Discovery of Novel Azetidine Amides as Potent Small-Molecule STAT3 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c01705

We optimized our previously reported proline-based STAT3 inhibitors into an exciting new series of (R)-azetidine-2-carboxamide analogues that have sub-micromolar potencies. 5a, 5o, and 8i have STAT3-inhibitory potencies (IC50) of 0.55, 0.38, and 0.34 μM, respectively, compared to potencies greater than 18 μM against STAT1 or STAT5 activity. Further modifications derived analogues, including 7e, 7f, 7g, and 9k, that addressed cell membrane permeability and other physicochemical issues. Isothermal titration calorimetry analysis confirmed high-affinity binding to STAT3, with KD of 880 nM (7g) and 960 nM (9k). 7g and 9k inhibited constitutive STAT3 phosphorylation and DNA-binding activity in human breast cancer, MDA-MB-231 or MDA-MB-468 cells. Furthermore, treatment of breast cancer cells with 7e, 7f, 7g, or 9k inhibited viable cells, with an EC50 of 0.9-1.9 μM, cell growth, and colony survival, and induced apoptosis while having relatively weaker effects on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively active STAT3.

Full text of DOI:10.1021/acs.jmedchem.0c01705

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Article
Discovery of Novel Azetidine Amides as Potent Small-Molecule
STAT3 Inhibitors
Christine Brotherton-Pleiss,# Peibin Yue,# Yinsong Zhu, Kayo Nakamura, Weiliang Chen, Wenzhen Fu,
Casie Kubota, Jasmine Chen, Felix Alonso-Valenteen, Simoun Mikhael, Lali Medina-Kauwe,
Marcus A. Tius, Francisco Lopez-Tapia,* and James Turkson*
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ABSTRACT: We optimized our previously reported proline-based
STAT3 inhibitors into an exciting new series of (R)-azetidine-2-
carboxamide analogues that have sub-micromolar potencies. 5a,
5o, and 8i have STAT3-inhibitory potencies (IC50) of 0.55, 0.38,
and 0.34 μM, respectively, compared to potencies greater than 18
μM against STAT1 or STAT5 activity. Further modifications
derived analogues, including 7e, 7f, 7g, and 9k, that addressed cell
membrane permeability and other physicochemical issues.
Isothermal titration calorimetry analysis confirmed high-affinity
binding to STAT3, with KD of 880 nM (7g) and 960 nM (9k). 7g
and 9k inhibited constitutive STAT3 phosphorylation and DNA-
binding activity in human breast cancer, MDA-MB-231 or MDA-MB-468 cells. Furthermore, treatment of breast cancer cells with
7e, 7f, 7g, or 9k inhibited viable cells, with an EC50 of 0.9−1.9 μM, cell growth, and colony survival, and induced apoptosis while
having relatively weaker effects on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively
active STAT3.
inhibitors of the STAT3 signaling pathway has emerged that
focuses on protein degradation. The PROTAC-STAT3
degraders, represented by SD-36, promoted STAT3 degrada-
tion with nanomolar potency, and SD-36 induced a complete
tumor growth inhibition in vivo in multiple tumor models.28,29
We have previously provided proof-of-concept for the in vivo
antitumor efficacy of the micromolar potent lead inhibitors, BP-
1-102, SH5-07, and SH4-54, which are based on the N-
methylglycinamide scaffold, with its two amine moieties
condensed with three different functionalities.24,26 We took
steps to address the challenges with potency and PK properties
and hence advance the development of these amino acid amide-
based inhibitors and recently published an extensive study on
the structure−activity relationship (SAR) analysis using an
iterative medicinal chemistry approach in which the amino acid
linker was varied along with the simultaneous optimization of
the three functionalities to improve potency and physicochem-
ical properties, and this led to new proline-based analogues.30
INTRODUCTION
■
The signal transducer and activator of transcription (STAT)
family of cytoplasmic transcription factors mediate the cellular
responses to cytokine and growth factors, including cell growth
and differentiation, inflammation, and immune responses.1−3
STAT activation is initiated upon receptor−ligand binding that
induces STAT recruitment to the receptor and STAT
phosphorylation by Janus kinases (JAKs) and Src family kinases.
Two phospho-STAT monomer proteins form STAT:STAT
dimers through a reciprocal pTyr-Src homology (SH)2 domain
interactions, translocate to the nucleus, and bind to specific
DNA-response elements in target gene promoters to induce
gene transcription.2−5
While normal STAT activation is rapid and transient, aberrant
activation of one family member, STAT3, is prevalent and has a
causal role in many human cancers.6,7 STAT3 is therefore a valid
and an attractive target for the development of novel anticancer
therapeutics.7−10 One of the main approaches to develop
inhibitors of STAT3 is focused on targeting the key pTyr:SH2
domain interaction and the STAT3:STAT3 dimerization
event.8,9 Several small-molecule inhibitors have been developed
that target the STAT3 SH2 domain and disrupt the
STAT3:STAT3 dimerization.11−26 However, there has been
limited success in the advancement of these STAT3 inhibitors
into clinical application due to low potency and pharmacokinetic
(PK) limitations and toxicity.27 More recently, a new class of
Received: September 28, 2020
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Figure 1. Azetidine analogues inhibit STAT3 DNA-binding activity in vitro. Nuclear extracts of equal total protein prepared from NIH3T3/v-Src
fibroblasts containing activated STAT3 were preincubated with increasing concentrations of the designated versions of azetidines (A) salicylic acids,
(B) benzoic acids, (C) methyl esters, (D) hydroxamic acids/methylamines, or (E) heterocycles for 30 min at room temperature prior to incubating
with the radiolabeled hSIE probe that binds STAT3 and performing EMSA analysis; bands corresponding to STAT3:DNA complexes in gel were
quantified using ImageJ and represented as a percent of control and plotted against the concentration of compounds, from which IC50 values were
determined. Positions of STAT3:DNA complexes in gel are labeled; control lanes (0) represent nuclear extracts pretreated with 10% DMSO. Data are
representative of two to three independent determinations.
With the focus on potency, we further extended the optimization
of the proline-based analogues into other cyclic amino acids and
have now derived more exciting new series of (R)-azetidine-2-
carboxamide analogues of BP-1-102, including 5a, 5o, and 8i,
which show sub-micromolar STAT3-inhibitory activity in vitro
(IC50 values of 0.52, 0.38, and 0.34 μM, respectively). To
improve membrane permeability, we further derived analogues
containing carboxylic acid surrogates, 7e, 7f, 7g, and 9k, which at
1 μM or less strongly inhibited the viability, anchorage-
dependent and independent growth, and colony formation of
MDA-MB-231 or MDA-MB-468 human breast cancer cells that
harbor aberrantly active STAT3.
RESULTS AND DISCUSSION
■
Progression from the Proline Linker: SAR of Early
Azetidine Analogues Shows Nanomolar Potency in
Disrupting STAT3 DNA-Binding Activity In Vitro. Pro-
gression from the previously reported proline linker (Supporting
Information Figure S1, 3)30 into other cyclic amino acid linkers
led us to discover more potent inhibitors of STAT3 activity, as
measured by DNA-binding activity/electrophoretic mobility
shift assay (EMSA). In this assay, nuclear extracts containing
active STAT3:STAT3 prepared from cancer cells are
preincubated with increasing concentrations of the compounds
at room temperature for 30 min, prior to incubation with the
B
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Figure 2. Structures of initial azetidine-based STAT3 inhibitors.
radiolabeled high-affinity sis-inducible element (hSIE) probe
(from the c-fos promoter) that binds active STAT3 and
subjecting to EMSA analysis.23,24,26 The basic premise is that
the binding of the high-affinity compounds to STAT3 inhibits
STAT3 DNA-binding activity, as shown in the
past.13,14,23,24,26,30 The bands corresponding to the DNA-
bound STAT3 are scanned, quantified by ImageJ, and
represented as percent of control (100%), which are plotted
against the concentration of the compounds. Representative
plots are shown in the Supporting Information Figure S2.
Although changing the 5-membered proline analogue, 3, to the
corresponding 6-membered, pipecolamide analogue, 4, de-
creased STAT3-inhibitory potency from EMSA IC50 2.4 μM for
3 to IC50 5.4 μM for 4 (Supporting Information Figure S3),
changing to the 4-membered azetidine-2-carboxamide analogue,
5a (Supporting Information Figure S3), gave over a 4-fold boost
in potency in vitro over proline, 3, against STAT3 DNA-binding
activity (Figure 1A). The concentration at which there is 50%
inhibition of STAT3 DNA-binding activity relative to the
dimethyl sulfoxide (DMSO)-treated control in the EMSA
analysis, IC50, is 0.52 μM for 5a. This result not only represented
over a log-order improvement in potency from the correspond-
ing glycine-based analogue, BP-1-102, but also represented one
of the first cases of small-molecule direct inhibitor of STAT3
DNA-binding activity in vitro, with sub-micromolar potency. To
confirm that the DNA-bound protein is STAT3, we performed
supershift analysis.31−33 The presence of the specific anti-
STAT3 antibody with the nuclear extract sample blocked and/
or caused a shift in the band for DNA:STAT3 complex
(Supporting Information Figure S4A). We also tested
napabucasin (BBI-608), the stem cell inhibitor, which is also
purported to inhibit STAT3 function,34 and another purported
STAT3 inhibitor, C188-9,35 both of which showed a minimal
direct effect on STAT3 activity up to 10 μM in the DNA-binding
assay/EMSA analysis (Supporting Information Figure S4B),
suggesting that these compounds have very little direct effects on
STAT3 DNA-binding activity. As previously observed with
other chiral inhibitors, such as 2 and 3 (Supporting Information
Figure S1),30 the (R)-enantiomer was more potent than the (S)-
enantiomer (EMSA IC50 of 0.52 μM for 5a vs 2.22 μM for 5b;
Figures 1A and 2), and changing the azetidine core from the
azetidine-2-carboxamide to the azetidine-3-carboxamide (5c)
(Figures 1A and 2) resulted in loss of activity. Our structure−
activity exploration thus focused on (R)-azetidine-2-carbox-
amides.
against tumor cells harboring constitutively active STAT3.
Based on our success in the previous work with the alanine and
proline analogues,30 we systematically varied the benzoic acid
and cyclohexylbenzyl moieties of the lead compound to
optimize potency and physicochemical properties (Figure 1
and Tables 1 and 2). Changes in the cyclohexyl group to
decrease lipophilicity were tolerated but with a variable effect on
potency (Table 1, entries: 3−6), suggesting that the increased
potency provided by the azetidine scaffold could allow for a
balancing of physicochemical properties while maintaining
sufficient potency. However, cyclohexyl remained the optimum.
Introduction of polarity by changing the phenyl ring of the
benzyl portion of the molecule to a heterocycle was successful.
Although replacement of the phenyl in the cyclohexylbenzyl
moiety with a 3-pyridyl resulted in a slight decrease in potency
(EMSA IC50 of 0.66 μM for 5m vs IC50 of 0.52 μM for 5a; Figure
1A and Table 1, entry 11), its replacement with the 2-pyridyl
analogue provided 50% boost in potency (EMSA IC50 of 0.38
μM for 5o; Figure 1A and Table 1, entry 13), potentially
indicating an additional binding interaction with the STAT3
protein. Moreover, increasing the polarity at this very lipophilic
region (i.e., the cyclohexylbenzyl moiety) results in better
molecular polarity distribution and thus may improve the drug-
like properties. Other modifications of the phenyl ring to
pyrazine, pyrimidine, or pyridazine (e.g., 5p, 5q, and 5r with
IC50 values of 0.46, 0.46, and 0.70 μM, respectively; Figure 1A
and Table 1) resulted in compounds retaining high affinity. In
this heterocyclic series as well, replacement of the cyclohexyl
with the less lipophilic cyclopentyl or tetrahydropyranyl resulted
in slightly less potent compounds (e.g., 5n, 5s, and 5t; Table 1).
Similar results were seen with the 5-fluorosalicylates (Table 1,
entries 7−10).
Benzoic acids, other than salicylic acids, led to less potent
compounds (Table 2). However, in the cyclohexylpyridylmethyl
subseries, the STAT3 potency was regained when the benzoic
acid group was substituted with fluorine at the 2- or 3-position
(Figure 1B and Table 2; compounds 6h and 6i with IC50 values
0.75 and 0.86 μM, respectively). Replacement of the benzoic
acid by a 4-oxazolecarboxylic acid or a 2-pyridinecarboxylic acid
led to compounds with weaker activity (Table 2, entries 4 and
11).
Analogues with Carboxylic Acid Motif Have Low
Cellular Activities. Given that constitutively active STAT3
promotes tumor cell proliferation and survival,27,36 we tested the
aforementioned, most active azetidine analogues for their effects
on the growth of the human breast cancer MDA-MB-231 and
MDA-MB-468 cells that harbor active STAT3.26,30 Despite their
sub-micromolar potency in the in vitro cell-free STAT3 DNA-
We took an iterative medicinal chemistry approach to design
and synthesize small-molecule STAT3 inhibitors of improved
physicochemical properties and that are potent and selective
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Table 1. SAR of Salicylic Acid-Based Analogues
a
Bands corresponding to STAT3:DNA complexes in gel were quantified using ImageJ and represented as a percent of control and plotted against
the concentration of compounds, from which IC50 values were determined.
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binding/EMSA assay (Tables 1 and 2), they showed weak
activity against the breast cancer cells up to 10 μM (Supporting
Information Figure S5A,B). This is presumably due to poor cell
membrane permeability afforded by the ionized polar
carboxylate group.37 Even tetrazole 6e (Table 2), which still
partially ionizes at physiological pH, showed weak cellular
activity at 10 μM. In this case, the concentration at which there is
loss of 50% of DMSO-treated control cell numbers relative to
the untreated, control cell numbers, EC50, is greater than 10 μM
against MDA-MB-231 cells.
Table 2. SAR of Benzoic Acid-Based Analogues
Carboxylate Methyl Esters, Phthalide, and Methyl
Amide Versions Improve Cellular Potency of the
Azetidine Analogues. To test whether the carboxylate
group was responsible for the low cellular activity, methyl esters
were prepared and evaluated (Figure 1C and Table 3). Not
surprisingly, the cell-free STAT3-inhibitory potencies of the
methyl ester versions were lower than their corresponding acid
analogues (Figure 1C and Table 3), highlighting the importance
of the acid motif for inhibiting STAT3 activity.23,24 By contrast,
Table 3. SAR of Ester Analogues
a
Bands corresponding to STAT3:DNA complexes in gel were
quantified using ImageJ and represented as a percent of control and
plotted against the concentration of compounds, from which IC50
values were determined.
a
Bands corresponding to STAT3:DNA complexes in gel were
quantified using ImageJ and represented as a percent of control and
plotted against the concentration of compounds, from which IC50
values were determined.
E
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Figure 3. In vitro cell viability and growth studies for effects of active azetidine analogs. (A) Human breast cancer MDA-MB-231 and MDA-MB-468
cells harboring aberrantly active STAT3 and (B) normal breast epithelial MCF-10A or breast cancer MCF-7 cells that do not and growing in 96-well
culture were treated once with 0−10 μM of the indicated STAT3 inhibitors, or (C) human breast cancer cells in 96-well culture were first treated with 1
μM 7g for 6 h followed by treatment with 0−100 nM docetaxel (Doc) or 0−20 μM cisplatin (Cis), or the cells were treated with docetaxel or cisplatin
alone. After 72-h culture, cells were harvested and subjected to CyQuant cell proliferation assay for the number of viable cells, which are plotted as %
cell viability against concentration from which EC50 values were derived; or (D) human breast cancer MDA-MB-231 or MCF-7 cells in a 6-well culture
plate were untreated or treated once with 2 μM 9k and every 24 h, cells were harvested and subjected to trypan blue exclusion/phase-contrast
microscopy for viable cell counts, which were plotted against the duration of treatment. Values are mean SEM of two to three studies each in three
replicates.
the ester versions generally showed stronger cellular activities
(Figure 3A and Table 3), presumably by facilitating cell
membrane permeability and functioning as prodrugs inside
cells. Thus, 7a, the methyl ester of salicylic acid 5a, had cell-free
EMSA potency of >4 μM (Figure 1C and Table 3), as compared
to 0.52 μM for 5a (Figure 1A and Table 1). However, 7a showed
good cellular activity against breast cancer cells following
treatment for 72 h, with EC50 values of 2.7 and 2.5 μM against
MDA-MB-231 and MDA-MB-468 viable cells, respectively
(Supporting Information Figure S5C and Table 3), as compared
with 5a, which showed no activity up to 10 μM (Supporting
Information Figure S5A). Similar results were seen when
comparing the methyl ester 7b with its corresponding acid, 6f
(EC50 against MDA-MB-231 cells after 72 h treatment was 4.4
μM for 7b vs > 10 μM for 6f) (Supporting Information Figure
S5B,C). The esters of the more active cyclohexylpyridylmethyl
analogues, 7c and 7d, also showed weaker STAT3-inhibitory
activities in the cell-free EMSA assay (Figure 1C and Table 3,
entries 3 and 4) than their corresponding acids (Table 1, entries
11 and 13), whereas they showed good activities against MDA-
MB-231 viable cells, with EC50 of 2.0 μM for 7c and 1.8 μM for
7d (Supporting Information Figure S5C and Table 3).
The interesting phthalide 7g showed one of the best activities
against MDA-MB-231 and MDA-MB-468 viable cells, with EC50
of 0.9 and 1.0 μM, respectively (Figure 3A and Tables 3 and 6).
The esters 7e (EC50 1.4 and 1.4 μM, respectively) and 7f (EC50
1.6 and 1.6 μM, respectively) also showed relatively better
cellular activities than their carboxylic acid versions against the
viable cell numbers of MDA-MB-231 and MDA-MB-468 cells
(Figure 3A and Tables 3 and 6). Notably, combined treatment
with 7g and docetaxel38 or cisplatin,39 both chemotherapeutic
agents used to treat TNBC, showed a strong shift of the dose−
response curves to the left indicative of an enhanced response
(Figure 3C). We note the apparent weaker cell-free EMSA
potencies of the methyl esters, which reflect their prodrug
properties, and is the reason the esters are more active in cells
than outside of cells. Under conditions that promote hydrolysis,
as is the case inside cells, the esters will be converted to
carboxylates. Moreover, the in vitro activities of these
compounds compare favorably to the parent lead compounds,
BP-1-102, SH5-07, and SH4-54, with IC50 of 6.8, 3.9, and 4.7
μM, respectively, and cellular activities at 10−20, 3.8, and 4.5
μM, respectively.24,26
With these promising results, our efforts continued also on
other bioisosteric replacements of the benzoic acid or salicylic
acid moieties that would enable penetration into cells. We first
concentrated our attention on benzohydroxamic acids, which
had shown promise in our previous work (e.g., SH5-07).26 The
first benzohydroxamic acids we prepared, 8a to 8d (Table 4),
showed only moderate inhibitory potencies against both the
STAT3 DNA-binding activity in the cell-free EMSA assay
(Figure 1D and Table 4) and the viable cell numbers of MDA-
MB-231 and MDA-MB-468 cells (Table 4). However, by
introducing the novel cyclohexylpyridylmethyl group, the
corresponding benzohydroxamic acid analogue 8i was found
to have the most potent STAT3-inhibitory activity in the whole
series in the EMSA assay, with an IC50 value of 0.34 μM (Figure
1D and Table 4, entry 9), though it did not have good cellular
activity (EC50 6.2 μM against MDA-MB-468; Table 4, entry 9).
Presumably, the increased polar surface area (PSA, 140 Å2)
mainly imparted by both the pyridyl ring in one vector and the
benzohydroxamic acid in the other vector had an impact on the
sluggish cell membrane permeability. Likewise, the benzohy-
droxamic acid-pyrazine analogue 8s gave a similar result (Figure
1D, Supporting Information Figure S5D, and Table 4, entry 19).
The O-methyl hydroxamic acid 8j (PSA 129 Å2, EMSA assay
IC50 0.51 μM; Table 4, entry 10) was similarly weak in the assay
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Table 4. SAR of Benzohydroxamic Acid and Salicylamide Analogues
a
Bands corresponding to STAT3:DNA complexes in gel were quantified using ImageJ and represented as a percent of control and plotted against
the concentration of compounds, from which IC50 values were determined. nd: not determined.
for viable cell numbers (Supporting Information Figure S5D and
Table 4, entry 10).
porting Information Figure S5C,E and Table 4, entry 17), and it
also showed a high STAT3-inhibitory potency (IC50 0.77 μM;
Figure 1D and Table 4, entry 17). The more polar primary
salicylamide 8p (PSA 133 Å2) showed relatively modest activity
against MDA-MB-231 and MDA-MB-468 cells, with EC50 2.9
and 2.6 μM, respectively (Table 4 and Supporting Information
Figure S5C), while strongly inhibiting STAT3 DNA-binding
activity in EMSA (IC50 0.66 μM).
Altogether, the replacement of the salicylic or benzoic acid
system with a methyl salicylate or benzoate (7a, 7b, 7c, 7d, 7e,
7f), phthalide (7g) (Table 3, Figure 3A, and Supporting
Information Figure S5C), or salicylamide (8p, 8q) (Table 4 and
Supporting Information Figure S5C) gave analogues that
showed variable cellular activities. Notably, 7e, 7f, and 7g
represent the best in the group to possess high cellular activities,
with an EC50 of 0.9−1.6 μM (Figure 3A and Table 6).
Variations of the sub-micromolar 3-fluorobenzoic acid
analogue, 6h, were explored. The 3-fluorobenzohydroxamic
acid, 8l, was weakly active against breast cancer cells (Supporting
Information Figure S5D and Table 4) while maintaining the
good STAT3-inhibitory potency in the EMSA assay (Figure 1D,
IC50 of 0.89 μM; Table 4, entry 12). We next sought to replace
the polar hydroxamic acid group while maintaining favorable
PSA and cLogP. The cutoff values that we followed, in
37
accordance with the literature, were PSA < 120 Å2 and
cLogP ≤ 5.40 The methyl salicylamide, 8q (PSA 119 Å2),
showed good activity against breast cancer cells in both viability
assays, with EC50 1.8 and 1.8 μM against MDA-MB-231 cells
and MDA-MB-468 cells, respectively, and trypan blue
exclusion/phase-contrast microscopy cell growth assay (Sup-
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Table 5. SAR of Benzo-Fused N-Heterocyclic Analogues
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Table 5. continued
a
Bands corresponding to STAT3:DNA complexes in gel were quantified using ImageJ and represented as a percent of control and plotted against
the concentration of compounds, from which IC50 values were determined. nd: not determined.
Comparatively, 7e, 7f, and 7g showed relatively weaker activities
against the normal human breast epithelial MCF-10A, with an
EC50 of 3.8−4.6 μM, and against the breast cancer MCF-7 cells
that do not harbor constitutively active STAT3, with an EC50 of
4.6−8.9 μM (Figure 3B and Table 6).
benzo-fused N-heterocyclic systems, which were also successful
in providing analogues with good cellular potency (Figure 1E
and Table 5). The benzo-fused N-heterocyclic replacement for
the salicylic acids allowed for further fine tuning of the cLogP
and especially PSA to achieve the desirable physicochemical
properties while maintaining potency. Initial results from the
isoquinolinone analogues were encouraging (Tables 5 and 6);
Additional specificity studies were conducted for the
inhibitors 7a, 7b, 7c, 8p, and 8q. The results showed that
these compounds are relatively weaker in inhibiting the number
of viable cells that do not harbor aberrantly active STAT3, with
EC50 4.6 and 7.2 μM (8p), and 3.8 and 4.6 μM (8q) against
MCF-7 or MCF-10A cells, respectively, and 4.3 μM (7a), 6.0
μM (7b) and 3.6 μM (7c) against MCF-10A cells that do not
harbor aberrantly active STAT3 (Supporting Information
Figure S5F and Table 4). Notably, treatment with 2 μM of 8q
showed no effect on the growth of MCF-7 cells as measured by
trypan blue exclusion/phase-contrast microscopy (Supporting
Information Figure S5E). These inhibitors therefore show
varied relative preferences against STAT3-dependent tumor
cells over cells that do not depend on STAT3 activity. Notably,
the carboxylic acid bioisosteres provided the first evidence that
strong cellular activity could be achieved.
Table 6. EMSA IC50 and EC50 Values of Select Esters and
Heterocycles
for example, compound 9b, with a PSA value of 99 Å2 showed
cellular activity against MDA-MB-231 cells, with an EC50 of 1.2
μM, while retaining STAT3-inhibitory potency (IC50 0.79 μM)
(Figure 1E and Table 5, entry 2). The corresponding
quinazolinone analogues were moderately active against
MDA-MB-231 and MDA-MB-468 cells (Table 5). This was
especially the case for the N-methyl variant 9g that was
moderately active against MDA-MB-231 and MDA-MB-468
Isosteric Replacement of Salicylic Acid Moiety with
Benzo-Fused N-Heterocyclic Systems Retained the In
Vitro Activity of Analogues and Greatly Improved Their
Cellular Activity. A concurrent approach was to incorporate
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cells (EC50 2.1 and 2.2 μM, respectively) while also maintaining
good STAT3-inhibitory activity, with an IC50 of 1.15 μM
(Figure 1E and Table 5, entry 7). As for the analogues with
benzo-fused 5-membered N-heterocycles, while the cellular
activity of benzotriazole 9r against MDA-MB-231 and MDA-
MB-468 was moderate (EC50 2.1 and 2.3, respectively), that of
9q was rather low (EC50 of 6.6 and 5.1 μM, respectively), despite
both having relatively good PSA values (107 and 119 Å2,
respectively; Table 5), a reflection perhaps of the more basic
pyridine over the pyrazine systems. The STAT3-inhibitory
potencies for both 9q and 9r, however, were high (IC50 0.61 and
0.63 μM, respectively; Figure 1E and Table 5, entries 17 and 18).
On the other hand, results for the isoindolinones 9i and 9j
showed moderate cellular activities (EC50 2.0 μM against MDA-
MB-231 and PSA 99 Å2 for both compounds), while 9i exhibited
better potency than 9j against STAT3 DNA-binding activity
(IC50 0.64 and 1.09 μM, respectively; Table 5, entries 9 and 10;
Supporting Information Table S1). On the other hand,
phthalimide 9k presented good inhibitory activities in the cell-
free STAT3 DNA-binding/EMSA (IC50 1.18 μM) (PSA 116 Å2;
Table 5, entry 11) and in the cell viability assays (EC50 1.7 and
1.9 μM against MDA-MB-231 and MDA-MB-468 cells,
respectively) (Figure 3A). Moreover, trypan blue exclusion/
phase-contrast microscopy showed that 2 μM 9k treatment of
MDA-MB-231 cells strongly inhibited cell growth (Figure 3D).
By contrast, the test of 9k on MCF-10A and MCF-7 cells that do
not harbor constitutively active STAT3 showed weaker effects
on both cell viability, with EC50 of 8.1 and 7.0 μM, respectively
(Figure 3B, Table 5, entry 11, and Table 6). Further, trypan blue
exclusion/phase-contrast microscopy showed that treatment
with 2 μM 9k of MCF-7 cells had no effect on cell growth
(Figure 3D). Furthermore, 72 h treatment of MCF-7 and MCF-
10A cells with 9a or 9d showed only moderate effects on viable
cell numbers (Table 5 and Supporting Information Figure S5F,
Table S1). Other systems such as indazole (9l, 9m, 9n, and 9o)
or benzimidazole (9p) did not look as promising (Table 5).
Comparatively, our inhibitors show better selectivity against
tumor cells harboring aberrantly active STAT3 than the
purported STAT3 inhibitors, napabucasin (BBI-608)34 or
C188-9.35 Treatments with increasing inhibitor concentrations
showed weak effects for C188-9 (EC50 25.7 μM) against MDA-
MB-231 cells but 2-fold stronger activity (EC50 13.75 μM)
against MCF-7 cells that do not harbor constitutively active
STAT3, while napabucasin showed strong effects against both
MDA-MB-231 (EC50 1.8 μM) and MCF-7 cells (EC50 1.49 μM)
(Supporting Information Figure S5G,H) suggesting the lack of
specificity for either inhibitor.
Figure 4. Isothermal titration calorimetry (ITC) measurements of 7g,
and 9k during incubation with STAT3. (A) Binding isotherm from the
integrated thermogram fit using the one-site model in the PEAQ-ITC
software generated from the titration of the inhibitors, 7g (red) and 9k
(blue), into STAT3. The KD was 880 and 960 nM, respectively, and (B)
the signature plots showing the thermodynamics parameters for each
titration reveal ΔH = −22.7 kJ/mol, ΔG = −34.6 kJ/mol, and −TΔS =
−11.9 kJ/mol for 7g and ΔH = −20.8 kJ/mol, ΔG = −34.4 kJ/mol, and
−TΔS = −13.6 kJ/mol for 9k. Data are representative of three
independent experiments.
Results together show that the azetidine inhibitors directly bind
with high affinity to STAT3.
Comparison of the Inhibition of STAT3 DNA-Binding
Activity over That of STAT1 and STAT5 In Vitro. To further
establish the specificity of select new analogues with potent
activity against STAT3, we investigated their effects on other
STAT family members, including STAT1 and STAT5 DNA-
binding activities in vitro, as previously reported.13,23,24,26
Nuclear extracts were prepared from epidermal growth factor
(EGF)-stimulated fibroblasts overexpressing the EGF receptor
(NIH3T3/EGFR) containing active STAT1, STAT3, and
STAT5. Extracts of equal total protein were incubated with
increasing or a single concentration of the azetidine compounds
prior to incubation with the radiolabeled hSIE probe that binds
STAT1 and STAT3 or the mammary gland factor element
(MGFe) that binds STAT1 and STAT5 and performing the
EMSA analysis. Results show that select azetidine analogues had
minimum effects on both STAT5 and STAT1 DNA-binding
activities (Figure 5 and Supporting Information Figure S6).
Excluding the esters of the carboxylic acids that in principle are
prodrugs, we selected and evaluated the selectivity of the
representative analogues from the other subgroups. All
compounds tested showed preferentially potent disruption of
the DNA-binding activity of STAT3:STAT3 homodimers ahead
of STAT1:STAT3 heterodimers, which were inhibited ahead of
STAT1:STAT1 homodimers or STAT5:STAT5 homodimers,
with potencies (IC50) of 0.52, 2.61, 12.0, and 9.3 μM (5a), 0.38,
1.46, >20, and >20 μM (5o), 1.08, 4.92, >20, and 17.5 μM (6f),
0.77, 3.14, >20, and >20 μM (8q), and 1.18, 4.71, >20, and >20
μM (9k) (Figure 5). Other compounds, including 5e, 5g, 5i, 5m,
5n, 7a, 7b, 8d, 8f, 8q, and 9k, also showed minimal effect on the
DNA-binding activity of STAT5:STAT5 or STAT1:STAT1
homodimers (Supporting Information Figure S6). Altogether,
Isothermal Titration Calorimetry (ITC) Studies of
Inhibitor Binding to STAT3. Given the enhanced potency
of the azetidine-based inhibitors against STAT3, we were
interested to determine their level of binding to the target in
vitro. We performed isothermal titration calorimetry (ITC)
studies, as previously described.41 The binding isotherm from
the integrated thermogram fit using the one-site model in the
PEAQ-ITC software generated from the titration of the
representative inhibitors, 7g (red) and 9k (blue), into STAT3
shows KD of 880 and 960 nM, respectively (Figure 4A). The
signature plots showing the thermodynamics parameters for
each titration reveal ΔH = −22.7 kJ/mol, ΔG = −34.6 kJ/mol,
and −TΔS = −11.9 kJ/mol for 7g and ΔH = −20.8 kJ/mol, ΔG
= −34.4 kJ/mol, and −TΔS = −13.6 kJ/mol for 9k (Figure 4B).
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Figure 5. Comparing the effect of new analogues on STAT1, STAT3, or STAT5 DNA-binding activity in vitro. Nuclear extracts of equal total protein
prepared from epidermal growth factor-stimulated NIH3T3/EGFR fibroblasts containing activated STAT1, STAT3, and STAT5 were preincubated
with increasing concentrations of the designated compounds for 30 min at room temperature prior to incubating with the radiolabeled hSIE probe that
binds STAT1 and STAT3 (upper panel) or the MGFe probe that binds STAT5 (bottom panel) and performing EMSA analysis. Positions of
STAT:DNA complexes in gel are labeled; control lanes (0) represent nuclear extracts preincubated with 10% DMSO. Data are representative of two to
three independent determinations.
these data show that active azetidine inhibitors tested have
preferential effects on the DNA-binding activity of STAT3 over
that of STAT1 or STAT5.
Analogues Inhibited Constitutive STAT3 Phosphor-
ylation and DNA-Binding Activity in Human Breast
Cancer Cells. The human breast cancer MDA-MB-231 and
MDA-MB-468 cells harbor aberrantly active STAT327,42 and are
sensitive to the azetidine inhibitors (Figure 3A). We were
interested to determine the effect of select active azetidine
analogues on the constitutive STAT3 signaling in the breast
cancer cells. Cells were treated with inhibitors, including 7g, 8q,
and 9k, at 1−5 μM for 0−24 h. Nuclear extracts were prepared
and subjected to DNA-binding activity/EMSA analysis, while
whole-cell lysates were prepared for sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE)/Western
blotting analysis to determine effects on intracellular STAT3
activity.23,24,26 Results showed that STAT3 DNA-binding
activity was inhibited to variable degrees in MDA-MB-468
cells by 7g, 8q, and 9k and in a time-dependent manner and as
early as 5 min for 7g and 9k and 6 h for 8q (Figure 6A and
Supporting Information Figure S7A). Similarly, treatment with
increasing concentrations of 7g, 8q, and 9k inhibited
pY705STAT3 in dose- and time-dependent manner in the
breast cancer cells (Figure 6B and Supporting Information
Figure S7B). For the 9k treatment condition, the pYSTAT3
bounced back by 24 h (Figure 6B-iii). Comparatively, treatment
of MDA-MB-468 and MDA-MB-231 cells for 2−3 h with the
purported STAT3 inhibitor, BBI-608,34 at 0.5−5 μM showed
weak to moderate effects on STAT3 DNA-binding activity or
pY705STAT3 (Supporting Information Figure S7C,D). For
nonspecific effects, immunoblotting analysis showed that
treatment of MDA-MB-231 cells with 1 or 3 μM 7g had no
measurable effect on the tyrosine kinases, EGFR, JAK2, and Src,
or on AKT and ERK1/2 (Supporting Information Figure S7E).
These results show that select cell-permeable azetidine
inhibitors are active at 1−3 μM against constitutive STAT3
induction in human breast cancer cells.
Figure 6. Effects of compounds on constitutive STAT3 activation in
tumor cells. (A) Nuclear extracts of equal total protein prepared from
the human breast cancer, MDA-MB-468 cells untreated (DMSO, 0) or
treated with 5 μM of the indicated analogues for 1−3 h were subjected
to STAT3 DNA-binding assay using the hSIE probe that binds STAT3,
and (B) immunoblotting analysis of whole-cell lysates of equal total
protein prepared from (i) and (ii) MDA-MB-231 cells untreated
(DMSO, 0) or treated with 1 or 3 μM of 7g for 3−24 h or (iii) MDA-
MB-468 cells untreated (DMSO, 0) or treated with 3 μM of 9k for 3−
24 h and probing for pY705STAT3, STAT3, or tubulin. Positions of
STAT3:DNA complex or proteins in gel are shown; control (0 or Con)
lane represents whole-cell lysates or nuclear extracts prepared from
0.05% DMSO-treated cells. Data are representative of two to three
independent determinations.
results indicate that at the concentration of 0.5 μM, 7g shows
significant inhibition of colony formation; however, 7e, 7f, and
9k only show minimal to moderate inhibition of colony
formation (Figure 7). Moreover, at the concentration of 1
μM, the results show a complete inhibition of colony formation
for 7g, a near-complete inhibition for 7e, and a significant
inhibition for 7f and 9k (Figure 7), whereas, 8q does not show
any inhibitory effect up to 1 μM (Supporting Information Figure
S8). These results indicate that selected azetidine-based STAT3
Azetidines Inhibited the Colony Survival of Human
Breast Cancer Cells. Human MDA-MB-231 breast cancer
cells in a single-cell culture were treated once with analogues 7e,
7f, 7g, 8q, or 9k at 0.5−1 μM and allowed to culture until
colonies were visible, which were stained and imaged. The
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Figure 7. Compounds 7e, 7f, 7g, and 9k inhibit the colony survival of human breast cancer cells in vitro. Human breast cancer MDA-MB-231 cells were
seeded as single-cell culture and treated once with 0−1 μM of the indicated compounds and allowed to culture until large colonies were visible, which
were stained with crystal violet and imaged. Data are representative of three independent determinations.
inhibitors attenuate the survival of cancer cells that harbor
constitutively active STAT3 at concentrations that inhibit
STAT3 activity.
aqueous solubility, as found by simulated gastric fluid (SGF) and
simulated intestinal fluid (SIF), above the standard cutoff of 60
μg/mL.47,48 For example, promising phthalide 7g has a solubility
at SGF of 116 μg/mL and at SIF of 200 μg/mL (Table S2).
Preliminary results from a human HLM MetID study of the
phthalide 7g showed both the parent compound and its
hydroxy-acid metabolite (Supporting Information Figure S9),
which is the result of hydrolytic lactone opening, and a
presumably more potent version in terms of directly inhibiting
STAT3 DNA-binding activity, suggesting that 7g and its
corresponding hydroxy-acid version exist in equilibrium.
Additional metabolites of 7g were also identified (Supporting
Information Figure S9).
Inhibition of STAT3-Regulated Genes and Induction
of Apoptosis. Consistent with the dysregulation of genes that
promote tumor cell growth, survival, and malignant pheno-
type,27 treatment of breast cancer cells with 1 or 3 μM 7g for 3−
24 h inhibited the expression of STAT3 target genes, c-Myc,
vascular endothelial growth factor (VEGF), Bcl-2, and survivin
(Figure 8) and induced poly (ADP-ribose) polymerase (PARP)
cleavage in parallel with the inhibition of pY705STAT3 (Figure
9).
CONCLUSIONS
■
Despite the strong validation of STAT3 as a target, as a
transcription factor, it has presented significant challenges for
drug discovery/development research due to the flat protein
surface that lacks deep pockets to target and design high-affinity
binders. Much of the earlier efforts to develop inhibitors of
STAT3 focused on targeting the SH2 domain to disrupt the
interactions with pTyr-containing binding sites and the
STAT3:STAT3 dimerization event.8,9 These efforts have
generated a number of small-molecule inhibitors,11−26,35 though
the clinical development of these inhibitors has been hampered
due to their low potency, unclear mechanisms of inhibition of
STAT3 signaling, and pharmacokinetic limitations among
others.27 New methods are required to design unique potent
small-molecule binders that can potentially overcome the
limitations. The nanomolar potent PROTAC-STAT3 de-
graders, including SD-36, are a new class of inhibitors, and the
proof-of-concept studies in which SD-36 inhibited the growth of
xenograft models of leukemia and lymphomas show that they
hold promise.28,29 In the current study, we have taken a different
strategy to address the challenge of potency by creating the new
azetidine class of inhibitors. The azetidine series marks a
significant advancement in the study of small-molecule STAT3
inhibitors. With the change from R-proline-amides to R-
Figure 8. Inhibition of STAT3 target gene expression in breast cancer
cells. Immunoblotting analysis of whole-cell lysates of equal total
protein prepared from MDA-MB-231 cells untreated (DMSO, C) or
treated with 1 or 3 μM of 7g for 3−24 h and probing for c-Myc Bcl-2,
VEGF, survivin, or tubulin. Positions of proteins in gel are shown;
control (C) lane represents whole-cell lysates prepared from 0.05%
DMSO-treated cells. Data are representative of two to three
independent determinations.
Initial Evaluation of Solubility and Metabolic Charac-
teristics of the Azetidine Compounds. The in vivo activity
of a drug is influenced by parameters, such as solubility,
permeability, and metabolism,43−45 which are dependent on its
physicochemical characteristic. With medicinal chemistry effort
focused on the optimization of the physicochemical features, we
conducted an industry-standard evaluation of solubility and
metabolism assays46 through the contract research organization
(CRO), Eurofins-CEREP. In general, compounds show good
Figure 9. Induction of apoptosis of human breast cancer cells. Human breast cancer, MDA-MB-231 and MDA-MB-468 cells in culture were treated
with 1 or 3 μM 7g for 0−24 h, whole-cell lysates were prepared, and samples of equal total protein were subjected to SDS/PAGE−Western blotting
analysis probing for pYSTAT3, STAT3, full-length PARP, cleaved PARP, and tubulin. Positions of proteins in gel are shown; control (0) lane
represents whole-cell lysates prepared from 0.05% DMSO-treated cells. Data are representative of two independent determinations.
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probes used were hSIE (high-affinity sis-inducible element from the c-
fos gene, m67 variant, 5′-AGCTTCATTTCCCGTAAATCCCTA)
that binds STAT1 and STAT3 and MGFe (mammary gland factor
element from the bovine β-casein gene promoter, 5′-AGATTTCTAG-
GAATTCAA) for STAT1 and STAT5 binding. Except where
indicated, nuclear extracts of equal total protein were preincubated
with compound for 30 min at room temperature prior to incubation
with the radiolabeled probe for 30 min at 30 °C before subjecting to
EMSA analysis. Where appropriate, bands corresponding to
STAT3:DNA complexes were scanned and quantified for each
concentration of compound using ImageJ and plotted as a percent of
control (DMSO) against the concentration of compound, from which
the IC50 values were derived.
Immunoblotting Analysis. Whole-cell lysate preparation and
immunoblotting analysis were performed as previously reported.23,24
Briefly, cultured cells treated or not were harvested and whole-cell
lysates were prepared in RIPA buffer. Samples of equal total protein
were subjected to SDS-PAGE and immunoblotting analysis. Primary
antibodies used were anti-STAT3, pY705STAT3, PARP, c-Myc, Bcl-2,
VEGF, survivin, tubulin, and GAPDH. All antibodies were purchased
from Cell Signaling Technology, Inc. (Danvers, MA), except GAPDH
from Santa Cruz Biotechnology (Dallas, Texas).
Cell Proliferation and Viability Assays. Studies were performed
as previously reported.23,24,26 Briefly, cultured cells in 6-well or 96-well
plates were treated with or without compounds for the indicated
concentrations, and cells were harvested every 24 h up to 96 h for viable
cell count by trypan blue exclusion phase-contrast microscopy, or after
72 h, cells were subjected to CyQuant cell proliferation assay following
the manufacturer’s instructions (Invitrogen/ThermoFisher Scientific).
In the case of the combination treatment, cells in culture were first
treated with azetidine inhibitor, 7g, for 6 h followed by treatment with
docetaxel or cisplatin and then harvested after a total of 72 h treatment
for CyQuant assay. Cell viability was normalized to the percentage of
the control groups.
Clonogenic Survival Assays. Colony survival assay was
performed as previously reported.24,26 Briefly, cells were seeded as
single-cell cultures in 6-well plates (250 cells per well), treated once the
next day with compounds at the indicated concentrations and allowed
to culture until large colonies were visible. Colonies were stained with
crystal violet for 4 h and photographed.
Isothermal Titration Calorimetry (ITC). The ITC experiment
was carried out as previously described41 with some modification using
Malvern Panalytical MicroCal PEAQ-ITC (United Kingdom). Studies
were done at 25 °C. Briefly, STAT3 inhibitors, previously suspended in
100% DMSO, were diluted in 20 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), 150 mM KCl buffer, so the
final DMSO was 5%. To avoid buffer mismatch, STAT3 in HEPES
buffer was diluted in HEPES buffer with 5% DMSO final concentration.
Three hundred microliter volumes (300 μL) of 3.0 μM STAT3 were
placed in the cell and titrated with 250 μM inhibitors. Titrations took
place by injecting 2 μL inhibitor in a 2.5 min injection for the titration
peak to return to the baseline. The KD was calculated using the
MicroCal PEAQ-ITC analysis software, as well as Prism GraphPad
software, using the one-site model. Control experiments were carried
out by titration of the inhibitors into buffer, buffer into STAT3, and
buffer into buffer. The three controls were used as a composite for the
ITC experiment to subtract the heat of dilution and background noise
from the measurements.
azetidine-2-carboxamides, the first analogues have been realized
with sub-micromolar potency in the STAT3 DNA-binding
activity/EMSA, i.e., salicylate 5a (EMSA IC50 0.55 μM) among
many others. Optimization of the salicylate series provided the
5-cyclohexyl-2-pyridinylmethyl analogues that led to the potent
salicylate analogue, 5o (EMSA IC50 0.38 μM), which
represented a log-order improvement in potency over earlier
published analogues.24,26 Further elaboration of the salicylic acid
portion of the molecule provided benzamides and benzo-fused
N-heterocycles, which maintained the sub-micromolar potency
in the in vitro STAT3 DNA-binding/EMSA assay and furnished
the most potent analogue in the whole azetidine series, i.e.,
benzohydroxamic acid 8i (EMSA IC50 0.34 μM). Having
overcome the potency barrier, we focused the medicinal
chemistry efforts on optimizing the physicochemical properties
while maintaining potency. We were able to trade some potency
for improved cell permeability, and several compounds showed
improved activity in cell-based assays over previous analogues.
In particular, ester versions of the carboxylic acid, including 7e
and 7f, or the lactone, 7g, or compounds that contained
carboxylic acid bioisosteres, such as salicylamides (8p and 8q)
and some of the benzo-fused N-heterocycle analogues (9b, 9f,
and 9k), showed good cellular activity. Notably, 7e, 7f, 7g, and
9k have the best cellular activities against breast tumor cells that
harbor aberrantly active STAT3, including inhibiting cell
growth, suppressing STAT3 target gene expression, and
inducing apoptosis. The current azetidine series of compounds
therefore are significantly improved over their leads, BP-1-102,
SH5-07, and SH4-54, which had EMSA IC50 of 6.8, 3.9, and 4.7
μM, respectively, and cellular activities at 10−20, 3.8, and 4.5
μM, respectively. They also compare more favorably to
napabucasin (BBI-608) and C188-9. The azetidine compounds
therefore represent promising new chemical entities in the
search for suitable small-molecule, direct STAT3 inhibitors for
further clinical development into new anticancer agents either as
standalone or in combination. Their utility in combination
therapy with chemotherapy, including docetaxel or cisplatin, is
demonstrated herein.
EXPERIMENTAL SECTION
■
Cell Lines and Reagents. The human breast cancer MDA-MB-468
and MCF-7 cell lines and the normal breast epithelial line MCF-10A
have been reported previously,24,26,31,32 and MDA-MB-231 was
obtained from the National Cancer Institute on December 2, 2015.
MCF-10A cells were grown in Dulbecco’s modified Eagle’s medium,
DMEM/F12 with 5% horse serum plus EGF (20 ng/mL), insulin (10
μg/mL), hydrocortisone (0.5 mg/mL), and 100 ng/mL cholera toxin.
All other cells were grown in DMEM plus 10% heat-inactivated fetal
bovine serum (FBS). Cell line authentication was done in December
2015 by American Type Culture Collection (ATCC) for MDA-MB-
468, which was found to be authentic. Mycoplasma test was conducted
on MDA-MB-231 and MCF-10A IDEXX BioAnalytics (Columbia,
MO) in Dec 2019. Both lines are negative, while the test has not been
done on MCF-7 and MDA-MB-468. Antibodies against STAT3,
pY705STAT3, pY1173EGFR, EGFR, pY1007/1008JAK2, JAK2,
pY416Src, Src, pS473AKT, AKT, pT202/Y204ERK1/2 (p44/42),
ERK1/2, full-length poly (ADP-ribose) polymerase (PARP), cleaved
PARP, tubulin, and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) were purchased from Cell Signaling Technology, Inc.
(Danvers, MA). Cisplatin and docetaxel were purchased from Sigma-
Aldrich (St. Louis, MO).
General Methods for Chemistry. All reagents and solvents were
purchased from commercial sources and used without further
purification. All moisture-sensitive reactions were performed under a
static atmosphere of nitrogen or argon in oven-dried glassware.
Tetrahydrofuran (THF), dichloromethane (DCM), diethyl ether
(Et2O), toluene, and dimethylformamide (DMF) used in the reactions
were dried by being passed through an SPS system. Other anhydrous
solvents were purchased from commercial sources. Thin-layer
chromatography (TLC) was performed on glass plates, 250−1000
μm. Flash column chromatography was performed on silica gel, 200−
Nuclear Extract Preparation, Gel Shift Assays, and Densito-
metric Analysis. Nuclear extract preparations and DNA-binding
activity/electrophoretic mobility shift assay (EMSA) were carried out
as previously described.13,14,23,24,26 The 33P-labeled oligonucleotide
1
400 mesh. H NMR spectra were obtained as CDCl3, CD3OD, or
(CD3)2SO solutions using an Agilent 300MHz NMR spectrometer
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̈
with an Agilent DD2 console, and chemical shifts were expressed in a
(ppm) using residual solvent (CDCl3, 7.26 ppm; CD3OD, 3.31 ppm;
and (CD3)2SO, 2.50 ppm) as the reference standard. When peak
multiplicities are reported, the following abbreviations are used: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br-s
(broadened singlet), dd (doublet of doublets), and dt (doublet of
triplets). Coupling constants, when reported, are reported in hertz
(Hz). All compounds were analyzed by LC/MS (liquid chromatog-
raphy/mass spectrometry) using an Agilent Triple Quad 640 LC/MS.
Ionization was generally achieved via electron spray (ESI) unless
otherwise indicated. The LC fraction detection consisted of a variable
wavelength detector, and all tested compounds had purity greater than
95%. High-resolution mass spectral (HRMS) data was obtained for all
tested compounds using either an Agilent 6200 LC/MSD TOF or an
Agilent 6545 Q-TOF LC/MS, and reported exact masses were
calculated based on an algorithm using MS (ESI) m/z for [M + H]+
and [M + Na]+ adducts and were within 5 ppm of the expected target
mass. Chiral molecules were analyzed by chiral HPLC using Chiralpak
AD-H or OD-H columns (4.6 mm × 250 mm, UV detection at 254 or
261nm); eluents used were hexane and i-PrOH.
Division of Oncology and Cedars-Sinai Cancer, Cedars-Sinai
Medical Center, Los Angeles, California 90048, United States
Yinsong Zhu − Department of Medicine, Division of Oncology
and Cedars-Sinai Cancer, Cedars-Sinai Medical Center, Los
Angeles, California 90048, United States
Kayo Nakamura − Department of Chemistry, University of
Hawaii, Manoa, Honolulu, Hawaii 9682, United States
Weiliang Chen − Department of Chemistry, University of
Hawaii, Manoa, Honolulu, Hawaii 9682, United States
Wenzhen Fu − Cancer Biology Program, University of Hawaii
Cancer Center, University of Hawaii, Manoa, Honolulu,
Hawaii 96813, United States; Department of Chemistry,
University of Hawaii, Manoa, Honolulu, Hawaii 9682, United
States
Casie Kubota − Cancer Biology Program, University of Hawaii
Cancer Center, University of Hawaii, Manoa, Honolulu,
Hawaii 96813, United States
Jasmine Chen − Cancer Biology Program, University of Hawaii
Cancer Center, University of Hawaii, Manoa, Honolulu,
Hawaii 96813, United States
Felix Alonso-Valenteen − Cedars-Sinai Cancer and
Department of Biomedical Sciences, Cedars-Sinai Medical
Center, Los Angeles, California 90048, United States
Simoun Mikhael − Cedars-Sinai Cancer and Department of
Biomedical Sciences, Cedars-Sinai Medical Center, Los
Angeles, California 90048, United States
Lali Medina-Kauwe − Cedars-Sinai Cancer and Department of
Biomedical Sciences, Cedars-Sinai Medical Center, Los
Angeles, California 90048, United States
Marcus A. Tius − Cancer Biology Program, University of
Hawaii Cancer Center, University of Hawaii, Manoa,
Honolulu, Hawaii 96813, United States; Medicinal Chemistry
Leader, Department of Chemistry, University of Hawaii,
Manoa, Honolulu, Hawaii 9682, United States; orcid.org/
0000-0002-7092-8993
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01705.
■
sı
Supplementary results and discussion; chemistry; early
leads (Figure S1); dose−response curves for the cell-free
EMSA analysis for the most active compounds (Figure
S2); EMSA IC50 and cell viability EC50 values for select
esters and heterocyclics (Table S1) (PDF)
Molecular formula strings (CSV)
Qualitative Analysis Report: Compound 5a (PDF)
Qualitative Analysis Report: Compound 5o (PDF)
Qualitative Analysis Report: Compound 7e (PDF)
Qualitative Analysis Report: Compound 7f (PDF)
Qualitative Analysis Report: Compound 7g (PDF)
Qualitative Analysis Report: Compound 8i (PDF)
Qualitative Analysis Report: Compound 9k (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01705
AUTHOR INFORMATION
Corresponding Authors
Francisco Lopez-Tapia − Cancer Biology Program, University
of Hawaii Cancer Center, University of Hawaii, Manoa,
Honolulu, Hawaii 96813, United States; Medicinal Chemistry
Leader, Department of Chemistry, University of Hawaii,
Manoa, Honolulu, Hawaii 9682, United States;
Author Contributions
■
#C.B.-P. and P.Y. authors contributed equally to this work.
Notes
The authors declare the following competing financial
interest(s): JT is a co-founder of Novella, LLC, which has
licensed STAT3 IP. The remaining authors have no competing
interests.
Email: francisco.lopez@cshs.org
James Turkson − Cancer Biology Program, University of
Hawaii Cancer Center, University of Hawaii, Manoa,
Honolulu, Hawaii 96813, United States; Department of
Medicine, Division of Oncology and Cedars-Sinai Cancer,
Cedars-Sinai Medical Center, Los Angeles, California 90048,
United States; orcid.org/0000-0002-6964-1602;
Phone: 310-423-6887; Email: james.turkson@cshs.org
ACKNOWLEDGMENTS
■
We thank all colleagues and members of our laboratory for the
stimulating discussions and Dr. Joel Kawakami and Mabel
Bernaldez of Chaminade University of Honolulu for their
contributions to the design of the Cover Art. This work was
supported by NIH/NCI R01 CA208851 (J.T.), LEIDOS
Biomedical Research/NCI Contract 19X122Q (J.T.), and
Cedars-Sinai Start-up funds (JT).
Authors
Christine Brotherton-Pleiss − Cancer Biology Program,
University of Hawaii Cancer Center, University of Hawaii,
Manoa, Honolulu, Hawaii 96813, United States; Medicinal
Chemistry Leader, Department of Chemistry, University of
Hawaii, Manoa, Honolulu, Hawaii 9682, United States
Peibin Yue − Cancer Biology Program, University of Hawaii
Cancer Center, University of Hawaii, Manoa, Honolulu,
Hawaii 96813, United States; Department of Medicine,
ABBREVIATIONS
■
STAT, signal transducer and activator of transcription; EMSA,
electrophoretic mobility shift assay; PARP, poly (ADP-ribose)
polymerase; VEGF, vascular endothelial growth factor;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase
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