2,4,5-Tris(alkoxyaryl)imidazoline derivatives as potent scaffold for novel p53-MDM2 interaction inhibitors: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2019.06.007

Imidazoline-based small molecule inhibitors of p53-MDM2 interaction intended for the treatment of p53 wild-type tumors are the promising structures for design of anticancer drugs. Based on fragment approach we have investigated a key role of substituents

Full text of DOI:10.1016/j.bmcl.2019.06.007

Accepted Manuscript
2,4,5-Tris(alkoxyaryl)imidazoline derivatives as potent scaffold for novel p53-
MDM2 interaction inhibitors: design, synthesis, and biological evaluation
Daniil R. Bazanov, Nikolay V. Pervushin, Victoria Yu. Savitskaya, Lada V.
Anikina, Marina V. Proskurnina, Natalia A. Lozinskaya, Gelina S. Kopeina
PII:
S0960-894X(19)30374-9
https://doi.org/10.1016/j.bmcl.2019.06.007
BMCL 26485
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 April 2019
4 June 2019
5 June 2019
Please cite this article as: Bazanov, D.R., Pervushin, N.V., Yu. Savitskaya, V., Anikina, L.V., Proskurnina, M.V.,
Lozinskaya, N.A., Kopeina, G.S., 2,4,5-Tris(alkoxyaryl)imidazoline derivatives as potent scaffold for novel p53-
MDM2 interaction inhibitors: design, synthesis, and biological evaluation, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.007
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
2,4,5-Tris(alkoxyaryl)imidazoline derivatives as potent scaffold for novel p53-
MDM2 interaction inhibitors: design, synthesis, and biological evaluation
Daniil R. Bazanova, Nikolay V. Pervushinb, Victoria Yu. Savitskayaa, Lada V. Anikinac, Marina V.
Proskurninaa,c, Natalia A. Lozinskayaa,c,* and Gelina S. Kopeina b,*
aDepartment of Chemistry, M. V. Lomonosov Moscow State University, 1, Leninskie Gory, 119992, Moscow, Russian Federation
bDepartment of Medicine, M.V. Lomonosov Moscow State University, 1, Leninskie Gory,119991 Moscow, Russian Federation
cInstitute of Physiologically Active Substances of RAS, 1 Northern Passage, 142432, Moscow Region, Russian Federation, *Corresponding author at: MV
Lomonosov Moscow State University, 119991 Moscow, Russia
E-mail address: lirroster@gmail.com (Kopeina G.S.), natalylozinskaya@mail.ru (Lozinskaya N.A.)
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Imidazoline-based small molecule inhibitors of p53-MDM2 interaction intended for the
treatment of p53 wild-type tumors are the promising structures for design of anticancer drugs.
Based on fragment approach we have investigated a key role of substituents in cis-imidazoline
core for biological activity of nutlin family compounds. Although the necessity of the
substituents in the phenyl rings of cis-imidazoline has been shown, there are no studies in which
the replacements of a halogen by other substituents have been investigated. A series of simple
cis-imidazoline derivatives containing halogen, hydroxy and alkoxy-substituents were
synthesized. The biological activity of the compounds was studied using assays of cytotoxicity
(MTT) and p53 level. It was found that the hydroxyl-derivatives were not cytotoxic whereas the
alkoxy analogues were the same or more active as halogen-substituted compounds in cell
viability test. The synthesized alkoxy derivatives induced an increase of p53 level and did not
promote necrotic cell death in the concentration up to 40 µM.
Keywords:
imidazolines
nutlin analogues
anticancer
synthetic design
fragmentary approach
p53-MDM2 system represents a unique class of protein−protein interaction (PPI) for target therapy in cancer studies1–3. p53 tumor
suppressor is a principal mediator of cell cycle arrest, senescence, and apoptosis in response to a broad array of cellular damage4. In
normal unstressed cells, p53 is a very unstable protein, the cellular level of which is low enough due to its degradation largely mediated
by MDM21. Through three inhibitory mechanisms, MDM2 functions as an effective antagonist of p532.
Because MDM2 plays a key role in inhibition of the p53 tumor suppressor functions and downregulates p53 through direct PPI.
MDM2 acts as an ubiquitin-protein ligase and targets p53 for proteasomal degradation. A blockage of the MDM2−p53 PPI releases p53
from MDM2, restoring the tumor suppressor functions of wild-type p53. Agents designed to block the MDM2−p53 interaction have a
therapeutic potential for the treatment of various cancers retaining wild-type p532. MDM2 binds p53 through its hydrophobic cleft and
its blockage by small molecules can reactivate p53 functions and
Phe subpocket
promotes apoptotic death of cancer cells5–8. A class of imidazoline
compounds, termed nutlins, interacts specifically with the p53-binding
Cl
Leu subpocket
O
Cl
N
O
O
pocket of MDM2 and inhibits the p53–MDM2 interaction9.
Importantly, nutlin analogues have being tested in clinical trials for
treatment of different cancers10. Nutlins’ molecular structure (fig. 1) is
capable to bind the site of MDM2, which is responsible for interaction
with N-terminal domain of p5311–15. The two cis-phenyl substituents
directly insert into two pockets of the binding site (Trp23 and Leu26),
whereas a third phenyl substituent indirectly reaches the third pocket
(Phe19) by means of an ortho-isopropoxy or ethoxy group5. Filling of
a small cavity of the Trp23 and Leu26 pockets usually with a halogen
seems to be a critical feature of an efficient MDM2 inhibitor5,6. The
fourth imidazoline substituent, the N-2-hydroxyethylpiperazine ring,
does not penetrate the p53-binding cleft directly but instead covers the
Phe19 pocket near the Met62 side chain of MDM2. This heterocyclic
motif likely increases water solubility of a compound5.
N
N
N
H
Cl
Kd = 20 M
Cl
O
N
Cap
N
Trp subpocket
RG7112
O
Kd = 0.22 M
S
Cl
Cl
RO
RO
N
N
N
R1
N
H
N
H
N
H
no binding
Kd = 26 M
MDM2 bindig ?
Fig. 1. MDM2 binding activity for Nutlin analog and its
fragments and proposal halogen atom substitution16.

Deconstruction of nutlins family into small fragments have shown that imidazoline fragment without substitution at N atom possess
binding MDM2 activity (fig 1)16. The necessity of halogen substituent and cis-configuration of aromatic rings at C4 and C5 position
was of great importance5,15. Although the necessity of the substituents in the aromatic ring has been shown, there are no studies in which
the replacement of a halogen in a nutlin molecule by another substituent would be studied. The reason for this is the lower availability
of other aryl-substituted cis-vicinal diamines, which are precursors in the synthesis of nutlins and their analogues. The aim of this study
is to predict potential MDM2 binding and an inhibition of MDM2-p53 interaction of nutlin analogues containing other substituents in
the aromatic cycle, such as alkoxy and hydroxy groups, by using of a simple cis-imidazoline fragment-based approach.
Synthetic approach was based on the reaction of aromatic aldehydes with ammonia solution, leading to synthesis of trimeric products
- 1,3,5-trisaryl-2,4-diaza-1,4-pentadienes. The latter, in turn, produced cis-imidazolines by the action of potassium t-butoxide or another
strong base in an aprotic solvent. The disrotatory closure of the pentadiene cycle led precisely to the cis product, which was indicated by
the chemical shifts of the C-H protons of the imidazoline ring. For the cis structures, a signal of C-H protons of the imidazoline ring was
about 5–5.5 ppm, while for the trans structures, a shift was observed at 4–5 ppm. It should be noted that during long-term treatment of
the cis product with a strong base such as potassium t-butoxide the isomerization to the more stable trans-isomer occured17.
The cleavage of the methoxy groups to obtain the derivatives 3r-v was carried out in presence of boron tribromide. We have found
that the higher reactivity of aryloxy groups in position 4 and 5 of imidazoline ring vs. position 3 can be used to obtain selectively
hydrolyzed compound 3s in kinetic conditions (low excess of BBr3 and short reaction time, Scheme 1).
R
R
CHO
NH3
24h
The cytotoxicity of the obtained imidazolines against lung carcinoma
1) tBuOK, THF
2) H2O
R
N
N
THF
OMe
NH
N
NH
Table 1. Cytotoxic activity of imidazoline derivatives 3a-v
R
R
R
1a-r
R
against A549 cancer cells.
R1
2a-r
3a-r
60-90%
R2
N
R2
N
H
2 eq. BBr3
R1
N
N
NH
Compound
3a
R
A549 (IC50, µM)
2-MeO (R1=R2)
3-MeO (R1=R2)
4-MeO (R1=R2)
2,3-diMeO (R1=R2)
2-EtO,3-MeO (R1=R2)
2,4-diMeO (R1=R2)
3,4-diMeO (R1=R2)
2,5-diMeO (R1=R2)
3,4,5-trisMeO (R1=R2)
4-EtO (R1=R2)
27.36±0.79
64.31±1.04
43.90±1.87
16.18±0.29
24.26±2.10
9.32±0.47
84.69±0.76
21.42±1.07
n.a.*
R1
R1
MeO
OMe
3c
reaction time:
1
3b
3c
2 eq. BBr3, 24 h
2
3s
4 h: , R =OH, R =OMe, 81%
1
2
3r,
24 h:
R =R =OH, 85%
Scheme 1. Synthesis of new cis-imidazoline derivatives.
3d
3e
cell line A549 was evaluated using MTT assay. The
cytotoxic activity of alkoxy substituted imidazolines was
comparable with those of halogen-substituted compounds
usually used as nutlin core (Fig. 1, Table 1). The absence of
alkoxy group in aryl substituents in position 4,5 of
imidazoline or its changing to hydroxy group led to
dramatic loss of cytotoxicity at all.
3f
3g
3h
3i
To investigate the possible binding mode for this set of
compounds, we selected the most active compound - 3c, 3f,
3j, 3m - to perform the molecular docking simulations18.
The aim of this work was to find a new imidazoline core
with prominent MDM2 binding activity and increased water
solubility. The water solubility is one of the crucial points
for effective drug design. The compounds 3p and 3l
containing halogen groups demonstrating significantly less
soluble in water then methoxy-containing imidazolines were
not tested. The compounds 3c and 3j were tested to evaluate
the role of substitution in the position 4 of aromatic ring.
The compound 3m was used as a reference because this
compound had the same substitution in 4,5 position of the
imidazoline core like the nutlins. However, the compound
3m did not have additional groups increasing binding
affinity to MDM2 so we compared biological activities of
"pure" methoxy and halogen-substituted imidazoline cores.
3j
13.26±0.37
67.49±0.04
10.68±0.18
20.25±1.88
164.41±13.74
13.72±1.02
9.05±0.20
107.83±4.65
n.a.*
3k
3l
3-MeO,4-EtO (R1=R2)
2-Cl (R1=R2)
3m
3n
3o
3p
3q
3r
4-Cl (R1=R2)
2,4-diCl (R1=R2)
3,4-diCl (R1=R2)
4-Br (R1=R2)
4-F (R1=R2)
4-OH (R1=R2)
3s
4-OH(R1),4-MeO(R2)
3-OH (R1=R2)
n.a.*
3t
310.60±16.67
n.a.*
3v
nutlin-3a
2,5-diOH (R1=R2)
15.1215
*n.a. means the absence of inhibition activity in concentration
range of 1.56-100 μM

The 3D structure of MDM2 for docking simulation was obtained from PDB (PDB id: 4HG7). The compounds successfully docked
inside the same active binding site of MDM2 where p53 peptide binds with a energy in a range of -6 to -9 Kcal/mol. As shown in Fig.
2, the obtained results were similar to those reported for nutlin-3a15. The cis-methoxyphenyl ring directly inserted into two pockets of
the binding site of MDM2 (Trp23 and Leu26) whereas the third
phenyl substituent indirectly reached the third pocket (Phe19).
We analyzed the abilitiy of synthesized derivatives to inhibit
MDM2-p53 interaction and stabilize p53. For this evaluation also
compounds 3c, 3f, 3j, 3m were selected. For study of biological
activity of the compounds lung adenocarcinoma cell line A549 was
chosen. Importantly, the basal level of p53 in A549 cells is low
enough. A treatment with nutlins or their analogues has been shown
to result in essential stabilization of p53 level in A54919. In contrast
to A549, nutlin-mediated p53 stabilization was not been detected in
many cell lines19. Moreover, nutlins and their analogues have been
shown not to induce apoptosis alone in A549 cells. It happens only
upon combination of nutlins with other agents, e.g. DNA-damaging
drug cisplatin20,21. In many other cell lines nutlins induce
apoptosis22,23, which leads to caspase- and calpain-dependent
cleavage of p53 and a drop of its level in the cell24–26. Consequently,
if nutlins or their analogs promote apoptosis induction, decrease of
p53 level interferes with nutlin-induced increase of this protein.
Accordingly, for correct estimation of p53 stabilization we used cell
line A549.
A549 cells were treated with the compounds 3c, 3f, 3j and 3m in
concentrations of 10, 20, 40, and 80 µM. These concentrations were
selected according to IC50. After treatment during 24 hours cells
were collected and p53 level was estimated using Western-blot
(WB) approach (fig.3). The analysis confirmed that all compounds
stabilized p53. 2,4-diMeO derivate 3f at 20 µM demonstrated the
best efficacy for p53 stabilization. Densitometric analysis of p53
bands normalized to glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) showed that the treatment with this compound led to 3.4-
fold increase of p53 level compared to non-treated cells. For agents
3c, 3j and 3m the most efficient concentration was also 20 µM,
which increased p53 level for 1.6, 2.3 and 2.4 times,
correspondingly (fig.3). Additionally, Nutlin-3a in concentration of
10, 20, 40, and 80 µM was tested as a positive control. According to
WB analysis, the treatment with Nutlin-3a led to 10.1-12-fold
increase of p53 level compared to non-treated cells A549.
Considering the fact that the synthesized molecules don’t comprise
all parts of nutlins, the efficacy of 2,4-diMeO derivate 3f was comparable to Nutlin-3a.
Fig. 3. The selected derivatives stabilized p53 level. Western
Blot analysis of total cellular lysates from A549 or RKO cells
upon treatment with indicated compounds. GAPDH was used
Fig. 2. Docked pose of (A) 3c, (B) 3m, (C) 3f, (D) 3j (blue
as a loading control. Designations: PARP full/cl – full form and
stick) overlaid with Nutlin 3a (green) in p53 binding site of
p89 fragment of PARP; GAPDH – glyceraldehyde 3-phosphate
MDM2 protein.
dehydrogenase; procasp-3 – procaspase-3; cl casp-3 – p19/17
fragments of caspase-3; p53/GAPDH – densitometric analysis
of p53P bands normalized to GAPDH. Data from 3 biological
replicates.

Additionally, colon carcinoma cell line RKO was used to
confirm the efficacy of 2,4-diMeO derivate 3f. According to WB
analysis, the treatment of RKO cells with the most efficient
compound 3f in concentration of 10 and 20 µM increased p53 level
for 2.8 and 3.8 times, correspondingly (Fig.3). The treatment of
RKO with Nutlin-3a in concentration of 10 and 20 µM led to 11.5-
10.6-fold increase of p53 level compared to non-treated cells RKO.
Taken together, we demonstrated that if cells (A549 and RKO) are
sensitive to Nutlin-3a, the treatment of these cell lines with the
most efficient 2,4-diMeO derivate also leads to pronounced
increase of p53 level.
To study whether these compounds might induce apoptosis
activation of death effector caspase-3 and cleavage of its substrate
– PARP (poly(ADP-ribose) polymerase 1) – were analyzed.
Importantly, Nutlin-3 in concentrations up to 20 µM was shown
not to induce apoptosis alone in A549 cells20,21. According to WB
analysis the compounds 3j and 3m in concentration of 40 µM
induced a generation of caspase-3 p17/19 active fragment and
subsequent cleavage of PARP (fig.3). The treatment with 80 µM of
these agents led to essential cell death (fig.4) and full degradation
of cellular proteins (fig 3). At the same time, the treatment of cells
with the compounds 3c and 3f led to week caspase-3 activation and
PARP cleavage even at concentration of 80 µM. Taken together, the
4-MeO- and 2,4-diMeO-derivatives 3c and 3f did not induce
apoptotic cell death in concentration up to 40 µM. In contrast, the
administration of 4-Cl- and 4-EtO-derivatives 3j and 3m at 40 µM
resulted in measurable activation of caspases and subsequent
apoptosis. Importantly, the treatment with 80 µM of 3j and 3m did
not increase p53 level but led to more pronounced cell death.
Accordingly, these compounds induced cell death due to non-
specific toxicity rather than p53 stabilization. Moreover, the
administration of 2,4-diMeO-derivate 3f led to the most efficient
stabilization of p53 level but not to apoptosis.
Collectively, our findings demonstrate that the selected
compounds are able to inhibit MDM2-p53 interaction and promote
p53 stabilization. According to WB analysis the synthesized
compounds at concentration of 20 µM promoted p53 stabilization to
different extents.
To evaluate apoptotic and necrotic cell death A549 cells were
treated with the compounds 3c, 3f, 3j and 3m in concentration of 10,
20, 40 and 80 µM for 24 hours. Then, the cells were stained with
Annexin V - FITC in combination with propidium iodide (PI) and
analyzed on a flow cytometer.
Double staining (Annexin V/PI +/+) was used to evaluate the
population of late apoptotic and secondary necrotic cells. Early apoptotic cells were stained with annexin V-FITC only (Annexin V/PI
+/-), and necrotic cells were stained with PI only (Annexin V/PI -/+)
Fig. 4. Flow cytometry (FC) analysis of A549 cells treated with
cisplatin, 3c, 3f, 3j and 3m. Cells were stained with the
conjugate of Annexin V-FITC and Propidium iodide (PI).
Designation for Annexin V/PI: –/–, viable cells; +/– apoptotic
cells; +/+ late apoptotic cells; +/- necrotic cells. (A) FC data for
control and treated cells. (B) Histograms of FC data for cells
treated with the compounds at different concentrations. All
experiments were performed at least three times. Results, if not
otherwise stated, are presented as mean ± standard deviation
(SD). *p < 0.05: significant difference compared to vehicle
treated sample (Mann-Whitney U test).
(fig. 4). As a positive control for triggering apoptosis the treatments
with 15 and 35 µM of cisplatin were used.
Cell death analysis using Annexin V-FITC/PI staining revealed
that the cisplatin treatment led to an increase of early and late
apoptotic populations – Annexin V/PI+/- and Annexin V/PI+/+,
correspondingly. In contrast to cisplatin, 4-MeO- and 2,4-diMeO-
derivatives 3c and 3f in concentrations up to 40 µM did not increase
apoptotic or necrotic populations (fig.4). The treatment with 80 µM
of these compounds only slightly enhanced Annexin V-FITC/PI
double staining of A549 cells. Obtained data were in accordance
with WB analysis of caspase-3 and PARP cleavage (fig.3). 4-Cl- and
4-EtO-derivatives 3j and 3m at 40 and 80 µM induced essential accumulation of necrotic PI-positive cells up to 70% (fig.4). This was
consistent with WB-detected prominent cleavage of cellular proteins, including PARP and caspase-3. This fact confirmed non-specific
toxicity of 4-Cl- and 4-EtO-derivatives 3j and 3m in concentrations higher than 40 µM.
Altogether, these data indicated that 4-MeO- and 2,4-diMeO- derivatives 3c and 3f did not induce necrotic cell death in
concentration up to 80 µM and were able to stabilize p53. In contrast, 4-Cl- and 4-EtO-derivatives 3j and 3m promoted necrotic cell
death and were less efficient for p53 stabilization. Additionally, the necessity of methoxy-group in the position 4 of imidazoline core

was investigated. A549 cells were treated with the compounds 3d and 3h in concentration of 10 and 20 µM. These compounds were
isomers of the derivatives 3f and 3c but didn’t have para-substituent in phenyl. The WB analysis confirmed that both compounds
demonstrated the lack of p53 stabilization that suggested the absence of MDM2-p53 interaction blocking activity (see fig. S1).
New cis-imidazoline derivatives containing methoxy and hydroxy groups were synthesized using the reaction of aromatic aldehydes
with ammonia. It was shown that the alkoxy substituents of obtained arylimidazolines had different reactivity and alkoxyaryl in position
4,5 of imidazoline ring could be hydrolyzed separately from alkoxy group of aryl in position 2. No one of the hydroxy-derivatives
including partially hydrolyzed showed antiproliferative activity whereas alkoxy-containing imidazolines were cytotoxic at a micromolar
range of concentrations. This fact shows the importance of the 4-alkoxyaryl substituent in positions 4 and 5 of imidazoline ring to retain
the cytotoxic activity comparable to 4-halogenaryl substituted imidazoline, which is usually used as nutlin core and has good water
solubility. The selected compounds demonstrated biological activity, promoting stabilization of p53 level in lung adenocarcinoma cells
A549.
Despite the fact that synthesized molecules did not comprise all parts of nutlins, these compounds were able to inhibit MDM2-p53
interaction and increased p53 level in cells. 2,4-diMeO derivate 3f has been shown to possess the best efficacy for p53 stabilization. The
treatment with this compound led to 3.5-3.8-fold increase of p53 level. This compound only slightly stimulated apoptosis and did not
induce necrotic death in A549 cells. These results are in accordance with other reports demonstrating that Nutlin-3 is not able to induce
apoptosis alone in A549 cells and can do that only in combination with other agents. At the same time the 4-Cl- and 4-EtO-derivatives
3j and 3m showed lower activity for p53 stabilization and induced necrotic cell death. According to the flow cytometry, these
compounds stimulated necrosis in concentrations higher than 20 µM.
Acknowledgments
This study was supported by the Russian Foundation for Basic Research (Projects No. 17-03-01320: Figures 1 and 2) and the
Russian Science Foundation (Projects 17-75-20102: Figures 3 and 4).
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2,4,5-Tris(alkoxyaryl)imidazoline derivatives
as potent scaffold for novel p53-MDM2
interaction inhibitors: design, synthesis, and
biological evaluation
Leave this area blank for abstract info.
Daniil R. Bazanov, Nikolay V. Pervushin, Victoria Yu. Savitskaya, Lada V. Anikina, Marina V. Proskurnina,
Natalia A. Lozinskaya and Gelina S. Kopeina
MDM2 bindig
AlkO
CHO
HO
Leu subpocket
1) NH3/THF
N
N
BBr3
R1
Ar1
2) tBuOK,THF
AlkO
N
H
N
H
AlkO
HO
Trp subpocket
no binding
non-cytotoxic
IC50 = 9.05 - 84.69 M (A549 cells)