Structure-activity relationship of the cinnamamide family of antibiotic potentiators for methicillin-resistant: Staphylococcus aureus (MRSA)

Article abstract of DOI:10.1039/c8md00479j

Methicillin-resistant Staphylococcus aureus (MRSA) is a global public health threat. MRSA has evolved a complex set of biochemical processes that mobilize the organism for inducible resistance on challenge by β-lactam antibiotics. Interfering pharmacologi

Full text of DOI:10.1039/c8md00479j

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RESEARCH ARTICLE
Structure–activity relationship of the cinnamamide
family of antibiotic potentiators for methicillin-
resistant Staphylococcus aureus (MRSA)†
Cite this: Med. Chem. Commun.,
2018, 9, 2008
*
Enrico Speri,
Jennifer Fishovitz
and Shahriar Mobashery
Methicillin-resistant Staphylococcus aureus (MRSA) is a global public health threat. MRSA has evolved a
complex set of biochemical processes that mobilize the organism for inducible resistance on challenge by
β-lactam antibiotics. Interfering pharmacologically with this machinery has the potential to reverse the
β-lactam-resistance phenotype, whereby susceptibility to obsolete antibiotics would be restored. We de-
scribe herein our discovery of one class of such agents, the cinnamamide family of antibiotic potentiators.
A hit compound of the class (compound 1) showed modest potentiation of the activity of oxacillin, a peni-
cillin antibiotic, against an MRSA strain. A total of 50 analogues of compound 1 were prepared and scre-
ened. Seven of these compounds showed more dramatic potentiation of the antibacterial activity, which
lowered the minimal-inhibitory concentrations (MICs) for the antibiotic by as much as 64- to 128-fold.
Received 24th September 2018,
Accepted 19th October 2018
DOI: 10.1039/c8md00479j
rsc.li/medchemcomm
inhibitory concentration (MIC) of oxacillin for strain
1. Introduction
MRSA252 (also known as strain USA200) from 256 μg mL−1 to
Methicillin-resistant Staphylococcus aureus (MRSA) has
evolved an elaborate scheme for manifestation of resistance
to β-lactam antibiotics. A key component of this process in-
volves sensing of the presence of the antibiotic by either the
BlaR1 or MecR1 antibiotic sensor/signal transducers, integral
membrane proteins, which then transduce the signal to the
cytoplasmic side of the membrane.1–4 During this process, a
cytoplasmic protease domain is activated, which degrades the
gene repressors BlaI or MecI, which leads to transcription of
genes blaZ and mecA for the resistance determinants
β-lactamase and penicillin-binding protein 2A, respectively.5–7
Furthermore, there are other contributors to the resistance re-
sponse, such as two-component systems.8,9 All in all, exposure
of the organism to the antibiotic creates a state of mobiliza-
tion, the outcome of which is broad resistance to virtually the
entire class of β-lactam antibiotics. These subjects have been
reviewed.10–12 The complexity of the system implies the poten-
tial for interference by small molecules at many steps of this
elaborate process that could reverse the antibiotic-resistance
phenotype.13 We describe one such class of compounds in
the present report.
128 μg mL−1 consistently, when it was present at 20 μM.
MRSA252 is a strain derived from the hospital-acquired
MRSA-16 (EMRSA-16) epidemic.14,15 Furthermore, 1 did not
show any antibacterial activity of its own, indicating that the
lowering of the MIC value for oxacillin likely involved inter-
ference with a step in the aforementioned elaborate process
of response by the organism to the antibiotic challenge. The
reduction in the MIC was a mere two-fold, but it was repro-
ducible, alerting us to explore further. We undertook to carry
out a structure–activity relationship (SAR) study for this com-
pound class, as we outline herein, with the intention of iden-
tifying more potent compounds.
We dissected the compound into four segments, referred
to as SAR1–4 (coloured boxes; Fig. 1), to evaluate the struc-
tural contributions of each segment of the template toward
the activity. As the actual target is presently unknown, the
search would be guided by generating structural diversity at
Screening of compounds within our labs for potentiators
of activity of oxacillin (OXA), a penicillin antibiotic (a
β-lactam), recently identified compound 1 as a modest poten-
tiator. Compound
1 was able to reduce the minimal-
Department of Chemistry and Biochemistry, University of Notre Dame, Notre
Dame, IN 46556, USA. E-mail: mobashery@nd.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00479j
Fig. 1 Compound 1 and the four sites for structural diversification:
SAR1 (red), SAR2 (blue), SAR3 (green), and SAR4 (purple).
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each of these SAR sites and screening for the potentiation
activity.
(in an SN2 reaction) or cinnamic acid (in a HATU-mediated
coupling reaction) were used, respectively (Fig. 2). Lastly, di-
versification in SAR4 was achieved using different alkylated
amines. A total of 50 compounds were synthesized in the di-
versification of the four SAR regions.
2. Results and discussion
Synthesis
Compound 1 was synthesized as reported in the literature.16
For synthetic expediency, we explored the possibility of
substituting the tertiary amine at the core of the molecule
with an amide. The SAR1 diversified the structure at the site
that is boxed in red. Scheme 1 outlines the synthetic route to
compound 6, but the strategy is general for other cinnamic
derivatives within the red box.
Briefly, the aniline amine of 2-aminobenzyl alcohol was
sulfonylated to give the sulfonamide 2. The alcohol in 2 was
oxidized to the aldehyde 3 and this underwent reductive
amination to the appropriate amine (in this case a methyl-
ated amine) to produce the intermediate 4. Compound 4 was
allowed to react with p-(trifluoromethyl)cinnamic acid using
the coupling reagent 1-[bisIJdimethylamino)methylene]-1H-
1,2,3-triazoloij4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU)18–20 to give 5. Compound 5 was N-alkylated with
2-iodoethanol21 to produce the final compound 6. By varia-
tion of the corresponding cinnamic derivatives in the red
box, we transformed compound 4 into a series of structures
diversified at this specific site.
Diversification at the SAR2 site took place as shown in
Scheme 2. The starting point was 2-nitrobenzyl alcohol. The
alcohol was activated with methanesulfonyl chloride, which
was in turn displaced by methylamine.22 The appropriate
cinnamyl derivative was introduced (intermediate 9) and the
nitro functionality was then reduced using zinc to intermedi-
ate 10. The SAR2 variable (boxed in blue) was introduced
using the desired sulfonyl chloride, in this case
4-fluorosulfonyl chloride, to produce compound 11.
N-Alkylation21 of 11 completed the synthesis, giving com-
pound 12.
SAR3 and SAR4 variables, boxed in green and purple in
Fig. 1, respectively, used the synthetic routes in either
Scheme 1 or Scheme 2 to achieve diversification at both sites.
To achieve the diversification in SAR3 as amines (SAR3-a)
and amides (SAR3-b), suitable cinnamyl methanesulfonate
Evaluation of the compounds
Each synthetic compound was tested alone to explore if it
would exhibit antibacterial activity of its own. None of the
compounds did, so we evaluated them as potentiators of
MRSA next. The activity of each compound was evaluated by
assessment of the minimal-inhibitory concentration (MIC)
for oxacillin (OXA) with strain MRSA252 in the presence of
the given synthetic sample with a fixed 20 μM concentration.
The MIC of OXA against strain MRSA252 in the absence of
any potentiator was 256 μg mL−1. These assessments are
given in Table 1. The compounds that lowered the MIC from
256 μg mL−1 to <8 μg mL−1 were deemed as active potentia-
tors (Table 1; given in blue). The values ≥8 μg mL−1 are in
red for less active compounds.
The results of SAR1 (on ring 1) indicated that the presence
of an electron-withdrawing group (EWG) at the para position
of the ring is beneficial. All of the seven most active com-
pounds (6 and 12–17) had a chloro atom or a trichloromethyl
moiety at the para position. When another chloro atom (26,
27 and 45) was introduced into the structure, either at the or-
tho or meta position, the potentiation activity was lost. A hy-
drogen atom or an electron-donating group (EDG), such as
methoxy or acetamide (35–37, 40, and 41), showed loss of po-
tentiation activity. SAR2 was explored as well with either
EWG or EDG substitutions. Compounds containing EWGs
and/or apolar groups preferred the para positions. 4-Fluoro
(12 and 17), 4-methyl (6 and 16), and 3-fluoro-4-methyl (14)
were well tolerated, all lowering the MIC for OXA to below
8 μg mL−1. The introduction of an isopropyl (28), nitro (29),
trifluoromethoxy (30), cyano (32), or trifluoromethyl (39)
group at the para position resulted in abrogation of the activ-
ity. When EDGs 4-methoxy (37) or 3,4-ethylenedioxy (31) were
introduced, the compounds did not show any potentiation.
While a simple mono-substituted phenyl ring (15) lowered
the MIC to 4 μg mL−1, the compounds with the benzene
Scheme 1 Synthetic route for SAR1 derivatives. (a) p-Toluenesulfonyl chloride, pyridine, DCM, reflux, overnight, yield 94%; (b) manganeseIJIV)
oxide,17 DCM, rt, 72 h, yield 85%; (c) methylamine 40 wt% in H2O, MeOH, 30 min, then NaBH4, 0 °C, 1 h, yield 98%; (d) 4-(trifluoromethyl)cinnamic
acid, HATU, DIPEA, DMF, rt, overnight, yield 71%; (e) 2-iodoethanol, K2CO3, DMF, 60 °C, 24 h, yield 62%.
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Scheme 2 Synthetic route for SAR2 derivatives. (a) Methanesulfonyl chloride, THF, rt, 2 h, yield 99%; (b) methylamine 40 wt% in H2O, 30 min, then
NaBH4, THF, rt, 1 h, yield 98%; (c) 4-(trifluoromethyl)cinnamic acid, HATU, DIPEA, DMF, rt, 24 h, yield 67%; (d) zinc, NH4Cl, acetone, reflux, 5 h,
yield 69%; (e) 4-fluorobenzenesulfonyl chloride, pyridine, DCM, 0 °C to rt, 24 h, yield 65%; (f) 2-iodoethanol, K2CO3, DMF, 60 °C, 24 h yield 61%.
bioisostere thiophene (33 and 34) did not show any activity.
SAR3 identified which X linker was more active between am-
ide and amine. Amines 1 and 37 to 45 showed modest to no
potentiation activity of OXA, although the MIC never went be-
low 8 μg mL−1. Amides 6 and 12 to 17 produced MIC values
in the range of 2–4 μg mL−1, indicating that a hydrogen-bond
acceptor might be beneficial. The SAR4 exploration aimed to
study the effect of different alkyl groups such as methyl (6
and 12 to 17), ethyl (20), cyclopropyl (21), isopropyl (22), and
isoamyl (23 and 24) or merely hydrogen (25) at the amide po-
sition. Only the methyl substitution resulted in potentiation
of the OXA activity. Interestingly, the ethyl alcohol chain in-
troduced during the last step of the synthesis in both
Schemes 1 and 2 was essential for the activity. Compound 11,
related to compound 12 minus the ethyl chain, was devoid of
activity.
Table 2 expands on the study at the R1 group (red box).
The explored chemical space involved the synthesis of a
cinnamyl bioisostere such as (4-chlorophenoxy)methyl (46) or
a reduced double bond in the cinnamyl moiety (47). The in-
troduction of heterocycles (48–51), naphthyl (52), phenyl (53
and 54), and ethenyl (55–57) moieties, an inverted amide (58)
and a meta instead of ortho connected compound (59) was
also explored, however all of these modifications were not
tolerated.
Notwithstanding the fact that there are no stereogenic
centers in these compounds, they appear to exhibit three-
dimensional attributes, as discerned from their NMR spectra.
The introduction of the N-ethyl alcohol chain, critical for the
biological function, in the last step of both the synthetic
schemes (red oval in Fig. 3) produced rigidity and particularly
slow rotation at the amide (blue oval in Fig. 3). This pro-
duced a higher-than-expected number of signals in the 1H-
NMR spectra. To demonstrate that this observation was due
to slow bond rotation, hence a conformational phenomenon,
multiple 1H-NMR spectra were acquired at increasing temper-
atures (starting at 25 °C with temperature increments of 10
°C) with a representative of the class (compound 16). Fig. 3
shows 8 different NMR spectra superposed between 2.8 ppm
and 3.3 ppm, the region where the methyl of the amide (cir-
cled in blue) is present. As shown in the spectrum at 25 °C,
Table
1 Minimal-inhibitory concentration (MIC) for oxacillin (OXA)
against strain MRSA252 in the presence of 20 μM potentiator. MIC values
of 8 μg mL−1 and above are given in red and those below in blue
SAR1
SAR2
SAR3
SAR4
MIC OXA
1
6
4-Cl
4-CF3
4-OMe
4-Me
X = CH2
X = CO
-Me
-Me
128
2
11
Same as 12 but without the lateral ethyl alcohol
chain
≥256
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
4-CF3
4-CF3
4-CF3
4-CF3
4-Cl
4-F
4-Et
3-F-4-Me
H
4-Me
4-F
4-Me
4-Me
4-Me
4-Me
4-Me
4-OMe
4-Me
4-Me
4-Me
4-Me
4-iPr
4-NO2
4-OCF3
3,4-Ethylenedioxy X = CO
4-CN
2-Thiophene
3-Thiophene
4-Me
4-Me
4-OMe
4-Me
4-CF3
4-CF3
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CH2
X = CH2
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Et
-C3H6
-iPr
-isobutyl
-isobutyl
-H
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
2
4
4
4
2
4
4-Cl
4-F
≥256
4-Me
4-CF3
4-CF3
4-CF3
4-Cl
64
≥256
≥256
≥256
128
128
≥256
≥256
16
4-Cl
4-CF3
2,4-Cl
3,4-Cl
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
4-Cl
32
≥256
32
64
32
X = CO
X = CO
X = CO
X = CO
X = CO
X = CH2
X = CH2
X = CH2
X = CH2
X = CH2
X = CH2
X = CH2
X = CH2
X = CH2
256
≥256
≥256
≥256
≥256
32
64
32
≥256
≥256
≥256
16
4-Cl
4-OMe
4-NHAc
H
4-Cl
4-Cl
H
H
4-Cl
4-Cl
4-Me
4-NHAc
-Benzyl
4-Cl
4-Cl
3,4-Cl
Fig. 2 Precursors for SAR3 exploration.
4-Me
32
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Table 2 Additional structures with more broad modification at the R1 position. MICs are given for oxacillin against strain MRSA252 in the presence of
20 μM potentiator
SAR1
SAR2
SAR3
SAR4
MIC OXA
46
47
48
49
50
51
52
53
54
55
56
57
(4-Chlorophenoxy)methyl
2-(4-Chlorophenyl)ethyl
(E)-5-(4-Chlorophenyl)-furan-2-ylethenyl
(E)-2-(3-Pyridinyl)ethenyl
(E)-2-(2-Furanyl)ethenyl
(E)-2-(5-Bromothiophenyl)ethenyl
6-Bromo-2-naphthyl
4-Chlorophenyl
Phenyl
Ethenyl
Ethenyl
Ethenyl
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-iPr
-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-CF3
4-Cl
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CO
X = CH2
X = CH2
X = CH2
X = CH2
≥256
≥256
≥256
≥256
≥256
≥256
≥256
≥256
≥256
≥256
≥256
≥256
4-OMe
4-Me
58 MIC = ≥256
59 MIC = ≥256
the methyl resonance is split into two peaks of roughly equal
intensity, which merge into one at 95 °C.
cycline; NRS386 (also known as strain USA700) a strain associ-
ated with both community and healthcare infections,23 and
NRS70 a resistant strain isolated in 1982.24 We maintained a
fixed 20 μM concentration of the synthetic compound through-
out these studies. Compounds 6, 12 and 16 showed an OXA
MIC of 2 μg mL−1 and compounds 13–15 and 17 exhibited an
OXA MIC of 4 μg mL−1 against MRSA252; 128-fold and 64-fold
lowering of MIC, respectively (Table 3). Among the additional
three strains, only NRS70 did not show pronounced potentia-
tion. In fact, the range of the MIC values against NRS100 and
NRS386 was essentially the same with a handful of exceptions.
The breadth of the potentiation
The potentiation activity of compounds 6 and 12–17 was stud-
ied with three additional MRSA strains: NRS100,
a
mecA-positive strain resistant to methicillin, oxacillin, and tetra-
3. Conclusion
We disclose here a class of cinnamamide potentiators of
β-lactam antibiotic activity against MRSA. The active
Table 3 MIC values of oxacillin (OXA) in the presence or absence of syn-
thetic compounds (given at 20 μM)
MIC OXA μg mL−1
Strain
No compd
6
12
13
14
15
16
17
Fig. 3 Compound 16 and its 1H-NMR signals between 2.8 and 3.3
ppm. Introduction of the hydroxyethyl moiety (red oval) introduced
conformational rigidity into the molecule, documented by the merger
of the methyl signals for the N-methylamide only at 95 °C.
MRSA252
NRS100
NRS386
NRS70
256
256
256
32
2
4
4
16
2
2
8
16
4
16
8
4
2
8
16
4
2
2
16
2
2
4
8
4
1
1
8
16
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potentiators that we have discovered lack antibacterial activ-
ity of their own. This study encompassed evaluation of 50 an-
alogues of the original hit molecule, which lowered the MIC
for oxacillin with strain MRSA252 from 256 μg mL−1 to 2 to 4
μg mL−1 for molecules 6 and 12 to 17. The target(s) for this
class of compounds need(s) to be elucidated, the identifica-
tion of which should stimulate further investigation of their
biological function.
HRMS (m/z): [M + Na]+, calcd for C14H15NNaO3S, 300.0665;
found, 300.0638.
N-(2-Formylphenyl)-4-methylbenzenesulfonamide (3)
Compound
2 (13.5 g, 48.7 mmol) was dissolved in
dichloromethane (DCM, 500 mL). Manganese dioxide (52.9 g,
608.5 mmol) is added in one portion to the solution. The re-
action mixture was stirred for 72 hours at room temperature
and the suspension was filtered through a pad of celite. The
filtrate was concentrated under reduced pressure to produce
4. Experimental methods
Minimal-inhibitory concentration (MIC) determination
1
a yellowish solid (11.40 g, 85%). H NMR (500 MHz, CDCl3) δ
ppm 10.79 (br. s., 1 H), 9.83 (s, 1 H), 7.78 (d, J = 8.3 Hz, 2 H),
7.69 (d, J = 8.3 Hz, 1 H), 7.59 (dd, J = 7.6, 1.5 Hz, 1 H), 7.51
(ddd, J = 8.6, 7.2, 1.6 Hz, 1 H), 7.20–7.30 (m, 2 H), 7.16 (td, J
= 7.6, 1.0 Hz, 1 H), 2.37 (s, 3 H). 13C NMR (125 MHz, CDCl3)
δ ppm 195.19, 144.38, 140.18, 136.64, 136.31, 136.02, 129.97,
127.49, 123.13, 122.09, 117.99, 21.75. HRMS (m/z): [M + Na]+,
calcd for C14H13NNaO3S, 298.0508; found, 298.0526.
MICs were determined with the broth microdilution method
following the Clinical & Laboratory Standards Institute (CLSI)
guidelines with slight modifications. Determination of MICs
was performed by two-fold serial dilution of each compound
in cation-adjusted Mueller-Hinton II broth (CAMHB II) with a
DMSO concentration of 2.4%. Experiments were carried out
in triplicate in 96-well plates with an inoculum of 5 × 105
CFU mL−1, followed by incubation at 36 °C for 16–20 h. The
MIC was recorded as the lowest concentration that inhibited
bacterial growth.
4-Methyl-N-(2-((methylamino)methyl)phenyl)-
benzenesulfonamide (4)
Compound 3 (6 g, 21.8 mmol) was dissolved in methanol
(600 mL). A 40% solution of methylamine in water (2.5 mL,
28.3 mmol) was added to the methanol solution. The reac-
tion was stirred for 30 min at room temperature and was
then chilled to ice–water temperature. Powdered sodium bo-
rohydride was added (412 mg, 10.9 mmol) in small portions
to the solution over 5 min. After 1 h the excess of borohy-
dride was neutralized with 250 mL of water and the methanol
was evaporated under reduced pressure. The remaining water
was washed with DCM and the layers were separated. The or-
ganic layer was dried over anhydrous Na2SO4, the suspension
was filtered and the filtrate was evaporated to give an off-
Syntheses
A detailed procedure for the synthesis of 6 (Scheme 1), 12
(Scheme 2), and 13 to 17 is given. The following procedures
are representative for all the 51 compounds (compound 1 plus
50 analogues; NMR and MS data for the complete set is given
in the ESI†). The purity of all the final compounds was found
to be ≥95% and it was determined by LC/MS using a Bruker-
Q II TOF electrospray mass spectrometer coupled with a
Dionex UltiMate 3000 ultra-high pressure liquid chromato-
graph. The solvents used were 0.1% formic acid in H2O (A)
and 0.1% formic acid in acetonitrile (B). Each sample run was
10 min as follows: 1 min of 10% B, 8 min of gradient to 100%
B, and 1 min of gradient back to 10% B. The flow rate was 0.4
mL min−1 and UV/vis detection was carried out at 240 nm. Pu-
rification of products was accomplished by silica-gel column
chromatography using a Teledyne ISCO CombiFlash Rf 200i.
1
white solid (6.22 g, 98%). H NMR (500 MHz, CDCl3) δ ppm
7.63–7.69 (m, 2 H), 7.50 (d, J = 7.8 Hz, 1 H), 7.17–7.25 (m, 3
H), 6.94–7.01 (m, 2 H), 3.47 (s, 2 H), 2.37 (s, 3 H), 2.35 (s, 3
H). 13C NMR (125 MHz, CDCl3) δ ppm 143.51, 138.38, 138.12,
129.77, 129.67, 128.70, 128.07, 127.02, 124.14, 121.47, 55.04,
35.62, 21.76. HRMS (m/z): [M + H]+, calcd for C15H19N2O2S,
291.1162; found, 291.1161.
N-(2-(Hydroxymethyl)phenyl)-4-methylbenzenesulfonamide (2)
(E)-N-Methyl-N-(2-((4-methylphenyl)sulfonamido)benzyl)-3-(4-
(trifluoromethyl)phenyl)acrylamide (5)
To a solution of 2-aminobenzyl alcohol (6.50 g, 52.8 mmol)
and pyridine (5.50 mL, 68.6 mmol) in dichloromethane
(DCM, 50 mL), p-toluenesulfonyl chloride (12.08 g, 58.1
mmol) was slowly added in small portions. The solution was
stirred overnight under reflux. Water (25 mL) was added to
the reaction mixture. The aqueous layer was separated and
the organic solution was washed with brine and then dried
over anhydrous Na2SO4. The filtered solution was evaporated
to afford an off-white solid (13.82 g, 94%). 1H NMR (500
MHz, CDCl3) δ ppm 7.89 (s, 1 H), 7.60–7.68 (m, 2 H), 7.43 (d,
J = 8.1 Hz, 1 H), 7.23–7.28 (m, 2 H), 7.21 (dd, J = 8.6, 0.5 Hz,
2 H), 7.05–7.11 (m, 2 H), 2.38 (s, 3 H) 4.39 (s, 2 H). 13C NMR
(125 MHz, CDCl3) δ ppm 144.01, 137.18, 136.63, 131.83,
129.58, 129.50, 129.25, 127.28, 125.57, 123.69, 64.15, 21.76.
To a solution of 4-(trifluoromethyl)cinnamic acid (162 mg, 0.8
mmol) in anhydrous N,N-dimethylformamide (DMF, 6 mL),
1-[bisIJdimethylamino)methylene]-1H-1,2,3-triazoloij4,5-b]-
pyridinium 3-oxid hexafluorophosphate (HATU, 316 mg, 0.8
mmol) was added and the solution was stirred for 10 min at
room temperature. Compound 4 (435 mg, 1.5 mmol) was
added and the solution was allowed to stir for an additional
20 min, at which point N,N-diisopropylethylamine (DIPEA,
0.39 mL, 2.3 mmol) was added and the solution was stirred
at room temperature overnight. The solution was diluted with
30 mL of diethyl ether and was washed once with water. The
organic layer was dried over anhydrous Na2SO4, the mixture
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was filtered and the filtrate was evaporated to dryness. The
solid material was purified on silica gel using a gradient of
3 : 7 EtOAc/hexane to 1 : 1 EtOAc/hexane, producing a white
129.92, 129.62, 125.53, 68.28, 37.98. HRMS (m/z): [M + H]+,
calcd for C8H13N2O5S, 249.0540; found, 249.0534.
1
solid (260 mg, 71%). H NMR (400 MHz, CDCl3) δ ppm 10.23
N-Methyl-1-(2-nitrophenyl)methanamine (8)
(s, 1 H), 7.86 (d, J = 15.3 Hz, 1 H), 7.50–7.77 (m, 7 H), 7.30 (t,
J = 7.1 Hz, 1 H), 7.16 (d, J = 7.5 Hz, 3 H), 7.06 (t, J = 7.5 Hz, 1
H), 6.86 (d, J = 15.3 Hz, 1 H), 4.21 (s, 2 H), 3.08 (s, 3 H), 2.33
(s, 3 H). 13C NMR (100 MHz, CDCl3) δ ppm 167.33, 143.51,
143.32, 138.46, 137.57, 136.95, 131.82, 131.58, 129.88, 129.69,
128.43, 128.13, 127.52, 127.33, 126.07, 125.46, 124.71, 123.41,
122.77, 120.04, 118.79, 49.56, 35.24, 21.76. HRMS (m/z): [M +
H]+, calcd for C25H24F3N2O3S, 489.1454; found, 489.1440.
Methylamine 40 wt% in water (112 mL, 1294.5 mmol) was
added dropwise over half an hour to a solution of compound 7
(30 g, 129.8 mmol) in tetrahydrofuran (THF, 140 mL) at room
temperature. TLC analysis showed that the reaction was com-
pleted within 2 h. The solvent was evaporated under reduced
pressure and the remaining oil was taken up in 100 mL
dichloromethane. To this solution were added water (100 mL)
and 37% hydrochloric acid until pH 4 was reached. The aque-
ous fraction was separated and washed with DCM and the or-
ganic portion was discarded. Fresh DCM was added to the aque-
ous solution. The mixture was treated with 10% NaOH until pH
11 was reached. The organic fraction was separated and saved.
The water fraction was then washed again with DCM. The com-
bined organic solution was dried over anhydrous Na2SO4, the
mixture was filtered and the filtrate was evaporated to dryness
to give a yellow oil (21.00 g, 98%). 1H NMR (500 MHz, CDCl3) δ
ppm 7.93 (dt, J = 8.1, 1.6 Hz, 1 H), 7.53–7.63 (m, 2 H), 7.36–7.43
(m, 1 H), 3.98 (d, J = 1.5 Hz, 2 H), 2.40–2.48 (m, 3 H), 1.60 (br.
s., 1 H). 13C NMR (126 MHz, CDCl3) δ ppm 147.28, 135.64,
135.35, 131.46, 128.15, 124.95, 52.99, 36.36. HRMS (m/z): [M +
H]+, calcd for C8H11N2O2, 167.0815; found, 167.0804.
(E)-N-(2-((N-(2-Hydroxyethyl)-4-
methylphenyl)sulfonamido)benzyl)-N-methyl-3-(4-
(trifluoromethyl)phenyl)acrylamide (6)
2-Iodoethanol (24 μL, 0.3 mmol) and potassium carbonate
(70 mg, 0.5 mmol) were added to a solution of compound 5
(100 mg, 0.2 mmol) in N,N-dimethylformamide (DMF, 2 mL).
The solution was heated at 60 °C for 24 h. The mixture was
then diluted with 5 mL water and was washed with diethyl
ether two times. The organic layer was dried over anhydrous
Na2SO4, the mixture was filtered and the filtrate was evapo-
rated to dryness. The crude product was purified by silica-gel
column chromatography in a gradient of 7 : 3 EtOAc/hexane
to 1 : 0 EtOAc/hexane, affording a white solid (66 mg, 62%).
1H NMR (500 MHz, DMSO-d6) δ ppm 8.00 (d, J = 8.1 Hz, 1 H),
7.80 (t, J = 8.4 Hz, 2 H), 7.69 (d, J = 8.3 Hz, 1 H), 7.66 (d, J =
7.1 Hz, 0.5 H), 7.63 (d, J = 7.1 Hz, 0.5 H), 7.50–7.58 (m, 2.5
H), 7.41–7.48 (m, 2 H), 7.29–7.39 (m, 1 H), 7.13–7.23 (m, 1.5
H), 7.03–7.13 (m, 1 H), 6.55 (dd, J = 4.2, 7.3 1 H), 5.13–5.19
(m, 0.5 H), 5.04–5.10 (m, 0.5 H), 4.92–5.01 (m, 1 H), 4.80–4.87
(m, 1 H), 3.82–3.96 (m, 1 H), 3.40–3.54 (m, 1 H), 3.26–3.34
(m, 1 H), 3.19–3.26 (m, 1 H), 3.15 (s, 1.5 H), 3.00 (s, 1.5 H)
2.42 (s, 3 H). 13C NMR (100 MHz, DMSO-d6) δ ppm 165.92,
165.89, 143.81, 143.63, 139.92, 139.82, 139.23, 139.00, 137.80,
137.57, 134.68, 134.35, 129.75, 129.70, 128.90, 128.68, 128.41,
127.79, 127.72, 127.67, 127.43, 127.37, 126.68, 125.95, 125.59,
125.56, 121.45, 120.89, 58.76, 58.56, 53.95, 53.76, 49.66,
47.19, 35.28, 34.51, 21.02. HRMS (m/z): [M + H]+, calcd for
C27H28F3N2O4S, 533.1716; found, 533.1749.
(E)-N-Methyl-N-(2-nitrobenzyl)-3-(4-
(trifluoromethyl)phenyl)acrylamide (9)
To
a solution of compound 8 (3.07 g, 23.1 mmol),
4-trifluoromethylcinnamic acid (4.00 g, 23.1 mmol) and HOBt
wetted with 14% water (3.75 g, 22.2 mmol) in N,N-
dimethylformamide
(100
mL),
were
added
N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
(EDC HCl, 4.26 g, 27.8 mmol) and subsequently N,N-
diisopropylethylamine (DIPEA, 8.1 mL, 46.5 mmol). The reac-
tion mixture was allowed to stir at room temperature for 24
h. The N,N-dimethylformamide was removed in vacuo and
DCM was added to the residue. The DCM was washed with
water, 5% aqueous HCl solution, 5% aqueous NaOH solution
and brine. The organic solution was dried over anhydrous
Na2SO4, the mixture was filtered and the filtrate was evapo-
rated to dryness. The crude was purified by column chroma-
tography on silica gel with elution in 1 : 1 EtOAc/hexane to
give a yellowish solid (5.62 g, 67%). 1H NMR (500 MHz,
CDCl3) δ ppm 8.19 (d, J = 7.8 Hz, 0.5 H), 8.05 (d, J = 7.8 Hz,
0.5 H), 7.78 (d, J = 15.2 Hz, 1 H), 7.57–7.74 (m, 3 H), 7.48–
7.57 (m, 2 H), 7.43 (t, J = 7.7 Hz, 1 H), 7.36 (d, J = 7.3 Hz, 1
H), 7.07 (d, J = 15.4 Hz, 0.5 H), 6.74 (d, J = 15.4 Hz, 0.5 H),
5.11 (s, 1 H), 5.07 (s, 1 H), 3.22 (s, 1.5 H), 3.11 (s, 1.5 H). 13C
NMR (126 MHz, CDCl3) δ ppm 167.23, 166.80, 148.82, 147.95,
142.56, 142.31, 138.65, 138.45, 134.80, 134.00, 133.12, 132.92,
131.73, 131.14, 129.30, 129.02, 128.98, 128.74, 128.37, 127.77,
126.06, 125.95, 125.42, 120.85, 119.36, 119.04, 51.74, 49.48,
36.37, 35.27. HRMS (m/z): [M + H]+, calcd for C18H16F3N2O3,
365.1108; found, 365.1125.
2-Nitrobenzyl methanesulfonate (7)
Methanesulfonyl chloride (12.64 mL, 163.3 mmol) was added
to a solution of 2-nitrobenzyl alcohol (20 g, 130.6 mmol) and
triethylamine (10 mL, 143.7 mmol) in tetrahydrofuran (THF, 50
mL). The reaction was stirred at room temperature for 2 h, at
which time, the solvent was removed by evaporation under re-
duced pressure. The residue was dissolved in dichloromethane
and was washed with water. The organic layer was dried over
Na2SO4, the mixture was filtered and the filtrate was evaporated
1
to dryness to give a pale-yellow solid (30.08 g, 99%). H NMR
(500 MHz, CDCl3) δ ppm 8.17 (d, J = 8.1 Hz, 1 H), 7.68–7.79 (m,
2 H), 7.53–7.59 (m, 1 H), 5.65 (s, 2 H), 3.12 (d, J = 0.5 Hz, 3 H).
13C NMR (126 MHz, CDCl3) δ ppm 147.20, 134.57, 130.37,
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(E)-N-(2-Aminobenzyl)-N-methyl-3-(4-
(trifluoromethyl)phenyl)acrylamide (10)
The solution was stirred at 60 °C for 24 h. The mixture was
then diluted with 5 mL water and was washed with diethyl
ether (2×). The combined organic layer was dried over
anhydrous Na2SO4, the mixture was filtered and the filtrate
was evaporated to dryness. The crude product was purified by
silica-gel column chromatography in a gradient of 7 : 3
EtOAc/hexane to 1 : 0 EtOAc/hexane, affording a white solid
A 5 M solution of NH4Cl in water (6.97 mL, 34.9 mmol) was
added to a solution of compound 9 (4.00 g, 11.0 mmol) in ac-
etone (75 mL) in a two-neck round-bottom flask. The solution
was heated to reflux, and then zinc (3.23 g, 49.4 mmol) was
added in small portions through the side neck to maintain a
moderate rate of reaction. The reaction mixture was refluxed
for 5 h. The solution was brought to room temperature. The
solid that formed during the reaction was filtered and
discarded. The filtrate was washed with water, and the or-
ganic fraction was dried over anhydrous Na2SO4. The mixture
was filtered and the filtrate was evaporated to give a yellow/
orange liquid (2.90 g, 69%). 1H NMR (500 MHz, CDCl3) δ
ppm 7.73 (d, J = 15.4 Hz, 1 H), 7.57–7.67 (m, 4 H), 7.12 (td, J
= 7.6, 1.50 Hz, 1 H), 7.08 (dd, J = 7.3, 1.2 Hz, 1 H), 6.96 (m, 1
H), 6.58–6.72 (m, 2 H), 4.63 (s, 4 H), 3.08 (s, 3 H). 13C NMR
(126 MHz, DMSO-d6) δ ppm 166.45, 146.47, 141.83, 138.77,
132.11, 131.56, 131.31, 129.73, 128.21, 127.42, 126.05, 126.02,
125.98, 125.95, 125.18, 123.07, 120.91, 119.96, 119.54, 117.24,
115.65, 49.24, 34.53. HRMS (m/z): [M + H]+, calcd for
C18H18F3N2O2, 335.1366; found, 533.1364.
1
(65 mg, 61%). H NMR (500 MHz, DMSO-d6) δ ppm 8.00 (d, J
= 8.1 Hz, 1 H), 7.61–7.85 (m, 6 H), 7.42–7.55 (m, 2.5 H), 7.29–
7.42 (m, 1 H), 7.03–7.25 (m, 2.5 H), 6.60 (dd, J = 7.3, 5.1 Hz, 1
H), 5.06–5.24 (m, 1 H), 4.95–5.05 (m, 1 H), 4.79–4.94 (m, 1
H), 3.83–3.99 (m, 1 H), 3.40–3.58 (m, 1 H), 3.23–3.40 (m, 1
H), 3.17 (s, 1.5 H), 3.02 (s, 1.5 H). 13C NMR (100 MHz,
DMSO-d6) δ ppm 165.93, 165.89, 163.49, 163.40, 139.98,
139.86, 139.25, 139.18, 139.03, 138.98, 137.51, 137.27, 133.86,
133.83, 133.52, 133.49, 130.93, 130.84, 130.77, 130.67, 129.46,
129.32, 129.11, 129.07, 129.07, 128.70, 128.63, 128.42, 127.89,
127.80, 127.53, 127.49, 126.78, 126.05, 125.61, 125.59, 121.43,
120.84, 116.67, 116.60, 116.43, 116.37, 58.66, 58.47, 54.01,
53.82, 49.63, 47.17, 35.29, 34.52. HRMS (m/z): [M + H]+, calcd
for C26H25F4N2O4S, 537.1466; found, 537.1444.
(E)-N-(2-((4-Ethyl-N-(2-
hydroxyethyl)phenyl)sulfonamido)benzyl)-N-methyl-3-(4-
(trifluoromethyl)phenyl)acrylamide (13)
(E)-N-(2-((4-Fluorophenyl)sulfonamido)benzyl)-N-methyl-3-(4-
(trifluoromethyl)phenyl)acrylamide (11)
This compound was prepared by the same procedure as for
12 and was purified with gradient silica gel column chroma-
tography from 7 : 3 EtOAc/hexane to 1 : 0 EtOAc/hexane,
affording a white solid (138 mg, 38%). 1H NMR (500 MHz,
DMSO-d6) δ ppm 7.99 (d, J = 8.07 Hz, 1 H), 7.79 (t, J = 8.3 Hz,
2 H), 7.69 (d, J = 8.1 Hz, 1 H), 7.60–7.68 (m, 1 H), 7.55 (d, J =
8.31 Hz, 1 H), 7.58 (d, J = 8.1 Hz, 1 H), 7.42–7.52 (m, 2.5 H),
7.29–7.41 (m, 1 H), 7.13–7.25 (m, 2.5 H), 7.03–7.13 (m, 1 H),
6.57 (dd, J = 7.6, 4.1 Hz, 1 H), 5.11–5.24 (m, 0.5 H), 5.01–5.11
(m, 0.5 H), 4.89–5.01 (m, 1 H), 4.72–4.88 (m, 1 H), 3.79–3.95
(m, 1 H), 3.40–3.58 (m, 1 H), 3.19–3.28 (m, 1 H), 3.14 (s, 1.5
H), 2.99 (s, 1.5 H), 2.64–2.78 (m, 2 H), 1.13–1.30 (m, 3 H). 13C
NMR (100 MHz, DMSO-d6) δ ppm 165.93, 165.90, 149.79,
149.62, 140.01, 139.87, 139.26, 139.22, 139.20, 139.02, 137.81,
137.58, 134.88, 134.58, 128.95, 128.74, 128.66, 128.60, 128.51,
128.46, 127.96, 127.82, 127.76, 127.43, 126.68, 125.97, 125.65,
125.62, 121.45, 120.85, 58.78, 58.58, 53.96, 53.76, 49.67,
47.20, 35.30, 34.57, 28.03, 15.03. HRMS (m/z): [M + H]+, calcd
for C28H30F3N2O4S, 547.1873; found, 547.1873.
Pyridine (0.44 ml, 5.4 mmol) was added to a solution of com-
pound 10 (300 mg, 0.9 mmol) in anhydrous DCM (6 mL) un-
der an argon atmosphere. The solution was chilled to ice–wa-
ter temperature and a solution of p-fluorobenzenesulfonyl
chloride (350 mg, 1.8 mmol) in anhydrous DCM (3 mL) was
added. Another equivalent of the sulfonyl chloride (175 mg,
0.9 mmol) in anhydrous DCM (1.5 mL) was added after 3 h
and the reaction was aged for an additional 24 hours. The so-
lution was washed with water, 5% HCl and brine. The or-
ganic layer was dried over anhydrous NaSO4, the suspension
was filtered and the filtrate was concentrated under reduced
pressure to dryness. The residue was purified on silica gel in
a gradient of 3 : 7 EtOAc/hexane to 1 : 1 EtOAc/hexane to af-
ford the title compound as a white solid (282 mg, 65%). 1H
NMR (500 MHz, CDCl3) δ ppm 10.31 (s, 1 H), 7.88 (s, 1 H),
7.77–7.86 (m, 2 H), 7.57–7.70 (m, 5 H), 7.29–7.38 (m, 1 H),
7.16 (dd, J = 7.4, 1.3 Hz, 1 H), 7.01–7.14 (m, 3 H), 6.75–6.90
(m, 1 H), 3.10 (s, 3 H), 4.18 (s, 2 H). 13C NMR (125 MHz,
CDCl3) δ ppm 167.42, 166.19, 164.17, 143.55, 138.30, 136.58,
136.54, 136.52, 131.81, 130.03, 129.99, 129.91, 128.40, 127.67,
127.28, 126.11, 126.09, 126.07, 125.11, 125.07, 123.81, 122.95,
120.79, 118.53, 116.35, 116.17, 49.50, 35.27. HRMS (m/z): [M +
H]+, calcd for C24H21F4N2O3S, 493.1204; found, 493.1219.
(E)-N-(2-((3-Fluoro-N-(2-hydroxyethyl)-4-
methylphenyl)sulfonamido)benzyl)-N-methyl-3-(4-
(trifluoromethyl)phenyl)acrylamide (14)
This compound was prepared by the same procedure as for
12 and was purified with gradient silica gel column chroma-
tography from 7 : 3 EtOAc/hexane to 1 : 0 EtOAc/hexane, giving
(E)-N-(2-((4-Fluoro-N-(2-
hydroxyethyl)phenyl)sulfonamido)benzyl)-N-methyl-3-(4-
(trifluoromethyl)phenyl)acrylamide (12)
1
a white solid (78 mg, 56%). H NMR (500 MHz, DMSO-d6) δ
2-Iodoethanol (24 μL, 0.3 mmol) and potassium carbonate
(70 mg, 0.5 mmol) were added to a solution of compound 11
(100 mg, 0.2 mmol) in N,N-dimethylformamide (DMF, 2 mL).
ppm 7.98 (d, J = 7.8 Hz, 1 H), 7.77 (t, J = 8.9 Hz, 2 H), 7.67 (d,
J = 8.1 Hz, 1.5 H), 7.63 (m, 1 H), 7.51–7.59 (m, 1 H), 7.45–7.51
(m, 1 H), 7.29–7.45 (m, 3 H), 7.02–7.25 (m, 2.5 H), 6.62 (d, J =
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7.6 Hz, 1 H), 5.11–5.22 (m, 0.5 H), 5.02–5.11 (m, 0.5 H), 4.92–
5.02 (m, 1 H), 4.71–4.91 (m, 1 H), 3.80–4.03 (m, 1 H), 3.49
(br. s., 1 H), 3.43 (br. s., 1 H), 3.22–3.38 (m, 3 H), 3.15 (s, 1.5
H), 2.99 (s, 1.5 H), 2.33 (s, 3 H). 13C NMR (100 MHz, DMSO-
d6) δ ppm 165.95, 165.91, 140.00, 139.90, 139.25, 139.21,
139.01, 137.45, 137.22, 136.76, 136.70, 132.61, 132.55, 130.85,
130.68, 130.51, 129.47, 129.12, 128.74, 128.70, 128.46, 127.89,
127.85, 127.55, 127.51, 126.78, 126.06, 125.65, 125.61, 123.91,
123.88, 123.76, 123.72, 121.43, 120.87, 114.50, 114.37, 114.25,
114.11, 58.69, 58.49, 54.08, 53.88, 49.63, 47.18, 35.30, 34.56,
14.37. HRMS (m/z): [M + H]+, calcd for C27H27F4N2O4S,
511.1622; found, 511.1606.
(E)-3-(4-Chlorophenyl)-N-(2-((4-fluoro-N-(2-
hydroxyethyl)phenyl)sulfonamido)benzyl)-N-methylacrylamide
(17)
This compound was prepared by the same procedure as for 12
and was purified with gradient silica gel column chromatogra-
phy from 7 : 3 EtOAc/hexane to 1 : 0 EtOAc/hexane, affording a
white solid (142 mg, 39%). 1H NMR (400 MHz, DMSO-d6) δ
ppm 7.80 (d, J = 7.9 Hz, 1 H), 7.66–7.77 (m, 2 H), 7.53–7.66
(m, 2 H), 7.43–7.53 (m, 3 H), 7.29–7.43 (m, 2.5 H), 7.20 (q, J =
7.5 Hz, 1 H), 6.95–7.15 (m, 1.5 H), 6.48–6.66 (m, 1 H), 5.02–
5.22 (m, 1 H), 4.91–5.02 (m, 1 H), 4.74–4.91 (m, 1 H), 3.75–
3.99 (m, 1 H), 3.40–3.59 (m, 1 H), 3.21–3.40 (m, 2 H), 3.14 (s,
1.5 H), 3.00 (s, 1.5 H). 13C NMR (100 MHz, DMSO-d6) δ ppm
166.12, 166.09, 140.36, 140.29, 139.31, 139.11, 137.46, 137.22,
134.07, 134.04, 133.89, 133.83, 130.90, 130.81, 130.74, 130.65,
129.80, 129.50, 129.04, 128.78, 128.60, 127.84, 127.44, 126.76,
126.00, 119.24, 118.70, 116.65, 116.58, 116.43, 116.36, 58.64,
58.45, 53.98, 53.81, 49.58, 47.13, 35.26, 34.49. HRMS (m/z): [M
+ H]+, calcd for C25H25ClN2O4S, 503.1202; found, 503.1205.
(E)-N-(2-(N-(2-Hydroxyethyl)phenylsulfonamido)benzyl)-N-
methyl-3-(4-(trifluoromethyl)phenyl)acrylamide (15)
This compound was prepared by the same procedure as for
12 and was purified with gradient silica gel column chroma-
tography from 7 : 3 EtOAc/hexane to 1 : 0 EtOAc/hexane,
affording a white solid (80 mg, 73%). 1H NMR (500 MHz,
DMSO-d6) δ ppm 8.00 (d, J = 7.6 Hz, 1 H), 7.72–7.87 (m, 3 H),
7.58–7.72 (m, 6 H), 7.44–7.56 (m, 0.5 H), 7.27–7.42 (m, 1 H),
7.02–7.25 (m, 2.5 H), 6.53 (t, J = 6.8 Hz, 1 H), 5.05–5.25 (m, 1
H), 4.93–5.04 (m, 1 H), 4.76–4.92 (m, 1 H), 3.80–4.04 (m, 1
H), 3.52 (br. s., 0.5 H), 3.46 (br. s., 0.5 H), 3.20–3.30 (m, 1.5
H), 3.16 (s, 1 H), 3.01 (s, 1.5 H). 13C NMR (100 MHz, DMSO-
d6) δ ppm 165.92, 165.89, 139.95, 139.84, 139.25, 139.02,
138.99, 137.64, 137.46, 137.40, 137.13, 133.40, 133.26, 129.45,
129.32, 129.26, 129.14, 128.96, 128.67, 128.53, 128.40, 127.73,
127.60, 127.45, 127.37, 126.71, 125.98, 125.59, 125.55, 125.51,
125.46, 125.36, 122.75, 122.65, 121.42, 120.85, 58.72, 58.52,
54.04, 53.84, 49.64, 47.18, 35.28, 34.50. HRMS (m/z): [M + H]+,
calcd for C26H26F3N2O4S, 519.1560; found, 519.1544.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by an NIH grant (AI104987). ES was
the recipient of an ECK Institute for Global Health graduate
student fellowship.
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(E)-3-(4-Chlorophenyl)-N-(2-((N-(2-hydroxyethyl)-4-
methylphenyl)sulfonamido)benzyl)-N-methylacrylamide (16)
This compound was prepared by the same procedure as for
12 and was purified with gradient silica gel column chroma-
tography from 7 : 3 EtOAc/hexane to 1 : 0 EtOAc/hexane, pro-
ducing a white solid (190 mg, 69%). 1H NMR (500 MHz,
CDCl3) δ ppm 7.80 (d, J = 8.5 Hz, 1 H), 7.61 (d, J = 8.3 Hz, 1
H), 7.58 (d, J = 6.6 Hz, 0.5 H), 7.55 (d, J = 7.3 Hz, 1.5 H), 7.52
(d, J = 8.3 Hz, 1 H), 7.49 (d, J = 8.6 Hz, 1 H), 7.44 (t, J = 7.7
Hz, 2 H), 7.35–7.41 (m, 1.5 H), 7.29–7.35 (m, 1 H), 7.13–7.23
(m, 1 H), 6.98–7.13 (m, 1.5 H), 6.56 (dd, J = 7.7, 3.1 Hz, 1 H),
5.01–5.16 (m, 1 H), 4.89–5.00 (m, 1 H), 4.73–4.89 (m, 1 H),
3.78–3.96 (m, 1 H), 3.40–3.55 (m, 1 H), 3.19–3.26 (m, 1 H),
3.14 (s, 1.5 H), 2.99 (s, 1.5 H), 2.42 (s, 3 H). 13C NMR (100
MHz, DMSO-d6) δ ppm 166.13, 166.11, 143.84, 143.67, 140.39,
140.32, 139.35, 139.15, 137.77, 137.53, 134.64, 134.35, 134.11,
134.07, 134.03, 133.92, 129.86, 129.81, 129.74, 129.56, 128.93,
128.81, 128.49, 127.82, 127.77, 127.70, 127.38, 126.68, 125.94,
119.26, 118.73, 58.76, 58.55, 53.96, 53.76, 49.63, 47.18, 35.28,
34.55, 21.08. HRMS (m/z): [M + H]+, calcd for C26H32N2O3S,
451.2050; found, 451.2068.
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