Shape-based virtual screening, synthesis and evaluation of novel pyrrolone derivatives as antiviral agents against HCV

Article abstract of DOI:10.1016/j.bmcl.2016.12.087

A ligand-based approach was applied to screen in silico a library of commercially available compounds, with the aim to find novel inhibitors of the HCV replication starting from the study of the viral NS3 helicase. Six structures were selected for evaluat

Full text of DOI:10.1016/j.bmcl.2016.12.087

Accepted Manuscript
Shape-based virtual screening, synthesis and evaluation of novel pyrrolone de-
rivatives as antiviral agents against HCV
Marcella Bassetto, Pieter Leyssen, Johan Neyts, Mark M. Yerukhimovich,
David N. Frick, Andrea Brancale
PII:
S0960-894X(16)31366-X
http://dx.doi.org/10.1016/j.bmcl.2016.12.087
BMCL 24576
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 November 2016
30 December 2016
31 December 2016
Please cite this article as: Bassetto, M., Leyssen, P., Neyts, J., Yerukhimovich, M.M., Frick, D.N., Brancale, A.,
Shape-based virtual screening, synthesis and evaluation of novel pyrrolone derivatives as antiviral agents against
HCV, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.087
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Shape-based virtual screening, synthesis and evaluation of
novel pyrrolone derivatives as antiviral agents against HCV
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Marcella Bassetto , Pieter Leyssen , Johan Neyts , Mark M. Yerukhimovich , David N. Frick ,
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Andrea Brancale
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Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, King Edward VII Avenue, Cardiff CF103NB, UK
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Rega Institute for Medical Research, University of Leuven, Belgium
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Department of Chemistry & Biochemistry, University of Wisconsin- Milwaukee, Milwaukee, Wisconsin 53211, United States
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Hepatitis C virus (HCV) is a major cause of chronic liver disease and affected individuals are at high
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risk of developing hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. There is no
vaccine available to prevent the infection and such vaccine is not soon expected. The standard of care
used to be a combination of pegylated interferon (pegIFN) and ribavirin, a therapy not specific for
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HCV and efficient in 50% of treated patients, with many associated side effects. However, new
interferon-free combinations of direct acting antivirals (DAAs) have revolutionized HCV prognosis
and treatment.
HCV ~9,000 nt single-stranded, positive-sense RNA genome encodes six non-structural proteins (NS2,
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NS3, NS4A, NS4B, NS5A and NS5B) essential for virus replication. Oral combination treatment
regimens with NS5B polymerase, NS3 protease and/or NS5A inhibitors have become the standard of
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HCV care, but they are associated with the development of resistant mutants
Due to these limitations, the development of new therapeutics is still needed.
and high costs.
HCV NS3 helicase is one of the most underexploited targets in the search for anti-HCV compounds,
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with very few specific inhibitors reported so far, none of which reached the clinical stage. The protein
main function, essential for viral replication, is the ATP-dependent unwinding of double-stranded RNA
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sequences.
Due to its fundamental role in HCV replication and the lack of inhibitors under
development, this enzyme was chosen as target for the computer-aided identification and synthesis of
viral replication inhibitors.
A successful computer-based approach for the identification of new biologically active compounds is
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represented by shape-complementarity search methods. Common docking-based techniques usually
estimate the ligand binding energy by a scoring procedure that emphasises electrostatics rather than
shape. In ligand-based methods instead, the shapes of known ligands are compared with unknown ones,
while the chemical properties of known molecules can be included in the comparison as
pharmacophoric or electrostatic models; for these approaches a higher accuracy in finding active
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compounds has been demonstrated. Ligand-based methods can evaluate two-dimensional similarities,
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such as fingerprints, or three-dimensional similarities, focusing on the molecule occupational volume.
While 2D comparison methods tend to find hits with the same functional groups of the query
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molecule, 3D search programs such as ROCS focus on the occupational volume associated with each
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Corresponding author
E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
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molecule, thus allowing the identification of novel lead structures with a higher level of scaffold
diversity.
While previous successful efforts in our research group focused on protein-based approaches for the
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identification of new antivirals,
in order to explore the potential of a shape-comparison method in
the search for novel anti-HCV agents, this technique was chosen to screen a library of commercially
available compounds. The structures of the dye soluble blue HT 1 and a series of reported compounds
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derived from 1 were used as starting point, being 1 the only known helicase inhibitor for which a
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crystal structure in complex with the enzyme is available (PDB ID 1ZJO, Figure 1).
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Figure 1: Crystal structure of 1 in complex with HCV NS3 helicase (PDB ID 1ZJO).
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While 1 is toxic to cells and its effect against the viral replication cannot be evaluated in cell-based
assays, several reported HCV NS3 helicase inhibitors have recently been tested in both an optimised
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enzymatic assay and a replicon cell-based assay: among them, one structural derivative of 1,
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triphenylmethane analogue 2 (Figure 2), has been confirmed to inhibit both the enzyme (IC50 17 +/- 7
mM) and the viral replication (~80% at 10 mM) without relevant cytotoxicity (~75% cell viability at 10
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mM).
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Figure 2: Chemical structures of 1 and 2.
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Due to its activity profile against the viral replication in cells, 2 was chosen as query for a shape-based
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screening of the SPECS library using ROCS 3D. In order to evaluate the shape complementarity
between the target query and screened compounds, shape-density overlapping volumes of
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superimposed molecules are considered by the program and scored according to a Tanimoto-like
overlapping value. The conformational state of the query molecule plays an essential role for hit
selection, since all analysed structures will be scored based on their overlapping with the target
conformation. Along with the conformation selection for the query, the evaluation of the different
three-dimensional states of the screened structures becomes crucial. First, a conformational search with
MOE2015.10 was performed for the creation of a conformational database for the screening
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compounds. A conformational analysis was also performed on 2, obtaining multiple low-energy
three-dimensional states. The conformational results found for 2 were then compared with the
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conformation of 1 in the 1ZJO crystal structure using MOE2015.10 Flexible Alignment tool. The
low-energy conformations found for 2 were rigidly aligned to 1, and the one corresponding to the best
alignment was chosen as query (Figure 3).
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B.
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Figure 3: A. Best conformational superimposition between 1, in pink, and 2, in green; B. Query generated from 2 in
vROCS3.1.2: the overall molecular shape is represented as grey surface, while the chemical features considered are an H-bond
acceptor (red sphere) and three hydrophobic/aromatic centres (green spheres).
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The conformational database obtained for the SPECS compounds was analysed against the selected
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conformation of 2 with vROCS3.1.2, considering both shape and electrostatics complementarity. All
screened conformations were ranked based on their similarity score with the given query. A selection
was made among the screened molecules to prioritise those with the highest similarity indexes
(Combo-shape Tanimoto score) and the biggest number of conformations matching the query. Six
compounds were finally selected, purchased and evaluated against the viral replication in a cell-based
assay (Supporting Information). Among them, 3a (Figure 4) was found to inhibit the viral replication
with an EC50 value of 38 µM (Table 1). Its scaffold was chosen for the preparation of a small series of
novel derivatives, with which to confirm the biological activity found.
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Figure 4: Chemical structure and best alignment found for 3a (aligned in purple against 2, in green).
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3a is characterised by a central pyrrolone nucleus, substituted in position 1 and 5 with two aromatic
rings, and linked in position 3 with a phenylidene system. A preliminary series of triphenyl-pyrrolone
derivatives was designed by varying the aromatic substituents on the 1-phenyl and 3-phenylidene rings.
This small series of compounds was envisaged to confirm the antiviral potential originally found for 3a
and to explore the effect of the aromatic substituents in the two substituted aromatic rings. In particular,
removal of the 4-ethyl carboxylate group on the 1-phenyl ring of 3a was envisaged to confirm its
relevance for antiviral activity (3b, 7b), along with its replacement with different electron withdrawing
(4-Br in 7d) and electron donating ( 4-OMe in 7c and 4-Phenyl in 7e) groups. Preliminary
modifications designed for the substituted 3-phenylidene ring of 3a included the removal of both
substituents (7a-e, 8a) or of the original 2-Br function only (8a), and their replacement with a 2,3-
dimethyl substitution (9a). All desired compounds were prepared according to an optimised two-step
synthetic pathway (Scheme 1).
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1
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01
02
03
R2
O
O
O
N
i
O
ii
O
OH
+
O
R1
R1
R1
4
5a-e
6a-e
3a-b, 7a-e, 8a, 9a
31-51%
29-57%
a R1= COOEt
b R1= H
c R1= OMe
d R1= Br
3a R1= COOEt; R2= 2-Br, 4-Me
3b R1= H; R2= 2-Br, 4-Me
7a R1= COOEt; R2= H
7b R1= H; R2= H
e R1= Ph
7c R1= OMe; R2= H
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d R1= Br; R2= H
e R1= Ph; R2= H
a R1= COOEt; R2= 4-Me
a R1= COOEt; R2= 2,3-Me
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Scheme 1: Reagents and conditions: i. AcONa, Ac O, 95 °C, 2h; ii. Substituted aniline, AcOH, rt, 4h, reflux, 22h.
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The strategy carried out began with the condensation of 3-benzoyl propanoic acid 4 with different para
substituted benzaldehydes 5a-e, with the formation of butenolide intermediates 6a-e through furanone
ring closure, followed by phenyl-amine displacement of the cyclic ester with the formation of the
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pyrrolone system in final products 3a-b, 7a-e, 8a and 9. According to literature procedures,
furanone intermediates 6a-e were obtained by heating the starting materials at 95 °C in acetic
anhydride in the presence of sodium acetate. The formation of the central butenolide ring is believed to
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follow a Perkin condensation between b-benzoylpropionic acid and the substituted aryl aldehyde,
which are thought to form an aldol-condensation product that subsequently undergoes internal
cyclisation with the formation of the furanone ring. In these conditions only one species, corresponding
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to the cis isomer, is formed. The desired final products were subsequently obtained by treating
intermediates 6a-e with differently substituted anilines, stirring the reaction mixture in glacial acetic
acid first at room temperature for 4 hours, and then heating to reflux for 22 hours. The furanone ring is
believed to open as a result of the ammonolysis of the starting compound by the aromatic amine,
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followed by cyclisation and dehydration events to give the final pyrrolone system.
All newly synthesised pyrrolones were evaluated for their potential antiviral activity in the HCV
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replicon and cytostatic assay (Table 1). The HCV protease inhibitor telaprevir (VX-950) was included
as positive control.
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Table 1: Antiviral effect of the test compounds on hepatitis C virus replication in the Huh5-2 replicon system and inhibition of
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NS3 helicase unwinding activity.
a,d; 37
b,d
c,d
e
Compound
EC (mM)
EC (mM)
CC (mM)
SI
Unwinding
5
0
90
50
f;
IC50 (µM)
1
6
3
3
3
7
7
7
7
7
8
9
a (SPECS)
38
149
>204
>4
>10
>58
4
n.d.
a (synthesised)
20.3±1.1
4.1±2.5
27.2±7.6
4.7±0.5
5.9±1.8
4.3±1.5
16.1±2.7
22.5±0.9
43.1±19.9
0.8±0.2
-
61.7±2.7
>205
>1000
>1000
>1000
>1000
438
b
a
b
c
12.4±6.3
>240
100
112
14±4.2
30.3±5.9
97.2±8.7
25.7±6.4
156±63.3
>244
6.4
18
6
21.6±1.8
d
e
11.6±0.8
>1000
>1000
>1000
>1000
n.d.
61.7
9.7
>11
2.1
58.8
-
a
a
81.1±13.1
-
-
-
-
90.8±26.8
47
(
VX-950)
Primuline
-
10±2
Aurintricarboxylic
-
-
-
0.3± 0.1
Acid
a
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133
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EC50 = 50% effective concentration (concentration at which 50% inhibition of virus replication is observed).
EC90 = 90% effective concentration (concentration at which 50% inhibition of virus replication is observed).
b
c
d
CC50 = 50% cytostatic/cytotoxic concentration (concentration at which 50% adverse effect is observed on the host cell).
The EC50, EC90 and CC50 values are the mean of at least 2 independent experiments, with standard deviations of ±10% of the
value quoted unless otherwise stated (mean value ± standard deviations).
e
SI = the ratio of CC50 to EC50
.
f
Concentration needed to reduce rates of helicase-catalysed DNA unwinding by 50%
n.d. Not determined
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Re-synthesised 3a confirmed its activity profile, with EC50, EC90 and CC50 values close to the ones
previously found for the batch acquired from SPECS. The presence of an ethyl ester function in the 3-
phenylidene ring does not appear to be essential for activity retention, since 3b, where this group is
removed, is the most active analogue found so far. A similar profile can be identified for derivatives
7b-e, where the 1-phenyl ring is unsubstituted and the ethyl ester group is replaced respectively with 4-
H, 4-Me, 4-Br and 4-phenyl groups. For analogs 7b-d however an increased cytotoxic effect can also
be observed. Activity retention found for 7a and 8a seems to indicate that also the presence of the 2-
bromo, 4-methyl substituent in the 1-phenyl ring is not essential for antiviral activity, while the
insertion of a methyl group in position 3 of this ring and the replacement of the original 2-bromo
substituent with an extra methyl group are detrimental, as can be observed for 9a.
All the final compounds synthesised were also tested for their potential interference with the HCV NS3
helicase activity (Table 1).Despite the fact that 7c shows some interference with the helicase
unwinding activity at high concentrations and 3b was found to competitively inhibit RNA binding with
an IC50 of 620 µM (data not shown), a trend for this effect cannot be identified, and it does not seem to
correlate with any specific structural feature. Moreover, the IC50 values observed are dramatically
higher than the range of activities found in the HCV replicon assay, and a correlation between the two
sets of data cannot be found. This evidence suggests that the antiviral effect of the pyrrolone structures
presented in this study might be due to a different target, viral or cellular, other than the HCV NS3
helicase. Additional studies aimed to further explore the structure-activity relationships associated to
the pyrrolone scaffold and to determine the biological target of the newly synthesised compounds are
ongoing and will be reported in due course. In particular, ongoing synthetic efforts are directed to
increase the water solubility of the novel antiviral scaffold, to further explore the effect of different
aromatic substitutions on the 1-phenyl, 3-phenylidene and 5-phenyl rings, to evaluate the importance of
the rigidity of the molecule with the preparation of unsaturated analogues, and to replace the central
pyrrolone nucleus with different heterocyclic systems.
In conclusion, starting from a ligand-based in silico approach, a substituted pyrrolone scaffold was
identified as hit for the inhibition of HCV replication in the subgenomic replicon assay. Its structure
was the starting point for the design and synthesis of a first series of new analogs, with which antiviral
potential was confirmed and improved. Different novel derivatives with EC50 values in the low
micromolar range were found. Preliminary structure-activity relationships could be identified for the
antiviral effect, while the first enzymatic evaluations performed seem to suggest that the antiviral
potential associated with the newly prepared compounds is not directly correlated with interference
with the NS3 helicase, but other viral or cellular targets might be involved. Further exploration of
modified novel derivatives and understanding the biological target of these structures are the current
focus of on-going investigations.
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