Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide

Article abstract of DOI:10.1021/acs.jmedchem.9b00274

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.

Full text of DOI:10.1021/acs.jmedchem.9b00274

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Article
Novel deazaflavin analogues potently inhibited tyrosyl DNA
phosphodiesterase 2 (TDP2) and strongly sensitized cancer cells
toward treatment with topoisomerase II (TOP2) poison etoposide
Jayakanth Kankanala, Carlos J. A. Ribeiro, Evgeny Kiselev, Azhar Ravji, Jessica
Williams, Jiashu Xie, Hideki Aihara, Yves Pommier, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00274 • Publication Date (Web): 18 Apr 2019
Downloaded from http://pubs.acs.org on April 18, 2019
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Novel deazaflavin analogues potently inhibited tyrosyl DNA
phosphodiesterase 2 (TDP2) and strongly sensitized cancer cells
toward treatment with topoisomerase II (TOP2) poison etoposide
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Jayakanth Kankanala a,#, Carlos J. A. Ribeiro a,#, Evgeny Kiselev b, Azhar Ravji b, Jessica Williams
a, Jiashu Xie a, Hideki Aihara c, Yves Pommier b and Zhengqiang Wang a, *
a) Center for Drug Design, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, United States.
b) Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, MD 20892, United States.
c) Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States.
Abstract
Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes
(TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2
(TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2 thus represents an
attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack
cellular potency and / or permeability. We report herein two novel subtypes of the deazaflavin
TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype
11), or to the N-3 site of the deazaflavin scaffold (subtype 12) we have generated novel analogues
with considerably improved biochemical potency and / or permeability. Importantly, many
analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b and 12h, exhibited much
stronger cancer cell sensitizing effect than the best reported previous analogue 4a toward the
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treatment with etoposide (ETP), suggesting that these analogues could serve as effective cellular
probes.
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Introduction
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Topoisomerase II (TOP2) manages DNA topology during essential DNA transactions such
as transcription and replication.1-2 Mechanistically, TOP2 cleaves DNA using its tyrosine residue
to generate a transient TOP2 cleavage complexes (TOP2cc) in which TOP2 is covalently linked
to the 5′ terminus of the DNA break via a tyrosyl phosphodiester bond. The TOP2cc is typically
re-ligated at the end of each catalytic cycle, creating a dynamic DNA equilibrium between the
resealed form and the TOP2cc. However, when the normally transient TOP2cc is trapped it
becomes abortive and the DNA is damaged. Clinically important TOP2 poisons work by this exact
mechanism as they bind to and stabilize the TOP2cc to prevent DNA re-ligation, resulting in the
accumulation of abortive TOP2cc.3-4 Multiple lines of recent evidence demonstrated that tyrosyl
DNA phosphodiesterase 2 (TDP2) repairs TOP2-mediated DNA damages, including the abortive
TOP2cc trapped by TOP2 poisons, and causes cellular resistance to TOP2 poisons: 1) in cultured
cells and animal models the lack of TDP2 led to enhanced cellular sensitivity to DNA breaks
induced by TOP2 poisons;5-9 2) up-regulation of TDP2 transcription through a gain-of-function
p53 mutation was linked to TOP2 poison resistance in human lung cancer.10 TOP2 poisons, such
as etoposide (ETP), teniposide and doxorubicin, are widely used for treating a wide range of
cancers, including lung cancer, testicular cancer, breast cancer and as a second line treatment
option for platinum-resistant ovarian cancers.11 Inhibiting TDP2 represents a mechanism-based
sensitizing approach which could allow these poisons to be used at lower and safer doses and
against cancers that are resistant to TOP2 poisons.
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In addition, TDP2 could be involved in the genome repair of certain DNA and RNA
viruses, such as hepatitis B virus (HBV)12 and picornaviruses.13-14 The genome replication of these
viruses is protein-primed via a tyrosine residue, which results in protein tyrosine-nucleic acid
adducts similar to the TOP2cc. The repair to remove the viral proteins at the 5 end of the viral
genomes is believed to be carried out by host DNA repair machineries, possibly TDP2. Therefore,
inhibiting TDP2 could also represent a novel antiviral approach.
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A few compounds have recently been reported as TDP2 inhibitors (Figure 1), including
benzopteridine-2,4-dione (1, Ro 08-2750),15 diaminoquinoline-2,8-dione (2, NSC111041),16
isoquinoline-1,3-diones (3),17 deazaflavins (4),15, 18-19 benzylidenepyrazolone (5, NSC375976),20
phenylacetylcystine (6, n = 0, NSC114532; n =1, NSC3198),20 quinazolinylaminopyrimidinone
(7),21 triazolopyridine (8),22 indenoisoquinolines (9),23-24 and furoquinolinediones (10)25 (Figure
1). Most of these inhibitors were identified through random screening of compound libraries using
biochemical assays.26 A few of them, such as 1 (reactive group), 2 (redox cycler), 5 (Michael
acceptor), and 6 (redox cycler), fit the profiles of pan-assay interference structure (PAINS)27 and
may not be appropriate for further development as TDP2 inhibitors. Compound 9 was reported as
triple inhibitors of TOP1/TDP1/TDP2.24 More importantly, these compounds generally inhibit
TDP2 only moderately with IC50 values in micromolar range. The exception is the deazaflavin
chemotype (4) which inhibited TDP2 in nanomolar range with a genuine structure-activity
relationship (SAR)15 and a defined binding mode,18 and sensitized cancer cells to TOP2 poison
ETP,19 representing by far the most potent and the best characterized TDP2 inhibitor type. The
challenge, however, is that the SAR revealed a strong dependence of TDP2 inhibition on the H-
bond donating ability of the substituent on N-10 phenyl, rendering potent compounds poorly
permeable.15 This is consistent with the observation that the sensitizing effect by the most potent
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analogue 4a (R = meta-tetrazole, chemotype 4) on DT40, chicken lymphoma cells, toward ETP
treatment was not as pronounced as predicted based on its nanomolar potency in biochemical
assays.19 Therefore, as potent as some of the reported 4 analogues are in biochemical assays, their
use as cellular probes to study cellular functions of TDP2 is limited.
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Figure 1. Reported TDP2 inhibitors. Deazaflavin 4 is the most potent and best characterized TDP2
inhibitor type.
Ideally, molecular probes enabling cellular studies should have balanced profiles of potent
inhibitory activity, good permeability and low cytotoxicity within the concentration range of
studies. Interestingly, Raoof et al. also reported that an inactive compound of the same deazaflavin
core exhibited good permeability and reduced efflux,15 suggesting that it is possible to develop the
deazaflavin core into useful probes. We report herein our medicinal chemistry efforts toward this
end. By chemically modifying the N-10 and N-3 sites (Figure 2), we have generated novel
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deazaflavin subtypes with enhanced potency, both biochemically and in cancer cells, and improved
permeability.
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Figure 2. Novel deazaflavin subtypes 11-12 designed to improve lipophilicity by adding a second
phenyl ring (B) directly off ring A (for subtype 11) or at N-3 site (for subtype 12).
Results and Discussion
Chemistry
Deazaflavin scaffold 4 was prepared according to reported procedures (Scheme 1A).15 The
synthesis involved reaction of 6-chlorouracil (13) with substituted amines (14) to provide 6-amino
intermediate 15 which upon reacting with 4-cyano-2-fluorobenzaldehyde (16) yielded the
deazaflavin core (4). The key to the synthesis of subtype 11 was the preparation of amine
intermediates 20-21 (Scheme 1B). Intermediates 20 were synthesized from an N-Boc protected 3
or 4-aminophenol (17) which was treated with substituted benzyl bromides (18) to yield
corresponding O-benzyl derivatives (19). The subsequent deprotection of the N-Boc with TFA
gave the aniline derivatives (20). On the other hand, amine intermediates 21 was synthesized via
an SNAr reaction of phenolic alcohol 22 with 4-nitrophenyl fluoride 23 followed by the reduction
of the nitro group. To advance the synthesis, amines 20 or 21 were reacted with 6-chlorouracil (13)
to give intermediate 25 which upon condensation with 4-cyano-2-fluorobenzaldehyde (16)
furnished subtype 11 (Scheme 1B).
Scheme 1. Synthesis of deazaflavin scaffold 4 and new subtypes 11-12
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aReagents and conditions: a) EtOH, 150 °C, 30 min, MW, 60-85%; b) DMF, 110 °C, MW, 30 min,
40-60%; c) K2CO3, DMF, r. t., 12 h; d) TFA, DCM, r. t., 6h, 60-75% over two steps; e) K2CO3,
DMF, 110ºC, 12h, 59%; f) 10% Pd/C, EtOAc, rt, o. n. 87%; g) EtOH, NaOH 1N, reflux, 3h, 85%
h) Cu(OAc)2, DMF, air, r. t., 24-48 h, 40-52%.
N3-substituted deazaflavin subtype 12 was efficiently synthesized in one step from scaffold
4 via a modified Chan-Lam coupling28-30 with substituted boronic acids 26 under the catalysis of
Cu(OAc)2 (Scheme 1C).
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Figure 3. Chemoselectivity of the Chan-Lam coupling reaction. Under reaction conditions, the
arylation occurred exclusively at the N-3 site to yield subtype 12 with O-arylated products 11 not
observed.
It is important to note that most of our analogues of subtype 12 bear a phenolic alcohol off
phenyl ring A, which could complicate their synthesis from the corresponding 4 via the Chan-Lam
coupling as both the NH at 3 position and the phenolic alcohol can be arylated under the Chan-
Lam condition (Figure 3). However, in all our synthesis the N-arylated compounds of subtype 12
were obtained as the sole products, and the formation of the O-arylated subtype 11 was not
observed (Figure 3). This complete chemoselectivity of N-arylation over O-arylation31 under
Chan-Lam condition was confirmed by synthesizing analogue 12b, 12j-k via an alternative linear
synthesis (Scheme 2). In this synthesis, phenyl ring B was introduced at the very beginning of the
synthesis as part of the starting urea 27. Cyclocondensation of 27 with diethylmalonate produced
the 1-phenylpyrimidine-trione (28) in excellent yield under reflux which upon treatment with
POCl3 in the presence of BnEt3NCl yielded the key intermediate 3-phenylpyrimidine-2,4-dione
(29).32-33 Reaction of 6-chloro intermediate (29) with anilines afforded 6-amination products in
moderate to excellent yields. The desired deazaflavin subtype 12 was constructed in moderate
yields by the subsequent amination and condensation reaction sequence.15 12b synthesized from
this linear route was found identical to that obtained via the Chan-Lam coupling. It is also
noteworthy that analogues 12j-k and 31 were synthesized via the linear route only.
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Scheme 2. Alternative linear synthesis for subtype 12
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aReagents and conditions: a) Diethylmalonate, NaOEt, EtOH, reflux, o. n., 86%; b) POCl3,
BnEt3NCl, 50 °C, 6 h, 70%; c) aniline derivative, EtOH, 150 °C, 30 min, MW, 55-80%; d) DMF,
110 °C, 30 min, MW, 50-69%.
Biology
To assess TDP2 inhibitory activity all final compounds were tested in dose-response
fashion using our recently developed fluorescence-based biochemical assay.21 Most analogues
were also evaluated for both the toxicity and sensitizing effect on chicken lymphoma DT40 cells
toward treatment with ETP. Permeability was measured for all final compounds in Parallel
Artificial Membrane Permeation Assay (PAMPA).34 Additional cytotoxicity studies were carried
out in HepG2 cells for all final compounds, and in HeLa cells for most analogues. These cells are
widely used for the infection of HBV and picornavirus, respectively.
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Table 1. TDP2 inhibitory activity and PAMPA permeability for the control series.
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PAMPA
TDP2 IC50
Compd
R
Pe (10-6
cm/s) b
(M) a
4ac
0.041 ± 0.003
0.003
4bc
4cc
4dc
4e
3 OH
4 OH
H
0.042 ± 0.004
0.14 ± 0.01
0.01
0.008
0.64 ± 0.02
1.0 ± 0.1
0.4
1.1
4 OCF3
4 OCHF2
0.88 ± 0.06
0.96 ± 0.02
0.3
0.5
4f
4g
1.5 ± 0.1
1.1
0.1
4h
31
--
0.32 ± 0.02
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a IC50: concentration of a compound producing 50% inhibition, expressed as mean ± standard
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b Pe: effective permeability coefficients determined using the PAMPA, measured in five replicates.
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c Previously reported analogues. Reported IC50 values are 0.04 M (4a), 0.05 M (4b), 0.09 M
(4c) and 0.50 M (4d).15
The first series of compounds (Table 1) served as a control series centered around analogue
4c. IC50 values from our own biochemical assay largely agreed with reported values15 (Table 1
footnote) and followed similar SAR trends in that 1) potent TDP2 inhibition by deazaflavin
subtype 4 generally required a polar substituent with H-bonding ability on phenyl ring A (4a-c);
removal (4d) or etherification (4e-h) of the OH group led to activity drop by 2-10 fold; 2)
analogues with a meta polar group on phenyl ring A demonstrated better potency than those with
a para polar group (4b vs 4c). Unfortunately, the polar group (OH or tetrazole) critical for target
binding also conferred extremely low PAMPA permeability (4a-c) with the effective permeability
coefficient (Pe) in the range of 0.003–0.01 (a Pe of ≥ 1.5 is considered indicative of high
permeability). PAMPA permeability was substantially improved upon removal (4d) or
etherification (4e-h) of the polar OH group (Table 1). Notably analogue 4f was designed to achieve
balanced potency and permeability based on the concept of lipophilic H-bond donor.35 Because
the H of the difluoromethoxy group (–OCHF2) is rendered acidic by strong electron-withdrawing
O and F atoms, presumably this functionality could provide the key H-bonding for potency while
improving permeability. However, significantly improved TDP2 inhibition was not observed with
4f when compared with other ether analogues, particularly 4e. On the other hand, methylation of
the 3-NH group (31 vs 4c) led to significantly improved permeability (13 fold) with only a
moderate loss of inhibitory activity (2 fold).
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These observations prompted us to explore more drastic structural modifications over the
original deazaflavin inhibitor type 4. Specifically, we decided to derivatize the polar group on
phenyl ring A and / or the NH group at 3 position of the deazaflavin core with a second phenyl
ring (ring B, Figure 2). We expected that such derivatization would improve permeability and
hypothesized that the added phenyl ring (B) could provide additional interactions with TDP2 to
possibly compensate for the loss of the polar interactions. As for the polar group on phenyl A, we
chose the OH group (4b-c) mainly due to its ability to confer high potency and easy synthetic
accessibility.
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Table 2. TDP2 inhibitory activity, PAMPA permeability and cytotoxicity for analogues from
phenolic OH modifications (11a-m).
TDP2 IC50
PAMPA Pe
(10-6 cm/s) b
Compd
R
(M) a
--
0.14 ± 0.01
0.20 ± 0.02
1.6 ± 0.2
0.008
1.2
4c
H
11a
11b
4 Cl
0.4
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4 Br
3 Cl
1.3 ± 0.0
0.99 ± 0.09
0.15 ± 0.01
0.13 ± 0.01
1.1 ± 0.1
>10
0.2
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2.8
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2.7
0.7
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11d
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11i
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3 CO2Me
3 CO2H
H
4 Cl
4 F
3.8 ± 0.3
0.93 ± 0.05
3 CO2Me
11j
3 CO2H
0.19 ± 0.01
0.041 ± 0.004
0.38 ± 0.02
0.30 ± 0.00
0.003
0.01
1.7
11k
4b
--
--
--
11l
2.0
11m
a IC50: concentration of a compound producing 50% inhibition, expressed as mean ± standard
deviation from three independent experiments performed in triplicate.
b Pe: effective permeability coefficients determined using the PAMPA, measured in five replicates.
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We first explored the derivatization of the OH group on phenyl ring A. Toward this end,
we synthesized a panel of benzyl ethers and phenyl ethers (subtype 11, Table 2). The most
prominent SAR with this series was that benzyl ethers exhibited considerably higher inhibitory
potency than the corresponding phenyl ethers (11a, IC50 = 0.196 M vs 11g, IC50 = 1.13 M; 11b,
IC50 = 1.60 M vs 11h, IC50 > 10 M; 11e, IC50 = 0.153 M vs 11j, IC50 = 0.934 M; 11f, IC50 =
10
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0.131 M vs 11k, IC50 = 0.186 M). Particularly intriguing are 11a and 11e which inhibited TDP2
with essentially the same potency as 4c but exhibited drastically improved permeability over 4c
(75 and 150 fold, respectively). Direct comparison between compounds with an ester functionality
and a carboxylic acid functionality (11e vs 11f; 11j vs 11k) further confirmed that a polar H-bond
donor benefits target binding but compromises permeability. These results validate our approach
of derivatizing the polar group, particularly the phenolic OH, for balanced potency and
permeability. In addition, the two analogues (11l-m) synthesized based on the meta phenolic
analogue 4b also showed strong TDP2 inhibition and markedly improved permeability, though the
IC50 values were much higher than 4b (8-10 fold).
Table 3. TDP2 inhibitory activity and PAMPA permeability for analogues from N-3 modifications
(12a-i).
PAMPA Pe
(10-6 cm/s) b
Compd
R
TDP2 IC50 (M) a
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--
0.14 ± 0.01
0.008
0.1
4c
H
0.033 ± 0.001
0.033 ± 0.002
0.070 ± 0.003
0.065 ± 0.003
0.038 ± 0.003
0.035 ± 0.000
0.042 ± 0.004
0.0073 ± 0.0003
0.020 ± 0.002
12a
12b
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4 Cl
4 F
0.08
0.06
0.05
0.03
0.01
0.01
0.04
0.07
12c
12d
12e
12f
4b
4 Br
3 CO2Me
3 CONH2
--
4 Cl
12g
12h
12i
3 Cl
3, 4 di-Cl
0.014 ± 0.000
0.03
a IC50: concentration of a compound producing 50% inhibition, expressed as mean ± standard
deviation from three independent experiments performed in triplicate.
b Pe: effective permeability coefficients determined using the PAMPA, measured in five replicates.
Next, we probed the impacts of introducing phenyl ring B to the N-3 site while retaining
the OH group on phenyl A. For this purpose, Chan-Lam products from both 4c (para phenolic
OH, 12a-f) and 4b (meta phenolic OH, 12g-i) were obtained. Comparing with subtype 11,
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analogues of this series all showed considerably better TDP2 inhibition. As far as SAR is
concerned, for analogues (12a-f) derived from para phenolic alcohol 4c, the R group on phenyl
ring B or its position of substitution did not significantly impact the inhibitory potency, with all
analogues showing 2-4 fold of improved potency over 4c. Similar level of potency improvement
(2-6 fold) was observed with analogues 12g-i over the meta phenolic alcohol 4b, though the site
of Cl substitution (3 vs 4) seems to have a more significant impact on potency. Moderate
improvement on the permeability (3-13 fold) over 4b-c was also observed with all analogues of
subtype 12, with the single exception of 12f. Nevertheless, the N-3 arylation generated a novel
deazaflavin subtype with improved TDP2 inhibition and PAMPA permeability.
10
11
12
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59
60
Table 4. TDP2 inhibitory activity and PAMPA permeability for additional analogues of subtype
12 with N-10 phenyl A modifications.
PAMPA Pe
(10-6 cm/s) b
TDP2 IC50 (M) a
Compd
R
4 OH
4 NHSO2Me
3 OH
0.033 ± 0.002
0.0065 ± 0.0004
0.0073 ± 0.0003
0.08
0.02
0.04
12b
12j
12g
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3 NHSO2Me
0.0061 ± 0.0006
0.27 ± 0.02
0.06
4.4
12k
12l
H
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a IC50: concentration of a compound producing 50% inhibition, expressed as mean ± standard
deviation from three independent experiments performed in triplicate.
b Pe: effective permeability coefficients determined using the PAMPA, measured in five replicates.
Finally, with the N-3 arylated with a para-chlorophenyl group, we examined the impact of
different phenyl ring A substituents for subtype 12 (Table 4). First of all, removing the polar
phenolic OH group of ring A resulted in a compound (12l) with much reduced inhibitory activity
(12l vs 12b /12g, Table 4), though the permeability of 12l was among the best of all compounds
tested. Secondly, replacement of the phenolic alcohol by another polar and H-bond enabling
NHSO2Me group generated analogues (12j-k) showing the highest potency of all compounds
tested so far, with IC50 values in single digit nM range for both para and meta substitution (Table
4). Although permeability remains poor for both, the values still reflect a moderate improvement
over 4b-c (3-6 fold). Overall, our data suggest that the N-3 arylation can be a highly productive
and desirable modification, and that the resulting subtype 12 represents the most active inhibitor
type of TDP2 to date.
High permeability conferred cytotoxicity. To gauge their potential as probes for cell-based studies,
all final compounds were evaluated in two cell lines for cytotoxicity: the HepG2 cells commonly
used for HBV infection36 and the HeLa cells permissible to picornavirus infection13-14 (Table 5).
Overall, a strong correlation between cytotoxicity and PAMPA permeability was observed. For
subtypes 4 and 11 no significant cytotoxicity was observed at concentrations up to 100 M for
compounds with a PAMP Pe ≤ 0.5, with 4f being the only exception; whereas compounds with a
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PAMP Pe > 0.5 all showed cytotoxicity (Table 5). For subtype 12 only analogue 12l exhibited high
permeability, and hence, cytotoxicity. A few other analogues (12b-c, 12d, 12i) also demonstrated
cytotoxicity despite their low permeability, although their extraordinary TDP2 inhibitory potency
indicates that cellular testing of these compounds most likely will not require a concentration range
close to the CC. Therefore, their potential as molecular probes for cellular studies should not be
overlooked.
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Table 5. Cytotoxicity evaluation in two cell lines.
CC50 (M)a
PAMPA
Pe (10-6
cm/s) b
CC50 (M)a
HepG2 HeLa
PAMPA
Pe (10-6
cm/s) b
Compd
Compd
HepG2
HeLa
4ad
4bd
4cd
4dd
4e
>100
>100
>100
>100
40
>100
0.003
0.01
0.008
0.4
14
8.7
12
9.4
2.7
0.7
11i
11j
--
--
>100
25
>100
30
0.003
1.7
11k
11l
--
28
1.1
2.0
11m
12a
12b
12c
9.0
21
85
79
0.3
50
>100
0.1
4f
>100
42
>100
39
0.5
41
46
45
32
0.08
0.06
4g
1.1
4h
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12d
>100
14
>100
12
0.1
1.2
0.4
0.2
0.4
0.6
0.01
2.8
0.8
49
>100
>100
85
67
--
0.05
0.03
0.01
0.04
0.07
31
11a
11b
11c
11d
11e
11f
11g
11h
12e
12f
12g
12h
12i
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>100
17
>100
--
--
65
14
12
82
90
29
29
42
62
0.03
0.02
>100
29
>100
--
>100
>100
33
>100
>100
25
12j
12k
12l
0.06
4.4
5.3
7.4
a CC50: concentration of a compound causing 50% cell death. ETP was used as control: CC50 = 15
M (HepG2) and 17 M (HeLa).
b Pe: effective permeability coefficients determined using the PAMPA, measured in five replicates.
Novel inhibitors strongly sensitized DT40 cells toward ETP treatment. The efficacy of TDP2
inhibitors in potentiating the cytotoxic action of TOP2 poison ETP was evaluated in DT40 chicken
lymphoma cells.37 Cell viability was assayed using the ATPlite luminescence assay (PerkinElmer).
DT40 cells were treated with combinations of ETP and TDP2 inhibitors.19 Treatment with only
the TDP2 inhibitor was used as control. It is expected that TDP2 inhibition alone should not elicit
cytotoxic effect. On the other hand, supplementation with a TDP2 inhibitor should amplify the
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cytotoxic effect associated with TOP2 poison ETP. Therefore, the decrease of cell viability with
increasing concentrations of the TDP2 inhibitor at constant ETP dose would reflects the
cooperative interaction of ETP and the TDP2 inhibitor in inducing toxicity. As shown in Figure 3,
previously reported deazaflavin analogue 4a and our compounds 12a and 12b were non-cytotoxic
up to 25M. Although showing toxicity at 25 M, compound 12h reached plateau in enhancing
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ETP action at concentrations below 10 M. As far as subtype 11 is concerned, compounds 11a
and 11e both showed general cytotoxicity at M concentrations (see supporting information)
which corroborated their cytotoxicity observed in HepG2 and HeLa cells. However, at
concentrations much lower than the cytotoxic concentrations (<25 nM), 11a and 11e both
sensitized DT40 to ETP treatment. This extraordinary sensitizing effect likely reflects their
balanced properties between TDP2 inhibition and PAMPA permeability (Table 2). Nevertheless,
all analogues of subtypes 11-12 shown in Figure 3 demonstrated stronger sensitizing effect when
compared with 4a, the best analogue previously reported. The improved sensitizing effect over 4a
was also observed with many other analogues (see supporting information). These results strongly
validate analogues of our new subtypes 11-12 as effective cellular probes.
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Figure 3. Effect of TDP2 inhibitors 4a, 12a, 12b, 12h, 11a and 11e on potentiating toxic action
of ETP.
The overall profiles of TDP2 inhibitory activity, permeability and cytotoxicity for selected
analogues of subtypes 11-12 are summarized in Table 6. These properties, along with their superior
cancer cell sensitizing effects as shown in Figure 3 and supporting information, strongly validate
our novel analogues as improved cellular probes over the best previous analogue 4a.
Table 6. TDP2 inhibitory activity, permeability and cytotoxicity profiles of selected analogues.
CC50 (M)c
PAMPA Pe (10-6
TDP2 IC50 (M) a
Compd
cm/s) b
HepG2
HeLa
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0.20 ± 0.02
0.15 ± 0.01
1.2
0.6
14
29
12
29
11a
11e
12a
12b
12g
12h
12j
12k
4ad
4bd
4cd
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0.033 ± 0.001
0.033 ± 0.002
0.0073 ± 0.0003
0.020 ± 0.002
0.0065 ± 0.0004
0.0061 ± 0.0006
0.041 ± 0.003
0.042 ± 0.004
0.14 ± 0.01
0.1
50
>100
45
0.08
0.04
0.07
0.02
0.06
0.003
0.01
0.008
41
85
65
82
90
>100
>100
>100
>100
>100
>100
>100
>100
--
--
a IC50: concentration of a compound producing 50% inhibition, expressed as mean ± standard
deviation from three independent experiments.
b Pe: effective permeability coefficients determined using the PAMPA, measured in five replicates.
a CC50: concentration of a compound causing 50% cell death.
d Previously reported15 analogues.
Molecular modeling
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The co-crystal structure18 of humanized mouse TDP2 catalytic domain bound to 4c (PDB
code: 5J42) elucidated the binding mode and mechanism of inhibition of deazaflavin inhibitors.
Key to inhibitor binding is an extensive H-bonding network involving i) the pyrimido-dione ring
of deazaflavin and the guanidine group of R266 and R268 ii) the phenolic hydroxyl group and the
backbone carbonyl of N264 via water molecule (Figure 4A).18 The structure also revealed empty
spaces within the binding pocket to accommodate an additional phenyl ring in pointed directions
(Figure 4B) to generate two novel subtypes: subtype 11 via the O-substitution (left arrow, Figure
4B) and subtype 12 via the N-substitution (right arrow, Figure 4B). Importantly, representative
analogues 11k and 12b are both docked nicely into the crystal structure and are predicted to interact
extensively with the side chains of R266 and R268 using its nitrogen and carbonyl oxygen atoms
within the pyrimido-dione core (Figure 4C-D), a binding feature also observed with compound 4c
(Figure 4A). In addition, the ether linker of 11k allows the molecule to extend the added phenyl
group into the active site and engage with active site residues H226, N264 and H349 of TDP2
using its phenyl substituent, the carboxylate group (Figure 4C). Additional docking with analogues
11a and 11e generated similar poses (SI, Figure S1), except that the additional methylene group
extends phenyl ring B further to be in closer contact with H349 (pi-pi interaction) and D262 (pi-
anion interaction) than phenyl ring B of 11k. The methyl carboxylate group in 11e also forms an
H-bond with R206. On the other hand, docking of 12b reveals a water mediated H-bond interaction
between the phenolic hydroxyl group and the side chains of N264 and R266 as observed for 4c.
Nevertheless, unique to subtype 12 is that the phenyl ring at N-3 position of pyrimido-dione is
oriented parallel to the R268, which could enable cation-π interactions (Figure 4D). Cation-π
interactions38 are considered vital in several physiological processes which could influence
biological structures and molecular recognition.39-40 These interactions are widely observed in
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protein-protein41 or protein-ligand40, 42 and protein-DNA43 complexes. In the biochemical assay,
all analogues with an N-3 aryl group (12a-i) showed much improved potency (2-6 fold) over 4c
or 4b, whereas compound 31 (N-Me analog) lacking the N-3 aryl ring was found to be 2-fold less
active over 4c. These SAR observations strongly reflect the critical binding benefits gained via the
predicted cation-π interaction between the N-3 aryl of subtype 12 and the R268 residue of TDP2.
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Figure 4. Molecular modeling of 11k and 12b. (A) Binding mode of 4c within the crystal structure
of catalytic domain of humanized mouse TDP2 (PDB code: 5J4218). (B) Potential vectors for
designing novel deazaflavin inhibitor types. (C) Predicted binding mode of 11k within the catalytic
domain of humanized mouse TDP2. (D) Predicted binding mode of 12b within the catalytic
domain of humanized mouse TDP2. Key residues are highlighted in green sticks. H-bond
interactions are depicted as black dotted lines. Cation- π and π-π interaction are represented as
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double headed arrow in black. Water molecule and magnesium ion were represented as red and
blue non-bonded sphere. All the residue numberings are based on the human TDP2.
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Conclusion
We have generated two novel deazaflavin subtypes (11 and 12) as inhibitor types of TDP2.
The synthesis of the N-3 aryl subtype featured a chemoselective Chan-Lam coupling. Most
analogues of these chemotypes showed improved biochemical potency and / or PAMPA
permeability. Many analogues of our new subtypes 11-12, particularly 11a, 11e, 12a, 12b and 12h,
demonstrated stronger cancer cell sensitizing effect than the best previous analogue 4a toward ETP
treatment. Molecular modeling revealed critical binding interactions provided by the added aryl
group, particularly the cation- interaction between the N-3 aryl and the guanidinium head of
R268. Further characterization of these improved deazaflavin analogues in expanded human
cancer cell lines, including ETP-resistant cell lines, is currently underway and data will be
published in due course.
Experimental section
Chemistry.
General procedures. All commercial chemicals were used as supplied unless otherwise indicated.
Flash chromatography was performed on a Teledyne Combiflash RF-200 with RediSep columns
(silica) and indicated mobile phase. All moisture sensitive reactions were performed under an inert
atmosphere of ultrapure argon with oven-dried glassware. 1H and 13C NMR spectra were recorded
on a Varian 600 MHz or Bruker 400 spectrometer. Mass data were acquired on an Agilent 6230
TOF LC/MS spectrometer capable of ESI and APCI ion sources. Analysis of sample purity was
performed on a Varian Prepstar SD-1 HPLC system with a Phenomenex Gemini, 5 μm C18 column
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(250 mm × 4.6 mm). HPLC conditions: solvent A = H2O, solvent B = MeCN; flow rate = 1.0
mL/min; compounds were eluted with a gradient of 20% MeCN/H2O to 100% MeCN for 30 min.
Purity was determined by total absorbance at 254 nm. All tested compounds have a purity ≥ 97%.
Deazaflavin analogues (4a-d) were reported earlier by Raoof et al.15
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General procedure for the synthesis of deazaflavin derivatives 4, 11, 12j-k, and 31.15
To a suspension of 15, 25, or 30 (0.1 g, 1.0 eq) in DMF (2 mL) was added 4-cyano-2-
fluorobenzaldehyde (16, 1.2 eq) and heated by microwave irradiation at 110 °C for 30 minutes.
Water (10 mL) was added to the reaction mixture, and the resulting precipitate was filtered, washed
with excess water and dried under vacuum. The crude product was purified by combiflash using
0-2% methanol in DCM and the obtained compound was triturated with methanol to furnish the
desired compound. Compounds 4 (Scheme 1A), 11 (Scheme 1B), 12j-k (Scheme 2), and 31
(Scheme 2) were synthesized starting with intermediate 15, 25, 30, and 30 respectively.
10-(3-Hydroxyphenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
1
carbonitrile (4b).15 Yellow solid, 75% yield; H NMR (600 MHz, DMSO-d6) δ 11.22 (s, 1H),
10.04 (s, 1H), 9.13 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.87 (dd, J = 8.2, 1.5 Hz, 1H), 7.49 (t, J = 8.0
Hz, 1H), 7.08 (s, 1H), 7.04 (dd, J = 8.2, 1.5 Hz, 1H), 6.86 – 6.77 (m, 2H). HRMS-ESI (+) m/z
calculated for C18H11N4O3, 331.0831 [M+H]+; found: 331.0832.
10-(4-Hydroxyphenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
1
carbonitrile (4c).15 Yellow solid, 68% yield; H NMR (600 MHz, DMSO-d6) δ 11.21 (s, 1H),
10.03 (s, 1H), 9.13 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.20 (d, J = 8.6 Hz,
2H), 7.12 (s, 1H), 7.03 (d, J = 8.6 Hz, 2H). HRMS-ESI (+) m/z calculated for C18H11N4O3,
331.0831 [M+H]+; found: 331.0834.
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2,4-Dioxo-10-phenyl-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-carbonitrile (4d).15
5
6
7
8
Yellow solid (70% yield). 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.17 (s, 1H), 8.42 (d,
J = 8.2 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.72 (t, J = 7.6 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.44
(d, J = 7.7 Hz, 2H), 7.00 (s, 1H). HRMS-ESI (+) m/z calculated for C18H11N4O2, 315.0877
[M+H]+; found: 315.0876.
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2,4-Dioxo-10-(4-(trifluoromethoxy)phenyl)-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (4e). Yellow solid, 72% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.27 (s, 1H), 9.16
(s, 1H), 8.41 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.60 (d, J =
13
7.5 Hz, 2H), 7.19 (s, 1H). C NMR (150 MHz, DMSO-d6) δ 161.0, 158.7, 155.8, 148.5, 140.9,
140.8, 135.2, 132.1, 130.4, 126.2, 123.3, 122.5, 120.6, 117.8, 117.4, 115.5. HRMS-ESI (+) m/z
calculated for C19H10F3N4O3, 399.0705 [M+H]+; found: 399.0707.
10-(4-(Difluoromethoxy)phenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (4f). Yellow solid, 62% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.15
(s, 1H), 8.40 (d, J = 8.2 Hz, 1H), 7.89 (dd, J = 8.1, 1.3 Hz, 1H), 7.56 (t, J = 78 Hz, 1H), 7.52 –
7.48 (m, 4H), 7.15 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ 161.4, 159.1, 156.2, 151.7, 141.4,
141.2, 133.4, 132.5, 130.3, 126.5, 123.6, 121.0, 120.2, 118.2, 118.1 (t, J = 256.5 Hz), 117.8, 115.8.
HRMS-ESI (+) m/z calculated for C19H11F2N4O3, 381.0799 [M+H]+; found: 381.0800.
10-(Benzo[d][1,3]dioxol-5-yl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (4g). Yellow solid, 66% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.24 (s, 1H), 9.14
(s, 1H), 8.39 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.19 (d, J = 8.2 Hz, 1H),
7.04 (d, J = 1.5 Hz, 1H), 6.88 (dd, J = 8.1, 1.6 Hz, 1H), 6.21 (d, J = 1.9 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ 161.3, 159.0, 156.1, 148.5, 148.0, 141.5, 140.9, 132.2, 130.0, 126.2, 123.3,
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121.6, 121.1, 117.9, 117.6, 115.6, 109.0, 108.9, 102.0. HRMS-ESI (+) m/z calculated for
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C19H11N4O4, 359.0780 [M+H]+; found: 359.0783.
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10-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-
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b]quinoline-8-carbonitrile (4h). Yellow solid, 73% yield; H NMR (600 MHz, DMSO-d6) δ
11.28 (s, 1H), 9.17 (s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.89 (dd, J = 8.1, 1.2 Hz, 1H), 7.71 (d, J =
8.5 Hz, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.49 (s, 1H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H). 13C NMR (100
MHz, DMSO-d6) δ 161.2, 159.0, 156.0, 143.7, 143.3, 141.3, 141.2, 132.6, 132.1, 126.5, 124.8,
123.3, 121.3, 117.8, 117.6, 115.8, 111.3, 111.0. HRMS-ESI (+) m/z calculated for C19H9F2N4O4,
395.0592 [M+H]+; found: 395.0596.
10-(4-(Benzyloxy)phenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (11a). Yellow solid, 75% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.14
(s, 1H), 8.39 (d, J = 8.2 Hz, 1H), 7.87 (dd, J = 8.1, 1.2 Hz, 1H), 7.53 (d, J = 7.4 Hz, 2H), 7.44 (t,
J = 7.5 Hz, 2H), 7.37-7.33 (m, 3H), 7.31 (d, J = 9.0 Hz, 2H), 7.12 (s, 1H), 5.23 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 161.3, 159.0, 158.8, 156.1, 141.5, 140.8, 136.5, 132.3, 129.3, 129.2,
128.3, 127.84, 127.7, 126.1, 123.4, 120.9, 118.0, 117.6, 116.1, 115.5, 69.6. HRMS-ESI (+) m/z
calculated for C25H17N4O3, 421.1301 [M+H]+; found: 421.1303.
10-(4-((4-Chlorobenzyl)oxy)phenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-
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b]quinoline-8-carbonitrile (11b). Yellow solid, 58% yield; H NMR (600 MHz, DMSO-d6) δ
11.23 (s, 1H), 9.15 (s, 1H), 8.40 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.56 (d, J = 8.1 Hz,
2H), 7.51 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.11 (s, 1H), 5.24
(s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 161.1, 158.8, 158.4, 155.9, 141.3, 140.6, 135.4, 132.2,
132.1, 129.3, 129.2, 128.2, 125.9, 125.1, 123.2, 120.7, 117.8, 117.4, 115.9, 115.3, 68.5. HRMS-
ESI (+) m/z calculated for C25H16ClN4O3, 455.0911 [M+H]+; found: 455.0912.
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10-(4-((4-Bromobenzyl) oxy) phenyl)-2, 4-dioxo-2, 3, 4, 10-tetrahydropyrimido [4, 5-b]
quinoline-8-carbonitrile (11c). Orange solid, 69% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.23
(s, 1H), 9.15 (s, 1H), 8.40 (d, J = 7.5 Hz, 1H), 7.88 (d, J = 7.4 Hz, 1H), 7.64 (d, J = 7.3 Hz, 2H),
7.50 (d, J = 7.2 Hz, 2H), 7.36 (d, J = 7.8 Hz, 2H), 7.31 (d, J = 7.0 Hz, 2H), 7.11 (s, 1H), 5.22 (s,
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2H). C NMR (150 MHz, DMSO-d6) δ 161.8, 159.5, 159.1, 156.5, 142.0, 141.4, 141.2, 136.5,
131.6, 130.4, 130.2, 129.8, 129.7, 123.9, 121.4, 118.5, 118.1, 116.8, 116.4, 115.9, 69.2. HRMS-
ESI (+) m/z calculated for C25H16BrN4O3, 499.0406 [M+H]+; found: 499.0407.
10-(4-((3-Chlorobenzyl)oxy)phenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinolone
-8-carbonitrile (11d). Yellow solid, 52% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.24 (s, 1H),
9.15 (s, 1H), 8.40 (d, J = 8.2 Hz, 1H), 7.88 (dd, J = 8.2, 1.2 Hz, 1H), 7.61 (s, 1H), 7.52 – 7.44 (m,
3H), 7.37 (d, J = 9.0 Hz, 2H), 7.32 (d, J = 9.0 Hz, 2H), 7.12 (s, 1H), 5.26 (s, 2H). 13C NMR (100
MHz, DMSO-d6) δ 162.2, 159.9, 159.4, 156.9, 142.4, 141.7, 140.0, 133.8, 133.1, 131.1, 130.2,
128.6, 128.2, 127.1, 126.9, 124.3, 121.7, 118.9, 118.4, 117.0, 116.3, 69.5. HRMS-ESI (+) m/z
calculated for C25H16ClN4O3, 455.0911 [M+H]+; found: 455.0915.
Methyl-3-((4-(8-cyano-2,4-dioxo-3,4-dihydropyrimido[4,5-b]quinolin-10(2H)-yl)phenoxy)
methyl)benzoate (11e). Yellow solid. 82% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H),
9.14 (s, 1H), 8.40 (d, J = 7.4 Hz, 1H), 8.13 (s, 1H), 7.97 (d, J = 7.4 Hz, 1H), 7.88 (d, J = 7.5 Hz,
1H), 7.82 (d, J = 7.4 Hz, 1H), 7.65 – 7.58 (m, 1H), 7.38-7.34 (m, 4H), 7.11 (s, 1H), 5.33 (s, 2H),
3.88 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.3, 161.7, 159.4, 159.0, 156.5, 142.0, 141.3,
137.8, 132.8, 132.7, 130.1, 129.8, 129.3, 129.0, 128.6, 126.5, 123.8, 121.3, 118.4, 118.0, 116.6,
115.9, 69.3, 52.5. HRMS-ESI (+) m/z calculated for C27H19N4O5, 479.1355 [M+H]+; found:
479.1357.
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3-((4-(8-cyano-2,4-dioxo-3,4-dihydropyrimido[4,5-b]quinolin-10(2H)-yl)phenoxy)
methyl)benzoic acid (11f). Yellow solid. 52% yield; 1H NMR (600 MHz, DMSO-d6) δ 13.05 (s,
1H), 11.23 (s, 1H), 9.15 (s, 1H), 8.40 (d, J = 8.2 Hz, 1H), 8.11 (s, 1H), 7.95 (d, J = 7.7 Hz, 1H),
7.88 (dd, J = 8.2, 1.1 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 8.9
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Hz, 2H), 7.33 (d, J = 9.0 Hz, 2H), 7.12 (s, 1H), 5.31 (s, 2H). C NMR (100 MHz, DMSO-d6) δ
167.5, 161.9, 159.6, 159.3, 156.7, 142.2, 141.4, 137.7, 132.9, 132.6, 131.5, 130.0, 129.3, 129.0,
126.7, 124.0, 121.5, 118.6, 118.2, 116.8, 116.1, 69.6. HRMS-ESI (+) m/z calculated for
C26H17N4O5, 465.1199 [M+H]+; found: 465.1202.
2,4-Dioxo-10-(4-phenoxyphenyl)-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (11g). Yellow solid, 79% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.15
(s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 7.26 (t, J = 7.9
Hz, 5H), 7.22 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ 161.4, 159.2, 157.9, 156.3, 155.4, 141.6,
141.1, 132.4, 131.3, 130.3, 130.1, 126.4, 124.5, 123.6, 121.1, 119.9, 119.1, 118.2, 117.8, 115.8.
HRMS-ESI (+) m/z calculated for C24H15N4O3, 407.1144 [M+H]+; found: 407.1146.
10-(4-(4-chlorophenoxy)phenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (11h). Yellow solid, 79% yield; 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.16
(s, 1H), 8.41 (d, J = 8.2 Hz, 1H), 7.90 (dd, J = 8.2, 1.3 Hz, 1H), 7.57 – 7.50 (m, 2H), 7.50 – 7.40
(m, 2H), 7.32 – 7.28 (m, 4H), 7.24 (s, 1H). 13C NMR (150 MHz, DMSO-d6) δ 161.2, 158.9, 157.2,
156.0, 154.2, 141.3, 140.8, 132.2, 131.4, 129.9, 129.8, 128.0, 126.2, 123.4, 121.3, 120.8, 119.1,
117.9, 117.5, 115.5. HRMS-ESI (+) m/z calculated for C24H14ClN4O3, 441.0754 [M+H]+; found:
441.0756.
10-(4-(4-Fluorophenoxy)phenyl)-2,4-dioxo-2,3,4,10-tetrahydropyrimido[4,5-b]quinoline-8-
carbonitrile (11i). Yellow solid, 81% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 9.15
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