Discovery, Optimization, and Characterization of Novel Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection

Article abstract of DOI:10.1021/acs.jmedchem.5b00752

Recently, we reported that chlorcyclizine (CCZ, Rac-2), an over-the-counter antihistamine piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we describe structure-activity relationship (SAR) efforts that resulted in the optimization of novel chlorcyclizine derivatives as anti-HCV agents. Several compounds exhibited EC₅₀ values below 10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus pathogenesis and host-virus interaction.

Full text of DOI:10.1021/acs.jmedchem.5b00752

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Article
Discovery, Optimization, and Characterization of Novel Chlorcyclizine
Derivatives for the Treatment of Hepatitis C Virus Infection
Shanshan He, Jingbo Xiao, Andrés E Dulcey, Billy Lin, Adam Rolt, Zongyi Hu, Xin Hu, Amy
Q Wang, Xin Xu, Noel Southall, Marc Ferrer, Wei Zheng, T Jake Liang, and Juan J Marugan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00752 • Publication Date (Web): 24 Nov 2015
Downloaded from http://pubs.acs.org on November 26, 2015
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Discovery, Optimization, and Characterization of
Novel Chlorcyclizine Derivatives for the Treatment
of Hepatitis C Virus Infection
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Shanshan He, §,† Jingbo Xiao, §,‡ Andrés E Dulcey, ‡ Billy Lin, † Adam Rolt, ‡ Zongyi Hu, † Xin
Hu, ‡ Amy Q. Wang, ‡ Xin Xu, ‡ Noel Southall, ‡ Marc Ferrer, ‡ Wei Zheng, ‡ T. Jake Liang,*,† and
Juan J. Marugan*,‡
†Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892
‡Division of PreꢀClinical Innovations, National Center for Advancing Translational Sciences,
National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850
KEYWORDS
hepatitis C virus, antihistamines, chlorcyclizine, antivirals
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ABSTRACT
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Recently we reported that chlorcyclizine (CCZ, Rac-2), an overꢀtheꢀcounter antihistamine
piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we
describe structureꢀactivity relationship (SAR) efforts that resulted in the optimization of novel
chlorcyclizine derivatives as antiꢀHCV agents. Several compounds exhibited EC50 values below
10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved
in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical
candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus
pathogenesis, and hostꢀvirus interaction.
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INTRODUCTION
Hepatitis C virus (HCV) is an enveloped positiveꢀstranded RNA virus of the Flaviviridae
family. The HCV replication cycle is initiated by virions entering the host cell via interaction
with cell surface receptors. Following pHꢀdependent fusion and uncoating, the HCV RNA
genome is translated. The resulting polyprotein undergoes proteolytic cleavage into structural
(core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins.
After RNA replication, HCV undergoes assembly, maturation and secretion processes.1, 2
HCV leads to acute and chronic inflammatory hepatic infections that often progress toward
chronic liver diseases, including cirrhosis, with an elevated risk of developing hepatocellular
carcinoma. There is no vaccine for HCV to date.3 Besides there being around 180 million people
chronically infected worldwide with HCV, the standard of care for many years has been limited
to interferon α (IFNꢀα) in combination with ribavirin (RBV).4, 5 This combination therapy is
partially effective with serious adverse effects. Drug discovery efforts during the past 20 years
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led to a new paradigm for HCV treatment, which is marked by the recent approval of multiple
directꢀacting antivirals (DAAs) for interferonꢀfree regimens.2, 6, 7 The DAAs inhibit certain
replication steps in the HCV replication cycle by directly targeting viral proteins, such as
NS3/4A proteases, NS5A or NS5B polymerase.8 Viral rebounds were often observed during
monotherapy of these DAAs due to selection of drugꢀresistant viral mutants. Thus, the clinical
application of DAAs is mostly limited to combination regimens, which face challenges including
additional side effects, complex administration and drugꢀdrug interactions.2 Sofosbuvir,9 a DAA
that has been approved for interferonꢀfree regimen, costs more than $80,000 during a typical 12ꢀ
week course of treatment alone, raising concerns about the affordability of DAAs in order to
globally impact the burden of HCV disease.10, 11 Hostꢀtargeting agents (HTAs), on the other
hand, inhibit host factors that are essential in the viral replication cycle. Promising hostꢀfactor
targets of HTAs include entry factors, components of viral replication complex cyclophilin A,
and miR122 that binds to viral RNA to facilitate replication.1 There is a higher genetic barrier to
develop resistance to HTAs.12 Moreover, HTAs can also be used as chemical probes to elucidate
antiꢀHCV mechanisms and hostꢀvirus interactions. However, few HTAs candidates are in the
antiꢀHCV drug discovery pipeline, possibly due to the nature of primary drug screen assays that
were mostly based on certain viral proteins or HCV replicons. Overall, there is still need to
improve the current therapeutic regimens by exploring novel antiꢀHCV targets and small
molecules.
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Recently, we reported the antiꢀHCV activity of chlorcyclizine (CCZ, Rac-2), discovered
through the screening of the NCGC Pharmaceutical Collection (NPC), in a cellꢀbased antiꢀHCV
quantitative highꢀthroughput screening (qHTS) platform.13ꢀ15 The hit compound chlorcylizine
HCl (CCZ (Rac-2), Figure 1) showed potent in vitro antiꢀHCV activity and preferable liver
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distribution in mouse models, as well as in vivo efficacy against HCV infection in Albꢀ
uPA/SCID chimeric mouse model engrafted with primary human hepatocytes.14 CCZ (Rac-2)
was also reported by ChamounꢀEmanuelli et al. to block HCV entry, possibly via a cholesterolꢀ
dependent pathway.16 However the target and precise mechanism of action of CCZ (Rac-2) in
inhibiting HCV entry remains unknown. Here, we present an SAR study aiming to optimize CCZ
(Rac-2) for an antiꢀHCV application focusing on the following features: generation of a nonꢀ
chiral lead, improvement of its antiꢀHCV potency, modulation of its physicochemical properties
to potentially reduce CNS exposure, reduction or elimination of its antihistamine activity, and
improvement of pharmacokinetic properties. The resulting lead compounds in this series,
represented by nonꢀchiral compound 30, exhibited increased antiꢀHCV activity and selectivity
(up to 19ꢀfold and 8ꢀfold, respectively) and improved in vivo pharmacokinetics properties. The
optimized lead compounds merit further preclinical development for the treatment of hepatitis C.
RESULTS
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The synthesis of CCZ analogs is displayed in Schemes 1ꢀ5. Scheme 1A shows the synthesis of
asymmetrical CCZ derivatives. Thus, following modified literature procedrues, the
corresponding aldehyde or ketone underwent reductive amination with commercially available
chiral (R)-1 or (S)-1, and NaBH3CN in the presence of acetic acid (compounds 10-15)28 or pꢀ
toluenesulfonic acid (compound (S)-18) using an alcoholic solvent, to afford the corresponding
Nꢀalkylated derivatives. Ti(OiPr)4 was required to carry out the reductive amination of the
cyclopentyl ((S)-16) and cyclohexyl ((S)-17) derivatives.17 Acylation of (S)-1 with acetyl
chloride afforded Nꢀacyl derivative (S)-19. Deuterated derivatives (R)-20 and (S)-20 where
synthesized from (R)-1 and (S)-1 with CD3I in the presence of aqueous NaOH. Likewise,
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trifluorinated derivatives (R)-21 and (S)-21 were prepared from the common chiral starting
material and trifluoroethyl triflate in the presence of K2CO3.
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Scheme 1B displays the synthesis of racemic CCZ analogs with a solubilizing polyethylene
glycol side chain. Starting from the commercially available hydroxyzine (Rac-5), introduction of
the phthalimido moiety via standard Mitsunobu conditions afforded Rac-22. Subsequent
hydrazine mediated deprotection afforded primary amine Rac-23. Elongation of the solubilizing
side chain was obtained by alkylating Rac-5 to produce NꢀBoc derivative Rac-24, which was
deprotected to the free amine Rac-25 and subsequently acylated to afford Rac-26 (Scheme 1B).
The synthesis of achiral dichlorcyclizine derivatives is displayed in scheme 2A. Starting from
dichlorobenzophenone 27, NaBH4 mediated reduction of the ketone, followed by chlorination
with SOCl2 afforded chloride 28, which was used to alkylate a variety of cyclic amine
derivatives following modified literature procedures to yield compounds 29-33 (Scheme 2A).29ꢀ31
Piperazine derivative 29 was further alkylated, following a modified literature procedure, to
produce the diehtylene glycolꢀcontaining compound 34,32 which was further elongated to the
pentaethylene glycol amine 35. To probe the halogen effect on the cyclizine scaffold, compounds
38 and 39 were prepared according to Scheme 2B, via reductive amination of acetaldehyde with
the corresponding piperazines 36 and 37 in the presence of NaBH3CN and acetic acid in
methanol.
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Cyclizine scaffold modification via exchange of the central tertiary carbon with the nitrogen
analog is displayed in Scheme 3A, where methyl piperidone underwent reductive amination with
the corresponding aniline 40 and 41 to afford the intermediate amines 42 and 43, which
subsequently underwent Nꢀarylation under Buchwald conditions to afford amines 44 and 45. The
last modification studied was the introduction of rigidity into the cyclizine scaffold. As displayed
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in Scheme 3B, this was accomplished by starting from the commercial bromofluorenone 46,
which was reduced to the alcohol with NaBH4, and subsequently converted to the chloride 47
with CaCl2 and concentrated HCl. Chloride 47 was used to alkylate Nꢀethyl piperazine to afford
the cyclizine rigid analog 48 (Scheme 3B).
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Tables 1ꢀ3 disclose the activity of synthesized compounds in our structural/chemical
modification study centered around CCZ (Rac-2). The antiꢀHCV activity was reported in EC50
values (concentration of compound inhibiting 50% of viral levels in comparison with DMSO
control) using the HCVꢀLuc (HCV JFHꢀ1 strain (GT2a) with insertion of the luciferase reporter
gene) infection assay. The cytotoxicity was measured by CC50 values (concentration of
compound exhibiting 50% cytotoxicity in comparison with DMSO control) evaluated with an
ATPlite assay in Huh7.5.1 cells, the same cell line used for measuring the antiviral activity. Thus,
the activities of the hit compound, racemic CCZ (Rac-2) and its enantiomers ((R)-2 and (S)-2)
were confirmed as having good selectivities (selective indices = 1132~1875, Table 1). NorꢀCCZ
(compound 1), a known in vivo metabolite of CCZ, showed comparable antiꢀHCV activity, but
with reduced selectivity (Table 1). It should be noted with interest norꢀCCZ’s lack of
antihistamine activity (Table 4).18ꢀ20
In Table 1, we addressed the effect of two types of structural modifications: (1) chiral
configurations, and (2) side chains off the piperazine ring. The enantiomers of compounds 1, 2,
10, 11, 12, 13, 15, 20, and 21 showed comparable EC50 and CC50 values, suggesting that the
chiral configuration of CCZ analogs does not have a major effect on the antiviral activity and
selectivity. With compounds 10, 11, 12, 13, and 18, we investigated the effect of the length of
the aliphatic chain on the antiꢀHCV activity. When the number of carbons was less than 4,
analogs showed comparable activity in the range of low doubleꢀdigit nanomolar and comparable
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selectivity to CCZ (Rac-2), regardless of whether the chain was linear or branched. Chains with
a carbon number of 4 led to decreased activity as shown for compounds 12 and 13. Surprisingly,
compound 16, having a cyclopentyl ring exhibited an improved activity (EC50 = 19 nM) while
compound 17, having a cyclohexyl ring, showed an activity in the expected range (EC50 = 177
nM), comparable to compounds 12 and 13. Additionally, acetylation of the piperazine ring
dramatically reduced the potency of analogue 19 to doubleꢀdigit micromolar value. Introduction
of a bulky substituted benzyl group also reduced the antiꢀHCV activity (compound 14, EC50 =
456 nM). Deuterium, introduced in the side chain of compounds 20 and 15 with the aim of
increasing their metabolic t1/2, did not impact their antiꢀHCV activity, and indeed lead to a
slightly improved t1/2 value (2.9 h for (R)-15 in comparison with 2.2 h for (R)-10 in rat
microsomal stability assay). Moreover, the trifluoromethyl group in compound 21 led to a
complete loss of activity, potentially indicating that the pKa value of the tertiary amine was
important for the antiꢀHCV activity.
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The side effects of CCZ (Rac-2) include drowsiness, dizziness and headache, attributed to its
ability to cross the bloodꢀbrain barrier and its CNS penetration. For this reason, first generation
antihistamine drugs were replaced by analogs like cetirizine, with reduced CNSꢀrelated side
effects.21 In order to explore the possibility of reducing the CNS penetration of our molecules,
we further expanded our synthetic efforts introducing oligoethylene glycol side chains. This
structural modification would increase the molecular weight, solubility, total polar surface area
and number of free rotatable bonds, also potentially leading toward longer pharmacokinetic halfꢀ
lives. By using ethylene glycol or polyethylene glycol as linker, we explore the effect of
introducing a series of functional groups at the terminal position. Compound Rac-3 having a
terminal carboxylic acid, completely lost the antiꢀHCV activity, potentially due to lack of cell
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permeability. Moderate activities in the range of tripleꢀdigit nanomolar to singleꢀdigit
micromolar were observed with carboxamideꢀsubstituted compounds Rac-4, Rac-22, Rac-24,
and Rac-26 (Table 1). Compounds Rac-5, Rac-23 and Rac-25, having hydroxyl or amino
groups at the chain terminal position, displayed high antiꢀHCV activity and moderate
cytotoxicity. It was noteworthy that a 6ꢀfold increased activity and retained selectivity was
observed with compound Rac-23 (EC50 values below 8 nM with selective indices above 1000)
(Table 1).
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Table 2 shows the activity of analogs exploring different phenyl ring substituents. Thus,
elimination of the paraꢀchloro substituent reduced the activity from doubleꢀdigit nanomolar
(compound Rac-2, EC50= 44 nM) to singleꢀdigit micromolar (compound 6, EC50= 1.14 ꢁM).
Introduction of an additional paraꢀcloro substituent in the other ring slightly increased the
activity (compound 30, EC50= 17 nM). Following similar trend, nonꢀchiral dichloro compounds
29, 31, 34 and 35 also showed increased activity (1.3 ~ 8.7ꢀfold) in comparison with the
corresponding monochloro analogues Rac-1, (S)-10, Rac-5, and Rac-25. In general, the
introduction of Br substituents led to comparable activity and selectivity to the corresponding Cl
substituted compounds as shown in Rac-36, Rac-37, Rac-38, and Rac-39.
Structural modifications to the piperazine core are displayed in Table 3, where compounds
Rac-2, 29, 6 and Rac-38 were included for comparison. Compound Rac-7, having a one carbon
extension of the piperazine ring, retained the activity but led to increased cytotoxicity. The
replacement of the piperazine ring with other ring structures in compounds 32, 33, and 9 led to a
dramatic loss of activity. In contrast, compounds 44, 45, and 8 retained the antiꢀHCV activity
and selectivity.
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Compound 48, which has a rigid cyclizine scaffold, showed an EC50 value that was more than 1
ꢁM, indicating that the conformation of the rings was important for the antiꢀHCV activity.
From our SAR studies, we selected a number of lead compounds based on their antiꢀHCV
activity, selectivity and structure diversity for an expanded characterization (Table 4). The
cytotoxicity of these molecules was further evaluated in HepG2 cells and primary human
hepatocytes. All compounds showed less than 1.8ꢀfold difference in CC50 values in these two cell
types in comparison with Huh7.5.1 cells (used for our antiꢀHCV activity assay), with the
exception of compound 31, which was less cytotoxic in HepG2 cells than in Huh7.5.1
(approximately 3ꢀfold higher CC50) (Table 4). The H1HR antagonistic activity of the chosen
compounds were evaluated by measuring their capacity to block the betaꢀarrestin internalization
signal induced by Histamine. The numeric value observed corresponds to the percentage of
activation induced by Histamine (250 nM) in the presence of 10 nM of compound, using CCZ
(Rac-2) and (S)-1 as the positive and negative controls respectively. Shown in Table 4, the
results of CCZ (Rac-2) and (S)-1 were consistent with previously reported results.14 Lead
compounds with R3 = H (compounds (S)-1 and 29) showed very low H1HR inhibition
(approximately 10%). Meanwhile when R3 = Me, Et or medium oligoethylene glycol chain, the
H1HR inhibitory effects were generally lower than that of CCZ (Rac-2) (compounds Rac-23,
30, 31, and 34), and (S)-10 showed approximately 4ꢀfold lower inhibition. Compound Rac-25
having a long oligoethylene glycol chain, showed more than 3ꢀfold lower inhibitory effect
towards H1HR.
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Additionally, HCV replication cycle assays were carried out to study the target stage of the
CCZ analogues in the HCV replication cycle. The lead compounds exhibited potent inhibition in
the HCV singleꢀcycle assay, in which singleꢀround infectious HCV (HCVsc) can infect
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hepatocytes but does not assemble into new virions (Table 4).13, 14 The activity suggested that the
CCZ analogues inhibited early steps of the HCV replication cycle prior to assembly. The
analogues were tested in the HCV pseudoparticle (HCVpp) assay and the HCV subgenomic
replicon assay,13 which detect whether the compounds target HCVpp entry and HCV replication,
respectively. The HCVpp assay applies defective retroviral particles that harbor the HCV
envelope glycoproteins to assess viral entry inhibition.13, 22ꢀ24 No significant inhibitory effect was
observed in the HCVpp (genotype 1a and 1b) assay with the lead compounds, except for Rac-23
possibly due to cytotoxicity (Table 4). To address viral specificity in the entry process, vesicular
stomatitis virus G pseudoparticle (VSVꢀGpp) and murine leukemia virus pseudoparticle
(MLVpp) were tested as control, in which no inhibitory effect was detected (Table 4). It is worth
noting that the lack of HCVpp inhibition of a compound does not necessarily exclude virus entry
as a mode of action. A compound could be targeting an entry step that is not otherwise captured
by the HCVpp system. Multiple HCV entry inhibitors were reported without HCVpp inhibitory
effect, including NPC1L1 antagonist ezetimibe and human apolipoprotein E peptides.25, 26 In the
genotype 2a HCV replicon cell line, all the lead compounds failed to reduce below 60% the
replicon activity, as compared tothe DMSO control, indicating that RNA replication was not the
target of these analogues (Table 4).
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Moreover, besides the in vitro physical properties of the chosen lead compounds, their in vitro
metabolic properties were also evaluated using human, mouse and rat microsomes. Compounds
29, 30, and 34 showed preferable human microsomal stability (t1/2 ≥ 30 min), while maintaining
reasonable solubility.
In vivo pharmacokinetics and tissue distribution levels of compound 30 was measured in mice
after a single dose of 10 mg/kg through intraperitoneal (i.p.) route (Table S1). The halfꢀlife in
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liver was 8.5 h, which was an improvement in comparison with the halfꢀlives of (S)ꢀCCZ
[compound (S)-2] and (S)ꢀnorꢀCCZ [compound (S)-1] (1.99 h and 4.7 h, respectively) measured
under the same conditions (Figure 2A and C).14 Preferential liver distribution was also observed,
as shown by the liver/plasma AUClast ratio of 16 (Figure 2C). Within 24 h after a single dose of
10 mg/kg, the liver concentration of compound 30 (56.9 ~ 5.29 ꢁM) was dramatically above its
in vitro EC50 values (0.017 ꢁM). To detect any potential hepatotoxicity effect, the alanine
transaminase (ALT) levels27 in the mouse serum were measured (Figure 2B). When treated with
compound 30, the ALT levels were around or below 80 U/L at most time points except when t =
2 and 4 h, which may suggest a potential transient mild liver toxicity. From these observations
we concluded that there was not a clear correlation between the ALT levels and liver
concentration of the compounds. To study whether the mild elevations of ALT level were due to
compounds or vehicle, a control study was carried out dosing vehicle only. The ALT level was
elevated at multiple time points with the treatment of vehicle only (Figure 2B). Overall, we
concluded that no clear hepatotoxicity was detected with the treatment of compound 30 in this
condition.
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To evaluate the capacity of these compounds to potentially impact other viruses in the
Flaviviridae family, their antiviral activity was tested against dengue virus. Both lead
compounds (S)-10 and 30 showed EC50 values in DENVꢀRVPs assay that were approximately
1000ꢀfold more than their EC50 values against HCVꢀLuc infection (Table 4), together with CC50
values consistent with observation when cells were infected with HCVꢀLuc (Table S1). The
selective indices were below 5 for both compounds against dengue virus. Furthermore, the
activity of (S)-10 was submitted for evaluation in the NIAID antiviral screen against 13 types of
viruses: hepatitis B virus, HCV replicon, herpes simplex virusꢀ1, human cytomegalovirus,
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vaccinia virus, dengue virus, influenza A (H1N1) virus, respiratory syncytial virus, SARS
coronavirus, poliovirus 3, Rift Valley fever virus, Tacaribe virus, and Venezuelan equine
encephalitis virus. Little or no antiviral activity was detected (selective index < 10 and/or EC50 ≥
1 ꢁM), suggesting that the antiviral effects and mechanism of these analogues were HCVꢀ
specific.
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DISCUSSION
Besides the advancement in developing efficacious treatments for chronic HCV infection, the
cost and side effects of current approved methods point to the direction of developing alternative
approaches to therapeutically intervene in the disease. In this sense, in our previous study, we
disclosed the in vitro and in vivo antiꢀHCV properties of chlorcyclizine (CCZ, Rac-2), a first
generation antihistamine compound approved for overꢀtheꢀcounter use.14 Its affordability and
established clinical safety profile make CCZ (Rac-2) an attractive candidate for repurposing
toward chronic HCV infection (https://clinicaltrials.gov/ct2/show/NCT02118012). Additionally,
we decided to carry out structural modifications, in vitro and in vivo studies to further optimize
this series with the aim of improving their antiꢀHCV profile. We focused the chemical
modifications on four major structural motifs: chirality, side chains off the piperazine ring,
substituents on the phenyl rings, and modifications on the piperazine core. As summarized in
Figure 3, chirality had little effect on the antiviral activity and selectivity. Dual paraꢀchloro
substitution on the aromatic rings led to more potent nonꢀchiral analogues. Furthermore, the
introduction of oligoethylene glycol off the piperazine ring nitrogen improved the activity
without compromising the selectivity. Most of the structure modifications in the piperazine core
were not tolerated, suggesting that its geometry and its pKa value were important for the antiꢀ
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HCV activity of the series. Selected lead compounds were also analyzed for additional
assessment of cytotoxicity in HepG2 hepatocytes and primary human hepatocytes. They showed
similar profiles as those observed in Huh7.5.1 cells, suggesting that the cytotoxicity evaluation in
Huh7.5.1 cells in parallel with HCV infection assay provides a good estimation of the
cytotoxicity in human hepatocytes. Overall, the lead compounds in Table 4 exhibited excellent
antiviral activity and selectivity in host cells (SI = 8608 ~ 201). One of the goals of this
medicinal chemistry study was to eliminate the antihistamine side effect of CCZ (Rac-2), which
was achieved with lead compounds having R3 = H or long ethylene glycol chains (compounds 29
and Rac-25).
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In the HCV replication cycle assays, the lead compounds showed similar inhibitory patterns as
CCZ (Rac-2), displaying a dramatic inhibition in early stage HCV infection (in HCVsc assay)
but no inhibition on HCVpp entry or in the HCV replicon system. Although the exact mechanism
of action of CCZ (Rac-2) is still under investigation, the observations here strongly suggest that
the lead compounds likely follow the same mechanism of action as CCZ (Rac-2), only with
improved activity and selectivity.
In vitro microsomal stability assays were carried out to evaluate the potential metabolic
stability of the lead compounds. Among compounds 29, 30, and 34 with t1/2 > 30 min in human
microsomes, compounds 30 was selected for in vivo pharmacokinetic mouse studies because of
its lower in vitro cytotoxicity. Compound 30 showed improved PK properties in comparison with
(S)ꢀCCZ ((S)-2), namely a longer halfꢀlife, while retaining a high liver/plasma ratio.
CONCLUSION
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Besides the progress in the treatment of chronic HCV infection, among other issues, the global
affordability is still an important factor to consider within the current treatment options.
Repurposing the overꢀtheꢀcount drug CCZ (Rac-2) may offer an affordable treatment for chronic
HCV infection.14 Additionally, we presented a chemical/structural modification study, which
resulted in optimized, nonꢀchiral wellꢀtolerated CCZ analogues with improved antiꢀHCV
potency and pharmacokinetic properties that are able to provide good coverage in the liver at
very reasonable doses. The lead compounds inhibited HCVsc infection without affecting
HCVpp entry or HCV replication in the replicon assay, which is similar to the findings for CCZ
(Rac-2), suggesting an unaltered mechanism of action. The lead compounds showing overall
improved properties, will be selected for in vivo antiꢀHCV efficacy studies and potentially for
further preclinical drug development efforts with the aim of moving additional compounds of
this series toward antiꢀHCV human clinical trials.
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EXPERIMENTAL SECTION
Chemistry. All air or moisture sensitive reactions were performed under positive pressure of
nitrogen with ovenꢀdried glassware. Anhydrous solvents such as dichloromethane, N,Nꢀ
dimethylformamide (DMF), acetonitrile, methanol and triethylamine were purchased from
SigmaꢀAldrich (St. Louis, MO). Preparative purification was performed on a Waters semiꢀ
preparative HPLC system (Waters Corp., Milford, MA). The column used was a Phenomenex
Luna C18 (5 micron, 30 x 75 mm; Phenomenex, Inc., Torrance, CA) at a flow rate of 45.0
mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1%
trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 min was used during the
purification. Fraction collection was triggered by UV detection at 220 nM. Analytical analysis
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was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7ꢀ
min gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water
(containing 0.05% trifluoroacetic acid) was used with an 8ꢀmin run time at a flow rate of 1.0
mL/min. Method 2: A 3ꢀmin gradient of 4% to 100% acetonitrile (containing 0.025%
trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5ꢀmin run
time at a flow rate of 1.0 mL/min. A Phenomenex Luna C18 column (3 micron, 3 x 75 mm) was
used at a temperature of 50 °C. Purity determination was performed using an Agilent diode array
detector for both Method 1 and Method 2. Mass determination was performed using an Agilent
6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were
recorded on Varian 400 MHz spectrometers (Agilent Technologies, Santa Clara, CA). Chemical
shifts are reported in ppm with undeuterated solvent (DMSO at 2.49 ppm) as internal standard
for DMSOꢀd6 solutions. All of the analogs tested in the biological assays have a purity of greater
than 95% based on both analytical methods.
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High resolution mass. spectrometry was recorded on Agilent 6210 TimeꢀofꢀFlight (TOF)
LC/MS system. Confirmation of molecular formula was accomplished using electrospray
ionization in the positive mode with the Agilent Masshunter software (Version B.02).
Enantiomerically pure compounds were purified to > 99% purity using supercritical fluid
chromatography (SFC) preparative systems at Lotus Separations, LLC (Princeton, NJ, USA).
Compounds Rac-1, (S)-1, and (R)-1 were purchased from Albany Molecular Research (Albany,
NY, USA). Compounds Rac-2, (S)-2, and (R)-2 were purchased from MP Biomedicals (Santa
Ana, CA, USA). Compounds Rac-3 and 6 were purchased from Prestwick Chemical (France).
Compound Rac-5 was purchased from TimTec (Newark, DE, USA). Compound Rac-7 was
purchased from Biomol (Germany). Compounds 8 and 9 were purchased from SigmaꢀAldrich
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(St. Louis, MO, USA). Compounds 10,28 12,28 29,29 30,30 32,31 33,31 and 34,32 were synthesized
by modified literature procedures. Compounds Rac-36 and Rac-37 were purchased from Vitasꢀ
M Laboratory (Netherlands).
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(S)-1-((4-Chlorophenyl)(phenyl)methyl)-4-ethylpiperazine ((S)-10). A solution of (S)-1 (50.0
mg, 0.174 mmol) in methanol (MeOH) (2.00 mL) was treated at room temperature with
acetaldehyde (38.4 mg, 0.872 mmol), NaBH3CN (32.9 mg, 0.523 mmol) and acetic acid (30.0
mL, 0.523 mmol). The reaction mixture was stirred at room temperature overnight and then
quenched with 1 N NaOH solution. The mixture was dried by blowing air, the residue was reꢀ
disolved in DMSO, filtered and purified by preparative HPLC to afford (S)-10 (47.0 mg, 63%) as
1
the TFA salt. H NMR (400 MHz, DMSOꢀd6) δ ppm 9.22 (s, 1H), 7.50 – 7.29 (m, 8H), 7.29 –
7.19 (m, 1H), 4.54 (s, 1H), 3.42 (d, J = 12.23 Hz, 2H), 3.18 – 3.09 (m, 2H), 3.04 (q, J = 11.21
Hz, 2H), 2.84 (d, J = 13.01 Hz, 2H), 2.21 (q, J = 11.50 Hz, 2H), 1.18 (t, J = 7.27 Hz, 3H); LCMS
RT (Method 1) = 4.566 min; RT (Method 2) = 3.035 min, m/z 315.1 [M + H+]; HRMS (ESI) m/z
calcd for C19H24ClN2 [M + H+] 315.1623, found 315.1637.
+
bis(4-Chlorophenyl)methanol. A solution of 27 (3.00 g, 11.9 mmol) in MeOH (15.0 mL) was
treated at 0 oC in portions with NaBH4 (0.678 g, 17.9 mmol). The reaction mixture was stirred at
o
0 C for 15 min, allowed to warm to room temperature and stirred for 2 h. The reaction was
quenched with ice, diluted with H2O and extracted with EtOAc. The organic layer was separated,
dried over MgSO4 and concentrated to give the title compound as a white solid (3.00 g, 99%),
which was used without further purification. 1H NMR (400 MHz, CDCl3) δ ppm 7.31 (d, J = 8.8
Hz, 4H), 7.28 (d, J = 8.7 Hz, 4H), 5.78 (d, J = 3.2 Hz, 1H), 2.26 (d, J = 3.5 Hz, 1H). LCMS RT
(Method 2) = 3.733 min, m/z 254.5 [M+H+].
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4,4'-(Chloromethylene)bis(chlorobenzene) (28). bis(4ꢀChlorophenyl)methanol (3.00 g, 11.8
mmol) was dissolved in CH2Cl2 (10.0 mL), to this was added 3ꢀ4 drops of DMF followed by
thionyl chloride (2.60 mL, 35.6 mmol). The resulting reaction mixture was allowed to stir at
room temperature for 45 min, after which TLC anlysis (20% EtOAc in Hex) showed completion.
Reaction mixture was concentrated under reduced pressure to afford 28 (2.50 g, 78%) as a white
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solid, which was used without further purification. H NMR (400 MHz, CDCl3) δ ppm 7.42 –
7.27 (m, 8H), 6.06 (s, 1H). LCMS RT (Method 2) = 3.932 min, m/z 272.6 [M+H+].
1-(bis(4-Chlorophenyl)methyl)piperazine (29). A solution of 28 (80.0 mg, 0.295 mmol) in
THF (10.0 mL) was treated with piperazine (38.1 mg, 0.442 mmol) followed by K2CO3 (81.0
mg, 0.589 mmol). A catalytic amount of tetrabutylammonium iodide (10.9 mg, 0.029 mmol) was
added to the mixture. The reaction mixture was refluxed for 8 h, after which LC/MS analysis
showed completion. The reaction mixture was concentrated and reꢀdisolved in EtOAc. The
organic layer was washed three times with saturated NaHCO3 solution, dried over MgSO4,
filtered and concentrated. The crude product was purified by preparative HPLC, to give 29 (80.0
1
mg, 63%) as the TFA salt. H NMR (400 MHz, DMSOꢀd6) δ ppm 8.50 (s, 2H), 7.43 (d, J = 8.7
Hz, 4H), 7.39 (d, J = 8.6 Hz, 4H), 4.56 (s, 1H), 3.11 (s, 4H), 2.46 (s, 4H). LCMS RT (Method 1)
+
= 4.760 min, m/z 322.7 [M + H+]; HRMS (ESI) m/z calcd for C17H19Cl2N2 [M + H+] 321.0920,
found 321.0930.
1-(bis(4-Chlorophenyl)methyl)-4-methylpiperazine (30). To a stirred solution of 28 (0.800 g,
2.95 mmol) in THF (10.0 mL) was added K2CO3 (0.814 g, 5.89 mmol), 1ꢀmethylpiperazine
(0.654 mL, 5.89 mmol) and catalytic potassium iodide (73.0 mg, 0.442 mmol). The reaction was
o
heated to 100 C for 48 h. The reaction mixture was partitioned between EtOAc and H2O, the
layers separated and the organic phase washed with brine, dried over MgSO4, filtered and
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concentrated. Crude mixture was purified by flash column chromatography: silica gel with a
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6
gradient of 0ꢀ5% MeOH in CH2Cl2 to afford 30 (603 mg, 61%) as a freeꢀbase oil, which was
7
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9
1
then mixed in a 1:1 ratio with oxalic acid to form the oxalate salt as a white powder. H NMR
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(400 MHz, DMSOꢀd6) δ ppm 7.41 (d, J = 8.6 Hz, 4H), 7.34 (d, J = 8.5 Hz, 4H), 4.33 (s, 1H),
2.32 (s, 4H), 2.27 (s, 4H), 2.14 (s, 3H). LCMS RT (Method 1) = 4.843 min,
+
m/z 336.9 [M + H+]; HRMS (ESI) m/z calcd for C18H21Cl2N2 [M + H+] 335.1076, found
335.1086.
1-(bis(4-Chlorophenyl)methyl)-4-ethylpiperazine, (31). A solution of 28 (160 mg, 0.589 mmol)
in THF (10.0 mL) was treated with 1ꢀethylpiperazine (101 mg, 0.884 mmol) followed by K2CO3
(163 mg, 1.18 mmol). A catalytic amount of tetrabutylammonium iodide (21.8 mg, 0.059 mmol)
o
was added, and the resulting reaction mixture was heated to 100 C for 48 hours. The reaction
mixture was partitioned between EtOAc and H2O, the layers separated and the organic phase
washed with brine, dried over MgSO4, filtered and concentrated. Crude mixture was purified by
flash column chromatography: silica gel with a gradient of 0ꢀ5% MeOH in CH2Cl2 to afford 31
(123 mg, 60%) as a freeꢀbase oil, which was then mixed in a 1:1 ratio with oxalic acid to form
the oxalate salt. 1H NMR (400 MHz, DMSOꢀd6) δ ppm 7.44 (d, J = 8.8 Hz, 4H), 7.40 (d, J = 8.8
Hz, 4H), 4.57 (s, 1H), 3.11 – 3.02 (m, 2H), 2.80 (s, 8H), 2.24 (s, 2H), 1.17 (t, J = 7.2 Hz, 3H).
LCMS RT (Method 1) = 5.029 min,
+
m/z 350.7 [M + H+]; HRMS (ESI) m/z calcd for C19H23Cl2N2 [M + H+] 349.1233, found
349.1239.
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2-(2-(2-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)ethyl)isoindoline-1,3-dione
(Rac-22). Et3N (0.279 mL, 2.00 mmol) was added to a solution of Rac-5 (250 mg, 0.667 mmol)
in THF (10.0 mL) at room temperature. The mixture was stirred for 15 min, then phthalimide
(147 mg, 1.000 mmol) and triphenylphosphine (262 mg, 1.00 mmol) were added to the mixture
followed by diisopropyl azodicarboxylate (0.130 mL, 0.667 mmol). The reaction mixture was
stirred at room temperature for 4 h, after which LCMS analysis showed product formation.
Reaction mixture was concentrated to dryness and residue purified by preparative HPLC to give
Rac-22 (239 mg, 58%) as the TFA salt. 1H NMR (400 MHz, DMSOꢀd6) δ ppm 9.42 (s, 1H), 7.72
(m, 4H), 7.46 (d, J = 8.4 Hz, 2H), 7.44 – 7.38 (m, 4H), 7.34 (t, J = 7.5 Hz, 2H), 7.25 (t, J = 7.4
Hz, 1H), 4.53 (s, 1H), 3.73 (d, J = 4.8 Hz, 4H), 3.58 (t, J = 5.2 Hz, 4H), 3.14 (d, J = 11.2 Hz,
2H), 3.04 – 2.97 (m, 2H), 2.82 (d, J = 12.8 Hz, 2H), 2.28 (m, 2H). LCMS RT (Method 1) = 5.205
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+
min, m/z 505.7 [M + H+]; HRMS (ESI) m/z calcd for C29H31ClN3O3 [M + H+] 504.2048, found
504.2043.
2-(2-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)ethanamine
(Rac-23).
Hydrazine (0.181 mL, 5.77 mmol) was added to a solution of Rac-22 (97.0 mg, 0.192 mmol) in
EtOH (3.00 mL). The reaction mixture was stirred at 60 °C for 3 h, after which LCMS analysis
showed completion. The reaction mixture was concentrated under reduced pressure and residue
1
purified by preparative HPLC, to give Rac-23 (58.0 mg, 63%) as the TFA salt. H NMR (400
MHz, DMSOꢀd6) δ ppm 9.39 (s, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.41 – 7.38 (m, 4H), 7.35 (t, J =
7.8 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 4.55 (s, 1H), 3.77 (d, J = 4.6 Hz, 2H), 3.55 (t, J = 5.0 Hz,
4H), 3.19 (d, J = 11.0 Hz, 2H), 3.09 – 2.95 (m, 2H), 2.80 (d, J = 11.5 Hz, 2H), 2.25 (m, 2H).
LCMS RT (Method 1) = 3.959 min,
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m/z 374.7 [M + H+]; HRMS (ESI) m/z calcd for C21H29ClN3O+ [M + H+] 374.1994, found
3
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6
374.2002.
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tert-Butyl
(14-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)-3,6,9,12-
tetraoxatetradecyl)carbamate (Rac-24). A solution of Rac-5 (250 mg, 0.558 mmol) in DMF
(5.00 mL) was treated with a 60% dispersion in mineral oil of NaH (89.0 mg, 2.23 mmol) at 0
oC. The reaction mixture was stirred at 0 oC for 10 min and room temperature for 30 min. To this
mixture was added a solution of tertꢀbutyl (2ꢀ(2ꢀ(2ꢀbromoethoxy)ethoxy)ethyl)carbamate (174
mg, 0.558 mmol) in DMF (1.00 mL) and the resulting mixture allowed to stir overnight. The
mixture was quenched with H2O and extracted with CH2Cl2. The organic layer was separated,
dried over MgSO4, filtered and concentrated. Crude residue was purified by preparative HPLC,
1
to give Rac-24 (220 mg, 55%) as the TFA salt. H NMR (400 MHz, CDCl3) δ ppm 7.45 – 7.37
(m, 4H), 7.37 – 7.18 (m, 5H), 4.44 (s, 1H), 3.86 (t, J = 4.4 Hz, 2H), 3.63 – 3.48 (m, 14H), 3.29
(s, 4H), 2.91 (s, 9H), 1.43 (s, 9H). 19F NMR (376 MHz, CDCl3) δ ppm ꢀ75.78. LCMS RT
(Method 1) = 5.372 min,
m/z 607.7 [M + H+]; HRMS (ESI) m/z calcd for C32H49ClN3O6 [M + H+] 606.3304, found
+
606.3307.
14-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-3,6,9,12-tetraoxatetradecan-1-amine
(Rac-25). A solution of Rac-24 (217 mg, 0.358 mmol) in CH2Cl2 (10.0 mL) was treated with
o
o
trifluoroacetic acid (5.00 mL) at 0 C. The reaction mixture was stirred at 0 C for 10 min and
room temperature for 30 min, after which LCMS analysis showed completion. The reaction
mixture was concentrated and the crude residue was purified by preparative HPLC, to give Rac-
25 (109 mg, 60%) as the TFA salt. 1H NMR (400 MHz, CDCl3) δ ppm 7.95 (s, 2H), 7.51 – 7.41
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(m, 4H), 7.38 – 7.25 (m, 4H), 4.57 (s, 1H), 3.79 (dd, J = 11.2, 6.6 Hz, 4H), 3.70 – 3.49 (m, 9H),
3.58 (s, 7H), 3.36 (d, J = 4.8 Hz, 2H), 3.17 (s, 3H), 3.00 (s, 5H). 19F NMR (376 MHz, CDCl3) δ ꢀ
75.78. LCMS RT (Method 1) = 3.916 min,
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+
m/z 507.2 [M + H+]; HRMS (ESI) m/z calcd for C27H41ClN3O4 [M + H+] 506.2780, found
506.2803.
Cells and viruses. Human hepatoma cell line Huh7.5.1 and other Huh7ꢀderived cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life technologies, Grand Island,
NY, USA) with 10% fetal bovine serum (FBS) (Life technologies, Grand Island, NY, USA) and
antibiotics in 5% CO2 at 37°C. HCVꢀLuc (HCV JFHꢀ1 strain with insertion of the luciferase
reporter gene) and pseudotyped viruses (HCVppꢀ1a, HCVppꢀ1b and VSVꢀGpp) were produced
as reported before. HCVꢀLuc (HCV JFHꢀ1 strain with insertion of the luciferase reporter gene),
HCVsc (singleꢀround infectious defective HCV particle) and pseudotyped viruses (HCVppꢀ1a,
HCVppꢀ1b and VSVꢀGpp) were produced as reported before.13, 22
HCV-Luc infection and ATPlite assays. Huh7.5.1 cells were plated in 96ꢀwell plates at 104
cells/well and incubated overnight. The cells were infected with HCVꢀLuc in the presence of
increasing concentration of compound of interest. Viral level was measured 48 h after treatment
using Renilla Luciferase assay system (Promega, Madison, WI, USA). ATPꢀbased cell viability
assay was carried out in parallel to evaluate the cytotoxicity with ATPlite assay kit (PerkinElmer,
Waltham, MA, USA). The concentration values that lead to 50% viral inhibition and cytotoxicity
(EC50
CC50) were calculated using nonlinear regression equation in GraphPad Prism 5.0
and
software (GraphPad Software Inc., La Jolla, CA, USA).
HCV Replication cycle assays. In HCVsc assay, Huh7.5.1 cells that were cultured in 96ꢀwell
plates (104 cells/well) overnight before infection with HCVsc in the presence of compound
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treatments. After 48 h of incubation, the viral level was detected by a luciferase assay, In HCV
subgenomic replicon assay, HCV replicon (GT 2a) cells were plated into 96ꢀwell plate at 104
cells/well and incubated overnight. The cells were treated with tested compounds for 48 h and
luciferase activity was measured. In HCVpp assays, Huh7.5.1 cells were seeded in 96ꢀwell plates
(104 cells/well) and cultured overnight. Then the cells were infected with HCVpp GT 1a, 1b,
VSVꢀGpp and MLVpp for 4 h in the present of compound treatment. The cells were then washed
and cultured for 48 h followed by a luciferase assay to detect the HCV entry. Positive controls
(cyclosporin A at 10 ꢁM and bafilomycin A1 at 10 nM) were tested in parallel.
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H1HR inhibition assay. PathHunter βꢀArrestin GPCR assay kit (DiscoveRx, Fremont, CA,
USA) was used following the antagonist procedure. The PathHunter cells were plated in white
96ꢀwell plates with clear bottom and cultured overnight. After incubation with the compound of
interest for 3 h, agonist histamine at 0.25 ꢁM were added and plates were incubated for
additional 2 h. Chemiluminescent signal was read after 60 min incubation with detection agent at
room temperature. %Antiꢀhistamine activity was calculated based on the result from histamineꢀ
treated wells.
DENV-RVPs assay and NIAID screen. Huh7.5.1 cells were plated in 96ꢀwell plates at 104
cells/well and cultured overnight. Dengue RVPs (Integral Molecular, Philadelphia, PA, USA)
was added to the cells with increasing concentrations of compound of interest. Dengue RVP
reproducibility was measured by luciferase signal 48 h after treatment. Lycorine HCl was tested
as positive control.33, 34 The nonꢀclinical and preꢀclinical services program offered by the
National
Institute
of
Allergy
and
Infectious
Diseases
(NIAID)
(http://www.niaid.nih.gov/labsandresources/resources/dmid/invitro/Pages/invitro.aspx) was used
for antiviral screen against the 13 viruses. The viruses include hepatitis B virus, HCV replicon,
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herpes simplex virusꢀ1, human cytomegalovirus, vaccinia virus, dengue virus, influenza A
(H1N1) virus, respiratory syncytial virus, SARS coronavirus, poliovirus 3, Rift Valley fever
virus, Tacaribe virus, and Venezuelan equine encephalitis virus.
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In vitro and In vivo pharmacokinetics properties. The in vitro microsomal stability was
measured by incubation of compounds with human/mouse/rat liver microsomes at 37°C in the
presence of the coꢀfactor, NADPH. The concentrations of compounds were measured by LCꢀ
MS/MS at 0, 5, 15, 30 and 45 minutes. halfꢀlife (t1/2) was calculated as described before 35.
The kinetic solubility of compounds was determined in phosphate buffer pH 7.4, using ꢁSOL
Evolution from pION Inc. (www.pionꢀinc.com), with a fully automated system of sample
preparation, sample analysis and data processing. The effective permeability of compounds was
determined via passive diffusion using stirring doubleꢀsink PAMPA (Parallel Artificial
Membrane Permeability Assay) method from pION Inc. (www.pionꢀinc.com), with a fully
automated system of sample preparation, sample analysis and data processing.
For in vivo pharmacokinetics, twentyꢀseven male CDꢀ1 mice (~35 g) were obtained from
Charles River Laboratories (Wilmington, MA). Mice were housed at the centralized animal
facilities at the NIH (Bethesda, MD) with a 12 h light dark cycle. The housing temperature and
relative humidity were controlled at 22°C and 55%, respectively. The animals had free access to
water and food. All experimental procedures were approved by the Animal Ethics Committee of
the NIH. A dosing concentration of 2 mg/mL of the appropriate compound was freshly prepared
in 50% PEG300 and 50% water. The pharmacokinetics was evaluated after single intraperitoneal
administration at 10 mg/kg. The blood and liver samples were collected at predose, 0.083, 0.25,
0.5, 1, 2, 4, 7 and 24 hr. Three samples (n = 3) were collected at each time point. The
concentrations of compound in plasma and liver were determined by ultraꢀperformance liquid
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chromatographyꢀmass spectrometry analysis (UPLCꢀMS/MS. The pharmacokinetic parameters
were calculated using the nonꢀcompartmental method (Model 200) of the pharmacokinetic
software package Phoenix WinNonlin, version 6.2 (Certara, St. Louis, MO, USA). The area
under the plasma and liver concentration versus time curve (AUC) was calculated using the
linear trapezoidal method. Where warranted, the slope of the apparent terminal phase was
estimated by log linear regression using at least 3 data points and the terminal rate constant (λ)
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was derived from the slope. AUC0ꢀ∞ was estimated as the sum of the AUC0ꢀt (where t is the time
of the last measurable concentration) and Ct/λ. The apparent terminal halfꢀlife (t ) was calculated
½
as 0.693/λ.
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ASSOCIATED CONTENT
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Supporting Information
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Antiviral activity of CCZ analogues against dengue virus, general chemistry methods and
compound characterization are included. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*For T.J.L.: phone, 301ꢀ496ꢀ1721; fax, 301ꢀ402ꢀ1612; Eꢀmail, jliang@nih.gov. For J.J.M.:
phone, 301ꢀ217ꢀ9198; fax, 301ꢀ217ꢀ5736; Eꢀmail: maruganj@mail.nih.gov.
Present Addresses
†If an author’s address is different than the one given in the affiliation line, this information may
be included here.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. §These authors contributed equally.
Funding Sources
The Intramural Research Program of the National Institute of Diabetes and Digestive and
Kidney Diseases and Molecular libraries funding.
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Notes
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T.J.L, M.F., S.H, X.H, Z.H, J.J.M., J.X., and W.Z are named as inventors on patent
applications related to this work: U.S. Provisional Patent Appl. 62/011,462, “Heterocyclic
compounds and methods of use thereof”. Other authors declare no competing interests.
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ACKNOWLEDGMENT
We thank S. Michael and M. Balcom for assistance in robotic control in qHTS; P. Shinn, M.
Itkin, and D. van Leer for help with compound management; and B. Zhang for technical
assistance.
ABBREVIATIONS USED
HCV, Hepatitis C virus; IFNꢀα, interferon α; RBV, ribavirin; DAAs, directꢀacting antivirals;
HTAs, Hostꢀtargeting agents; NPC, NCGC Pharmaceutical Collection; qHTS, highꢀthroughput
screening; CCZ, chlorcylizine HCl; SAR, structureꢀactivity relationship; DMSO, dimethyl
sulfoxide; HCVꢀLuc, HCV JFHꢀ1 strain with insertion of the luciferase reporter gene; EC50, the
concentration of compound that inhibited 50% of virus level of DMSO; CC50, the concentration
of compound that exhibited 50% of cytotoxicity of DMSO; H1HR, H1ꢀhistamine receptor;
HCVsc, singleꢀround infectious defective HCV particles; HCVpp, HCV pseudoparticle; ADME,
absorption, distribution, metabolism, and excretion; i.p., intraperitoneal; DMF, N,Nꢀ
dimethylformamide; LC/MS, liquid chromatography–mass spectrometry; TOF, TimeꢀofꢀFlight;
TFA, trifluoroacetic acid; SFC, supercritical fluid chromatography; THF, tetrahydrofuran;
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DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; NIAID, National
Institute of Allergy and Infectious Diseases.
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