Discovery of new LXRβ agonists as glioblastoma inhibitors

Discovery of new LXRβ agonists as glioblastoma inhibitors

Journal Pre-proof Discovery of new LXRβ agonists as glioblastoma inhibitors Hao Chen, Ziyang Chen, Zizhen Zhang, Yali Li, Shushu Zhang, Fuqiang Jiang,...

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Journal Pre-proof Discovery of new LXRβ agonists as glioblastoma inhibitors Hao Chen, Ziyang Chen, Zizhen Zhang, Yali Li, Shushu Zhang, Fuqiang Jiang, Junkang Wei, Peng Ding, Huihao Zhou, Qiong Gu, Jun Xu PII:

S0223-5234(20)30207-5

DOI:

https://doi.org/10.1016/j.ejmech.2020.112240

Reference:

EJMECH 112240

To appear in:

European Journal of Medicinal Chemistry

Received Date: 31 December 2019 Revised Date:

12 March 2020

Accepted Date: 13 March 2020

Please cite this article as: H. Chen, Z. Chen, Z. Zhang, Y. Li, S. Zhang, F. Jiang, J. Wei, P. Ding, H. Zhou, Q. Gu, J. Xu, Discovery of new LXRβ agonists as glioblastoma inhibitors, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112240. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Discovery of New LXRβ Agonists as Glioblastoma Inhibitors Hao Chen‡†, Ziyang Chen‡†, Zizhen Zhang‡†, Yali Li†, Shushu Zhang†, Fuqiang Jiang†, Junkang Wei†, Peng Ding†, Huihao Zhou *†, Qiong Gu*† and Jun Xu*†



Guangdong Provincial Key laboratory of New Drug Design and Evaluation,

Research Center for Drug Discovery at School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China

Abstract: Discovery and optimization of selective liver X receptor β (LXRβ) agonists are challenging due to the high homology of LXRα and LXRβ in the ligand binding domain (LBD). There is only one different residue (Val versus Ile) at the LBD of LXRs. With machine learning methods, we identified pan LXR agonists with a novel scaffold (spiro[pyrrolidine-3,3'-oxindole]). Then, we figured out the mechanism of LXR isoform selectivity from co-crystal structures. Based on the mechanism and the new scaffold, LXRβ selective agonists were designed and synthesized. This led to the discovery of LXRβ agonists 4-7rr, 4-13 and 4-13rr with IC50 values ranging from 1.78 to 6.36 µM against glioblastoma in vitro. Treatment with 50 mg/kg/day of 4-13 for 15 days significantly reduced tumor growth using an in vivo xenograft glioblastoma model.

Key words: nuclear receptor; glioblastoma inhibitor; spiro[pyrrolidine-3,3'-oxindole]; structure-based drug design.

1. Introduction The LXRs (LXRα and LXRβ) [1-4] are known as nuclear oxysterol receptors and recognized as potential targets for the disease in which lipids have a central role, such as atherosclerosis, inflammation, Alzheimer's disease and Parkinson's disease [5-9]. Recently, LXR agonists are used to treat cancer by regulating target genes, like APOE, ABCA1, IDOL and ABCG1 [9-12]. LXRβ selective agonist, LXR-623, was reported as an effective agent against glioblastoma (GBM), a human malignancy in brain with high mortality [11]. GBM cells do not synthesize cholesterol, and rely on exogenous cholesterol for survival[13]. LXR-623 activates LXRβ and up-regulates genes ABCA1 and IDOL, effluxes intracellular cholesterol and inhibits uptake of extracellular cholesterol leading to GBM cell death. Thus, LXRβ selective agonist becomes a novel strategy against GBM. LXR can be activated by endogenous ligands such as oxycholesterols or other natural or synthetic ligands [2, 14]. LXRβ is not organ-specifically expressed, while LXRα is mainly expressed in specific organs/tissues such as liver, intestine, adipose tissues, and macrophages. Activation of LXRα may induce substantial increases of triglycerides (TGs) in liver and plasma, and LXRβ selective agonists may avoid these side effects. Therefore LXRβ selective agonists are demanding [15-18]. Several LXRβ agonists, such as 61X (PDB code: 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 in ligand binding domain (LBD) and flexible pocket. There is only one residue difference in the proximity of the pocket: Val263(α) versus Ile277(β) [21]. The volume of ligand-binding pocket is 560-680 Å3 for LXR agonist T0901317 and 980-1090 Å3 for LXR agonist GW3965 [22, 23]. This allows LXRs to accommodate structurally diverse ligands, but it makes it difficult to identify novel and LXRβ selective agonists through structure-based drug design [24]. Previously, we have successfully identified farnesoid X receptor (FXR) agonists with support vector machine (SVM) and naïve Bayesian (NB) methods [25]. Here, we identified LXR agonists with a novel scaffold using the machine-learning based

virtual screening platform, and obtained co-crystal structures of LXR-ligands, from which we articulated the mechanism of LXR isoform selectivity. Based on these results, we further optimized the leads, and resulted in a potent compound that significantly inhibits GBM cells through a cholesterol-regulation pathway. O S

F

F3 C

O S N

F

O

N

F O

O

Cl O

N HO

F

O O

CF3 CF3

N N Cl

OH F

F

F N

F

N CF3

CF3

T0901317 LXRα/β (nM/nM)a SI =23/17

GW3965 LXRα/β (nM/nM) SI =200/40

LXR-623 LXRα/β (nM/nM) SI =179/24

61X LXRα/β (nM/nM) SI =40/2.8

WAY-40 LXRα/β (nM/nM) SI =668/20

Fig. 1. The known LXR agonists.

2. Results and discussion 2.1. Discovery of LXR agonists As shown in Fig. 2A, from ChEMBL and BindingDB databases, we collected 964 known LXR ligands, and created machine models by SVM and NB approaches, which were used to screen 9500 compounds from our in-house compound library, and got 59 biologically confirmed hits (Fig. 2A and Fig. S5 in Supporting Information), among which 13 LXR agonists were highly potent (Fig. 2B and 2C). Of them, compounds

4,

5

and

13

have

common

novel

scaffold

(spiro

[pyrrolidine-3,3'-oxindole]). The detailed machine learning protocol were given in the Hit Discovery Section in Supporting Information. Compound 4 is a non-selective LXR agonist. It exhibits most potent activity with EC50LXRα = 1.97 µM and EC50LXRβ = 1.59 µM (SI = 1.2, Table 2), and suppresses GBM-related U87EGRvIII cells with IC50 of 22.6 µM. Compound 4 has two enantiomers, which were separated by chiral chromatography resulting in R,Renantiomer 4rr and S,S-enantiomer 4ss. Further experiments demonstrated that 4ss displayed superior activities (EC50LXRβ (4ss) = 0.63 µM, SI = 1.3; EC50LXRβ (4rr) > 10 µM, Table 2). In order to convert the hits (4rr and 4ss) into LXRβ selective agonists, both enantiomers were incubated with LXRβ. The co-crystal structure of 4ss-LXRβ

complex was acquired (PDB code: 6K9H). A 4ss-LXRβ binding mode is depicted in Fig. 2D. 4ss is surrounded by helices H3, H11 and H12 (which is responsible for the LXR agonism) [26, 27]. The pocket is divided by 4ss into two chambers, A and B. In chamber A, 4ss activates LXRβ by reinforcing the stacking interaction between His435 and Trp457 [21]. In chamber B, however, 4ss has more space which can be used to establish more interactions between the ligand and receptor at H1, Ile277/H3 and β-sheet [28].

Fig. 2. Discovery of 13 LXR agonists, their LXRβ agonistic activities and the representative binding mode (crystal structure). (A) Flow-chart of screening (B) Thirteen LXR agonists. (C) Thirteen LXRβ agonistic activities measured by luciferase reporter assays in transiently transfected HEK293T cells. 5 h after transfection, cells were treated with tested compounds (1 µΜ) for 20 h. *P < 0.05, **P < 0.01 and ***P < 0.001. (D) The binding mode of 4ss (an enantiomer derived from compound 4) acquired by X-ray crystallography.

2.2. Mechanism of LXRβ agonist selectivity To elucidate the mechanism of the LXRβ agonist selectivity, we compared the 4ss-LXRβ against 61X-LXRβ (an LXRβ selective agonist, SI = 14, PDB code: 3KFC) and LX2-LXRα (an LXRβ selective agonist, SI = 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 interaction between His435 and Trp457 in chamber A. However, 4ss, 61X and LX2 perturb the residues in chamber B in

different ways. LXRs residues in chamber B are divided into two groups: (1) the primary layer, in which Leu260/Leu274 (LXRα/β), 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 indirectly with the ligand.

Fig. 3. Mechanism of LXRβ agonist selectivity. (A) The structural perturbation of non-selective LXR agonist 4ss in LXRβ. (B) The structural perturbation of LXRβ selective agonist in LXRβ. (C) The structural perturbation of LXRβ selective agonist in LXRα. Four different residues between LXRα and LXRβ are underlined.

The selective ligand (61X) has stronger Van der Waals (VDW) interactions on residues in the primary layer than the non-selective ligand (4ss) but it also induces a salt-bridge (red dotted line between Asp245 and Lys248) and hydrogen bondings (red dotted lines Gln246-Val249 and Asp245-Lsy248) in the secondary layer. However, 4ss, a non-selective ligand, is unable to induce a similar perturbation on the residues in the secondary layer. Consequently, 61X and chamber B are more compact than 4ss and chamber B between their primary and secondary layers. 61X-LXRβ complex is significantly stabilized and, the selectivity of 61X to LXRβ is 14. When the LXRβ

selective agonist LX2 (SI = 4) binds to LXRα, it induces the similar side-chain conformation changes in the primary layer, and a gap between the primary and secondary layers. Thus, LX2-LXRα complex is destabilized as we have predicted. We further analyzed the major clusters of 61X-LXRβ and LX2-LXRα complexes with molecular dynamics (MD) simulations for 125 nanoseconds (ns) (Fig. S4 in Supporting Information). The LXRβ selective agonists consistently make chamber B of LXRβ more compact than non-selective LXR agonists do. Thus, the selectivity mechanism of LXRβ agonists is associated with the chamber B compactness induced by a ligand at the LBD of LXR.

2.3. LXRβ selective agonists design. To design LXRβ selective agonists based on 4ss, we superimposed 4ss on 61X, and then docked 4ss into the pocket previously occupied by 61X (the implementation of docking was described in the Supporting Information). The chamber B for the compounds in the spiro[pyrrolidine-3,3'-oxindole] library consists of a “hydrophilic site” formed by Glu281, Glu315 and Arg319, and a “hydrophobic site” formed by Leu274, Ile277 and Leu330. Fragments that are responsible to the LXRβ selectivity are listed in Table 1, where fragments F6 − F10 reside at “hydrophobic site” and “hydrophilic site”, and have better LXRβ selectivity than fragments F1 – F5 [8, 20, 23, 29-34]. The different linkers can assemble these fragments onto meta-position of the phenyl in the spiro[pyrrolidine-3,3'-oxindole] scaffold. In the chamber A, when acyl and sulfonyl interact with His435, their side chains or rings can contact with H12 residues for LXR agonism. The library was constructed by enumerating compounds from the bioisosteres. Then, the library compounds were docked into the selected ligand binding pocket (PDB code: 1PQ6, 3KFC and 6K9H) for virtual screening. These compounds were ordered and chosen for the followed synthesis, according to their docking scores (MOE and Schrödinger programs) and contributions to the interactions with His435 and the residues at hydrophobic and hydrophilic sites. The design process of converting 4ss to LXRβ selective agonist library is depicted in Fig. 4.

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

SIb

ID

F1

LX2/3FC6

14/4

F2

Q4K/5AVL 510/160c

ID

a

Fragments a

Fragments

PDB code

SI

F6

61X/3KFC

40/2.8

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

The functional fragments of LXRβ selective agonist. The groups (in red) contact with the

hydrophilic site. The groups (in green) contact with the hydrophobic site. b α/β selectivity index based on the reported Ki values (nM/nM). c LXRα/β selectivity index based on the reported EC50 values (nM/nM). d The LXRβ selective agonist with proven binding mode.

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

2.4. Synthesis The intermediate S-2 was derived from tryptamine, and synthesized under Pictet-Spengler reaction conditions, and this was followed by N-bromosuccinimide (NBS)- mediated rearrangement to generate enantiomer-containing 2'S, 3S- and 2'R, 3R-isoforms over 3 steps [35, 36]. Subsequently, the bromine in the intermediate was exchanged for a boronic acid pinacol (Bpin) or an NH2 group for subsequent coupling and acylation reactions which combine the designed aromatic fragments (Fig. 5 and

Scheme 1). Other acyl groups replacing the Boc (t-butyloxy carbonyl) moieties were used to produce 20 desired compounds, in which active racemic analogues were isolated and confirmed as R,R and S,S-isomers with their X-ray structures (PDB code: 6K9G and 6K9H) and specific optical rotations (Fig. S1, Supporting Information).

Scheme 1. Synthetic route for spiro[pyrrolidine-3,3'-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, N'-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.

2.5. Structure-activity relationships for spiro[pyrrolidine-3,3'-oxindole] derivatives Twenty compounds were assayed for LXR agonism (Fig. 5 and Table 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 π-π stacking interaction with Phe329 in chamber B. The R1 containing methylsulfonyl boosted the agonistic activity to the same level as that of GW3965, because the methylsulfonyl moiety can match the “hydrophobic site” better in chamber B than derivatives with methyl, chlorine and methylsulfonamide. Then, we found that it is necessary

to

keep

a

0-1

atom

unit

as

the

linker

between

the

spiro[pyrrolidine-3,3'-oxindole] and the aryl substituent. Compounds 4-7, 4-13 and 4-14 share the same R1 moiety, but 4-14 reduced LXR agonism, because of its amide-linker exceeding this limit. In chamber A, the Boc and 3,3-dimethylbutanoyl

moieties retain in the scaffold resulting in LXR agonism. When R3 or R4 is a methyl group, the substituent clashed with Leu274, Met312 and Thr316 leading to reduction of the activation (Fig. 6).

A

4-15 to 4-20

4-1 to 4-14 Comp

R1

Comp

R1

Comp

R1

R2

R3

R4

H

H

H

H

H

4-Me

Me

5-F

Me

5-F

Me

5-F

CH2OH

4-1

4-8

4-15

4-2

4-9

4-16

4-3

4-10

4-17

4-4

4-11

4-18

SO2Me

CH2OH SO2Me

O

4-5

4-12

F

4-19 F

4-6

4-13

4-7

4-14

4-20

B

Fig. 5. LXR transcriptional activity of synthetic derivatives. (A) Structures of synthetic derivatives and positive controls. Substituents in red were introduced to match hydrophilic site in chamber B of ligand-binding pocket. Substituents in green for hydrophobic site in chamber B of ligand-binding pocket. (B) LXR transcriptional activity was measured by luciferase reporter assays in transiently transfected HEK293T cells. 5 h after transfection, cells were treated with tested compounds (1 µΜ) for 20 h. *P < 0.05, **P < 0.01 and ***P < 0.001.

Subsequently, the R,R- and S,S-isomers were prepared from these active

compounds and assayed for their EC50 and Ki values and anti-GBM activity (Table 2). Most of derivatives, including the enantiomers and their chiral isomers, showed better selectivity than compound 4. The R,R-isomers of 4-7 to 4-15 had an obvious advantage in activation of LXRβ with an EC50 of 16 − 92 nM and 2.9 − 5.0 fold selectivity (Table 2), and Ki values ranging between 1.0 − 101 nM, suggesting that the R,R-configuration is more beneficial to ligand binding with LXRβ 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 for 61X is also 2.0 [28], and SI for WAY-40 was 3.2 [28]. As listed in Table 2, our synthesized compounds had improved SI values. It is worth to note that the SI values for 4-5rr, 4-7rr, 4-8rr and 4-15rr ranged from 3.2 to 5.0.

Table 2. LXR agonism and anti-glioblastoma activities for the synthesized compounds. ID# 4

LXRα EC50 (µM)a (% efficacy)

b

1.968±0.417 (82 ± 15)

LXRβ EC50 (µM)a (% efficacy)

b

1.589±0.413 (74 ± 10)

LXRα/β c

selectivity 1.2 h

LXRβ Ki (nM)d

U87EGFRvIII IC50 (µM)e

NTf

22.63 ± 3.74

1700 ± 300

NT

4rr

>10

>10

--

4ss

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

NA

4-5rr

1.176 ± 0.028 (63±2)

0.369 ± 0.129 (52±6)

3.2

264.7 ± 48.0

NA

4-6rr

1.071 ± 0.029 (79±5)

0.680 ± 0.305 (67±3)

1.6

148.6 ± 33.7

NA

4-7

0.372 ± 0.174 (79±10)

0.174 ± 0.032 (74±9)

2.1

NT

7.05 ± 2.47

4-7rr

0.145 ± 0.018 (81±3)

0.035 ± 0.014 (65±4)

4.1

1.0 ± 0.4

6.36 ± 2.82

4-7ss

0.94 ± 0.063 (82±11)

0.43 ± 0.110 (81±17)

2.8

134.9 ± 32.5

NA

4-8

0.708 ± 0.169 (84±23)

0.341 ± 0.193 (70±9)

2.1

NT

13.27 ± 3.07

4-8rr

0.174 ± 0.088 (80±12)

0.052 ± 0.014 (67±2)

3.3

37.2 ± 15.0

7.53 ± 0.71

4-8ss

1.746 ± 0.370 (68±16)

0.905 ± 0.139 (73±4)

1.9

1200 ± 400

NA

4-9

0.251 ± 0.046 (83±18)

0.103 ± 0.051 (54±6)

2.5

NT

8.49 ± 0.36

4-9rr

0.274 ± 0.008 (80±13)

0.092 ± 0.017 (48±1)

2.9

67.7 ± 16.1

7.78 ± 1.90

4-9ss

0.270 ± 0.043 (81±10)

0.281 ± 0.015 (54±1)

1.0

132.5 ± 20.3

NA

4-13

0.277 ± 0.066 (75±8)

0.095 ± 0.012 (85±23)

2.9

NT

3.75 ± 1.22

4-13rr

0.270 ± 0.023 (74±4)

0.088 ± 0.018 (86±11)

3.1

101 ±19.5

1.78 ± 0.43

4-13ss

0.928 ± 0.210 (80±16)

0.474 ± 0.172 (75±18)

2.0

234.3 ± 36.6

NA

4-15

0.214 ± 0.068 (67±4)

0.067 ± 0.016 (76±12)

3.1

NT

NA

4-15rr

0.080 ± 0.020 (73±5)

0.016 ± 0.006 (66±2)

5.0

7.3 ± 4.5

NA

4-15ss

0.161 ± 0.063 (66±3)

0.083 ± 0.023 (71±7)

1.9

70.4 ± 12.8

NA

T0901317

0.098± 0.044 (88±1)

0.108± 0.047(100±11)

0.9

41.3 ± 7.0

NT

GW3965

0.401± 0.155 (100±19)

0.228± 0.045(100±17)

1.8

2.2 ± 1.9

3.65 ± 0.32

LXR-623 a

0.431 ± 0.124 (92±8)

0.212 ± 0.010 (80±2)

2.0

14.2 ± 3.1

6.14 ± 1.04

The EC50 value was measured by transient transfection and luciferase reporter assays. b %

efficacy is related to positive control GW3965. c LXRα/β selectivity is the ratio of the EC50 value of LXRα to that of LXRβ. The reported agonists T0901317, GW3965, LXR-623, 61X, (5.96µM /2.94µM, SI = 2.0 [28]) and WAY-40 (8.44µM /2.67µM, SI = 3.2 [28]) as positive control. High selectivity (SI > 3.0), medium selectivity (SI = 2.0 to 3.0). measured by fluorescence polarization experiments.

e

d

LXRβ Ki was

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.

Table 2 indicates the LXRβ agonism is consistent with the anti-GBM activity. Compounds 4-7, 4-9, 4-13, and their R,R-isomers demonstrated more GBM suppression than S,S-isomers did. Compound 4-13rr and racemate 4-13 efficiently inhibit U87EGFRvIII cell line with IC50 of 1.78 and 3.75 µM. Compounds 4-13rr and 4-13 had higher LXRβ agonism efficacy (86% and 74%) and increased inhibition against GBM. Combining Table 2 and Fig. 5, we summarized a SAR map for our LXRβ selective agonists (Fig. 6A). The fragments that contribute to the selectivity are listed in Fig. 6B.

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

2.6. Binding mode analyses To validate the lead optimization result, we obtained the co-crystal structure of 4-7rr-LXRβ complex (PDB code: 6K9G). Using this structure complex and 4ss-LXRβ complex (PDB code: 6K9H), we created four binding complex models (4rr-LXRβ, 4ss-LXRβ, 4-7rr-LXRβ, and 4-7ss-LXRβ). In order to investigate the relations of chirality and activities, these models were experienced MD simulations for 125 ns [28]. As shown in Fig. 7, 4rr and 4ss, 4-7rr and 4-7ss are mirror enantiomers at the spiro[pyrrolidine-3,3'-oxindole] scaffold, resulting 4ss/4-7rr to form hydrogen bonding with His435 in chamber A, and no such hydrogen bond for 4rr/4-7ss. Thus, we explain why the mirror enantiomers have different LXR agonism behavior. When superimposing 4rr-LXRβ and 4ss-LXRβ (Fig. 7A), and 4-7rr-LXRβ and 4-7ss-LXRβ (Fig. 7B), we find that 4-7rr/4-7ss directly establishes π-π stacking with Phe329, hydrogen bonding with Leu330, and VDW interactions with Leu274, Ile277 and Leu330 in the primary layer. Fig. 7B reveals that the compactness of the residues in the binding pocket is significantly increased by introducing 4-7rr or 4-7ss in comparison with Fig. 8A. The red dotted lines indicate interactions newly established by 4-7rr or 4-7ss.

Fig. 7. Binding mode analyses for LXRβ agonist selectivity mechanism. (A) Non-selective LXR agonists (4ss-LXRβ and 4rr-LXRβ) have less compactness of the primary and secondary layers. 4ss formed hydrogen bond with His435 (red dotted line). The distance between 4rr and His435 is out of the hydrogen bonding (B) Selective LXR agonists (4-7ss-LXRβ and 4-7rr-LXRβ) have stronger compactness of the primary and secondary layers. 4-7rr formed hydrogen bond with His435. The distance between 4-7ss and His435 is out of the hydrogen bonding.

2.7. 4-13rr inhibits growth of GBM cells As shown in Table 2, 4-13rr (IC50=1.78 ± 0.43µM) is the best GBM cell inhibitor, and it was further studied in cell-based activities and inhibitory mechanisms [11]. LXR agonist LXR-623 was recently found to be of great potential in GBM treatment [11]. In vitro assays demonstrated 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 µM, respectively, compared with LXR-623 IC50 values of 6.14, 3.06, and 8.30 µM (Fig. 8A). And 4-13rr showed much lower toxicity to HEK293T cells and normal human astrocytes HA1800 (Fig. 8B).

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 of 4-13rr in U87EGFRvIII, U251 and A172 cells. (B) 4-13rr showed lower toxicity to HEK293T cells and normal human astrocytes HA1800.

2.8. 4-13rr regulates LXR-mediated cholesterol uptake and efflux As shown in Fig. 9A, compound 4-13rr dose-dependently upregulated the down-stream genes ABCA1, IDOL, ABCG1, APOE and SREBP-1c in U87EGFRvIII cells. With U87EGFRvIII-based assays, we observed that both 4-13rr and LXR-623 inhibited LDL uptake due to the upregulation of IDOL (Fig. 9B and 9C), promoted cholesterol 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 α and β isoforms separately, we were able to conclude that the absence of LXRβ blocked the 4-13rr-mediated suppression in U87EGFRvIII cells, indicating that compound 4-13rr suppressed GBM through activation of LXRβ (Fig. 9F, 9G and 9H).

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 µM, unless otherwise 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) LXRα and LXRβ were knocked down by RNAi separately. (H) LXRβ knock-down blocked the 4-13rr-mediated suppression of U87EGFRvIII. *P < 0.05, **P < 0.01 and ***P < 0.001.

2.9. Pharmacokinetics of compound 4-13 Compound 4-13rr and its racemate 4-13 were more potent than LXR-623 in vitro, therefore, 4-13 was further validated in in vivo models [11]. The in vivo (using Sprague-Dawley (SD) rats) pharmacokinetic (PK) study of compound 4-13 was conducted. The SD rats (male, n = 6) were treated with intravenous administration at 10 mg/kg dose, respectively. Plasma samples were collected at a period of 24 h, and concentration values of compound 4-13 were measured with LC-MS. The PK parameters were summarized in Table 3, which indicated that the T1/2 of compound 4-13 was 2.98 h, the Cmax was 1010 ± 174 ng/mL at Tmax 0.22 h, and AUC0-t was 1249 ± 51 h·ng/mL. Table 3. Pharmacokinetic parameters of compound 4-13 Tmax (h) mean ± SD

2.10.

0.22 ± 0.08

Cmax (ng/mL) 1010 ± 174

T1/2 (h) 2.98 ± 0.50

AUC0-t

AUC0-∞

(h·ng/mL)

(h·ng/mL)

1249 ± 51

1407 ± 93

MRTINF (h) 3.48 ± 0.44

Antitumor activity of 4-13 in vivo

Compound 4-13 (U87EGFRvIII IC50 = 3.75 µM) was validated in GBM xenograft models. Male nude mice were randomly divided into three groups (n = 6) and intraperitoneally treated with 4-13 at 50 mg/kg/day for 15 continuous days. The results are depicted in Fig. 10. The in vivo data demonstrated that the size of GBM tumors were significantly controlled, the efficacies of 4-13, GW3965 and LXR-623 were comparable (Fig. 10A~10C) and trends suggested that 4-13 could recover the body weight of the experimental mice (Fig. 10D). Serum assays demonstrated that 4-13, but not GW3965 and LXR-623, reduced adverse effects on triglycerides levels in blood (Fig. 10E).

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 tumor size reached 40 mm3, mice were treated with GW3965 (40 mg/kg/day, i.g.), LXR-623 (40 mg/kg/day, i.g.) or 4-13 (50 mg/kg/day, i.p.) for 15 continuous days. (A) Isolated tumor tissues 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), glutamic oxaloacetic transaminase (AST) and alkaline phosphatase (ALP) after administration for 15 days. *P < 0.05 and **P < 0.01.

3. Conclusion It is challenging to discover selective regulators for a protein target while it has a high homological isoforms at a binding pocket. Conventionally, people turn to explore allosteric sites or scaffold hopping approaches. In this study, however, we demonstrate a new strategy, which consists of following steps: (1) identifying hits with new scaffold using machine learning based screening; (2) optimizing the hits for an isoform selectivity based on the results of elucidating the selectivity mechanism, which is (in our case study) the pocket compactness-based LXRβ selectivity mechanism from co-crystal structures.

This study may open a new door to develop a selective agent for the target with multiple homologs. In the binding pocket, we should concern not only the direct interactions between a ligand and the key residues, but also the pocket compactness that is induced 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 Supporting Information.

4.2. Synthesis All chemical reagents and organic solvent were purchased from J&K Chemicals. 1

H NMR and

13

C NMR spectra were recorded on a Bruker Avance 400/500 MHz

NMR spectrometer for hydrogen and 100/125 MHz for carbon. Low resolution electrospray ionization (ESI) mass spectra were recorded on a Agilent 6120 single quadrupole LC/MS system using reverse-phase conditions (methanol/water, 0.05% formic acid). 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 (methanol-water, 85: 15, 1.5 mL/min) to separate the compounds. HPLC Purity was determined by analysis method on a Shimadzu HPLC system. Column: Agilent SB-C18; 5µm 4.6×250 mm. Solvent: 75% acetonitrile, 25% water. Flow rate 1.0 mL/min. All of 20 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: 85% methanol, 15% water. Flow rate 0.8 mL/min. The ee values of 18 chiral compounds exceeded 99% (Fig. S1). Specific rotations were detected on an AntonPaa MCP200 (solvent: methanol, Fig. S1) 4.2.1. General preparation for S-1a, S-1b and S-1c 3-Bromobenzaldehyde (6.5 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 200 mL DCM 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 50 mL EtOAc, and subsequently precipitated by addition of 200 mL hexane/EtOAc (v/v: 1/1), filtered, and washed with hexane twice to obtain the products S-1a, S-1b and 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) δ 7.72 (dt, J = 7.7, 1.7 Hz, 1H, H-4'), 7.64 (t, J = 1.7 Hz, 1H, H-2'), 7.58 (dt, J = 8.0, 1.1 Hz, 1H, H-5), 7.45 (t, J = 7.7 Hz, 1H, H-5'), 7.41 (dt, J = 7.7, 1.7 Hz, 1H, H-6'), 7.34 (dt, J = 8.1, 1.1 Hz, 1H, H-8), 7.20 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H, H-7), 7.12 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H, H-6). 5.94 (d, J = 1.7 Hz, 1H, H-1), 3.69 – 3.60 (m, 1H, H-3a), 3.60 – 3.53 (m, 1H, H-3b), 3.30 – 3.22 (m, 1H, H-4a), 3.24 – 3.18 (m, 1H, H-4b).

13

C NMR (100

MHz, CD3OD) δ 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 [M+H] +.

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) δ 7.69 – 7.67 (m, 2H), 7.54 (dd, J = 7.9, 1.6 Hz, 1H), 7.44 – 7.39 (m, 1H), 7.33 (dd, J = 7.9, 1.6 Hz, 1H), 7.14 (dt, J = 7.9, 1.6 Hz, 1H), 7.09 – 7.05 (m, 1H), 5.94 (s, 1H), 3.50 – 3.45 (m, 1H), 3.44 – 3.39 (m, 1H), 3.19 – 3.16 (m, 1H), 3.06 – 3.03 (m, 1H, 1H), 2.41 (s, 3H). ESI-MS m/z: 341.2, 343.2 [M+H] +. 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) δ 7.61 – 7.50 (m, 2H), 7.46 (d, J = 1.6 Hz, 1H), 7.33 (d, J = 7.8 Hz, 1H), 7.23 – 7.15 (m, 2H), 7.10 (t, J = 7.8 Hz, 1H), 5.95 (s, 1H), 3.65 – 3.61 (m, 1H), 3.58 – 3.55 (m, 1H), 3.24 – 3.20 (m, 1H), 3.18 – 3.15 (m, 1H).13C NMR (100 MHz, CD3OD) δ 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-MS m/z: 345.2, 347.2 [M+H] +.

4.2.2. Conversion of S-2a to S-2c NBS (9.8 g, 55 mmol) was added in batches to a solution of S-1 in THF/H2O (v/v: 4/1, 500 mL) with saturated NaHCO3 which was chilled in an ice bath, then dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford the crude product. TEA (21 mL, 150 mmol, 3.0 equiv) and di-t-butyl dicarbonate (10.9 mL, 50 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 anhydrous Na2SO4, filtered and concentrated in vacuo to afford the crude product which was dissolved in EtOAc, and diluted with hexane/EtOAc (3/1) for precipitation. 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-3,3'-pyrrolidine]-1'carboxylate (S-2a) White solid (11g, yield: 49.6%). 1H NMR (500 MHz, DMSO-d6) δ 10.33 (s, 1H, NH), 7.39 (dd, J = 7.8, 1.8 Hz, 1H, H-4''), 7.19 (t, J = 7.8 Hz, 1H, H-5''), 7.14 (brs, 1H, H-2''), 7.09 (t, J = 7.7 Hz, 1H, H-6), 7.00 (d, J = 7.7 Hz, 1H, H-4), 6.79 (d, J = 7.7 Hz, 1H, H-7), 6.68 (t, J = 7.7 Hz, 1H, H-5), 6.19 (d, J = 7.8 Hz, 1H, H-6''), 4.93 (s, 1H, H-2'), 4.00 – 3.87 (m, 2H, H-5'), 2.28 – 2.20 (m, 2H, H-4'), 1.28 (s, 9H, Boc-H). 13C NMR (100 MHz, CDCl3) δ 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. for C22H23BrN2O3[M+Na]+ 465.0784, found 465.0776.

4.2.2.2. tert-Butyl-2'-(3-bromo-4-methylphenyl)-2-oxospiro[indoline-3,3'pyrrolidine]-1'-carboxylate (S-2b) White solid (10.2 g, yield: 40.7%). 1H NMR (400 MHz, CDCl3) δ 9.28 (s, 1H), 7.08 (brs, 1H), 7.02 (dt, J = 7.7, 1.2 Hz, 1H), 6.94 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.73 – 6.56 (m, 2H), 6.23 (brs, 1H), 4.96 (brs, 1H), 4.14 – 3.99 (m, 1H), 3.88 (brs, 1H), 2.26 – 2.19 (m, 4H), 1.57 – 0.96 (m, 9H).13C NMR (100 MHz, CDCl3) δ 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[M+Na]+ 479.0941, found 479.0932.

4.2.2.3. tert-Butyl-2'-(3-bromo-5-fluorophenyl)-2-oxospiro[indoline-3,3'pyrrolidine]-1'- carboxylate (S-2c) White solid (13 g, yield: 51.2%). 1H NMR (500 MHz, CDCl3) δ 8.63 (s, 1H), 7.06 (td, J = 7.8, 1.2 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.89 – 6.48 (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) δ 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[M+Na]+ 483.0690, found 483.0674.

4.2.3. General preparation of S-3a, S-3b and S-3c Bis(pinacolato)diboron (2.54 g, 10 mmol, 1.1 equiv.), potassium acetate (0.98 g, 10 mmol, 1.1 euqiv.) and Pd(dppf)2Cl2 (328mg, 0.45 mmol, 0.05 equiv.) were added to a solution of S-2 (9 mmol, 1.0 equiv.) in 2,4-dioxane (60 mL) under an N2 atmosphere, and stirred at 85°C overnight. The mixture was cooled in an ice bath, and this was followed by addition of saturated NH4Cl to quench the reaction. The mixture was extracted with 100 mL EtOAc thrice, washed by brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (Biotage SP-1, 330 g SiO2 column, gradient elution from 0 - 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-3,3'-pyrrolidine]-1'-carboxylate (S-3a) Colourless solid (3.8 g, yield: 85%). 1H NMR (500 MHz, CDCl3) δ 7.75 (brs, 1H, H-4''), 7.53 (brs, 1H, H-5''), 7.09 (brs, 1H, H-2''), 6.97 (t, J = 7.7 Hz, 1H, H-6), 6.67 (d, J = 7.7 Hz, 1H, H-7), 6.58 (brs, 1H, H-5), 6.14 (brs, 1H, H-6''), 5.02 (s, 1H, H-2'), 4.10 – 4.06 (m, 1H, H-5'a), 3.93 (m, 1H, H-5'b), 2.24 (m, 2H, H-4'), 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-3,3'-pyrrolidine]-1'-carboxylate (S-3b) White solid (3.0 g, yield: 66%). 1H NMR (400 MHz, CDCl3) δ 7.66 (brs, 1H),

6.98 (t, J = 7.8 Hz, 1H), 6.89 (brs, 1H), 6.78 (brs, 1H), 6.67 (d, J = 7.7 Hz, 1H), 6.61 (brs, 1H), 6.15 (brs, 1H), 4.98 (s, 1H), 4.06 – 4.05 (m, 1H), 3.90 (brs, 1H), 2.39 – 2.38 (m, 2H), 2.24 – 2.21 (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 4-12, 4-15 to 4-20 Compounds 4-1 to 4-12, 4-15 to 4-20 were synthesized by the general method described here. Bromobenzene (0.22 mmol, 1.1 equiv.), potassium carbonate (30 mg, 0.22 mmol, 1.1 equiv.) and Pd(dppf)2Cl2 (7 mg, 0.01 mmol, 0.05 euiqv.) were added under an N2 atmosphere to a solution of S-3 (100 mg, 0.2 mmol, 1.0 equiv.) in 3 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 by addition of saturated NH4Cl to quench the reaction. The mixture was extracted with 20 mL EtOAc thrice, washed with brine, dried over anhydrous 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 20-60% EtOAc) to afford the derivatives 4-1 to 4-12, 4-15 and 4-20. 4.2.4.1.

tert-Butyl-2'-(4'-(hydroxymethyl)-[1,1'-biphenyl]-3-yl)-2-oxospiro[indoline-

3,3'- pyrrolidine]-1'-carboxylate (4-1) White solid (50 mg, yield: 53%). 1H NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 7.46 – 7.08 (m, 6H), 6.96 (dt, J = 7.8, 1.2 Hz, 1H, H-6), 6.69 (d, J = 7.8 Hz, 1H, H-7), 6.58 (brs, 1H, H-5), 6.17 – 5.81 (m, 1H, H-6''), 5.05 (s, 1H, H-2'), 4.62 (s, 2H, Bn-CH2), 4.18 – 4.07 (m, 1H, H-5'a), 4.05 – 3.93 (m, 1H, H-5'b), 2.25 – 2.16 (m, 2H, H-4'), 1.43 (brs, 3H, Boc-H), 1.21 – 1.07 (m, 6H, Boc-H). 13C NMR (100 MHz, CDCl3) δ 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. for C29H30N2O4[M+Na]+ 493.2098, found 493.2089.

4.2.4.2. tert-Butyl-2'-(4'-(methoxycarbonyl)-[1,1'-biphenyl]-3-yl)-2-oxospiro[indoline3,3'-pyrrolidine]-1'-carb-oxylate (4-2) White solid (60 mg, yield: 60%). 1H NMR (400 MHz, CDCl3) δ 9.21 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.33 (m, 2H), 7.22 (s, 1H), 7.09 (s, 1H), 7.00 (t, J = 7.9 Hz, 1H), 6.77 (d, J = 7.9 Hz, 1H), 6.60 (s, 1H), 6.20 (brs, 1H), 5.84 (brs, 1H), 5.15 (s, 1H),

4.20 – 4.04 (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) δ 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[M+Na]+ 521.2047, found521.2051.

4.2.4.3.

tert-Butyl-2'-(4'-(hydroxymethyl)-3',5'-dimethyl-1,1'-biphenyl]-3-yl)-2-

oxospiro [indoline-3,3'-pyrrolidine]-1'-carboxylate (4-3) White solid (48 mg, yield: 48%). 1H NMR (400 MHz, CD3OD) δ7.32 (dt, J = 7.7, 1.40 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.11 – 6.79 (m, 4H), 6.73 (d, J = 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 = 10.4, 8.0 Hz, 1H), 3.89 (q, J = 10.7, 8.0 Hz, 1H), 2.33 (s, 6H), 2.23 (overlapped, 2H), 1.40 (brs, 3H), 1.09 (brs, 6H).

13

C NMR (100 MHz, CD3OD) δ 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[M+Na]+ 521.2411, found 521.2411.

4.2.4.4.

tert-Butyl-2'-(3',5'-dimethyl-4'-(methylsulfonamido)-[1,1'-biphenyl]-3-yl)-2-

oxospiro[indoline- 3,3'-pyrrolidine]-1'-carboxylate (4-4) White solid (43 mg, yield: 38%). 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.23 – 7.08 (overlapped, 2H), 7.00 (t, J = 7.6 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.61 (brs, 1H), 6.56 (s, 1H), 6.14 (brs, 1H), 5.87 (brs, 1H), 5.05 (s, 1H), 4.16 – 4.03 (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) δ 179.8, 153.7, 139.6 (overlapped), 138.8 (overlapped), 136.6, 131.1, 127.5 (overlapped), 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[M+Na]+ 584.2190, found 584.2179.

4.2.4.5.

tert-Butyl-2'-(3'-chloro-4'-(dimethylcarbamoyl)-[1,1'-biphenyl]-3-yl)-2-

oxospiro[indoline-3,3'- pyrrolidine]-1'-carboxylate (4-5) White solid (68 mg, yield: 62%). 1H NMR (400 MHz, CDCl3) δ 9.10 (s, 1H), 7.24 (overlapped, 5H), 7.12 – 6.82 (m, 2H), 6.73 (d, J = 7.7 Hz, 1H), 6.61 (s, 1H),

6.20 (s, 1H), 5.85 (s, 1H), 5.08 (s, 1H), 4.11 (dt, J = 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). 13

C NMR (100 MHz, CDCl3) δ 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, found 544.1999.

4.2.4.6.

tert-Butyl-2'-(3'-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)-2-oxospiro[indoline-

3,3'- pyrrolidine]- 1'-carboxylate (4-6) White solid (54 mg, yield: 52%). 1H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H), 7.81 (t, J = 7.8 Hz, 2H), 7.65 – 7.41 (m, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.25 (s, 1H), 7.05 – 6.86 (m, 3H), 6.78 (d, J = 7.8 Hz, 1H), 6.72 (d, J = 7.6 Hz, 1H), 6.63 (s, 1H), 5.11 (s, 1H), 4.18 – 4.04 (m, 1H), 3.94 (brs, 1H), 2.27 (overlapped, 2H), 1.43 (brs, 3H), 1.10 (brs, 6H).

13

C NMR (100 MHz, CDCl3) δ 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[M+Na]+ 541.1768, found 541.1761.

4.2.4.7. tert-Butyl-2'-(4'-(hydroxymethyl)-3'-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)-2oxospiro[indoline- 3,3'-pyrrolidine]-1'-carboxylate (4-7) White solid (39 mg, yield: 36%). 1H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.94 (s, 1H), 7.50 (s, 2H), 7.30 (d, J = 7.7 Hz, 1H), 7.23 (s, 1H), 7.00 (t, J = 7.7 Hz, 1H), 6.73 (d, J = 7.7 Hz, 1H), 6.66 (s, 1H), 6.35 (s, 1H), 5.99 (s, 1H), 5.13 (s, 1H), 4.91-4.87 (m, 2H), 4.12 (q, J = 8.8, 7.2 Hz, 1H), 3.93 (brs, 1H), 3.13 (s, 3H), 2.35 (s, 1H), 2.27 – 2.09 (m, 1H), 1.43 (brs, 3H), 1.14 (brs, 6H).

13

C NMR (100 MHz,

DMSO-d6) δ 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. for C30H32N2O6S [M-H]- 547.1908, found 547.1900.

4.2.4.8. tert-Butyl-2'-(4'-(methoxycarbonyl)-3'-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)2-oxospiro [indoline-3,3'-pyrrolidine]-1'-carboxylate (4-8)

White solid (51 mg, yield: 44%). 1H NMR (500 MHz, CDCl3) δ 8.45 (s, 1H), 8.04 (s, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.57 (m, 1H), 7.33 (d, J = 7.5 Hz, 1H), 7.26 (m, 1H), 7.01 (t, J = 7.7 Hz, 1H), 6.75 (d, J = 7.7 Hz, 1H), 6.65 (brs, 1H), 6.31 (brs, 1H), 5.98 (brs, 1H), 5.13 (brs, 1H), 4.13 (d, J = 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) δ 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 [M+Na]+ 599.1822, found 599.1803.

4.2.4.9. tert-Butyl-2'-(3'-fluoro-4'-(hydroxymethyl)-5'-(methylsulfonyl)-[1,1'-biphenyl] -3-yl)-2-oxospiro [indoline-3,3'-pyrrolidine]-1'-carboxylate (4-9) White solid (35 mg, yield: 31%). 1H NMR (400 MHz, CD3OD) δ 7.97 (s, 1H), 7.57 (overlapped, 2H), 7.46 (brs, 1H), 7.25 (brs, 1H), 7.13 (d, J = 7.7 Hz, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.73 (s, 1H), 6.35 (brs, 1H), 6.08 (brs, 1H), 5.16 (s, 1H), 5.13 (d, J = 2.0 Hz, 2H), 4.22 – 4.13 (m, 1H), 4.07 (dt, J = 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) δ 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 [M+Na]+ 589.1779, found 589.1762.

4.2.4.10. tert-Butyl-2'-(4'-(hydroxymethyl)-3'-(methylsulfonamido)-[1,1'-biphenyl]-3yl)-2- oxospiro [indoline-3,3'-pyrrolidine]-1'-carboxylate (4-10) White solid (44 mg, yield: 39%). 1H NMR (400 MHz, CD3OD) δ 7.52 (overlapped, 2H), 7.46 – 7.27 (m, 3H), 7.19 (m, 1H), 7.12 (dt, J = 7.7, 1.2 Hz, 1H), 7.04 – 6.95 (m, 1H), 6.87 (d, J = 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, J = 11.1, 7.5 Hz, 1H), 4.04 (dt, J = 11.1, 7.5 Hz, 1H), 3.25(s, 3H), 2.26-2.10 (m, 2H), 1.43 (brs, 3H), 1.07 (brs, 6H). 13C NMR (100 MHz, CD3OD) δ 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[M+H]+ 564.2163, found 564.2153.

4.2.4.11. tert-Butyl-2'-(4'-(methoxycarbonyl)-3'-(methylsulfonamido)-[1,1'-biphenyl]3-yl)-2-oxospiro[indoline-3,3'-pyrrolidine]-1'-carboxylate (4-11) White solid (67 mg, yield: 67%). 1H NMR (400 MHz, CD3OD) δ 10.43 (s, 1H), 8.60 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.04 (t, J = 7.7 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.64 (s, 1H), 6.44 (s, 1H), 6.15 (s, 1H), 5.17 (brs, 1H), 4.23 – 4.07 (m, 1H), 3.92 (d, J = 8.1 Hz, 1H), 3.87 (s, 3H), 2.23 – 2.04 (m, 2H), 1.42 (brs, 3H), 1.10 (brs, 6H). 13

C NMR (100 MHz, CD3OD) δ 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. for C31H33N3O7S[M+Na]+ 614.1931, found 614.1912.

4.2.4.12.

tert-Butyl-2'-(3'-methyl-5'-(methylsulfonamido)-[1,1'-biphenyl]-3-yl)-2-

oxospiro[indoline-3,3'- pyrrolidine]-1'-carboxylate (4-12) White solid (50 mg, yield: 50%). 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 7.65 (s, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.22 – 7.11 (m, 1H), 6.97 (m, 3H), 6.74 (d, J = 7.8 Hz, 1H), 6.62 (s, 1H), 6.29 (brs, 1H), 6.00 (brs, 1H), 5.11 (s, 1H), 4.18 – 4.07 (m, 1H), 3.94 (brs, 1H), 2.97 (s, 3H), 2.28 (s, 3H), 2.23 – 2.13 (m, 1H), 2.05 (brs, 1H), 1.42 (s, 3H), 1.14 (brs, 6H). 13C NMR (100 MHz, CDCl3) δ 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 [M+Na]+ 570.2033, found 570.2027.

4.2.4.13.

tert-Butyl-2'-(4'-(hydroxymethyl)-6-methyl-3'-(methylsulfonyl)-[1,1'-

biphenyl]- 3-yl)-2-oxospiro [indoline-3,3'-pyrrolidine]-1'-carboxylate (4-17) White solid (50 mg, yield: 85%). 1H NMR (400 MHz, CD3OD) δ 7.65 (d, J = 8.3 Hz, 2H), 7.37 (s, 1H), 7.13 (s, 1H), 7.03 (t, J = 7.7 Hz, 1H), 6.91 (brs, 1H), 6.72 (d, J = 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 = 10.6, 6.4 Hz, 1H), 3.85 (dt, J = 10.6, 6.4Hz, 1H), 3.12 (s, 3H), 2.20 (brs, 1H), 2.09 (s, 3H), 1.19 – 1.13 (m, 9H).

13

C NMR (100 MHz, CD3OD) δ

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[M+Na]+ 585.2030, found 585.2016.

4.2.5. Compounds 4-15 and 4-16 Compounds 4-15 and 4-16 were synthesized by the following general method. HCl (0.4 mL, 4.1 equiv., 4M in 2,4-dioxane) was added to a solution of 4-7 or 4-8 (0.37 mmol, 1.0 equiv.) in 5 mL DCM, and stirred at RT for 1 h. The mixture was concentrated in vacuo and the residue was dissolved in DCM (5 mL) and Et3N (0.2 mL, 1.48 mmol, 4 equiv.) was added and the mixture was stirred for 5 min in an ice bath. Then anhydride (0.37 mmol, 1 equiv.) was added to the mixture, which was stirred for 5 h. The mixture was diluted with DCM, washed with aq. NaHCO3 and brine, dried over anhydrous 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 - 60% EtOAc) to afford 4-15 and 4-16.

4.2.5.1.

1'-(3,3-Dimethyl-butanoyl)-2'-(4'-(hydroxymethyl)-3'-(methylsulfonyl)-[1,1'-

biphenyl]-3-yl)spiro[indoline-3,3'-pyrrolidin]- 2-one (4-15) White solid (55 mg, yield: 27%). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.92 (s, 1H), 7.49 (s, 2H), 7.32 (brs, 1H), 7.25 (s, 1H), 7.02 (t, J = 7.8 Hz, 1H), 6.94 (s, 1H), 6.75 (d, J = 7.8 Hz, 1H), 6.57 (t, J = 7.8 Hz, 1H), 5.83 (s, 1H), 5.44 (s, 1H), 4.88 (d, J = 6.6 Hz, 2H), 4.47 – 4.19 (m, 1H), 4.19 (brs, 1H), 3.17 – 3.10 (m, 3H), 2.36 (brs, 2H), 1.35 (s, 9H).

13

C NMR (125 MHz, CDCl3) δ 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[M+Na]+ 569.2081, found 569.2074.

4.2.5.2.

2'-(4'-(Hydroxymethyl)-3'-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)-1'-(5-oxo-

pyrrolidine-2-carbonyl)spiro-[indoline-3,3'-pyrrolidin]-2-one (4-16) White solid (10 mg, 5%). 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.64 (s, 1H), 7.58 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.43 – 7.34 (m, 1H), 7.32 (brs, 1H), 7.06 (m, 1H), 6.97 (s, 1H), 6.81 (d, J = 8.1 Hz, 1H), 6.54 (t, J = 7.6 Hz, 1H), 5.28 (m, 1H), 5.23 (s, 1H), 4.63 (s, 2H), 4.28 (m, 1H), 4.17 (m, 1H), 4.10

– 4.03 (m, 1H), 4.01 (s, 1H), 3.05 (s, 3H), 2.61 (p, J = 8.9 Hz, 1H), 2.50 (dd, J = 13.1, 9.2 Hz, 1H), 2.42 – 2.21 (m, 1H), 2.20 – 2.08 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 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. for C31H29N3O7S[M-H]- 586.1653, found 586.1639.

4.2.6. Compounds 4-18 to 4-20 For compounds 4-18 to 4-20, intermediates were prepared by coupling followed by amidation. Then K2CO3 (14 mg, 0.1 mmol, 1 equiv.) and iodomethane (0.02 mL, 0.32 mmol, 3.2 equiv.) were added to a solution of intermediate (0.1 mmol, 1 equiv.) in DMF and stirred for 5 h. The mixture was then concentrated in vacuo. The residue was purified by silica gel flash chromatography (Biotage SP-1, 20 g SiO2 column, gradient elution from 0 - 50% EtOAc) to afford the 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-3,3'- pyrrolidine]-1'-carboxylate (4-18) White solid (40 mg, yield: 68%). 1H NMR (500 MHz, CDCl3) δ 7.64 (d, J = 8.1 Hz, 1H), 7.28 (brs, 2H), 7.18 (overlapped, 3H), 7.11 (d, J = 3.0 Hz, 1H), 6.78 (overlapped, 3H), 6.51 (d, J = 2.9 Hz, 1H), 6.12 (s, 1H), 5.15 (s, 1H), 4.29 – 4.18 (m, 1H), 4.06-3.95 (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) δ 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. for C32H32FN3O3[M+Na]+ 548.2320, found 548.2306.

4.2.6.2.1'-(2,4-difluorobenzoyl)-2'-(5-fluoro-4'-(methoxymethyl)-3'-(methylsulfonyl)-[ 1,1'-biphenyl]-3-yl)-1-methylspiro[indoline-3,3'-pyrrolidin]-2-one (4-19) White solid (42 mg, yield: 65%). 1H NMR (500 MHz, CDCl3) δ 8.05 (s, 1H), 7.64 (d, J = 3.0 Hz, 3H), 7.22 (t, J = 7.8 Hz, 1H), 7.15-7.12 (m, 2H), 7.04 (dt, J = 8.3, 2.4 Hz, 1H), 6.97 (dt, J = 8.3, 2.4 Hz, 1H), 6.85 (d, J = 7.8 Hz, 3H), 6.38 (d, J = 7.8 Hz, 1H), 5.63 (s, 1H), 4.89 (s, 2H), 4.12 (dt, J = 10.2, 7.2 Hz, 1H), 4.04 (dt, J = 10.2, 7.2

Hz, 1H), 3.51 (s, 3H), 3.30 (s, 3H), 3.21 (s, 3H), 2.43 (dt, J = 13.4, 7.2 Hz, 1H), 2.32 (dt, J = 13.4, 7.2 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 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. for C34H29F3N2O5S [M+Na]+ 657.1641, found 657.1631.

4.2.6.3. 2'-(5-Fluoro-4'-(methoxymethyl)-3'-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)-1'(isopropylsulfonyl)-1-methylspiro[indoline-3,3'-pyrrolidin]-2-one (4-20) White solid (30 mg, yield: 50%). 1H NMR (500 MHz, CDCl3) δ 7.91 (s, 1H), 7.53 (s, 2H), 7.11 (t, J = 7.7 Hz, 1H), 7.06 (dt, J = 7.7, 1.9 Hz, 1H), 6.90 (s, 1H), 6.81 (s, 1H), 6.73 (d, J = 7.7 Hz, 1H), 6.68 (t, J = 7.7 Hz, 1H), 6.07 (d, J = 7.7 Hz, 1H), 5.09 (s, 1H), 4.79 (s, 2H), 4.23 (dt, J = 9.6, 6.1 Hz, 1H), 3.96 (dt, J = 9.6, 6.1 Hz, 1H), 3.42 (s, 3H), 3.35-3.31 (m, 1H), 3.18 (s, 3H), 3.12 (s, 3H), 2.35 – 2.27 (m, 2H), 1.41 (d, J = 2.3 Hz, 3H), 1.40 (d, J = 2.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 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. for C30H33FN2O6S2[M+Na]+ 623.1656, found 601.1632.

4.2.7. tert-Butyl-2'-(3-aminophenyl)-2-oxospiro-[indoline-3,3'-pyrrolidine]-1'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,N'-dimethyl-1,2-ethanediamine (10 µL, 0.1 mmol 0.05 equiv.) and NaN3 (325 mg, 5 mmol, 2.5 equiv. in 5 mL water) were added to the mixture under an N2 atmosphere, and stirred at 85 °C overnight. The mixture was diluted with EtOAc followed by addition of saturated NH4Cl to quench the reaction. The organic mixture was washed with saturated NH4Cl and H2O thrice, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in EtOAc (5 mL), and subsequently precipitated by addition of hexane/EtOAc (v/v: 1/1, 5 mL), filtered, and washed with hexane twice to obtain the product as a light yellow solid (600 mg, yield: 79%). 1H

NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 7.04 (dt, J = 7.7, 1.2 Hz, 1H), 6.85 (s, 1H), 6.75 (d, J = 7.7 Hz, 1H), 6.60 (t, J = 7.7 Hz, 1H), 6.40 (d, J = 7.7 Hz, 1H), 6.30 – 5.79 (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), 1.30 – 0.98 (m, 7H). ESI-MS: 380.2 [M+H] +.

4.2.7.1. tert-Butyl-2'-(3-((4-(hydroxymethyl)-3-(methylsulfonyl)phenyl)amino)phenyl)-2-oxospiro[indoline-3,3'-pyrrolidine]-1'-carboxylate (4-13) S-4 (500 mg, 1.32 mmol, 1.0 equiv.) was dissolved 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, 0.05 equiv.) 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. The mixture was cooled in an ice bath, followed by addition of saturated NH4Cl to quench reaction. The mixture was extracted with 100 mL EtOAc thrice, washed with brine, dried over anhydrous 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 30% EtOAc to 50% EtOAc) to afford the title compound, as a white solid (512 mg, yield: 85%). 1H NMR (400 MHz, CD3OD) δ 7.67 (s, 1H), 7.43 (s, 1H), 7.22 (brs, 1H), 7.15 (t, J = 7.7 Hz, 1H), 7.01- 6.90 (overlapped, 3H), 6.75 (t, J = 7.7 Hz, 1H), 6.28 (s, 1H), 6.09 (s, 1H), 4.96 (overlapped, 3H), 4.09 (q, J = 8.8 Hz, 1H), 4.01 – 3.88 (m, 1H), 3.26 (s, 3H), 2.53 – 2.38 (m, 1H), 2.26 (d, J = 13.8 Hz, 1H), 1.52 (brs, 3H), 1.28 (brs, 6H). 13C NMR (100 MHz, CD3OD) δ 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. for C30H33N3O6S [M+Na]+ 586.1982, found 586.1998.

4.2.7.2. tert-Butyl-2'-(3-(4-(hydroxymethyl)-3-(methylsulfonyl)benzamido)phenyl)- 2oxospiro[indoline-3,3'-pyrrolidine]-1'-carboxylate (4-14) S-4 (100 mg, 0.26 mmol, 1.0 equiv.) was dissolved in DMF (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.) and DIPEA (60 µL, 0.32 mmol, 1.2 equiv.) were added to the mixture and stirred overnight. The mixture was diluted with 50 mL EtOAc,

washed with H2O, dried over anhydrous Na2SO4, filtered and concentrated 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) was added to the mixture and stirred for 5 min. Water was added to quench the reaction, and the mixture was extracted with 10 mL EtOAc thrice, washed with brine, dried over anhydrous 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 30 - 50% EtOAc) to afford the title compound, as a white solid (48 mg, yield: 25%). 1H NMR (400 MHz, CD3OD) δ 8.42 (s, 1H), 8.12 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.12 (s, 1H), 6.97 (dt, J = 7.7, 1.2 Hz, 1H), 6.85 (s, 1H), 6.71 (d, J = 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, J = 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) δ 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 [M+H] +.

4.3. In vitro and in vivo experiments section 4.3.1. Cell culture The human glioma cell lines U87EGFRvIII and U251 were provided by Paul S. Mischel (University of California, San Diego, USA). The human glioma cell line A172 was provided by by Musheng Zeng (Sun Yat-Sen University, Guangzhou, China). HEK293T cells and all GBM cells were maintained in DMEM (Gibco) supplemented

with

10%

fetal

bovine

serum

(HyClone)

and

1%

penicillin/streptomycin. The normal human astrocytes HA1800 were maintained in Astrocyte Medium (ScienCell). All cell lines were maintained at 37 °C in 5% CO2.

4.3.2. Transient transfection and dual 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

µg

pGL3/(DR-4)-c-fos-FF-luc, 0.13 µg pCMV/Renilla-luc, 1.3 µg pSG5/hRXRα and 1.3 µg pSG5/hLXRα or pSG5/hLXRβ. 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 [25]. 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).

4.3.3. Quantitative real-time PCR Cells were seeded in 6-well plates in 5% FBS for 24 h, then changed to 1% lipoprotein deficient serum (LPDS) medium and treated with tested compound for 48 h. Human LPDS was purchased from Yiyuan Biotechnologies (Guangzhou, China). Total RNA was extracted by using RNAiso plus (TaKaRa) according to its protocol. 1 µg 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 β-actin, which was used as an endogenous control. The following qRT-PCR primers were used: β-actin Forward: 5’-CTCTTCCAGCCTTCCTTCCT-3’ β-actin Reverse: 5’-TGTTGGCGTACAGGTCTTTG-3’ LXRα Forward: 5’-GTTATAACCGGGAAGACTTTGC-3’ LXRα Reverse: 5’-AAACTCGGCATCATTGAGTTG-3’ LXRβ Forward: 5’-TTTGAGGGTATTTGAGTAGCGG-3’ LXRβ Reverse: 5’-CTCTCGCGGAGTGAACTAC-3’ ABCA1 Forward: 5’-AACAGTTTGTGGCCCTTTTG-3’ ABCA1 Reverse: 5’-AGTTCCAGGCTGGGGTACTT-3’ ABCG1 Forward: 5’-ATTCAGGGACCTTTCCTATTCGG-3’ ABCG1 Reverse: 5’-CTCACCACTATTGAACTTCCCG-3’ IDOL Forward: 5’-CGAGGACTGCCTCAACCA-3’ IDOL Reverse: 5’-TGCAGTCCAAAATAGTCAACTTCT-3’ ApoE Forward: 5’-TGGGTCGCTTTTGGGATTAC-3’ ApoE Reverse: 5’-TTCAACTCCTTCATGGTCTCG-3’ SREBP-1c Forward: 5’-GGAGGGGTAGGGCCAACGGCCT-3’ SREBP-1c Reverse: 5’-CATGTCTTCGAAAGTGCAATCC-3’

4.3.4. Cell proliferation and viability assay Cells 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) (Yeasen Biotechnology, Shanghai, China).

4.3.5. LDL uptake Cells were seeded at 1 × 105 per well in 6-well plates in 5% FBS overnight, then changed to 1% LPDS medium with 2 µg/ml Dil-LDL (Yiyuan Biotechnologies, Guangzhou, China). After treated with tested compounds for 48 h, the cells were washed

twice

with

phosphate-buffered

saline

(PBS)

and

fixed

in

4%

paraformaldehyde for 30 min, then stained by DAPI for imaging. Cell imaging was performed on EVOS FL Auto Cell Imaging System (Invitrogen) and fluorescence intensity was quantified by ImageJ.

4.3.6. Cholesterol efflux Cells were seeded at were seeded in 96-well plates at 4 × 104 per well for 12 h. After being labelled with 0.5 µM 22-NBD-cholesterol in the presence of tested compounds for 24 h, cells were washed twice with PBS and incubated for 4 h in DMEM containing 15 µg/ml ApoA1 (Sino Biological, Beijing, China). Then the cholesterol in the medium and cells was assayed using a microplate reader respectively (Flex Station 3, excitation 485 nm, emission 535 nm) [37].

4.3.7. Cellular cholesterol measurement Cells were placed in 10 cm dishes. After treated with tested compounds for 48 h, 400 µl RIPA lysis buffer per dish was added and cells were lysed with a probe sonicator. Protein concentration was determined by a BCA assay (Pierce). Then, MeOH (250 µl) and CHCl3 (750 µl) were added sequentially, vortexing after each addition. The samples were centrifuged (3000 rpm, 4 °C, 10 min) to obtain separate phases. The CHCl3 (lower) phase was evaporated to dryness and measured using Total-Cholesterol Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

4.3.8. siRNA transfection 50 nM siRNA negative control (scramble), LXRα or LXRβ was transfected into U87EGFRvIII using Lipofectamine 3000 (Invitrogen) in 1% LPDS for 24 h. siRNA negative control (scramble), LXRα and LXRβ were purchased from GenePharma (Suzhou, China). The following siRNAs were used: siRNA negative control (scramble) sense: 5’-UUCUCCGAACGUGUCACGUTT-3’ siRNA negative control (scramble) antisense: 5’-ACGUUGACACGUUCGGAGAATT-3’ siRNA LXRα sense: 5’-CACGGAUGCUAAUGAAACUTT-3’ siRNA LXRα antisense: 5’-AGUUUCAUUAGCAUCCGUGTT-3’ siRNA LXRβ sense: 5’-CCCAGAUCCCGAAGAGGAATT-3’ siRNA LXRβ antisense: 5’- UUCCUCUUCGGGAUCUGGGTT-3’

4.3.9. Cloning, expression and purification of LXRβ For fluorescence polarization assays and protein crystallization, human LXRβ-LBD was prepared as reported with minor modifications [38]. Briefly, LXRβ-LBD (residues 215-461) was modified at the C-terminus by adding a peptide (687HHKILHRLLQDSSS699) from co-activator SRC2 (SRC2-2). To reduce the surface entropy and the potential protein aggregation, four mutations (Q259A, R261G, D262S, R264S) were performed [38]. The modified LXRβ-LBD DNA coding sequence was inserted into pET28a (+) plasmid (Novagen). LXRβ-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen). The bacteria were grown in Luria–Bertani broth at 37 °C till absorption at 600 nm reached around 0.6. Then 0.25mM of isopropyl β-D-1-thiogalactopyanoside (IPTG) was added, and the bacteria were further cultured at 18 °C overnight before harvest by centrifugation 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 centrifuged at 18000 rpm for 30 min, and then the supernatant was loaded onto a pre-equilibrated Ni-NTA column (5 mL resin). The impurity was washed with 50 mL of lysis buffer, followed by elution with imidazole step procedure. Fractions

were analyzed by SDS-PAGE and fractions containing LXRβ-LBD were concentrated and exchanged to gel filtration buffer (20 mM Tris pH8.5, 200 mM NaCl, 5% glycerol and 5 mM β-mercaptoethanol). The LXRβ-LBD protein was further purified with the HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare). Peak fractions were analyzed by SDS-PAGE, pooled, concentrated to 20 mg/ml and stored at -80°C in the storage buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 5 mM β-mercaptoethanol).

4.3.10. FP binding assay Ki was determined by a FP-based competition assay developed in our lab. FP experiments were conducted with a Victor X5 microplate reader (Perkin-Elmer) using black NBS polystyrene 384-well microplates (Corning). Final concentrations of 400 nM LXRβ and 10 nM hyodeoxycholic acid-based fluorescent tracer were used in the competition assays. Compounds to be determined were diluted using the FP buffer (50 mM Tris, pH 8.0, 400 mM NaCl and 5 mM 2-mercaptoethanol). And assays were performed in triplicate. The final concentration of DMSO in the reactions was less than 2% (v/v). The microplate was shaken gently (100 rpm) for 10 min at 25°C using an orbital shaker and centrifuged at 1500 rpm for 1 min. After equilibration at room temperature for 30 min, the FP values were recorded using excitation and emission filters of 485 nm and 535 nm, respectively. Graphpad Prism 6.0 was used to calculate the Ki by fitting the curve of the FP value (FPread) versus the concentration of LXR modulators as reported [39].

4.3.11. Crystallography 4ss or 4-7rr (100 mM in DMSO) was diluted with protein storage buffer (20 mM Tris pH 8.0, 150 mM NaCl, and 5 mM β-mercaptoethanol) and mixed with the LBD domain of human LXRβ (10 mg/mL) at a final fragment concentration of 2 mM and a final DMSO concentration of 2% (v/v). The protein and fragment were incubated at 4°C overnight, and the precipitant was removed by centrifuging at 11000 g for 10 minutes at 4°C. Crystallization was performed using sitting vapor-diffusion method by mixing 1 µl of LXRβ-LBD/fragment complex with 1µL of reservoir solution (0.1 M Tris pH 8.5, 22% (w/v) PEG3350) and then equilibrating against 100 µL of reservoir solution. Crystallization plates were placed at 8°C for 2-5 days before flat rod crystals would appear.

The diffraction data were collected at 100 K at the beamline BL17U1 of Shanghai Synchrotron Radiation Facility (SSRF) [40] and processed using HKL-3000 [41]. The structure was solved by the molecular replacement method with Phaser [42], and the reported structure of LXRβ-LBD (PDB code: 5HJP) was used as the searching model [43]. Then iterative refinements of the structure models were carried out using Coot and Refmac5 [44]. The atomic coordinates and structure factors (accession code 6K9G and 6K9H) have been deposited in the Protein Data Bank. The LXRβ 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 in Table S2.

4.3.12. Pharmacokinetics Analysis of 4-13 Compound 4-13 was suspended in normal saline for intragastric administration. The SD rats (male, n = 6) were treated by intravenous administration at 10 mg/kg dose, and the blood samples were taken at various time points during a 24 h period. The concentration of compounds in the blood was analysed by LC-MS (Thermo chromatographic system and mass spectrometer, TSQ quantum access max, Finnigan, USA).

4.3.13. Xenograft model 5 × 105 U87EGFRvIII cells were implanted into 4-week-old male BALB/c nu/nu mice for subcutaneous (s.c.) xenograft studies. After tumor size reached 40 mm3, mice were randomly divided into 4 groups (n=6), including the control group and treatment group treated with GW3965 (40 mg/kg/day, i.g.), LXR-623 (40 mg/kg/day, i.g.) or 4-13 (50 mg/kg/day, i.p.) for 15 continuous days. Tumor growth was monitored with calipers on the days indicted throughout of course of the experiment.4,17 All experiments were conducted after approval by the Laboratory Animal Center at Sun Yet-Sun University.

Supporting information Details of Hits Discovery Section, Fig. S1-S6 and Tables S1-S4.

Author information Corresponding author [email protected] [email protected] [email protected] Author contributions ‡

These authors contributed equally.

Notes The authors declare no competing financial interests.

Acknowledgment This work has been funded in part of the National Key R&D Program of China (2017YFB02034043),

Science

and

Technology

Program

of

Guanghzou

(201604020109), National Natural Science Foundation of China (81773636), and Guangdong Provincial Key Lab. of Construction Foundation (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.

Abbreviations SBDD, Structure-based drug design; TFA, trifluoroacetic acid; DCM, dichloromethane;

THF,

bis(pinacolato)diboron;

tetrahydrofuran; DPPF,

Boc

t-butyloxy

carbonyl;

(Bpin)2,

1,1'-ferrocenebis(diphenylphosphine);

DBA,

dibenzylideneacetone; X-Phos, 2-(dicyclohexylphosphino)-2',4',6'-tri-i-propyl-1,1'biphenyl;

HATU,

dimethylazanium;

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

N,N-diisopropylethylamine;

DMF,

N,N-Dimethylformamide; ABCA1, ATP-binding cassette transporter A1; IDOL, inducible degrader of LDLR; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; ABCG1, ATP-binding cassette transporter G1; APOE, apolipoprotein E; SREBP-1c, Sterol-regulatory element binding protein 1c; FP,

fluorescence polarization; AUC: area under the curve; MRT: mean residence time.

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Table of contents Discovery of New LXRβ Agonists as Glioblastoma Inhibitors

Highlight: LXRβ selective agonists with a common novel scaffold are identified with in silico and in vitro experiments. The leads were optimized and resulted in nanomolar EC50 (16 − 92 nM) agents with structure-based drug design approaches. The potent compound significantly inhibits glioblastoma cells through a cholesterol-regulation pathway.

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: