4-Hydroxy-: N -[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide: A novel inhibitor of the canonical NF-κB cascade

Article abstract of DOI:10.1039/c7md00278e

The NF-κB signaling pathway is a validated oncological target. Here, we applied scaffold hopping to IMD-0354, a presumed IKKβ inhibitor, and identified 4-hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide (4) as a nM-inhibitor of t

Full text of DOI:10.1039/c7md00278e

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4-Hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-
carboxamide: a novel inhibitor of the canonical NF-kB cascade
Agnese C. Pippione,a Antonella Federico,a Alex Ducime,a Stefano Sainas,a Donatella Boschi,a
Alessandro Barge,a Elisa Lupino,b Marco Piccinini,b Michael Kubbutat,c Jean-Marie Contreras,d
Christophe Morice,d Salam Al-Karadaghie and Marco L. Lolli.a*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract. NF-κB signaling pathway is a validated oncological target. The efforts aimed at discovering new NF-κB inhibitors, and
Here, we applied scaffold hopping to IMD-0354, a presumed IKKβ particularly inhibitors of IKKβ, have been intensified in recent
inhibitor,
and
identified
4-hydroxy-N-[3,5- years.5 Recently, the X-ray structures of IKKβ in a ligand-free
bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide (4) as a form and in complex with an inhibitor have been reported,6, 7
nM-inhibitor of the NF-κB pathway. However, both 4 and IMD- thus providing new opportunities for the design of new potent
0354, being potent inhibitors of the canonical NF-κB pathway, were inhibitors.1 Although no IKKβ inhibitors have reached the
found to be inactive in human IKKβ enzyme assays.
human pharmacopoeia yet, some interesting molecules have
been studied. Among those is PS-1145 (Figure 1), a β-carboline
analogue known to potently and selectively inhibit the
endogenous IKK complex with an IC50 of 150 nM.8-10 Other
NF-κB is a ubiquitously expressed family of transcription
factors, known to be key regulators of immune response, cell
proliferation, cell death and inflammation.1 NF-kB dimers are
normally inhibited in the cytoplasm of resting cells by proteins
called IκB. Following cell stimulation, the inhibitory IκB
proteins are rapidly phosphorylated by the so-called IKK
kinases, and subsequently degraded by the 26S proteasome.
This is followed by the translocation of the transcription
factors to the nuclei and subsequent activation of the
corresponding gene expression (the so-called canonical
pathway of NF-κB activation). NF-κB signaling has been found
to be constitutively activated in a variety of malignancies,
leading to uncontrolled apoptosis, cell cycle deregulation and
metastatic growth.1 These observations validated the NF-κB
pathway as an oncologic target, in particular in breast2 and
thyroid cancer.3 The trimeric IKK complex involved in the
activation of the canonical NF-κB pathway contains two
catalytic subunits, IKKα and IKKβ kinase, and a regulatory
protein IKKγ (also called NEMO). In a parallel, so-called non-
canonical pathway,4 TNF-receptor superfamily members
selectively activate a different set of kinases, NF-κB-inducing
kinase (NIK) and IκB kinase 1 (IKK1).4
11
examples include, BMS-345541 which acts by allosterically
inhibiting IKKβ, and IMD-0354, which reached the stage of
clinical trials12 (Figure 1). IMD-0354 was designed by the
Institute of Medicinal Molecular Design Inc (Tokyo, Japan) and
is claimed to be a selective inhibitor of IKKβ.13 The compound
has completed phase 1 clinical trials as an anti-inflammatory,
anti-allergic and anti–microbial agent,14 while its prodrug,
called IMD-104115 was evaluated in clinical trials that are
currently awaiting proof-of-concept results (NCT00883584).14
Figure 1: PS-1145, BMS-345541 and IMD-0354 as examples of
IKKβ inhibitors.
The biological activity of IMD-0354 has been described in a
variety of assays related to metabolic diseases and
cardiovascular diseases.15-27 In particular, it has been reported
that IMD-0354 is able to suppresses neoplastic proliferation of
human mast cells with constitutively activated c-kit receptor,28
and inhibits the growth of human breast cancer cells MDA-MB-
231, HMC1-8 and MCF-7.29
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Bio(iso)steric replacement is a widely used approach in
medicinal chemistry, aimed at improving the characteristics of
a lead compound, such as bioavailability, selectivity, and
potency.30 As an example of the use of the method, we
recently designed a new generation of potent hDHODH
inhibitors31 using hydroxylated azoles. In these systems, the
substitution of the azole ring allowed fine-tuning of the
accessible chemical space,32 thus increasing the probability of
triggering the desired change in biological activity. Here, we
applied a similar approach to the phenolic substructure in
F
F
F
OH
Cl
O
F
F
N
H
F
IMD-0354
F
F
F
F
F
F
F
F
F
F
F
F
O
O
O
O
HO
F
HO
HO
F
F
HO
F
N
H
N
H
N
H
N
F
F
F
F
N
H
N
N
N
F
N
N
N
F
F
N
F
N
O
S
10
4
1
7
F
F
F
F
F
F
F
F
F
F
F
F
IMD-0354 to design new IKKβ inhibitors. The compounds
1 to
O
O
O
O
HO
F
HO
HO
HO
F
N
H
11 (Figure 2) have been designed using four different acidic
azoles: hydroxyoxadiazole, hydroxythiadiazole, hydroxytriazole
and hydroxypyrazole. The four hydroxyazole systems have
been selected to bioisosterically modulate the phenolic moiety
of IMD-0354 by their different acidic and lipophilic
properties.33 Notably, hydroxytriazole and hydroxypyrazole
scaffolds can be modified by the addition of protruding
substituents, which may be designed to occupy the
surrounding chemical space in several directions. Schemes 1
and 2 outline the synthetic methodologies used for the
N
N
N
H
F
H
H
N
N
F
F
F
N
N
N
N
N
N
S
N
11
O
2
5
8
F
F
F
F
F
F
F
F
F
O
O
O
HO
H2N
O
F
F
F
15
N
N
H
N
H
N
F
H
N
S
F
F
N
F
N
N
F
N
F
N
H
O
3
9
6
Figure 2: The bioisosteric scaffold hopping strategy applied to
IMD-0354 using hydroxyoxadiazole, hydroxythiadiazole,
hydroxytriazole and hydroxypyrazole scaffolds.
In some cases, a benzyloxy-protected azolecarboxylate (12, 16,
preparation of the target compounds 1 - 11
.
20) was transformed into the corresponding acyl chloride,
which was allowed to react with the appropriate aniline, thus
obtaining the corresponding amide (compounds 13, 17 and
21). The removal of the protecting benzyl group was
accomplished by applying room pressure hydrogenation
conditions (1, 2, 7, 8, 10, 11). In the case of triazole analogue
9
(scheme 2), the coupling reaction was conducted on the p-
methoxybenzyl protected precursor 18, previously obtained
from its corresponding ethyl ester.33 The resulting amide 19
was then deprotected under acidic conditions (TFA).
Scheme 1. Synthesis of 1,2,5-oxadiazole and thiadiazole analogues 1 -
: i) a) oxalyl chloride, DMF, dry THF, b) substituted aniline, dry
6
pyridine, dry THF; ii) H2, Pd/C, dry THF; iii) CH3I, Cs2CO3, dry THF; iv)
HBTU, DMAP, 3,5-bis(trifluoromethyl)aniline, dry DMF.
Scheme 2. Synthesis of triazole and pyrazole analogues (7 – 11). i) a)
oxalyl chloride, DMF, dry THF; b) 3,5-bis(trifluoromethyl)aniline, dry
pyridine, dry THF; ii) H2, Pd/C, dry THF; iii) TFA, 45°C.
Other compounds (4, 5 and 3, scheme 1) were obtained by
coupling unprotected 4-hydroxythiadiazole-3-carboxylic acyl
chloride of the corresponding acid 1434 or commercial
2 | J. Name., 2012, 00, 1-3
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available 4-aminofurazan-3-carboxylic acid 15 with the Indeed, IMD-0354 was found to inhibit the activated
corresponding aniline. Compound was then methylated with expression of NF-kB in a dose-dependent manner in HepG2
methyl iodide to obtain compound . Synthetic procedures cells transfected with pFLAG-CMV-IKKβ (S177E/S181E) vector,
4
6
and spectral characterization of the final compounds
shown in the supplementary material.
1
-
11 are and subsequent verification of IκBα degradation by Western
blot analysis of cytosolic phospho-IκBα. The authors
The designed compounds 1 – 11 (Figure 2) were evaluated concluded17 that the results were consistent with IKKβ
both by enzymatic and cellular assays and compared to IMD- inhibition although IMD-0354 was not assayed on isolated
035435 and PS-1145,36 the latter used as IKKβ reference enzyme. In recent years, Azucena Gomez-Cabrero et al39
inhibitor8 (Table 1).
defined IMD-0354 as “an indirect inhibitor of NF-κB”, probably
due to the lack of strong evidence supporting an IKKβ related
mechanism. In order to get a broader overview of the action of
IMD-0354, we assayed both IMD-0354 and PS-1145, as well as
IKKβ
IC50 ± SE
IKKα
IC50 ± SE
(µM)
(% inhib at
100 μM) a
IKKε
IC50 ± SE
(µM)
(% inhib at
100 μM) a
NIK
IC50 ± SE
(µM)
(% inhib at
100 μM) a
IC50 (µM) on
IκBα
(µM)
(% inhib at
100 μM)a
Compound
degradation
assayb
compounds
1 - 11, against the other three kinases involved in
the canonical and non-canonical NF-κB activation pathways
(IKKα, IKKε and NIK). In this assay the newly synthesized
compounds and IMD-0354 were found to be essentially
>100
(5.43)
>100
(3.45)
>100
(30.5)
>100
(13.8)
0.218±0.007
IMD-0354
0.087±0.005
>100
>100
>100
0.186±0.008
6.93±0.12
PS-1145
1
inactive (only modest activity of compounds
μM range was observed, Table 1).
1, 3 and 5 in the
>100
(12.2)
>100
(18.6)
>100
(19.3)
58.5±0.4
Since IMD-0354 has been described as potent inhibitor of the
NF-ĸB pathway in cellular assays, we also evaluated the ability
of IMD-0354 and compounds 1 - 11 to block the NF-κB
pathway. In these experiments the capacity of the compounds
to inhibit the degradation of IκBα after inflammatory stimulus
was evaluated in Jurkat cells. In agreement with earlier
results,17 these assays showed IMD-0354 to be a potent NF-κB
inhibitor with IC50 = 0.218 μM (Table 2). Among the new
>100
(2.06)
>100
(-2.5)
>100
(32.1)
>100
(11.8)
1.72±0.11
>100
2
>100
(31.59)
>100
(29.3)
>100
(-9.2)
44.8±0.8
3
>100
(-10.32)
>100
(25.2)
>100
(30.2)
>100
(4.34)
0.143±0.005
>100
4
>100
(-0.94)
>100
(25.8)
>100
(-1.1)
35.5±0.6
5
>100
(8.51)
>100
(24.1)
>100
(21.9)
>100
(-1.9)
>100
6
compounds, the hydroxythiadiazole 4 was the most active,
with IC50 of 0.143 μM (Table 1 and Figure 3). Compounds 1, 2,
>100
(5.19)
>100
(46.9)
>100
(6.9)
>100
(9.1)
>100
7
10 and 11 were active in the low μM range.
>100
(11.2)
>100
(13.1)
>100
(12.1)
58.5±0.3
>100
8
>100
(5.14)
>100
(24.5)
>100
(15.3)
>100
(-9.5)
>100
9
>100
(12.7)
>100
(12.1)
>100
(17.2)
>100
(7.23)
25.9±0.4
1.32±0.21
10
11
>100
(13.9)
>100
(13.2)
>100
(14.2)
>100
(24.1)
Table 1. The effects of IMD-0354, PS-1145 and the designed
compounds on: a) ATP-based kinase assays for IKKβ, IKKα, IKKε
b)
and NIK (expressed as IC50 value, μM); IĸBα degradation
assay in Jurkat cells (expressed as IC50 value, μM). All
experiments were performed in triplicate, and data represent
means ± standard deviation (SD).
At the enzymatic level, the activity was assessed on
recombinant human IKKβ. Surprisingly, the IMD-0354 lead, as
well as the designed compounds 1 - 11, were found to be
inactive in the assays (Table 1). This result does not agree with
the earlier proposed mechanism of action of IMD-0354, which
assumes that the compound is a potent inhibitor of IKKβ.28, 29,
Figure 3. Effects of compound
4 on IκBα degradation in Jurkat
cells stimulated by TNF-α. IκBα protein expression was
evaluated by immunoblotting. One representative immunoblot
of three independent experiments is shown. Graphs represent
IκBα relative band intensity quantified by densitometric
analysis after normalization using β-actin as reference. Values
are means ± SD of three independent experiments.
37
In particular, its molecular structure was originally designed
by analysing the binding mode of aspirin to IKKβ at the APB
IKK-2 binding site,28 and based on that it was suggested to
compete with ATP for binding to IKKβ.38 Based on an NF-κB-
IKKβ reporter assay that uses a constitutively active IKKβ
mutant,17 the compound’s mechanism of action was suggested
to involve the inhibition of phosphorylation of IκB.
The effects of PS-1145, IMD-0354 and compound
4 on NF-κB
gene reporter assay in Jurkat (TIB-152, ATCC) and MDA-MB-
231 (ACC-732, DSMZ) cells were also evaluated (Figure 4).
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Figure 5. Effects of compound 4, IMD-0354, PS-1145, aspirin
(ASA) and indomethacin (Indo) on IκBα degradation in THP-1
cells stimulated by LPS. IκBα protein expression was evaluated
by immunoblotting. One representative immunoblot of three
independent experiments is shown.
These results also agree with earlier experiments, which
showed that IMD-0354 is able to specifically block the NF-ĸB
pathway when induced by proinflammatory cytokines, such as
TNFα and IL-1β.15,
29
The presented experiments show that
compound
conditions of TNFα activation. However, similarly to IMD-0354,
compound does not inhibit IKKβ at enzymatic level (Table 1).
4 potently blocks the NF-kB cascade, particularly in
4
For a better understanding of the possible role of IKKβ in the
IMD-0354 mechanism of action, we assayed the compound
against the trimeric IKKβ-IKKα-Nemo complex, isolated from
Jurkat cells treated with TNFα. In this assay, IMD-0354 showed
Figure 4. Effects of PS-1145, IMD-0354 and compound
4 on
NF-κB gene reporter assay in Jurkat and MDA-MB-231 cells.
All three compounds were found active on TNFα-activated
Jurkat cells 6h post-treatment, with compound having higher
potency than IMD-0354 and PS-1145 (residual activity at 1 μM:
IMD-0354, 23.48 %; PS-1145, 38.14 %; , 15.91 %). However,
both compounds and IMD-0354 were almost completely
inactive on MDA-MB-231 cells 6h post-treatment (residual
activity at 10 μM: IMD-0354, 98.99 %; compound , 96.08 %)
and showed an appreciable activity 24h post-treatment
(residual activity at 10 μM: IMD-0354, 58.27 %; compound
only weak activity (40
% inhibition at 100 µM, see
4
Supplementary), which does not explain the claimed potency
of the compound on the NF-κB cascade. Moreover, in Jurkat
cells exposed to IMD-0354 and treated with TNFα, the IKKs of
the trimeric complex were found to be phosphorylated
(Supplementary), indicating that the TNFα stimulus did reach
them. These results suggest that the mechanism of inhibition
of the canonical NF-κB pathway by IMD-0354 is probably more
complex than it was thought. However, the elucidation of this
mechanism is outside the scope of this publication.
4
4
4
4,
63.01 %). Conversely, 10 µM PS-1145 showed potent activity
not only at 24h but also at 6h (residual activity at 10 μM:
19.52% (6 h) and 8.67% (24 h)). The different inhibitory activity
Finally, we also studied antiproliferative activity and
cytotoxicity of compounds
4 on MDA-MB-231 cells, comparing
showed of IMD-0354 and compound
4 in gene reporter assay,
them to IMD-0354 and PS-1145 (Table 2). Quantitation of DNA
content and a fluorescent assay to assess cell membrane
integrity were used to evaluate cell proliferation and
cytotoxicity , respectively.
carried-out in Jurkat and MDA-MB-231 cells, is probably a
consequence of the activation status of the NF-kB pathway. In
fact, in Jurkat cells the NF-κB pathway was activated through
the treatment with TNFα whilst in MDA-MB-231 cells the NF-
κB signaling pathway was reported to be constitutively
activated and driven by both IKKβ and IKKα.40
Antiproliferative effect
IC50 ± SE (µM)
Cytotoxicity
IC50 ± SE (µM)
Compound
The anti-inflammatory effects of compound
4
were evaluated
in LPS-stimulated THP-1 (ACC-16, DSMZ) in which the canonical
4
1.29±0.08
0.64±0.04
0.90±0.07
53±2
0.85±0.01
>100
NF-κB signaling pathway controls the expression of several
pro-inflammatory cytokines.41 Compound
IMD-0354
PS-1145
4 prevented in a
dose-dependent manner the LPS-induced degradation of IκBα
(Figure 5).
Table 2. Antiproliferative and cytotoxic effects of compound 4,
IMD-0354 and PS-1145 on MDA-MB-231 cells. Cells were
exposed to inhibitors for 72 h. Data obtained for IMD-0354
agree with previously reported results.29 Data obtained for PS-
4 | J. Name., 2012, 00, 1-3
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1145 agree with previously reported antiproliferation assays in
MDA-MB-231 cells.40
It can be seen from the data in Table 2 that compound
4 has
10.
antiproliferative effect in the low µM range (1.29 µM), slightly
lower than that of the two lead compounds. On the other
hand, this compound is not cytotoxic as IMD-0354, showing an
effect more reminiscent of that of PS-1145, able to block the
NF-kB cascade without significant cytotoxicity. In contrast to
11.
12.
13.
compound
4 and PS-1145, IMD-0354 has similar IC50 in both
proliferation and cytotoxicity assays.
In conclusion, here we introduce the hydroxythiadiazole
4 as a
14.
15.
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nanomolar inhibitor of the canonical NF-kB cascade. The
compound was designed through bioisosteric scaffold
hopping approach applied to the phenolic moiety of IMD-0354.
When compared to IMD-0354, compound showed similar
a
4
mode of action, although with higher potency in blocking the
16.
17.
18.
NF-kB cascade on Jurkat cells and lower cytotoxicity on MDA-
MB-231 cells. Both
4 and the lead IMD0354 were found to be
inactive in IKKβ enzymatic assays, although both being able to
inhibit the canonical NF-kB pathway after TNFα or LPS
stimulus.
Conflict of interest
19.
20.
Y. Onai, J.-i. Suzuki, Y. Maejima, G. Haraguchi, S. Muto, A.
Itai and M. Isobe, Am. J. Physiol., 2007, 292, H530-H538.
H. Ogawa, M. Azuma, S. Muto, Y. Nishioka, A. Honjo, T.
Tezuka, H. Uehara, K. Izumi, A. Itai and S. Sone, Clinical &
Experimental Allergy, 2011, 41, 104-115.
The authors declare no conflicts of interest.
Acknowledgements
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J. Wei, M. Shi, W.-Q. Wu, H. Xu, T. Wang, N. Wang, J.-L.
Ma and Y.-G. Wang, World J. Gastroenterol., 2011, 17,
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This research was financially supported by the TAKTIC
Translational Kinase Tumour Inhibitor discovery Consortium
FP7-SME-2012 grant 315746. Authors wish to thank dr. Livio
Stevanato for performing all the NMR experiments and for
maintenance of the instrument.
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Limite 20 parole:
Hydroxythiadiazole 4, obtained applying scaffold hopping to IMDꢀ0354, blocks the canonical NFꢀκB pathway although
is inactive on IKKβ enzyme.
Figura 8 cm x 4 cm.