156.1,
144.6, 143.3, 135.6, 135.1, 130.1, 126.1, 124.8, 119.3, 116.2, 115.3,
113.9, 111.1, 105.8, 60.1, 30.4, 29.9, 21.0, 14.6. Rf: 0.45 (30% EtOAc
in Hexane). HRMS (ESI-TOF): C21H20BrNO5S ([M+H]+) requires
478.0318, found 478.0317
21. Ethyl 6-bromo-5-hydroxy-2-(((3-hydroxyphenyl)thio)methyl)-1-
methyl-1H-indole-3-carboxylate 8. Sodium carbonate (106 mg, 1.00
mmol, 2.0 eq.) was added to a stirred solution of meta-hydroxy indole
8a (240 mg, 0.502 mmol, 1.0 eq.) in methanol (40 mL) and left to stir
for 2h. The solution was then filtered and the solvent removed in
vacuo. The product was re-dissolved in ethyl acetate (10 mL) and
washed once with water (10 mL) before drying (Na2SO4) and
concentrating in vacuo to give the title product as a white solid, which
could be used without further purification (160 mg, 67%). NMR: δH
(600 MHz, MeOD) 7.60 (s, 1H, H7), 7.58 (s, 1H, H4), 7.07 (dd, J =