N-(1-Benzyl-3,5-dimethyl?1H?pyrazol-4-yl)benzamides: Antiproliferative activity and effects on mTORC1 and autophagy

Article abstract of DOI:10.1021/acsmedchemlett.6b00392

Guided by antiproliferative activity in MIA PaCa-2 cells, we have performed preliminary structure?activity relationship studies on N-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides. Two selected compounds showed submicromolar antiproliferative activity

Full text of DOI:10.1021/acsmedchemlett.6b00392

Letter
pubs.acs.org/acsmedchemlett
N‑(1-Benzyl-3,5-dimethyl‑1H‑pyrazol-4-yl)benzamides:
Antiproliferative Activity and Effects on mTORC1 and Autophagy
Teng Ai,†,∥ Rose Willett,‡,∥ Jessica Williams,† Rui Ding,† Daniel J. Wilson,† Jiashu Xie,† Do-Hyung Kim,§
Rosa Puertollano,‡ and Liqiang Chen*,†
†Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, Minnesota 55455, United States
‡Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
20892, United States
§Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, United
States
S
* Supporting Information
ABSTRACT: Guided by antiproliferative activity in MIA PaCa-2 cells, we
have performed preliminary structure−activity relationship studies on N-
(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides. Two selected com-
pounds showed submicromolar antiproliferative activity and good
metabolic stability. Both compounds reduced mTORC1 activity and
increased autophagy at the basal level. In addition, they disrupted
autophagic flux by interfering with mTORC1 reactivation and clearance
of LC3-II under starvation/refeed conditions, as evidenced by accumu-
lation of LC3-II and abnormal LC3 labeled punctae. Therefore, N-(1-
benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides may represent a new class
of autophagy modulators that possesses potent anticancer activity and
potentially a novel mechanism of action.
KEYWORDS: Autophagy, autophagy modulator, mTOR, pancreatic cancer, anticancer agents
acroautophagy (hereafter referred to as autophagy) is a
mTORC1, which in turn increases autophagy by unblocking
Mconserved cellular process through which cytosolic ULK1. In addition to its role in autophagy, mTORC1 also
phosphorylates and regulates substrates including ribosomal
protein S6 kinase beta-1 (P70S6K1, hereafter referred to as
P70) and 4E-binding protein 1 (4EBP1), and levels of
phosphorylated substrates are routinely used to gauge
mTORC1 activity.
components, such as proteins and organelles, are sequestered,
degraded, and recycled.1 The autophagy machinery requires a
number of autophagy-related (Atg) proteins and functional
complexes, including the UNC51-like kinase 1 (ULK1)
complex, the class III phosphoinositide 3-kinase (PI3K)
complex, the Atg12-Atg5-Atg16 ubiquitin-like conjugation
system, and the microtubule-associated protein light chain 3
(LC3)-phosphatidylethanolamine (PE) conjugation system.
Among these autophagy components, LC3-II, which is the
PE lipidated form of LC3-I, plays a key role. LC3-II is
generated after a series of processing by the Atg4-Atg7-Atg3
conjugation machinery. Since it is closely involved in
autophagy, the level of LC3-II is commonly used as a marker
to monitor autophagy.2
Autophagy is regulated by a network of complex signaling
pathways, where the class I phosphatidylinositol 3-kinase-AKT-
mammalian target of rapamycin complex 1 (PI3K-AKT-
mTORC1) pathway is a well-established negative regulator.
While a basal level of autophagy is required to maintain cellular
homeostasis, it is upregulated in response to lack of nutrients,
unfavorable energy status, attenuated growth signaling, virus/
bacteria invasion, and a number of cellular stresses including
endoplasmic reticulum stress, oxidative stress, and hypoxia.3
Activation of autophagy is generally accomplished by inhibiting
The precise role of autophagy in tumorigenesis and cancer
therapy still remains to be defined due to the heterogeneous
nature of cancers and the complexity of the autophagy
machinery.4 Studies have shown that autophagy plays different
or even opposite roles depending on the cancer type and the
stage of tumor progression. Currently, it is generally accepted
that autophagy behaves as a tumor suppressor in normal cells in
part because it removes damaged cellular components.
Nevertheless, increasing evidence shows that autophagy is a
cytoprotective mechanism often exploited by established cancer
cells to cope with their harsh microenvironment and cellular
stresses induced by chemotherapies. Inhibition of autophagy
has been proposed as a promising strategy to combat pancreatic
ductal adenocarcinoma (PDAC), the most lethal cancer among
major malignancies. It has been shown that autophagy is
Received: October 6, 2016
Accepted: November 28, 2016
Published: November 28, 2016
© XXXX American Chemical Society
A
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elevated and required for de novo tumor growth and that
inhibition of autophagy leads to robust tumor regression;5,6
however, the status of p53 may determine the outcome of
autophagy inhibition.7,8 Despite the complexity of autophagy as
a therapeutic target, these studies suggest that disabling the pro-
survival autophagy machinery might represent a viable
approach to promote cell death and reduce drug resistance in
refractory cancers such as pancreatic cancer.3,9
a tyrosine kinase inhibitor that has been approved for use in
combination with gemcitabine. Moreover, our initial examina-
tion of compound 1’s effect on autophagy suggested that it
disrupted the autophagy pathway. Taken together, these results
suggested that compound 1 could serve as a starting point for
SAR studies and the subsequent exploration of improved
compounds for their effects on autophagy.
First, we investigated ring A by introducing a functional
group, as represented by R1, R2, and R3, at the para, meta, and
ortho positions, respectively. Because an amide provided an
opportunity to serve as both a hydrogen bond donor and a
hydrogen bond acceptor, we chose to first explore amide
functionalities (Table 1). When a primary amide was
introduced, a negative effect was observed for a substitution
at the para (compound 2) or ortho (compound 4) position.
However, compound 3, which contained a primary amide at the
meta position, retained activity nearly identical to that of parent
compound 1, suggesting that the meta position might be more
amenable to structural modifications. Indeed, incorporation of a
methyl amide as seen in compound 5 slightly improved
anticancer activity in comparison with its primary amide
counterpart. To further explore amide substitutions, we
prepared compounds 6−9, which contained a dimethyl, ethyl,
isopropyl, and phenyl amide, respectively, at the meta position.
Comparable activities exhibited by these compounds indicated
that further structural elaboration beyond a methyl amide was
not productive.
Because of their potential application as anticancer agents
used alone or in combination with other cancer therapeutics,
autophagy inhibitors have been actively pursued even though
early inhibitors generally lack selectivity or target the lysosomal
function.10 Chloroquine and hydrochloroquine, two lysosomo-
tropic agents, have been commonly used as autophagy
inhibitors. However, it has been proposed that chloroquine’s
anticancer property is independent of its effect on autoph-
agy.11,12 These findings underscore the importance of
discovering new and specific autophagy inhibitors, preferably
those with a novel mechanism of action. In recent years, in
addition to the continued search for improved lysosomotropic
autophagy inhibitors,13−16 there has been a growing list of
selective autophagy inhibitors, especially those targeting Beclin
1,17 ULK1 kinase,18−20 Vps34 (Class III PI3K),21−23 or
Atg4B.24 Herein we report our discovery of N-(1-benzyl-3,5-
dimethyl-1H-pyrazol-4-yl)benzamides that possess antiprolifer-
ative activity and elicit differential effects on autophagy under
different nutrient conditions. Structure−activity relationship
(SAR) studies of this class of compounds, assessment of in vitro
absorption, distribution, metabolism, and excretion (ADME)
properties, and evaluation of effects on mTORC1 and
autophagy are presented.
With a methyl amide fixed at the meta position of ring A, we
proceeded to investigate the phenyl ring D (Table 2).
Compound 10, in which the phenyl ring was replaced with a
saturated cyclohexyl group, showed reduced activity, high-
lighting the importance of an aromatic ring. Subsequently, we
studied the substituents on ring D by modifying parent
compound 5. To that end, we performed a substitution scan at
During our search for active compounds in pancreatic cancer
cells, we identified compound 1, which possessed an EC50 value
of 10 μM in MIA PaCa-2, a pancreatic cancer line (Table 1).
When compared with known autophagy inhibitors, compound
1 was more active than 3-methyladenine (3 mA) and spautin-
117 while possessing an antiproliferative capacity similar to that
of chloroquine. Furthermore, it was more potent than erlotinib,
a
Table 2. SAR Studies: Substituents on Ring D
a
Table 1. SAR Studies: Substituents on Ring A
EC50
(μM)
compd
R1
R2
R3
compd
R1
R2
R3
EC50 (μM)
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
14
C(O)NH2
>100
9.2
Me
H
H
H
2.1
6.2
2.6
8.5
6.2
8.0
6.3
5.9
4.2
10
H
H
H
H
H
H
H
C(O)NH2
H
Me
H
H
C(O)NH2
42
H
Me
H
C(O)NHMe
C(O)N(Me)2
C(O)NHEt
C(O)NHiPr
C(O)NHPh
H
H
H
H
H
6.2
OMe
H
H
6.4
OMe
H
H
11
H
OMe
H
8.9
Cl
H
6.4
H
Cl
H
H
gemcitabine
erlotinib
0.0015
27
H
Cl
CF3
H
H
H
3 mA
>100
14
CF3
H
H
2.3
0.80
0.62
chloroquine
spautin-1
H
CF3
CF3
20
Me
H
a
a
EC50 values were determined in triplicate in MIA PaCa-2 cells.
EC50 values were determined in triplicate in MIA PaCa-2 cells.
B
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the ortho, meta, and para positions, as represented by R1, R2,
and R3, respectively. To gauge the impact of electron-donating
or -withdrawing groups, we used groups with different
electrosteric properties, including Me, OMe, Cl, and CF3.
First, we conducted a methyl scan on ring D (compounds 11−
13), which revealed that a methyl group at the ortho or para
position enhanced activity, while one at the meta position had
no effect. When a similar scan was performed using a methoxy
(14−16) or chloro (17−19) group, the resulting compounds
failed to significantly improve anticancer activity relative to
compound 5. Remarkably, introducing a trifluoromethyl group
at the para position led to compound 22, which possessed a
submicromolar EC50 value. Because a methyl group at the ortho
position resulted in an increase in activity as seen for
compound 11, we chose to investigate a potential additive
effect by preparing compound 23, which featured a
trifluoromethyl and methyl group at the para and ortho
position, respectively. Compound 23 exhibited an EC50 value of
0.62 μM without significant improvement over 22. In summary,
a trifluoromethyl group was the preferred substituent at the
para position of ring D, while a methyl at the ortho position
could be beneficial.
Figure 1. Induction of basal autophagy. ARPE-19 cells were treated for
4 h with DMSO, 23, or 22 without starvation. (A) Fold increase of
LC3-II/actin ratios normalized to the DMSO control (n = 3). (B)
Representative Western blot of LC-3 after treatment with 23 or 22 (25
μM). (C) Representative Western blot of LC-3 after treatment with 23
or 22 (50 μM). (D) Representative immunofluorescence detection of
LC3 punctae after treatment with 23 or 22 (25 μM). Scale bar is 10
μm. CD63 was used as a late endosomal/lysosomal marker.
To evaluate in vitro ADME properties of selected
compounds, we tested compounds 22 and 23 for their
metabolic stabilities (Table S1). Both compounds possessed
excellent stability in mouse and human plasma as well as very
good stability in mouse and human liver microsomes.
Furthermore, both compounds showed good aqueous solubility
with 23 being 2-fold more soluble than 22. This enhanced
solubility could be explained by reduced coplanarity of ring D
(in relation to the oxymethyl moiety within the phenoxymethyl
linker) induced by the extra methyl group on that ring in
compound 23. Taken together, compounds 23 and 22 showed
both relatively good in vitro ADME properties and antiprolifer-
ative activity; therefore, their effects on autophagy were
examined.
late endosomal/lysosomal marker. Limited colocalization
between LC3 and CD63 in normal (Figure 1D) and starvation
conditions (Figure 3C,D) indicated that the LC3-positive
punctae corresponded to autophagosomes instead of degrada-
tion-deficient autolysosomes. Enhanced colocalization between
LC3 and CD63 was observed in bafilomycin-treated ARPE-19
cells and LC3/CD63-positive structures corresponded to
degradation-deficient autolysosomes (Figure S2).
Because mTORC1 is a negative regulator of autophagy, we
tested for mTORC1 activity in cells treated with compound 23
or 22. Basal mTORC1 activity was measured by the
phosphorylation level of the mTORC1 substrate P70 (Figure
2). Levels of phosphorylated P70/total P70 were decreased
Effects on autophagy were studied in ARPE-19, a human
retinal pigment epithelial cell line. Markedly higher EC50 values
of compounds 23 (7.1 μM) and 22 (47.9 μM) in ARPE-19
cells indicated that both compounds were less cytotoxic in
noncancer ARPE-19 cells than in MIA PaCa-2 pancreatic
cancer cells. This finding is not unexpected since cancer cells
often rely on autophagy and are hence more vulnerable to
disruption of the autophagy machinery.
Basal autophagy was measured by treating ARPE-19 cells
with increasing concentrations of compound 23 or 22 (or
DMSO control) for 4 h in complete medium. Autophagy
induction was quantified by measuring the fold increase in
LC3-II/actin ratio compared to control cells.2 A low dose (10
μM) of both compounds resulted in significant increase of
LC3-II conversion compared to control (Figure 1A and Figure
S1). This response was dose dependent for compound 23 as
judged by enhanced effects at the higher concentrations of 25
μM (Figure 1A,B) and 50 μM (Figure 1A,C). Compound 23
was slightly more effective at LC3 turnover (3.3-fold increase
compared to control) at 50 μM, while compound 22 peaked at
25 μM (2.7-fold increase compared to control). This
observation was consistent with compound 23’s slightly higher
antiproliferative ability and improved aqueous solubility.
Furthermore, in line with the biochemical data, an increase in
the number of LC3 labeled punctae was detected in cells
treated with 25 μM of either compound 23 or 22 (Figure 1D).
In our immunofluorescence experiment, CD63 was used as a
Figure 2. Decreased basal mTORC1 activity. ARPE-19 cells were
treated for 4 h with DMSO, 23, or 22 without starvation. (A) Level of
phosphorylated P70/total P70 after treatment with increasing
concentrations of 23 or 22 vs DMSO (100%). (B) Representative
Western blot of phosphorylated P70 after treatment with 23 or 22 (25
μM). #, nonspecific; ##, phosphorylated P85 S6 kinase (an isoform of
P70 S6 kinase).
after treatment with 10 μM compound 23 or 22 (Figure 2A).
Similar to the induction of basal autophagy, the decrease in
mTORC1 activity was also dose dependent. Compound 23 was
more responsive than compound 22, decreasing mTORC1
activity as much as 78% compared to control treated cells,
whereas compound 22 peaked at a decrease of only 46%
(Figure 2A,B and Figure S3). Taken together, these results
suggested that compounds 23 and 22 reduced basal mTORC1
activity, thereby inducing an increase in autophagy.
C
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To better understand the effects of compounds 23 and 22 on
autophagy, we performed an autophagic flux assay2 and used
bafilomycin to block the late fusion/degradation stage of
autophagy. ARPE-19 cells were pretreated for 30 min with a
low dose (10 μM) of compound 23 or 22 (or DMSO). Cells
were then incubated for 4 h with compound 23 or 22 in Earle’s
balanced salt solution (EBSS, starvation medium) or EBSS with
100 nM bafilomycin and the fold increase in LC3-II/actin was
quantified (Figure 3A,B and Figure S4). Under starvation
accumulation after refeed was more pronounced when treated
with increasing concentrations (25 and 50 μM) of either
compound (Figures S5 and S6). Lingering LC3-II was also
observed in cells treated with 25 μM compound 23 or 22 under
starvation/refeed conditions, where abnormal LC3 punctae
were still detected even after 1 h recovery with complete
medium (Figure 3D), suggesting that there was a problem with
the clearance of LC3 punctae in compound treated cells.
Autophagic flux was also measured in the presence of 100
nM bafilomycin in full medium. However, no statistically
significant reduction in autophagic flux was observed (Figure
S7). While further studies are needed, this finding could be
partly due to the fact that the basal autophagy level in ARPE-19
cells is not particularly high and suffers from consequent
experimental variability.
The impact of these two compounds on mTORC1 activity
was assessed under the same starvation/refeed conditions as
described above. Treatment of cells with 10 μM compound 23
resulted in reduced mTORC1 reactivation following starvation
and subsequent refeed, as judged by the phosphorylation levels
of P70 and 4EBP1, two mTORC1 specific substrates (Figure
4A,B). No significant effects were observed for compound 22 at
Figure 3. Impaired autophagy flux. ARPE-19 cells were treated with
DMSO, 23, or 22 under starvation (4 h EBSS), starvation/refeed (4 h
EBSS followed by 1 h complete medium recovery), and starvation/
bafilomycin (4 h EBSS containing 100 nM bafilomycin) conditions (n
= 3). (A) Fold increase LC3-II/actin normalized to DMSO control
after treatment with 23 or 22 (10 μM) under starvation/refeed
conditions. All the three controls were fixed at value = 1. (B)
Representative Western blot of LC3 after treatment with 23 or 22 (10
μM). (C) Representative immunofluorescence detection of LC3
punctae after treatment with 23 or 22 (25 μM) under starvation
conditions. (D) Representative immunofluorescence detections of
LC3 punctae after treatment with 23 or 22 (25 μM) under starvation/
refeed conditions. Scale bar is 10 μm. CD63 was used as a late
endosomal/lysosomal marker.
Figure 4. Reduced mTORC1 reactivation under refeed conditions.
ARPE-19 cells were treated with DMSO, 23, or 22 (10 μM) under
starvation (4 h EBSS) and starvation/refed (4 h EBSS followed by 1 h
complete medium recovery) conditions. (A) Level of phosphorylated
P70 normalized to control after treatment with 23 or 22 (10 μM)
under starvation/refeed conditions (n = 3). (B) Representative
Western blot of P70, phosphorylated P70, 4E-BP1, and phosphory-
lated 4E-BP1. #, P85 S6 kinase (an isoform of P70 S6 kinase); ##,
nonspecific; ###, phosphorylated P85 S6 kinase.
conditions, cells treated with compounds had a decrease in LC3
turnover compared to DMSO treated cells (1.7-fold decrease
for 23 and 1.2-fold decrease for 22; Figure 3A). Treatment with
bafilomycin induced a strong increase in LC3-II/actin both in
control and compound-treated cells, indicating that compounds
23 and 22 did not inhibit fusion between autophagosomes and
lysosomes. However, the LC3-II/actin ratios were decreased in
compound-treated cells, suggesting that autophagosome
degradation might be affected at some extent by the
compounds. Moreover, upon treatment with 25 μM of either
compound under starvation conditions, LC3 labeled structures
were enlarged and nonuniform in shape, whereas DMSO
treated cells had uniform LC3 labeled punctae that were evenly
distributed throughout the cell periphery (Figure 3C).
To measure autophagy under starvation/refeed conditions,
cells starved for 4 h in EBSS were then incubated in complete
medium with either compound or DMSO for 1 h to recover
from autophagy induction (refeed conditions). DMSO treated
cells refed for 1 h had a near complete recovery in LC3
turnover to basal levels, whereas cells treated with 10 μM of
compound 23 were not able to recover LC3 turnover (1.7-fold
increase compared to DMSO treatment; Figure 3A). LC3-II
10 μM. However, treatment with higher concentrations of
either compound led to reduced mTORC1 reactivation
(Figures S5 and S6). Taken together, these results suggested
that both compounds 23 and 22 disrupted autophagic flux by
inhibiting mTORC1 under basal conditions and interfering
with its reactivation under refeed conditions.
The synthesis of N-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-
yl)benzamides is straightforward (Scheme 1). Alkylation of
pyrazole 24 with benzyl bromide followed by reduction of the
nitro group with NaBH4 in the presence of NiCl2 gave amine
25a, which in turn underwent an amide coupling reaction to
afford compound 1. An identical sequence also produced
methyl esters 26b−d, which were then converted into the
corresponding primary amides 2−4 after treatment with
methanolic ammonia. Upon treatment with methylamine,
methyl ester 26c was transformed into methyl amide 5.
Hydrolysis of 26c gave carboxylic acid 27, which was used to
D
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a
tempting to speculate that these compounds might be able to
selectively target solid tumor cells that are under metabolic
stress and hypoxia due to poor vasculature and impeded
delivery of nutrients and oxygen. The harsh tumor micro-
environment has been shown to induce autophagy as a survival
mechanism, which might prove to be a weakness that can be
selectively disrupted by compound 23 under nutrient-deficient
conditions. At the same time, compound 23 might spare
normal cells by allowing or even prompting basal autophagy
that is expected to reduce tumorigenesis. Therefore, at least in
principle, a compound like 23 is expected to be advantageous
over a conventional autophagy inhibitor that unselectively
suppresses autophagy in both normal and cancerous cells.
Taken together, N-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)-
benzamides may hold promise as a new class of anticancer
agents against PDAC. Further SAR studies and elucidation of
their mechanism of action are therefore warranted.
Scheme 1
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00392.
In vitro ADME properties of compounds 22 and 23;
Western blots of ARPE-19 cells treated with compounds
23 or 22 under basal, starvation, and starvation/refeed
conditions; synthesis of phenol 33; and experimental
methods (PDF)
a
Reagents and conditions: (a) ArCH2Br, Cs2CO3, DMF; (b) NaBH4,
NiCl2·6H2O, MeOH; (c) 4-(phenoxymethyl)benzoic acid, EDC,
DMF/CH2Cl2; (d) 7 N methanolic NH3, 70 °C; (e) MeNH2,
EtOH, 70 °C; (f) NaOH, MeOH, H2O; (g) amine, HBTU, Et3N,
DMF/CH2Cl2; (h) methyl 3-(bromomethyl)benzoate, Cs2CO3, DMF;
(i) for 10, 4-((cyclohexyloxy)methyl)benzoic acid, HBTU, Et3N; for
30, 4-(chloromethyl)benzoyl chloride, Et3N, CH2Cl2; (j) ArOH,
Cs2CO3, DMF, 50 °C.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 612-624-2575. Fax: 612-624-8154. E-mail: chenx462@
umn.edu.
ORCID
Liqiang Chen: 0000-0002-4229-863X
prepare compounds 6−9 under standard HBTU-mediated
amide formation conditions.
Author Contributions
∥These authors contributed equally to this work.
To prepare compounds 10−23, pyrazole 24 was alkylated
with methyl 3-(bromomethyl)benzoate followed by treatment
with methylamine to give 28, which was then reduced to amine
29. Routine amide coupling between amine 29 and 4-
((cyclohexyloxy)methyl)benzoic acid gave 10. However,
amine 29 was acylated with 4-(chloromethyl)benzoyl chloride
to give intermediate 30, whose benzyl chloride moiety
underwent nucleophilic substitutions with a set of substituted
phenol to afford compounds 11−23 in a range of yields.
Preparation of phenol 33, which was needed for compound 23,
was depicted in Scheme S1. Commercially available bromide 31
was converted into boronic pinacolate ester 32, which was
subsequently oxidized with mCPBA to give 33 in good yields.
In summary, guided by antiproliferative activity in MIA
PaCa-2 cells, our preliminary SAR studies on the N-(1-benzyl-
3,5-dimethyl-1H-pyrazol-4-yl)benzamide core structure have
led to compounds 23 and 22, which show submicromolar
antiproliferative activity and good metabolic stability. We have
also demonstrated that both compounds increase basal
autophagy but impair autophagic flux under starvation and
starvation/refeed conditions. To our best knowledge, no
compound with such differential activities on autophagy has
been reported previously. Therefore, compound 23 and its
analogues might represent a new class of autophagy modulators
that possess remarkable anticancer activity. Furthermore, it is
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Center for Drug Design in the
Academic Health Center of the University of Minnesota (to L.
C.). R.W. and R.P. were supported by the Intramural Research
Program of the National Institutes of Health, National Heart,
Lung, and Blood Institute (NHLBI). D.-H.K. was supported by
the National Institute of General Medical Sciences (NIGMS)
(R01GM097057). We thank Dr. Huaqing Cui for the initial
synthesis of compound 1.
ABBREVIATIONS
■
PDAC, pancreatic ductal adenocarcinoma; Atg, autophagy-
related; ULK1, UNC51-like kinase 1; PI3K, phosphoinositide
3-kinase; LC3, microtubule-associated protein light chain 3; PE,
phosphatidylethanolamine; mTORC1, mammalian target of
rapamycin complex 1; P70S6K1 (P70), ribosomal protein S6
kinase beta-1; 4EBP1, 4E-binding protein 1; SAR, structure−
activity relationship; ADME, absorption, distribution, metabo-
lism, and excretion; 3 mA, 3-methyladenine; EBSS, Earle’s
balanced salt solution
E
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DOI: 10.1021/acsmedchemlett.6b00392
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