Article pubs.acs.org/jmc
New Disubstituted Quindoline Derivatives Inhibiting Burkitt’s Lymphoma Cell Proliferation by Impeding c‑MYC Transcription Hui-Yun Liu,† Ai-Chun Chen,† Qi-Kun Yin, Zeng Li, Su-Mei Huang, Gang Du, Jin-Hui He, Li-Peng Zan, Shi-Ke Wang, Yao-Hao Xu, Jia-Heng Tan, Tian-Miao Ou, Ding Li, Lian-Quan Gu, and Zhi-Shu Huang* Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China S Supporting Information *
ABSTRACT: The c-MYC oncogene is overactivated during Burkitt’s lymphoma pathogenesis. Targeting c-MYC to inhibit its transcriptional activity has emerged as an effective anticancer strategy. We synthesized four series of disubstituted quindoline derivatives by introducing the second cationic amino side chain and 5-N-methyl group based on a previous study of SYUIQ-5 (1) as c-MYC promoter G-quadruplex ligands. The in vitro evaluations showed that all new compounds exhibited higher stabilities and binding affinities, and most of them had better selectivity (over duplex DNA) for the c-MYC G-quadruplex compared to 1. Moreover, the new ligands prevented NM23-H2, a transcription factor, from effectively binding to the c-MYC G-quadruplex. Further studies showed that the selected ligand, 7a4, down-regulated c-MYC transcription by targeting promoter G-quadruplex and disrupting the NM23-H2/c-MYC interaction in RAJI cells. 7a4 could inhibit Burkitt’s lymphoma cell proliferation through cell cycle arrest and apoptosis and suppress tumor growth in a human Burkitt’s lymphoma xenograft.
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INTRODUCTION The c-MYC oncoprotein is a transcription factor that plays essential roles in controlling cell proliferation, growth, and apoptosis and in triggering carcinogenic progression.1,2 Deregulated c-MYC expression is associated with multiple human cancers, including Burkitt’s lymphoma. The c-MYC protein is significantly stabilized in several Burkitt’s lymphomaderived cell lines, and it contributes to the pathogenesis of Burkitt’s lymphoma.3 The validity of c-MYC as a molecular target in oncology has been demonstrated.4 Strategies that aim to target c-MYC, including those that interfere with c-MYC synthesis, stability, and transcriptional activity, have emerged as effective anticancer therapies.5 The transcriptional regulation of c-MYC expression is complex and involves multiple promoters and transcriptional start sites.6 The nuclease hypersensitive element III1 (NHE III1) is a highly conserved polypurine/polypyrimidine tract that consists of a 27-bp sequence (Pu27 and Py27) that is located 115−142 base pairs upstream of the P1 promoter, and it is required for 80−95% of c-MYC transcription. The NHE III1 is guanine-rich, and it folds into a specific DNA secondary structure, known as the G-quadruplex, under physiological conditions.7−9 The formation of the DNA G-quadruplex structure to repress c-MYC transcription has been well established.10,11 These non-B-DNA structures lead to chromatin unfolding and are substrates for transcription factors that cooperate to form a docking platform to assemble the transcriptionally engaged RNA polymerase II.12 Multiple © 2017 American Chemical Society
human protein factors bind to the c-MYC G-quadruplex structure and modulate gene expression in vitro and in vivo, including the NM23-H2 metastasis suppressor protein,7,13−15 cellular nucleic acid binding protein (CNBP),16 heterogeneous ribonucleoprotein particle proteins (hnRNP) A1, A2, B1, and K,17 nuclease-sensitive element protein 1 (NSEP-1),18 and specificity protein 1 (Sp1),19 among others. Because NM23-H2 activates the transcription of c-MYC gene by binding to singlestranded DNA within the c-MYC NHE III1 promoter, blocking the interaction between NM23-H2 and c-MYC NHE III1 to inhibit c-MYC transcription is a promising cancer treatment strategy.10,20,21 Stabilization of the G-quadruplex or i-motif structures that form within the c-MYC NHE III1 promoter prevents NM23H2 from activating c-MYC gene expression.20 Thus, regulation of the c-MYC G-quadruplex structural equilibrium with small molecules, whose binding interferes with transcription factor binding, has become a promising new cancer chemotherapeutic approach. Various small molecules have been synthesized and tested for their abilities to stabilize the c-MYC promoter Gquadruplex, including quindolines derivatives,22−26 berberine derivatives,27−29 porphyrin derivatives,30−33 and others. In our previous studies, we used quindoline (Figure 1) as the parent structure for designing and synthesizing compound 1 (SYUIQ5, Figure 1),34 a quindoline derivative. We observed that 1 Received: January 24, 2017 Published: June 12, 2017 5438
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
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Figure 1. Structures of quindoline, quindoline derivative 1, and new disubstituted quindoline derivatives.
Scheme 1. Synthesis of the Disubstituted Quinoline Derivativesa
Reaction conditions: (a) BBr3, CH2Cl2, rt, 16 h; (b) PPh3, DIAD, hydroxyamines or hydroxy ester, THF, rt, 24 h; (c1) phenol, 55 °C, 6 h, (c2) N,N-dialkylalkylamines, 120 °C, 8 h; (d) 4-methylbenzenesulfonic acid, alkylamines, 120 °C, 12 h; (e) sulfolane, iodomethane, 120 °C, 0.5 h; (f) alkylamines, ethylene glycol diethyl ether, 120 °C, 0.5 h. a
prevented NM23-H2 from binding to the c-MYC promoter Gquadruplex and inhibited cancer cell proliferation.24 However, its binding selectivity for the G-quadruplex versus double stranded DNA remained unremarkable and has prompted further studies toward developing more effective quindoline inhibitors for cancer therapy. Previous studies have shown that modifications, including a suitable second side chain35−37 or positive charge,38 to the G-
quadruplex ligands could effectively improve G-quadruplex binding selectivity and binding affinity of compounds. To improve the binding affinity, selectivity, and stabilization activity of the quindoline derivatives toward the G-quadruplex, we designed and synthesized the following four series of disubstituted quindoline analogs (Figure 1 and Scheme 1): series I, compounds that contained a second alkylamino side chain at the 7-, 8-, or 9-position of the quindoline ring; series II, 5439
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Figure 2. Disubstituted quindoline derivatives as c-MYC G4 ligands to restrict NM23-H2/c-MYC binding. (A) The stabilization temperatures (ΔTm) of 0.2 μM FPu22T in Tris-HCl buffer (10 mM, pH 7.4) containing 2 mM KCl that was enhanced with 2 μM disubstituted quindoline derivatives were determined by FRET. Ligand 1 was used as the control. (B) Binding affinity determination of 10 μM FAM labeled Pu27 to the 0−20 μM disubstituted quindoline derivatives and 1 in Tris-HCl buffer (10 mM, pH 7.4) containing 150 mM KCl by MST. The data were analyzed using NT Analysis 1.4.23 with the Hill model. The error bars represent the standard error of the mean (SEM) calculated from three replicates. (C) Binding selectivities of the disubstituted quindoline derivatives and 1 between the c-MYC G-quadruplex and duplex DNA. The binding affinities of ligands were tested by MST. (D) Inhibition of the NM23-H2 (0.5 μM) and Pu27 (1.5 μM) interaction in response to the 10 μM disubstituted quindoline derivatives and 1 by EMSA. The error bars represent the standard error of the mean calculated from three replicates.
derivatives are shown in Scheme 1. The preparation method for the key intermediates of 11-chloroquindoline 1a−1c has been previously reported.38−40 Compounds 2a−2c were prepared by low-temperature demethylation of compounds 1a−1c, respectively, with boron tribromide. Side chains were subsequently introduced to the 7-, 8- or 9-position of the benzofuro[3,2b]quinoline ring using hydramines by the Mitsunobu reaction to generate compounds 3a−3g and 4. 41 By reacting compounds 3a−3g with substituted alkylamines, the 7a1− 7a8 (7,11-, 8,11-, or 9,11-disubstituted quindoline) analogs were generated as final products through phenylate intermediates. Compounds 7b1−7b5 were produced from 4 after reactions with 1.7 equiv of 4-methylbenzenesulfonic acid and alkylamines. The N-5 methylation of 11-chloroquindolines 4 and 3a−3c for intermediates 5 and 6a−6c was achieved with iodomethane in the presence of sulfolane.38 The substitution reactions for compounds 5 and 6 with various alkylamines generated the 11-amino-5-N-methylquindoline derivatives as final products (7c1−7c10 and 7d1−7d4, respectively). All compounds were purified by reprecipitation and exhibited appropriate analytical and spectroscopic properties that were in full accordance with their assigned structures (see Supporting Information). The purified compounds were used for physical and cell-based studies. Characterization of Disubstituted Quindoline Derivatives as c-MYC G-Quadruplex Ligands and NM23-H2/GQuadruplex Binding Inhibitors. Stabilization of G-quadruplex DNA was assessed by fluorescence resonance energy
compounds that contained expanded side chains at their 7positions through amido linkage insertions; series III, compounds that contained single positive charges through methylation at the 5-N position of the quindoline; series IV, compounds that contained two positive charges through methylations at the 5-N position of the quindoline and on the nitrogen atom at the end of the side chain. Our fluorescence resonance energy transfer (FRET)-, circular dichroism (CD)-, and microscale thermophoresis (MST)-based analyses showed that the stabilizing ability, binding affinity, and G-quadruplex selectivity over duplex DNA of these compounds toward the c-MYC G-quadruplex were significantly increased comparing to compound 1. The outcome for disrupting the interaction between NM23-H2 and the c-MYC G-quadruplex was also evaluated in vitro. On the basis of a structure−activity relationship illustration, 7a4 (a 7,11-disubstituted quindoline derivative) became a promising candidate for further studies. 7a4 effectively down-regulated cMYC transcription and expression by stabilizing the c-MYC Gquadruplex and disrupting NM23-H2 binding to DNA in Burkitt’s lymphoma cells. Proliferation of these cancer cells was also inhibited by 7a4 through cell cycle arrest and apoptosis. Moreover, 7a4 exhibited the inhibition activity in a human Burkitt’s lymphoma xenograft.
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RESULTS Synthesis of the Disubstituted Quindoline Derivatives. The synthetic routes for the disubstituted quindoline 5440
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all newly synthesized compounds could induce c-MYC Gquadruplex formation. Microscale thermophoresis (MST) was used to further examine the ligand G-quadruplex binding affinities and selectivities toward the G-quadruplex and duplex DNA. MST is a useful technique for showing a ligand’s binding affinity for G-quadruplex DNA and for revealing the selectivity differences between multiple ligands for G-quadruplex DNA versus double stranded DNA.44 The c-MYC promoter G-quadruplex sequence and duplex DNA containing a FAM label at 5′ (FAM-Pu27 and FAM-duplex) were used. The data presented in Table S2 and Figure 2B demonstrated that all disubstituted quindoline analogs exhibited stronger G-quadruplex binding activities than 1, the monosubstituted analog. The binding constant (KD) to the G-quadruplex for each ligand in series I was within 1.3−4.9 μM, which was lower than that of 1 (7.8 μM). The binding activity of each series II compound was stronger than or equivalent to the 7,11-disubstituted quindoline derivatives in series I. These results were consistent with previously reported conclusions.45 Specifically, the introduction of a side chain onto the quindoline skeleton was beneficial for its interaction with the loops or grooves of the G-quadruplex, which increased the binding activity of the G-quadruplex. The overall binding activity to the G-quadruplex by the series III ligand group, which was characterized by a single positive charge at N5 of the quindoline ring, was enhanced. Specifically, the KD values for 7c4 and 7c5 were 0.8 μM and 0.7 μM, respectively. These results were predictable because the introduction of a steady positive charge at the 5-N position benefited the electrostatic and π-stacking interactions by reducing of the electron density of the ligand’s aromatic core.38 Surprisingly, the series IV binding activity was not enhanced further when a positive charge was introduced to the side chain. Further, we observed that most of the designed ligands showed a significant improvement in specificity for the quadruplex over the duplex DNA compared with compound 1 (KDduplex/KDc‑MYC = 1.02) (Figure 2C and Table S2). The series I ligand group showed a distinct selectivity for the Gquadruplex structure, with KDduplex/KDc‑MYC values ranging from 2.5 to 10.1. Similar results were obtained for the ligands in series II (KDduplex/KDc‑MYC values ranging from 2.9 to 9.7). Additionally, all series III ligands were more tightly bound to the c-MYC G-quadruplex than those in series I and II. However, the binding affinity of series III to the duplex DNA was also increased, which reduced the group’s selectivity for the Gquadruplex. Surprisingly, the second positive charge to be introduced to the side chain (series IV) did not increase the binding affinity for the G-quadruplex but on the contrary increase the binding affinity to the duplex DNA. Altogether, the FRET and MST experimental results indicated that the second side chain strongly increased the ligand’s binding ability and selectivity for the G-quadruplex DNA over the duplex DNA. A positive charge at the 5-N position of the quindoline ring increased the ligand’s binding ability to both the G-quadruplex and the duplex DNA while maintaining or reducing its selectivity for the G-quadruplex. A positive charge on the end of the side chain had no beneficial effect toward the ligand’s binding affinity or selectivity to the G-quadruplex. The newly synthesized ligands were also tested for their abilities to prevent the NM23-H3 transcription factor from binding to the c-MYC promoter G-rich sequence (Pu27). The electrophoretic mobility shift assay (EMSA) was used to assess the inhibitory effects of the ligands on the NM23-H2/Pu27
transfer (FRET), which is a proven approach for effectively screening G-quadruplex-binding ligands. The stabilizing activity of a candidate was evidenced by a melting temperature (ΔTm, in °C) increase that was imparted by the ligand’s presence.41−43 The promoter G-quadruplex sequence Pu22 (a shortened sequence of Pu27) of c-MYC containing a 6-carboxyfluorescein (FAM) label at 5′- and 6-carboxytetramethylrhodamine (TAMRA) label at 3′ (FPu22T) was used. The FRET results (Table S1, Figure 2A, and Figure S1) showed that all ligands strongly increased the stability of the c-MYC G-quadruplex, with ΔTm values ranging from 13.5 to 36.7 °C, and the stabilizing abilities of most ligands were superior to that of 1. A comparison of the ΔTm values between each disubstituted quindoline derivative revealed that four structural factors influenced the G-quadruplex stabilization activities of the ligands. First, the position (i.e., the 7-, 8-, or 9-position) of the second side chain in the benzene ring affected the compound’s activity. With the same substituent, two 7,11disubstituted derivatives (7a2 and 7a3) and two 8,11disubstituted derivatives (7a5 and 7a6) exhibited higher ΔTm values (22.1−25.2 °C) than those for two 9,11-disubstituted derivatives (7a7 and 7a8: 18.6 and 13.5 °C, respectively). Second, introducing a positive charge to the nitrogen atom at the 5-position of the quindoline moiety or amino acid side chain played an important role. For example, 7b2 and 7c2 had identical side chains, and 7c2 (35.6 °C) with a single positive charge showed an obviously higher stabilizing activity toward the G-quadruplexes than 7b2 (21.2 °C), which lacked the positive charge. And ligands 7d1−7d4 that had two positive charges (on the quindoline ring system and on the second side chain) all showed a quite strong stabilizing ability (33.1−36.7 °C). Third, a comparison within series III showed that the length of the side chain was also an important factor. 7c1 (n = 2), 7c2 (n = 3), and 7c3 (n = 4) had identical amino groups at the ends of their side chains, but 7c2 showed the strongest ability toward G-quadruplex stabilization because it contained the appropriate side chain length. Similar observations were made relative to 7c6 (n = 2, ΔTm = 28.4 °C) and 7c7 (n = 3, ΔTm = 36.1 °C), or 7c8 (n = 2, ΔTm = 30.0 °C) and 7c9 (n = 3, ΔTm = 35.2 °C). Finally, the basic group type at the end of the side chains might also influence the ligand’s activity. Compared with 7c1, 7c4, 7c6, 7c8, and 7c10, which had identically long side chains (n = 2), the basic group at the end of the side chains showed an activity trend toward Gquadruplex stabilization as follows: diethylamino > dimethylamino > N-methylpiperazino > piperidino > morpholino. In a word, the second side chain and the positive charges both enhanced the quindoline compound’s effect on G-quadruplex stabilization. To investigate the binding ability of the synthetic ligands for the c-MYC G-quadruplex, we performed circular dichroism (CD) spectroscopy. The CD spectra for Pu27 showed a major positive band at 260 nm and a negative band at 240 nm in the potassium solution, which were typical features of a parallel Gquadruplex. When the derivatives were added to the Pu27 solution without K+, both major bands were strongly enhanced, indicating that the ligands induced a higher number of parallel G-quadruplexes formation (Figure S2A). However, when the derivatives were added to the Pu27 solution with K+, the CD curves displayed no changes, suggesting that the c-MYC G-rich sequences formed into the G-quadruplex structures completely in the K+ solution (Figure S2B). The CD results indicated that 5441
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Figure 3. Characterization of 7a4 for preventing NM23-H3 binding to the c-MYC G-quadruplex. (A) CD spectrum of 5 μM Pu27 annealed with 7a4 in 10 mM Tris-HCl buffer without K+, pH 7.4. 7a4 induced c-MYC G-quadruplex formation in a concentration-dependent manner. (B) The correlation of inhibition percentage and 7a4 concentration was calculated by EMSA. Each mean value with SEM was calculated from three replicates. (C) The intervention effect of 7a4 on the NM23-H2 (250 nM) and Pu27 (100 nM) interaction was validated by ELISA. Mean value with SEM was calculated from three replicates. au, arbitrary units.
interaction according to the previous study.21 The band from the NM23-H2/Pu27 mixture without compound was set as negative control and 100% DNA−protein interaction intensity. The inhibition ability of NM23-H2/Pu27 interaction by the compounds could be evaluated according to the decreased complex band intensity. As shown in Figures 2D and S3, most of the reformatory compounds showed higher inhibition percentage than 1. Compound 7a4 showed the highest inhibition percentage for preventing NM23-H2 binding to Pu27 (inhibition percentage of >80% at 10 μM). Nevertheless, there was no apparent correlation between the derivative’s binding or stabilizing efficiency relative to the G-quadruplex DNA and its ability to interfere with the NM23-H2/DNA interaction. Finally, the inhibitory activity of new derivatives and 1 on Burkitt’s lymphoma cell proliferation was evaluated using MTT assay, and the result is shown in Table S3. Compounds in series I and series II inhibit the RAJI cell proliferation sufficiently, and the half maximal inhibitory concentration (IC50) ranged from 1 μM to 4 μM. However, the IC50 values of most of the compounds in series III and series IV were over 20 μM. Given the fact that large molecular polarity is a negative factor for small molecules to penetrate cell membrane, the poor inhibition activity of compounds in series III and series IV (positive charges introduced at N5 of the quindoline ring and the second side chain) might be from the low cellular uptake. Further Characterization of 7a4 for Preventing NM23H3 Binding to the c-MYC G-Quadruplex. When the results described above were integrated, we found that compound 7a4 from series I displayed the strongest characteristics, including stabilizing ability (ΔTm = 26.6 °C), binding affinity (KDc‑MYC = 1.3 μM), and selectivity (KDduplex/KDc‑MYC = 8.0) for the c-MYC G-quadruplex DNA and inhibition of NM23-H2 binding to the c-MYC G-rich sequence (inhibition percentage = 80.5%). To increase our understanding, the 7a4 molecular recognition mechanism for the c-MYC G-quadruplex DNA was also
studied. The NMR structure of 1 with c-MYC G-quadruplex DNA (Pu22) has been reported. Thus, molecular docking of ligand 7a4 was carried out based on the NMR structure.23 As shown in Figure S4, 7a4 could interact with both 5′- and 3′terminal of c-MYC G-quadruplex DNA like 1. The crescent aromatic core of 7a4 stacks onto two guanine residues of both G-quartet with the 5-N electropositive center overlapped by the cation channel of the quadruplex, which plays the role of the potassium ion. 7a4 has two amino acid side chains that form a V-shape and stretch toward two grooves. These might cause the much stronger binding energy (ΔG = −17.6 kcal/mol) of 7a4 toward the 3′-terminal of c-MYC G-quadruplex DNA than 1 (ΔG = −8.7 kcal/mol). Overall, the binding of 7a4 toward cMYC G-quadruplex DNA was stronger than that of 1. These interactions explained the perfect binding affinities and stabilizing abilities of 7a4 on the G-quadruplex compared to 1. These results are in accordance with the experimental results described above. Furthermore, 7a4 induced c-MYC G-quadruplex formation in a concentration-dependent manner (Figure 3A). Additionally, 7a4 sufficiently inhibited NM23-H2/Pu27 binding, and the EMSA-based half maximal inhibitory concentration (IC50) was 1.56 μM (Figure 3B and Figure S5). The 7a4 effect toward disrupting NM23-H2/Pu27 binding was also verified by enzyme-linked immunosorbent assay (ELISA).21 In this assay, the binding of NM23-H2 and Pu27 was revealed by the absorbance intensity at 450 nm. As shown in Figure 3C, with the 7a4 concentration increasing, the absorbance intensity at 450 nm was decreased obviously, indicating 7a4 interrupted NM23-H2/Pu27 binding sufficiently. This result provided further evidence that 7a4 was an effective NM23-H2/c-MYC binding inhibitor. Down-Regulation of c-MYC Transcription and Expression in Burkitt’s Lymphoma Cells by 7a4 via Stabilization of the c-MYC Promoter G-Quadruplex and Disruption of NM23-H2 Binding. Multiple properties 5442
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Figure 4. 7a4 directly targeted the c-MYC promoter G-quadruplex in cells. The exon specific assay was performed in the RAJI (A) and CA46 (B) cell lines. The NHE III1 element of the c-MYC gene was removed together with the P1 and P2 promoters in CA46 cells, while RAJI cells retained this element following translocation. In this figure, the cell samples without compound treatment were set as control. The experiment was repeated three times. Two-tailed unpaired Student’s t tests were applied for statistical analysis. The data were expressed as the mean ± SEM. p < 0.05 was considered significant: (∗∗) p < 0.01, (∗∗∗) p < 0.001 compared with the untreated groups.
of the disubstituted quindoline derivatives were evaluated, and the candidate compound, 7a4, showed the strongest characteristics in vitro. We asked whether 7a4 targeted the c-MYC promoter, stabilized the G-quadruplex, and inhibited gene transcription in cells. Two Burkitt’s lymphoma cell lines (RAJI and CA46) were used in the exon specific assay,46 which was considered a necessary experiment for evaluating the ligands that directly targeted c-MYC G-quadruplex in vivo. c-MYC gene nontranslocated (NT) cells coexist with translocated (T) cells in the CA46 cell line.25 If the mechanism of c-MYC transcriptional modulation is mediated through Gquadruplex stabilization, a preferential decrease should be evident in exon 1 but not in exon 2. However, the whole c-MYC gene is translocated in the RAJI cell line, including the P1 and P2 promoters with the NHE III1 element. Thus, the changes observed in exons 1 and 2 in RAJI cells should be indistinguishable from one another. Our results showed a significant, dose-dependent transcriptional down-regulation of c-MYC exons 1 and 2 in RAJI cells by 7a4 with an inconsequential effect on NM23-H2 transcription (Figure 4A). In CA46 cells, c-MYC exon 1 exhibited a low transcription level that was down-regulated further by 7a4, while c-MYC exon 2 exhibited a high transcription level, which was minimally affected by 7a4 (Figure 4B). These results indicated that the 7a4 ligand might directly target the c-MYC promoter Gquadruplex in cells. As the results showed, compound 1 presented the larger activity on the down-regulation of exon 1 and exon 2 expressions at the same treating concentration with 7a4. This result might be mainly caused by the larger cytotoxicity of compound 1 on RAJI and CA46 cells than 7a4 (Table S4). Meanwhile, compound 1 showed less selectivity between RAJI and CA46 cells compared to 7a4, which indicated compound 1 might act on the other target in cell.
To further verify the action mechanism of 7a4 in Burkitt’s lymphoma cells, we chose other two lymphoma cell lines with high (CCRF-CEM) or low (U266B1) c-MYC transcription level separately, which was identified by RT-PCR detection (Figure S6). MTT assay was used to evaluate the antiproliferation effects of 7a4 on the three lymphoma cells (Table 1). After 7a4 treatment, RAJI cell exhibited much more Table 1. IC50 (μM) Values of 7a4 on Different Lymphoma Cells from MTT Assay cell line IC50 (μM)
RAJI
CCRF-CEM
U266B1
4.7 ± 0.2
18.1 ± 2.8
23.0 ± 2.6
effective proliferation inhibition (IC50 = 4.7 ± 0.2 μM) than CCRF-CEM (IC50 = 18.1 ± 2.8 μM) and U266B1 (IC50 = 23.0 ± 2.6 μM). Furthermore, 7a4 treatment resulted in a great decreasing effect on c-MYC transcription in Burkitt’s lymphoma cells (RAJI) but presented slight effects on that in U266B1 cell (Figures S7A and S7B), which supported that the 7a4 ligand targets the c-MYC promoter G-quadruplex in cells. Besides, in CCRF-CEM cell, the c-MYC transcription level was decreased slightly (less than 20%) compared to the RAJI cell group (decreased more than 80%) by 7a4 at the concentration of 2 μM, which suggested that 7a4 acted in Burkitt’s lymphoma effectively and selectively (Figures S7A and S7C). Altogether, these results demonstrated that 7a4 selectively inhibited Burkitt’s lymphoma cell proliferation by interfering with cMYC transcription. In addition to c-MYC transcriptional regulation, we tested whether c-MYC protein expression in RAJI cells was modulated by the 7a4 ligand. RAJI cells that were treated with 7a4 for 6, 12, 24, and 48 h were collected for Western blot analyses. As 5443
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Figure 5. Down-regulation of c-MYC expression by 7a4. (A) c-MYC expression detection in RAJI cells at various 7a4 (1 μM) treatment time-points. (B) c-MYC expression detection in RAJI cells after treatments with different concentrations of 7a4 for 6 h. (C) The binding level of NM23-H2 to cMYC promoter NHE III1 was detected by ChIP-PCR. In this figure, the cell samples without compound treatment were set as control. The error bars represent the standard error of the mean (SEM) calculated from three replicates. Two-tailed unpaired Student’s t tests were applied for statistical analysis: (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 compared with control.
Figure 6. 7a4 inhibits Burkitt’s lymphoma cell proliferation by inducing cell cycle arrest at G0/G1 and apoptosis. (A) The long-term proliferation assay was used to evaluate the anticancer effect of 7a4. (B) The 7a4-treated RAJI cell arrest at G0/G1 was detected by flow cytometry. (C) Early/ late apoptosis of RAJI cells that were induced with 7a4 for 12 or 48 h was detected. (D) The biomarkers for G0/G1 arrest were examined by Western blot, and the results were consistent with the results in (B). (E) The biomarkers for cell apoptosis were examined by Western blot, and the results were consistent with the results in (C).
shown in Figure 5A, the c-MYC protein was down-regulated to its lowest level after the 6 h 7a4 treatment, and the downregulation was maintained for 24 h. c-MYC protein expression subsequently recovered toward its original baseline level. We hypothesized that the c-MYC protein recovery was due to cellular metabolism of 7a4. Therefore, the uptake assay was performed at different time points. As shown in Figure S8, the 7a4 concentration in RAJI cells reached its highest point at 6 h and gradually descended thereafter. This result explained the time-dependent regulation of c-MYC expression by 7a4 in cells. Moreover, when the RAJI cells were treated with various concentrations of 7a4 for 6 h, the c-MYC protein level significantly decreased in a dose-dependent manner (Figure 5B). These results demonstrated that 7a4 specifically inhibited c-MYC expression in the cells with the c-MYC non-
translocation. Additionally, NM23-H2 expression did not change with the 7a4 treatment (Figure 5A and Figure 5B) during any of these processes, which indicated that the c-MYC expression down-regulation was due to interference with the NM23-H2 function and not to its reduced expression. The EMSA- and ELISA-based results indicated that the 7a4 ligand prevented NM23-H2 from binding to the c-MYC G-rich sequence by stabilizing the G-quadruplex structure in vitro. We subsequently explored the cellular mechanisms behind the 7a4mediated down-regulation of c-MYC transcription and expression. The chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR) was applied to assess the disruption in NM23-H2/c-MYC binding by 7a4 in RAJI cells. In the 7a4 treatment group, the bound c-MYC G-rich fragment was significantly reduced compared to the control (Figure 5C), 5444
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Figure 7. Evaluation of the anti-Burkitt’s lymphoma activity of 7a4 in vivo. Compound 7a4, doxorubicin, and saline were administered by ip injection to NOD/SCID mice with human Burkitt’s lymphoma established by using RAJI cells. Negative controls were injected with saline. The positive control group was treated with doxorubicin by ip injection at a dose of 1 mg/kg, once in 2 days for 2 weeks. Compound 7a4 was similarly administered to mice, once a day, at a dose of 15 and 30 mg/kg, respectively. The tumor volume (A) and body weight (C) were recorded every day. The tumor weight (B) and other tissues weight (D) were evaluated when the treatment ended. Two-tailed unpaired Student’s t tests were applied for tumor weight statistical analysis: (∗) p < 0.05, (∗∗) p < 0.01 compared to control.
suggesting that 7a4 might target and stabilize the c-MYC promoter G-quadruplex structure to inhibit NM23-H2 binding in RAJI cells. These findings supported the observed 7a4mediated reduction in c-MYC expression that resulted from NM23-H2 inhibition. 7a4 Inhibits Burkitt’s Lymphoma Cell Proliferation by Inducing Cell Cycle Arrest at G0/G1 and Apoptosis. We used a long-term proliferation assay to test whether the 7a4mediated suppression of oncogenic c-MYC expression had an antitumor effect on Burkitt’s lymphoma cells. RAJI cells were treated with indicated concentrations of 7a4, and the cell counts and viabilities were determined every 3 days. Incubations were complete when growth arrest was observed. As shown in Figure 6A, RAJI cell proliferation was significantly suppressed by 7a4, and the suppression activity was concentration-dependent. Because the c-MYC oncoprotein has been associated with cell cycle arrest47 and cell apoptosis,48 we investigated whether these cellular processes promoted the RAJI cell proliferation suppression by 7a4. The RAJI cell cycles, with or without the 7a4 treatment, were observed by flow cytometry. The results shown in Figure 6B indicated that the cell cycle spectrum clearly changed as the compound’s action time progressed. The cells were arrested at G0/G1 (G0/G1, 60.2%) after the 12 h 7a4 treatment. The arresting effect was subsequently reduced, which might have been caused by cellular metabolism of 7a4 (Figure S8). A subG1 peak was observed after 24 h of 7a4. Therefore, cells that were treated with or without 7a4 were examined for apoptosis by PI and annexin V staining. Early apoptosis was evident in RAJI cells by 12 h and late apoptosis was observed at 48 h (Figure 6C), which indicated that 7a4 inhibited RAJI proliferation by inducing apoptosis. Moreover, we analyzed markers for cell cycle arrest at G0/G1 and apoptosis markers in cells that were treated with 7a4 for 12 h by Western blot (Figure 6D and Figure 6E). The results showed a clear, dose-
dependent down-regulation of the following G0/G1 biomarkers: cyclin D1, cyclin D3, CDK4, and CDK6. Besides, the up-regulation of p27, a cyclin-dependent kinase inhibitor, also supported the G0/G1 phase stalling. Furthermore, two apoptosis marker proteins, cleaved PARP and cleaved caspase9, were up-regulated in 7a4-treated cells relative to the untreated group; this was consistent with our previous data. Together, these results indicated that the proliferation inhibition by 7a4 in RAJI cells occurred through cell cycle arrest at G0/G1 and apoptosis. Compound 7a4 Inhibits Tumor Growth in a Human Burkitt’s Lymphoma Xenograft. 7a4 presented the effective anti-Burkitt’s lymphoma activity in vitro, and it prompted us to investigate the potential of anti-Burkitt’s lymphoma proliferation by 7a4 in vivo. A human Burkitt’s lymphoma (RAJI) xenograft model in NOD/SCID mice was established according to the method reported.49 And the mice were divided into four groups followed by treating saline (negative control), doxorubicin (positive control, at a dose of 1 mg/kg), and 7a4 (two experimental groups, at a dose of 30 and 15 mg/kg) for 2 weeks, respectively. After treatment, the tumors were collected and evaluated. The negative control group had an average tumor volume of >900 mm3 after 2 weeks. In contrast, the mice from the three experimental groups treated with 30 and 15 mg/ kg compound 7a4 had an average tumor volume of 300 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.67 (s, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.92−7.87 (m, 1H), 7.86−7.80 (m, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H). ESI-MS m/z: 270 [M + 1]+. General Method for the Preparation of the Compounds 3a− 3g and 4. Compound 2a, 2b, or 2c (0.8 g, 3.0 mmol), PPh3 (2.4 g, 9.0 mmol), and hydroxyamines (for 3a−3c) or hydroxy ester (for 4) (9.0 mmol) were placed in a reaction flask and dissolved in dry THF (6 mL). To the solution cooled to 0 °C was added dropwise a solution of DIAD (1.8 mL, 9 mmol) in dry THF under nitrogen. The resulting yellow solution was allowed to warm to room temperature and stirred for 24 h. The solvent was removed in vacuo, and the residue was subjected to column chromatography with ethyl acetate as an eluent to afford 3a−3c (or 4) as a white solid. 2-((11-Chlorobenzofuro[3,2-b]quinolin-7-yl)oxy)-N,N-dimethylethan-1-amine (3a). The 1H NMR data of 3a was the same as described.51 ESI-MS m/z: 341 [M + H]+. 3-((11-Chlorobenzofuro[3,2-b]quinolin-7-yl)oxy)-N,N-dimethylpropan-1-amine (3b). The 1H NMR data of 3b was the same as described.51 ESI-MS m/z: 355 [M + H]+. 11-Chloro-7-((4-methylpiperazin-1-yl)methoxy)benzofuro[3,2-b]quinoline (3c). Mp 195−197 °C. 1H NMR (400 MHz, CDCl3): δ 8.17 (dd, J = 17.1, 8.2 Hz, 2H), 7.66−7.61 (m, 2H), 7.58−7.52 (m, 1H), 7.42 (d, J = 9.0 Hz, 1H), 7.10 (dd, J = 8.2, 1.8 Hz, 1H), 3.83 (d, J = 5.6 Hz, 2H), 2.83−2.69 (m, 4H), 2.36 (s, 3H), 1.92 (m, 4H), 1.75−1.59 (m, 1H). (ESI-MS m/z: 382 [M + H]+. 2-((11-Chlorobenzofuro[3,2-b]quinolin-8-yl)oxy)-N,N-dimethylethan-1-amine (3d). Mp 193−194 °C. 1H NMR (400 MHz, CDCl3): δ 8.27 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 7.75−7.67 (m, 2H), 7.65−7.58 (m, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.23 (dd, J = 9.0, 2.7 Hz, 1H), 4.16 (t, J = 5.6 Hz, 2H), 2.76 (t, J = 5.6 Hz, 2H), 2.32 (s, 6H). ESI-MS m/z: 341 [M + 1]+. 3-((11-Chlorobenzofuro[3,2-b]quinolin-8-yl)oxy)-N,N-dimethylpropan-1-amine (3e). Mp 185−187 °C. 1H NMR (400 MHz, CDCl3): δ 8.31 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 8.6 Hz, 1H), 7.76 (dd, J = 10.3, 4.8 Hz, 2H), 7.66 (dd, J = 8.2, 7.0 Hz, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.23 (dd, J = 9.0, 2.6 Hz, 1H), 4.15 (t, J = 6.4 Hz, 2H), 2.55− 2.49 (m, 2H), 2.30 (s, 6H), 2.08−1.99 (m, 2H). ESI-MS m/z: 355 [M + 1]+. 2-((11-Chlorobenzofuro[3,2-b]quinolin-9-yl)oxy)-N,N-dimethylethan-1-amine (3f). Mp 197−198 °C. 1H NMR (400 MHz, CDCl3): δ
DISCUSSION AND CONCLUSION
In our study, we explored a new strategy for Burkitt’s lymphoma chemotherapy by synthesizing 27 new disubstituted quindoline derivatives to target the oncogenic c-MYC promoter G-quadruplex and interfere with transcription factor binding (specifically NM23-H2) to this G-quadruplex DNA, with subsequent inhibition of c-MYC gene transcription. Several experiments were performed to evaluate the structure−activity relationships of these new compounds. The results indicated that the 7,11- or 8,11-disubstituted derivatives with the appropriate side chain length and amino groups at the ends of their side chains exhibited stronger stabilization abilities, binding affinities, and selectivities for the c-MYC G-quadruplex. Although the 5-N-methyl group on the quindoline ring was important for G-quadruplex binding, the poor binding selectivity for the G-quadruplex relative to the duplex DNA rendered this group of compounds unsuitable for further testing in cells. In addition to its inhibitory effect on NM23H2/c-MYC binding, compound 7a4 exhibited the strongest characteristics among the new compounds and was selected for further investigations in cells. The 7a4 ligand effectively and selectively targeted and inhibited the c-MYC transcriptional program in RAJI cells. NM23-H2 binding to the c-MYC promoter was prevented, and the c-MYC mRNA product was down-regulated. Consequently, the c-MYC protein was down-regulated in a time-dependent manner. The action time for 7a4 in the RAJI Burkitt’s lymphoma cell line was within 24 h, and then the c-MYC protein subsequently recovered due to the gradual cellular metabolism of 7a4. This phenomenon might have been caused by feedback regulation in the exposed cells, where the c-MYC protein was overexpressed to promote survival by enabling cells to escape death.50 The c-MYC protein was down-regulated in RAJI cells that were exposed to compound 7a4, and the proliferation inhibition of the RAJI cells occurred through cell cycle arrest at G0/G1 and apoptosis. The c-MYC protein is a transcription factor, and its target genes participate in the cell cycle, apoptosis, survival, protein synthesis, cell adhesion, the cytoskeleton, and metabolism. Different target genes are regulated under specific conditions for specific cell types.51 Thus, the likely participation of the c-MYC protein in the RAJI cell division and antiapoptosis processes suggests that downregulation of the c-MYC protein in RAJI cells would cause cell cycle arrest at G0/G1 and apoptosis. In summary, 7a4 effectively bound to and stabilized Gquadruplex structure of c-MYC promoter, interfered with NM23-H2 binding to the c-MYC promoter, down-regulated cMYC gene transcription and translation, inhibited Burkitt’s lymphoma cell proliferation, and promoted cell death. And the anti-Burkitt’s lymphoma activity of 7a4 was furtherly confirmed in vivo. Our results provide a new strategy for anti-Burkitt’s lymphoma therapeutics that involves modulating transcription of the c-MYC oncogene by stabilizing its promoter secondary structure and inhibiting binding by its transcription factor. 5446
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
Journal of Medicinal Chemistry
Article
56.2, 43.5, 43.3, 40.1. HRMS (ESI): m/z, calcd for (C23H28N4O2) [M − H]− 391.2139, found 391.2134. HPLC purity 99.8%. N1-(7-(2-(Dimethylamino)ethoxy)benzofuro[3,2-b]quinolin-11yl)-N3,N3-dimethylpropane-1,3-diamine (7a2). 3a was used as starting materials. A mixture of 3a (170.5 mg, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane-1,3diamine (102 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a2 (137 mg, 68%). Mp 181−173 °C. 1H NMR (400 MHz, D2O): δ 7.92 (d, J = 8.6 Hz, 1H), 7.77 (t, J = 7.4 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 9.2 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.35 (dd, J = 9.2, 2.4 Hz, 1H), 7.25 (d, J = 1.8 Hz, 1H), 4.36−4.29 (m, 2H), 3.95 (t, J = 7.0 Hz, 2H), 3.69−3.62 (m, 2H), 3.30−3.22 (m, 2H), 3.00 (s, 6H), 2.83 (s, 6H), 2.22−2.10 (m, 2H). 13 C NMR (101 MHz, D2O): δ 154.2, 152.4, 141.8, 136.0, 135.5, 133.2, 130.9, 125.8, 122.9, 122.3, 118.8, 115.4, 114.5, 114.1, 103.8, 62.5, 56.3, 55.0, 43.2, 42.9, 42.5, 25.4. HRMS (ESI): m/z, calcd for [M − H]− (C24H30N4O2) requires 405.2296, found 405.2301. HPLC purity 99.7%. N1-(7-(3-(Dimethylamino)propoxy)benzofuro[3,2-b]quinolin-11yl)-N3,N3-dimethylpropane-1,3-diamine (7a3). 3b was used as starting materials. A mixture of 3b (177.5 mg, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane-1,3diamine (102 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a3 (123 mg, 59%). Mp 175−177 °C. 1H NMR (400 MHz, D2O): δ 7.49 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.23 (d, J = 9.0 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 7.4 Hz, 1H), 6.80 (dd, J = 8.8, 1.8 Hz, 1H), 6.40 (s, 1H), 3.69 (t, J = 7.4 Hz, 2H), 3.50−3.43 (m, 2H), 3.24 (td, J = 13.2, 8.4 Hz, 4H), 2.89 (d, J = 6.6 Hz, 12H), 2.11 (dd, J = 15.0, 7.8 Hz, 2H), 2.05−1.96 (m, 2H). 13 C NMR (101 MHz, D2O): δ 154.5, 151.7, 141.2, 135.2, 135.0, 133.0, 130.3, 125.6, 122.6, 122.2, 118.4, 114.6, 114.0, 113.6, 102.7, 65.3, 55.1, 55.0, 43.0, 42.6, 25.4, 24.0. HRMS (ESI): m/z, calcd for [M − H]− (C25H32N4O2) requires 419.2453, found 419.2451. HPLC purity 99.9%. N 1 ,N 1 -Dimethyl-N 3 -(7-((1-methylpiperidin-4-yl)methoxy)benzofuro[3,2-b]quinolin-11-yl)propane-1,3-diamine (7a4). 3c was used as starting materials. A mixture of 3c (177.5 g, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane1,3-diamine (102 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a4 (1.18 g, 53%). Mp 148−150 °C. 1H NMR (400 MHz, D2O): δ 7.71 (d, J = 8.2 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 9.2 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.01 (d, J = 9.2 Hz, 1H), 6.70 (s, 1H), 3.88−3.82 (m, 2H), 3.49 (d, J = 4.8 Hz, 2H), 3.28 (t, J = 7.8 Hz, 2H), 3.05 (t, J = 12.0 Hz, 2H), 2.86 (d, J = 5.8 Hz, 9H), 2.10 (dt, J = 37.6, 15.2 Hz, 7H), 1.59 (dd, J = 24.2, 10.8 Hz, 2H). 13C NMR (101 MHz, D2O): δ 154.7, 151.6, 141.3, 135.20, 135.0, 133.0, 130.3, 125.7, 123.0, 122.6, 118.4, 114.6, 114.0, 113.6, 102.6, 71.4, 55.0, 54.2, 43.3, 43.0, 42.6, 32.8, 26.2, 25.4. HRMS (ESI): m/z, calcd for [M − H]− (C27H34N4O2) requires 445.2609, found 445.2608. HPLC purity 99.6%. N1-(8-(2-(Dimethylamino)ethoxy)benzofuro[3,2-b]quinolin-11yl)-N3,N3-dimethylpropane-1,3-diamine (7a5). 3d was used as starting materials. A mixture of 3d (190.5 mg, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane-1,3diamine (102 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a5 (143 mg, 71%). Mp 177−178 °C. 1H NMR (400 MHz, D2O): δ 7.70 (d, J = 8.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.20 (dd, J = 17.0, 7.8 Hz, 2H), 7.08 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 4.31−4.21 (m, 2H), 3.83 (t, J = 6.7 Hz, 2H), 3.71−3.64 (m, 2H), 3.34−3.27 (m, 2H), 3.06 (s, 6H), 2.91 (s, 6H), 2.23−2.13 (m, 2H). 13C NMR (101 MHz, D2O): δ 145.8, 142.8, 141.7, 135.6, 135.3, 133.1, 130.1, 125.9, 125.7, 122.5, 118.5, 116.3, 115.5, 114.3, 114.0, 63.5, 56.3, 55.0, 43.9, 43.0, 42.8, 24.8. HRMS (ESI): m/z, calcd for [M − H]− (C24H30N4O2) requires 405.2296, found 405.2301. HPLC purity 98.9%. N1-(8-(3-(Dimethylamino)propoxy)benzofuro[3,2-b]quinolin-11yl)-N3,N3-dimethylpropane-1,3-diamine (7a6). 3e was used as starting materials. A mixture of 3e (170.5 g, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane-1,3diamine (102 mg, 1 mmol) was added and the mixture was heated at
8.23 (dd, J = 18.0, 8.8 Hz, 2H), 7.85 (d, J = 7.7 Hz, 1H), 7.72−7.65 (m, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 4.38 (t, J = 5.6 Hz, 2H), 2.94 (t, J = 5.6 Hz, 2H), 2.44 (s, 6H). ESI-MS m/z: 341 [M + 1]+. 3-((11-Chlorobenzofuro[3,2-b]quinolin-9-yl)oxy)-N,N-dimethylpropan-1-amine (3g). Mp 180−181 °C. 1H NMR (400 MHz, CDCl3): δ 8.29 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.66−7.60 (m, 1H), 7.32 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 2.56 (t, J = 7.2 Hz, 2H), 2.27 (s, 6H), 2.09 (m, 2H). ESI-MS m/z: 355 [M + 1]+. Methyl 2-((11-Chlorobenzofuro[3,2-b]quinolin-7-yl)oxy)acetate (4). 1H NMR (400 MHz, CDCl3): δ 8.38 (d, J = 8.2 Hz, 1H), 8.31 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 7.0 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.75−7.69 (m, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.38 (dd, J = 9.0, 2.7 Hz, 1H), 4.81 (s, 2H), 3.87 (s, 3H). ESI-MS m/z: 343 [M + H]+. General Method for the Preparation of the Compounds 5 and 6a−c. The N-5 methylation of 11-chloroquindoline 4 (or 3a− 3c) with iodomethane was achieved in the presence of sulfolane with an excellent yield (88−93%). A mixture of 4 (or 3a−3c) (5 mmol) and 10 g of iodomethane was heated at 120 °C for 0.5 h. The mixture was cooled to room temperature and was poured to 50 mL of ether. Then the product 5 (or 6a−6c) would be precipitated in the ether. The products were filtered out of the ether and then dried under the infrared lamp. The product was used without any purification and was a pale yellow solid. 11-Iodo-5-methyl-7-(2-(trimethylammonio)ethoxy)benzofuro[3,2-b]quinolin-5-ium Iodide (6a). 1H NMR (400 MHz, DMSO-d6): δ 8.83 (d, J = 9.0 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.32 (t, J = 8.0 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H), 8.20 (d, J = 9.2 Hz, 1H), 8.12 (t, J = 7.8 Hz, 1H), 7.81 (dd, J = 9.2, 2.3 Hz, 1H), 4.93 (s, 3H), 4.75 (t, J = 3.6 Hz, 2H), 3.92 (t, J = 3.6 Hz, 2H), 3.27 (s, 9H). ESI-MS m/z: 231 [M − 2I]2+/2. 11-Iodo-5-methyl-7-(3-(trimethylammonio)propoxy)benzofuro[3,2-b]quinolin-5-ium Iodide (6b). Mp 282.9−285.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.83 (d, J = 8.5 Hz, 1H), 8.61 (d, J = 7.5 Hz, 1H), 8.34 (t, J = 7.2 Hz, 1H), 8.23−8.11 (m, 2H), 7.86 (d, J = 10.1 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 4.92 (s, 3H), 4.31 (d, J = 30.7 Hz, 2H), 3.66−3.53 (m, 2H), 3.17 (s, 9H), 2.29 (s, 2H). ESI-MS m/z: 238 [M − 2I]2+/2. 7-((1,1-Dimethylpiperidin-1-ium-4-yl)methoxy)-11-iodo-5methylbenzofuro[3,2-b]quinolin-5-ium Iodide (6c). Mp 273.8−275.9 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.82 (d, J = 8.7 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.37−8.28 (m, 1H), 8.22−8.11 (m, 3H), 7.76 (dd, J = 13.4, 7.7 Hz, 1H), 4.92 (s, 3H), 4.19 (dd, J = 32.2, 5.6 Hz, 2H), 3.65−3.41 (m, 4H), 3.13 (s, 3H), 2.84 (s, 3H), 2.19−1.99 (m, 4H), 1.58 (d, J = 12.4 Hz, 1H). ESI-MS m/z: 205 [M − 2I]2+/2. General Method for the Preparation of the Compounds 7a1−7a8. A mixture of 3 (0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then 5 mmol of alkylamines was added and the mixture was heated at 120 °C for 4 h. The mixture was cooled to room temperature and was poured into 50 mL of water. The pH of the mixture was adjusted to 10 with a 40% NaOH solution, and the organic product was extracted with chloroform, washed with saturated NaCl solution and water, and then dried with Na2SO4. After concentration, the product was purified by column chromatography on silica gel. Further purification was carried out by recrystallization to give a pale yellow solid. N1-(7-(2-(Dimethylamino)ethoxy)benzofuro[3,2-b]quinolin-11yl)-N2,N2-dimethylethane-1,2-diamine (7a1). 3a was used as starting materials. A mixture of 3a (170.5 mg, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylethane-1,2-diamine (880 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a1 (125 mg, 64%). Mp 204−206 °C. 1H NMR (400 MHz, D2O): δ 7.99 (d, J = 8.2 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.38 (dd, J = 9.2, 1.2 Hz, 1H), 7.28 (s, 1H), 4.31 (t, J = 6.4 Hz, 4H), 3.69− 3.62 (m, 2H), 3.56 (t, J = 6.4 Hz, 2H), 2.99 (d, J = 11.4 Hz, 12H). 13C NMR (101 MHz, D2O): δ 154.3, 152.6, 141.5, 136.2, 135.2, 133.3, 130.9, 126.1, 123.2, 122.3, 118.6, 115.2, 114.4, 114.2, 104.0, 62.5, 56.6, 5447
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
Journal of Medicinal Chemistry
Article
85 °C. 1H NMR (400 MHz, CD3OD): δ 8.07 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 2.6 Hz, 1H), 7.56 (t, J = 7.1 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.36 (t, J = 7.1 Hz, 1H), 7.24 (dd, J = 9.0, 2.6 Hz, 1H), 4.56 (s, 2H), 3.94 (t, J = 6.9 Hz, 2H), 3.25 (t, J = 6.8 Hz, 2H), 2.50−2.39 (m, 2H), 2.27−2.20 (m, 2H), 2.19 (s, 6H), 2.07 (s, 6H), 1.95−1.84 (m, 2H), 1.65 (dd, J = 14.6, 7.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 167.6, 154.1, 152.3, 146.6, 146.0, 135.1, 133.2, 128.8, 127.8, 123.5, 122.9, 122.1, 119.1, 117.8, 112.8, 104.9, 67.8, 56.4, 44.5, 44.4, 42.9, 36.7, 30.5, 27.7, 26.2. HRMS (ESI): m/z, calcd for [M + H]+ (C27H35N5O3) 478.2813, found 478.2806. HPLC purity 97.8%. N-(2-(Diethylamino)ethyl)-2-((11-((2-(diethylamino)ethyl)amino)benzofuro[3,2-b]quinolin-7-yl)oxy)acetamide (7b3). Compound 4 (241 mg, 0.5 mmol) reacted with 4-methylbenzenesulfonic acid (146.2 mg, 0.85 mmol) in N,N-diethylethane-1,2-diamine (580 mg, 5 mmol) at 120 °C to afford 7b3 (141.4 mg, 56%). Mp 67−69 °C. 1 H NMR (400 MHz, CDCl3): δ 8.16 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 2.6 Hz, 1H), 7.65 (dd, J = 11.2, 4.1 Hz, 1H), 7.49 (s, 1H), 7.47 (s, 1H), 7.23 (dd, J = 8.9, 2.7 Hz, 1H), 4.64 (s, 2H), 4.13 (d, J = 5.2 Hz, 2H), 3.45 (dd, J = 11.5, 5.7 Hz, 2H), 2.97−2.90 (m, 2H), 2.72 (q, J = 7.1 Hz, 4H), 2.65 (t, J = 6.0 Hz, 2H), 2.58 (q, J = 7.1 Hz, 4H), 1.14 (t, J = 7.1 Hz, 6H), 1.03 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 168.1, 153.7, 153.4, 146.8, 146.7, 134.9, 134.6, 129.4, 127.9, 124.2, 123.7, 120.4, 118.6, 118.2, 112.6, 105.7, 68.3, 52.2, 51.3, 46.9, 46.7, 42.1, 36.5, 11.9, 11.8. HRMS (ESI): m/z, calcd for [M + H]+ (C29H39N5O3) requires 506.3126, found 506.3117. HPLC purity 97.0%. N-(3-(Diethylamino)propyl)-2-((11-((3-(diethylamino)propyl)amino)benzofuro[3,2-b]quinolin-7-yl)oxy)acetamide (7b4). Compound 4 (241 mg, 0.5 mmol) reacted with 4-methylbenzenesulfonic acid (146.2 mg, 0.85 mmol) in N1,N1-diethylpropane-1,3-diamine (650 mg, 5 mmol) at 120 °C to afford 7b4 (175.9 mg, 66%). Mp 86−88 °C. 1 H NMR (400 MHz, CD3OD): δ 8.00 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.52 (t, J = 7.1 Hz, 1H), 7.39− 7.27 (m, 2H), 7.17 (dd, J = 8.9, 2.5 Hz, 1H), 4.51 (s, 2H), 3.83 (t, J = 6.6 Hz, 2H), 3.22 (dd, J = 3.7, 2.0 Hz, 2H), 2.61−2.52 (m, 2H), 2.47 (dd, J = 14.2, 7.1 Hz, 4H), 2.41−2.30 (m, 6H), 1.91−1.75 (m, 2H), 1.69−1.56 (m, 2H), 0.93 (t, J = 7.1 Hz, 6H), 0.86 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, CD3OD): δ 171.0, 155.5, 154.4, 147.5, 147.2, 137.6, 134.9, 129.5, 128.8, 124.7, 124.3, 122.7, 120.4, 119.2, 113.9, 106.4, 69.0, 58.2, 58.1, 45.3, 45.1, 44.6, 38.4, 29.3, 27.7. HRMS (ESI): m/z, calcd for [M + H]+ (C31H43N5O3) requires 534.3439, found 534.3435. HPLC purity 97.1%. N-(2-(Piperidin-1-yl)ethyl)-2-((11-((2-(piperidin-1-yl)ethyl)amino)benzofuro[3,2-b]quinolin-7-yl)oxy)acetamide (7b5). Compound 4 (241 mg, 0.5 mmol) reacted with 4-methylbenzenesulfonic acid (146.2 mg, 0.85 mmol) in 2-(piperidin-1-yl)ethanamine (640 mg, 5 mmol) at 120 °C to afford 7b5 (153 mg, 58%). Mp 94−96 °C. 1H NMR (400 MHz, CD3OD): δ 8.13 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 2.4 Hz, 1H), 7.68−7.62 (m, 1H), 7.46 (dd, J = 18.4, 8.2 Hz, 2H), 7.30 (dd, J = 9.0, 2.6 Hz, 1H), 4.62 (s, 2H), 4.07 (t, J = 7.0 Hz, 2H), 3.47 (t, J = 6.7 Hz, 2H), 2.73 (t, J = 7.0 Hz, 2H), 2.56 (s, 4H), 2.54−2.50 (m, 2H), 2.45 (s, 4H), 1.68−1.52 (m, 8H), 1.50 (d, J = 4.5 Hz, 2H), 1.43 (d, J = 5.0 Hz, 2H). 13C NMR (101 MHz, CD3OD): δ 170.8, 155.4, 154.4, 147.5, 147.2, 137.3, 134.8, 129.5, 128.9, 124.7, 124.3, 122.6, 120.3, 119.2, 113.7, 106.4, 68.9, 60.4, 58.8, 55.7, 55.5, 42.7, 37.0, 26.7, 26.6, 25.2, 25.1. HRMS (ESI): m/z, calcd for [M + H]+ (C31H39N5O3) requires 530.31152, found 530.31257. HPLC purity 99.7%. General Method for the Preparation of the Compounds 7c1−7c10 and 7d1−7d4. The substitution reaction of compounds 5 (or 6a−6c) with various alkylamines gave the final products of 11amino-5-N-methylquindoline derivatives 7c1−7c10 (or 7d1−7d4) (Scheme 1). 5 (or 6a−6c) (0.5 mmol) dissolved in 4 mL of ethylene glycol diethyl ether and reacted with 5 mM alkylamines at 120 °C for 0.5 h. The mixture was cooled to room temperature and was poured into 30 mL of ether. Then the products 7c1−7c10 (or 7d1−7d4) would be precipitated in the ether. The products were filtered out of the ether and then dried under the infrared lamp. Further purification
120 °C for 4 h to offer 7a6 (136 mg, 65%). Mp 166−168 °C. 1H NMR (400 MHz, D2O): δ 7.48 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.01 (dd, J = 12.2, 7.6 Hz, 2H), 6.83 (t, J = 7.6 Hz, 1H), 6.44 (d, J = 7.8 Hz, 1H), 3.59 (t, J = 7.4 Hz, 4H), 3.32−3.25 (m, 2H), 3.24−3.17 (m, 2H), 2.90 (d, J = 13.8 Hz, 12H), 2.14−2.01 (m, 4H). 13C NMR (101 MHz, D2O): δ 145.3, 143.2, 141.3, 135.2, 135.0, 133.0, 129.6, 125.8, 125.6, 122.4, 118.2, 115.6, 115.1, 113.9, 112.9, 65.7, 55.0, 54.6, 43.0, 43.0, 42.9, 24.8, 24.1. HRMS (ESI): m/z, calcd for [M − H]− (C25H32N4O2) requires 419.2452, found 419.2453. HPLC purity 98.9%. N1-(9-(2-(Dimethylamino)ethoxy)benzofuro[3,2-b]quinolin-11yl)-N3,N3-dimethylpropane-1,3-diamine (7a7). 3f was used as starting materials. A mixture of 3f (177.5 mg, 0.5 mmol) and 1 g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane-1,3diamine (102 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a7 (115 mg, 57%). Mp 175−176 °C. 1H NMR (400 MHz, D2O): δ 7.93 (d, J = 8.6 Hz, 1H), 7.83−7.74 (m, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 9.2 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 7.34 (dd, J = 9.2, 2.6 Hz, 1H), 7.26 (d, J = 2.6 Hz, 1H), 4.35−4.30 (m, 2H), 3.95 (t, J = 6.8 Hz, 2H), 3.67−3.62 (m, 2H), 3.27−3.20 (m, 2H), 2.98 (s, 6H), 2.80 (s, 6H), 2.21−2.08 (m, 2H). 13 C NMR (101 MHz, D2O): δ 154.1, 152.3, 141.6, 135.7, 135.3, 133.1, 130.7, 125.7, 122.9, 122.2, 118.6, 115.2, 114.2, 114.0, 103.6, 62.4, 56.2, 55.0, 43.2, 42.9, 42.5, 25.4. HRMS (ESI): m/z, calcd for [M − H]− (C24H30N4O2) requires 405.2296, found 405.2312. HPLC purity 99.6%. N1-(9-(3-(Dimethylamino)propoxy)benzofuro[3,2-b]quinolin-11yl)-N3,N3-dimethylpropane-1,3-diamine (7a8). 3g was used as starting materials.. A mixture of 3g (170.5 mg, 0.5 mmol) and 1g of phenol was heated at 55 °C for 6 h, then N1,N1-dimethylpropane-1,3diamine (102 mg, 1 mmol) was added and the mixture was heated at 120 °C for 4 h to offer 7a8 (108 mg, 52%). Mp 168−169 °C. 1H NMR (400 MHz, D2O): δ 7.83 (d, J = 8.4 Hz, 1H), 7.74−7.69 (m, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 9.2 Hz, 1H), 7.44−7.39 (m, 1H), 7.14 (dd, J = 9.2, 2.4 Hz, 1H), 6.97 (s, 1H), 3.89 (dd, J = 9.5, 4.6 Hz, 4H), 3.37−3.30 (m, 2H), 3.28−3.21 (m, 2H), 2.92 (s, 6H), 2.82 (s, 6H), 2.22−2.09 (m, 4H). 13C NMR (101 MHz, D2O): δ 154.6, 151.8, 141.4, 135.5, 135.2, 133.0, 130.5, 125.7, 122.6, 122.3, 118.6, 114.9, 114.2, 113.7, 102.9, 65.4, 55.2, 55.0, 43.0, 42.9, 42.5, 25.4, 24.0. HRMS (ESI): m/z, calcd for [M − H]− (C25H32N4O2) 419.2452, found 419.2466. HPLC purity 99.7%. General Method for the Preparation of the Compounds 7b1−7b5. Compound 4 reacted with 1.7 equiv of 4-methylbenzenesulfonic acid in 10 equiv of alkylamines at 120 °C for 12 h to afford 7b1−7b5. The mixture was cooled to room temperature and was poured into 50 mL of water, and the organic product was extracted with chloroform and then dried with Na2SO4. After concentration, the product was purified by column chromatography on silica gel. Further purification was carried out by recrystallization to give a pale yellow solid. N-(2-(Dimethylamino)ethyl)-2-((11-((2-(dimethylamino)ethyl)amino)benzofuro[3,2-b]quinolin-7-yl)oxy)acetamide (7b1). Compound 4 (241 mg, 0.5 mmol) reacted with 4-methylbenzenesulfonic acid (146.2 mg, 0.85 mmol) in N1,N1-dimethylethane-1,2-diamine (440 mg, 5 mmol) at 120 °C to afford 7b1 (134.7 mg, 60%). Mp 89− 91 °C. 1H NMR (400 MHz, CD3OD): δ 8.09 (d, J = 8.6 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 2.6 Hz, 1H), 7.60−7.54 (m, 1H), 7.47 (d, J = 9.0 Hz, 1H), 7.39−7.34 (m, 1H), 7.26 (dd, J = 9.0, 2.7 Hz, 1H), 4.56 (s, 2H), 4.04 (t, J = 7.0 Hz, 2H), 3.37 (t, J = 6.7 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.44 (t, J = 6.7 Hz, 2H), 2.29 (d, J = 11.4 Hz, 6H), 2.19 (s, 6H). 13C NMR (101 MHz, DMSO-d6): δ 167.5, 154.1, 152.3, 146.6, 146.04, 135.0, 133.2, 128.9, 127.8, 123.5, 123.1, 122.1, 119.2, 117.8, 112.7, 105.1, 67.9, 59.6, 58.0, 45.4, 45.1, 42.4, 36.4. HRMS (ESI): m/z, calcd for [M + H]+ (C25H31N5O3) requires 450.2500, found 450.2497. HPLC purity 98.9%. N-(3-(Dimethylamino)propyl)-2-((11-((3-(dimethylamino)propyl)amino)benzofuro[3,2-b]quinolin-7-yl)oxy)acetamide (7b2). Compound 4 (241 mg, 0.5 mmol) reacted with 4-methylbenzenesulfonic acid (146.2 mg, 0.85 mmol) in N1,N1-dimethylpropane-1,3-diamine (510 mg, 5 mmol) at 120 °C to afford 7b2 (147.7 mg, 62%). Mp 84− 5448
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
Journal of Medicinal Chemistry
Article
4.51 (s, 2H), 4.37 (s, 3H), 4.06 (t, J = 6.3 Hz, 2H), 3.15−3.09 (m, 2H), 2.55 (t, J = 6.5 Hz, 2H), 2.43 (dd, J = 13.7, 6.6 Hz, 4H), 2.36− 2.25 (m, 6H), 1.94−1.81 (m, 2H), 1.58−1.44 (m, 2H), 0.85 (t, J = 7.1 Hz, 6H), 0.77 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 167.4, 155.0, 152.7, 142.9, 138.3, 133.7, 132.8, 125.6, 124.6, 122.1, 117.4, 117.2, 116.9, 114.1, 108.0, 100.0, 68.6, 52.4, 51.3, 47.7, 46.8, 39.1, 38.6, 26.1, 25.1, 11.6, 11.0. HRMS (ESI): m/z, calcd for [M − I]+ (C32H46IN5O3) requires 548.3595, found 548.3667. HPLC purity 99.8%. 5-Methyl-11-((2-morpholinoethyl)amino)-7-(2-((2morpholinoethyl)amino)-2-oxoethoxy)benzofuro[3,2-b]quinolin-5ium Iodide (7c6). Compound 5 (241 mg, 0.5 mmol) reacted with 2morpholinoethanamine (650 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c6 (184 mg, 67.3%). Mp 215−216 °C. 1H NMR (400 MHz, CDCl3): δ 9.02 (d, J = 9.0 Hz, 1H), 8.04 (s, 1H), 7.95 (d, J = 6.2 Hz, 2H), 7.69 (dd, J = 18.1, 8.3 Hz, 1H), 7.57 (s, 1H), 7.48 (d, J = 9.4 Hz, 1H), 4.78 (s, 2H), 4.60 (s, 3H), 4.36 (t, J = 5.7 Hz, 2H), 3.78−3.72 (m, 4H), 3.72−3.66 (m, 4H), 3.59−3.52 (m, 2H), 2.98 (t, J = 6.3 Hz, 2H), 2.67 (d, J = 4.2 Hz, 6H), 2.59 (s, 4H). 13 C NMR (101 MHz, DMSO-d6): δ 167.2, 154.5, 151.9, 142.3, 138.0, 138.0, 133.2, 132.2, 125.3, 123.9, 122.4, 118.0, 116.9, 116.4, 113.9, 107.8, 68.0, 66.2, 66.1, 58.2, 57.2, 53.4, 53.2, 4.7, 37.5, 35.5. HRMS (ESI): m/z, calcd for [M − I]+ (C30H38IN5O5) requires 548.2867, found 548.2815. HPLC purity 99.0%. 5-Methyl-11-((3-morpholinopropyl)amino)-7-(2-((3morpholinopropyl)amino)-2-oxoethoxy)benzofuro[3,2-b]quinolin5-ium Iodide (7c7). Compound 5 (241 mg, 0.5 mmol) reacted with 3morpholinopropan-1-amine (720 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c7 (127 mg, 44.3%). Mp 195−197 °C. 1H NMR (400 MHz, CDCl3): δ 8.96 (d, J = 8.2 Hz, 1H), 8.08 (s, 1H), 7.94 (s, 2H), 7.64 (dd, J = 8.7, 1.7 Hz, 2H), 7.44 (d, J = 9.2 Hz, 1H), 4.76 (s, 2H), 4.59 (s, 3H), 4.40 (t, J = 5.8 Hz, 2H), 3.76 (s, 4H), 3.73 (d, J = 3.2 Hz, 4H), 3.49 (dd, J = 11.7, 7.2 Hz, 2H), 2.78−2.68 (m, 2H), 2.60 (s, 4H), 2.49 (d, J = 2.8 Hz, 8H), 2.18−2.11 (m, 2H), 1.91−1.78 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 167.2, 154.6, 152.0, 142.3, 138.3, 138.0, 133.3, 132.1, 125.3, 124.0, 122.5, 118.0, 117.0, 116.3, 114.0, 108.0, 68.1, 66.1, 66.1, 55.9, 55.7, 53.2, 44.2, 37.5, 36.9, 26.7, 25.9. HRMS (ESI): m/z, calcd for [M − I]+ (C32H42IN5O5) requires 576.3180, found 576.3134. HPLC purity 95.7%. 5-Methyl-7-(2-oxo-2-((2-(piperidin-1-yl)ethyl)amino)ethoxy)-11((2-(piperidin-1-yl)ethyl)amino)benzofuro[3,2-b]quinolin-5-ium Iodide (7c8). Compound 5 (241 mg, 0.5 mmol) reacted with 2(piperidin-1-yl)ethanamine (640 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c8 (81 mg, 30.0%). Mp 231−233 °C. 1H NMR (400 MHz, CDCl3): δ 8.84 (d, J = 8.3 Hz, 1H), 8.05 (s, 1H), 7.99 (d, J = 8.9 Hz, 1H), 7.97−7.90 (m, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 9.1 Hz, 1H), 4.77 (s, 2H), 4.61 (s, 3H), 4.32 (t, J = 6.3 Hz, 2H), 3.53 (d, J = 5.6 Hz, 2H), 2.96−2.89 (m, 2H), 2.64 (dd, J = 12.8, 5.4 Hz, 6H), 2.52 (s, 4H), 1.67−1.51 (m, 8H), 1.47 (s, 4H). 13C NMR (101 MHz, CDCl3): δ 166.6, 153.9, 151.6, 141.4, 137.1, 137.0, 132.8, 131.5, 124.9, 124.3, 121.1, 116.0, 115.9, 115.6, 113.0, 107.2, 76.4, 76.1, 75.8, 67.5, 56.7, 56.3, 53.4, 53.3, 34.8, 25.0, 24.7, 23.1. HRMS (ESI): m/z, calcd for [M − I]+ (C32H42IN5O3) requires 544.3282, found 544.3292. HPLC purity 97.8%. 5-Methyl-7-(2-oxo-2-((3-(piperidin-1-yl)propyl)amino)ethoxy)-11((3-(piperidin-1-yl)propyl)amino)benzofuro[3,2-b]quinolin-5-ium Iodide (7c9). Compound 5 (241 mg, 0.5 mmol) reacted with 3(piperidin-1-yl)propan-1-amine (710 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c9 (205 mg, 72%). Mp 192−193 °C. 1H NMR (400 MHz, CD3OD): δ 8.56 (d, J = 8.2 Hz, 1H), 8.32 (d, J = 9.0 Hz, 1H), 8.15−8.07 (m, 1H), 8.06 (s, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 9.0 Hz, 1H), 4.77 (s, 2H), 4.63 (s, 3H), 4.29 (t, J = 6.8 Hz, 2H), 3.39 (t, J = 6.7 Hz, 2H), 2.74−2.49 (m, 12H), 2.22−2.12 (m, 2H), 1.86 (dd, J = 15.1, 7.3 Hz, 2H), 1.66 (s, 8H), 1.53 (s, 4H). 13C NMR (101 MHz, CD3OD): δ 156.2, 154.4, 144.0, 139.7, 134.9, 133.9, 132.4, 129.9, 126.9, 125.3, 123.6, 118.8, 118.5, 118.0, 115.6, 109.4, 69.3, 66.7, 60.2, 56.8, 56.5, 55.0, 54.7, 47.9, 45.2, 38.8, 37.6, 31.7, 30.7, 27.2, 26.0, 25.4, 25.0, 23.9, 23.5, 20.3, 14.1, 9.5, 8.6. HRMS (ESI): m/z, calcd for [M − I]+ (C34H46N5O3) 572.3595, found 572.3585. HPLC purity 95.3%.
was carried out by recrystallization or was purified by column chromatography on silica gel to give a pale yellow or white solid. 11-((2-(Dimethylamino)ethyl)amino)-7-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethoxy)-5-methylbenzofuro[3,2-b]quinolin-5ium Iodide (7c1). Compound 5 (241 mg, 0.5 mmol) reacted with N,N-diethylethane-1,2-diamine (440 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c1 (101 mg, 43.8%). Mp 132−135 °C. 1H NMR (400 MHz, CDCl3): δ 8.86 (d, J = 11.8 Hz, 1H), 8.08 (s, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.93−7.84 (m, 1H), 7.67− 7.59 (m, 2H), 7.43 (d, J = 8.7 Hz, 1H), 4.75 (s, 2H), 4.59 (s, 3H), 4.34−4.27 (m, 2H), 3.55−3.47 (m, 2H), 2.91−2.83 (m, 2H), 2.61− 2.55 (m, 2H), 2.41 (s, 6H), 2.31 (s, 6H). 13C NMR (101 MHz, DMSO-d6): δ 167.2, 154.5, 151.9, 142.2, 138.0, 138.0, 133.2, 132.2, 125.2, 124.0, 122.4, 117.9, 117.0, 116.5, 113.9, 107.8, 68.0, 59.0, 58.0, 45.3, 45.1, 43.5, 37.4, 36.4. HRMS (ESI): m/z, calcd for [M − I]+ (C26H34N5O3) requires 464.2662, found 464.2656. HPLC purity 96.4%. 11-((3-(Dimethylamino)propyl)amino)-7-(2-((3-(dimethylamino)propyl)amino)-2-oxoethoxy)-5-methylbenzofuro[3,2-b]quinolin-5ium Iodide (7c2). Compound 5 (241 mg, 0.5 mmol) reacted with 3(dimethylamino)-1-propylamine (510 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c2 (130 mg, 53%). Mp 211−212 °C. 1H NMR (400 MHz, CD3OD): δ 8.46 (d, J = 8.7 Hz, 1H), 8.32 (d, J = 8.7 Hz, 1H), 8.10 (t, J = 8.3 Hz, 1H), 8.03 (s, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.80 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 9.3 Hz, 1H), 4.75 (s, 2H), 4.63 (s, 3H), 4.30 (t, J = 7.0 Hz, 2H), 3.36 (t, J = 6.9 Hz, 2H), 2.65 (t, J = 6.7 Hz, 2H), 2.40 (d, J = 7.6 Hz, 2H), 2.36 (s, 6H), 2.25 (s, 6H), 2.15−2.09 (m, 2H), 1.81−1.74 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 167.1, 154.7, 152.0, 142.2, 138.1, 137.9, 133.1, 132.4, 125.2, 123.9, 122.2, 117.9, 117.1, 116.7, 114.0, 107.8, 68.1, 56.9, 56.8, 45.0, 44.7, 37.4, 37.0, 27.5, 26.9. HRMS (ESI): m/z, calcd for [M − I]+ (C28H38IN5O3) requires 492.2969, found 492.3008. HPLC purity 97.7%. 11-((4-(Dimethylamino)butyl)amino)-7-(2-((4-(dimethylamino)butyl)amino)-2-oxoethoxy)-5-methylbenzofuro[3,2-b]quinolin-5ium Iodide (7c3). Compound 5 (241 mg, 0.5 mmol) reacted with N1,N1-dimethylbutane-1,4-diamine (580 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c3 (171 mg, 66%). Mp 158−160 °C. 1H NMR (400 MHz, CDCl3): δ 8.89 (d, J = 10.3 Hz, 1H), 8.17 (s, 1H), 7.98 (t, J = 8.2 Hz, 2H), 7.77 (s, 1H), 7.69 (d, J = 9.3 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 4.80 (s, 2H), 4.64 (s, 3H), 4.28 (t, J = 4.8 Hz, 2H), 3.43 (dd, J = 8.4, 4.2 Hz, 2H), 2.69 (s, 4H), 2.45 (d, J = 3.2 Hz, 12H), 2.07 (dd, J = 12.8, 7.0 Hz, 4H), 1.95−1.87 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 167.1, 154.7, 152.0, 142.0, 138.4, 138.2, 133.8, 131.9, 125.6, 124.1, 122.5, 118.3, 116.8, 116.1, 113.9, 107.9, 68.0, 57.5, 57.3, 43.8, 43.4, 37.8, 37.6, 27.7, 26.5, 22.9, 22.6. HRMS (ESI): m/z, calcd for [M − I]+ (C30H42N5O3) 520.3282, found 520.3270. HPLC purity 95. 5%. 11-((2-(Diethylamino)ethyl)amino)-7-(2-((2-(diethylamino)ethyl)amino)-2-oxoethoxy)-5- methylbenzofuro[3,2-b]quinolin-5-ium Iodide (7c4). Compound 5 (241 mg, 0.5 mmol) reacted with N,Ndiethylethane-1,2-diamine (580 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c4 (119 mg, 46%). Mp 186−188 °C. 1H NMR (400 MHz, CDCl3): δ 8.93 (d, J = 8.9 Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.70 (t, J = 8.4 Hz, 2H), 7.59 (s, 1H), 7.46 (d, J = 11.4 Hz, 1H), 4.77 (s, 2H), 4.61 (s, 3H), 4.34 (t, J = 6.4 Hz, 2H), 3.47 (dd, J = 11.1, 5.0 Hz, 2H), 3.03 (t, J = 7.0 Hz, 2H), 2.71 (dd, J = 14.3, 7.0 Hz, 6H), 2.63 (dd, J = 14.2, 7.0 Hz, 4H), 1.07 (dd, J = 14.7, 7.2 Hz, 12H). 13C NMR (101 MHz, DMSO-d6): δ 167.2, 154.5, 151.8, 142.4, 138.0, 137.9, 133.2, 132.2, 125.2, 123.9, 122.4, 118.0, 117.0, 116.4, 113.8, 107.9, 68.0, 52.6, 51.3, 46.8, 46.6, 43.8,37.5, 36.5, 12.0, 11.7. HRMS (ESI): m/z calcd for [M − I]+ (C30H42IN5O3) requires 520.3282, found 520.3350. HPLC purity 97.2%. 11-((3-(Diethylamino)propyl)amino)-7-(2-((3-(diethylamino)propyl)amino)-2-oxoethoxy)-5-methylbenzofuro[3,2-b]quinolin-5ium Iodide (7c5). Compound 5 (241 mg, 0.5 mmol) reacted with N,N-diethylpropane-1,3-diamine (650 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c5 (152 mg, 55.7%). Mp 210−211 °C. 1H NMR (400 MHz, CD3OD): δ 8.23 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.89−7.82 (m, 1H), 7.78 (s, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.40 (d, J = 9.2 Hz, 1H), 5449
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
Journal of Medicinal Chemistry
Article
92%; mp 258−259 °C. 1H NMR (400 MHz, CD3OD): δ 8.44 (d, J = 7.9 Hz, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.09 (t, J = 8.2 Hz, 1H), 7.95 (s, 1H), 7.82 (d, J = 9.5 Hz, 1H), 7.79 (t, J = 7.1 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 4.63 (s, 3H), 4.27 (t, J = 6.6 Hz, 2H), 4.24 (d, J = 5.2 Hz, 2H), 3.63 (d, J = 11.1 Hz, 2H), 3.53 (t, J = 10.3 Hz, 2H), 3.25 (s, 3H), 3.22 (s, 3H), 2.65 (t, J = 6.4 Hz, 2H), 2.36 (s, 6H), 2.27 (s, 1H), 2.20− 2.01 (m, 6H). 13C NMR (101 MHz, DMSO-d6) δ 154.3, 152.0, 142.6, 138.1, 137.8, 133.3, 132.4, 125.4, 124.0, 122.5, 118.2, 117.1, 116.5, 114.0, 108.1, 63.1, 53.3, 52.6, 46.9, 43.8, 37.7, 37.4, 31.4, 23.9, 20.0. HRMS (ESI): m/z, calcd for [M − 2I]2+ (C29H40N4O2) requires 238.1570, found 238.1577. HPLC purity 99.2%. Materials. All oligomers/primers used in this study (Table S5) were purchased from Invitrogen. Stock solutions of all the derivatives (10 mM) were made using DMSO (10%) or double-distilled deionized water. Further dilutions to working concentrations were made with double-distilled deionized water. All other chemicals or solvents were of analytical grade or better. Fluorescence Resonance Energy Transfer (FRET) Assays. FRET assay was carried out on a real-time PCR apparatus following previously published procedures.38 The fluorescently labeled oligonucleotide FPu22T (5′-FAM-TGAGGGTGGGTAGGGTGGGTAATAMRA-3′) was used as the FRET probes. Fluorescence melting curves were determined with a Roche LightCycler 2 real-time PCR machine, using a total reaction volume of 20 μL, with 0.2 μM labeled oligonucleotide in Tris-HCl buffer (10 mM, pH 7.4) containing 2 mM KCl with or without 2 μM disubstituted quindoline derivatives and 1. Fluorescence readings with excitation at 470 nm and detection at 530 nm were taken at intervals of 1 °C over the range 37−99 °C, with a constant temperature being maintained for 30 s prior to each reading to ensure a stable value. The melting of the G-quadruplex was monitored alone or in the presence of various concentrations of compounds. Final analysis of the data was carried out using Origin8.0 (OriginLab Corp.). Microscale Thermophoresis (MST) Assay. The fluorescently labeled oligonucleotides (FAM-Pu27, 5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′, FAM-duplex, 5′-CGCGCGCGTTTTCGCGCGCG-3′) were diluted from stock to the required concentration (10 μM) in 10 mM Tris-HCl buffer, pH 7.4, in the presence of 150 mM KCl. Then the mix was annealed by heating at 95 °C for 5 min, gradually cooled to room temperature, and incubated at 4 °C overnight. The concentration of ligands was varied from 0 to 20 μM (or 200 μM for tested the KD of duplex DNA). A 12-point dilution series was prepared for each DNA. After incubation, the samples were loaded into MST standard-treated glass capillaries, and MST analysis was performed using a Monolith NT.115 instrument (NanoTemper). Circular Dichroism (CD) Measurements. CD experiments were performed on a Chirascan circular dichroism spectrophotometer (Applied Photophysics). A quartz cuvette with 4 mm path length was used for the spectra recorded over a wavelength range of 230−400 at 1 nm bandwidth, 1 nm step size, and 0.5 s per point. The oligomer cMYC (Pu27, 5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′) was diluted from stock to the required concentration (5 μM) in 10 mM Tris-HCl buffer, pH 7.4, in the absence or presence of 100 mM KCl and then annealed by heating at 95 °C for 5 min, gradually cooled to room temperature, and incubated at 4 °C overnight. A buffer baseline was collected in the same cuvette and subtracted from the sample spectra. Final analysis of the data was carried out using Origin 8.0 (OriginLab Corp.). Recombinant Protein and Purification. The wild-type NM23H2 expression was provided by Dr. Edith Postel’s laboratory,53 and the expression was carried out as described.21 Briefly, the BL21 bacteria transfected with pET-28a-NM23-H2 recombinant were inoculated in 5 mL of LB media containing kanamycin and grown overnight at 37 °C at 200 rpm in the incubator. Inoculate 1 mL of the cell culture in 100 mL of LB media containing kanamycin, and incubate it at 37 °C at 250 rpm for 3 h. Add 1 mM IPTG to the media and incubate overnight at 16 °C at 200 rpm to induce the NM23-H2 protein expression. The cell culture was collected and spun down for 30 min at 4 °C at 4000 rpm. Resuspend the pellet in 10 mL of start buffer (20 mM Na3PO4, 500 mM NaCl, pH 7.4). Spin down for 10 min at 14 000 rpm at 4 °C after
5-Methyl-11-((2-(4-methylpiperazin-1-yl)ethyl)amino)-7-(2-((2(4-methylpiperazin-1-yl)- ethyl)amino)-2-oxoethoxy)benzofuro[3,2b]quinolin-5-ium Iodide (7c10). Compound 5 (241 mg, 0.5 mmol) reacted with 2-(4-methylpiperazin-1-yl)ethylamine (715 mg, 5 mmol) in ethylene glycol diethyl ether (4 mL) at 120 °C to afford 7c10 (183 mg, 64%). Mp 177−179 °C. 1H NMR (400 MHz, CD3OD): δ 8.46 (d, J = 8.3 Hz, 1H), 8.23 (d, J = 8.9 Hz, 1H), 8.04−7.97 (m, 1H), 7.95 (s, 1H), 7.79 (d, J = 9.2 Hz, 1H), 7.73−7.66 (m, 1H), 7.56 (dd, J = 9.2, 2.2 Hz, 1H), 4.67 (s, 2H), 4.54 (s, 3H), 4.26 (t, J = 6.5 Hz, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.85 (t, J = 6.5 Hz, 2H), 2.81−2.48 (m, 18H), 2.40 (s, 3H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 167.3, 154.5, 152.0, 142.2, 138.3, 138.0, 133.4, 132.2, 125.4, 124.0, 122.5 118.1, 117.0, 116.2, 114.0, 107.9, 68.0, 57.3, 56.2, 53.9, 53.7, 51.5, 51.2, 44.4, 44.2, 42.7, 37.7, 35.7. HRMS (ESI): m/z, calcd for [M − I]+ (C32H44IN7O3) requires 574.3500, found 574.3489. HPLC purity 97.9%. 11-((2-(Dimethylamino)ethyl)amino)-5-methyl-7-(2(trimethylammonio)ethoxy)benzofuro- [3,2-b]quinolin-5-ium Iodide (7d1). Compound 6a reacted with 10 equiv N1,N1-dimethylethane-1,2-diamine in ethylene glycol diethyl ether at 120 °C (30 min) to afford 7d1. The 7d1 was precipitated after ether titrated in the solution, and the desired product was obtained as a yellow solid. Yield 93%; mp 245−246 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.68 (d, J = 8.2 Hz, 1H), 8.42 (d, J = 8.8 Hz, 1H), 8.10 (d, J = 9.2 Hz, 2H), 7.96 (d, J = 9.1 Hz, 1H), 7.80 (t, J = 7.5 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 4.72 (t, J = 2.6 Hz, 2H), 4.58 (s, 3H), 4.21 (t, J = 5.8 Hz, 2H), 3.91 (t, J = 4.6 Hz, 2H), 3.27 (s, 9H), 2.78 (t, J = 6.0 Hz, 2H), 2.33 (s, 6H). 13 C NMR (101 MHz, DMSO-d6): δ 154.3, 152.0, 142.2, 138.2, 137.9, 133.3, 132.1, 125.3, 124.0, 122.6, 118.0, 116.9, 116.3, 114.1, 108.0, 64.1, 63.2, 58.6, 53.4, 45.0, 43.0, 37.9. HRMS (ESI): m/z, calcd for [M − 2I]2+ (C25H34N4O2) 211.1335, found 211.1329. HPLC purity 99.4%. 11-((3-(Dimethylamino)propyl)amino)-5-methyl-7-(2(trimethylammonio)ethoxy)benzofuro[3,2-b]quinolin-5-ium Iodide (7d2). Compound 6a reacted with 10 equiv N1,N1-dimethylpropane1,3-diamine in ethylene glycol diethyl ether at 120 °C (30 min) to afford 7d2. The 7d2 was precipitated after ether titrated in the solution, and the desired product was obtained as a yellow solid. Yield 92%; mp 242−243 °C. 1H NMR (400 MHz, CD3OD): δ 8.61 (d, J = 8.5 Hz, 1H), 8.35 (d, J = 9.0 Hz, 1H), 8.15−8.08 (m, 2H), 7.92 (d, J = 9.2 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.66 (dd, J = 0.8 Hz, 1H), 4.77 (t, J = 2.0 Hz, 2H), 4.68 (s, 3H), 4.39 (t, J = 6.8 Hz, 2H), 4.00 (t, J = 4.2 Hz, 2H), 3.37 (s, 9H), 3.36 (s, J = 2.6 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.49 (s, 6H). 13C NMR (101 MHz, DMSO-d6): δ 154.3, 152.2, 142.3, 138.3, 138.0, 133.3, 132.3, 125.4, 123.9, 122.5, 118.1, 117.0, 116.4, 114.2, 108.0, 64.1, 63.1, 56.3, 53.3, 44.5, 44.2, 37.8, 27.0. HRMS (ESI): m/z, calcd for [M − 2I]2+ (C26H36I2N4O2) 218.1414, found 218.1411. HPLC purity 98.9%. 11-((3-(Dimethylamino)propyl)amino)-5-methyl-7-(3(trimethylammonio)propoxy)benzofuro[3,2-b]quinolin-5-ium Iodide (7d3). Compound 6b reacted with 10 equiv N 1 ,N 1 dimethylpropane-1,3-diamine in ethylene glycol diethyl ether at 120 °C (30 min) to afford 7d3. The 7d3 was precipitated after ether titrated in the solution, and the desired product was obtained as a yellow solid. Yield 92%; mp 246−247 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.57 (d, J = 8.4 Hz, 1H), 8.39 (d, J = 8.9 Hz, 1H), 8.08 (t, J = 7.6 Hz, 1H), 7.98 (d, J = 1.9 Hz, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.80 (t, J = 8.4 Hz, 1H), 7.56 (dd, J = 9.2, 2.1 Hz, 1H), 4.54 (s, 3H), 4.29 (t, J = 5.6 Hz, 2H), 4.14 (t, J = 6.9 Hz, 2H), 3.64−3.57 (m, 2H), 3.17 (s, 9H), 2.64 (t, J = 4.9 Hz, 2H), 2.34 (s, 6H), 2.30−2.24 (m, 2H), 2.01 (t, J = 6.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6): δ 152.0, 151.2, 142.2, 140.8, 138.6, 138.0, 133.3, 125.4, 123.9, 122.3, 118.1, 117.1, 116.3, 114.1, 107.6, 66.1, 62.9, 56.0, 52.4, 44.3, 44.0, 37.7, 26.8, 22.6. HRMS (ESI): m/z, calcd for [M − 2I]2+ (C27H38N4O2) requires 225.1492, found 225.1493. HPLC purity 99.6%. 11-((3-(Dimethylamino)propyl)amino)-7-((1,1-dimethylpiperidin1-ium-4-yl)methoxy)-5- methylbenzofuro[3,2-b]quinolin-5-ium Iodide (7d4). Compound 6c reacted with 10 equiv N1,N1-dimethylpropane-1,3-diamine in ethylene glycol diethyl ether at 120 °C (30 min) to afford 7d4. The 7d4 was precipitated after ether titrated in the solution, and the desired product was obtained as a yellow solid. Yield 5450
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
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plate (1 × 106 cells per well) separately and incubated overnight. 7a4 was added at final concentration of 4, 2, 1, 0.5 μM and DMSO used as control. After incubation for 12 h, cells were harvested, and the RNA was extracted according to the manufacturer’s instructions. Total RNA was used as a template for reverse transcription using the following protocol: each 25 μL reaction contained 5 μL of 5× M-MLV buffer, 1.25 μL of 2.5 mM dNTP, 2 μL of 100 pM oligo dT18 primer, 1 μL of M-MLV reverse transcriptase, 0.625 μL of 40 U/μL RNase inhibitor, DEPC treated water (DEPC H2O), and 2 μg of total RNA. Briefly, RNA and oligo dT18 primer were incubated at 70 °C for 10 min and then immediately placed on ice. Next, the other components were added and incubated at 42 °C for 1 h and then at 70 °C for 15 min. The reacted solution was stored at −20 °C. NM23-H2, c-MYC, and βactin were amplified by using a real-time PCR apparatus (Roche Light Cycler 2), and the PCR products were analyzed with electrophoresis on 1.5% agarose gel at 120 V for 40 min. MTT Assay. RAJI, CA46, CCRF-CEM, and U266B1 cells were seeded at a density of 5 × 103 cells with 100 μL of culture medium per well. And the cells were incubated in the presence or absence of the indicated concentrations of the 7a4/1 for 12 h; for testing the RAJI cell proliferation inhibition by all the synthesized compounds, the drug treating time was set as 48 h; the control group was administered the same volume of DMSO as the test compounds. Next, add the 0.5 mg/ mL MTT solution into the culture medium and incubate for 4 h at 37 °C. Then, add the lysis solution to each well and incubate at 37 °C overnight. The absorbance was measured at 570 nm using a microplate reader (Biotek, USA). Each assay was carried out in triplicate, and the cytotoxicity was evaluated based on the percentage of cell survival in a dose-dependent manner with regard to the negative control. The final IC50 values were calculated by using the GraphPad Prism 5. Western Blotting. After 7a4 treatment, RAJI cell was lysed with 1% NP-40 lysis buffer. Equal amounts of protein (50 μg/lane) were resolved on a 3−12% SDS−polyacrylamide gel. The proteins were then transferred to nitrocellulose membranes and probed with human anti-NM23-H2 antibody (1:1,000; Santa Cruz Biotechnology), anti-cMYC antibody (1:1000; Cell Signaling Technology), and antiGAPDH antibody (1:1000, abclone). Signal detection was carried out using a secondary antibody conjugated to horseradish peroxidase (1:3000; Bio-Rad) and an enhanced chemiluminescence kit (Cell Signaling Technology). Chromatin Immunoprecipitation. RAJI and CA46 cells were treated with 1 μM 7a4 for 6 h and collected. Chromatin immunoprecipitation (ChIP) experiments were performed using the Pierce agarose ChIP kit (no. 26158, Thermo Scientific) according to the manufacturer’s protocol. In brief, the cells were fixed with 1% formaldehyde and lysed. 10% of the lysate was removed for use as an input. The rest of the lysate was immunoprecipitated with NM23-H2 antibody (SantaCruz, sc-100400) and then pulled down by protein A beads. Normal rabbit IgG (SantaCruz, sc-3888) was used as the negative control. The experiment was repeated twice. The immunoprecipitated DNA samples were then separately analyzed by PCR using c-MYC-ChIP-A and c-MYC-ChIP-S primers (Supporting Information Table S1). Long-Term Cell Proliferation Experiment. Long-term proliferation experiments were carried out using the RAJI cell line. Cells (1.0 × 105) were grown in six-well plates and exposed to a subcytotoxic concentration of a ligand or an equivalent volume of 0.1% DMSO every 3 days. The cells in control and drug-exposed flasks were counted and flasks reseeded with 1.0 × 105 cells. This process was continued for 15 days. Cell-Cycle Detection. The RAJI cell was treated with compounds or DMSO was washed in PBS and fixed with 70% ethanol, then centrifuged and resuspended in a staining solution (50 μg/mL PI, 75 KU/mL RNase A in PBS) for 30 min at room temperature in dark. Cells were analyzed by flow cytometry using an EPICS XL flow cytometer (Beckman Coulter, USA). For each analysis, 10 000 events were collected. The cell cycle distribution was analyzed by EXPO32 ADC software. FITC Annexin V/PI Apoptosis Detection. The cells treated with compounds or control medium and were washed in PBS and detected
the ultrasonic disruption. The supernatant was purified by nickel affinity column, washed repeatedly with washing buffer (20 mM Na3PO4, 500 mM NaCl, 40 mM imidazole, pH 7.4). NM23-H2 protein was eluted (elution buffer: 20 mM Na3PO4, 500 mM NaCl, 500 mM imidazole, pH 7.4) and concentrated by using ultrafiltration column. The protein concentration was determined by BCA assay. And the purity of the protein was verified by SDS−PAGE. Electrophoretic Mobility Shift Assay (EMSA). Labeled Pu27 (1.5 μM) substrates were annealed and mixed with recombinant NM23-H2 (0.5 μM) for 1 h at 37 °C in a total volume of 20 μL. Reactions containing 10 μM G-quadruplex-interactive agents or 5% DMSO were incubated for 1 h at 25 °C before the addition of NM23H2. To separate the protein−DNA complexes from free DNA, the reactions were electrophoresed on 5% native polyacrylamide gels in the presence of 0.5× TBE at 4 °C for ∼1 h at 150 V. Then a gel imaging system with a UV light was used for band visualization. The gray intensity of protein−DNA complex was measured by Quantity One. We set the gray level of the complex in the control lane (without compounds) as 100%, and the later lanes were compared to the second lane to obtain the relative level of the protein−DNA complex. The IC50 value was evaluated via a Hill model in Origin 8. All gel mobility shift assays were done three times. Representative gels are shown in the figures. Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA experiment was performed according to the previous study.21 The streptavidin labeled 96-well high bind plates (Roche) were used in the ELISA assays. The 5′-biotin-labeled c-MYC Pu27 was diluted in 200 μL of Tris-HCl buffer (10 mM, pH 7.4) containing 100 mM KCl at a concentration of 100 nM and then annealed by heating to 95 °C for 5 min followed by cooling to room temperature. To prepare the 7a4 and NM23-H2 mixed samples, the concentration of 7a4 protein was initially 50 μM and half-diluted eight times in blocking buffer (3% BSA in ELISA buffer: 50 mM KH2PO4, 100 mM KCl, pH 7.4), followed by incubating with 250 nM NM23-H2 at 25 °C for 1 h, respectively. Equal volume DMSO was used as control. After that, each compound/ protein mixture was incubated with the labeled DNA in the well at 4 °C overnight, washed three times by washing buffer (ELISA buffer containing 0.1% Tween-20), and incubated with primary antibody (SantaCruz, sc-100400) at 4 °C overnight, washed three times by washing buffer, incubated with secondary HRP-conjugated antibody (CST, 7076s) for 2 h at 37 °C, and then washed three times with washing buffer. TMB Chromogen solution (Life Technologies) was added to each well for color developing, and the reaction was stopped by addition of 50 μL of 1 M H2SO4. Absorbance was read at 450 nm. The ftting curve was obtained using GraphPad Prism 5. Exon Specific Assay. In this test, RAJI and CA46 were used and treated by 7a4 for 12 h in various concentrations. The total RNA was extracted followed by reverse transcription. The amplification of exon 1 is used to monitor the effects on the nontranslocated, G-quadruplexmaintaining c-MYC gene, while amplification of exon 2 will predominantly represent c-MYC expression from the translocated, G-quadruplex-lost chromosome. To determine if the c-MYC-lowering effect of compound 4 is mediated through the c-MYC G-quadruplex and to further examine the mechanism of compound 12, we tested the compounds in this exon-specific assay in RAJI and CA46 cells. Total RNA was used as a template for reverse transcription using the following protocol: each 20 μL reaction contained 1× M-MLV buffer, 2.5 μM dNTP, 100 pM oligo dT18 primer, 1 μL of M-MLV reverse transcriptase, DEPC in water (DEPC−H2O), and 2 μg of total RNA. Briefly, RNA and oligo dT18 primer were incubated at 70 °C for 10 min and then immediately placed on ice. Next, the other components were added and incubated at 42 °C for 1 h and then at 92 °C for 15 min. Finally, the reacted solution was stored at −20 °C. Both c-MYC two exons, NM23-H2 and β-actin were amplified by using a real-time PCR apparatus (Roche LightCycler 2), and the PCR products were analyzed with electrophoresis on 1% agarose gel at 120 V for 20 min. Reverse Transcription Polymerase Chain Reaction (RT-PCR). RAJI, CCRF-CEM, and U266B1 cells were cultivated at 37 °C in RPMI 1640 medium (Life Technologies, Scotland) with 10% fetal calf serum (HyClone, U.S.A.). The four cell lines were plated in six-well 5451
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
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using the FITC annexin V/PI apoptosis detection kit (KeyGEN). Briefly, RAJI cell was digested and resuspended in binding buffer. FITC annexin V and PI were added, and the cells were disturbed by gently vortexing the samples prior to incubation for 15 min in the dark. Emitted florescence was quantitated by Epics Elite flow cytometry (Beckman-Coulter). Xenograft Animal Model and Drug Treatments. NOD/SCID male and female nude mice were obtained from SJA Lab Animal. RAJI cells in the logarithmic phase were harvested, pelleted by centrifugation at 800g for 5 min, and resuspended in 1640 medium with 10% serum to the density of 5 × 106 cells per 100 μL. The cell suspension was then subcutaneously implanted into mouse underarms region. The tumor implanted in mice grows into almost 1000 mm3 after 1 week of feeding. These mice are separated into five groups randomly: negative control, compound 7a4-treated (treating concentrations, 30 and 15 mg/kg), and positive control (1.0 mg/kg doxorubicin-treated). Compound 7a4, doxorubicin, and saline are administered by ip injection to the xenografts animal model. The positive control group was treated with doxorubicin by ip injection once in 2 days. Compound 7a4 was similarly administered to mice, once a day, at a different dose, respectively. The tumor volume and body weight were recorded every day. After treatment for 2 weeks, tumor tissues are collected and tested for weight and volume.
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hnRNP, heterogeneous ribonucleoprotein particle proteins; MST, microscale thermophoresis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHE III1, nuclease hypersensitive element III1; NSEP-1, nuclease-sensitive element protein 1; RT-PCR, reverse transcription polymerase chain reaction; Sp1, specificity protein 1; TAMRA, 6carboxytetramethylrhodamine
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(1) Marcu, K. B. Regulation of expression of the c-myc protooncogene. BioEssays 1987, 6, 28−32. (2) Pelengaris, S.; Khan, M.; Evan, G. I. Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 2002, 109, 321−334. (3) Gregory, M. A.; Hann, S. R. c-Myc proteolysis by the ubiquitinproteasome pathway: stabilization of c-Myc in Burkitt’s lymphoma cells. Mol. Cell. Biol. 2000, 20, 2423−2435. (4) Soucek, L.; Whitfield, J.; Martins, C. P.; Finch, A. J.; Murphy, D. J.; Sodir, N. M.; Karnezis, A. N.; Swigart, L. B.; Nasi, S.; Evan, G. I. Modelling Myc inhibition as a cancer therapy. Nature 2008, 455, 679− 683. (5) Huang, H.; Weng, H.; Zhou, H.; Qu, L. Attacking c-Myc: targeted and combined therapies for cancer. Curr. Pharm. Des. 2014, 20, 6543−6554. (6) Marcu, K. B.; Patel, A. J.; Yang, Y. Differential regulation of the cMYC P1 and P2 promoters in the absence of functional tumor suppressors: implications for mechanisms of deregulated MYC transcription. Curr. Top. Microbiol. Immunol. 1997, 224, 47−56. (7) Berberich, S. J.; Postel, E. H. PuF/NM23-H2/NDPK-B transactivates a human c-myc promoter-CAT gene via a functional nuclease hypersensitive element. Oncogene 1995, 10, 2343−2347. (8) Simonsson, T.; Pecinka, P.; Kubista, M. DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res. 1998, 26, 1167−1172. (9) Simonsson, T.; Sjoback, R. DNA tetraplex formation studied with fluorescence resonance energy transfer. J. Biol. Chem. 1999, 274, 17379−17383. (10) Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11593−11598. (11) Yang, D.; Hurley, L. H. Structure of the biologically relevant Gquadruplex in the c-MYC promoter. Nucleosides, Nucleotides Nucleic Acids 2006, 25, 951−968. (12) Michelotti, E. F.; Michelotti, G. A.; Aronsohn, A. I.; Levens, D. Heterogeneous nuclear ribonucleoprotein K is a transcription factor. Mol. Cell. Biol. 1996, 16, 2350−2360. (13) Postel, E. H.; Mango, S. E.; Flint, S. J. A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor. Mol. Cell. Biol. 1989, 9, 5123−5133. (14) Postel, E. H.; Berberich, S. J.; Flint, S. J.; Ferrone, C. A. Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science 1993, 261, 478−480. (15) Thakur, R. K.; Kumar, P.; Halder, K.; Verma, A.; Kar, A.; Parent, J. L.; Basundra, R.; Kumar, A.; Chowdhury, S. Metastases suppressor NM23-H2 interaction with G-quadruplex DNA within c-MYC promoter nuclease hypersensitive element induces c-MYC expression. Nucleic Acids Res. 2009, 37, 172−183. (16) Michelotti, E. F.; Tomonaga, T.; Krutzsch, H.; Levens, D. Cellular nucleic acid binding protein regulates the CT element of the human c-myc protooncogene. J. Biol. Chem. 1995, 270, 9494−9499. (17) Takimoto, M.; Tomonaga, T.; Matunis, M.; Avigan, M.; Krutzsch, H.; Dreyfuss, G.; Levens, D. Specific binding of heterogeneous ribonucleoprotein particle protein K to the human cmyc promoter, in vitro. J. Biol. Chem. 1993, 268, 18249−18258. (18) Kolluri, R.; Torrey, T. A.; Kinniburgh, A. J. A CT promoter element binding protein: definition of a double-strand and a novel
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00099. Synthesis of intermediates and final products, FRET data, CD spectrum, MST data, molecular modeling data, EMSA for testing intervention of NM23-H2/c-MYC binding by disubstituted quindoline derivatives, MTT data, RT-PCR data for evaluating effects of 7a4 on cMYC transcription in Burkitt’s lymphoma cells, cellular uptake of 7a4 in RAJI cell (PDF) Molecular formula strings and some data (CSV)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-20-39943056. Fax: + 86-20-39943056. E-mail:
[email protected].. ORCID
Tian-Miao Ou: 0000-0002-8176-4576 Zhi-Shu Huang: 0000-0002-6211-5482 Author Contributions †
H.-Y.L. and A.-C.C. contributed equally.
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), the Science Foundation of Guangzhou (Grant 2010U1-E00531-1), and Guangdong Provincial Key Laboratory of Construction Foundation (Grant 2011A060901014) for financial support of this study.
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ABBREVIATIONS USED CD, circular dichroism; ChIP-PCR, chromatin immunoprecipitation polymerase chain reaction; CNBP, cellular nucleic acid binding protein; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FAM, 6-carboxyfluorescein; FRET, fluorescence resonance energy transfer; 5452
DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
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DOI: 10.1021/acs.jmedchem.7b00099 J. Med. Chem. 2017, 60, 5438−5454
