Optimization of small-molecule inhibitors of influenza virus polymerase: From thiophene-3-carboxamide to polyamido scaffolds

Article abstract of DOI:10.1021/jm500300r

Influenza virus infections represent a serious concern to public health, being characterized by high morbidity and significant mortality. To date, compounds targeting the viral ion-channel M2 or the viral neuraminidase are the drugs available for treatment of influenza, but the emergence of drug-resistant viral mutants renders the search for novel targets and their possible inhibitors a major priority. Recently, we demonstrated that the viral RNA-dependent RNA polymerase (RdRP) complex can be an optimal target of protein-protein disruption by small molecules, with thiophene-3-carboxamide derivatives emerging as promising candidates for the development of new anti-influenza drugs with broad-spectrum activity. Here, we report a further dissection of the thiophene-3-carboxamide structure. By using a GRID molecular interaction field (MIF)-based scaffold-hopping approach, more potent and nontoxic polyamido derivatives were identified, highlighting a new space in the chemical variability of RdRP inhibitors. Finally, a possible pharmacophoric model highlighting the key features required for RdRP inhibition is proposed.

Full text of DOI:10.1021/jm500300r

Article
pubs.acs.org/jmc
Optimization of Small-Molecule Inhibitors of Influenza Virus
Polymerase: From Thiophene-3-Carboxamide to Polyamido Scaffolds
Susan Lepri,§,# Giulio Nannetti,⊥,# Giulia Muratore,⊥ Gabriele Cruciani,§ Renzo Ruzziconi,§
Beatrice Mercorelli,⊥ Giorgio Palu,⊥ Arianna Loregian,*,⊥,∥ and Laura Goracci*,§,∥
̀
§Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy
⊥Department of Molecular Medicine, University of Padua, 35121 Padua, Italy
S
* Supporting Information
ABSTRACT: Influenza virus infections represent a serious
concern to public health, being characterized by high morbidity
and significant mortality. To date, compounds targeting the
viral ion-channel M2 or the viral neuraminidase are the drugs
available for treatment of influenza, but the emergence of drug-
resistant viral mutants renders the search for novel targets and
their possible inhibitors a major priority. Recently, we
demonstrated that the viral RNA-dependent RNA polymerase
(RdRP) complex can be an optimal target of protein−protein disruption by small molecules, with thiophene-3-carboxamide
derivatives emerging as promising candidates for the development of new anti-influenza drugs with broad-spectrum activity.
Here, we report a further dissection of the thiophene-3-carboxamide structure. By using a GRID molecular interaction field
(MIF)-based scaffold-hopping approach, more potent and nontoxic polyamido derivatives were identified, highlighting a new
space in the chemical variability of RdRP inhibitors. Finally, a possible pharmacophoric model highlighting the key features
required for RdRP inhibition is proposed.
identification of novel antiviral strategies.10 Recently, the viral
INTRODUCTION
■
polymerase attracted attention as a new target for the
Influenza A (FluA) and B (FluB) viruses are responsible for
hundreds of thousands of deaths each year, especially among
development of novel anti-influenza compounds.11−18 This
protein is a heterotrimeric complex formed by the PB1, PB2,
high-risk population groups (infants, elderly, people with
and PA subunits. The correct noncovalent assembly of the
immune deficiency).1 The prophylactic, yearly reformulated
three subunits is essential for viral RNA synthesis.19 It is known
vaccine and two classes of drugs are currently the only available
that all three subunits, PB1, PB2, and PA, are necessary for both
anti-influenza therapeutic options.2 The first class is repre-
sented by the M2 viral ion-channel inhibitors and includes the
transcription and replication of the viral genome.20,21 PB1
possesses polymerase activity, PB2 is responsible for cap-
adamantanes (i.e., amantadine and rimantadine). The M2
binding of host cell pre-mRNAs, whereas PA contains the
inhibitors are only effective against FluA, and a significant
increase of virus resistance to this class of compounds has been
observed in recent years. Currently, all circulating FluA virus
endonuclease domain and is implicated in RNA replication by
cleaving capped host pre-mRNAs. The main advantage in
strains appear to be resistant to M2 inhibitors.3,4 The second
exploiting the viral RNA polymerase as a target for drug design
is that, in contrast to the viral glycoproteins, it is highly
class of antiviral compounds targets the viral neuraminidase
conserved among different viral strains,19 and thus RNA
(NA). NA inhibitors block the release of virions after budding
from the host cell.5,6 Zanamivir was the first NA inhibitor
polymerase inhibitors are expected to possess a broad antiviral
activity.22
commercially developed.7 It was discovered in 1989 using a
Just as the rational design of NA inhibitors became possible
once the neuraminidase X-ray structure became available,
target-guided design and exemplifies one of the first successful
uses of this approach in drug discovery. The computational
today, the design of potential inhibitors of the influenza virus
approach was based on the GRID force field, developed at the
University of Oxford by Peter Goodford.8 The discovery of
RNA polymerase is facilitated by the recent elucidation of its
structure at atomic level.23−25 In particular, two recently
zanamivir was published in 1993 with more than a thousand
published X-ray structures of a C-terminal domain of PA bound
citations to date. Although limited by poor bioavailability,
to an N-terminal peptide of PB123,24 have clarified the
zanamivir is still on the market, even though the orally active
oseltamivir is usually preferred. Nowadays, NA inhibitors
represent the only class of antiviral drugs to treat both FluA
molecular details of the PA−PB1 interaction: an N-terminal
310 helix from PB1 binds into a hydrophobic groove in the C-
and FluB infections. However, resistance episodes were recently
observed for oseltamivir in several Flu strains.3,9 For these
Received: February 25, 2014
Published: May 1, 2014
reasons, increasing efforts have been devoted to the
© 2014 American Chemical Society
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terminus of PA, with relatively few residues driving the PA−
PB1 binding. More recently, the PB1−PB2 interaction domain
was also explored by X-ray crystallography.25 Although the use
of the PB1−PB2 interface as a target for the design of possible
RdRP inhibitors is still under investigation,26 the tailored design
of small molecules that inhibit the formation of the PA−PB1
complex formation has already been demonstrated to be a
promising strategy toward a new class of anti-influenza
drugs.11−18 Attempts to inhibit the formation of the PA−PB1
complex using peptides have also been reported.27,28 However,
whether it is better to use peptides or small molecules to inhibit
protein−protein interaction is still a topic of much debate. On
the one hand, peptides can better mimic the protein interaction
showing high target selectivity and affinity.29 On the other
hand, small molecules usually possess better pharmacokinetic
properties. Furthermore, there are only very few examples of
unmodified peptides that have reached the market as drugs, due
to their proteolytic instability or lack of cell permeation.
Recently, we reported an in silico screening of 3 million small
molecule structures to search for inhibitors of the PA−PB1
interaction, using the X-ray structure of the PA subunit
reported by He et al.24 as a template.13 As a result, the N-(3-
carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-7-(di-
fluoromethyl)-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxa-
mide, named as 1 in the present study (Figure 1, compound 5
RESULTS AND DISCUSSION
■
Design of Compound 1 Derivatives. Chemically,
compound 1 is a pyrazolo[1,5-a]pyrimidine carrying a N-
cyclopentathiophene carboxamide moiety at the C-3′ position,
a phenyl substituent at the C-5′ position, and a difluoromethyl
group at the C-7′ position (Figure 1). In this study, the
thiophenecarboxamide moiety was preserved since it was
recently identified to be a promising scaffold for anti-Flu
compounds.16 As a first attempt, the pyrazolo[1,5-a]pyrimidine
(scaffold I, Table 1) was also maintained, while the
difluoromethyl group, the cyclopentathiophene amide moiety,
and the phenyl substituent at the C-5′ position were modified
(compounds 2−6, Table 1). The selective incorporation of
fluorine atom(s) or fluoroalkyl group(s) (such as CF3, CHF2,
and CH2F) into organic molecules has become a trend in life-
sciences-related applications.30,31 Many studies have shown that
a fluorine atom(s) or fluoroalkyl group(s) can bring several
beneficial effects in bioactive molecules, such as the enhance-
ment of metabolic stability, lipophilicity, bioavailability, and
binding affinity.32−39 Among the fluoroalkyl groups, the
difluoromethyl (CHF2) group in compound 1 is of particular
interest, because it is known to be isosteric and isopolar to a
carbinol (CH2OH) unit and, despite its lipophilic nature, can
also act as a hydrogen bond donor.40 By replacing the
difluoromethyl group with the trifluoromethyl group (com-
pounds 2, 4−6, Table 1), we wanted to investigate whether or
not the hydrogen-bond donor capability of the CHF2 group
could be crucial in the interaction of compound 1 with PA.
Recent studies on new RdRP inhibitors bearing a cyclo-
heptathiophene moiety revealed that the size of the aliphatic
ring fused with thiophene was critical for the inhibitory effect.16
Therefore, we also tried to increase the size of the ring by
replacing the cyclopentane with a cyclohexane ring (com-
pounds 3−6, Table 1).
The last modification for scaffold I was the introduction of a
hydrophobic or a hydrophilic substituent at the para-position of
the phenyl ring (compounds 5 and 6 in Table 1, respectively).
The major structural modifications were performed by
replacement of the pyrazolo[1,5-a]pyrimidine (scaffold I,
Table 1) with a triazolopyrimidine or pyridine (scaffolds II
and III in Table 1, respectively). The replacement of the carbon
atom in the 3-position of the pyrazolo[1,5-a]pyrimidine with a
nitrogen atom (as in scaffold II) forces the amidic bond to
move from C-3′ to C-2′, leading to a more linear compound (7,
Table 1).
Compound 8 was synthesized from scaffold III (Table 1). Its
chemical structure shares some common features with a good
inhibitor recently published,16 in which a 2-pyridyl-amide is
bound to a cycloheptathiophene moiety at the C-2 position.
Scaffold Hopping Approach. A second strategy for the
hit optimization was the use of compound 1 as a template for
scaffold hopping, i.e., to search for new molecules endowed
with similar chemical features but characterized by a different
scaffold. This approach complements the classical structure−
activity relationship (SAR) study reported in the previous
section. Indeed, while structural modification on the same
scaffold provides information on possible critical interactions
for activity within a chemical series, the aim of a scaffold
hopping approach is to move from a hit compound toward
different scaffolds in the chemical space. Thus, if new active
compounds with a different structure emerge, their common
features can be related to activity.
Figure 1. Chemical structure of compound 1, an inhibitor of the PA−
PB1 interaction.
in ref 12), was shown to inhibit the PA−PB1 complex
formation in vitro (with an IC50 of 25.4
3.9 μM) and to
reduce the catalytic activity of the viral polymerase (with an
IC50 of 31.4 4.2 μM). In addition, compound 1 exhibited
antiviral activity against FluA in infected cells at EC50 values
around 100 μM, without showing significant cytotoxicity (CC50
> 250 μM, evaluated in MDCK and 293T cell lines).13
Because of its promising potency and lack of cytotoxicity, we
decided to give compound 1 further consideration.
In the present study, the optimization of compound 1 by
using two different approaches is reported. A number of
analogues of compound 1 were first synthesized to study the
structure−antiviral activity relationship using a classical
medicinal chemistry approach. In particular, the thiophene-3-
carboxamide moiety was maintained, recently emerging as a
promising scaffold for the design of RdRP inhibitors.16 Second,
using compound 1 as a template, we applied a scaffold hopping
approach to identify novel scaffolds for PA−PB1 complex
inhibitors. The biological evaluation of the novel candidates is
reported in terms of cytotoxicity, PA−PB1 binding disruption,
and activity against Flu virus replication. Finally, a possible
pharmacophore for PA−PB1 complex inhibitors is reported
here for the first time.
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Table 1. Summary of Activities of Synthesized Analogues of Compound 1 against FluA
Cytotoxicity (MTT) assay
ELISA PA−PB1
FluA minireplicon FluA plaque reduction
d
structure
interaction assay
assay
assay
CC50 (μM)
a
b
c
comp
scaffold
n
Rf
R
IC50 (μM)
EC50 (μM)
EC50 (μM)
293T cells MDCK cells
1
I
I
1
1
2
2
2
2
2
2
CHF2
H
H
27.2 3.6
44.8 3.3
91.2 7.4
120.4 15.6
43.0 9.9
135.3 14.2
7.5 0.7
>200
29.5 5.4
27.5 3.5
94.2 8.5
70.5 6.6
63.0 9.9
ND
75.5 8.8
57.0 15.5
66.0 8.5
>100
>250
>250
>250
>250
2
CF3
CHF2
CF3
3
I
H
>250
>250
4
I
H
>250
>250
5
I
CF3
Me
OMe
H
100.7 14.0
ND
>250
>250
6
I
CF3
40.4 3.3
>250
51.3 3.1
>250
7
II
III
CF3
9.2 2.3
ND
23.7 9.1
ND
8
>250
45.2 3.1
18.3 2.5
>250
9
35.1 4.3
30.5 6.2
>200
ND
ND
12.4 2.3
>250
10
45.9 4.4
ND
18.7 3.8
ND
11
30.1 4.1
>250
20.2 3.3
>250
12
PB11−15-Tat peptide
RBV
>200
>100
>100
37.2 5.6
ND
20.9 6.7
24.9 5.1
50.5 13.8
15.6 6.3
>250
>250
>250
>250
a
Activity of the compounds in ELISA PA−PB1 interaction assays. The IC50 value represents the compound concentration that reduces by 50% the
b
interaction between PA and PB1. Activity of the compounds in minireplicon assays. The EC50 value represents the compound concentration that
reduces by 50% the catalytic activity of FluA polymerase. Activity of the compounds in plaque reduction assays with the FluA PR8 strain. The EC50
value represents the compound concentration that inhibits 50% of plaque formation. Activity of the compounds in MTT assays. The CC50 value
c
d
represents the compound concentration that causes a decrease of cell viability of 50%. All the reported values represent the means SD of data
derived from at least three independent experiments in duplicate. ND: not determined.
As mentioned above, a structure-based virtual screening on 3
million compounds using the X-ray structure of the PA subunit
as a template was previously used to identify 293 possible PA−
PB1 complex inhibitors. Of the 293 hits, only 32 compounds
(including our hit compound) were acquired and tested.13 In
this study, starting with compound 1 as a template, we screened
the 293 compounds using the FLAP algorithm41 to evaluate
their similarity based on the GRID molecular interaction fields
(MIFs).8,42 The subset of 293 compounds were thus ranked by
their similarity to the template. On the basis of this similarity
scoring and taking into consideration their availability, cost, and
druggability, four commercially available compounds (9−12 in
Figure 2) were acquired and tested. Interestingly none of them
possess a thiophene-3-carboxamide scaffold.
Chemistry. Synthesis of 2-Aminotiophene-3-
[substituted]carboxamide Compounds. The synthesis of
Figure 2. Structures of four analogues of compound 1, according to
compound 1 and its analogues 2−8 is illustrated in Scheme
1. Knoevenagel condensation of cyanoacetamide with cyclo-
pentanone or cyclohexanone followed by sulfur-promoted
cyclization gave the suitable thiophene carboxamide derivatives
13a−b, respectively.43 The final amidopyrazoles 1−8 were
obtained by oxalyl chloride-promoted condensation of amino-
thiophenes 13a−b with the appropriate carboxylic acid,
obtained by alkaline hydrolysis of the respective ethyl esters.
The esters for the synthesis of products 5−8 were acquired
from SPECS (www.specs.net), whereas the esters 14a−b were
prepared by condensing the commercially available ethyl 3-
amino-1H-pyrazole-4-carboxylate with fluorinated 1,3-diones44
(Scheme 2). The triazole derivative 7 and the nicotinamide 8
the FLAP similarity score.
were analogously prepared by coupling amino-thiophene 13b
either with the chloride of the commercially available 5-phenyl-
7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidine-2-carbox-
ylic acid or with nicotinoyl chloride, respectively.
Synthesis of Polyamido Derivatives. A number of
derivatives of compound 10 were also synthesized. The
polyamido-sulfonamides 18−22 were prepared according to
literature procedures or their modification (Scheme 3). Thus,
the nucleophilic attack of either sec-butylamine or aniline at C-4
of isatoic anhydride, followed by the elimination of carbon
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a
Scheme 1
a
Reagents and conditions: (i) S8, morpholine, EtOH, 60 °C;43 (ii) 1. Suitable carboxylic acid (see Experimental Section), oxalyl chloride, DCM,
DMF cat.; 2. Pyridine, DCM. For R and Rf definition see Table 1.
a
Scheme 2
a
Reagents and conditions: (i) AcOH reflux;44 (ii) NaOH, EtOH, H2O.
a
presence of triethylamine, afforded the bisamide 17a−c.
Butyllithium-promoted bromine−lithium exchange in THF at
−78 °C, followed by addition of sulfur dioxide at −60 °C,
converted the bromo derivatives 17a−c in the corresponding
lithium sulfinate.46 The latter were treated with N-chlorosucci-
nimide to give the expected sulfonyl chlorides, which were
smoothly converted into the target sulfonamide 10 and 18−22
in 20−25% overall yield by reaction with the suitable amine in
acetone at 50 °C.48
Scheme 3
Biological Evaluation. Our hit compound 1, previously
tested as provided from the vendor,13 was synthesized and
retested, giving comparable results (Table 1).
The synthesized compounds 2−8 and the acquired
compounds 9−12 were tested in ELISA to determine their
inhibitory activity on PA−PB1 interaction. The PB1(1−15)−
Tat peptide13 was used as a positive control. In addition, we
evaluated the ability of the compounds to inhibit the activity of
FluA RNA polymerase by a minireplicon assay in transfected
HEK 293T cells,49 while the antiviral activity in FluA virus-
infected MDCK cells was evaluated by plaque reduction assays
(PRA) using the A/PR/8/34 (PR8) strain. In these assays,
ribavirin (RBV), a known inhibitor of RNA viruses polymer-
ase,50 was used as a positive control. Moreover, MTT
cytotoxicity assays in MDCK and HEK293T cell lines using
RBV as a reference compound were also performed to exclude
a
Reagents and conditions: (i) sec-butylamine (for 16a) or aniline (for
16b), DMF, 100 °C;45 (ii) 3-bromobenzoyl chloride (for 17a) or 3-
bromo-4-methyl-benzoyl chloride (for 17b−c) Et3N, toluene, reflux;
(iii) 1. n-BuLi, −78 °C, THF; 2. SO2, −60 °C, THF;47 3. NCS, DCM;
4. ArNH2, Et3N, acetone, 50 °C.48 For Ar see Table 2.
dioxide from the carbamic acid intermediate, gave the
anthranilamides 16a−b, respectively, in excellent yield.45,46
The condensation of the anthranilamides 16a−b with 4-
substituted 3-bromobenzoyl chloride in refluxing toluene, in the
4340
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Table 2. Summary of Activities of Analogues of Compound 10 against FluA Virus
a
Activity of the compounds in ELISA PA−PB1 interaction assays. The IC50 value represents the compound concentration that reduces the
b
interaction between PA and PB1 by 50%. Activity of the compounds in minireplicon assays. The EC50 value represents the compound
concentration that reduces the catalytic activity of FluA polymerase by 50%. Activity of the compounds in plaque reduction assays with the FluA
PR8 strain. The EC50 value represents the compound concentration that inhibits 50% of plaque formation. Activity of the compounds in MTT
c
d
assays. The CC50 value represents the compound concentration that causes a decrease of cell viability of 50%. All the reported values represent the
means SD of data derived from at least three independent experiments in duplicate.
cytotoxic compounds. Antiviral activity and toxicity data for all
the tested compounds are reported in Table 1.
the FluA minireplicon assay was observed. The inhibitory
efficacy in PRA was also slightly improved, even though
compound 5 resulted to be a weaker inhibitor with respect to
the hit compound. This finding is in agreement with our
previous results,13,16 indicating that hydrophobic interactions
are favorable in designing RdRP inhibitors. When a polar
substituent such as a methoxy group was introduced instead of
a methyl group to give compound 6, a less active compound in
the ELISA assay was obtained. This compound also resulted to
be toxic in cell-based assays. Compound 6 appeared to be the
only cytotoxic compound in the scaffold I series.
Even though the optimization of the scaffold I did not bring
successful results, the replacement of the pyrazolo[1,5-
a]pyrimidine with a triazolopyrimidine (scaffold II) to give
compound 7 in Table 1 proved to be very effective. Indeed,
compound 7, which bears the CF3 substituent and cyclohexane,
resulted to be about 3-fold more efficient than compound 1 in
inhibiting both PA−PB1 complex formation and viral
Modifications made using scaffold I (compounds 2−6) did
not give any significant improvement in the antiviral activity.
However, a comparison of the results for 1 and 2, differing in
the fluorinated substituent only, reveals that the H-bond donor
capability of the CHF2 moiety seems not to be critical for
inhibition. On the other hand, the replacement of the
cyclopentathiophene moiety of compound 1 with the cyclo-
hexathiophene moiety to give compound 3 induced a 3-fold
increase of the IC50 value in the ELISA and of the EC50 value in
the FluA minireplicon assay, although the activity of compound
3 against FluA replication is very similar to those of compounds
1 and 2. Surprisingly, the additional substitution of CHF2 with
CF3 (compound 4) led to a decreased activity in all the tests
performed. When a methyl group was added in the para-
position of the phenyl ring of compound 4 to give compound 5,
a decrease of the IC50 value in the ELISA and the EC50 value in
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Table 3. Evaluation of Broad-Spectrum Activities of Most Active Compounds
a
a
PRA vs FluA (EC50, μM)
PRA vs FluB (EC50, μM)
H1N1
H3N2
compound A/Solomon Island/3/06 A/Padova/30/11 A/Parma/24/09 (oseltamivir-resistant) A/WSN/67/05
B/Lee/40
B/Malaysia/2506/04
1
>100
>100
95.5 5.6
22.9 1.5
25.0 2.6
22.1 2.1
43.0 3.6
>100
>100
>100
7
23.3 3.0
17.5 2.6
16.5 1.7
22.5 3.5
20.6 2.8
22.6 5.3
16.7 4.2
37.6 6.6
20.0 2.3
15.9 3.2
10.8 2.1
23.5 2.8
33.5 4.2
21.8 1.4
15.5 2.1
25.5 3.7
25.9 1.8
29.6 4.0
19.8 4.2
26.3 5.5
10
18
19
a
Activity of the compounds in plaque reduction assays with the different FluA and FluB strains. The EC50 value represents the compound
concentration that inhibits 50% of plaque formation. All the reported values represent the means
independent experiments in duplicate.
SD of data derived from at least three
replication. Purification difficulties were encountered during the
synthesis of the cyclopentane analogue, and thus further
investigation of this scaffold would require an optimization of
the reaction conditions. The replacement of the pyrazolo[1,5-
a]pyrimidine with a pyridine to give scaffold III in Table 1 led
to a cytotoxic compound (compound 8) with no activity
according to the ELISA.
Addition of a further hydrophobic moiety on the sulfonamide
part, as in compound 20, did not affect cytotoxicity, but had a
detrimental effect on the antiviral activity. On the other hand,
replacement of the sec-butyl group in the terminal amido
moiety with a phenyl ring, as in compound 21, resulted in
toxicity in MCDK cells. This compound is only modestly active
in ELISA; thus its good antiviral activity in PRA could be due to
its toxicity profile. Finally, compound 22, which held a hydroxyl
group in para-position of sulfonamide portion, showed a
dramatic decrease in activity.
To test the specificity of the inhibitory activity, the four most
active compounds (compounds 7, 10, 18, and 19) and hit
compound 1 as a reference were tested by ELISA for inhibition
of an unrelated protein−protein interaction, i.e., the interaction
between the UL54 and UL44 subunits of human cytomegalo-
virus (HCMV) DNA polymerase complex as previously
described.52,53 None of the tested compounds exhibited a
dose-dependent inhibitory activity up to a concentration of 200
μM. In contrast, compound AL5, previously shown to inhibit
the interactions between the HCMV DNA polymerase
subunits,53 did interfere with UL54−UL44 binding (see Figure
S1 in the Supporting Information).
The antiviral effect of the four most active compounds 7, 10,
18, and 19 was finally investigated against a number of clinical
isolates of FluA other than PR8, of both H1N1 and H3N2
subtypes, including a swine-derived pandemic strain and an
oseltamivir-resistant virus. In addition, the same compounds
were also tested for their ability to inhibit the replication of two
FluB strains (B/Lee/40 and B/Malaysia/2506/04). The hit
compound 1 was also included as a reference compound. The
biological data for the evaluation of broad-spectrum activity are
reported in Table 3.
Compounds 7, 10, 18, and 19 displayed a similar antiviral
activity against all FluA and FluB strains, with EC50 values
ranging from 10.8 to 43.0 μM and significantly higher than
those of compound 1, confirming the successful optimization of
the hit compound.
Concerning the four compounds acquired following the
scaffold hopping approach, the polyamido derivative 10 was
found to be a good RdRP inhibitor. Indeed, compound 10 not
only exhibited an inhibitory activity against the PA−PB1
interaction comparable to that of the hit compound but also
proved to be 4-fold more potent than compound 1 in blocking
FluA replication. Of the other three acquired compounds (9,
11, 12), compounds 9 and 11 resulted to be cytotoxic, although
an IC50 of 35.1 4.3 μM was obtained for compound 9 in the
ELISA assays. Thus, these compounds were not further
investigated. Compound 12 was found to be not cytotoxic
but also inactive. To further investigate the new polyamido
scaffold, compound 10 and five analogues were synthesized.
Tests on the resynthesized compound 10 gave comparable
inhibitory effects to those obtained using the commercial
sample both in ELISA (IC50 values of 28.7 μM and 30.5 μM,
respectively) and in PRA (EC50 values of 19.3 μM and 18.7
μM, respectively).
The derivatives of compound 10 were aimed at tuning the
hydrophobicity and their size. Compounds 18 and 19, which
differ from the reference compound for the lack of one or all
methyl substituents, respectively, were synthesized first. Methyl
groups are not likely to be involved in specific interaction with
protein residues, but enhancing the overall hydrophobicity
could play a role in RdRP inhibitory efficacy, since it is known
that the PA cavity is mainly hydrophobic.13,16,51 Compounds
20 and 21 were then synthesized by adding an additional
aromatic ring to the two edges of compound 10. These
compounds served to preliminary investigate whether larger
and more hydrophobic compounds could display a similar
inhibitory activity. To mimic a “peptide-like” feature, an amide
linkage was used, which was typical of the polyamido scaffold of
compound 10. Finally, compound 22 was synthesized as an
attempt to explore the effect of the introduction of polar
substituents. The structures and biological data for the five
polyamido derivatives are reported in Table 2.
Preliminary ADME studies (water solubility, permeability,
and metabolic stability) were performed for compounds 7 and
19, which were the most promising compounds. Methods for
these studies are reported in the Supporting Information.
Concerning water solubility, compounds 7 and 19 showed a
solubility of 2.4 and 1.0 μg/mL, respectively, measured by
NMR as previously reported.54
Removal of one (18) or all (19) methyl groups gave results
comparable to the lead 10, so we assumed that their role was
not crucial for preservation of antiviral activity. However,
compound 19 resulted in the most efficient in inhibiting the
PA−PB1 interaction, giving an IC50 value of 9.2 1.5 μM in
the ELISA.
A preliminary PAMPA permeability assay55 was also
performed, with compound 19 resulting to be in the range of
medium/high permeability, while compound 7 fell in the “low
permeability” class. Finally, metabolic stability was evaluated in
HLM after 30 min incubation. Compound 7 was metabolically
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Figure 3. Structure of the five RdRP inhibitors used for pharmacophore generation. aCompound 1 in ref 13; bcompound 19 in ref 16; ccompound 6
in ref 16.
Figure 4. Pharmacophore for RdRP inhibitors targeting the PA−PB1 complex generated by FLAP. (A) Structure of the pharmacophore. The shape
is reported as a wireframe surface, while the pharmacophoric points and GRID MIFs are reported as solid spheres and surfaces, respectively. Color-
code: green = hydrophobic; red = H-bond acceptor; blue = H-bond donor. (B−F) compounds 7, 19, 23, 24, 25 aligned to the pharmacophore,
respectively.
stable in HLM, while compound 19 underwent through
aromatic hydroxylation at the benzenesulfonamide moiety to
give the monohydroxylated metabolite. After 30 min, substrate
was at 50% abundance. The metabolic stability of compound 1
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was also measured, and aliphatic hydroxylation at the
cyclopentane ring was observed, with compound 1 reduced
to 37%, highlighting the better stability of compounds 19 and 7
with respect to the hit compound. Although the reported
ADME profile is still very preliminary, it suggests that both
compounds are too lipophilic, and further lead optimization will
be aimed at finding the best compromise between lipophilicity
(which is important for activity) and solubility.
Pharmacophore Generation. Our recent studies, aimed at
discovering new small-molecule antiviral compounds targeting
the PA−PB1 subunit interaction of the viral RdRP, led to the
identification of five compounds that possess very different
chemical structures but similar inhibitory activity against the
replication of FluA and FluB. Compounds 7 and 19 were found
to be the most potent RdRP inhibitors. Three other
compounds were previously reported by some of us to be
active against the influenza polymerase complex.13,16 The
chemical structures of the five compounds are reported in
Figure 3. When a number of active compounds with different
scaffolds are identified, the most natural step to advance their
improvement is to attempt to generate a possible pharmaco-
phore. A pharmacophoric model was therefore generated by
alignment of the five compounds using the FLAPpharm
algorithm.56
The pharmacophoric model is reported in Figure 4A, while
the structures of the five compounds aligned to the
pharmacophore are reported in Figure 4B−F. As shown in
Figure 4A, a pharmacophore generated by FLAP is composed
of three entities: a shape (the wireframe surface), a number of
common atomic features indicating pharmacophoric points
(the spheres), and a number of regions (the solid surfaces)
indicating the most conserved GRID MIFs. The latter point is
very important since GRID MIFs provide information on the
possible interaction of a ligand with a target. Conserved GRID
MIFs among aligned active compounds are therefore likely to
be related to the activity. In Figure 4, the green MIFs are
related to hydrophobic interactions, while blue and red MIFs
refer to H-bond donor and H-bond acceptor interactions,
respectively. The same color-code is also used for the
pharmacophoric points.
donor group to match the GRID acceptor field (see Figure S2
in the Supporting Information). This result, in addition to the
variability of the NH groups previously discussed, suggests that
the H-bond acceptor GRID MIF region could be less critical for
binding.
CONCLUSIONS
■
In recent years, the viral RdRP has proved to be an attractive
target in the design of small molecules capable of inhibiting
influenza virus replication. A few RdRP inhibitors have been
published so far, and their antiviral activity is sometimes
associated with cytotoxic effects.15,17 The aim of this study was
to further dissect the chemical space of the RdRP inhibitors
targeting the PA−PB1 complex. The previously published
compound 1 was selected as a hit compound for further
optimization by two complementary approaches: (1) the design
and the synthesis of derivatives of compound 1, to investigate
the structural features that might be responsible for PA−PB1
complex disruption; (2) a scaffold hopping approach to identify
novel scaffolds for PA−PB1 complex inhibitors. In the
modulation of the chemical structure of compound 1, the
thiophene-3-carboxamide moiety, which emerged as a favorable
scaffold in the design of RdRP inhibitors,16 was always
preserved. However, when the pyrazolo[1,5-a]pyrimidine was
replaced with a triazolopyrimidine to give compound 7, a 3-fold
more potent PA−PB1 inhibitor was obtained with respect to
compound 1, inhibiting the physical interaction between the
two viral subunits with an IC50 value of 7.5 0.7 μM. This
finding suggests that the synthesis of new derivatives having a
more linear shape might be helpful to improve the inhibitory
effect. As a result of the scaffold hopping approach, we
identified the sulphonamide compound 10, which was acquired
and assayed. Despite the significantly different polyamido
scaffold, compound 10 produced a good RdRP inhibitory effect
targeting the PA−PB1 complex. The synthesis of the slightly
different compound 19 led to an inhibitory activity comparable
to that of compound 7. Both compounds 7 and 19 were found
to be not cytotoxic in the two cell lines used for testing, and
they were also able to inhibit a number of FluA and FluB
strains.
According to the pharmacophore GRID MIFs, extended and
quite planar hydrophobic moieties represent a common feature
among the RdRP inhibitors (green solid surface in Figure 4A).
In addition, one H-bond donor and one H-bond acceptor MIF
emerged upon alignment of the five compounds (blue and red
solid surfaces in Figure 4A, respectively). By observing the
correspondence of the GRID MIFs, the pharmacophoric points
(spheres), and the chemical structures of the aligned
compounds, it becomes clear that in all compounds a carbonyl
moiety is responsible for the H-bond donor MIF, acting as a H-
bond acceptor group, while the red MIFs are generated by an
NH group acting as a H-bond donor. In this case, the nature of
the NH moiety seems to be variable, ranging from amines,
anilines, amides, and sulfonamides. To further validate our
pharmacophoric hypothesis, five other recently published RdRP
inhibitors targeting the PA−PB1 complex were selected and
aligned on the pharmacophore. Among them, three inhibitors
were benzofurazan derivatives (compounds 8c and 47e in ref 17
and compound 7e in ref 15), and the remaining two
compounds were 3-cyano-4,6-diphenyl pyiridine derivatives
(compounds 11 and 12 in ref 18). Compounds 11, 12, and 8c
were able to fit all the pharmacophoric regions previously
described, while compounds 47e and 7e only lack the H-bond
The discovery of two very different scaffolds with similar
activities prompted us to generate a pharmacophoric model by
also taking into consideration three other recently published
RdRP inhibitors that have been proven to be active against
FluA and FluB. On the basis of this model, all the selected
active compounds were shown to possess an extended and
quite planar hydrophobic moiety. In addition, the alignment of
the molecular structures revealed that they have a common
carbonyl group which is able to act as a H-bond acceptor, and
in the opposite site an NH group of varying chemical nature
which is able to act as a H-bond donating group. The proposed
pharmacophore could be useful to design novel RdRP
inhibitors, focusing the attention on similar interaction
capabilities rather than on similar scaffolds.
EXPERIMENTAL SECTION
■
Computational Methods. The search for novel scaffolds for
RdRP inhibitors and pharmacophore generation were performed using
the FLAP algorithm.41,56 The FLAP software is developed and licensed
by Molecular Discovery Ltd. (www.moldiscovery.com). In the scaffold
hopping study, a database containing the best 293 hits previously
selected as possible PA−PB1 interaction inhibitors13 was prepared. All
protomeric forms in at least 20% abundance at pH 7.4 were generated
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for each compound using the MoKa algorithm.57,58 A number of 50
conformers for each protomer was also generated to mimic flexibility.
Then a ligand-based virtual screening was performed using compound
1 as a template. Thus, in this study FLAP algorithm was not used to
perform de novo virtual screening but only to rescore the best hits
found by a structure-based approach13 for their similarity to our hit
compound. The 293 candidates were ranked by the GloB-Sum
descriptor, and four compounds having a similarity score >1.5 and
different scaffolds were selected. Their availability, cost, and
druggability were also taken into consideration. Chemical structures
for the 293 compounds and their FLAP similarity scores are available
as Supporting Information. During pharmacophore generation, the
FLAPpharm algorithm56 in the FLAP package was used. The five
selected compounds 7, 19, 23, 24, and 25 modeled generating a
number of 30 conformers were automatically aligned by the software.
The same procedure was used to align the five RdRP inhibitors used to
validate the model.
Purities of the Acquired Compounds. Compounds 9−12 were
purchased from Ambinter (www.ambinter.com, codes: Amb1938148,
Amb10767907, Amb6474013, Amb1646026, respectively). Purity of
the acquired compounds was determined by UHPLC on Agilent
Technologies 6540 UHD Accurate Mass Q-TOF LC/MS, HPLC 1290
Infinity with DAD detector and evaluated to be higher than 95%.
UHPLC conditions to assess the purity of acquired or final
compounds were as follows: column, Phenomenex AERIS Peptide
1.2 mm × 1000 mm (1.7 μm); flow rate, 0.8 mL/min; acquisition
time, 20 min; DAD 190−650 nm; oven temperature, 45 °C; gradient
of acetonitrile in water containing 0.1% of formic acid (0−100% in 20
min).
and dried under a vacuum to afford a pale yellow solid (14% yield):
mp 170−172 °C (lit.59 mp 169−171 °C); H NMR (DMSO-d6) δ
1
7.15 (s, 2H), 6.47 (s, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.63 (t, J = 7.2 Hz,
2H), 2.26 (p, J = 7.1 Hz, 2H); 13C NMR (DMSO-d6) δ 168.2, 166.1,
140.4, 119.8, 103.5, 30.4, 28.8, 27.6; HRMS: calcd for C8H10N2OS
183.0592 (M + H+), found 183.0590 (M + H+).
2-Amino-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide
(13b).43 The title compound was synthesized according to the
procedure used for 13a from cyclohexanone, to give a pale pink solid
in 81% yield; mp 187−190 °C (lit.60 mp 190 °C); 1H NMR (CDCl3)
δ 6.16 (s, 2H), 5.52 (s, 2H), 2.69−2.60 (m, 2H), 2.59−2.42 (m, 2H),
1.92−1.68 (m, 4H); 13C NMR (CDCl3) δ 168.4, 160.6, 129.0, 118.5,
107.4, 27.0, 24.4, 22.8 (2C); HRMS: calcd for C9H12N2OS 197.0749
(M + H+), found 197.0749 (M + H+).
Ethyl 7-(difluoromethyl)-5-phenylpyrazolo[1,5-a]pyrimidine-3-
carboxylate (14a).44 A mixture of 4,4-difluoro-1-phenyl-1,3-butane-
dione (1.50 g, 7.6 mmol) and ethyl 3-amino-1H-pyrazole-4-
carboxylate (1.17 g, 7.6 mmol) was refluxed in glacial acetic acid (3
mL) overnight. After cooling to room temperature, the resulting
precipitate was filtered off, washed with water and dried. Trituration
with petroleum ether gave a white solid (1.71 g, 71% yield) which was
characterized as follows: mp 130−132 °C (lit.44 mp 137 °C); 1H NMR
(CDCl3) δ 8.60 (s, 1H), 8.34−8.19 (m, 2H), 7.74 (s, 1H), 7.63−7.54
(m, 3H), 7.40 (t, J = 53 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 1.47 (t, J =
7.2 Hz, 3H); 13C NMR (CDCl3) δ 162.3, 159.2, 148.3, 148.0, 139.7 (t,
J = 28 Hz), 135.8, 131.8, 129.2 (2C), 127.8 (2C), 108.0 (t, J = 242
Hz), 104.0, 103.3 (t, J = 4.9 Hz), 60.5, 14.5; 19F NMR (CDCl3) δ
−125.00 (d, J = 54 Hz, 2F); HRMS: calcd for C16H13F2N3O2 318.1054
(M + H+), found 318.1055 (M + H+).
Ethyl 7-(trifluoromethyl)-5-phenylpyrazolo[1,5-a]pyrimidine-3-
carboxylate (14b).44 The title compound was synthesized according
to the procedure used for ester 14a, but starting from 4,4,4-trifluoro-1-
phenyl-1,3-butanedione instead of 4,4-difluoro-1-phenyl-1,3-butane-
dione, to give compound 14b in 77% yield. Yellow solid, mp 110−112
°C (lit.44 mp 111−113 °C); 1H NMR (CDCl3) δ 8.74 (s, 1H), 8.42−
8.37 (m, 2H), 8.31 (s, 1H), 7.69−7.55 (m, 3H), 4.35 (q, J = 7.0 Hz,
2H), 1.38 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3) δ 161.8, 158.8,
148.4, 148.3, 135.6, 134.3 (q, J = 37 Hz), 132.4, 129.6 (2C), 128.3
(2C), 119.2 (q, J = 275 Hz), 106.7 (q, J = 3.4 Hz), 103.7, 60.5, 14.4;
19F NMR (CDCl3) δ −68.64 (3F); HRMS: calcd for C16H12F3N3O2
Chemistry. Nuclear magnetic resonance (NMR) spectra were
recorded on Bruker Advance II 400 MHz spectrometer at room
temperature with tetramethylsilane and trichlorofluoromethane as
internal standard. Chemical shifts (δ) are reported in parts per million
(ppm), and peak multiplicity are reported as s (singlet), d (doublet), t
(triplet), q (quartet), p (pentet), hept (heptet), m (multiplet), or br s
(broad singlet). HRMS spectra were registered on Agilent
Technologies 6540 UHD Accurate Mass Q-TOF LC/MS, HPLC
1290 Infinity. Purities of the final compounds were determined by
UHPLC as described above for acquired compounds and were ≥98%
pure. Methyl 5-(p-tolyl)-7-(trifluoromethyl)pyrazolo[1,5-a]-
pyrimidine-3-carboxylate, 5-(4-methoxyphenyl)-7-(trifluoromethyl)-
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid, 5-phenyl-7-(trifluoro-
methyl)-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylic acid were ac-
quired from Specs and used without further purification. All other
commercial products were acquired from Sigma-Aldrich; aniline and
2,5-dimethylaniline were freshly distilled before use.
336.0960 (M + H+), found 336.0960 (M + H+).
7-(Difluoromethyl)-5-phenylpyrazolo[1,5-a]pyrimidine-3-carbox-
ylic Acid (15a).44 Ester 14a (0.50 g, 1.6 mmol) was added to a KOH
(0.50 g) dissolved in ethanol/water (10:1, 16.5 mL), and the resulting
mixture was refluxed for 3 h. Upon cooling at room temperature, 5 N
hydrochloric acid was added until complete precipitation of the
product, which was filtered off and washed with water. After drying
under a vacuum, a white solid in 78% yield was obtained; mp 225−230
5-(p-Tolyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carbox-
ylic acid was obtained by ordinary hydrolysis of the corresponding
methyl ester with potassium hydroxide in methanol.
1
°C; H NMR (DMSO-d6) δ 12.61 (s, 1H), 8.68 (s, 1H), 8.49−8.37
(m, 2H), 8.12 (s, 1H), 7.66 (t, J = 53 Hz, 1H), 7.66−7.56 (m, 3H);
2-(sec-Butylamino)- and 2-(phenylamino)benzoic acid 16a−b were
prepared by heating anthranilic anhydride (20.0 mmol) with sec-
butylamine and aniline respectively, in DMF at 100 °C for 14 h.45,46 3-
Bromo-4-methylbenzoyl chloride was obtained by refluxing the
corresponding acid in SOCl2 for 4 h. After evaporation of the reagent
excess at reduced pressure, the crude product was used without further
purification. N-(3-Aminophenyl)benzamide was prepared by reacting
benzoyl chloride with tert-butyl 3-aminophenylcarbamate followed by
amino group deprotection upon heating at 140 °C. Tetrahydrofuran
was distilled from sodium wire after the characteristic blue color of in
situ generated sodium biphenyl ketyl (benzophenone-sodium “radical
anion”) had been found to persist. Air- and moisture-sensitive
compounds were stored in Schlenk tubes or Schlenk burets. They
were handled under an atmosphere of 99.995% pure nitrogen, using
appropriate glassware.
13C NMR (DMSO-d6) δ 168.2, 163.5, 152.5, 145.0 (t, J = 27 Hz),
140.8, 136.9 (2C), 134.3 (2C), 133.1, 133.0 (t, J = 9.9 Hz), 109.2 (t, J
= 254 Hz), 109.1, 108.8; 19F NMR (DMSO-d6) δ −67.50 (2F);
HRMS: calcd for C14H9F2N3O2 290.0741 (M + H+), found 290.0744
(M + H+).
7-(Trifluoromethyl)-5-phenylpyrazolo[1,5-a]pyrimidine-3-carbox-
ylic acid (15b).44 The title compound was prepared following the
same procedure as for acid 17a but using ester 14b. A white solid in
55% yield was obtained; mp 252−253 °C (lit.44 mp 250−251 °C); 1H
NMR (DMSO-d6) δ 12.65 (s, 1H), 8.74 (s, 1H), 8.45−8.39 (m, 2H),
8.35 (s, 1H), 7.68−7.53 (m, 3H); 13C NMR (DMSO-d6) δ 163.3,
158.6, 148.9 (2C), 148.3 (2C), 135.7, 134.3 (q, J = 37 Hz), 132.4,
129.6, 128.4, 120.0 (q, J = 279 Hz), 106.7 (q, J = 3.3 Hz), 104.5; 19F
NMR (DMSO-d6) δ −67.50 (3F); HRMS: calcd for C14H8F3N3O2
308.0647 (M + H+), found 308.0649 (M + H+).
2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxa-
mide (13a).43 Cyclopentanone (8.57 g, 0.11 mol) and morpholine
(9.74 g, 0.11 mol) were added dropwise to a stirred suspension of
sulfur (3.27 g, 0.10 mol, 1.0 equiv) and cyanoacetamide (8.60 g, 0.10
mol) in ethanol (100 mL), and the mixture was stirred at 60 °C
overnight. After cooling, the solid was filtered off, washed with MeOH,
General Procedure To Prepare Amides 1−8. Oxalyl chloride
(1.5 mmol) was added to a solution of the suitable carboxylic acid
15a−b (1.0 mmol) and DMF (0.1 mmol) in dry DCM (3 mL), and
the mixture was kept 2 h at 25 °C while stirring. After the solvent was
evaporated at reduced pressure, the residue was dissolved in dry
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dichloromethane (4 mL), the appropriate 2-amino-thiophene-3-
carboxamide (1.0 mmol) and pyridine (2.0 mmol) were added, and
the mixture was made to react at room temperature for 16 h. The
solvent was evaporated at reduced pressure, and the residue was
washed in sequence with diethyl ether (2 × 5 mL), 2 M sodium
hydroxide, 2 M hydrochloric acid, and water. Finally, the residue was
dried under a vacuum and chromatographed on silica gel (eluent, 9:1
dichloromethane/methanol mixture).
NMR (DMSO-d6)
δ
−67.42 (s, 3F); HRMS: calcd for
C24H20F3N5O3S 516.1312 (M + H+), found 516.1318 (M + H+).
2-({[5-Phenyl-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-
2-yl]carbonyl}amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car-
1
boxamide (7). Yield, 75%. Yellow crystals, mp >270 °C; H NMR
(DMSO-d6) δ 13.3 (s, 1H), 8.6 (s, 1H), 8.5−8.4 (m, 2H), 7.7 (m,
4H), 7.0 (s, 1H), 2.7 (dt, J = 32 and 6.0 Hz, 4H), 2.0−1.5 (m, 4H);
13C NMR (DMSO-d6) δ 167.7, 163.1, 158.9, 156.3, 155.1, 141.7,
135.3, 135.3 (q, J = 38 Hz), 133.1, 129.9, 129.8 (2C), 128.8 (2C),
128.0, 119.5 (q, J = 250 Hz), 118.0, 109.1, 25.7, 24.4, 22.9, 22.8; 19F
NMR (DMSO-d6): δ −67.48 (s, 3F). HRMS: calcd for
C22H17F3N6O2S 487.1164 (M + H+), found 487.1161 (M + H+).
2-(3-Pyridinecarbonylamino)-4,5,6,7-tetrahydrobenzo[b]-
thiophene-3-carboxamide (8). Yield, 71%. Yellow crystals, mp 233−
235 °C; 1H NMR (DMSO-d6) δ 13.06 (s, 1H), 9.05 (s, 1H), 8.81 (d, J
= 3.9 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.75 (bs, 1H), 7.63 (dd, J =
7.5 and 5.0 Hz, 1H), 7.15 (bs, 1H), 2.74 (bs, 2H), 2.66 (bs, 2H), 1.75
(bs, 4H); 13C NMR (DMSO-d6) δ 168.2, 161.5, 153.4, 148.6, 142.9,
135.4, 129.8, 128.7, 127.3, 124.6, 117.2, 25.6, 24.4, 22.9, 22.8; HRMS:
calcd for C15H15N3O2S 302.0963 (M + H+), found 302.0965 (M +
H+).
2-(3-Bromobenzamido)-N-(sec-butyl)benzamide (17a). A mixture
of 3-bromobenzoyl chloride (700 mg, 3.1 mmol), 2-amino-N-(sec-
butyl)benzamide (16a) (600 mg, 3.1 mmol), and triethylamine (0.56
mL, 4.0 mmol) in toluene (40 mL) was kept at 110 °C for 14 h. After
solvent evaporation at reduced pressure, the crude product was
crystallized from methanol to obtain a white solid (81% yield)
exhibiting the following properties: mp 133−134 °C; 1H NMR
(CDCl3) δ 12.16 (s, 1H), 8.71 (d, J = 8.8 Hz, 1H), 8.19 (s, 1H), 7.90
(d, J = 8.3 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.59−7.45 (m, 2H), 7.38
(t, J = 7.9 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 6.30 (bd, J = 8.5 Hz, 1H),
4.13 (hept, J = 6.9 Hz, 1H), 1.61 (q, J = 7.2 Hz, 2H), 1.27 (d, J = 6.6
Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3) δ 168.6, 164.1,
139.4, 136.9, 134.8, 132.6, 131.1, 130.3, 126.6, 125.4, 123.2, 123.1,
121.6, 121.0, 47.3, 29.7, 20.4, 10.5; HRMS calcd for C18H1979BrN2O2
375.0703 (M + H+), found 375.0715 (M + H+).
N-(3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-7-
(difluoromethyl)-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxa-
mide (1). Yield, 69%. Yellow crystals, mp >270 °C; 1H NMR (DMSO-
d6) δ 12.82 (s, 1H), 8.95−8.67 (m, 3H), 8.17 (s, 1H), 7.67 (t, J = 53
Hz, 1H), 7.87−7.42 (m, 3H), 7.54 (bs, 1H), 6.77 (s, 1H), 2.97 (t, J =
7.2 Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H), 2.39 (p, J = 7.5 Hz, 2H); 13C
NMR (DMSO-d6) δ 167.2, 159.6, 158.2, 148.3, 148.2, 145.8, 140.4 (t,
J = 27 Hz), 139.6, 135.4, 132.8, 132.3, 129.6 (2C), 129.4 (2C), 112.3,
109.6 (t, J = 253 Hz), 105.2, 104.7 (t, J = 5.0 Hz), 29.7, 28.8, 28.2; 19F
NMR (DMSO-d6) δ −124.47 (d, J = 52 Hz, 2F); HRMS: calcd for
C22H17F2N5O2S 454.1149 (M + H+), found 454.11496 (M + H+).
N-(3-Carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-5-
phenyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxa-
mide (2). Yield, 49%. Yellow crystals, mp >270 °C; 1H NMR (DMSO-
d6) δ 12.84 (s, 1H), 9.00−8.78 (m, 3H), 8.38 (s, 1H), 7.96−7.40 (m,
4H), 6.79 (bs, 1H), 2.97 (t, J = 7.0 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H),
2.44−2.27 (m, 2H); 13C NMR (DMSO-d6) δ 167.4, 159.4, 158.0,
148.5, 148.3, 146.4, 139.0, 135.1, 134.6 (q, J = 37 Hz), 132.8, 132.5,
129.7 (2C), 129.6 (2C), 119.8 (q, J = 273 Hz), 112.6, 106.9, 105.7,
29.6, 28.2, 28.0; 19F NMR (DMSO-d6) δ −67.50 (3F); HRMS: calcd
for C22H16F3N5O2S 472.1055 (M + H+), found 472.1051 (M + H+).
2-({[5-Phenyl-7-(difluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]-
carbonyl}amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxa-
mide (3). Yield, 59% Yellow crystals, mp >270 °C; 1H NMR (DMSO-
d6) δ 12.37 (s, 1H), 8.84−8.77 (m, 3H), 8.18 (s, 1H), 7.68 (t, J = 52
Hz, 1H), 7.74−7.58 (m, 3H), 7.54 (s, 1H), 7.04 (s, 1H), 3.02−2.70
(m, 2H), 2.70−2.61 (m, 2H), 1.94−1.64 (m, 4H); 13C NMR (DMSO-
d6) δ 167.4, 159.5, 158.1, 148.2, 145.8, 142.0, 140.5 (t, J = 27 Hz),
135.4, 132.3, 129.7 (2C), 129.4 (2C), 129.3, 126.9, 117.6, 108.9 (t, J =
257 Hz), 105.3, 104.7 (t, J = 4.9 Hz), 25.6, 24.2, 23.0, 22.9; 19F NMR
(DMSO-d6) δ −124.95 (d, J = 52 Hz, 2F); HRMS: calcd for
C23H19F2N5O2S 468.1306 (M + H+), found 468.1309 (M + H+).
2-({[5-Phenyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]-
carbonyl}amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxa-
mide (4). Yield, 59%. Orange crystals, mp >270 °C; 1H NMR
(DMSO-d6) δ 12.42 (s, 1H), 8.85 (s, 3H), 8.40 (s, 1H), 7.67 (m, 4H),
7.08 (bs, 1H), 2.78 (bs, 2H), 2.68 (bs, 2H), 1.77 (s, 4H). 13C NMR
(DMSO-d6) δ 167.4, 159.4, 158.0, 148.5, 146.4, 142.0, 135.1, 134.6 (q,
J = 37 Hz), 132.5, 129.7 (2C), 129.6 (2C), 129.4, 127.0, 119.8 (q, J =
273 Hz), 117.6, 106.9, 105.7, 25.7, 24.2, 23.0, 22.9; 19F NMR (DMSO-
d6) δ −67.48 (s, 3F). HRMS: calcd for C23H18F3N5O2S 486.1206 (M
+ H+), found 486.1215 (M + H+).
2-(3-Bromo-4-methylbenzamido)-N-(sec-butyl)benzamide (17b).
The title compound was prepared from 3-bromo-4-methylbenzoyl
chloride and 2-amino-N-(sec-butyl)benzamide (16a), analogously to
1
17a, to give a white solid in 80% yield; mp 136−137 °C. H NMR
(CDCl3) δ 12.07 (s, 1H), 8.72 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 2.0 Hz,
1H), 7.81 (dd, J = 7.8 and 2.1 Hz, 1H), 7.57−7.29 (m, 3H), 7.08 (t, J
= 6.5 and 5.5 Hz, 1H), 6.15 (bd, J = 9.5 Hz, 1H), 4.13 (hept, J = 7.0
Hz, 1H), 2.46 (d, J = 4.0 Hz, 3H), 1.60 (p, J = 7.3 Hz, 2H), 1.25 (d, J
= 6.6 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3) δ 168.6,
164.1, 142.0, 139.6, 134.2, 132.6, 132.0, 131.0, 126.4, 125.6, 125.4,
123.0, 121.6, 121.0, 47.3, 29.7, 23.1, 20.4, 10.5; HRMS calcd for
C19H2179BrN2O2 389.0859 (M + H+), found 389.0854 (M + H+).
2-(3-Bromo-4-methylbenzamido)-N-(phenyl)benzamide (17c).
The title compound was prepared according to the procedure used
for 17b, from 3-bromo-4-methylbenzoyl chloride and 2-amino-N-
(phenyl)benzamide (16b), to give a pale pink solid product in 88%
yield; mp 215 °C (dec.); 1H NMR (DMSO-d6) δ 11.55 (s, 1H), 10.53
(s, 1H), 8.33 (d, J = 8.5 Hz, 1H), 8.08 (s, 1H), 7.90 (d, J = 7.9, 1H),
7.79 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.61 (t, J = 7.9 Hz,
1H), 7.55 (d, J = 7.8 Hz, 1H), 7.44−7.27 (m, 3H), 7.20−7.05 (m,
1H), 2.41 (s, 3H); 13C NMR (DMSO-d6) δ 167.7, 163.6, 142.1, 139.1,
138.6, 134.5, 132.5, 131.9, 131.4, 129.5, 129.1 (2C), 126.5, 124.9,
124.6, 124.4, 124.1, 122.3, 121.4 (2C), 23.0; HRMS: calcd for
C21H1779BrN2O2 409.0546 (M + H+), found 409.0550 (M + H+).
General Procedure To Prepare Polyamido Products 10, 18−
22.47,48 Butyllithium (20.3 mL, 1.48 M in hexanes, 30 mmol) was
added dropwise at −75 °C to a solution of the appropriate bromo
derivative (17a−c) (10 mmol) in tetrahydrofuran (100 mL), and the
mixture was made to react for 10 min, under stirring, before the bath
temperature was allowed to rise to −60 °C. Sulfur dioxide was bubbled
into the mixture until its pH value reached 6−7. The cold bath was
removed, and the temperature was allowed to rise to 25 °C. Hexane
(20 mL) was added and the formed white precipitate was filtered,
washed with hexane, and dried at 50 °C. The dry solid was suspended
2-({[5-(p-Tolyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]-
carbonyl}amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxa-
mide (5). Yield, 47%. Yellow crystals, mp >270 °C; 1H NMR (DMSO-
d6) δ 12.36 (s, 1H), 8.81 (s, 1H), 8.75 (d, J = 8.2 Hz, 2H), 8.36 (s,
1H), 7.59 (bs, 1H), 7.51 (d, J = 8.2 Hz, 2H), 7.07 (bs, 1H), 2.72 (bs,
2H), 2.67 (bs, 2H), 2.42 (s, 3H), 1.73 (bs, 4H); 13C NMR (DMSO-
d6) δ 167.4, 159.3, 158.0, 148.4, 146.4, 142.9, 142.0, 134.5 (q, J = 38
Hz), 132.4, 130.3 (2C), 129.6 (2C), 129.3, 126.9, 119.8 (q, J = 275
Hz), 117.6, 106.7, 105.5, 25.6, 24.2, 23.1, 23.0, 21.6; 19F NMR
(DMSO-d6) δ −67.46 (s, 3F). HRMS: calcd for C24H20F3N5O2S
500.1363 (M + H+), found 500.1371 (M + H+).
2-({[5-(4-Methoxyphenyl)-7-(trifluoromethyl)pyrazolo-[1,5-a]-
pyrimidin-3-yl]carbonyl}amino)-4,5,6,7-tetrahydrobenzo[b]-
thiophene-3-carboxamide (6). Yield, 85%. Orange crystals, mp >270
1
°C; H NMR (DMSO-d6) δ 12.37 (s, 1H), 8.58 (d, J = 9.0 Hz, 2H),
8.79 (s, 1H), 8.32 (s, 1H), 7.62 (bs, 1H), 7.25 (d, J = 9.0 Hz, 2H), 7.07
(bs, 1H), 3.92 (s, 3H), 2.78 (bs, 2H), 2.68 (bs, 2H), 1.78 (bs, 4H). 13C
NMR (DMSO-d6) δ 167.4, 163.3, 159.0, 158.0, 148.3, 146.4, 142.1,
134.3 (q, J = 37 Hz), 131.7 (2C), 129.3, 127.6, 126.9, 119.8 (q, J = 369
Hz), 117.5, 115.1 (2C), 106.4, 105.2, 56.1, 25.6, 24.2, 23.1, 23.0; 19F
4346
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Article
in dichloromethane (20 mL), the mixture was cooled at 0 °C, and N-
chlorosuccinimide (1.9 g, 14 mmol, 1.4 equiv) was added in portions.
The mixture was made to react for 15 min at 0 °C, while stirring, and
then it was kept at room temperature for further 25 min. The resulting
suspension was filtered off on Celite, the solvent was evaporated at
reduced pressure, and the solid residue was dissolved in acetone (20
mL). Triethylamine (1.4 mL, 10 mmol) and the suitable amine (10
mmol) were added, and the mixture was kept at room temperature for
12 h while stirring. After solvent evaporation at reduced pressure,
chromatography of the residue on silica gel allowed collection of the
pure product.
2.57 (s, 3H), 2.09 (s, 3H), 1.97 (s, 3H); 13C NMR (DMSO-d6) δ
167.7, 163.6, 141.4, 140.3, 139.0, 138.6, 136.0, 134.6, 133.7, 132.8,
132.6, 131.8, 131.0, 130.8, 129.4, 129.1 (2C), 128.6, 128.0, 127.9,
124.7, 124.1, 124.0, 122.2, 121.5 (2C), 20.8, 20.6, 17.6; HRMS: calcd
for C29H27N3O4S 514.1801 (M + H+), found 514.1797 (M + H+).
3-[2-(sec-Butylcarbamoyl)phenylcarbamoyl]-N-(4-
hydroxyphenyl)benzenesulfonamide (22). Yield, 26%. White solid,
1
mp 202 °C (dec.). H NMR (DMSO-d6) δ 12.61 (s, 1H), 9.88 (s,
1H), 9.28 (s, 1H), 8.59−8.49 (m, 2H), 8.28 (s, 1H), 8.07 (d, J = 8.0
Hz, 1H), 7.89−7.70 (m, 3H), 7.56 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6
Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 8.7 Hz, 2H), 3.98 (hept,
J = 6.5 Hz, 1H), 1.59−1.44 (m, 2H), 1.14 (d, J = 6.7 Hz, 3H), 0.86 (t,
J = 7.4 Hz, 3H). 13C NMR (DMSO-d6) δ 167.8, 162.9, 155.0, 140.5,
138.8, 135.3, 132.1, 130.1, 130.0, 129.9, 128.3, 128.0, 125.9, 124.3
(2C), 123.2, 121.2, 120.6, 115.6 (2C), 46.6, 28.6, 19.9, 10.7; HRMS:
calcd for C24H25N3O5S 468.5986 (M + H+), found 468.1599 (M +
H+).
Biology. Compounds and Peptide. Each test compound was
dissolved in DMSO 100%. RBV (1-D-ribofuranosyl-1,2,4-triazole-3-
carboxamide) was obtained from Roche. The PB1(1−15)-Tat peptide
was synthesized and purified by the Peptide Facility of CRIBI
Biotechnology Center (University of Padua, Padua, Italy). This
peptide contains the first 15 N-terminal amino acids of PB1 protein
conjugated to the C-terminal sequence of HIV Tat protein (amino
acids 47−59), which allows intracellular delivery.
Plasmids. Plasmids pcDNA-PB1, pcDNA-PB2, pcDNA-PA, and
pcDNA-NP, containing cDNA copies of the influenza A/PR/8/34
virus PB1, PB2, PA, and NP genes, respectively, were created as
described elsewhere49 and kindly provided by P. Digard (Roslin
Institute, University of Edinburgh, United Kingdom). Plasmid pPolI-
Flu-ffLuc, which contains an influenza virus-based luciferase
minireplicon vRNA under the control of the human RNA polymerase
I promoter, was provided by L. Tiley (University of Cambridge,
United Kingdom). Plasmid pRL-SV40 expressing the Renilla luciferase
was purchased from Promega.
Cells and Virus. Mardin-Darby canine kidney (MDCK) and human
embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Life Biotechnologies) supple-
mented with 10% (v/v) fetal bovine serum (FBS, Life Technologies)
and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin,
Life Technologies) at 37 °C in a humidified atmosphere with 5% CO2.
Influenza A/PR/8/34 virus (H1N1, Cambridge lineage) was obtained
from P. Digard (Roslin Institute, University of Edinburgh, United
Kingdom). The FluA viruses A/Wisconsin/67/05 and A/Solomon
Island/3/06, and the influenza B/Malaysia/2506/4 virus were
provided by R. Cusinato (Clinical Microbiology and Virology Unit,
Padua University Hospital, Padua, Italy). Influenza B/Lee/40 virus was
obtained from W. S. Barclay (Imperial College, London, United
Kingdom). The clinical isolate A/Parma/24/09 was kindly provided
3-[2-(sec-Butylcarbamoyl)phenylcarbamoyl]-2-methyl-N-(2,5-
dimethylphenyl)benzenesulfonamide (10). Yield, 19%. White
1
rhombic crystals, mp 186−188 °C: H NMR (DMSO-d6) δ 12.61
(s, 1H), 9.71 (s, 1H), 8.70−8.39 (m, 2H), 8.27 (s, 1H), 8.01 (d, J =
7.5, 1H), 7.85−7.70 (m, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.55 (t, J = 7.9
Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 7.7 Hz, 1H), 6.90 (d, J =
8.1 Hz, 1H), 6.75 (s, 1H), 3.97 (hept, J = 7.2 Hz, 1H), 2.58 (s, 3H),
2.12 (s, 3H), 1.98 (s, 3H), 1.61−1.53 (m, 2H), 1.13 (d, J = 6.2 Hz,
3H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6) δ 168.3, 163.4,
142.1, 139.2, 135.9, 135.8, 134.7, 132.5, 131.5, 131.0, 130.6, 130.5,
130.2, 128.8, 127.8, 127.7, 126.2, 123.8, 121.8, 121.2, 47.0, 29.1, 23.1,
20.9, 20.4, 17.6, 11.1; HRMS: calcd for C27H31N3O4S 494.2114 (M +
H+), found 494.2111 (M + H+).
3-[2-(sec-Butylcarbamoyl)phenylcarbamoyl]-N-(2,5-
dimethylphenyl)benzene-sulfonamide (18). Yield, 18%. White
1
rhombic crystals, mp 183−184 °C. H NMR (DMSO-d6) δ 12.61
(s, 1H), 9.67 (s, 1H), 8.70−8.39 (m, 2H), 8.25 (s, 1H), 8.11 (dt, J =
7.5 and 1.6 Hz, 1H), 7.89−7.73 (m, 3H), 7.56 (t, J = 7.9 Hz, 1H), 7.22
(t, J = 7.8 Hz, 1H), 6.99 (d, J = 7.7 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H),
6.76 (d, J = 2.3 Hz, 1H), 3.97 (hept, J = 7.4 Hz, 1H), 2.13 (s, 3H),
1.91 (s, 3H), 1.61−1.53 (m, 2H), 1.13 (d, J = 6.6 Hz, 3H), 0.85 (t, J =
7.5 Hz, 3H); 13C NMR (DMSO-d6) δ 168.3, 163.4, 142.1, 139.2,
135.9, 135.8, 134.7, 132.5, 131.5, 131.0, 130.6, 130.5, 130.2, 128.8,
127.8, 127.7, 126.2, 123.8, 121.8, 121.2, 47.0, 29.1, 20.9, 20.4, 17.6,
11.1; HRMS: calcd for C26H29N3O4S 480.5986 (M + H+), found
480.1956 (M + H+).
3-[2-(sec-Butylcarbamoyl)phenylcarbamoyl]-N-phenylbenzene-
1
sulfonamide (19). Yield, 21%. White crystals, mp 207−208 °C. H
NMR (DMSO-d6) δ 12.64 (s, 1H), 10.46 (s, 1H), 8.62−8.48 (m, 2H),
8.35 (t, J = 1.8 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.00−7.90 (m, 1H),
7.85 (dd, J = 7.9 and 1.5 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.56 (ddd, J
= 8.6, 7.4, and 1.5 Hz, 1H), 7.26−7.16 (m, 3H), 7.16−7.07 (m, 2H),
7.01 (tt, J = 7.1 and 1.2 Hz, 1H), 3.97 (hept, J = 6.8 Hz, 1H), 1.60−
1.45 (m, 2H), 1.13 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H); 13C
NMR (DMSO-d6) δ 168.3, 163.2, 140.9, 139.2, 137.8, 135.9, 132.5,
131.0, 130.7, 130.3, 129.7 (2C), 128.8, 126.3, 124.8, 123.8, 121.7,
121.1, 120.7 (2C), 47.1, 29.1, 20.4, 11.2; HRMS: calcd for
C24H25N3O4S 452.5454 (M + H+), found 452.1651 (M + H+).
5-[2-(sec-Butylcarbamoyl)phenylcarbamoyl]-2-methyl-N-[(3-ben-
zamido)-phenyl]benzenesulfonamide (20). Yield, 22%. White
̀
by I. Donatelli (Istituto Superiore di Sanita, Rome, Italy); the local
strain A/Padova/30/2011 of the pandemic FluA H1N1 virus was
donated by C. Salata and A. Calistri (University of Padua, Padua,
Italy). All influenza viruses were propagated in MDCK cells.
1
crystalline, mp 155−157 °C. H NMR (DMSO-d6) δ 12.57 (s, 1H),
10.63 (s, 1H), 10.20 (s, 1H), 8.66−8.48 (m, 3H), 7.97 (dd, J = 7.9 and
1.9 Hz, 1H), 7.89−7.80 (m, 3H), 7.69 (t, J = 2.0 Hz, 1H), 7.66−7.50
(m, 3H), 7.47−7.40 (m, 2H), 7.40−7.35 (m, 1H), 7.26−7.13 (m, 2H),
6.87 (ddd, J = 8.2, 2.1, and 1.0 Hz, 1H), 3.96 (hept, J = 7.2 Hz, 1H),
2.68 (s, 3H), 1.62−1.41 (m, 2H), 1.12 (d, J = 6.6 Hz, 3H), 0.85 (t, J =
7.4 Hz, 3H). 13C NMR (DMSO-d6) δ 168.4, 166.0, 163.5, 141.3,
140.4, 139.4, 138.8, 138.1, 135.2, 133.8, 133.2, 132.5, 132.0, 131.1,
129.7, 129.4, 128.8, 128.7 (2C), 128.1 (2C), 123.5, 121.7, 121.0, 116.1,
114.6, 111.5, 47.1, 29.1, 20.4, 20.3, 11.1; HRMS: calcd for
C32H32N4O5S 585.6927 (M + H+), found 585.2173 (M + H+).
3-(N-(2,5-Dimethylphenyl)sulfamoyl)-4-methyl-N-(2-
(phenylcarbamoyl)phenyl)benzamide (21). Yield, 24%. White
Protein Expression and Purification. Escherichia coli-expressed,
purified GST and GST-PB1(1−25) proteins were obtained as previously
described.13,61,62 The 6His-PA(239−716) protein was expressed in E. coli
strain BL21(DE3)pLysS and purified as described.13,16
PA−PB1 Interaction Enzyme-Linked Immunosorbent Assay
(ELISA). To analyze the ability of each test compound to dissociate
the PA−PB1 interaction in vitro, a procedure already described13 was
followed. Briefly, microtiter plates (Nuova Aptca) were coated with
400 ng of purified 6His-PA(239−716) for 3 h at 37 °C and then blocked
with 2% BSA (Sigma) in PBS for 1 h at 37 °C. After washing with PBS
plus 0.3% Tween 20, 200 ng of GST-PB1(1−25), or of GST alone as a
control, in the absence or the presence of test compounds at various
concentrations (10, 50, 100, 200 μM) were added and incubated O/N
at room temperature. After washing, the interaction between 6His-
PA(239−716) and GST-PB1(1−25) was detected by an with anti-GST
monoclonal antibody conjugated to horseradish peroxidase (HRP)
(GenScript; diluted 1:3000 in PBS plus 2% FBS). Following washes
1
rhombic crystals, mp 235 °C (dec.); H NMR (DMSO-d6) δ 11.70
(s, 1H), 10.53 (s, 1H), 9.70 (s, 1H), 8.35 (dd, J = 8.3 and 1.3 Hz, 1H),
8.29 (d, J = 1.9 Hz, 1H), 8.01 (dd, J = 8.0 and 2.0 Hz, 1H), 7.94−7.88
(m, 1H), 7.72 (d, J = 7.6 Hz, 2H), 7.61 (dd, J = 7.8 and 6.0 Hz, 2H),
7.36 (t, J = 7.9 Hz, 2H), 7.31 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.3 Hz,
1H), 7.00 (d, J = 7.7 Hz, 1H), 6.89 (d, J = 7.9 Hz, 1H), 6.75 (s, 1H),
4347
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Journal of Medicinal Chemistry
Article
with PBS plus 0.3% Tween 20, the chromogenic substrate 3,3′,5,5′
tetramethylbenzidine (TMB, KPL) was added, and the consequent
color development was measured at 450 nm by an ELISA plate reader
(Tecan Sunrise). Values obtained from the samples treated with only
DMSO were used to set as 100% of PA−PB1 interaction.
Cytotoxicity Assays. Cytotoxicity of compounds was tested in
MDCK and HEK 293T cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) method, as previously
reported.12 Briefly, HEK 293T or MDCK cells (2 × 104 per well)
were cultured in 96-well plates at 37 °C for 24 or 48 h, respectively, in
DMEM containing serially diluted compounds (from 250 to 1.9 μM).
Then, MTT solution (5 mg/mL in PBS) was added to each well and
plates were incubated for 4 h at 37 °C. Successively, a solubilization
solution was added to lyse cells. After 3 h of further incubation at 37
°C, absorbance was read at 620 nm using an ELISA plate reader
(Tecan Sunrise).
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*(L.G.) Phone, +39 075 585 5632; fax, +39 075 45646; e-mail,
laura.goracci@unipg.it.
*(A.L.) Phone, +39 049 8272363; fax, +39 049 8272355; e-
mail, arianna.loregian@unipd.it.
Author Contributions
#S.L. and G.N. contributed equally to this work.
Author Contributions
∥L.G. and A.L. contributed equally to this work.
Plaque Reduction Assays (PRA). The experiments were carried out
as previously described.13 Briefly, in a 12-well plates format, a
confluent monolayer of MDCK cells were infected with all FluA or
FluB virus assessed at approximately 40 PFU/well in DMEM
supplemented with 1 μg/mL of TPCK-treated trypsin (Worthington
Biochemical Corporation) and 0.14% BSA and incubated for 1 h at 37
°C. The influenza viruses infection was performed in the presence of
different concentrations of test compounds or solvent (DMSO) as a
control. Serum-free medium containing 1 μg/mL of TPCK-treated
trypsin, 0.14% BSA, 1.2% Avicel, and DMSO or test compounds at the
indicated concentrations was added after 1 h of virus adsorption. After
48 h of incubation, cells were fixed with 4% formaldehyde and stained
with 0.1% toluidine blue. Viral plaques were counted, and the mean
plaque number in the DMSO-treated control was set at 100%.
Minireplicon Assays. HEK 293T cells were seeded at 105 per well
on 24-well plates. After 24 h, cells were transiently transfected, in the
presence of the test compounds at different concentrations or DMSO,
with pcDNA-PB1, pcDNA-PB2, pcDNA-PA, pcDNA-NP plasmids
(100 ng/well of each) along with pPolI-Flu-ffLuc plasmid (50 ng/
well) as described elsewhere.13 In addition, a plasmid constitutively
expressing Renilla luciferase, pRL-SV40 (50 ng/well), was included in
the transfection mixture to normalize variations in transfection
efficiency. After 4 h, the medium was replaced with fresh DMEM
containing DMSO or test compounds. At 24 h post-transfection, cells
were lysed, and the relative firefly and Renilla luciferase activities were
determined using the Dual Luciferase Assay Kit from Promega and a
luminescence counter (Victor X2, PerkinElmer). The luciferase
activities of the samples treated with DMSO (no test compound)
were set as 100%.
UL54−UL44 Interaction ELISA. This assay was conducted as
previously described,53 with minor modifications. Briefly, microtiter
plates were coated with 0.2 mg of purified baculovirus-expressed
HCMV UL54 and blocked with 2% BSA (Sigma) in PBS for 1 h. After
washing, 0.5 mg of purified baculovirus-expressed UL44, mixed with
each compound at different concentrations or with DMSO alone at a
final 1% concentration (no compound), was added and incubated for 1
h at 37 °C. Following further washes, the wells were incubated with
monoclonal antibody (mAb) YL1/2, which recognizes the EEF
epitope inserted at the C terminus of UL44,52 for 1 h at 37 °C. Plates
were then washed and incubated with horseradish peroxidase (HRP)-
conjugated antirat antibody (Sigma). Following washes with PBS plus
0.3% Tween 20, the chromogenic substrate 3,3′,5,5′ tetramethylbenzi-
dine (TMB) (KPL) was added, and absorbance was read at 450 nm on
an ELISA plate reader (Tecan Sunrise).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank P. Digard, R. Cusinato, A. Calistri, C. Salata, I.
Donatelli, and W. S. Barclay for FluA and FluB viruses; P.
Digard and L. Tiley for plasmids; A. Valeri for collaboration in
metabolic stability assay; and R. Alvarez and S. Wendelspiess at
F. Hoffmann-La Roche Ltd. for permeability assays. We also
thank P. Digard for helpful suggestions. This work was
supported by Italian Ministry of Health and Istituto Superiore
̀
Sanita, Progetto Finalizzato 2009 “Studio e Sviluppo di Nuovi
Farmaci Antivirali Contro Infezioni da Virus Influenzale A-
H1N1” (to V.C., A.L., G.P., and O.T.) and by ESCMID
Research Grant 2013 (to B.M.).
ABBREVIATIONS
■
NA, neuraminidase; CC50, concentration that causes a decrease
of cell viability of 50%; Flu, influenza virus; FluA, influenza A
virus; FluB, influenza B virus; HEK, human embryonic kidney;
HLM, human liver microsomes; PAMPA, parallel artificial
membrane permeability assay; MDCK, Mardin−Darby canine
kidney; NP, nucleoprotein; PA, polymerase acidic protein; PB1,
polymerase basic protein 1; PB2, polymerase basic protein 2;
PRA, plaque reduction assays; RBV, ribavirin; RdRP, RNA-
dependent RNA polymerase; RNP, ribonucleoprotein; SAR,
structure−activity relationship; ADME, absorption, distribu-
tion, metabolism and excretion; HCMV, human cytomegalo-
virus
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ASSOCIATED CONTENT
■
S
* Supporting Information
Table containing structures of the 293 compounds used for
ligand-based virtual screening and their FLAP similarity score.
Validation of the pharmacophore using literature data. Methods
for preliminary ADME assays. Figure reporting data of
compounds activity in the UL54−UL44 interaction ELISA.
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