Diphenylpyrroles: Novel p53 activators

Article abstract of DOI:10.1016/j.ejmech.2014.05.082

Cellular tumor antigen p53 is crucial for cancer prevention via different mechanisms. E3 ubiquitin-protein ligase HDM2 binds to p53, blocks its ability to activate transcription, and therefore acts as a negative regulator. Blocking p53 binding site on HDM2 was believed to generate efficient antitumor agents. So far, limited scaffolds were reported with HDM2 antagonist activity. Herein, diphenylpyrroles were introduced and evaluated as a novel scaffold in the field of p53 activators. An efficient synthesis of novel 3-heteroaryl-pyrroles is described via reactions of E-3-(dimethylamino)-1-(2-methyl-4,5-diphenyl-1H- pyrrol-3-yl)prop-2-en-1-one or E-1-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)-3- morpholinoprop-2-en-1-one with hydrazine hydrate, phenyl hydrazine, hydroxylamine, various heterocyclic amines and active methylene compounds.

Full text of DOI:10.1016/j.ejmech.2014.05.082

Accepted Manuscript
Diphenylpyrroles: Novel p53 Activators
Sobhi M. Gomha , Taha M.A. Eldebss , Mohamed M. Abdulla , Abdelrahman S.
Mayhoub
PII:
S0223-5234(14)00514-5
10.1016/j.ejmech.2014.05.082
EJMECH 7040
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 May 2014
Revised Date: 23 May 2014
Accepted Date: 31 May 2014
Please cite this article as: S.M. Gomha, T.M.A. Eldebss, M.M. Abdulla, A.S. Mayhoub,
Diphenylpyrroles: Novel p53 Activators, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.082.
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ACCEPTED MANUSCRIPT
•
•
•
•
Blocking p53 binding site on HDM2 was believed to generate efficient
antitumor agents
Diphenylpyrroles were introduced and evaluated as a novel scaffold in the
field of p53 activators
New 4,5-diphenyl-3-heteroaryl-pyrroles were synthesized via reactions of
enaminones with C- and N-nucleophiles.
Diphenylpyrroles scaffold has the advantage of synthetic protocol
accessibility

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Graphical Abstract
________________________________________________________
Diphenylpyrroles: Novel p53 Activators
Sobhi M. Gomhaa*, Taha M. A. Eldebss,a Mohamed M. Abdullab and Abdelrahman S.
Mayhoubc
An efficient synthesis of novel 4,5-diphenyl-3-heteroaryl-pyrroles were
synthesized and evaluated as a novel scaffold in the field of p53 activators.

ACCEPTED MANUSCRIPT
Diphenylpyrroles: Novel p53 Activators
Sobhi M. Gomhaa*, Taha M. A. Eldebss,a Mohamed M. Abdullab and Abdelrahman S. Mayhoubc
aDepartment of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
bResearch Unit, Saco Pharm. Co., 6th October City, Egypt
cDepartment of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, 11884 Egypt
Abstract: Cellular tumor antigen p53 is crucial for cancer prevention via different mechanisms. E3
ubiquitin-protein ligase HDM2 binds to p53, blocks its ability to activate transcription, and therefore
acts as a negative regulator. Blocking p53 binding site on HDM2 was believed to generate efficient
antitumor agents. So far, limited scaffolds were reported with HDM2 antagonist activity. Herein,
diphenylpyrroles were introduced and evaluated as a novel scaffold in the field of p53 activators. An
efficient synthesis of novel 3-heteroaryl-pyrroles is described via reactions of E-3-(dimethylamino)-1-
(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)prop-2-en-1-one or E-1-(2-methyl-4,5-diphenyl-1H-pyrrol-3-
yl)-3-morpholinoprop-2-en-1-one with hydrazine hydrate, phenyl hydrazine, hydroxylamine, various
heterocyclic amines and active methylene compounds.
Keywords: Pyrroles, triazolopyrimidine, benzimidazopyrimidine, pyrazolopyrimidine, and p53-specific
ubiquitin E3 ligase HDM2.
1. Introduction
The tumor suppressor protein p53 is central to mammalian cells protection mechanism against
malignant transformation. So far, p53 is inactivated in a large proportion of cancer cells [1]. p53 is
regulated by another protein called human double minute 2 (HDM2). HDM2 binds to p53 at its
transactivating domain and block its ability to activate transcription. Therefore, it acts as a negative
regulator to p53 suppressor protein [2, 3]. Moreover, the oncogene HDM2 and p53 are linked in a
negative feed-back loop in which p53 activates HDM2 [4]. HDM2 is acting as a p53-specific ubiquitin
E3 ligase and thus promoting degradation of p53 protein through the ubiquitin-proteosome system [5].
In the same vein, overexpression of p53 negative regulator HDM2 was observed in many human
malignancies [6-15].
*Corresponding author, Tel: + 20100 1649576, Fax: + (202) 35727556
E-mail: s.m.gomha@hotmail.com
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Recently, medicinal chemist adopted a strategy for the reactivation of the pro-apoptotic p53
activities, where HDM2 is overexpressed, is therefore to interrupt the p53-HDM2 feed-back loop,
either by blocking the protein–protein interaction between the p53 N-terminal domain and HDM2, or
by inhibiting the E3 ligase activity of HDM2 [16]. Several small molecules have been so far reported as
p53-pathway modulators [17].The capability of 5-deazaflavin derivatives to activate the tumor
suppressor p53 in cancer cells was reported through inhibition of the p53-specific ubiquitin E3 ligase
HDM2 (Chart 1) [18-20]. In addition, some diphenylimidazolidines, glucocorticoids and engineered
cyclotides have been reported to play the same role [21, 22].
Chart 1. Chemical structures of some reported HDM2 ligase inhibitors.
Research Design: Aiming to expand the reported tumor suppressor p53 activators, we are reporting
herein 2,3-diphenylpyrrole as a novel scaffold in the field of interrupting p53-HDM2 signaling
pathway. Diphenylimidazolidine derivatives (Chart 1), reported by L. T. Vassilev, ranked among the
most reported potent p53 activators [23]. The structures of all reported diphenylimidazolidines were
built using Sybyl-X, aligned and a pharmacophore model was generated using Discovery Studio 3.1 as
briefly shown in Chart 2 (ring substitutions and side chains were ignored at this stage). With this model
in hand, a library of mono- and diphenyl-substituted 5-memebered compounds was virtually screened
and the top scored structures were assayed for their p53-activation properties. These efforts collectively
afforded compound 7 (Chart 2) that has a closely related pharmacophore (Chart 2).
Chart 2. Work Design
Chart 3. Alignment of reported (black) and newly (red) derived structures: The diphenyl rings of the
lead structure (black) were reported to interact with MDM2 (PDB: 1rv1), leaving the other two groups
facing p53.
The two phenyl rings of the lead diphenylimidazolidine were reported to interact with MDM2
protein, while the substituents at imidazolidine 2 and 3 positions were reported to interrelate with p53
[23]. Since diphenyl moieties of lead and newly presented structures are well-aligned together as
shown in Chart 3, they are intuitively expected to bind similarly keeping the aromatic system at
pyrrole-3 position facing the binding HDM2-pocket at p53. Our focus in this report was directed
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towards modifying the aromatic system at pyrrole-3 position to comprehensively cover the structure-
activity-relationships of this diphenylpyrrole as a new p53 activator.
2. Results and discussion
2.1. Chemistry
The starting E-3-(dimethylamino)-1-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)prop-2-en-1-one
(3) was prepared as previously described [24] via condensation of the pyrazole derivative 1 [25] with
dimethylformamide dimethyl acetal (DMF-DMA, 2). Treatment of the enaminone 3 with morpholine
under reflux condition afforded enaminone 4 as shown in Scheme 1.
Scheme 1: Synthesis of compounds 3-5
Treatment of 3 or 4 with N-nucleophile such as hydrazine hydrate and phenyl hydrazine in
absolute ethanol under reflux gave 5-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)-1H-pyrazole (6) and 5-
(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazole (7), respectively (Scheme 2) [26, 27].
Similar treatment of 3 or 4 with hydroxylamine hydrochloride in heated ethanol in the presence of
potassium carbonate afforded only one isolable product that was identified, on the basis of its spectral
and elemental analyses, as the isoxazole derivative 4 (Scheme 2). The structures of the products 6, 7
1
13
and 8 were confirmed by their spectral (MS, IR, H- and C NMR) and elemental analyses data (see
Experimental section). For example, their IR spectra revealed the absence of the carbonyl group present
in the spectrum of the enaminone 3 or 4. The 1H NMR spectrum of 6 showed two characteristic signals
at δ 6.38 with J value of 7.2 Hz, and 7.67 with the same J value of the pyrazole ring protons. Structure
8 was assigned for the reaction products on the basis of the 1H NMR spectral data in which a resonance
for H-4 and H-5 of isoxazole appeared typically at δ 6.38 and 8.37ppm, respectively [28].
Compounds 6-8 were supported by the alternate chemical synthesis via the reaction
of sodium salt of 3-acetyl-2-methyl-4,5-diphenyl-1H-pyrrole (5) with hydrazine hydrate,
phenyl hydrazine, and hydroxylamine hydrochloride in ethanol containing few drops of acetic acid,
respectively (Scheme 1).
Scheme 2: Synthesis of pyrazoles 6, 7 and isoxazole 8
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The reactivity of the enaminone 3 or the morpholinyl derivative 4 towards some heterocyclic
amines were also examined. Thus, reaction of 3 or 4 with 5-amino-1H-pyrazole-4-carbonitrile 9 in
acetic acid under reflux yielded the respective pyrazolo[1,5-a]pyrimidine derivative 10 (Scheme 3).
Similar treatment of 3 or 4 with 5-amino-1,2,4-triazole (11), 5-amino-1H-tetrazole (13), 2-
aminobenzimidazole (15) and 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (17) under the same
reaction conditions afforded the respective 1,2,4-triazolo[1,5-a]pyrimidine 12, tetrazolo[1,5-
a]pyrimidine 14, benzimidazo[1,2-a]pyrimidine 16 and 2,3-dihydropyrido[2,3-d]pyrimidinone 18
derivatives, respectively (Scheme 3).
Scheme 3: Synthesis of fused pyrimidines 10, 12, 14, 16 and 18
Pyrimidine-containing derivatives 10, 12, 14, 16 and 18 were prepared by allowing enaminone
3 or 4 to react with a set of amine-containing heterocycles. The reaction passed through two main steps:
fist, Michael-type addition of the exocyclic amine followed by in situ tandem elimination of
1
dimethylamine (Scheme 4). The H NMR spectrum of each final isolated products; i.e. 10, 12, 14 and
16, revealed two doublets in the aromatic regions δ 8.09- 8.16 and 8.59- 8.76 with J value around 4.5
Hz assignable to the two vicinal protons of the pyrimidine ring [29-31].
Scheme 4: Mechanism of synthesis of fused pyrimidines 10, 12, 14, and 16
Next, pyridinyl analogues were prepared via reactions of the enaminone 3 or 4 with C-
nucleophile such as active methylene compounds. Thus, reaction of 3 or 4 with acetylacetone 19a,
ethyl acetoacetate 19b and ethyl benzoylacetate 19c in acetic acid/ammonium acetate yielded 2-(pyrrol-
3-yl) pyridine derivatives 20a-c, respectively (Scheme 5). As also depicted in Scheme 5, the formation
of 20 seems to start with Michael addition of the active methylene 19 to the activated double bond of 3
followed by tandem elimination of dimethylamine and condensation with ammonia. The other possible
1
isomeric structure 21 was discarded on the basis of spectral data. For example, the H NMR spectrum
of each products 20a-c exhibited two doublet signals in the regions of δ 7.75-7.76 and 8.32-8.39 due to
pyridine H-3 and H-4, respectively with J value around 8.2 Hz. The latter coupling constant value is
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characteristic for ortho H-3 and H-4 pyridine coupling, and much higher than the values of H-2 and H-
3 (J = 4-6 Hz) [32].
Scheme 5: Synthesis of pyridines 20a-c
Reactions of 3 or 4 with malononitrile 22a and ethyl cyanoacetate 22b in the presence of
sodium ethoxide afforded products 23a and 23b, respectively. The assignments of their structures were
based on their spectral and elemental analyses data (see Experimental section). Furthermore, the
structure of 23a was confirmed by an alternative synthesis; i.e. by reaction of 3 with cyanoacetamide
24a under the same reaction conditions (Scheme 6). As discussed before, the formation of 23 was
suggested to start with Michael addition of the active methylene compound 22 to the activated double
bond of 3 followed by tandem cyclization, elimination of dimethylamine and Dimroth type
rearrangement of the formed pyran intermediate to give 23 as the end product (Scheme 6).
.
Scheme 6: Synthesis of pyridinones 23a,b
2.2. Pharmacology
Synthesized compounds were assayed for inhibition of p53 ubiquitination by incubation with
GST-tagged HDM2, immobilized on glutathione-Sepharose, p53, ubiquitin, as well as E1 and E2
(UbcH5B) ligases, in an assay buffer containing ATP as detailed in references [33, 34]. The reaction
products were then resolved by SDS-PAGE and p53 ubiquitination, and were quantified by Western
blotting using an anti-p53 antibody [35].
Tested compounds that pass the preliminary assay; i.e. compounds that showed some activity at
concentrations below 100 µM were then subjected to determination of half-maximal inhibition
concentration (IC50) of p53 ubiquitination using essentially the same assay methodology, except that a
fluorescently labelled form of ubiquitin was used. After completion of the ubiquitination reaction,
excess fluorescent ubiquitin was removed from the immobilized p53-HDM2 complex by
centrifugation, and incorporation of ubiquitin was measured by fluorescence spectroscopy (Table 1).
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Table 1: IC50 of p53 ubiquitination of the newly synthesized compounds (in vitro)
.
Figure 3. Hypothetical binding mode of compound 16 (colored yellow) and the reference
diphenylimidazoline compound (colored light gray) in MDM2 protein (PDB: 1t4f).
To rationalize the observed HMD2 inhibition results, chemical structures of all biologically-
tested compounds were built using Sybyl-X, subjected to energy minimization and docked within p53-
binding site on HDM2 (PDB: 1t4f). The calculated binding mode of the most potent derivative,
compound 16, was presented in Figure 3 (colored yellow) as a representative example together with the
reported diphenylimidazolidine derivative as a reference (colored light grey). Briefly, there is great
similarity between the binding modes of both as hypothesized earlier. In more details, there is complete
overlap between phenyl ring at pyrole position-2 and the phenyl ring of imidazolidine at positon-5. So
far, both phenyl rings was calculated to be docked with a lipophililc pocket close to Leu56. The
terminal benzene ring of the fused aromatic system at pyrrole postion-3 was calculated to interact
favorably with the phenyl ring of Tyr66 and the lipothilic side chain of Ile60 via face to face π-π
interaction (Figure 3).
Our model sheds lights on how diphenylpyrroles interact with HDM2; however calculating the
binding modes of our inhibitors while both proteins interacting with each other is beyond our
computational facilities. The reference compound was found to occupy the pocket normally occupied
by Phe19, Try23, and Leu26 of p53 [23]. Analogously, we are expecting same type of interactions in
case of our newly designed ligands; i.e. the phenyl ring at pyrrole position-4, in addition to the aromatic
system at positon-3 is expected to interact with such lipophilic residues.
Finally, by examining of the SAR at pyrrole postion-3, we came up with some conclusions. The
benzo[4,5]imidazo[1,2-a]pyrimidine linked to pyrrole ring increases the activity more than the 2-
thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one.
The
2-thioxo-2,3-dihydropyrido[2,3-d]
pyrimidin-4(1H)-one increases the activity more than the pyridine. The pyridinone provide more
activity profile than the pyridine. The pyridine moiety give rise to higher activity than the tetrazolo[1,5-
a]pyrimidine one. The tetrazolo[1,5-a]pyrimidine moiety give rise to higher activity than the
pyrazolo[1,5-a] pyrimidine. For pyridine derivatives 23a, b: the ester group provides more activity than
the cyano group. For derivatives 20a-c: the phenyl moiety gives higher activity than the methyl one and
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the ester give higher activity than the acetyl one. Isoxazole moiety increases the activity more than the
pyrazole one.
3. Conclusion
In the investigation described above, a 4,5-diphenyl-3-heteroaryl-pyrroles were introduced as a
new class of p53 activators. The scaffold has the advantage of synthetic protocol accessibility. Briefly,
target compounds were prepared via reactions of E-3-(dimethylamino)-1-(2-methyl-4,5-diphenyl-1H-
pyrrol-3-yl)prop-2-en-1-one or E-1-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)-3-morpholinoprop-2-en-1-
one with C- and N-nucleophiles.
4. Experimental protocols
4.1. Chemistry
4.1.1. General
All melting points were measured on a Gallenkamp melting point apparatus. The infrared spectra were
recorded in potassium bromide disks on a Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared
spectrophotometers. The NMR spectra were recorded on a Varian Mercury VX-300 NMR
1
13
spectrometer. H (300 MHz) and C NMR (75.46 MHz) were run in deuterated dimethylsulphoxide
(DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a
Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 e.V. Elemental analyses were carried out at
the Microanalytical Center of Cairo University, Giza, Egypt.
4.1.2. Reactions of enaminone 3 or 4 with hydrazines. To a solution of the enaminone 3 or 4 (1
mmol) in EtOH (10 mL) was added hydrazine hydrate (1 mL) or phenylhydrazine (1 mL) and the
mixture was heated under reflux for 2 h. The reaction mixture was acidified by HCl / ice mixture and
the formed product was filtered and crystallized from EtOH to give the respective pyrazoles 6 and 7.
4.1.2.1. 5-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)-1H-pyrazole (6). Yield 88%, mp 232 °C; IR (KBr) υ:
3398, 3214 (2NH) cm–1; 1H NMR (DMSO-d6) δ: 1.73 (s, 3H, CH3), 6.37 (d, 1H, pyrazole- H4), 7.10–
7.26 (m, 10H, Ar-H), 7.67 (d, 1H, pyrazole-H5), 10.81 (br., s, 1H, NH, D2O exchangeable), 11.16 (br.,
s, 1H, NH, D2O exchangeable) ppm; MS m/z (%): 300 (M++1, 77), 299 (M+, 100), 210 (93), 176 (72),
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100 (80), 77 (60). Anal. Calcd for C20H17N3 (299.37): C, 80.24; H, 5.72; N, 14.04. Found: C, 80.24; H,
5.72; N, 14.04%.
4.1.2.2. 5-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)-1-phenyl-1H-pyrazole (7): Yield 82%, mp 243-245
1
°C; IR (KBr) υ: 3306 (NH) cm–1; H NMR (DMSO-d6) δ: 1.83 (s, 3H, CH3), 6.71 (d, 1H, pyrazole H-
4), 7.12–7.44 (m, 15H, Ar-H), 7.68 (d, 1H, pyrazole-H5), 11.35 (br., s, 1H, NH, D2O exchangeable);
13C NMR (DMSO-d6): δ 13.7 (CH3), 109.3, 112.3, 117.8, 123.1, 125.7, 126.1, 126.3, 126.4, 126.5,
127.7, 128.0, 128.1, 128.3, 128.4, 129.0, 130.7, 136.2, 139.6, 145.9 (Ar-C) ppm; MS m/z (%): 375
(M+, 55), 318 (52), 256 (57), 172 (48), 92 (85), 77 (100). Anal. Calcd for C26H21N3 (375.47): C, 83.17;
H, 5.64; N, 11.19. Found: C, 83.11; H, 5.48; N, 11.04%.
4.1.3. Alternative synthesis of compound 6. A solution of sodium (2-methyl-4,5-diphenyl-1H-pyrrol-
3-yl)-3-oxoprop-1-en-1-olate (5) (0.650 g, 2 mmol) and hydrazine hydrate (1 mL) in acetic acid (10
mL) was refluxed for 2 hr. After completion of the reaction, the hot reaction mixture was cooled and
the solid products collected by filtration and recrystallized from EtOH gave product 6 in 80% yield,
which were identical in all aspects (m.p., mixed m.p. and spectra) with those obtained from reaction of
enaminone 3 or 4 with hydrazine hydrate.
4.1.4. Synthesis of 3-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)isoxazole (8)
Hydroxylamine
hydrochloride (0.07 g, 1 mmol) was added to a mixture of enaminone 3 or 4 (1 mmol) and anhydrous
potassium carbonate (0.5 g) in absolute EtOH (20 mL). The mixture was heated under reflux for 5 h
and poured onto water. The solid product was filtered and crystallized from ethanol to give compound
1
8 in 86% yield, mp: 228-230 °C; IR (KBr) υ: 3251 (NH) cm–1; H NMR (DMSO-d6) δ: 1.82 (s, 3H,
CH3), 6.42(d, 1H, isoxazole- H4), 7.19-7.45 (m, 10H, Ar-H), 8.42 (d, 1H, isoxazole-H5), 11.66 (br., s,
1H, NH, D2O exchangeable) ppm; MS m/z (%): 301 (M++1, 78), 300 (M+, 52), 271 (82), 154 (95), 77
(100). Anal. Calcd for C20H16N2O (300.35): C, 79.98; H, 5.37; N, 9.33. Found: C, 79.69; H, 5.32; N,
9.18%.
4.1.5. Reactions of enaminone 3 or 4 with heterocyclic amines.
General procedure: A mixture of enaminone 3 or 4 (5 mmol) and the appropriate 5-amino-1H-
pyrazole-4-carbonitrile 9, aminotriazole 11, aminotetrazole 13, 2-aminobenzimidazole 15, or 6-amino-
2-thioxo-2,3-dihydropyrimidin-4(1H)-one 17 (5 mmol) in acetic acid (20 mL) was refluxed for 6 h. The
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reaction mixture was cooled and diluted with MeOH and the solid product was collected by filtration
and recrystallized from the suitable solvent gave products 10, 12, 14, 16 or 18, respectively. The
synthesized compounds together with their physical and spectral data are listed below.
4.1.5.1. 7-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile (10): Yield
1
76%, mp 262-264 °C; IR (KBr) υ: 3311 (NH), 2229 (CN) cm–1; H NMR (DMSO-d6) δ: 1.83 (s, 3H,
CH3), 7.07-7.56 (m, 10H, Ar-H), 8.34(1H, d, pyrimidine-H4), 8.42(1H, s, pyrazole-H), 9.59(1H, d,
pyrimidine-H6), 11.59 (br., s, 1H, NH, D2O exchangeable) ppm; MS m/z (%): 375 (M+, 52), 331 (97),
218 (100), 189 (94), 102 (92), 87 (94), 64 (100). Anal. Calcd for C24H17N5 (375.43): C, 76.78; H, 4.56;
N, 18.65. Found: C, 76.58; H, 4.66; N, 18.41%.
4.1.5.2. 7-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (12): Yield 78%,
1
mp 303-305 °C; IR (KBr) υ: 3249 (NH) cm−1; H NMR (DMSO-d6) δ: 1.83 (s, 3H, CH3), 7.03–7.44
(m, 10H, Ar-H), 8.14(1H, d, pyrimidine-H), 8.59(1H, d, pyrimidine-H), 9.03(1H, s, triazole-H), 11.58
13
(br., s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6): δ 13.0 (CH3), 116.1, 117.5, 118.7, 122.0,
125.6, 126.0, 126.5, 126.8, 128.0, 128.2, 128.9, 129.6, 129.7, 130.7, 132.1, 135.1, 137.0, 144.3, 146.4
(Ar-C) ppm; MS m/z (%): 352 (M++1, 49), 351 (M+, 100), 260 (83), 218 (73), 189 (67), 77 (78). Anal.
Calcd for C22H17N5 (351.40): C, 75.19; H, 4.88; N, 19.93. Found: C, 75.10; H, 4.76; N, 19.76%.
4.1.5.3. 7-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)tetrazolo[1,5-a]pyrimidine (14): Yield 76%, mp 256-
1
257 °C; IR (KBr) υ: 3266 (NH) cm−1; H NMR (DMSO-d6) δ: 1.75 (s, 3H, CH3), 7.12–7.37 (m, 10H,
Ar-H), 8.14(1H, d, pyrimidine-H), 8.52(1H, d, pyrimidine-H), 11.59 (br., s, 1H, NH, D2O
exchangeable) ppm; MS m/z (%): 353 (M++1, 68), 352 (M+, 100), 264 (86), 244 (86), 203 (89), 129
(79), 77 (76). Anal. Calcd for C21H16N6 (352.39): C, 71.58; H, 4.58; N, 23.85. Found: C, 71.62; H,
4.51; N, 23.62%.
4.1.5.4. 4-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)benzo[4,5]imidazo[1,2-a]pyrimidine (16): Yield
1
76%, mp 256-257 °C; IR (KBr) υ: 3251 (NH) cm−1; H NMR (DMSO-d6) δ: 1.75 (s, 3H, CH3), 7.12–
7.37 (m, 14H, Ar-H), 8.14(1H, d, pyrimidine-H), 8.59 (1H, d, pyrimidine-H), 11.59 (br., s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6): δ 12.5 (CH3), 108.2, 114.4, 117.8, 122.0, 125.1, 125.7,
126.0, 126.5, 126.8, 126.9, 127.8, 128.1, 128.2, 128.7, 129.5, 130.7, 132.1, 135.1, 137.0, 139.8, 140.3,
145.9(Ar-C) ppm; MS m/z (%): 401 (M++1, 67), 400 (M+, 100), 355 (67), 296 (85), 229 (58), 128 (69),
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64 (60). Anal. Calcd for C27H20N4 (400.47): C, 80.98; H, 5.03; N, 13.99. Found: C, 80.77; H, 5.01; N,
13.67%.
4.1.5.5. 5-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-
one (18): Yield 72%, mp 326-328 °C; IR (KBr) υ: 3423, 3316, 3203 (3 NH), 1668 (CO)cm−1; 1H NMR
(DMSO-d6) δ: 1.75 (s, 3H, CH3), 7.11–7.37 (m, 11H, Ar-H, NH), 7.59 (d, 1H, pyridine-H3), 8.38 (d,
1H, pyridine- H2), 11.59, 12.46 (br., s, 2H, 2NH, D2O exchangeable) ppm; MS m/z (%): 411 (M++1,
73), 410 (M+, 85), 362 (86), 271 (100), 220 (97), 181 (97), 87 (81). Anal. Calcd for C24H18N4OS
(410.49): C, 70.22; H, 4.42; N, 13.65. Found: C, 70.01; H, 4.38; N, 13.36%.
4.1.6. Alternative synthesis of compounds 10. A solution of sodium (2-methyl-4,5-diphenyl-1H-
pyrrol-3-yl)-3-oxoprop-1-en-1-olate (5) (0.650 g, 2 mmol) and 3-amino-4-cyanopyrazole 9 (0.216 g, 2
mmol) and piperidine acetate (1 mL) in water (3 mL) was refluxed for 10 min. After completion of the
reaction, the hot reaction mixture was neutralized with acetic acid (1.5 mL), then cooled and the solid
products collected by filtration and recrystallized from EtOH gave product 10 in 84% yield, which
were identical in all aspects (m.p., mixed m.p. and spectra) with those obtained from reaction of
enaminone 3 or 4 with 9.
4.1.7. Preparation of compounds 20a-c. To a solution of 3 or 4 (5 mmol) in glacial acetic acid (20
mL) was added the appropriate active methylene compound (acetylacetone 19a or ethyl acetoacetate
19b or ethyl benzoylacetate 19c) (5 mmol) and ammonium acetate (0.5 g, 6 mmol). The reaction
mixture was heated under reflux for 20-30 hours. The reaction was followed by TLC. The reaction
mixture poured into cold water, the solid product that precipitated was filtered off and crystallized from
ethanol to give the respective product 13. The compounds 13a-c prepared together with their physical
constant are given bellow.
4.1.7.1. 1-(2-Methyl-6-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)pyridin-3-yl)ethanone (20a). Yield 70%,
1
mp 243-245 °C; IR (KBr) υ: 3262 (NH), 1708 (CO)cm−1; H NMR (DMSO-d6) δ: 1.83 (s, 3H, CH3),
2.48 (s, 3H, CH3), 2.68 (s, 3H, CH3), 7.19–7.47 (m, 10H, Ar-H), 7.72 (d, 1H, pyridine-H3), 8.35 (d,
13
1H, pyridine- H2), 11.67 (br., s, 1H, 1NH, D2O exchangeable); C NMR (DMSO-d6): δ 13.7 (CH3),
28.7 (CH3), 30.4 (CH3), 109.3, 112.1, 112.3, 117.8, 119.7, 122.0, 126.0, 126.5, 126.8, 128.1, 128.2,
130.7, 132.1, 135.1, 137.0, 145.0, 146.3 (Ar-C), 194.4 (C=O) ppm; MS m/z (%): 367 (M++1, 20), 366
10

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(M+, 32), 260 (100), 230 (56), 189 (33), 102 (37), 77 (84). Anal. Calcd for C25H22N2O (366.45): C,
81.94; H, 6.05; N, 7.64. Found: C, 81.68; H, 6.01; N, 7.56%.
4.1.7.2. Ethyl 2-methyl-6-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)nicotinate (20b). Yield 74%, mp 214-
1
216 °C; IR (KBr) υ: 3212 (NH), 1723 (CO)cm−1; H NMR (DMSO-d6) δ: 1.35 (t, 3H, CH3), 1.82 (s,
3H, CH3), 2.57 (s, 3H, CH3), 4.29 (q, 2H, CH2), 7.19–7.51 (m, 10H, Ar-H), 7.57 (d, 1H, pyridine-H3),
13
8.38 (d, 1H, pyridine- H2), 11.67 (br., s, 1H, 1NH, D2O exchangeable) ppm; C NMR (DMSO-d6): δ
10.5(CH3),13.7(CH3), 30.4 (CH3), 66.0 (CH2), 114.9, 116.5, 118.6, 122.0, 126.0, 126.5, 126.8, 128.1,
128.2, 129.1, 1296, 130.1, 130.6, 132.1, 135.1, 137.0, 140.1 (Ar-C), 173.4 (C=O) ppm; MS m/z (%):
397 (M++1, 47), 396 (M+, 96), 248 (81), 157 (74), 136 (83), 77 (100). Anal. Calcd for C26H24N2O2
(396.48): C, 78.76; H, 6.10; N, 7.07. Found: C, 78.65; H, 6.03; N, 7.02%.
4.1.7.3. Ethyl 6-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)-2-phenylnicotinate (20c). Yield 71%, mp 266
1
°C; IR (KBr) υ: 3248 (NH), 1717 (CO)cm−1; H NMR (DMSO-d6) δ: 1.24(t, 3H, CH3), 1.76 (s, 3H,
CH3), 4.29 (q, 2H, CH2), 7.11–7.39 (m, 15H, Ar-H), 7.86 (d, 1H, pyridine-H3), 8.49 (d, 1H, pyridine-
H2), 11.56 (br., s, 1H, 1NH, D2O exchangeable) ppm; MS m/z (%): 459 (M++1, 34), 458 (M+, 100),
324(81), 203 (67), 157 (56), 77 (78). Anal. Calcd for C31H26N2O2 (458.55): C, 81.20; H, 5.72; N, 6.11.
Found: C, 81.03; H, 5.58; N, 6.03%.
4.1.8. Preparation of compounds 23a,b. To compound 3 or 4 (5 mmol) in solution of sodium
ethoxide in ethanol (0.12 g of sodium metal in 20 mL absolute ethanol) was added malononitrile 22a or
ethyl cyanoacetate 22b (5 mmol). The reaction mixture was refluxed for 5 h., and then poured into ice-
cold water. The solution was acidified with HCl (1 M) and the solid that deposited was filtered and
crystallized from ethanol to give the respective compounds 23a, b.
4.1.8.1. 6-(2-Methyl-4,5-diphenyl-1H-pyrrol-3-yl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (23a).
1
Yield 75%, mp 203-205 °C; IR (KBr) υ: 3406, 3212 (2NH), 2214 (CN), 1657 (CO)cm−1; H NMR
(DMSO-d6) δ: 1.82 (s, 3H, CH3), 7.19–7.61 (m, 10H, Ar-H), 8.01 (d, 1H, pyridine-H), 8.57 (d, 1H,
pyridine-H), 11.66 (br., s, 1H, 1NH, D2O exchangeable), 12.29 (br., s, 1H, 1NH, D2O exchangeable)
ppm; MS m/z (%): 352 (M++1, 57), 351 (M+, 76), 270 (100), 210 (79), 144 (80), 101 (61), 63 (71).
Anal. Calcd for C23H17N3O (351.40): C, 78.61; H, 4.88; N, 11.96. Found: C, 78.43; H, 4.39; N,
11.72%.
11

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4.1.8.2. Ethyl 6-(2-methyl-4,5-diphenyl-1H-pyrrol-3-yl)-2-oxo-1,2-dihydropyridine-3-carboxylate
1
(23b). Yield 77%, mp 189-191 °C; IR (KBr) υ: 3387, 3231 (2NH),1712, 1657 (2CO)cm−1; H NMR
(DMSO-d6) δ: 1.34 (t, 3H, CH3), 1.82 (s, 3H, CH3), 4.24 (q, 2H, CH2), 7.11–7.56 (m, 10H, Ar-H), 8.05
(d, 1H, pyridine-H), 8.56 (d, 1H, pyridine-H), 11.56 (br., s, 1H, 1NH, D2O exchangeable), 12.13 (br., s,
1H, 1NH, D2O exchangeable) ppm; MS m/z (%): 352 (M++1, 57), 351 (M+, 76), 270 (100), 210 (79),
144 (80), 101 (61), 63 (71). Anal. Calcd for C25H22N2O3 (398.45): C, 75.36; H, 5.57; N, 7.03. Found:
C, 75.23; H, 5.53; N, 6.91%.
4.1.9. Alternative synthesis of compound 23a. To a solution of 3 (2.11 g, 5 mmole) in ethanolic
sodium ethoxide solution (0.12 g. 5 mmol of sodium metal in 20 ml absolute ethanol) was added
cyanoacetamide 24a (0.42 g, 5 mmol). The reaction mixture was refluxed for 20 h., and the solid that
precipitated was filtered off and washed with water then crystallized from ethanol to give compound
23a which was found identical in all respects with that compound produced from reaction of compound
3 with malononitrile.
4.2. Pharmacology
4.2.1. Biological assay In vitro ubiquitination assay. The expression of recombinant glutathione-S-
transferase-tagged MDM2 (GST-MDM2) was induced in 25 mL culture of exponentially growing
Escherichia coli BL21 cells (OD600 0.6) by 1 mM isopropyl-thio-β-D-galactoside for 3 h. Glutathione-
S-transferase-MDM2 was purified on glutathione-sepharose beads (Amersham). The beads were
washed with 50 mM Tris (pH 7.5).
Fluorescent ubiquitin (5 µg; Invitrogen), 50 ng mammalian E1 (Enzo), 200 ng human recombinant
UbcH5B E2 (Enzo) and 200 ng His-p53 (Enzo) were mixed with reaction buffer [50 mM Tris pH 8, 2
mM dithiothreitol, 5 mM MgCl2, 2 mM adenosine triphosphate (ATP)]. A dose titration of each MPD
compound or dimethyl sulfoxide (DMSO) was added to the mixture and the mixture was pipetted onto
°
GS4b-MDM2 beads. The reaction was incubated at 37 C, shaking at 1200 r.p.m. for 1 h and then
stopped by the addition of 3X sodium dodecyl sulfate sample buffer. Free fluorescent ubiquitin was
washed off and total fluorescent ubiquitin signal was measured on a monochromator plate reader
(Safire).
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4.2.2. For non-fluorescent in vitro ubiquitination assays: the procedure was as above except 5 µg
unlabeled ubiquitin (Enzo) was used. Ten micrometers of each tested compound or DMSO was added
to the mixture and then pipetted onto GS4b-MDM2 beads. After incubation, as described previously,
reaction products were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
analyzed by western blotting with anti-p53 DO-1. For the MDM2 RING autoubiquitination assay,
GS4b-MDM2 RING beads were prepared as above and used in place of the full-length MDM2.
Abbreviations: ARF, alternative reading frame; DMEM, Dulbecco’s modified Eagle’s medium;
DMSO, dimethyl sulfoxide, HLI98, HDM2 ligase inhibitor 98 class.
For the MDM2 autoubiquitination assay, the procedure was performed as detailed above except that no
His-p53 was added to the reaction and western blotting was with Ab1 and Ab2 antibodies.
For the Cbl autoubiquitination assay, bacterially expressed full-length Cbl was used in a reaction with
the previously described quantities of E1, E2 and ubiquitin. Ubiquitinated Cbl was detected by western
blotting using antiubiquitin antibody (Sigma clone 6C1).
4.3. Molecular Modeling. The structures of newly synthesized compounds were built with Sybyl-X
software and minimized to 0.01kcal/mol by the Powell method, using Gasteiger-Hückel charges and
the Tripos force field. The proteins coordinates have been downloaded from Protein Data Bank website
(PDB IDs: 1t4f). The water molecules and all other substructures including p53 residues were removed.
The hydrogen atoms were added and the energy of the protein was minimized using the Amber force
field with Amber charges. The energy-optimized lignads were docked into the binding site (close to
atomno. 654) in the protein using GOLD [36]. The parameters were set as the default values for
GOLD. The maximum distance between hydrogen bond donors and acceptors for hydrogen bonding
was set to 3.5 Å. After docking, the top three poses conformations of each lignad were merged into the
ligand-free protein. The new ligand–protein complexes were subsequently subjected to energy
minimization using the Amber force field with Amber charges. The energy minimization, in all cases,
was performed using the Powell method with a 0.05 kcal/(mol Å) energy gradient convergence
criterion and a distance dependent dielectric function.
The authors have declared no conflict of interest.
13

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Table 1: IC50 of p53 ubiquitination of the newly synthesized compounds (in vitro).
Compound No
IC50 of p53 ubiquitination
0.99
0.97
6
7
0.94
8
0.91
10
0.89
12
0.84
14
0.36
16
0.44
18
0.81
20a
0.78
20b
0.61
20c
23a
0.57
0.55
23b
Diph. Imidazole
0.26(17)

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CH3
OH
R2
H3C
N
R1
Cl
Cl
O
N
O
N
N
X
HN
N
HN
N
O
N
OMe
N
N
O
O
O
N
H
H3C
O
N
N
O
NH
O2N
Flavin (X = N)
Dazaflavin (X = CH)
CH3
HL198
Diphenylimidazoline
HL1373
Chart 1. Chemical structures of some reported HDM2 ligase inhibitors.
Chart 2. Work Design
OH
Cl
N
O
N
N
N
N
p53
OMe
MDM2
N
N
O
H
Cl
1

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Chart 3. Alignment of reported (black) and newly (red) derived structures. The diphenyl rings of the
lead structure (black) were reported to interact with MDM2 (PDB: 1rv1), leaving the other two
groups facing p53.
Figure 3. Hypothetical binding mode of compound 16 (colored yellow) and the reference
diphenylimidazoline compound (colored light gray) in MDM2 protein (PDB: 1t4f).
2

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Scheme 1: Synthesis of compounds 3-5
1

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Scheme 2: Synthesis of pyrazoles 6, 7 and isoxazole 8
2

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Scheme 3: Synthesis of fused pyrimidines 10, 12, 14, 16 and 18
3

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HN
HN
H2N
O
O
Y
N
Ph
Ph
H
9,11,13,15
-HY
CH3
N
Ph
Ph
CH3
N
N
H
H
3 or 4
Y = NMe2 or
O
HN
N
N
HO
Ph
Ph
N
-H2O
Ph
CH3
CH3
Ph
N
N
H
H
10,12,14,16
Scheme 4: Mechanism of synthesis of fused pyrimidines 10, 12, 14, and 16
4

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Scheme 5: Synthesis of pyridines 20a-c
5

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Scheme 6: Synthesis of pyridinones 23a,b
6

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Diphenylpyrroles: Novel p53 Activators
Sobhi M. Gomhaa*, Taha M. A. Eldebss,a Mohamed M. Abdullab and Abdelrahman S. Mayhoubc
aDepartment of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
bResearch Unit, Saco Pharm. Co., 6th October City, Egypt
cDepartment of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, 11884 Egypt
Table of Contents
Page
1H NMR spectrum of compound 6
1H NMR spectrum of compound 7
13C NMR spectrum of compound 7
1H NMR spectrum of compound 8
1H NMR spectrum of compound 10
1H NMR spectrum of compound 12
13C NMR spectrum of compound 12
1H NMR spectrum of compound 16
13C NMR spectrum of compound 16
1H NMR spectrum of compound 18
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
1H NMR spectrum of compound 20a S12
13C NMR spectrum of compound 20a S13
1H NMR spectrum of compound 20b S14
13C NMR spectrum of compound 20b S15
1H NMR spectrum of compound 20c S16
1H NMR spectrum of compound 23a S17
S1