Design, synthesis and biological evaluation of nitrogen-containing macrocyclic bisbibenzyl derivatives as potent anticancer agents by targeting the lysosome

European Journal of Medicinal Chemistry 136 (2017) 603e618

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

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Design, synthesis and biological evaluation of nitrogen-containing macrocyclic bisbibenzyl derivatives as potent anticancer agents by targeting the lysosome Bin Sun a, b, 1, Jun Liu b, 1, Yun Gao b, Hong-bo Zheng b, Lin Li b, Qing-wen Hu b, Hui-qing Yuan c, Hong-xiang Lou a, b, * a b c

National Glycoengineering Research Center, Shandong University, Jinan, 250012, PR China Key Laboratory of Natural Products & Chemical Biology, Ministry of Education, Shandong University, Jinan, 250012, PR China School of Medicine, Shandong University, Jinan, 250012, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 March 2017 Received in revised form 15 May 2017 Accepted 24 May 2017 Available online 25 May 2017

A series of novel nitrogen-containing macrocyclic bisbibenzyl derivatives was designed, synthesized, and evaluated for antiproliferative activity against three anthropic cancer cell lines. Among these novel molecules, the tri-O-alkylated compound 18a displayed the most potent anticancer activity against the A549, MCF-7, and k562 cancer cell lines, with IC50 values of 0.51, 0.23, and 0.19 mM, respectively, which were obviously superior to those of the parent compound riccardin D, and were 3e10-fold better than those of the clinical used drug ADR. The bis-Mannich derivative 11b also exhibited significantly enhanced antiproliferative potency, with submicromolar IC50 values. Structure-activity relationship analyses of these newly synthesized compounds were also performed. Mechanistic studies indicated that these compounds could target the lysosome to induce lysosomal membrane permeabilization, and could also induce cell death that displayed features characteristic of both apoptosis and necrosis. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Bisbibenzyls Anticancer activity Nitrogen-containing derivatives Lysosome

1. Introduction Lysosomes are acidic intracellular organelles that are involved in several cellular processes, including receptor degradation, autophagy, apoptosis, post-translational protein maturation, and the extracellular release of active enzymes [1e3]. They control the recycling of large amounts of cellular organelles and macromolecules through the actions of more than 50 acid hydrolytic enzymes. Interestingly, lysosomes in cancer cells are larger, less stable, more numerous, and exhibit greater cathepsin activity than those in normal cells [3,4]. Additionally, weakly basic drugs have been found to accumulate specifically in lysosomes, after which they can no longer easily diffuse out of the vesicles because they become protonated [5,6]. This accumulation of weakly basic drugs could lead to changes in osmolality within the lysosomes, which results in

* Corresponding author. National Glycoengineering Research Center, Key Laboratory of Natural Products & Chemical Biology, Ministry of Education, Shandong University, Jinan, 250012, PR China. E-mail address: [email protected] (H.-x. Lou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2017.05.050 0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

lysosomal swelling and rupture that leads to lysosomal membrane permeabilization (LMP) [7]. LMP usually causes lysosomal proteases to leak into the cytosol and trigger initiation of either the apoptotic or necrotic cell death pathways [8,9]. Therefore, the lysosome is a critical target for anticancer therapy [10,11], and agents that interact with lysosomes show promise for the development of novel and potent anticancer drugs. Macrocyclic bisbibenzyls are a series of phenolic natural products that are mainly found in liverworts [12]. These natural products exhibit versatile biological activities, including antifungal, antibacterial, antiviral, anti-mitotic, antioxidant, cytotoxic, musclerelaxing, LXR-modulating, and NOS-inhibiting activities [13e17]. Therefore, bisbibenzyls are of great interest to natural product researchers because of their potential applications as pharmacological agents. Riccardin D, a macrocyclic bisbibenzyl isolated from Marchantia polymorpha L., has been shown to exhibit robust anticancer activity [18,19]. In our previous study, we prepared two Mannich base derivatives of riccardin DdRDN-1 and RDN-2 (Fig. 1)dthat exhibited markedly improved anticancer activity compared with the parent riccardin D. Interestingly, both molecules were found to accumulate in the lysosomes. Mechanistic

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Fig. 1. The chemical structures of riccardin D, RDN-1 and RDN-2.

studies indicated that both compounds could induce LMP and cathepsin release from the lysosomes into the cytosol, and furthermore could induce cell death that displayed features characteristic to both apoptosis and necrosis. We then predicted that the introduction of basic groups to riccardin D might improve its anticancer activity by promoting lysosome targeting [20,21]. Based on the findings described above, a series of riccardin D derivatives with weakly basic groups, especially groups that contained nitrogen atoms, was designed. Our modification strategy for riccardin D is shown in Fig. 2. We first modified the Mannich side chains of RDN-1 and RDN-2, and changed the methylamino groups to either ethylamino, pyrrolidinyl, piperidinyl, morpholino, thiomorpholino, 4-hydroxypiperidinyl, 4-(4-methoxyphenyl)piperazinyl, 4-methylpiperazinyl, bis((dibutylamino)methyl) amino, or bis(2-methoxyethyl)amino groups to evaluate how the Mannich side chains influence anticancer activities. Second, we directly introduced a nitrogen atom into the arene ring of riccardin D and prepared some derivatives based on the structure of aniline to determine the effect of these substitutions on the cytotoxic activity of riccardin D. Finally, we focused on the modification of phenolic groups of riccardin D by introducing O-alkylation of different nitrogen-containing electrophiles, and the resulting mono- and triO-alkylated derivatives were obtained for structure-activity relationship (SAR) studies. Herein, we describe the synthesis of a series of novel nitrogencontaining riccardin D derivatives and the evaluation of these compounds for antiproliferative activities using the MTT assay. The anticancer mechanisms of two representative derivatives were also investigated using flow cytometry and confocal microscopy. Together, these findings indicated that these novel weakly basic derivatives of macrocyclic bisbibenzyl represent a novel class of

potent anticancer agents that target lysosomes. 2. Results and discussion 2.1. Chemistry Riccardin D was prepared in 11 steps with a satisfactory yield following a procedure reported previously [22], and was used as a staring material for further structural modifications. The general procedure is outlined in Scheme 1. Mannich reactions of riccardin D were carried out using different secondary amines [23]. The reaction of riccardin D, amine, and formaldehyde at a molar ratio of 1:1.5:1.5 in ethanol yielded the mono-Mannich derivatives 10ae10m as the main product at a moderate yield. The position of the Mannich side chain was then determined based on a NOE spectrum. As shown in Fig. 3, correlations between H2-130 and H-50 were determined for representative compound 10l (Figs. S25 and S26), which indicated that mono-Mannich derivatives have Mannich side-chains on the 40 -position of arene B. The substitution pattern in the mono-Mannich derivatives was consistent with that of RDN-1, which might occur because of the stereo-hindrance effect was reduced (compared with 2-position on arene D) and a higher electron density (compared with 13-position on arene C) was present at the 40 -position of arene B. When the quantity of the secondary amine and formaldehyde were increased to 3 and 3 equivalents, respectively, the bis-Mannich derivatives 11ae11m were obtained as the main products. Basic groups were introduced at the 40 -position on arene B and 13-position on arene C, which was confirmed by the NOE spectrum of the representative derivative 11l. As shown in Fig. 3, correlations of H2-130 with H-50 , and H2-170 with H-14 were determined (Figs. S53 and S54, respectively).

Fig. 2. Modification strategy for riccardin D derivatives.

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Scheme 1. Synthesis of riccardin D. Reagents and conditions: (a) i. CuO, K2CO3, Py, reflux, 80%; ii. LiAlH4, THF, 40  C to r.t., 92%; iii. PPh3HBr, MeCN, reflux, 92%; (b) Pd(PPh3)4, toluene, EtOH, Na2CO3, reflux, 75%; (c) K2CO3, 18-crown-6, DCM, reflux, 90%; (d) i. Pd/C (10%), H2, Et3N, EtOAc, r.t., 95%; ii. HCl/THF (1:1), r.t., 93%; iii. PPh3HBr, MeCN, reflux, 97%; (e) NaOMe, DCM, r.t., 89%; (f) i. Pd/C (10%), H2, EtOAc, r.t., 92%; ii. BBr3, DCM, 40  C to r.t., 86%.

Fig. 3. Key NOESY correlations (dashed blue arrows) for compounds 10l and 11l. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Derivative 12 with a nitro group at 13-position was prepared from riccardin D and nitric acid at a moderate yield. The nitro group of compound 12 was reduced by hydrogen over Pd/C to yield

compound 13, which was underwent reductive methylation to generate derivative 14 [24]. Compound 13 was acetylated by acetyl chloride to give derivative 15, and the selective deacetylation of

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compound 15 by sodium hydroxide provided derivative 16 [25,26]. The alkylation of riccardin D with selected amino-alkyl chloride electrophiles [e.g., 2-chloro-N,N-dimethylethanamine hydrochloride] at a molar ratio of 1:1.5 in acetone gave the mono-alkylated derivatives 17ae17h as the main product in the presence of potassium carbonate [27]. The structural assignment of representative compound 17a was determined based on the NOE spectrum. As shown in Fig. 4, correlations of H2-130 with H-40 , and H3-150 with H2 were determined for compound 17a (Fig. S69), which indicated that the monoalkylated derivatives have basic side chains on arene B. When the quantity of amino-alkyl chloride electrophiles and potassium carbonate were increased to 5 and 10 equivalents, respectively, the tri-alkylated derivatives 18ae18h were obtained at a good yield. 2.2. Biological evaluations 2.2.1. In vitro antiproliferative activity and SAR analysis The cytotoxic activity of the synthesized analogues (10ae10m, 11ae11m, 12e16, 17ae17h, and 18ae18h) was evaluated in parallel in three human cancer cell lines (A549, MCF-7 and k562) using adriamycin (ADR), riccardin D (RD), RDN-1, and RDN-2 as reference compounds. Cells were treated with each compound for 48 h, and viability was assessed using the MTT method. Results are summarized in Table 1. Among these novel molecules, the tri-O-alkylated derivative 18a exhibited the most potent anticancer activity against the A549, MCF-7, and k562 cell lines, with IC50 values of 0.51, 0.23, and 0.19 mM, respectively, which was clearly superior to those of the parent compound riccardin D (IC50 ¼ 28.14, 18.31, and 17.56 mM, respectively). Surprisingly, these values were three- to ten-fold better than those of the clinically used drug ADR (IC50 ¼ 1.45, 2.30, and 0.61 mM, respectively). Additionally, three other tri-Oalkylated derivatives, 18b, 18d and 18e, also exhibited significant antiproliferative activities, with IC50 values ranging from 0.24 to 0.97 mM. Moreover, the bis-Mannich derivative 11b displayed

excellent cytotoxic activity against the A549, MCF-7, and k562 cell lines, with IC50 values of 0.99, 0.43, and 0.80 mM, respectively, which were comparable to those of ADR. The SARs of these novel riccardin D analogues were analyzed and are summarized below. 2.2.1.1. Effects of the Mannich side chains. As shown in Scheme 2, for the first step we introduced different Mannich side chains to the 40 -position of riccardin D, and then prepared 13 mono-Mannich derivatives, 10ae10m. The anticancer activities of these compounds are shown in Table 1. Compounds 10ae10c, 10f, 10h and 10j were substituted with less bulky Mannich side chains, such as ethylamino, pyrrolidinyl, piperidinyl, 4-hydroxypiperidinyl, and 4methylpiperazinyl groups, and exhibited improved anticancer activities compared with riccardin D and RDN-1, with IC50 values ranging from 2.26 to 9.19 mM. By contrast, changing the piperidinyl group to a morpholino or thiomorpholino group led to diminished cytotoxic activity (e.g., 10d and 10e). To our surprise, when the more sterically bulky Mannich side chains, such as 4-(4methoxyphenyl)piperazinyl, bis((dibutylamino)methyl)amino, and bis(2-methoxyethyl)amino groups, were introduced into riccardin D, the resulting derivatives 10g, 10i and 10ke10m showed reduced antiproliferative potency, with IC50 values greater than 20 mM. We hypothesized that the receptor binding site for the Mannich side chains was relatively small in size so that it could barely accommodate large substituents. In addition, 13 bis-Mannich derivatives, 11ae11m, were then prepared and evaluated for anticancer activities. As shown in Table 1, the bis-Mannich derivatives with less bulky substituents displayed obviously enhanced cytotoxic activity compared with mono-Mannich derivatives (11a vs. 10a, 11b vs. 10b, 11c vs. 10c, 11e vs. 10e, 11f vs. 10f, 11h vs. 10h, and 11j vs. 10j). Moreover, compound 11b had two pyrrolidinyl groups and was the most potent compound among all of the Mannich derivatives that we tested. However, the introduction of bulky basic groups led to bis-Mannich derivatives 11g, 11i, and 11ke11m that did not have anticancer activity. We then found the following cytotoxic activity trend based on the small Mannich side chains: pyrrolidinyl > piperidinyl ¼ ethylamino > 4-methylpiperazinyl > 4-hydroxypiperidinyl > (2hydroxyethyl)(methyl)amino > thiomorpholino > morpholino. Further increasing the size of the basic side chains resulted in total loss of activity, which suggested the existence of a relatively small binding pocket for the receptor around the Mannich base moiety, with a pyrrolidinyl group representing the optimal size. These findings indicated that the presence of a less bulky Mannich base at the 40 -position of riccardin D is critical for anticancer activity, and that the introduction of a second Mannich side chain could dramatically enhance the activity, perhaps because of the increased total basicity of the molecule. These data are consistent with our prediction mentioned above. 2.2.1.2. Effects of the aniline substituents. To obtain more SAR information, the nitrogen atom was directly introduced into the arene ring of riccardin D and, as shown in Scheme 3, a series of derivatives based on aniline were obtained (compounds 12e16). However, all of these derivatives displayed reduced anticancer activity compared with riccardin D, with an IC50 value greater than 20 mM. This trend might occur because of p-p conjugate interactions between the nitrogen atom and arene ring, which led to a subtle effect on the pKa of aniline and a reduction in basicity.

Fig. 4. Key NOESY correlations (dashed blue arrows) for compound 17a. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.2.1.3. Effects of basic ether side chains. After confirming the effects of Mannich side chains and an aniline moiety on anticancer activity, we then focused on modifying riccardin D by O-alkylation

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Table 1 In Vitro cytotoxicity of riccardin D derivatives against three cancer cell lines. Cpd.

10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k 10l 10m 11a 11b 11c 11d 11e 11f 11g 11h 11i 11j 11k 11l 11m

IC50 (mM)a

Cpd.

A549

MCF-7

k562

5.74 ± 1.01 2.26 ± 0.36 7.12 ± 1.22 >20 16.28 ± 3.25 5.74 ± 1.07 >20 9.07 ± 2.31 >20 5.59 ± 0.98 >20 >20 >20 3.78 ± 0.72 0.99 ± 0.24 4.85 ± 0.96 >20 10.21 ± 1.71 3.26 ± 0.62 >20 5.21 ± 1.02 >20 2.16 ± 0.38 >20 >20 >20

7.59 ± 1.69 2.41 ± 0.55 5.07 ± 1.52 >20 15.77 ± 3.98 9.05 ± 2.15 >20 8.28 ± 2.00 >20 6.02 ± 1.74 >20 >20 >20 1.05 ± 0.16 0.43 ± 0.09 2.04 ± 0.54 >20 9.25 ± 2.00 3.95 ± 0.75 >20 6.00 ± 1.57 >20 2.89 ± 0.49 >20 >20 >20

7.86 ± 2.01 4.82 ± 1.60 5.88 ± 1.24 >20 13.44 ± 2.96 7.07 ± 1.13 >20 9.19 ± 2.07 >20 3.31 ± 0.58 >20 >20 >20 1.86 ± 0.37 0.80 ± 0.20 0.66 ± 0.17 >20 8.35 ± 1.39 2.56 ± 0.35 >20 5.67 ± 1.17 >20 3.32 ± 0.55 >20 >20 >20

12 13 14 15 16 17a 17b 17c 17d 17e 17f 17g 17h 18a 18b 18c 18d 18e 18f 18g 18h RDb RDN-1 RDN-2 ADR

IC50 (mM)a A549

MCF-7

k562

>20 >20 >20 >20 >20 1.30 ± 0.21 1.87 ± 0.34 2.02 ± 0.41 2.13 ± 0.52 2.98 ± 0.67 7.49 ± 1.58 1.99 ± 0.29 4.53 ± 1.20 0.51 ± 0.11 0.78 ± 0.14 1.04 ± 0.26 0.97 ± 0.14 0.53 ± 0.11 5.23 ± 1.23 1.44 ± 0.32 1.67 ± 0.35 28.14 ± 5.11 13.25 ± 2.35 4.81 ± 0.92 1.45 ± 0.27

>20 >20 >20 >20 >20 2.38 ± 0.34 2.73 ± 0.25 2.42 ± 0.19 2.49 ± 0.46 3.03 ± 0.50 5.02 ± 1.02 3.25 ± 0.38 3.68 ± 0.50 0.23 ± 0.06 0.56 ± 0.16 0.88 ± 0.25 0.24 ± 0.02 0.66 ± 0.15 4.19 ± 0.59 3.05 ± 0.50 2.12 ± 0.44 18.31 ± 3.14 12.54 ± 2.01 4.22 ± 0.96 2.30 ± 0.44

>20 >20 >20 >20 >20 1.56 ± 0.33 2.23 ± 0.40 2.22 ± 0.31 2.31 ± 0.44 2.73 ± 0.28 10.05 ± 1.25 2.61 ± 0.38 2.94 ± 0.49 0.19 ± 0.02 0.65 ± 0.11 1.79 ± 0.44 0.56 ± 0.12 0.26 ± 0.04 3.43 ± 0.58 2.50 ± 0.41 2.13 ± 0.37 17.56 ± 3.20 10.14 ± 1.65 2.84 ± 0.39 0.61 ± 0.20

a The IC50 values (mM) indicate the concentrations corresponding to 50% inhibition of the growth of the indicated cell line growth; Mean values are based on three independent experiments. b RD represent riccardin D.

Scheme 2. Synthesis of aminomethylation derivatives of riccardin D (10a-10m, 11a-11m). Reagents and conditions: (a) HNR1R2 (1.5 eq), 37% HCHO (1.5 eq), EtOH, reflux, 62%e75%; (b) HNR1R2 (3 eq), 37% HCHO (3 eq), EtOH, reflux, 51%e70%.

of the hydroxyl group with a series of nitrogen-containing electrophiles to evaluate the effects of these aminoalkyl side chains. As shown in Scheme 4, 16 mono- and tri-O-alkylated derivatives were prepared; the anticancer activities of these compounds are presented in Table 1. Surprisingly, when the modification pattern was changed from aminomethylation to O-alkylation, the “effect of large size” disappeared, and all of the O-alkylated derivatives displayed potent cytotoxic activity. Introduction of aminoalkyl side chains to the C-30 position on arene B yielded the mono-alkylated derivatives 17ae17h, which exhibited markedly improved anticancer activity compared with riccardin D, with IC50 values ranging from 1.30 to 10.05 mM. Compound 17a, with the (dimethylamino)

ethyl group, was found to be the most potent agent among the mono-alkylated series, with IC50 values of 1.30 mM (for A549), 2.38 mM (for MCF-7) and 1.56 mM (for K562), respectively. When the (diethylamino)ethyl, five-membered (pyrrolidin-1-yl)ethyl, sixmembered (piperidin-1-yl)ethyl, or seven-membered (azepan-1yl)ethyl groups were introduced to the C-30 position on arene B, the anticancer activities of the resulting compounds 17b, 17c, 17d, and 17e, respectively, were slightly diminished compared with 17a. Unexpectedly, the cytotoxic activity of compound 17f, which had a six-membered (morpholino-1-yl)ethyl group at position C-30 of arene B, was markedly reduced compared with compound 17d (which contained a cyclic six-membered aminoalkyl side chain).

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Scheme 3. Synthesis of amination derivatives of riccardin D 12e16. Reagents and conditions: (a) HNO3, AcOH, DCM, 24 h, 65%; (b) H2, Pd/C (10%), MeOH, r.t., 8 h, 80%; (c) HCHO, NaBH4, THF, 2 h, 54%; (d) AcCl, Et3N, DCM, 0  C to r.t., 92%; (e) 1 N NaOH, 0  C, 30 min, 64%.

Scheme 4. Synthesis of alkylation derivatives of riccardin D (17a-17h, and 18a-18h). Reagents and conditions: (a) RCl$HCl (1.5 eq), K2CO3 (5 eq), acetone, 60  C, 66%e78%; (b) RCl$HCl (5 eq), K2CO3 (10 eq), acetone, 60  C, 83%e90%.

Additionally, when the ethyl linkage in the aminoalkyl side chain of compounds 17a and 17c was expanded to three carbon atoms, the resulting compounds 17g and 17h also displayed reduced activity. Further introduction of aminoalkyl side chains resulted in tri-Oalkylated derivatives 18ae18h. Among these derivatives, compound 18a, bearing three (dimethylamino)ethyl groups, was found to be the most potent agent with IC50 values of 0.51 mM (for A549), 0.23 mM (for MCF-7) and 0.19 mM (for K562), respectively. Interestingly, when the dimethylamino groups of 18a were changed to larger diethylamino or pyrrolidin-1-yl groups, the resulting compounds 18b and 18c displayed diminished activity. However, further increasing the size to piperidin-1-yl and azepan-1-yl groups reversed this trend in activity, and led to derivatives 18d and 18e that exhibited more potent anticancer activity compared with 18b and 18c. The introduction of morpholino-1-yl groups or extending the ethyl linkage in the aminoalkyl side chains also resulted in decreased activity (18f vs. 18d, 18g vs. 18a, and 18h vs. 18c), which is consistent with our observations of the mono-alkylated series described above. Notably, all of the tri-O-alkylated derivatives displayed more potent antiproliferative activity than the corresponding mono-O-alkylated derivatives (18a vs. 17a, 18b vs. 17b, 18c vs. 17c, 18d vs. 17d, 18e vs. 17e, 18f vs. 17f, 18g vs. 17g, and 18h vs. 17h), perhaps because the loss of acidic phenol groups and the increase in basic centers occurred simultaneously, resulting in

improved pKa values of the molecules. The antiproliferative activity of the most potent compounds 11b and 18a was representative of the Mannich and O-alkylated series, respectively. These compounds were further assessed in A549 cells using the xCELLigence system; hydroxycholoroquine (HCQ) was used as a positive control. As shown in Fig. 5, compound 11b inhibited A549 cell proliferation within 5 h and markedly reduced the number of cells within 20 h, which is similar to the actions of HCQ. After treatment for ~60 h, the effects of 11b were similar to those of HCQ. The cell growth curve shows that compound 11b inhibited A549 cell proliferation and, subsequently, induced cell death. 2.2.2. Riccardin D derivatives induce lysosomal membrane permeabilization (LMP) After the evaluating the antiproliferative activities, we extended our work to a mechanistic and phenotypic study of the effects of riccardin D derivatives on lysosomal stability by applying confocal microscopy and flow cytometry. Two lysosomotropic fluorescence probes, AO and LysoTracker Red, were used to determine the effect of compounds 11b and 18a on the lysosomal stability of A549 cancer cells. AO, a metachromatic fluorophore, is retained in its charged form (AOHþ) by proton trapping inside of lysosomes and emits red fluorescence.

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Fig. 5. A549 cells were treated with compound 11b and 18a as indicated. Cells were then plated 2000 cells/well into E-plate 16 and analyzed using a xCELLigence RTCA DP instrument. The results are representative of three independent experiments.

Lysosomal rupture in these AO-loaded cells results in diminished red fluorescence. LysoTracker Red, a fluorescent weak base, could accumulate in acidic organelles and successfully stain lysosomes in living cells. The LysoTracker Red and AO-uptake in lysosomes results were monitored by confocal microscopy and flow cytometry. As shown in Fig. 6, after treatment with compounds 11b or 18a for 6 h, lysosomal rupture could be observed, as indicated by increased numbers of “pale cells,” i.e., cells with reduced numbers of LysoTracker Red- and AO-accumulating lysosomes. Our data demonstrated that both of the riccardin derivatives 11b and 18a could induce lysosomal membrane permeabilization in A549 cancer cells. 2.2.3. Riccardin D derivatives trigger apoptosis and necrosis in A549 cells The significant antiproliferative activity of riccardin D derivatives and their obvious lysosomal rupture properties encouraged us to future investigate the underlying biological mechanism. We examined the effect of compounds 11b and 18a on apoptosis and necrosis in A549 cells using microscopy and flow cytometry. As shown in Fig. 7A, after treatment with compounds 11b or 18a, morphological changes in A549 cells were observed using DAPI staining. Both derivatives induced marked morphological alterations characteristic of apoptosis, including cell shrinkage and granular apoptotic body formation. Additionally, A549 cells treated with specified doses of 11b and 18a were stained with Annexin VFITC and PI, respectively, and then were analyzed by flow cytometry to assess whether both derivatives induced cell death in cancer cells via apoptosis or necrosis. Annexin V-FITCþ/PI- represents a hallmark of early apoptosis. Annexin V-FITCþ/PIþ staining represents the end-stage of apoptosis or necrosis. As shown in Fig. 7B, treatment with compounds 11b or 18a caused significant increases in the fraction of apoptotic and necrotic A549 cancer cells, respectively. 3. Conclusions In this present study, a series of novel riccardin D derivatives was designed, synthesized and evaluated for antiproliferative activity against three anthropic cancer cell lines, resulting in the discovery of several novel and potent antitumor agents. Among these molecules, the tri-O-alkylated compound 18a displayed the most potent anticancer activity, with IC50 values that ranged from 0.19 to 0.51 mM. This activity was obviously superior to that of parent riccardin D, and was 3e10-fold better than the clinically used drug ADR. The bis-Mannich derivative 11b also exhibited significantly improved antiproliferative potency, with an IC50 value in the submicromolar range. Our mechanistic studies indicated that

both Mannich and O-alkylated derivatives could target the lysosome and induce lysosomal membrane permeabilization, which could lead to cell death that displayed features characteristic to both apoptosis and necrosis. Together, these data indicated that the introduction of nitrogen-containing groups to riccardin D, which increased the total basicity of the molecule, is critical for the improved cytotoxicity. This activity might occur because the basic derivatives accumulate in acidic lysosomes, which further results in the death of cancer cells. In conclusion, this present study discovered several potent nitrogen-containing bisbibenzyl derivatives, and our systematic research has yielded novel molecular scaffolds for the further development of antitumor agents that target lysosomes. 4. Experimental section 4.1. Chemistry Chemicals were commercially available and used as received without further purification. Solvents (THF, Acetone, MeCN and DCM) were dried and freshly distilled before use according to procedures reported in the literature. Reactions were monitored by thin-layer chromatography using Merck plates with fluorescent indicator. Column chromatography was carried out on silica gel or alumina (200e300 mesh). Melting points were determined in capillary tubes using a Mel-Temp apparatus and are not corrected. Infrared spectra were obtained as films on salt plates using CHCl3 as the solvent except where otherwise specified, using a Perkin-Elmer Spectrum One FT-IR spectrometer, and are baseline-corrected. The NMR spectra were recorded on a Bruker Spectrospin spectrometer at 600 MHz (13C NMR at 150 MHz) using TMS as an internal standard. The chemical shifts are reported in parts per million (ppm d) referenced to the residual 1H resonance of the solvent (CDCl3, 7.26 ppm). Abbreviations used in the splitting pattern were as follows: s ¼ singlet, d ¼ doublet, t ¼ triplet, quin ¼ quintet, m ¼ multiplet, and br ¼ broad. Mass spectral analyses were performed on SHIMASZU LCMS 2020 at the Shandong UniversityChemical Analysis Center. All HRMS spectra (ESI) were obtained on a LTQ Orbitrap mass spectrometer. 4.1.1. General procedure 1 (GP 1) for the Mannich reaction A mixture of riccardin D, secondary amine and aqueous formaldehyde (37%) in ethanol was stirred under reflux for 24 h. The solvent was then removed in vacuo and the residue was purified by flash column chromatography, eluting with a gradient mixture of EtOAc/MeOH/ aqueous ammonia (93:5:2 to 80:18:2, V/V), to yield the pure product (compounds 10a-10m and 11a-11m) as a solid in moderate yield.

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Fig. 6. Compounds 11b and 18a induce lysosomal membrane permeabilization (LMP) in cancer cells. (A) Lysosomal rupture assayed based on LysoTracker Red- and AO-uptake methods. A549 cells were treated with 11b and 18a for 6 h, and then were exposed to LysoTracker Red and AO as described in the experimental section. Cells were measured by confocal microscopy. (B) A549 cells were treated with 11b and 18a for 6 h, exposed to LysoTracker Red and AO, and then finally measured by flow cytometry.

4.1.2. General procedure 2 (GP 2) for the alkylation of phenol A mixture of riccardin D, alkyl chloride hydrochloride and potassium carbonate in acetone was stirred under reflux for 24 h. The insoluble material was then filtered off and the filtrate was concentrated to provide the residue that was purified by flash column chromatography, eluting with a gradient mixture of MeOH/ EtOAc/aqueous ammonia (93:5:2 to 80:18:2, V/V), to yield the pure product (compounds 17a-17f and 18a-18f) as a solid in good yield.

4.1.3. Riccardin D Riccardin D (5 g) was prepared from compounds 1, 2, 4, and 5 in 11 steps with satisfied yield by following the procedure reported previously [22]. White solid. mp 152e153  C; 1H NMR (CDCl3) d 7.36

(t, J ¼ 7.9 Hz, 1H), 7.08 (dd, J ¼ 7.7, 1.1 Hz, 1H), 6.97e6.89 (m, 3H), 6.86e6.83 (m, 3H), 6.78 (d, J ¼ 8.0 Hz, 1H), 6.75 (dd, J ¼ 8.1, 1.9 Hz, 1H), 6.53 (d, J ¼ 1.4 Hz, 1H), 6.32 (dd, J ¼ 7.7, 1.5 Hz, 1H), 5.60 (s, 1H), 5.42 (d, J ¼ 1.9 Hz, 1H), 4.87 (s, 1H), 4.77 (s, 1H), 3.04e2.96 (m, 2H), 2.87e2.92 (m, 1H), 2.83e2.72 (m, 2H), 2.69e2.56 (m, 3H); MS (ESI) 425 (MþH)þ. 4.1.4. 40 -((Diethylamino)methyl)-riccardin D (10a) This compound was prepared from riccardin D (60 mg, 0.142 mmol), diethylamine (15 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 66% yield. White solid; mp 110e1110  C. IR (film) 3368, 1588, 1521, 1463, 1365, 1150, 956 cm1; 1H NMR (CDCl3) d7.08 (d, J ¼ 7.8 Hz, 1H), 6.96 (d,

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611

Fig. 7. Analysis of apoptosis and necrosis in compound 11b- and 18a-treated A549 cancer cell lines. (A) A549 cells were treated with various concentrations of 11b or 18a for 8 h, stained with DAPI, and finally measured by microscopy. (B) A549 cells were treated with various concentrations of 11b or 18a for 8 h. Cells were collected and stained with FITCconjugated Annexin V and PI and analyzed using flow cytometry. The mean percentages of Annexin V/(PIþ plus PI) cells obtained from three independent experiments with the indicated cell lines ± SD are shown.

J ¼ 7.8 Hz, 1H), 6.93e6.86 (m, 4H), 6.85e6.77 (m, 2H), 6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.54 (dd, J ¼ 7.8, 1.8 Hz, 1H), 6.29 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 3.87 (q, J ¼ 14.3 Hz, 2H), 3.01e2.89 (m, 3H), 2.86e2.76 (m, 3H), 2.69 (q, J ¼ 7.2 Hz, 4H), 2.65e2.50 (m, 2H), 1.11 (t, J ¼ 7.2 Hz, 6H); 13C NMR (CDCl3) d 155.2, 153.3, 152.8, 146.7, 143.6, 143.3, 142.1, 140.4, 133.3, 132.1, 129.7, 128.9, 128.5, 124.5, 122.5, 122.0, 121.8, 121.6, 120.9, 119.4, 118.7, 116.2, 114.9, 56.4, 46.2, 38.1, 37.7, 36.8, 34.5, 10.7; MS (ESI) 510 (MþH)þ; HRMS (ESI) calcd for C33H36NO4 510.2639; found 510.2633 (MþH) þ. 4.1.5. 40 -((Pyrrolidin-1-yl)methyl)-riccardin D (10b) This compound was prepared from riccardin D (60 mg, 0.142 mmol), pyrrolidine (15 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 69% yield. White solid; mp 125e126  C. IR (film) 3351, 1571, 1510, 1449, 1360, 1141, 950 cm1; 1H NMR (CDCl3) d 7.20 (d, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 6.7 Hz, 1H), 6.89 (d, J ¼ 7.3 Hz, 2H), 6.86 (d, J ¼ 7.7 Hz, 1H), 6.80 (d, J ¼ 7.8 Hz, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.50 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.35 (d, J ¼ 1.7 Hz, 1H), 5.37 (d, J ¼ 2.0 Hz, 1H), 4.09 (d, J ¼ 13.6 Hz, 1H), 3.92 (d, J ¼ 13.6 Hz, 1H), 2.99e2.74 (m, 9H), 2.73e2.47 (m, 3H), 1.91e1.89 (m, 4H); 13C NMR (CDCl3) d 154.3, 153.3, 152.8, 146.7, 144.3, 143.4, 142.6, 140.3, 133.2, 132.0, 129.7, 129.1, 128.9, 124.5, 122.5, 122.2, 122.1, 120.5, 119.1, 116.1, 114.9, 61.6, 53.3, 38.0, 37.7, 36.8, 34.7, 23.5; MS (ESI) 508 (MþH)þ; HRMS (ESI) calcd for C33H34NO4 508.2482; found 508.2488 (MþH) þ. 4.1.6. 40 -((Piperidin-1-yl)methyl)-riccardin D (10c) This compound was prepared from riccardin D (60 mg, 0.142 mmol), piperidine (17 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 62% yield. White solid; mp 134e1350  C. IR (film) 3374, 1590, 1522, 1469, 1363, 1159, 910 cm1; 1H NMR (CDCl3) d 7.22 (s, 1H), 6.99 (d, J ¼ 7.9 Hz, 1H), 6.91 (d, J ¼ 7.9 Hz, 1H), 6.89 (d, J ¼ 8.0 Hz, 2H), 6.85 (d, J ¼ 7.7 Hz, 1H), 6.82e6.78 (m, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.49 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.37 (s, 1H), 5.37 (d, J ¼ 1.9 Hz, 1H), 3.93e3.87 (m, 2H), 3.21e2.89 (m, 4H), 2.89e2.81 (m, 2H), 2.80e2.76 (m, 2H), 2.70e2.29 (m, 4H), 1.90e1.30 (m, 6H); 13C NMR (CDCl3) d 154.3, 153.2, 152.7, 149.1, 146.7, 143.3, 142.8, 140.3, 133.2, 131.9, 129.6, 128.9, 124.7, 122.6, 122.4, 122.1, 122.0, 121.2, 119.0, 116.1, 114.9, 60.4, 53.4, 37.9, 37.7, 36.7, 34.7, 24.4, 23.1, 14.2; MS (ESI) 522 (MþH)þ; HRMS (ESI) calcd for C34H36NO4 522.2639; found 522.2645 (MþH) þ.

4.1.7. 40 -(Morpholinomethyl)-riccardin D (10d) This compound was prepared from riccardin D (60 mg, 0.142 mmol), morpholine (18 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 65% yield. White solid; mp 130e131  C. IR (film) 3359, 1580, 1527, 1424, 1351, 1139, 941 cm1; 1H NMR (CDCl3) d 7.14 (d, J ¼ 8.2 Hz, 1H), 6.99 (d, J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 2H), 6.89e6.85 (m, 2H), 6.83e6.77 (m, 2H), 6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.51 (dd, J ¼ 7.8, 1.8 Hz, 1H), 6.32 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 3.83e3.68 (m, 6H), 2.98e2.75 (m, 7H), 2.70e2.51 (m, 5H); 13C NMR (CDCl3) d 156.6, 154.3, 153.7, 153.0, 152.8, 146.7, 146.0, 143.3, 140.3, 133.2, 132.0, 129.6, 129.0, 124.1, 122.5, 122.3, 122.1, 121.2, 118.8, 116.1, 114.9, 66.2, 60.4, 52.7, 38.0, 37.7, 36.8, 34.6; MS (ESI) 524 (MþH)þ; HRMS (ESI) calcd for C33H34NO5 524.2431; found 524.2438 (MþH) þ. 4.1.8. 40 -(Thiomorpholinomethyl)-riccardin D (10e) This compound was prepared from riccardin D (60 mg, 0.142 mmol), thiomorpholine (20 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 63% yield. White solid; mp 142e143  C. IR (film) 3357, 1571, 1525, 1467, 1354, 1138, 935 cm1; 1H NMR (600 MHz, CDCl3) d 7.16 (s, 1H), 7.00 (d, J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 1H), 6.91e6.87 (m, 2H), 6.86 (d, J ¼ 7.7 Hz, 1H), 6.81 (d, J ¼ 8.1 Hz, 2H), 6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.54e6.45 (m, 1H), 6.34 (s, 1H), 5.37 (d, J ¼ 1.9 Hz, 1H), 3.99e3.73 (m, 2H), 3.12e2.90 (m, 5H), 2.90e2.74 (m, 6H), 2.75e2.47 (m, 5H); 13 C NMR (CDCl3) d 154.3, 153.0, 152.7, 146.6, 143.3, 140.3, 133.2, 131.9, 129.5, 129.0, 124.3, 122.5, 122.1, 122.0, 121.3, 118.7, 116.1, 114.9, 60.4, 54.2, 37.9, 37.7, 36.7, 34.6; MS (ESI) 540 (MþH)þ; HRMS (ESI) calcd for C33H34NO4S 540.2203; found 540.2207 (MþH) þ. 4.1.9. 40 -((4-Hydroxypiperidin-1-yl)methyl)-riccardin D (10f) This compound was prepared from riccardin D (60 mg, 0.142 mmol), piperidin-4-ol (22 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 62% yield. White solid; mp 177e178  C. IR (film) 3360, 1578, 1531, 1473, 1356, 1149, 976 cm1; 1H NMR (CD3OD) d 7.07 (d, J ¼ 7.8 Hz, 1H), 6.93 (d, J ¼ 7.8 Hz, 2H), 6.86 (d, J ¼ 7.9 Hz, 1H), 6.79 (d, J ¼ 7.9 Hz, 2H), 6.77e6.73 (m, 2H), 6.68 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.31 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.24 (d, J ¼ 1.6 Hz, 1H), 5.44 (d, J ¼ 2.0 Hz, 1H), 3.84 (s, 2H), 3.73e3.68 (m, 1H), 3.00e2.85 (m, 5H), 2.80e2.55 (m, 5H), 2.49e2.38 (m, 2H), 1.97e1.79 (m, 2H), 1.62e1.53 (m, 2H); 13C NMR (CD3OD) d 156.3, 155.4, 154.8, 148.9, 145.3, 145.0, 142.9, 141.7, 134.3, 133.9, 130.6, 130.1, 129.6, 127.0, 123.5, 123.1, 122.9, 122.2, 122.1, 121.0,

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119.4, 118.2, 118.0, 116.9, 61.5, 51.2, 49.5, 49.3, 38.9, 38.0, 35.9, 34.6; MS (ESI) 538 (MþH)þ; HRMS (ESI) calcd for C34H36NO5 538.2588; found 538.2582 (MþH) þ. 4.1.10. 40 -((4-(4-Methoxyphenyl)piperazin-1-yl)methyl)-riccardin D (10g) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(4-methoxyphenyl)- piperazine (41 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 63% yield. White solid; mp 147e148  C. IR (film) 3358, 1577, 1531, 1459, 1345, 1140, 979 cm1; 1H NMR (600 MHz, CDCl3) d 7.19 (s, 1H), 7.01 (d, J ¼ 7.8 Hz, 1H), 6.96e6.84 (m, 6H), 6.84e6.76 (m, 4H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.51 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.34 (s, 1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 3.99 (d, J ¼ 13.6 Hz, 1H), 3.85e3.78 (m, 1H), 3.75 (s, 3H), 3.38e3.02 (m, 4H), 3.02e2.69 (m, 9H), 2.69e2.30 (m, 3H); 13C NMR (CDCl3) d 154.4, 153.1, 152.8, 146.7, 143.4, 142.5, 140.3, 133.2, 132.0, 129.6, 129.0, 124.3, 122.5, 122.4, 122.1, 122.0, 121.2, 119.0, 118.8, 116.1, 114.9, 114.6, 114.5, 60.1, 55.5, 52.4, 50.1, 38.0, 37.7, 36.7, 34.6; MS (ESI) 629 (MþH)þ; HRMS (ESI) calcd for C40H41N2O5 629.3010; found 629.3015 (MþH) þ. 4.1.11. 40 -{[(2-Hydroxyethyl)(methyl)amino]methyl}-riccardin D (10h) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 2-(methylamino)ethanol (16 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 63% yield. White solid; mp 126e127  C. IR (film) 3360, 1581, 1527, 1440, 1325, 1100, 907 cm1; 1H NMR (CD3OD) d 7.02 (d, J ¼ 7.8 Hz, 1H), 6.88 (d, J ¼ 7.8 Hz, 2H), 6.81 (d, J ¼ 7.8 Hz, 1H), 6.76 (d, J ¼ 3.1 Hz, 1H), 6.74 (d, J ¼ 2.7 Hz, 1H), 6.71 (d, J ¼ 8.5 Hz, 2H), 6.63 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.26 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.20 (d, J ¼ 1.6 Hz, 1H), 5.39 (d, J ¼ 2.0 Hz, 1H), 3.77 (s, 2H), 3.65 (t, J ¼ 5.7 Hz, 2H), 2.94e2.44 (m, 10H), 2.34 (s, 3H); 13C NMR (CD3OD) d 156.4, 155.3, 154.7, 148.9, 145.2, 144.9, 142.8, 141.7, 134.3, 134.0, 130.6, 130.1, 129.5, 127.1, 123.5, 123.1, 122.9, 122.4, 122.1, 121.0, 120.1, 118.2, 116.8, 61.9, 59.7, 59.5, 41.7, 38.9, 38.8, 38.0, 35.9; MS (ESI) 512 (MþH)þ; HRMS (ESI) calcd for C32H33NO5 512.2431; found 512.2436 (MþH) þ. 4.1.12. 40 -((4-Benzylpiperazin-1-yl)methyl)-riccardin D (10i) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-benzylpiperazine (36 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 69% yield. White solid; mp 140e141  C. IR (film) 3375, 1566, 1535, 1444, 1325, 1110, 906 cm1; 1H NMR (CDCl3) d 7.41e7.25 (m, 5H), 7.07 (d, J ¼ 7.8 Hz, 1H), 6.97 (d, J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 1H), 6.90e6.87 (m, 1H), 6.85 (dd, J ¼ 8.2, 2.3 Hz, 1H), 6.83e6.76 (m, 2H), 6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.29 (d, J ¼ 1.7 Hz, 1H), 5.36 (d, J ¼ 2.0 Hz, 1H), 3.89 (d, J ¼ 14.0 Hz, 1H), 3.70 (d, J ¼ 14.1 Hz, 1H), 3.61e3.50 (m, 2H), 3.11e2.86 (m, 4H), 2.86e2.75 (m, 4H), 2.74e2.14 (m, 8H); 13C NMR (CDCl3) d 154.5, 153.0, 152.7, 146.7, 143.8, 143.3, 142.2, 140.3, 133.2, 132.0, 129.6, 129.4, 128.9, 128.8, 128.4, 128.3, 124.1, 122.5, 122.1, 122.0, 121.0, 118.9, 116.1, 114.9, 62.5, 61.1, 60.4, 52.3, 38.1, 37.7, 36.8, 34.4; MS (ESI) 613 (MþH)þ; HRMS (ESI) calcd for C40H41N2O4 613.3061; found 613.3067 (MþH) þ . 4.1.13. 40 -((4-Methylpiperazin-1-yl)methyl)-riccardin D (10j) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-methylpiperazine (22 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 73% yield. White solid; mp 144e145  C. IR (film) 3359, 1578, 1524, 1460, 1265, 1210, 977 cm1; 1H NMR (CDCl3) d 7.07 (d, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.91e6.89 (m, 2H), 6.88e6.84 (m, 2H), 6.82e6.76 (m, 2H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.50 (dd, J ¼ 7.8,

1.7 Hz, 1H), 6.29 (d, J ¼ 1.6 Hz, 1H), 5.36 (d, J ¼ 1.9 Hz, 1H), 3.92 (d, J ¼ 14.0 Hz, 1H), 3.73 (d, J ¼ 13.9 Hz, 1H), 3.16e2.89 (m, 5H), 2.89e2.81 (m, 4H), 2.79 (d, J ¼ 10.6 Hz, 3H), 2.68e2.49 (m, 4H), 2.45 (s, 3H); 13C NMR (CDCl3) d 154.4, 152.9, 152.7, 146.6, 144.2, 143.3, 142.3, 140.3, 133.2, 132.0, 129.5, 129.0, 129.1, 129.0, 124.0, 122.5, 122.3, 122.2, 122.0, 121.0, 118.6, 117.7, 116.1, 114.9, 65.3, 60.9, 60.4, 42.0, 38.0, 37.7, 36.8, 34.5; MS (ESI) 537 (MþH)þ; HRMS (ESI) calcd for C34H37N2O4 537.2748; found 537.2743 (MþH) þ. 4.1.14. 40 -((Bis(2-methoxyethyl)amino)methyl)-riccardin D (10l) This compound was prepared from riccardin D (60 mg, 0.142 mmol), bis(2-methoxyethyl)amine (29 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 75% yield. Colorless oil; IR (film) 3371, 1599, 1521, 1403, 1371, 1250 cm1; 1H NMR (CDCl3) d 7.08 (d, J ¼ 7.8 Hz, 1H), 6.96 (d, J ¼ 7.8 Hz, 1H), 6.92 (d, J ¼ 7.8 Hz, 2H), 6.88 (d, J ¼ 7.8 Hz, 2H), 6.82e6.79 (m, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.56 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.27 (d, J ¼ 1.7 Hz, 1H), 5.37 (d, J ¼ 2.0 Hz, 1H), 4.08 (d, J ¼ 14.0 Hz, 1H), 3.86 (d, J ¼ 14.0 Hz, 1H), 3.61e3.50 (m, 4H), 3.30 (s, 6H), 3.02e2.74 (m, 10H), 2.62e2.50 (m, 2H); 13C NMR (CDCl3) d 154.4, 153.4, 152.7, 146.7, 143.8, 143.3, 141.9, 140.4, 133.3, 132.0, 129.8, 128.7, 124.9, 122.5, 122.1, 121.9, 121.6, 120.7, 119.9, 119.0, 116.2, 114.8, 69.8, 58.8, 58.4, 53.4, 38.0, 37.7, 36.7, 34.3; MS (ESI) 570 (MþH)þ; HRMS (ESI) calcd for C35H40NO6 570.2850; found 570.2855 (MþH) þ. 4.1.15. 40 -((4-(Tert-butoxycarbonyl)piperazin-1-yl)methyl)riccardin D (10m) This compound was prepared from riccardin D (60 mg, 0.142 mmol), tert-butyl piperazine-1- carboxylate (40 mg, 0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 66% yield. White solid; mp 158e159  C. IR (film) 3362, 1578, 1522, 1478, 1360, 1151, 946 cm1; 1H NMR (600 MHz, CDCl3) d 7.07 (d, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 2H), 6.89e6.84 (m, 2H), 6.83e6.77 (m, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.30 (d, J ¼ 1.7 Hz, 1H), 5.37 (d, J ¼ 1.9 Hz, 1H), 3.89 (d, J ¼ 14.0 Hz, 1H), 3.66 (d, J ¼ 13.9 Hz, 1H), 3.57e3.05 (m, 4H), 2.97e2.78 (m, 6H), 2.67e2.35 (m, 6H), 1.44 (s, 9H); 13C NMR (CDCl3) d 154.5, 154.4, 152.9, 152.7, 146.7, 144.0, 143.3, 142.3, 140.3, 133.3, 132.0, 129.6, 129.0, 128.9, 128.2, 124.0, 122.5, 122.2, 122.1, 121.0, 118.7, 118.1, 116.1, 114.9, 80.1, 61.6, 52.3, 38.1, 37.8, 36.8, 34.4, 28.4; MS (ESI) 623 (MþH)þ; HRMS (ESI) calcd for C38H43N2O6 623.3116; found 623.3120 (MþH) þ. 4.1.16. 13, 40 -Bis((diethylamino)methyl)-riccardin D (11a) This compound was prepared from riccardin D (60 mg, 0.142 mmol), diethylamine (30 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 70% yield. White solid; mp 132e133  C. IR (film) 3369, 1587, 1520, 1464, 1366, 1140, 961 cm1; 1H NMR (CDCl3) d 7.05 (d, J ¼ 7.7 Hz, 1H), 6.94 (d, J ¼ 7.8 Hz, 1H), 6.90 (d, J ¼ 7.6 Hz, 1H), 6.89e6.86 (m, 1H), 6.84 (d, J ¼ 8.3 Hz, 3H), 6.53 (dd, J ¼ 7.8, 1.8 Hz, 1H), 6.52 (s, 1H), 6.32 (d, J ¼ 1.6 Hz, 1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 3.89 (dd, J ¼ 14.2, 4.8 Hz, 2H), 3.82 (dd, J ¼ 14.2, 9.5 Hz, 2H), 2.98e2.87 (m, 3H), 2.83e2.70 (m, 7H), 2.66 (q, J ¼ 7.2 Hz, 4H), 2.60e2.55 (m, 1H), 2.51 (dd, J ¼ 12.2, 10.6 Hz, 1H), 1.20 (t, J ¼ 7.2 Hz, 6H), 1.09 (t, J ¼ 7.2 Hz, 6H); 13C NMR (CDCl3) d 155.3, 153.5, 153.2, 147.8, 144.9, 143.6, 142.0, 139.7, 132.1, 131.6, 129.4, 128.6, 128.3, 124.3, 122.8, 122.2, 121.7, 121.6, 121.3, 120.8, 119.1, 118.8, 116.1, 65.3, 56.5, 46.3, 46.1, 38.1, 37.7, 36.7, 34.5, 10.8; MS (ESI) 595 (MþH)þ; HRMS (ESI) calcd for C38H47N2O4 595.3530; found 595.3537 (MþH) þ. 4.1.17. 13, 40 -Bis((pyrrolidin-1-yl)methyl)-riccardin D (11b) This compound was prepared from riccardin D (60 mg, 0.142 mmol), pyrrolidine (30 mg, 0.424 mmol), and aqueous

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formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 53% yield. White solid; mp 133e134  C. IR (film) 3360, 1578, 1525, 1461, 1360, 1158, 936 cm1; 1H NMR (CDCl3) d 7.12 (d, J ¼ 7.7 Hz, 1H), 6.96 (d, J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 7.7 Hz, 2H), 6.86e6.81 (m, 3H), 6.74 (s, 1H), 6.53 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.27 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 1.9 Hz, 1H), 4.15e4.03 (m, 3H), 3.82 (d, J ¼ 13.8 Hz, 1H), 3.03e2.90 (m, 6H), 2.85e2.77 (m, 3H), 2.74 (s, 4H), 2.58e2.30 (m, 3H), 2.03e1.90 (m, 4H), 1.87e1.75 (m, 4H); 13C NMR (CDCl3) d 154.8, 153.2, 147.6, 143.9, 143.7, 142.0, 140.8, 140.1, 132.5, 132.2, 129.6, 128.7, 128.3, 124.4, 122.7, 122.1, 122.0, 121.4, 120.8, 119.0, 118.9, 117.0, 64.8, 58.0, 53.3, 53.2, 38.1, 37.7, 36.7, 34.5, 23.6, 23.5; MS (ESI) 591 (MþH)þ; HRMS (ESI) calcd for C38H43N2O4 591.3217; found 591.3222 (MþH) þ. 4.1.18. 13, 40 -Bis((piperidin-1-yl)methyl)-riccardin D (11c) This compound was prepared from riccardin D (60 mg, 0.142 mmol), piperidine (34 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 57% yield. White solid; mp 156e157  C. IR (film) 3366, 1584, 1529, 1464, 1369, 1159, 959 cm1; 1H NMR (CDCl3) d 7.05 (d, J ¼ 7.8 Hz, 1H), 6.94 (d, J ¼ 7.8 Hz, 1H), 6.90 (d, J ¼ 7.7 Hz, 1H), 6.88 (dd, J ¼ 7.4, 2.3 Hz, 1H), 6.86e6.79 (m, 3H), 6.54 (dd, J ¼ 7.7, 1.8 Hz, 2H), 6.32 (d, J ¼ 1.6 Hz, 1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 3.83 (d, J ¼ 14.1 Hz, 2H), 3.67 (d, J ¼ 14.1 Hz, 2H), 3.02e2.85 (m, 4H), 2.85e2.74 (m, 4H), 2.73e2.61 (m, 2H), 2.58 (t, J ¼ 11.4 Hz, 2H), 2.52 (t, J ¼ 11.4 Hz, 2H), 2.46e2.16 (m, 2H), 1.83e1.67 (m, 4H), 1.59e1.35 (m, 8H); 13C NMR (CDCl3) d 155.0, 153.4, 153.1, 147.7, 144.6, 143.6, 142.1, 139.7, 132.1, 131.7, 129.4, 128.7, 128.5, 124.2, 122.7, 122.2, 121.8, 121.5, 120.8, 119.0, 118.4, 116.2, 61.8, 61.7, 53.7, 38.1, 37.7, 36.7, 34.4, 25.4, 25.3, 23.8, 23.7, 14.2; MS (ESI) 619 (MþH)þ; HRMS (ESI) calcd for C40H47N2O4 619.3530; found 619.3536 (MþH) þ. 4.1.19. 13, 40 -Bis(morpholinomethyl)-riccardin D (11d) This compound was prepared from riccardin D (60 mg, 0.142 mmol), morpholine (36 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 60% yield. White solid; mp 138e139  C. IR (film) 3360, 1581, 1532, 1456, 1377, 1159, 969 cm1; 1H NMR (CDCl3) d 7.10 (d, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 2H), 6.87e6.78 (m, 3H), 6.59 (s, 1H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.32 (d, J ¼ 1.7 Hz, 1H), 5.37 (d, J ¼ 2.0 Hz, 1H), 3.93e3.82 (m, 6H), 3.82e3.58 (m, 6H), 3.06e2.68 (m, 10H), 2.68e2.43 (m, 6H); 13C NMR (CDCl3) d 154.4, 153.3, 153.0, 147.8, 144.2, 144.1, 142.2, 139.8, 132.3, 132.1, 129.4, 129.1, 128.8, 124.1, 122.7, 122.3, 121.1, 121.0, 118.6, 117.6, 116.6, 66.4, 66.3, 61.6, 60.4, 52.8, 52.7, 38.1, 37.6, 36.7, 34.5; MS (ESI) 623 (MþH)þ; HRMS (ESI) calcd for C38H43N2O6 623.3116; found 623.3121 (MþH) þ. 4.1.20. 13, 40 -Bis(thiomorpholinomethyl)-riccardin D (11e) This compound was prepared from riccardin D (60 mg, 0.142 mmol), thiomorpholine (39 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 53% yield. White solid; mp 159e160  C. IR (film) 3361, 1580, 1531, 1474, 1360, 1142, 955 cm1; 1H NMR (600 MHz, CDCl3) d 7.10 (t, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.7 Hz, 1H), 6.91 (t, J ¼ 7.2 Hz, 1H), 6.89e6.87 (m, 1H), 6.87e6.82 (m, 2H), 6.82e6.77 (m, 1H), 6.73 (dd, J ¼ 8.1, 1.9 Hz, 1H), 6.52 (dt, J ¼ 8.1, 1.9 Hz, 1H), 6.31 (dd, J ¼ 5.0, 1.6 Hz, 1H), 5.37 (d, J ¼ 2.4 Hz, 1H), 3.91e3.87 (m, 2H), 3.76e3.72 (m, 2H), 3.57e2.99 (m, 5H), 2.98e2.86 (m, 8H), 2.85e2.77 (m, 4H), 2.77e2.62 (m, 5H), 2.62e2.43 (m, 2H); 13C NMR (CDCl3) d 154.5, 152.9, 152.7, 147.7, 146.6, 143.3, 142.3, 142.2, 140.3, 133.2, 132.1, 132.0, 129.5, 128.9, 124.1, 122.6, 122.5, 122.2, 122.1, 121.1, 120.9, 118.8, 118.7, 116.1, 114.9, 61.9, 60.4, 54.3, 54.2, 45.3, 38.0, 37.7, 36.7, 34.4; MS (ESI) 655 (MþH)þ; HRMS (ESI) calcd for C38H43N2O4S2 655.2659; found 655.2652 (MþH) þ.

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4.1.21. 13, 40 -Bis((4-hydroxypiperidin-1-yl)methyl)-riccardin D (11f) This compound was prepared from riccardin D (60 mg, 0.142 mmol), piperidin-4-ol (44 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 65% yield. White solid; mp 157e158  C. IR (film) 3362, 1580, 1520, 1469, 1364, 1155, 965 cm1; 1H NMR (CD3OD) d 7.07 (d, J ¼ 7.8 Hz, 1H), 6.95 (d, J ¼ 8.1 Hz, 1H), 6.93 (d, J ¼ 8.1 Hz, 1H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.79 (d, J ¼ 7.7 Hz, 1H), 6.77e6.70 (m, 2H), 6.61 (d, J ¼ 1.9 Hz, 1H), 6.31 (dd, J ¼ 7.7, 1.6 Hz, 1H), 6.24 (d, J ¼ 1.6 Hz, 1H), 5.40 (d, J ¼ 1.8 Hz, 1H), 3.85 (s, 2H), 3.80 (s, 2H), 3.77e3.63 (m, 2H), 3.11e2.81 (m, 7H), 2.81e2.27 (m, 9H), 2.01e1.87 (m, 4H), 1.74e1.45 (m, 4H); 13C NMR (CD3OD) d 156.3, 155.4, 154.6, 148.9, 145.4, 144.9, 142.8, 141.8, 134.0, 133.6, 130.6, 130.2, 129.6, 127.0, 123.5, 123.4, 123.1, 122.2, 121.0, 118.0, 117.7, 61.6, 60.3, 51.4, 51.1, 50.6, 49.6, 49.2, 48.9, 38.9, 38.8, 38.0, 35.9; MS (ESI) 651 (MþH)þ; HRMS (ESI) calcd for C40H47N2O6 651.3429; found 651.3432 (MþH) þ. 4.1.22. 13, 40 -Bis((4-(4-methoxyphenyl)piperazin-1-yl)methyl)riccardin D (11g) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(4-methoxyphenyl)- piperazine (82 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 54% yield. White solid; mp 149e150  C. IR (film) 3368, 1589, 1522, 1464, 1365, 1151, 951 cm1; 1H NMR (600 MHz, CDCl3) d 7.11 (d, J ¼ 7.8 Hz, 1H), 6.99 (d, J ¼ 7.8 Hz, 1H), 6.96e6.88 (m, 4H), 6.88e6.73 (m, 9H), 6.60e6.53 (m, 2H), 6.34 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 1.9 Hz, 1H), 3.98e3.83 (m, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 3.74 (s, 1H), 3.22 (s, 4H), 3.09 (br, 4H), 3.00e2.73 (m, 13H), 2.70e2.45 (m, 3H); 13C NMR (CDCl3) d 154.6, 154.3, 153.4, 153.1, 147.9, 145.2, 145.1, 144.4, 144.1, 142.2, 139.8, 132.1, 132.0, 129.4, 128.9, 128.8, 124.1, 122.8, 122.3, 122.2, 121.3, 120.9, 118.7, 118.1, 116.4, 114.5, 114.4, 61.3, 55.6, 55.5, 52.6, 50.5, 38.1, 37.7, 36.7, 34.5; MS (ESI) 833 (MþH)þ; HRMS (ESI) calcd for C52H57N4O6 833.4273; found 833.4270 (MþH) þ. 4.1.23. 13, 40 -Bis{[(2-hydroxyethyl)(methyl)amino]methyl}riccardin D (11h) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 2-(methylamino)ethanol (32 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 59% yield. White solid; mp 132e133  C. IR (film) 3368, 1581, 1527, 1464, 1361, 1154, 954 cm1; 1H NMR (CD3OD) d 7.06 (d, J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 7.8 Hz, 2H), 6.85 (d, J ¼ 7.8 Hz, 1H), 6.78 (d, J ¼ 7.7 Hz, 1H), 6.74 (d, J ¼ 7.9 Hz, 2H), 6.58 (d, J ¼ 2.0 Hz, 1H), 6.28 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.23 (d, J ¼ 1.6 Hz, 1H), 5.38 (d, J ¼ 1.9 Hz, 1H), 3.82 (s, 2H), 3.80 (s, 2H), 3.76 (t, J ¼ 5.8 Hz, 2H), 3.68 (t, J ¼ 5.7 Hz, 2H), 2.94e2.84 (m, 3H), 2.77e2.52 (m, 9H), 2.41 (s, 3H), 2.37 (s, 3H); 13C NMR (CD3OD) d 156.5, 155.4, 154.7, 149.0, 145.4, 144.8, 142.7, 141.7, 134.0, 133.5, 130.6, 130.1, 129.4, 127.1, 123.7, 123.5, 123.1, 122.5, 122.0, 121.0, 120.3, 118.2, 117.6, 62.1, 60.9, 59.8, 59.7, 59.6, 58.3, 41.9, 41.7, 38.9, 38.8, 38.0, 35.9; MS (ESI) 599 (MþH)þ; HRMS (ESI) calcd for C36H43N2O6 599.3116; found 599.3110 (MþH) þ . 4.1.24. 13, 40 -Bis((4-benzylpiperazin-1-yl)methyl)-riccardin D (11i) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-benzylpiperazine (72 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 60% yield. White solid; mp 148e149  C. IR (film) 3362, 1578, 1532, 1460, 1366, 1154, 946 cm1; 1H NMR (CDCl3) d 7.37e7.27 (m, 9H), 7.25e7.22 (m, 1H), 7.06 (d, J ¼ 7.9 Hz, 1H), 6.95 (d, J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 7.7 Hz, 1H), 6.87 (d, J ¼ 2.1 Hz, 1H), 6.83e6.81 (m, 3H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.48 (d, J ¼ 2.1 Hz, 1H), 6.32 (d, J ¼ 1.6 Hz, 1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 3.87 (d, J ¼ 14.1 Hz, 1H), 3.82

614

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(d, J ¼ 13.8 Hz, 1H), 3.75 (d, J ¼ 13.9 Hz, 1H), 3.69 (d, J ¼ 14.1 Hz, 1H), 3.59 (s, 2H), 3.49 (s, 2H), 3.02e2.89 (m, 4H), 2.89e2.73 (m, 8H), 2.72e2.39 (m, 12H); 13C NMR (CDCl3) d 154.7, 153.4, 153.0, 147.8, 144.5, 143.9, 142.2, 139.6, 132.1, 131.7, 129.4, 129.3, 129.2, 128.7, 128.4, 128.3, 127.4, 127.3, 124.0, 122.8, 122.3, 122.0, 121.3, 120.9, 118.8, 118.2, 116.2, 62.7, 61.3, 60.9, 60.4, 52.6, 52.4, 52.3, 52.2, 38.1, 37.6, 36.7, 34.5; MS (ESI) 801 (MþH)þ; HRMS (ESI) calcd for C52H57N4O4 801.4374; found 801.4377 (MþH) þ. 4.1.25. 13, 40 -Bis((4-methylpiperazin-1-yl)methyl)-riccardin D (11j) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-methylpiperazine (44 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 51% yield. White solid; mp 139e140  C. IR (film) 3360, 1588, 1527, 1466, 1368, 1157, 950 cm1; 1H NMR (CDCl3) d 7.06 (d, J ¼ 7.8 Hz, 1H), 6.96 (d, J ¼ 7.8 Hz, 1H), 6.92e6.86 (M, 2H), 6.85e6.78 (m, 3H), 6.52 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.47 (d, J ¼ 2.0 Hz, 1H), 6.32 (d, J ¼ 1.7 Hz, 1H), 5.34 (d, J ¼ 2.0 Hz, 1H), 3.89 (d, J ¼ 13.9 Hz, 1H), 3.81 (d, J ¼ 13.8 Hz, 1H), 3.76 (d, J ¼ 13.9 Hz, 1H), 3.70 (d, J ¼ 14.0 Hz, 1H), 3.17e2.85 (m, 5H), 2.85e2.72 (m, 7H), 2.72e2.57 (m, 6H), 2.57e2.45 (m, 4H), 2.42 (s, 3H), 2.40e2.34 (m, 1H), 2.31 (d, J ¼ 2.0 Hz, 3H), 2.29e2.15 (m, 1H); 13C NMR (CDCl3) d 154.0, 153.4, 153.0, 147.8, 144.3, 144.0, 142.2, 139.7, 132.1, 131.9, 130.9, 129.3, 128.8, 124.0, 122.7, 122.3, 122.1, 121.4, 121.3, 120.9, 118.7, 118.1, 116.3, 65.0, 61.2, 54.7, 54.4, 51.9, 51.8, 45.5, 45.3, 38.1, 37.7, 36.7, 34.5; MS (ESI) 649 (MþH)þ; HRMS (ESI) calcd for C40H49N4O4 649.3748; found 649.3742 (MþH) þ. 4.1.26. 13, 40 -Bis((dibutylamino)methyl)-riccardin D (11k) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-methylpiperazine (44 mg, 0.424 mmol), and aqueous formaldehyde (55 mg, 0.424 mmol) by following GP 1 in 68% yield. Colorless oil. IR (film) 3367, 1589, 1523, 1464, 1368, 1157, 956 cm1; 1H NMR (600 MHz, CDCl3) d 6.93 (d, J ¼ 8.1 Hz, 2H), 6.88 (d, J ¼ 8.0 Hz, 1H), 6.86e6.82 (m, 2H), 6.66 (t, J ¼ 2.0 Hz, 1H), 6.62 (d, J ¼ 8.2 Hz, 2H), 6.47 (dd, J ¼ 8.2, 2.7 Hz, 1H), 6.21 (d, J ¼ 7.5 Hz, 1H), 5.44 (d, J ¼ 1.9 Hz, 1H), 3.80e3.75 (m, 3H), 3.00 (s, 4H), 2.75 (q, J ¼ 8.1, 7.6 Hz, 4H), 2.56 (t, J ¼ 7.6 Hz, 3H), 2.48 (dd, J ¼ 9.2, 6.4 Hz, 4H), 1.67e1.51 (m, 4H), 1.51e1.40 (m, 5H), 1.34 (q, J ¼ 7.5 Hz, 4H), 1.30e1.20 (m, 5H), 0.91 (t, J ¼ 7.3 Hz, 6H), 0.86 (t, J ¼ 7.3 Hz, 6H); 13C NMR (CDCl3) d 157.8, 153.5, 150.8, 147.1, 144.9, 142.0, 139.9, 138.5, 135.7, 129.4, 127.7, 124.4, 122.3, 121.6, 120.8, 119.8, 115.7, 115.5, 112.1, 57.9, 53.2, 36.6, 36.0, 34.8, 30.2, 28.3, 26.9, 20.7, 20.6, 14.0, 13.9; MS (ESI) 707 (MþH)þ; HRMS (ESI) calcd for C46H63N2O4 707.4782; found 707.4788 (MþH) þ. 4.1.27. 13, 40 -Bis((Bis(2-methoxyethyl)amino)methyl)-riccardin D (11l) This compound was prepared from riccardin D (60 mg, 0.142 mmol), bis(2-methoxyethyl)amine (60 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 70% yield. Colorless oil. IR (film) 3371, 1582, 1514, 1459, 1359, 1148, 957 cm1; 1H NMR (CDCl3) d 7.04 (d, J ¼ 7.8 Hz, 1H), 6.94 (d, J ¼ 7.8 Hz, 1H), 6.88 (d, J ¼ 7.8 Hz, 2H), 6.856.83 (m, 3H), 6.56 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.48 (d, J ¼ 1.9 Hz, 1H), 6.28 (d, J ¼ 1.7 Hz, 1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 4.03 (d, J ¼ 14.0 Hz, 1H), 3.97 (d, J ¼ 13.9 Hz, 1H), 3.87 (d, J ¼ 13.9 Hz, 1H), 3.81 (d, J ¼ 14.1 Hz, 1H), 3.60 (t, J ¼ 5.5 Hz, 4H), 3.56e3.45 (m, 4H), 3.36 (s, 6H), 3.29 (s, 6H), 2.95e2.84 (m, 9H), 2.84e2.71 (m, 5H), 2.60e2.37 (m, 2H); 13C NMR (CDCl3) d 154.6, 153.7, 153.5, 148.1, 144.6, 143.8, 141.9, 139.7, 132.1, 131.7, 129.5, 128.5, 124.8, 123.0, 122.2, 122.0, 121.8, 121.4, 120.6, 119.7, 119.4, 116.4, 70.4, 70.1, 58.8, 58.7, 58.2, 53.5, 53.4, 38.1, 37.7, 36.7, 34.4; MS (ESI) 715 (MþH)þ; HRMS (ESI) calcd for C45H55N2O8 715.3953; found 715.3957 (MþH) þ.

4.1.28. 13, 40 -Bis ((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)riccardin D (11m) This compound was prepared from riccardin D (60 mg, 0.142 mmol), tert-butyl piperazine-1- carboxylate (79 mg, 0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 59% yield. White solid; mp 125e126  C. IR (film) 3369, 1582, 1521, 1461, 1366, 1157, 947 cm1; 1H NMR (600 MHz, CDCl3) d 7.06 (d, J ¼ 7.8 Hz, 1H), 6.97 (d, J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 7.7 Hz, 1H), 6.87e6.83 (m, 4H), 6.52 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.46 (s, 1H), 6.33 (d, J ¼ 1.7 Hz, 1H), 5.35 (d, J ¼ 2.1 Hz, 1H), 3.89 (d, J ¼ 14.0 Hz, 1H), 3.79 (d, J ¼ 13.8 Hz, 1H), 3.73 (d, J ¼ 13.7 Hz, 1H), 3.67 (d, J ¼ 14.0 Hz, 1H), 3.59e3.34 (m, 7H), 2.97e2.71 (m, 7H), 2.68e2.44 (m, 10H), 1.47 (s, 9H), 1.44 (s, 9H); 13C NMR (CDCl3) d 154.5, 154.4, 153.0, 147.8, 144.2, 144.1, 142.3, 140.4, 139.8, 132.1, 131.8, 129.4, 128.8, 127.4, 122.3, 121.0, 120.9, 118.6, 117.9, 114.9, 113.7, 80.1, 65.4, 52.3, 42.0, 38.0, 37.6, 36.7, 34.5, 30.2, 29.1, 28.4, 28.3; MS (ESI) 821 (MþH)þ; HRMS (ESI) calcd for C48H61N4O8 821.4484; found 821.4489 (MþH) þ. 4.1.29. 13-Nitro-riccardin D (12) To a solution of riccardin D (50 mg, 0.12 mmol) in DCM (0.7 mL) was added the mixture of nitric acid in acetic acid (5.3 mL HNO3 in 0.5 mL acetic acid) at 0  C. The reaction was stirred at room temperature for 24 h and poured into ice-water. The organic phase was isolated and the aqueous solution was extract with DCM. The combined organic phase then washed with brine, dried, and evaporated. The residue was purified by silica gel column chromatography to give compound 12 as yellow solid, yield 65%; m.p. 88e89  C. IR (film) 3388, 1596, 1529, 1478, 1360, 1150, 949 cm1; 1H NMR (CDCl3) d 10.72 (s, 1H), 7.63 (s, 1H), 7.38 (t, J ¼ 7.8 Hz, 1H), 7.28 (s, 1H), 7.10 (d, J ¼ 7.7 Hz, 1H), 6.96 (d, J ¼ 8.1 Hz, 1H), 6.93 (d, J ¼ 8.0 Hz, 1H), 6.88 (m, 3H), 6.84 (d, J ¼ 8.1 Hz, 1H), 6.56 (s, 1H), 6.38 (d, J ¼ 7.6 Hz, 1H), 5.81 (s, 1H), 4.96 (s, 1H), 4.94 (s, 1H), 3.06e2.57 (m, 8H); 13C NMR (CDCl3) d 153.5, 153.2, 152.3, 149.7, 144.3, 143.7, 143.0, 140.6, 134.0, 132.3, 132.0, 130.5, 129.7, 129.5, 123.1, 122.9, 122.4, 122.3, 122.0, 121.2, 117.7, 117.1, 116.5, 113.6, 39.8, 37.9, 37.3, 36.7, 35.0; MS (ESI) 492 (MþNa)þ; HRMS (ESI) calcd for C28H23NNaO6 492.1418, found: 492.1414 [MþNa]þ. 4.1.30. 13-Amino-riccardin D (13) Compound 12 (98 mg, 0.21 mmol) and Pd/C (10 mg) was dissolved in MeOH (3 mL) and hydrogen gas was bubbled through the mixture for 8 h. DCM was added and the catalyst was removed by filtration, The filtrate was evaporated and the residue was puried with silica gel column chromatography to give compound 13 (92 mg, yield 80%) as red solid; m.p. 84e85  C. IR (film) 3392, 1592, 1515, 1462, 1354, 1151, 966 cm1; 1H NMR (CDCl3) d 7.34 (t, J ¼ 7.9 Hz, 1H), 7.07 (d, J ¼ 7.7 Hz, 1H), 6.91 (d, J ¼ 7.8 Hz, 2H), 6.82 (m, 3H), 6.76 (d, J ¼ 7.9 Hz, 1H), 6.49 (s, 1H), 6.34 (d, J ¼ 7.6 Hz, 1H), 6.28 (s, 1H), 4.85 (s, 1H), 3.06e2.82 (m, 3H), 2.76e2.65 (m, 2H), 2.60 (m, 3H); 13C NMR (CDCl3) d 153.6, 153.0, 152.8, 146.8, 144.0, 143.9, 140.2, 134.4, 132.6, 131.4, 131.1, 130.2, 129.2, 129.1, 122.8, 122.6, 122.2, 121.7, 117.2, 117.0, 113.3, 109.8, 107.2, 46.4, 37.8, 37.6, 36.9, 35.0; MS (ESI) 440 (MþH)þ; HRMS (ESI) calcd for C28H26NO4 440.1856, found: 440.1851 [MþH]þ. 4.1.31. 60 -Dimethylamino-riccardin D (14) A suspension of compound 13 (30 mg, 0.07 mmol) and NaBH4 in THF was added slowly to a mixture of 3 M H2SO4 and formaldehyde at 0  C. The reaction was allowed to stirred for 1 h and quenched with 1 mL H2O. KOH then added and stirred for 0.5 h, and solution was extract ether. The organic phase was dried and evaporated for silica gel column chromatography to give compound 14 (17 mg, 54%) as white solid; m.p. 99e100  C. IR (film) 3352, 1581, 1511, 1463, 1360 cm1; 1H NMR (CDCl3) d 7.35 (t, J ¼ 7.9 Hz, 1H), 7.07 (d,

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J ¼ 7.7 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 2H), 6.82 (m, 4H), 6.59 (s, 1H), 6.53 (s, 1H), 6.36 (d, J ¼ 7.6 Hz, 1H), 5.22 (s, 1H), 3.00e2.86 (m, 3H), 2.78 (s, 6H), 2.77e2.71 (m, 2H), 2.68e2.58 (m, 3H); 13C NMR (CDCl3) d 153.6, 153.1, 152.9, 146.8, 143.9, 140.9, 139.9, 137.5, 131.9, 131.5, 130.2, 129.2, 129.1, 122.9, 122.7, 122.5, 122.3, 121.5, 117.1, 117.1, 113.3, 112.8, 112.2, 44.4, 37.8, 37.5, 37.0, 35.0; MS (ESI) 468 (MþH) þ; HRMS (ESI) calcd for C30H30NO4 468.2169, found: 468.2166 [MþH]þ. 4.1.32. N-Acetamide-acetyl-riccardin D (15) Acetyl chloride (33.7 mL) was added dropwise to a solution of compound 13 (47 mg, 0.107 mmol) and Et3N (104 mL) in DCM (2 mL) at 0  C. The reaction was allowed to room temperature for 1 h and dilute HCl was added. The organic phase was isolated and the aqueous solution was extract with DCM. The combined organic phase then washed with brine, dried, and evaporated. The residue was purified with silica gel column chromatography to give compound 15 (60 mg, yield 80%) as white solid; m.p. 114e115  C. IR (film) 1759, 1591, 1505, 1457, 1194, 1150, 956 cm1; 1H NMR (CDCl3) d 7.82 (s, 1H), 7.42 (t, J ¼ 7.8 Hz, 1H), 7.35 (d, J ¼ 7.8 Hz, 1H), 7.22 (s, 1H), 6.97 (d, J ¼ 7.9 Hz, 1H), 6.90 (m, 2H), 6.83 (d, J ¼ 7.9 Hz, 1H), 6.73 (m, 2H), 6.60 (d, J ¼ 9.5 Hz, 2H), 2.92 (m, 3H), 2.77 (m, 5H), 2.40 (s, 3H), 2.22 (s, 3H), 2.04 (s, 3H), 1.83 (s, 3H). 13C NMR (CDCl3) d 169.7, 168.5, 168.3, 168.1, 152.9, 150.7, 148.7, 147.6, 143.7, 141.6, 140.2, 139.4, 132.0, 131.2, 129.8, 129.3, 129.2, 129.0, 128.1, 127.6, 125.9, 124.9, 123.3, 122.4, 122.1, 119.9, 114.4, 113.1, 37.7, 37.4, 37.0, 34.6, 24.9, 21.0, 20.7, 20.5; MS (ESI) 630 (MþNa) þ; HRMS (ESI) calcd for C36H37N2O8 625.2544 found: 625.2552 [MþNH4]þ. 4.1.33. N-riccardin D-acetamide (16) The solution of 1 N NaOH (0.5 mL) was added to a suspension of compound 15 (50 mg, 0.1 mmol) in MeOH (0.5 mL) at 0  C. The solution was quenched after stirring for 0.5 h with hydrochloric acid. Then DCM was added and isolated. The organic phase was dried and evaporated for silica gel column chromatography to give compound 16 (25 mg, 64%) as white solid, m.p. 117e118  C. IR (film) 3368, 1760, 1588, 1515, 1453, 1360, 1151 cm1; 1H NMR (600 MHz, (CD3)2CO) d 8.96 (s, 1H), 8.65 (s, 1H), 7.73 (s, 1H), 7.20 (m, 3H), 6.97 (m, 2H), 6.87 (d, J ¼ 6.9 Hz, 1H), 6.80 (d, J ¼ 7.5 Hz, 1H), 6.73 (m, 3H), 6.28 (m, 2H), 5.18 (s, 1H), 2.92e2.83 (m, 3H), 2.73 (m, 1H), 2.60 (m, 5H), 2.21 (s, 3H). 13C NMR (151 MHz, (CD3)2CO) d 170.2, 155.5, 155.0, 154.3, 149.5, 144.9, 142.5, 141.3, 136.0, 133.7, 133.6, 132.9, 130.2, 123.0, 129.3, 128.4, 126.0, 123.0, 122.8, 122.5, 122.4, 121.1, 117.7, 115.1, 113.7, 38.4, 38.3, 35.6, 23.9; MS (ESI) 504 (MþNa)þ; HRMS (ESI) calcd for C30H27NNaO5 504.1781, found: 504.1775 [MþNa]þ. 4.1.34. 30 -(2-(Dimethylamino)ethoxy)-riccardin D (17a) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 2-chloro-N,N- dimethylethanamine hydrochloride (31 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 66% yield. White solid; mp 162e163  C. IR (film) 3368, 1589, 1520, 1465, 1360, 1142, 955 cm1; 1H NMR (CDCl3) d 7.35 (t, J ¼ 7.9 Hz, 1H), 7.12 (d, J ¼ 7.8 Hz, 1H), 7.02 (d, J ¼ 7.9 Hz, 1H), 6.96e6.93 (m, 2H), 6.89 (d, J ¼ 7.6 Hz, 1H), 6.86e6.77 (m, 3H), 6.75 (dd, J ¼ 8.1, 1.6 Hz, 1H), 6.65 (d, J ¼ 7.6 Hz, 1H), 6.21 (s, 1H), 5.38 (d, J ¼ 1.6 Hz, 1H), 4.17e4.02 (m, 2H), 3.07e2.96 (m, 2H), 2.96e2.80 (m, 4H), 2.70e2.61 (m, 1H), 2.61e2.53 (m, 2H), 2.42 (t, J ¼ 12.0 Hz, 1H), 2.29 (s, 6H); 13C NMR (CDCl3) d 156.1, 154.7, 152.8, 146.8, 143.8, 143.4, 141.4, 140.6, 133.5, 131.5, 130.5, 128.5, 128.3, 127.8, 123.6, 123.3, 122.8, 122.0, 121.5, 121.1, 116.3, 114.8, 108.9, 65.1, 58.0, 45.1, 37.8, 37.6, 37.1, 34.2; MS (ESI) 496 (MþH)þ; HRMS (ESI) calcd for C32H34NO4 496.2482; found 496.2480 (MþH) þ. 4.1.35. 30 -(2-(Diethylamino)ethoxy)-riccardin D (17b) This compound was prepared from riccardin D (60 mg,

615

0.142 mmol), 2-chloro-N,N- diethylethanamine hydrochloride (37 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 78% yield. White solid; mp 198e199  C. IR (film) 3370, 1581, 1524, 1464, 1360, 1151, 959 cm1; 1H NMR (CDCl3) d 7.38 (t, J ¼ 7.9 Hz, 1H), 7.16 (d, J ¼ 7.7 Hz, 1H), 6.99 (d, J ¼ 7.3 Hz, 1H), 6.93 (d, J ¼ 7.9 Hz, 2H), 6.94e6.82 (m, 4H), 6.74 (d, J ¼ 7.5 Hz, 1H), 6.51 (d, J ¼ 6.1 Hz, 1H), 6.28 (s, 1H), 5.40 (s, 1H), 4.30e4.20 (m, 2H), 2.98e2.90 (m, 4H), 2.82 (d, J ¼ 10.5 Hz, 2H), 2.70 (s, 4H), 2.63 (t, J ¼ 11.1 Hz, 1H), 2.50 (t, J ¼ 11.1 Hz, 1H), 1.27 (s, 6H); 13C NMR (CDCl3) d 155.8, 153.8, 152.7, 146.8, 144.4, 143.4, 141.8, 140.5, 133.4, 131.7, 130.0, 129.0, 128.8, 124.1, 122.7, 122.0, 121.9, 120.9, 116.1, 114.8, 109.6, 64.5, 50.9, 46.8, 37.6, 36.9, 34.5, 9.4; MS (ESI) 524 (MþH)þ; HRMS (ESI) calcd for C34H38NO4 524.2795; found 524.2798 (MþH) þ. 4.1.36. 30 -(2-(Pyrrolidin-1-yl)ethoxy)-riccardin D (17c) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (37 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 70% yield. White solid; mp 224e225  C. IR (film) 3369, 1585, 1524, 1463, 1366, 1150, 951 cm1; 1H NMR (CDCl3) d 7.33 (t, J ¼ 7.9 Hz, 1H), 7.10 (dd, J ¼ 7.8, 1.1 Hz, 1H), 7.00 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.91 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.90 (d, J ¼ 7.8, 1H), 6.84 (d, J ¼ 7.6 Hz, 1H), 6.83e6.74 (m, 3H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.57 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.20 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 4.24e4.03 (m, 2H), 3.01e2.75 (m, 8H), 2.72e2.47 (m, 5H), 2.46e2.33 (m, 1H), 1.90e1.75 (m, 4H).13C NMR (CDCl3) d 156.0, 154.6, 152.7, 146.9, 144.0, 143.4, 141.5, 140.6, 133.5, 131.6, 130.3, 128.7, 128.5, 127.6, 123.6, 122.7, 121.9, 121.6, 120.9, 116.1, 114.8, 109.1, 65.9, 54.6, 54.1, 53.4, 50.8, 37.8, 37.6, 37.0, 34.4, 23.3; MS (ESI) 522 (MþH)þ; HRMS (ESI) calcd for C34H36NO4 522.2639; found 522.2635 (MþH) þ. 4.1.37. 30 -(2-(Piperidin-1-yl)ethoxy)-riccardin D (17d) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(2-chloroethyl)piperidine hydrochloride (42 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 76% yield. White solid; mp 220e221  C. IR (film) 3369, 1585, 1524, 1465, 1364, 1151, 950 cm1; 1H NMR (CDCl3) d 7.36 (t, J ¼ 8.0 Hz, 1H), 7.13 (d, J ¼ 7.8 Hz, 1H), 7.02 (d, J ¼ 7.4 Hz, 1H), 6.93 (d, J ¼ 8.0 Hz, 2H), 6.84 (d, J ¼ 7.6 Hz, 1H), 6.83e6.78 (m, 3H), 6.74 (dd, J ¼ 8.1, 1.5 Hz, 1H), 6.55 (d, J ¼ 7.6 Hz, 1H), 6.29 (s, 1H), 5.40 (d, J ¼ 1.5 Hz, 1H), 4.30e4.25 (m, 1H), 4.15e4.12 (m, 1H), 3.03e2.71 (m, 8H), 2.65e2.40 (m, 6H), 1.75e1.62 (m, 4H), 1.50e1.42 (m, 2H); 13C NMR (CDCl3) d 155.9, 154.2, 152.7, 146.9, 144.1, 143.4, 141.7, 140.6, 133.5, 131.7, 130.2, 128.8, 128.7, 127.2, 123.9, 122.7, 121.7, 120.9, 116.0, 114.8, 109.4, 64.4, 57.4, 54.5, 37.8, 37.6, 37.0, 34.4, 29.7, 24.3; MS (ESI) 536 (MþH)þ; HRMS (ESI) calcd for C35H38NO4 536.2795; found 536.2790 (MþH) þ. 4.1.38. 30 -(2-(Azepan-1-yl)ethoxy)-riccardin D (17e) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(2-chloroethyl)azepane hydrochloride (42 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 72% yield. White solid; mp 189e190  C. IR (film) 3364, 1582, 1520, 1464, 1365, 1155, 957 cm1; 1H NMR (CDCl3) d 7.34 (t, J ¼ 7.9 Hz, 1H), 7.11 (d, J ¼ 7.5 Hz, 1H), 6.98 (d, J ¼ 7.2 Hz, 1H), 6.92 (d, J ¼ 8.0 Hz, 2H), 6.87e6.76 (m, 4H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.56 (d, J ¼ 7.9 Hz, 1H), 6.23 (s, 1H), 5.38 (s, 1H), 4.23e4.02 (m, 2H), 3.02e2.79 (m, 8H), 2.71 (s, 4H), 2.63e2.40 (m, 2H), 1.64e1.58 (m, 8H); 13C NMR (CDCl3) d 155.9, 154.3, 152.7, 146.8, 144.1, 143.4, 141.6, 140.5, 133.5, 131.7, 130.2, 128.7, 128.6, 127.1, 123.8, 122.8, 122.0, 121.7, 121.6, 120.9, 116.2, 114.8, 65.1, 55.6, 55.4, 37.8, 37.7, 37.0, 34.4, 27.0, 26.9; MS (ESI) 550 (MþH)þ; HRMS (ESI) calcd for C36H40NO4 550.2952; found 550.2946 (MþH) þ.

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4.1.39. 30 -(2-Morpholinoethoxy)-riccardin D (17f) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 4-(2-chloroethyl)- morpholine hydrochloride (40 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 70% yield. White solid; mp 160e161  C. IR (film) 3367, 1587, 1515, 1460, 1355, 1157, 950 cm1; 1H NMR (CDCl3) d 7.36 (t, J ¼ 8.0 Hz, 1H), 7.14 (d, J ¼ 7.7 Hz, 1H), 6.97 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.92 (d, J ¼ 8.0 Hz, 2H), 6.84e6.78 (m, 3H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.53 (d, J ¼ 7.8 Hz, 1H), 6.26 (s, 1H), 5.65 (s, 1H), 5.36 (d, J ¼ 1.8 Hz, 1H), 4.52e4.05 (m, 2H), 3.75 (s, 4H), 3.06e2.74 (m, 7H), 2.73e2.14 (m, 7H); 13C NMR (CDCl3) d 156.1, 155.6, 153.6, 152.7, 146.8, 143.4, 141.9, 140.5, 133.2, 131.8, 130.0, 128.9, 128.8, 124.3, 122.7, 122.0, 121.9, 121.0, 116.0, 114.9, 64.5, 61.1, 57.4, 53.5, 37.7, 37.6, 37.0, 34.5; MS (ESI) 538 (MþH)þ; HRMS (ESI) calcd for C34H36NO5 538.2588; found 538.2582 (MþH) þ. 4.1.40. 30 -(3-(Diethylamino)propoxy)-riccardin D (17g) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 3-chloro-N,N- dimethylpropan-1-amine hydrochloride (35 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 71% yield. White solid; mp 186e187  C. IR (film) 3365, 1588, 1523, 1465, 1365, 1156, 949 cm1; 1 H NMR (CDCl3) d 7.36 (t, J ¼ 7.9 Hz, 1H), 7.12 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.94e6.87 (m, 4H), 6.84 (dd, J ¼ 8.3, 1.2 Hz, 1H), 6.79 (d, J ¼ 7.9 Hz, 2H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.43 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.27 (d, J ¼ 1.7 Hz, 1H), 5.40 (d, J ¼ 2.0 Hz, 1H), 4.02e3.97 (m, 1H), 3.90e3.86 (m, 1H), 2.98e2.85 (m, 4H), 2.82e2.71 (m, 3H), 2.70e2.48 (m, 3H), 2.21 (s, 6H), 1.85e1.74 (m, 2H); 13C NMR (CDCl3) d 156.2, 153.2, 152.8, 146.8, 144.5, 143.5, 141.8, 140.4, 133.2, 132.0, 129.6, 129.3, 126.0, 123.8, 122.6, 122.0, 121.3, 120.6, 118.0, 116.1, 115.0, 110.3, 66.6, 55.8, 44.7, 37.8, 37.7, 36.8, 34.6, 26.9; MS (ESI) 510 (MþH)þ; HRMS (ESI) calcd for C33H36NO4 510.2639; found 510.2646 (MþH) þ. 4.1.41. 30 -(3-(Pyrrolidin-1-yl)propoxy)-riccardin D (17h) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(3-chloropropyl)- pyrrolidine hydrochloride (39 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by following GP 2 in 73% yield. White solid; mp 197e198  C. IR (film) 3368, 1588, 1528, 1464, 1365, 1153, 954 cm1; 1 H NMR (CDCl3) d 7.38 (t, J ¼ 8.0 Hz, 1H), 7.15 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.94e6.88 (m, 3H), 6.84 (dd, J ¼ 8.2, 1.1 Hz, 1H), 6.82e6.77 (m, 3H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.42 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.29 (d, J ¼ 1.7 Hz, 1H), 5.40 (d, J ¼ 2.0 Hz, 1H), 4.09e4.05 (m, 1H), 3.92e3.87 (m, 1H), 3.00e2.84 (m, 6H), 2.79e2.61 (m, 6H), 2.58e2.48 (m, 2H), 2.00e1.88 (m, 6H); 13C NMR (CDCl3) d 155.9, 153.3, 152.6, 146.8, 144.6, 143.5, 141.8, 140.5, 133.1, 132.2, 129.5, 129.0, 125.8, 124.2, 122.6, 122.2, 122.0, 120.4, 117.3, 116.0, 114.9, 110.7, 65.9, 53.7, 52.6, 37.8, 37.7, 36.9, 34.7, 26.9, 23.4; MS (ESI) 536 (MþH)þ; HRMS (ESI) calcd for C35H38NO4 536.2795; found 536.2790 (MþH) þ. 4.1.42. 3, 12, 30 -Tri(2-(dimethylamino)ethoxy)-riccardin D (18a) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 2-chloro-N,N- dimethylethanamine hydrochloride (102 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 83% yield. White solid; mp 77e78  C. IR (film) 1587, 1521, 1465, 1361, 1142, 959 cm1; 1H NMR (CDCl3) d 7.28 (t, J ¼ 7.9 Hz, 1H), 7.03 (dd, J ¼ 7.7, 1.1 Hz, 1H), 6.90 (d, J ¼ 8.0 Hz, 2H), 6.81 (d, J ¼ 8.0, 2H), 6.78e6.74 (m, 2H), 6.73e6.68 (m, 2H), 6.37 (d, J ¼ 1.6 Hz, 1H), 6.28 (dd, J ¼ 7.6, 1.6 Hz, 1H), 5.47 (d, J ¼ 2.0 Hz, 1H), 4.20 (t, J ¼ 6.3 Hz, 2H), 3.97e3.76 (m, 4H), 2.99e2.84 (m, 3H), 2.82 (t, J ¼ 6.2 Hz, 2H), 2.80e2.56 (m, 5H), 2.52 (q, J ¼ 6.5 Hz, 2H), 2.44 (td, J ¼ 6.0, 2.7 Hz, 2H), 2.37 (s, 6H), 2.17 (s, 6H), 2.08 (s, 6H); 13C NMR (CDCl3) d 156.5, 156.0, 153.1, 149.8, 146.2,

143.8, 141.3, 140.1, 134.9, 132.9, 130.9, 129.2, 128.8, 128.2, 127.9, 123.2, 122.6, 122.3, 121.5, 121.1, 117.1, 114.7, 112.8, 109.6, 67.9, 67.2, 66.9, 58.3, 58.2, 57.9, 46.0, 45.9, 45.8, 37.8, 37.6, 36.9, 35.2; MS (ESI) 638 (MþH)þ; HRMS (ESI) calcd for C40H52N3O4 638.3952; found 638.3959 (MþH) þ. 4.1.43. 3, 12, 30 -Tri(2-(diethylamino)ethoxy)-riccardin D (18b) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 2-chloro-N,N- diethylethanamine hydrochloride (122 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 86% yield. White solid; mp 89e90  C. IR (film) 1587, 1522, 1465, 1361, 1150, 962 cm1; 1H NMR (CDCl3) d 7.23 (t, J ¼ 8.0 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.84 (d, J ¼ 8.2 Hz, 2H), 6.77 (d, J ¼ 6.6 Hz, 1H), 6.712e6.68 (m, 5H), 6.36 (s, 1H), 6.20 (d, J ¼ 7.6 Hz, 1H), 5.42 (d, J ¼ 1.7 Hz, 1H), 4.24 (t, J ¼ 6.0 Hz, 2H), 4.07e3.97 (m, 4H), 3.13e3.06 (m, 2H), 2.96e2.84 (m, 7H), 2.81 (q, J ¼ 7.1 Hz, 4H), 2.78e2.71 (m, 1H), 2.72e2.65 (m, 1H), 2.61 (q, J ¼ 7.1 Hz, 4H), 2.58e2.52 (m, 2H), 2.49 (q, J ¼ 7.1 Hz, 4H), 2.44e2.41 (m, 1H), 1.12 (t, J ¼ 7.2 Hz, 6H), 0.92 (t, J ¼ 7.2 Hz, 6H), 0.88 (t, J ¼ 7.2 Hz, 6H); 13C NMR (CDCl3) d 155.8, 155.5, 153.0, 149.3, 145.7, 144.0, 141.7, 140.1, 134.8, 132.7, 129.5, 129.1, 128.5, 127.3, 123.0, 122.8, 122.1, 121.7, 121.6, 117.0, 114.3, 112.2, 109.4, 66.6, 65.3, 65.2, 51.3, 51.0, 50.8, 47.6, 47.5, 47.4, 37.7, 37.4, 36.6, 35.1, 10.8, 10.1, 10.0; MS (ESI) 722 (MþH)þ; HRMS (ESI) calcd for C46H64N3O4 722.4891; found 722.4898 (MþH) þ. 4.1.44. 3, 12, 30 - Tri (2-(pyrrolidin-1-yl)ethoxy)-riccardin D (18c) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (120 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 90% yield. White solid; mp 82e83  C. IR (film) 1586, 1525, 1465, 1364, 1151, 954 cm1; 1H NMR (CDCl3) d 7.27 (t, J ¼ 7.7 Hz, 1H), 7.03 (d, J ¼ 7.7 Hz, 1H), 6.91 (d, J ¼ 8.2 Hz, 1H), 6.89e6.86 (m, 2H), 6.80 (d, J ¼ 8.2 Hz, 1H), 6.78e6.73 (m, 3H), 6.71 (d, J ¼ 8.2 Hz, 1H), 6.32 (s, 1H), 6.30 (dd, J ¼ 8.5, 2.0 Hz, 1H), 5.50 (d, J ¼ 2.0 Hz, 1H), 4.39 (t, J ¼ 5.6 Hz, 2H), 4.32e4.08 (m, 4H), 3.40e3.26 (m, 2H), 3.24 (t, J ¼ 5.6 Hz, 2H), 3.11e3.02 (m, 6H), 2.94e2.76 (m, 8H), 2.72e2.51 (m, 8H), 1.97e1.87 (m, 4H), 1.87e1.78 (m, 4H), 1.77e1.60 (m, 4H); 13C NMR (CDCl3) d 155.5, 155.2, 153.1, 149.4, 145.7, 144.0, 141.8, 140.1, 135.1, 132.8, 129.5, 129.2, 128.6, 127.1, 123.1, 123.0, 122.2, 121.8, 121.6, 117.3, 115.1, 112.1, 109.0, 67.1, 64.8, 64.6, 54.5, 54.4, 54.3, 54.2, 54.1, 54.0, 37.6, 37.3, 36.7, 35.2, 23.3, 23.1, 23.0. MS (ESI) 716 (MþH)þ; HRMS (ESI) calcd for C46H58N3O4 716.4422; found 716.4416 (MþH) þ. 4.1.45. 3, 12, 30 - Tri (2-(piperidin-1-yl)ethoxy)-riccardin D (18d) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(2-chloroethyl)piperidine hydrochloride (130 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 88% yield. White solid; mp 95e96  C. IR (film) 1586, 1525, 1464, 1365, 1150, 951 cm1; 1H NMR (CDCl3) d 7.21 (t, J ¼ 8.0 Hz, 1H), 6.96 (d, J ¼ 7.7 Hz, 1H), 6.83 (d, J ¼ 8.2 Hz, 2H), 6.77 (d, J ¼ 7.4 Hz, 1H), 6.74 (d, J ¼ 7.6 Hz, 1H), 6.69 (d, J ¼ 7.9 Hz, 2H), 6.66e6.65 (m, 2H), 6.30 (s, 1H), 6.24 (d, J ¼ 7.6 Hz, 1H), 5.41 (d, J ¼ 1.7 Hz, 1H), 4.18 (t, J ¼ 6.3 Hz, 2H), 3.95e3.76 (m, 4H), 2.87e2.80 (m, 5H), 2.75e2.68 (m, 1H), 2.66e2.56 (m, 4H), 2.56e2.48 (m, 8H), 2.28 (s, 4H), 2.16 (s, 4H), 1.61e1.52 (m, 4H), 1.45 (dt, J ¼ 11.1, 5.6 Hz, 4H), 1.38 (dt, J ¼ 11.4, 5.6 Hz, 4H), 1.36e1.22 (m, 6H); 13C NMR (CDCl3) d 156.4, 156.0, 153.0, 149.5, 146.1, 143.9, 141.4, 140.2, 134.7, 132.8, 129.4, 129.3, 128.2, 127.8, 123.2, 122.6, 122.3, 121.5, 121.0, 117.0, 114.2, 112.7, 109.4, 67.1, 66.6, 66.4, 57.9, 57.8, 57.5, 55.0, 54.9, 54.5, 37.9, 37.6, 36.9, 35.2, 25.8, 25.7, 25.6, 24.1, 23.9, 23.8. MS (ESI) 758 (MþH)þ; HRMS (ESI) calcd for C49H64N3O4 758.4891; found 758.4898 (MþH) þ.

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4.1.46. 3, 120 , 30 -Tri(2-(azepan-1-yl)ethoxy)-riccardin D (18e) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(2-chloroethyl)azepane hydrochloride (140 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 84% yield. White solid; mp 88e89  C. IR (film) 1585, 1522, 1463, 1365, 1152, 956 cm1; 1H NMR (CDCl3) d 7.28 (t, J ¼ 7.8, 1H), 7.02 (dd, J ¼ 7.8, 1.0 Hz, 1H), 6.91 (d, J ¼ 8.2 Hz, 2H), 6.84e6.80 (m, 2H), 6.77e6.72 (m, 4H), 6.37 (d, J ¼ 1.5 Hz, 1H), 6.30 (dd, J ¼ 7.6, 1.5 Hz, 1H), 5.47 (d, J ¼ 2.1 Hz, 1H), 4.21 (t, J ¼ 6.6 Hz, 2H), 4.01e3.76 (m, 4H), 3.05 (t, J ¼ 6.6 Hz, 2H), 2.97e2.86 (m, 3H), 2.85e2.79 (m, 4H), 2.78e2.72 (m, 3H), 2.70e2.65 (m, 4H), 2.66e2.59 (m, 2H), 2.58e2.57 (m, 4H), 2.47e2.45 (m, 4H), 1.69e1.66 (m, 4H), 1.62e1.57 (m, 4H), 1.52 (s, 8H), 1.46 (s, 8H); 13C NMR (CDCl3) d 156.6, 156.1, 153.1, 149.6, 146.2, 143.8, 141.3, 140.1, 134.6, 132.8, 129.3, 128.1, 127.8, 123.2, 122.4, 122.3, 121.4, 120.9, 117.0, 114.2, 112.7, 109.3, 67.9, 67.0, 66.8, 56.9, 56.6, 56.4, 56.0, 55.9, 55.5, 37.9, 37.6, 36.9, 35.2, 28.0, 27.9, 27.8, 27.0, 26.9; MS (ESI) 800 (MþH)þ; HRMS (ESI) calcd for C52H70N3O4 800.5361; found 800.5354 (MþH) þ . 4.1.47. 3, 12, 30 -Tri(2-morpholinoethoxy)-riccardin D (18f) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 4-(2-chloroethyl)- morpholine hydrochloride (130 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 80% yield. White solid; mp 80e81  C. IR (film) 1588, 1515, 1461, 1356, 1157, 951 cm1; 1H NMR (CDCl3) d 7.29 (t, J ¼ 7.9 Hz, 1H), 7.04 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.92 (d, J ¼ 8.2 Hz, 2H), 6.85 (d, J ¼ 7.5 Hz, 1H), 6.81 (d, J ¼ 7.6 Hz, 1H), 6.77e6.73 (m, 4H), 6.38 (d, J ¼ 1.5 Hz, 1H), 6.30 (dd, J ¼ 7.6, 1.5 Hz, 1H), 5.48 (d, J ¼ 2.1 Hz, 1H), 4.30 (s, 2H), 4.03 (s, 4H), 3.78 (t, J ¼ 4.6 Hz, 4H), 3.64 (s, 4H), 3.56 (s, 4H), 3.05e2.86 (m, 5H), 2.82e2.55 (m, 11H), 2.45 (s, 4H), 2.29 (s, 4H); 13C NMR (CDCl3) d 153.1, 149.7, 146.0, 144.0, 141.9, 141.6, 140.2, 125.1, 132.8, 129.5, 128.5, 127.6, 124.9, 123.3, 122.9, 122.2, 121.7, 121.4, 117.1, 115.1, 112.5, 109.3, 67.3, 66.6, 66.5, 66.2, 66.0, 57.6, 57.5, 57.2, 54.0, 53.8, 53.5, 37.8, 37.5, 36.9, 35.2. MS (ESI) 764 (MþH)þ; HRMS (ESI) calcd for C46H58N3O7 764.4269; found 764.4262 (MþH) þ. 4.1.48. 3, 12, 30 - Tri (3-(diethylamino)propoxy)-riccardin D (18g) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 3-chloro-N,N- dimethylpropan-1-amine hydrochloride (113 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 87% yield. White solid; mp 93e94  C. IR (film) 1588, 1520, 1465, 1366, 1156, 950 cm1; 1H NMR (CDCl3) d 7.32 (t, J ¼ 7.9 Hz, 1H), 7.10 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 1H), 6.89e6.77 (m, 4H), 6.76 (dd, J ¼ 8.2, 2.0 Hz, 2H), 6.71e6.61 (m, 1H), 6.41 (dd, J ¼ 7.6, 1.5 Hz, 1H), 6.29 (d, J ¼ 1.5 Hz, 1H), 5.43 (d, J ¼ 2.0 Hz, 1H), 4.20 (t, J ¼ 5.9 Hz, 2H), 4.09e3.98 (m, 1H), 3.92 (t, J ¼ 5.4 Hz, 2H), 3.76e3.70 (m, 1H), 3.18e3.02 (m, 2H), 3.00e2.78 (m, 5H), 2.75e2.68 (m, 3H), 2.66 (s, 6H), 2.63e2.59 (m, 1H), 2.57 (s, 6H), 2.47 (s, 6H), 2.35e2.11 (m, 3H), 2.09e1.84 (m, 6H); 13 C NMR (CDCl3) d 156.1, 155.6, 153.0, 149.6, 145.8, 143.7, 141.6, 140.1, 135.1, 132.4, 129.4, 128.6, 128.1, 123.7, 123.0, 122.2, 121.9, 121.0, 117.3, 115.6, 113.1, 111.0, 67.4, 66.2, 65.0, 55.9, 54.9, 54.8, 43.7, 43.0, 42.8, 37.8, 37.6, 37.1, 34.9, 25.5, 24.8, 24.6. MS (ESI) 680 (MþH)þ; HRMS (ESI) calcd for C43H58N3O4 680.4422; found 680.4430 (MþH) þ. 4.1.49. 3, 12, 30 - Tri (3-(pyrrolidin-1-yl)propoxy)-riccardin D (18h) This compound was prepared from riccardin D (60 mg, 0.142 mmol), 1-(3-chloropropyl)- pyrrolidine hydrochloride (131 mg, 0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by following GP 2 in 90% yield. White solid; mp 86e87  C. IR (film) 1587, 1528, 1464, 1364, 1155, 954 cm1; 1H NMR (CDCl3) d 7.31 (t, J ¼ 7.9 Hz, 1H), 7.10 (d, J ¼ 7.7 Hz, 1H), 6.90 (d, J ¼ 8.2 Hz, 1H), 6.85 (d, J ¼ 8.5 Hz, 2H), 6.84e6.81 (m, 2H),

617

6.79e6.72 (m, 2H), 6.65 (s, 1H), 6.39 (d, J ¼ 7.5 Hz, 1H), 6.59 (d, J ¼ 2.1 Hz, 1H), 5.40 (d, J ¼ 1.9 Hz, 1H), 4.20 (t, J ¼ 5.5 Hz, 2H), 4.06e3.96 (m, 1H), 3.94e3.91 (m, 2H), 3.81e3.76 (m, 2H), 3.69e3.65 (m, 2H), 3.58e3.47 (m, 2H), 3.42e3.36 (m, 3H), 3.10e3.04 (m, 2H), 2.95e2.81 (m, 5H), 2.77e2.49 (m, 11H), 2.45e2.40 (m, 2H), 2.22e2.11 (m, 4H), 2.09e1.86 (m, 13H); 13C NMR (CDCl3) d 156.0, 155.6, 152.9, 149.5, 145.6, 143.7, 141.5, 140.0, 135.1, 132.4, 129.5, 128.6, 128.2, 123.9, 123.1, 122.2, 121.9, 121.0, 117.3, 115.7, 113.2, 111.5, 67.2, 66.5, 65.1, 53.8, 53.7, 53.5, 53.4, 53.3, 53.2, 53.1, 53.0, 37.9, 37.6, 37.1, 34.9, 26.0, 25.8, 25.7, 23.4, 23.3, 23.2, 22.4; MS (ESI) 758 (MþH)þ; HRMS (ESI) calcd for C49H64N3O4 758.4891; found 758.4898 (MþH) þ. 4.1.50. Antiproliferative studies Human non-small cell lung cancer A549 cell lines and human breast adenocarcinoma cell line MCF-7 were purchased from the Shanghai Institute for Biological Sciences (SIBS), China Academy of Sciences (China). Human myelogenous leukemia k562 cell line was purchased from the Department of Pharmacology, Institute of Hematology of the Chinese Academy of Medical Sciences, Tianjin (China). The cells were cultured in RPMI-1640 (HyClone) medium containing 10% FBS (Sijiqing Company, Ltd.), 100 units/mL of penicillin G, and 100 mg/mL of streptomycin in a stable environment with 37  C and 5% CO2. The antiproliferative activity of the riccardin D derivatives on the three tumor cell lines was measured by the MTT method. Briefly, after treatment with the candidate drug for 48 h, the absorbance of the soluble MTT product was measured at 570 nm. All experiments were performed at least three times. 4.1.51. AO and LysoTracker uptake We used LysoTracker (50 nm, Molecular Probes) or AO (5 mg/mL, Sigma-Aldrich) for lysosomal integrity assays. Flow cytometry was done on a FACScan cytometry (FACSCalibur, Becton Dickinson, USA). Data were analyzed using CELLQUEST software (Verity Software House, Topsham, Maine, USA). 4.1.52. DAPI staining We put 10-mm round glass cover slips in 24-well plates. A549 cells were seeded on slips at a density of 5  104/mL. After incubation for 24 h, cells were treated with compounds 11b and 18a for 8 h. After the cells were washed with the cold PBS, cold-methyl alcohol/acetone (1:1) was used to fix cells. Then washed by PBS and stained with DAPI (4 mg/mL) for 10 min at room temperature. Cover slips containing the cells were then washed with PBS-TX (10 mL PBSþ10 mL 10% Tritonx-100) three times and mounted using mounting medium (PBS:Glycerol ¼ 1:1 [v/v]) and analyzed by fluorescence microscopy. 4.1.53. Apoptosis detection Surface exposure of phosphatidylserine in apoptotic cells was quantitatively detected using Annexin V/FITC and PI apoptosis detection kit (Becton Dickinson, USA). Briefly, cells (5  105 per well) were seeded into 6-well plates and then treated with varying concentrations of compound 11b and 18a, respectively. After 8 h, the cells were harvested and washed twice with ice-cold PBS (0.01 M, pH7.2). After 5 min of centrifuging at 200 g, Annexin V/FITC and PI double-staining were performed according to manufacturer's instruction. Cell apoptosis was analyzed on a FACScan flow cytometry (Becton Dickinson, USA). Annexin V-positive, PInegative cells were scored as apoptotic. Double-stained cells were considered either as necrotic or as late apoptotic. Acknowledgment This work was supported by grant from the National Natural

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