Aryl Sulfonamide Inhibits Entry and Replication of Diverse Influenza Viruses via the Hemagglutinin Protein

Article abstract of DOI:10.1021/acs.jmedchem.1c00304

Influenza viruses cause approximately half a million deaths every year worldwide. Vaccines are available but partially effective, and the number of antiviral medications is limited. Thus, it is crucial to develop therapeutic strategies to counteract this major pathogen. Influenza viruses enter the host cell via their hemagglutinin (HA) proteins. The HA subtypes of influenza A virus are phylogenetically classified into groups 1 and 2. Here, we identified an inhibitor of the HA protein, a tertiary aryl sulfonamide, that prevents influenza virus entry and replication. This compound shows potent antiviral activity against diverse H1N1, H5N1, and H3N2 influenza viruses encoding HA proteins from both groups 1 and 2. Synthesis of derivatives of this aryl sulfonamide identified moieties important for antiviral activity. This compound may be considered as a lead for drug development with the intent to be used alone or in combination with other influenza A virus antivirals to enhance pan-subtype efficacy.

Full text of DOI:10.1021/acs.jmedchem.1c00304

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Article
Aryl Sulfonamide Inhibits Entry and Replication of Diverse Influenza
Viruses via the Hemagglutinin Protein
Kris White, Matthew Esparza, Jue Liang, Prasanna Bhat, Jacinth Naidoo, Briana L. McGovern,
Michael A. P. Williams, Busola R. Alabi, Jerry Shay, Hanspeter Niederstrasser, Bruce Posner,
Adolfo García-Sastre, Joseph Ready, and Beatriz M. A. Fontoura*
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ABSTRACT: Influenza viruses cause approximately half a million
deaths every year worldwide. Vaccines are available but partially
effective, and the number of antiviral medications is limited. Thus, it is
crucial to develop therapeutic strategies to counteract this major
pathogen. Influenza viruses enter the host cell via their hemagglutinin
(HA) proteins. The HA subtypes of influenza A virus are
phylogenetically classified into groups 1 and 2. Here, we identified
an inhibitor of the HA protein, a tertiary aryl sulfonamide, that
prevents influenza virus entry and replication. This compound shows
potent antiviral activity against diverse H1N1, H5N1, and H3N2
influenza viruses encoding HA proteins from both groups 1 and 2.
Synthesis of derivatives of this aryl sulfonamide identified moieties
important for antiviral activity. This compound may be considered as a
lead for drug development with the intent to be used alone or in
combination with other influenza A virus antivirals to enhance pan-subtype efficacy.
H4, H7, H10, H14, and H15). HA protein forms a trimer, and
each monomer has two subunits (HA1 and HA2) linked by a
disulfide bond. The HA1 subunit functions in receptor binding
located in the head region of the protein. The HA2 subunit
forms, together with part of the HA1 subunit, the stem region
of the HA and is the mediator of the viral membrane fusion
with the endosomal membrane. The low endosomal pH
triggers this fusion event by inducing conformation changes in
HA, that expose its fusion peptide, and culminates with the
release of the viral genome into the cytoplasm. This step has
been the target of several HA inhibitors4−17 that prevent fusion
of HA proteins from either group 1 or group 2 but not both.
Here, we identified an aryl sulfonamide as an inhibitor of
influenza A virus entry that targets the HA protein, preventing
viral membrane fusion. Importantly, this compound prevents
replication of influenza A viruses that encode HA proteins
from both groups 1 and 2, including H1N1, H5N1, and H3N2,
at non-toxic concentrations. This HA inhibitor therefore
targets diverse influenza A viruses.
INTRODUCTION
■
Influenza viruses are responsible for up to 646,000 deaths
worldwide per year,1 and few therapeutic options are available
to counteract the infection. These viruses enter the host cell via
interactions of their hemagglutinin (HA) proteins present in
the virus envelope with sialic aids that modify glycoproteins in
the host plasma membrane.2 They are internalized via
endocytosis, and the eight viral ribonucleoproteins (vRNPs),
which constitute the segmented viral genome, are released into
the cytoplasm upon fusion of the viral and endosomal
membranes. The vRNPs are subsequently imported into the
nucleus through the nuclear pore complex and transcribed into
mRNAs, among which are two mRNAs that undergo
alternative splicing. The viral mRNAs are exported from the
nucleus and translated in the cytoplasm. vRNPs are also
templates for virus replication in the nucleus. vRNPs are
transcribed into complementary ribonucleoproteins (cRNPs),
which are then transcribed back into vRNPs. Newly generated
vRNPs are exported from the nucleus to the cytoplasm where
they, together with viral proteins, assemble viral particles that
bud from the host cell.2
Received: February 16, 2021
One key step for chemical inhibition of influenza virus
replication is entry into the host cell, which involves the HA
protein. The HA subtypes of influenza A virus3 are
phylogenetically divided into group 1 (H1, H2, H5, H6, H8,
H9, H11, H12, H13, H16, H17, and H18) and group 2 (H3,
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Figure 1. Compound 1 reduces viral mRNA levels. (A) Structure of compound 1. (B) smRNA-FISH followed by fluorescence microscopy was
performed with probes to detect M mRNA in A549 cells infected with WSN at multiplicity of infection (MOI) 2 for 8 h. (C) Cells were treated as
in B except that smRNA-FISH was performed with probes to detect HA mRNA. (D) Cells were treated as in B except that smRNA-FISH was
performed with probes to detect NS mRNA. (E) RNA-FISH and smRNA-FISH followed by fluorescence microscopy were performed in cells
treated with 0.1% DMSO or 1 μM compound 1 to detect poly(A) RNA and GAPDH mRNA, respectively, in uninfected cells. (F−I) A549 cells
were treated with 0.1% DMSO or 0.5 or 1 μM compound 1 for 1 h before infection with A/WSN/33 at MOI 2. Infection was performed for 8 h in
the absence and presence of compound. Purified RNA from total cell lysates was collected and subjected to qPCR to measure viral HA (F), NS1
(G), M1 (H), and M2 (I) mRNA levels. Three independent experiments were performed for all data sets. Graphs are mean SD. *p < 0.05, **p <
0.01, ***p < 0.001, ****p < 0.0001.
autophagy during infection.21 The NS mRNA encodes the
NS1 and NS2 proteins. NS1 protein is a multifunctional
RESULTS AND DISCUSSION
■
We have recently reported a chemical screen that identified
virulence factor that inhibits host gene expression and
inhibitors of influenza A virus mRNA processing and nuclear
immunity,22 whereas NS2 functions in nuclear export of viral
export.18 Here, we reveal a hit in the screen, a tertiary aryl
RNAs2 and in viral RNA replication.23 While this compound
sulfonamide (2,5-dichloro-N-ethyl-N-(o-tolyl)-
benzenesulfonamide) (Figure 1A), also termed here com-
pound 1. This aryl sulfonamide reduced influenza virus (A/
WSN/33) mRNA levels, as detected by single-molecule RNA
decreased viral mRNA levels (Figure 1B−D), it did not have a
significant effect on cellular bulk poly(A) RNA or GAPDH, as
shown by RNA-FISH and smRNA-FISH, respectively (Figure
1E). The effect of compound 1 on viral mRNA levels detected
in situ hybridization (smRNA-FISH) (Figure 1B−D). We
assessed the viral M, HA, and NS mRNAs. HA mRNA, as
mentioned above, encodes the HA protein involved in viral
entry into the host cell.2 M mRNA encodes the M1 and M2
proteins, which are involved in viral trafficking and
budding.2,19,20 M2 has also a role in the inhibition of
by imaging was further corroborated by quantitative real-time
PCR (qPCR) (Figure 1F−I), which again detected reduced
levels of viral mRNAs. The experiments mentioned above were
performed in A549 cells, which are lung epithelial cells derived
from adenocarcinoma. Thus, we have also tested the effect of
compound 1 in primary human bronchial epithelial cells
B
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Figure 2. Compound 1 decreases viral protein levels. (A) Dose-dependent inhibition of WSN-PB2-Nanoluc virus by compound 1. HCT116 cells
were incubated with compound 1 for 2 h and then infected with WSN-PB2-Nanoluc with a MOI of 0.02. After 24 h of infection, cells were
harvested in Renilla lysis buffer, and luminescence was measured using the Nano-Glo substrate. Luminescence was normalized to protein levels, and
IC50 was calculated using GraphPad Prism 7. (B) A549 cells were pre-treated with either 0.1% DMSO or 1 μM compound 1 before infection with
A/WSN/33 at MOI 2 for 8 h. Cell lysates were subjected to western blot analysis to detect the depicted viral proteins. β-Actin was used as a loading
control. This blot is a representative of three independent experiments.
Figure 3. Influenza virus entry is inhibited by compound 1. (A) A549 cells were incubated with 1 μM compound 1 for 1 h before infection or for 1
h post-infection. Infection was performed with WSN at MOI 2 for 8 h. Cells were then subjected to smRNA-FISH with probes to detect viral M
mRNA. (B) A549 cells were incubated with 1 μM compound 1 for 1 h before infection or for 1 h or 2 h post-infection. Infection was performed
with WSN at MOI 2 for 8 h. Cells were lysed and then subjected to western blot analysis to detect the depicted viral proteins.
infected with influenza virus. As shown in Figure S1,
compound 1 also robustly decreased viral mRNA levels in
primary cells. Taken together, these results suggest that this
compound could be interfering with viral entry or viral mRNA
transcription and/or processing. Consequently, less viral
proteins were expressed. In fact, we show that cells infected
with WSN-PB2-Nanoluc virus, in which Nano luciferase is
fused with the viral polymerase subunit PB2 protein and then
released by cleavage at a T2A site, show decreased levels of
Nanoluc in a dose-dependent manner with an IC50 = 16.79 nM
(Figure 2A). Additionally, western blot analysis of cells
infected with WSN also depicted reduced levels of influenza
virus proteins (Figure 2B).
Next, we tested whether viral entry is inhibited by
compound 1. Cells were incubated with compound for 1 h
before infection, or compound 1 was added 1 h post-infection.
After 8 h post-infection, cells were subjected to smRNA-FISH
to detect viral M mRNA (Figure 3A). We found that the viral
M mRNA was expressed at very low levels when compound 1
was added prior to infection, whereas viral M mRNA was
robustly expressed when cells were incubated with compound
1 after infection (Figure 3A). The lack of antiviral activity of
the compound when used shortly post-infection was confirmed
by western blot against the viral proteins NP and M1 (Figure
3B). These results indicate that compound 1 likely inhibits
viral entry. Then, we tested whether compound 1 would target
a viral protein. We therefore sought to determine whether
compound 1 would yield viral mutants that could resist
infection in the presence of the highest compound
concentration that still generates enough viruses for passaging
at the same MOI. After four passages at MOI 0.01, resistant
WSN viruses were identified by increases in viral titers in the
presence of compound 1 similar to dimethyl sulfoxide
(DMSO) controls and isolated in seven independent experi-
ments. Three virus plaques were purified from each
independent resistance experiment, and viral RNA was purified
and submitted for Illumina sequencing (Figure 4A). We found
that all sequenced viruses contained mutations in the HA gene,
indicating that HA is likely the target of compound 1 (Figure
4B). Interestingly, the identified mutations in the resistant
virus were spread across the HA stem region formed by part of
HA1 and by the ectodomain of HA2 and did not highlight a
single binding pocket (Figure 4C). This scattered resistance
mutation phenotype in the HA gene is actually well established
in our previous work24 and work from several other
groups8−11,14 to result from escape from the antiviral activity
of HA fusion inhibitors. In fact, in our previous work, we
identified the same exact M59I (HA2) and K321T (HA1)
mutations to cause resistance to the fusion inhibitor S20.24
These fusion inhibitors are shown to stabilize the prefusion
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Figure 4. Mutations within the HA protein cause resistance to the inhibitory effects of compound 1. (A) Schematic representation of the assays for
the identification of viral mutants resistant to compound 1 (0.5 μM) effect on viral replication. (B) Table depicting the viral RNA segment in which
the mutations where identified, showing that the HA RNA was the target of mutations at the positions listed. (C) Crystal structure of A/PR/8/34
HA (PDB 1RU7) protein showing the HA1 (red) and HA2 (orange) subunits. Residue positions where compound 1 escape mutations occurred
are indicated in teal (HA1) and green (HA2). (D) Suspension of chicken erythrocytes was incubated with WSN on ice in the absence of presence
of compound 1 at the indicated concentrations. The mixture was then acidified with a pH of 5. The suspension was incubated at 37 °C for 30 min
and assayed at 340 nM for NADH released upon lysis of the erythrocytes, as a measurement of fusion. Data are expressed as percentage relative to
the DMSO control, and means of triplicates SD are shown.
conformation of HA24 and prevent fusion from occurring, and
it is thought that these resistance mutations counteract the
inhibition through destabilization of the prefusion complex.
Mutations in similarly scattered, but not identical, positions
were also found to cause resistance to the well-established
fusion inhibitor arbidol,25 which also was shown to stabilize the
prefusion conformation of the HA protein. This indicates that
this resistance pattern is a paradigm of this class of fusion
inhibitors, and it can be considered as strong evidence of the
HA prefusion stabilizing mechanism. In support of this
hypothesis, a resistance mutation identified in this study
through selective pressure from compound 1, R47G (HA2),
occurs in the same position where a positively charged amino
acid has been associated with the prefusion stability of the HA
protein.26 Therefore, this set of resistance mutations induced
by compound 1 is suggestive of an HA fusion inhibitor that
may stabilize the prefusion conformation of influenza HA,
preventing the HA conformational change required for viral
entry. To test this possibility, we performed a hemolysis assay,
which measures the ability of influenza virus to induce
membrane fusion and lysis of red blood cells under low-pH
conditions. We found that compound 1 displayed a robust
inhibition of fusion induced at a pH of 5 (Figure 4D).
Together these results demonstrate that compound 1 targets
the HA protein and thereby prevents HA-mediated membrane
fusion.
We next determined the breadth of compound 1 inhibition
against multiple subtypes of influenza A virus. We tested an
inhibitory, but non-toxic, concentration of compound 1 for its
ability to reduce the viral titers from infections performed with
wild-type A/WSN/1933 (H1N1), A/Vietnam/2506/2004
(H5N1), or A/Panama/1999 (H3N2) in A549 cells. We
found that compound 1 was capable of reducing viral titers by
a minimum of four logs against all three subtypes of influenza
A virus (Figure 5), indicating that it has antiviral activity
against influenza viruses with either group 1 or group 2 HAs.
Additionally, we used smRNA-FISH to test the effect of
compound 1 and two of its active analogues (12 and 21) on
different H1N1 (A/California/07/2009) and H3N2 (A/
Wyoming/03/2003) strains from the ones mentioned above.
Cells were infected at MOI of 1. We found that these three
compounds robustly reduced the number of cells infected with
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activity (32) but at a 10-fold lower level than 21. The
positioning of the halides was also important as other
arrangements were either inactive (33, 35, 36, and 37) or
>10-fold less active than 1−55 (e.g., 34). Similarly, a brief
exploration of other halogen combinations did not identify
analogues with improved potency relative to dichloride 1−55
(see 38, 39, and 40).
Table 3 highlights our efforts to improve metabolic stability
and solubility of compound 1 analogues by replacing the N-
aryl ring with an N-heteroaryl substituent. Unfortunately,
analogues 41−48 proved inactive.
Finally, several analogues were synthesized to make
substantial changes to the scaffold or to provide reagents for
affinity purification (Figure 6). For example, 49 and 50
cyclized the N-alkyl and N-aryl ring in the context of an
indoline or tetrahydroquinoline, respectively. Analogue 51
replaced the sulfonyl linker with an amide. All of these were
inactive under the assay conditions. Likewise, a pair of alkyne-
diazarines (52 and 53) as potential photocross-linkers were not
active. Encouragingly, however, a simple analogue featuring an
aryl azide and an alkyne (54) retained robust activity and could
provide a tool for photocross-linking experiments in future
studies. In summary, the initial analogues demonstrate (a) a
steep structure−activity relationship with regard to the aryl
sulfonyl group, (b) the requirement for both N-alkyl and N-
aryl groups on the central nitrogen, (c) the incompatibility of
heteroaryl rings or highly polar groups as nitrogen substituents,
and (d) the beneficial effects of 1,4-disubstitution on the N-
aryl ring.
Figure 5. Compound 1 inhibits replication of diverse influenza
viruses. A549 cells were infected with influenza H1N1 A/WSN/1933
virus (MOI = 0.01, 24 h), H5N1 A/Vietnam/1203/2004 HaLo virus
(MOI = 0.01, 24 h), or H3N2 A/Panama/1999 (MOI = 0.1, 48 h) in
the absence or presence of 25 μM of compound 1, and viral titers
were determined by standard plaque assay. The limit of detection for
viral titers is indicated with a dotted line. Means SD (error bars) of
three independent experiments are indicated (**p < 0.01 and ***p <
0.001).
A/California/07/2009 (Figure S2A). In the case of A/
Wyoming/03/2003, while compounds 1 and 21 slightly
inhibited the number of infected cells, compound 12 more
effectively decreased infection (Figure S2B) at non-toxic
concentrations (Figure S2C). Taken together, compound 1
and its analogues can inhibit influenza viruses from group 1
and group 2 HAs with different potency.
Structure−activity relationship: To explore the structural
requirements for activity, we prepared a series of derivatives of
compound 1, starting with modifications to the aniline moiety
(Table 1). Removing or shrinking the N-ethyl group
substantially diminished activity (2, 3). Allyl and propargyl
groups maintained activity (4, 5), albeit at a decreased level,
and an N-isopropyl analogue (6) had greater than 10-fold loss
of activity compared to compound 1. On the aryl ring,
removing the 2-methyl group cost >10× (7), but moving it to
the para position was well tolerated (11). Chloride and nitrile
were less effective substituents than the methyl group (8, 9),
but fluorine was similarly potent (10). Disubstituted aryl rings
proved to be among the most active analogues. For example,
the 2-methyl-4-chlorophenyl analogues 12, 13, and 14 were
generally more potent than the corresponding mono-
substituted matched pairs (1 vs 12; 4 vs 13; 5 vs 14). In an
attempt to remove potential metabolic liabilities, we
synthesized the N−CH2-cPr derivative 15, but it was >10×
less active than the N-ethyl version (12). Similarly, we
attempted to introduce polarity on the N-alkyl group to
reduce log P and improve drug-like properties. However,
analogues 16, 17, 18, and 19 displayed lower activity. Among
dihalogenated analogues, the difluoro analogue 21 proved
equipotent to compound 1 while replacing or blocking
potential sites of metabolism. Trisubstituted derivatives (22
and 23) were less active. Finally, we attempted to introduce
polarity and functional handles at the para position of the
phenyl ring with mixed success. An ester maintained activity
(23), whereas the corresponding acid, alcohol, and amides
were inactive (24, 26−29).
CONCLUSIONS
■
We identified an inhibitor of influenza A virus replication that
targets the HA protein of both group 1 (H1 and H5) and
group 2 (H3). H1N1 and H3N2 strains co-circulate during the
same season and can cause severe disease. Moreover, sporadic
human infections with highly pathogenic avian H5 viruses
resulting in severe disease are still taking place since their
emergence in 1997. What separates compound 1 from those
fusion inhibitors identified previously in the literature is that
compound 1 shows potent antiviral activity against both group
1 and group 2 influenza viruses, while most other HA fusion
inhibitors have been group specific. Therefore, this compound
warrants further study as a lead for drug development of anti-
influenza therapy and may also be used in combination with
other antivirals for increasing pan-subtype efficacy. Future
mechanistic studies may broaden our understanding on the
activity of this compound toward its target and provide a
platform for additional therapeutic strategies.
EXPERIMENTAL SECTION
■
Cell Culture. Human lung adenocarcinoma epithelial cells (A549)
and MDCK cells, obtained from American Type Culture Collection
(ATCC), were maintained in high-glucose Dulbecco’s modified Eagle
medium (Gibco), 10% FBS (Sigma), and 100 units/mL Pen/Strep
antibiotics at 37 °C with 5% CO2. HCT116 cells were obtained from
ATCC and cultured in McCoy’s 5a medium modified with 10% FBS
and antibiotics as mentioned above. Primary human bronchial
epithelial cells were cultured, as previously described.27
Viruses. Influenza A viruses (A/WSN/33, A/Vietnam/1203/04,
A/Panama/99, A/California/07/2009, and A/Wyoming/03/2003)
were generated in embryonated eggs or in MDCK cells after growth
from a clonal population of virus at low MOI to avoid accumulation
of defective virus particles. A/Vietnam/1203/2004 (H5N1) has a
mutated polybasic cleavage site in the HA segment (HAlo).28 In
Building off of compound 21, we explored modification of
the aryl sulfonamide (Table 2). The two chlorides were
important as the phenyl sulfonamide (30) and 2-Cl (31)
analogues were inactive. The 3-Cl derivative maintained
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a
Table 1. Aniline Modifications of Compound 1
a
A549 cells were infected with A/WSN/33 at MOI 0.01. After 24 h post-infection, cells were subjected to immunofluorescence microscopy using
antibody against the influenza virus NP protein. Percent infection was quantified by dividing the number of NP-positive cells by the total number of
cells. Cytotoxicity was also performed using MTT assay concurrent with immunostaining.
MDCK cells, virus was amplified at MOI 0.1−0.001 in infection
media containing EMEM (ATCC, 30-2003), 10 mM HEPES
(Gibco), 0.125% bovine serum albumin (BSA) (Gibco), and 0.5
μg/mL TPCK trypsin (Worthington Biomedical Corporation). Cells
were incubated with virus for 1 h at 37 °C and then washed before
amplification in infection media. After cell death was observed at 48−
72 h post-infection, supernatants were centrifuged at 1000g for 10 min
to remove cell debris, aliquoted, and stored at −80 °C. All virus stocks
are controlled for an appropriate ratio of HA/PFU titer and
sequenced by RNAseq to confirm the full sequence of the virus.
WSN PB2-WSN-Nanoluc encodes Nano luciferase fused with PB2.29
Viral Replication and Cytotoxicity Assays. A549 cells were
infected with each viral strain at MOI 0.01. After 24 h post-infection
with A/WSN/33 and A/Vietnam/1203/04, or 48 h with A/Panama/
99, cells were fixed with 4% formaldehyde for 30 min. Cells were
briefly washed with phosphate buffered saline (PBS) and then
permeabilized with 0.1% Triton X-100 in PBS for 15 min. Blocking
occurred at room temperature for 1 h with 0.5% BSA in PBS followed
by incubation with the NP antibody (HT103, a gift from Thomas
Moran) in 0.5% BSA in PBS for 1 h at room temperature. Cells were
washed with PBS 2× and incubated with a fluorescently labeled
secondary antibody, Alexa-fluor-488 (Invitrogen), in 0.5% BSA in PBS
with DAPI for 45 min at room temperature. Two washes with PBS
F
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a
a
Table 2. Sulfonyl Modifications
Table 3. Heterocyclic Aniline Replacements
a
A549 cells were infected with A/WSN/33 at MOI 0.01. After 24 h
post-infection, cells were subjected to immunofluorescence micros-
copy using antibody against the influenza virus NP protein. Percent
infection was quantified by dividing the number of NP-positive cells
by the total number of cells. Cytotoxicity was also performed using
MTT assay concurrent with immunostaining.
a
A549 cells were infected with A/WSN/33 at MOI 0.01. After 24 h
post-infection, cells were subjected to immunofluorescence micros-
copy using antibody against the influenza virus NP protein. Percent
infection was quantified by dividing the number of NP-positive cells
by the total number of cells. Cytotoxicity was also performed using
MTT assay concurrent with immunostaining.
were performed before imaging the cells on a Celigo Image cytometer.
Percent infection was quantified by dividing the number of NP-
positive cells by the total number of cells. Cytotoxicity was also
performed using MTT assay (Roche), according to the manufac-
turer’s instructions, concurrent with immunostaining. IC50, as shown
in Figures 2A and 6, was estimated using WSN-PB2-Nanoluc virus.
Cells were incubated with compounds for 2 h and then infected with
WSN-PB2-Nanoluc with a MOI of 0.02. After 24 h of infection, cells
were harvested in Renilla lysis buffer (Promega), and luminescence
was measured using the Nano-Glow substrate (Promega) in a
microplate reader (BMG Labtech). The lysate protein concentration
was measured using the Bradford reagent (Bio-Rad). Luminescence
readings were normalized to protein levels wherever mentioned.
Using GraphPad Prism 7, (inhibitor) versus response-variable slope
(four parameters) equation was selected for nonlinear regression
analysis to determine the IC50. As shown in Figure 6, CC50 was
calculated using CellTiter-Glo (Promega), according to the
manufacturer’s instructions. As shown in Figure S1, CellTiter-Glo
(Promega) was used to assess cell viability by measuring cellular ATP
levels.
Figure 6. Modified scaffolds and probes for target identification. A549
cells were treated with various concentrations of compound 1 and its
analogues before infection. After 2 h of compound treatment, cells
were infected with WSN-PB2-Nanoluc virus at MOI 0.02 for 24 h and
then harvested in Renilla lysis buffer. Luminescence was measured for
equal volume of lysates using the Nano-Glo substrate, and IC50 was
determined. CC50 was determined using CellTiter-Glo.
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smRNA-FISH. smRNA-FISH was performed, as previously
described,30 which includes the sequences of M1 and NS1 probes
except for the HA probes that are listed below. Briefly, cells were
grown on glass coverslips (Fisherbrand, Fisher Scientific) coated with
1 mL of 0.1% gelatin (Sigma-Aldrich). Cells were fixed with 4%
paraformaldehyde (PFA, Electron Microscopy Sciences) in PBS for
15 min before incubation in 70% ethanol for 12 h at 4 °C. Coverslips
were then placed in wash buffer for 5 min, containing nuclease free
water, 2× SSC Buffer (Sigma), and 10% formamide (Sigma). The
coverslips were then removed and incubated in hybridization buffer
containing the FISH probe. Hybridization occurred at 37 °C for 4 h,
then cells were washed with wash buffer for 30 min at 37 °C.
Coverslips were then washed twice for 5 min in PBS and stained with
1 μg/mL Hoechst 33258 (Molecular Probes/Life Technologies) for
10 min. Coverslips were briefly washed with PBS before mounting in
the ProLong Gold antifade reagent (Life Technologies).
saturated ammonium chloride solution three times and then washed
with 5 mL of distilled water. The DCM layer was dried over sodium
sulfate, filtered, and concentrated. The residue was purified by
filtration through a small plug of silica gel, eluting with DCM.
Step 2. The required alkyl bromide (0.30 mmol, 3.0 equiv) was
added to a mixture of N-aryl sulfonamide from step 1 (0.10 mmol, 1.0
equiv) and potassium carbonate (16.6 mg, 0.12 mmol, 1.2 equiv) in 1
mL of acetone. The mixture was sealed in a 20 mL scintillation vial
and stirred at 60 °C overnight. Acetone and excess bromide were
removed under vacuum. The crude residue was resuspended in 10 mL
of DCM and washed with 2 mL of saturated ammonium chloride
solution three times and then washed with 2 mL of distilled water.
The DCM layer was dried over sodium sulfate, filtered, and
concentrated. The resulting residue was either loaded to a silica gel
plug and eluted by DCM only or further purified by silica gel
chromatography using DCM and hexane to afford the desired
product.
RNA Purification and RT-qPCR. Total RNA was isolated from
A549 cells using the RNeasy Plus Mini kit (Qiagen) and reverse
transcribed into cDNA by SuperScript II reverse transcriptase
(Invitrogen), each according to the manufacturers’ protocols. Samples
were then amplified in a LightCycler 480 qPCR system (Roche) using
SYBR Green I (Roche) and sequence-specific primers.
2,5-Dichloro-N-(o-tolyl)benzenesulfonamide (2). 2,5-Dichloro-N-
(o-tolyl)benzenesulfonamide (2) was prepared according to the
general procedure using o-toluidine (0.43 mL, 4.0 mmol). Purification
by automated flash chromatography in 100% DCM gave 1.19 g of the
1
title compound in 93% yield. H NMR (400 MHz, chloroform-d): δ
8.00 (dd, J = 1.8, 1.0 Hz, 1H), 7.50−7.44 (m, 2H), 7.19−7.13 (m,
1H), 7.09 (td, J = 5.7, 4.0 Hz, 3H), 6.79 (s, 1H), 2.30 (s, 3H). ESI-
MS (m/z): 316.0 [M + H]+.
RT-PCR primer sequences:
M1 Forward-ATCAGACATGAGAACAGAATGG.
Reverse-TGCCTGGCCTGACTAGCAATATC.
2,5-Dichloro-N-methyl-N-(o-tolyl)benzenesulfonamide (3). 2,5-
Dichloro-N-methyl-N-(o-tolyl)benzenesulfonamide (3) was prepared
according to the general procedure from 2 (77.3 mg, 0.24 mmol) to
give 77.1 mg of the title compound in 96% yield. 1H NMR (400 MHz,
chloroform-d): δ 7.82 (d, J = 2.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H),
7.44 (dd, J = 8.5, 2.5 Hz, 1H), 7.26 (dd, J = 7.7, 1.9 Hz, 1H), 7.22 (td,
J = 7.4, 1.3 Hz, 1H), 7.06 (td, J = 7.5, 1.9 Hz, 1H), 6.81 (dd, J = 7.9,
1.3 Hz, 1H), 3.40 (s, 3H), 2.33 (s, 3H). 13C NMR (101 MHz,
chloroform-d): δ 139.2, 138.8, 138.7, 133.5, 133.2, 133.1, 132.3,
131.8, 130.4, 129.0, 128.8, 127.0, 40.4, 18.1. ESI-MS (m/z): 330.0 [M
+ H]+.
M2 Forward: CGAGGTCGAAACGCCTATCAGAAAC.
Reverse: CCAATGATATTTGCTGCAATGACGAG.
18S Forward: GTAACCCGTTGAACCCCATT.
Reverse: CCATCCAATCGGTAGTAGCG.
Selection of Compound 1-Resistant Influenza Viruses. The
concentration of compound 1 required for maximum virus inhibition
(three logs), while maintaining enough virus production for
subsequent passages, was determined (0.5 μM compound 1). A549
cells were infected with WSN at an MOI of 0.01 for 24 h at 37 °C
under compound 1 treatment. The supernatant was then collected
and titered by plaque assay. If the recovered compound 1-treated
virus did not show increased viral titer similar to that of the DMSO-
treated control, the virus was passaged again by using the same
method. Once increased titers in the presence of compound 1 were
detected for two consecutive passages, the viruses were plaque
purified. Following plaque purification, all eight genome segments
were sequenced using the libraries prepared with the NEBNext Ultra
II RNA Library Prep Kit for Illumina (New England BioLabs) and
sequenced on a NextSeq 550 system (Illumina). Sequencing from the
DMSO-passaged control virus was used to generate the FASTA file to
which all compound 1-treated samples were compared using the
Integrative Genomics Viewer (Version 2.5.0) to detect escape
mutations.
N-Allyl-2,5-dichloro-N-(o-tolyl)benzenesulfonamide (4). N-Allyl-
2,5-dichloro-N-(o-tolyl)benzenesulfonamide (4) was prepared ac-
cording to the general procedure from 2 (74.7 mg, 0.24 mmol) to give
1
81.5 mg of the title compound in 97% yield. H NMR (400 MHz,
chloroform-d): δ 7.83 (d, J = 2.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H),
7.47 (dd, J = 8.5, 2.5 Hz, 1H), 7.28−7.22 (m, 2H), 7.11 (ddd, J = 8.7,
6.1, 2.9 Hz, 1H), 6.92−6.87 (m, 1H), 5.90 (ddt, J = 17.0, 10.0, 6.9 Hz,
1H), 5.15−5.01 (m, 2H), 4.44 (d, J = 6.5 Hz, 2H), 2.29 (s, 3H). 13C
NMR (101 MHz, chloroform-d): δ 139.4, 139.30, 136.4, 133.5, 133.1,
133.1, 132.8, 132.3, 131.6, 130.7, 130.3, 128.9, 126.5, 119.9, 56.0,
18.3. ESI-MS (m/z): 356.0 [M + H]+.
2,5-Dichloro-N-(prop-2-yn-1-yl)-N-(o-tolyl)benzenesulfonamide
(5). 2,5-Dichloro-N-(prop-2-yn-1-yl)-N-(o-tolyl)benzenesulfonamide
(5) was prepared according to the general procedure from 2 (75
mg, 0.24 mmol) to give 80.5 mg of the title compound in 94% yield.
1H NMR (400 MHz, chloroform-d): δ 7.85 (d, J = 2.4 Hz, 1H), 7.50
Hemolysis Inhibition Assay. Fresh chicken erythrocytes (RBCs)
were used in hemolysis inhibition assay, as previously described.24
Compounds. Compound 1 was initially purchased from ChemDiv
(Y030-0868) and synthesized in-house.
(d, J = 8.5 Hz, 1H), 7.46 (dd, J = 8.5, 2.4 Hz, 1H), 7.30−7.25 (m,
2H), 7.12 (dt, J = 8.8, 4.3 Hz, 1H), 7.05−7.00 (m, 1H), 4.77 (s, 1H),
4.51 (s, 1H), 2.31 (s, 3H), 2.26 (t, J = 2.5 Hz, 1H). 13C NMR (101
MHz, chloroform-d): δ 139.38, 139.01, 136.2, 133.8, 133.2, 133.1,
132.3, 131.7, 130.7, 130.5, 129.5, 126.7, 78.1, 74.1, 42.5, 18.1. ESI-MS
(m/z): 354.0 [M + H]+.
2,5-Dichloro-N-isopropyl-N-(o-tolyl)benzenesulfonamide (6).
2,5-Dichloro-N-isopropyl-N-(o-tolyl)benzenesulfonamide (6) was
prepared according to the general procedure from 2 (75 mg, 0.24
mmol). After overnight stirring, the conversion to the desired product
was slow when monitored by LC−MS. The reaction was condensed,
and acetonitrile (2.4 mL) and K2CO3 (42.2 mg, 0.31 mmol) were
added, and the reaction was heated to 80 °C overnight and went to
completion. The crude reaction mixture was condensed to dryness
and purified by automated flash chromatography in 100% DCM to
give 73.8 mg of the title compound in 87% yield. 1H NMR (400 MHz,
chloroform-d): δ 7.75 (d, J = 2.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H),
7.39 (dd, J = 8.5, 2.5 Hz, 1H), 7.27−7.20 (m, 2H), 7.13−7.06 (m,
Chemical Methods. General. All tested compounds have purity
of >95%, as judged by HPLC analysis (UV detection at 210 nM).
Chemical shifts δ are measured in parts per million, and spectra were
referenced using the residual solvent peak. The following abbrevia-
tions are used: singlet (s), doublet (d), triplet (t), quartet (q), double
doublet (dd), quintet (quin), multiplet (m), and broad signal (bs).
Mass spectra (m/z) were recorded on an Agilent LC−MS 1290
Infinity using ESI ionization. All chemicals were used as received
unless otherwise noted. Compounds 1 and 10 were purchased from
ChemDiv.
General Procedure for the Synthesis of N-Alkyl-N-aryl-sulfona-
mides. Step 1. Pyridine (87.0 mg, 3.3 mmol, 1.1 equiv) was added to
a mixture of aryl sulfonyl chloride (3.0 mmol, 1.0 equiv) and
substituted aniline (3.0 mmol, 1.0 equiv) in 9 mL of anhydrous
dichloromethane (DCM). The mixture was sealed in a 20 mL
scintillation vial and stirred at room temperature overnight. DCM and
excess pyridine were removed under vacuum. The crude white
powder was resuspended in 10 mL of DCM and washed with 5 mL of
H
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1H), 6.95−6.90 (m, 1H), 4.84 (p, J = 6.7 Hz, 1H), 2.15 (s, 3H), 1.12
(dd, J = 14.1, 6.7 Hz, 6H). 13C NMR (101 MHz, chloroform-d): δ
141.0, 140.1, 133.34, 133.31, 133.13, 133.07, 132.96, 132.1, 131.7,
130.3, 129.1, 126.0, 53.5, 22.8, 21.7, 18.9. ESI-MS (m/z): 358.0 [M +
H]+.
2,5-Dichloro-N-ethyl-N-phenylbenzenesulfonamide (7). Step 1.
2,5-Dichloro-N-phenylbenzenesulfonamide was prepared according to
the general procedure using aniline (0.37 mL, 4.1 mmol). The title
compound was isolated from flash chromatography in 100% DCM to
give 1.17 g in 94% yield. 1H NMR (400 MHz, chloroform-d): δ 7.98
(dd, J = 1.9, 1.0 Hz, 1H), 7.43 (d, J = 1.8 Hz, 2H), 7.26 (dd, J = 8.8,
6.9 Hz, 2H), 7.18−7.10 (m, 3H), 7.04 (s, 1H). ESI-MS (m/z): 300.0
[M−H]−.
Step 2. Compound 7 was prepared according to the general
procedure using the sulfonamide from step 1 (76.1 mg, 0.25 mmol) to
give 81.1 mg of the title compound in 98% yield. 1H NMR (400 MHz,
chloroform-d): δ 7.78 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H),
7.36 (dd, J = 8.5, 2.4 Hz, 1H), 7.31−7.22 (m, 3H), 7.19−7.13 (m,
2H), 3.88 (q, J = 7.1 Hz, 2H), 1.13 (dd, J = 7.5, 6.7 Hz, 3H). 13C
NMR (101 MHz, chloroform-d): δ 138.6, 137.7, 133.53, 133.01,
132.95, 132.3, 130.4, 129.5, 129.4, 128.4, 47.7, 14.8. ESI-MS (m/z):
330.0 [M + H]+.
2,5-Dichloro-N-(2-chlorophenyl)-N-ethylbenzenesulfonamide
(8). 2,5-Dichloro-N-(2-chlorophenyl)-N-ethylbenzenesulfonamide
(8) was prepared according to the general procedure to give 78.2
mg of the title compound in 95% yield. 1H NMR (400 MHz,
chloroform-d): δ 7.71−7.66 (m, 1H), 7.36−7.22 (m, 4H), 7.21−7.14
(m, 2H), 3.85 (d, J = 171.6 Hz, 2H), 1.05 (td, J = 7.1, 1.2 Hz, 3H).
13C NMR (101 MHz, chloroform-d): δ 139.7, 134.9, 134.54, 134.46,
2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-ethylbenzenesulfo-
namide (12). 2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-ethylben-
zenesulfonamide (12) was prepared according to the general
procedure as an oil, 78.1 mg; 92% yield. 1H NMR (400 MHz,
chloroform-d): δ 7.78 (d, J = 2.3 Hz, 1H), 7.49−7.40 (m, 2H), 7.23
(d, J = 2.5 Hz, 1H), 7.07 (dd, J = 8.5, 2.5 Hz, 1H), 6.82 (d, J = 8.5 Hz,
1H), 3.82 (dp, J = 20.7, 6.9 Hz, 2H), 2.23 (s, 3H), 1.10 (t, J = 7.1 Hz,
3H). 13C NMR (101 MHz, chloroform-d): δ 141.5, 139.0, 134.9,
134.5, 133.6, 133.3, 133.2, 132.2, 131.7, 131.5, 130.3, 126.9, 47.9,
18.2, 14.3. ESI-MS (m/z): 378.0 [M + H]+.
N-Allyl-2,5-dichloro-N-(4-chloro-2-methylphenyl)-
benzenesulfonamide (13). N-Allyl-2,5-dichloro-N-(4-chloro-2-
methylphenyl)benzenesulfonamide (13) was prepared according to
1
the general procedure as an oil, 79.3 mg; 93% yield. H NMR (400
MHz, chloroform-d): δ 7.79 (d, J = 2.3 Hz, 1H), 7.50−7.42 (m, 2H),
7.21 (d, J = 2.5 Hz, 1H), 7.04 (dd, J = 8.5, 2.5 Hz, 1H), 6.80 (d, J =
8.5 Hz, 1H), 5.82 (ddt, J = 17.0, 10.0, 6.9 Hz, 1H), 5.09−4.95 (m,
2H), 4.35 (d, J = 6.7 Hz, 2H), 2.21 (s, 3H). 13C NMR (101 MHz,
chloroform-d): δ 141.3, 139.1, 135.0, 134.6, 133.8, 133.3, 133.2,
132.4, 132.3, 132.0, 131.5, 130.3, 126.8, 120.4, 56.0, 18.9. ESI-MS
(m/z): 390.0 [M + H]+.
2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-(prop-2-yn-1-yl)-
benzenesulfonamide (14). 2,5-Dichloro-N-(4-chloro-2-methylphen-
yl)-N-(prop-2-yn-1-yl)benzenesulfonamide (14) was prepared ac-
1
cording to the general procedure as an oil, 51.2 mg in 61% yield. H
NMR (400 MHz, chloroform-d): δ 7.83 (d, J = 2.1 Hz, 1H), 7.52−
7.43 (m, 2H), 7.25 (d, J = 2.5 Hz, 1H), 7.08 (dd, J = 8.5, 2.5 Hz, 1H),
6.96 (d, J = 8.5 Hz, 1H), 4.85−4.33 (m, 2H), 2.25 (d, J = 2.7 Hz,
4H). 13C NMR (101 MHz, chloroform-d): δ 141.4, 138.8, 135.3,
134.8, 134.0, 133.3, 133.2, 132.3, 132.0, 131.6, 130.5, 127.0, 77.8,
74.4, 42.5, 18.1. ESI-MS (m/z): 388.0 [M + H]+.
133.45, 133.1, 132.8, 131.7, 130.8, 130.8, 130.2, 127.5, 47.0, 14.6. ESI-
MS (m/z): 364.0 [M + H]+.
2 , 5 - D i c h l o r o - N - ( 4 - c h l o r o - 2 - m e t h y l p h e n y l ) - N -
(cyclopropylmethyl)benzenesulfonamide (15). 2,5-Dichloro-N-(4-
chloro-2-methylphenyl)-N-(cyclopropylmethyl)benzenesulfonamide
(15) was prepared according to the general procedure to afford 38.8
2,5-Dichloro-N-(2-cyanophenyl)-N-ethylbenzenesulfonamide
(9). Step 1. 2,5-Dichlorobenzenesulfonyl chloride (1.0 g, 4.1 mmol)
was slowly added to a solution of 2-aminobenzonitrile (0.484 g, 4.1
mmol) in pyridine (14 mL) with a catalytic amount of
dimethylaminopyridine. The reaction was heated to 60 °C overnight.
The cooled reaction mixture was diluted with Et2O and washed
several times with water and then brine. The organic layer was dried
over Na2SO4, filtered, and condensed. The crude mixture was purified
by automated flash chromatography in 100% DCM to give 345.8 mg
of 2,5-dichloro-N-(2-cyanophenyl)benzenesulfonamide in 47% yield.
1H NMR (400 MHz, chloroform-d): δ 8.10−8.05 (m, 1H), 7.62 (dt, J
1
mg of colorless oil as the product, yield 95%. H NMR (CDCl3, 400
MHz): δ ppm 7.78 (d, J = 2.3 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.42
(ddd, J = 8.5, 2.4, 1.0 Hz, 1H), 7.23 (d, J = 2.5 Hz, 1H), 7.05 (dd, J =
8.5, 2.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 3.69 (dd, J = 14.0, 7.5 Hz,
1H), 3.57 (dd, J = 14.1, 7.0 Hz, 1H), 2.27 (s, 3H), 0.91 (ddd, J =
17.2, 7.9, 3.9 Hz, 1H), 0.44 (q, J = 7.3, 6.7 Hz, 2H), 0.10 (ddd, J =
39.0, 9.3, 4.4 Hz, 2H). 13C NMR (CDCl3, 101 MHz): δ ppm 141.5,
139.0, 135.5, 134.4, 133.5, 133.1, 133.0, 132.2, 131.7, 131.3, 130.1,
126.7, 58.2, 18.1, 10.4, 4.2, 4.0. ESI-MS m/z = 404.0 [M + H]+.
2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-(2-methoxyethyl)-
benzenesulfonamide (16). 2,5-Dichloro-N-(4-chloro-2-methylphen-
yl)-N-(2-methoxyethyl)benzenesulfonamide (16) was prepared ac-
cording to the general procedure to afford 34.0 mg of colorless oil as
the product, yield 82%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.82 (t,
J = 1.8 Hz, 1H), 7.49−7.39 (m, 2H), 7.22 (t, J = 1.7 Hz, 1H), 7.06
(dd, J = 8.7, 2.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 3.94 (tq, J = 20.2,
8.4, 6.9 Hz, 2H), 3.40 (td, J = 5.7, 1.4 Hz, 2H), 3.27 (d, J = 1.4 Hz,
3H), 2.24 (s, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 141.4,
140.5, 138.8, 135.2, 134.5, 133.5, 133.1, 132.2, 131.6, 131.4, 130.2,
126.8, 69.8, 58.6, 52.0, 17.9. ESI-MS m/z = 408.0 [M + H]+.
2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-(2-hydroxyethyl)-
benzenesulfonamide (17). 2,5-Dichloro-N-(4-chloro-2-methylphen-
yl)-N-(2-hydroxyethyl)benzenesulfonamide (17) was prepared ac-
cording to the general procedure to afford 34.0 mg of colorless oil as
the product, yield 59%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.81 (d,
J = 2.3 Hz, 1H), 7.51−7.41 (m, 2H), 7.22 (d, J = 2.6 Hz, 1H), 7.10
(dd, J = 8.5, 2.5 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 4.52 (s, 1H), 3.92
(dt, J = 8.2, 5.6 Hz, 2H), 3.67 (q, J = 5.5 Hz, 2H), 2.21 (s, 3H). 13C
NMR (CDCl3, 101 MHz): δ ppm 140.9, 138.7, 135.0, 134.7, 133.7,
133.2, 133.1, 132.2, 132.0, 131.5, 130.1, 127.0, 60.1, 54.9, 17.9. ESI-
MS m/z = 808.9 [2M + Na]+.
= 8.1, 1.1 Hz, 1H), 7.59−7.44 (m, 5H), 7.19 (td, J = 7.6, 1.1 Hz, 1H).
ESI-MS (m/z): 325.0 [M−H]−.
Step 2. Compound 9 was prepared following the general procedure
using 2,5-dichloro-N-(2-cyanophenyl)benzenesulfonamide (75.0 mg,
1
0.23 mmol) to give 48.0 mg of the title compound in 59% yield. H
NMR (400 MHz, chloroform-d): δ 7.82 (d, J = 2.3 Hz, 1H), 7.68−
7.60 (m, 2H), 7.51−7.42 (m, 4H), 3.98 (q, J = 7.1 Hz, 2H), 1.23−
1.14 (m, 3H). 13C NMR (101 MHz, chloroform-d): δ 140.3, 138.5,
134.2, 134.0, 133.71, 133.66, 133.2, 133.13, 133.09, 131.7, 131.0,
129.3, 115.9, 114.5, 48.0, 14.6. ESI-MS (m/z): 355.0 [M + H]+.
2,5-Dichloro-N-ethyl-N-(p-tolyl)benzenesulfonamide (11). Step
1. 2,5-Dichloro-N-(p-tolyl)benzenesulfonamide was prepared accord-
ing to the general procedure using p-toluidine (111.9 mg, 1.0 mmol).
The crude product was further purified by trituration in DCM and
1
hexane to afford 286 mg of white solid as the product, yield 90%. H
NMR (CDCl3, 400 MHz): δ ppm 7.93 (d, J = 2.1 Hz, 1H), 7.46−7.37
(m, 2H), 7.06−6.93 (m, 5H), 2.25 (s, 3H). 13C NMR (CDCl3, 101
MHz): δ ppm 137.6, 136.4, 133.9, 133.5, 132.5, 132.4, 131.7, 130.1,
129.4, 122.6, 20.9. ESI-MS m/z = 314.0 [M−H]+.
Step 2. Compound 11 was prepared from the sulfonamide from
step 1 (32.0 mg, 0.10 mmol) following the general procedure to
1
provide 33.7 mg of yellow oil as the product, yield 97%. H NMR
(CDCl3, 400 MHz): δ ppm 7.81 (d, J = 2.5 Hz, 1H), 7.46−7.34 (m,
2H), 7.07 (q, J = 8.4 Hz, 4H), 3.87 (q, J = 7.1 Hz, 2H), 2.31 (s, 3H),
1.14 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 138.7,
138.4, 134.8, 133.4, 132.9, 132.8, 132.2, 130.3, 130.0, 129.3, 47.7,
21.1, 14.7. ESI-MS m/z = 344.0 [M + H]+.
2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-(2-fluoroethyl)-
benzenesulfonamide (18). To a stirring solution of 17 in 1 mL of
DCM cooled to 0 °C under nitrogen, Morph-Dast (48.8 μL, 0.40
mmol, 4.0 equiv) was added dropwise. The reaction mixture was
I
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warmed to room temperature and allowed to stir overnight until the
starting material was consumed. Upon completion of the reaction,
Morph-Dast was quenched slowly by saturated sodium bicarbonate
solution. The reaction mixture was diluted with water and extracted
with DCM. The combined organics were washed with brine, dried
over sodium sulfate, and then concentrated. The crude product was
purified by silica gel chromatography using 50% of DCM in hexane to
Hz), 133.6, 133.1, 132.9, 131.8, 130.6, 121.1 (d, J = 16.2 Hz), 111.9
(dd, J = 22.3, 3.7 Hz), 105.0 (dd, J = 26.4, 24.0 Hz), 46.9 (d, J = 2.4
Hz), 14.4. ESI-MS m/z = 366.0 [M + H]+.
2,5-Dichloro-N-(2,4-dichloro-6-methylphenyl)-N-ethylbenzene-
sulfonamide (22). Step 1. 2,5-Dichloro-N-(2,4-dichloro-6-
methylphenyl)benzenesulfonamide was prepared according to the
general procedure using 2,4-dichloro-6-methylaniline (177.1 mg, 1.0
mmol, 1.0 equiv) as the aniline. 4-(Dimethylamino)pyridine was
added in the catalytic amount (3.40 mg, 0.024 mmol, 0.024 equiv),
and the reaction mixture was heated to 60 °C for 9 days. The crude
was further purified by silica gel chromatography using 100% of DCM
in hexane to afford 246 mg of white solid as the product, yield 65%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.87 (t, J = 1.4 Hz, 1H), 7.48
1
afford 19.5 mg of colorless oil as the product, yield 49%. H NMR
(CDCl3, 400 MHz): δ ppm 7.78 (d, J = 2.2 Hz, 1H), 7.51−7.41 (m,
2H), 7.24 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.5, 2.4 Hz, 1H), 6.88 (d,
J = 8.5 Hz, 1H), 4.58−4.38 (m, 2H), 4.18−3.99 (m, 2H), 2.25 (s,
3H). 13C NMR (CDCl3, 101 MHz): δ ppm 141.3, 138.5, 134.94,
134.86, 133.8, 133.2, 133.1, 132.3, 131.61, 131.60, 130.2, 127.0, 80.97
(d, J = 173.1 Hz), 52.83 (d, J = 21.4 Hz), 17.91 (d, J = 1.7 Hz). ESI-
MS m/z = 430.0 [M + Cl]−.
(d, J = 1.4 Hz, 2H), 7.18 (ddd, J = 8.8, 2.5, 0.8 Hz, 2H), 6.73 (s, br,
1H), 2.44 (s, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 142.9,
140.1, 134.3, 133.9, 133.8, 133.3, 132.6, 130.4, 130.2, 130.0, 129.7,
127.2, 19.8. ESI-MS m/z = 382.0 [M−H]+.
2,5-Dichloro-N-(4-chloro-2-methylphenyl)-N-(difluoromethyl)-
benzenesulfonamide (19). A well-stirred slurry of 2,5-dichloro-N-(4-
chloro-2-methylphenyl)benzenesulfonamide (40.9 mg, 0.12 mmol,
1.0 equiv), potassium carbonate (51.3 mg, 0.35 mmol, 2.9 equiv), and
sodium chlorodifluoroacetate (53.5 mg, 0.35 mmol, 2.9 equiv) in 2
mL of acetonitrile was heated to 60 °C for 72 h. The resulting slurry
was then concentrated in vacuo, diluted with 2 mL of DCM and 2 mL
of water. The organic phase was separated, dried, and concentrated in
vacuo. The crude mixture was further purified by silica gel
chromatography using DCM to afford 41.6 mg of white solid as the
product, yield 89%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.75 (t, J =
1.4 Hz, 1H), 7.58−7.39 (m, 3H), 7.30 (d, J = 2.5 Hz, 1H), 7.06 (dd, J
= 8.4, 2.5 Hz, 1H), 6.69 (dd, J = 8.5, 1.6 Hz, 1H), 2.23 (s, 3H). 13C
NMR (CDCl3, 101 MHz): δ ppm 143.1, 136.6, 136.6, 134.9, 133.5,
133.5, 133.4, 132.5, 131.8, 130.4, 127.3, 126.7, 111.3 (dd, J = 252.1,
246.9 Hz), 18.1 (d, J = 3.3 Hz). ESI-MS: compound did not ionize.
2,5-Dichloro-N-(4-chloro-2-fluorophenyl)-N-ethylbenzenesulfo-
namide (20). Step 1. 2,5-Dichloro-N-(4-chloro-2-fluorophenyl)-
benzenesulfonamide was prepared according to the general procedure
using 4-chloro-2-fluoroaniline (33.6 mg, 0.20 mmol, 1.0 equiv), yield
97%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.97 (d, J = 2.5 Hz, 1H),
7.51−7.41 (m, 3H), 7.22 (s, 1H), 7.07 (dt, J = 8.6, 1.8 Hz, 1H), 7.03
(dd, J = 9.9, 2.2 Hz, 1H). 13C NMR (CDCl3, 101 MHz): δ ppm 153.8
(d, J = 249.3 Hz), 137.3, 134.4, 133.4, 132.9, 131.6 (d, J = 9.6 Hz),
131.4, 130.0, 125.3 (d, J = 3.8 Hz), 124.1, 122.3 (d, J = 12.5 Hz),
116.6 (d, J = 22.8 Hz). ESI-MS m/z = 352.0 [M−H]+.
Step 2. The title compound was prepared according to the general
procedure to afford 39.1 mg of colorless oil as the product with
1
quantitative yield. H NMR (CDCl3, 400 MHz): δ ppm 7.87 (d, J =
1.9 Hz, 1H), 7.43 (s, 1H), 7.21 (d, J = 4.9 Hz, 1H), 4.14 (dq, J = 14.4,
7.3 Hz, 1H), 3.73 (dq, J = 14.1, 7.1 Hz, 1H), 2.43 (s, 3H), 1.14 (t, J =
7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 144.5, 140.2,
135.9, 134.7, 133.4, 133.1, 133.0, 132.8, 131.5, 130.6, 130.1, 128.0,
46.6, 20.0, 14.3. ESI-MS m/z = 434.2 [M + Na]+.
2,5-Dichloro-N-(4-chloro-2,6-difluorophenyl)-N-ethylbenzene-
sulfonamide (23). Step 1. 2,5-Dichloro-N-(4-chloro-2,6-
difluorophenyl)benzenesulfonamide was prepared according to the
general procedure using 4-chloro-2,6-difluoroaniline (249 mg, 1.0
mmol, 1.0 equiv) as the aniline. 4-(Dimethylamino)pyridine was
added in the catalytic amount (3.40 mg, 0.024 mmol, 0.024 equiv),
and the reaction mixture was heated to 70 °C for 7 days. The crude
was further purified by silica gel chromatography using 50% of DCM
in hexane to afford 203 mg of white flake as the product, yield 54%.
1H NMR (CDCl3, 101 MHz): δ ppm 7.93 (t, J = 1.5 Hz, 1H), 7.56−
7.46 (m, 2H), 7.01−6.91 (m, 2H). 13C NMR (CDCl3, 400 MHz): δ
158.7 (t, J = 255.7, 5.1 Hz), 138.6, 134.7 (t, J = 12.5 Hz), 134.1,
133.2, 132.7, 130.6, 130.2, 113.5−113.1 (m), 111.3 (t, J = 16.5 Hz).
ESI-MS m/z = 370.0 [M−H]+.
Step 2. The title compound was prepared according to the general
procedure to afford 21.0 mg of colorless oil as the product, yield 98%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.93 (d, J = 2.1 Hz, 1H), 7.52−
7.41 (m, 2H), 6.99 (d, J = 7.6 Hz, 2H), 3.78 (q, J = 7.2 Hz, 2H), 1.15
(t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.2 (d, J
= 5.4 Hz), 159.7 (d, J = 5.4 Hz), 137.4 (d, J = 282.5 Hz), 136.0 (d, J =
25.6 Hz), 133.7, 133.2, 132.9, 131.5, 130.6, 113.5 (dd, J = 24.6, 3.4
Hz), 46.6, 14.1. ESI-MS m/z = 421.9 [M + Na]+.
Step 2. The title compound was prepared according to the general
procedure to afford 38.1 mg of colorless oil as the product, yield 98%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.86 (t, J = 2.1 Hz, 1H), 7.49−
7.39 (m, 2H), 7.29 (t, J = 8.3 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.08
(dd, J = 10.0, 2.1 Hz, 1H), 3.82 (q, J = 7.1 Hz, 2H), 1.14 (t, J = 7.1
Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 159.7 (d, J = 256.3
Hz), 138.6, 135.6 (d, J = 9.9 Hz), 134.1 (d, J = 1.4 Hz), 133.7, 133.1,
132.9, 131.8, 130.6, 125.1 (d, J = 3.7 Hz), 123.6 (d, J = 12.1 Hz),
117.5 (d, J = 23.6 Hz), 46.8 (d, J = 2.5 Hz), 14.4. ESI-MS m/z =
382.0 [M + H]+.
Ethyl 2-(4-((2,5-Dichloro-N-ethylphenyl)sulfonamido)phenyl)-
acetate (24). Step 1. Ethyl 2-(4-((2,5-dichlorophenyl)sulfonamido)-
phenyl)acetate was prepared according to the general procedure using
2,5-dichlorobenzenesulfonyl chloride (248 mg, 3.0 mmol, 1.0 equiv)
as sulfonyl chloride and ethyl (4-aminophenyl)acetate (540.4 mg, 3.0
2,5-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(21). Step 1. 2,5-Dichloro-N-(2,4-difluorophenyl)benzenesulfonamide
was prepared according to the general procedure using 2,4-
difluoroaniline (244 mg, 1.0 mmol, 1.0 equiv) as the aniline, yield
1
mmol, 1.0 equiv) as the aniline, yield 95%. H NMR (CDCl3, 400
MHz): δ ppm 7.97 (t, J = 1.4 Hz, 1H), 7.42 (d, J = 1.4 Hz, 2H), 7.22
(s, br, 1H), 7.15 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 4.12 (q,
J = 7.2 Hz, 2H), 3.52 (s, 2H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR
(CDCl3, 101 MHz): δ ppm 171.4, 137.6, 134.2, 134.0, 133.5, 132.6,
132.0, 131.7, 130.4, 129.5, 121.9, 61.0, 40.6, 14.1. ESI-MS m/z =
386.1 [M−H]+.
Step 2. The title compound was prepared according to the general
procedure to afford 809 mg of yellow oil as the product, yield 98%. 1H
NMR (CDCl3, 400 MHz): δ ppm 7.81 (d, J = 2.4 Hz, 1H), 7.42 (d, J
= 8.4 Hz, 1H), 7.38 (dd, J = 8.5, 2.4 Hz, 1H), 7.21 (d, J = 8.5 Hz,
2H), 7.12 (d, J = 8.5 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.87 (q, J =
7.1 Hz, 2H), 3.56 (s, 2H), 1.22 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz,
3H). 13C NMR (CDCl3, 101 MHz): δ ppm 171.1, 138.5, 136.5,
134.4, 133.4, 132.9, 132.8, 132.2, 130.3, 130.2, 129.4, 61.0, 47.6, 40.9,
14.7, 14.1. ESI-MS m/z = 416.1 [M + H]+.
1
98%. H NMR (CDCl3, 400 MHz): δ ppm 7.92 (t, J = 1.5 Hz, 1H),
7.49 (td, J = 9.0, 5.7 Hz, 1H), 7.45 (d, J = 1.5 Hz, 2H), 7.15 (s, br,
1H), 6.83 (dddd, J = 9.2, 7.8, 2.9, 1.6 Hz, 1H), 6.75 (ddd, J = 10.7,
8.2, 2.8 Hz, 1H). 13C NMR (CDCl3, 101 MHz): δ ppm 160.5 (dd, J =
249.4, 11.2 Hz), 154.9 (dd, J = 249.1, 12.3 Hz), 137.5, 134.3, 133.4,
132.8, 131.3, 130.1, 126.0 (dd, J = 9.7, 1.7 Hz), 119.5 (dd, J = 12.9,
3.9 Hz), 112.0 (dd, J = 22.4, 3.9 Hz), 104.4 (dd, J = 26.8, 23.4 Hz).
ESI-MS m/z = 336.0 [M−H]+.
Step 2. The title compound was prepared according to the general
procedure to afford 36.0 mg of colorless oil as the product, yield 99%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.84 (d, J = 2.2 Hz, 1H), 7.50−
7.39 (m, 2H), 7.32 (q, J = 7.9, 7.3 Hz, 1H), 6.88 (t, J = 8.0 Hz, 1H),
6.79 (t, J = 9.5 Hz, 1H), 3.83 (q, J = 6.0, 4.9 Hz, 2H), 1.14 (t, J = 6.8
Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.7 (dd, J = 252.1,
11.5 Hz), 160.2 (dd, J = 255.5, 12.5 Hz), 138.6, 134.4 (d, J = 10.0
2-(4-((2,5-Dichloro-N-ethylphenyl)sulfonamido)phenyl)acetic
Acid (25). Lithium hydroxide monohydrate (25.6 mg, 1.0 mmol, 5.0
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equiv) was added to compound 23 (85.3 mg, 0.2 mmol, 1.0 equiv) in
a mixed solvent of 1.2 mL of tetrahydrofuran, 0.8 mL of methanol,
and 0.4 mL of distilled water. The mixture was stirred at room
temperature overnight. The reaction was quenched by adding 1 M
HCl after TLC showed complete disappearance of starting material.
The crude material was concentrated in vacuo and further purified by
silica gel chromatography using 5% of methanol in DCM to afford
chromatography using 5% methanol in DCM to afford 1.2 mg of
white solid as the product, yield 49%. 1H NMR (CDCl3, 400 MHz): δ
ppm 7.82 (d, J = 2.4 Hz, 1H), 7.49−7.36 (m, 2H), 7.12 (s, 4H), 5.42
(s, br, 1H), 3.87 (q, J = 7.1 Hz, 2H), 3.48 (q, J = 6.7 Hz, 2H), 2.79 (t,
J = 7.1 Hz, 2H), 1.94 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3, 101 MHz): δ ppm 170.0, 139.2, 138.5, 136.0, 133.4, 132.9,
132.2, 130.4, 129.62, 129.60, 47.6, 40.4, 35.2, 23.3, 14.7. ESI-MS m/z
= 415.1 [M + H]+.
1
71.5 mg of white solid as the product, yield 90%. H NMR (CDCl3,
400 MHz): δ ppm 7.80 (d, J = 2.4 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H),
7.37 (dd, J = 8.5, 2.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.14 (d, J =
8.4 Hz, 2H), 3.86 (q, J = 7.1 Hz, 2H), 3.60 (s, 2H), 1.13 (t, J = 7.1
Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 177.2, 138.5, 136.9,
133.5, 133.5, 133.0, 132.9, 132.2, 130.4, 130.4, 129.5, 47.6, 40.5, 14.7.
ESI-MS m/z = 388.0 [M + H+].
2,5-Dichloro-N-ethyl-N-(4-(2-(4-methylpiperazin-1-yl)-2-
oxoethyl)phenyl)benzenesulfonamide (29). To a solution of acid 25
(9.50 mg, 0.025 mmol, 1.0 equiv) in 0.3 mL of dimethylformamide
were added 1-methylpiperazine (2.80 mg, 0.028 mmol, 1.1 equiv),
HATU (10.5 mg, 0.028 mmol, 1.1 equiv), and N,N-diisopropylethyl-
amine (8.7 μL, 0.050 mmol, 2.0 equiv). The mixture was sealed in a
LC−MS vial and stirred at room temperature for 2 h. The crude
mixture was purified by silica gel chromatography using 5% methanol
in chloroform to afford 6.7 mg of white solid as the product, yield
57%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.81 (d, J = 2.5 Hz, 1H),
7.48−7.36 (m, 2H), 7.16 (q, J = 8.5 Hz, 4H), 3.88 (q, J = 7.1 Hz,
2H), 3.69 (s, 2H), 3.64 (t, J = 5.1 Hz, 2H), 3.42 (t, J = 5.1 Hz, 2H),
2.35 (t, J = 5.1 Hz, 2H), 2.26 (s, 3H), 2.21 (t, J = 5.0 Hz, 2H), 1.15 (t,
J = 7.1 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 168.9, 138.5,
136.3, 135.4, 133.4, 132.9, 132.9, 132.2, 130.4, 129.7, 129.6, 54.8,
54.5, 47.6, 45.9, 41.7, 40.3, 14.7. ESI-MS m/z = 470.1 [M + H]+.
2,5-Dichloro-N-ethyl-N-(4-(2-(4-methylpiperazin-1-yl)-2-
oxoethyl)phenyl)benzenesulfonamide (29). To a solution of acid 25
(9.50 mg, 0.025 mmol, 1.0 equiv) in 0.3 mL of dimethylformamide
were added 1-methylpiperazine (2.80 mg, 0.028 mmol, 1.1 equiv),
HATU (10.5 mg, 0.028 mmol, 1.1 equiv), and N,N-diisopropylethyl-
amine (8.7 μL, 0.050 mmol, 2.0 equiv). The mixture was sealed in a
LC−MS vial and stirred at room temperature for 2 h. The crude was
purified by silica gel chromatography using 5% methanol in
chloroform to afford 6.7 mg of white solid as the product, yield
57%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.81 (d, J = 2.5 Hz, 1H),
7.48−7.36 (m, 2H), 7.16 (q, J = 8.5 Hz, 4H), 3.88 (q, J = 7.1 Hz,
2H), 3.69 (s, 2H), 3.64 (t, J = 5.1 Hz, 2H), 3.42 (t, J = 5.1 Hz, 2H),
2.35 (t, J = 5.1 Hz, 2H), 2.26 (s, 3H), 2.21 (t, J = 5.0 Hz, 2H), 1.15 (t,
J = 7.1 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 168.9, 138.5,
136.3, 135.4, 133.4, 132.9, 132.9, 132.2, 130.4, 129.7, 129.6, 54.8,
54.5, 47.6, 45.9, 41.7, 40.3, 14.7. ESI-MS m/z = 470.1 [M + H]+.
N-(2,4-Difluorophenyl)-N-ethylbenzenesulfonamide (30). N-
(2,4-Difluorophenyl)-N-ethylbenzenesulfonamide (30) was prepared
according to the general procedure using benzenesulfonyl chloride
(17.7 mg, 0.10 mmol, 1.0 equiv) and N-ethyl-2,4-difluoroaniline (23.6
mg with 70% purity, 0.10 mmol, 1.0 equiv) to afford 29.6 mg of
colorless oil as the product, yield 79%. 1H NMR (CDCl3, 400 MHz):
δ ppm 7.73−7.66 (m, 2H), 7.63−7.54 (m, 1H), 7.52−7.43 (m, 2H),
7.20 (td, J = 8.6, 6.0 Hz, 1H), 6.86 (dddd, J = 9.0, 7.8, 2.9, 1.4 Hz,
1H), 6.79 (ddd, J = 10.3, 8.4, 2.8 Hz, 1H), 3.60 (q, J = 7.2 Hz, 2H),
1.08 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.4
(dd, J = 251.4, 11.4 Hz), 160.2 (dd, J = 255.3, 12.6 Hz), 139.0, 133.6
(dd, J = 10.2, 2.4 Hz), 132.8, 128.9, 127.4, 122.1 (dd, J = 12.1, 4.1
Hz), 111.7 (dd, J = 22.2, 3.8 Hz), 105.0 (dd, J = 26.2, 24.2 Hz), 45.4
(d, J = 2.7 Hz), 14.1. ESI-MS m/z = 298.1 [M + H]+.
2,5-Dichloro-N-ethyl-N-(4-(2-hydroxyethyl)phenyl)-
benzenesulfonamide (26). Ethyl 2-(4-((2,5-dichloro-N-ethylphenyl)-
sulfonamido)phenyl)acetate (24, 160 mg, 0.4 mmol, 1.0 equiv) in 2.4
mL of tetrahydrofuran was treated dropwise with 2.0 M lithium
aluminum hydride in tetrahydrofuran (0.2 mL, 0.4 mmol, 1.0 equiv).
After stirring for 2 h at room temperature, the reaction mixture was
carefully quenched by 17.3 μL of water, 17.3 μL of 15% NaOH, and
then 52.0 μL of water. The mixture was stirred for 20 min and filtered
through a plug of celite to remove the solids. The celite plug was
rinsed repeatedly with ethyl acetate. The crude mixture was
concentrated in vacuo and further purified by silica gel chromatog-
raphy using 5% of acetone in DCM to afford 91.9 mg of white solid as
the product, yield 42%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.80 (d,
J = 2.4 Hz, 1H), 7.46−7.34 (m, 2H), 7.15 (d, J = 8.4 Hz, 2H), 7.10
(d, J = 8.5 Hz, 2H), 3.85 (q, J = 6.9 Hz, 2H), 3.82−3.76 (m, 2H),
2.81 (t, J = 6.6 Hz, 2H), 1.13 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3,
101 MHz): δ ppm 139.1, 138.5, 135.8, 133.4, 132.9, 132.9, 132.2,
130.3, 129.9, 129.5, 63.3, 47.6, 38.7, 14.7. ESI-MS m/z = 374.0 [M +
H]+.
N-(4-(2-Aminoethyl)phenyl)-2,5-dichloro-N-ethylbenzenesulfo-
namide (27). Step 1. To a mixture of alcohol 26 (37.2 mg, 0.10
mmol, 1.0 equiv) in 2.6 mL of tetrahydrofuran were added diethyl
azodicarboxylate (38.8 mg, 0.20 mmol, 2.0 equiv), triphenylphosphine
(54.7 mg, 0.20 mmol, 2.0 equiv), and diphenylphosphoryl azide (61.4
mg, 0.20 mmol, 2.0 equiv). The mixture was stirred at room
temperature for 5 h. After removal of solvent, the residue was purified
by silica gel chromatography using 50% DCM in hexane to afford 22.0
mg of N-(4-(2-azidoethyl)phenyl)-2,5-dichloro-N-ethylbenzenesulfo-
1
namide as a colorless oil, yield 55%. H NMR (CDCl3, 400 MHz): δ
ppm 7.81 (s, 1H), 7.48−7.34 (m, 2H), 7.15 (q, J = 8.5, 8.0 Hz, 4H),
3.88 (q, J = 7.1 Hz, 2H), 3.48 (t, J = 7.1 Hz, 2H), 2.86 (t, J = 7.2 Hz,
2H), 1.15 (td, J = 7.1, 1.5 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ
ppm 138.5, 138.4, 136.2, 133.4, 132.9, 132.8, 132.3, 130.3, 129.7,
129.6, 52.1, 47.6, 34.9, 14.7. ESI-MS m/z = 399.0 [M + H]+.
Step 2. To a solution of the azide from step 1 (8.40 mg, 0.020
mmol, 1.0 equiv) in 0.3 mL of methanol was added 10% Pd−C (0.6
mg). The resulting mixture was stirred for 4 h under a hydrogen
atmosphere. The mixture was filtered through a pad of celite, and the
filtrate was concentrated. The residue was purified by silica gel
chromatography using 15% methanol in chloroform to afford 3.7 mg
1
of colorless oil as the product, yield 47%. H NMR (CDCl3, 400
2-Chloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(31). 2-Chloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(31) was prepared according to the general procedure using 2-
chloro-benzenesulfonyl chloride (21.1 mg, 0.10 mmol, 1.0 equiv) and
N-ethyl-2,4-difluoroaniline (23.6 mg with 70% purity, 0.10 mmol, 1.0
equiv) to afford 29.3 mg of colorless oil as the product, yield 77%. 1H
NMR (CDCl3, 400 MHz): δ ppm 7.84 (d, J = 8.0 Hz, 1H), 7.52 (d, J
= 8.0 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.32−7.24 (m, 2H), 6.83 (t, J
= 8.3 Hz, 1H), 6.76 (td, J = 9.5, 8.4, 2.8 Hz, 1H), 3.84 (q, J = 7.2 Hz,
2H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm
162.5 (dd, J = 251.6, 11.4 Hz), 160.3 (dd, J = 255.6, 12.6 Hz), 137.2,
134.4 (dd, J = 10.3, 2.2 Hz), 133.7, 132.3, 132.1 (d, J = 13.3 Hz),
126.7, 121.4 (dd, J = 12.3, 4.2 Hz), 111.7 (dd, J = 22.2, 3.8 Hz), 104.9
(dd, J = 26.2, 24.1 Hz), 46.6 (d, J = 2.5 Hz), 14.4. ESI-MS m/z =
332.0 [M + H]+.
MHz): δ ppm 7.81 (d, J = 2.4 Hz, 1H), 7.47−7.35 (m, 2H), 7.18−
7.06 (m, 4H), 3.87 (q, J = 7.1 Hz, 2H), 2.94 (t, J = 6.9 Hz, 2H), 2.71
(t, J = 6.9 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 101
MHz): δ ppm 140.2, 138.6, 135.6, 133.4, 132.9, 132.8, 132.2, 130.3,
129.7, 129.5, 47.6, 43.2, 39.5, 14.7. ESI-MS m/z = 373.1 [M + H]+.
N-(4-((2,5-Dichloro-N-ethylphenyl)sulfonamido)phenethyl)-
acetamide (28). Acetyl chloride (0.700 mg, 0.0088 mmol, 1.5 equiv)
was added to a stirred solution of amine 27 (2.20 mg, 0.0059 mmol,
1.0 equiv) and solid sodium bicarbonate (2.20 mg, 0.0088 mmol, 1.5
equiv) in 0.75 mL of chloroform. The resulting mixture was stirred at
room temperature overnight. The reaction mixture was diluted with
DCM (1 mL) and water (2 mL) and stirred for 30 min. The aqueous
and organic layers were partitioned, and the aqueous layer was
extracted with DCM (2 × 1 mL). The combined organic layers were
dried over sodium sulfate. The residue was purified by silica gel
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3-Chloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(32). 3-Chloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(32) was prepared according to the general procedure using 3-
chlorobenzenesulfonyl chloride (21.1 mg, 0.10 mmol, 1.0 equiv) and
N-ethyl-2,4-difluoroaniline (23.6 mg with 70% purity, 0.10 mmol, 1.0
equiv) as the aniline to afford 25.9 mg of colorless oil as the product,
5.9 Hz, 1H), 6.91 (t, J = 8.3 Hz, 1H), 6.83 (ddd, J = 10.8, 8.4, 2.8 Hz,
1H), 3.60 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3, 101 MHz): δ ppm 162.6 (dd, J = 252.2, 11.5 Hz), 159.9 (dd,
J = 255.1, 12.7 Hz), 139.0, 137.6, 133.8 (dd, J = 10.1, 2.3 Hz), 133.6,
131.0, 129.2, 126.4, 121.5 (dd, J = 12.0, 4.2 Hz), 112.0 (dd, J = 22.2,
3.7 Hz), 105.2 (dd, J = 26.3, 24.1 Hz), 45.8 (d, J = 2.8 Hz), 14.1. ESI-
MS m/z = 366.0 [M + H]+.
1
yield 70%. H NMR (CDCl3, 400 MHz): δ ppm 7.69 (t, J = 1.9 Hz,
1H), 7.58−7.52 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.22 (td, J = 8.7,
6.0 Hz, 1H), 6.88 (dddd, J = 9.0, 7.7, 2.8, 1.4 Hz, 1H), 6.81 (ddd, J =
10.4, 8.4, 2.8 Hz, 1H), 3.60 (q, J = 7.2 Hz, 2H), 1.09 (td, J = 7.1, 0.6
Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.6 (dd, J = 11.5
Hz), 160.0 (dd, J = 12.6 Hz), 140.8, 135.1, 133.7 (dd, J = 10.1, 2.3
Hz), 132.9, 130.2, 127.4, 125.4, 121.7 (dd, J = 12.1, 4.2 Hz), 111.9
(dd, J = 22.2, 3.8 Hz), 105.1 (dd, J = 26.2, 24.1 Hz), 45.7 (d, J = 2.7
Hz), 14.1. ESI-MS m/z = 332.0 [M + H]+.
2,4-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(33). 2,4-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfona-
mide (33) was prepared according to the general procedure using
2,4-dichloro-benzenesulfonyl chloride (24.1 mg, 0.10 mmol, 1.0
equiv) and N-ethyl-2,4-difluoroaniline (28.3 mg with 70% purity, 0.10
mmol, 1.0 equiv) to afford 26.3 mg of colorless oil as the product,
yield 73%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.76 (d, J = 8.6 Hz,
1H), 7.53 (d, J = 2.1 Hz, 1H), 7.31 (td, J = 8.7, 6.0 Hz, 1H), 7.25 (dd,
J = 8.6, 2.1 Hz, 1H), 6.86 (dddd, J = 9.1, 7.8, 2.9, 1.5 Hz, 1H), 6.78
(ddd, J = 10.3, 8.4, 2.8 Hz, 1H), 3.83 (q, J = 7.1 Hz, 2H), 1.13 (td, J =
7.2, 0.7 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.6 (dd, J =
252.1, 11.4 Hz), 160.2 (dd, J = 255.5, 12.6 Hz), 139.5, 135.9, 134.5
(dd, J = 10.1, 2.2 Hz), 133.3, 133.0, 131.8, 127.0, 121.1 (dd, J = 12.3,
4.1 Hz), 111.8 (dd, J = 22.2, 3.8 Hz), 105.0 (dd, J = 26.2, 24.1 Hz),
46.8 (d, J = 2.5 Hz), 14.4. ESI-MS m/z = 366.0 [M + H]+.
2,3-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(37). 2,3-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfona-
mide (37) was prepared according to the general procedure using
2,3-dichlorobenzenesulfonyl chloride (23.8 mg, 0.10 mmol, 1.0 equiv)
and N-ethyl-2,4-difluoroaniline (22.0 mg with 70% purity, 0.10 mmol,
1.0 equiv) to afford 21.5 mg of colorless oil as the product, yield 61%.
1H NMR (CDCl3, 101 MHz): δ ppm 7.78 (dd, J = 8.0, 1.6 Hz, 1H),
7.64 (dd, J = 8.0, 1.6 Hz, 1H), 7.30 (td, J = 8.7, 6.0 Hz, 1H), 7.22 (t, J
= 8.0 Hz, 1H), 6.85 (tdd, J = 9.0, 2.9, 1.4 Hz, 1H), 6.77 (ddd, J =
10.1, 8.4, 2.8 Hz, 1H), 3.84 (q, J = 7.1 Hz, 2H), 1.14 (t, J = 7.1 Hz,
3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.6 (dd, J = 252.1, 11.3
Hz), 160.2 (dd, J = 255.6, 12.5 Hz), 139.3, 135.7, 134.5, 134.4 (d, J =
8.0, 2.1 Hz), 130.8, 130.5, 126.9, 121.1 (dd, J = 12.4, 4.2 Hz), 111.8
(dd, J = 22.2, 3.8 Hz), 105.0 (dd, J = 26.2, 24.1 Hz), 46.9 (d, J = 2.1
Hz), 14.4. ESI-MS m/z = 366.0 [M + H]+.
2-Chloro-N-(2,4-difluorophenyl)-N-ethyl-5-fluorobenzenesulfo-
namide (38). 2-Chloro-N-(2,4-difluorophenyl)-N-ethyl-5-fluoroben-
zenesulfonamide (38) was prepared according to the general
procedure using 2-chloro-5-fluorobenzenesulfonyl chloride (24.4
mg, 0.10 mmol, 1.0 equiv) and N-ethyl-2,4-difluoroaniline (22.2 mg
with 70% purity, 0.10 mmol, 1.0 equiv) to afford 30.6 mg of colorless
1
oil as the product, yield 82%. H NMR (CDCl3, 400 MHz): δ ppm
7.58 (dd, J = 8.2, 3.1 Hz, 1H), 7.50 (dd, J = 8.8, 4.7 Hz, 1H), 7.31 (td,
J = 8.6, 6.1 Hz, 1H), 7.18 (ddd, J = 8.7, 7.3, 3.1 Hz, 1H), 6.87 (td, J =
8.4, 7.7, 2.9 Hz, 1H), 6.79 (ddd, J = 11.2, 8.5, 2.9 Hz, 1H), 3.85 (q, J
= 7.2 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 101
MHz): δ ppm 163.9 (d, J = 11.4 Hz), 161.5 (t, J = 11.8 Hz), 159.0 (t,
J = 6.3 Hz), 138.9 (d, J = 6.5 Hz), 134.4 (dd, J = 10.2, 2.2 Hz), 133.4
(d, J = 7.5 Hz), 127.4 (d, J = 3.7 Hz), 121.1 (dd, J = 12.4, 4.1 Hz),
120.8 (d, J = 22.6 Hz), 119.3 (d, J = 26.3 Hz), 111.9 (dd, J = 22.3, 3.8
Hz), 105.0 (dd, J = 26.3, 24.1 Hz), 46.9 (d, J = 2.4 Hz), 14.4. ESI-MS
m/z = 350.0 [M + H]+.
3,5-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(34). 3,5-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfona-
mide (34) was prepared according to the general procedure using
3,5-dichloro-benzenesulfonyl chloride (27.3 mg, 0.10 mmol, 1.0
equiv) and N-ethyl-2,4-difluoroaniline (29.7 mg with 70% purity, 0.10
mmol, 1.0 equiv). The reaction mixture was heated to 70 °C for 3
days. The crude was further purified by silica gel chromatography
using DCM to afford 27.6 mg of white solid as the product, yield 68%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.56 (s, 3H), 7.26 (td, J = 8.7,
5.9 Hz, 1H), 6.92 (dddd, J = 9.0, 7.7, 2.8, 1.4 Hz, 1H), 6.85 (ddd, J =
10.9, 8.4, 2.8 Hz, 1H), 3.61 (q, J = 7.2 Hz, 2H), 1.11 (t, J = 7.2 Hz,
3H). 13C NMR (CDCl3, 101 MHz): δ ppm 162.7 (dd, J = 252.4, 11.5
Hz), 159.9 (dd, J = 255.1, 12.7 Hz), 142.0, 136.0, 133.9 (dd, J = 10.2,
2.3 Hz), 132.7, 125.6, 121.4 (dd, J = 12.1, 4.1 Hz), 112.1 (dd, J =
22.2, 3.8 Hz), 105.2 (dd, J = 26.3, 24.1 Hz), 45.9 (d, J = 2.7 Hz), 14.1.
ESI-MS m/z = 365.9 [M + H]+.
2,6-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(35). 2,6-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfona-
mide (35) was prepared according to the general procedure using
2,6-dichlorobenzenesulfonyl chloride (27.9 mg, 0.10 mmol, 1.0 equiv)
and N-ethyl-2,4-difluoroaniline (24.5 mg with 70% purity, 0.10 mmol,
1.0 equiv) to afford 27.2 mg of white solid as the product, yield 65%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.45−7.33 (m, 3H), 7.33−7.23
(m, 1H), 6.87 (td, J = 9.1, 3.0 Hz, 1H), 6.77 (ddd, J = 10.9, 8.3, 2.9
Hz, 1H), 3.90 (q, J = 7.2 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H). 13C NMR
(CDCl3, 101 MHz): δ ppm 162.7 (dd, J = 252.0, 11.5 Hz), 160.4 (dd,
J = 255.5, 12.7 Hz), 136.1, 135.2, 134.7 (dd, J = 10.1, 2.2 Hz), 132.6,
131.5, 121.0 (dd, J = 12.3, 4.0 Hz), 111.8 (dd, J = 22.2, 3.8 Hz), 104.9
(dd, J = 26.3, 24.1 Hz), 46.6 (d, J = 2.4 Hz), 14.3. ESI-MS m/z =
366.0 [M + H]+.
3,4-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfonamide
(36). 3,4-Dichloro-N-(2,4-difluorophenyl)-N-ethylbenzenesulfona-
mide (36) was prepared according to the general procedure using
3,4-dichloro-benzenesulfonyl chloride (26.6 mg, 0.10 mmol, 1.0
equiv) and N-ethyl-2,4-difluoroaniline (28.0 mg with 70% purity, 0.10
mmol, 1.0 equiv). The reaction mixture was heated to 70 °C for 3
days. The crude was further purified by silica gel chromatography
using DCM to afford 33.4 mg of white solid as the product, yield 84%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.79 (d, J = 2.0 Hz, 1H), 7.56
5-Chloro-N-(2,4-difluorophenyl)-N-ethyl-2-fluorobenzenesulfo-
namide (39). 5-Chloro-N-(2,4-difluorophenyl)-N-ethyl-2-fluoroben-
zenesulfonamide (39) was prepared according to the general
procedure using 5-chloro-2-fluorobenzenesulphonyl chloride (23.6
mg, 0.10 mmol, 1.0 equiv) and N-ethyl-2,4-difluoroaniline (22.8 mg
with 70% purity, 0.10 mmol, 1.0 equiv) to afford 31.7 mg of colorless
1
oil as the product, yield 88%. H NMR (CDCl3, 400 MHz): δ ppm
7.68 (dd, J = 6.0, 2.7 Hz, 1H), 7.50 (dt, J = 8.9, 3.5 Hz, 1H), 7.31 (td,
J = 8.7, 5.9 Hz, 1H), 7.17 (t, J = 9.1 Hz, 1H), 6.89 (td, J = 8.4, 7.8, 3.0
Hz, 1H), 6.80 (ddd, J = 10.9, 8.4, 2.8 Hz, 1H), 3.77 (q, J = 7.2 Hz,
2H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm
162.7 (dd, J = 252.1, 11.6 Hz), 160.1 (dd, J = 254.5, 12.3 Hz), 157.5
(d, J = 256.5 Hz), 134.8 (d, J = 8.5 Hz), 134.4 (dd, J = 10.2, 2.3 Hz),
130.5, 129.5 (d, J = 3.7 Hz), 129.1 (d, J = 16.4 Hz), 121.1 (dd, J =
12.3, 4.2 Hz), 118.5 (d, J = 23.7 Hz), 112.0 (dd, J = 22.2, 3.7 Hz),
105.0 (dd, J = 26.4, 24.0 Hz), 46.2 (t, J = 3.1 Hz), 14.4. ESI-MS m/z
= 350.0 [M + H]+.
N-(2,4-Difluorophenyl)-N-ethyl-2,5-difluorobenzenesulfonamide
(40). N-(2,4-Difluorophenyl)-N-ethyl-2,5-difluorobenzenesulfona-
mide (40) was prepared according to the general procedure using
2,5-difluorobenzenesulfonyl chloride (27.5 mg, 0.10 mmol, 1.0 equiv)
and N-ethyl-2,4-difluoroaniline (24.7 mg with 70% purity, 0.10 mmol,
1.0 equiv) to afford 32.8 mg of colorless oil as the product, yield 76%.
1H NMR (CDCl3, 400 MHz): δ ppm 7.40 (ddd, J = 8.2, 5.2, 3.2 Hz,
1H), 7.30 (td, J = 8.7, 5.9 Hz, 1H), 7.21 (dtd, J = 18.0, 9.1, 4.9 Hz,
2H), 6.89 (td, J = 8.4, 7.6, 2.9 Hz, 1H), 6.80 (ddd, J = 10.8, 8.4, 2.6
Hz, 1H), 3.78 (q, J = 7.2 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3, 101 MHz): δ ppm 162.7 (dd, J = 252.1, 11.5 Hz), 160.1 (dd,
J = 255.0, 12.7 Hz), 156.29 (t, J = 2.6 Hz), 156.26 (dd, J = 497.8, 2.8
Hz), 134.3 (dd, J = 10.2, 2.3 Hz), 128.9 (dd, J = 17.5, 6.9 Hz), 121.6
(dd, J = 24.0, 8.6 Hz), 121.2 (dd, J = 12.4, 4.1 Hz), 118.5 (dd, J =
(d, J = 8.4 Hz, 1H), 7.50 (dd, J = 8.4, 2.1 Hz, 1H), 7.26 (td, J = 8.7,
L
https://doi.org/10.1021/acs.jmedchem.1c00304
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
24.8, 7.9 Hz), 117.6 (d, J = 26.9 Hz), 111.9 (dd, J = 22.3, 3.7 Hz),
105.0 (dd, J = 26.3, 24.0 Hz), 46.2 (t, J = 3.1 Hz), 14.4. ESI-MS m/z
= 334.0 [M + H]+.
procedure using 2-methylpyrimidin-5-amine (12.6 mg, 0.10 mmol, 1.0
equiv) to afford 15.5 mg of white solid, yield 46%. 1H NMR (CDCl3,
400 MHz): δ ppm 8.05 (d, J = 1.6 Hz, 1H), 7.57 (d, J = 9.5 Hz, 1H),
7.32−7.25 (m, 3H), 2.33 (s, 3H). ESI-MS m/z = 317.9 [M + H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (15.5 mg, 0.050 mmol,
1.0 equiv) and 1-bromoethane to afford 13.0 mg of yellow solid as the
2 , 5 - D i c h l o r o - N - e t h y l - N - ( 4 - m e t h y l p y r i d i n - 3 - y l ) -
benzenesulfonamide (41). Step 1. To a scintillation vial equipped
with a stir bar and Teflon-lined lid containing 3-amino-4-
methylpyridine (441.9 mg, 4.1 mmol), N-methylimidazole (0.36
mL, 4.5 mmol), and DCM (14 mL) was slowly added 2,5-
dichlorobenzenesulfonyl chloride (1.0 g, 4.1 mmol), and the reaction
was stirred overnight. The reaction was condensed to dryness and
purified by automated flash chromatography in 100% DCM to give
261.5 mg of 2,5-dichloro-N-(4-methylpyridin-3-yl)-
benzenesulfonamide in 20% yield. 1H NMR (400 MHz, chloro-
form-d): δ 8.32 (d, J = 4.9 Hz, 1H), 8.15 (s, 1H), 7.92 (d, J = 2.1 Hz,
1H), 7.55−7.47 (m, 2H), 7.15 (d, J = 4.9 Hz, 1H), 6.91 (s, 1H), 2.38
(s, 3H). ESI-MS (m/z): 317.0 [M + H]+.
1
product, yield 77%. H NMR (CDCl3, 400 MHz): δ ppm 8.28−8.16
(m, 2H), 7.38 (m, 2H), 7.25 (d, J = 9.5 Hz, 1H), 4.33 (q, J = 7.2 Hz,
2H), 2.42 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 101
MHz): δ ppm 153.7, 149.0, 142.2, 132.8, 132.6, 132.29, 132.28, 130.4,
129.5, 127.0, 51.3, 20.9, 13.2. ESI-MS m/z = 346.0 [M + H]+.
2 , 5 - D i c h l o r o - N - e t h y l - N - ( 6 - m e t h y l p y r i d i n - 3 - y l ) -
benzenesulfonamide (45). Step 1. 2,5-Dichloro-N-(6-methylpyridin-
3-yl)benzenesulfonamide was synthesized according to the general
procedure using 5-amino-2-methylpyridin (11.1 mg, 0.10 mmol, 1.0
equiv) e to afford 26.2 mg of white solid as the product, yield 86%. 1H
NMR (CDCl3, 400 MHz): δ ppm 8.10 (d, J = 2.7 Hz, 1H), 7.93 (d, J
= 1.8 Hz, 1H), 7.48 (dd, J = 8.4, 2.7 Hz, 1H), 7.40−7.36 (m, 2H),
7.02 (d, J = 8.3 Hz, 1H), 2.40 (s, 3H). 13C NMR (CDCl3, 101 MHz):
δ ppm 155.1, 141.7, 137.6, 134.0, 133.3, 132.8, 131.5, 130.7, 129.9,
129.8, 123.9, 23.3. ESI-MS m/z = 317.0 [M + H]+.
Step 2. Potassium carbonate (34.7 mg, 0.25 mmol), followed by
iodoethane (34.4 mL, 0.25 mmol), was added to a solution of the
sulfonamide from step 1 (44.6 mg, 0.14 mmol) in acetone (1.4 mL).
The reaction was stirred at 60 °C overnight, then condensed to
dryness and purified by automated flash chromatography in 100%
1
DCM to give 28.5 mg of the title compound in 59% yield. H NMR
(400 MHz, chloroform-d): δ 8.49 (d, J = 1.7 Hz, 1H), 8.19 (d, J = 2.4
Hz, 1H), 7.53 (dd, J = 5.9, 1.7 Hz, 1H), 7.34−7.28 (m, 2H), 7.25 (dd,
J = 8.4, 2.5 Hz, 1H), 4.26 (q, J = 7.4 Hz, 2H), 2.36 (s, 3H), 1.55 (t, J
= 7.4 Hz, 3H). 13C NMR (101 MHz, chloroform-d): δ 150.0, 144.0,
132.6, 132.3, 131.2, 130.5, 130.1, 129.9, 128.3, 127.0, 56.6, 19.6, 16.7.
ESI-MS (m/z): 345.0 [M + H]+.
2 , 5 - D i c h l o r o - N - e t h y l - N - (5 - m e t h y l p y r a z i n - 2 - y l ) -
benzenesulfonamide (42). Step 1. 2,5-Dichloro-N-(5-methylpyrazin-
2-yl)benzenesulfonamide was synthesized according to the general
procedure using 5-methylpyrazin-2-amine (12.2 mg, 0.10 mmol, 1.0
equiv) to afford 28.6 mg of white solid as the product, yield 89%. 1H
NMR (CDCl3, 400 MHz): δ ppm 8.31 (s, 1H), 8.15 (d, J = 2.4 Hz,
1H), 8.03 (s, 1H), 7.41 (dd, J = 8.6, 2.5 Hz, 1H), 7.34 (d, J = 8.5 Hz,
1H), 2.39 (s, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 148.6,
144.9, 141.4, 138.1, 134.2, 133.6, 133.3, 132.9, 131.9, 130.1, 20.4. ESI-
MS m/z = 317.9 [M + H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (28.6 mg, 0.10 mmol,
1.0 equiv) and 1-bromoethane. The reaction was heated at 70 °C for
5 days. The crude was further purified by a silica gel plug and eluted
by DCM to afford 16.0 mg of white solid as the product, yield 51%.
1H NMR (CDCl3, 400 MHz): δ ppm 8.67 (d, J = 1.4 Hz, 1H), 8.17
(d, J = 1.4 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.5, 2.5 Hz,
1H), 7.37 (d, J = 8.5 Hz, 1H), 3.98 (q, J = 7.0 Hz, 2H), 2.54 (s, 3H),
1.19 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 150.9,
145.8, 141.9, 141.3, 138.2, 133.8, 133.2, 133.2, 131.7, 130.4, 43.8,
20.9, 14.3. ESI-MS m/z = 346.0 [M + H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (26.2 mg, 0.10 mmol,
1.0 equiv) and 1-bromoethane to afford 26.1 mg of colorless oil as the
product, yield 92%. 1H NMR (CDCl3, 400 MHz): δ ppm 8.23 (d, J =
2.6 Hz, 1H), 7.83 (d, J = 2.3 Hz, 1H), 7.48 (dd, J = 8.3, 2.6 Hz, 1H),
7.44 (d, J = 8.6 Hz, 1H), 7.40 (dd, J = 8.4, 2.3 Hz, 1H), 7.13 (d, J =
8.3 Hz, 1H), 3.87 (q, J = 7.1 Hz, 2H), 2.52 (s, 3H), 1.15 (t, J = 7.1
Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 158.5, 149.5, 138.1,
137.5, 133.8, 133.2, 133.0, 132.2, 131.9, 130.3, 123.6, 47.7, 24.1, 14.6.
ESI-MS m/z = 345.0 [M + H]+.
2,5-Dichloro-N-(5-chloropyridin-2-yl)-N-ethylbenzenesulfona-
mide (46). Step 1. 2,5-Dichloro-N-(5-chloropyridin-2-yl)-
benzenesulfonamide was synthesized according to the general
procedure using 2-amino-5-chloropyridine (14.0 mg, 0.10 mmol, 1.0
equiv) to afford 16.0 mg of white solid as the product, yield 47%. 1H
NMR (d4-MeOH, 400 MHz): δ ppm 8.17 (d, J = 2.6 Hz, 1H), 8.05
(d, J = 2.5 Hz, 1H), 7.66 (dd, J = 8.8, 2.7 Hz, 1H), 7.56 (dd, J = 8.5,
2.5 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H). ESI-
MS m/z = 336.9 [M + H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (16.0 mg, 0.050 mmol,
1.0 equiv) and 1-bromoethane to afford 7.5 mg of colorless oil as the
product, yield 43%. 1H NMR (CDCl3, 400 MHz): δ ppm 8.27 (d, J =
2.6 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.64 (dd, J = 8.7, 2.6 Hz, 1H),
7.45 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 8.5, 2.4 Hz, 1H), 7.37 (d, J =
8.5 Hz, 1H), 4.00 (q, J = 7.0 Hz, 2H), 1.19 (t, J = 7.0 Hz, 3H). 13C
NMR (CDCl3, 101 MHz): δ ppm 149.8, 146.9, 138.4, 137.5, 133.7,
133.2, 133.1, 131.7, 130.5, 129.1, 121.4, 43.8, 14.4. ESI-MS m/z =
364.9 [M + H]+.
2,5-Dichloro-N-(3-chloropyridin-4-yl)-N-ethylbenzenesulfona-
mide (47). Step 1. 2,5-Dichloro-N-(3-chloropyridin-4-yl)-
benzenesulfonamide was synthesized according to the general
procedure using 2 4-amino-3-chloropyridine (13.6 mg, 0.10 mmol,
1.0 equiv) to afford 24.1 mg of white solid as the product, yield 71%.
1H NMR (CDCl3, 400 MHz): δ ppm 8.14 (s, 1H), 8.04 (d, J = 2.6
2,5-Dichloro-N-ethyl-N-(2-methylpyrimidin-5-yl)-
benzenesulfonamide (43). Step 1. 2,5-Dichloro-N-(2-methylpyrimi-
din-5-yl)benzenesulfonamide was synthesized according to the
general procedure using 2-methylpyrimidin-5-amine (11.6 mg, 0.10
mmol, 1.0 equiv) to afford 29.3 mg of white solid as the product, yield
87%. 1H NMR (CDCl3, 400 MHz): δ ppm 8.42 (s, 2H), 7.97 (d, J =
2.0 Hz, 1H), 7.45−7.34 (m, 2H), 2.58 (s, 3H). 13C NMR (CDCl3,
101 MHz): δ ppm 164.3, 158.9, 149.5, 137.5, 134.4, 133.6, 133.0,
131.5, 129.8, 110.0, 24.9. ESI-MS m/z = 317.9 [M + H]+,
C11H9Cl2N3O2S requires 317.0.
Hz, 1H), 7.36 (d, J = 1.5 Hz, 1H), 7.28 (q, J = 7.9, 7.3 Hz, 2H), 6.75
(s, 1H). ESI-MS m/z = 336.9 [M + H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (29.3 mg, 0.10 mmol,
1.0 equiv) to afford 30.7 mg of yellow solid as the product, yield 96%.
1H NMR (CDCl3, 400 MHz): δ ppm 8.48 (s, 2H), 7.89 (s, 1H), 7.45
(d, J = 1.5 Hz, 2H), 3.84 (q, J = 7.2 Hz, 2H), 2.72 (s, 3H), 1.17 (t, J =
7.1 Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 178.3, 167.6,
157.1, 137.7, 134.2, 133.4, 133.2, 132.2, 130.8, 130.2, 47.5, 25.7, 14.5.
ESI-MS m/z = 346.0 [M + H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (11.4 mg, 0.050 mmol,
1.0 equiv) and 1-bromoethane to afford 5.9 mg of white solid as the
product, yield 48%. 1H NMR (CDCl3, 400 MHz): δ ppm 8.13 (d, J =
2.1 Hz, 1H), 7.85 (s, 1H), 7.54 (s, 2H), 7.30 (d, J = 3.2 Hz, 2H), 3.99
(q, J = 7.3 Hz, 2H), 1.43 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3, 101
MHz): δ ppm 158.5, 141.7, 138.3, 138.1, 132.6, 132.4, 132.2, 130.7,
129.5, 125.0, 113.9, 53.3, 16.0. ESI-MS m/z = 365.0 [M + H]+.
2 , 5 - D i c h l o r o - N - e t h y l - N - ( 3 - fl u o r o p y r i d i n - 4 - y l ) -
benzenesulfonamide (48). Step 1. 2,5-Dichloro-N-(3-fluoropyridin-
4-yl)benzenesulfonamide was synthesized according to the general
2,5-Dichloro-N-ethyl-N-(6-methylpyridazin-3-yl)-
benzenesulfonamide (44). Step 1. 2,5-Dichloro-N-(6-methylpyrida-
zin-3-yl)benzenesulfonamide was synthesized according to the general
M
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procedure using 4-amino-3-fluoropyridine (12.6 mg, 0.10 mmol, 1.0
equiv) to afford 16.6 mg of white solid as the product, yield 46%. 1H
NMR (d4-MeOH, 400 MHz): δ ppm 8.84 (t, J = 7.8 Hz, 1H), 8.19 (d,
J = 5.0 Hz, 1H), 8.15 (d, J = 2.2 Hz, 1H), 7.94 (d, J = 6.7 Hz, 1H),
7.56 (d, J = 7.3 Hz, 1H), 7.46 (s, 1H). ESI-MS m/z = 320.9 [M +
H]+.
Step 2. The title compound was prepared according to the general
procedure using the sulfonamide from step 1 (8.3 mg, 0.050 mmol,
1.0 equiv) and 1-bromoethane to afford 7.6 mg of colorless oil as the
product, yield 84%. 1H NMR (CDCl3, 400 MHz): δ ppm 8.14 (d, J =
2.0 Hz, 1H), 7.74 (dd, J = 5.8, 2.0 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H),
7.55 (dd, J = 7.4, 2.0 Hz, 1H), 7.32 (d, J = 2.3 Hz, 2H), 4.05 (q, J =
7.3 Hz, 2H), 1.47 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3, 101 MHz):
δ ppm 153.1, 151.9 (d, J = 258.5 Hz), 141.7, 137.4 (d, J = 1.4 Hz),
132.6, 132.6, 132.3, 130.8, 129.6, 126.9 (d, J = 36.5 Hz), 115.1 (d, J =
5.7 Hz), 53.5, 16.0. ESI-MS m/z = 349.0 [M + H]+.
133.2, 132.2, 131.6, 131.5, 130.2, 127.0, 82.4, 69.5, 47.6, 32.7, 31.9,
26.1, 18.0, 13.2. ESI-MS m/z = 503.9 [M + Na]+.
2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl 2-(4-((2,5-Dichloro-N-
ethylphenyl)sulfonamido)phenyl)acetate (53). To a solution of acid
24 (7.8 mg, 0.020 mmol, 1.0 equiv) in 0.1 mL of dimethylformamide
were added potassium carbonate (5.3 mg, 0.040 mmol, 2.0 equiv) and
3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine (5.5 mg, 0.022 mmol,
1.1 equiv).31 The reaction mixture was stirred at room temperature
overnight. The crude mixture was purified by a silica gel plug and
eluted by 50% of ethyl acetate in hexane to afford 8.7 mg of off-white
solid as the product, yield 85%. 1H NMR (CDCl3, 400 MHz): δ ppm
7.82 (t, J = 2.0 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.39 (dt, J = 8.5, 2.0
Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 3.99 (t, J
= 6.3 Hz, 2H), 3.88 (q, J = 7.1 Hz, 2H), 3.62 (s, 2H), 2.03−1.94 (m,
3H), 1.72 (t, J = 6.3 Hz, 2H), 1.61 (t, J = 7.5 Hz, 2H), 1.15 (t, J = 7.1
Hz, 3H). 13C NMR (CDCl3, 101 MHz): δ ppm 170.8, 138.5, 136.6,
134.0, 133.5, 132.9, 132.9, 132.2, 130.3, 129.5, 82.5, 69.4, 59.7, 47.6,
40.8, 32.2, 32.1, 26.2, 14.7, 13.2. ESI-MS m/z = 507.9 [M + H]+.
N-(3-Azidophenyl)-2,5-dichloro-N-(prop-2-yn-1-yl)-
benzenesulfonamide (54). N-(3-Azidophenyl)-2,5-dichloro-N-
(prop-2-yn-1-yl)benzenesulfonamide (54) was synthesized according
to the general procedure to afford 15.8 mg of off-white solid as the
product, yield 82%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.91 (dd, J
= 2.0, 1.0 Hz, 1H), 7.46 (d, J = 1.9 Hz, 2H), 7.30 (t, J = 8.0 Hz, 1H),
7.05 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 7.00 (ddd, J = 8.1, 2.3, 0.9 Hz,
1H), 6.96 (t, J = 2.1 Hz, 1H), 4.61 (d, J = 2.5 Hz, 2H), 2.29 (t, J = 2.5
Hz, 1H). 13C NMR (CDCl3, 101 MHz): δ ppm 141.2, 139.4, 138.1,
134.0, 133.2, 133.0, 132.3, 130.5, 130.5, 125.1, 119.7, 119.3, 77.9,
74.2, 42.2. ESI-MS m/z = 380.9 [M + Na]+.
5-Chloro-1-((2,5-dichlorophenyl)sulfonyl)indoline (49). 5-
Chloro-1-((2,5-dichlorophenyl)sulfonyl)indoline (49) was synthe-
sized according to the general procedure using 5-chloro-2,3-
dihydro-(1H)-indole (30.7 mg, 0.20 mmol, 1.0 equiv). The reaction
mixture was heated to 70 °C for 5 days. The crude was further
purified by silica gel chromatography using 66% of DCM in hexane to
1
afford 70.6 mg of white solid as the product, yield 98.5%. H NMR
(CDCl3, 400 MHz): δ ppm 8.13 (d, J = 2.8 Hz, 1H), 7.48−7.35 (m,
2H), 7.18 (d, J = 8.6 Hz, 1H), 7.12−7.01 (m, 2H), 4.20 (td, J = 8.3,
3.9 Hz, 2H), 3.08 (t, J = 8.5 Hz, 2H). 13C NMR (CDCl3, 101 MHz):
δ ppm 139.9, 137.8, 134.0, 133.3, 133.1, 133.1, 132.0, 130.5, 128.7,
127.5, 125.5, 114.8, 50.7, 27.8. ESI-MS m/z = 362.0 [M + H]+.
6-Chloro-1-((2,5-dichlorophenyl)sulfonyl)-1,2,3,4-tetrahydroqui-
noline (50). 6-Chloro-1-((2,5-dichlorophenyl)sulfonyl)-1,2,3,4-tetra-
hydroquinoline (50) was synthesized according to the general
procedure using 6-chloro-1,2,3,4-tetrahydroquinoline (33.6 mg, 0.20
mmol, 1.0 equiv). The reaction mixture was heated to 60 °C for 7
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00304.
■
sı
1
days to afford 23.2 mg of white solid as the product, yield 31%. H
NMR (CDCl3, 400 MHz): δ ppm 8.12 (d, J = 2.5 Hz, 1H), 7.46 (dd,
J = 8.5, 2.5 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 9.6 Hz,
1H), 7.07 (s, br, 2H), 3.85 (t, J = 6.0 Hz, 2H), 2.72 (t, J = 6.7 Hz,
2H), 1.89 (p, J = 6.5 Hz, 2H). 13C NMR (CDCl3, 101 MHz): δ ppm
139.3, 135.4, 133.9, 133.3, 133.3, 131.8, 131.5, 130.5, 129.9, 129.0,
126.5, 124.0, 46.5, 26.7, 22.2. ESI-MS m/z = 376.0 [M + H]+.
2,5-Dichloro-N-(4-chloro-2-methylphenyl)benzamide (51). BOP-
Cl (bis(2-oxo-3-oxazolidinyl)phosphinic chloride, 196 mg, 0.75
mmol, 1.5 equiv) was added to a stirred solution of 2,5-
dichlorobenzoic acid (99.0 mg, 0.50 mmol, 1.0 equiv) in 5 mL of
THF and i-Pr2NEt (129 mg, 1.0 mmol, 2.0 equiv). The mixture was
stirred for 1 h at room temperature. 4-Chloro-2-methylaniline (86.0
mg, 0.55 mmol, 1.1 equiv) was added and allowed to react for
overnight at 50 °C under nitrogen. After completion, the crude
mixture was poured onto ice. The precipitation was collected by
filtration and washed by water to afford 157 mg of off-white solid,
yield 91%. 1H NMR (CDCl3, 400 MHz): δ ppm 7.70−7.63 (m, 2H),
7.35 (s, br, 2H), 7.17 (d, J = 8.6 Hz, 2H), 2.26 (s, 3H). 13C NMR
(CDCl3, 101 MHz): δ ppm 164.3, 136.5, 133.4, 133.3, 133.1, 131.4,
131.4, 130.4, 129.6, 128.9, 126.5, 125.8, 125.7, 17.8. ESI-MS m/z =
314.0 [M + H]+.
NMR spectra for new compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
■
Beatriz M. A. Fontoura − Department of Cell Biology,
University of Texas Southwestern Medical Center, Dallas,
Texas 75390, United States; orcid.org/0000-0001-8468-
5315; Phone: (214) 648-4207; Email: beatriz.fontoura@
utsouthwestern.edu
Authors
Kris White − Department of Microbiology, Icahn School of
Medicine at Mount Sinai, New York, New York 10029,
United States; Global Health and Emerging Pathogens
Institute, Icahn School of Medicine at Mount Sinai, New
York, New York 10029, United States
Matthew Esparza − Department of Cell Biology, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Jue Liang − Department of Biochemistry, University of Texas
Southwestern Medical Center, Dallas, Texas 75390, United
States
Prasanna Bhat − Department of Cell Biology, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Jacinth Naidoo − Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Briana L. McGovern − Department of Microbiology, Icahn
School of Medicine at Mount Sinai, New York, New York
10029, United States; Global Health and Emerging
N-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-2,5-dichloro-N-(4-
chloro-2-methylphenyl)benzenesulfonamide (52). N-(2-(3-(But-3-
yn-1-yl)-3H-diazirin-3-yl)ethyl)-2,5-dichloro-N-(4-chloro-2-
methylphenyl)benzenesulfonamide (52) was synthesized according to
the general procedure, alkylating with 3-(but-3-yn-1-yl)-3-(2-
iodoethyl)-3H-diazirine (6.9 mg, 0.024 mmol, 1.2 equiv) in the
second step.31 The crude mixture was further purified by a silica gel
column and eluted by 20% of ethyl acetate in hexane to afford 6.3 mg
1
of off-white solid as the product, yield 73%. H NMR (CDCl3, 400
MHz): δ ppm 7.75 (t, J = 2.2 Hz, 1H), 7.51−7.40 (m, 2H), 7.22 (s,
1H), 7.07 (dt, J = 8.6, 2.3 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 3.65 (t, J
= 9.2 Hz, 2H), 2.17 (s, 3H), 2.03−1.91 (m, 3H), 1.69 (dt, J = 16.4,
7.3 Hz, 1H), 1.61 (t, J = 7.2 Hz, 2H), 1.56 (s, 1H). 13C NMR
(CDCl3, 101 MHz): δ ppm 140.9, 138.5, 134.8, 134.5, 133.8, 133.3,
N
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Pathogens Institute, Icahn School of Medicine at Mount Sinai,
New York, New York 10029, United States
Michael A. P. Williams − Department of Microbiology, Icahn
School of Medicine at Mount Sinai, New York, New York
10029, United States; Global Health and Emerging
Pathogens Institute, Icahn School of Medicine at Mount Sinai,
New York, New York 10029, United States
Busola R. Alabi − Department of Cell Biology, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Jerry Shay − Department of Cell Biology, University of Texas
Southwestern Medical Center, Dallas, Texas 75390, United
States
HPLC, high-performance liquid chromatography; IC50, 50%
inhibitory concentration; LC−MS, liquid chromatography−
mass spectrometry; M1, matrix protein 1; M2, matrix protein
2; MDCK cells, Madin−Darby Canine Kidney; min, minutes;
MOI, multiplicity of infection; mRNA, messenger RNA; NMR,
nuclear magnetic resonance; NP, nucleoprotein; NS, non-
structural protein; NS1, non-structural protein 1; NS2, non-
structural protein 2; PB1, polymerase basic subunit 1; PB2,
polymerase basic subunit 2; PBS, phosphate buffered saline;
PFA, paraformaldehyde; PFU, plaque forming units; poly(A)
RNA, polyadenylated RNA; ppm, parts per million; qPCR,
quantitative real-time PCR; RBC, red blood cells; RNA-FISH,
RNA fluorescent in situ hybridization; RNAseq, RNA
sequencing; smRNA-FISH, single molecule RNA fluorescent
in situ hybridization; SSC buffer, saline-sodium citrate buffer;
UV, ultraviolet; vRNPs, viral ribonucleoproteins
Hanspeter Niederstrasser − Department of Biochemistry,
University of Texas Southwestern Medical Center, Dallas,
Texas 75390, United States
Bruce Posner − Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States
Adolfo García-Sastre − Department of Microbiology, Icahn
School of Medicine at Mount Sinai, New York, New York
10029, United States; Global Health and Emerging
Pathogens Institute, Department of Medicine, Division of
Infectious Diseases, and The Tisch Cancer Institute, Icahn
School of Medicine at Mount Sinai, New York, New York
10029, United States
REFERENCES
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Joseph Ready − Department of Biochemistry, University of
Texas Southwestern Medical Center, Dallas, Texas 75390,
United States; orcid.org/0000-0003-1305-9581
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00304
Author Contributions
K.W. and M.E. contributed equally to this work. Conceptu-
alization: B.M.A.F. and M.E.; investigation: K.W., M.E., J.L.,
and P.B.; resources: B.M.A.F., A.G.-S., and J.R.; writing (review
and editing): B.M.A.F., K.W., J.R., and J.L.; and funding:
B.M.A.F., A.G.-S., and J.R.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH Grants R33 AI119304-01
(to B.M.A.F. and A.G.-S.), R01AI125524-01 (to B.M.A.F. and
A.G.-S.), and R01 AI154635, and the Welch Foundation (I-
1612, to J.R.). This work was also partly supported by the
Center for Research in Influenza Pathogenesis (CRIP), a
NIAID funded Center of Excellence for Influenza Research
and Surveillance (CEIRS, contract # HHSN272201400008C)
to A.G.-S. We thank Richard Cadagan for excellent technical
assistance.
ABBREVIATIONS
■
A/PR/8/34, Influenza A/Puerto Rico/8/1934; A/WSN/33,
Influenza A/Wisconsin/1933; ATCC, American Type Culture
Collection; BSA, bovine serum albumin; CC50, 50% cytotox-
icity concentration; cDNA, complementary DNA; cmpd,
compound; cRNPs, complementary ribonucleoproteins;
DCM, dichloromethane; DMSO, dimethyl sulfoxide; EMEM,
Eagle’s minimum essential medium; ESI, electrospray ioniza-
tion; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; h, hours; HA, hemagglutinin;
O
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