Synthesis and anti-HCV activity of 4-hydroxyamino α-pyranone carboxamide analogues

Article abstract of DOI:10.1021/ml400432f

High genetic variability in hepatitis C virus (HCV), emergence of drug resistant viruses and side effects demand the requirement for development of new scaffolds to show an alternate mechanism. Herein, we report discovery of new scaffold I based on 4-hydr

Full text of DOI:10.1021/ml400432f

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Anti-HCV Activity of 4‑Hydroxyamino α‑Pyranone
Carboxamide Analogues
Ananda Kumar Konreddy,† Massaki Toyama,‡ Wataru Ito,‡ Chandralata Bal,† Masanori Baba,‡
and Ashoke Sharon*,†
†Department of Applied Chemistry, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India
‡Division of Antiviral Chemotherapy, Center for Chronic Viral Diseases, Kagoshima University, Kagoshima, Japan
S
* Supporting Information
ABSTRACT: High genetic variability in hepatitis C virus (HCV), emergence of drug resistant viruses and side effects demand
the requirement for development of new scaffolds to show an alternate mechanism. Herein, we report discovery of new scaffold I
based on 4-hydroxyamino α-pyranone carboxamide as promising anti-HCV agents. A comprehensive structure−activity
relationship (SAR) was explored with several newly synthesized compounds. In all promising compounds (17−19, 21−22, 24−
25, and 49) with EC50 ranging 0.15 to 0.40 μM, the aryl group at C-6 position of α-pyranone were unsubstituted. In particular,
25 demonstrated potential anti-HCV activity with EC50 of 0.18 μM in cell based HCV replicon system with lower cytotoxicity
(CC50 > 20 μM) and provided a new scaffold for anti-HCV drug development. Further investigations, including biochemical
characterization, are yet to be performed to elucidate its possible mode of action.
KEYWORDS: Hepatitis C virus, non-nucleoside inhibitor, α-pyranone carboxamide
The current standard of care (SoC) for HCV treatment
includes one of the approved protease inhibitors combined with
pegylated interferon-α (PegIFN) and ribavirin (RBV).
epatitis C virus (HCV) is a single stranded 9.6 kb RNA
Hvirus of the Flaviviridae family predominantly infecting
hepatocytes. HCV infection is silent and in long-term may lead
to serious liver disease, including fibrosis and cirrhosis followed
by hepatocellular carcinoma.1,2 The prophylactic treatment has
been of very limited success,3 and so far no vaccine is available.4
A serious health threat has been realized, and a series of
investigations are underway to discover direct acting antivirals
(DAAs) as promising anti-HCV agents.5
The potential molecules 1−4 (Figure 1) belonging to DAAs
target mainly three viral enzymes: (i) protease NS3/4A, (ii)
replicase factor NS5A, and (iii) HCV RNA-dependent RNA
polymerase (HCV RdRp) NS5B.6 The medicinal chemistry
approaches with breakthrough exploration in HCV biology7
were translated recently into first generation DAAs, boceprevir8
and telaprevir9 (protease inhibitors) approved by US FDA.
Several new generation protease inhibitors are under serious
investigations to combat the challenges associated with anti-
HCV therapy.10,11
The new regimen improves sustained viral response (SVR)
rates to ∼75%;8,9 however, it also adds side effect burden to
patients. The use of PegIFN/RBV is associated with severe side
effects followed by treatment discontinuation and contra-
indication.12 Overall, drug-resistance, drug−drug interactions,
and side effects are major concerns,13 which demand effective
PegIFN/RBV free regimen. In addition to recent success in
protease inhibitors,10,11 parallel research efforts are in progress
to discover nucleoside inhibitors (NIs) to find novel DAAs to
target HCV RdRp.14,15 Similarly, several non-nucleoside
allosteric inhibitors are in development phase, which may
provide a choice of combination with other DAAs to discover
Special Issue: HCV Therapies
Received: October 30, 2013
Accepted: December 3, 2013
Published: December 3, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Figure 1. Chemical structures of potential scaffold as DAAs for HCV infection.
Figure 2. Structures of promising non-nucleosides (5−7) DAAs against HCV infection. Hybridization of α-pyranone motif (red), hydroxyl (blue),
and carboxamide motif (green) in prototypes I.
PegIFN/RBV free regimen in future.16 Recently several non-
nucleosides (Figure 2) had shown promising results and
opened a scope for the development of new anti-HCV drug
candidates.17
pyridine-3-yloxy)methaniminium hexafluorophosphate] as a
coupling reagent yielding α-pyranone carboxamides (11) in
good yield as per our previous reported procedure.21 The
methylthio group at C-4 of amide derivatives (11) was replaced
with a hydroxyamino to yield target compounds I (12−53) as
shown in Scheme 1 and Table 1.
All the synthesized compounds were adequately charac-
terized by spectroscopic methods. One of the potential
compounds (25) was further confirmed by single crystal X-
ray diffraction analysis. Figure 3 represents an ellipsoidal plot of
25 as an ORTEP diagram with 30% probability level. One
chloroform molecule (crystallization solvent) within the
asymmetric unit of crystal structure may be attributed due to
the presence of strong intermolecular H-bonding between
chloroform and compound.
The presence of α-pyranone motif18 (red) in 5 (Figure 2)
was considered as a major core in our molecular design process.
Further, hydroxyl (blue, 5 and 6)19 and carboxamide motif
(green)20 in 7 and in several other promising anti-HCV
molecules16 prompted us to combine these motifs on core α-
pyranone ring for synthesis of prototypes I to access their
potential as possible anti-HCV candidates. In continuation to
our antiviral discovery effort based on pyranone carboxamide
series,21 here, we describe the identification of a new series
based on pyranone scaffold having excellent activity against
HCV.
Versatile ketene dithioacetals (9)22 were synthesized from
appropriate acetophenones (8). Ketene intermediates (9) were
converted into key intermediates α-pyranone acids (10)
followed by coupling with appropriate amines using HATU
[(dimethylamino)-N,N-dimethyl(3H-1,2,3-triazolo[4,5-b]-
The anti-HCV assay of 12−53 was carried out in cells
harboring subgenomic HCV RNA replicons (genotype 1b)
with a luciferase reporter (LucNeo#2).23 The carboxamide
motifs in 12−29 are aromatic, 30−48 are aliphatic, and 49−53
are benzyl. Anti-HCV activity was determined by their ability to
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ACS Medicinal Chemistry Letters
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Scheme 1. Synthesis of 4-Hydroxyamino α-Pyranone
Carboxamide-Based Analogues I
Table 1. Anti-HCV Activity of Synthesized Compounds I
a
a
Reagents and conditions: (i) NaH, CS2, CH3I, THF, 0 °C to rt, 4 h;
(ii) NaH, diethylmalonate, dioxane, 0−110 °C, 6 h; (iii) R2NH2,
HATU, DMF, rt, 1 h; (iv) NH2OH.HCl, NaHCO3, ethanol, 80 °C, 4
h.
reduce luciferase activity in HCV RNA replicons. The
cytotoxicity was evaluated by the reduction in the number of
viable cells using a tetrazolium dye method. EC50 and CC50
were calculated, and results are summarized in Table 1.
The compounds 17−19, 21−22, 24−25, and 49 (highlighted
in Table 1) were significantly active with their EC50s ranging
between 0.15 to 0.40 μM along with low cytotoxicity (CC50
>
20 μM). Interestingly, R1 = H in all of them, and except for 49,
all are aromatic carboxamides. Telaprevir, a US FDA approved
NS3 protease inhibitor and nesbuvir (HCV-796), a NS5B non-
nucleoside inhibitor, were used as reference for comparison.
The selectivity index (SI), which is a parameter of preferential
antiviral activity of a compound in relation to its cytotoxicity
(CC50/EC50), of 12−53 are also tabulated in Table 1. The SIs
of 21 and 25 were >110, while for telaprevir and nesbuvir, they
were 44.4 and 57.1, respectively, which makes them suitable
candidates for further optimization. The anti-HCV activity of
25 is shown in Figure 4.
The ability of 25 to inhibit HCV RNA was evaluated by an
intracellular HCV RNA quantitative assay at various concen-
trations. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), one of the housekeeping genes expressed at a
relatively constant level in experimental conditions, was used as
an index of cell viability. HCV RNA and GAPDH RNA were
quantitatively measured simultaneously by real-time PCR. The
results are shown in Figure 5.
Levels of GAPDH RNA were almost constant at
concentrations of 25 up to 10 μM. In contrast, the EC50 and
EC90 of 25 to inhibit HCV RNA were 0.077 and 0.45 μM,
respectively.
In conclusion, a new series of 4-hydroxyamino α-pyranone
carboxamide analogues were prepared, and their structure−
activity relationships (SAR) as inhibitors of HCV replication
were studied. Eight out of 42 synthesized compounds showed
significant anti-HCV activity with lower cytotoxicity. Among
them, 21 and 25 had significantly higher SI (>110) in
comparison to recently approved drug telaprevir (SI = 44.4),
which reveals the promising discovery of novel scaffold I for
anti-HCV drug development. Further, biochemical investiga-
tions are needed to elucidate its possible mode of action.
X-ray Crystal Structure Data for Compound 25.
a
The 50% cytotoxic concentration, determined by the reduction of
b
viable cell number. The 50% effective concentration, determined by
the inhibition of luciferase activity. Ratio of CC50 to EC50.
c
C H N O .CHCl , M = 792.04, triclinic, space group: P1, a
̅
38 32
4
8
3
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Anti-HCV Activity. Cells harboring subgenomic HCV RNA
replicons with a luciferase reporter (LucNeo#2)24 (50,000
cells/mL) were suspended in Dylbecco’s modified Eagle
medium (DMEM) supplemented with 10% FBS, antibiotics,
and 1 mg/mL of G418 (a protein synthesis inhibitor). After an
incubation of 24 h, the cells were further incubated for 3 days in
fresh culture media containing various concentrations of test
compounds but not containing the G418. For a luciferase assay,
the cells were washed with PBS, followed by treatment with a
lysis buffer. The cell lysate (25 μL) was transferred to a
microtiter plate, and luciferase assay reagent (100 μL) was
dispensed into each well. Luciferase activity was measured with
a luminometer. For cell viability assay, a viable cell detection
reagent (Tetra Color ONE, Seikagaku Corporation) (10 μL)
containing a tetrazolium salt (2-(2-methoxy-4-nitrophenyl)-3-
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, mono-
sodium salt: WST-8) was dispensed into each well. After an
incubation of 1 h at 37 °C, specific absorbance (at 450 nm) was
measured with a microplate reader.
Figure 3. ORTEP diagram of 25 along with solvent of crystallization
(CHCl3) at 30% probability level.
HCV RNA Quantitative Assay. Full genome HCV RNA
replicon cells (NNC#2) (30,000 cells/mL) were suspended in
DMEM supplemented with 10% FBS and 1 mg/mL of G418
for 24 h.25 The cells were incubated in fresh culture media
containing various concentrations of 25 but not containing the
G418 for another 3 days. The cells were washed with PBS and
treated with a lysis buffer containing deoxyribonuclease. The
cell lysate (10 μL) was subjected to a reverse transcription
reaction, followed by quantitative determination of HCV RNA
and GAPDH RNA simultaneously by real-time PCR.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, characterization data, and NMR-mass
spectrum for all new compounds. X-ray structure cif data for
compound 25. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
Figure 4. Anti-HCV activity of 25 in subgenomic HCV RNA replicon
cells (LucNeo#2). The cell viability and luciferase activity are shown in
bars with blue and brown colors, respectively.
S
AUTHOR INFORMATION
Corresponding Author
*(A.S.) Tel: 0651-2276531. Fax: 0651-2275401. E-mail:
asharon@bitmesra.ac.in.
■
Author Contributions
The manuscript was written through contributions of all
authors.
Funding
Financial support in terms of professional development grant
and institute fellowship for Ph.D, scholar was received from
Birla Institute of Technology (BIT), Ranchi, India. Grant to
establish initial infrastructure for antihepatitis research was
supported by DST and UGC, New Delhi.
Figure 5. HCV and GAPDH RNA quantitative assays of compound
25. The HCV and GAPDH RNA levels in full genome HCV RNA
replicon cells (NNC#2) are shown in bars with yellow and blue colors,
respectively. EC50, EC90, and CC50 were 0.077, 0.45, and >10 μM,
respectively.
Notes
The authors declare no competing financial interest.
= 10.547 (1), b = 14.464 (2), c = 14.485 (2) Å, α = 62.635
(15), β = 70.780 (10), γ = 86.674 (9), V = 1841.8 (4) Å3, T =
150 (2) K, Z = 2, μ = 0.31 mm−1, F(000) = 820.0, Dc = 1.428
Mg m−1, yellow rectangular crystal, crystal size, 0.32 × 0.28 ×
0.24 mm, 14276 reflections measured, 6483 unique, R1 =
0.0604 for 4032 Fo > 4σ(Fo), and 0.1015 for all 6483 data and
493 parameters. Unit cell determination and intensity data
collection (2θ = 50°) was performed with 99.8% completeness
at 150 (2) K. Structure solutions by direct methods and
refinements by full-matrix least-squares methods on F2.
ACKNOWLEDGMENTS
■
A.S. and C.B. acknowledge the financial support from BIT in
terms of faculty development grant. A.S. and C.B. extend their
acknowledgement to DST (SR/FT/CS-001/2009 & SR/FT/
CS-069/2009) and UGC (37-110/2009 & 39-705/2010),
Delhi, for providing an initial grant to establish antihepatitis
research & infrastructure. A.K. thanks BIT Mesra, Ranchi for
the Institute Fellowship. We acknowledge Dr. Reddy’s Institute
of Life Sciences, Hyderabad, for NMR-Mass Facility and CIF,
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ABBREVIATIONS
■
HCV, hepatitis C virus; RdRp, RNA-dependent RNA polymer-
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