Synthesis of 5-deazaflavin derivatives and their activation of p53 in cells

Article abstract of DOI:10.1016/j.bmc.2006.10.011

A family of 5-deazaflavin derivatives has been synthesised using a two-step convergent strategy. The biological activity of these compounds was evaluated in cells, by assessing their ability to stabilize and activate p53. These compounds may act as low molecular weight inhibitors of the E3 activity of HMD2 in tumours that retain wild-type p53. Importantly, we have demonstrated that the nitro group present in all three of the original lead compounds [1-3 (HL198C-E)] is not essential for observation of this biological activity.

Full text of DOI:10.1016/j.bmc.2006.10.011

Bioorganic & Medicinal Chemistry 15 (2007) 77–86
Synthesis of 5-deazaflavin derivatives and their activation
of p53 in cells
Jennifer M. Wilson,a Graham Henderson,a Fiona Black,a Andrew Sutherland,a
Robert L. Ludwig,b Karen H. Vousdenb and David J. Robinsa,*
aWestCHEM, Department of Chemistry, The Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
bThe Beatson Institute for Cancer Research, Switchback Road, Glasgow G61 1BD, UK
Received 12 May 2006; revised 5 October 2006; accepted 10 October 2006
Available online 12 October 2006
Abstract—A family of 5-deazaflavin derivatives has been synthesised using a two-step convergent strategy. The biological activity of
these compounds was evaluated in cells, by assessing their ability to stabilize and activate p53. These compounds may act as low
molecular weight inhibitors of the E3 activity of HMD2 in tumours that retain wild-type p53. Importantly, we have demonstrated
that the nitro group present in all three of the original lead compounds [1–3 (HL198C-E)] is not essential for observation of this
biological activity.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
oncogenes’ activation or perturbations in ribosome
assembly such as ARF and L11, which interact with
HDM2 to inhibit its E3 activity.8–14 In cancer cells that
retain wild-type p53 activity these pathways are often
defective and thus the p53 protein cannot be stabilized
and activated in response to stress.
The p53 protein plays a crucial role in preventing cancer
development by inducing both cell cycle arrest and
apoptosis (programmed cell death) in response to onco-
genic stress.1 While both of these responses may contrib-
ute to the inhibition of tumour development, evidence
suggests that activation of p53 may cause tumour specif-
ic killing, whilst inducing a potentially reversible cell
cycle arrest in normal tissue.2–6
In an effort to identify small molecule inhibitors of the
E3 activity of HDM2 in tumours that retain wild-type
p53, we have previously carried out a high-throughput
assay.15 From a library of 10,000 compounds, forty
inhibited HDM2 autoubiquitylation by more than
50%. Further testing using an in vitro gel-based assay
identified a family of three compounds 1–3 (HL198C-
E) (Fig. 1) with the 5-deazaflavin structural motif which
significantly inhibited HDM2 autoubiquitylation. We
now report the synthesis and biological evaluation of a
small library of 5-deazaflavins to probe the structure–ac-
tivity relationship of this series of compounds for their
ability to stabilize and activate p53. This ability may
be due to the inhibition of HDM2 autodegradation by
the new compounds.
In unstressed cells, to allow normal proliferation, the
activity and stability of the p53 protein is tightly regulat-
ed.7 One of the key regulators of p53 stability is HDM2,
a ubiquitin ligase (E3) that targets the p53 protein for
degradation through the proteasome. In response to
tumourigenic stress, such as DNA damage or oncogene
activation, degradation of p53 by HDM2 is inhibited,
resulting in a rapid increase in the concentration of the
p53 protein in cells. Several pathways which inhibit
the degradation of p53 by HDM2 have been reported,
including the activation of DNA damage-induced
kinases, which phosphorylate p53 and HDM2; and the
induction of proteins involved in the response to
2. Chemistry
Keywords: Deazaflavins; Heterocycle synthesis; Autoubiquitylation
inhibitors; p53; Cell cycle arrest.
Our initial aim was to develop a short efficient synthesis
of the three lead compounds 1–3. The first stage of this
involved the reaction of 2,4,6-trichloropyrimidine 4 with
*
Corresponding author. Tel.: +44 141 330 4378; fax: +44 141 330
4888; e-mail: D.Robins@chem.gla.ac.uk
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.011

78
J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
Figure 1. Structures of original lead compounds 1–3.
sodium hydroxide to give 6-chlorouracil 5 in 71% yield
and this was used as the key intermediate for the prepa-
ration of all the 5-deazaflavins in this study. The next
stage involved a two-step convergent approach where
6-chlorouracil 5 was fused at melt temperature with
the corresponding aryl amine followed by heating the
resulting 6-N-aryl-aminouracils with 2-chloro-5-nitro-
benzaldehyde 6 in DMF (Scheme 1).16,17 This gave 10-
(3-chlorophenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-
2,4-dione (1), 10-(4-chlorophenyl)-7-nitro-10H-pyrimi-
do[4,5-b]quinoline-2,4-dione (2) and 10-(4-methylphe-
nyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione (3)
in 26%, 22% and 79% yields, respectively, over the two
steps.
Having developed a short route to the lead compounds
that are 7-nitro-5-deazaflavins, we sought to probe the
effect of size and electronic character of the substituents
on the aniline portion of the molecule. A series of com-
pounds (Fig. 2) was prepared using the two-step conver-
gent approach described above. Here the various phenyl
substituents were incorporated by reaction of 6-chloro-
uracil 5 with the appropriately substituted aniline. This
allowed the preparation of various fluorinated com-
pounds substituted at the o-, m- and p-positions (8, 10
and 11), as well as the o-chloro compound (7) and an
analogue with no substituent on the phenyl ring (9).
All of the compounds prepared so far contain a 7-nitro
group. It cannot be ruled out that part of the mecha-
nism of action of these compounds may involve oxida-
tive stress processes.18 Accordingly, to probe this and
to elucidate further the structure–activity relationship
of these 5-deazaflavins, compounds 12–29 (Fig. 3)
without the 7-nitro group were prepared using the
two step convergent approach described above. Thus,
a series of compounds was prepared containing either
a chloro-, fluoro- or the strongly electronegative tri-
fluoromethyl group at the 6-, 7-, 8- or 9-positions
and with either no substituent, chloro-, fluoro- or
Scheme 1. Reagents and conditions: (i) NaOH, H2O, D, (71%); (ii)
neat, D; (iii) DMF, D, (1, 26%), (2, 22%), (3, 79%).
Figure 2. Structures of the 7-nitro-5-deazaflavin analogues (yields from 6-chlorouracil 5).

J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
79
Figure 3. Structures of the 5-deazaflavin analogues (yields from 6-chlorouracil 5).
methyl group in the aniline portion of the molecule. It
is of interest to note that treatment of 6-N-aryl-amino-
uracils with 2-chloro-6-fluorobenzaldehyde produced
the 6-chlorodeazaflavins 12–18 rather than the corre-
sponding 6-fluoro derivatives. These reactions follow
an addition–elimination process of nucleophilic aro-
matic substitution and fluoride is displaced rather than
chloride due to the increased electronegativity of fluo-
rine—a decrease in the electron density at the C–F
bond results in faster attack there by the nucleophile
compared to the C–Cl bond.19,20 Finally, to probe fur-
ther the size of the binding pocket the tetraazanaph-
thacene analogue 30 was also prepared.
analysed by Western blotting with anti-p53 DO-1
(Pharmingen), anti-HDM2 AB1/AB2 (Oncogene Sci-
ence), anti-phospho-p53 (serine 15) (Cell Signaling)
and anti-21WAF1/CIP1 (Santa Cruz Biotechnology) anti-
bodies. Blots were also probed with an anti-Cdk4 anti-
body (Santa Cruz Biotechnology) to monitor protein
loading. The effects of some of the compounds on cell
cycle progression and cell death were assessed by flow
cytometry, as previously described.21
4. Results and discussion
Each of the 5-deazaflavin analogues was analysed for
the ability to stabilize endogenous HDM2 and p53 in
primary human pigment epithelial cells (RPE). After
treating the cells with 1–20 lM of each compound for
24 h, HDM2 and p53 levels were assessed by Western
blotting (Fig. 4 and Table 1). Several of the new com-
pounds showed activity in the stabilization of p53 com-
parable to that of the previously described 5-
deazaflavin 1. Three of the analogues, 7, 14 and 15,
3. Biology
Protein expression levels were assessed by Western blot-
ting, after incubation of primary human pigment epithe-
lial (RPE) cells with 1–20 lM of each compound for
24 h. Proteins from whole cell extracts were separated
by SDS 12% polyacrylamide gel electrophoresis and

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J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
Figure 4. Western blots of protein expression levels after treatment of cells with 2.5 lM deazaflavin analogues or 0.1 lg/ml adriamycin.
reproducibly showed stronger p53 stabilization and
activity at lower concentrations than 1 (1 lM compared
to 5 lM). Although stabilization of p53 with adriamy-
cin is accompanied by activation of DNA damage-in-
duced kinases that phosphorylate p53 at serine 15,
the p53 stabilized by the 5-deazaflavin compounds
was not clearly phosphorylated at this residue
(Fig. 4). These results support a role for the com-
pounds in stabilizing p53 by inhibition of HDM2, rath-
er than the activation of a DNA damage response. The
original lead compounds each contained a nitro group
that could be predicted to be reduced to a nitro anion
radical, which could then interact with DNA to induce
DNA damage. The failure to detect p53 phosphoryla-
tion would argue against this mode of action, but more
compellingly we found that some analogues without
the 7-nitro group retained biological activity (Table
1). These included compounds 13–17 with a 6-chloro
group together with compound 26 that has a 7-trifluo-
romethyl group.
Table 1. Stabilization of p53 measured by Western blotting in RPE
cells treated with the analogues at the indicated concentrations for 24 h
Compound
1 lM
5–10 lM
1
7
ꢀ
+
+
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
8
ND
ND
ND
ND
ND
ꢀ
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
+
++
++
+
+
+
ꢀ
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ND
ND
ND
ND
ND
ꢀ
ND
ND
ꢀ
Although the principal function of HDM2 in regulating
p53 activity is through ubiquitination and destabiliza-
tion of the p53 protein,22 previous studies have suggest-
ed that the interaction of HDM2 with p53 can inhibit
the transcriptional activity of p53.23 To confirm that
the p53 stabilized following treatment with the deazafla-
vin analogues is transcriptionally active, we looked for
increased expression of the p53-inducible target gene
product, p21WAF1/CIP1 (Fig. 4 and Table 2). As seen
for the original compounds 1–3, stabilization of p53
ND
ND
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ND
Results of three experiments are summarised to indicate no elevation
of p53 levels (ꢀ), increase in p53 comparable with that seen with 5 lM
compound 1 (+), or increase in p53 levels in excess of that seen with
5 lM compound 1 (++).
ND, not determined.

J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
Table 2. Cell cycle effects of analogues in RKO and RKO E6 cells
81
Compound
Stabilization of p53
Induction
of p21 (RPE cells)
Cell cycle/apoptosis
in RKO cells
Cell cycle/apoptosis
in RKO/E6 cells
1
7
+
+
+
+
+
ꢀ
ꢀ
+
G1/apoptosis
G2/apoptosis
G2/apoptosis
G1/apoptosis
G1/apoptosis
None
None
+
Apoptosis
Apoptosis
None
13
14
15
23
29
ND
+
+
None
None
None
ꢀ
ꢀ
None
p53 and p21WAF1/CIP1 levels were assessed as increasing (+) or not increasing (ꢀ) following treatment.
are less potent. However, in the case of compound 13
the weaker activity is likely to reflect a strong apoptotic
response and the death of cells treated with this com-
pound (see below).
RKO
RKO/E6
0.8%
0.9%
G1 = 61%
The consequence of p53 activation is the induction of
cell cycle arrest and apoptosis, and so we investigated
the effects of some of the novel deazaflavin analogues
on cell cycle progression and cell death (Fig. 5 and Table
2). In RKO cells, a colon cancer cell line that retains
wild-type p53, all of the analogues that showed an abil-
ity to stabilize p53 also caused cell cycle perturbations
and induced apoptosis. Control compounds that did
not stabilize p53 (23 and 29) had no effect on cell cycle
progression and did not induce significant levels of cell
death. Interestingly, two clear types of responses were
seen following treatment of these cells with the active
analogues. Compounds 1, 14 and 15 induced a clear
G1 arrest, and some apoptosis, a pattern that was simi-
lar but less pronounced following treatment with com-
pounds 16, 17 and 26. By contrast, 7 and 13 induced
significant accumulation of cells in G2/M and substan-
tial apoptosis. To determine which of these effects is
dependent on p53, we examined the response of RKO/
E6 cells—RKO cells expressing the HPV16 E6 protein
that inactivates endogenous p53. The compounds that
activated a G1 arrest and slight apoptosis in the RKO
cells (1, 14 and 15) had no effect in RKO/E6 cells, indi-
cating that their activity is a reflection of the stabiliza-
tion and activation of p53. However, the compounds
that induced a G2/M accumulation and strong apopto-
sis in RKO cells (7 and 13) retained the strong apoptotic
activity in the RKO/E6 cells, suggesting that these com-
pounds show significant off target effects and can kill
cells through p53-independent pathways. While there
is a clear differential between these two groups of com-
pounds at these concentrations, at higher concentrations
all the compounds show some off target activities, as
also described for the original compounds 1–3.
DMSO
FL2-H
FL2-H
10.0%
2.4%
G1 = 75%
1
FL2-H
FL2-H
37.5%
33.9%
7
FL2-H
FL2-H
13.7%
3.4%
G1 = 73%
15
FL2-H
FL2-H
0.8%
0.9%
G1 = 62%
29
FL2-H
FL2-H
Thus a family of 5-deazaflavin derivatives has been syn-
thesised using a two-step convergent strategy and their
ability to stabilize and activate p53 has been evaluated
in cells. It is difficult to draw any conclusions about
the structure–activity relationships from the biological
data for these new compounds, but the most important
new finding is that the nitro group present in all three of
the original compounds 1–3 is not essential for their
ability to stabilize and activate p53. Compounds 14
and 15 with the 7-nitro group replaced with a 6-chloro
Figure 5. Effect of deazaflavin analogues on cell cycle progression and
cell death.
by the active analogues identified in this study resulted
in the increased expression of p21WAF1/CIP1, indicating
that the compounds both stabilize and activate p53.
Some of the active compounds showed a clearly weaker
ability to activate p53 than others, suggesting that they

82
J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
group were better at stabilization of p53 and were effec-
tive at lower concentrations than the original lead com-
pounds 1–3. These new low molecular weight
compounds that may inhibit the E3 activity of HMD2
in tumours that retain wild-type p53 should be useful
in further biological studies.
(CH), 128.2 (CH), 128.4 (C), 130.1 (CH), 134.4 (C),
150.7 (C), 152.1 (C), 164.2 (C); Anal. Calcd for
C10H8ClN3O2: C, 50.54; H, 3.39; N, 17.68. Found: C,
50.42; H, 3.26; N, 17.45; m/z (CI): 238.0 (MH+, 91%).
5.1.5. 6-(3-Chlorophenylamino)-1H-pyrimidine-2,4-dione.
Yield 99%; mp 328–329 °C; 1H NMR (DMSO-d6,
400 MHz): 4.76 (s, 1H), 7.18 (dt, 2H, J = 8.0, 2.0 Hz),
7.25 (d, 1H, J = 2.0 Hz), 7.39 (t, 1H, J = 8.0 Hz), 8.53
(s, 1H), 10.41 (s, 1H), 10.50 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 77.6 (CH), 121.1 (CH), 122.1
(CH), 124.4 (CH), 131.4 (CH), 133.9 (C), 140.2 (C),
151.2 (C), 152.1 (C), 164.7 (C); Anal. Calcd for
C10H8ClN3O2: C, 50.54; H, 3.39; N, 17.68. Found: C,
50.39; H, 3.25; N, 17.55.
5. Experimental
5.1. General experimental considerations
All reactions were performed under a nitrogen atmo-
sphere unless otherwise noted. Reagents and starting
materials were obtained from commercial sources and
1
used as received. H NMR and 13C NMR spectra were
recorded on a Bruker DPX 400 spectrometer with chem-
ical shift values in parts per million relative to residual
chloroform (dH 7.28 and dC 77.2) as standard. Infrared
spectra were recorded on a JASCO FTIR 410 spectrom-
eter and mass spectra were obtained using a JEOL JMS-
700 spectrometer. Melting points were determined on a
Reichert platform melting point apparatus.
5.1.6. 6-(4-Chlorophenylamino)-1H-pyrimidine-2,4-dione.
Yield 43%; mp (dec) 344–346 °C; IR (KBr): 3195 (NH),
1
1754 (CO), 1616 (C@C) cmꢀ1; H NMR (DMSO-d6,
400 MHz): 4.73 (s, 1H), 7.23 (AA0BB0 system, 2H,
J = 8.6 Hz), 7.43 (AA0BB0 system, 2H, J = 8.6 Hz),
8.42 (s, 1H), 10.32 (s, 1H), 10.55 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 76.6 (CH), 124.1 (CH), 128.2
(C), 129.3 (CH), 137.1 (C), 150.9 (C), 151.9 (C), 164.4
(C); Anal. Calcd for C10H8ClN3O2: C, 50.54; H, 3.39;
N, 17.68. Found: C, 50.33; H, 3.22; N, 17.44; m/z (CI):
238.04 (MH+, 3%).
5.1.1. 6-Chlorouracil 5. 2,4,6-Trichloropyrimidine
(15.0 g, 8.2 mmol) was added to a stirring solution of
sodium hydroxide (13.2 g, 0.33 mol) and water
(135 cm3). The reaction mixture was heated under reflux
for 1 h. The solution was cooled and the pH was adjust-
ed to ꢁ2 to 3 using concentrated hydrochloric acid
(18 cm3). The mixture was stored at 0 °C overnight.
The white precipitate was filtered and recrystallised from
water (9.1 g, 71%); mp 287–289 °C (lit.24 295–298 °C);
1H NMR (DMSO-d6, 400 MHz): 5.76 (s, 1H), 11.33 (s,
1H), 12.09 (s, 1H); m/z (FAB): 147.05 (MH+, 86%).
5.1.7. 6-(4-Methylphenylamino)-1H-primidine-2,4-dione.
Yield 91%; mp 321–323 °C; IR (KBr): 3273 (NH),
1
1772 (CO), 1634 (C@C) cmꢀ1; H NMR (DMSO-d6,
400 MHz): 2.29 (s, 3H), 4.61 (s, 1H), 7.10 (AA0BB0
system, 2H, J = 8.4 Hz), 7.20 (AA0BB0 system, 2H,
J = 8.4 Hz), 8.23 (s, 1H), 10.18 (s, 1H), 10.45 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 20.4 (CH3),
75.2 (CH), 123.1 (CH), 129.8 (CH), 134.1 (C), 135.1
(C), 150.8 (C), 152.5 (C), 164.3 (C); Anal. Calcd for
C11H11N3O2: C, 60.82; H, 5.10; N, 19.34. Found: C,
60.68; H, 5.07; N, 19.10; m/z (CI): 217.04 (MH+,
10%).
5.1.2. General procedure A. 6-Chlorouracil (1 equiv) and
aniline (3 equiv) were heated at 170 °C for 20 min. The
mixture was then cooled and diethyl ether (20 cm3)
was added. The mixture was sonicated for 15 min. The
white suspension was filtered and washed with water
(15 cm3), methanol (15 cm3) and diethyl ether (15 cm3).
The white solid was placed in a desiccator and dried
overnight using silica and P2O5.
5.1.8. 6-(2-Fluorophenylamino)pyrimidine-2,4-dione. Yield
84%; mp > 360 °C; IR (KBr): 3247 (NH), 3092 (NH),
1
1736 (CO), 1626 (C@C), 1547 cmꢀ1; H NMR (DMSO-
d6, 400 MHz): 4.45 (s, 1H), 7.21–7.42 (m, 4H), 8.20 (s,
1H), 10.32 (s, 1H), 10.55 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): 76.1 (CH), 116.3 (CH), 117.9 (CH), 120.4
(CH), 125.2 (CH), 127.6 (C), 150.6 (C), 152.2 (C), 158.6
(C), 164.2 (C); m/z (FAB): 222.1 (MH+, 1%).
5.1.3. 6-Phenylamino-1H-pyrimidine-2,4-dione. Yield
84%; mp 332–333 °C; IR (KBr): 3414 (NH), 1818
(CO), 1807 (CO) cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 4.70 (s, 1H), 7.15–7.23 (m, 3H), 7.41 (t,
2H, J = 7.8 Hz), 8.28 (s, 1H), 10.20 (s, 1H), 10.50 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 75.8 (CH),
122.7 (CH), 124.7 (CH), 129.4 (CH), 137.9 (C), 150.8
(C), 152.2 (C), 164.4 (C); HRMS (FAB+): m/z found
204.0771, calcd C10H10N3O2 (MH+) 204.0773.
5.1.9.
6-(3-Fluorophenylamino)pyrimidine-2,4-dione.
Yield 75%; mp 321–322 °C; IR (KBr): 3322 (NH),
1
3231 (NH), 1752 (CO), 1604 (C@C) cmꢀ1; H NMR
(DMSO-d6, 400 MHz): 4.66 (s, 1H), 7.00 (dt, 2H,
J = 8.0, 2.1 Hz), 7.38–7.42 (m, 2H), 8.75 (s, 1H), 10.60
(br s, 1H); 13C NMR (DMSO-d6, 100 MHz):
76.8 (CH), 107.6 (CH), 109.1 (CH), 111.3 (CH), 131.0
(CH), 131.2 (CH), 139.8 (CH), 150.7 (CH), 151.7
(CH), 164.6 (C); m/z (FAB): 221.1 (MH+, 4%).
5.1.4. 6-(2-Chlorophenylamino)-1H-pyrimidine-2,4-dione.
Yield 60%; mp (dec) 321–323 °C; IR (KBr): 3202 (NH),
1731 (CO), 1632 (C@C), 1540 cmꢀ1; 1H NMR (DMSO-
d6, 400 MHz): 4.37 (s, 1H), 7.29 (ddd, 1H, J = 8.0, 8.0,
1.6 Hz), 7.41 (ddd, 1H, J = 8.0, 8.0, 1.3 Hz), 7.46 (dd,
1H, J = 8.0, 1.6 Hz), 7.59 (dd, 1H, J = 1.3, 8.0 Hz),
8.10 (s, 1H) 10.38 (s, 1H), 10.55 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 76.1 (CH), 126.8 (CH), 127.4
5.1.10.
6-(4-Fluorophenylamino)pyrimidine-2,4-dione.
Yield 76%; mp 339–340 °C; IR (KBr): 3391 (NH),
1
1752 (CO), 1680 (CO), 1577 (C@C) cmꢀ1; H NMR

J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
83
(DMSO-d6, 400 MHz): 4.53 (s, 1H), 7.20–7.27 (m, 4H),
8.22 (s, 1H), 10.25 (s, 1H), 10.45 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 75.5 (CH), 115.9 (CH), 116.2
(CH), 125.3 (CH), 125.5 (CH), 134.8 (CH), 150.8 (C),
152.7 (C), 164.3 (C); m/z (FAB): 222.1 (MH+, 100%).
(CH), 143.3 (C), 145.1 (C), 156.6 (C), 159.7 (C), 161.7
(C); m/z (FAB): 369.2 (MH+, 5%).
5.1.16. 10-(4-Methylphenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (3). Yield 87%; mp > 380 °C;
IR (KBr): 3408 (NH), 1716 (CO), 1614 (C@C),
5.1.11. 6-(4-Bromophenylamino)pyrimidine-2,4-dione. Yield
80%; mp 339–340 °C; IR (KBr): 3263 (NH), 1766 (CO),
1485 (NO2), 1340 (NO2) cmꢀ1 1H NMR (DMSO-
;
d6, 400 MHz): 2.47 (s, 3H), 6.91 (d, 1H,
J = 9.5 Hz), 7.42 (m, 4H), 8.44 (dd, 1H, J = 9.5,
2.6 Hz), 9.20 (d, 1H, J = 2.6 Hz), 9.38 (s, 1H),
11.26 (s, 1H); 13C NMR (DMSO-d6, 100 MHz):
21.2 (CH3), 117.9 (C), 119.0 (CH), 120.8 (C), 127.3
(CH), 128.3 (2 CH), 128.6 (CH), 131.2 (2 CH),
135.0 (C), 139.7 (C), 142.4 (CH), 143.2 (C), 145.6
(C), 156.7 (C), 159.9 (C), 161.8 (C); m/z (FAB):
349.3 (MH+, 56%).
1616 (C@C), 1554 cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 4.73 (s, 1H), 7.16 (AA0BB0 system, 2H,
J = 8.6 Hz), 7.55 (AA0BB0 system, 2H, J = 8.6 Hz), 8.40
(s, 1H), 10.28 (s, 1H), 10.51 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz): 77.1 (CH), 116.6 (C), 124.7 (CH), 132.6
(CH), 138.0 (C), 151.2 (C), 152.1 (C), 164.7 (C); Anal.
Calcd for C11H11BrN3O2: C, 42.58; H, 2.86; N, 14.89.
Found: C, 42.44; H, 2.66; N, 14.63.
5.1.12. 6-(4-Nitrophenylamino)pyrimidine-2,4-dione. Yield
48%; mp 328–329 °C; IR (KBr): 3270 (NH), 3103
(NH), 1734 (CO), 1624 (C@C), 1570 (NO2), 1344
5.1.17.
10-(2-Chlorophenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (7). Yield 66%; mp 326–328 °C;
IR (KBr): 3144 (NH), 2826 (CH), 1683 (CO), 1617
(NO2) cmꢀ1; H NMR (DMSO-d6, 400 MHz): 5.12 (s,
(C@C) cmꢀ1 1H NMR (DMSO-d6, 400 MHz): 6.88
;
1
1H), 7.34 (AA0BB0 system, 2H, J = 9.1 Hz), 8.21
(AA0BB0 system, 2H, J = 9.1 Hz), 9.20 (s, 1H), 10.60 (s,
1H), 10.70 (s, 1H); 13C NMR (DMSO-d6, 100 MHz):
81.8 (CH), 119.7 (CH), 125.8 (CH), 142.0 (C), 146.0
(C), 150.3 (C), 151.1 (C), 165.5 (C). HRMS (EI): m/z
found 248.0543, calcd C10H8N4O4 (MH+) 248.0546. m/z
(EI): 248.0 (MH+, 6%).
(d, 1H, J = 9.6 Hz), 7.56–7.70 (m, 3H), 7.81–7.89 (m,
1H), 8.48 (dd, 1H, J = 9.6, 2.8 Hz), 9.27 (s, 1H), 9.32
(d, 1H, J = 2.8 Hz), 11.43 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz): 117.2 (C), 117.7 (CH), 120.4 (C), 127.5
(CH), 129.0 (CH), 129.5 (CH), 130.4 (CH), 130.9
(CH), 131.6 (C), 131.9 (CH), 134.2 (C), 142.7 (CH),
143.3 (C). 143.8 (C), 156.2 (C), 158.8 (C), 161.1 (C);
HRMS (FAB): m/z found 369.0391, calcd C17H9N4O4Cl
(MH+) 369.0388.
5.1.13. General procedure B. A suspension of substituted
uracil (1 equiv) and DMF (15 cm3) was prepared. The
chlorobenzaldehyde (1.2 equiv) was added and the reac-
tion mixture was heated at 110 °C for 90 min. The yel-
low transparent solution was cooled and water
(25 cm3) was added. The resulting precipitate was fil-
tered and washed with water (15 cm3). The yellow solid
was placed in a desiccator and dried overnight using sil-
ica and P2O5.
5.1.18.
10-(2-Fluorophenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (8). Yield 60%; mp > 360 °C; IR
(KBr): 2558 (NH), 1685 (CO), 1630 (C@C),
1571 cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.94 (d,
1
1H, J = 9.6 Hz), 7.52–7.61 (m, 4H), 8.45 (dd, 1H,
J = 9.6, 2.8 Hz), 9.21 (d, 1H, J = 2.8 Hz), 9.31 (s, 1H),
11.35 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): 117.5
(C), 118.5 (CH), 120.4 (CH), 127.2 (CH), 128.3 (CH),
129.7 (CH), 130.4 (CH), 137.2 (C), 145.1 (C), 156.3
(C), 161.4 (C); Anal. Calcd for C17H9FN4O4: C, 57.96;
H, 2.58; N, 15.90. Found: C, 57.49; H, 2.65, N, 15.43;
m/z (FAB): 353.2 (MH+, 5%).
5.1.14.
10-(3-Chlorophenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (1). Yield 26%; mp 368–369 °C;
IR (KBr): 3400 (NH), 1714 (CO), 1614 (C@C), 1571,
1
1510 (NO2), 1340 (NO2) cmꢀ1; H NMR (DMSO-d6,
400 MHz): 6.95 (d, 1H, J = 9.5 Hz), 7.49 (t, 1H,
J = 4.6 Hz), 7.69 (s, 1H), 7.76 (dd, 2H, J = 4.7,
1.9 Hz), 8.45 (dd, 1H, J = 9.5, 2.7 Hz), 9.24 (d, 1H,
J = 2.7 Hz), 9.32 (s, 1H), 11.46 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 117.8 (CH), 118.9 (CH), 120.8
(C), 127.6 (CH), 127.7 (CH), 128.9 (CH), 130.3 (CH),
132.5 (CH), 134.8 (C), 138.8 (C), 142.7 (CH), 143.3
(C), 145.2 (C), 156.6 (C), 159.6 (C), 161.5 (C), 162.7
(C); m/z (EI): 368.4 (M+, 1%).
5.1.19. 7-Nitro-10-phenyl-10H-pyrimido[4,5-b]quinoline-
2,4-dione (9). Yield 84%; mp > 360 °C; IR (KBr): 3220
(NH), 1722 (CO), 1610 (C@C) cmꢀ1
;
1H NMR
(DMSO-d6, 400 MHz): 6.87 (d, 1 H, J = 9.2 Hz), 7.47–
7.75 (m, 2H), 7.65–7.75 (m, 3H), 8.45 (dd, 1H, J = 9.2,
2.4 Hz), 9.23 (d, 1H, J = 2.4 Hz), 9.31 (s, 1H), 11.32 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 117.5 (C),
118.5 (CH), 120.4 (C), 127.2 (CH), 128.3 (CH), 128.4
(CH), 129.7 (CH), 130.4 (CH), 137.2 (C), 142.1 (C),
142.9 (C), 145.1 (C), 156.3 (C), 159.5 (C), 161.4 (C);
Anal. Calcd for C17H10N4O4: C, 61.08; H, 3.02; N,
16.76. Found: C, 60.69; H, 2.75; N, 16.39; m/z (FAB):
335.2 (MH+, 13%).
5.1.15.
10-(4-Chlorophenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (2). Yield 52%; mp > 380 °C; IR
(KBr): 3072 (NH), 1716 (CO) 1570 (C@C), 1484
(NO2), 1344 (NO2) cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 6.96 (d, 1H, J = 9.5 Hz), 7.53 (d, 2 H,
J = 6.7 Hz), 7.80 (d, 2H, J = 6.7 Hz), 8.43 (dd, 1H,
J = 2.7 Hz), 9.22 (d, 1H, J = 2.7 Hz), 9.31 (s, 1H),
11.31 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): 117.6
(C), 118.9 (CH), 121.2 (C), 127.5 (CH), 128.7 (CH),
130.7 (CH), 130.9 (CH), 134.8 (C), 136.5 (C), 142.6
5.1.20.
10-(3-Fluorophenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (10). Yield 63%; mp 371–372 °C;
IR (KBr): 3467 (NH), 1707 (CO), 1612 (C@C),
1522 cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.93 (d,
1H, J = 9.4 Hz), 7.50 (t, 1H, J = 4.5 Hz), 7.65 (dd, 1H,
1

84
J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
J = 4.5, 2.0 Hz), 7.9 (br s, 1H), 8.40 (dd, 1H, J = 9.4,
2.6 Hz), 9.11 (d, 1H, J = 2.6 Hz), 9.3 (s, 1H), 11.3 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 115.9 (CH),
118.5 (CH), 120.3 (CH), 127.1 (CH), 128.4 (CH), 132.2
(CH), 132.5 (CH), 138.3 (CH), 142.3 (CH), 142.9 (C),
156.2 (C), 159.3 (CH), 161.3 (C), 161.6 (C), 161.6 (C),
162.5 (C), 164.1 (C); Anal. Calcd for C17H9FN4O4: C,
57.96; H, 2.58; N, 15.90. Found: C, 57.53; H, 2.22, N,
15.43; m/z (FAB): 353.2 (MH+, 2%).
5.1.25. 6-Chloro-10-(4-methylphenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (15). Yield 44%; mp > 360 °C; IR
(KBr): 3131 (NH), 3000 (NH), 1707 (CO), 1656
1
(C@C); H NMR (DMSO-d6, 400 MHz): 2.49 (s, 3H),
6.75 (d, 1H, J = 9.2 Hz), 7.30 (AA0BB0 system, 2H,
J = 8.2 Hz), 7.50 (AA0BB0 system, 2H, J = 8.2 Hz),
7.66–7.73 (m, 2H, 7-H), 9.02 (s, 1H), 11.24 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): 20.8 (CH3), 116.9
(CH), 118.4 (C), 124.8 (CH), 125.3 (C), 127.9 (CH),
130.7 (CH), 133.7 (C), 134.9 (C), 135.1 (CH), 136.4
(CH), 139.1 (C) 143.3 (C), 156.3 (C), 158.6 (C), 161.6
(C); HRMS (FAB): m/z found 338.0700, calcd
C18H12N3O2Cl (MH+) 338.0696.
5.1.21.
10-(4-Fluorophenyl)-7-nitro-10H-pyrimido[4,5-
b]quinoline-2,4-dione (11). Yield 74%; mp > 360 °C; IR
(KBr): 3051 (NH), 1709 (CO), 1613 (C@C),
1
1569 cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.94 (d,
1H, J = 9.6 Hz), 7.52–7.61 (m, 4H), 8.45 (dd, 1H,
J = 9.6, 2.8 Hz), 9.21 (d, 1H, J = 2.8 Hz), 9.31 (s, 1H),
11.35 (s, 1H); 13C NMR (DMSO-d6, 100 MHz): 117.8
(CH, d, J = 23 Hz), 118.9 (CH), 120.8 (C), 127.6 (CH),
128.7 (CH), 131.3 (CH, d, J = 9.2 Hz), 133.8 (C), 138.0
(C), 142.6 (CH), 143.3 (C), 145.6 (C), 156.6 (C), 160.0
(C), 162.7 (C, d, J = 246 Hz), 161.74 (C); mz (FAB):
353.2 (MH+, 8%).
5.1.26. 6-Chloro-10-(2-fluorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (16). Yield 58%; mp 323–324 °C;
IR (KBr): 3523 (NH), 1694 (CO), 1629 (CO), 1504,
1
1229, 754 cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.81
(s, 1H), 7.55–7.71 (m, 4H), 7.72–7.81 (m, 3H), 11.38
(s, 1H); 13C NMR (DMSO-d6, 100 MHz): 116.3 (CH),
117.2 (CH), 118.3 (CH), 119.8 (CH), 120.5 (CH), 124.6
(CH), 124.7 (C), 128.3 (CH), 129.6 (C), 130.1 (CH),
132.6 (C), 140.6 (C), 148.9 (C), 159.8 (C), 160.3 (C),
162.2 (C), 164.8 (C); Anal. Calcd for C17H9FClN3O2:
C, 59.75; H, 2.65; N, 12.30; Found: C, 59.39; H, 2.42;
N, 12.45; m/z (FAB): 342.1 (MH+, 3%).
5.1.22.
6-Chloro-10-phenyl-10H-pyrimido[4,5-b]quino-
line-2,4-dione (12). Yield 94%; mp (dec) 338–340 °C;
IR (KBr): 3381 (NH), 3234 (NH), 1702 (CO), 1669
1
(CO) cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.70 (dd,
1H, J = 8.0, 1.2 Hz), 7.43–7.45 (m, 2H, 7-H), 7.63–7.71
(m, 5H), 9.04 (s, 1H), 11.27 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 116.7 (C), 116.8 (CH), 118.4
(C), 124.9 (CH), 128.3 (CH), 129.5 (CH), 130.3 (CH),
133.7 (C), 135.2 (CH), 136.5 (CH), 137.6 (C), 143.1
(C), 156.3 (C), 158.5 (C), 161.6 (C); HRMS (FAB):
m/z found 324.0536, calcd C17H10N3O2Cl (MH+)
324.0540.
5.1.27. 6-Chloro-10-(3-fluorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (17). Yield 95%; mp 326–328 °C;
IR (KBr): 3475 (NH), 1707 (CO), 1671 (CO), 1522
1
(C@C) cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.81 (s,
1H), 7.55–7.71 (m, 4H), 7.72–7.81 (m, 3H), 11.38 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 104.3 (CH),
107.6 (CH), 114.3 (CH), 116.8 (CH), 117.5 (CH), 125.1
(C), 129.1 (CH), 130.2 (CH), 131.3 (CH), 132.3 (C),
136.4 (C), 138.5 (CH), 141.5 (C), 158.6 (C), 161.1 (C),
162.4 (C), 164.7 (C); Anal. Calcd for C17H9FClN3O2:
C, 59.75; H, 2.65; N, 12.30. Found; C, 59.51; H, 2.43;
N, 12.67; m/z (FAB): 342.1 (MH+, 3%).
5.1.23. 6-Chloro-10-(2-chlorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (13). Yield 36%; mp 305–307 °C;
IR (KBr): 3142 (NH), 2801 (NH), 1709 (CO), 1685
(CO), 1607 (C@C) cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 6.72 (d, 1 H, J = 10.8 Hz), 7.62–7.66 (m,
1H), 7.68–7.79 (m, 4H), 7.85–7.96 (m, 1H), 9.89 (s,
1H), 10.33 (s, 1H); 13C NMR (DMSO-d6, 100 MHz):
115.9 (CH), 116.5 (C), 118.4 (C), 125.4 (CH), 129.5
(CH), 130.4 (CH), 130.8 (CH), 131.0 (C), 131.7 (CH),
134.1 (C), 134.5 (C), 135.9 (CH), 137.2 (C), 141.9
(C), 156.2 (C), 157.9 (C), 161.3 (C); HRMS (FAB):
m/z found 358.0151, calcd C17H9N3O2Cl2 (MH+)
358.0150.
5.1.28. 6-Chloro-10-(4-fluorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (18). Yield 41%; mp > 360 °C; IR
(KBr): 3273 (NH), 1717 (CO), 1650 (C@C),
1578 cmꢀ1;1H NMR (DMSO-d6, 400 MHz): 6.93 (d,
1H, J = 9.4 Hz), 7.50 (t, 1H, J = 4.3 Hz), 7.65 (dd, 1H,
J = 4.3, 2.0 Hz), 7.9 (s, 1H), 8.40 (dd, 1H, J = 9.4,
2.6 Hz), 9.11 (1H, d, J = 7.2 Hz), 9.3 (1H, s), 11.3 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 116.3 (CH),
117.8 (CH), 119.5 (CH), 121.6 (CH), 123.6 (C), 127.9
(CH), 131.2 (CH), 133.5 (C), 135.2 (CH), 136.2 (C),
139.5 (C), 143.2 (C), 158.3 (C), 162.2 (C), 164.2 (C);
mz (FAB): 342.1 (MH+, 2%).
5.1.24. 6-Chloro-10-(4-chlorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (14). Yield 64%; mp (dec) 220–
222 °C; IR (KBr): 3254 (NH), 1667 (CO), 1602 (C@C)
1
cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.55 (d, 1 H,
5.1.29.
8-Chloro-10-phenyl-10H-pyrimido[4,5-b]quino-
J = 8.8 Hz), 7.50 (AA0BB0 system, 2H, J = 8.4 Hz),
7.59–7.65 (m, 2H), 7.79 (AA0BB0 system, 2H,
J = 8.4 Hz), 9.07 (s, 1H), 11.29 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz): 116.7 (C), 116.8 (CH), 118.5
(C), 124.9 (CH), 130.3 (CH), 130.4 (CH), 133.8 (C),
134.2 (C), 135.3 (CH), 136.3 (C), 136.7 (CH), 142.9
(C). 156.2 (C), 158.6 (C), 161.5 (C); HRMS (FAB):
m/z found 358.0152, calcd C17H9N3O2Cl2 (MH+)
358.0150.
line-2,4-dione (19). Yield 71%; mp > 360°C; IR (KBr):
3335 (NH), 1700 (CO), 1659 (CO); H NMR (DMSO-
1
d6, 400 MHz): 6.50 (d, 1 H, J = 1.6 Hz), 7.38–7.40 (m,
2H), 7.53 (dd, 1H, J = 8.8, 1.6 Hz), 7.57–7.65 (m, 3H),
8.21 (d, 1H, J = 8.8 Hz), 9.08 (s, 1H), 11.10 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): 115.9 (C), 116.1
(CH), 119.8 (C), 124.7 (CH), 128.3 (CH), 129.6 (C),
130.4 (CH), 133.2 (CH), 137.1 (C), 139.3 (C), 141.7
(CH), 142.5 (C), 156.3 (C), 158.5 (C), 161.7 (C); Anal.

J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
85
Calcd for C17H20ClN3O2: C, 63.07; H, 3.11; N, 12.98.
Found: C, 62.85; H, 3.11; N, 12.75; m/z (FAB): 324.2
(MH+, 100%).
400 MHz): 6.81 (s, 1H), 7.55–7.71 (m, 4H), 7.73–7.81
(m, 3H), 11.38 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): 115.9 (CH), 118.9 (CH), 119.2 (CH), 120.1
(CH), 120.9 (C), 124.2 (CH), 124.5 (CH), 124.9 (C),
125.1 (CH), 129.3 (CH), 133.7 (C), 135.4 (C), 140.2
(C), 143.8 (C), 159.1 (C), 160.3 (C), 163.5 (C), 169.3
(C); Anal. Calcd for C18H9F4N3O2: C, 57.61; H, 2.42;
N, 11.20. Found; C, 57.91; H, 2.80; N, 11.54; m/z
(FAB): 376.1 (MH+, 1%).
5.1.30. 6-Fluoro-10-(2-fluorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (20). Yield 79%; mp 333–335 °C;
IR (KBr): 3434 (NH), 1715 (CO), 1668 (CO), 1565
1
(C@C) cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.81 (s,
1H), 7.55–7.71 (m, 4H), 7.72–7.81 (m, 3H), 11.38 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 102.1 (CH),
109.5 (C), 112.2 (CH), 115.9 (CH), 118.5 (CH), 121.7
(CH), 124.3 (CH), 127.1 (CH), 128.3 (C), 129.1 (CH),
135.6 (C), 143.2 (C), 157.8 (C), 159.7 (C), 160.3 (C),
161.2 (C), 169.4 (C); Anal. Calcd for C17H9F2N3O2: C,
62.77; H, 2.79; N, 12.92. Found; C, 62.51; H, 2.42; N,
13.28; m/z (FAB): 326.3 (MH+, 2%).
5.1.35.
10-(3-Fluorophenyl)-7-(trifluoromethyl)-10H-
pyrimido[4,5-b]quinoline-2,4-dione (25). Yield 39%; mp
326–328 °C; IR (KBr): 3263 (NH), 1707 (CO), 1671
(CO), 1522 (C@C) cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 6.81 (s, 1H), 7.55–7.71 (m, 4H), 7.73–7.81
(m, 3H), 11.38 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): 105.9 (CH), 106.3 (CH), 110.7 (CH), 115.6
(CH), 119.2 (C), 121.3 (CH), 123.2 (C), 124.2 (CH),
126.3 (CH), 127.1 (C), 129.8 (CH), 135.2 (C), 136.9
(C), 143.8 (C), 159.9 (C), 163.2 (C), 164.2 (C), 169.7
(C); Anal. Calcd for C18H9F4N3O2: C, 57.61; H, 2.42;
N, 11.20. Found: C, 57.34; H, 2.79; N, 11.67; m/z
(FAB): 376.1 (MH+, 1%).
5.1.31. 6-Fluoro-10-(3-fluorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (21). Yield 75%; mp 337–339 °C;
IR (KBr): 3439 (NH), 1710 (CO), 1677 (CO), 1531
1
(C@C) cmꢀ1; H NMR (DMSO-d6, 400 MHz): 6.81 (s,
1H), 7.55–7.71 (m, 4H), 7.72–7.81 (m, 3H), 11.38 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 101.3 (CH),
104.3 (CH), 107.2 (CH), 109.6 (C), 112.5 (CH), 113.4
(CH), 125.6 (CH), 128.6 (CH), 130.3 (CH), 137.6 (C),
139.5 (C), 142.7 (C), 157.2 (C), 158.6 (C), 161.3 (C),
164.2 (C), 169.8 (C); Anal. Calcd for C17H9F2N3O2: C,
62.77; H, 2.79; N, 12.92. Found: C, 63.19; H, 3.01, N,
12.58; m/z (FAB): 326.3 (MH+, 1%).
5.1.36.
10-(4-Fluorophenyl)-7-(trifluoromethyl)-10H-
pyrimido[4,5-b]quinoline-2,4-dione (26). Yield 43%; mp
361–363 °C; IR (KBr): 3545 (NH), 1707 (CO), 1661
(CO), 1525 (C@C) cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 6.81 (s, 1H), 7.55–7.71 (m, 4H), 7.73–7.81
(m, 3H), 11.38 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): 116.4 (CH), 118.3 (C), 120.1 (C), 120.6
(CH), 123.8 (CH), 124.4 (CH), 124.7 (C), 133.7 (C),
135.3 (CH), 136.6 (CH), 138.8 (C), 142.8 (C), 156.2
(C), 158.4 (C), 161.5 (C), 164.0 (C); Anal. Calcd for
C18H9F4N3O2: C, 57.61; H, 2.42; N, 11.20. Found: C,
57.37; H, 2.71; N, 10.94; m/z (FAB): 376.1 (MH+, 1%).
5.1.32. 6-Fluoro-10-(4-fluorophenyl)-10H-pyrimido[4,5-
b]quinoline-2,4-dione (22). Yield 72%; mp 334–336 °C;
IR (KBr): 3475 (NH), 1707 (CO), 1671 (CO), 1522
(C@C) cmꢀ1 1H NMR (DMSO-d6, 400 MHz): 6.60
;
(d, 1 H, J = 8.8 Hz), 7.49–7.55 (m, 4H), 7.72 (m, 1H),
8.90 (s, 1H), 9.11 (d, 1H, J = 8.4 Hz), 11.25 (s, 1H);
13C NMR (DMSO-d6, 100 MHz): 103.2 (CH), 109.5
(C), 113.7 (CH), 115.9 (2 CH), 118.9 (2 CH), 125.6
(CH), 129.7 (CH), 139.6 (C), 134.8 (C), 142.9 (C),
151.2 (C), 156.9 (C), 157.9 (C), 163.5 (C), 169.1 (C);
Anal. Calcd for C17H9F2N3O2: C, 62.77; H, 2.79; N,
12.92. Found; C, 62.49; H, 2.91; N, 12.63; m/z (FAB):
326.2 (MH+, 3%).
5.1.37.
10-(2-Fluorophenyl)-9-(trifluoromethyl)-10H-
pyrimido[4,5-b]quinoline-2,4-dione (27). Yield 57%;
mp > 360 °C; IR (KBr): 3297 (NH), 1695 (CO), 1647
(CO), 1532 (C@C) cmꢀ1
;
1H NMR (DMSO-d6,
400 MHz): 5.50 (s, 1H), 7.00–7.40 (m, 4H), 7.65–7.90
(m, 3H), 11.01 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): 115.9 (C), 116.1 (CH), 117.3 (CH), 118.4
(C), 119.3 (CH), 120.4 (CH), 122.2 (CH), 125.2 (CH),
127.6 (CH), 127.9 (C), 129.2 (C), 129.6 (CH), 135.2
(C), 143.9 (C), 159.6 (C), 159.9 (C), 163.7 (C), 169.3
(C); Anal. Calcd for C18H9F4N3O2: C, 57.61; H, 2.42;
N, 11.20. Found; C, 57.34; H, 2.79; N, 11.37; m/z
(FAB): 376.1 (MH+, 3%).
5.1.33.
10-(4-Chlorophenyl)-7-(trifluoromethyl)-10H-
pyrimido[4,5-b]quinoline-2,4-dione (23). Yield 83%; mp
342–343 °C; IR (KBr): 3155 (NH), 1690 (CO), 1618
(C@C) cmꢀ1 1H NMR (DMSO-d6, 400 MHz): 6.97
;
(d, 1H, J = 9.2 Hz), 7.54 (AA0BB0 system, 2H,
J = 8.6 Hz), 7.79 (AA0BB0 system, 2H, J = 8.6 Hz),
8.01 (dd, 1H, J = 9.2, 2.0 Hz), 8.54 (d, 1H, J = 2.0 Hz),
9.22 (s, 1H), 11.27 (s, 1H); 13C NMR (DMSO-d6,
100 MHz): 117.0 (C), 118.4 (CH), 120.6 (C), 123.3 (C),
124.9 (CF3, J = 33 Hz), 128.9 (CH), 130.5 (CH), 131.2
(CH), 132.8 (CH), 134.3 (C), 136.1 (C), 142.1 (CH),
143.6 (C). 156.3 (C), 159.8 (C), 161.5 (C); Anal. Calcd
for C, 55.19; H, 2.31; N, 10.73. Found: C, 54.99; H,
2.25; N, 10.59; m/z (FAB): 392.00 (MH+, 100%).
5.1.38.
10-(4-Fluorophenyl)-9-(trifluoromethyl)-10H-
pyrimido[4,5-b]quinoline-2,4-dione (28). Yield 80%; mp
327–329 °C; IR (KBr): 3296 (NH), 1699 (CO), 1597
1
(C@C) cmꢀ1; H NMR (DMSO-d6, 400 MHz): 5.50 (s,
1H), 7.00–7.40 (m, 4H), 7.65–7.90 (m, 3H), 11.01 (s,
1H); 13C NMR (DMSO-d6, 100 MHz): 115.8 (C),
116.2 (CH), 117.9 (C), 118.9 (CH), 120.5 (CH), 124.6
(CH), 127.9 (C), 128.1 (CH), 129.4 (CH), 134.5 (C),
137.4 (C), 142.2 (C), 149.3 (C), 157.2 (C), 158.1 (C),
161.0 (C); Anal. Calcd for C18H9F4N3O2: C, 57.61; H,
2.42; N, 11.20. Found; C, 57.34; H, 2.79; N, 11.37; m/z
(FAB): 376.1 (MH+, 2%).
5.1.34.
10-(2-Fluorophenyl)-7-(trifluoromethyl)-10H-
pyrimido[4,5-b]quinoline-2,4-dione (24). Yield 49%;
mp > 360 °C; IR (KBr): 3523 (NH), 1694 (CO), 1651
(CO), 1504 (C@C) cmꢀ1
;
1H NMR (DMSO-d6,

86
J. M. Wilson et al. / Bioorg. Med. Chem. 15 (2007) 77–86
4. Fazeli, A.; Steen, R. G.; Dickinson, S. L.; Bautista, D.;
10-(4-Methylphenyl)-6-(trifluoromethyl)-10H-
5.1.39.
Dietrich, W. F.; Bronson, R. T.; Bresalier, R. S.; Lander,
E. S.; Costa, J.; Weinberg, R. A. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 10199.
pyrimido-[4,5-b]quinoline-2,4-dione (29). Yield 40%;
mp > 360 °C; IR (KBr): 3145 (NH), 3006 (CH), 1714
(CO), 1670 (CO) cmꢀ1
;
1H NMR (DMSO-d6,
5. Liu, G.; Parant, J. M.; Lang, G.; Chau, P.; Chavez-Reyes,
A.; El-Naggar, A. K.; Multani, A.; Chang, S.; Lozano, G.
Nat. Genet. 2004, 36, 63.
6. Ryan, K. M.; O’Prey, J.; Vousden, K. H. Cancer Res.
2004, 64, 4415.
7. Michael, D.; Oren, M. Semin. Cancer Biol. 2003, 13, 49.
8. Ljungman, M. Neoplasia 2000, 2, 208.
9. Meek, D. W.; Knippschild, U. Cancer Res. 2003, 1,
1017.
10. Dai, M. S.; Zeng, S. X.; Jin, Y.; Sun, X. X.; David, L.; Lu,
H. Mol. Biol. Cell 2004, 24, 7654.
11. Jin, A.; Itahana, K.; O’Keefe, K.; Zhang, Y. Mol. Cell.
Biol. 2004, 24, 7669.
400 MHz): 2.47 (s, 3H), 6.91 (d, 1H, J = 9.2 Hz), 7.32
(AA0BB0 system, 2H, J = 8.2 Hz), 7.50 (AA0BB0 system,
2H, J = 8.2 Hz), 8.00 (dd, 1H, J = 9.2, 2.0 Hz), 9.22 (br
s, 1H), 10.33 (s, 1H), 11.23 (s, 1H); 13C NMR (DMSO-
d6, 100 MHz): 20.8 (CH), 117.0 (CH), 118.5 (CH), 120.5
(C), 122.4 (C), 124.4 (CF3, q, J = 33 Hz), 128.0 (CH),
128.8 (CH), 130.3 (CH), 130.8 (CH), 134.7 (C), 139.1
(C), 141.9 (CH), 143.9 (C). 156.4 (C), 159.3 (C), 161.6
(C); Anal. Calcd for C, 61.46; H, 3.26; N, 11.32. Found:
C, 61.34; H, 3.09; N, 11.24; mz (FAB): 372.10 (MH+,
93%).
12. Lohrum, M. A.; Ludwig, R. L.; Kubbutat, M. H. G.;
Hanlon, M.; Vousden, K. H. Cancer Cell 2003, 3, 577.
13. Sherr, C. J.; Weber, J. D. Curr. Opin. Genet. Dev. 2000, 10,
94.
5.1.40. 12-Phenyl-12H-1,3,11,12-tetraazanaphthacene-
2,4-dione 30. Yield 74%; mp (dec) 353–357 °C; IR
1
(KBr): 3230 (NH), 1713 (CO), 1628 (C@C) cmꢀ1; H
NMR (DMSO-d6, 400 MHz): 7.40–7.42 (m, 2H), 7.56–
7.58 (m, 1H), 7.62–7.68 (m, 4H), 7.85–7.87 (m, 1H),
8.32 (d, 1H, J = 7.6 Hz), 9.21 (s, 1H), 9.34 (s, 1H),
11.3 (s, 1H); Anal. Calcd for C20H12N4O2: C, 70.58;
H, 3.55; N, 16.46. Found: C, 70.47; H, 3.46; N, 16.34;
m/z (FAB): 341.2 (MH+, 1%).
14. Zhang, Y.; Wolf, G. W.; Bhat, K.; Jin, A.; Allio, T.;
Burkhart, W. A.; Xiong, Y. Mol. Cell. Biol. 2003, 23,
8902.
15. 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.
16. Nagamatsu, T.; Hashiguchi, Y.; Yoneda, F. J. Chem. Soc.,
Perkin Trans. 1 1984, 561.
Acknowledgments
17. Yoneda, F.; Shinozuka, K.; Tsukuda, K. J. Heterocycl.
Chem. 1979, 16, 1365.
We thank the University of Glasgow for a Scholarship
(J.M.W.) and CRUK for supporting R.L.L., K.H.V.
and J.M.W.
18. Martindale, J. L.; Holbrook, N. J. J. Cell. Physiol. 2002,
192, 119.
19. Loudon, J. D.; Shulman, N. J. J. Chem. Soc. 1941, 772.
20. Suhr, H. Chem. Ber. 1963, 97, 3268.
21. Rowan, S.; Ludwig, R. L.; Haupt, Y.; Bates, S.; Lu, X.;
Oren, M.; Vousden, K. H. EMBO J. 1996, 15, 827.
22. Marine, J. C.; Francoz, S.; Maetens, M.; Wahl, G.;
Toledo, F.; Lozano, G. Cell Death Diff. 2006, 13, 927.
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References and notes
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Hoffman, R. M.; Lowe, S. W. Cancer Cell 2002, 1, 289.
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Am. Chem. Soc. 1980, 102, 5036.