5-Deazaflavin derivatives as inhibitors of p53 ubiquitination by HDM2

Article abstract of DOI:10.1016/j.bmc.2013.09.038

Based on previous reports of certain 5-deazaflavin derivatives being capable of activating the tumour suppressor p53 in cancer cells through inhibition of the p53-specific ubiquitin E3 ligase HDM2, we have conducted an structure-activity relationship (SAR) analysis through systematic modification of the 5-deazaflavin template. This analysis shows that HDM2-inhibitory activity depends on a combination of factors. The most active compounds (e.g., 15) contain a trifluoromethyl or chloro substituent at the deazaflavin C9 position and this activity depends to a large extent on the presence of at least one additional halogen or methyl substituent of the phenyl group at N10. Our SAR results, in combination with the HDM2 RING domain receptor recognition model we present, form the basis for the design of drug-like and potent activators of p53 for potential cancer therapy.

Full text of DOI:10.1016/j.bmc.2013.09.038

Bioorganic & Medicinal Chemistry 21 (2013) 6868–6877
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-Deazaflavin derivatives as inhibitors of p53 ubiquitination by
HDM2 q
Michael P. Dickens a, Patricia Roxburgh b, Andreas Hock b, Mokdad Mezna b, Barrie Kellam a,
Karen H. Vousden b, Peter M. Fischer a,
⇑
a School of Pharmacy & Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
b The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2013
Revised 12 September 2013
Accepted 16 September 2013
Available online 25 September 2013
Based on previous reports of certain 5-deazaflavin derivatives being capable of activating the tumour
suppressor p53 in cancer cells through inhibition of the p53-specific ubiquitin E3 ligase HDM2, we have
conducted an structure–activity relationship (SAR) analysis through systematic modification of the 5-
deazaflavin template. This analysis shows that HDM2-inhibitory activity depends on a combination of
factors. The most active compounds (e.g., 15) contain a trifluoromethyl or chloro substituent at the deaza-
flavin C9 position and this activity depends to a large extent on the presence of at least one additional
halogen or methyl substituent of the phenyl group at N10. Our SAR results, in combination with the
HDM2 RING domain receptor recognition model we present, form the basis for the design of drug-like
and potent activators of p53 for potential cancer therapy.
Keywords:
Cancer
HDM2–p53
Ubiquitination inhibitors
Ubiquitin E3 ligase
Deazaflavin
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
HLI98 compounds, has also been reported to inhibit the E3 ubiqui-
tin ligase activity of HDM2 and thereby selectively to kill cancer
The tumour suppressor p53 is central to the protection of mam-
malian cells against genetic damage and p53 is dysfunctional in a
large proportion of cancer cells.1 The oncogene HDM2 and p53
are linked in a negative feed-back loop in which p53 activates
HDM2, the latter acting as a p53-specific ubiquitin E3 ligase and
thus promoting degradation of p53 protein through the ubiqui-
tin–proteasome system.2 Tumours that retain wild-type p53 fre-
quently show aberrations in p53 regulation, most commonly
cells in a p53-dependent manner.6 Whereas 7-nitro-5-deazaflavins
inhibit both HDM2-mediated p53 ubiquitination and auto-ubiqui-
tination, other reported compounds that target the ubiquitin E3 li-
gase activity of HDM2 apparently are selective for p53
ubiquitination.7
Using surface plasmon resonance spectroscopy we found that
active, but not inactive 5-deazaflavin analogues, bound to the
HDM2 RING domain. In cellular assays, active 5-deazaflavin com-
pounds inhibited p53 ubiquitination, stabilised p53, and induced
the expression of p53 targets.9
The flavin and 5-deazaflavin (also known as 5-deaza-isoalloxa-
zine or pyrimido[4,5-b]quinoline-2,4(3H,10H)-dione (Fig. 1)) sub-
structures are found in a number of naturally occurring redox
cofactors.10,11 Various synthetic 5-deazaflavin derivatives have
been reported to possess antibacterial, antiparasitic, and antican-
cer pharmacological activities, although the molecular targets for
these activities remain unknown in most cases.12,13
Because of the high reduction potential of nitro-5-deazaflavins,
they easily undergo biological one-electron reduction to generate
nitro anion radicals, which, when present in flat heteroaromatic
systems that can interact with DNA,14 are known to induce cyto-
toxicity through DNA damage.15–17 It has been shown that certain
nitro-5-deazaflavins have antitumour activities18 and that 6- and
8-nitro derivatives (containing long-chain alkyl groups at N10 in-
stead of the phenyl group present in HLI98 compounds) generate
through overexpression of the p53 negative regulator HDM2.3
A
strategy for the reactivation of the pro-apoptotic p53 activities in
such tumours 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 li-
gase activity of HDM2.4
We have previously reported on a family of 7-nitro-5-deaza-fla-
vin compounds, which were discovered in a screen for inhibitors of
HDM2 E3 activity.5 This group of compounds was named the
HDM2 ligase inhibitor 98 class (HLI98; Fig. 1). A compound known
as HLI373 (Fig. 1), whose structure differs significantly from active
q
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.
⇑
Corresponding author. Tel.: +44 115 846 6242; fax: +44 115 951 3412.
E-mail address: peter.fischer@nottingham.ac.uk (P.M. Fischer).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.038

M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
6869
With regards to the HLI98 class of compounds as leads for phar-
macological p53 reactivation, we were interested in abolishing the
possibility of their promiscuous cytotoxic activity through biore-
ductive activation and DNA damage. Preliminary SAR studies indi-
cated that the cellular activity of 10-aryl-5-deazaflavins was in fact
not due to the C7-nitro function, since both cell-inactive 7-nitro
derivatives and cell-active analogues devoid of the 7-nitro group
were observed.8 Here we report on the synthesis and structure–
activity relationships (SARs) with respect to HDM2 E3 ligase inhi-
bition and cellular activity of an extensive set of 5-deazaflavin
derivatives.
2. Chemistry
The 5-deazaflavins 5–96 (Table 1 & Table S1) were prepared as
shown in Scheme 1. 6-Chlorouracil 2, prepared by hydrolysis of
2,4,6-trichloropyrimidine 1,22 was fused with the appropriate ani-
lines or other primary amines to afford the 6-anilinouracils 3a and
6-(ar)alkyluracils 3b in moderate to high yield.23 These were then
submitted to cyclocondensation with 2-halobenzaldehydes 4.8,24,25
The latter were chosen depending on commercial availability. The
6-chlorodeazaflavins could be prepared unambiguously using 2-
chloro-6-fluorobenzaldehyde, due to the enhanced reactivity of
the ortho-fluoro compared to the ortho-chloro group in the cyclo-
condensation reaction.
Figure 1. The chemical structures of flavin and 5-deazaflavin (numbering system
indicated in blue), as well as derivatives of 5-deazaflavin known as HDM2 ligase
inhibitors (HLI).5,6,8
stable one-electron reduction products with selective cytotoxicity
against hypoxic cells.19 Furthermore, direct interaction between a
5-deazaflavin–oligonucleotide conjugate and DNA has been
demonstrated.20,21
Table 1
Inhibition of in vitro ubiquitination of p53 by HDM2 by substituted 10-aryl (I) and 10-alkyldeazaflavins (II)a
Compd
Prescreen
IC50 (lM)
R1
R2
R3
R4
R5
R6
R7
R8
R9
5 (I)
6 (I)
7 (I)
8 (I)
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
NO2
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
CF3
H
H
H
H
H
H
H
H
H
NO2
H
NO2
CF3
H
CF3
H
H
F
H
H
H
H
H
H
H
H
H
H
Cl
Cl
F
H
Me
H
—
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Bn
—
—
—
—
—
—
—
—
—
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
Active
>100
>75
>50
H
H
H
F
Cl
Cl
H
H
H
Cl
Cl
Cl
Cl
H
Cl
H
F
17.5b
>100
19.6b
>100
>100
>100
11.9b
1.5b
9 (I)
10 (I)
11 (I)
12 (I)
13 (I)
14 (I)
15 (I)
16 (I)
17 (I)
18 (I)
19 (I)
20 (I)
21 (II)
22 (I)
23 (I)
24 (I)
25 (I)
26 (I)
27 (I)
28 (I)
29 (I)
30 (I)
F
H
H
H
H
H
H
H
H
H
H
—
H
F
H
H
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Cl
H
H
Cl
Cl
10.7b
12.3b
13.1b
8.4b
H
Me
—
H
32b
>100
>100
>100
>100
15.0b
>100
>100
H
H
H
H
H
Me
H
H
Cl
Cl
H
H
Cl
H
Cl
H
Br
Br
H
H
H
>100b
>100
49.5b
H
Cl
a
For explanation of prescreen refer to text; for summary of inactive compounds refer to Table S1.
IC50 values correspond to those reported previously9 for compounds 8, 10, 14–20, 25, 28, and 30; corresponding to MPD compounds 19, 20, 37, 131, 162, 134, 137, 140,
b
146, 32, 39, and 159, respectively, in Ref. 9.

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M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
Figure 2. Example of p53 ubiquitination assay for selected compounds (refer
Table 1). Western blots with anti-p53 antibody of SDS–PAGE separation of reaction
mixtures after incubation of immobilised HDM2 with test compounds or diluent
only (C) and p53, ubiquitin, and ubiquitin ligases (-E2 indicates reactions omitting
E2 ligase). Prescreen at 50–250 lM for compounds with different activity levels (a)
and titration of compound 15 (b). The positions of p53, mono-ubiquitinated (Ub-
53), and poly-ubiquitinated p53 (Ubn-p53) are indicated.
lised p53–HDM2 complex by centrifugation, and incorporation of
ubiquitin was measured by fluorescence spectroscopy.
Based on the original HLI98 7-nitro-5-deazaflavins, we exam-
ined the role of the nitro substituent in the benzene ring of the tri-
cyclic deazaflavin system (Table 1 & Table S1). As expected,5,8 we
confirmed the activity of the 7-nitro derivative containing a para-
chloro-substituted 10-phenyl group (6). However, we also found
that introduction of a nitro group at C9 resulted in moderately ac-
tive compounds in the context of either a 10-para-chlorophenyl (7)
or 10-ortho-fluorophenyl group (5). Substituents on the 10-phenyl
group appeared to be important, since the 7-nitro derivative con-
taining an unsubstituted 10-phenyl group (32; refer Table S1 for
summary of inactive compounds) was inactive.
As mentioned previously, we were interested in removing the
nitro group altogether and therefore turned our attention to
replacing this group with alternative substituents. The trifluoro-
methyl group has similar steric and electronic properties to the ni-
tro group and we examined a number of 5-deazaflavin derivatives
incorporating this function. We observed that in the absence of
substituents on the 10-phenyl group, introduction of a trifluoro-
methyl group in the benzene ring of the 5-deazaflavin tricyclic sys-
tem only afforded an active compound in the case of the 9-
trifluoromethyl derivative 8.
Scheme 1. Synthesis of 5-deazaflavin derivatives 5–96. For definition of substit-
uents R1–9 refer to Table 1.
Where no 2-halobenzaldehyde but 2-hydroxybenzaldehyde
starting materials were available, 2-tosylbenzaldehydes (4,
X = OTs) were prepared and used successfully in the deazaflavin
cyclocondensation reaction. Other benzaldehydes were prepared
by literature procedures. Thus 2-fluoro-6-nitrobenzaldehyde was
prepared from 2-fluoro-6-nitrotoluene by the nitrone method,26
whereas 2-chloro-4-nitrobenzaldehyde27 was prepared from
methyl 2-chlorobenzoate by (iBu2AlH)2 reduction.28 3-Formyl-2-
hydroxybenzonitrile was obtained by Reimer-Tiemann ortho-
formylation of 2-hydroxybenzonitrile.29
3. Results and discussion
Test compounds were assayed for inhibition of p53 ubiquitina-
tion9,30 by incubation with GST-tagged HDM2, immobilised on glu-
tathione-Sepharose, p53, ubiquitin, as well as E1 and E2 (UbcH5B)
ligases, in an assay buffer containing ATP. The reaction products
were then resolved by SDS–PAGE and p53 ubiquitination was
quantitated by Western blotting using an anti-p53 antibody
(Fig. 2).31 Test compounds that were active as inhibitors at concen-
In the case of analogues with the 6-, 7-, 8-, or 9-trifluoro-methyl
groups in combination with either the 10-ortho-fluorophenyl or
10-para-chlorophenyl substitutions, similar results were observed
insofar as only the 9-trifluoromethyl derivatives 8 and 10 showed
significant biological activity, whereas the compounds with the tri-
fluoromethyl group at other positions were less active (9, 11–13).
The 9-trifluoromethyl compounds 8 and 10 were observed to have
significantly enhanced potency compared to the corresponding 9-
nitro compounds 5 and 7. Further elaboration of the halogen sub-
stituents on the 10-phenyl group in the context of the 9-trifluoro-
methyl-5-deazaflavin system revealed that chloro and fluoro
groups were in fact also tolerated in the meta- and para-positions
(14–18), but not in the ortho-position in the case of the larger
trations below 100 lM in this assay (labelled ‘prescreen’ in Table 1)
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 reac-
tion, excess fluorescent ubiquitin was removed from the immobi-

M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
6871
chloro group. The most potent compound in this series was the
meta-chloro derivative 15.
compounds. Only two of these were reported by Wilson et al.8 (10
and 18, corresponding to compounds 27 and 28, respectively, in
Wilson et al.8), and both were found to be inactive in cells. To re-
solve this apparent paradox, we compared our compound analyses
with those reported by Wilson et al.8 We found that spectroscopic
data matched well in some cases but detected discrepancies for
those compounds where the pharmacological data is at variance
(i.e. 10 and 18, corresponding to compounds 27 and 28, respec-
tively, in Wilson et al.8). In these cases there is poor correlation be-
tween the 13C NMR spectra and in each case the 1H NMR
description in Wilson et al.8 lacks the characteristic singlet peak
for the C5-H resonance in the d 9.0–9.3 region, which we observe
in the spectra of all our compounds. During subsequent biological
testing of the compounds synthesised here, we noted a cumulative
loss of activity for several of the active compounds, suggesting that
they may show instability. We therefore believe that this instabil-
ity of these compounds may account for the apparent discrepan-
cies in biological activity and spectroscopic data.
Both 5-deazaflavins (X = CH, Fig. 1) and flavins (X = N) contain a
redox system and undergo facile hydride transfer reduction and
the reduced 1,5-dihydro species are oxidised back readily.11,33 In
the case of 5-deazaflavins, which contain an electrophilic CH func-
tion at C5, nucleophilic attack at that position is comparatively fac-
ile. Electron withdrawing substituents such as nitro,
trifluoromethyl, etc. in the tricyclic system enhance the reactivity
of the redox system34 and it would therefore be important to de-
velop potent analogues that have neutral or electron-donating sub-
stituents in the benzo ring, since these should be more stable.
Apart from extending the SARs of the 5-deazaflavins through syn-
thesis and testing of more analogues, the most important aspect
would be to modulate the electrophilic reactivity of the system.
This inherent property is undesirable because reactivity, especially
as far as interference with physiological redox systems is con-
cerned, might lead to poor bioavailability and potentially toxicity.
It should be noted, however, that even flavins, which are more
reactive than 5-deazaflavins, appear to have drug-like properties
potential, based on the fact that certain flavin derivatives with
antimalarial activity and close structural similarity to the 5-deazaf-
lavins of the current report, have been shown to possess in vivo
activity by both the parenteral and oral administration routes in
a malaria mouse model.13,35
Despite the availability of structural information on the HDM2
RING domain,36–39 its E3 ligase catalytic mechanism remains un-
clear since neither the nucleotide-binding site nor the active site
have been delineated. The structural basis of the inhibition of the
HDM2 E3 ligase activity by our compounds thus also remains
uncertain. It has been shown that the extreme C-terminal tripep-
tide of HDM2 is critical for the HDM2 E3 ligase activity40,41 and a
structural solution of an HDM2–HDMX RING domain heterodi-
mer38 shows that this tripeptide binds a groove of the partner pro-
tein, thus forming a composite binding site for the E2-ubiquitin
complex (Fig. 3a & b). Since this interaction is apparently required
for HDM2 E3 ligase activity both in trans and in cis,40 it is possible
that nonselective inhibitors, i.e. compounds such as our 5-deazaf-
lavins that do not distinguish between inhibition of HDM2 auto-
ubiquitination and ubiquitination of p53,9 target this E2-binding
site.
We therefore extracted the coordinates of the HDM2 RING
domain38 and docked bioactive 5-deazaflavins to this receptor by
defining the entire domain as the receptor site. We found that all
plausible and highly scoring poses put the ligands exactly where
the tripeptide tail in the above complex structure is (Fig. 3c). In fact
it can be observed that the 5-deazaflavins appear to mimic the ter-
minal residues of the peptide, with the substituted fused phenyl
ring of the 5-deazaflavin system occupying the position of the Ile
residue, the pyrimidinedione ring in the position of the Ala residue,
A similar situation was revealed when the 10-phenyl halogen
substituents in 9-trifluoromethyl-5-deazaflavins were replaced
with methyl groups. Of the three 10-toluyl derivatives assayed,
the meta-isomer 19 was most active, followed by the less potent
para-isomer 20. As in the nitro series, modification of the active
9-trifluoro-methyl-10-aryl-5-deazaflavin core by alkylation at N3
(62 and 63; Table S1) abolished activity, and the only replacement
of the 10-aryl group that was tolerated was with a benzyl group
(21).
We next examined the effect of groups other than the strongly
electron-withdrawing nitro and trifluoromethyl functions on the
biological activity of 5-deazaflavin compounds. The weakly elec-
tron-withdrawing chloro substituent in the contexts of an unsub-
stituted, ortho-fluoro- or para-chloro-substituted 10-phenyl
group resulted in poorly active (22–24) or inactive derivatives,
with the exception of the combination of chloro substituents at
both C9 and at the para-position of the 10-phenyl group, which
afforded a compound (25) with reasonable activity. This situation
is similar to the SARs in the 9-trifluoro-methyl series; however,
here alternative halogen substitutions in the 10-phenyl group were
less well tolerated (26 and 27).
Replacement of the electron-withdrawing nitro, trifluoro-
methyl, or chloro substituents in the benzene ring of the 5-deaza-
flavin ring system with the electron-releasing and sterically
undemanding methyl group resulted in inactive compounds when
the 10-phenyl group was unsubstituted. Unlike in the case of the 9-
trifluoromethyl series, in the otherwise active context of the para-
chloro- or ortho-fluoro-substituted 10-phenyl group, this resulted
in a clear loss or decline in biological activity in all cases, with only
compound 28 retaining marginal activity.
In the case of the more strongly electron-releasing hydroxyl
group at positions C9 of the deazaflavin system, no biological activ-
ity was obtained, regardless of 10-phenyl substitution. However, 9-
bromo (29 and 30) and 9-fluoro derivatives were also compara-
tively inactive, and the same was true for both derivatives with
the strongly electron-withdrawing cyano group at C9 (95 and 96;
Table S1).
Overall these results suggest that the SARs for inhibition of
HDM2 E3 ubiquitin ligase activity in substituted 5-deazaflavin
derivatives depend on a combination of factors. The most active
compounds contain a trifluoromethyl or chloro substituent at C9
and this activity depends to a large extent on the presence of at
least one additional halogen or methyl substituent of the phenyl
group at N10. Although we have not yet tackled modification of
the heterocyclic rings in the 5-deazaflavin system extensively,
SARs in that region appear to be quite restrictive and neither
replacement of the (substituted) 10-phenyl nor the N3-H appear
to be tolerated. Selectivity of the 5-deazaflavins described here
for the HDM2 E3 ubiquitin ligase has been demonstrated previ-
ously9 using similar assay systems for inhibition of auto-ubiquiti-
nation of either HDM2 or the related RING E3 ubiquitin ligase
CBL.32
Some of the compound structures described here coincide with
those reported earlier by Wilson et al.8 and in general there is
agreement between the in vitro activity described here and the
activity of the relevant compounds in cells, as measured by Wilson
et al.8 However, two 6-chloro-5-deazaflavin derivatives described
by Wilson et al. (24 and 69, corresponding to compounds 14 and
16, respectively, in Wilson et al.8) were found to activate p53 in
cells, while our data indicate that these compounds cannot directly
inhibit HDM2. These results suggest that these compounds induce
p53 through an indirect mechanism in cells, and that the cell activ-
ity is an off-target effect. In the in vitro assays described here, we
show that the 9-trifluoromethyl-5-deazaflavins are the most active

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M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
Melting points were recorded using Gallenkamp and Bibby Stuart
Scientific (SMP3) apparatus and are uncorrected. NMR spectra
were recorded on a Bruker-AV 400 instrument at 400.1 MHz (1H)
or 75 MHz (13C) with compound solutions in DMSO-d6 (unless
otherwise indicated). Chemical shifts (d) are reported in ppm with
reference to the chemical shift of residual solvent or the internal
standard SiMe4. Mass spectra (TOF-ES) were recorded using a
Waters 2795 Separation Module and Micromass LCT platform.
Infrared spectra were recorded using Avatar 360 Nicolet FT-IR
and Nicolet IR200 FT-IR spectrophotometers. A Waters 2525 Binary
Gradient Module was used for analytical RP-HPLC with a Waters
2487 Dual Wavelength Absorbance UV Detector. Analytical
RP-HPLC was used to confirm purity for all compounds by chro-
matogram integration at 254 nm, using two systems. System A:
Phenomenex Onyx monolithic reversed phase C18 column
(100 Â 3.0 mm) with a flow rate of 3 mL/min and using isocratic
elution with 7:3 H2O–MeCN. System B: Phenomenex Kromasil
reversed phase C18 column (250 Â 4.6 mm) with a flow rate of
1 mL/min and using isocratic elution with 1:1 H2O–MeOH. Biolog-
ical assays were carried out as described.9
5.2. General method for the preparation of 6-anilinouracils (3a)
To 6-chlorouracil (2, 1 equiv) was added the appropriate aniline
(6 equiv) and the mixture was heated at 180–200 °C for 1.5–3 h. It
was then cooled and the residue was triturated with Et2O. Precip-
itated product was filtered and washed on a sinter with Et2O and
H2O. After drying, the crude product was crystallised from EtOH.
In this manner pure 6-anilinouracils 3a were obtained, whose
analytical details were in accord with the literature. E.g. 6-[(3-
methylphenyl)amino]-1,2,3,4-tetrahydropyrimidine-2,4-dione
(3a, R5,7 = H, R6 = Me) in 71% yield as an off-white solid with mp
303–304 °C (lit.43 302–303 °C); IR (KBr): 3211 (NH), 3053 (NH),
Figure 3. HDM2–HDMX RING domain heterodimer complex (constructed from
PDB entry 2VJF). (a) Secondary structure cartoon with HDM2 in green and HDMX in
cyan. The two zinc ions in each RING domain are shown as spheres with the
coordinating residue side chains as sticks. A composite of one face of HDM2,
together with the C-terminus of HDMX (key residues shown with side chains and
labelled), forms the likely interaction site with the E2 ubiquitin conjugating
enzyme. (b) Interaction of the C-terminus of HDMX (cyan) with HDM2 (green CPK
surface). (c) Predominant docked poses of compounds HLI98C (magenta) and 15
(yellow).
1734 (C@O), 1625 (C@C) cmÀ1 1H NMR: d 2.30 (3H, s, Me), 4.68
;
(1H, s, C5-H), 6.95–7.02 (3H, m, Ph-H), 7.26 (1H, t, J = 7.5 Hz, Ph
5-H), 8.16 (1H, s, NH), 10.10 (1H, s, N1-H), 10.43 (1H, s, N3-H);
13C NMR: d 17.78 (CH3), 75.21 (CH), 126.66 (CH), 127.07 (CH),
127.32 (CH), 131.45 (CH), 134.21 (Cq), 136.12 (Cq), 151.29 (Cq),
153.54 (Cq), 164.70 (Cq); HRMS (ESI+): calcd for C11H12N3O2
[M+H]+ 218.0930, found 218.0923.
and the 10-aryl group projecting towards the Phe residue. The
main determinant of activity, i.e. a 9-trifluoromethyl group, may
be due to favourable interactions of that group with a pocket delin-
eated by HDM2 residues Leu430 and Leu458. Substitution at C9 of
the 5-deazaflavin system also imposes rotational limitations on
the 10-aryl group, thus locking this group into conformations
which positions its halogen substituents, which are important for
activity, for interactions with either His457 or Leu430–Pro431. While
these finding are suggestive, it remains to be shown experimen-
tally that HDM2-inhibitory 5-deazaflavins compete directly with
the C-terminal peptide.
5.3. General method for the preparation of 2-tosylbenzalde-
hydes (4, X = OTs)
A mixture of the appropriate salicylaldehyde (1 equiv) and Na2-
CO3 (4 equiv) in Me2CO (5 mL/mmol salicylaldehyde) was stirred
for 30 min under N2, when tosyl chloride (2 equiv) in Me2CO
(2 mL/mmol) was added. The solution was heated under reflux
for 5 h, cooled, and concentrated under reduced pressure. The res-
idue was purified by flash chromatography (2:1 hexane–Et2O) to
afford the product.
4. Conclusion
Following our recent elucidation of the fact that bioactive 5-
deazaflavins stabilize and activate p53 by binding directly to the
HDM2 RING domain,9 here we have presented a comprehensive
SAR analysis of 5-deazaflavin as inhibitors of p53 ubiquitination
by the p53-specific ubiquitin E3 ligase HDM2. Together with the
structural model of the mode of interaction of 5-deazaflavins with
HDM2, it should now be possible to design and develop drug-like
and potent inhibitors of HDM2 E3 ligase activity for potential ther-
apeutic application in oncology.
5.4. 2-Formyl-5-methylphenyl 4-methylbenzene-1-sulfonate (4,
R1,2,4 = H, R3 = Me, X = OTs)
Prepared using general method 5.3 from 2-hydroxy-4-methyl-
benzaldehyde (0.50 g, 3.7 mmol). White solid (0.63 g, 59%). Mp
93–94 °C; 1H NMR: d 2.36 (3H, s, Me), 2.42 (3H, s, Me), 7.06 (1H,
s, C3-H), 7.33 (1H, d, J = 7.8 Hz, C5-H), 7.48 (2H, d, J = 8.2 Hz, Ph
3-H), 7.70 (1H, d, J = 7.8 Hz, C6-H), 7.76 (2H, d, J = 8.2 Hz, Ph 2-
H), 9.83 (1H, s, CHO); 13C NMR: d 21.65 (CH3), 21.66 (CH3),
124.44 (CH), 126.96 (Cq), 128.86 (CH), 128.96 (CH), 129.31 (CH),
130.85 (CH), 130.90 (Cq), 146.95 (Cq), 147.74 (Cq), 150.51 (Cq),
187.45 (CH).
5. Experimental section
5.1. General
Silica gel (Merck Kieselgel 60, 230–400) flash column chroma-
tography was carried out as described42 or using Biotage Argonaut
Flash Master II and Biotage Flash Master Personal instruments.

M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
6873
5.5. 2-Bromo-6-formylphenyl 4-methylbenzene-1-sulfonate (4,
R1–3 = H, R4 = Br, X = OTs)
tR 1.63 min (98.4%, A), 4.75 min (100%, B); HRMS (ESI+): calcd for
C17H10ClN4O4 [M+H]+ 369.0391, found 369.0419.
Prepared using general method 5.3 from 3-bromo-2-hydroxy-
benzaldehyde (0.57 g, 2.8 mmol). White solid (0.82 g, 82%). Mp
79–91 °C; 1H NMR: d 2.46 (3H, s, Me), 7.44–7.58 (3H, m), 7.77–
7.88 (3H, m), 8.05 (1H, d, J = 7.3 Hz, C4-H), 9.86 (1H, s, CHO); 13C
NMR: d 21.74 (CH3), 118.58 (Cq), 128.40 (CH), 129.17 (CH),
129.94 (CH), 131.02 (CH), 131.28 (Cq), 132.40 (Cq), 139.98 (CH),
147.44 (Cq), 147.65 (Cq), 187.53 (CH).
5.10. 10-Phenyl-9-(trifluoromethyl)-2H,3H,4H,10H-pyrimido
[4,5-b]quinoline-2,4-dione (8)
Prepared using general method 5.7 from 3a (R5–7 = H; 58 mg,
0.28 mmol)46 and 4 (R1–3 = H, R4 = CF3, X = F; 65 mg, 0.34 mmol).
Yellow solid (23.2 mg, 23%). Mp 312–314 °C; IR (KBr): 3.424
(NH), 1.716 (C@O), 1.659 (C@O), 1.618 (C@C) cmÀ1 1H NMR: d
;
7.30–7.36 (2H, m, Ph 2-H), 7.45–7.51 (3H, m, Ph-H), 7.66 (1H, t,
J = 7.7 Hz, C7-H), 8.18 (1H, dd, 4J = 1.4 Hz, 3J = 7.7 Hz, C8-H), 8.48
(1H, dd, 4J = 1.4 Hz, 3J = 7.7 Hz, C6-H), 9.11 (1H, s, C5-H), 11.23
(1H, s, N3-H); 13C NMR: d 116.79 (Cq), 120.91 (CH), 124.26 (Cq),
124.94 (CH), 128.44 (CH), 129.58 (CH), 129.90 (CH), 131.20 (CH),
136.79 (Cq), 139.46 (Cq), 139.67 (Cq), 143.32(CH), 156.80 (Cq),
161.18 (Cq), 162.07 (Cq); anal. RP-HPLC: tR 2.12 min (100%, A),
5.20 min (100%, B); HRMS (ESI+): calcd for C18H11F3N3O2 [M+H]+
358.0803, found 358.0765.
5.6. 2-Cyano-6-formylphenyl 4-methylbenzene-1-sulfonate (4,
R1–3 = H, R4 = CN, X = OTs)
Prepared using general method 5.3 from 3-formyl-2-hydrox-
ybenzonitrile (0.34 g, 1.7 mmol).29 White solid (0.48 g, 94%). Mp
84–86 °C; 1H NMR: d 2.46 (3H, s, Me), 7.55 (2H, d, J = 8.5 Hz, Ph
3-H), 7.75 (1H, t, J = 7.6 Hz, C5-H), 7.82 (2H, d, J = 8.5 Hz, Ph 2-H),
8.15 (1H, dd, J = 1.8, 7.6 Hz, C6-H), 8.28 (1H, dd, J = 1.8, 7.6 Hz,
C4-H), 9.84 (1H, s, CHO); 13C NMR: d 21.76 (CH3), 109.73 (Cq),
114.46 (Cq), 129.27 (CH), 129.55 (CH), 129.95 (Cq), 131.22 (Cq),
131.25 (CH), 133.78 (CH), 140.01 (CH), 148.06 (Cq), 150.68 (Cq),
186.55 (CH).
5.11. 10-(2-Fluorophenyl)-8-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (9)
Prepared using general method 5.7 from 3a (R5 = F, R6,7 = H;
5.7. General method for the preparation of 10-aryl-5-deaza-
flavins using 2-halobenzaldehydes or 2-tosylbenzaldehydes
59 mg, 0.27 mmol)8 and (R1,2,4 = H, R3 = CF3, X = F; 61 mg,
4
0.32 mmol). Yellow solid (39.1 mg, 39%). Mp 315–318 °C; IR
(KBr): 3.423 (NH), 1.708 (C@O), 1.672 (C@O), 1.622 (C@C) cmÀ1
;
A mixture of a 6-(arylamino)pyrimidine-2,4(1H,3H)-dione 3a
(1 equiv) and a 2-halo- or 2-tosyl-benzaldehyde 4 (X = F, Cl, or
OTs; 1.2 equiv) in DMF (10 mL/mmol 3a) was heated under reflux
for 4 h. After cooling, the solution was evaporated under reduced
pressure and the residue was purified by flash chromatography
(5:95 MeOH–CH2Cl2) to afford the product.
1H NMR: d 6.90 (1H, s, C9-H), 7.56 (1H, t, J = 7.6 Hz, Ph 5-H),
7.62–7.69 (2H, m, Ph-H), 7.73–7.80(1H, m, Ph-H), 7.89 (1H, d,
J = 8.4 Hz, C7-H), 8.51 (1H, d, J = 8.4 Hz, C6-H), 9.23 (1H, s, C5-H),
11.34 (1H, s, N3-H); 13C NMR: d 112.97 (CH, d, J = 4.77 Hz),
117.88 (CH, d, J = 18.40 Hz), 118.28 (Cq), 121.10 (CH, d,
J = 3.62 Hz), 123.86 (Cq), 124.29 (Cq, d, J = 13.29 Hz), 126.81 (CH,
d, J = 3.52 Hz), 130.94 (CH), 133.06 (CH, d, J = 8.09 Hz), 133.93
(CH), 134.27 (Cq), 141.12 (Cq), 142.34 (CH), 156.63 (Cq), 157.58
(Cq, d, J = 251.92 Hz), 159.17 (Cq), 161.77 (Cq); anal. RP-HPLC: tR
3.59 min (97.3%, A), 6.42 min (100%, B); HRMS (ESI+): calcd for C18-
H10F4N3O2 [M+H]+ 376.0709, found 376.0705.
5.8. 10-(2-Fluorophenyl)-9-nitro-2H,3H,4H,10H-pyrimido[4, 5-
b]quinoline-2,4-dione (5)
Prepared using general method 5.7 from 3a (R5 = F, R6,7 = H;
62 mg, 0.28 mmol) and 4 (R1–3 = H, R4 = NO2, X = OTs; 108 mg,
0.34 mmol).44,45 Yellow solid (23.0 mg, 23%). Mp: 305–307 °C; IR
(KBr): 3.425 (NH), 1.712 (C@O), 1.679 (C@O), 1.619 (C@C), 1.487
5.12. 10-(2-Fluorophenyl)-9-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (10)
(NO2), 1.328 (NO2) cmÀ1 1H NMR: d 7.25 (1H, t, J = 7.5 Hz, Ph 5-
;
H), 7.35–7.44 (2H, m, Ph-H), 7.56–7.74 (2H, m, C7-H & Ph-H),
8.17 (1H, d, J = 7.3 Hz, C8-H), 8.50 (1H, d, J = 7.3 Hz, C6-H), 9.19
(1H, s, C5-H), 11.36 (1H, s, N3-H); 13C NMR: d 116.72 (CH, d,
J = 19.6 Hz), 117.13 (Cq), 124.05 (Cq), 125.11 (CH), 125.21 (Cq),
125.34 (Cq), 125.36 (CH), 130.03 (CH), 132.60 (CH), 133.12 (CH,
d, J = 8.0 Hz), 136.17 (CH), 140.85 (Cq), 142.95 (CH), 156.53 (Cq),
158.52 (Cq, d, J = 252.4 Hz), 159.51 (Cq), 161.71 (Cq); anal. RP-
HPLC: tR 1.10 min (100%, A), 3.98 min (97.6%, B); HRMS (ESI+):
calcd for C17H10FN4O4 [M+H]+ 353.0681, found 353.0676.
Prepared using general method 5.7 from 3a (R5 = F, R6,7 = H;
59 mg, 0.27 mmol)8 and
4
(R1–3 = H, R4 = CF3, X = F; 62 mg,
0.32 mmol). Yellow solid (65.7 mg, 66%). Mp: 292–293 °C (lit.8
>360 °C); IR (KBr): 3.418 (NH), 1.703 (C@O), 1.679 (C@O), 1,631
(C@C) cmÀ1 1H NMR: d 7.25–7.31(2H, m, Ph-H), 7.42 (1H, t,
;
J = 9.7 Hz, Ph 5-H), 7.55–7.63 (1H, m, Ph-H), 7.69 (1H, t,
J = 7.9 Hz, C7-H), 8.23 (1H, dd, 4J = 1.3 Hz, 3J = 7.9 Hz, C8-H), 8.52
(1H, dd, 4J = 1.3 Hz, 3J = 7.9 Hz, C6-H), 9.15 (1H, s, C5-H), 11.32
(1H, s, N3-H); 13C NMR: d 115.80 (CH, d, J = 20.1 Hz), 116.05
(CH), 123.89 (Cq), 124.64 (Cq), 126.75 (CH, d, J = 3.6 Hz), 129.70
(CH), 130.95 (CH), 131.14 (Cq), 131.99 (CH, d, J = 7.64 Hz), 135.36
(Cq), 136.91 (Cq), 139.57 (Cq), 143.28 (CH), 156.26 (Cq), 158.77
(Cq, d, J = 251.8 Hz), 159.99 (Cq), 161.45 (Cq); anal. RP-HPLC: tR
2.52 min (98.0%, A), 5.62 min (97.5%, B); HRMS (ESI+): calcd for C18-
H10F4N3O2 [M+H]+ 376.0709, found 376.0729.
5.9. 10-(4-Chlorophenyl)-9-nitro-2H,3H,4H,10H-pyrimido[4, 5-
b]quinoline-2,4-dione (7)
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
64 mg, 0.27 mmol) and 4 (R1–3 = H, R4 = NO2, X = OTs; 104 mg,
0.32 mmol).44,45 Yellow solid (23.8 mg, 24%). Mp 336–338 °C; IR
(KBr): 3.422 (NH), 1.706 (C@O), 1.672 (C@O), 1.620 (C@C), 1.427
5.13. 10-(4-Chlorophenyl)-6-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (11)
(NO2), 1,352 (NO2) cmÀ1
;
1H NMR: d 7.41 (2H, d, J = 8.6 Hz, Ph 2-
H), 7.55 (2H, d, J = 8.6 Hz, Ph 3-H), 7.64 (1H, t, J = 7.8 Hz, C7-H),
8.13 (1H, d, J = 7.8 Hz, C8-H), 8.48 (1H, d, J = 7.8 Hz, C6-H), 9.17
(1H, s, C5-H), 11.30 (1H, s, N3-H); 13C NMR: d 117.45 (Cq),
123.98 (Cq), 124.87 (CH), 129.21 (CH), 131.40 (CH), 132.03 (CH),
132.52 (Cq), 134.63 (Cq), 135.89 (CH), 136.24 (Cq), 141.09 (Cq),
142.62 (CH), 156.53 (Cq), 160.37 (Cq), 161.86 (Cq); anal. RP-HPLC:
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
61 mg, 0.26 mmol)25 and
4
(R1 = CF3, R2–4 = H, X = F; 60 mg,
0.31 mmol). Yellow solid (11.1 mg, 11%). Mp 304–306 °C; IR
(KBr): 3.410 (NH), 1.716 (C = O), 1.670 (C@O), 1624 (C@C) cmÀ1
1H NMR: d 7.16 (1H, d, J = 8.0 Hz, C9-H), 7.52 (2H, d, J = 8.8 Hz,
;

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M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
Ph 2-H), 7.80 (2H, d, J = 8.8 Hz, Ph 3-H), 7.88 (1H, t, J = 8.0 Hz, C8-
H), 7.97 (1H, d, J = 8.0 Hz, C7-H), 8.86 (1H, s, C5-H), 11.33 (1H, s,
N3-H); 13C NMR: d 116.89 (Cq), 118.04 (Cq), 122.94 (CH), 123.27
(CH), 127.48 (Cq), 130.86 (CH), 130.99 (CH), 134.66 (CH), 134.83
(Cq), 135.64 (CH), 136.70 (Cq), 143.28 (Cq), 156.53 (Cq), 158.80
(Cq), 161.88 (Cq); anal. RP-HPLC: tR 3.98 min (100%, A), 7.47 min
(96.2%, B); HRMS (ESI+): calcd for C18H10ClF3N3O2 [M+H]+
392.0414, found 392.0439.
(KBr): 3.486 (NH), 1.705 (C@O), 1.655 (C@O), 1.609 (C@C) cmÀ1
;
1H NMR: d 7.46 (1H, dd, 4J = 1.1 Hz, 3J = 8.6 Hz, Ph 6-H), 7.69 (1H,
t, J = 7.8 Hz, C7-H), 7.73 (1H, d, 4J = 1.1 Hz, Ph 2-H), 7.81 (1H, d,
J = 8.6 Hz, Ph 5-H), 8.23 (1H, dd, 4J = 1.6 Hz, 3J = 7.8 Hz, C8-H),
8.50 (1H, dd, 4J = 1.6 Hz, 3J = 7.8 Hz, C6-H), 9.12 (1H, s,C5-H),
11.30 (1H, s, N3-H); 13C NMR: d 116.71 (Cq), 118.33 (Cq), 121.67
(Cq), 124.46 (Cq), 125.13 (CH), 130.37 (CH), 130.94 (Cq), 132.28
(CH), 132.58 (Cq), 133.14 (CH), 135.71 (CH), 137.30 (CH), 139.84
(Cq), 143.71 (CH), 156.61 (Cq), 161.14 (Cq), 161.88 (Cq); anal. RP-
HPLC: tR 6.40 min (99.4%, A), 6.35 min (98.7%, B) HRMS (ESI+): calcd
for C18H9Cl2F3N3O2 [M+H]+ 426.0024, found 426.0034.
5.14. 10-(4-Chlorophenyl)-8-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (13)
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
84 mg, 0.35 mmol)25 and 4 (R1,2,4 = H, R3 = CF3, X = F; 81 mg,
0.42 mmol). Yellow solid (70.8 mg, 51%). Mp 348–349 °C (dec); IR
5.18. 10-(4-Fluorophenyl)-9-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (18)
(KBr): 3.430 (NH), 1.716 (C@O), 1.683 (C@O), 1.650 (C@C) cmÀ1
;
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = F;
1H NMR: d 6.89 (1H, s, C9-H), 7.54 (2H, d, J = 8.7 Hz, Ph 2-H),
7.81(2H, d, J = 8.7 Hz, Ph 3-H), 7.86 (1H, d, J = 8.2 Hz, C7-H), 8.49
(1H, d, J = 8.2 Hz, C6-H), 9.22 (1H, s, C5-H), 11.28 (1H, s, N3-H);
13C NMR: d 118.40 (CH), 120.69 (CH), 120.72 (CH), 122.36 (Cq),
123.96 (Cq), 130.94 (CH), 131.06 (CH), 133.59 (CH), 134.92 (Cq),
136.31 (Cq), 141.79 (Cq), 141.94 (CH), 156.67 (Cq), 159.58 (Cq),
161.97 (Cq); anal. RP-HPLC: tR 6.18 min (100%, A), 8.60 min
(97.4%, B). HRMS (ESI+): calcd for C18H10ClF3N3O2 [M+H]+
392.0414, found 392.0374.
58 mg, 0.26 mmol)8 and (R1–3 = H, R4 = CF3, X = F; 60 mg,
4
0.31 mmol). Yellow solid (45.5 mg, 46%). Mp: >350 °C (lit.8 327–
329 °C); IR (KBr): 3.420 (NH), 1.716 (C@O), 1.660 (C@O), 1.620
(C@C) cmÀ1 1H NMR: d 7.29–7.36 (2H, m, Ph 2-H), 7.38–7.45
;
(2H, m, Ph 3-H), 7.67 (1H, t, J = 7.4 Hz, C7-H), 8.20 (1H, d,
J = 7.4 Hz, C8-H), 8.49 (1H, d, J = 7.4 Hz, C6-H), 9.11 (1H, s, C5-H),
11.24 (1H, s, N3-H); 13C NMR: d 115.41 (CH, d, J = 23.0 Hz),
116.76 (Cq), 118.60 (Cq), 118.91 (Cq), 124.34 (Cq), 124.88 (CH),
133.52 (CH, d, J = 9.3 Hz), 135.57 (CH), 136.97 (CH), 140.44 (Cq),
143.42 (CH), 156.75 (Cq), 162.00 (Cq), 161.31 (Cq), 162.27 (Cq, d,
J = 247.93 Hz); anal. RP-HPLC: tR 2.25 min (100%, A), 27.65 min
(100%, B); HRMS (ESI+): calcd for C18H10F4N3O2 [M+H]+ 376.0709,
found 376.0719.
5.15. 10-(4-Chlorophenyl)-9-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (14)
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
61 mg, 0.26 mmol)25 and
4
(R1–3 = H, R4 = CF3, X = F; 60 mg,
5.19. 10-(3-Methylphenyl)-9-(trifluoromethyl)-2H,3H,4H, 10H-
0.31 mmol). Yellow solid (28.3 mg, 28%). Mp 372–375 °C; IR
pyrimido[4,5-b]quinoline-2,4-dione (19)
(KBr): 3.483 (NH), 1.715 (C@O), 1.659 (C@O), 1.618 (C@C) cmÀ1
;
1H NMR: d 7.39 (2H, d, J = 8.6 Hz, Ph 2-H), 7.56 (2H, d, J = 8.6 Hz,
Ph 3-H), 7.67 (1H, t, J = 7.7 Hz, C7-H), 8.20 (1H, dd, 4J = 1.3 Hz,
3J = 7.7 Hz, C8-H), 8.49 (1H, dd, 4J = 1.3 Hz, 3J = 7.7 Hz, C6-H), 9.11
(1H, s, C5-H),11.25 (1H, s, N3-H); 13C NMR: d 116.76 (Cq), 118.70
(Cq), 124.38 (Cq), 124.98 (CH), 128.57 (CH), 133.16 (CH), 134.23
(Cq), 135.60 (CH), 137.03 (CH), 139.24 (Cq), 140.15 (Cq), 143.47
(CH), 156.69 (Cq), 161.14 (Cq), 161.97 (Cq); anal. RP-HPLC: tR
4.37 min (100%, A), 7.17 min (98.2%, B); HRMS (ESI+): calcd for C18-
H10ClF3N3O2 [M+H]+ 392.0414, found 392.0416.
Prepared using general method 5.7 from 3a (R5,7 = H, R6 = Me;
58 mg, 0.27 mmol)43 and
4
(R1–3 = H, R4 = CF3, X = F; 62 mg,
0.32 mmol). Yellow solid (38.6 mg, 39%). Mp 296–298 °C (dec); IR
(KBr): 3.474 (NH), 1.716 (C@O), 1.657 (C@O), 1.619 (C@C) cmÀ1
;
1H NMR: d 2.31 (3H, s, Me), 7.08 (1H, s, Ph 2-H), 7.18 (1H, d,
J = 7.6 Hz, Ph 4-H), 7.29 (1H, d, J = 7.6 Hz, Ph 3-H), 7.36 (1H, t,
J = 7.6 Hz, Ph 5-H), 7.66 (1H, t, J = 7.6 Hz, C7-H), 8.18 (1H, dd,
4J = 1.5 Hz, 3J = 7.6 Hz, C8-H), 8.47 (1H, dd, 4J = 1.5 Hz, 3J = 7.6 Hz,
C6-H), 9.10 (1H, s, C5-H), 11.22 (1H, s, N3-H); 13C NMR: d 21.78
(CH3), 116.71 (Cq), 118.95 (Cq), 119.27 (Cq), 124.28 (Cq), 124.82
(CH), 128.12 (CH), 128.66 (CH), 130.10 (CH), 131.11 (CH), 135.51
(CH), 136.79 (CH), 137.96 (Cq), 140.49 (Cq), 143.29 (CH), 156.85
(Cq), 161.23 (Cq), 162.05 (Cq); anal. RP-HPLC: tR 3.00 min (100%,
A), 4.64 min (99.7%, B); HRMS (ESI+): calcd for C19H13F3N3O2
[M+H]+ 372.0960, found 372.0954.
5.16. 10-(3-Chlorophenyl)-9-(trifluoromethyl)-2H,3H,4H, 10H-
pyrimido[4,5-b]quinoline-2,4-dione (15)
Prepared using general method 5.7 from 3a (R5,7 = H, R6 = Cl;
60 mg, 0.25 mmol)8 and
4
(R1–3 = H, R4 = CF3, X = F; 58 mg,
0.3 mmol). Yellow solid (54.6 mg, 55%). Mp: 342–343 °C (dec); IR
(KBr): 3.421 (NH), 1.706 (C@O), 1.672 (C@O), 1.623 (C@C) cmÀ1
;
5.20. 10-(4-Methylphenyl)-9-(trifluoromethyl)-2H,3H,4H, 10H-
1H NMR: d 7.37 (1H, d, J = 7.7 Hz, Ph-H), 7.48–7.62 (3H, m, Ph-H),
7.68 (1H, t, J = 7.5 Hz, C7-H), 8.21 (1H, d, J = 7.5 Hz, C8-H), 8.49
(1H, d, J = 7.5 Hz, C6-H), 9.11 (1H, s, C5-H), 11.28 (1H, s, N3-H);
13C NMR: d 116.76 (Cq), 124.45 (Cq), 125.03 (CH), 129.72 (CH),
130.04 (CH), 130.44 (CH), 131.19 (CH), 132.58 (Cq), 135.61 (CH),
135.66 (Cq), 137.06 (CH), 140.04 (Cq), 141.39 (Cq), 143.56 (CH),
156.70 (Cq), 161.16 (Cq), 161.95 (Cq); anal. RP-HPLC: tR 3.54 min
(97.8%, A), 8.69 min (98.9%, B); HRMS (ESI+): calcd for C18H10ClF3-
N3O2 [M+H]+ 392.0408, found 392.0404.
pyrimido[4,5-b]quinoline-2,4-dione (20)
Prepared using method 5.7 from 3a (R5,6 = H, R7 = Me; 59 mg,
0.27 mmol)8 and 4 (R1–3 = H, R4 = CF3, X = F; 62 mg, 0.32 mmol).
Yellow solid (52.1 mg, 52%). Mp 344–346 °C (dec); IR (KBr):
3.438 (NH), 1.706 (C@O), 1.681 (C@O), 1.622 (C@C) cmÀ1 1H
;
NMR: d 2.38 (3H, s, Me), 7.19 (2H, d, J = 8.4 Hz, Ph 3-H), 7.27 (2H,
d, J = 8.4 Hz, Ph 2-H), 7.65 (1H, t, J = 7.9 Hz, C7-H), 8.17 (1H, dd,
4J = 1.3 Hz, 3J = 7.9 Hz, C8-H), 8.47 (1H, dd, 4J = 1.3 Hz, 3J = 7.9 Hz,
C6-H), 9.09 (1H, s, C5-H), 11.21 (1H, s, N3-H); 13C NMR: d 21.27
(CH3), 116.72 (Cq), 118.94 (Cq), 124.30 (Cq), 124.80 (CH), 128.94
(CH), 130.92 (CH), 135.52 (CH), 136.79 (CH), 138.18 (Cq), 139.12
(Cq), 140.56 (Cq), 143.28 (CH), 156.80 (Cq), 161.29 (Cq), 162.07
(Cq); anal. RP-HPLC: tR 3.13 min (100%, A), 8.57 min (99.7%, B);
HRMS (ESI+): calcd for C19H13F3N3O2 [M+H]+ 372.0960, found
372.0957.
5.17. 10-(3,4-Dichlorophenyl)-9-(trifluoromethyl)-2H,3H,4H,
10H-pyrimido[4,5-b]quinoline-2,4-dione (16)
Prepared using general method 5.7 from 3a (R5 = H, R6,7 = Cl;
64 mg, 0.24 mmol)47 and
4
(R1–3 = H, R4 = CF3, X = F; 55 mg,
0.29 mmol). Yellow solid (62.3 mg, 62%). Mp 320–322 °C; IR

M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
6875
5.21. 10-Benzyl-9-(trifluoromethyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (21)
(99.6%; A), 6.05 min (100%, B); HRMS (ESI+): calcd for C17H10Cl2N3-
O2 [M+H]+ 358.0150, found 358.0155.
Prepared using method 5.7 from 3b (R9 = Bn; 56 mg,
0.26 mmol)48 and 4 (R1–3 = H, R4 = CF3, X = F; 60 mg, 0.31 mmol).
Yellow solid (11.5 mg, 12%). Mp 235–237 °C; IR (KBr): 3.416
5.25. 9-Chloro-10-(4-chlorophenyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (25)
(NH), 1.706 (C@O), 1.664 (C@O), 1.615 (C@C) cmÀ1
;
1H NMR: d
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
5.87 (2H, s, CH2), 6.81 (2H, d, J = 6.7 Hz, Ph 2-H), 7.13–7.21 (3H,
m, Ph-H), 7.68 (1H, t, J = 7.8 Hz, C7-H), 8.31 (1H, dd, 4J = 1.3 Hz,
3J = 7.8 Hz, C8-H), 8.47 (1H, d, J = 7.8 Hz, C6-H), 9.02 (1H, s, C5-
H), 11.26 (1H, s, N3-H); 13C NMR: d 54.41 (CH2), 117.14 (Cq),
118.17 (Cq), 124.54 (Cq), 125.08 (CH), 126.70 (CH), 127.62 (CH),
128.86 (CH), 135.99 (CH), 136.69 (Cq), 137.34 (CH), 139.88 (Cq),
142.47 (CH), 156.73 (Cq), 160.83 (Cq), 162.02 (Cq); anal. RP-HPLC:
tR 2.34 min (97.5%, A), 8.22 min (96.8%, B); HRMS (ESI+): calcd for
65 mg, 0.27 mmol)25 and (R1–3 = H, R4 = Cl, X = F; 51 mg,
4
0.32 mmol). Yellow solid (14.6 mg, 15%). Mp > 350 °C; IR (KBr):
3.415 (NH), 1.715 (C@O), 1.655 (C@O), 1.613 (C@C) cmÀ1 1H
;
NMR: d 7.43–7.52 (3H, m, C7-H & Ph 2-H), 7.60 (2H, d, J = 8.8 Hz,
Ph 3-H), 7.85 (1H, dd, 4J = 1.5 Hz, 3J = 7.9 Hz, C8-H), 8.28 (1H, dd,
4J = 1.5 Hz, 3J = 7.9 Hz, C6-H), 9.09 (1H, s, C5-H), 11.22 (1H, s, N3-
H); 13C NMR: d 116.48 (Cq), 121.00 (Cq), 124.59 (Cq), 125.77
(CH), 129.18 (CH), 132.22 (CH), 132.45 (CH), 134.12 (Cq), 137.53
(Cq), 138.57 (Cq), 139.25 (CH), 143.33 (CH), 156.63 (Cq), 160.81
(Cq), 162.04 (Cq); anal. RP-HPLC: tR 3.10 min (100%; A), 6.34 min
(97.2%; B); HRMS (ESI+): calcd for C17H10Cl2N3O2 [M+H]+
358.0150, found 358.0156.
C
19H13F3N3O2 [M+H]+ 372.0960, found 372.0973.
5.22. 9-Chloro-10-phenyl-2H,3H,4H,10H-pyrimido[4,5-
b]quinoline-2,4-dione (22)
Prepared using method 5.7 from 3a (R5–7 = H; 62 mg,
0.31 mmol)46 and 4 (R1–3 = H, R4 = CF3, X = F; 59 mg, 0.37 mmol).
Yellow solid (38.8 mg, 39%). Mp > 350 °C. IR (KBr): 3.441 (NH),
5.26. 9-Chloro-10-(3-chlorophenyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (26)
1.717 (C@O), 1.659 (C@O), 1.615 (C@C) cmÀ1 1H NMR: d 7.37
;
Prepared using general method 5.7 from 3a (R5,7 = H, R6 = Cl,
(2H, m, Ph 2-H), 7.45–7.54 (3H, m, Ph-H), 7.48 (1H, t, J = 7.8 Hz,
C7-H), 7.83 (1H, dd, 4J = 1.5 Hz, 3J = 7.8 Hz, C8-H), 8.23 (1H, dd,
4J = 1.5 Hz, 3J = 7.8 Hz, C6-H), 9.10 (1H, s, C5-H), 11.18 (1H, s, N3-
H); 13C NMR: d 116.42 (Cq), 121.22 (Cq), 124.57 (Cq), 125.70
(CH), 129.10 (CH), 129.45 (CH), 130.33 (CH), 132.34 (CH), 137.67
(Cq), 139.26 (CH), 139.62 (Cq), 143.26 (CH), 156.75 (Cq), 160.75
(Cq), 162.08 (Cq); anal. RP-HPLC: tR 1.53 min (97.2%, A), 4.59 min
(100%, B); HRMS (ESI+): calcd for C17H11ClN3O2 [M+H]+ 324.0540,
found 324.0513.
65 mg, 0.27 mmol)8 and
4
(R1–3 = H, R4 = Cl, X = F; 51 mg,
0.32 mmol). Yellow solid (29.3 mg, 30%). Mp > 350 °C; IR (KBr):
3.416 (NH), 1.715 (C@O), 1.655 (C@O), 1.613 (C@C) cmÀ1 1H
;
NMR: d 7.41–7.45 (1H, m, Ph-H), 7.49 (1H, t, J = 7.6 Hz, Ph 5-H),
7.56 (1H, t, J = 8.1 Hz, C7-H), 7.60–7.64 (2H, m, Ph-H), 7.86 (1H,
dd, 4J = 1.2 Hz, 3J = 8.1 Hz, C8-H), 8.25 (1H, dd, 4J = 1.2 Hz,
3J = 8.1 Hz, C6-H), 9.10 (1H, s, C5-H), 11.21 (1H, s, N3-H); 13C
NMR: d 116.39 (Cq), 120.84 (Cq), 124.56 (Cq), 125.82 (CH),
129.40 (CH), 129.60 (CH), 130.35 (CH), 130.62 (CH), 132.46 (CH),
133.31 (Cq), 137.39 (Cq), 139.32 (CH), 140.86 (Cq), 143.46 (CH),
156.62 (Cq), 160.75 (Cq), 161.96 (Cq); anal. RP-HPLC: tR 2.48 min
(100%; A), 8.60 min (98.3%; B); HRMS (ESI+): calcd for C17H10Cl2N3-
O2 [M+H]+ 358.0150, found 358.0151.
5.23. 7-Chloro-10-(2-fluorophenyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (23)
Prepared using method 5.7 from 3a (R5 = F, R6,7 = H; 65 mg,
0.29 mmol)8 and 4 (R1–3 = H, R2 = Cl, X = Cl; 55 mg, 0.35 mmol).
Yellow solid (79.1 mg, 79%). Mp 329–330 °C; IR (KBr): 3.427
5.27. 9-Chloro-10-(3-fluorophenyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (27)
(NH), 1.707 (C@O), 1.652 (C@O), 1.615 (C@C) cmÀ1 1H NMR: d
;
6.84 (1H, d, J = 9.1 Hz, C9-H), 7.51–7.66 (3H, m, Ph-H), 7.71–7.77
(1H, m, Ph-H), 7.80 (1H, dd, 3J = 2.5 Hz, 4J = 9.1 Hz, C8-H), 8.41
(1H, d, 4J = 2.5 Hz, C6-H), 9.12 (1H, s, C5-H), 11.26 (1H, s, N3-H);
13C NMR: d 117.05 (CH), 117.75 (CH, d, J = 19.1 Hz), 118.85 (CH),
122.46 (Cq), 124.72 (Cq, d, J = 13.6 Hz), 126.68 (CH), 129.30 (Cq),
130.67 (CH), 130.93 (CH), 132.75 (CH, d, J = 7.5 Hz), 135.54 (CH),
140.08 (Cq), 142.25 (CH), 157.72 (Cq), 157.57 (Cq, d,
J = 251.6 Hz), 158.79 (Cq), 161.92 (Cq); anal. RP-HPLC: tR
2.05 min (100%, A), 5.34 min (98.6%, B); HRMS (ESI+): calcd for C17-
H10ClFN3O2 [M+H]+ 342.0446, found 342.0399.
Prepared using general method 5.7 from 3a (R5,7 = H, R6 = F;
65 mg, 0.29 mmol)8 and
4
(R1–3 = H, R4 = Cl, X = F; 55 mg,
0.35 mmol). Yellow solid (71.0 mg, 71%). Mp > 365 °C; IR (KBr):
3.417 (NH), 1.717 (C@O), 1.658 (C@O), 1.614 (C@C) cmÀ1 1H
;
NMR: d 7.28 (1H, d, J = 7.8 Hz, Ph 6-H), 7.38–7.45 (2H, m, Ph-H),
7.49 (1H, t, J = 7.8 Hz, C7-H), 7.53–7.60 (1H, m, Ph-H), 7.86 (1H,
dd, 4J = 1.3 Hz, 3J = 7.8 Hz, C8-H), 8.25 (1H, dd, 4J = 1.3 Hz,
3J = 7.8 Hz, C6-H), 9.10 (1H, s, C5-H), 11.21 (1H, s, N3-H); 13C
NMR: d 116.38 (Cq), 116.59 (CH, d, J = 21.2 Hz), 118.11 (CH, d,
J = 23.8 Hz), 120.94 (Cq), 124.55 (Cq), 125.82 (CH), 126.86 (CH, d,
J = 2.9 Hz), 130.58 (CH, d, J = 8.8 Hz), 132.45 (CH), 137.43 (Cq),
139.33 (CH), 140.88 (Cq, d, J = 10.9 Hz), 143.42 (CH), 156.64 (Cq),
160.73 (Cq), 161.96 (Cq), 162.35 (Cq, d, J = 243.7 Hz); anal. RP-
HPLC: tR 1.63 min (97.0%; A), 19.62 min (98.1%; B); HRMS (ESI+):
calcd for C17H10ClFN3O2 [M+H]+ 342.0446, found 342.0453.
5.24. 6-Chloro-10-(4-chlorophenyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (24)
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
66 mg, 0.28 mmol)25 and
4
(R1 = Cl, R2–4 = H, X = F; 53 mg,
0.34 mmol). Yellow solid, (37.6 mg, 38%). Mp: 331–332 °C (lit.8
220–222 °C, dec); IR (KBr): 3.422 (NH), 1.703 (C@O), 1.672
(C@O), 1.607 (C@C) cmÀ1 1H NMR: d 6.77 (1H, dd, 4J = 2.3 Hz,
;
5.28. 10-(4-Chlorophenyl)-8-methyl-2H,3H,4H,10H-pyrimido
3J = 7.1 Hz, C9-H), 7.48 (2H, d, J = 8.7 Hz, Ph 2-H), 7.68–7.71 (2H,
m, C7-H & C8-H), 7.78 (2H, d, J = 8.7 Hz, Ph 3-H), 9.01 (1H, s, C5-
H), 11.27 (1H, s, N3-H); 13C NMR: d 117.22 (Cq), 117.30 (CH),
118.97 (Cq), 125.42 (CH), 130.86 (CH), 130.90 (CH), 134.25 (Cq),
134.72 (Cq), 135.78 (CH), 136.85 (Cq), 137.08 (CH), 143.50 (Cq),
156.69 (Cq), 159.08 (Cq), 162.04 (Cq); anal. RP-HPLC: tR 2.50 min
[4,5-b]quinoline-2,4-dione (28)
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl;
71 mg, 0.3 mmol)25 and 4 (R1,2,4 = H, R3 = Me, X = OTs; 105 mg,
0.36 mmol). Yellow solid (24.3 mg, 24%). Mp 346–347 °C (dec); IR
(KBr): 3.441 (NH), 1.694 (C@O), 1.663 (C@O), 1.602 (C@C) cmÀ1
;

6876
M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
1H NMR: d 2.36 (3H, s, Me), 6.57 (1H, s, C9-H), 7.36 (1H, d,
J = 8.1 Hz, C7-H), 7.47 (2H, d, J = 8.5 Hz, Ph 2-H), 7.76 (2H, d,
J = 8.5 Hz, Ph 3-H), 8.12 (1H, d, J = 8.1 Hz, C6-H), 9.08 (1H, s, C5-
H), 11.04 (1H, s, N3-H); 13C NMR: d 22.58 (CH3), 115.06 (Cq),
117.06 (CH), 119.64 (Cq), 126.66 (CH), 130.84 (CH), 130.99 (CH),
131.79 (CH), 134.46 (Cq), 136.95 (Cq), 142.29 (CH), 142.81 (Cq),
147.11 (Cq), 156.80 (Cq), 159.33 (Cq), 162.47 (Cq); anal. RP-HPLC:
tR 2.07 min (98.9%; A), 8.40 min (99.8%; B); HRMS (ESI+): calcd for
Acknowledgments
We acknowledge the Grant of a CRUK studentship to MPD by
the Beatson Institute for Cancer Research, Glasgow. We thank
Christophe Fromont for assistance with analytical work.
Supplementary data
C
18H13ClN3O2 [M+H]+ 338.0696, found 338.0718.
Supplementary data (supplementary Table S1, summarising
inactive compounds 31–96. 1H and 13C NMR spectra for com-
pounds 10, 18, and 69 (with analytical and pharmacological data
at variance with Wilson et al.8). Preparative and analytical data
for pharmacologically inactive compounds 31–96) associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2013.09.038.
5.29. 9-Bromo-10-phenyl-2H,3H,4H,10H-pyrimido[4,5b]-qui-
noline-2,4-dione (29)
Prepared using general method 5.7 from 3a (R5–7 = H; 56 mg,
0.27 mmol) and 4 (R1–3 = H, R4 = Br, X = OTs; 115 mg, 0.32 mmol).
Yellow solid (29.3 mg, 29%). Mp > 350 °C; IR (KBr): 3.413 (NH),
1.717 (C@O), 1.658 (C@O), 1.614 (C@C) cmÀ1 1H NMR: d 7.37–
;
References and notes
7.42 (3H, m), 7.51–7.55 (3H, m), 8.06 (1H, dd, 4J = 1.5 Hz,
3J = 7.8 Hz, C8-H), 8.27 (1H, dd, 4J = 1.5 Hz, 3J = 7.8 Hz, C6-H), 9.06
(1H, s, C5-H), 11.16 (1H, s, N3-H); 13C NMR: d 109.28 (Cq),
116.31 (Cq), 124.76 (Cq), 126.06 (CH), 129.11 (CH), 129.63 (CH),
131.02 (Cq), 131.15 (CH), 132.87 (CH), 138.75 (CH), 138.88 (Cq),
143.19 (CH), 156.73 (Cq), 160.73 (Cq), 162.06 (Cq); anal. RP-HPLC:
tR 1.55 min (98.5%; A), 21.07 min (99.2%; B); HRMS (ESI+): calcd for
1. Vousden, K. H.; Lane, D. P. Nat. Rev. Mol. Cell. Biol. 2007, 8, 275.
2. Fang, S.; Jensen, J. P.; Ludwig, R. L.; Vousden, K. H.; Weissman, A. M. J. Biol.
Chem. 2000, 275, 8945.
3. Kubbutat, M. H.; Jones, S. N.; Vousden, K. H. Nature 1997, 387, 299.
4. Dickens, M. P.; Fitzgerald, R.; Fischer, P. M. Semin. Cancer Biol. 2010, 20, 10.
5. Yang, Y.; Ludwig, R. L.; Jensen, J. P.; Pierre, S. A.; Medaglia, M. V.; Davydov, I. V.;
Safiran, Y. J.; Oberoi, P.; Kenten, J. H.; Phillips, A. C.; Weissman, A. M.; Vousden,
K. H. Cancer Cell 2005, 7, 547.
C
17H11BrN3O2 [M+H]+ 368.0035, found 367.9998.
6. Kitagaki, J.; Agama, K. K.; Pommier, Y.; Yang, Y.; Weissman, A. M. Mol. Cancer
Ther. 2008, 7, 2445.
7. Macchiarulo, A.; Giacche, N.; Mancini, F.; Puxeddu, E.; Moretti, F.; Pellicciari, R.
Expert Opin. Ther. Pat. 2011, 21, 287.
8. Wilson, J. M.; Henderson, G.; Black, F.; Sutherland, A.; Ludwig, R. L.; Vousden, K.
H.; Robins, D. J. Bioorg. Med. Chem. 2007, 15, 77.
5.30. 9-Bromo-10-(4-chlorophenyl)-2H,3H,4H,10H-pyrimido-
[4,5-b]quinoline-2,4-dione (30)
9. Roxburgh, P.; Hock, A. K.; Dickens, M. P.; Mezna, M.; Fischer, P. M.; Vousden, K.
H. Carcinogenesis 2012, 33, 791.
10. Walsh, C. Acc. Chem. Res. 1986, 19, 216.
11. Hemmerich, P.; Massey, V.; Fenner, H. FEBS Lett. 1977, 84, 5.
12. Shrestha, A. R.; Ali, H. I.; Ashida, N.; Nagamatsu, T. Bioorg. Med. Chem. 2008, 16,
9161.
Prepared using general method 5.7 from 3a (R5,6 = H, R7 = Cl,
60 mg, 0.25 mmol)25 and 4 (R1–3 = H, R4 = Br, X = OTs; 107 mg,
0.3 mmol). Yellow solid (36.4 mg, 36%). Mp > 350 °C; IR (KBr):
3.439 (NH), 1.714 (C@O), 1.655 (C@O), 1.610 (C@C) cmÀ1 1H
;
13. Cowden, W. B.; Clark, I. A.; Hunt, N. H. J. Med. Chem. 1988, 31, 799.
14. Burgstaller, P.; Hermann, T.; Huber, C.; Westhof, E.; Famulok, M. Nucleic Acids
Res. 1997, 25, 4018.
15. Tocher, J. H.; Edwards, D. I. Free Radic. Res. Commun. 1992, 16, 19.
16. Declerck, P. J.; De Ranter, C. J.; Volckaert, G. FEBS Lett. 1983, 164, 145.
17. Knight, R. C.; Skolimowski, I. M.; Edwards, D. I. Biochem. Pharmacol. 1978, 27,
2089.
18. Kawamoto, T.; Ikeuchi, Y.; Hiraki, J.; Eikyu, Y.; Shimizu, K.; Tomishima, M.;
Bessho, K.; Yoneda, F.; Mikata, Y., et al Bioorg. Med. Chem. Lett. 1995, 5, 2109.
19. Kawamoto, T.; Ikeuchi, Y.; Hiraki, J.; Eikyu, Y.; Shimizu, K.; Tomishima, M.;
Bessho, K.; Yoneda, F.; Mikata, Y.; Nishida, M.; Ikehara, K. Bioorg. Med. Chem.
Lett. 1995, 5, 2115.
NMR: d 7.40 (1H, t, J = 7.7 Hz, C7-H), 7.44 (2H, d, J = 8.7 Hz, Ph 2-
H), 7.61 (2H, d, J = 8.7 Hz, Ph 3-H), 8.08 (1H, dd, 4J = 1.5 Hz,
3J = 7.7 Hz, C8-H), 8.27 (1H, dd, 4J = 1.5 Hz, 3J = 7.7 Hz, C6-H), 9.06
(1H, s, C5-H), 11.19 (1H, s, N3-H); 13C NMR: d 109.12 (Cq),
116.30 (Cq), 124.76 (Cq), 126.15 (CH), 129.18 (CH), 132.95 (CH),
132.96 (CH), 134.33 (Cq), 137.72 (Cq), 138.72 (Cq), 143.19 (CH),
143.34 (CH), 156.68 (Cq), 160.84 (Cq), 162.04 (Cq); anal. RP-HPLC:
tR 2.73 min (97.6%; A), 4.17 min (100%; B); HRMS (ESI+): calcd for
C
17H10BrClN3O2 [M+H]+ 401.9645, found 401.9613.
20. Nakamura, Y.; Akiyama, T.; Bessho, K.; Yoneda, F. Chem. Pharm. Bull. 1993, 41,
1315.
21. Nakamura, Y.; Akiyama, T.; Yoneda, Y.; Tanaka, K.; Yoneda, F. Chem. Pharm. Bull.
1993, 41, 778.
5.31. Molecular modeling
22. Cresswell, R. M.; Wood, H. C. S. J. Chem. Soc. 1960, 4768.
23. Yoneda, F.; Sakuma, Y.; Ichiba, M.; Shinomura, K. J. Am. Chem. Soc. 1976, 98,
830.
24. Nagamatsu, T.; Hashiguchi, Y.; Higuchi, M.; Yoneda, F. J. Chem. Soc. Chem.
Commun. 1982, 1085.
25. Nagamatsu, T.; Hashiguchi, Y.; Yoneda, F. J. Chem. Soc. Perkin Trans. 1984, 1,
561.
26. Kalir, A. Org. Synth. 1966, 46, 81.
27. Goh, S. H.; Kam, T. S. J. J. Chem. Soc. Perkin Trans. 1981, 1, 423.
28. Zakharkin, L. I.; Khorlina, I. M. Tetrahedron Lett. 1962, 14, 619.
29. Ferguson, L. N.; Calvin, M. J. Am. Chem. Soc. 1950, 72, 4324.
30. Davydov, I. V.; Woods, D.; Safiran, Y. J.; Oberoi, P.; Fearnhead, H. O.; Fang, S.;
Jensen, J. P.; Weissman, A. M.; Kenten, J. H.; Vousden, K. H. J. Biomol. Screen.
2004, 9, 695.
Flexible docking was carried out using OMEGA, version 2.1.6,
OEDocking, version 3.0.0, and VIDA, version 4.1.2, OpenEye Scien-
tific Software, Inc., Santa Fe, NM, USA, www.eyesopen.com, 2012.
Chain A (E3 ubiquitin-protein ligase domain of MDM2, residues
428–491) from PDB entry 2VJF was extracted and a docking recep-
tor was constructed using the OEDocking application Make Recep-
tor. The structure of the entire chain was set as the ligand box
(23.0 Å Â 29.7 Å Â 25.7 Å; 17,513 Å2) and molecular cavity detec-
tion was used with default settings. The balanced binding site
shape potential resulting from this included all protein surface cav-
ities (total contour 2315 Å2), with an inner contour (53 Å2) encom-
passing the binding site of the C-terminus of HDMX (chain B
488FIA490) observed in 2VJF. Multiconformer databases of the bioac-
tive compounds (Table 1) were created (OMEGA; default settings),
and these were docked to the above receptor with default settings
and no constraints. For each compound up to 10 binding poses
were ranked using the Chemgauss 4 scoring function. The majority
(>80%) of docking poses with scores <-3 kcal/mol placed the ligand
at the C-terminal tripeptide HDMX binding site observed in 2VJF.
31. Midgley, C. A.; Fisher, C. J.; Bartek, J.; Vojtesek, B.; Lane, D.; Barnes, D. M. J. Cell
Sci. 1992, 101, 183.
32. Weissman, A. M. Nat. Rev. Mol. Cell. Biol. 2001, 2, 169.
33. Wessiak, A.; Bruice, T. C. J. Am. Chem. Soc. 1983, 105, 4809.
34. Link, P. A. J.; Van der Plas, H. C.; Mueller, F. Photochem. Photobiol. 1987, 45, 557.
35. Cowden, W.B.; Clark, I.A. PCT Int. Pat. Appl. Publ. WO 8804658, 1988, 31 pp.
36. Popowicz, G. M.; Czarna, A.; Rothweiler, U.; Sszwagierczak, A.; Krajewski, M.;
Weber, L.; Holak, T. A. Cell Cycle 2007, 6, 2386.
37. Popowicz, G. M.; Czarna, A.; Holak, T. A. Cell Cycle 2008, 7, 2441.
38. Linke, K.; Mace, P. D.; Smith, C. A.; Vaux, D. L.; Silke, J.; Day, C. L. Cell Death Diff.
2008, 15, 841.

M. P. Dickens et al. / Bioorg. Med. Chem. 21 (2013) 6868–6877
6877
39. Kostic, M.; Matt, T.; Martinez-Yamout, M. A.; Dyson, H. J.; Wright, P. E. J. Mol.
Biol. 2006, 363, 433.
40. Poyurovsky, M. V.; Priest, C.; Kentsis, A.; Borden, K. L.; Pan, Z. Q.; Pavletich, N.;
Prives, C. EMBO J. 2007, 26, 90.
41. Uldrijan, S.; Pannekoek, W. J.; Vousden, K. H. EMBO J. 2007, 26, 102.
42. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
43. Baker, B. R.; Rzeszotarski, W. J. Med. Chem. 1968, 11, 639.
44. Allan, D.; Loudon, J. D. J. Chem. Soc. 1949, 821.
45. Balasubrahmanyam, S. N.; Radhakrishna, A. S.; Boulton, A. J.; Kan-Woon, T. J.
Org. Chem. 1977, 42, 897.
46. Fang, W.-P.; Cheng, Y.-T.; Cheng, Y.-R.; Cherng, Y.-J. Tetrahedron 2005, 61, 3107.
47. Wright, G. E.; Brown, N. C. J. Med. Chem. 1980, 23, 34.
48. Pfleiderer, W.; Nubel, G. Justus Liebigs Ann. Chem. 1960, 631, 168.