N-aryl-piperidine-4-carboxamides as a novel class of potent inhibitors of MALT1 proteolytic activity

Article abstract of DOI:10.1016/j.bmcl.2018.05.017

Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell acti

Full text of DOI:10.1016/j.bmcl.2018.05.017

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-aryl-piperidine-4-carboxamides as a novel class of potent inhibitors of
MALT1 proteolytic activity
⇑
Achim Schlapbach , Laszlo Revesz, Carole Pissot Soldermann, Thomas Zoller, Catherine H. Régnier,
Frédéric Bornancin, Thomas Radimerski, Jutta Blank, Ansgar Schuffenhauer, Martin Renatus, Paulus Erbel,
Samu Melkko, Richard Heng, Oliver Simic, Ralf Endres, Markus Wartmann, Jean Quancard
Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were
elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds
showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-
Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as
Received 20 February 2018
Revised 16 April 2018
Accepted 8 May 2018
Available online xxxx
B-cell lymphomas with a dysregulated NF-jB pathway. Initially, rat pharmacokinetic properties of this
compound series were dominated by very high clearance which could be linked to amide cleavage.
Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the iden-
tification of compounds with improved PK properties.
Keywords:
MALT1
Paracaspase
Protease inhibitors
Autoimmune disease
B-cell lymphoma
Ó 2018 Elsevier Ltd. All rights reserved.
The NF-
j
B pathway is of chief importance in immunity and
the potential to dampen NF-
autoimmune diseases.9
jB signaling in B-cell lymphomas and
cancer biology.1 Attempts to target this pathway by small molecule
inhibitors have had little success so far, essentially because NF-
j
B
Following the discovery of MALT10s protease function and its
key role in lymphocyte signaling3 a high throughput screen was
initiated to search for compounds which inhibit the cleavage of
an artificial MALT1 substrate.10 In addition to several known,
mostly irreversible, cysteine protease inhibitors, compound 1
devoid of any classical covalent warhead functionality was discov-
ered (Fig. 1). We observed that 1 not only inhibited the MALT1 pro-
is required for tissue homeostasis, in particular in the liver.2 As a
consequence, full blockade of this pathway turned out to be asso-
ciated with toxicities which prevented therapeutic use of this con-
cept.2 Mucosa-associated lymphoid tissue lymphoma translocation
protein 1 (MALT1) plays a key role in NF-jB pathway activation, in
particular in T and B lymphocytes.3,4 Upon antigen receptor stimu-
lation, MALT1 is recruited to CARD11 (CARMA1) together with
BCL10 to form the ‘CBM’ complex that triggers inhibitor of
kappa-B kinase (IKK) activation. Akin to the structurally related
caspases, MALT1 (aka paracaspase 1) possesses an original cysteine
protease domain. However, in contrast to caspases, MALT1 cleaves
substrates at arginine residues.5 X-ray crystal structures of the
MALT1 catalytic domain show a dimeric organization in a classical
caspase-like fold.6 Several studies using mouse models of MALT1
deficiencies have shown that MALT1 plays a role in autoimmune
diseases.7 Furthermore, first MALT1 inhibitors and their activity
in vitro and in animal models have been disclosed.8 Collectively,
this provides evidence to support MALT1 as an attractive novel
protease target for development of small molecule inhibitors with
tease function in vitro with an IC50 of 1.6
displayed activity in a cellular reporter gene assay (RGA), measur-
ing NF- B activation in a HEK293 reporter cell line stably trans-
lM, but that it also
j
fected with the fusion protein cIAP2-MALT1.10,11 Furthermore,
compound 1 inhibited the cleavage of one of the MALT1 substrates,
BCL10 in the OCI-Ly3 cell line which is characterized by constitu-
tive CBM assembly, resulting in high spontaneous MALT1 activ-
ity.12,13 Based on these results an optimization campaign was
initiated.
For SAR studies the molecule was derivatized in three major
parts, the piperidine substituents, the core piperidine moiety and
the amide substituent. Substituted benzenes and 6-ring heterocy-
cles were tolerated as piperidine substituents (Table 1). Aliphatic
N-substituents or a benzyl group led to inactive molecules (com-
pounds 4 and 5). The pyrimidine as it is present in the hit structure
could be replaced by a phenyl-, a pyridyl- or pyridazyl-ring (com-
pounds 2, 3 and 10). Substitution in meta-position led to reduced
⇑
Corresponding author.
E-mail address: achim.schlapbach@novartis.com (A. Schlapbach).
https://doi.org/10.1016/j.bmcl.2018.05.017
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Schlapbach A., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.05.017

2
A. Schlapbach et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
potency (Table 2). Lipophilic substituents, e.g. a chlorine in the
meta-position increased potency tremendously (compound 13).
The para-position was very tolerant of modifications and several
substitutions could lead to further potency increase. As such a para
fluoro- or methoxy-group yielded potent inhibitors (8 and 14,
respectively) and a triazole (1,2,3 or 1,2,4) was found to be the
most beneficial para-substituent (compounds 15–18). The triazole
substituent could be further substituted, e.g. by an additional
amide as in 18. Ortho-substituents were not tolerated (not shown),
and the phenyl ring could be replaced by a 3-pyridyl-substituent
without major impact on potency, e.g. 16 vs. 17.
With potent MALT1 inhibitors at hand we turned our attention
to analyzing their cellular effects. To assess the effects on T cell
activation, selected compounds were tested using an IL-2 reporter
gene assay in Jurkat T cells after stimulation with PMA/anti-CD28
monoclonal antibody (mAb).10 In line with the compounds’ effect
on the protease function of MALT1, the IL-2 reporter gene was
inhibited in the 50–100 nM range (Table 3).
In addition, compounds inhibited OCI-Ly3 cell proliferation
with IC50 values in the low micromolar range, whereas prolifera-
tion of BJAB cells, which do not display constitutive MALT1/NF-
kB activity was not affected, indicating that the anti-proliferative
effect is linked to MALT1 inhibition14 (Table 3).
Fig. 1. Chemical structure and properties of HTS hit compound 1.
potency (compound 6); however a significant increase in potency
was achieved by adding ortho-substituents, in particular lipophilic
substituents like chlorine (compound 8). Interestingly, the same
increase in potency could be achieved by addition of an ortho
amino group (compound 9). With the exception of compound 8
which appears to be unexpectedly potent in our biochemical assay,
biochemical activity generally translated well into cellular potency
as measured in a NF-jB reporter gene assay, or more directly, by
Off-target activity of compound 17 was assessed in an extended
measuring inhibition of cleavage of the MALT1 substrate BCL10.
An aromatic group was required as amide substituent, as com-
pound 11 containing a cyclohexyl substituent was devoid of any
activity while the unsubstituted phenyl derivative 12 retained
selectivity panel. Apart from weak activity (8 lM) on Cox-2, no sig-
nificant activity was found in a panel of 64 receptors, transporters
and enzymes. In particular, only very high micromolar activity was
noted in a panel of cysteine proteases.15
Table 1
SAR around the piperidine substituent.
R1
MALT1 biochemical IC50
1.62
(lM)
MALT1 cellular NF-
3.1
jB RGA IC50
(l
M)
MALT1 cellular BCL10 cleavage EC50
5.4
(lM)
1
2
3
4
0.82
1.90
>100
3.2
9.2
n.d.
n.d.
n.d.
n.d.
5
6
>100
9.0
n.d.
n.d.
n.d.
n.d.
7
0.42
2.7
2.1
8
0.028
0.10
0.10
1.35
0.42
0.29
1.5
9
0.54
0.40
10
For detailed assay descriptions see [10,13]. The IC50 values are averages of at least 2 separate determinations.
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A. Schlapbach et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
Table 2
SAR around the amide substituent.
R2
X
MALT1 biochemical
IC50 M)
MALT1 cellular NF-
jB
MALT1 cellular BCL10
(
l
RGAIC50
(
lM)
cleavageEC50
(lM)
11
12
13
Cl
Cl
Cl
>100
n.d.
n.d.
4.7
n.d.
n.d.
3.4
1.5
0.21
1.59
1.35
8
Cl
Cl
Cl
0.028
0.013
0.004
14
15
0.19
0.09
0.45
0.23
16
17
18
NH2
NH2
Cl
0.007
0.035
0.021
0.09
0.11
0.14
0.56
0.19
0.21
For detailed assay descriptions see [10,13]. The IC50 values are averages of at least 2 separate determinations.
This high clearance could be attributed to cleavage of the central
amide bond in vivo, as high levels of acid 24 (see Scheme 2) were
detected in blood samples upon re-analysis.17 Furthermore com-
pounds were stable in plasma and whole blood, indicating that
the proteolytic activity responsible for this amide cleavage was
not present in blood. In order to establish an in vitro assay to better
mimic the in vivo stability we tested selected structurally diverse
compounds in rat hepatocytes which might contain enzymes
responsible for the amide cleavage.18 Indeed, rat hepatocyte clear-
ance correlated much better with in vivo clearance for this diverse
set of compounds (Fig. 2).
Thus, compound 18 showed a much higher turn-over in hepato-
cytes, better reflecting the in vivo situation. Compound 19 dis-
played high turnover in both in vitro systems which translated to
very high clearance in vivo. For compound 20 low clearance was
found in microsomes as well as in heptocytes translating into good
Table 3
Cellular profile of selected MALT1 inhibitors.
15
16
17
NF-
BCL10 cleavage, EC50
OCY-Ly3 proliferation, EC50
BJAB proliferation, EC50 M]
IL-2 RGA, IC50 M]
j
B RGA, IC50
[l
M]
[lM]
0.09
0.23
1.40
>10
0.09
0.56
3.60
>10
0.11
0.19
2.0
>10
n.d.
[lM]
[l
[l
0.05
0.08
The high selectivity and the non-protease-inhibitor-like struc-
ture prompted us to investigate the binding mode of this inhibitor
class. By SPR (surface plasmon resonance spectroscopy) reversible
binding could be demonstrated. Similar as shown for Phenothiazin
derivatives16 binding of compound 17 to an E397A mutant MALT1
was reduced, which suggests these inhibitors also bind in the
recently discovered allosteric pocket at the interface of the cat-
alytic and the Ig3 domain.16
Next, we evaluated the compounds with respect to their ADME
profile and pharmacokinetic properties. In general compounds
were characterized by good permeability and moderate to high
microsomal in vitro clearance (Table 4).
Solubility was low to moderate at neutral pH and higher at pH4
as expected for these slightly basic molecules. Rat pharmacokinet-
ics revealed very high in vivo clearance, e.g. for compounds 17 and
18. In particular the in vivo clearance of 18 was higher than what
could be expected from the in vitro microsomal clearance alone.
PK properties with a moderate rat in vivo clearance of 43 mlꢀminꢁ1
-
kgꢁ1, bioavailability of 46% but a short half-life of 0.5 h. Most
remarkable was the shift from moderate clearance in microsomes
to low clearance in hepatocytes for compound 21. The in vitro-
in vivo correlation for compound 21 using hepatocytes was much
better, translating to a rat in vivo clearance of 56 mlꢀminꢁ1kgꢁ1
,
moderate bioavailability of 46% and a half-life of 2.4 h (Table 4).
While there is no clear rationale for this improvement based on
the chemical structures, the in vitro hepatocyte assay could be used
to further optimize in vivo clearance. However, compounds with
good in vivo exposure, e.g. 20 and 21 were only moderately active
Please cite this article in press as: Schlapbach A., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.05.017

4
A. Schlapbach et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 4
In vitro ADME and in vivo parameters for selected compounds.
17
18
19
20
21
MALT1 IC50/NF-jB RGA [
l
M]
0.035/0.11
0.021/0.14
0.054/3.7
0.184/0.64
0.340/3.0
in vitro
Solubility pH4/6.8 [
l
M]
48/<4
19.4
ꢁ4.4
102
8/<4
8.2
ꢁ5.3
59.8
228
<4/<4
n.d.
ꢁ4.0
81.0
173
170/7
n.d.
ꢁ6.2
29.0
4.1
690/16
7.2
ꢁ4.2
60.2
18.8
Caco-2 Papp A-B [10ꢁ6cm/s]
PAMPA Permeability, log Pe [cm/s]
RLM Cl [mLꢀminꢁ1ꢀkgꢁ1
]
Rat hepatocyte Cl [mLꢀminꢁ1ꢀ106cells]
in vivo (rat)
n.d.
CL [mLꢀminꢁ1ꢀkgꢁ1
]
259
4.1
0.3
161
n.d.
282
7.5
0.6
125
n.d.
320
21.4
2.0
143
n.d.
43
56
Vss [L/kg]
1.1
0.5
914
46
8.7
2.4
623
46
t1/2 [h]
AUC i.v., (1 mg/kg) [nMꢀh]
BAV [%]
Fig. 2. Correlation of microsomal clearance, hepatocyte clearance and rat in vivo clearance.
Scheme 1. Synthesis of compounds 1–10, reagents and conditions: a) TBTU, DIPEA, DMF r.t. (91%); b) 4 N HCl in dioxane, r.t. (100%); c) see table; d) aldehyde, NaBH3CN,
MeOH/AcOH,DIPEA, r.t. (39–40%).
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A. Schlapbach et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Scheme 2. Synthesis of compounds 11–21, reagents and conditions: a) piperidine-4-carboxylic acid, DIPEA, NMP, 120–180 °C (microwave) (82–92%); b) aniline/
aminopyridine, HATU, DIPEA, DMF,r.t., or EDC, DCM, r.t. (29–75%); c) SnCl2, 2 N HCl, ethanol, reflux (62–94%); d) 1,2,3-triazole, NaH, DMF, 0 °C (24–35%), e) SnCl2, 2 N HCl,
ethanol, reflux (83–91%); f) N-methyl-1H-1,2,4-triazole-3-carboxamide, K2CO3, DMF, r.t. (93%); g) Fe, NH4Cl, ethanol, reflux (69–84%); h) 3,5-dimethyl-1,2,4-triazole, NaH,
DMF, 0 °C (68%).
on MALT1 which did not allow to progress a compound into in vivo
efficacy studies and the optimization of this scaffold was
discontinued.
Compounds were synthesized using the reaction sequences
depicted in Schemes 1 and 2.
could serve as good in vitro tool compounds to study MALT1 biol-
ogy. However, it was not possible to identify a compound combin-
ing sufficient potency and good pharmacokinetic properties which
would allow to further progress this compound series.
Amide coupling of Boc-piperidine carboxylic acid with anilines
provided amides 22, which upon de-protection yielded the
piperidines 23. Finally, the piperidine substituent was installed
using Buchwald-type couplings,19 direct SNAr or reductive amina-
tion to provide compounds 1–10.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.05.017.
Variations to the amide substituent could be best achieved
using the route shown in Scheme 2.
References
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reduction of the nitro group yielded the final compounds
(Scheme 2). Non-commercial anilines and aminopyridines were
prepared by nucleophilic substitution of fluoro- or chloro-
nitrobenzenes and pyridines respectively, followed by nitro reduc-
tion using either SnCl2 or iron.
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protease inhibitors which display high potency in biochemical
and cellular assays. Compounds showed activity in a mechanistic
T cell activation assay (IL-2 RGA) in Jurkat T cells as well as in
the lymphoma cell line OCI-Ly3, which suggests potential use of
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compounds with improved PK properties were identified. With
the excellent potency and selectivity profile these compounds
Please cite this article in press as: Schlapbach A., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.05.017

6
A. Schlapbach et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
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14. To monitor the selectivity of MALT1 inhibitors with respect to suppression of
proliferation of OCI-Ly3 compared to BJAB cell models, cells were incubated
with serially diluted compounds and viability determined after 72 hours by
measuring cellular ATP using
a Cell TiterGlo assay kit (Promega). The
concentration (IC50) at which half-maximal reduction of ATP levels relative
to vehicle-treated controls were achieved was calculated using 4-parametric
curve-fitting (XLfit, V4.3.2) of the dose-response curves.
15. Compound 17 was tested in dose-response mode up to 30
transporters and enzymes. Activity on the following proteases was tested up to
100 M (IC50 [ M]): caspase3 (46), cathepsins B (94), C (88), G (>100), K (57), L
lM for receptors,
l
l
13. MALT1-dependent BCL10 cleavage is assessed in a capture ELISA format using
the MSD (Meso Scale Discovery) platform. Briefly, 2x105 cells, per well/90mL in
a 96-well round bottom plate, are treated with 10 mL of 3-fold serial compound
dilutions, starting at 30mM. The final vehicle concentration is 0.3% DMSO in all
wells. After 24h incubation, cell pellet lysis is achieved by addition of 100mL
MSD-lysis buffer/well and incubation on ice for 20 min. Inhibition of target
cleavage is then assessed by transferring 50mL of lysate to the MSD plate pre-
coated with anti-uncleaved BCL10 rabbit mAb (EP606Y/ab33905, Abcam). After
2h of incubation at RT, wells are exposed for 1h to an anti-BCL10 mAb (331.3/
sc-5273, Santa Cruz Biotechnology). Immune complexes are detected by a
SULFO-TAGTM anti-mouse secondary detection antibody incubated for 1 h at RT.
Light emission at 620nm triggered by application of electric current is recorded
on a MSD Sector Imager 6000. The effect of a particular test compound on Bcl10
cleavage inhibition is expressed relative to the effect of Z-VRPR-FMK; set as
100%. EC50 values (mM) are determined using 4-parametric curve-fitting
(XLfit4 software, V2.2.2).
(51), S (99), trypsin (>100), chemotrypsin (>100), chymase (>100), factor VIIa
(>100), IXa (>100), Xa (>100), XIa (>100), XIIa (>100), kallikrein5 (>100), 7
(>100), MT-SP1 (>100), neutrophil elastase (>100), elastase1 (>100), proteinase
3 (>100), thrombin (>100), MMP8 (>100).
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one experiments in permanently cannulated rats. Up to 7 compounds were
dosed simultaneously at 1.0 mg/kg, i.v., in N-methyl-2-pyrrolidone,
polyethylene glycol 200 (30%, 70%), and at 3.0 mg/kg, p.o. as suspension in
carboxymethylcellulose, water, Tween80, (0.5%, 99%, 0.5%). Upon re-analysis
the acid fragment could be detected in circulation in significant concentrations
in several PK experiments from this compound series.
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Please cite this article in press as: Schlapbach A., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.05.017