Structure-Activity Relationship Study of QL47: A Broad-Spectrum Antiviral Agent

Article abstract of DOI:10.1021/acsmedchemlett.7b00008

Here we report the structure-activity relationship (SAR) investigations of QL-XII-47 (QL47), a compound that possesses broad-spectrum antiviral activity against dengue virus and other RNA viruses. A medicinal chemistry campaign initiated from QL47, a previously reported covalent BTK inhibitor, to derive YKL-04-085, which is devoid of any kinase activity when screened against a panel of 468 kinases and with improved pharmacokinetic properties. Both QL47 and YKL-04-085 are potent inhibitors of viral translation and exhibit cellular antiviral activity at 35-fold lower concentrations relative to inhibition of host-cell proliferation.

Full text of DOI:10.1021/acsmedchemlett.7b00008

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Letter
Structure-Activity Relationship study of
QL47 – a Broad-spectrum Antiviral agent
Yanke Liang, Melissanne De Wispelaere, Margot Carocci, Qingsong
Liu, Jinhua Wang, Priscilla L. Yang, and Nathanael S Gray
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00008 • Publication Date (Web): 03 Feb 2017
Downloaded from http://pubs.acs.org on February 4, 2017
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Structure-Activity Relationship study of QL47 – a Broad-spectrum
Antiviral agent
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Yanke Liang,†,‡,ǁ Melissanne de Wispelaere,§,ǁ Margot Carocci,§,ǁ Qingsong Liu,†,‡ Jinhua Wang,†,‡ Priscilla L. Yang,*,§
Nathanael S. Gray*,†,‡
†Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.
‡Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,
USA.
§Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
ǁThese authors contributed equally to this work.
KEYWORDS. Degue virus, broad-spectrum antiviral, QL47, structure-activity relationship.
ABSTRACT: Here we report the structure-activity relationship (SAR) investigations of QL-XII-47 (QL47), a compound that pos-
sesses broad-spectrum antiviral activity against dengue virus and other RNA viruses. A medicinal chemistry campaign initiated from
QL47, a previously reported covalent BTK inhibitor, to derive YKL-04-085 which is devoid of any kinase activity when screened
against a panel of 468 kinases and with improved pharmacokinetic properties. Both QL47 and YKL-04-085 are potent inhibitors of
viral translation and exhibit cellular anti-viral activity at 35-fold lower concentrations relative to inhibition of host-cell proliferation.
candidates. We pursued a complementary approach to identify
compounds that target host-factors that are essential for viral
Introduction
replication. The potential advantage of this approach is that vi-
Dengue virus (DENV) is a significant human pathogen for
ral resistance might emerge more slowly because there is not a
which no effective drugs or vaccines currently exist. DENV is
direct route to viral resistance arising from mutations that block
a plus-stranded RNA virus and a member of the Flaviviridae
drug-binding to a viral target. A second advantage is that host-
family. The four DENV serotypes (DENV-1, -2, -3, -4) are de-
directed antivirals might possess broader spectrum anti-viral ac-
fined by their immuno-reactivity and differ from one another by
tivity if they target processes commonly utilized by different
25%-40% at the amino acid level.1 Due to the large number of
viruses.9
people at risk for infection, DENV is the most widespread mos-
quito-borne viral disease affecting humans today. An estimated
2.5 billion people live in areas at risk for epidemic transmission,
DENV infection that resulted in the identification of inhibitors
Previously we have implemented cellular phenotypic screens of
and an estimated 390 million DENV infections occur annually
(95% credible interval 284-528 million), of which 96 million
(67-136) manifest clinically.2 Infection with DENV is respon-
sible for disease ranging from dengue fever to the much more
severe and life-threatening dengue hemorrhagic fever (DHF)
and dengue shock syndrome (DSS) that are characterized by
vascular leakage. An estimated 500,000 cases of DHF/DSS oc-
cur annually and are associated with 2.5% fatality although fa-
tality rates for DHF/DSS can exceed 20% if untreated.3 Since
both the number of DENV infections and the proportion of in-
fections resulting in DHF have escalated in recent years, the ab-
sence of anti-DENV therapeutics or an anti-DENV vaccine is a
growing public health concern.
of host signaling and lipid metabolism.10-11 For example, inhi-
bition of the tyrosine kinase Fyn results in a block in DENV
RNA replication.10, 12 We extended this screening to a large col-
lection of cysteine-reactive, covalent kinase inhibitors that have
the potential to target a subset of the approximately 150 kinases
that possess a cysteine near the ATP-binding site of the kinase.
From this screen (details to be published elsewhere), we identi-
fied a tricyclic quinoline compound, QL-XII-47 (QL47), previ-
ously identified as a potent and covalent inhibitor of BTK and
other Tec-family kinases.13 Here we describe a medicinal chem-
istry campaign focused on the goal of understanding the struc-
tural features required for antiviral activity of this scaffold rel-
ative to inhibition of host-cell kinases. Surprisingly, we discov-
ered compounds such as YKL-04-085, which lack the critical
quinoline nitrogen required for hydrogen bonding to the kinase
hinge and that are devoid of any kinase activity, retain the po-
tent anti-viral activity. We demonstrate that this series of com-
pounds inhibit DENV infection at concentrations that are not
cytotoxic and also inhibit the replication of other RNA viruses
in cell culture.
Classical antiviral approaches involve development of so-called
‘direct-acting’ antivirals that target essential, virally encoded
proteins, which in the case of DENV would include the viral
polymerase and protease.4 Despite tremendous success in de-
veloping antivirals that target the analogous enzymes of HIV
and HCV,5-8 to-date efforts to develop antivirals against the den-
gue polymerase or protease have not yielded promising clinical
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Results and Discussion
revealed how this scaffold binds to the ATP-binding cleft (Fig-
ure S1).28 These tricyclic quinolines use the quinoline N nitro-
gen to form an essential hydrogen bond to the kinase hinge seg-
ment while the two side chains extend towards the back and the
front of the ATP binding site. The acrylamide arm of QL47 ex-
tends towards Cys481 of BTK located at the entrance of the
ATP-binding site.13
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To identify new host-targeted inhibitors of DENV, we used two
different approaches to screen small molecule libraries. Since a
detailed account of the two screens is described elsewhere (de
Wispelaere, et al. in review), we present here a qualitative de-
scription as background. A first screen was based on the utili-
zation of DENV reporter viral particles (RVP), which are com-
posed of DENV2 E and prM/M proteins and contain a nucle-
ocapsid formed by DENV2 C and a subgenomic replicon of the
closely related West Nile virus (WNV), another Flavivirus14. In
a second screen, we employed a previously characterized HEK-
293T derived stable cell line encoding a GFP tagged DENV2
replicon, the T-REx-293-DGZ cell line.14 In both screens, the
expression levels of the reporter gene encoded by either the
RVP-packaged WNV replicon, or the DENV2 replicon, were
measured after treatment with a library of small molecules.
We first examined whether QL47 acrylamide moiety, which is
required for covalent inhibition of BTK, is essential for the an-
tiviral activity. We used QL47R, in which the acrylamide moi-
ety was replaced with a nonreactive propyl amide functional
group (Figure 4A). QL47R did not inhibit DENV2 at concen-
trations below 10 µM (Figure 4B), suggesting the acrylamide
moiety is essential for antiviral activity. Consistent with the in-
terpretation that QL47 covalently modifies its target, we also
found that the full anti-DENV activity was recapitulated when
cells were pre-treated with QL47 for six hours followed by ex-
tensive washing prior to DENV infection (de Wispelaere, et al.
in review).
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The library of small molecules screened was a collection of
acrylamide-modified, ATP-site directed heterocycles designed
to target cysteine residues located in or around the kinase ATP-
site. This library contains experimentally validated covalent in-
hibitors of EGFR,15 BTK,16 BMX,17 BLK,16 TEC,16 JAK318,
FGFR1,2,3,4,19 JNK1,2,3,20 CDK7,20 CDK12,21 CDK1321 as
well as potential inhibitors of a subset of the more than 150 ki-
nases that possess a potentially reactive cysteine.22 In addition,
proteomic studies have demonstrated that these compounds can
target other non-kinase targets either due to the presence of a
hyper-reactive cysteine or due to the presence of a suitable bind-
ing pocket and an adjacent cysteine residue.23
We next extended the SAR study to other regions of QL47. The
quinolinyl analogues of QL47 were prepared based on the re-
ported synthetic routes for QL47,13 Torin1,29 and BEZ235,30
with variations of substituents used in the schemes (Scheme
1A). Starting material S1 was reacted with the appropriate ani-
line, followed by a series of reduction, oxidation, and ring-clos-
ing reactions to give rise to S2. Intermediate S2 was coupled
with boronic acids, followed by reduction and acrylation to pro-
vide compounds 2-13. To prepare 6-membered urea analogues,
intermediate S3 underwent a reductive amination reaction, fol-
lowed by urea formation using triphosgene. A similar sequence
of reactions was followed to obtain compound 16 from interme-
diate S4. 5-Membered urea analogues 17-18 were prepared us-
ing a similar strategy as has been reported for BEZ23530 from
intermediate S5. We have also prepared naphthyl analogues of
QL47 that lack a nitrogen that is required for a critical H-bond
to the kinase hinge segment (Scheme 1B). Commercially avail-
able 7-chlorotetralone S7 was converted to aldehyde S8 via
Vilsmeire-Haack-type reaction, followed by dehydrogenation
with DBU. Buchwald coupling was performed to provide com-
pound S9, which was subjected to a ring-closing reaction em-
ploying triethyl phosphonoacetate in basic conditions to pro-
vide common intermediate S10. Suzuki coupling with different
aromatic boronic acids, followed by reduction of the nitro group
provided S11, which was finally reacted with acryloyl chloride
or crotonyl chloride to give rise to the naphthyl analogues 17-
22 and YKL-04-085.
The screen resulted in the identification of a series of small mol-
ecules belonging to a class of tricyclic quinoline inhibitors in-
cluding QL-XII-47 (QL47, Figure 1A), a previously reported
inhibitor of BTK and other TEC-family kinases.13 Among the
screening hits, QL47 exhibited the most potent antiviral activ-
ity, with IC90 value against DENV2 of 0.343 µM, and a desira-
ble window between potency and cytotoxicity (Figure 1B). In
order to determine whether BTK/BMX kinases are the pharma-
cologically relevant targets for viral replication, we further
evaluated other known BTK/BMX kinase inhibitors, CGI-
1746,24 Ibrutinib,16 and BMX-IN-1,25 However, none of these
inhibitors showed comparable antiviral activity (Figure 2), in-
dicating BTK/BMX are not likely to be the pharmacologically
relevant targets of QL47. To further investigate the potent anti-
viral activity of QL47, we examined the inhibition of other un-
related viruses and observed that QL47 exhibited broad-spec-
trum antiviral activity against other Flaviviruses, such as West
Nile virus (WNV), as well as against Enteroviruses, Vesiculo-
viruses, Pneumoviruses (Figure 3), amongst others. The potent
inhibition of a variety of viruses by QL47 indicates that the mo-
lecular target of QL47 might be a host cell factor commonly
utilized in the infectious cycle of these viruses. Despite the risk
of side effects, targeting host cell proteins could be used as a
complementary approach to direct-targeting of essential viral
proteins.
To assess antiviral activity, all compounds were screened in an
antiviral assay at concentrations of 2 and 10 µM. The levels of
DENV production at 24h post-infection/treatment were meas-
ured using a viral focus-forming assay, and the antiviral potency
for each compound was expressed as a log-fold change relative
to a DMSO treatment. Mechanism of action studies, to be re-
ported elsewhere, demonstrated that QL47 affects steady-state
abundance of viral proteins in DENV-infected cells and trans-
Encouraged by the broad spectrum antiviral activity of QL47,
we sought to elucidate the structure-activity relationships re-
quired to impart antiviral activity, and to improve the poor phar-
macokinetic properties of QL47 (Table S1). QL47 was origi-
nally developed as a covalent BTK inhibitor using structure-
based design starting from the highly potent mTOR inhibitor,
Torin2.26-27 Torin2 has been co-crystallized with mTOR, which
lation of
a
DENV-derived reporter replicon RNA
[DENV2(GVD)] electroporated into cells (de Wispelaere, et al.
in review). To evaluate the effect of these drugs on viral protein
accumulation, cells were transfected with a non-replicative
DENV2(GVD) replicon, which encodes a luciferase reporter
gene.12 The levels of luciferase accumulation after treatment
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with 2 and 10 µM of all compounds was therefore assessed to
The SAR indicated that an extended conjugated system from
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evaluate their ability to inhibit the accumulation of DENV viral
proteins in vivo. The cytotoxicity of compounds was assessed
using a cell viability assay and expressed as the concentration
of drug required to kill 50% of the cells (CC50). Finally, as an
initial assessment of in vitro pharmacokinetic properties of the
compounds, the half-life of compounds (T1/2) when incubated
with mouse liver microsome (MLM) is reported, comparing to
QL47, which exhibits a MLM half-life of only 1.2 min.
methylpyrazole to acrylamide is required to preserve antiviral
activity. Combining the hypothesis that the kinase targets of
QL47, BTK/BMX, are not likely responsible for the effects on
viral replication, we were interested to assess naphthyl ana-
logues without the critical kinase hinge-interacting nitrogen
atom. The antiviral activity, inhibition of reporter replicon
translation, and MLM stability for naphthyl analogues are
shown in Table 2. As expected, compounds 17-19 exhibited po-
tent viral inhibition at 10 µM, suggesting that the molecular tar-
get of these compounds might not be a protein kinase. Among
these compounds, 17, with a methylpyrazole substituent,
demonstrated potency comparable to QL47, although its MLM
half-life was only marginally improved to 4 min. We also tested
hydrogenated analogues 20 and 21, which exhibited no antiviral
activity, further confirming the necessity of a conjugated ring
system to achieve viral inhibition. Intriguingly, when a dime-
thylamine tail was installed at the acrylamide site, the antiviral
activity of compound 22 and YKL-04-085 was greatly im-
proved at 2 µM. More promisingly, the MLM half-life of com-
pound YKL-04-085 was significantly improved to 15.6 min.
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We initially explored modifications to the methylpyrazole moi-
ety of QL47 because it has previously been determined to be a
critical determinant of kinase selectivity. A series of QL47 an-
alogs were prepared including compounds such as 1 (Table 1),
but they did not improve the poor mouse microsomal stability
exhibited by QL47. We hypothesized that the metabolic insta-
bility might derive from the easily oxidized indoline moiety. To
address this hypothesis, we turned our attention to the open-ring
analogues of QL47 (Table 1, compound 2-9). Methylpyrazole-
substituted analogue 4 showed only modest improvement of mi-
crosomal stability, but exhibited significantly reduced antiviral
activity at 2 µM. Compounds 2 and 3, which bear other mono-
cyclic aromatic moieties, showed antiviral activity, effects on
DENV2(GVD) reporter replicon activity (a proxy for transla-
tion of the viral RNA), and microsomal stability comparable to
4. We next examined bicyclic aromatic substituents at this po-
sition in an effort to regain the antiviral activity exhibited by
QL47. Although compounds 5 and 6 showed improved viral in-
hibition at 2 µM, the stability of the compounds decreased,
probably due to the extended conjugated ring systems. Indeed,
when non-conjugated ring systems were used (compounds 7-9),
the stability was moderately improved. However, these com-
pounds again completely lost viral inhibition even at 10 µM,
suggesting a conjugated ring system is required to achieve anti-
viral activity.
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To further characterize compound YKL-04-085, it was
screened against a panel of 468 kinases and disease relevant
mutant variants using an active site-directed competition bind-
ing assay at 10 µM drug concentration (Figure 5A). Only a
handful of kinases, such as PIMs and DDRs, exhibited more
than 80% binding with YKL-04-085. We further determined the
IC50 values for these kinases, as well as BTK/BMX (Figure 5B),
and none of them showed significant inhibition by YKL-04-
085, which confirmed YKL-04-085 is devoid of any kinase ac-
tivity due to the lack of the critical hinge-contacting nitrogen.
The anti-DENV2 IC90 value of YKL-04-085 was determined to
be 0.555 µM, and the inhibitor exhibited more than 35-fold win-
dow between viral inhibition and cytotoxicity (Figure 5C). In
addition, YKL-04-085 demonstrated good inhibition of transla-
tion of the DENV2(GVD) reporter replicon in cellulo (Figure
5D). Encouraged by the potent inhibition of DENV2 and im-
proved metabolic stability of YKL-04-085, we then evaluated
the mouse PK properties to determine whether it is a suitable
candidate for future in vivo efficacy studies (Table 3). Although
the compound displayed high clearance via intravenous (IV)
route and low bioavailability, intraperitoneal (IP) administra-
tion gave acceptable plasma drug exposure. Furthermore, a 40
mg/kg once daily IP dosing regime was both well-tolerated and
sufficient to maintain plasma concentrations of YKL-04-085
above the IC90 value measured in cellulo. (Figure S2).
Next, we evaluated the effects of the substituents at the position
ortho to the acrylamide (Table 1, compounds 10-13). Com-
pound 10 demonstrated comparable potency to QL47, presum-
ably due to the intra-molecular hydrogen bonding of the ortho-
fluoro group, which forms a pseudo-5-membered ring. Com-
pound 10 also showed potent in vivo translation inhibition and
antiviral activity, but the half-life of the compound was only
marginally improved to 4.5 min. The ortho-ether analogue 11
exhibited reduced antiviral activity and translation inhibition.
The benzamide analogue 12 showed moderate activity at 2 µM,
while the benzoic acid analogue 13 did not inhibit the virus at
all, which might due to the poor cellular permeability.
Conclusion
The unsuccessful effort to improve microsomal stability by var-
ying methylpyrazole and acrylamide moieties prompted us to
examine the tricyclic quinolinyl core of QL47 (Table 1, com-
pound 14-16). Interestingly, compounds containing a 6-mem-
bered urea moiety, such as 14, only showed minimal antiviral
activity at 10 µM, suggesting that this region of the molecule is
a critical determinant for antiviral activity. We further evaluated
5-membered methylurea analogues 15 and 16. Both displayed
increased antiviral activity and inhibition of DENV(GVD) re-
porter replicon translation, probably due to the restoration of
some conjugated features of the tricyclic core. However, none
of these compounds exhibited substantially improved metabolic
stability.
In conclusion, we report the identification, characterization and
preliminary structure-activity relationships (SAR) for a novel
class of covalent inhibitors of DENV. Preliminary mechanistic
studies of QL47 suggest that its antiviral activity is correlated
with effects on the steady-state abundance of viral proteins,
likely due to an effect on translation of the viral RNA genome.
The correlation of anti-DENV activity with effects on transla-
tion of the DENV2(GVD) reporter replicon in the SAR studies
reported here provide further support that effects on translation
of the viral genome are a key contributor to the antiviral activity
of these compounds. Nevertheless, there are several outliers that
fall outside this correlation, indicating that additional mecha-
nisms may contribute to the overall antiviral activity of these
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Figure 4. A. Chemical structures of QL47 and QL47R. B. Huh7 cells were infected
compounds. While the precise molecular target of this series of
compounds is currently under investigation, KINOMEscan of
QL47 and YKL-04-085 suggest that protein kinases are un-
likely to be targets. Lead optimization yielded compound YKL-
04-085, which might be an appropriate candidate for further in
vivo studies to test efficacy of this class of compounds. Further
optimization of the lead compound, target identification, and in
vivo efficacy studies are the subject of on-going research.
with DENV2 at MOI of 1 then treated 2 μM with each of the indicated small molecules.
The antiviral activity of each small molecule against DENV2 was measured by titra-
tion of the infectious particles that had been secreted to culture supernatants at 24h
post-infection. A representative experiment out of n = 2 repeats is shown. The assays
were performed on duplicate experimental samples, and error bars represent the stand-
ard deviation of the experiment. FFU, focus forming unit.
1
2
3
4
5
6
7
8
A.
B.
105
104
103
102
101
2µM
10µM
Figure 1. A. Chemical structure of QL47. B. Huh7 cells were infected with DENV2
at MOI of 1 and then treated with a range of QL47 concentrations. Cell viability (black)
and viral yield (red) were measured 24 hours later and are plotted here as a percentage
of the DMSO (vehicle) control. CC50 and IC90 values – respectively, corresponding to
the concentrations of QL47 that lead to 50% cytotoxicity and to a 1-log10 unit reduction
in the number of infectious progeny virions produced – were determined by non-linear
regression (see Materials and Methods). A representative experiment out of n ≥ 2
repeats is shown. The assays were performed on duplicate experimental samples, and
error bars represent the standard deviation of the experiment.
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DMSO
QL47
QL47R
Figure 5. A. DiscoverX KINOMEscan selectivity profile of YKL-04-085. Compound
was screened at 10 µM concentration against 468 kinases and mutants. Scores for pri-
mary screen hits were reported as a percent of the DMSO control (% control). The
lower the score the lower the Kd is likely to be. Scores are related to the probability of
being a hit but are not strictly an affinity measurement. Scores for kinase targets which
showed more than 80% binding are shown in table. B. Enzymatic IC50s of YKL-04-
085. IC50s against DDR1/2 were obtained with LanthaScreen binding assays, while
IC50s against PIM1/2 and BTK/BMX were obtained with Z’-Lyte activity assays.
A.
B.
150
100
50
antiviral activity
IC90= 0.343 mM
cytotoxicity
A.
B.
CC50= 67 mM
0
-3
-2
-1
0
1
2
3
4
5
Log10[QL47] mM
Figure 2. Huh7 cells were infected with DENV2 at MOI of 1 and then treated with
each of the indicated small molecules. The antiviral activity of each small molecule
against DENV2 was measured by titration of the infectious particles that had been
secreted to culture supernatants at 24h post-infection. A representative experiment out
of n = 2 repeats is shown. The assays were performed on duplicate experimental sam-
ples, and error bars represent the standard deviation of the experiment. FFU, focus
forming unit.
C.
D.
DV2(GVD) replicon
150
100
50
antiviral activity
IC90= 0.555 mM
2µM
2.5µM
106
100
50
0
10µM
cytotoxicity
5µM
105
CC90= 20 mM
10µM
104
103
102
101
0
-5
-4
-3
-2
-1
0
1
2
3
L
m
7
5
8
O
4
S
/
L
0
-
4
M
Log10[YKL-04-085] mM
g
µ
Q
D
0
-
0
3
L
K
X
Y
H
C
DMSO QL47
CGI-1746
Ibrutinib
BMX-IN-1
Scheme 1. Synthesis of quinolinyl and naphthyl analogues
of QL47
Figure 3. Huh7 cells were infected with West Nile virus (Kunjin strain) at a MOI of
1, then treated with 2 μM of QL47. The infectious virus released to the supernatants at
24h post-infection was quantified by PFA in BHK-21 cells. Vero cells were infected
with poliovirus (PV) at a MOI of 1, then treated with 4 μM of QL47. The infectious
virus released to the supernatants at 12h post-infection was quantified by PFA in Vero
cells. Vero cells were infected with vesicular stomatitis virus (VSV) (Indiana strain)
at a MOI of 1, then treated with 4 μM of QL47. The infectious virus released to the
supernatants at 6h post-infection was quantified by PFA in BHK-21 cells.
A
Vero cells were infected with RSV (A2 strain) at a MOI of 0.01, then treated with 2.5
μM of QL47. The number of plaque formed at 7 days post-infection were counted and
the results were plotted relative to DMSO-treated samples. For each virus, a repre-
sentative experiment out of n ≥ 2 repeats is shown. The assays were performed on
duplicate experimental samples, and error bars represent the standard deviation of the
experiment. FFU, focus forming unit. PFU, plaque forming unit.
Enterovirus
PV
Pneumovirus
RSV
Vesiculovirus
VSV
107
Flavivirus
WNV
107
106
105
104
103
107
106
105
104
100
50
0
106
105
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102
O
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µ
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4
S
µ
S
S
µ
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4
2
M
M
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2
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M
D
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4
4
7
L
L
L
4
Q
L
Q
Q
Q
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Page 5 of 6
ACS Medicinal Chemistry Letters
Reagent and conditions: (a) 4-methyl-3-nitroaniline, dioxane, 100 oC; (b) NaBH4,
Table 2. Structure-activity relationship of naphthyl ana-
logues
EtOH, 0 oC - rt; (c) MnO2, DCM, rt; (d) triethyl phosphonoacetate, K2CO3, EtOH, 100
oC; (e) R-B(OH)2, Pd(PPh3)2Cl2, tBuXPhos, Na2CO3, dioxane, H2O, 80 oC; (f) SnCl2,
EtOAc, 70 oC; (g) acryloyl chloride, THF/NaHCO3 1:1, 0 oC. (h) 2,4-dimethoxyben-
zylamine, Na(OAc)3BH, AcOH, THF, rt; (i) triphosgene, DIEA, DCM, 0 oC; (j) TFA,
DCM, rt; (k) NaH, MeI, THF, 0 oC.
1
2
3
4
Antiviral
activity
∆Log10
FFU
(DMSO/co
mpound)
Luciferase ac-
tivity: cellular
translation of
DENV2(GV
D)
Micro-
somal
stability
T1/2
Com-
pound
CC50
(µM)
R
5
B
(% DMSO)
(min)
6
7
2
10
2
10
µM
µM
µM
µM
8
9
17
18
19
4
4
4
4
76
39
82
95
79
94
>160
29
4
4
9
2.0
1.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
75
20
0.5
1.0
49
60
>160
nda
21
22
0.5
3.3
4
0.1
4
77
17
65
88
66
95
>160
21
nd
Reagent and conditions: (a) i. PBr3, DMF, CHCl3, 70 oC; ii. DDQ, toluene, 110 oC; (b)
4-methyl-3-nitroaniline, Pd2(dba)3, BINAP, Cs2CO3, toluene, 80 oC; (c) triethyl phos-
phonoacetate, K2CO3, EtOH, 100 oC; (d) i. Ar-B(OH)2, Pd(PPh3)2Cl2, tBuXPhos,
Na2CO3, dioxane, H2O, 80 oC; ii. SnCl2, EtOAc, 70 oC; (e) acryloyl chloride,
THF/NaHCO3 1:1, 0 oC; (f) i. 4-bromocrotonyl chloride, DIEA, acetonitrile, 0 oC; ii.
NHMe2, NMP, 50 oC.
6.3
15.6
YKL-
04-085
a nd = Not determined.
4
12
Table 3. In vivo pharmacokinetic properties of YKL-04-
085.
Table 1. Structure-activity relationship of QL47 and quino-
linyl analogues
Dose
(mg/
kg)
AUClast
(ng
hr/mL)
AUCinf
(ng
hr/mL)
CL
(mL/mi
n/kg)
T1/2
(hrs)
Tmax
(hrs)
Cmax
Vss
(L/kg)
F
(%)
Route
x
x
(ng/mL)
Luciferase ac-
Antiviral ac-
tivity: cellular
translation of
DENV2(GV
i.v.
p.o.
i.p.
i.p.
2
1.07
1.53
461.71
40.41
161.33
95.40
167.28
117.89
1213.28
8554.01
199.26
9.58
tivity ∆Log10
FFU
(DMSO/com
pound)
Micro-
somal
stability
T1/2
Com-
pound
CC50
(µM)
10
10
50
1.00
0.25
0.08
12
R
D)
(%
DMSO)
(min)
786.78
4505.75
1112.65
8222.94
2
10
2
10
µM
µM
µM
µM
98
QL47
4
4
94
68
1.2
ASSOCIATED CONTENT
Supporting Information
1
2
3
4
0
1.9
4
81
44
29
40
100
75
13
2.3
6.4
6.5
4.1
0.8
0.8
1.0
75
4
76
77
Supporting Information containing supplemental figures, tables,
materials and methods, experimental procedures, and compound
characterization (PDF) is available free of charge on the ACS Pub-
lications website.
4
34
> 160
5
6
7
8
2.7
2.8
0.1
0.1
4
82
32
0
95
94
23
2
> 160
46
1.3
2.7
9
4
1.2
0
103
44
AUTHOR INFORMATION
Corresponding Author
0
5
9
0
0.4
23
24
107
7.9
* Nathanael S. Gray. E-mail: nathanael_gray@dfci.harvard.edu
* Priscilla L. Yang. E-mail: priscilla_yang@hms.harvard.edu
10
11
12
4
4
4
4
97
39
4
99
90
93
105
99
4.5
nda
nd
1.1
2.0
83
ACKNOWLEDGMENT
13
0
0
3
19
>160
68
nd
We gratefully acknowledge Dr. Tinghu Zhang for the synthesis of
relevant tool compounds and helpful discussions; and Dr. Michael
Cameron at the Scripps Research Institute for conducting in vitro
mouse microsome study and in vivo tolerability study. This work
was funded by the Linde program in chemical biology.
14
0.1
0.8
49
53
2
15
2.5
2.0
4
4
94
77
99
98
79
2
2
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