Design, synthesis and biological evaluation of novel antitumor spirotetrahydrothiopyran–oxindole derivatives as potent p53-MDM2 inhibitors

Bioorganic & Medicinal Chemistry 25 (2017) 5268–5277

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Design, synthesis and biological evaluation of novel antitumor spirotetrahydrothiopyran–oxindole derivatives as potent p53-MDM2 inhibitors Changjin Ji a,b,d, Shengzheng Wang c,d, Shuqiang Chen b,d, Shipeng He a, Yan Jiang b, Zhenyuan Miao b, Jian Li a,⇑, Chunquan Sheng b,⇑ a

School of Pharmacy, East China University of Science & Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China c Department of Medicinal Chemistry, School of Pharmacy, Fourth Military Medical University, 169 Changle West Road, Xi’an 710032, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 4 May 2017 Revised 17 July 2017 Accepted 26 July 2017 Available online 28 July 2017 Keywords: Spirooxindole p53–MDM2 inhibitors Antitumor activity Organocatalytic cascade reaction

a b s t r a c t p53–MDM2 protein-protein interaction is a promising target for novel antitumor drug development. Previously, we identified a new class of spirotetrahydrothiopyran–oxindole p53–MDM2 inhibitors by novel organocatalytic enantioselective cascade reactions. Herein, a series of new derivatives were designed, synthesized and assayed to investigate the structure-activity relationships. Among them, compound B14 bearing a novel spiroindole–thiopyranopyridone scaffold exhibited potent MDM2 inhibitory activity as well as antitumor activity, which could effectively induce the apoptosis of A549 cancer cells. It represents a promising lead compound for the development of novel antitumor agents. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction The p53 tumor suppressor is a potent transcription factor that plays a crucial role in DNA repair, differentiation, senescence, apoptosis, and cell-cycle arrest.1 It has been one of the most intensively studied tumor suppressors over the past 20 years. In
50% human cancers, p53 is mutated and inactivated, implying the importance of disabling p53 function during tumorigenesis.2 For the remaining half of the cancers, p53 retains its wild type form, whose and abundance and activity are tightly regulated under normal physiologic conditions. By directly binding to the N-terminus of p53 and promoting p53 ubiquitination and degradation, murine double-minute 2 (MDM2) oncogene is an essential negative regulator of the p53 tumor suppressor.3 Moreover, MDM2 functions as an E3 ligase that ubiquitinates p53 and promotes its proteasomal degradation.4 As a result, stabilization and activation of p53 function by the disruption of the p53–MDM2 interaction has been explored as an attractive approach for the treatment of cancers.5 Up to now, numerous small molecule inhibitors of p53–MDM2 interaction have been reported that significantly reduce the tumor

growth both in vitro and in vivo, and several of them have advanced into clinical trials for cancer therapy (Fig. 1A).6 Continuing our work in discovering potent p53–MDM2 inhibitors,7 our group developed a one-step organocatalytic enantioselective Michael–Michael cascade reaction to construct a novel spirotetrahydrothiopyran–oxindole scaffold (Fig. 1B).8 Interestingly, the new scaffold displayed good antitumor activity, which was further confirmed as a new type of p53–MDM2 inhibitor. Particularly, compound 1 (Fig. 1B) showed potent MDM2 inhibitory activity as well as antitumor potency, which represented a promising lead compound for antitumor drug discovery. Thus, it is highly desirable to investigate the structure-activity relationship (SAR) of this type of novel scaffold. In order to achieve deeper understanding of the chemical space around this novel spirooxindole scaffold, a series of new derivatives were designed, synthesized and assayed. Most target compounds showed improved antitumor activity and new SAR information of this scaffold was also obtained.

2. Chemistry ⇑ Corresponding authors. d

E-mail addresses: [email protected] (J. Li), [email protected] (C. Sheng). These three authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bmc.2017.07.049 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.

The synthetic route for the target compounds is shown in Scheme 1. Lead compound 1 was prepared according to the procedures from our previous study.8 Staring from 1, derivatives A1 and

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Fig. 1. (A) Representative p53–MDM2 inhibitors in clinical trials. (B) Design rationale of the spirotetrahydrothiopyran–oxindoles as novel p53–MDM2 inhibitors.

A2 were prepared by reductive amination. Then, intramolecular ring closing reaction was performed to obtain derivatives B1–B14 in the presence of CF3COOH at room temperature. For investigating the effect of the side chain’s polarity on the antitumor activity, compound C1 was prepared by reducing the ester and aldehyde groups of 1 to the hydroxy groups in the presence of diisobutylaluminium hydride (DIBAL-H).

ester, and bromophenyl groups bound to the Leu26, Phe19, and Trp23 hot spots of MDM2, respectively (Fig. 1B). Because the aldehyde and ester group in lead compound 1 were unstable, they were transformed to afford a series of new derivatives (Fig. 1C). First, the aldehyde group was replaced by aromatic amine groups (A1-A2). Then, a novel spiroindole-thiopyranopyridone scaffold (B1–B14) was designed by ring closure between the ester group and the amine side chain. Finally, both ester and aldehyde group were reduced to the hydroxyl group (C1).

3. Results and discussion 3.2. In vitro antitumor activity and structure-activity relationships 3.1. Design of new spirotetrahydrothiopyran–oxindole derivatives Molecular docking simulation indicated that compound 1 was well-fitted into the MDM2 binding cavity with its oxindole, methyl

The target compounds were assayed for in vitro antitumor activity against the three types of human cancer cell lines with p53 in its wild type (A549 lung carcinoma cell, HCT116 colon cancer cell,

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Scheme 1. Reagents and conditions: (a) FeCl3, AcCl, DCM, rt, yield = 33.0%; (b) (Et)3N, DCM, N2, rt, yield 63.6%; (c) 20 mol% catalyst, DCM, PhCO2H, rt, 3 d, yield 61.0%; (d) AcOH, MeOH, rt, 12 h; (e) NaBH3 CN, rt, 1 h, two steps (d and e) yields 15.5–17.2%; (f) CF3COOH, DCM, rt, 3 h, three steps (d to f) yields 12.5–40.0%; (g) DIBAL-H, DCM, 78 °C–rt, N2, 2 h, yield 41.2%.

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and MCF-7 breast cancer cell) using the standard CCK-8 method. Nutlin-39 (a representative p53-MDM2 inhibitor) and lead compound 1 were used as reference drugs. As summarized in Table 1, derivatives A1–A2 and B1–B14 generally displayed better in vitro antitumor activity than Nutlin-3 and lead compound 1. For the A549 cell line, these compounds displayed potent antitumor activity with IC50 values in the range of 2.3–6.3 lM, which were more active than Nutlin-3 (IC50 = 10.0 lM). Meanwhile, they also showed good antitumor activity against the MCF-7 cell line with IC50 values ranging from 1.8 lM to 6.9 lM, whereas Nutlin-3 had an IC50 value of 14.9 lM. For the HCT116 cell line, most derivatives displayed decreased activities (IC50 range: 2.4–10.5 lM). Among the synthesized compounds, B13 and B14 with larger bulky hydrophobic groups were the most active spirooxindoles with IC50 values in the range of 1.8–4.3 lM against all the three cancer cell lines. In contrast, compound C1, bearing two hydroxy groups in the side chain, showed decreased in vitro antitumor activity (IC50 range: 13.5–19.1 lM), indicating that the hydrophilic side chains were not beneficial for improving the antitumor activity. Taken together, these results highlighted the importance of a hydrophobic side chain on the antitumor activity.

3.3. p53-MDM2 inhibitory activity and binding mode All the synthesized compounds were assayed for the MDM2 inhibitory activity using the fluorescence polarization assay.7 b As shown in Table 1, these compounds exhibited moderate to good binding affinity to MDM2 with the range of KD values from 2.0 lM to 11.7 lM. Moreover, B13 and B14 exhibited the best binding affinity with KD values of 2.0 lM and 4.2 lM (Fig. 2), respectively, which were more potent than lead compound 1 (KD = 4.9 lM). However, they were still less potent than Nutlin-3 (KD = 0.15 lM). Furthermore, we investigated whether B14 inhibited the p53-MDM2 interaction in the cancer cells by Western blotting analysis. Similar to nutlin-3, compound B14 showed a dosedependent up-regulation of p53 and MDM2 expressions in A549 cells. (Fig. 3). To investigate the binding modes between compounds B13 and B14 with MDM2, molecular docking was performed using our well-established protocols.8 As depicted in Fig. 4, both compounds were observed to be well-fitted into the p53-binding domain of MDM2. The oxindole part was located into the Leu26 pocket of MDM2 and formed hydrophobic interactions with surrounding

residues such as Leu54, Ile99, Tyr100 and His96. Moreover, the tetrahydrothiopyran-lactam ring was located into the Phe19 pocket and formed hydrophobic interactions with Met62 and Tyr67. Meanwhile, additional hydrophobic interaction was observed between the hydrophobic side chain attached to the lactam ring and Met62 and Gly58. Notably, the p-bromophenyl group was only partly inserted into the Trp23 pocket of MDM2, which remained to be further optimized to improve the binding affinity. In addition, no hydrogen bonds were observed, which should be considered for lead optimization. 3.4. Effect of compound B14 on apoptosis in A549 cells To test the effect of novel spirooxindole derivatives on the induction of apoptosis, compound B14 was evaluated in A549 cancer cells by fluorescence-activated cell sorting. Nutlin-3 was used as the control (Fig. 5). After treating with 5 lM or 10 lM of compound B14 for 48 h, the percentage of apoptotic cells was 18.48% and 34.11%, respectively, which was comparable to Nutlin-3 (22.12% for 5 lM and 32.00% for 10 lM). In contrast, the percentage of apoptotic cells in the untreated control group and DMSO groups was 9.67% and 9.14%, respectively. These results indicated that this novel spirooxindole scaffold could effectively induce the apoptosis of A549 cancer cells. 4. Conclusions In summary, a series of novel antitumor oxindole–spirotetrahydrothiopyran derivatives were designed and synthesized. Most of them showed better antitumor activity than Nutlin-3 and lead compound 1. In particular, compounds B13 and B14 exerted good antitumor activity by inhibition of p53–MDM2 protein-protein interaction. Moreover, compound B14 could effectively induce the apoptosis of cancer cells. The obtained SAR and binding mode can provide useful information for lead optimization. 5. Experimental section 5.1. Chemistry All reagents and solvents were reagent grade or were purified by standard methods before use. 1H NMR and 13C NMR spectra were recorded on Bruker AVANCE600 spectrometer (Bruker Com-

Table 1 In vitro antitumor activity of the target compounds (MDM2 KD and cell IC50, lM).

a

Compounds

KDa

A549a

MCF-7a

HCT116a

A1 A2 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 C1 1 Nutlin-3

11.7 ± 4.4 3.4 ± 0.6 4.1 ± 1.2 4.7 ± 0.3 4.2 ± 0.9 4.1 ± 0.8 3.4 ± 0.2 3.4 ± 0.3 4.0 ± 0.7 5.2 ± 1.8 6.4 ± 0.7 4.8 ± 1.1 3.0 ± 0.6 3.0 ± 0.7 2.0 ± 0.7 4.2 ± 1.4 10.1 ± 2.6 4.9 ± 1.6 0.15 ± 0.04

4.2 ± 0.7 3.5 ± 0.2 2.3 ± 0.6 3.5 ± 0.6 4.8 ± 0.9 4.2 ± 0.2 4.4 ± 0.8 2.6 ± 0.1 4.5 ± 0.4 4.6 ± 0.5 4.3 ± 0.5 2.5 ± 0.2 4.1 ± 0.6 6.3 ± 1.2 2.6 ± 0.4 2.9 ± 0.2 16.5 ± 1.6 11.2 ± 0.8 10.0 ± 0.8

3.6 ± 0.4 2.8 ± 0.2 4.6 ± 0.9 3.8 ± 0.7 4.2 ± 0.1 4.0 ± 0.4 4.0 ± 0.4 3.2 ± 0.2 3.6 ± 0.3 3.0 ± 0.6 3.0 ± 0.5 4.4 ± 0.8 6.9 ± 1.0 5.9 ± 1.3 2.4 ± 0.4 1.8 ± 0.5 13.5 ± 1.0 8.5 ± 0.7 14.9 ± 0.6

10.5 ± 1.7 8.6 ± 0.7 9.3 ± 0.7 9.5 ± 1.1 8.0 ± 0.9 8.6 ± 0.8 10.5 ± 1.9 10.4 ± 2.3 8.5 ± 0.6 5.1 ± 0.7 8.3 ± 0.6 9.8 ± 0.4 5.6 ± 0.9 8.8 ± 1.5 4.3 ± 0.6 2.4 ± 0.1 19.1 ± 2.6 17.3 ± 2.2 28.4 ± 2.5

Activity data are reported as the mean and standard errors of at least three runs.

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Fig. 2. MDM2 binding affinity of Nutlin-3 (A), lead compound 1 (B), compounds B13 (C) and B14 (D).

entific Instrument Co., LTD) with a 0.8 mL cell (c given in g/100 mL). Lead compound 1 was prepared according to our previously reported procedure.8

Fig. 3. Cellular activity of nutlin-3 and B14 for the p53 pathway activation to upregulate p53 and MDM2 detected by Western Blot in A549 cells.

pany, Germany), using TMS as an internal standard and CDCl3 or DMSO-d6 as solvents. Multiplicities were given as: s (singlet), d (doublet), t (triplet), dd (double of doublet) or m (multiplets). Chemical shifts (d values) and coupling constants (J values) are given in ppm and Hz, respectively. High resolution mass spectrometry (HRMS) was recorded on an Agilent 6538 UHD Accurate-Mass Q-TOF LC/MS spectrometer. TLC analysis was carried out on silica gel plates GF254 (Qingdao Haiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 60G (Qingdao Haiyang Chemical, China). Enantioselectivities were determined by High performance liquid chromatography (HPLC) analysis employing a Daicel Chiralpak AD-H. Optical rotations were measured in DCM on a SGW-1 automatic polarimeter (Shanghai Precision Sci-

5.1.1. General procedure for the preparation of compounds A1–A2 To a stirred solution of lead compound 1 (138 mg, 0.25 mmol, 1.0 equiv) in MeOH (10 mL) was added amine (0.375 mmol, 1.5 equiv) and AcOH (2 drops, catalytic amount). The resulted solution was stirred at room temperature overnight. Then sodium cyanoborohydride (48 mg, 0.75 mmol, 3.0 equiv) was added and the resulting mixture was stirred at room temperature for another 1 h. Then the solvent was evaporated under the reduced pressure. The residue was diluted with EtOAc (40 mL) and H2O (40 mL). The organic layer was separated, washed with saturated NaHCO3 (30 mL) and saturated NaCl (30 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, DCM/ MeOH = 100:1, v/v). 5.1.2. Methyl 2-((3R,30 S,40 S,50 S)-4-bromo-30 -(4-bromophenyl)-40 -(((2fluorophenyl)amino)methyl)-2-oxo-30 ,40 ,50 ,60 -tetrahydrospiro[indoline3,20 -thiopyran]-50 -yl) acetate (A1) White solid (25 mg), yield: 15.5%. Rf = 0.35 (DCM/MeOH = 100:5); 1H NMR (600 MHz, CDCl3) d 7.27–7.32 (m, 1H), 7.21–7.26 (m, 1H), 7.12–7.18 (m, 1H), 7.10 (d, J = 7.8 Hz, 1H), 6.90–6.96 (m, 3H), 6.82 (t, J = 7.8 Hz, 1H), 6.54–6.59 (m, 1H), 6.52 (d, J = 7.8 Hz, 1H), 6.17 (d, J = 8.4, 1H), 4.52 (d, J = 12.0 Hz, 1H), 3.87 (dd, J = 13.2, 11.4 Hz, 1H), 3.07–3.13 (m, 1H), 3.02 (dd, J = 13.2, 2.4 Hz,

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Fig. 4. Schematic representation of the proposed binding mode for compound B13 (A) and B14 (B) in the p53 binding domain of MDM2. Three key hotpots of MDM2 are labeled red color. The figures were generated using PyMol (http://pymol.sourceforge.net/) and Schrodinger.

1H), 2.78–2.87 (m, 2H), 2.64–2.72 (m, 1H), 2.58 (dd, J = 13.8, 3.0 Hz, 1H), 2.45 (dd, J = 15.6, 7.2 Hz, 1H). 13C NMR (150 MHz, CDCl3) d 177.21, 172.61, 152.28 (d, JC-F = 237.0 Hz, 1H), 150.99, 141.42, 137.55, 136.53, 136.49 (d, JC-F = 12.0 Hz, 1H), 132.41, 131.74, 131.11, 130.51, 127.61, 127.51, 126.75, 124.38, 121.31, 119.67, 116.82 (d, JC-F = 6.6 Hz, 1H), 114.28 (d, JC-F = 18.3 Hz, 1H), 112.44, 108.85, 52.08, 51.90, 46.44, 43.20, 39.19, 38.94, 37.64, 28.91; HRMS (ESI): C28H25Br2FN2O3S requires m/z 645.9937, found 644.9939 [MH]; [a]25 D = +62.7 (c = 0.27 in DCM). 5.1.3. Methyl 2-((3R,30 S,40 S,50 S)-4-bromo-30 -(4-bromophenyl)-40 -(((3fluorophenyl)amino)methyl)-2-oxo-30 ,40 ,50 ,60 -tetrahydrospiro[indoline3,20 -thiopyran]-50 -yl) acetate (A2) White solid (30 mg), yield: 17.2%. Rf = 0.35 (DCM/MeOH = 100:5); 1H NMR (600 MHz, CDCl3) d 7.53 (s, 1H), 7.29–7.34 (m, 1H), 7.21–7.26 (m, 1H), 7.10 (t, J = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.98 (q, J = 7.8 Hz, 1H), 6.94 (t, J = 7.8 Hz, 1H), 6.54 (d, J = 7.8 Hz, 1H), 6.31 (td, J = 8.4, 1.8 Hz, 1H), 6.11 (dd, J = 8.4,

1.8 Hz, 1H), 5.97 (d, J = 12.0 Hz, 1H), 4.49 (d, J = 11.4 Hz, 1H), 3.86 (t, J = 12.6 Hz, 2H), 3.66 (s, 3H), 3.05 (t, J = 10.8 Hz, 1H), 2.94 (d, J = 13.2 Hz, 1H), 2.80–2.84 (m, 1H), 2.78 (dd, J = 15.6, 4.8 Hz, 1H), 2.60 (dd, J = 13.8, 3.0 Hz, 1H), 2.47 (dd, J = 15.6, 7.2 Hz, 1H). 13 C NMR (150 MHz, CDCl3) d 177.26, 172.92, 163.93 (d, JC-F = 241.5 Hz, 1H), 149.94 (d, JC-F = 10.5 Hz, 1H), 149.98, 149.91, 141.45, 137.51, 132.54, 131.75, 131.15, 130.54, 130.03 (d, JC-F = 10.0 Hz, 1H), 130.00, 127.62, 127.49, 126.76, 121.39, 119.66, 109.16, 108.89, 104.02 (d, JC-F = 21.4 Hz, 1H), 99.94 (d, JC-F = 25.0 Hz, 1H), 56.42, 52.12, 51.99, 46.54, 43.68, 39.24, 38.95, 37.66, 29.02; HRMS (ESI): C28H25Br2FN2O3S requires m/z 645.9937, found 644.9939 [MH]; [a]25 D = +121.1 (c = 0.12 in DCM). 5.1.4. General procedure for the preparation of compounds B1–B14 To a stirred solution of lead compound 1 (166 mg, 0.3 mmol, 1.0 equiv) in MeOH (10 mL) were added amine (0.45 mmol, 1.5 equiv) and AcOH (2 drops, catalytic amount). The resulting solution was

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Fig. 5. Nutlin-3 and compound B14 induced cell apoptosis. A549 cells were treated with DMSO or Nutlin-3 (5 lM or 10 lM) or B14 (5 lM or 10 lM) for 48 h. Apoptosis was examined by flow cytometry (n = 3). Representative photographs from three independent experiments are displayed.

stirred at room temperature overnight. Then sodium cyanoborohydride (57 mg, 0.9 mmol, 3.0 equiv) was added and the mixture was stirred at room temperature for another 1 h. Then the solvent was evaporated under reduced pressure. The residue was diluted with EtOAc (40 mL) and H2O (40 mL). The organic layer was separated, washed with saturated NaHCO3 (30 mL) and saturated NaCl solution (30 mL), dried over anhydrous Na2SO4 and concentrated under the reduced pressure. The residue was used for the next step without purification. It was diluted with DCM (10 mL) and TFA (0.75 mL) was added and the resulting solution was stirred at room temperature for 3 h. Then the reaction mixture was diluted with of DCM (20 mL), washed with saturated NaHCO3 (20 mL) and NaCl (20 mL) solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, DCM/MeOH = 100:2, v/v). 5.1.5. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(2-fluorophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c] pyridine]-2,70 (10 H)-dione (B1) White solid (60 mg), yield: 32.5%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.80 (s, 1H), 7.46 (dd, J = 8.4, 1.8 Hz, 1H), 7.26–7.33 (m, 3H), 7.22 (td, J = 7.8, 1.8 Hz, 2H), 7.16 (m, 1H), 7.12 (dd, J = 8.4, 0.6 Hz, 1H), 7.05 (t, J = 8.4 Hz, 1H), 7.03 (dd, J = 8.4, 1.8 Hz, 1H), 6.63 (dd, J = 7.8, 0.6 Hz, 1H), 4.12 (d, J = 10.8 Hz, 1H), 3.59–3.65 (m, 1H), 3.33–3.38 (m, 1H), 3.29–3.34 (m, 1H), 2.68–2.75 (m, 3H), 2.39–2.46 (m, 1H),

2.32–2.39 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.72, 168.47, 157.70 (d, JC-F = 165.8 Hz, 1H), 141.79, 136.23, 132.20, 131.95, 131.26, 130.76, 129.66 (d, JC-F = 8.6 Hz, 1H), 129.25 (d, JC-F = 4.9 Hz, 1H), 129.08, 127.55, 127.19, 126.14, 124.73, 121.74, 119.56, 116.73 (d, JC-F = 13.2 Hz, 1H), 109.09, 63.54, 55.68, 52.05, 47.07, 38.49, 37.80, 35.49, 28.62; HRMS (ESI): C27H21Br2FN2O2S requires m/z 613.9675, found 612.9676 [MH]; [a]25 D = +48.2 (c = 0.40 in DCM). 5.1.6. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(3-fluorophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a-hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c] pyridine]-2,70 (10 H)-dione (B2) White solid (63 mg), yield: 34.0%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, CDCl3) d 7.48 (s, 1H), 7.32 (dd, J = 8.4, 1.8 Hz, 1H), 7.24–7.29 (m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 7.10 (dd, J = 8.4, 1.8 Hz, 1H), 7.00 (dd, J = 8.4, 1.8 Hz, 1H), 6.97 (t, J = 7.8 Hz, 1H), 6.85–6.94 (m, 3H), 6.55 (d, J = 7.2 Hz, 1H), 4.16 (d, J = 10.8 Hz, 1H), 3.78 (t, J = 12.0 Hz, 1H), 3.36–3.47 (m, 1H), 3.26 (t, J = 12.0 Hz, 1H), 3.04 (dd, J = 12.0, 4.2 Hz, 1H), 2.83 (t, J = 16.2 Hz, 1H), 2.56 (d, J = 13.8 Hz, 2H), 2.40–2.51 (m, 1H). 13C NMR (150 MHz, CDCl3): d 176.62, 168.38, 162.84 (d, JC-F = 163.9 Hz, 1H), 143.87 (d, JC-F = 6.4 Hz, 1H), 141.73, 136.22, 132.30, 131.98, 131.39, 130.83, 130.26 (d, JC-F = 9.0 Hz, 1H), 127.66, 127.12, 126.18, 121.88, 121.82, 119.65, 114.04 (d, JC-F = 6.0 Hz, 1H), 113.82 (d, JC-F = 6.0 Hz, 1H), 109.05, 56.07, 52.09, 47.17, 38.99, 37.98, 35.72, 28.56; HRMS (ESI): C27H21Br2FN2O2S

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requires m/z 613.9675, found 612.9677 [MH]; [a]25 D = +61.3 (c = 0.25 in DCM). 5.1.7. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-chlorophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c] pyridine]-2,70 (10 H)-dione (B3) White solid (53 mg), yield: 27.9%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.75 (s, 1H), 7.40 (dd, J = 8.4, 1.8 Hz, 1H), 7.31–7.36 (m, 2H), 7.24 (dd, J = 8.4, 2.4 Hz, 1H), 7.16–7.21 (m, 3H), 7.16–7.21 (m, 3H), 7.09 (dd, J = 8.4, 1.2 Hz, 1H), 7.02 (t, J = 7.8 Hz, 1H), 6.98–7.01 (m, 1H), 6.60 (dd, J = 7.8, 1.2 Hz, 1H), 4.08 (d, J = 10.8 Hz, 1H), 3.54–3.60 (m, 1H), 3.40 (t, J = 10.8 Hz, 1H), 3.25–3.30 (m, 1H), 2.70 (dd, J = 10.8, 4.8 Hz, 1H), 2.61–2.67 (m, 2H), 2.36–2.44 (m, 1H), 30–2.36 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.67, 168.48, 141.71, 140.84, 136.18, 132.65, 132.26, 131.91, 131.38, 130.81, 129.39, 127.61, 127.51, 127.04, 126.09, 121.85, 119.55, 109.09, 56.09, 52.02, 47.09, 38.79, 37.90, 35.64, 28.57; HRMS (ESI): C27H21Br2ClN2O2S requires m/z 629.9379, found 629.9379 [MH]; [a]25 D = +88.2 (c = 0.17 in DCM). 5.1.8. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -phenyl-40 ,4a0 , 50 ,60 ,80 ,8a0 -hexahydro-spiro[indoline-3,30 -thiopyrano[4,3-c]pyridine]2,70 (10 H)-dione (B4) White solid (51 mg), yield: 28.4%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.77 (s, 1H), 7.42 (dd, J = 8.4, 2.4 Hz, 1H), 7.30 (t, J = 7.8 Hz, 2H), 7.27 (dd, J = 8.4, 1.8 Hz, 1H), 7.21 (dd, J = 8.4, 1.8 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 7.15 (dd, J = 8.4, 0.6 Hz, 2H), 7.11 (dd, J = 7.8, 1.2 Hz, 1H), 7.04 (t, J = 7.8 Hz, 1H), 7.02 (dd, J = 7.8, 2.4 Hz, 1H), 6.62 (dd, J = 7.8, 0.6 Hz, 1H), 4.10 (d, J = 11.4 Hz, 1H), 3.60 (t, J = 12.0 Hz, 1H), 3.41 (t, J = 11.4 Hz, 1H), 3.26–3.34 (m, 1H), 2.74 (dd, J = 11.4, 4.8 Hz, 1H), 2.63–2.70 (m, 2H), 2.38–2.44 (m, 1H), 2.31–2.37 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.84, 168.54, 142.40, 141.90, 136.28, 132.19, 131.93, 131.27, 130.74, 129.26, 127.46, 127.10, 126.25, 121.74, 119.48, 109.14, 56.24, 52.08, 47.11, 38.81, 37.89, 35.61, 28.57; HRMS (ESI): C27H22Br2N2O2S requires m/z 595.9769, found 594.9767 [MH]; [a]25 D = +85.8 (c = 0.31 in DCM). 5.1.9. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-fluorophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c] pyridine]-2,70 (10 H)-dione (B5) White solid (74 mg), yield: 40.0%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.78 (s, 1H), 7.42 (dd, J = 8.4, 2.4 Hz, 1H), 7.27 (dd, J = 8.4, 2.4 Hz, 1H), 7.18–7.23 (m, 3H), 7.10–7.15 (m, 3H), 7.04 (t, J = 7.8 Hz, 1H), 7.01 (dd, J = 8.4, 2.4 Hz, 1H), 6.62 (dd, J = 7.8, 0.6 Hz, 1H), 4.10 (d, J = 10.8 Hz, 1H), 3.59 (t, J = 12.0 Hz, 1H), 3.39 (t, J = 11.4 Hz, 1H), 3.27–3.34 (m, 1H), 2.61–2.73 (m, 3H), 2.38–2.43 (m, 1H), 2.30–2.36 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.84, 168.70, 162.04, 160.40, 141.91, 138.26, 136.24, 132.21, 131.94, 131.35, 130.78, 128.02, 127.96, 127.51, 127.07, 126.10, 121.79, 119.49, 116.24, 116.09, 109.16, 56.42, 52.05, 47.10, 38.75, 37.86, 35.62, 28.55; HRMS (ESI): C27H21Br2FN2O2S requires m/z 613.9675, found 612.9674 [MH]; [a]25 D = +87.2 (c = 0.27 in DCM). 5.1.10. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-ethoxyphenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3c]pyridine]-2,70 (10 H)-dione (B6) White solid (66 mg), yield: 34.3%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.77 (s, 1H), 7.43 (dd, J = 8.4, 1.8 Hz, 1H), 7.26 (dd, J = 7.8, 1.8 Hz, 1H), 7.21 (dd, J = 8.4, 1.8 Hz, 1H), 7.11 (dd, J = 7.8, 1.2 Hz, 1H), 6.99–7.06 (m, 3H), 6.82 (d, J = 9.0 Hz, 2H), 6.62 (dd, J = 7.8, 1.2 Hz, 1H), 4.04 (d, J = 10.8 Hz, 1H), 3.96 (q, J = 7.2, 1H), 3.58 (t, J = 12.6 Hz, 1H), 3.31–3.36 (m, 1H), 3.24–3.32 (m, 1H), 2.68 (td, J = 12.6, 3.6 Hz,

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2H), 2.63 (dd, J = 10.8, 4.8 Hz, 1H), 2.34–2.43 (m, 1H), 2.27–2.34 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) d 176.93, 168.84, 157.78, 142.05, 136.37, 135.00, 132.16, 131.95, 131.27, 130.71, 127.35, 127.15, 126.14, 121.70, 119.42, 115.07, 109.22, 63.60, 56.63, 52.07, 47.16, 38.75, 37.86, 35.58, 28.57, 14.78; HRMS (ESI): C29H26Br2N2O3S requires m/z 640.0031, found 639.0030 [MH]; [a]25 D = +84.0 (c = 0.20 in DCM). 5.1.11. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(3-ethoxy phenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3c]pyridine]-2,70 (10 H)-dione (B7) White solid (69 mg), yield: 35.8%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.78 (s, 1H), 7.44 (dd, J = 8.4, 2.4 Hz, 1H), 7.28 (dd, J = 8.4, 2.4 Hz, 1H), 7.23 (dd, J = 7.8, 1.8 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 7.12 (dd, J = 7.8, 0.6 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.4, 1.8 Hz, 1H), 6.73–6.77 (m, 2H), 6.69–6.71 (m, 1H), 6.63 (dd, J = 7.8, 0.6 Hz, 1H), 4.11 (d, J = 10.8 Hz, 1H), 3.96 (qd, J = 7.2, 1.8 Hz, 1H), 3.57–3.62 (m, 1H), 3.40 (t, J = 11.4 Hz, 1H), 3.28–3.31 (m, 1H), 2.70–2.75 (m, 1H), 2.63–2.70 (m, 2H), 2.37–2.43 (m, 1H), 2.31–2.37 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) d 176.64, 168.29, 159.57, 143.56, 141.68, 136.25, 132.22, 131.94, 131.31, 130.76, 129.91, 127.59, 127.11, 126.17, 121.77, 119.58, 118.30, 113.22, 112.89, 109.04, 63.54, 56.25, 52.05, 47.12, 38.89, 37.91, 35.62, 28.61, 14.75; HRMS (ESI): C29H26Br2N2O3S requires m/z 640.0031, found 639.0030 [MH]; [a]25 D = +74.1 (c = 0.25 in DCM). 5.1.12. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(p-tolyl)-40 , 4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c]pyridine]2,70 (10 H)-dione (B8) White solid (72 mg), yield: 39.2%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.78 (s, 1H), 7.43 (dd, J = 8.4, 2.4 Hz, 1H), 7.27 (dd, J = 8.4, 2.4 Hz, 1H), 7.22 (dd, J = 7.8, 1.8 Hz, 1H), 7.19 (t, J = 9.0 Hz, 3H), 7.12 (dd, J = 7.8, 0.6 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 7.01–7.06 (m, 4H), 6.63 (d, J = 7.2 Hz, 1H), 4.11 (d, J = 10.8 Hz, 1H), 3.57–3.63 (m, 1H), 3.37 (t, J = 11.4 Hz, 1H), 3.25–3.32 (m, 1H), 2.62–2.72 (m, 3H), 2.36–2.44 (m, 1H), 2.30–2.36 (m, 1H), 2.24 (s, 3H). 13C NMR (150 MHz, CDCl3) d 176.74, 168.50, 141.77, 139.87, 136.99, 136.30, 132.20, 131.93, 131.29, 130.74, 129.93, 127.54, 127.13, 126.18, 126.07, 121.74, 119.54, 109.08, 56.36, 52.06, 47.15, 38.84, 37.93, 35.61, 28.62, 21.01; HRMS (ESI): C28H24Br2N2O2S requires m/z 609.9925, found 608.9923 [MH]; [a]25 D = +87.9 (c = 0.21 in DCM). 5.1.13. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-methoxyphenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3c]pyridine]-2,70 (10 H)-dione (B9) White solid (55 mg), yield: 29.2%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.77 (s, 1H), 7.43 (dd, J = 8.4, 2.4 Hz, 1H), 7.26 (dd, J = 8.4, 2.4 Hz, 1H), 7.21 (dd, J = 8.4, 2.4 Hz, 1H), 7.11 (dd, J = 7.4, 0.6 Hz, 1H), 7.00–7.07 (m, 4H), 6.84 (d, J = 9.0 Hz, 2H), 6.62 (dd, J = 7.8, 1.2 Hz, 1H), 4.09 (d, J = 10.8 Hz, 1H), 3.59 (dd, J = 13.2, 11.4 Hz, 1H), 3.33 (t, J = 11.4, 1H), 3.25–3.32 (m, 1H), 2.61–2.72 (m, 4H), 2.35–2.44 (m, 1H), 2.28–2.35 (m, 1H). 13C NMR (150 MHz, CDCl3) d176.68, 168.64, 158.35, 141.79, 136.33, 135.24, 132.19, 131.94, 131.29, 130.74, 127.52, 127.39, 127.13, 126.17, 121.73, 119.53, 114.56, 109.08, 56.58, 55.40, 52.04, 47.16, 37.90, 35.60, 28.61; HRMS (ESI): C28H24Br2N2O3S requires m/z 625.9874, found 624.9872 [MH]; [a]25 D = +56.4 (c = 0.30 in DCM). 5.1.14. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(2,4-difluorophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c] pyridine]-2,70 (10 H)-dione (B10) White solid (46 mg), yield: 24.2%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6)] d 10.80 (s, 1H), 7.46 (dd,

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J = 8.4, 2.4 Hz, 1H), 7.35–7.40 (m, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.25– 7.30 (m, 2H), 7.22 (dd, J = 8.4, 2.4 Hz, 1H), 7.12 (dd, J = 8.4, 0.6 Hz, 1H), 7.01–7.08 (m, 3H), 6.63 (dd, J = 8.4, 0.6 Hz, 1H), 4.11 (d, J = 10.8 Hz, 1H), 3.58–3.64 (m, 1H), 3.33–3.38 (m, 1H), 3.28–3.33 (m, 1H), 2.66–2.74 (m, 3H), 2.38–2.45 (m, 1H), 2.32–2.38 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.86, 168.74, 161.07, 161.00, 141.92, 136.20, 132.21, 131.95, 131.31, 130.79, 129.92, 129.85, 127.52, 127.13, 126.09, 121.78, 119.52, 111.88, 109.15, 105.17, 55.81, 52.04, 47.07, 38.42, 37.75, 37.75, 28.62; HRMS (ESI): C27H20Br2F2N2O2S requires m/z 631.9580, found 630.9581 [MH]; [a]25 D = +132.5 (c = 0.15 in DCM). 5.1.15. (3R,40 S,4a0 S,8a0 S)-60 -([1,10 -Biphenyl]-4-yl)-4-bromo-40 -(4-bromophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano [4,3-c]pyridine]-2,70 (10 H)-dione (B11) White solid (54 mg), yield: 26.7%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.76 (s, 1H), 7.59 (d, J = 7.2 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 7.8 Hz, 3H), 7.33 (d, J = 7.8 Hz, 1H), 7.27 (dd, J = 8.4, 1.8 Hz, 1H), 7.23 (d, J = 7.8 Hz, 2H), 7.20 (dd, J = 7.8, 1.8 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.02 (dd, J = 8.4, 2.4 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 4.11 (d, J = 11.4 Hz, 1H), 3.68 (d, J = 4.8 Hz, 4H), 3.56–3.63 (m, 1H), 3.45 (t, J = 12.0 Hz, 1H), 3.33 (td, J = 10.8, 4.8 Hz, 1H), 2.78 (dd, J = 12.0, 4.8 Hz, 1H), 2.68 (d, J = 3.6 Hz, 1H), 2.65 (t, J = 3.6 Hz, 1H), 2.39– 2.46 (m, 1H), 2.32–2.39 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.94, 168.75, 142.03, 141.48, 140.31, 140.11, 136.33, 132.22, 131.97, 131.32, 130.75, 128.74, 128.01, 127.42, 127.19, 126.45, 126.12, 121.76, 119.45, 109.21, 56.23, 47.13, 38.85, 37.90, 35.63, 28.56; HRMS (ESI): C33H26Br2N2O2S requires m/z 672.0082, found 671.0082 [MH]; [a]25 D = +79.8 (c = 0.27 in DCM). 5.1.16. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-(4-methylpiperazin-1-yl)phenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 thiopyrano[4,3-c]pyridine]-2,70 (10 H)-dione (B12) White solid (45 mg), yield: 21.6%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.75 (s, 1H), 7.41 (dd, J = 8.4, 2.4 Hz, 1H), 7.24 (dd, J = 8.4, 2.4 Hz, 1H), 7.19 (dd, J = 8.4, 2.4 Hz, 1H), 7.09 (dd, J = 8.4, 0.6 Hz, 1H), 7.01 (t, J = 7.8 Hz, 1H), 6.99 (dd, J = 8.4, 2.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 6.60 (dd, J = 7.8, 0.6 Hz, 1H), 4.07 (d, J = 11.2 Hz, 1H), 3.53–3.59 (m, 1H), 3.27–3.29 (m, 1H), 3.22–3.27 (m, 1H), 3.00–3.01 (m, 4H), 2.58–2.68 (m, 3H), 2.39–2.47 (m, 4H), 2.32– 2.39 (m, 1H), 2.25–2.31 (m, 1H), 2.21 (s, 3H). 13C NMR (150 MHz, CDCl3) d176.89, 168.90, 146.75, 142.06, 136.46, 132.13, 131.97, 131.25, 130.67, 130.16, 127.33, 127.21, 127.13, 126.21, 121.65, 119.43, 111.66, 109.19, 56.80, 52.07, 47.20, 44.33, 38.81, 37.87, 35.56, 28.61; HRMS (ESI): C32H32Br2N4O2S requires m/z 694.0613, found 693.0612 [MH]; [a]25 D = +62.6 (c = 0.23 in DCM). 5.1.17. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-morpholinophenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano [4,3-c]pyridine]-2,70 (10 H)-dione (B13) White solid (41 mg), yield: 20.1%. Rf = 0.28 (DCM/MeOH = 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.75 (s, 1H), 7.41 (dd, J = 8.4, 2.4 Hz, 1H), 7.24 (dd, J = 8.4, 2.4 Hz, 1H), 7.19 (dd, J = 8.4, 2.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (t, J = 7.8 Hz, 1H), 6.98– 7.00 (m, 1H), 6.96 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 9.0 Hz, 2H), 6.60 (d, J = 7.8 Hz, 1H), 4.07 (d, J = 10.2 Hz, 1H), 3.68 (d, J = 4.8 Hz, 4H), 3.53–3.59 (m, 1H), 3.27–3.29 (m, 1H), 3.22–3.27 (m, 1H), 3.02 (d, J = 4.8 Hz, 4H), 2.58–2.68 (m, 3H), 2.32–2.39 (m, 1H), 2.25–2.31 (m, 1H). 13C NMR (150 MHz, CDCl3, TMS): d 176.86, 168.80, 149.97, 136.41, 134.41, 132.15, 131.26, 130.71, 127.38, 127.17, 126.96, 126.15, 121.68, 119.44, 116.04, 109.18, 66.79, 56.51, 52.05, 49.07, 47.15, 38.77, 37.88, 35.57, 28.59; HRMS (ESI): C31H29Br2N3O3S requires m/z 681.0296, found 680.0298 [MH]; [a]25 D = +82.6 (c = 0.39 in DCM).

5.1.18. (3R,40 S,4a0 S,8a0 S)-4-Bromo-40 -(4-bromophenyl)-60 -(4-(diethylamino)phenyl)-40 ,4a0 ,50 ,60 ,80 ,8a0 -hexahydrospiro[indoline-3,30 -thiopyrano[4,3-c]pyridine]-2,70 (10 H)-dione (B14) White solid (25 mg), yield: 12.5%. Rf = 0.28 (DCM /MeOH 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.74 (s, 1H), 7.41 (dd, J = 8.4, 2.4 Hz, 1H), 7.24 (dd, J = 8.4, 2.4 Hz, 1H), 7.19 (dd, J = 8.4, 2.4 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.01 (t, J = 7.8 Hz, 1H), 6.99 (dd, J = 8.4, 2.4 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H), 6.60 (d, J = 7.8 Hz, 1H), 6.50 (d, J = 9.0 Hz, 2H), 4.06 (d, J = 10.2 Hz, 1H), 3.53–3.59 (m, 1H), 3.21–3.28 (m, 6H), 2.66–2.69 (m, 1H), 2.64 (dd, J = 13.2, 3.6 Hz, 1H), 2.60 (dd, J = 16.8, 4.2 Hz, 1H), 2.30–2.39 (m, 1H), 2.23–2.29 (m, 1H), 1.02 (d, J = 7.8 Hz, 6H); 13C NMR (150 MHz, CDCl3,) d 176.56, 168.61, 146.70, 141.67, 136.39, 132.14, 131.95, 131.24, 130.70, 130.21, 127.53, 127.18, 127.11, 126.23, 121.66, 119.57, 111.63, 108.98, 56.75, 52.02, 47.17, 44.33, 38.86, 37.90, 35.55, 28.64, 12.57; HRMS (ESI): C31H31Br2N3O2S requires m/z 667.0504, found 666.0502 [MH]; [a]25 D = +61.7 (c = 0.31 in DCM). HPLC (Chiralpak AD, 0.46 cm I.D.  25 cm L  5 lm, 25 °C, i-propanol/hexane = 30: 70, flow rate 0.8 mL/min, k = 254 nm): tmajor = 24.65 min, tminor = 31.79 min, ee >99%. 5.1.19. Preparation of (3R,30 S,40 S,50 S)-4-bromo-30 -(4-bromophen-yl)50 -(2-hydroxyethyl)-40 -(hydroxymethyl)-30 ,4 0 ,50 ,60 -tetrahydrospiro [indoline-3,2 0 -thiopyran]-2-one (C1) To a stirred solution of lead compound 1 (150 mg, 0.28 mmol, 1.0 equiv) in anhydrous DCM (15 mL) was added DIBAL-H (1.12 mL, 1 M in hexane, 1.12 mmol, 4.0 equiv) dropwise at 78 °C under N2. The resulting solution was stirred at 78 °C for 1 h and then was warmed to room temperature and stirred for another 1 h. The reaction was quenched by the addition of saturated NH4Cl solution (30 mL). Then the mixture was separated and the organic layer was washed with saturated NaCl (30 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, DCM/MeOH = 100:5, v/v) to afforded compound C1 (70 mg, 41.2% yield) as a white solid. Rf = 0.15 (DCM/MeOH 100:5); 1H NMR (600 MHz, DMSO-d6) d 10.59 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 8.4, 1.2 Hz, 1H), 7.03–7.07 (m, 1H), 6.99 (t, J = 7.8 Hz, 1H), 6.57 (dd, J = 7.8, 1.2 Hz, 1H), 4.48 (t, J = 4.8 Hz, 1H), 4.44 (d, J = 11.4 Hz, 1H), 4.22 (t, J = 4.2 Hz, 1H), 3.50–3.62 (m, 4H), 2.76–2.81 (m, 1H), 2.58 (dd, J = 12.6, 3.0 Hz, 1H), 2.58 (d, J = 10.8 Hz, 1H), 2.15–2.22 (m, 1H), 1.93–1.99 (m, 1H), 1.39–1.46 (m, 1H). 13C NMR (150 MHz, CDCl3) d 176.68, 168.64, 158.35, 141.79, 136.33, 135.24, 132.19, 131.94, 131.29, 130.74, 127.52, 127.39, 127.13, 126.17, 121.73, 119.53, 114.56, 109.08, 56.58, 55.40, 52.04, 47.16, 38.80, 37.90, 35.60, 28.61; HRMS (ESI): C21H21Br2NO3S requires m/z 524.9609, found 523.9609 [MH]; [a]25 D = +246.7 (c = 0.08 in DCM). 5.2. Biological screening 5.2.1. Cell cultures A549 cells and MCF-7 cells were cultured in DMEM medium (Hyclone) with 10% fetal calf serum (Gibco), 100 U/ml penicillin and 100 lg/ml streptomycin (Invitrogen-Gibco, Karlsruhe, Germany), and HCT116 cells were cultured in McCoy’s 5A medium (Gibco) with 10% fetal calf serum (Gibco), 100 U/ml penicillin and 100 lg/ml streptomycin (Invitrogen-Gibco, Karlsruhe, Germany). Cells were maintained in a humidified incubator with 5% CO2 at 37 °C. 5.2.2. In vitro cytotoxicity assay Cells were plated in 96-well microtiter plates at a density of 8  103/well and incubated in a humidified atmosphere with 5% CO2 at 37 °C for 24 h. Test compounds were added onto triplicate wells with different concentrations and 0.1% DMSO for control.

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After they had been incubated for 24 h, 10 lL of CCK8 was added to each well and the plate was incubated for additional 1 h. The absorbance (OD) was read on a WellscanMK-2 microplate reader (Labsystems) at 450 nm. The concentration causing 50% inhibition of cell growth (IC50) was determined by the Logit method.10 All experiments were performed three times. 5.2.3. Fluorescence polarization binding assay Briefly, the fluorescence polarization experiments were read on Biotek Synergy H2 with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in black, 96-well plates with nonbinding surface (Corning #3993) at room temperature. The background fluorescence intensities of blank samples containing the reference buffer were subtracted, and the steady-state fluorescence polarization was calculated using the equation P = 1000  (Intparallel  Gintperpedicular)/(Intparallel + GIntperpedicular). The correction factor G (G = 0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP). The dose-dependent binding experiments were carried out with serial dilution in DMSO of compounds. A 5 lL sample of the test sample, preincubated (for 30 min) MDM2 binding domain (1–118) (10 nM), and PMDM6-F peptide (Anaspec) (10 nM) in assay buffer (100 mM potassium phosphate, pH 7.5; 100 lg/ mL bovine gamma globulin; 0.02% sodium azide) were added to microplates to produce a final volume of 115 lL. For each assay, the controls contained the MDM2 binding domain and PMDM6-F. Plates were read at 1 h after mixing all assay components. Binding constant (KD) was determined by fitting inhibition curves using GraphPad Prism software. Nutlin-3 (Sigma-Aldrich) was used as reference compound for validating the assay in each plate. 5.2.4. Western blots A549 cell lines were plated at a density of 1  105 cells/mL in 6well plates (Corning). Cells were incubated for 18 h with indicated concentration of compound B14 and washed with cold-ice PBS for 2 times and 60 lL ice-cold lysis buffer containing 1% protease inhibitors (Roche). Cells were scraped off after 30 min on ice and centrifuged for 12,000 rpm for 15 min at 4 °C. The protein extract was denatured and run on 10% SDS-PAGE gels. The gels were blotted onto NC membranes (Whatman) and blocked with 5% BSA Buffer (5% Albumin Bovine V from Bovine serum in TBST) for 2 h at room temperature. The primary antibodies used for Western bolting were: p53 (Abcam, ab7757), MDM2 (Abcam, ab16895), GAPDH. Then, the membranes were infrared secondary antibodies. After washing with TBST, blots were scanned on a LI-COR Odyssey imaging system. The protein levels were quantified by the gray values of the bands in the resulting images using the control group as the standard.

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5.2.5. Apoptosis detection assay A549 cells (5  105 cells/mL) were seeded in six-well plates and treated with compounds at a concentration of 5 lM for 48 h. The cells were then harvested by trypsinization and washed twice with cold PBS. After centrifugation and removal of the supernatants, cells were resuspended in 400 lL of 1 binding buffer, which was then added to 5 lL of annexin V-FITC and incubated at room temperature for 15 min. After adding 10 lL of PI, the cells were incubated at room temperature for another 15 min in the dark. The stained cells were analyzed by a flow cytometer (BD Accuri C6). 5.2.5. Docking studies The initial three dimensional geometric coordinates of the X-ray crystal structure of MDM2 (PDB code: 4OAS)11 was downloaded from the Protein Data Bank (PDB) (http://www.pdb.org/pdb/ home/home.do). Then we used GOLD 5.1 with GoldScore fitness function to dock compounds B13 and B14 into the binding pocket of MDM2 using the protocol as described earlier.8 Acknowledgements This work was supported by National Natural Science Foundation of China (21602252 to S.W., 81373331 to Z.M.), National Key R&D Program of China (2017YFA0506000 to C. S.) and Natural Science Basic Research Plan in Shaanxi Province (2016JQ8006 to S.W.). References 1. (a) Levine AJ. Cell. 1997;88:323–331; (b) Vogelstein B, Lane D, Levine AJ. Nature. 2000;408:307–310; (c) Vousden KH, Lane DP. Nat Rev Mol Cell Biol. 2007;8:275–283. 2. (a) Chene P. Nat Rev Cancer. 2003;3:102–109; (b) Hainaut P, Hollstein M. Adv Cancer Res. 2000;77:81–137. 3. (a) Li Q, Lozano G. Clin Cancer Res. 2013;19:34–41; (b) Boeckler FM, Joerger AC, Jaggi G, et al. Proc Natl Acad Sci USA. 2008;105:10360–10365. 4. (a) Haupt Y, Maya R, Kazaz A, et al. Nature. 1997;387:296–299; (b) Bai L, Wang S. Annu Rev Med. 2014;65:139–155. 5. (a) Wade M, Li YC, Wahl GM. Nat Rev Cancer. 2013;13:83–96; (b) Popowicz GM, Domling A, Holak TA. Angew Chem Int Ed Engl. 2011;50:2680–2688; (c) Cheok CF, Verma CS, Baselga J, et al. Nat Rev Clin Oncol. 2011;8:25–37. 6. (a) Wang W, Hu Y. Med Res Rev. 2012;32:1159–1196; (b) Zhao Y, Aguilar A, Bernard D, et al. J Med Chem. 2015;58:1038–1052; (c) Siu L, Italiano A, Miller Jr W, et al. J Clin Oncol. 2014;32:2535; (d) Ray-Coquard I, Blay JY, Italiano A, et al. Lancet Oncol. 2012;13: 1133–1140. 7. (a) Zhuang C, Miao Z, Wu Y, et al. J Med Chem. 2014;57:567–577; (b) Zhuang C, Miao Z, Zhu L, et al. J Med Chem. 2012;55:9630–9642; (c) Li J, Wu Y, Guo Z, et al. Bioorg Med Chem Lett. 2014;24:2648–2650. 8. Wang S, Jiang Y, Wu S, et al. Org Lett. 2016;18:1028–1031. 9. Vassilev LT, Vu BT, Graves B, et al. Science. 2004;303:844–848. 10. Huang H, Chen Q, Ku X, et al. J Med Chem. 2010;53:3048–3064. 11. Sun D, Li Z, Rew Y, et al. J Med Chem. 2014;57:1454–1472.