Discovery of novel liver X receptor inverse agonists as lipogenesis inhibitors

Discovery of novel liver X receptor inverse agonists as lipogenesis inhibitors

European Journal of Medicinal Chemistry 206 (2020) 112793 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal ...

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European Journal of Medicinal Chemistry 206 (2020) 112793

Contents lists available at ScienceDirect

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

Research paper

Discovery of novel liver X receptor inverse agonists as lipogenesis inhibitors Ziyang Chen a, b, 1, Hao Chen a, 1, Zizhen Zhang a, Peng Ding a, Xin Yan a, Yanwen Li c, Songxuan Zhang a, Qiong Gu a, Huihao Zhou a, **, Jun Xu a, c, * a b c

Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China Shenzhen Pingle Orthopaedic Hospital (Shenzhen Pingshan Traditional Chinese Medicine Hospital), Shenzhen, 518118, China School of Biotechnology and Health Sciences, Wuyi University, Jiangmen, 529020, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2020 Received in revised form 26 August 2020 Accepted 26 August 2020 Available online 6 September 2020

Based on the co-crystal structures of LXRb and its agonists (spiro [pyrrolidine-3,30 -oxindole] derivatives) discovered by us previously, we designed and synthesized a compound library to explore the agonistic activities. The library was screened with luciferase reporter assays, interestingly, it resulted in the discovery of 10 LXR inverse agonists besides 5 LXR agonists. To clarify the mechanism of the actions, we conducted molecular dynamics (MD) simulations on the LXR and inverse agonists complexes, and revealed that H3, H11 and H12 configurations are the key to turn on agonism or inverse agonism status for LXR. Binding tightly with H3, pushing H11 out and destabilizing H12 could form a bigger hydrophobic groove to accommodate NCOR1 to turn on LXR inverse agonism. The inverse agonist 10rr was further studied, and found as a lipogenesis inhibitor through down-regulating LXR target genes SREBP-1c, ACC, FAS and SCD-1, and demonstrated lipid-lowering effects in 3T3-L1 cells, HepG2 cells and mice with Triton WR-1339-induced hyperlipidemia. Therefore, we have proved that LXR inverse agonists can be promising agents for hyperlipidemia treatment. © 2020 Elsevier Masson SAS. All rights reserved.

Keywords: Liver X receptor Inverse agonists Lipogenesis Hyperlipidemia

1. Introduction Liver X receptors (LXRs), LXRa (NR1H3) and LXRb (NR1H2), are members of the nuclear receptor superfamily [1,2]. LXRa is expressed in liver, intestine, kidney, adipose tissue and macrophage while LXRb is expressed ubiquitously [1e3]. LXRs involved in regulating the metabolism of cholesterol, lipid, and glucose [4e6]. LXR agonists induce reverse cholesterol transport, inhibit inflammation, and are potential anti-atherogenic agents [5e8]. For decades, many LXR agonists were discovered, but were not clinically useful due to the lipogenesis side effects, such as hepatic steatosis and the elevation of plasma triglycerides [5]. Synthetic LXR agonists promote lipogenesis through up-regulating the expression of sterol-regulatory element binding proteins 1c

* Corresponding author. Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China., ** Corresponding author. E-mail addresses: [email protected] (H. Zhou), [email protected] com (J. Xu). 1 These authors contributed equally. https://doi.org/10.1016/j.ejmech.2020.112793 0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

(SREBP-1c), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) [9]. Therefore, there is a need for novel LXR agonists without these side effects. The lipogenesis side effect of synthetic LXR agonists is likely an opportunity for LXR inverse agonists, which are reported infrequently. Based on structural modification of GSK2033, Burris and colleagues developed a liver-selective LXR inverse agonist SR9238 [10,11] to improve fatty liver conditions, and an inverse agonist SR9243 [12,13], which provides systemic exposure to inhibit tumors by targeting the Warburg effect and lipogenesis. The lipogenesis was inhibited through the down-regulation of the expression of SREBP-1c, FAS and SCD-1 and the plasma lipid levels were lowered [10e13]. LXR inverse agonists show promise as lipogenesis inhibitors for hyperlipidemia treatment. In addition, Burton and co-workers synthesized two steroidal LXR inverse agonists, 27-norcholestenoic acid [14] and 25,25difluoro-27-norcholestenoic acid [15]. They proved that these inverse agonists disrupted the key binding of His435 and Trp457, and changed the conformation of H11-loop-H12 [14e16]. Recently, we reported new spiro [pyrrolidine-3,30 -oxindole] derivatives as LXR agonists [17] (Fig. 1). In order to optimize the

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European Journal of Medicinal Chemistry 206 (2020) 112793

Fig. 1. Chemical structures of spiro [pyrrolidine-3,30 -oxindole] derivatives 1ss and 2rr.

residues in Chamber B. The 1ss-series compounds were synthesized with Scheme 1. The intermediate 1 was prepared under Pictet-Spengler reaction conditions followed by N-bromosuccinimide (NBS)-mediated rearrangement to generate enantiomers containing the ()-20 S,3S- and (þ)-20 R,3R-isoforms [18,19]. After acylation, 1-chloro-3iodobenzene was coupled with the spiro [pyrrolidine-3,30 -oxindole] core under conditions of the Ullmann coupling reaction. The chlorine in the core was converted to a boronic acid pinacol (Bpin) for a subsequent coupling reaction combining it with the designed aromatic fragments. The active racemic analogues were isolated and confirmed to be R,R and S,S-isomers by their specific optical rotation.

new LXR agonists, we designed and synthesized a library of spiro [pyrrolidine-3,30 -oxindole] derivatives based on the two co-crystal structures of the LXRb-ligand complexes (PDB access codes: 6K9M and 6K9G). Interestingly, besides 5 LXR agonists, 10 LXR inverse agonists were discovered. Then, we explored the lipid-lowering activity of these LXR inverse agonists and validated their molecular mechanism of action.

2. Results and discussion 2.1. Design and synthesis of a library of derivative compounds Based on the co-crystal structures, we divided the ligandbinding pocket (LBP) of LXR into Chambers A and B, and realized that compound 1ss occupied Chamber A (Fig. 2: 6K9M) and compound 2rr occupied Chambers A and B (Fig. 2: 6K9G). Both ligands were able to keep His435-Trp457 bonded for LXRb agonism. We hypothesized that increasing the ligand interactions at both Chambers A and B while keeping His435-Trp457 bonded will improve the LXR agonism. Therefore, based on the 1ss scaffold, we designed new ligands by adding a bulky group at R1 and polar groups at R2 (Fig. 2: 1ss-series) [17]. Thus, the 1ss-series would occupy Chambers A and B, and establish more interactions with

2.2. Bioassay results of compounds against LXRs The 1-series compounds (Table 1) were assayed for their LXRa/b transcriptional activities with cell-based luciferase reporter experiments. As shown in Fig. 3, five compounds (8, 9, 17, 18 and 19) were confirmed as LXR agonists, and interestingly, 10 compounds were found as LXR inverse agonists. Specifically, compounds 10e13 were selective inverse agonists toward LXRb although they had inverse

Fig. 2. The designed spiro [pyrrolidine-3,30 -oxindole] derivatives based on the co-crystal structure of LXRb-1ss complexes. Functional groups at R1 were designed to create more interactions in Chamber B; Functional groups at R2 were designed to keep the His435-Trp457 interaction.

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Scheme 1. Synthetic route to spiro [pyrrolidine-3,30 -oxindole] derivatives. (a) iso-Butyraldehyde, TFA, DCM. (b) NBS, THF/water, 0  C. (c) Boc2O, TEA, DCM. (d) BrR1, NaH, MeCN. (e) NaOH, EtOH/water, 80  C. (f) Aryl bromides or 1-chloro-3-iodobenzene, K2CO3, cat. CuI, cat. N,N0 -dimethyl-1,2-ethanediamine, toluene, 90  C. (g) DIBAL-H, DCM, 78  C. (h) Pd2 (DBA)3, X-Phos, (Bpin)2, K2CO3, toluene, 95  C. (i) BrR1, Pd (DPPF)Cl2, K2CO3, 2,4-Dioxane, 85  C. (j) HCl, DCM. (k) R2OR2, pyridine, DCM. (l) LiOH, THF/MeOH/water. The structures were shown in Table 1.

Fig. 3. LXR transcriptional activities for 1ss-series. LXRa/b transcriptional activities were measured by luciferase reporter assays in transiently transfected HEK293T cells. 5 h after transfection, cells were treated with 1ss-series compounds at 10 mМ for 20 h. Data are represented as means ± SD of three independent experiments. * (or #) P < 0.05, ** (or ##) P < 0.01 and *** (or ###) P < 0.001, ns, not significant. The two-tailed unpaired Student’s t-test was used for comparisons of means between two groups, the significant difference is indicated by "*". ANOVA was used for comparison of means between >3 groups with Tukey’s hoc test for multiple comparisons, the significant difference is indicated by "#"

2.3. Structure and activity relations (SAR) of LXRb agonists and inverse agonists

agonism toward LXRa as well. Their IC50 values are listed in Table 2. To understand the roles of chiral centers on the LXRb inverse agonism, the inverse agonist (10) of LXRb (IC50 ¼ 0.39 mM) was separated into two isomers (10rr, 10ss) with chiral chromatography. The LXRb activity of the isomers has IC50 values of 0.36 mM for 10rr (R,R-isoform) and 1.04 mM for 10ss (S,S-isoform).

According to IC50 and % inverse agonism values in Table 2, the SAR for LXRb regulators are summarized and depicted in Fig. 4. R1 and R2 were modified based on their binding potential at LBP 3

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Table 1 Chemical structures designed and synthesized based on 1ss scaffold.

more binding with the key residues at LBP is desired. An aromatic ring was such a linker, resulting in molecules with increased both agonistic and inverse agonistic activities. Modifying R1 resulted in compounds 17e21, among which 18 and 19 are agonists, 20 (IC50 LXRb ¼ 1.43 mM) and 21 (IC50 LXRb ¼ 4.57 mM) are inverse agonists. This means that the polarity of R2 contribute to the switch of agonism and inverse agonism. When a polar, bulky and rigid moiety was placed at R2, the agonistic activity would be decreased (17), and the inverse agonistic activity would be increased (20). With hydrogen bond donor at R1, compounds 10e13, 15 and 16 became LXR inverse agonists. However, 18 and 19 are agonists due to hydrogen acceptor at R1 although the substitutes at R2 are the same as the ones in compounds 11 and 12. This suggests that modifying R1 (hydrogen bond donor) can result in switching the ligand from an agonist (18 or 19) to an inverse agonist (11 or 12). In contrast with 15 and 16, compound 10 had much better inverse agonistic activity, whose methylsulfonyl group at R1 interacted with more residues (such as Leu330, Leu274 and Ile277) in Chamber B than the chlorine and hydrogen did (Fig. 9). The (2-(methylsulfonyl)phenyl)methanol moiety combined with the tbutoxycarbonyl (Boc) at R2 affording active compound 10 with an IC50 value of 0.39 mM.

Table 2 LXR transcriptional inverse agonistic activity. Compd.

LXRa

LXRb

IC50 (mM) SR9238 10 10rr 10ss 11 12 13 14 15 16 20 21 22

0.16 3.23 2.25 9.06 8.57 7.54 8.16 NA NA NA 2.59 3.56 NA

± ± ± ± ± ± ±

a

0.02 0.43 0.58 1.41 3.63 3.48 2.26

± 1.12 ± 0.50

% inverse agonism 93 ± 77 ± 81 ± 56 ± 46 ± 55 ± 50 ± NA NA NA 75 ± 66 ± NA

5 5 2 2 1 1 19

17 1

b

IC50 (mM)a

% inverse agonismb

0.10 ± 0.01 0.39 ± 0.01 0.36 ± 0.05 1.04 ± 0.01 1.79 ± 0.25 2.40 ± 0.36 3.44 ± 0.63 10.08 ± 0.79 6.97 ± 2.42 8.64 ± 4.39 1.43 ± 0.07 4.57 ± 0.09 >20

78 83 85 75 77 78 59 36 49 41 61 56 47

± ± ± ± ± ± ± ± ± ± ± ± ±

15 4 4 10 13 12 3 13 18 13 16 7 5

a The IC50 value was measured by transient transfection and luciferase reporter assays. Results are given as the mean of at least two independent experiments. b % inverse agonism is the maximal inhibition rate relative to control. NA ¼ not active.

in LXRb. Compounds 3e9 demonstrated weaker activity due to lack of binding with the key residues in the LBP. A linker connecting the spiro [pyrrolidine-3,30 -oxindole] core with fragments to establish 4

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Fig. 4. SAR for LXRb agonists and inverse agonists. The substituent groups are in the order of descending activity according to IC50 and % inverse agonism values in Table 2 a The inverse agonist group A, including compounds 10e13, 15 and 16. b The inverse agonist group B, including 20 and 21. c The agonist group including 18 and 19. d HBD: hydrogen bond donor. e HBA: hydrogen bond acceptor.

2.4. 10rr regulates LXR as an inverse agonist

demonstrated that 10rr down-regulated LXR target genes, SREBP1c, ACC, FAS and SCD-1 to inhibit lipogenesis in both 3T3-L1 (Fig. 7A) and HepG2 (Fig. 7B) cells.

As shown in Fig. 5A, 10rr was not toxic at 10 mM or less in HEK293T cells. Using SR9238 as the positive control of an LXR inverse agonist, luciferase reporter assays showed that 10rr dosedependently inhibited LXR transactivation activity in the absence (Fig. 5B1 and 5B2) or presence (Fig. 5B3 and 5B4) of the LXR agonist GW3965, suggesting that 10rr is an LXR inverse agonist, and more selective to LXRb. In order to confirm the binding of 10rr to LXRb, we conducted a fluorescence polarization assay, which demonstrated that 10rr dose-dependently inhibited the binding of FITC-hyodeoxycholic acid to LXRb with a Ki value of 2.3 mM (Fig. 5C), suggesting that 10rr binds to LXRb directly.

2.6. Compound 10 improved hyperlipidemia in vivo Triton WR-1339, a non-ionic detergent, is usually used to induce acute hyperlipidemia in animals. This is a fast and simple animal model to screen hypolipidemic compounds in vivo. A WR-1339induced hyperlipidemia model was created by injecting Triton WR-1339 into C57BL/6 mice in order to study the effects of compound 10 on hyperlipidemia in vivo. The results are depicted in Fig. 8. Fig. 8AeC demonstrate that both compound 10 and the positive control (Fenofibrate) significantly decreased serum triglycerides (TG), total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) comparing with the model group. In addition, compound 10 decreased triglyceride levels in the liver tissue (Fig. 7D and E). Therefore, compound 10, as a lipogenesis inhibitor, can be a promising agent to treat hyperlipidemia and fatty liver disease. LXR agonists exhibit anti-atherogenic properties by upregulating ATP-binding cassette (ABC) transporters, which increases reverse cholesterol transport (RCT) to decrease the blood levels of TC [5]. Thus, considering LXR inverse agonists may have adverse effects on RCT and TC, SR9238 was developed as a liverselective LXR inverse agonist to avoid the suppression of LXR target genes outside the liver [10]. However, SR9238 decreased plasma TC levels by inhibiting SREBP-2 translocation [10]. Moreover, SR9243 which provides systemic exposure also decreased

2.5. 10rr inhibited lipogenesis Cell viability assays showed that 10rr was not toxic at a concentration of 10 mM in 3T3-L1 and HepG2 cells (Fig. 6A and D). With triglycerides assay and Oil Red staining methods, we found that 10rr dose-dependently decreased lipid accumulation in 3T3-L1 adipocytes with an IC50 of 0.27 mM (Fig. 6B and C). And 10rr also inhibited accumulation of triglycerides induced either by highglucose medium (Fig. 6E) or by GW3965 (Fig. 6F) in human hepatoma HepG2 cells. ACC, FAS and SCD-1 are key enzymes during de novo lipogenesis and regulated by SREBP-1c and LXR directly [9]. To test whether 10rr inhibits lipogenesis through this mechanism, we detected the mRNA expression of these LXR target genes. The results

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Fig. 5. 10rr is an LXR inverse agonist which directly binds to LXRb. A positive control, SR9238, was tested at 3 mM. (A) Cytotoxicity of 10rr in HEK293T cells measured by MTT assays. (B) 10rr inhibited LXR transactivation activity dose-dependently in the presence or absence of LXR agonist GW3965. (C) 10rr (Ki ¼ 2.3 ± 1.5 mM) inhibited the binding of FITChyodeoxycholic acid to LXRb. GW3965 (Ki ¼ 2.2 ± 1.9 nM) and SR9238 (Ki ¼ 352 ± 45 nM) were tested as positive controls [20]. Data are represented as means ± SD of three independent experiments and were analyzed using Student’s t-tests. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 6. Lipid-lowering effect of 10rr in 3T3-L1 adipocytes and HepG2 cells. A positive control, SR9238, was tested at 3 mM. (A) Cytotoxicity of 10rr in 3T3-L1 cells measured by MTT assays. (B) 10rr decreased triglyceride content in 3T3-L1 adipocytes as shown by a triglyceride assay, with an IC50 of 0.27 mM. (C) 10rr decreased triglyceride content in 3T3-L1 adipocytes by Oil Red-O staining, with 40  objective (magnification, 400  ). UND, undifferentiated group. Control, differentiated group without compounds. (D) Cytotoxicity of 10rr in HepG2 cells measured by MTT assays. 10rr inhibited triglyceride accumulation induced by (E) high-glucose medium or (F) by GW3965. Data are represented as means ± SD of three independent experiments and were analyzed using Student’s t-tests. (AeE) *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control. (F) ##P < 0.01 compared with control. *P < 0.05, **P < 0.01 compared with 1 mМ GW3965. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 7. Effects of 10rr on mRNA expression of LXR target genes. A positive control, SR9238, was tested at 3 mM. (A) Effects of 10rr on the mRNA expression of Srebp-1c, Acc, Fas and Scd-1 in 3T3-L1 cells. (B) Effects of 10rr on the mRNA expression of SREBP-1c, ACC, FAS and SCD-1 in HepG2 cells. Data are represented as means ± SD of three independent experiments and were analyzed using Student’s t-tests. *P < 0.05, **P < 0.01 and *** (or ###) P < 0.001 compared with control (or undifferentiated, UND).

resulting in 3 clusters (details are depicted in Figs. S2 and S3 in the Supporting Information). The proportionate populations of the major clusters for LXRb-18rr and LXRb-10rr are 87.8% and 96.0%, respectively. The surface models generated from the major clusters of LXRb-18rr and LXRb-10rr are depicted in Fig. 9A and C, in which the co-activator (NCOA1, for agonism) and co-repressor (NCOR1, for inverse agonism) are docked in the activation function 2 (AF-2) domain (The docking results were displayed in Figs. S4 and S5 in the Supporting Information) [22e24]. As shown in Fig. 9A and B, NCOA1 is located at a hydrophobic groove surrounded by H3, H4, and H12 in LXRb-18rr complex, residues Lys287 (at H3) and Glu455 (at H12) function as a “charge clamp” to coordinate the two termini of the helix of NCOA1. In contrast, when NCOR1 was docked, the longer helix of NCOR1 (17

plasma TG and TC levels. Our studies further demonstrated that the LXR inverse agonist decreased both TG and TC levels in vivo, and can be a therapeutic agent against hyperlipidemia. 2.7. H3, H11 and H12 configurations are the key to turn on the agonism or inverse agonism status for LXR In order to elucidate the bioactivity data of LXRb agonists and inverse agonists, we conducted molecular dynamics (MD) simulations [21] for 125 ns on the complexes of LXRb-18rr and LXRb-10rr starting from the coordinates derived from co-crystal structure (PDB code: 6K9G). 1250 representative conformations were extracted using averages from trajectories of the period of 0e125 ns simulation, 7

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Fig. 8. Lipid-lowering effect of 10 in mice with Triton WR-1339-induced hyperlipidemia. (AeC) 10 decreased the triglycerides (TG), total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) contents in serum. 10 decreased TG content in the liver tissues by (D) triglycerides assay and (E) Oil Red-O staining (magnification, 200  and 400  ). Data are represented as means ± SD of 6 mice per group and were analyzed using Student’s t-tests. * (or #) P < 0.05, ** (or ##) P < 0.01 and *** (or ###) P < 0.001 compared with model group (or control group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

bond with Ser278 in Chamber B as 10rr does. Similarly, it pushes H11 out and destabilizes H12 (helix lost) in Chamber A, leading to LXR inverse agonism (Fig. 10B). However, 20rr forms a p-p stacking with Phe329 and no hydrogen bonds with Ser278 in Chamber B, destabilizing H12 (helix lost) in a more directly way (Fig. 10A). In Fig. 9C and D, the distance between Arg261 (Ca, in H3) and Gln445 (Ca, in H11) in the LXRb complexes, indicates the range of motion for H3 and H11. The distance ranges in 125 ns MD trajectories were assayed (Fig. S6 in the Supporting Information). The frequency distribution of the distance ranges in a period of 60e125 ns, were showed in Fig. 11. When 10rr interacted with H3 more (Fig. 9G), the distance range of LXRb-10rr was larger than LXRb-20rr and LXRb-21rr, in favor of LXRb-10rr complex in inverse agonistic state. Additionally, LXRb-18rr complex shortened the distance to keep H3 and H11 configurations in agonistic state. To evaluate the SAR further, the RMSF (Root Mean Square Fluctuation) of small molecular skeleton for 10rr, 18rr, 20rr and 21rr in LXRb in a period of 60  125 ns was provided in Fig. S7. The fluctuation range of the Boc moiety (10rr) was smaller than other R2 moieties in Chamber A. When a spiro carbon atom of spiro [pyrrolidine-3,30 oxindole] is a reference, the Boc moiety had less range of motion than t-butylcarbamothioyl group of 20rr and propane-2-sulfonyl group of 21rr (Fig. S8A, S8C and S8D). However, 3,3-dimethylbutanoyl of 18rr had large range of motion (Fig. S8B). These suggested that the polarized and rigid R2 promote the conformational changes of H11 and H12 in an LXR inverse agonistic state.

residues in NCOR1 compared with 11 residues in NCOA1) was unacceptable to this hydrophobic groove and the “charge clamp” of the LXRb-18rr complex (Fig. S4) [22]. Fig. 9C-F shows that 10rr (as LXR inverse agonist) pushes H11 away from H3 and destabilizes the helix conformation of H12 (helix lost), resulting in a bigger hydrophobic groove for accommodating NCOR1. As shown in Fig. 9G, 10rr has strong binding to H3 (Leu274, Ile277, Ser278 and Glu281) in Chamber B with DE of 12.59 kcal/ mol; while 18rr (an LXR agonist) weakly binds to H3 with DE of 7.59 kcal/mol (Fig. 9G and Table 3). The Boc moiety occupies Chamber A and has hydrophobic interactions with H11 and H12. 10rr pushes H11 away from H3, resulting a reduce of DE (14.35 kcal/mol) for H11 (for LXR-18rr, the DE is 10.27 kcal/mol) (Fig. 9F and Table 3). And H12 is destabilized with DE of 3.40 kcal/ mol for LXR-10rr compared with 5.38 kcal/mol for LXR-18rr (Fig. 9E and F and Table 3). Consequently, the conformation changes of H3, H11 and H12 contribute to form a hydrophobic groove preferring NCOR1, leading to LXR inverse agonism. However, 18rr stabilizes NCOA1 in the groove, leading to LXR agonism. H3, H11 and H12 configurations are the key to turn on the agonism or inverse agonism status for LXR. To further study the function of H3, H11 and H12 to regulate LXR agonism and inverse agonism, LXRb-20rr and LXRb-21rr were conducted with MD simulations and compared with LXRb-18rr (Fig. 10). 20rr and 21rr have few interactions with residues in Chamber B. 21rr forms a p-p stacking with Phe329 and a hydrogen 8

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Fig. 9. Ligands 10rr and 18rr induce conformational changes of H3, H11 and H12. (A) The surface model generated from the major clusters of LXRb-18rr. (B) NCOA1 is stably docked in a hydrophobic groove of LXRb-18rr formed by H3, H4 and H12. (C) The surface model generated from the major clusters of LXRb-10rr. (D) NCOR1 is stably docked in the groove of LXRb-10rr. (E, F) Superimposition of LXRb-10rr and LXRb-18rr (red ribbons for LXRb-10rr, blue ribbons for LXRb-18rr). 10rr pushes H11 out and destabilizes H12 in Chamber A. (G) In Chamber B, 10rr has more interactions than 18rr with the residues in H3 (blue dotted line for p-p stacking, yellow dotted line for hydrogen bonding, green dotted line for van der Waals interaction). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Table 3 DE values for significant substructures.

LXRb-10rr LXRb-18rr

a

kcal/mol.

H3 in Chamber B DE

Chamber B DE

H11 DE

H12 DE

12.59 ± 0.48a 7.59 ± 0.57

20.77 ± 0.57 12.40 ± 0.63

14.35 ± 0.85 10.27 ± 1.97

3.40 ± 1.54 5.38 ± 2.11

Fig. 10. Ligands 20rr and 21rr induce conformational changes of H11 and H12 (red ribbons for LXRb-20rr or LXRb-21rr, blue ribbons for LXRb-18rr). (A) 20rr forms a p-p stacking with Phe329 in Chamber B, and destabilizes H12 in Chamber A. (B) 21rr forms a p-p stacking with Phe329 and a hydrogen bond with Ser278 in Chamber B, pushes H11 out and destabilizes H12 in Chamber A. (blue dotted line for p-p stacking, yellow dotted line for hydrogen bonding). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 11. The frequency distribution of distance between Arg261 (Ca, in H3) and Gln445 (Ca, in H11) for LXRb-10rr, LXRb-18rr, LXRb-20rr, LXRb-21rr complexes and Apo LXRb in a period of 60  125 ns MD trajectories. The peak value of each curve is labeled.

People’s Republic of China) were used for column chromatography by Biotage SP-1 (Biotage Inc). Semipreparative chiral HPLC separation was performed on an LC-20AT Shimadzu liquid chromatography system with an SPD-M20A diode array detector. All solvents were of chromatographic grade (Fisher Scientific UK Ltd.). Enantiomers were separated by semipreparative chiral HPLC (MeOH/ H2O, 80:20, 1.5 mL/min). HPLC purity was determined by analysis method on a Shimadzu HPLC system. Column: Agilent SB-C18; 5 mm 4.6  250 mm. Solvent: 75% acetonitrile, 25% water. Flow rate 1.0 mL/min. All derivatives had purification values of >95%. The ee values were assayed by analysis method on a Shimadzu HPLC system. Column: Phenomenex, Lux 5u Cellulose-3; 4.6  250 mm. Solvent: 80% methanol, 20% water. Specific rotations were measured on an AntonPaa MCP200 (solvent: MeOH).

Hence, LXR inverse agonism involve in binding tightly with H3, pushing H11 out and destabilizing H12. These conformational changes induce a bigger hydrophobic groove to accommodate NCOR1, leading to LXR inverse agonism. 3. Conclusion Based on co-crystal structures of LXRb and its agonists discovered in our lab, we designed and synthesized a library of the agonist scaffold (spiro [pyrrolidine-3,30 -oxindole]) to explore the SAR. We, unexpectedly, discovered LXR inverse agonists. Based upon the in silico and in vitro experiments, we figured out the SAR for this series compounds, but also revealed that H3, H11 and H12 configurations are the key to LXR inverse agonism. Further in vitro and in vivo experiments proved that the inverse agonists inhibited lipogenesis through down-regulation of LXR target genes SREBP-1c, ACC, FAS and SCD-1, and exerted lipidlowering effects. LXR inverse agonists can be promising agents for hyperlipidemia treatment. This work can be a good example to discover inverse agonists for other nuclear receptors.

4.1.1. General preparation of S-1 Iso-Butyraldehyde (5 mL, 55 mmol, 1.1 equiv.) and TFA (7.5 mL, 100 mmol, 2.0 equiv.) were added dropwise to the solution of tryptamine (8.0 g, 50 mmol, 1.0 equiv.) in DCM (200 mL) in an ice bath and stirred at room temperature (RT) overnight. Then, the mixture was concentrated in vacuo to remove the DCM and TFA. The residue was dissolved in EtOAc (50 mL), and subsequently precipitated by addition of 200 mL hexane/EtOAc (v/v: 1/1), filtered, and washed twice with hexane to obtain the product 1-isopropyl2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (S-1) as a white solid (9.8 g, yield: 92%). 1H NMR (400 MHz, CD3OD) d 7.48 (dd, J ¼ 8.0, 1.0 Hz, 1H), 7.37 (dt, J ¼ 8.2, 1.0 Hz, 1H), 7.16 (dd, J ¼ 8.2, 7.0, 1.0 Hz, 1H), 7.06 (dd, J ¼ 8.0, 7.0, 1.0 Hz, 1H), 4.67 (d, J ¼ 4.0 Hz, 1H), 3.75 (dd, J ¼ 12.7, 5.7, 3.3 Hz, 1H), 3.45 (dd, J ¼ 12.7, 9.7, 5.7 Hz, 1H), 3.18e3.00 (m, 2H), 2.64 (pd, J ¼ 7.1, 4.0 Hz, 1H), 1.25 (d, J ¼ 7.1 Hz, 3H), 0.97 (d, J ¼ 7.1 Hz, 3H). ESI-MS m/z: 215.3 [M þ H]þ.

4. Experimental section 4.1. Chemistry All chemical reagents and organic solvents were purchased from J&K Chemicals. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400/500 MHz NMR spectrometer for hydrogen and 100/125 MHz for carbon. HRESIMS were acquired on a Shimadzu LCMS-IT-TOF. Low resolution electrospray ionization (ESI) mass spectra were recorded on an Agilent 6120 single quadrupole LC/MS system using reverse-phase conditions (MeOH/H2O, 0.05% formic acid). Silica gel (200e300 Mesh Marine Chemical Ltd., Qingdao,

4.1.2. General preparation of 1 NBS (9.0 g, 51 mmol, 1.1 equiv.) was added in batches to a 10

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which was purified by flash chromatography (Biotage, 20 g silica gel, gradient elution from 20 to 60% EtOAc). 4-(1’-(tert-Butoxycarbonyl)-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidin]-1-yl) butanoic acid (5) as a white solid (80 mg, yield: 77%). 1H NMR (400 MHz, CDCl3) d 7.40e7.17 (m, 2H), 7.02 (t, J ¼ 7.8 Hz, 1H), 6.94 (d, J ¼ 7.8 Hz, 1H), 4.03 (s, 1H), 3.90e3.87 (m, 1H), 3.72e3.68 (m, 3H), 3.48 (dt, J ¼ 11.2, 7.0 Hz, 1H), 2.33 (s, 2H), 2.16 (t, J ¼ 7.0 Hz, 2H), 2.04e1.84 (m, 3H), 1.47 (s, 9H), 1.22 (t, J ¼ 7.00 Hz, 2H), 0.97 (d, J ¼ 6.7 Hz, 3H), 0.64 (d, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 179.47, 156.39, 142.95, 128.64, 124.92, 122.13, 108.69, 79.84, 67.83, 58.16, 45.77, 39.11, 34.99, 30.67, 21.58, 18.84. HRMS (ESI) m/z: calcd. for C23H32N2O5, 417.2384, found 417.2380.

solution of S-1 in THF/H2O (v/v: 4/1, 500 mL) which was chilled in an ice bath, stirred for 4 h. Then the mixture was neutralized with saturated NaHCO3. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product. TEA (19 mL, 140 mmol, 3.0 equiv.) and di-tert-butyl dicarbonate (10.0 mL, 46 mmol, 1.0 equiv.) were added dropwise to a solution of the crude product in DCM cooled in an ice bath, and the mixture was stirred at RT for 0.5 h. The mixture was then diluted with DCM, and washed with saturated NaHCO3, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product, which was dissolved in EtOAc, and diluted with hexane/ EtOAc (v/v: 3/1) for precipitation. Then, the mixture was filtered and washed with hexane/EtOAc (v/v: 3/1) to afford 8.0 g of tertbutyl-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (1), as a white solid (yield: 44%). 1H NMR (400 MHz, CD3OD) d 9.45 (s, 1H), 7.24e7.18 (m, 1H), 7.15 (dt, J ¼ 7.7, 1.2 Hz, 1H), 6.93 (dt, J ¼ 7.7, 1.2 Hz, 1H), 6.86 (d, J ¼ 7.7 Hz, 1H), 4.01e3.90 (m, 1H), 3.91 (d, J ¼ 6.1 Hz, 1H), 3.48e3.40 (m, 1H), 2.20e2.10 (m, 2H), 2.00e1.86 (m, 1H), 1.42 (s, 9H), 0.94 (d, J ¼ 6.8 Hz, 3H), 0.62 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 182.37, 156.33, 141.51, 128.97, 128.43, 125.01, 121.99, 110.23, 79.60, 67.64, 56.63, 45.64, 42.20, 34.76, 30.74, 28.49, 21.82, 18.86. ESI-MS m/z: 231.2 [M þ H -Boc]þ。1ss,

4.1.5. General preparation of S-2, 6 and 8 1-Chloro-3-iodobenzene (3.4 mL, 27.5 mmol, 1.1 equiv.) or the aryl bromide (R1Br, for 7 and 9), potassium carbonate (3.8 g, 27.5 mmol, 1.1 equiv.) N,N-dimethylethane-1,2-diamine (0.15 mL, 1.25 mmol, 0.05 equiv.) and CuI (240 mg, 1.25 mmol, 0.05 equiv.) were added to a solution of 1 (8.3 g, 25 mmol, 1 equiv.) in toluene (100 mL) under an N2 atmosphere, and stirred at 90  C overnight. The mixture was then diluted with EtOAc, and washed with saturated aq. NH4Cl, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product, which was purified by flash chromatography (Biotage, gradient elution from 20 to 50% EtOAc) to afford the product. Tert-butyl-1-(3-chlorophenyl)-20 -isopropyl2-oxospiro [indoline-3,30 -pyrrolidine]-10 -carboxylate (S-2) as a white solid (6.4 g, 58%). 1H NMR (400 MHz, CDCl3) d 7.37e7.32 (m, 2H), 7.32e7.28 (m, 1H), 7.23 (m, 2H), 7.14 (dt, J ¼ 7.7, 1.2 Hz, 1H), 7.00 (t, J ¼ 7.7 Hz, 1H), 6.75 (d, J ¼ 7.7 Hz, 1H), 3.97e3.96 (m, 2H), 3.46 (dt, J ¼ 11.2, 6.7 Hz, 1H), 2.21 (dd, J ¼ 8.4, 5.5 Hz, 2H), 2.01e1.99 (m, 1H), 1.39 (s, 9H), 0.94 (d, J ¼ 6.7 Hz, 3H), 0.63 (d, J ¼ 6.7 Hz, 3H). ESI-MS m/z: 441.3 [M þ H]þ.

20 ½a20 D ¼ 24.4 (c ¼ 0.16), 1rr, ½aD ¼ 24.3 (c ¼ 0.16).

4.1.3. General preparation of 3e4 NaH (24 mg, 0.6 mmol 1.2 equiv.) was added to a solution of 1 (165 mg, 0.5 mmol, 1.0 equiv.) in MeCN (5 mL) which was chilled in an ice bath, stirred for 0.5 h. Then R1Br (0.6 mmol, 1.2 equiv.) in MeCN was added to the mixture stirred overnight. Then the mixture was neutralized by aq. NH4Cl. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product, which was purified by flash chromatography (Biotage, 20 g silica gel, gradient elution from 0 to 50% EtOAc).

4.1.5.1. tert-Butyl-20 -isopropyl-1-(4-(methoxycarbonyl)phenyl)-2oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (6). A white solid (120 mg, yield: 52%). 1H NMR (400 MHz, CDCl3) d 7.48 (t, J ¼ 7.8 Hz, 1H), 7.40 (d, J ¼ 7.6 Hz, 1H), 7.34 (d, J ¼ 7.6 Hz, 3H), 7.25 (dt, J ¼ 7.8, 1.3 Hz, 1H), 7.10 (dt, J ¼ 7.6, 1.3 Hz, 1H), 6.88 (d, J ¼ 7.8 Hz, 1H), 4.13 (s, 1H), 4.09 (d, J ¼ 6.3 Hz, 1H), 3.73 (s, 3H), 3.70 (s, 2H), 3.58 (dd, J ¼ 11.2, 7.9, 5.7 Hz, 1H), 2.34e2.32 (m, 2H), 2.13e2.11 (m, 1H), 1.50 (s, 9H), 1.05 (d, J ¼ 6.8 Hz, 3H), 0.74 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.39, 170.50, 155.10, 142.65, 134.52, 133.56, 128.67, 127.89, 127.67, 127.33, 126.46, 124.28, 124.16, 121.62, 108.53, 78.61, 67.15, 55.26, 51.11, 44.99, 39.84, 34.69, 29.75, 27.46, 20.47, 17.97. HRMS (ESI) m/z: calcd. for C27H32N2O5, 465.2384, found 465.2378.

4.1.3.1. tert-Butyl-1-(3-chloropropyl)-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (3). A white solid (180 mg, yield 89%). 1H NMR (400 MHz, CDCl3) d 7.35e7.31 (m, 2H), 7.07 (dt, J ¼ 7.7, 1.1 Hz, 1H), 6.96 (d, J ¼ 7.7 Hz, 1H), 3.94e3.84 (m, 4H), 3.60 (t, J ¼ 6.3 Hz, 2H), 3.53 (dt, J ¼ 11.2, 6.3 Hz, 1H), 2.22e2.15 (m, 4H), 2.08e1.94 (m, 1H), 1.69 (brs, 2H), 1.51 (s, 9H), 1.00 (d, J ¼ 6.7 Hz, 3H), 0.67 (d, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 179.31, 156.17, 143.03, 128.50, 125.09, 122.13, 108.21, 79.60, 67.91, 56.19, 45.88, 42.20, 37.41, 35.19, 30.74, 30.43, 28.47, 21.51, 18.89. HRMS (ESI) m/z: calcd. for C22H31ClN2O3, 407.2096, found 407.2075. 4.1.3.2. tert-Butyl-1-(3-cyanopropyl)-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (4). A white solid (165 mg, yield 83%). 1H NMR (400 MHz, CD3OD) d 7.31e7.23 (m, 2H), 7.02 (dt, J ¼ 7.7, 1.1 Hz, 1H), 6.86 (d, J ¼ 7.7 Hz, 1H), 5.26 (s, 1H), 3.94e3.91 (m, 1H), 3.82e3.71 (m, 3H), 3.44e3.41 (m, 1H), 2.39 (t, J ¼ 7.2 Hz, 2H), 2.26e2.16 (m, 1H), 2.15e2.11 (m, 1H), 1.99 (q, J ¼ 7.0 Hz, 2H), 1.96e1.90 (m, 1H), 1.41 (s, 9H), 0.93 (d, J ¼ 6.8 Hz, 3H), 0.58 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CD3OD) d 180.50, 157.06, 142.97, 129.31, 128.77, 125.81, 123.24, 119.63, 108.79, 80.70, 68.40, 53.97, 46.05, 39.15, 35.09, 31.18, 28.77, 24.09, 22.22, 19.19, 15.25. HRMS (ESI) m/z: calcd. for C23H31N3O3, 398.2438, found 398.2412.

4.1.5.2. tert-Butyl-20 -isopropyl-1-(4-(methoxycarbonyl)-3-(methylsulfonyl)phenyl)-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (8). A white solid (125 mg, yield: 46%). 1H NMR (400 MHz, CDCl3) d 8.15 (d, J ¼ 2.0 Hz, 1H), 7.81 (d, J ¼ 8.1 Hz, 1H), 7.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 7.41e7.27 (m, 1H), 7.28e7.12 (m, 1H), 7.18e7.00 (m, 1H), 6.84 (d, J ¼ 7.9 Hz, 1H), 4.09e3.94 (m, 3H), 3.92 (s, 3H), 3.48 (dt, J ¼ 11.1, 6.5 Hz, 1H), 3.34 (s, 3H), 2.35e2.19 (m, 1H), 2.02e2.00 (m, 1H), 1.84 (s, 1H), 1.41 (s, 9H), 0.97 (d, J ¼ 6.7 Hz, 3H), 0.66 (d, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 178.28, 166.63, 156.08, 142.01, 140.91, 137.26, 131.57, 131.24, 131.13, 128.73, 128.24, 127.38, 125.58, 123.64, 109.32, 79.77, 68.12, 53.35, 45.63, 44.84, 35.30, 30.73, 28.44, 21.81, 18.87. HRMS (ESI) m/z: calcd. for C28H34N2O7S, 543.2159, found 543.2148.

4.1.4. Preparation of 5 To the solution of 4 (100 mg, 0.25 mmol, 1.0 equiv.) in EtOH, was added 1 mL aq NaOH (2 M in water), stirred at 80  C for 2 h. Then the mixture was diluted with EtOAC and neutralized by aq. NH4Cl. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product,

4.1.6. Preparation of 7 and 9 To the solution of 6 or 8 (0.25 mmol, 1 equiv.) in 2 mL DCM, was added DIBAL-H (0.75 mL, 0.75 mol, 3.0 equiv. 1 M in toluene) at 78  C, and stirred for 2 h. Then the mixture was diluted with 11

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times with 20 mL EtOAc, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (Biotage SP-1, 20 g SiO2 column, gradient elution from 20 to 60% EtOAc) to afford the derivatives. Then, the Boc moiety was replace by other R2. The Boc derivative (0.25 mmol, 1.0 equiv.) was dissolved in DCM. HCl (1 mL, 1 M in DCM) was added to the mixture, stirred for 2 h. Then the mixture was concentrated in vacuo to afford the crude. To the solution of the crude in DCM/pyridine (v/v: 2/1), was added by R2OR2 (0.25 mmol, 1.0 equiv.) and stirred overnight. The mixture was diluted with DCM, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (Biotage SP-1, 20 g SiO2 column, gradient elution from 20 to 60% EtOAc).

DCM and neutralized by aq. NH4Cl. The organic layer was washed with water, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product, which was purified by flash chromatography (Biotage, 20 g silica gel, gradient elution from 20 to 60% EtOAc). 4.1.6.1. tert-Butyl-1-(4-(hydroxymethyl)phenyl)-2 0 -isopropyl-2oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (7). A white solid (35 mg, yield: 32%). 1H NMR (400 MHz, CDCl3) d 7.38 (t, J ¼ 7.7 Hz, 1H), 7.30 (dd, J ¼ 7.7, 1.2 Hz, 1H), 7.28e7.20 (m, 3H), 7.14 (dt, J ¼ 7.7, 1.2 Hz, 1H), 7.00 (dt, J ¼ 7.6, 1.1 Hz, 1H), 6.77 (d, J ¼ 7.8 Hz, 1H), 4.00e3.98 (m, 1H), 3.99 (d, J ¼ 6.4 Hz, 2H), 3.60 (s, 2H), 3.50e3.46 (m, 1H), 2.25e2.20 (m, 2H), 2.03e2.01 (m, 1H), 1.40 (s, 9H), 0.95 (d, J ¼ 6.8 Hz, 3H), 0.64 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.52, 174.74, 155.21, 142.60, 134.14, 133.50, 128.69, 128.06, 127.59, 127.38, 126.62, 124.38, 124.14, 121.69, 108.61, 78.84, 67.13, 55.20, 44.97, 39.69, 34.59, 29.73, 27.45, 20.49, 17.95. HRMS (ESI) m/ z: calcd. for C26H32N2O4, 437.2435, found 437.2430.

4.1.8.1. tert-Butyl-1-(4’-(hydroxymethyl)-3’-(methylsulfonyl)-[1,10 biphenyl]-3-yl)-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 carboxylate (10). A white solid (60 mg, yield: 58%). 1H NMR (400 MHz, CDCl3) d 8.14 (d, J ¼ 1.9 Hz, 1H), 7.74 (dd, J ¼ 7.9, 1.9 Hz, 1H), 7.59 (d, J ¼ 7.9 Hz, 1H), 7.57e7.47 (m, 3H), 7.34 (t, J ¼ 6.5 Hz, 2H), 7.16 (t, J ¼ 7.7 Hz, 1H), 7.02 (t, J ¼ 7.7 Hz, 1H), 6.77 (d, J ¼ 7.9 Hz, 1H), 4.90 (d, J ¼ 6.5 Hz, 2H), 4.11e3.86 (m, 2H), 3.63 (t, J ¼ 6.5 Hz, 1H), 3.50e3.47 (m, 1H), 3.09 (s, 3H), 2.39e2.20 (m, 2H), 2.04 (q, J ¼ 6.6 Hz, 1H), 1.37 (s, 9H), 0.97 (d, J ¼ 6.8 Hz, 3H), 0.68 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.63, 155.20, 142.50, 139.39, 139.36, 138.68, 137.77, 134.10, 131.45, 130.71, 129.36, 127.49, 127.35, 127.15, 125.75, 125.43, 124.33, 124.30, 121.89, 108.43, 78.74, 66.99, 61.10, 55.24, 44.82, 43.96, 34.42, 29.76, 27.43, 20.74.17.91. HRMS (ESI) m/z: calcd. for C33H38N2O6S, 613.2343, found

4.1.6.2. tert-Butyl-1-(4-(hydroxymethyl)-3-(methylsulfonyl)phenyl)20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (9). A white solid (110 mg, yield: 43%). 1H NMR (400 MHz, CDCl3) d 8.03 (d, J ¼ 2.1 Hz, 1H), 7.69 (d, J ¼ 8.1 Hz, 2H), 7.64 (dd, J ¼ 8.1, 2.1 Hz, 1H), 7.34 (dd, J ¼ 7.6, 1.2 Hz, 1H), 7.26e7.13 (m, 1H), 7.06 (dt, J ¼ 7.6, 1.2 Hz, 1H), 6.78 (d, J ¼ 7.9 Hz, 1H), 4.94 (s, 2H), 4.13e3.90 (m, 3H), 3.48 (dt, J ¼ 11.2, 6.6 Hz, 1H), 3.14 (s, 3H), 2.26 (t, J ¼ 7.3 Hz, 2H), 2.07e1.83 (m, 1H), 1.41 (s, 9H), 0.98 (d, J ¼ 6.8 Hz, 3H), 0.67 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.50, 155.17, 141.59, 138.78, 138.73, 133.83, 131.52, 131.19, 127.67, 127.26, 126.70, 124.45, 122.34, 108.30, 78.82, 67.12, 61.32, 55.21, 44.71, 44.04, 34.31, 29.75, 27.45, 20.76, 17.88. HRMS (ESI) m/z: calcd. for C27H34N2O6S, 537.2030, found 537.2041.

20 613.2350.10ss, ½a20 D ¼ 49.1 (c ¼ 0.20), ee: 100%; 10rr, ½aD ¼ 45.6 (c ¼ 0.22), ee: 100%.

4.1.7. General preparation of S-3 Bis(pinacolato)diboron (2.8 g, 11 mmol, 1.1 equiv.), potassium carbonate (1.52 g, 11 mmol, 1.1 equiv.), X-Phos (240 mg, 0.5 mmol, 0.05 equiv.) and Pd2 (DBA)3 (460 mg, 0.5 mmol, 0.05 equiv.) were added to a solution of S-2 (4.4 g, 10 mmol, 1.0 equiv.) in toluene (60 mL) under an N2 atmosphere, and stirred at 95  C overnight. The mixture was cooled in an ice bath, and this was followed by addition of saturated aq. NH4Cl to quench the reaction. The mixture was extracted three times with 100 mL EtOAc, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo to afford the crude product which was dissolved in EtOAc, and diluted with hexane/EtOAc (v/v: 1/1) for precipitation. Then, the mixture was filtered and washed with hexane/EtOAc (v/v: 1/1) to afford 3.2 g of tert-butyl-20 -isopropyl-2-oxo-1-(3-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl) spiro[indoline-3,30 -pyrrolidine]-10 carboxylate (S-3), as a white solid (3.2 g, 61%). 1H NMR (400 MHz, CDCl3) d 7.76 (d, J ¼ 7.6 Hz, 1H), 7.72 (d, J ¼ 2.0 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.38 (d, J ¼ 7.8 Hz, 1H), 7.30 (d, J ¼ 7.6 Hz, 1H), 7.13 (t, J ¼ 7.8 Hz, 1H), 6.99 (t, J ¼ 7.6 Hz, 1H), 6.69 (d, J ¼ 7.8 Hz, 1H), 4.08e3.95 (m, 2H), 3.48 (dt, J ¼ 11.2, 6.7 Hz, 1H), 2.23e2.21 (m, 2H), 2.02 (q, J ¼ 6.8 Hz, 1H), 1.40 (s, 9H), 1.26 (s, 12H), 0.96 (d, J ¼ 6.8 Hz, 3H), 0.66 (d, J ¼ 6.8 Hz, 3H).

4.1.8.2. 1’-(3,3-dimethylbutanoyl)-1-(4’-(hydroxymethyl)-3’-(methylsulfonyl)-[1,10 -biphenyl]-3-yl)-20 -isopropylspiro[indoline-3,30 -pyrrolidin]-2-one (11). A white solid (70 mg, yield: 48%). 1H NMR (400 MHz, CDCl3) d 8.15 (d, J ¼ 1.8 Hz, 1H), 7.75 (dd, J ¼ 7.9, 1.8 Hz, 1H), 7.59e7.52 (m, 5H), 7.39e7.27 (m, 2H), 7.24e7.11 (m, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.78 (d, J ¼ 7.8 Hz, 1H), 4.91 (s, 2H), 4.42 (d, J ¼ 6.0 Hz, 1H), 4.03 (q, J ¼ 8.8 Hz, 1H), 3.66 (dt, J ¼ 9.5, 3.2 Hz, 1H), 3.39 (d, J ¼ 13.0 Hz, 1H), 3.12 (s, 3H), 2.45 (dt, J ¼ 13.0, 8.6 Hz, 1H), 2.40e2.32 (m, 1H), 2.19 (d, J ¼ 13.0 Hz, 1H), 2.08 (q, J ¼ 6.8 Hz, 1H), 1.02 (s, 9H), 0.97 (d, J ¼ 6.8 Hz, 3H), 0.72 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.50, 171.70, 142.77, 139.65, 139.37, 138.51, 137.97, 134.06, 131.58, 131.09, 131.00, 129.34, 127.66, 127.58, 127.46, 127.32, 126.64, 125.81, 125.54, 124.45, 124.25, 121.82, 108.49, 65.36, 61.42, 53.97, 45.75, 45.43, 44.09, 33.34, 30.36, 29.68, 29.09, 21.35, 18.06. HRMS (ESI) m/z: calcd. for C34H40N2O5S, 611.2550, found 611.2562. 4.1.8.3. 1-(4’-(hydroxymethyl)-3’-(methylsulfonyl)-[1,10 -biphenyl]-3yl)-20 -isopropyl-10 -pivaloylspiro[indoline-3,30 -pyrrolidin]-2-one (12). A white solid (60 mg, yield: 42%). 1H NMR (500 MHz, CDCl3) d 8.15 (d, J ¼ 1.8 Hz, 1H), 7.75 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H), 7.57e7.49 (m, 3H), 7.35 (dd, J ¼ 7.1, 3.1 Hz, 2H), 7.23e7.14 (m, 1H), 7.04 (t, J ¼ 7.5 Hz, 1H), 6.76 (d, J ¼ 7.8 Hz, 1H), 4.90 (s, 2H), 4.56 (d, J ¼ 6.8 Hz, 1H), 4.23 (dt, J ¼ 10.4, 7.5 Hz, 1H), 3.77 (dt, J ¼ 10.4, 7.2 Hz, 1H), 3.40 (s, 1H), 3.12 (s, 3H), 2.40 (t, J ¼ 7.2 Hz, 2H), 2.07 (h, J ¼ 6.8 Hz, 1H), 1.26 (s, 9H), 0.90 (d, J ¼ 6.8 Hz, 3H), 0.65 (d, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 177.67, 177.11, 142.79, 139.65, 139.39, 138.47, 137.93, 134.13, 131.59, 131.00, 129.40, 127.57, 127.32, 127.12, 125.86, 125.70, 124.54, 124.14, 121.74, 108.46, 67.08, 61.42, 53.33, 45.32, 44.07, 38.44, 34.42, 29.30, 27.00, 20.88, 17.68. HRMS (ESI) m/z: calcd. for C33H38N2O5S, 597.2394, found 597.2388.

4.1.8. Synthesis of compounds 10 to 24 Compounds 10 to 24 were synthesized by the general method described here. Aryl bromide (0.25 mmol, 1.1 equiv.), potassium carbonate (35 mg, 0.25 mmol, 1.1 equiv.) and Pd (DPPF)Cl2 (20 mg, 0.025 mmol, 0.1 equiv.) were added under an N2 atmosphere to a solution of S-3 (100 mg, 0.2 mmol, 1.0 equiv.) in 2,4-dioxane (3 mL), and the mixture was stirred at 85  C overnight. The mixture was cooled in an ice bath, and this was followed by addition of saturated aq. NH4Cl to quench the reaction. The mixture was extracted three 12

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European Journal of Medicinal Chemistry 206 (2020) 112793

4.1.8.4. 1-(4’-(hydroxymethyl)-3’-(methylsulfonyl)-[1,10 -biphenyl]-3yl)-20 -isopropyl-1’-(2,2,2-trifluoroacetyl)spiro[indoline-3,30 -pyrrolidin]-2-one (13). A white solid (83 mg, yield: 57%). 1H NMR (500 MHz, CDCl3) d 8.17 (d, J ¼ 3.1 Hz, 1H), 7.78 (d, J ¼ 6.5 Hz, 1H), 7.58 (m, 4H), 7.36 (d, J ¼ 7.6 Hz, 2H), 7.25e7.20 (m, 1H), 7.08 (d, J ¼ 6.5 Hz, 1H), 6.81 (d, J ¼ 7.1 Hz, 1H), 4.91 (d, J ¼ 7.2 Hz, 1H), 4.42 (t, J ¼ 4.5 Hz, 1H), 4.23 (q, J ¼ 9.3 Hz, 1H), 3.88e3.79 (m, 1H), 3.63 (t, J ¼ 5.6 Hz, 1H), 3.13 (s, 3H), 3.12e3.05 (m, 2H), 2.49 (dd, J ¼ 8.6, 3.5 Hz, 1H), 2.44 (dd, J ¼ 8.6, 4.1 Hz, 1H), 2.08e2.06 (m, 1H), 0.99 (d, J ¼ 6.6 Hz, 3H), 0.75 (d, J ¼ 6.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 178.00, 157.75(q), 143.80, 140.77, 140.49, 139.41, 139.13, 134.78, 132.68, 132.19, 130.51, 129.18, 128.49, 127.08, 126.71, 126.51, 125.41, 125.31, 123.19, 116.55 (q), 109.85, 68.44, 62.65, 54.50, 45.17, 45.04, 34.38, 30.35, 22.02, 18.58. HRMS (ESI) m/z: calcd. for C30H29F3N2O5S, 609.1641, found 609.1639.

139.48, 134.89, 130.00, 128.79, 128.37, 127.48, 127.39, 126.71, 125.45, 125.24, 122.66, 109.52, 79.79, 64.99, 56.23, 46.06, 35.76, 30.80, 29.70, 28.48, 21.49, 19.00. HRMS (ESI) m/z: calcd. for C32H36N2O4, 535.2567, found 535.2571. 4.1.8.8. tert-Butyl-1-(3 0 -chloro-4’-(dimethylcarbamoyl)-[1,1 0 biphenyl]-3-yl)-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 carboxylate (17). A white solid (80 mg, yield: 55%). 1H NMR (400 MHz, CDCl3) d 7.62 (s, 1H), 7.61e7.56 (m, 3H), 7.54 (d, J ¼ 7.8 Hz, 1H), 7.41 (t, J ¼ 7.1 Hz, 2H), 7.36 (d, J ¼ 7.8 Hz, 1H), 7.24 (t, J ¼ 7.8 Hz, 1H), 7.09 (t, J ¼ 7.5 Hz, 1H), 6.87 (d, J ¼ 7.8 Hz, 1H), 4.09 (q, J ¼ 7.1 Hz, 2H), 3.56 (dt, J ¼ 12.2, 6.8 Hz, 1H), 3.14 (s, 3H), 2.90 (s, 3H), 2.40e2.26 (m, 2H), 2.10 (h, J ¼ 6.7 Hz, 1H), 1.47 (s, 9H), 1.04 (d, J ¼ 6.7 Hz, 3H), 0.74 (d, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 178.51, 168.16, 156.11, 143.51, 142.24, 140.67, 135.45, 135.11, 130.86, 130.24, 128.55, 128.45, 128.27, 128.14, 126.63, 126.20, 126.07, 125.35, 125.30, 122.84, 109.41, 79.62, 74.81, 68.13, 60.33, 56.23, 45.92, 38.11, 34.68, 30.77, 28.47, 21.62. HRMS (ESI) m/z: calcd. for C34H38ClN3O4, 588.2624, found 588.2617.

4.1.8.5. 3’-(1’-(tert-butoxycarbonyl)-20 -isopropyl-2-oxospiro[indoline-3,3 0 -pyrrolidin]-1-yl)-3-(methylsulfonyl)-[1,1 0 -biphenyl]-4carboxylic acid (14). The intermediate, tert-butyl 20 -isopropyl-1(4’-(methoxycarbonyl)-3’-(methylsulfonyl)-[1,10 -biphenyl]-3-yl)-2oxospiro [indoline-3,30 -pyrrolidine]-10 -carboxylate, was synthesized by the preparation method 4.1.8., without further purification. The crude was dissolved in THF/MeOH/water (v/v/v: 2/1/1). LiOH (12 mg, 0.5 mmol, 2.2 equiv.) was added to the mixture, stirred for 5 h. The mixture was cooled in an ice bath, and this was followed by addition of saturated aq. NH4Cl to quench the reaction. The mixture was extracted three times with 20 mL EtOAc, washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (Biotage SP-1, 20 g SiO2 column, gradient elution from 20 to 60% EtOAc) to afford the product, as a white solid (78 mg, yield: 52%). 1H NMR (500 MHz, CDCl3) d 8.27 (d, J ¼ 1.6 Hz, 1H), 7.86e7.74 (m, 2H), 7.66e7.53 (m, 3H), 7.41 (dd, J ¼ 5.5, 3.8 Hz, 1H), 7.35 (d, J ¼ 7.5 Hz, 1H), 7.22e7.13 (m, 1H), 7.04 (t, J ¼ 7.6 Hz, 1H), 6.78 (d, J ¼ 7.9 Hz, 1H), 4.12e3.93 (m, 3H), 3.52 (dt, J ¼ 12.0, 6.7 Hz, 1H), 3.34 (s, 3H), 2.37e2.23 (m, 1H), 2.11e2.01 (m, 1H), 1.41 (s, 9H), 0.99 (d, J ¼ 6.8 Hz, 3H), 0.70 (d, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 178.85, 168.70, 156.43, 143.48, 143.32, 140.08, 139.89, 135.20, 131.82, 131.79, 131.14, 130.53, 128.57, 128.51, 128.48, 127.04, 125.67, 125.38, 123.04, 109.54, 109.49, 75.59, 67.99, 56.45, 45.86, 44.93, 30.80, 28.52, 28.45, 21.76, 18.93. HRMS (ESI) m/z: calcd. for C33H36N2O7S, 627.2135, found 627.2151.

4.1.8.9. 3-Chloro-3’-(1’-(3,3-dimethylbutanoyl)-2 0 -isopropyl-2oxospiro[indoline-3,3 0 -pyrrolidin]-1-yl)-N,N-dimethyl-[1,1 0 biphenyl]-4-carboxamide (18). A white solid (85 mg, yield: 58%). 1H NMR (400 MHz, CDCl3) d 7.62 (d, J ¼ 1.7 Hz, 1H), 7.60e7.57 (m, 3H), 7.54 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.42e7.40 (m, 2H), 7.37 (d, J ¼ 7.9 Hz, 1H), 7.29e7.22 (m, 1H), 7.11 (dt, J ¼ 7.6, 1.1 Hz, 1H), 6.87 (d, J ¼ 7.9 Hz, 1H), 4.48 (d, J ¼ 6.1 Hz, 1H), 4.10 (dt, J ¼ 10.1, 8.3 Hz, 1H), 3.72 (dt, J ¼ 9.5, 3.4 Hz, 1H), 3.16 (s, 3H), 2.91 (s, 3H), 2.51 (dt, J ¼ 13.1, 8.7 Hz, 1H), 2.41e2.36 (m, 1H), 2.37 (d, J ¼ 14.2 Hz, 1H), 2.28 (d, J ¼ 14.2 Hz, 1H), 2.15 (h, J ¼ 6.8 Hz, 1H), 1.11 (s, 9H), 1.04 (d, J ¼ 6.8 Hz, 3H), 0.78 (d, J ¼ 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.42, 171.59, 167.22, 142.78, 141.23, 139.73, 134.39, 133.93, 129.85, 129.21, 127.60, 127.23, 127.19, 126.69, 125.70, 125.31, 125.09, 124.40, 124.23, 121.74, 108.46, 65.33, 53.94, 45.72, 45.39, 37.13, 33.69, 33.32, 30.34, 29.68, 29.08, 21.31, 18.05. HRMS (ESI) m/z: calcd. for C35H40ClN3O3, 608.2650, found 608.2638. 4.1.8.10. 3-Chloro-3’-(-20 -isopropyl-2-oxo-10 -pivaloylspiro[indoline3,30 -pyrrolidin]-1-yl)-N,N-dimethyl-[1,10 -biphenyl]-4-carboxamide (19). A white solid (80 mg, yield: 56%). 1H NMR (500 MHz, CDCl3) d 7.64e7.57 (m, 1H), 7.55 (d, J ¼ 1.6 Hz, 1H), 7.51 (t, J ¼ 6.8 Hz, 2H), 7.47 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.39 (dt, J ¼ 7.8, 2.7 Hz, 1H), 7.34 (dd, J ¼ 7.0, 2.7 Hz, 2H), 7.30 (d, J ¼ 7.8 Hz, 1H), 7.22e7.15 (m, 1H), 7.04 (t, J ¼ 7.8 Hz, 1H), 4.57 (d, J ¼ 6.8 Hz, 1H), 4.23 (dt, J ¼ 10.5, 7.2 Hz, 1H), 3.77 (dt, J ¼ 10.5, 7.0 Hz, 1H), 3.09 (s, 3H), 2.85 (s, 3H), 2.38 (t, J ¼ 7.2 Hz, 2H), 2.07 (h, J ¼ 6.8 Hz, 1H), 1.28 (s, 9H), 0.91 (d, J ¼ 6.8 Hz, 3H), 0.64 (d, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 177.57, 177.00, 167.22, 142.81, 141.25, 139.76, 134.38, 134.04, 129.27, 127.53, 127.51, 127.23, 127.21, 127.19, 125.76, 125.50, 125.11, 124.49, 124.13, 121.66, 108.43, 67.10, 53.33, 45.32, 38.44, 37.14, 34.45, 33.69, 29.30, 27.01, 20.82, 17.68. HRMS (ESI) m/z: calcd. for C34H38ClN3O3, 572.2674, found 572.2704.

4.1.8.6. tert-Butyl-1-(30 -chloro-4’-(hydroxymethyl)-[1,10 -biphenyl]3-yl)-20 -isopropyl-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (15). A white solid (62 mg, yield: 45%). 1H NMR (400 MHz, CDCl3) d 7.58e7.49 (m, 4H), 7.48 (s, 1H), 7.44 (dd, J ¼ 7.9, 1.8 Hz, 1H), 7.33 (dt, J ¼ 6.0, 3.3 Hz, 2H), 7.184 (t, J ¼ 6.8 Hz 1H), 7.03 (t, J ¼ 6.8 Hz 1H), 6.81 (d, J ¼ 7.9 Hz, 1H), 4.75 (s, 2H), 4.03 (d, J ¼ 6.6 Hz, 2H), 3.50 (dt, J ¼ 11.5, 7.2 Hz, 1H), 2.37e2.14 (m, 1H), 2.07e2.05 (m, 2H), 1.41 (s, 9H), 0.98 (d, J ¼ 6.7 Hz, 3H), 0.68 (d, J ¼ 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.50, 155.15, 142.61, 140.09, 139.85, 136.58, 133.96, 132.09, 129.13, 128.04, 127.61, 127.39, 126.86, 125.62, 124.94, 124.75, 124.26, 124.20, 121.73, 108.43, 78.72, 61.51, 56.40, 49.84, 44.96, 34.60, 29.78, 27.45, 20.55, 17.95. HRMS (ESI) m/z: calcd. for C32H35ClN2O4, 569.2178, found 569.2188.

4.1.8.11. 3’-(-1’-(tert-butylcarbamothioyl)-20 -isopropyl-2-oxospiro [indoline-3,3 0 -pyrrol-idin]-1-yl)-3-chloro-N,N-dimethyl-[1,1 0 biphenyl]-4-carboxamide (20). A white solid (50 mg, yield: 34%). 1H NMR (400 MHz, CDCl3) d 7.65e7.63 (m, 3H), 7.58e7.53 (m, 1H), 7.45 (s, 1H), 7.40 (d, J ¼ 2.8 Hz, 1H), 7.38 (t, J ¼ 3.5 Hz, 1H), 7.29 (d, J ¼ 2.8 Hz, 1H), 7.28e7.23 (m, 1H), 7.12 (d, J ¼ 7.3 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 4.80 (d, J ¼ 7.0 Hz, 1H), 4.33 (q, J ¼ 8.4 Hz, 1H), 3.41e3.37 (m, 1H), 3.75 (dt, J ¼ 9.5, 3.8 Hz, 1H), 3.17 (s, 3H), 2.93 (s, 3H), 2.56e2.47 (m, 1H), 2.39e2.27 (m, 1H), 2.05 (brs, 6H), 1.58 (brs, 3H), 0.83 (d, J ¼ 6.7 Hz, 3H), 0.60 (d, J ¼ 6.7 Hz, 3H)$13C NMR (100 MHz, CDCl3) d 179.80, 176.78, 167.20, 142.65, 141.58, 139.71, 134.45, 133.95, 131.22, 129.87, 129.23, 127.77, 127.26, 127.17, 126.61,

4.1.8.7. tert-Butyl-1-(4’-(hydroxymethyl)-[1,10 -biphenyl]-3-yl)-20 isopropyl-2-oxospiro-[indoline-3,30 -pyrrolidine]-10 -carboxylate (16). A white solid (86 mg, yield: 67%). 1H NMR (400 MHz, CDCl3) d 7.67e7.55 (m, 5H), 7.47 (d, J ¼ 7.8 Hz, 2H), 7.43 (d, J ¼ 7.6 Hz, 1H), 7.40 (dd, J ¼ 7.6, 1.7 Hz, 1H), 7.26 (d, J ¼ 7.8 Hz, 1H), 7.12 (t, J ¼ 7.5 Hz, 1H), 6.92 (d, J ¼ 7.9 Hz, 1H), 4.77 (s, 2H), 4.13 (d, J ¼ 7.1 Hz, 2H), 3.60 (dt, J ¼ 12.0, 6.8 Hz, 1H), 2.38 (m, 2H), 2.14 (h, J ¼ 6.6 Hz, 1H), 1.51 (d, J ¼ 1.5 Hz, 9H), 1.07 (d, J ¼ 6.6 Hz, 3H), 0.76 (d, J ¼ 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 178.51, 156.16, 143.79, 142.50, 140.49, 13

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121.39, 120.10, 118.17, 108.63, 106.97, 99.81, 67.17, 53.37, 45.41, 38.46, 34.60, 31.97, 29.33, 27.03, 20.76, 17.71. HRMS (ESI) m/z: calcd. for C34H37N3O2, 542.2778, found 542.2779.

125.70, 125.06, 124.33, 124.03, 122.08, 108.53, 73.91, 68.21, 55.48, 54.79, 53.15, 47.12, 44.25, 40.02, 37.12, 33.69, 28.26. HRMS (ESI) m/z: calcd. for C34H39ClN4O2S,604.2555, found 604.2557. 4.1.8.12. 3-Chloro-3’-(-20 -isopropyl-1’-(isopropylsulfonyl)-2-oxospiro [indoline-3,3 0 -pyrrol-idin]-1-yl)-N,N-dimethyl-[1,1 0 -biphenyl]-4carboxamide (21). A white solid (75 mg, yield: 50%). 1H NMR (400 MHz, CDCl3) d 7.68e7.61 (m, 3H), 7.59 (d, J ¼ 2.1 Hz, 1H), 7.56 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 2H), 7.40 (d, J ¼ 7.8 Hz, 1H), 7.32 (t, J ¼ 7.8 Hz, 1H), 7.16 (t, J ¼ 7.6 Hz, 1H), 6.96 (d, J ¼ 7.8 Hz, 1H), 4.45 (dd, J ¼ 11.0, 8.6 Hz, 1H), 4.10 (d, J ¼ 8.6 Hz, 1H), 3.36 (dt, J ¼ 11.0, 5.6 Hz, 1H), 3.18 (s, 3H), 2.94 (s, 3H), 2.47 (dt, J ¼ 11.0, 8.6 Hz, 1H), 2.03e1.93 (m, 2H), 1.97 (d, J ¼ 7.2 Hz, 1H), 1.92 (s, 6H), 1.09 (d, J ¼ 6.7 Hz, 3H), 0.60 (d, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 176.48, 168.18, 142.56, 142.21, 140.89, 135.56, 135.13, 130.94, 130.42, 130.37, 128.40, 128.32, 128.20, 126.90, 126.09, 125.97, 125.14, 125.06, 122.97, 109.84, 84.38, 75.11, 56.94, 40.96, 40.42, 38.14, 34.72, 31.56, 28.36, 27.67, 22.09, 19.02. HRMS (ESI) m/z: calcd. for C32H36ClN3O4S, 616.2007, found 616.2001.

4.2. Molecular docking The crystal structure of LXRb with the native ligand was obtained from the RCSB Protein Data Bank (PDB code: 6K9G). The initial three-dimensional structures of the compounds (10rr, 18rr, 20rr and 21rr) were computed and optimized using B3LYP/6-31G* method by the Gaussian 09 program [25,26]. Two different virtual screening programs (MOE and Schrӧdinger) were employed to generate the initial receptor-ligand complexes [27]. All the parameters were set to the default values. For each compound, 10 conformations were sampled in short time molecular dynamic simulations to test the stability of the model. MM-GBSA energies were calculated to rescore the binding complexes. The top-3 lowest energy conformers were extracted and for extended molecular dynamic simulations. The co-activator (NCOA1) and co-repressor (NCOR1) were obtained from co-crystal structure (PDB codes: 6K9G and 4WVD, respectively). The conformation of the LXRb with compounds 10rr and 18rr were computed by Method 4.3. We employed the docking program of Schrӧdinger to prepare the NCOA1-LXRb and NCOR1-LXRb complexes. The residues Lys287, Glu455 and Asp458 at LXRb were defined as an attractive term to constraint on the docking process. The docking results were shown in Figs. S3 and S4 in Supporting Information.

4.1.8.13. tert-Butyl-2 0 -isopropyl-1-(3-(1-methyl-1H-indol-6-yl) phenyl)-2-oxospiro[indoline-3,30 -pyrrolidine]-10 -carboxylate (22). A white solid (68 mg, yield: 51%). 1H NMR (400 MHz, CDCl3) d 7.76e7.71 (m, 3H), 7.62 (t, J ¼ 7.8 Hz, 1H), 7.57 (s, 1H), 7.45 (d, J ¼ 7.6 Hz, 1H), 7.43e7.40 (m, 1H), 7.38 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.32e7.19 (m, 1H), 7.18e7.05 (m, 2H), 6.95 (d, J ¼ 7.9 Hz, 1H), 6.54 (d, J ¼ 3.1 Hz, 1H), 4.17 (d, J ¼ 5.8 Hz, 2H), 3.87 (s, 3H), 3.62 (dd, J ¼ 11.3, 7.8, 5.8 Hz, 1H), 2.42e2.40 (m, 2H), 2.17 (h, J ¼ 6.7 Hz, 1H), 1.53 (s, 9H), 1.10 (d, J ¼ 6.7 Hz, 3H), 0.80 (d, J ¼ 6.7 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 177.49, 155.15, 143.23, 142.95, 136.15, 133.72, 132.93, 128.84, 128.77, 127.73, 127.34, 127.14, 126.11, 124.51, 124.16, 123.70, 121.53, 120.12, 118.15, 108.59, 106.89, 99.83, 78.63, 67.16, 55.44, 45.04, 34.71, 31.91, 29.78, 27.47, 20.49, 17.98. HRMS (ESI) m/z: calcd. for C34H37N3O4, 558.2727, found 558.2742.

4.3. Molecular dynamic (MD) simulation AMBER 12 [28,29] was employed for LXRb-ligand complex MD simulations. For each compound, a best LXRb-ligand complex produced by the docking program was used as the initial conformation for the MD simulations with ff99SB (for the protein) and Generalized Amber Force Field (for the ligand) [30] force field. The partial atomic charges of the ligands were obtained from the restrained electrostatic potential (RESP) charges based on HF/631G* calculations with the Gaussian 09 program [25]. Each system was solvated in a truncated octahedron box of TIP3P water molecules [31] with a margin distance of 10 Å. The receptor-ligand complexes were neutralized by adding sodium or chlorine counter ions. For each receptor-ligand complex, three steps of energy minimization were performed before heating. The first step involved 2000 cycles of steepest descent minimization and 2000 cycles of conjugated gradient minimization, with all complex atoms constrained by a restricting potential of 50 kcal/mol/Å. Solvent molecules were not restrained. All heavy atoms were subject to 5 kcal/ mol/Å constraints during 2500 cycles of steepest descent minimization and 2500 cycles of conjugated gradient minimization. Finally, 5000 cycles of steepest descent minimization and 5000 cycles of conjugated gradient minimization were carried out without constraints. After the minimization, the system was heated from 0 to 300 K in 50 ps (ps) using Langevin dynamics at constant volume, and then equilibrated for 500 ps at a constant pressure of 1 atm. A weak constraint of 10 kcal/mol/Å was used to restrain all of the heavy atoms in the receptor-ligand complexes during the heating steps. A production simulation run for 20 ns was performed using the NVT ensemble under a target temperature of 300 K. All bonds involving hydrogen atoms were constrained by SHAKE algorithm and a time step of 2.0 fs was applied for all of the simulations [21]. The interaction between each ligand and protein residue was computed

4.1.8.14. 1’-(3,3-Dimethylbutanoyl)-20 -isopropyl-1-(3-(1-methyl-1Hindol-6-yl)phenyl)-spiro[indoline-3,30 -pyrrolidin]-2-one (23). A white solid (50 mg, yield: 36%). 1H NMR (500 MHz, CDCl3) d 7.66e7.60 (m, 2H), 7.60e7.58 (m, 1H), 7.50 (t, J ¼ 7.8 Hz, 1H), 7.47 (s, 1H), 7.33 (d, J ¼ 7.6 Hz, 1H), 7.30 (dd, J ¼ 8.1, 1.4 Hz, 1H), 7.25 (d, J ¼ 8.1 Hz, 1H), 7.17 (d, J ¼ 7.8 Hz, 1H), 7.06e6.92 (m, 2H), 6.82 (d, J ¼ 7.9 Hz, 1H), 6.43 (d, J ¼ 3.0 Hz, 1H), 4.44 (d, J ¼ 6.1 Hz, 1H), 3.78 (s, 3H), 3.69e3.65 (m, 1H), 3.63 (brs, 1H), 2.44 (dt, J ¼ 13.3, 8.9 Hz, 1H), 2.38e2.30 (m, 2H), 2.29 (d, J ¼ 13.3 Hz, 1H), 2.23 (d, J ¼ 13.3 Hz, 1H), 2.11e2.09 (m, 1H), 1.05 (s, 9H), 0.98 (d, J ¼ 6.7 Hz, 3H), 0.73 (d, J ¼ 6.7 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 177.42, 170.14, 143.23, 136.14, 133.57, 132.91, 128.85, 128.76, 127.52, 127.11, 126.79, 126.20, 124.54, 124.11, 123.85, 121.47, 120.10, 118.16, 108.65, 106.96, 99.80, 65.36, 53.96, 45.74, 45.44, 33.45, 31.97, 30.34, 29.70, 21.28, 20.03, 18.07. HRMS (ESI) m/z: calcd. for C35H39N3O2, 556.2934, found 556.2924. 4.1.8.15. 20 -isopropyl-1-(3-(1-methyl-1H-indol-6-yl)phenyl)-10 -pivaloylspiro[indoline-3,30 -pyrrolidin]-2-one (24). A white solid (60 mg, yield: 46%). 1H NMR (500 MHz, CDCl3) d 7.63e7.60 (m, 2H), 7.59 (d, J ¼ 6.8 Hz, 1H), 7.50 (t, J ¼ 7.8 Hz, 1H), 7.46 (s, 1H), 7.34 (d, J ¼ 7.6 Hz, 1H), 7.30 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.25 (d, J ¼ 7.6 Hz, 1H), 7.16 (d, J ¼ 7.6 Hz, 1H), 7.06e6.96 (m, 2H), 6.81 (d, J ¼ 7.9 Hz, 1H), 6.43 (d, J ¼ 3.0 Hz, 1H), 4.59 (d, J ¼ 6.8 Hz, 1H), 4.27e4.23 (m, 1H), 3.78 (overlapped, 4H), 2.44e2.35 (m, 2H), 2.08 (h, J ¼ 6.8 Hz, 1H), 1.29 (s, 9H), 0.91 (d, J ¼ 6.8 Hz, 3H), 0.66 (d, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) d 177.56, 161.42, 143.26, 136.14, 133.66, 132.95, 128.90, 128.75, 127.43, 127.33, 127.10, 126.25, 124.63, 124.00, 123.98,

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European Journal of Medicinal Chemistry 206 (2020) 112793

4.8. Quantitative real-time PCR

using the MM/GBSA (molecular mechanics generalized Born surface area) decomposition process applied in the mm pbsa program from AMBER16 [21].

Total RNA was extracted by using RNAiso plus (TaKaRa) according to its protocol. 1 mg of total RNA was reverse transcribed into cDNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO), and quantitative real-time PCR amplification was performed using SYBR Green Realtime PCR Master Mix (TOYOBO). Gene expression was normalized to that of b-actin, which was used as an endogenous control.

4.4. Cell culture and treatment HEK293T, 3T3-L1 and HepG2 cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin at 37  C and 5% CO2. 3T3-L1 adipocytes differentiation was performed as previously described [32]. Briefly, 2 days after full confluence (day 0), cells were cultured in differentiation medium containing 2 mg/mL insulin, 0.1 mg/mL dexamethasone, 0.5 mM 3-isobutyl-1methylxanthine (IBMX), 10 ng/mL biotin, 10% fetal bovine serum and 1% penicillin/streptomycin for 3 days (day 3). Then, the cells were cultured in medium containing 2 mg/mL insulin, 10% fetal bovine serum and 1% penicillin/streptomycin for another 3 days (day 6). Finally, cells were collected for TG assay, Oil Red-O staining and RNA extraction. To induce lipogenesis by GW3965, HepG2 cells were seeded in 48-well plates at 1  105 per well overnight and then treated with GW3965 and tested compounds for 4 days. To induce lipogenesis by a high-glucose medium, HepG2 cells were grown in 12-well plates. After cells grown to 70% confluence, the cells were quenched overnight in serum-free DMEM containing 1 g/L D-glucose. Then, media was changed to serum-free DMEM containing 4.5 g/L Dglucose, and the cells were treated with tested compounds for 48 h. To analyze mRNA expression, HepG2 cells were seeded in 6-well plates at 8  106 per well overnight and then treated with tested compounds for 24 h.

4.9. Lipid content measurement Cells were collected and washed twice with phosphate-buffered saline (PBS), then ultrasonicated in ice-cold PBS. The triglyceride content of lysates was measured by Triglycerides Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein concentration was determined by BCA assay using the BCA kit (Pierce) according to the manufacturer’s instructions. Liver tissues were homogenized and extracted with CHCl3eMeOH (1:2). The samples were centrifuged (3000 r/min, 4  C, 10 min). The chloroform phase was evaporated to dryness and measured using the Triglyceride Assay Kit and the Total-Cholesterol Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum was separated by centrifugation (3000 r/min, 4  C, 10 min). Serum triglycerides (TG), total cholesterol (TC), and lowdensity lipoprotein-cholesterol (LDL-C) were measured using TG, TC and LDL-C assay kits purchased from Nanjing Jiancheng Bioengineering Institute. 4.10. Oil Red-O staining

4.5. Animal studies

After fixed with 4% paraformaldehyde for 1 h, cells were stained by Oil Red-O for 1 h at RT. Liver tissues were fixed in formalin and stained by Oil Red-O according to a standard procedure. Imaging was performed on EVOS FL Auto Cell Imaging System (Invitrogen).

6-week-old female C57BL/6 mice were randomly divided into 4 groups (n ¼ 6), the control group, hyperlipidemia model group, fenofibrate group (100 mg/kg, ig) and a treatment group in which mice were treated with 10 (100 mg/kg, ig), administered by gastric lavage for 8 days. 24 h before the last administration, acute hyperlipidemia was induced by an intramuscular injection of Triton WR-1339 (1500 mg/kg). Then 1 h after the last administration, blood was drawn from eyeballs of the mice which were then sacrificed.

4.11. Presentation of data and statistical analysis Data are represented as means ± SD. The two-tailed unpaired Student’s t-test was used for comparisons of means between two groups. ANOVA was used for comparison of means between >3 groups with Tukey’s hoc test for multiple comparisons. P < 0.05 was considered statistically significant.

4.6. Transient transfection and luciferase reporter assays HEK293T cells were seeded in 96-well plates at 2  104 per well for 24 h. The cells were transfected using Lipofectamine 3000 (Gibco) with 6.5 mg pGL3/(DR-4)-c-foseFFeluc, 0.13 mg pCMV/ Renilla-luc, 1.3 mg pSG5/hRXRa and 1.3 mg pSG5/hLXRa or pSG5/ hLXRb. The 5 h after transfection, cells were treated with tested compounds for 20 h. Cells were lysed and assayed for firefly and renilla luciferase activities using Dual Luciferase Reporter Assay System kit (Progema) following the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase for each well. The plasmids were gifts from Qing Song (University of Science and Technology, Beijing, China).

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work has been funded in part by the National Key Research and Development Program of China (2017YFB0203403), the Science and Technology Program of Guangzhou (201604020109), the National Natural Science Foundation of China (81773636 and 81903540) and the Guangdong Provincial Key Lab of New Drug Design and Evaluation (Grant 2011A060901014). We thank the staff of BL17U1 and BL19U1 beamlines at Shanghai Synchrotron Radiation Facility, Shanghai, People’s Republic of China, for assistance during data collection.

4.7. Cell viability assay HEK293T, 3T3-L1 or HepG2 cells were seeded in 96-well plates at 5  103, 2  103 or 1  105 cells per well respectively for 24 h. After treated with tested compounds for 48 h, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) cell proliferation assay. 15

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Appendix A. Supplementary data

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Fig. S1-S4, quantitative real-time PCR primers, fluorescence polarization assay, crystallography and spectrum copies. Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2020.112793. Abbreviations ABC ACC Boc Bpin DBA DCM DPPF FAS HBA HBD IBMX LBP LBD LDL-C LXRs MD MM/GBSA NBS RCT RT SAR SCD-1 SREBP-1c TC TFA TG THF X-Phos

ATP-binding cassette acetyl-CoA carboxylase t-butyloxy carbonyl boronic acid pinacol dibenzylideneacetone dichloromethane 1,10 -ferrocenebis(diphenylphosphine) fatty acid synthase hydrogen bond acceptor hydrogen bond donor 3-isobutyl-1-methylxanthine ligand-binding pocket ligand-binding domain low-density lipoprotein-cholesterol liver X receptors molecular dynamics molecular mechanics generalized Born surface area N-bromosuccinimide reverse cholesterol transport room temperature structure-activity relationship stearoyl-CoA desaturase-1 sterol-regulatory element binding proteins 1c total cholesterol trifluoroacetic acid triglycerides tetrahydrofuran 2-(dicyclohexylphosphino)-20 ,40 ,60 -tri-i-propyl-1,10 biphenyl

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