Quinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids

Quinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids | 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 Article Quinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids Jing-Ping Liou, Iin Narwanti, Zih-Yao Yu, Bidyadhar Sethy, Pei-Ling Zheng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6282946/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cell division cycle 25 (CDC25) phosphatases have emerged as critical regulators of cell cycle progression and genomic stability, making them compelling therapeutic targets in oncology. Building on the established CDC25 inhibitor NSC663284 , we strategically designed, synthesized, and evaluated a novel series of derivatives with diverse alkylamino side chains to enhance potency and selectivity. Among them, derivatives featuring 2-(4-methylpiperidin-1-yl)ethylamino ( D3a and D3b ) or 2-(dimethylamino)ethylamino groups ( D11a and D11b ) demonstrated remarkable anti-cancer efficacy, exhibiting potent apoptotic induction and broad-spectrum growth inhibition. These compounds displayed IC 50 values between 0.21 to 1.22 μM in leukemia cells and from 0.13 to 1.5 μM in CRC cells, surpassing the activity of NSC663284 . Mechanistically, these derivatives effectively inhibited CDC25 phosphatase activity in vitro and disrupted CDC25-mediated dephosphorylation of CDK1 in cells, leading to cell cycle arrest and catastrophic genomic instability. Treatment with these compounds induced rapid and extensive double-stranded DNA breaks, highlighting their potential to drive irreversible cancer cell death. Importantly, their therapeutic potential was further validated in CRC patient-derived tumor organoids, offering clinically relevant insights into patient-specific drug responses and underscoring their translational significance. Our findings establish a new class of CDC25 inhibitors with superior anti-cancer activity and mechanistic precision, paving the way for next-generation therapeutics targeting leukemia and CRC. Biological sciences/Drug discovery/Drug delivery Biological sciences/Cancer/Cancer therapy/Drug development CDC25 inhibitors cancer therapeutics cell cycle arrest genomic instability patient-derived organoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Leukemia is a type of hematological malignancy characterized by progressive aberrant growth of leukocytes [ 1 ]. In general, leukemia is classified into four subtypes determined by the cell lineage (lymphocytic or myeloid) and the degree of maturation arrest (acute or chronic), all of which are risk factors for people of all ages. These subtypes comprise acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) [ 2 , 3 ]. Globally, leukemia contributed to 2.5% and 31.1% of all new cancer incidences and deaths, respectively [ 4 ]. Despite the progress achieved in developing targeted therapies for various types of leukemia, the presence of cytotoxicity, therapy resistance, and relapse remain a substantial obstacle that impedes the effectiveness of targeted therapy in the treatment of leukemia [ 5 – 7 ]. Hence, novel drugs with improved anti-leukemia capabilities are required. CDC25 phosphatases are members of the dual-specific protein tyrosine phosphatase (PTP) family, which play a crucial role in cell cycle regulation [ 8 ]. In human genome, three isoforms of CDC25 phosphatases have been characterized, namely: CDC25A, CDC25B, and CDC25C [ 9 ]. The activation of cyclin-dependent kinases (CDKs) is crucial for cell cycle progression [ 10 ]. CDC25s are responsible for removing phosphate groups from CDK/cyclin complexes at the Thr14 and/or Tyr15 residues, thereby activating CDK/cyclin and facilitating cell cycle progression [ 11 , 12 ]. It appears that CDC25s act at different stages of the cell cycle on different CDK/cyclin complexes. CDC25A appears to be implicated in controlling the G 1 /S and G 2 /M transitions, whereas CDC25B and CDC25C appear to be more involved in the regulation of the G 2 /M transition [ 13 ]. CDC25s malfunction is often correlated to aberrant cell cycle progression, which contribute to tumorigenesis [ 14 ]. CDC25 overexpression has been identified in several high-grade human malignancies and linked to poor prognosis [ 15 ]. In addition, the role of CDC25s in cancer development including leukemia has been investigated in previous literature. Increased expression of CDC25A could promote cell proliferation in human AML [ 16 – 18 ]. Inactivation of CDC25A has also been correlated with antiproliferative, proapoptotic, and cytotoxic effects in various leukemia cell lines [ 19 – 21 ]. AML cell growth seems to be decreased by CDC25B inhibition [ 22 , 23 ]. CDC25C appears to have a significant role in determining the characteristics of AML cells [ 24 ]. Therefore, CDC25s could be considered an attractive potential therapeutic target in leukemia [ 16 , 25 ]. Different attempts to develop CDC25 inhibitors have been extensively investigated for over two decades [ 26 – 34 ]. And quinone-based derivatives remain the most effective of these inhibitors. Representative quinone-based molecules as CDC25 inhibitors are presented in Fig. 1 . Menadione (Vitamin K3, VK3) demonstrated a potent CDC25 inhibition, inducing cell cycle arrest and apoptosis on cervical carcinoma cells [ 35 ]. Compound 2 ( M2N12 ), was identified to be potent against CDC25 isoforms [ 36 ]. Compound 3 ( M5N36 ) is a selective CDC25C inhibitor with promising anti-growth activity and desirable predicted physicochemical properties [ 37 ]. A quinoid bearing 2-(dimethylamino)ethylamino moiety, compound 4 ( BN-82685 ), as a potent in vitro and in vivo CDC25 inhibitor. Compound 4 revealed specifically inhibited CDK1 Tyr-15 dephosphorylation and induced cell cycle arrest, leading to antitumor growth in Mia PaCa-2 xenograft mice models [ 38 ]. Compound 5 ( IRC-083864 ) bearing two quinone scaffolds displayed a selective CDC25 inhibitor. Also, compound 5 revealed potential antiproliferative activity, strongly inhibited cell cycle progression, and revealed tumor growth inhibition in both pancreatic and prostate tumor xenograft models [ 32 ]. In another investigation, compound NSC663284 ( 6a ) was found to be a highly effective CDC25 inhibitor [ 39 ]. However, many CDC25 inhibitors with different scaffolds have been identified, manifesting the potent therapeutic utility of these compounds. The development of small-molecule inhibitors that specifically target CDC25 is still in its early stages and has limited biochemical potency. Therefore, an effort to develop potent CDC25 inhibitors would be useful for developing new anticancer therapeutics. In our previous study, we emphasized the therapeutic potential of CDC25 inhibitors and how they disrupt cell cycle progression in colorectal cancers (CRCs) [ 40 ]. Some derivatives that incorporated quinoline-5,8-dione core scaffold and morpholinoalkylamino at the C-6 position demonstrated potent cytotoxicity. Driven by compound NSC663284 ( 6a ) and 6b [ 40 ] with its promising anticancer activity, in current study, we thus designed, synthesized, and evaluated a series of NSC663284 derivatives as anti-leukemia and -CRC agents. The quinoline-5,8-dione moiety was maintained, and then various side chains including heterocyclic/non-heterocyclic moieties at the C-7 and C-6 positions were introduced (Fig. 2 ). Antiproliferative activity were examined in various leukemia and CRC cell lines and validated in the CRC cancer organoid model. Further mechanism of action of these synthesized compounds on CDC25 inhibition, cell cycle, apoptosis, and DNA damage was investigated. Result and discussion Chemistry The synthesis route of NSC663284 derivatives is summarized in Scheme 1 . Starting with the commercially available 5-nitroquinolin-8-ol ( A ) was converted to 5-aminoquinolin-8-ol ( B ) intermediate through catalytic hydrogenation [ 41 ]. Further, intermediate 6,7-dichloroquinoline-5,8-dione ( C ) was prepared by oxidizing intermediate B with NaClO 3 /HCl [ 42 ]. Subsequently, the intermediate C was reacted with various amines to afford C7-substituted products 6a, D1a-D12a and C6-substituted products 6b, D1b-D12b . Compounds D6a and D6b were obtained from the removal of the Boc protecting group of compounds D8a and D8b , respectively, which was accomplished by using trifluoroacetic acid in anhydrous dichloromethane. In addition, as described in the experimental procedure, to synthesize D5a and D5b , the spirocyclic amine was initially synthesized. This spirocyclic amine intermediate was obtained from a nucleophilic substitution reaction of the commercially available 1,4-dioxa-8-azaspiro[4.5]decane with 2-chloroacetonitrile to give nitrile intermediate, followed by nitrile intermediate reduction by lithium alumunium hydride to afford the 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine. The intermediate C was treated with the 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine to afford D5a and D5b . Meanwhile, to synthesize D12a and D12b , 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) was synthesized first by using 2,2'-azanediylbis(ethan-1-ol) and 2-bromoacetonitrile to give nitrile intermediate which was reduced to give 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol). Further, the treatment of intermediate C with 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) afforded D12a and D12b . Based on the structure of the key intermediates C , two –Cl atoms attached in the quinoline-5,8-dione scaffold (at C-7 and C-6 positions) have identical electronic properties, therefore these two –Cl atoms were replaced by nucleophile amines to generate C-7 and C-6 substituted products [ 43 ]. Here, we observed that the solvent used influenced the yield of each regioisomer. The regiochemistry of the products was determined according to the reported literature by assessing the proton chemical shifts (δ H ) difference at 2 and 4 positions [ 44 ]. In all instances, the 7-substituted and 6-substituted products were separated through silica column chromatography (SCC), even though their Rf values overlapped. Furthermore, all the synthesized compounds were characterized by NMR ( 1 H- and 13 C-NMR), melting point, and HRMS. The purity of all final target compounds was found to be ≥ 95%. Cytotoxicity evaluation on CRC cancer cells CDC25 has been suggested as a target for early diagnosis and clinical treatment of different types of cancer [ 11 , 14 ]. Our earlier study also highlighted the therapeutic potential of CDC25 inhibitors and their interference with cell cycle progression in colorectal cancers (CRCs) [ 40 ]. In this work, we initially performed cytotoxicity evaluation of all the newly designed and synthesized derivatives D1a-D12a , and D1b-D12b against CRC cell lines (HCT116 and DLD-1) by using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium (MTS) assay after 72 h incubation at 2 µM concentration, compared to the reference compound NSC663284 (6a) . In an attempt to develop an active potent compound, an exploration of the structural modification of 6a at the C-7 or C-6 positions of the quinoline-5,8-dione core scaffold was conducted. Hence, our attention was directed towards substituting the 2-morpholinoethylamino with different motifs. The results revealed that some alterations of the side chain of compound 6a augmented the cytotoxicity against HCT116 and DLD-1 cells (Table S1). Notably, the introduction of 2-thiomorpholinoethylamino at the C-6 position ( D1b ) demonstrated a considerable improvement in cytotoxic effect against HCT116 and DLD-1 cells. Substituting 2-morpholinoethylamino with a hydrophobic pyrrolidin-1-yl moiety ( D2a, D2b ) was also beneficial for the cytotoxicity. Similarly, the side chain modification particularly with 2-(4-methylpiperidin-1-yl)ethylamino ( D3a, D3b ) at the C-7 and C-6 position showed a pronounced effect against HCT116 and DLD-1 cells. Subsequently, more structural modifications by introducing different side chains were also investigated. However, replacement of 2-morpholinoethylamino moiety with 2-(4-hydroxypiperidin-1-yl)ethylamino ( D4a , D4b ) diminished cytotoxic effect against HCT116 and DLD-1 cells. Next, azaspiro and piperazine containing compound was also evaluated. The introduction of 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethylamino and 2-(4-methylpiperazin-1-yl)ethylamino at the C-6 position ( D5b , D7b ) exhibited a potent activity against both HCT116 and DLD-1 cells. Further, D6a and D6b containing 2-(piperazin-1-yl)ethylamino revealed a weak activity, implying that a polar side chain might be unfavorable. Meanwhile, changing the piperazin-1-yl ( D6b ) to 4-methylpiperazin-1-yl ( D7b ), led to increased cytotoxicity. IC 50 determination of the selected derivatives in CRC cells The half maximal inhibitory concentration (IC 50 ) values of the selected compounds against CRC cell lines HCT116 and DLD-1 revealed varying degrees of cytotoxic activity (Table S2). Several derivatives exhibited potent inhibitory effects on both cell lines. Notably, compound D1b bearing 2-thiomorpholinoethylamino at the C-6 position demonstrated significant activity, with IC 50 values of 0.48 µM in HCT116 and 0.38 µM in DLD-1 cells. Additionally, compounds D2a , D2b, D3a, D3b, D5b , and D7b displayed moderate potency, particularly in HCT116, with IC 50 values of 1.43, 0.88, 1.50, 1.13, 0.83, and 0.83 µM, respectively. Compounds bearing 2-(pyrrolidin-1-yl)ethyl)amino ( D2a ) at C-7 and 2-(4-methylpiperidin-1-yl)ethylamino ( D3a and D3b) at the C-7 and C-6 positions, exhibited potent inhibitory activity against the DLD-1 cell with IC 50 values of 0.76, 0.74 and 0.65 µM, respectively. Furthermore, compounds conjugated with 2-(pyrrolidin-1-yl)ethyl)amino ( D2b ), 2-(piperazin-1-yl)ethyl)amino ( D5b ), and 2-(4-methylpiperazin-1-yl)ethyl)amino ( D7b) at C-6 positions exhibited moderate inhibitory activity against the DLD-1 cell with IC 50 values 1.29, 0.84, and 1.77 µM, respectively. Interestingly, D11a bearing 2-(dimethylamino)ethylamino at the C-7 position exhibited selective potency, being more effective against DLD-1 (0.56 µM) than HCT116 (1.14 µM). Among selected compounds, compound D11b bearing 2-(dimethylamino)ethylamino at the C-6 position exhibited the highest potency, with the lowest IC 50 values of 0.17 µM in HCT116 and 0.13 µM in DLD-1, indicating its strong potency in inhibiting cell proliferation. Overall, D1b and D11b emerges as the most promising compounds, warranting further investigation for their potential therapeutic application in CRC treatment. Cytotoxicity evaluation on leukemia cells Given that CDC25 inhibitors have demonstrated promise in leukemia development [ 25 ], we also assayed the cytotoxicity of newly synthesized compounds in various types of blood cell lines isolated from leukemias, including HL-60 (acute promyelocytic leukemia), KG-1 (acute myelogenous leukemia), MV-4-11 (biphenotypic B-myelomonocytic leukemia), and K-562 (chronic myelogenous leukemia). We treated cells with 2 µM of compounds for 72 hours, followed by MTS assay. The results presented in Table 1 revealed that several derivatives delivered high toxicity to all cell lines. Compound NSC663284 ( 6a ) bearing 2-morpholinoethylamino at the C-7 position showed higher cytotoxicity in the KG-1 cell line and moderate cytotoxicity in the three tested cell lines (HL-60, MV4-11, and K562 cell lines). The regiomer compound 6b bearing 2-morpholinoethylamino at the C-6 position exhibited more pronounced cytotoxicity against the tested cell lines, as compared to compound 6a . As shown in Table 1 , replacing 2-morpholinoethylamino moiety with 2-thiomorpholinoethylamino ( D1a, D1b ) showed almost similar results to 6a and 6b . Compound D1a , which incorporated 2-thiomorpholinoethylamino at the C-7 position, showed potent cytotoxicity in the KG-1 cell line. However, D1b showed a similar potency with 6b , suggesting that the replacement of 2-morpholinoethylamino moiety with 2-thiomorpholinoethylamino at the C-6 position retained the activity. Interestingly, the substitution of 2-morpholinoethylamino moiety by a hydrophobic moiety such as pyrrolidin-1-yl moiety ( D2a and D2b ) led to improved cytotoxicity. Further, more structural variations by introducing a piperidinyl moiety on the C-7 and C-6 positions were explored. Notably, an increase in cytotoxicity was remarked in the replacement of 2-morpholinoethylamino ( 6a, 6b ) with 2-(4-methylpiperidin-1-yl)ethylamino ( D3a, D3b ). When compared to 6a , the cytotoxicity of D3a and D3b was significantly improved against all the tested leukemia cell lines, indicating that the substitution of 2-morpholinoethylamino with 2-(4-methylpiperidin-1-yl)ethylamino moiety at the C-7 and C-6 position appeared to be beneficial for the activity. The introduction of 2-(4-hydroxypiperidin-1-yl)ethylamino at the C-7 position ( D4a ) exhibited potent cytotoxicity against the three tested cell lines (KG-1, HL-60, and MV4-11), reducing cell viability by > 80%. Meanwhile, compound D4b bearing 2-(4-hydroxypiperidin-1-yl)ethylamino at the C-6 position showed lower potency compared to D4a . Next, 1,4-dioxa-8-azaspiro and piperazinyl-containing compounds were evaluated. Compound D5a bearing a 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethylamino at the C-7 position revealed higher cytotoxicity against KG-1, HL-60, and MV4-11 cell lines, meanwhile, its regioisomer ( D5b ) exhibited potency towards MV4-11 and K562 cell lines. Compound D6a and D6b were found to be less potent than 6a , indicating that 2-(piperazin-1-yl)ethylamino as a polar side chain was not beneficial for the activity. Notably, D7a and D7b which incorporated 2-(4-methylpiperazin-1-yl)ethylamino side chain revealed high potency, indicating that the replacement of 2-morpholinoethylamino with 2-(4-methylpiperazin-1-yl)ethylamino motifs favored for the activity. Compound D7a and D7b demonstrated broad antileukemia activities in KG-1, HL-60, MV4-11, and K562 cell lines, reducing cell viability by > 80%. Meanwhile, D8a, D8b , and D9a-D9b represent a small series of derivatives bearing bulky substituents. Substitution of the 2-morpholinoethylamino moiety by a bulkier tert-butyl piperazine-1-carboxylate motifs at the C-7 and C-6 positions ( D8a and D8b ) led to reduced cytotoxicity when compared to compound 6a . Moreover, the incorporation of tert-butyl (1-(2-aminoethyl)piperidin-4-yl)carbamate moiety at the C-7 and C-6 positions exhibited better cytotoxicity (compare D9a and D9b with 6a , D8a , and D8b) . Further, the effect of the replacement of 2-morpholinoethylamino with various acyclic side chains such as 2-((2-hydroxyethyl)(methyl)amino)ethylamino, 2-(dimethylamino)ethylamino, and 2-(bis(2-hydroxyethyl)amino)ethylamino moieties was also explore. The introduction of 2-((2-hydroxyethyl)(methyl)amino)ethylamino ( D10a, D10b ) slightly maintained the cytotoxicity against KG-1, HL-60, and MV4-11 cell lines. Interestingly, D11a and D11b bearing 2-(dimethylamino)ethylamino at the C-7 and C-6 positions led to more pronounced cytotoxicity against all the tested leukemia cell lines (compared to compound 6a ), suggesting that the substitution of 2-morpholinoethylamino with hydrophobic alkyl side chain 2-(dimethylamino)ethylamino was found to be essential for the activity. However, a decrease in cytotoxicity was observed when 2-(bis(2-hydroxyethyl)amino)ethylamino moiety ( D12a, D12b ) was introduced. Taken together, the structure-activity relationship (SAR) revealed that 2-(4-methylpiperidin-1-yl)ethylamino and 2-(dimethylamino)ethylamino moieties either both at the C-7 or C-6 positions were favorable for the activity. In general, our cytotoxicity screen demonstrated that D3a , D3b , D11a , and D11b displayed considerable effectiveness across all leukemia cell lines and proved to be even more toxic than 6b , the most potent compound reported in earlier study [ 40 ]. Table 1 Cytotoxicity of all synthesized compounds Compound KG-1 HL-60 MV4-11 K562 NSC663284 (6a) 20.1 ± 6.2 46.5 ± 2 46.4 ± 2.9 89.7 ± 15.4 6b 24.7 ± 1.4 36.3 ± 3.8 3.4 ± 2.7 7.3 ± 3.8 D1a 17.04 ± 4.5 25.14 ± 6.6 43.3 ± 2.9 77.4 ± 6.8 D1b 15.4 ± 2.6 27.4 ± 1.5 1.5 ± 0.95 0.37 ± 0.96 D2a 1.28 ± 1.4 3.3 ± 2.8 13.5 ± 3.8 73.9 ± 9.8 D2b 11.6 ± 0.98 31.8 ± 4.7 BDL 11.4 ± 3.7 D3a BDL BDL BDL 5.9 ± 4.6 D3b BDL 0.15 ± 0.19 BDL 0.2 ± 0.27 D4a BDL BDL 1.8 ± 1.9 55.1 ± 10.7 D4b 42.3 ± 5 76.3 ± 3.4 30.6 ± 2.3 91.4 ± 11.3 D5a 1.02 ± 1.05 0.16 ± 0.26 13.6 ± 3 42.2 ± 10.1 D5b 32.6 ± 3.2 44.6 ± 3.1 2.5 ± 1.2 12.9 ± 8.8 D6a 74.7 ± 4.8 83.7 ± 1.5 64 ± 1.6 98.6 ± 4.9 D6b 63.4 ± 5.3 65.4 ± 3.9 51.6 ± 2.1 97.7 ± 7.6 D7a 1.24 ± 1.08 3.4 ± 2.5 14.2 ± 3.3 87.4 ± 9.1 D7b 14.7 ± 1.8 27.2 ± 4.4 3.5 ± 3.4 34.9 ± 7.9 D8a 53.3 ± 8.8 58.3 ± 3.5 50.4 ± 6.9 99.5 ± 9.5 D8b 65.2 ± 8 56.5 ± 8.04 12.5 ± 3.4 16.9 ± 8.5 D9a BDL 0.01 ± 0.01 2.3 ± 3.2 40.9 ± 9.3 D9b 17.3 ± 4.8 22.1 ± 6.7 1 ± 1.4 17.9 ± 9.6 D10a BDL BDL 0.25 ± 0.25 59.31 ± 8.1 D10b 27.5 ± 4.8 42.2 ± 11.9 28.9 ± 1.9 89.7 ± 8.6 D11a BDL BDL BDL 9.1 ± 3.9 D11b 0.04 ± 0.08 0.05 ± 0.04 BDL 0.07 ± 0.1 D12a 74.3 ± 8.9 67.3 ± 7.5 48.2 ± 5.3 86.2 ± 7.8 D12b 60.2 ± 8.9 80 ± 1.7 47.2 ± 2.5 85.7 ± 2.4 DMSO 100 ± 3.1 100 ± 2.8 100 ± 3.4 100 ± 3.1 Cells were treated with 2 µM of indicated compounds for 72 h and the percentages of surviving cells were determined by MTS assay. Results are shown as means with SDs from at least two independent experiments ( n ≥ 5). BDL = Below Detection Limit. IC 50 determination of the selected derivatives in leukemia cells As mentioned above, all the prepared compounds were examined for their cytotoxicity. The screening outputs revealed that side chain modifications with either a 2-(4-methylpiperidin-1-yl)ethylamino and 2-(dimethylamino)ethylamino moieties are essential for anti-leukemic activity. Hence, the growth inhibitory activity of selected derivatives was examined and an IC 50 for the selected compounds is described in Table 2 . We found that the selected compounds exhibited high efficacy against tested cell lines, with an IC 50 value close to or below 1 µM. Compound D3a and D3b bearing a 2-(4-methylpiperidin-1-yl)ethylamino side chain at the C-7 and C-6 positions, respectively, retained potent activity against all investigated leukemia cell lines with a sub-micromolar to low single single-digit micromolar with IC 50 values ranging between 0.23 and 1.22 µM. Compound D11a , bearing 2-(dimethylamino)ethylamino as a side chain at the C-7 position, showed potency against the leukemia cell lines (IC 50 values ranging between 0.32–0.98 µM). Compound D11b , bearing 2-(dimethylamino)ethylamino at the C-6 position showed comparable antiproliferative activity to D11a with IC 50 values ranging between 0.21 and 0.61 µM. Taken together, our inhibitory activity evaluation against leukemia cell lines revealed that D3a and D11a were more potent in HL-60 and KG-1 cells, whereas D11b was most toxic to MV-4-11 and K-562 cells. Table 2 IC 50 values for selected compounds against a set of leukemia cell lines IC 50 (µM) D3a D3b D11a D11b HL-60 0.23 ± 0.02 0.93 ± 0.21 0.32 ± 0.05 0.42 ± 0.06 KG-1 0.45 ± 0.12 1.22 ± 0.14 0.46 ± 0.06 0.61 ± 0.06 MV4-11 0.73 ± 0.05 0.64 ± 0.05 0.67 ± 0.02 0.21 ± 0.04 K562 1.01 ± 0.07 0.97 ± 0.11 0.98 ± 0.06 0.49 ± 0.15 Various leukemia cell lines were treated with indicated compounds for 72 h and the values of half maximal inhibitory concentration (IC 50 ) were determined by MTS assay. Results are shown as means with SDs from at least two independent experiments ( n ≥ 6). Apoptosis We utilized flow cytometry to analyze the cell status at the endpoint of compound treatment. Our results showed that treating HL-60 cells with the most potent compounds, D3a and D11a , significantly increased the sub-G 1 population (Fig. 3 A), which is indicative of DNA fragmentation. In contrast, we did not observe any effect from the reference compound 6a under the same conditions (Fig. 3 A). Similarly, D1b and D11b , the most toxic compounds to CRC cells, also led to an increase in the sub-G 1 population in HCT116 cells (Figure S1). We further examined the effect of compound treatment in apoptosis pathways and found that the treatment of D3a or D11a induced the cleavage of pro-apoptotic Caspase-3 and − 9, while reduced the levels of anti-apoptotic proteins Mcl-1 and Bcl-2, in HL-60 cells (Fig. 3 B). These findings indicate that the selected derivatives possess strong cytotoxicity and are capable to trigger apoptosis, albeit with variable potencies across different cell lines. CDC25 inhibitory activity Cell cycle progression highly relies on the activation of cyclin-dependent kinases (CDKs) [ 10 ]. The activation of CDK requires the removal of the inhibitory phosphorylation at Tyr15 by the phosphatase CDC25 [ 12 ]. Our previous study demonstrated that 6b nearly prohibit G 2 /M transition by preventing CDC25-mediated CDK1 dephosphorylation in CRC cells [ 40 ]. In light of this, we investigated the potential CDC25 inhibitory activity of D3a and D11a . The in vitro enzymatic analysis revealed that the modification of 2-morpholinoethylamino moiety on NSC663284 ( 6a ) structure with 2-(4-methylpiperidin-1-yl)ethylamino and 2-(dimethylamino)ethylamino moieties (compounds D3a and D11a) enhanced the CDC25-inhibitory activity from the IC 50 value of 0.11 µM ( 6a ) to 0.065 µM and 0.068 µM, respectively (Fig. 4 ). CDC25C inhibition analysis of indicated compounds using CDC25C Human Phosphatase Enzymatic LeadHunter Assay. Percentages of inhibition (left) and IC 50 values from two individual assays (right) are shown. CDK1 dephosphorylation and mitosis progression To validate the enhanced CDC25-inhibitory activity of compounds D3a and D11a , we monitored the phosphorylation dynamics of CDK1 at Tyr15 in HL-60 cells following treatment with these compounds. We used thymidine to synchronize cells in the early S phase and assessed the phosphorylation level of CDK1 at Tyr15 from the S phase through to mitosis (Fig. 5 ). We observed that most cells entered the G 2 phase four hours after being released from the thymidine block (T4), during which CDK1 phosphorylation was high and only a faint mitotic signal was detected, indicated by the phosphorylation of cyclin B1, histone H3, and a mitotic protein identified by the MPM2 antibody (Fig. 5 and S2). To assess CDK1 dephosphorylation, we subsequently treated these cells with mitotic inhibitors colchicine or paclitaxel (Taxol) to prevent CDK1 re-phosphorylation after nuclear division. At T8, these cells accumulated in mitosis, exhibiting a low level of CDK1 phosphorylation and increased mitotic signals (Fig. 5 and S1). Notably, cells co-treated with D3a or D11a demonstrated higher CDK1 phosphorylation levels, coupled with lower mitotic signals (Fig. 5 and S2). Similar outcomes were observed in HCT116 cells treated with D1b , D3b , or D11b (Figure S3). Importantly, these compounds affected cell cycle progression with varying efficacies (Figure S4). Our findings suggest that our compounds are potent CDC25 inhibitors that disrupt cell cycle progression. Analysis of cell cycle progression and mitotic signaling of thymidine-synchronized HL-60 cells co-treated with 0.5 µM of colchicine and 0.75 µM of indicated compounds. Experimental design (left upper) and representative results of FACS (left lower) and Western blot (right) from one of two biological replicates are shown. Asyn: Asynchronous; cpd: Compound; Col: Colchicine; Thy: Thymidine. Molecular docking study The selected compounds D3a , and D11a together with the reference compound 6a were further investigated the protein-drug interaction for the CDC25C crystal protein (PDB ID: 3OP3). The ligands 6a , D3a , and D11a were docked into the coordinates centered on the active site of the CDC25C protein using Discovery Studio Client 2022 (Biovia, CA, USA). Their binding poses were selected based on the highest LibDock score and protein-ligand interactions analyzed. The eminent interactions between the active pocket of CDC25C and compounds 6a , D3a , or D11a are predicted by their respective 3D (Fig. 5 A, 5 B, 5 D, 5 E, 5 G, and 5 H) and 2D (Fig. 5 C, 5 F, and 5 I) cartoon pictures. Importantly, various residues make the hydrogen (H)-bonding interactions to the quinoline-5, 8-dione moiety of D3a , and D11a similar to the reference compound 6a . Notably, compound D3a makes three conventional H-bondings with Glu378, Phe379, and Arg383, whereas compound D11a interacts with the active site via four H-bonding such as Cys377, Glu382, Arg383, and Gly384. Furthermore, in addition to the H-bonding, other interactions such as Pi-Pi T-shaped (Phe379), Pi-anion (Glu382), Pi-sulphur (Cys377, Met435) were able to preserve the CDC25C activity of compounds D3a and D11a as compared with 6a . However, the side chains (methyl-piperidine and N, N -dimethyl amine) of the compounds D3a and D11a were bound at the catalytic site via alkyl and pi-alkyl (Lys298, His299, Tyr304, and Pro385) and van der Waals (Ile411, Leu412 and Pro385) interactions respectively, as well a H-bonding with N, N -dimethyl amine ( D11a ) and Cys377 residues distant from the core catalytic region. Also, it can be seen that methyl-piperidine and N, N -dimethyl amine side chain motif contribute to D3a and D11a binding and stabilization at the hydrophobic pocket of CDC25C core active site (Fig. 5 J and 5 K). Therefore, D3a and D11a remained deeply buried at the core active catalytic site of CDC25C by maintaining H-bonding, Pi-Pi stacked, Pi-anion, Pi-sulphur, and as well van der Waals interactions which implied distinct compactness to favor its better activity. H4K20 methylation NSC663284 ( 6a ) and 6b have also been shown to target SETD8, an enzyme that methylates histone H4 at the site of Lys20 (H4K20) [ 40 , 45 , 46 ]. Disruption of SETD8-mediated H4K20 methylation leads to genome instability and cell cycle delay [ 40 , 46 ]. We thus assayed the level of H4K20 methylation in HL-60 cells treated with the identified compounds D3a and D11a . Consistent with the previous studies [ 47 , 48 ], H4K20 mono-methylation remarkably reduced before genome replication (T0) and gradually accumulated in S and G 2 /M phases (T4 and T6). The treatment of D3a or D11a impeded this progressive H4K20 methylation, while 6a showed a slight effect (Figure S5). Consistently, D11b also significantly blocked H4K20 methylation in HCT116 cells (Figure S6). These results argue that the new NSC663284 derivatives harbor inhibitory activities toward multiple pathways. Double-stranded DNA break CDK activity and H4K20 methylation play essential roles in maintaining genome stability [ 49 – 53 ]. Our earlier study found that 6b effectively inhibited CDK1Y15 dephosphorylation and H4K20 mono-methylation, leading to a DNA damage response [ 40 ]. In this study, we showed that the treatment of D3a or D11a induced significant DNA damage in HL-60 cells, particularly in double-stranded DNA break (DSB) pathways, as evidenced by phosphorylations of ATM, CHK2, KAP1, and RPA2 at Ser4/Ser8 (Fig. 7 A). Notably, D11b , the most potent NSC663284 derivative in leukemia K-562 and MV-4-11 cells (Table 2 ), as well as CRC cells (Table S2), also induces a higher level of DNA damage in K-562 and HCT116 cells (Figure S7). To determine if these compounds induce DSB, we extracted genomic DNAs from compound-treated cells and subjected them to gel electrophoresis. We observed clear ladder patterns in the genomic DNA samples of treated cells (Fig. 7 B), indicating that these compounds can rapidly trigger DNA fragmentation. Our findings suggest that these compounds are promising candidates for cancer therapeutics by inducing genome instability and catastrophe. Pharmacokinetic and drug-likeness properties Pharmacokinetic and drug-likeness properties of all compounds were predicted using the free online SwissADME software [ 54 ] and the results are presented in Table S3. The selected compounds, D1b, D3a, D3b, D11a , and D11b , adhered to Lipinski's rule criteria and exhibited favorable physicochemical attributes. Compounds 6a, 6b, D1b, D3a, D3b, D11a , and D11b have molecular weights (MW) ranging from 279.72 to 333.81 g/mol. The selected compounds have one H-bond donor (HBD), 4 rotatable bonds (RB), and 4 to 5 H-bond acceptors (HBA). The topological polar surface area (TPSA) values range from 62.30 to 71.53 Å2. The CLogP varies from 1.03 to 2.18. D1b, D3a, D3b, D11a , and D11b were in the soluble class. In addition, these compounds are predicted to have a high probability of gastrointestinal absorption (GIA) and be able to diffuse through the blood-brain barrier (BBB). Of interest, the selected compounds do not exhibit violation of either the Lipinski, Veber, Egan, or Muegge filters, which means that these compounds have the potential to be absorbed through the gastrointestinal tract (Table S5). Therefore, D1b , D3a, D3b, D11a , and D11b display desirable pharmacokinetic properties and are adequate for more advanced investigations. Patient-derived organoid model To evaluate its potential translation into clinical practice, we determined the efficacy of D11b in suppressing the proliferation of colorectal cancer (CRC) patient-derived organoids (PDOs). As shown in Fig. 8 A, CRC organoids treated with D11b exhibited decreased proliferation, as demonstrated by endpoint images and corresponding proliferation rate line graphs. The inhibition of proliferation was dose-dependent and statistically significant ( n = 3, P < 0.05). Invasion assays further confirmed the compound’s inhibitory effect (Fig. 8 B). Organoids treated with different concentrations of D11b displayed reduced invasion ability. Statistical analysis revealed a significant reduction in invasion with increasing concentrations of D11b . These findings highlight the potential clinical relevance of the identified compound as a therapeutic candidate for colorectal cancer. By effectively inhibiting both proliferation and invasion of CRC PDOs, the compound may offer a novel treatment strategy that targets the aggressive and metastatic behavior of CRC tumors. Conclusion Focusing on developing CDC25 inhibitors, we have designed, synthesized, and evaluated a series of NSC663284 derivatives containing various alkylamino side chains at the C-7 and C-6 positions of the quinoline-5,8-dione scaffold. In the SAR study, we explored the structural modification of the lead compound, NSC663284 ( 6a ), by substituting 2-morpholinoethylamino moiety with various alkylamino side chains. Modifying 2-morpholinoethylamino moiety with either 2-(4-methylpiperidin-1-yl)ethylamino or 2-(dimethylamino)ethylamino moieties improved the activity. D3a, D3b, D11a , and D11b showed remarkable cytotoxicity against the tested leukemia and CRC cell lines with IC 50 close to or below 1 µM. In vitro enzymatic assay of D3a and D11a demonstrated a potent CDC25-inhibitory activity with the IC 50 values of 0.065 µM and 0.068 µM, respectively. Mechanistically, the identified compounds impaired CDK1 dephosphorylation and slowed G 2 /M progression, ultimately leading to apoptosis, revealed by cell accumulation in the sub-G 1 phase, cleavage of pro-apoptotic Caspase-3 and − 9 and reduction of anti-apoptotic protein Mcl-1 and Bcl-2. The evaluation of genome integrity further demonstrated that these compounds possess a genotoxicity that triggered double-stranded DNA breaks. In silico SwissADME prediction confirmed the drug-like properties and the druggability of the selected compound. Notably, we demonstrated that D11b effectively inhibits the proliferation and invasion of CRC PDOs in a dose-dependent manner. These results underscore the compound’s potential as a therapeutic agent for CRC, with significant implications for reducing tumor progression and metastasis. Given the promising in vitro efficacy, further investigations, including preclinical and clinical trials, are warranted to evaluate the compound’s therapeutic potential and translational feasibility in clinical settings. This work highlights the importance of integrating medicinal chemistry and organoid models to identify and develop novel, targeted cancer therapies. Experimental Section Chemistry General procedure Silica column chromatography (SCC) was conducted using 230–400 mesh silica gel (Siliaflash® P60). The reaction progress was monitored by Thin Layer Chromatography (TLC), which was conducted on silica gel plate (ALUGRAM ® Xtra SILG, Macherey-Nagel, Germany) and detected under UV light at 254 nm (UVGL-25, Analytik Jena US). The spectra of the compound including 1 H and 13 C spectra were obtained using a Bruker 300 MHz and an Agilent 600 MHz spectrometer. The purity of all final compounds was determined by HPLC (Agilent 1260 Infinity II, Agilent Technologies, Germany) using a Dikma (Diamonsil 5 µm C18x150x4.6 mm) column. HPLC analysis conditions are as follows: (1) ACN and water containing NH 4 OAc 10 mM with HCOOH 0.1% as a solvent system with a flow rate of 0.5 mL/min; or (2) ACN and water containing NH 4 OAc 10 mM as a solvent system with a flow rate of 0.4 mL/min. And purity of the final compounds was found to be > 95%. General procedure for the preparation of compounds 6a, D1a-D12a and 6b, D1b-D12b The appropriate amines were added to a well-stirred solution of 6,7-dichloroquinoline-5,8-dione C and DIPEA in dry THF or DCM. The resulting mixture was stirred at room temperature for 2–7 h, after which TLC analysis revealed the absence of starting materials. The reaction mixture was then quenched with water. Dichloromethane was used for extraction, and the combined organic phase was dried by adding anhydrous magnesium sulfate. Under reduced pressure, the organic phase was evaporated using a rotary evaporator, and the resulting residue was then purified by silica column chromatography (SCC) using the appropriate eluent to afford the corresponding compounds 6a, D1a-D12a (24–94%) and 6b, D1b-D12b (7–90%). 6 -Chloro-7-((2-morpholinoethyl)amino)quinoline-5,8-dione ( 6a ) and 7-Chloro-6-((2-morpholinoethyl)amino)quinoline-5,8-dione ( 6b ) The synthesis and characterization of compounds 6a and 6b were reported previously [ 40 ]. 6-Chloro-7-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione () and 7-chloro-6-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione () 6-Chloro-7-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione ( D1a ) and 7-chloro-6-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione ( D1b ) Following the general procedure, compounds D1a and D1b were synthesized by using 4-(2-aminoethyl)thiomorpholine (176 mg, 1.2 mmol), 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol), and DIPEA (208 µL, 1.2 mmol) in dry THF (9 mL). Purification by SCC using n -hexane: EA: MeOH = 4: 1: 0.3 as a solvent system to afford pure D1a and D1b as a red solid in 59% and 15% yield, respectively. Data for 7-substituted product D1a : m.p 166–167°C; 1 H NMR (600 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.7, 1.8 Hz, 1H), 8.45 (dd, J = 7.8, 1.7 Hz, 1H), 7.64 (dd, J = 7.8, 4.6 Hz, 1H), 7.07 (br s, 1H), 3.98 (q, J = 5.7 Hz, 2H), 2.83 (s, 4H), 2.73 (s, 6H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.4, 153.4, 146.3, 145.4, 134.7, 129.9, 128.4, 57.2, 54.7, 41.0, 28.0; HRMS (ESI) for C 15 H 17 N 3 O 2 SCl [M + H] + : calcd, 338.0730; found, 338.0742. HPLC purity = 97.3% (t r = 8.891 min). Data for 6-substituted product D1b : m.p 159–160°C; 1 H NMR (600 MHz, CDCl 3 ) δ H 8.99 (dd, J = 4.8, 1.7 Hz, 1H), 8.33 (dd, J = 7.9, 1.7 Hz, 1H), 7.57 (dd, J = 7.8, 4.7 Hz, 1H), 6.95 (br s, 1H), 3.94 (q, J = 5.7 Hz, 2H), 2.81–2.74 (m, 4H), 2.76–2.69 (m, 4H), 2.68 (t, J = 6.0 Hz, 2H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.3, 175.1, 155.3, 148.6, 144.3, 134.7, 127.0, 126.6, 56.9, 54.6, 41.0, 28.2; HRMS (ESI) for C 15 H 17 N 3 O 2 SCl [M + H] + : calcd, 338.0730; found, 338.0739. HPLC purity = 99.1% (t r = 8.179 min). 6-Chloro-7-((2-(pyrrolidin-1-yl)ethyl)amino)quinoline-5,8-dione ( D2a) and 7-chloro-6-((2-(pyrrolidin-1-yl)ethyl)amino)quinoline-5,8-dione ( D2b ) Following the general procedure, compounds D2a and D2b were synthesized by using N-(2-aminoethyl)pyrrolidine (140 µL, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol) in DCM (3 mL) and MeOH (2 mL). Purification by SCC using n -hexane: EA: MeOH = 4: 2 : 0.3 as a solvent system to afford pure compounds D2b and D2b as red solid in 33% and 3% yield, respectively. Data for 7-substituted product D2a : m.p. 115–116°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.7, 1.7 Hz, 1H), 8.46 (dd, J = 7.9, 1.7 Hz, 1H), 7.63 (dd, J = 7.9, 4.7 Hz, 1H), 6.97 (br s, 1H), 3.97 (q, J = 5.8 Hz, 2H), 2.78 (t, J = 6.1 Hz, 2H), 2.63–2.51 (m, 4H), 1.87–1.70 (m, 4H); 13 C NMR (151 MHz, CDCl 3 ) δ C 178.9, 175.4, 153.4, 146.4, 145.8, 134.7, 129.9, 128.4, 55.0, 53.9, 43.3, 23.8; HRMS (ESI) for C 15 H 17 N 3 O 2 Cl [M + H] + : calcd, 306.1009; found, 306.0999. HPLC purity = 98.7% (t r = 10.816 min). Data for 6-substituted product D2b : m.p 129–130°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.98 (dd, J = 4.7, 1.8 Hz, 1H), 8.32 (dd, J = 7.8, 1.8 Hz, 1H), 7.56 (dd, J = 7.8, 4.7 Hz, 1H), 6.92 (br s, 1H), 3.96 (q, J = 5.6 Hz, 2H), 2.79 (t, J = 6.0 Hz, 2H), 2.64–2.53 (m, 4H), 1.87–1.72 (m, 4H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.2, 175.1, 155.2, 148.7, 144.6, 134.7, 127.0, 126.5, 54.7, 53.7, 43.4, 23.8; HRMS (ESI) for C 15 H 17 N 3 O 2 Cl [M + H] + : calcd, 306.1009; found, 306.1003. HPLC purity = 97.9% (t r = 11.450 min). 6-Chloro-7-((2-(4-methylpiperidin-1-yl)ethyl)amino)quinoline-5,8-dione ( D3a ) and 7-chloro-6-((2-(4-methylpiperidin-1-yl)ethyl)amino)quinoline-5,8-dione ( D3b ) Following the general procedure, compounds D3a and D3b were synthesized by using 2-(4-methylpiperidin-1-yl)ethan-1-amine (158 mg, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol), and DIPEA (192 µL, 1.1 mmol) in dry THF (9 mL). Purification by SCC using n -hexane: EA: MeOH = 4: 1: 0.3 as a solvent system to afford pure compounds D3a and D3b as a red solid in 52% and 6% yield, respectively. Data for 7-substituted product D3a : m.p. 118–119°C; 1 H NMR (600 MHz, CDCl 3 ) δ H 8.89 (dd, J = 4.7, 1.7 Hz, 1H), 8.45 (dd, J = 7.9, 1.7 Hz, 1H), 7.63 (dd, J = 7.9, 4.7 Hz, 1H), 7.26 (br s, 1H), 4.02–3.89 (m, 2H), 2.94–2.83 (m, 2H), 2.68 (t, J = 6.2 Hz, 2H), 2.19–2.06 (m, 2H), 1.68–1.59 (m, 2H), 1.44–1.35 (m, 1H), 1.35–1.27 (m, 2H), 0.92 (d, J = 6.3 Hz, 3H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.3, 153.3, 146.4, 145.7, 134.7, 130.0, 128.3, 56.7, 53.5, 41.5, 34.2, 30.7, 21.8; HRMS (ESI) for C 17 H 21 N 3 O 2 Cl [M + H] + : calcd, 334.1322; found, 334.1333. HPLC purity = 98.7% (t r = 9.602 min). Data for 6-substituted product D3b : m.p. 140–141°C; 1 H NMR (600 MHz, CDCl 3 ) δ H 9.00 (dd, J = 4.7, 1.7 Hz, 1H), 8.34 (dd, J = 7.8, 1.8 Hz, 1H), 7.56 (dd, J = 7.8, 4.7 Hz, 1H), 7.11 (br s, 1H), 3.97–3.91 (m, 2H), 2.88–2.82 (m, 2H), 2.63 (t, J = 6.0 Hz, 2H), 2.66–2.61 (m, 2H), 1.68–1.62 (m, 2H), 1.45–1.34 (m, 1H), 1.31–1.21 (m, 2H), 0.94 (d, J = 6.5 Hz, 3H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.4, 175.0, 155.2, 148.7, 144.6, 134.7, 127.1, 126.5, 56.6, 53.5, 41.5, 34.5, 30.9, 21.9; HRMS (ESI) for C 17 H 21 N 3 O 2 Cl [M + H] + : calcd, 334.1322; found, 334.1332. HPLC purity = 96.5%% (t r = 8.763 min). 6-Chloro-7-((2-(4-hydroxypiperidin-1-yl)ethyl)amino)quinoline-5,8-dione ( D4a ) and 7-chloro-6-((2-(4-hydroxypiperidin-1-yl)ethyl)amino)quinoline-5,8-dione ( D4a ) 1-(2-Aminoethyl)piperidin-4-ol (216 mg, 1.5 mmol) was added to a stirred solution of 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol) and DIPEA (209 µL, 1.2 mmol) in dry DCM (9 mL). The resulting mixture was stirred at rt for 7 h. Under reduced pressure, the solvent was removed using a rotary evaporator. The resulting residue was then purified by SCC using n -hexane: EA: 2: 2: 0.2 to afford pure compounds D4a and D4b in 39% and 45% yield, respectively. Data for 7-substituted product D4a : m.p 155–156°C; 1 H NMR (300 MHz, MeOD) δ H 8.84 (dd, J = 4.8, 1.7 Hz, 1H), 8.46 (dd, J = 7.8, 1.7 Hz, 1H), 7.79 (dd, J = 7.9, 4.8 Hz, 1H), 3.98 (t, J = 6.3 Hz, 2H), 3.68–3.56 (m, 1H), 2.93–2.82 (m, 2H), 2.70 (t, J = 6.4 Hz, 2H), 2.37–2.24 (m, 2H), 1.90–1.79 (m, 2H), 1.62–1.49 (m, 2H); 13 C NMR (151 MHz, MeOD) δ C 179.6, 176.5, 153.9, 147.6, 135.8, 131.0, 129.9, 68.2, 58.4, 52.0, 42.7, 35.0; HRMS (ESI) for C 16 H 19 N 3 O 3 Cl [M + H] + : calcd, 336.1115; found, 336.1122. HPLC purity = 99.2% (t r = 11.494 min). Data for 6-substituted product D4b : m.p 122–123°C; 1 H NMR (300 MHz, MeOD) δ H 8.89 (dd, J = 4.9, 1.6 Hz, 1H), 8.42 (dd, J = 7.8, 1.7 Hz, 1H), 7.73 (dd, J = 7.9, 4.8 Hz, 1H), 3.99 (t, J = 6.4 Hz, 2H), 3.73–3.58 (m, 1H), 2.97–2.86 (m, 2H), 2.75 (t, J = 6.4 Hz, 2H), 2.46–2.28 (m, 2H), 1.94–1.82 (m, 2H), 1.67–1.49 (m, 2H); 13 C NMR (151 MHz, MeOD) δ C 180.8, 176.2, 155.4, 149.2, 148.3, 136.2, 128.8, 128.4, 67.8, 58.2, 51.9, 42.4, 34.8; HRMS (ESI) for C 16 H 19 N 3 O 3 Cl [M + H] + : calcd, 336.1115; found, 336.1127. HPLC purity = 95.1% (t r = 10.987 min). Synthesis of intermediates 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)acetonitrile To a stirred a mixture of 1,4-dioxa-8-azaspiro[4.5]decane (716 mg, 5.0 mmol) and DIPEA (954 µL, 5.48 mmol) in toluene (9 mL) was added dropwise 2-chloroacetonitrile (331 µL, 5.24 mmol). The mixture was then allowed to stir for 2 hours at 80°C. The mixture was allowed to cool to room temperature. The organic solvent was evaporated by a rotary evaporator to get a residue. The residue was then quenched with water. Dichloromethane was used for extraction, and the organic phase was dried by adding anhydrous magnesium sulfate. Under reduced pressure, the organic phase was evaporated using a rotary evaporator to obtain a crude product in 87%. 1 H NMR (300 MHz, MeOD) δ H 3.95 (s, 4H), 3.66 (s, 2H), 2.72–2.62 (m, 4H), 1.81–1.70 (m, 4H). Synthesis of intermediates 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine To a round-bottom flask, a mixture of 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)acetonitrile (790 mg, 4.34 mmol) in dry THF (10 mL) was placed, then it was equipped with a rubber septum. The flask was degasified and then flushed with nitrogen gas. Lithium aluminum hydride 2.4 M in THF (2.7 mL, 6.50 mmol) was added dropwise through a syringe and kept stirring for 1 hour under an ice bath. Then, the resulting reaction solution was allowed to stir at room temperature overnight. Under the cooling condition, the mixture was added with water (5 drops) and 1.5 mL of NaOH (aq), then kept stirring vigorously for 30 minutes. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. Purification by SCC using gradient eluent of DCM: MeOH = 3: 1◊ 3: 2◊ 3: 3 to afford the desired compound in 75% yield. 1 H NMR (300 MHz, CDCl 3 ) δ H 3.93 (s, 4H), 2.77 (t, J = 6.2 Hz, 2H), 2.57–2.46 (m, 4H), 2.42 (t, J = 6.2 Hz, 2H), 1.77 (s, 2H), 1.75–1.67 (m, 4H). 7-((2-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)ethyl)amino)-6-chloroquinoline-5,8-dione ( D5a ) and 6-((2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethyl)amino)-7-chloroquinoline-5,8-dione ( D5b ) Following the general procedure, compounds D5a and D5b were synthesized using2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine (436 mg, 2.34 mmol), C (445 mg, 1.95 mmol) and DIPEA (436 µL, 2.34 mmol) in dry THF (15 mL). Purification by SCC using n -hexane: EA: MeOH = 20: 20: 1 as a solvent system to afford pure compounds D5a and D5b as red solid in 30% and 46%, respectively. Data for 7-substituted product D5a : m.p 162–163°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.7, 1.7 Hz, 1H), 8.45 (dd, J = 7.9, 1.7 Hz, 1H), 7.63 (dd, J = 7.9, 4.7 Hz, 1H), 7.13 (br s, 1H), 4.10–3.83 (m, 6H), 2.71 (t, J = 6.0 Hz, 2H), 2.68–2.57 (m, 4H), 1.83–1.74 (m, 4H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.2, 153.4, 146.4, 145.5, 134.7, 129.9, 128.4, 106.9, 64.5, 56.2, 51.1, 41.6, 34.8; HRMS (ESI) for C 18 H 21 N 3 O 4 Cl [M + H] + : calcd, 378.1221; found, 378.1220. HPLC purity = 97.4% (t r = 8.930 min). Data for 6-substituted product D5b : m.p 161–162°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 9.00 (dd, J = 4.8, 1.8 Hz, 1H), 8.33 (dd, J = 7.9, 1.8 Hz, 1H), 7.57 (dd, J = 7.8, 4.7 Hz, 1H), 7.07 (br s, 1H), 4.02–3.91 (m, 6H), 2.72 (t, J = 6.0 Hz, 2H), 2.67–2.61 (m, 4H), 1.85–1.75 (m, 4H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.2, 175.1, 155.2, 148.6, 144.4, 134.7, 127.0, 126.5, 107.1, 64.4, 55.8, 50.9, 41.5, 35.0; HRMS (ESI) for C 18 H 21 N 3 O 4 Cl [M + H] + : calcd, 378.1221; found, 378.1205. HPLC purity = 98.4% (t r = 8.089 min). 6-Chloro-7-((2-(piperazin-1-yl)ethyl)amino)quinoline-5,8-dione ( D6a ) and 7-Chloro-6-((2-(piperazin-1-yl)ethyl)amino)quinoline-5,8-dione ( D6b ) To a solution of D8a (180 mg, 0.43 mmol) in dry DCM (6 mL) was dropwise added trifluoroacetic acid (2 mL) in an ice bath. The mixture was stirred for 30 minutes under ice bath, then allowed to stir at room temperature for 2 hours. After confirming the completion of the reaction, the solvent was removed by a rotary evaporator to obtain the crude product. Purification by SCC using DCM: MeOH = 30: 5 as a solvent system to afford pure compound D6a as a red solid in 94% yield. Data for 7-substituted product D6a : m.p 154–155°C; 1 H NMR (300 MHz, MeOD) δ H 8.86 (dd, J = 4.8, 1.7 Hz, 1H), 8.46 (dd, J = 7.9, 1.6 Hz, 1H), 7.81 (dd, J = 7.9, 4.7 Hz, 1H), 4.01 (t, J = 6.1 Hz, 2H), 3.26–3.17 (m, 4H), 2.85–2.73 (m, 6H); 13 C NMR (151 MHz, MeOD) δ C 179.9, 176.5, 154.0, 147.5, 135.8, 130.9, 130.0, 58.3, 50.5, 44.9, 42.3. HRMS (ESI) for C 15 H 18 N 4 O 2 Cl [M + H] + : calcd, 321.1118; found, 321.1130. HPLC purity = 99.1% (t r = 10.715 min). The synthetic procedure of compound D6b is similar to that of compound D6a using D8b (180 mg, 0.43 mmol in dry DCM (6 mL) and trifluoroacetic acid (2 mL). Purification by SCC using DCM: MeOH = 30: 2 as a solvent system to afford pure compound D6b as a red solid in 90% yield. Data for 6-substituted product D6b : m.p 173–174°C; 1 H NMR (600 MHz, DMSO) δ H 8.95 (dd, J = 4.7, 1.7 Hz, 1H), 8.33 (dd, J = 7.8, 1.7 Hz, 1H), 7.75 (dd, J = 7.9, 4.7 Hz, 1H), 7.32 (br s, 1H), 3.85 (q, J = 6.4 Hz, 2H), 3.04 (t, J = 5.1 Hz, 4H), 2.73–2.58 (m, 6H); 13 C NMR (151 MHz, DMSO) δ C 179.9, 174.0, 154.5, 147.7, 145.4, 134.4, 127.3, 126.9, 56.8, 49.0, 42.9, 40.9; HRMS (ESI) for C 15 H 18 N 4 O 2 Cl [M + H] + : calcd, 321.1118; found, 321.1118. HPLC purity = 98.9% (t r = 9.852 min). 6-Chloro-7-((2-(4-methylpiperazin-1-yl)ethyl)amino)quinoline-5,8-dione ( D7a ) and 7-chloro-6-((2-(4-methylpiperazin-1-yl)ethyl)amino)quinoline-5,8-dione ( D7b ) Following the general procedure, compounds D7a and D7b were synthesized by using 2-(4-methylpiperazin-1-yl)ethylamine (158 mg, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol) and DIPEA (192 µL, 1.1 mmol) in dry THF (9 mL). Purification by SCC using n -hexane: EA: MeOH = 3: 5: 0.1 as a solvent system to afford pure compounds D7a and D7b as red solid in 60% and 6% yield, respectively. Data for 7-substituted product D7a : m.p 130–131°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.91 (dd, J = 4.7, 1.7 Hz, 1H), 8.47 (dd, J = 7.9, 1.7 Hz, 1H), 7.65 (dd, J = 7.9, 4.7 Hz, 1H), 7.09 (br s, 1H), 3.97 (q, J = 5.9 Hz, 2H), 2.72 (t, J = 6.0 Hz, 2H), 2.65 (br s, 8H), 2.39 (s, 3H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.4, 153.4, 146.3, 145.5, 134.7, 130.0, 128.4, 56.1, 55.2, 52.3, 46.0, 41.3; HRMS (ESI) for C 16 H 20 N 4 O 2 Cl [M + H] + : calcd, 335.1275; found, 335.1277. HPLC purity = 99.0% (t r = 11.096 min). Data for 6-substituted product D7b : m.p. 193–194°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.99 (dd, J = 4.7, 1.8 Hz, 1H), 8.33 (dd, J = 7.8, 1.8 Hz, 1H), 7.57 (dd, J = 7.9, 4.7 Hz, 1H), 7.01 (br s, 1H), 3.95 (q, J = 5.9 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H), 2.59 (br s, 8H), 2.31 (s, 3H); 13 C NMR (151 MHz, CDCl 3 ) δ H 180.3, 175.1, 155.2, 148.7, 144.4, 134.7, 127.0, 126.5, 56.2, 55.2, 52.5, 46.1, 41.2; HRMS (ESI) for C 16 H 20 N 4 O 2 Cl [M + H] + : calcd, 335.1275; found, 335.1278. HPLC purity = 97.4% (t r = 11.171 min). tert-butyl 4-(2-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)ethyl)piperazine-1-carboxylate ( D8a ) and tert-butyl 4-(2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)ethyl)piperazine-1-carboxylate ( D8b ) Following the general procedure, compounds D8a and D8b were synthesized by using tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (275 mg, 1.2 mmol), 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol), and DIPEA (209 µL, 1.2 mmol) in dry THF (9 mL). Purification by SCC using n -hexane: EA: MeOH = 2: 1: 0.2 as a solvent system to afford pure compounds D8a and D8b as a red solid in 68% and 28% yield, respectively. Data for 7-substituted product D8a : m.p 168–169°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.7, 1.7 Hz, 1H), 8.46 (dd, J = 7.9, 1.7 Hz, 1H), 7.64 (dd, J = 7.9, 4.7 Hz, 1H), 7.03 (br s, 1H), 4.00 (q, J = 5.7 Hz, 3H), 3.53–3.43 (m, 4H), 2.72 (t, J = 5.5, 2H), 2.49 (br s, 4H), 1.47 (s, 9H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.4, 154.7, 153.5, 146.3, 145.4, 134.7, 129.9, 128.4, 80.0, 56.6, 52.5, 43.9, 43.2, 41.1, 28.5; HRMS (ESI) for C 20 H 26 N 4 O 4 Cl [M + H] + : calcd, 421.1643; found, 421.1654. HPLC purity = 99.0% (t r = 13.053 min). Data for 6-substituted product D8b : m.p 151–152°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 9.00 (dd, J = 4.8, 1.7 Hz, 1H), 8.33 (dd, J = 7.8, 1.8 Hz, 1H), 7.57 (dd, J = 7.8, 4.7 Hz, 1H), 6.95 (br s, 1H), 3.97 (q, J = 5.7 Hz, 2H), 3.48 (t, J = 5.1 Hz, 5H), 2.70 (t, J = 5.9 Hz, 2H), 2.47 (br s, 4H), 1.45 (s, 9H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.3, 175.1, 155.3, 154.8, 148.6, 144.3, 134.7, 127.0, 126.6, 80.0, 56.3, 52.4, 44.2, 43.4, 41.1, 28.5; HRMS (ESI) for C 20 H 26 N 4 O 4 Cl [M + H] + : calcd, 421.1643; found, 421.1643. HPLC purity = 99.0% (t r = 12.610 min). tert-butyl (1-(2-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)ethyl)piperidin-4-yl)carbamate ( D9a ) and tert-butyl (1-(2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)ethyl)piperidin-4-yl)carbamate ( D9b ) Following the general procedure, compounds D9a and D9b were synthesized by using tert-butyl (1-(2-aminoethyl)piperidin-4-yl)carbamate (383 mg, 1.57 mmol), 6,7-dichloroquinoline-5,8-dione C (299 mg, 1.31 mmol), and DIPEA (273 µL, 1.57 mmol) in dry THF (9 mL). Purification by SCC using n -hexane: EA: MeOH = 1: 2: 0.2 as a solvent system to afford pure compounds D9a and D9b as red solid in 71% and 17%, respectively. Data for 7-substituted product D9a : m.p 167–168°C; 1 H NMR (600 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.7, 1.7 Hz, 1H), 8.46 (dd, J = 7.8, 1.7 Hz, 1H), 7.64 (dd, J = 7.8, 4.6 Hz, 1H), 7.18 (br s, 1H), 4.46 (br s, 1H), 3.94 (q, J = 5.7 Hz, 2H), 3.50 (br s, 1H), 2.85–2.80 (m, 2H), 2.66 (t, J = 6.0 Hz, 2H), 2.26–2.18 (m, 2H), 1.99–1.93 (m, 2H), 1.51–1.41 (m, 2H), 1.44 (s, 9H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.4, 155.3, 153.4, 146.3, 145.4, 134.7, 130.0, 128.4, 79.5, 56.0, 51.9, 47.8, 41.5, 32.8, 28.5; HRMS (ESI) for C 21 H 28 N 4 O 4 Cl [M + H] + : calcd, 435.1799; found, 435.1794. HPLC purity = 97.1% (t r = 10.923 min). Data for 6-substituted product D9b : m.p 80–81°C; 1 H NMR (600 MHz, CDCl 3 ) δ H 9.00 (dd, J = 4.7, 1.7 Hz, 1H), 8.33 (dd, J = 7.9, 1.7 Hz, 1H), 7.57 (dd, J = 7.8, 4.7 Hz, 1H), 7.03 (br s, 1H), 4.48 (br s, 1H), 3.94 (q, J = 5.7 Hz, 2H), 3.51 (br s, 1H), 2.87–2.82 (m, 2H), 2.67 (t, J = 6.0 Hz, 2H), 2.27–2.20 (m, 2H), 2.01–1.95 (m, 2H), 1.52–1.45 (m, 2H), 1.44 (s, 9H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.3, 175.1, 155.3, 155.2, 148.6, 144.4, 134.7, 127.0, 126.5, 79.5, 56.1, 51.9, 47.8, 41.4, 32.8, 28.5; HRMS (ESI) for C 21 H 28 N 4 O 4 Cl [M + H] + : calcd, 435.1799; found, 435.1798. HPLC purity = 98.5% (t r = 15.522 min). 6-Chloro-7-((2-((2-hydroxyethyl)(methyl)amino)ethyl)amino)quinoline-5,8-dione ( D10a ) and 7-chloro-6-((2-((2-hydroxyethyl)(methyl)amino)ethyl)amino)quinoline-5,8-dione ( D10b ) A mixture of 2-(2-aminoethylamino)ethanol (142 mg, 1.2 mmol) and 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol) in dry THF (9 mL) was stirred at room temperature for 6 h. The mixture was subsequently concentrated under reduced pressure. The resulting residue was purified by SCC using n -hexane: EA: MeOH = 1: 3: 0.1 as a solvent system to afford pure compounds D10a and D10b as red solid in 24% and 27%, respectively. Data for 7-substituted product D10a : 108–109°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.7, 1.7 Hz, 1H), 8.46 (dd, J = 7.9, 1.7 Hz, 1H), 7.64 (dd, J = 7.9, 4.7 Hz, 1H), 6.93 (br s, 1H), 4.01 (q, J = 5.8 Hz, 2H), 3.72 (t, J = 5.1 Hz, 2H), 2.82 (t, J = 6.0 Hz, 2H), 2.69 (t, J = 5.2 Hz, 2H), 2.39 (s, 3H); 13 C NMR (151 MHz, CDCl 3 ) δ C 179.1, 175.6, 153.5, 146.2, 145.0, 134.8, 130.0, 128.5, 59.3, 59.2, 56.9, 42.1, 41.6; HRMS (ESI) for C 14 H 17 N 3 O 3 Cl [M + H] + : calcd, 310.0958; found, 310.0955. HPLC purity = 97.1% (t r = 11.488 min). Data for 6-substituted product D10b : m.p 136–137°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 9.00 (dd, J = 4.7, 1.7 Hz, 1H), 8.34 (dd, J = 7.8, 1.7 Hz, 1H), 7.57 (dd, J = 7.9, 4.7 Hz, 1H), 6.85 (br s, 1H), 3.98 (q, J = 5.6 Hz, 2H), 3.70 (t, J = 5.2 Hz, 2H), 2.76 (t, J = 5.9 Hz, 2H), 2.65 (t, J = 5.3 Hz, 2H), 2.34 (s, 3H); 13 C NMR (151 MHz, CDCl 3 ) δ C 180.4, 175.3, 155.4, 148.7, 144.1, 134.8, 126.9, 126.6, 59.3, 59.2, 56.7, 41.9, 41.4; HRMS (ESI) for C 14 H 17 N 3 O 3 Cl [M + H] + : calcd, 310.0958; found, 310.0962. HPLC purity = 95.8% (t r = 11.038 min). 6-Chloro-7-((2-(dimethylamino)ethyl)amino)quinoline-5,8-dione ( D11a ) and 7-chloro-6-((2-(dimethylamino)ethyl)amino)quinoline-5,8-dione ( D11b ) Following the general procedure, compounds D11a and D11b were synthesized by using unsym-dimethyl-ethylenediamine (120 µL, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione C (228 mg, 1.0 mmol) and DIPEA (192 µL, 1.1 mmol) in dry THF (9 mL). Purification by SCC using n -hexane: EA: MeOH = 3: 5: 0.3 as a solvent system to afford pure compounds D11a and D11b as a red solid in 37% and 7% yield, respectively. Data for 7-substituted product D11a : m.p. 133–134°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 8.90 (dd, J = 4.8, 1.7 Hz, 1H), 8.46 (dd, J = 8.1, 1.7 Hz, 1H), 7.63 (dd, J = 7.9, 4.6 Hz, 1H), 6.90 (br s, 1H), 3.95 (q, J = 5.7 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2H), 2.29 (s, 6H); 13 C NMR (151 MHz, CDCl 3 ) δ C 178.8, 175.4, 153.4, 146.4, 145.8, 134.7, 129.9, 128.3, 58.1, 45.0, 42.4; HRMS (ESI) for C 13 H 15 N 3 O 2 Cl [M + H] + : calcd, 280.0853; found, 280.0858. HPLC purity = 99.2% (t r = 11.934 min). Data for 6-substituted product D11b : m.p. 132–133°C; 1 H NMR (300 MHz, CDCl 3 ) δ H 9.00 (dd, J = 4.7, 1.7 Hz, 1H), 8.33 (dd, J = 7.8, 1.8 Hz, 1H), 7.57 (dd, J = 7.8, 4.7 Hz, 1H), 6.89 (br s, 1H), 3.93 (q, J = 5.6 Hz, 2H), 2.59 (t, J = 5.9 Hz, 2H), 2.30 (s, 6H); 13 C NMR (151 MHz, CDCl 3 ) δ H 180.2, 175.1, 155.2, 148.7, 144.6, 134.7, 127.1, 126.5, 57.9, 45.0, 42.2; HRMS (ESI) for C 13 H 15 N 3 O 2 Cl [M + H] + : calcd, 280.0853; found, 280.0862. HPLC purity = 97.1% (t r = 11.520 min). Synthesis of intermediate 2-(dis(2-hydroxyethyl)amino)acetonitrile 2,2'-Azanediylbis(ethan-1-ol) (2.5 g, 23.8 mmol) and TEA (7 mL, 50.2 mmol) was mixed in anhydrous methanol (15 mL), followed by the addition of 2-bromoacetonitrile (3.9 g, 32.5 mmol) dropwise under an ice-bath. Then, the reaction mixture was allowed to stir at room temperature for 3 h. The mixture was concentrated under reduced pressure to remove the solvent and then was added dichloromethane (15 mL). The insoluble was filtered off, and the filtrate was concentrated under reduced pressure to obtain a residue. Purification by SCC using DCM: MeOH = 3: 0.2 to give the desired compound in 50% yield. 1 H NMR (300 MHz, CDCl 3 ) δ H 3.33–2.84 (m, 4H), 1.43–1.31 (m, 6H). Synthesis of intermediate 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) To a suspension of 2-(bis(2-hydroxyethyl)amino)acetonitrile (730 mg, 5.06 mmol) in dry THF (8 mL) was added 3.5 mL of LiAlH 4 2.4 in THF solution dropwise while stirring under an ice-bath. The mixture was stirred at 0°C for 1 h, then slowly increased to room temperature overnight. The reaction mixture was added 5 drops of water under an ice bath, then quenched with NaOH 1.0 N solution (1.5 mL). The resulting mixture was filtered using a pad of celite, then the filtrate was collected, followed by the removal of the solvent under reduced pressure. Purification by SCC using DCM: MeOH = 4: 1 to give the desired compound in 28% yield. 1 H NMR (300 MHz, MeOD) δ H 3.64–3.54 (m, 4H), 2.75–2.56 (m, 8H). 7-((2-(bis(2-Hydroxyethyl)amino)ethyl)amino)-6-chloroquinoline-5,8-dione ( D12a ) and 6-((2-(bis(2-hydroxyethyl)amino)ethyl)amino)-7-chloroquinoline-5,8-dione ( D12b ) A mixture of 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) (178 mg, 1.20 mmol) and C (150 mg, 0.66 mmol) in dry THF (9 mL) was stirred at rt for 7 h. The reaction mixture was concentrated using a rotary evaporator. The resulting residue was then purified by SCC using n -hexane: EA: MeOH = 2: 2: 0.3 ◊ 0.2: 0.2: 0.4 ◊ 2: 2: 0.5 as a solvent system to afford pure compounds D12a and D12b as a red solid in 38% and 39% yield, respectively. Data for 7-substituted product D12a : m.p 105–106°C; 1 H NMR (600 MHz, MeOD) δ H 8.84 (dd, J = 4.7, 1.6 Hz, 1H), 8.45 (dd, J = 7.9, 1.6 Hz, 1H), 7.79 (dd, J = 7.8, 4.7 Hz, 1H), 3.97 (t, J = 6.2 Hz, 2H), 3.65 (t, J = 5.8 Hz, 4H), 2.93 (t, J = 6.2 Hz, 2H), 2.77 (t, J = 5.8 Hz, 4H); 13 C NMR (151 MHz, MeOD) δ C 179.8, 177.0, 153.8, 147.5, 135.8, 131.0, 129.9, 60.9, 57.6, 56.0, 43.6; HRMS (ESI) for C 15 H 19 N 3 O 4 Cl [M + H] + : calcd, 340.1064; found, 340.1064. HPLC purity = 99.2% (tr = 11.633 min). Data for 6-substituted product D12b : m.p 146–147°C; 1 H NMR (600 MHz, MeOD) δ H 8.89 (dd, J = 4.8, 1.6 Hz, 1H), 8.42 (dd, J = 7.9, 1.7 Hz, 1H), 7.73 (dd, J = 7.8, 4.7 Hz, 1H), 4.00 (t, J = 6.2 Hz, 2H), 3.68 (t, J = 5.6 Hz, 4H), 3.01 (t, J = 6.4 Hz, 2H), 2.85 (t, J = 5.7 Hz, 4H); 13 C NMR (151 MHz, MeOD) δ C 180.9, 176.3, 155.4, 149.2, 147.1, 136.2, 128.8, 128.3, 60.4, 57.5, 55.9, 43.2; HRMS (ESI) for C 15 H 19 N 3 O 4 Cl [M + H] + : calcd, 340.1064 ; found, 340.1064. HPLC purity = 96.9% (t r = 10.981 min). Molecular docking study The molecular docking simulation was performed with the Discovery Studio Client 2022 (Biovia Corp. CA, USA) to elucidate the interaction of the selected derivatives into the CDC25C active site. The CDC25C crystal protein (PDB ID: 3OP3) was downloaded from RSCB protein data bank website, and imported to the Discovery Studio (DS) Client 2022. The crystal protein was cleaned by removing unwanted ligands and water molecules, and hydrogen was added before the dock. Compounds 6a , D3a , and D11a were used here and their chemical structures were drawn using Chem3D Professional 15.0 software (PerkinElmer Informatics, Inc. CA, USA). The compounds were energy minimized at root-mean-square gradient tolerance of 0.01 and saved as SD files for further operation. The LibDock molecular docking method was used to conduct the experiment after protein setup by defining the binding site from the active site of CDC25C to dock all the prepared ligands ( 6a , D3a , and D11a ). Based on the highest LibDock score of respective compounds, their binding interactions with CDC25C crystal protein were analyzed. Biology Most materials and methodologies of biological evaluations have been applied in our previous report. Please refer to reference [ 40 ]. Cell culture HL-60 promyeloblasts from acute promyelocytic leukemia and KG-1 macrophage from acute myelogenous leukemia were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (11875-093, Gibco). MV-4-11 macrophages from biphenotypic B-myelomonocytic leukemia and K-562 lymphoblast from chronic myelogenous leukemia were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (12200-036, Gibco), with 1.5g/L and 3g/L sodium bicarbonate, respectively. All media were supplemented with 10% fetal bovine serum (F0926, Sigma) and 1% penicillin-streptomycin-glutamine (10378-016, Gibco) in a humidified atmosphere at 37℃ and 5% CO 2 . Chemicals and antibodies Colchicine and Paclitaxel (Taxol) were purchased from Sigma (C9754) and Cytoskeleton, Inc. (TXD01), 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 H3 (ab1791; Abcam); anti-Ataxia-telangiectasia mutated kinase (ATM) pS1981 (560007, BD Pharmingen™); 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-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-histone H3 (ab1791, Abcam); anti-Induced myeloid leukemia cell differentiation protein (Mcl-1)(ab32087, Abcam); anti-caspase 3 (NB100-56708, Novus Biologicals); anti-B-cell leukemia/lymphoma 2 (Bcl-2)(#3498, Cell Signaling); anti-Caspase 9 (#9502, Cell Signaling); anti-histone H4K20me1 (07–1570; Millipore); anti-histone H4 (ab31830; Abcam). Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse (115-035-003; Jackson ImmunoResearch Labs); anti-rabbit (111-035-003; Jackson ImmunoResearch Labs) antibodies. MTS assay Cells were seeded in 96-well plates and treated with indicated compounds for 72 h. The surviving cells were determined by incubating them with 0.2 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium (MTS; Abcam, ab223881) and measuring the absorbance at 490 nm by using a PerkinElmer VICTOR3™ Multilabel Plate Reader. The concentration of 50% growth inhibition (IC 50 ) was calculated using the CompuSyn software. Flow cytometry Cells were collected, fixed with 70% ice-cold ethanol, washed with cold phosphate-buffered saline containing 1% FBS, and 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 minutes. DNA content was analyzed using the Becton Dickinson FACSCalibur Flow Cytometer and the cell-cycle profiles were plotted with the FlowJo software. Western blotting Cells were lysed with Laemmli sample buffer (LSB), and proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membranes. After blocking with 5% skim milk, indicated proteins were probed with specific primary antibodies, followed by hybridization with HRP-conjugated species-specific secondary antibodies. Signals were developed using enhanced chemiluminescence substrates (Bio-Rad) and detected by using the Invitrogen iBright FL-1500 system. In vitro CDC25C activity assay Enzyme inhibition assays were performed by the Eurofins Panlabs, Inc. Compounds were pre-incubated with human CDC25C enzyme purified from E. coli in the reaction buffer (50 mM Tris-HCl, pH 8.5, 0.1% BSA, 1mM DTT, 100 mM NaCl) for 15 minutes and then mixed with 15.0 µM of 3-O-methylfluorescein phosphate for 60 minutes. 3-Omethylfluorescein signals were quantified and the percentages of inhibition were plotted using GraphPad Prism 6. Genomic DNA purification Cells were washed with PBS and resuspended in DNA extraction lysis buffer (10mM Tris-HCl, pH8.0, 10mM EDTA, and 1% SDS) containing 400 µg/ml of proteinase K (Worthington Biochemical; 39450-01-6). After overnight incubation at 56 ℃, lysates were purified using Phenol: Chloroform: Isoamyl Alcohol (25:24:1, v/v; Invitrogen™; 15593031). The supernatant was treated with RNase A (250µg/ml) at 37℃ for 1h, followed by the second purification using Phenol: Chloroform: Isoamyl Alcohol. Genomic DNAs were finally precipitated by ethanol and dissolved in pure water, followed by 2% agarose gel separation and signal detection using the Invitrogen iBright FL-1500 system. Cell proliferation assay PDOs were seeded at a density of 5×10 4 in 10 µL of rBM in 24-well plates and cultured for 3 days. To assess the growth of the PDOs, 72 h after seeding, the cultures were treated with increasing concentrations of D3a , D3b , D11a , and D11b , followed by a cell proliferation assay using the Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer's protocol. In brief, 10 µL of CCK-8 reagent was added to 200 µL of culture medium and incubated for 2 h at 37°C in a 5% CO2. The absorbance at 450 nm was then measured using a SpectraMax iD5 microplate reader. Dual-chamber invasion assay 100 CRC organoids were cultured and plated on Transwell inserts (BD Biosciences) coated with a thin layer of collagen type I (BD Biosciences) under serum free medium overnight. In the presence of 10% FBS incubated for 24 h to allow invasion through the collagen. After the incubation, cells that invaded the insert membrane were fixed, stained with SYTOX Green (Invitrogen), and counted under a fluorescence microscope. 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. Acknowledgment This research were supported by the National Science and Technology Council of Taiwan (grant no. 112-2113-M-038-001). This work was also supported by the Higher Education Sprout Project by the Ministry of Education (MOE) of Taiwan (grant no. DP2-TMU-112-C-03). We sincerely thank Dr. Li-Jung Juan (Genomics Research Center, Academia Sinica) for providing experimental resources and Richa Upadhyay for performing ADME prediction analysis. 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Sci Rep 7:42717. https://doi.org/10.1038/srep42717 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations There is no conflict of interest Supplementary Files Scheme1.jpeg Scheme 1. Synthetic approaches to compounds 6a, D1a-D12a and 6b, D1b-D12b. Reagents and conditions: (i) Pd/C 5% (w/w), H 2 , MeOH, rt, 8 h; (ii) conc. HCl, NaClO 3 , 40-50 °C, 2 h; (iii) corresponding amines, with or without DIPEA, THF, DCM or MeOH, rt, 2-7 h, D8a (D8b) is converted to D6a (D6b): TFA, DCM, 0 °C to rt, 2 h. Supplementaryfile20250322JP.docx Quinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids Cite Share Download PDF Status: Posted Version 1 posted 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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Tsai","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kelvin","middleName":"K.","lastName":"Tsai","suffix":""},{"id":479449837,"identity":"a7aa97f5-5fc8-498f-9bbe-ceef7efd5fc9","order_by":7,"name":"Sung-Bau Lee","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sung-Bau","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-03-22 10:20:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6282946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6282946/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86319784,"identity":"8a5667e8-5a11-488b-b2ed-82a165ce1367","added_by":"auto","created_at":"2025-07-09 09:29:12","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203445,"visible":true,"origin":"","legend":"\u003cp\u003eQuinone-based molecules as CDC25 inhibitors\u003c/p\u003e","description":"","filename":"1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/e1659ec3b16ae50863c22913.jpeg"},{"id":86319773,"identity":"04d38534-d992-49fc-9242-811da19eb5e1","added_by":"auto","created_at":"2025-07-09 09:29:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":463360,"visible":true,"origin":"","legend":"\u003cp\u003eStructural modification of compound \u003cstrong\u003eNSC663284 \u003c/strong\u003e(\u003cstrong\u003e6a\u003c/strong\u003e) by introducing various side chains at the C-7 and C-6 positions\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/3f6698ddfa37e33074c59ad6.jpeg"},{"id":86319722,"identity":"38bc5833-3d09-47c2-93f2-a4d52771be32","added_by":"auto","created_at":"2025-07-09 09:28:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD3a and D11a trigger apoptosis in HL-60 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Analysis of cell cycle profiles of HL-60 cells treated with 1 μM indicated compounds for 72 h. Percentages of the sub-G\u003csub\u003e1\u003c/sub\u003e population from at least three biological replicates are shown and displayed with means and SDs (\u003cem\u003en\u003c/em\u003e ≥ 3). (B) Analysis of apoptotic proteins in HL-60 cells treated with 1 μM of indicated compounds for 72h. Representative results from one of two biological replicates are shown. Data were collected from different sets of gel electrophoresis with equal loading of the same samples. p-Casp: Precursor caspase; a-Casp: Active caspase.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/ae3eb3ae3fc886bb7675664f.png"},{"id":86319774,"identity":"2692f5d3-e689-43f2-a8fe-c4e791163568","added_by":"auto","created_at":"2025-07-09 09:29:00","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD3a and D11a suppress CDC25C activity.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/dfd892a8f80eb62e17db5f64.jpeg"},{"id":86319781,"identity":"af1780f3-9e55-4035-a30f-7356d6b8b2de","added_by":"auto","created_at":"2025-07-09 09:29:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD3a and D11a impede CDK1 dephosphorylation and mitosis progression in HL-60 cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/727c76719867a6eac58a9c77.png"},{"id":86319778,"identity":"b7ae7559-ee09-462e-b453-31fdeb309976","added_by":"auto","created_at":"2025-07-09 09:29:08","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1314498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking and binding interaction prediction of compounds 6a, D3a, and D11a in the CDC25C crystal protein (PDB ID: 3OP3).\u003c/strong\u003e \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e crystal protein 3D interactions are present in the respective figures (A), (B), (D), (E), (G), and (H). 2D interaction images of compounds \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e are presented in figure (C), (F), and (I) respectively. (J) and (K) represents the merged docking pose of compounds \u003cstrong\u003e6a\u003c/strong\u003e (orange), \u003cstrong\u003eD3a\u003c/strong\u003e (purple), and \u003cstrong\u003eD11a\u003c/strong\u003e(green) at the active site of the CDC25C crystal protein.\u003c/p\u003e","description":"","filename":"6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/cafb204d4da15c667d1a65e4.jpeg"},{"id":86319772,"identity":"028129fd-e486-4013-b762-eedea21df856","added_by":"auto","created_at":"2025-07-09 09:28:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":89924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD3a and D11a trigger DNA damage and induce DSBs in HL-60 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eAnalysis of DNA damage signaling in HL-60 cells treated with 1 μM of indicated compounds for 4h. \u003cstrong\u003e(B) \u003c/strong\u003eGel electrophoresis of genomic DNAs isolated from HL-60 cells treated with 1 μM of indicated compounds for 4 and 8 h. Representative results from one of two biological replicates are shown. bp: Base pair\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/7518a92df8872c326c37cd18.png"},{"id":86319787,"identity":"eb80cc5f-7461-4a6c-9637-130e4e3068f9","added_by":"auto","created_at":"2025-07-09 09:29:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":466851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eD11b reduces the proliferation and invasion abilities of CRC PDOs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eAnalysis of cell proliferation of CRC cancer organoids treated with indicated concentrations of \u003cstrong\u003eD11b\u003c/strong\u003e. Representative images of PDOs at the endpoint (upper) and the line graphs of the proliferation rate (lower) are shown and displayed with means and SEMs (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003e(B)\u003c/strong\u003e Analysis of the invasion ability of CRC cancer organoids treated with indicated concentrations of \u003cstrong\u003eD11b\u003c/strong\u003e. Representative images (upper) and quantification results (lower) are shown and displayed with means and SEMs (\u003cem\u003en\u003c/em\u003e = 3). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/65c7f049b85f86a20727cc27.png"},{"id":86320279,"identity":"51f65243-8a3e-4c6e-b395-809b3d4afc5f","added_by":"auto","created_at":"2025-07-09 09:37:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5187174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/f284d758-cc5e-414c-85f5-f454251c8654.pdf"},{"id":86319776,"identity":"7913dd8f-6b6f-4035-87ed-c38640c6b8df","added_by":"auto","created_at":"2025-07-09 09:29:02","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":468781,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1.\u003cstrong\u003e \u003c/strong\u003eSynthetic approaches to compounds \u003cstrong\u003e6a, D1a-D12a \u003c/strong\u003eand \u003cstrong\u003e6b, D1b-D12b.\u003c/strong\u003e Reagents and conditions: (i) Pd/C 5% (w/w), H\u003csub\u003e2\u003c/sub\u003e, MeOH, rt, 8 h; (ii) conc. HCl, NaClO\u003csub\u003e3\u003c/sub\u003e, 40-50 °C, 2 h; (iii) corresponding amines, with or without DIPEA, THF, DCM or MeOH, rt, 2-7 h, \u003cstrong\u003eD8a \u003c/strong\u003e(\u003cstrong\u003eD8b\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eis\u003cstrong\u003e \u003c/strong\u003econverted to\u003cstrong\u003e D6a \u003c/strong\u003e(\u003cstrong\u003eD6b\u003c/strong\u003e): TFA, DCM, 0 °C to rt, 2 h.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Scheme1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/dbaaa23147f4942656390667.jpeg"},{"id":86319791,"identity":"0c4c12df-b6cb-4369-bb54-dc3bf960d2de","added_by":"auto","created_at":"2025-07-09 09:29:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8732232,"visible":true,"origin":"","legend":"\u003cp\u003eQuinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids\u003c/p\u003e","description":"","filename":"Supplementaryfile20250322JP.docx","url":"https://assets-eu.researchsquare.com/files/rs-6282946/v1/a06d539dafc875a7727a53be.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Quinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeukemia is a type of hematological malignancy characterized by progressive aberrant growth of leukocytes [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. In general, leukemia is classified into four subtypes determined by the cell lineage (lymphocytic or myeloid) and the degree of maturation arrest (acute or chronic), all of which are risk factors for people of all ages. These subtypes comprise acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Globally, leukemia contributed to 2.5% and 31.1% of all new cancer incidences and deaths, respectively [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite the progress achieved in developing targeted therapies for various types of leukemia, the presence of cytotoxicity, therapy resistance, and relapse remain a substantial obstacle that impedes the effectiveness of targeted therapy in the treatment of leukemia [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Hence, novel drugs with improved anti-leukemia capabilities are required.\u003c/p\u003e\n\u003cp\u003eCDC25 phosphatases are members of the dual-specific protein tyrosine phosphatase (PTP) family, which play a crucial role in cell cycle regulation [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. In human genome, three isoforms of CDC25 phosphatases have been characterized, namely: CDC25A, CDC25B, and CDC25C [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. The activation of cyclin-dependent kinases (CDKs) is crucial for cell cycle progression [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. CDC25s are responsible for removing phosphate groups from CDK/cyclin complexes at the Thr14 and/or Tyr15 residues, thereby activating CDK/cyclin and facilitating cell cycle progression [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. It appears that CDC25s act at different stages of the cell cycle on different CDK/cyclin complexes. CDC25A appears to be implicated in controlling the G\u003csub\u003e1\u003c/sub\u003e/S and G\u003csub\u003e2\u003c/sub\u003e/M transitions, whereas CDC25B and CDC25C appear to be more involved in the regulation of the G\u003csub\u003e2\u003c/sub\u003e/M transition [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. CDC25s malfunction is often correlated to aberrant cell cycle progression, which contribute to tumorigenesis [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. CDC25 overexpression has been identified in several high-grade human malignancies and linked to poor prognosis [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, the role of CDC25s in cancer development including leukemia has been investigated in previous literature. Increased expression of CDC25A could promote cell proliferation in human AML [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Inactivation of CDC25A has also been correlated with antiproliferative, proapoptotic, and cytotoxic effects in various leukemia cell lines [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. AML cell growth seems to be decreased by CDC25B inhibition [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. CDC25C appears to have a significant role in determining the characteristics of AML cells [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, CDC25s could be considered an attractive potential therapeutic target in leukemia [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eDifferent attempts to develop CDC25 inhibitors have been extensively investigated for over two decades [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. And quinone-based derivatives remain the most effective of these inhibitors. Representative quinone-based molecules as CDC25 inhibitors are presented in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Menadione (Vitamin K3, VK3) demonstrated a potent CDC25 inhibition, inducing cell cycle arrest and apoptosis on cervical carcinoma cells [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Compound \u003cstrong\u003e2\u003c/strong\u003e (\u003cstrong\u003eM2N12\u003c/strong\u003e), was identified to be potent against CDC25 isoforms [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. Compound \u003cstrong\u003e3\u003c/strong\u003e (\u003cstrong\u003eM5N36\u003c/strong\u003e) is a selective CDC25C inhibitor with promising anti-growth activity and desirable predicted physicochemical properties [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. A quinoid bearing 2-(dimethylamino)ethylamino moiety, compound \u003cstrong\u003e4\u003c/strong\u003e (\u003cstrong\u003eBN-82685\u003c/strong\u003e), as a potent in vitro and in vivo CDC25 inhibitor. Compound \u003cstrong\u003e4\u003c/strong\u003e revealed specifically inhibited CDK1 Tyr-15 dephosphorylation and induced cell cycle arrest, leading to antitumor growth in Mia PaCa-2 xenograft mice models [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. Compound \u003cstrong\u003e5\u003c/strong\u003e (\u003cstrong\u003eIRC-083864\u003c/strong\u003e) bearing two quinone scaffolds displayed a selective CDC25 inhibitor. Also, compound \u003cstrong\u003e5\u003c/strong\u003e revealed potential antiproliferative activity, strongly inhibited cell cycle progression, and revealed tumor growth inhibition in both pancreatic and prostate tumor xenograft models [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. In another investigation, compound \u003cstrong\u003eNSC663284\u003c/strong\u003e (\u003cstrong\u003e6a\u003c/strong\u003e) was found to be a highly effective CDC25 inhibitor [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, many CDC25 inhibitors with different scaffolds have been identified, manifesting the potent therapeutic utility of these compounds. The development of small-molecule inhibitors that specifically target CDC25 is still in its early stages and has limited biochemical potency. Therefore, an effort to develop potent CDC25 inhibitors would be useful for developing new anticancer therapeutics.\u003c/p\u003e\n\u003cp\u003eIn our previous study, we emphasized the therapeutic potential of CDC25 inhibitors and how they disrupt cell cycle progression in colorectal cancers (CRCs) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Some derivatives that incorporated quinoline-5,8-dione core scaffold and morpholinoalkylamino at the C-6 position demonstrated potent cytotoxicity. Driven by compound \u003cstrong\u003eNSC663284\u003c/strong\u003e (\u003cstrong\u003e6a\u003c/strong\u003e) and \u003cstrong\u003e6b\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] with its promising anticancer activity, in current study, we thus designed, synthesized, and evaluated a series of \u003cstrong\u003eNSC663284\u003c/strong\u003e derivatives as anti-leukemia and -CRC agents. The quinoline-5,8-dione moiety was maintained, and then various side chains including heterocyclic/non-heterocyclic moieties at the C-7 and C-6 positions were introduced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Antiproliferative activity were examined in various leukemia and CRC cell lines and validated in the CRC cancer organoid model. Further mechanism of action of these synthesized compounds on CDC25 inhibition, cell cycle, apoptosis, and DNA damage was investigated.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Result and discussion","content":"\u003cp\u003e\u003cstrong\u003eChemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis route of \u003cstrong\u003eNSC663284\u003c/strong\u003e derivatives is summarized in Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Starting with the commercially available 5-nitroquinolin-8-ol (\u003cstrong\u003eA\u003c/strong\u003e) was converted to 5-aminoquinolin-8-ol (\u003cstrong\u003eB\u003c/strong\u003e) intermediate through catalytic hydrogenation [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Further, intermediate 6,7-dichloroquinoline-5,8-dione (\u003cstrong\u003eC\u003c/strong\u003e) was prepared by oxidizing intermediate \u003cstrong\u003eB\u003c/strong\u003e with NaClO\u003csub\u003e3\u003c/sub\u003e/HCl [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Subsequently, the intermediate \u003cstrong\u003eC\u003c/strong\u003e was reacted with various amines to afford C7-substituted products \u003cstrong\u003e6a, D1a-D12a\u003c/strong\u003e and C6-substituted products \u003cstrong\u003e6b, D1b-D12b\u003c/strong\u003e. Compounds \u003cstrong\u003eD6a\u003c/strong\u003e and \u003cstrong\u003eD6b\u003c/strong\u003e were obtained from the removal of the Boc protecting group of compounds \u003cstrong\u003eD8a\u003c/strong\u003e and \u003cstrong\u003eD8b\u003c/strong\u003e, respectively, which was accomplished by using trifluoroacetic acid in anhydrous dichloromethane. In addition, as described in the experimental procedure, to synthesize \u003cstrong\u003eD5a\u003c/strong\u003e and \u003cstrong\u003eD5b\u003c/strong\u003e, the spirocyclic amine was initially synthesized. This spirocyclic amine intermediate was obtained from a nucleophilic substitution reaction of the commercially available 1,4-dioxa-8-azaspiro[4.5]decane with 2-chloroacetonitrile to give nitrile intermediate, followed by nitrile intermediate reduction by lithium alumunium hydride to afford the 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine. The intermediate \u003cstrong\u003eC\u003c/strong\u003e was treated with the 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine to afford \u003cstrong\u003eD5a\u003c/strong\u003e and \u003cstrong\u003eD5b\u003c/strong\u003e. Meanwhile, to synthesize \u003cstrong\u003eD12a\u003c/strong\u003e and \u003cstrong\u003eD12b\u003c/strong\u003e, 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) was synthesized first by using 2,2'-azanediylbis(ethan-1-ol) and 2-bromoacetonitrile to give nitrile intermediate which was reduced to give 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol). Further, the treatment of intermediate \u003cstrong\u003eC\u003c/strong\u003e with 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) afforded \u003cstrong\u003eD12a\u003c/strong\u003e and \u003cstrong\u003eD12b\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBased on the structure of the key intermediates \u003cstrong\u003eC\u003c/strong\u003e, two \u0026ndash;Cl atoms attached in the quinoline-5,8-dione scaffold (at C-7 and C-6 positions) have identical electronic properties, therefore these two \u0026ndash;Cl atoms were replaced by nucleophile amines to generate C-7 and C-6 substituted products [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Here, we observed that the solvent used influenced the yield of each regioisomer. The regiochemistry of the products was determined according to the reported literature by assessing the proton chemical shifts (\u0026delta;\u003csub\u003eH\u003c/sub\u003e) difference at 2 and 4 positions [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. In all instances, the 7-substituted and 6-substituted products were separated through silica column chromatography (SCC), even though their Rf values overlapped. Furthermore, all the synthesized compounds were characterized by NMR (\u003csup\u003e1\u003c/sup\u003eH- and \u003csup\u003e13\u003c/sup\u003eC-NMR), melting point, and HRMS. The purity of all final target compounds was found to be \u0026ge;\u0026thinsp;95%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity evaluation on CRC cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCDC25 has been suggested as a target for early diagnosis and clinical treatment of different types of cancer [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our earlier study also highlighted the therapeutic potential of CDC25 inhibitors and their interference with cell cycle progression in colorectal cancers (CRCs) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this work, we initially performed cytotoxicity evaluation of all the newly designed and synthesized derivatives \u003cstrong\u003eD1a-D12a\u003c/strong\u003e, and \u003cstrong\u003eD1b-D12b\u003c/strong\u003e against CRC cell lines (HCT116 and DLD-1) by using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2\u003cem\u003eH\u003c/em\u003e-tetrazolium (MTS) assay after 72 h incubation at 2 \u0026micro;M concentration, compared to the reference compound \u003cstrong\u003eNSC663284 (6a)\u003c/strong\u003e. In an attempt to develop an active potent compound, an exploration of the structural modification of \u003cstrong\u003e6a\u003c/strong\u003e at the C-7 or C-6 positions of the quinoline-5,8-dione core scaffold was conducted. Hence, our attention was directed towards substituting the 2-morpholinoethylamino with different motifs. The results revealed that some alterations of the side chain of compound \u003cstrong\u003e6a\u003c/strong\u003e augmented the cytotoxicity against HCT116 and DLD-1 cells (Table S1). Notably, the introduction of 2-thiomorpholinoethylamino at the C-6 position (\u003cstrong\u003eD1b\u003c/strong\u003e) demonstrated a considerable improvement in cytotoxic effect against HCT116 and DLD-1 cells. Substituting 2-morpholinoethylamino with a hydrophobic pyrrolidin-1-yl moiety (\u003cstrong\u003eD2a, D2b\u003c/strong\u003e) was also beneficial for the cytotoxicity. Similarly, the side chain modification particularly with 2-(4-methylpiperidin-1-yl)ethylamino (\u003cstrong\u003eD3a, D3b\u003c/strong\u003e) at the C-7 and C-6 position showed a pronounced effect against HCT116 and DLD-1 cells. Subsequently, more structural modifications by introducing different side chains were also investigated. However, replacement of 2-morpholinoethylamino moiety with 2-(4-hydroxypiperidin-1-yl)ethylamino (\u003cstrong\u003eD4a\u003c/strong\u003e, \u003cstrong\u003eD4b\u003c/strong\u003e) diminished cytotoxic effect against HCT116 and DLD-1 cells. Next, azaspiro and piperazine containing compound was also evaluated. The introduction of 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethylamino and 2-(4-methylpiperazin-1-yl)ethylamino at the C-6 position (\u003cstrong\u003eD5b\u003c/strong\u003e, \u003cstrong\u003eD7b\u003c/strong\u003e) exhibited a potent activity against both HCT116 and DLD-1 cells. Further, \u003cstrong\u003eD6a\u003c/strong\u003e and \u003cstrong\u003eD6b\u003c/strong\u003e containing 2-(piperazin-1-yl)ethylamino revealed a weak activity, implying that a polar side chain might be unfavorable. Meanwhile, changing the piperazin-1-yl (\u003cstrong\u003eD6b\u003c/strong\u003e) to 4-methylpiperazin-1-yl (\u003cstrong\u003eD7b\u003c/strong\u003e), led to increased cytotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003edetermination of the selected derivatives in CRC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values of the selected compounds against CRC cell lines HCT116 and DLD-1 revealed varying degrees of cytotoxic activity (Table S2). Several derivatives exhibited potent inhibitory effects on both cell lines. Notably, compound \u003cstrong\u003eD1b\u003c/strong\u003e bearing 2-thiomorpholinoethylamino at the C-6 position demonstrated significant activity, with IC\u003csub\u003e50\u003c/sub\u003e values of 0.48 \u0026micro;M in HCT116 and 0.38 \u0026micro;M in DLD-1 cells. Additionally, compounds \u003cstrong\u003eD2a\u003c/strong\u003e, \u003cstrong\u003eD2b, D3a, D3b, D5b\u003c/strong\u003e, and \u003cstrong\u003eD7b\u003c/strong\u003e displayed moderate potency, particularly in HCT116, with IC\u003csub\u003e50\u003c/sub\u003e values of 1.43, 0.88, 1.50, 1.13, 0.83, and 0.83 \u0026micro;M, respectively. Compounds bearing 2-(pyrrolidin-1-yl)ethyl)amino (\u003cstrong\u003eD2a\u003c/strong\u003e) at C-7 and 2-(4-methylpiperidin-1-yl)ethylamino (\u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD3b)\u003c/strong\u003e at the C-7 and C-6 positions, exhibited potent inhibitory activity against the DLD-1 cell with IC\u003csub\u003e50\u003c/sub\u003e values of 0.76, 0.74 and 0.65 \u0026micro;M, respectively. Furthermore, compounds conjugated with 2-(pyrrolidin-1-yl)ethyl)amino (\u003cstrong\u003eD2b\u003c/strong\u003e), 2-(piperazin-1-yl)ethyl)amino (\u003cstrong\u003eD5b\u003c/strong\u003e), and 2-(4-methylpiperazin-1-yl)ethyl)amino (\u003cstrong\u003eD7b)\u003c/strong\u003e at C-6 positions exhibited moderate inhibitory activity against the DLD-1 cell with IC\u003csub\u003e50\u003c/sub\u003e values 1.29, 0.84, and 1.77 \u0026micro;M, respectively. Interestingly, \u003cstrong\u003eD11a\u003c/strong\u003e bearing 2-(dimethylamino)ethylamino at the C-7 position exhibited selective potency, being more effective against DLD-1 (0.56 \u0026micro;M) than HCT116 (1.14 \u0026micro;M). Among selected compounds, compound \u003cstrong\u003eD11b\u003c/strong\u003e bearing 2-(dimethylamino)ethylamino at the C-6 position exhibited the highest potency, with the lowest IC\u003csub\u003e50\u003c/sub\u003e values of 0.17 \u0026micro;M in HCT116 and 0.13 \u0026micro;M in DLD-1, indicating its strong potency in inhibiting cell proliferation. Overall, \u003cstrong\u003eD1b\u003c/strong\u003e and \u003cstrong\u003eD11b\u003c/strong\u003e emerges as the most promising compounds, warranting further investigation for their potential therapeutic application in CRC treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity evaluation on leukemia cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that CDC25 inhibitors have demonstrated promise in leukemia development [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], we also assayed the cytotoxicity of newly synthesized compounds in various types of blood cell lines isolated from leukemias, including HL-60 (acute promyelocytic leukemia), KG-1 (acute myelogenous leukemia), MV-4-11 (biphenotypic B-myelomonocytic leukemia), and K-562 (chronic myelogenous leukemia). We treated cells with 2 \u0026micro;M of compounds for 72 hours, followed by MTS assay. The results presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e revealed that several derivatives delivered high toxicity to all cell lines. Compound \u003cstrong\u003eNSC663284\u003c/strong\u003e (\u003cstrong\u003e6a\u003c/strong\u003e) bearing 2-morpholinoethylamino at the C-7 position showed higher cytotoxicity in the KG-1 cell line and moderate cytotoxicity in the three tested cell lines (HL-60, MV4-11, and K562 cell lines). The regiomer compound \u003cstrong\u003e6b\u003c/strong\u003e bearing 2-morpholinoethylamino at the C-6 position exhibited more pronounced cytotoxicity against the tested cell lines, as compared to compound \u003cstrong\u003e6a\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, replacing 2-morpholinoethylamino moiety with 2-thiomorpholinoethylamino (\u003cstrong\u003eD1a, D1b\u003c/strong\u003e) showed almost similar results to \u003cstrong\u003e6a\u003c/strong\u003e and \u003cstrong\u003e6b\u003c/strong\u003e. Compound \u003cstrong\u003eD1a\u003c/strong\u003e, which incorporated 2-thiomorpholinoethylamino at the C-7 position, showed potent cytotoxicity in the KG-1 cell line. However, \u003cstrong\u003eD1b\u003c/strong\u003e showed a similar potency with \u003cstrong\u003e6b\u003c/strong\u003e, suggesting that the replacement of 2-morpholinoethylamino moiety with 2-thiomorpholinoethylamino at the C-6 position retained the activity. Interestingly, the substitution of 2-morpholinoethylamino moiety by a hydrophobic moiety such as pyrrolidin-1-yl moiety (\u003cstrong\u003eD2a\u003c/strong\u003e and \u003cstrong\u003eD2b\u003c/strong\u003e) led to improved cytotoxicity. Further, more structural variations by introducing a piperidinyl moiety on the C-7 and C-6 positions were explored. Notably, an increase in cytotoxicity was remarked in the replacement of 2-morpholinoethylamino (\u003cstrong\u003e6a, 6b\u003c/strong\u003e) with 2-(4-methylpiperidin-1-yl)ethylamino (\u003cstrong\u003eD3a, D3b\u003c/strong\u003e). When compared to \u003cstrong\u003e6a\u003c/strong\u003e, the cytotoxicity of \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD3b\u003c/strong\u003e was significantly improved against all the tested leukemia cell lines, indicating that the substitution of 2-morpholinoethylamino with 2-(4-methylpiperidin-1-yl)ethylamino moiety at the C-7 and C-6 position appeared to be beneficial for the activity. The introduction of 2-(4-hydroxypiperidin-1-yl)ethylamino at the C-7 position (\u003cstrong\u003eD4a\u003c/strong\u003e) exhibited potent cytotoxicity against the three tested cell lines (KG-1, HL-60, and MV4-11), reducing cell viability by \u0026gt;\u0026thinsp;80%. Meanwhile, compound \u003cstrong\u003eD4b\u003c/strong\u003e bearing 2-(4-hydroxypiperidin-1-yl)ethylamino at the C-6 position showed lower potency compared to \u003cstrong\u003eD4a\u003c/strong\u003e. Next, 1,4-dioxa-8-azaspiro and piperazinyl-containing compounds were evaluated. Compound \u003cstrong\u003eD5a\u003c/strong\u003e bearing a 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethylamino at the C-7 position revealed higher cytotoxicity against KG-1, HL-60, and MV4-11 cell lines, meanwhile, its regioisomer (\u003cstrong\u003eD5b\u003c/strong\u003e) exhibited potency towards MV4-11 and K562 cell lines. Compound \u003cstrong\u003eD6a\u003c/strong\u003e and \u003cstrong\u003eD6b\u003c/strong\u003e were found to be less potent than \u003cstrong\u003e6a\u003c/strong\u003e, indicating that 2-(piperazin-1-yl)ethylamino as a polar side chain was not beneficial for the activity. Notably, \u003cstrong\u003eD7a\u003c/strong\u003e and \u003cstrong\u003eD7b\u003c/strong\u003e which incorporated 2-(4-methylpiperazin-1-yl)ethylamino side chain revealed high potency, indicating that the replacement of 2-morpholinoethylamino with 2-(4-methylpiperazin-1-yl)ethylamino motifs favored for the activity. Compound \u003cstrong\u003eD7a\u003c/strong\u003e and \u003cstrong\u003eD7b\u003c/strong\u003e demonstrated broad antileukemia activities in KG-1, HL-60, MV4-11, and K562 cell lines, reducing cell viability by \u0026gt;\u0026thinsp;80%. Meanwhile, \u003cstrong\u003eD8a, D8b\u003c/strong\u003e, and \u003cstrong\u003eD9a-D9b\u003c/strong\u003e represent a small series of derivatives bearing bulky substituents. Substitution of the 2-morpholinoethylamino moiety by a bulkier tert-butyl piperazine-1-carboxylate motifs at the C-7 and C-6 positions (\u003cstrong\u003eD8a\u003c/strong\u003e and \u003cstrong\u003eD8b\u003c/strong\u003e) led to reduced cytotoxicity when compared to compound \u003cstrong\u003e6a\u003c/strong\u003e. Moreover, the incorporation of tert-butyl (1-(2-aminoethyl)piperidin-4-yl)carbamate moiety at the C-7 and C-6 positions exhibited better cytotoxicity (compare \u003cstrong\u003eD9a\u003c/strong\u003e and \u003cstrong\u003eD9b\u003c/strong\u003e with \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD8a\u003c/strong\u003e, and \u003cstrong\u003eD8b)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFurther, the effect of the replacement of 2-morpholinoethylamino with various acyclic side chains such as 2-((2-hydroxyethyl)(methyl)amino)ethylamino, 2-(dimethylamino)ethylamino, and 2-(bis(2-hydroxyethyl)amino)ethylamino moieties was also explore. The introduction of 2-((2-hydroxyethyl)(methyl)amino)ethylamino (\u003cstrong\u003eD10a, D10b\u003c/strong\u003e) slightly maintained the cytotoxicity against KG-1, HL-60, and MV4-11 cell lines. Interestingly, \u003cstrong\u003eD11a\u003c/strong\u003e and \u003cstrong\u003eD11b\u003c/strong\u003e bearing 2-(dimethylamino)ethylamino at the C-7 and C-6 positions led to more pronounced cytotoxicity against all the tested leukemia cell lines (compared to compound \u003cstrong\u003e6a\u003c/strong\u003e), suggesting that the substitution of 2-morpholinoethylamino with hydrophobic alkyl side chain 2-(dimethylamino)ethylamino was found to be essential for the activity. However, a decrease in cytotoxicity was observed when 2-(bis(2-hydroxyethyl)amino)ethylamino moiety (\u003cstrong\u003eD12a, D12b\u003c/strong\u003e) was introduced. Taken together, the structure-activity relationship (SAR) revealed that 2-(4-methylpiperidin-1-yl)ethylamino and 2-(dimethylamino)ethylamino moieties either both at the C-7 or C-6 positions were favorable for the activity. In general, our cytotoxicity screen demonstrated that \u003cstrong\u003eD3a\u003c/strong\u003e, \u003cstrong\u003eD3b\u003c/strong\u003e, \u003cstrong\u003eD11a\u003c/strong\u003e, and \u003cstrong\u003eD11b\u003c/strong\u003e displayed considerable effectiveness across all leukemia cell lines and proved to be even more toxic than \u003cstrong\u003e6b\u003c/strong\u003e, the most potent compound reported in earlier study [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCytotoxicity of all synthesized compounds\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\u003ccolgroup\u003e\u003c/colgroup\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCompound\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKG-1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHL-60\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMV4-11\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eK562\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eNSC663284 (6a)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e89.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e6b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD1a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17.04\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25.14\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e43.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e77.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD1b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD2a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e73.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD2b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD3a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD3b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD4a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e55.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD4b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e91.4\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD5a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e42.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD5b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e44.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD6a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e83.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e98.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD6b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e63.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e51.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e97.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD7a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e87.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD7b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD8a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e99.5\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD8b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD9a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e40.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD9b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD10a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e59.31\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD10b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42.2\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e89.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD11a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD11b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBDL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD12a\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e48.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD12b\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e85.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDMSO\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCells were treated with 2 \u0026micro;M of indicated compounds for 72 h and the percentages of surviving cells were determined by MTS assay. Results are shown as means with SDs from at least two independent experiments (\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;5). BDL\u0026thinsp;=\u0026thinsp;Below Detection Limit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003edetermination of the selected derivatives in leukemia cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs mentioned above, all the prepared compounds were examined for their cytotoxicity. The screening outputs revealed that side chain modifications with either a 2-(4-methylpiperidin-1-yl)ethylamino and 2-(dimethylamino)ethylamino moieties are essential for anti-leukemic activity. Hence, the growth inhibitory activity of selected derivatives was examined and an IC\u003csub\u003e50\u003c/sub\u003e for the selected compounds is described in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. We found that the selected compounds exhibited high efficacy against tested cell lines, with an IC\u003csub\u003e50\u003c/sub\u003e value close to or below 1 \u0026micro;M. Compound \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD3b\u003c/strong\u003e bearing a 2-(4-methylpiperidin-1-yl)ethylamino side chain at the C-7 and C-6 positions, respectively, retained potent activity against all investigated leukemia cell lines with a sub-micromolar to low single single-digit micromolar with IC\u003csub\u003e50\u003c/sub\u003e values ranging between 0.23 and 1.22 \u0026micro;M. Compound \u003cstrong\u003eD11a\u003c/strong\u003e, bearing 2-(dimethylamino)ethylamino as a side chain at the C-7 position, showed potency against the leukemia cell lines (IC\u003csub\u003e50\u003c/sub\u003e values ranging between 0.32\u0026ndash;0.98 \u0026micro;M). Compound \u003cstrong\u003eD11b\u003c/strong\u003e, bearing 2-(dimethylamino)ethylamino at the C-6 position showed comparable antiproliferative activity to \u003cstrong\u003eD11a\u003c/strong\u003e with IC\u003csub\u003e50\u003c/sub\u003e values ranging between 0.21 and 0.61 \u0026micro;M. Taken together, our inhibitory activity evaluation against leukemia cell lines revealed that \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e were more potent in HL-60 and KG-1 cells, whereas \u003cstrong\u003eD11b\u003c/strong\u003e was most toxic to MV-4-11 and K-562 cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e values for selected compounds against a set of leukemia cell lines\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\u003ccolgroup\u003e\u003c/colgroup\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth colspan=\"4\" align=\"left\"\u003e\n\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD3a\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD3b\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD11a\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eD11b\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHL-60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKG-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMV4-11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eK562\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eVarious leukemia cell lines were treated with indicated compounds for 72 h and the values of half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) were determined by MTS assay. Results are shown as means with SDs from at least two independent experiments (\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe utilized flow cytometry to analyze the cell status at the endpoint of compound treatment. Our results showed that treating HL-60 cells with the most potent compounds, \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e, significantly increased the sub-G\u003csub\u003e1\u003c/sub\u003e population (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), which is indicative of DNA fragmentation. In contrast, we did not observe any effect from the reference compound \u003cstrong\u003e6a\u003c/strong\u003e under the same conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, \u003cstrong\u003eD1b\u003c/strong\u003e and \u003cstrong\u003eD11b\u003c/strong\u003e, the most toxic compounds to CRC cells, also led to an increase in the sub-G\u003csub\u003e1\u003c/sub\u003e population in HCT116 cells (Figure S1). We further examined the effect of compound treatment in apoptosis pathways and found that the treatment of \u003cstrong\u003eD3a\u003c/strong\u003e or \u003cstrong\u003eD11a\u003c/strong\u003e induced the cleavage of pro-apoptotic Caspase-3 and \u0026minus;\u0026thinsp;9, while reduced the levels of anti-apoptotic proteins Mcl-1 and Bcl-2, in HL-60 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings indicate that the selected derivatives possess strong cytotoxicity and are capable to trigger apoptosis, albeit with variable potencies across different cell lines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDC25 inhibitory activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell cycle progression highly relies on the activation of cyclin-dependent kinases (CDKs)\u003c/p\u003e\n\u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. The activation of CDK requires the removal of the inhibitory phosphorylation at Tyr15 by the phosphatase CDC25 [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our previous study demonstrated that \u003cstrong\u003e6b\u003c/strong\u003e nearly prohibit G\u003csub\u003e2\u003c/sub\u003e/M transition by preventing CDC25-mediated CDK1 dephosphorylation in CRC cells [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. In light of this, we investigated the potential CDC25 inhibitory activity of \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e. The in vitro enzymatic analysis revealed that the modification of 2-morpholinoethylamino moiety on \u003cstrong\u003eNSC663284\u003c/strong\u003e (\u003cstrong\u003e6a\u003c/strong\u003e) structure with 2-(4-methylpiperidin-1-yl)ethylamino and 2-(dimethylamino)ethylamino moieties (compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a)\u003c/strong\u003e enhanced the CDC25-inhibitory activity from the IC\u003csub\u003e50\u003c/sub\u003e value of 0.11 \u0026micro;M (\u003cstrong\u003e6a\u003c/strong\u003e) to 0.065 \u0026micro;M and 0.068 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eCDC25C inhibition analysis of indicated compounds using CDC25C Human Phosphatase Enzymatic LeadHunter Assay. Percentages of inhibition (left) and IC\u003csub\u003e50\u003c/sub\u003e values from two individual assays (right) are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDK1 dephosphorylation and mitosis progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the enhanced CDC25-inhibitory activity of compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e, we monitored the phosphorylation dynamics of CDK1 at Tyr15 in HL-60 cells following treatment with these compounds. We used thymidine to synchronize cells in the early S phase and assessed the phosphorylation level of CDK1 at Tyr15 from the S phase through to mitosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). We observed that most cells entered the G\u003csub\u003e2\u003c/sub\u003e phase four hours after being released from the thymidine block (T4), during which CDK1 phosphorylation was high and only a faint mitotic signal was detected, indicated by the phosphorylation of cyclin B1, histone H3, and a mitotic protein identified by the MPM2 antibody (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and S2). To assess CDK1 dephosphorylation, we subsequently treated these cells with mitotic inhibitors colchicine or paclitaxel (Taxol) to prevent CDK1 re-phosphorylation after nuclear division. At T8, these cells accumulated in mitosis, exhibiting a low level of CDK1 phosphorylation and increased mitotic signals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and S1). Notably, cells co-treated with \u003cstrong\u003eD3a\u003c/strong\u003e or \u003cstrong\u003eD11a\u003c/strong\u003e demonstrated higher CDK1 phosphorylation levels, coupled with lower mitotic signals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and S2). Similar outcomes were observed in HCT116 cells treated with \u003cstrong\u003eD1b\u003c/strong\u003e, \u003cstrong\u003eD3b\u003c/strong\u003e, or \u003cstrong\u003eD11b\u003c/strong\u003e (Figure S3). Importantly, these compounds affected cell cycle progression with varying efficacies (Figure S4). Our findings suggest that our compounds are potent CDC25 inhibitors that disrupt cell cycle progression.\u003c/p\u003e\n\u003cp\u003eAnalysis of cell cycle progression and mitotic signaling of thymidine-synchronized HL-60 cells co-treated with 0.5 \u0026micro;M of colchicine and 0.75 \u0026micro;M of indicated compounds. Experimental design (left upper) and representative results of FACS (left lower) and Western blot (right) from one of two biological replicates are shown. Asyn: Asynchronous; cpd: Compound; Col: Colchicine; Thy: Thymidine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selected compounds \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e together with the reference compound \u003cstrong\u003e6a\u003c/strong\u003e were further investigated the protein-drug interaction for the CDC25C crystal protein (PDB ID: 3OP3). The ligands \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e were docked into the coordinates centered on the active site of the CDC25C protein using Discovery Studio Client 2022 (Biovia, CA, USA). Their binding poses were selected based on the highest LibDock score and protein-ligand interactions analyzed. The eminent interactions between the active pocket of CDC25C and compounds \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD3a\u003c/strong\u003e, or \u003cstrong\u003eD11a\u003c/strong\u003e are predicted by their respective 3D (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG, and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH) and 2D (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF, and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI) cartoon pictures. Importantly, various residues make the hydrogen (H)-bonding interactions to the quinoline-5, 8-dione moiety of \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e similar to the reference compound \u003cstrong\u003e6a\u003c/strong\u003e. Notably, compound \u003cstrong\u003eD3a\u003c/strong\u003e makes three conventional H-bondings with Glu378, Phe379, and Arg383, whereas compound \u003cstrong\u003eD11a\u003c/strong\u003e interacts with the active site via four H-bonding such as Cys377, Glu382, Arg383, and Gly384. Furthermore, in addition to the H-bonding, other interactions such as Pi-Pi T-shaped (Phe379), Pi-anion (Glu382), Pi-sulphur (Cys377, Met435) were able to preserve the CDC25C activity of compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e as compared with \u003cstrong\u003e6a\u003c/strong\u003e. However, the side chains (methyl-piperidine and \u003cem\u003eN, N\u003c/em\u003e-dimethyl amine) of the compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e were bound at the catalytic site via alkyl and pi-alkyl (Lys298, His299, Tyr304, and Pro385) and van der Waals (Ile411, Leu412 and Pro385) interactions respectively, as well a H-bonding with \u003cem\u003eN, N\u003c/em\u003e-dimethyl amine (\u003cstrong\u003eD11a\u003c/strong\u003e) and Cys377 residues distant from the core catalytic region. Also, it can be seen that methyl-piperidine and \u003cem\u003eN, N\u003c/em\u003e-dimethyl amine side chain motif contribute to \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e binding and stabilization at the hydrophobic pocket of CDC25C core active site (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eJ and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eK). Therefore, \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e remained deeply buried at the core active catalytic site of CDC25C by maintaining H-bonding, Pi-Pi stacked, Pi-anion, Pi-sulphur, and as well van der Waals interactions which implied distinct compactness to favor its better activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH4K20 methylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNSC663284\u003c/strong\u003e (\u003cstrong\u003e6a\u003c/strong\u003e) and \u003cstrong\u003e6b\u003c/strong\u003e have also been shown to target SETD8, an enzyme that methylates histone H4 at the site of Lys20 (H4K20) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. Disruption of SETD8-mediated H4K20 methylation leads to genome instability and cell cycle delay [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. We thus assayed the level of H4K20 methylation in HL-60 cells treated with the identified compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD11a\u003c/strong\u003e. Consistent with the previous studies [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], H4K20 mono-methylation remarkably reduced before genome replication (T0) and gradually accumulated in S and G\u003csub\u003e2\u003c/sub\u003e/M phases (T4 and T6). The treatment of \u003cstrong\u003eD3a\u003c/strong\u003e or \u003cstrong\u003eD11a\u003c/strong\u003e impeded this progressive H4K20 methylation, while \u003cstrong\u003e6a\u003c/strong\u003e showed a slight effect (Figure S5). Consistently, \u003cstrong\u003eD11b\u003c/strong\u003e also significantly blocked H4K20 methylation in HCT116 cells (Figure S6). These results argue that the new \u003cstrong\u003eNSC663284\u003c/strong\u003e derivatives harbor inhibitory activities toward multiple pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDouble-stranded DNA break\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCDK activity and H4K20 methylation play essential roles in maintaining genome stability [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Our earlier study found that \u003cstrong\u003e6b\u003c/strong\u003e effectively inhibited CDK1Y15 dephosphorylation and H4K20 mono-methylation, leading to a DNA damage response [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, we showed that the treatment of \u003cstrong\u003eD3a\u003c/strong\u003e or \u003cstrong\u003eD11a\u003c/strong\u003e induced significant DNA damage in HL-60 cells, particularly in double-stranded DNA break (DSB) pathways, as evidenced by phosphorylations of ATM, CHK2, KAP1, and RPA2 at Ser4/Ser8 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Notably, \u003cstrong\u003eD11b\u003c/strong\u003e, the most potent \u003cstrong\u003eNSC663284\u003c/strong\u003e derivative in leukemia K-562 and MV-4-11 cells (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), as well as CRC cells (Table S2), also induces a higher level of DNA damage in K-562 and HCT116 cells (Figure S7). To determine if these compounds induce DSB, we extracted genomic DNAs from compound-treated cells and subjected them to gel electrophoresis. We observed clear ladder patterns in the genomic DNA samples of treated cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating that these compounds can rapidly trigger DNA fragmentation. Our findings suggest that these compounds are promising candidates for cancer therapeutics by inducing genome instability and catastrophe.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetic and drug-likeness properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePharmacokinetic and drug-likeness properties of all compounds were predicted using the free online SwissADME software [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e] and the results are presented in Table S3. The selected compounds, \u003cstrong\u003eD1b, D3a, D3b, D11a\u003c/strong\u003e, and \u003cstrong\u003eD11b\u003c/strong\u003e, adhered to Lipinski's rule criteria and exhibited favorable physicochemical attributes. Compounds \u003cstrong\u003e6a, 6b, D1b, D3a, D3b, D11a\u003c/strong\u003e, and \u003cstrong\u003eD11b\u003c/strong\u003e have molecular weights (MW) ranging from 279.72 to 333.81 g/mol. The selected compounds have one H-bond donor (HBD), 4 rotatable bonds (RB), and 4 to 5 H-bond acceptors (HBA). The topological polar surface area (TPSA) values range from 62.30 to 71.53 \u0026Aring;2. The CLogP varies from 1.03 to 2.18. \u003cstrong\u003eD1b, D3a, D3b, D11a\u003c/strong\u003e, and \u003cstrong\u003eD11b\u003c/strong\u003e were in the soluble class. In addition, these compounds are predicted to have a high probability of gastrointestinal absorption (GIA) and be able to diffuse through the blood-brain barrier (BBB). Of interest, the selected compounds do not exhibit violation of either the Lipinski, Veber, Egan, or Muegge filters, which means that these compounds have the potential to be absorbed through the gastrointestinal tract (Table S5). Therefore, \u003cstrong\u003eD1b\u003c/strong\u003e, \u003cstrong\u003eD3a, D3b, D11a\u003c/strong\u003e, and \u003cstrong\u003eD11b\u003c/strong\u003e display desirable pharmacokinetic properties and are adequate for more advanced investigations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient-derived organoid model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate its potential translation into clinical practice, we determined the efficacy of \u003cstrong\u003eD11b\u003c/strong\u003e in suppressing the proliferation of colorectal cancer (CRC) patient-derived organoids (PDOs). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA, CRC organoids treated with \u003cstrong\u003eD11b\u003c/strong\u003e exhibited decreased proliferation, as demonstrated by endpoint images and corresponding proliferation rate line graphs. The inhibition of proliferation was dose-dependent and statistically significant (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Invasion assays further confirmed the compound\u0026rsquo;s inhibitory effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB). Organoids treated with different concentrations of \u003cstrong\u003eD11b\u003c/strong\u003e displayed reduced invasion ability. Statistical analysis revealed a significant reduction in invasion with increasing concentrations of \u003cstrong\u003eD11b\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThese findings highlight the potential clinical relevance of the identified compound as a therapeutic candidate for colorectal cancer. By effectively inhibiting both proliferation and invasion of CRC PDOs, the compound may offer a novel treatment strategy that targets the aggressive and metastatic behavior of CRC tumors.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFocusing on developing CDC25 inhibitors, we have designed, synthesized, and evaluated a series of \u003cb\u003eNSC663284\u003c/b\u003e derivatives containing various alkylamino side chains at the C-7 and C-6 positions of the quinoline-5,8-dione scaffold. In the SAR study, we explored the structural modification of the lead compound, \u003cb\u003eNSC663284\u003c/b\u003e (\u003cb\u003e6a\u003c/b\u003e), by substituting 2-morpholinoethylamino moiety with various alkylamino side chains. Modifying 2-morpholinoethylamino moiety with either 2-(4-methylpiperidin-1-yl)ethylamino or 2-(dimethylamino)ethylamino moieties improved the activity. \u003cb\u003eD3a, D3b, D11a\u003c/b\u003e, and \u003cb\u003eD11b\u003c/b\u003e showed remarkable cytotoxicity against the tested leukemia and CRC cell lines with IC\u003csub\u003e50\u003c/sub\u003e close to or below 1 \u0026micro;M. In vitro enzymatic assay of \u003cb\u003eD3a\u003c/b\u003e and \u003cb\u003eD11a\u003c/b\u003e demonstrated a potent CDC25-inhibitory activity with the IC\u003csub\u003e50\u003c/sub\u003e values of 0.065 \u0026micro;M and 0.068 \u0026micro;M, respectively. Mechanistically, the identified compounds impaired CDK1 dephosphorylation and slowed G\u003csub\u003e2\u003c/sub\u003e/M progression, ultimately leading to apoptosis, revealed by cell accumulation in the sub-G\u003csub\u003e1\u003c/sub\u003e phase, cleavage of pro-apoptotic Caspase-3 and \u0026minus;\u0026thinsp;9 and reduction of anti-apoptotic protein Mcl-1 and Bcl-2. The evaluation of genome integrity further demonstrated that these compounds possess a genotoxicity that triggered double-stranded DNA breaks. In silico SwissADME prediction confirmed the drug-like properties and the druggability of the selected compound. Notably, we demonstrated that \u003cb\u003eD11b\u003c/b\u003e effectively inhibits the proliferation and invasion of CRC PDOs in a dose-dependent manner. These results underscore the compound\u0026rsquo;s potential as a therapeutic agent for CRC, with significant implications for reducing tumor progression and metastasis. Given the promising in vitro efficacy, further investigations, including preclinical and clinical trials, are warranted to evaluate the compound\u0026rsquo;s therapeutic potential and translational feasibility in clinical settings. This work highlights the importance of integrating medicinal chemistry and organoid models to identify and develop novel, targeted cancer therapies.\u003c/p\u003e"},{"header":"Experimental Section","content":"\u003cp\u003e\u003cstrong\u003eChemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSilica column chromatography (SCC) was conducted using 230\u0026ndash;400 mesh silica gel (Siliaflash\u0026reg; P60). The reaction progress was monitored by Thin Layer Chromatography (TLC), which was conducted on silica gel plate (ALUGRAM\u003csup\u003e\u0026reg;\u003c/sup\u003e Xtra SILG, Macherey-Nagel, Germany) and detected under UV light at 254 nm (UVGL-25, Analytik Jena US). The spectra of the compound including \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC spectra were obtained using a Bruker 300 MHz and an Agilent 600 MHz spectrometer. The purity of all final compounds was determined by HPLC (Agilent 1260 Infinity II, Agilent Technologies, Germany) using a Dikma (Diamonsil 5 \u0026micro;m C18x150x4.6 mm) column. HPLC analysis conditions are as follows: (1) ACN and water containing NH\u003csub\u003e4\u003c/sub\u003eOAc 10 mM with HCOOH 0.1% as a solvent system with a flow rate of 0.5 mL/min; or (2) ACN and water containing NH\u003csub\u003e4\u003c/sub\u003eOAc 10 mM as a solvent system with a flow rate of 0.4 mL/min. And purity of the final compounds was found to be \u0026gt;\u0026thinsp;95%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure for the preparation of compounds 6a, D1a-D12a and 6b, D1b-D12b\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe appropriate amines were added to a well-stirred solution of 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e and DIPEA in dry THF or DCM. The resulting mixture was stirred at room temperature for 2\u0026ndash;7 h, after which TLC analysis revealed the absence of starting materials. The reaction mixture was then quenched with water. Dichloromethane was used for extraction, and the combined organic phase was dried by adding anhydrous magnesium sulfate. Under reduced pressure, the organic phase was evaporated using a rotary evaporator, and the resulting residue was then purified by silica column chromatography (SCC) using the appropriate eluent to afford the corresponding compounds \u003cstrong\u003e6a, D1a-D12a\u003c/strong\u003e (24\u0026ndash;94%) and \u003cstrong\u003e6b, D1b-D12b\u003c/strong\u003e (7\u0026ndash;90%).\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003e6\u003cem\u003e-Chloro-7-((2-morpholinoethyl)amino)quinoline-5,8-dione (\u003c/em\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003cem\u003e) and 7-Chloro-6-((2-morpholinoethyl)amino)quinoline-5,8-dione\u003c/em\u003e (\u003cstrong\u003e6b\u003c/strong\u003e)\u003c/div\u003e\n\u003cp\u003eThe synthesis and characterization of compounds \u003cstrong\u003e6a\u003c/strong\u003e and \u003cstrong\u003e6b\u003c/strong\u003e were reported previously [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione () and 7-chloro-6-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione ()\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003e6-Chloro-7-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD1a\u003c/strong\u003e) and 7-chloro-6-((2-thiomorpholinoethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD1b\u003c/strong\u003e)\u003c/div\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD1a\u003c/strong\u003e and \u003cstrong\u003eD1b\u003c/strong\u003e were synthesized by using 4-(2-aminoethyl)thiomorpholine (176 mg, 1.2 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol), and DIPEA (208 \u0026micro;L, 1.2 mmol) in dry THF (9 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;4: 1: 0.3 as a solvent system to afford pure \u003cstrong\u003eD1a\u003c/strong\u003e and \u003cstrong\u003eD1b\u003c/strong\u003e as a red solid in 59% and 15% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD1a\u003c/strong\u003e: m.p 166\u0026ndash;167\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.8 Hz, 1H), 8.45 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.7 Hz, 1H), 7.64 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.6 Hz, 1H), 7.07 (br s, 1H), 3.98 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 2.83 (s, 4H), 2.73 (s, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.4, 153.4, 146.3, 145.4, 134.7, 129.9, 128.4, 57.2, 54.7, 41.0, 28.0; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eSCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 338.0730; found, 338.0742. HPLC purity\u0026thinsp;=\u0026thinsp;97.3% (t\u003csub\u003er\u003c/sub\u003e = 8.891 min). Data for 6-substituted product \u003cstrong\u003eD1b\u003c/strong\u003e: m.p 159\u0026ndash;160\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.99 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.7 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 6.95 (br s, 1H), 3.94 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 2.81\u0026ndash;2.74 (m, 4H), 2.76\u0026ndash;2.69 (m, 4H), 2.68 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.3, 175.1, 155.3, 148.6, 144.3, 134.7, 127.0, 126.6, 56.9, 54.6, 41.0, 28.2; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eSCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 338.0730; found, 338.0739. HPLC purity\u0026thinsp;=\u0026thinsp;99.1% (t\u003csub\u003er\u003c/sub\u003e = 8.179 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-(pyrrolidin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD2a)\u003c/strong\u003e and 7-chloro-6-((2-(pyrrolidin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD2b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD2a\u003c/strong\u003e and \u003cstrong\u003eD2b\u003c/strong\u003e were synthesized by using N-(2-aminoethyl)pyrrolidine (140 \u0026micro;L, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol) in DCM (3 mL) and MeOH (2 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;4: 2 : 0.3 as a solvent system to afford pure compounds \u003cstrong\u003eD2b\u003c/strong\u003e and \u003cstrong\u003eD2b\u003c/strong\u003e as red solid in 33% and 3% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD2a\u003c/strong\u003e: m.p. 115\u0026ndash;116\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.63 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 6.97 (br s, 1H), 3.97 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2H), 2.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 2H), 2.63\u0026ndash;2.51 (m, 4H), 1.87\u0026ndash;1.70 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 178.9, 175.4, 153.4, 146.4, 145.8, 134.7, 129.9, 128.4, 55.0, 53.9, 43.3, 23.8; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 306.1009; found, 306.0999. HPLC purity\u0026thinsp;=\u0026thinsp;98.7% (t\u003csub\u003er\u003c/sub\u003e = 10.816 min). Data for 6-substituted product \u003cstrong\u003eD2b\u003c/strong\u003e: m.p 129\u0026ndash;130\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.8 Hz, 1H), 8.32 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.8 Hz, 1H), 7.56 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 6.92 (br s, 1H), 3.96 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 2H), 2.79 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.64\u0026ndash;2.53 (m, 4H), 1.87\u0026ndash;1.72 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.2, 175.1, 155.2, 148.7, 144.6, 134.7, 127.0, 126.5, 54.7, 53.7, 43.4, 23.8; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 306.1009; found, 306.1003. HPLC purity\u0026thinsp;=\u0026thinsp;97.9% (t\u003csub\u003er\u003c/sub\u003e = 11.450 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-(4-methylpiperidin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD3a\u003c/strong\u003e) and 7-chloro-6-((2-(4-methylpiperidin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD3b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD3b\u003c/strong\u003e were synthesized by using 2-(4-methylpiperidin-1-yl)ethan-1-amine (158 mg, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol), and DIPEA (192 \u0026micro;L, 1.1 mmol) in dry THF (9 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;4: 1: 0.3 as a solvent system to afford pure compounds \u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD3b\u003c/strong\u003e as a red solid in 52% and 6% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD3a\u003c/strong\u003e: m.p. 118\u0026ndash;119\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.89 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.45 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.63 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.26 (br s, 1H), 4.02\u0026ndash;3.89 (m, 2H), 2.94\u0026ndash;2.83 (m, 2H), 2.68 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 2.19\u0026ndash;2.06 (m, 2H), 1.68\u0026ndash;1.59 (m, 2H), 1.44\u0026ndash;1.35 (m, 1H), 1.35\u0026ndash;1.27 (m, 2H), 0.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.3, 153.3, 146.4, 145.7, 134.7, 130.0, 128.3, 56.7, 53.5, 41.5, 34.2, 30.7, 21.8; HRMS (ESI) for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 334.1322; found, 334.1333. HPLC purity\u0026thinsp;=\u0026thinsp;98.7% (t\u003csub\u003er\u003c/sub\u003e = 9.602 min). Data for 6-substituted product \u003cstrong\u003eD3b\u003c/strong\u003e: m.p. 140\u0026ndash;141\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.34 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.8 Hz, 1H), 7.56 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 7.11 (br s, 1H), 3.97\u0026ndash;3.91 (m, 2H), 2.88\u0026ndash;2.82 (m, 2H), 2.63 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.66\u0026ndash;2.61 (m, 2H), 1.68\u0026ndash;1.62 (m, 2H), 1.45\u0026ndash;1.34 (m, 1H), 1.31\u0026ndash;1.21 (m, 2H), 0.94 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.4, 175.0, 155.2, 148.7, 144.6, 134.7, 127.1, 126.5, 56.6, 53.5, 41.5, 34.5, 30.9, 21.9; HRMS (ESI) for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 334.1322; found, 334.1332. HPLC purity\u0026thinsp;=\u0026thinsp;96.5%% (t\u003csub\u003er\u003c/sub\u003e = 8.763 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-(4-hydroxypiperidin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD4a\u003c/strong\u003e) and 7-chloro-6-((2-(4-hydroxypiperidin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD4a\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003e1-(2-Aminoethyl)piperidin-4-ol (216 mg, 1.5 mmol) was added to a stirred solution of 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol) and DIPEA (209 \u0026micro;L, 1.2 mmol) in dry DCM (9 mL). The resulting mixture was stirred at rt for 7 h. Under reduced pressure, the solvent was removed using a rotary evaporator. The resulting residue was then purified by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: 2: 2: 0.2 to afford pure compounds \u003cstrong\u003eD4a\u003c/strong\u003e and \u003cstrong\u003eD4b\u003c/strong\u003e in 39% and 45% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD4a\u003c/strong\u003e: m.p 155\u0026ndash;156\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.84 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.7 Hz, 1H), 7.79 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.8 Hz, 1H), 3.98 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3 Hz, 2H), 3.68\u0026ndash;3.56 (m, 1H), 2.93\u0026ndash;2.82 (m, 2H), 2.70 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H), 2.37\u0026ndash;2.24 (m, 2H), 1.90\u0026ndash;1.79 (m, 2H), 1.62\u0026ndash;1.49 (m, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.6, 176.5, 153.9, 147.6, 135.8, 131.0, 129.9, 68.2, 58.4, 52.0, 42.7, 35.0; HRMS (ESI) for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 336.1115; found, 336.1122. HPLC purity\u0026thinsp;=\u0026thinsp;99.2% (t\u003csub\u003er\u003c/sub\u003e = 11.494 min). Data for 6-substituted product \u003cstrong\u003eD4b\u003c/strong\u003e: m.p 122\u0026ndash;123\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.89 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.9, 1.6 Hz, 1H), 8.42 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.7 Hz, 1H), 7.73 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.8 Hz, 1H), 3.99 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H), 3.73\u0026ndash;3.58 (m, 1H), 2.97\u0026ndash;2.86 (m, 2H), 2.75 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H), 2.46\u0026ndash;2.28 (m, 2H), 1.94\u0026ndash;1.82 (m, 2H), 1.67\u0026ndash;1.49 (m, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.8, 176.2, 155.4, 149.2, 148.3, 136.2, 128.8, 128.4, 67.8, 58.2, 51.9, 42.4, 34.8; HRMS (ESI) for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 336.1115; found, 336.1127. HPLC purity\u0026thinsp;=\u0026thinsp;95.1% (t\u003csub\u003er\u003c/sub\u003e = 10.987 min).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of intermediates 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)acetonitrile\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo a stirred a mixture of 1,4-dioxa-8-azaspiro[4.5]decane (716 mg, 5.0 mmol) and DIPEA (954 \u0026micro;L, 5.48 mmol) in toluene (9 mL) was added dropwise 2-chloroacetonitrile (331 \u0026micro;L, 5.24 mmol). The mixture was then allowed to stir for 2 hours at 80\u0026deg;C. The mixture was allowed to cool to room temperature. The organic solvent was evaporated by a rotary evaporator to get a residue. The residue was then quenched with water. Dichloromethane was used for extraction, and the organic phase was dried by adding anhydrous magnesium sulfate. Under reduced pressure, the organic phase was evaporated using a rotary evaporator to obtain a crude product in 87%. \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 3.95 (s, 4H), 3.66 (s, 2H), 2.72\u0026ndash;2.62 (m, 4H), 1.81\u0026ndash;1.70 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of intermediates 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo a round-bottom flask, a mixture of 2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)acetonitrile (790 mg, 4.34 mmol) in dry THF (10 mL) was placed, then it was equipped with a rubber septum. The flask was degasified and then flushed with nitrogen gas. Lithium aluminum hydride 2.4 M in THF (2.7 mL, 6.50 mmol) was added dropwise through a syringe and kept stirring for 1 hour under an ice bath. Then, the resulting reaction solution was allowed to stir at room temperature overnight. Under the cooling condition, the mixture was added with water (5 drops) and 1.5 mL of NaOH (aq), then kept stirring vigorously for 30 minutes. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. Purification by SCC using gradient eluent of DCM: MeOH\u0026thinsp;=\u0026thinsp;3: 1\u0026loz; 3: 2\u0026loz; 3: 3 to afford the desired compound in 75% yield. \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 3.93 (s, 4H), 2.77 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 2.57\u0026ndash;2.46 (m, 4H), 2.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 1.77 (s, 2H), 1.75\u0026ndash;1.67 (m, 4H).\u003c/p\u003e\n\u003cp\u003e7-((2-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)ethyl)amino)-6-chloroquinoline-5,8-dione (\u003cstrong\u003eD5a\u003c/strong\u003e) and 6-((2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethyl)amino)-7-chloroquinoline-5,8-dione (\u003cstrong\u003eD5b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD5a\u003c/strong\u003e and \u003cstrong\u003eD5b\u003c/strong\u003e were synthesized using2-(1,4-dioxa-8-azaspiro[4.5]decan-8-yl)ethan-1-amine (436 mg, 2.34 mmol), \u003cstrong\u003eC\u003c/strong\u003e (445 mg, 1.95 mmol) and DIPEA (436 \u0026micro;L, 2.34 mmol) in dry THF (15 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;20: 20: 1 as a solvent system to afford pure compounds \u003cstrong\u003eD5a\u003c/strong\u003e and \u003cstrong\u003eD5b\u003c/strong\u003e as red solid in 30% and 46%, respectively. Data for 7-substituted product \u003cstrong\u003eD5a\u003c/strong\u003e: m.p 162\u0026ndash;163\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.45 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.63 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.13 (br s, 1H), 4.10\u0026ndash;3.83 (m, 6H), 2.71 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.68\u0026ndash;2.57 (m, 4H), 1.83\u0026ndash;1.74 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.2, 153.4, 146.4, 145.5, 134.7, 129.9, 128.4, 106.9, 64.5, 56.2, 51.1, 41.6, 34.8; HRMS (ESI) for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 378.1221; found, 378.1220. HPLC purity\u0026thinsp;=\u0026thinsp;97.4% (t\u003csub\u003er\u003c/sub\u003e = 8.930 min). Data for 6-substituted product \u003cstrong\u003eD5b\u003c/strong\u003e: m.p 161\u0026ndash;162\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.8 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.8 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 7.07 (br s, 1H), 4.02\u0026ndash;3.91 (m, 6H), 2.72 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.67\u0026ndash;2.61 (m, 4H), 1.85\u0026ndash;1.75 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.2, 175.1, 155.2, 148.6, 144.4, 134.7, 127.0, 126.5, 107.1, 64.4, 55.8, 50.9, 41.5, 35.0; HRMS (ESI) for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 378.1221; found, 378.1205. HPLC purity\u0026thinsp;=\u0026thinsp;98.4% (t\u003csub\u003er\u003c/sub\u003e = 8.089 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-(piperazin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD6a\u003c/strong\u003e) and 7-Chloro-6-((2-(piperazin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD6b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eTo a solution of \u003cstrong\u003eD8a\u003c/strong\u003e (180 mg, 0.43 mmol) in dry DCM (6 mL) was dropwise added trifluoroacetic acid (2 mL) in an ice bath. The mixture was stirred for 30 minutes under ice bath, then allowed to stir at room temperature for 2 hours. After confirming the completion of the reaction, the solvent was removed by a rotary evaporator to obtain the crude product. Purification by SCC using DCM: MeOH\u0026thinsp;=\u0026thinsp;30: 5 as a solvent system to afford pure compound \u003cstrong\u003eD6a\u003c/strong\u003e as a red solid in 94% yield. Data for 7-substituted product \u003cstrong\u003eD6a\u003c/strong\u003e: m.p 154\u0026ndash;155\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.86 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.6 Hz, 1H), 7.81 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 4.01 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 2H), 3.26\u0026ndash;3.17 (m, 4H), 2.85\u0026ndash;2.73 (m, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.9, 176.5, 154.0, 147.5, 135.8, 130.9, 130.0, 58.3, 50.5, 44.9, 42.3. HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 321.1118; found, 321.1130. HPLC purity\u0026thinsp;=\u0026thinsp;99.1% (t\u003csub\u003er\u003c/sub\u003e = 10.715 min).\u003c/p\u003e\n\u003cp\u003eThe synthetic procedure of compound \u003cstrong\u003eD6b\u003c/strong\u003e is similar to that of compound \u003cstrong\u003eD6a\u003c/strong\u003e using \u003cstrong\u003eD8b\u003c/strong\u003e (180 mg, 0.43 mmol in dry DCM (6 mL) and trifluoroacetic acid (2 mL). Purification by SCC using DCM: MeOH\u0026thinsp;=\u0026thinsp;30: 2 as a solvent system to afford pure compound \u003cstrong\u003eD6b\u003c/strong\u003e as a red solid in 90% yield. Data for 6-substituted product \u003cstrong\u003eD6b\u003c/strong\u003e: m.p 173\u0026ndash;174\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.95 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.7 Hz, 1H), 7.75 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.32 (br s, 1H), 3.85 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H), 3.04 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.1 Hz, 4H), 2.73\u0026ndash;2.58 (m, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.9, 174.0, 154.5, 147.7, 145.4, 134.4, 127.3, 126.9, 56.8, 49.0, 42.9, 40.9; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 321.1118; found, 321.1118. HPLC purity\u0026thinsp;=\u0026thinsp;98.9% (t\u003csub\u003er\u003c/sub\u003e = 9.852 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-(4-methylpiperazin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD7a\u003c/strong\u003e) and 7-chloro-6-((2-(4-methylpiperazin-1-yl)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD7b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD7a\u003c/strong\u003e and \u003cstrong\u003eD7b\u003c/strong\u003e were synthesized by using 2-(4-methylpiperazin-1-yl)ethylamine (158 mg, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol) and DIPEA (192 \u0026micro;L, 1.1 mmol) in dry THF (9 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;3: 5: 0.1 as a solvent system to afford pure compounds \u003cstrong\u003eD7a\u003c/strong\u003e and \u003cstrong\u003eD7b\u003c/strong\u003e as red solid in 60% and 6% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD7a\u003c/strong\u003e: m.p 130\u0026ndash;131\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.91 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.47 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.65 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.09 (br s, 1H), 3.97 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 2.72 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.65 (br s, 8H), 2.39 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.4, 153.4, 146.3, 145.5, 134.7, 130.0, 128.4, 56.1, 55.2, 52.3, 46.0, 41.3; HRMS (ESI) for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 335.1275; found, 335.1277. HPLC purity\u0026thinsp;=\u0026thinsp;99.0% (t\u003csub\u003er\u003c/sub\u003e = 11.096 min). Data for 6-substituted product \u003cstrong\u003eD7b\u003c/strong\u003e: m.p. 193\u0026ndash;194\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.99 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.8 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.8 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.01 (br s, 1H), 3.95 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 2.68 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.59 (br s, 8H), 2.31 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 180.3, 175.1, 155.2, 148.7, 144.4, 134.7, 127.0, 126.5, 56.2, 55.2, 52.5, 46.1, 41.2; HRMS (ESI) for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 335.1275; found, 335.1278. HPLC purity\u0026thinsp;=\u0026thinsp;97.4% (t\u003csub\u003er\u003c/sub\u003e = 11.171 min).\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003e\u003cem\u003etert-butyl 4-(2-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)ethyl)piperazine-1-carboxylate (\u003c/em\u003e\u003cstrong\u003eD8a\u003c/strong\u003e\u003cem\u003e) and tert-butyl 4-(2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)ethyl)piperazine-1-carboxylate (\u003c/em\u003e\u003cstrong\u003eD8b\u003c/strong\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD8a\u003c/strong\u003e and \u003cstrong\u003eD8b\u003c/strong\u003e were synthesized by using tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (275 mg, 1.2 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol), and DIPEA (209 \u0026micro;L, 1.2 mmol) in dry THF (9 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;2: 1: 0.2 as a solvent system to afford pure compounds \u003cstrong\u003eD8a\u003c/strong\u003e and \u003cstrong\u003eD8b\u003c/strong\u003e as a red solid in 68% and 28% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD8a\u003c/strong\u003e: m.p 168\u0026ndash;169\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.64 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.03 (br s, 1H), 4.00 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 3H), 3.53\u0026ndash;3.43 (m, 4H), 2.72 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5, 2H), 2.49 (br s, 4H), 1.47 (s, 9H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.4, 154.7, 153.5, 146.3, 145.4, 134.7, 129.9, 128.4, 80.0, 56.6, 52.5, 43.9, 43.2, 41.1, 28.5; HRMS (ESI) for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 421.1643; found, 421.1654. HPLC purity\u0026thinsp;=\u0026thinsp;99.0% (t\u003csub\u003er\u003c/sub\u003e = 13.053 min). Data for 6-substituted product \u003cstrong\u003eD8b\u003c/strong\u003e: m.p 151\u0026ndash;152\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.7 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.8 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 6.95 (br s, 1H), 3.97 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 3.48 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.1 Hz, 5H), 2.70 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 2.47 (br s, 4H), 1.45 (s, 9H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.3, 175.1, 155.3, 154.8, 148.6, 144.3, 134.7, 127.0, 126.6, 80.0, 56.3, 52.4, 44.2, 43.4, 41.1, 28.5; HRMS (ESI) for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 421.1643; found, 421.1643. HPLC purity\u0026thinsp;=\u0026thinsp;99.0% (t\u003csub\u003er\u003c/sub\u003e = 12.610 min).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003etert-butyl (1-(2-((6-Chloro-5,8-dioxo-5,8-dihydroquinolin-7-yl)amino)ethyl)piperidin-4-yl)carbamate (\u003c/em\u003e\u003cstrong\u003eD9a\u003c/strong\u003e\u003cem\u003e) and tert-butyl (1-(2-((7-chloro-5,8-dioxo-5,8-dihydroquinolin-6-yl)amino)ethyl)piperidin-4-yl)carbamate (\u003c/em\u003e\u003cstrong\u003eD9b\u003c/strong\u003e\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD9a\u003c/strong\u003e and \u003cstrong\u003eD9b\u003c/strong\u003e were synthesized by using tert-butyl (1-(2-aminoethyl)piperidin-4-yl)carbamate (383 mg, 1.57 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (299 mg, 1.31 mmol), and DIPEA (273 \u0026micro;L, 1.57 mmol) in dry THF (9 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;1: 2: 0.2 as a solvent system to afford pure compounds \u003cstrong\u003eD9a\u003c/strong\u003e and \u003cstrong\u003eD9b\u003c/strong\u003e as red solid in 71% and 17%, respectively. Data for 7-substituted product \u003cstrong\u003eD9a\u003c/strong\u003e: m.p 167\u0026ndash;168\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.7 Hz, 1H), 7.64 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.6 Hz, 1H), 7.18 (br s, 1H), 4.46 (br s, 1H), 3.94 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 3.50 (br s, 1H), 2.85\u0026ndash;2.80 (m, 2H), 2.66 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.26\u0026ndash;2.18 (m, 2H), 1.99\u0026ndash;1.93 (m, 2H), 1.51\u0026ndash;1.41 (m, 2H), 1.44 (s, 9H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.4, 155.3, 153.4, 146.3, 145.4, 134.7, 130.0, 128.4, 79.5, 56.0, 51.9, 47.8, 41.5, 32.8, 28.5; HRMS (ESI) for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 435.1799; found, 435.1794. HPLC purity\u0026thinsp;=\u0026thinsp;97.1% (t\u003csub\u003er\u003c/sub\u003e = 10.923 min). Data for 6-substituted product \u003cstrong\u003eD9b\u003c/strong\u003e: m.p 80\u0026ndash;81\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 7.03 (br s, 1H), 4.48 (br s, 1H), 3.94 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 3.51 (br s, 1H), 2.87\u0026ndash;2.82 (m, 2H), 2.67 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.27\u0026ndash;2.20 (m, 2H), 2.01\u0026ndash;1.95 (m, 2H), 1.52\u0026ndash;1.45 (m, 2H), 1.44 (s, 9H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.3, 175.1, 155.3, 155.2, 148.6, 144.4, 134.7, 127.0, 126.5, 79.5, 56.1, 51.9, 47.8, 41.4, 32.8, 28.5; HRMS (ESI) for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 435.1799; found, 435.1798. HPLC purity\u0026thinsp;=\u0026thinsp;98.5% (t\u003csub\u003er\u003c/sub\u003e = 15.522 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-((2-hydroxyethyl)(methyl)amino)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD10a\u003c/strong\u003e) and 7-chloro-6-((2-((2-hydroxyethyl)(methyl)amino)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD10b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eA mixture of 2-(2-aminoethylamino)ethanol (142 mg, 1.2 mmol) and 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol) in dry THF (9 mL) was stirred at room temperature for 6 h. The mixture was subsequently concentrated under reduced pressure. The resulting residue was purified by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;1: 3: 0.1 as a solvent system to afford pure compounds \u003cstrong\u003eD10a\u003c/strong\u003e and \u003cstrong\u003eD10b\u003c/strong\u003e as red solid in 24% and 27%, respectively. Data for 7-substituted product \u003cstrong\u003eD10a\u003c/strong\u003e: 108\u0026ndash;109\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.64 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 6.93 (br s, 1H), 4.01 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2H), 3.72 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.1 Hz, 2H), 2.82 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.69 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.2 Hz, 2H), 2.39 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.1, 175.6, 153.5, 146.2, 145.0, 134.8, 130.0, 128.5, 59.3, 59.2, 56.9, 42.1, 41.6; HRMS (ESI) for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 310.0958; found, 310.0955. HPLC purity\u0026thinsp;=\u0026thinsp;97.1% (t\u003csub\u003er\u003c/sub\u003e = 11.488 min). Data for 6-substituted product \u003cstrong\u003eD10b\u003c/strong\u003e: m.p 136\u0026ndash;137\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.34 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.7 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 6.85 (br s, 1H), 3.98 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 2H), 3.70 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.2 Hz, 2H), 2.76 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 2.65 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.3 Hz, 2H), 2.34 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.4, 175.3, 155.4, 148.7, 144.1, 134.8, 126.9, 126.6, 59.3, 59.2, 56.7, 41.9, 41.4; HRMS (ESI) for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 310.0958; found, 310.0962. HPLC purity\u0026thinsp;=\u0026thinsp;95.8% (t\u003csub\u003er\u003c/sub\u003e = 11.038 min).\u003c/p\u003e\n\u003cp\u003e6-Chloro-7-((2-(dimethylamino)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD11a\u003c/strong\u003e) and 7-chloro-6-((2-(dimethylamino)ethyl)amino)quinoline-5,8-dione (\u003cstrong\u003eD11b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eFollowing the general procedure, compounds \u003cstrong\u003eD11a\u003c/strong\u003e and \u003cstrong\u003eD11b\u003c/strong\u003e were synthesized by using unsym-dimethyl-ethylenediamine (120 \u0026micro;L, 1.1 mmol), 6,7-dichloroquinoline-5,8-dione \u003cstrong\u003eC\u003c/strong\u003e (228 mg, 1.0 mmol) and DIPEA (192 \u0026micro;L, 1.1 mmol) in dry THF (9 mL). Purification by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;3: 5: 0.3 as a solvent system to afford pure compounds \u003cstrong\u003eD11a\u003c/strong\u003e and \u003cstrong\u003eD11b\u003c/strong\u003e as a red solid in 37% and 7% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD11a\u003c/strong\u003e: m.p. 133\u0026ndash;134\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.90 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.7 Hz, 1H), 8.46 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 1.7 Hz, 1H), 7.63 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.6 Hz, 1H), 6.90 (br s, 1H), 3.95 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 2.59 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 2H), 2.29 (s, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 178.8, 175.4, 153.4, 146.4, 145.8, 134.7, 129.9, 128.3, 58.1, 45.0, 42.4; HRMS (ESI) for C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 280.0853; found, 280.0858. HPLC purity\u0026thinsp;=\u0026thinsp;99.2% (t\u003csub\u003er\u003c/sub\u003e = 11.934 min). Data for 6-substituted product \u003cstrong\u003eD11b\u003c/strong\u003e: m.p. 132\u0026ndash;133\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 9.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.33 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.8 Hz, 1H), 7.57 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 6.89 (br s, 1H), 3.93 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 2H), 2.59 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H), 2.30 (s, 6H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 180.2, 175.1, 155.2, 148.7, 144.6, 134.7, 127.1, 126.5, 57.9, 45.0, 42.2; HRMS (ESI) for C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 280.0853; found, 280.0862. HPLC purity\u0026thinsp;=\u0026thinsp;97.1% (t\u003csub\u003er\u003c/sub\u003e = 11.520 min).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of intermediate 2-(dis(2-hydroxyethyl)amino)acetonitrile\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e2,2'-Azanediylbis(ethan-1-ol) (2.5 g, 23.8 mmol) and TEA (7 mL, 50.2 mmol) was mixed in anhydrous methanol (15 mL), followed by the addition of 2-bromoacetonitrile (3.9 g, 32.5 mmol) dropwise under an ice-bath. Then, the reaction mixture was allowed to stir at room temperature for 3 h. The mixture was concentrated under reduced pressure to remove the solvent and then was added dichloromethane (15 mL). The insoluble was filtered off, and the filtrate was concentrated under reduced pressure to obtain a residue. Purification by SCC using DCM: MeOH\u0026thinsp;=\u0026thinsp;3: 0.2 to give the desired compound in 50% yield. \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 3.33\u0026ndash;2.84 (m, 4H), 1.43\u0026ndash;1.31 (m, 6H).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of intermediate 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo a suspension of 2-(bis(2-hydroxyethyl)amino)acetonitrile (730 mg, 5.06 mmol) in dry THF (8 mL) was added 3.5 mL of LiAlH\u003csub\u003e4\u003c/sub\u003e 2.4 in THF solution dropwise while stirring under an ice-bath. The mixture was stirred at 0\u0026deg;C for 1 h, then slowly increased to room temperature overnight. The reaction mixture was added 5 drops of water under an ice bath, then quenched with NaOH 1.0 N solution (1.5 mL). The resulting mixture was filtered using a pad of celite, then the filtrate was collected, followed by the removal of the solvent under reduced pressure. Purification by SCC using DCM: MeOH\u0026thinsp;=\u0026thinsp;4: 1 to give the desired compound in 28% yield. \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 3.64\u0026ndash;3.54 (m, 4H), 2.75\u0026ndash;2.56 (m, 8H).\u003c/p\u003e\n\u003cp\u003e7-((2-(bis(2-Hydroxyethyl)amino)ethyl)amino)-6-chloroquinoline-5,8-dione (\u003cstrong\u003eD12a\u003c/strong\u003e) and 6-((2-(bis(2-hydroxyethyl)amino)ethyl)amino)-7-chloroquinoline-5,8-dione (\u003cstrong\u003eD12b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eA mixture of 2,2'-((2-aminoethyl)azanediyl)bis(ethan-1-ol) (178 mg, 1.20 mmol) and \u003cstrong\u003eC\u003c/strong\u003e (150 mg, 0.66 mmol) in dry THF (9 mL) was stirred at rt for 7 h. The reaction mixture was concentrated using a rotary evaporator. The resulting residue was then purified by SCC using \u003cem\u003en\u003c/em\u003e-hexane: EA: MeOH\u0026thinsp;=\u0026thinsp;2: 2: 0.3 \u0026loz; 0.2: 0.2: 0.4 \u0026loz; 2: 2: 0.5 as a solvent system to afford pure compounds \u003cstrong\u003eD12a\u003c/strong\u003e and \u003cstrong\u003eD12b\u003c/strong\u003e as a red solid in 38% and 39% yield, respectively. Data for 7-substituted product \u003cstrong\u003eD12a\u003c/strong\u003e: m.p 105\u0026ndash;106\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.84 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.6 Hz, 1H), 8.45 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.6 Hz, 1H), 7.79 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 3.97 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 3.65 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 4H), 2.93 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 2.77 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 179.8, 177.0, 153.8, 147.5, 135.8, 131.0, 129.9, 60.9, 57.6, 56.0, 43.6; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 340.1064; found, 340.1064. HPLC purity\u0026thinsp;=\u0026thinsp;99.2% (tr\u0026thinsp;=\u0026thinsp;11.633 min). Data for 6-substituted product \u003cstrong\u003eD12b\u003c/strong\u003e: m.p 146\u0026ndash;147\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eH\u003c/em\u003e\u003c/sub\u003e 8.89 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 1.6 Hz, 1H), 8.42 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7 Hz, 1H), 7.73 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 4.7 Hz, 1H), 4.00 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 2H), 3.68 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 4H), 3.01 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H), 2.85 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, MeOD) \u0026delta;\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e 180.9, 176.3, 155.4, 149.2, 147.1, 136.2, 128.8, 128.3, 60.4, 57.5, 55.9, 43.2; HRMS (ESI) for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e: calcd, 340.1064 ; found, 340.1064. HPLC purity\u0026thinsp;=\u0026thinsp;96.9% (t\u003csub\u003er\u003c/sub\u003e = 10.981 min).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular docking simulation was performed with the Discovery Studio Client 2022 (Biovia Corp. CA, USA) to elucidate the interaction of the selected derivatives into the CDC25C active site. The CDC25C crystal protein (PDB ID: 3OP3) was downloaded from RSCB protein data bank website, and imported to the Discovery Studio (DS) Client 2022. The crystal protein was cleaned by removing unwanted ligands and water molecules, and hydrogen was added before the dock. Compounds \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e were used here and their chemical structures were drawn using Chem3D Professional 15.0 software (PerkinElmer Informatics, Inc. CA, USA). The compounds were energy minimized at root-mean-square gradient tolerance of 0.01 and saved as SD files for further operation. The LibDock molecular docking method was used to conduct the experiment after protein setup by defining the binding site from the active site of CDC25C to dock all the prepared ligands (\u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003eD3a\u003c/strong\u003e, and \u003cstrong\u003eD11a\u003c/strong\u003e). Based on the highest LibDock score of respective compounds, their binding interactions with CDC25C crystal protein were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost materials and methodologies of biological evaluations have been applied in our previous report. Please refer to reference [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHL-60 promyeloblasts from acute promyelocytic leukemia and KG-1 macrophage from acute myelogenous leukemia were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (11875-093, Gibco). MV-4-11 macrophages from biphenotypic B-myelomonocytic leukemia and K-562 lymphoblast from chronic myelogenous leukemia were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (12200-036, Gibco), with 1.5g/L and 3g/L sodium bicarbonate, respectively. All media were supplemented with 10% fetal bovine serum (F0926, Sigma) and 1% penicillin-streptomycin-glutamine (10378-016, Gibco) in a humidified atmosphere at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemicals and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColchicine and Paclitaxel (Taxol) were purchased from Sigma (C9754) and Cytoskeleton, Inc. (TXD01), 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 H3 (ab1791; Abcam); anti-Ataxia-telangiectasia mutated kinase (ATM) pS1981 (560007, BD Pharmingen\u0026trade;); 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-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-histone H3 (ab1791, Abcam); anti-Induced myeloid leukemia cell differentiation protein (Mcl-1)(ab32087, Abcam); anti-caspase 3 (NB100-56708, Novus Biologicals); anti-B-cell leukemia/lymphoma 2 (Bcl-2)(#3498, Cell Signaling); anti-Caspase 9 (#9502, Cell Signaling); anti-histone H4K20me1 (07\u0026ndash;1570; Millipore); anti-histone H4 (ab31830; Abcam). Secondary antibodies: Horseradish peroxidase (HRP)-conjugated goat anti-mouse (115-035-003; Jackson ImmunoResearch Labs); anti-rabbit (111-035-003; Jackson ImmunoResearch Labs) antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMTS assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 96-well plates and treated with indicated compounds for 72 h. The surviving cells were determined by incubating them with 0.2 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2\u003cem\u003eH\u003c/em\u003e-tetrazolium (MTS; Abcam, ab223881) and measuring the absorbance at 490 nm by using a PerkinElmer VICTOR3\u0026trade; Multilabel Plate Reader. The concentration of 50% growth inhibition (IC\u003csub\u003e50\u003c/sub\u003e) was calculated using the CompuSyn software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were collected, fixed with 70% ice-cold ethanol, washed with cold phosphate-buffered saline containing 1% FBS, and 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\u0026deg;C for 30 minutes. DNA content was analyzed using the Becton Dickinson FACSCalibur Flow Cytometer and the cell-cycle profiles were plotted with the FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed with Laemmli sample buffer (LSB), and proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membranes. After blocking with 5% skim milk, indicated proteins were probed with specific primary antibodies, followed by hybridization with HRP-conjugated species-specific secondary antibodies. Signals were developed using enhanced chemiluminescence substrates (Bio-Rad) and detected by using the Invitrogen iBright FL-1500 system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003eCDC25C activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzyme inhibition assays were performed by the Eurofins Panlabs, Inc. Compounds were pre-incubated with human CDC25C enzyme purified from E. coli in the reaction buffer (50 mM Tris-HCl, pH 8.5, 0.1% BSA, 1mM DTT, 100 mM NaCl) for 15 minutes and then mixed with 15.0 \u0026micro;M of 3-O-methylfluorescein phosphate for 60 minutes. 3-Omethylfluorescein signals were quantified and the percentages of inhibition were plotted using GraphPad Prism 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic DNA purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were washed with PBS and resuspended in DNA extraction lysis buffer (10mM Tris-HCl, pH8.0, 10mM EDTA, and 1% SDS) containing 400 \u0026micro;g/ml of proteinase K (Worthington Biochemical; 39450-01-6). After overnight incubation at 56 ℃, lysates were purified using Phenol: Chloroform: Isoamyl Alcohol (25:24:1, v/v; Invitrogen\u0026trade;; 15593031). The supernatant was treated with RNase A (250\u0026micro;g/ml) at 37℃ for 1h, followed by the second purification using Phenol: Chloroform: Isoamyl Alcohol. Genomic DNAs were finally precipitated by ethanol and dissolved in pure water, followed by 2% agarose gel separation and signal detection using the Invitrogen iBright FL-1500 system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003ePDOs were seeded at a density of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e in 10 \u0026micro;L of rBM in 24-well plates and cultured for 3 days. To assess the growth of the PDOs, 72 h after seeding, the cultures were treated with increasing concentrations of \u003cstrong\u003eD3a\u003c/strong\u003e, \u003cstrong\u003eD3b\u003c/strong\u003e, \u003cstrong\u003eD11a\u003c/strong\u003e, and \u003cstrong\u003eD11b\u003c/strong\u003e, followed by a cell proliferation assay using the Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer's protocol. In brief, 10 \u0026micro;L of CCK-8 reagent was added to 200 \u0026micro;L of culture medium and incubated for 2 h at 37\u0026deg;C in a 5% CO2. The absorbance at 450 nm was then measured using a SpectraMax iD5 microplate reader.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eDual-chamber invasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e100 CRC organoids were cultured and plated on Transwell inserts (BD Biosciences) coated with a thin layer of collagen type I (BD Biosciences) under serum free medium overnight. In the presence of 10% FBS incubated for 24 h to allow invasion through the collagen. After the incubation, cells that invaded the insert membrane were fixed, stained with SYTOX Green (Invitrogen), and counted under a fluorescence microscope.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\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\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research were supported by the National Science and Technology Council of Taiwan (grant no. 112-2113-M-038-001). This work was also supported by the Higher Education Sprout Project by the Ministry of Education (MOE) of Taiwan (grant no. DP2-TMU-112-C-03). We sincerely thank Dr. Li-Jung Juan (Genomics Research Center, Academia Sinica) for providing experimental resources and Richa Upadhyay for performing ADME prediction analysis. The technical support provided by the Core Facilities of Taipei Medical University and Academia Sinica was also appreciated.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDana SMmA, Meghdadi M, Kakhki SK, Khademi R (2024) Anti-leukemia effects of ginsenoside monomer: A narrative review of pharmacodynamics study. 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Sci Rep 7:42717. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep42717\u003c/span\u003e\u003cspan address=\"10.1038/srep42717\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CDC25 inhibitors, cancer therapeutics, cell cycle arrest, genomic instability, patient-derived organoids","lastPublishedDoi":"10.21203/rs.3.rs-6282946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6282946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCell division cycle 25 (CDC25) phosphatases have emerged as critical regulators of cell cycle progression and genomic stability, making them compelling therapeutic targets in oncology. Building on the established CDC25 inhibitor \u003cstrong\u003eNSC663284\u003c/strong\u003e, we strategically designed, synthesized, and evaluated a novel series of derivatives with diverse alkylamino side chains to enhance potency and selectivity. Among them, derivatives featuring 2-(4-methylpiperidin-1-yl)ethylamino (\u003cstrong\u003eD3a\u003c/strong\u003e and \u003cstrong\u003eD3b\u003c/strong\u003e) or 2-(dimethylamino)ethylamino groups (\u003cstrong\u003eD11a\u003c/strong\u003e and \u003cstrong\u003eD11b\u003c/strong\u003e) demonstrated remarkable anti-cancer efficacy, exhibiting potent apoptotic induction and broad-spectrum growth inhibition. These compounds displayed IC\u003csub\u003e50\u003c/sub\u003e values between 0.21 to 1.22 μM in leukemia cells and from 0.13 to 1.5 μM in CRC cells, surpassing the activity of \u003cstrong\u003eNSC663284\u003c/strong\u003e. Mechanistically, these derivatives effectively inhibited CDC25 phosphatase activity in vitro and disrupted CDC25-mediated dephosphorylation of CDK1 in cells, leading to cell cycle arrest and catastrophic genomic instability. Treatment with these compounds induced rapid and extensive double-stranded DNA breaks, highlighting their potential to drive irreversible cancer cell death. Importantly, their therapeutic potential was further validated in CRC patient-derived tumor organoids, offering clinically relevant insights into patient-specific drug responses and underscoring their translational significance. Our findings establish a new class of CDC25 inhibitors with superior anti-cancer activity and mechanistic precision, paving the way for next-generation therapeutics targeting leukemia and CRC.\u003c/p\u003e","manuscriptTitle":"Quinoline-5,8-Dione CDC25 Inhibitors: Potent Anti-Cancer Agents in Leukemia and Patient-Derived Colorectal Organoids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 09:27:52","doi":"10.21203/rs.3.rs-6282946/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"346f4243-a240-43f5-adb8-edceeacb327b","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50908161,"name":"Biological sciences/Drug discovery/Drug delivery"},{"id":50908162,"name":"Biological sciences/Cancer/Cancer therapy/Drug development"}],"tags":[],"updatedAt":"2025-07-09T09:27:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-09 09:27:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6282946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6282946","identity":"rs-6282946","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}