Small-molecule inhibitors of cytokine-mediated STAT1 signal transduction in β-cells with improved aqueous solubility

Article abstract of DOI:10.1021/jm400397x

We previously reported the discovery of BRD0476 (1), a small molecule generated by diversity-oriented synthesis that suppresses cytokine-induced β-cell apoptosis. Herein, we report the synthesis and biological evaluation of 1 and analogues with improved aqueous solubility. By replacing naphthyl with quinoline moieties, we prepared active analogues with up to a 1400-fold increase in solubility from 1. In addition, we demonstrated that 1 and analogues inhibit STAT1 signal transduction induced by IFN-γ.

Full text of DOI:10.1021/jm400397x

Brief Article
pubs.acs.org/jmc
Small-Molecule Inhibitors of Cytokine-Mediated STAT1 Signal
Transduction in β‑Cells with Improved Aqueous Solubility
Stephen S. Scully,† Alicia J. Tang,† Morten Lundh,†,‡ Carrie M. Mosher,† Kedar M. Perkins,†
and Bridget K. Wagner*,†
†Chemical Biology Program and Chemical Biology Platform, The Broad Institute of Harvard and MIT, 7 Cambridge Center,
Cambridge, Massachusetts 02142, United States
‡Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark
S
* Supporting Information
ABSTRACT: We previously reported the discovery of BRD0476 (1), a small molecule generated by diversity-oriented synthesis
that suppresses cytokine-induced β-cell apoptosis. Herein, we report the synthesis and biological evaluation of 1 and analogues
with improved aqueous solubility. By replacing naphthyl with quinoline moieties, we prepared active analogues with up to a
1400-fold increase in solubility from 1. In addition, we demonstrated that 1 and analogues inhibit STAT1 signal transduction
induced by IFN-γ.
with a highly functionalized and stereochemically rich medium-
sized (8-membered) lactam ring.13,14
INTRODUCTION
■
Type-1 diabetes (T1D) is a chronic metabolic disorder that
78000 children worldwide develop each year.1 This disease is
characterized by autoimmune destruction of pancreatic β-cells,
resulting in decreased insulin production. The secretion of pro-
inflammatory cytokines by macrophages in the pancreatic islets
of Langerhans is believed to trigger intracellular signaling
cascades leading to β-cell apoptosis.2 Small molecules that
restore β-cell viability in the presence of cytokines may have
potential to augment insulin replacement therapy. However,
relatively few small molecules are known to possess protective
effects from cytokines.3−6
In a campaign to discover new therapies and probes for T1D,
our group developed a phenotypic assay using the rat INS-1E
β-cell line to screen for small-molecule suppressors of apoptosis
in the presence of the cytokines tumor necrosis factor-α (TNF-
α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ).7 High-
throughput screening identified several hits that increased β-cell
viability. Subsequent medicinal chemistry optimization of a
screening hit belonging to a library derived from diversity-
oriented synthesis (DOS)8−10 led to the discovery of BRD0476
(1, ML18711) (Figure 1), a small molecule with submicromolar
activity (EC50 = 0.78 μM).12 1 represents a novel chemotype
1 exhibits poor aqueous solubility, which would adversely
affect the bioavailability of 1 in animals and limit its use in vivo.
In this study, we describe our efforts toward the synthesis and
biological evaluation of analogues of 1 with improved solubility.
In addition, we show that the biological activity of 1 and
analogues largely results from inhibiting cell-signaling pathways
activated by IFN-γ.
RESULTS AND DISCUSSION
■
To rapidly access 1 and urea analogues, we developed an
efficient, solution-phase synthesis of aniline 4 in four steps from
2, a DOS-generated intermediate previously prepared using an
aldol-based “build/couple/pair” strategy (Scheme 1).12,13
Treatment of 2 with TBSOTf resulted in a protecting group
switch from a N-Boc-carbamate to a N-silyl-carbamate
intermediate, which was desilylated (TBAF) to afford a
secondary crude amine. Direct cleavage of the Boc-carbamate
to a free amine under acidic conditions was not feasible due to
competing deprotection of the p-methoxybenzyl (PMB) ether.
Capping the amine intermediate with 1,4-benzodioxan-6-
sulfonyl chloride provided sulfonamide 3 in 74% isolated
yield for three steps. Hydrogenation (H2, Pd/C) of the nitro
moiety generated 4 in gram quantities.
Two strategies were pursued to install the urea moiety for 1
and analogues. One strategy involved addition of isocyanates
directly to aniline 4. However, these reactions were sluggish
and required microwave heating under basic conditions to
achieve moderate yields at best. Furthermore, quinolyl
isocyanates were not commericially available for the preparation
of quinoline urea analogues, and their syntheses proved
problematic using standard protocols for isocyanate formation.
Figure 1. DOS-generated 1 suppresses cytokine-induced β-cell
apoptosis.
Received: March 8, 2013
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of Advanced Aniline Intermediate 4
Table 1. SAR and Solubility for 1 and Analogues
a
Reagents and conditions: (a) TBSOTf, 2,6-lutidine, CH2Cl2; (b)
TBAF, AcOH, THF; (c) 1,4-benzodioxan-6-sulfonyl chloride, 2,6-
lutidine, CH2Cl2, 74% yield (3 steps); (d) H2, Pd/C, EtOH, 91% yield.
Instead, the urea moiety could be installed using a two-step
protocol by subjecting 4 to diphosgene (ClCO2CCl3) to form
isocyanate intermediate 5,15 followed by addition of various aryl
and alkyl amines (Scheme 2). Isocyanate 5 did not require
a
Scheme 2. General Synthesis of 1 and Urea Analogues
a
IC50 values were determined using three replicates in the caspase-3/7
b
assay in INS-1E β-cells. Maximum activity is reported as % rescue
from cytokine-induced caspase-3/7 activation. Solubility was
measured in a phosphate buffered saline solution (pH 7.4). n.d.,
c
d
not determined.
caspase-3/7 activity was measured as a more direct readout of
apoptosis. 1 demonstrated a small protective effect with
standard concentrations of cytokines in the caspase assay
(condition A, Figure 2). Lowering the concentrations of IL-1β
and TNF-α by 20- and 10-fold, respectively, as well as
increasing IFN-γ 4-fold resulted in higher rescue efficacy of 1,
despite lowering caspase-3/7 activity for this mixture
(condition B). Further increasing the concentration of IFN-γ
to 500 ng/mL resulted in equal levels of cytokine-induced
caspase-3/7 activation but elicited the strongest protective
effects from 1, reducing caspase activity by 74% (condition C).
Using these cytokine concentrations, 1 generated a dose−
response with an IC50 value of 1.66 μM and a maximum activity
of 71% (Table 1). We used this assay for the biological
evaluation of additional analogues.
a
Reagents and conditions: (a) diphosgene (ClCO2CCl3), proton
sponge, CH2Cl2, 96% yield; (b) R2NH, PhMe, 36−99% yield; (c)
DDQ, pH 7.4 buffer, CH2Cl2, 63% and 50% yield (for 1 and 7b,
respectively); (d) TFA, CH2Cl2, 44−87% yield (for 7a,c−i).
purification for further reactions and could be stored at −20 °C
for relatively short periods of time (at least one week).
Moreover, addition of amines to 5 was facile and proceeded at
room temperature under neutral conditions to afford PMB-
protected urea derivatives 6 in moderate to excellent yields
(36−99%). Deprotection of the PMB ether with DDQ or TFA
provided 1 and urea analogues 7 (see Table 1 for complete
structures).
Using a thermodynamic solubility assay, the aqueous
solubility of 1 was determined to be 0.1 μM in phosphate
buffered saline (pH 7.4) (Table 1). This low solubility may be
attributed to the lipophilic naphthyl moiety. In previous SAR
In our earlier report,12 we determined the potency and
maximum activity of 1 and analogues using the CellTiter-Glo
viability assay with INS-1E β-cells.16 In the present study,
B
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Journal of Medicinal Chemistry
Brief Article
STAT1 has previously been shown to regulate apoptosis in
INS-1E β-cells.22,23 Binding of IFN-γ to cell surface receptors
leads to activation of JAK1/JAK2, which subsequently
phosphorylate cytoplasmic STAT1 at Tyr701. Upon phosphor-
ylation, STAT1 dimerizes and translocates to the nucleus to
regulate gene transcription.
When INS-1E β-cells were treated with IFN-γ, rapid
phosphorylation of STAT1 at 30 min was observed by Western
blotting (Figure 3). In addition to Tyr701, phosphorylation was
Figure 2. Optimization of cytokine concentrations for the caspase-3/7
assay in INS-1E β-cells. Effects of cytokines and compounds were
measured after 48 h. Data represent the mean standard error for
three independent experiments with six replicates.
Figure 3. Regulation of STAT1 phosphorylation by IFN-γ, 1, and
analogues in INS-1E β-cells. Effects of cytokines and compounds were
analyzed by Western blot after 30 min.
studies, this group was found to be a crucial structural
requirement in suppressing cytokine-induced β-cell apoptosis.12
An approach to increase aqueous solubility while retaining the
naphthyl moiety involved disrupting molecular planarity
between the naphthyl and urea groups to lower crystal packing
energy of solutes.17 1-Naphthylmethyl urea 7a was designed to
disrupt planarity by interrupting delocalization of the naphthyl-
and urea-conjugated π system with a methylene spacer.
However, 7a did not show enhanced solubility and was inactive.
N-Methyl-N-1-naphthyl urea 7b was also designed to
increase aqueous solubility by disrupting molecular planarity.
The addition of the methyl group to the urea nitrogen may
prevent access to planar conformations, which should be
hindered by a steric interaction between the N-methyl and
C(2)- or C(8)-hydrogen of the quinoline moiety. In fact, 7b
exhibited a 110-fold increase in solubility (11.2 μM), which
correlated to a decreased melting point for 7b (144−146 °C)
compared to 1 (170−172 °C). However, 7b was less efficacious
in inhibiting apoptosis (maximum activity = 15%)
Alternatively, quinoline analogues 7c−i should retain the
steric properties of the naphthyl urea moiety crucial for
biological activity while increasing aqueous solubility. Intro-
duction of nitrogen atoms would decrease the lipophilicity of
the naphthyl component and provide an additional hydrogen
bond acceptor that should enhance solubility.18,19 Indeed,
quinoline analogues 7c−i demonstrated an increase in solubility
in which 5-quinolyl urea 7f showed a remarkable 1400-fold
increase in solubility compared to 1. 7f was equally potent
(1.62 μM) to 1 in the caspase assay but had a lower maximum
activity (41%). Likewise, the exceptionally soluble 4-isoquinolyl
(7d) and 5-isoquinolyl (7g) ureas exhibited similar biological
activity. 1-Isoquinolyl (7c) and 8-quinolyl (7i) ureas were less
soluble (0.7 and 1.8 μM, respectively) than other quinoline
analogues, which may be due to hydrogen bonding of the urea
hydrogen (N−H) and quinoline nitrogen. However, 7i
possessed the best activity among the quinoline analogues,
inhibiting apoptosis similarly to 1 with a favorable maximum
activity of 69%.
also observed at Ser727 (data not shown).20,21 Co-treatment
with IFN-γ and 1 led to a complete reduction of STAT1
phosphorylation, while inactive analogue 7a did not result in
inhibition. Further, total STAT1 levels were slightly increased
by IFN-γ alone or in combination with 1 and analogues. Taken
together, these results suggest that STAT1 phosphorylation
may be an excellent biomarker for compound activity. To this
end, the 8-quinolyl urea analogue 7i with improved aqueous
solubility also significantly decreased levels of pSTAT1 in an
analogous manner to 1.
To examine the effect of inhibiting STAT1 phosphorylation
on subsequent nuclear translocation, cells were treated with
IFN-γ in the presence or absence of compounds, fixed, and
immunofluorescence was performed to monitor subcellular
localization of STAT1 (Figure 4).24 STAT1 was located in both
the cytoplasm and nucleus of untreated cells, while treatment
with IFN-γ induced a substantial accumulation of STAT1 in the
nucleus. Remarkably, 1 and 7i nearly abolished this nuclear
localization of STAT1. As a negative control, 7a did not inhibit
nuclear translocation of STAT1 in the presence of IFN-γ.
CONCLUSION
■
In summary, we developed an efficient synthesis of 1 and
analogues which suppress cytokine-induced β-cell apoptosis.
Quinoline-containing analogues showed improved aqueous
solubility from 1, of which 8-quinolyl urea 7i retained optimal
biological activity. In addition, we have demonstrated that these
small molecules inhibit the JAK-STAT signaling pathway by
inhibiting IFN-γ-induced STAT1 phosphorylation and nuclear
translocation. Studies to improve the pharmacokinetic proper-
ties of 1 and 7i, as well as develop a quantitative
immunofluorescence assay for STAT1 nuclear translocation,
are ongoing and will be described in subsequent reports.
EXPERIMENTAL SECTION
■
Chemistry. The purity of all compounds was >95% as determined
by UPLC-MS. The synthesis of 3, 4, and 7i is described below. See
Supporting Information for detailed syntheses and spectral character-
ization of all compounds.
As indicated in the optimization of cytokine concentrations
for the caspase-3/7 assay, 1 and active analogues may directly
perturb cell-signaling pathways induced by IFN-γ. This
cytokine activates the JAK/STAT signaling pathway,20,21 and
N-(((2R,3R)-5-((S)-1-((4-Methoxybenzyl)oxy)propan-2-yl)-3-
methyl-10-nitro-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b][1,5]-
C
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Journal of Medicinal Chemistry
Brief Article
Figure 4. Regulation of STAT1 nuclear translocation by IFN-γ, 1, and analogues in INS-1E β-cells. Effects of cytokines and compounds were
assessed after 30 min by immunofluorescence imaging.
oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo[b][1,4]-
dioxine-6-sulfonamide (3). TBSOTf (1.98 mL, 8.61 mmol) was
added dropwise to a solution of N-Boc-protected amine 212 (1.6 g,
2.87 mmol) and 2,6-lutidine (1.35 mL, 11.59 mmol) in CH2Cl2 (29
mL) at rt. The reaction mixture was stirred at rt for 1 h, diluted with
satd NH4Cl, and extracted into CH2Cl2. The organic layers were dried
over MgSO4, filtered, and concentrated in vacuo to provide a N-silyl
carbamate intermediate. LCMS (ESI+) m/z: 616.39 (M + H). This
crude intermediate was dissolved in THF (57 mL) and cooled to 0 °C.
To the resulting solution was added AcOH (0.181 mL, 3.16 mmol)
followed by dropwise addition of TBAF (1.0 M in THF, 3.16 mL, 3.16
mmol). The reaction mixture was further stirred for 30 min at 0 °C,
diluted with satd NH4Cl, and extracted into EtOAc. The organic layers
were washed with brine, dried over MgSO4, filtered, and concentrated
in vacuo to provide a secondary amine. LCMS (ESI+) m/z: 458.41 (M
+ H). This crude intermediate and 2,6-lutidine (1.67 mL, 14.35 mmol)
were dissolved in CH2Cl2 (29 mL), and 1,4-benzodioxan-6-sulfonyl
chloride (808 mg, 3.44 mmol) was added to the resulting solution at
rt. The reaction mixture was further stirred for 20 h at rt, diluted with
satd NH4Cl, and extracted into CH2Cl2. The organic layers were dried
over MgSO4, filtered, and concentrated in vacuo. Purification on silica
gel (gradient of 10−50% EtOAc in hexanes) provided 1.39 g (2.12
N-(((2R,3R)-5-((S)-1-Hydroxypropan-2-yl)-3-methyl-6-oxo-
10-(3-(quinolin-8-yl)ureido)-3,4,5,6-tetrahydro-2H-benzo[b]-
[1,5]oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo[b]-
[1,4]dioxine-6-sulfonamide (7i). To a solution of disphosgene (11
μL, 0.095 mmol) in CH2Cl2 (0.5 mL) at 0 °C was added dropwise a
solution of aniline 4 (100 mg, 0.160 mmol) and proton sponge (67
mg, 0.314 mmol) in CH2Cl2 (0.5 mL). The reaction mixture was
warmed to rt, stirred for 15 min, and concentrated in vacuo. The
residue was redissolved in CH2Cl2, washed with 1 M HCl and 1 M
NaOH, dried over MgSO4, filtered, and concentrated in vacuo to
provide 120 mg (0.184 mmol, 96% yield) of isocyanate 5. LCMS
(ESI+) m/z: 652.25 (M + H). Crude 5 (80 mg, 0.123 mmol) and 8-
aminoquinoline (89 mg, 0.615 mmol) were dissolved in toluene (2.1
mL) and stirred at rt for 2.5 h. The reaction mixture was concentrated
in vacuo and directly purified on silica gel (gradient of 0−5% MeOH
in CH2Cl2) to provide 88 mg (0.111 mmol, 90% yield) of quinolyl
urea 6i. LCMS (ESI+) m/z: 796.37 (M + H). To a solution of 6i (53
mg, 0.067 mmol) in CH2Cl2 (2.3 mL) was added TFA (1.1 mL)
dropwise at rt. The reaction mixture was further stirred at rt for 15
min, concentrated in vacuo, and directly purified on silica gel (gradient
of 0−5% MeOH in CH2Cl2) to provide the 39 mg of 7i (0.058 mmol,
87% yield). 1H NMR (300 MHz, CDCl3) δ 9.63 (s, 1H), 8.86 (d, J =
2.7 Hz, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.18 (s,
1H), 8.07 (dd, J = 6.6 Hz, 3.0 Hz, 1H) 7.53 (ovrlp m, 3H), 7.32 (d, J =
1.8 Hz, 1H), 7.26 (ovrlp dd, J = 8.1 Hz, 1.5 Hz, 1H), 7.16 (ovrlp m,
2H), 6.85 (d, J = 8.4 Hz, 1H), 5.65 (br s, 1H), 4.22 (ovrlp m, 4H),
4.10 (m, 1H), 3.78 (dd, J = 11.1 Hz, 2.1 Hz, 1H), 3.79−3.47 (ovrlp m,
4H), 3.31 (d, J = 13.5 Hz, 1H), 3.02 (ovrlp d, J = 15.6 Hz, 1H), 2.95
(ovrlp s, 3H), 2.26 (m, 1H), 1.41 (d, J = 6.9 Hz, 3H), 0.83 (d, J = 6.3
Hz, 3H). HRMS (ESI+) m/z calcd for C34H38N5O8S (M + H),
676.2441; found, 676.2441.
1
mmol, 74% yield for 3 steps) of 3 as a white foam. H NMR (300
MHz, CDCl3) δ 7.86 (dd, J = 8.1, 1.2 Hz, 1H), 7.70 (dd, J = 7.5, 1.5
Hz, 1H), 7.30−7.21 (ovrlp m, 5H), 6.95 (d, J = 8.4 Hz, 1H), 6.84 (d, J
= 8.7 Hz, 2H), 4.55 (d, J = 11.7 Hz, 1H), 4.47 (d, J = 11.7 Hz, 1H),
4.39 (m, 1H), 4.29 (m, 4H), 3.90−3.77 (ovrlp m, 3H), 3.77 (ovrlp s,
3H), 3.66 (dd, J = 9.9, 4.5 Hz, 1H), 3.38−3.20 (ovrlp m, 2H), 3.11 (d,
J = 12.6 Hz, 1H), 2.81 (s, 3H) 2.70 (m, 1H), 1.38 (d, J = 6.9 Hz, 3H),
0.86 (d, J = 6.6 Hz, 3H). LCMS (ESI+) m/z: 656.38 (M + H).
N-(((2R,3R)-10-Amino-5-((S)-1-((4-methoxybenzyl)oxy)-
propan-2-yl)-3-methyl-6-oxo-3,4,5,6-tetrahydro-2H-benzo[b]-
[1,5]oxazocin-2-yl)methyl)-N-methyl-2,3-dihydrobenzo[b]-
[1,4]dioxine-6-sulfonamide (4). Under hydrogen (H2) atmosphere,
a solution of 3 (1.39 g, 2.12 mmol) and palladium (10% on activated
carbon, 226 mg, 0.212 mmol) in EtOH (42 mL) was stirred at 35 °C
for 3.5 h. The reaction mixture was cooled, filtered, and washed
through a pad of Celite and concentrated in vacuo. Purification on
silica gel (gradient of 10−60% EtOAc in hexanes) provided 1.21 g
ASSOCIATED CONTENT
■
S
* Supporting Information
New compound characterization and procedures for compound
synthesis, solubility determination, and biological assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
(1.93 mmol, 91% yield) of 4 as a white foam. H NMR (300 MHz,
CDCl3) δ 7.34−7.26 (ovrlp m, 4H), 7.02 (d, J = 8.4 Hz, 1H), 6.97 (d,
J = 7.5 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 7.8 Hz, 2H),
4.67 (m, 1H), 4.54 (d, J = 11.4 Hz, 1H), 4.47−4.43 (ovrlp m, 3H),
4.36−4.33 (ovrlp m, 4H), 3.81−3.74 (ovrlp m, 2H), 3.79 (ovrlp s,
3H), 3.67 (dd, J = 10.2, 4.5 Hz, 1H), 3.49 (dd, J = 15.6, 10.5 Hz, 1H),
3.30 (d, J = 13.8 Hz, 1H), 3.06 (d, J = 15.0 Hz, 1H), 2.94−2.86 (ovrlp
m, 1H), 2.90 (ovrlp s, 3H), 2.01 (m, 1H), 1.32 (d, J = 6.9 Hz, 3H),
0.83 (d, J = 6.9 Hz, 3H). HRMS (ESI+) m/z calcd for C32H39N3O8SNa
(M + Na), 648.2356; found, 648.2352.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (617) 714-7363. Fax: (617) 714-8943. E-mail:
bwagner@broadinstitute.org.
Notes
The authors declare no competing financial interest.
D
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ACKNOWLEDGMENTS
■
Financial support from the NIH-NIDDK (Type 1 Diabetes
Pathfinder Award to B.K.W.) is gratefully acknowledged. K.P.
was sponsored in part by a NIH/NIGMS MARC U*STAR T34
08663 National Research Award and the Howard Hughes
Medical Institute (HHMI) Undergraduate Science Education
Program at UMBC. We thank Tamara Gilbert for experimental
assistance as well as Dr. Danny Chou, Dr. Jeremy Duvall, and
Prof. Stuart Schreiber (Broad Institute) for helpful discussions.
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in small molecule drug discovery programs by disruption of molecular
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ABBREVIATIONS USED
■
JAK, Janus kinase; STAT, signal transducer and activation of
transcription; T1D, type-1 diabetes
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(11) This compound is registered with the NIH Molecular Libraries
Program (probe ML187) and is available upon request.
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Suh, B.-C.; Forbeck, E. M.; Faloon, P.; Wagner, B. K.; Marcaurelle, L.
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(13) Marcaurelle, L. A.; Comer, E.; Dandapani, S.; Duvall, J. R.;
Gerard, B.; Kesavan, S.; Lee, M. D.; Liu, H.; Lowe, J. T.; Marie, J.-C.;
Mulrooney, C. A.; Pandya, B. A.; Rowley, A.; Ryba, T. D.; Suh, B.-C.;
Wei, J.; Young, D. W.; Akella, L. B.; Ross, N. T.; Zhang, Y.-L.; Fass, D.
M.; Reis, S. A.; Zhao, W.-N.; Haggarty, S. J.; Palmer, M.; Foley, M. A.
An aldol-based build/couple/pair strategy for the synthesis of
medium- and large-sized rings: discovery of macrocyclic histone
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(14) Gerard, B.; Duvall, J. R.; Lowe, J. T.; Murillo, T.; Wei, J.; Akella,
L. B.; Marcaurelle, L. A. Synthesis of a stereochemically diverse library
of medium-sized lactam and sultams via S(N)Ar cycloetherification.
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simple method for the preparation of isocyanates from aliphatic
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