Artificial Macrocycles as Potent p53-MDM2 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00219

Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53-MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73, Val93, and Lys94 yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence polarization (FP) assay and ¹H-¹⁵N two-dimensional HSQC nuclear magnetic resonance experiments.

Full text of DOI:10.1021/acsmedchemlett.7b00219

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Letter
Artificial macrocycles as potent p53-MDM2 inhibitors
Natalia Estrada-Ortiz, Constantinos G. Neochoritis, Aleksandra Twarda-
Clapa, Bogdan Musielak, Tad A Holak, and Alexander Dömling
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00219 • Publication Date (Web): 20 Sep 2017
Downloaded from http://pubs.acs.org on September 21, 2017
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Artificial macrocycles as potent p53ꢀMDM2 inhibitors
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†
, ‡
†, ‡
#, ⊥
Natalia EstradaꢀOrtiz, Constantinos G. Neochoritis, Aleksandra TwardaꢀClapa, Bogdan Musieꢀ
§
⊥,
§
†*
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lak, Tad A. Holak, Alexander Dömling
†
Department of Drug Design, University of Groningen, A. Deusinglaan 1, Groningen 9700AV, The Netherlands.
#
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30ꢀ387 Krakow, Poland.
⊥
Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30ꢀ387 Krakow, Poland.
§
Department of Chemistry, Jagiellonian University, Ingardena 3, 30ꢀ060 Krakow, Poland.
KEYWORDS: Macrocycles, Ugi reaction, p53, MDM2, protein-protein interaction, cancer
ABSTRACT: Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial
macrocyclic inhibitors of the p53ꢀMDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled
peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73 Val93 and Lys94 yielding
derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence
1
15
polarization (FP) assay and Hꢀ N 2D HSQC NMR experiments.
In nature, macrocycles are not uncommon and often exhibꢀ
it interesting biological activities. Often macrocyclic comꢀ
undergoing phase I and II clinical trials in patients suffering of
solid tumors, lymphoma and myeloid leukemias (ClinicalTriꢀ
als.gov ID: NCT02264613 and NCT02909972).
Here we propose a novel series of non peptidic artificial
macrocyclic compounds that inhibit the p53ꢀMDM2 interacꢀ
tion, which might have a different activity profile from the
currently available scaffolds. Our synthesis strategy is shown
in Figure 1. Based on the Ugi scaffold, we introduced two
terminal eneꢀfunctionalities, via the carboxylic acid and the
isocyanide component and we were able to cyclize the comꢀ
1
pounds have distinct advantages over their open chain analoꢀ
2
gous including higher affinity and selectivity, preferable
entropic signature, better membrane permeation and oral bioaꢀ
1
,2
vailability or higher stability. Thus, macrocycles can inꢀ
1
crease affinity and selectivity for a specific target. In spite of
their potential, macrocycles pose considerable synthesis probꢀ
lems and also the accurate prediction of their conformation
1
,2
makes it difficult to predict activity.
1
5, 16
The tumor suppressor protein p53 is involved in controlꢀ
ling pathways of cell cycle, apoptosis, angiogenesis metaboꢀ
pounds by ring closing metathesis (RCM, Figure 1).
3
,4
lism, senescence and autophagia. TP53 gene is one of the
5
most frequently mutated gene in a multitude of human cancer.
Additionally, in multiple cases where TP53 is intact, p53’s
function is impaired by its negative regulators: Mouse double
minute 2 and 4 homologs (MDM2 and MDMX), due to ampliꢀ
3ꢀ5
fication or enhanced expression of their coding genes. Mulꢀ
Figure 1. Macrocyclization strategy to inhibit the p53ꢀMDM2
interaction.
tiple potent and selective compound classes to inhibit the p53ꢀ
MDM2 interaction have been discovered, described and evalꢀ
uated in early clinical trials. However, the pharmacokinetic
6
Our previously introduced three and fourꢀpoint pharmacoꢀ
phore models were the basis of the discovery and development
of the current inhibitors. Thus, we used as the starting point
our formerly described αꢀaminoacylamide (YH300, shown in
and pharmacodynamics properties of the studied scaffolds
could still be optimized to minimize the side effects. Thus, the
discovery of new p53ꢀMDM2 inhibitors with diverse strucꢀ
6ꢀ8
tures to improve their properties is still of importance. The
three finger pharmacophore model for p53ꢀMDM2 is recogꢀ
nized as responsible for the binding of small molecules and
cyan sticks, Figure 2) with a K of 600 nM and its crystal
i
1
3
structure in complex with MDM2 receptor (PDB ID 4MDN).
Accordingly, the 6ꢀchloroindoleꢀ2ꢀcarboxylic acid was used as
an ‘anchor’ in order to mimic the Trp23 amino acid and conꢀ
strain the position of other substituents and three additional
binding sites were defined, Phe19, Leu26 and the induced
Leu26 subpocket.
9
,10
peptides to the MDM2. We already described several series
of potent p53ꢀMDM2 antagonists, proposing an extended four
finger model; the intrinsically disordered MDM2 Nꢀterminus
is ordered by certain small molecules, which can be obtained
1
1,12
by multicomponent reaction chemistry,
crystallization.
as shown by coꢀ
Recently, several macrocyclic stapled pepꢀ
1
3,14
tides have been described with great affinity towards MDM2
6
and MDMX. ALRNꢀ6924 (Aileron Therapeutics) is currently
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Figure 2. Four and threeꢀpoint pharmacophore modelling of macrocyclic compounds; Left: YH300 ꢀPDB ID: 4MDNꢀ (cyan lines),
Tyr67, Gln72, His73 Val93 and Lys94 (blue sticks), proposed macrocycle to explore the fourꢀpharmacophore point (pink sticks).
Right: SAHꢀp53ꢀ8 stapledꢀpeptide ꢀPDB ID: 3V3Bꢀ (cyan lines), Tyr67, Gln72, His73, Val93 and Lys94 (blue sticks), proposed
macrocycle to explore the threeꢀpharmacophore point (pink sticks).
Our design of the macrocyclic linker aims to bind on top
of the loop linking α2’ and α1’ helices, covering a large hyꢀ
drophobic surface area formed by Tyr67, Gln72, His73, Val93
and Lys94 (Figure 2) and potentially increasing the affinity to
the receptor. Moreover, we reasoned that the addition of a
methyl group on the ring could mimic the tertꢀbutyl group of
Aldehyde 6 was synthesized from the 6ꢀchloroꢀindole derivaꢀ
17,18
tive using the VilsmeierꢀHaack formylation reaction.
For
the preliminary SAR analysis of the Leu26 and the induced
pockets, the benzylamines 5 were used as commercially availꢀ
able (5bꢀh) or obtained via Williamson ether synthesis (5a).
Probing both the Phe19 pocket and the larger hydrophobic
surface area formed by Tyr67, Gln72, His73, Val93 and
Lys94, the isocyanides 7a,b were synthesized from the correꢀ
sponding formamides, whereas the carboxylic acids are comꢀ
mercially available. In particular, the amine 5a with the oxyꢀ
gen linker that designed to probe the induced pocket was synꢀ
thesized through a Williamson ether synthesis of the protected
4ꢀhydroxybenzylamine with the 3,4ꢀbenzyl chloride under
basic conditions (Supporting information, Scheme 1). The
isocyanides 7a,b were synthesized from the formamides via
the revised LeuckartꢀWallach reaction of the corresponding
13
YH300, likely leading to increased affinity.
The retrosynthetic plan of the designed macrocycle 1 foreꢀ
sees (Scheme 1), a ring closing metathesis reaction (RCM),
followed by a classical Ugi fourꢀcomponent (Uꢀ4CR). The Ugi
adduct is formed of the anchoring 6ꢀchloroꢀ3ꢀcarboxaldehyde
6
, suitably substituted benzylamines 5 and the longꢀchained
aliphatic carboxylic acids 8 along with the isocyanides 7 inꢀ
corporating terminal double bonds.
19
carbonyl compounds (SI, Scheme 2).
Scheme 1. Retrosynthetic plan based on a Uꢀ4CR and a ring closing metathesis
disconnection point
CHO
CO
N
H
n
n
n
O
O
O
NH2
O
m
O
m
O
m
X
Et
2
X
X
X
N
N
N
R1
R1
R1
Cl
N
N
N
H
H
H
RCM
U-4CR
5
6
+
CO H
CO Et
CO Et
2
2
2
N
H
N
H
N
H
R1
Cl
Cl
Cl
1
3
4
NC
2
CO H
m
n
7
8
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Next, we proceeded in the Ugi fourꢀcomponent reaction.
the mixture to hydrogenation on Pd/C isolating compounds 2.
Last step was the ester hydrolysis obtaining the final screening
compounds 1 (Scheme 2). Performing a preliminary SAR, we
built a small library of macrocycles of various ring sizes (12,
13, 18, 19 and 24 number of atoms) targeting the hydrophobic
region around Tyr67, Gln72, His73 Val93 and Lys94. We
maintained the anchoring indole group for the Trp23 and the
phenyl group for Leu26 and further explored the induced
pocket with the extended dichlorobenzyloxy moiety.
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Equimolar mixture of the substituted benzylamine 5, aldehyde
6, isocyanide 7 and carboxylic acid 8 in 2,2,2ꢀtrifluoroethanol
(TFE) was irradiated at 120 C for 1 h in a microwave oven
o
yielding compounds 4 (Scheme 2). Afterwards, we successfulꢀ
nd
ly performed the ring closing metathesis with the 2 generaꢀ
TM
tion of Grubbs catalyst in dichloromethane (DCM) affordꢀ
ing compounds 3 as mixture of isomers (E and Z). Due to the
fact that the existence of the double bond gives rise to two
possible isomers and most importantly reduces the flexibility
of
the
macrocycle,
we
decided
to
subject
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Scheme 2. Synthesis of the macrocycle library based on the U-4CR/RCM strategy
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Two complementary assays based on independent physiꢀ
cochemical principles, fluorescence polarization (FP) and Hꢀ
N 2D HSQC NMR were used to measure affinity and to
exclude false positive hits. Fluorescence polarization (FP)
plex. Particularly, Val93 is clearly involved in the interaction,
as its cross peak shifted between titration steps for MDM2:1j
molar ratios equal to 2:1 and 1:1. After 1:1 step the peak reꢀ
mained in the same position. NMR titration also confirmed the
tight binding of 1j, as e.g. for Arg29 NMR signal splitting was
observed (Figure 3), which indicated strong interaction with
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assay was employed to determine the inhibitory affinities (K )
i
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0
of the macrocycles against MDM2 as previously described
and the results are presented in Table 1.
MDM2 at K below 1 ꢁM (and a slow chemical exchange).
d
Examining both the three and fourꢀfinger pharmacophore
model, it seems that most of the obtained macrocycles are
active towards MDM2, many of them demonstrating an affiniꢀ
ty below 1 µM. Although it is a preliminary SAR study, we
can conclude that the ideal ring size for the fourꢀfinger model,
seems to be around 18 (entry 3). The affinity improves while
increasing the size from 12 (1a, entry 1) to 13 (1b, entry 2)
and eventually 18 (1c, entry 3) atom ring size. Moreover,
compound 1c has an affinity of 100 nΜ as a mixture of diaꢀ
stereomers, whereas the larger 24ꢀmembered macrocycle 1k
(entry 11) shows decreased activity (1.91 ꢁM).
Table 1. Results of the evaluation of inhibitory activity of
macrocyclic inhibitors toward MDM2 using FP assay.
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Entry Compound Ring size
K (µM)
i
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1a
12
0.35
2
3
4
5
1b
1c
1d
1e
13
18
18
18
0.32
0.10
N/A
5.3
In addition, we focused on the threeꢀfingerꢀpharmacophore
model, which characterizes the vast majority of the currently
6
available smallꢀmolecule MDM2 inhibitors. We synthesized
6
7
8
9
1f
1g
1h
1i
18
18
18
18
N/A
1.3
various macrocycles with a different substitution pattern. The
position of halogens on the phenyl group seems to play a
significant role since the para fluoro (1d, entry 4) or chloro
substituted (1f, entry 6) derivatives are only slightly or not
active at all. On the contrary, the addition of a second chlorine
in oꢀ or mꢀposition influenced the binding mode; the 2,4ꢀ
dichloro derivative (1e) showed activity of 5.3 ꢁΜ (entry 5)
and the corresponding 3,4ꢀdichloro compound 1h (entry 8)
displayed an activity of 80 nM. Whereas placing the donor
group ꢀOMe in compound 1g (entry 7), improved the affinity
with 1.3 ꢁΜ. Nonetheless, mꢀOMe substitution keeping the
same ring size of 18 (1i, entry 9), did not show any significant
difference, exhibiting an activity of 1.8 ꢁΜ. Due to our previꢀ
0.08
1.8
0
.14 (racꢀ1j)
10
1j
19
0.09 {(+)ꢀ1ja}
0.70 {(ꢀ)ꢀ1jb}
11
1k
3j
24
19
24
12
24
1.9
0.34
2.5
1
2
13
3k
2a
2k
1
4
3.0
2
1
ous results, where we were able to synthesize fluorinated
1
5
N/A
phenyls as compound 9 with K up to 100 nM, we employed
i
the 3,4,5ꢀtrifluorobenzylamine and we synthesized compound
j (entry 10) which exhibited an interesting affinity as a raceꢀ
N/A: not active; all measurements performed in duplicates; standard
errors up to 10%.
1
mic mixture of 140 nM. Enantiomeric separation of the raceꢀ
mic mixture via chiral SFC provided the enantiomers (+)ꢀ1ja
and (ꢀ)ꢀ1jb with affinities of 90 nM and 700 nΜ, respectively.
As it was expected, the separated enantiomers showed a sigꢀ
nificant increase of the activity compared to the racemic mixꢀ
ture.
The existence of the double bond in the macrocycles 3j
and 3k, as anticipated, reduced significantly the affinity to 340
nM and 2.5 ꢁΜ respectively (entries 12, 13), compared with
the corresponding hydrogenated compounds 1j (K = 140 nM)
i
and 1k (K = 1.9 ꢁΜ). The corresponding esters, 2a and 2k
i
(entries 14, 15), are orders of magnitude less reactive or inacꢀ
tive comparing with the acids in accordance with our previous
1
1,12,22ꢀ25
experience (entries 1, 11).
Interestingly, the acyclic
Ugiꢀadduct 4j was also proven to be practically inactive both
in the ester and acid forms (SI, Table S1, entry 15). Moreover,
changing the anchor to a nonꢀsubstituted indole moiety (comꢀ
pound 10) or to the 3ꢀ or 4ꢀphenyl moiety (compound 11),
resulted in nearly no activity.
15
Figure 3. Spectrum of the Nꢀlabeled MDM2 (blue) superimꢀ
posed with spectrum after addition of 1j in MDM2/1j molar ratio
equal to 2:1 (red) and 1:1 (green). The closeꢀup view shows seꢀ
lected peaks assigned to Val93 and Arg29. For Arg29, NMR
1
15
signal splitting indicates strong interaction at K below 1 ꢁM.
The expected ligandꢀinduced perturbations in Hꢀ N 2D
HSQC NMR spectra were indeed observed (Figure 3). The
d
15
Nꢀlabeled MDM2 was titrated with increasing concentration
of the compound. Since all cross peaks in the MDM2 specꢀ
26
trum were assigned to particular amino acid residues, it was
possible to analyze the interaction within the MDM2/1j comꢀ
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Three of the macrocyclic compounds (1c, 1h and 1j) obꢀ
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tained, demonstrated improved binding affinities (K ~100
i
nM) over the lead acyclic molecule, YH300 (K = 600 nM). In
i
order to rationalize the tight receptor ligand interaction, we
2
7
exploit modelling studies using MOLOC based on the HSQC
binding data having as template a known coꢀcrystal structure
2
1
(
PDB ID: 3TU1) and the small network analysis using Scorꢀ
28
pion software (Figure 4). It revealed the existence of van der
Waals interactions of the aliphatic handle with Tyr67 and
His73, the expected alignment of the 6ꢀchloroꢀindole moiety
of the designed compounds with the Trp23 pocket, whereas
the 3,4,5ꢀtrifluorophenyl ring occupied the Leu26 hydroꢀ
p53
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p53
phobic pocket. Moreover, the πꢀπ interaction of His96 with the
3
,4,5ꢀtrifluorophenyl fragment and several van der Waals
interactions with Leu54, Ile61, Phe86, Phe91, Val93, His96
and Tyr100 are depicted. These findings support our initial
hypothesis of the divergent hydrophobic handle position comꢀ
Figure 5. Physicochemical properties of the synthesized macroꢀ
cycles, compared with the oral macrocycle marketed drugs on a
hexagon radar graph. The dark gray area contains the low limits
of the oral macrocycle drugs, whereas the light gray the highest
limits.
2
9ꢀ34
pared to staple peptides shown before,
suggesting a new
approach to improve and diversify the extensive collection of
MDM2/X inhibitors.
To analyze and compare the physicochemical properties of
our newly synthesized macrocycles with the orally available
35
ASSOCIATED CONTENT
Supporting Information
macrocycle drugs, we plotted molecular weight, clogP, topoꢀ
logical polar surface area (TPSA), number of hydrogen bond
donors (HBDs) and hydrogen bond acceptors (HBAs) as well
as the number of rotatable bonds (RB) (Figure 5). Interestingꢀ
ly, the values of the properties in most of our macrocycles are
set in the appropriate range, demonstrating the significance of
this new strategy to develop potentially oral bioavailable macꢀ
rocycles targeting p53ꢀMDM2 interaction. In the case of 1c
and 1k, clogP, goes off the limits as expected since we were
targeting a very lipophilic surface. However, this will be overꢀ
come in the future with a strategy to incorporate heteroatoms
The Supporting Information is available free of charge on the
ACS Publications website. General procedures, characterization
data, screening results exemplary copies of NMR (file type, i.e.,
PDF) are included.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +31503633307; fax: +31503637582; Eꢀmail address:
a.s.s.domling@rug.nl (A. Dömling).
(oxygens) on both the acid and isocyanide linker. After this
initial SAR study, we will in the future synthesize libraries of
novel macrocycles as potent p53ꢀMDM2 inhibitors with highꢀ
er diversity and complexity.
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script. / ‡ These authors contributed equally.
Funding Sources
Research in the area of p53ꢀMDM2/X interactions in the Dömling
research group was supported by the US National Institutes of
Health (NIH) (2R01GM097082ꢀ05). Funding has been received
from the European Union's Horizon 2020 research and innovation
programme under MSC ITN “Accelerated Early stage drug dISꢀ
covery” (AEGIS, grant agreement No 675555) and CoFund
ALERT (grant agreement No 665250). N.E.O. was supported by
the Department of Sciences, Technology, and Innovationꢀ
COLCIENCIAS (Colombia).
Figure 4. Small network analysis of 1j (white sticks) modelled
into the MDM2 receptor (PDB ID: 3TU1, cyan sticks): πꢀπ and
van der Waals interactions are shown in orange and yellow dotted
lines, respectively.
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tideꢀAnalogue with Human Mdm2 and Mdmx. Cell Cycle
Georget. Tex 2009, 8 (8), 1176–1184.
Huang, Y.; Wolf, S.; Koes, D.; Popowicz, G. M.; Camacho,
C. J.; Holak, T. A.; Dömling, A. Exhaustive Fluorine Scanꢀ
ning toward Potent p53–Mdm2 Antagonists. ChemMedChem
REFERENCES
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
1)
Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The
Exploration of Macrocycles for Drug DiscoveryꢀAn Underꢀ
exploited Structural Class. Nat. Rev. Drug Discov. 2008, 7
(7), 608–624.
(
(
21)
22)
2
012, 7 (1), 49–52.
(
(
2)
3)
Giordanetto, F.; Kihlberg, J. Macrocyclic Drugs and Clinical
Candidates: What Can Medicinal Chemists Learn from Their
Properties? J. Med. Chem. 2014, 57(2), 278ꢀ295.
Lane, D. P. p53, Guardian of the Genome. Nature 1992, 358
(6381), 15–16.
Neochoritis, C. G.; Wang, K.; EstradaꢀOrtiz, N.; Herdtweck,
E.; Kubica, K.; Twarda, A.; Zak, K. M.; Holak, T. A.; Dömꢀ
ling,
p53/MDM2/MDMX Antagonists. Bioorg. Med. Chem. Lett.
015, 25 (24), 5661–5666.
A.
2,3′ꢀBis(1′HꢀIndole)
Heterocycles:
New
2
(4)
Klein, C.; Vassilev, L. T. Targeting the p53ꢀMDM2 Interacꢀ
tion to Treat Cancer. Br. J. Cancer 2004, 91 (8), 1415–1419.
Vogelstein, B.; Lane, D.; Levine, A. J. Surfing the p53
Network. Nature 2000, 408 (6810), 307–310.
EstradaꢀOrtiz, N.; Neochoritis, C. G.; Dömling, A. How To
Design a Successful p53ꢀMDM2/X Interaction Inhibitor: A
Thorough Overview Based on Crystal Structures.
ChemMedChem 2016, 11 (8), 757–772.
(
23)
Czarna, A.; Beck, B.; Srivastava, S.; Popowicz, G. M.; Wolf,
S.; Huang, Y.; Bista, M.; Holak, T. A.; Dömling, A. Robust
Generation of Lead Compounds for ProteinꢀProtein Interacꢀ
tions by Computational and MCR Chemistry: p53/Hdm2
Antagonists. Angew. Chem. Int. Ed Engl. 2010, 49 (31),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
5)
6)
5
352–5356.
(
(
24)
25)
Huang, Y.; Wolf, S.; Bista, M.; Meireles, L.; Camacho, C.;
Holak, T. A.; Dömling, A. 1,4ꢀThienodiazepineꢀ2,5ꢀDiones
via MCR (I): Synthesis, Virtual Space and p53ꢀMdm2 Activꢀ
ity. Chem. Biol. Drug Des. 2010, 76 (2), 116–129.
Srivastava, S.; Beck, B.; Wang, W.; Czarna, A.; Holak, T.
A.; Dömling, A. Rapid and Efficient Hydrophilicity Tuning
of p53/mdm2 Antagonists. J. Comb. Chem. 2009, 11 (4),
(
(
7)
8)
Khoo, K. H.; Verma, C. S.; Lane, D. P. Drugging the p53
Pathway: Understanding the Route to Clinical Efficacy. Nat.
Rev. Drug Discov. 2014, 13 (3), 217–236.
Zhao, Y.; Aguilar, A.; Bernard, D.; Wang, S. Smallꢀ
Molecule Inhibitors of the MDM2–p53 Protein–Protein Inꢀ
teraction (MDM2 Inhibitors) in Clinical Trials for Cancer
Treatment. J. Med. Chem. 2015, 58 (3), 1038–1052.
Chen, J.; Marechal, V.; Levine, A. J. Mapping of the p53
and Mdmꢀ2 Interaction Domains. Mol. Cell. Biol. 1993, 13
6
31–639.
(26)
Rehm, T.; Huber, R.; Holak, T. A. Application of NMR in
Structural Proteomics: Screening for Proteins Amenable to
Structural Analysis. Structure 2002, 10 (12), 1613–1618.
Gerber, P. R.; Müller, K. MAB, a Generally Applicable
Molecular Force Field for Structure Modelling in Medicinal
Chemistry. J. Comput. Aided Mol. Des. 1995, 9 (3), 251–
268.
Kuhn, B.; Fuchs, J. E.; Reutlinger, M.; Stahl, M.; Taylor, N.
R. Rationalizing Tight Ligand Binding through Cooperative
Interaction Networks. J. Chem. Inf. Model. 2011, 51 (12),
(
9)
(
7), 4107–4114.
(
27)
(10)
Picksley, S. M.; Vojtesek, B.; Sparks, A.; Lane, D. P. Immuꢀ
nochemical Analysis of the Interaction of p53 with MDM2;
Fine Mapping of the MDM2 Binding Site on p53 Using Synꢀ
thetic Peptides. Oncogene 1994, 9 (9), 2523–2529.
(28)
(
(
11)
12)
Shaabani, S.; Neochoritis, C. G.; TwardaꢀClapa, A.;
Musielak, B.; Holak, T. A.; Dömling, A. Scaffold Hopping
via ANCHOR.QUERY: βꢀLactams as Potent p53ꢀMDM2
Antagonists. MedChemComm 2017, 8, 1046ꢀ1052.
Surmiak, E.; Neochoritis, C. G.; Musielak, B.; Twardaꢀ
Clapa, A.; Kurpiewska, K.; Dubin, G.; Camacho, C.; Holak,
T. A.; Dömling, A. Rational Design and Synthesis of 1,5ꢀ
Disubstituted Tetrazoles as Potent Inhibitors of the MDM2ꢀ
p53 Interaction. Eur. J. Med. Chem. 2017, 126, 384–407.
Bista, M.; Wolf, S.; Khoury, K.; Kowalska, K.; Huang, Y.;
Wrona, E.; Arciniega, M.; Popowicz, G. M.; Holak, T. A.;
Dömling, A. Transient Protein States in Designing Inhibitors
of the MDM2ꢀp53 Interaction. Structure 2013, 21 (12),
3
180–3198.
(
29)
Phan, J.; Li, Z.; Kasprzak, A.; Li, B.; Sebti, S.; Guida, W.;
Schönbrunn, E.; Chen, J. StructureꢀBased Design of High
Affinity Peptides Inhibiting the Interaction of p53 with
MDM2 and MDMX. J. Biol. Chem. 2010, 285 (3), 2174–
2
183.
(
(
30)
31)
Baek, S.; Kutchukian, P. S.; Verdine, G. L.; Huber, R.;
Holak, T. A.; Lee, K. W.; Popowicz, G. M. Structure of the
Stapled p53 Peptide Bound to Mdm2. J. Am. Chem. Soc.
2012, 134 (1), 103–106.
Pazgier, M.; Liu, M.; Zou, G.; Yuan, W.; Li, C.; Li, C.; Li,
J.; Monbo, J.; Zella, D.; Tarasov, S. G.; Lu, W. Structural
Basis for HighꢀAffinity Peptide Inhibition of p53 Interacꢀ
tions with MDM2 and MDMX. Proc. Natl. Acad. Sci. 2009,
(13)
(14)
2
143–2151.
Bauer, M. R.; Boeckler, F. M. Hitting a Moving Target:
Targeting Transient Protein States. Structure 2013, 21 (12),
2
095–2097.
1
06 (12), 4665–4670.
(
15)
Beck, B.; Larbig, G.; Mejat, B.; MagninꢀLachaux, M.; Piꢀ
card, A.; Herdtweck, E.; Dömling, A. Short and Diverse
Route toward Complex Natural Productꢀlike Macrocycles.
Org. Lett. 2003, 5 (7), 1047–1050.
(32)
Zhan, C.; Zhao, L.; Wei, X.; Wu, X.; Chen, X.; Yuan, W.;
Lu, W.ꢀY.; Pazgier, M.; Lu, W. An Ultrahigh Affinity Dꢀ
Peptide Antagonist Of MDM2. J. Med. Chem. 2012, 55 (13),
6
237–6241.
(16)
Wessjohann, L. A.; Rivera, D. G.; Vercillo, O. E. Multiple
Multicomponent Macrocyclizations (MiBs): A Strategic Deꢀ
velopment toward Macrocycle Diversity. Chem. Rev. 2009,
(
(
(
33)
34)
35)
Liu, M.; Li, C.; Pazgier, M.; Li, C.; Mao, Y.; Lv, Y.; Gu, B.;
Wei, G.; Yuan, W.; Zhan, C.; Lu, W.ꢀY.; Lu, W. DꢀPeptide
Inhibitors of the p53ꢀMDM2 Interaction for Targeted Moꢀ
lecular Therapy of Malignant Neoplasms. Proc. Natl. Acad.
Sci. U. S. A. 2010, 107 (32), 14321–14326.
Chee, S. M. Q.; Wongsantichon, J.; Soo Tng, Q.; Robinson,
R.; Joseph, T. L.; Verma, C.; Lane, D. P.; Brown, C. J.;
Ghadessy, F. J. Structure of a Stapled Peptide Antagonist
Bound to NutlinꢀResistant Mdm2. PLoS ONE 2014, 9 (8),
e104914.
1
09 (2), 796–814.
(
17)
Dömling, A. P53ꢀmdm2 Antagonists. Patent Application
WO 2012/033525 A3, 2012.
(18)
Dömling, A.; Holak, T. Novel p53ꢀmdm2/p53ꢀmdm4 Anꢀ
tagonists to Treat Proliferative Disease. Patent application
WO2011106650 A3, 2012.
(
(
19)
20)
Neochoritis, C. G.; ZarganesꢀTzitzikas, T.; Stotani, S.;
Dömling, A.; Herdtweck, E.; Khoury, K.; Dömling, A.
Leuckart–Wallach Route Toward Isocyanides and Some
Applications. ACS Comb. Sci. 2015, 17 (9), 493–499.
Czarna, A.; Popowicz, G. M.; Pecak, A.; Wolf, S.; Dubin,
G.; Holak, T. A. High Affinity Interaction of the p53 Pepꢀ
Villar, E. A.; Beglov, D.; Chennamadhavuni, S.; Jr, J. A. P.;
Kozakov, D.; Vajda, S.; Whitty, A. How Proteins Bind Macꢀ
rocycles. Nat. Chem. Biol. 2014, 10 (9), 723–731.
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Lay summary
In nature, macrocycles are not uncommon and often exhibit interesting biological activities as preferable entropic signaꢀ
ture, better membrane permeation and oral bioavailability or higher stability. The tumor suppressor protein p53 is involved in
controlling pathways of cell cycle, apoptosis, angiogenesis metabolism, senescence and autophagia. Multiple potent and
selective compound classes to inhibit the p53ꢀMDM2 interaction have been discovered, described and evaluated in early
clinical trials.
Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial
macrocyclic inhibitors of the p53ꢀMDM2 interaction was designed and synthesized. These macrocycles, alternatively to
stapled peptides, target the large hydrophobic surface area formed by Tyr67, Gln72, His73 Val93 and Lys94 yielding derivaꢀ
tives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence
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polarization (FP) assay and Hꢀ N 2D HSQC NMR experiments.
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