StructureâActivity Relationship Study of Permethyl Ningalin B

Article pubs.acs.org/jmc

Structure−Activity Relationship Study of Permethyl Ningalin B Analogues as P‑Glycoprotein Chemosensitizers Jin Wen Bin,†,∥ Iris L. K. Wong,‡,§,∥ Xuesen Hu,‡,§ Zhang Xiao Yu,† Li Fu Xing,† Tao Jiang,† Larry M. C. Chow,*,‡,§ and Wan Sheng Biao*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003 Shandong, China ‡ Department of Applied Biology and Chemical Technology and the State Key Laboratory for Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Special Administrative Region, China § State Key Laboratory in Chinese Medicine and Molecular Pharmacology, Shenzhen, 518057 Guangdong, China S Supporting Information *

ABSTRACT: A novel series of permethyl ningalin B analogues were synthesized and evaluated for their Pglycoprotein (P-gp)-modulating activities in a P-gp-overexpressing breast cancer cell line (LCC6MDR). Compounds 35 and 37, which possess one methoxy group and one benzyloxy group at aryl ring C, displayed the most potent Pgp-modulating activity. A 1 μM concentration of 35 and 37 resensitized LCC6MDR cells toward paclitaxel by 42.7-fold, with respective EC50 values of 93.5 and 110.0 nM. Their mechanism of P-gp modulation is associated with an increase in intracellular drug accumulation. Their advantages also include low cytotoxicity (IC50 for L929 fibroblast >100 μM) and high therapeutic indexes (>909 after normalization with their EC50 values). 35 is not a substrate of P-gp. They are potentially dual-selective modulators for both P-gp and breast cancer resistance protein transporters. The present study demonstrates that these new compounds can be employed as effective and safe modulators of Pgp-mediated drug resistance in cancer cells.

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curcumin,25 kaempferol,26 quercetin,27 quercetin pentamethyl ether,28 myricetin,29 epigallocatechin gallate,30,31 lamallarine K, lamallarine I, lamallarine O, permethyl lamallarine D, permethyl ningalin D, and permethyl ningalin B have been discovered as nontoxic MDR modulators.32 Further structural modification on flavonoids33,34 and marine natural phenolic products ningalins35−38 has been demonstrated to significantly enhance their P-gp-modulating activity. Our previous structural modification of permethyl ningalin B35 discovered that the N-substituted 3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione compounds which mimic permethyl ningalin B are safe and potent P-gp inhibitors (compounds 1−8 in Figure 1). These results suggest that partial or full methylation of phenol rings might significantly improve P-gp-modulating activity. To further investigate the structure−activity relationship of permethyl ningalin B analogues, we have herein synthesized a series of novel Nsubstituted 3,4-diaryl-1H-pyrrole-2,5-dione compounds by introducing two or three methoxy groups at the A or B aryl ring (Figure 1) and various substituents at the N atom. Here we report a new series of permethyl ningalin B analogues that are effective and safe in modulating P-gp.

INTRODUCTION Multidrug resistance (MDR) severely hampers the efficacy of cancer chemotherapy. In addition to established anticancer drugs such as anthracyclines, vinca alkaloids, or topoisomerase inhibitors,1−4 MDR could also occur against other novel cytostatic agents such as monoclonal antibodies, tyrosine receptor kinase inhibitors, or proteasome inhibitors.5−7 One of the major causes of MDR is the overexpression of the drug efflux pump, the ATP-binding cassette (ABC) superfamily membrane proteins, which transport anticancer drugs out of the cells and result in drug resistance.7,8 Three ABC transporters are associated with MDR, namely, P-glycoprotein (P-gp, ABCB1), multidrug-resistance-related protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2).9 P-gp is probably the best characterized ABC transporter. It pumps a variety of hydrophobic anticancer drugs out of the cells, resulting in a lowered intracellular drug accumulation.10−15 Numerous P-gp inhibitors have been developed with the goal of reversing cancer MDR, and they can be broadly divided into three generations.7,8,16,17 However, only a few nontoxic and specific P-gp inhibitors have been found,18 and none of these inhibitors can be used clinically.19 The availability of the crystal structure of P-gp homologues20,21 facilitates the discovery of safer P-gp inhibitors.22−24 In the past several years, many natural phenolic compounds and their methyl ethers such as © 2013 American Chemical Society

Received: June 20, 2013 Published: October 30, 2013 9057

dx.doi.org/10.1021/jm400930e | J. Med. Chem. 2013, 56, 9057−9070

Journal of Medicinal Chemistry

Article

Figure 1. P-gp inhibitors of permethyl ningalin B analogues.

Scheme 1. Synthetic Routes of Compounds 14a, 14b, and 14ca

a Reagents and conditions: (a) (CH2)6N4, CHCl3, rt, 2 h; (b) HBr, CH3OH, rt, 2 d; (c) EDCI, DMAP, dry CH2Cl2, 3,4,5-trimethoxybenzoic acid or 3,4-dimethoxybenzoic acid, rt, overnight; (d) NaH, (TBS)Cl, DMF, 0 °C to rt, N2, 2 h; (e) t-BuOK, t-BuOH, N2 to O2, 4 h.

Scheme 2. Synthetic Routes of Compounds 15−24a

a Reagents and conditions: (a) K2CO3, DMF, 3,4,5-triethoxybenzyl methanesulfonate, 3,4,5-triisopropoxybenzyl methanesulfonate, 3,4,5trimethoxybenzyl methanesulfonate, 3,4-dimethoxybenzyl methanesulfonate, or 4-methoxybenzyl methanesulfonate, rt, N2, overnight; (b) K2CO3, DMF, 3,4,5-trimethoxyphenethyl methanesulfonate, 3,4-dimethoxyphenethyl methanesulfonate, or 4-methoxyphenethyl methanesulfonate, rt, N2, overnight; (c) Ph3P, DIAD, dry THF, 2-(3,4,5-tris(allyloxy)phenyl)methanol, 0 °C to rt, N2, overnight.

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Scheme 3. Synthetic Routes of Compounds 28−40a

a Reagents and conditions: (a) K2CO3, DMF, 60 °C, N2, 12 h; (b) K2CO3, DMF, ethyl 2-bromoacetate or tert-butyl 2-bromoacetate, 60 °C, N2, 12 h; (c) TFA, CH2Cl2, rt, 2 d.

Scheme 4. Synthetic Routes of Compounds 42 and 44a

a

Reagents and conditions: (a) K2CO3, DMF, 60 °C, N2, 12 h.

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RESULTS AND DISCUSSION Chemistry. Three 3,4-diaryl-1H-pyrrole-2,5-dione intermediates, 14a, 14b, and 14c, were designed to investigate the effect of methoxy substitution at the two aryl rings (A and B rings shown in Scheme 1). The synthesis of 3,4-bis(3,4dimethoxyphenyl)-1H-pyrrole-2,5-dione (14a) has been reported previously.35 Using a similar strategy, 14b and 14c were prepared as shown in Scheme 1. Ammoniation of 2-bromo-1(3,4-dimethoxyphenyl)ethanone (9a) or 2-bromo-1-(3,4,5trimethoxyphenyl)ethanone (9b) with hexamethylenetetramine, followed by addition of concentrated hydrobromic acid in absolute methanol, provided 2-amino-1-(3,4dimethoxyphenyl)ethanone hydrobromide (11a) or 2-amino1-(3,4,5-trimethoxyphenyl)ethanone hydrobromide (11b), respectively. Coupling of 11a or 11b with 2-(3,4,5trimethoxyphenyl)acetic acid, catalyzed by EDCI and HOBt, resulted in related compound 12b or 12c. Subsequent cyclization of 12b or 12c, catalyzed by t-BuOK in t-BuOH under nitrogen protection, afforded 14b or 14c with yields of 31.2% and 24.7%, respectively. To improve the cyclization

yield, compound 12b or 12c was reacted with tertbutylchlorodimethylsilane39 to produce intermediate 13b or 13c, which was then cyclized to give 14b or 14c. The yields of 14b and 14c were increased to 44.5% and 36.4% via these two steps. However, purification of intermediates 13b and 13c is difficult because of their instability. In Scheme 2, we synthesized compounds 15−24 on the basis of our previously published P-gp modulators 1−5. 35 Intermediate 14a was coupled with 3,4,5-triethoxybenzyl methanesulfonate or 3,4,5-triisopropoxybenzyl methanesulfonate in the presence of K2CO3 in DMF and afforded compound 15 or 16, respectively. Similarly, coupling of 14b with 4methoxybenzyl methanesulfonate, 3,4-dimethoxybenzyl methanesulfonate, or 3,4,5-trimethoxybenzyl methanesulfonate gave related compound 17, 18, or 19. In the same reaction conditions, 14b was reacted with 4-methoxyphenethyl methanesulfonate, 3,4-dimethoxyphenethyl methanesulfonate, or 3,4,5-trimethoxyphenethyl methanesulfonate to produce compounds 20−21. The Mitsunobu method35 was adopted in the synthesis of target compounds 23 and 24, which were obtained 9059



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Table 1. P-gp-Modulating Activity of Permethyl Ningalin B Analoguesa no. of methoxy groups on aryl ring A

no. of methoxy groups on aryl ring B

1 2 3 17 18 19 6 7 8 20 21 22 15 16 23 24 39a

di di di di di di di di di di di di di di di di di

di di di tri tri tri di di di tri tri tri di di di tri di

39b

di

di

40 42 44 4 5 28 29 30 31 32 33 34 35

di di di di di di di di di tri tri tri di

di di di di di di tri tri tri tri tri tri di

36 37

di di

di tri

38 verapamil LCC6MDRc LCC6c

di

tri

series I

II

III

compd

no. of functional groups on aryl ring C monomethoxy dimethoxy trimethoxy monomethoxy dimethoxy trimethoxy monomethoxy dimethoxy trimethoxy monomethoxy dimethoxy trimethoxy triethoxy triisopropoxy tris(allyloxy) tris(allyoxy)

monomethoxy dimethoxy trimethoxy monomethoxy dimethoxy trimethoxy monomethoxy dimethoxy trimethoxy monomethoxy and mono(benzyloxy) mono(benzyloxy) monomethoxy and mono(benzyloxy) mono(benzyloxy)

type of linker methylene methylene methylene methylene methylene methylene bismethylene bismethylene bismethylene bismethylene bismethylene bismethylene methylene methylene methylene methylene (ethoxycarbonyl) methylene ((tert-butyloxy)carbonyl) methylene carboxylmethylene heterocyclic methylene heterocyclic methylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene carbonylmethylene

IC50 of paclitaxel (nM)

8.3 ± 0.5 11 ± 2.3 20 ± 3.7

12 32 103 78 18 40 22 21

± ± ± ± ± ± ± ±

1.5 6.6 2.7 2.9 2.9 8.8 2.4 6.5

RF 9.3b 12.4b 18.2b 18.0 13.2 7.4 10.7b 6.9b 4.5b 12.7 4.6 1.5 1.9 8.5 3.8 6.8 7.0

45 ± 3.7

3.3

159 ± 7.8 88 ± 17 123 ± 3.8

0.9 1.7 1.2 9.9b 8.2b 9.3 7.2 5.1 3.8 4.9 3.3 1.8 42.7

16 21 29 39 31 45 83 3.5

± ± ± ± ± ± ± ±

0.4 1.2 0.2 1.1 0.6 6.4 1.8 0.3

carbonylmethylene carbonylmethylene

14 ± 0.6 3.5 ± 0.3

10.4 42.7

carbonylmethylene

9.1 33 149 2.6

± ± ± ±

16.8 4.5 1.0 57.4

1 3.5 11 0.5

a

The relative fold (RF) represents the fold change in drug sensitivity. RF = (IC50 without modulator)/(IC50 with modulator). The IC50 value from LCC6MDR containing no modulators was used for normalization (RF = 1.0). A known P-glycoprotein modulator, verapamil, is included for comparison. N = 2−3 independent experiments, and values are presented as the mean ± standard error of the mean. bThese RF values have been published.35 cNo modulator was used in LCC6MDR and LCC6cells.

by coupling of intermediate 14a or 14b with 2-(3,4dimethoxyphenyl)ethanol in the presence of PPh3 and DIAD in anhydrous THF. In Scheme 3, we synthesized compounds 28−40 on the basis of the most potent P-gp modulators we reported previously (compounds 6−8 shown in Figure 1).35 Intermediates 14a, 14b, and 14c reacted with diverse substituted 2-bromo-1phenylethanones 9a, 9b, and 25−27 in the presence of K2CO3 in DMF to produce compounds 28−38, respectively. The target compounds 39a and 39b were obtained through coupling of 14a with ethyl 2-bromoacetate and tert-butyl 2bromoacetate, respectively, in the same conditions. Compound 40 was only produced by hydrolysis of 39b by trifluoroacetic acid, but not from 39a by base.

In Scheme 4, compounds 42 and 44 containing the heterocycle residues were obtained through reaction of 14a with 41 or 43 in the presence of K2CO3 in DMF. Biological Evaluation. P-gp-Modulating Activity of Permethyl Ningalin B Analogues. The P-gp-transfected breast cancer cell line MDA435/LCC6MDR and its parent MDA435/ LCC6 were employed in this study. We found that LCC6MDR cells were about 57.4-fold more resistant to paclitaxel than their parental LCC6 cells (Table 1). MDR reversal activity of permethyl ningalin B analogues was compared by measuring the relative fold (RF), defined as the ratio of IC50 without modulator to IC50 with modulator. A relatively low concentration of permethyl ningalin B analogues (1 μM) was used because of their high potency. Verapamil, a 9060



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Figure 2. Chemical structures of series I compounds.

Figure 3. Chemical structures of series II compounds.

known P-gp modulator, displayed a moderate activity with an RF of 4.5 (Table 1). Structure−Activity Relationship Study of Permethyl Ningalin B Analogues. A total of 34 analogues were divided into 3 series for investigating their SARs. In the present study, these new permethyl ningalin B analogues were further designed by (1) increasing the number of methoxy substituents at ring B, (2) changing the polarity and length of the linking chain, and (3) substituting ring C with different functional groups. Their structures are shown in Figures 2−4. Compounds 1−8 shown in Table 1 have been reported previously.35 In series I (Figure 2), compounds 17−22 possess an additional methoxy group at ring B as compared to the previous generation of analogues 1−3 and 6−8. Compounds 17 (RF = 18.0) and 20 (RF = 12.7) (Table 1) with a monomethoxy group at aryl ring C exhibited a better reversal activity on MDR cancer cells when compared to compounds 1 (RF = 9.3) and 6 (RF = 10.7). However, no improved or diminished P-gpmodulating activity was observed among the di- or trimethoxysubstituted (at ring C) compounds 18 (RF = 13.2), 19 (RF = 7.4), 21 (RF = 4.6), and 22 (RF = 1.5) as compared to their respective parent compounds 2 (RF = 12.4), 3 (RF = 18.2), 7 (RF = 6.9), and 8 (RF = 4.5). This result suggests that the number of methoxy substituents at rings B and C plays an important factor in determining P-gp-modulating activity.

Among these new synthetic compounds in series I, 17 (RF = 18.0) displayed the most potent P-gp-modulating effect, which was about 4-fold stronger than that of verapamil (RF = 4.5). It is possible that monomethoxy substitution at the C ring could decrease the overall bulkiness of permethyl ningalin B analogues, which already possess di- or trimethoxy substituents at rings A and B and therefore increase the binding affinity of permethyl ningalin B analogues toward P-gp. To investigate the effect of different substituents at the N atom of 3,4-diarylpyrrole-2,5-dione, compounds 15, 16, 23, 24, 39a, 39b, 40, 42, and 44 (shown in Figure 3) with various Nsubstituted moieties in series II were prepared and studied. When the three methoxy groups of compound 3 (RF = 18.2) at ring C were replaced by ethoxy, isopropoxy, or allyloxy groups, respectively, the resulting compounds 15 (RF = 1.9), 16 (RF = 8.5), and 23 (RF = 3.8) displayed poorer P-gp-modulating activity. This result suggests that ethoxy, isopropoxy, or allyloxy substitutions at ring C are undesirable. Compound 24 (RF = 6.8) with an additional methoxy group at the B ring was more potent than compound 23 (RF = 3.8). When the N atom of 3,4-diarylpyrrole-2,5-dione was substituted by (ethoxycarbonyl)methylene or ((tert-butyloxy)carbonyl)methylene, the resulting compounds 39a and 39b showed only moderate P-gp-modulating activity with RF values of 7.0 and 3.3, respectively. The more polar compound 40 with a carboxylic group at the linker had no P-gp-modulating activity. 9061



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Figure 4. Chemical structures of series III compounds.

Figure 5. Alignments of compound 35 with 36 and compound 37 with 38.

Compounds 42 and 44, which possess heterocylic moieties at the linker, exhibited very weak or no P-gp-modulating activity. This suggests that polar or bulky linkers are not favored. Compounds 4, 5, and 28−38 in series III (Figure 4) contain a carbonylmethylene group linking ring C. Compounds 4, 5, and 28 contain dimethoxy substituents at the A and B rings, but have different numbers of methoxy substitutions at the C ring. The latter did not have a significant effect on the P-gpmodulating activity as compounds 4, 5 and 28, containing one, two, and three methoxy substitutions, have similar RF values of 8.2−9.9. In contrast, the RF values of the methylene-linked (RF(1−3) = 9.3−18.2) or bismethylene-linked (RF(6−8) = 10.7−4.5) permethyl ningalin B analogues showed positive and negative relationships with the number of methoxy substituents at aryl ring C, respectively. The P-gp-modulating activity of analogues containing two methoxy groups at the A ring and three methoxy groups at the B ring is dependent on the length of the linking chain. The analogues with the shortest linker, methylene, 17 (RF = 18.0, with one methoxy group at the C ring), 18 (RF = 13.2, with two methoxy groups at the C ring), and 19 (RF = 7.4, with three methoxy groups at the C ring), displayed stronger P-gp-modulating activity as compared to the compounds with longer linkers such as bismethylene (RF(20−

22) = 12.7−1.5) and carbonylmethylene (RF(29−31) = 7.2− 3.8). Compounds 4, 5, and 28 (RF = 9.9−9.3) with two methoxy groups at the A and B rings showed higher P-gp-modulating activity than compounds 29−31 (RF = 7.2−3.8) with two methoxy groups at the A ring and three methoxy groups at the B ring. Compounds 32−34 (RF = 4.9−1.8) with three methoxy substituents at both rings A and B showed the weakest P-gpmodulating activity. In general, compounds 29−34 with trimethoxy groups at the B ring displayed the tendency that increasing the methoxy substitution at the C ring decreases their P-gp-modulating activity. Overall, the linking chain length and the number of methoxy substituents at the B and C rings are critical factors in controlling the P-gp-modulating potency of permethyl ningalin B analogues. Interestingly, compounds 35 and 37 with a benzyloxy group and a methoxy group at the C ring showed the most potent Pgp-modulating activity with a high RF value of 42.7. They are about 5.2- to 8.4-fold stronger than the relative compounds 5 (RF = 8.2) and 30 (RF = 5.1). Compound 38 (RF = 16.8) with a monobenzyloxy group consistently showed higher P-gpmodulating activity than compound 29 (RF = 7.2). However, compound 36 (RF = 10.4) had an RF value similar to that of the respective compound 4 (RF = 9.9). This result suggests that 9062



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addition of a benzyloxy group at the C ring significantly improves the P-gp-modulating activity of permethyl ningalin B analogues. After energy optimization and alignment, it was shown that compounds 35 and 36 have similar confirmations; the same is true for compounds 37 and 38 (Figure 5). This result suggests that the methoxy groups at the C ring of compounds 35 and 37 are important pharmacophore groups and significantly improve the P-gp-modulating activity of permethyl ningalin B analogues. P-gp substrates are usually amphipathic and lipid soluble.8 log P and the polar surface area (PSA) may affect the P-gpmodulating activity.40 We did not find significant correlation between cLogP and RF values for our compounds (Table 2 and Table 2. Calculated cLogP and PSA of Permethyl Ningalin B Analogues compd

cLogP

PSA (Å2)

compd

cLogP

PSA (Å2)

35 37 3 17 38 18 20 2 6 36 4 1 28 16 5 19 29 39a

4.62 4.26 2.25 2.51 4.59 2.25 3.14 2.61 3.50 4.94 3.18 2.87 2.46 4.76 2.85 1.89 2.82 2.20

118.8 110.9 98.9 109.0 109.6 110.0 90.8 90.1 77.1 111.6 116.9 91.4 123.7 84.2 114.2 107.7 122.8 105.8

7 24 30 32 21 8 23 31 33 39b 15 34 42 22 44 40 ningalin B pemethyl ningalin B

3.24 4.21 2.50 2.46 3.24 2.88 4.57 2.10 2.14 2.91 3.84 1.74 0.91 2.88 1.27

76.4 112.1 116.5 111.1 85.8 79.9 87.2 115.0 118.2 95.4 83.2 116.6 192.4 85.1 157.9 169.8 427.5 151.5

3.15 5.19

Figure 6. Comparison of cLogP or PSA with the RF of permethyl ningalin B analogues.

Figure 6). It has been reported that 52% of P-gp substrates have a topological polar surface area (tPSA) over 90 Å2.41 We have calculated PSA values for our compounds and found that most of them have PSA values over 90 Å2. However, we did not observe any correlations between the PSA and RF values (Table 2 and Figure 6). The PSA values of the most potent Pgp inhibitors 35 and 37 are 118.7 and 110.9 Å2, respectively (Table 2). Together, our results suggest that there is no correlation between cLogP or PSA and P-gp-modulating activities in our permethyl ningalin B analogues. EC50 of Permethyl Ningalin B Analogues for Reversing Paclitaxel Drug Resistance in LCC6MDR and Their Effect on DOX Accumulation. Among the 26 compounds, 35 and 37 exhibited the strongest P-gp-modulating activity (RF = 42.7 at 1.0 μM) and did not show any significant cytotoxicity to normal fibroblasts (IC50 of L929 >100 μM) (Table 3). The EC50 values for 35 and 37 in reversing paclitaxel resistance were 93.5 and 110.0 nM, respectively (Table 3). They were about 4.1−4.8-fold more potent than that of verapamil (EC50 = 445.7 nM) and about 2.9−3.4-fold less potent than that of cyclosporine A (EC50 = 32.0 nM). In terms of therapeutic index, compounds 35 (>1069.5) and 37 (>909.1) were as safe as cyclosporine A (1059.4) and at least 4.5-fold safer than verapamil (>200.1). We investigated whether the P-gp -modulating activity of 35 and 37 is associated with a concomitant increase in drug

accumulation. DOX accumulation in parental LCC6 cells was 2.4-fold (P < 0.0001) higher than that of LCC6MDR cells (Figure 7). Treatment of parental LCC6 cells with 1 μM 35, 37, or verapamil did not increase the intracellular DOX accumulation. On the contrary, treatment of P-gp-overexpressing LCC6MDR cells with 1 μM 35, 37, or verapamil resulted in a 2.2-fold (P < 0.005), 2.3-fold (P < 0.005), or 1.8-fold increase in intracellular DOX accumulation (Figure 7). A 1 μM concentration of 35, 37, and verapamil also reversed DOX resistance with RF values of 5.6, 5.6, and 12.7, respectively. These results suggested that modulation of DOX resistance by 35 and 37 is due to the inhibition of the drug transport activity of P-gp. We have determined whether compound 35 is a P-gp substrate. We have measured the intracellular accumulation of compound 35 in both LCC6 and LCC6MDR cells and found that they accumulated similar levels of compound 35 (Figure 8). This result suggested that 35 is not a substrate of P-gp and might directly inhibit the transporter activity of P-gp without competition with DOX. MRP1- and BCRP-Modulating Activity of Compounds 35 and 37. In addition to P-gp, MRP1 and BCRP are the other two ABC transporters that can also confer MDR in cancer. We have investigated the selectivity of 35 and 37 toward MRP1 and BCRP. The MRP1-transfected human ovarian carcinoma cells 9063



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Table 3. EC50 (nM) for Reversing the Paclitaxel Drug Resistance and Therapeutic Index in LCC6MDRa cytotoxicity (IC50, μM) compd

LCC6

LCC6MDR

L929

35 37 verapamil cyclosporine A

>100 >100 63.8 ± 0.1 2.8 ± 0.6

>100 >100 63.9 ± 1.7 8.3 ± 1.5

>100 >100 89.2 ± 8.2 33.9 ± 5.2

EC50 (nM) in LCC6MDR 93.5 110.0 445.7 32.0

± ± ± ±

therapeutic index

8.3 5.3 40.7 1.0

>1069.5 >909.1 200.1 1059.4

EC50 values are presented as the mean ± standard error of the mean. N = 2−5 independent experiments. The therapeutic index, a ratio of cytotoxicity to EC50, acts as a parameter for drug safety.

a

(2008/MRP1) were 9.1-fold more resistant to DOX than their parental 2008/P cells (Table 4). At 1 μM, neither compound 35 nor compound 37 has any significant MRP1-modulating activity in 2008/MRP1 (Table 4, RF = 1.1 and 1.2, respectively). The positive control, a flavonoid dimer 4e,42 reversed MRP1-mediated DOX resistance with an RF value of 14.0 at 1 μM (Table 4). The BCRP-transfected human embryonic kidney cell line (HEK293/R2) was about 30.3-fold more resistant to topotecan than its parental HEK293/pcDNA3.1 cells. At 1 μM, compounds 35 and 37 have moderate BCRP-modulating activity in HEK293/R2 cells with RF = 6.9 and 8.2, respectively. The positive control, Ko143, has an RF of 20.0 (Table 4). These data highly suggest that compounds 35 and 37 can modulate both P-gp and BCRP.

Figure 7. Effect of 35 and 37 on DOX accumulation in LCC6MDR. LCC6 or LCC6MDR cells were incubated with 20 μM DOX for 150 min at 37 °C with or without 1 μM 35, 37, or verapamil. DMSO (0.1%) was used as a negative control. After incubation, the cells were lysed, and the supernatant was used to measure the DOX level by spectrofluorometry. N = 3−5 independent experiments. The results are presented as the mean ± standard error of the mean. P values were calculated using Student’s t test.

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CONCLUSION In this study, we synthesized and evaluated a series of novel Nsubstituted 3,4-diaryl-1H-pyrrole-2,5-dione compounds by introducing two or three methoxy groups at the A or B aryl ring and various substituents at the N atom. These synthetic compounds exhibited promising P-gp-modulating activity in Pgp-overexpressing breast cancer cell lines (LCC6MDR) without causing any cytotoxicity toward normal cells (IC50 > 100 μM). The SAR study suggests that increasing methoxy groups at the B ring only showed low to moderate P-gpmodulating activity. Importantly, an addition of a benzyloxy group at the aryl C ring significantly enhances the P-gpmodulating activity. A 1 μM concentration of compounds 35 and 37, which possess one methoxy group and one benzyloxy group, at the aryl ring C, displayed the most potent P-gpmodulating activity and resensitized LCC6MDR cells toward paclitaxel by 42.7-fold with EC50 values of 93.5 and 110.0 nM, respectively. Their mechanism of P-gp modulation is associated with an increase in intracellular drug accumulation. Compound 35 is not a substrate of the P-gp transporter. Their significant advantages include their noncytotoxicity (IC50 for L929 >100

Figure 8. Intracellular accumulation of 35 in LCC6 and LCC6MDR. LCC6 and LCC6MDR cells were incubated with either 20 or 200 μM 35. Intracellular accumulation of 35 was measured by HPLC. N = 2 independent experiments. The data are represented as the mean ± standard error of the mean.

Table 4. MRP1- and BCRP-Modulating Activity of Compounds 35 and 37a MRP1-modulating activity cell line + treatment 2008/MRP1 2008/P 2008/MRP1 + 35 2008/MRP1 + 37 2008/MRP1 + 4e

BCRP-modulating activity

IC50 of DOX (nM)

RF

cell line + treatment

± ± ± ± ±

1.0 9.1 1.1 1.2 14.0

HEK293/R2 HEK293/pcDNA3.1 HEK293/R2 + 35 HEK293/R2 + 37 HEK293/R2 + Ko143

574.7 63.0 542.8 485.1 41.1

9.6 5.0 105.8 78.8 12.6

IC50 of topotecan (nM)

RF

± ± ± ± ±

1.0 30.3 6.9 8.2 20.0

479.3 15.8 69.9 58.7 24

21.5 1.5 2.0 1.0 1.9

The MRP1- and BCRP-modulating activity of 35 and 37 at 1 μM was investigated using 2008/MRP1 and HEK293/R2 cells which overexpress MRP1 and BCRP, respectively. Their IC50 values toward DOX and topotecan were determined in the presence of compound 35 or 37 or positive controls (flavonoid dimer 4e and Ko143) and normalized to that in their absence (RF = 1.0). The 2008/P and HEK293/pcDNA3.1 cells are untransfected controls. N = 2−4 independent experiments, and the values are presented as the mean ± standard error of the mean. a

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μM) and their therapeutic indexes (>909 after normalization with their EC50 values). They are potentially dual-selective modulators for both P-gp and BCRP transporters. The present study demonstrates that these synthetic analogues of permethyl ningalin B can be employed as effective and safe modulators of P-gp-mediated drug resistance in cancer cells.

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the preparation of compound 12b, but using compound 11b as the starting material, the desired compound 12c was obtained: yield 46.4%; mp 131−133 °C; ESI-MS m/z [M + H]+ 434.1; 1H NMR (CDCl3, 500 MHz) δ 7.04 (s, 2 H), 6.79 (s, 1 H), 6.44 (s, 2 H), 4.58 (d, J = 4.3 Hz, 2H), 3.73 (m, 18 H), 3.48 (s, 2 H); 13C NMR (CDCl3, 125 MHz) δ 190.6, 170.8, 153.2, 152.8, 142.8, 136.6, 130.1, 129.1, 105.9, 104.8, 60.5, 60.3, 55.8, 55.6, 45.9, 43.2. 3-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1Hpyrrole-2,5-dione (14b). To a mixture solution of compound 12b (1.0 g, 2.48 mmol) in anhydrous DMF (20 mL) was slowly added NaH (0.30 g, 12.25 mmol) at 0 °C with stirring, and then to the resulting solution was added (TBS)Cl (2.00 g, 12.25 mmol). After addition of (TBS)Cl, the solution was warmed to room temperature and reacted until TLC showed the reaction was complete. The solution was diluted with CH2Cl2 (50 mL), washed with brine, and dried over anhydrous MgSO4. The volatiles were then removed in vacuo, and the crude compound 13b was used without further purification. Under a N2 atmosphere, t-BuOK (1.00 g, 8.91 mmol) was added to a stirring solution of compound 13b in t-BuOH (20 mL) at 0 °C. Then the reaction was allowed to slowly warm to room temperature. After 2−3 h, the reaction solution was exposed to air. After 2 h, the resulting reaction solution was poured into ice-cold water (100 mL), and then the pH was adjusted to 6−7 by adding 2 N hydrochloric acid to give a thick suspension. The above suspension was filtered and purified by flash chromatography on silica gel to afford compound 14b: yield 440 mg, 44.5%; mp 164−166 °C; 1H NMR (CDCl3, 600 MHz) δ 8.18 (s, 1 H), 7.24 (dd, J = 1.7, 8.2 Hz, 1 H), 7.02 (s, J = 1.7 Hz, 1 H), 6.85 (d, J = 8.2 Hz, 1 H), 6.74 (s, 2 H), 3.88 (s, 3 H), 3.86 (s, 3 H), 3.71 (s, 6 H), 3.89 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 174.9, 153.6, 150.2, 150.1, 148.7, 137.9, 131.0, 127.9, 125.5, 120.3, 111.0, 106.7, 60.8, 56.2, 55.9, 48.1; HRMS m/z calcd for (C21H22O7N + H)+ 400.1391, found 400.1389. 3,4-Bis(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (14c). Following the procedure for the preparation of compound 14b, but using compound 12b as the starting material, the desired compound 14c was obtained: yield 36.4%; mp 155−158 °C; 1H NMR (CDCl3, 600 MHz) δ 7.94 (s, 1 H), 6.77 (s, 4 H), 3.86 (s, 6 H), 3.71 (s, 12 H); 13 C NMR (CDCl3, 150 MHz) δ 170.6, 153.3, 139.7, 136.1, 123.7, 107.4, 107.3, 61.1, 61.0, 56.2, 56.1; HRMS m/z calcd for (C22H24O8N + H)+ 430.1496, found 430.1505. 1-(3,4,5-Triethoxybenzyl)-3,4-bis(3,4-dimethoxyphenyl)-1Hpyrrole-2,5-dione (15). A mixture of compound 14a (150 mg, 0.41 mmol), 3,4,5-trimethoxybenzyl methanesulfonate (165 mg, 0.52 mmol), and K2CO3 (250 mg, 1.76 mmol) in anhydrous DMF (10 mL) was stirred at room temperature under a N2 atmosphere overnight. The resulting solution was poured into water (40 mL), extracted with EtOAc, washed with brine, and dried over anhydrous MgSO4, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel (EtOAc/PE = 1/1, v/v) to afford the desired compound 15: yield 92 mg, 35.8%; mp 131−133 °C; 1H NMR (CDCl3, 600 MHz) δ 7.20 (dd, J = 8.2, 2.2 Hz, 2 H), 7.02 (d, J = 2.2 Hz, 2 H), 6.85 (d, J = 8.2 Hz, 2 H), 6.68 (s, 2 H), 4.66 (s, 2 H), 4.08−4.04 (q, J = 7.1 Hz, 4 H), 4.03−4.00 (q, J = 7.1 Hz 2 H), 3.89 (s, 6 H), 3.86 (s, 6 H), 1.41 (t, J = 7.1 Hz, 6 H), 1.34 (t, J = 7.1 Hz, 3 H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 153.0, 150.5, 148.8, 134.3, 131.8, 123.6, 121.5, 112.7, 111.0, 107.8, 68.9, 64.8, 56.0, 55.9, 42.3, 15.7, 15.0; HRMS m/z calcd for (C33H38O9N + H)+ 592.2541, found 592.2538. 1-(3,4,5-Triisopropoxybenzyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (16). Using 3,4,5-triisopropoxybenzyl methanesulfonate as the starting material, the title compound 16 was synthesized according to the approach for the preparation of compound 15: yield 84.6%; mp 126−128 °C; 1H NMR (CDCl3, 600 MHz) δ 7.19 (dd, J = 8.2, 2.2 Hz, 2 H), 7.02 (d, J = 2.2 Hz, 2 H), 6.85 (d, J = 8.2 Hz, 2 H), 6.66 (s, 2 H), 4.65 (s, 2 H), 4.56−4.50 (m, 2 H), 4.03−4.00 (m, 2 H), 3.89 (s, 6 H), 3.69 (s, 6 H), 1.31 (d, J = 6.6 Hz, 12 H), 1.27 (d, J = 6.6 Hz, 6 H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 152.3, 150.4, 148.7, 134.3, 131.8, 123.6, 121.6, 112.7, 111.1, 75.7, 71.3, 56.0, 22.9, 22.3; HRMS m/z calcd for (C36H44O9N + H)+ 634.3011, found 634.3013.

EXPERIMENTAL SECTION

General Information. All moisture-sensitive reactions were conducted under a nitrogen atmosphere in anhydrous, freshly distilled solvents. Starting materials and reagents, unless otherwise stated, were of commercial grade and were used without further purification. Solvents were dried according to standard procedures. 9-(2Bromoethyl)-9H-purin-6-amine (41),43 9-(2-bromoethyl)-1,3-dimethyl-1H-purine-2,6(3H,9H)-dione (43),44 3,4-bis(3,4-trimethoxyphenyl)-1H-pyrrole-2,5-dione (14a),35 2-bromo-1-(3,4-dimethoxyphenyl)ethanone (9a), 45 2-bromo-1-(3,4,5-trimethoxyphenyl)ethanone (9b),45 and 2-bromo-1-(4-methoxyphenyl)ethanone (25)45 were synthesized as described previously. TLC chromatography was performed on aluminum sheets (silica gel 60-F254, E. Merck). 1H (600 MHz) and 13C (150 MHz) NMR experiments were determined on an instrument with CDCl3 as the solvent. Chemical shifts are given in parts per million (δ) with TMS as the internal standard and coupling constants (J) in hertz. High-resolution (ESI) MS spectra were performed with a QTOF-2 Micromass spectrometer. In addition to NMR and high-resolution (ESI) MS, HPLC analysis was used to determine the purity (>95%) of the compounds. The compounds were dissolved in methanol (1.5 mL). A reversed-phase Diamonsil C18 (2) (4.6 × 150 mm) column attached to a Gilson 322 pump coupled to a Gilson UV−vis-152 detector was used. Each sample was injected at a volume of 20 μL and eluted with methanol, and the flow rate was 1 mL/min. Melting points were determined with a micro melting point apparatus, MP-500D, and are uncorrected. 2-Amino-1-(3,4,5-trimethoxyphenyl)ethanone Hydrobromide (11b). To a 250 mL round-bottomed flask was added a solution of compound 9b (4.70 g, 16.5 mmol) in chloroform (70 mL) at room temperature, followed by addition of hexamethylenetetramine (2.78 g, 19.8 mmol). After 2 h, the precipitate was filtered, washed with chloroform, and dried in vacuo to yield the desired compound 10b (4.66 g) as a white solid with insufficient purity to be used directly in the next step. To a mixture solution of 10b (4.66 g) in absolute methanol (80 mL) was slowly added concentrated hydrobromic acid (65 mL) at 0 °C with stirring; the reaction was allowed to slowly warm to room temperature. After 3−4 days, a thick suspension was obtained. The resulting suspension was filtered, washed with ethyl ether and acetone, and dried in vacuo to afford the desired compound 11b (2.72 g, 12.1 mmol) as a white solid: yield 73.3%; mp 233−235 °C; ESI-MS m/z [M + H]+ 226.1. N -(2-(3,4-Dimethoxyphenyl)-2-oxoethyl)-2-(3,4,5trimethoxyphenyl)acetamide (12b). A mixture solution of 2(3,4,5-trimethoxyphenyl)acetic acid (3.39 g, 15.0 mmol), EDC (2.96 g, 15.0 mmol), and HOBt (2.00 g, 15.0 mmol) in dry CH2Cl2 (30 mL) was cooled to 0 °C with stirring for 30 min. Then to the resulting solution were added 11a (2.75 g, 10.0 mmol) and DMAP (4.89 g, 40.0 mmol), and the reaction was stirred overnight while being allowed to warm to room temperature naturally. The solution was diluted with CH2Cl2 (20 mL), washed with brine, and dried over anhydrous MgSO4. The solvent was evaporated to dryness to give the crude residue. Recrystallization from EtOAc and CH2Cl2 gave the desired product 12b (2.18 g, 5.4 mmol) as a white solid: yield 54.1%; mp 145−148 °C; ESI-MS m/z [M + H]+ 404.2; 1H NMR (CDCl3, 600 MHz) δ 7.55 (dd, J = 8.3, 1.7 Hz, 1 H), 7.43 (d, J = 1.7 Hz, 1 H), 6.87 (d, J = 8.3 Hz, 1 H), 6.70 (s, 1 H), 6.52 (s, 2 H), 4.67 (d, J = 4.4 Hz, 2 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 3.85 (s, 6 H), 3.81 (s, 3 H), 3.57(s, 2 H); 13C NMR (CDCl3, 150 MHz) δ 192.5, 170.0, 154.1, 153.5, 149.2, 137.2, 130.2, 127.4, 122.6, 122.6, 111.3, 109.8, 106.3, 60.9, 56.2, 56.1, 46.1, 43.9. N-(2-Oxo-2-(3,4,5-trimethoxyphenyl)ethyl)-2-(3,4,5trimethoxyphenyl)acetamide (12c). Following the procedure for 9065



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General Procedure for the Preparation of Compounds 17− 22. Under a N2 atmosphere, to a solution of compound 14b (100 mg, 0.25 mmol) and K2CO3 (138 mg, 1.00 mmol) in dry DMF (10 mL) was added 1.2 equiv of the corresponding methanesulfonate derivate at room temperature. The solution was stirred overnight. The solution was poured into ice−water (100 mL), and the resulting mixture was extracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4. The solvent was evaporated to dryness, and the resulting residue was purified by flash chromatography to afford the title compounds 17−22. The physical and spectral data for 17−22 are listed below. Data for 3-(3,4-dimethoxyphenyl)-1-(4-methoxybenzyl)-4-(3,4,5trimethoxyphenyl)-1H-pyrrole-2,5-dione (17): yield 46.9%; mp 155− 157 °C; 1H NMR (CDCl3, 600 MHz) δ 7.40 (m, 2 H), 7.22 (m, 1 H), 7.04 (d, J = 1.6 Hz, 1 H), 6.86 (m, 3 H), 6.75 (s, 2 H), 4.72 (s, 2 H), 3.88 (s, 3 H), 3.85 (s, 3 H), 3.77 (s, 3 H), 3.70 (s, 9 H); 13C NMR (CDCl3, 150 MHz) δ 170.7, 159.3, 153.2, 150.6, 148.7, 135.2, 134.1, 130.3, 128.8, 124.3, 123.9, 121.2, 114.1, 112.7, 111.0, 107.2, 61.0, 56.2, 56.0, 55.9, 55.4, 41.4; HRMS m/z calcd for (C36H44O9N + H)+ 520.1966, found 520.1963. Data for 1-(3,4-dimethoxybenzyl)-3-(3,4-dimethoxyphenyl)-4(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (18): yield 48.2%; mp 152−154 °C; 1H NMR (CDCl3, 600 MHz) δ 7.22 (dd, J = 2.2, 8.3 Hz, 1 H), 7.03−7.00 (m, 3 H), 6.84−6.80 (m, 2 H), 6.74 (s, 2 H), 4.71 (s, 2 H), 3.88 (s, 3 H), 3.87 (s, 3 H), 3.85 (s, 3 H), 3.84 (s, 3 H), 3.69 (m, 9 H) ; 13C NMR (CDCl3, 150 MHz) δ 170.7, 153.2, 150.6, 148.9, 148.7, 148.6, 139.3, 135.2, 134.0, 129.1, 124.2, 123.8, 121.4, 121.1, 112.6, 112.2, 111.0, 110.9, 107.1, 60.9, 56.1, 55.9, 55.8, 41.8; HRMS m/z calcd for (C30H32O9N + H)+ 550.2072, found 550.2071. Data for 3-(3,4-dimethoxyphenyl)-1-(3,4,5-trimethoxybenzyl)-4(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (19): yield 49.1%; mp 154−156 °C; 1H NMR (CDCl3, 600 MHz) δ 7.23 (dd, J = 1.7, 8.2 Hz, 1 H), 7.02 (d, J = 1.7 Hz, 1 H), 6.86 (d, J = 8.2 Hz, 1 H), 6.74 (s, 2 H), 6.70 (s, 2 H), 4.69 (s, 2 H), 3.89 (s, 3 H), 3.85 (s, 9 H), 3.81 (s, 3 H), 3.70 (s, 6 H), 3.69 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 170.7, 153.3, 153.2, 150.6, 148.7, 139.4, 137.7, 135.3, 134.1, 132.2, 128.7, 124.2, 123.9, 121.1, 112.7, 110.9, 107.2, 106.2, 60.9, 56.3, 56.1, 56.0, 55.8, 42.3; HRMS m/z calcd for (C31H34O10N + H)+ 580.2177, found 580.2188. Data for 3-(3,4-dimethoxyphenyl)-1-(4-methoxyphenethyl)-4(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (20): yield 47.1%; mp 85−88 °C; 1H NMR (CDCl3, 600 MHz) δ 7.22 (dd, J = 7.7, 2.2 Hz, 1 H), 7.19 (d, J = 7.7 Hz, 2 H), 7.04 (s, 1 H), 6.86−6.84 (m, 3 H), 6.74 (s, 2 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.83 (t, J = 7.7 Hz, 2 H), 3.78 (s, 3 H), 3.72 (s, 9 H), 2.93 (t, J = 7.7 Hz, 2 H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 158.4, 153.2, 150.6, 148.9, 148.7, 139.4, 135.1, 134.0, 130.2, 129.9, 124.3, 123.8, 121.1, 114.0, 112.7, 110.9, 107.2, 61.0, 56.2, 56.1, 55.8, 55.3, 39.9, 33.9; HRMS m/z calcd for (C30H32O8N + H)+ 534.2122, found 534.2119. Data for 1-(3,4-dimethoxyphenethyl)-3-(3,4-dimethoxyphenyl)4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (21): yield 47.4%; mp 95−98 °C; 1H NMR (CDCl3, 600 MHz) δ 7.21 (dd, J = 8.8, 2.2 Hz, 1 H), 7.03 (d, J = 2.2 Hz, 1 H), 6.86 (d, J = 8.8 Hz, 1 H), 6.81 (s, 2 H), 6.76 (s, 1 H), 6.73 (s, 2 H), 3.89 (s, 3 H), 3.86−3.83 (m, 11 H), 3.71 (s, 9H), 2.94 (t, J = 7.7 Hz, 2 H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 153.2, 150.6, 148.9, 148.7, 147.8, 139.4, 135.1, 134.0, 130.6, 124.2, 123.8, 121.1, 120.9, 112.6, 112.0, 111.4, 110.9, 107.1, 61.0, 56.1, 55.9, 39.8, 34.3; HRMS m/z calcd for (C31H34O9N + H)+ 564.2228, found 564.2219. Data for 3-(3,4-dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-4(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (22): yield 45.2%; mp 172−174 °C; 1H NMR (CDCl3, 600 MHz) δ 7.20 (dd, J = 8.3, 2.2 Hz, 1 H), 7.02 (d, J = 2.2 Hz, 1 H), 6.85 (d, J = 8.3 Hz, 1 H), 6.74 (s, 2 H), 6.46 (s, 2 H), 3.89 (s, 3 H), 3.86−3.83 (m, 11 H), 3.80 (s, 3 H), 3.70 (s, 9 H), 2.94 (t, J = 7.7 Hz, 2 H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 153.2, 150.6, 148.7, 139.4, 136.6, 135.2, 134.0, 133.7, 124.2, 123.8, 121.1, 112.6, 111.0, 107.1, 105.8, 61.0, 60.9, 56.1, 56.0, 55.8, 39.5, 35.0; HRMS m/z calcd for (C32H36O10N + H)+ 594.2334, found 594.2349. 3,4-Bis(3,4-dimethoxyphenyl)-1-(3,4,5-tris(allyloxy)benzyl)1H-pyrrole-2,5-dione (23). Under a N2 atmosphere, DIAD was

added dropwise to a mixture of (3,4,5-tris(allyloxy)phenyl)methanol (150 mg, 0.41 mmol), PPh3 (323 mg, 1.23 mmol), and compound 14a (150 mg, 0.41 mmol) in dry THF with stirring at 0 °C. The suspension was heated to room temperature and stirred overnight until TLC showed the reaction has complete. The resulting reaction mixture was diluted with CH2Cl2 (50 mL) before the organic phase was washed twice with water (50 mL) and once with brine (50 mL). The organic phase was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc/P = 1/2, v/v) to yield the desired compound 23: yield 121 mg, 47.3%; mp 81−85 °C; 1 H NMR (CDCl3, 600 MHz) δ 7.20 (dd, J = 8.2, 2.2 Hz, 2 H), 7.03 (d, J = 2.2 Hz, 2 H), 6.86 (d, J = 8.2 Hz, 2 H), 6.69 (s, 2 H), 6.01−6.12 (m, 3 H), 5.42 (dd, J = 1.1, 17.0 Hz, 2 H), 5.33 (dd, J = 1.1, 17.0 Hz, 1 H), 5.25 (dd, J = 1.1, 10.4 Hz, 2 H), 5.17 (dd, J = 1.1, 10.4 Hz, 1 H), 4.66 (s, 2 H), 4.57 (dd, J = 1.6, 6.7 Hz, 4 H), 4.51 (dd, J = 1.6, 6.7 Hz, 2 H), 3.89 (s, 6 H), 3.70 (s, 6 H); 13C NMR (CDCl3, 150 MHz) δ 170.9, 152.7, 150.5, 148.8, 137.6, 134.7, 134.3, 133.3, 132.0, 123.7, 121.5, 117.6, 112.7, 111.0, 108.4, 74.2, 70.1, 56.1, 56.0, 55.9, 42.2; HRMS m/z calcd for (C36H38O9N + H)+ 628.2541, found 628.2557. 3-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1(3,4,5-tris(allyloxy)benzyl)-1H-pyrrole-2,5-dione (24). Using compound 14b as the starting material, the title compound 24 was synthesized according to the approach for the preparation of compound 23: yield 46.3%; mp 101−103 °C; 1H NMR (CDCl3, 600 MHz) δ 7.24 (dd, J = 7.7, 2.2 Hz, 1 H), 7.03 (d, J = 1.8 Hz, 1 H), 6.86 (d, J = 8.8 Hz, 1 H), 6.75 (s, 2 H), 6.69 (s, 2 H), 6.12 (m, 3 H), 5.43 (dd, J = 1.1, 17.0 Hz, 2 H), 5.33 (dd, J = 1.1, 17.0 Hz, 1 H), 5.26 (dd, J = 1.1, 10.4 Hz, 2 H), 5.18 (dd, J = 1.1, 10.4 Hz, 1 H), 4.66 (s, 2 H), 4.57 (d, J = 5.5 Hz, 4 H), 4.52 (d, J = 5.5 Hz, 2 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 3.71 (s, 6 H), 3.70 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 170.7, 153.3, 152.7, 150.7, 148.7, 139.5, 137.7, 135.6, 134.7, 134.2, 133.4, 131.9, 123.9, 121.2, 117.6, 112.7, 111.0, 108.4, 107.2, 74.2, 70.0, 56.2, 56.0, 55.9, 42.2; HRMS m/z calcd for (C37H40O10N + H)+ 658.2647, found 658.2644. General Procedure for the Preparation of Compounds 28− 38, 39a, and 39b. Appropriate diversely substituted 2-bromo-1phenylethanone 9a, 9b, or 25−27 (0.49 mmol) was added to a suspension of the corresponding pyrrole-2,5-dione 14a, 14b, or 14c (0.41 mmol) and K2CO3 (226 mg, 1.64 mmol) in DMF (10 mL) at room temperature under a N2 atmosphere. The suspension was stirred overnight until TLC showed the reaction has complete. The resulting solution was poured into water (40 mL), extracted with CH2Cl2 (40 mL), washed with brine (40 mL), and dried over anhydrous MgSO4, and the solvent was removed under reduced pressure. Purification by flash column chromatography on silica gel (EtOAc/PE = 1/1, v/v) gave the desired compounds 28−38, 39a, and 39b. The physical and spectral data for 28−38, 39a, and 39b are listed below. Data for 3,4-bis(3,4-dimethoxyphenyl)-1-(2-oxo-2-(3,4,5trimethoxyphenyl)ethyl)-1H-pyrrole-2,5-dione (28): yield 88.0%; mp 99−101 °C; 1H NMR (CDCl3, 600 MHz) δ 7.28−7.25 (m, 4 H), 7.10 (d, J = 1.7 Hz, 2 H), 6.88 (d, J = 8.8 Hz, 2 H), 5.06 (s, 2 H), 3.95 (s, 3 H), 3.93 (s, 6 H), 3.91 (s, 6 H), 3.72 (s, 6 H); 13C NMR (CDCl3, 150 MHz) δ 190.6, 170.9, 153.3, 150.5, 148.7, 143.4, 134.7, 129.6, 123.7, 121.4, 112.7, 111.0, 105.7, 61.1, 56.4, 56.0, 55.9, 44.4; HRMS m/z calcd for (C31H32O10N + H)+ 578.2021, found 578.2022. Data for 1-(2-(4-methoxyphenyl)-2-oxoethyl)-4-(3,4,5-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (29): yield 75.4%; mp 77−80 °C; 1H NMR (CDCl3, 600 MHz) δ 7.97 (d, J = 8.8 Hz, 2 H), 7.26−7.25 (m, 2 H), 6.96 (d, J = 8.8 Hz, 2 H), 6.85 (d, J = 8.2 Hz, 1 H), 6.79 (s, 2 H), 5.01 (s, 2 H), 3.87(s, 3 H), 3.86 (s, 3 H), 3.85 (s, 3 H), 3.70 (s, 6 H), 3.69 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 189.8, 170.7, 164.2, 153.2, 150.6, 148.6, 139.4, 135.7, 134.5, 130.5, 127.4, 124.2, 123.9, 121.2, 114.1, 112.8, 110.9, 107.2, 60.9, 56.1, 55.9, 55.6, 44.2; HRMS m/z calcd for (C30H30O9N + H)+ 548.1915, found 548.1916. Data for 1-(2-(3,4-dimethoxyphenyl)-2-oxoethyl)-4-(3,4,5-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (30): yield 78.1%; mp 144−147 °C; 1H NMR (CDCl3, 600 MHz) δ 7.63 (dd, J = 8.3, 1.7 Hz, 1 H), 7.50 (d, J = 1.7 Hz, 1 H), 7.27−7.25 (m, 1 9066



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H), 7.08 (d, J = 1.7 Hz, 1 H), 6.91 (d, J = 8.2 Hz, 1 H), 6.85 (d, J = 8.3 Hz, 1 H), 6.79 (s, 2 H), 5.03 (s, 2 H), 3.94 (s, 3 H), 3.90 (s, 3 H), 3.87 (s, 3 H), 3.85 (s, 3 H), 3.70 (s, 6 H), 3.69 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 190.0, 170.7, 154.2, 153.2, 150.7, 149.4, 148.7, 139.5, 135.7, 134.7, 127.6, 124.2, 123.9, 122.8, 121.2, 112.8, 111.0, 110.3, 107.4, 61.0, 56.2, 55.8, 55.6, 44.2; HRMS m/z calcd for (C31H32O10N + H)+ 578.2021, found 578.2020. Data for 1-(2-(3,4,5-trimethoxyphenyl)-2-oxoethyl)-4-(3,4,5-trimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (31): yield 81.0%; mp 137−140 °C; 1H NMR (CDCl3, 600 MHz) δ 7.29−7.26 (m, 1 H), 7.23 (s, 2 H), 7.08 (d, J = 2.2 Hz, 1 H), 6.86 (d, J = 8.3 Hz, 1 H), 6.80 (s, 2 H), 5.03 (s, 2 H), 3.92 (s, 3 H), 3.90 (s, 6 H), 3.88 (s, 3 H), 3.86 (s, 3 H), 3.71 (s, 6 H), 3.70 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 190.5, 170.6, 153.4, 153.3, 150.7, 148.7, 143.6, 135.7, 134.6, 129.6, 124.2, 124.0, 121.2, 112.9, 111.0, 107.4, 107.3, 105.8, 61.0, 56.5, 56.4, 56.2, 44.4; HRMS m/z calcd for (C32H34O11N + H)+ 608.2126, found 608.2131. Data for 1-(2-(4-methoxyphenyl)-2-oxoethyl)-3,4-bis(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (32): yield 75.0%; mp 122−125 °C; 1H NMR (CDCl3, 600 MHz) δ 7.97 (d, J = 8.8 Hz, 2 H), 6.96 (d, J = 8.8 Hz, 2 H), 6.81 (s, 4 H), 5.02 (s, 2 H), 3.86 (s, 3 H), 3.85 (s, 6 H), 3.70 (s, 12 H); 13C NMR (CDCl3, 150 MHz) δ 189.7, 170.5, 164.3, 153.1, 139.5, 135.6, 130.5, 127.3, 123.9, 114.1, 107.3, 61.0, 56.1, 55.6, 44.2; HRMS m/z calcd for (C31H32O10N + H)+ 578.2021, found 578.2020. Data for 1-(2-(3,4-dimethoxyphenyl)-2-oxoethyl)-3,4-bis(3,4,5trimethoxyphenyl)-1H-pyrrole-2,5-dione (33): yield 77.2%; mp 125−127 °C; 1H NMR (CDCl3, 600 MHz) δ 7.65 (dd, J = 1.1, 8.8 Hz, 1 H), 7.52 (d, J = 1.1 Hz, 1 H), 6.93 (d, J = 8.8 Hz, 1 H), 6.82 (s, 4 H), 5.05 (s, 2 H), 3.97 (s, 3 H), 3.92 (s, 3 H), 3.85 (s, 6 H), 3.71 (s, 12 H); 13C NMR (CDCl3, 150 MHz) δ 190.0, 170.5, 154.2, 153.2, 149.4, 139.7, 135.7, 127.6, 123.9, 122.9, 110.3, 107.5, 107.4, 61.1, 60.9, 56.2, 56.1, 56.0, 44.2; HRMS m/z calcd for (C32H34O11N + H)+ 608.2126, found 608.2133. Data for 1-(2-(3,4,5-trimethoxyphenyl)-2-oxoethyl)-3,4-bis(3,4,5trimethoxyphenyl)-1H-pyrrole-2,5-dione (34): yield 78.0%; mp 172− 174 °C; 1H NMR (CDCl3, 600 MHz) δ 7.23 (s, 2 H), 6.82 (s, 4 H), 5.05 (s, 2 H), 3.93 (s, 3 H), 3.91 (s, 6 H), 3.85 (s, 6 H), 3.71 (s, 12 H); 13C NMR (CDCl3, 150 MHz) δ 190.4, 170.4, 153.3, 153.2, 143.6, 139.7, 135.7, 129.5, 123.8, 107.5, 107.4, 105.7, 61.1, 56.5, 56.4, 56.2, 56.1, 44.5; HRMS m/z calcd for (C33H36O12N + H)+ 638.2232, found 638.2234. Data for 1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3,4bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (35): yield 90.7%; mp 115−119 °C; 1H NMR (CDCl3, 600 MHz) δ 7.57−7.54 (m, 2 H), 7.44−7.32 (m, 5 H), 7.34−7.32 (m, 2 H), 7.24 (d, J = 8.8 Hz, 2 H), 7.09 (s, 2 H), 6.94 (d, J = 8.8 Hz, 1 H), 6.86 (d, J = 8.8 Hz, 2 H), 5.25 (s, 2 H), 5.02 (s, 2 H), 3.93 (s, 3 H), 3.90 (s, 6 H), 3.73 (s, 6 H); 13C NMR (CDCl3, 150 MHz) δ 190.6, 170.9, 153.3, 150.5, 148.7, 143.4, 134.7, 129.6, 123.7, 121.4, 112.7, 111.0, 105.7, 61.1, 56.4, 56.0, 55.9, 44.4; HRMS m/z calcd for (C36H34O9N + H)+ 624.2228, found 624.2236. Data for 1-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (36): yield 88.1%; mp 174− 176 °C; 1H NMR (CDCl3, 600 MHz) δ 7.99 (d, J = 8.8 Hz, 2 H), 7.44−7.39 (m, 4 H), 7.37−7.34 (m, 1 H), 7.24−7.22(m, 2 H), 7.09 (d, J = 2.2 Hz, 2 H), 7.06 (d, J = 8.8 Hz, 2 H), 6.86 (d, J = 8.8 Hz, 2 H), 5.15 (s, 2 H), 5.03 (s, 2 H), 3.90 (s, 6 H), 3.71 (s, 6 H); 13C NMR (CDCl3, 150 MHz) δ 190.1, 170.7, 153.3, 150.7, 149.9, 148.7, 139.5, 136.1, 135.8, 134.6, 128.9, 128.3, 127.3, 124.0, 122.6, 121.3, 112.8, 112.4, 111.0, 110.7, 107.3, 70.9, 61.0, 58.6, 56.2, 56.0, 55.9, 44.2; HRMS m/z calcd for (C35H32O8N + H)+594.2122, found 594.2128. Data for 1-(2-(4-(benzyloxy)-3-methoxyphenyl)-2-oxoethyl)-3(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5dione (37): yield 89.0%; mp 161−163 °C; 1H NMR (CDCl3, 600 MHz) δ 7.57−7.54 (m, 2 H), 7.44−7.31 (m, 5 H), 7.28−7.26 (m, 1 H), 7.10 (s, 1 H), 6.94 (d, J = 7.7 Hz, 1 H), 6.86 (d, J = 8.8 Hz, 1 H), 6.80 (s, 2 H), 5.25 (s, 2 H), 5.02 (s, 2 H), 3.93 (s, 3 H), 3.90 (s, 3 H), 3.87 (s, 3H), 3.71 (s, 9 H); 13C NMR (CDCl3, 150 MHz) δ 190.1, 170.7, 153.3, 150.7, 149.9, 148.7, 139.5, 136.1, 135.8, 134.6, 128.9, 128.3, 127.3, 124.0, 122.6, 121.3, 112.8, 112.4, 111.0, 110.7, 107.3,

70.9, 61.0, 58.6, 56.2, 56.0, 55.9, 44.2; HRMS m/z calcd for (C37H36O10N + H)+ 654.2334, found 654.2343. Data for 1-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)-3-(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione (38): yield 92.4%; mp 142−145 °C; 1H NMR (CDCl3, 600 MHz) δ 7.99 (d, J = 8.8 Hz, 2 H), 7.44−7.39 (m, 4 H), 7.37−7.34 (m, 1 H), 7.28−7.27(m, 1 H), 7.10 (d, J = 2.2 Hz, 1 H), 7.06 (d, J = 8.8 Hz, 2 H), 6.87 (d, J = 8.8 Hz, 1 H), 6.80 (s, 2H), 5.16 (s, 2 H), 5.03 (s, 2 H), 3.90 (s, 3 H), 3.88 (s, 3 H), 3.72 (s, 9 H); 13C NMR (CDCl3, 150 MHz) δ 189.8, 170.7, 163.4, 153.3, 150.6, 148.7, 139.5, 136.0, 135.7, 134.5, 130.5, 128.8, 128.4, 127.6, 124.3, 123.9, 121.2, 115.0, 114.0, 112.8, 110.9, 107.3, 70.3, 61.0, 56.2, 56.0, 55.9, 44.2; HRMS m/z calcd for (C36H34O9N + H)+ 624.2228, found 624.2223. Data for ethyl 2-(3,4-bis(3,4-dimethoxyphenyl)-2,5-dioxo-2,5dihydro-1H-pyrrole-1-yl)acetate (39a): yield 88.4%; mp 131−133 °C; 1H NMR (CDCl3, 600 MHz) δ 7.21 (dd, J = 2.2, 8.8 Hz, 2 H), 7.05 (d, J = 2.2 Hz, 2 H), 6.85 (d, J = 8.2 Hz, 2H), 4.36 (s, 2 H), 4.24 (q, J = 7.1 Hz, 2 H), 3.89 (s, 6 H), 3.70 (s, 6 H), 1.30 (t, J = 7.1 Hz, 3 H); 13C NMR (CDCl3, 150 MHz) δ 170.4, 167.6, 150.5, 148.7, 134.5, 123.7, 121.3, 112.6, 111.0, 61.9, 56.0, 55.9, 39.2, 14.2; HRMS m/z calcd for (C24H26O8N + H)+ 456.1653, found 456.1661. Data for tert-butyl 2-(3,4-bis(3,4-dimethoxyphenyl)-2,5-dioxo2,5-dihydro-1H-pyrrol-1-yl)acetate (39b): yield 87.8%; mp 125−127 °C; 1H NMR (CDCl3, 600 MHz) δ 7.17 (dd, J = 1.7, 8.2 Hz, 2 H), 7.00 (d, J = 1.7 Hz, 2 H), 6.80 (d, J = 8.2 Hz, 2 H), 4.22 (s, 2 H), 3.83 (s, 6 H), 3.65 (s, 6 H), 1.42 (s, 9 H); 13C NMR (CDCl3, 150 MHz) δ 170.3, 166.5, 150.3, 148.5, 134.3, 123.5, 121.2, 112.5, 110.8, 82.6, 55.8, 55.6, 39.7, 27.9; HRMS m/z calcd for (C26H30O8N + H)+ 484.1966, found 484.1977. 2-(3,4-Bis(3,4-dimethoxyphenyl)-2,5-dioxo-2,5-dihydro-1Hpyrrol-1-yl)acetic Acid (40). A solution of compound 39b (483 mg, 1 mmol) in a mixture of CH2Cl2 (10 mL) and trifluoroacetic acid (0.8 mL) was stirred for 4 h. After concentration under vacuum, the volatiles were removed under reduced pressure. The residue was purified by column chromatography on silica gel (gradient elution with EtOAc/MeOH = 95/5 to 85/15) to yield the title compound 40: yield 235 mg, 55% yield; mp 176−178 °C; 1H NMR (CDCl3, 600 MHz) δ 7.22 (dd, J = 1.7, 8.3 Hz, 2 H), 7.05 (d, J = 1.7 Hz, 2 H), 6.85 (d, J = 8.3 Hz, 2 H), 4.42 (s, 2 H), 3.90 (s, 6 H), 3.70 (s, 6 H); 13C NMR (CDCl3, 150 MHz) δ 170.3, 150.6, 148.8, 134.5, 123.7, 121.2, 112.6, 111.0, 56.0, 55.9, 29.8; HRMS m/z calcd for (C37H36O10N + H)+ 428.1340, found 428.1337. 1-(2-(6-Amino-9H-purin-9-yl)ethyl)-3,4-bis(3,4-dimethoxyphenyl)-1H-pyrrole-2,5-dione (42). A suspension of compound 14a (369 mg, 1 mmol), 9-(2-bromoethyl)-9H-purin-6-amine (41) (280 mg, 1 mmol), and K2CO3 (276 mg, 2 mmol) was stirred in anhydrous DMF (10 mL) and heated to 60 °C overnight. The solution was poured into ice−water (100 mL), and the resulting mixture was extracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4. The solvent was evaporated to dryness, and the resulting residue was purified by flash chromatography to afford the title compound 42: yield 228 mg, 43.1%; mp 319 °C dec; 1H NMR (CDCl3, 600 MHz) δ 8.27 (s, 1 H), 7.79 (s, 1 H), 7.06 (dd, J = 2.2, 8.2 Hz, 2 H), 6.89 (d, J = 2.2 Hz, 2 H), 6.83 (d, J = 8.2 Hz, 2 H), 4.52 (m, 2 H), 4.10 (m, 2 H), 3.89 (s, 6 H), 3.70 (s, 6 H); 13C NMR (CDCl3, 150 MHz) δ 170.6, 155.3, 153.2, 150.5, 148.7, 140.4, 134.3, 123.5, 121.1, 112.4, 111.0, 56.0, 55.8, 42.0, 38.1; HRMS m/z calcd for (C27H27O6N6 + H)+ 531.1987, found 531.1983. 9-(2-(3,4-Bis(3,4-dimethoxyphenyl)-2,5-dioxo-2,5-dihydro1H-pyrrol-1-yl)ethyl)-1,3-dimethyl-1H-purine-1H-2,6(3H,9H)dione (44). Following the procedure for the preparation of compound 42, but using 9-(2-bromoethyl)-1,3-dimethyl-1H-purine2,6(3H,9H)-dione (43) as the starting material, the target compound 44 was obtained: yield 46.6%; mp 162−164 °C; 1H NMR (CDCl3, 600 MHz) δ 7.45(s, 1 H), 7.13 (dd, J = 2.2, 8.2 Hz, 2 H), 6.95 (d, J = 2.2 Hz, 2 H), 6.84 (d, J = 8.2 Hz, 2 H), 4.58 (m, 2 H), 4.10 (m, 2 H), 3.89 (s, 6 H), 3.68 (s, 6 H), 3.54 (s, 3 H), 3.39 (s, 3 H); 13C NMR (CDCl3, 150 MHz) δ 170.6, 151.7, 150.6, 148.8, 141.1, 134.1, 123.6, 121.0, 112.4, 111.1, 77.3, 77.1, 76.9, 56.0, 55.9, 45.6, 38.6, 29.9, 28.1; HRMS m/z calcd for (C29H30O8N5 + H)+ 576.2089, found 576.2087. 9067



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μL and eluted with methanol with the flow rate of 1 mL/min. The wavelength used was 272 nm, and the retention time was 3.2 min. Calculated Molecular Descriptors. Calculated descriptors such as cLog P and PSA were determined with SYBYL-X 2.0. The structures of compounds 35−38 were built and energy minimized under the Tripos force field at 0.05 kcal/(mol Å). The Gasteiger−Huckel method was used to calculate the charges. Energy minimization was performed by the Powell method with 2000 iterations.

Materials for Biological Studies. DMSO, verapamil, doxorubicin (DOX), topotecan, and paclitaxel were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), trypsin−ethylenediaminetetracetic acid (EDTA), and penicillin/streptomycin were from Gibco BRL. Fetal bovine serum (FBS) was from Hyclone Laboratories. 2-(4,5-Dimethylthiazol-2-yl)-5-[3-(carboxymethoxy)phenyl]-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) were purchased from Promega. Human breast cancer cell lines MDA435/LCC6 and MDA435/LCC6MDR were kindly provided by Dr. Robert Clarke (Georgetown University, Washington, DC). The human ovarian carcinoma cell lines 2008/P and 2008/MRP1 were generous gifts from Prof. P. Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The HEK293/pcDNA3.1 and HEK293/R2 cell lines were kindly provided by Dr. Kenneth To (The Chinese University of Hong Kong, Hong Kong). The L929 cell line was purchased from ATCC. Cell Culture. The MDA435/LCC6, MDA435/LCC6MDR, and L929 cell lines were cultured in supplemented DMEM medium with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The 2008/P, 2008/MRP1, HEK293/pcDNA3.1, and HEK293/R2 cells were cultured in RPMI 1640 medium containing heat-inactivated 10% FBS, 100 U/mL penicillin, and 100 μg/mLof streptomycin. They were maintained at 37 °C in a humidified atmosphere with 5% CO2. The cells were split constantly after a confluent monolayer was formed. To split the cells, the plate was washed briefly with phosphate-buffered saline (PBS) and the cells were treated with 0.05% trypsin−EDTA and harvested by centrifugation. Cell Proliferation Assay. A total of 6000 cells of LCC6 or LCC6MDR and paclitaxel were mixed with or without modulators to a final volume of 200 μL in each well of a 96-well plate. A total of 4000 cells of 2008/P or 2008/MRP1 and DOX were coincubated with or without modulators to a final volume of 200 μL. A total of 4500 cells of HEK293/pcDAN3.1 or HEK293/R2 and topotecan were coincubated with or without modulators to a final volume of 200 μL. The plate were then incubated for 5 days at 37 °C. The CellTiter 96 AQueous Assay (Promega) was used to measure the cell proliferation according to the manufacturer’s instructions. MTS (2 mg/mL) and PMS (0.92 mg/mL) were mixed in a ratio of 20:1. An aliquot (10 μL) of the freshly prepared MTS/PMS mixture was added into each well, and the plate was incubated for 2 h at 37 °C. Optical absorbance at 490 nm was then recorded with a microplate absorbance reader (Bio-Rad). IC50 values were calculated from the dose−response curves of MTS assays (Prism 4.0). Cytotoxicity Assay. A total of 10 000 cells of LCC6, LCC6MDR, or L929 were mixed with a series concentration of permethyl ningalin B analogues to a final volume of 100 μL in each well of a 96-well plate. The plate was then incubated for 3 days at 37 °C. The 50% inhibitory concentration (IC50) of permethyl ningalin B analogues was determined using the MTS proliferation assay as described previously.35 Intracellular DOX Accumulation. A total of 1 × 106 cells of LCC6 or LCC6MDR were added to an Eppendorf tube containing 20 μM DOX and different concentrations of permethyl ningalin B analogues at 37 °C for 150 min. DMSO (0.1%) in place of permethyl ningalin B analogues was used as a negative control. After incubation, the cells were spun down, washed with cold PBS, pH 7.4, and lysed with lysis buffer (0.75 M HCl, 0.2% Triton-X100 in 2-propanol). The lysate was spun down, and the supernatant was saved. The fluorescence level of DOX was determined by a fluorescence spectrophotometer (BMG Technologies) using excitation and emission wavelengths of 460 and 610 nm.35 Intracellular Accumulation of Permethyl Ningalin B Analogue 35. A total of 3 × 106 cells of LCC6 or LCC6MDR were incubated with 20 or 200 μM 35 at 37 °C for 150 min. After incubation, the cells were pelleted, washed with cold PBS, pH 7.4, resuspended with 100 μL of 100% methanol, and lysed by 5 min of vortexing. The lysate was spun down, and the supernatant was saved for HPLC measurement. A reversed-phase Diamonsil C18 (4.6 × 150 mm) column attached to a Gilson 322 pump coupled to a Gilson UV− vis-152 detector was used. Each sample was injected at a volume of 20

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ASSOCIATED CONTENT

* Supporting Information S

1 H NMR spectra and 13C NMR spectra of all compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: (852)-34008662. Fax: (852)-23649932. E-mail: larry. [email protected]. *Phone: (86)-82031087. Fax: (86)-82033054. E-mail: [email protected] Author Contributions ∥

J.W.B. and I.L.K.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by the Natural Science Foundation of Shandong Province (Grant ZR2011HM009), National Natural Science Foundation of China (NSFC Grant 81172926), General Research Fund (Grant B-Q16G) of the Research Grant Council of Hong Kong, and Special Fund for Marine Scientific Research in the Public Interest of China (Grant 201005024).

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ABBREVIATIONS USED MDR, multidrug resistance; ABC, ATP-binding cassette; P-gp, P-glycoprotein; MRP1, multidrug-resistance-related protein 1; BCRP, breast cancer resistance protein; EDCI, N-[3(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; DIAD, diisopropyl azodicarboxylate; TFA, trifluoroacetic acid; RF, relative fold; DOX, doxorubicin; PSA, polar surface area; cLogP, calculated log P value; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; MTS, 2-(4,5dimethylthiazol-2-yl-)-5-[3- (carboxymethoxy)phenyl]-2-(4-sulfophenyl)-2H-tetrazolium; PMS, phenazine methosulfate

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