Discovery of Mcl-1 inhibitors based on a thiazolidine-2,4-dione scaffold

Article abstract of DOI:10.1016/j.bmcl.2017.11.023

Inspired by a rhodanine-based dual inhibitor of Bcl-x_L and Mcl-1, a focused library of analogues was prepared wherein the rhodanine core was replaced with a less promiscuous thiazolidine-2,4-dione scaffold. Compounds were initially evaluated for their abilities to inhibit Mcl-1. The most potent compound 12b inhibited Mcl-1 with a K_i of 155 nM. Further investigation revealed comparable inhibition of Bcl-x_L (K_i = 90 nM), indicating that the dual inhibitory profile of the initial rhodanine lead had been retained upon switching the heterocycle core.

Full text of DOI:10.1016/j.bmcl.2017.11.023

Accepted Manuscript
Discovery of Mcl-1 inhibitors based on a thiazolidine-2,4-dione scaffold
Ellis Whiting, Mithun R. Raje, Jay. Chauhan, Paul T. Wilder, Daniel Van Eker,
Samuel J. Hughes, Nathan G. Bowen, Gregory E.A. Vickers, Ian C. Fenimore,
Steven Fletcher
PII:
S0960-894X(17)31111-3
https://doi.org/10.1016/j.bmcl.2017.11.023
BMCL 25429
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
23 August 2017
8 November 2017
10 November 2017
Please cite this article as: Whiting, E., Raje, M.R., Chauhan, Jay., Wilder, P.T., Van Eker, D., Hughes, S.J., Bowen,
N.G., Vickers, G.E.A., Fenimore, I.C., Fletcher, S., Discovery of Mcl-1 inhibitors based on a thiazolidine-2,4-dione
scaffold, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.11.023
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Ellis Whiting, Mithun R. Raje, Jay Chauhan, Paul T. Wilder, Daniel Van Eker, Samuel J. Hughes, Nathan G.
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of Mcl-1 inhibitors based on a thiazolidine-2,4-dione scaffold
a†
b†
b
c
a
a
Ellis Whiting , Mithun R. Raje , Jay. Chauhan , Paul T. Wilder , Daniel Van Eker , Samuel J. Hughes ,
a
a
d
b
Nathan G. Bowen, Gregory E. A. Vickers, Ian C. Fenimore and Steven Fletcher
a
School of Chemistry, University of Cardiff, Cardiff, UK, CF10 3AT
b
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine St., Baltimore, MD 21201
Department of Biochemistry, University of Maryland School of Medicine, 22 S. Greene St., Baltimore, MD 21201
c
d
Eberly College of Science, Penn State University, 517 Thomas St., State College, PA 16803
steven.fletcher@rx.umaryland.edu; Tel.: 410-706-6361; Fax.: 410-706-5017
†
Contributed equally
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
L
Inspired by a rhodanine-based dual inhibitor of Bcl-x and Mcl-1, a focused library of analogues
was prepared wherein the rhodanine core was replaced with a less promiscuous thiazolidine-2,4-
dione scaffold. Compounds were initially evaluated for their abilities to inhibit Mcl-1. The most
Accepted
Available online
potent compound 12b inhibited Mcl-1 with a K
comparable inhibition of Bcl-x (K = 90 nM), indicating that the dual inhibitory profile had
been retained upon switching the heterocycle core.
i
of 155 nM. Further investigation revealed
L
i
Keywords:
Mcl-1
Bcl-xL
2
009 Elsevier Ltd. All rights reserved.
apoptosis
cancer
protein-protein interaction
The breakdown of apoptosis, a programmed cell-death
mechanism, is one of the hallmarks of cancer and a crucial step in
hydrophobic grooves on the surfaces of the pro-survival proteins;
specifically, conserved hydrophobic side chains along one face of
the -helix at positions i, i+3/4, i+7 and i+11 probe into sub-
pockets p1 through p4. In addition, a conserved aspartate on the
opposing face of the BH3 -helix engages in a salt bridge
interaction with a conserved arginine (Arg263 (Mcl-1), Arg139
1
-3
tumorigenesis.
Apoptosis is triggered by either cytotoxic
stimuli, such as growth factor deprivation, UV and γ-irradiation,
and stress (called the intrinsic or mitochondrial pathway) or by
binding of an external ligand to a member of the tumor necrosis
factor (TNF) receptor family (called the extrinsic or death
receptor pathway). The intrinsic pathway is strictly regulated by
1
0,11
(Bcl-x ) and Arg143 (Bcl-2)).
L
4
Irregularities in the mitochondrial apoptotic pathway are a
the B-cell lymphoma 2 (Bcl-2) family of proteins, which exerts
12
root cause of cancer. The overexpression of Bcl-2-like proteins
its influence over cell fate primarily by interactions between three
distinct subfamilies. These include the BH3-only family
members such as Bad, Bid, Bim, Noxa, and Puma (death
initiators), the pro-survival Bcl-2-like proteins such as Bcl-2
13
or the loss of Bax or Bak function contributes to tumorigenesis
by boosting the survival of cells that would otherwise undergo
apoptosis. In addition, defects in apoptosis contribute to the
5
resistance of diverse tumors to cytotoxic drugs. Consequently,
itself, Bcl-x , Bcl-w, and Mcl-1 (cell guardians), and the pro-
L
small molecules disrupting the protein-protein interactions (PPIs)
by binding to the hydrophobic cleft of Bcl-2-like survival
proteins would be highly valuable as they are predicted to release
apoptotic proteins such as Bax, Bak, and Bok (effector proteins).
The Bcl-2 family of proteins is commonly described as a
5
tripartite apoptotic switch. External cytotoxic stress signals
pro-apoptotic proteins and restore normal levels of
activate BH3-only members of the protein family and induce
apoptosis by two distinct, but not mutually exclusive,
10,11,14
apoptosis.
Specifically, chemical agents that mimic the BH3
6
-7
domains of the pro-apoptotic Bcl-2 family members might
furnish novel antineoplastics, enhancing the chemical weaponry
mechanisms: sequestration of the pro-survival Bcl-2 family
8
proteins which liberates the pro-apoptotic proteins Bax and Bak
15
in the war on cancer. In fact, this has been an intense area of
through their BH3 domains or by direct activation of Bax and
11
9
research for over a decade. Two of the best characterized BH3
Bak. The pro-survival and pro-apoptotic Bcl-2 proteins
mimetics are ABT-737 and its orally available derivative ABT-
neutralize each other’s function to ensure apoptosis is tightly
regulated. This is accomplished through the molecular
recognition of the BH3 -helix of pro-apoptotic proteins by
2
63 (navitoclax), reported by Abbott Laboratories through a
combination of NMR-based screening and structure-based

1
6,17
design.
restores the ability of cancer cells to undergo apoptosis.
antagonizes the anti-apoptotic proteins Bcl-2, Bcl-x , and Bcl-w,
ABT-263 is a first-in-class Bcl-2 family inhibitor that
GOLD to determine a putative binding mode of 3 using Mcl-1
extracted from PDB ID: 4HW2. One low-energy docked
1
7,18
24
It
solution is presented in Figure 2. In the docking experiment, the
only constraint was that the binding site was centered 10Å radius
about the N of the guanidinium function of Arg263, which is
near the middle of the BH3 binding crevice. As depicted, the
carboxylic acid and one of the TZD ring carbonyls form
electrostatic interactions with Arg263 (2.7 Å), recapitulating the
salt bridge interaction between Arg263 and a conserved aspartic
acid found on one face of the BH3 -helices of pro-apoptotic
Bcl-2 proteins. In addition, the 4-bromophenyl moiety points
down into the hydrophobic p2 pocket, engaging in van der Waals
interactions with various hydrophobic residues that include
Val243, Leu246 and Met250. The S-isobutyl group of 3 appears
to interact with the tetramethylene side chain of Arg263 and the
methyl of Thr266. Interestingly, the docking solution suggests
that groups larger than isobutyl of corresponding R-enantiomers
may be able to engage in interactions with His224, Ala227 and
Phe228. We hypothesized that expanding the bromophenyl
L
1
7,18
thereby releasing Bax and Bak.
subset of anti-apoptotic Bcl-2 protein family has produced
encouraging clinical outcomes. Indeed, the Bcl-2 selective
inhibitor ABT-199
approval for the treatment of patients with chronic lymphocytic
leukemia who harbor a 17p chromosomal deletion. Within the
last few years, Mcl-1 selective inhibitors have begun to
This selective inhibition of a
1
9
2
0
(venetoclax) recently received FDA
2
1
2
2-28
29
emerge,
and at least one clinical trial has been initiated.
Many cancer cells are resistant to ABT-737 and ABT-263
due to existing high levels, or compensatory upregulation, of
30,31
Mcl-1.
Downregulation of Mcl-1 by siRNA, miRNA, or Noxa
upregulation overcomes this resistance and sensitizes cancer cells
towards ABT-263.
Mcl-1 and Bcl-2/Bcl-x /Bcl-w may be therapeutically more
beneficial,
3
2-33
This suggests that the dual inhibition of
L
either
through
engineering
drugs
with
34-
polypharmacological profiles, so-called “pan-Bcl-2” inhibitors,
4
0
or through adjuvant therapies. Thus, the expansion of small
molecule chemical artillery to target single or multiple anti-
apoptotic proteins involved in cancer progression continues to be
an unmet medical need and is urgently required. Herein, we
report the discovery of potent Mcl-1 inhibitors based on a
thiazolidine-2,4-dione scaffold. Selected molecules were also
O
O
O
N
N
S
OH
O
S
OH
Br
N
O
O
S
S
BH3I-1 (1)
2
O
N
O
S
OH
Br
O
moiety of 3 will afford more potent inhibitors owing to greater
3
occupation of the p2 pockets of Mcl-1 and Bcl-x , as has been
L
1
1,17,24
evaluated for their abilities to inhibit Bcl-xL.
described previously.
Figure 1: Lead rhodanine Mcl-1 inhibitors (1 and 2) and a novel
thiazolidine-2,4-dione derivative (3).
Figure 2: A low-energy GOLD docking solution of 3 with Mcl-1
extracted from PDB ID: 4HW2. Ligand and protein coloured by atom type.
Black labels indicate Mcl-1 residues, purple labels describe sub-pockets
within the BH3-binding crevice.
Previously, Yuan’s group screened a chemical library of
1
6,320 compounds and discovered the rhodanine derivative
BH3I-1 (1, Figure 1) as an inhibitor of Bcl-x with a K of 2.4
L
i
4
1

M. In a subsequent report, Bernardo and colleagues described
The TZD target molecules were synthesized according to a
short and straightforward synthetic pathway (Schemes 1 and 2).
Briefly, the methyl ester of the appropriate amino acid 4a–4h,
was condensed with thioglycolic acid in the presence of
carbonyl-1,1-diimidazole (CDI) to construct the N-substituted
TZD rings of compounds 5a–5h. Knoevenagel condensations of
compounds 5a–5h with various aldehydes delivered compounds
7a–7h, 9b–9c and 11b–11i. Finally, the methyl esters of 7a–7i,
a library of analogues of BH3I-1 that are dual inhibitors of Bcl-xL
and Mcl-1, the most potent of which is 2 with K ’s of 3.7 M
i
4
2
(
Bcl-x ) and 7.6 M (Mcl-1). Rhodanines have gained notoriety
L
as pan-assay interference compounds (PAINS), and are the
amongst the worst offenders; to some degree, this has been
attributed to their exocyclic sulfur atom that permits access to
multiple hydrogen bonding interactions with limited restriction
43
on geometries. Towards the discovery of less promiscuous
9
b–9c and 11b–11h were hydrolyzed under acid catalyzed
conditions to furnish the final molecules 8a–8c, 3, 8e–8h, 10b–
0c and 12b–12h, respectively. As depicted in Scheme 3,
inhibitors of Mcl-1 (and Bcl-x ), we considered adjusting the
L
rhodanine core of BH3I-1 to a thiazolidine-2,4-dione (TZD), as
exemplified by compound 3. It should be noted that we are not
necessarily condemning BH3I-1. However, devoid of an
exocyclic sulfur atom, it was surmised that TZDs would begin
with inherently improved selectivity profiles relative to the
corresponding rhodanines, and thus represent improved leads for
optimization, through access to fewer, and geometrically more
restricted, hydrogen bonds. We enlisted the modeling package
1
primary carboxamide 13 was generated through activation of
carboxylic acid 8h with HBTU followed by quenching with
NH Cl. Deletion of the N-substitution of compounds 12b–12h
4
and reduction of the double bond of 12d were accomplished
through the de novo synthesis described in Scheme 3 to yield 15
and 18, respectively.

a: R = (S)-isobutyl
b: R = (S)-benzyl
c: R = (S)-methyl
d: R = H
e: R = (R)-isobutyl
f: R = (S)-isopropyl
g: R = (R)-methyl
h: R = (R)-benzyl
resulted in improved inhibitory activity of Mcl-1, both in the
meta and para positions, although a polar hydroxyl in the para
position was not tolerated. Increasing the size of the peripheral
aryl group of compounds 8f to 8g resulted in improved inhibition
but the addition of a bromine atom into the internal phenyl ring
OH
O
R
HS
R
O
O
N
HCl.H2N
S
O
O
CDI, DIPEA
O
O
CH2Cl2, RT, 16 h
4
a-4h
53-64%
5a-5h
O
a: R = H
b: R = 3-Br
c: R = 4-tert-Bu
d: R = 4-Br
e: R = 4-OH
(8g) resulted in a slight drop in activity. Substitution of the (S)-
O
H
R
O
N
R
isobutyl groups of compounds 8b and 8c with (S)-benzyl groups
generated inhibitors with greater activities. For example,
N
S
6
S
O
O
f: R = 4-OPh
g: R = 4-(4-chloro-3,5-dimethyl)phenoxy
O
O
cat. piperidine
O
O
Y
5a
EtOH, reflux, 16 h
h: R = 3-Br, 4-(4-chloro-3,5-dimethyl)phenoxy homologue 10b (K = 290 nM) was almost ten-fold more potent
i
3
1-54%
than 8b (K = 2.13 M). In the series of 4-(4-chloro-3,5-
H2SO4/AcOH
00 oC, 4 h
8-72%
7a-7i (Y = Me)
i
1
dimethylphenyl) derivatives 12b–12h, the larger the N-
4
8a-8c, 3, 8e-8h (Y = H)
substituent, the more active the compound, with 12b the most
O
potent of the series (K = 155 nM). Another observation is that in
O
H
R
i
O
N
R
every case, S-enantiomers exhibited greater affinities than their
R-counterparts, in contrast to our speculation with the docking
result for 3. For example, compound 12b bound Mcl-1 five times
more strongly than enantiomer 12h, and 8g was more than twice
as active as enantiomer 12e. Given that the chiral centre
appeared to impact binding affinities, it was important to
determine their optical integrities. To this end, we prepared
diastereomeric amides of enantiomers 12b and 12h with
enantiomerically-pure (S)-phenethylamine (1.1 eq of each of
N
b: R = 3-Br
c: R = 4-tert-Bu
S
6
O
S
O
O
O
cat. piperidine
O
O
Y
EtOH, reflux, 16 h
5b
3
8-40%
H2SO4/AcOH
oC, 4 h
9b, 9c (Y = Me)
100
6
4-70%
10b, 10c (Y = H)
Scheme 1
O
NH
cat. piperidine
EtOH, reflux, 16 h Cl
O
1
HBTU, HOAt, DIPEA in DMF, rt, 1 h). Analysis of the H NMR
Cl
S
O
NH
O
+
spectra of the products indicated an approximate 2:1 inseparable
mixture of two amides, suggesting 33% racemization; efforts will
be made to obtain enantiomerically-pure compounds in a follow-
up full paper so that the effect of the chiral centre can be more
carefully scrutinized. Methyl ester 7g and carboxamide 13
exhibited no binding affinities to Mcl-1, consistent with the
requirement for a salt bridge interaction with Arg263. In line
with this finding, complete removal of the N-substituent, as in 15,
also resulted in an inactive inhibitor. Finally, reduction of the
double bond of 12d yielded inhibitor 18 with a four-fold lower
binding affinity, which may be ascribed to the generation of a
more flexible inhibitor that suffered a greater entropic penalty
upon binding. This finding is especially significant, since two
S
6
1%
O
O
O
6
h
14
15
O
O
LiBH4
THF/pyridine
Br
Cl
O
NH
S
8
0 oC, 8 h
4%
O
K2CO3
DMF, 60 oC, 1 h
O
6
16
6
4%
O
O
Cl
TFA/CH2Cl2
RT, 3 h
Cl
N
O
N
S
O
S
OH
O
O
O
O
O
70%
17
18
Scheme 2
O
R
N
cat. piperidine
O
N
O
R
O
Cl
EtOH, reflux, 16 h
Cl
O
S
+
S
O
O
34-57%
O
O
Y
O
O
5
b-5h
6h
H2SO4/AcOH
00 oC, 4 h
5-66%
11b-11h (Y = Me)
12b-12h (Y = H)
1
5
O
HBTU, NH4Cl, DIPEA
DMF, rt, 16 h
O
N
Cl
Cl
N
S
OH
S
NH2
50%
O
O
O
O
O
O
8h
13
Scheme 3
The abilities of our inhibitors to bind Mcl-1 were quantified
by a fluorescence polarization competition assay (FPCA), as
2
8
described by us previously. Briefly, compounds were titrated
into 96-well plates in order to compete with a fluorescently-
labeled Bak-BH3 peptide for binding to Mcl-1. The IC50 values
resulting from the changes in fluorescence polarization were then
features that may render molecules such as BH3I-1
promiscuous have been modified or removed: the rhodanine
core has been replaced, and the heterocycle is no longer aromatic.
4
3
4
4
converted to K values using the Nikolovska-Coleska equation.
Figure 3: A low-energy GOLD docking solution of 12b with Mcl-1
extracted from PDB ID: 4HW2. Ligand and protein coloured by atom type.
Black labels indicate Mcl-1 residues, purple labels describe sub-pockets
within the BH3-binding crevice.
i
For further details, please see the Experimental Section.
First, as a point of reference the K for the binding of BH3I-1
i
to Mcl-1 was determined to be 44 M using the previously
4
2
45
reported IC50 data and an online calculator associated with
reference 44. As indicated in Table 1, compound 8a exhibited no
inhibition of Mcl-1. However, the introduction of hydrophobic
groups into the unsubstituted phenyl ring of compound 8a
We then evaluated the binding affinities of two of the most
potent compounds to Bcl-x ; 8g and 12b bound with K values of
L
i
60 nM and 90 nM, respectively. These improved binding
affinities to Bcl-x over Mcl-1 is mirrored by similar findings
L

1
72-371
with related rhodanine-based inhibitors, and suggests the core
scaffold modulation has not adversely affected the dual inhibitory
competition assay with Mcl-1
and fluorescein isocyanate (FITC)-labeled
Bak-BH3 peptide were converted to K
i
values using the Nikolovska-Coleska
4
2
44
profiles of the original rhodanine lead. GOLD docking studies
were performed with 12b, as shown in Figure 3, again with PDB
ID: 4HW2. A semi-transparent surfacing has been utilized to
indicate that the sizeable p2 pocket (deepest point Met250) is
capable of accommodating the large 4-chloro-3,5-dimethylphenyl
moiety). Although the salt bridge between the ligand’s carboxylic
acid and Arg263 is now much weaker (4.3 Å), GOLD is not
capable of taking into account the flexibility of the protein.
Finally, direct binding of our inhibitors to Mcl-1 was confirmed
equation.
In summary, starting from the rhodanine-derived lead BH3I-1,
we have developed a focused library of thiazolidine-2,4-dione-
based dual inhibitors of Mcl-1 and Bcl-x . Compounds were
readily accessed in three steps, and their inhibitory activities were
initially evaluated against Mcl-1. In general, greater inhibition of
Mcl-1 was realized when larger hydrophobic groups were
substituted off the benzylidene aryl moiety. Likewise, large
hydrophobic groups at the chiral carbon elicited more potent
inhibition. The most potent compound, 12b, inhibited Mcl-1 with
L
1
15
by an orthogonal technique. 2D H- N heteronuclear single
1
5
quantum coherence (HSQC) spectra of N-Mcl-1 were collected
in the absence (black) and presence (red) of 12b, and overlaid in
Figure 4. As illustrated, there were multiple chemical shift
perturbations indicating that 12b bound directly to Mcl-1.
However, due to broad loss of peaks from intermediate chemical
exchange, we cannot determine specifically where the compound
is interacting.
a K of 155 nM. Further investigation revealed the dual inhibitory
i
profile of the parent rhodanines had been retained as 12b
inhibited Bcl-x with a K of 90 nM. Rhodanine derivatives have
L
i
become infamous as PAINS compounds. Herein, we have
replaced the promiscuous scaffold with a reputably more
selective thiazolidine-2,4-dione core,
compounds with affinities of more than an order of magnitude
greater than the lead BH3I-1. Armed in the knowledge that
switching the heterocyclic core is tolerated, further work is now
43
and discovered
Table 1: Mcl-1 inhibitory structure–activity relationships of substituted
a,b
a
thiazolidine-2,4-diones.
IC50 data from
a
fluorescence polarization
O
X
S
N
R
O
Compound
X
R
Ki ( M)
Compound
X
R
Ki ( M)
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
8
8
a
b
>100
12b
0.155 ± 0.018
1.94 ± 0.19
OH
OH
OH
OH
OH
OH
OH
OH
OMe
NH2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
1
1
2c
2d
5
a
2.13 ± 0.97
2.22 ± 1.22
OH
OH
O
O
Br
2.34 ± 0.25
8
c
1
1
1
2e
2f
1.00 ± 0.11
1.19 ± 0.08
2.78 ± 0.30
3
8
8
8.78 ± 3.27
OH
OH
OH
Br
O
O
O
e
f
>100
HO
2g
12.7 ± 2.9
O
1
2h
0.878 ± 0.088
Cl
Cl
8
8
g
h
0.419 ± 0.053
0.791 ± 0.125
OH
OH
O
O
O
O
7
g
>100
1
1
3
5
>100
>100
Br
Br
O
H
Cl
Cl
1
1
0b
0c
OH
OH
0.290 ± 0.054
0.845 ± 0.193
O
O
O
O
18
10.2 ± 3.6
OH
O

1
1
5. Lessene, G.; Czabotar, P. E.; Colman, P. M. Nat. Rev. Drug
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1
15
Figure 4: 2D H- N HSQC NMR spectra overlay of apo-
Mcl-1 (black) and 12b-bound Mcl-1 (red). The NMR samples
1
5
contained 131 mM N-labeled Mcl-1, (182 mM 12b), 20 mM
HEPES, pH 6.8, 50 mM NaCl, 3 mM dithiothreitol, 20% D O,
2
and 5% D -DMSO.
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Acknowledgements
We thank the University of Maryland School of Pharmacy and
the Center for Biomolecular Therapeutics, University of
Maryland School of Medicine for financial support of this
research.
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