Discovery of Indole Derivatives as Novel and Potent Dengue Virus Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00913

3-Acyl-indole derivative 1 was identified as a novel dengue virus (DENV) inhibitor from a DENV serotype 2 (DENV-2) phenotypic antiviral screen. Extensive SAR studies led to the discovery of new derivatives with improved DENV-2 potency as well as activity in nanomolar to micromolar range against the other DENV serotypes. In addition to the potency, physicochemical properties and metabolic stability in rat and human microsomes were improved during the optimization process. Chiral separation of the racemic mixtures showed a clear preference for one of the two enantiomers. Furthermore, rat pharmacokinetics of two compounds will be discussed in more detail, demonstrating the potential of this new series of pan-serotype-DENV inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b00913

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Article
Discovery of Indole Derivatives as Novel and Potent Dengue Virus Inhibitors
Dorothée Bardiot, Mohamed Koukni, Wim Smets, Gunter Carlens, Suzanne
Kaptein, Kai Dallmeier, Patrick Chaltin, Johan Neyts, and Arnaud Marchand
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00913 • Publication Date (Web): 27 Aug 2018
Downloaded from http://pubs.acs.org on August 28, 2018
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Discovery of Indole Derivatives as Novel and Potent
Dengue Virus Inhibitors
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Dorothée Bardiota, Mohamed Kouknia, Wim Smetsa, Gunter Carlensa, Suzanne Kapteinc, Kai
Dallmeierc, Patrick Chaltina,b, Johan Neytsc and Arnaud Marchanda,*
b
aCistim Leuven vzw, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, Belgium; Centre for
Drug Design and Discovery, Bioincubator 2, Gaston Geenslaan 2, 3001 Leuven, Belgium;
cLaboratory of Virology, Rega Institute for Medical Research, KULeuven, Herestraat 49 ꢀ box
1030, 3000 Leuven, Belgium
ABSTRACT
3ꢀAcylꢀindole derivative 1 was identified as a novel dengue virus (DENV) inhibitor from a
DENV serotype 2 (DENVꢀ2) phenotypic antiviral screen. Extensive SAR studies led to the
discovery of new derivatives with improved DENVꢀ2 potency as well as activity in nanomolar to
micromolar range against the other DENV serotypes. In addition to the potency,
physicochemical properties and metabolic stability in rat and human microsomes were improved
during the optimization process.
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Chiral separation of the racemic mixtures showed a clear preference for one of the two
enantiomers. Furthermore, rat pharmacokinetics of 2 compounds will be discussed in more detail
demonstrating the potential of this new series of panꢀserotypeꢀDENV inhibitors.
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INTRODUCTION
Infections with dengue virus (DENV) can result in dengue fever that can further develop into
severe dengue such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS); a
potentially lethal condition if left untreated. Annually, an estimated 390 million infections with
DENV occur in tropical and subꢀtropical areas worldwide, of which roughly a quarter (~96
million) develop symptoms which is typically a painful, debilitating disease with high fever,
headache, retroꢀorbital pain, general malaise, rash, nausea, vomiting and joint/bone/muscle pain
(‘breakbone’ fever)1. A proportion of the cases (~500 000) progresses into severe dengue for
which treatment is limited to symptomatic alleviation and supportive care and an estimated 22
000 people succumb to DENV infections each year. Because of the increase in the geographical
distribution of the DENV vector (the Aedes mosquito), the urbanization and globalization, the
incidence and prevalence of dengue cases have exploded in the last decades and are expected to
rise even more2ꢀ4. Currently, ~2.5ꢀ3.0 billion people living in (sub)tropical regions are at risk to
become infected with DENV4. The virus forms a tremendous health and socioꢀeconomic burden
in the affected regions and extra measures to control the dramatic spread of the virus are urgently
needed. Recently a DENV vaccine (CYDꢀTDV from SanofiꢀPasteur) has been licensed in a
number of countries but it has rather limited efficacy and some safety issues5ꢀ6. Because of that,
CYDꢀTDV is contraꢀindicated for use in individuals with a seronegative status as well as in
young children (<5 years), which are also thought to be primarily seronegative for dengue. Via a
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process called antibodyꢀdependent enhancement (ADE), the vaccine is believed to sensitize
naive recipients, resulting in an enhanced DENV infection when exposed to DENV for the first
time7. Vaccination could then lead to an opposite effect than for which it was originally
designed, i.e. more cases of hospitalized dengue (DHS and DSS) instead of fewer cases. Hence, a
dengue vaccine or dengue antiviral should be equally effective against all four DENV serotypes6.
Potent inhibitors of the virus could serve multiple purposes such as prophylactic treatment for
travelers to and people living in dengueꢀendemic regions, household prophylaxis when there is
an index case and treatment of patients with an ongoing DENV infection. Despite efforts
performed by different research groups8ꢀ13, there are still no antiviral drugs available for the
treatment and/or prophylaxis of DENV infections. The clinical development of the majority of
these dengue molecules was halted because of limited or low efficacy against some of the four
dengue serotypes12, limited solubility13 or other pharmacokinetic and/or safety issues in
animals10. In an effort to identify novel small molecules acting as dengue virus inhibitors, a
mediumꢀthroughput phenotypic antiviral screening using a cytopathic effect (CPE) reduction
assay against DENVꢀ2 (‘Rega’ lab strain) was performed with the CD3 (Centre for Drug Design
and Discovery, KULeuven) compound library. This effort led to the identification of 2ꢀ((3,4ꢀ
dimethoxyphenyl)amino)ꢀ1ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀphenylethanꢀ1ꢀone (Compound (1), Figure 1) as a
novel dengue virus inhibitor. Using a virus yield reduction assay (RTꢀqPCR readout) as
confirmatory assay, compound 1 demonstrated potent and genuine in vitro antiviral activity
against DENVꢀ2 (EC50 = 0.078 µM) and was shown to significantly reduce viral RNA
replication (3.8log10) at nonꢀtoxic concentrations (data not shown), indicating that the antiviral
activity is selective and is thus not associated with an adverse/pleiotropic effect on the host cell.
In terms of mechanism of action, selection of in vitro drugꢀresistant variants led to the
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identification of multiple mutations in the viral NS4B sequence that are required to be present
together to result in phenotypic resistance. A manuscript describing the mechanism of action
studies for this class of compounds is in preparation and will be published elsewhere. As the
potential molecular target (NS4B) of compound 1 does not exert any enzymatic activity and the
structure of this membrane associated protein has not been solved, the optimization of the
antiviral activity was performed by systematic modifications of the constituent parts of
compound 1 (Figure 2) using the DENVꢀ2 virus yield reduction assay with RTꢀqPCR readout for
activity determination. In this paper, we describe how SAR investigations led to compounds with
nanomolar to micromolar activity against the 4 DENV serotypes. In addition, we report on
further optimization of the series resulting in compounds with a good pharmacokinetic profile
and improved oral bioavailability in rat.
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CHEMISTRY
Two synthetic routes were developed to prepare the desired ketoindole derivatives 1 and 10ꢀ
56, as depicted in Scheme 1. The first synthetic route was based on the crossꢀcoupling of
14
aldehydes with imines catalyzed by an Nꢀheterocyclic carbene as described by Li et al.
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Starting from imines 4 (generally synthesized by heating of an aldehyde 2 and an amine 3) and
BOCꢀprotected indoleꢀ3ꢀcarboxaldehydes 5, this oneꢀpot procedure allowed us to generate a
diverse library of final compounds bearing modifications on R1, R2, and R3. During the
establishment of the condensation reaction between the imines 4 and the aldehydes 5, it appeared
that the protection of the indole NH was required. The BOC group turned to be the most suitable
protecting group as it could be removed easily in a few minutes at 160 °C under microwave
irradiation (MW) without obvious decomposition. It also seemed to activate the aldehyde in 3ꢀ
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position, resulting in better yields than other protection groups such as benzyl. However, this
Umpolung reaction failed to give the desired products when using alkylꢀ or cycloalkylꢀaldehydes
2 or some electronꢀrich anilines 3 such as dimethoxyanilines. Therefore, a new threeꢀstep
synthesis approach was developed to access other compounds of interest. FriedelꢀCrafts reaction
with acyl chloride 7 in the presence of Et2AlCl15 as Lewis acid proceeded regioselectively in 3ꢀ
position of unprotected indoles 6 to give 3ꢀacylindole derivatives 8 in good yields. Bromination
in αꢀposition of the carbonyl group with phenyltrimethylammonium tribromide16 followed by
bromine displacement with amines 3 provided the desired final compounds. Scheme 2 illustrates
the synthesis of compounds 61ꢀ62, 76ꢀ79 and 91ꢀ92. Compounds 61 and 62 with an
hydroxymethyl group were prepared by acylation of 59 and 60 with αꢀchlorophenylacetyl
chloride in pyridine17 followed by chlorine displacement with 3,5ꢀdiꢀmethoxyaniline under
microwave irradiation. It is worth mentioning that the acylation conditions used in Scheme 1 led
to a complex mixture of compounds. The hydroxyethylꢀindoles 67 and 68 were prepared from
indole carboxaldehydes 63 and 64 by a Wittig reaction to get the corresponding vinyl indoles
followed by hydroboration. Further conversions of the hydroxyl group in dimethylamino or
methyl sulfone were performed on the 3ꢀacylindole 73. The hydroxypropyl chain on the indole
was constructed in 3 steps comprising a HornerꢀWadsworthꢀEmmons reaction of indole
carboxaldehydes 63 and 80 to get the methyl acrylate derivatives 81 and 82, followed by the
reduction of the double bond by hydrogenation and ester reduction with LiBH4. Scheme 3
outlines the synthesis of the anilines substituted with alkoxy chains containing a hydroxy or an
amino group. Selective monoꢀdemethylation of 3,5ꢀdimethoxyaniline 93 with dodecanethiol and
NaOH in NMP18 provided 3ꢀaminoꢀ5ꢀmethoxyphenol 94. OꢀAlkylation with bromoalkyls or
alkylsulfonates afforded the desired anilines.
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RESULTS AND DISCUSSION
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Early structureꢀactivity relationship (SAR) investigation. The SAR was explored with the
conceptual division of hit 1 into three distinct structural subunits: the 3,4ꢀdiꢀmethoxyaniline, the
phenyl head part and the indole core (Figure 2). The DENVꢀ2 antiviral activity of all newly
synthesized compounds was determined together with their cellular cytotoxicity.
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First, we investigated aniline modifications (Table 1) by varying substituents or substitution
patterns on the aromatic ring (10ꢀ21). Aniline replacement with heteroaromatic or cycloalkyl
amines was also investigated (22ꢀ28). A survey of the dimethoxy anilines revealed that the
substitution pattern was essential for the activity. Indeed, the 3,5ꢀdimethoxyaniline analogue 12
showed a tenꢀfold increase of antiviral activity compared to 1 and was amongst the most potent
compounds within the series (EC50 = 0.007 µM), while the 2,5ꢀ and 2,3ꢀdimethoxyaniline
derivatives only showed submicromolar antiviral activity (10ꢀ11). In addition to the beneficial
impact on potency, the 3,5ꢀdimethoxyaniline moiety offers the advantage of lacking a methoxy
group in para position of the amine, which could pose a safety concern if metabolized in
iminoquinone.19ꢀ20 Combination of a 3ꢀmethoxy and another substituent in 5ꢀposition on the
aniline, such as an ethoxy 13 or a fluorine 14, was well tolerated whereas the 3,5ꢀdifluoroaniline
derivative 15 showed a 85ꢀfold decrease in potency compared to 14. This result clearly indicates
that at least one methoxy group in meta position of the aniline is required to get DENVꢀ2 double
digit nanomolar potency. Moreover, the crucial role of a methoxy in meta position of the aniline
was confirmed with monoꢀsubstituted aniline derivatives. Indeed, the substitution of the meta
methoxy (compound 16) by a trifluoromethoxy, cyano, fluoro or methyl sulfone (17ꢀ20) was
associated with a decrease of antiviral activity (12ꢀ to 90ꢀfold, respectively). Similarly, the key
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role of one meta methoxy substituent was demonstrated in the pyridyl series (compounds 22 and
23). Furthermore, other heteroarylamines, even those bearing a methoxy group (24ꢀ27),
cycloalkylamines such as 28 and alkylamines (data not shown) were associated with a reduced
potency.
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We next investigated modifications around the phenyl head part (Table 2). To evaluate the
effect of substituents on the phenyl ring, we synthesized compounds 29ꢀ35. Introduction of one
methyl 29, fluorine 32 or cyano 34 in paraꢀposition led to a 2ꢀ to 3ꢀfold improvement in activity
compared to 16. The nature of the substituents on this para position proved to be important for
potency and could lead to a strong reduction in activity as demonstrated by the methyl sulfone
derivative 35. Comparing the activities of the orthoꢀ, metaꢀ or paraꢀmethyl derivatives 29ꢀ31,
paraꢀsubstitution seemed to be preferred, orthoꢀsubstitution was well tolerated whereas metaꢀ
substitution led to a 12ꢀfold decrease in activity. We also investigated the effect of sixꢀmembered
(36; 38ꢀ41), fiveꢀmembered (42ꢀ44) and bicyclic (45ꢀ47) heteroaromatic rings. 3ꢀ or 4ꢀPyridyl
derivatives 36 and 39 were the 6ꢀmembered heteroaromatic rings best tolerated and substitution
of the 3ꢀpyridyl with a methyl (38) further increased the activity. Bioisosteric replacement21ꢀ22 of
the phenyl ring by a thienyl 42 or a furyl 43 led to compounds with similar potency whereas
other fiveꢀmembered heteroaryl rings such as oxadiazole 44 were less active. An interesting
improvement in potency could be achieved with some bicycles. Benzoxazole and
imidazopyridine derivatives 45 and 46 were significantly more potent than the phenyl derivative
16 with antiviral activities in the low nanomolar range. Combining the modifications of the
phenyl head part with the aniline associated with the best potency (3,5ꢀdimethoxyaniline) led to
at least a 5ꢀfold improvement in activity as observed with compounds 33 and 37.
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A variety of analogues (48ꢀ56) were synthesized to evaluate the effect of substitution in 4ꢀ, 5ꢀ,
6ꢀ and 7ꢀposition of the indole core (Table 3). Introduction of halogens, methoxy or methyl
groups was well tolerated in all positions. Direct matched pair analysis of 4ꢀ and 6ꢀchloro and
fluoro substituted indoles (48, 49, 52 and 53) showed a clear preference for chlorine over
fluorine. The most potent compound with a 6ꢀchloroindole 53 presented an EC50 value of 0.013
µM against DENVꢀ2 with a high SI (1000). Combining a substituted indole with the 3,5ꢀ
dimethoxyaniline yielded a significantly more potent compound with a 5ꢀfold improvement in
activity (50 and 51).
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Having identified key modifications on the three structural subunits which resulted in about
20ꢀfold improved potency (1 vs. 12), we turned our attention to the chiral center and its influence
on the DENVꢀ2 antiviral activity. Considering the difficulties to synthesize the compounds in an
enantioselective manner, we decided to use chiral HPLC to separate the enantiomers from a
racemic mixture. To this end, 12 was selected as test compound and was screened on several
chiral columns. A good separation could be achieved (see Experimental Part) leading to both
enantiomers 12a and 12b which were evaluated in our DENVꢀ2 assay together with 12 (Table 4).
Albeit having a similar cytotoxicity, the two enantiomers showed a marked difference in potency
with the (+)ꢀenantiomer 12a being 300ꢀfold more potent than the (ꢀ)ꢀenantiomer 12b. Until now,
we were not able to determine the absolute configuration of the most active enantiomer.
Broadꢀspectrum evaluation. One challenge in the development of smallꢀmolecule dengue
inhibitors is that compounds should be potent against the four dengue serotypes23. The antiviral
effect of the hit 1 and a selection of potent compounds against the DENVꢀ2 ‘Rega’ lab strain
(12a, 37 and 51) was evaluated against clinical isolates or labꢀadapted strains representing the
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four dengue serotypes (Table 5). While 1 showed potent activity against DENVꢀ2, it was only
weakly active against the three other tested dengue serotypes with EC50 values in the micromolar
range. Interestingly, compounds showing improved DENVꢀ2 activities also presented an
improved profile against DENVꢀ1 and DENVꢀ3. However, the DENVꢀ4 activity remained in the
micromolar range despite the noticeable improvements achieved against the other 3 serotypes.
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In vitro ADMET and PK. Compounds were selected based on their antiviral potency and
chemical diversity for a detailed evaluation of their in vitro ADMET properties (Table 6).
Compound 12 displayed low solubility and moderate permeability. Metabolic stability was
different across the species with high stability in human liver microsomes (HLM) and moderate
stability in rat liver microsomes (RLM). 12 did not show significant hERG inhibition. However,
there was a consistent single digit micromolar inhibition of CYP1A2, 2C19 and 2C9 (IC50
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10µM for 2D6 and 3A4). The two enantiomers 12a and 12b showed a similar profile regarding
solubility, permeability, plasma protein binding, CYP inhibition and metabolic stability in HLM.
However, the most potent enantiomer 12a presented the best metabolic stability in RLM (37%
turnover vs 91% for 12b and 63% for the racemate 12). A significant solubility improvement was
observed for compound 23 due to the introduction of the 3ꢀaminoꢀ5ꢀmethoxypyridine instead of
the 3,5ꢀdimethoxyaniline. Unfortunately, this gain in solubility was associated with a more
pronounced hERG inhibition (IC50 = 3.5 µM) as well as a strong CYP3A4 inhibition (IC50 = 0.05
µM). Compound 45 with a benzoxazole instead of the phenyl head part proved to be very potent
against DENVꢀ2 (EC50 = 0.011 µM) but its further development was hampered because of its
unfavorable ADMET profile (poor solubility and poor metabolic stability in RLM).
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Since most of the compounds contain an aniline moiety, we decided to evaluate the potential
for mutagenicity.24ꢀ26 In that respect, several active compounds bearing more or less electronꢀrich
moieties (1, 12, 16, 23 and 51) were evaluated in an Ames test in presence or absence of human
liver S9 fraction and all results proved to be negative (data not shown).
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Overall 12a presented the most interesting profile (antiviral activity and ADMET) and was
therefore selected for rat pharmacokinetics (Table 7). Despite a moderate turnover in RLM
(37%), the clearance after iv administration turned out to be high and we therefore decided to
check the stability of the compound in rat hepatocytes to see whether phase II metabolism could
possibly explain the discrepancy between our in vitro and in vivo data. Although 12a was devoid
of polar groups, it appeared to be quickly turned over in rat hepatocytes (98% of compound
metabolized after 1 h), which could explain the rapid clearance observed upon iv dosing. Oral
dosing led to a very low Cmax and exposure respective to the given dose, which could probably
be attributed to a poor absorption. Consequently, a low oral bioavailability (7%) was calculated.
Improvement of oral bioavailability. The limited oral bioavailability could be improved by
increasing the absorption or by reducing the firstꢀpass effect or by a combination of both. To
overcome absorption issue in the series, we decided to introduce polar chains on the scaffold
since we suspected that the low absorption could be limited by the low aqueous solubility.
Since the SAR revealed that substituents on the indole core were well tolerated, we first
focused on introducing alkyl chains bearing hydroxy, amino or sulfonyl groups. Table 8
summarizes the biological activities of all compounds bearing polar chains on the indole as well
as solubility and HLM data for the most potent compounds. Introduction of hydroxyalkyl chains
in 4ꢀposition led to decreased potency. The 5ꢀposition seemed to be the most suitable position for
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the polar chains as the 3 compounds 62, 76 and 92 showed interesting activity with EC50 values
ranging between 0.096 µM and 0.181 µM. The most active compound 92 with a hydroxypropyl
chain displayed an improved solubility compared to 12 but also a lower metabolic stability in
HLM. Compound 77 with a hydroxyethyl chain in 7ꢀposition presented a promising activity
below 0.1 µM but unfortunately also exhibited limited solubility. Derivatives 78 and 79 with a
dimethylamino or methylsulfonyl group on the chain were less active. Being unable to maintain
an acceptable level of potency and a better aqueous solubility with polar side chains on the
indole, we decided to look at other parts of the compounds.
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As compound 13 with a 3ꢀethoxyꢀ5ꢀmethoxyꢀaniline and compound 12 with a 3,5ꢀdiꢀ
methoxyaniline showed similar activity (Table 1), we thought that combining one methoxy
group and an elongated polar chain on the aniline could lead to potent compounds with better
aqueous solubility. Except for compound 102 bearing a dimethylamino group, all the synthesized
compounds with a polar chain on the aniline showed DENVꢀ2 activities below 0.1 µM (Table 9).
Amongst the hydroxylated chains, the most promising group seemed to be the glycol (100) with
a positive impact on solubility and metabolic stability. Compared to 12, compound 100 displayed
a 6ꢀfold better solubility, a similar high stability in HLM and an improved stability in RLM
while maintaining a decent antiviral potency. Activity and metabolic stability could be further
improved when combining the aniline with the glycol chain and one of the best head part
identified in early SAR (paraꢀfluoroꢀphenyl) in compound 101 but with a slight decrease in
solubility. This clearly indicated that there is still room for optimization to get compounds with
better antiviral and ADME properties. Following the good profile of 100, its enantiomers were
separated by chiral SFC. The most active enantiomer 100a showed potent antiviral activity with
a DENVꢀ2 EC50 value of 0.012 µM and a better metabolic stability in RLM than the racemate
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(21% turnover). The metabolic stability of 100a was also evaluated in rat hepatocytes and was
found to be moderate with 60% metabolized after 60 minutes. Based on its improved ADME
profile compared to 12a (see table 6), 100a was selected for rat PK (Table 7). As anticipated, its
improved solubility and metabolic stability translated into an interesting rat PK profile. The low
turnover in RLM and moderate stability in rat hepatocytes correlated with a moderate iv
clearance. The oral dose gave a 9ꢀfold increase in Cmax compared to 12a and a 19ꢀfold increase
in AUC with an oral bioavailability of 64%. These results clearly demonstrate the importance of
identifying compounds with an acceptable aqueous solubility and high metabolic stability to
reach acceptable levels of exposure in vivo. The PK profile of 100a shows the potential for these
novel dengue inhibitors in view of further optimization and in vivo studies.
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CONCLUSION
In summary, we discovered and optimized a new class of indole derivatives as potent and
selective inhibitors of dengue virus replication. Throughout a systematic SAR exploration,
potency was improved leading to compounds with in vitro activity in the nanomolar to
micromolar range against each of the four dengue serotypes. Chiral separation of two racemic
mixtures by HPLC or SFC revealed a marked difference in potency between the two
enantiomers. Despite a first rather unfavorable iv and oral rat PK profile further optimization
focusing on improving solubility and metabolic stability led to the identification of 100a which
has an interesting PK profile in rats. Based on these favorable results, further optimization
aiming at the improvement of the panꢀserotypic coverage as well as the ADMET and PK
properties has been initiated. The results (SAR, PK and in vivo studies) will be reported in
further studies.
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EXPERIMENTAL PART
8
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Chemistry. All reagents and solvents were purchased from commercial sources and used
without further purification. Flash chromatography purifications were performed on Biotage
prepacked silica gel columns using Biotage Isolera or SP4 instruments. TLC was carried out with
MachereyꢀNagel Alugram Sil G/UV254 plates. TLC plates were revealed with UV light, KMnO4,
pꢀanisaldehyde or ninhydrine solutions. 1H NMR spectra were recorded on a 300 MHz Avance, a
400 MHz Ascend or a 500 MHz Bruker spectrometer. Proton chemical shifts are reported in
parts per million (δ) using TMS as a standard. Electrospray mass (ESI) measurements were
obtained on a Brucker Esquire 6000 mass spectrometer. The purity of all compounds screened in
biological assays was > 95 %. Purity was determined by LCꢀMS recorded on a system consisting
of a Dionex Ultimate 3000 HPLC equipped with a PDA detector and a Brucker Esquire 6000
mass spectrometer, using a Cꢀ18 column (SunFire C18, 3.5 µm, 3.0 x 100 mm or XBridge C18,
3.5 µm, 3.0 x 100 mm). The enantiomer separations mentioned in this experimental part were
carried out by chiral HPLC or chiral SFC. Optical rotations were measured on a PerkinꢀElmer
341 polarimeter with a sodium lamp and reported as follows: [α]DT (λ, c g/100ml, solvent, TºC).
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General procedure for FriedelꢀCrafts reaction. Exemplified for 1ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ
phenylethanone (8; R1 = Ph, R3 = H). To a solution of 1Hꢀindole (2.5 g; 21.3 mmol) in CH2Cl2
(80 mL) cooled at 0 °C was added dropwise a 1M Et2AlCl solution in hexane (32 mL; 32.0
mmol). After 30 min at 0 °C, a solution of phenylacetyl chloride (4.3 mL; 32.3 mmol) in CH2Cl2
(80 mL) was added. The reaction mixture was stirred at 0 °C for 3 h. 1M Rochelle salt solution
was added slowly at 0 °C and the reaction mixture was stirred at rt for 2 h. The phases were
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separated. The organic phase was washed with sat. NaHCO3, water and brine, dried over
Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with
EtOAc to give 3.2 g (65%) of the title compound as a white powder. ESI/APCI(+): 236 (M+H).
9
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ESI/APCI(ꢀ): 234 (MꢀH). H NMR (300 MHz, DMSOꢀd6) δ ppm 12.01 (br s, 1H), 8.53 (s, 1H),
8.16 (d, J = 7.2 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.25 ꢀ 7.41 (m, 4H), 7.11 ꢀ 7.25 (m, 3H), 4.16
(s, 2H).
General procedure for bromination reaction. Exemplified for 2ꢀbromoꢀ1ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ
phenylethanone (9; R1 = Ph, R3 = H). To a solution of 8 (3.2 g; 13.8 mmol) in THF (140 mL)
cooled at 0 °C was added dropwise a solution of phenyltrimethylammonium tribromide (5.7 g;
15.2 mmol) in THF (70 mL). The reaction mixture was stirred at 0 °C for 1 h and at rt overnight.
The solids were filtered and washed with EtOAc. The filtrate was concentrated under reduced
pressure. The residue was triturated with EtOAc to give 3.7 g (85%) of the title compound as a
white solid. ESI/APCI(+): 314, 316 (M+H). ESI/APCI(ꢀ): 312, 314 (MꢀH). 1H NMR (300 MHz,
DMSOꢀd6) δ ppm 12.21 (br s, 1H), 8.67 (d, J = 3.0 Hz, 1H), 8.19 (d, J = 6.4 Hz, 1H), 7.68 (d, J =
6.4 Hz, 1H), 7.14 ꢀ 7.43 (m, 7H), 6.84 (s, 1H).
General procedure for bromine displacement. Exemplified for 1ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ((3ꢀ
methoxyphenyl)amino)ꢀ2ꢀphenylethanone (16). A mixture of 9 (0.032 g; 0.102 mmol) and mꢀ
anisidine (0.063 mL; 0.563 mmol) in acetonitrile (0.5 mL) was irradiated in a microwave oven at
100 °C for 15 min. The reaction mixture was concentrated. The residue was partitioned between
EtOAc and 1N HCl. The phases were separated. The organic phase was washed with sat.
NaHCO3, water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of EtOAc (20% to 60%) in
heptane furnished 0.030 g (83%) of the title product as a white powder. ESI/APCI(+): 357
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(M+H). ESI/APCI(ꢀ): 355 (MꢀH). H NMR (300 MHz, DMSOꢀd6) δ ppm 12.14 (br s, 1H), 8.89
(s, 1H), 8.16 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 7.5 Hz, 2H), 7.46 (d, J = 7.5 Hz, 1H), 7.29 (t, J =
7.6 Hz, 2H), 7.09 ꢀ 7.25 (m, 3H), 6.91 (t, J = 8.1 Hz, 1H), 6.38 ꢀ 6.50 (m, 2H), 6.35 (d, J = 7.9
Hz, 1H), 6.10 (d, J = 7.9 Hz, 2H), 3.62 (s, 3H).
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General procedure for Umpolung reaction. Exemplified for 1ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀ((3ꢀ
methoxyphenyl)amino)ꢀ2ꢀ(pyrazolo[1,5ꢀa]pyridinꢀ2ꢀyl)ethanꢀ1ꢀone (46). A solution of 3ꢀ
methoxyꢀNꢀ(pyrazolo[1,5ꢀa]pyridinꢀ2ꢀylmethylene)aniline in ethanol was prepared by heating
pyrazolo[1,5ꢀa]pyridineꢀ2ꢀcarbaldehyde (0.087 g; 0.595 mmol) and 3ꢀmethoxyaniline (0.067
mL; 0.599 mmol) in EtOH (1 mL) at 60 °C for 6 h.
To a solution of 3ꢀbenzylꢀ5ꢀ(2ꢀhydroxyethyl)ꢀ4ꢀmethylthiazolꢀ3ꢀium chloride (0.082 g; 0.304
mmol) in EtOH (0.7 mL) heated at 70 °C was added NEt3 (0.060 mL; 0.430 mmol). After 5 min
stirring, tertꢀbutyl 3ꢀformylꢀ1Hꢀindoleꢀ1ꢀcarboxylate (0.147 g; 0.600 mmol) and the solution of
imine prepared above (0.599 mmol) were added. The reaction mixture was stirred at 70 °C
overnight, after which the reaction mixture was irradiated in a microwave oven at 160 °C for 4
min. The reaction mixture was concentrated under reduced pressure. Purification by flash
chromatography on silica gel using a gradient of EtOAc (10% to 60%) in heptane followed by
precipitation from CH2Cl2 furnished 0.055 g (23%) of the title compound as a yellow solid.
1
ESI/APCI(+): 397 (M+H). ESI/APCI(ꢀ): 395 (MꢀH). H NMR (300 MHz, DMSOꢀd6) δ ppm
12.07 (br s, 1H), 8.77 (s, 1H), 8.59 (d, J = 7.1 Hz, 1H), 8.18 (d, J = 7.1 Hz, 1H), 7.59 (d, J = 8.6
Hz, 1H), 7.47 (d, J = 7.1 Hz, 1H), 7.09 ꢀ 7.26 (m, 3H), 6.94 (t, J = 8.3 Hz, 1H), 6.81 (t, J = 7.1
Hz, 1H), 6.65 (s, 1H), 6.41 ꢀ 6.50 (m, 2H), 6.21 ꢀ 6.34 (m, 2H), 6.14 (d, J = 7.6 Hz, 1H), 3.64 (s,
3H).
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Enantiomers separation of 12. Racemic 12 was subjected to chiral HPLC purification
(ChiralPak IC column; eluent: nꢀheptane/dichloromethane/ethanol/diethylamine: 50/50/1/0.1) to
provide 12a (retention time = 8.1 min) and 12b (retention time = 17.9 min). 12a: ESI/APCI(+):
9
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387 (M+H). ESI/APCI(ꢀ): 385 (MꢀH). H NMR (500 MHz, DMSOꢀd6) δ ppm 12.14 (br s, 1H),
8.89 (s, 1H), 8.15 (d, J = 7.3 Hz, 1H), 7.63 (d, J = 7.6 Hz, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.28 (t,
J = 7.6 Hz, 2H), 7.13 ꢀ 7.23 (m, 3H), 6.35 (d, J = 8.2 Hz, 1H), 6.07 (d, J = 8.2 Hz, 1H), 6.04 (d, J
20
= 1.9 Hz, 2H), 5.70 (t, J = 2.0 Hz, 1H), 3.61 (s, 6H). ee > 99%. [α]D = +44.2° (589 nm, c 0.251
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w/v %, DMF, 20°C). 12b: ESI/APCI(+): 387 (M+H). ESI/APCI(ꢀ): 385 (MꢀH). H NMR (500
MHz, DMSOꢀd6) δ ppm 12.14 (br s, 1H), 8.89 (s, 1H), 8.15 (dd, J = 7.3, 1.1 Hz, 1H), 7.63 (d, J =
7.3 Hz, 2H), 7.46 (d, J = 7.6 Hz, 1H), 7.28 (t, J = 7.7 Hz, 2H), 7.12 ꢀ 7.23 (m, 3H), 6.35 (d, J =
8.2 Hz, 1H), 6.07 (d, J = 7.9 Hz, 1H), 6.04 (d, J = 2.2 Hz, 2H), 5.71 (t, J = 2.0 Hz, 1H), 3.61 (s,
6H). ee > 99%. [α]D20 = ꢀ44.1° (589 nm, c 0.213, DMF, 20°C).
General procedure for acylation with αꢀchlorophenylacetyl chloride followed by chlorine
displacement. Exemplified for 2ꢀ((3,5ꢀdimethoxyphenyl)amino)ꢀ1ꢀ(4ꢀ(hydroxymethyl)ꢀ1Hꢀ
indolꢀ3ꢀyl)ꢀ2ꢀphenylethanone (61). To a solution of 59 (1.0 g; 3.8 mmol) and pyridine (0.300
mL; 3.7 mmol) in toluene (10 mL) heated at 55 °C was added dropwise αꢀchlorophenylacetyl
chloride (0.552 mL; 3.8 mmol). The reaction mixture was heated at 55 °C for 1 h. After cooling
to rt, water and MeOH were added. The reaction mixture was stirred at rt for 1 h. The reaction
mixture was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4,
filtered and concentrated under reduced pressure. Purification by flash chromatography on silica
gel using a gradient of EtOAc (15% to 70%) in heptane furnished 0.648 g (41%) of 1ꢀ(4ꢀ(((tertꢀ
butyldimethylsilyl)oxy)methyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀchloroꢀ2ꢀphenylethanone as a brown oil and
0.317 g (28%) of 2ꢀchloroꢀ1ꢀ(4ꢀ(hydroxymethyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀphenylethanone.
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2ꢀChloroꢀ1ꢀ(4ꢀ(hydroxymethyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀphenylethanone. ESI/APCI(ꢀ): 298 (MꢀH). H
NMR (400 MHz, DMSOꢀd6) δ ppm 12.26 (br s, 1H), 8.67 (d, J = 3.4 Hz, 1H), 7.58 (d, J = 7.0
Hz, 2H), 7.26 ꢀ 7.41 (m, 5H), 7.21 (t, J = 7.7 Hz, 1H), 6.88 (s, 1H), 5.08 (dd, J = 14.7, 5.8 Hz,
1H), 4.97 (t, J = 5.7 Hz, 1H), 4.79 (dd, J = 14.7, 5.8 Hz, 1H).
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A mixture of 2ꢀchloroꢀ1ꢀ(4ꢀ(hydroxymethyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀphenylethanone (0.150 g; 0.500
mmol) and 3,5ꢀdimethoxyaniline (0.767 g; 5.0 mmol) in CH3CN (3 mL) was irradiated in a
microwave oven at 130 °C for 15 min. The reaction mixture was concentrated. The residue was
partitioned between EtOAc and 1N HCl. The phases were separated. The organic phase was
washed with sat. NaHCO3, water and brine, dried over Na2SO4, filtered and concentrated under
reduced pressure. Purification by flash chromatography on silica gel using a gradient of EtOAc
(2% to 40%) in heptane followed by precipitation from CH2Cl2 furnished 0.028 g (14%) of the
1
title product as a white powder. ESI/APCI(+): 417 (M+H). ESI/APCI(ꢀ): 415 (MꢀH). H NMR
(300 MHz, DMSOꢀd6) δ ppm 12.24 (br s, 1H), 8.96 (s, 1H), 7.60 (d, J = 7.5 Hz, 2H), 7.25 ꢀ 7.43
(m, 4H), 7.13 ꢀ 7.25 (m, 2H), 6.36 (d, J = 7.9 Hz, 1H), 6.17 (d, J = 7.9 Hz, 1H), 6.08 (s, 2H),
4.91 ꢀ 5.17 (m, 2H), 4.65 (dd, J = 14.1, 5.5 Hz, 1H), 3.64 (s, 6H).
2ꢀ(1HꢀIndolꢀ7ꢀyl)ethanol (68). To a suspension of 64 (1.00 g; 6.9 mmol) and
methyltriphenylphosphonium bromide (2.96 g; 8.3 mmol) in THF (40 mL) cooled at 0 °C was
added dropwise a solution of tBuOK (1.17 g; 10.4 mmol) in THF (10 mL). The reaction mixture
was stirred at rt overnight. The reaction mixture was poured into a mixture ice/water and was
extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, filtered and
concentrated under reduced pressure. Purification by flash chromatography on silica gel using a
gradient of EtOAc (10% to 50%) in heptane furnished 0.914 g of 66 as a pink oil. ESI/APCI(+):
144 (M+H).
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To a solution of 66 (0.914 g; 6.4 mmol) in THF (37 mL) cooled at 0 °C was added dropwise a
1M boraneꢀtetrahydrofuran complex solution in THF (6.4 mL ; 6.4 mmol). The reaction mixture
was stirred at rt for 2.5 h. 10% NaOH (3.1 mL; 7.8 mmol) and 30% H2O2 (0.869 mL; 9.8 mmol)
were added. The reaction mixture was stirred at rt for 4 h. Sat. NH4Cl was added. After 5 min
stirring at rt, the reaction mixture was extracted with EtOAc. The organic phase was washed with
water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of EtOAc (20% to 70%) in
heptane furnished 0.490 g (44% over two steps) of the title product as a brown oil. ESI/APCI(+):
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162 (M+H). ESI/APCI(ꢀ): 160 (MꢀH). H NMR (300 MHz, DMSOꢀd6) δ ppm 11.00 (br s, 1H),
7.37 (t, J = 4.3 Hz, 1H), 7.29 (t, J = 2.8 Hz, 1H), 6.90 (d, J = 4.5 Hz, 2H), 6.35 ꢀ 6.47 (m, 1H),
4.67 (t, J = 4.9 Hz, 1H), 3.71 (q, 2H), 3.00 (t, J = 7.2 Hz, 2H).
7ꢀ(2ꢀ((tertꢀButyldimethylsilyl)oxy)ethyl)ꢀ1Hꢀindole (70). To a solution of 68 (0.200 g; 1.2
mmol) in THF (4.6 mL) were added DBU (0.280 mL; 1.9 mmol) and TBDMSCl (0.412 g; 2.7
mmol). After 4 h at rt, the reaction mixture was diluted with EtOAc and washed with water. The
phases were separated. The organic phase was washed with brine, dried over Na2SO4, filtered
and concentrated under reduced pressure. Purification by flash chromatography on silica gel
using a gradient of EtOAc (20% to 80%) in heptane furnished 0.288 g (84%) of the title product
1
as a brown oil. ESI/APCI(+): 276 (M+H). H NMR (300 MHz, DMSOꢀd6) δ ppm 11.02 (br s,
1H), 7.37 (t, J = 4.5 Hz, 1H), 7.29 (t, J = 2.8 Hz, 1H), 6.90 (d, J = 4.5 Hz, 2H), 6.34 – 6.46 (m,
1H), 3.87 (t, J = 6.7 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H), 0.91 (s, 9H), ꢀ0.10 (s, 6H).
1ꢀ(7ꢀ(2ꢀ((tertꢀButyldimethylsilyl)oxy)ethyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀphenylethanone (72). This
compound was synthesized in a similar manner as described for 8. ESI/APCI(+): 394 (M+H).
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ESI/APCI(ꢀ): 392 (MꢀH). H NMR (300 MHz, DMSOꢀd6) δ ppm 11.96 (br s, 1H), 8.64 (s, 1H),
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8.01 (d, J = 7.2 Hz, 1H), 7.24 ꢀ7.42 (m, 4H), 7.21 (d, J = 7.2 Hz, 1H), 6.96 ꢀ 7.14 (m, 2H), 4.15
(s, 2H), 3.85 (t, J = 6.6 Hz, 2H), 3.05 (t, J = 6.6 Hz, 2H), 0.77 (s, 9H), ꢀ0.15 (s, 6H).
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2ꢀ((3,5ꢀDimethoxyphenyl)amino)ꢀ1ꢀ(7ꢀ(2ꢀhydroxyethyl)ꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2ꢀphenylethanone
(77). This compound was synthesized in two steps following general procedures for bromination
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and bromine displacement. ESI/APCI(+): 431 (M+H). ESI/APCI(ꢀ): 429 (MꢀH). H NMR (300
MHz, DMSOꢀd6) δ ppm 12.04 (br s, 1H), 8.86 (s, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 7.5
Hz, 2H), 7.28 (t, J = 7.1 Hz, 2H), 7.13 ꢀ 7.22 (m, 1H), 6.99 ꢀ 7.13 (m, 2H), 6.33 (d, J = 7.9 Hz,
1H), 6.10 (d, J = 7.9 Hz, 1H), 6.05 (d, J = 1.9 Hz, 2H), 5.71 (s, 1H), 4.69 (br s, 1H), 3.69 (t, J =
6.8 Hz, 2H), 3.61 (s, 6H), 3.00 (t, J = 6.8 Hz, 2H).
3ꢀAminoꢀ5ꢀmethoxyphenol (94). To a solution of 93 (2.00 g; 13.1 mmol) in NMP (10 mL) were
added dodecanethiol (5.28 g; 26.1 mmol) and NaOH (1.15 g; 28.2 mmol). The reaction mixture
was irradiated at 160 °C for 1 h in a microwave oven. The reaction mixture was allowed to cool
to rt. The reaction mixture was diluted with sat. NaH2PO4 and brine and extracted with EtOAc.
The organic phase was washed with brine, dried over MgSO4, filtered and concentrated under
reduced pressure to give a biphasic system. CH3CN was added and both phases were separated.
The CH3CN layer was concentrated under reduced pressure. Purification by flash
chromatography on silica gel using a gradient of EtOAc (25% to 75%) in heptane furnished 1.43
1
g (79%) of the title compound as a beige solid. ESI/APCI (+): 140 (M+H). H NMR (300 MHz,
DMSOꢀd6) δ ppm 8.88 (s, 1H), 5.61 (br s, 2H), 5.53 (s, 1H), 4.91 (br s, 2H), 3.58 (s, 3H).
3ꢀ(2ꢀ(tertꢀButoxy)ethoxy)ꢀ5ꢀmethoxyaniline (95). To a solution of 94 (2.79 g; 20.5 mmol) in
dry DMF (90 mL) cooled at 0 °C was added portionwise NaH (60% dispersion in oil, 1.76 g;
44.1 mmol). A solution of 2ꢀ(tertꢀbutoxy)ethyl 4ꢀmethylbenzenesulfonate (5.46 g; 20.1 mmol) in
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dry DMF (10 mL) was then added dropwise and the reaction mixture was stirred at rt overnight.
Water was added and the reaction mixture was extracted with EtOAc. The organic phase was
dried with Na2SO4, filtered and concentrated. Purification by flash column chromatography on
silica gel using a gradient of EtOAc (5% to 50%) in heptane furnished 2.30 g (48%) of the title
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compound as a brown oil. ESI/APCI(+): 240 (M+H). H NMR (300 MHz, CDCl3) δ ppm 5.96
(d, J = 2.4 Hz, 1H), 5.91 (t, J = 2.0 Hz, 1H), 5.88 (t, J = 2.0 Hz, 1H), 4.03 (t, J = 5.6 Hz, 2H),
3.75 (s, 3H), 3.70 (t, J = 5.6 Hz, 2H), 1.25 (s, 9H).
2ꢀ(3ꢀaminoꢀ5ꢀmethoxyphenoxy)ethanꢀ1ꢀol (96). A solution of 95 (0.400 g; 1.7 mmol) in a 4N
HCl solution in dioxane (4.6 mL; 18.4 mmol) was stirred at rt overnight. The reaction mixture
was basified with 2N NaOH and extracted with EtOAc. The organic phase was washed with
water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure.
Purification by flash chromatography on silica gel using a gradient of MeOH (0% to 10%) in
CH2Cl2 furnished 0.296 g (97%) of the title product as a white powder. ESI/APCI(+): 184
1
(M+H). H NMR (300 MHz, DMSOꢀd6) δ ppm 5.72 – 5.80 (m, 2H), 5.68 (t, J = 2.0 Hz, 1H),
5.06 (s, 2H), 4.82 (t, J = 5.8 Hz, 1H), 3.84 (t, J = 5.2 Hz, 2H), 3.66 (t, J = 5.2 Hz, 2H), 3.63 (s,
3H).
Enantiomers separation of 100. Racemic 100 was subjected to chiral SFC purification
(ChiralPak IC column; mobile phase: 60% CO2, 40% MeOH) to provide 100a and 100b. 100a:
1
ESI/APCI(+): 417 (M+H). ESI/APCI(ꢀ): 415 (MꢀH). H NMR (500 MHz, DMSOꢀd6) δ ppm
12.14 (br s, 1H), 8.90 (s, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.62 (br d, J = 7.6 Hz, 2H), 7.46 (d, J =
7.9 Hz, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.12 ꢀ 7.24 (m, 3H), 6.34 (d, J = 7.9 Hz, 1H), 5.99 – 6.12
(m, 3H), 5.70 (s, 1H), 4.79 (br s, 1H), 3.83 (dq, J =10.3, 5.1 Hz, 2H), 3.55 ꢀ 3.71 (m, 5H).
20
Retention time: 1.9 min. ee > 99%. [α]D = +39.7° (589 nm, c 0.292, DMF, 20°C). 100b:
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ESI/APCI(+):417 (M+H). ESI/APCI(ꢀ): 415 (MꢀH). H NMR (500 MHz, DMSOꢀd6) δ ppm
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12.15 (br s, 1H), 8.90 (s, 1H), 8.15 (br d, J = 7.6 Hz, 1H), 7.62 (br d, J = 7.6 Hz, 2H), 7.46 (br d,
J = 7.9 Hz, 1H), 7.28 (br t, J = 7.4 Hz, 2H), 7.13 ꢀ 7.24 (m, 3H), 6.34 (br d, J = 8.2 Hz, 1H), 6.01
– 6.12 (m, 3H), 5.70 (s, 1H), 4.79 (br s, 1H), 3.77 – 3.90 (m, 2H), 3.55 ꢀ 3.70 (m, 5H). Retention
time: 2.8 min. ee > 99%. [α]D20 = ꢀ39.9° (589 nm , c 0.283, DMF, 20°C).
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Biological methods.
Cells and virus. The following DENV strains were used: DENVꢀ1 Djibouti strain
D1/H/IMTSSA/98/606 (GenBank accession number AF298808; kindly provided by Dr. X. de
Lamballerie, AixꢀMarseille Université, Marseille, France), DENV serotype 2 (DENVꢀ2) ‘Rega’
lab strain (kindly provided by Dr. V. Deubel, formerly at Institute Pasteur, Lyon, France),
DENVꢀ3 strain H87 (KU050695; kindly provided by Dr. X. de Lamballerie), DENVꢀ4
DAKHD34460 (AF051107, only partial sequences available; kindly provided by Dr. X. de
Lamballerie). DENVꢀ2 was initially cultured on Vero cells (African Green monkey kidney cells,
obtained from the European Collection of Cell Cultures [ECACC]), but later we switched to
C6/36 mosquito cells (from Aedes albopictus; ATCC CRLꢀ1660). All other DENV serotypes
were grown in C6/36 cells. Vero cells were grown in Dulbecco's Modified Eagle Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 1% Lꢀglutamine, and 1% sodium
bicarbonate. C6/36 cells were cultured in Leibovitz’s Lꢀ15 medium (ThermoꢀFisher Scientific)
supplemented with 10% FBS, 1% nonessential amino acids (NEAA; ThermoꢀFisher Scientific),
1% HEPES buffer (ThermoꢀFisher Scientific), and 1% penicillin (100 U/mL)/streptomycin (100
ꢁg/mL) solution (ThermoꢀFisher Scientific) at 28 °C in the absence of CO2.
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Cytopathic effect (CPE)ꢀreduction assay. Vero cells were seeded in 96ꢀwell plates at a density
of 7×103 cells/well. One day later, cells were infected with DENVꢀ2 ‘Rega’ lab strain (MOI =
0.05) diluted in assay medium (MEM supplemented with 2% FBS, 0.075% sodium bicarbonate,
and 2 mM Lꢀglutamine) and in the presence of a 5ꢀfold serial dilution of the compounds. After
incubating for 7 days, the medium was removed and cells were fixed with ethanol and stained
with a 1% methylene blue solution. For confirmation of hits, CPEꢀbased assays were performed
using the same protocol, but using a 2ꢀfold serial dilution. Also, a newly synthesized batch for
each potential hit compound was prepared.
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Virus yield reduction assay. Vero cells were seeded in 96ꢀwell plates at a density of 5×104
cells/well. One day later, cells were infected with DENV (MOI = 0.01) diluted in assay medium.
After one hour of incubation, input virus was removed and, after rinsing the cells 3 times,
replaced with assay medium containing a 3ꢀ or 5ꢀfold serial dilution of the compound. After an
incubation period of 4 days (for DENVꢀ2 and DENVꢀ4) to 7 days (for DENVꢀ1 and DENVꢀ3),
supernatant was harvested and the viral RNA load was determined by realꢀtime quantitative RTꢀ
PCR, as described previously27. To determine a possible cytotoxic/ꢀstatic effect of the
compound, the same protocol was followed except that virus infection was omitted. Read out
was performed using the MTS/PMS method (Promega), as described previously28. The 50%
effective concentration (EC50) and the 50% cytotoxic concentration (CC50), which are defined as
the compound concentration that is required to inhibit viral RNA replication by 50% respectively
to inhibit cell growth by 50%, were determined using logarithmic interpolation.
Assay Interference Compounds
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All compounds for biological testing were screened against known assay interference
chemotypes29 by the authors. No flags were reported.
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Animals and Ethics
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All experiments were performed in the on site Association for Assessment and Accreditation of
Laboratory Animal Care approved rodent facility according to the applicable animal welfare
guidelines and legislation, and experimental procedures were approved by the institutional ethics
committee.
AUTHOR INFORMATION
Corresponding Author
*Tel: +3216852604; fax: +3216852601; eꢀmail: arnaud.marchand@cistim.be
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Funding Sources
This work was supported by The Wellcome Trust [089328/Z/09].
ACKNOWLEDGMENT
We want to thank Agnès Calleja for the analytical support and the chiral HPLC separation of
enantiomers 12a and 12b. We thank Michel Carpentier for is assistance with the chiral SFC
separation of compound 100. We also acknowledge Mieke Flament, Ruben Pholien, Carolien De
Keyzer and Annelies De Ceulaer for their excellent assistance in the acquisition of the antiviral
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data. Finally, we also want to thank Bart stoops for providing ADMET data (table 6 and 9) as
well rat PK data for compounds 12a and 100a.
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ABBREVIATIONS
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DENV, dengue virus; SI, selectivity index; MW, microwave irradiation; SAR, structureꢀactivity
relationships; HLM, human liver microsomes; RLM, rat liver microsomes; SFC, supercritical
fluid chromatography.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the ACS Publications website.
Experimental and analytical data of key intermediates.
Molecular Formula Strings with DENVꢀ2 EC50 and CC50.
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Figure 1. Structure and properties of hit compound 1
Figure 2. Decomposition of 1 in three structural subunits to explore SAR
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