Synthesis and biological evaluation of glaucocalyxin A derivatives as potential anticancer agents

European Journal of Medicinal Chemistry 86 (2014) 235e241

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

Original article

Synthesis and biological evaluation of glaucocalyxin A derivatives as potential anticancer agents Jing Yang, Yanli Liu, Chengwen Xue, Wei Yu, Jian Zhang, Chunhua Qiao* College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2014 Received in revised form 28 July 2014 Accepted 20 August 2014 Available online 21 August 2014

A series of Mannich base type derivatives of Glaucocalyxin A (GLA) were designed and prepared. The cytotoxicity of these compounds was evaluated against six tumor cell lines (SMMC-7721, B16, SGC-7901, A549, KB, HL-60). Most compounds exhibited potent antiproliferative effects with low micromolar IC50 values. Compound 1 with para methyl benzyl amine moiety and compound 16 with cyclohexylamine moiety displayed the highest inhibition efficacy. Significantly, the cytotoxicity of compound 1 was much lower than GLA against the normal human liver cell (HL-7702). The in vitro stability assay revealed that transformation of GLA to Mannich base type derivatives improved the compound stability in rat plasma. Finally, decomposition product analysis supported that compound 1 could act as a prodrug and release GLA in the intracellular environment. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Glaucocalyxin A Glaucocalyxin A derivatives Antitumor Prodrug Structure activity relationship

1. Introduction Natural products play a highly significant role in the drug discovery and development process, and this was particularly evident in the areas of cancer and infectious diseases [1]. Natural tetracyclic diterpenoids represents an important class of natural products. Among them, ent-kaurane diterpenoids with exo-methylene cyclopentanone in the D-ring, such as oridonin (1), phyllostachysin F (2) and Glaucocalyxin A (GLA, 3, Fig. 1) are the representative members of this family. These diterpenoids were reported to exhibit variable pharmacological activities including antitumor, antibacterial, and anti-inflammatory effects [2e5]. The common structural feature of these diterpenoids include: oxygen- and stereochemistry-rich chemical skeletons, and a, b-unsaturated ketone moiety in the D-ring. The a-methylene cyclopentanone system in D-ring was believed to be associated with their variety of bioactivities [6]. As an important member of ent-kaurane diterpenoids, Glaucocalyxin A (GLA) has been reported to exhibit a wide range of biological activities such as antitumor effects [7e9], inhibition of platelet activating factor (PAF) e induced platelet aggregation [10], immunosuppressive activity [11], antioxidative and DNA damage protecting activity [12]. In particular, GLA has been reported to

inhibit the proliferation of a wide variety of tumor cells [13]. However, like other tetracyclic diterpenoids, clinical application of GLA was limited by its high metabolic rate and poor bioavailability [14]. So far, no research effort was reported to address these issues. We speculated that the a,b-unsaturated ketone in GLA molecule might act as a Michael addition acceptor and react with universal nucleophiles in the biological system, resulting in the observed high toxicity and metabolic rate. To test this hypothesis, the reactive a-methylene cyclopentanone group was masked by converting GLA to its corresponding Mannich base derivatives. We hope this transformation would decrease the compound toxicity while retaining the antiproliferation capability. Based on the above rationale, sixteen Mannich base type GLA derivatives were prepared, their antiproliferative ability was evaluated against six cancer cell lines. For the most potent compound 1, its activity to induce cell apoptosis was investigated, the in vitro chemical stability and reactivity with thiol nucleophile was examined. These studies identified a new GLA derivative with low toxicity and good metabolic stability profile compared to the parent compound GLA. 2. Results and discussion 2.1. Chemistry

* Corresponding author. E-mail address: [email protected] (C. Qiao). http://dx.doi.org/10.1016/j.ejmech.2014.08.061 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

The synthesis of compounds 1e16 was depicted in Scheme 1. Briefly, the Michael addition reaction between GLA and different

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J. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 235e241

HO

OH

HC B

A

O

H

D OH

O OH

H

20

OH Oridonin, 1

1 3

O OH

H

9

2

OH O

4

5

10

H 18

Phyllostachysin F, 2

12

11

13

14

H

15

8 7

OH

6

17 16

OH O

19

Glaucocalyxin A, 3

Fig. 1. Structures of oridonin, phyllostachysin F and glaucocalyxin A.

NH R OH

H O

H

OH

O

R-NH2

OH

H CH3OH, Silica, reflux 35%-90% 1 2 3 4 5 6 7 8 9

O

R = C6H5-4-CH3 R = C6H5-3-CH3 R = C6H5-2-CH3 R = C6H5-4-F R = C6H5-2-F R = C6H5-4-Cl R = C6H5-2-Cl R = C6H5-4-Br R = C6H5-2-Br

H 10 11 12 13 14 15 16

OH

O

R = C6H5-4-OCH3 R = C6H5-4-COCH3 R = C6H5-3-CN R = C6H5-4-SO2C6H5 R = C6H5-4-COOH R = C6H5 R = (CH2)5CH

Scheme 1. Synthesis of compounds 1e16.

amines afforded sixteen novel Mannich base type GLA derivatives. An appropriate amount of silica was added as catalyst to increase the reaction rates [15]. Depending on the nature of the amine reactants, the yield of the reaction is 35e90%. All the target compounds were fully characterized by 1H NMR, 13C NMR and high resolution mass spectrometry (HRMS).

2.2. Anti-proliferative activity and SAR study Applying the MTT colorimetric assay [16], the antiproliferative activity of these compounds against six tumor cell lines SMMC7721 (human liver carcinoma), B16 (mice melanoma), SGC-7901 (human gastric carcinoma), A549 (human lung carcinoma), KB (human oral epidermoid carcinoma), HL-60 (human promyelocytic leukemia) and a normal cell line (HL-7702) were evaluated and the results were summarized in Table 1. The parent compound GLA and 5-fluorouracil (5-FU) were used as positive control in each panel. As shown in Table 1, all Mannich base type GLA derivatives demonstrated higher antiproliferative potency than the positive control 5-FU. Some compounds exhibited similar potency compared to GLA. Among them, compound 1 and 16 displayed higher potency than other GLA derivatives against most cancer cell lines. Compared with other aniline derivatives, compound 15 without substituent group on the aniline displayed the lowest activity. Apparently, compounds with electron e donating groups on the amine moiety (compounds 1, 10) exhibited higher potency than compounds with electron e withdrawing groups (compounds 4e9 and 11e14). The result also revealed that compounds with para e substituent on the arylamine exhibited higher cycotoxicity, compared with the corresponding compounds bearing ortho e

substituted arylmines, for example: the activity of compound 1 is higher than that of compound 3. Likewise, the same trend could be noticed from compound 6 vs 7, and compound 8 vs 9. However, this conclusion did not apply to fluorine substituted compound 4 and compound 5. Compounds with meta e or ortho e substituents displayed similar antiproliferative activity (compound 2 vs 3). It was noticed that compound 16 with cyclohexylamine in the structure also displayed high inhibition potency. However, further GLA derivatives based on 16 were not pursued considering any saturated amine, other than aromatic amine, would greatly change the compound basicity. Overall, these GLA derivatives were discovered to exhibit higher antiproliferative efficacy against KB cell line. To test whether or not these Mannich base type derivatives of GLA would exhibit the expected low toxicity, compounds 1, 14, and 16 were incubated with normal human liver cell HL-7702, their cytotoxicity was evaluated and compared to that against liver cancer cell line SMMC-7721 [16]. The result was listed in Table 2. The IC50 values of 1 and 14 were obviously lower against liver cancer cell line than that against normal human liver cell line HL7702. Compound 16 exhibited similar inhibitory potency against normal human liver cell vs human cancer liver cell, which could be due to a great physical property change.

2.3. Induction of apoptosis Based on the antiproliferative data, the most potent compound 1 was selected for further mechanistic study. We investigated whether or not the observed growth inhibition in the KB cell was attributed to apoptosis. The KB cell was treated with a variable

J. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 235e241

237

Table 1 The IC50 values (mM) of GLA derivatives against cancer and normal cell lines. Compd

Cytotoxicity ( IC50, mM) SMMC-7721

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 GLA 5-FU

12.15 20.70 22.22 19.76 14.51 14.12 32.33 18.07 28.43 18.04 26.10 19.11 14.06 16.77 48.02 15.23 8.11 67.70

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.29 1.61 0.75 1.48 0.16 0.98 7.97 1.39 0.66 0.57 1.37 0.58 0.66 1.37 3.10 0.71 0.30 1.79

B16 8.54 14.40 19.18 12.86 9.59 9.40 16.74 10.91 15.24 12.07 18.32 18.17 10.52 6.79 11.47 5.66 4.84 17.85

SGC-7901 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.54 0.64 0.57 0.26 0.41 0.55 1.35 1.42 1.87 1.36 0.50 0.67 0.95 0.20 0.89 0.53 0.31 1.56

16.14 38.74 38.44 34.24 24.28 21.19 28.61 25.37 33.47 22.44 31.88 24.41 15.45 22.26 32.57 20.67 11.70 59.90

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

A549

1.98 2.89 2.30 4.65 2.06 1.55 1.85 2.18 3.38 3.87 2.50 2.33 0.43 0.70 0.91 5.31 1.19 14.75

10.61 28.30 24.65 22.66 20.61 15.83 21.59 19.23 32.58 13.60 21.83 21.28 13.69 11.71 37.19 11.12 7.50 51.64

KB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.35 4.57 3.56 1.77 4.82 1.15 0.77 1.86 4.63 1.08 1.47 0.72 0.28 1.04 2.56 0.94 0.59 11.11

7.22 29.62 23.37 12.33 11.23 6.54 11.01 6.31 13.93 6.41 12.64 8.08 5.64 6.13 30.96 11.28 6.47 53.52

HL-60 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.44 1.19 2.65 2.39 1.71 0.25 0.85 0.27 0.77 0.29 1.45 0.81 0.27 0.84 7.06 0.84 0.23 4.46

8.92 13.48 18.13 17.77 15.61 14.07 23.03 14.56 12.53 5.70 11.71 23.24 15.28 10.54 15.08 9.35 5.52 27.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.97 1.51 3.91 2.05 4.12 0.28 2.97 0.88 2.17 0.81 3.32 3.68 2.56 1.35 3.37 1.55 0.04 0.64

*The data were the mean ± SD obtained from three independent experiments.

concentration of compound 1 (10, 20 and 40 mM), GLA was used as control. As determined by flow cytometry analysis [17], the percentage of apoptotic cells was shown in Fig. 2. Compound 1 induced KB cell apoptosis in a dose-dependent manner. After 48 h treatment, the observed apoptotic cell percentage were 17.79% ± 1.16, 24.50% ± 1.28 and 37.43% ± 1.43 (early and late apoptosis) at the indicated concentration. While the control group (no drug treatment) displayed 11.55% ± 0.89 apoptotic percentage, indicating the marked effect of compound 1 to induce apoptosis. Treatment of KB cells with GLA also induced apoptosis in a dose-dependent manner. The induced apoptosis rates by GLA at 5, 10 and 20 mM were 14.66 ± 1.88%, 21.08% ± 2.31, and 52.13% ± 4.71, as compared to 10.88% ± 1.89 in the untreated control group. These results indicated that the antiproliferative activity of GLA and its Mannich base type derivative 1 was partially mediated by their ability to induce cell apoptosis. 2.4. In vitro stability study To study the stability of these GLA derivatives, compound 1 was chosen as the representative. Its stability in PBS buffer (pH 7.4) and in 80% rat plasma [18] was examined. Again, GLA was used as control. The result was shown in Table 3. After 24 h of incubation at 37
C, the remaining percentage of both compound 1 and GLA were observed to be close to hundred percent, indicating they were stable in PBS buffer. In rat plasma solution, a remaining percentage of 86% of compound 1 was detected; By comparison, 24.2% of GLA was recorded after 24 h of incubation. The stability study demonstrated that the Mannich base derivative 1 was more stable than GLA in rat plasma. 2.5. Identification of compound 1 as prodrug To examine whether these GLA derivatives behave as prodrug, compound 1 was again selected as the representative to investigate its stability and reactivity in KB cell lysate containing nucleophile cysteine (20 mM). The formation of cysteine conjugates was evaluated by LCeHRMS (Fig. S1). It was observed that GLA quickly reacted with cysteine and the corresponding conjugate peak ([MþH]þ ¼ 454.2214, tR ¼ 2.52 min) was detected by high

resolution mass spectrum analysis. Incubation of compound 1 with the same cell lysate, a peak with molecular mass corresponding to GLA ([MþH]þ ¼ 333.2035, tR ¼ 5.75 min) was detected first. Given prolonged incubation time, the peak corresponding to GLA and cysteine adduct ([MþH]þ ¼ 454.2219, tR ¼ 2.42 min) was observed. This result suggested that GLA would react readily with a thiocontaining nucleophile, like cysteine or glutathione, in the intracellular system. Accordingly, the experimental result supported the prodrug design rationale: the Mannich base type GLA derivative could release GLA first, subsequently, form a nucleophilic addition product.

3. Conclusion In conclusion, we reported here a new series of Mannich base type GLA derivatives (compounds 1e16) which were prepared from reactions of GLA with different amines. The antiproliferative study against six cancer cell lines discovered compounds with slightly higher or equivalent potency compared to GLA. The structure activity relationship (SAR) as revealed by IC50 values showed that the amine moiety exerted influence on the compound inhibition potency; for the substituted aniline, the electronegativity property as well as the substitute position on the phenyl ring played a role on the compound inhibition ability. Specifically, Compound 1, 14, and 16 exhibited higher cytotoxicity against six tumor cell lines. It was demonstrated that the compound antiproliferative effect was partially mediated by its induction of cell apoptosis. Significantly, derivatives 1 and 14 displayed lower cytotoxicity than GLA against a normal human liver cell (HL-7702). The in vitro stability assay in rat plasma demonstrated the GLA derivative with improved stability (compound 1 vs GLA). The result from high resolution mass spectrum analysis supported a prodrug design rationale: these Mannich base type GLA derivatives could release GLA in the intracellular environment. However, whether or not these compounds would interact directly with any biomolecules was unknown. Taken together, our results discovered a GLA derivative compound 1 with potent antiproliferative activity and low toxicity. Further study regarding to the compound in vivo antitumor activity is ongoing and will be reported in due course.

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Table 2 Comparison of the IC50 values of compound 1, 14 and 16 against normal human liver cell HL-7702 vs cancer liver cell line SMMC-7721. Compound

a

4.1. General methods

Cytotoxicity ( IC50, mM)a SMMC-7721

1 14 16 GLA

4. Experimental

12.15 16.77 15.23 8.71

± ± ± ±

0.19 1.37 0.71 1.21

HL-7702 21.10 35.39 8.95 8.62

± ± ± ±

3.37 5.89 0.75 0.27

The data were the mean ± SD obtained from three independent experiments.

Thin layer chromatography (TLC) analysis was carried on Yellow Sea Sil G/UV 450 on polyester plates using different solvents system. Flash chromatography was carried on silica gel using petroleum as the initial elution followed by different elution mixtures (dichloromethane/methanol). 1H NMR spectrum and 13C NMR spectrum were recorded at 25
C on 300 MHz or 400 MHz. 1H NMR

Fig. 2. Induction of apoptosis on the KB cells by compound 1 and GLA. (A) Flow cytometry analysis of apoptotic KB cells induced by compound 1 at different concentrations. (B) Flow cytometry analysis of apoptotic KB cells induced by GLA at different concentrations. (C) Apoptotic ratio of different concentrations of compound 1 and GLA in KB cells. The values are means ± SE of at least three independent experiments. ((*) p < 0.05, (**) p < 0.01 comparing to vehicle-treated control.).

Table 3 The stability of compound 1 and GLA in PBS buffer and in 80% rat plasma. Conditions

Percentage of compound remaining after 24 h Compound 1

GLA

PBS buffer (pH 7.4) 80% rat plasma

104.2 ± 0.6 86.4 ± 1.0

109.4 ± 2.1 24.2 ± 0.2

Percentage of the remaining parent compounds were determined by HPLC analysis after 24 h incubation at 37
C. Mean ± SD, n ¼ 3.

spectrum was recorded using TMS as the internal reference, 13C NMR spectrum was recorded using the residual solvent as the internal reference. Chemical shifts (d) are given in parts per million (ppm). Coupling constants (J) are reported as Hertz. NMR abbreviations are used as follows: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet. The solutions are taken from a concentrated sample dissolved in CDCl3 and DMSO-d6. Analytical HPLC was conducted on SHIMADZU LC-20AD. Prior to biological evaluation, all compounds were determined to be >95% pure using appropriate analytical methods (MeOH/H2O 60e75% v/v) based on the peak area percentage.

J. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 235e241

4.2. Analytical method The compound purity analysis was afforded with the Prominence UFLC from SHIMADZU. And the column was Inertsil® ODS-SP, 5 mm, 4.6  150 mm. Mobile phases consisted of water and methanol at a flow rate of 1 mL min1 (MeOH/H2O 75% v/v, MeOH/H2O 70% v/v, MeOH/H2O 65% v/v, MeOH/H2O 60% v/v). The elution time was 30 min. Injection volume: 20 mL. The corresponding HPLC file was showed in Supporting information. The in vitro stability analysis was afforded with the Prominence UFLC from SHIMADZU using external standard method. And the column was Inertsil® ODS-SP, 5 mm, 4.6  150 mm. Eluent A: water þ 0.1% TFA; Eluent B: methanol. T (30 min): 50% A: 50% B. For LC-HRMS analysis, HPLC column was a Durashell C18 (Bonna-Agela Technologies, USA), 3 mm, 50  4.6 mm. The analysis was afforded with the Agilent Technologies 6540 UHD AccurateMass Q-TOF LC/MS. Mobile phases consisted of water (þ0.1% FA) and methanol at a flow rate of 1.4 mL min1. Injection volume: 10 mL. Conditions chosen were the following: Eluent A: water þ 0.1% FA; Eluent B: methanol. T (0 min): 95% A: 5% B; T (4 min) 5% A: 95% B; T (5 min) 5% A: 95% B; T (6 min) 95% A: 5% B; T (8 min) 95% A: 5% B. Detection was in positive polarity. 4.3. General procedure to synthesize compounds 1e16 4.3.1. Preparation procedures for compound 1e16 Compound glaucocalyxin A (50 mg, 0.15 mmol), corresponding arylamine 0.45 mmol and Silica 150 mg were suspended in methanol (2 mL) and heated at reflux at 70e80
C for 12e48 h in an inert atmosphere of N2. Solvent was evaporated from the filtrate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (MeOH/CH2Cl2, 1/ 200e1/50) to afford the title compounds (yields 35e90%) as white solids. aniline4.3.1.1. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-methyl) 3,15-dione. Compound 1. White solid, 51% yield, Rf ¼ 0.25 (50:1 DCM/Methanol): 1H NMR (400 MHz, CDCl3) d 7.01 (d, J ¼ 8.0 Hz, 2H, H-30 , 50 ), 6.62 (d, J ¼ 8.0 Hz, 2H, H-20 , 60 ), 5.03 (s, 1H), 4.91 (s, 1H, H14a), 4.20 (d, J ¼ 8.0 Hz, 1H, H-7b), 3.46 (d, J ¼ 4.0 Hz, 1H, H-16), 3.27e3.14 (m, 2H, H-17), 2.63 (s, 1H, H-13), 2.57e2.37 (m, 2H, H-2), 2.24 (s, 3H, H-70 ), 1.93e1.52 (m, 8H), 1.41e1.28 (m, 3H), 1.11 (s, 3H, H-20), 1.07 (s, 6H, H-18,19); 13C NMR (100 MHz, CDCl3) d 221.1 (C15), 216.8 (C-3), 145.7 (C-10 ), 129.9 (C-30 , 50 ), 127.6 (C-40 ), 113.8 (C-20 , 60 ), 75.2 (C-14), 74.3 (C-7), 61.6 (C-8), 53.2 (C-16), 51.7 (C-5), 47.8 (C17), 46.8 (C-4), 41.4 (C-13), 40.6 (C-8), 38.6 (C-10), 38.0 (C-1), 33.6 (C-2), 29.8 (C-12), 29.6 (C-6), 27.8 (C-18), 24.9 (C-70 ), 20.9 (C-11), 20.6 (C-19), 18.4 (C-20); HRMS (ESI) m/z calcd for C27H37NO4 [MþH]þ 440.2795, found 440.2798 (error e 0.68 ppm). aniline4.3.1.2. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(3-methyl) 3,15-dione (compound 2). White solid, 79% yield, Rf ¼ 0.25 (50:1 DCM/Methanol): 1H NMR (400 MHz, CDCl3) d 7.08 (t, J ¼ 7.6 Hz, 1H, H-50 ), 6.57 (d, J ¼ 6.8 Hz, 1H, H-20 ), 6.51 (s, 2H, H-40 , 60 ), 5.01 (s, 1H), 4.92 (s, 1H, H-14a), 4.22 (d, J ¼ 9.2 Hz, 1H, H-7b), 3.53e3.41 (m, 1H, H-16), 3.28e3.20 (m, 2H, H-17), 2.64 (s, 1H, H-13), 2.57e2.37 (m, 2H, H-2), 2.28 (s, 3H, H-70 ), 1.93e1.54 (m, 9H), 1.41e1.29 (m, 3H), 1.12 (s, 3H, H-20), 1.07 (s, 6H, H-18, 19); 13C NMR (75 MHz, DMSO) d 219.3 (C-15), 216.1 (C-3), 148.5 (C-10 ), 137.9 (C-30 ), 128.8 (C-50 ), 116.8 (C-40 ), 112.7 (C-20 ), 109.4 (C-60 ), 74.2 (C-14), 73.0 (C-7), 60.3 (C9), 52.8 (C-5), 50.3(C-16), 47.6 (C-4), 46.0 (C-17), 37.9 (C-10), 37.3 (C1), 33.3 (C-2), 29.9 (C-12), 27.1 (C-6), 24.2 (C-18), 21.4 (C-70 ), 20.6 (C19), 17.7 (C-20); HRMS (ESI) m/z calcd for C27H37NO4 [MþNa]þ 462.2615, found 462.2615 (error 0 ppm).

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4.3.1.3. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(2-methyl) aniline3,15-dione (compound 3). White solid, 50% yield, Rf ¼ 0.25 (50:1 DCM/Methanol): 1H NMR (300 MHz, CDCl3) d 7.17e7.05 (m, 2H, H30 , 50 ), 6.73e6.62 (m, 2H, H-40 , 60 ), 4.95 (s, 1H, H-14a), 4.85 (s, 1H), 4.30e4.22 (m, 1H, H-7b), 3.54e3.45 (m, 1H, H-16), 3.36e3.23 (m, 2H, H-17), 2.86 (d, J ¼ 4.2 Hz, 1H), 2.69 (s, 1H, H-13), 2.62e2.37 (m, 3H, H-2), 2.19 (s, 3H, H-70 ), 1.97e1.80 (m, 5H), 1.72e1.64 (m, 2H), 1.42e1.31 (m, 3H), 1.14 (s, 3H, H-20), 1.09 (s, 6H, H-18, 19); 13C NMR (75 MHz, DMSO) d 219.8 (C-15), 216.2 (C-3), 146.1 (C-10 ), 129.9 (C30 ), 126.9 (C-20 ), 122.1(C-50 ), 116.0 (C-40 ), 109.0 (C-60 ), 74.3 (C-14), 73.0 (C-7), 60.4 (C-9), 52.8 (C-16), 50.3 (C-5), 47.2 (C-13), 46.0 (C17), 37.9 (C-10), 37.3 (C-1), 33.4 (C-2), 30.0 (C-12), 27.1 (C-6), 24.2 (C-18), 20.6 (C-19), 17.7 (C-70 ), 17.6 (C-20),; HRMS (ESI) m/z calcd for C27H37NO4 [MþNa]þ 462.2615, found 462.2634 (error 6.06 ppm). 4.3.1.4. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-fluorine) aniline3,15-dione (compound 4). White solid, 90% yield, Rf ¼ 0.20 (50:1 DCM/Methanol): 1H NMR (400 MHz, CDCl3) d 6.90 (t, J ¼ 8.0 Hz, 2H, H-20 ,60 ), 6.69e6.61 (m, 2H, H-30 , 50 ), 5.17 (s, 1H), 4.92 (s, 1H, H-14a), 4.27e4.13 (m, 1H, H-7b), 3.77 (s, 1H), 3.42e3.38 (m, 1H, H-16), 3.27e3.12 (m, 2H, H-17), 2.63 (s, 1H, H-13), 2.59e2.37 (m, 2H, H-2), 1.97e1.51 (m, 9H), 1.41e1.27 (m, 3H), 1.11 (s, 3H, H-20), 1.07 (s, 6H, H-18,19); 13C NMR (75 MHz, CDCl3) d 221.2 (C-15), 216.8 (C-3), 144.3 (C-10 ), 116.0 (C-20 ), 115.7 (C-60 ), 114.6 (C-30 ), 114.6 (C-50 ), 75.1 (C-14), 74.2 (C-7), 61.6 (C-9), 53.1 (C-16), 51.6 (C-5), 47.6 (C-13), 46.8 (C-17), 41.8 (C-8), 40.6 (C-4), 38.5 (C-10), 38.0 (C-1), 33.6 (C-2), 29.6 (C-12), 27.8 (C-6), 24.8 (C-18), 20.9 (C-19), 18.4 (C-20); HRMS (ESI) m/z calcd for C26H34FNO4 [MþNa]þ 466.2371, found 466.2364 (error 1.50 ppm). 4.3.1.5. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(2-fluorine) aniline3,15-dione (compound 5). White solid, 35% yield, Rf ¼ 0.23 (50:1 DCM/Methanol): 1H NMR (300 MHz, CDCl3) d 7.03e6.90 (m, 2H, H30 , 50 ), 6.72 (t, J ¼ 9.0 Hz, 1H, H-60 ), 6.64 (dd, J ¼ 12.0, 6.0 Hz, 1H, H40 ), 5.15 (s, 1H), 4.93 (s, 1H, H-14a), 4.43 (s, 1H), 4.26e4.18 (m, 1H, H7b), 3.69 (s, 1H), 3.60e3.48 (m, 1H, H-16), 3.29e3.19 (m, 2H, H-17), 2.66 (s, 1H, H-13), 2.58e2.36 (m, 2H, H-2), 1.99e1.49 (m, 9H), 1.41e1.28 (m, 3H), 1.12 (s, 3H, H-20), 1.07 (s, 6H, H-18,19); 13C NMR (75 MHz, DMSO) d 219.5 (C-15), 216.3 (C-3), 136.43 (d, J ¼ 11.4 Hz, C10 ), 124.90 (C-50 ), 115.75 (d, J ¼ 6.9 Hz, C-40 ), 114.45 (d, J ¼ 18.0 Hz,C60 ), 111.7 (C-30 ), 74.2 (C-14), 73.0 (C-7), 60.3 (C-9), 52.8 (C-16), 50.3 (C-5), 47.3 (C-13), 46.0 (C-17), 37.9 (C-10), 37.3 (C-1), 33.4 (C-2), 30.0 (C-12), 27.1 (C-6), 24.2 (C-18), 20.7 (C-19), 17.8 (C-20); HRMS (ESI) m/z calcd for C26H34FNO4 [MþNa]þ 466.2377, found 466.2364 (error 2.79 ppm). 4.3.1.6. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-chlorine) aniline3,15-dione (compound 6). White solid, 90% yield, Rf ¼ 0.26 (50:1 DCM/Methanol): 1H NMR (400 MHz, DMSO) d 7.10 (d, J ¼ 8.4 Hz, 2H, 30 , 50 ), 6.60 (d, J ¼ 8.8 Hz, 2H, H-20 , 60 ), 4.79 (s, 1H, H-14a), 3.95e3.88 (m, 1H, H-7b), 3.40 (d, J ¼ 10.0 Hz, 1H, H-16), 3.11e2.96 (m, 2H, H17a, 13), 2.40e2.32 (m, 1H, H-17b), 1.85e1.47 (m, 7H), 1.38e1.13 (m, 3H), 1.03 (s, 3H, H-20), 0.99 (s, 3H, H-18), 0.97 (s, 3H, H-19); 13C NMR (100 MHz, DMSO) d 219.1 (C-15), 216.2 (C-3), 147.4 (C-10 ), 128.7 (C-30 ,50 ), 119.1 (C-40 ), 113.4 (C-20 ,60 ), 74.2 (C-14), 73.0 (C-7), 60.2 (C-9), 52.8 (C-16), 50.3 (C-5), 47.5 (C-13), 46.0 (C-17), 37.9 (C10), 37.3 (C-1), 33.4 (C-2), 30.0 (C-12), 27.1 (C-6), 24.2 (C-18), 20.7 (C-19), 17.8 (C-20); HRMS (ESI) m/z calcd for C26H34ClNO4 [MþNa]þ 482.2069, found 482.2069 (error 0 ppm). 4.3.1.7. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(2-chlorine) aniline3,15-dione (compound 7). White solid, 81% yield, Rf ¼ 0.27 (50:1 DCM/Methanol): 1H NMR (300 MHz, CDC3) d 7.25 (s, 1H, H-30 ), 7.19e7.11 (m, 1H, H-50 ), 6.73e6.62 (m, 2H, H-40 , 60 ), 4.95 (s, 1H), 4.92 (s, 1H, H-14a), 4.80 (s, 1H), 4.26 (s, 1H, H-7b), 3.60 (s, 1H, H-16), 3.27

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(s, 2H, H-17), 2.89 (s, 1H), 2.71 (s, 1H, H-13), 2.60e2.39 (m, 2H, H-2), 1.97e1.79 (m, 5H), 1.72e1.55 (m, 5H), 1.34 (s, 2H), 1.13 (s, 3H, H-20), 1.09 (s, 6H, H-18, 19); 13C NMR (75 MHz, DMSO) d 219.8 (C-15), 216.2 (C-3), 143.8 (C-10 ), 129.1 (C-30 ), 128.2 (C-50 ), 118.3 (C-20 ), 116.8 (C-40 ), 111.1(C-60 ), 74.3 (C-14), 73.0 (C-7), 60.4 (C-9), 52.8 (C-16), 50.3 (C-5), 47.0 (C-13), 46.0 (C-17), 37.9 (C-10), 37.3 (C-1), 33.4 (C-2), 30.0 (C-12), 27.1 (C-6), 24.2 (C-18), 20.6 (C-19), 17.7 (C-20); HRMS (ESI) m/z calcd for C26H34ClNO4 [MþNa]þ 482.2075, found 482.2069 (error 1.24 ppm). 4.3.1.8. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-bromine) aniline3,15-dione (compound 8). White solid, 60% yield, Rf ¼ 0.28 (50:1 DCM/Methanol): 1H NMR (400 MHz, DMSO) d 7.21 (d, J ¼ 8.4 Hz, 2H, H-30 ,50 ), 6.56 (d, J ¼ 8.4 Hz, 2H, H-20 , 60 ), 6.01 (s, 1H), 5.94 (d, J ¼ 5.2 Hz, 1H), 5.84 (d, J ¼ 5.6 Hz, 1H), 4.80 (s, 1H, H-14a), 3.96e3.87 (m, 1H, H-7b), 3.43e3.37 (m, 1H, H-16), 3.08e2.97 (m, 2H, H-17), 2.41e2.30 (m, 1H, H-13), 1.84e1.48 (m, 8H), 1.41e1.06 (m, 4H), 1.03 (s, 3H, H-20), 0.99 (s, 3H, H-18), 0.97 (s, 3H, H-19); 13C NMR (100 MHz, DMSO) d 219.1 (C-15), 216.2 (C-3), 147.8 (C-10 ), 131.5 (C30 , 50 ), 114.0 (C-20 ,60 ), 106.5 (C-40 ), 74.2 (C-14), 73.0 (C-7), 60.2 (C-9), 52.8 (C-16), 50.3 (C-5), 47.5 (C-13), 46.0 (C-17), 37.9 (C-10), 37.3 (C1), 33.4 (C-2), 30.0 (C-12), 27.1 (C-6), 24.2 (C-18), 20.7 (C-19), 17.8 (C-20); HRMS (ESI) m/z calcd for C26H34BrNO4 [MþNa]þ 526.1563, found 526.1578 (error 2.85 ppm). 4.3.1.9. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(2-bromine) aniline3,15-dione (compound 9). White solid, 60% yield, Rf ¼ 0.29 (50:1 DCM/Methanol): 1H NMR (400 MHz, DMSO) d 7.42 (d, J ¼ 7.6 Hz, 1H, H-30 ), 7.20 (t, J ¼ 7.6 Hz, 1H, H-50 ), 6.75 (d, J ¼ 8.0 Hz, 1H, H-60 ), 6.55 (t, J ¼ 7.6 Hz, 1H, H-40 ), 6.01 (s, 1H), 5.83 (d, J ¼ 5.6 Hz, 1H), 5.36 (s, 1H), 4.79 (s, 1H, H-14a), 3.96e3.86 (m, 1H, H-7b), 3.50e3.40 (m, 1H, H-16), 3.10e3.01 (m, 1H, H-17a), 2.46 (s, 1H, H-13), 2.40e2.33 (m, 1H, H-17b), 1.96e1.42 (m, 8H), 1.41e1.09 (m, 4H), 1.03 (s, 3H, H-20), 0.99 (s, 3H, H-18), 0.97 (s, 3H, H-19); 13C NMR (100 MHz, CDCl3) d 220.3 (C-15), 216.7 (C-3), 144.8 (C-10 ), 132.7 (C-30 ), 128.7 (C-5), 118.4 (C-40 ), 111.5(C-60 ), 110.3 (C-20 ), 75.1 (C-14), 74.5 (C-7), 61.7 (C9), 53.2 (C-16), 51.7 (C-5), 47.7 (C-13), 46.8 (C-17), 40.6 (C-4), 40.4 (C-8), 38.6 (C-10), 38.0 (C-1), 33.6 (C-2), 29.7 (C-12), 27.9 (C-6), 24.8 (C-18), 21.0 (C-19), 18.4 (C-20); HRMS (ESI) m/z calcd for C26H34BrNO4 [MþNa]þ 526.1563, found 526.1578 (error 2.85 ppm). 4.3.1.10. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-methoxy) aniline3,15-dione (compound 10). White solid, 53% yield, Rf ¼ 0.24 (50:1 DCM/Methanol): 1H NMR (400 MHz, CDCl3) d 6.80 (d, J ¼ 8.8 Hz, 2H, H-20 , 60 ), 6.70 (d, J ¼ 8.8 Hz, 2H, H-30 , 50 ), 4.91 (s, 1H, H-14a), 4.19 (d, J ¼ 8.4 Hz, 1H, H-7b), 3.75 (s, 3H, H-70 ), 3.46e3.37 (m, 1H, H-16), 3.19 (d, J ¼ 6.8 Hz, 2H, H-17), 2.63 (s, 1H, H-13), 2.57e2.37 (m, 2H, H2), 1.97e1.48 (m, 8H), 1.42e1.23 (m, 3H), 1.11 (s, 3H, H-20), 1.06 (s, 6H, H-18, 19); 13C NMR (75 MHz, DMSO) d 219.5 (C-15), 216.3 (C-3), 150.9 (C-40 ), 142.7 (C-10 ), 114.7 (C-20 ,60 ), 113.3 (C-30 ,5), 74.3 (C-14), 73.0 (C-13), 60.3 (C-9), 55.4 (C-70 ), 52.8 (C-16), 50.3 (C-5), 47.6 (C13), 46.1 (C-17), 37.9 (C-10), 37.3 (C-1), 33.4 (C-2), 30.0 (C-12), 27.1 (C-6), 24.2 (C-18), 20.7 (C-19), 17.8 (C-20); HRMS (ESI) m/z calcd for C27H37NO5 [MþNa]þ 478.2564, found 478.2563 (error 0.21 ppm). 4.3.1.11. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-acetyl) aniline3,15-dione (compound 11). White solid, 36% yield, Rf ¼ 0.20 (50:1 DCM/Methanol): 1H NMR (400 MHz, CDCl3) d 7.81 (d, J ¼ 8.8 Hz, 2H, H-30 , 50 ), 6.59 (d, J ¼ 8.8 Hz, 2H, H-20 , 60 ), 5.12 (s, 1H), 4.95 (s, 1H, H14a), 4.87 (s, 1H), 4.24 (d, J ¼ 11.6 Hz, 1H, H-7b), 3.59e3.45 (m, 2H, H-17a), 3.36e3.27 (m, 1H, H-16), 3.22e3.16 (m, 1H, H-17b), 2.65 (s, 1H, H-13), 2.60e2.52 (m, 1H, H-2a), 2.50 (s, 3H, H-80 ), 2.48e2.40 (m, 1H, H-2b), 1.95e1.76 (m, 5H), 1.70e1.57 (m, 5H), 1.42e1.30 (m, 3H), 1.13 (s, 3H, H-20), 1.08 (s, 6H, H-18, 19); 13C NMR (75 MHz, DMSO) d 218.8 (C-15), 216.2 (C-3), 195.1 (C-70 ), 152.6 (C-10 ), 130.6 (C-30 , 50 ),

125.1 (C-40 ), 110.8 (C-20 , 60 ), 74.2 (C-14), 73.0 (C-7), 60.2 (C-9), 52.8 (C-16), 50.3 (C-5), 47.6 (C-13), 46.0 (C-17), 37.9 (C-10), 37.3 (C-1), 33.4 (C-2), 29.9 (C-12), 27.1 (C-6), 25.9 (C-80 ), 24.2 (C-18), 20.6 (C19), 17.7 (C-20); HRMS (ESI) m/z calcd for C28H37NO5 [MþNa]þ 490.2564, found 490.2582 (error 3.67 ppm). 4.3.1.12. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(3-cyan) aniline3,15-dione (compound 12). White solid, 78% yield, Rf ¼ 0.43 (20:1 DCM/Methanol): 1H NMR (300 MHz, CDCl3) d 7.24e7.10 (m, 1H, H50 ), 7.01e6.94 (m, 1H, H-40 ), 6.87e6.79 (m, 2H, H-60 , 20 ), 5.08 (s, 1H), 4.94 (s, 1H, H-14a), 4.22 (d, J ¼ 14.0 Hz, 1H, H-7b), 3.51e3.38 (m, 2H, H-16), 3.29e3.11 (m, 2H, H-17), 2.65 (s, 1H, H-13), 2.58e2.36 (m, 2H, H-2), 1.97e1.53 (m, 9H), 1.42e1.28 (m, 3H), 1.12 (d, J ¼ 2.8 Hz, 3H, H20), 1.07 (d, J ¼ 2.8 Hz, 6H, H-18, 19); 13C NMR (75 MHz, DMSO) d 218.8 (C-15), 216.2 (C-3), 149.1 (C-10 ), 130.1 (C-50 ), 119.6 (C-60 ), 119.0 (C-40 ), 117.0 (C-20 ), 113.8 (C-70 ), 111.8 (C-30 ), 74.2 (C-14), 73.0 (C-7), 60.2 (C-9), 52.8 (C-16), 50.3 (C-5), 47.5 (C-13), 46.0 (C-17), 37.9 (C-10), 37.3 (C-1), 33.3 (C-2), 29.9 (C-12), 27.1 (C-6), 24.2 (C-18), 20.6 (C-19), 17.7 (C-20); HRMS (ESI) m/z calcd for C27H34N2O4 [MþNa]þ 473.2411, found 473.2428 (error 3.59 ppm). 4.3.1.13. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-phenysulfonyl) aniline-3,15-dione (compound 13). White solid, 78% yield, Rf ¼ 0.45 (20:1 DCM/Methanol): 1H NMR (300 MHz, CDCl3) d 7.92 (d, J ¼ 10.8 Hz, 2H, H-80 , 120 ), 7.57e7.44 (m, 3H, H-90 , 100 , 110 ), 7.27e7.14 (m, 3H, H-20 , 30 , 50 ), 6.75 (d, J ¼ 10.8 Hz, 1H, H-60 ), 4.94 (s, 1H, H14a), 4.42 (s, 1H), 4.17 (d, J ¼ 12 Hz, 1H, H-7b), 3.50e3.40 (m, 1H, H16), 3.27e3.08 (m, 2H, H-17), 2.64 (s, 1H, H-13), 2.54e2.35 (m, 2H, H-2), 1.94e1.51 (m, 8H), 1.42e1.27 (m, 3H), 1.09 (s, 3H, H-20), 1.04 (s, 6H, H-18,19); 13C NMR (75 MHz, CDCl3) d 220.4 (C-15), 217.0 (C-3), 148.8 (C-10 ), 142.1 (C-70 ), 141.7 (C-40 ), 133.2 (C-100 ), 130.3 (C-90 ), 129.3 (C-110 ), 127.6 (C-30 ,50 ), 117.4 (C-80 ), 116.3 (C-120 ), 111.0 (C-20 ,60 ), 75.2 (C-14), 74.1 (C-7), 61.3 (C-9), 53.3 (C-16), 51.6 (C-5), 47.7 (C-13), 46.8 (C-17), 40.5 (C-8), 38.5 (C-10), 38.0 (C-1), 33.6 (C-2), 29.8 (C12), 27.8 (C-6), 24.8 (C-18), 20.9 (C-19), 18.3 (C-20); HRMS (ESI) m/z calcd for C32H39NO6S [MþNa]þ 588.2407, found 588.239 (error 2.89 ppm). 4.3.1.14. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-(4-carboxyl) aniline3,15-dione (compound 14). White solid, 43% yield, Rf ¼ 0.10 (20:1 DCM/Methanol): 1H NMR (400 MHz, DMSO) d 12.01 (s, 1H, H-70 ), 7.68 (d, J ¼ 8.4 Hz, 2H, H-30 , 50 ), 6.61 (d, J ¼ 8.4 Hz, 2H, H-20 , 60 ), 6.56 (s, 1H), 6.02 (s, 1H), 5.85 (d, J ¼ 4.0 Hz, 1H), 4.80 (s, 1H, H-14a), 3.93 (d, J ¼ 6.0 Hz, 1H, H-7b), 3.56e3.44 (m, 1H, H-16), 3.19e3.00 (m, 2H, H-17), 2.42e2.29 (m, 1H, H-13), 1.86e1.47 (m, 7H), 1.40e1.29 (m, 1H), 1.23e1.10 (m, 2H), 1.03 (s, 3H, H-20), 0.99 (s, 3H, H-18), 0.97 (s, 3H, H-19); 13C NMR (75 MHz, DMSO) d 218.9 (C-15), 216.2 (C-3), 167.5 (C-70 ), 152.2 (C-10 ), 131.2 (C-30 ,50 ), 117.2 (C-40 ), 110.9 (C-20 , 60 ), 74.2 (C-14), 73.0 (C-7), 60.3 (C-9), 52.8 (C-16), 50.3 (C-5), 47.6 (C13), 46.0 (C-17), 37.9 (C-10), 37.3 (C-1), 33.4 (C-2), 29.9 (C-12), 27.1 (C-6), 24.2 (C-18), 20.7 (C-19), 17.8 (C-20); HRMS (ESI) m/z calcd for C27H35NO6 [MþNa]þ 492.2357, found 492.2358 (error 0.2 ppm). 4.3.1.15. (5b,7a,9b,10a)-7,14-Dihydroxykaur-17-aniline-3,15-dione (compound 15). White solid, 61% yield, Rf ¼ 0.20 (100:1 DCM/ Methanol): 1H NMR (400 MHz, CDCl3) d 7.19 (t, J ¼ 7.6 Hz, 2H, H-30 , 50 ), 6.74 (t, J ¼ 7.2 Hz, 1H, H-40 ), 6.68 (d, J ¼ 8.0 Hz, 2H, H-20 , 60 ), 5.06 (s, 1H, HeOH), 4.92 (s, 1H, H-14a), 4.21 (dd, J ¼ 11.2, 3.2 Hz, 1H, H7b), 3.55 (s, 1H, HeOH), 3.48 (dd, J ¼ 11.2, 6.8 Hz, 1H, H-16), 3.28e3.18 (m, 2H, H-17), 2.64 (s, 1H, H-13), 2.58e2.49 (m, 1H, H-2), 2.47e2.38 (m, 1H, H-2), 1.93e1.86 (m, 2H), 1.84e1.76 (m, 3H), 1.69e1.53 (m, 3H), 1.41e1.29 (m, 3H, H-20), 1.12 (s, 3H, H-18), 1.07 (s, 6H, H-19); 13C NMR (100 MHz, CDCl3) d 221.0 (C-15), 217.5 (C-3), 207.9, 147.9 (C-10 ), 147.4, 129.3 (C-30 , 50 ), 118.3, 118.1 (C-40 ), 113.4 (C20 , 60 ), 75.0 (C-14), 74.9, 73.7 (C-7), 73.4, 61.1 (C-9), 53.1 (C-8), 52.8,

J. Yang et al. / European Journal of Medicinal Chemistry 86 (2014) 235e241

51.4 (C-5), 47.7 (C-16), 46.7 (C-4), 45.9, 40.8 (C-17), 40.4 (C-13), 38.7, 38.3 (C-10), 38.0, 37.9 (C-1), 33.7, 33.5 (C-2), 30.7, 29.7 (C-12), 29.6, 29.3, 27.6 (C-6), 24.7 (C 18), 22.7, 20.9, 20.8 (C-11), 18.3 (C-19), 18.2, 18.1 (C-20), 18.0; LCeMS m/z 426.1 [MþH]. 4.3.1.16. (5 b ,7 a ,9 b ,10 a )-7,14-Dihydroxykaur-17-cyclohexyl-3,15dione (compound 16). White solid, 42% yield, Rf ¼ 0.20 (20:1 DCM/ Methanol): 1H NMR (400 MHz, CDCl3) d 4.98 (s, 1H, H-14a), 3.98 (dd, J ¼ 11.6, 2.8 Hz, 1H, H-7b), 3.40e3.28 (m, 2H, H-17), 3.18 (d, J ¼ 6.0 Hz, 1H, H-16), 3.00e2.92 (m, 2H, H-20 ), 2.54e2.36 (m, 2H, H10, 13), 2.23 (t, J ¼ 11.6 Hz, 2H, H- 60 ), 1.95e1.80 (m, 5H), 1.73e1.52 (m, 6H), 1.56 (d, J ¼ 13.8 Hz, 2H), 1.38e1.14 (m, 6H), 1.08 (s, 3H, H20), 1.05 (d, J ¼ 3.2 Hz, 6H, H-18, 19); 13C NMR (100 MHz, CDCl3) d 218.5 (C-14), 216.6 (C-7), 75.2 (C-9), 73.3 (C-8), 60.5 (C-10 ), 58.8 (C5), 53.3 (C-16), 51.8 (C-17), 46.8 (C-4), 46.5 (C-13), 41.3 (C-10), 40.0 (C-1), 38.4 (C-1), 37.9 (C-2), 33.6 (C-20 , 60 ), 29.5 (C-12), 29.4 (C-6), 28.9 (C-40 ), 27.7 (C-18), 24.9 (C-30 , 50 ), 24.6 (C-11), 20.9 (C-19), 18.2 (C-20). LCeMS m/z 432.2 [MþH]. 4.4. MTT assay in vitro The antiproliferative activities of GLA derivatives were determined using a standard (MTT) e based colorimetric assay. Briefly, cell lines were seeded at a density of 3  103 cells/well in 96-well microtiter plates. After 24 h, exponentially growing cells were exposed to the indicated compounds at final concentrations ranging from 1.25 to 40 mM. After 48 h, cell survival was determined by the addition of an MTT solution (20 mL of 5 mg mL1 MTT in PBS). After 6 h, the medium was removed by aspiration. The cells were dissolved in 150 mL DMSO and optical absorbance was measured at 570 nm on an Elx800 BioTek microplate reader. Survival ratios were expressed in percentages with respect to untreated cells. IC50 values were determined from replicates of 3 wells from at least three independent experiments. 4.5. Analysis of cellular apoptosis KB cells were incubated in 6-well plates (2.5  105 cells/mL). Cells were then treated with compound 1 and GLA at different concentrations for 48 h, and then both adherent and floating cells were collected, washed with PBS for 2 times. Resuspended cells were incubated with 500 mL binding buffer, 5 mL of Annexin V and 5 mL of PI at room temperature for 20 min. Apoptosis was measured immediately using the BD FACS calibur Flow Cytometer. 4.6. In vitro stability assays Chemical stability was tested under physiological pH conditions (10 mM PBS, pH 7.4), at 37
C. Stock solutions of compound 1 and GLA were prepared in DMSO, and each sample was incubated at a final concentration of 1 mM in pre-warmed buffered solutions (final concentration of DMSO: 1% v/v). At regular time points, aliquots were sampled and immediately injected into the HPLC system. For rat plasma stability assays, rat plasma was quickly thawed and diluted to 80% (v/v) with 0.1 M PBS, pH 7.4. Compound stock solution in DMSO was added (final compound concentration: 1.0 mM, DMSO concentration: 1% v/v) and maintained at 37
C. Aliquots of solution were sampled, two volumes of acetonitrile were added and the mixture was centrifuged (4
C, 10,000 g, 10 min) and analyzed by HPLC.

241

4.7. Analysis of compound 1 as prodrug The reactivity of compound 1 and GLA in KB cell lysate was tested in the presence of cysteine to trap thiol-reactive compounds. KB cells (5  106) were lysed mechanically by the freeze/thaw method. Stock solutions (12.5 mM) of compound 1 and GLA were prepared in DMSO immediately before use and added to cell lysate at a final concentration of 500 mM (equal percentage of DMSO in controls), where 10 mM cysteine had been previously added. Incubations were carried out at 37
C. At stated time points (t ¼ 30 min and 1 h) aliquots were sampled, a double volume of acetonitrile was added, and the resulting solutions were centrifuged (4
C, 10,000 g, 10 min) and directly analyzed by LC-HRMS.

Acknowledgments This work was financially supported by grants from the Priority Academic Program Development of Jiangsu Higher Education Institutions, Chinese National Science & Technology Major Project on “Key New Drug Creation and Manufacturing Program” (2009ZX09103-129). The Project is also co-sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry to Chunhua Qiao, Jiangsu province 333 project funding to Chunhua Qiao, and “CXZZ13_0843” to Jing Yang from Jiangsu Province.

Appendix A. Supporting information Supporting information related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.08.061.

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