N -Acetyl-3-aminopyrazoles block the non-canonical NF-kB cascade by selectively inhibiting NIK

Article abstract of DOI:10.1039/c8md00068a

NF-κB-inducing kinase (NIK), an oncogenic drug target that is associated with various cancers, is a central signalling component of the non-canonical pathway. A blind screening process, which established that amino pyrazole related scaffolds have an effect on IKKbeta, led to a hit-to-lead optimization process that identified the aminopyrazole 3a as a low μM selective NIK inhibitor. Compound 3a effectively inhibited the NIK-dependent activation of the NF-κB pathway in tumour cells, confirming its selective inhibitory profile.

Full text of DOI:10.1039/c8md00068a

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Sainas, A. Federico, E. LUPINO, M. Piccinini, M. Kubbutat, J. M. Contreras, C. Morice, A. Barge, A. DUCIME,
D. boschi, S. Al-Karadaghi and M. L. L. LOLLI, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00068A.
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RESEARCH ARTICLE
N-Acetyl-3-aminopyrazoles block the non canonical NF-kB cascade
by selectively inhibiting NIK
Agnese C. Pippione,a Stefano Sainas,a Antonella Federico,a Elisa Lupino,b Marco Piccinini,b Michael
Kubbutat,c Jean-Marie Contreras,d Christophe Morice,d Alessandro Barge,a Alex Ducime,a Donatella
Boschi,a Salam Al-Karadaghie and Marco L. Lolli.a*
+Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract. NFꢀκBꢀinducing kinase (NIK), an oncogenic drug target thus potentially influence multiple human diseases, including a
that is associated with various cancers, is a central signalling number of cancers.7 The crystal structure of the NIK catalytic
component of the nonꢀcanonical pathway. A blind screening process, domain has recently been resolved,8 allowing potent,
which established that amino pyrazole related scaffolds have an conformationally restricted NIK inhibitors to be identified
effect on IKKbeta, led to a hitꢀtoꢀlead optimization process that
identified aminopyrazole 3a as a low ꢁM selective NIK inhibitor.
Compound 3a effectively inhibited the NIKꢀdependent activation of
the NFꢀκB pathway in tumour cells, confirming its selective
inhibitory profile.
Acting as
a key regulator of immune response, cell
proliferation, cell death and inflammation, NFꢀκB is
a
(Figure 1).6, 9
ubiquitously expressed family of transcription factors known to
be constitutively activated in a variety of malignancies,
resulting in uncontrolled apoptosis, cell cycle deregulation and
metastatic growth.1 These observations have led to the NFꢀκB
pathway being validated as target, in particular in breast2 and
thyroid cancers.3 The nonꢀcanonical NFꢀκB pathway is an
important component of NFꢀκB signalling and predominantly
Figure 1: Examples of selective NIK inhibitors A9 and B.6 PSꢀ1145
structure of potent IKKβ inhibitor.10
With the aim of identifying new chemical entities11 that can
block the NFꢀkB cascades, the authors has recently described 4ꢀ
hydroxyꢀNꢀ[3,5ꢀbis(trifluoromethyl)phenyl]ꢀ1,2,5ꢀthiadiazoleꢀ
targets the activation of the p52/RelB NFꢀκB complex.4,
5
3ꢀcarboxamide12, as a nMꢀinhibitor of the NFꢀκB pathway.
13
Specifically, the nonꢀcanonical NFꢀκB pathway is involved in
secondary lymphoid organ development, B cell survival and
maturation, dendritic cell activation, lymphocyte recruitment
and bone metabolism.6 NFꢀκBꢀinducing kinase (NIK), is a
central signalling component of the nonꢀcanonical pathway that
integrates signals from a subset of TNF receptor family
members and activates a downstream kinase, IκB kinaseꢀα
(IKKα), triggering p100 phosphorylation and processing. The
specific kinase inhibition activity shown by NIK may provide a
means to directly inhibit the nonꢀclassical NFꢀκB pathway and
Following that experience, in this work, a blind screening was
conducted on the four kinases, IKKβ, IKKα, IKKε and NIK,
that characterise the NFꢀκB pathway in order to find novel
chemotypes. The study involved 2320 compounds from three
dedicated Prestwick libraries (Prestwick Chemical Library
(1200 cpds), Prestwick Pyridazine Library (400 cpds) and
Prestwick Fragment Library (720 cpds)). An aminopyrazole
®
(
2a, Figure 2), which is able to weakly (microM range) but
selectively inhibit IKKβ, was identified from among the 10
most promising hits. Starting from this compound, a hitꢀtoꢀlead
optimization process was directed to improve its potency while,
simultaneously, retain the observed selectivity. A schematic
representation of compound 2a modulation is presented in
Figure 2.
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Scheme 1. General synthesis scheme of the lead compounds with
general structure formula and . Reaction conditions: i) dry THF,
2,
3
4
NaH, reflux, overnight; ii) NH2NH2·2HCl, 3Å sieves, dry EtOH, reflux,
overnight; iii) R3COCl, dry pyridine, dry THF, rt, or appropriate
anhydride, dry THF, rt, from 2h to overnight.
The treatment of compound 2a with acetic anhydride gave two
monoꢀacetylated products; 3a (main product), and 4a. The
functionalization of the two annular nitrogen atoms was
established using the 1H NMR spectra of the two products
obtained; broad singlet signals with
δ values of 5.47 ppm and
6.75 ppm, which correspond to the NH2 protons, were observed
in both spectra (Figure 3).
Figure
2: A schematic description of the compounds
investigated in the hitꢀtoꢀlead process applied to 2a
.
Of the 44 aminopyrazole derivatives involved in this study, 39
were synthesised using the general procedures described in
Scheme 1, in which a Claisen condensation between the
appropriate ethyl ester and a benzyl cyanide gave the 3ꢀoxoꢀ2ꢀ
phenylpropionitriles, structure type
allowed to react, according to
procedure,14 with an excess of hydrazine dihydrochloride to
yield the desired 3ꢀaminopyrazoles (structure type ).
Acetylated product types and were prepared from these
compounds. The 3ꢀaminopyrazoles, type can be
1
. These systems, had been
a
previouslyꢀreported
2
3
4
2
,
acetylated/alkylated in three positions; the primary exocyclic
NH2 group and the two annular nitrogen atoms. The variations
in the regiochemistry of the acylation reactions of 3ꢀ
aminopyrazoles have been described in several studies,15ꢀ18
although 5ꢀmethylꢀ1Hꢀpyrazolꢀ3ꢀamines, substituted with a
phenylꢀsubstituted group in the 4 position, have never been
used as a substrate, to the best of our knowledge. Nevertheless,
it appears that regioselective substitution is heavily dependent
on the nature of the electrophilic reagent,15 as well as the
reaction conditions and substrate.19ꢀ22
Figure 3: 1HꢀNMR spectra of starting material 2a and acylated
3a ꢀ 4a.
2 | J. Name., 2012, 00, 1-3
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The two regioisomers were distinguished by comparing their
NH2 proton signals with a similar isomeric couple described in
the literature (compounds
C and D
, Figure 4).17 The authors
observed the presence of two intramolecular NꢀHꢀꢀꢀO hydrogen
bonds between the 5ꢀexoamino group and the carbonyl of the
N1ꢀacetyl group in compound
being downfield compared to compound
C
, resulting in its NH2 signal
Scheme 3: Methylation of 2a to give compounds 5a and 5b
Reaction conditions: i) Cs2CO3, CH3I, dry THF, rt, overnight
.
D.
Notably,
a
.
downfield chemical shift in the pyrazolic CH3 signal, which
was probably due to the shielding effect of the vicinal CH3CO
An effort to increase conversion yields, which were low for
both regioisomers (19 and 11 %), by adding more than 1
group, can also be seen in compound 4a
.
equivalent of methyl iodide resulted in dimethylated
/
polymethylated mixtures that were difficult to resolve. The two
regioisomers (5a and 5b), were easily resolved by flash
chromatography and distinguished using 2DꢀNMR (see
Supplementary).
The designed compounds (Figure 2), were biologically evaluated
both at enzymatic and cellular levels and compared with Amgen
compound A (Figure 1),9 and PSꢀ1145,23, 24, which were used as NIK
and IKKβ reference inhibitors respectively (Table 1).
Figure 4: chemical shift of compounds described by
KusakiewiczꢀDawid et al.17
In order to modulate the active isomer 3a, other 1ꢀacylꢀ5ꢀ
aminopirazoles (see Figure 2, Scheme 1), were synthesised
under Scheme 1 conditions. If the appropriate anhydrides were
not commercially available, the corresponding and more
accessible acyl chlorides were involved. As for 2a, acylation
generally gave type
of compound type
3
as the main product. In some cases, traces
were isolated and characterized (see
4
Supplementary). In only two cases (compounds 6a,b), did the
acylation of the primary exocyclic NH2 group occur (Scheme
2).
Scheme 2: Synthesis of compounds 6a and 6b. Reaction conditions:
i) pyridineꢀ2ꢀcarbonyl chloride or trifluoroacetic anhydride, dry
pyridine, dry THF, rt, overnight
.
Surprisingly, when compound 2a was treated with pyridineꢀ2ꢀ
carbonyl chloride, in an attempt to obtain the corresponding 2ꢀ
pyridinyl regioisomer 3, only carboxamide 6a was produced in
modest yield. On the other hand, the synthesis of 6b should be
carried out using trifluoroacetic anhydride instead of acetic
anhydride. Pierce et al. have observed the same acylation trend
in a number of 3,4ꢀdiarylꢀ5ꢀaminopyrazoles.19
In an attempt to modulate 2a via the insertion of an alkyl group at
endocyclic N positions of the pyrazole ring, it was reacted with 1.0
equivalent of methyl iodide, producing a mixture of compounds 5a
and 5b (Scheme 3).
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In order to complete the biological investigation, we assayed all
the compounds against the other three kinases that are involved
in the canonical and nonꢀcanonical NFꢀκB activation pathways
(IKKα, IKKε and NIK). The first group contains compounds
IKKβ
IKKα
IKKε
NIK
CPD
IC50 (ꢁM) IC50 (ꢁM) IC50 (ꢁM) IC50 (ꢁM)
PSꢀ1145 0.09±0.01
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
89.7
>100
>100
>100
>100
>100
>100
>100
>100
93.4
>100
0.07±0.01
>100
(
2b ꢀ 2o), that were derived from 2a via modulation of the
Cpd A
2a
2b
2c
2d
2e
2f
>100
50.9±3.2
>100
phenyl moiety. In 2b, the attempt to remove both the chlorine
atoms present in 2a led to inactivity towards all four kinases.
The same behaviour was observed when both the chlorine
atoms present in 2a are replaced with lipophilic/bulky
>100
>100
>100
42.8±1.3
>100
>100
substituents (cpds 2c
2f 2j was investigated and it was observed that the presence of
a trifluoromethyl group on 2i was beneficial for its IKKβ
activity. 2j was two times more active against IKKβ (IC50
ꢀ 2e). The meta substitution of compounds
>100
ꢀ
>100
>100
2g
2h
2i
>100
>100
=
88.0±2.4
>100
>100
>100
>100
>100
>100
>100
15.4
>100
22.4 ꢁM), than 2a, but retains the same selectivity profile.
Similar behaviour was observed in 2o (IC50 = 22.9 ꢁM), which
bears two meta trifluoromethyl groups. The presence of a single
>100
2j
22.4±1.8
>100
>100
2k
2l
>100
meta substitution (cpds 2k
ꢀ 2n), does not generally appear to
37.9±3.3
60.1
61.1
be beneficial for activity. The only interesting behaviour here
was observed in 2l, which showed NIK, as well as IKKβ,
activity. Inverting the phenyl and methyl moieties in compound
2p, which is an isomer of 2a, did not improve IKKβ activity,
although selectivity was retained. The major breakthrough in
the series was obtained when 3(5)ꢀaminopyrazoles were
acetylated/alkylated in one of the three nitrogen positions.
While the substitution of the exocyclic NH2 (cpds 6a and 6b),
2m
2n
2o
2p
3a
3b
3c
3d
3e
3f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
34.9±2.3
>100
22.9±3.5
75.7±2.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
22.9±3.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8.4±1.3
27.4±3.2
>100
>100
>100
>100
24.3±4.3
2.9±1.1
41.0±1.2
3.3±0.4
23.0±1.4
>100
and N1 acylation (cpds 4a, 4e and 4i), led to a complete loss of
>100
activity, it became evident that the N(2) acetyl analogue 3a was
the most interesting compound in the entire series due to its
selective NIK profile in the low ꢁM range (IC50 = 8.4 ꢁM). As
the presence of the acetyl moiety was required for activity (the
N(2) methyl analogue 5b was found to be inactive), the acyl
substituent was investigated further (cpds 3b ꢀ 3r), leading to
the cyclobutyl analogue 3e (IC50 = 2.9 ꢁM), and the cyclohexyl
analogue 3g (IC50 = 3.32 ꢁM), being observed as the most
potent NIK inhibitors.
>100
3g
3h
3i
>100
>100
>100
3j
>100
58.2±4.1
>100
3l
>100
3k
3m
3n
3o
3p
3q
3r
3s
>100
>100
>100
>100
The cellular activity of the compounds with a NIK selective
profile was evaluated using a gene reporter assay to measure
NFꢀkB activation in EJM cells (ACCꢀ560, DSMZ); a multiple
myeloma cell line characterized by constitutively high levels of
nuclear NFꢀκB as a consequence of NIK gene amplification.25
The inhibitory activity of each compound was determined at the
highest concentration resulting in ≥ 80 % cell viability on the
Dual Luciferase reporter assay (Table 2). Cell viability was
determined using a Promega CellTiter Glo luminescent assay.
In order to check the selectivity of the compounds, gene
reporter assays were carried out in SKBr3 (HTBꢀ30, ATCC)
and MDAꢀMBꢀ231 (ACCꢀ732, DSMZ), two cell lines in which
constitutive activation of nuclear NFꢀκB is unrelated to NIK.26,
>100
>100
>100
39.6±0.9
>100
>100
>100
>100
>100
>100
13.6±1.6
>100
>100
4a
4e
4i
>100
>100
>100
>100
>100
4s
47.7±4.0
>100
12.5±2.1
>100
5a
5b
6a
6b
>100
>100
>100
>100
27
All the compounds were found to inhibit the NFꢀκB activity
>100
in EJM cells, but not in MDAꢀMBꢀ231 and SKBr3 cells, thus
indicating their potential for NIKꢀselective inhibitory activity in
cells. Compound 3a was also tested against two purified NIK
kinase domains. The first was mouse NIK kinase domain
(mNIK) comprising amino acids 345ꢀ675 of in which V480
was mutated to L, originating the so called humanized KDꢀ
mNIK. The second one was human NIK kinase domain (hNIK)
comprising amino acids 330ꢀ680 in which S549 was mutated to
Table 1. The effects of PSꢀ1145, Amgen compound
A and the
designed compounds on: ATPꢀbased kinase assays for IKKβ,
IKKα, IKKε and NIK (expressed as IC50 value, ꢁM). All
experiments were performed in triplicate and data represent
means ± SD
4 | J. Name., 2012, 00, 1-3
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D, originating the constitutively active form. Compound 3a did NIK in a panel of 44 kinases. It also displayed acceptable log D
not show appreciable inhibitory activity against either domain and solubility parameters. Further experiments will be
(data not shown). Further investigations are in progress to performed to elucidate the binding mode of 3a and improve its
identify the mode of action of compound 3a
.
metabolic stability.
NFꢀkB reporter assay ꢀ % of inhibition (concentration)
IC50
(µM)
IC50
(µM)
Cpd
MDAa
EJMb
SKBr3c
Kinase
Kinase
87.9±3.3
(50 ꢁM)
AMPKꢀalpha1
aa1ꢀ550
89.3±4.3 (20 ꢁM)
3.6±0.2 (50 ꢁM)
PSꢀ1145
Cpd A
2l
> 100 JAK1
> 100
7.1±0.4 (1 ꢁM)
29.9±3.4 (50 ꢁM)
ꢀ0.77±0.1 (100 ꢁM)
10.3±1.3 (100 ꢁM)
5.8±0.3 (50 ꢁM)
93.5±2.4 (1 ꢁM)
10.4±1.1 (50 ꢁM)
83.4±3.6 (25 ꢁM)
66.8±2.2 (50 ꢁM)
68.9±3.8 (100 ꢁM)
96.2±4.1 (25 ꢁM)
94.7±3.4 (25 ꢁM)
75.2±2.8 (50 ꢁM)
2.3±0.1 (1 ꢁM)
4.2±0.7 (100 ꢁM)
3.9±0.2 (100 ꢁM)
AXL
> 100 MAP3K11
> 100 MAP4K2
> 100 MEK1 wt
> 100 MELK
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
BꢀRAF wt
CAMKK1
3a
ꢀ3.14±0.1 (100 ꢁM) CDK1/CycB1
3b
CHK2
> 100 MET wt
> 100 MST1
3.66±0.1 (50ꢁM)
5.2±0.2 (100 ꢁM)
3.7±1.3 (100 ꢁM)
0.31±0.1 (100ꢁM)
3d
CK1ꢀalpha1
CK2ꢀalpha1
CLK1
2.36±0.2 (100 ꢁM)
2.36±0.6 (100 ꢁM)
13.8±1.1 (100 ꢁM)
3e
> 100 mTOR
3g
> 100 NEK6
3h
COT
> 100 p38ꢀalpha
> 100 PAK1
CSK
Table 2. The effects of PSꢀ1145, Amgen compound
designed compounds on the NFꢀκB signalling pathway. NFꢀκB
activation was assessed using a Dual Luciferase reporter assay
A and the
DAPK1
> 100 PCTAIRE1/CycY
> 100 PDGFRꢀalpha wt
> 100 PDK1
DYRK1A
EGFꢀR wt
EPHA6
a)
b)
c)
in
MDAꢀMBꢀ231,
EJM and
SKBr3 cells. Data are
> 100 PIM1
expressed as % of inhibition at the highest concentration tested,
indicated in brackets. All experiments were performed in
triplicate and data represent means ± SD
ERK2
> 100 PKCꢀalpha
> 100 PLK1
FGFꢀR1 wt
FLT3 wt
GSK3ꢀbeta
HIPK1
> 100 SRC (GSTꢀHISꢀtag)
> 100 SRPK2
Given 3a was found to be selective towards the other kinases
involved in NFꢀκB activation, we investigated its activity
towards a larger kinase panel (Table 4). The determination of
IC50 values for 44 representative kinases showed that 3a had a
very good selectivity profile versus NIK (IC50 kinases > 100
µM).
> 100 SYK
INSꢀR
> 100 TAOK2
> 100 TLK1
IRAK4 (untagged)
Table 4. IC50 profiling for 3a against 44 protein kinases
Conflict of interest
The preliminary ADME profile of 3a was evaluated as the final
step in this work. Whereras 3a showed acceptable log D and
solubility parameters at this stage, the relative metabolic
instability of the compound was highlighted and must be
considered in any future exploration.
The authors declare no competing interest.
Acknowledgements
This research has been financially supported by TAKTIC
Translational Kinase Tumour Inhibitor discovery Consortium
FP7ꢀSMEꢀ2012 grant 315746. The authors wish to thank Dr.
Annalisa Costale for performing all UPLC analyses, Dr. Livio
Stevanato for performing all NMR experiments and for
maintaining the instrument and, finally, Dr. Dale James
Matthew Lawson for proofreading the final manuscript.
Cpd 3a
Metabolic stability a
log D c
Kinetic solubility d
1% b
4.09
12 µM
a)
Table 3. Early ADME profiling for 3a
.
Human liver
microsome (obtained by Xenotech: Xtreme 200 Human Liver
b)
Microsomes) 1 µM, 37 °C, NADPH cofactor;
cpd found
Notes and references
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instability or a nonꢀNADPH dependent enzymatic degradation;
1.
2.
3.
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d)
PBS pH 7.4 / octanol;
temperature.
PBS pH 7.4, 60 min, room
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cascade by selectively inhibiting NIK. Compound 3a is the
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t. H. Obtained from Sigma (code: P6624, , ).
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Resulted from a hit-to-lead optimization, the aminopyrazole 3a is a NIK inhibitor selective over 44 kinases.
Figure (8 cm x 4 cm)