4-benzylideneisoquinoline-1,3(2H,4H)-diones as tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors

Article abstract of DOI:10.1007/s00044-020-02662-w

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (Top2) mediated DNA damages, including double-strand breaks (DSBs) that underpin the anticancer mechanism of clinical Top2 poisons such as etoposide (ETP). Inhibition of TDP2 could sensitize

Full text of DOI:10.1007/s00044-020-02662-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02662-w
ORIGINAL RESEARCH
4-benzylideneisoquinoline-1,3(2H,4H)-diones as tyrosyl DNA
phosphodiesterase 2 (TDP2) inhibitors
1
Sameera Senaweera1 Tianyu He1 Haixi Cui1 Hideki Aihara2 Zhengqiang Wang
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Received: 11 September 2020 / Accepted: 3 November 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (Top2) mediated DNA damages, including double-strand breaks
(DSBs) that underpin the anticancer mechanism of clinical Top2 poisons such as etoposide (ETP). Inhibition of TDP2 could sensitize
cancer cells toward Top2 poisons by increasing Top2 cleavage complex. We have previously identified isoquinoline-1,3-dione as a
selective inhibitor type of TDP2. However, the reported structure-activity relationship (SAR) was limited to simple substitutions on the
isoquinoline-1,3-dione core. Herein, we report the extended SAR consisting of the synthesis and testing of a total of 50 analogs
featuring N-2 and C-4 modifications. Major SAR observations include the loss of potency upon N-2 substitution, the lack of inhibition
with C-4 enamine analogs (subtype 11), or any other C-4 modifications (subtypes 13-15) except for the benzylidene substitution
(subtype 12), where eight analogs showed low micromolar potency. The best analog, 12q, inhibited TDP2 with an IC50 of 4.8 μM.
Molecular modeling was performed to help understand the observed SAR trends. Overall, these SAR observations which could
significantly benefit future work on the design of improved TDP2 inhibitors.
Graphical Abstract
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Keywords Tyrosyl-DNA phosphodiesterase 2 (TDP2) Anti-cancer benzylideneisoquinoline-1 3(2H 4H)-dione
Introduction
The cellular DNA replication machinery creates topologically
strained structures such as supercoils and tangles. Topoisomerase
II (Top2) unwinds, unknots, and untangles the genetic material
Supplementary information The online version of this article (https://
to enable important DNA transactions [1]. Mechanistically, Top2
doi.org/10.1007/s00044-020-02662-w) contains supplementary
cleaves DNA using its tyrosine residue to generate a transient
material, which is available to authorized users.
Top2 cleavage complex (Top2cc) in which Top2 is covalently
linked to the 5′ terminus of the DNA via a phosphotyrosyl bond
[2]. Top2-targeting drugs, particularly Top2 poisons, such as
* Zhengqiang Wang
wangx472@umn.edu
1
etoposide (ETP), teniposide, and doxorubicin, are extensively
used for treating a wide range of cancers, and as a second line
treatment option for platinum-resistant ovarian cancers [3].
Pharmacologically these poisons work by binding to and
Center for Drug Design, College of Pharmacy, University of
Minnesota, Minneapolis, MN 55455, USA
2
Department of Biochemistry, Molecular Biology and Biophysics,
University of Minnesota, Minneapolis, MN 55455, USA

Medicinal Chemistry Research
Fig. 1 Structures of known TDP2 inhibitors
stabilizing the Top2cc to cause DNA double-strand breaks
(DSBs) [4, 5], and their efficacy hinges largely on the abundance
of trapped Top2-DNA crosslinks. However, the DSBs typically
trigger host DNA repair pathways which can ultimately lead to
reduced sensitivity. Importantly, tyrosyl-DNA phosphodiesterase
2 (TDP2) is the only known human enzyme that can selectively
cleave the 5′ phosphotyrosyl covalent bond to initiate the repair
of the abortive Top2cc [6]. Evidence supporting TDP2 as an
important component of the repair machinery of Top2-mediated
DNA damages [7–9] includes the observations that TDP2-
deleted chicken lymphoma DT40 cells were hypersensitive to
TOP2 poison ETP [6], and that the up-regulation of TDP2
transcription was linked to ETP resistance in human lung cancer
[10]. Inhibiting TDP2 could sensitize cancers that are resistant to
Top2 poisons. In addition, long-term use of Top2 poisons is
associated with treatment-induced secondary malignancy [3],
such as leukemia. TDP2 inhibition could allow these poisons to
be used at lower and safer doses to mitigate this safety issue.
Therefore, there is a need for continued medicinal chemistry
efforts identifying potent TDP2 inhibitors.
Since the phosphodiesterase activity of TDP2 was discovered
in 2009 [6], a few TDP2 inhibitor scaffolds have been reported
(Fig. 1) [11, 12]. Although many of them are moderately active,
poorly characterized, and/or lacking drug-like properties, the
deazaflavin (2) chemotype exhibited nanomolar biochemical
potency [13], well characterized mechanism of inhibition [14],
and strong cancer cell sensitizing effects [15, 16]. However, the
observed sensitization was also correlated with the ability of
deazaflavins to increase ETP intracellular concentration [17],
likely by impacting efflux. This adds to the mechanistic com-
plexity of the deazaflavin inhibitor type and calls for further
efforts to develop new chemotypes that sensitize cancers cells by
specifically targeting TDP2. In addition, deazaflavins inhibit
TDP2 by occupying the DNA-binding groove instead of directly
inactivating the catalytic activity [14]. It is desirable to identify
small molecules that inhibit TDP2 with a mechanism of action
different from that of deazaflavins. We have previously identi-
fied isoquinoline-1,3-dione as a TDP2 inhibitor type [18].
Despite the moderate potency, this chemotype is compact in size
(molecular weight < 200 Da) and tractable synthetically, ren-
dering it highly amenable toward expanded SAR via new
structural modifications.
Selective inhibition of TDP2 by isoquinoline-1,3-diones
(Fig. 2, 8) was first disclosed in 2016 [18]. The report was
mainly focused on SAR around a benzene ring off the
isoquinoline-1,3-dione core (subtype 9). The effect of C-4
functionalization toward potency was not investigated.
Herein, we report the expanded SAR of the isoquinoline-
1,3-diones chemotype based on available synthesis. The
effort resulted in the identification of 4-benzylideneisoqui-
noline-1,3(2H,4H)-diones as a new TDP2 inhibitor subtype.
The expanded SAR started with the N-2 NH, where its
importance for the activity was probed using unsubstituted (X =
H), oxidized (X = OH), and differently substituted
(X = Me, OBn) analogs (Fig. 2, subtype 10). The focus of the
current SAR, however, was on synthetic C-4 derivatives. In that
vein, five different series of compounds were synthesized and
tested: (1) olefinic C-4 substituents with a nitrogen linker (sub-
type 11); (2) olefinic C-4 substituents with a carbon linker
(subtype 12); (3) benzylic and phenyl substituents at C-4 (sub-
type 13); (4) cyclization between C-4 and C-3 carbonyl group
(subtype 14); and (5) substituents on both the benzene ring and
C-4 position (subtype 15).
Results and discussion
Chemistry
The synthesis of analogs is depicted in Scheme 1. We
commenced our SAR by synthesizing isoquinoline-1,3-
dione core structure (8) using commercially available and

Medicinal Chemistry Research
Fig. 2 Comparison of SAR investigated previously vs. this work
cheap homophthalic acid (16) and conc. ammonium
hydroxide (Scheme 1) [19]. To probe the importance of free
N–H group, we decided to substitute the N-2 NH with Me,
OBn, and OH (subtype 10). Methylated analog (10a) and
O-benzylated analog (10b) was synthesized by using
methylamine and benzyloxyamine in the place of ammo-
nium hydroxide, correspondingly. BBr3 mediated benzyl
deprotection of 10b yielded N-hydroxy derivative, 10c.
Isoquinoline-1,3-diones with olefinic C-4 substituents pos-
sessing nitrogen linker (subtype 11) were obtained in a two-
step synthetic sequence employing the synthesis of reactive
intermediate 17 followed by a displacement of OEt group
with corresponding amines [20]. The final products of
subtype 11 were exclusive Z-isomers. This is presumably
due to the favorable H-bonding between C-3 carbonyl
group and the NH of the nitrogen linker. The types of
amines incorporated in the isoquinoline-1,3-dione core
include alkyl amines, aryl amines, benzyl amines, and 2-
arylethyl amines. Next, isoquinoline-1,3-diones with ole-
finic C-4 substituents possessing aromatics, heteroaro-
matics, and alkyl groups were examined (subtype 12).
Synthesis of such compounds was achieved by the Knoe-
venagel condensation between the isoquinoline-1,3-dione
core (8) and the corresponding aldehyde using a modified
literature procedure [21]. Under these thermodynamic
reaction conditions, typically the thermodynamically more
stable Z-isomer was obtained as the sole product [22], with
a few exceptions where the isolated product contained both
Z- (major) and E- (minor) isomers as an inseparable mixture
as indicated by both 1H and 13C NMR. The Z-configuration
of the major product was confirmed by 1D-NOESY NMR
(see supporting information).
obtain the subtype 13. However, attempts to reduce pre-
viously synthesized olefinic analogs (12) using standard
reduction conditions only yielded unwanted byproducts.
Therefore, linear synthesis of such analogs was performed
by methylation of homopthalic acid followed by a Knoe-
venagel condensation to install olefinic moiety at C-4
position to yield compound 19. Unlike the compound 12,
the catalytic reduction on intermediate 19 to generate the
diester compound with the desired benzylic alkyl sub-
stituent (20) was feasible. Intermediate 20 was then sapo-
nified and cyclized to obtain the core structure 13a.
Synthesis of 4-aryl isoquinolinedione derivatives (13d)
were furnished by palladium-catalyzed coupling of aryl
bromides with isoquinoline-1,3-diones [23]. When OMe
group is present on an arene, it was demethylated using
BBr3 mediated demethylation to afford the corresponding
hydroxy derivatives 13b and 13 g. Tertiary C-4 was then
fully substituted by an oxidation (13c). All analogs of
subtype 13 were isolated as racemates. Then, we turned our
attention to further modifying our core structure to acquire a
tricyclic structure. We sought to preserve the free N–H and
at least one carbonyl group of the core structure for
anticipated metal binding at the active site. This left us with
the option to cyclize between C-4 and C-3 carbonyl group
to obtain subtype 14. Synthesis of compound 14a was
commenced by making para-methoxybenzyl (PMB) pro-
tected isoquinoline-1,3-diones (21) followed by a Regitz
diazo transfer to produce compound 22 [24]. Then, 1,3‐
oxazole moiety was installed by a nitrile cycloaddition to an
α‐diazocarbonyl moiety to yield oxazolo[5,4‐c]isoquinolin‐
5(4H)‐one scaffold, 14a [25]. PMB deprotection of 14a was
carried out to recover the desired 14b with the free N–H
group. Finally, we synthesized a couple of analogs with
substituents on both the benzene ring and the C-4 position
Then, we turned our attention to isoquinolines sub-
stituted with benzylic and phenyl substituents at C-4 to

Medicinal Chemistry Research
Scheme 1 Synthesis of different analogs investigated in this study
of isoquinoline-1,3-diones (subtype 15). Synthesis up to the
compound 27 was carried out following a literature proce-
dure followed by a Knoevenagel condensation using
selected aldehydes to obtain the subtype 15.
reference which had a previously reported IC50 value of
25 μM and had a comparable IC50 of 22 μM in our bio-
chemical assay. Importantly, when the NH is substituted
with either Me (10a) or OBn (10b), the resulting com-
pounds completely lost activity (Table 1), suggesting that
the NH may be essential for target binding, possibly by
serving as a H-bond donor. However, the N–OH analog
(10c) did not inhibit TDP2 either, indicating that TDP2
inhibition may entail an NH specific binding interaction,
but not a metal chelating functional group, unlike the
inhibition of some other Mg2+ dependent DNA proces-
sing enzymes.
Biology
All final compounds and selected synthetic intermediates
were evaluated in our in-house fluorescence-based bio-
chemical assay [26]. The importance of the free NH
group was assessed using a small series of compounds
depicted in Table 1. Compound 8 was used as the

Medicinal Chemistry Research
Table 1 SAR of N-substituted derivatives of isoquinoline-1,3-dione
subtype 12. To that end, we further extended our SAR to
analogs bearing non-olefinic substituents at the C-4 of iso-
quinoline-1,3-diones (subtype 13). Specifically, the SAR
was probed with a small but a diverse library of compounds
with a tertiary C-4 (13a-13c) and a few compounds with a
fully substituted C-4 (13d-13g) via an oxidation (Table 4).
Unfortunately, none of these analogs displayed significant
TDP2 inhibition. Similarly, oxazolo[5,4‐c]isoquinolin‐5
(4H)‐one scaffold (14a-14b) and its analogs with sub-
stituents on both the benzene ring and C-4 position (15a-
15c) were all inactive.
Molecular modeling
Our SAR observations raised a few questions concerning the
binding mode of these compounds: (1) the loss of potency
when the NH of isoquinoline-1,3-diones was substituted with
OH; (2) inactive nature of subtype 11 compared to subtype 12;
and (3) important ligand-protein interactions rendering 12q
most potent of all. To address these questions, we performed
detailed docking analysis. Despite the availability of numerous
mouse TDP2 (mTDP2) and a handful of human TDP2
(hTDP2) crystal structures with bound ligands, a high resolu-
tion hTDP2 structure with a metal ion in the active site remains
elusive. Therefore, we decided to construct a homology model
of hTDP2 based on the available crystal structure of mouse
TDP2 (mTDP2; PDB code: 4GZ1), which shares a high degree
of sequence identity (65%) with hTDP2 [18]. Also, it has been
shown that the catalytic and inhibitory mechanisms of mTDP2
mimic those of hTDP2 with few key mutations in the relative
proximity of the catalytic site [27]. With the hTDP2 model,
docking analysis was performed using Glide XP to understand
the observed SAR.
We commenced our molecular modeling by reprodu-
cing the binding pose of 1,3-dioxo-1,2,3,4-tetra-
hydroisoquinoline-7-carboxylic acid (compound 40, ref
[18]). In accordance with the published binding mode, the
carbonyl group at position 1 forms an H-bond with the
backbone NH of N264. The NH at position 2 and carbonyl
group at position 3 coordinate to the E152 and Mg2+
respectively (Fig. 3, panel A). Binding mode comparison
of 8 and 10c revealed that the potency of 8 may arise from
the Mg2+ coordination by the C-3 carbonyl group (Fig. 3,
panel B). Similarly, 12k, which is predicted to bind to the
active site Mg2+, resulted in inhibition, whereas 11b pre-
fers a binding pose where the core is flipped and binds
away from the Mg2+, resulting in no inhibition at con-
centrations up to 50 μM (Fig. 3, panel C). Finally, the
docking of the most potent compound, 12q, revealed a
Mg2+ binding by the C-1 carbonyl, as well as H-bonding
interactions of the NH, the C-3 carbonyl, and the carbox-
ylate group on the terminal phenyl with E152, E236, and
Y178, respectively (Fig. 3, panel D).
Next, we tested the potency of the compounds with
olefinic C-4 substituents with a nitrogen linker (Table 2).
Similar compounds have previously been identified as
potential antitumor agents, which potently inhibit cyclin-
dependent kinase 4 [20]. Our analogs covers a wide range
of amine headpieces including the ones with potential
reversible and irreversible covalent warheads (11 h–11 l).
Unfortunately, none of those compounds showed inhibitory
potency in our TDP2 biochemical assay at 50 μM.
We then turned our attention to the C-4 substituted
analogs with no nitrogen linker (subtype 12) and IC50
values are presented in Table 3. Compound 12c where the R
group is pyridine, showed a reasonable potency. Sub-
stituents such as pyrroles (12d and 12e) and thiophenes (12
f and 12 g) showed no activity at 50 μM. However, com-
pounds 12 h (R = indazole) and 12i (R = indole) had IC50
values 19 μM and 13 μM respectively. Observed potency
reveals that H-bond donor/acceptor moieties play a role
when the inhibitor binds with TDP2. To probe that phe-
nomenon, compounds 12h-12k were synthesized and tes-
ted. Among those, compound 12k which possesses 4-
hydroxybenzylidene showed an improved potency (IC50
=
10 μM). The effect of the substitution position at the ben-
zylidene was also significant. Regiochemistry of the pinacol
boronate in compounds 12 l and 12 m conferred drastic
difference in observed potency. Among the compounds
tested, the best potency of 4.8 μM was obtained when
benzylidene is substituted with a carboxy moiety at the 4-
position (12q).
Given that compounds with olefinic C-4 substituents
demonstrated significant TDP2 inhibiotion, it was logical to
explore the effect of the reduction of the double bond of the

Medicinal Chemistry Research
Table 2 TDP2 inhibition by subtype 11
Conclusion
Material and methods
Based on a systematic SAR of the isoquinoline-1,3-diones
core structure, we have identified benzylideneisoquinoline-1,3
(2H,4H)-dione as a new TDP2 inhibitor subtype. The best
analog of the class, compound 12q, showed an IC50 of 4.8 μM
in our biochemical assay. We further explored the effect of N-
substitution of the isoquinoline-1,3-diones core. SAR, along
with molecular modeling, revealed a binding mode where
both the NH and the flanking carbonyls of the isoquinoline-
1,3-dione core could play important roles in H-bonding and
Mg2+ coordination. Although current study identified com-
pounds with moderate potency only, the insight gained herein
may facilitate the design of improved TDP2 inhibitors.
Chemistry
General Procedures. All commercial chemicals were used
as supplied unless otherwise indicated. Flash chromato-
graphy was performed on a Teledyne Combiflash RF-200
with RediSep silica columns (silica) and indicated mobile
phase. Moisture sensitive reactions were performed under
an inert atmosphere of ultrapure argon with oven-dried
glassware. H and 13C NMR spectra were recorded on a
Varian 600 MHz spectrometer. Mass data were acquired on
an Agilent TOF II TOS/MS spectrometer capable of ESI
and APCI ion sources.
1

Medicinal Chemistry Research
Table 3 TDP2 inhibition by subtype 12
Synthesis of compound 10b
A solution of homophthalic acid (78 mg, 1.0 equiv,
0.43 mmol) and O-benzylhydroxylamine (2.0 equiv,
0.86 mmol) in 1,2-dichlorobenzene (1 mL) was heated in a
microwave reactor (Biotage initiator+) at 110 °C for
45 mins. Reaction was concentrated and it was purified by
column chromatography with hexane and ethyl acetate to
obtain 10b as a white solid (yield = 75%). Spectral data of
the isolated product matched with literature data [28].
Synthesis of compound 10c
A solution of 10b (50 mg, 1.0 equiv, 0.19 mmol) in DCM
(1 mL) was added BBr3 (4.0 equiv, 0.75 mmol) and the
reaction was stirred at room temperature for overnight.
Water (2 mL) was added to the reaction and it was extracted
with EtOAc (3x). Combined organic layer was dried over
Na2SO4, filtered, and concentrated. The crude was purified
by column chromatography with DCM and MeOH to obtain
10c as a white solid (yield = 36%). Spectral data of the
isolated product matched with literature data [28].
General procedure for synthesis of subtype 11 (11a - 11l)
Intermediate 17 was prepared by following a literature proce-
dure [29]. In a pressure vial, intermediate 17 (75 mg, 1.0 equiv)
was added followed by the amine (1.1 equiv), Et3N (1.5 equiv)
and DMF (1.1 mL, 0.3 M). The vial was capped, and it was
heated at 115 °C for 1 h in an oil bath. After the reaction time,
the reaction was allowed to cool down to room temperature and
volatiles were concentrated in vacuo to obtain a crude solid.
The solid was washed with methanol (2x), ether (2x) and dried
to yield the products 11a - 11 l.
(Z)-4-(((4-fluorobenzyl)amino)methylene)
isoquinoline-1,3(2H,4H)-dione (11a)
1H NMR (600 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.62 (dt, J
= 13.3, 6.4 Hz, 1H), 8.64 (d, J = 13.3 Hz, 1H), 7.97 (dd, J =
8.0, 1.5 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.55 (ddd, J = 8.6,
7.0, 1.5 Hz, 1H), 7.47 – 7.43 (m, 2H), 7.22 (t, J = 8.9 Hz, 2H),
7.17 (t, J = 7.5 Hz, 1H), 4.68 (d, J = 6.4 Hz, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 166.1, 163.9, 161.6 (d, J = 243.3 Hz),
153.9, 137.7, 134.7 (d, J = 3.1 Hz), 132.9, 129.7, 129.7, 127.6,
123.0, 120.2, 118.6, 115.5, 115.4, 92.2, 51.5. HRMS (ESI−) m/
z calcd for C17H12FN2O2 295.0888, found 295.0900.
Synthesis of compound 10a
In a round bottom flask equipped with a reflux condenser,
homopthalic acid (0.5 g, 1.0 equiv, 2.77 mmol) was dis-
solved in 1,2-dichlorobenzene (2 mL). To the mixture, 40%
solution MeNH2 in H2O (2.0 equiv, 5.55 mmol) was added
and it was refluxed at 110 °C for 1 h. Then the condenser
was removed, and the mixture was heated up to 210 °C until
most of the solvent is dried off. The crude was purified by
column chromatography with hexane and ethyl acetate to
obtain 10a as a white solid (yield = 85%). Spectral data of
the isolated product matched with literature data [25].
(Z)-4-(((4-hydroxybenzyl)amino)methylene)
isoquinoline-1,3(2H,4H)-dione (11b)
1H NMR (600 MHz, DMSO-d6) δ 10.95 (s, 1H), 10.59 (dt,
J = 13.2, 6.3 Hz, 1H), 9.44 (s, 1H), 8.62 (d, J = 13.3 Hz,

Medicinal Chemistry Research
Table 4 Lack of TDP2 inhibition by subtypes 13-15
1H), 7.97 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.55
(t, J = 7.7 Hz, 1H), 7.20 (d, J = 8.1 Hz, 2H), 7.16 (t, J =
7.5 Hz, 1H), 6.76 (d, J = 8.1 Hz, 2H), 4.58 (d, J = 6.2 Hz,
2H). 13C NMR (151 MHz, DMSO-d6) δ 166.1, 163.9,
157.0, 153.7, 137.8, 132.9, 129.0 (2 C), 128.3, 127.6,
122.9, 120.2, 118.6, 115.4 (2 C), 91.9, 52.0. HRMS (ESI−)
m/z calcd for C17H13N2O3 293.0932, found 293.0940.
(Z)-4-(isoindolin-2-ylmethylene)isoquinoline-1,3
(2H,4H)-dione (11e)
1H NMR (600 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.44 (s,
1H), 8.02 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.58
(d, J = 7.7 Hz, 2H), 7.37 (d, J = 7.5 Hz, 1H), 7.33 (t, J =
7.9 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.19 (t, J = 7.4 Hz,
1H), 4.18 (t, J = 7.8 Hz, 2H), 3.24 (t, J = 7.9 Hz, 2H). 13C
NMR (151 MHz, DMSO-d6) δ 164.8, 161.4, 145.2, 144.4,
138.5, 133.5, 133.4, 128.3, 128.0, 125.8, 125.8, 124.4,
121.6, 121.3, 113.5, 98.6, 55.1, 29.0. HRMS (ESI−) m/z
calcd for C18H13N2O2 289.0983, found 289.0985.
(Z)-4-((cyclopropylamino)methylene)isoquinoline-
1,3(2H,4H)-dione (11c)
1H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.54 (d,
J = 13.1 Hz, 1H), 8.46 (d, J = 13.0 Hz, 1H), 7.97 (d, J =
9.2 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.54 (t, J = 7.0 Hz,
1H), 7.17 (t, J = 7.5 Hz, 1H), 3.12 (dd, J = 8.0, 4.2 Hz,
1H), 0.84 (dt, J = 15.3, 4.2 Hz, 4H). 13C NMR
(151 MHz, DMSO-d6) δ 166.1, 163.8, 153.4, 137.3,
132.9, 127.5, 123.0, 120.2, 118.8, 92.3, 30.2, 6.4 (2 C).
HRMS (ESI−) m/z calcd for C13H11N2O2 227.0826,
found 227.0832.
(Z)-4-((thiazol-2-ylamino)methylene)isoquinoline-
1,3(2H,4H)-dione (11f)
1H NMR (600 MHz, DMSO-d6) δ 12.57 (d, J = 11.8 Hz, 1H),
11.47 (s, 1H), 8.85 (d, J = 11.8 Hz, 1H), 8.05 (d, J = 7.8 Hz,
1H), 7.95 (d, J = 8.2 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.51 (d,
J = 3.4 Hz, 1H), 7.42 – 7.31 (m, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 166.9, 164.2, 162.1, 142.0, 139.9, 135.9, 134.1,
128.2, 125.9, 122.3, 120.5, 115.7, 98.8. HRMS (ESI−) m/z
calcd for C13H8N3O2S 270.0343, found 270.0350.
(Z)-4-((morpholinoamino)methylene)isoquinoline-
1,3(2H,4H)-dione (11d)
1H NMR (600 MHz, DMSO-d6) δ 11.07 (s, 1H), 10.90 (d, J
= 10.8 Hz, 1H), 8.49 (d, J = 10.9 Hz, 1H), 7.97 (d, J =
8.1 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.52 (t, J = 8.4 Hz,
1H), 7.18 (t, J = 7.5 Hz, 1H), 3.70 (t, J = 4.5 Hz, 4H), 2.97
(t, J = 4.7 Hz, 4H). 13C NMR (151 MHz, DMSO-d6) δ
165.9, 163.9, 151.6, 137.2, 133.1, 127.5, 123.3, 120.5,
119.1, 91.1, 65.7 (2 C), 56.0 (2 C). HRMS (ESI−) m/z calcd
for C14H14N3O3 272.1041, found 272.1052.
(Z)-4-(((3-(2H-tetrazol-5-yl)phenyl)amino)methylene)
isoquinoline-1,3(2H,4H)-dione (11g)
1H NMR (600 MHz, DMSO-d6) δ 12.50 (d, J = 12.4 Hz,
1H), 11.40 (s, 1H), 8.97 (d, J = 12.4 Hz, 1H), 8.21 (d, J =
8.3 Hz, 1H), 8.12 (t, J = 1.9 Hz, 1H), 8.05 (dd, J = 8.0,
1.5 Hz, 1H), 7.82 (t, J = 7.9 Hz, 2H), 7.65 (td, J = 8.0,
5.2 Hz, 2H), 7.32 (t, J = 7.5 Hz, 1H). 13C NMR (151 MHz,

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DMSO-d6) δ 167.0, 163.8, 162.5, 144.4, 140.1, 136.3,
133.2, 130.7, 127.6, 125.9, 124.5, 122.7, 121.2, 120.1,
119.8, 115.9, 96.4. HRMS (ESI−) m/z calcd for
C17H11N6O2 331.0949, found 331.0957.
= 8.1 Hz, 2H), 7.96 (dd, J = 7.9, 1.5 Hz, 1H), 7.70 (t, J =
7.7 Hz, 3H), 7.56–7.41 (m, 1H), 7.15 (t, J = 7.5 Hz, 1H),
3.78 (q, J = 6.9 Hz, 2H), 3.09 (dd, J = 7.4, 4.9 Hz, 2H). 13C
NMR (151 MHz, DMSO-d6) δ 166.0, 163.9, 153.9, 148.4,
137.7, 132.8, 130.9, 129.6, 129.5, 128.5, 127.5, 122.8,
120.1, 118.5, 91.9, 49.4, 45.5, 36.6, 8.5. HRMS (ESI+) m/z
calcd for C18H16FN2O4S 375.0809, found 375.0814.
(Z)-4-(((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl)amino)methylene)isoquinoline-1,3(2H,4H)-
dione (11h)
(Z)-4-(((4-(methylsulfonyl)phenyl)amino)methylene)
isoquinoline-1,3(2H,4H)-dione (11l)
1H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H), 10.65 (dt, J
= 13.0, 6.4 Hz, 1H), 8.65 (d, J = 13.2 Hz, 1H), 7.97 (dd, J =
8.0, 1.5 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 7.9 Hz,
2H), 7.55 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.39 (d, J = 7.7 Hz,
2H), 7.17 (t, J = 7.5 Hz, 1H), 4.73 (d, J = 6.3 Hz, 2H), 1.28 (s,
12H). 13C NMR (151 MHz, DMSO-d6) δ 166.1, 163.9, 154.0,
141.7, 137.7, 134.8 (2 C), 132.9, 127.6, 126.9 (2 C), 123.0,
120.2, 118.6, 92.3, 83.6 (2 C), 52.2, 24.6 (4 C). HRMS (ESI−)
m/z calcd for C23H24BN2O4 403.1835, found 403.1841.
1H NMR (600 MHz, DMSO-d6) δ 12.44 (d, J = 12.3 Hz,
1H), 11.46 (s, 1H), 8.94 (d, J = 12.2 Hz, 1H), 8.23 (d, J =
8.3 Hz, 1H), 8.05 (dd, J = 7.9, 1.5 Hz, 1H), 7.93 (d, J =
8.7 Hz, 2H), 7.81 (d, J = 8.8 Hz, 2H), 7.66 (ddd, J = 8.5,
7.1, 1.6 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 3.23 (s, 3H). 13
C
NMR (151 MHz, DMSO-d6) δ 167.0, 163.7, 143.6, 143.5,
135.9, 135.5, 133.2, 128.7 (2 C), 127.6, 125.0, 121.4,
120.5, 117.5 (2 C), 97.7, 43.7. HRMS (ESI−) m/z calcd for
C17H13N2O4S 341.0602, found 341.0608.
(Z)-4-(((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzyl)amino)methylene)isoquinoline-1,3(2H,4H)-
dione (11i)
General procedure A for Knoevenagel condensation
(12b - 12v)
1H NMR (600 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.64 (dt, J
= 13.1, 6.5 Hz, 1H), 8.67 (d, J = 13.2 Hz, 1H), 8.03 – 7.94 (m,
1H), 7.84 (d, J = 8.3 Hz, 1H), 7.73 (s, 1H), 7.62 (d, J = 7.3 Hz,
1H), 7.59–7.51 (m, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.17 (t, J =
7.5 Hz, 1H), 4.71 (d, J = 6.3 Hz, 2H), 1.29 (s, 12H). 13C NMR
(151 MHz, DMSO-d6) δ 166.5, 164.4, 154.4, 138.3, 138.2,
134.2, 134.2, 133.4, 131.3, 128.7 (2 C), 128.0, 123.4, 120.7,
119.1, 92.6, 84.2 (2 C), 52.8, 25.1(4 C). HRMS (ESI−) m/z
calcd for C23H24BN2O4 403.1835, found 403.1840.
In a pressure vial, compound 8 (100 mg, 0.62 mmol, 1.0
equiv) was dissolved in a mixture of ethanol:benzene
(3:2 v/v, 1 mL) and piperidine (0.25 equiv) was added.
The mixture was stirred for 10 min at room temperature
before slow addition of the corresponding aldehyde (1.0
equiv). The vial was capped, and it was heated at 85 °C
for 2 h in an oil bath. After the reaction time, the reaction
was allowed to cool down to room temperature and
volatiles were concentrated in vacuo to obtain a crude
solid. The solid was washed with ethanol (2x), ether (2x)
and dried to yield the products 12b - 12 v.
Note: In some cases, the isolated product contained both
E- and Z- isomers in an inseparable mixture as observed by
both 1H and 13C NMR. When possible, NMR data for
isomers are reported separately. Further attempts were not
made to determine the isomeric ratio.
(Z)-4-(((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)amino)methylene)isoquinoline-1,3
(2H,4H)-dione (11j)
1H NMR (600 MHz, DMSO-d6) δ 12.49 (d, J = 12.5 Hz, 1H),
11.34 (s, 1H), 8.94 (d, J = 12.6 Hz, 1H), 8.20 (d, J = 8.3 Hz,
1H), 8.03 (dd, J = 7.9, 1.6 Hz, 1H), 7.75 (dt, J = 7.2, 2.3 Hz,
1H), 7.66 (d, J = 2.5 Hz, 1H), 7.65–7.60 (m, 1H), 7.50–7.43
(m, 2H), 7.29 (t, J = 7.5 Hz, 1H), 1.32 (s, 12H). 13C NMR
(151 MHz, DMSO-d6) δ 166.9, 163.7, 162.3, 144.9, 138.9,
136.5, 133.1, 130.5, 129.3, 127.5, 124.2, 123.3, 121.0, 120.3,
120.2, 95.7, 83.9, 24.6. HRMS (ESI−) m/z calcd for
C22H22BN2O4 289.1678, found 289.1680.
4-benzylideneisoquinoline-1,3(2H,4H)-dione (12b)
1H NMR (600 MHz, DMSO-d6) δ 11.63 (s, 1H), 8.09 (s,
1H), 8.07 (dd, J = 7.9, 1.4 Hz, 1H), 7.93 – 6.65 (m, 8H).
13C NMR of the major isomer (151 MHz, DMSO-d6) δ
165.32, 163.99, 142.86, 135.30, 132.55, 132.43, 130.61,
129.46, 129.02 (2 C), 129.00, 128.41 (2 C), 127.97, 127.56,
126.78. 13C NMR of the minor isomer (151 MHz, DMSO-
d6) δ 164.15, 163.45, 143.95, 135.93, 135.26, 133.82,
129.27, 128.46, 128.30, 127.65, 125.68, 125.47, 124.27,
124.11, 123.65. HRMS (ESI−) m/z calcd for C16H10NO2
248.0717, found 248.0724.
(Z)-4-(2-(((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-
ylidene)methyl)amino)ethyl)benzenesulfonyl
fluoride (11k)
1H NMR (600 MHz, DMSO-d6) δ 10.95 (s, 1H), 10.39 (dt,
J = 13.0, 6.3 Hz, 1H), 8.40 (d, J = 13.2 Hz, 1H), 8.10 (d, J

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Fig. 3 Predicted optimal binding modes of (a) 1,3-dioxo-1,2,3,4-tet-
rahydroisoquinoline-7-carboxylic acid; [18] (b) overlay of compounds
8 and 10c; (c) overlay of compounds 11b and 12k; (d) 12q within
active site of hTDP2 (numbering was based on hTDP2 sequence). The
active site residues are shown as orange sticks and metal ions as red
spheres. Important interactions are depicted as black dotted lines.
Valency and hydrogen atoms are omitted for clarity. Homology model
(hTDP2) was built using the crystal structure of mTDP2 (PDB
code: 4GZ1)
4-(pyridin-4-ylmethylene)isoquinoline-1,3(2H,4H)-
dione (12c)
143.8, 139.3, 133.9, 132.7, 129.5, 128.1, 127.8, 127.1, 125.9,
123.9, 123.3, 122.5. 13C NMR of the minor isomer (151 MHz,
DMSO-d6) δ 164.0, 163.2, 149.0, 144.3, 140.2, 134.7, 131.8,
129.5, 128.1, 127.8, 126.8, 124.4. HRMS (ESI−) m/z calcd for
C15H9N2O2 249.0670, found 249.0681.
1H NMR (600 MHz, DMSO-d6) δ 11.72 (s, 1H, minor), 11.50
(s, 1H, major), 8.66–8.63 (m, 2H, minor), 8.60 – 8.58 (m, 2H,
major), 8.20 (s, 2H, minor), 8.19 (d, J = 2.1 Hz, 2H, major),
8.11–8.08 (m, 1H, major), 7.99 (s, 1H, minor), 7.82 – 7.76 (m,
1H, major), 7.61 (t, J = 7.6 Hz, 1H, major), 7.52 (t, J =
7.6 Hz, 1H, minor), 7.50 – 7.47 (m, 2H, major), 7.46 – 7.41
(m, 1H, minor), 7.27 (d, J = 8.1 Hz, 1H, minor). 13C NMR of
the major isomer (151 MHz, DMSO-d6) δ 164.9, 163.9, 150.4,
4-((1H-pyrrol-2-yl)methylene)isoquinoline-1,3
(2H,4H)-dione (12d)
1H NMR (600 MHz, DMSO-d6) δ 12.78 (s, 1H), 11.59 (s, 1H),
8.18 (d, J = 6.9 Hz, 2H), 8.08 (dd, J = 7.9, 1.5 Hz, 1H), 7.77 –

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7.68 (m, 1H), 7.47 – 7.41 (m, 2H), 7.18 (dt, J = 3.7, 1.8 Hz,
1H), 6.44 (dt, J = 4.2, 2.2 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 165.7, 164.1, 137.2, 133.6, 132.7, 128.9, 128.0,
127.7, 126.4, 125.7, 122.6, 122.2, 112.2, 111.3. HRMS (ESI−)
m/z calcd for C14H9N2O2 237.0670, found 237.0681.
136.6, 134.8, 133.8, 129.9, 128.0, 127.6, 127.2, 125.3, 123.8,
123.5, 122.6, 122.3, 109.0. HRMS (ESI−) m/z calcd for
C17H12NO3 278.0823, found 278.0833.
4-((1H-indol-5-yl)methylene)isoquinoline-1,3
(2H,4H)-dione (12i)
4-((1-methyl-1H-pyrrol-2-yl)methylene)isoquinoline-
1,3(2H,4H)-dione (12e)
1H NMR (600 MHz, DMSO-d6) δ 11.52 (s, 1H), 11.38 (s, 1H),
8.21 (s, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.81 (s, 1H), 7.77 (d, J
= 8.1 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.42–7.36 (m, 3H),
7.26 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 2.5 Hz, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 165.7, 164.1, 145.7, 136.7, 133.5,
132.3, 128.5, 127.9, 127.8, 126.7, 126.4, 125.4, 125.3, 122.4,
122.3, 122.1, 111.9, 102.1. HRMS (ESI−) m/z calcd for
C18H11N2O2 287.0826, found 287.0839.
1H NMR (600 MHz, DMSO-d6) δ 11.66 (s, 1H), 9.72 (d, J
= 8.1 Hz, 1H), 8.12 (dd, J = 7.8, 1.5 Hz, 1H), 8.00 (s, 1H),
7.71 (t, J = 8.4 Hz, 1H), 7.59–7.54 (m, 2H), 7.33 (s, 1H),
3.86 (s, 3H). 13C NMR of the major isomer (151 MHz,
DMSO-d6) δ 165.9, 163.9, 141.5, 132.9, 132.6, 130.1,
129.1, 129.0, 127.4, 126.3, 125.6, 124.6, 123.0 (2 C), 33.5.
4-(thiophen-2-ylmethylene)isoquinoline-1,3(2H,4H)-
4-(4-hydroxybenzylidene)isoquinoline-1,3(2H,4H)-
dione (12f)
dione (12k)
1H NMR (600 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.64 (s,
1H), 8.29 (d, J = 8.1 Hz, 1H), 8.10 (s, 1H), 8.03 (t, J =
4.5 Hz, 2H), 7.77 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.5 Hz,
1H), 7.31 (t, J = 4.2 Hz, 1H). 13C NMR (151 MHz, DMSO-
d6) δ 164.5, 163.9, 142.0, 137.8, 137.4, 136.6, 136.0, 133.7,
127.8, 127.7, 127.4, 123.7, 122.8, 116.2. HRMS (ESI−) m/z
calcd for C14H8NO2S 254.0281, found 254.0292.
1H NMR (600 MHz, DMSO-d6) δ 11.50 (s, 1H, major
isomer), 11.36 (s, 1H, minor isomer), 10.10 (s, 1H),
8.15 – 7.92 (m, 3H), 7.81 – 7.69 (m, 1H), 7.55 – 7.33 (m,
3H), 6.80 (dd, J = 8.8, 3.8 Hz, 2H). Aromatic signals of
both isomers are overlapped and have been integrated
together. 13C NMR of the major isomer (151 MHz,
DMSO-d6) δ 166.06, 164.50, 143.97, 135.41, 133.67,
132.85, 131.81, 129.03, 128.30, 126.84, 125.89, 125.59,
123.80, 122.96, 116.23, 115.12. 13C NMR of the minor
isomer (151 MHz, DMSO-d6) δ 164.67, 164.25, 145.44,
137.49, 134.17, 128.76, 128.06, 128.01, 126.01, 124.08,
120.96, 116.23, 115.12. HRMS (ESI−) m/z calcd for
C16H10NO3 264.0666, found 264.0676.
4-(thiophen-3-ylmethylene)isoquinoline-1,3(2H,4H)-
dione (12g)
1H NMR (600 MHz, DMSO-d6) δ 11.50 (s, 1H, major and
minor), 8.67 (d, J = 3.0 Hz, 2H, minor), 8.20–7.97 (m, 3H,,
major and minor), 7.88–7.64 (m, 2H, major and minor),
7.63–7.48 (m, 2H, major and minor), 7.21 (d, J = 5.0 Hz, 1H,
minor). 13C NMR (151 MHz, DMSO-d6) δ 165.7 (minor),
164.1, 164.0 (minor), 164.0, 136.8, 136.7, 136.5 (minor),
136.3, 135.7 (minor), 135.0, 133.7, 132.9 (minor), 132.6, 131.9
(minor), 130.7, 129.0 (minor), 128.0 (minor), 127.9, 127.7
(minor), 127.5 (minor), 127.0, 126.7 (minor), 125.5, 125.4,
124.0 (minor), 123.9 (minor), 123.3, 121.0 (minor). HRMS
(ESI−) m/z calcd for C14H8NO2S 254.0281, found 254.0292.
4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
benzylidene)isoquinoline-1,3(2H,4H)-dione (12l)
1H NMR (600 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.07 (d, J =
7.8 Hz, 2H), 7.71 (d, J = 7.7 Hz, 2H), 7.68 (s, minor isomer),
7.49 (d, J = 7.9 Hz, 3H), 7.45 – 7.39 (m, 2H), 1.31 (s, 12H).
13C NMR (151 MHz, DMSO-d6) δ 165.2, 163.9, 142.3,
138.3, 134.9, 133.5, 132.5, 132.4, 129.5, 129.2, 128.0, 127.8,
126.9, 126.0, 125.7, 83.9, 24.7. Only the major product’s
peaks were assigned. HRMS (ESI−) m/z calcd for
C22H21BNO4 374.1569, found 374.1571.
4-((1H-indazol-5-yl)methylene)isoquinoline-1,3
(2H,4H)-dione (12h)
1H NMR (600 MHz, DMSO-d6) δ 13.29 (s, 1H), 11.58 (s,
1H), 8.20 (s, 1H), 8.13 (s, 1H), 8.07 (dd, J = 7.8, 1.4 Hz, 1H),
8.02 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.7 Hz,
1H), 7.45 (t, J = 7.6 Hz, 2H), 7.41 – 7.37 (m, 1H). 13C NMR
of the major isomer (151 MHz, DMSO-d6) δ 165.5, 164.1,
144.0, 140.0, 134.5, 133.0, 132.4, 128.8, 127.9, 127.0, 126.6,
126.6, 125.6, 123.9, 123.2, 122.7, 110.7. 13C NMR of the
minor isomer (151 MHz, DMSO-d6) δ 164.2, 163.7, 145.4,
4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
benzylidene)isoquinoline-1,3(2H,4H)-dione (12m)
1H NMR of both isomers (600 MHz, DMSO-d6) δ 11.57 (s,
1H), 8.27 – 7.84 (m, 3H), 7.79 – 7.65 (m, 2H), 7.63 – 7.54 (m,
1H), 7.52 – 7.33 (m, 3H), 1.31 (s, minor), 1.27 (s, 12H). 13C
NMR (151 MHz, DMSO-d6) δ 165.7, 164.4, 144.2, 143.0,
137.1, 136.3, 135.7, 135.4, 134.6, 134.3, 133.0, 132.8, 131.9,

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129.5, 129.1, 128.5, 128.1, 127.3, 126.1, 124.1, 84.3, 25.1.
HRMS (ESI−) m/z calcd for C22H21BNO4 374.1569, found
374.1572.
attempts were made to assign the minor isomer separately.
13C NMR of the major isomer (151 MHz, DMSO-d6) δ
166.9, 165.2, 164.0, 141.5, 139.9, 133.9, 132.6, 131.3, 129.9
(2 C), 129.3, 128.6 (2 C), 128.1, 127.1, 125.8, 123.8. 13C
NMR of the minor isomer (151 MHz, DMSO-d6) δ 167.1,
164.1, 163.3, 142.5, 140.3, 135.4, 132.2, 130.7, 130.0 (2 C),
128.9, 128.5(2 C), 127.7, 126.7, 125.6, 124.3. HRMS (ESI−)
m/z calcd for C17H10NO4 292.0615, found 292.0625.
4-(3,4-dihydroxybenzylidene)isoquinoline-1,3
(2H,4H)-dione (12n)
1H NMR (600 MHz, DMSO-d6) δ 11.48 (s, 1H), 9.42 (s,
2H), 8.04 (d, J = 7.3 Hz, 1H), 7.91 (s, 1H), 7.84 (d, J =
7.6 Hz, 1H), 7.47 (t, J = 7.9 Hz, 2H), 6.96 (s, 1H), 6.89 (d,
J = 8.2 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H). Signals of the
minor isomer is overlapped with the major isomer and no
attempts were made to assign the minor isomer separately.
13C NMR (151 MHz, DMSO-d6) δ 165.6, 164.0, 147.9,
145.5, 143.9, 133.7, 133.2, 132.4, 128.5, 127.7, 126.6,
125.6, 125.4, 122.2, 116.0, 115.9. HRMS (ESI−) m/z calcd
for C16H10NO4 280.0615, found 280.0624.
4-(4-bromobenzylidene)isoquinoline-1,3(2H,4H)-
dione (12r)
1H NMR (600 MHz, DMSO-d6) δ 11.61 (s, 1H), 8.04 (d, J
= 7.8 Hz, 1H), 7.95 (s, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.46
(t, J = 8.1 Hz, 1H), 7.44 – 7.40 (m, 4H). 13C NMR of the
major isomer (151 MHz, DMSO-d6) δ 165.7, 164.4, 141.7,
134.9, 134.3, 133.0, 132.5, 131.2, 131.0, 129.6, 128.5,
127.3, 126.5, 126.2, 124.2, 123.2. HRMS (ESI−) m/z calcd
for C16H9BrNO2 325.9822, found 325.9835.
4-(4-methoxybenzylidene)isoquinoline-1,3(2H,4H)-
dione (12o)
4-(4-fluorobenzylidene)isoquinoline-1,3(2H,4H)-
dione (12s)
1H NMR (600 MHz, DMSO-d6) δ 11.54 (s, 1H), 8.18 – 7.92
(m, 3H), 7.82 – 7.35 (m, 4H), 6.99 (d, J = 8.4 Hz, 2H), 3.81
(s, 3H). Both isomers were integrated together. 13C NMR of
the major isomer (151 MHz, DMSO-d6) δ 165.5, 164.0,
160.5, 142.9, 134.3, 133.0, 132.4, 131.0, 128.7, 127.9,
126.9, 126.5, 125.5, 123.5, 114.4,113.2, 55.3. 13C NMR of
the minor isomer (151 MHz, DMSO-d6) δ 164.2, 163.8,
160.9, 144.3, 136.7, 133.7, 127.9, 127.6, 127.1, 123.8,
121.6, 55.3. Some fully substituted carbons of the minor
product did not appear. HRMS (ESI−) m/z calcd for
C17H10N3O2 288.0779, found 288.0790.
1H NMR (600 MHz, DMSO-d6) δ 11.62 (s, 1H), 8.24–7.80
(m, 2H), 7.64 – 7.37 (m, 5H), 7.34–7.11 (m, 2H). Both major
and minor products were integrated and reported together. 13
C
NMR of both isomers (151 MHz, DMSO-d6) δ 165.3, 164.0,
163.5, 161.7, 142.8, 141.7, 135.9, 133.8, 133.4, 133.4, 132.6,
132.5, 131.6, 131.6, 131.1, 131.0, 129.0, 128.5, 128.0, 127.6,
126.8, 125.8, 125.6, 124.1, 123.7, 116.2, 116.0, 114.6, 114.5.
HRMS (ESI−) m/z calcd for C16H9FNO2 266.0623, found
266.0632.
2-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)
methyl)benzenesulfonate sodium (12p)
N-(4-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-
ylidene)methyl)phenyl)acetamide (12u)
1H NMR (600 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.62 (s, 1H),
8.03 (dd, J = 7.9, 1.5 Hz, 1H), 7.87 (dd, J = 7.8, 1.3 Hz, 1H),
7.39 (t, J = 7.2 Hz, 2H), 7.31 – 7.27 (m, 1H), 7.25 (td, J = 7.5,
1.3 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H).
13C NMR of the major isomer (151 MHz, DMSO-d6) δ 165.7,
164.7, 147.0, 146.4, 134.0, 133.7, 132.7, 129.6, 129.0, 128.8,
128.6, 128.4, 128.1, 127.5, 125.8, 124.3. HRMS (ESI−) m/z
calcd for C16H10NO5S- 328.0280, found 328.0285.
1H NMR (600 MHz, DMSO-d6) δ 11.56 (s, 1H), 10.17 (s,
1H), 8.06 (dd, J = 6.9, 2.3 Hz, 1H), 7.99 (s, 1H), 7.66 (d, J =
7.3 Hz, 1H), 7.63 (d, J = 8.7 Hz, 2H), 7.50 – 7.43 (m, 4H),
2.07 (s, 3H). 13C NMR of the major isomer (151 MHz,
DMSO-d6) δ 168.7, 165.5, 164.0, 142.8, 140.8, 132.9, 132.4,
129.9, 129.1, 128.8, 127.9, 127.6, 126.6, 125.6, 124.0, 123.5,
118.8, 24.1. 13C NMR of the minor isomer (151 MHz,
DMSO-d6) δ 164.2, 163.7, 144.1, 141.0, 136.5, 133.8, 132.9,
129.3, 128.0, 123.9, 122.3, 117.6, 24.1. HRMS (ESI−) m/z
calcd for C18H13N2O3 305.0932, found 305.0941.
4-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)
methyl)benzoic acid (12q)
4-(cyclopropylmethylene)isoquinoline-1,3(2H,4H)-
dione (12v)
1H NMR (600 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.11 – 8.04
(m, 2H), 7.94 (d, J = 8.1 Hz, 2H), 7.90 (d, J = 8.1 Hz, 1H),
7.54 (d, J = 7.8 Hz, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.39 (t, J
= 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H). Signals of the
minor isomer is overlapped with the major isomer and no
1H NMR of the major isomer (600 MHz, DMSO-d6) δ
11.33 (s, 1H), 8.03 (dd, J = 7.9, 1.5 Hz, 1H), 7.92 (d, J =
8.2 Hz, 1H), 7.65 (t, J = 8.4 Hz, 1H), 7.45 (t, J = 7.5 Hz,

Medicinal Chemistry Research
1H), 6.82 (d, J = 10.9 Hz, 1H), 3.36 (dtt, J = 12.1, 8.2,
4.5 Hz, 1H), 1.19 (dq, J = 6.8, 4.0 Hz, 2H), 0.95 (dq, J =
6.8, 3.8 Hz, 2H). 13C NMR of the major isomer (151 MHz,
DMSO-d6) δ 165.0, 164.0, 157.3, 135.6, 133.5, 127.5,
127.3, 126.3, 122.7, 122.3, 121.5, 14.2, 11.1. 13C NMR of
the minor isomer (151 MHz, DMSO-d6) δ 165.2, 164.1,
154.3, 133.9, 133.5, 128.0, 127.9, 124.7, 123.6, 14.0, 11.1.
Some signals of the minor isomer did not show up. HRMS
(ESI−) m/z calcd for C13H10NO2 212.0717, found 212.0723.
125.3, 113.8 (2 C), 55.2, 48.5, 42.2. HRMS (ESI−) m/z
calcd for C17H14NO3 280.0979, found 280.0987.
Synthesis of compounds 13b and 13g: general
procedure for demethylation of OMe group using
BBr3
Compound containing aromatic methoxy group (13a or
13d, 1.0 equiv) was dissolved in DCM (0.2 M). It was
added BBr3 (1.0 M solution in DCM, 4.0 equiv) and the
reaction was stirred at room temperature for overnight. The
reaction was quenched by adding MeOH (2 mL) slowly.
The mixture was concentrated in vacuo. The crude material
was purified by column chromatography with DCM and
MeOH to obtain corresponding demethylated products (13b
and 13 g) as white solids (yield = 60–65%).
Synthesis of compounds 13a
Compounds of the subtype 13 was prepared starting from
commercially available homopthalic acid (16) following
literature procedures. First, di-methylation of homopthalic
acid was carried out using SOCl2 in MeOH to obtain 18
(yield = quant.) [30]. Knoevenagel condensation of 18 with
p-anisaldehyde was performed to synthesize 19 (yield =
80%) [31].
4-(4-hydroxybenzyl)isoquinoline-1,3(2H,4H)-dione
(13b)
Compound 19 (0.78 g, 2.4 mmol, 1.0 equiv) was then
subjected to catalytic hydrogenation using Pd/C (250 mg) in
MeOH (20 mL). After 2 h, the reaction was filtered through
a pad of celite and the MeOH layer was concentrated in
vacuo to obtain the intermediate 20 as a colorless oil (yield
= quant.) which was carried out to the next step with no
further purification.
Intermediate 20 (0.3 g, 0.91 mmol) was dissolved in MeOH
(10 mL) and 10% KOH(aq). The reaction was refluxed for 3 h
before it was cooled down to room temperature. MeOH was
removed in vacuo and the aqueous layer was acidified with
2 M HCl until pH=1. The aqueous layer was extracted with
EtOAc, washed with H2O, brine, dried over Na2SO4 and
concentrated to obtain the saponified intermediate. The inter-
mediate was then dissolved in 1,2-dichlorobenzene (5 mL) in a
round bottom flask equipped with a reflux condenser. To the
mixture, conc. NH4OH (5 mL) was added and it was refluxed
at 110 °C for 1 h. Then the condenser was removed, and the
mixture was heated up to 210 °C until most of the solvent is
dried off. The crude was purified by column chromatography
with hexane and ethyl acetate to obtain 13a as a white solid
(yield = 35%).
1H NMR (600 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.15 (s,
1H), 7.86 (d, J = 7.8 Hz, 1H), 7.67 (td, J = 7.5, 1.4 Hz, 1H),
7.49 (d, J = 7.7 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 6.45 (q, J
= 8.4 Hz, 4H), 4.21 (t, J = 5.2 Hz, 1H), 3.28 (dd, J = 13.7,
5.3 Hz, 1H), 3.22 – 3.13 (m, 1H). 13C NMR (151 MHz,
Methanol-d4) δ 176.1, 166.6, 157.5, 141.4, 141.3, 134.8,
131.5, 129.8, 129.3, 129.2, 128.6, 128.5, 127.6, 126.9,
115.8, 42.9. HRMS (ESI−) m/z calcd for C16H12NO3
266.0823, found 266.0831.
4-(3-hydroxyphenyl)isoquinoline-1,3(2H,4H)-dione
(13g)
1H NMR (600 MHz, DMSO-d6) δ 11.44 (s, 1H), 9.44 (s,
1H), 8.09 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.49
(t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.11 (t, J =
7.8 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.59 (d, J = 7.6 Hz,
1H), 6.52 (s, 1H), 5.15 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 172.1, 165.0, 157.6, 141.0, 140.4, 133.9,
129.9, 128.7, 127.6, 127.3, 124.6, 119.3, 115.2, 114.5,
51.5. HRMS (ESI−) m/z calcd for C15H10NO3 252.0666,
found 252.0676.
4-(4-methoxybenzyl)isoquinoline-1,3(2H,4H)-dione
(13a)
Synthesis of compounds 13c – 13f
1H NMR (600 MHz, Chloroform-d) δ 8.08 (dd, J = 7.8,
1.5 Hz, 1H), 7.91 (s, 1H), 7.61 (td, J = 7.6, 1.6 Hz, 1H),
7.44 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.7 Hz, 1H), 6.64 (d, J
= 1.6 Hz, 4H), 4.15 (t, J = 5.6 Hz, 1H), 3.72 (s, 3H), 3.40
(dd, J = 13.6, 6.2 Hz, 1H), 3.29 (dd, J = 13.6, 4.8 Hz, 1H).
13C NMR (151 MHz, Chloroform-d) δ 173.3, 164.3, 158.9,
139.4, 133.9, 130.5 (2 C), 128.5, 128.0, 127.9, 127.4,
Compound 13c was prepared following a literature proce-
dure [32]. In a round bottom flask equipped with a magnetic
stirrer, the C-4 substituted isoquinoline-1,3-diones (13b,
13d) (1.0 equiv) was dissolved in MeCN (1 mL) and
CsCO3 (0.2 equiv) was added. The reaction was stirred at
room temperature for overnight. Then the volatiles were
removed and the crude was purified by column

Medicinal Chemistry Research
chromatography with hexane and ethyl acetate to obtain 13c
and 13e as white solids (yields = 50–60%).
Compounds 13d and 13 f were prepared following a
literature procedure [23].
Synthesis of compound 14a
Compound 21 was synthesized following a literature pro-
cedure [25]. It was subjected to an aqueous-phase diazo
transfer reaction following the reported protocol to obtain
compound 22 [24]. Rh(II)-catalyzed cycloaddition of 22
with benzonitrile following a reported procedure delivered
14a as a white solid (yield = 50%) [25].
4-hydroxy-4-(4-hydroxybenzyl)isoquinoline-1,3
(2H,4H)-dione (13c)
1H NMR (600 MHz, DMSO-d6) δ 11.15 (s, 1H), 9.21 (s,
1H), 7.80 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.64
(d, J = 7.9 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 6.46 (s, 1H),
6.41 (d, J = 8.0 Hz, 2H), 6.30 (d, J = 8.0 Hz, 2H), 3.15 (d,
J = 13.2 Hz, 1H), 2.96 (d, J = 12.8 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6) δ 175.5, 163.6, 156.1, 142.8, 133.5,
130.6 (2 C), 128.0, 126.7, 126.3, 124.8, 123.9, 114.5 (2 C),
75.0, 50.1. HRMS (ESI−) m/z calcd for C16H12NO4
282.0772, found 282.0779.
4-(4-methoxybenzyl)-2-phenyloxazolo[5,4-c]
isoquinolin-5(4H)-one (14a)
1H NMR (600 MHz, Chloroform-d) δ 8.50 (d, J = 8.1 Hz,
1H), 8.15 – 8.08 (m, 3H), 7.80 – 7.74 (m, 1H), 7.56 – 7.47
(m, 6H), 6.87 (dd, J = 8.7, 1.2 Hz, 2H), 5.47 (s, 2H), 3.76
(s, 3H). 13 C NMR (151 MHz, Chloroform-d) δ 160.8,
159.6, 156.4, 146.3, 133.4, 131.2, 130.6, 130.3 (2 C),
129.7, 129.1 (2 C), 128.1, 127.0, 126.2 (2 C), 126.1, 123.8,
121.0, 116.6, 114.3 (2 C), 55.4, 46.2. HRMS (ESI−) m/z
calcd for C15H10NO3 381.1245, found 381.1247.
4-phenylisoquinoline-1,3(2H,4H)-dione (13d)
1H NMR (600 MHz, DMSO-d6) δ 11.47 (s, 1H), 8.10 (d, J
= 7.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.49 (t, J = 7.6 Hz,
1H), 7.33 (t, J = 7.5 Hz, 2H), 7.29 (d, J = 7.3 Hz, 1H), 7.17
(d, J = 7.5 Hz, 2H), 7.12 (d, J = 7.8 Hz, 1H), 5.28 (s, 1H).
13C NMR (151 MHz, dmso) δ 172.2, 164.9, 140.4, 139.9,
133.9, 128.9 (2 C), 128.7, 128.7 (2 C), 127.6, 127.4, 127.3,
124.7, 51.5. HRMS (ESI−) m/z calcd for C15H10NO2
236.0717, found 236.0726.
General procedure for synthesis of subtype 15 (15a
– 15c)
Compound 27 was prepared following a literature proce-
dure [18]. Then, the compound 27 was subjected to the
General procedure A for Knoevenagel Condensation to
obtain 15a – 15c (yield = 37–50%).
4-hydroxy-4-phenylisoquinoline-1,3(2H,4H)-dione
7-(3-chlorophenyl)-4-(4-hydroxybenzylidene)
(13e)
isoquinoline-1,3(2H,4H)-dione (15a)
1H NMR (600 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.08 (d, J
= 7.8 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz,
1H), 7.48 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.5 Hz, 2H), 7.25
(t, J = 9.5 Hz, 3H), 7.02 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 174.2, 164.2, 144.2, 143.0, 134.4, 128.4,
128.3 (2 C), 127.8, 127.4, 126.9, 125.2 (2 C), 124.4, 74.9.
HRMS (ESI−) m/z calcd for C15H10NO3 252.0666, found
252.0674.
1H NMR (600 MHz, DMSO-d6) δ 11.59 (s, 1H), 11.45 (s,
minor isomer), 10.16 (s, 1H), 8.29 (t, J = 2.8 Hz, 1H),
8.25 – 8.05 (m, 1H), 8.03 – 7.82 (m, 3H), 7.79 – 7.73 (m,
1H), 7.72 – 7.41 (m, 4H), 6.82 (d, J = 8.4 Hz, 2H). Peaks
for both isomers were integrated together. 13C NMR
(101 MHz, DMSO-d6) δ 165.4, 164.0, 163.9, 163.7, 160.2,
159.5, 145.3, 143.9, 140.6, 140.4, 138.3, 137.4, 136.6,
135.2, 133.9, 132.8, 131.9, 131.5, 130.9, 130.6, 128.0,
127.9, 127.1, 126.3, 126.3, 126.1, 125.5, 125.5, 125.3,
125.3, 125.1, 124.3, 124.1, 122.0, 119.9, 115.8, 114.7. Due
to the complexicity and overlapping of NMR signals,
attempts were not made to assign major and minor isomers
separately. HRMS (ESI−) m/z calcd for C22H13ClNO3
374.0589, found 374.0593.
4-(3-methoxyphenyl)isoquinoline-1,3(2H,4H)-dione
(13f)
1H NMR (600 MHz, DMSO-d6) δ 11.46 (s, 1H), 8.09 (d, J
= 7.9 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.6 Hz,
1H), 7.24 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 6.86
(dd, J = 8.2, 2.5 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.66 (d,
J = 7.6 Hz, 1H), 5.25 (s, 1H), 3.72 (s, 3H). 13C NMR
(151 MHz, DMSO-d6) δ 172.0, 164.9, 159.4, 141.2, 140.3,
133.9, 130.0, 128.7, 127.6, 127.3, 124.6, 120.6, 115.1,
112.5, 55.1, 51.4. HRMS (ESI−) m/z calcd for C16H12NO3
266.0823, found 266.0830.
7-(3-chlorophenyl)-4-(4-fluorobenzylidene)
isoquinoline-1,3(2H,4H)-dione (15b)
1H NMR (600 MHz, DMSO-d6) δ 11.72 (s, 1H), 8.32 (d, J
= 2.2 Hz, 1H), 8.09 (s, 1H), 7.84 (dd, J = 8.5, 2.2 Hz, 1H),
7.80 (t, J = 1.9 Hz, 1H), 7.72 (dt, J = 7.8, 1.4 Hz, 1H),

Medicinal Chemistry Research
7.63 – 7.59 (m, 2H), 7.57 – 7.46 (m, 3H), 7.31 (t, J =
8.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.7,
164.4, 164.3, 163.9, 161.8, 143.7, 142.5, 141.0, 140.7,
139.2, 138.8, 136.0, 134.5, 134.1, 134.0, 132.6, 132.5,
132.1, 132.1, 131.7, 131.6, 131.5, 131.2, 128.7, 128.6,
128.0, 126.9, 126.9, 126.8, 126.2, 125.9, 125.8, 125.7,
125.2, 124.1, 116.8, 116.6, 115.2, 115.0. Due to the com-
plexicity and overlapping of NMR signals, attempts were
not made to assign major and minor isomers separately.
HRMS (ESI+) m/z calcd for C22H14ClFNO2 378.0692,
found 378.0691.
determinations represent the means of three independent
experiments performed in triplicate.
Molecular modeling
Selected Isoquinoline-1,3-diones were docked into the hTDP2.
The hTDP2 model was built with SWISS-MODEL using
mTDP2 structure as a template. The mTDP2 crystal structure
(PDB:4GZ1) was trimmed by removing one of the TDP2
monomer chains, bound DNA, and water molecules. The
trimmed mTDP2 structure and hTDP2 sequence (UniProtKB -
O95551) were submitted to SWISS-MODEL for homology
modeling. The magnesium ion was incorporated by super-
position of the model and 4GZ1 monomer (trimmed) followed
by the deletion of the protein chain of 4GZ1 in Schrodinger
Maestro. Resulting mTDP2 model containing the metal ion
was minimized using protein preparation wizard in Maestro.
Receptor Grid Generation function in Maestro was used to
create the grid box (enclosing box size= 15 Å) centroid to the
active site residues E152, D261, and H351. Ligand structures
were prepared in Maestro Ligprep function using OPLS3e
force field. The docking of the prepared ligands was performed
with Glide using Extra Precision setting in Maestro Ligand
docking function. All figures showing binding modes were
generated using PyMol (The PyMOL Molecular Graphics
System, Schrödinger LLC.)
7-(3-chlorophenyl)-4-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzylidene)isoquinoline-1,3
(2H,4H)-dione (15c)
1H NMR (600 MHz, DMSO-d6) δ 11.74 (s, 1H), 8.32 (d, J
= 2.2 Hz, 1H), 8.12 (s, 1H), 7.83 – 7.79 (m, 2H), 7.76 –
7.66 (m, 3H), 7.56 – 7.45 (m, 5H), 1.32 (s, 12H). Only the
major isomer is integrated. 13C NMR (101 MHz, DMSO-d6)
δ 173.8, 165.1, 163.8, 163.6, 163.3, 143.7, 142.6, 140.4,
140.2, 140.2, 138.8, 138.5, 138.4, 138.3, 138.1, 137.9,
137.4, 135.2, 134.9, 134.0, 133.9, 133.9, 133.5, 132.0,
131.9, 131.0, 130.9, 130.7, 129.6, 128.2, 128.1, 127.9,
127.7, 126.4, 126.4, 126.4, 126.3, 125.6, 125.6, 125.4,
125.4, 125.3, 124.8, 124.7, 124.5, 83.9, 83.8, 83.6, 24.7,
24.6. Due to the complexicity and overlapping of NMR
signals, attempts were not made to assign major and minor
isomers separately. HRMS (ESI−) m/z calcd for
C28H24BClNO4 484.1487, found 484.1489.
Acknowledgements This research was supported by the University of
Minnesota AHC Faculty Research Development Grant (to ZW and
HA), and the National Institutes of Health grant GM118047 (to HA).
We thank the Center for Drug Design, University of Minnesota for the
support, and the Minnesota Supercomputing Institute (Minneapolis,
USA) for molecular modeling resources.
Biology. 14M_zTDP2 fluorescence-based
biochemical assay
Compliance with ethical standards
IC50 values of the analogs were determined using our in-
house biochemical assay [26]. The reaction buffer used was
composed by 50 mM Tris-HCl pH 7.4, 10 mM MgCl2,
80 mM KCl, 0.05% (v/v) Tween-20, and 1 mM DTT. To a
black 384-well plate, 10 µL of compound solution (in
reaction buffer, concentration 2-fold higher than the tested
concentration, and final DMSO concentration of 2.5%) was
added, followed by addition of 5 µL of 14M_TDP2 enzyme
(25 pM, final concentration of 6.25 pM). After a pre-
incubation period of 10 min, 5 µL of substrate 5′-(6-FAM-
NHS)(5′-tyrosine)GATCT(3′-BHQ-1)-3′ (4 µM, final con-
centration of 1 µM) was added, and the reaction was
allowed to proceed at 25 °C for 60 min. The fluorescence of
the product was measured using a SpectraMax M5e
(Molecular Devices) (λex 285 nm; λem 325 nm; λcutoff
315 nm) in kinetic-mode at 25 °C for 60 minutes. IC50
experiments data using 12 concentrations of inhibitor and
vehicle-alone were fitted by GraphPad Prism software. IC50
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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