Design, Synthesis, and Evaluation of New Selective NM23-H2 Binders

Article pubs.acs.org/jmc

Design, Synthesis, and Evaluation of New Selective NM23-H2 Binders as c‑MYC Transcription Inhibitors via Disruption of the NM23-H2/GQuadruplex Interaction Yu-Qing Wang, Zhou-Li Huang, Shuo-Bin Chen, Chen-Xi Wang, Chan Shan, Qi-Kun Yin, Tian-Miao Ou, Ding Li, Lian-Quan Gu, Jia-Heng Tan,* and Zhi-Shu Huang* School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China Downloaded via IOWA STATE UNIV on January 3, 2019 at 06:00:21 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: c-MYC is one of the important human proto-oncogenes, and transcriptional factor NM23-H2 can activate c-MYC transcription by recognizing the G-quadruplex in the promoter of the gene. Small molecules that inhibit c-MYC transcription by disrupting the NM23-H2/G-quadruplex interaction might be a promising strategy for developing selective anticancer agents. In recent studies, we developed a series of isaindigotone derivatives, which can bind to G-quadruplex and NM23-H2, thus downregulating c-MYC (J. Med. Chem. 2017, 60, 1292−1308). Herein, a series of novel isaindigotone derivatives were designed, synthesized, and screened for NM23-H2 selective binding ligands. Among them, compound 37 showed a high specific binding affinity to NM23-H2, effectively disrupting the interaction of NM23-H2 with G-quadruplex, and it strongly down-regulated cMYC transcription. Furthermore, 37 induced cell cycle arrest and apoptosis, and it exhibited good tumor growth inhibition in a mouse xenograft model. This work provides a new strategy to modulate c-MYC transcription for the development of selective anticancer drugs.

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INTRODUCTION

down-regulating c-MYC transcription is a feasible strategy to expand cancer therapy. The NM23-H2 protein comes from the human NM23 family, and NM23-H2 has been known to function as both a nucleoside diphosphate kinase (NDPK) and transcriptional factor.11−13 NM23-H2 has been reported to play important roles in many physiological processes, such as gene transcription, DNA repair, oncogenesis, and cellular proliferation.14−17 In fact, aberrant expression of NM23-H2 has been observed in many tumors, such as hepatocellular carcinoma, chronic myeloid leukemia, and breast cancer,16,18,19 indicating NM23-H2 as an ideal target for cancer treatment. Notably, NM23-H2 has been identified as a transcription factor of the cMYC oncogene, which activates c-MYC transcription by recognizing and unfolding the structural and functional element (G-quadruplex) located in the promoter region of the gene.7,20−22 Hence, disruption of the NM23-H2/c-MYC Gquadruplex interaction may be a feasible strategy for down-

Oncogenes driving tumorigenesis have been proposed as promising therapeutic targets for cancers treating.1 The human c-MYC gene is one of the most well-known oncogenes; it encodes a multifunctional transcription factor that regulates the expression of genes involved in cell growth, proliferation, differentiation, and apoptosis.2,3 The aberrant overexpression of c-MYC is associated with various human cancers, and numerous murine cancer models are c-MYC-dependent.4 Anti-c-MYC therapeutic strategies involve multiple routine approaches, including the prevention of c-MYC binding to its partner proteins or target genes, and c-MYC gene transcription inhibition.5−8 Because c-MYC does not have any cavities that small molecules can easily bind into, the design of direct inhibitors is very complicated.9 The development of effective therapeutic strategies for the inhibition of c-MYC gene transcription has been challenging. Additionally, the inhibition of the bromodomain and extra-terminal (BET) family BRD4 protein can suppress c-MYC transcription and lead to tumor inhibition preclinically in vivo.6,10 This finding indicates that © 2017 American Chemical Society

Received: March 19, 2017 Published: July 17, 2017 6924

DOI: 10.1021/acs.jmedchem.7b00421 J. Med. Chem. 2017, 60, 6924−6941

Journal of Medicinal Chemistry

Article

Figure 1. Structures of compound 1, 2, and the new isaindigotone derivatives.

Table 1. Structures of the New Synthesized Isaindigotone Derivatives (12−56)

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Scheme 1. Synthesis of the Isaindigotone Derivativesa

a Reagents and conditions: (a) pyrrolidin-2-one, POCl3, reflux, 24 h, 84%; (b) R1(CH2)nNH2, 100 °C, 24 h, 52−78%; (c) aldehydes, DMF, TMSCl, 100 °C, 24−48 h, 40−88%.

fluorine atom with different N-nucleophiles of the primary amines to obtain intermediates 5−11. Treatment of 5−11 with different aldehydes via the Knoevenagel reaction gave the target compounds 12−56.31 The structure of the absolute configuration of compound 37 was finally identified using single crystal X-ray crystallographic analysis via anomalous scattering of Cu Kα radiation (Figure S1 in Supporting Information). Binding Affinities of the Derivatives for NM23-H2 Protein and SAR Study. To investigate the binding affinities of the synthesized compounds for the NM23-H2 protein, surface plasmon resonance (SPR) experiments were performed (Figure S2A). The dissociation constants (KD) were achieved from the equilibrium fitting mode. As shown in Table 2, most

regulating c-MYC transcription. Several studies have been performed to develop compounds that disrupt the interaction, and most of the compounds are G-quadruplex ligands.20,23 Gquadruplex structural motifs can form anywhere in the genome in G-quadruplex-forming sequences, whereas single-stranded G-rich DNA is exposed during replication, transcription, or recombination.24,25 The G-quadruplex ligands may lack targeting specificity and have side effects in oncotherapy. In recent studies, we developed isaindigotone derivative 1 (SYSUID-01) and 2 (SYSU-ID-19d) with two amino side chains (Figure 1), which bind to both G-quadruplex DNA and the NM23-H2 protein26 and can also down-regulate c-MYC transcription by disrupting the NM23-H2/G-quadruplex interaction. Therefore, further design and synthesis of highly specific NM23-H2 binding ligands to enhance the target selectivity will supply new and important information for cMYC transcription inhibition strategies. In this study, to explore novel NM23-H2-specific ligands, a series of new isaindigotone derivatives were designed based on the structures of our previous isaindigotone derivatives with two amino side chains (Figure 1). First, we retained the fivecarbon aliphatic ring fused to the pyrrolo[2,1-b]quinazoline moiety because our previous research confirmed that its derivatives exhibit higher binding affinities with the NM23H2 protein than compounds fusing six-carbon aliphatic ring.26 Second, introducing two amino side chains to the chromophore has been proven to be an effective way to obtain high Gquadruplex DNA binding and stabilizing potency.27,28 To remove the G-quadruplex ligand properties of the derivatives, only one positive amino side chain at the 6-position was introduced for the requirement of solubility. Meanwhile, we replaced another amino side chain at the 4′-position of the styrene ring with a variety of substituents, such as alkyl, hydroxyl, alkoxy, alkamino, fluoro, trifluoromethyl, and so on. Therefore, a series of new (E)-6-amino-3-benzylidene-7-fluoro2,3-dihydropyrrolo[2,1-b]quinazoline-9(1H)-one derivatives (12−56) were synthesized based on the structural modification described above (Figure 1 and Table 1). Their biological activities and structure−activity relationships were analyzed. After a screening assay, the most outstanding compound was further investigated, including activity of the control of c-MYC gene transcription, cell cycle arrest and apoptosis-inducing activities in SiHa cells, and inhibition of tumor growth in a mouse xenograft model of cervical squamous cancer.

Table 2. Binding Affinity of the Derivatives to NM23-H2 Proteina

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compd

KD/μM with SPRb

KD/μM with MSTc

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

d d d d d 13.8 d 15.4 14.6 13.9 d 17.1 16.3 4.6 16.7 8.5 10.1 20.6 12.4 9.6 25.6 9.2 13.3 15.8

61.6 59.4 55.3 e e 20.8 48.5 23.5 20.6 11.7 72.8 25.1 28.2 3.4 25.8 10.8 18.9 21.0 16.3 15.4 8.9 10.7 16.5 19.2

compd

KD/μM with SPRb

KD/μM with MSTc

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 1 2

16.8 3.1 13.2 d d d d d d 38.0 28.4 18.4 18.5 14.3 d d d 8.1 11.5 12.2 d 10.4 19.8

18.4 2.0 16.1 44.2 e e e e e e 25.6 22.3 23.6 16.9 68.0 27.5 45.7 6.4 10.8 7.6 56.0 24.6 18.0

a

The equilibrium dissociation constants (KD) were determined with the SPR assay and MST assay. bThe KD values determined using the SPR assay were achieved from the equilibrium fitting mode of the Analysis module in the ProteOn XPR36 software. cThe KD values determined using the MST assay were achieved in the Analysis module of the NT Analysis software using the Hill model. dNo significant binding was found at a concentration up to 40 μM ligand. eThe KD values of the compound were not obtained by fitting.

RESULTS AND DISCUSSION Chemistry. The facile synthetic pathway for the isaindigotone derivatives is shown in Scheme 1. Intermediate 4 was obtained by the reaction of 2-amino-4,5-difluorobenzoic acid (3) and pyrrolidin-2-one in the presence of POCl3.29,30 The next step was the nucleophilic substitution of the 6-position 6926



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(MST) assay was also performed to verify and validate the binding activity of the compounds (Table 2 and Figure S3), and the structure−activity relationship was consistent with the SPR assay. To gain more details on the interaction mode of compound 37 with NM23-H2 protein, we further performed molecular docking simulations of compound 37 with NM23-H2 (PDB code 3BBB) using Schrodinger Maestro 9.3. As shown in Figure 2, compound 37 was well-fitted into the narrow, slightly

of the derivatives bound to NM23-H2 protein showed moderate to strong binding affinity, and the abilities of some ligands were superior than 1 and 2. The structure−activity relationship was further explored as described below. First, we investigated the effect of the single substituent of R4 on the styrene portion, which includes a series of analogs that were designed and synthesized with different R4 substituents. As shown in Table 2, in the series of 6-(3-diethylaminopropyl)amino substituted compounds (12−20) and in the series of 6-(3-morpholinopropyl)amino substituted compounds (39−47), the binding potency of the derivatives is very sensitive to the type of R4 substituent. Compounds 19, 20, 46, 47 with an alkoxy group (4′-OCH3 or 4′-OCH2Ph) obviously exhibited better activity than other substituents (alkamino, alkyl, or fluoro groups) except compound 17, and compounds 20 and 47 with the 4′-benzyloxy group exhibited a slightly stronger binding affinity than that of compounds 19 and 46 with the 4′-methoxy group. Similarly, the 6-(3-dimethylaminopropyl)amino substituted compounds (28−32) with different types of alkoxy groups also exhibited considerable activity. All of the above results indicated that introducing an alkoxy group at the 4′-position of the 3-benzylidene-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one moiety was important for increasing the binding affinity. We also synthesized a series of compounds with two or more alkoxy groups at the 2′-, 3′-, 4′-, or 5′-position of the phenyl ring. As shown in Table 2, upon comparison of the activities of 2′,4′-disubstituted derivative 25 and 3′,4′-disubstituted derivative 26 with 4′-substituted derivative 20, we found that a hydroxy group introduced at the 2′-position seems to be more beneficial for increasing the activity of the compounds than introduction at the 3′-position. These results were also found for other series of compounds, such as for compounds 37 (2′-OH-4′-OCH2Ph, KD = 3.1 μM) and 38 (3′-OH-4′-OCH2Ph, KD = 13.2 μM) and for compounds 49 (2′-OH-4′-OCH2Ph, KD = 14.3 μM) to 47 (4′-OCH2Ph, KD = 18.4 μM). Amino side chain substituents at the 6-position of 2,3dihydropyrrolo[2,1-b]quinazolin- 9(1H)-one also have substantial effects on the binding activity. A series of analogs (25, 37, 49, 53−55) that contain the 4-(benzyloxy)-2-hydroxybenzylidene group at the 3-position of the moiety were analyzed. As shown in Table 2, the introduction of an open-ring basic terminal group, i.e., a dimethylamino group (compound 37) or diethylamino group (compound 25), exerted a remarkable improvement in the binding activity with KD values of 3.1 and 4.6 μM, respectively, compared with the corresponding closed-ring basic terminal group containing compounds 49 (14.3 μM) and 54 (11.5 μM). We also observed that the length of the amino side chain at the 6-position had an effect on the activity. For example, for compounds that had the same terminal side chain, the binding affinity of 37 (n = 3) was better than that of 53 (n = 2), and the binding affinity of 49 (n = 3) was better than that of 56 (n = 0). These results indicated that the length and terminal basic groups of the side chain at the 6-position have important impacts on the compounds regarding their binding activities. In conclusion, the new isaindigotone derivatives synthesized via structural modification were confirmed as NM23-H2 protein binders, and some compounds exhibited a significant binding affinity to NM23-H2 with a KD value below 10 μM. Compound 37 exhibited the best binding affinity (3.1 μM, Figure S2) and was stronger than the previous compounds 1 (10.4 μM) and 2 (19.8 μM). The microscale thermophoresis

Figure 2. Predicted binding mode of compound 37 with NM23-H2: (A, B) binding mode of compound 37 bound to one subunit of NM23-H2 (PDB code 3BBB); (C) binding affinity of compound 37 to wild-type and mutant NM23-H2 determined by SPR and MST. “-a” in the table means the KD value was not obtained by fitting.

curved pocket that the dinucleotide possessed as previously reported.7 Compound 37 undergoes hydrogen bonding with residues in the channel of the protein active site (Gly113 and Asp121), hydrophobic interactions with His118 and Lys66, and π−π stacking with Phe60 and Tyr67. To further validate the binding modes, five single amino acid mutant NM23-H2 proteins (D54A, F60A, G113F, H118F, and D121A) were further investigated.7 The SPR experiment and MST experiment results showed that compound 37 could still bind to the D54A, G113F, and H118F mutants with a reduced binding affinity compared to the wild-type NM23-H2 while losing binding affinity to the F60A and D121A mutant (Figure 2C and Figure S2). These results indicated that compound 37 binds to the NM23-H2 active pocket, and Phe60 as well as Asp121 showed the most important roles. In addition, we also examined the binding affinities of Gquadruplex DNA to wild-type and mutant NM23-H2 proteins. As shown in Table S1, the KD value of pu22 binding to the wild-type NM23-H2 was 0.45 μM, while the binding affinity was reduced for the D54A and H118F mutant. For F60A, G113F, and D121A mutants, pu22 lost binding affinity. To some extent, these results were consistent with the behavior of compound 37 binding to the wild-type and mutant NM23-H2 proteins, indicating 37 might have the potential to occupy some binding sites of pu22 toward NM23-H2 and disrupt the NM23H2/G-quadruplex interaction accordingly. Stabilizing and Binding Activities of Derivatives on the c-MYC G-Quadruplex. We evaluated the binding affinities of the synthesized isaindigotone derivatives to NM23-H2 protein, but we are also interested in the stabilizing and binding activities of the compounds to G-quadruplex DNA. A fluorescence resonance energy transfer (FRET) assay was performed to evaluate the stabilizing activity of the compounds on c-MYC G-quadruplex DNA (Table S2), and the results 6927



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Figure 3. Intervening effects of the isaindigotone derivatives on the interaction of NM23-H2 with the c-MYC G-quadruplex determined by ELISA assays. In total, 100 nM 5′-biotin-labeled c-MYC Pu22 was annealed in 200 μL of Tris-HCl buffer (10 mM, pH 7.4) containing 100 mM KCl. The absorbance reading was performed at 450 nm, and the KD values were fitted and calculated from the original data in Origin 8. Compound 1 was used as the reference compound. The KD of NM23-H2-G-quadruplex binding in the presence of the compounds (KDwith compound) was normalized by the KD of the NM23-H2-DNA interaction without the compound (KDwithout compound) and columned. The data were obtained from three individual experiments and are expressed as the mean ± SD.

affinity to NM23-H2 protein (KD = 3.1 μM) and disrupting ability (KD ratio of 4.1) (Figure S5). Compared with reference compound 2, for which the KD ratio was determined to be 3.8 μM and the KD value with NM23-H2 protein was 19.8 μM, compound 37 showed a better binding ability to the protein and a slightly better disrupting activity on the protein/DNA interaction than the previous compound 2. Electrophoretic mobility shift assay (EMSA) was also performed, elucidating that compound 37 was able to inhibit the interaction of NM23H2 and DNA in a dose dependent manner (Figure S6). In addition, we evaluated the cell proliferation inhibitory activities of the compounds on various human cancer cell lines, and the IC50 values are listed in Table S6. The results showed that most of the compounds displayed significant antiproliferative activities against several human cancer cell lines derived from different tissues or organs, including the human epithelial cervical cancer cell line HeLa, human cervical squamous cancer cell line SiHa, human ovarian cancer cell line A2780, non-smallcell-lung cancer cell line A549, human malignant myeloid cell line K562, human acute leukemic cell line HL-60, and malignant lymphoma cell line RAJI. Compound 37 exhibited a wide anticancer spectrum and outstanding antiproliferation activity with an IC50 of 1.78 μM in SiHa cells. Down-Regulation of c-MYC Transcription by Compound 37 by Disrupting the NM23-H2/G-Quadruplex Interaction in Cells. On the basis of the SPR, MST, and ELISA assay data, compound 37 showed good potential for NM23-H2 selective binding, NM23-H2/c-MYC G-quadruplex interaction disruption, and cancer cell proliferation suppression. To determine whether compound 37 could disrupt the interaction of NM23-H2/c-MYC G-quadruplex in cancer cells, we further applied chromatin immunoprecipitation (ChIP) assays. As shown in Figure 4A, the ChIP band indicated the quantity of immunoprecipitated c-MYC G-quadruplex fragments that bonded to the NM23-H2 protein in the cells. In comparison to the control group, compound 37 reduced the cMYC immunoprecipitated fragment in a dose-dependent manner. Related to the in vitro assay results, we confirmed that compound 37 disrupted the interaction of NM23-H2 protein with the c-MYC G-quadruplex in SiHa cells.

(Table S3) showed that most of the compounds exhibited a weak stabilization effect on the G-quadruplex, while compound 37 had no meaningful stabilizing activity. Additionally, a surface plasmon resonance (SPR) assay was performed to investigate the binding activity and selectivity of the compounds to the cMYC G-quadruplex (Pu22), the telomeric G-quadruplex (Htg21), and duplex DNA (hairpin 18). As shown in Table S4, most of the compounds similarly exhibited a weak activity, and compound 37 had an unmeasurable binding affinity to the c-MYC G-quadruplex. Other tested DNAs at a concentration of 40 μM compound indicated that this series of isaindigotone derivatives had no or weak stabilizing and binding activities to G-quadruplex DNA. Disrupting Activity of the Derivatives to the NM23H2/c-MYC G-Quadruplex Interaction. Since the major objective of this study was to develop disrupting ligands for the NM23-H2/c-MYC G-quadruplex interaction (Figure S4), the disrupting activities of newly synthesized NM23-H2 binders were identified using enzyme-linked immunosorbent assay (ELISA) methods. The affinity of NM23-H2 bound to Gquadruplex (KD = 0.38 ± 0.047 μM) was similar to a previous report.7 Then, the KD values of NM23-H2/G-quadruplex interaction were tested in the presence of the compounds (Table S5). We visualized the results as shown in Figure 3, where the KD ratio (KD ratio = KDwith compound/KDwithout compound) is the y-axis of the column graph. A higher ratio represents a stronger disrupting ability of the compounds to the NM23-H2/ c-MYC G-quadruplex interaction. Compared with compound 1 (KD ratio of 2.3), some compounds maintained (17, 18, 21, 36, and 47) (KD ratio in the range of 2.1−2.6) or increased (20, 25−27, 31, 37, 38, and 53−55) (KD ratio of ≥3.0) the disrupting potency. Meanwhile, most of the compounds (20, 25, 27, 31, 37, 38, and 53−55) with a higher KD ratio had a stronger binding with NM23-H2 protein (KD < 15 μM, Table 2), and those compounds with a poor binding ability to the protein exhibited a weak disrupting ability, which indicated a good correlation between the binding affinity of the compounds on NM23-H2 and the disrupting activity toward the NM23-H2/c-MYC G-quadruplex interaction. Among all of the new synthetic compounds, compound 37 possessed the best activities in terms of both the binding 6928



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affinity but differ in G-quadurplex binding. Accordingly, compound 28 and our previously reported compound 20d26 were chosen. Compounds 28 (KD = 10.1 μM) and 20d (KD = 7.6 μM) have similar NM23-H2 binding affinity, but their binding affinities to G-quadruplex were different (28 (KD = 35.8 μM) and 20d (KD = 3.8 μM)). As shown in Figure S8, 20d showed better c-MYC inhibition activity compared with 28, which indicated that enhancing the G-quadruplex binding affinity of compounds might make contribution to c-MYC inhibition. Regulation of the Cell Cycle by Compound 37. In proliferating cancer cells, a decreasing expression of c-MYC may induce cell cycle arrest and eventual apoptosis.16,20,32,33 To determine the effect of 37 on the cell cycle, SiHa and primary cultured mouse mesangial (the proliferation does not depend on c-MYC expression) cell lines treated with 37 were analyzed using flow cytometry. As shown in Figure 5A, the G0/G1 phase cell cycle arrest was significantly increased with SiHa cells treated with 37 for 3, 6, and 9 h compared with the control group. When SiHa cells were treated with 37 at concentrations of 0, 0.25, 0.5, 1.0, and 2.0 μM for 9 h of incubation, the percentage of cells in the G0/G1 phase increased dosedependently, along with concomitant losses in the G2/M phase (Figure 5B). When the incubation time was extended to 12, 24, 36, and 48 h, the G0/G1 cell cycle arrest decreased and the SUB-G1 peak (a characteristic hypodiploid peak) increased significantly and time-dependently, which indicated that most cells underwent DNA fragmentation (Figure 5A).34 For primary cultured mouse mesangial cells, where proliferation is independent of c-MYC, the cells showed no cell cycle arrest at the same concentrations (Figure S9). Moreover, we also investigated the expression of proteins in the cell cycle regulation pathway via Western blot after incubation with 37. As Figure 5C shows, cyclins D1, CDK4, and CDK6 were significantly down-regulated. These results indicated that 37 induced cell cycle arrest at the G0/G1 phase in a dose- and time-dependent manner in SiHa cells. Since the proliferation of primary cultured normal cells does not depend on c-MYC expression, this suggested that the cell cycle arrest by 37 in SiHa cells may be due to the regulation of c-MYC transcription. Induction of Cell Apoptosis by Compound 37. Because an obvious SUB-G1 peak was observed in the cell cycle analysis results above (Figure 5A), 37-treated SiHa cells and primary cultured mouse mesangial cells were also analyzed using flow cytometry after double-staining for FITC-labeled annexin-V binding to phosphatidylserine of the membrane and propidium iodide (PI) binding for cellular DNA.35 As shown in Figure 6A, after SiHa cells were treated with 37 at different concentrations of 0, 0.25, 0.5, 1.0, and 2.0 μM for 48 h, both early and late cell apoptosis was induced. The percentages of early apoptosis cells were 11.8%, 80.7%, and 4.8%, and the percentages of late apoptosis cells were 5.1%, 10.6%, and 90.3%, respectively. In addition, as shown via the Western blot assay, a dose-dependent increase in apoptosisrelated proteins, such as cleaved caspase-3 and cleaved PARP, was observed in SiHa cells after treatment with 37 (Figure 6B). Nevertheless, we did not observe apoptosis of the primary cultured mouse mesangial cells at the same concentration of 37 (Figure S10). Thus, the induction of apoptosis in SiHa cells by 37 may be related to the regulation of the c-MYC gene. Evaluation of Antitumor Activity by Compound 37 in Cells. Because compound 37 prevented NM23-H2 protein from binding to the c-MYC promoter, which led to the

Figure 4. Effects of compound 37 on the interaction of NM23-H2 with the c-MYC G-quadruplex and c-MYC transcription and expression in SiHa cells. (A) ChIP results of NM23-H2-DNA binding in SiHa cells treated with compound 37 for 9 h. The bands of the PCR products were quantified via Quantity One. The NM23-H2 ChIP groups were normalized using the input groups, and the sample without compound was set at 100%. All of the experiments were repeated three times: (∗∗∗) P < 0.001 compared with control group. (B) mRNA level of c-MYC and the NM23-H2 gene in SiHa cells treated with compound 37 for 9 h determined by RT-PCR. β-Actin was used as the internal control. The bands of amplified products were quantified via Quantity One. The values of c-MYC and NM23-H2 were normalized using β-actin, and the values of the sample without ligand were set at 100%. All of the experiments were repeated three times: (∗∗∗) P < 0.001 compared with control group. (C) Expression level of c-MYC and NM23-H2 protein in SiHa cells treated with compound 37 for 48 h, determined via Western blot. β-Actin was used as the internal control. All of the experiments were repeated three times: (∗∗) P < 0.01, (∗∗∗) P < 0.001 compared with control group.

NM23-H2 protein can activate c-MYC transcription by specifically binding and unwinding the G-quadruplex in the promoter,20,21 and disruption of the NM23-H2/c-MYC Gquadruplex interaction by compounds should down-regulate cMYC transcription.7,26 Subsequently, we used semiquantitative reverse transcription (RT-PCR) to examine the effect of compound 37 on the c-MYC transcription level. SiHa cells were treated with 37 at concentrations of 0, 0.5, 1.0, and 2.0 μM for 9 h of incubation. The mRNA level of c-MYC decreased in a dose-dependent manner, and the mRNA level of NM23-H2 was not affected (Figure 4B). Meanwhile, 37 also repressed the translation level of the c-MYC gene at 1.0 and 2.0 μM concentrations according to the Western blot assay (Figure 4C). Additionally, compared with reference compounds 1 and 2, 37 showed better c-MYC transcription and expression inhibitory activity (Figure S7). These data collectively suggested that the NM23-H2 binding ligand 37 can disrupt the NM23-H2/G-quadruplex interaction mechanistically and that the anticancer effect may be correlated with its ability to decrease c-MYC transcription and expression. As G-quadurplex stabilization has been pursued for c-MYC inhibition, it would be interesting to see a comparative study in efficacy between compounds that have similar NM23-H2 6929



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Figure 5. Cell cycle arrest of SiHa cells with or without treatment of compound 37. SiHa cells were plated in six-well plates for 24 h and treated without or with compound 37 at a concentration of 2 μM for 3, 6, 9, 12, 24, 36, and 48 h (A) and at concentrations of 0, 0.25, 0.5, 1.0, and 2.0 μM for 9 h of incubation (B). Then, the cells were harvested to determine the PI-stained DNA content via flow cytometry, and the total proteins were harvested for the Western blot analysis to detect the expression of cyclin D1, CDK4, and CDK6 (C). Quantitative analysis of the percentage of cells in each cell cycle phase was done using EXPO32 ADC analysis software. The protein bands were quantified using Quantity One. The experiments were performed at least three times: (∗∗) P < 0.01, (∗∗∗) P < 0.001, compared with control group. The results of representative experiments are shown.

of cancer cell proliferation by compound 37 may be due to the inhibition of c-MYC transcription. Then, we further studied the ability of compound 37 to inhibit colony formation. SiHa cells and primary cultured mouse mesangial cells were treated with various concentrations of 37 (0.5, 0.25, 0.125, 0.0625, and 0.03125 μM, and a DMSO group was used as the negative control) for 14 days. As Figure 7D and Figure S11 show, SiHa cells were more sensitive to 37 at any concentration than primary cultured mouse mesangial cells, which is consistent with the results of the RTCA assay. Additionally, almost no colonies formed in SiHa cells with 0.5 μM compound 37. In addition, because the inhibition of NM23-H2 activity was associated with inhibition of metastasis,37 a determination of whether 37 regulates cell migration or not was also performed. With an increase in concentration of 37, cell migration of SiHa cells significantly decreased in the wound scrape model (Figure 7E and Figure S12). Time course and concentration gradient analyses of the migrated cells showed that the inhibition of cell migration in SiHa cells was dose- and time-dependent. Thus, these results indicated that compound 37 had potent antitumor activity and may be related to the regulation of c-MYC gene transcription, which leads to the possibility of developing c-MYC transcription inhibitors by

transcriptional down-regulation of the oncogene, we explored the antitumor activity of 37. To compare the effects of 37 on the viability and proliferation of different cells, impedancebased real-time cell analyzer (RTCA) measurements were performed in SiHa cell lines and in primary cultured mouse mesangial cell lines (Figure 7A and Figure 7B). All of the cells exhibited similar responses during the first 24 h, and after 37 treatment, a significant proliferation arrest appeared in SiHa cells in a dose-dependent manner. No obvious effects on primary cultured mouse mesangial cells at concentrations of 0.5, 1.0, and 2.0 μM 37 were observed. To be noted, 37 exhibits slight antiproliferative activity against primary cultured mouse mesangial cells at a high concentration. This phenomenon might due to that NM23-H2 does not exclusively regulate cMYC transcription. As has been reported, NM23-H2 can also participate in the regulation of other genes, including myeloperoxidase, CD11b, and CCR5.36 Additionally, NM23H2-siRNA knockdown has been reported to have antitumor growth properties.16 Thus, the NM23-H2 siRNA knockdown SiHa cells were treated with 37 after incubation with siRNA for 24 h, and the cell growth was not inhibited even with doses up to 2 μM 37 (Figure 7C). This differential behavior of the proliferation in SiHa cells further indicated that the inhibition 6930



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Figure 6. Induction of apoptosis in SiHa cells by compound 37. SiHa cells were treated with compound 37 at concentrations of 0, 0.5, 1.0, and 2.0 μM for 48 h. Then, the cells were trypsinized, harvested, and stained with annexin V-FITC and PI solution for flow cytometry (A) or lysed to harvest the total proteins for the Western blot analysis to detect the levels of the apoptosis-related proteins caspase-3, cleaved caspase-3, c-PARP, and cleaved-PARP (B). Quantitative analysis of the percentage of cells in each cell cycle phase was performed using EXPO32 ADC analysis software. The protein bands were quantified using Quantity One. The experiments were performed at least three times: (∗∗) P < 0.01, (∗∗∗) P < 0.001, compared with control group. The results of representative experiments are shown.

Figure 7. Antitumor activity of compound 37 in cells. RTCA profiling of SiHa cells (A) and primary cultured mouse mesangial cells (B) with different concentrations of compound 37. (C) RTCA profiling of SiHa cells with siRNA interference and compound 37 treatment. (D) Colony formation assays of compound 37 with different inhibition effects of cell proliferation between SiHa cells and primary cultured mouse mesangial cells. (E) Effects of compound 37 on the migration of SiHa cells. Cells were damaged by mechanical scraping. Representative monolayer images of cell migration in the wound scrape model at 0, 24, 48, and 96 h are shown. RTCA plots were generated using RTCA software 1.1.2. The areas of migrated cells were evaluated using Image Pro6. All of the experiments were repeated three times: (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001, compared with control group.

disrupting the interaction of NM23-H2 with c-MYC Gquadruplex. Inhibition of Tumor Growth by Compound 37 in Vivo. To further demonstrate that the NM23-H2 inhibitors can be useful for treating cancers, we tested compound 37 in a SiHa xenograft mouse model of human cervical squamous cancer. SiHa tumors were established by subcutaneous injection of

SiHa cells into the right armpit of immunocompromised mice. When the tumor size reached approximately 50 mm3, the mice were randomly divided into four groups (the vehicle-treated group, the compound 2.5 mg/kg treated group, the compound 5.0 mg/kg treated group, and the doxorubicin 1.0 mg/kg treated group, n = 8/group) and treated via intraperitoneal (ip) injection daily, and the tumors were collected after 3 weeks of 6931



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Figure 8. Compound 37 inhibits tumor growth in a Siha xenograft model in vivo. After treatment with compound 37 at 2.5 mg/kg or 5 mg/kg or doxorubicin at 1.0 mg/kg for 3 weeks, the mice were sacrificed, and the tumors were weighed. (A) Images of excised tumors from each group. (B) Tumor volume of the mice in each group during the observation period. (C) Weights of the excised tumors from each group. (D) Body weight of the mice in each group at the end of the observation period. The data are presented as the mean ± SEM: (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001, significantly different compared with the vehicle group by t test, n = 8.

results showed that the derivatives are a new class of NM23H2-selective binders and have no effect on the stabilization and binding of G-quadruplex DNA. The SAR analysis indicated the key structural features of the isaindigotone derivatives as selective NM23-H2 ligands, including a long amino side chain (especially the terminal group of dimethylamino group) at the 6-position and an alkoxy substituent at the 4′-position (especially 4′-benzyloxy) of the 3-benzylidene-7-fluoro-2,3dihydropyrrolo[2,1-b]quinazolin-9(1H)-one moiety. This result in the SAR analysis led to the discovery of compound 37 with good activity for further study. Moreover, further studies showed that compound 37 had significant binding affinity to NM23-H2 and disrupting activity of the NM23-H2/c-MYC G-quadruplex interaction in vitro and in cells. The inhibition of oncogene c-MYC transcription by 37 induced many cellular events, such as cell cycle G0/G1 phase arrest, cellular apoptosis, proliferation inhibition, and migration inhibition. In addition, compound 37 exhibited a potent antiproliferation activity in many cancer cell lines and exhibited a good antitumor ability via inhibition of cervical squamous cancer growth in BALB/C-nu/nu mice with a SiHa xenograft via the suppression of proliferation. Our study reported the design and discovery of new NM23H2-selective binding ligands that may disrupt the interaction of the transcription factor NM23-H2 with the c-MYC promoter Gquadruplex to inhibit oncogene transcription. This work provides a new strategy for modulating c-MYC transcription for the development of selective anticancer drugs.

treatment and analyzed. As shown in Figure 8A and Figure 8B, compared with the vehicle group (mean, 511.6 mg; Figure 8C), the treatment with compound 37 at 2.5 mg/kg and 5.0 mg/kg resulted in a statistically significant reduction in tumor weight with a tumor growth inhibition (TGI) of 48.0% and 64.8%, respectively (mean values of 265.9 mg and 180.1 mg, P < 0.001, respectively). The wide-spectrum antitumor antibiotic doxorubicin was used as the reference molecule (TGI = 67.6%, mean, 165.9 mg, P < 0.001). In addition, treatment with 37 at 2.5 mg/ kg and 5.0 mg/kg resulted in a significant decrease in the final tumor volume (means of 332.6 mm3 and 272.1 mm3; P < 0.01 and P < 0.05, respectively) when compared to the vehicle group (mean, 812.8 mm3; Figure 8C). Additionally, the data showed that treatment with 37 presented a time-dependent inhibition in tumor growth. During the experiment, all of the animals appeared healthy with no visible signs of pain, distress, or discomfort. There was no significant difference in body weight change among the vehicle group and the 37-treated groups, indicating that it was tolerated well at these doses (Figure 8D). Although the treatment of doxorubicin caused good tumor growth inhibition, its toxicity was apparent as evidenced by body weight loss. All of the data showed that compound 37 exhibited a good antitumor ability for the inhibition of cervical squamous cancer growth in BALB/C-nu/nu mice with SiHa xenografts via the suppression of proliferation.

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CONCLUSION In this study, we reported the design and synthesis of a series of new isaindigotone derivatives as NM23-H2-selective binding agents to disrupt the interaction of NM23-H2 with the c-MYC G-quadruplex and down-regulate the oncogene c-MYC transcription. A series of biological evaluations from the molecular to the animal level were performed. We determined the binding affinities of derivatives to NM23H2 and c-MYC promoter G-quadruplex DNA and the disruption activities of derivatives for the NM23-H2/c-MYC G-quadruplex interaction, and the SAR was summarized. The

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EXPERIMENTAL SECTION

Chemical Synthesis. All chemicals were purchased from commercial sources unless otherwise specified. All the solvents were of analytical reagent grade and were used without further purification. 1 H and 13C NMR spectra were recorded in DMSO-d6, MeOD, or CDCl3 with a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard and coupling constants in hertz. Chemical shifts are reported in parts per million 6932



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(ppm) relative to residual DMSO-d6 (δ = 2.50, 1H; δ = 40.0, 13C), MeOD (δ = 3.31, 1H; δ = 49.0, 13C) and CHCl3 (δ = 7.26, 1H; δ = 77.0, 13C). High resolution mass spectra (HRMS) were recorded on Shimadzu LCMS-IT-TOF. Flash column chromatography was performed with silica gel (200−300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The purities of synthesized compounds were confirmed to be higher than 95% by analytical HPLC performed with a dual pump Shimadzu LC-20AB system equipped with a Ultimate XB-C18 column (4.6 mm × 250 mm, 5 μm) and eluted with methanol/water (15:85) containing 0.1% TFA at a flow rate of 0.4 mL/min. General Method for the Synthesis of Compounds 5−11. A mixture of intermediate compound 4 (2.22 g, 10 mmol, 1 equiv) and aliphatic amines (20 mmol, 2 equiv) was introduced into a 20 mL sealed tube. The mixture was stirred at 100 °C for 12 h and monitored by TLC. The mixture was cooled to room temperature, 10 mL of ethyl ether was added, filtered, washed with ethyl ether, and dried under vacuum to afford the solid products 5−11. 6-((3-(Diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (5). Pale yellow solid, 2.3 g (yield 69%). 1 H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 11.7 Hz, 1H), 6.72 (s, 1H), 6.67 (d, J = 7.7 Hz, 1H), 4.19−4.12 (m, 2H), 3.31 (dd, J = 10.6, 5.8 Hz, 2H), 3.11 (t, J = 7.9 Hz, 2H), 2.63−2.59 (m, 2H), 2.55 (q, J = 7.1 Hz, 4H), 2.30−2.19 (m, 2H), 1.89−1.81 (m, 2H), 1.06 (t, J = 7.1 Hz, 6H). 6-((3-(Dimethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (6). Pale yellow solid, 2.1 g (yield 66%). 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 11.7 Hz, 1H), 6.63 (d, J = 7.7 Hz, 1H), 4.12−4.05 (m, 2H), 3.24 (dd, J = 11.6, 6.3 Hz, 2H), 3.04 (t, J = 7.9 Hz, 2H), 2.37 (t, J = 6.4 Hz, 4H), 2.19 (s, 6H), 1.77 (dt, J = 12.8, 6.2 Hz, 2H). 7-Fluoro-6-((3-morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1b]quinazolin-9(1H)-one (7). Milk white solid, 1.8 g (yield 52%). 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 11.7 Hz, 1H), 6.62 (d, J = 7.7 Hz, 1H), 4.13−4.05 (m, 2H), 3.72−3.67 (m, 2H), 3.66−3.61 (m, 2H), 3.25 (dd, J = 11.1, 5.8 Hz, 2H), 3.04 (t, J = 7.9 Hz, 4H), 2.51−2.46 (m, 3H), 2.24−2.12 (m, 4H), 1.86−1.75 (m, 2H). 6-((2-(Dimethylamino)ethyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin -9(1H)-one (8). Milk white solid, 1.7 g (yield 58%). 1 H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 11.5 Hz, 1H), 6.61 (d, J = 7.4 Hz, 1H), 4.22−4.01 (m, 2H), 3.44 (dd, J = 12.6, 6.5 Hz, 2H), 3.14 (t, J = 7.8 Hz, 2H), 2.47 (t, J = 6.5 Hz, 4H), 2.18 (s, 6H). 7-Fluoro-6-((2-(piperidin-1-yl)ethyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (9). Milk white solid, 2.4 g (yield 73%). 1 H NMR (400 MHz, CDCl3) δ 7.75 (dd, J = 11.6, 4.6 Hz, 1H), 6.71 (d, J = 7.5 Hz, 1H), 5.32 (s, 1H), 4.16 (t, J = 7.0 Hz, 2H), 3.33−3.20 (m, 2H), 3.12 (dd, J = 9.6, 5.9 Hz, 2H), 2.64 (t, J = 5.6 Hz, 2H), 2.42 (s, 4H), 2.30−2.21 (m, 2H), 1.59 (d, J = 4.8 Hz, 4H), 1.46 (d, J = 3.3 Hz, 2H). 7-Fluoro-6-((3-(pyrrolidin-1-yl)propyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (10). Pale yellow solid, 1.9 g (yield 57%). 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 11.7 Hz, 1H), 6.70 (d, J = 7.7 Hz, 1H), 4.22−4.12 (m, 2H), 3.33 (dd, J = 11.1, 6.0 Hz, 2H), 3.13 (t, J = 7.9 Hz, 2H), 2.68 (t, J = 6.2 Hz, 2H), 2.56 (t, J = 5.9 Hz, 5H), 2.31−2.22 (m, 2H), 1.90 (dt, J = 12.4, 6.2 Hz, 2H), 1.85− 1.80 (m, 4H). 7-Fluoro-6-morpholino-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (11). Pale yellow solid, 1.9 g (yield 66%). 1H NMR (400 MHz, CDCl3) δ 7.84−7.72 (m, 1H), 7.03 (d, J = 7.8 Hz, 1H), 4.24− 4.13 (m, 2H), 3.95−3.83 (m, 4H), 3.27−3.19 (m, 4H), 3.14 (t, J = 8.0 Hz, 2H), 2.34−2.22 (m, 2H). General Method for the Synthesis of Compounds 12−26. A solution of the intermediate compound 5 (0.3323 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube, and the mixture was heated (100 °C) for 24 h and monitored by TLC. The solution was cooled to room temperature, filtered, washed with alcohol. And then the crude products were purified by flash column chromatography to afford solid compounds.

(E)-6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-(4-fluorobenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (12). Pale yellow solid, 0.2674 g (yield 61%). 1H NMR (400 MHz, CDCl3) δ 7.78−7.71 (m, 2H), 7.52 (dd, J = 8.5, 5.5 Hz, 2H), 7.13 (dd, J = 15.8, 7.2 Hz, 2H), 6.76 (d, J = 7.7 Hz, 1H), 6.71 (s, 1H), 4.23 (t, J = 8.0 Hz, 2H), 3.34 (d, J = 4.6 Hz, 2H), 3.22 (dd, J = 9.6, 4.3 Hz, 2H), 2.65−2.59 (m, 2H), 2.55 (q, J = 7.1 Hz, 4H), 1.86 (dt, J = 11.8, 5.9 Hz, 2H), 1.06 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 162.7 (d, J = 250.8 Hz), 160.5, 157.7 (d, J = 544.8 Hz), 152.0, 149.6, 148.7, 143.5, 143.3, 131.5 (2C), 128.4, 116.0, 115.8, 109.7 (d, J = 20.9 Hz), 109.3 (d, J = 7.7 Hz), 105.4, 52.7, 46.8 (2C), 43.8, 43.7, 25.5, 25.1, 11.6 (2C). ESI-HRMS [M + Na]+ m/z = 461.2231, calcd for C25H28N4OF2, 461.2353. Purity: 97.6% by HPLC. (E) -6-((3 -(Di et hylamino )prop yl)ami no)-7-fl uoro -3 -(4 (trifluoromethyl)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (13). Pale yellow solid, 0.3201 g (yield 82%). 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 2.7 Hz, 1H), 7.76 (d, J = 11.7 Hz, 1H), 7.69 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 8.3 Hz, 2H), 6.86 (s, 1H), 6.78 (d, J = 7.7 Hz, 1H), 4.27 (t, J = 7.1 Hz, 2H), 3.36 (dd, J = 10.7, 5.4 Hz, 2H), 3.29 (dd, J = 9.7, 4.2 Hz, 2H), 2.64 (t, J = 5.5 Hz, 2H), 2.58 (dd, J = 13.8, 6.8 Hz, 4H), 1.92−1.84 (m, 2H), 1.08 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.3 (d, J = 3.3 Hz), 154.6, 152.0, 149.6, 148.5, 143.4 (d, J = 13.9 Hz), 138.9, 134.7, 131.1−129.8 (m), 129.6 (2C), 127.8, 125.7 (d, J = 3.7 Hz), 123.9 (d, J = 272.1 Hz), 109.7 (d, J = 20.9 Hz), 105.6 (d, J = 4.0 Hz), 66.8 (2C), 58.1, 53.7 (2C), 43.8, 43.5, 25.6, 23.9. ESI-HRMS [M + H]+ m/z = 489.2199, calcd for C26H28N4OF4, 489.2283. Purity: 99.4% by HPLC. (E) -6-((3 -(Di et hylamino )prop yl)ami no)-7-fl uoro -3 -(4 morpholinobenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (14). Pale yellow solid, 0.3840 g (yield 76%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 11.7 Hz, 1H), 7.69 (s, 1H), 7.49 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 6.76 (d, J = 7.7 Hz, 1H), 4.23 (t, J = 6.9 Hz, 2H), 3.89−3.84 (m, 4H), 3.39−3.32 (m, 2H), 3.28−3.21 (m, 6H), 2.69−2.52 (m, 6H), 1.93−1.83 (m, 2H), 1.08 (t, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.5, 155.7, 151.1, 150.7 (d, J = 121.7 Hz), 148.9, 143.3 (d, J = 14.0 Hz), 131.1 (2C), 129.5, 128.4, 126.9, 114.7 (2C), 109.6 (d, J = 20.7 Hz), 109.1 (d, J = 7.6 Hz), 105.4 (d, J = 4.1 Hz), 66.7, 52.8, 48.1, 46.8 (2C), 43.8, 43.68, 25.6, 25.2, 11.7 (2C). ESI-HRMS [M + H]+ m/z = 506.2853, calcd for C29H36N5O2F, 506.2919. Purity: 99.1% by HPLC. (E)-6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-(4-(piperidin1yl)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (15). Pale yellow solid, 0.4281 g (yield 85%). 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H), 7.68 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 7.7 Hz, 1H), 6.59 (s, 1H), 4.22 (t, J = 7.0 Hz, 2H), 3.37−3.18 (m, 8H), 2.65−2.60 (m, 2H), 2.56 (dd, J = 13.9, 6.9 Hz, 4H), 1.91−1.82 (m, 2H), 1.70 (s, 4H), 1.63 (d, J = 4.2 Hz, 2H), 1.06 (t, J = 7.0 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.7 (d, J = 3.3 Hz), 155.9, 151.7, 150.9 (t, J = 121.6 Hz), 149.0, 143.3 (d, J = 13.9 Hz), 131.3 (2C), 129.9, 127.3, 125.6, 115.0 (2C), 109.6 (d, J = 20.8 Hz), 109.1 (d, J = 7.6 Hz), 105.4 (d, J = 4.0 Hz), 52.7, 49.2 (2C), 46.9 (2C), 43.9, 43.6, 25.6, 25.5 (2C), 25.2, 24.3, 11.6 (2C). ESIHRMS [M + H]+ m/z = 504.3060, calcd for C30H38N5OF, 504.3139. Purity: 99.0% by HPLC. (E)-6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-(4-isopropylbenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (16). Pale yellow solid, 0.2738 g (yield 74%). 1H NMR (400 MHz, CDCl3) δ 7.79−7.72 (m, 2H), 7.50 (d, J = 7.5 Hz, 2H), 7.30 (d, J = 7.7 Hz, 2H), 6.78 (d, J = 7.5 Hz, 1H), 6.72 (s, 1H), 4.24 (t, J = 6.7 Hz, 2H), 3.40−3.32 (m, 2H), 3.31−3.22 (m, 2H), 3.05−2.78 (m, 1H), 2.67−2.54 (m, 6H), 1.94−1.82 (m, 2H), 1.28 (d, J = 6.7 Hz, 6H), 1.08 (t, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.5 (d, J = 3.5 Hz), 155.3, 150.7 (d, J = 87.8 Hz), 149.8, 148.8, 143.4 (d, J = 14.0 Hz), 133.3, 131.0, 129.8 (2C), 129.6, 126.9 (2C), 109.6 (d, J = 20.8 Hz), 109.2 (d, J = 7.7 Hz), 105.4 (d, J = 4.2 Hz), 52.8, 46.9 (2C), 43.8, 43.7, 34.0, 25.6, 25.2, 23.8 (2C), 11.7 (2C). ESI-HRMS [M + H]+ m/z = 463.2795, calcd for C28H35N4OF, 463.2881. Purity: 99.1% by HPLC. 6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-((E)-4-((E)-2-(pyridin-2-yl)vinyl)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (17). Pale yellow solid, 0.3298 g (yield 63%). 1H NMR (400 MHz, MeOD) δ 8.41 (d, J = 4.7 Hz, 1H), 7.72−7.63 (m, 2H), 6933



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9.5, 5.4 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.81 (dd, J = 7.6, 2.4 Hz, 1H), 6.66 (s, 1H), 4.20 (dd, J = 12.4, 5.2 Hz, 2H), 3.91 (s, 3H), 3.38− 3.31 (m, 2H), 3.21 (td, J = 7.2, 2.7 Hz, 2H), 2.66−2.60 (m, 2H), 2.60−2.52 (m, 4H), 1.92−1.82 (m, 2H), 1.07 (td, J = 7.0, 3.6 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.6, 158.0, 153.6 (d, J = 338.9 Hz), 149.5, 148.8, 143.4, 143.2, 132.0, 130.1, 129.0, 124.7, 120.3, 110.8, 109.6 (d, J = 20.8 Hz), 109.3 (d, J = 7.6 Hz), 105.7, 55.5, 52.8, 46.8 (2C), 43.8, 43.7, 25.7, 25.2, 11.7 (2C). ESI-HRMS [M + H]+ m/z = 451.2431, calcd for C26H31N4O2F, 451.2515. Purity: 97.8% by HPLC. (E)-6-((3-(Diethylamino)propyl)amino)-3-(3,4-dimethoxybenzylidene)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (23). Pale yellow solid, 0.2643 g (yield 55%). 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 11.7 Hz, 1H), 7.74 (t, J = 2.7 Hz, 1H), 7.11 (d, J = 1.9 Hz, 1H), 7.04 (d, J = 1.9 Hz, 1H), 6.97 (s, 1H), 6.80 (s, 1H), 6.65 (s, 1H), 4.31−4.25 (m, 2H), 3.96 (d, J = 1.6 Hz, 6H), 3.51−3.34 (m, 4H), 2.72−2.58 (m, 6H), 1.97−1.89 (m, 2H), 1.12 (dd, J = 12.6, 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.5 (d, J = 3.6 Hz), 155.5 (d, J = 2.1 Hz), 150.3 (t, J = 123.1 Hz), 149.7, 148.9, 148.8, 143.6 (d, J = 12.9 Hz), 129.7, 129.6, 128.7, 123.0, 112.7, 111.2, 109.7 (d, J = 21.4 Hz), 109.4 (d, J = 7.5 Hz), 105.5 (d, J = 3.7 Hz), 58.3, 55.9, 55.9, 45.4 (2C), 43.9, 42.8, 25.7, 25.5, 11.5 (2C). ESIHRMS [M + H]+ m/z = 481.2537, calcd for C27H33N4O3F, 481.2602. Purity: 98.5% by HPLC. (E)-6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-(3,4,5trimethoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (24). Pale yellow solid, 0.4539 g (yield 89%). 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 11.6 Hz, 1H), 7.69 (t, J = 2.6 Hz, 1H), 6.79 (s, 2H), 6.76 (d, J = 7.7 Hz, 1H), 4.28−4.23 (m, 2H), 3.91 (d, J = 1.8 Hz, 9H), 3.35 (t, J = 5.8 Hz, 2H), 3.31−3.24 (m, 2H), 2.65 (t, J = 6.0 Hz, 2H), 2.59 (q, J = 7.1 Hz, 4H), 1.89 (dt, J = 12.0, 6.0 Hz, 2H), 1.08 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.5, 155.2, 153.3 (2C), 151.6, 150.7 (d, J = 241.9 Hz), 148.7, 143.4 (d, J = 14.0 Hz), 138.9, 131.2, 130.9, 129.8, 109.7 (d, J = 20.9 Hz), 109.3 (d, J = 7.6 Hz), 107.1 (2C), 105.3, 61.0, 56.2 (2C), 52.6, 46.8 (2C), 43.9, 43.5, 25.5, 25.0, 11.5 (2C). ESI-HRMS [M + H]+ m/z = 511.2642, calcd for C28H35N4O4F, 511.2718. Purity: 97.9% by HPLC. (E)-3-(4-(Benzyloxy)-2-hydroxybenzylidene)-6-((3(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (25). Pale yellow solid, 0.3744 g (yield 69%). 1H NMR (400 MHz, DMSO-d6) δ 7.96 (t, J = 2.6 Hz, 1H), 7.55 (d, J = 11.9 Hz, 1H), 7.47−7.44 (m, 2H), 7.43−7.38 (m, 2H), 7.35 (dt, J = 5.3, 2.1 Hz, 1H), 7.30 (s, 1H), 6.87−6.82 (m, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 5.11 (s, 2H), 4.10−4.05 (m, 2H), 3.29−3.24 (m, 4H), 3.18−3.12 (m, 2H), 2.49−2.45 (m, 4H), 1.75 (dq, J = 13.1, 6.4 Hz, 2H), 1.05− 0.91 (m, 6H). 13C NMR (101 MHz, DMSO-d6) δ 159.9, 159.3, 157.9, 155.8, 148.5 (d, J = 21.9 Hz), 142.5 (dd, J = 12.1, 8.2 Hz), 142.4, 136.8, 129.7, 128.4 (2C), 128.3 (2C), 127.8, 127.6, 122.9, 115.9, 108.9 (d, J = 24.6 Hz), 108.1 (d, J = 6.6 Hz), 106.2, 105.2, 101.9, 69.1, 50.4, 46.3 (2C), 43.7, 41.1, 24.9, 24.4, 10.8 (2C). ESI-HRMS [M + H]+ m/z = 543.2693, calcd for C32H35N4O3F, 543.2768. Purity: 99.7% by HPLC. (E)-3-(4-(Benzyloxy)-3-hydroxybenzylidene)-6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (26). Pale yellow solid, 0.3418 g (yield 63%). 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 11.7 Hz, 1H), 7.66 (s, 1H), 7.46−7.34 (m, 5H), 7.17 (d, J = 1.0 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.57 (s, 1H), 5.14 (s, 2H), 4.20 (t, J = 7.2 Hz, 2H), 3.34 (s, 2H), 3.21 (t, J = 6.0 Hz, 2H), 2.63 (t, J = 5.9 Hz, 2H), 2.58 (dd, J = 14.2, 7.1 Hz, 4H), 1.92−1.82 (m, 2H), 1.07 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.6 (d, J = 3.3 Hz), 155.5, 150.7 (d, J = 244.0 Hz), 148.8, 146.5, 145.9, 143.3 (d, J = 14.0 Hz), 135.9, 130.0, 129.6, 129.5, 128.8 (2C), 128.6, 127.8 (2C), 123.0, 115.6, 112.2, 109.7 (d, J = 20.6 Hz), 109.2 (d, J = 7.6 Hz), 105.4 (d, J = 3.9 Hz), 71.1, 52.6, 46.8 (2C), 43.9, 43.5, 25.5, 25.1, 11.5 (2C). ESI-HRMS [M + H]+ m/z = 543.2693, calcd for C32H35N4O3F, 543.2771. Purity: 99.1% by HPLC. General Method for the Synthesis of Compounds 27−38. A solution of the intermediate compound 6 (0.3042 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube, and the mixture was heated (100 °C) for 24 h and monitored by TLC. The mixture

7.57 (t, J = 8.6 Hz, 3H), 7.54−7.46 (m, 4H), 7.16 (dd, J = 17.0, 11.0 Hz, 2H), 6.76 (d, J = 7.9 Hz, 1H), 4.12 (t, J = 7.0 Hz, 2H), 3.26 (t, J = 6.9 Hz, 2H), 2.52 (dt, J = 21.8, 7.1 Hz, 6H), 1.84−1.75 (m, 2H), 1.26−1.16 (m, 2H), 0.98 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, MeOD) δ 160.9, 155.7, 155.6, 150.8 (d, J = 244.0 Hz), 148.9, 148.8, 143.8−143.6 (m), 137.2, 137.0, 135.8, 132.4, 132.2, 129.9 (2C), 129.2, 128.4, 127.1, 126.3, 122.2, 121.9, 108.8 (d, J = 21.3 Hz), 108.6 (d, J = 5.0 Hz), 105.1 (d, J = 4.3 Hz), 100.0, 51.1, 46.7 (2C), 43.9, 41.7, 25.3, 25.2, 10.3 (2C). ESI-HRMS [M + H]+ m/z = 524.2747, calcd for C32H34N5OF, 524.2821. Purity: 99.7% by HPLC. (E)-3-(4-(Diethylamino)benzylidene)-6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (18). Pale yellow solid, 0.3568 g (yield 72%). 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 7.76 (d, J = 11.7 Hz, 1H), 7.35 (d, J = 9.4 Hz, 2H), 6.91 (d, J = 7.9 Hz, 2H), 6.00 (s, 1H), 4.25−4.22 (m, 2H), 3.34− 3.31 (m, 2H), 3.19−3.09 (m, 2H), 3.109−3.05 (m, 4H), 2.59−2.52 (m, 6H), 1.82−1.80 (m, 2H), 1.06−1.03 (m, 6H), 0.94−0.90 (m, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.6, 156.2, 150.3 (d, J = 237.5 Hz), 149.0, 147.9, 143.1 (d, J = 12.9 Hz), 131.7, 130.3 (2C), 125.5, 122.8, 111.2 (2C), 109.5 (dd, J = 19.4, 3.3 Hz), 109.2 (d, J = 7.5 Hz), 105.4 (d, J = 3.7 Hz), 58.0, 53.8, 44.4, 43.9 (2C), 43.4 (2C), 25.5, 24.1, 13.2 (2C), 12.6 (2C). ESI-HRMS [M + H]+ m/z = 492.3060, calcd for C32H33N4O4F, 492.3133. Purity: 95.2% by HPLC. (E)-6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-(4-methoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (19). Pale yellow solid, 0.2021 g (yield 45%). 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.65−7.57 (m, J = 10.4 Hz, 2H), 7.42 (t, J = 7.1 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 7.9 Hz, 1H), 4.15−4.07 (m, 2H), 3.89 (s, 3H), 3.36 (t, J = 6.6 Hz, 2H), 3.23−3.18 (m, 2H), 3.12 (qd, J = 12.2, 6.0 Hz, 6H), 2.06−1.96 (m, 2H), 1.23 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.5, 158.0, 153.6 (d, J = 337.6 Hz), 149.5, 148.8, 143.3 (d, J = 14.0 Hz), 132.0, 130.1 (2C), 128.9, 120.3, 110.8 (2C), 109.6 (d, J = 20.8 Hz), 109.3 (d, J = 7.7 Hz), 105.7 (d, J = 5.4 Hz), 55.5, 52.7, 46.9 (2C), 43.8, 43.6, 25.7, 25.3, 11.7 (2C). ESI-HRMS [M + H]+ m/z = 451.2431, calcd for C26H31N4O2F, 451.2512. Purity: 98.2% by HPLC. (E)-3-(4-(Benzyloxy)benzylidene)-6-((3-(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (20). Pale yellow solid, 0.4470 g (yield 85%). 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 11.7 Hz, 1H), 7.67−7.64 (m, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.37 (t, J = 7.3 Hz, 2H), 7.31 (dd, J = 8.2, 5.8 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 7.7 Hz, 1H), 6.54 (s, 1H), 5.07 (s, 2H), 4.22−4.09 (m, 2H), 3.31 (dd, J = 10.5, 5.6 Hz, 2H), 3.12 (t, J = 5.9 Hz, 2H), 2.61−2.56 (m, 2H), 2.53 (q, J = 7.1 Hz, 4H), 1.83 (dd, J = 11.7, 5.8 Hz, 2H), 1.04 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.5, 159.2, 155.4, 150.6 (d, J = 243.8 Hz), 148.8, 143.3 (d, J = 14.0 Hz), 136.6, 131.3 (2C), 129.5, 129.2, 128.7, 128.6 (2C), 128.1, 127.4 (2C), 115.2 (2C), 109.6 (d, J = 20.8 Hz), 109.2 (d, J = 7.7 Hz), 105.4 (d, J = 4.0 Hz), 70.0, 52.7, 46.8 (2C), 43.8, 43.6, 25.5, 25.2, 11.7 (2C). ESI-HRMS [M + H]+ m/z = 527.2744, calcd for C32H35N4O2F, 527.2823. Purity: 97.6% by HPLC. (E)-3-(4-(Diethylamino)-2-hydroxybenzylidene)-6-((3(diethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (21). Pale yellow solid, 0.2380 g (yield 47%). 1 H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 7.76 (d, J = 11.7 Hz, 1H), 7.35 (d, J = 9.0 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.17 (d, J = 8.9 Hz, 1H), 5.98 (d, J = 2.3 Hz, 1H), 4.27−4.22 (m, 2H), 3.34−3.25 (m, 2H), 3.22−3.15 (m, 2H), 3.10−3.01 (m, 4H), 2.65−2.49 (m, 6H), 1.89−1.75 (m, 2H), 1.06 (t, J = 6.9 Hz, 6H), 0.91 (t, J = 6.9 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.5, 159.1, 149.4 (t, J = 117.9 Hz), 148.0, 143.6 (d, J = 28.1 Hz), 140.9, 130.1 (2C), 128.3, 128.2, 127.4, 121.8, 112.1 (2C), 109.9 (d, J = 21.3 Hz), 103.8 (d, J = 10.9 Hz), 98.6, 52.8, 46.8, 44.4 (2C), 44.3 (2C), 43.8, 29.7, 25.5, 24.8, 12.5 (2C), 11.6 (2C). ESI- HRMS[M + H]+ m/z = 508.3082, calcd for C29H38N5O2F, 508.3102. Purity: 95.9% by HPLC. (E)-6-((3-(Diethylamino)propyl)amino)-7-fluoro-3-(2-methoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (22). Pale yellow solid, 0.3061 g (yield 68%). 1H NMR (400 MHz, CDCl3) δ 8.11 (t, J = 2.5 Hz, 1H), 7.75 (dd, J = 11.7, 3.2 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.34 (dd, J = 11.3, 4.4 Hz, 1H), 7.00 (dd, J = 6934



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was cooled to room temperature, filtered, washed with alcohol. And then the crude product was purified by flash column chromatography to afford solid compounds. 6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-((E)-4-((E)-2(pyridin-2-yl)vinyl)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (27). Pale yellow solid, 0.2726 g (yield 55%). 1 H NMR (400 MHz, MeOD) δ 8.61 (d, J = 5.2 Hz, 1H), 8.20 (t, J = 7.8 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.9 Hz, 3H), 7.66− 7.59 (m, 4H), 7.36 (d, J = 16.4 Hz, 2H), 6.94 (d, J = 7.3 Hz, 1H), 4.25 (t, J = 6.8 Hz, 2H), 3.49 (t, J = 6.4 Hz, 2H), 3.39−3.34 (m, 4H), 2.97 (s, 6H), 2.20 (dd, J = 14.4, 6.9 Hz, 2H), 1.37−1.29 (m, 2H). 13C NMR (101 MHz, MeOD) δ 160.5, 152.8, 150.7 (d, J = 244.6 Hz), 149.5, 148.9, 148.1, 144.4, 142.0, 136.9, 136.1, 134.3 (dd, J = 15.2, 8.5 Hz), 133.0 (2C), 130.1, 129.2 (2C), 127.6−127.3 (m), 123.5, 123.2, 123.0, 109.3 (d, J = 8.1 Hz), 109.1 (d, J = 11.7 Hz), 105.2 (d, J = 2.7 Hz), 55.8, 44.1, 42.4 (2C), 39.6, 25.3, 23.7. ESI-HRMS [M + H]+ m/z = 496.2482, calcd for C30H30N5OF, 496.2498. Purity: 96.2% by HPLC. (E)-4-((6-((3-(Dimethylamino)propyl)amino)-7-fluoro-9-oxo-1,2dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)phenyl Acetate (28). Yellow solid, 0.2198 g (yield 61%). 1H NMR (400 MHz, MeOD) δ 7.74 (d, J = 11.4 Hz, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 7.1 Hz, 1H), 6.99 (s, 1H), 3.91 (t, J = 6.5 Hz, 2H), 3.52 (t, J = 6.9 Hz, 2H), 3.45−3.38 (m, 4H), 2.97 (s, 6H), 2.94 (s, 3H), 2.13−2.03 (m, 2H). 13C NMR (101 MHz, MeOD) δ 171.9, 161.1, 157.9, 157.4, 150.8 (d, J = 247.2 Hz), 144.4 (d, J = 16.3 Hz), 139.6, 133.4 (2C), 132.3, 125.8, 122.5, 116.1 (2C), 110.5 (d, J = 22.4 Hz), 106.8 (d, J = 7.9 Hz), 98.2, 55.6, 46.8, 42.4 (2C), 39.8, 34.4, 25.3, 23.5. ESI-HRMS [M + H]+ m/z = 451.2067, calcd for C25H27N4O3F, 451.2124. Purity: 97.0% by HPLC. (E)-6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-(4-propoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (29). Yellow solid, 0.3830 g (yield 85%). 1H NMR (400 MHz, MeOD) δ 8.37 (s, 1H), 7.65−7.53 (m, 3H), 7.18 (d, J = 7.0 Hz, 1H), 6.93 (d, J = 8.2 Hz, 2H), 4.46 (t, J = 6.9 Hz, 2H), 3.96 (t, J = 6.3 Hz, 2H), 3.50− 3.34 (m, 6H), 2.99 (s, 6H), 2.25−2.12 (m, 2H), 1.89−1.77 (m, 2H), 1.08 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 162.1, 157.8 (d, J = 5.4 Hz), 157.6 (d, J = 2.7 Hz), 151.6−146.0 (m), 149.5, 144.3 (d, J = 4.0 Hz), 138.7, 133.0 (2C), 126.8, 123.8, 115.1 (2C), 110.3 (d, J = 17.3 Hz), 106.9 (d, J = 6.9 Hz), 99.9, 69.8, 55.6, 42.5 (2C), 39.8, 34.1, 25.2, 23.5, 22.1, 9.2. ESI-HRMS [M + H]+ m/z = 451.2431, calcd for C26H31N4O2F, 451.2505. Purity: 99.0% by HPLC. (E)-6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-(4-phenoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (30). Yellow solid, 0.2791 g (yield 72%). 1H NMR (400 MHz, MeOD) δ 8.36 (s, 1H), 7.49 (t, J = 9.7 Hz, 3H), 7.43 (t, J = 7.9 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H), 7.11−7.05 (m, 3H), 6.91 (d, J = 8.6 Hz, 2H), 4.45 (t, J = 6.9 Hz, 2H), 3.42−3.31 (m, 6H), 2.96 (s, 6H), 2.22−2.08 (m, 2H). 13 C NMR (101 MHz, MeOD) δ 160.6, 157.5, 157.2 (d, J = 6.1 Hz), 155.2 (d, J = 4.2 Hz), 150.6 (d, J = 245.1 Hz), 146.9, 144.1, 144.0, 137.8, 132.9 (2C), 129.9 (2C), 128.5, 125.1, 124.6, 119.9 (2C), 117.6 (2C), 110.1 (d, J = 20.9 Hz), 106.6 (d, J = 5.7 Hz), 97.7 (d, J = 3.7 Hz), 55.4, 44.7, 42.3 (2C), 39.6, 25.2, 23.2. ESI-HRMS [M + H]+ m/z = 485.2275, calcd for C29H29N4O2F, 485.2344. Purity: 99.8% by HPLC. (E)-6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-(4isopropoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (31). Yellow solid, 0.2920 g (yield 81%). 1H NMR (400 MHz, MeOD) δ 8.39 (s, 1H), 7.70−7.59 (m, 3H), 7.24 (d, J = 7.1 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 4.72 (dt, J = 12.1, 6.0 Hz, 1H), 4.44 (t, J = 7.0 Hz, 2H), 3.47 (t, J = 6.9 Hz, 2H), 3.44−3.34 (m, 4H), 2.98 (s, 6H), 2.26−2.15 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H). 13C NMR (101 MHz, MeOD) δ 161.0, 157.8, 157.4, 150.8 (d, J = 247.4 Hz), 144.3 (d, J = 14.0 Hz), 139.0, 138.3, 133.0 (C), 126.6, 123.5, 116.1 (2C), 110.4 (d, J = 22.3 Hz), 106.8 (d, J = 8.7 Hz), 98.3, 70.4, 55.5, 46.8, 42.4 (2C), 39.8, 25.2, 23.4, 20.8 (2C). ESI-HRMS [M + H]+ m/z = 451.2431, calcd for C26H31N4O2F, 451.2497. Purity: 99.8% by HPLC. (E)-6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-(4-(prop-2yn-1-yloxy)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (32). Milk white solid, 0.3260 g (yield 73%). 1H NMR (400 MHz, DMSO-d6) δ 7.57 (dd, J = 18.0, 10.8 Hz, 4H), 7.07 (dd, J = 16.8, 8.2 Hz, 2H), 6.76 (d, J = 7.8 Hz, 1H), 6.40 (s, 1H), 4.79 (d, J = 10.0 Hz,

2H), 4.13−4.04 (m, 2H), 3.30−3.23 (m, 2H), 3.22−3.16 (m, 2H), 2.49 (s, 1H), 2.36 (t, J = 6.5 Hz, 2H), 2.18 (s, 6H), 1.81−1.72 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.8 (d, J = 3.2 Hz), 158.6 (d, J = 40.9 Hz), 155.8, 150.3 (d, J = 242.1 Hz), 149.0, 143.3 (d, J = 13.8 Hz), 131.7 (2C), 129.5, 128.4, 121.9, 115.6 (2C), 109.5 (d, J = 20.4 Hz), 108.9 (d, J = 7.2 Hz), 105.9 (d, J = 3.9 Hz), 70.5, 63.9, 57.6, 52.5, 45.6, 44.2, 41.7 (2C), 26.5, 25.5. ESI-HRMS [M + K]+ m/z = 485.2118, calcd for C26H27N4O2FK, 485.2343. Purity: 98.2% by HPLC. (E)-3-(3,4-Dihydroxybenzylidene)-6-((3-(dimethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (33). Pale yellow solid, 0.2844 g (yield 67%). 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 11.8 Hz, 1H), 7.49 (s, 1H), 7.09 (s, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.84 (dd, J = 11.3, 8.2 Hz, 2H), 6.61 (s, 1H), 4.16−4.08 (m, 2H), 3.14 (dd, J = 14.9, 7.7 Hz, 4H), 2.74 (s, 6H), 2.06−1.95 (m, 2H), 1.42−1.21 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 160.5 (d, J = 3.4 Hz), 155.5 (d, J = 1.9 Hz), 150.3 (d, J = 121.8 Hz), 149.7, 148.9, 148.8, 143.6 (d, J = 13.8 Hz), 129.7, 129.6, 128.7, 123.0, 112.7, 111.2, 109.7 (d, J = 20.8 Hz), 109.4 (d, J = 7.6 Hz), 105.5 (d, J = 3.8 Hz), 58.3, 45.4, 43.9, 42.8 (2C), 25.8, 25.5. ESIHRMS [M + H]+ m/z = 425.1911, calcd for C23H25N4O3F, 425.1983. Purity: 97.2% by HPLC. (E)-6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-(4-hydroxy3,5-dimethoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (34). Pale yellow solid, 0.2624 g (yield 56%). 1H NMR (400 MHz, MeOD) δ 7.63 (dd, J = 19.7, 12.3 Hz, 2H), 6.86 (d, J = 14.2 Hz, 3H), 4.26−4.15 (m, 2H), 3.92 (d, J = 5.2 Hz, 6H), 3.45 (dd, J = 12.3, 6.0 Hz, 2H), 3.31−3.19 (m, 4H), 2.96 (s, 6H), 2.24−2.12 (m, 2H). 13C NMR (101 MHz, MeOD) δ 160.8, 156.2, 150.6 (d, J = 242.7 Hz), 148.7, 148.3 (2C), 142.9 (d, J = 14.5 Hz), 137.7, 130.5, 129.0, 126.7, 109.2 (d, J = 7.9 Hz), 109.1 (d, J = 5.4 Hz), 108.0 (2C), 105.3 (d, J = 2.9 Hz), 55.9, 55.8, 44.0, 42.4 (2C), 39.6, 24.9, 23.7, 16.8. ESIHRMS [M + H]+ m/z = 469.2173, calcd for C25H29N4O4F, 469.2242. Purity: 99.7% by HPLC. (E)-3-(3,4-Dimethoxybenzylidene)-6-((3-(dimethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (35). Pale yellow solid, 0.3484 g (yield 77%). 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 11.7 Hz, 1H), 7.70 (t, J = 2.5 Hz, 1H), 7.17 (dd, J = 8.4, 1.5 Hz, 1H), 7.08 (d, J = 1.5 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 5.96 (s, 1H), 4.28−4.21 (m, 2H), 3.93 (d, J = 2.9 Hz, 6H), 3.35 (dd, J = 11.5, 6.1 Hz, 2H), 3.29−3.22 (m, 2H), 2.49 (t, J = 6.3 Hz, 2H), 2.30 (s, 6H), 1.88 (p, J = 6.3 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 160.6 (d, J = 3.4 Hz), 155.5 (d, J = 1.9 Hz), 150.3 (t, J = 121.8 Hz), 149.7, 148.9, 148.8, 143.6 (d, J = 13.8 Hz), 129.7, 129.6, 128.7, 123.1, 112.8, 111.2, 109.7 (d, J = 20.8 Hz), 109.4 (d, J = 7.6 Hz), 105.6 (d, J = 3.8 Hz), 58.3, 55.9, 55.9, 45.4, 43.9, 42.8 (2C), 25.8, 25.5. ESI-HRMS [M + H]+ m/z = 453.2224, calcd for C25H29N4O3F, 453.2292. Purity: 99.6% by HPLC. (E)-6-((3-(Dimethylamino)propyl)amino)-7-fluoro-3-(3,4,5trimethoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (36). Pale yellow solid, 0.3281 g (yield 68%). 1H NMR (400 MHz, DMSO-d6) δ 7.71 (s, 1H), 7.60 (d, J = 11.9 Hz, 1H), 6.94−6.83 (m, 3H), 4.14 (t, J = 6.9 Hz, 2H), 3.86 (s, 6H), 3.75 (s, 2H), 3.38− 3.32 (m, 4H), 3.16 (s, 2H), 2.75 (s, 6H), 2.11−1.99 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.6 (d, J = 3.1 Hz), 156.0, 153.6 (2C), 149.9 (t, J = 144.2 Hz), 149.5, 148.5, 142.7 (d, J = 14.1 Hz), 132.2, 131.3, 129.8, 109.8 (d, J = 20.5 Hz), 109.3 (d, J = 7.1 Hz), 108.3 (2C), 105.8 (d, J = 2.4 Hz), 61.0, 60.7, 56.7, 55.2, 44.6, 42.7 (2C), 25.4, 23.5. ESI-HRMS [M + H]+ m/z = 483.2329, calcd for C26H31N4O4F, 483.2399. Purity: 99.7% by HPLC. (E)-3-(4-(Benzyloxy)-2-hydroxybenzylidene)-6-((3(dimethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (37). Pale yellow solid, 0.3805 g (yield 74%). 1 H NMR (400 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.56 (d, J = 11.7 Hz, 1H), 7.50−7.29 (m, 6H), 6.86 (d, J = 7.7 Hz, 1H), 6.64 (d, J = 10.9 Hz, 1H), 6.58 (d, J = 8.5 Hz, 1H), 5.10 (s, 2H), 4.12−4.03 (m, 2H), 3.34 (d, J = 5.9 Hz, 2H), 3.14 (m, 4H), 2.75 (s, 6H), 2.06−1.95 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.4, 159.8 (d, J = 3.0 Hz), 158.5, 153.8 (d, J = 517.8 Hz), 148.9, 142.8, 142.7, 137.3, 130.1, 128.9 (2C), 128.7, 128.4, 128.1 (2C), 123.4, 116.4, 109.6 (d, J = 20.1 Hz), 108.9 (d, J = 7.2 Hz), 106.7, 105.9 (d, J = 3.6 Hz), 102.5, 69.6, 54.9, 6935



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44.2, 42.5 (2C), 25.4, 23.5. ESI-HRMS [M + H]+ m/z = 515.2380, calcd for C30H31N4O3F, 515.2456. Purity: 98.9% by HPLC. (E)-3-(4-(Benzyloxy)-3-hydroxybenzylidene)-6-((3(dimethylamino)propyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (38). Pale yellow solid, 0.2985 g (yield 58%). 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (t, J = 2.6 Hz, 1H), 7.58 (d, J = 11.9 Hz, 1H), 7.48−7.44 (m, 2H), 7.43−7.38 (m, 2H), 7.35 (dt, J = 5.1, 2.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.64 (s, 1H), 6.60 (dd, J = 8.6, 2.5 Hz, 2H), 5.11 (s, 2H), 4.15−4.05 (m, 2H), 3.30−3.22 (m, 2H), 3.19−3.09 (m, 4H), 2.75 (s, 6H), 2.08−1.93 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.5, 159.8 (d, J = 3.0 Hz), 158.5, 153.8 (d, J = 517.8 Hz), 148.9, 142.8, 142.7, 137.3, 130.7, 128.9 (2C), 128.7, 128.4 (2C), 128.1, 123.4, 116.4, 109.6 (d, J = 20.2 Hz), 108.7 (d, J = 7.3 Hz), 106.7, 105.8 (d, J = 3.4 Hz), 102.5, 69.6, 54.9, 44.2, 42.5 (2C), 25.5, 23.5. ESI-HRMS [M + H]+ m/z = 515.2380, calcd for C30H31N4O3F, 515.2458. Purity: 99.4% by HPLC. General Method for the Synthesis of Compounds 39−49. A solution of the intermediate compound 7 (0.3462 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube, and the mixture was heated (100 °C) for 24 h and monitered by TLC. The mixture was cooled to room temperature, filtered, washed with alcohol. And then the crude products were purified by flash column chromatography to afford solid compounds. (E)-7-Fluoro-3-(4-fluorobenzylidene)-6-((3-morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (39). Pale yellow solid, 0.3484 g (yield 77%). 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 11.7 Hz, 1H), 7.73 (t, J = 2.5 Hz, 1H), 7.53 (dd, J = 8.7, 5.4 Hz, 2H), 7.13 (t, J = 8.6 Hz, 2H), 6.80 (d, J = 7.7 Hz, 1H), 6.46 (s, 1H), 4.28−4.22 (m, 2H), 3.85−3.75 (m, 4H), 3.42−3.33 (m, 2H), 3.24 (t, J = 5.9 Hz, 2H), 2.68−2.47 (m, 6H), 1.97−1.87 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 163.9, 161.4, 160.4 (d, J = 3.2 Hz), 155.1, 150.7 (d, J = 243.8 Hz), 148.7, 143.3 (d, J = 13.9 Hz), 131.6, 131.4, 128.4, 116.0, 115.8, 109.8 (d, J = 20.8 Hz), 109.5 (d, J = 7.7 Hz), 105.6 (d, J = 4.0 Hz), 66.9 (2C), 58.1, 53.8 (2C), 43.8, 43.4, 25.5, 24.0. ESIHRMS [M + H]+ m/z = 453.2095, calcd for C25H26N4O2F, 453.2024. Purity: 96.5% by HPLC. (E)-7-Fluoro-6-((3-morpholinopropyl)amino)-3-(4(trifluoromethyl)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (40). Pale yellow solid, 0.3568 g (yield 71%). 1H NMR (400 MHz, CDCl3) δ 7.77−7.71 (m, 2H), 7.67 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.2 Hz, 2H), 6.77 (d, J = 7.6 Hz, 1H), 6.64 (s, 1H), 4.24 (t, J = 7.0 Hz, 2H), 3.84−3.74 (m, 4H), 3.41−3.32 (m, 2H), 3.23 (t, J = 5.5 Hz, 2H), 2.57 (dd, J = 17.1, 11.5 Hz, 6H), 1.96−1.86 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 160.3 (d, J = 3.4 Hz), 154.6, 150.8 (d, J = 244.5 Hz), 148.5, 143.3 (d, J = 14.0 Hz), 138.9, 134.7, 130.7−129.8 (m), 129.6 (2C), 127.8, 125.7 (d, J = 3.7 Hz, 2C), 109.7 (d, J = 20.8 Hz), 109.5 (d, J = 7.7 Hz), 105.5 (d, J = 4.0 Hz), 66.9 (2C), 58.1, 53.8 (2C), 43.8, 43.5, 25.6, 23.8. ESI-HRMS [M + H]+ m/z = 503.2057, calcd for C26H26N4O2F4, 503.1992. Purity: 95.5% by HPLC. (E)-7-Fluoro-3-(4-morpholinobenzylidene)-6-((3-morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (41). Pale yellow solid, 0.3325 g (yield 64%). 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 11.7 Hz, 1H), 7.70 (t, J = 2.4 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 6.79 (d, J = 7.7 Hz, 1H), 6.44 (s, 1H), 4.28−4.21 (m, 2H), 3.91−3.84 (m, 4H), 3.84−3.74 (m, 4H), 3.36 (dd, J = 9.9, 4.9 Hz, 2H), 3.25 (dd, J = 9.6, 4.8 Hz, 6H), 2.68− 2.42 (m, 6H), 1.92 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 160.6 (d, J = 3.3 Hz), 155.8, 151.8, 150.8 (t, J = 121.6 Hz), 148.9, 143.3, 143.1, 134.8, 131.2 (2C), 128.3, 114.8 (2C), 109.7 (d, J = 20.8 Hz), 109.4 (d, J = 7.6 Hz), 105.5 (d, J = 3.8 Hz), 66.9 (2C), 66.7 (2C), 58.0, 53.8 (2C), 48.2 (2C), 43.9, 43.3, 25.6, 24.2. ESI-HRMS [M + H]+ m/z = 520.2718, calcd for C29H34N5O3F, 520.2646. Purity: 98.1% by HPLC. (E)-7-Fluoro-6-((3-morpholinopropyl)amino)-3-(4-(piperidin-1yl)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (42). Pale yellow solid, 0.4090 g (yield 79%). 1H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 11.2 Hz, 1H), 7.64 (s, 1H), 7.42 (d, J = 7.5 Hz, 1H), 6.90 (d, J = 7.9 Hz, 2H), 6.76 (d, J = 7.0 Hz, 1H), 6.39 (s, 1H), 4.27−4.12 (m, 2H), 3.78 (m, 4H), 3.43−3.22 (m, 6H), 3.17 (s, 2H), 2.64−2.41 (m, 6H), 1.89 (s, 2H), 1.67 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 160.6, 155.9, 150.9 (t, J = 134.1 Hz), 149.2, 148.9, 143.1 (d,

J = 12.1 Hz), 131.2 (2C), 129.8, 127.2, 114.9 (2C), 113.2, 109.6 (d, J = 20.1 Hz), 109.2 (d, J = 6.8 Hz), 105.4, 66.8 (2C), 58.0, 53.7 (2C), 49.1 (2C), 48.4, 43.9, 43.4, 25.5 (2C), 24.3, 24.0. ESI-HRMS [M + H]+ m/ z = 518.2909, calcd for C30H36N5O2F, 518.2853. Purity: 98.3% by HPLC. (E)-7-Fluoro-3-(4-isopropylbenzylidene)-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (43). Pale yellow solid, 0.2955 g (yield 62%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 12.3 Hz, 2H), 7.48 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 7.9 Hz, 1H), 6.78 (d, J = 7.7 Hz, 1H), 6.50 (s, 1H), 4.22 (t, J = 7.1 Hz, 2H), 3.78 (t, J = 7.0 Hz, 4H), 3.36 (dd, J = 10.4, 5.2 Hz, 2H), 3.23 (t, J = 6.2 Hz, 2H), 2.95 (dt, J = 13.8, 6.9 Hz, 1H), 2.62−2.48 (m, 6H), 1.91 (t, J = 5.6 Hz, 2H), 1.28 (d, J = 6.9 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 160.5 (d, J = 3.4 Hz), 155.4 (d, J = 1.8 Hz), 150.4 (t, J = 121.8 Hz), 149.9, 148.7, 143.2 (d, J = 13.9 Hz), 133.3, 130.9, 129.8 (2C), 129.7, 126.9 (2C), 109.7 (d, J = 20.8 Hz), 109.5 (d, J = 7.6 Hz), 105.5 (d, J = 3.9 Hz), 66.9 (2C), 58.1, 53.8 (2C), 43.9, 43.4, 34.0, 25.6, 24.0, 23.8 (2C). ESI-HRMS [M + H]+ m/ z = 477.2653, calcd for C28H33N4O2F, 477.2588. Purity: 99.2% by HPLC. (E)-3-(4-(Diethylamino)benzylidene)-7-fluoro-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (44). Pale yellow solid, 0.2427 g (yield 48%). 1H NMR (400 MHz, CDCl3) δ 7.78−7.69 (m, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.1 Hz, 2H), 6.76 (t, J = 7.2 Hz, 1H), 6.68 (t, J = 7.7 Hz, 2H), 6.37 (s, 1H), 4.26−4.12 (m, 2H), 3.78 (d, J = 3.8 Hz, 4H), 3.46− 3.31 (m, 6H), 3.25−3.10 (m, 2H), 2.61−2.44 (m, J = 14.3, 8.6 Hz, 6H), 1.89 (d, J = 5.2 Hz, 2H), 1.20 (dd, J = 11.6, 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.6, 156.3, 150.4 (d, J = 241.0 Hz), 149.0, 147.9, 143.1 (d, J = 13.7 Hz), 131.7 (2C), 130.3, 125.6, 122.8, 111.3 (2C), 109.6 (dd, J = 20.4, 3.3 Hz), 109.2 (d, J = 7.6 Hz), 105.4 (d, J = 3.8 Hz), 66.9 (2C), 58.0, 53.8 (2C), 44.4, 43.9 (2C), 43.4, 25.5, 24.1, 12.6 (2C). ESI-HRMS [M + H]+ m/z = 506.2853, calcd for C29H30N5O2F, 506.2919. Purity: 97.9% by HPLC. (E)-4-((7-Fluoro-6-((3-morpholinopropyl)amino)-9-oxo-1,2dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)benzaldehyde (45). Pale yellow solid, 0.3145 g (yield 68%). 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 7.98 (s, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.79−7.66 (m, 3H), 7.60 (t, J = 7.5 Hz, 1H), 6.77 (d, J = 7.5 Hz, 1H), 6.60 (s, 1H), 4.24 (t, J = 6.9 Hz, 2H), 3.78 (s, 4H), 3.42−3.30 (m, 2H), 3.29−3.20 (m, 2H), 2.66−2.42 (m, 6H), 1.91 (t, J = 4.9 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 191.7, 160.3 (d, J = 3.3 Hz), 154.7, 150.8 (d, J = 244.2 Hz), 148.5, 143.3 (d, J = 14.0 Hz), 135.3 (2C), 134.0, 129.8 (2C), 129.7, 129.6, 127.8, 109.7 (d, J = 20.9 Hz), 109.5 (d, J = 7.8 Hz), 105.5 (d, J = 4.0 Hz), 66.9 (2C), 58.1, 53.8 (2C), 43.9, 43.5, 25.6, 23.9. ESI-HRMS [M + H]+ m/z = 463.2134, calcd for C26H27N4O3F, 463.2067. Purity: 97.9% by HPLC. (E)-7-Fluoro-3-(4-methoxybenzylidene)-6-((3-morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (46). Pale yellow solid, 0.3484 g (yield 75%). 1H NMR (400 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 5.4 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 7.6 Hz, 1H), 6.75 (s, 1H), 4.19−4.10 (m, 2H), 3.96 (dd, J = 12.4, 2.6 Hz, 3H), 3.83 (s, 4H), 3.43 (d, J = 12.0 Hz, 3H), 3.34 (t, J = 6.3 Hz, 2H), 3.27−3.16 (m, 4H), 3.07 (dd, J = 21.0, 9.1 Hz, 3H), 2.53 (d, J = 5.6 Hz, 2H), 2.13−2.01 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 160.5, 158.0, 153.6 (d, J = 337.6 Hz), 149.5, 148.8, 143.3 (d, J = 14.0 Hz), 132.0, 130.1, 128.9 (2C), 124., 120.3, 110.8 (2C), 109.6 (d, J = 20.8 Hz), 109.3 (d, J = 7.7 Hz), 105.7 (d, J = 4.2 Hz), 55.5, 52.7, 46.9 (2C), 43.8, 43.6, 25.7, 25.8, 11.7 (2C). ESI-HRMS [M + H]+ m/z = 465.2296, calcd for C26H29N4O3F, 465.2224. Purity: 95.8% by HPLC. (E)-3-(4-(Benzyloxy)benzylidene)-7-fluoro-6-((3-morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (47). Pale yellow solid, 0.3189 g (yield 59%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 9.8 Hz, 1H), 7.71 (s, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.46−7.30 (m, 5H), 7.03 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 7.6 Hz, 1H), 6.42 (s, 1H), 5.11 (s, 2H), 4.22 (t, J = 6.9 Hz, 2H), 3.78 (s, 4H), 3.42− 3.31 (m, 2H), 3.27−3.17 (m, 2H), 2.62−2.43 (m, 6H), 1.89 (t, J = 4.8 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 160.5 (d, J = 3.4 Hz), 159.2, 155.6 (d, J = 1.8 Hz), 150.6 (d, J = 243.1 Hz), 148.8, 143.2, 143.1, 136.5, 131.3 (2C), 130.3, 129.4, 128.6 (2C), 127.8 (2C), 127.4 (2C), 6936



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(s, 6H), 1.20 (t, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 160.7 (d, J = 11.8 Hz), 148.9, 148.5 (d, J = 213.8 Hz), 142.8 (d, J = 11.8 Hz), 131.8 (2C), 130.4, 125.4, 122.8, 111.3 (2C), 109.9−109.5 (m), 105.9 (d, J = 3.9 Hz), 57.4, 45.1 (2C), 44.4 (2C), 43.9, 40.2, 25.6, 12.6 (2C). ESI-HRMS [M + H]+ m/z = 450.2650, calcd for C26H32N5OF, 450.2591. Purity: 98.8% by HPLC. (E)-3-(4-(Benzyloxy)-2-hydroxybenzylidene)-6-((2(dimethylamino)ethyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (53). Pale yellow solid, 0.3154 g (yield 63%). 1 H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.75 (d, J = 11.4 Hz, 1H), 7.37−7.31 (m, 2H), 7.29−7.28 (m, 4H), 6.83 (d, J = 6.9 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 6.44 (s, 1H), 5.20 (s, 1H), 4.94 (s, 2H), 4.21−4.18 (m, 2H), 3.16 (d, J = 25.2 Hz, 4H), 2.57 (m, 2H), 2.39 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 159.9, 159.3, 157.9, 155.8, 148.5 (d, J = 22.9 Hz), 142.7, 142.6 (d, J = 12.1 Hz), 142.4, 136.8, 129.7, 128.4 (2C), 128.3 (2C), 127.6, 122.9, 115.9, 108.9 (d, J = 25.6 Hz), 108.1 (d, J = 6.8 Hz), 106.2, 105.2, 101.9, 69.1, 50.4, 46.2 (2C), 43.7, 41.1, 24.9. ESI-HRMS [M + H]+ m/z = 501.2224, calcd for C29H29N4O3F, 501.2001. Purity: 98.7% by HPLC. Synthesis of Compounds 54−56. (E)-3-(4-(Benzyloxy)-2hydroxybenzylidene)-7-fluoro-6-((3-(pyrrolidin-1-yl)propyl)amino)2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (54). A solution of the intermediate compound 9 (0.3304 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube and heated (100 °C) for 24 h followed by TLC. The solution was cooled to room temperature, filtered, and washed with alcohol. And then the crude product was purified by using flash column chromatography to afford solid compound. Pale yellow solid, 0.2595 g (yield 48%). 1H NMR (400 MHz, DMSO-d6) δ 7.96 (t, J = 2.7 Hz, 1H), 7.55 (d, J = 11.9 Hz, 1H), 7.47−7.44 (m, 2H), 7.43−7.38 (m, 2H), 7.35 (dt, J = 5.2, 2.1 Hz, 1H), 7.30 (s, 1H), 6.87−6.82 (m, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 5.11 (s, 2H), 4.10−4.05 (m, 2H), 3.29−3.24 (m, 4H), 3.18−3.12 (m, 2H), 2.49−2.45 (m, 4H), 1.75 (dq, J = 13.1, 6.4 Hz, 2H), 1.03− 0.89 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 159.9, 159.3, 157.9, 155.8, 148.5 (d, J = 21.9 Hz), 142.6, 142.5 (d, J = 12.2 Hz), 142.4, 136.8, 129.7 (2C), 128.4, 128.3 (2C), 127.8, 127.6, 122.9, 115.9, 108.9 (d, J = 24.6 Hz), 108.1 (d, J = 6.6 Hz), 106.1, 105.2, 101.9, 69.1, 50.4, 46.3 (2C), 43.7, 41.1, 24.9, 24.3, 10.8 (2C). ESI-HRMS [M + H]+ m/z = 541.2537, calcd for C32H33N4O3F, 541.2611. Purity: 98.1% by HPLC. (E)-3-(4-(Benzyloxy)-2-hydroxybenzylidene)-7-fluoro-6-((2-(piperidin-1-yl)ethyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (55). A solution of the intermediate compound 10 (0.3304 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube, and the mixture was heated (100 °C) for 24 h and monitored by TLC. The mixture was cooled to room temperature, filtered, washed with alcohol. And then the crude product was purified by using flash column chromatography to afford solid compound. Pale yellow solid, 0.2865 g (yield 53%). 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.76 (d, J = 11.3 Hz, 1H), 7.46−7.33 (m, 2H), 7.33−7.26 (m, 3H), 6.83 (d, J = 6.9 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 6.44 (s, 1H), 5.20 (s, 1H), 4.94 (s, 2H), 4.25−4.13 (m, 2H), 3.16 (d, J = 26.2 Hz, 4H), 2.57 (s, 2H), 2.39 (s, 4H), 1.57 (s, 4H), 1.43 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 160.9, 160.3, 156.6, 155.0 (d, J = 111.1 Hz), 151.6, 143.1 (d, J = 13.7 Hz), 136.6, 129.9, 128.5 (2C), 127.9, 127.1 (2C), 125.6, 116.9, 110.0 (d, J = 21.2 Hz), 107.6, 105.4 (d, J = 8.3 Hz), 102.9, 100.0, 70.0, 56.6, 54.2 (2C), 44.1, 39.5, 25.8 (2C), 25.5, 24.2. ESI-HRMS [M + H]+ m/z = 541.2537, calcd for C32H33N4O3F, 541.2606. Purity: 98.9% by HPLC. (E)-3-(4-(Benzyloxy)-2-hydroxybenzylidene)-7-fluoro-6-morpholino-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (56). A solution of the intermediate compound 11 (0.2893 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube, and the mixture was heated (100 °C) for 24 h and monitoredby TLC. The mixture was cooled to room temperature, filtered, washed with alcohol. And then the crude product was purified by flash column chromatography to afford solid compound. Pale yellow solid, 0.3147 g (yield 63%). 1H NMR (400

115.2, 109.7 (d, J = 20.9 Hz), 109.5 (d, J = 7.9 Hz), 105.6 (d, J = 3.7 Hz), 70.1 (2C), 53.7 (2C), 50.9, 43.9, 43.2, 29.7, 25.5, 24.2. ESIHRMS [M + H]+ m/z = 541.2607, calcd for C32H33N4O3F, 541.2537. Purity: 95.4% by HPLC. (E)-7-Fluoro-6-((3-morpholinopropyl)amino)-3-(3,4,5trimethoxybenzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (48). Pale yellow solid, 0.4092 g (yield 78%). 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 11.7 Hz, 1H), 7.61 (t, J = 2.5 Hz, 1H), 6.71 (s, 2H), 6.42 (s, 1H), 4.22−4.13 (m, 2H), 3.84 (s, 9H), 3.71 (t, J = 4.5 Hz, 4H), 3.28 (dd, J = 10.8, 5.7 Hz, 2H), 3.22−3.15 (m, 2H), 2.54−2.48 (m, 2H), 2.45 (s, 2H), 1.83 (dt, J = 11.9, 6.0 Hz, 2H), 1.73 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 160.5 (d, J = 3.3 Hz), 155.2 (d, J = 1.5 Hz), 153.3 (2C), 150.7 (d, J = 244.0 Hz), 148.7, 143.3, 143.2, 138.8, 131.2, 130.9, 129.8, 109.7 (d, J = 20.8 Hz), 109.4 (d, J = 7.7 Hz), 107.0 (2C), 105.4 (d, J = 4.0 Hz), 66.8 (2C), 61.0, 58.1, 56.2 (2C), 53.7 (2C), 43.9, 43.4, 25.4, 23.9. ESI-HRMS [M + H]+ m/z = 525.2512, calcd for C28H33N4O5F, 525.2435. Purity: 98.7% by HPLC. (E)-3-(4-(Benzyloxy)-2-hydroxybenzylidene)-7-fluoro-6-((3morpholinopropyl)amino)-2,3-dihydropyrrolo[2,1-b]quinazolin9(1H)-one (49). Pale yellow solid, 0.2785 g (yield 50%). 1H NMR (400 MHz, MeOD) δ 8.41 (s, 1H), 7.62 (dd, J = 8.3 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.37−7.17 (m, 7H), 6.98 (t, J = 6.2 Hz, 1H), 6.56 (d, J = 8.5 Hz, 1H), 6.50 (s, 1H), 5.04 (s, 1H), 4.00−3.90 (m, 4H), 3.90− 3.77 (m, 4H), 3.43 (dd, J = 14.7, 8.1 Hz, 7H), 3.30−3.23 (m, 5H), 3.17−2.97 (m, 5H), 2.22−2.06 (m, 4H). 13C NMR (101 MHz, MeOD) δ 163.5, 157.8 (d, J = 57.8 Hz), 157.5, 144.4 (d, J = 14.0 Hz), 136.6, 134.0, 131.1, 128.2 (2C), 127.7, 127.1 (2C), 121.9, 115.1, 110.5 (d, J = 22.1 Hz), 108.9 (d, J = 99.2 Hz), 107.6, 106.8, 102.3, 101.9, 98.1, 98.1, 70.0, 63.6 (2C), 54.9, 52.0 (2C), 46.5, 39.8, 25.6, 22.5. ESIHRMS [M + H]+ m/z = 557.2486, calcd for C32H33N4O4F, 557.2559. Purity: 98.5% by HPLC. General Method for the Synthesis of Compounds 50−53. A solution of the intermediate compound 8 (0.2903 g, 1 mmol) and aldehydes (1.1 mmol) in 2 mL of DMSO, 10 mL of trimethylchlorosilane was introduced into a 20 mL sealed tube, and the mixture was heated (100 °C) for 24 h and minitored by TLC. The mixture was cooled to room temperature, filtered, washed with alcohol. And then the crude product was purified by flash column chromatography to afford solid compounds. (E)-4-((6-((2-(Dimethylamino)ethyl)amino)-7-fluoro-9-oxo-1,2dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)benzaldehyde (50). Pale yellow solid, 0.2886 g (yield 71%). 1H NMR (400 MHz, CDCl3) δ 10.05 (s, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.83− 7.77 (m, 2H), 7.70 (d, J = 8.1 Hz, 2H), 6.83 (d, J = 7.7 Hz, 1H), 4.28 (t, J = 7.1 Hz, 2H), 3.37−3.26 (m, 4H), 2.65 (t, J = 5.9 Hz, 2H), 2.30 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 191.5, 160.3, 153.3 (d, J = 271.8 Hz), 149.5, 148.4, 143.1 (d, J = 13.8 Hz), 141.4, 135.7, 135.4, 130.1, 130.0, 128.3 (2C), 110.1−109.8 (m), 106.2 (d, J = 3.8 Hz), 99.9, 57.3, 45.1 (2C), 43.9, 40.2, 25.9. ESI-HRMS [M + H]+ m/z = 407.1805, calcd for C23H23N4O2F, 407.1882. Purity: 98.1% by HPLC. (E)-6-((2-(Dimethylamino)ethyl)amino)-7-fluoro-3-(4-(prop-2-yn1-yloxy)benzylidene)-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (51). Pale yellow solid, 0.2811 g (yield 65%). 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 11.6 Hz, 1H), 7.71 (s, 1H), 7.51 (dd, J = 8.5, 4.8 Hz, 2H), 7.04 (d, J = 8.7 Hz, 1H), 6.98 (dd, J = 8.5, 5.5 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 5.18 (s, 1H), 4.69 (d, J = 43.9 Hz, 2H), 4.24 (t, J = 7.2 Hz, 2H), 3.26 (dt, J = 15.5, 5.7 Hz, 4H), 2.63 (t, J = 5.9 Hz, 2H), 2.29 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 159.8 (d, J = 3.2 Hz), 158.6 (d, J = 40.9 Hz), 155.9, 150.4 (d, J = 244.2 Hz), 149.0, 143.3 (d, J = 12.9 Hz), 131.6 (2C), 129.5, 128.4, 121.9, 115.6 (2C), 109.4 (d, J = 20.1 Hz), 108.9 (d, J = 7.3 Hz), 105.9 (d, J = 4.0 Hz), 70.5, 63.9, 57.6, 52.5, 45.6 (2C), 44.2, 41.7, 26.5. ESI-HRMS [M + H]+ m/z = 433.1962, calcd for C25H25N4O2F, 433.2039. Purity: 99.8% by HPLC. (E)-3-(4-(Diethylamino)benzylidene)-6-((2-(dimethylamino)ethyl)amino)-7-fluoro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)one (52). Pale yellow solid, 0.2427 g (yield 54%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 11.7 Hz, 1H), 7.66 (s, 1H), 7.44 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 7.7 Hz, 1H), 6.69 (d, J = 8.6 Hz, 2H), 5.13 (s, 1H), 4.21 (t, J = 7.2 Hz, 2H), 3.40 (q, J = 6.8 Hz, 4H), 3.28 (dd, J = 10.5, 5.2 Hz, 2H), 3.23−3.15 (m, 2H), 2.63 (t, J = 5.8 Hz, 2H), 2.29 6937



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MHz, DMSO-d6) δ 8.13 (s, 1H), 7.61 (d, J = 11.5 Hz, 1H), 7.50−7.43 (m, 3H), 7.41 (dd, J = 7.1, 5.6 Hz, 2H), 7.35 (dt, J = 5.2, 2.1 Hz, 1H), 6.97 (d, J = 7.7 Hz, 1H), 6.65 (d, J = 2.4 Hz, 1H), 6.61 (dd, J = 8.7, 2.4 Hz, 1H), 5.12 (s, 2H), 4.18−4.08 (m, 2H), 3.95 (d, J = 12.4 Hz, 2H), 3.81 (t, J = 11.7 Hz, 2H), 3.19 (d, J = 5.0 Hz, 4H), 3.06 (dd, J = 20.9, 9.1 Hz, 2H). 13C NMR (101 MHz, MeOD) δ 163.5, δ 157.7 (d, J = 58.8 Hz), 157.5, 144.3 (d, J = 14.2 Hz), 136.6, 134.0, 131.0 (2C), 128.2, 127.7 (2C), 127.1, 121.9, 115.1, 110.5 (d, J = 21.6 Hz), 108.9 (d, J = 94.2 Hz), 107.7, 106.8, 102.3, 101.9, 98.1, 98.1, 70.0, 63.6 (2C), 52.0 (2C), 46.6, 25.6. ESI-HRMS [M + H]+ m/z = 500.1907, calcd for C29H26N3O4F, 500.1968. Purity: 98.8% by HPLC. Biological Assay. Surface Plasmon Resonance. SPR measurements were performed on a ProteOn XPR36 protein interaction array system (Bio-Rad Laboratories, Hercules, CA) using a GLH sensor chip. In a typical experiment, the chips were activated simultaneously by injecting 200 μL of a freshly mixed solution of EDAC and SulfoNHS. Immediately after activation, 180 μL of NM23-H2 protein (100 μg/mL in sodium acetate) or streptavidin (100 μg/mL in sodium acetate) solution was injected into the channels. For deactivation, 150 μL of ethanolamine hydrochloride (1 M, pH 8.5) was injected into the relevant channels simultaneously. 100 μL of the biotin labeled oligonucleotide was injected into the streptavidin channels. The protein was immobilized (∼12 000 RU) in channel 1, leaving channel 2 as a blank; the DNA samples were then captured (∼14 000 RU) in channels 3 and 4, leaving the fifth channel as a blank. Ligand solutions (at 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM) were prepared with running buffer by serial dilutions from stock solutions. NM23-H2 protein, biotinylated duplex DNA, and biotinylated pu22 were folded in filtered and degassed running buffer (10 mM HEPES, 150 mM NaCl, 1.65 mM EDTA, 0.5 mM MgCl2, pH 7.4, 0.005% Tween-20 for protein and 50 mM Tris-HCl, 100 mM KCl, pH 7.4, 0.005% Tween-20 for DNA). The ligand was injected simultaneously at a flow rate of 25 μL/min for 200−300 s during the association phase, which was followed by a 400 s dissociation phase at 25 °C. The GLH chip was regenerated with a short injection of 1 M NaCl between consecutive measurements. The final graphs were obtained by subtracting blank sensorgrams from the NM23-H2 protein, duplex or quadruplex sensorgrams. Data were analyzed with ProteOn manager software, using the Langmuir model for fitting kinetic data. Microscale Thermophoresis Experiments. Proteins were Nterminally labeled for microscale thermophoresis (MST) using the Monolith NT protein labeling kit RED-NHS (NanoTemper Technologies, München, Germany), according to the instructions of the manufacturer. Briefly, NM23-H2 proteins at concentrations of 20 μM were incubated with 2× dye at a ratio of 1:1 in labeling buffer (50 mM HEPES, pH 6.0, 150 mM NaCl, 50 mM MgCl2) in the dark at room temperature for 30 min. Free dye was removed using gel filtration columns (manufacturer supplied), and the protein eluted in 0.5 mL of binding buffer (25 mM Tris-HCl, pH 8.0, 300 mM NaCl). MST experiments were carried out on a blue/red Monolith NT.115 (NanoTemper Technologies) using the red filter set. To determine the KD values of NM23-H2 protein and compounds, 200 nM NT-647NHS labeled NM23-H2 proteins was incubated with increasing concentrations of compounds (initially 500 μM and half-diluted 15 times) for 30 min at room temperature in binding buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20). The samples were then centrifuged (10 000g, 1 min) to remove any aggregates before being loaded into microscale thermophoresis (MST) grade glass capillaries. Binding data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA) to determine the KD values, and the KD is the numeric equivalent of the concentration of compounds when the response is half of the plateau response (Rmax) on the fiting curve. Enzyme-Linked Immunosorbent Assays. The streptaWell High Bind plates (Roche) were used in the ELISA assays. ELISAs for binding affinity and specificity were performed using standard methods. Briefly, biotinylated pu22 was bound to streptavidin-coated plates followed by incubation with the protein samples at 37 °C for 3 h. To test the compounds’ inhibitory activity for pu22 and NM23-H2 protein, the protein samples were prepared in advance: the

concentration of NM23-H2 protein was initially 4000 nM and halfdiluted nine times in blocking buffer. After addition of DMSO or 20 μM compounds, the samples were incubated at 25 °C for 1 h. Then, detection was achieved with a primary antibody (SantaCruz, sc100400) at 4 °C overnight, secondary HRP-conjugated antibody (CST, 7076s) for 2 h at 37 °C, and TMB (3,3′,5,5′-tetramethylbenzidine, HRP substrate, Life Technologies). Signal intensity was measured at 450 nm on a monochromators based multimode microplate reader (INFINITE M1000). The fitting curve was obtained using GraphPad Prism 5 via Hill fiting, and the KD is the numeric equivalent of the concentration of NM23-H2 when the absorbance is half of the plateau absorbance (Amax) on the fiting curve. G-quadruplex oligonucleotide pu22 was annealed in Tris-HCl buffer (10 mM, pH 7.4) containing 100 mM KCl by slow cooling from 95 °C to room temperature. Molecular Modeling Studies. The structure of human NM23-H2 protein was generated based on their X-ray structures (PDB code 3BBB) using Protein Preparation Wizard in Schrodinger Maestro 9.3 (Schrodinger, LLC, New York, NY, USA). The residues were corrected for physiological pH. The ligand structure compound 37 obtained was prepared using LigPrep module (Maestro 9.3) by submission of ligands in SDF format as an input. Docking analyses were performed with the prepared compound 37 and NM23-H2 structure using the Glide module (Maestro 9.3). The enclosing box center of the docking site was placed in the centroid of the original ligand in chain E7, and the dimensions of the box were set to 20 Å × 20 Å× 20 Å. All other parameters accepted default settings. The docking results were visualized using the Discovery Studio 2.5 client software package. Cell Culture and Treatments. Human epithelial cervical cancer cell line HeLa, human cervical squamous cancer cell line SiHa, human ovarian cancer cell line A2780, non-small-cell lung cancer cell line A549, and primary cultured mouse mesangial cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), at 37 °C humidified atmosphere with 5% CO2. Human malignant myeloid cell lines K562, human acute leukemic cell lines HL-60, malignant lymphoma cell lines RAJI were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), at 37 °C humidified atmosphere with 5% CO2. For the drug treatment experiments, the compound was added to cells at 24 h after plating and the cells were harvested for various experiments after 3−72 h of treatment. MTT Assay. The antiproliferative and cytotoxic effects of the target compound on human cancer cell lines and normal human cells, respectively, were examined by MTT (3-(4,5-dimethyl-2-thiazolyl)2,5- diphenyl-2H-tetrazolium bromide) assay. Briefly, when the cells reached the logarithmic phase, 5000 cells/well were seeded into 96well plates. 24 h after being seeded, the cells were treated with different concentrations of the test compounds for 48 h; each experiment was performed in triplicate. Afterward, MTT (Sigma, USA) was added to each well with final concentration of 0.5 mg/mL, and the cells were incubated for another 4 h. Then, the medium was discarded and 100 μL of DMSO was added to each well to dissolve the blue crystal. The absorbance at 570 nm was measured by a microplate reader (BioTek, USA), and cell viability after drug intervention was normalized with vehicle treated. The IC50 (50% growth inhibitory concentration) values were calculated with GraphPad Prism version 5.0. RNA Extraction and RT-PCR. RNA was extracted using TRIzol reagent (BioTeke Corporation) as per the manufacturer’s protocol. RNA was quantitated spectrophotometrically. For cDNA synthesis, 1.5 μg of total RNA was used as a template for reverse transcription using M-MLV RTase (Takara). 1.5 μg of total RNA, 2.0 μL of Oligo(dT)18 primer (50 μM), and added EDPC water to 15 μL were heated at 70 °C for 10 min and placed in ice hold at 4 °C. To the solution was added 10 μL of a mix containing (final concentrations) the following: 1× reverse transcriptase M-MLV buffer; 0.2 μM dNTP mixture; 8 U/ μL of M-MLV reverse transcriptase; 1 U/μL of recombinant RNase inhibitor. The reactions were incubated for 1 h at 42 °C and stopped by heating at 70 °C for 15 min, finally holding at 4 °C. Store the cDNA samples at −20 °C, or proceed directly to PCR reactions. 6938



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The primers used in the PCR were listed in Supporting Information Table S2. The PCR reactions were performed with 2.5 μL of 10× Dream Taq Green buffer, 0.5 μL of dNTP mixture (2.5 μM), 1.0 μL of cDNA mixture, 0.5 μL of primer sense (10 μM), 0.5 μL of primer antisense (10 μM), 0.25 μL of Dream TaqDNA polymerase, and added water to 25 μL. The PCR cycle was 5 min at 95 °C, 30 cycles 30 s at 95 °C, 30 s at 58 °C, 60 s at 72 °C and stopped by heating at 72 °C for 10 min, finally holding at 10 °C. The amplified products were also separated on a 1.5% agarose gel, and photos were taken on a Gel Doc 2000 imager system. All the above experiments were performed at least three times across multiple repeats of the treatments. Western Blot Assay. SiHa cells (2.0 × 105 cells) were incubated in six-well plates with or without compound 37 at various concentrations for 24 or 48 h. After incubation, cells were washed once with PBS and lysed with extraction buffer (50 mM glucose, 25 mM Tris-HCl, pH 8, 10 mM EDTA, 1 mM PMSF) at 4 °C for 30 min, intermittently vortexed every 5 min, and centrifuged at 15 000 rpm at 4 °C for 15 min to harvest the supernatant. The supernatant was transferred into another tube, and the concentration of protein was calculated via a BCA protein assay kit (Thermo Fisher Scientific). The protein extracts were reconstituted in loading buffer containing 50 mM Tris-HCl, pH 6.8, 6 M urea, 6% 2-mercaptoethanol, 3% SDS, 0.003% bromophenol blue, and the mixture was boiled at 95 °C for 5 min. An equal amount of the proteins (30 μg) were electrophoresed on 10% SDS−PAGE and transferred to a nitrocellulose membrane at 80 V for 2 h. The membranes were blocked for 1 h with 5% solution nonfat dry milk in TBS, 1% Tween-20 at room temperature. Membranes with the samples were overnight incubated at 4 °C with primary antibodies. After three washes in TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 2 h. For the detection of the proteins we used Super SignalWest PICO and FEMTO (Thermo Fisher Scientific Pierce). Exposure length depends on the antibodies used. Primary antibodies used for Western blot in this study were β-actin (Cell Signaling Technology, 4970S), NM23-H2 (Santa Cruz, sc-100400), caspase-3 (Cell Signaling Technology, 9665S), PARP (Cell Signaling Technology, 9542S), Cyclin D1(Cell Signaling Technology, 2922S), and c-MYC (abcam, ab32072). Secondary antibodies used were horseradish peroxidase-conjugated anti-mouse (Cell Signaling Technology, 7076S) or anti-rabbit (Cell Signaling Technology, 7074S). Chromatin Immunoprecipitation. Chromatin immunoprecipitation (ChIP) experiments were performed using a Pierce Agarose ChIP kit (Thermo Fisher Scientific 26156) according to the manufacturer’s protocol. After 9 h of incubation with or without compound 37 at various concentrations, antibody against the NM23H2 epitope (3 μg, Santa Cruz, sc-100400) was used to immunoprecipitate chromatin in Siha cells. Normal rabbit IgG (SantaCruz, sc-3888) was used as the negative control. ChIP was performed overnight at 4 °C, and immune complexes were collected using the Protein A/G Plus Agarose in the kit and washed extensively. The DNA was extracted from immunoprecipitated chromatin and amplifid by PCR using c-MYC-ChIP-A and c-MYC-ChIP-S primers (Supporting Information Table S2). The amplified products were also separated on a 1.5% agarose gel, and photos were taken on a Gel Doc 2000 imager system. Cell Cycle Analysis. For flow cytometric analysis of DNA content, SiHa cells were plated in six-well plates (2.0 × 105 cells) and incubated with or without compound 37 at various concentrations for 3, 6, 9, 12, 24, 36, 48 h. At the end of the treatment, the cells were collected and washed twice with PBS. Next, the cells were harvested and fixed in icecold 70% ethanol overnight. The cells were pelleted and resuspended in 1 mL of PBS containing 20 μg/mL of RNase A at 37 °C for 30 min. The cells were then incubated with the DNA staining solution PI solution, and flow cytometry analysis was carried out using a Epics Elite flow cytometry (Beckman Coulter, Epics XL) at 488 nm. The data were analyzed by EXPO32 ADC analysis software. Apoptosis Analysis. For flow cytometric analysis of DNA content, SiHa cells were plated in six-well plates (2.0 × 105 cells) and incubated with or without compound 37 at various concentrations for 24 or 48 h to induce cell apoptosis. Apoptosis detection was

performed using the FITC annexin V/PI apoptosis detection kit (Beyotime, China) according to the manufacturer’s instructions. At the end of the treatment, the cells were collected and washed twice with PBS. FITC annexin V (5 μL) and PI (10 μL) were mixed in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2 at pH 7.4), added, and incubated for 30 min at room temperature in the dark. And flow cytometry analysis was carried out using an Epics Elite flow cytometry instrument (Beckman Coulter, Epics XL) at 488 nm. The data were analyzed by EXPO32 ADC analysis software. Real-Time Cellular Activity Assay. Compound-mediated cytotoxicity was monitored using a real-time cell analyzer multiplate (RTCA) instrument, xCELLigence RTCA DP instrument (Roche Applied Science, Indianapolis, IN), which was placed in a humidified incubator at 37 °C and 5% CO2. This system monitors cellular events (cell viability, cell number, cell morphology, and degree of adhesion) in real time, measuring electrical impedance across interdigitated microelectrodes integrated on the bottom of tissue culture E-Plates 16well plates (Roche Applied Science, Indianapolis, IN). The evaluation of cellular events was performed on SiHa and primary cultured mouse mesangial cell lines according to previous descriptions. For time-dependent cell response profiling, 50 μL of medium was added to 16-well electric plates (E-Plates) to obtain background readings followed by the addition of 100 μL of cell suspension at the density of 20 000 cells/mL. After seeding, cells were allowed to settle for 20 min at room temperature before being inserted into the xCELLigence RTCA DP instrument. After 24 h, the cells were treated with or without compound 37 at various concentrations. The cells were monitored every 2 min for duration of 2 h after compound addition to capture the short-term response and for every 15 min from 2 h after compound addition until the end of the treatment to capture the long-term response. For siRNA interference, after 24 h precultured, SiHa cells were transfected with 30 nM NM23-H2 siRNA for 6 h, then different concentrations of compound 37 were added, and impedance was continuously measured until the end of the treatment. The amount of DMSO was kept constant between the individual conditions. Results were plotted using the RTCA software 1.2 (Roche Applied Science, Indianapolis, IN). The data expressed in cell index (CI) units were exported to Microsoft Excel software (Microsoft, Redmond, WA) for mathematical analysis and normalization. The data were normalized to a starting CI of 1.0 at the time point immediately prior to compound addition. RNAi Knockdown. NM23-H2-specific siRNA (Supporting Information Table S2) and siNO581522147 negative control siRNAs were purchased from Guangzhou RiboBio Co., Ltd. Transfection of siRNA duplexes was accomplished using Lipo3000 (Invitrogen) based on the manufacturer’s protocol. Cells were incubated with siRNA for 72 h. Cells were harvested for various experiments 24, 48, 72 h after transfection with the siRNA. In the RTCA experiment, cells were incubated with or without compound 37 at various concentrations after the siRNA incubated at 24 h. In Vitro Wound Scrape Assay. SiHa cervical squamous cancer cells of each group were incubated in 12-well plates. A small wound area was made in the confluent monolayer with a 200 μL pipet tip in a lengthwise stripe. Cells were then washed twice with PBS and exposed to 37 at final concentrations of 2, 1, 0.5, 0.25 μM and DMSO control treatment in serum-free DMEM medium at 37 °C in a 5% CO2 incubator. Images were captured at different times from 0 to 96 h. Wound width was measured at a ×100 magnification using a FL Auto Imaging System EVOS microscope (Life Technologies). Ten measurements were determined at random intervals along the wound length using Image Pro6. This experiment was carried out in triplicate. Colony Formation Assay. SiHa cervical squamous cancer cells and primary cultured mouse mesangial cells were seeded on six-well plates (300/well) and exposed to 37 at final concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125 μM and DMSO control treatment at 37 °C in a 5% CO2 incubator. DMEM was replaced, and different concentrations of 37 were added every 2 days. Cells were fixed with methanol and dyed with crystal violet after cultured 14 days. 6939



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Evaluation of in Vivo Antitumor Activity. Female BALB/c nude mice (5 weeks old) were purchased from and housed at the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China) and maintained in pathogen-free conditions (12 h light−dark cycle at 24 ± 1 °C with 60−70% humidity and provided with food and water ad libitum). SiHa cells were harvested during log-phase growth and were resuspended in FBS-free DMEM medium at 8 × 107 cells/ mL. Each mouse was injected subcutaneously in the right flank with 1 × 107 cells with a 22-gauge needle. Tumor growth was examined twice a week after implantation until the tumor volume reached approximately 50 mm3. The volume of the tumor mass was measured with an electronic caliper and calculated as 1/2 × length × width2 in mm3. The mice were randomly divided into four groups of eight animals and treated intraperitoneally (ip) with various regimens every other day for the entire period of observation (3 weeks). Mice in the control group were treated with an equivalent volume of vehicle, the 37treated group at a dose of 2.5 or 5 mg/kg body weight, and the doxorubicin-treated group at a dose of 1 mg/kg. The tumor size and the body weight of the mice were measured every day after drug treatment, and growth curves were plotted using average tumor volume within each experimental group. At the end of the observation period, the animals were euthanized by cervical dislocation, and the tumors were removed and weighed. The rate of inhibition (IR) was calculated according to the formula: IR = (1 − Mean tumor weight of the experimental group/Mean tumor weight of the control group) × 100%.

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Provincial Key Laboratory of Construction Foundation (Grant 2011A060901014) for financial support of this study.

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ABBREVIATIONS USED c-caspase 3, cleaved caspase 3; c-PARP, cleaved PARP; SPR, surface plasmon resonance; CHIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunesorbent assay; FRET, fluorescence resonance energy transfer; MST, microscale thermophoresis; NHE III1, nuclease hypersensitive element; RTCA, real-time cellular analysis; SAR, structure−activity relationship

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00421. Additional experimental results, 1H and 13C NMR spectra, HRMS and HPLC assay data for final compounds (PDF) Molecular formula strings and some data (CSV) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*J.-H.T.: phone, 8620-39943053; e-mail, [email protected]. edu.cn. *Z.-S.H.: phone, 8620-39943056; e-mail, [email protected]. edu.cn. ORCID

Tian-Miao Ou: 0000-0002-8176-4576 Zhi-Shu Huang: 0000-0002-6211-5482 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Natural Science Foundation of China (Grants 81330077 and 21172272 for Z.-S.H., Grant 21672268 for J.H.T., and Grant 21472252 for D.L.), the Guangdong Provincial Science and Technology Development Special Foundation (Public Interest Research and Capacity Building) (Grant 2016A020217004 for Z.-S.H.), the Guangdong Provincial Natural Science Foundation for Distinguished Young Scholars (Grant 2015A030306004 for J.-H.T.), and Guangdong 6940



Article

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Design, Synthesis, and Evaluation of New Selective NM23-H2 Binders

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