Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer

Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer Bidyadhar Sethy, Richa Upadhyay, Iin Narwanti, Zih-Yao Yu, Sung-Bau Lee, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4661784/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Oct, 2024 Read the published version in Apoptosis → Version 1 posted 16 You are reading this latest preprint version Abstract Triple-negative breast cancer (TNBC) poses a significant challenge for treatment due to its aggressive nature and the lack of effective therapies. This study developed dual inhibitors against cell division cycle 25 (CDC25) and histone deacetylases (HDACs) for TNBC treatment. CDC25 phosphatases are crucial for activating cyclin-dependent kinases (CDKs), the master regulators of cell cycle progression. HDACs regulate various biological processes by deacetylating histone and non-histone proteins, affecting gene expression, chromatin structure, cell differentiation, and proliferation. Dysregulations of HDACs and CDC25s are associated with several human malignancies. We generated a group of dual inhibitors for CDC25 and HDAC by combining the molecular structures of CDC25 (quinoline-5,8-dione) and HDAC (hydroxamic acid or benzamide) pharmacophores. The newly developed compounds were evaluated against solid-tumor, leukemia, and non-malignant breast epithelial cells. Among the synthesized compounds, 18A emerged as a potent inhibitor, demonstrating significant cytotoxicity against TNBC cells, superior to its effects on other cancer types while sparing non-malignant cells. 18A possessed similar HDAC inhibitory activity as Entinostat and potently suppressed the CDC25 activity in cells. Additionally, 18A hindered the progression of S and G 2 /M phases, caused DNA damage, and induced apoptosis. These findings suggest that 18A holds promise as a targeted therapy for TNBC and warrants further preclinical development. CDC25 inhibitor HDAC inhibitor dual-target Triple-Negative Breast Cancer genome instability apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Cancer remains a significant global health burden, with nearly 20 million new cases reported in 2022, leading to approximately 9.7 million cancer-related deaths worldwide. Among all cancer types, breast cancer stands out as one of the most prevalent, with 2.3 million new cases in 2022, making it the second most commonly diagnosed cancer globally. Within the spectrum of breast cancer subtypes, triple-negative breast cancer (TNBC) accounts for approximately 10–15% of all breast cancer cases. [ 1 ]. TNBC is characterized by the absence of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2) expressions, presenting as a particularly aggressive form of the disease with a poorer prognosis compared to other subtypes. Based on gene expression profiling, TNBCs are subdivided into seven classes: immunomodulatory (IM), luminal androgen receptor (LAR), mesenchymal-like (M), mesenchymal stem-like (MSL), basal-like 1 (BL1), basal-like 2 (BL2), and unstable (UNS). Research efforts are ongoing to develop targeted therapies tailored to address the unique challenges posed by TNBC and improve treatment outcomes for individuals affected by this subtype. Conventional chemotherapy and radiation are the primary systemic therapeutic strategies, and no other FDA-approved targeted therapies are yet available for TNBC. Due to these features and the lack of targeted therapies, numerous attempts have been made to discover potential molecular targets for TNBC [ 2 ]. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are crucial enzymes in the epigenetic regulation of gene expression through post-translational modifications of histone proteins. HDACs remove acetyl groups from histones, leading to chromatin condensation and gene repression, whereas HATs add acetyl groups, resulting in chromatin relaxation and gene activation. [ 3 ]. Besides histones, HDACs deacetylate non-histone substrates, including polyamines, tubulin, structural maintenance of chromosomes (SMC) proteins, p53, and Hsp90. This deacetylation activity of HDACs affects gene expression, chromatin structure, and cellular processes such as differentiation and proliferation, thereby significantly regulating multiple biological functions [ 4 , 5 ]. In humans, HDACs comprise a family of 18 genes which are divided into four classes: The "classical" HDACs with a zinc-dependent active site are Class I (HDAC1, HDAC2, HDAC3, and HDAC8), Class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), and Class IV (HDAC11) [ 6 ]. Class I HDAC1-3 mainly deacetylates histones H3 and H4 but also targets non-histone proteins [ 7 ]. It is generally agreed that up-regulation of HDACs in malignant tumor cells leads to the deregulation of homeostasis, differentiation, cell apoptosis, and inactivation of tumor suppressors [ 8 ]. Since epigenetic changes are highly implicated in tumorigenesis, inhibitors of histone deacetylases could represent a promising therapeutic strategy [ 9 ]. Four HDAC inhibitors have been approved by the U.S. Food and Drug Administration (FDA): vorinostat (SAHA; 1 ), romidepsin (FK228), belinostat (PXD-101; 2 ), and panobinostat (LBH589; 3 ) for the treatment of hematological malignancies (Fig. 1 ) [ 10 ]. The China FDA has also authorized the chidamide (CS055; 4 ) to treat peripheral T-cell lymphoma [ 11 ]. Entinostat (MS-275; 5 ) is a widely used benzamide group inhibitor targeting HDAC1-3. It has been demonstrated to be helpful in treating hematological malignancies, while its inhibitory effectiveness against solid tumors is modest [ 12 ]. Many clinical researches have shown that HDAC inhibitors exploit synergistic effects with other anticancer agents. However, multicomponent drugs raised the risks involved in complex pharmacokinetic properties, unpredictable drug-drug interactions, and different drug solubilities. Developing HDAC-based multitargeting drugs may thus provide an effective and practical strategy to overcome the limitations of single and multicomponent agents [ 13 ]. One notable factor contributing to the development of malignant tumors is the disruption of cell cycle control. Disordered cell cycle, unlimited proliferation, and mutations in tumor suppressors and oncogenes are common characteristics all malignancies share, including TNBCs [ 14 ]. Cell division cycle 25 (CDC25) phosphatases are central to the regulation of the cell cycle, in which CDC25 dephosphorylates the threonine and tyrosine residues of cyclin-dependent kinase (CDK)/cyclin complexes, promoting CDK activation and, thus, cell cycle progression. CDC25 dysregulation has been implicated in various malignancies, highlighting their potential as therapeutic targets in cancer treatment [ 15 ]. The CDC25 family comprises three isoforms - CDC25A, CDC25B, and CDC25C. These isoforms function on distinct CDK/cyclin complexes at various cell cycle phases. CDC25A mainly activates the CDK2/cyclin E and CDK2/cyclin A complexes during the G1/S transition, while Cdc25B and C primarily regulate the CDK1/cyclinB1 activity to promote the progression of mitosis [ 16 ]. Targeting CDC25 has shown promise in preclinical studies, with inhibitors demonstrating the ability to induce cell cycle arrest and promote apoptosis in cancer cells [ 17 ]. Figure 1 also shows the typical quinone moieties carrying CDC25 inhibitors. The naphthoquinone derivative, UPD-140 (6) , inhibits CDC25 activity in HeLa cells, with an IC 50 value of 1.2 µM [ 18 ]. Compound Cpd5 (7) is a thioether derivative that inhibits CDC25 and can impede G 1 and G 2 /M progression [ 19 ]. Through high-throughput screening (HTS), a sulfur-containing vitamin K analog, NSC95397 (8) , was found to be a potent irreversible CDC25 inhibitor but also targeting MKK7 and other kinases [ 20 ]. Moreover, compounds NSC668394 (9) and NSC663284/6a (11) were shown to be sub-micromolar IC 50 (0.64 and 0.25 µM, respectively) [ 21 ]. NSC663284/6a (11) is one of the most potent Cdc25 inhibitors reported, has been shown to block cell cycle progression of tumor cells at both G1 and G2/M, and causes hyperphosphorylation of Cdk1/Cdc2 [ 22 ]. In addition, Jing research group discovered a quinoline-5, 8-dione derivative M2N12 (10) , which potently inhibits CDC25C with IC 50 of 0.09 µM and also exhibited remarkable suppression on KB-VIN cell growth (IC 50 = 6.81 µM) [ 23 ]. Furthermore, Narwanti group conducted a study that identified 6-ragiomeric quinoline-5, 8-dione containing compound 6b (12) as a potent CDC25 inhibitor that induces genome instability and kills cancer cells through apoptotic pathways [ 24 ]. In recent years, advancements have made it possible to target multiple pathways implicated in the progression of TNBC therapy with combinatorial techniques that combine numerous targeted therapies [ 25 ]. A dual (multiple)-target therapies show high efficacy and can reduce the therapeutic doses and side effects compared to single-target drug therapy [ 26 ]. A promising approach to drug design is the incorporation of HDAC inhibitory pharmacophores (hydroxamate or o -phenylenediamine) into the structure of the inhibitor for another target to create HDAC-based dual or multiple inhibitors [ 27 – 29 ]. Among these molecules, the HDAC/CDK dual inhibitors have shown promising enzymatic inhibitory and anti-proliferative activities [ 29 ]. As mentioned earlier, various cyclin-dependent kinases (CDKs) activated upon removal of phosphate residues by CDC25 resulted in cell cycle progression. According to recent research, CDK inhibition makes cancer cells more susceptible to HDAC inhibitors, which may cause cancer cell death [ 30 ]. Therefore, we hypothesized that combining CDC25 and HDAC inhibitors may statistically limit the growth of cancer cells. In the present study, potential CDC25-HDAC dual inhibitors have been designed and synthesized by adopting the principle of molecular hybridization of the potential CDC25-HDAC pharmacophore, and the structure-activity relationships (SARs) were explored. All the hybrid molecules were screened against various cancer cell lines, and the CDC25-HDAC inhibitory activities of the selected compounds in cells were further examined. The effects on the cell cycle progression, DNA damage, and apoptosis were biologically investigated herein. 2. Chemistry 2.1. Study rationale design To guide the design of potent anti-TNBC agents with a thorough understanding of related proteins, such as CDC25 and HDAC in TNBC progression, we applied the principle of molecular hybridization combining structural features of CDC25 and HDAC inhibitors to create dual-target molecules. According to the classical HDAC inhibitors model, HDAC inhibitors should have a zinc-binding group (ZBG), an aromatic or aliphatic linker, and a capping group to effectively inhibit the hydrolysis of acetyl-lysine residues on histone tails and non-histone proteins (Figure 2) . The ZBG is a hydrophilic domain, including hydroxamic acid and 2-aminobenzamide groups interacting with the active zinc cation (Zn2+) at the HDAC site. Furthermore, as discussed earlier, the cap group is a crucial unit susceptible to chemical modifications to enhance selectivity and assisted pharmacokinetic properties. Here, we introduced quinoline-5, 8-dione moiety as the cap, which may cause of improving the effectivity by interacting with the aromatic amino acid residues located either close to the outer domain of the active site or at the external surface of the enzyme, as well as by facilitating its ability to form redox biological reaction and DNA damage. Therefore, based on the principle of molecular hybridization, by considering the structural analogous of the core scaffold of CDC25 inhibitors (quinoline-5, 8-dione moiety), we designed and synthesized multiple inhibitors of CDC25-HDAC combined via different aliphatic or aromatic linkers with hydroxamic acid or benzamide moieties (Figure 2), which may provide reasonable pharmaceutical properties and expecting improved efficacy for TNBC treatment. 2.2. Synthesis The synthesis of all the target compounds was depicted in Scheme 1-3 . Scheme 1 illustrates the synthesis of the N -benzyl linker-based hydroxamic acid containing target compounds 13A , 13B , 14A , and 14B . The synthesis starts with a condensation reaction using a commercially available starting material, phthalic anhydride and para ( p )-phenylenediamine, to produce 21 . A substitution reaction was generated for 22 and 23 using the intermediates methyl 4-(bromomethyl) benzoate and 20, respectively. Before this, we synthesized the intermediate 20 by utilizing methyl 2-bromo-4-methylbenzoate, which was subjected to Suzuki-coupling with phenylboronic acid to produce 19 , and then radical bromination of 19 gave the intermediate 20 . Subsequently, lithium hydroxide assisted ester hydrolysis of 22 and 23 made the carboxylic acid product 24 and 25 , respectively, which upon amidation using O -(tetrahydro-2H-pyran-2-yl) hydroxylamine (NH 2 -OTHP) to yield 26 and 27 . A hydrazine hydrate-based phthalimide deprotection reaction has also generated free aniline intermediates ( 28 and 29 ). These intermediates are utilized to react with 6, 7-dichloroquinoline-5, 8-dione to get the regioisomers of respected anilines such as 30A/31A (7-regiomer) and 30B/31B (6-regiomer), which are upon OTHP deprotection furnished the target hydroxamic acid products 13A , 14A , 13B , and 14B , respectively. Furthermore, we introduced various long-chain linker-based hydroxamic acids ( 15A , 15B , 16A , 16B , 17A , and 17B ) by adopting a general synthetic protocol for synthesizing the target compounds presented in Scheme 2 . Firstly, we followed a similar methodology such as EDC/HOBt assisted amidation of alkoxyalkanoic acids ( 32 , 33 , and 34 ) with NH 2 -OTHP to generate 35 , 36 , and 37 , then lithium hydroxide-based ester hydrolysis to yield 38 , 39 , and 40 , and those are upon amidation with 21 afforded the intermediates 41 , 42 , and 43 , respectively. Next, the aniline intermediates ( 44 , 45 , and 46 ) were generated by the deprotection of respective phthalimides using hydrazine hydrate. Further, the free aniline intermediates were subjected to a substitution reaction with 6,7-dichloroquinoline-5,8-dione to produce their respective 7-, 6-regiomer intermediates ( 47A , 47B , 48A , 48B , 49A , and 49B ), which upon acid catalyzed OTHP deprotection to afford the desired products 15A , 15B , 16A , 16B , 17A , and 17B respectively. In addition, according to our design strategy, we further introduced o -phenylenediamine as the zinc-binding motif, and the synthetic route to the target compounds is depicted in Scheme 3 . The intermediate 50 was synthesized using the commercially available starting material tert-butyl (4-aminophenyl) carbamate and methyl 4-(bromomethyl)benzoate. The intermediate 50 was subjected to ester hydrolysis to obtain 51 , which proceeded to an amidation reaction using o -phenylenediamine catalyzed by EDC/HOBt to generate 52 . Subsequently, 53 was obtained upon BOC-deprotection of 52 , which was further subjected to react with 6, 7-dichloroquinoline-5, 8-dione to give their respective 7-, 6-regiomer target compounds 18A and 18B respectively. All the new compounds were characterized and determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and the purity was confirmed >95% using high-pressure liquid chromatography (HPLC) and the respective figures were attached in the supplementary file. 2.3. Cytotoxicity To evaluate potential of compounds 13A-18A and 13B-18B for cancer treatment, we initially tested their cytotoxicity on six different cell lines derived from solid tumors. These included triple-negative breast cancer (MDA-MB-231, MDA-MB-436), HER2-negative breast cancer (MCF-7), pancreatic adenocarcinoma (PANC-1), glioblastoma (U87MG), and colorectal cancer (CRC; DLD-1). Cells were treated with the compounds at 2 µM for 48 hours, and their viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In these assays, we also included compounds 6a , 6b [ 24 ] , MS-275, and SAHA as reference controls for comparison. The results depicted in Figure 3 and S1 reveal intriguing details about the cytotoxicity of all the synthesized compounds. Notably, among the compounds tested, 18A showed a significant reduction in cell viability of DLD-1 cells, comparable to the effect of 6b (Figure S1A), the compound identified as a potent CDC25 inhibitor specifically targeting CRC cells in our previous study [24]. Additionally, 18A exhibited superior cytotoxic activity against triple-negative breast cancer cells (MDA-MB-231 and MDA-MB-436) compared to HER-2 negative breast cancer cells (MCF-7) (Figure 3A-C). Furthermore, we observed lower cytotoxicity of 18A in U87MG and PANC-1 cells (Figure S1B-C). In contrast to 18A , the remaining compounds in the series displayed relatively weak cytotoxic effects across all tested cell lines. Most importantly, we extended our investigation to assess the effect of 18A on a non-malignant breast epithelial cell line and found that 18A exhibit minimal cytotoxicity to the M10 cells (Figure 3D). These findings underscore the potential selectivity of 18A for TNBC cells over non-TNBC and normal breast cells. We further conducted a study on the impact of synthesized compounds on leukemia cell lines, including HL-60 (acute promyelocytic leukemia), K-562 (chronic myelogenous leukemia), MV4-11 (biphenotypic B-myelomonocytic leukemia) and KG-1 (acute myelogenous leukemia). Cells were treated with the compounds at 0.4µM, 1µM, 0.3µM and 0.5µM for 48 hours. We exploited the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) assay for evaluation. Our analysis revealed that 18A was particularly effective against HL60 and K562, but it did not show differential toxicity in KG-1 and MV4-11 compared with reference compounds (Figure S1D-G). Our results showed that subtle structural modifications within this series significantly impact cytotoxicity. The cytotoxicity results in MDA-MB-436 cells revealed that the compounds bearing N-benzyl ( 13A ) and N-phenyl benzoyl ( 14A ) hydroxamic acid motif with quinoline-5, 8-dione conjugated via aromatic linkers possess better antiproliferative effects than its regioisomers 13B and 14B (Figure 3B). Further modification of the hybrid molecules by employing the aliphatic chain linker ( 15A - 17A and 15B - 17B ) weaker cytotoxic effects in all the selected solid tumor and leukemia cell lines (Figure S1). It is worth noting that the benzamide derivatives such as compound 18A , linking via N-benzyl aromatic to the quinoline-5, 8-dione scaffold, demonstrated the best antiproliferative effects against TNBCs (MDA-MB-231 and MDA-MB-436) among all synthesized compounds as well as the reference HDAC ( 1 , and 4 ) and CDC25 ( 11 , and 12 ) inhibitors (Figure 3). These results suggest that TNBCs are more sensitive to treatment with the hybrid molecule 18A as a first-in-class CDC25-HDAC dual inhibitor, compared to other solid tumor and leukemia cells, and warrant further investigation into its mechanism of action. 2.4. HDAC inhibitory activity Given that 18A was the most toxic of the synthesized compounds, we determined its inhibitory activity towards the potential target, HDACs. Our results indicated that treatment with 18A dose-dependently increased the acetylation levels of histone H3/H4 (Figure 4), which are the canonical substrates of the class I HDACs. The increased level was similar to that observed in the group treated with the reference compound MS-275 (Figure 4). In contrast, the treatment did not significantly change SMC3 and alpha-tubulin acetylation, indicating that 18A may not target HDAC8 and HDAC6. These findings strongly suggest that 18A is a class I HDAC inhibitor possessing comparable activity and specificity as MS-275 in cells. 2.5. HDAC isoform inhibition assay We next conducted a detailed analysis of the selective inhibitory properties of 18A against the HDAC 1, 2, and 3 isoforms in vitro. The compelling results outlined in Table 1 demonstrate that 18A outperformed the reference compound MS-275, exhibiting a significantly stronger inhibition of the HDAC1 isoform with IC50 values of 67.5 nM, compared to 212.5 nM for MS-275. Notably, both 18A and MS-275 displayed comparable inhibitory activities against the HDAC2 and HDAC3 isoforms. These findings emphatically support the classification of 18A as a potent class I HDAC inhibitor. Table 1: Inhibitory activities against HDAC1-3 of MS-275 and 18A. IC 50 (nM) 18A MS-275 HDAC1 67.5 212.5 HDAC2 415 451.5 HDAC3 715.5 898 2.6. CDC25 inhibitory activity We next analyzed the impact of 18A treatment on the activity of another potential target, CDC25s. We used a high concentration of thymidine to synchronize cells in the early-S phase (Figure 5; T0) and allowed the cells to progress into mitosis after being released from the thymidine block. To minimize the potential secondary effect from defective cell-cycle progression caused by compound treatment, we treated cells with compounds 8 hours after thymidine removal (T8), at which most cells had completed genome replication and accumulated in the G 2 phase . We showed that the phosphorylation levels of CDK1 at tyrosine 15 (CDK1Y15) at T0 and T8 were comparably high (Figure 5), consistent with the presence of cells in S and G 2 phases, respectively. To avoid re-phosphorylation of CDK1 after cell division, the tubulin destabilizing agent, nocodazole, was added to hinder mitosis progression. We found that CDK1Y15 phosphorylation dropped from T8 to T14, indicating that cells moved to and arrested at mitosis (Figure 5). 18A treatment resulted in an accumulation of CDK1Y15 phosphorylation and reduction of phosphorylation levels of mitotic markers cyclin B1 at Ser126, histone H3 at Ser10, and mitotic proteins recognized by the MPM2 antibody (Figure 5). This phenomenon was not observed in cells treated with 6a and 6b under the same conditions. We also obtained similar observations from cells arrested in mitosis by Taxol and colchicine (Figure S2). These results indicate that 18A impedes CDK1 dephosphorylation, most likely by downregulating CDC25 activity. Intriguingly, 18A treatment at a higher concentration (5 μM) unexpectedly increased phosphorylation levels of proteins that could also be detected by the MPM2 antibody (Figure 5), plausibly due to activation of an unknown pathway, which remains further investigation. 2.7. Cell cycle analysis After finding that 18A effectively inhibits CDK1 dephosphorylation (Figure 5 and S2), we decided to study its impact on cell cycle progression. We treated synchronized cells with 18A at T2 and used flow cytometry to monitor the cell cycle progression from the S phase to the subsequent G 1 phase (Figure 6; T2-T16). Our results revealed that treating cells with 2 μM of 18A slowed down the progression of the S phase and delayed the exit from mitosis. Treating cells with 4 μM of 18A resulted in almost complete arrest of the cell cycle in the S phase (Figure 6). To further investigate the direct effect of 18A on G 2 /M phase progression, we treated cells at T8 and found that 18A impaired G 2 /M phase progression in a dose-dependent manner (Figure S3). Based on these results, we conclude that 18A suppresses CDC25 activity, disrupting CDK activity, and ultimately interfering with cell cycle progression. 2.8. Genome stability High CDK activity has been reported to be required for efficient DNA repair processes, such as homologous recombination and checkpoint responses. CDC25 inhibition, which limits CDK activation, may impede HR and thus accumulate DNA lesions [31]. Our previous study showing DNA damage induction by 6b argued this hypothesis [24]. Moreover, HDAC inhibition alters chromatin structure, potentially compromising DNA integrity and repair mechanisms [32] . Therefore, we assessed the effect of 18A on genome stability. Our data revealed that the treatment of 18A remarkably increased phosphorylation levels of H2AX phosphorylation at S139 (γH2AX) and double-stranded breaks DNA markers, including phosphorylation of KRAB-associated protein-1 (KAP-1) at S824, replication protein A2 (RPA2) at S4/S8, and CHK2 at T68, as compared to reference compounds MS-275 and 6a (Figure 7). The treatment of 18A at 2 µM resulted in ataxia telangiectasia mutated (ATR) activation, revealed by RPA2 phosphorylation at S33 and CHK1 phosphorylation at S345. However, CHK1 phosphorylation was dramatically reduced when cells were treated with the higher concentration (5 μM) of 18A (Figure 7). These findings suggest that 18A is a potent DNA damage-inducing agent. 2.9. Apoptosis The flow cytometry analysis indicated that treatment with 18A resulted in an increased population of sub-G1 cells after 48 h, and this effect was dependent on the dosage (Figure 8A). Additionally, we examined the levels of proteins involved in apoptosis pathways and observed a significant increase in active caspases-3, -8, and -9 in cells treated with 18A. (Figure 8B). These changes were less pronounced in cells treated with 6a and MS-275 (Figure 8B). Our findings suggest that 18A is a potent dual inhibitor of CDC25 and HDAC, leading to cancer cell death through an apoptotic pathway. 3. Conclusion In this study, a series of novel CDC25-HDAC dual inhibitors were designed, synthesized, and biologically evaluated. Our biological evaluation demonstrates the potential of 18A , a benzamide derivative linking N-benzyl aromatic to the quinoline-5, 8-dione scaffold, exhibits potent cytotoxicity against TNBC cell lines, particularly MDA-MB-231 and MDA-MB-436 cells. Importantly, 18A exhibited minimal cytotoxicity towards non-malignant breast cell lines, highlighting its selectivity for cancerous cells. Our evidence suggests that simultaneously targeting CDC25 and HDACs may offer a promising therapeutic avenue for TNBC. Compound 18A displayed excellent HDAC1 inhibitory potency, surpassing the reference control MS-275. Meanwhile, the activities against HDAC2 and HDAC3 between the two compounds were comparable. In cell-based analysis, 18A treatment effectively increased the acetylation of histones H3/H4, indicating that 18A is a selective class I HDAC inhibitor. Moreover, 18A sufficiently inhibited CDC25 activity, leading to the accumulation of phosphorylated CDK1. 18A treatment resulted in the downregulation of mitotic markers and cell cycle arrest at S and G 2 /M phases, suggesting that 18A is a cell-cycle inhibitor by disrupting the CDC25/CDK pathway. Additionally, 18A perturbed genome stability and induced apoptotic cell death in TNBC cells, as evidenced by high levels of DNA damage response, increased sub-G 1 population, and upregulation of apoptosis pathways. The observed cytotoxicity, induction of cell cycle arrest, DNA damage, and apoptosis highlight the multifaceted mechanism of action of 18A in TNBC cells. These findings suggest that 18A holds promise as a targeted therapy for TNBC, providing a new approach to addressing the lack of effective treatments for this aggressive subtype of breast cancer. 4. Experimental Section 4.1. Chemistry All the chemicals, solvents and reagents used here without further purification, unless stated otherwise. The chemical reactions were monitored by Merk thin-layer chromatography (TLC) of silica gel 60 F254 aluminum plates. TLC plates were visualized under UV light at 254 nm using (UV visualizer name, company). For the concentration of organic solvents, Buchi rotary evaporator was used. Compounds were purified using normal phase column chromatography on silica gel (Merk Kieselgel 60, No. 9385, 230-400 mesh ASTM). 1 H and 13 C spectra were performed using Bruker DRX-500 spectrometer (operated at 300 MHz) and 500 MHz NMR instruments respectively, CDCl3 with tetramethylsilane used as internal standard, CD 3 OD and d6 -DMSO were also used as d-solvents. Chemical shifts (δ) are expressed in parts per million (ppm) scale, reference to the residual solvent peaks (CDCl3: 1 H 7.26 and 13 C 77.16, CD 3 OD: 1 H 3.31 and 13 C 49.00, d6 -DMSO: 1 H 2.50 and 13 C 39.52 and d6 -Acetone: 1 H 2.05 and 13 C 29.84, 206.26 etc. Signal multiplicity are expressed as: br.s (broad singlet), s (singlet), d (doublet), t (triplet), q (quartet), p (pentet) and m (multiplet). To process NMR spectra, topspin software was used. Coupling constant ( J ) values are calculated in hertz (Hz). Melting point of all final compounds measured using Fargo MP-2D apparatus and are uncorrected. The high-resolution mass spectra were measured by JEOL (JMS-700) electron impact (EI) mass spectrometer. All the final compounds were checked (>95 %) and determined by HPLC (Agilent 1260 Infinity II, Agilent Technologies, Germany) using a Dikma (Diamonsil 5 µm C18x150x4.6 mm) column. HPLC analysis conditions used ACN (mobile phase A) and water containing NH 4 OAc 10 mM with HCOOH 0.1% as a solvent system (mobile phase B) with a flow rate of 0.5 mL/min. 4.1.1. Methyl 5-methyl-[1,1'-biphenyl]-2-carboxylate ( 19) The synthetic procedure of compound 19 was conducted by following a procedure by Jeffrey et al. A mixture of methyl 2-bromobenzoate (6.5 g, 28.4 mmol), phenylboronic acid (5.36 g, 43.9 mmol), Pd(PPh 3 ) 4 (1.314 g, 3.68 mmol), K 2 CO 3 (10.64 g, 71.0 mmol) in anhydrous DMF (65 mL) was heated at 110°C for 24 h under argon (Ar) atmosphere. The reaction mixture was cooled to room temperature, added with water then extracted with EA. The organic layer was combined, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure to obtain the residue. The resulting residue was purified by silica column chromatography ( n -hexane: EA = 100: 1) to give compound 19 in 97% yield. Compound 19 : 1 H NMR (300 MHz, Chloroform- d ) δ H 7.77 (d, J = 7.8 Hz, 1H), 7.40 – 7.34 (m, 3H), 7.32 – 7.29 (m, 2H), 7.24 – 7.19 (m, 2H), 3.63 (s, 3H), 2.42 (s, 3H). 4.1.2. Methyl 5-(bromomethyl)-[1,1'-biphenyl]-2-carboxylate ( 20 ) Adapting a procedure by Jeffrey et al., mixture of compound 19 (2.26 g, 10.0 mmol), N-bromosuccinimide (1.87 g, 10.5 mmol), 1,1′-azobis(cyclohexanecarbonitrile) (30 mg, 0.12 mmol) in carbon tetrachloride (40 mL) was refluxed at 85-90°C under argon for 23 h. The reaction mixture was cooled to room temperature, then extracted with EA. The organic layer was combined, dried over magnesium sulfate, and then concentrated under reduced pressure to obtain the residue. The resulting residue was purified by silica column chromatography ( n -hexane: 100 à n -hexane: EA = 250:1 à 100: 1) to give compound 20 in 83% yield. Compound 20 : 1 H NMR (300 MHz, Chloroform- d ) δ H 7.81 (d, J = 7.8 Hz, 1H), 7.45 – 7.36 (m, 5H), 7.34 – 7.26 (m, 2H), 4.51 (s, 2H), 3.64 (s, 3H). 4.1.3. 2-(4-aminophenyl)isoindoline-1,3-dione ( 21 ) A mixture of phthalic anhydride (14.812 g, 10 mmol) was dissolved in DMF (7 mL), and p-phenylenediamine (10.814 g, 10 mmol) was added in batches. The mixture was stirred under reflux at 150 o C and maintained for an overnight reaction. The reaction was monitored by TLC, after completion of the reaction, ice/water was poured into it, a green solid was precipitated, and collected after suction filtration to obtain compound 21 (65% yield). Compound 21 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 7.92 – 7.85 (m, 4H), 7.02 – 6.99 (m, 2H), 6.64 – 6.61 (m, 2H), 5.32 (s, 2H). 4.1.4. Methyl 4-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)benzoate ( 22 ) A mixture of compound 21 (238 mg, 1 mmol), methyl 4-(bromomethyl)benzoate (229 mg, 1 mmol), and K 2 CO 3 (138 mg, 1 mmol) in anhydrous DMF (3 mL) was stirred at room temperature for 24 h. After confirming the completion of the reaction using TLC, the mixture was quenched with water (5 mL). Then EA (10 mL) was added, and an orange solid was precipitated, which was filtered using suction to obtain compound 22 (67.3% yield). Compound 22 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 7.94 – 7.84 (m, 6H), 7.51 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 6.64 (t, J = 7.2 Hz, 1H), 6.62 (d, J = 7.2 Hz, 2H), 4.40 (d, J = 6.0 Hz, 2H), 3.82 (s, 3H). 4.1.5. Methyl 5-(((4-(1,3-dioxoisoindolin-2-yl) phenyl) amino) methyl)-[1,1'-biphenyl]-2- carboxylate (23) A mixture of compound 21 (955 mg, 4 mmol), 20 (1220 mg, 4 mmol), and K 2 CO 3 (552 mg, 4 mmol) in anhydrous DMF (10 mL) was stirred at room temperature for 24 h. TLC was monitored, and after confirming the reaction completion, the mixture was quenched with water (20 mL). Then EA (20 mL) was added, and a white solid was precipitated, which was filtered using suction to obtain compound 23 (56.4% yield). Compound 23 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 7.92 – 7.84 (m, 4H), 7.72 (d, J = 7.8 Hz, 1H), 7.48 – 7.35 (m, 5H), 7.28 – 7.7.26 (m, 2H), 7.08 – 7.05 (m, 2H), 6.68 – 6.65 (m, 3H), 4.41 (d, J = 6.3 Hz, 2H), 3.55 (s, 3H). 4.1.6. 4-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)benzoic acid ( 24 ) and 5-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid ( 25 ) To a solution of compound 22 or 23 ( 2 mmol) in MeOH (8 mL) was added lithium hydroxide 1.0 M (aq) (7 mL), and the reaction mixture was heated from room temperature to 55 o C and stirred for overnight. The solvent was reduced by half using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath to adjust pH 4-5 and then EA was added, a brown solid precipitate was collected after suction filtration to obtain the desired product 24 or 25 respectively (85-90 % yield). The compound was directly used for the next steps without purification. Compound 24 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 12.87 (br.s, 1H), 9.91 (s, 1H), 7.90 – 7.87 (m, 2H), 7.82 – 7.79 (m, 1H), 7.63 – 7.45 (m, 5H), 7.36 (d, J = 9.0 Hz, 2H), 6.55 (d, J = 8.7 Hz, 2H), 4.34 (s, 2H). Compound 25 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 7.99 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.63 – 7.51 (m, 3H), 7.44 – 7.29 (m, 10H), 6.65 – 6.62 (m, 2H), 4.41 (s, 2H). 4.1.7. 4-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide ( 26 ) and 5-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1'-biphenyl]-2-carboxamide ( 27 ) A mixture of 24 (500 mg, 1.34 mmol, 1 equv.) or 25 (580 mg, 1.29 mmol, 1 equv.), O-(tetrahydro-2 H -pyran-3-yl)hydroxylamine (1.2 equv.), EDC. HCl (1.5 equv.), HOBt (1.2 equv), and N-methylmorpholine (1.5 equv.) in anhydrous DMF (5 mL) were stirred at room temperature for 3-4 h. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA: MeOH = 2: 2: 0.1) to give the desired compound 26 or 27 respectively with 55-60% yield. Compound 26 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 11.54 (s, 1H), 7.92 – 7.84 (m, 4H), 7.71 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.05 – 7.02 (m, 2H), 6.68 – 6.59 (m, 3H), 4.97 (s, 1H), 4.37 (d, J = 6.0 Hz, 2H), 4.05 – 3.99 (m, 1H), 3.52 – 3.48 (m, 1H), 1.70 (br.s, 3H), 1.53 (br.s, 3H). Compound 27 : 1 H NMR (300 MHz, CDCl 3 ) δ H 8.09 (s, 1H), 7.92 – 7.90 (m, 2H), 7.77 – 7.74 (m, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.44 – 7.39 (m, 8H), 7.21 – 7.18 (m, 2H), 6.74 (d, J = 8.7 Hz, 2H), 4.75 (s, 1H), 4.44 (s, 2H), 3.63 – 3.56 (m, 1H), 3.42 – 3.36 (m, 1H), 1.73 (br.s, 3H), 1.49 (br.s, 3H) 4.1.8. 4-(((4-aminophenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide ( 28 ) and 5-(((4-aminophenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1'-biphenyl]-2-carboxamide ( 29 ) A solution of 26 or 27 (1 mmol, 1 equv), and hydrazine hydrate (7 equv) in ethanol (10 mL) was stirred at 65-70 o C for 2-3 h under argon (Ar) gas. The resultant precipitate was filtered off and washed several times with ethanol, and the filtrate was collected and dried under vacuum to obtain the desired product 28 or 29 respectively with 65-70% yield. Compound 28 : 1 H NMR (300 MHz, CDCl3) δ H 9.02 (br.s, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 6.60 – 6.55 (m, 2H), 6.50 – 6.45 (m, 2H), 5.06 (s, 1H), 4.30 (s, 2H), 4.03 – 3.96 (m, 1H), 3.67 – 3.60 (m, 1H), 3.21 (br.s, 2H), 1.86 (br.s, 3H), 1.60 (br.s, 3H). Compound 29 : 1 H NMR (300 MHz, DMSO- d6 ) δ H 11.78 (s, 1H), 7.79 – 7.70 (m, 8H), 6.77 (s, 4H), 5.87 (s, 1H), 5.27 (s, 1H), 4.63 (d, J = 5.7 Hz, 2H), 4.34 – 4.27 (m, 1H), 3.87 – 3.84 (m, 1H), 2.02 (br.s, 3H), 1.89 (br.s, 3H). 4.1.9. 4-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide ( 30A ) and 4-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide ( 30B ) A mixture of 6,7-dichloroquinoline-5,8-dione (228 mg, 1 mmol), 28 (341 mg, 1 mmol), and DIPEA (175 µL, 1 mmol) in anhydrous DCM (9 mL) was stirred at room temperature for overnight. The reaction mixture was quenched with water (30 mL) and extracted using DCM (50 mL×2). The organic layer was separated, dried over anhydrous MgSO 4 and evaporated in a vacuum. The residue was applied to silica column chromatography ( n -hexane: EA: MeOH = 1: 1: 0.1) to give compounds 30A and 30B . Compound 30A (purple solid, yield = 36 %): 1 H NMR (300 MHz, CDCl 3 ) δ H 8.96 (dd, J = 4.8, 1.8 Hz, 1H), 8.78 (s, 1H), 8.50 (dd, J = 7.8, 1.8 Hz, 1H), 7.80 (s, 1H), 7.74 (d, J = 8.4, 2H), 7.68 (dd, J = 8.1, 4.8 Hz, 1H), 7.44 (d, J = 8.1, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.59 – 6.54 (m, 2H), 5.08 (t, J = 3.3, 1H), 4.42 (d, J = 4.2, 2H), 4.27 (s, 1H), 4.03 – 3.97 (m, 1H), 3.68 – 3.64 (m, 1H), 1.88 (br.s, 3H), 1.62 (br.s, 3H). Compound 30B (purple solid, yield = 31 %): 1 H NMR (300 MHz, Methanol- d 4 ) δ H 8.91 (dd, J = 4.8, 1.8 Hz, 1H), 8.46 (dd, J = 7.8, 1.8 Hz, 1H), 7.77 – 7.72 (m, 3H), 7.49 (d, J = 8.4, 2H 6.95 – 6.92 (m, 2H), 6.60 – 6.57 (m, 2H), 5.05 (br. s, 1H), 4.2 (s, 2H), 4.16 – 4.09 (m, 1H), 3.64 – 3.60 (m, 1H), 1.91 – 1.78 (m, 3H), 1.62 (br.s, 3H). 4.1.10. 5-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1'-biphenyl]-2-carboxamide ( 31A ) and 5-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1'-biphenyl]-2-carboxamide ( 31B ) Following the procedure for preparation of 30A and 30B , addition of 6,7-dichloroquinoline-5,8-dione (228 mg, 1 mmol), 29 (417 mg, 1 mmol), and DIPEA (175 µL, 1 mmol) in anhydrous DCM (9 mL) was stirred at room temperature for overnight, which gave compound 31A and 31B. Compound 31A (purple solid, yield = 43%): 1 H NMR (300 MHz, CDCl 3 ) δ H 8.95 (dd, J = 4.8, 1.8 Hz, 1H), 8.49 (dd, J = 7.8, 1.8 Hz, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.70 – 7.66 (m, 2H), 7.43 – 7.38 (m, 7H), 6.95 (d, J = 8.7, 2H), 6.58 (d, J = 8.7 Hz, 2H), 4.75 (s, 1H), 4.43 (s, 2H), 4.31 (br. s, 1H), 3.58 – 3.54 (m, 1H), 3.40 – 3.36 (m, 1H), 1.73 (br.s, 3H), 1.49 (br.s, 3H). Compound 31B (purple solid, yield = 41%): 1 H NMR (300 MHz, CDCl 3 ) δ H 9.04 (dd, J = 4.5, 1.8 Hz, 1H), 8.40 (dd, J = 7.8, 1.8 Hz, 1H), 7.96 (s, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.63 – 7.59 (m, 2H), 7.44 – 7.39 (m, 7H), 6.95 (d, J = 8.7, 2H), 6.58 (d, J = 8.7 Hz, 2H), 4.75 (s, 1H), 4.44 (s, 2H), 4.30 (br. s, 1H), 3.61 – 3.54 (m, 1H), 3.40 – 3.36 (m, 1H), 1.74 (br.s, 3H), 1.50 (br.s, 3H). 4.1.11. 4-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-hydroxybenzamide ( 13A ) To a mixture of 30A (190 mg, 0.36 mmol) in dry THF (4 mL) and MeOH (2 mL) was added hydrochloric acid (0.5 mL) under an ice bath, stirred for 2 h, and then allowed to stir at room temperature for 30 min. The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM (30 mL) and a small amount of methanol (5 mL) to afford a precipitate. The precipitate was then washed with DCM and ether to give compound 13A . Compound 13A : m.p. = 192-193 °C, purple solid, yield = 84%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 11.21 (br. s, 1H), 9.32 (s, 1H), 8.92 (dd, J = 4.8, 1.8 Hz, 1H), 8.35 (dd, J = 7.8, 1.8 Hz, 1H), 7.81 (dd, J = 7.8, 4.8 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 6.96 (d, J = 8.7, 2H), 6.75 (d, J = 8.7 Hz, 2H), 4.39 (s, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 192.9, 178.4, 175.9, 163.8, 153.2, 146.6, 144.0, 133.9, 132.0, 129.5, 129.3, 128.6, 128.4, 127.6, 126.9, 125.2, 124.7, 122.9, 49.3. HRMS (ESI) calcd for C 23 H 18 O 4 N 4 Cl [M+H] + 449.1011; found 449.1012. HPLC purity = 99.5% (t r = 13.821 min.). 4.1.12. 4-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-hydroxybenzamide ( 13B ) Following the procedure for preparation of 13A , a mixture of 30B (160 mg, 0.30 mmol) in dry THF (4 mL) and MeOH (2 mL) followed by the addition of hydrochloric acid (0.5 mL) and stirred for 3 h under an ice-bath to get the desired compound 13B . Compound 13B : m.p. = 180-181 °C, purple solid, yield = 90%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 11.19 (br. s, 1H), 9.23 (s, 1H), 8.96 (dd, J = 4.8, 1.8 Hz, 1H), 8.35 (dd, J = 7.8, 1.8 Hz, 1H), 7.76 (dd, J = 7.8, 4.8 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 6.96 (d, J = 8.4, 2H), 6.75 (d, J = 7.2 Hz, 2H), 4.39 (s, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 192.9, 179.9, 175.1, 163.8, 154.3, 147.7, 142.8, 134.6, 129.5, 128.7, 127.7, 127.5, 127.2, 126.9, 125.1, 124.6, 122.9, 54.9. HRMS (ESI) calcd for C 23 H 18 O 4 N 4 Cl [M+H] + 449.1011; found 449.1012. HPLC purity = 99.3% (t r = 13.316 min.). 4.1.13. 5-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-hydroxy-[1,1'-biphenyl]-2-carboxamide ( 14A ) Following the procedure for preparation of 13A , a mixture of 31A (260 mg, 0.43 mmol) in dry THF (4 mL) and MeOH (2 mL) followed by the addition of hydrochloric acid (0.5 mL) and stirred for 2 h under an ice-bath to get the desired compound 14A . Compound 14A : m.p. = 159-160 °C, purple solid, yield = 92%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.75 (br. s, 1H), 9.20 (br. s, 1H), 8.92 – 8.32 (m, 2H), 8.33 (dd, J = 7.8, 1.8 Hz, 1H), 7.80 (dd, J = 7.8, 1.5 Hz, 1H), 7.39 – 7.31 (m, 8H), 6.88 (d, J = 8.7, 2H), 6.54 (d, J = 8.7 Hz, 2H), 6.46 (t, J = 6.3 Hz, 1H), 4.35 (d, J = 6.0 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 178.5, 175.5, 166.1, 153.0, 146.4, 146.3, 144.1, 142.0, 140.2, 139.6, 133.8, 132.8, 129.5, 128.8, 128.4, 128.3, 128.2, 127.4, 127.2, 126.0, 125.7, 111.5, 110.4, 46.1. HRMS (ESI) calcd for C 29 H 22 O 4 N 4 Cl [M+H] + 525.1324; found 525.1327. HPLC purity = 96.2% (t r = 16.199 min.). 4.1.14. 5-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-hydroxy-[1,1'-biphenyl]-2-carboxamide ( 14B ) Following the procedure for preparation of 13A , a mixture of 31B (250 mg, 0.41 mmol) in dry THF (4 mL) and MeOH (2 mL) followed by the addition of hydrochloric acid (0.5 mL) and stirred for 3 h under an ice-bath to get the desired compound 14B . Compound 14B : m.p. = 220-221°C, purple solid, yield = 94%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.75 (br. s, 1H), 9.15 (br. s, 1H), 8.97 – 8.93 (m, 2H), 8.34 (dd, J = 7.8, 1.8 Hz, 1H), 7.75 (dd, J = 7.8, 4.8 Hz, 1H), 7.40 – 7.32 (m, 8H), 6.89 (d, J = 8.7, 2H), 6.55 (d, J = 9.0 Hz, 2H), 6.44 (t, J = 6.3 Hz, 1H), 4.36 (d, J = 6.0 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 192.9, 179.8, 175.1, 165.8, 165.1, 154.3, 147.7, 139.8, 139.6, 134.6, 131.4, 131.1, 129.3, 128.5, 128.4, 128.3, 128.2, 127.8, 127.6, 124.8, 124.6, 123.0, 116.7, 114.3, 54.9. HRMS (ESI) calcd for C 29 H 22 O 4 N 4 Cl [M+H] + 525.1324; found 525.1325. HPLC purity = 97.7% (t r = 15.689 min.). 4.1.15. 8-Oxo-8-(((tetrahydro-2H-pyran-3-yl)oxy)amino)octanoic acid ( 38 ) A mixture of monomethyl suberate 32 (941 mg, 5.0 mmol), O -(tetrahydro-2 H -pyran-3-yl) hydroxylamine (879 mg, 7.5 mmol), EDC. HCl (1.43 g, 7.5 mmol), HOBt (811 mg, 6.0 mmol), and N-methylmorpholine (1.38 mL, 12.5 mmol) in anhydrous DMF (5 mL) was stirred at room temperature overnight. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA = 2: 1) to give compound 35 with 90% yield. Compound 35 : 1 H NMR (300 MHz, Chloroform- d ) δ H 8.63 (s, 1H), 4.92 (br s, 1H), 3.99 – 3.79 (m, 1H), 3.64 (s, 3H), 3.63 – 3.56 (m, 1H), 2.28 (t, J = 7.5 Hz, 2H), 2.09 (br s, 2H), 1.86 – 1.73 (m, 3H), 1.67 – 1.54 (m, 7H), 1.38 – 1.27 (m, 4H). To a solution of compound 35 (1.0 g, 3.48 mmol) in (15 mL) was added lithium hydroxide 1.0 M (aq) (10 mL), and the reaction mixture was stirred at heated at 50°C overnight. The solvent was removed by using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath and then extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to give compound 38 in 98% yield. The compound was directly used for further steps without purification. Compound 38 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 11.96 (s, 1H), 10.87 (s, 1H), 4.79 (br s, 1H), 3.99 – 3.83 (m, 1H), 3.57 – 3.43 (m, 1H), 2.18 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.3 Hz, 2H), 1.72 – 1.57 (m, 3H), 1.56 – 1.41 (m, 7H), 1.30 – 1.19 (m, 4H). 4.1.16. 9-Oxo-9-(((tetrahydro-2H-pyran-3-yl)oxy)amino)nonanoic acid ( 39) A mixture of monomethyl azelate 33 (1.7 g, 8.41 mmol), O -(tetrahydro-2 H -pyran-3-yl)hydroxylamine (985 mg, 8.41 mmol), EDC. HCl (2.418 g, 12.6 mmol), HOBt (1.364 mg, 10.1 mmol), and N-methylmorpholine (2.31 mL, 21.0 mmol) in anhydrous DMF (7 mL) were stirred at room temperature overnight. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA = 2: 1) to give compound 36 with 98% yield. Compound 36 : 1 H NMR (300 MHz, Chloroform- d ) δ H 8.72 (s, 1H), 4.91 (s, 1H), 4.04 – 3.86 (m, 1H), 3.63 (s, 3H), 3.62 – 3.52 (m, 1H), 2.28 (t, J = 7.4 Hz, 3H), 2.08 (br s, 2H), 1.82 – 1.74 (m, 3H), 1.64 – 1.53 (m, 7H), 1.33 – 1.26 (m, 6H). To a solution of compound 36 (2.483 g, 8.24 mmol) in MeOH (10 mL) was added lithium hydroxide 1.0 M (aq) (20 mL), and the reaction mixture was stirred at heated at 50°C overnight. The solvent was removed by using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath and then extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to give compound 39 in 97% yield. The compound was directly used for further steps without purification. Compound 39 : 1 H NMR (300 MHz, Chloroform- d ) δ H 4.93 (br s, 1H), 4.05 – 3.84 (m, 1H), 3.76 – 3.56 (m, 1H), 2.34 (t, J = 7.4 Hz, 2H), 2.11 (br s, 2H), 1.87 – 1.74 (m, 3H), 1.68 – 1.57 (m, 7H), 1.36 – 1.30 (m, 6H). 4.1.17. 10-Oxo-10-(((tetrahydro-2H-pyran-3-yl)oxy)amino)decanoic acid ( 40 ) A mixture of monomethyl sebacate 34 (850 mg, 3.93 mmol), O-(tetrahydro-2 H -pyran-3-yl)hydroxylamine (460 mg, 3.93 mmol), EDC. HCl (1.13 g, 5.89 mmol), HOBt (638 mg, 4.72 mmol), and N-methylmorpholine (1.08 mL, 9.82 mmol) in anhydrous DMF (5 mL) was stirred at room temperature overnight. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA = 2: 1) to give compound 37 with 83% yield. Compound 37 : 1 H NMR (300 MHz, Chloroform- d ) δ H 4.91 (br s, 1H), 3.98 – 3.86 (m, 1H), 3.64 (s, 3H), 3.61 – 3.54 (m, 1H), 2.27 (t, J = 7.5 Hz, 2H), 2.08 (br s, 2H), 1.85 – 1.73 (m, 3H), 1.69 – 1.53 (m, 7H), 1.30 – 1.23 (m, 8H). To a solution of compound 37 ( 1.07 g, 3.93 mmol) in dioxane (15 mL) was added lithium hydroxide 1.0 M (aq) (7 mL), and the reaction mixture was stirred at room temperature for overnight. The solvent was removed by using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath and then extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated by using a rotary evaporator to give compound 40 in 98% yield. The compound was directly used for further steps without purification. Compound 40 : 1 H NMR (300 MHz, Chloroform- d ) δ H 4.92 (br s, 1H), 4.02 – 3.89 (m, 1H), 3.68 – 3.56 (m, 1H), 2.32 (t, J = 7.4 Hz, 2H), 2.14 (br s, 2H), 1.85 – 1.74 (m, 3H), 1.69 – 1.58 (m, 7H), 1.33 – 1.27 (m, 8H). 4.1.18. N 1 -(4-(1,3-dioxoisoindolin-2-yl)phenyl)-N 8 -((tetrahydro-2H-pyran-3-yl)oxy)octanediamide ( 41 ) A mixture of 8-oxo-8-(((tetrahydro-2 H -pyran-3-yl)oxy)amino)octanoic acid 38 (550 mg, 2.01 mmol), 2-(4-aminophenyl)isoindoline-1,3-dione 21 (400 mg, 1.68 mmol), EDC. HCl (480 mg, 2.50 mmol), HOBt (271 mg, 2.0 mmol), and N-methylmorpholine (460 µL, 4.17 mmol) in anhydrous DMF (5 mL) was stirred at room temperature overnight. The mixture was quenched with water, then added EA. The white-formed precipitate was filtered by vacuum filtration to obtain the crude product. The crude product was washed with n -hexane and EA to give compound 41 with 83% yield. Compound 41 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.89 (s, 1H), 10.05 (s, 1H), 8.07 – 7.83 (m, 4H), 7.83 – 7.68 (m, 2H), 7.47 – 7.31 (m, 2H), 4.81 (br s, 1H), 4.03 – 3.84 (m, 1H), 3.59 – 3.46 (m, 1H), 2.33 (t, J = 7.4 Hz, 2H), 2.00 (d, J = 7.3 Hz, 1H), 1.76 – 1.45 (m, 10H), 1.41 – 1.21 (m, 4H). 4.1.19. N 1 -(4-(1,3-Dioxoisoindolin-2-yl)phenyl)-N 9 -((tetrahydro-2H-pyran-3-yl)oxy)nonanediamide ( 42 ) The preparation of compound 42 is similar to that of compound 41 by using 9-oxo-9-(((tetrahydro-2 H -pyran-3-yl)oxy)amino)nonanoic acid ( 39 ) (658 mg, 2.29 mmol), 2-(4-aminophenyl)isoindoline-1,3-dione ( 21 ) ( 694 mg, 2.91 mmol), EDC. HCl (659 mg, 3.44 mmol), HOBt (372 mg, 2.75 mmol), and N-methylmorpholine (629 µL, 5.72 mmol) in anhydrous DMF (5 mL). Compound 42 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.88 (s, 1H), 10.05 (s, 1H), 7.98 – 7.89 (m, 4H), 7.75 – 7.67 (m, 2H), 7.40 – 7.30 (m, 2H), 4.79 (br s, 1H), 3.97 – 3.86 (m, 1H), 3.57 – 3.43 (m, 1H), 2.33 (t, J = 7.3 Hz, 2H), 1.98 (t, J = 7.5 Hz, 2H), 1.70 – 1.41 (m, 10H), 1.38 – 1.25 (m, 6H). 4.1.20. N 1 -(4-(1-Methylene-3-oxoisoindolin-2-yl)phenyl)-N 10 -((tetrahydro-2H-pyran-3-yl)oxy)decanediamide ( 43 ) The preparation of compound 43 is similar to that of compound 41 by using 10-oxo-10-(((tetrahydro-2 H -pyran-3-yl)oxy)amino)decanoic acid 40 (420 mg, 1.39 mmol), 2-(4-aminophenyl)isoindoline-1,3-dione 21 (365 mg, 1.53 mmol), EDC. HCl (401 mg, 2.09 mmol), HOBt (226 mg, 1.67 mmol), and N-methylmorpholine (383 µL, 3.48 mmol) in anhydrous DMF (5 mL). Compound 43 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.88 (s, 1H), 10.04 (s, 1H), 8.00 – 7.87 (m, 4H), 7.76 – 7.65 (m, 2H), 7.45 – 7.31 (m, 2H), 4.79 (br s, 1H), 4.05 – 3.79 (m, 1H), 3.61 – 3.44 (m, 1H), 2.33 (t, J = 7.4 Hz, 2H), 1.97 (t, J = 7.3 Hz, 2H), 1.73 – 1.39 (m, 10H), 1.35 – 1.23 (m, 8H). 4.1.21. N 1 -(4-Aminophenyl)-N 8 -((tetrahydro-2H-pyran-3-yl)oxy)octanediamide ( 44 ) Compound 41 (508 mg, 1.0 mmol) and ethanol (10 mL) were placed in a round-bottom flask. Then, hydrazine 64% (253 µL, 5.0 mmol) was added to the reaction mixture. The flask was degassed and flushed with an argon gas. The reaction mixture was then heated at 50-60 °C for 4-5 h. The white precipitate was observed during this reaction. After confirming the completion of the reaction by TLC, the reaction was cooled to room temperature, then the white precipitate was filtered by vacuum filtration. The filtrate was collected and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (DCM: MeOH = 4: 0.1) to give compound 44 in 70% yield. Compound 44 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.88 (s, 1H), 9.39 (s, 1H), 7.25 – 7.14 (m, 2H), 6.53 – 6.42 (m, 2H), 4.79 (s, 3H), 3.94 – 3.88 (m, 1H), 3.54 – 3.44 (m, 1H), 2.19 (t, J = 7.4 Hz, 2H), 1.97 (t, J = 7.3 Hz, 2H), 1.71 – 1.46 (m, 10H), 1.30 – 1.21 (m, 4H). 4.1.22. N 1 -(4-Aminophenyl)-N 9 -((tetrahydro-2H-pyran-3-yl)oxy)nonanediamide ( 45 ) The preparation of compound 45 is similar to that of the compound 44 by using N 1 -(4-(1,3-dioxoisoindolin-2-yl)phenyl)- N 9 -((tetrahydro-2 H -pyran-3-yl)oxy)nonanediamide 42 (451 mg, 0.89 mmol) and hydrazine 64% (269 μL, 5.33 mmol) in ethanol (10 mL). Purification was accomplished by silica column chromatography (DCM: MeOH = 3: 0.1) to give compound 45 in 91% yield. Compound 45 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.88 (s, 1H), 9.39 (s, 1H), 7.29 – 7.15 (m, 2H), 6.57 – 6.39 (m, 2H), 4.79 (s, 3H), 3.91 (s, 1H), 3.58 – 3.44 (m, 1H), 2.19 (t, J = 7.4 Hz, 2H), 1.97 (t, J = 7.2 Hz, 2H), 1.69 – 1.45 (m, 10H), 1.32 – 1.19 (m, 6H). 4.1.23. N 1 -(4-Aminophenyl)-N 10 -((tetrahydro-2H-pyran-3-yl)oxy)decanediamide ( 46 ) The preparation of compound 46 is similar to that of the compound 44 by using N 1 -(4-(1-methylene-3-oxoisoindolin-2-yl)phenyl)- N 10 -((tetrahydro-2 H -pyran-3-yl)oxy)decanediamide 43 (470 mg, 0.9 mmol), hydrazine 64% (270 μL, 5.4 mmol) in ethanol (10 mL). Purification was accomplished by silica column chromatography (DCM: MeOH = 5: 0.1) to give compound 46 in 97% yield. Compound 46 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.88 (s, 1H), 9.39 (s, 1H), 7.34 – 7.10 (m, 2H), 6.57 – 6.39 (m, 2H), 4.79 (s, 3H), 3.99 – 3.80 (m, 1H), 3.55 – 3.40 (m, 1H), 2.19 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.2 Hz, 2H), 1.79 – 1.44 (m, 10H), 1.32 – 1.20 (m, 8H). 4.1.24. N 1 -(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N 8 -((tetrahydro-2H-pyran-2-yl)oxy)octanediamide ( 47A ) and N 1 -(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N 8 -((tetrahydro-2H-pyran-2-yl)oxy)octanediamide ( 47B ) A mixture of 6,7-dichloroquinoline-5,8-dione (190 mg, 0.83 mmol), N 1 -(4-aminophenyl)- N 8 -((tetrahydro-2 H -pyran-3-yl)oxy)octanediamide 44 (252 mg, 0.69 mmol), and DIPEA (133 µL, 0.76 mmol) in anhydrous DCM was stirred at room temperature overnight. The reaction mixture was concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatoraphy ( n -hexane: EA: MeOH = 1: 2: 0.2) to give compounds 47A and 47B . Compound 47A (purple solid, yield = 20%): 1 H NMR (300 MHz, Methanol- d 4 ) δ H 8.90 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (dd, J = 7.9, 4.8 Hz, 1H), 7.61 – 7.51 (m, 2H), 7.19 – 7.07 (m, 2H), 4.89 (br s, 1H), 4.06 – 3.93 (m, 1H), 3.65 – 3.55 (m, 1H), 2.38 (t, J = 7.4 Hz, 2H), 2.13 (t, J = 7.3 Hz, 2H), 1.81 – 1.56 (m, 10H), 1.46 – 1.37 (m, 4H). Compound 47B (purple solid, yield = 20%): 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.89 (s, 1H), 9.90 (s, 1H), 8.98 (dd, J = 4.8, 1.5 Hz, 1H), 8.37 (dd, J = 8.1, 1.3 Hz, 1H), 7.78 (dd, J = 7.9, 4.7 Hz, 1H), 7.61 – 7.49 (m, 2H), 7.24 – 6.93 (m, 2H), 4.80 (br s, 1H), 4.08 – 3.79 (m, 1H), 3.53 – 3.49 (m, 1H), 2.29 (t, J = 7.4 Hz, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.66 – 1.48 (m, 10H), 1.32 – 1.25 (m, 4H). 4.1.25. N 1 -(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N 9 -((tetrahydro-2H-pyran-2-yl)oxy)nonanediamide ( 48A ) and N 1 -(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N 9 -((tetrahydro-2H-pyran-3-yl)oxy)nonanediamide ( 48B ) A mixture of 6,7-dichloroquinoline-5,8-dione (151 mg, 0.66 mmol), N 1 -(4-aminophenyl)- N 9 -((tetrahydro-2 H -pyran-3-yl)oxy)nonanediamide 45 (167 mg, 0.45 mmol), and DIPEA (86 µL, 0.49 mmol) in anhydrous DCM was stirred at room temperature overnight. The reaction mixture was concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA: MeOH = 1: 2: 0.2) to give compounds 48A and 48B . Compound 48A (purple solid, yield = 41%): 1 H NMR (300 MHz, Methanol- d 4 ) δ H 8.90 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (dd, J = 7.9, 4.8 Hz, 1H), 7.62 – 7.51 (m, 2H), 7.18 – 7.08 (m, 2H), 4.07 – 3.93 (m, 1H), 3.65 – 3.53 (m, 1H), 2.38 (t, J = 7.5 Hz, 2H), 2.12 (t, J = 7.3 Hz, 2H), 1.81 – 1.54 (m, 10H), 1.45 – 1.35 (m, 6H). Compound 48B (purple solid, yield 40%): 1 H NMR (300 MHz, Methanol- d 4 ) 1 H NMR (300 MHz, Methanol- d 4 ) δ H 8.93 (dd, J = 4.8, 1.7 Hz, 1H), 8.50 (dd, J = 7.9, 1.7 Hz, 1H), 7.78 (dd, J = 7.9, 4.8 Hz, 1H), 7.61 – 7.51 (m, 2H), 7.18 – 7.08 (m, 2H), 4.06 – 3.93 (m, 1H), 3.60 – 3.54 (m, 1H), 2.38 (t, J = 7.5 Hz, 2H), 2.12 (t, J = 7.4 Hz, 2H), 1.83 – 1.54 (m, 10H), 1.45 – 1.34 (m, 6H). 4.1.26. N 1 -(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N 10 -((tetrahydro-2H-pyran-2-yl)oxy)decanediamide ( 49A ) and N 1 -(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N 10 -((tetrahydro-2H-pyran-2-yl)oxy)decanediamide ( 49B ) A mixture of 6,7-dichloroquinoline-5,8-dione (228 mg, 1.0 mmol), N 1 -(4-aminophenyl)- N 10 -((tetrahydro-2 H -pyran-3-yl)oxy)decanediamide 46 (383 mg, 1.01 mmol), and DIPEA (192 µL, 1.11 mmol) in anhydrous THF was stirred at room temperature overnight. The reaction mixture was concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA: MeOH = 1: 2: 0.2) to give compounds 49A and 49B . Compound 49A (purple solid, yield = 42%): 1 H NMR (300 MHz, Methanol- d 4 ) δ H 8.90 (dd, J = 4.8, 1.7 Hz, 1H), 8.51 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (dd, J = 7.9, 4.8 Hz, 1H), 7.63 – 7.50 (m, 2H), 7.18 – 7.08 (m, 2H), 4.88 (br s, 1H), 4.05 – 3.94 (m, 1H), 3.65 – 3.53 (m, 1H), 2.38 (t, J = 7.4 Hz, 2H), 2.11 (t, J = 7.4 Hz, 2H), 1.83 – 1.56 (m, 10H), 1.37 (s, 8H). Compound 49B (purple solid, yield =40%): 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.88 (s, 1H), 9.88 (s, 1H), 9.30 (br s, 1H), 8.98 (dd, J = 4.7, 1.6 Hz, 1H), 8.37 (dd, J = 7.8, 1.1 Hz, 1H), 7.78 (dd, J = 7.8, 4.7 Hz, 1H), 7.57 – 7.48 (m, 2H), 7.11 – 7.02 (m, 2H), 4.79 (br s, 1H), 3.94 – 3.88 (m, 1H), 3.53 – 3.43 (m, 1H), 2.29 (t, J = 7.3 Hz, 2H), 1.97 (t, J = 7.2 Hz, 2H), 1.73 – 1.42 (m, 10H), 1.34 – 1.22 (m, 8H). 4.1.27. N 1 -(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N 8 -hydroxyoctanediamide (15A) compound 47A (110 mg, 0.198 mmol) was dissolved in dry THF (4 mL) and MeOH (1 mL). While stirring under an ice bath, concentrated hydrochloric acid (0.6 mL) was added to it dropwise. The reaction mixture was stirred for 2-3 h. The solvent was removed by using a rotary evaporator to obtain the crude product, then it was purified by silica column chromatography (DCM: MeOH =3: 0.3 à 3: 0.4 à 3: 0.5) to give compound 15A . Compound 15A : m.p. = 69-70 °C, purple solid, yield = 81%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.36 (s, 1H), 9.98 (s, 1H), 8.95 (dd, J = 4.7, 1.7 Hz, 1H), 8.65 (s, 1H), 8.37 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (dd, J = 7.9, 4.7 Hz, 1H), 7.57 – 7.52 (m, 2H), 7.10 – 7.05 (m, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.60 – 1.47 (m, 4H), 1.35 – 1.28 (m, 4H); 13 C NMR (151 MHz, DMSO- d 6 ) δ C 178.4, 176.0, 171.2, 169.1, 153.2, 146.6, 144.0, 136.4, 133.9, 133.6, 129.7, 129.3, 128.4, 124.7, 118.5, 112.4, 36.3, 32.2, 28.4 (2C), 25.0 (2C). HRMS (ESI) calcd for C 23 H 24 O 5 N 4 Cl [M+H] + 471.1435; found 471.1437. HPLC purity = 95.5% (t r = 12.607 min.). 4.1.28. N 1 -(4-((7-Chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N 8 -hydroxyoctanediamide ( 15B ) To a mixture N 1 -(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)- N 8 -((tetrahydro-2 H -pyran-2-yl)oxy)octanediamide 47B (320 mg, 0.58 mmol) in dry THF (3 mL) and MeOH (2 mL) was added hydrochloric acid (0.6 mL). The mixture was stirred under an ice bath for 2 h, and then allowed to stir at room temperature for 30 min. The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM and small amount of methanol to afford a precipitate. The precipitate was then washed with DCM and ether to give compound 15B . Compound 15B : m.p. = 225-226 °C, purple solid, yield = 54%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.32 (s, 1H), 9.89 (s, 1H), 9.32 (s, 1H), 8.98 (dd, J = 4.7, 1.7 Hz, 1H), 8.64 (s, 1H), 8.37 (dd, J = 7.9, 1.7 Hz, 1H), 7.78 (dd, J = 7.9, 4.7 Hz, 1H), 7.56 – 7.51 (m, 2H), 7.10 – 7.05 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.60 – 1.47 (m, 4H), 1.31 – 1.28 (m, 4H); 13 C NMR (151 MHz, DMSO- d 6 ) δ C 179.9, 175.0, 171.2, 169.1, 154.4, 147.8, 142.9, 136.5, 134.4, 133.5, 127.5, 127.2, 124.6, 118.5, 114.4, 36.3, 32.2, 28.4 (2C), 25.0 (2C). HRMS (ESI) calcd for C 23 H 24 O 5 N 4 Cl [M+H] + 471.1435; found 471.1431. HPLC purity = 95.3% (t r = 12.123 min.). 4.1.29. N 1 -(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N 9 -hydroxynonanediamide (16A) The synthesis method of 16A is similar to that of compound 15A by using N 1 -(4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)- N 9 -((tetrahydro-2 H -pyran-2-yl)oxy)nonanediamide 48A (105 mg, 0.185 mmol). The crude product was purified by silica column chromatography (DCM: MeOH =3: 0.3 à 3: 0.4 à 3: 0.5) to give compound 16A . Compound 16A : m.p. = 184-185 °C, purple solid, yield = 92%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.36 (br s, 1H), 9.99 (s, 1H), 9.24 (br s, 1H), 8.94 (dd, J = 4.7, 1.7 Hz, 1H), 8.65 (br s, 1H), 8.37 (dd, J = 7.8, 1.7 Hz, 1H), 7.83 (dd, J = 7.9, 4.7 Hz, 1H), 7.57 – 7.52 (m, 2H), 7.09 – 7.04 (m, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.60 – 1.46 (m, 4H), 1.32 – 1.25 (m, 6H); 13 C NMR (151 MHz, DMSO- d 6 ) δ C 178.4, 176.0, 171.2, 169.1, 153.2, 146.6, 144.1, 136.4, 133.9, 133.6, 129.3, 128.3, 124.7, 118.5, 112.4, 36.3, 32.2, 28.6, 28.5, 28.4, 25.1(2C). HRMS (ESI) calcd for C 24 H 26 O 5 N 4 Cl [M+H] + 485.1592; found 485.1585. HPLC purity = 97.0% (t r = 13.443 min.). 4.1.30. N 1 -(4-((7-Chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N 9 -hydroxynonanediamide ( 16B ) The synthesis method of compound 16B is similar to that of compound 15A by using N 1 -(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)- N 9 -((tetrahydro-2 H -pyran-3-yl)oxy)nonanediamide 48B (171 mg, 0.30 mmol). The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM and small amount of methanol to afford a precipitate. The precipitate was then washed with DCM and ether to give compound 16B . Compound 16B : m.p. = 222-223 °C, purple solid, yield = 42%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.31 (s, 1H), 9.88 (s, 1H), 9.32 (s, 1H), 8.98 (dd, J = 4.7, 1.7 Hz, 1H), 8.62 (br s, 1H), 8.37 (dd, J = 7.8, 1.7 Hz, 1H), 7.78 (dd, J = 7.9, 4.7 Hz, 1H), 7.56 – 7.51 (m, 2H), 7.10 – 7.05 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.60 – 1.46 (m, 4H), 1.32 – 1.28 (m, 6H); 13 C NMR (151 MHz, DMSO- d 6 ) δ C 180.0, 175.2, 171.1, 169.1, 154.4, 147.8, 142.8, 136.3, 134.4, 133.6, 127.5, 127.1, 124.7, 118.5, 114.5, 36.4, 32.2, 28.6, 28.5, 28.4, 25.1(2C). HRMS (ESI) calcd for C 24 H 26 O 5 N 4 Cl [M+H] + 485.1592; found 485.1585. HPLC purity = 95.1% (t r = 15.340 min.). 4.1.31. N 1 -(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N 10 -hydroxydecanediamide ( 17A ) The synthesis method of compound 17A is similar to that of compound 15A by using N 1 -(4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)- N 10 -((tetrahydro-2 H -pyran-2-yl)oxy)decanediamide 49A (250 mg, 0.43 mmol). The crude product was purified by silica column chromatography (DCM: MeOH = 3: 0.1 à 3: 0.2) to give compound 17A . Compound 17A : m.p. = 190-191 °C, purple solid, yield = 95%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.31 (s, 1H), 9.88 (s, 1H), 9.40 (br s, 1H), 8.94 (dd, J = 4.6, 1.6 Hz, 1H), 8.64 (s, 1H), 8.37 (dd, J = 7.9, 1.7 Hz, 1H), 7.83 (dd, J = 7.8, 4.7 Hz, 1H), 7.55 – 7.51 (m, 2H), 7.10 – 7.05 (m, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.93 (t, J = 7.3 Hz, 2H), 1.61 – 1.46 (m, 4H), 1.32 – 1.26 (m, 8H); 13 C NMR (151 MHz, DMSO- d 6 ) δ C 178.4, 176.0, 171.1, 169.1, 153.2, 146.6, 144.1, 136.3, 133.9, 133.6, 129.3, 128.3, 124.7, 118.5, 112.4, 36.4, 32.2, 28.7(2C), 28.6(2C), 25.1(2C). HRMS (ESI) calcd for C 25 H 28 O 5 N 4 Cl [M+H] + 499.1748; found 499.1748. HPLC purity = 97.5% (t r = 14.274 min.). 4.1.32. N 1 -(4-((7-Chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N 10 -hydroxydecanediamide ( 17B ) The synthesis method of compound 17B is similar to that of compound 15A by using N 1 -(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)- N 10 -((tetrahydro-2 H -pyran-2-yl)oxy)decanediamide 49B (230 mg, 0.39 mmol). The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM and a small amount of methanol to afford a precipitate. The precipitate was then washed with dichloromethane and ether to give compound 17B . Compound 17B : m.p. = 215-216 °C, purple solid, yield = 47%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.31 (s, 1H), 9.88 (s, 1H), 9.24 (br s, 1H), 8.98 (dd, J = 4.7, 1.7 Hz, 1H), 8.64 (s, 1H), 8.37 (dd, J = 7.8, 1.7 Hz, 1H), 7.78 (dd, J = 7.9, 4.7 Hz, 1H), 7.56 – 7.51 (m, 2H), 7.10 – 7.05 (m, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.93 (t, J = 7.3 Hz, 2H), 1.61 – 1.46 (m, 4H), 1.27 – 1.23 (m, 8H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 180.0, 175.2, 171.2, 169.1, 154.4, 147.8, 142.9, 136.3, 134.4, 133.7, 129.7, 127.5, 124.7, 118.5, 114.5, 36.4, 32.3, 28.7(2C), 28.6(2C), 25.1(2C). HRMS (ESI) calcd for C 25 H 28 O 5 N 4 Cl [M+H] + 499.1748; found 499.1741. HPLC purity = 95.2% (t r = 13.780 min.). 4.1.33. 4-(((4-((tert-butoxycarbonyl)amino)phenyl)amino)methyl)benzoic acid ( 51 ) A mixture of methyl 4-(bromomethyl)benzoate (750 mg, 3.27 mmol), tert-butyl (4-aminophenyl)carbamate (818 mg, 3.93 mmol), K 2 CO 3 (678 mg, 4.91 mmol) in anhydrous DMF (5 mL) was stirred at room temperature for 5 h. After confirming the completion of the reaction, the mixture was quenched with water and then extracted with ethyl acetate (EA). The organic layer was combined, dried over MgSO 4 , filtered, and concentrated using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA = 4: 1) to give compound 50 . Next, compound 50 (800 mg, 2.24 mmol) was placed in a round-bottom flask, and dissolved in methanol (10 mL). Then, 10 mL of LiOH aqueous solution (1.0 M) was added. The reaction mixture was refluxed at 60 °C for 3 h. The solvent was reduced by half by using a rotary evaporator, and then HCl 1.0 N was added to adjust the pH to 2-3. The resulting mixture was then extracted with EA, dried over MgSO 4 , filtered, and concentrated using a rotary evaporator to obtain a residue. The residue was then purified by silica column chromatography (DCM: MeOH = 4: 0.1) to give compound 51 in 54% yield. Compound 51 : 1 H NMR (300 MHz, Methanol- d 4 ) δ H 7.96 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H), 6.57 – 6.54 (m, 2H), 4.36 (s, 2H), 1.48 (s, 9H). 4.1.34. tert-butyl (4-((4-((2-Aminophenyl)carbamoyl)benzyl)amino)phenyl)carbamate ( 52 ) A mixture of 4-(((4-((tert-butoxycarbonyl)amino)phenyl)amino)methyl)benzoic acid 51 (300 mg, 0.88 mmol), o -phenylenediamine (107 mg, 1.06 mmol), EDC.HCl (203 mmol, 1.06 mmol), HOBt (178 mg, 1.32 mmol), N-methylmorpholine (242 µL, 2.2 mmol) in anhydrous DMF (4 mL) was stirred at room temperature for 5 h. The mixture was quenched with water and then extracted with EA. The organic layer was combined, dried over MgSO 4 , filtered, and concentrated using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography ( n -hexane: EA = 2: 3) to give compound 52 in 86% yield. Compound 52 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 9.58 (s, 1H), 8.77 (s, 1H), 7.92 – 7.89 (m, 2H), 7.47 – 7.44 (m, 2H), 7.17 – 7.07 (m, 3H), 6.99 – 6.94 (m, 1H), 6.78 – 6.76 (m, 1H), 6.61 – 6.56 (m, 1H), 6.49 – 6.46 (m, 2H), 6.07 (t, J = 6.2 Hz, 1H), 4.87 (s, 2H), 4.31 (d, J = 6.2 Hz, 2H), 1.43 (s, 9H). 4.1.35. N-(2-Aminophenyl)-4-(((4-aminophenyl)amino)methyl)benzamide ( 53 ) Tert-butyl (4-((4-((2-Aminophenyl)carbamoyl)benzyl)amino)phenyl)carbamate 52 (327 mg, 0.76 mmol) was dissolved in anhydrous DCM (6 mL). About 2 mL of TFA was added dropwise under an ice bath. The resulting mixture was then stirred at room temperature for 2 h. After confirming the completion of the reaction, the solvent was removed using a rotary evaporator to obtain the crude product. It was triturated by the addition of ether and hexane to give compound 53 in 90% yield. Compound 53 : 1 H NMR (300 MHz, DMSO- d 6 ) δ H 10.19 (s, 1H), 7.97 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 7.5 Hz, 1H), 7.25 – 7.15 (m, 4H), 7.04 (d, J = 8.7 Hz, 2H), 6.64 (d, J = 8.7 Hz, 2H), 4.39 (s, 2H). 4.1.36. N-(2-aminophenyl)-4-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)benzamide ( 18A ) and N-(2-aminophenyl)-4-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)benzamide ( 18B ) A mixture of N -(2-aminophenyl)-4-(((4-aminophenyl)amino)methyl)benzamide 53 (200 mg, 0.60 mmol), 6,7-dichloroquinoline-5,8-dione (100 mg, 0.44 mmol), DIPEA (300 µL, 1.72 mmol) in MeOH (5 mL) was stirred at room temperature for 7 h. The mixture was concentrated under reduced pressure to obtain the crude product. The resulting crude product was purified by silica column chromatography (DCM: MeOH = 60: 1) to obtain the 7-, 6-substituted product 18A and 18B respectively. Compound 18A : m.p = 182-183 o C, purple solid, yield = 23%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 9.60 (s, 1H), 9.26 (s, 1H), 8.92 (dd, J = 4.7, 1.7 Hz, 1H), 8.35 (dd, J = 7.9, 1.7 Hz, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.80 (dd, J = 8.1, 4.8 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 6.6 Hz, 1H), 7.00 – 6.94 (m, 1H), 6.89 (d, J = 8.7 Hz, 2H), 6.77 (dd, J = 7.8, 1.2 Hz, 1H), 6.62 – 6.52 (m, 3H), 6.44 (t, J = 6.3 Hz, 1H), 4.88 (s, 2H), 4.37 (d, J = 5.7 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 178.5, 175.5, 165.2, 153.0, 146.4 (2C), 144.0 (2C), 143.0, 133.8, 133.1, 129.5, 128.4, 127.8, 127.4, 126.9, 126.6, 126.4, 125.9, 123.4, 116.2, 116.1, 111.5, 110.4, 46.3. HRMS (ESI) calcd for C 29 H 23 O 3 N 5 Cl [M+H] + 524.1489; found 524.1483. HPLC purity = 97.3% (t r = 17.818 min.). Compound 18B : m.p = 185-186 o C, purple solid, yield = 60%; 1 H NMR (300 MHz, DMSO- d 6 ) δ H 9.61 (s, 1H), 9.16 (s, 1H), 8.96 (dd, J = 4.8, 1.7 Hz, 1H), 8.35 (dd, J = 7.8, 1.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.75 (dd, J = 8.1, 4.8 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.16 (d, J = 7.8 Hz, 1H), 6.99 – 6.94 (m, 1H), 6.91 – 6.87 (m, 2H), 6.77 (dd, J = 8.1, 1.2 Hz, 1H), 6.62 – 6.52 (m, 3H), 6.44 (t, J = 6.1 Hz, 1H), 4.88 (s, 2H), 4.37 (d, J = 6.0 Hz, 2H). 13 C NMR (151 MHz, DMSO- d 6 ) δ C 180.1, 174.8, 165.2, 154.4, 148.0, 146.4, 144.0, 143.1, 142.8, 134.3, 133.1, 127.8, 127.4, 127.3, 126.9, 126.6, 126.4, 125.9, 123.4, 116.3, 116.1, 112.5, 111.5, 46.3. HRMS (ESI) calcd for C 29 H 23 O 3 N 5 Cl [M+H] + 524.1489; found 524.1476. HPLC purity = 98.2% (t r = 17.259 min.). 4.2 Biology 4.2.1 Cell culture Triple-negative breast cancer MDA-MB-231 and Pancreatic ductal adenocarcinoma PANC-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (12800-017; Gibco). Triple-negative breast cancer cells MDA-MB-436 were cultured in Dulbecco’s Modified Eagle Medium F12 (DMEM F12) medium (41300-039; Gibco). Human epidermal growth factor receptor-2 negative breast adenocarcinoma MCF-7 and gliobastoma U87MG cells were cultured in alpha modified Eagle's minimum essential medium (α-MEM) (11900-024; Gibco). Human leukemia cell lines KG-1, HL-60 and colorectal adenocarcinoma DLD-1 were cultured in Roswell Park Memorial Institute (RPMI) 1640 (31800-022; Gibco). Human leukemia cell lines MV4-11 and K-562 were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (12200-036; Gibco), containing 1.5g/L and 3g/L sodium bicarbonate respectively. All cell lines were cultured in medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine (PSG; 10378-016; Gibco) at 37 ◦C in a humidified atmosphere. 4.2.2. Chemicals and antibodies Colchicine, Paclitaxel (Taxol) and Nocodazole were purchased from Sigma (C9754), Cytoskeleton, Inc. (TXD01) and Sigma (31430-18-9) respectively. Primary antibodies: anti-phospho-Ser/Thr-Pro-mitotic protein monoclonal 2 (MPM2) (05–368; Millipore); anti-cyclin B1 pS126 (ab55184; Abcam); anti-Cyclin-dependent kinase 1 pY15 (CDK1 pY15) (GTX1281550; GeneTex); anti-histone H3 pS10 (06–570; Millipore); anti-Histone 3 (ab1791; Abcam); anti-acetyl-Histone 3 lysine 9 (07e352; Millipore); anti-Histone 4 (ab10158; Abcam); antiacetyl-Histone 4 lysine 5/8/12/16 (06e866; Millipore); anti-SMC3 (A300-060A; Bethyl); anti-acetyl-SMC3 lysine 105/106 (MABE1073; Millipore); anti-a-tubulin (T5168; Sigma); anti-acetyl-a-Tubulin lysine 40 (T7451; Sigma); anti- KRAB domain-associated protein 1 (KAP1) pS824 (ab70369; Abcam); anti-Checkpoint kinase 2 (CHK2) pT68 (2661; Cell Signaling Technology); anti-CHK1 pS345 (2348; Cell Signaling Technology); anti-CHK1 (G-4) (sc-8408; Santa Cruz); anti-Replication Protein A2 (RPA2) pS33 (A300-246A; Bethyl); anti-RPA2 pS4/S8 (A300- 245A; Bethyl); anti-γH2AX (05–636; Millipore); anti-H2AX (2595; Cell Signaling Technology); anti-actin (MAB1501; Millipore); anti-Caspase-3 (NB100-56708; Novus); anti-Caspase-8 (9746; Cell Signaling); anti-Caspase-9 (9502; Cell Signaling); anti-induced myeloid leukemia cell differentiation protein (Mcl-1)(ab32087, Abcam); anti-B-cell leukemia/lymphoma 2(Bcl-2)(3498; Cell Signaling). Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse (115-035-003; Jackson ImmunoResearch Labs); anti-rabbit (111-035- 003; Jackson ImmunoResearch Labs) antibodies. 4.2.3. MTT assay Cells were seeded in 96-well plates and treated with compounds for 48h, followed by incubation with 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, M2128) at 37 ◦C for 2-3 h. Formazan crystals were then dissolved in dimethyl sulfoxide (DMSO). The number of surviving attached cells was determined by measuring the absorbance at 562 nm (PerkinElmer VICTOR3 TM multilabel plate reader). 4.2.4. MTS assay Cells were seeded in 96-well plates and treated with compounds for 48h. Surviving cells were determined by incubation with 0.2 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Abcam, ab223881), and absorbance at 490 nm was measured by using PerkinElmer VICTOR3TM multilabel plate reader. 4.2.5. Thymidine synchronization Cells were incubated with 4 mM of thymidine (Sigma, T1895) for 20–24 h to enrich cells at the early-S phase. Cells were then washed with fresh medium twice and incubated with culture medium for recovery from thymidine block. 4.2.6. Flow cytometry Cells were collected and fixed with 70% ice-cold ethanol. After washing with cold phosphate-buffered saline containing 1% FBS, cells were incubated with 0.05 mg/ml of propidium iodide (PI; Sigma, P4170) and 0.25 mg/ml ribonuclease A (RNase A; Sigma, R6513) at 37 ◦C for 30 min. DNA content was analyzed by using Becton Dickinson FACSCanto TM II Flow Cytometer, and cell-cycle profiles were plotted with the FlowJo software. 4.2.7. Western blotting analysis Cells were lysed in Laemmli sample buffer (60 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, and 10% glycerol). Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membranes. After blocking with 5% skim milk, proteins were probed with specific primary antibodies and then HRP-conjugated species-specific secondary antibodies, followed by signal detection using enhanced chemiluminescence substrates (Bio-Rad). Images were obtained by using the iBright FL-1500 system. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Bidyadhar Sethy, and Iin Narwanti performed the research, designed, synthesized, analyzed data, and wrote the manuscript. Richa Upadhyay performed the mechanistic experiments, analyzed bio-results and wrote the manuscript. Zih-Yao Yu analyzed and technical supported biological experiments. Sung-Bau Lee designed, supervised, and helped to revise the manuscript and gave final approval for the version to be published. Jing-Ping Liou designed, supervised the study, analyzed and revised the manuscript. All the authors read and approved the final manuscript and declares no competing financial interest. Acknowledgment. This research was supported by the National Science and Technology Council of Taiwan (grant no. 111-2113-M-038-004, 112-2113-M-038-001, 112-2320-B-038-004). This work was supported of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. (grant no. DP2-TMU-113-C-03). References Ferlay J, Colombet M, Soerjomataram I, Parkin DM, Pineros M, Znaor A, Bray F (2021) Cancer statistics for the year 2020: An overview. Int J Cancer Zhao S, Zuo WJ, Shao ZM, Jiang YZ (2020) Molecular subtypes and precision treatment of triple-negative breast cancer. Ann Transl Med 8:499 Verza FA, Das U, Fachin AL, Dimmock JR, Marins M (2020) Roles of Histone Deacetylases and Inhibitors in Anticancer Therapy. 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Supplementary Files Supplimentary20240630.pdf floatimage1.png Graphical Abstract: floatimage4.png Scheme 1; Synthesis of 13A, 13B, 14A, and 14B (Reagents and conditions): (a) Phenylboronic acid, Pd(PPh 3 ) 4 , K 2 CO 3 , DMF, 110 o C, Inert (Ar), 16 h, (b) NBS, AICN, CCl 4 , 90 o C, Inert (Ar), 5 h, (c) p -Phenylenediamine, DMF, 140 o C Reflux, o.n., (d) methyl 4-(bromomethyl)benzoate or P1-M2, K 2 CO 3 , DMF, rt, 1-2 days., (e) LiOH (1M), MeOH, 55 o C, o.n., (f) NH 2 -OTHP, EDC.HCl, HOBt, NMM, DMF, rt, 3 h, (g) NH 2 -NH 2 .H 2 O, Ethanol, Ar-gas, 60-65 o C, Reflux, 3-4 h, (h) 6,7-dichloroquinoline-5,8-dione, THF, DIPEA, rt, 1 h, (i) 10% HCl in MeOH, 0 o C - rt, 3 h. floatimage5.png Scheme 2; Synthesis of 15A, 15B, 16A, 16B, 17A and 17B (Reagents and conditions): (a) NH 2 -OTHP, EDC.HCl, HOBt, NMM, DMF, rt, o.n. (b) LiOH (1M), MeOH, rt, o.n., (c) 38, or 39, or 40 respectively, EDC.HCl, HOBt, NMM, DMF, rt, 5-6 h (d) NH 2 -NH 2 .H 2 O, Ethanol, Ar-gas, 60-65 o C, Reflux, 3-4 h, (e) 6,7-dichloroquinoline-5,8-dione, DCM, DIPEA, rt, 1 h, (f) 10% HCl in MeOH, 0 o C - rt, 3 h. floatimage6.png Scheme 3; Synthesis of 18A and 18B (Reagents and conditions): (a) Methyl 4-(bromomethyl) benzoate, K 2 CO 3 , DMF, rt, 5 h (b) LiOH 1.0 M in MeOH, 60 o C, 3 h, (c) o -Phenylenediamine, EDC.HCl, HOBt, NMM, DMF, rt, o.n., (d) 20% TFA in DCM, 0 o C-rt, 2h, (e) 6,7-dichloroquinoline-5,8-dione, DIPEA, MeOH, rt, 7h. Cite Share Download PDF Status: Published Journal Publication published 12 Oct, 2024 Read the published version in Apoptosis → Version 1 posted Editorial decision: Revision requested 22 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviews received at journal 16 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviews received at journal 06 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers agreed at journal 06 Jul, 2024 Reviewers invited by journal 06 Jul, 2024 Editor assigned by journal 30 Jun, 2024 Submission checks completed at journal 30 Jun, 2024 First submitted to journal 30 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4661784","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329993401,"identity":"60a96187-12ad-4302-9807-782566c4dcc4","order_by":0,"name":"Bidyadhar Sethy","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bidyadhar","middleName":"","lastName":"Sethy","suffix":""},{"id":329993402,"identity":"56858338-e5a8-4869-8ff9-d1ff46f95bc5","order_by":1,"name":"Richa Upadhyay","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Richa","middleName":"","lastName":"Upadhyay","suffix":""},{"id":329993405,"identity":"3e765177-bea1-47a6-850f-efe9b910e3ad","order_by":2,"name":"Iin Narwanti","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Iin","middleName":"","lastName":"Narwanti","suffix":""},{"id":329993406,"identity":"1ea7ec88-49ad-41da-a65a-e717300d43c1","order_by":3,"name":"Zih-Yao Yu","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zih-Yao","middleName":"","lastName":"Yu","suffix":""},{"id":329993408,"identity":"7da083b3-61a2-4ae9-b4cd-5b2f2100562a","order_by":4,"name":"Sung-Bau Lee","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sung-Bau","middleName":"","lastName":"Lee","suffix":""},{"id":329993410,"identity":"6a0b0a89-98db-4aa1-9b8b-4a086fcdadde","order_by":5,"name":"Jing-Ping Liou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBACxgbmxgcQZgLRWhibDQ7AtBwgUlObBGlamGc3tlV/zDnMwM+eY8D8sY0YO+YcbLtxcNthBsmeNwYMB4nSMiMRosXgRg5QyzYitRSAtNiTpIUBbIsE0VrmHGyWOLstnUfizLOCA2f/EaHFcHbzwQ+V26zl+NuTNz6oOEOMlhkQmgdEHCBCAwODvARRykbBKBgFo2BEAwAIXj4wMUru3gAAAABJRU5ErkJggg==","orcid":"","institution":"Taipei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jing-Ping","middleName":"","lastName":"Liou","suffix":""}],"badges":[],"createdAt":"2024-06-30 08:06:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4661784/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4661784/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10495-024-02023-7","type":"published","date":"2024-10-12T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60981726,"identity":"d9e0d886-3a82-4033-836b-f34890ea81f0","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55848,"visible":true,"origin":"","legend":"\u003cp\u003eHydroxyamide and benzamide containing HDAC inhibitors (\u003cstrong\u003e1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) for the treatment of various cancers in clinical and the chemical structure of quinone-based CDC25 inhibitors (\u003cstrong\u003e6\u003c/strong\u003e-\u003cstrong\u003e12\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/1ea444f0fb6cc58f9200bf2c.png"},{"id":60981727,"identity":"98332ce0-e934-4889-be75-ba8e4a297f22","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70637,"visible":true,"origin":"","legend":"\u003cp\u003eDesign rationale of dual CDC25-HDAC inhibitors based on the chemical structure of quinoline-5, 8-dione as CDC25 inhibitor (NSC663284/\u003cstrong\u003e6a\u003c/strong\u003e) and hydroxamic acid/benzamide pharmacophores as HDAC inhibitors.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/3d7d0cb8947474b708f87cc2.png"},{"id":60982535,"identity":"62a0ca73-15e0-4bc6-a0e2-efe0362e79fe","added_by":"auto","created_at":"2024-07-24 09:24:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity screening of all the synthetics in various breast cancer cell lines and non-malignant breast epithelial cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) MDA-MB-231, (B) MDA-MB-436, (C) MCF-7 and (D) M10 cells were treated with 2 µM of indicated compounds for 48 h. The percentages of surviving cells were determined by the MTT assay. Results are shown as means with SDs from at least two independent experiments (\u003cem\u003en\u003c/em\u003e≥6).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/9187399c57d13b7fe4cb5a62.png"},{"id":60981733,"identity":"1166d867-9804-4524-9227-df639649c973","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDAC-inhibitory activity of 18A in cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were treated with various concentrations of indicated compounds for 6 h. Cell lysates were harvested and subjected to Western blot analysis for acetylation levels of different HDAC substrates. Representative results from one of two biological repeats are shown.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/18788b2fd4510d42585f3728.png"},{"id":60981729,"identity":"164f23a0-cf49-463d-a90e-5ed8c4fe66ce","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e18A treatment prevents dephosphorylation of CDK1 and activation of mitotic signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThymidine-synchronized MDA-MB-231 cells were co-treated with 100 ng/ml of nocodazole and various concentrations of indicated compounds and subjected to cell cycle and mitotic signaling analyses.Experimental design (upper), representative cell cycle profiles (lower left) and western blot results (lower right) from one of two biological replicates are shown. Asyn: Asynchronous; Cpd: Compound; Noco: Nocodazole.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/f073ca7ff239f4342751ae8b.png"},{"id":60982536,"identity":"8d93aa1f-6566-4dd4-9d83-4e08bb57d1c6","added_by":"auto","created_at":"2024-07-24 09:24:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e18A treatment impairs cell cycle progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThymidine-synchronized MDA-MB-231 cells were treated with various concentrations of indicated compounds and subjected to cell cycle analyses by flow cytometry. Experimental design (upper) and representative result from one of two biological replicates (lower) is shown. Asyn: Asynchronous; Cpd: Compound.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/ea393fa5692f3773423bae78.png"},{"id":60982538,"identity":"3e0cd8b6-577b-42ca-bee7-b239d8bb5bad","added_by":"auto","created_at":"2024-07-24 09:24:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":71724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e18A\u003c/strong\u003e \u003cstrong\u003etriggers DNA damage\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were treated with 2 and 5 μM of indicated compounds for 6 h and subjected to western blot analysis of DNA damage markers. Representative results from one of two biological repeats are shown.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/a8f0d8e42d5ad5249668e2ac.png"},{"id":60981730,"identity":"f55b3128-9b08-4341-a474-d5bc4617d2f3","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":68543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e18A treatment triggers apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eMDA-MB-231 cells were treated with various concentrations of indicated compounds for 48 h. Cell cycle profiles were analyzed by flow cytometry. \u003cstrong\u003e(B) \u003c/strong\u003eMDA-MB-231 cells were treated with 2 µM of indicated compounds for 72 h. Cell lysates were harvested and subjected to Western blot analysis for indicated apoptotic proteins. Results from one of the two biological repeats are shown.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/6c7ac64b278862fd739c743a.png"},{"id":66597122,"identity":"0e5adb77-4075-428e-ba19-bdb18baea6ac","added_by":"auto","created_at":"2024-10-14 16:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1946627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/0ea578f1-8929-4ef9-a288-fdbe6e5d0fbd.pdf"},{"id":60984017,"identity":"321958e3-2d50-479e-b8d2-5da597a16274","added_by":"auto","created_at":"2024-07-24 09:40:43","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3532238,"visible":true,"origin":"","legend":"","description":"","filename":"Supplimentary20240630.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/87b3e2b8f5f7859497330212.pdf"},{"id":60981737,"identity":"2078ba23-4422-4a62-9f78-3680181374a2","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":404706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract:\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/f41476e9a675faa496cad766.png"},{"id":60981738,"identity":"0fd1eb9f-f4df-4247-8340-8d8fb021d312","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":210842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1;\u003c/strong\u003e Synthesis of \u003cstrong\u003e13A\u003c/strong\u003e,\u003cstrong\u003e 13B\u003c/strong\u003e,\u003cstrong\u003e 14A\u003c/strong\u003e, and\u003cstrong\u003e 14B\u003c/strong\u003e (Reagents and conditions): (a) Phenylboronic acid, Pd(PPh\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003e, K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, DMF, 110 \u003csup\u003eo\u003c/sup\u003eC, Inert (Ar), 16 h, (b) NBS, AICN, CCl\u003csub\u003e4\u003c/sub\u003e, 90 \u003csup\u003eo\u003c/sup\u003eC, Inert (Ar), 5 h, (c) \u003cem\u003ep\u003c/em\u003e-Phenylenediamine, DMF, 140 \u003csup\u003eo\u003c/sup\u003eC Reflux, o.n., (d) methyl 4-(bromomethyl)benzoate or P1-M2, K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, DMF, rt, 1-2 days., (e) LiOH (1M), MeOH, 55 \u003csup\u003eo\u003c/sup\u003eC, o.n., (f) NH\u003csub\u003e2\u003c/sub\u003e-OTHP, EDC.HCl, HOBt, NMM, DMF, rt, 3 h, (g) NH\u003csub\u003e2\u003c/sub\u003e-NH\u003csub\u003e2\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO, Ethanol, Ar-gas, 60-65 \u003csup\u003eo\u003c/sup\u003eC, Reflux, 3-4 h, (h) 6,7-dichloroquinoline-5,8-dione, THF, DIPEA, rt, 1 h, (i) 10% HCl in MeOH, 0 \u003csup\u003eo\u003c/sup\u003eC - rt, 3 h.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/32ed88c389179b9d00d518a6.png"},{"id":60981736,"identity":"0af53014-9a98-4e51-b476-c86dba895940","added_by":"auto","created_at":"2024-07-24 09:16:43","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":191276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2;\u003c/strong\u003e Synthesis of \u003cstrong\u003e15A\u003c/strong\u003e, \u003cstrong\u003e15B\u003c/strong\u003e, \u003cstrong\u003e16A\u003c/strong\u003e, \u003cstrong\u003e16B\u003c/strong\u003e, \u003cstrong\u003e17A\u003c/strong\u003e and \u003cstrong\u003e17B\u003c/strong\u003e (Reagents and conditions): (a) NH\u003csub\u003e2\u003c/sub\u003e-OTHP, EDC.HCl, HOBt, NMM, DMF, rt, o.n. (b) LiOH (1M), MeOH, rt, o.n., (c) \u003cstrong\u003e38\u003c/strong\u003e, or \u003cstrong\u003e39\u003c/strong\u003e, or \u003cstrong\u003e40\u003c/strong\u003e respectively, EDC.HCl, HOBt, NMM, DMF, rt, 5-6 h (d) NH\u003csub\u003e2\u003c/sub\u003e-NH\u003csub\u003e2\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO, Ethanol, Ar-gas, 60-65 \u003csup\u003eo\u003c/sup\u003eC, Reflux, 3-4 h, (e) 6,7-dichloroquinoline-5,8-dione, DCM, DIPEA, rt, 1 h, (f) 10% HCl in MeOH, 0 \u003csup\u003eo\u003c/sup\u003eC - rt, 3 h.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/5d1b75db0389b2e050f58092.png"},{"id":60983484,"identity":"ef344231-928d-4f38-b0b0-dcc1eff8ccdc","added_by":"auto","created_at":"2024-07-24 09:32:43","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":135773,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;\u003cstrong\u003eScheme 3\u003c/strong\u003e; Synthesis of \u003cstrong\u003e18A\u003c/strong\u003e and \u003cstrong\u003e18B\u003c/strong\u003e (Reagents and conditions): (a) Methyl 4-(bromomethyl) benzoate, K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, DMF, rt, 5 h (b) LiOH 1.0 M in MeOH, 60 \u003csup\u003eo\u003c/sup\u003eC, 3 h, (c) \u003cem\u003eo\u003c/em\u003e-Phenylenediamine, EDC.HCl, HOBt, NMM, DMF, rt, o.n., (d) 20% TFA in DCM, 0 \u003csup\u003eo\u003c/sup\u003eC-rt, 2h, (e) 6,7-dichloroquinoline-5,8-dione, DIPEA, MeOH, rt, 7h.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4661784/v1/3dd53bc0bcfa23ddb87ded7e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer remains a significant global health burden, with nearly 20\u0026nbsp;million new cases reported in 2022, leading to approximately 9.7\u0026nbsp;million cancer-related deaths worldwide. Among all cancer types, breast cancer stands out as one of the most prevalent, with 2.3\u0026nbsp;million new cases in 2022, making it the second most commonly diagnosed cancer globally. Within the spectrum of breast cancer subtypes, triple-negative breast cancer (TNBC) accounts for approximately 10\u0026ndash;15% of all breast cancer cases. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. TNBC is characterized by the absence of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2) expressions, presenting as a particularly aggressive form of the disease with a poorer prognosis compared to other subtypes. Based on gene expression profiling, TNBCs are subdivided into seven classes: immunomodulatory (IM), luminal androgen receptor (LAR), mesenchymal-like (M), mesenchymal stem-like (MSL), basal-like 1 (BL1), basal-like 2 (BL2), and unstable (UNS). Research efforts are ongoing to develop targeted therapies tailored to address the unique challenges posed by TNBC and improve treatment outcomes for individuals affected by this subtype. Conventional chemotherapy and radiation are the primary systemic therapeutic strategies, and no other FDA-approved targeted therapies are yet available for TNBC. Due to these features and the lack of targeted therapies, numerous attempts have been made to discover potential molecular targets for TNBC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistone deacetylases (HDACs) and histone acetyltransferases (HATs) are crucial enzymes in the epigenetic regulation of gene expression through post-translational modifications of histone proteins. HDACs remove acetyl groups from histones, leading to chromatin condensation and gene repression, whereas HATs add acetyl groups, resulting in chromatin relaxation and gene activation. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Besides histones, HDACs deacetylate non-histone substrates, including polyamines, tubulin, structural maintenance of chromosomes (SMC) proteins, p53, and Hsp90. This deacetylation activity of HDACs affects gene expression, chromatin structure, and cellular processes such as differentiation and proliferation, thereby significantly regulating multiple biological functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In humans, HDACs comprise a family of 18 genes which are divided into four classes: The \"classical\" HDACs with a zinc-dependent active site are Class I (HDAC1, HDAC2, HDAC3, and HDAC8), Class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), and Class IV (HDAC11) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Class I HDAC1-3 mainly deacetylates histones H3 and H4 but also targets non-histone proteins [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is generally agreed that up-regulation of HDACs in malignant tumor cells leads to the deregulation of homeostasis, differentiation, cell apoptosis, and inactivation of tumor suppressors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Since epigenetic changes are highly implicated in tumorigenesis, inhibitors of histone deacetylases could represent a promising therapeutic strategy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Four HDAC inhibitors have been approved by the U.S. Food and Drug Administration (FDA): vorinostat (SAHA; \u003cb\u003e1\u003c/b\u003e), romidepsin (FK228), belinostat (PXD-101; \u003cb\u003e2\u003c/b\u003e), and panobinostat (LBH589; \u003cb\u003e3\u003c/b\u003e) for the treatment of hematological malignancies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The China FDA has also authorized the chidamide (CS055; \u003cb\u003e4\u003c/b\u003e) to treat peripheral T-cell lymphoma [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Entinostat (MS-275; \u003cb\u003e5\u003c/b\u003e) is a widely used benzamide group inhibitor targeting HDAC1-3. It has been demonstrated to be helpful in treating hematological malignancies, while its inhibitory effectiveness against solid tumors is modest [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Many clinical researches have shown that HDAC inhibitors exploit synergistic effects with other anticancer agents. However, multicomponent drugs raised the risks involved in complex pharmacokinetic properties, unpredictable drug-drug interactions, and different drug solubilities. Developing HDAC-based multitargeting drugs may thus provide an effective and practical strategy to overcome the limitations of single and multicomponent agents [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne notable factor contributing to the development of malignant tumors is the disruption of cell cycle control. Disordered cell cycle, unlimited proliferation, and mutations in tumor suppressors and oncogenes are common characteristics all malignancies share, including TNBCs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Cell division cycle 25 (CDC25) phosphatases are central to the regulation of the cell cycle, in which CDC25 dephosphorylates the threonine and tyrosine residues of cyclin-dependent kinase (CDK)/cyclin complexes, promoting CDK activation and, thus, cell cycle progression. CDC25 dysregulation has been implicated in various malignancies, highlighting their potential as therapeutic targets in cancer treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The CDC25 family comprises three isoforms - CDC25A, CDC25B, and CDC25C. These isoforms function on distinct CDK/cyclin complexes at various cell cycle phases. CDC25A mainly activates the CDK2/cyclin E and CDK2/cyclin A complexes during the G1/S transition, while Cdc25B and C primarily regulate the CDK1/cyclinB1 activity to promote the progression of mitosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Targeting CDC25 has shown promise in preclinical studies, with inhibitors demonstrating the ability to induce cell cycle arrest and promote apoptosis in cancer cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also shows the typical quinone moieties carrying CDC25 inhibitors. The naphthoquinone derivative, UPD-140 \u003cb\u003e(6)\u003c/b\u003e, inhibits CDC25 activity in HeLa cells, with an IC\u003csub\u003e50\u003c/sub\u003e value of 1.2 \u0026micro;M [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compound Cpd5 \u003cb\u003e(7)\u003c/b\u003e is a thioether derivative that inhibits CDC25 and can impede G\u003csub\u003e1\u003c/sub\u003e and G\u003csub\u003e2\u003c/sub\u003e/M progression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Through high-throughput screening (HTS), a sulfur-containing vitamin K analog, NSC95397 \u003cb\u003e(8)\u003c/b\u003e, was found to be a potent irreversible CDC25 inhibitor but also targeting MKK7 and other kinases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, compounds NSC668394 \u003cb\u003e(9)\u003c/b\u003e and NSC663284/6a \u003cb\u003e(11)\u003c/b\u003e were shown to be sub-micromolar IC\u003csub\u003e50\u003c/sub\u003e (0.64 and 0.25 \u0026micro;M, respectively) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. NSC663284/6a \u003cb\u003e(11)\u003c/b\u003e is one of the most potent Cdc25 inhibitors reported, has been shown to block cell cycle progression of tumor cells at both G1 and G2/M, and causes hyperphosphorylation of Cdk1/Cdc2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, Jing research group discovered a quinoline-5, 8-dione derivative M2N12 \u003cb\u003e(10)\u003c/b\u003e, which potently inhibits CDC25C with IC\u003csub\u003e50\u003c/sub\u003e of 0.09 \u0026micro;M and also exhibited remarkable suppression on KB-VIN cell growth (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.81 \u0026micro;M) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, Narwanti group conducted a study that identified 6-ragiomeric quinoline-5, 8-dione containing compound 6b \u003cb\u003e(12)\u003c/b\u003e as a potent CDC25 inhibitor that induces genome instability and kills cancer cells through apoptotic pathways [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, advancements have made it possible to target multiple pathways implicated in the progression of TNBC therapy with combinatorial techniques that combine numerous targeted therapies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A dual (multiple)-target therapies show high efficacy and can reduce the therapeutic doses and side effects compared to single-target drug therapy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A promising approach to drug design is the incorporation of HDAC inhibitory pharmacophores (hydroxamate or \u003cem\u003eo\u003c/em\u003e-phenylenediamine) into the structure of the inhibitor for another target to create HDAC-based dual or multiple inhibitors [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among these molecules, the HDAC/CDK dual inhibitors have shown promising enzymatic inhibitory and anti-proliferative activities [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As mentioned earlier, various cyclin-dependent kinases (CDKs) activated upon removal of phosphate residues by CDC25 resulted in cell cycle progression. According to recent research, CDK inhibition makes cancer cells more susceptible to HDAC inhibitors, which may cause cancer cell death [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, we hypothesized that combining CDC25 and HDAC inhibitors may statistically limit the growth of cancer cells. In the present study, potential CDC25-HDAC dual inhibitors have been designed and synthesized by adopting the principle of molecular hybridization of the potential CDC25-HDAC pharmacophore, and the structure-activity relationships (SARs) were explored. All the hybrid molecules were screened against various cancer cell lines, and the CDC25-HDAC inhibitory activities of the selected compounds in cells were further examined. The effects on the cell cycle progression, DNA damage, and apoptosis were biologically investigated herein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Chemistry","content":"\u003cp\u003e\u003cstrong\u003e2.1. Study rationale design\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo guide the design of potent anti-TNBC agents with a thorough understanding of related proteins, such as CDC25 and HDAC in TNBC progression, we applied the principle of molecular hybridization combining structural features of CDC25 and HDAC inhibitors to create dual-target molecules. According to the classical HDAC inhibitors model, HDAC inhibitors should have a zinc-binding group (ZBG), an aromatic or aliphatic linker, and a capping group to effectively inhibit the hydrolysis of acetyl-lysine residues on histone tails and non-histone proteins \u003cstrong\u003e(Figure 2)\u003c/strong\u003e. The ZBG is a hydrophilic domain, including hydroxamic acid and 2-aminobenzamide groups interacting with the active zinc cation (Zn2+) at the HDAC site. Furthermore, as discussed earlier, the cap group is a crucial unit susceptible to chemical modifications to enhance selectivity and assisted pharmacokinetic properties. Here, we introduced quinoline-5, 8-dione moiety as the cap, which may cause of improving the effectivity by interacting with the aromatic amino acid residues located either close to the outer domain of the active site or at the external surface of the enzyme, as well as by facilitating its ability to form redox biological reaction and DNA damage. Therefore, based on the principle of molecular hybridization, by considering the structural analogous of the core scaffold of CDC25 inhibitors (quinoline-5, 8-dione moiety),\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003ewe designed and synthesized multiple inhibitors of CDC25-HDAC combined via different aliphatic or aromatic linkers with hydroxamic acid or benzamide moieties (Figure 2), which may provide reasonable pharmaceutical properties\u0026nbsp;\u003c/strong\u003e\u003c/strong\u003eand expecting improved efficacy \u003cstrong\u003efor TNBC treatment.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Synthesis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis of all the target compounds was depicted in \u003cstrong\u003eScheme 1-3\u003c/strong\u003e. \u003cstrong\u003eScheme 1\u003c/strong\u003e illustrates the synthesis of the \u003cem\u003eN\u003c/em\u003e-benzyl linker-based hydroxamic acid containing target compounds \u003cstrong\u003e13A\u003c/strong\u003e, \u003cstrong\u003e13B\u003c/strong\u003e, \u003cstrong\u003e14A\u003c/strong\u003e, and \u003cstrong\u003e14B\u003c/strong\u003e. The synthesis starts with a condensation reaction using a commercially available starting material, phthalic anhydride and para (\u003cem\u003ep\u003c/em\u003e)-phenylenediamine, to produce \u003cstrong\u003e21\u003c/strong\u003e. A substitution reaction was generated for\u003cstrong\u003e\u0026nbsp;22\u003c/strong\u003e and \u003cstrong\u003e23\u003c/strong\u003e using the intermediates methyl 4-(bromomethyl) benzoate and \u003cstrong\u003e20,\u0026nbsp;\u003c/strong\u003erespectively. Before this, we synthesized the intermediate \u003cstrong\u003e20\u003c/strong\u003e by utilizing methyl 2-bromo-4-methylbenzoate, which was subjected to Suzuki-coupling with phenylboronic acid to produce \u003cstrong\u003e19\u003c/strong\u003e, and then radical bromination of \u003cstrong\u003e19\u003c/strong\u003e gave the intermediate \u003cstrong\u003e20\u003c/strong\u003e. Subsequently, lithium hydroxide assisted ester hydrolysis of \u003cstrong\u003e22\u003c/strong\u003e and \u003cstrong\u003e23\u003c/strong\u003e made the carboxylic acid product \u003cstrong\u003e24\u003c/strong\u003e and \u003cstrong\u003e25\u003c/strong\u003e, respectively, which upon amidation using \u003cem\u003eO\u003c/em\u003e-(tetrahydro-2H-pyran-2-yl) hydroxylamine (NH\u003csub\u003e2\u003c/sub\u003e-OTHP) to yield \u003cstrong\u003e26\u003c/strong\u003e and \u003cstrong\u003e27\u003c/strong\u003e. A hydrazine hydrate-based phthalimide deprotection reaction has also generated free aniline intermediates (\u003cstrong\u003e28\u003c/strong\u003e and \u003cstrong\u003e29\u003c/strong\u003e). These intermediates are utilized to react with 6, 7-dichloroquinoline-5, 8-dione to get the regioisomers of respected anilines such as \u003cstrong\u003e30A/31A\u003c/strong\u003e (7-regiomer) and \u003cstrong\u003e30B/31B\u003c/strong\u003e (6-regiomer), which are upon OTHP deprotection furnished the target hydroxamic acid products \u003cstrong\u003e13A\u003c/strong\u003e,\u003cstrong\u003e14A\u003c/strong\u003e, \u003cstrong\u003e13B\u003c/strong\u003e, and \u003cstrong\u003e14B\u003c/strong\u003e, respectively.\u003c/p\u003e\n\u003cp\u003eFurthermore, we introduced various long-chain linker-based hydroxamic acids (\u003cstrong\u003e15A\u003c/strong\u003e, \u003cstrong\u003e15B\u003c/strong\u003e, \u003cstrong\u003e16A\u003c/strong\u003e, \u003cstrong\u003e16B\u003c/strong\u003e, \u003cstrong\u003e17A\u003c/strong\u003e, and \u003cstrong\u003e17B\u003c/strong\u003e) by adopting a general synthetic protocol for synthesizing the target compounds presented in \u003cstrong\u003eScheme 2\u003c/strong\u003e. Firstly, we followed a similar methodology such as EDC/HOBt assisted amidation of alkoxyalkanoic acids (\u003cstrong\u003e32\u003c/strong\u003e, \u003cstrong\u003e33\u003c/strong\u003e, and \u003cstrong\u003e34\u003c/strong\u003e) with NH\u003csub\u003e2\u003c/sub\u003e-OTHP to generate \u003cstrong\u003e35\u003c/strong\u003e, \u003cstrong\u003e36\u003c/strong\u003e, and \u003cstrong\u003e37\u003c/strong\u003e, then lithium hydroxide-based ester hydrolysis to yield \u003cstrong\u003e38\u003c/strong\u003e, \u003cstrong\u003e39\u003c/strong\u003e, and \u003cstrong\u003e40\u003c/strong\u003e, and those are upon amidation with \u003cstrong\u003e21\u003c/strong\u003e afforded the intermediates \u003cstrong\u003e41\u003c/strong\u003e, \u003cstrong\u003e42\u003c/strong\u003e, and \u003cstrong\u003e43\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erespectively. Next, the aniline intermediates (\u003cstrong\u003e44\u003c/strong\u003e, \u003cstrong\u003e45\u003c/strong\u003e, and \u003cstrong\u003e46\u003c/strong\u003e) were generated by the deprotection of respective phthalimides using hydrazine hydrate. Further, the free aniline intermediates were subjected to a substitution reaction with 6,7-dichloroquinoline-5,8-dione to produce their respective 7-, 6-regiomer intermediates (\u003cstrong\u003e47A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;47B\u003c/strong\u003e, \u003cstrong\u003e48A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;48B\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;49A\u003c/strong\u003e, and \u003cstrong\u003e49B\u003c/strong\u003e), which upon acid catalyzed OTHP deprotection to afford the desired products \u003cstrong\u003e15A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;15B\u003c/strong\u003e, \u003cstrong\u003e16A\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;16B\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;17A\u003c/strong\u003e, and \u003cstrong\u003e17B\u003c/strong\u003e respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, according to our design strategy, we further introduced \u003cem\u003eo\u003c/em\u003e-phenylenediamine as the zinc-binding motif, and the synthetic route to the target compounds is depicted in \u003cstrong\u003eScheme 3\u003c/strong\u003e. The intermediate \u003cstrong\u003e50\u003c/strong\u003e was synthesized using the commercially available starting material tert-butyl (4-aminophenyl) carbamate and methyl 4-(bromomethyl)benzoate. The intermediate \u003cstrong\u003e50\u003c/strong\u003e was subjected to ester hydrolysis to obtain \u003cstrong\u003e51\u003c/strong\u003e, which proceeded to an amidation reaction using\u003cem\u003e\u0026nbsp;o\u003c/em\u003e-phenylenediamine catalyzed by EDC/HOBt to generate \u003cstrong\u003e52\u003c/strong\u003e. Subsequently, \u003cstrong\u003e53\u0026nbsp;\u003c/strong\u003ewas obtained upon BOC-deprotection of \u003cstrong\u003e52\u003c/strong\u003e, which was further subjected to react with 6, 7-dichloroquinoline-5, 8-dione to give their respective 7-, 6-regiomer target compounds \u003cstrong\u003e18A\u003c/strong\u003e and \u003cstrong\u003e18B\u003c/strong\u003e respectively. All the new compounds were characterized and determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and the purity was confirmed \u0026gt;95% using high-pressure liquid chromatography (HPLC) and the respective figures were attached in the supplementary file.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate potential of compounds \u003cstrong\u003e13A-18A\u003c/strong\u003e and \u003cstrong\u003e13B-18B\u003c/strong\u003e for cancer treatment, we initially tested their cytotoxicity on six different cell lines derived from solid tumors. These included triple-negative breast cancer (MDA-MB-231, MDA-MB-436), HER2-negative breast cancer (MCF-7), pancreatic adenocarcinoma (PANC-1), glioblastoma (U87MG), and colorectal cancer (CRC; DLD-1). Cells were treated with the compounds at 2 \u0026micro;M for 48 hours, and their viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In these assays, we also included compounds \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003e6b\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e[\u003c/strong\u003e24\u003cstrong\u003e]\u003c/strong\u003e, MS-275, and SAHA as reference controls for comparison.\u003c/p\u003e\n\u003cp\u003eThe results depicted in Figure 3 and S1 reveal intriguing details about the cytotoxicity of all the synthesized compounds. Notably, among the compounds tested, \u003cstrong\u003e18A\u003c/strong\u003e showed a significant reduction in cell viability of DLD-1 cells, comparable to the effect of \u003cstrong\u003e6b\u003c/strong\u003e (Figure S1A), the compound identified as a potent CDC25 inhibitor specifically targeting CRC cells in our previous study [24]. Additionally, \u003cstrong\u003e18A\u003c/strong\u003e exhibited superior cytotoxic activity against triple-negative breast cancer cells (MDA-MB-231 and MDA-MB-436) compared to HER-2 negative breast cancer cells (MCF-7) (Figure 3A-C). Furthermore, we observed lower cytotoxicity of \u003cstrong\u003e18A\u003c/strong\u003e in U87MG and PANC-1 cells (Figure S1B-C). In contrast to \u003cstrong\u003e18A\u003c/strong\u003e, the remaining compounds in the series displayed relatively weak cytotoxic effects across all tested cell lines. Most importantly, we extended our investigation to assess the effect of \u003cstrong\u003e18A\u003c/strong\u003e on a non-malignant breast epithelial cell line and found that \u003cstrong\u003e18A\u003c/strong\u003e exhibit minimal cytotoxicity to the M10 cells (Figure 3D). These findings underscore the potential selectivity of \u003cstrong\u003e18A\u003c/strong\u003e for TNBC cells over non-TNBC and normal breast cells.\u003c/p\u003e\n\u003cp\u003eWe further conducted a study on the impact of synthesized compounds on leukemia cell lines, including HL-60 (acute promyelocytic leukemia), K-562 (chronic myelogenous leukemia), MV4-11 (biphenotypic B-myelomonocytic leukemia) and KG-1 (acute myelogenous leukemia). Cells were treated with the compounds at 0.4\u0026micro;M, 1\u0026micro;M, 0.3\u0026micro;M and 0.5\u0026micro;M for 48 hours. We exploited the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) assay for evaluation. Our analysis revealed that \u003cstrong\u003e18A\u003c/strong\u003e was particularly effective against HL60 and K562, but it did not show differential toxicity in KG-1 and MV4-11 compared with reference compounds (Figure S1D-G).\u003c/p\u003e\n\u003cp\u003eOur results showed that subtle structural modifications within this series significantly impact cytotoxicity. The cytotoxicity results in MDA-MB-436 cells revealed that the compounds bearing N-benzyl (\u003cstrong\u003e13A\u003c/strong\u003e) and N-phenyl benzoyl (\u003cstrong\u003e14A\u003c/strong\u003e) hydroxamic acid motif with quinoline-5, 8-dione conjugated via aromatic linkers possess better antiproliferative effects than its regioisomers \u003cstrong\u003e13B\u003c/strong\u003e and \u003cstrong\u003e14B\u003c/strong\u003e (Figure 3B). Further modification of the hybrid molecules by employing the aliphatic chain linker (\u003cstrong\u003e15A\u003c/strong\u003e-\u003cstrong\u003e17A\u003c/strong\u003e and \u003cstrong\u003e15B\u003c/strong\u003e-\u003cstrong\u003e17B\u003c/strong\u003e) weaker cytotoxic effects in all the selected solid tumor and leukemia cell lines (Figure S1). It is worth noting that the benzamide derivatives such as compound \u003cstrong\u003e18A\u003c/strong\u003e, linking via N-benzyl aromatic to the quinoline-5, 8-dione scaffold, demonstrated the best antiproliferative effects against TNBCs (MDA-MB-231 and MDA-MB-436) among all synthesized compounds as well as the reference HDAC (\u003cstrong\u003e1\u003c/strong\u003e, and \u003cstrong\u003e4\u003c/strong\u003e) and CDC25 (\u003cstrong\u003e11\u003c/strong\u003e, and \u003cstrong\u003e12\u003c/strong\u003e) inhibitors (Figure 3). These results suggest that TNBCs are more sensitive to treatment with the hybrid molecule \u003cstrong\u003e18A\u003c/strong\u003e as a first-in-class CDC25-HDAC dual inhibitor, compared to other solid tumor and leukemia cells, and warrant further investigation into its mechanism of action.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. HDAC inhibitory activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that \u003cstrong\u003e18A\u0026nbsp;\u003c/strong\u003ewas the most toxic of the synthesized compounds, we determined its inhibitory activity towards the potential target, HDACs. Our results indicated that treatment with \u003cstrong\u003e18A\u003c/strong\u003e dose-dependently increased the acetylation levels of histone H3/H4 (Figure 4), which are the canonical substrates of the class I HDACs. The increased level was similar to that observed in the group treated with the reference compound MS-275 (Figure 4). In contrast, the treatment did not significantly change SMC3 and alpha-tubulin acetylation, indicating that \u003cstrong\u003e18A\u003c/strong\u003e may not target HDAC8 and HDAC6. These findings strongly suggest that \u003cstrong\u003e18A\u003c/strong\u003e is a class I HDAC inhibitor possessing comparable activity and specificity as MS-275 in cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. HDAC isoform inhibition assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next conducted a detailed analysis of the selective inhibitory properties of \u003cstrong\u003e18A\u003c/strong\u003e against the HDAC 1, 2, and 3 isoforms in vitro. The compelling results outlined in Table 1 demonstrate that \u003cstrong\u003e18A\u003c/strong\u003e outperformed the reference compound MS-275, exhibiting a significantly stronger inhibition of the HDAC1 isoform with IC50 values of 67.5 nM, compared to 212.5 nM for MS-275. Notably, both \u003cstrong\u003e18A\u003c/strong\u003e and MS-275 displayed comparable inhibitory activities against the HDAC2 and HDAC3 isoforms. These findings emphatically support the classification of \u003cstrong\u003e18A\u003c/strong\u003e as a potent class I HDAC inhibitor.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Inhibitory activities against HDAC1-3 of MS-275 and 18A.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.903225806451616%\" rowspan=\"2\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"67.09677419354838%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e18A\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS-275\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.903225806451616%\" valign=\"top\"\u003e\n \u003cp\u003eHDAC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.54838709677419%\" valign=\"top\"\u003e\n \u003cp\u003e67.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.54838709677419%\" valign=\"top\"\u003e\n \u003cp\u003e212.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.903225806451616%\" valign=\"top\"\u003e\n \u003cp\u003eHDAC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.54838709677419%\" valign=\"top\"\u003e\n \u003cp\u003e415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.54838709677419%\" valign=\"top\"\u003e\n \u003cp\u003e451.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.903225806451616%\" valign=\"top\"\u003e\n \u003cp\u003eHDAC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.54838709677419%\" valign=\"top\"\u003e\n \u003cp\u003e715.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.54838709677419%\" valign=\"top\"\u003e\n \u003cp\u003e898\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. CDC25 inhibitory activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next analyzed the impact of \u003cstrong\u003e18A\u003c/strong\u003e treatment on the activity of another potential target, CDC25s. We used a high concentration of thymidine to synchronize cells in the early-S phase (Figure 5; T0) and allowed the cells to progress into mitosis after being released from the thymidine block. To minimize the potential secondary effect from defective cell-cycle progression caused by compound treatment, we treated cells with compounds 8 hours after thymidine removal (T8), at which most cells had completed genome replication and accumulated in the G\u003csub\u003e2\u003c/sub\u003e phase\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eWe showed that the phosphorylation levels of CDK1 at\u0026nbsp;tyrosine\u0026nbsp;15 (CDK1Y15) at T0 and T8 were comparably high (Figure 5), consistent\u0026nbsp;with\u0026nbsp;the presence of cells in S and G\u003csub\u003e2\u003c/sub\u003e phases, respectively. To avoid re-phosphorylation of CDK1 after cell division, the tubulin destabilizing agent, nocodazole, was added to hinder mitosis progression. We found that CDK1Y15 phosphorylation dropped from T8 to T14, indicating that cells moved to and arrested at mitosis (Figure 5). \u003cstrong\u003e18A\u003c/strong\u003e treatment resulted in an accumulation of CDK1Y15 phosphorylation and reduction of phosphorylation levels of mitotic markers cyclin B1 at Ser126, histone H3 at Ser10, and mitotic proteins recognized by the MPM2 antibody (Figure 5). This phenomenon was not observed in cells treated with \u003cstrong\u003e6a\u003c/strong\u003e and \u003cstrong\u003e6b\u0026nbsp;\u003c/strong\u003eunder the same conditions. We also obtained similar observations from cells arrested in mitosis by Taxol and colchicine (Figure S2). These results indicate that \u003cstrong\u003e18A\u003c/strong\u003e impedes CDK1 dephosphorylation, most likely by downregulating CDC25 activity. Intriguingly, \u003cstrong\u003e18A\u003c/strong\u003e treatment at a higher concentration (5 \u0026mu;M) unexpectedly increased phosphorylation levels of proteins that could also be detected by the MPM2 antibody (Figure 5), plausibly due to activation of an unknown pathway, which remains further investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Cell cycle analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter finding that \u003cstrong\u003e18A\u003c/strong\u003e effectively inhibits CDK1 dephosphorylation (Figure 5 and S2), we decided to study its impact on cell cycle progression. We treated synchronized cells with \u003cstrong\u003e18A\u003c/strong\u003e at T2 and used flow cytometry to monitor the cell cycle progression from the S phase to the subsequent G\u003csub\u003e1\u003c/sub\u003e phase (Figure 6; T2-T16). Our results revealed that treating cells with 2 \u0026mu;M of \u003cstrong\u003e18A\u003c/strong\u003e slowed down the progression of the S phase and delayed the exit from mitosis. Treating cells with 4 \u0026mu;M of \u003cstrong\u003e18A\u003c/strong\u003e resulted in almost complete arrest of the cell cycle in the S phase (Figure 6).\u003c/p\u003e\n\u003cp\u003eTo further investigate the direct effect of \u003cstrong\u003e18A\u003c/strong\u003e on G\u003csub\u003e2\u003c/sub\u003e/M phase progression, we treated cells at T8 and found that \u003cstrong\u003e18A\u003c/strong\u003e impaired G\u003csub\u003e2\u003c/sub\u003e/M phase progression in a dose-dependent manner (Figure S3). Based on these results, we conclude that \u003cstrong\u003e18A\u003c/strong\u003e suppresses CDC25 activity, disrupting CDK activity, and ultimately interfering with cell cycle progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Genome stability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh CDK activity has been reported to be required for efficient DNA repair processes, such as homologous recombination and checkpoint responses. CDC25 inhibition, which limits CDK activation, may impede HR and thus accumulate DNA lesions [31]. Our previous study showing DNA damage induction by \u003cstrong\u003e6b\u003c/strong\u003e argued this hypothesis [24]. Moreover, HDAC inhibition alters chromatin structure, potentially compromising DNA integrity and repair mechanisms [32]\u003cstrong\u003e.\u003c/strong\u003e Therefore, we assessed the effect of \u003cstrong\u003e18A\u003c/strong\u003e on genome stability. Our data revealed that the treatment of \u003cstrong\u003e18A\u003c/strong\u003e remarkably increased phosphorylation levels of H2AX phosphorylation at S139 (\u0026gamma;H2AX) and double-stranded breaks DNA markers, including phosphorylation of KRAB-associated protein-1 (KAP-1) at S824, replication protein A2 (RPA2) at S4/S8, and CHK2 at T68, as compared to reference compounds MS-275 and \u003cstrong\u003e6a\u003c/strong\u003e (Figure 7).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe treatment of \u003cstrong\u003e18A\u003c/strong\u003e at 2 \u0026micro;M resulted in ataxia telangiectasia mutated (ATR) activation, revealed by RPA2 phosphorylation at S33 and CHK1 phosphorylation at S345. However, CHK1 phosphorylation was dramatically reduced when cells were treated with the higher concentration (5 \u0026mu;M) of \u003cstrong\u003e18A\u003c/strong\u003e (Figure 7).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThese findings suggest that \u003cstrong\u003e18A\u003c/strong\u003e is a potent DNA damage-inducing agent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e2.9. Apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe flow cytometry analysis indicated that treatment with \u003cstrong\u003e18A\u003c/strong\u003e resulted in an increased population of sub-G1 cells after 48 h, and this effect was dependent on the dosage (Figure 8A). Additionally, we examined the levels of proteins involved in apoptosis pathways and observed a significant increase in active caspases-3, -8, and -9 in cells treated with \u003cstrong\u003e18A.\u0026nbsp;\u003c/strong\u003e(Figure 8B). These changes were less pronounced in cells treated with \u003cstrong\u003e6a\u003c/strong\u003e and MS-275 (Figure 8B). Our findings suggest that \u003cstrong\u003e18A\u003c/strong\u003e is a potent dual inhibitor of CDC25 and HDAC, leading to cancer cell death through an apoptotic pathway.\u003c/p\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn this study, a series of novel CDC25-HDAC dual inhibitors were designed, synthesized, and biologically evaluated. Our biological evaluation demonstrates the potential of \u003cb\u003e18A\u003c/b\u003e, a benzamide derivative linking N-benzyl aromatic to the quinoline-5, 8-dione scaffold, exhibits potent cytotoxicity against TNBC cell lines, particularly MDA-MB-231 and MDA-MB-436 cells. Importantly, \u003cb\u003e18A\u003c/b\u003e exhibited minimal cytotoxicity towards non-malignant breast cell lines, highlighting its selectivity for cancerous cells. Our evidence suggests that simultaneously targeting CDC25 and HDACs may offer a promising therapeutic avenue for TNBC.\u003c/p\u003e \u003cp\u003eCompound \u003cb\u003e18A\u003c/b\u003e displayed excellent HDAC1 inhibitory potency, surpassing the reference control MS-275. Meanwhile, the activities against HDAC2 and HDAC3 between the two compounds were comparable. In cell-based analysis, \u003cb\u003e18A\u003c/b\u003e treatment effectively increased the acetylation of histones H3/H4, indicating that \u003cb\u003e18A\u003c/b\u003e is a selective class I HDAC inhibitor. Moreover, \u003cb\u003e18A\u003c/b\u003e sufficiently inhibited CDC25 activity, leading to the accumulation of phosphorylated CDK1. \u003cb\u003e18A\u003c/b\u003e treatment resulted in the downregulation of mitotic markers and cell cycle arrest at S and G\u003csub\u003e2\u003c/sub\u003e/M phases, suggesting that \u003cb\u003e18A\u003c/b\u003e is a cell-cycle inhibitor by disrupting the CDC25/CDK pathway. Additionally, \u003cb\u003e18A\u003c/b\u003e perturbed genome stability and induced apoptotic cell death in TNBC cells, as evidenced by high levels of DNA damage response, increased sub-G\u003csub\u003e1\u003c/sub\u003e population, and upregulation of apoptosis pathways. The observed cytotoxicity, induction of cell cycle arrest, DNA damage, and apoptosis highlight the multifaceted mechanism of action of \u003cb\u003e18A\u003c/b\u003e in TNBC cells. These findings suggest that \u003cb\u003e18A\u003c/b\u003e holds promise as a targeted therapy for TNBC, providing a new approach to addressing the lack of effective treatments for this aggressive subtype of breast cancer.\u003c/p\u003e"},{"header":"4. Experimental Section","content":"\u003cp\u003e\u003cstrong\u003e4.1. Chemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the chemicals, solvents and reagents used here without further purification, unless stated otherwise. The chemical reactions were monitored by Merk thin-layer chromatography (TLC) of silica gel 60 F254 aluminum plates. TLC plates were visualized under UV light at 254 nm using (UV visualizer name, company). For the concentration of organic solvents, Buchi rotary evaporator was used. Compounds were purified using normal phase column chromatography on silica gel (Merk Kieselgel 60, No. 9385, 230-400 mesh ASTM). \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC spectra were performed using Bruker DRX-500 spectrometer (operated at 300 MHz) and 500 MHz NMR instruments respectively, CDCl3 with tetramethylsilane used as internal standard, CD\u003csub\u003e3\u003c/sub\u003eOD and \u003cem\u003ed6\u003c/em\u003e-DMSO were also used as d-solvents. Chemical shifts (\u0026delta;) are expressed in parts per million (ppm) scale, reference to the residual solvent peaks (CDCl3: \u003csup\u003e1\u003c/sup\u003eH 7.26 and \u003csup\u003e13\u003c/sup\u003eC 77.16, CD\u003csub\u003e3\u003c/sub\u003eOD: \u003csup\u003e1\u003c/sup\u003eH 3.31 and \u003csup\u003e13\u003c/sup\u003eC 49.00, \u003cem\u003ed6\u003c/em\u003e-DMSO: \u003csup\u003e1\u003c/sup\u003eH 2.50 and \u003csup\u003e13\u003c/sup\u003eC 39.52 and \u003cem\u003ed6\u003c/em\u003e-Acetone: \u003csup\u003e1\u003c/sup\u003eH 2.05 and \u003csup\u003e13\u003c/sup\u003eC 29.84, 206.26 etc. Signal multiplicity are expressed as: br.s (broad singlet), s (singlet), d (doublet), t (triplet), q (quartet), p (pentet) and m (multiplet). To process NMR spectra, topspin software was used. Coupling constant (\u003cem\u003eJ\u003c/em\u003e) values are calculated in hertz (Hz). Melting point of all final compounds measured using\u0026nbsp;Fargo MP-2D apparatus and are uncorrected. The high-resolution mass spectra were measured by JEOL (JMS-700) electron impact (EI) mass spectrometer. All the final compounds were checked (\u0026gt;95 %) and determined by\u0026nbsp;HPLC (Agilent 1260 Infinity II, Agilent Technologies, Germany) using a Dikma (Diamonsil 5 \u0026micro;m C18x150x4.6 mm) column. HPLC analysis conditions used\u0026nbsp;ACN (mobile phase A) and water containing NH\u003csub\u003e4\u003c/sub\u003eOAc 10 mM with HCOOH 0.1% as a solvent system (mobile phase B) with a flow rate of 0.5 mL/min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.1.\u0026nbsp;\u003c/em\u003e\u003cem\u003eMethyl 5-methyl-[1,1\u0026apos;-biphenyl]-2-carboxylate (\u003cstrong\u003e19)\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe synthetic procedure of compound \u003cstrong\u003e19\u003c/strong\u003e was conducted by following a procedure by Jeffrey et al. A mixture of methyl 2-bromobenzoate (6.5 g, 28.4 mmol), phenylboronic acid (5.36 g, 43.9 mmol), Pd(PPh\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003e (1.314 g, 3.68 mmol), K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (10.64 g, 71.0 mmol) in anhydrous DMF (65 mL) was heated at 110\u0026deg;C for 24 h under argon (Ar) atmosphere. The reaction mixture was cooled to room temperature, added with water then extracted with EA. The organic layer was combined, dried over anhydrous magnesium sulfate, and then concentrated\u0026nbsp;under reduced pressure to obtain the residue. The resulting residue was purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA = 100: 1) to give compound\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e19\u0026nbsp;\u003c/strong\u003ein 97% yield. Compound \u003cstrong\u003e19\u003c/strong\u003e:\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.77 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 7.40 \u0026ndash; 7.34 (m, 3H), 7.32 \u0026ndash; 7.29 (m, 2H), 7.24 \u0026ndash; 7.19 (m, 2H), 3.63 (s, 3H), 2.42 (s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.2.\u0026nbsp;\u003c/em\u003e\u003cem\u003eMethyl 5-(bromomethyl)-[1,1\u0026apos;-biphenyl]-2-carboxylate (\u003cstrong\u003e20\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdapting a procedure by Jeffrey et al., mixture of compound \u003cstrong\u003e19\u0026nbsp;\u003c/strong\u003e(2.26 g, 10.0 mmol), N-bromosuccinimide (1.87 g, 10.5 mmol), 1,1\u0026prime;-azobis(cyclohexanecarbonitrile) (30 mg, 0.12 mmol) in carbon tetrachloride (40 mL) was refluxed at 85-90\u0026deg;C under argon for 23 h. The reaction mixture was cooled to room temperature, then extracted with EA.\u0026nbsp;The organic layer was combined, dried over magnesium sulfate, and then concentrated\u0026nbsp;under reduced pressure to obtain the residue. The resulting residue was purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: 100\u0026nbsp;\u0026agrave;\u0026nbsp;\u003cem\u003en\u003c/em\u003e-hexane: EA = 250:1\u0026nbsp;\u0026agrave;\u0026nbsp;100: 1) to give compound\u003cstrong\u003e\u0026nbsp;20\u003c/strong\u003e in 83% yield. Compound \u003cstrong\u003e20\u003c/strong\u003e:\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.81 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 7.45 \u0026ndash; 7.36 (m, 5H), 7.34 \u0026ndash; 7.26 (m, 2H), 4.51 (s, 2H), 3.64 (s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.3. 2-(4-aminophenyl)isoindoline-1,3-dione (\u003cstrong\u003e21\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of phthalic anhydride (14.812 g, 10 mmol) was dissolved in DMF (7 mL), and p-phenylenediamine (10.814 g, 10 mmol) was added in batches. The mixture was stirred under reflux at 150 \u003csup\u003eo\u003c/sup\u003eC and maintained for an overnight reaction. The reaction was monitored by TLC, after completion of the reaction, ice/water was poured into it, a green solid was precipitated, and collected after suction filtration to obtain compound \u003cstrong\u003e21\u003c/strong\u003e (65% yield). \u0026nbsp;Compound\u0026nbsp;\u003cstrong\u003e21\u003c/strong\u003e:\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.92 \u0026ndash; 7.85 (m, 4H), 7.02 \u0026ndash; 6.99 (m, 2H), 6.64 \u0026ndash; 6.61 (m, 2H), 5.32 (s, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.4.\u003c/em\u003e \u003cem\u003eMethyl 4-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)benzoate (\u003cstrong\u003e22\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of compound \u003cstrong\u003e21\u003c/strong\u003e (238 mg, 1 mmol), methyl 4-(bromomethyl)benzoate (229 mg, 1 mmol), and K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (138 mg, 1 mmol) in anhydrous DMF (3 mL) was stirred at room temperature for 24 h. After confirming the completion of the reaction using TLC, the mixture was quenched with water (5 mL). Then EA (10 mL) was added, and an orange solid was precipitated, which was filtered using suction to obtain compound \u003cstrong\u003e22\u003c/strong\u003e (67.3% yield). \u0026nbsp;Compound\u0026nbsp;\u003cstrong\u003e22\u003c/strong\u003e:\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.94 \u0026ndash; 7.84 (m, 6H), 7.51 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.05 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 6.64 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 1H), 6.62 (d, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 4.40 (d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 2H), 3.82 (s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.5. Methyl 5-(((4-(1,3-dioxoisoindolin-2-yl) phenyl) amino) methyl)-[1,1\u0026apos;-biphenyl]-2- carboxylate \u003cstrong\u003e(23)\u003c/strong\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of compound \u003cstrong\u003e21\u003c/strong\u003e (955 mg, 4 mmol), \u003cstrong\u003e20\u003c/strong\u003e (1220 mg, 4 mmol), and K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (552 mg, 4 mmol) in anhydrous DMF (10 mL) was stirred at room temperature for 24 h. TLC was monitored, and after confirming the reaction completion, the mixture was quenched with water (20 mL). Then EA (20 mL) was added, and a white solid was precipitated, which was filtered using suction to obtain compound \u003cstrong\u003e23\u003c/strong\u003e (56.4% yield). Compound \u003cstrong\u003e23\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.92 \u0026ndash; 7.84 (m, 4H), 7.72 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 7.48 \u0026ndash; 7.35 (m, 5H), 7.28 \u0026ndash; 7.7.26 (m, 2H), 7.08 \u0026ndash; 7.05 (m, 2H), 6.68 \u0026ndash; 6.65 (m, 3H), 4.41 (d, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 2H), 3.55 (s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.6. 4-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)benzoic acid (\u003cstrong\u003e24\u003c/strong\u003e) and 5-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)-[1,1\u0026apos;-biphenyl]-2-carboxylic acid (\u003cstrong\u003e25\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo a solution of compound \u003cstrong\u003e22\u0026nbsp;\u003c/strong\u003eor\u003cstrong\u003e\u0026nbsp;23 (\u003c/strong\u003e2 mmol) in MeOH (8 mL) was added lithium hydroxide 1.0 M (aq) (7 mL), and the reaction mixture was heated from room temperature to 55 \u003csup\u003eo\u003c/sup\u003eC and stirred for overnight. The solvent was reduced by half using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath to adjust pH 4-5 and then EA was added, a brown solid precipitate was collected after suction filtration to obtain the desired product \u003cstrong\u003e24\u0026nbsp;\u003c/strong\u003eor\u003cstrong\u003e\u0026nbsp;25\u0026nbsp;\u003c/strong\u003erespectively (85-90 % yield). The compound was directly used for the next steps without purification.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e24\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 12.87 (br.s, 1H), 9.91 (s, 1H), 7.90 \u0026ndash; 7.87 (m, 2H), \u0026nbsp; 7.82 \u0026ndash; 7.79 (m, 1H), 7.63 \u0026ndash; 7.45 (m, 5H), 7.36 (d, \u003cem\u003eJ\u003c/em\u003e = 9.0 Hz, 2H), 6.55 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 4.34 (s, 2H).\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e25\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.99 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.75 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.63 \u0026ndash; 7.51 (m, 3H), 7.44 \u0026ndash; 7.29 (m, 10H), 6.65 \u0026ndash; 6.62 (m, 2H), 4.41 (s, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.7. 4-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (\u003cstrong\u003e26\u003c/strong\u003e) and 5-(((4-(1,3-dioxoisoindolin-2-yl)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1\u0026apos;-biphenyl]-2-carboxamide (\u003cstrong\u003e27\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of \u003cstrong\u003e24\u0026nbsp;\u003c/strong\u003e(500 mg, 1.34 mmol, 1 equv.) or \u003cstrong\u003e25\u003c/strong\u003e (580 mg, 1.29 mmol, 1 equv.), O-(tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)hydroxylamine (1.2 equv.), EDC. HCl (1.5 equv.), HOBt (1.2 equv), and N-methylmorpholine (1.5 equv.) in anhydrous DMF (5 mL) were stirred at room temperature for 3-4 h. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH = 2: 2: 0.1) to give the desired compound \u003cstrong\u003e26\u003c/strong\u003e or \u003cstrong\u003e27\u003c/strong\u003e respectively with 55-60% yield.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e26\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 11.54 (s, 1H), 7.92 \u0026ndash; 7.84 (m, 4H), 7.71 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.45 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.05 \u0026ndash; 7.02 (m, 2H), 6.68 \u0026ndash; 6.59 (m, 3H), 4.97 (s, 1H), 4.37 (d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 2H), 4.05 \u0026ndash; 3.99 (m, 1H), 3.52 \u0026ndash; 3.48 (m, 1H), 1.70 (br.s, 3H), 1.53 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e27\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.09 (s, 1H), 7.92 \u0026ndash; 7.90 (m, 2H), 7.77 \u0026ndash; 7.74 (m, 2H), 7.65 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 7.44 \u0026ndash; 7.39 (m, 8H), 7.21 \u0026ndash; 7.18 (m, 2H), 6.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 4.75 (s, 1H), 4.44 (s, 2H), 3.63 \u0026ndash; 3.56 (m, 1H), 3.42 \u0026ndash; 3.36 (m, 1H), 1.73 (br.s, 3H), 1.49 (br.s, 3H)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.8. 4-(((4-aminophenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (\u003cstrong\u003e28\u003c/strong\u003e) and 5-(((4-aminophenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1\u0026apos;-biphenyl]-2-carboxamide (\u003cstrong\u003e29\u003c/strong\u003e)\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA solution of \u003cstrong\u003e26\u003c/strong\u003e or \u003cstrong\u003e27\u003c/strong\u003e (1 mmol, 1 equv), and hydrazine hydrate (7 equv) in ethanol (10 mL) was stirred at 65-70 \u003csup\u003eo\u003c/sup\u003eC for 2-3 h under argon (Ar) gas. The resultant precipitate was filtered off and washed several times with ethanol, and the filtrate was collected and dried under vacuum to obtain the desired product \u003cstrong\u003e28\u0026nbsp;\u003c/strong\u003eor \u003cstrong\u003e29\u003c/strong\u003e respectively with 65-70% yield.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e28\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl3) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 9.02 (br.s, 1H), 7.69 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.39 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), \u0026nbsp;6.60 \u0026ndash; 6.55 (m, 2H), 6.50 \u0026ndash; 6.45 (m, 2H), 5.06 (s, 1H), 4.30 (s, 2H), 4.03 \u0026ndash; 3.96 (m, 1H), 3.67 \u0026ndash; 3.60 (m, 1H), 3.21 (br.s, 2H), 1.86 (br.s, 3H), 1.60 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e29\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 11.78 (s, 1H), 7.79 \u0026ndash; 7.70 (m, 8H), 6.77 (s, 4H), 5.87 (s, 1H), 5.27 (s, 1H), 4.63 (d, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 2H), 4.34 \u0026ndash; 4.27 (m, 1H), 3.87 \u0026ndash; 3.84 (m, 1H), 2.02 (br.s, 3H), 1.89 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.9. 4-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (\u003cstrong\u003e30A\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;4-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (\u003cstrong\u003e30B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 6,7-dichloroquinoline-5,8-dione (228 mg, 1 mmol), \u003cstrong\u003e28\u0026nbsp;\u003c/strong\u003e(341 mg, 1 mmol), and DIPEA (175 \u0026micro;L, 1 mmol) in anhydrous DCM (9 mL) was stirred at room temperature for overnight. The reaction mixture was quenched with water (30 mL) and extracted using DCM (50 mL\u0026times;2). The organic layer was separated, dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e and evaporated in a vacuum. The residue was applied to silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH = 1: 1: 0.1) to give compounds \u003cstrong\u003e30A\u003c/strong\u003e and \u003cstrong\u003e30B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e30A\u003c/strong\u003e (purple solid, yield = 36 %): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.96 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.8 Hz, 1H), 8.78 (s, 1H), 8.50 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.80 (s, 1H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4, 2H), 7.68 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.1, 4.8 Hz, 1H), 7.44 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1, 2H), 6.95 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 6.59 \u0026ndash; 6.54 (m, 2H), 5.08 (t, \u003cem\u003eJ\u003c/em\u003e = 3.3, 1H), 4.42 (d, \u003cem\u003eJ\u003c/em\u003e = 4.2, 2H), 4.27 (s, 1H), 4.03 \u0026ndash; 3.97 (m, 1H), 3.68 \u0026ndash; 3.64 (m, 1H), \u0026nbsp;1.88 (br.s, 3H), 1.62 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e30B\u003c/strong\u003e (purple solid, yield = 31 %): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.91 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.8 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.77 \u0026ndash; 7.72 (m, 3H), 7.49 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4, 2H 6.95 \u0026ndash; 6.92 (m, 2H), 6.60 \u0026ndash; 6.57 (m, 2H), 5.05 (br. s, 1H), 4.2 (s, 2H), 4.16 \u0026ndash; 4.09 (m, 1H), 3.64 \u0026ndash; 3.60 (m, 1H), \u0026nbsp;1.91 \u0026ndash; 1.78 (m, 3H), 1.62 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.10. 5-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1\u0026apos;-biphenyl]-2-carboxamide (\u003cstrong\u003e31A\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;5-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-[1,1\u0026apos;-biphenyl]-2-carboxamide (\u003cstrong\u003e31B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the procedure for preparation of \u003cstrong\u003e30A\u003c/strong\u003e and \u003cstrong\u003e30B\u003c/strong\u003e, addition of 6,7-dichloroquinoline-5,8-dione (228 mg, 1 mmol), \u003cstrong\u003e29\u0026nbsp;\u003c/strong\u003e(417 mg, 1 mmol), and DIPEA (175 \u0026micro;L, 1 mmol) in anhydrous DCM (9 mL) was stirred at room temperature for overnight, which gave compound \u003cstrong\u003e31A\u003c/strong\u003e and \u003cstrong\u003e31B.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e31A\u003c/strong\u003e (purple solid, yield = 43%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.95 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.8 Hz, 1H), 8.49 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.70 \u0026ndash; 7.66 (m, 2H), 7.43 \u0026ndash; 7.38 (m, 7H), 6.95 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7, 2H), 6.58 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 4.75 (s, 1H), 4.43 (s, 2H), 4.31 (br. s, 1H), 3.58 \u0026ndash; 3.54 (m, 1H), 3.40 \u0026ndash; 3.36 (m, 1H), \u0026nbsp;1.73 (br.s, 3H), 1.49 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e31B\u003c/strong\u003e (purple solid, yield = 41%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 9.04 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.5, 1.8 Hz, 1H), 8.40 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.96 (s, 1H), 7.69 (d, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 1H), 7.63 \u0026ndash; 7.59 (m, 2H), 7.44 \u0026ndash; 7.39 (m, 7H), 6.95 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7, 2H), 6.58 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 4.75 (s, 1H), 4.44 (s, 2H), 4.30 (br. s, 1H), 3.61 \u0026ndash; 3.54 (m, 1H), 3.40 \u0026ndash; 3.36 (m, 1H), \u0026nbsp;1.74 (br.s, 3H), 1.50 (br.s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.11. 4-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-hydroxybenzamide (\u003cstrong\u003e13A\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo a mixture of \u003cstrong\u003e30A\u0026nbsp;\u003c/strong\u003e(190 mg, 0.36 mmol)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein dry THF (4 mL) and MeOH (2 mL) was added hydrochloric acid (0.5 mL) under an ice bath, stirred for 2 h, and then allowed to stir at room temperature for 30 min. The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM (30 mL) and a small amount of methanol (5 mL) to afford a precipitate. The precipitate was then washed with DCM and ether to give compound \u003cstrong\u003e13A\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e13A\u003c/strong\u003e: m.p. = 192-193 \u0026deg;C, purple solid, yield = 84%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 11.21 (br. s, 1H), 9.32 (s, 1H), 8.92 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.8 Hz, 1H), 8.35 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.81 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 4.8 Hz, 1H), 7.70 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.45 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 6.96 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7, 2H), 6.75 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 4.39 (s, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u0026nbsp;\u003c/sub\u003e\u003c/em\u003e192.9, 178.4, 175.9, 163.8, 153.2, 146.6, 144.0, 133.9, 132.0, 129.5, 129.3, 128.6, 128.4, 127.6, 126.9, 125.2, 124.7, 122.9, 49.3. HRMS (ESI) calcd for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 449.1011; found 449.1012. HPLC purity = 99.5% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 13.821 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.12. 4-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-hydroxybenzamide (\u003cstrong\u003e13B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the procedure for preparation of \u003cstrong\u003e13A\u003c/strong\u003e, a mixture of \u003cstrong\u003e30B\u003c/strong\u003e (160 mg, 0.30 mmol) in dry THF (4 mL) and MeOH (2 mL) followed by the addition of hydrochloric acid (0.5 mL) and stirred for 3 h under an ice-bath to get the desired compound \u003cstrong\u003e13B\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e13B\u003c/strong\u003e: m.p. = 180-181 \u0026deg;C, purple solid, yield = 90%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 11.19 (br. s, 1H), 9.23 (s, 1H), 8.96 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.8 Hz, 1H), 8.35 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.76 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 4.8 Hz, 1H), 7.71 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.45 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 6.96 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4, 2H), 6.75 (d, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 4.39 (s, 2H).\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 192.9, 179.9, 175.1, 163.8, 154.3, 147.7, 142.8, 134.6, 129.5, 128.7, 127.7, 127.5, 127.2, 126.9, 125.1, 124.6, 122.9, 54.9. HRMS (ESI) calcd for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 449.1011; found 449.1012. HPLC purity = 99.3% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 13.316 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.13. 5-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)-N-hydroxy-[1,1\u0026apos;-biphenyl]-2-carboxamide (\u003cstrong\u003e14A\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the procedure for preparation of \u003cstrong\u003e13A\u003c/strong\u003e, a mixture of \u003cstrong\u003e31A\u003c/strong\u003e (260 mg, 0.43 mmol) in dry THF (4 mL) and MeOH (2 mL) followed by the addition of hydrochloric acid (0.5 mL) and stirred for 2 h under an ice-bath to get the desired compound \u003cstrong\u003e14A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e14A\u003c/strong\u003e: m.p. = 159-160 \u0026deg;C, purple solid, yield = 92%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.75 (br. s, 1H), 9.20 (br. s, 1H), 8.92 \u0026ndash; 8.32 (m, 2H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.80 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.5 Hz, 1H), 7.39 \u0026ndash; 7.31 (m, 8H), 6.88 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7, 2H), 6.54 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 6.46 (t, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 1H), 4.35 (d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 2H).\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 178.5, 175.5, 166.1, 153.0, 146.4, 146.3, 144.1, 142.0, 140.2, 139.6, 133.8, 132.8, 129.5, 128.8, 128.4, 128.3, 128.2, 127.4, 127.2, 126.0, 125.7, 111.5, 110.4, 46.1. HRMS (ESI) calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 525.1324; found 525.1327. HPLC purity = 96.2% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 16.199 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.14. 5-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)-N-hydroxy-[1,1\u0026apos;-biphenyl]-2-carboxamide (\u003cstrong\u003e14B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the procedure for preparation of \u003cstrong\u003e13A\u003c/strong\u003e, a mixture of \u003cstrong\u003e31B\u003c/strong\u003e (250 mg, 0.41 mmol) in dry THF (4 mL) and MeOH (2 mL) followed by the addition of hydrochloric acid (0.5 mL) and stirred for 3 h under an ice-bath to get the desired compound \u003cstrong\u003e14B\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e14B\u003c/strong\u003e: m.p. = 220-221\u0026deg;C, purple solid, yield = 94%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.75 (br. s, 1H), 9.15 (br. s, 1H), 8.97 \u0026ndash; 8.93 (m, 2H), 8.34 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.75 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 4.8 Hz, 1H), 7.40 \u0026ndash; 7.32 (m, 8H), 6.89 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7, 2H), 6.55 (d, \u003cem\u003eJ\u003c/em\u003e = 9.0 Hz, 2H), 6.44 (t, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 1H), 4.36 (d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 2H).\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 192.9, 179.8, 175.1, 165.8, 165.1, 154.3, 147.7, 139.8, 139.6, 134.6, 131.4, 131.1, 129.3, 128.5, 128.4, 128.3, 128.2, 127.8, 127.6, 124.8, 124.6, 123.0, 116.7, 114.3, 54.9. HRMS (ESI) calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl [M+H]\u003csup\u003e+\u003c/sup\u003e 525.1324; found 525.1325. HPLC purity = 97.7% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 15.689 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.15. 8-Oxo-8-(((tetrahydro-2H-pyran-3-yl)oxy)amino)octanoic acid (\u003cstrong\u003e38\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of monomethyl suberate \u003cstrong\u003e32\u0026nbsp;\u003c/strong\u003e(941 mg, 5.0 mmol), \u003cem\u003eO\u003c/em\u003e-(tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl) hydroxylamine (879 mg, 7.5 mmol), EDC. HCl (1.43 g, 7.5 mmol), HOBt (811 mg, 6.0 mmol), and N-methylmorpholine (1.38 mL, 12.5 mmol) in anhydrous DMF (5 mL) was stirred at room temperature overnight. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA = 2: 1) to give compound \u003cstrong\u003e35\u003c/strong\u003e with 90% yield. Compound \u003cstrong\u003e35\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.63 (s, 1H), 4.92 (br s, 1H), 3.99 \u0026ndash; 3.79 (m, 1H), 3.64 (s, 3H), 3.63 \u0026ndash; 3.56 (m, 1H), 2.28 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 2.09 (br s, 2H), 1.86 \u0026ndash; 1.73 (m, 3H), 1.67 \u0026ndash; 1.54 (m, 7H), 1.38 \u0026ndash; 1.27 (m, 4H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo a solution of compound \u003cstrong\u003e35\u0026nbsp;\u003c/strong\u003e(1.0 g, 3.48 mmol) in (15 mL) was added lithium hydroxide 1.0 M (aq) (10 mL), and the reaction mixture was stirred at heated at 50\u0026deg;C overnight. The solvent was removed by using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath and then extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to give compound \u003cstrong\u003e38\u0026nbsp;\u003c/strong\u003ein 98% yield. The compound was directly used for further steps without purification. Compound \u003cstrong\u003e38\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 11.96 (s, 1H), 10.87 (s, 1H), 4.79 (br s, 1H), 3.99 \u0026ndash; 3.83 (m, 1H), 3.57 \u0026ndash; 3.43 (m, 1H), 2.18 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.96 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.72 \u0026ndash; 1.57 (m, 3H), 1.56 \u0026ndash; 1.41 (m, 7H), 1.30 \u0026ndash; 1.19 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.16. 9-Oxo-9-(((tetrahydro-2H-pyran-3-yl)oxy)amino)nonanoic acid (\u003cstrong\u003e39)\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of monomethyl azelate \u003cstrong\u003e33\u0026nbsp;\u003c/strong\u003e(1.7 g, 8.41 mmol), \u003cem\u003eO\u003c/em\u003e-(tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)hydroxylamine (985 mg, 8.41 mmol), EDC. HCl (2.418 g, 12.6 mmol), HOBt (1.364 mg, 10.1 mmol), and N-methylmorpholine (2.31 mL, 21.0 mmol) in anhydrous DMF (7 mL) were stirred at room temperature overnight. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA = 2: 1) to give compound \u003cstrong\u003e36\u003c/strong\u003e with 98% yield. Compound \u003cstrong\u003e36\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.72 (s, 1H), 4.91 (s, 1H), 4.04 \u0026ndash; 3.86 (m, 1H), 3.63 (s, 3H), 3.62 \u0026ndash; 3.52 (m, 1H), 2.28 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 3H), 2.08 (br s, 2H), 1.82 \u0026ndash; 1.74 (m, 3H), 1.64 \u0026ndash; 1.53 (m, 7H), 1.33 \u0026ndash; 1.26 (m, 6H).\u003c/p\u003e\n\u003cp\u003eTo a solution of compound \u003cstrong\u003e36\u0026nbsp;\u003c/strong\u003e(2.483 g, 8.24 mmol) in MeOH (10 mL) was added lithium hydroxide 1.0 M (aq) (20 mL), and the reaction mixture was stirred at heated at 50\u0026deg;C overnight. The solvent was removed by using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath and then extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to give compound \u003cstrong\u003e39\u003c/strong\u003e in 97% yield. The compound was directly used for further steps without purification. Compound \u003cstrong\u003e39\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 4.93 (br s, 1H), 4.05 \u0026ndash; 3.84 (m, 1H), 3.76 \u0026ndash; 3.56 (m, 1H), 2.34 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 2.11 (br s, 2H), 1.87 \u0026ndash; 1.74 (m, 3H), 1.68 \u0026ndash; 1.57 (m, 7H), 1.36 \u0026ndash; 1.30 (m, 6H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.17. 10-Oxo-10-(((tetrahydro-2H-pyran-3-yl)oxy)amino)decanoic acid (\u003cstrong\u003e40\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of monomethyl sebacate \u003cstrong\u003e34\u0026nbsp;\u003c/strong\u003e(850 mg, 3.93 mmol), O-(tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)hydroxylamine (460 mg, 3.93 mmol), EDC. HCl (1.13 g, 5.89 mmol), HOBt (638 mg, 4.72 mmol), and N-methylmorpholine (1.08 mL, 9.82 mmol) in anhydrous DMF (5 mL) was stirred at room temperature overnight. The reaction mixture was quenched with water and extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA = 2: 1) to give compound \u003cstrong\u003e37\u0026nbsp;\u003c/strong\u003ewith 83% yield. Compound \u003cstrong\u003e37\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 4.91 (br s, 1H), 3.98 \u0026ndash; 3.86 (m, 1H), 3.64 (s, 3H), 3.61 \u0026ndash; 3.54 (m, 1H), 2.27 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 2.08 (br s, 2H), 1.85 \u0026ndash; 1.73 (m, 3H), 1.69 \u0026ndash; 1.53 (m, 7H), 1.30 \u0026ndash; 1.23 (m, 8H).\u003c/p\u003e\n\u003cp\u003eTo a solution of compound \u003cstrong\u003e37 (\u003c/strong\u003e1.07 g, 3.93 mmol) in dioxane (15 mL) was added lithium hydroxide 1.0 M (aq) (7 mL), and the reaction mixture was stirred at room temperature for overnight. The solvent was removed by using a rotary evaporator to obtain a residue. The resulting residue was acidified using HCl 5% under an ice bath and then extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated by using a rotary evaporator to give compound \u003cstrong\u003e40\u003c/strong\u003e in 98% yield. The compound was directly used for further steps without purification. Compound \u003cstrong\u003e40\u003c/strong\u003e: \u0026nbsp; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 4.92 (br s, 1H), 4.02 \u0026ndash; 3.89 (m, 1H), 3.68 \u0026ndash; 3.56 (m, 1H), 2.32 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 2.14 (br s, 2H), 1.85 \u0026ndash; 1.74 (m, 3H), 1.69 \u0026ndash; 1.58 (m, 7H), 1.33 \u0026ndash; 1.27 (m, 8H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.18. N\u003csup\u003e1\u003c/sup\u003e-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-N\u003csup\u003e8\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)octanediamide (\u003cstrong\u003e41\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 8-oxo-8-(((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)amino)octanoic acid \u003cstrong\u003e38\u0026nbsp;\u003c/strong\u003e(550 mg, 2.01 mmol), 2-(4-aminophenyl)isoindoline-1,3-dione \u003cstrong\u003e21\u0026nbsp;\u003c/strong\u003e(400 mg, 1.68 mmol), EDC. HCl (480 mg, 2.50 mmol), HOBt (271 mg, 2.0 mmol), and N-methylmorpholine (460 \u0026micro;L, 4.17 mmol) in anhydrous DMF (5 mL) was stirred at room temperature overnight. The mixture was quenched with water, then added EA. The white-formed precipitate was filtered by vacuum filtration to obtain the crude product. The crude product was washed with \u003cem\u003en\u003c/em\u003e-hexane and EA to give compound \u003cstrong\u003e41\u003c/strong\u003e with 83% yield. Compound \u003cstrong\u003e41\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.89 (s, 1H), 10.05 (s, 1H), 8.07 \u0026ndash; 7.83 (m, 4H), 7.83 \u0026ndash; 7.68 (m, 2H), 7.47 \u0026ndash; 7.31 (m, 2H), 4.81 (br s, 1H), 4.03 \u0026ndash; 3.84 (m, 1H), 3.59 \u0026ndash; 3.46 (m, 1H), 2.33 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 2.00 (d, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 1H), 1.76 \u0026ndash; 1.45 (m, 10H), 1.41 \u0026ndash; 1.21 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.19. N\u003csup\u003e1\u003c/sup\u003e-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)-N\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)nonanediamide (\u003cstrong\u003e42\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of compound \u003cstrong\u003e42\u003c/strong\u003e is similar to that of compound \u003cstrong\u003e41\u003c/strong\u003e by using 9-oxo-9-(((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)amino)nonanoic acid (\u003cstrong\u003e39\u003c/strong\u003e)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(658 mg, 2.29 mmol), 2-(4-aminophenyl)isoindoline-1,3-dione (\u003cstrong\u003e21\u003c/strong\u003e)\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e694 mg, 2.91 mmol), EDC. HCl (659 mg, 3.44 mmol), HOBt (372 mg, 2.75 mmol), and N-methylmorpholine (629 \u0026micro;L, 5.72 mmol) in anhydrous DMF (5 mL). Compound \u003cstrong\u003e42\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.88 (s, 1H), 10.05 (s, 1H), 7.98 \u0026ndash; 7.89 (m, 4H), 7.75 \u0026ndash; 7.67 (m, 2H), 7.40 \u0026ndash; 7.30 (m, 2H), 4.79 (br s, 1H), 3.97 \u0026ndash; 3.86 (m, 1H), 3.57 \u0026ndash; 3.43 (m, 1H), 2.33 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.98 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 1.70 \u0026ndash; 1.41 (m, 10H), 1.38 \u0026ndash; 1.25 (m, 6H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.20. N\u003csup\u003e1\u003c/sup\u003e-(4-(1-Methylene-3-oxoisoindolin-2-yl)phenyl)-N\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)decanediamide (\u003cstrong\u003e43\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of compound \u003cstrong\u003e43\u003c/strong\u003e is similar to that of compound \u003cstrong\u003e41\u003c/strong\u003e by using 10-oxo-10-(((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)amino)decanoic acid \u003cstrong\u003e40\u0026nbsp;\u003c/strong\u003e(420 mg, 1.39 mmol), 2-(4-aminophenyl)isoindoline-1,3-dione \u003cstrong\u003e21\u0026nbsp;\u003c/strong\u003e(365 mg, 1.53 mmol), EDC. HCl (401 mg, 2.09 mmol), HOBt (226 mg, 1.67 mmol), and N-methylmorpholine (383 \u0026micro;L, 3.48 mmol) in anhydrous DMF (5 mL). Compound \u003cstrong\u003e43\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.88 (s, 1H), 10.04 (s, 1H), 8.00 \u0026ndash; 7.87 (m, 4H), 7.76 \u0026ndash; 7.65 (m, 2H), 7.45 \u0026ndash; 7.31 (m, 2H), 4.79 (br s, 1H), 4.05 \u0026ndash; 3.79 (m, 1H), 3.61 \u0026ndash; 3.44 (m, 1H), 2.33 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.97 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.73 \u0026ndash; 1.39 (m, 10H), 1.35 \u0026ndash; 1.23 (m, 8H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.21. N\u003csup\u003e1\u003c/sup\u003e-(4-Aminophenyl)-N\u003csup\u003e8\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)octanediamide (\u003cstrong\u003e44\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e41\u003c/strong\u003e (508 mg, 1.0 mmol) and ethanol (10 mL) were placed in a round-bottom flask. Then, hydrazine 64% (253 \u0026micro;L, 5.0 mmol) was added to the reaction mixture. The flask was degassed and flushed with an argon gas. The reaction mixture was then heated at 50-60 \u0026deg;C for 4-5 h. The white precipitate was observed during this reaction. After confirming the completion of the reaction by TLC, the reaction was cooled to room temperature, then the white precipitate was filtered by vacuum filtration. The filtrate was collected and concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (DCM: MeOH = 4: 0.1) to give compound \u003cstrong\u003e44\u003c/strong\u003e in 70% yield. Compound \u003cstrong\u003e44\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.88 (s, 1H), 9.39 (s, 1H), 7.25 \u0026ndash; 7.14 (m, 2H), 6.53 \u0026ndash; 6.42 (m, 2H), 4.79 (s, 3H), 3.94 \u0026ndash; 3.88 (m, 1H), 3.54 \u0026ndash; 3.44 (m, 1H), 2.19 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.97 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.71 \u0026ndash; 1.46 (m, 10H), 1.30 \u0026ndash; 1.21 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.22. N\u003csup\u003e1\u003c/sup\u003e-(4-Aminophenyl)-N\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)nonanediamide (\u003cstrong\u003e45\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of compound \u003cstrong\u003e45\u0026nbsp;\u003c/strong\u003eis similar to that of the compound \u003cstrong\u003e44\u003c/strong\u003e by using \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-(1,3-dioxoisoindolin-2-yl)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)nonanediamide \u003cstrong\u003e42\u003c/strong\u003e (451 mg, 0.89 mmol) and hydrazine 64% (269 \u0026mu;L, 5.33 mmol) in ethanol (10 mL). Purification was accomplished by silica column chromatography (DCM: MeOH = 3: 0.1) to give compound \u003cstrong\u003e45\u003c/strong\u003e in 91% yield. Compound \u003cstrong\u003e45\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.88 (s, 1H), 9.39 (s, 1H), 7.29 \u0026ndash; 7.15 (m, 2H), 6.57 \u0026ndash; 6.39 (m, 2H), 4.79 (s, 3H), 3.91 (s, 1H), 3.58 \u0026ndash; 3.44 (m, 1H), 2.19 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.97 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 1.69 \u0026ndash; 1.45 (m, 10H), 1.32 \u0026ndash; 1.19 (m, 6H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.23. N\u003csup\u003e1\u003c/sup\u003e-(4-Aminophenyl)-N\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)decanediamide (\u003cstrong\u003e46\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of compound \u003cstrong\u003e46\u0026nbsp;\u003c/strong\u003eis similar to that of the compound \u003cstrong\u003e44\u003c/strong\u003e by using \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-(1-methylene-3-oxoisoindolin-2-yl)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)decanediamide \u003cstrong\u003e43\u0026nbsp;\u003c/strong\u003e(470 mg, 0.9 mmol), hydrazine 64% (270 \u0026mu;L, 5.4 mmol) in ethanol (10 mL). Purification was accomplished by silica column chromatography (DCM: MeOH = 5: 0.1) to give compound \u003cstrong\u003e46\u003c/strong\u003e in 97% yield. Compound \u003cstrong\u003e46\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.88 (s, 1H), 9.39 (s, 1H), 7.34 \u0026ndash; 7.10 (m, 2H), 6.57 \u0026ndash; 6.39 (m, 2H), 4.79 (s, 3H), 3.99 \u0026ndash; 3.80 (m, 1H), 3.55 \u0026ndash; 3.40 (m, 1H), 2.19 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.96 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 1.79 \u0026ndash; 1.44 (m, 10H), 1.32 \u0026ndash; 1.20 (m, 8H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.24. N\u003csup\u003e1\u003c/sup\u003e-(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N\u003csup\u003e8\u003c/sup\u003e-((tetrahydro-2H-pyran-2-yl)oxy)octanediamide (\u003cstrong\u003e47A\u003c/strong\u003e) and N\u003csup\u003e1\u003c/sup\u003e-(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N\u003csup\u003e8\u003c/sup\u003e-((tetrahydro-2H-pyran-2-yl)oxy)octanediamide (\u003cstrong\u003e47B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 6,7-dichloroquinoline-5,8-dione (190 mg, 0.83 mmol), \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-aminophenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)octanediamide \u003cstrong\u003e44\u0026nbsp;\u003c/strong\u003e(252 mg, 0.69 mmol), and DIPEA (133 \u0026micro;L, 0.76 mmol) in anhydrous DCM was stirred at room temperature overnight. The reaction mixture was concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatoraphy (\u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH = 1: 2: 0.2) to give compounds \u003cstrong\u003e47A\u003c/strong\u003e and \u003cstrong\u003e47B\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Compound \u003cstrong\u003e47A\u003c/strong\u003e (purple solid, yield = 20%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.7 Hz, 1H), 8.51 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.83 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.8 Hz, 1H), 7.61 \u0026ndash; 7.51 (m, 2H), 7.19 \u0026ndash; 7.07 (m, 2H), 4.89 (br s, 1H), 4.06 \u0026ndash; 3.93 (m, 1H), 3.65 \u0026ndash; 3.55 (m, 1H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 2.13 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.81 \u0026ndash; 1.56 (m, 10H), 1.46 \u0026ndash; 1.37 (m, 4H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e47B\u003c/strong\u003e (purple solid, yield = 20%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.89 (s, 1H), 9.90 (s, 1H), 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.5 Hz, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.1, 1.3 Hz, 1H), 7.78 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.7 Hz, 1H), 7.61 \u0026ndash; 7.49 (m, 2H), 7.24 \u0026ndash; 6.93 (m, 2H), 4.80 (br s, 1H), 4.08 \u0026ndash; 3.79 (m, 1H), 3.53 \u0026ndash; 3.49 (m, 1H), 2.29 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.98 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.66 \u0026ndash; 1.48 (m, 10H), 1.32 \u0026ndash; 1.25 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.25. N\u003csup\u003e1\u003c/sup\u003e-(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2H-pyran-2-yl)oxy)nonanediamide (\u003cstrong\u003e48A\u003c/strong\u003e) and N\u003csup\u003e1\u003c/sup\u003e-(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2H-pyran-3-yl)oxy)nonanediamide (\u003cstrong\u003e48B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 6,7-dichloroquinoline-5,8-dione (151 mg, 0.66 mmol), \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-aminophenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)nonanediamide \u003cstrong\u003e45\u0026nbsp;\u003c/strong\u003e(167 mg, 0.45 mmol), and DIPEA (86 \u0026micro;L, 0.49 mmol) in anhydrous DCM was stirred at room temperature overnight. The reaction mixture was concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH = 1: 2: 0.2) to give compounds \u003cstrong\u003e48A\u003c/strong\u003e and \u003cstrong\u003e48B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e48A\u003c/strong\u003e (purple solid, yield = 41%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.7 Hz, 1H), 8.51 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.83 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.8 Hz, 1H), 7.62 \u0026ndash; 7.51 (m, 2H), 7.18 \u0026ndash; 7.08 (m, 2H), 4.07 \u0026ndash; 3.93 (m, 1H), 3.65 \u0026ndash; 3.53 (m, 1H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 2.12 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.81 \u0026ndash; 1.54 (m, 10H), 1.45 \u0026ndash; 1.35 (m, 6H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e48B\u0026nbsp;\u003c/strong\u003e(purple solid, yield 40%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.93 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.7 Hz, 1H), 8.50 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.78 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.8 Hz, 1H), 7.61 \u0026ndash; 7.51 (m, 2H), 7.18 \u0026ndash; 7.08 (m, 2H), 4.06 \u0026ndash; 3.93 (m, 1H), 3.60 \u0026ndash; 3.54 (m, 1H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 2.12 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.83 \u0026ndash; 1.54 (m, 10H), 1.45 \u0026ndash; 1.34 (m, 6H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.26. N\u003csup\u003e1\u003c/sup\u003e-(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2H-pyran-2-yl)oxy)decanediamide (\u003cstrong\u003e49A\u003c/strong\u003e) and N\u003csup\u003e1\u003c/sup\u003e-(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2H-pyran-2-yl)oxy)decanediamide (\u003cstrong\u003e49B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 6,7-dichloroquinoline-5,8-dione (228 mg, 1.0 mmol), \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-aminophenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)decanediamide \u003cstrong\u003e46\u0026nbsp;\u003c/strong\u003e(383 mg, 1.01 mmol), and DIPEA (192 \u0026micro;L, 1.11 mmol) in anhydrous THF was stirred at room temperature overnight. The reaction mixture was concentrated by using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH = 1: 2: 0.2) to give compounds \u003cstrong\u003e49A\u003c/strong\u003e and \u003cstrong\u003e49B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e49A\u003c/strong\u003e (purple solid, yield = 42%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.7 Hz, 1H), 8.51 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.83 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.8 Hz, 1H), 7.63 \u0026ndash; 7.50 (m, 2H), 7.18 \u0026ndash; 7.08 (m, 2H), 4.88 (br s, 1H), 4.05 \u0026ndash; 3.94 (m, 1H), 3.65 \u0026ndash; 3.53 (m, 1H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 2.11 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.83 \u0026ndash; 1.56 (m, 10H), 1.37 (s, 8H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e49B\u0026nbsp;\u003c/strong\u003e(purple solid, yield =40%): \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.88 (s, 1H), 9.88 (s, 1H), 9.30 (br s, 1H), 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.6 Hz, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.1 Hz, 1H), 7.78 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 4.7 Hz, 1H), 7.57 \u0026ndash; 7.48 (m, 2H), 7.11 \u0026ndash; 7.02 (m, 2H), 4.79 (br s, 1H), 3.94 \u0026ndash; 3.88 (m, 1H), 3.53 \u0026ndash; 3.43 (m, 1H), 2.29 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.97 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 1.73 \u0026ndash; 1.42 (m, 10H), 1.34 \u0026ndash; 1.22 (m, 8H).\u003c/p\u003e\n\u003cp\u003e4.1.27. \u003cem\u003eN\u003csup\u003e1\u003c/sup\u003e-(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N\u003csup\u003e8\u003c/sup\u003e-hydroxyoctanediamide (15A)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ecompound 47A (110 mg, 0.198 mmol) was dissolved in dry THF (4 mL) and MeOH (1 mL). While stirring under an ice bath, concentrated hydrochloric acid (0.6 mL) was added to it dropwise. The reaction mixture was stirred for 2-3 h. The solvent was removed by using a rotary evaporator to obtain the crude product, then it was purified by silica column chromatography (DCM: MeOH =3: 0.3 \u0026nbsp;\u0026agrave;\u0026nbsp;3: 0.4\u0026nbsp;\u0026agrave;\u0026nbsp;3: 0.5) to give compound \u003cstrong\u003e15A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e15A\u003c/strong\u003e: m.p. = 69-70 \u0026deg;C, purple solid, yield = 81%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.36 (s, 1H), 9.98 (s, 1H), 8.95 \u0026nbsp;(dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.7 Hz, 1H), 8.65 (s, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.83 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.7 Hz, 1H), 7.57 \u0026ndash; 7.52 (m, 2H), 7.10 \u0026ndash; 7.05 (m, 2H), 2.30 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 1.94 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.60 \u0026ndash; 1.47 (m, 4H), 1.35 \u0026ndash; 1.28 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 178.4, 176.0, 171.2, 169.1, 153.2, 146.6, 144.0, 136.4, 133.9, 133.6, 129.7, 129.3, 128.4, 124.7, 118.5, 112.4, 36.3, 32.2, 28.4 (2C), 25.0 (2C). HRMS (ESI) calcd for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 471.1435; found 471.1437. HPLC purity = 95.5% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 12.607 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.28. N\u003csup\u003e1\u003c/sup\u003e-(4-((7-Chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N\u003csup\u003e8\u003c/sup\u003e-hydroxyoctanediamide (\u003cstrong\u003e15B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo a mixture \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-2-yl)oxy)octanediamide \u003cstrong\u003e47B\u003c/strong\u003e (320 mg, 0.58 mmol) in dry THF (3 mL) and MeOH (2 mL) was added hydrochloric acid (0.6 mL). The mixture was stirred under an ice bath for 2 h, and then allowed to stir at room temperature for 30 min. The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM and small amount of methanol to afford a precipitate. The precipitate was then washed with DCM and ether to give compound \u003cstrong\u003e15B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e15B\u003c/strong\u003e: m.p. = 225-226 \u0026deg;C, purple solid, yield = 54%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.32 (s, 1H), 9.89 (s, 1H), 9.32 (s, 1H), 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.7 Hz, 1H), 8.64 (s, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.78 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.7 Hz, 1H), 7.56 \u0026ndash; 7.51 (m, 2H), 7.10 \u0026ndash; 7.05 (m, 2H), 2.29 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.94 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.60 \u0026ndash; 1.47 (m, 4H), 1.31 \u0026ndash; 1.28 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 179.9, 175.0, 171.2, 169.1, 154.4, 147.8, 142.9, 136.5, 134.4, 133.5, 127.5, 127.2, 124.6, 118.5, 114.4, 36.3, 32.2, 28.4 (2C), 25.0 (2C). HRMS (ESI) calcd for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 471.1435; found 471.1431. HPLC purity = 95.3% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 12.123 min.).\u003c/p\u003e\n\u003cp\u003e4.1.29. \u003cem\u003eN\u003csup\u003e1\u003c/sup\u003e-(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N\u003csup\u003e9\u003c/sup\u003e-hydroxynonanediamide (16A)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis method of \u003cstrong\u003e16A\u003c/strong\u003e is similar to that of compound \u003cstrong\u003e15A\u0026nbsp;\u003c/strong\u003eby using \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-2-yl)oxy)nonanediamide \u003cstrong\u003e48A\u003c/strong\u003e (105 mg, 0.185 mmol). The crude product was purified by silica column chromatography (DCM: MeOH =3: 0.3 \u0026nbsp;\u0026agrave;\u0026nbsp;3: 0.4\u0026nbsp;\u0026agrave;\u0026nbsp;3: 0.5) to give compound \u003cstrong\u003e16A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e16A\u003c/strong\u003e: m.p. = 184-185 \u0026deg;C, purple solid, yield = 92%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.36 (br s, 1H), 9.99 (s, 1H), 9.24 (br s, 1H), 8.94 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.7 Hz, 1H), 8.65 (br s, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.7 Hz, 1H), 7.83 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.7 Hz, 1H), 7.57 \u0026ndash; 7.52 (m, 2H), 7.09 \u0026ndash; 7.04 (m, 2H), 2.30 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.94 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.60 \u0026ndash; 1.46 (m, 4H), 1.32 \u0026ndash; 1.25 (m, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 178.4, 176.0, 171.2, 169.1, 153.2, 146.6, 144.1, 136.4, 133.9, 133.6, 129.3, 128.3, 124.7, 118.5, 112.4, 36.3, 32.2, 28.6, 28.5, 28.4, 25.1(2C). HRMS (ESI) calcd for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 485.1592; found 485.1585. HPLC purity = 97.0% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 13.443 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;4.1.30. N\u003csup\u003e1\u003c/sup\u003e-(4-((7-Chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N\u003csup\u003e9\u003c/sup\u003e-hydroxynonanediamide (\u003cstrong\u003e16B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis method of compound\u003cstrong\u003e\u0026nbsp;16B\u003c/strong\u003e is similar to that of compound \u003cstrong\u003e15A\u003c/strong\u003e by using \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-3-yl)oxy)nonanediamide \u003cstrong\u003e48B\u003c/strong\u003e (171 mg, 0.30 mmol). The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM and small amount of methanol to afford a precipitate. The precipitate was then washed with DCM and ether to give compound \u003cstrong\u003e16B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e16B\u003c/strong\u003e: m.p. = 222-223 \u0026deg;C, purple solid, yield = 42%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.31 (s, 1H), 9.88 (s, 1H), 9.32 (s, 1H), 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.7 Hz, 1H), 8.62 (br s, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.7 Hz, 1H), 7.78 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.7 Hz, 1H), 7.56 \u0026ndash; 7.51 (m, 2H), 7.10 \u0026ndash; 7.05 (m, 2H), 2.29 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.94 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.60 \u0026ndash; 1.46 (m, 4H), 1.32 \u0026ndash; 1.28 (m, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 180.0, 175.2, 171.1, 169.1, 154.4, 147.8, 142.8, 136.3, 134.4, 133.6, 127.5, 127.1, 124.7, 118.5, 114.5, 36.4, 32.2, 28.6, 28.5, 28.4, 25.1(2C). HRMS (ESI) calcd for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 485.1592; found 485.1585. HPLC purity = 95.1% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 15.340 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.31. N\u003csup\u003e1\u003c/sup\u003e-(4-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-N\u003csup\u003e10\u003c/sup\u003e-hydroxydecanediamide (\u003cstrong\u003e17A\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis method of compound\u003cstrong\u003e\u0026nbsp;17A\u003c/strong\u003e is similar to that of compound \u003cstrong\u003e15A\u003c/strong\u003e by using \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-2-yl)oxy)decanediamide \u003cstrong\u003e49A\u003c/strong\u003e (250 mg, 0.43 mmol). The crude product was purified by silica column chromatography (DCM: MeOH = 3: 0.1 \u0026nbsp;\u0026agrave;\u0026nbsp;3: 0.2) to give compound \u003cstrong\u003e17A\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e17A\u003c/strong\u003e: m.p. = 190-191 \u0026deg;C, purple solid, yield = 95%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.31 (s, 1H), 9.88 (s, 1H), 9.40 (br s, 1H), 8.94 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.6, 1.6 Hz, 1H), 8.64 (s, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.83 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 4.7 Hz, 1H), 7.55 \u0026ndash; 7.51 (m, 2H), 7.10 \u0026ndash; 7.05 (m, 2H), 2.29 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.93 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.61 \u0026ndash; 1.46 (m, 4H), 1.32 \u0026ndash; 1.26 (m, 8H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 178.4, 176.0, 171.1, 169.1, 153.2, 146.6, 144.1, 136.3, 133.9, 133.6, 129.3, 128.3, 124.7, 118.5, 112.4, 36.4, 32.2, 28.7(2C), 28.6(2C), 25.1(2C). HRMS (ESI) calcd for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 499.1748; found 499.1748. HPLC purity = 97.5% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 14.274 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.32. N\u003csup\u003e1\u003c/sup\u003e-(4-((7-Chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-N\u003csup\u003e10\u003c/sup\u003e-hydroxydecanediamide (\u003cstrong\u003e17B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis method of compound \u003cstrong\u003e17B\u003c/strong\u003e is similar to that of compound \u003cstrong\u003e15A\u0026nbsp;\u003c/strong\u003eby using \u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-(4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)-\u003cem\u003eN\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e-((tetrahydro-2\u003cem\u003eH\u003c/em\u003e-pyran-2-yl)oxy)decanediamide \u003cstrong\u003e49B\u003c/strong\u003e (230 mg, 0.39 mmol). The solvent was removed by using a rotary evaporator to obtain a crude product, and then dried under vacuum. The crude product was added DCM and a small amount of methanol to afford a precipitate. The precipitate was then washed with dichloromethane and ether to give compound \u003cstrong\u003e17B\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e17B\u003c/strong\u003e: m.p. = 215-216 \u0026deg;C, purple solid, yield = 47%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.31 (s, 1H), 9.88 (s, 1H), 9.24 (br s, 1H), 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.7 Hz, 1H), 8.64 (s, 1H), 8.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.7 Hz, 1H), 7.78 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 4.7 Hz, 1H), 7.56 \u0026ndash; 7.51 (m, 2H), 7.10 \u0026ndash; 7.05 (m, 2H), 2.29 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.93 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.61 \u0026ndash; 1.46 (m, 4H), 1.27 \u0026ndash; 1.23 (m, 8H). \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 180.0, 175.2, 171.2, 169.1, 154.4, 147.8, 142.9, 136.3, 134.4, 133.7, 129.7, 127.5, 124.7, 118.5, 114.5, 36.4, 32.3, 28.7(2C), 28.6(2C), 25.1(2C). HRMS (ESI) calcd for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eCl\u003csub\u003e\u0026nbsp;\u003c/sub\u003e[M+H]\u003csup\u003e+\u003c/sup\u003e 499.1748; found 499.1741. HPLC purity = 95.2% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 13.780 min.).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.33. 4-(((4-((tert-butoxycarbonyl)amino)phenyl)amino)methyl)benzoic acid (\u003cstrong\u003e51\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of methyl 4-(bromomethyl)benzoate (750 mg, 3.27 mmol), tert-butyl (4-aminophenyl)carbamate (818 mg, 3.93 mmol), K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (678 mg, 4.91 mmol) in anhydrous DMF (5 mL) was stirred at room temperature for 5 h. After confirming the completion of the reaction, the mixture was quenched with water and then extracted with ethyl acetate (EA). The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA = 4: 1) to give compound \u003cstrong\u003e50\u003c/strong\u003e. Next, compound \u003cstrong\u003e50\u003c/strong\u003e (800 mg, 2.24 mmol) was placed in a round-bottom flask, and dissolved in methanol (10 mL). Then, 10 mL of LiOH aqueous solution (1.0 M) was added. The reaction mixture was refluxed at 60 \u0026deg;C for 3 h. The solvent was reduced by half by using a rotary evaporator, and then HCl 1.0 N was added to adjust the pH to 2-3. The resulting mixture was then extracted with EA, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated using a rotary evaporator to obtain a residue. The residue was then purified by silica column chromatography (DCM: MeOH = 4: 0.1) to give compound \u003cstrong\u003e51\u0026nbsp;\u003c/strong\u003ein 54% yield.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e51\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 7.96 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.45 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.07 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 6.57 \u0026ndash; 6.54 (m, 2H), 4.36 (s, 2H), 1.48 (s, 9H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.34. tert-butyl (4-((4-((2-Aminophenyl)carbamoyl)benzyl)amino)phenyl)carbamate\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003e\u003cem\u003e52\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e)\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of 4-(((4-((tert-butoxycarbonyl)amino)phenyl)amino)methyl)benzoic acid\u003cstrong\u003e\u0026nbsp;51\u003c/strong\u003e (300 mg, 0.88 mmol), \u003cem\u003eo\u003c/em\u003e-phenylenediamine (107 mg, 1.06 mmol), EDC.HCl (203 mmol, 1.06 mmol), HOBt (178 mg, 1.32 mmol), N-methylmorpholine (242 \u0026micro;L, 2.2 mmol) in anhydrous DMF (4 mL) was stirred at room temperature for 5 h. The mixture was quenched with water and then extracted with EA. The organic layer was combined, dried over MgSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated using a rotary evaporator to obtain a residue. The resulting residue was then purified by silica column chromatography (\u003cem\u003en\u003c/em\u003e-hexane: EA = 2: 3) to give compound \u003cstrong\u003e52\u0026nbsp;\u003c/strong\u003ein 86% yield. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e52\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 9.58 (s, 1H), 8.77 (s, 1H), 7.92 \u0026ndash; 7.89 (m, 2H), 7.47 \u0026ndash; 7.44 (m, 2H), 7.17 \u0026ndash; 7.07 (m, 3H), 6.99 \u0026ndash; 6.94 (m, 1H), 6.78 \u0026ndash; 6.76 (m, 1H), 6.61 \u0026ndash; 6.56 (m, 1H), 6.49 \u0026ndash; 6.46 (m, 2H), 6.07 (t, \u003cem\u003eJ\u003c/em\u003e = 6.2 Hz, 1H), 4.87 (s, 2H), 4.31 (d, \u003cem\u003eJ\u003c/em\u003e = 6.2 Hz, 2H), 1.43 (s, 9H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.35. N-(2-Aminophenyl)-4-(((4-aminophenyl)amino)methyl)benzamide (\u003cstrong\u003e53\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTert-butyl (4-((4-((2-Aminophenyl)carbamoyl)benzyl)amino)phenyl)carbamate \u003cstrong\u003e52\u003c/strong\u003e (327 mg, 0.76 mmol) was dissolved in anhydrous DCM (6 mL). About 2 mL of TFA was added dropwise under an ice bath. The resulting mixture was then stirred at room temperature for 2 h. After confirming the completion of the reaction, the solvent was removed using a rotary evaporator to obtain the crude product. It was triturated by the addition of ether and hexane to give compound \u003cstrong\u003e53\u0026nbsp;\u003c/strong\u003ein 90% yield.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e53\u003c/strong\u003e: \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 10.19 (s, 1H), 7.97 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.50 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.38 (d, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 1H), 7.25 \u0026ndash; 7.15 (m, 4H), 7.04 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 6.64 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 4.39 (s, 2H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.1.36. N-(2-aminophenyl)-4-(((4-((6-chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)phenyl)amino)methyl)benzamide (\u003cstrong\u003e18A\u003c/strong\u003e)\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;N-(2-aminophenyl)-4-(((4-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)phenyl)amino)methyl)benzamide (\u003cstrong\u003e18B\u003c/strong\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA mixture of \u003cem\u003eN\u003c/em\u003e-(2-aminophenyl)-4-(((4-aminophenyl)amino)methyl)benzamide \u003cstrong\u003e53\u0026nbsp;\u003c/strong\u003e(200 mg, 0.60 mmol), 6,7-dichloroquinoline-5,8-dione (100 mg, 0.44 mmol), DIPEA (300 \u0026micro;L, 1.72 mmol) in MeOH (5 mL) was stirred at room temperature for 7 h. The mixture was concentrated under reduced pressure to obtain the crude product. The resulting crude product was purified by silica column chromatography (DCM: MeOH = 60: 1) to obtain the 7-, 6-substituted product \u003cstrong\u003e18A\u003c/strong\u003e and \u003cstrong\u003e18B\u0026nbsp;\u003c/strong\u003erespectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompound\u003cstrong\u003e\u0026nbsp;18A\u003c/strong\u003e: m.p = 182-183 \u003csup\u003eo\u003c/sup\u003eC, purple solid, yield = 23%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 9.60 (s, 1H), 9.26 (s, 1H), 8.92 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.7, 1.7 Hz, 1H), 8.35 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.94 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.80 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.1, 4.8 Hz, 1H), 7.49 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e = 6.6 Hz, 1H), 7.00 \u0026ndash; 6.94 (m, 1H), 6.89 (d, \u003cem\u003eJ\u003c/em\u003e = 8.7 Hz, 2H), 6.77 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.2 Hz, 1H), 6.62 \u0026ndash; 6.52 (m, 3H), 6.44 (t, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 1H), 4.88 (s, 2H), 4.37 (d, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u0026nbsp;\u003c/sub\u003e\u003c/em\u003e178.5, 175.5, 165.2, 153.0, 146.4 (2C), 144.0 (2C), 143.0, 133.8, 133.1, 129.5, 128.4, 127.8, 127.4, 126.9, 126.6, 126.4, 125.9, 123.4, 116.2, 116.1, 111.5, 110.4, 46.3. HRMS (ESI) calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eCl [M+H]\u003csup\u003e+\u003c/sup\u003e 524.1489; found 524.1483. HPLC purity = 97.3% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 17.818 min.).\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e18B\u003c/strong\u003e: m.p = 185-186 \u003csup\u003eo\u003c/sup\u003eC, purple solid, yield = 60%; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eH\u003c/sub\u003e\u003c/em\u003e 9.61 (s, 1H), 9.16 (s, 1H), 8.96 (dd, \u003cem\u003eJ\u003c/em\u003e = 4.8, 1.7 Hz, 1H), 8.35 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.8 Hz, 1H), 7.93 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.75 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.1, 4.8 Hz, 1H), 7.49 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 6.99 \u0026ndash; 6.94 (m, 1H), 6.91 \u0026ndash; 6.87 (m, 2H), 6.77 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.1, 1.2 Hz, 1H), 6.62 \u0026ndash; 6.52 (m, 3H), 6.44 (t, \u003cem\u003eJ\u003c/em\u003e = 6.1 Hz, 1H), 4.88 (s, 2H), 4.37 (d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 2H).\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR (151 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta;\u003cem\u003e\u003csub\u003eC\u003c/sub\u003e\u003c/em\u003e 180.1, 174.8, 165.2, 154.4, 148.0, 146.4, 144.0, 143.1, 142.8, 134.3, 133.1, 127.8, 127.4, 127.3, 126.9, 126.6, 126.4, 125.9, 123.4, 116.3, 116.1, 112.5, 111.5, 46.3. HRMS (ESI) calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eCl [M+H]\u003csup\u003e+\u003c/sup\u003e 524.1489; found 524.1476. HPLC purity = 98.2% (t\u003csub\u003er\u0026nbsp;\u003c/sub\u003e= 17.259 min.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Biology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.1 Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTriple-negative breast cancer MDA-MB-231 and Pancreatic ductal adenocarcinoma PANC-1 cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (12800-017; Gibco). Triple-negative breast cancer cells MDA-MB-436 were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium F12 (DMEM F12) medium (41300-039; Gibco). Human epidermal growth factor receptor-2 negative breast adenocarcinoma MCF-7 and gliobastoma U87MG cells were cultured in alpha modified\u0026nbsp;Eagle\u0026apos;s minimum essential medium (\u0026alpha;-MEM)\u0026nbsp;(11900-024; Gibco).\u0026nbsp;Human leukemia cell lines KG-1, HL-60 and colorectal adenocarcinoma DLD-1 were cultured in Roswell Park Memorial Institute (RPMI) 1640 (31800-022; Gibco). Human leukemia cell lines MV4-11 and K-562 were cultured in Iscove\u0026rsquo;s modified Dulbecco\u0026rsquo;s medium (IMDM) (12200-036; Gibco), containing 1.5g/L and 3g/L sodium bicarbonate respectively. All cell lines were cultured in medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine (PSG; 10378-016; Gibco) at 37 ◦C in a humidified atmosphere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2. Chemicals and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColchicine, Paclitaxel (Taxol) and Nocodazole were purchased from Sigma (C9754), Cytoskeleton, Inc. (TXD01) and Sigma (31430-18-9)\u0026nbsp;respectively. Primary antibodies: anti-phospho-Ser/Thr-Pro-mitotic protein monoclonal 2 (MPM2) (05\u0026ndash;368; Millipore); anti-cyclin B1 pS126 (ab55184; Abcam); anti-Cyclin-dependent kinase 1 pY15 (CDK1 pY15) (GTX1281550; GeneTex); anti-histone H3 pS10 (06\u0026ndash;570; Millipore); anti-Histone 3 (ab1791; Abcam); anti-acetyl-Histone 3 lysine 9 (07e352; Millipore); anti-Histone 4 (ab10158; Abcam); antiacetyl-Histone 4 lysine 5/8/12/16 (06e866; Millipore); anti-SMC3 (A300-060A; Bethyl); anti-acetyl-SMC3 lysine 105/106 (MABE1073; Millipore); anti-a-tubulin (T5168; Sigma); anti-acetyl-a-Tubulin lysine 40 (T7451; Sigma); anti- KRAB domain-associated protein 1 (KAP1) pS824 (ab70369; Abcam); anti-Checkpoint kinase 2 (CHK2) pT68 (2661; Cell Signaling Technology); anti-CHK1 pS345 (2348; Cell Signaling Technology); anti-CHK1 (G-4) (sc-8408; Santa Cruz); anti-Replication Protein A2 (RPA2) pS33 (A300-246A; Bethyl); anti-RPA2 pS4/S8 (A300- 245A; Bethyl); anti-\u0026gamma;H2AX (05\u0026ndash;636; Millipore); anti-H2AX (2595; Cell Signaling Technology); anti-actin (MAB1501; Millipore); anti-Caspase-3 (NB100-56708; Novus); anti-Caspase-8 (9746; Cell Signaling); anti-Caspase-9 (9502; Cell Signaling); anti-induced myeloid leukemia cell differentiation protein (Mcl-1)(ab32087, Abcam); anti-B-cell leukemia/lymphoma 2(Bcl-2)(3498; Cell Signaling). Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse (115-035-003; Jackson ImmunoResearch Labs); anti-rabbit (111-035- 003; Jackson ImmunoResearch Labs) antibodies.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3. MTT assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 96-well plates and treated with compounds for 48h, followed by incubation with 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, M2128) at 37 ◦C for 2-3 h. Formazan crystals were then dissolved in dimethyl sulfoxide (DMSO). The number of surviving attached cells was determined by measuring the absorbance at 562 nm (PerkinElmer VICTOR3\u003csup\u003eTM\u003c/sup\u003e multilabel plate reader).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.4. MTS assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 96-well plates and treated with compounds for 48h. Surviving cells were determined by incubation with 0.2 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Abcam, ab223881), and absorbance at 490 nm was measured by using PerkinElmer VICTOR3TM multilabel plate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.5. Thymidine synchronization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were incubated with 4 mM of thymidine (Sigma, T1895) for 20\u0026ndash;24 h to enrich cells at the early-S phase. Cells were then washed with fresh medium twice and incubated with culture medium for recovery from thymidine block.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.6. Flow cytometry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were collected and fixed with 70% ice-cold ethanol. After washing with cold phosphate-buffered saline containing 1% FBS, cells were incubated with 0.05 mg/ml of propidium iodide (PI; Sigma, P4170) and 0.25 mg/ml ribonuclease A (RNase A; Sigma, R6513) at 37 ◦C for 30 min. DNA content was analyzed by using Becton Dickinson FACSCanto\u003csup\u003eTM\u003c/sup\u003eII Flow Cytometer, and cell-cycle profiles were plotted with the FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.7. Western blotting analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in Laemmli sample buffer (60 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, and 10% glycerol). Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membranes. After blocking with 5% skim milk, proteins were probed with specific primary antibodies and then HRP-conjugated species-specific secondary antibodies, followed by signal detection using enhanced chemiluminescence substrates (Bio-Rad). Images were obtained by using the iBright FL-1500 system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBidyadhar Sethy, and Iin Narwanti performed the research, designed, synthesized, analyzed data, and wrote the manuscript. Richa Upadhyay performed the mechanistic experiments, analyzed bio-results and wrote the manuscript. Zih-Yao Yu analyzed and technical supported biological experiments. Sung-Bau Lee designed, supervised, and helped to revise the manuscript and gave final approval for the version to be published. Jing-Ping Liou designed, supervised the study, analyzed and revised the manuscript. All the authors read and approved the final manuscript and declares no competing financial interest.\u003c/p\u003e\u003ch2\u003eAcknowledgment.\u003c/h2\u003e \u003cp\u003eThis research was supported by the National Science and Technology Council of Taiwan (grant no. 111-2113-M-038-004, 112-2113-M-038-001, 112-2320-B-038-004). This work was supported of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. (grant no. DP2-TMU-113-C-03).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerlay J, Colombet M, Soerjomataram I, Parkin DM, Pineros M, Znaor A, Bray F (2021) Cancer statistics for the year 2020: An overview. Int J Cancer\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao S, Zuo WJ, Shao ZM, Jiang YZ (2020) Molecular subtypes and precision treatment of triple-negative breast cancer. 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Eur J Med Chem 222:113588\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Wei Y, Wang X, Ma L, Li X, Sun Y, Wu Y, Zhang L, Wang J, Li M, Zhang K, Wei M, Yang G, Yang C (2023) Discovery of novel and bioavailable histone deacetylases and cyclin-dependent kinases dual inhibitor to impair the stemness of leukemia cells. Eur J Med Chem 249:115140\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoos A, Gahr BM, Park DD, Braun V, Buhler A, Rottbauer W, Just S (2023) Hdac1-deficiency affects the cell cycle axis Cdc25-Cdk1 causing impaired G2/M phase progression and reduced cardiomyocyte proliferation in zebrafish. Biochem Biophys Res Commun 665:98\u0026ndash;106\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerqueira A, Santamar\u0026iacute;a D, Mart\u0026iacute;nez-Pastor B, Cuadrado M, Fern\u0026aacute;ndez-Capetillo O, Barbacid M (2009) Overall Cdk activity modulates the DNA damage response in mammalian cells. J Cell Biol 187:773\u0026ndash;780\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEot-Houllier G, Fulcrand G, Magnaghi-Jaulin L, Jaulin C (2009) Histone deacetylase inhibitors and genomic instability. Cancer Lett 274:169\u0026ndash;176\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"apoptosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appt","sideBox":"Learn more about [Apoptosis](http://link.springer.com/journal/10495)","snPcode":"10495","submissionUrl":"https://submission.nature.com/new-submission/10495/3","title":"Apoptosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CDC25 inhibitor, HDAC inhibitor, dual-target, Triple-Negative Breast Cancer, genome instability, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4661784/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4661784/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriple-negative breast cancer (TNBC) poses a significant challenge for treatment due to its aggressive nature and the lack of effective therapies. This study developed dual inhibitors against cell division cycle 25 (CDC25) and histone deacetylases (HDACs) for TNBC treatment. CDC25 phosphatases are crucial for activating cyclin-dependent kinases (CDKs), the master regulators of cell cycle progression. HDACs regulate various biological processes by deacetylating histone and non-histone proteins, affecting gene expression, chromatin structure, cell differentiation, and proliferation. Dysregulations of HDACs and CDC25s are associated with several human malignancies. We generated a group of dual inhibitors for CDC25 and HDAC by combining the molecular structures of CDC25 (quinoline-5,8-dione) and HDAC (hydroxamic acid or benzamide) pharmacophores. The newly developed compounds were evaluated against solid-tumor, leukemia, and non-malignant breast epithelial cells. Among the synthesized compounds, \u003cstrong\u003e18A\u003c/strong\u003e emerged as a potent inhibitor, demonstrating significant cytotoxicity against TNBC cells, superior to its effects on other cancer types while sparing non-malignant cells.\u003cstrong\u003e18A\u003c/strong\u003e possessed similar HDAC inhibitory activity as Entinostat and potently suppressed the CDC25 activity in cells. Additionally, \u003cstrong\u003e18A \u003c/strong\u003ehindered the progression of S and G\u003csub\u003e2\u003c/sub\u003e/M phases, caused DNA damage, and induced apoptosis. These findings suggest that \u003cstrong\u003e18A\u003c/strong\u003e holds promise as a targeted therapy for TNBC and warrants further preclinical development.\u003c/p\u003e","manuscriptTitle":"Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 09:16:38","doi":"10.21203/rs.3.rs-4661784/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-22T07:27:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-18T05:55:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-16T11:41:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307899676183798322698625315895779882462","date":"2024-07-08T13:15:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143740647148347674680830444826798220893","date":"2024-07-08T11:49:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58722426235660759953751531416222700718","date":"2024-07-08T11:09:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165690286612153618846806558954198151593","date":"2024-07-08T09:34:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266871166758888286382752056890800020308","date":"2024-07-07T06:18:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46240791798685249757337786335059905422","date":"2024-07-07T01:09:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-06T14:41:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161971327919226948755240702194234278013","date":"2024-07-06T11:57:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297041336091161987350858117676275539275","date":"2024-07-06T10:17:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-06T09:01:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-01T02:38:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T02:37:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Apoptosis","date":"2024-06-30T08:03:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"apoptosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appt","sideBox":"Learn more about [Apoptosis](http://link.springer.com/journal/10495)","snPcode":"10495","submissionUrl":"https://submission.nature.com/new-submission/10495/3","title":"Apoptosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"346f4243-a240-43f5-adb8-edceeacb327b","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:00:31+00:00","versionOfRecord":{"articleIdentity":"rs-4661784","link":"https://doi.org/10.1007/s10495-024-02023-7","journal":{"identity":"apoptosis","isVorOnly":false,"title":"Apoptosis"},"publishedOn":"2024-10-12 15:57:19","publishedOnDateReadable":"October 12th, 2024"},"versionCreatedAt":"2024-07-24 09:16:38","video":"","vorDoi":"10.1007/s10495-024-02023-7","vorDoiUrl":"https://doi.org/10.1007/s10495-024-02023-7","workflowStages":[]},"version":"v1","identity":"rs-4661784","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4661784","identity":"rs-4661784","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}