Diaryl- and triaryl-pyrrole derivatives: Inhibitors of the MDM2-p53 and MDMX-p53 protein-protein interactions

Article abstract of DOI:10.1039/c3md00161j

Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)- ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carbonitrile as an MDM2-p53 inhibitor (IC₅₀ = 12.3 μM). MDM2-p53 and MDMX-p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5- diphenyl-1H-pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC₅₀ = 0.11 μM; MDMX IC₅₀ = 4.2 μM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H, 3H,5H)-trione (MDM2 IC₅₀ = 0.15 μM; MDMX IC₅₀ = 4.2 μM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX-p53 activity but not for MDM2-p53 inhibition. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3md00161j

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Diaryl- and triaryl-pyrrole derivatives: inhibitors of the
MDM2–p53 and MDMX–p53 protein–protein
interactions†
Cite this: Med. Chem. Commun., 2013,
4, 1297
Tim J. Blackburn,a Shafiq Ahmed,b Christopher R. Coxon,a Junfeng Liu,b
Xiaohong Lu,b Bernard T. Golding,a Roger J. Griffin,a Claire Hutton,b David R. Newell,b
a
a
a
b
b
*
Stephen Ojo, Anna F. Watson, Andrey Zaytzev, Yan Zhao, John Lunec
a
*
and Ian R. Hardcastle
Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2,5-dimethyl-1H-
pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile as an MDM2–p53 inhibitor (IC50
12.3 mM). MDM2–p53 and MDMX–p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1H-pyrrol-
3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC50 ¼ 0.11 mM; MDMX IC50
4.2 mM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-
trione (MDM2 IC50 ¼ 0.15 mM; MDMX IC50 ¼ 4.2 mM), and cellular activity consistent with p53 activation in
MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for
MDMX–p53 activity but not for MDM2–p53 inhibition.
¼
¼
Received 11th June 2013
Accepted 16th July 2013
DOI: 10.1039/c3md00161j
www.rsc.org/medchemcomm
The MDM2 and MDMX proteins regulate the activity of p53
with different and non-redundant mechanisms.8 In addition to
Introduction
The tumor suppressor protein p53 functions as a molecular
sensor in diverse signalling pathways resulting from cellular
stresses, such as DNA damage, oncogene activation and
possibly hypoxia.1 Abrogation of the function of the tumor
suppressor p53 is a key feature of the molecular pathology in
many cancers. Mutation of the TP53 gene occurs in approxi-
mately 50% of common adult sporadic cancers, resulting in
inactive protein.2,3 Alternatively, p53 may be silenced by the
overexpression of the regulatory proteins MDM2 or MDMX
(MDM4).4–6 MDM2 amplication has been reported to occur in
approximately 11% of all tumors and the paralogue MDMX has
been reported to be amplied in brain (11%), breast (5–40%),
and so tissue tumors (17%). Overexpression of MDMX has also
been observed in a wider range of tumor types, including uterus
(15%), testes (27%), melanoma (65%), stomach/small intestine
(43%), and lung (18%).6,7
the MDM2 gene being a target for p53-dependent transcription,
MDM2 regulates p53 in an autoregulatory negative feedback loop
by binding to the p53 transactivation domain, and acting as an
E3-ligase for polyubiquitination of p53 to promote p53 degrada-
tion by the ubiquitin-mediated proteosomal pathway.9–12 MDMX
also inhibits p53 transcriptional activity, but does not act as an E3
ligase independently of MDM2, and its expression is not p53
dependent.13 Furthermore, MDMX–MDM2 heterodimers have
enhanced E3 ligase activity over MDM2 alone and may be an
important mechanism of p53 regulation.
The MDM2–p53 binding interaction is amenable to small-
molecule inhibition, as it consists of a relatively deep binding
groove on the surface of the MDM2 protein into which an
amphipathic helix of p53 binds.14 A number of potent MDM2–
p53 inhibitors have been reported based on diverse chemo-
types,15 such as the cis-imidazoline RG-7112 (IC50 ¼ 12 nM),16
spirooxindoles, e.g. MI-888 (IC50 ¼ 6.8 nM),17 and the substituted
piperidone AM-8553 (IC50 ¼ 2.2 nM),18 and have demonstrated
cellular activity consistent with inhibition of MDM2–p53 binding
and in vivo antitumor activity. However, these series lack signif-
icant potency against MDMX,19 and overexpression of MDMX
offers a possible mechanism of resistance to such MDM2–p53
inhibitors. For this reason compounds able to inhibit both
interactions have great signicance.20
aNewcastle Cancer Centre, Northern Institute for Cancer Research and School of
Chemistry, Newcastle University, Bedson Building, Newcastle, NE1 7RU, UK. E-mail:
Ian.Hardcastle@ncl.ac.uk; Fax: +44 (0)191 8591; Tel: +44 (0)191 222 6645
bNewcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle
University, Paul O'Gorman Building, Medical School, Framlington Place, Newcastle,
NE2 4HH, UK. E-mail: John.Lunec@ncl.ac.uk; Fax: +44 (0)191 4301; Tel: +44 (0)191
246 4420
† Electronic supplementary information (ESI) available: Experimental details for
compound synthesis, analytical data for all compounds and intermediates.
Details for the biological evaluation. Further details for the modeling. Table of
combustion analysis data. See DOI: 10.1039/c3md00161j
To date, there have been few reports of small-molecule
MDMX inhibitors. The 5-oxo-pyrazolylidene SJ-172552 was
identied in an MDMX high-throughput uorescence
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polarisation assay and showed selective MDMX inhibition, the triarylpyrroles with a small series of the related diarylpyrrole
through a complex, irreversible mechanism.21,22 The 3-imida- MDM2–p53 inhibitors demonstrates key structural requirements
zolyl indole (1a) is a mixed MDM2–, MDMX–p53 inhibitor for mixed MDM2 and MDMX inhibition in this series.
(MDM2 IC50 ¼ 0.19 mM; MDMX IC50 ¼ 20 mM), and has provided
the rst X-ray crystal structure of MDMX bound to a small-
molecule ligand.19 A series of MDM2–p53 inhibitory pyrrolidone
derivatives, e.g. (2a, MDM2 ¼ 0.26 mM, MDMX ¼ 2.7 mM; and 2b,
MDM2 ¼ 1.3 mM, MDMX ¼ 2.1 mM), also show modest MDMX
activity in addition to MDM2 inhibition.23 The indolyl hydan-
toins, e.g. RO-5963 (MDM2 ¼ 17 nM, MDMX ¼ 25 nM), are the
most potent MDM2–p53 and MDMX–p53 inhibitors reported to
date.24
Identication of MDM2–p53 inhibitors by screening
A pilot set of 800 structurally diverse compounds, obtained from
the Cancer Research UK screening collection was studied in an
MDM2–p53 ELISA protein–protein binding assay, at 5 and 20 mM
concentrations.25 Follow up IC50 determinations on active
compounds revealed pyrrole 3 as a hit, with an IC50 of 12.3 ꢀ
1.5 mM against MDM2–p53, which also demonstrated dose-
dependent cellular activity by Western blotting for MDM2 and
In this paper, we describe the discovery, structure–activity p53 induction. A series of 96 related analogues was purchased,
relationships (SARs) and cellular activity of triarylpyrrole based on similarity searching and visual inspection, and
compounds with promising inhibitory activity against both screened for MDM2 activity. Twelve compounds (4a–l) showed
MDM2–p53 and MDMX–p53. Comparison of molecular models of promising MDM2–p53 inhibitory activity with IC50 values in the
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Table 1 MDM2 structure–activity relationships for purchased pyrroles 4a–l
Table 2 MDM2 and MDMX structure–activity relationships for triarylpyrroles 4c
and 4m–z
MDM2 IC50
(nM)
Compound R1
R2
R3
R4
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-ClPh
Me
Me
Ph
Ph
Ph
Ph
Ph
Me Ph
Me
Me
H
H
H
H
H
H
Ph
H
Ph
720 ꢀ 100
3300 ꢀ 700
120 ꢀ 20
230 ꢀ 44
163 ꢀ 17
256 ꢀ 39
Insol.
199 ꢀ 16
8400 ꢀ 900
4700 ꢀ 200
700 ꢀ 20
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-ClPh
4-BrPh
4-MePh
4-MePh
Compound R1 R2
X
Y
MDM2 IC50 (mM)a MDMX IC50 (mM)a
4c
4m
4n
4o
4p
4q
4r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Br
OMe
t-Bu
CN
CN
NO2
NO2
S
0.11 ꢀ 0.03
0.30 ꢀ 0.03
0.18 ꢀ 0.07
1.9 ꢀ 0.3
4.2 ꢀ 1.2
nd
nd
3-ClPh
O
O
O
O
O
S
O
S
S
O
S
S
O
S
3,4-diMePh Ph
Me
Me
Ph
Ph
H
13 ꢀ 7
1.9 ꢀ 0.3
nd
H
H
H
4.7 ꢀ 1.9
7.0 ꢀ 3.0
0.90 ꢀ 0.42
0.68 ꢀ 0.18
0.63 ꢀ 0.12
nd
4j
4k
0.20 ꢀ 0.07d
0.15 ꢀ 0.06
0.17 ꢀ 0.09d
0.30 ꢀ 0.12c
0.89 ꢀ 0.04
0.26 ꢀ 0.05
0.11 ꢀ 0.02d
0.34 ꢀ 0.08
0.073 ꢀ 0.002
4-EtO2CPh Ph
H
4s
4t
4u
4v
4w
4x
4y
4z
Et Et Cl
Et Et Br
Et Et Br
74%b
0.12–8.4 mM range (Table 1). Pyrroles bearing N2-aryl groups with
chloro-, bromo-, or ethoxylcarbonyl 4-substituents showed the
greatest potency. Pyrroles with 2,5-diphenyl substitution (4c, 4d)
were 5–10 fold more potent than the comparable 2,5-dimethyl
analogues (4a, 4b). Substitution on the thiobarbituric acid moiety
had a modest negative effect on potency.
nd
Me
Me
Me
H
H
H
Cl
Br
Br
28 ꢀ 23
35 ꢀ 20
nd
a
c
n ¼ 3, from resynthesised material. b % inhibition at 50 mM. n ¼ 6.
n ¼ 4; nd ¼ not determined.
d
Synthesis and structure–activity relationships
Formylation of the pyrroles under Vilsmeier–Haack conditions
proceeded smoothly with microwave heating to give 3-for-
mylpyrroles 6 in good yields. Condensation of aldehydes 6 with
the required barbituric or thiobarbituric acid was carrꢁied out in
ethanol at room temperature or in acetic acid at 120 C to give
the pyrrole barbiturates (4a–w). The use of mono-N-methyl
barbituric or thiobarbituric acid gave 4x–z as inseparable
mixtures of regioisomers.
In order to validate and further explore the SAR, a selection of
2,5-symmetrically substituted pyrroles 5 was prepared from
1,2-dibenzoylethane and the appropriate 4-substituted aniline,
using triuoroethanol (TFE) as solvent and triuoroacetic acid
(TFA) as catalyst under microwave heating (Scheme 1).26–29
The use of TFE-TFA with microwave heating is especially
advantageous over conventional procedures30 as it ensures
clean, homogeneous reactions that proceed in high yields.
Scheme 1 Synthesis of substituted triarylpyrroles 4a–z. Reagents and conditions: (a) TFA, TFE, MW, 150 ꢁC, 20 min; (b) POCl3 DMF, 0–70 ꢁC, MW, 1 h; (c) EtOH, rt, 12 h,
or AcOH, 120 ꢁC, 2 h.
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Scheme 2 Synthesis of substituted triarylpyrroles 7 and 7. Reagents and conditions: (a) toluene, piperidine, acetic acid, D; (b) NaBH4, EtOH.
MDM2 inhibitory SARs for a series of analogues of 4c were
determined, and selected compounds were assayed for MDMX–
p53 inhibitory activity (Table 2). Unexpectedly, pyrrole 4c was
shown to be a low micromolar inhibitor of the MDMX–p53
interaction. Comparision of the MDM2 inhibitory activity of
matched pairs of thiobarbituric acid and barbituric acid deriva-
tives shows a 3–20 fold reduction in potency for the oxo-deriva-
tives (e.g. 4m and 4c, 4q and 4r, 4v and 4w, 4y and 4z), with the
exception of the 4-nitro derivatives 4s and 4t that were equi-
potent. The 4-N-aryl substitutents had a profound inuence on
potency for MDM2–p53. Thus, potency was conferred by chloro-
or bromo-substituents (4c, 4m, and 4n) or electron-withdrawing
Scheme 3 Synthesis of substituted triarylpyrroles 10a–f. Reagents and condi-
tions: (a) toluene, piperidine, acetic acid, D.
groups e.g. nitro or cyano (4q–t). In contrast, larger or electron-
donating groups gave poor MDM2 inhibition, e.g. OCH3, t-Bu
(4o and 4p). The 4-aryl substituent also signicantly inuenced
activity against MDMX, with 4-nitro- (4s and 4t), 4-cyano- (4q and
4r), or 4-chloro- (4c) N-phenyl substituents conferring the greatest inactive, whereas the malonic acid derivative 10b showed
inhibitory potency. The N,N-diethylbarbituric acid or thio- modest activity against MDM2–p53. The malonamide derivative
barbituric acid derivatives (4u,v) were 3–4 fold less potent against 10c showed similar activity to 10b, whereas the N-alkyl malo-
MDM2 compared with their unsubstituted analogues, whereas namide derivatives 10d and 10e showed a 3- and 6-fold loss of
the mono-N-methyl analogues (4x,y) were equipotent with their potency against MDM2–p53. The monocarboxylic acid analogue
parents. Similarly, N-alkyl substitution on the barbituric acid or 10f was only weakly active against MDM2–p53 and inactive
thiobarbituric acid moiety (4u–z) resulted in a signicant loss of against MDMX–p53. These results suggest that binding to
MDMX–p53 activity. In all cases reduced solubility was observed MDM2 requires at least one H-bond donor in the 4-substituent.
for the N-alkyl derivatives.
A series of 2-alkyl-1,5-diarylpyrroles 11 was designed to probe
A series of derivatives with alternative substituents to the the SAR about the pyrrole for MDM2 and MDMX inhibition
barbituric acid was prepared to explore the SAR for this moiety (Table 4). Their synthesis required the preparation of 1,4-dike-
for both MDM2 and MDMX inhibition. Compound 7 was tones 12 via a Stetter reaction followed by cyclisation with the
prepared by heating aldehyde 5b with Meldrum's acid 8 in appropriate aniline (Scheme 4).31 Formylation of pyrrole 13 gave
toluene with piperidine acetate as catalyst (Scheme 2). Reduc- an inseparable mixture of isomers (14) that was reacted with
tion of 7 to 9 was achieved with sodium borohydride in ethanol. barbituric acid affording a mixture of 3- and 4-isomers 11 that
The malonic acid derivatives 10a–e were prepared by conden- were only separable by HPLC (e.g. X ¼ Cl). The limited practi-
sation of the required malonic acid, ester or amide with alde- cality of this route prompted the search for a method capable of
hyde 5a (Scheme 3). The reaction with malonic acid gave the yielding either regioisomer, as required. Thus, b-ketoesters 15,
decarboxylated product 10f in addition to 10b.
prepared from Meldrum's acid 12, were subjected to a tandem
The Meldrum's acid derivative 7 was >100-fold less potent homologation/addition sequence mediated by the Furukawa
than 8c against MDM2–p53, whereas the reduced derivative 9 reagent,32 with oxidation of the intermediate providing the
suffered a less signicant 20-fold reduction in potency (Table 3). a-ester 1,4-diketones 16 (Scheme 5).27 Regiospecic synthesis of
The acyclic derivatives 10a–f all lacked signicant MDMX–p53 3-, and 4-substituted pyrroles was achieved by variation of the
activity, and showed reduced MDM2–p53 potency compared R-groups on the b-ketoester and the aldehyde, providing both
with 4c. The dimethyl malonate derivative 10a was essentially regioisomeric a-ester 1,4-diketones 16 that were combined with
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Table 3 MDM2 and MDMX structure–activity relationships for triarylpyrroles 7,
9, and 10a–f
MDM2 IC50 MDMX IC50
Compound Structure R
X
Y
(mM)a
(mM)a
7
9
A
B
C
C
C
C
C
D
Me Br O
Me Br O
17 ꢀ 4b
nd
Scheme 4 Synthesis of substituted diarylpyrrole 11a. Reagents and conditions:
(a) 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride, NEt3, RT; (b) TFA,
TFE, MW, 150 ꢁC, 20 min; (c) POCl3, DMF, 0–70 ꢁC MW, 1 h; (d) barbituric acid,
AcOH, 120 ꢁC.
2.9 ꢀ 0.7b nd
10a
10b
10c
10d
10e
10f
—
—
—
—
—
—
Cl OMe
Cl OH
Cl NH2
Cl NHMe
>50
>50
>50
30c
30c
42c
>50
2.9 ꢀ 0.2
2.5 ꢀ 0.2
7.6 ꢀ 0.4
Cl NH(CH2)2OH 15 ꢀ 3
Cl OH
17 ꢀ 3
a
n ¼ 3. b n ¼ 5. c n ¼ 1; nd ¼ not determined.
the required aniline. This is a versatile and efficient approach to
a variety of 1,2,3-trisubstituted pyrroles. The aldehydes 17 were
prepared from the esters 18 by DIBAL-H reduction to the
Table 4 MDM2 and MDMX structure–activity relationships for diarylpyrroles
11a–g
Scheme 5 Synthesis of substituted diarylpyrroles 11b–g. Reagents and condi-
tions: (a) pyridine, R1COCl, DCM, 0 ꢁC; (b) EtOH, toluene, reflux; (c) CH2I2, Et2Zn,
R2CHO DCM 0 ꢁC; (d) PCC, DCM, R.T.; (e) TFA, TFE, MW, 150 ꢁC or PTSA, toluene,
ꢁ
˚
Dean–Stark; (f) DIBAL-H, DCM, ꢂ78 C; (g) TPAP, NMO, 4 A MS, DCM, rt; (h) thi-
obarbituric acid, AcOH, 120 ꢁC-rt.
corresponding alcohols 19 followed by oxidation.33 Condensa-
tion with thiobarbituric acid gave the desired non-symmetri-
cally substituted pyrroles 11b–g.
a
b
MDM2 IC50
(mM)
MDMX IC50
(mM)
Compound Isomer
R1
X
Y
Substitution of the 2-phenyl residue with a methyl group
resulted in a greater than 3-fold loss of MDM2 inhibitory
potency for the mixture of regioisomers 11a (Table 4).
Replacement of the 5-phenyl with a tert-butyl group (11b,c) gave
a small loss of MDM2 inhibitory potency, independent of the
position of the thiobarbituric acid residue, but resulted in a
>200 fold loss of potency for MDMX. Similarly, the 2- or 5-
cyclopropyl derivatives retained modest MDM2 inhibitory
11a
11b
11c
11d
11e
11f
Mixture Me
Cl
Br
Cl
O
S
S
S
S
S
S
>1
nd
963
3
3
4
4
3
3
t-Bu
t-Bu
CyPr Cl
CyPr Br
CyPr Cl
CyPr Br
0.76 ꢀ 0.27c
1.1 ꢀ 0.7c
1.6 ꢀ 1.7
1.6 ꢀ 1.6
2.1 ꢀ 2.7
2.2 ꢀ 2.7
1684
3486
3428
4916
5322
11g
a
n ¼ 3. b n ¼ 1. c n ¼ 4; nd ¼ not determined.
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activity, independent of the position of the thiobarbituric acid
residue, but were inactive against MDMX.
MDM2 and MDMX modeling studies
Previously, we have demonstrated that docking ligands into the
MDM2 binding pocket can yield multiple low energy solu-
tions.34 For this reason, we opted to generate representative
binding modes for the pyrroles from the X-ray co-crystal struc-
tures of MDM2 and MDMX with structurally similar ligands, i.e.
imidazole derivatives (1a,b), by superposition and replacement
of the ligand.19 Pyrrole (4c) with mixed MDM2 and MDMX
potency, and two MDM2 selective pyrroles (11c and 11d) were
aligned in the ligands 1a in MDM2 (pdb: 3LBK) and 1b in
MDMX (pdb: 3LBJ), using the ligand builder function and the
CCP4 ‘cprodrg’ plugin within COOT.35,36 The high degree of
structural overlap between the original ligand and the modeled
compound in these models gives condence that the binding
modes are reasonable.
The binding modes for 4c in both MDM2 and MDMX (Fig. 1)
show good overlap with two of the aryl substituents of the
imidazole series (1a,b). In particular, the N-4-chlorophenyl ring
occupies the pocket normally lled by Trp23 of p53 for both
MDM2 and MDMX,14 overlaying the chloroindole ring of 1a or 1b.
The 5-phenyl ring of the pyrrole occupies the Phe19 pocket with
good overlap with the 1-phenyl ring of 1a or 1b. The remaining
phenyl ring is accommodated by the Leu26 pocket with a less well
dened overlap and different vector compared with the original
ligands. The thiobarbituric acid group projects away from the
protein surface into the space occupied by the carboxylic acid
residue of 1a and the amide group of 1b, suggesting that these
groups may act, in part, as a hydrophilic cap.37
Fig. 2 Modeled binding mode of 1,2-diarylpyrrole 11c (green) overlayed with
MDM2 (blue mesh) and MDMX (solid pink).
bound to MDMX and the acid group of 1b bound to MDM2, and
raises the possibility that water mediated H-bonding to the
protein backbone may be important for affinity.
The modelling of 11c into MDM2 and MDMX shows the N-4-
chlorophenyl group occupying the Trp23 pocket as seen for 4c
(Fig. 2). The t-butyl residue is positioned into the Phe19 binding
pocket, which is occupied by a number of alkyl substituents in
recent MDM2 X-ray structures, e.g. the MI-series (3LBL)19 and
the AM-8553 series (4ERE).18 The MDMX structure also places
the t-butyl group into the Phe19 pocket, but the 5-phenyl ring no
longer makes a good interaction with Leu26 pocket which
appears to be broader and shallower than for MDM2. This
observation may explain the dramatic loss in MDMX potency for
this series, compared with the retention of MDM2 potency.
Interestingly, the model of 11d (ESI†), demonstrates the same
arrangement of substituents, regardless of the positioning of
the thiobarbituric acid moiety on the pyrrole ring.
The MDM2 binding mode model is consistent with the
observed SARs, as the Trp23 pocket of MDM2 shows a strong
preference for haloaromatic groups as seen in the X-ray struc-
tures of high-affinity ligands. The preference for haloaromatic
groups in the MDMX Trp23 binding-pocket is not as well
established as for MDM2 due to the smaller number of depos-
ited structures; however, the SARs in this series suggest that the
pocket is similar to that in MDM2.
Cellular activity of pyrrole inhibitors
Growth inhibitory activity was determined for selected pyrroles in
a panel of cell lines with dened MDM2, MDMX and p53 status
(Table 5). The SJSA-1 osteosarcoma line has amplied MDM2 and
wild-type p53, and is paired with the SN40R2 line (MDM2
amplied, p53 mutant), an SJSA-1 derived line that is resistant to
Nutlin-3a due to p53 mutation. For comparison, the MRK-NU-1
breast cancer cell line has amplied MDMX and wild-type p53.
Compounds showed growth inhibitory activity in the 2–10 mM
The role of the barbituric acid or thiobarbituric acid group is
less well explained by the models. The positioning of the groups
in the models is consistent with that seen for the amide of 1a
Table 5 Growth inhibitory activity of pyrroles 4c, 4r, 4t, 4x in a panel of cell lines
with defined MDM2, MDMX and p53 statusa
GI50 (mM)
Compound
SJSA-1
SN40R2b
MRK-NU-1
4cc
4rc
4tc
4xc
2.3 ꢀ 0.2
5.1 ꢀ 0.9
4.7 ꢀ 1.0
5.5 ꢀ 0.6
2.8 ꢀ 0.6
5.9 ꢀ 0.3
5.2 ꢀ 0.4
6.3 ꢀ 0.4
2.3 ꢀ 0.3
6.1 ꢀ 0.7
4.9 ꢀ 0.6
7.0 ꢀ 1.0
Fig. 1 Modeled binding mode of 4c (green) overlayed with: (A) 1b (magenta) in
MDM2 (light blue); (B) 1a (purple) in MDMX (pink), binding pockets for p53
residues are indicated.
n ¼ 3. b p53 null. c Resynthesised material.
a
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Fig. 3 Cellular activity of 3, 4c and 4d for p53 pathway activation detected by western blotting in cell lines treated with increasing concentrations (mM) for 4 h: (A)
SJSA-1 cells; (B) A2780 cells; and (C) A2780CP70 cells.
range without a strong correlation to either MDM2 or MDMX p53-dependent growth inhibitory activity and the poor physi-
inhibitory activity. Disappointingly, the compounds were equally cochemical properties for this series presents a substantial
growth inhibitory in all cell lines regardless of their potency vs. challenge for their further development as drugs; however, they
MDM2 or MDMX, and the MDM2 and MDMX status of the cell represent interesting dual MDM2/MDMX ligands for both
line. Importantly, the p53 mutant SN40R2 line was equally structural and mechanistic studies.
sensitive to the pyrroles 4c, 4r, 4t and 4x, in contrast to the 2–100
fold difference in activity reported for potent MDM2 inhibitors.
Cell lines with dened MDM2 and p53 status were treated
with increasing concentrations (0.1, 0.2 and 5 mM) of pyrroles 3,
Abbreviations
DCM
DIBAL-H
DIPEA
DMF
DMSO
ELISA
MDM
NMO
PCC
Dichloromethane
4c and 4d to investigate the transcriptional activation of p53
and the subsequent induction of p53-dependent proteins by
Western blotting. In the SJSA-1 line (MDM2 amplied, p53wt)
induction of MDM2, p53 and p21 was clearly visible at 5 mM for
each compound (Fig. 3). In the A2780 line (p53wt) induction of
MDM2 and p21 is observed for 4c and 4d, but not for 3. Higher
levels of p53 obscured any induction in this case. In contrast, in
the A2780 CP70 line (p53 mutant) no induction of MDM2, p53,
and p21 was observed. In contrast to the growth inhibition data,
these results clearly demonstrate a p53-dependent cellular
response to the pyrroles.
Di-isopropylaluminium hydride
Diisopropylethyl amine
N,N-Dimethylformamide
Dimethyl sulfoxide
Enzyme-linked immunosorbent assay
Murine double minute
N-Methylmorpholine-N-oxide
Pyridinium chlorochromate
p-Toluenesulfonic acid
Structure–activity relationship
Sulforhodamine B
PTSA
SAR
SRB
Compounds with N-phenylpyrrole or alkylidene barbituric
acid groups have been identied as ‘frequent-hitters’ in HTS
campaigns.38 With this in mind, it is likely that, despite the
TFA
TFE
THF
Triuoroacetic acid
2,2,2-Triuoroethanol
Tetrahydrofuran
ability of these compounds to activate p53 dependent cellular TPAP
Tetrapropylammonium perruthenate
Wild-type.
wt
processes, the modest growth inhibition seen for this series is
the result of additional off-target activity.
Conclusions
Acknowledgements
To date, the majority of potent MDM2–p53 inhibitors are highly
selective for MDM2 over MDMX, and there are very few pub-
lished inhibitors of MDM2 that retain signicant potency for
MDMX. The imidazole series, e.g. 1a, is reported to have weak
MDMX activity. The hydantoin derivative RO-5963 is reported to
be a potent dual MDM2 and MDMX inhibitor, but with an
unusual binding mode and cellular mechanism of action. We
have identied a series of 1,2,5-triarylpyrroles that display
MDM2–p53 and MDMX–p53 inhibitory activity in an cell-free
ELISA MDM2–p53 binding assay, and are able to activate p53-
dependent gene transcription in whole cells. Modeling studies
suggest a binding mode which is consistent with other reported
MDM2 inhibitors, and that offers insight into the structural
requirements for the design of compounds able to inhibit
both MDM2–p53 and MDMX–p53. However, the lack of
The authors thank: Cancer Research UK and the EU FP6 DeP-
PICT for funding; Carlo Bawn and Karen Haggerty for expert
technical assistance; Martin Noble for helpful discussions. Use
of the EPSRC Mass Spectrometry Service at the University of
Wales (Swansea) is gratefully acknowledged.
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