Hitting on the move: Targeting intrinsically disordered protein states of the MDM2-p53 interaction

Article abstract of DOI:10.1016/j.ejmech.2019.111588

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal ‘‘lid’’ segment of MDM2 from the proximity of the p53-complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds.

Full text of DOI:10.1016/j.ejmech.2019.111588

Journal Pre-proof
Hitting on the move: Targeting intrinsically disordered protein states of the MDM2-p53
interaction
Constantinos G. Neochoritis, Jack Atmaj, Aleksandra Twarda-Clapa, Ewa Surmiak,
Lukasz Skalniak, Lisa-Maria Köhler, Damian Muszak, Katarzyna Kurpiewska, Justyna
Kalinowska-Tłuścik, Barbara Beck, Tad A. Holak, Alexander Dömling
PII:
S0223-5234(19)30722-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.111588
EJMECH 111588
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 May 2019
Revised Date: 22 July 2019
Accepted Date: 4 August 2019
Please cite this article as: C.G. Neochoritis, J. Atmaj, A. Twarda-Clapa, E. Surmiak, L. Skalniak, L.-
M. Köhler, D. Muszak, K. Kurpiewska, J. Kalinowska-Tłuścik, B. Beck, T.A. Holak, A. Dömling, Hitting
on the move: Targeting intrinsically disordered protein states of the MDM2-p53 interaction, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111588.
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Hitting on the move: targeting intrinsically disordered protein states of
the MDM2-p53 interaction
a,†
a,b
b
b
Constantinos G. Neochoritis, Jack Atmaj, Aleksandra Twarda-Clapa, Ewa Surmiak, Lukasz
b
c
b
b
Skalniak, Lisa-Maria Köhler, Damian Muszak, Katarzyna Kurpiewska, Justyna Kalinowska-
b
c
b
a,*
Tłuścik, Barbara Beck Tad A. Holak, Alexander Dömling
a
Department of Pharmacy, Drug Design group, University of Groningen, Antonius Deusinglaan 1, 9700 AD
Groningen, The Netherlands
b
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Krakow, Poland
c
Department of Internal Medicine III, Klinikum der Universität München, Munich, Germany; Clinical Co-Operation
Group Immunotherapy at the Helmholtz Institute Munich, Munich, Germany
†
Current affiliation: Department of Chemistry, University of Crete, 70013, Heraklion, Greece
1

ABSTRACT
Intrinsically disordered proteins are an emerging class of proteins without a folded structure and
currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell
division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53
recognition is a dynamic and multistage process that amongst other, employs the dissociation of
a transient α-helical N-terminal ‘‘lid’’ segment of MDM2 from the proximity of the p53-
complementary interface. Several small molecule inhibitors have been reported to inhibit the
formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19,
Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point
pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the
binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including
chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-
specific anti-cancer activity towards p53-wild-type cancer cells was observed for several
representative compounds.
Keywords: Intrinsically disordered proteins, p53-MDM2, 4-point pharmacophore model, Ugi
1
15
reaction, SAR analysis, AnchorQuery, H- N 2D HSQC NMR, cancer
2

INTRODUCTION
Drug discovery remains slow, expensive and unreliable. Although immense progress in
individual whole genome sequencing for personalized medicine has provided a wealth of novel
drug targets, very few small molecule drugs for post-genomic identified targets are currently in
development.[1–3] A major hurdle to the efficient and fast generation of novel drugs is the slow
and traditional approach performed in early drug discovery and development. The rationale
behind modern drug design is based on all the theoretical and experimental knowledge of the
particular system under investigation. Of outmost importance is considering the dynamical
nature of the proteins. Proteins are flexible entities, thus move. The determined conformations of
a protein often differ in its ligand-bound and unbound forms. Protein conformational changes
could open new ligand binding sites, whose exploration is a key to fully access the efficacy of a
drug as well as to identify non-specific targeting with possible undesired effects.[3–7]
Intrinsically disordered protein states (IDPs) are an emerging family of proteins whose most
characteristic feature is that they don’t present a folded structure, and thus challenge the
sequence-structure-function paradigm.[8–10] This lack of stable structure, over the entire protein
length or in some regions (Intrinsically Disordered Regions or IDRs), provides them a structural
plasticity which is not achievable by ordered proteins that is essential to carry out their cellular
function.
The tumor-suppressor protein p53 is the principal regulator of cell division and growth, as it is
able to control genes that are implicated in cell-cycle control, apoptosis, angiogenesis,
senescence and autophagia.[11–13] The TP53 gene is one of the most frequently mutated genes
in a multitude of human cancer.[14] Additionally, in most cases where TP53 is intact, p53’s
function is impaired by its negative regulators, mouse double minute 2 and 4 homologues
3

(MDM2 and MDMX), due to amplification or enhanced expression of their coding
genes.[11,12,14] MDM2 functions as an E3 ligase leading to p53-ubiquitilation and then
proteasomal inactivation. The MDM2-p53 recognition is a dynamic and multistage process that
employs the binding-induced folding of p53,[15–18] the stepwise p53 induced MDM2 binding
pocket formation starting with pocking of p53-Trp23 into MDM2,[19] the rearrangement of the
Leu26 subpocket of MDM2 by a twist of the Tyr100 ring from the ‘‘closed’’ to the ‘‘open’’
(anti)conformations,[15,20–23] and the dissociation of a transient α-helical N-terminal ‘‘lid’’
segment of MDM2 (residues 19–23) from the proximity of the p53-complementary
interface.[24–28] Several potent small molecules are currently in clinical trials, which are direct
antagonists of the p53-MDM2 interaction and which mimic the p53 hot-spot triad
1
9
23
26
Phe Trp Leu described with the classical three finger pharmacophore model.[29–31] In more
detail, the great majority of the small molecule inhibitors of the p53-MDM2 interaction target the
same “closed” Tyr100 state and are incapable of reaching the N-terminal “lid” segment, an
intrinsically disordered region of MDM2 (Figure 1,A).[32–36] However some time ago,[37] we
were able to discover, for the first time, an inhibitor that could bound to the “open” Tyr100
conformation leading to a four point pharmacophore interaction (Figure 1,B).[38]
A.
B.
Figure 1. (A) Alignment of the two co-crystal structures demonstrating the closed (cartoon, magenta) and open lid
gray surface); (B) Alignment of the two inhibitors YH119 (PDB ID 3TJ2) and YH300 (PDB ID 4MDN).
(
4

In this way, we were able to transit from a 3-point pharmacophore model (YH119, PDB ID
TJ2) to a 4-point (YH300, PDB ID 4MDN), increasing the binding affinity by almost three
3
times (Figure 2,A). The co-crystal structure with the inhibitor YH300 surprisingly, but clearly,
indicated that the 4-chlorobenzyl phenyl ether moiety filled the Leu26 and an induced subpocket,
the tert-butyl group occupied the Phe19 pocket and the anchoring 6-chloroindole, by default,
filled the Trp23 pocket (Figure 2,B). Herein, with all this information in hand, we performed a
thorough SAR analysis (Figure 2,C), mostly focusing on the Leu26 and the newly presented
th
induced sub cleft (4 pharmacophore) which was evaluated with two different biorthogonal
assays, followed by cell-based studies using p53-wild-type and p53-deleted cells. The results
were rationalized by further modeling studies.
A.
B.
C.
5

Figure 2. (A) The transition from the 3-point to 4-point interaction point with increasing binding affinity; (B) the
binding mode of the YH300; (C) SAR analysis studies evaluated with two different biorthogonal assays presented in
the current manuscript.
RESULTS AND DISCUSSION
Design and synthesis
In continuation of our previous work on successfully developing potent inhibitors of the p53-
MDM2/X interaction, we demonstrate an effective strategy of scaffold hopping (Figure 3).[39–
4
4] As a design tool we used our open access pharmacophore-based virtual screening web-
platform AnchorQuery™ (http://anchorquery.csb.pitt.edu),[16,45] which is a specialized
pharmacophore search technology that brings interactive virtual screening of novel protein-
protein inhibitors to the desktop. It leverages the concept of anchors, amino acid residues that
bury a large amount of solvent accessible surface area at the protein-protein
interface.[16,39,43,45] Every compound in our multi-component reaction (MCR) accessible
virtual library contains an anchor analog, a functional group that is a chemical mimic of a
specific amino acid. AnchorQuery™ pharmacophore queries always include an anchor feature in
addition to the standard hydrophobic, ionic and hydrogen bond donors. The utilization of
convergent MCR chemistry allowed us for a rapid SAR analysis and discovery of potent
compounds that could eventually serve as an excellent starting point for further development of
pharmaceutically relevant p53-MDM2 interaction inhibitors.
6

Figure 3. Potent inhibitors of p53-MDM2/X interaction discovered by our group; Scaffold hopping employing
different MCR scaffolds via AnchorQuery™. The light blue color on the axis corresponds to the design and
synthesis of inhibitors based on the 3-point interaction mode, whereas the dark blue on the 4-point.
We examined the possibility of a halogen bond, therefore we employed a variety of electron
donating and withdrawing substituents and we utilized different heteroatoms or linkers’ size. In
addition, in order to verify our hypothesis and the co-crystal structure of YH300 (PDB ID
4
MDN), we modified the anchoring indole moiety, we replaced the formyl with acetyl group and
we deformylated our adducts to examine how the binding affinity would be affected. We also
probed the Phe19 pocket by replacing the tert-butyl moiety with other bulky, aliphatic groups.
Finally, we performed hydrolysis and amidation reactions of the ester group (Figure 2,C). Hence,
an equimolar mixture of suitable substituted benzylamines 1, the 6-chloro-indole carboxaldehyde
2
, the corresponding isocyanides 3 and carboxylic acids 4, was stirred in MeOH at rt for 48 h
yielding the Ugi-4CR adducts 5. Τhe importance of the carboxylate moiety was taken into
account, as found in previous MDM2 binders, since -CO H group contributes to the formation of
2
7

several hydrogen bonds with water molecules interacting with MDM2.[37,39,46] For that
reason, the hydrolysis with LiOH in water-ethanol (1:1) of the ester group was followed,
affording the targeted compounds 6 (Scheme 1).
Scheme 1. Reactions and conditions of the general synthetic strategy; i) rt, 48 h, MeOH (1 M), ii) LiOH (10.0 eq),
rt, 24 h, H O-EtOH (1:1).
2
Probe of Leu26 and induced pocket
The cocrystal structure of MDM2-YH300 (PDB ID 4MDN) revealed that the large 4-
chlorobenzyl phenyl ether filled the enlarged Leu26 new subpocket (induced pocket, blue color,
Figure 4,A). Due to the presence of several carbonyl oxygens of a series of amino acids (Gln24,
Ala21, Glu23, Pro20, Ile19, His96 and Ile99) in a range of 3.5-4.0 Å from the benzyl group, we
suspected the possibility of involving halogen bonding (Figure 4,B).[47] Thus, we started our
structure-activity relation studies by performing an exhaustive halogen scanning around the
benzyloxy ring.
8

A.
B.
Figure 4. (A) A close-up view of the YH300 with the 4 subpockets visible; (B) Halogen bonding to 6 (red dotted
lines) and surrounding carbonyl oxygens of amino acids in a 3.5-4.0 Å range.
To explore this Leu26 and its induced subpocket, we utilized the elongated halogen-substituted
benzylamines 1a-t which were easily accessible either via Williamson or Mitsunobu reaction
from the corresponding 4-hydroxybenzylamine (Scheme 2,A). For easier handling and
purification, the corresponding elongated benzylamines were used as hydrochloric or triflic salts
in the next steps (Scheme 2,A,B). The aldehyde 2 was synthesized from the 6-chloro-indole
derivative using the Vilsmeier-Haack formylation reaction.[41–43]
Scheme 2. (A) Reactions and conditions: i) (Boc)
2
3 2 2 2 2 3
O, NaHCO , rt, MeOH, or (Boc) O, rt, H O, ii) RCH X, K CO ,
o
reflux, MeCN or iii) RCH OH, DIAD or DEAD, PPh , 0 C-rt, THF; (B) Molecular geometry observed in the
2
3
crystal structures of 1m (CCDC 1848075), showing the atom labeling scheme. Displacement ellipsoids of non-
hydrogen atoms are drawn at the 30% probability level. H atoms are presented as small spheres with an arbitrary
radius; (C) Reactions and conditions: i) NaH, rt, DMF; ii) LiAlH , reflux, THF; (D) Molecular geometry observed in
4
the crystal structures of 1sa (CCDC 1848076), showing the atom labeling scheme. Displacement ellipsoids of non-
hydrogen atoms are drawn at the 30% probability level. H atoms are presented as small spheres with an arbitrary
radius
9

A.
B.
H
2
N
BocHN
2
H N
ii or iii
i
R
OH
OH
O
1a-ai, 36-95%
C.
D.
F
F
F
F
Ph
O
F
F
i, ii
OH
+
O
F
NH
2
CN
1s, 79%
CN
Consequently, the U-4CR of the benzylamines 1a-t, 6-chloro-indole carboxaldehyde 2, tert-butyl
isocyanide and formic acid afforded compounds 5a-t which were hydrolyzed towards the acids
6
a-t (Scheme 3). We synthesized not only the corresponding para-substituted fluoro, bromo and
iodo-based derivatives (e.g. 6a, 6k, 6n) but also on all the other positions, including few
disubstituted halogen compounds (e.g. 6d, 6h, 6j). In addition, we enriched our SARs studies by
3
employing -CF substituted derivatives (6r) and suitably halogen-substituted derivatives on the
phenoxy group (Leu26 pocket, Figure 4, magenta color, Scheme 2,C) as indicated by the crystal
structure analysis (Figure 4,B e.g. 6s). We also synthesized the non-halogen substituted
derivative 5t which served as a “control” compound in order to compare the binding affinities.
10

Scheme 3. Reactions and conditions: i) rt, 48 h, MeOH (1 M), ii) LiOH (10.0 eq), rt, 24 h, H O-EtOH (1:1)
2
11

Two complementary assays based on independent physicochemical principles, HSQC NMR
and fluorescence polarization (FP) were used to exclude false positive hits. FP assay was
employed to determine the inhibitory affinities (K ) of the derivatives against MDM2/X as
i
previously described.[48] Halogen bonding has been known for decades, but in drug design the
utility of these interactions is rather a random finding than rational. The halogen bond can be
formed between a halogen and any accessible Lewis base in the binding pocket, where in most of
the cases the most prominent Lewis base is the backbone carbonyl oxygen of an amino acid. Side
chain oxygens of serine, threonine, tyrosine, aspartate, glutamate, asparagine and glutamine and
nitrogen of histidine have been also occasionally engaged in halogen bonding.
The FP results from the halogen dancing exercise (Table 1) indicate the possibility of halogen
bond existence. It can be seen that the binding affinity increases from the p-substituted fluorine
derivative to the corresponding iodine i.e. K = 1.02 µΜ, 0.60 µΜ, 0.39 µΜ and 0.32 µΜ,
i
respectively (entries 1, 5, 11, 16). The same correlation applies to the o-substituted halogens (o-
F<o-Cl<o-Br<o-I), giving even higher binding affinities (K = 0.74 µΜ, 0.36 µΜ, 0.07 µΜ and
i
0
.05 µΜ, respectively (entries 3, 7, 13, 18)). Clearly, these trends show a size-dependent activity,
driven by the σ-hole, relationship being in accordance with literature.[47,49]
In m-substituted derivatives this trend, by the exception of 6o (entry 17, K = 0.43 µΜ), can also
i
be identified. Thus, the affinity of m-F derivative (6b) is similar to m-Cl (6f, entries 2 and 6),
which is increased by the m-disubstituted (6d, entry 4) and m-Br (6l, entry 12), i.e. K = 0.6 µΜ,
i
0
.42 µΜ and 0.30 µΜ, respectively. Moreover, the substitution of R = H (6t, entry 22, K = 0.76
i
µΜ) by heavier halogens (Br, Cl, I) both in para, meta and ortho position enhances the halogen
bond as expected and increases the affinity.
12

The combination of other disubstituted chloro derivatives as the meta and para substituted (6h,
entry 8) or ortho and para substituted (6i, entry 9) gave increased binding affinities comparing
with the corresponding para (6e, entry 5) and meta (6f, entry 6) monosubstituted derivatives, i.e
K = 0.26 µΜ and 0.37 µΜ compared to 0.60 µΜ and 0.63 µΜ, respectively. The di-ortho
i
substituted chloro derivative (6j, entry 10) found to be rather inactive (K = 4.7 µΜ).
i
3
Other halogenated ligands as the –CF moieties were also employed (entries 19 and 20) showing
affinities of 0.24 µΜ and 1.6 µΜ for the mono ortho-substituted and di meta-substituted
derivative, respectively. Again, the halogenated substituent on ortho position gave the highest
binding affinity similar to ortho-substituted chloro derivative. The difluoro substituted on the
phenoxy group derivative (6s) resulted in loss of the activity (entry 21). Figure 5 shows a binding
affinity plot in comparison with the σ-hole of the used halogen (see also SI, Figure S4).
The ortho-substituted bromo (6m) and iodo derivatives (6p) have shown the strongest inhibition
with K of 70 nM and 34 nM, respectively. It has also to be stated that the strength of halogen
i
bonds is really dependent on the geometry of the interaction. Sometimes, stronger type of
interactions or networks of synergistic interactions may displace the halogen from its favored
orientation in terms of distance (between the halogen and Lewis base), σ-hole angle (between the
covalent bond of the halogen and the halogen bond with the Lewis base) and the spatial
orientation (torsion angle based on the bonding situation of the Lewis base).[47]
13

Figure 5. Binding affinity dependence on the σ-hole of the halogen; The trend of F<Cl<Br<I is observed in the
para- (blue), meta- (red) and ortho-substitution (green). The blue spot depicts compound 6t (R = H) whereas other
disubstituted compounds are also depicted. Ortho-substitution with heavy halogens (Br, I) gave the best binding
affinities.
Compounds 5a-t (ester form) were found to be rather inactive in accordance with our previous
observations.[41–44] Due to both the plausible metabolic instability and the relatively higher
molecular weight of the iodo compound 6p, compound 6m was selected to be further
investigated with the two enantiomers separated. Indeed, the compound 5m (ester form) was
subjected to chiral separation providing compound (+)-5m (enantiomer 1) and (-)-5m
(enantiomer 2), which were subsequently hydrolyzed to the corresponding acids.
Scheme 4. Reactions and conditions: i) chiral separation by supercritical fluid chromatography, ii) LiOH (10.0 eq),
rt, 24 h, H O-EtOH (1:1). Enantiomer 1 was eluted first by the chiral column and enantiomer 2 followed (see
2
Supporting information, SI), stereochemistry is randomly assigned.
As expected, one enantiomer showed increased binding affinity comparing with the racemic
mixture (K = 0.07 µΜ) when it was tested by the FP assay. The enantiomer 1 ((+)-6m)
i
demonstrated a K of 0.140 µΜ and the enantiomer 2 ((-)-6m) a K of 34 nM (entries 14 and 15).
i
i
14

Table 1. Activities of Ugi-4CR based inhibitors of p53-MDM2/X interaction.
Yield of
Yield of
K [µM] for
i
Entry
Code
Structure
a
compound 5 (%) compound 6 (%)
MDM2
1
6a
70
71
76
68
1.02
0
.62
2
6b
6c
6d
(
6.30)
3
4
5
6
68
59
49
62
71
68
75
50
0.74
0
.42
(
6.03)
6
e
0.60
(
YH300)
0
.63
6f
(
6.21)
15

0
.36
7
8
6g
6h
69
71
60
65
(
(
4.67)
0
.26
4.73)
0
.37
9
6i
6j
68
45
59
58
(
7.05)
1
0
1
4.67
0
.39
1
6k
53
67
(
6.01)
Br
O
0
.30
O
1
1
2
3
6l
70
70
53
49
HN
N
(3.63)
O
CO2H
Cl
N
H
0
.07
6m
(5.45)
16

0
.14
1
1
4
5
(+)-6m
-
-
Enantiomer 1
(
-)-6m
0.03
(
DN285)
Enantiomer 2
0
.32
1
1
6
7
6n
46
45
50
40
(
(
4.74)
0
.43
6o
4.74)
0
.05
1
1
2
8
9
0
6p
6q
6r
40
65
64
50
72
73
(
(
6.39)
0
.24
4.01)
1.63
17

2
2
1
2
6s
6t
65
53
65
72
11.6
0
.76
(
5.36)
a
Data in parentheses refers to MDMX protein whenever the compound showed any significant activity (all
compounds were tested against MDM2 and MDMX). K values were calculated based on fluorescence polarization
i
binding assay (see SI).
Besides the halogen scan, we synthesized derivatives with a variety of electron withdrawing and
donating groups (EWG and EDG); we employed aromatic moieties as biphenyl, naphthalene,
heterocycles (pyridine and thiophene derivatives), aliphatic rings (cyclohexyl, pentyl) and other
donating groups as –Me or –OMe. Furthermore, we examined strong withdrawing groups as –
CN or –NO . Finally, we evaluated the size of the linker by adjusting the length of the benzyl
2
phenyl ether group (Scheme 5).
18

Scheme 5. Reactions and conditions: i) rt, 48 h, MeOH (1 M), ii) LiOH (10.0 eq), rt, 24 h, H O-EtOH (1:1).
2
O
O
O
O
O
O
O
O
O
O
O
O
O
HN
HN
HN
HN
HN
N
N
N
N
N
O
O
O
O
O
CO2H
CO2H
CO2H
CO2H
2
CO H
Cl
N
Cl
N
H
Cl
N
H
Cl
N
H
Cl
N
H
H
6u
6v
6w
6x
6y
O2N
S
NC
O
O
O
O
O
O
O
O
N
O
O
HN
HN
HN
HN
N
HN
N
N
N
O
O
O
O
O
CO2H
CO2H
CO2H
CO2H
CO2H
Cl
N
H
Cl
N
Cl
N
Cl
N
H
Cl
N
H
H
H
6z
6aa
6ac
6ad
6ab
N
N
N
Br
O
O
O
O
O
O
O
O
O
O
N
HN
HN
HN
N
HN
N
HN
N
N
O
O
O
O
O
CO2H
CO2H
CO2H
CO2H
CO2H
Cl
Cl
N
H
N
Cl
Cl
N
N
Cl
N
H
H
H
H
6af
6ag
6ah
6ai
6ae
19

th
Extending towards the 4 pharmacophore point with biphenyl or naphthalene substituents (6u,v,
entries 1 and 2, Table 2), resulted also in relatively good binders with K of 0.57 µΜ and 0.42
i
µΜ, respectively. Similar results were also obtained with EDG as 3-methoxy (6w, entry 3) or
2
,3-dimethoxy substituents (6x, entry 4), giving K of 0.50 µΜ and 0.52 µΜ, respectively.
i
Aliphatic substituents as cyclohexyl or cyclopentyl and strong EWG as 4-cyano or 4-nitro
resulted in an actual loss of activity (entries 6-9). Replacement of the benzylic phenyl group with
heterocycles as 3-thiophene (entry 10) and pyridine moieties (entries 11-13) abolished the
activity or did not improve the affinity. It seems that the halogen substitution is ideal to target the
fourth pharmacophore point. Next, we wanted to examine the size of the linkers; thus we added
one more methylene on the benzyloxy phenyl group yielding compound 6ah (entry 14). Indeed,
the affinity was increased with a K of 0.26 µΜ compared with compound 6t (K = 0.76 µΜ).
i
i
However, this was not the case when we increased by one methylene the linker next to the
phenol moiety, resulting compound 6ai (entry 15, K = 4 µΜ) with loss of activity in comparison
i
with compound 6k (K = 0.39 µΜ).
i
Table 2. Activities of Ugi-4CR based inhibitors of p53-MDM2/X interaction.
Yield of
Yield of
K [µM] for
i
Entry
Code
Structure
a
compound 5 (%)
compound 6 (%)
MDM2
0
.57
1
6u
54
65
55
65
(
(
3.96)
0
.42
2
6v
5.78)
20

0
.50
3
4
6w
70
71
70
71
(
5.74)
6x
0.52
5
6
6y
6z
39
65
39
65
0.72
5.28
7
8
6aa
6ab
89
41
89
41
1.68
2.24
9
6ac
85
85
N/A
21

S
N/A
O
(the ester form 5ad
was found
O
1
1
0
1
6ad
6ae
61
41
60
41
HN
N
to be active, K =
i
O
CO2H
Cl
N
3.68 µΜ)
H
11.9
1
1
2
3
6af
50
50
50
50
N/A
2.54
6ag
1
1
4
5
6ah
6ai
57
51
49
55
0.26
4.23
a
Data in parentheses refers to MDMX protein whenever the compound showed any significant activity (all
compounds were tested against MDM2 and MDMX). N/A - no activity against MDM2 protein. K values were
i
calculated based on fluorescence polarization binding assay (see SI).
22

Replacement of oxygen as heteroatom linker
In continuation of our exploration of the Leu26 and the induced subpocket, we replaced the
oxygen-linker to a nitrogen (Scheme 6); A -NH in that position, as hydrogen donor, could be
beneficial. We performed an U-4CR with the 4-nitrobenzylamine, followed by an in situ
reductive amination yielding, after hydrolysis of the ester group, compound 8. However, the
replacement of the oxygen linker with the hydrogen bond donor -NH resulted in loss of activity
i i
(SI, entry 39, K = 1.9 µΜ compared with compound 6k, K = 0.39 µΜ). Moreover, compound 8
while in solution, was found to be susceptible to degradation.
Scheme 6. Reactions and conditions: i) rt, 48 h, MeOH (1.0 M), ii) Zn/CH CO H, 4-bromobenzaldehyde, rt, MeOH
3
2
2
iii) LiOH (10.0 eq), rt, 24 h, H O-EtOH (1:1).
Probe of Trp23 (change in anchor)
Initially, indole fragment was taken as starting point to mimic tryptophan residue, where an
important hydrogen bond was observed. The starting point for our antagonist discoveries was the
anchoring side chain of tryptophan embedded in a deep hydrophobic pocket formed by the
residues Leu57, Phe86 and Ile99 using ANCHOR.QUERY.[37,39,50,51] Indeed, the -NH group
of the 6-chloroindole group participates in a hydrogen bond with the carbonyl oxygen of Leu54
and the indole scaffold assumes an orientation almost identical to the Trp side chain in the native
p53−MDM2 complex, while the chlorine atom stabilizes the interaction at the bottom of the
23

hydrophobic cleft. Studies by Garcia-Echeverria et al.[39] on a p53-derived linear octapeptide
showed that a Trp23 to (6-Cl) Trp substitution gave rise to a 63-fold increase in affinity for
MDM2. Accordingly, we synthesized few examples to prove our rationale (Figure 4,A, yellow
color). Firstly, we synthesized a methylated on the indole –NH derivative (9) by using the
corresponding N-methylated 6-chloroindole aldehyde in the Ugi reaction. Secondly, we
completely switched the indole core to other chloro-substituted phenyl rings (10, 11, Figure 6).
The absence of the free –NH of the indole scaffold (due to the methylation), led to total loss of
activity. Likewise, the replacement of the indole core with 3- or 4-chlorosubstituted phenyl
groups as anchors resulted in no binding.
Cl
O
HN
N
O
Cl
1
1, 90%
Figure 6. Probe of the subpocket Trp23; Change of the anchor with the methylated 3-indole carboxaldehyde (9), p-
chlorobenazaldehyde (10) and m-chlorobenzaldehyde (11, CCDC 1847731).
Amidation
As it was aforementioned, the ester form of the majority of the compounds that were tested
was found inactive to the contrary of the corresponding acids. Τo potentially improve the
properties of our compounds, we also converted the ester group of the derivative 5m into the
corresponding better water soluble amide 12 with a one-step DBU-catalyzed amidation
procedure (Scheme 7).[44,52] In our previous studies, amidation of the indole-2-carboxylate
24

offered improved water solubility.[51] However, the corresponding amidation with the
morpholino moiety resulted in loss of activity (8 µΜ).
Scheme 7. Reactions and conditions: (i) 2-Morpholinoethylamine, DBU, reflux overnight, THF.
Probe of Phe19
The cocrystal structure of YH300 revealed that the Phe19 subpocket is occupied by the tert-
butyl group with van der Waals contacts with Ile61 and Val93 (Figure 7). By close inspection of
the crystal structure, it seems that a bulky aliphatic group is necessary to address all these
hydrophobic interactions (Figure 4,A, red color). Therefore, we probed this particular subpocket
by employing bulky, aliphatic isocyanides as 1- and 2-adamantyl moieties and camphor (exo-
endo mixture) (Figure 7). These isocyanides were prepared by the classical dehydration of the
3
corresponding formamide using POCl .[53,54]
25

Figure 7. Probe of the subpocket Phe19; Utilization of bulky, aliphatic isocyanides.
The replacement of tert-butyl group with 1-adamantyl resulted almost in loss of activity (13, 17,
entries 1 and 5, Table 3) but the replacement with 2-adamantyl gave a binding affinity of 6 nM
(
14, entry 2) compared with compound 6m (K = 70 nM). It seems that this bulky moiety
i
stabilizes the extended hydrophobic region by the His73, Val93, and Lys94. Increased binding
affinities were also achieved by employing the camphor moiety in compounds 15 (entry 3) and
1
6 (entry 4), giving K of 61 nM and 6 nM compared with compounds 6k and 6m respectively.
i
Table 3. Activities of Ugi-4CR based inhibitors of p53-MDM2/X interaction.
Yield of ester Yield of acid
K [µM] for
i
Entry
Code
Structure
a
(
%)
(%)
MDM2
1
13
40
74
43
3.44
1
4
0.006
(1.68)
2
89
(
DN389)
26

0
.06
3
4
15
47
51
53
55
(
1.55)
1
6
0.006
(1.95)
(
DN381)
5
17
44
46
N/A
a
Data in parentheses refers to MDMX protein whenever the compound showed any significant activity (all
compounds were tested against MDM2 and MDMX). N/A - no activity against MDM2 protein. K
calculated based on fluorescence polarization binding assay (see SI).
i
values were
Exploitation of acid moiety/deformylation
The formyl group does not participate in any sub cleft of the MDM2 and it’s rather exposed to
the hydrophilic part of the protein. For this reason, in order to exploit the effect of this particular
group, we initially cleaved it (18, 19) by treating it with HCl (5N in dioxane) and then
substituted with a methyl group (20, 21). We synthesized both the corresponding esters and acids
(Scheme 8,A). Moreover, we exchanged the formyl to the acetyl group by simply changing the
acid component in our U-4CR (22, 23, Scheme 8,B).
27

Scheme 8. (A) Reactions and conditions: i) HCl (5 N in dioxane), reflux, H O, 1 h, ii) LiOH (10.0 eq), rt, 24 h,
2
H O-EtOH (1:1), iii) MeI, K CO , THF, rt, overnight; (B) Replacement of the formyl group with acetyl
2
2
3
A.
B.
Deformylation, leaving a free –NH group, resulted in decrease of the affinity (K = 1.85 µΜ, SI,
i
entry 50). In addition, replacement of the formyl group either by a methyl or acetyl group
resulted in loss of activity. Although the formyl group does not seem to participate in the binding
mode of the current small molecules, it is exposed to the hydrophilic surface of the pocket and
stabilizes better the interaction of the small molecule and MDM2.
28