WO2018112128A1

WO2018112128A1 - Arbidol analogs with improved influenza hemagglutinin potency

Info

Publication number: WO2018112128A1
Application number: PCT/US2017/066275
Authority: WO
Inventors: Ian A. Wilson; Dennis W. WOLAN; Zoë V .F. WRIGHT; Rameshwar U. KADAM; Nicholas C. WU
Original assignee: The Scripps Research Institute
Priority date: 2016-12-16
Filing date: 2017-12-14
Publication date: 2018-06-21

Abstract

The invention provides a series of analogs of arbidol having enhanced binding activity with respect to influenza hemagglutinin. Accordingly, the invention can provide a method of inhibiting the bioactivity of viral hemagglutinin activity, which is an essential step in the entry of infectious viral particles into host cells. The invention also can provide a method of treatment of influence, comprising administering an effective amount of a compound of formula (A), wherein X is S or O, to a patient afflicted therewith.

Description

Arbidoi Analogs with improved irtfiuenza Hemagglutinin Potency

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. provisional application serial number 62/435,232, filed Dec 16,. 2016,. the disclosure of which is incorporated by reference, herein in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under 1 R56 Al 1.17675-01 awarded by the National institute of Allergy and infectious Diseases. The government has certain rights in the. invention.

BACKGROUND influenza-is a highly-contagious respiratory viral infection which annually affects -5-20% of the US population leading to nearly 200,000 influenza-related hospitalizations per year and approximately 3,000-49,000 deaths per annum. The 2008 pandemic demonstrated the speed with which the infection can spread across the globe and highlighted the eonsiderable limitations on current protection strategies. Whilst vaccines have produced. some control over the risk of Infection, rapid antigenic drift makes the selection of strains an annual challenge. There_, are currently four licensed drugs available for treatment use in the US once, infection occurs; the 2 ion channel inhibitors- Amantadine (Symmetrel^®) and Rimantadine (Fiumadine®), and the neuraminidase (NA) inhibitors Osetlamivir (Tamiflu^®) and Zanamivir (Reienza^¾¾. However, as new strains emerge showing resistance to these drugs, the quest for small molecule therapeutics with novel modes of action becomes more urgent. We herein describe our efforts towards the optimization of the antiviral drug Arbidoi against influenza with the hope of

developing a new drug capable of treating; the infection.

Arbidoi (Umifenovir) is a broad-spectrum antiviral against a number of viruses, including influenza, Ebola and hepatitis B & C, Despite the lack of a m_.echani_.sm-of-action against any target virus, Arbidoi is clinically used- in Russia and China and is. currently in phase IV US clinical trials {clinicaitriais.gov/ct27show/NC-T01651663}. One major drawback to the use of Arbidoi is. the large dose required to achieve therapeutic efficiency. Several groups have tried to improve the therapeutic potential by changing the substituents decorating the indole core (Figure 1). These have included changes to the nitrogen substltueni, hydroxy grou in position 5, the bromo grou in position 6, and changes to the thiophenol in position 2. However, despite the large number of structure-activity relationship studies carried out to date, none of the compounds have shown a vast improvement in efficacy over their parent compound Arbidoi. We recently published structures of Arbidoi in complex wit the influenza viral fusion protein hemagglutinin (HA) from pandemic 1968: H3N2 and the recent 2013 H7N9 viruses. Our data shows that Arbidoi binds a conserved hydrophobic cavity at the interface of the HA protomers within the upper stem region. The structures demonstrate the. molecular mechanism of antiviral activity, as non-covalent interactions to Arbidoi stabilize the pre-fusicn conformation of HA and prevent the conformational rearrangement required fo membrane fusion and subsequent infection of the host cell Using our structural data, we: observed that_. Arbidoi binds tightly to the pocket along the edge of the indole core at positions 1 , 5 and 6 (Figure 1_.) and explains why altering groups at these positions had a deleterious effect on the antiviral efficacy of those molecules. Our data aiso. showed that there was additional space within the pocket not fully- optimized for binding to the protein by the amino group in position 4 and the thiophenol group at position 2. Using our structural data, we designed and synihesiseti several molecules to take advantage of ^'this, to improve Arbidoi binding. We herein report the first Arbidoi analogue with significantly- improved binding to HA in comparison to the parent compound. We then compared this compound to Arbidoi in a fusion assay and demonstrated both molecules exhibit the same inhibitory effect on membrane fusion, Our study provides new insights into how to manipulate compounds to bind to the influenza virus and presents excising evidence for a possible new influenza therapeutic.

S U !A ,!R

The invention provides, in various embodiments, a method of inhibiting the bioactivily of a virai hemagglutinin (HA) molecule that is displayed on the susface of the influenza virus, comprising contacting the hemagglutinin molecule -with an effective concentration of a

wherein X is S or O. For instance, the compound of formula (A) can be the compound of formula 11

Further, in various em odiments, the invention provides a method of treating or preventing an influenza viral infection in a patient, comprising administering to the patient an rmula (A)

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Structure of Arbidoi.

Figure 2: Synthesis of Arbidoi and Analogues. Reagents and Conditions: {!) acetic anhydride, pyridine (il) Mel, NaH, D F (iii) 8r₂, CC (iv) thiophehbi, OH, MeOH (v) ^'Ν,Ν,Ν',- Ν'- tetramethyidiaminomethane, 1 ,4-dioxane (yi) mine, 1 ,4-dioxane (vfi) R"SH, KOH, MeOH Figure 3: Analogues of Arbidoi

Figure 4: BLi measurements of Arbidoi Analogues

Figure 5: Additional Analogues of Arbidoi based on 11. Compound 20 could not be synihesised using the synthetic route described as amine addition to the phenol was observed as well as amine addition to the indole.

Figure 6: Trypsin susceptibiisiy (TS) assay performed on compound 11 to determine if binding to. HA would have a similar effect to that of Arbidoi DETAILED DESCRIPTION

Synthesis of Arbidoi Analogues:

Our structural data showed that there was space. In .the -binding pocket to accommodate changes to both the thiophenoi group at position 2 and the amine group at position 4. To investigate the SAR, we optimised the original route to synthesize Arbidoi to allow multiple analogues to be made from the common dibrominated intermediate (2) (Figure 2).

Crystaiiographic data highlighted the importance of a water molecule in the binding pocket in the .meta position with respect to the thiophenoi group. To. exploit this potential interaction with HA, we added either an amino or hydroxy -group at the meta position, as well as extending the size of the ring to investigate the effect of increased conjugation.. To investigate the importance of the amino group, these analogues were synthesized with and without the presence of the amine- in position 4 on the indole, as well as replacing the amine with a piperazine to see if any further interactions towards the back of the binding pocket could be beneficial.

The synthesis began with the orthogonal protection and double bromination of the commercially available indole core (1 ) to give 2. Here the synthesis diverged to provide two separate sets of analogues; in route 1, thiophenoi is added to give intermediate 3, followed ^'by reaction with /V.^ A/'^W'-t^'etramethyidiaminomethane to give Arbidoi (4). Reaction with two piperazine analogues gave compound^'s 14 and 15. in route 2, various thiols were reacted to give intermediates 8 ~ 10. These were again reacted with N,N,N^!..N'~

tetramethyidiamtnometh-an-e to generate analogues 11 - 13 (Figure 3),

Evaluation of Kinetics using Βίο-tayer Interferometry:

To investigate the binding affinity of the compounds to hemagglutinin, Ka was determined by bioiayerinterferomeiry (BLI) using an Octet Red instrument (ForteBio),

Biotinylated HAs, purified as- described previously, were, used for these measurements-. HAs at -10-50 ί-sg ml^"1 in 1 ^χ kinetics buffer (1 ^χ PBS, pH 7.4, 0.01% BSA and 0.002% Tween 20) were loaded onto streptavidin-eoated biosensors and incubated with varying concentrations of small molecule in solution. The ratio- of fcn to k determines the % reported here.

Arbidoi Is a broad-spectrum antiviral which can be used, to treat influenza infection from both group 1 and group 2 viruses. To test the efficacy of our Arbidoi analogues, each compound was assessed for binding against one HA from group 1 (PR8; A/Puerto Rico/8/1934) and one from group 2 (HK68: A/Hong Kong/1 /68).

As with previous Arbidol-based compounds, many of our analogues did not show an improved affinity for HA over the parent compound; increasing the size of the group at indole position 4 from an amine to a piperaztne did not improve binding to H3 and indeed showed a decreased Kd fo Hl (Figure 4, Entries 9 and 10). Similarly, increasing the size -of the^' aromatic ring did not generate improved interactions, nor did the addition of a meta-NHs with respect to the thiol (Figure 4, Entries 4, 5, 7, and 8). Of particular interest was the observation that the binding affinity appeared to improve slightly when the amino group at indole position 4 on

Arbido! was removed, which was not predicted by the structural data (Figure 4, Entries 1 and 2), The most interesting analogue of all was compound 11 (Figure 4, Entry 6) which showed a 100- fold increase in binding with respect to Arbsdoi against HI and a 1000-fold increase in binding again&t H3. it is Interesting to note, that contrary to- compounds 3 and 4, here removal of the amine at position 4 decreases binding, with compound 8 showing only a threefold increase in binding against HI and a fivefold increase in binding against H3 (Figure 4, Entry 3).

Evaluation of the Structure-Based Drug Design Strategy:

Ou crystallography data predicted that b putting a hydroxy group in the meta-po.sillon, it would be possible to increase the binding by displacing a water molecule in the binding pocket. To show that our -approach to structure-based drug design was correct, the next compounds to be synthesised were the.orthor- and: para-hydroxy analogues (Figure 5). It was predicted that these would have a decrease in binding affinity with respect to the meta analogue as the compounds would, not be able to: form the hydrogen bond with Leu-98..

i was also decided. to test the efficacy of the meia-rnethoxy compound, which has shown promise in the treatment of Hepatitis B. To further investigate the effect of the amine substitution in_. position 4, analogues_, were . synthesised with and without this moiety. Compound 20 could not be synthesised using the designed route as double addition with the amine adding to both the indole and the para-hydroxy ring was observed {Figure 5).

The compounds were again tested in the HA assay using bio-layer interferometry with HI and H3_. representing group 1 and group 2 viruses. As predicted by our crystal structure, no binding was observed with eithe the ortho- or para-hydroxy substituted rings. The most interesting result is that replacing the meta-hydroxy group- ith a roethoxy substituent also did not exhibit any binding. Investigation into the Mechanism of Action;

The mechanism-of-action of Arbidol is to prevent the low pH induced conformational change in HA through stabilization of the pre-fusipn conformation of HA._. Trypsin susceptibilit (IS) assay ^'was perf ormed on compound 11 to see if binding to HA woufd have a similar effect to Arbidol. At pH 5, HA is readily converted into a protease-susceptible post-fusion form {Figure 6, Lanes 1 and 2), Arbidol has been shown to prevent conversion of P^{' '}8 HA at pH 5 and thereby prevent^' trypsin digestion. Compound 11 was found to show the -same effect (Figure 6, Lane 4), which ^'was consistent with the result observed for the fusion-inhibiting stem targeting antibody GR911 Fab (Figure 6, Lane 3).

With the assistance of structure- based drug design, we have been able to further probe the structure-activity relationship of the binding site of Arbidol on the influenza HA_. fusion protein. Previous work by other groups has shown that many substitutions do not lead to increased binding affinity of analogues with respect to Arbidol. A com arison of the previous structural changes- with our crystal -data showed that ail previous attem ts^, at_. improvement interfered with key: interactions the molecule makes within the hydrophobic binding pocket, in particular,_, substitutions at indole positions 5 and 6 interfered with the binding to Y9 and K310, Our work aimed to take advantage of a structured water molecule bound next to Arbidol within our HA co- complex structures, with the hope that exploiting these key interactions would increase the binding of Arbidol analogues. Replacement of the thiophenoj ri g with a meta- hydroxythiophenol to form 11, generated an impressive 100-fold and 1000-fold increase in affinity against H1 and H3, respectively. To see if the initial prediction based on the structure was correct, subsequently synthesized ortho- and para-substituted miophenois had no detectable binding capacity, as measured by bio-layer interferometry assay. The meta-methoxy compounds 20 and.21, which showed -promise against Hepatitis B were also assessed and lacked ability to. interact with HAs H1 and H3, Our highly improved Arbidol analogue and results clearly show that the replacement of the structured wafer molecule with a hydrogen bond donating moiety that interacts directly with, the HA binding pocket vastly - increases binding affinity and broadly to H I and- H3 HAs. Our optimized Arbidol represents the first compound with such a dramatic increase: in affinity.

The Trypsin susceptibility assay (TS) confirmed that compound 11 acted in the same way as ArbidoL namely by -stabilizing the pre-fusion complex and thus preventing fusion from occurring. Currently, the only treatments for influenza involv either inhibiting the M2 Ion channel inhibitors or neuraminidase. These mechanisms have been shown to be prone to antigenic drift and indeed resistance to these drugs has already been reported in th clinic. Arbidoi has been shown to have both a novel mechanism of action and a novel binding site but needs to be admiriistered in fairly high doses to achieve therapeutic concentrations in blood plasma. As compound 11 shows a much higher binding affinity in the BLi assay, it is possible that the dosage required to reach peak therapeutic efficiency would be far less with respect to the parent compound.

Documents cited

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Pec_.heur .Ei, etal. {2016) The Synthetic antiviral drug arbidoi inhibits giobaiiy prevalent pathogenic viruses. J. Virol. 90{δ):308δ~3092.

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Balzarini, J., et ai: (2014) Structural Analogs of Uniifenovir. 1 « Synthesis and .biological activity of ethyl S-hydrpxy-1 -methyS-2~(/f ar;s-2-phersylcyclopropyl)-1 H~indole-3~carboxyiate. Chemistry of

Heterocyclic Compounds, 50 4):489-495,

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Ghai, H„ et ai. (2006) Synthesis and in vitro anti-hepatitis B virus activities of. some, ethyl 6- bromo-5_.-hydroxy-1 H-lndofer3~carboxyiates. Bioorg. Med. Cham., 14:911-917,

Kadsm, R, U. & Wi!son, I. A. (2016) Structural, basis of influenza virus fusion inhibition by the antiviral drug arbidoi, in press. Ekieri, D. C; Kashyap, A. K.; Steel, J.: Rubrum, A,; Bhabha, G.; Khayat, R.; Lee, J, H.: Dillon, . A.; O'Neii, . E.; Faynboym A. .; Horowitz, M,; ^'Horowitz, L.;- Ward,_. A. B.; Paiese, P.; Webby, R.; Lerrser, R. A.; Bhatt, R. R.; Wilson, I. A., Nature, 489, 526-532 (20/12)

Ekiert DC, et a!. (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324(5924): 46-251 ,

Dreyfus C, et al. (20 2) Highly conserved protective epitopes on influenza B viruses. Science 337(6100): 1343-1348.

Chai, H.; Zhao, Y.: Zhao, C.; Gong, P., Bioorg. Med. Chem., 14, 911-917 (20D6)

Brancato, V,; Peduto, A.; Wharton, S.; Martin, S.; More, V.; Di ola, A.; SV assa, A.; Perfetto, B.; Dorinarumrrsa, G.; Schlraldi, C; Tufa no, M. A.; de Rosa, .; Fiiosa, R.; Hay, A,_., Antiviral Research, 99, 125-135 (2013)

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Ekiert, D. G.: Kashyap, A. K.; Steel, J„; Rubrum, A.; Bhabha, G.; Khayat R.; Lee, J. H.: Dillon, M. A.; O'Neii. R. £.; Faynboym A, ,; Horowitz.,_. .;· Horowitz, L; Ward, A. B.; Patese, P..;_. Webby. .; Lefner,_. R, A.; Bhatt, R. R.: Wilson, I. A., Nature.. 489, 528-532 (2012)

All patents and publications referred to herein are Incorporated by reference herein to the same extent as if each individual publication was specifically and Individ uaiiy indicated to be incorporated by ref rence in Its entirety.

EXAMPLES

c

Original Synthesis

internfsediate 8 Afbki ;i (soW as the HGi salt)

Intermediate A Com ound 5 Co!Tspoisf¾d 6 fVfatersals and Methods:

General Experimenta : 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 Miliipore Mil HQ® water purifier with Q-Gard® 2 column and 0,22 μΜ filter from mif!ipore or used directly from a bottle of HPLC-grade water. Ail reactions were carried out in closed systems under Argon. NM.R spectra were recorded using a Bruker AVili HD-600, DRX-500, AVI 11-400 and DPX-40G spectrometer (600 MHz, 500 MHz, 400^' MHz and 400 MHz, respectively) and ail samples were dissolved in deuiera ted chloroform unless otherwise stated. Offline data processing was carried put using the Mest^'reN.ova software^". Chemical shifts (5) are given in; ppm units- relative to tetramethylsilane and coupling constants (J) are measured in Hertz.

Proton (1 H) NMR multiplicities are shown as s (singlet), d (doublet), t (triplet), q (quartet), m (mu!tiplet)^', dd (double doublet), dt {double triplet), dq (doublet of quartets), dt (doublet of triplets), ti (triplet of triplets), br s (broad singlet), br d (broad doublet). MS refers to low resolution mass spectrometry and H RMS. refers to high resolution mass spectrometry.

Eiectrospray 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 rn- and reverse phase column .chromatography was carried out using silica gel 60 silanized (53-200 μηι). Thin layer chromatography (TLC) was performed on aluminium backed ^' Sigma-Aldrich TLC plates with. FIM fluorescent indicator: Developed plates were air dried and analysed under a. UV light or by staining with the appropriate indicator.

Arfojdel Core Synthesis

Ethyl^' 5-aceioxy-2-methyM tf-indo¼^»3^«earboxylate 1a: Acetic anhydride (25,9 ml, 274 mmc- 20 eq.) was added to a stirred solution of ethyl 5-hydroxy-2-methy|-1 W-lndole-3-carbo ylate 1 (3.00 g, 13.6 mmoi, 1 ,0 eq.) in pyridine (3,32 mL, 41 .1 mmoi, 3.0 eq,) and the reaction heated to reflux. After 1 h, the reaction was allowed to cool back to rt before pouring- the mixture into a solution of aqueous saturated sodium bicarbonate (40 mL). The product .was. extracted with ethyl acetate (3 x 40 ml) and the combined organic layers^, were washed with wate (40 mL),

to yield the product as a white solid which was used without. further purification (3.4 g, 96%). NM : δ» (40Q MHz, CDCls) 8.34 (1 H, s; H), 7.75 (1H, s, «»}, 7.21 (1 H , d, J 8.5, He), 6.89 (1 H, d, J 8.5, ₇\ 4.38 (2H, q, J 7.1 , C0₂CH₂CH₃), 2.71 (3H, e, CiCH₃), 2.34 (3H, s, Cq_aCH₃), 1.43 (3H, t, J 7.1 , CO₂0H₂C«₃). «c (100 MHz, CQCb) 170.8 (CG2CH3}, 165.8 (COaEt), 1.45.9 (Gs), 145.3 (G₂), 132.4 (C₈). 127,9 {C₃), 116.3 (C₆), 113.8 (C<>, 111.2 <G₇), 99,8 (C), 59.7 (Ρρ₂ΡΗ₂ΡΗ₃), 21.4 {CiCH₃). 14,8 {PQ₂PH₂CB₃}, 14.4 (CO2CH3). R_f: 0,31 (40% ethyl aceiaie in hexane), HR S. (ESI-TGF):

found 262.1074.

Ethyl 5-acetoxy-1₃2-dimethyi-1H-m iofe-3-carboxy at® 1b: Protected indole 1b (1.35 g, 5.17 mmoi, 1 eq.) was dissolved in DMF (15 mL). To this solution, methyl iodide (.0.965 ml, 15.5 mmoi, 3.0 eq.) was added and the resulting mixture was cooled on ice. Sodium hydride {0.186 g, 7.75 mmoi, 1.5 eq.) was added and the reaction was left to stir on ice for 1.5 h. After this time, a small amount of water (5,0 n l.) as added to the reaction and the solvents removed in vacuo. The resultant brown oil was then purified directly by column chromatography (30% ethyl acetate in petrol) to yield the title compound as a pale yel ow solid (1.50 g, 95%}. HWH: ¾ (500 MHz.. CDCI3) 7.79 (s, 1 H, H₄)_> 7.26 (m, 1 H, H₆), 6.96 (ddd, J = .8.8, 2.4, 0.8 Hz, 1H, H₇), 4,38 (f, * 7.1 Hz, 2H, COZCWJCHS),. 3.69 (s, 3H, NCH.j), 2.77 {d, J = 1.3 Hz, 3H, ΑΐβΗ₃), 2.33 (s, 3H, COaC s), 1.43 (t_s J-~ 7.1 Hz, 3H, C0₂CH₂C,¾). 5_C (150 MHz, CDCi₃) 170.5 (CO2CH3), 166.0 (COaEt), 146.5 (C₅)_t 146.0 (<½>, 134.5 (C₈), 127.2 (C₃), 116.2 (Ce), 114.0 {€«}, 109.6 (C₇), 104.4 (C_f), 59.6 {COsCHaCHa), 29.9 (NCH3}, 21.3 (C CH₃), 14.8 (C0₂CH₂CH₃), 12.1 (C0₂CH₃). _fi 0.4 {30% ethyi acetate in hex-arse),. HR S (ESt-TGF):

276,1230, found 276.1229.

Ethyi 8-bromo-2»(bromome hyS)-5~hydroxy-1-met.hy ~1 ^Mndole-S-carbox late 2: Bromine (558 p.L, 10.9 mmoi, 3.0 eq.) was. added to a stirred solution of protected indole {1b, 1 ,00 g, 3,63 mmoi, 1.0 eq.) in carbon tetrachloride (100 mL). After refluxing for 16 h, the reaction was cooled and aqueous sodium thlosuiphate (10%. w/v, -100 mL) was added and left to stir for 20 mfrv until the orange colour disappeared. After this time, the organic layer was separated, washed with water (2 x 100 mL), dried (Na₂S0 and concentrated in vacuo to yield a pale yellow solid, which was used without further purification {1 ,40 g, 99%). UMR δ_Η (400 MHz, PDCb) 7.86 {1 H, s, b l 7,54 (1 H, s, H , 5.05- (2H, s, C½Br), 4.41 {2H, q, J 7.1 , COaCHaCHg), .3.69 (3H, .s, HCH3), 2.39 (3H, s, GO2CW3), 1.45 (3H, t J 7,1 , C0₂CH₂CH-i), 5_C {100 MHz, CDCi₃)

169.4 (GQ₂CH-3), 164.3 (C0₂Ei), 143.4 (C₅), 142.6 (C₂), 35.5 (C₃), 125.7 {G₈}, 116.3 (C₄), 1 13.9 (G_?), 1 12.0 (C₆), 05.6 (C,), 60,3 (Ρ0₂ΡΒ₂ΡΗ₃), 30.1 (NPHs), 20.9 (C0₂CH₃), 20.6 (CiCHaBr),

1.4.5 <PQ₂pH₂PH₃), R_f: 0.75 CH₂PI₂). HRMS (ESI-TOF): Ρ_1δΗι^· ₅0₃ΝΒΓ _.([ +H]⁺) requires 431.944 , found 43 .9441 , Ethyl 6-bromo-5~h droxy~i -methy!-2-({phenylt io)methyi)-1 W ndole»3«carboxy1aie 3: Thiophenoi (99.8 pL, 0.972 rnol, 1 .0 eq.) was added to a solution of potassium hydroxide (164 mg, 2,92 -mmol, 3.0 eq.) in methanol {2 ml) and left to stir at room temperature for 15 min. Afte this time, the solution was cooled on ice and bromo indole 2 (880 nig, 0,972 mmol, 10 eq.) in CH2.CJ2 (5 mL) was added. The reaction was left to stir for 3 h before neutralisation with acetic acid. The solvent was removed in vacuo and columned directly (20% EiOAc in petrol) to yield the title product as a pale yellow solid {362 mg, 86%). UUR δ_Η (600 MHz, CDCb) 7.74 (s, 1 H , Hr), 7.43 (s, 1H, H₄), 7.36 (dq, J - 5.2, 3.4, 2.4 Hz, 2H, Hw), 7.25 <dd, J ~ 5.2, 1.9 Hz, 3H, H„ and 5.33 (s, 2Ή, SCH₂), 4,29 (q, J ~ 7.3 Hz, 2H, COaCHaCHs), 3.60.(d, J * 18,1 Hz, 3H, NGH₃), 1 ,38 (t, * 7.3 Hz, 3H, CQjCHzCHs), 6c (150 MHz, CDCb) 165.1 , 147.7, 144,2, 134.1 , 132.9, 129.0, 1.27.9, 127.1 , 1 12.5, 107.5, 106.9, 105.8, 59.9, 31.1 , 30.3, 29.8, 14.7. _f: 0.35 {20%^". EiOAc in petrol). HRMS (ES1-TOF)-:. C^H^BrNOaS ([M÷H}⁺) requires 420.0263, found 420.0260.

ArbidoS [EthyH 6~bromo-4^(dimeihyiamino)metliyl)~S~hydr xy~1 ~m¾thyl-2- {{pheny!thio)methyl -1 f/-indole-3-carboxylate] 4:^; Indole 3 (200 mg, 0,476 mmof, 1.0 eq.) and N, N, N'_f W^? etramethyldi.amtnometha.ne (1-95 pL, 1.43 mmol, 3,0 eq.) were dissolved in 1 ,4- dsoxane (2 mi.). The reaction was. heated to reflux for 3,5 h before removing the solvent in vacuo. The reaction was then re-dissolved in ethyl acetate and 1 M HCI.was added to the solution causing the title product to crash .out as a pale yellow solid (1 17 mg, 51%). NMR-: δ» (500 MHz, MeOD) 7.87 (s, I N, H₇), 7.39 (dd, J ~ 7.4, 2.2 Hz, 2H, H_w), 7.35 ~ 7.31 (m, 3H, Hu and ««). 4,87 (s, 2H , SCtf*), 4.71 (s, 2H, C^N e?), 4.33 (q, J = 7.1 Hz, 2H, C0₂C¾CB₃), 3,63 (s, 3H, HCHs), 2.97 (s, 6H, U(CH₃ )_> 1.39 (t, J - 7.1 Hz, 3H, C0₂C:H₂C¾}, S_c (150 MHz, MeOD) 169,7 C0₂Et), 152.7 (<¾}, 149.0 (C), 13.7.7 (C_f0), 136,4 (<¾), 136.0 (<¾, 132.3 (C»._}), 131.3 (CH), 129.3 (C_?2), 11.9.8 (C>-),. 11:3.1 (C2), 111.3 (Cs), 108.3 (0,), 64.2. (CO2CH2CH3), 57.4 (CH₂NMe₂), 45.4 {CH₂N(CR3)2); 33.7 (CHjSPh), 32,9 (NCH₃), 16.6 (CO2CH2CH3). R_f: 0.25

(EtOAc). HR S .(ESI-TOF): G₂₂H₂sBrN₂0₃S ({M+H]*) requires 477.0842, found 477,0844.

Ethyl 5»aceiGxy«8 ro o«2^((3«hydr0xyph

carboxy ate 8a: 3-hydrpxy htophenol. (117 pL, 1 , 15 mmol, 1.0 eq.) was added to a solution of sodium carbonate (367 mg, 3.46 mmoi, 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 TOO °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%). HMR: δ« (500 MHz,. CDC ) 7.85 (s, 1H, Hi), 7.56 (s, 1 H, H₄), 7.12 (t, J ~ 7.9 Η-ζ,. ΙΗ, ml 6.95 - 6.90. (m, . 1 H, μ 6-78 (s, 1 H, Hw)_f 6.75-6.71 (m, 1H,.H,₂}, 4.69 (s, 2H, SCH₂), 4.30 {q, J == 7.4 Hz, 3H, CO-aCHaCHs), -3.66 (s, 3H. NG?¾}, 2.4Q (s, 3H, CGCHs), 1.38 (t, J = 7.4 Hz, 3H, C0₂CH₂CH₃). 6_C (150 MHz, CDCta) 169,8, 65,0, 156,1 , 144.6, 143,3, 135.6, 135.1 , 130.1, 126. _: 124.8, 119,3, 116.2, 115.3, 113.9, 11 1.1, 105.8, 60.1 , 30.4, 29.9, 21.0, 14.6. R_f OAS (30% EtOAc in Hexane). HRMS^' (ESS-TOF): C₂iHa₀Sr O_sS ( +Hf) requires 473,0318, found 478.0317.

Ethyl 6-bromo<"S~h droxy- (^^

carboxyiate 8: Sodium carbonate (106 mg. 1.00 mmoi-, 2.0 eq.) was -added to a stirred solution of meta-hydfoxy indole 8a (240 mg, 0.502 mmoi, 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 (40

and concentrating in vacuo to give the title product as a white soltd, which could be used without further purification (160 mg, 67%), NMR-: ©_H (600 MHz, MeOD) 7.60 (s, 1H, Hi), 7.58 (s, 1 H, H₄), 7.07 (dd, J * 8.2, 7,7 Hz, 1H, H₁₃), 6.83 - 6.81 (m, ίΗ,, Η,*), 6.79 (ddd, J ~ 7.7, 1.8, 0.9, 1 H, H ), 6,7.0 (dd. - 8.2, 1.8, 0.9 Hz, 1 H, ½), 4.70 (s, 2H, SCH₂), 4,26 (q, J ^« 7.1 Hz, 2H,

COaCHzCHs), 3.64 (s, 3H, NCHj), 1.39 (t, J = 7.1 Hz, 3H, COaCHaCHj). δς (150 MHz, MeOD) 166,9, 158.9, 150.6, 145.5, 136.4, 133.6, 131.0,. 130.7, 128.0, 1.24.9, 120.5, 116.0, 1 14.8, 107.9, 104.8, 60.8, 30.5, 30.4, 14.8. _f 0.45 (1 %

([M÷H]⁺) requires 436.0213, found 436.0215.

Ethyl 2^»{{{3^«amtnophenyi)thio}m8thyl)^«6^«bromo*.5^^

carboxyiate_. 9: 3-aminoihiophenol (54.3 yL,.0.51 mmoi, 1 ,0 eq.) was. added to a solution of potassium hydroxide (86 mg, 1.53 mmoi, 3.0 eq .) in methanol (2 ml) and left to stir at room temperature for 15 min. After this time, the solution was cooled on ice and bromo indole 2 (200 mg, 0,511 mmoi, 1.0 eq.) in CH₂CI₂ (5 ml) was added- The reaction was left ^'to- stir for 3 before neutralisation with acetic-acid. The solvent was removed in vacuo and purified directly fay preparative TLC (1% MeOH in CH₂Ci₂) to yield the title product as a pale yellow solid (138 mg, 62%), NMR: « (500 MHz, CDCI₃) 7.74 (d, J = 1.8 Hz, 1H, Hi), .7.42 (d, J = 1.8 Hz., 1H, H₄% 7.03 (t, J =. 8.1 Hz, I K, Hia), 6,75 (d, J ~ 7.7 Hz, 1H, H ), 6.68 (s, 1 H, Hu), 6.55 (d, J = 6.1 Hz, 1 H, Hii), 4,68 (d, J - 1.9 Hz, 2H, SCW₂), 4.35™ 4.30 (m, 2H, GOC¾GH₃),. 3,60 (d, J = 1.9 Hz, 3H, HCHa), 1.40 (id, J - ? .1 , 1.8 Hz, 3H, ^'COCH₂CH₃>^'.-5c (150 MHz, CDCb) 1-66.9, 150.6, 149.6, 146.0, 136.0, 133.5, 1_.30.5, 128.1 , 123.1 , 120.2, 117.6, 115.8, 114.8, 07.9, 104.6, 68.1 , 60.8, 30.4, 1 .8. _f: 0.85 {1% MeOH in CH2CI2), HRMS (ESI-TOF):

requires 435.0372, found 435.0370,

Ethyl 2~({{3~armnQphenyi}thio}methy }-6^^

carboxy^aie 10: 2-napthaleneihiol (82.0 mg, 0,511 mmot, 1.0 eq.) was added to a solution of potassium hydroxide {86 mg, 1.53 mmol, 3.0 eq.) in methanol (2 mL.) and left to stir at room temperature for 15 m.in. After this time, the solution was cooled on ice and bromo indole .2 (200 mg, 0.51 mmo!, 1.0 eq.) in CH₂Ci₂ (5 mL}^'was added. The reaction was left to stir for 3 h oefore neutralisation with acetic acid. The solvent was removed .in vacuo and purified directly by preparative TLC (1% MeOH in CH2CI2) to yield the title product as a pale yellow solid (118 mg, 50%). N R: δκ (6QQ MHz, DMSO) 9.77 (s, 1 H, 0,% 7.83 (d, J ~ 1.8 Hz, 1H, Ar), 7.81 - 7.79 (m, 1 H, Ar), 7,75 (d, J 8.6 Hz, 1Ή, Ar), 7.72 - 7,70 {m, 1H, Ar), 7,66 (s, 1 H, Hz), 7.46 (s, 1 K, ¾}, 7.45 - 7,40 (m, 2H, Ar), 7.34 (dd, J ~ 8.5, 1.9 Hz, 1H, Ar), 4.82 s, 2H, QH&Ph), 4.04 (q, J » 7.1 Hz, 2H, CC¾C½CH₃), 3.63 (s, 3H, NC.%), 1.14 ({, J ~ 7.1 Hz, 3H, CO_¾CH₂CH₃). 5c (150 MHz, DMSO) 164.3, 149.3, 143.4, 133,2, 132.0, 131.7, 131,6, 128.9, 128.4, 128,4, 127.7, 127,2, 126.8, 126,3, 1:26.0, 114.3, 106.5, 1_.06.3, 103.3, 59.2, 30.3, 28.1 , 14.3. R_f: 0.75 (1% MeOH in CH₂Cf₂). HR.MS (ESI-TGP): CasHaBr QsS (fM+Hf) requires 470.0420, found 470.0420.

methyl- W-indofe~3~carboxy!3te 11 ; Meta-hydroxy indole 8 (30.0 mg, 0.069 mmoi, 1 ,0 eq,) and N, N,N\ Λ/'-tetramethyidtaminomethane (47.0 μΐ, 0.344 mmol, 5.0 eq.) were dissolved in CHaCia (30 mL). The reaction was heated to reflux for 3.5 h before removing the solvent in vacuo to, yield the iifie product as a pale yellow solid (34 mg. 99%). R; 5H (500 MHz, CDC ) 7.47 (s, 1H, ₇), 7.12 (t, J ^» 7.9 Hz, 1H, H₁₃), 6.90 (d, J = 7,9 Hz, 1 H, H_t4), 6.90 (d, J ~ 7.9 Hz, 1 H, H_i2), 6.66 (s, 1 H, Hip), 4.41 (s, 2H, CH₂NMe₂), 4.34 (s, 2H, C.H₂SPh), 4.15 (q, J * 7.1 Hz, 2H, CO2CH2CH3), 3.60 {s, 3H. NC.W3), 2.55 (s, 6H, CHaN^CH^z),_. 1.33 - 1.21 (m, 3H,

C0₂CH CW₃). 6.c (_.150 MHz, CDC¾) 165.9, 156.7, 150.9, 142,6, 135,1, 132.2, 131.0, 130,0, 128.9, 124.6, 124.3, 19.3, 115.5, 113.4, 108.6, 106.3, 60.8, 58.7, 44,0, 30.4, 29.9, 14.3, R_f: 0.15 (10% MeOH in CH2CI2). HRMS (ESS-TOF): C22H₂sBr 204S ([M+H } requires 493,0791 , found 493.0792,

methyM ff-irs o!e-S-carfeoxyiate 12: eia-amino Indole 14 104 mg, 0.238. mmot, 1.0 eq.) and V, Α/,ΛΓ, A/' eti¾rnethyidfami omethane (1.95 μΐ, 1 ,43 mmoi, 3.0 eq.) were dissolved in 1 ,4- dioxane (1.5 ml). The reaction was heaied to reflux for 3.5 h before removing the solvent in vacuo. The product was purified using preparative TIC to yield a paie yellow solid {13 mg, 11 %). NMR: δ_Β (500 MHz, MeOD.) 7.61 s, 1H, H?), 7,00 (t, J ^« 7.8 Hz, 1 H, H13), 8,69 (t, J « .2.0 Hz, i_.H, Hio), 6.61 ~ 6,66 (m, 2H, H and H_M), 4.57 (s, 2H, CWiS^'P ), 4.40 (s, 2H, CWzNMez}, 4.24 (q. J - 7.2 Hz, 2H, CQ2CH2CH3), 3.61 (s, 3H, NCH₃), 2.55 (s, 6H, CH₂N{CH₃}₂X 1.36 (t, J * 7.1 Hz, 3H, C0₂GHgCH₃}. 5_C (150 MHz.. eOD) 167.6, 149.7, 144.5, 136.0, 132.6, 130.6, 129.8, 126.1, 123.0, 120.2, 115.9, 114,8, 112.5, 111.4, 1 10.7, 61.5, 59.8, 43.5, 31.2, 30,5, 14,6. R*: 0.35 (10% MeOH in CH₂C!₂}. H MS (ESi-TOF): C_?2H₂eBrN30_aS ([M+H]^*) requires 492.0951 , found 492.0951.

Ethyl S^romo-4~({d1methyla inQ)m

yifh¾}iTiethyl}-lW-indoie-3--Garbox late 13: Maphthatenyl indole 10 (1 12 mg, 0.238 mmoi, 1.0 eq.) and ^ /V^A/'-tetramethyidraminornethane (195 μί_, 1 .43. mmoi, 3.0 eq.) were dissolved in 1 ,4-dioxane ( 1.5 mL). The reaction was heated, to reflux for 3.5 h .before removing the solvent in vacuo. The product was purified using preparative TIC to yield a pale yellow solid (26 mg, 20%). .NMR: 5H (600 MHz, MeOD) 7.83 ~ 7.60 (m, 1H, Ar), 7.74 (d, J ~ 7.8 Hz, 2H, Ar), 7.68 - 7.65 (m, 1H, Ar), 7.60 (s, 1 H, r), 7.49 -7.43 (m, 2H, Ar), 7,34 (dd. J * 8.6, 1 ,7 Hz, IH. Ar), 4.69 (s, 2H, C½SPh), 4,27 {s, 2H, C½ Me₂), 4.04 ~ 3,98 (m, 2H, CQzOHiC z), 3.65 (s, 3H, NCW₃), 2,45 (s, 6H, Ο^ ίΟΗ, ), 1.21 - 1.15 (m, 3H, COzCR^Cfk)-. 5c (150 MHz, .MeOD) 167.3, 154.9, 143.8, 135.0, 134.1, 133.2, 132.6, 132.6, 131.3, 129.5, 128.7, 128.5, 127.6, 127.6, 125.9, 1 14.6, 1.12.6, 110.8, 106.6, 61.4, 60,0, 43.5, 31,0, 30.8, 30,6, 14.5. R_f: 0.65 {10% MeOH in CH₂C¾), H MS (ESI-TOF): Caj /BrNaOaS ([M+H}⁺} requires 527.0998_» found 527.0996.

Boc piparaxinyl arbidoi fEthyS S-acetoxy»6-bronTO-4-{(4-iefi-butoxycarbonyi)pipera2in-1» yl)methyl}-1-methyl-2-((phenylth^^ 14; Arbidoi 4 (20 mg,

0.0419 mmol, 1.0 eq.) and 1-boc~plperazlne (2 q, 0.126 mrrsol, 3.0 eq. ) were dissolved In 1 ,4- dioxane. (2 mL) and ref!uxed overnight. The solvent was then removed in vacuo and the reaction columned directly to yield the title product as a yellow solid (20 mg, 77%). MMR: 5H (400 MHz, MeOD) 7.55 (s, 1 H, Hi), 7.30 - 7,20 (m, 5H, SPh), 4.54 (s, 2H, CHaSPh), 4,25 - 4.07 (m, 4H_S CH2NR2 and CQ2CH2CH3), 3.58 (d₍ J - 8.2 Hz, 3H, HCH₃), 3.47 (br s, 4H, BocN(CH₂)₂), 2.53 (brs, 4H, BoeN(CH₂C«₂)2). 1.44 (s, 9B, Boc), 1.30 - 1.26 (m, 3H, CO2CH2CH3). 6_C (150 MHz, MeOD) 167.2, 156.2, 151.3, 144.0, 1.35.4, 134.3, 134.1 , 133.6,. 130.1 , 129.9,. 129.0, 128.9, 125,6, 114.8, 114.3, 1 3.8, 108.7, 107.9, 167.1 , 81 ,5, 61 ,5, 60.7, 58.6, 53.0, 30.9, 30.6, 28.6, 14.8, 14.6. Hfi 0.5 (30% EtQAo in petrol). HUMS (ESI-TOF):

{(M + H ) requires 618.1632, found 618.1631.

l}met yi)-1- ethyl-2-i(pheF^lths0}^^{^} 15; Arfaidoi 4 (20 mg,

0,0419 mmoi, 1.0 eq.) and 1-(2-{(trimethyisiiyr)oxy)ethy!)piperazine (25 mg, 0.126 moi, 3.0 eq.) were dissolved in 1 ,4-dioxane (2 mL) and refluxed overnight. The solvent was then removed in vacuo and the reaction columned directly to. yield the title product as a yellow solid (12 mg, 51 %). HMR δ_Η (400 MHz, MeOD) 7,56 (s, 1.H, H_?), 7.22 - 7.30 (m_f SH, SPh), 4.57 (s, 2H, CHzSPh), 4.14 - 4.19 (m, 4H, CH2NR2 and 0Ο₂Ο/¾0Η₃), 3.68 (t, J ~ 5.9 Hz, 2 ,_. CH£>H), 3,60 (s, 3H₍ NCWs), 2.53 - 2.70 (m, 10H, piperazine ring and - e_.H_zCH?OH),_. 134 - 1.30 (rn, 3H, C0₂CH₂C,¾). «c (150 MHz, eOD) 167.2, 151.7. 143.9, 135.4, 134.2, 1 33.4, 130.1 , 129.0, 125.5, 1 14.1 , 1 13.6, 108,9, 107.0, 61.5, 61.1 , 59.8, 59.1 , 54.3, 52.9, 30.9, 30.5, 14,7. R_f: 0.15 (S% MeOH in CH2CI2),

found 562.1368,

Ethyl 6"^ro 0^»5-hydrG y"2^«{ (2 iydroxy

car boxy ate 18: 2-hydroxythiophenol (26.0 μΐ, 0.256 mmoi, 1.0 eq.) was added to a solution of sodium carbonate (81 ,0 mg, 0.767 mmoi, 3.0 eq.) and promo indole 2 (1 00 mg, 0.256 mmoi, 1 .0 eq.) in ethyl acetate {2 mL). The reaction was heated to 50 °C and stirred for 2 h before cooling and removing the solvent in vacuo. The product was then re-dissolved in methanol (2 ml.) and potassium hydroxide (21 ,5 mg, 0.384 mmoi, 1.5 eq ,} was added. The reaction was stirred at room temperature for 3 h before direct purification by preparative TLC (2% MeOH in CH2CI2) to yield the title product as. a white solid (20,5 mg, 1.8%), HMR δ» (500 MHz,. MeOD) 7.69 (s, 1 H„ Hr), 7.54 (s, I N, Η₄), 7Λ7 {Χ, J - 7.7 Hz, 1 , H₁₂), 7.08 (d, J - 7,7 Hz, 1H, Η»), 6,85 (d, -7.7 Hz, 1H, M_i3), 6.66 (t, J - 7.7 Hz, 1 H, H_i5), 4.58 (s, 2H, SCH_?), 4.24 (q, J - 7.1 Hz, 2H,

GQ₂CW₂CH₃), 3.59 {s, 3H, NCH₃), . 1.40 {!, J - 7.1 Hz, 3H,. C0₂CH₂C¾). S_c ( 150 MHz, MeOD) 161.3, 146.4, 143.3, 134.8, 132,0, 130.4, 129.4, 123.7, 23,0, 121,8, 12Q.S, 1 14.4, 113.6, 110.0, 102.7, 60.4, 30.0, 29,2, 15.0. R_f: 0.6 (2% MeOH in CHyC ). HUMS (ESI-TOF): CnjHi_eBrN0₄S ([M+Hf) requires 436.0213, found 436.0212.

Ethyl 6-bromo-5-hydrpxy-2-(({4-hydr xyphen l)thio}methyi)

carboxylase 17; 4 iydroxythiophenol (26.0 pL, 0.256 mmoi, 1 .0 eq.) was added to a solution of .sodium carbonate (81.0 mg, 0,767 mmoi, 3.0 eq.) and bromo indole 2 (100 mg, 0.256 mmoi, 1.0 eq.) in ethyl acetate (2 mL). The reaction was heated to 50 °C. and stirred for 2 h before cooling and removing the solvent . in vacuo. The product was then re-dissolved in methanol {2 mL) and potassium hydroxide (21 ,5 mg, 0,384 mmo!, 1,5 eq.) was added. The reaction was stirred at room temperature for 3 h before direct purification by preparative TLC (2% MeOH in CH2CI₂) to yield the title product as a white solid (2.5 mg, 2%). R: δ_Η (600 MHz, DMSO) 7.65 (s, i H, Hi), 7,49 (s, 1 H, H₄), 7.03 (d, J » 8,7 Hz, 2H , H12), 6.58 (d, J - 8.7 Hz, 2_.H H13), 4,52 (s, 2H, SC ½), 4.08 (q, J * 7.2 Hz, 2H, CO2CH2CH3), 3.51 (s, 3H, NGW₃), 1 ,23 <t_s J * 7.1 Hz, 3H, C0₂CH₂C¾). Sc (150 MHz, DMSO) 164.2, 157.9, 49.2, 144.3, 135,7, 131.4, 126.1 , 1214, 15.9, 1 14.1 , 106.3, 102.9, 79.2, 59.0, 55.4, 30.0, 14.3, R_f; 0,5 (2% MeOH in CHsCfa). HRMS- {ESI-TOF): Ci?H,₈BrN04S^: ([ + ) requires 436.0213, found 436.0213.

carboxyf ate 18a: 3~methpxythiophenoi (14.6 μ^'!_, 0.118 mmo!, 1.0 eg.) was added to a solution of sodium carbonate (37.4 mg, 0.353 rrtmoi, 3.Q eq.) and bromo indoles 2 (46.0 mg, 0.118 rornoi, .0 eq.) in dry ethyl acetate (20 ml). The reaction was heated to 50 °C and stirred for 2 h before addition of water. The organic layer was separated, dried (Na^O ) and concentrated .in vacuo. The compound was purified by column chromatography (20% EtOAc in Hexanes) to produce the title product as a white solid (34 mg, 59%). UMR; δ_Η (600 MHz, DMSO) 7,92 (s, 1 H, Hi), 7.6.6 (s, 1 H, ¾), 7.13 (t, J = 7.9 Hz, 1H, /½), 6.8.7 - 6.84 (m, 1 H, Hi4, 6.79■- 6.74 (m, 2H, Hw and^' /½), 4.77 (s, 2H, SCffe), 4,13 (q, J ^ 7.1 Hz, 2H, C0₂CW₂CHs}, 3,70 (s, 3H, NCHj), 3.58 {s, 3H, SPhOCH}), 2,27 {s, 3H, COCH₃),- 1.20 (t, J ~ 7.1 Hz, 3H, CO2CH2CW3). ¾ (150 MHz, DMSO) 169.1 , 163.9, 159.4, 144.9, 142.6, 135.1 , 135.Q, 129,9, 125,2, 123,3, 116.2, 115,1 , 114.7, 113.5, 110.3, 104,3, 59.5, 55.1 , 30.6, 28.1 , 20.7, 14,3. R_f: 0.4 (20% EtOAc in Hexane). HR S (ESI- OF): C₂2H₂₂BrNO_sS ([ +H]^*} requires 492.0475, found 492.0472.

Ethyl 6-brGmo-5~hy r0xy«2~{¾^

carboxyla e 18: Sodium carbonate (41.3 mg, 0.390 mmol, 2.0 eq.) was added to a stirred solution of meta-methoxy indole 18a (96,0 mg, 0.195 mmot, 1.0 eq.) in methanol (10 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 (NazSC ) a d concentrating in vacuo to give the title product as a white solid, which could he used without further purification (80 mg, 91%}, NMR: δ_Η (600 MHz, GDCis) 7.73 (s, 1H, Ηή, 7,41 (s, 1 H, ¾), 7.17 - 7.13 (m, ½), 7.07 (irn, . 1 H, . H )_t 6,96 (dt, J ~ 7.7, 1.3, I H, _i0)_x 6.85 (m, 1H, W_i2), 4.71 (s, 2H, SC fc), 4.30 \ - J = 7.1 Hz, 2H, G0₂CH₂CH₃)₍ 3,63 (s, 3H, NCHs), 1.41 (t, J - 7.1 Hz, 3H GOsCH₂G ¾). 5c (150 MHz, CDCfe) 165,2, 159,8, 147.7, 135.4, 132.6, 129.8, 12.7.2, 124.6, 119.7, 1 17,3, 114.1 , 112.5, 107.5, 107.3, 105.0, 59.9, 55.4, 29.9, 29.6, 14,7 R_f:. 0.55 (1% MeOH in CH₂C!₂}. HR S ESl-TOF): C₂₀H_?oBrN:0₄S <[M*Hf) requires 450.0369, found 450,0367.

Ethyl 6-brorno^{(dimethySam o)meihy)^

meetyMW-in^'iioIe-3-ca^'rbox iiate 19? Ortho-hydroxy indqie_:16 (13.5 mg, 0,0309 rnmol, 1,0 eq.) and N, , A^J, W-tetramethyldiaminomethane (12.7 μί, 0.0928 mmoi, 3,0 eq.) were dissolved in 1,4-dioxane (2.0 mL). The reaction was heated io reflux for 3,5 h before removing the solvent in vacuo to yield the title product as a white solid (13 mg, 85 %). HMR: ¾_H (500 MHz, MeOD) 7.53 (s, 1Ή, H₇), 7.19 - 7,11 (m, 1H,

7.6, .7 Hz, 1H, #«), 6 -86 - 6.78 (m, TH, H ), 6.63 (dt, J - 13.7, 7.6 Hz, 1H, H_f5), 4.48 (s, 2H, C½Sph), 4.34 (s, 2H, CM>NMe₂), 4.22 (dq, J * 10.8, 7,1, 6.3 Hz, 2H, C0₂GH₂CHs), 3.56 (s, 3H, HCH₃), 2.49 (d, J* 11.4 Hz, 6H, CH₂N(CW₃)₂), 1.42 ~ 1.37 (m, 3H, COzCH^CHs. S_c (I SO MHz, MeOD) 167.5, 160.4, 155.0, 144,6, 137.0, 136.6, 132.5, 131.6, 130.6, 126.1, 120,3, 116.8, 114.5, 112.6, 110.6, 106.0, 68.1, 61 ,4, 60.1, 43.5, 29.9, 14.6. R_f: 0.4 (5% MeOH in CH₂CI₂), HRMS (ES1-TOF): CaHasB NzO-iS ([M+Hj⁺) requires 493,0791, found 493.0793.

Ethyl 63rom0-4"{{d!m@thyiamifiO)met^^

methyi-1 f -indoie-S-car oxyiaie 21 : Sodium carbonate (17,5 mg, 0165 mmoi, 3.0 eq.) was added to a stirred solution of meta-rnethoxy indole 18 (27.0 mg, 0.055 mmoi, 1.0 eq.) in- ethyl acetate (8 mi..} and methanol (1 mL). The solution was left to stir for 3h before filtering and removing the solvent in vacuo. The compound was then re-dissoived in 1 ,4-dioxane (5 mL) and W,MW', VHeramethy!diaminOfn^'ethane (5.5 ,uL, 0.04 mmoi, 3.0 eq.) as added. The reaction was heated to reflux overnight before removing the solvent in vacuo. Purification by preparative TLC (5% MeOH In CH2CI2) yielded the title product as a pale yellow solid (7 mg, 24%). NMR: δ_Μ (600 MHz, GD<¾) 7.44 <s, 1H, tt₇), 7.19 (t, J =7.9 Hz, 1H, H_i5), 6.96 (rn, 1H, H_i4), 6.82 (m, 1H, W₁₀), 6.77 (m, 1H, 12), 4.52 (s, 2H, C½SPh), 4.21 (qd, J = 7.2, 0,8 Hz, 2H, CO2CH2CH3), 4.1? (s, 2H, CftNMej), 3.66 (s, 3H, HQH₃), 3.58 (s, 3H, OC fc), 2.38 (s, 6H, G ₂H{GH₃k)..1.34 (m, 3H, CO2CH2CH3).5c (150 MHz, CDCis) 165.6, 159.9, 151.7, 141.7, 135.5,131.9, 129.9, 124.7, 11 .5,_: 114.2, 113.1, 1 2.6, 108.6.106.2, 60.5, 59.9, 55.3, 44.2, 30.5, 29.9, 14.5. f¾: 0.35 (5% MeOH in CM2CI2). HUMS

((M+H ) requires 523.0897, found 523.0894. Biological Procedures

OCTET Bio-Layer fnierferomelry Data and Testing: Κ was determined by BLJ using an Octet Red instrument (ForteBio). Biofinylated HAs, purified as described previously", were used for these measurements. HAs at -10-60 pg m!^"1 m i x kinetics buffer (1 * PBS, pH 7.4, 0.01 % BSA and 0.002% Tween 20). ere loaded onto streptavid in- coated biosensors and incubated with varying concentrations of small molecule in solution.. All binding data were collected -at 30. °C. The experiments comprised five steps: (1 ) baseline acquisition {60 s); (2) HA loading onto sensor (1800 s); (3) second baseline acquisition (120 s); (4). association of small molecule for the measurement of fen (180 s); and (5) dissociation of small molecule for the. measurement of feff (18Q s). Baseline and dissociation steps were carried out in. buffer only. The ratio of f _w- to fe« determines the % reported here.

Results of OCTET Biolayer Inierferometry Test

See Figure 4.

Claims

CLAIMS What, is claimed is:

1. A method of inhibiting the bioactivity of a v!rai hemagglutinin (HA) molecule that is displayed on the surface of the Influenza virus, comprising contacting the hemagglutinin molecule with an effective- concentration of a compound of formula (A)

wherein X is S or 0.

The method of claim 1. wherein .the compound of formula (A) is the compound of formula

3. A method of treating or preventing an influenza viral infection in a patient, comprising administering to the patient an effective: dose: of a compound of formula (A)

The method of claim 3, wherein the compound of formula (A) is the compound of formula