Disrupting the Conserved Salt Bridge in the Trimerization of Influenza A Nucleoprotein

Article abstract of DOI:10.1021/acs.jmedchem.9b01244

Antiviral drug resistance in influenza infections has been a major threat to public health. To develop a broad-spectrum inhibitor of influenza to combat the problem of drug resistance, we previously identified the highly conserved E339?R416 salt bridge of the nucleoprotein trimer as a target and compound 1 as an inhibitor disrupting the salt bridge with an EC₅₀ = 2.7 μM against influenza A (A/WSN/1933). We have further modified this compound via a structure-based approach and performed antiviral activity screening to identify compounds 29 and 30 with EC₅₀ values of 110 and 120 nM, respectively, and without measurable host cell cytotoxicity. Compared to the clinically used neuraminidase inhibitors, these two compounds showed better activity profiles against drug-resistant influenza A strains, as well as influenza B, and improved survival of influenza-infected mice.

Full text of DOI:10.1021/acs.jmedchem.9b01244

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Article
Disrupting the Conserved Salt Bridge in the
Trimerization of Influenza A Nucleoprotein
Jennifer Woodring, Shao-Hung Lu, Larissa Krasnova, Shih-Chi Wang,
Jhih-Bin Chen, Chiu-Chun Chou, Yi-Chou Huang, Ting-Jen Cheng, Ying-
Ta Wu, Yu-Hou Chen, Jim-Min Fang, Ming-Daw Tsai, and Chi-Huey Wong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01244 • Publication Date (Web): 26 Nov 2019
Downloaded from pubs.acs.org on November 27, 2019
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Disrupting the Conserved Salt Bridge in the Trimerization of Influ-
enza A Nucleoprotein
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Jennifer L. Woodring,† Shao‐Hung Lu,‡, ǁ Larissa Krasnova,† Shih‐Chi Wang,‡ Jhih‐Bin Chen,‡, ǁ Chiu‐
Chun Chou,ǁ Yi‐Chou Huang,ǁ Ting‐Jen Rachel Cheng,‡ Ying‐Ta Wu,‡ Yu‐Hou Chen, Jim‐Min Fang,‡, ǁ
Ming‐Daw Tsai and Chi‐Huey Wong.†, ‡*
†Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United
States.
‡Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
ǁDepartment of Chemistry, National Taiwan University, Taipei 106, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
JLW and SHL had equal contributions
ABSTRACT: Antiviral drug resistance in influenza infections has been a major threat to public health. In order to
develop a broad‐spectrum inhibitor of influenza to combat the problem of drug resistance, we previously identified the
highly conserved E339…R416 salt bridge of the nucleoprotein trimer as a target, and compound 1 as an inhibitor dis‐
rupting the salt bridge with an EC50 = 2.7 µM against influenza A (A/WSN/1933). We have further modified this com‐
pound via a structure‐based approach and performed an antiviral activity screen to identify compounds 29 and 30 with
EC50 values of 110 and 120 nM respectively and without measurable host cell cytotoxicity. Compared to the clinically
used neuraminidase inhibitors, these two compounds showed better activity profiles against drug‐resistant influenza
A strains, as well as influenza B, and improved survival of influenza‐infected mice.
the cytoplasm during viral assembly and matura‐
tion, and has a tail‐loop region that helps the NP
form trimers through the highly conserved
E339…R416 salt bridge (Figure 1A).13‐16 Through
high‐throughput campaigns performed by us and
others, some small molecules were found to inhibit
the influenza RNA polymerase activity by promot‐
ing aggregation of NP17 or by blocking the transport
of NP to the nucleus.18 However, resistance to the in‐
hibitor of RNA polymerase emerges quickly so tar‐
geting the highly conserved salt bridge involved in
NP trimerization would be advantageous. The con‐
firmed hits from our initial primary screening of
anti‐influenza compounds were selected for further
investigation. Through secondary assays and vir‐
tual screening using pharmacophore mapping,
compound 1 was identified as an effective inhibitor
of the E339…R416 salt bridge in the protein‐protein
interaction of influenza NP trimerization.19 It was
 INTRODUCTION
Influenza viruses infect millions of people every
year.1 Although several antiviral drugs and vaccines
targeting specific viral proteins have been devel‐
oped (e.g., the Neuraminidase inhibitors Zanamivir
and Oseltamivir,2 and the cap‐dependent endonu‐
clease inhibitor baloxavir marboxil3), these drugs
are only effective when taken at the onset of infec‐
tion and their use in the clinic has encountered drug
resistance.4‐6 Vaccines require annual renewal and
have limited coverage across various subtypes and
mutations.7 To tackle the problem of drug re‐
sistance in influenza infections, a novel influenza
target called the nucleoprotein (NP) was reported.8‐
12
This protein encapsidates the viral genome, in‐
cluding RNA and RNA polymerase, as well as plays
an essential role in viral replication. It accumulates
in the nucleus during early infection, distributes in
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hypothesized that compound 1 obstructs the guan‐
2B and 2C, and their syntheses are outlined in
idino group of R416 to interact with the carboxyl
group of E339; thus, disrupting the salt bridge to
prevent NP from trimerization (Figure 1B, see Fig‐
ure S1 for the PDB model). Compound 1 (Figure 2A)
has a modest antiviral potency with an EC50 of 2.7
µM yet exhibits MDCK toxicity.19 In order to further
improve the potency and selectivity, we describe
here the identification of the key pharmacophore
associated with the antiviral activity of compound 1
and its optimization via structure‐ and activity‐
based drug discovery approaches to identify new
lead compounds. Given the challenges in developing
effective inhibitors against protein‐protein interac‐
tions and the comprehensive set of inhibitors iden‐
tified in this study, we believe this work represents
a new direction toward the solution of drug re‐
sistance in influenza infection.
Scheme 1, as well as in the Supporting Information.
Of the 9 total libraries, only the compounds with cy‐
clobutenedione (3), cyclic guanidine (5), aniline (7),
amide (8), and propyl‐morpholine substitutions
(10) had a wide range of activities (Table S1‐S11,
and Figure S1). The thiazole replacements (9) and
arginine mimics (4) only gave 1 or 2 compounds, re‐
spectively, with any measurable potency. Neither
the glutamic acid set (2) nor the benzimidazole se‐
ries (6) gave any active compounds. In general,
modifications made to the secondary amine of the
aniline, thiazole ring, amide bond, or propyl chain
did not produce any significant increase in activity,
as compared to compound 1.
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Figure 1.
(A) Molecular modeling of the
E339…R416 salt bridge, which is essential for NP
trimerization, in the protein‐protein interaction
within the tail‐loop region (shown as a green rib‐
bon), and (B) the complex that compound 1 is
docked into the tail‐loop binding pocket.
 RESULTS AND DISCUSSION
Our approach to the structure‐activity‐relation‐
ship (SAR) study of compound 1 focused on two de‐
sign strategies: (1) structure‐based design, where
the arginine and glutamic acid mimics were incor‐
porated into the compound to specifically interact
with the E339 or R416 residues in the salt bridge
formed during influenza NP trimerization, and (2)
activity‐based screening using the virus‐induced
cytopathic effects, where each functional group or
motif of the compound was individually modified
and the antiviral activity of the analogue was deter‐
mined directly through measuring the cell death in‐
duced by infection of influenza A/WSN/1933
(H1N1) virus. For each of these design strategies,
several libraries were pursued, as shown in Figure
Figure 2. (A) Structure and activity of the hit com‐
pound 1 identified by high‐throughput screening,
(B) the 5 libraries pursued for the structure‐based
approach in which the replacements acted as argi‐
nine or glutamic acid mimics to target the
E339…R416 salt bridge, and (C) the 4 libraries pur‐
sued under the activity‐based screening approach
in which each moiety of compound 1 was modified
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and the analogs were analyzed for their impact on
(see Table S1‒S11). From these libraries, we iden‐
5
cell death induced by infection of influenza virus.
tified the best aniline (EC50 <5 µM) and propyl‐mor‐
pholine (EC50 <1 µM) replacements, which were
used to design cross‐over compounds combining
these best moieties in order to further improve the
potency.
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Overall, the aniline (7) and propyl‐morpholine sub‐
stitutions (10) libraries generated more active com‐
pounds than any other sets and were the only series
to have compounds with sub‐micromolar potencies
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Scheme 1. General synthetic schemes.
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Table 1. Potency and host cell cytotoxicity of cross‐over compounds containing motifs from the best aniline
(EC50 = <5 µM) and best morpholine replacement (EC50 = <1 µM) libraries.
Compound
1
X
Cl
Y
Z
H
R
Morpholine
OiPr
EC50 (µM)
2.7
CC50 (µM)
35.6
100
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
11
Cl
H
0.7
12
Cl
H
OEt
0.2
>100
41
13
Cl
Cl
Cl
Cl
H
Morpholine
OiPr
1.5
14
Cl
0.6
>100
>100
>100
>100
>100
47
15
Cl
OEt
0.4
16
H
Morpholine
OiPr
3.1
17
H
H
1.2
18
H
H
OEt
1.3
19
CH3
CH3
CH3
CH3
CH3
CH3
F
H
Morpholine
OiPr
3.7
20
H
1.2
>100
52
21
H
OEt
1.0
22
CH3
CH3
CH3
H
Morpholine
OiPr
3.1
45.7
>100
>100
45
23
1.3
24
OEt
2.3
25
Morpholine
OiPr
3.2
26
F
H
0.5
>100
>100
52
27
F
H
OEt
0.6
28
F
F
Morpholine
OiPr
0.8
29
F
F
0.11
0.12
>100
>100
30
F
F
OEt
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To explore the proper carbon distance for the linker
(Table S7 and Figure S2). Especially, the para‐chlo‐
5
of these two groups, we synthesized five com‐
pounds bearing hydroxyl group with different dis‐
tance chain length (compounds S167‐S171 in Ta‐
ble S10) and showed that only 3‐carbon distance
compound (the compound S168) had the measura‐
ble activity. Furthermore, the linker tailing with ox‐
ygen atom improved the binding affinity (see Table
S10, compounds S172‐S177). In every case, replac‐
ing the morpholine (R) with an ether group im‐
proved the potency. Both outcomes were aligned
with the observation from the molecular model
(Figure 1B), in which the side chain guanidino
group of Arg267 and the backbone alpha‐amino
group of Asp340 create an environment preferring
electronegative hydrogen‐bonding acceptor atoms
at the linker end; the cleft formed by Phe338 and
Tyr487 suggesting a hydrophobic tailing group af‐
ter the 3‐carbon linker. Therefore, a series of ethers
were synthesized in the study of propyl‐morpholine
substitutions. Among the sub‐micromolar com‐
pounds, the ethers in the propyl‐morpholine substi‐
tutions, such as OiPr and OEt groups, were in gen‐
eral obtaining better affinity than compounds with
morpholine, yet exhibited less MDCK host cell cyto‐
toxicity (Table 1 and Table S10). Since the ether re‐
placements resulted in compounds with both
higher potency and selectivity, the ethers were pre‐
ferred over the amines and solely used in the cross‐
over compound series.
rine, but not the mata‐chlorine, on the aniline ring
of Compound 29 and 30 could increase the binding
by forming halogen‐bonding interactions with
Arg389 and Ala387; the two fluorine atoms by
forming weak hydrogen bonding interactions with
Phe304, Trp330, and Ala387 (see Figure S2). It is
noted that the potencies for the aliphatic anilines
improved slightly by a factor of 2‐3 when incorpo‐
rating the ether group (e.g. 16 vs. 17 and 18),
whereas the potencies for the halogen anilines im‐
proved the activity more greatly compared to the
morpholines (e.g. 2.5‐fold for 14 and 3.8‐fold for 15
compared to 13, 7.3‐fold for 29 and 6.7‐fold for 30
compared to 28).
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Because of their high potency and great selectivity
profile, both compounds 29 and 30 were selected
for additional studies. These compounds, along
with compound 1 and three neuraminidase inhibi‐
tors (Oseltamivir, Zanamivir, and Peramivir), were
screened against a panel of 9 influenza strains (Ta‐
ble S12). The comparison of the anti‐influenza ac‐
tivities (pEC50 values) for compound 1, compound
29, and compound 30 to each of the neuraminidase
inhibitors is displayed in the heat map in Figure 3.
In most cases, compound 1 did not perform as well
as the other analogs across the influenza virus
strains, but the two new compounds 29 and 30 had
better potencies than neuraminidase inhibitors for
almost all tested H1N1 influenza virus strains, one
H5N1 recombinant virus and one influenza B strain
virus (denoted by darker shades of blue).
The results for the cross‐over compounds can be
found in Table 1. The chlorine moiety at the 3‐po‐
sition of the aniline (X) could be substituted with a
hydrogen, fluorine, or methyl with little effect on
the potency. Any changes to the chlorine at the 4‐
position (Y) were not tolerated in the assay. If sym‐
metry was created by adding a functional group at
the 5‐position of the aniline ring (Z), it maintained
or improved the potency when compared to the
corresponding aniline without symmetry (as seen
in 1 vs. 13, 19 vs. 22, and 25 vs. 28). In every case,
replacing the morpholine (R) with an isopropyl or
ethyl ether improved the potency. There are two
reasons why replacing the morpholine (R) with an
isopropyl or ethyl ether would improve the potency.
However, for the ether derivatives, only the anilines
with a halogen (F, Cl) and not an aliphatic group (H,
CH3) at the 3‐position, exhibited sub‐micromolar
potencies. Our modeling also indicated that halide
anilines are better than other aryl substitutions
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challenges and recovered gradually after 10 days
post virus‐infection.
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(A)
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Days Post Infection
Days Post Infection
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Days Post Infection
Days Post Infection
Figure 4. Representative virus challenge studies at
2x LD50 A/WSN/1933 (H1N1) for (A) compound 29
as a disulfate salt, and (B) compound 30 as a
dimesylate salt, both with dosing at 20 mg/kg/day
for 5 days. Control mice received phosphate‐buff‐
ered saline containing 40% polyethylene glycol 400
on the same schedule.
Figure 3. Heat map detailing the pEC50 values for
neuraminidase inhibitors Oseltamivir, Zanamivir,
and Peramivir, as well as compound 1, compound
29, and compound 30. Red color denotes low pEC50
values with potencies in the micromolar range,
while the blue color denotes higher pEC50 values
with potencies in the nanomolar range. Gray color
denotes no measurable activity for that influenza
strain.
Docking of compound 29 in the E339…R416 bind‐
ing site (Figure 5) gives a more detailed analysis of
how this compound disrupts the protein‐protein in‐
teraction. The carboxyl group of the Glu339 residue,
which forms the salt bridge with the tail‐loop
Arg416 residue, exerts at least two hydrogen bond‐
ing interactions with both secondary amines of this
compound. As for halogen‐bonding interactions,
the chlorine on the aniline non‐covalently binds
with the Arg389 and Ala387 residues. In compound
29, one fluorine interacts with the Arg389 residues
(with longer distances compared to the interaction
with chlorine), while the other fluorine interacts
with the Phe304 and Trp330 residues (Figure S2).
Additionally, the aromaticity of the thiazole ring
could undergo π‐π stacking with Phe458, and the
new ethyl ether is oriented towards the hydropho‐
bic cleft formed by Phe338 and Tyr487.
In addition to cell‐based assays using a panel of in‐
fluenza virus strains, compounds 29 and 30 were
also tested in an animal virus challenge model to de‐
termine their in vivo efficacy. This virus challenge
model has been successfully used to evaluate the ac‐
tivities of oseltamivir, zanamivir, and their ana‐
logues.20,21,22 The Balb/c mice were subjected to
two times the lethal dose (2x LD50) of the influenza
A/WSN/1933 (H1N1) virus. Due to poor aqueous
solubility, compound 29 was screened as a disulfate
salt and compound 30 as a dimesylate salt. The
compounds were dosed intraperitoneally for 5 days
at concentrations of 20 mg/kg/day. The resulting
survival rates post infection are shown in Figure 4.
The survival rate was extended to 30% for com‐
pound 29 and to 70% for compound 30. Also, the
body weight of the treated mice dropped upon virus
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suggest that compounds 29 and 30 have a signifi‐
cant potential for further development into a new
class of antiviral therapeutics targeting the highly
conserved E339…R416 salt bridge in the trimeriza‐
tion of the influenza nucleoprotein.
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 EXPERIMENTAL SECTION
General Methods for Compound Synthesis. Rea‐
gents were purchased from Alfa Aesar, Acros, Chem
Impex, Combi‐Blocks, Fisher, Oakwood, Sigma‐Al‐
drich, Strem, or TCI chemical companies and used
as received. Solvents were purchased from Acros,
Fisher, Sigma‐Aldrich, or VWR chemical companies
and used as received (no extra drying, distillation or
special handling practices were employed). Thin
layer chromatography (TLC) analyses were per‐
formed on EMD 250 μm Silica Gel 60 F254 plates
and visualized by short wave UV light (λmax = 254
nm), staining only when indicated for individual
compounds. Microwave reactions were performed
on a Biotage Initiator+ system. Flash column chro‐
matography was performed using a Biotage
Isolera™ One flash system with silica gel cartridges:
KP‐Sil (50 μm irregular silica), HP‐Sphere™ (25 mi‐
cron spherical silica) and KP‐C18‐HS. Purest frac‐
tions from chromatography provided the percent
yields reported and any impure fractions were dis‐
carded. These analogs were determined to be
>95% purity by LCMS and NMR analyses prior to bi‐
ological testing.
Figure 5. Modeling of compound 29 involved in
the disruption of the highly conserved E339…R416
salt bridge in the protein‐protein interaction during
NP trimerization (tail‐loop shown as green ribbon).
The influenza nucleoprotein (PDB code 2IQH) was
used. The 3D model of the binding site shows the
halogen‐bonding interactions between the fluorine
and chlorine atoms of the aniline ring with Phe304,
Trp330, Ala387 and Arg389 residues, the hydrogen‐
bond interactions between the Glu339 residue and
the secondary amines of compound 29, and the hy‐
drophobic interaction between the isopropyl ether
portion of the molecule and the well‐defined hydro‐
phobic cleft.
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Further analysis based on NP mass distribution by
analytical ultracentrifuge showed that binding of
compounds 29 and 30 causes inhibition of NP oli‐
gomerization and trimerization, indicating the
same mode of action as compound 1 (Figure S5). In
addition, both compounds 29 and 30 are more ef‐
fective than compound 1 on disruption of NP‐NP in‐
teraction (Figure S5).
Compound Analysis. Optical rotations were ob‐
tained on a Perkin Elmer 341 Polarimeter with a so‐
dium D line (λ = 589 nm) light source and a cylin‐
drical glass cell with path length of 1.0 dm. Melting
points were determined on a Barnstead Electro‐
thermal 1A9300 apparatus. All newly synthesized
compounds were deemed >95% pure by LCMS and
NMR analysis. For MS analysis, low‐resolution
LCMS analysis was performed using an Agilent
1100 series reverse‐phase LC/MSD and Agilent
Zorbax 5 µM C8 4.6 x 50 mm column, with an Ag‐
ilent G1956B single‐quadrupole mass spectrometer
(electrospray ionization positive mode). Mobile
phases consisted of water/0.1% formic acid and ac‐
etonitrile/0.1% formic acid. Gradient started out at
95% aqueous and ramped to 95% organic in 7
minutes. Stop time was set to 10 min, with a four
minute post time. High‐resolution LCMS analysis
 CONCLUSION
In conclusion, the E339…R416 salt bridge of the
nucleoprotein is a novel drug target for the develop‐
ment of inhibitors against the influenza A virus.
From the SAR study of analog 1, we developed com‐
pounds 29 and 30 with a 23‐fold enhancement in
potency and no measurable host cell cytotoxicity ef‐
fect. These two novel compounds were also more
active than three commercially developed neuram‐
inidase inhibitors against a set of mutated influenza
strains and prolonged the survival rate of infected
mice in the animal challenge studies. Our results
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was performed using an Agilent G1969A ESI‐TOF
synthesis (Scheme 1). The respective thiourea
(electrospray ionization – time of flight) mass spec‐
trometer with a capillary voltage of 4000V and 200
µL/min flow rate. Data also acquired in positive
mode. Samples were directly infused, with the mo‐
bile phase set to 90:10 (acetonitrile/0.1% formic
acid: water 0.1% formic acid). NMR spectra were
recorded on Bruker DRX‐500, DRX‐600 and AV‐400
instruments. The chemical shifts (δ) are expressed
in parts per million (ppm) relative to residual sig‐
nals of (CD3)2CO (1H NMR = 2.05 ppm, 13C NMR 29.8,
206.3 ppm), CDCl3 (1H NMR = 7.26 ppm, 13C NMR =
77.2 ppm) CD2Cl2 (1H NMR = 5.32 ppm, 13C NMR
53.8 ppm), (CD3)2NCDO (1H NMR = 2.75, 2.92, 8.03
ppm, 13C NMR 29.8, 34.9, 163.2 ppm), (CD3)2SO (1H
NMR = 2.50 ppm, 13C NMR = 39.5 ppm), and CD3OD
(1H NMR = 3.31 ppm, 13C NMR 49.0 ppm) as internal
standards. Spectra were analyzed using ACD Labs
and MestraNova NMR processing software.
(1.0 equiv) was dissolved in ethanol (0.47 M). To
this solution was added 80% ethyl bromopyruvate
(1.0 equiv) and the reaction proceeded in the mi‐
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crowave for 30 minutes at 150 C. The reaction
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mixture was concentrated under reduced pressure
until dry. To the crude ester compound was added
a 1:1 solution of MeOH and 2M aqueous NaOH (0.2
M) and the mixture stirred at room temperature for
12 hours. The mixture was neutralized to a pH of 5
with 1 M HCl and the resulting precipitate was col‐
lected by vacuum filtration to obtain the desired
product.
General procedure D – HATU amide couplings
(Scheme 1).
In a flask, 2‐((3,4‐dichloro‐
phenyl)amino)thiazole‐4‐carboxylic acid or a simi‐
lar carboxylic acid (1.0 equiv) and HATU (1.0 equiv)
were suspended in dichloromethane (0.10 M).
While stirring, DIPEA (2.0 equiv), and the respec‐
tive amine (1.0 equiv) were added to the suspen‐
sion. The mixture was stirred at room temperature
for 12 hours, diluted with water, and the organic
layer was extracted 3 times with dichloromethane.
The combined organic layers were evaporated un‐
der reduced pressure and purified by reversed‐
phase chromatography (water/acetonitrile) to ob‐
tain the final products. Any variances are denoted
with each compound.
Synthesis and Characterization of Compounds 1,
11 ‒ 30
General procedure A – Synthesis of ethyl 3‐thi‐
oureidocarboxylates (Scheme 1).23 The respec‐
tive aniline (1.0 equiv) was dissolved in anhydrous
acetone (0.15 M) and then ethoxycarbonyl isothio‐
cyanate (1.1 equiv) was added. The mixture was
heated at 40 oC for 20 minutes and then room tem‐
perature for 12 hours. The reaction mixture was
concentrated under reduced pressure until dry.
Then 10 mL of anhydrous cyclohexane was added
and the mixture sonicated. The precipitate was col‐
lected by vacuum filtration to obtain the product.
General procedure B – Synthesis of thioureas
from thioureidocarboxylates (Scheme 1). The
respective thioureidocarboxylate (1.0 equiv) was
dissolved in 10.0 equivalents of a 70% solution of
ethylamine in water. The mixture stirred at room
temperature for 12 hours, diluted with methanol,
and concentrated under reduced pressure until dry.
Then 10 mL of anhydrous cyclohexane was added
and the mixture sonicated. The precipitate was col‐
lected by vacuum filtration to obtain the product.
Alternatively, 50.0 equivalents of 28‐30% ammo‐
nium hydroxide can be used in lieu of aqueous
ethylamine. However, the total reaction time for
ammonium hydroxide was 2‐3 days.
General procedure E – Synthesis of thioureas
from anilines (Scheme 1). To a solution of aniline
(1.0 equiv) in 1M HCl (0.4 M) was added potassium
thiocyanate (4.6 equiv) under argon at room tem‐
perature. The mixture was stirred at 110 oC for 15
hours. The precipitate was filtered and washed with
cold water to afford the desired thiourea, which
was used without further purification.
General procedure
F – Synthesis of 2‐
(amino)thiazole‐4‐carboxylic acids from thiou‐
reas (Scheme 1). To a solution of the thiourea (1
equiv.) in CH3CN (0.3 M) was added bromopyruvic
acid (1 equiv.) under argon at room temperature.
The mixture was stirred at 80 oC for 18 hours. The
precipitate was filtered and washed with cold
CH3CN to afford the desired intermediate which
was used without further purification.
General procedure C – Synthesis of 2‐
(amino)thiazole‐4‐carboxylic acids via ester
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General procedure
G
–
HOBt couplings
vacuum filtration to obtain the final desired prod‐
uct.
(Scheme 1). To a solution of the carboxylic acid
(1.0 equiv) in DMF and N‐methylmorpholine (0.05
M, 1:0.04 DMF:N‐methylmorpholine) were added
1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide
(4.4 equiv) and hydroxybenzotriazole (4.4 equiv)
under argon at room temperature. The mixture was
stirred at room temperature for 10 minutes then
amine (1.0 equiv) was added slowly. The mixture
was stirred at room temperature for 10 hours. After
evaporation in vacuo to remove DMF and N‐methyl‐
morpholine, the reaction mixture was acidified
with 1 M HCl and extracted with water and CH2Cl2.
The aqueous layer was basified with 1 M NaOH and
extracted with CH2Cl2. The organic layer was then
separated, dried over anhydrous magnesium sul‐
fate. After concentrated under reduced pressure,
the residue was purified by SiO2 column chroma‐
tography to afford the amide compound.
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Ethyl
3‐(3,4‐dichlorophenyl)thioureidocar‐
boxylate. Scheme 1, general procedure A, using
3,4‐dichloroaniline. White solid, 98% yield (226.3
mg). Rf = 0.74 (100% DCM). m.p. 125.6‐134.8 oC.
1H NMR (600 MHz, (CD3)2SO) δ ppm 11.54 (s, 1 H),
11.39 (s, 1 H), 8.01 (d, J = 1.5 Hz, 1 H), 7.64 (d, J = 8.7
Hz, 1 H), 7.55 (dd, J = 8.5 Hz, 1.8 Hz, 1 H), 4.21 (q, J
= 6.9 Hz, 2 H), 1.26 (t, J = 7.1 Hz, 3 H). 13C NMR (151
MHz, (CD3)2SO) δ ppm 179.1, 153.3, 138.2, 130.6,
128.1, 126.4, 125.1, 62.1, 14.1. HRMS (ESI‐TOF)
calcd for C10H11Cl2N2O2S: 292.9913, found 292.9912
[M+H]+.
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Ethyl 3‐(3,4,5‐trichlorophenyl)thioureidocar‐
boxy‐late. Scheme 1, general procedure A, using
3,4,5‐dichloroaniline. Off‐white solid, 87% yield
(328.9 mg). Rf = 0.81 (100% DCM). m.p. 141.4‐
1
General procedure H – Amide coupling reac‐
tions with 2‐chlorothiazoles (Scheme 1). In a
flask, 2‐chlorothiazole‐4‐carboxylic acid or similar
2‐chlorothiazole (1.0 equiv) and HATU (1.0 equiv)
were suspended in dichloromethane (0.12 M). To
this mixture was added DIPEA (2.0 equiv) and then
the amine (1.0 equiv). The mixture stirred at room
temperature for 12 hours. The reaction mixture
was diluted with water and extracted with di‐
chloromethane three times. The organic layers
were combined and concentrated under reduced
pressure. The product was purified by reversed‐
phase chromatography (water/acetonitrile).
145.5 oC. H NMR (600 MHz, (CD3)2SO) δ ppm 11.55
(s, 1 H), 11.48 (s, 1 H), 7.97 (s, 2 H), 4.22 (q, J = 7.0
Hz, 2 H), 1.26 (t, J = 7.1 Hz, 3 H). 13C NMR (151 MHz,
(CD3)2SO) δ ppm 179.3, 153.2, 138.3, 132.3, 126.8,
125.5, 62.2, 14.1. HRMS (ESI‐TOF) calcd for
C10H10Cl3N2O2S: 326.9523, found 326.9526 [M+H]+.
Ethyl
3‐(4‐chlorophenyl)thioureidocarbox‐
ylate. Scheme 1, general procedure A, using 4‐
chloroaniline. Pale yellow solid, 91% yield (423.2
mg). Rf = 0.70 (100% DCM). m.p. 121.9‐129.7 oC.
1H NMR (600 MHz, (CD3)2SO) δ ppm 11.51 (s, 1 H),
11.30 (s, 1 H), 7.62 (d, J = 8.5 Hz, 2 H), 7.44 (d, J = 8.5
Hz, 2 H), 4.21 (q, J = 7.1 Hz, 2 H), 1.26 (t, J = 6.9 Hz,
3 H). 13C NMR (151 MHz, (CD3)2SO) δ ppm 178.9,
153.5, 137.1, 130.1, 128.5, 126.4, 62.1, 14.1. HRMS
(ESI‐TOF) calcd for C10H12ClN2O2S: 259.0302, found
259.0303 [M+H]+.
General procedure I – Buchwald coupling reac‐
tions (Scheme 1).24 For each reaction, a mixture of
2‐chloro‐N‐(3‐morpholinopropyl)thiazole‐4‐car‐
boxamide or similar 2‐chlorothiazole (1.0 equiv),
palladium (II) acetate (0.1 equiv), xantphos (0.2
equiv), potassium carbonate (3.0 equiv), the re‐
spective aniline (1.2 equiv), and anhydrous 1,4‐di‐
oxane (0.09 M) was refluxed at 105 oC for 12 hours.
Precipitates were filtered off via vacuum filtration
and discarded. The remaining filtrate was concen‐
trated under reduced pressure and purified by re‐
versed‐phase chromatography (water/acetoni‐
trile). If aniline starting material remained after the
column, the crude product was suspended in cyclo‐
hexane, sonicated, and the precipitate collected by
Ethyl 3‐(4‐chloro‐3‐fluorophenyl)thioureido‐
carboxylate. Scheme 1, general procedure A, us‐
ing 4‐chloro‐3‐fluoroaniline. Pale yellow solid,
99% yield (485.0 mg). Rf = 0.69 (100% DCM). m.p.
o
167.4‐168.9 C. 1H NMR (600 MHz, (CD3)2SO) δ
ppm 11.59 (s, 1 H), 11.39 (s, 1 H), 7.90 (dd, J = 11.1,
2.3 Hz, 1 H), 7.60 (t, J = 8.6 Hz, 1 H), 7.43 (m, 1 H),
4.22 (q, J = 7.2 Hz, 2 H), 1.26 (t, J = 7.1 Hz, 3 H). 13C
NMR (151 MHz, (CD3)2SO) δ ppm 178.9, 156.5 (d, J
= 244.3 Hz), 153.4, 138.6 (d, J = 9.9 Hz), 130.2, 121.7
(d, J = 3.3 Hz), 116.3 (d, J = 17.6 Hz), 112.9 (d, J =
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25.3 Hz), 62.2, 14.1. HRMS (ESI‐TOF) calcd for
C10H11ClFN2O2S: 277.0208, found 277.0209 [M+H]+.
fluorophenyl)thioureidocarboxylate. Pale yellow
solid, 96% yield (653.3 mg). Rf = 0.42 (5% MeOH in
o
DCM). m.p. 164.9‐181.7 C. 1H NMR (600 MHz,
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Ethyl 3‐(4‐chloro‐3,5‐difluorophenyl)thiou‐
reido‐carboxylate. Scheme 1, general procedure
A, using 4‐chloro‐3,5‐difluoroaniline. Brown solid,
89% yield (421.1 mg). Rf = 0.76 (100% DCM). m.p.
(CD3)2SO) δ ppm 10.04 (s, 1 H), 7.79 (br. s, 2 H), 7.82
(dd, J = 11.7, 2.2 Hz, 1 H), 7.48 (t, J = 8.7 Hz, 1 H),
7.23 (ddd, J = 8.7, 2.3, 1.0 Hz, 1 H). 13C NMR (151
MHz, (CD3)2SO) δ ppm 181.2, 156.7 (d, J = 244.3 Hz),
140.1 (d, J = 8.8 Hz), 130.3, 119.4 (d, J = 3.3 Hz),
114.0 (d, J = 17.6 Hz), 110.7 (d, J = 25.3 Hz). HRMS
(ESI‐TOF) calcd for C7H7ClFN2S: 204.9997, found
204.9997 [M+H]+.
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o
148.9‐156.3 C. 1H NMR (600 MHz, (CD3)2SO) δ
ppm 11.65 (s, 1 H), 11.48 (br. s, 1 H), 7.76 (m, 2 H),
4.22 (q, J = 7.2 Hz, 2 H), 1.26 (t, J = 7.2 Hz, 3 H). 13C
NMR (151 MHz, (CD3)2SO) δ ppm 179.0, 157.4 (dd,
J = 247.6, 5.5), 153.3, 138.5 (t, J = 13.2 Hz), 108.7
(m), 105.0 (t, J = 20.9 Hz), 62.2, 14.1. HRMS (ESI‐
TOF) calcd for C10H10ClF2N2O2S: 295.0114, found
295.0115 [M+H]+.
1‐(4‐Chloro‐3,5‐difluorophenyl)thiourea.
Scheme 1, general procedure B, using ethyl 3‐(4‐
chloro‐3,5‐difluorophenyl)thioureidocarboxylate.
Brown solid, 97% yield (279.0 mg). Rf = 0.51 (5%
1
1‐(3,4‐Dichlorophenyl)thiourea. Scheme 1,
general procedure B, using ethyl 3‐(3,4‐dichloro‐
MeOH in DCM). m.p. 159.9‐164.4 oC. H NMR (600
MHz, (CD3)2SO) δ ppm 10.09 (s, 1 H), 8.13 (br. s, 1
H), 7.56 (m, 2 H), 7.48 (br. s, 1 H). 13C NMR (151
MHz, (CD3)2SO) δ ppm 181.2, 157.5 (dd, J = 244.3,
5.5 Hz), 140.1 (t, J = 13.2 Hz), 105.8 (m), 102.1 (t, J
= 22.0 Hz). HRMS (ESI‐TOF) calcd for C7H6ClF2N2S:
222.9903, found 222.9904 [M+H]+.
phenyl)thioureidocarboxylate.
Off‐white solid,
96% yield (77.8 mg). Rf = 0.48 (5% MeOH in DCM).
1
m.p. 214.4‐218.3 oC. H NMR (600 MHz, (CD3)2SO)
δ ppm 9.87 (s, 1 H), 7.91 (d, J = 2.3 Hz, 1 H), 7.99 (br.
s, 1 H), 7.54 (d, J = 8.7 Hz, 1 H), 7.38 (dd, J = 8.7, 2.6
Hz, 1 H), 7.39 (br. s, 1 H). 13C NMR (151 MHz,
(CD3)2SO) δ ppm 181.3, 139.5, 130.6, 130.3, 125.7,
123.9, 122.7. HRMS (ESI‐TOF) calcd for C7H7Cl2N2S:
220.9701, found 220.9702 [M+H]+.
2‐((3,4‐Dichlorophenyl)amino)thiazole‐4‐
carboxylic acid. Scheme 1, general procedure C,
using 1‐(3,4‐dichlorophenyl)thiourea. Off‐white
solid, 97% yield (3.1539 g). Decomposes at 288.9
1‐(3,4,5‐Trichlorophenyl)thiourea. Scheme 1,
general procedure B, using ethyl 3‐(3,4,5‐trichloro‐
phenyl)thioureidocarboxylate. Pale yellow solid,
99% yield (232.1 mg). Rf = 0.58 (5% MeOH in DCM).
1
oC. H NMR (500 MHz, (CD3)2SO) δ ppm 10.76 (br.
s, 1 H), 8.16 (m, 1 H), 7.79 (s, 1 H), 7.58 (m, 2 H). 13C
NMR (151 MHz, (CD3)2SO) δ ppm 162.1, 162.1,
143.4, 140.8, 131.1, 130.7, 122.3, 119.1, 117.8,
117.0. HRMS (ESI‐TOF) calcd for C10H7Cl2N2O2S:
288.9600, found 288.9602 [M+H]+.
1
m.p. 179.5‐181.5 oC. H NMR (600 MHz, (CD3)2SO)
δ ppm 10.00 (s, 1 H), 8.23 (br. s, 1 H), 7.83 (s, 2 H),
7.55 (br. s, 1 H). 13C NMR (151 MHz, (CD3)2SO) δ
ppm 181.3, 139.7, 132.3, 124.0, 122.4. HRMS (ESI‐
TOF) calcd for C7H6Cl3N2S: 254.9312, found
254.9315 [M+H]+.
2‐((3,4,5‐Trichlorophenyl)amino)thiazole‐4‐
carboxylic acid. Scheme 1, general procedure C,
using 1‐(3,4,5‐trichlorophenyl)thiourea.
Light
1‐(4‐Chlorophenyl)thiourea. Scheme 1, gen‐
eral procedure B, using ethyl 3‐(4‐chloro‐
phenyl)thioureidocarboxylate. Pale yellow solid,
98% yield (665.4 mg). Rf = 0.55 (5% MeOH in DCM).
brown solid, 99% yield (585.5 mg). Decomposes at
1
271.8 oC. H NMR (600 MHz, (CD3)2SO) δ ppm 12.82
(br. s, 1 H), 11.26 (s, 1 H), 8.02 (s, 2 H), 7.82 (s, 1 H).
13C NMR (151 MHz, (CD3)2SO) δ ppm 161.9, 161.8,
143.3, 140.7, 132.9, 120.7, 119.8, 116.8. HRMS
(ESI‐TOF) calcd for C10H6Cl3N2O2S: 322.9210, found
322.9211 [M+H]+.
1
m.p. 174.7‐178.9 oC. H NMR (600 MHz, (CD3)2SO)
δ ppm 9.83 (s, 1 H), 7.59 (m, 2 H), 7.49 (m, 2 H), 7.37
(m, 2 H). 13C NMR (151 MHz, (CD3)2SO) δ ppm 181.2,
138.3, 128.4, 128.0, 124.5. HRMS (ESI‐TOF) calcd
for C7H8ClN2S: 187.0091, found 187.0092 [M+H]+.
Scheme 1, general procedure E and F, using 3,4,5‐
trichloroaniline. White solid, 81% yield (1.3 g).
1‐(4‐Chloro‐3‐fluorophenyl)thiourea. Scheme
1, general procedure B, using ethyl 3‐(4‐chloro‐3‐
2‐((4‐Chlorophenyl)amino)thiazole‐4‐carbox‐
ylic acid. Scheme 1, general procedure C, using 1‐
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(4‐chlorophenyl)thiourea. Brown solid, 76% yield
(289.1 mg). Decomposes at 242.7 oC. H NMR (600
MHz, (CD3)2SO) δ ppm 12.73 (br. s, 1 H), 10.50 (s, 1
H), 7.74 (s, 1 H), 7.71 (m, 2 H), 7.37 (m, 2 H). 13C
NMR (151 MHz, (CD3)2SO) δ ppm 162.5, 162.1,
143.4, 139.7, 128.8, 124.8, 118.6, 118.4. HRMS
(ESI‐TOF) calcd for C10H8ClN2O2S: 254.9990, found
254.9991 [M+H]+.
2‐Chloro‐N‐(3‐isopropoxypropyl)thiazole‐4‐
carbo‐xamide. Scheme 1, general procedure H,
using 2‐chlorothiazole‐4‐carboxylic acid and 3‐iso‐
propoxypropylamine. Yellow oil, 74% yield (545.7
mg). Rf = 0.57 (5% MeOH in DCM). 1H NMR (500
MHz, CDCl3) δ ppm 7.97 (s, 1 H), 7.92 (br. s, 1 H),
3.58 (m, 5 H), 1.86 (quint, J = 5.9 Hz, 2 H), 1.22 (d, J
= 6.2 Hz, 6 H). 13C NMR (151 MHz, CDCl3) δ ppm
159.7, 151.4, 148.8, 124.4, 71.9, 67.2, 38.6, 29.1,
22.0. HRMS (ESI‐TOF) calcd for C10H16ClN2O2S:
263.0615, found 263.0617 [M+H]+.
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Scheme 1, general procedure E and F, using 4‐
chloroaniline. White solid, 96% yield (437 mg).
2‐((4‐Chloro‐3‐fluorophenyl)amino)thiazole‐
4‐carboxylic acid. Scheme 1, general procedure C,
using 1‐(4‐chloro‐3‐fluorophenyl)thiourea. Brown
solid, 88% yield (405.9 mg). Decomposes at 233.9
2‐Chloro‐N‐(3‐ethoxypropyl)thiazole‐4‐car‐
box‐amide. Scheme 1, general procedure H, using
2‐chlorothiazole‐4‐carboxylic acid and 3‐ethoxy‐
propylamine. Off‐white solid, 78% yield (547.3
mg). Rf = 0.36 (5% MeOH in DCM). m.p. 76.7‐79.4
1
oC. H NMR (600 MHz, (CD3)2SO) δ ppm 12.73 (br.
s, 1 H), 10.98 (br. s, 1 H), 8.02 (dd, J = 12.2, 2.2 Hz, 1
H), 7.78 (s, 1 H), 7.49 (t, J = 8.7 Hz, 1 H), 7.37 (d, J =
8.7 Hz, 1 H). 13C NMR (151 MHz, (CD3)2SO) δ ppm
162.1, 162.0, 157.2 (d, J = 243.2 Hz), 143.4, 141.3 (d,
J = 11.0 Hz), 130.5, 119.2, 113.9 (d, J = 3.3 Hz), 110.4
(d, J = 17.6 Hz), 104.9 (d, J = 26.4 Hz). HRMS (ESI‐
TOF) calcd for C10H7ClFN2O2S: 272.9895, found
272.9897 [M+H]+.
1
oC. H NMR (500 MHz, CDCl3) δ ppm 7.97 (s, 1 H),
7.82 (br. s, 1 H), 3.54 (m, 6 H), 1.87 (quin, J = 5.9 Hz,
2 H), 1.28 (t, J = 7.0 Hz, 3 H). 13C NMR (151 MHz,
CDCl3) δ ppm 159.7, 151.4, 148.7, 124.5, 69.6, 66.5,
38.3, 29.0, 15.2. HRMS (ESI‐TOF) calcd for
C9H14ClN2O2S: 249.0459, found 249.0457 [M+H]+.
2‐((4‐Chloro‐3,5‐difluorophenyl)amino)thia‐
zole‐4‐carboxylic acid. Scheme 1, general proce‐
dure C, using 1‐(4‐chloro‐3,5‐difluorophenyl)thiou‐
rea. Light brown solid, 96% yield (4.7515 g). De‐
N‐(3‐Morpholinopropyl)‐2‐(3,4‐dichloro‐
phenyl‐amino)thiazole‐4‐carboxamide
Scheme 1, general procedure G, using 2‐((3,4‐di‐
chlorophenyl)amino)thiazole‐4‐carboxylic acid
(1).
1
composes at 290.6 oC. H NMR (600 MHz, (CD3)2SO)
and 3‐morpholinopropylamine. Column chroma‐
tography (MeOH/CH2Cl2, 1:39 to 1:19). White solid,
72% yield (299 mg). Rf = 0.33 (MeOH/CH2Cl2 =
1:19). m.p. 108–109 oC. IR vmax (neat) 2920, 2850,
δ ppm 12.85 (br. s, 1 H), 11.50 (s, 1 H), 7.82 (s, 1 H),
7.71 (d, J = 10.0 Hz, 2 H). 13C NMR (151 MHz,
(CD3)2SO) δ ppm 161.9 (d, J = 8.8 Hz), 158.9 (d, J =
5.5 Hz), 157.3 (d, J = 5.5 Hz), 143.2, 141.1 (t, J = 13.8
Hz), 119.7, 100.7 (d, J = 28.6 Hz), 98.9 (t, J = 22.0 Hz).
HRMS (ESI‐TOF) calcd for C10H6ClF2N2O2S:
290.9801, found 290.9802 [M+H]+.
1
1620, 1550, 1475 cm−1. H NMR (600 MHz, CDCl3)
δ ppm 9.34 (s, 1 H), 7.76 (d, J = 2.4 Hz, 1 H), 7.72 (t,
J = 6.6 Hz, 1 H), 7.40 (s, 1 H), 7.33 (dd, J = 9.0, 2.4 Hz,
1 H), 7.28 (d, J = 9.0 Hz. 1 H), 3.61 (t, J = 4.2 Hz, 4 H),
3.49 (q, J = 6.6 Hz, 2 H), 2.41–2.37 (m, 6 H), 1.74
(quint, J = 6.6 Hz, 2 H). 13C NMR (150 MHz, CDCl3) δ
ppm 163.2, 161.6, 145.6, 139.9, 132.5, 130.4, 124.9,
119.2, 117.0, 113.6, 66.6 (× 2), 56.7, 53.5 (× 2), 38.3,
2‐Chloro‐N‐(3‐morpholinopropyl)thiazole‐4‐
carbo‐xamide. Scheme 1, general procedure H,
using 2‐chlorothiazole‐4‐carboxylic acid and N‐(3‐
aminopropyl)morpholine. Pale orange solid, 71%
yield (697.0 mg). Rf = 0.43 (5% MeOH in DCM). m.p.
25.8.
HRMS (ESI) calcd for C17H21Cl2N4O2S:
415.0757, found: 415.0837 [M + H]+.
1
75.8‐78.6 oC. H NMR (600 MHz, CDCl3) δ ppm 8.72
(br. s, 1 H), 7.98 (s, 1 H), 3.87 (t, J = 4.6 Hz, 4 H), 3.55
(q, J = 5.9 Hz, 2 H), 2.54 (m, 6 H), 1.79 (quint, J = 6.1
Hz, 2 H). 13C NMR (151 MHz, CDCl3) δ ppm 159.9,
151.5, 148.8, 124.5, 66.6, 58.4, 53.8, 39.8, 24.4.
HRMS (ESI‐TOF) calcd for C11H17ClN3O2S: 290.0724,
found 290.0726 [M+H]+.
2‐((3,4‐Dichlorophenyl)amino)‐N‐(3‐iso‐
propoxy‐propyl)thiazole‐4‐carboxamide (11).
Scheme 1, general procedure D, using 2‐((3,4‐di‐
chlorophenyl)amino)thiazole‐4‐carboxylic
acid
and 3‐isopropoxypropylamine. Light orange solid,
23% yield (66.9 mg). Rf = 0.51 (5% MeOH in DCM).
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o
m.p. 119.7‐124.1 C. 1H NMR (600 MHz, CDCl3) δ
ppm 8.28 (br.s., 1 H), 7.70 (d, J = 2.6 Hz, 1 H), 7.56 (t,
J = 5.8 Hz, 1 H), 7.47 (s, 1 H), 7.39 (d, J = 8.7 Hz, 1 H),
7.34 (dd, J = 9.0, 2.6 Hz, 1 H), 3.58 (m, 5 H), 1.88
(quint, J = 6.2 Hz, 2 H), 1.15 (d, J = 6.2 Hz, 6 H). 13C
NMR (151 MHz, CDCl3) δ ppm 163.1, 161.4, 146.1,
139.7, 132.9, 130.8, 125.7, 119.6, 117.3, 113.5, 71.8,
66.3, 37.8, 29.7, 22.1. HRMS (ESI‐TOF) calcd for
C16H20Cl2N3O2S: 388.0648, found 388.0651 [M+H]+.
ppm 8.33 (br. s, 1 H), 7.64 (s, 2 H), 7.56 (m, 1 H),
7.51 (s, 1 H), 3.58 (m, 5 H), 1.90 (quint, J = 6.2 Hz, 2
H), 1.15 (d, J = 5.9 Hz, 6 H). 13C NMR (151 MHz,
CDCl3) δ ppm 162.5, 161.3, 146.2, 139.4, 134.4,
124.4, 117.8, 114.0, 71.8, 66.3, 37.9, 29.6, 22.1.
HRMS (ESI‐TOF) calcd C16H19Cl3N3O2S: 422.0258,
found 422.0265 [M+H]+.
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13
14
15
16
17
18
19
20
21
22
23
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N‐(3‐Ethoxypropyl)‐2‐((3,4,5‐trichlorophenyl)
amino)thiazole‐4‐carboxamide (15). Scheme 1,
general procedure D, using 2‐((3,4,5‐trichloro‐
phenyl)amino)thiazole‐4‐carboxylic acid and 3‐
ethoxypropylamine. Light brown solid, 25% yield
(52.6 mg). Rf = 0.46 (5% MeOH in DCM). m.p.
2‐((3,4‐Dichlorophenyl)amino)‐N‐(3‐ethoxy‐
propyl) thiazole‐4‐carboxamide (12). Scheme 1,
general procedure D, using 2‐((3,4‐dichloro‐
phenyl)amino)thiazole‐4‐carboxylic acid and 3‐
ethoxypropylamine. Off‐white solid, 37% yield
(109.9 mg). Rf = 0.51 (5% MeOH in DCM). m.p.
o
171.6‐177.0 C. 1H NMR (600 MHz, (CD3)2CO) δ
ppm 9.76 (br. s, 1 H), 7.92 (s, 2 H), 7.79 (m, 1 H),
7.55 (s, 1 H), 3.52 (t, J = 6.0 Hz, 2 H), 3.46 (m, 4 H),
1.84 (quint, J = 6.4 Hz, 2 H), 1.11 (t, J = 6.9 Hz, 3 H).
13C NMR (151 MHz, (CD3)2CO) δ ppm 153.5, 151.5,
138.1, 131.7, 124.7, 113.3, 108.5, 104.8, 59.4, 56.9,
139.1‐143.7 C. 1H NMR (600 MHz, CDCl3) δ ppm
o
8.42 (br. s, 1 H), 7.72 (d, J = 2.3 Hz, 1 H), 7.59 (t, J =
5.7 Hz, 1 H), 7.47 (s, 1 H), 7.38 (m, 2 H), 3.58 (m, 4
H), 3.51 (q, J = 7.0 Hz, 2 H), 1.90 (quint, J = 6.2 Hz, 2
H), 1.19 (t, J = 6.9 Hz, 3 H). 13C NMR (151 MHz, CDCl3)
δ ppm 163.1, 161.4, 146.1, 139.7, 132.9, 130.7,
125.6, 119.5, 117.2, 113.5, 68.9, 66.4, 37.8, 29.4,
15.1. HRMS (ESI‐TOF) calcd for C15H18Cl2N3O2S:
374.0491, found 374.0495 [M+H]+.
28.0, 20.8, 5.7.
HRMS (ESI‐TOF) calcd for
C15H17Cl3N3O2S: 408.0102, found 408.0101 [M+H]+.
2‐(4‐Chlorophenylamino)‐N‐(3‐morpho‐
linopropyl)
thiazole‐4‐carboxamide
(16).
Scheme 1, general procedure G, using 2‐((4‐chloro‐
phenyl)amino)thiazole‐4‐carboxylic acid and N‐(3‐
aminopropyl)morpholine. Silica gel column chro‐
matography (CH2Cl2/2.5‐5% MeOH). White solid,
72% yield (299 mg). Rf = 0.31 (MeOH/CH2Cl2, 1:19).
2‐(3,4,5‐Trichlorophenylamino)‐N‐(3‐
morpholino‐propyl)thiazole‐4‐carboxamide
(13). Scheme 1, general procedure G, using 2‐
((3,4,5‐trichlorophenyl)amino)thiazole‐4‐
1
carboxylic acid and N‐(3‐aminopropyl)morpholine.
Silica gel column chromatography (CH2Cl2/2.5‐5%
MeOH). White solid, 79% yield (708 mg). Rf = 0.34
m.p. 130–131 oC. H NMR (600 MHz, CDCl3) δ ppm
8.94 (s, 1 H), 7.73 (t, J = 6.6 Hz, 1 H), 7.41 (d, J = 8.4
Hz, 2 H), 7.21 (d, J = 8.4 Hz, 2 H), 3.63 (t, J = 4.2 Hz,
4 H), 3.46 (q, J = 6.6 Hz, 2 H), 2.38 (m, 6 H), 1.72
(quint, J = 6.6 Hz, 2 H). 13C NMR (150 MHz, CDCl3) δ
ppm 163.9, 161.6, 145.7, 138.9, 129.0, 127.3, 119.3,
113.0, 66.6, 56.7, 53.5, 38.2, 25.8. HRMS (ESI‐TOF)
calcd for C17H22ClN4O2S: 381.1147, found 381.1143
[M+H]+.
(MeOH/CH2Cl2, 1:19). m.p. 164–165 C. 1H NMR
o
(600 MHz, CDCl3) δ ppm 9.07 (s, 1 H), 7.75 (t, J = 6.0
Hz, 1 H), 7.65 (s, 2 H), 7.46 (s, 1 H), 3.66 (m, 4 H),
3.54 (q, J = 6.0 Hz, 2 H), 2.45 (m, 6 H), 1.79 (quint, J
= 6.0 Hz, 2 H). 13C NMR (150 MHz, CDCl3) δ ppm
162.7, 161.5, 145.9, 139.6, 134.2, 124.0, 117.6,
114.2, 66.8, 57.0, 53.7, 38.5, 25.8. HRMS (ESI‐TOF)
calcd for C17H20Cl3N4O2S: 449.0367, found 449.0376
[M+H]+.
2‐((4‐Chlorophenyl)amino)‐N‐(3‐isopropoxy‐
propyl) thiazole‐4‐carboxamide (17). Scheme 1,
general procedure D, using 2‐((4‐chloro‐
phenyl)amino)thiazole‐4‐carboxylic acid and 3‐iso‐
propoxypropylamine. Off‐white solid, 28% yield
(110.3 mg). Rf = 0.54 (5% MeOH in DCM). m.p.
N‐(3‐Isopropoxypropyl)‐2‐((3,4,5‐trichloro‐
phenyl)amino)thiazole‐4‐carboxamide
Scheme 1, general procedure D, using 2‐((3,4,5‐tri‐
chlorophenyl)amino)thiazole‐4‐carboxylic acid
(14).
o
148.7‐151.8 C. 1H NMR (600 MHz, CDCl3) δ ppm
and 3‐isopropoxypropylamine. Light red solid,
7.80 (s, 1 H), 7.54 (t, J = 5.5 Hz, 1 H), 7.44 (s, 1 H),
7.39 (m, 2 H), 7.31 (m, 2 H), 3.57 (m, 5 H), 1.87
(quint, J = 6.2 Hz, 2 H), 1.17 (d, J = 6.2 Hz, 6 H). 13C
53% yield (168.5 mg). Rf = 0.51 (5% MeOH in DCM).
o
m.p. 173.9‐178.5 C. 1H NMR (600 MHz, CDCl3) δ
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NMR (151 MHz, CDCl3) δ ppm 163.8, 161.4, 146.2,
6.2 Hz, 6 H). 13C NMR (151 MHz, (CD3)2CO) δ ppm
5
138.7, 129.4, 128.1, 119.6, 112.9, 71.7, 66.4, 37.7,
29.7, 22.1. HRMS (ESI‐TOF) calcd for C16H21ClN3O2S
[M+H]+: 354.1037, found 354.1040.
154.5, 151.7, 138.0, 130.9, 127.3, 120.3, 117.4,
111.0, 107.7, 103.4, 62.2, 56.9, 28.0, 21.1, 12.6, 10.5.
HRMS (ESI‐TOF) calcd for C17H23ClN3O2S: 368.1194,
found 368.1194 [M+H]+.
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2‐((4‐Chlorophenyl)amino)‐N‐(3‐ethoxypro‐
pyl) thiazole‐4‐carboxamide (18). Scheme 1,
general procedure D, using 2‐((4‐chloro‐
phenyl)amino)thiazole‐4‐carboxylic acid and 3‐
2‐((4‐Chloro‐3‐methylphenyl)amino)‐N‐(3‐
ethoxypropyl)thiazole‐4‐carboxamide(21).
Scheme 1, general procedure I, using 2‐chloro‐N‐(3‐
ethoxypropylamine.
Orange solid, 49% yield
ethoxypropyl)thiazole‐4‐carboxamide
and
4‐
(126.2 mg). Rf = 0.21 (5% MeOH in DCM). m.p.
chloro‐3‐methylaniline. Orange solid, 33% yield
o
138.2‐142.5 C. 1H NMR (600 MHz, (CD3)2CO) δ
(91.8 mg). Rf = 0.53 (5% MeOH in DCM). m.p.
o
ppm 9.62 (br. s, 1 H), 7.84 (br. s, 1 H), 7.76 (m, 2 H),
7.47 (s, 1 H), 7.30 (m, 2 H), 3.50 (t, J = 6.0 Hz, 2 H),
3.46 (m, 4 H), 1.83 (quint, J = 6.5 Hz, 2 H), 1.13 (t, J
= 6.9 Hz, 3 H). 13C NMR (151 MHz, (CD3)2SO) δ ppm
162.7, 160.5, 146.1, 139.7, 128.7, 124.7, 118.7,
113.2, 67.9, 65.4, 36.6, 29.6, 15.1. HRMS (ESI‐TOF)
calcd for C15H19ClN3O2S: 340.0881, found 340.0881
[M+H]+.
108.8‐112.6 C. 1H NMR (500 MHz, (CD3)2CO) δ
ppm 9.55 (br. s, 1 H), 7.85 (m, 1 H), 7.65 (dd, J = 8.6,
2.4 Hz, 1 H), 7.59 (s, 1 H), 7.47 (s, 1 H), 7.27 (d, J =
8.7 Hz, 1 H), 3.51 (t, J = 6.2 Hz, 2 H), 3.46 (m, 4 H),
2.34 (s, 3 H), 1.84 (quint, J = 6.4 Hz, 2 H), 1.12 (t, J =
6.9 Hz, 3 H). 13C NMR (151 MHz, (CD3)2CO) δ ppm
164.4, 161.8, 147.8, 140.8, 137.2, 130.2, 127.3,
120.8, 117.6, 113.4, 69.4, 66.7, 38.0, 30.7, 20.4, 15.6.
HRMS (ESI‐TOF) calcd for C16H21ClN3O2S: 354.1037,
found 354.1038 [M+H]+.
2‐((4‐Chloro‐3‐methylphenyl)amino)‐N‐(3‐
morpholinopropyl)thiazole‐4‐carboxamide
(19). Scheme 1, general procedure I, using 2‐
chloro‐N‐(3‐morpholinopropyl)thiazole‐4‐carbox‐
amide and 4‐chloro‐3‐methylaniline. Red solid,
45% yield (99.2 mg). Rf = 0.38 (5% MeOH in DCM).
m.p. 67.8‐79.4 oC. 1H NMR (600 MHz, (CD3)2CO) δ
ppm 9.59 (br. s, 1 H), 7.92 (t, J = 5.3 Hz, 1 H), 7.65
(dd, J = 8.7, 2.6 Hz, 1 H), 7.56 (d, J = 2.3 Hz, 1 H), 7.49
(s, 1 H), 7.27 (d, J = 8.5 Hz, 1 H), 3.58 (m, 4 H), 3.45
(q, J = 6.7 Hz, 2 H), 2.39 (m, 6 H), 2.33 (s, 3 H), 1.77
(quin, J = 6.9 Hz, 2 H). 13C NMR (151 MHz, (CD3)2CO)
δ ppm 164.5, 161.9, 147.7, 140.8, 137.2, 130.2,
127.4, 120.9, 117.7, 113.6, 67.4, 57.5, 54.7, 38.6,
27.2, 20.4. HRMS (ESI‐TOF) calcd for C18H24ClN4O2S:
395.1303, found 395.1305 [M+H]+.
2‐((4‐Chloro‐3,5‐dimethylphenyl)amino)‐N‐
(3‐morpholinopropyl)thiazole‐4‐carboxamide
(22). Scheme 1, general procedure I, using 2‐
chloro‐N‐(3‐morpholinopropyl)thiazole‐4‐carbox‐
amide and 4‐chloro‐3,5‐dimethylaniline. Pale yel‐
low solid, 34% yield (79.5 mg). Rf = 0.19 (5% MeOH
1
in DCM). m.p. 137.5‐142.3 oC. H NMR (600 MHz,
CDCl3) δ ppm 7.87 (m, 1 H), 7.53 (br. s, 1 H), 7.43 (s,
1 H), 7.10 (s, 2 H), 3.75 (m, 4 H), 3.52 (q, J = 6.3 Hz,
2 H), 2.47 (m, 6 H), 2.38 (s, 6 H), 1.78 (quint, J = 6.6,
2 H). 13C NMR (151 MHz, CDCl3) δ ppm 164.6, 161.4,
146.1, 137.7, 137.4, 129.3, 118.8, 112.6, 66.8, 57.3,
53.7, 38.6, 26.8, 21.0. HRMS (ESI‐TOF) calcd for
C19H26ClN4O2S: 409.1459, found 409.1458 [M+H]+.
2‐((4‐Chloro‐3‐methylphenyl)amino)‐N‐(3‐
isopropoxypropyl)thiazole‐4‐carboxamide
(20). Scheme 1, general procedure I, using 2‐
chloro‐N‐(3‐isopropoxypropyl)thiazole‐4‐carbox‐
amide and 4‐chloro‐3‐methylaniline. Orange solid,
33% yield (95.4 mg). Rf = 0.49 (5% MeOH in DCM).
2‐((4‐Chloro‐3,5‐dimethylphenyl)amino)‐N‐
(3‐isopropoxypropyl)thiazole‐4‐carboxamide
(23). Scheme 1, general procedure I, using 2‐
chloro‐N‐(3‐isopropoxypropyl)thiazole‐4‐carbox‐
amide and 4‐chloro‐3,5‐dimethylaniline. Pale yel‐
low solid, 51% yield (93.0 mg). Rf = 0.28 (5% MeOH
1
1
m.p. 127.0‐130.9 oC. H NMR (600 MHz, (CD3)2CO)
in DCM). m.p. 147.7‐151.0 oC. H NMR (500 MHz,
δ ppm 9.48 (br. s, 1 H), 7.79 (t, J = 5.1 Hz, 1 H), 7.64
(dd, J = 8.7, 2.6 Hz, 1 H), 7.59 (d, J = 2.3 Hz, 1 H), 7.46
(s, 1 H), 7.27 (d, J = 8.7 Hz, 1 H), 3.55 (spt, J = 6.2 Hz,
1 H), 3.51 (t, J = 6.0 Hz, 2 H), 3.46 (q, J = 6.7 Hz, 2 H),
2.34 (s, 3 H), 1.82 (quint, J = 6.4 Hz, 2 H), 1.09 (d, J =
(CD3)2CO) δ ppm 9.35 (br. s, 1 H), 7.74 (m, 1 H), 7.49
(s, 2 H), 7.44 (s, 1 H), 3.55 (m, 3 H), 3.46 (q, J = 6.7
Hz, 2 H), 2.35 (s, 6 H), 1.83 (quint, J = 6.4 Hz, 2 H),
1.09 (d, J = 6.2 Hz, 6 H). 13C NMR (151 MHz,
(CD3)2CO) δ ppm 164.5, 161.7, 147.9, 140.1, 137.5,
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127.9, 118.7, 113.1, 72.1, 66.9, 38.0, 31.0, 22.5, 21.1.
106.3 (d, J = 26.4 Hz), 71.8, 66.2, 37.8, 29.7, 22.0.
HRMS (ESI‐TOF) calcd for C18H25ClN3O2S: 382.1350,
found 382.1350 [M+H]+.
HRMS (ESI‐TOF) calcd for C16H20ClFN3O2S:
372.0943, found 372.0948 [M+H]+.
6
7
8
9
2‐((4‐Chloro‐3,5‐dimethylphenyl)amino)‐N‐
(3‐ethoxypropyl)thiazole‐4‐carboxamide (24).
Scheme 1, general procedure I, using 2‐chloro‐N‐(3‐
2‐((4‐Chloro‐3‐fluorophenyl)amino)‐N‐(3‐
ethoxypropyl)thiazole‐4‐carboxamide(27).
Scheme 1, general procedure D, using 2‐((4‐chloro‐
3‐fluorophenyl)amino)thiazole‐4‐carboxylic acid
and 3‐ethoxypropylamine. Orange solid, 57% yield
(166.1 mg). Rf = 0.25 (5% MeOH in DCM). m.p.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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60
ethoxypropyl)thiazole‐4‐carboxamide
and
4‐
chloro‐3,5‐dimethylaniline. Pale yellow solid, 29%
yield (53.8 mg). Rf = 0.49 (5% MeOH in DCM). m.p.
o
O
137.1‐142.0 C. 1H NMR (500 MHz, (CD3)2CO) δ
150.6‐154.8 C. 1H NMR (600 MHz, (CD3)2SO) δ
ppm 9.35 (br. s, 1 H), 7.76 (m, 1 H), 7.50 (s, 2 H),
7.44 (s, 1 H), 3.53 (t, J = 6.2 Hz, 2 H), 3.46 (m, 4 H),
2.35 (s, 6 H), 1.85 (quint, J = 6.4 Hz, 2 H), 1.11 (t, J =
6.9 Hz, 3 H). 13C NMR (151 MHz, (CD3)2CO) δ ppm
164.5, 161.6, 147.9, 140.1, 137.5, 127.9, 118.7,
113.1, 69.4, 66.7, 37.9, 30.6, 21.1, 15.6. HRMS (ESI‐
TOF) calcd for C17H23ClN3O2S: 368.1194, found
368.1192 [M+H]+.
ppm 10.63 (br. s, 1 H), 8.19 (t, J = 5.9 Hz, 1 H), 7.93
(dd, J = 12.1, 2.8 Hz, 1 H), 7.55 (s, 1 H), 7.45 (t, J = 8.5
Hz, 1 H), 7.38 (dd, J = 8.9, 2.2 Hz, 1 H), 3.42 (m, 4 H),
3.32 (q, J = 6.4 Hz, 2 H), 1.76 (quint, J = 6.6 Hz, 2 H),
1.09 (t, J = 7.1 Hz, 3 H). 13C NMR (151 MHz, (CD3)2SO)
δ ppm 162.3, 160.5, 157.3 (d, J = 244.3 Hz), 146.3,
141.1 (d, J = 9.9 Hz), 130.5, 114.1 (d, J = 3.3 Hz),
113.8, 110.6 (d, J = 17.6 Hz), 105.2 (d, J = 26.4 Hz),
67.9, 65.4, 36.6, 29.5, 15.1. HRMS (ESI‐TOF) calcd
for C15H18ClFN3O2S: 358.0787, found 358.0788
[M+H]+.
2‐((4‐Chloro‐3‐fluorophenyl)amino)‐N‐(3‐
morpholinopropyl)thiazole‐4‐carboxamide
(25). Scheme 1, general procedure I, using 2‐
chloro‐N‐(3‐morpholinopropyl)thiazole‐4‐carbox‐
amide and 4‐chloro‐3‐fluoroaniline. Dark orange
solid, 48% yield (81.3 mg). Rf = 0.35 (5% MeOH in
2‐((4‐Chloro‐3,5‐difluorophenyl)amino)‐N‐
(3‐morpholinopropyl)thiazole‐4‐carboxamide
(28). Scheme 1, general procedure D, using 2‐((4‐
chloro‐3,5‐difluorophenyl)amino)thiazole‐4‐car‐
boxylic acid and N‐(3‐aminopropyl)morpholine.
Orange solid, 61% yield (145.6 mg). Rf = 0.43 (5%
o
DCM). m.p. 58.0‐69.3 C. 1H NMR (600 MHz,
(CD3)2CO) δ ppm 9.80 (br. s, 1 H), 7.97 (br. s, 1 H),
7.89 (dd, J = 11.7, 2.2 Hz, 1 H), 7.54 (s, 1 H), 7.40 (m,
2 H), 3.59 (m, 4 H), 3.45 (q, J = 6.7 Hz, 2 H), 2.40 (m,
6 H), 1.78 (quint, J = 6.7 Hz, 2 H). 13C NMR (151 MHz,
(CD3)2CO) δ ppm 163.9, 161.6, 158.1 (d, J = 244.3
Hz), 148.0, 142.4 (d, J = 12.1 Hz), 131.5, 115.2 (d, J =
3.3 Hz), 114.3, 112.8 (d, J = 17.6 Hz), 106.6 (d, J =
26.4 Hz), 67.4, 57.5, 54.7, 38.5, 27.3. HRMS (ESI‐
TOF) calcd for C17H21ClFN4O2S: 399.1052, found
399.1049 [M+H]+.
1
MeOH in DCM). m.p. 139.9‐146.3 oC. H NMR (600
MHz, (CD3)2CO) δ ppm 8.01 (m, 1 H), 7.57 (m, 3 H),
3.58 (m, 4 H), 3.45 (q, J = 6.9 Hz, 2 H), 2.40 (m, 6 H),
1.77 (quint, J = 6.9 Hz, 2 H). 13C NMR (151 MHz,
(CD3)2CO) δ ppm 163.5, 161.5 (m), 160.6 (d, J = 5.5
Hz), 159.0 (d, J = 5.5 Hz), 148.1, 142.1 (t, J = 13.2 Hz),
114.8, 102.2 (m), 102.0 (m), 101.3 (t, J = 22.0 Hz),
67.5, 57.5, 54.7, 38.5, 27.3. HRMS (ESI‐TOF) calcd
for C17H20ClF2N4O2S: 417.0958, found 417.0958
[M+H]+.
2‐((4‐Chloro‐3‐fluorophenyl)amino)‐N‐(3‐iso‐
propoxypropyl)thiazole‐4‐carboxamide(26).
Scheme 1, general procedure D, using 2‐((4‐chloro‐
3‐fluorophenyl)amino)thiazole‐4‐carboxylic acid
and 3‐isopropoxypropylamine. Light brown solid,
59% yield (251.0 mg). Rf = 0.43 (5% MeOH in DCM).
2‐((4‐Chloro‐3,5‐difluorophenyl)amino)‐N‐
(3‐isopropoxypropyl)thiazole‐4‐carboxamide
(29). Scheme 1, general procedure D, using 2‐((4‐
chloro‐3,5‐difluorophenyl)amino)thiazole‐4‐car‐
boxylic acid and 3‐isopropoxypropylamine. Light
brown solid, 72% yield (205.3 mg). Rf = 0.40 (5%
o
m.p. 143.9‐147.1 C. 1H NMR (600 MHz, CDCl3) δ
ppm 8.65 (m, 1 H), 7.57 (m, 2 H), 7.47 (m, 1 H), 7.32
(m, 1 H), 7.16 (m, 1 H), 3.57 (m, 5 H), 1.88 (quint, J
= 6.2 Hz, 2 H), 1.15 (m, 6 H). 13C NMR (151 MHz,
CDCl3) δ ppm 163.1 (m), 161.5 (m), 159.1, 157.4,
146.0 (d, J = 4.4 Hz), 140.3 (m), 130.7, 114.1, 113.6,
1
MeOH in DCM). m.p. 149.9‐154.8 oC. H NMR (600
MHz, (CD3)2CO) δ ppm 9.96 (br. s, 1 H), 7.95 (m, 1
H), 7.58 (m, 3 H), 3.55 (spt, J = 6.1 Hz, 1 H), 3.50 (t, J
= 6.1 Hz, 2 H), 3.46 (q, J = 6.9 Hz, 2 H), 1.81 (quint, J
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= 6.7 Hz, 2 H), 1.09 (d, J = 6.2 Hz, 6 H). 13C NMR (151
Nam/1194/2004 (H5N1) (NA wt), Influenza RG14
5
MHz, (CD3)2SO) δ ppm 162.0, 160.4, 159.0 (d, J = 5.5
Hz), 157.4 (d, J = 5.5 Hz), 146.4, 140.8 (t, J = 13.2 Hz),
114.3, 101.1 (d, J = 28.6 Hz), 99.2 (t, J = 22.0 Hz),
70.7, 65.4, 36.6, 29.8, 22.0. HRMS (ESI‐TOF) calcd
for C16H19ClF2N3O2S: 390.0849, found 390.0850
[M+H]+. For synthesis of the salt compound used in
in vivo studies, anhydrous 1,4‐dioxane (0.13 M) was
added to 2‐((4‐chloro‐3,5‐difluorophenyl)amino)‐
N‐(3‐isopropoxypropyl)thiazole‐4‐carboxamide
(compound 29, 1.0 equiv) and heated or sonicated
until completely dissolved. While stirring, concen‐
trated sulfuric acid (2.0 equiv) was added dropwise
and the mixture stirred at room temperature for 2
hours. The final product (dark orange solid, 99%
yield) was obtained by concentrating the mixture
under reduced pressure until completely dry.
(H5N1) (NA H274Y) were produced by using plas‐
mids provided by Dr. King‐Song Jeng (Institute of
Molecular Biology, Academia Sinica). The attenu‐
ated reassortant H1N1 influenza viruses A/Califor‐
nia/7/2009 was from the National Institute for Bi‐
ological Standards and Control. Influenza A/Missis‐
sippi (A/New Caledonia/20/99‐like) (wt), Influ‐
enza A/Mississippi (A/New Caledonia/20/99‐like)
(H274Y), A/Perth (H1N1pdm09) (wt), and A/Perth
(H1N1pdm09) (H275Y) were obtained from World
Health Organization (WHO). Influenza B/Florida
were kindly provided from Dr. Che Ma (Genomics
Research Center, Academia Sinica). All viruses were
cultured either by using Madin−Darby canine kid‐
ney (MDCK) cells or in the allantoic cavities of 10‐
day‐old embryonated chicken eggs for 72 hours.
MDCK cells were obtained from American Type Cul‐
ture Collection (Manassas, VA), and were grown in
DMEM (Dulbecco’s Modified Eagle Medium, Gib‐
coBRL) containing 10% fetal bovine serum (Gib‐
coBRL) and penicillin−streptomycin (GibcoBRL) at
37 °C under 5% CO2. All cell culture reagents were
obtained from Invitrogen Inc. (Carlsbad, CA, USA).
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2‐((4‐Chloro‐3,5‐difluorophenyl)amino)‐N‐
(3‐ethoxypropyl)thiazole‐4‐carboxamide (30).
Scheme 1, general procedure D, using 2‐((4‐chloro‐
3,5‐difluorophenyl)amino)thiazole‐4‐carboxylic
acid and 3‐ethoxypropylamine. Off‐white solid,
43% yield (128.2 mg). Rf = 0.37 (5% MeOH in DCM).
1
m.p. 180.2‐183.9 oC. H NMR (600 MHz, (CD3)2SO)
δ ppm 10.8 (s, 1 H), 8.27 (t, J = 5.9 Hz, 1 H), 7.59 (m,
3 H), 3.41 (m, 4 H), 3.33 (q, J = 6.7 Hz, 2 H), 1.76
(quint, J = 6.6 Hz, 2 H), 1.09 (t, J = 7.1 Hz, 3 H). 13C
NMR (151 MHz, (CD3)2SO) δ ppm 162.0, 160.4,
159.0 (d, J = 5.5 Hz), 157.3 (d, J = 5.5 Hz), 146.4,
140.7 (t, J = 13.2 Hz), 114.4, 101.1 (m), 101.0 (m),
99.2 (t, J = 22.0 Hz), 67.9, 65.4, 36.6, 29.5, 15.1.
HRMS (ESI‐TOF) calcd for C15H17ClF2N3O2S:
376.0693, found 376.0692 [M+H]+. For synthesis of
the salt compounds used in in vivo studies, anhy‐
drous ethyl acetate (0.05 M) was added to 2‐((4‐
chloro‐3,5‐difluorophenyl)amino)‐N‐(3‐ethoxy‐
propyl)thiazole‐4‐carboxamide (compound 30, 1.0
equiv) and heated or sonicated until completely dis‐
solved. While stirring, methane sulfonic acid (2.0
equiv) was added dropwise and the mixture stirred
at room temperature for 2 hours. The final product
(orange solid, 98% yield) was obtained by concen‐
trating the mixture under reduced pressure until
completely dry.
Determination of EC50 and CC50 values of small
molecules. The anti‐influenza activities of com‐
pounds were measured by the EC50 values, which
were the concentrations of tested molecules for
50% protection of the influenza virus infection‐me‐
diated cytopathic effects (CPE). Fifty microliters of
diluted influenza virus at 100 TCID50 was mixed
with equal volumes of compounds at varied concen‐
trations. The mixtures were used to infect 100 μL of
MDCK cells at 1 × 105 cells/mL in 96 wells. After 48
hours of incubation at 37 °C under 5% CO2, the cy‐
topathic effects were determined with CellTiter 96
AQueous Non‐Radioactive Cell Proliferation Assay
reagent (Promega). The EC50 values were deter‐
mined by fitting the curve of percent CPE versus the
concentrations of compounds using Graph Pad
Prism 4. The CC50 values (50% cytotoxic concentra‐
tions) of compounds to MDCK cells were deter‐
mined by the procedures analogous to the EC50 de‐
termination but without virus infection.
Viruses, cells, and biological reagents. Recom‐
binant virus Influenza A/WSN/1933 (H1N1) (NA
wt), Influenza A/WSN/1933 (H1N1) (NA H274Y),
Influenza RG14 (H5N1) harboring the hemaggluti‐
Animal experiments for virus challenges. Fe‐
male BALB/c mice (18−20 g) were obtained from
Charles River Laboratories or National Laboratory
Animal Center (Taiwan). All animal experiments
were evaluated and approved by the Institutional
nin
and
neuraminidase
from
A/Viet
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Animal Care and Use Committee of Academia Sinica,
Corresponding Author
Taiwan. The mice were quarantined for 48−72
hours before use. The mice were anesthetized by in‐
traperitoneal injection of zoletil (or keta‐
mine/xylazine) and inoculated intranasally with 25
μL of infectious influenza virus. The test com‐
pounds were dissolved in phosphate‐buffered sa‐
line containing 40% polyethylene glycol 400 and
administered to mice through intraperitoneal injec‐
tion at the indicated dosages twice daily for 5 days.
Control mice received phosphate‐buffered saline
containing 40% polyethylene glycol 400 on the
same schedule. Ten mice per test group were used
throughout the studies. Four hours after the first
dose of drug, mice were inoculated with influenza
virus at 2X mouse LD50. Mice were observed daily
for 14 days for survival and body weight.
Molecular Modeling. All molecular modeling
docking was performed on programs within the
Schrodinger Suite. The influenza nucleoprotein
(PDB code 2IQH) was refined by Prime
(Schrodinger) and the A‐chain monomer was ap‐
plied as the target for molecular docking. A grid box
was generated by using Glide (Schrodinger) to sur‐
round the tail‐loop pocket of the desired compound.
The compound was prepared by using LigPrep
(Schrodinger) before molecular docking by using
Glide. The protein‐protein complex was obtained
and refined further by using Prime and the VSGB
solvation model. The OPLS_2005 force field was ap‐
plied in all procedures.
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*Email:wong@scripps.edu,
chwong@gate.sinica.edu.tw. Tel: +886‐2‐27899929,
Fax:+886‐2‐27899927
Notes
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The authors declare no conflict of interest.
 ACKNOWLEDGMENT
This work was supported by the National Institutes
of Health (AI130227‐01A1), Academia Sinica [AS‐
SUMMIT‐108] and Ministry of Science and Technol‐
ogy [MOST 108‐3114‐Y‐001‐002], and the Kwang
Hua Foundation.
 ABBREVIATIONS USED
CDI, carbonyldiimidazole; DCC, N,N'‐dicyclohexyl‐
carbodiimide; DCM, dichloromethane; DIPEA, N,N‐
diisopropylethylamine; DMF, dimethylformamide;
DMSO, dimethylsulfoxide; EDC, 1‐ethyl‐3‐(3‐dime‐
thylaminopropyl)carbodiimide
hydrochloride;
EtOAc, ethyl acetate; EtOH, ethanol; HATU, 1‐
[bis(dimethylamino)methylene]‐1H‐1,2,3‐tria‐
zolo[4,5‐b]pyridinium 3‐oxid hexafluorophosphate;
HBTU,
2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetra‐
methyluronium hexafluorophosphate; HMDS, hexa‐
methyldisilazane; HOBt, hydroxybenzotriazole;
LAH, lithium aluminum hydride; LCMS, liquid chro‐
matography mass spectrometry; MeOh, methanol;
MW, microwave; NMM, N‐methylmorpholine; NMR,
nuclear magnetic resonance; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TLC, thin layer chroma‐
tography; T3P, propylphosphonic anhydride;
Xantphos, 4,5‐bis(diphenylphosphino)‐9,9‐dime‐
thylxanthene.
 ASSOCIATED CONTENT
Supporting Information
Supplementary tables detailing all compound struc‐
tures and inhibition data for all viral strains tested,
supplementary figures detailing molecular binding
and analytical ultracentrifugation, methods for bio‐
logical experiments and molecular modeling, and
details on the synthesis and characterization of
compounds are included in the supporting infor‐
mation. A document including the molecular for‐
mula strings for each analog is also available. The
Supporting Information is available free of charge
on the ACS Publications website.
 REFERENCES
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EC50 = 0.11 M / CC50 > 100 M
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