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European Journal of Medicinal Chemistry 194 (2020) 112240

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Research paper

Discovery of new LXRb agonists as glioblastoma inhibitors

Hao Chen 1, Ziyang Chen 1, Zizhen Zhang 1, Yali Li, Shushu Zhang, Fuqiang Jiang,Junkang Wei, Peng Ding, Huihao Zhou**, Qiong Gu***, Jun Xu*

Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, Research Center for Drug Discovery at School of Pharmaceutical Sciences, SunYat-Sen University, Guangzhou, 510006, China

a r t i c l e i n f o

Article history:Received 31 December 2019Received in revised form12 March 2020Accepted 13 March 2020Available online 17 March 2020

Keywords:Nuclear receptorGlioblastoma inhibitorSpiro[pyrrolidine-3,30-oxindole]Structure-based drug design

* Corresponding author.** Corresponding author.*** Corresponding author.

E-mail addresses: zhuihao@mail.sysu.edu.cn (H. Zhcn (Q. Gu), junxu@biochemomes.com (J. Xu).

1 These authors contributed equally.

https://doi.org/10.1016/j.ejmech.2020.1122400223-5234/© 2020 Elsevier Masson SAS. All rights re

a b s t r a c t

Discovery and optimization of selective liver X receptor b (LXRb) agonists are challenging due to the highhomology of LXRa and LXRb in the ligand-binding domain. There is only one different residue (Val versusIle) at the ligand-binding pocket of LXRs. With machine learning methods, we identified pan LXR ago-nists with a novel scaffold (spiro[pyrrolidine-3,30-oxindole]). Then, we figured out the mechanism of LXRisoform selectivity from co-crystal structures. Based on the mechanism and the new scaffold, LXRb se-lective agonists were designed and synthesized. This led to the discovery of LXRb agonists 4-7rr, 4e13and 4-13rr with IC50 values ranging from 1.78 to 6.36 mM against glioblastoma in vitro. Treatment with50 mg/kg/day of 4-13 for 15 days significantly reduced tumor growth using an in vivo xenograft glio-blastoma model.

© 2020 Elsevier Masson SAS. All rights reserved.

1. Introduction

The LXRs (LXRa and LXRb) [1e4] are known as nuclear oxysterolreceptors and recognized as potential targets for the disease inwhich lipids have a central role, such as atherosclerosis, inflam-mation, Alzheimer’s disease and Parkinson’s disease [5e9].Recently, LXR agonists are used to treat cancer by regulating targetgenes, like APOE, ABCA1, IDOL and ABCG1 [9e12]. LXRb selectiveagonist, LXR-623, was reported as an effective agent against glio-blastoma (GBM), a human malignancy in brain with high mortality[11]. GBM cells do not synthesize cholesterol, and rely on exoge-nous cholesterol for survival [13]. LXR-623 activates LXRb and up-regulates genes ABCA1 and IDOL, effluxes intracellular cholesteroland inhibits uptake of extracellular cholesterol leading to GBM celldeath. Thus, LXRb selective agonist becomes a novel strategyagainst GBM.

LXR can be activated by endogenous ligands such as oxy-cholesterols or other natural or synthetic ligands [2,14]. LXRb is notorgan-specifically expressed, while LXRa is mainly expressed in

ou), guqiong@mail.sysu.edu.

served.

specific organs/tissues such as liver, intestine, adipose tissues, andmacrophages. Activation of LXRa may induce substantial increasesof triglycerides (TGs) in liver and plasma, and LXRb selective ago-nists may avoid these side effects. Therefore LXRb selective agonistsare demanding [15e18]. Several LXRb agonists, such as 61X (PDBcode: 3KFC) and WAY-40, are regarded as high selective agents(Fig. 1), but the mechanism of the selectivity is not articulated yet[19,20]. The reason is that LXR isoforms have high homology inligand-binding domain (LBD) and flexible pocket. There is only oneresidue difference in the proximity of the pocket: Val263(a) versusIle277(b) [21]. The volume of ligand-binding pocket is 560e680 Å3

for LXR agonist T0901317 and 980e1090 Å3 for LXR agonistGW3965 [22,23]. This allows LXRs to accommodate structurallydiverse ligands, but it makes it difficult to identify novel and LXRbselective agonists through structure-based drug design [24].

Previously, we have successfully identified farnesoid X receptor(FXR) agonists with support vector machine (SVM) and naïveBayesian (NB) methods [25]. Here, we identified LXR agonists witha novel scaffold using themachine-learning based virtual screeningplatform, and obtained co-crystal structures of LXR-ligands, fromwhich we articulated the mechanism of LXR isoform selectivity.Based on these results, we further optimized the leads, and resultedin a potent compound that significantly inhibits GBM cells througha cholesterol-regulation pathway.

Fig. 1. The known LXR agonists. a a/b selectivity index based on the reported Ki values (nM/nM).

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 1122402

2. Results and discussion

2.1. Discovery of LXR agonists

As shown in Fig. 2A, from ChEMBL and BindingDB databases, wecollected 964 known LXR ligands, and created machine models bySVM and NB approaches, which were used to screen 9500 com-pounds from our in-house compound library, and got 59 biologi-cally confirmed hits (Fig. 2A and Fig. S5 in Supporting Information),among which 13 LXR agonists were highly potent (Fig. 2B and C). Ofthem, compounds 4, 5 and 13 have common novel scaffold (spiro[pyrrolidine-3,30-oxindole]). The detailed machine learning proto-col were given in the Hit Discovery Section in SupportingInformation.

Compound 4 is a non-selective LXR agonist. It exhibits mostpotent activity with EC50LXRa ¼ 1.97 mM and EC50LXRb ¼ 1.59 mM(SI ¼ 1.2, Table 2), and suppresses GBM-related U87EGRvIII cells

Fig. 2. Discovery of 13 LXR agonists, their LXRb agonistic activities and the representative binThirteen LXRb agonistic activities measured by luciferase reporter assays in transiently transfmМ) for 20 h. *P < 0.05, **P < 0.01 and ***P < 0.001. (D) The binding mode of 4ss (an ena

with IC50 of 22.6 mM. Compound 4 has two enantiomers, whichwere separated by chiral chromatography resulting in R,R-enan-tiomer 4rr and S,S-enantiomer 4ss. Further experiments demon-strated that 4ss displayed superior activities (EC50LXRb(4ss) ¼ 0.63 mM, SI ¼ 1.3; EC50LXRb (4rr) > 10 mM, Table 2).

In order to convert the hits (4rr and 4ss) into LXRb selectiveagonists, both enantiomers were incubated with LXRb. The co-crystal structure of 4ss-LXRb complex was acquired (PDB code:6K9H). A 4ss-LXRb binding mode is depicted in Fig. 2D. 4ss is sur-rounded by helices H3, H11 and H12 (which is responsible for theLXR agonism) [26,27]. The pocket is divided by 4ss into twochambers, A and B. In chamber A, 4ss activates LXRb by reinforcingthe stacking interaction between His435 and Trp457 [21]. Inchamber B, however, 4ss has more space which can be used toestablish more interactions between the ligand and receptor at H1,Ile277/H3 and b-sheet [28].

ding mode (crystal structure). (A) Flow-chart of screening (B) Thirteen LXR agonists. (C)ected HEK293T cells. 5 h after transfection, cells were treated with tested compounds (1ntiomer derived from compound 4) acquired by X-ray crystallography.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 3

2.2. Mechanism of LXRb agonist selectivity

To elucidate the mechanism of the LXRb agonist selectivity, wecompared the 4ss-LXRb against 61X-LXRb (an LXRb selectiveagonist, SI ¼ 14, PDB code: 3KFC) and LX2-LXRa (an LXRb selectiveagonist, SI ¼ 4, PDB code: 3FC6) [20,29].

As shown in Fig. 3, three ligands occupy the chamber A in LBD,and induce LXR agonism by supporting the stacking interactionbetween His435 and Trp457 in chamber A. However, 4ss, 61X andLX2 perturb the residues in chamber B in different ways. LXRsresidues in chamber B are divided into two groups: (1) the primarylayer, in which Leu260/Leu274 (LXRa/b), Val263/Ile277, Phe315/Phe329, and Leu316/Leu330 interact with the ligand directly; and(2) the secondary layer in which Asp231/Asp245, Arg232/Gln246,Leu233/Pro247, Arg234/Lys248 and Val235/Val249 interact indi-rectly with the ligand.

The selective ligand (61X) has stronger van der Waals (VDW)interactions on residues in the primary layer than the non-selectiveligand (4ss) but it also induces a salt-bridge (red dotted line be-tween Asp245 and Lys248) and hydrogen bondings (red dottedlines Gln246-Val249 and Asp245-Lsy248) in the secondary layer.However, 4ss, a non-selective ligand, is unable to induce a similarperturbation on the residues in the secondary layer. Consequently,61X and chamber B are more compact than 4ss and chamber Bbetween their primary and secondary layers. 61X-LXRb complex issignificantly stabilized, and the selectivity of 61X to LXRb is 14.When the LXRb selective agonist LX2 (SI ¼ 4) binds to LXRa, itinduces the similar side-chain conformation changes in the pri-mary layer, and a gap between the primary and secondary layers.Thus, LX2-LXRa complex is destabilized as we have predicted. Wefurther analyzed the major clusters of 61X-LXRb and LX2-LXRacomplexes with molecular dynamics (MD) simulations for

Fig. 3. Mechanism of LXRb agonist selectivity. (A) The structural perturbation of non-selectiLXRb. (C) The structural perturbation of LXRb selective agonist in LXRa. Four different resid

125 nanoseconds (ns) (Fig. S4 in the Supporting Information). TheLXRb selective agonists consistently make chamber B of LXRbmorecompact than non-selective LXR agonists do. Thus, the selectivitymechanism of LXRb agonists is associated with the chamber Bcompactness induced by a ligand at the LBD of LXR.

2.3. LXRb selective agonists design

To design LXRb selective agonists based on 4ss, we super-imposed 4ss on 61X, and then docked 4ss into the pocket previ-ously occupied by 61X (the implementation of docking wasdescribed in the Supporting Information). The chamber B for thecompounds in the spiro[pyrrolidine-3,30-oxindole] library consistsof a “hydrophilic site” formed by Glu281, Glu315 and Arg319, and a“hydrophobic site” formed by Leu274, Ile277 and Leu330. Frag-ments that are responsible to the LXRb selectivity are listed inTable 1, where fragments F6 � F10 reside at “hydrophobic site” and“hydrophilic site”, and have better LXRb selectivity than fragmentsF1 - F5 [8,20,23,29e34]. The different linkers can assemble thesefragments onto meta-position of the phenyl in the spiro[pyrroli-dine-3,30-oxindole] scaffold. In the chamber A, when acyl andsulfonyl interact with His435, their side chains or rings can contactwith H12 residues for LXR agonism. The library was constructed byenumerating compounds from the bioisosteres. Then, the librarycompounds were docked into the selected ligand binding pocket(PDB code: 1PQ6, 3KFC and 6K9H) for virtual screening. Thesecompounds were ordered and chosen for the followed synthesis,according to their docking scores (MOE and Schr€odinger programs)and contributions to the interactions with His435 and the residuesat hydrophobic and hydrophilic sites. The design process of con-verting 4ss to LXRb selective agonist library is depicted in Fig. 4.

ve LXR agonist 4ss in LXRb. (B) The structural perturbation of LXRb selective agonist inues between LXRa and LXRb are underlined.

Table 1Privileged fragments that interact with the key residues in chamber B.

ID Fragments a PDB code SIb ID Fragments PDB code SI

F1 LX2/3FC6 14/4 F6 61X/3KFC 40/2.8

F2 Q4K/5AVL 510/160c F7 60X/5JY3 53/10

F3 965/1PQ6 200/50 F8 67S/5I4V 81/3

F4 4KM/5AVI 870/180 F9 WAY-40 d 668/20

F5 LXR-623 d 179/24 F10 668/5HJP 1900/40

a The functional fragments of LXRb selective agonist. The groups (in red) contact with the hydrophilic site. The groups (in green) contact with the hydrophobic site.b a/b selectivity index based on the reported Ki values (nM/nM).c LXRa/b selectivity index based on the reported EC50 values (nM/nM).d The LXRb selective agonist with proven binding mode.

Fig. 4. The process of structure-based LXRb selective agonist design.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 1122404

2.4. Synthesis

The intermediate S-2 was derived from tryptamine, and syn-thesized under Pictet-Spengler reaction conditions, and this wasfollowed by N-bromosuccinimide (NBS)- mediated rearrangementto generate enantiomer-containing 20S, 3S- and 20R, 3R-isoformsover 3 steps [35,36]. Subsequently, the bromine in the intermediatewas exchanged for a boronic acid pinacol (Bpin) or an NH2 group forsubsequent coupling and acylation reactions which combine thedesigned aromatic fragments (Fig. 5 and Scheme 1). Other acylgroups replacing the Boc (t-butyloxy carbonyl) moieties were usedto produce 20 desired compounds, in which active racemic ana-logues were isolated and confirmed as R,R and S,S-isomers withtheir X-ray structures (PDB code: 6K9G and 6K9H) and specificoptical rotations (Fig. S1, Supporting Information).

2.5. Structure-activity relationships for spiro[pyrrolidine-3,30-oxindole] derivatives

Twenty compounds were assayed for LXR agonism (Fig. 5 andTable 2). When R1 is a benzyl alcohol, derivatives showed improved

LXR agonism, suggesting that hydrogen bond donor can match the“hydrophilic site”, and that benzyl can form a p-p stacking inter-actionwith Phe329 in chamber B. The R1 containingmethylsulfonylboosted the agonistic activity to the same level as that of GW3965,because the methylsulfonyl moiety can match the “hydrophobicsite” better in chamber B than derivatives withmethyl, chlorine andmethylsulfonamide. Then, we found that it is necessary to keep a0e1 atom unit as the linker between the spiro[pyrrolidine-3,30-oxindole] and the aryl substituent. Compounds 4e7, 4e13 and 4e14share the same R1 moiety, but 4e14 reduced LXR agonism, becauseof its amide-linker exceeding this limit. In chamber A, the Boc and3,3-dimethylbutanoyl moieties retain in the scaffold resulting inLXR agonism. When R3 or R4 is a methyl group, the substituentclashed with Leu274, Met312 and Thr316 leading to reduction ofthe activation (Fig. 6).

Subsequently, the R,R- and S,S-isomers were prepared fromthese active compounds and assayed for their EC50 and Ki valuesand anti-GBM activity (Table 2). Most of derivatives, including theenantiomers and their chiral isomers, showed better selectivitythan compound 4. The R,R-isomers of 4-7 to 4e15 had an obviousadvantage in activation of LXRb with an EC50 of 16e92 nM and

Fig. 5. LXR transcriptional activity of synthetic derivatives. (A) Structures of synthetic derivatives. Substituents in red were introduced to match hydrophilic site in chamber B ofligand-binding pocket. Substituents in green for hydrophobic site in chamber B of ligand-binding pocket. (B) LXR transcriptional activity was measured by luciferase reporter assaysin transiently transfected HEK293T cells. 5 h after transfection, cells were treated with tested compounds (1 mМ) for 20 h. *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretationof the references to colour in this figure legend, the reader is referred to the Web version of this article.)

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 5

2.9e5.0 fold selectivity (Table 2), and Ki values ranging between 1.0and 101 nM, suggesting that the R,R-configuration is more benefi-cial to ligand binding with LXRb rather than the S,S-configuration,whose EC50 values are 3.1 � 5.4 times more than R,R-configuration.Our experiments demonstrated that SI for LXR-623 was 2.0, SI for61X is also 2.0 [28], and SI for WAY-40 was 3.2 [28]. As listed inTable 2, our synthesized compounds had improved SI values. It isworth to note that the SI values for 4-5rr, 4-7rr, 4-8rr and 4-15rrranged from 3.2 to 5.0.

Table 2 indicates the LXRb agonism is consistent with the anti-GBM activity. Compounds 4e7, 4e9, 4e13, and their R,R-isomersdemonstrated more GBM suppression than S,S-isomers did. Com-pound 4-13rr and racemate 4e13 efficiently inhibit U87EGFRvIIIcell line with IC50 of 1.78 and 3.75 mM. Compounds 4-13rr and 4e13

had higher LXRb agonism efficacy (86% and 74%) and increasedinhibition against GBM.

Combining Table 2 and Fig. 5, we summarized a SARmap for ourLXRb selective agonists (Fig. 6A). The fragments that contribute tothe selectivity are listed in Fig. 6B.

2.6. Binding mode analyses

To validate the lead optimization result, we obtained the co-crystal structure of 4-7rr-LXRb complex (PDB code: 6K9G). Usingthis structure complex and 4ss-LXRb complex (PDB code: 6K9H),we created four binding complex models (4rr-LXRb, 4ss-LXRb, 4-7rr-LXRb, and 4-7ss-LXRb). In order to investigate the relations ofchirality and activities, these models were experienced MD

Scheme 1. Synthetic route for spiro[pyrrolidine-3,30-oxindole] derivatives.(a) Benzaldehydes, TFA, DCM. (b) NBS, cat. TFA, THF/water. (c) Boc2O, NEt3, DCM. (d) Pd(DPPF)2Cl2, (Bpin)2, KOAc, 2,4-dioxane, 85 �C. (e) BrR1, Pd(DPPF)2Cl2, Na2CO3, KF, 2,4-dioxane/water, 85 �C. (f) HCl, DCM. (g) R2OR2, NEt3, DCM. (h) MeI, K2CO3, DMF. (i) NaN3, CuI, sodium ascorbate, N, N0-dimethyl-1,2-ethanediamine, DMSO, 85 �C. (j) BrR1, Pd2(DBA)3, X-Phos,K2CO3, i-BuOH, 85 �C. (k) R1OH, HATU, DIPEA, DMF. (l) NaBH4, MeOH, 0 �C.

Table 2LXR agonism and anti-glioblastoma activities for the synthesized compounds.

ID# LXRa EC50 (mM)a

(% efficacy)bLXRb EC50 (mM)a

(% efficacy)bLXRa/b selectivityc LXRb Ki (nM)d U87EGFRvIII

IC50 (mM)e

4 1.968 ± 0.417 (82 ± 15) 1.589 ± 0.413 (74 ± 10) 1.2 NTf 22.63 ± 3.744rr >10 >10 –h 1700 ± 300 NT4ss 0.843 ± 0.276 (88 ± 10) 0.632 ± 0.216 (82 ± 8) 1.3 600.0 ± 52.4 NA g

4-1rr 1.954 ± 0.327 (84 ± 7) 0.445 ± 0.135 (65 ± 7) 4.4 195.8 ± 57.1 NA4-5rr 1.176 ± 0.028 (63 ± 2) 0.369 ± 0.129 (52 ± 6) 3.2 264.7 ± 48.0 NA4-6rr 1.071 ± 0.029 (79 ± 5) 0.680 ± 0.305 (67 ± 3) 1.6 148.6 ± 33.7 NA4e7 0.372 ± 0.174 (79 ± 10) 0.174 ± 0.032 (74 ± 9) 2.1 NT 7.05 ± 2.474-7rr 0.145 ± 0.018 (81 ± 3) 0.035 ± 0.014 (65 ± 4) 4.1 1.0 ± 0.4 6.36 ± 2.824-7ss 0.94 ± 0.063 (82 ± 11) 0.43 ± 0.110 (81 ± 17) 2.8 134.9 ± 32.5 NA4e8 0.708 ± 0.169 (84 ± 23) 0.341 ± 0.193 (70 ± 9) 2.1 NT 13.27 ± 3.074-8rr 0.174 ± 0.088 (80 ± 12) 0.052 ± 0.014 (67 ± 2) 3.3 37.2 ± 15.0 7.53 ± 0.714-8ss 1.746 ± 0.370 (68 ± 16) 0.905 ± 0.139 (73 ± 4) 1.9 1200 ± 400 NA4e9 0.251 ± 0.046 (83 ± 18) 0.103 ± 0.051 (54 ± 6) 2.5 NT 8.49 ± 0.364-9rr 0.274 ± 0.008 (80 ± 13) 0.092 ± 0.017 (48 ± 1) 2.9 67.7 ± 16.1 7.78 ± 1.904-9ss 0.270 ± 0.043 (81 ± 10) 0.281 ± 0.015 (54 ± 1) 1.0 132.5 ± 20.3 NA4e13 0.277 ± 0.066 (75 ± 8) 0.095 ± 0.012 (85 ± 23) 2.9 NT 3.75 ± 1.224-13rr 0.270 ± 0.023 (74 ± 4) 0.088 ± 0.018 (86 ± 11) 3.1 101 ± 19.5 1.78 ± 0.434-13ss 0.928 ± 0.210 (80 ± 16) 0.474 ± 0.172 (75 ± 18) 2.0 234.3 ± 36.6 NA4e15 0.214 ± 0.068 (67 ± 4) 0.067 ± 0.016 (76 ± 12) 3.1 NT NA4-15rr 0.080 ± 0.020 (73 ± 5) 0.016 ± 0.006 (66 ± 2) 5.0 7.3 ± 4.5 NA4-15ss 0.161 ± 0.063 (66 ± 3) 0.083 ± 0.023 (71 ± 7) 1.9 70.4 ± 12.8 NAT0901317 0.098 ± 0.044 (88 ± 1) 0.108 ± 0.047(100 ± 11) 0.9 41.3 ± 7.0 NTGW3965 0.401 ± 0.155 (100 ± 19) 0.228 ± 0.045(100 ± 17) 1.8 2.2 ± 1.9 3.65 ± 0.32LXR-623 0.431 ± 0.124 (92 ± 8) 0.212 ± 0.010 (80 ± 2) 2.0 14.2 ± 3.1 6.14 ± 1.04

a The EC50 value was measured by transient transfection and luciferase reporter assays.b % efficacy is related to positive control GW3965.c LXRa/b selectivity is the ratio of the EC50 value of LXRa to that of LXRb. The reported agonists T0901317, GW3965, LXR-623, 61X, (5.96 mM/2.94 mM, SI ¼ 2.0 [28]) and

WAY-40 (8.44 mM/2.67 mM, SI ¼ 3.2 [28]) as positive control. High selectivity (SI > 3.0), medium selectivity (SI ¼ 2.0 to 3.0).d LXRb Ki was measured by fluorescence polarization experiments.e The IC50 of U87EGFRvIII was determined by cell viability assay in U87EGFRvIII cells.f Not tested.g Not active.h Cannot calculate. Assay results are the average of at least three independent experiments.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 1122406

simulations for 125 ns [28]. As shown in Fig. 7, 4rr and 4ss, 4-7rrand 4-7ss are mirror enantiomers at the spiro[pyrrolidine-3,30-oxindole] scaffold, resulting 4ss/4-7rr to form hydrogen bondingwith His435 in chamber A, and no such hydrogen bond for 4rr/4-7ss. Thus, we explain why the mirror enantiomers have differentLXR agonism behavior.

When superimposing 4rr-LXRb and 4ss-LXRb (Fig. 7A), and 4-7rr-LXRb and 4-7ss-LXRb (Fig. 7B), we find that 4-7rr/4-7ss directlyestablishes p-p stacking with Phe329, hydrogen bonding withLeu330, and VDW interactions with Leu274, Ile277 and Leu330 in

the primary layer. Fig. 7B reveals that the compactness of the res-idues in the binding pocket is significantly increased by introducing4-7rr or 4-7ss in comparison with Fig. 7A. The red dotted linesindicate interactions newly established by 4-7rr or 4-7ss.

2.7. 4-13rr inhibits growth of GBM cells

As shown in Table 2, 4-13rr (IC50 ¼ 1.78 ± 0.43 mM) is the bestGBM cell inhibitor, and it was further studied in cell-based activitiesand inhibitory mechanisms [11]. LXR agonist LXR-623was recently

Fig. 6. SAR for spiro[pyrrolidine-3,30-oxindole] derivatives. (A) SAR map for the spiro-scaffold, based on 4-7rr-LXRb complex (PDB code: 6K9G) and 4-13rr-LXRb dockingmodel. Both 4-7rr and 4-13rr have similar interactions with the key residues. (B) Thefragments ordered according to their contribution to the selectivity.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 7

found to be of great potential in GBM treatment [11]. In vitro assaysdemonstrated that 4-13rr is a potent inhibitor of U87EGFRvIII,U251, and A172 cell lines with IC50 values of 1.78, 2.23, and 6.03 mM,respectively, compared with LXR-623 IC50 values of 6.14, 3.06, and8.30 mM (Fig. 8A). And 4-13rr showed much lower toxicity toHEK293T cells and normal human astrocytes HA1800 (Fig. 8B).

Fig. 7. Binding mode analyses for LXRb agonist selectivity mechanism. (A) Non-selective LXRlayers. 4ss formed hydrogen bond with His435 (red dotted line). The distance between 4rr an7rr-LXRb) have stronger compactness of the primary and secondary layers. 4-7rr formed hydbonding. (For interpretation of the references to colour in this figure legend, the reader is

2.8. 4-13rr regulates LXR-mediated cholesterol uptake and efflux

As shown in Fig. 9A, compound 4-13rr dose-dependentlyupregulated the down-stream genes ABCA1, IDOL, ABCG1, APOEand SREBP-1c in U87EGFRvIII cells. With U87EGFRvIII-based assays,we observed that both 4-13rr and LXR-623 inhibited LDL uptakedue to the upregulation of IDOL (Fig. 9B and 9C), promotedcholesterol efflux to ApoA1 due to the upregulation of ABCA1(Fig. 9D and S6D) and reduced intracellular cholesterol levels(Fig. 9E and S6E). With knock-down a and b isoforms separately, wewere able to conclude that the absence of LXRb blocked the 4-13rr-mediated suppression in U87EGFRvIII cells, indicating that com-pound 4-13rr suppressed GBM through activation of LXRb (Fig. 9F,9G and 9H).

2.9. Pharmacokinetics of compound 4-13

Compound 4-13rr and its racemate 4e13weremore potent thanLXR-623 in vitro, therefore, 4e13 was further validated in in vivomodels [11]. The in vivo (using Sprague-Dawley (SD) rats) phar-macokinetic (PK) study of compound 4e13 was conducted. The SDrats (male, n ¼ 6) were treated with intravenous administration at10 mg/kg dose, respectively. Plasma samples were collected at aperiod of 24 h, and concentration values of compound 4e13 weremeasured with LC-MS. The PK parameters were summarized inTable 3, which indicated that the T1/2 of compound 4e13was 2.98 h,the Cmax was 1010 ± 174 ng/mL at Tmax 0.22 h, and AUC0-t was1249 ± 51 h ng/mL.

2.10. Antitumor activity of 4-13 in vivo

Compound 4e13 (U87EGFRvIII IC50 ¼ 3.75 mM) was validated inGBM xenograft models. Male nude mice were randomly dividedinto three groups (n ¼ 6) and intraperitoneally treated with 4-13 at50 mg/kg/day for 15 continuous days. The results are depicted inFig. 10. The in vivo data demonstrated that the size of GBM tumorswere significantly controlled, the efficacies of 4-13, GW3965 andLXR-623 were comparable (Fig. 10A, B and C) and trends suggestedthat 4e13 could recover the body weight of the experimental mice

agonists (4ss-LXRb and 4rr-LXRb) have less compactness of the primary and secondaryd His435 is out of the hydrogen bonding (B) Selective LXR agonists (4-7ss-LXRb and 4-rogen bond with His435. The distance between 4-7ss and His435 is out of the hydrogenreferred to the Web version of this article.)

Fig. 8. 4-13rr showed anti-glioblastoma activity in U87EGFRvIII, U251 and A172 cells. Cells were treated with tested compounds for 7 days. (A, C) The anti-glioblastoma activity of4-13rr in U87EGFRvIII, U251 and A172 cells. (B) 4-13rr showed lower toxicity to HEK293T cells and normal human astrocytes HA1800.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 1122408

(Fig. 10D). Serum assays demonstrated that 4e13, but not GW3965and LXR-623, reduced adverse effects on triglycerides levels inblood (Fig. 10E).

3. Conclusion

It is challenging to discover selective regulators for a proteintarget while it has a high homological isoforms at a binding pocket.Conventionally, people turn to explore allosteric sites or scaffoldhopping approaches.

In this study, however, we demonstrate a new strategy, whichconsists of following steps:

(1) identifying hits with new scaffold using machine learningbased screening;

(2) optimizing the hits for an isoform selectivity based on theresults of elucidating the selectivity mechanism, which is (inour case study) the pocket compactness-based LXRb selec-tivity mechanism from co-crystal structures.

This study may open a new door to develop a selective agent forthe target with multiple homologs. In the binding pocket, weshould concern not only the direct interactions between a ligandand the key residues, but also the pocket compactness that isinduced by such interactions, which may consist of multiple

interactive layers.

4. Experimental procedures

4.1. Hit discovery section

The details are in the Hit Discovery Section in the SupportingInformation.

4.2. Synthesis

All chemical reagents and organic solvent were purchased fromJ&K Chemicals. 1H NMR and 13C NMR spectra were recorded on aBruker Avance 400/500 MHz NMR spectrometer for hydrogen and100/125 MHz for carbon. Low resolution electrospray ionization(ESI) mass spectra were recorded on a Agilent 6120 single quad-rupole LC/MS system using reverse-phase conditions (methanol/water, 0.05% formic acid). Semipreparative chiral HPLC separationwas performed on an LC-20AT Shimadzu liquid chromatographysystem with an SPD-M20A diode array detector. All solvents wereof chromatographic grade (Fisher Scientific UK Ltd.). Enantiomerswere separated by semipreparative chiral HPLC (methanol-water,85: 15, 1.5 mL/min) to separate the compounds. HPLC Purity wasdetermined by analysis method on a Shimadzu HPLC system. Col-umn: Agilent SB-C18; 5 mm 4.6 � 250 mm. Solvent: 75%

Fig. 9. 4-13rr regulated LXR-mediated cholesterol uptake and efflux, leading to glioblastoma cell death in U87EGFRvIII cells. LXR-623 and 4-13rr were tested at 5 mM, unlessotherwise specified. (A) 4-13rr upregulated the mRNA expression of LXR target genes ABCA1, IDOL, ABCG1, APOE and SREBP-1c. (B, C) 4-13rr inhibited LDL uptake (magnification,400 � ). (D) 4-13rr promoted cholesterol efflux to ApoA1. (E) 4-13rr reduced cellular cholesterol levels. (F, G) LXRa and LXRb were knocked down by RNAi separately. (H) LXRbknock-down blocked the 4-13rr-mediated suppression of U87EGFRvIII. *P < 0.05, **P < 0.01 and ***P < 0.001.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 9

Table 3Pharmacokinetic parameters of compound 4-13.

Tmax (h) Cmax (ng/mL) T1/2 (h) AUC0-t (h∙ng/mL) AUC0-∞ (h∙ng/mL) MRTINF (h)

mean ± SD 0.22 ± 0.08 1010 ± 174 2.98 ± 0.50 1249 ± 51 1407 ± 93 3.48 ± 0.44

Fig. 10. Antitumor activity of 4-13 in the U87EGFRvIII xenograft model. 5 � 105 U87EGFRvIII cells implanted subcutaneously into 4-week-old male BALB/c nu/nu mice. After tumorsize reached 40 mm3, mice were treated with GW3965 (40 mg/kg/day, i.g.), LXR-623 (40 mg/kg/day, i.g.) or 4e13 (50 mg/kg/day, i.p.) for 15 continuous days. (A) Isolated tumortissues after administration for 15 days. (B) Tumor size, (C) relative tumor size and (D) body weight. (E) Serum levels of triglycerides (TG), alanine aminotransferase (ALT), glutamicoxaloacetic transaminase (AST) and alkaline phosphatase (ALP) after administration for 15 days. *P < 0.05 and **P < 0.01.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 11224010

acetonitrile, 25% water. Flow rate 1.0 mL/min. All of 20 derivativeshad purification values of >95%. The ee values were assayed byanalysis method on a Shimadzu HPLC system. Column: Phenom-enex, Lux 5u Cellulose-3; 4.6 � 250 mm. Solvent: 85% methanol,15% water. Flow rate 0.8 mL/min. The ee values of 18 chiral com-pounds exceeded 99% (Fig. S1). Specific rotations were detected onan AntonPaa MCP200 (solvent: methanol, Fig. S1).

4.2.1. General preparation for S-1a, S-1b and S-1c3-Bromobenzaldehyde (6.5 mL, 55 mmol, 1.1 equiv.) and TFA

(7.5 mL, 100 mmol, 2.0 equiv.) were added dropwise to the solutionof tryptamine (8.0 g, 50 mmol, 1.0 equiv.) in 200 mL DCM in an icebath and stirred at room temperature (RT) overnight. Then, themixture was concentrated in vacuo to remove the DCM and TFA.The residue was dissolved in 50 mL EtOAc, and subsequently

precipitated by addition of 200mL hexane/EtOAc (v/v: 1/1), filtered,and washed with hexane twice to obtain the products S-1a, S-1band S-1c.

4.2.1.1. 1-(3-Bromophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (S-1a). White solid (16 g, yield: 66%); 1H NMR (400 MHz,CD3OD) d 7.72 (dt, J ¼ 7.7, 1.7 Hz, 1H, H-40), 7.64 (t, J ¼ 1.7 Hz, 1H, H-20), 7.58 (dt, J¼ 8.0, 1.1 Hz,1H, H-5), 7.45 (t, J¼ 7.7 Hz,1H, H-50), 7.41(dt, J ¼ 7.7, 1.7 Hz, 1H, H-60), 7.34 (dt, J ¼ 8.1, 1.1 Hz, 1H, H-8), 7.20(ddd, J¼ 8.1, 7.0, 1.1 Hz, 1H, H-7), 7.12 (ddd, J¼ 8.0, 7.0, 1.1 Hz, 1H, H-6). 5.94 (d, J ¼ 1.7 Hz, 1H, H-1), 3.69e3.60 (m, 1H, H-3a), 3.60e3.53(m,1H, H-3b), 3.30e3.22 (m,1H, H-4a), 3.24e3.18 (m,1H, H-4b). 13CNMR (100 MHz, CD3OD) d 138.6, 137.9, 134.6, 133.8, 132.2, 129.7,127.8, 127.2, 124.2, 123.9, 120.8, 119.3, 112.5, 109.3, 57.6, 42.3, 19.5.ESI-MS m/z: 327.2, 329.2 [MþH] þ.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 11

4.2.1.2. 1-(3-Bromo-4-methylphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (S-1b). White solid (15 g, yield: 60%); 1H NMR(500 MHz, DMSO‑d6) d 7.69e7.67 (m, 2H), 7.54 (dd, J ¼ 7.9, 1.6 Hz,1H), 7.44e7.39 (m, 1H), 7.33 (dd, J ¼ 7.9, 1.6 Hz, 1H), 7.14 (dt, J ¼ 7.9,1.6 Hz, 1H), 7.09e7.05 (m, 1H), 5.94 (s, 1H), 3.50e3.45 (m, 1H),3.44e3.39 (m,1H), 3.19e3.16 (m,1H), 3.06e3.03 (m,1H,1H), 2.41 (s,3H). ESI-MS m/z: 341.2, 343.2 [MþH] þ.

4.2.1.3. 1-(3-Bromo-5-fluorophenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (S-1c). White solid (12 g, yield: 48%); 1H NMR(400 MHz, CD3OD) d 7.61e7.50 (m, 2H), 7.46 (d, J ¼ 1.6 Hz, 1H), 7.33(d, J ¼ 7.8 Hz, 1H), 7.23e7.15 (m, 2H), 7.10 (t, J ¼ 7.8 Hz, 1H), 5.95 (s,1H), 3.65e3.61 (m, 1H), 3.58e3.55 (m, 1H), 3.24e3.20 (m, 1H),3.18e3.15 (m, 1H).13C NMR (100 MHz, CD3OD) d 164.1, 161.6, 138.3,138.2, 137.2, 128.7, 128.7, 125.9, 125.7, 123.2, 123.1, 122.7, 120.8,120.5,119.5,118.0,115.8,115.6,111.2,108.1, 55.6, 41.0,18.1. ESI-MSm/z: 345.2, 347.2 [MþH] þ.

4.2.2. Conversion of S-2a to S-2cNBS (9.8 g, 55mmol) was added in batches to a solution of S-1 in

THF/H2O (v/v: 4/1, 500 mL) with saturated NaHCO3 which waschilled in an ice bath, then dried over anhydrous Na2SO4, filteredand concentrated in vacuo to afford the crude product. TEA (21 mL,150mmol, 3.0 equiv) and di-t-butyl dicarbonate (10.9mL, 50mmol,1.0 equiv.) were added dropwise to a solution of the crude productin DCM cooled in an ice bath, and the mixture was stirred at RT for0.5 h. The mixture was then diluted with DCM, and washed withsaturated NaHCO3, dried over anhydrous Na2SO4, filtered andconcentrated in vacuo to afford the crude product which was dis-solved in EtOAc, and diluted with hexane/EtOAc (3/1) for precipi-tation. Then, the mixture was filtered and washed with hexane/EtOAc (3/1) to afford S-2a to S-2c.

4.2.2.1. tert-Butyl-2’-(3-bromophenyl)-2-oxospiro[indoline-3,30-pyr-rolidine]-10- carboxylate (S-2a). White solid (11g, yield: 49.6%). 1HNMR (500 MHz, DMSO‑d6) d 10.33 (s, 1H, NH), 7.39 (dd, J ¼ 7.8,1.8 Hz, 1H, H-400), 7.19 (t, J ¼ 7.8 Hz, 1H, H-500), 7.14 (brs, 1H, H-200),7.09 (t, J ¼ 7.7 Hz, 1H, H-6), 7.00 (d, J ¼ 7.7 Hz, 1H, H-4), 6.79 (d,J ¼ 7.7 Hz, 1H, H-7), 6.68 (t, J ¼ 7.7 Hz, 1H, H-5), 6.19 (d, J ¼ 7.8 Hz,1H, H-600), 4.93 (s, 1H, H-20), 4.00e3.87 (m, 2H, H-50), 2.28e2.20 (m,2H, H-40), 1.28 (s, 9H, Boc-H). 13C NMR (100 MHz, CDCl3) d 153.86,141.30, 139.88, 129.40, 128.42, 127.54, 124.31, 120.89, 108.80, 79.52,65.75, 57.66, 44.90, 32.61, 27.06. HRMS (ESI) m/z: calcd. forC22H23BrN2O3[MþNa]þ 465.0784, found 465.0776.

4.2.2.2. tert-Butyl-2’-(3-bromo-4-methylphenyl)-2-oxospiro[indo-line-3,30- pyrrolidine]-10-carboxylate (S-2b). White solid (10.2 g,yield: 40.7%). 1H NMR (400MHz, CDCl3) d 9.28 (s, 1H), 7.08 (brs,1H),7.02 (dt, J¼ 7.7,1.2 Hz,1H), 6.94 (d, J¼ 7.7 Hz,1H), 6.76 (d, J¼ 7.8 Hz,1H), 6.73e6.56 (m, 2H), 6.23 (brs, 1H), 4.96 (brs, 1H), 4.14e3.99 (m,1H), 3.88 (brs, 1H), 2.26e2.19 (m, 4H), 1.57e0.96 (m, 9H).13C NMR(100 MHz, CDCl3) d 179.7, 153.6, 139.7, 138.3, 135.6, 129.4, 129.1,127.4, 127.1, 124.7, 124.4, 123.3, 120.8, 108.8, 79.1, 65.3, 57.6, 44.8,32.6, 27.1, 21.5. HRMS (ESI) m/z: calcd. for C23H25BrN2O3[MþNa]þ

479.0941, found 479.0932.

4.2.2.3. tert-Butyl-2’-(3-bromo-5-fluorophenyl)-2-oxospiro[indoline-3,30- pyrrolidine]-10- carboxylate (S-2c). White solid (13 g, yield:51.2%). 1H NMR (500 MHz, CDCl3) d 8.63 (s, 1H), 7.06 (td, J ¼ 7.8,1.2 Hz,1H), 6.96 (d, J¼ 7.8 Hz,1H), 6.89e6.48 (m, 3H), 6.41 (brs,1H),4.99 (s, 1H), 4.08 (brs, 1H), 3.88 (brs, 1H), 2.35 (brs, 1H), 2.15 (brs,1H), 1.43 (brs, 3H), 1.16 (brs, 6H).13C NMR (100 MHz, CDCl3) d 179.1,162.4, 159.9, 153.6, 143.2, 139.5, 130.5, 127.7, 127.4, 127.0, 124.5,124.1, 121.1, 117.0, 116.7, 111.5, 109.0, 79.5, 65.6, 57.8, 45.0, 32.8, 27.1.HRMS (ESI) m/z: calcd. for C22H22BrFN2O3[MþNa]þ 483.0690,

found 483.0674.

4.2.3. General preparation of S-3a, S-3b and S-3cBis(pinacolato)diboron (2.54 g, 10 mmol, 1.1 equiv.), potassium

acetate (0.98 g, 10 mmol, 1.1 euqiv.) and Pd(dppf)2Cl2 (328 mg,0.45mmol, 0.05 equiv.) were added to a solution of S-2 (9 mmol, 1.0equiv.) in 2,4-dioxane (60 mL) under an N2 atmosphere, and stirredat 85 �C overnight. The mixture was cooled in an ice bath, and thiswas followed by addition of saturated NH4Cl to quench the reaction.The mixture was extracted with 100 mL EtOAc thrice, washed bybrine, dried over anhydrous Na2SO4, filtered and concentrated invacuo. The residue was purified by silica gel flash chromatography(Biotage SP-1, 330 g SiO2 column, gradient elution from 0 to 32%EtOAc) to afford S-3a, S-3b and S-3c.

4.2.3.1. tert-Butyl-2-oxo-2 ’-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) spiro[indoline-3,30-pyrrolidine]-10-carboxylate (S-3a). Colourless solid (3.8 g, yield: 85%). 1H NMR(500 MHz, CDCl3) d 7.75 (brs, 1H, H-400), 7.53 (brs, 1H, H-500), 7.09(brs, 1H, H-200), 6.97 (t, J¼ 7.7 Hz, 1H, H-6), 6.67 (d, J¼ 7.7 Hz, 1H, H-7), 6.58 (brs, 1H, H-5), 6.14 (brs, 1H, H-600), 5.02 (s, 1H, H-20),4.10e4.06 (m, 1H, H-50a), 3.93 (m, 1H, H-50b), 2.24 (m, 2H, H-40),1.24 (s, 6H, Bpin-H), 1.22 (s, 6H, Bpin-H), 1.17 (brs, 3H, Boc-H), 1.08(brs, 6H, Boc-H).

4.2.3.2. tert-Butyl-2’-(4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (S-3b). White solid (3.0 g, yield: 66%). 1H NMR(400 MHz, CDCl3) d 7.66 (brs, 1H), 6.98 (t, J ¼ 7.8 Hz, 1H), 6.89 (brs,1H), 6.78 (brs, 1H), 6.67 (d, J ¼ 7.7 Hz, 1H), 6.61 (brs, 1H), 6.15 (brs,1H), 4.98 (s, 1H), 4.06e4.05 (m, 1H), 3.90 (brs, 1H), 2.39e2.38 (m,2H), 2.24e2.21 (m, 3H), 1.23 (s, 6H), 1.21 (s, 6H), 1.18 (brs,3H), 1.10(brs, 6H).

4.2.4. General preparation of 4-1 to 4e12, 4e15 to 4-20Compounds 4-1 to 4e12, 4e15 to 4e20were synthesized by the

general method described here. Bromobenzene (0.22 mmol, 1.1equiv.), potassium carbonate (30 mg, 0.22 mmol, 1.1 equiv.) andPd(dppf)2Cl2 (7 mg, 0.01 mmol, 0.05 euiqv.) were added under anN2 atmosphere to a solution of S-3 (100 mg, 0.2 mmol, 1.0 equiv.) in3 mL 2,4-dioxane, and the mixture was stirred at 85 �C overnight.The mixture was cooled in an ice bath, and this was followed byaddition of saturated NH4Cl to quench the reaction. The mixturewas extracted with 20 mL EtOAc thrice, washed with brine, driedover anhydrous Na2SO4, filtered and concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (Biotage SP-1, 20 g SiO2 column, gradient elution from 20 to 60% EtOAc) toafford the derivatives 4-1 to 4e12, 4e15 and 4e20.

4.2.4.1. tert-Butyl-2’-(4’-(hydroxymethyl)-[1,10-biphenyl]-3-yl)-2-oxospiro[indoline- 3,30- pyrrolidine]-10-carboxylate (4-1).White solid (50 mg, yield: 53%). 1H NMR (400 MHz, CDCl3) d 9.11 (s,1H), 7.46e7.08 (m, 6H), 6.96 (dt, J ¼ 7.8, 1.2 Hz, 1H, H-6), 6.69 (d,J¼ 7.8 Hz,1H, H-7), 6.58 (brs,1H, H-5), 6.17e5.81 (m,1H, H-600), 5.05(s, 1H, H-20), 4.62 (s, 2H, Bn-CH2), 4.18e4.07 (m, 1H, H-50a),4.05e3.93 (m, 1H, H-50b), 2.25e2.16 (m, 2H, H-40), 1.43 (brs, 3H,Boc-H), 1.21e1.07 (m, 6H, Boc-H). 13C NMR (100 MHz, CDCl3)d 179.8, 153.8, 139.7, 139.6, 139.5, 139.3, 139.2, 139.1, 127.3� 2, 127.2,126.5, 126.4� 2,126.1� 2,125.0,124.4, 120.8, 108.7, 79.0, 66.3, 63.8,57.6, 44.8, 32.5, 27.1 � 3. HRMS (ESI) m/z: calcd. forC29H30N2O4[MþNa]þ 493.2098, found 493.2089.

4.2.4.2. tert-Butyl-2’-(4’-(methoxycarbonyl)-[1,10-biphenyl]-3-yl)-2-oxospiro[indoline- 3,30-pyrrolidine]-10-carb-oxylate (4-2).White solid (60 mg, yield: 60%). 1H NMR (400MHz, CDCl3) d 9.21 (s,

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 11224012

1H), 7.95 (d, J ¼ 7.9 Hz, 2H), 7.33 (m, 2H), 7.22 (s, 1H), 7.09 (s, 1H),7.00 (t, J ¼ 7.9 Hz, 1H), 6.77 (d, J ¼ 7.9 Hz, 1H), 6.60 (s, 1H), 6.20 (brs,1H), 5.84 (brs, 1H), 5.15 (s, 1H), 4.20e4.04 (m, 1H), 3.95 (brs, 1H),3.84 (s, 3H), 2.23 (brs, 2H), 1.43 (brs, 3H), 1.17 (brs, 6H). 13C NMR(100 MHz, CDCl3) d 179.9, 166.0, 153.7, 144.5, 139.9, 139.7, 139.9,138.4,129.0� 2,127.8,127.4, 127.4� 2,127.2,125.9� 2,125.3,124.4,120.7, 108.8, 79.0, 66.2, 57.6, 51.1, 44.9, 32.5, 27.1� 3. HRMS (ESI)m/z: calcd. for C30H30N2O5[MþNa]þ 521.2047, found521.2051.

4.2.4.3. tert-Butyl-2’-(4’-(hydroxymethyl)-3 0,5 0-dimethyl-1,10-biphenyl]-3-yl)-2- oxospiro [indoline-3,30-pyrrolidine]-10-carboxylate(4-3). White solid (48 mg, yield: 48%). 1H NMR (400 MHz, CD3OD)d7.32 (dt, J ¼ 7.7, 1.40 Hz, 1H), 7.20 (d, J ¼ 7.7 Hz, 1H), 7.11e6.79 (m,4H), 6.73 (d, J ¼ 7.8 Hz, 1H), 6.55 (brs, 1H), 6.11 (brs, 1H), 5.81 (brs,1H), 4.94 (s, 1H), 4.60 (s, 2H), 3.99 (dt, J ¼ 10.4, 8.0 Hz, 1H), 3.89 (q,J ¼ 10.7, 8.0 Hz, 1H), 2.33 (s, 6H), 2.23 (overlapped, 2H), 1.40 (brs,3H), 1.09 (brs, 6H). 13C NMR (100 MHz, CD3OD) d 181.4, 155.1, 141.5,140.6, 140.4, 140.1, 137.7 � 2, 136.7, 135.7, 128.3, 128.0, 128.0, 126.4,125.7, 125.3, 121.3, 109.3, 80.0, 67.3, 57.4, 54.7, 45.6, 33.0, 27.1 � 3,18.3 � 2. HRMS (ESI) m/z: calcd. for C31H34N2O4[MþNa]þ 521.2411,found 521.2411.

4.2.4.4. tert-Butyl-2’-(30,50-dimethyl-4’-(methylsulfonamido)-[1,10-biphenyl]-3-yl)-2- oxospiro[indoline- 3,30-pyrrolidine]-10-carboxylate(4-4). White solid (43 mg, yield: 38%). 1H NMR (400 MHz, CDCl3)d 8.81 (s, 1H), 7.26 (d, J ¼ 7.6 Hz, 1H), 7.23e7.08 (overlapped, 2H),7.00 (t, J ¼ 7.6 Hz, 1H), 6.76 (d, J ¼ 7.7 Hz, 1H), 6.61 (brs, 1H), 6.56 (s,1H), 6.14 (brs, 1H), 5.87 (brs, 1H), 5.05 (s, 1H), 4.16e4.03 (m, 1H),3.93 (brs, 1H), 3.03 (s, 3H), 2.36 (s, 6H), 2.25 (overlapped, 2H), 1.43(brs, 3H), 1.09 (brs, 6H). 13C NMR (100 MHz, CDCl3) d 179.8, 153.7,139.6 (overlapped), 138.8 (overlapped), 136.6, 131.1, 127.5 (over-lapped), 126.6, 125.2, 125.1, 124.5, 120.9, 108.7, 79.0, 66.2, 57.6, 44.8,41.0, 32.5, 27.1� 3,18.4� 2. HRMS (ESI)m/z: calcd. for C31H35N3O5S[MþNa]þ 584.2190, found 584.2179.

4.2.4.5. tert-Butyl-2’-(30-chloro-4’-(dimethylcarbamoyl)-[1,10-biphenyl]-3-yl)-2- oxospiro[indoline-3,30- pyrrolidine]-10-carboxylate(4e5). White solid (68 mg, yield: 62%). 1H NMR (400 MHz, CDCl3)d 9.10 (s, 1H), 7.24 (overlapped, 5H), 7.12e6.82 (m, 2H), 6.73 (d,J¼ 7.7 Hz,1H), 6.61 (s, 1H), 6.20 (s, 1H), 5.85 (s, 1H), 5.08 (s, 1H), 4.11(dt, J¼ 10.8, 7.6 Hz,1H), 3.94 (brs, 1H), 3.08 (s, 3H), 2.83 (s, 3H), 2.29(brs, 1H), 2.21 (brs, 1H), 1.44 (brs, 3H), 1.09 (brs, 6H). 13C NMR(100 MHz, CDCl3) d 179.5, 167.4, 153.7, 142.3, 142.2, 139.8, 137.6,137.5, 133.8, 129.6, 127.5, 127.4 � 2, 127.0 � 2, 125.1, 125.1, 124.8,124.3, 120.7, 108.8, 79.0, 66.3, 57.6, 45.0, 37.2, 33.7, 32.5, 27.1 � 3.HRMS (ESI) m/z: calcd for C31H32ClN3O4[M-H]- 544.2009, found544.1999.

4.2.4.6. tert-Butyl-2’-(3’-(methylsulfonyl)-[1,10-biphenyl]-3-yl)-2-oxospiro[indoline- 3,30- pyrrolidine]- 10-carboxylate (4e6).White solid (54 mg, yield: 52%). 1H NMR (400 MHz, CDCl3) d 9.19 (s,1H), 7.81 (t, J ¼ 7.8 Hz, 2H), 7.65e7.41 (m, 2H), 7.32 (d, J ¼ 7.7 Hz,1H), 7.25 (s, 1H), 7.05e6.86 (m, 3H), 6.78 (d, J ¼ 7.8 Hz, 1H), 6.72 (d,J¼ 7.6 Hz, 1H), 6.63 (s, 1H), 5.11 (s, 1H), 4.18e4.04 (m,1H), 3.94 (brs,1H), 2.27 (overlapped, 2H), 1.43 (brs, 3H), 1.10 (brs, 6H). 13C NMR(100 MHz, CDCl3) d 179.6, 153.8, 141.6, 140.0(overlapped, 2), 139.7,137.5, 131.2, 128.8, 127.6, 127.5 � 2, 127.1, 125.3, 124.9, 124.7,124.4 � 2, 120.7, 108.9, 79.1, 66.3, 57.6, 45.0, 43.5, 32.6, 27.1 � 3.HRMS (ESI) m/z: calcd for C29H30N2O5S[MþNa]þ 541.1768, found541.1761.

4.2.4.7. tert-Butyl-2’-(4’-(hydroxymethyl)-3’-(methylsulfonyl)-[1,10-biphenyl]-3-yl)-2- oxospiro[indoline- 3,30-pyrrolidine]-10-carboxylate(4e7). White solid (39 mg, yield: 36%). 1H NMR (400 MHz, CDCl3)d 8.17 (s, 1H), 7.94 (s, 1H), 7.50 (s, 2H), 7.30 (d, J¼ 7.7 Hz, 1H), 7.23 (s,

1H), 7.00 (t, J ¼ 7.7 Hz, 1H), 6.73 (d, J ¼ 7.7 Hz, 1H), 6.66 (s, 1H), 6.35(s, 1H), 5.99 (s, 1H), 5.13 (s, 1H), 4.91e4.87 (m, 2H), 4.12 (q, J ¼ 8.8,7.2 Hz, 1H), 3.93 (brs, 1H), 3.13 (s, 3H), 2.35 (s, 1H), 2.27e2.09 (m,1H), 1.43 (brs, 3H), 1.14 (brs, 6H). 13C NMR (100 MHz, DMSO‑d6)d 179.9, 154.1, 142.3, 141.1, 139.9, 138.6, 138.2, 132.0, 130.3, 129.1,128.6, 127.0, 126.2, 125.4, 121.2, 109.7, 79.2, 67.0, 60.0, 58.1, 56.5,55.3, 46.0, 44.5, 33.8, 28.3 � 3. HRMS (ESI) m/z: calcd. forC30H32N2O6S [M-H]- 547.1908, found 547.1900.

4.2.4.8. tert-Butyl-2’-(4’-(methoxycarbonyl)-3’-(methylsulfonyl)-[1,10-biphenyl]-3-yl)- 2-oxospiro [indoline-3,30-pyrrolidine]-10-carboxylate (4e8). White solid (51 mg, yield: 44%). 1H NMR(500 MHz, CDCl3) d 8.45 (s, 1H), 8.04 (s, 1H), 7.66 (d, J ¼ 7.7 Hz, 1H),7.57 (m, 1H), 7.33 (d, J ¼ 7.5 Hz, 1H), 7.26 (m, 1H), 7.01 (t, J ¼ 7.7 Hz,1H), 6.75 (d, J ¼ 7.7 Hz, 1H), 6.65 (brs, 1H), 6.31 (brs, 1H), 5.98 (brs,1H), 5.13 (brs, 1H), 4.13 (d, J ¼ 9.2 Hz, 1H), 3.91 (overlapped, 4H),3.34 (s, 3H), 2.36 (brs, 1H), 2.20 (s, 1H), 1.44 (brs, 3H), 1.09 (brs, 6H).13C NMR (125 MHz, CDCl3) d 166.2, 153.7, 143.4, 139.5, 139.4, 138.8,136.6 (br), 130.5, 130.1, 129.5, 127.7, 127.5, 127.4, 125.4, 125.2, 124.3,120.9, 108.7, 79.1, 66.4, 57.8, 52.2, 45.2, 43.9, 32.7, 27.1 � 3. HRMS(ESI) m/z: calcd. for C31H32N2O7S [MþNa]þ 599.1822, found599.1803.

4.2.4.9. tert-Butyl-2’-(30-fluoro-4’-(hydroxymethyl)-5’-(methyl-sulfonyl)-[1,10-biphenyl] -3-yl)-2-oxospiro [indoline-3,30-pyrrolidine]-10-carboxylate (4e9). White solid (35 mg, yield: 31%). 1H NMR(400 MHz, CD3OD) d 7.97 (s, 1H), 7.57 (overlapped, 2H), 7.46 (brs,1H), 7.25 (brs, 1H), 7.13 (d, J ¼ 7.7 Hz, 1H), 6.87 (d, J ¼ 7.8 Hz, 1H),6.73 (s, 1H), 6.35 (brs, 1H), 6.08 (brs, 1H), 5.16 (s, 1H), 5.13 (d,J¼ 2.0 Hz, 2H), 4.22e4.13 (m,1H), 4.07 (dt, J¼ 10.9, 6.2 Hz,1H), 3.40(s, 4H), 2.38 (overlapped, 2H), 1.42 (brs, 3H), 1.16 (brs, 6H). 13C NMR(100 MHz, CD3OD) d 182.3, 164.6, 162.1, 156.4, 144.6, 144.5, 143.0,143.0,142.8,138.7,130.0,129.7,128.0,127.3,127.1,126.5,124.7,124.7,122.8, 122.7, 120.3, 120.1, 110.8, 81.5, 68.6, 58.3, 53.9, 53.8, 47.2, 45.8,34.4, 28.5 � 3. HRMS (ESI) m/z: calcd. for C30H31FN2O6S [MþNa]þ

589.1779, found 589.1762.

4.2.4.10. tert-Butyl-2’-(4’-(hydroxymethyl)-3’-(methylsulfonamido)-[1,10-biphenyl]-3- yl)-2- oxospiro [indoline-3,30-pyrrolidine]-10-carboxylate (4e10). White solid (44 mg, yield: 39%). 1H NMR(400 MHz, CD3OD) d 7.52 (overlapped, 2H), 7.46e7.27 (m, 3H), 7.19(m, 1H), 7.12 (dt, J ¼ 7.7, 1.2 Hz, 1H), 7.04e6.95 (m, 1H), 6.87 (d,J¼ 7.7 Hz,1H), 6.71 (s, 1H), 6.26 (s, 1H), 6.02 (s, 1H), 5.12 (s, 1H), 4.82(s, 2H), 4.15 (dt, J¼ 11.1, 7.5 Hz,1H), 4.04 (dt, J¼ 11.1, 7.5 Hz,1H), 3.25(s, 3H), 2.26e2.10 (m, 2H), 1.43 (brs, 3H), 1.07 (brs, 6H). 13C NMR(100 MHz, CD3OD) d 175.1, 156.4, 142.9, 142.8, 142.8, 142.6, 141.1,137.1,135.6,130.4,129.6,129.5,127.2,127.1,126.6,125.7,125.6,123.8,122.7, 110.7, 81.5, 62.4, 54.8, 47.0, 40.0, 34.4, 28.5� 3. HRMS (ESI)m/z: calcd. for C30H33N3O6S[MþH]þ 564.2163, found 564.2153.

4.2.4.11. tert-Butyl-2 ’-(4 ’-(methoxycarbonyl)-3 ’-(methyl-sulfonamido)-[1,10-biphenyl]- 3-yl)-2-oxospiro[indoline-3,30-pyrroli-dine]-10-carboxylate (4e11). White solid (67 mg, yield: 67%). 1HNMR (400 MHz, CD3OD) d 10.43 (s, 1H), 8.60 (s, 1H), 7.96 (d,J¼ 8.4 Hz,1H), 7.67 (s, 1H), 7.28 (d, J¼ 7.7 Hz,1H), 7.20 (d, J¼ 1.2 Hz,1H), 7.04 (t, J¼ 7.7 Hz, 1H), 6.95 (t, J¼ 7.8 Hz, 1H), 6.72 (d, J¼ 7.8 Hz,1H), 6.64 (s, 1H), 6.44 (s, 1H), 6.15 (s, 1H), 5.17 (brs, 1H), 4.23e4.07(m,1H), 3.92 (d, J¼ 8.1 Hz, 1H), 3.87 (s, 3H), 2.23e2.04 (m, 2H), 1.42(brs, 3H), 1.10 (brs, 6H). 13C NMR (100 MHz, CD3OD) d 175.1, 167.3,153.8, 146.6, 140.1, 139.7, 139.4 (overlapped), 137.6, 131.1, 130.9,128.9, 127.7, 127.3, 125.1, 124.2, 120.9, 120.4, 115.4, 113.0, 108.7, 79.1,66.6, 58.1, 51.6, 45.5, 39.0, 32.8, 27.1 � 3. HRMS (ESI) m/z: calcd. forC31H33N3O7S[MþNa]þ 614.1931, found 614.1912.

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 13

4.2.4.12. tert-Butyl-2’-(30-methyl-5’-(methylsulfonamido)-[1,10-biphenyl]-3-yl)-2- oxospiro[indoline-3,30- pyrrolidine]-10-carboxylate(4e12). White solid (50 mg, yield: 50%). 1H NMR (400 MHz, CDCl3)d 8.78 (s, 1H), 7.65 (s, 1H), 7.25 (d, J ¼ 7.6 Hz, 1H), 7.22e7.11 (m, 1H),6.97 (m, 3H), 6.74 (d, J¼ 7.8 Hz, 1H), 6.62 (s, 1H), 6.29 (brs, 1H), 6.00(brs, 1H), 5.11 (s, 1H), 4.18e4.07 (m, 1H), 3.94 (brs, 1H), 2.97 (s, 3H),2.28 (s, 3H), 2.23e2.13 (m, 1H), 2.05 (brs, 1H), 1.42 (s, 3H), 1.14 (brs,6H). 13C NMR (100 MHz, CDCl3) d 179.4, 153.9, 141.4 (br), 139.5 (br),139.0 (br), 138.7 (br), 136.5, 127.6, 127.3 � 2, 127.2, 125.0, 124.3,123.5, 120.9, 118.8, 115.3, 108.8, 79.1, 66.5, 57.9, 45.2, 38.2, 32.6,27.1 � 3, 20.5. HRMS (ESI) m/z: calcd. for C30H33N3O5S [MþNa]þ

570.2033, found 570.2027.

4.2.4.13. tert-Butyl-2’-(4’-(hydroxymethyl)-6-methyl-3’-(methyl-sulfonyl)-[1,10- biphenyl]- 3-yl)-2-oxospiro [indoline-3,30-pyrroli-dine]-10-carboxylate (4e17). White solid (50 mg, yield: 85%). 1HNMR (400MHz, CD3OD) d 7.65 (d, J¼ 8.3 Hz, 2H), 7.37 (s,1H), 7.13 (s,1H), 7.03 (t, J ¼ 7.7 Hz, 1H), 6.91 (brs, 1H), 6.72 (d, J ¼ 7.8 Hz, 1H),6.67 (s,1H), 6.53 (s, 1H), 6.13 (s, 1H), 5.96 (s,1H), 4.95 (s, 2H), 4.90 (s,1H), 3.97 (dt, J ¼ 10.6, 6.4 Hz, 1H), 3.85 (dt, J ¼ 10.6, 6.4Hz, 1H), 3.12(s, 3H), 2.20 (brs, 1H), 2.09 (s, 3H), 1.19e1.13 (m, 9H). 13C NMR(100 MHz, CD3OD) d 170.1, 159.0, 145.7, 145.4, 143.5, 143.0, 141.8,138.1, 133.8, 133.7, 133.3, 132.2, 129.2, 129.1, 128.5,125.3, 117.8, 113.6,84.0, 70.7, 64.4, 56.1, 49.6, 47.7, 36.8, 31.1, 22.6. HRMS (ESI) m/z:calcd. for C31H34N2O6S[MþNa]þ 585.2030, found 585.2016.

4.2.5. Compounds 4e15 and 4-16Compounds 4e15 and 4e16 were synthesized by the following

general method. HCl (0.4 mL, 4.1 equiv., 4M in 2,4-dioxane) wasadded to a solution of 4-7 or 4e8 (0.37 mmol, 1.0 equiv.) in 5 mLDCM, and stirred at RT for 1 h. The mixture was concentrated invacuo and the residue was dissolved in DCM (5 mL) and Et3N(0.2mL,1.48mmol, 4 equiv.) was added and themixturewas stirredfor 5 min in an ice bath. Then anhydride (0.37 mmol, 1 equiv.) wasadded to the mixture, which was stirred for 5 h. The mixture wasdiluted with DCM, washed with aq. NaHCO3 and brine, dried overanhydrous Na2SO4, filtered and concentrated in vacuo. The residuewas purified by silica gel flash chromatography (Biotage SP-1, 20 gSiO2 column, gradient elution from 20 to 60% EtOAc) to afford 4e15and 4e16.

4.2.5.1. 1’-(3,3-Dimethyl-butanoyl)-2’-(4’-(hydroxymethyl)-3’-(methylsulfonyl)-[1,10- biphenyl]-3-yl)spiro[indoline-3,30-pyrrolidin]-2-one (4e15). White solid (55 mg, yield: 27%). 1H NMR (400 MHz,CDCl3) d 8.01 (s, 1H), 7.92 (s, 1H), 7.49 (s, 2H), 7.32 (brs, 1H), 7.25 (s,1H), 7.02 (t, J ¼ 7.8 Hz, 1H), 6.94 (s, 1H), 6.75 (d, J ¼ 7.8 Hz, 1H), 6.57(t, J ¼ 7.8 Hz, 1H), 5.83 (s, 1H), 5.44 (s, 1H), 4.88 (d, J ¼ 6.6 Hz, 2H),4.47e4.19 (m, 1H), 4.19 (brs, 1H), 3.17e3.10 (m, 3H), 2.36 (brs, 2H),1.35 (s, 9H). 13C NMR (125 MHz, CDCl3) d 181.2, 172.4, 141.7, 140.8,140.2, 139.8, 138.5, 138.3, 131.9, 130.4, 129.4, 128.6, 128.4, 127.5,127.3, 127.3, 126.0, 125.2, 121.2, 109.5, 66.4, 60.5, 56.5, 53.5, 46.3,43.8, 33.1, 29.3 � 3. HRMS (ESI) m/z: calcd. for C31H34N2O5S[MþNa]þ 569.2081, found 569.2074.

4.2.5.2. 2’-(4’-(Hydroxymethyl)-3’-(methylsulfonyl)-[1,10-biphenyl]-3-yl)-1’-(5-oxo- pyrrolidine-2-carbonyl)spiro-[indoline-3,30-pyrroli-din]-2-one (4e16). White solid (10 mg, 5%). 1H NMR (400 MHz,CDCl3) d 8.00 (s, 1H), 7.64 (s, 1H), 7.58 (s, 1H), 7.53 (d, J¼ 7.6 Hz, 1H),7.48 (d, J ¼ 7.8 Hz, 1H), 7.43e7.34 (m, 1H), 7.32 (brs, 1H), 7.06 (m,1H), 6.97 (s, 1H), 6.81 (d, J ¼ 8.1 Hz, 1H), 6.54 (t, J ¼ 7.6 Hz, 1H), 5.28(m, 1H), 5.23 (s, 1H), 4.63 (s, 2H), 4.28 (m, 1H), 4.17 (m, 1H),4.10e4.03 (m, 1H), 4.01 (s, 1H), 3.05 (s, 3H), 2.61 (p, J ¼ 8.9 Hz, 1H),2.50 (dd, J¼ 13.1, 9.2 Hz, 1H), 2.42e2.21 (m,1H), 2.20e2.08 (m,1H).13C NMR (100 MHz, CDCl3) d 179.1, 172.2, 170.7, 167.2, 144.0, 140.1,139.9, 139.1, 138.3, 131.9, 131.6, 130.8, 130.5, 129.4, 129.2, 128.6,

128.3, 127.2, 125.2, 124.9, 124.3, 116.4, 69.5, 66.4, 56.0, 53.2, 46.3,45.2, 45.0, 33.5, 29.5, 29.2, 26.8. HRMS (ESI) m/z: calcd. forC31H29N3O7S[M-H]- 586.1653, found 586.1639.

4.2.6. Compounds 4e18 to 4-20For compounds 4e18 to 4e20, intermediates were prepared by

coupling followed by amidation. Then K2CO3 (14 mg, 0.1 mmol, 1equiv.) and iodomethane (0.02 mL, 0.32 mmol, 3.2 equiv.) wereadded to a solution of intermediate (0.1 mmol, 1 equiv.) in DMF andstirred for 5 h. The mixture was then concentrated in vacuo. Theresidue was purified by silica gel flash chromatography (Biotage SP-1, 20 g SiO2 column, gradient elution from 0 to 50% EtOAc) to affordthe title compound.

4.2.6.1. tert-Butyl-2’-(3-fluoro-5-(1-methyl-1H-indol-6-yl)phenyl)-1-methyl-2-oxo- spiro[indoline-3,30- pyrrolidine]-10-carboxylate(4e18). White solid (40 mg, yield: 68%). 1H NMR (500 MHz, CDCl3)d 7.64 (d, J¼ 8.1 Hz,1H), 7.28 (brs, 2H), 7.18 (overlapped, 3H), 7.11 (d,J¼ 3.0 Hz,1H), 6.78 (overlapped, 3H), 6.51 (d, J¼ 2.9 Hz,1H), 6.12 (s,1H), 5.15 (s, 1H), 4.29e4.18 (m, 1H), 4.06e3.95 (m, 1H), 3.83 (s, 3H),3.26 (s, 3H), 2.40 (brs, 1H), 2.32 (brs, 1H), 1.55 (brs, 3H), 1.15 (brs,6H). 13C NMR (125 MHz, CDCl3) d 171.2, 164.1, 162.0, 154.7, 146.4,144.6, 144.2, 143.4, 143.0, 137.0, 133.6, 129.9, 128.5, 128.2, 125.2,122.1, 121.1, 119.0, 113.4, 113.0, 107.9, 100.9, 80.1, 67.2, 58.4, 46.0,33.6, 32.9, 28.1 � 3, 26.4. HRMS (ESI) m/z: calcd. forC32H32FN3O3[MþNa]þ 548.2320, found 548.2306.

4.2.6.2. 1’-(2,4-difluorobenzoyl)-2’-(5-fluoro-4’-(methoxymethyl)-3’-(methylsulfonyl)-[1,10-biphenyl]-3-yl)-1-methylspiro[indoline-3,30-pyrrolidin]-2-one (4e19). White solid (42 mg, yield: 65%). 1H NMR(500 MHz, CDCl3) d 8.05 (s, 1H), 7.64 (d, J ¼ 3.0 Hz, 3H), 7.22 (t,J ¼ 7.8 Hz, 1H), 7.15e7.12 (m, 2H), 7.04 (dt, J ¼ 8.3, 2.4 Hz, 1H), 6.97(dt, J ¼ 8.3, 2.4 Hz, 1H), 6.85 (d, J ¼ 7.8 Hz, 3H), 6.38 (d, J ¼ 7.8 Hz,1H), 5.63 (s, 1H), 4.89 (s, 2H), 4.12 (dt, J ¼ 10.2, 7.2 Hz, 1H), 4.04 (dt,J ¼ 10.2, 7.2 Hz, 1H), 3.51 (s, 3H), 3.30 (s, 3H), 3.21 (s, 3H), 2.43 (dt,J ¼ 13.4, 7.2 Hz, 1H), 2.32 (dt, J ¼ 13.4, 7.2 Hz, 1H). 13C NMR(125 MHz, CDCl3) d 176.5, 164.3, 164.0, 163.9, 163.0, 162.0, 161.9,161.0, 159.1, 159.0, 157.1, 157.0, 142.3, 140.3,140.2,139.6, 139.5, 139.4,135.8, 130.9, 130.6, 130.0, 129.9, 129.8, 128.0, 127.3, 126.1, 124.0,121.2, 120.4, 120.2, 112.4, 112.2, 111.5, 111.4, 107.3, 103.4, 70.4, 65.5,57.7, 55.7, 46.5, 44.0, 33.1, 25.5. HRMS (ESI) m/z: calcd. forC34H29F3N2O5S [MþNa]þ 657.1641, found 657.1631.

4.2.6.3. 2’-(5-Fluoro-4’-(methoxymethyl)-3’-(methylsulfonyl)-[1,10-biphenyl]-3-yl)-1’- (isopropylsulfonyl)-1-methylspiro[indoline-3,30-pyrrolidin]-2-one (4e20). White solid (30 mg, yield: 50%). 1H NMR(500 MHz, CDCl3) d 7.91 (s, 1H), 7.53 (s, 2H), 7.11 (t, J ¼ 7.7 Hz, 1H),7.06 (dt, J ¼ 7.7, 1.9 Hz, 1H), 6.90 (s, 1H), 6.81 (s, 1H), 6.73 (d,J¼ 7.7 Hz,1H), 6.68 (t, J¼ 7.7 Hz,1H), 6.07 (d, J¼ 7.7 Hz,1H), 5.09 (s,1H), 4.79 (s, 2H), 4.23 (dt, J¼ 9.6, 6.1 Hz,1H), 3.96 (dt, J¼ 9.6, 6.1 Hz,1H), 3.42 (s, 3H), 3.35e3.31 (m, 1H), 3.18 (s, 3H), 3.12 (s, 3H),2.35e2.27 (m, 2H),1.41 (d, J¼ 2.3 Hz, 3H),1.40 (d, J¼ 2.3 Hz, 3H). 13CNMR (125 MHz, CDCl3) d 176.5, 162.9, 160.9, 142.3, 142.3, 142.2,139.4, 139.3, 139.3,139.3,138.1, 135.8,131.0, 130.7, 128.1, 127.2, 125.7,123.9, 121.2, 120.8, 112.9, 112.7, 112.6, 112.4, 107.3, 70.4, 67.6, 57.7,57.1, 52.7, 47.0, 44.0, 33.1, 25.4, 16.1, 15.9. HRMS (ESI) m/z: calcd. forC30H33FN2O6S2[MþNa]þ 623.1656, found 601.1632.

4.2.7. tert-Butyl-2’-(3-aminophenyl)-2-oxospiro-[indoline-3,30-pyrrolidine]-10- carboxylate (S-4)

S-2a (886 mg, 2 mmol) was dissolved in 15 mL DMSO. Then CuI(114 mg, 0.6 mmol, 0.3 equiv.) sodium ascorbate (300 mg, 2 mmol,1equiv.), N,N0-dimethyl-1,2-ethanediamine (10 mL, 0.1 mmol 0.05equiv.) and NaN3 (325 mg, 5 mmol, 2.5 equiv. in 5 mL water) wereadded to the mixture under an N2 atmosphere, and stirred at 85 �C

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 11224014

overnight. The mixture was diluted with EtOAc followed by addi-tion of saturated NH4Cl to quench the reaction. The organic mixturewas washed with saturated NH4Cl and H2O thrice, dried overanhydrous Na2SO4, filtered and concentrated in vacuo. The residuewas dissolved in EtOAc (5 mL), and subsequently precipitated byaddition of hexane/EtOAc (v/v: 1/1, 5 mL), filtered, and washed withhexane twice to obtain the product as a light yellow solid (600 mg,yield: 79%). 1H NMR (400 MHz, DMSO‑d6) d 10.41 (s, 1H), 7.04 (dt,J ¼ 7.7, 1.2 Hz, 1H), 6.85 (s, 1H), 6.75 (d, J ¼ 7.7 Hz, 1H), 6.60 (t,J ¼ 7.7 Hz, 1H), 6.40 (d, J ¼ 7.7 Hz, 1H), 6.30e5.79 (m, 2H), 4.95 (s,3H), 4.66 (s, 1H), 3.81 (brs, 3H), 2.25 (s, 2H), 2.13 (brs, 1H), 1.42 (s,5H), 1.30e0.98 (m, 7H). ESI-MS: 380.2 [MþH] þ.

4.2.7.1. tert-Butyl-2’-(3-((4-(hydroxymethyl)-3-(methylsulfonyl)phenyl)amino)- phenyl)-2-oxospiro[indoline-3,30-pyrrolidine]-10-carboxylate (4e13). S-4 (500 mg, 1.32 mmol, 1.0 equiv.) was dis-solved in tBuOH (8 mL). (4-bromo-2-(methylsulfonyl)phenyl)methanol (384 mg, 1.45 mmol, 1.1 equiv.), potassium carbonate(276 mg, 1.98 mmol, 1.5 equiv.) and X-Phos (33 mg, 0.07 mmol, 0.05equiv.) and tris(dibenzylideneacetone)dipalladium (60 mg,0.07 mmol, 0.05 equiv.) were added to the mixture under an N2

atmosphere, and the mixture was stirred at 85 �C overnight. Themixturewas cooled in an ice bath, followed by addition of saturatedNH4Cl to quench reaction. The mixture was extracted with 100 mLEtOAc thrice, washed with brine, dried over anhydrous Na2SO4,filtered and concentrated in vacuo. The residue was purified bysilica gel flash chromatography (Biotage SP-1, 20 g SiO2 column,gradient elution from 30% EtOAc to 50% EtOAc) to afford the titlecompound, as awhite solid (512mg, yield: 85%). 1H NMR (400MHz,CD3OD) d 7.67 (s, 1H), 7.43 (s, 1H), 7.22 (brs, 1H), 7.15 (t, J ¼ 7.7 Hz,1H), 7.01e6.90 (overlapped, 3H), 6.75 (t, J ¼ 7.7 Hz, 1H), 6.28 (s, 1H),6.09 (s, 1H), 4.96 (overlapped, 3H), 4.09 (q, J ¼ 8.8 Hz, 1H),4.01e3.88 (m, 1H), 3.26 (s, 3H), 2.53e2.38 (m, 1H), 2.26 (d,J ¼ 13.8 Hz, 1H), 1.52 (brs, 3H), 1.28 (brs, 6H). 13C NMR (100 MHz,CD3OD) d 185.3, 158.9, 148.3, 145.9, 145.4, 142.9, 135.8, 134.2, 132.7,132.2,132.1� 2,129.2� 2,125.5,123.8,121.7, 120.3,113.2, 84.0, 71.0,64.5, 62.1, 49.3, 47.9, 37.0, 31.1 � 3. HRMS (ESI) m/z: calcd. forC30H33N3O6S [MþNa]þ 586.1982, found 586.1998.

4.2.7.2. tert-Butyl-2’-(3-(4-(hydroxymethyl)-3-(methylsulfonyl)ben-zamido)phenyl)- 2- oxospiro[indoline-3,30-pyrrolidine]-10-carbox-ylate (4e14). S-4 (100 mg, 0.26 mmol, 1.0 equiv.) was dissolved inDMF (5 mL). 4-formyl-3-(methylsulfonyl)benzoic acid (73 mg,0.32 mmol, 1.2 equiv.), HATU (120 mg, 0.32 mmol, 1.2 equiv.) andDIPEA (60 mL, 0.32 mmol, 1.2 equiv.) were added to the mixture andstirred overnight. The mixture was diluted with 50 mL EtOAc,washed with H2O, dried over anhydrous Na2SO4, filtered andconcentrated in vacuo. The residue was dissolved in MeOH (5 mL)cooled in an ice-bath. Then NaBH4 (50 mg, 1.3 mmol, 5.0 equiv) wasadded to the mixture and stirred for 5 min. Water was added toquench the reaction, and the mixture was extracted with 10 mLEtOAc thrice, washed with brine, dried over anhydrous Na2SO4,filtered and concentrated in vacuo. The residue was purified bysilica gel flash chromatography (Biotage SP-1, 20 g SiO2 column,gradient elution from 30 to 50% EtOAc) to afford the title com-pound, as a white solid (48 mg, yield: 25%). 1H NMR (400 MHz,CD3OD) d 8.42 (s, 1H), 8.12 (s, 1H), 7.83 (d, J ¼ 7.7 Hz, 1H), 7.60 (d,J ¼ 7.7 Hz, 1H), 7.12 (s, 1H), 6.97 (dt, J ¼ 7.7, 1.2 Hz, 1H), 6.85 (s, 1H),6.71 (d, J ¼ 7.7 Hz, 1H), 6.55 (s, 2H), 6.07 (s, 1H), 5.84 (s, 1H), 5.00 (s,2H), 4.88 (brs,1H), 3.97 (q, J¼ 8.4 Hz,1H), 3.15 (s, 3H), 2.35 (brs,1H),2.14 (brs, 2H), 1.39 (brs, 3H), 1.14 (brs, 6H). 13C NMR (100 MHz,CD3OD) d 178.0, 162.2, 155.0, 148.8, 144.8, 144.4, 141.5, 138.1, 134.7,134.3, 132.4, 132.4 � 2, 128.9 � 2, 125.2, 123.5, 121.7, 120.3, 109.2,80.0, 76.6, 60.4, 58.0, 49.3, 43.4, 29.4, 27.0� 3. ESI-MS: 592.2 [MþH]þ.

4.3. In vitro and in vivo experiments section

4.3.1. Cell cultureThe human glioma cell lines U87EGFRvIII and U251 were pro-

vided by Paul S. Mischel (University of California, San Diego, USA).The human glioma cell line A172 was provided by Musheng Zeng(Sun Yat-Sen University, Guangzhou, China). HEK293T cells and allGBM cells were maintained in DMEM (Gibco) supplemented with10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin.The normal human astrocytes HA1800 were maintained in Astro-cyte Medium (ScienCell). All cell lines were maintained at 37 �C in5% CO2.

4.3.2. Transient transfection and dual luciferase reporter assaysHEK293T 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-fos-FF-luc, 0.13 mg pCMV/Renilla-luc, 1.3 mg pSG5/hRXRa and 1.3 mg pSG5/hLXRa or pSG5/hLXRb. 5 h after transfection, cells were treated with tested com-pounds for 20 h. Cells were lysed and assayed for firefly and renillaluciferase activities using Dual Luciferase Reporter Assay System kit(Progema) following the manufacturer’s instructions [25]. Fireflyluciferase activity was normalized to Renilla luciferase for eachwell. The plasmids were gifts from Qing Song (University of Scienceand Technology, Beijing, China).

4.3.3. Quantitative real-time PCRCells were seeded in 6-well plates in 5% FBS for 24 h, then

changed to 1% lipoprotein deficient serum (LPDS) medium andtreated with tested compound for 48 h. Human LPDS was pur-chased fromYiyuan Biotechnologies (Guangzhou, China). Total RNAwas extracted by using RNAiso plus (TaKaRa) according to its pro-tocol. 1 mg of total RNAwas reverse transcribed into cDNA using theReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO),and quantitative real-time PCR amplification was performed usingSYBR Green Realtime PCR Master Mix (TOYOBO). Gene expressionwas normalized to b-actin, which was used as an endogenouscontrol. The following qRT-PCR primers were used:

b-actin Forward: 50-CTCTTCCAGCCTTCCTTCCT-3’b-actin Reverse: 50-TGTTGGCGTACAGGTCTTTG-3’LXRa Forward: 50-GTTATAACCGGGAAGACTTTGC-3’LXRa Reverse: 50-AAACTCGGCATCATTGAGTTG-3’LXRb Forward: 50-TTTGAGGGTATTTGAGTAGCGG-3’LXRb Reverse: 50-CTCTCGCGGAGTGAACTAC-3’ABCA1 Forward: 50-AACAGTTTGTGGCCCTTTTG-3’ABCA1 Reverse: 50-AGTTCCAGGCTGGGGTACTT-3’ABCG1 Forward: 50-ATTCAGGGACCTTTCCTATTCGG-3’ABCG1 Reverse: 50-CTCACCACTATTGAACTTCCCG-3’IDOL Forward: 50-CGAGGACTGCCTCAACCA-3’IDOL Reverse: 50-TGCAGTCCAAAATAGTCAACTTCT-3’ApoE Forward: 50-TGGGTCGCTTTTGGGATTAC-3’ApoE Reverse: 50-TTCAACTCCTTCATGGTCTCG-3’SREBP-1c Forward: 50-GGAGGGGTAGGGCCAACGGCCT-3’SREBP-1c Reverse: 50-CATGTCTTCGAAAGTGCAATCC-3’

4.3.4. Cell proliferation and viability assayCells were seeded in 96-well plates at 1.5 � 103 cells per well in

1% LPDS for 24 h. After treating with tested compounds for 7 days,cells were tested with a Cell Counting Kit (CCK-8) (YeasenBiotechnology, Shanghai, China).

4.3.5. LDL uptakeCells were seeded at 1 � 105 per well in 6-well plates in 5% FBS

overnight, then changed to 1% LPDS medium with 2 mg/ml Dil-LDL(Yiyuan Biotechnologies, Guangzhou, China). After treated with

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 112240 15

tested compounds for 48 h, the cells were washed twice withphosphate-buffered saline (PBS) and fixed in 4% paraformaldehydefor 30 min, then stained by DAPI for imaging. Cell imaging wasperformed on EVOS FL Auto Cell Imaging System (Invitrogen) andfluorescence intensity was quantified by ImageJ.

4.3.6. Cholesterol effluxCells were seeded at were seeded in 96-well plates at 4 � 104

per well for 12 h. After being labelled with 0.5 mM 22-NBD-cholesterol in the presence of tested compounds for 24 h, cells werewashed twice with PBS and incubated for 4 h in DMEM containing15 mg/ml ApoA1 (Sino Biological, Beijing, China). Then the choles-terol in the medium and cells was assayed using a microplatereader respectively (Flex Station 3, excitation 485 nm, emission535 nm) [37].

4.3.7. Cellular cholesterol measurementCells were placed in 10 cm dishes. After treated with tested

compounds for 48 h, 400 ml RIPA lysis buffer per dish was addedand cells were lysed with a probe sonicator. Protein concentrationwas determined by a BCA assay (Pierce). Then, MeOH (250 ml) andCHCl3 (750 ml) were added sequentially, vortexing after eachaddition. The samples were centrifuged (3000 rpm, 4 �C, 10 min) toobtain separate phases. The CHCl3 (lower) phase was evaporated todryness and measured using Total-Cholesterol Assay Kit (NanjingJiancheng Bioengineering Institute, Nanjing, China).

4.3.8. siRNA transfection50 nM siRNA negative control (scramble), LXRa or LXRb was

transfected into U87EGFRvIII using Lipofectamine 3000 (Invi-trogen) in 1% LPDS for 24 h siRNA negative control (scramble), LXRaand LXRb were purchased from GenePharma (Suzhou, China). Thefollowing siRNAs were used:

siRNA negative control (scramble) sense:50-UUCUCCGAACGUGUCACGUTT-3’siRNA negative control (scramble) antisense:50-ACGUUGACACGUUCGGAGAATT-3’siRNA LXRa sense: 50-CACGGAUGCUAAUGAAACUTT-3’siRNA LXRa antisense: 50-AGUUUCAUUAGCAUCCGUGTT-3’siRNA LXRb sense: 50-CCCAGAUCCCGAAGAGGAATT-3’siRNA LXRb antisense: 50- UUCCUCUUCGGGAUCUGGGTT-3’

4.3.9. Cloning, expression and purification of LXRbFor fluorescence polarization assays and protein crystallization,

human LXRb-LBD was prepared as reported with minor modifica-tions [38]. Briefly, LXRb-LBD (residues 215e461) was modified atthe C-terminus by adding a peptide (687HHKILHRLLQDSSS699) fromco-activator SRC2 (SRC2-2). To reduce the surface entropy and thepotential protein aggregation, four mutations (Q259A, R261G,D262S, R264S) were performed [38]. The modified LXRb-LBD DNAcoding sequence was inserted into pET28a (þ) plasmid (Novagen).

LXRb-LBD were overexpressed in Escherichia coli BL21 (DE3)cells (Novagen). The bacteria were grown in LuriaeBertani broth at37 �C till absorption at 600 nm reached around 0.6. Then 0.25 mMof isopropyl b-D-1-thiogalactopyanoside (IPTG) was added, and thebacteria were further cultured at 18 �C overnight before harvest bycentrifugation at 5000 rpm (rotor JLA 9.1000, Avanti J-E, Beckman).Bacterial cells were resuspended with the lysis buffer (50 mM Tris-HCl pH 8.5, 400 mM NaCl, 5% glycerol, 20 mM imidazole, 2 mM 2-mercaptoethanol) and lysed by sonication. The lysate was centri-fuged at 18,000 rpm for 30 min, and then the supernatant wasloaded onto a pre-equilibrated Ni-NTA column (5 mL resin). Theimpurity was washedwith 50mL of lysis buffer, followed by elutionwith imidazole step procedure. Fractions were analyzed by SDS-PAGE and fractions containing LXRb-LBD were concentrated and

exchanged to gel filtration buffer (20 mM Tris pH8.5, 200 mMNaCl,5% glycerol and 5 mM b-mercaptoethanol). The LXRb-LBD proteinwas further purified with the HiLoad 16/60 Superdex 200 prepgrade column (GE Healthcare). Peak fractions were analyzed bySDS-PAGE, pooled, concentrated to 20 mg/ml and stored at �80 �Cin the storage buffer (20mMTris pH 8.0,150mMNaCl, and 5mM b-mercaptoethanol).

4.3.10. FP binding assayKi was determined by a FP-based competition assay developed

in our lab. FP experiments were conducted with a Victor X5microplate reader (PerkinElmer) using black NBS polystyrene 384-well microplates (Corning). Final concentrations of 400 nM LXRband 10 nM hyodeoxycholic acid-based fluorescent tracer were usedin the competition assays. Compounds to be determined werediluted using the FP buffer (50 mM Tris, pH 8.0, 400 mM NaCl and5 mM 2-mercaptoethanol). And assays were performed in tripli-cate. The final concentration of DMSO in the reactions was less than2% (v/v). The microplate was shaken gently (100 rpm) for 10 min at25 �C using an orbital shaker and centrifuged at 1500 rpm for 1min.After equilibration at room temperature for 30 min, the FP valueswere recorded using excitation and emission filters of 485 nm and535 nm, respectively. Graphpad Prism 6.0 was used to calculate theKi by fitting the curve of the FP value (FPread) versus the concen-tration of LXR modulators as reported [39].

4.3.11. Crystallography4ss or 4-7rr (100mM in DMSO)was dilutedwith protein storage

buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 5 mM b-mercap-toethanol) and mixed with the LBD domain of human LXRb (10 mg/mL) at a final fragment concentration of 2 mM and a final DMSOconcentration of 2% (v/v). The protein and fragment were incubatedat 4 �C overnight, and the precipitant was removed by centrifugingat 11,000 g for 10 min at 4 �C. Crystallization was performed usingsitting vapor-diffusion method by mixing 1 ml of LXRb-LBD/frag-ment complexwith 1 mL of reservoir solution (0.1M Tris pH 8.5, 22%(w/v) PEG3350) and then equilibrating against 100 mL of reservoirsolution. Crystallization plates were placed at 8 �C for 2e5 daysbefore flat rod crystals would appear.

The diffraction data were collected at 100 K at the beamlineBL17U1 of Shanghai Synchrotron Radiation Facility (SSRF) [40] andprocessed using HKL-3000 [41]. The structure was solved by themolecular replacement method with Phaser [42], and the reportedstructure of LXRb-LBD (PDB code: 5HJP) was used as the searchingmodel [43]. Then iterative refinements of the structure modelswere carried out using Coot and Refmac5 [44]. The atomic co-ordinates and structure factors (accession code 6K9G and 6K9H)have been deposited in the Protein Data Bank.

The LXRb protein binding with 4ss and 4-7rr was co-crystallized. Their PDB codes are 6K9H (ligand 4ss) and 6K9G(ligand 4-7rr). Their refinement statistics data were showed inTable S2.

4.3.12. Pharmacokinetics analysis of 4-13Compound 4e13 was suspended in normal saline for intra-

gastric administration. The SD rats (male, n ¼ 6) were treated byintravenous administration at 10 mg/kg dose, and the blood sam-ples were taken at various time points during a 24 h period. Theconcentration of compounds in the blood was analyzed by LC-MS(Thermo chromatographic system and mass spectrometer, TSQquantum access max, Finnigan, USA).

4.3.13. Xenograft model5 � 105 U87EGFRvIII cells were implanted into 4-week-old male

BALB/c nu/nu mice for subcutaneous (s.c.) xenograft studies. After

H. Chen et al. / European Journal of Medicinal Chemistry 194 (2020) 11224016

tumor size reached 40 mm3, mice were randomly divided into 4groups (n ¼ 6), including the control group and treatment grouptreated with GW3965 (40 mg/kg/day, i.g.), LXR-623 (40 mg/kg/day,i.g.) or 4e13 (50 mg/kg/day, i.p.) for 15 continuous days. Tumorgrowth was monitored with calipers on the days indictedthroughout of course of the experiment. All experiments wereconducted after approval by the Laboratory Animal Center at SunYet-Sun University.

Declaration of competing interest

The authors declare that they have no known competingfinancial interests or personal relationships that could haveappeared to influence the work reported in this paper.

Acknowledgment

This work has been funded in part of the National Key R&DProgram of China (2017YFB02034043), Science and TechnologyProgram of Guanghzou (201604020109), National Natural ScienceFoundation of China (81773636), and Guangdong Provincial KeyLab. of Construction Foundation (2011A060901014). We thank thestaff of BL17U1 and BL19U1 beamlines at Shanghai SynchrotronRadiation Facility, Shanghai, People’s Republic of China, for assis-tance during data collection.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.ejmech.2020.112240.

Abbreviations

SBDD Structure-based drug designTFA trifluoroacetic acidDCM dichloromethaneTHF tetrahydrofuranBoc t-butyloxy carbonyl(Bpin)2 bis(pinacolato)diboronDPPF 1,10-ferrocenebis(diphenylphosphine)DBA dibenzylideneacetoneX-Phos 2-(dicyclohexylphosphino)-20,40,60-tri-i-propyl-1,10-

biphenylHATU [dimethylamino(triazolo[4,5-b]pyridin-3-yloxy)

methylidene]- dimethylazaniumDIPEA N,N-diisopropylethylamineDMF N,N-DimethylformamideABCA1 ATP-binding cassette transporter A1IDOL inducible degrader of LDLRLDL low density lipoproteinLDLR low density lipoprotein receptorABCG1 ATP-binding cassette transporter G1APOE apolipoprotein ESREBP-1c Sterol-regulatory element binding protein 1cFP fluorescence polarizationAUC area under the curveMRT mean residence time

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