Chemical Synthesis Enables Structural Reengineering of Aglaroxin C Leading to Inhibition Bias for Hepatitis C Viral Infection

Article abstract of DOI:10.1021/jacs.8b11477

As a unique rocaglate (flavagline) natural product, aglaroxin C displays intriguing biological activity by inhibiting hepatitis C viral entry. To further elucidate structure-activity relationships and diversify the pyrimidinone scaffold, we report a concise synthesis of aglaroxin C utilizing a highly regioselective pyrimidinone condensation. We have prepared more than 40 aglaroxin C analogues utilizing various amidine condensation partners. Through biological evaluation of analogues, we have discovered two lead compounds, CMLD012043 and CMLD012044, which show preferential bias for the inhibition of hepatitis C viral entry vs translation inhibition. Overall, the study demonstrates the power of chemical synthesis to produce natural product variants with both target inhibition bias and improved therapeutic indexes.

Full text of DOI:10.1021/jacs.8b11477

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Article
Chemical Synthesis Enables Structural Reengineering of
Aglaroxin C Leading to Inhibition Bias for HCV Infection
Wenhan Zhang, Shufeng Liu, Rayelle I Maiga, Jerry Pelletier,
Lauren E. Brown, Tony T. Wang, and John A. Porco
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11477 • Publication Date (Web): 27 Dec 2018
Downloaded from http://pubs.acs.org on January 2, 2019
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Chemical Synthesis Enables Structural Reengineering of Aglaroxin
C Leading to Inhibition Bias for HCV Infection
Wenhan Zhang,a‡ Shufeng Liu,b‡ Rayelle I. Maiga,c Jerry Pelletier,c, d, e Lauren E. Brown,a Tony T.
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Wang,b* and John A. Porco, Jr.a*
a Department of Chemistry, Center for Molecular Discovery (BU-CMD), Boston University, Boston, MA 02215, USA
b Laboratory of Vector-borne Viral Diseases, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug
Administration, Silver Spring, MD 20903, USA
c Department of Biochemistry, d Department of Oncology, e Rosalind & Morris Goodman Cancer Research Centre, McGill University,
Montreal, Quebec, H3G1Y6, Canada
KEYWORDS aglaroxin, natural product synthesis, late-stage modification, viral entry, HCV, rocaglate, natural product analogues,
inhibition bias.
ABSTRACT: As a unique rocaglate (flavagline) natural product, aglaroxin C displays intriguing biological activity by inhibiting
HCV viral entry. To further elucidate structure-activity relationships and diversify the pyrimidinone scaffold, we report a concise
synthesis of aglaroxin C utilizing a highly regioselective pyrimidinone condensation. We have prepared more than forty aglaroxin C
analogues utilizing various amidine condensation partners. Through biological evaluation of analogues, we have discovered two lead
compounds, CMLD012043 and CMLD012044, which show preferential bias for the inhibition of HCV viral entry vs. translation
inhibition. Overall, the study demonstrates the power of chemical synthesis to produce natural product variants with both target
inhibition bias and improved therapeutic indexes.
INTRODUCTION
Rocaglates (flavagline) natural products were first isolated
from the dried roots and stems of Aglaia elliptifolia Merr. (fam-
ily Meliaceae) in 1982.1 Since then, over thirty rocaglate natural
products have been identified with unique structures and intri-
guing biological activities.2, 3, 4 For instance, silvestrol (1, Fig-
ure 1)5 was identified as an excellent translation inhibitor for
cancer chemotherapy,6 whereas other related natural products
and analogues (2-4) displayed similar activities.7 In our previ-
ous studies, translation inhibition was found to be associated
with inhibition of the DEAD box RNA helicase eIF4A.8 Owing
to the interesting structures and biological activities of
rocaglates, a number of total syntheses9 and medicinal chemis-
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try studies10, have been disclosed. Our group has a long-
standing interest in the synthesis of rocaglates and derivatives
as well as investigations of their biology.6, 7, 8, 9g, i, k, o-q, 10a, c, e-f In
2015, we reported the enantioselective synthesis of aglaiastatin
(5) and aglaroxin C (6) through biomimetic kinetic resolution.9p
Subsequent studies revealed that 5 was a promising translation
inhibitor. In contrast, 6, containing a fully substituted pyrimidi-
none core, showed only moderate translation inhibition.9p In
separate studies, we discovered that 6 inhibited hepatitis C viral
(HCV) entry into host cells at a low µM concentration, poten-
tially through inhibition of the prohibitins (PHBs) as viral entry
factors.12, 13 Notably, prohibitins 1 and 2 have been reported as
general viral entry factors for other viruses including dengue
virus type 2 (DENV-2)14 and Chikungunya.15 We were further
encouraged by the observation that 6 did not induce cytotoxicity
by translation inhibition at a concentration that is effective for
inhibition of viral entry, providing a promising therapeutic in-
dex (TI) for potential treatment of HCV infection.12
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Our goal was to reengineer the structure of aglaroxin C (6) to
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HCV is a widespread viral pathogen. The recent estimated
number of viral carriers is 143 million worldwide,16 and over 2%
of the population in North America has been infected. In addi-
tion, chronic HCV infection causes severe liver diseases in car-
riers, including liver cancer and cirrhosis.17 In 2015, over a half
million people died from diseases due to HCV infection.18 Re-
cently, direct-acting antiviral agents (DAAs) have become
available to cure HCV infection19 by inhibiting the function of
non-structural (NS) viral proteins. However, treatment effects
of DAAs vary across different genotypes of HCV, and HCV
potentially may develop resistance to these agents.20 For exam-
ple, the NS3 protease inhibitor simeprevir only treats genotypes
1 and 4 of HCV, and several signature resistance mutations have
been identified against this treatment.21 Sofosbuvir, a top NS5B
inhibitor, failed in HCV treatment which was associated with
resistance mutations (Figure SI1).22 Accordingly, discovery of
alternative treatments for HCV is sorely needed. As PHB-
mediated cellular signaling pathways12 are required by all HCV
genotypes to infect host cells, it is conceivable that a small mol-
ecule such as aglaroxin C (6) may block infection from multiple
HCV genotypes.23 Additionally, targeting the host component
for viral entry may create higher genetic barriers against re-
sistance.24 Lastly, aglaroxin C and analogues may also be valu-
able for dissecting the mechanisms of HCV entry and may in-
hibit other viruses sharing the same entry mechanism.
increase activity against HCV entry while minimizing transla-
tion inhibition, which may lead to undesired cytotoxicity.5, 6, 7, 8
As the oxidation state change between 6 and aglaiastatin (5) led
to different biological profiles, we speculated that the pyrimidi-
none subunit of 6 may be important for inhibition of HCV viral
entry vs. translation inhibition. Our first-generation synthesis of
aglaroxin C (6) utilized a key intermediate, keto-rocaglate 9,25
which was synthesized through the excited state intramolecular
proton transfer (ESIPT) [3+2] cycloaddition between 3-hy-
droxyflavone 7 and methyl cinnamate followed by α-ketol shift
of the aglain intermediate 8 (Scheme 1).9i However, the late
stage pyrimidinone synthesis in our first-generation synthetic
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route is not ideal for rapid analog synthesis, affording 6 in low methods for the synthesis of pyrimidinones and pyrimidines, in-
yield on a multi-milligram scale (Scheme 1).26 We also envi-
sioned that stepwise pyrimidinone synthesis may suffer from
poor functional group tolerance in analogues due to the use of
both acidic and oxidative conditions. Moreover, use of tethered
aminoacetals such as 10 in the ester-amide exchange to produce
intermediate 11 considerably narrows the diversity of accessi-
ble analogues due to limited availability of such building blocks.
We therefore considered a streamlined synthesis of aglaroxin
analogues through late stage, one-step pyrimidinone formation
(Scheme 2).
cluding the Traube synthesis of purines,29 which inspired us to
consider late stage construction of the pyrimidinone core of
aglaroxin analogues.30 However, we anticipated that condensa-
tion between the structurally complex substrate 9 and amidines
13 would be challenging. In particular, the highly ionizable ter-
tiary, benzylic alcohol adjacent to the ketoester moiety may pro-
vide unexpected reactivity under the reaction conditions.
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Our initial model study for pyrimidinone formation em-
ployed benzamidine as a simplified condensation partner. Mi-
crowave conditions (Table 1, entry 1) produced minimal
amounts of pyrimidinone 12a in an unsatisfactory yield of 28%
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RESULTS AND DISCUSSION
1
(determined by H NMR analysis) due to low conversion.9p
Development of a Direct Pyrimidinone Formation. Our
second-generation synthetic route (Scheme 2), which relies on
condensation of keto-rocaglate (9) with commercially available
amidines 13, was expected to flexibly provide analogues (12)
for biological experiments. Pyrimidinones have served as bio-
logically important substructures in both drugs and natural
products.27, 28 There are many well-established and practical
Thermal conditions (entry 2) resulted in full consumption of 9,
while cyclopentapyrimidinedione 16 and 3,5-dimethox-
yphloroglucinol were found as the major identified side prod-
ucts. The formation of fragmentation product 16 was found to
correlate with the amount of 4-dimethylaminopyridine (DMAP)
employed; reduction to 0.3 equivalents of DMAP minimized
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production of 16 (entry 3) but also led to formation of an unex-
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pected cyclization product, oxazoline 15. The structure of 15
was confirmed by single crystal X-ray crystal structure analy-
sis.26 Reduced equivalents of the benzamidine reaction partner
resulted in an incomplete reaction, with increased production of
decarboxylation product 14 and retro-Nazarov products 17 (en-
tries 4 & 5).26 We next found that diluting the reaction eight-
fold improved the yield of 12a (entry 6). Increasing both tem-
perature and reaction time under dilute conditions eliminated
the production of 15, but also led to a lower yield of 12a in favor
of significant amounts of 16 (entry 7). Gratifyingly, by reducing
the reaction time to 45 minutes, we obtained a 90% NMR yield
of 12a with minimal fragmentation to 16 (entry 8); ultimately,
an 81% isolated yield of 12a was obtained on a 100-mg scale.
Interestingly, we found that the model reaction gave the almost
identical yield in absence of DMAP under the optimized condi-
tions (entry 9), but we acheived better reproducibility when am-
idine hydrochloride salt was used with DMAP (vide infra).
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Proposed Mechanistic Pathway. According to trends ob-
served during reaction optimization, we propose the mechanis-
tic pathway depicted in Scheme 3. We postulate that water gen-
erated from the pyrimidinone condensation may hydrolyze 9 to
the β-keto-acid intermediate 21, which undergoes decarboxyla-
tion to ketone 14. When no nucleophile was used, 14 and retro-
Nazarov product 17 were obtained. Presumably DMAP may
promote intramolecular proton transfer to 19 followed by the
extrusion of water to afford the retro-Nazarov precursor 20,
whereas the formed water triggered decarboxylation of 9 (an
alternative mechanism is shown in the SI). In the presence of
the amidine, the desired formation of hemiaminals 22 and epi-
22 appears to compete with decarboxylation and retro-Nazarov
reaction, which can be enhanced through use of increased
equivalents of the amidine. Based on the Katrizky mechanism
determined for the Traube reaction of β-keto esters and ami-
dines,31 two possible pathways may be envisioned for the for-
mation of pyrimidinone 12a. In one mechanism, hemiaminal
epi-22, generated from disfavored concave-addition of the am-
idine to 9,9i may cyclize to dihydropyrimidinone 25 followed by
extrusion of water. In contrast, hemiaminal 22, obtained from
amidine addition to the convex face of 9, has an anti-relation-
ship between the aminal and ester thereby preventing direct cy-
clization. Instead, loss of water would produce the observed
enamine 23, which we propose then cyclizes to 12a through ex-
trusion of methanol generating imidoyl ketene 24 followed by
a 6π-electrocyclization to pyrimidinone 12a.32 To support the
formation of hemiaminal 22, the oxazoline byproduct 15 was
formed through cyclization of the tertiary alcohol of 22 to the
amidine carbon followed by extrusion of ammonia (blue arrow).
The formation of 22 is also supported by our isolation of
enamine 23 from a 250 mg scale reaction, where a 28% yield of
23 was found to precipitate after 10 minutes; we subsequently
found that 23 can also be synthesized in 60% yield using 9 and
benzamidine at 60 ℃ in toluene.26 Interestingly, isolated 23 was
found to solely produce 12a with no observed formation of 15
when 23 was resubjected to the reaction conditions. We ration-
alize that the sp3-hybridized hemiaminal carbon of 22 allows for
a conformation necessary for intramolecular cyclization,
whereas the sp2-hybridized enamine carbon of 23 prevents a
similar cyclization. This conforms to the observation that ele-
vated temperatures facilitate extrusion of water generating
enamine 23 and minimize formation of byproduct 15. In con-
trast, 15 did not generate 12a under the reaction conditions. In
a control experiment, DMAP was found to accelerate the frag-
mentation of 12a into 16 and 3,5-dimethoxyphloroglucinol.26
Second-Generation Synthesis of Aglaroxin C. After deter-
mining optimal conditions for direct pyrimidinone formation,
we next sought to apply these conditions to synthesize aglaroxin
C (6). In this case, the requisite pyrrolidin-2-imine was com-
mercially available as an HCl salt (Table 2). Initial attempts us-
ing stepwise free-basing protocols failed due to the high-water
solubility of the amidine; accordingly, we focused on in-situ
free-basing conditions for optimization studies. Using excess
base (entries 1 and 2), no conversion of substrate 9 was ob-
served after refluxing for 12 h. As 9 exists as mixture of
enol/keto isomers, presumably excess base favors formation of
an unreactive enolate. In contrast, use of excess pyrrolidin-2-
imine HCl salt (entry 3) did not induce pyrimidinone formation.
We assume that DMAP is protonated by the amidine salt which
negated its function. When a slight excess of amidine vs. Na-
OMe (entry 4) was used, we obtained 6 in 19% isolated yield.
Consistent with our earlier optimization efforts, use of in-
creased concentration and 12 h reaction time resulted in a com-
plex reaction mixture containing fragmentation products (entry
5). Finally, use of 3.0 equiv. of amidine salt and 2.95 equiv. of
NaOMe along with a catalytic amount of DMAP (40 mol%, 130
oC) afforded 6 in 76% isolated yield on a 100-mg scale (entry
6).
Synthesis of Aglaroxin Analogues. With reliable annulation
protocols available using both amidines and amidine salts, we
next synthesized a library of aglaroxin analogues (Table 3). In
general, direct pyrimidinone formation was found to tolerate
both aromatic and aliphatic amidines. We observed consistent
yields for formation of C-substituted pyrimidinones (12b-12f).
For instance, simple aliphatic amidines reacted with 9 to afford
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C-Me (12b) and C-Bn (12c) pyrimidinones. Hindered amidines
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such as iso-butanamidine and cyclohexylcarboxamidine af- As a comparison to the direct condensation with unsymmet-
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forded the desired products 12d and 12e in excellent yields. In-
terestingly, condensation of carbomethoxyformamidine with 9
was followed by an unexpected decarboxylation to produce the
non-substituted product 12f in 53% yield. Moreover, we discov-
ered that the pyrimidinone condensation could tolerate various
functional groups, including ester 12g (41%), cyclopropane 12h
(91%), and methoxyl methylene 12i (52%). Ester exchange was
observed when ethyl amidinoacetate was employed, producing
compound 12g. Additionally, guanidine-type reaction partners
successfully underwent condensation, yielding products such as
12j (90% yield).
rical amidines, we also attempted alkylations of 12a to produce
compounds 12ai-aj (Table 4) 12al-an (see Supporting Infor-
mation).26 Unfortunately, all attempted alkylations displayed
poor chemo- and regioselectivity, favoring the undesired O-al-
kylation products such as O-methoxypyrimidine 12ai.
BIOLOGICAL STUDIES
Structure-Activity Relationships. We next evaluated the li-
brary of (±) -aglaroxin analogues and side products against HCV
infection, and also tested their corresponding cytotoxicity in hu-
man hepatic (Huh) 7.5.1 cells.26 In comparison to aglaroxin C
(6), C-alkyl substituted pyrimidinones 12b-d and 12h (Table 3)
exhibited increased inhibition against HCV infection, whereas
12e-g had decreased activities. Nevertheless, the C-aryl substi-
tuted products (12a, k-w) showed a promising increase of inhi-
bition of HCV infection with similar or reduced cytotoxicity in
comparison to 6. Excitingly, 12l (CMLD012043) and 12s
(CMLD012044) showed a three-fold greater inhibition of HCV
infection in comparison to 6, while 12l and 12s exhibited rela-
tively low cytotoxicity to Huh-7.5.1 cells (less than 50% cell
death at 200 μM). Thus, the two lead compounds (12l and 12s)
provided excellent selectivity indexes (SI) in inhibiting HCV
infection (vide infra). Notably, among all the C-heteroaryl sub-
stituted aglaroxin analogues (12x-12ac), only 12y and 12ab dis-
played slightly increased inhibition of HCV infection relative to
6. The six-membered ring analog 12ad and guanidine-type ad-
ducts 12j and 12af were found to have moderate antiviral activ-
ity.
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Preliminary biological studies of the analog set indicated that
C-aryl pyrimidinones consistently possessed good inhibition of
HCV infection with low cytotoxicity (vide infra).26 Accordingly,
we synthesized additional C-aryl pyrimidinone analogues with
a variety of substitution patterns, with an initial focus on para-
substituted aryl benzamidines (Table 3, 12k-r). Generally, con-
densations tolerated both electron-rich and deficient benzami-
dines; however, we found that greater amounts of fragmentation
products were generated using electron-deficient amidines (cf.
compounds 12k-n 66-83% yield) with compound 12o, which
was generated in 53% yield along with the corresponding frag-
mentation products. Compound 12m was further subjected to
hydrogenolysis to afford the free phenol which may serve as a
potential handle for further modifications.26 As expected, ana-
logues including para-halides (12p-12r) were synthesized in
70-80% yields. Such compounds may also allow for late stage
functionalization via aminations and SNAr reactions. We found
that meta-substituted benzamidines were also workable, afford-
ing products (12s-12u) using the standard protocol in reasona-
ble yields (71%-73%). Using the sterically hindered ortho-sub-
stituted benzamidines, 12v and 12w were synthesized in mod-
erate yields (54% and 66%). Six C-heteroaryl substituted ana-
logues (12x-12ac) were also synthesized; in these cases, we ob-
served substantial fragmentation of the desired products. Only
12x and 12z were obtained in reasonable yields (92% and 77%,
respectively), while compounds 12y and 12aa-12ac were ob-
tained in yields averaging 50%.
To further understand structure-activity relationships (SAR)
among aglaroxin analogues, we highlight ten compounds in Ta-
ble 4 along with dose-response curves depicting their effective-
ness in both HCV infection and cytotoxicity assays. As a bench-
mark compound, 6 was found to inhibit HCV infection with an
EC50 of 1.3 μM and showed a CC50 of 12 μM. Of note, EC50 and
CC50 values were calculated according to the fitted sigmoid
curves, which are reported as absolute values (indicating the
concentration of compounds providing 50% inhibition and 50%
cell death, respectively). Next, we compared the EC50 of cyclo-
hexyl- (12e) and phenyl-substituted (12a) pyrimidinones and
found that C-aryl substitutions led to improved potency against
viral infection (420 nM vs. 2.5 μM). The C-3 epimer (12ah) of
12a was found to be inactive against HCV infection and no cy-
totoxicity. It is apparent that a syn-relationship of the two aryl
rings on the cyclopenta[b]benzofuran core is crucial for HCV
inhibition.
As we found that aglaroxin C (6, Table 2) was synthesized
regioselectively using an unsymmetrical amidine, we also
tested additional unsymmetrical amidines in the pyrimidinone
formation. Among all products formed, we found that the N-
substituent was situated exclusively on the nitrogen adjacent to
the pyrimidinone carbonyl (12ad-af). During the synthesis of
12ae, a small amount of oxazoline 15 was obtained as the only
observable side product. This result supports our mechanistic
proposal (cf. Scheme 3) wherein the less sterically hindered, un-
substituted nitrogen likely engages in initial hemiaminal for-
mation with 9. Along these lines, we also synthesized 12ac, a
ring-expanded analog of 6, in 69% yield. Unlike 12ad, adducts
12ae and 12af were synthesized in 24 and 33% yields, respec-
tively; these reactions generated significant amounts of retro-
Nazarov and decarboxylated products, suggestive of lower
overall reactivities among the N-substituted amidines.26 We
also optimized the yield of 12ae to 39% by increasing the reac-
tion concentration to 0.2 M. Finally, using exo-keto-rocaglate
as the starting material, we synthesized compounds 12ah and
12ag, the C-3 epimers of 12a and 6, in 77 and 52% yields, re-
spectively.
Based on the observation that N-methylated isomer 12ae was
one-fold more active than 12a (EC50 0.2 μM vs. 0.42 μM) with
similar cytotoxicities (CC50 7.3 μM vs. 8.1 μM),33 we next uti-
lized methylation to evaluate other sites for introduction of tar-
get identification tags. In contrast to N-methylation, we ob-
served a decrease in inhibition of HCV infection (EC50 = 9.2
μM) with the O-methylated pyrimidine 12ai. The doubly meth-
ylated product 12aj was also found inactive and non-toxic. Fi-
nally, we studied the impact of substituting the conjugated C-
aryl group of the pyrimidinone (cf. 12l, 12o, and 12s). Com-
pound 12o, with an electron-withdrawing CF3-group, had
higher cytotoxicity (CC50 = 10 μM), and only a one-fold better
EC50 (0.24 μM) for HCV inhibition than 12l and 12s (EC50
~
0.5 μM). The two lead compounds 12l and 12s displayed im-
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proved EC50s in the viral infection assay but displayed low cy-
curve (AUC) by integration,34 an alternative method for accu-
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totoxicity; neither compound reached 50% cell death up to 200
μM concentration (maximum cytotoxicity plateau: 40% and 35%
cell death for 12l and 12s, respectively). Of note, we observed
a similar plateauing of cytotoxicity using aglaroxin C (6) and its
analogues (12e, 12a, and 12ae). In contrast, the 4’-trifluoro-
methylphenyl analog 12o achieved close to 100% cell death.
For a more reliable comparison of cytotoxicities among these
compounds, we also calculated the area under the cytotoxicity
rately quantifying low cytotoxicity.35 In this manner, AUC0.2-200
analysis indicates that 12e, 12a, and 12ai share similar cytotox-
icities to 6, whereas 12l and 12s exhibit relatively lower cyto-
toxicities. Extending this analysis to compare the SI among the
analogues, we next calculated the AUC0.2-20(EC/CC) ratios, de-
termining that 12ae, 12l, 12o, and 12s had wider therapeutic
windows (0.6 to greater than 2-fold increase in the SI) than 6.
Based on the performance of 12l and 12s using these metrics,
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we utilized these two lead compounds for further biological in- this system, translation of the Firefly luciferase (FLuc) transla-
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vestigations including mechanism studies.
tion is cap-dependent whereas Renilla luciferase (RLuc) trans-
lation is dependent upon the HCV internal ribosome entry site
(IRES) (Fig. 3A). It was observed that translation inhibition of
firefly luciferase by 6 and analogues 12l and 12s were minimal
To further characterize the mode of action of the two lead
compounds 12l and 12s, we independently synthesized each of
their respective enantiomers and investigated their biological
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in comparison with CR-1-31-B (Figure 1, 4), a highly potent
rocaglate translation inhibitor showing strong inhibition of cap-
dependent protein synthesis (Figure 3B).10c This data suggests
that HCV mRNA translation is not inhibited by 6 and its ana-
logues 12l and 12s.
activities in both the HCV viral infection and cytotoxicity as-
says (Table 5). As expected, only (+)-12l and (+)-12s displayed
HCV inhibition, whereas the other enantiomers (-)-12l, and (-)-
12s were found to be inactive and non-toxic.26 Interestingly, we
noticed that maximum cytotoxicity plateau of both active enan-
tiomer (+)-12l and (+)-12s increased substantially compared to
their racemic counterparts (±)-12l and (±)-12s. To verify this
result, a secondary MTS cell viability assay was also performed
on racemic compounds (±)-12k, (±)-12l, and (±)-12o, and
showed nearly identical toxicity curves as were observed in the
CellTiter-Glo assay.26 While these results warrant further inves-
tigation, they suggest a potential rationale for the observed SI
enhancement in which the “inactive” enantiomers may reduce
or otherwise mitigate the cytotoxic effects of the active species
through an as-yet undefined mechanism.
Aglaroxin Analogues Do Not Affect HCV RNA Replica-
tion and Translation. We subsequently evaluated the ability of
the aglaroxin C analogues to inhibit HCV RNA replication and
mRNA translation using an HCV replicon system which har-
bors a full-length HCV Genotype 1b RNA genome.36 In repli-
con cells, HCV RNA replicates and is translated into viral pro-
teins without forming infectious viruses. Hence, this system
permits accurate assessment of any effects on viral RNA repli-
cation and translation. As shown in Figure 2, compounds (+)-
12l and (+)-12s when used at 2 µM for 3 h did not inhibit HCV
RNA replication or protein synthesis. This data implies that the
observed inhibitory effects of (+)-12l and (+)-12s were unlikely
due to inhibition of viral RNA replication or mRNA translation.
To corroborate these findings, we assembled an in vitro trans-
lation inhibition assay where a bicistronic reporter mRNA was
programmed for translation in Krebs-2 extracts (Figure 3).37 In
Aglaroxin Analogues Inhibit HCV Entry. As aglaroxin C
was previously reported to inhibit HCV entry,12 we carried out
two sets of experiments to test whether compounds 12l and 12s
also inhibit viral entry. Firstly, we generated infectious lentivi-
ral pseudotypes bearing glycoproteins of HCV (HCVpp),
Chikungunya virus (CHIKVpp), Ebola virus (Ebolapp), and ve-
sicular stomatitis virus (VSVpp). Compositionally, these
pseudotyped viruses differ only in viral envelopes because they
are packaged using the same lentiviral reporter construct with a
separate construct expressing specified viral envelope protein.
Hence, the only difference among these pseudoviral particles is
the mode of entry, which is dictated by the particular viral gly-
coprotein found on the viral envelopes. Compounds 12l and 12s,
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as both the racemates and single (+)-enantiomers, specifically synthesize a library of over forty aglaroxin C analogues. Among
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inhibited HCVpp and CHIKVpp but not Ebolapp and VSVpp
(Figure 4, A and B). Interestingly, like rocaglamide, compounds
(+)-12l and (+)-12s as well as their racemic versions also inhib-
ited Dengue virus infection (Figure 4C). It is worth mentioning
that HCV, Dengue virus, and CHIKV all utilize PHBs to enter
cells; further studies are warranted to determine whether com-
pounds 12l and 12s directly target PHBs. Secondly, to confirm
inhibition of HCV entry, we performed time-of-addition exper-
newly synthesized analogues, we successfully demonstrated
SAR for inhibition of HCV infection and identified two aryl py-
rimidinone lead compounds, 12l and 12s, which have low cyto-
toxicities to Huh 7.5.1 cells. Additional biological studies with
12l and 12s indicate that the mechanism of inhibition of HCV
infection is through inhibition of HCV viral entry, rather than
by blocking viral replication and translation. Finally, 12l and
12s are also effective against infection of other viruses includ-
ing Dengue and Chikungunya, both of which have been found
to use prohibitins (PHBs) as an entry factor. These studies illus-
trate the power of chemical synthesis to bias inhibition of HCV
viral entry vs. translation inhibition and improve properties in-
cluding therapeutic index. Further studies toward target identi-
fication of 12l, 12s, and related compounds is currently in pro-
gress and will be reported in due course.
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures and characterization data for all new
compounds described herein, including a CIF file for compound
15. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* porco@bu.edu
* tony.wang@fda.hhs.gov
Author Contributions
‡These authors contributed equally.
ACKNOWLEDGMENT
We thank the National Institutes of Health (R01 DK088787 and
R35 GM-118173) and the Canadian Institutes of Health Re-
search (FDN-148366) for research support, Dr. Jeffrey Bacon
(Boston University) for X-ray crystal structure analyses, Dr.
Norman Lee (Boston University) for high-resolution mass spec-
trometry data, and Nicholas Grigoriadis (Boston University) for
preliminary studies NMR (CHE-0619339) and MS
(CHE0443618) facilities at Boston University are supported by
the NSF. Research at the BU-CMD was supported by NIH grant
GM-067041. We thank Dr. Kyle Reichl (Boston University) for
proofreading the manuscript.
iments using the lead compounds 12l and 12s wherein com-
pounds were added at different times relative to when the virus
was added to cells. Similar to 6, 12l and 12s displayed maximal
anti-HCV activity when added together with the virus, but par-
tially lost their activity when added 3 h after infection was ini-
tiated (Figure 4D). This finding confirmed that 12l and 12s are
preferentially inhibiting viral entry.
CONCLUSION
In summary, we have developed a second-generation syn-
thesis of aglaroxin C using late-stage, direct pyrimidinone for-
mation of a keto-rocaglate scaffold. Using this method, we have
used commercially available amidines as reaction partners to
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