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

Sameera Senaweera; Tianyu He; Haixi Cui; Hideki Aihara; Zhengqiang Wang

doi:10.1007/s00044-020-02662-w

. Author manuscript; available in PMC: 2022 Feb 1.

Published in final edited form as: Med Chem Res. 2020 Nov 19;30(2):371–386. doi: 10.1007/s00044-020-02662-w

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

Sameera Senaweera ¹, Tianyu He ¹, Haixi Cui ¹, Hideki Aihara ², Zhengqiang Wang ¹

PMCID: PMC7993140 NIHMSID: NIHMS1648473 PMID: 33776385

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.

Keywords: Tyrosyl-DNA phosphodiesterase 2 (TDP2); Anti-cancer; benzylideneisoquinoline-1,3(2H,4H)-dione

Graphical Abstract

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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 to enable important DNA transactions.[1] Mechanistically, Top2 cleaves DNA using its tyrosine residue to generate a transient 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 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 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 etoposide,[6] and that the up-regulation of TDP2 transcription was linked to etoposide 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 (Figure 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 complexity 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 identified 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, rendering it highly amenable toward expanded SAR via new structural modifications.

Selective inhibition of TDP2 by isoquinoline-1,3-diones (Figure 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 towards 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-benzylideneisoquinoline-1,3(2H,4H)-diones as a new TDP2 inhibitor subtype.

The expanded SAR started with the N-2 NH , where the its importance for the activity was probed using unsubstituted (X=H), oxidized (X=OH), and differently substituted (X=Me, OBn) analogs (Figure 2, subtype 10). The focus of the current SAR, however, was on synthetic C-4 derivatives. In that vein, four different series of compounds were synthesized and tested: (1) olefinic C-4 substituents with a nitrogen linker (subtype 11); (2) olefinic C-4 substituents with a carbon linker (subtype 12); (3) benzylic and phenyl substituents at C-4 (subtype 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 cheap homophthalic acid (16) and cone, 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 ammonium hydroxide, correspondingly. BBr₃ mediated benzyl deprotection of 10b yielded N-hydroxy derivative, 10c. Isoquinoline-1,3-diones with olefinic C-4 substituents possessing 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 olefinic C-4 substituents possessing aromatics, heteroaromatics, and alkyl groups were examined (subtype 12). Synthesis of such compounds was achieved by the Knoevenagel 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).

Then, we turned our attention to isoquinolines substituted with benzylic and phenyl substituents at C-4 to obtain the subtype 13. However, attempts to reduce previously 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 Knoevenagel 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 substituent (20) was feasible. Intermediate 20 was then saponified 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 13g. 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) protected 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 ɑ - 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 of isoquinoline-1,3-diones (subtype 15). Synthesis up to the compound 27 was carried out following a literature procedure followed by a Knoevenagel condensation using selected aldehydes to obtain the subtype 15.

Biology

All final compounds and selected synthetic intermediates were evaluated in our in-house fluorescence-based biochemical 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 reference which had a previously reported IC₅₀ value of 25 μM and had a comparable IC50 of 22 μM in our biochemical assay. Importantly, when the NH is substituted with either Me (10a) or OBn (10b), the resulting compounds 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 processing enzymes.

Table 1.

SAR of N-substituted derivatives of isoquinoline-1,3-dione

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^aIC₅₀: concentration of a compound production 50% inhibition, expressed as mean ± standard deviation from at least three independent experiments.

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 (CDK4).[20] Our analogs covers a wide range of amine headpieces including the ones with potential reversible and irreversible covalent warheads (11h - 11l). Unfortunately, none of those compounds showed inhibitory potency in our TDP2 biochemical assay at 50 μM.

Table 2.

TDP2 inhibition by subtype 11

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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. Substituents such as pyrroles (12d and 12e) and thiophenes (12f and 12g) showed no activity at 50 μM. However, compounds 12h (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 phenomenon, compounds 12h-12k were synthesized and tested. Among those, compound 12k which possesses 4-hydroxybenzylidene showed an improved potency (IC50 =10 μM). The effect of the substitution position at the benzylidene was also significant. Regiochemistry of the pinacol boronate in compounds 12l and 12m 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).

Table 3.

TDP2 inhibition by subtype 12

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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 subtype 12. To that end, we further extended our SAR to analogs bearing non-olefinic substituents at the C-4 of isoquinoline-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 substituents on both the benzene ring and C-4 position (15a-15c) were all inactive.

Table 4.

Lack of TDP2 inhibition by subtypes 13-15

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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 resolution 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 reproducing the binding pose of 1,3-dioxo-l,2,3,4-tetrahydroisoquinoline-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 (Figure 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 (Figure 3, panel B). Similarly, 12k, which is predicted to bind to the active site Mg²⁺, resulted in inhibition, whereas 11b prefers a binding pose where the core is flipped and binds away from the Mg²⁺, resulting in no inhibition at concentrations up to 50 μM (Figure 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 carboxylate group on the terminal phenyl with E152, E236, and Y178, respectively (Figure 3, panel D).

Figure 3. — Predicted optimal binding modes of (A) 1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-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).

Conclusion

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 compounds with moderate potency only, the insight gained herein may facilitate the design of improved TDP2 inhibitors.

Material and Methods

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 silica columns (silica) and indicated mobile phase. 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 spectrometer. Mass data were acquired on an Agilent TOF II TOS/MS spectrometer capable of ESI and APCI ion sources.

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 dissolved 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 hr. 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]

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 procedure.[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 hr 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 - 11l.

(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.

(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, 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 (2C), 128.3, 127.6, 122.9, 120.2, 118.6, 115.4 (2C), 91.9, 52.0. HRMS (ESI⁻) m/z calcd for C₁₇H₁₃N₂O₃ 293.0932, found 293.0940.

(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 (2C). HRMS (ESI⁻) m/z calcd for C₁₃H₁₁N₂O₂ 227.0826, found 227.0832.

(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 (2C), 56.0 (2C). HRMS (ESI⁻) m/z calcd for C₁₄H₁₄N₃O₃ 272.1041, found 272.1052.

(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-((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-(((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, 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.

(Z)-4-(((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11h)

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 (2C), 132.9, 127.6, 126.9 (2C), 123.0, 120.2, 118.6, 92.3, 83.6 (2C), 52.2, 24.6 (4C). HRMS (ESI⁻) m/z calcd for C₂₃H₂₄BN₂O₄ 403.1835, found 403.1841.

(Z)-4-(((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11i)

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). ¹³CNMR (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 (2C), 128.0, 123.4, 120.7, 119.1, 92.6, 84.2 (2C), 52.8, 25.1(4C). HRMS (ESI⁻) m/z calcd for C₂₃H₂₄BN₂O₄ 403.1835, found 403.1840.

(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.

(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 = 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-(methylsulfonyl)phenyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11l)

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). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.0, 163.7, 143.6, 143.5, 135.9, 135.5, 133.2, 128.7 (2C), 127.6, 125.0, 121.4, 120.5, 117.5 (2C), 97.7, 43.7. HRMS (ESI⁻) m/z calcd for C₁₇H₁₃N₂O₄S 341.0602, found 341.0608.

General procedure A for Knoevenagel Condensation (12b - 12v)

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 minutes 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 hrs 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 - 12v.

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.

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 (2C), 129.00, 128.41 (2C), 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.

4-(pyridin-4-ylmethylene)isoquinoline-1,3(2H,4H)-dione (12c)

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, 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.

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 – 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.

4-((1-methyl-1H-pyrrol-2-yl)methylene)isoquinoline-1,3(2H,4H)-dione (12e)

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 (2C), 33.5.

4-(thiophen-2-ylmethylene)isoquinoline-1,3(2H,4H)-dione (12f)

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.

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-((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, 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)

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.

4-(4-hydroxybenzylidene)isoquinoline-1,3(2H,4H)-dione (12k)

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-(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-(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, 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.

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-methoxybenzylidene)isoquinoline-1,3(2H,4H)-dione (12o)

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.

2-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)benzenesulfonate sodium (12p)

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.

4-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)benzoic acid (12q)

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 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 (2C), 129.3, 128.6 (2C), 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 (2C), 128.9, 128.5(2C), 127.7, 126.7, 125.6, 124.3. HRMS (ESI⁻) m/z calcd for C₁₇H₁₀NO₄ 292.0615, found 292.0625.

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-fluorobenzylidene)isoquinoline-1,3(2H,4H)-dione (12s)

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. ¹³C 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.

N-(4-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)phenyl)acetamide (12u)

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-(cyclopropylmethylene)isoquinoline-1,3(2H,4H)-dione (12v)

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, 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.

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]

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 intermediate 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 hr. 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%).

4-(4-methoxybenzyl)isoquinoline-1,3(2H,4H)-dione (13a)

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 (2C), 128.5, 128.0, 127.9, 127.4, 125.3, 113.8 (2C), 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 13g) as white solids (yield = 60-65%).

4-(4-hydroxybenzyl)isoquinoline-1,3(2H,4H)-dione (13b)

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.

Synthesis of compounds 13c – 13f

Compound 13c was prepared following a literature procedure. [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 chromatography with hexane and ethyl acetate to obtain 13c and 13e as white solids (yields = 50 - 60%).

Compounds 13d and 13f were prepared following a literature procedure. [23]

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 (2C), 128.0, 126.7, 126.3, 124.8, 123.9, 114.5 (2C), 75.0, 50.1. HRMS (ESI⁻) m/z calcd for C₁₆H₁₂NO₄ 282.0772, found 282.0779.

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 (2C), 128.7, 128.7 (2C), 127.6, 127.4, 127.3, 124.7, 51.5. HRMS (ESI⁻) m/z calcd for C₁₅H₁₀NO₂ 236.0717, found 236.0726.

4-hydroxy-4-phenylisoquinoline-1,3(2H,4H)-dione (13e)

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, 128.3, 128.3 (2C), 127.8, 127.4, 126.9, 125.2 (2C), 124.4, 74.9. HRMS (ESI⁻) m/z calcd for C₁₅H₁₀NO₃ 252.0666, found 252.0674.

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.

Synthesis of compound 14a

Compound 21 was synthesized following a literature procedure.[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-(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). 13C NMR (151 MHz, Chloroform-d) δ 160.8, 159.6, 156.4, 146.3, 133.4, 131.2, 130.6, 130.3 (2C), 129.7, 129.1 (2C), 128.1, 127.0, 126.2 (2C), 126.1, 123.8, 121.0, 116.6, 114.3 (2C), 55.4, 46.2. HRMS (ESI−) m/z calcd for C15H10NO3 381.1245, found 381.1247.

General procedure for synthesis of subtype 15 (15a – 15c)

Compound 27 was prepared following a literature procedure. [18] Then, the compound 27 was subjected to the General procedure A for Knoevenagel Condensation to obtain 15a – 15c (yield = 37% - 50%).

7-(3-chlorophenyl)-4-(4-hydroxybenzylidene)isoquinoline-1,3(2H,4H)-dione (15a)

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 (ESr) m/z calcd for C₂₂H₁₃ClNO₃ 374.0589, found 374.0593.

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), 7.63 – 7.59 (m, 2H), 7.55 – 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, 125.8, 125.7, 125.2, 124.1, 116.8, 116.6, 115.2, 115.0. 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 C₂₂H₁₄ClFNO₂ 378.0692, found 378.0691.

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.

Biology.

14M_zTDP2 fluorescence-based biochemical assay

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 minutes, 5 μL of substrate 5’-(6-FAM-NHS)(5’-tyrosine)GATCT(3’-BHQ-1)-3’ (4 μM, final concentration of 1 μM) was added, and the reaction was allowed to proceed at 25 °C for 60 minutes. The fluorescence of the product was measured using a SpectraMax M5e (Molecular Devices) (λex 285 nm; λem 325 nm; λcutoff 315nm) 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 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 superposition 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, Schrodinger LLC.)

Supplementary Material

44_2020_2662_MOESM1_ESM

NIHMS1648473-supplement-44_2020_2662_MOESM1_ESM.pdf^{(9.6MB, pdf)}

Acknowledgments

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.

Footnotes

Supplementary information: The online version of this article contains supplementary material, which is available to authorized users.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

44_2020_2662_MOESM1_ESM

NIHMS1648473-supplement-44_2020_2662_MOESM1_ESM.pdf^{(9.6MB, pdf)}

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[R23] 23.Yang Y, Li Y, Cheng C, Yang G, Zhang J, Zhang Y et al. Synthesis of 4-Aryl Isoquinolinedione Derivatives by a Palladium-Catalyzed Coupling Reaction of Aryl Halides with Isoquinoline-1,3(2iT,4/7)-diones. J Org Chem. 2018;83(6):3348–53. doi: 10.1021/acs.joc.7b02961. [DOI] [PubMed] [Google Scholar]

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PERMALINK

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

Sameera Senaweera

Tianyu He

Haixi Cui

Hideki Aihara

Zhengqiang Wang

Abstract

Graphical Abstract

Introduction

Figure 1.

Figure 2.

Results and Discussion

Chemistry

Scheme 1.

Biology

Table 1.

Table 2.

Table 3.

Table 4.

Molecular modeling

Figure 3.

Conclusion

Material and Methods

Chemistry.

General Procedures.

Synthesis of compound 10a

Synthesis of compound 10b

Synthesis of compound 10c

General procedure for synthesis of subtype 11 (11a - 11l)

(Z)-4-(((4-fluorobenzyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11a)

(Z)-4-(((4-hydroxybenzyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11b)

(Z)-4-((cyclopropylamino)methylene)isoquinoline-1,3(2H,4H)-dione (11c)

(Z)-4-((morpholinoamino)methylene)isoquinoline-1,3(2H,4H)-dione (11d)

(Z)-4-(isoindolin-2-ylmethylene)isoquinoline-1,3(2H,4H)-dione (11e)

(Z)-4-((thiazol-2-ylamino)methylene)isoquinoline-1,3(2H,4H)-dione (11f)

(Z)-4-(((3-(2H-tetrazol-5-yl)phenyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11g)

(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-(((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11i)

(Z)-4-(((3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11j)

(Z)-4-(2-(((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)amino)ethyl)benzenesulfonyl fluoride (11k)

(Z)-4-(((4-(methylsulfonyl)phenyl)amino)methylene)isoquinoline-1,3(2H,4H)-dione (11l)

General procedure A for Knoevenagel Condensation (12b - 12v)

4-benzylideneisoquinoline-1,3(2H,4H)-dione (12b)

4-(pyridin-4-ylmethylene)isoquinoline-1,3(2H,4H)-dione (12c)

4-((1H-pyrrol-2-yl)methylene)isoquinoline-1,3(2H,4H)-dione (12d)

4-((1-methyl-1H-pyrrol-2-yl)methylene)isoquinoline-1,3(2H,4H)-dione (12e)

4-(thiophen-2-ylmethylene)isoquinoline-1,3(2H,4H)-dione (12f)

4-(thiophen-3-ylmethylene)isoquinoline-1,3(2H,4H)-dione (12g)

4-((1H-indazol-5-yl)methylene)isoquinoline-1,3(2H,4H)-dione (12h)

4-((1H-indol-5-yl)methylene)isoquinoline-1,3(2H,4H)-dione (12i)

4-(4-hydroxybenzylidene)isoquinoline-1,3(2H,4H)-dione (12k)

4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isoquinoline-1,3(2H,4H)-dione (12l)

4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isoquinoline-1,3(2H,4H)-dione (12m)

4-(3,4-dihydroxybenzylidene)isoquinoline-1,3(2H,4H)-dione (12n)

4-(4-methoxybenzylidene)isoquinoline-1,3(2H,4H)-dione (12o)

2-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)benzenesulfonate sodium (12p)

4-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)benzoic acid (12q)

4-(4-bromobenzylidene)isoquinoline-1,3(2H,4H)-dione (12r)

4-(4-fluorobenzylidene)isoquinoline-1,3(2H,4H)-dione (12s)

N-(4-((1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene)methyl)phenyl)acetamide (12u)

4-(cyclopropylmethylene)isoquinoline-1,3(2H,4H)-dione (12v)

Synthesis of compounds 13a

4-(4-methoxybenzyl)isoquinoline-1,3(2H,4H)-dione (13a)

Synthesis of compounds 13b and 13g: General procedure for demethylation of OMe group using BBr3

4-(4-hydroxybenzyl)isoquinoline-1,3(2H,4H)-dione (13b)

4-(3-hydroxyphenyl)isoquinoline-1,3(2H,4H)-dione (13g)

Synthesis of compounds 13c – 13f

4-hydroxy-4-(4-hydroxybenzyl)isoquinoline-1,3(2H,4H)-dione (13c)

4-phenylisoquinoline-1,3(2H,4H)-dione (13d)

4-hydroxy-4-phenylisoquinoline-1,3(2H,4H)-dione (13e)

4-(3-methoxyphenyl)isoquinoline-1,3(2H,4H)-dione (13f)

Synthesis of compound 14a

4-(4-methoxybenzyl)-2-phenyloxazolo[5,4-c]isoquinolin-5(4H)-one (14a)

General procedure for synthesis of subtype 15 (15a – 15c)

7-(3-chlorophenyl)-4-(4-hydroxybenzylidene)isoquinoline-1,3(2H,4H)-dione (15a)

7-(3-chlorophenyl)-4-(4-fluorobenzylidene)isoquinoline-1,3(2H,4H)-dione (15b)

7-(3-chlorophenyl)-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isoquinoline-1,3(2H,4H)-dione (15c)

Biology.

14M_zTDP2 fluorescence-based biochemical assay

Synthesis of compounds 13b and 13g: General procedure for demethylation of OMe group using BBr₃