Systematic study of the glutathione (GSH) reactivity of N-arylacrylamides: 1. Effects of aryl substitution

Article abstract of DOI:10.1021/acs.jmedchem.5b01018

Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relationships among N-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between ¹H and ¹³C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion.

Full text of DOI:10.1021/acs.jmedchem.5b01018

Article
pubs.acs.org/jmc
Systematic Study of the Glutathione (GSH) Reactivity of
N‑Arylacrylamides: 1. Effects of Aryl Substitution
Victor J. Cee,* Laurie P. Volak, Yuping Chen, Michael D. Bartberger,* Chris Tegley, Tara Arvedson,
John McCarter, Andrew S. Tasker, and Christopher Fotsch
Departments of Medicinal Chemistry, Pharmacokinetics and Drug Metabolism, Molecular Structure, Discovery Technologies, and
Oncology Research, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States
S
* Supporting Information
ABSTRACT: Success in the design of targeted covalent
inhibitors depends in part on a knowledge of the factors
influencing electrophile reactivity. In an effort to further
develop an understanding of structure−reactivity relationships
among N-arylacrylamides, we determined glutathione (GSH)
reaction rates for a family of N-arylacrylamides independently
substituted at ortho-, meta-, and para-positions with 11
different groups common to inhibitor design. We find that
substituent effects on reaction rates show a linear Hammett
correlation for ortho-, meta-, and para-substitution. In
1
addition, we note a correlation between H and 13C NMR
chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate
measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation
parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic
candidates in a prospective fashion.
INTRODUCTION
■
Recent FDA approvals of covalent inhibitors targeting non-
catalytic cysteine residues of kinases BTK (ibrutinib) and EGFR
(afatinib) for the treatment of mantle cell lymphoma/chronic
lymphocytic leukemia and non-small-cell lung cancer, respec-
tively, have fueled a renewed interest in targeted covalent
inhibitors of cysteine-containing proteins.1 To fully achieve the
potential of targeted covalent inhibitors, strategies to maximize
Figure 1. N-Arylacrylamides. Indicated substituents are independently o
(ortho), m (meta), and p (para).
target protein engagement and minimize off-target reactions are
necessary. Key to such strategies will be an understanding of the
factors influencing electrophile reactivity. Although the acryl-
amide functional group has become a widely used electrophile in
cysteine-targeted covalent inhibitor design,2 the relative
reactivity of structurally diverse acrylamides had not been
described until very recently.3 N-Arylacrylamides are featured in
a number of targeted covalent inhibitors undergoing clinical
testing including AZD9291,3b rociletinib (CO-1686),4 and
spebrutinib (AVL-292/CC-292).5 In an effort to further develop
an understanding of structure−reactivity relationships among N-
arylacrylamides, we determined glutathione (GSH) reaction
rates for a family of N-arylacrylamides independently substituted
at ortho-, meta-, and para-positions with 11 different groups
common to inhibitor design.
ortho-, meta-, and para-positions (2o−12o, 2m−12m, and
2p−12p, respectively). The acrylamides were either procured
from commercial sources or synthesized from the corresponding
anilines and acryloyl chloride. NMR spectra (1H and 13C) were
obtained for all acrylamides, and for determining correlations
between GSH reactivity and chemical shift, we found it
convenient to focus on the chemical shifts of the proton defined
as Hb1 (the most upfield doublet of doublets in the acrylamide
system) and the carbon defined as Cb (easily identified in a
distortionless enhancement by polarization transfer6 (DEPT)
experiment as the only carbon bound to two hydrogen atoms).
Experimental GSH Reaction Rates and Correlations.
For determination of rates of reaction with GSH, the acrylamide
reactant (1 μM) was incubated at 37 °C with 5 mM GSH in 67
RESULTS AND DISCUSSION
■
The acrylamides chosen for study are shown in Figure 1 and
include the parent N-phenylacrylamide (1) as well as N-
arylacrylamides containing a wide-range of substituents at
Received: June 29, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b01018
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Journal of Medicinal Chemistry
Article
Table 1. Half-Lives and Reaction Rates for Benchmark Electron-Rich and Electron-Deficient N-Arylacrylamides
a
b
compd
R
N
GSH t1/2 (min)
kpseudo1st (min−1 × 10−3
)
log kGSH (M−1 s−1
)
1
H
4
179 18
247 18
7.90 0.20
3.86 0.43
2.80 0.21
87.7 2.3
−1.89 0.05
−2.03 0.03
−0.534 0.01
11p
12p
para-NMe2
para-NO2
2
3
a
b
Number of independent determinations. kGSH is defined as in ref 3b: kpseudo1st/[GSH], with [GSH] = 5 mM.
a
Table 2. Reaction Rates, Half-Lives, and NMR Chemical Shifts for N-Arylacrylamides
b
c
kpseudo1st (min−1 × 10−3
)
log kGSH (M−1 s−1
)
Hb1 δ (ppm)
Cb δ (ppm)
−
compd
R
Hammett σm or σp
GSH t1/2 (min)
d
d
d
1
H
F
179
3.86
−1.89
5.75
5.79
5.78
5.74
5.78
5.65
5.83
5.84
5.71
5.74
5.72
5.83
5.79
5.79
5.73
5.81
5.78
5.79
5.82
5.75
5.76
5.71
5.84
5.75
5.77
5.72
5.82
5.77
5.81
5.83
5.71
5.74
5.67
5.86
126.8
127.5
127.4
126.4
127.2
126.4
127.4
128.1
126.5
126.7
126.4
128.2
127.4
127.5
126.6
127.6
126.9
127.3
127.9
126.9
127
2o
−0.03
0.19
110
48.3
311
63.5
223
29.9
25.8
6.29
14.4
2.23
10.9
3.11
23.2
26.9
−1.68
−1.32
−2.13
−1.44
−1.98
−1.11
−1.05
−1.92
−1.50
−1.55
3o
Cl
4o
Me
−0.17
0.65
5o
CF3
Ph
6o
0.02
7o
CO2Me
CN
0.64
8o
1.00
e
e
e
9o
OMe
SMe
NMe2
NO2
F
−0.26
0.06
194
3.58
10o
11o
12o
2m
3m
4m
5m
6m
7m
8m
9m
10m
11m
12m
2p
72.6
9.55
e
e
e
−0.12
1.27
82.7
8.38
e
e
e
6.00
115
−0.416
0.337
0.373
−0.069
0.43
71.6
68.1
213
61.8
103
116
41.4
116
87.3
9.68
10.2
3.25
11.2
6.74
5.99
16.7
6.00
7.94
−1.49
−1.47
−1.97
−1.43
−1.65
−1.70
−1.25
−1.70
−1.58
Cl
Me
CF3
Ph
0.06
CO2Me
CN
0.36
0.56
OMe
SMe
NMe2
NO2
F
0.115
0.15
f
f
f
−0.15
0.71
152
4.56
−1.82
−1.04
126.4
128
e
e
e
25.5
27.2
−0.03
0.19
159
92.5
265
39.5
117
23.3
17.3
342
136
4.36
7.49
2.62
17.5
5.90
29.7
40.1
2.03
5.09
−1.84
−1.60
−2.06
−1.23
−1.71
−1.00
−0.874
−2.17
−1.77
126.9
127.2
126.5
127.8
126.9
127.8
128.2
126.2
126.7
125.6
128.7
3p
Cl
4p
Me
−0.17
0.65
5p
CF3
Ph
6p
0.02
7p
CO2Me
CN
0.64
8p
1.00
9p
OMe
SMe
NMe2
NO2
−0.26
0.06
10p
11p
12p
e
e
e
−0.12
1.27
247
2.80
−2.03
f
f
f
7.90
87.7
−0.534
a
b
−
Data represent single determinations unless otherwise indicated. For ortho- and para-, σp is listed; for meta, σm is listed; values were obtained
from ref 6. kGSH is defined as in ref 3b: kpseudo1st/[GSH], with [GSH] = 5 mM. Average of four determinations. Average of two determinations.
Average of three determinations. For statistical information, see the Supporting Information.
c
d
e
f
mM potassium phosphate buffer, pH 7.4 (1.0−1.5% DMSO),
and the samples were analyzed with an LC/MS system that
consisted of a Leap autosampler and an AB Sciex TripleTOF
5600 mass spectrometer. The concentration of GSH was chosen
to approximate intracellular concentrations of GSH (reported7
range 0.5−10 mM). Samples were directly injected from the
incubation immediately after GSH was added to the mixture (less
than 2 min after the acrylamide reactants were dosed into buffer)
and then at predetermined intervals over 120 min. The analytes
were separated with a Waters Xselect HSS T3 2.5 μm (2.1 mm ×
30 mm) column, and the mobile phase consisted of 0.1% formic
acid in water/acetonitrile with a linear gradient of organic phase
increasing from 5% to 95% over the 1 min run time. To increase
throughput, compounds were tested in pools of 10−15
compounds, and three controls were routinely included in each
pool. Compounds were added as 10 mM DMSO solutions, and
the final DMSO content varied from 1.0% to 1.5% depending on
the number of compounds pooled. From the mass spectrum,
peak area ratios (peak area analyte/peak area of a stable internal
standard) were calculated and the percent compound remaining
was determined relative to time zero. Rates were calculated by
fitting to a pseudo-first-order kinetic equation to natural log
transformed percent remaining data (eq 1). Half-lives were
calculated according to eq 2.
ln([acrylamide]) = −kpseudo1stt + ln([acrylamide0])
(1)
t1/2 = 0.693/kpseudo1st
(2)
B
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Article
−
Figure 2. Rate of GSH addition is correlated with Hammett sigma values. (a) Ortho-substituted 2o−12o, plot of log kGSH (M−1 s−1) vs σp . (b) Meta-
−
substituted 2m−12m, plot of log kGSH (M−1 s−1) vs σm. (c) Para-substitued 2p−12p, plot of log kGSH (M−1 s−1) vs σp .
Figure 3. GSH reaction rate increases with increasing chemical shift. (a) Acrylamide proton (Hb1) chemical shift (400 MHz, DMSO-d6). (b) Acrylamide
carbon (Cb) chemical shift (100 MHz, DMSO-d6). Red, green, and purple coloration correspond to ortho-, meta-, and para-substitution patterns,
respectively. Parent denotes N-phenylacrylamide 1.
The maximum half-life that could be determined with a 120
min incubation was set at 512 min based on the ability to
confidently detect 15% parent depletion, taking all uncertainty in
the measurement into consideration. A control experiment in
which GSH was not present was simultaneously conducted to
confirm that the observed disappearance of acrylamide reactant
was GSH-dependent. All compounds reported herein showed
only GSH-dependent disappearance. The reproducibility and
stability of the assay were confirmed by multiple determinations
on different days for GSH reaction with N-phenylacrylamide (1)
as well as the electron-rich N-(4-(dimethylamino)phenyl)-
acrylamide (11p) and electron-poor N-(4-nitrophenyl)-
acrylamide (12p) (Table 1). Low variability was observed across
different determinations, as evidenced by the low standard
deviations. An additional confirmatory experiment was per-
formed in which the rate of formation of the GSH-adduct was
measured for N-phenylacrylamide (1). The rate of GSH-adduct
formation was observed to closely match the rate of N-
phenylacrylamide disappearance (Figure SI-1). The mean GSH
half-life for N-phenylacrylamide (1, 179 min) under these
conditions is between that reported by Flanagan et al.3a (53 min,
1 mM electrophile, 10 mM GSH, 100 mM, pH 7.4 phosphate
buffer, 10% acetonitrile) and Ward et al.3b (299 min, 50 μM
electrophile, 4.6 mM GSH, pH 7.4 phosphate buffer; phosphate
concentration not indicated). We suspect that differences in
experimental parameters such as concentration, cosolvent, and
ionic strength are responsible for differences in the measured
value for N-phenylacrylamide and stress that the relative trends
observed under a given set of conditions will have the most value.
The full set of N-arylacrylamides (Figure 1) was then
evaluated, and the data are presented in Table 2. The aryl-ring
substituents are observed to have the largest impact on GSH t1/2
at the ortho- and para-positions (GSH t1/2 range of 6.00−311 and
7.90−342 min, respectively) and a less dramatic impact at the
meta-position (GSH t1/2 range of 25.5−213 min). The impact of
the individual substituents on GSH reactivity was evaluated by
fitting the data to the Hammett equation (eq 3) and determining
the set of Hammett σ values (also known as substituent
constants) that provided the best linear fit to the data (Figure 2).
From the best linear fit to the data, the reaction constant ρ, a
measure of the sensitivity of the reaction to substituent effects
relative to the substituent constants, is obtained from the slope,
while the intercept provides the constant c, which is equivalent to
the log of the rate of the unsubstituted reference compound.
log k = ρσ + c
(3)
We find that the best fit is obtained by using σp− for both ortho
(Figure 2a, R2 = 0.80) and para (Figure 2c, R2 = 0.98)
substituents and σm for meta-substituents (Figure 2b, R2 = 0.83).
The derived values for ρ are near unity (ortho, 0.85; meta, 0.89;
para, 1.0), and the intercept c (ortho, −1.7 M−1 s−1; meta, −1.7
M−1 s−1; para, −1.8 M−1 s−1) is nearly identical to log kGSH of the
unsubstituted N-phenylacrylamide (compound 1, Table 2,
log kGSH − 1.89 M−1 s−1). The ρ values presented here are
lower than that found in a study published recently by Ward et
al.3b (5.56), and we believe the discrepancy may be due to the
smaller data set used in the latter analysis (one meta- and five
para-substituents were studied), as well as the use of a single
substituent constant for all substituents, regardless of position.
−
The observation that σp (derived from ionization of phenols)
provides an improved correlation relative to σp (derived from
ionization of benzoic acids) indicates that through-resonance
effects are significant in the addition of GSH to ortho- and para-
N-arylacrylamides; through resonance is generally not consid-
ered for meta-substituents.8 It is also notable that ortho-
substituted N-phenylacrylamides are well-modeled by a simple
Hammett correlation, and consideration of steric and proximity
polar effects unique to ortho-substituents as advanced by the
C
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Fujita−Nishioka equations9 appears to be unnecessary in this
case. The reason for this may be that in the case of N-
arylacrylamides, the reacting center is separated from the
aromatic system by three atoms and is therefore less affected
by steric and proximity polar effects.
We observe a reasonable correlation between proton and
carbon chemical shift (Hb1 and Cb, respectively, as defined in
Figure 1) and GSH reaction rate values (Figure 3) for this group
of closely related N-arylacrylamides, with R2 values of 0.71 and
0.80, respectively. Examination of the ortho-, meta-, and para-
subsets indicates that the correlation between chemical shift and
reaction rate is somewhat better for the meta- and para-subsets
than the ortho-subset, an outcome that was also observed in the
correlation to Hammett σ values (Figure 2).10 The finding that
downfield chemical shifts are associated with increased reactivity
suggests that the relative electron deficiency of the acrylamide is
responsible for both effects.11 While the correlation observed
among the structurally similar acrylamide electrophiles reported
in this study does not appear to hold as well as substrate diversity
increases (i.e., substituted and/or non-arylacrylamides),3a our
results suggest that NMR chemical shifts may still provide utility
as a rapid screening metric within a homologous series of
substrates.
To more readily visualize these data, we present a heat map of
substituent effects on GSH reaction rates relative to N-
phenylacrylamide (Table 3). Values are the fold-change in
Table 3. Heatmap of Fold-Change in kGSH Relative to N-
Phenylacrylamide (1)
a
Figure 4. Transition structures and relevant geometric parameters for
methanethiolate addition to N-phenylacrylamide 1 at the IEFPCM-
B3LYP/6-311+G(d,p) level of theory: (a) top and side views of TS-syn-
1; (b) top and side views of TS-syn-2; (c) single view of TS-syn-3; (d)
top and side views of TS-anti-1; (e) single view of TS-anti-2. See Table
4 for comparison of relative energies and geometric parameters
determined by the IEFPCM versus SMD solvation models. Interatomic
distances are in ångstroms, and angles are in degrees.
for a syn-type orientation of the methanethiolate nucleophile is in
accord with high-level CBS-QB3 calculations by Krenske and
Houk on model enone−methanethiolate systems.16 A prior
study reporting a theoretical analysis of methanethiolate addition
to acrylamides at the same level of theory appeared to utilize an
anti-type transition structure to the s-cis conformer (Figure 4d,
TS-anti-1 (s-cis)).3a
a
Values are the fold-change in reaction rate relative to N-phenyl-
acrylamide. Color scale is shown as a gradient with green = 0.5
(minimum) and red = 26 (maximum).
Activation parameters (determined in the typical fashion,
utilizing the lowest-energy conformation of the TS compared to
the lowest-energy s-cis conformer of the reactant) were calculated
for the family of acrylamides in Table 2. In all cases, a TS-syn-1-
like TS was found to be most favored. These results are reported
in Table 5.
reaction rate relative to N-phenylacrylamide (1), with values of
<1 indicating a slower rate of reaction with GSH and with values
>1 indicating a faster rate of reaction with GSH. For
polysubstituted N-arylacrylamides, we expect the substituent
effects presented in Table 3 to be additive provided that the
substituents are able to adopt conformations similar to the simple
monosubstituted N-arylacrylamides.12
Theoretical Analysis: Transition Structures and Acti-
vation Parameters. The addition of methanethiolate anion to
N-phenylacrylamide (1) was studied at the B3LYP/6-311+G-
(d,p) level of theory with the inclusion of solvent effects via the
IEFPCM-SMD13 method, and a number of transition structures
for methanethiolate addition to both s-cis and s-trans N-
phenylacrylamide and aryl-substituted analogs have been located
(Figure 4 and Table 4).14,15 A syn-like orientation of the
nucleophile with respect to the acrylamide CC bond is
preferred (Figure 4a, TS-syn-1 (s-cis)). The general preference
The computed activation parameters Ea and ΔG⧧, determined
in this fashion, exhibit a reasonably good correlation (R2 = 0.83
and 0.75, respectively) with experimental reaction rates (Figure
5). Similar methodology has been successfully applied to the
reaction of nitriles with cysteine, suggesting that the determi-
nation of activation parameters with density functional theory
will have broad utility in the prediction of reaction rates of sulfur
nucleophiles with covalent warheads.17 Inspection of the
individual data points for ortho-, meta-, and para-substitution
indicates a somewhat tighter correlation for the last two
substitution patterns, as was found for both Hammett constants
and chemical shift values (Figures 2 and 3, respectively).18
D
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Journal of Medicinal Chemistry
Article
Table 4. Comparison of IEFPCM and SMD-IEFPCM Energetic and Geometric Data for Methanethiolate Addition Transition
a
Structures of 1
b
b
TS
r(CC) (Å)
r(C−S) (Å)
∠CCS (deg)
φ(C−C−S−C) (deg)
Erel (kcal/mol)
ΔGrel (kcal/mol)
cd
,
TS-syn-1
1.420
(1.394)
1.422
e
2.176
(2.376)
2.171
e
115.9
(114.0)
116.2
e
−20.9
(−16.6)
23.3
0
0
(0)
0.2
e
(0)
0.7
e
cd
,
TS-syn-2
TS-syn-3
TS-anti-1
TS-anti-2
e
cd
,
1.430
(1.400)
1.402
(1.388)
1.411
(1.392)
2.144
(2.347)
2.291
(2.429)
2.248
(2.410)
116.4
(113.8)
116.3
(117.7)
115.9
(115.8)
−33.1
(−22.8)
177.0
(175.1)
168.1
(160.1)
0.4
(0.6)
1.4
(1.8)
1.9
(2.4)
1.5
(0.4)
0.2
(0.7)
1.9
(0.8)
cdf
, ,
cd
,
a
Data for fully optimized transition structures independently optimized at the B3LYP/6-311+G(d,p) level of theory using both implicit solvation
b
models. All transition structures characterized by Hessian and internal reaction coordinate (IRC) analysis. Temperature and frequency scale factors
of 310.15 K and 0.9877, respectively, were utilized for zero-point energy and entropic contributions in order to attempt consistency with ref 3a.
c
d
e
Data for IEFPCM solvation model in top row (see Figure 4). Data for IEFPCM-SMD solvent model parenthesized in italics in bottom row. TS-
f
syn-2 converges to TS-syn-1 on the IEFPCM-SMD-B3LYP/6-311+G(d,p) potential energy surface. Transition structure reported for
methanethiolate addition to α,β-unsubstituted acrylamides in ref 3a.
a
Table 5. Calculated Activation Energies (Ea) and Free Energies (ΔG⧧) in Implicit Water Solvent (B3LYP/6-311+G(d,p)
b
b
b
b
compd
R
Ea (kcal/mol)
ΔG⧧ (kcal/mol)
compd
R
Ea (kcal/mol)
ΔG⧧ (kcal/mol)
c
c
1
H
F
8.9 (4.4)
19.2 (15.5)
7m
8m
9m
10m
11m
12m
2p
CO2Me
CN
8.0
6.9
8.6
8.4
9.6
6.7
8.7
8.1
9.7
6.8
8.5
6.6
6.0
10.0
8.4
11.1
4.5
19.1
17.5
19.5
19.3
20.7
17.6
19.8
19.1
20.7
18.0
19.7
17.9
17.1
20.8
19.0
22.4
16.0
2o
7.6
7.3
8.9
6.7
8.8
7.6
5.7
9.4
8.4
9.9
4.4
7.5
7.6
9.0
7.2
8.7
18.2
18.0
20.3
17.7
19.8
18.8
17.3
20.2
19.4
21.3
15.4
18.3
18.5
19.7
17.8
19.8
3o
Cl
OMe
SMe
NMe2
NO2
F
4o
Me
5o
CF3
Ph
6o
7o
CO2Me
CN
OMe
SMe
NMe2
NO2
F
8o
3p
Cl
9o
4p
Me
10o
11o
12o
2m
3m
4m
5m
6m
5p
CF3
6p
Ph
7p
CO2Me
CN
8p
Cl
9p
OMe
SMe
NMe2
NO2
Me
10p
11p
12p
CF3
Ph
a
Data for fully optimized transition structures independently optimized at the IEFPCM-B3LYP/6-311+G(d,p) level of theory. IEFPCM utilized due
b
to favorable geometric convergence behavior. Temperature and frequency scale factors of 310.15 K and 0.9877, respectively, were utilized for zero-
point energy and entropic contributions in order to attempt consistency with ref 3a. Data for IEFPCM-SMD evaluation of 1 parenthesized in italics.
c
Figure 5. Graph of log kGSH versus computed (a) activation energy (Ea) and (b) activation free energy (ΔG⧧) for systems in Table 5 at the IEFPCM-
B3LYP/6-311+G(d,p) level of theory. Red, green, and purple coloration correspond to ortho-, meta-, and para-substitution patterns, respectively.
Parent denotes N-phenylacrylamide 1.
arylacrylamides independently substituted at ortho-, meta-, and
para-positions with 11 different groups common to inhibitor
design. We find that substituent effects on reaction rates show a
linear Hammett correlation for ortho-, meta-, and para-
CONCLUSIONS
■
In an effort to further develop an understanding of structure−
reactivity relationships among N-arylacrylamides, we determined
glutathione (GSH) reaction rates for a family of N-
E
DOI: 10.1021/acs.jmedchem.5b01018
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Article
substitution. In addition, we note a correlation between 1H and
13C NMR chemical shifts of the acrylamide with GSH reaction
rates, suggesting that NMR chemical shifts may indeed be a
convenient surrogate measure of relative acrylamide reactivity.
Our density functional theory calculations characterize the
nature of the transition structures as involving a preferential syn-
like orientation of the methanethiolate nucleophile upon
addition to the α,β-unsubstituted N-arylacrylamides utilized in
this study. A correlation between computed activation
parameters and experimentally determined reaction rates was
observed, validating the use of such methodology for the
screening of synthetic candidates in a prospective fashion. We
believe that this comprehensive study elucidates the key
structure−reactivity relationships for N-arylacrylamides and
will allow for more informed strategies for targeted covalent
inhibitor design in three ways: (1) the experimental reaction
rates presented here may be used to select N-aryl substituents
that will provide a desired level of reactivity; (2) proton and
carbon NMR chemical shifts may be used to quickly estimate the
reactivity of compounds that have already been prepared; (3)
DFT-computed activation energies for methanethiolate addition,
still quite modest in terms of CPU demand (on the order of a few
hours per system on a typical workstation), can be used to
prospectively estimate the reactivity of designed compounds
containing N-aryl substituents that have not been addressed by
this study. Additional studies involving α- and/or β-substituted
acrylamides will be reported in due course.
127.5, 115.1, 115.0, 110.0, 109.8, 106.2, 106.0. m/z (ESI, +ve ion) 166.1
(M + H)+.
N-(3-Fluorophenyl)acrylamide (2m). 1H NMR (400 MHz,
DMSO-d6) δ 10.33 (br s, 1H), 7.68 (d, J = 11.74 Hz, 1H), 7.32−7.39
(m, 2H), 6.86−6.93 (m, 1H), 6.42 (dd, J = 9.98, 17.02 Hz, 1H), 6.28
(dd, J = 2.05, 16.92 Hz, 1H), 5.79 (dd, J = 1.96, 9.98 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6) δ 163.4, 163.3, 160.9, 140.7, 140.6, 131.5, 130.4,
130.3, 127.4, 115.1, 115.0, 110.0, 109.8, 106.2, 106.0. m/z (ESI, +ve ion)
166.1 (M + H)+.
N-(4-Fluorophenyl)acrylamide (2p). 1H NMR (400 MHz,
DMSO-d6) δ 10.17 (br s, 1H), 7.65−7.72 (m, 2H), 7.16 (t, J = 8.41
Hz, 2H), 6.41 (dd, J = 10.07, 16.92 Hz, 1H), 6.26 (dd, J = 2.05, 16.92 Hz,
1H), 5.75 (dd, J = 2.15, 9.98 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 163.0, 159.3, 156.9, 135.4, 135.4, 131.7, 126.9, 121.1, 121.0, 115.4,
115.2. m/z (ESI, +ve ion) 166.1 (M + H)+.
N-(2-Chlorophenyl)acrylamide (3o). 1H NMR (400 MHz,
DMSO-d6) δ 9.71 (br s, 1H), 7.78 (d, J = 7.62 Hz, 1H), 7.51 (dd, J =
1.37, 8.02 Hz, 1H), 7.34 (dt, J = 1.57, 7.73 Hz, 1H), 7.21 (dt, J = 1.76,
7.73 Hz, 1H), 6.62 (dd, J = 10.17, 17.02 Hz, 1H), 6.28 (dd, J = 1.96,
17.02 Hz, 1H), 5.78 (dd, J = 1.96, 10.37 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 163.4, 134.7, 131.3, 129.4, 127.3, 126.6, 126.4, 126.3. m/z
(ESI, +ve ion) 182 (M + H)+.
N-(3-Chlorophenyl)acrylamide (3m). 1H NMR (400 MHz,
DMSO-d6) δ 10.31 (s, 1H), 7.90 (t, J = 1.96 Hz, 1H), 7.51 (d, J =
7.86 Hz, 1H), 7.35 (t, J = 8.12 Hz, 1H), 7.13 (ddd, J = 0.88, 2.10, 7.97 Hz,
1H), 6.42 (dd, J = 9.98, 17.02 Hz, 1H), 6.28 (dd, J = 1.96, 17.02 Hz, 1H),
5.79 (dd, J = 2.05, 10.07 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
163.4, 140.4, 133.0, 131.5, 130.4, 127.5, 123.1, 118.8, 117.7. m/z (ESI,
+ve ion) 182.1 (M + H)+.
N-(4-Chlorophenyl)acrylamide (3p). 1H NMR (400 MHz,
DMSO-d6) δ 10.26 (s, 1H), 7.64−7.75 (m, 2H), 7.31−7.45 (m, 2H),
6.42 (dd, J = 10.17, 17.02 Hz, 1H), 6.27 (dd, J = 2.05, 16.92 Hz, 1H),
5.77 (dd, J = 2.05, 10.07 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
163.2, 137.9, 131.6, 128.6, 127.2, 127.0, 120.8. m/z (ESI, +ve ion) 182.1
(M + H)+.
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. Anhydrous
solvents were obtained from Aldrich and used directly. All reactions
involving air- or moisture-sensitive reagents were performed under a
nitrogen or argon atmosphere. Silica gel chromatography was performed
using medium pressure liquid chromatography (MPLC) on a
CombiFlash Companion (Teledyne Isco) with RediSep normal-phase
silica gel (35−60 μm) columns and UV detection at 254 nm. All
compounds were purified to ≥95% purity as determined by Agilent
1100 series high performance liquid chromatography (HPLC) with UV
detection at 254 nm (Phenomenex Synergi, 2 mm × 50 mm, 3 min, 1.0
mL/min flow rate, 0−95% 0.1% TFA in CH3CN/0.1% TFA in H2O).
NMR spectra were determined with a Bruker AV-400 (400 MHz) at
ambient temperature. Low-resolution mass spectral (MS) data were
determined on an Agilent 1100 series LCMS with UV detection at 254
nm and a low resonance electrospray mode (ESI). Chemical shifts are
reported in ppm from the solvent resonance (DMSO-d6, 2.50 ppm).
Data are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling
constants, and number of protons.
N-(2-Methylphenyl)acrylamide (4o). 1H NMR (400 MHz,
DMSO-d6) δ 9.46 (br s, 1H), 7.47 (d, J = 7.82 Hz, 1H), 7.07−7.24
(m, 3H), 6.54 (dd, J = 10.27, 17.12 Hz, 1H), 6.24 (dd, J = 1.96, 17.02 Hz,
1H), 5.74 (dd, J = 2.15, 10.17 Hz, 1H), 2.21 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 163.2, 136.0, 131.8, 131.6, 130.3, 126.4, 125.9,
125.2, 124.9, 124.8, 17.9. m/z (ESI, +ve ion) 162.1 (M + H)+.
N-(3-Methylphenyl)acrylamide (4m). 1H NMR (500 MHz,
DMSO-d6) δ ppm 10.03 (br s, 1 H), 7.50 (s, 1 H), 7.45 (d, J = 8.0
Hz, 1 H), 7.19 (t, J = 7.9 Hz, 1 H), 6.88 (d, J = 7.4 Hz, 1 H), 6.43 (dd, J =
17.0, 10.2 Hz, 1 H), 6.24 (dd, J = 17.0, 1.9 Hz, 1 H), 5.73 (dd, J = 10.1,
1.9 Hz, 1 H), 2.28 (s, 3 H). 13C NMR (101 MHz, DMSO-d6) δ 163.0,
138.9, 137.9, 131.9, 128.6, 126.6, 124.1, 119.8, 116.5, 21.2. m/z (ESI,
+ve ion) 162.1 (M + H)+.
N-(4-Methylphenyl)acrylamide (4p). 1H NMR (500 MHz,
DMSO-d6) δ ppm 10.02 (br s, 1 H), 7.55 (m, J = 8.3 Hz, 2 H), 7.12
(m, J = 8.2 Hz, 2 H), 6.42 (dd, J = 17.0, 10.2 Hz, 1 H), 6.23 (dd, J = 17.0,
1.9 Hz, 1 H), 5.72 (dd, J = 10.1, 1.9 Hz, 1 H), 2.25 (s, 3 H). 13C NMR
(101 MHz, DMSO-d6) δ 162.9, 136.5, 132.4, 131.9, 129.1, 126.5, 119.3,
20.4. m/z (ESI, +ve ion) 162.1 (M + H)+.
General Procedure for Synthesis of Noncommercial Acryl-
amides. To a solution of aniline (1 mmol) in 4 mL DCM at 0 °C was
added N,N-diisopropylethylamine (2 mmol) and acryloyl chloride (1
mmol). Upon completion, the reaction was concentrated and was
purified by silica gel chromatography. The product-containing fractions
were combined and concentrated to give the desired acrylamides.
N-Phenylacrylamide (1). 1H NMR (400 MHz, DMSO-d6) δ 10.12
(br s, 1H), 7.67 (d, J = 7.82 Hz, 2H), 7.33 (t, J = 7.92 Hz, 2H), 7.07 (t, J =
7.16 Hz, 1H), 6.45 (dd, J = 9.98, 17.02 Hz, 1H), 6.23−6.30 (m, 1H),
5.76 (dd, J = 1.86, 10.07 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
163.1, 139.0, 131.9, 128.7, 126.8, 123.5, 119.3. m/z (ESI, +ve ion) 148.1
(M + H)+.
N-(2-Trifluoromethylphenyl)acrylamide (5o). 1H NMR (400
MHz, DMSO-d6) δ 9.76 (s, 1H), 7.75 (t, J = 7.70 Hz, 1H), 7.69 (d, J =
7.71 Hz, 1H), 7.54 (d, J = 7.71 Hz, 1H), 7.47 (t, J = 7.70 Hz, 1H), 6.53
(dd, J = 10.17, 17.02 Hz, 1H), 6.25 (dd, J = 1.96, 17.02 Hz, 1H), 5.78
(dd, J = 1.96, 10.17 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 164.2,
135.1, 132.9, 131.0, 130.1, 127.2, 126.8, 126.3, 126.3, 124.9, 124.7, 122.2.
m/z (ESI, +ve ion) 216.1 (M + H)+.
1
N-(3-Trifluoromethylphenyl)acrylamide (5m). H NMR (400
MHz, DMSO-d6) δ 10.46 (s, 1H), 8.17 (s, 1H), 7.85 (d, J = 8.41 Hz,
1H), 7.57 (t, J = 7.92 Hz, 1H), 7.42 (d, J = 7.82 Hz, 1H), 6.43 (dd, J =
9.98, 17.02 Hz, 1H), 6.27−6.34 (m, 1H), 5.81 (dd, J = 2.05, 10.07 Hz,
1H). 13C NMR (101 MHz, DMSO-d6) δ 163.6, 139.7, 131.4, 130.0,
129.5 (q, J = 32 Hz), 127.6, 124.0 (q, J = 271 Hz), 122.8, 119.7, 115.3.
m/z (ESI, +ve ion) 216.1 (M + H)+.
N-(2-Fluorophenyl)acrylamide (2o). 1H NMR (400 MHz,
DMSO-d6) δ 10.33 (br s, 1H), 7.68 (d, J = 11.76 Hz, 1H), 7.32−7.39
(m, 2H), 6.86−6.94 (m, 1H), 6.42 (dd, J = 9.98, 17.02 Hz, 1H), 6.25−
6.32 (m, 1H), 5.79 (dd, J = 1.96, 9.98 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 163.4, 163.3, 160.9, 140.7, 140.6, 131.5, 130.4, 130.3,
N-(4-Trifluoromethylphenyl)acrylamide (5p). 1H NMR (400
MHz, DMSO-d6) δ 10.47 (s, 1H), 7.85−7.90 (m, J = 8.41 Hz, 2H),
F
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Journal of Medicinal Chemistry
Article
7.66−7.71 (m, J = 8.61 Hz, 2H), 6.46 (dd, J = 9.98, 17.02 Hz, 1H), 6.28−
6.35 (m, 1H), 5.82 (dd, J = 1.86, 10.07 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 163.6, 142.5, 131.4, 127.8, 126.0, 125.7, 123.6, 123.3,
123.0, 119.2. m/z (ESI, +ve ion) 216.1 (M + H)+.
J = 7.24 Hz, 1H), 6.43 (dd, J = 10.07, 16.92 Hz, 1H), 6.25 (dd, J = 1.66,
16.92 Hz, 1H), 5.75 (dd, J = 1.76, 10.17 Hz, 1H), 3.73 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 163.1, 159.5, 140.1, 131.8, 129.5, 126.8, 111.6,
108.9, 105.1, 54.9. m/z (ESI, +ve ion) 178.1 (M + H)+.
1
N-(4-Methoxyphenyl)acrylamide (9p). 1H NMR (400 MHz,
DMSO-d6) δ 9.98 (s, 1H), 7.54−7.61 (m, J = 9.00 Hz, 2H), 6.87−6.93
(m, J = 8.80 Hz, 2H), 6.40 (dd, J = 10.17, 17.02 Hz, 1H), 6.22 (dd, J =
N-([1,1′-Biphenyl]-2-yl)acrylamide (6o). H NMR (500 MHz,
DMSO-d6) δ ppm 9.44 (br s, 1 H), 7.53 (d, J = 7.4 Hz, 1 H), 7.29−7.45
(m, 7 H), 6.23−6.39 (m, 1 H), 6.15 (d, J = 15.6 Hz, 1 H), 5.65 (d, J =
10.1 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 163.8, 138.8, 136.6,
134.4, 131.6, 130.3, 128.7, 128.3, 127.6, 127.2, 126.4, 126.1. m/z (ESI,
+ve ion) 224.0 (M + H)+.
1.96, 16.82 Hz, 1H), 5.71 (dd, J = 1.96, 9.98 Hz, 1H), 3.73 (s, 3H). 13
C
NMR (101 MHz, DMSO-d6) δ 162.6, 155.3, 132.1, 131.9, 126.2, 120.7,
113.9, 55.1. m/z (ESI, +ve ion) 178.2 (M + H)+.
1
N-([1,1′-Biphenyl]-3-yl)acrylamide (6m). H NMR (500 MHz,
N-(2-(Methylthio)phenyl)acrylamide (10o). 1H NMR (500
MHz, DMSO-d6) δ ppm 9.51 (br s, 1 H), 7.43 (d, J = 7.4 Hz, 1 H),
7.35 (d, J = 7.8 Hz, 1 H), 7.12−7.29 (m, 2 H), 6.40−6.61 (m, 1 H), 6.23
DMSO-d6) δ ppm 10.23 (br s, 1 H), 8.00 (s, 1 H), 7.58−7.68 (m, 3 H),
7.33−7.51 (m, 5 H), 6.47 (dd, J = 17.0, 10.2 Hz, 1 H), 6.29 (dd, J = 16.9,
1.8 Hz, 1 H), 5.78 (dd, J = 10.2, 1.8 Hz, 1 H). 13C NMR (101 MHz,
DMSO-d6) δ 163.2, 140.8, 140.1, 139.5, 131.8, 129.3, 128.9, 127.5,
126.9, 126.6, 121.9, 118.3, 117.6. m/z (ESI, +ve ion) 224.0 (M + H)+.
(dd, J = 17.0, 1.8 Hz, 1 H), 5.74 (d, J = 10.1 Hz, 1 H), 2.41 (s, 3 H). 13
C
NMR (101 MHz, DMSO-d6) δ 163.5, 134.9, 133.4, 131.5, 126.7, 126.3,
126.1, 125.2, 15.1. m/z (ESI, +ve ion) 194.0 (M + H)+.
1
N-([1,1′-Biphenyl]-4-yl)acrylamide (6p). H NMR (400 MHz,
N-(3-(Methylthio)phenyl)acrylamide (10m). 1H NMR (500
MHz, DMSO-d6) δ ppm 10.14 (br s, 1 H), 7.66 (s, 1 H), 7.40 (d, J =
7.5 Hz, 1 H), 7.26 (t, J = 7.9 Hz, 1 H), 6.96 (d, J = 7.2 Hz, 1 H), 6.42 (dd,
J = 17.0, 10.1 Hz, 1 H), 6.26 (dd, J = 17.0, 1.8 Hz, 1 H), 5.76 (dd, J = 10.1,
1.8 Hz, 1 H). 13C NMR (101 MHz, DMSO-d6) δ 163.2, 139.5, 138.6,
131.7, 129.2, 127.0, 120.8, 116.3, 115.8, 40.4, 14.6. m/z (ESI, +ve ion)
194.0 (M + H)+.
DMSO-d6) δ 10.23 (s, 1H), 7.77 (d, J = 7.98 Hz, 2H), 7.62−7.68 (m,
4H), 7.44 (t, J = 7.16 Hz, 2H), 7.27−7.38 (m, 1H), 6.46 (dd, J = 10.07,
16.92 Hz, 1H), 6.28 (dd, J = 1.96, 17.02 Hz, 1H), 5.75−5.80 (m, 1H).
13C NMR (101 MHz, DMSO-d6) δ 163.1, 139.6, 138.4, 135.1, 131.8,
128.8, 127.0, 126.9, 126.9, 126.2, 119.7. m/z (ESI, +ve ion) 224.4 (M +
H)+.
Methyl 2-Acrylamidobenzoate (7o). 1H NMR (400 MHz,
DMSO-d6) δ 10.77 (s, 1H), 8.30 (dd, J = 1.37, 8.41 Hz, 1H), 7.92
(dd, J = 1.66, 7.92 Hz, 1H), 7.62 (t, J = 7.81 Hz, 1H), 7.22 (t, J = 7.49 Hz,
1H), 6.44 (dd, J = 10.17, 17.02 Hz, 1H), 6.27 (dd, J = 1.56, 17.02 Hz,
1H), 5.83 (dd, J = 1.76, 10.17 Hz, 1H), 3.85 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 167.5, 163.3, 139.2, 139.2, 133.8, 132.1, 130.5,
127.4, 123.5, 121.4, 118.2, 52.4. m/z (ESI, +ve ion) 206.1 (M + H)+.
Methyl 3-Acrylamidobenzoate (7m). 1H NMR (400 MHz,
DMSO-d6) δ 10.36 (s, 1H), 8.34 (s, 1H), 7.93 (dd, J = 1.08, 8.12 Hz,
1H), 7.66 (d, J = 7.39 Hz, 1H), 7.47 (t, J = 7.92 Hz, 1H), 6.43 (dd, J =
9.98, 17.02 Hz, 1H), 6.29 (dd, J = 2.05, 16.92 Hz, 1H), 5.79 (dd, J = 1.96,
9.98 Hz, 1H), 3.86 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 166.0,
163.3, 139.3, 131.6, 130.1, 129.2, 127.3, 124.0, 123.7, 119.8, 52.2. m/z
(ESI, +ve ion) 206.1 (M + H)+.
N-(4-(Methylthio)phenyl)acrylamide (10p). 1H NMR (400
MHz, DMSO-d6) δ 10.13 (s, 1H), 7.60−7.66 (m, J = 8.61 Hz, 2H),
7.20−7.29 (m, J = 8.61 Hz, 2H), 6.42 (dd, J = 9.98, 17.02 Hz, 1H), 6.21−
6.30 (m, 1H), 5.74 (dd, J = 1.86, 10.07 Hz, 1H), 2.45 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 163.0, 136.4, 132.1, 131.8, 127.0, 126.7, 119.9,
15.4. m/z (ESI, +ve ion) 194.1 (M + H)+.
N-(2-(Dimethylamino)phenyl)acrylamide (11o). 1H NMR (400
MHz, DMSO-d6) δ 9.28 (br s, 1H), 7.88−8.01 (m, 1H), 6.99−7.17 (m,
3H), 6.73 (dd, J = 10.17, 17.02 Hz, 1H), 6.24 (dd, J = 1.96, 17.02 Hz,
1H), 5.72 (dd, J = 2.15, 10.17 Hz, 1H), 2.61 (s, 6H). 13C NMR (101
MHz, DMSO-d6) δ 163.2, 144.8, 132.2, 131.7, 126.4, 124.5, 122.8,
122.6, 119.1, 43.6. m/z (ESI, +ve ion) 191.1 (M + H)+.
N-(3-(Dimethylamino)phenyl)acrylamide (11m). 1H NMR
(500 MHz, DMSO-d6) δ ppm 9.92 (br s, 1 H), 7.06−7.13 (m, 2 H),
6.99 (d, J = 7.8 Hz, 1 H), 6.39−6.48 (m, 2 H), 6.23 (dd, J = 17.0, 1.9 Hz,
1 H), 5.71 (dd, J = 10.1, 1.9 Hz, 1 H), 2.87 (s, 6 H). 13C NMR (101
MHz, DMSO-d6) δ 162.9, 150.7, 139.7, 132.1, 129.0, 126.4, 108.0,
107.6, 103.4, 40.1. m/z (ESI, +ve ion) 191.1 (M + H)+.
Methyl 4-Acrylamidobenzoate (7p). 1H NMR (400 MHz,
DMSO-d6) δ 10.46 (s, 1H), 7.91−7.96 (m, J = 8.61 Hz, 2H), 7.78−
7.83 (m, J = 8.80 Hz, 2H), 6.46 (dd, J = 10.17, 17.02 Hz, 1H), 6.30 (dd, J
= 1.76, 17.02 Hz, 1H), 5.81 (dd, J = 1.86, 10.07 Hz, 1H), 3.83 (s, 3H).
13C NMR (101 MHz, DMSO-d6) δ 165.7, 163.5, 143.3, 131.5, 130.2,
127.8, 124.1, 118.7, 51.9. m/z (ESI, +ve ion) 206.1 (M + H)+.
N-(2-Cyanophenyl)acrylamide (8o). 1H NMR (400 MHz,
DMSO-d6) δ 10.36 (s, 1H), 7.83 (dd, J = 1.66, 7.73 Hz, 1H), 7.62−
7.74 (m, 2H), 7.37 (t, J = 7.28 Hz, 1H), 6.52 (dd, J = 10.17, 17.21 Hz,
1H), 6.32 (dd, J = 1.86, 17.12 Hz, 1H), 5.84 (dd, J = 1.76, 10.17 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 163.5, 139.9, 133.8, 133.2, 130.8,
128.1, 125.9, 125.6, 116.8, 107.3. m/z (ESI, +ve ion) 173.1 (M + H)+.
N-(3-Cyanophenyl)acrylamide (8m). 1H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1H), 8.17 (s, 1H), 7.86 (t, J = 3.93 Hz, 1H),
7.54 (s, 1H), 7.55 (d, J = 5.94 Hz, 2H), 6.43 (dd, J = 9.88, 16.92 Hz, 1H),
6.28−6.34 (m, 1H), 5.82 (dd, J = 1.96, 9.98 Hz, 1H). 13C NMR (101
MHz, DMSO-d6) δ 163.6, 139.7, 131.3, 130.3, 127.9, 127.0, 123.9,
122.0, 118.6, 111.6. m/z (ESI, +ve ion) 173.1 (M + H)+.
N-(4-(Dimethylamino)phenyl)acrylamide (11p). 1H NMR (400
MHz, DMSO-d6) δ 9.83 (s, 1H), 7.45−7.52 (m, J = 9.00 Hz, 2H), 6.66−
6.74 (m, J = 9.19 Hz, 2H), 6.39 (dd, J = 10.07, 16.92 Hz, 1H), 6.19 (dd, J
= 2.05, 16.92 Hz, 1H), 5.67 (dd, J = 2.05, 10.07 Hz, 1H), 2.85 (s, 6H).
13C NMR (101 MHz, DMSO-d6) δ 162.3, 147.1, 132.1, 128.8, 125.6,
120.6, 112.6, 40.4. m/z (ESI, +ve ion) 191.1 (M + H)+.
N-(2-Nitrophenyl)acrylamide (12o). 1H NMR (400 MHz,
DMSO-d6) δ 10.47 (br s, 1H), 7.96 (d, J = 7.80 Hz, 1H), 7.65−7.74
(m, 2H), 7.39 (ddd, J = 1.76, 6.90, 8.36 Hz, 1H), 6.46 (dd, J = 10.17,
17.02 Hz, 1H), 6.24−6.31 (m, 1H), 5.83 (dd, J = 1.96, 10.17 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 163.3, 142.7, 133.9, 130.8, 128.2,
125.4, 125.4, 124.9. m/z (ESI, +ve ion) 193.1 (M + H)+.
N-(4-Cyanophenyl)acrylamide (8p). 1H NMR (400 MHz,
DMSO-d6) δ 10.55 (s, 1H), 7.76−7.87 (m, 4H), 6.45 (dd, J = 10.07,
16.92 Hz, 1H), 6.28−6.36 (m, 1H), 5.83 (dd, J = 1.86, 10.07 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 163.7, 143.2, 133.2, 131.3, 128.2,
119.4, 119.0, 105.2. m/z (ESI, +ve ion) 173.1 (M + H)+.
N-(3-Nitrophenyl)acrylamide (12m). 1H NMR (400 MHz,
DMSO-d6) δ 10.61 (s, 1H), 8.70 (t, J = 2.15 Hz, 1H), 7.98 (d, J =
7.80 Hz, 1H), 7.92 (d, J = 7.79 Hz, 1H), 7.62 (t, J = 8.22 Hz, 1H), 6.44
(dd, J = 9.98, 17.02 Hz, 1H), 6.33 (dd, J = 1.96, 17.02 Hz, 1H), 5.84 (dd,
J = 2.05, 9.88 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 147.9,
140.1, 131.2, 130.2, 128.0, 125.2, 117.9, 113.4. m/z (ESI, +ve ion) 193.1
(M + H)+.
N-(2-Methoxyphenyl)acrylamide (9o). 1H NMR (400 MHz,
DMSO-d6) δ 9.34 (br s, 1H), 8.03 (d, J = 8.02 Hz, 1H), 7.03−7.12 (m,
2H), 6.92 (t, J = 7.61 Hz, 1H), 6.70 (dd, J = 10.17, 17.02 Hz, 1H), 6.23
(dd, J = 2.15, 17.02 Hz, 1H), 5.71 (dd, J = 2.15, 10.17 Hz, 1H), 3.84 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 163.2, 149.7, 149.7, 132.1,
127.0, 126.5, 124.6, 122.1, 120.2, 111.1, 55.6. m/z (ESI, +ve ion) 178.1
(M + H)+.
N-(4-Nitrophenyl)acrylamide (12p). 1H NMR (400 MHz,
DMSO-d6) δ 10.73 (s, 1H), 8.21−8.28 (m, J = 9.19 Hz, 2H), 7.90−
7.95 (m, J = 9.19 Hz, 2H), 6.48 (dd, J = 9.98, 17.02 Hz, 1H), 6.35 (dd, J =
1.66, 16.92 Hz, 1H), 5.85−5.90 (m, 1H). 13C NMR (101 MHz, DMSO-
d6) δ 163.8, 145.2, 142.4, 131.2, 128.5, 125.0, 119.1. m/z (ESI, +ve ion)
193.1 (M + H)+.
N-(3-Methoxyphenyl)acrylamide (9m). 1H NMR (400 MHz,
DMSO-d6) δ 10.10 (br s, 1H), 7.38 (s, 1H), 7.17−7.25 (m, 2H), 6.65 (d,
G
DOI: 10.1021/acs.jmedchem.5b01018
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Schuurmann, G. Prediction of Michael-type acceptor reactivity toward
glutathione. Chem. Res. Toxicol. 2010, 23, 1576−1585.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01018.
■
S
(4) Walter, A. O.; Sjin, R. T. T.; Haringsma, H. J.; Ohashi, K.; Sun, J.;
Lee, K.; Dubrovskiy, A.; Labenski, M.; Zhu, Z.; Wang, Z.; Sheetos, M.;
St. Martin, T.; Karp, R.; van Kalken, D.; Chaturvedi, P.; Niu, D.; Nacht,
M.; Petter, R. C.; Westlin, W.; Lin, K.; Jaw-Tsai, S.; Raponi, M.; Van
Dyke, T.; Etter, J.; Weaver, Z.; Pao, W.; Singh, J.; Simmons, A. D.;
Hardning, T. C.; Allen, A. Discovery of a Mutant-Selective Covalent
Inhibitor of EGFR that Overcomes T790M-Mediated Resistance in
NSCLC. Cancer Discovery 2013, 3, 1404−1415.
Figure SI-1; GSH assay protocol; statistical analysis of
experimental GSH data for acrylamides 1, 9o, 11o, 12o,
11m, 12m, 11p, 12p (multiple determinations); NMR
spectra of acrylamides 1−12; coordinates of optimized
transition structures for acrylamides 1−12 (PDF)
(5) Evans, E. K.; Tester, R.; Aslanian, S.; Karp, R.; Sheets, M.; Labenski,
M. T.; Witowski, S. R.; Lounsbury, H.; Chaturvedi, P.; Mazdiyasni, H.;
Zhu, Z.; Nacht, M.; Freed, M. I.; Petter, R. C.; Dubrovskiy, A.; Singh, J.;
Westlin, W. F. Inhibition of Btk with CC-292 Provides Early
Pharmacodynamic Assessment of Activity in Mice and Humans. J.
Pharmacol. Exp. Ther. 2013, 346, 219−228.
AUTHOR INFORMATION
Corresponding Authors
*V.J.C.: e-mail, vcee@amgen.com; phone, 805-313-5500.
*M.D.B.: e-mail, mbartber@amgen.com; phone, 805-447-3446.
Author Contributions
■
(6) Balci, M. 15: Multiple-Pulse NMR Experiments. In Basic 1H- and
13C-NMR Spectroscopy; Balci, M., Ed.; Elsevier Science: Amsterdam,
2005; pp 339−375.
(7) Lushchak, V. I. Glutathione Homeostasis and Functions: Potential
Targets for Medical Interventions. J. Amino Acids 2012, 2012, 1−26.
(8) Hansch, C.; Leo, A. Electronic Effects on Organic Reactions. In
Exploring QSAR Fundamentals and Applications in Chemistry and Biology;
ACS Professional Reference Books: Washington, DC, 1995; pp 1−24.
(9) Fujita, T.; Nishioka, T. The Analysis of the Ortho Effect. In Progress
in Physical Organic Chemistry; Taft, R. W., Ed.; John Wiley & Sons, Inc.:
New York, 1976; Vol 12, pp 49−89.
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Useful discussions with Dr. Hua Gao are acknowledged with
thanks.
■
(10) Analysis of ortho-, meta-, and para-subsets showed R2 of 0.61,
0.81, and 0.89, respectively, for Hb1 chemical shift and 0.73, 0.85, and
0.91, respectively, for Cb chemical shift.
(11) For a successful application of 13C NMR chemical shifts in the
understanding of the Michael acceptor potential of alkylidene
pyrazolones and alkylidene benzothiazinones see the following:
Cusack, K. P.; Arnold, L. D.; Barberis, C. E.; Chen, H.; Ericsson, A.
M.; Gaza-Bulseco, G. S.; Gordon, T. D.; Grinnell, C. M.; Harsch, A.;
Pellegrini, M.; Tarcsa, E. A 13C NMR approach to categorizing potential
limitations of α,β-unsaturated carbonyl systems in drug-like molecules.
Bioorg. Med. Chem. Lett. 2004, 14, 5503−5507.
(12) Preliminary assessment of polysubstituted N-arylacrylamides
supports this assertion. For example, the GSH reaction rate of N-(3,5-
difluorophenyl)acrylamide is predicted from Table 3 to be 2.5 + 2.5 = 5-
fold faster than the parent N-phenylacrylamide; the measured GSH t1/2
of 37.8 min indicates a 4.7-fold increase in rate.
(13) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Universal solvation
model based on solute electron density and on a continuum model of
the solvent defined by the bulk dielectric constant and atomic surface
tensions. J. Phys. Chem. B 2009, 113 (18), 6378−6396.
(14) Our geometric and energetic results for 1 at the IEFPCM-SMD-
B3LYP/6-311+G(d,p) level, and for all systems at the IEFPCM-
B3LYP/6-311+G(d,p) level, are provided in Supporting Information.
(15) The standard (non-SMD) IEFPCM solvation model yielded
uniformly smoother geometric convergence behavior in our hands and
was thus used throughout this study.
ABBREVIATIONS USED
■
B3LYP, Becke three-parameter Lee−Yang−Parr hybrid density
functional; BTK, Bruton’s tyrosine kinase; DEPT, distortionless
enhancement by polarization transfer; DMSO, dimethylsulf-
oxide; EGFR, epidermal growth factor receptor; GSH,
glutathione; IEFPCM, integral-equation-formalism polarizable
continuum model; SMD, solvation model density; TS, transition
structure
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H
DOI: 10.1021/acs.jmedchem.5b01018
J. Med. Chem. XXXX, XXX, XXX−XXX