Discovery of Novel Antitumor Dibenzocyclooctatetraene Derivatives and Related Biphenyls as Potent Inhibitors of NF-\xce\xbaB Signaling Pathway
Several dibenzocyclooctatetraene derivatives (5-7) and related biphenyls (8-11) were designed, synthesized, and evaluated for inhibition of cancer cell growth and the NF-\xce\xbaB signaling pathway. Compound 5a, a dibenzocyclooctatetraene succinimide, was discovered as a potent inhibitor of the NF-\xce\xbaB signaling pathway with significant antitumor activity against several human tumor cell lines (GI50 1.38\xe2\x80\x931.45 \xce\xbcM) and was more potent than paclitaxel against the drug-resistant KBvin cell line. Compound 5a also inhibited LPS-induced NF-\xce\xbaB activation in RAW264.7 cells with an IC50 value of 0.52 \xce\xbcM, prevented I\xce\xbaB-\xce\xb1 degradation and p65 nuclear translocation, and suppressed LPS-induced NO production in a dose-dependent manner. The antitumor data in cellular assays indicated that relative positions and types of substituents on the dibenzocyclooctatetraene or acyclic biphenyl as well as torsional angles between the two phenyls are of primary importance to antitumor activity.
1. Instruction<!>2. Chemistry<!>3. Results and Discussion<!>4. Conclusions<!>5.1 Chemistry<!>5.1.1 General process of Suzuki cross-coupling to prepare mono- or di-formylbiphenyls (4a-f)<!>5.1.1.1 2,2\xe2\x80\xb2-Diformyl-4\xe2\x80\xb2,5\xe2\x80\xb2-methylenedioxy-4,5,6-trimethoxybiphenyl (4a)<!>5.1.1.2 2,2\xe2\x80\xb2-Diformyl-4,4\xe2\x80\xb2,5,5\xe2\x80\xb2,6-pentamethoxybiphenyl (4b)<!>5.1.1.3.2,2\xe2\x80\xb2-Diformyl-4,5,6-trimethoxybiphenyl (4c)<!>5.1.1.4. 2-Carbomethoxy-2\xe2\x80\xb2-formyl-4,5,6-trimethoxybiphenyl (4d)<!>5.1.1.5. 2,2\xe2\x80\xb2-Diformyl-4,5-methylenedioxy-6-methoxybiphenyl (4e)<!>5.1.1.6. 2,2\xe2\x80\xb2-Diformyl-5,6-methylenedioxy-4-methoxybiphenyl (4f)<!>5.1.2 General procedure for preparing dibenzocyclooctatetraenes 5a-c<!>5.1.2.1 (5Z,7Z)-10,11-Methylenedioxy-1,2,3-trimethoxydibenzocyclooctatetraene-6,7-succinimide (5a)<!>5.1.2.2 (5Z,7Z)-1,2,3,10,11-Pentamethoxydibenzocyclooctatetraene-6,7-succinimide (5b)<!>5.1.2.3 (5Z,7Z)-1,2,3-Trimethoxydibenzocyclooctatetraene-6,7-succinimide (5c)<!>5.1.3.1 (5Z,7Z)-6,7-Dicarbomethoxy-10,11-methylenedioxy-1,2,3-trimethoxydibenzo-cyclooctatetraene (6a)<!>5.1.3.2 (5Z,7Z)-6,7-Dicarbomethoxy-1,2,3,10,11-pentamethoxydibenzocyclooc tatetraene (6b)<!>5.1.4 (5Z, 7Z)-6,7-Dicarboxy-10,11-methylenedioxy-1,2,3-trimethoxydibenzocyclooctatetraene (7)<!>5.1.5 General procedure for preparing nitrovinylbiphenyls (8d, 8e, 9a, 9d, 10a, 11a, and 11b)<!>5.1.5.1. (E)-2,2\xe2\x80\xb2-Dinitrovinyl-4,5,6-trimethoxybiphenyl (8d)<!>5.1.5.2. (E)-2-Carbomethoxy-2\xe2\x80\xb2-(2-methylnitrovinyl)-4,5,6-trimethoxybiphenyl (8e)<!>5.1.5.3 (E)-2,2-Di(2-methylnitrovinnyl)-4\xe2\x80\xb2,5\xe2\x80\xb2-methylenedioxy-4,5,6-trimethoxybiphenyl (9a)<!>5.1.5.4. (E)-2,2\xe2\x80\xb2-Dinitrovinyl-4,5\xe2\x80\xb2-methylenedioxy-4,5,6-trimethoxybiphenyl (9d)<!>5.1.5.5. (E)-2,2\xe2\x80\xb2-Di(2-methylnitrovinyl)-4,4\xe2\x80\xb2,5,5\xe2\x80\xb2,6-pentamethoxybiphenyl (10a)<!>5.1.5.6 (E)-2,2\xe2\x80\xb2-Di(2-methylnitrovinyl)-6-methoxy-4,5-methylenedioxybiphenyl (11a)<!>5.1.5 7 (E)-2,2\xe2\x80\xb2-Di(2-methylnitrovinyl)-4-methoxy-5,6-methylenedioxybiphenyl (11b)<!>5.1.6. General procedure of preparing (2-carboethoxy)cyanovinylbiphenyls (8b, 8f, 9b, 10b)<!>5.1.6.1. (E)-2,2\xe2\x80\xb2-Di(2-carbomethoxy)cyanovinyl-4,5,6-trimethoxybiphenyl (8b)<!>5.1.6.2. 2-Carbomethoxy-2\xe2\x80\xb2-(2-carbomethoxy)cyanovinyl-4,5,6-trimethoxybiphenyl (8f)<!>5.1.6.3. (E)-2,2\xe2\x80\xb2-Di(2-carbomethoxy)cyanovinyl-4\xe2\x80\xb2,5\xe2\x80\xb2-methylenedioxy-4,5,6-trimethoxybiphenyl (9b)<!>5.1.6.4. (E)-2,2\xe2\x80\xb2-Di(2-carbomethoxy)cyanovinyl-4,4\xe2\x80\xb2,5,5\xe2\x80\xb2,6-pentamethoxybiphenyl (10b)<!>5.1.7. General procedure for preparing 2,2\xe2\x80\xb2-dicyanovinyl)biphenyls (8c, 8g, 9c, and 10c)<!>5.1.7.1. 2,2\xe2\x80\xb2-Di(2,2\xe2\x80\xb2-dicyanovinyl)-4,5,6-trimethoxybiphenyl (8c)<!>5.1.7.2. 2-Carbomethoxy-2\xe2\x80\xb2-(2,2-dicyanovinyl)-4,5,6-trimethoxybiphenyl (8g)<!>5.1.7.3. 2,2\xe2\x80\xb2-Di(2,2\xe2\x80\xb2-dicyanovinyl)-4\xe2\x80\xb2,5\xe2\x80\xb2-methylenedioxy-4,5,6-trimethoxybiphenyl (9c)<!>5.1.7.4. 2,2\xe2\x80\xb2-Di(2,2\xe2\x80\xb2-dicyanovinyl)-4\xe2\x80\xb2,5\xe2\x80\xb2,4,5,6-pentamethoxybiphenyl (10c)<!>5.2.1. Anti-tumor activity against tumor cell lines in HTCL assay<!>5.2.2. NF-\xce\xbaB luciferase reporter gene assays<!>5.2.3. Protein isolation and Western blot analysis<!>5.2.4 Nitric oxide determination<!>5.3. Conformation search method
<p>Despite many technological advances during the past two decades, natural products remain one of the most important sources of novel leads for new drugs. Dibenzocyclooctadiene lignans are major constituents of the traditional Chinese medicinal plant Schizandra chinensis, and show diverse biological activities,1,2 including antiviral, hepatoprotective, anticancer, and anti-inflammatory effects. Various synthetic studies on natural dibenzocyclooctadiene lignans were reported during the 1970s,3,4 and recently, interest in this area has revived.5,6,7 During our prior attempts to synthesize the natural product gomisin G,8 we produced and evaluated a series of unsymmetrical biphenyls for cytotoxicity against several human cancer cell lines, resulting in the discovery of a new lead compound, 1 (Figure 1).9 Compound 1 showed promising cytotoxicity against human A549 (lung), DU145 (prostate), KB (nasopharyngeal), and drug-resistant KBvin cancer cell lines with low GI50 values of 0.12, 0.29, 0.41 and 0.51 μM, respectively. Notably, lead 1 displayed similar potencies against KB and paclitaxel-resistant KBvin cell lines, while the anticancer drug paclitaxel exhibited significantly reduced potency against KBvin compared with KB cells (GI50 1800 and 8 nM, respectively). In a continuing study to explore novel antitumor agents, gomisin G analogs (5-7) with a dibenzocyclooctatetraene skeleton were synthesized from unsymmetrical 2,2′-diformylbiphenyls. Meanwhile, new biphenyls (8-11) based on lead 1 were also obtained from formylbiphenyl intermediates. Structurally, the biphenyl compounds are analogous to dibenzocyclooctatetraenes lacking a C6-C7 bond to form the cyclooctatetranene ring. Various 2,2′-substituents (R1 and R2) with a conjugated double-bond were introduced and the biphenyl A- and B-tings were modified with different patterns of methoxy and methylenedioxy groups, which are commonly found in natural products. Steric compression between the two bulky 2,2′-groups could affect torsion angles between the two phenyl rings of the biphenyl series, which could in turn affect the biological activity. Thus, we were interested in whether correlations would be found between activities of the two series, acyclic biphenyls and dibenzocyclooctatetraenes. To evaluate antitumor activity, newly synthesized compounds were initially evaluated in human tumor cell lines (HTCL) assays, and active compounds were then tested for inhibition of the nuclear factor kappa B (NF-κB) signaling pathway, as aberrant NF-κB regulation is observed in various hematological malignancies and solid tumors.10,11 Continuing activation of NF-κB in tumor cells turns on expression of genes that maintain cell proliferation and protect the cell from death via apoptosis. Thus, blocking NF-κB and its signaling pathway can cause tumor cells to stop proliferating, to die, or to be more sensitive to the action of antitumor agents, providing promising target(s) and approaches for anticancer therapy.12 Herein, we report the synthesis of dibenzocyclooctatetraene derivatives and related biphenyl compounds, their antitumor activity against a HTCL panel, inhibitory activity against the NF-κB signaling pathway, SAR analysis, and results on mechanism studies using the most active compound 5a as a probe.</p><!><p>Chemical syntheses of the two series of target compounds are described in Schemes 1 and 2, respectively. The key intermediate unsymmetrical mono- and di-formylbiphenyls (4) were synthesized by a Suzuki cross-coupling reaction between a phenylboronic acid (2) and a bromobenzene (3). As shown in Scheme 1, 6-formyl-2,3,4-trimethoxyphenylboronic acid (2a) was coupled individually with 2-bromopiperonal (3a), 2-bromoveratraldehyde (3b), or 2-bromobenzaldehyde (3c) using Pd(dppf)Cl2 (5% mol) as catalyst in the presence of anhydrous Cs2CO3 to produce 2,2′-diformylbiphenyls 4a-c, respectively. Subsequently, 4a-c were reacted with a stable phosphorane Wittig reagent formed by Bu3P and maleimide,13 followed by an intramolecular Knoevenagel condensation to afford dibenzocyclooctatetraene succinimides 5a-c, respectively. In a similar manner, dibenzocyclooctatetraene compounds 6a and 6b were prepared by Wittig reaction of dimethyl maleate and Bu3P with diformylbiphenyl 4a and 4b, respectively, and a subsequent intramolecular cyclization via Knoevenagel condensation in the presence of piperidine and HOAc in benzene under microwave irradiation. Wittig reagents prepared using Bu3P are easily purified, because a water-soluble phosphine oxide Bu3P=O is produced. Succinimide compound 5a was further hydrolyzed under basic conditions to afford 6,7-dicarboxylic dibenzocyclooctatetraene analog 7. Syntheses of unsymmetrical biphenyls with bulky 2,2′-conjugated substituents (8-11) are shown in Scheme 2. A Suzuki cross-coupling reaction was used to prepare formylbiphenyl intermediates 4d-f from commercially available 2-formylphenylboronic acid (2b) with methyl 2-bromo-3,4,5- trimethoxybenzoate (3d), 2-bromo-3-methoxy-4,5-methylenedioxybenzaldehyde (3e), or 2-bromo-3,4-methylenedioxy-5-methoxybenzaldehyde (3f), respectively. Next, Knoevenagel condensation of formylbiphenyls 4a-f with nitroethane, nitromethane, ethyl cyanoacetate, or malononitrile, respectively, yielded corresponding unsymmetrical biphenyls 8-11 with 2-and/or 2′-substituents containing a conjugated double bond. Series 8 compounds have no other substituents on the phenyl B-ring, while series 9 and 10 have a methylenedioxy (9a-d) or two methoxy groups (10a-c) at the 4′- and 5′-positions on the B-ring, respectively. In addition, modifications on the A-ring of 1 produced a pair of isomers 11a and 11b, in which a methylenedioxy plus a methoxy group replaced the original three methoxy groups on the phenyl A-ring. All new compounds were identified by mass and nuclear magnetic resonance spectra.</p><!><p>According to procedures described previously,14,15 the newly synthesized dibenzocyclooctadienes (5-7) and unsymmetrical biphenyl compounds (8–11) were tested against a HTCL panel, including A549, DU145, KB, and KBvin cell lines, with paclitaxel as the positive control. In vitro anticancer activity (GI50) was determined using the established SRB (sulforhodamine B) method.16 To explore the possible biological target and mechanism of action, active compounds were screened initially at 10 μM in RAW264.7 cells to evaluate NF-κB inhibitory activity. Compounds that showed greater than 98% inhibition at this concentration were further evaluated at serial concentrations to obtain corresponding IC50 values in parallel with positive control BAY 11-7086. Related data of the two compound series are shown in Tables 1 and 2. Interestingly, dibenzocyclooctatetraene succinimide compounds 5a-c showed promising inhibitory activity in the above assays. The most potent compound 5a exhibited low GI50 values (1.38–1.47 μM) against cancer cell proliferation in the tested HTCL panel and a submicromolar IC50 value of 0.52 μM in the NF-κB signaling pathway assay. However, when the succinimide ring was opened, dibenzocyclooctatetraene 6,7-dicarboethoxy compounds 6a, 6b, or 6,7-dicarboxylate compound 7 were at least 10-fold less potent in comparison with 5a, suggesting that the succinimide moiety might be important for antitumor activity. In addition, comparison of 5a with 5b and 6a with 6b indicate that a methylenedioxy on the phenyl ring (B-ring) is more favorable than two methoxy groups at the same positions.</p><p>The new series of unsymmetrical biphenyls extended our prior studies and provided more SAR information. The series 8 compounds were designed to have the same biphenyl scaffold as lead 1, but different 2,2′-substituents, including nitrovinyl, cyanovinyl, and ester groups. Compound 8d with 2,2′-dinitrovinyl substitution showed potent antitumor activity with GI50 values ranging from 2.09 to 4.60 μM in the HTCL panel, but was less potent than 1. Compounds 8e with a 2-carbomethoxy and a 2′-(2-methyl-2- nitro)vinyl group and 8b with 2,2′-di-(2-carboethoxy-2-cyano)vinyl groups exhibited moderate potency with low micromolar GI50 values ranging from 6.17 to 6.95 μM and 6.53 to 8.45 μM, respectively but were less potent than 8d. In contrast, 8c, 8f, and 8g without a nitrovinyl group at the 2,2′-position were inactive (GI50 >20 μM).</p><p>The same potency pattern was observed in series 9 and 10 biphenyls, which contain a 4′,5′-methylenedioxy or 4′,5′-dimethoxy substitution, respectively, on the phenyl B-ring. As expected, compound 9a with 2,2′-di-(2-methyl-2-nitro)vinyl groups showed low micromolar GI50 values ranging from 0.72 to 1.51 μM in the cellular assays, which were similar to those of lead 1 and 5a. Compound 9a was more potent than 2,2′-dinitrovinyl 9d (GI50 5.49–9.09 μM), di-2-carboethoxy-2-cyanovinyl 9b (GI50 >22 μM), and 2,2′-dicyanovinyl 9c (GI50 >40 μM). Similarly, 10a was also more potent than 10b and 10c.</p><p>Di-(2-methyl-2-nitro)vinyl biphenyls 11a and 11b each have with one methylenedioxy (positions 4/5 in 11a and 5/6 in 11b) and one methoxy group (position 6 in 11a and 4 in 11b) on the A-ring, rather than the three methoxy groups found in lead 1. Neither compound was more potent than 1 in the antitumor cellular assays. However, compound 11a displayed selective potency against A549 and DU145 cell proliferation (GI50 4.02 and 5.02 μM respectively) compared with KB and KBvin (GI50 13.6 and 12.31μM), while 11b was less potent against all four cell lines (GI50 13.89–19.82 μM).</p><p>In summary, the cellular assay results on the biphenyls indicated that (1) 2,2′-substituents significantly affect antitumor potency regardless of other substituents on the biphenyl ring system, (2) conjugated nitrovinyl substituents at the 2,2′-positions are more favorable than other substituents on these positions, which is consistent with prior literature,17 and (3) trimethoxy substitution on the biphenyl A-ring is preferred compared with both methoxy and methylenedioxy groups at different positions based on a limited data set.</p><p>Active dibenzocyclooctatetraene (5a-c) and nitrovinylbiphenyls (1, 8d, 8e, 9a, 9d, 10a, 11a and 11b) were further assayed for inhibitory effects on the NF-κB signaling pathway in RAW264.7 cells, which are stably transfected with a luciferase reporter gene controlled by NF-κB activation.18 Interestingly, the compounds exhibited promising inhibitory activity in this assay with IC50 values ranging from 0.52 to 2.16 μM. The inhibitory potency was comparable or greater than that of positive control BAY 11-7086 (IC50 1.72 μM), a pharmacological inhibitor of IκB kinase, indicating that these active compounds do interfere with the NF-κB signaling pathway. Consequently, dibenzocyclooctatetraene succinimide 5a was tested in parallel with positive control BAY 11-7082 in further assays based on the NF-κB signaling pathway. Firstly, we found that 5a indeed suppressed lipopolysaccharide (LPS)-induced NF-κB activation dose-dependently As shown in Fig 2, RAW264.7 cells, stably expressing the NF-κB luciferase reporter gene, were stimulated with LPS to increase luciferase activity in the presence or absence of 5a before measurement of luciferase activity. The cells treated with 10 μM of 5a completely inhibited LPS-induced NF-κB activation comparable to BAY11-7082 at 20 μM. Next, the underlying mechanisms of action of 5a were investigated. As a pharmacological inhibitor of the actions of IκB kinase, BAY 11-7082 can prevent the translocation of free NF-κB to the nucleus and, thus, was used as a reference in Western blotting to confirm the action of 5a. As shown in Fig. 3, the p65 subunit of NF-κB, a major component in NF-κB complex activation, was translocated into the nucleus after LPS challenge. However, pretreatment with 5a substantially inhibited translocation of p65 in a dose-dependent manner. Since IκB protein degradation is an essential step for NF-κB activation induced by LPS, Figure 3 also illustrates that LPS stimulation induced a marked degradation of IκB, while this degradation was suppressed by 5a in a dose-dependent manner. On the other hand, nitric oxide (NO) is known to function as a pro-inflammatory mediator in the pathogenesis of inflammation. Overproduction of NO may have detrimental consequences and seems to be involved in the pathophysiology of various human diseases. As shown in Fig 4, RAW264.7 cells without LPS stimulation produced minimal NO in the supernatant, while cells released significantly higher levels of NO in response to LPS exposure. When RAW264.7 cells were treated with 5a, the elevation of NO release was significantly reduced relative to the dose. The reduction in LPS-induced NO production to 5a at a concentration of 2.5 μM was comparable with the decrease in response to a higher concentration (20 μM) of BAY 11-7082. Based on the results, we suggest that 5a is a more effective NO inhibitor than BAY 11-7082.</p><p>Because molecular binding orientation is related to biological activity, we wanted to observe changes in the biphenyl torsion angle, C2-C1-C1′-C6′ (Fig 5), as well as determine possible relationships between dibenzocyclooctatetraenes and acyclic biphenyls. Therefore, even though the actual biological target(s) has not been identified, we performed conformational searches on the two compound series using Discovery Studio 3.0. For the minimized conformers of dibenzocyclooctatetraenes, the biphenyl torsion angles of C2-C1-C1′-C6′ ranged from 110° to 130 ° (defined as in Fig 5), irrespective to whe ther the compound was active or inactive. For example, active compound 5a shows a torsional angle of 129.8° and inactive 6b has a 114.1° angle. Considering the steric compression of the 2,2′-bulky groups, we postulated that the C2-C1-C1′-C6′ torsional angle in the acyclic biphenyls would deviate from those of related dibenzocyclooctatetraenes. Interestingly, we found that active dinitrovinylbiphenyl 9a displayed a torsional angle of 122.2° similar to that of active 5a, while the torsional angle of inactive 2,2′-dicyanovinyl biphenyl 9c (101.4°) did not fall in the above range. The conformers of representative compounds are shown in Figure 5. We suggest that the torsional angle between the two phenyl rings, either in dibenzocyclooctatetraenes or biphenyls, may impact the molecular antitumor activity because molecular binding orientations can affect interactions between small molecules and their biological target.</p><!><p>This study discovered new dibenzocyclooctatetraene succinimides (5a-c) and related unsymmetric 2- and/or 2′-nitrovinyl biphenyls (8b, 8d-e, 9a, 9d, 10a, 11a) with promising cytotoxic (GI50 < 10 μM in a HTCL panel) and NF-κB inhibitory (IC50 values 0.52–3.02 μM) activities. The results suggest that these antitumor agents are potential NF-κB signaling pathway inhibitors. In mechanism of action studies based on NF-κB signaling pathway assays, the most potent compound 5a suppressed lipopolysaccharide (LPS)-induced NF-κB activation in RAW264.7 cells (IC50 0.52 μM), inhibited LPS-induced degradation of IκBα and nuclear translocation of p65, and significantly blocked LPS-induced nitric oxide (NO) production at low concentrations (0.6–2.5 μM). The results demonstrated that 5a has an inhibitory profile similar to that of IKK inhibitor BAY 11-7082, implying 5a may reduce LPS-induced NF-κB activation via inhibition of IKK activities. The antitumor data in cellular assays indicated that the relative positions and types of substituents either on the dibenzocyclooctatetraene or the acyclic biphenyl as well as the torsional angles between the two phenyls are of primary importance to antitumor activity.</p><!><p>Melting points were measured with a RY-1 melting apparatus without correction. The proton nuclear magnetic resonance (1H NMR) spectra were measured on a JNM-ECA-400 (400 MHz) spectrometer using tetramethylsilane (TMS) as internal standard. The solvent used was CDCl3 unless indicated. Mass spectra (MS) were measured on API-150EX mass spectrometer with electrospray ionization connected with an Agilent 1100 system. Thin-layer chromatography (TLC) was performed on silica gel GF254 plates. Silica gel GF254 and H (200-300 mesh) from Qingdao Haiyang Chemical Company was used for TLC, preparative TLC, and column chromatography respectively. Medium-pressure column chromatography was performed using a CombiFlash® companion system from ISCO, Inc. to purify target compounds. All chemicals were obtained from Beijing Chemical Works or Sigma-Aldrich, Inc.</p><!><p>A mixture of a substituted bromobenzene (1 equiv) and a phenylboronic acid (1.5 equiv) in the presence of Pd(dppf)Cl2 (5% mol) and Cs2CO3 (2.0 equiv) in 1,2-dimethoxyethane (DME, ca 10 mL) was heated to 80 °C for 8–12 h under N2 protection. After cooling to room temperature, the mixture was diluted with EtOAc and filtered through celite. The filtrate was washed with water and brine, successively, and dried over Na2SO4. After removal of solvent in vacuo, the residue was purified by a flash column (gradual elution: EtOAc/petroleum ether 0 ∼ 40%) to afford corresponding formylbiphenyls.</p><!><p>Starting with 2-bromopiperonal (3a, 300 mg, 1.31 mmol) and 6-formyl-2,3,4-trimethoxyphenyl boronic acid (2a, 470 mg, 1.96 mmol) to afford 210 mg of 4a, 47% yield, white solid, 138-140 °C; 1H NMR δ ppm 9.64 & 9.58 (each 1H, s, CHO), 7.51 (1H, s, ArH-3′), 7.38 (1H, s, ArH-3), 6.74 (1H, s, ArH-6′), 6.15 (2H, s, OCH2O), 4.00 & 3.98 (each 3H, s, OCH3), 3.63 (3H, s, OCH3); MS m/z (%) 367.1 (M + 23, 40), 299.3 (M – 45, 100).</p><!><p>Starting with 2-bromo veratraldehyde (3b, 200 mg, 0.82 mmol) and 2a (310 mg, 1.29 mmol) to afford 207 mg of 4b, 70% yield, white solid, mp 142-144 °C; 1H NMR δ ppm 9.65 & 9.52 (each 1H, s, CHO), 7.76 (1H, s, ArH-3′), 7.40 (1H, s, ArH-3), 6.73 (1H, s, ArH-6′), 4.02 ∼ 3.94 (12H, ms, OCH3 × 4), 3.61 (3H, s, OCH3); MS m/z (%) 383.3 (M + 23, 50), 315.2 (M – 45, 100).</p><!><p>Starting with 2-bromobenzaldehyde (3c, 200 mg, 1.08 mmol) and 2a (390 mg, 1.62 mmol) to afford 300 mg of 4c, 92% yield, pale-yellow oil; 1H NMR δ ppm 9.85 & 9.60 (each 1H, s, CHO), 8.09 (1H, d, J = 8.0 Hz, ArH-3′), 7.67 & 7.60 (each 1H, t, J = 8.0 Hz, ArH-4′, 5′), 7.42 (1H, s, ArH-3), 7.32 (1H, d, J = 8.0 Hz, ArH-6), 4.01 & 3.99 (each 3H, s, OCH3), 3.55 (3H, s, OCH3); MS m/z (%) 323.3 (M + 23, 10), 255.3 (M – 45, 100).</p><!><p>Starting with methyl 2-bromo-3,4,5-trimethoxybenzoate (3d, 500 mg, 1.64 mmol) and 2-formylphenylboronic acid (2b, 295 mg, 1.97 mmol) to afford 326 mg of 4d, 60% yield, pale-yellow oil. 1H NMR δ ppm 9.84 (1H, s, CHO), 8.23 (1H, d, J = 8.0 Hz, ArH-3′), 7.60 & 7. 50 (each 1H, t, J = 8.0 Hz, ArH-4′, 5′), 7.37 (1H, s, ArH-3), 7.19 (1H, d, J = 8.0 Hz, ArH-6′), 3.97, 3.96, 3.58, and 3.54 (12H, ms, OCH3 × 4); MS m/z (%) 271.3 (M – 59, 40), 256.3 (M – 64, 100).</p><!><p>Starting with 2-bromo-3-methoxy-4,5-methylenedioxybenzaldehyde (3e, 130 mg, 0.5 mmol) and 2b (150 mg, 1 mmol) to afford 98 mg of 4e, 68% yield, white solid, mp 125-126 °C; 1H-NMR δ ppm 9.86 and 9.47 (each 1H, s, CHO), 8.06 (1H, d, J = 7.6 Hz, ArH-3′), 7.67 (1H, t, J = 7.6 Hz, ArH-4′), 7.64 (1H, t, J = 7.6 Hz, ArH-5′), 7.27 (1H, s, ArH-3), 7.25 (1H, d, J = 7.6 Hz, ArH-6′), 6.13 (2H, s, CH2), 3.83 (3H, s, OMe); MS m/z (%) 307.3 (M + 23, 45), 239.4 (M – 45, 100).</p><!><p>Starting with 2-bromo-3,4-methylenedioxy-5-methoxybenzaldehyde 3f (129 mg, 0.5 mmol) and 2b (150 mg, 1.0 mmol) to afford 100 mg of 4f, 70% yield, white solid, mp 112-114 °C; 1H-NMR δ ppm 9.95 and 9.68 (each 1H, s, CHO), 8.07 (1H, dd, J = 1.6 & 7.6 Hz, ArH-3′), 7.70 (1H, t, J = 7.6 Hz, ArH-4′), 7.61 (1H, t, J = 7.6 Hz, ArH-5′), 7.39 (1H, s, ArH-3), 7.37 (1H, dd, J = 1.6 & 7.6 Hz, ArH-6′), 6.13 (2H, s, OCH2O), 3.83 (3H, s, OMe), MS m/z (%) 307.5 (M + 23, 20).</p><!><p>A mixture of 2,2′-diformylbiphenyl (4a, 4b, or 4c, 1 equiv) and maleimide (1.5 equiv) in anhydrous THF (5-15 mL) in the presence of Bu3P (1.5 equiv) was heated to reflux for 8–12 h under N2 protection. After removal of solvent, the residue was dissolved in HOAc (3–15 mL) and 1–3 drops of piperidine were added. The mixture was heated to 100 °C for 4–6 h with TLC monitoring, then poured into ice-water. The solid was filtered and washed with water to neutral, dried, and purified by a flash column (gradual elution: EtOAc/H2Cl2 0 ∼ 20%) to afford corresponding desired pure product.</p><!><p>Starting with 4a (100 mg, 0.29 mmol) to afford 5a, purple solid, 74 mg, 62% yield, mp 266–268 °C; 1H NMR δ ppm 9.01 (1H, s, =CH), 7.77 (1H, s, =CH), 7.50 (1H, br, NH), 7.23 & 7.18 (each 1H, s, ArH-5 or 12), 6.85 (1H, s, ArH-9), 6.13 (2H, s, OCH2O), 4.04, 3.99, and 3.96 (each 3H, s, OCH3); 13C NMR (DMSO-d6) δ 172.33, 172.20, 151.89, 151.35, 149.00, 146.59, 144.92, 142.50, 130.21, 129.59, 126.83, 126.56, 125.70, 123.18, 118.79, 106.35, 104.10, 103.50, 101.81, 60.94, 60.48, 55.80; MS m/z (%) 408.5 (M + 1, 100).</p><!><p>Starting with 4b (1.0 g, 2.47 mmol) to afford 530 mg of 5b, 51% yield, orange solid, 231–132 °C, 1H NMR (DMSO-d6) δ ppm 11.14 (1H, s, NH), 8.97 (1H, s, =CH), 7.82 (1H, s, =CH), 7.47 and 7.32 (each 1H, s, ArH), 7.13 (1H, s, ArH), 4.02 ∼ 3.92 (15H, ms, OCH3 × 5); MS m/z (%) 424.2 (M + 1, 100).</p><!><p>Starting with 4c (300 mg, 1 mmol) to afford 5c, orange solid, 130 mg, 31% yield, 212–214 °C; 1H NMR δ ppm 9.54 (1H, d, J = 8.4 Hz, ArH), 7.89 (2H, m, ArH), 7.70 (1H, t, J = 8.4 Hz, ArH-5), 7.60 (2H, m, ArH, NH), 7.20 & 6.88 (each 1H, s, =CH), 4.06, 4.02, and 3.97 (each 3H, s, OCH3 × 3); 13C NMR (DMSO-d6) δ 172.66, 172.55, 152.86, 152.53, 145.27, 143.25, 131.20, 130.61, 130.48, 129.95, 129.90, 128.79, 127.57, 126.73, 126.65, 125.45, 118.95, 104.33, 61.33, 60.86, 56.28; MS m/z (%) 386.2 (M + 23, 70), 364.3 (M + 1, 100).</p><!><p>To a solution of 4a (50 mg, 0.15 mmol) in THF (2–3 mL) was added dimethyl maleate (0.07 mL, 0.56 mmol) and Bu3P (0.14 mL, 0.56 mmol) in a sealed tube (10 mL). The mixture was heated to 100 °C under microwave irradiation for 2 h, and then cooled to room temperature, diluted with EtOAc, and washed with water several times. After removal of solvent in vacuo, the residue was purified on a flash column (gradual elution: EtOAc/petroleum ether 0 ∼ 60%). The obtained intermediate was re-dissolved in dried benzene (ca. 10 mL) and 3 drops of piperidine and HOAc were added. The mixture was heated to reflux for 24 h under N2 protection equipped with a distillation trap to remove water produced during reaction. After removal of solvent in vacuo, the residue was purified on a flash column (gradual elution: EtOAc/petroleum ether 0 ∼ 50%) and crystallized from MeOH to afford 30 mg of 6a, 45% yield, pale-yellow needles, 146–148 °C. 1H NMR δ ppm 8.99 (1H, s, =CH), 7.57 (1H, s, =CH), 7.40 & 7.19 (each 1H, s, ArH), 6.27 (1H, s, ArH), 6.11 (2H, s, OCH2O), 4.03, 3.99 and 3.98 (each 3H, s, OCH3 × 3), 3.85 and 3.84 (each 3H, s, OCH3 × 2); MS m/z (%) 455.4 (M + 1, 50), 423.4 (M – 31, 100).</p><!><p>The procedure was the same as that of 6a Starting with 4b (100 mg, 0.28 mmol) to afford 54 mg of 6b, 41% yield, pale-yellow needles, 126–128 °C. 1H NMR δ ppm 9.16 (1H, s, =CH), 7.57 (1H, s, =CH), 7.56, 7.09 and 6.29 (each 1H, s, ArH), 4.10 and 4.05 (each 3H, s, OCH3), 4.03 ∼ 4.00 (9H, ms, OCH3 × 3), 3.86 and 3.84 (each 3H, s, CO2CH3 × 2); MS m/z (%) 471.5 (M + 1, 40), 439.4 (M – 31, 100).</p><!><p>A solution of 5a (50 mg, 0.12 mmol) in THF (2-3 mL) in the presence of a few drops of aq NaOH (10%) was heated to 50 °C for 1 h. The mixture was then poured into ice-water, acidified pH to 1–2 with aq HCl (5%), extracted with EtOAc three times, and washed with water to neutral. After removal of solvent in vacuo, residue was purified on a flash column (gradual elution: MeOH/CH2Cl2 with 3% HOAc 0 ∼ 20%) to afford 7 as a brown solid, 28 mg, 54% yield, mp >200 °C (des); 1H NMR (DMSO-d6) δ ppm 8.82 (1H, s, =CH), 7.67 (1H, s, =CH), 7.50, 7.36, 7.36 and 6.27 (each 1H, s, ArH), 6.20 (2H, s, OCH2O), 3.90 (6H, s, OCH3 × 2), 3.88 (3H, s, OCH3); MS m/z (%) 427.2 (M + 1, 100).</p><!><p>A mixture of a diformyl or monoformylbiphenyl compound (4, 100 mg) and nitroethane or nitromethane (1 mL, excess) in toluene (5–7 mL) in the presence of ice-cold HOAc (0.03 mL) and n-butylamine (0.02 mL) was heated at 100–120 °C for 3–4 days. The mixture was poured into ice-water, extracted with EtOAc, and dried over Na2SO4. After removal of solvent in vacuo, the residue was purified by thin plate chromatography (TLC) eluted with CH2Cl2 or benzene/EtOAc (100:2) to obtain desired products.</p><!><p>Starting with 4c (100 mg) and nitromethane (excess) to afford 8d in 70% yield, yellow solid, mp 148–150 °C; 1H-NMR δ ppm 8.16 (1H, d, J = 7.6 Hz, =CH), 7.92 (1H, d, J = 7.6 Hz, ArH), 7.50-7.55 (3H, m, ArH), 6.72 (1H, s, ArH), 5.47 (1H, dt, J = 2.0 and 8.4 Hz, =CH), 4.06-4.18 (2H, m, =CHNO2), 3.94, 3.92 and 3.47 (each 3H, s, OMe); MS m/z (%) 326.1 (M + 1, 100).</p><!><p>Starting with 4c (100 mg) and nitroethane (excess) to afford 8e in 57% yield, yellow oil; 1H-NMR δ ppm 7.76 (1H, s, =CH), 7.43 (2H, m, ArH × 2), 7.31 (3H, m, ArH × 2, and =CH), 7.30 (1H, s, ArH-3), 3.96 (3H, s, OCH3), 3.93 (3H, s, OCH3), 3.55 (3H, s, OCH3), 3.53 (3H, s, OCH3), 2.33 (3H, s, CH3); MS m/z (%) 388.2 (M + 1, 60).</p><!><p>Starting with 4a (100 mg) and nitroethane (excess) to afford 9a in 37% yield, yellow solid, mp 98–99 °C; 1H-NMR δ ppm 8.05 (1H, s, CH=), 7.47 (1H, s, CH=), 6.87 (1H, s, ArH), 6.75 (1H, s, ArH), 6.66 (1H, s, ArH), 6.13 (2H, s, OCH2O), 3.95 ∼ 3.90 (9H, ms, OCH3), 2.50 (3H, s, CH3), 2.29 (3H, s, CH3); MS m/z (%) 459.2 (M + 1, 95), 384.2 (100).</p><!><p>Starting with 4a (100 mg) and nitromethane (excess) to afford 9d in 72% yield, yellow solid, mp 114–115 °C; 1H-NMR δ ppm 8.04 (1H, s, ArH), 7.24 (2H, d, J = 12.8 Hz, =CH), 6.83 (2H, d, J = 12.8 Hz, =CH), 6.05 and 6.00 (each 1H, s, ArH), 4.28 (2H, m, OCH2O), 4.05, 3.96, and 3.51 (each 3H, s, OMe); MS m/z (%) 431.1 (M + 1, 10), 453.2 (M + Na, 50), 370.0 (100).</p><!><p>Starting with 4b (100 mg) and nitroethane (excess) to afford 10a in 27% yield, yellow oil; 1H-NMR δ ppm 7.53 (1H, s, =CH), 7.36 (1H, s, =CH), 6.75-6.87 (2H, m, ArH), 6.59 (1H, s, ArH), 3.87-3.97 (18H, ms, OCH3 × 5, =CCH3), 3.60 (3H, s, CH3); MS m/z (%) 497.3 (M + Na, 100).</p><!><p>Starting with 4e (95 mg, 0.33 mmol) and nitroethane (10 mL, excess) to give 54 mg of 11a, 41% yield, yellow oil; 1H NMR δ ppm 7.74 (1H, s, =CH), 7.46 ∼ 7.21 (4H, m, ArH), 6.69 (1H, s, =CH), 6.02 (1H, s, ArH), 4.62 (2H, s, OCH2O), 3.79 (3H, s, OCH3), 2.36 (3H, s, CH3), 1.97 (3H, s, CH3); MS m/z (%) 399.2 (M + 1, 20).</p><!><p>Starting with 4f (50 mg, 0.18 mmol) and nitroethane (10 mL) to give 40 mg of 11b, 57% yield, yellow oil, 1H-NMR δ ppm 7.68 (1H, s, =CH), 7.51 ∼ 7.36 (4H, m, ArH), 6.61 (1H, s, =CH), 6.07 (1H, s, ArH), 5.98 (2H, s, OCH2O), 3.98 (3H, s, OCH3), 2.30 (3H, s, CH3), 2.26 (3H, s, CH3); MS m/z (%) 399.2 (M + 1, 30).</p><!><p>A solution of a formylbiphenyl 4 (50 mg) and ethyl cyanoacetate (0.1 mL) in HOAc (3 mL) in the presence of 3 drops of piperidine was heated to 100 °C with stirring under N2 protection for 6–8 h. The mixture was poured into ice-water, extracted with EtOAc, and washed with water to neutral. After removal of solvent in vacuo, the residue was simply washed with MeOH or purified on a flash column (gradual elution: EtOAc/petroleum ether 0 ∼ 40%) or thin plate chromatography (elution: cyclohexane/EtOAc 5:2) to afford pure product.</p><!><p>Starting with 4c (50 mg) and ethyl cyanoacetate (0.1 mL) to give 8b in 50% yield, pale yellow oil; 1H NMR δ ppm 8.40 (1H, m, ArH-3′), 7.93 (1H, s, ArH-3), 7.84 & 7.73 (each 1H, s, =CH), 7.60 (2H, m, ArH-4′, 5′), 7.23 (1H, m, ArH-6′), 4.28 (4H, m, OCH2 × 2), 4.01, 4.00 & 3.54 (each 3H, s, OCH3 × 3), 1.32 (6H, m, CH3 × 2); MS m/z (%) 513.5 (M + 23, 50), 445.2 (M – 45, 100).</p><!><p>Starting with 4d (50 mg) and ethyl cyanoacetate (0.1 mL) to give 8f in 58% yield, colorless oil; 1H NMR δ ppm 8.40 (1H, d, J = 8.0 Hz, ArH-3′), 8.06 (1H, s, =CH), 7.52 (2H, m, ArH-4′, 5′), 7.35 (1H, s, ArH-3), 7.21 (1H, d, J = 8.0 Hz, ArH-6′), 4.28 (2H, q, J = 8.0 Hz, CH2), 3.97 and 3.95 (each 3H, s, OCH3 × 2), 3.53 (6H, s, OCH3 × 2), 1.32 (3H, t, J = 8.0 Hz, CH3); MS m/z (%) 448.4 (M + 23, 20), 348.2 (M – 77, 100).</p><!><p>Starting with 4a (50 mg) and ethyl cyanoacetate (0.1 mL) to give 9b in 50% yield, white needles, mp 66–68 °C; 1H NMR δ ppm 7.90 (1H, s, ArH), 6.81 (1H, s, ArH), 6.63 (1H, s, ArH), 6.03 and 6.01 (each 1H, s, =CH), 4.52 and 4.44 (each 2H, m, OCH2 × 2), 3.92 (6H, s, OCH3 × 2), 3.80 ∼ 3.70 (5H, m, OCH2O, OCH3), 1.44 (6H, t, CH2CH3 × 2); MS m/z (%) 557.3 (M + 23, 95), 552.5 (M + 18, 100), 535.4 (M + 1, 80).</p><!><p>Starting with 4b (50 mg) and ethyl cyanoacetate (0.1 mL) to give 10b in 52% yield, white solid, mp 96–98 °C; 1H NMR δ ppm 8.16 (1H, s, ArH-3′), 7.84 (1H, s, ArH-3), 7.81 and 7.74 (each 1H, s, =CH × 2), 6.68 (1H, s, ArH-6′), 4.30 ∼ 4.26 (4H, m, OCH2 × 2), 4.04 (3H, s, OCH3), 4.01 (6H, s, OCH3 × 2), 3.91 and 3.58 (each 3H, s, OCH3 × 2), 1.32 (6H, t, J = 8.0 Hz, CH3 × 2); 13C NMR (CDCl3) δ 162.72, 161.12, 153.73, 152.62, 152.49, 151.48, 149.17, 146.26, 132.80, 129.23, 126.32, 124.22, 116.28, 115.76, 114.38, 110.28, 107.59, 103.24, 100.64, 67.78, 62.55, 61.36, 61.09, 56.37, 56.30, 14.17, 14.13; MS m/z (%) 551.4 (M + 1, 20), 573.5 (M + 23, 50).</p><!><p>A solution of di- or mono-formylbiphenyl 4 (1 equiv) and malononitrile (4 equiv) in CH3CN (5 mL) in the presence of NH4OAc (100 mg) was heated to 60 °C with stirring under N2 protection for 6–8 h. The mixture was poured into ice-water, extracted with EtOAc, washed with water and brine successively. After removal of solvent in vacuo, the residue was purified on a flash column (gradual elution: EtOAc/petroleum ether 0 ∼ 40%) to afford desired pure product.</p><!><p>Starting with 4b (50 mg) and malononitrile to provide 8c in 47% yield, white solid, mp 178–180 °C; 1H NMR δ ppm 8.34 (1H, d, J = 8.0 Hz, ArH-3′), 7.81 (1H, s, ArH-3), 7.71 ∼ 7.67 (2H, m, ArH-4′, 5′), 7.47 (1H, s, =CH), 7.25 (1H, s, =CH), 7.22 (1H, d, J = 8.0 Hz, ArH-6′), 4.06, 4.02, and 3.53 (each 3H, s, OCH3 × 3); MS m/z (%) 419.3 (M + 23, 100), 397.2 (M + 1, 10).</p><!><p>Starting with 4d (50 mg) and malononitrile to provide 8g in 70% yield, white solid, mp 102–104 °C; 1H NMR δ ppm 8.30 (1H, d, J = 8.0 Hz, ArH-3′), 7.61 ∼ 7.51 (3H, m, ArH), 7.38 (1H, s, =CH), 7.23 (1H, d, J = 8.0 Hz, ArH-6′), 3.99, 3.98, 3.58 and 3.54 (each 3H, s, OCH3 × 4); MS m/z (%) 401.4 (M + 23, 50), 288.2 (M – 90, 100).</p><!><p>Starting with 4a (50 mg) and malononitrileto provide 9c in 58% yield, light yellow solid, mp 172–174 °C; 1H NMR δ ppm 7.94 (1H, s, ArH-3′), 7.80 (1H, s, =CH), 7.24 (1H, s, =CH), 7.18 and 6.65 (each 1H, s, ArH), 6.25 (2H, d, J = 8.0 Hz, OCH2O), 4.05 and 4.02 (each 3H, s, OCH3 × 2), 3.63 (3H, s, OCH3); MS m/z (%) 463.3 (M + 23, 90), 458.4 (M + 18, 100).</p><!><p>Starting with 4b (50 mg) and malononitrile to provide 10c in 40% yield, light yellow solid, mp 103–104 °C; 1H NMR δ ppm 8.07 (1H, s, ArH-3′), 7.81 (1H, s, ArH-3), 7.23 (1H, s, =CH), 7.21 (1H, s, =CH), 6.62 (1H, s, ArH-6′), 4.07 ∼ 4.02 (9H, ms, OCH3 × 3), 3.96 (3H, s, OCH3), 3.61 (3H, s, OCH3-6); MS m/z (%) 479.2 (M + 23, 95).</p><!><p>Target compounds were assayed for in vitro anticancer activity by using the SRB method according to procedures described previously14, 15, 19 The panel of cell lines included human lung carcinoma (A-549), epidermoid carcinoma of the nasopharynx (KB), P-gp-expressing epidermoid carcinoma of the nasopharynx (KBvin), and prostate cancer (DU145). The cytotoxic effects of each compound were expressed as GI50 values, which represent the molar drug concentrations required to cause 50% tumor cell growth inhibition.</p><!><p>The effect of the tested compounds on LPS-induced NF-κB activity was measured using RAW264.7 cells stably transfected with an NF-κB luciferase reporter construct. Cells were seeded at 104 cells per well in a 96-well plate overnight for attachment, then cells were pretreated with 5a or BAY11-7082 for 30 min before being stimulated with LPS for 6 h. The treated cells were harvested and lysed, and the firefly luciferase activities in lysates were measured using the Promega Luciferase Assay System according to manufacturer's instructions (Promega).</p><!><p>RAW264.7 cells were seeded in petri-dishes and incubated overnight for attachment. Cells were incubated with LPS (1 μg/mL) for 15 min. In order to study the effects of the inhibitor, cells were pretreated with either 5a (2.5, 5, and 10 μM) or BAY11-7082 (20 μM) for 30 min prior to treatment with LPS. The total protein was isolated from cells in radio-immunoprecipitation assay buffer containing protease and phosphatase inhibitors. The nuclear and cytosolic extracts were isolated by using the Nuclear Extraction Kit (Cayman) according to the manufacturer's recommendations. The proteins were then subjected to Western blot analysis. Briefly equal amounts of protein were resolved with SDS-PAGE and transferred to PVDF membranes (Roche). The membranes were probed with primary antibodies overnight at 4 °C and incubated for 1 h with secondary peroxidase-conjugated antibod y at room temperature. The signals on the membrane were visualized by Lumiglo reagent (Cell Signaling) and exposed to X-ray film (Fuji Photo Film).</p><!><p>The measurement of NO2- in the cell culture medium was carried out by using Griess reagents I and II (Beyotime), as a measure of NO production. Briefly RAW264.7 cells were seeded in petri-dishes and incubated overnight for attachment. Cells were stimulated by LPS (1 μg/mL) for 18 h. In order to study the effects of the inhibitor, cells were pretreated with either 5a (0.6, 1.25 and 2.5 μM) or BAY 11-7082 (20 μM) for 30 min prior to treatment with LPS. After treatment, the cells culture medium was collected and centrifuged (69×g/5 min). Griess reagents I and II were equilibrated to room temperature before use. Fifty-microliter volumes of cell culture supernatants were reacted with Griess reagent I (50 μL) and Griess reagent II (50 μL). Finally, absorbance was measured at 540 nm.</p><!><p>Conformation searching was performed by Discovery Studio 3.0 software (Accelrys, Inc.). The default atom types and parameters supplied with Discovery Studio 3.0 were used throughout the conformation searching. Before search, all target compounds were assigned to the same configuration where the phenyl-A ring rotates clockwise and the phenyl-B ring rotates counter-clockwise along the biphenyl axis. Small molecules were prepared using the ligand preparation tool available in the software and then systematic search conformation method was deployed by generate conformations tool with default CHARMm force-field. The indicated torsion angle and absolute energy of the global minimum were measured.</p>