8-Triazolylpurines: Towards fluorescent inhibitors of the MDM2/p53 interaction

Article abstract of DOI:10.1371/journal.pone.0124423

Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an α-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.

Full text of DOI:10.1371/journal.pone.0124423

RESEARCH ARTICLE
8-Triazolylpurines: Towards Fluorescent
Inhibitors of the MDM2/p53 Interaction
Mariell Pettersson1☯, David Bliman1☯, Jimmy Jacobsson1, Jesper R. Nilsson2, Jaeki Min3,
Luigi Iconaru4, R. Kiplin Guy3, Richard W. Kriwacki4, Joakim Andréasson2,
Morten Grøtli1*
1 Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden,
2 Department of Chemical and Biological Engineering/Chemistry and Biochemistry, Chalmers University of
Technology, Gothenburg, Sweden, 3 Department of Chemical Biology and Therapeutics, St. Jude Children’s
Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee, United States of America,
4 Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place,
Memphis, Tennessee, United States of America
a11111
☯ These authors contributed equally to this work.
* grotli@chem.gu.se
Abstract
OPEN ACCESS
Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein in-
teraction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regula-
tory pathways in a cell, and the ability to control and modulate PPIs is therefore of great
significance to basic biology, where controlled disruption of protein networks is key to under-
standing network connectivity and function. We have designed and synthesised two series
of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed
based on low energy conformations but did not display any biological activity in a biochemi-
cal fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conforma-
tion studies demonstrated that such molecules could mimic the topography of an α-helix,
docking studies indicated that the same compounds were not optimal as inhibitors for the
MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combi-
nation of docking studies and analysis of recently published inhibitors. The best compound
displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluores-
cence polarisation assay. In order to evaluate the applicability of these compounds as bio-
logically active and intrinsically fluorescent probes, their absorption/emission properties
were measured. The compounds display fluorescent properties with quantum yields up to
50%.
Citation: Pettersson M, Bliman D, Jacobsson J,
Nilsson JR, Min J, Iconaru L, et al. (2015) 8-
Triazolylpurines: Towards Fluorescent Inhibitors of
the MDM2/p53 Interaction. PLoS ONE 10(5):
e0124423. doi:10.1371/journal.pone.0124423
Academic Editor: A Ganesan, University of East
Anglia, UNITED KINGDOM
Received: December 31, 2014
Accepted: March 2, 2015
Published: May 5, 2015
Copyright: © 2015 Pettersson et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the Swedish
Research Council (project # 62120083533), the
American Lebanese Syrian Associated Charities
(ALSAC) and St Jude Children's Research Hospital.
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Introduction
Protein-protein interactions (PPIs) mediate virtually all important biological regulatory path-
ways [1], and the ability to control and modulate PPIs is therefore of great significance to basic
biology, where the controlled disruption of PPI networks is key to understanding network
Competing Interests: The authors have declared
that no competing interests exist.
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8-Triazolylpurines as MDM2/p53 Inhibitors
connectivity and function. It is also becoming increasingly clear that the modulation of PPIs of-
fers enormous opportunities in drug discovery for medical diagnostics and treatment.
Designing small molecule inhibitors of PPIs poses a substantial challenge due to PPIs gener-
ally shallow interaction sites and large surface area as compared with more traditionally tar-
geted enzyme active sites [2]. However, small regions consisting of a collection of residues that
constitute the majority of the free binding energy have been identified and are generally re-
ferred to as “hot spots” [3]. These hot spots are often amino acid residues protruding from one
face of an α-helix at the interaction surface [4]. A mimetic that reproduces the key interactions
of the α-helix should bind to the target binding site of the α-helix.
Tumour protein p53 is crucial in multicellular organisms, where it regulates the cell cycle
and functions as a tumour suppressor [5, 6]. All known tumour cells either mutate the p53
gene, or use internal cell p53 modulators like MDM2 and MDMX to disable its function. Re-
leasing functional p53 from inhibition by MDM2 and MDMX should, in principle, provide an
efficient, nongenotoxic means of cancer therapy.
The p53 protein binds to MDM2 and MDMX using a short helix with a “hot spot triad”
consisting of p53’s Trp23, Leu26, and Phe19 [7]. A number of elegant examples of nonpeptidic
α-helix mimetics that inhibit the MDM2/p53 interaction by targeting these hot spots have
been published [8]. These inhibitors can be divided into three subcategories: type I, II and III
[9]. Type I inhibitors include stabilised oligomers that are designed to mimic the α-helical to-
pography. The second type of inhibitors, functional mimetics, are based on scaffolds that place
substituents in the spatial orientation of the parent helix, but the scaffolds themselves are not
designed to mimic the α-helix topography. Notable examples of type II inhibitors include the
nutlins [10], piperidinones [11] and spiroindolines [12]. These scaffolds vary widely in struc-
ture but share the common denominator that they can arrange the substituents in analogy with
the i, i+4 and i+7 amino acid side chains of an α-helical structure. In the third category of in-
hibitors, the nonpeptidic scaffold mimics the topography of an α-helix with substituents pro-
truding in the positions of the i, i+4 and i+7 amino acid side chains of an α-helical structure.
Examples from type III include terphenyl [13, 14], and 8-amido-pyrrolopyrimidines[15].
Purines widely occur in nature [16] and are recognised as privileged structures in drug dis-
covery [17]. We have previously developed 8-(triazolyl) purines as fluorescent base analogues
[18, 19] and hypothesised that 8-(triazolyl)purines with substituents in the 2- and 9-positions
would project their substituents in analogy with the residues of one face of an α-helix (Fig 1).
7-Deazapurines have previously been reported as inhibitors for MDM2/p53 [15]. Since triazoles
are recognised as bioisosters of amide bonds [20–22], we chose to evaluate 8-(triazolyl) purines
as α-helix mimetics. Furthermore, given the fluorescent properties of 8-triazolylpurines that
were previously reported by our group, we expected 2,9-substituted 8-triazolylpurines to display
similar characteristics. Compounds that display both biological activity and intrinsically fluores-
cent properties would be extremely useful for all applications in which intracellular localisation
of the bio-relevant molecule is of interest.
Here, we have evaluated a series of 2,9-substituted 8-(triazolyl)purines as α-helix mimetics.
The UV/vis absorption and fluorescence properties of these compounds have also
been characterised.
Results and Discussion
Type III inhibitors
Design. Comparison of low energy conformations of 8-(triazolyl)purine derivatives with
an idealised α-helix (Fig 1c) indicated that the scaffold arranges substituents in the N2, 9 and
on the triazole analogous to the i, i+4, i+7 residues (Fig 1).
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 1. Overview of 8-(triazolyl)purine design. Schematic representation of a) an α-helix with residues
present on one face of the helix (i, i+4 and i+7) denoted as black spheres, and b) the 8-triazolyl scaffold with
R-groups denoted as black spheres. c) Superimposition of Ala-helix (black), with low energy conformations of
8-(1,4-triazolyl)-purine (turquoise) and 8-(1,5-triazolyl)-purine (yellow).
doi:10.1371/journal.pone.0124423.g001
The regiochemistry of the triazole can be controlled by the choice of catalyst, which provides
a possibility to access compounds with structural diversity. Performing the cyclisation in the
presence of a Cu(I) catalyst results in the 1,4-triazole [23]. Changing the catalyst system to Ru
(II) results in the 1,5-triazole with high regioselectivity [24]. The 1,5-regioisomer can alterna-
tively be obtained with Me4N+OH- as catalyst in DMSO [25]. Alkyl- and benzylazides can be
obtained from readily available alkylhalides, enabling chemical diversity for the introduction of
the R3 substituent. The terminal alkyne required for the cyclisation could be accessible through
a palladium-catalyzed Sonagashira-type C-C coupling. Activation of the 8-position with a ha-
lide would set it up for the C-C coupling. The R4 substituent was intended to be introduced by
a nucleophilic aromatic substitution (SNAr). The precedence in the literature [26] for this type
of reaction in the 6-position of purine derivatives and the commercial availability of 6-chloro-
purines made this strategy a reasonable choice. Possible strategies for the introduction of sub-
stituents in the N2 and 9-positions include nucleophilic substitution of alkyl halides, reductive
amination (N2) or alternatively Mitsunobu-type reactions with primary and secondary
alcohols.
Synthesis. The i, i+4 and i+7 residues on the α-helix of p53, that are generally recognised
as key residues for MDM2 binding, are all hydrophobic (leucine, tryptophan and phenylala-
nine), which led to the selection of a series of hydrophobic substituents in R1-3 for our com-
pounds. The initial route to 2,6,8,9-substituted purines started with alkylation of the 9 position
using alkyl or benzyl halide followed by the introduction of a substituent in the N2 position via
reductive amination. While the alkylation in the 9-position produced reasonable yields using
3-bromo-2-methylpropene (73%), benzyl bromide (59%) and phenethyl bromide (93%) [27],
the reductive amination using NaCNBH3 or NaBH(OAc)3 failed due to the low reactivity of
the 2-amino substituent. The major product generated in the reaction was the alcohol product
from reduction of the corresponding aldehyde. We then turned to alkylation of the N2 position
despite expected problems with bis-substitution. As expected, alkylation with aryl halides, po-
tassium iodide and potassium carbonate as base at N2 resulted in a mixture of mono- and bis-
substitution. Although these species were easily separated by column chromatography, this
route failed to generate sufficient quantities of the 2,9-substituted intermediate. It was therefore
decided to investigate alternative strategies to achieve these substitutions.
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 2. Synthesis of compound 1–6. Reaction conditions: (a) R1OH, DIAD, Ph3P, THF. (b) R2OH, ADDP,
nBu3P, THF. (c) DMF, MW, 180°C. (d) PyBr3, CH2Cl2. (e) (i) Pd(II)Cl2(PPh3)2, Cu(I)I, Amberlite IRA-67, TMS-
acetylene, MW, 110°C. (ii) PS-F, THF.
doi:10.1371/journal.pone.0124423.g002
Fletcher et al. have published a synthesis of 2,9-substituted guanines starting from 6-chloro-
2-amino-purine [28, 29]. The synthesis involves Boc protection in the N2-position (1) in order
to activate it for Mitsunobu coupling (Fig 2). The regioselectivity in the two consecutive Mitsu-
nobu couplings relies on the greater reactivity of the 9-position. This protocol proved success-
ful and allowed us to gain access to 3a-c. Yields were typically high in both couplings, ranging
from 74–91% (2a-c) for reactions in the 9-position and 82–94% (3a-c) on the exocyclic
N2-amine.
The chloro substituent in the 6-position of compound 3 is susceptible to nucleophilic aro-
matic substitution (Fig 2). The dimethylamino substituent was introduced by heating 3a-e in
DMF at 180°C [30]. These reaction conditions also remove the Boc protecting group in the
N2-position, resulting in 4a-c in 82–95% yield.
Regarding the next step, activating the 8-position towards C-C-cross coupling by introduc-
tion of a bromide, we recently published a procedure for 8-bromination of electron-rich purine
derivative using PyBr3 in DCM [27]. This procedure afforded 5a-e in high yields, between 84
and 93%. It is worth mentioning that the bromination is regiospecific and does not react with
aromatic substituents.
The terminal alkyne needed for the cyclization was introduced by a Sonogashira-type C-C
cross coupling catalysed by PdCl2(PPh3)2 and CuI. Using a solid supported tertiary amine
(AmberliteTM IRA67) as base facilitates the workup, and deprotection of the trimethylsilyl
group can be carried out on the crude product after filtration to remove the base. Trimethylsilyl
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 3. Synthesis of 7a-c and 8a-b. Reaction conditions: (a) NaN3, R3-X, Cu(I)I, NaAsc, DMEDA, DMF. (b) Cp*RuCl(PPh3)2, MW, 120°C, DMF.
doi:10.1371/journal.pone.0124423.g003
removal using polymer supported fluoride (PS-F) in THF afforded 6a-e in yields of 57–74%
over two steps (Fig 2). Here again, the use of a polymer-supported reagent facilitates the work-
up of the reaction.
1,4-triazoles can be formed with high regioselectivity using a Cu(I) catalyst, a reaction re-
ferred to as Copper Catalysed Azide Alkyne Cyclisation (CuAAC). 7a was obtained by reacting
alkyne 6b with benzyl azide in the presence of CuI, sodium ascorbate and DMEDA at room
temperature overnight (Fig 3). To avoid the need to isolate low molecular weight, potentially
explosive alkyl azides, isobutyl azide was formed immediately prior to use from isobutyl bro-
mide and sodium azide in DMF. Alkyne, CuI, sodium ascorbate and DMEDA were then
added to the azide solution. 1,4-triazolepurines 7a–c were isolated in 55–82% yields (Fig 3).
1,5-Triazoles are accessible by ruthenium-catalysed cyclisation of azides and alkynes [24] and a
method for sequential preformation of low molecular weight azides has been reported [31]. Re-
acting alkyne 6b with benzyl azide afforded 8a in 22% yield and when 1,5 cyclization with iso-
butylazide was attempted, the expected product (8b) was isolated in very low yield after
extensive purification (Fig 3). The low yield can be attributed, at least in part, to difficulties in
separating the product from unidentified by-products.
Evaluation against MDM2/p53. Compounds 7a-c and 8a-b were evaluated as MDM2 in-
hibitors in a fluorescence polarisation (FP) assay that measures displacement of a wild-type
p53 peptide tagged with a fluorescent probe (Texas Red) bound to MDM2. Unfortunately, the
compounds showed no activity at the concentrations tested (up to 47 μM). However, these re-
sults prompted us to investigate the solution conformation.
Solution conformation analysis. The ability of 8-triazolylpurines to act as α-helix
mimetics is dependent on the conformation of the triazole moiety in relation to the purine
since this determines the relative positions of the R1 and R3 substituents. The conformation of
8a in solution was determined by a combined NMR spectroscopic and computational analysis
utilizing the NMR Analysis of Molecular Flexibility (NAMFIS) algorithm [32]. NAMFIS has
previously been employed for description of the solution conformations of oligopeptides [33]
and small molecules [34], and to evaluate the effect of interstrand hydrogen bonding on β-
hairpin stability of cyclic peptides [35]. Experimental proton-proton distances were derived
from a NOESY buildup using the initial rate approximation with five mixing times between 80
and 800 ms, ensuring linearity. Theoretically available conformations were predicted using
Monte Carlo conformational searches along with Molecular Mechanics Minimisation
(MCMM). In order to ensure that the entire conformational space available for the compound
was sampled, the conformational search was performed with two different force fields (AmberÃ
and OPLS-2005) and three different solvent models (chloroform, water and octanol). The con-
formational ensembles were combined and the redundant conformations were removed. Using
the NAMFIS algorithm the conformations existing in solution were selected based on the ex-
perimentally obtained distances (NOE).
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 4. Conformational analysis of 8a. a) NOE correlations (green arrows) in proximity of the triazole ring
observed for 8a. b) Sideview and c) topview of one of the conformations found in the NAMFIS-analysis. (All 6
NAMFIS conformations can be found in Fig. D and E in S1 File).
doi:10.1371/journal.pone.0124423.g004
NAMFIS analysis identified an ensemble of 6 conformations in solution, including confor-
mations that place the substituent in the same spatial orientation as the side chains of an ideal-
ised α-helix (Fig 4). The R2 substituent was omitted from the analysis since the goal of the
conformation analysis was to determine the distance and relative orientation of the R1 and R3
substituents. The distance and orientation between R2 and R1 is determined by the rigid and
flat purine ring system. An analysis of this part of the molecule would add little or no informa-
tion about the relative positions of the R1 and R3 substituents.
The solution conformations for the 1,4-regioisomer (7a) were elucidated using the same
method. The experimental distances between protons on the triazole ring and the R1 and R3
substituents (see Fig 5) were determined by running NOE-buildups with 5 mixing times (80,
200, 400, 600, 800 and 1000 ms). As only two NOE correlations from the triazole H-X are
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 5. NOE correlations observed for 7a. NOE correlations in proximity of the triazole ring observed for 7a.
doi:10.1371/journal.pone.0124423.g005
observed for the 1,4-regioisomer (Fig 5), the conformational ensemble could not be reliably de-
termined (overfitting).
The aqueous solubility of compound 7c was measured and shown to be low (0.3 μM at pH
7.4). Although these compounds are suspected (and supposed) to be lipophilic, the solubility
was too low to be practical for biological studies. A more hydrophilic substituent in the 6-posi-
tion could increase the aqueous solubility of the compounds.
In summary, the solution conformational studies showed that the 8-triazolylpurines scaffold
should be able to place the R1 and R3 substituents in a spatial orientation that mimics the α-
helical conformation. The analysis did not, however, reveal why this series was inactive against
MDM2/p53 in the FP assay. Since the compounds were inactive as inhibitors against MDM2/
p53 interaction at the concentrations evaluated and also showed poor solubility, re-evaluation
of the design was undertaken, opting for type II inhibitors.
Type II inhibitors
Design. During our work with the 8-triazolylpurines, new crystal structures of MDM2 co-
crystallised with highly potent inhibitors were published [11, 36–38]. Analysis of these and pre-
viously published crystal structures [10] of ligand/MDM2 complexes encouraged us to examine
two main changes in our design. First, the size of one or two of the hydrophobic substituents
should be decreased. Second, several of the published binders of MDM2 have a hydrophilic
substituent pointing out towards the solvent/hydrophilic surface of MDM2 enabling hydrogen
bonding and/or ionic interactions with the His96 and Lys94 residues of MDM2. The 6-position
of our purine derivatives was suitable for the introduction of such functional groups. A series
of 8-(triazolyl)purine derivatives were docked into the α-helix binding site of MDM2 (PDB
code: 4HBM), using the Schrödinger software package (Glide XP mode), to find a suitable sub-
stitution pattern on the 8-(triazolyl)purine ring system. This crystal complex was chosen due
to the high affinity and potency of the co-crystallized piperidinones. The models suggest that
decreasing the size of the R3 substituent to a methyl group, R2 to a propyl and R1 to an indanyl
should improve the fit between the ligand and MDM2. The indanyl is a more rigid isoster of
phenethyl. In addition, the introduction of a more hydrophilic group such as an acid or ester
could give rise to hydrogen bonding and/or ionic interactions. An example where R1 = indanyl,
R2 = propyl, R3 = methyl, and R4 = NHCH2COOH is shown in Fig 6b. A series of purines with
R1-3 substituents with varying size and shape were selected for further evaluation of inhibitors.
Compound 8a (type III) was also docked in the grid built from the ligand binding site of
MDM2 (PDB code: 4HBM). As expected, the R1-3 substituents of a type III inhibitor do not fit
into Phe-Trp-Leu-pockets of a crystal structure derived from a type II inhibitor/MDM2
complex.
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 6. Small molecule inhibitors bound to MDM2. a) Crystal structure of a piperidinone-based inhibitor bound to MDM2 (PDB code: 4HBM)[37] and b) type
II inhibitor (R1 = indanyl, R2 = propyl, R3 = methyl and R4 = NHCH2COOH) docked into the MDM2 binding site.
doi:10.1371/journal.pone.0124423.g006
The syntheses of our second series of compounds are shown in Fig 7 Introduction of R1 and
R2 substituents and dimethylamine in the 6-position was performed as described above; 10a-d
and 11a-b were isolated in 75–94% and 87–94% yield, respectively. When the N2-position was
substituted with a p-chlorobenzyl, milder conditions for the introduction of dimethylamine
Fig 7. Synthesis of 9–13. Reaction conditions: (a) R1OH, DIAD, Ph3P, THF. (b) R2OH, ADDP, nBu3P, THF. (c) R4 = NMe2, DMF, MW, 180°C. R2 = p-ClBn,
R4 = NMe2, (i) 5.6 M NMe2 in ethanol. (ii) TFA/CH2Cl2. R4 = NHCH2CO2Et, (i) NH2CH2CO2Et, Et3N, ethanol, 100°C. (ii) TFA/CH2Cl2, R4 = SCH2CO2Et, (i)
SHCH2CO2Et, NaH, toluene, 70°C. (ii) TFA/CH2Cl2. (d) PyBr3, CH2Cl2. For R4 = NH2, (i) NH4OH (aq.), dioxane, 100°C. (ii) TFA/CH2Cl2 (iii) PyBr3, CH2Cl2.
(e) (i) Pd(II)Cl2(PPh3)2, Cu(I)I, Amberlite IRA-67, TMS-acetylene, MW, 110°C. (ii) PS-F, THF.
doi:10.1371/journal.pone.0124423.g007
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8-Triazolylpurines as MDM2/p53 Inhibitors
were used to avoid aromatic substitution in the p-chloro position. Running the reaction with
dimethylamine in ethanol (5.6 M) at 80°C gave regioselective substitution of the 6-chloro and
subsequent Boc removal with TFA in DCM gave 11c in 95% yield over two steps. For the intro-
duction of an ester in the 6-postion, the 6-chloropurine derivatives were reacted with thioglycol
ethylester and glycine ethylester under basic conditions followed by Boc removal (TFA/DCM)
to give 11d-g in 60–98% yields over two steps. Next, treatment of 11a-g with PyrBr3 provided
12a-g in 35–96% yield, where the lower yields of 35% was obtained for bromination of com-
pound 11g. Sulphur in the 6-position gives lower yields compared with nitrogen. An alterna-
tive to decrease the lipophilicity of these compounds would be to introduce a primary amine in
the 6-position. Treatment of the 6-chloroderivatives with ammonium hydroxide gave the
6-aminoderivative. Bromination using PyBr3 in DCM did not give the desired product which
was assumed to be due to the electron withdrawing effect of the Boc group in the N2-position.
Removing the Boc group with TFA in DCM prior to the bromination was necessary to facilitate
the success of the reaction. The amination, deprotection and bromination sequence can be run
conveniently without any intermediate purification to afford 12h and 12i in 51 and 73% yield
over three steps, respectively.
The Sonsogashira coupling followed the same protocol as above to give 13a-i (Fig 7). Fur-
ther, the same protocol for the 1,4-triazole formation was again employed to provide 14a-l
(Fig 8), and as described above for the synthesis of isobutylazide, methylazide was formed
in situ. The highest yields in the cyclisation were obtained when the azide formation was run in
room temperature followed by cyclisation at 60°C. Low yields were isolated if the reaction mix-
ture vial was flushed with nitrogen after azide formation which is assumed to be due to the vol-
atility of methylazide.
These new compounds were also evaluated for their inhibitory effect on MDM2 in a fluores-
cence polarisation assay. Of the 12 new compounds, 10 were inactive towards p53/MDM2 at
concentrations up to 100 μM. Compounds 14f and 14j (Fig 9) displayed IC50 values of 10
(Fig. P in S1 File) and 56 μM, respectively. Compound 14f was further evaluated using Water-
LOGSY[39] which confirmed binding to MDM2 (Fig. Q in S1 File). These results verify that
with this scaffold, compounds with larger substituents in the R1 and R2-position do not bind to
MDM2. This validates the redesign and fits with the observation that many of the published
low nM inhibitors of the p53/MDM2 interaction are typically less extended than the model
p53 α-helix. One explanation for the low potency observed for the type II 8-(triazolyl)purine
Fig 8. Synthesis of 14a-l. Reaction conditions: (a) NaN3, R3-X, Cu(I)I, NaAsc, DMEDA, DMF.
doi:10.1371/journal.pone.0124423.g008
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 9. 8-(triazolyl)purine inhibitors of MDM2/p53.
doi:10.1371/journal.pone.0124423.g009
derivatives could be that while the methyl on the triazole fits into one of the hydrophobic inter-
action sites as observed in the docking studies, this fit might bury the polar triazole as well, re-
sulting in a desolvation penalty. Furthermore, the active compounds 14f and 14j lack the
ability of the ionic interaction formed between the carboxylic acid of the model inhibitor
(piperidinones) and the Lys94 residue of MDM2. Recently published docking studies of known
p53/MDM2 inhibitors highlight the challenges posed by using docking as a tool for developing
inhibitors against this target.[40]
Photophysical characterisation. Intrinsically fluorescent α-helix mimetics could be used
for imaging and localization of the compounds in cells. We have previously reported the fluo-
rescent properties of 6,9-substituted 8-triazolylpurines [18] and anticipated that the structural-
ly similar 2,6,9-substituted derivatives would have similar behaviour. UV/vis absorption and
fluorescence measurements were undertaken to investigate this possibility. The data are pre-
sented in Table 1.
Table 1. Absorption and fluorescent properties of 8-(triazolyl)purine derivatives in methanol solution.
Entry
Compound
R1
R2
R3
R4
λabs,max (nm)
λem,max (nm)
ΦF (%)
1
14a
14k
7c
2-indanyl
phenethyl
cyclopentylmethyl
2-indanyl
cyclopentylmethyl
Bn
Bn
Bn
Bn
nPr
Bn
Bn
nPr
Bn
Bn
nPr
nPr
iBu
Bn
iBu
Bn
Bn
1,4-Me
1,4-Me
1,4-Bn
1,4-Me
1,4-Me
1,4-Me
1,4-Me
1,4-Me
1,4-Bn
1,4-Bn
1,4-Me
1,4-Bn
1,4-iBu
1,5-Bn
1,5-iBu
1,4-Bn
NMe2
310
315
317
311
316
314
311
311
318
316
313
318
316
322
320
340
376
376
377
378
376
375
377
377
381
382
380
378
378
437
422
398
2
2
NMe2
2
3
NMe2
2
4
14b
14l
14i
14j
14d
14g
14e
14f
7a
NMe2
1
5
NMe2
2
6
NH2
7
7
2-indanyl
2-indanyl
Bn
NH2
4
8
NHCH2CO2Et
NHCH2CO2Et
NHCH2CO2Et
NHCH2CO2Et
NMe2
8
9
10
4
10
11
12
13
14
15
16
2-indanyl
2-indanyl
1-naphtyl
phenethyl
1-naphtyl
phenethyl
Bn
5
2
7b
NMe2
5
8a
NMe2
37
51
2
8b
NMe2
14h
SCH2CO2Et
λabs, max and λem, max are the wavelengths of absorption and emission maxima, respectively, and ΦF is the fluorescence quantum yield
doi:10.1371/journal.pone.0124423.t001
PLOS ONE | DOI:10.1371/journal.pone.0124423 May 5, 2015
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8-Triazolylpurines as MDM2/p53 Inhibitors
Fig 10. Photophysical characterization of 14b, 14f, 14g, 14h and 14i. Normalised absorption (solid lines)
and emission (dashed lines) spectra of a selection of the investigated compounds (the full set of absorption
and emission spectra can be found in the SI).
doi:10.1371/journal.pone.0124423.g010
The 8-triazolyl compounds (Table 1, entries 1–15) have absorption maxima in the range
310–322 nm, with the exception of 14h (entry 16), which bears a sulphur substituent in the
6-position. The absorption maximum of this compound is redshifted to 340 nm. Compounds
with a 6-dimethylamino substituent, such as R4 and a 1,4-triazole, all have low fluorescence
quantum yields between 1 and 2% (entries 1–5,12–13). Changing from tertiary to primary or
secondary amines as R4 substituent gives higher quantum yield (5–10%). A selection of absorp-
tion and emission spectra is shown in Fig 10.
The substitution pattern on the triazole and in the 9 and N2 position seems to have little ef-
fect on the quantum yield. This is expected since none of the substituents in these positions
changes the conjugation of the heterocyclic core. Interestingly, changing the regioisomerism of
the triazole from 1,4 to 1,5 (entries 12–15) results in a 10-20-fold increase of the quantum
yield. In addition, the Stokes shift increases from 62 to 102 nm (entries 13 and 15). To the best
of our knowledge, this is the first time this regioisomeric effect has been observed for fluores-
cent triazoles and it might have implications in the field of fluorescent base analogs. The in-
crease in quantum yield observed when shifting from the 1,4 to the 1,5-regioisomer could
possibly originate from increased steric hindrance and a higher rotational barrier of the pu-
rine-triazole bond leading to less rotational flexibility in the excited state, decreasing the rate of
non-radiative deactivation. To establish the influence of the triazole on the fluorescent proper-
ties, 5a and 15–16 bearing a proton, bromine, and an alkyne in the 8-position, respectively,
were also measured (data not shown). These compounds all displayed very weak fluorescent
properties and demonstrate that the 8-triazole substituent is necessary to observe the higher
quantum yields.
Materials and Methods
A representative synthesis example of 6-(ethoxy-carbonyl-methylamino)-9-(2-indanyl)-8-
(1-methyl-1H-1,2,3-triazol-4-yl)-2-(1-propylamino)purine (14f) is described below.
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8-Triazolylpurines as MDM2/p53 Inhibitors
2-Amino-9-tert-butoxycarbonyl-6-chloropurine
Following a published procedure[29] with minor modifications, 2-amino-6-chloropurine
(2.50 g, 14.7 mmol) and di-tert-butyl dicarbonate (3.21 g, 14.7 mmol) were dissolved in anhy-
drous DMSO (50 ml). The pale yellow solution was cooled in an ice bath with vigorous stirring
and the ice bath was removed just as the DMSO started to freeze. DMAP (90 mg, 0.74 mmol)
was added and the solution was stirred under nitrogen at room temperature for 8 h. Additional
di-tert-butyl dicarbonate (220 mg, 1.00 mmol) was added and the reaction mixture was stirred
at room temperature overnight. Full consumption of starting material and formation of one
new spot was confirmed by TLC (10% methanol in CHCl3).The reaction mixture was diluted
with water (450 ml) and extracted with ethyl acetate (3 x 150 ml). The organic phases were
washed with water (5 x 100 ml), dried over Na2SO4, filtered and evaporated to give the ex-
pected product as a white solid (3640 mg, 92%) which was used without further purification in
the next step. 1H NMR (CDCl3): 8.13 (s, 1H), 5.92 (br s, 2H), 1.66 (s, 9H); 13C NMR (CDCl3):
160.6, 153.4, 152.4, 145.7, 140.2, 125.5, 87.2, 28.0.
2-tert-Butoxycarbonylamino-6-chloropurine (1)
Sodium hydride (715 mg, 17.9 mmol, 60% mineral oil dispersion) was added to a stirred solu-
tion of 2-amino-9-tert-butoxycarbonyl-6-chloropurine, (3490 mg, 12.9 mmol) in dry THF
(120 ml) at room temperature under nitrogen. The white suspension was stirred at room tem-
perature for 2 h. Full conversion of starting material was confirmed by TLC (70% ethyl acetate
in pentane). The reaction was cooled to 0°C and quenched with brine (5 ml); a white precipi-
tate was observed after the addition. The mixture was then allowed to reach room temperature.
The THF was removed under reduced pressure (note: save 10–20 ml THF, to simplify the ex-
traction). CHCl3 (200 ml) and distilled water (800 ml) were added (everything did not dis-
solve). The pH on the aqueous phase was determined to be 14. The phases were separated and
extracted with CHCl3 (3 x 100 ml, note: wait approximately 10 min between every extraction)
and these organic phases were discarded. Sat. NaHCO3 (aq.) was added to the aqueous phase,
whereupon a white precipitate was observed; the aqueous phase was extracted with CHCl3 (3 x
100 ml). The combined organic phases were washed with brine. The solvent was removed
under reduced pressure to yield 1 as a white solid in quantitative yield. 1H and 13C NMR corre-
sponds with previously published results[27]. 1H NMR (CDCl3): 13.53 (s, 1H), 8.44 (s, 1H),
7.78 (s, 1H), 1.58 (s, 9H); 13C NMR (CDCl3): 153.3, 151.6, 151.3, 151.0, 145.5, 128.3, 82.4, 28.3.
General procedure A: Mitsunobu reaction in the 9-position
The alkylation was performed following a published procedure[29] with minor modifications.
1 (1.0 eq.) was dissolved in dry THF under nitrogen in oven-dried round-bottomed flask and
alcohol (1.1–2.5 eq.) and PPh3 (1.1–2.5 eq.) was added. The nitrogen flow was temporarily re-
moved when solid alcohols were added. When all of the PPh3 was dissolved, DIAD (1.0–2.5
eq.), were added drop wise. The reaction was stirred at room temperature under nitrogen, until
TLC indicated full consumption of the starting material, unless otherwise noted. The solvent
was removed under reduced pressure and the crude product was purified by flash column
chromatography or automated flash column chromatography.
6-Chloro-9-(2-indanyl)-2-(tert-butoxycarbonylamino)purine (9a)
Compound 9a was synthesised following general procedure A at 0°C for 2 h from 1 (816 mg,
3.03 mmol), 2-indanol (1031 mg, 7.60 mmol), PPh3 (1953 mg, 7.44 mmol) and DIAD (1.45 ml,
7.36 mmol) as substrates in dry THF (42 ml). The crude product was purified by flash column
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8-Triazolylpurines as MDM2/p53 Inhibitors
chromatography (30–70% ethyl acetate in pentane) to provide 9a as a white solid (970 mg,
83%). 1H NMR (CDCl3): 7.73 (s, 1H), 7.53 (br s, 1H), 7.34–7.25 (m, 4H), 5.55 (tt, J 7.4, 4.3 Hz,
1H), 3.63 (dd, J 16.5, 7.4 Hz, 2H), 3.26 (dd, J 16.4, 4.3 Hz, 2H), 1.54 (s, 9H); 13C NMR (CDCl3):
152.6, 152.3, 151.1, 150.2, 141.9, 139.6, 127.6, 127.5, 124.8, 81.7, 55.0, 40.1, 28.2. HRMS m/z
[M + H]+ calculated for C19H20ClN5O2: 386.1384. Found: 386.1351.
General procedure B: Mitsunobu reaction in the 2-position
The alkylation was performed following a published procedure[29] with minor modifications.
The purine was dissolved in dry THF under nitrogen in an oven-dried round-bottomed flask,
PBu3 (2.4–2.6 eq.), alcohol (2.4–2.6 eq.) and ADDP (2.5–2.6 eq.) was added in that order. The
nitrogen flow was temporarily suspended when solid alcohols and ADDP were added. The re-
action was stirred at room temperature, until TLC indicated full consumption of the starting
material, unless otherwise noted. The obtained white precipitate was filtered off, and washed
with THF, and the solvent was removed under reduced pressure. The crude product was puri-
fied by flash column chromatography or automated flash column chromatography.
6-Chloro-9-(2-indanyl)-2-(N-propyl-tert-butoxycarbonylamino)purine
(10b)
Compound 10b was synthesised following general procedure B from 9a (2930 mg, 7.59 mmol),
n-propanol (1.45 ml, 19.40 mmol), PBu3 (4.8 ml, 18.26 mmol) and ADDP (4790 mg, 18.99
mmol) in dry THF (144 ml). The reaction was stirred at room temperature for 24 h. The reac-
tion was monitored by TLC (50% ethyl acetate in pentane). The crude product was purified by
flash column chromatography (25–100% ethyl acetate in pentane) to provide compound 10b
as a light yellow sticky solid (2450 mg, 75%). Starting material (727 mg) was recovered after
column chromatography. 1H NMR (CDCl3): 7.84 (s, 1H), 7.33–7.24 (m, 4H), 5.45 (tt, J 7.6,
5.0 Hz, 1H), 3.89–3.85 (m, 2H), 3.60 (dd, J 16.3, 7.5 Hz, 3H), 3.36 (dd, J 16.3, 5.0 Hz, 2H), 1.67
(sext, J 7.5 Hz, 2H), 1.50 (s, 9H), 0.88 (t, J 7.5, 3H); 13C NMR (CDCl3): 155.3, 154.0, 152.4,
150.5, 142.8, 139.6, 128.4, 127.7, 124.9, 81.4, 55.5, 50.2, 39.8, 28.3, 22.2, 11.4. HRMS m/z
[M + H]+ calculated for C22H26ClN5O2: 428.1853. Found: 428.1865.
6-(Ethoxy-carbonyl-methylamino)-9-(2-indanyl)-2-(1-propylamino)purine
(11e)
The reaction was run in two batches, which were then pooled prior to work-up and purifica-
tion. 10b (250 mg, 1.17 mmol) was suspended in ethanol (15 ml, 99.7%) in two oven-dried mi-
crowave vials. Glycine ethyl ester hydrochloride (245 mg, 1.76 mmol) and triethyl amine
(0.41 ml, 2.94 mmol) were added and the reactions were heated to 100°C whereupon the start-
ing materials dissolved. The reaction was left at 100°C overnight. TLC (5% methanol in
CH2Cl2) indicated full consumption of the starting material. The two batches were pooled and
the solvent was removed under reduced pressure. The crude product was dissolved in CH2Cl2
(13 ml), and TFA (4 ml) was added. The reaction mixture was stirred at room temperature for
3 h, after which full deprotection was confirmed by LCMS. The reaction mixture was cooled on
ice and diluted with distilled water (20 ml) and then basified with 6 M NaOH (aq.). The aque-
ous phase was extracted with CH2Cl2 (4 x 20 ml). The combined organic phases were washed
with brine and dried over MgSO4, filtered, and the solvent was removed under reduced pres-
sure. The crude product was purified by automated flash column chromatography (5% metha-
nol in CH2Cl2) to provide compound 11e as a light yellow oil/foam (451 mg, 98%). 1H NMR
(CDCl3): 7.31 (s, 1H), 7.30–7.18 (m, 4H), 6.25 (br s, 1H), 5.24–5.31 (m, 1H), 4.86 (t, J 5.9 Hz,
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8-Triazolylpurines as MDM2/p53 Inhibitors
1H), 4.31 (d, J 5.4 Hz, 2H), 4.20 (q, J 7.1 Hz, 2H), 3.50 (dd, J 16.2, 7.6 Hz, 2H), 3.38–3.25 (m,
4H), 1.59 (sext, J 7.3 Hz, 2H), 1.26 (t, J 7.1 Hz, 3H), 0.95 (t, J 7.4 Hz, 3H); 13C NMR (CDCl3):
170.7, 159.6, 154.5, 151.8, 140.5, 135.1, 127.2, 124.7, 114.4, 61.2, 54.2, 43.8, 42.7, 39.8, 23.2,
14.3, 11.7. HRMS m/z [M + H]+ calculated for C21H25N6O2: 395.2195. Found: 395.2195.
General procedure D: 8-bromination of 2,6,9-substituted purines
Purine (1.0 eq.) was dissolved in dry CH2Cl2 in oven-dried glassware under nitrogen. The ni-
trogen flow was temporarily suspended when pyridinium tribromide (PyrBr3) (1.1–1.4 eq.)
was added in one portion. The reaction mixture was stirred at room temperature under nitro-
gen until full conversion was observed by TLC or LCMS. The reaction was quenched with 10%
Na2S2O3 (aq.); the colour changed from yellow to colorless. The pH was adjusted to 9–12 with
15% NaOH (aq.). The phases were separated and the aqueous phase was extracted with
CH2Cl2 (x 3). The organic phases were pooled, washed with brine, dried over Na2SO4 and fil-
tered. The solvent was removed under reduced pressure, and the crude product was purified by
flash column chromatography.
8-Bromo-6-(ethoxy-carbonyl-methylamino)-9-(2-indanyl)-2-
(1-propylamino)purine (12e)
Compound 12e was synthesised following general procedure D from 11e (418 mg, 1.06 mmol)
and PyrBr3 (410 mg, 1.21 mmol) in dry CH2Cl2 (17 ml). The reaction was stirred at room tem-
perature for 5 h, whereupon full consumption of starting material was observed by LCMS. The
crude product was purified by automated flash column chromatography (20–30% ethyl acetate
in pentane) to provide 12e as a colorless oil (479 mg, 96%). 1H NMR (CDCl3): 7.26–7.19 (m,
4H), 5.92 (t, J 5.3 Hz, 1H), 5.34 (quin, J 9.1 Hz, 1H), 4.77 (t, J 5.9 Hz, 1H), 4.30 (d, J 5.5 Hz,
2H), 4.23 (q, J 7.1 Hz, 2H), 3.96 (dd, J 15.5, 9.2 Hz, 2H), 3.26 (dd, J 15.4, 9.0 Hz, 2H), 3.14 (q, J
6.6 Hz, 2H), 1.48 (sext, J 7.4 Hz, 2H), 1.29 (t, J 7.1 Hz, 3H), 0.84 (t, J 7.4 Hz, 3H); 13C NMR
(CDCl3): 170.5, 158.8, 153.4, 153.1, 140.8, 126.9, 124.5, 122.2, 115.3, 61.4, 56.6, 43.6, 42.6, 37.0,
23.0, 14.4, 11.6. HRMS m/z [M + H]+ calculated for C21H25BrN6O2: 473.1300. Found:
473.1331.
6-(Ethoxy-carbonyl-methylamino)-8-ethynyl-9-(2-indanyl)-2-
(1-propylamino)purine (13e)
PdCl2(PPh3)2 (28 mg, 0.040 mmol), CuI (31 mg, 0.16 mmol), and amberlite IRA-67 (718 mg,
4.02 mmol) were added to a solution of 12e (380 mg, 0.803 mmol) in dry THF (12 ml). The
vial was capped, nitrogen was bubbled through the reaction mixture and ethynyltrimethylsi-
lane (450 μl, 3.25 mmol) was added. The yellow reaction mixture was heated in a microwave
reactor at 110°C for 50 min. Full consumption of 12e was confirmed by TLC (2% methanol in
CHCl3). The reaction mixture was filtered through a short plug of silica that was eluted with
THF. The solution was concentrated and the volume was adjusted to 8 ml. Polymer-supported
fluoride (422 mg) was added and the reaction mixture was stirred under nitrogen at room tem-
perature for 19 h. The polymer was filtered off, washed with THF and CH2Cl2 and the solvents
were removed under reduced pressure. Purification by flash column chromatography (first col-
umn eluted with 2% methanol in CH2Cl2 and second column with 33% ethyl acetate in pen-
tane) provided 13e as a yellow solid (200 mg, 60%). 1H NMR (CDCl3): 7.26–7.17 (m, 4H), 6.11
(s, 1H), 5.47 (quin, J 9.0 Hz, 1H), 4.84 (t, J 5.8 Hz, 1H), 4.31 (br s, 2H), 4.23 (q, J 7.1 Hz, 2H),
3.91 (dd, J 15.5, 9.3 Hz, 2H), 3.35 (s, 1H), 3.30 (dd, J 15.5, 8.8 Hz, 3H), 3.20 (q, J 6.7 Hz, 2H),
1.51 (sext, J 7.3 Hz, 2H), 1.28 (t, J 7.1 Hz, 3H), 0.87 (t, J 7.4 Hz, 3H); 13C NMR (CDCl3): 170.4,
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8-Triazolylpurines as MDM2/p53 Inhibitors
159.9, 154.4, 151.7, 141.0, 129.7, 126.9, 124.5, 114.8, 83.0, 74.3, 61.4, 55.4, 43.6, 42.6, 37.6, 23.1,
14.3, 11.6. HRMS m/z [M + H]+ calculated for C23H26N6O2: 419.2195. Found: 419.2206.
6-(Ethoxy-carbonyl-methylamino)-9-(2-indanyl)-8-(1-methyl-1H-
1,2,3-triazol-4-yl)-2-(1-propylamino)purine (14f)
Methyl iodide (24 μl, 0.39 mmol) and sodium azide (25 mg, 0.38 mmol) in dry DMF (2 ml) were
stirred at room temperature for 3 h in a microwave vial. The formed methyl azide solution was
then transferred to a microwave vial containing 13e (82 mg, 0.20 mmol), sodium ascorbate
(12 mg, 0.06 mmol), CuI (7 mg, 0.04 mmol) and DMEDA (5 μl, 0.05 mmol) in dry DMF
(1.5 ml). The reaction mixture was heated at 60°C for 3 h and then at room temperature for 15 h.
Full consumption of 13e was confirmed by TLC (10% methanol in CHCl3). The reaction mixture
was poured into ethyl acetate (25 ml), extracted with water (3 x 20 ml), dried over Na2SO4 and
the solvents were removed under reduced pressure. Caution: Keep aqueous phase slightly basic
(0.5 M NaOH (aq.) was added to the aqueous phase) to avoid hydrazoic acid formation. Purifica-
tion by flash column chromatography (ethyl acetate) provided 14f as a yellow solid (81 mg,
87%). 1H NMR (CDCl3): 8.11 (br s, 1H), 7.25–7.13 (m, 4H), 6.31 (br s, 1H), 6.16 (br s, 1H), 4.75
(br s, 4.75, 1H), 4.33 (s, 2H), 4.24 (q, J 7.1 Hz, 2H),4.14 (s, 3H), 4.05 (dd, J 14.9, 8.5 Hz, 2H), 3.32
(dd, J 14.8, 8.9 Hz, 2H), 3.12 (q, J 6.6 Hz, 2H), 1.46 (q, J 7.2 Hz, 1H), 1.30 (t, J 7.0 Hz, 3H), 0.81
(t, J 7.4 Hz, 3H); 13C NMR (CDCl3): 170.9, 158.9, 154.4, 153.4, 141.6, 140.9, 139.0, 126.5, 125.4,
124.4, 114.7, 61.2, 56.1, 43.6, 42.5, 37.2, 36.8, 23.0, 14.3, 11.5. Note: Weighting functions were ap-
plied to the 1H NMR spectra to obtain J couplings (sine bell: 2°, sine square: 88°). HRMS m/z [M
+ H]+ calculated for C24H29N9O2: 476.2522. Found: 476.2485.
Conclusion
An efficient strategy for the preparation of 2,6,9-substituted 8-triazolylpurines has been devel-
oped. The target compounds are obtained in excellent yields by a convenient multistep proce-
dure. Conformation studies by solution phase NMR spectroscopy demonstrated that
2,6,9-substituted 8-triazolylpurines could mimic the topography of an α-helix and could be po-
tentially useful as MDM2/p53 inhibitors. However, our study clearly indicates that this is not
sufficient for in vitro activity as the first series of compounds were inactive at the tested concen-
trations in a biochemical fluorescence polarisation assay targeting MDM2/p53. Further itera-
tive design-synthesis-testing cycles using docking as the main design tool resulted in a one
micromolar inhibitor (14f, 10 μM). Binding of 14f to MDM2 was confirmed by WaterLOGSY
measurements. Furthermore, the 2,6,9-substituted 8-triazolylpurines displayed moderate to
high fluorescence quantum yields in the range of 5–51%. The combination of fluorescent prop-
erties with inhibitory effect on the MDM2-p53 interaction could potentially be useful for real-
time imaging as well as for the development of in vitro displacement assays for MDM2/p53.
Developments in this direction are currently ongoing in our laboratories.
Supporting Information
S1 File. Describes synthesis and characterisation including Proton (1H) and Carbon (13C)
NMR spectra of all compounds synthesised. In addition, experimental procedures for the bio-
logical evaluation and photophysical characterisation are included. A more specific table of
contents can be located in this document.
(PDF)
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8-Triazolylpurines as MDM2/p53 Inhibitors
Acknowledgments
We thank Emma Rova Danelius and Mate Erdelyi for their valuable help with the
NAMFIS analysis.
Author Contributions
Conceived and designed the experiments: MP DB JM MG. Performed the experiments: MP DB
JRN JJ LI JM. Analyzed the data: MP DB JJ JRN JM LI RKG RWK JA MG. Contributed re-
agents/materials/analysis tools: RKG RWK JA MG. Wrote the paper: MP DB MG.
References
1. Stites WE. Protein—Protein Interactions: Interface Structure, Binding Thermodynamics, and Mutational
Analysis. Chem. Rev. 1997; 97: 1233–1250. PMID: 11851449
2. Jones S, Thornton JM. Principles of protein-protein interactions. P. Natl. Acad. Sci. USA 1996; 93: 13–20.
3. Keskin O, Gursoy A, Ma B, Nussinov R. Principles of Protein—Protein Interactions: What are the Pre-
ferred Ways For Proteins To Interact? Chem. Rev. 2008; 108: 1225–1244. doi: 10.1021/cr040409x
PMID: 18355092
4. Jochim AL, Arora PS. Systematic Analysis of Helical Protein Interfaces Reveals Targets for Synthetic
Inhibitors. ACS Chem. Biol. 2010; 5: 919–923. doi: 10.1021/cb1001747 PMID: 20712375
5. Vousden KH, Prives C. Blinded by the Light: The Growing Complexity of p53. Cell 2009; 137: 413–431.
doi: 10.1016/j.cell.2009.04.037 PMID: 19410540
6. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408: 307–310. PMID:
11099028
7. Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, et al. Structure of the MDM2 Oncoprotein
Bound to the p53 Tumor Suppressor Transactivation Domain. Science 1996; 274: 948–953. PMID:
8875929
8. Aeluri M, Chamakuri S, Dasari B, Guduru SKR, Jimmidi R, Jogula S, et al. Small Molecule Modulators
of Protein—Protein Interactions: Selected Case Studies. Chem. Rev. 2014; 114: 4640–4694. doi: 10.
1021/cr4004049 PMID: 24673632
9. Azzarito V, Long K, Murphy NS, Wilson AJ. Inhibition of [alpha]-helix-mediated protein-protein interac-
tions using designed molecules. Nat. Chem. 2013; 5: 161–173. doi: 10.1038/nchem.1568 PMID:
23422557
10. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In Vivo Activation of the p53
Pathway by Small-Molecule Antagonists of MDM2. Science 2004; 303: 844–848. PMID: 14704432
11. Sun D, Li Z, Rew Y, Gribble M, Bartberger MD, Beck HP, et al. Discovery of AMG 232, a Potent, Selec-
tive, and Orally Bioavailable MDM2–p53 Inhibitor in Clinical Development. J. Med. Chem. 2014; 57:
1454–1472. doi: 10.1021/jm401753e PMID: 24456472
12. Zhao Y, Yu S, Sun W, Liu L, Lu J, McEachern D, et al. A Potent Small-Molecule Inhibitor of the MDM2–
p53 Interaction (MI-888) Achieved Complete and Durable Tumor Regression in Mice. J. Med. Chem
2013; 56: 5553–5561. doi: 10.1021/jm4005708 PMID: 23786219
13. Orner BP, Ernst JT, Hamilton AD. Toward Proteomimetics: Terphenyl Derivatives as Structural and
Functional Mimics of Extended Regions of an α-Helix. J. Am. Chem. Soc. 2001; 123: 5382–5383.
PMID: 11457415
14. Yin H, Lee G-i, Park HS, Payne GA, Rodriguez JM, Sebti SM, et al. Terphenyl-Based Helical Mimetics
That Disrupt the p53/HDM2 Interaction. Angew. Chem. Ger. Edit. 2005; 117: 2764–2767.
15. Lee JH, Zhang Q, Jo S, Chai SC, Oh M, Im W, et al. Novel Pyrrolopyrimidine-Based α-Helix Mimetics:
Cell-Permeable Inhibitors of Protein—Protein Interactions. J. Am. Chem. Soc. 2010; 133: 676–679.
16. Rosemeyer H. The Chemodiversity of Purine as a Constituent of Natural Products. Chem. Biodivers.
2004; 1: 361–401. PMID: 17191854
17. Horton DA, Bourne GT, Smythe ML. The Combinatorial Synthesis of Bicyclic Privileged Structures or
Privileged Substructures. Chem. Rev. 2003; 103: 893–930. PMID: 12630855
18. Dyrager C, Börjesson K, Dinér P, Elf A, Albinsson B, Wilhelmsson M, et al. Synthesis and Photophysi-
cal Characterisation of Fluorescent 8-(1H-1,2,3-Triazol-4-yl)adenosine Derivatives. Eur. J. Org. Chem.
2009; 2009: 1515–1521.
PLOS ONE | DOI:10.1371/journal.pone.0124423 May 5, 2015
16 / 17

8-Triazolylpurines as MDM2/p53 Inhibitors
19. Dierckx A, Dinér P, El-Sagheer AH, Kumar JD, Brown T, Grøtli M, et al. Characterization of photophysi-
cal and base-mimicking properties of a novel fluorescent adenine analogue in DNA. Nucleic Acids Res.
2011; 39: 4513–4524. doi: 10.1093/nar/gkr010 PMID: 21278417
20. Pedersen DS, Abell A. 1,2,3-Triazoles in Peptidomimetic Chemistry. Eur. J. Org. Chem. 2011; 2011:
2399–2411.
21. Davis MR, Singh EK, Wahyudi H, Alexander LD, Kunicki JB, Nazarova LA, et al. Synthesis of sansalva-
mide A peptidomimetics: triazole, oxazole, thiazole, and pseudoproline containing compounds. Tetra-
hedron 2012; 68: 1029–1051. PMID: 22287031
22. Horne WS, Yadav MK, Stout CD, Ghadiri MR. Heterocyclic Peptide Backbone Modifications in an α-
Helical Coiled Coil. J. Am. Chem. Soc. 2004; 126: 15366–15367. PMID: 15563148
23. Meldal M, Tornøe CW. Cu-Catalyzed Azide-Alkyne Cycloaddition. Chem. Rev. 2008; 108: 2952–3015.
doi: 10.1021/cr0783479 PMID: 18698735
24. Zhang L, Chen XG, Xue P, Sun HH, Williams ID, Sharpless KB, et al. Ruthenium-catalyzed cycloaddi-
tion of alkynes and organic azides. J. Am. Chem. Soc. 2005; 127: 15998–15999. PMID: 16287266
25. Kwok SW, Fotsing JR, Fraser RJ, Rodionov VO, Fokin VV. Transition-Metal-Free Catalytic Synthesis
of 1,5-Diaryl-1,2,3-triazoles. Org. Lett. 2010; 12: 4217–4219. doi: 10.1021/ol101568d PMID: 20825167
26. Chang Y-T, Gray NS, Rosania GR, Sutherlin DP, Kwon S, et al. Synthesis and application of functional-
ly diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. Chem. Biol. 1999; 6: 361–375. PMID:
10375538
27. Bliman D, Pettersson M, Bood M, Grøtli M. 8-Bromination of 2,6,9-trisubstituted purines with pyridinium
tribromide. Tetrahedron Lett. 2014; 55: 2929–2931.
28. Fletcher S, Shahani VM, Lough AJ, Gunning PT. Concise access to N9-mono-, N2-mono- and N2,N9-
di-substituted guanines via efficient Mitsunobu reactions. Tetrahedron 2010; 66: 4621–4632.
29. Fletcher S, Shahani VM, Gunning PT. Facile and efficient access to 2,6,9-tri-substituted purines
through sequential N9, N2 Mitsunobu reactions. Tetrahedron Lett. 2009; 50: 4258–4261.
30. Čechová L, Jansa P, Šála M, Dračínský M, Holý A, Janeba Z. The optimized microwave-assisted de-
composition of formamides and its synthetic utility in the amination reactions of purines. Tetrahedron
2011; 67: 866–871.
31. Johansson JR, Lincoln P, Nordén B, Kann N. Sequential One-Pot Ruthenium-Catalyzed Azide—Alkyne
Cycloaddition from Primary Alkyl Halides and Sodium Azide. J. Org. Chem. 2011; 76: 2355–2359. doi:
10.1021/jo200134a PMID: 21388208
32. Cicero DO, Barbato G, Bazzo R. NMR Analysis of Molecular Flexibility in Solution: A New Method for
the Study of Complex Distributions of Rapidly Exchanging Conformations. Application to a 13-Residue
Peptide with an 8-Residue Loop. J. Am. Chem. Soc. 1995; 117: 1027–1033.
33. Koivisto JJ, Kumpulainen ETT, Koskinen AMP. Conformational ensembles of flexible [small beta]-turn
mimetics in DMSO-d6. Org. Biomol. Chem. 2010; 8: 2103–2116. doi: 10.1039/b921794k PMID:
20401387
34. Fridén-Saxin M, Seifert T, Hansen LK, Grøtli M, Erdelyi M, Luthman K. Proline-mediated formation of
novel chroman-4-one tetrahydropyrimidines. Tetrahedron 2012; 68: 7035–7040.
35. Danelius E, Brath U, Erdélyi M. Insight into β-Hairpin Stability: Interstrand Hydrogen Bonding. Synlett.
2013; 24: 2407–2410.
36. Gonzalez-Lopez de Turiso F, Sun D, Rew Y, Bartberger MD, Beck HP, Canon J, et al. Rational Design
and Binding Mode Duality of MDM2–p53 Inhibitors. J. Med. Chem. 2013; 56: 4053–4070. doi: 10.1021/
jm400293z PMID: 23597064
37. Michelsen K, Jordan JB, Lewis J, Long AM, Yang E, Rew Y. Ordering of the N-Terminus of Human
MDM2 by Small Molecule Inhibitors. J. Am. Chem. Soc. 2012; 134: 17059–17067. doi: 10.1021/
ja305839b PMID: 22991965
38. Rew Y, Sun D, Gonzalez-Lopez De Turiso F, Bartberger MD, Beck HP, Canon J, et al. Structure-Based
Design of Novel Inhibitors of the MDM2–p53 Interaction. J. Med. Chem. 2012; 55: 4936–4954. doi: 10.
1021/jm300354j PMID: 22524527
39. Dalvit C, Fogliatto G, Stewart A, Veronesi M, Stockman B. WaterLOGSY as a method for primary NMR
screening: Practical aspects and range of applicability. J. Biomol. NMR 2001; 21: 349–359. PMID:
11824754
40. Bharatham N, Bharatham K, Shelat AA, Bashford D. Ligand Binding Mode Prediction by Docking:
Mdm2/Mdmx Inhibitors as a Case Study. J. Chem. Inf. Model. 2013; 54: 648–659.
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