Phthalazino[1,2-b]quinazolinones as p53 Activators: Cell Cycle Arrest

Article pubs.acs.org/jmc

Phthalazino[1,2‑b]quinazolinones as p53 Activators: Cell Cycle Arrest, Apoptotic Response and Bak−Bcl-xl Complex Reorganization in Bladder Cancer Cells Guo-Hai Zhang,†,‡,# Jing-Mei Yuan,†,# Gang Qian,† Chen-Xi Gu,† Kai Wei,† Dong-Liang Mo,† Jiang-Ke Qin,† Yan Peng,† Zu-Ping Zhou,‡ Cheng-Xue Pan,*,† and Gui-Fa Su*,† †

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, 15 Yu Cai Road, Guilin 541004, China ‡ Guangxi Universities Key Laboratory of Stem Cell and Biopharmaceutical Technology, School of Life Sciences, Guangxi Normal University, 15 Yu Cai Road, Guilin 541004, China S Supporting Information *

ABSTRACT: p53 inactivation is a clinically defined characteristic for cancer treatment-nonresponsiveness. It is therefore highly desirable to develop anticancer agents by restoring p53 function. 1 Herein the synthesized phthalazino[1,2-b]quinazolinones were discovered as p53 activators in bladder cancer cells. 10-Bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5da) was identified as the most promising candidate in view of both its anticancer activity and mechanisms of action. 5da exhibited strong anticancer activity on a broad range of cancer cell lines and significantly reduced tumor growth in xenograft models at doses as low as 6 mg/ kg. Furthermore, 5da caused cell cycle arrest at S/G2 phase, induced apoptosis, changed cell size, and led to cell death by increasing the proportion of sub-G1 cells. Molecular mechanism studies suggested that accumulation of phospho-p53 in mitochondria after 5da treatment resulted in conformational activation of Bak, thereby evoking cell apoptosis, finally leading to irreversible cancer cell inhibition. Our present studies furnish new insights into the molecular interactions and anticancer mechanisms of phospho-p53-dependent quinazolinone compound.

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INTRODUCTION Bladder cancer (BC) continues to be the second most common malignancy of urinary system worldwide with an overall fiveyear recurrence rate up to 78%.2−4 Especially for patients suffering from muscle-invasive tumors, high mortality is a serious health threat due to distant metastases.5,6 Chemotherapy is commonly adopted to delay relapse and prolong survival,7 however, no more than half of the muscle-invasive BC patients respond to chemotherapy.7−9 Furthermore, the problem of easy recurrence remains to be unsolved.10 Therefore, it is very important to explore novel chemotherapeutic agents with treatment and prevention functions and elucidate the mechanisms of increasing treatment responsiveness of bladder cancer cells. Inactivation of the p53 tumor suppressor pathway is a common feature of human cancers, furnishing an attractive avenue for cancer therapies based on restoring p53 function in established tumors.11,12 It has been proposed that p53 activation promoted cancer cell killing.13,14 Moreover, activation of p53 pathway can restrict malignant transformation by triggering apoptosis or cell cycle arrest.15 It is therefore highly desirable to restore p53 function by persistent p53 activation with small-molecule compounds for BC therapy. Quinazolinone is a key structural motif in many natural products, as well as in synthetic pharmaceutical compounds. © 2017 American Chemical Society

Quinazolinone derivatives exhibit diverse pharmacological activities and have been identified as enzyme inhibitors,16−18 antileishmanial agents,19 receptor antagonists,20,21 anti-inflammatory agents,22,23 and anticancer agents.24−26 A recent study27 aimed to identify p53 activator with potent antitumor activity documented that 5-(3-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one (BMH-7,27 5ab, Figure 1) could regulate the p53 signaling pathway and exhibit anticancer activities in vitro and in vivo. However, the synthesis and further structure−activity relationship studies of this scaffold have not yet been reported in the literature. To obtain increased efficacy, in the present study, we designed and synthesized a series of phthalazino[1,2-b]quinazolinone derivatives (5) and identified 10-bromo-5-(2-dimethylaminoethylamino)phthalazino[1,2-b]quinazolin-8-one (5da, Figure 1) as the most promising one in view of both the anticancer activity and the mechanisms of action. We were able to demonstrate clearly that 5da could induce phospho-p53 mitochondria-targeted accumulation with complicated regulatory process in cell apoptosis and proliferation inhibition. Received: January 4, 2017 Published: July 26, 2017 6853

DOI: 10.1021/acs.jmedchem.6b01769 J. Med. Chem. 2017, 60, 6853−6866

Journal of Medicinal Chemistry

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Figure 1. 5da is not a pan assay interference compound (PAINS). (A) Structures of 5ab and 5da. (B) Effects of 5da on absorption of light in 490 nm in T24 cells. (C) Gel electrophoresis of pBR322 DNA after being incubated with 5da at the concentrations of 1−10 μM for 1 h in Tris-HCl buffer. DMSO 1% + plasmid in lane 1 as negative control, EB + plasmid in lane 2 as positive control. (D) Immunoblotting analysis of γH2AX related to 5da-induced DNA damage. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibody against γH2AX. (E) Emission spectra of 5da. (F) Effects of 5da on membrane disruption in T24 cells. EB: Ethidium bromide.

Scheme 1. Synthesis of Phthalazino[1,2-b]quinazolin-8-one Derivatives 5aa−5ee

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RESULTS

substituted 5-chlorophthalazino[1,2-b]quinazolin-8-ones (4) in 50−72% yields. Finally, 4 reacted with amine derivatives in toluene furnished the target compounds 5. The structure of 10fluoro-5-(2-dimethylaminoethylamino)phthalazino[1,2-b]quinazolin-8-one (5ba) was further unambiguously determined by the X-ray crystallography (detailed parameters are included in the Supporting Information). In Vitro Biological Evaluations. Compounds 5 were evaluated in vitro against a panel of five cancer cell lines

Chemistry. Synthesis of phthalazino[1,2-b]quinazolinone derivatives 5 was described in Scheme 1 and Scheme 2. First, commercially available anthranilic acids were converted to the corresponding anthranilic hydrazides (2) in two steps according to literature methods.28 Then 2 and phthalic anhydride in glycol were heated in oil bath to provide 10-substituted 6Hphthalazino[1,2-b]quinazolin-5,8-diones (3) in high yield. Treatment of 3 with phosphorus oxychloride gave 106854



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explored. The results indicated that in general aminoalkylamine side chains could give better antiproliferative activity than 4methyl-N-piperazinyl (series 5xe), and if the 5-substituent was replaced by a N-morpholinyl (series 5xd), alkyl (5dk), or ωalkoxycarbonyl substituted alkyl (5dl and 5dm), the compounds almost lost the cytotoxicity. The tail of the aminoalkylamine side chain also significantly affected the antiproliferative activity. Compound with dimethylamino at the tail (5da) are more potent than with diethylamino (5df) or diiospropylamino (5dg). A tail with pyrrolidinyl and pyridyl (compounds 5dh and 5di) also result in potent antiproliferative activity. When the tail of the aminoalkylamine was replaced by a N-morpholinyl (5dc, 5dj), the cytotoxicity of the compounds would decrease. The length of the side chain also showed notable influence to cytotoxicity. A link of aminoethyl is more favorable than an aminopropyl (5da vs 5db, 5dj vs 5dc). The data in Table 1 also suggested that replacing the hydrogen (H) atom (5ax series) at the 10-position of the phthalazino[1,2-b]quinazolin-8-one skeleton with a halogen atom (5bx−5dx series) or a methyl (5ex series) could increase the cytotoxicity. Among them, the Br substitution (5dx series)

Scheme 2. Synthesis of Phthalazino[1,2-b]quinazolin-8-one Derivatives 5df−5dm

including T24, HepG2, NCI-H460, MGC-803, and HeLa using microculture tetrazolium (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT) assay. The IC50 values derived from dose−response curves were summarized in Table 1. As shown in Table 1, many of the compounds showed potent cytotoxicity to all the tested cell lines. The data indicated that the substituent on the 5-position of the phthalazino[1,2b]quinazolin-8-one skeleton had a very important effect on activity. Thirteen different substituents on the 5-position were

Table 1. IC50 of Quinazolinone Compounds 5 against Multiple Cancer Cell Lines IC50 (μM)a

a

compd

T24

HepG2

NCI-H460

MGC-803

HeLa

5aa 5ba 5ca 5da 5ea 5ab 5bb 5cb 5db 5eb 5ac 5bc 5cc 5dc 5ec 5ad 5bd 5cd 5dd 5ed 5ae 5be 5ce 5de 5ee 5df 5dg 5dh 5di 5dj 5dk 5dl 5dm HCPTb

7.47 ± 1.62 4.62 ± 0.80 3.46 ± 0.41 3.09 ± 0.31 2.12 ± 0.24 14.54 ± 2.93 8.90 ± 1.93 9.83 ± 1.56 3.25 ± 0.33 7.28 ± 2.08 27.95 ± 5.19 18.84 ± 4.18 8.85 ± 2.12 9.03 ± 2.05 9.78 ± 1.64 >100 >100 >100 >100 >100 33.81 ± 6.68 34.84 ± 6.22 30.73 ± 8.06 77.02 ± 15.28 60.48 ± 12.38 10.69 ± 2.47 12.71 ± 2.76 6.43 ± 1.24 3.81 ± 0.47 10.75 ± 1.86 >100 >100 >100 5.53 ± 1.24

10.24 ± 2.41 4.68 ± 1.07 4.89 ± 0.84 3.80 ± 0.74 4.97 ± 1.17 11.46 ± 2.86 10.69 ± 2.38 8.78 ± 1.62 2.20 ± 0.31 7.82 ± 1.92 30.48 ± 5.81 45.09 ± 8.87 18.34 ± 2.65 12.82 ± 3.26 24.09 ± 5.12 92.29 ± 19.60 >100 >100 >100 >100 39.42 ± 8.03 51.89 ± 11.01 57.56 ± 12.22 41.13 ± 8.71 55.19 ± 13.61 2.99 ± 0.31 11.68 ± 2.18 5.21 ± 0.43 4.14 ± 0.86 11.28 ± 2.54 >100 >100 >100 5.52 ± 0.96

12.13 ± 3.36 7.49 ± 1.41 5.11 ± 0.72 5.03 ± 0.55 5.69 ± 1.33 16.97 ± 4.10 11.18 ± 2.24 10.5 ± 2.44 5.48 ± 1.18 9.70 ± 2.30 40.19 ± 7.15 52.02 ± 10.20 19.93 ± 2.43 15.07 ± 3.90 21.61 ± 4.12 >100 >100 >100 >100 >100 31.35 ± 5.47 77.72 ± 14.41 70.36 ± 16.28 43.70 ± 9.07 70.56 ± 16.64 5.06 ± 1.74 13.61 ± 2.47 6.84 ± 1.22 7.85 ± 2.53 7.85 ± 1.66 >100 >100 >100 4.91 ± 1.02

8.71 ± 2.06 6.60 ± 2.21 4.82 ± 0.41 4.29 ± 0.72 5.65 ± 1.13 10.66 ± 1.83 32.15 ± 5.66 13.88 ± 2.14 3.35 ± 0.56 5.24 ± 1.17 14.49 ± 1.93 79.08 ± 14.02 23.14 ± 4.69 7.21 ± 1.24 20.67 ± 4.20 >100 >100 >100 >100 >100 44.45 ± 7.20 26.11 ± 7.06 66.14 ± 14.30 40.39 ± 6.71 34.18 ± 5.19 7.26 ± 1.39 10.60 ± 2.11 4.15 ± 0.46 4.48 ± 0.69 9.90 ± 1.07 >100 >100 >100 15.19 ± 2.81

12.96 ± 3.11 7.92 ± 1.26 5.60 ± 1.12 6.10 ± 1.48 3.41 ± 1.06 20.19 ± 5.42 22.09 ± 5.06 15.65 ± 3.09 7.13 ± 1.64 13.62 ± 3.94 53.82 ± 9.01 60.58 ± 11.13 34.56 ± 6.70 17.40 ± 4.64 24.23 ± 5.86 >100 >100 >100 >100 >100 50.38 ± 9.94 44.89 ± 10.07 >100 73.5 ± 14.33 85.00 ± 19.88 8.81 ± 2.66 11.38 ± 2.57 3.71 ± 0.53 2.01 ± 0.83 8.80 ± 1.55 >100 >100 >100 29.16 ± 6.19

Each IC50 value was calculated from three independent experiments performed in triplicate. b10-Hydroxycamptothecin. 6855



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Figure 2. Effects of 5da on cell cycle distribution and cell size in T24 and J82 cells. (A) Regulation of cell cycle arrest by 5da was conducted by FACS analysis with PI staining in T24 cells. (B) Distribution of cell cycles in 5da-treated T24 cells was plotted. Mean ± SD was from three independent measurements. (C) 5da reduced the cell size in T24 cells. Cells were treated for 48 h with DMSO or 5 μM of 5da and analyzed by FACS for relative cell size. Histograms of the forward scatter (FSC-H) for the treated cells and mean FSC-H values are shown. (D) Regulation of cell cycle arrest by 5da was conducted by FACS analysis with PI staining in J82 cells. (E) 5da and 5ab significantly activated p53 with little influence on 6856



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Figure 2. continued its expression. T24 cells were incubated with indicated concentrations of 5da and 5ab for 24 h. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against p53 and p-p53 (s392). The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. (F) IC50 values of 5da in DU145, PC-3, and LnCap cancer cells. (G) Knock down p53 for inhibition sensitivity study. T24 cells were pre-exposed to p53-siRNA or scRNA for 24 h and then treated with indicated concentrations of 5da for 48 h. (H) Immunoblotting analysis of p53 activation induced by 5da. Nutlin-3 was used as a positive control. (I) Immunoblotting analysis of proteins related to 5da-induced cell cycle arrest. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against proteins indicated. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. **p < 0.01.

the S/G2 phase arrest-induced growth inhibition and cell size reduction. To investigate the mechanism of 5da-induced cell cycle arrest, we first checked the p53 expression and p53 phosphorylation after 5da treatment, as the 5da analogue 5ab could pharmacologically activate the p53 pathway to exhibit anticancer activity.27 As shown in Figure 2E, both 5da and 5ab could induce p53 activation by promoting p53 phosphorylation in a dose-dependent manner along with little impact on p53 expression. To find out whether the anticancer effect of 5da against T24 cancer cells was due to p53 activation, we examined the anticancer activities of 5da in DU145 cancer cells (p53 mutation cell line), p53-null PC-3 cancer cells, and LnCap cancer cells (wild-type p53). Our results documented that the anticancer activities of 5da in DU145 cancer cells and PC-3 cancer cells were obviously attenuated compared to that in wtp53 LnCap cancer cells (Figure 2F). To further explore that the inhibitory effect of 5da on cell proliferation was mainly due to p53 activation, p53-siRNA was used to ablate endogenous p53. Both cells (p53 present and absent) were exposed to different concentrations of 5da for 48 h. In scRNA-treated T24 cells and control group, 5da treatment dramatically inhibited cell proliferation in a dose-dependent manner, while in p53-ablated T24 cells, the sensitivity of T24 cells to 5da was significantly decreased (Figure 2G). Furthermore, to provide more direct evidence on p53 activation, we also studied the inhibition of interactions between p53 and MDM2 after 5da treatment. As depicted in Figure 2H, both 5da and nutlin-3 effectively activated p53 in T24 cells, evident by robust increase of p21 protein. 5da was even more potent than nutlin-3, the first MDM2 inhibitor reported by Vassilev and colleagues.30 Both 5da and nutlin-3 could decrease MDM2 protein in T24 cells, but almost no effect on the expression of p53. These results suggested that 5da exhibited toxicity for cancerous was predominantly due to p53 activation. Generally speaking, p53 activation is a common response to distinct cellular stresses, which induces cell cycle arrest or apoptosis by governing the downstream signaling molecules.31 Because p21 is the direct target of p53 regulatory circuit, we investigated the cyclinE−CDK2 complexes, cyclinD−CDK4 complexes, and cyclin-dependent kinase (CDK) inhibitor p21 variance due to their functions in cell cycle progression. Cyclin E binds to CDK2, which is required for the transition from G1 to S phase during cell division.32 Our results showed that 5da could induce significant increase of cyclinE−CDK2 complexes to promote G1 to S phase transition (Figure 2I). However, nearly no change was found in the expression of cyclin D and CDK4. Instead, 5da could up-regulate p21 expression in a dosedependent manner (Figure 2I). These results suggested that S/ G2 arrest induced by 5da resulted from the inhibition of CDK activity by p21 rather than the expression reduction of cyclin D and CDK4.

gave the most potency to most of the tested cell lines in general, as exemplified by 5da. The anticancer activities of 5da in vitro were superior to the first-line chemotherapeutic agent 10-hydroxycamptothecin (HCPT), and this compound was chosen to further investigate the mechanism of its cytotoxic effect. 5da Is Not a Pan Assay Interference Compound (PAINS). Prior to study the anticancer mechanism of 5da, firm experiments had been carried out to check its apparent activity was not an artifact. 5da and its analogues are known as quinoline class of putative PAINS; their absorption of light in the range 570−620 nm would interfere the effective IC50 values in turn.29 To check the potential interference effect, we tested the absorption change of light in 490 nm in T24 cells after 5da treatment. As shown in Figure 1B, 5da treatment (doses from 1 to 10 μM, 1 h) did not affect the absorption of light in 490 nm (P > 0.05). Another mechanism of assay interference of such class putative PAINS may be to cause DNA damage. To test the possibility, DNA relaxation assay was used to investigate the interaction between 5da and DNA directly. After concentrations (1−10 μM) of 5da treatment, there was nearly no difference either in conformational changes of DNA or in the mobility rate of DNA compared with the negative control group, while in the positive control group (EB group), the migration rate of DNA was significantly inhibited (Figure 1C). Moreover, a biomarker, γH2AX, was adopted to detect DNA damage. As depicted in Figure 1D, no DNA damage was detected after 5da treatment. Furthermore, 5da containing a fluorophore could emit fluorescence in the range 420−450 nm (Figure 1E), thus leading to other mechanisms of assay interference. To test the possibility, T24 cells were treated with 5da (doses from 1 to 10 μM, 1 h), stained with PI, and examined by FACS analysis. As shown in Figure 1F, 5da neither triggered obvious membrane disruption nor affected the distribution of cells in the FITC channel to further ensure the reliability of the data based on fluorescence analysis in the following experiments. Collectively, these results suggested that 5da is not PAINS. 5da Induces Cell Cycle Arrest and Cell Size Change in T24 Cancer Cells. To study the potential mechanistic pathways responsible for cell proliferation inhibition by 5da, we tested the change of cell cycle in T24 and J82 cancer cell lines. As shown in Figure 2A, D, 5da treatment (doses from 1 to 10 μM, 48 h) clearly increased the cell proportion of S/G2 phase and sub-G1 phase (a specific phase of cell death), while the cell proportions in G1 phase were markedly reduced (Figure 2B, D). Consistent with the effects on cell cycle arrest, 5da induced a more profound reduction in cell size (Figure 2C, left). The relative cell size (mean FSC-H) of 5da-treated cells were 87.7% of control cells in T24 cell line (Figure 2C, right). Collectively, these results documented the antiproliferation potency of 5da in a broad spectrum of cancer cell lines were potentially due to 6857



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Figure 3. 5da induces cell apoptosis through the intrinsic apoptotic pathway. (A) Induction of apoptosis by 5da was examined by FACS analysis with PI and FITC-annexin V staining in T24 cells. (B) 5da induced apoptosis by triggering caspase-3 activities in T24 cells. Arrow showed the caspase-3-activited cells. (C) AIF translocated to the nucleus after treatment by 5da. Images were acquired using a Carl Zeiss LSM 710 microscope (magnification 400×). (D) Immunoblotting analysis of proteins related to the mitochondria-mediated intrinsic apoptotic pathway evoked by 5da. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against proteins indicated. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading.

5da Induces Cell Apoptosis through the Intrinsic Apoptotic Pathway. To gain further insight into the mechanisms of tumor suppression activity of 5da, we wanted to confirm first whether the sub-G1 phase cells induced by 5da were due to apoptosis. The 5da-treated cells were therefore investigated with PI and FITC-annexin V staining for apoptosis identification. As depicted in Figure 3A, cells treated by 5da

displayed obvious cell apoptosis in T24 cells in a dosedependent manner. Compared to vehicle-treated cells, 5da induced 4.22% (1 μM), 26.4% (5 μM), and 29.4% (10 μM) of apoptotic cells, respectively. To confirm the proapoptotic effect of 5da in T24 cells, cell apoptosis with activated caspase-3 was also studied. As shown in Figure 3B, the peaks for cells with activated caspase-3 were gradually increased in the 5da-treated 6858



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cells compared with the control group. The proportions of caspase-3 activated cells after the 5da treatment were 7.0% (1 μM), 17.6% (5 μM), and 50.0% (10 μM) in the T24 cells. Furthermore, after the treatment of 5da, Apoptosis-inducing factor (AIF) was released from mitochondria and translocated to the nucleus to trigger apoptosis (Figure 3C). The observations prompted us to verify which death effectors in the mitochondria were dynamic participants in the death signaling because AIF is a mitochondrial protein.33 Bcl-2 family proteins were first chosen to examine how the abundance of likely candidate proteins varied after 5da treatment. As shown in Figure 3D, the most increase was in the levels of Bim, Bax, and Bak, while Bcl-xl and Bcl-2 showed insignificant change. These results collectively indicated that 5da-induced cell apoptosis were mainly through mitochondrial-mediated intrinsic apoptotic pathway. 5da Induces Conformational Activation of Bak for Cell Apoptosis by Mitochondrial Accumulation of Phosphop53. Conformational activation of Bak characterized by Nterminal exposure is a distinct marker of the sensitized mitochondrial apoptotic pathway.34,35 Having confirmed the activated intrinsic apoptotic pathway, we sought to find out whether 5da could conformationally activate Bak, thus defining a framework to understand the mechanism for apoptotic signals cascading. Immunohistochemistry method was adopted to examine the Bak activation. As depicted in Figure 4A, almost no activated Bak could be observed in the control group, reflecting the fact that Bak without activation could not play the role of death effector even in the case of high abundance. However, after the treatment by 5da, conformationally altered Bak could be captured by N-terminal Bak antibody, and such phenomenon was more obvious when the drug concentration increased. The results indicated that 5da could conformationally activate Bak for cell apoptosis. Conformational activation of Bak is a dissociation step from antiapoptotic Bcl-2 family members to form Bak homodimers, eventually leading to generate the pore forming activity.36 Previous studies have shown that antiapoptotic Bcl-2 family member Bcl-xl controls the conformational status of Bak by way of forming a Bak−Bcl-xl complex.34,35 That fact raised the possibility that 5da-induced Bak activation may be due to disrupting the Bak−Bcl-xl interaction. To answer such a question, coimmunoprecipitation analysis was adopted to study the two proteins of interests. As expected, 5da could disrupt the Bak−Bcl-xl interaction (Figure 4C) and without affecting the expression of Bcl-xl (Figure 3D). Having established Bak activation by 5da-induced Bak−Bclxl complex perturbation, what interested us most was how 5da predominantly affected cells to transfer proapoptotic stimuli. 5da has been confirmed to be a p53 activator in our study. We found that 5da did not affect p53 expression but significantly elevated p53 phosphorylation (Figure 2G) and phospho-p53 mainly accumulated in mitochondria (Figure 4B). Previous studies have shown that activated p53 always was translocated into mitochondria to trigger cell apoptosis through the transcription-independent p53 pathways.36,37 It is therefore quite reasonable for us to study the potential role of p53 in 5da-induced Bak activation. To analyze p53’s ability to perturb Bak−Bcl-xl interaction after 5da treatment, we coimmunoprecipitated phospho-p53 with Bcl-xl and Bcl-xl with Bak in T24 cells. As shown in Figure 4C, p53 exhibited direct interaction and high affinity for Bcl-xl after 5da treatment for 24 h, which was consistent with the previous report,38 while Bak dissociated

Figure 4. 5da-induced mitochondrial accumulation of phospho-p53 conformationally activated Bak by reorganizing the Bak−Bcl-xl complex. (A) 5da induced conformational Bak activation. Cells were immunostained with antibodies for active Bak. Images were acquired using a Carl Zeiss LSM 710 microscope (magnification 400×). (B) Phospho-p53 targeting accumulation in mitochondria after treatment by 5da. T24 cells treated by 5da were subjected to subcellular fractionation, and immunoblotting was performed with cytoplasm (cytosol) and mitochondria (mito) fractions. β-Actin and cox IV were used as cytosolic and mitochondrial marker proteins, respectively. (C) Status of Bak-Bcl-xl interaction and phospho-p53-Bcl-xl interaction in T24 cells were treated with 5 μM of 5da for 24 h.

from the Bak−Bcl-xl complex. The results suggested that 5dainduced mitochondrial accumulation of phospho-p53 promoted Bak activation even when it stayed bound to Bcl-xl. 5da Shows Potential Anticancer Potency in Vivo with Low Toxicity. The potential clinical utility of 5da was finally examined in HepG2 xenograft tumors in nude mice. In the HepG2 xenograft models, mice with tumors at the volume of 80−200 mm3 were randomized into treatment and vehicle control groups (n = 6/group) and treated via ip injection of 5da or HCPT, respectively, every other day. The tumor-bearing mice treated by 5da (6 or 12 mg/kg/2 days) showed significant tumor growth inhibition over the treatment period compared with those in vehicle-treated, tumor-bearing mice (Figure 5A− C). The results of tumor growth inhibition (TGI) identical to vehicle control group reached to 43.6% (5da, 6 mg/kg/2 days), 61.3% (5da, 12 mg/kg/2 days), and 71.6% (HCPT, 6 mg/kg/2 days), respectively (P < 0.001). In all experiments, except for a slight body weight decrease in HCPT-treated animals, no differences were found between the control group and 5datreated animals in clinical signs of toxicity and food consumption (Figure 5D). To consistent with the in vitro findings, we also checked the expression level of p53 and Bak as well as anticancer activity in the T24 xenograft models. As depicted in Figure 5E, the results of tumor growth inhibition (TGI) identical to vehicle control group reached to 44.8% 6859



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Figure 5. In vivo anticancer activity of 5da in HepG2 and T24 xenograft mice. (A) Tumor volume of mice from each group during the observation period. Tumor-bearing mice were administered the vehicle (negative control), 5da (6 or 12 mg/kg per 2 days), or HCPT (6 mg/kg per 2 days, positive control). (B) Images of the excised tumors from each group. (D) Body weights of the mice recorded at the end of the treatments. (C,E) Weight of the excised tumors from HepG2 xenograft model (C) and T24 xenograft model (E). Data were presented as the mean ± SD. Error bars represented SD, n = 6, *p < 0.05, and **p < 0.01, vs control. (F) Immunoblotting analysis of p53 and Bak in vivo after 5da treatment.

apoptosis by regulating the related targets based on mitochondria-targeted accumulation of phospho-p53. Cell cycle arrest is a common response to p53 activation.39,40 In our effort to elaborate its molecular mechanism, we found that 5da could induce significant cell cycle arrest in S/G2 phase after treatment for 24 h with consistent variance in G2-related target p21. p21 is a direct target of p53-induced transcriptional program.41 5da could up-regulate p21 expression for cell cycle response but had no influence on cyclin D and CDK4, two other functional proteins in S and G2 phase progression. We further examined the cell apoptosis evoked by 5da in T24 cells. We found that 5da induced cell apoptosis identified by membrane blebbing, caspase-3 activation, and AIF release from mitochondria. Mitochondria-mediated intrinsic apoptotic pathway is always involved in small-molecule compoundinduced cell apoptosis.42 With the observations of cell apoptosis and mitochondrial special protein AIF release, we verified

(5da, 6 mg/kg/2 days) and 54.2% (5da, 12 mg/kg/2 days), respectively (P < 0.001). Consistent with the in vitro findings, 5da induced p53 activation in vivo along with little impact on p53 expression (Figure 5F). However, high dose administration (5da, 12 mg/kg/2 days) could significantly decrease the Bak expression in vivo. This may be related to too much dosage, as in vitro studies (Figure 3D), the Bak was also down-regulated in T24 cells after treatment with 5da (10 μM).

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DISCUSSION AND CONCLUSIONS In this study, a new series of p53 activators based on the phthalazino[1,2-b]quinazolinone motif have been designed and synthesized. Some of the analogues exhibit promising anticancer activities. Our results indicated that 5da could effectively suppress cell proliferation in T24 cancer cells and obviously changed the cell size. Moreover, we verified that the antitumor effect of 5da is likely due to the induction of 6860



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purification. 3a, yield 89%, mp 288 °C (dec); 3b, yield 82%, mp 193 °C (dec); 3c, yield 64%, mp 166−167 °C; 3d, yield 83%, mp 202 °C (dec); 3e, yield 92%, mp 266 °C (dec). General Procedure for Synthesis of 10-Substituted 5Chlorophthalazino[1,2-b]quinazolin-8-ones (4). 3 (0.01 mol) was mixted with redistilled POCl3 (10 mL) and heated in 90 °C oil bath under nitrogen atmosphere for about 6 h. After completion (monitored by TLC), the excess POCl3 was removed by reduced distillation and ice−water was poured in. The precipitate was filtered off and washing successively with water and methanol to give paleyellow solid 4, which were used directly in next step. 4a, yield 89%, mp 156−158 °C (dec); 4b, yield 82%, mp 143−145 °C; 4c, yield 64%, mp 187−189 °C (dec); 4d, yield 83%, mp 202 °C (dec); 4e, yield 92%, mp 163−166 °C. General Procedure for Synthesis of 5,10-Disubstituted Phthalazino[1,2-b]quinazolin-8-ones (5). 4 (5 mmol) and amine (10 mmol) in toluene (10 mL) was heated in 90 °C oil bath for 12 h until the reaction completed (monitored by TLC). Then the solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (eluent: EtOAc/DCM/methanol = 9/3/1) to afford 5 as pale-yellow or white solid. 5-(2-Dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin8-one (5aa). Following the general procedure, 4a (1.41 g, 5 mmol) and 2-dimethylaminoethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.05 g, 63% yield), mp 219−221 °C. 1H NMR (CD3OD) δ 8.74 (dd, 1H, J = 6.2, 3.0 Hz), 8.23 (dd, 1H, J = 8.1, 0.9 Hz), 7.94 (dd, 1H, J = 6.1, 3.0 Hz), 7.77−7.69 (m, 3H), 7.67 (d, 1H, J = 7.7 Hz), 7.45−7.35 (m, 1H), 3.75 (t, 2H, J = 6.4 Hz), 2.79 (t, 2H, J = 6.4 Hz), 2.41 (s, 6H). 13C NMR (125 MHz, CD3OD) δ 159.0, 149.2, 146.2, 142.8, 133.6, 132.5, 131.8, 128.6, 126.9, 126.3, 126.2, 125.5, 122.1, 121.4, 119.4, 57.5, 44.1, 38.6. HRMS (ESI) m/z: calcd for C19H20N5O [M + H]+ 334.1668, found 334.1660. RP-C18-HPLC: 97.342%, tR = 17.897 min. 5-(3-Dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one (5ab). Following the general procedure, 4a (1.41 g, 5 mmol) and 3-dimethylaminopropylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (0.96 g, 55% yield), mp 158−159 °C. 1H NMR (500 MHz, CDCl3) δ 8.88 (d, J = 7.6 Hz, 1H), 8.24 (dd, J = 7.6, 3.5 Hz, 2H), 7.98−7.90 (m, 2H), 7.83 (t, J = 7.5 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.68 (s, 1H), 7.51 (t, J = 7.4 Hz, 1H), 3.51 (dd, J = 12.5, 6.6 Hz, 2H), 2.38 (t, J = 6.8 Hz, 2H), 2.20 (s, 6H,), 1.90 (t, J = 7.0, 2H). 13C NMR (125 MHz, DMSOd6) δ 157.8, 149.0, 146.3, 143.6, 134.2, 133.4, 132.7, 129.0, 127.5, 127.1, 126.7, 126.0, 123.3, 122.0, 120.3, 57.7, 45.7, 40.5, 26.2. HRMS (ESI) m/z: calcd for C20H22N5O [M + H]+ 348.1824, found 348.1816. RP-C18-HPLC: 95.985%, tR = 21.009 min. 5-(3-Morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin8-one (5ac). Following the general procedure, 4a (1.41 g, 5 mmol) and 3-morpholinopropan-1-amine (1.44 g, 10 mmol) were used and the desired product was obtained as a pale-yellow solid (1.57 g, 81% yield), mp 158−162 °C. 1H NMR (500 MHz, DMSO-d6) δ 8.86 (dd, J = 7.5, 1.2 Hz, 1H), 8.25 (dd, J = 19.9, 7.7 Hz, 2H), 7.96−7.89 (m, 2H), 7.81 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.60 (s, 1H), 7.49 (t, J = 7.5 Hz, 1H), 3.74 (t, J = 4.5 Hz, 2H), 3.62 (t, J = 4.1 Hz, 4H), 3.00 (t, J = 4.3 Hz, 2H), 2.42 (t, J = 6.8 Hz, 4H), 1.92 (q, J = 7.0 Hz, 2H). 13C NMR (125 MHz, DMSO-d6) δ 157.7, 149.0, 146.3, 143.6, 134.1, 133.3, 132.7, 129.0, 127.5, 127.0, 126.7, 126.0, 123.4, 122.0, 120.3, 66.6, 56.8, 53.8, 39.5, 25.1. HRMS (ESI) m/z: calcd for C22H24N5O2 [M + H]+ 390.1930, found 390.1922. RP-C18-HPLC: 99.293%, tR = 18.011 min. 5-(Morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one (5ad). Following the general procedure, 4a (1.41 g, 5 mmol) and morpholine (0.87 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.49 g, 90% yield), mp 221−222 °C. 1H NMR (500 MHz, CDCl3) δ 8.98 (dd, J = 7.9, 1.4 Hz, 1H), 8.46 (dd, J = 8.0, 0.8 Hz, 1H), 7.93 (dd, J = 7.7, 1.3 Hz, 1H), 7.85−7.77 (m, 4H), 7.49 (ddd, J = 8.0, 6.7, 1.5 Hz, 1H), 3.98 (t, J = 4.6 Hz, 4H), 3.50 (t, J = 4.6 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ 158.7, 154.5, 146.6, 143.6, 134.4, 132.5, 132.4, 130.9, 127.7, 127.5, 127.3, 126.2, 125.2, 122.7,

which death effectors in the mitochondria were dynamic participants in the death signaling and found the increase of proapoptotic proteins in Bax, Bim, and Bak. Conformational activation of Bak is a distinct marker of the sensitized mitochondrial apoptotic pathway.34,35 Here we found that 5da-induced mitochondrial accumulation of phospho-p53 conformationally activated Bak by reorganizing the Bak−Bcl-xl complex. Bcl-xl controls conformational status of Bak by physical interaction,35 and conformational activation of Bak is a dissociation step from Bcl-xl to form Bak homodimers.36 Coimmunoprecipitation analysis suggested that phospho-p53 exhibited direct interaction and high affinity for Bcl-xl after 5da treatment, while Bak passively dissociated from Bak−Bcl-xl complex. Although 5da was proven valuable as a chemotherapeutic agent for BC therapy with the relatively clear molecular mechanism, the exact mechanism of how 5da interferes with p53 activation is still unclear. Further study should be carried out to verify if 5da interacts with p53 directly or indirectly. Moreover, pharmacodynamic evaluation in vitro and in vivo suggested that 5da was more potent than HCPT in vitro but less potent in vivo. The significant difference of activity between in vitro and in vivo indicated bioavailability of 5da may be an important factor to affect its biological activity, which restricted its clinical application. In conclusion, we present here a small-molecule platform that enables a switch to trigger cell apoptosis by manipulating p53 apoptotic signaling pathway, emphasized the molecular regulatory networks for pharmacological characterization. These results provide strong evidence for the preclinical efficacy of 5da as a promising anticancer agent in BC therapy or an efficient adjuvant in p53 restoration-mediated anticancer therapy.

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EXPERIMENTAL SECTION

Materials. All reagents were purchased from commercial sources and were used without further purification unless otherwise noted. All reactions were carried out under an atmosphere of dry nitrogen. Melting points were recorded on WRS-IA apparatus without correction. NMR spectra were recorded in DMSO-d6, CDCl3, or CD3OD on Bruker Advance (500 or 400 MHz) with TMS as internal standard. HRMS were measured in EI or ESI mode and the mass analyzer of the HRMS was TOF. Flash column chromatography was performed on silica gel (200−300 mesh). Propidium iodide (PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO). Antiphospho p53 (s392), anti-p53, anti-p21, anti-Bim, anti-Bcl-2, anti-Bcl-xl, anti-AIF, antilamin B, anticox IV, antiactin, and anti-Bax antibodies were purchased from Abcam (Cambridge, MA). Anti-Bak antibody and caspase-3 activity fluorescence detection kits were purchased from Calbiochem. Antirabbit and -mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All synthetic compounds 5 were analyzed by HPLC (254 nm). The system was equipped with a C18 5 μm Shimadzu-GL ODS-3 column (4.6 mm × 250 mm) with a flow rate of 0.7 mL/min. HPLC analysis showed that the purity of all compounds were >95%, and their spectra could be found in Supporting Information. Compounds 5 were stored at −20 °C at a concentration of 10 mM in dimethyl sulfoxide (DMSO). General Procedure for Synthesis of 10-Substituted 6HPhthalazino[1,2-b]quinazolin-5,8-diones (3). Phthalic anhydride (1.48 g, 0.01 mol) and hydrazide 2 (0.01 mol) in glycol (15 mL) was heated in 150 °C oil bath for 2 h until the reaction completed (monitored by TLC). When the mixture was cooled, the solid was vacuum filtered and washed with ethyl acetate (10 mL × 2) to give pale-yellow solid 3, which were used in next step without further 6861



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120.6, 66.8, 51.5. HRMS (ESI) m/z: calcd for C19H17N4O2 [M + H]+ 333.1352, found 333.1348. RP-C18-HPLC: 98.812%, tR = 30.408 min. 5-(4-Methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5ae). Following the general procedure, 4a (1.41 g, 5 mmol) and 1methylpiperazine (1.00 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.21 g, 70% yield), mp 197−198 °C. 1H NMR (500 MHz, CDCl3) δ 8.98 (dd, J = 7.8, 1.4 Hz, 1H), 8.47 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 (dd, J = 7.8, 1.2 Hz, 1H), 7.84−7.77 (m, 4H), 7.49 (ddd, J = 8.1, 6.8, 1.4 Hz, 1H), 3.55 (s, 4H), 2.72 (s, 4H), 2.41 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 158.8, 154.5, 146.7, 143.7, 134.3, 132.3, 132.3, 130.9, 127.7, 127.5, 127.3, 126.1, 125.4, 123.0, 120.7, 54.9, 50.8, 46.3. HRMS (ESI) m/z: calcd for C20H20N5O [M + H]+ 346.1668, found 346.1660. RP-C18-HPLC: 99.696%, tR = 22.346 min. 10-Fluoro-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5ba). Following the general procedure, 4b (1.50 g, 5 mmol) and 2-dimethylaminoethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.51 g, 86% yield), mp 125−226 °C. 1H NMR (500 MHz, CD3OD) δ 8.66 (dd, J = 6.1, 3.3 Hz, 1H), 7.95 (dd, J = 5.8, 3.3 Hz, 1H), 7.82 (dd, J = 6.1, 3.2 Hz, 2H), 7.74 (dd, J = 8.7, 2.9 Hz, 1H), 7.65 (dd, J = 9.0, 4.9 Hz, 1H), 7.51 (td, J = 8.6, 2.9 Hz, 1H), 3.82 (t, J = 5.7 Hz, 2H), 3.13 (t, J = 5.5 Hz, 2H), 2.76 (s, 6H). 13C NMR (125 MHz, CD3OD) δ 162.5, 160.5, 159.5, 151.1, 144.3, 143.1, 134.0, 133.6, 131.2, 129.6, 127.5, 123.6, 122.3, 121.4, 111.6, 59.8, 45.1, 39.8. HRMS (ESI) m/z: calcd for C19H19FN5O [M + H]+ 352.1574, found 352.1567. RP-C18HPLC: 99.268%, tR = 18.222 min. 10-Fluoro-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one (5bb). Following the general procedure, 4b (1.50 g, 5 mmol) and 3-dimethylaminopropylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.61 g, 88% yield), mp 198−200 °C. 1H NMR (500 MHz, CD3OD) δ 8.62−8.57 (m, 1H), 7.86−7.82 (m, 1H), 7.75−7.70 (m, 2H), 7.69 (dd, J = 8.8, 2.9 Hz, 1H), 7.58 (dd, J = 9.0, 4.9 Hz, 1H), 7.43 (td, J = 8.6, 3.0 Hz, 1H), 3.60 (t, J = 6.7 Hz, 2H), 2.76 (t, J = 7.0 Hz, 2H), 2.53 (s, 6H), 2.07−1.98 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 162.5, 160.5, 159.4, 151.4, 144.3, 143.4, 133.9, 131.1, 129.7, 127.5, 124.0, 123.4, 122.6, 121.6, 111.6, 57.6, 44.9, 40.6, 27.1. HRMS (ESI) m/z: calcd for C20H21FN5O [M + H]+ 366.1730, found 366.1725. RP-C18HPLC: 95.914%, tR = 18.265 min. 10-Fluoro-5-3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one (5bc). Following the general procedure, 4b (1.50 g, 5 mmol) and 3-morpholinopropan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.67 g, 82% yield), mp 232−233 °C. 1H NMR (500 MHz, CDCl3) δ 8.96 (dd, J = 7.3, 2.0 Hz, 1H), 8.05 (dd, J = 8.7, 3.0 Hz, 1H), 7.86 (dd, J = 6.3, 2.6 Hz, 1H), 7.83−7.77 (m, 3H), 7.54 (s, 1H), 7.51−7.46 (m, 1H), 3.84 (t, J = 4.5 Hz, 4H), 3.79 (dd, J = 10.3, 5.6 Hz, 2H), 2.67 (t, J = 5.5 Hz, 2H), 2.61 (s, 4H), 2.00−1.91 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 161.4, 159.4, 158.1, 149.2, 143.2, 142.7, 132.4, 132.1, 129.8, 129.5, 127.3, 122.9, 121.9, 121.6, 111.9, 67.1, 59.5, 54.1, 43.3, 23.3. HRMS (ESI) m/z: calcd for C22H23FN5O2 [M + H]+ 408.1836, found 408.1830. RP-C18-HPLC: 98.134%, tR = 18.534 min. 10-Fluoro-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8-one (5bd). Following the general procedure, 4b (1.50 g, 5 mmol) and morpholine (0.87 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.40 g, 80% yield), mp 277−278 °C. 1 H NMR (500 MHz, CDCl3) δ 8.94 (dd, J = 7.7, 1.6 Hz, 1H), 8.06 (dd, J = 8.6, 3.0 Hz, 1H), 7.94 (dd, J = 7.8, 1.4 Hz, 1H), 7.86−7.78 (m, 3H), 7.54−7.49 (m, 1H), 3.98 (t, J = 4.6 Hz, 4H), 3.50 (t, J = 4.6 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ 161.5, 159.6, 158.1, 154.8, 143.4, 143.0, 132.6, 130.8, 130.0, 127.2, 125.3, 123.4, 122.6, 121.7, 112.2, 66.7, 51.5. HRMS (ESI) m/z: calcd for C19H16FN4O2 [M + H]+ 351.1257, found 351.1254. RP-C18-HPLC: 97.205%, tR = 21.819 min. 10-Fluoro-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5be). Following the general procedure, 4b (1.50 g, 5 mmol) and 1-methylpiperazine (1.00 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.45 g, 80% yield), mp 208−209 °C. 1H NMR (500 MHz, CDCl3) δ 8.94 (dd, J = 7.7, 1.2 Hz, 1H), 8.06 (dd, J = 8.6, 2.9 Hz, 1H), 7.93 (dd, J = 6.5, 1.2

Hz, 1H), 7.85−7.81 (m, 3H), 7.55−7.48 (m, 1H), 3.56 (s, 4H), 2.73 (s, 4H), 2.42 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 161.5, 159.5, 158.1, 154.8, 143.4, 143.1, 132.4, 130.8, 129.9, 127.2, 125.4, 123.4, 122.8, 121.7, 112.2, 54.7, 50.7, 46.2. HRMS (ESI) m/z: calcd for C20H19FN5O [M + H]+ 364.1574, found 364.1567. RP-C18-HPLC: 95.026%, tR = 25.709 min. 10-Chloro-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5ca). Following the general procedure, 4c (1.58 g, 5 mmol) and 2-dimethylaminoethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (0.81 g, 44% yield), mp 294−297 °C. 1H NMR (500 MHz, CD3OD) δ 8.67−8.64 (m, 1H), 7.98 (dd, J = 5.6, 2.3 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 7.90−7.83 (m, 2H), 7.63 (dd, J = 8.8, 2.4 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 3.81 (t, J = 4.4, 2H), 3.49 (t, J = 4.4, 2H), 3.09 (s, 6H). 13 C NMR (125 MHz, CD3OD) δ 158.0, 150.6, 144.6, 142.1, 134.4, 133.2, 133.1, 131.5, 129.1, 128.2, 126.3, 125.1, 122.9, 120.6, 119.5, 60.0, 43.0, 38.2. HRMS (ESI) m/z: calcd for C19H19ClN5O [M + H]+ 368.1278, found 368.1273. RP-C18-HPLC: 98.536%, tR = 19.701 min. 10-Chloro-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one (5cb). Following the general procedure, 4c (1.58 g, 5 mmol) and 3-dimethylaminopropylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.45 g, 76% yield), mp 224−226 °C. 1H NMR (500 MHz, CD3OD) δ 8.78−8.75 (m, 1H), 8.11 (d, J = 2.3 Hz, 1H), 8.09−8.04 (m, 1H), 7.93−7.84 (m, 2H), 7.69 (dd, J = 8.8, 2.4 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 3.74 (t, J = 6.1 Hz, 2H), 3.21 (t, J = 6.3 Hz, 2H), 2.93 (s, 6H), 2.29−2.15 (m, 2H). 13C NMR (500 MHz, CD3OD) δ 158.0, 151.6, 144.7, 142.6, 134.3, 133.1, 132.7, 131.3, 129.1, 128.4, 126.4, 125.2, 122.6, 121.1, 119.8, 53.6, 42.2, 37.5, 25.7. HRMS (ESI) m/z: calcd for C20H21ClN5O [M + H]+ 382.1435, found 382.1431. RP-C18-HPLC: 96.601%, tR = 19.947 min. 10-Chloro-5-(3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one (5cc). Following the general procedure, 4c (1.58 g, 5 mmol) and 3-morpholinopropan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.70 g, 80% yield), mp 231−233 °C. 1H NMR (500 MHz, CDCl3) δ 8.98− 8.93 (m, 1H), 8.38 (d, J = 2.4 Hz, 1H), 7.90−7.85 (m, 2H), 7.83−7.79 (m, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.66 (dd, J = 8.8, 2.4 Hz, 1H), 7.55 (s, 1H), 3.85 (t, J = 4.6 Hz, 4H), 3.79 (dd, J = 10.3, 5.7 Hz, 2H), 2.70− 2.67 (m, 2H), 2.62 (s, 4H), 2.00−1.93 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 157.0, 148.5, 144.2, 142.8, 133.5, 131.8, 131.4, 130.7, 128.6, 128.3, 126.7, 125.8, 121.2, 121.1, 120.6, 66.3, 58.7, 53.4, 42.4, 22.5. HRMS (ESI) m/z: calcd for C22H23ClN5O2 [M + H]+ 424.1540, found 424.1533. RP-C18-HPLC: 97.240%, tR = 20.421 min. 10-Chloro-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8one (5cd). Following the general procedure, 4c (1.58 g, 5 mmol) and morpholine (0.87 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.71 g, 93% yield), mp 258−259 °C. 1 H NMR (500 MHz, CDCl3) δ 8.99 (dd, J = 7.7, 1.4 Hz, 1H), 8.42 (d, J = 2.4 Hz, 1H), 7.97 (dd, J = 7.2, 1.8 Hz, 1H), 7.86−7.82 (m, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.73 (dd, J = 8.8, 2.4 Hz, 1H), 3.99 (t, J = 4.6, 4H), 3.52 (t, J = 4.5, 4H). 13C NMR (125 MHz, CDCl3) δ 157.6, 154.7, 145.0, 143.7, 134.8, 132.5, 132.5, 131.8, 130.6, 129.0, 127.3, 126.7, 125.1, 122.6, 121.4, 66.6, 51.4. HRMS (ESI) m/z: calcd for C19H16ClN4O2 [M + H]+ 367.0962, found 367.0937. RP-C18-HPLC: 97.101%, tR = 34.262 min. 10-Chloro-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5ce). Following the general procedure, 4c (1.58 g, 5 mmol) and 1-methyl-piperazine (1.00 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.71 g, 90% yield), mp 206−208 °C. 1H NMR (500 MHz, CDCl3) δ 8.37 (d, J = 2.4 Hz, 1H), 7.92 (dd, J = 5.6, 2.1 Hz, 1H), 7.82−7.79 (m, 2H), 7.74 (d, J = 8.7 Hz, 1H), 7.69 (dd, J = 8.7, 2.4 Hz, 1H), 3.54 (s, 4H), 2.71 (s, 4H), 2.41 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 157.8, 154.8, 145.1, 143.9, 134.8, 132.5, 132.4, 131.8, 130.7, 129.1, 127.3, 126.8, 125.4, 122.9, 121.5, 54.9, 50.8, 46.3. HRMS (ESI) m/z: calcd for C20H19ClN5O [M + H]+ 380.1278, found 380.1270. RP-C18-HPLC: 98.399%, tR = 19.247 min. 10-Bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5da). Following the general procedure, 4d (1.80 g, 6862



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10-Methyl-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one (5eb). Following the general procedure, 4e (1.48 g, 5 mmol) and 3-dimethylaminopropylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.30 g, 72% yield), mp 191−193 °C. 1H NMR (500 MHz, CD3OD) δ 8.77−8.65 (m, 1H), 7.97−7.91 (m, 2H), 7.80−7.75 (m, 2H), 7.53 (d, J = 1.7 Hz, 2H), 3.68 (t, J = 6.4 Hz, 2H), 3.00 (t, J = 6.5 Hz, 2H), 2.74 (s, 6H), 2.43 (s, 3H), 2.17−2.06 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 160.2, 152.0, 145.6, 143.1, 137.6, 136.8, 133.8, 133.6, 130.1, 128.3, 127.6, 126.7, 123.6, 122.5, 120.2, 56.2, 44.2, 39.7, 27.2, 21.5. HRMS (ESI) m/z: calcd for C21H24N5O [M + H]+ 362.1981, found 362.1960. RP-C18-HPLC: 97.174%, tR = 19.311 min. 10-Methyl-5-(3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one (5ec). Following the general procedure, 4e (1.48 g, 5 mmol) and 3-morpholinopropan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.70 g, 84% yield), mp 189−191 °C. 1H NMR (500 MHz, CDCl3) δ 8.99− 8.92 (m, 1H), 8.22 (d, J = 0.6 Hz, 1H), 7.98−7.91 (m, 1H), 7.81−7.76 (m, 2H), 7.71 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 8.4, 2.1 Hz, 1H), 7.48 (s, 1H), 3.89 (t, J = 4.6 Hz, 4H), 3.80 (d, J = 4.5 Hz, 2H), 2.81−2.75 (m, 2H), 2.72 (s, 4H), 2.52 (s, 3H), 2.06−1.97 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 158.6, 148.9, 144.4, 142.5, 135.9, 135.4, 132.1, 131.9, 129.5, 127.1, 127.0, 126.5, 121.9, 121.7, 120.1, 66.4, 58.6, 53.6, 42.2, 23.1, 21.4. HRMS (ESI) m/z: calcd for C23H26N5O2 [M + H]+ 404.2086, found 404.2077. RP-C18-HPLC: 97.628%, tR = 19.219 min. 10-Methyl-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8one (5ed). Following the general procedure, 4e (1.48 g, 5 mmol) and morpholine (0.87 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.51 g, 87% yield), mp 217−218 °C. 1 H NMR (500 MHz, CDCl3) δ 8.98 (dd, J = 7.9, 1.1 Hz, 1H), 8.26 (s, 1H), 7.94 (dd, J = 7.8, 1.1 Hz, 1H), 7.87−7.76 (m, 2H), 7.75 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 8.4, 2.0 Hz, 1H), 3.99 (t, J = 6.0 Hz, 4H), 3.51 (t, J = 6.5 Hz, 4H), 2.54 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 167.8, 158.8, 154.5, 144.7, 143.0, 136.5, 136.1, 132.4, 131.0, 129.0, 127.3, 126.9, 125.2, 122.7, 120.4, 66.8, 65.7, 51.5. HRMS (ESI) m/z: calcd for C20H19N4O2 [M + H]+ 347.1508, found 347.1503. RP-C18HPLC: 99.167%, tR = 25.378 min. 10-Methyl-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5ee). Following the general procedure, 4e (1.48 g, 5 mmol) and 1-methylpiperazine (1.00 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.51 g, 84% yield), mp 217−219 °C. 1H NMR (500 MHz, CDCl3) δ 8.96 (dd, J = 7.6, 1.7 Hz, 1H), 8.25 (s, 1H), 7.92 (dd, J = 7.6, 1.1 Hz, 1H), 7.83− 7.76 (m, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.62 (dd, J = 8.4, 2.1 Hz, 1H), 3.56 (s, 4H), 2.74 (s, 4H), 2.54 (s, 3H), 2.43 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 158.8, 154.5, 144.7, 143.0, 136.4, 136.0, 132.3, 132.1, 131.0, 127.3, 127.1, 126.9, 125.3, 122.8, 120.4, 54.9, 50.7, 46.2, 21.6. HRMS (ESI) m/z: calcd for C21H22N5O [M + H]+ 360.1824, found 360.1817. RP-C18-HPLC: 97.873%, tR = 18.897 min. 10-Bromo-5-((2-(diethylamino)ethyl)amino)-8H-phthalazino[1,2b]quinazolin-8-one (5df). Following the general procedure, 4d (0.72 g, 2 mmol) and N,N-diethylethane-1,2-diamine (10 mmol) were used, and the desired product was obtained as a white solid (0.59 g, 67% yield), mp 175−176 °C. 1H NMR (400 MHz, CD3OD) δ 8.76 (dd, J = 6.3, 3.0 Hz, 1H), 8.21 (d, J = 2.3 Hz, 1H), 8.06 (dt, J = 7.4, 3.2 Hz, 1H), 7.93−7.89 (m, 2H), 7.83 (dd, J = 8.8, 2.3 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 3.84 (dd, J = 5.3, 3.1 Hz, 2H), 3.50−3.47 (m, 2H), 3.42 (dd, J = 6.9, 3.1 Hz, 4H), 1.43 (t, J = 7.3 Hz, 6H). 13C NMR (100 MHz, CD3OD) δ 159.4, 152.1, 146.6, 144.1, 138.6, 134.6, 134.5, 130.7, 130.0, 129.9, 127.9, 124.3, 122.3, 121.6, 120.5, 55.7, 49.9, 39.9, 9.0. HRMS (ESI) m/z: calcd for C21H22BrN5O [M + H]+ 440.1086, found 440.1071. RP-C18-HPLC: 98.067%, tR = 23.019 min. 1 0 - B ro m o - 5 - ( ( 2 - ( d i i s o p r o p y l a m i n o ) e t h y l ) a m i n o ) - 8 H phthalazino[1,2-b]quinazolin-8-one (5dg). Following the general procedure, 4d (0.72 g, 2 mmol) and N,N-diisopropyl-ethane-1,2diamine (10 mmol) were used, and the desired product was obtained as a white solid (0.66 g, 72% yield), mp 180−182 °C. 1H NMR (400 MHz, CD3OD) δ 8.96−8.93 (m, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.14− 8.12 (m, 1H), 8.00−7.92 (m, 3H), 7.75 (d, J = 8.8 Hz, 1H), 3.92 (s, 4H), 3.55 (s, 2H), 1.50 (d, J = 32.2 Hz, 12H). 13C NMR (100 MHz,

5 mmol) and 2-dimethylaminoethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.40 g, 68% yield), mp 141−146 °C. 1H NMR (500 MHz, CD3OD) δ 8.43 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.69−7.62 (m, 2H), 7.56 (dd, J = 8.6, 2.1 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 3.69 (t, J = 5.8 Hz, 2H), 2.93 (t, J = 5.8 Hz, 2H), 2.57 (s, 6H). 13 C NMR (125 MHz, CD3OD) δ 161.4, 153.4, 148.5, 146.6, 140.4, 136.6, 135.9, 132.7, 132.3, 131.9, 130.1, 126.1, 124.9, 124.2, 122.5, 61.9, 47.9, 42.4. HRMS (ESI) m/z: calcd for C19H19BrN5O [M + H]+ 412.0773, found 412.0769. RP-C18-HPLC: 98.463%, tR = 13.635 min. 10-Bromo-5-(2-dimethylamino-propylamino)phthalazino[1,2-b]quinazolin-8-one (5db). Following the general procedure, 4d (1.80 g, 5 mmol) and 3-dimethylaminopropylamine (1.02 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.60 g, 75% yield), mp 215−217 °C. 1H NMR (500 MHz, DMSO-d6) δ 8.84 (d, J = 7.5 Hz, 1H), 8.27 (s, 1H), 8.22 (d, J = 7.5 Hz, 1H), 7.98− 7.88 (m, 3H), 7.70 (d, J = 8.7 Hz, 2H), 3.51 (dd, J = 11.3, 5.9 Hz, 2H), 2.39 (t, J = 6.5 Hz, 2H), 2.21 (s, 6H), 1.94−1.85 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 156.6, 149.2, 145.2, 144.1, 136.9, 133.6, 132.7, 129.9, 128.9, 128.8, 126.8, 123.3, 122.0, 121.7, 118.2, 57.8, 56.5, 45.7, 26.2. HRMS (ESI) m/z: calcd for C20H21BrN5O [M + H]+ 426.0929, found 426.0922. RP-C18-HPLC: 95.188%, tR = 23.679 min. 10-Bromo-5(3-morpholin-4-yl-propylamino)phthalazino[1,2-b]quinazolin-8-one (5dc). Following the general procedure, 4d (1.80 g, 5 mmol) and 3-morpholinopropan-1-amine (1.44 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.90 g, 81% yield), mp 236−238 °C. 1H NMR (500 MHz, CDCl3) δ 8.98− 8.92 (m, 1H), 8.53 (d, J = 2.2 Hz, 1H), 7.86−7.81 (m, 3H), 7.79 (dd, J = 9.0, 2.3 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.57 (s, 1H), 3.84 (t, J = 4.5 Hz, 4H), 3.78 (dd, J = 9.7, 4.9 Hz, 2H), 2.68−2.64 (m, 2H), 2.60 (s, 4H), 1.98−1.89 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 157.5, 149.1, 145.1, 143.5, 136.7, 132.4, 132.0, 129.6, 129.2, 129.0, 127.3, 121.8, 121.6, 119.0, 67.1, 59.5, 54.0, 43.3, 23.1. HRMS (ESI) m/z: calcd for C22H23BrN5O2 [M + H]+ 468.1035, found 468.1030. RPC18-HPLC: 98.748%, tR = 20.902 min. 10-Bromo-5-(morpholin-4-yl)phthalazino[1,2-b]quinazolin-8one (5dd). Following the general procedure, 4d (1.80 g, 5 mmol) and morpholine (0.87 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.40 g, 68% yield), mp 254−255 °C. 1 H NMR (500 MHz, CDCl3) δ 8.99 (dd, J = 7.7, 1.7 Hz, 1H), 8.58 (d, J = 2.3 Hz, 1H), 7.97 (dd, J = 6.2, 1.5 Hz, 1H), 7.89−7.84 (m, 3H), 7.72 (d, J = 8.7 Hz, 1H), 3.99 (t, J = 4.6 Hz, 4H), 3.52 (t, J = 4.6 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ 156.7, 153.8, 144.5, 143.0, 136.6, 131.7, 131.7, 129.8, 129.0, 128.4, 126.5, 124.3, 121.8, 120.9, 118.7, 65.8, 50.5. HRMS (ESI) m/z: calcd for C19H16BrN4O2 [M + H]+ 411.0457, found 411.0453. RP-C18-HPLC: 98.795%, tR = 33.722 min. 10-Bromo-5-(4-methyl-piperazin-1-yl)phthalazino[1,2-b]quinazolin-8-one (5de). Following the general procedure, 4d (1.80 g, 5 mmol) and 1-methylpiperazine (1.00 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.21 g, 57% yield), mp 220−221 °C. 1H NMR (500 MHz, CDCl3) δ 8.94 (dd, J = 7.3, 2.0 Hz, 1H), 8.55 (d, J = 2.3 Hz, 1H), 7.93 (dd, J = 7.2, 2.0 Hz, 1H), 7.85−7.80 (m, 3H), 7.68 (d, J = 8.7 Hz, 1H), 3.56 (s, 4H), 2.73 (s, 4H), 2.42 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 157.6, 154.8, 145.5, 144.1, 137.5, 132.6, 132.5, 130.7, 130.0, 129.3, 127.3, 125.5, 123.0, 121.9, 119.5, 54.8, 50.8, 46.3. HRMS (ESI) m/z: calcd for C20H19BrN5O [M + H]+ 424.0773, found 424.0768. RP-C18-HPLC: 98.476%, tR = 19.826 min. 10-Methyl-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5ea). Following the general procedure, 4e (1.48 g, 5 mmol) and 2-dimethylaminoethanamine (0.88 g, 10 mmol) were used, and the desired product was obtained as a pale-yellow solid (1.18 g, 68% yield), mp 124 °C (dec). 1H NMR (500 MHz, CD3OD) δ 8.33−8.30 (m, 1H), 7.66−7.62 (m, 1H), 7.57 (s, 1H), 7.55−7.51 (m, 2H), 7.22 (dd, J = 8.4, 1.7 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 3.61 (t, J = 5.6 Hz, 2H), 2.99 (t, J = 5.3 Hz, 2H), 2.63 (s, 6H), 2.24 (s, 3H). 13C NMR (125 MHz, CD3OD) δ 162.3, 153.2, 147.7, 145.1, 139.7, 139.0, 136.0, 135.8, 132.1, 130.6, 129.8, 129.0, 126.0, 124.5, 122.4, 62.4, 47.7, 42.3, 24.1. HRMS (ESI) m/z: calcd for C20H22N5O [M + H]+ 348.1824, found 348.1819. RP-C18-HPLC: 97.492%, tR = 18.891 min. 6863



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CD3OD) δ 157.8, 145.3, 143.0, 137.2, 133.3, 133.2, 129.4, 128.8, 128.6, 126.7, 122.8, 120.8, 120.5, 119.2, 41.2. HRMS (ESI) m/z: calcd for C23H28BrN5O [M + H]− 468.1400, found 468.1386. RP-C18HPLC: 97.821%, tR = 20.967 min. 10-Bromo-5-((2-(pyrrolidin-1-yl)ethyl)amino)-8H-phthalazino[1,2-b]quinazolin-8-one (5dh). Following the general procedure, 4d (0.72 g, 2 mmol) and 2-pyrrolidin-1-yl-ethylamine (10 mmol) were used, and the desired product was obtained as a white solid (0.48 g, 56% yield), mp 189 °C (dec). 1H NMR (400 MHz, CD3OD) δ 8.85− 8.83 (m, 1H), 8.24 (s, 1H), 8.07−8.05 (m, 1H), 7.97 (dd, J = 6.3, 2.6 Hz, 2H), 7.89 (dd, J = 8.8, 2.2 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 3.93 (s, 2H), 3.80−3.78 (m, 2H), 3.57−3.55 (m, 2H), 3.18 (s, 2H), 2.38 (s, 2H), 2.25 (s, 2H). 13C NMR (125 MHz, CD3OD) δ 159.6, 151.9, 146.5, 144.0, 138.6, 134.7, 130.7, 129.9, 127.9, 124.3, 122.3, 121.5, 120.6, 119.1, 59.3, 56.0, 40.4, 24.7. HRMS (ESI) m/z: calcd for C21H20BrN5O [M + H]+ 438.0930, found 438.0916. RP-C18-HPLC: 98.078%, tR = 24.160 min. 10-Bromo-5-((2-(piperidin-1-yl)ethyl)amino)-8H-phthalazino[1,2b]quinazolin-8-one (5di). Following the general procedure, 4d (0.72 g, 2 mmol) and 2-piperidin-1-yl-ethylamine (10 mmol) were used, and the desired product was obtained as a pale-yellow solid (0.57 g, 63% yield), mp 196 (dec) °C. 1H NMR (500 MHz, CD3OD) δ 8.66 (d, J = 4.3 Hz, 1H), 8.12 (d, J = 1.2 Hz, 1H), 7.97 (d, J = 5.6 Hz, 1H), 7.86 (dd, J = 37.9, 5.4 Hz, 3H), 7.52 (d, J = 8.6 Hz, 1H), 3.76 (d, J = 47.4 Hz, 4H), 3.42 (s, 2H), 3.05 (s, 2H), 2.17 (t, J = 128.4 Hz, 6H). 13C NMR (125 MHz, CD3OD) δ 158.9, 151.4, 146.3, 143.8, 138.4, 134.6, 134.3, 130.5, 129.7, 129.6, 127.7, 124.2, 122.1, 121.3, 120.5, 56.0, 54.9, 38.3, 23.2, 22.9. HRMS (ESI) m/z: calcd for C22H22BrN5O [M + H]+ 452.1086, found 452.1073. RP-C18-HPLC: 98.323%, tR = 20.567 min. 10-Bromo-5-((2-morpholinoethyl)amino)-8H-phthalazino[1,2-b]quinazolin-8-one (5dj). Following the general procedure, 4d (0.72 g, 2 mmol) and 2-morpholin-4-yl-ethylamine (10 mmol) were used, and the desired product was obtained as a pale-yellow solid (87% yield), mp 157−158 °C. 1H NMR (400 MHz, CDCl3 (0.6 mL) + CD3OD (a drop)) δ 9.00 (s, 1H), 8.57 (s, 1H), 8.05−7.52 (m, 6H), 3.80 (s, 6H), 2.83 (s, 2H), 2.63 (s, 4H). 13C NMR (100 MHz, CDCl3 (0.6 mL) + CD3OD (a drop)) δ 158.0, 149.3, 145.0, 143.3, 137.1, 133.0, 132.3, 129.3, 129.0, 128.7, 126.9, 121.9, 121.5, 120.9, 119.2, 66.2, 56.9, 53.1, 37.2. HRMS (ESI) m/z: calcd for C21H20BrN5O2 [M + H]+ 454.0879, found 454.0866. RP-C18-HPLC: 98.656%, tR = 20.295 min. 10-Bromo-5-(butylamino)-8H-phthalazino[1,2-b]quinazolin-8one (5dk). Following the general procedure, 4d (0.72 g, 2 mmol) and n-butylamine (20 mmol) were used, and the desired product was obtained as a pale-yellow solid (0.37 g, 47% yield), mp 120−122 °C. 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J = 7.6 Hz, 1H), 8.32−8.27 (m, 2H), 7.98−7.91 (m, 3H), 7.71 (d, J = 8.8 Hz, 1H), 7.54 (s, 1H), 3.52 (dd, J = 12.0, 6.4 Hz, 2H), 1.78−1.71 (m, 2H), 1.47 (dd, J = 14.8, 7.4 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSOd6) δ 156.7, 149.2, 145.2, 144.1, 136.9, 133.6, 132.8, 129.9, 129.0, 128.9, 126.8, 123.5, 122.0, 121.7, 118.1, 41.5, 30.7, 20.5, 14.3. HRMS (ESI) m/z: calcd for C19H17BrN4O [M + H]+ 397.0664, found 397.0654. RP-C18-HPLC: 95.885%, tR = 20.930 min. Ethyl-3-((10-bromo-8-oxo-8H-phthalazino[1,2-b]quinazolin-5yl)amino)propanoate (5dl). Following the general procedure, 4d (0.72 g, 2 mmol) and ethyl 3-aminopropanoate (10 mmol) were used, and the desired product was obtained as a pale-yellow solid (0.41 g, 47% yield), mp 251 °C (dec). 1H NMR (400 MHz, CDCl3) δ 9.03 (dd, J = 6.2, 3.0 Hz, 1H), 8.57 (d, J = 2.3 Hz, 1H), 7.88 (ddd, J = 8.4, 6.7, 4.0 Hz, 3H), 7.75−7.70 (m, 2H), 6.13 (t, J = 5.5 Hz, 1H), 4.23 (q, J = 7.1 Hz, 2H), 4.02 (dd, J = 11.5, 5.7 Hz, 2H), 2.86−2.83 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 173.8, 157.6, 148.5, 145.1, 143.7, 137.2, 133.1, 132.5, 129.8, 129.3, 129.1, 127.4, 121.7, 121.6, 121.5, 119.4, 61.0, 37.4, 33.0, 14.3. HRMS (ESI) m/z: calcd for C20H17BrN4O3 [M + H]+ 441.0562, found 441.0551. RPC18-HPLC: 98.729%, tR = 20.561 min. Ethyl (10-Bromo-8-oxo-8H-phthalazino[1,2-b]quinazolin-5-yl)glycinate (5dm). Following the general procedure, 4d (0.72 g, 2 mmol) and ethyl glycinate (10 mmol) were used, and the desired product was obtained as a pale-yellow solid (0.44 g, 51% yield), mp 188 °C (dec). 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 6.2, 2.3

Hz, 1H), 8.57 (d, J = 2.2 Hz, 1H), 7.85−7.80 (m, 4H), 7.72 (d, J = 8.7 Hz, 1H), 4.48 (d, J = 4.3 Hz, 2H), 4.37 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 171.2, 157.4, 148.1, 145.1, 143.6, 137.2, 133.0, 132.6, 129.8, 129.3, 129.2, 127.4, 121.7, 121.6, 121.1, 119.4, 62.0, 43.8, 14.4. HRMS (ESI) m/z: calcd for C19H15BrN4O3 [M + H]+ 427.0406, found 427.0396. RP-C18-HPLC: 97.193%, tR = 27.823 min. Cell Culture and Cancer Cell Lines. T24, MGC-803, NCI-H460, HepG2, HeLa, DU145, PC-3, LnCap, and J82 cell lines were purchased from ATCC (Manassas, VA). All cell lines were cultured in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS). Cell Viability Assay. Cell proliferation was tested using the MTT assay. MTT (5 mg·mL−1) was added into the wells, and the plates were incubated at 37 °C for 4 h. The MTT assay was stopped by adding dimethyl sulfoxide (150 mL per well) and mixed for 10 min vigorously before measuring absorbance at 490 nm in a multiwell plate reader. Cell viability was calculated using the following formula: cell viability (%) = (A490e/A490c) × 100%, A490e and A490c represented the absorbance values from the experimental and control groups, respectively. DNA Relaxation Assay. The pBR322 DNA were incubated with a range of concentrations of 5da (1−10 μM) in the solutions (5 mM Tris-HCl, 50 mM NaCl buffer, pH 7.2) for 1 h. The samples were electrophoresed in a 1.5% agarose gel and stained with 0.5 μg·mL−1 ethidium bromide before detection. Membrane Disruption Assay. Membrane disruption analyses were carried out by flow cytometry. T24 cells were harvested from adherent cultures by trypsinization. Following centrifugation at 1000 rpm for 5 min, cells were washed with PBS and centrifuged as before. Cellular pellets were resuspended in 50 μg/mL propidium iodide (Sigma) in PBS for nucleic acids staining and immediately analyzed on a flow cytometer equipped with a 488 nm argon laser (Becton− Dickinson). Analyses of Cell Cycle and Cell Size. Cell cycle and cell size analyses were performed by flow cytometry. T24 and HepG2 cells were harvested from adherent cultures by trypsinization. Following centrifugation at 1000 rpm for 5 min, cells were washed with PBS and then fixed with 70% ethanol in PBS. Fixed cells were collected and washed with PBS and resuspended in 50 μg/mL propidium iodide (Sigma) for nucleic acids staining. Cell cycle distribution was analyzed in a flow cytometer using ModFit LT software, and mean FSC-H population was used to determine relative cell size. Apoptosis Assay. Cell apoptosis analysis was performed by annexin V-FITC/PI apoptosis detection kit (Becton−Dickinson, USA). Cells were harvested in cold PBS and collected by centrifugation for 5 min at 1000g. Cells were resuspended at a density of 1 × 106 cells/mL in 1× binding buffer, stained with PI and FITClabeled annexin V for 20 min, and immediately analyzed on a flow cytometer equipped with a 488 nm argon laser (Becton−Dickinson). In Vitro Assay of Caspase-3 Activity. The assay was carried out based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate FITC-DEVD-FMK (for caspase-3). The control and 5da-treated cells were harvested at a density of 1 × 106 cells/mL in RPMI 1640 medium. Then 300 μL each of the treated and control cultures were incubated with 1 μL of FITC-DEVD-FMK in a 37 °C incubator with 5% CO2 for 1 h. The analysis was performed using a flow cytometer equipped with a 488 nm argon laser. Results are represented as the percent change of the activity compared to the control. Fluorescence Confocal Microscopy. T24 cells were cultured on glass slide treated with compound 5da. Then 48 h after treatment, the cells were washed with PBS twice, fixed in 4% paraformaldehyde, and permeablized with 1% Triton X-100. The cells were washed for three times, and a final concentration of 0.5 μg/mL Hoechst 33258 (Sigma) was included to stain the nuclei. The images were visualized with a Zeiss 710 fluorescence confocal microscope. For immunostaining, 48 h after treatment, the cells were washed with PBS twice, fixed with 4% paraformaldehyde, and incubated with primary antibodies followed by the addition of Cy3-conjugated secondary antibodies. 6864



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Western Blot Analysis. T24 cells (3 × 105) were cultured in each well of 6-well plates to 85−95% confluence. The cells were exposed to 5da (0−10 μM) for 24 h and then washed once with ice-cold PBS and extracted with the sample buffer. The cell extracts were separated by polyacrylamide-SDS gels, transferred to nitrocellulose membrane, and probed with primary antibodies as indicated. The membrane was incubated with antirabbit IgG (AP-linked) and detected by an ECL Western blot system (Kodak, USA). Coimmunoprecipitation Assay. The CoIP assay was performed as described.33,34 Briefly, cells were lysed in 300 μL of IP-lysis buffer with protein inhibitor mixture (Roche). Immunoprecipitations were performed using the Catch and Release v2.0 Reversible Immunoprecipitation System kit (Millipore). Columns were washed according to the protocol, and the reaction mix for immunoprecipitation contained 2000 μg of protein, 2 μg of IP-antibody, and antibody capture affinity ligand. In Vivo Xenograft Model Assay. Pathogen-free male BALB/C nude mice aged 6 weeks (Changzhou Cavens Experimental Animal Co., Ltd., Changzhou, China) were used to establish the HCC and bladder xenograft model. The mice were raised under controlled environmental conditions (12 h light-dark cycle at 24 °C and 60−85% humidity). Solid tumors were constructed by subcutaneous injection of 5 × 106 HepG2 cells into the flank region of the nude mice (n = 6). The tumor-bearing mice were treated ip with vehicle (5% DMSO in saline, v/v) or with 6 or 12 mg kg−1 of 5da per 2 days. 10Hydroxycamptothecin (HCPT, 6 mg·kg−1, per 2 days) was used as a positive control. The tumor size and body weight of the mice were measured three times a week. The tumor size was determined by measuring the length (l) and width (w) and calculating the volume (V = lw2/2). Statistical Analysis. All data were shown as mean ± standard deviation (SD) using two-tailed Student t tests and one-way ANOVA with Bonferroni multiple comparison post-test. P less than 0.05 was considered as significant difference.

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Ministry of Education of China (IRT_16R15), the Natural Science Foundation of Guangxi Province (2014GXNSFAA118053, 2015GXNSFDA139009), the Foundation of State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEMR2014-A01, CMEMR2012-A04, CMEMR2011-17, CMEMR2016-A04).

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ABBREVIATIONS USED BC, bladder cancer; HCC, hepatocellular carcinoma; Bak, Bcl2antagonist/killer; Bax, Bcl2-associated X protein; Bim, BCL2like 1; AIF, apoptosis-inducing factor; CoIP, coimmunoprecipitation; PI, propidium iodide; EB, ethidium bromide; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FSC-H, forward scatter height; FMK, fluoromethyl ketone; CDK, cyclin-dependent kinase; HCPT, 10-hydroxy-camptothecin; PAINS, pan assay interference compounds

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01769. 1 H and 13C NMR spectra, HRMS spectra, HPLC spectra of all targeted compounds; X-ray crystallography of 5ba (PDF) Molecular formula strings (CSV)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*For G.F.S.: phone/fax, +86 773 5826869; E-mail, gfysglgx@ 163.com. *For C.X.P.: phone/fax, +86 773 5826869; E-mail, [email protected]. ORCID

Dong-Liang Mo: 0000-0002-4005-2249 Gui-Fa Su: 0000-0003-3128-2381 Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Professor Erkang Fan (University of Washington) and Professor Ke Ding (Jinan University, PR China) for helpful discussions and are grateful for financial support from the National Natural Science Foundation of China (21462008, 81673473, 81401912, and 21162002), 6865



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Phthalazino[1,2-b]quinazolinones as p53 Activators: Cell Cycle Arrest

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