Discovery of a Potent Grp94 Selective Inhibitor with Anti-inflammatory

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Discovery of a Potent Grp94 Selective Inhibitor with Antiinflammatory Efficacy in a Mouse Model of Ulcerative Colitis Fen Jiang, An-ping Guo, Jia-chen Xu, Qi-Dong You, and Xiao-Li Xu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00800 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Journal of Medicinal Chemistry

Discovery of a Potent Grp94 Selective Inhibitor with Anti-inflammatory Efficacy in a Mouse Model of Ulcerative Colitis Fen Jianga,b, An-ping Guoa,b, Jia-chen Xua,b, Qi-Dong Youa,b* and Xiao-Li Xua,b* a

State Key Laboratory of Natural Medicines, and Jiang Su Key Laboratory of Drug Design and

Optimization, China Pharmaceutical University, Nanjing 210009, China b

Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing

210009, China

ABSTRACT As the endoplasmic reticulum paralogue of Hsp90, Grp94 chaperones a small set of client proteins associated with some diseases, including cancer, primary open-angle glaucoma and inflammatory disorders. Grp94-selective inhibition has been a potential therapeutic strategy for these diseases. In this study, inspired by the conclusion that ligand-induced “Phe199 shift” effect is the structural basis of Grp94-selective inhibition, a series of novel Grp94 selective inhibitors incorporating “benzamide” moiety were developed. Among which, Compound 54 manifested the most potent Grp94 inhibitory activity with an IC50 value of 2 nM and over 1000 folds selectivity to Grp94 against Hsp90α. In a DSS-induced mouse model of ulcerative colitis (UC), compound 54 exhibited significant anti-inflammatory efficacy. This work provides a potent Grp94 selective inhibitor as probe compound for the biological study of Grp94, and represents the first study which confirms the potential therapeutic efficacy of Grp94-selective inhibitors against UC.

INTRODUCTION

ACS Paragon Plus Environment

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As an important family of molecular chaperones, heat shock proteins 90 (Hsp90s) are crucial for the correct folding, stability, and function of a wide range of client proteins.1, 2 Hsp90s in higher eukaryotes include four isoforms: Hsp90α and Hsp90β in cytoplasm, glucose-regulated protein 94 (Grp94) in endoplasmic reticulum (ER) and tumor necrosis factor receptor-associated protein-1 (Trap1) in mitochondria, which have specific biological functions and chaperone different client proteins.3 More than 500 client proteins of Hsp90s have been discovered, and many of them are involved in the initiation and progression of certain diseases.3-5 Inhibiting the chaperone function of Hsp90s can induce the degradation of disease-related client proteins and then block disease-dependent signaling pathways.6 Thus, Hsp90 has emerged as an attractive target.7-9 To date, many Hsp90 inhibitors have entered clinical trials for the treatment of cancer.10 However, these inhibitors suffer from prevalent adverse effects (ocular toxicity, gastrointestinal disorders, etc.), which have seriously limited their clinical development.11,

12

The adverse effects are partially due to the pan-Hsp90 inhibitory activity. The

indiscriminate inhibition effects of the four isoforms destabilize a large number of client proteins and may lead to the various clinical adverse effects.3 Compared with the pan-Hsp90 inhibitors, isoform-selective inhibitors only affect the isoform-specific clients, endowing them with more favorable safety profiles and more clinical potential.3, 13-17 Recently, the development of Hsp90 isoform-selective inhibitors has attracted more attention. As the ER paralogue of Hsp90, Grp94 chaperones a small set of client proteins, most of which are cell surface or secretory proteins, including integrins, toll-like receptors (TLRs), insulin-like growth factors (IGFs), Her2, and Wnt coreceptor LRP6.18 The promoting effects of Grp94 and its client proteins on some types of cancer and primary open-angle glaucoma (POAG) have been revealed.19-25 As


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potential therapeutic agents for these diseases, Grp94-selective inhibitors have been pursued by medicinal chemists in recent years. Despite the high sequence and structural homology among the four Hsp90 isoforms, the 5-amino acid (QEDGQ) insertion of Grp94 leads to the presence of Grp94-specific hydrophobic pockets Site 2 and Site 3, which can be utilized in the design of Grp94-selective inhibitors.3 To date, three classes of Grp94-selective inhibitors have been developed (Figure 1). The purine (PU)-class compound NECA (1) was the first identified Grp94-selective inhibitor, which bound to Grp94 with a Kd value of 0.2 μM without exhibiting affinity for cytoplasmic Hsp90α/β.26 The Grp94 selectivity of compound 1 could be attributed to the occupation of the Grp94-specific Site 3 pocket by the N-ethyl moiety.27 Because of the off-target adenosine receptor-activating effect, further study of compound 1 was limited. Compound library screening by the group of Gabriela Chiosis identified another PU-based compound, PU-H54 (2), featuring moderate Grp94-selective inhibitory activity over Hsp90α. Structural analysis revealed that Grp94 could adopt a ligand-induced conformation rearrangement to expose an isoform-unique Site 2 hydrophobic pocket. The favored occupation of this newly exposed Site 2 pocket resulted in the Grp94 selectivity of compound 2.28 Further structural modification led to PU-WS13 (3), which exhibited improved Grp94 inhibitory activity (FP IC50: 0.22 μM) and selectivity (over 120-fold).29 The excellent works by Gabriela Chiosis’s group suggest that the ligand-induced Site 2 pocket can be utilized in the design of Grp94-selective inhibitors. Based on the cis-amide binding pose of radamide (RDA, 4), Brian S. J. Blagg’s group developed resorcinol-based Grp94-selective inhibitors. Replacing the amide linkage of compound 4 with the cis-amide bioisostere imidazole led to BnIm (5) possessing 12-fold selectivity for Grp94 over Hsp90α (FP IC50: 1.1 μM vs 13.1 μM).30,

31

The hydrophobic benzyl moiety of compound 5 was predicted to be inserted into the



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same pocket (Site 3) occupied by the N-ethyl moiety of compound 1. Structural modification focused on the benzyl moiety of compound 5 resulted the 2-ethoxy substituted KUNG29 (6), which exhibited significantly improved Grp94 inhibition activity (FP IC50: 0.2 μM) and selectivity (41-fold).32 The structure of 6 soaked into apo-Grp94 crystal was recently determined and interpreted as placing the 2-ethoxybenzyl moiety into the Site 3 pocket.33 Modification of the imidazole group into a phenyl ring and further SAR explorations gave compound 7, which exhibited 73-fold selectivity for Grp94.34 Recently, the work of Daniel T. Gewirth’s group indicated that compound 5 was actually not Grp94 selective (ITC Kd: Grp94 vs Hsp90α, 1.38 μM vs 0.62 μM). Additionally, the solved cocrystal structure of 5:Grp94 complex revealed a quite different binding mode from previous interpretations. When 5 was bound to Grp94, the resorcinol moiety was flipped by 180° and adopted a “chloro-out” conformation. The methyl ester group moved the Phe199 residue away and sat in the mouth of the newly exposed Site 2 pocket. This Phe199 shift effect was demonstrated to result the Grp94-selective inhibitory activity. Replacing the 3-chloro substituent with another methyl ester group afforded the bis-methyl ester-containing compound 8, which exhibited excellent Grp94 selectivity with a 4.2 μM Grp94 affinity (ITC Kd) and no detectable Hsp90α binding.35 Compound 8 can serve as a lead compound for further optimization to improve the Grp94 inhibitory activity. KUNG94 (9) is another resorcinol-based Grp94-selective inhibitor. Despite its potent Grp94 inhibition (FP IC50: 8 nM), further application of 9 is limited because of the remaining potent Hsp90α affinity (FP IC50: 77 nM).36 The benzamide-containing Grp94-selective inhibitor ACO1 (10) was developed by Brian S. J. Blagg’s group using structure-based design strategies. The hydroxyl-substituted benzyl moiety was introduced to occupy the Grp94-specific Site 2 pocket. Compound 10 demonstrated an IC50 of 0.44 μM for Grp94 and greater than 200-fold


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selectivity over Hsp90α.37 The potential efficacy of these Grp94-selective inhibitors against cancer and POAG has been confirmed.

Figure 1. Structures and selectivity profiles of the reported Grp94-selective inhibitors.

In addition to cancer and POAG, Grp94 is also extensively involved in intestinal inflammation.38, 39 Ulcerative colitis (UC) is a chronic intestinal inflammatory disease that may result from sustained



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stimulation of the mucosal immune system by endogenous luminal microbes in susceptible hosts.40 Grp94 has been reported to play multifaceted roles in the development of UC. Zihai Li’s group revealed that Grp94 was essential for maintaining gut homeostasis through direct regulation of the canonical Wnt signaling pathway. Conditional Grp94 knockout (KO) mice and intestinal tissue-specific Grp94 KO mice suffered from serious intestinal lesions.41 Another recent study by the same group demonstrated that Grp94 was essential for CD11c+ cells to induce regulatory T cells and maintain gut homeostasis. CD11c+ cell–specific Grp94-deficient mice developed spontaneous colitis and were highly susceptible to dextran sulfate sodium (DSS) induced colitis.42 The above results suggested that Grp94 has beneficial roles in maintaining gut homeostasis. However, in another study, macrophage-specific Grp94 KO mice were more resistant to DSS-induced colitis, indicted that Grp94 has harmful roles in exacerbating intestinal inﬂammation.43 Additionally, as the client proteins of Grp94, pattern recognition receptor TLRs expressed in epithelial cells and immune cells are crucial for the recognition of invading bacteria and activation of intestinal immune responses. Considering that Grp94 and TLRs are upregulated in UC patients, Grp94 may have some promoting effects on the inflammatory conditions of UC.44-46 Inhibiting the chaperone function of Grp94 can reduce the TLRs expression in the intestinal mucosa, which may block the bacteria-stimulated immune responses and then ameliorate the inflammatory symptoms of UC. In general, the comprehensive biological roles of Grp94 in the pathogenesis of UC are still unclear, and the potential therapeutic efficacy of Grp94 selective inhibitors against UC has not been explored. The currently developed Grp94-selective inhibitors can serve as useful chemical tools for Grp94 study. However, their activity or selectivity is still not high enough. We believe that more potent and selective inhibitors would be more useful. Here, we report the discovery of a potent and selective Grp94


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inhibitor and its anti-inflammatory efficacy in a DSS-induced mouse model of UC.

CHEMISTRY Compounds 42−70 were synthesized according to Scheme 1. Briefly, commercial reagent 12 was coupled with various aryl iodides or aryl bromides in dioxane using Pd(PPh3)4 as the catalyst and Cs2CO3 as the base to generate intermediates 13−41. Then, the intermediates were reacted with trans-4-aminocyclohexanol in the presence of DIPEA in DMSO at 120 ºC for 24 h. Subsequently, the cyano group was hydrolyzed into a carbamoyl moiety in a mixture of ethanol, 1 M NaOH and 30% H2O2 to afford the desired compounds. Scheme 1. Synthetic Route for Compounds 42−70a F

OH

NC a O

B

12 a

F NC

O HN

b,c H2N

O Ar

Ar

13-41

42-70

Reagents and conditions: (a) aryl iodide or aryl bromide, Pd(PPh3)4, Cs2CO3, dioxane, 90 ºC, overnight; (b)

trans-4-aminocyclohexanol, DIPEA, DMSO, 120 ºC, 24 h; (c) ethanol, 1 M NaOH, 30% H2O2, 30 ºC, overnight.

Compounds 71−86 were synthesized according to Scheme 2. Intermediate 25 was reacted with different amines in the presence of DIPEA in DMSO at 120 ºC for 24 h, and then the cyano group hydrolyzed into a carbamoyl moiety to afford compounds 71−78. Intermediate 25 was reacted with 4-amino-1-Boc-piperidine, followed by hydrolysis of the cyano group and removal of the Boc protecting



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group to give compound 79. For compounds 80−86 featuring amino acid side chains, 79 was condensed with different Boc-protected amino acids using Bop as a coupling agent, and then the Boc protecting group was removed to yield the desired compounds. Scheme 2. Synthetic Route for Compounds 71−86a O

R

H2N

71-78 a,c O NH F

O

NC

HN

O

H2N

R1

HN

H2N e,f

b,c,d

25 a

N

79

80-86

Reagents and conditions: (a) different amines, DIPEA, 120 ºC, 24 h; (b) 4-amino-1-Boc-piperidine, DIPEA, 120 ºC,

24 h; (c) ethanol, 1 M NaOH, 30% H2O2, 30 ºC, overnight; (d) TFA, DCM, rt, 48 h; (e) different Boc-protected amino acids, BOP, DIPEA, DMF, rt, 5 h; (f) TFA, DCM, rt, 36 h.

RESULTS AND DISCUSSION Structure-based design of lead compound 42 To discover novel Grp94-selective inhibitors, we should determine how a compound can discriminate between different Hsp90 isoforms to achieve Grp94-selective inhibition. The research of Gabriela Chiosis’s group provides us some structural insights.29 When bound to Hsp90α (Figure 2A, referred to


Page 9 of 83


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as “Binding mode A”, PDB: 3O0I), compound 2 adopts a “Forward bent” conformation, the 8-aryl group inserts into the Site 1 hydrophobic pocket, and the Phe138 residue adopts a “plane” conformation. When bound to Grp94 (Figure 2B, referred to as “Binding mode B”, PDB: 3O2F), compound 2 adopts a “Backwards bent” conformation, the 8-aryl group inserts into the newly exposed Site 2 hydrophobic pocket, and the Phe199 residue (equivalent to Phe138 in Hsp90α) adopts an “oblique” conformation. In contrast, in the Apo-Grp94 ATP-binding pocket (Figure 2C, PDB: 1YT2), Phe199 adopts the same “plane” conformation as the Phe138 in the 2:Hsp90α complex. Superposition of the crystal structures clearly reveals that Phe199 in the 2:Grp94 complex shifts to some degree compared with Phe199 in Apo-Grp94 (Figure 2D). This ligand-induced “Phe199 shift” effect leads to the conformational change in Grp94 and exposure of the Grp94-specific Site 2 hydrophobic pocket. Based on these analyses, we conclude that the ligand-induced “Phe199 shift” effect is the structural basis of Grp94-selective inhibition. This conclusion has recently been confirmed by Daniel T. Gewirth’s group via the cocrystallization method. 35 Compound 2 and further optimized compound 3 selectively bind to Grp94 by means of conformational changes in both the compounds and protein. Therefore, the Grp94 selectivity profiles are dependent on the conformation that the compounds prefer to adopt when bind to Grp94. We hypothesize that compounds incorporating rigid moieties to induce the “Phe199 shift” should possess more favorable Grp94 selectivity.


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Figure 2. Structural basis of the Grp94-selective inhibitory activity of PU-class compounds. (A) Compound 2 adopts “Binding mode A” to bind with Hsp90α (PDB: 3O0I). Phe138 is shown as a pink stick. (B) Compound 2 adopts “Binding mode B” to bind with Grp94 (PDB: 3O2F). Phe199 is shown as a gray stick. (C) ATP binding pocket of Apo-Grp94N (PDB: 1YT2). Phe199 is shown in blue stick. (D) Superimposition of the 2:Grp94 complex and Apo-Grp94N. The “Phe199 shift” effect is indicated by a small magenta arrow, and the conformational change in Grp94 (H5 helix rearrangement) is indicated by a large magenta arrow.

By comparing the three kinds of fragments that interact with the Asp149 and Thr245 residues in the Grp94 ATP-binding pocket, we find that the benzamide fragment is suitable as the starting point for the design of Grp94-selective inhibitors. The benzene ring provides an appropriate position for the introduction of rigid moieties. SNX0723 (11) is a benzamide Hsp90 inhibitor that binds with Grp94 via


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“Binding mode A” (PDB: 4NH9), in which the Phe199 residue adopts a “plane” conformation (Figure 3A). Superposition of the 11:Grp94 complex and 2:Grp94 complex clearly reveals that the meta position to the carbamoyl group (3-position, numbered according to compound 11), which points toward Phe199, is ideal for the introduction of rigid moieties to induce the “Phe199 shift” (Figure 3B). Considering the strictly hydrophobic property of the induced Site 2 pocket, a rigid and hydrophobic phenyl ring B was introduced. We expected that phenyl ring B could induce the “Phe199 shift” and occupy the newly exposed Site 2 hydrophobic pocket. These efforts led to the design of compound 42, the Grp94 and Hsp90α inhibitory activity of which was determined. Encouragingly, compound 42 had moderate Grp94 inhibitory activity with an IC50 value of 2.77 μM without exhibiting obvious Hsp90α inhibition at 100 μM, indicating a favorable Grp94 selectivity profile.


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Figure 3. Design strategies for Grp94-selective inhibitors. (A) Benzamide Hsp90 inhibitor 11 adopts “Binding mode A” to bind with Grp94 (PDB: 4NH9). Compound 11 is shown as a pink stick. (B) Superposition of the 11:Grp94 complex and 2:Grp94 complex. Compound 2 is shown as a yellow stick, the meta position to the carbamoyl group in 11 is indicated by a red arrow, and the “Phe199 shift” is indicated by a magenta arrow. (C) Predicted binding mode of compound 42. Compound 42 and the residues in the Grp94 active site are shown as green and gray sticks, respectively. Conserved water molecules are shown as red spheres, and H-bonds are indicated by magenta dashed lines. (D) Superimposition of the 42:Grp94 complex and 2:Grp94 complex.

Compound 42 docked well into the Grp94 ATP-binding pocket (Figure 3C). The carbamoyl group formed tight H-bonds with Asp149, Thr245 and two conserved water molecules. These key H-bond interactions

anchored

42

into

the

ATP-binding

pocket.

The

hydroxyl

group

on

the

trans-aminocyclohexanol moiety formed another H-bond with the Lys114 residue. The newly introduced phenyl ring B inserted into the Site 2 pocket and formed hydrophobic interactions with the surrounding residues. The superposition of compound 42 and compound 2 in the Grp94 ATP-binding pocket showed that phenyl ring B of compound 42 was well overlapped with the 8-aryl group of compound 2. Modification of the phenyl B Compound 42 is a promising lead compound for further optimization, as it possesses favorable Grp94-selective inhibitory activity and ligand efficiency (0.33). Docking analysis reveals that phenyl ring B in 42 does not fully occupy the deep Site 2 pocket and that hydrophobic moieties can be introduced to fit the pocket more snugly to improve the Grp94 affinity. In addition, substituents on phenyl ring B may modulate the dihedral angle between the two benzene rings, and then generate some


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influence on the Grp94-selective inhibitory activity. Considering the above two factors, we conducted structure modification of 42 focused on the phenyl B. Various single hydrophobic substituents (methyl, chlorine, methoxy, ethyl) were introduced at the 2’-, 3’-, 4’-pisition of phenyl B, leading to compounds 43−53. For the 2’- and 3’- position, the methyl (43, 44), chlorine (46, 47) and ethyl (52) substituted compounds exhibited retained or moderately improved Grp94 inhibitory activity. In contrast, methoxy-substituted compounds 49 and 50 exhibited obviously decreased Grp94 inhibition, possibly due to the presence of the hydrophilic oxygen atoms, which were detrimental to the hydrophobic interactions with the Site 2 pocket. Compared with their 2’or 3’- substituted analogues, the 4’-substituted compounds (45, 48, 51, 53) displayed significantly improved Grp94 inhibitory activity, especially ethyl substituted 53, which had an IC50 value of 7 nM. For a given substituent, the Grp94-selective inhibition displayed a trend of 4’-subtituted > 3’-subtituted > 2’-subtituted. The above results indicated that hydrophobic substituents at the 4’- position were favorable for Grp94 inhibition. This finding was coincident with the docking analysis, which indicated that the 4’-position pointed toward the hydrophobic bottom of the Site 2 pocket and that hydrophobic substituents could form additional hydrophobic interactions with the surrounding residues. Inspired by the above SARs, we introduced several other larger hydrophobic substituents (isopropyl, propyl, tert-butyl, ethoxy) to afford compounds 54−57. The 4’-isopropyl-substituted compound (54) exhibited further improved Grp94 inhibition compared with ethyl-substituted 53, providing an IC50 value of 2 nM. Compounds incorporating propyl (55), tert-butyl (56), and ethoxy (57) groups, however, were less potent. These results revealed that among the mono-substituted compounds, the 4’-isopropyl group was the most favorable substituent.



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We next expanded the scope of our investigations to explore the SARs of disubstituted and trisubstituted compounds. The 2’,3’-, 2’,4’-, 2’,6’-, and 3’,4’-dimethyl-substituted compounds (58−61) and 2’,4’,6’- trimethyl-substituted 63 exhibited 2−6-fold improvements in activity compared with lead compound 42. Additionally, the 2’,3’-, 2’,6’-, and 3’,4’-dichloro compounds (64−66) displayed better activity than the corresponding dimethyl-substituted compounds (64 vs 58, 65 vs 60, 66 vs 61), indicating that dichloro substitutions at these positions were more favorable than dimethyl substitutions. However, the 3’,5’- dimethyl (62) and 3’,5’-dichloro (67) compounds exhibited dramatically decreased activity, which was most likely caused by steric interference with the surrounding residues. To further explore the volume and plasticity of the Site 2 pocket, phenyl ring B was replaced by larger moieties. The Grp94 inhibitory activity of 1-naphthyl-substituted compound 68 sharply dropped to zero, while the 2-naphthyl compound 69 exhibited ~5-fold decreased activity. Introducing another aryl (pyrrolyl) at the 4’-position led to compound 70, which exhibited no Grp94 inhibition. These results revealed that Site 2 pocket had limited volume and moiety tolerance. All the above mentioned compounds exhibited no Hsp90α inhibitory activity at 100 μM, indicating excellent Grp94 selectivity of the biphenyl structure. In general, through structure modification focused on phenyl ring B, we obtained compound 54 featuring a 4’-isopropyl group as the most potent Grp94-selective inhibitor. Additionally, the SARs of phenyl ring B matched the hydrophobic property of the Site 2 pocket, which confirmed the rationality of our design strategy. Table 1. The Grp94 inhibitory activity of compounds 42−70


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OH O

HN

H2N Ar

Grp94 FP activity a Structure Compd.

Percent inhibition Ar =

IC50 (μM) (100 μM)

42

93 %

2.77 ± 0.08

43

90 %

1.81 ± 0.13

44

100 %

1.16 ± 0.19

45

100 %

0.34 ± 0.03

100 %

2.72 ± 0.85

100 %

2.03 ± 0.56

100 %

0.17 ± 0.02

89 %

11.47 ± 0.51

100 %

4.75 ± 0.04

Cl

46

47 Cl

48 Cl

49

OCH3

50 OCH3



51

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100 %

0.50 ± 0.07

52

92 %

2.06 ± 0.14

53

100 %

0.007 ± 0.001

54

100 %

0.002 ± 0.001

55

100 %

0.053 ± 0.015

56

100 %

0.79 ± 0.09

100 %

0.49 ± 0.04

58

81 %

0.46 ± 0.23

59

91 %

1.22 ± 0.26

60

100 %

1.42 ± 0.38

61

100 %

0.71 ± 0.11

62

72 %

19.67 ± 1.04

OCH3

57 O


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63

100 %

1.11 ± 0.29

100 %

0.31 ± 0.06

100 %

0.14 ± 0.01

100%

0.60 ± 0.01

66 %

24.66 ± 5.78

68

0

－

69

75 %

12.64 ± 0.88

0

－

AT13387

100 %

0.020 ± 0.001

SNX-2112

100 %

0.097 ± 0.006

Cl

64

Cl

65

Cl

Cl

66

Cl Cl

67 Cl

Cl

70 N

a Values

shown are the mean ± SD (n = 3). All compounds exhibited no Hsp90α inhibition at 100 μM (data not shown).

Pan-Hsp90 inhibitors AT13387 and SNX-2112, which were selected as positive controls, have Hsp90 inhibitory activities (IC50) of 0.022 μM and 0.034 μM, respectively.

Binding mode analysis of compound 54 The binding mode of compound 54 was analyzed by molecular docking methods. As shown in Figure 4, compound 54 fitted well into the Grp94 ATP-binding pocket snugly (The protein structure of Grp94 was



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extracted from PDB: 3O2F). Direct and water-mediated H-bonds were formed between the carbamoyl moiety and Asp149, Thr245. The hydroxyl group on the trans-aminocyclohexanol moiety formed another H-bond with the Lys114 residue. The 4’-isopropyl-substituted phenyl ring B tightly occupied the hydrophobic Site 2 pocket. Compared with lead compound 42, compound 54 inserted the newly introduced 4’-isopropyl group into the bottom of Site 2 pocket and formed additional hydrophobic interactions with the Leu104, Phe199, Ala202, Phe203, Val209 and Leu249 residues. These additional hydrophobic interactions contributed to the potent Grp94 inhibitory activity of compound 54.

Figure 4. Predicted binding mode of compound 54. The Protein structure of Grp94 was extracted from PDB: 3O2F. Active site of Grp94 is surfaced in the hydrophobic state, compound 54 and the surrounded residues are shown as green and gray sticks, respectively. Conserved water molecules are shown as red spheres, and H-bonds are indicated by magenta dashed lines.

SAR study of the 4-position amino side chains (R position)


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We next focused our attention on the 4-position of benzene ring A. Docking analysis (Figure 4) showed that the 4-position directed into a pocket formed by both hydrophobic residues (Ala111, Val152, Met154, Leu191, Phe195) and polar residues (Lys114, Glu158, Asn162), indicating that substituents at this position should have some influence on Grp94 inhibitory activity and selectivity. In the discovery of benzamide Hsp90 inhibitors, amino side chains were demonstrated as favorable substituents for the same position.47 To explore the SARs of the 4-position for Grp94, various amino side chains were introduced to afford compounds 71−86. Compound 71 without a hydroxyl group displayed seriously reduced Grp94 inhibitory activity, suggesting that the H-bond between the hydroxyl group and Lys114 was important. The isobutylamine analogue (72) and cyclopentylamine analogue (73) were even less potent than 71. Moreover, the cyclohexylmethanamine analogue (74) exhibited no Grp94 inhibition. Compared with the above mentioned compounds, H-bond acceptor-bearing analogues 75−78 exhibited improved activity, especially compounds 77 and 78, which featured six-membered ring side chains. The oxygen atom or carbonyl group in the side chains could form H-bonds with the Lys114 or Glu158 residue. Compound 79 without an acetyl group exhibited reduced activity compared with compound 78, confirming the importance of the H-bond acceptor. The above results indicated that H-bond acceptor-bearing six-membered ring side chains were favorable substituents for the 4-position. Various amino acids were then introduced on the piperidine NH of compound 79 while maintaining the carbonyl group as an H-bond acceptor to afford compounds 80−86. Among them, compounds 80, 82 and 86 bearing linear amino acids had Grp94 inhibitory activity in the range from 150 nM to 220 nM, while the α-position substituted analogues (81, 83−85) were less potent. Compounds 71−86 also exhibited no Hsp90α



Page 20 of 83

inhibitory activity at 100 μM. Taken together, the results of these extensive investigations revealed the SARs of the 4-position and demonstrated the importance of H-bond acceptors on the amino side chains. Table 2. The Grp94 inhibitory activity of compounds 71−86 O

R

H2N

Grp94 FP activity a Structure Compd.

Percent inhibition R=

IC50 (μM) (100 μM)

71

N H

72

N H

73

N H

74

N H

75

N H

O

97 %

0.37 ± 0.01

96 %

2.32 ± 0.09

89 %

3.11 ± 0.50

No inhibition

－

96 %

0.93 ± 0.13

95 %

0.13 ± 0.01

100 %

0.073 ± 0.012

100 %

0.055 ± 0.009

100 %

0.47 ± 0.02

100 %

0.21 ± 0.01

O

76

N H

O

77

N H O N

78 N H

NH

79

N H O

80

N

NH2

N H


Page 21 of 83 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O

81

NH2

N

88 %

0.37 ± 0.08

96 %

0.22 ± 0.05

91 %

0.45 ± 0.01

89 %

0.48 ± 0.04

92 %

0.47 ± 0.06

100 %

0.15 ± 0.01

N H O

82

NH2

N N H O

83

NH2

N N H

O NH2

N

84 N H

O N

85

NH2

N H O

86

N

NH2

N H

a Values

shown are the mean ± SD (n = 3). All compounds exhibit no Hsp90α inhibition at 100 μM.

In the above discovery process, compound 54 was established as the most potent Grp94 inhibitor with an IC50 value of 2 nM in an FP assay. Additionally, 54 exhibited no obvious Hsp90α inhibition at 100 μM, indicating significant Grp94 selectivity (the inhibition curves are shown in Figure 5A). The remarkable Grp94 affinity and selectivity of compound 54 was further evaluated by using BLI assay. As shown in Figure 5B/C, compound 54 bound to Grp94 and Hsp90α with KD values of 19.6 nM and 20.6 μM, respectively. These results confirmed the more than 1000 folds selectivity of 54 to Grp94 over Hsp90α. Considering that FP assay is a probe competitive binding method and the IC50 values determined are partially depended on the intrinsic affinity of the fluorescent probe to the two isoforms, it is reasonable that no Hsp90 inhibition was detected for compound 54 in the FP assay.35 In general, compound 54 exhibited excellent Grp94 selectivity both in FP assay and BLI assay, and the selectivity profile is high enough for it as a probe to study the Grp94-specific biological functions. Additionally, in



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NMR-based Carr-Purcell-Meiboom-Gill (CPMG) experiment, the NMR signals of compound 54 were dose-dependently attenuated after Grp94N protein was added, which indicated the interaction between compound 54 and Grp94.


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Figure 5. Selectivity of compound 54 to Grp94 and Hsp90α. (A) The inhibition curves of compound 54 to inhibit Grp94 and Hsp90α. The mP values for negative control and blank control in the determination of Hsp90α are 428 and 318, respectively. The results are shown as mean ± SD, n = 3. (B) BLI dose−response curves reflecting the direct binding of 54 to Grp94. (C) BLI dose−response curves reflecting the direct binding of 54 to Hsp90α. (D) NMR-based CPMG determination of direct binding of 54 to Grp94. NMR spectra of 54 at different concentrations of Grp94N are shown in different colors.

Molecular dynamics (MD) simulation to analyze the structural basis of Grp94 selectivity As mentioned above, Grp94 isoform-selective binding is a dynamic process that involves a “Phe199 shift” and conformational changes in the Grp94 protein. To further understand this dynamic process and confirm the structural basis of the Grp94 isoform selectivity of our compounds, MD simulation was conducted. In ligand-free Apo-Grp94N (Figure 6A), Phe199 adopts a “plane” conformation, while in compound 2-bound Grp94N (Figure 6D), Phe199 adopts an “oblique” conformation. After compound 2 was removed from Grp94N, a 20 ns MD simulation was performed (Figure 6B/C). The results revealed that the Phe199 returned to the “plane” conformation, similar to the Phe199 in Apo-Grp94N, indicating that the “plane” conformation was favored for Phe199 in ligand-free Grp94N. Meanwhile, the 54:Grp94N docking complex was also subjected to a 20 ns MD simulation. In the resultant complex (Figure 6E/F), the Phe199 residue still remained in the “oblique” conformation, which was well overlapped with Phe199 in the 2:Grp94N complex. In general, these MD simulation results helped us understand that the ligand-induced “Phe199 shift” effect was the structural basis of Grp94-selective inhibition.


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Figure 6. MD simulation to analyze the structural basis of Grp94-selective inhibition. (A) ATP binding pocket of Apo-Grp94N (PDB: 1YT2), Phe199 is shown in blue stick. (B) MD simulation results (20 ns) of the compound 2 removed Grp94N (PDB: 3O2F), Phe199 is shown in gray stick. (C) Superimposition of A and B. (D) ATP binding pocket of compound 2 bound Grp94N (PDB: 3O2F), Phe199 and compound 2 are shown in green and yellow sticks, respectively. (E) MD simulation results (20 ns) of the 54:Grp94N docking complex, Phe199 and compound 54 are shown in pink and navy blue sticks, respectively. (F) Superimposition of D and E.

Confirming the Grp94-selective inhibitory activity of compound 54 in cells Grp94-specific clients include a subset of integrin subunits such as integrin α2 and integrin αL. Their maturation and trafficking to the cell surface are dependent on the Grp94 chaperone function but have no association with cytoplasmic Hsp90α. Inhibiting or knocking down Grp94 in cells will decrease cell


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surface secretion of integrins. Moreover, unlike Hsp90α inhibition, Grp94-selective inhibition does not induce heat shock response and has no influence on the Hsp70 expression level. To characterize the Grp94-selective inhibitory activity of compound 54 in cells, Western blot analysis was conducted to evaluate the expression levels of the selected biomarkers in 54-treated panc-1 cells. As shown in Figure 7A, treatment with 54 significantly downregulated the cell surface expression levels of integrin α2 and integrin αL in a dose-dependent manner. Additionally, no modulation of Hsp70 and Akt (Hsp90α specific client) was observed. In contrast, the pan-Hsp90 inhibitor AT13387 obviously induced upregulation of Hsp70 and degradation of Akt. These results confirmed the Grp94-selective inhibitory activity of compound 54 in cells.

Figure 7. Western blot analysis of some biomarkers in 54 treated panc-1 cells. (A) Integrin α2 and integrin αL levels in cell surface protein extracts after treatment with 54 at indicated concentrations for 36 h. (B) Hsp70 and Akt levels in total protein extracts after treatment with 54 at indicated concentrations for 36 h. AT13387 (AT) is used as the reference. β-actin is used as the control for protein loading.

Anti-inflammatory efficacy of compound 54 in a DSS-induced mouse model of UC DSS-induced colitis can simulate the disease characteristics of human UC, including weight loss, diarrhea, rectal bleeding, intestinal mucosal damage and pro-inflammatory cytokine elevation in serum and colonic tissues. This model has been widely used to study the pathogenesis of UC and screen


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potential therapeutic agents. To explore the exact biological roles of Grp94 in UC pathogenesis and the potential anti-inflammatory efficacy of Grp94-selective inhibitors against UC, compound 54 was applied to the DSS-induced mouse model of UC. In this study, C57BL/6 mice were given 3% DSS drinking water, and then typical symptoms of UC (weight loss, diarrhea, and rectal bleeding) were observed, indicating that the model was successfully established. Mice in the two treatment groups were co-administrated 54 at 10 mg/kg or 30 mg/kg dosage. As shown in Figure 8A, compared with those of the “Normal Control” group mice, the body weights of the “Disease Control” group mice were seriously decreased from day 3. Clearly, administration of 54 slowed down the body weight loss. Disease activity index (DAI), another indicator used to evaluate the disease severity of UC mice, is calculated by scoring changes in weight loss, stool consistency, and stool blood.48, 49 As shown in Figure 8B, DAI scores of the “Disease Control” group mice were significantly increased from day 3, while for the 54-treated mice, DAI scores were significantly increased from day 5. At day 7, DAI scores of the 54-treated mice were significantly lower than those of the “Disease Control” group mice, indicating that 54 treatment ameliorated the symptoms of the UC mice. Additionally, at day 7, three mice in “Disease Control” group were dead, one mouse in 10 mg/kg treatment group was dead, and no mice in 30 mg/kg treatment group were dead (data not shown). All the above results confirmed the treatment efficacy of 54 against the DSS-induced UC in mice. Significant colon shortening, which is another obvious symptom, was observed in DSS-induced colitis. However, administration of 54 did not attenuate the colon shortening (Figure 8C). Additionally, histological observation showed that colonic tissue damage in the treated mice was not obviously reduced (Figure 8D). These results suggested that 54 could not protect the colonic tissue from the



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DSS-induced damage. Pro-inflammatory cytokines such as TNF-α and IL-6 are important factors involved in UC pathogenesis and are often elevated in UC patients. To further confirm the anti-inflammatory efficacy of 54 against DSS-induced UC, the levels of the cytokine TNF-α and IL-6 were determined. As shown in Figure 8E, DSS administration led to significant elevation of TNF-α and IL-6 in serum and colonic tissues. As expected, treatment with 54 significantly reduced the cytokine levels in a dose-dependent manner. TLRs are typical Grp94-specific clients that play important roles in the pathogenesis of UC. As the best-investigated pattern recognition receptor, TLRs can recognize the pathogen-associated molecular patterns (PAMPs) of pathogenic microorganisms in the intestine. Upon PAMP stimulation, TLRs will trigger the activation of some inflammatory signaling pathways, such as the NF-κB signaling pathway, and then lead to inflammatory responses. In the anti-inflammatory mechanism study, the TLR2 and TLR9 levels in colonic tissues were analyzed by Western blot assay. As shown in Figure 8F, DSS administration significantly upregulated TLR2 and TLR9 expressions in colonic tissues of the mice. Compared with the “Disease Control” group mice, the 54-treated mice had reduced TLR2 and TLR9 levels in colonic tissues. Moreover, the NF-κB subunit p65 in colonic tissues of the UC mice was also significantly upregulated, indicting the activation of the NF-κB signaling pathway. Interestingly, treatment with 54 significantly reduced the p65 expression in colonic tissues, especially those in the 30 mg/kg dose group. To further confirm the Grp94 selectivity of 54 in vivo, the Hsp70 and Hsp90 client protein Akt levels in colonic tissues were evaluated. The Akt expression level in colonic tissues of the DSS-administrated mice was greatly increased, suggesting that Akt-related signaling pathways were also activated in UC mice, as reported in previous studies.50, 51 Compared with the “Disease Control” group,


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Hsp70 and Akt expression levels in colonic tissues of the 54-treated mice were not affected, indicating no Hsp90α inhibition. The above results demonstrated that compound 54 could inhibit the chaperone function of Grp94 selectively in vivo, then led to the degradation of some TLRs and blockage of the TLR-dependent NF-κB signaling pathway.

Figure 8. Anti-inflammatory efficacy of compound 54 in DSS-induced UC mice (n = 8). (A) Body weight changes of the mice during treatment. (B) DAI score changes during treatment. (C) Colon length of each group at day 7. (D)


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Representative H&E images of the colon samples. Scale bar, 200 μm. (E) TNFα and IL-6 levels in the serum and colonic tissues of each group of mice (n = 5, 5 samples were analyzed). *p < 0.05; **p < 0.01; ***p < 0.001, compared with the “Disease Control” group. (F) Western blot analysis of the biomarkers in colonic tissues. β-actin is used as the control for protein loading.

Safety evaluation of compound 54 in health mice. Safety is concerned in the development of therapeutic agents for chronic inflammation. To evaluate the safety of compound 54, a subacute toxicity assay was carried out. Healthy C57BL/6 mice were administrated compound 54 (ip, qd) at three different dosages (30 mg/kg, 60 mg/kg and 90 mg/kg) for two weeks. During the treatment, no behavioral abnormalities and obvious body weight loos were observed (Figure 9A). Additionally, hematoxylin-eosin (HE) staining of the harvested organs showed no organ damage in the 54 treated mice (Figure 9B). These results confirmed the safety of compound 54 as a potential UC treatment agent.


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Figure 9. Subacute toxicity evaluation of compound 54 in healthy C57BL/6 mice (n = 8). The mice were treated with vehicle or three different dosages of compound 54. (A) Body weight change of each group during the treatment. (B) H&E staining analysis for the different organs of the mice in each group. Scale bar, 50 μm.

CONCLUSION In this paper, we described the structure-based discovery of a potent Grp94 selective inhibitor. Based on the 2:Grp94N cocrystal structure, we conclude that the ligand-induced “Phe199 shift” effect is the


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structural basis of Grp94-selective inhibition. Inspired by this conclusion, we selected the “benzamide” moiety as the active fragment and introduced a rigid “phenyl” ring at the meta position to the carbamoyl moiety to shift the Phe199 residue. Encouragingly, these efforts led to the discovery of lead compound 42, which exhibited moderate Grp94-selective inhibitory activity with an IC50 value of 2.77 μM. A systematic SAR study provided compound 54, which manifested significantly improved Grp94 inhibitory activity with an IC50 value of 2 nM without exhibiting obvious Hsp90α inhibition even at 100 μM. BLI assay revealed a 19.6 nM binding affinity of 54 to Grp94 and over 1000 folds selectivity against Hsp90α. Docking and MD simulation revealed that compound 54 could induce the “Phe199 shift” and suitably occupied the Grp94-specific Site 2 pocket. At cellular level, treatment with compound 54 downregulated the Grp94-specific client proteins and exhibited no influence on the expression of Hsp70 and Akt (client protein of Hsp90). The discovery of compound 54 confirmed that the ligand-induced Site 2 pocket could be utilized for the design of novel Grp94-selective inhibitors. Then, compound 54 was used as a tool compound to explore Grp94 inhibitory effects in a DSS-induced mouse model of UC. The results showed that 54 could ameliorate the inflammatory symptoms of UC mice and reduce the levels of pro-inflammatory cytokines (TNF-α and IL-6) in serum and colonic tissues. Western blot analysis showed that 54 treatment downregulated TLR2 and TLR9 in colonic tissues and inhibited TLR-dependent NF-κB pathway activation. Additionally, no modulation on the expression levels of Hsp70 and Akt was observed. These results indicated that the anti-inflammatory efficacy of compound 54 in UC model was resulted from Grp94-selective inhibition. This work represents the first study which confirms the therapeutic efficacy of Grp94-selective inhibitors against UC. As the most potent Grp94 selective inhibitor, compound 54 can be further used in the exploration of


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Grp94 biological functions.

EXPERIMENTAL SECTION 1. Biology. 1.1. Protein expression and purification. Recombinant canine Grp94NΔ41(residues 69−337; residues 286−328 were replaced by four glycine residues) and human Hsp90αN (residues 1-236) were expressed as GST- and His-tagged fusions in E. coli BL21star (DE3) as previously reported.28,

32, 35

The GST- and His-tagged proteins were purified

using GSTrapTM HP Column (GE Healthcare, Sweden, 17528202) and HisTrapTM HP Column (GE Healthcare, Sweden, 17524802) according to standard protocols, respectively. 1.2. FP competition assay. FP competition assay was used to evaluate the inhibition activities of the compounds to Grp94N and Hsp90αN. The compounds (10 mM in DMSO) were prepared into various concentration solutions by serial diluted in assay buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl2, PH 7.4, 0.01% NP40, 2 mM DTT, 0.1 mg/mL BGG). The fluorescent geldanamycin (GM-FITC, #BML-EI361-0001) and purified Grp94N and Hsp90αN were diluted into the needed concentrations in assay buffer. The experiments were conducted in 384 well black flat-bottomed polystyrene plates (Corning #3575). For each assay, equal volumes of the diluted compounds (20 μL), Grp94N or Hsp90αN (15 nM final) and GM-FITC solution (6 nM final) were added into the plate wells orderly and yielded a final volume of 60 μL. Plates were covered and rocked for 4 h at 4 °C in dark and then the FP values were detected using a SpectraMax



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multimode microplate reader (Molecular Devices) with excitation and emission wavelength at 485 and 535 nm, respectively. For each assay, the FP values of blank controls (GM-FITC only) were recorded as Pmin, the FP values of negative controls (GM-FITC and protein) were recorded as Pmax and the FP values of test wells (compounds, GM-FITC and protein) were recorded as Ptest. The inhibition rate of the compounds at each concentration was calculated using the equation as follows: inhibition rate (%) = [1 − (Ptest − Pmin) / (Pmax − Pmin)] × 100%. IC50 values were calculated using Graphpad Prim 6.0 software. 1.3. BLI assay. Biolayer interferometry (BLI) assay was performed based on the protocols previously described.52 The interaction between the ligand and protein was measured in 96-well black microplate (#655209, Greiner) by biolayer interferometry using an Octet Red 96 instrument (FortéBio, MenloPark, CA, USA). His-tagged Grp94N and Hsp90N protein were biotinylated in 1 x PBS (pH 7.4) with one molar ratio using EZ link sulfo-NHS-LC-biotinylation kit (#21340, Thermo Pierce) as the manufacturer’s instructions. Excess biotin reagent was removed by overnight dialysis in PBS. Super Streptavidin (SSA) biosensors tips (FortéBio, Inc., Menlo Park, CA) were prewetted with BLI kinetics buffer (PBS, 0.05% BSA, 0.01% Tween-20) for 10 mins to establish a baseline before immobilization. Then biotinylated protein was bound by dipping SSA sensors into biotinylated Grp94N (a final concentration of 400 nM) or Hsp90N (a final concentration of 400 nM). Then four concentrations of compound 54 prepared in BLI kinetics buffer was immobilized onto SSA sensors. All of the binding data were collected at 30 °C. The experiments comprised five steps: (1) baseline acquisition (60s), (2) protein loading onto the sensor (1000s), (3) second baseline acquisition (120s), (4) association of compound for the measurement of Kon (300s), and (5) dissociation of compound for the measurement of Koff (300s). Baseline and dissociation


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steps were performed in the buffer only. The association and dissociation plot and kinetic constants (Kon and Koff) were step corrected, reference corrected, fit globally to a 1:1 binding model and automatically obtained with Octet data analysis software (7.1). Equilibrium dissociation constants (KD) were calculated by the ratio of Koff to Kon. 1.4. NMR based CPMG assay. NMR-based CPMG assay was applied to verify the 54:Grp94N interaction. The assay was conducted at 25 °C on a Bruker Avance III-600 MHz (proton frequency) spectrometer equipped with a cryogenically cooled probe (Bruker biospin, Germany). Samples containing 40 μM 54, 40 μM 54 in addition to 0.02 μM, 0.03 μM or 0.04 μM Grp94N in assay buffer (5% DMSO in PBS) were used in NMR data acquisition. 1.5. Induction of UC mice model and 54 Treatment. The studies were conducted according to protocols approved by Institutional Animal Care and Use Committee of China Pharmaceutical University. C57BL/6 mice (Male, 20−22 g) were randomly divided into four groups (n = 8). Mice in “Normal control” group were supplied with distilled water, the other three groups were given 3% DSS (36−50 KD) orally in drinking water for 7 days. For the 54-treated groups, two dosages of compound 54 (10 mg/kg, 30 mg/kg) were co-administrated (ip, qd) with DSS, while mice in the “Normal control” group and “Disease control” group were treated with vehicle. The body weight, stool consistency and rectal bleeding of the mice were recorded every day. Disease activity index (DAI) scores were calculated according to the above three factors to quantify the severity of colitis, and the detailed scoring criteria were illustrated in supporting information (Table S1). After the mice were sacrificed at day 7, serum and colons were collected. The serum was used for the determination of


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cytokines. Section of distal colons were fixed in 4% formaldehyde and embedded in paraffin for sectioning, the sections were subjected to H&E staining analysis. Other parts of the colons were immediately flash-frozen in liquid nitrogen. 1.6. Cytokines determination. Cytokine (TNFα and IL-6) expression levels in serum and colonic tissues were determined using ELISA method. For colonic tissues, homogenates were prepared and the supernatants were used for determination. Mouse TNFα ELISA kit (BOSTER Biological Technology co.ltd, China, EK0527) and IL-6 ELISA kit (BOSTER Biological Technology co.ltd, China, EK0411) were used following the manufacturer’s instructions. 1.7. Western blot. Biomarker expression levels were analyzed by Western blot. For the biomarkers in 54-treated cells, Panc-1 cells seeded in petri dishes were treated with 54 for 36 h at indicated concentrations. Surface protein was extracted using commercially kit (KeyGEN BioTECH, China, KGBSP002) according to the instructions. In general, the harvested cells were lysed in 1 ml cold buffer A and centrifuged to remove the supernatant as the cytoplasmic protein. Then 500 μl cold buffer B was added into the residue, lysed and centrifuged to remove the supernatant as the nuclear protein. After that, 500 μl cold buffer C was added to the obtained precipitates, then lysed and centrifuged to obtain the supernatant as the surface protein, which was used for the analysis of Integrin α2 and integrin αL located on cell surface. Total protein was extracted for the analysis of Hsp70 and Akt levels using the commercially kit (KeyGEN BioTECH, China, KGP250). Cells were lysed in the lysis buffer and centrifugated to obtain the supernatant as the total protein. For biomarkers in colonic tissues, the tissues were homogenized and


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lysed in lysis buffer, and the supernatants were used. Before using, protease and phosphatase inhibitors were added into the above mentioned buffers. The protein concentrations were determined using BCA kit (Thermo, Waltham, MA). Equal amounts of the protein extracts were separated by SDS-PAGE and then transferred onto PVDF membranes (PerkinElmer, Northwalk, CT, USA). After blocking the nonspecific binding sites with 1% BSA for 1 h, membranes were incubated at 37 °C for 1 h and then at 4 °C overnight with the following primary antibodies: Anti-Integrin α2 (Santa Cruz Biotechnology, Inc., USA, sc-74466), Anti-Integrin αL (Santa Cruz Biotechnology, Inc., USA, sc-374172), Anti-Hsp70 (Santa Cruz Biotechnology, Inc., USA, sc-24), Anti-Akt (Cell Signaling Technology, Inc., USA, #2920), Anti-TLR2 (Abcam, UK, ab209217), Anti-TLR9 (Santa Cruz Biotechnology, Inc., USA, sc-52966), Anti-p65 (Cell Signaling Technology, Inc., USA, #8242) and anti-actin (Proteintech Group, Inc., China). Then the membranes were washed and treated with a DyLight 800 labeled secondary antibody at 37 °C for 1 h and scanned using the Odyssey infrared imaging System (LI-COR, Lincoln, Nebraska, USA). 1.8. Subacute toxicity evaluation The studies were conducted according to protocols approved by Institutional Animal Care and Use Committee of China Pharmaceutical University. C57BL/6 mice (Male, 20−22 g) were randomly divided into four groups (n = 8). Mice in the three treatment groups were treated (ip, qd) with 54 in different dosages (30 mg/kg, 60 mg/kg and 90 mg/kg) for two weeks. Mice in the control group were treated (ip, qd) with vehicle. Body weight was measured every other day. The first day was recorded as Day 0. At Day 14, the mice were sacrificed and the heart, lung, spleen, liver and kidney organs were collected. The organs were fixed in 4% formaldehyde and embedded in paraffin for sectioning, the sections were subjected to H&E staining analysis.


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2. Docking. Molecular docking studies were carried out using Discovery Studio (DS) 4.0 software. The 2:Grp94N complex (PDB code: 3O2F) downloaded from the Protein Data Bank was chosen for docking studies. The protein was prepared and the conserved water molecules HOH22, HOH441 were kept for docking. Protein residues around compound 2 (radius 11.0 Å) that completely covered the ATP binding site were defined as the “SBD_Site_Sphere”. CHARMm force field was applied for the compounds. Then docking studies were conducted using “CDOCKER” module and the docking parameters were used as default. After completion of each docking calculation, the docking poses were analyzed. 3. Molecular dynamics (MD) simulation. Molecular dynamics simulations were carried out for Grp94N (PDB code: 3O2F, compound 2 was removed) and 54:Grp94N docking complex to analyze the structural basis of the Grp94 selectivity. All simulations were performed using Amber14 package, and gaff force field and ff14SB force field were used to generate the topology and coordinate files. The protein was solvated in a rectangular parallelepiped water box filled with TIP3P water molecules with a 11 Å water cap. Sodium ions were added to neutralize the system. Prior to MD simulations, two minimization steps were performed. In the first step, the protein was fixed and the water molecules were solely minimized (5000 cycles, 1500 cycles steepest descent were proceeded firstly). In the second step, the entire system was minimized (5000 cycles, 2000 cycles steepest descent were proceeded firstly). The equilibration process including two stages. In the heating stage, the system was annealed from 100 to 300 K over 100 ps at constant volume conditions. In the equilibration stage, the system was equilibrated at constant pressure over 100 ps. Then 20 ns simulations of the systems were performed and atomic coordinates, energies, and


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temperatures were sampled every 20 ps. The cutoff for the nonbonded interaction was 10 Å, PME method was used to calculate the long-range electrostatics, and SHAKE was employed to keep all bonds involving hydrogen atoms rigid. After completion, the results were analyzed. 4. Chemistry. General methods. All solvents and reagents were obtained commercially. Solvents were dried according to standard procedures, air and moisture sensitive reactions were performed under nitrogen. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (GF254) and visualized under UV light. Melting points were determined with a Melt-Temp II apparatus. The 1H NMR and

13C

NMR spectra were measured on a Bruker AV-300 instrument using deuterated solvents

with tetramethylsilane (TMS) as internal standard. ESI-mass and high resolution mass spectra (HRMS) were recorded on a Water Q-Tof micro mass spectrometer. The purity (≥ 95%) of the compounds was verified by the HPLC study performed on Agilent C18 (4.6 mm × 150 mm, 3.5μm) column using a mixture of solvent methanol/water at a flow rate of 0.5 mL/min and monitoring by UV absorption at 254 nm. General procedures for the synthesis of intermediates 13−41. 3-Cyano-4-fluorophenylboronic acid pinacol ester (12) and aryl iodide or aryl bromide (1.5 eq) was dissolved into dioxane, then Pd(PPh3)4 (0.1 eq) and Cs2CO3 (2.0 eq) were added. After reacted at 90 °C overnight under nitrogen atmosphere, the mixture was filtered through celite and the filtrate was concentrated. The residue was re-dissolved into 20 mL EA, then washed with water (20 mL × 2) and brine (20 mL × 2). The EA layer was dried over anhydrous Na2SO4, concentrated and purified by normal phase column chromatography with petroleum ether to afford intermediates 13−41.



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4-fluoro-[1,1'-biphenyl]-3-carbonitrile (13). Intermediate 13 was prepared from 12 (247 mg, 1.0 mmol) and iodobenzene (306 mg, 1.5 mmol) according to the general procedures. Colorless oil (168 mg, 85.3%). 1H NMR (300 MHz, Chloroform-d) δ 7.86−7.81 (m, 2H), 7.61−7.46 (m, 5H), 7.32 (m, 1H). 4-fluoro-2'-methyl-[1,1'-biphenyl]-3-carbonitrile (14). Intermediate 14 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-2-methylbenzene (327 mg, 1.5 mmol) according to the general procedures. Colorless oil (130 mg, 61.6%). 1H NMR (300 MHz, Chloroform-d) δ 7.63−7.57 (m, 2H), 7.34−7.30 (m, 4H), 7.20 (m, 1H), 2.28 (s, 3H). 4-fluoro-3'-methyl-[1,1'-biphenyl]-3-carbonitrile (15). Intermediate 15 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-3-methylbenzene (327 mg, 1.5 mmol) according to the general procedures. Light brown oil (180 mg, 85.2%). 1H NMR (300 MHz, Chloroform-d) δ 7.85−7.80 (m, 2H), 7.43−7.26 (m, 5H), 2.47 (s, 3H). 4-fluoro-4'-methyl-[1,1'-biphenyl]-3-carbonitrile (16). Intermediate 16 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-4-methylbenzene (327 mg, 1.5 mmol) according to the general procedures. Colorless oil (140 mg, 66.3%). 1H NMR (300 MHz, DMSO-d6) δ 8.22 (dd, J = 6.2, 2.4 Hz, 1H), 8.08−8.03 (m, 1H), 7.63−7.56 (m, 3H), 7.29 (d, J = 8.1 Hz, 2H), 2.34 (s, 3H). 2'-chloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (17). Intermediate 17 was prepared from 12 (247 mg, 1.0 mmol) and 1-chloro-2-iodobenzene (358 mg, 1.5 mmol) according to the general procedures. White solid (100 mg, 43.3%). 1H NMR (300 MHz, DMSO-d6) δ 7.91 (dd, J = 6.2, 2.4 Hz, 1H), 7.77−7.71 (m, 1H), 7.54−7.46 (m, 2H), 7.35−7.31 (m, 3H). 3'-chloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (18). Intermediate 18 was prepared from 12 (247 mg, 1.0 mmol) and 1-chloro-3-iodobenzene (358 mg, 1.5 mmol) according to the general procedures.


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Colorless oil (160 mg, 69.3%). 1H NMR (300 MHz, DMSO-d6) δ 8.33 (dd, J = 6.1, 2.5 Hz, 1H), 8.16−8.11 (m, 1H), 7.85−7.84 (m, 1H), 7.72−7.69 (m, 1H), 7.63 (t, J = 9.1 Hz, 1H), 7.54−7.46 (m, 2H). 4'-chloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (19). Intermediate 19 was prepared from 12 (247 mg, 1.0 mmol) and 1-chloro-4-iodobenzene (358 mg, 1.5 mmol) according to the general procedures. Colorless oil (192 mg, 82.9%). 1H NMR (300 MHz, DMSO-d6) δ 8.15 (dd, J = 6.2, 2.5 Hz, 1H), 7.99−7.94 (m, 1H), 7.63 (d, J = 8.6 Hz, 2H), 7.49 (t, J = 9.1 Hz, 1H), 7.41 (d, J = 8.6 Hz, 2H). 4-fluoro-2'-methoxy-[1,1'-biphenyl]-3-carbonitrile (20). Intermediate 20 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-2-methoxybenzene (351 mg, 1.5 mmol) according to the general procedures. Colorless oil (197 mg, 86.7%). 1H NMR (300 MHz, DMSO-d6) δ 8.00 (dd, J = 6.3, 2.3 Hz, 1H), 7.91−7.86 (m, 1H), 7.56 (t, J = 9.1 Hz, 1H), 7.43−7.33 (m, 2H), 7.14 (d, J = 7.8 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 3.78 (s, 3H). 4-fluoro-3'-methoxy-[1,1'-biphenyl]-3-carbonitrile (21). Intermediate 21 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-3-methoxybenzene (351 mg, 1.5 mmol) according to the general procedures. Light brown oil (181 mg, 79.7%). 1H NMR (300 MHz, DMSO-d6) δ 8.19 (dd, J = 6.2, 2.5 Hz, 1H), 8.04−7.98 (m, 1H), 7.52 (t, J = 9.0 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H), 7.21−7.18 (m, 2H), 6.91−6.87 (m, 1H), 3.74 (s, 3H). 4-fluoro-4'-methoxy-[1,1'-biphenyl]-3-carbonitrile (22). Intermediate 22 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-4-methoxybenzene (351 mg, 1.5 mmol) according to the general procedures. Colorless oil (189 mg, 93.2%). 1H NMR (300 MHz, DMSO-d6) δ 8.10 (dd, J = 6.2, 2.5 Hz, 1H), 7.97−7.91 (m, 1H), 7.61−7.56 (m, 2H), 7.48 (t, J = 9.0 Hz, 1H), 6.97−6.92 (m, 2H), 3.71 (s, 3H). 2'-ethyl-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (23). Intermediate 23 was prepared from 12 (247 mg,


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1.0 mmol) and 1-ethyl-2-iodobenzene (348 mg, 1.5 mmol) according to the general procedures. Colorless oil (119 mg, 53.0%). 1H NMR (300 MHz, DMSO-d6) δ 7.90 (dd, J = 6.3, 2.3 Hz, 1H), 7.75−7.70 (m, 1H), 7.59 (t, J = 9.0 Hz, 1H), 7.40−7.34 (m, 2H), 7.31−7.23 (m, 1H), 7.19−7.16 (m, 1H), 2.55−2.51 (m, 2H), 1.02 (t, J = 7.5 Hz, 3H). 4'-ethyl-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (24). Intermediate 24 was prepared from 12 (247 mg, 1.0 mmol) and 1-ethyl-4-iodobenzene (348 mg, 1.5 mmol) according to the general procedures. Colorless oil (192 mg, 85.3%). 1H NMR (300 MHz, DMSO-d6) δ 8.20 (dd, J = 6.1, 2.4 Hz, 1H), 8.07−8.02 (m, 1H), 7.65−7.55 (m, 3H), 7.34−7.30 (m, 2H), 2.64 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). 4-fluoro-4'-isopropyl-[1,1'-biphenyl]-3-carbonitrile (25). Intermediate 25 was prepared from 12 (1.98 g, 8.0 mmol) and 1-bromo-4-isopropylbenzene (2.39 g, 12 mmol) according to the general procedures. Colorless oil (1.35 g, 70.6%). 1H NMR (300 MHz, DMSO-d6) δ 8.19 (dd, J = 6.2, 2.5 Hz, 1H), 8.07−8.02 (m, 1H), 7.64−7.55 (m, 3H), 7.34 (d, J = 8.2 Hz, 2H), 2.98−2.86 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H). 4-fluoro-4'-propyl-[1,1'-biphenyl]-3-carbonitrile (26). Intermediate 26 was prepared from 12 (247 mg, 1.0 mmol) and 1-bromo-4-propylbenzene (299 mg, 1.5 mmol) according to the general procedures. Colorless oil (228 mg, 95.4%). 1H NMR (300 MHz, Chloroform-d) δ 7.82−7.76 (m, 2H), 7.45−7.43 (m, 2H), 7.29 (d, J = 8.6 Hz, 3H), 2.66 (t, J = 7.6 Hz, 2H), 1.76−1.64 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). 4'-(tert-butyl)-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (27). Intermediate 27 was prepared from 12 (247 mg, 1.0 mmol) and 1-tert-butyl-4-iodobenzene (390 mg, 1.5 mmol) according to the general procedures. Colorless oil (186 mg, 73.5%). 1H NMR (300 MHz, DMSO-d6) δ 8.21 (dd, J = 6.2, 2.4 Hz,


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1H), 8.09−8.03 (m, 1H), 7.67−7.57 (m, 3H), 7.52−7.47 (m, 2H), 1.31 (s, 9H). 4'-ethoxy-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (28). Intermediate 28 was prepared from 12 (247 mg, 1.0 mmol) and 4-iodophenetole (372 mg, 1.5 mmol) according to the general procedures. Colorless oil (100 mg, 41.5%). 1H NMR (300 MHz, DMSO-d6) δ 8.18 (dd, J = 6.1, 2.5 Hz, 1H), 8.05−8.00 (m, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.56 (t, J = 9.1 Hz, 1H), 7.02 ((d, J = 8.8 Hz, 2H), 4.08 (q, J = 7.0 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H). 4-fluoro-2',3'-dimethyl-[1,1'-biphenyl]-3-carbonitrile (29). Intermediate 29 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-2,3-dimethylbenzene (348 mg, 1.5 mmol) according to the general procedures. Colorless oil (179 mg, 80.0%). 1H NMR (300 MHz, DMSO-d6) δ 7.87 (dd, J = 6.3, 2.3 Hz, 1H), 7.73−7.68 (m, 1H), 7.58 (t, J = 9.0 Hz, 1H), 7.40−7.38 (m, 1H), 7.25−7.13 (m, 2H), 2.29 (s, 3H), 2.08 (s, 3H). 4-fluoro-2',4'-dimethyl-[1,1'-biphenyl]-3-carbonitrile (30). Intermediate 30 was prepared from 12 (247 mg, 1.0 mmol) and 4-iodo-m-xylene (348 mg, 1.5 mmol) according to the general procedures. Light yellow oil (200 mg, 88.9%). 1H NMR (300 MHz, Chloroform-d) δ 7.60−7.55 (m, 2H), 7.33−7.24 (m, 1H), 7.20−7.08 (m, 3H), 2.41 (s, 3H), 2.25 (s, 3H). 4-fluoro-2',6'-dimethyl-[1,1'-biphenyl]-3-carbonitrile (31). Intermediate 31 was prepared from 12 (247 mg, 1.0 mmol) and 2-iodo-1,3-dimethylbenzene (348 mg, 1.5 mmol) according to the general procedures. Colorless oil (149 mg, 66.1%). 1H NMR (300 MHz, DMSO-d6) δ 7.80−7.77 (m, 1H), 7.64−7.57 (m, 2H), 7.23−7.18 (m, 1H), 7.15−7.12 (m, 2H), 1.96 (s, 6H). 4-fluoro-3',4'-dimethyl-[1,1'-biphenyl]-3-carbonitrile (32). Intermediate 32 was prepared from 12 (247 mg, 1.0 mmol) and 4-iodo-1,2-dimethylbenzene (348 mg, 1.5 mmol) according to the general


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procedures. Colorless oil (210 mg, 93.3%). 1H NMR (300 MHz, DMSO-d6) δ 8.11 (dd, J = 6.2, 2.5 Hz, 1H), 7.98−7.93 (m, 1H), 7.52−7.43 (m, 2H), 7.37−7.32 (m, 1H), 7.14 (d, J = 7.9 Hz, 1H), 2.19 (s, 3H), 2.16 (s, 3H). 4-fluoro-3',5'-dimethyl-[1,1'-biphenyl]-3-carbonitrile (33). Intermediate 33 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodo-3,5-dimethylbenzene (348 mg, 1.5 mmol) according to the general procedures. Colorless oil (170 mg, 75.5%). 1H NMR (300 MHz, DMSO-d6) δ 8.21 (dd, J = 6.2, 2.4 Hz, 1H), 8.07−8.02 (m, 1H), 7.58 (t, J = 9.1 Hz, 1H), 7.33 (s, 2H), 7.04 (s, 1H), 2.33 (s, 6H). 4-fluoro-2',4',6'-trimethyl-[1,1'-biphenyl]-3-carbonitrile (34). Intermediate 34 was prepared from 12 (247 mg, 1.0 mmol) and 2,4,6-trimethyliodobenzene (369 mg, 1.5 mmol) according to the general procedures. Colorless oil (196 mg, 82.0%). 1H NMR (300 MHz, DMSO-d6) δ 7.74 (dd, J = 6.3, 2.0 Hz, 1H), 7.62−7.52 (m, 2H), 6.95 (s, 2H), 2.26 (s, 3H), 1.92 (s, 6H). 2',3'-dichloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (35). Intermediate 35 was prepared from 12 (247 mg, 1.0 mmol) and 1,2-dichloro-3-iodobenzene (409 mg, 1.5 mmol) according to the general procedures. Colorless oil (60 mg, 22.6%). 1H NMR (300 MHz, DMSO-d6) δ 7.95 (dd, J = 6.2, 2.3 Hz, 1H), 7.78−7.73 (m, 1H), 7.60 (dd, J = 7.5, 2.1 Hz, 1H), 7.52 (t, J = 9.1 Hz, 1H), 7.37−7.29 (m, 2H). 2',6'-dichloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (36). Intermediate 36 was prepared from 12 (247 mg, 1.0 mmol) and 1,3-dichloro-2-iodobenzene (409 mg, 1.5 mmol) according to the general procedures. Colorless oil (100 mg, 37.7%). 1H NMR (300 MHz, DMSO-d6) δ 8.02 (dd, J = 6.2, 2.3 Hz, 1H), 7.79−7.74 (m, 1H), 7.71−7.62 (m, 3H), 7.52−7.46 (m, 1H). 3',4'-dichloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (37). Intermediate 37 was prepared from 12 (247 mg, 1.0 mmol) and 1,2-dichloro-4-iodobenzene (409 mg, 1.5 mmol) according to the general


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procedures. Colorless oil (113 mg, 42.6%). 1H NMR (300 MHz, DMSO-d6) δ 8.38 (dd, J = 6.1, 2.5 Hz, 1H), 8.20−8.15 (m, 1H), 8.08 (t, J = 1.3 Hz, 1H), 7.76 (d, J = 1.3 Hz, 2H), 7.65 (t, J = 9.1 Hz, 1H). 3',5'-dichloro-4-fluoro-[1,1'-biphenyl]-3-carbonitrile (38). Intermediate 38 was prepared from 12 (247 mg, 1.0 mmol) and 1,3-dichloro-5-iodobenzene (409 mg, 1.5 mmol) according to the general procedures. Colorless oil (138 mg, 51.9%). 1H NMR (300 MHz, DMSO-d6) δ 8.40 (dd, J = 6.1, 2.5 Hz, 1H), 8.22−8.17 (m, 1H), 7.86 (d, J = 1.9 Hz, 2H), 7.67−7.61 (m, 2H). 2-fluoro-5-(naphthalen-1-yl)benzonitrile (39). Intermediate 39 was prepared from 12 (247 mg, 1.0 mmol) and 1-iodonaphthalene (381 mg, 1.5 mmol) according to the general procedures. Colorless oil (215 mg, 87.0%). 1H NMR (300 MHz, DMSO-d6) δ 8.42 (dd, J = 6.2, 2.4 Hz, 1H), 8.33 (d, J = 1.9 Hz, 1H), 8.28−8.22 (m, 1H), 8.05−7.95 (m, 3H), 7.92−7.88 (m, 1H), 7.67 (t, J = 9.1 Hz, 1H), 7.61−7.52 (m, 2H). 2-fluoro-5-(naphthalen-2-yl)benzonitrile (40). Intermediate 40 was prepared from 12 (247 mg, 1.0 mmol) and 2-iodonaphthalene (381 mg, 1.5 mmol) according to the general procedures. Colorless oil (168 mg, 68.0%). 1H NMR (300 MHz, DMSO-d6) δ 8.08−8.00 (m, 3H), 7.91−7.86 (m, 1H), 7.73−7.68 (m, 2H), 7.65−7.52 (m, 3H), 7.50−7.46 (m, 1H). 4-fluoro-4'-(1H-pyrrol-1-yl)-[1,1'-biphenyl]-3-carbonitrile (41). Intermediate 41 was prepared from 12 (247 mg, 1.0 mmol) and 1-(4-iodophenyl)pyrrole (404 mg, 1.5 mmol) according to the general procedures. Light yellow oil (173 mg, 66.0%). 1H NMR (300 MHz, DMSO-d6) δ 8.30 (dd, J = 6.2, 2.5 Hz, 1H), 8.16−8.10(m, 1H), 7.85−7.81 (m, 2H), 7.73−7.69 (m, 2H), 7.62 (t, J = 9.1 Hz, 1H), 7.46 (t, J = 2.2 Hz, 2H), 6.30 (t, J = 2.2 Hz, 2H). General procedures for the synthesis of compounds 42−70


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Intermediates 13−41 were dissolved into 5 mL DMSO, trans-4-aminocyclohexanol (4.5 eq) and DIPEA (9.0 eq) were added. The mixture was heated at 120 °C for 24 h and then poured into 20 mL water and extracted with 20 mL EA for two times. The EA layer was combined and dried over anhydrous Na2SO4. After concentration, the residue was dissolved into 20 mL ethanol, 2 mL 1M NaOH solution and 2 mL 30% H2O2 were added. Then the mixture was stirred at 30 °C overnight. After removing ethanol under reduced pressure, the residue was dissolved into 20 mL EA and washed with water (20 mL × 2) and brine (20 mL × 1). The EA layer was dried over anhydrous Na2SO4 and then concentrated. The residue was triturated with DCM to afford compounds 42−70. 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (42). Compound 42 was prepared from intermediate 13 (168 mg, 0.85 mmol) according to the general procedures. White solid. 78 mg, yield: 29.6%. mp 189−191 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.17 (d, J = 7.7 Hz, 1H), 8.02 (s, 1H), 7.90 (s, 1H), 7.64 (d, J = 7.7 Hz, 2H), 7.58 (d, J = 8.7 Hz, 1H), 7.39 (t, J = 7.6 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.17 (s, 1H), 6.79 (d, J = 8.8 Hz, 1H), 4.57 (s, 1H), 3.52−3.43 (m, 1H), 3.39−3.33 (m, 1H), 2.01−1.96 (m, 2H), 1.85−1.81 (m, 2H), 1.38−1.14 (m, 4H). HRMS (ESI): calcd for C19H23N2O2 [M+H]+ 311.1681, found 311.1757. 13C NMR (75 MHz, DMSO-d6) δ 171.65, 148.37, 139.92, 130.61, 128.65, 127.29, 125.91, 125.57, 125.37, 113.93, 112.15, 68.03, 49.43, 33.45, 30.25. Purity: 95.68% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-2'-methyl-[1,1'-biphenyl]-3-carboxamide (43). Compound 43 was prepared from intermediate 14 (130 mg, 0.62 mmol) according to the general procedures. White solid. 85 mg, yield: 42.3%. mp 186−188 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.19 (d, J = 7.6 Hz, 1H), 7.86 (s, 1H), 7.58 (s, 1H), 7.26−7.21 (m, 5H), 7.12 (s, 1H), 6.75 (d, J = 8.7 Hz, 1H), 4.60 (s, 1H),


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3.52−3.35 (m, 1H), 3.32−3.27 (m, 1H), 2.26 (s, 3H), 2.01−1.98 (m, 2H), 1.85−1.81 (m, 2H), 1.38−1.30 (m, 2H), 1.26−1.15 (m, 2H). HRMS (ESI): calcd for C20H25N2O2 [M + H]+ 325.1838, found 325.1915. Purity: 97.07% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-3'-methyl-[1,1'-biphenyl]-3-carboxamide (44). Compound 44 was prepared from intermediate 15 (180 mg, 0.85 mmol) according to the general procedures. White solid. 52 mg, yield: 18.9%. mp 179−181 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.16 (d, J = 7.7 Hz, 1H), 8.02 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.56 (dd, J = 8.7, 1.7 Hz, 1H), 7.47(s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.19 (s, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.78 (d, J = 8.9 Hz, 1H), 4.58 (d, J = 4.1 Hz, 1H), 3.52−3.44 (m, 1H), 3.40−3.34 (m, 1H), 2.35 (s, 3H), 2.00−1.96 (m, 2H), 1.85−1.80 (m, 2H), 1.39−1.29 (m, 2H), 1.27−1.14 (m, 2H). HRMS (ESI): calcd for C20H25N2O2 [M + H]+ 325.1838, found 325.1916. Purity: 97.15% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-4'-methyl-[1,1'-biphenyl]-3-carboxamide (45). Compound 45 was prepared from intermediate 16 (140 mg, 0.66 mmol) according to the general procedures. White solid. 34 mg, yield: 15.9%. mp 219−222 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.13 (d, J = 7.7 Hz, 1H), 8.01 (s, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 3H), 7.20 (d, J = 8.0 Hz, 3H), 6.77 (d, J = 8.9 Hz, 1H), 4.58 (d, J = 4.1 Hz, 1H), 3.51−3.42 (m, 1H), 3.34−3.26 (m, 1H), 2.30 (s, 3H), 2.00−1.95 (m, 2H), 1.84−1.81 (m, 2H), 1.38−1.30 (m, 2H), 1.29−1.16 (m, 2H). HRMS (ESI): calcd for C20H25N2O2 [M + H]+ 325.1838, found 325.1913.

13C

NMR (75 MHz, DMSO-d6) δ 171.67, 148.15, 137.03, 134.97,

130.38, 129.23, 126.95, 125.39, 125.35, 113.90, 112.12, 68.04, 49.41, 33.46, 30.26, 20.59. Purity: 95.22% by HPLC (MeOH/H2O = 90:10). 2'-chloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (46). Compound



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46 was prepared from intermediate 17 (100 mg, 0.43 mmol) according to the general procedures. White solid. 33 mg, yield: 22.3%. mp 196−198 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.22 (d, J = 7.7 Hz, 1H), 7.87 (s, 1H), 7.66 (d, J = 2.1 Hz, 1H), 7.51 (dd, J = 7.8, 1.2 Hz, 1H), 7.45−7.28 (m, 4H), 7.14 (s, 1H), 6.77 (d, J = 8.9 Hz, 1H), 4.58 (d, J = 4.2 Hz, 1H), 3.51−3.42 (m, 1H), 3.39−3.35 (m, 1H), 2.01−1.96 (m, 2H), 1.85−1.80 (m, 2H), 1.39−1.31 (m, 2H), 1.26−1.14 (m, 2H). HRMS (ESI): calcd for C19H22ClN2O2 [M + H]+ 345.1292, found 345.1367. Purity: 96.52% by HPLC (MeOH/H2O = 90:10). 3'-chloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (47). Compound 47 was prepared from intermediate 18 (160 mg, 0.69 mmol) according to the general procedures. White solid. 36 mg, yield: 15.2%. mp 189−191 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.29 (d, J = 7.7 Hz, 1H), 8.08 (s, 1H), 7.92 (d, J = 2.3 Hz, 1H), 7.76 (t, J = 1.7 Hz, 1H), 7.61 (dd, J = 8.7, 1.7 Hz, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.29−7.26 (m, 1H), 7.22 (s, 1H), 6.79 (d, J = 8.9 Hz, 1H), 4.59 (d, J = 4.1 Hz, 1H), 3.52−3.44 (m, 1H), 3.42−3.37 (m, 1H), 2.00−1.95 (m, 2H), 1.85−1.79 (m, 2H), 1.39−1.14 (m, 4H). HRMS (ESI): calcd for C19H22ClN2O2 [M + H]+ 345.1292, found 345.1368. Purity: 95.33% by HPLC (MeOH/H2O = 90:10). 4'-chloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (48). Compound 48 was prepared from intermediate 19 (192 mg, 0.83 mmol) according to the general procedures. White solid. 150 mg, yield: 52.5%. mp﹥250 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J = 7.7 Hz, 1H), 8.04 (s, 1H), 7.90 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.58 (dd, J = 8.8, 2.2 Hz, 1H), 7.44 (d, J = 8.6 Hz, 2H), 7.21 (s, 1H), 6.79 (d, J = 8.9 Hz, 1H), 4.59 (d, J = 4.1 Hz, 1H), 3.51−3.44 (m, 1H), 3.42−3.35 (m, 1H), 2.00−1.96 (m, 2H), 1.85−1.80 (m, 2H), 1.39−1.14 (m, 4H). HRMS (ESI): calcd for C19H22ClN2O2 [M + H]+ 345.1292, found 345.1371. 13C NMR (75 MHz, DMSO-d6) δ 171.55, 148.61,


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138.72, 130.53, 128.55, 127.26, 127.16, 123.84, 113.83, 112.19, 68.00, 49.39, 33.42, 30.21. Purity: 97.02% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-2'-methoxy-[1,1'-biphenyl]-3-carboxamide

(49).

Compound 49 was prepared from intermediate 20 (197 mg, 0.87 mmol) according to the general procedures. White solid. 97 mg, yield: 32.8%. mp 179−181 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.06 (d, J = 7.7 Hz, 1H), 7.83 (s, 1H), 7.66 (d, J = 2.1 Hz, 1H), 7.42 (dd, J = 8.7, 1.7 Hz, 1H), 7.32−7.23 (m, 2H), 7.06 (s, 1H), 7.03−6.95 (m, 2H), 6.72 (d, J = 8.8 Hz, 1H), 4.57 (d, J = 4.1 Hz, 1H), 3.74 (s, 3H), 3.52−3.43 (m, 1H), 3.31−3.26 (m, 1H), 2.01−1.97 (m, 2H), 1.84−1.81 (m, 2H), 1.38−1.27 (m, 2H), 1.24−1.15 (m, 2H). HRMS (ESI): calcd for C20H25N2O3 [M + H]+ 341.1787, found 341.1865. Purity: 97.90% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-3'-methoxy-[1,1'-biphenyl]-3-carboxamide

(50).

Compound 50 was prepared from intermediate 21 (181 mg, 0.80 mmol) according to the general procedures. Light yellow solid. 91 mg, yield: 33.5%. mp 196−198 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.10 (d, J = 7.7 Hz, 1H), 7.94 (s, 1H), 7.79 (d, J = 2.3 Hz, 1H), 7.49 (dd, J = 8.8, 2.1 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.13−7.10 (m, 3H), 6.74−6.67 (m, 2H), 4.47 (d, J = 4.2 Hz, 1H), 3.72 (s, 3H), 3.43−3.35 (m, 1H), 3.32−3.24 (m, 1H), 1.92−1.88 (m, 2H), 1.75−1.71 (m, 2H), 1.30−1.06 (m, 4H). HRMS (ESI): calcd for C20H25N2O3 [M + H]+ 341.1787, found 341.1857. Purity: 96.33% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-4'-methoxy-[1,1'-biphenyl]-3-carboxamide

(51).

Compound 51 was prepared from intermediate 22 (189 mg, 0.83 mmol) according to the general procedures. Light yellow solid. 60 mg, yield: 21.3%. mp 209−211 °C. 1H NMR (300 MHz, DMSO-d6)



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δ 7.99 (d, J = 7.7 Hz, 1H), 7.90 (s, 1H), 7.73 (d, J = 2.3 Hz, 1H), 7.51−7.45 (d, J = 8.8, 2H), 7.42 (dd, J = 8.7, 2.2 Hz, 1H), 7.05 (s, 1H), 6.87 (d, J = 8.9, 2H), 6.67 (d, J = 8.9 Hz, 1H), 4.47 (d, J = 4.2 Hz, 1H), 3.68 (s, 3H), 3.43−3.37 (m, 1H), 3.30−3.24 (m, 1H), 1.91−1.88 (m, 2H), 1.76−1.71 (m, 2H), 1.30−1.03 (m, 4H). HRMS (ESI): calcd for C20H25N2O3 [M + H]+ 341.1787, found 341.1855. 13C NMR (75 MHz, DMSO-d6) δ 171.69, 157.82, 147.85, 132.49, 130.24, 126.74, 126.66, 125.32, 114.08, 113.95, 112.15, 68.05, 55.07, 49.43, 33.46, 30.27. Purity: 95.29% by HPLC (MeOH/H2O = 90:10). 2'-ethyl-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (52). Compound 52 was prepared from intermediate 23 (119 mg, 0.53 mmol) according to the general procedures. White solid. 125 mg, yield: 69.8%. mp 180−182 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.17 (d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.55 (d, J = 2.1 Hz, 1H), 7.30−7.14 (m, 5H), 7.08 (s, 1H), 6.75 (d, J = 8.7 Hz, 1H), 4.57 (d, J = 4.2 Hz, 1H), 3.52−3.43 (m, 1H), 3.31−3.23 (m, 1H), 2.58 (q, J = 7.5 Hz, 2H), 2.01−1.97 (m, 2H), 1.85−1.81 (m, 2H), 1.39−1.14 (m, 4H), 1.05 (t, J = 7.5 Hz, 3H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2069. Purity: 97.85% by HPLC (MeOH/H2O = 90:10). 4'-ethyl-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (53). Compound 53 was prepared from intermediate 24 (192 mg, 0.85 mmol) according to the general procedures. White solid. 55 mg, yield: 19.2%. mp 172−174 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.12 (d, J = 7.8 Hz, 1H), 7.98 (s, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.55 (d, J = 8.2 Hz, 3H), 7.22 (d, J = 7.9 Hz, 2H), 7.12 (s, 1H), 6.77 (d, J = 8.9 Hz, 1H), 4.54 (d, J = 4.2 Hz, 1H), 3.52−3.43 (m, 1H), 3.39−3.30 (m, 1H), 2.61 (q, J = 7.6 Hz, 2H), 2.01−1.96 (m, 2H), 1.85−1.80 (m, 2H), 1.39−1.14 (m, 7H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2063.

13C

NMR (75 MHz, DMSO-d6) δ 171.68, 148.16,

141.43, 137.37, 130.45, 128.05, 127.07, 125.53, 125.46, 113.92, 112.12, 68.04, 49.43, 33.46, 30.26,


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27.75, 15.74. Purity: 95.59% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide

(54).

Compound 54 was prepared from intermediate 25 (200 mg, 0.84 mmol) according to the general procedures. White solid. 122 mg, yield: 41.3%. mp 197−199 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.14 (d, J = 7.7 Hz, 1H), 8.01 (s, 1H), 7.86 (d, J = 2.3 Hz, 1H), 7.55 (d, J = 8.5 Hz, 3H), 7.26 (d, J = 7.9 Hz, 2H), 7.18 (s, 1H), 6.78 (d, J = 8.8 Hz, 1H), 4.60 (d, J = 4.1 Hz, 1H), 3.52−3.44 (m, 1H), 3.33−3.26 (m, 1H), 2.94−2.85 (m, 1H), 2.00−1.97 (m, 2H), 1.85−1.81 (m, 2H), 1.39−1.28 (m, 2H), 1.22 (d, J = 6.9 Hz, 6H), 1.17−1.14 (m, 2H). HRMS (ESI): calcd for C22H29N2O2 [M + H]+ 353.2151, found 353.2227. 13C NMR (75 MHz, DMSO-d6) δ 171.69, 148.16, 146.06, 137.59, 130.48, 127.16, 126.54, 125.59, 125.55, 113.91, 112.10, 68.04, 49.44, 33.46, 33.01, 30.27, 23.91. Purity: 96.14% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-4'-propyl-[1,1'-biphenyl]-3-carboxamide (55). Compound 55 was prepared from intermediate 26 (228 mg, 0.95 mmol) according to the general procedures. White solid. 80 mg, yield: 23.9%. mp 196−198 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.13 (d, J = 7.7 Hz, 1H), 7.99 (s, 1H), 7.87 (d, J = 2.3 Hz, 1H), 7.56 (d, J = 8.2 Hz, 3H), 7.21 (d, J = 7.9 Hz, 2H), 7.14 (s, 1H), 6.78 (d, J = 8.9 Hz, 1H), 4.55 (d, J = 4.2 Hz, 1H), 3.53−3.43 (m, 1H), 3.39−3.33 (m, 1H), 2.56 (t, J = 7.4 Hz, 2H), 2.00−1.97 (m, 2H), 1.85−1.80 (m, 2H), 1.67−1.54 (m, 2H), 1.31−1.14 (m, 4H), 0.91 (t, J = 7.3 Hz, 3H). HRMS (ESI): calcd for C22H29N2O2 [M + H]+ 353.2151, found 353.2218. Purity: 96.80% by HPLC (MeOH/H2O = 90:10). 4'-(tert-butyl)-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide

(56).

Compound 56 was prepared from intermediate 27 (186 mg, 0.74 mmol) according to the general



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procedures. Light yellow solid. 48 mg, yield: 17.7%. mp 199−201 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.12 (d, J = 7.7 Hz, 1H), 7.97 (s, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.55 (d, J = 8.3 Hz, 3H), 7.40 (d, J = 8.4 Hz, 2H), 7.12 (s, 1H), 6.78 (d, J = 8.9 Hz, 1H), 4.54 (d, J = 4.2 Hz, 1H), 3.52−3.43 (m, 1H), 3.39−3.31 (m, 1H), 2.01−1.97 (m, 2H), 1.85−1.81 (m, 2H), 1.38−1.15 (m, 13H). HRMS (ESI): calcd for C23H31N2O2 [M + H]+ 367.2307, found 367.2380.

13C

NMR (75 MHz, DMSO-d6) δ 171.67, 148.27,

148.16, 137.17, 130.49, 127.19, 125.46, 125.37, 125.33, 113.92, 112.11, 68.03, 49.43, 34.08, 33.45, 31.13, 30.26. Purity: 95.78% by HPLC (MeOH/H2O = 90:10). 4'-ethoxy-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide (57). Compound 57 was prepared from intermediate 28 (100 mg, 0.41 mmol) according to the general procedures. White solid. 66 mg, yield: 45.5%. mp﹥250 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.08 (d, J = 7.7 Hz, 1H), 7.98 (s, 1H), 7.82 (s, 1H), 7.57−7.50 (m, 3H), 7.12 (s, 1H), 6.94 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.8 Hz, 1H), 4.54 (d, J = 4.1 Hz, 1H), 4.04 (q, J = 6.8 Hz, 2H), 3.52−3.44 (m, 1H), 3.40−3.30 (m, 1H), 2.00−1.96 (m, 2H), 1.85−1.80 (m, 2H), 1.33 (t, J = 6.9 Hz, 3H), 1.26−1.13 (m, 4H). HRMS (ESI): calcd for C21H27N2O3 [M + H]+ 355.1943, found 355.2009. Purity: 99.01% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-2',3'-dimethyl-[1,1'-biphenyl]-3-carboxamide

(58).

Compound 58 was prepared from intermediate 29 (179 mg, 0.80 mmol) according to the general procedures. White solid. 47 mg, yield: 17.4%. mp 222−224 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.16 (d, J = 7.6 Hz, 1H), 7.82 (s, 1H), 7.53 (d, J = 2.1 Hz, 1H), 7.18 (dd, J = 8.5, 2.1 Hz, 1H), 7.09 (d, J = 5.1 Hz, 2H), 7.06−7.02 (m, 1H), 6.74 (d, J = 8.7 Hz, 1H), 4.58 (d, J = 4.2 Hz, 1H), 3.52−3.43 (m, 1H), 3.32−3.25 (m, 1H), 2.27 (s, 3H), 2.12 (s, 3H), 2.01−1.97 (m, 2H), 1.85−1.81 (m, 2H), 1.38−1.14 (m, 4H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2069.


13C

NMR (75 MHz,

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DMSO-d6) δ 171.62, 147.79, 141.35, 136.60, 133.49, 133.41, 129.77, 128.01, 127.56, 127.03, 125.14, 113.16, 111.17, 68.07, 49.47, 33.50, 30.33, 20.41, 16.77. Purity: 95.44% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-2',4'-dimethyl-[1,1'-biphenyl]-3-carboxamide

(59).

Compound 59 was prepared from intermediate 30 (200 mg, 0.89 mmol) according to the general procedures. White solid. 67 mg, yield: 22.3%. mp 192−194 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.14 (d, J = 7.7 Hz, 1H), 7.83 (s, 1H), 7.54 (d, J = 2.0 Hz, 1H), 7.21 (dd, J = 8.5, 2.1 Hz, 1H), 7.10−7.00 (m, 4H), 6.73 (d, J = 8.8 Hz, 1H), 4.58 (d, J = 4.2 Hz, 1H), 3.53−3.42 (m, 1H), 3.32−3.26 (m, 1H), 2.28 (s, 3H), 2.21 (s, 3H), 2.00−1.97 (m, 2H), 1.85−1.80 (m, 2H), 1.38−1.14 (m, 4H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2070. Purity: 98.99% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-2',6'-dimethyl-[1,1'-biphenyl]-3-carboxamide

(60).

Compound 60 was prepared from intermediate 31 (149 mg, 0.66 mmol) according to the general procedures. Light yellow solid. 86 mg, yield: 38.6%. mp 212−214 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.25 (d, J = 7.5 Hz, 1H), 7.77 (s, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.08 (s, 3H), 7.01 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.6 Hz, 1H), 4.57 (d, J = 4.2 Hz, 1H), 3.52−3.44 (m, 1H), 3.31−3.26 (m, 1H), 2.01 (s, 6H), 1.99−1.94 (m, 2H), 1.86−1.82 (m, 2H), 1.39−1.30 (m, 2H), 1.26−1.15 (m, 2H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2068. Purity: 97.78% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-3',4'-dimethyl-[1,1'-biphenyl]-3-carboxamide

(61).

Compound 61 was prepared from intermediate 32 (210 mg, 0.93 mmol) according to the general procedures. White solid. 38 mg, yield: 12.1%. mp 172−174 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.03 (d, J = 7.7 Hz, 1H), 7.90 (s, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 7.35 (s, 1H), 7.26 (d,



Page 54 of 83

J = 7.8 Hz, 1H), 7.06−7.03 (m, 2H), 6.67 (d, J = 8.8 Hz, 1H), 4.47 (d, J = 4.1 Hz, 1H), 3.43−3.33 (m, 2H), 2.18 (s, 3H), 2.13 (s, 3H), 1.92−1.88 (m, 2H), 1.76−1.72 (m, 2H), 1.30−1.04 (m, 4H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2060. Purity: 96.68% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-3',5'-dimethyl-[1,1'-biphenyl]-3-carboxamide

(62).

Compound 62 was prepared from intermediate 33 (170 mg, 0.76 mmol) according to the general procedures. White solid. 118 mg, yield: 45.9%. mp 209−211 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.15 (d, J = 7.7 Hz, 1H), 8.01 (s, 1H), 7.85 (d, J = 2.3 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.25 (s, 2H), 7.19 (s, 1H), 6.87 (s, 1H), 6.76 (d, J = 8.7 Hz, 1H), 4.59 (d, J = 4.1 Hz, 1H), 3.53−3.42 (m, 1H), 3.32−3.25 (m, 1H), 2.30 (s, 6H), 2.00−1.96 (m, 2H), 1.85−1.80 (m, 2H), 1.38−1.30 (m, 2H), 1.27−1.13 (m, 2H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2068. Purity: 96.86% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-2',4',6'-trimethyl-[1,1'-biphenyl]-3-carboxamide

(63).

Compound 63 was prepared from intermediate 34 (196 mg, 0.82 mmol) according to the general procedures.

White solid. 126 mg, yield: 43.7%. mp 202−204 °C. 1H NMR (300 MHz, DMSO-d6) δ

8.21 (d, J = 7.6 Hz, 1H), 7.73 (s, 1H), 7.36 (d, J = 2.1 Hz, 1H), 6.98 (dd, J = 8.5, 2.0 Hz, 2H), 6.89 (s, 2H), 6.75 (d, J = 8.7 Hz, 1H), 4.54 (d, J = 4.2 Hz, 1H), 3.52−3.43 (m, 1H), 3.36−3.31 (m, 1H), 2.24 (s, 3H), 2.03−1.98 (m, 2H), 1.97 (s, 6H), 1.86−1.82 (m, 2H), 1.39−1.15 (m, 4H). HRMS (ESI): calcd for C22H29N2O2 [M + H]+ 353.2151, found 353.2220.

13C

NMR (75 MHz, DMSO-d6) δ 171.61, 147.77,

138.26, 135.85, 135.36, 133.39, 129.51, 127.78, 125.28, 113.08, 111.54, 68.12, 49.53, 33.55, 30.44, 20.68, 20.58. Purity: 98.75% by HPLC (MeOH/H2O = 90:10).


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2',3'-dichloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide

(64).

Compound 64 was prepared from intermediate 35 (60 mg, 0.23 mmol) according to the general procedures. White solid. 31 mg, yield: 35.6%. mp 229−231 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.29 (d, J = 7.6 Hz, 1H), 7.86 (s, 1H), 7.67 (d, J = 2.2 Hz, 1H), 7.58 (t, J = 4.8 Hz, 1H), 7.40−7.35 (m, 3H), 7.16 (s, 1H), 6.78 (d, J = 9.0 Hz, 1H), 4.59 (d, J = 4.2 Hz, 1H), 3.52−3.44 (m, 1H), 3.40−3.36 (m, 1H), 2.00−1.97 (m, 2H), 1.85−1.80 (m, 2H), 1.39−1.29 (m, 2H), 1.25−1.14 (m, 2H). HRMS (ESI): calcd for C19H21Cl2N2O2 [M + H]+ 379.0902, found 379.0981. Purity: 95.04% by HPLC (MeOH/H2O = 90:10). 2',6'-dichloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide

(65).

Compound 65 was prepared from intermediate 36 (100 mg, 0.38 mmol) according to the general procedures. Light yellow solid. 37 mg, yield: 25.8%. mp 189−191 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.46 (d, J = 7.5 Hz, 1H), 7.78 (s, 1H), 7.56−7.54 (m, 3H), 7.39−7.34 (m, 1H), 7.18−7.07 (m, 2H), 6.78 (d, J = 8.9 Hz, 1H), 4.58 (d, J = 4.2 Hz, 1H), 3.52−3.45 (m, 1H), 3.32−3.27 (m, 1H), 2.01−1.97 (m, 2H), 1.85−1.82 (m, 2H), 1.38−1.27 (m, 2H), 1.23−1.15 (m, 2H). HRMS (ESI): calcd for C19H21Cl2N2O2 [M + H]+ 379.0902, found 379.0978.

13C

NMR (75 MHz, DMSO-d6) δ 171.30, 148.84, 138.62, 134.71,

133.74, 130.40, 129.52, 128.33, 121.21, 112.48, 111.18, 68.02, 49.42, 33.44, 30.29. Purity: 99.51% by HPLC (MeOH/H2O = 90:10). 3',4'-dichloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide

(66).

Compound 66 was prepared from intermediate 37 (113 mg, 0.42 mmol) according to the general procedures. Light yellow solid. 42 mg, yield: 26.4%. mp 222−224 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.34 (d, J = 7.7 Hz, 1H), 8.05 (s, 1H), 7.95 (dd, J = 8.5, 2.1 Hz, 2H), 7.68−7.60 (m, 3H), 7.22 (s, 1H), 6.80 (d, J = 8.9 Hz, 1H), 4.54 (d, J = 4.2 Hz, 1H), 3.52−3.44 (m, 1H), 3.39−3.33 (m, 1H), 2.00−1.96 (m,



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2H), 1.85−1.79 (m, 2H), 1.40−1.20 (m, 4H). HRMS (ESI): calcd for C19H21Cl2N2O2 [M + H]+ 379.0902, found 379.0970. Purity: 97.47% by HPLC (MeOH/H2O = 90:10). 3',5'-dichloro-4-(((1r,4r)-4-hydroxycyclohexyl)amino)-[1,1'-biphenyl]-3-carboxamide

(67).

Compound 67 was prepared from intermediate 38 (138 mg, 0.52 mmol) according to the general procedures. White solid. 170 mg, yield: 86.5%. mp 236−238 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.41 (d, J = 7.7 Hz, 1H), 8.09 (s, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.75 (d, J = 1.8 Hz, 2H), 7.67 (dd, J = 8.8, 2.2 Hz, 1H), 7.41 (t, J = 1.9 Hz, 1H), 7.26 (s, 1H), 6.79 (d, J = 8.9 Hz, 1H), 4.58 (d, J = 4.2 Hz, 1H), 3.52−3.38 (m, 2H), 2.00−1.96 (m, 2H), 1.85−1.81 (m, 2H), 1.40−1.15 (m, 4H). HRMS (ESI): calcd for C19H21Cl2N2O2 [M + H]+ 379.0902, found 379.0981. Purity: 99.39% by HPLC (MeOH/H2O = 90:10). 2-(((1r,4r)-4-hydroxycyclohexyl)amino)-5-(naphthalen-1-yl)benzamide (68). Compound 68 was prepared from intermediate 39 (215 mg, 0.87 mmol) according to the general procedures. White solid. 126 mg, yield: 40.3%. mp﹥250 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.22 (d, J = 7.7 Hz, 1H), 7.98−7.95 (m, 1H), 7.91−7.87 (m, 3H), 7.74 (d, J = 2.1 Hz, 1H), 7.57−7.43 (m, 4H), 7.38 (dd, J = 8.5, 2.1 Hz, 1H), 7.12 (s, 1H), 6.86 (d, J = 8.7 Hz, 1H), 4.59 (d, J = 4.1 Hz, 1H), 3.54−3.47 (m, 1H), 3.42−3.36 (m, 1H), 2.06−2.01 (m, 2H), 1.87−1.83 (m, 2H), 1.40−1.17 (m, 4H). HRMS (ESI): calcd for C23H25N2O2 [M + H]+ 361.1838, found 361.1907. Purity: 96.02% by HPLC (MeOH/H2O = 90:10). 2-(((1r,4r)-4-hydroxycyclohexyl)amino)-5-(naphthalen-2-yl)benzamide (69). Compound 69 was prepared from intermediate 40 (168 mg, 0.68 mmol) according to the general procedures. Light yellow solid. 48 mg, yield: 19.6%. mp 196−198 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.24 (d, J = 7.6 Hz, 1H), 8.16 (s, 1H), 8.10 (s, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.96−7.87 (m, 4H), 7.75 (dd, J = 8.8, 2.2 Hz, 1H), 7.53−7.42 (m, 2H), 7.24 (s, 1H), 6.84 (d, J = 9.0 Hz, 1H), 4.59 (d, J = 3.8 Hz, 1H), 3.52−3.45 (m, 1H),


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3.42−3.37 (m, 1H), 2.02−1.98 (m, 2H), 1.86−1.82 (m, 2H), 1.40−1.15 (m, 4H). HRMS (ESI): calcd for C23H25N2O2 [M + H]+ 361.1838, found 361.1905. Purity: 96.53% by HPLC (MeOH/H2O = 90:10). 4-(((1r,4r)-4-hydroxycyclohexyl)amino)-4'-(1H-pyrrol-1-yl)-[1,1'-biphenyl]-3-carboxamide

(70).

Compound 70 was prepared from intermediate 41 (173 mg, 0.66 mmol) according to the general procedures. Light yellow solid. 80 mg, yield: 32.3%. mp﹥250 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.20 (d, J = 7.3 Hz, 1H), 8.03 (s, 1H), 7.94 (s, 1H), 7.76−7.73 (m, 2H), 7.64−7.59 (m, 3H), 7.39 (s, 2H), 7.16 (s, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.28 (s, 2H), 4.54 (d, J = 3.3 Hz, 1H), 3.55−3.47 (m, 1H), 3.38−3.36 (m, 1H), 2.03−2.00 (m, 2H), 1.87−1.83 (m, 2H), 1.41−1.17 (m, 4H). HRMS (ESI): calcd for C23H26N3O2 [M + H]+ 376.1947, found 376.2013. Purity: 99.22% by HPLC (MeOH/H2O = 90:10). General procedures for the synthesis of compounds 71−78 Intermediate 25 was dissolved into 5 mL DMSO, different amines (4.5−10 eq) and DIPEA (9.0 eq) were added. The mixture was heated at 120 °C for 24 h and then poured into 20 mL water and extracted with 20 mL EA for two times. The EA layer was combined and dried over anhydrous Na2SO4. After concentration, the residue was dissolved into 20 mL ethanol, 2 mL 1M NaOH solution and 2 mL 30% H2O2 were added. Then the mixture was stirred at 30 °C overnight. After removing ethanol under reduced pressure, the residue was dissolved into 20 mL EA and washed with water (20 mL × 2) and brine (20 mL × 1). The EA layer was dried over anhydrous Na2SO4 and then concentrated. The residue was triturated with DCM to afford compounds 71−78. 4-(cyclohexylamino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide (71). Compound 71 was prepared from intermediate 25 (150 mg, 0.63 mmol) and cyclohexylamine (279 mg, 2.82 mmol) according to the general procedures. Light yellow solid, 75 mg, yield: 35.4%. mp 197−198 °C. 1H NMR (300 MHz,



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Chloroform-d) δ 7.78 (d, J = 7.6 Hz, 1H), 7.49−7.44 (m, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.18 (s, 2H), 6.71 (d, J = 8.7 Hz, 1H), 5.58 (s, 2H), 3.38−3.28 (m, 1H), 2.93−2.80 (m, 1H), 1.98−1.94 (m, 2H), 1.74−1.69 (m, 2H), 1.57−1.49 (m, 2H), 1.39−1.26 (m, 4H), 1.21 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C22H29N2O [M + H]+ 337.2202, found 337.2279. Purity: 97.80% by HPLC (MeOH 100%). 4-(isobutylamino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide (72). Compound 72 was prepared from intermediate 25 (150 mg, 0.63 mmol) and isobutylamine (461 mg, 6.3 mmol) according to the general procedures. Light yellow solid, 107 mg, yield: 54.8%. mp 166−168 °C. 1H NMR (300 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.60−7.56 (m, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.28 (s, 2H), 6.81 (d, J = 8.6 Hz, 1H), 5.74 (s, 2H), 3.05 (d, J = 6.8 Hz, 2H), 3.01−2.91 (m, 1H), 2.08−1.95 (m, 1H), 1.30 (d, J = 7.0 Hz, 6H), 1.05 (d, J = 6.6 Hz, 6H). HRMS (ESI): calcd for C20H27N2O [M + H]+ 311.2045, found 311.2116. Purity: 95.00% by HPLC (MeOH/H2O = 90:10). 4-(cyclopentylamino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide (73). Compound 73 was prepared from intermediate 25 (150 mg, 0.63 mmol) and cyclopentylamine (240 mg, 2.82 mmol) according to the general procedures. White solid, 108 mg, yield: 53.2%. mp 169−171 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.50−7.47 (m, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.18 (s, 2H), 6.75 (d, J = 8.6 Hz, 1H), 5.62 (s, 2H), 3.83−3.75 (m, 1H), 2.91−2.82 (m, 1H), 2.02−1.92 (m, 2H), 1.76−1.66 (m, 2H), 1.60−1.48 (m, 4H), 1.21 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C21H27N2O [M + H]+ 323.2045, found 323.2122. Purity: 98.55% by HPLC (MeOH/H2O = 90:10). 4-((cyclohexylmethyl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide (74). Compound 74 was prepared from intermediate 25 (150 mg, 0.63 mmol) and cyclohexylmethanamine (319 mg, 2.82 mmol) according to the general procedures. Light yellow solid, 80 mg, yield: 36.3%. mp 248−249 °C. 1H NMR


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(300 MHz, DMSO-d6) δ 8.27 (t, J = 5.5 Hz, 1H), 8.00 (s, 1H), 7.87 (d, J = 2.3 Hz, 1H), 7.56 (d, J = 8.3 Hz, 3H), 7.26 (d, J = 8.2 Hz, 2H), 7.15 (s, 1H), 6.73 (d, J = 8.7 Hz, 1H), 3.00 (t, J = 6.1 Hz, 2H), 2.94−2.85 (m, 1H), 1.79−1.50 (m, 7H), 1.25−1.18 (m, 8H), 1.04−0.96 (m, 2H). HRMS (ESI): calcd for C23H31N2O [M + H]+ 351.2358, found 351.2436. Purity: 98.19% by HPLC (MeOH 100%). 4'-isopropyl-4-((2-methoxyethyl)amino)-[1,1'-biphenyl]-3-carboxamide (75). Compound 75 was prepared from intermediate 25 (150 mg, 0.63 mmol) and 2-methoxyethylamine (212 mg, 2.82 mmol) according to the general procedures. Light yellow solid, 118 mg, yield: 60.0%. mp 169−170 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.62−7.58 (m, 2H), 7.47 (d, J = 8.9 Hz, 2H), 7.28 (s, 2H), 6.88 (d, J = 8.6 Hz, 1H), 5.80 (s, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.46−3.42 (m, 5H), 3.00−2.91 (m, 1H), 1.30 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C19H25N2O2 [M + H]+ 313.1838, found 313.1913.

13C

NMR (75

MHz, DMSO-d6) δ 171.98, 149.28, 146.63, 138.03, 130.93, 127.50, 127.06, 126.48, 126.12, 114.94, 112.06, 70.99, 58.54, 42.42, 33.51, 24.40. Purity: 96.31% by HPLC (MeOH/H2O = 90:10). (S)-4'-isopropyl-4-((tetrahydrofuran-3-yl)amino)-[1,1'-biphenyl]-3-carboxamide (76). Compound 76 was prepared from intermediate 25 (150 mg, 0.63 mmol) and (S)-3-Aminotetrahydrofuran hydrochloride (348 mg, 2.82 mmol) according to the general procedures. White solid, 10 mg, yield: 4.9%. mp 224−226 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.23 (d, J = 6.6 Hz, 1H), 7.96 (s, 1H), 7.80 (d, J = 2.2 Hz, 1H), 7.50−7.47 (m, 3H), 7.19−7.14 (m, 3H), 6.68 (d, J = 8.7 Hz, 1H), 4.08−4.00 (m, 1H), 3.85−3.80 (m, 1H), 3.77−3.62 (m, 2H), 3.43 (dd, J = 8.8, 3.2 Hz, 1H), 2.85−2.76 (m, 1H), 2.23−2.11 (m, 1H), 1.69−1.59 (m, 1H), 1.13 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C20H25N2O2 [M + H]+ 325.1838, found 325.1915. Purity: 96.57% by HPLC (MeOH/H2O = 90:10). 4'-isopropyl-4-((tetrahydro-2H-pyran-4-yl)amino)-[1,1'-biphenyl]-3-carboxamide (77). Compound



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77 was prepared from intermediate 25 (150 mg, 0.63 mmol) and 4-aminotetrahydropyran (285 mg, 2.82 mmol) according to the general procedures. White solid, 83 mg, yield: 40.0%. mp 198−199 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.16 (d, J = 7.6 Hz, 1H), 7.94 (s, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.47 (d, J = 7.8 Hz, 3H), 7.17 (d, J = 8.3 Hz, 2H), 7.11 (s, 1H), 6.75 (d, J = 8.8 Hz, 1H), 3.77−3.73 (m, 2H), 3.58−3.49 (m, 1H), 3.38 (t, J = 10.3 Hz, 2H), 2.85−2.76 (m, 1H), 1.87−1.83 (m, 2H), 1.35−1.22 (m, 2H), 1.13 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C21H27N2O2 [M + H]+ 339.1994, found 339.2073. 13C NMR (75 MHz, DMSO-d6) δ 171.66, 147.73, 146.15, 137.52, 130.47, 127.21, 126.55, 125.95, 125.64, 114.18, 112.21, 65.57, 47.04, 33.02, 32.82, 23.90. Purity: 97.83% by HPLC (MeOH 100%). 4-((1-acetylpiperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide (78). Compound 78 was prepared from intermediate 25 (150 mg, 0.63 mmol) and 1-acetyl-4-aminopiperidine (401 mg, 2.82 mmol) according to the general procedures. Light yellow solid, 50 mg, yield: 20.9%. mp 203−204 °C. 1H

NMR (300 MHz, DMSO-d6) δ 8.18 (d, J = 7.9 Hz, 1H), 7.96 (s, 1H), 7.80 (d, J = 2.3 Hz, 1H), 7.47

(d, J = 8.2 Hz, 3H), 7.17 (d, J = 8.0 Hz, 2H), 7.12 (s, 1H), 6.77 (d, J = 8.9 Hz, 1H), 4.04−3.99 (m, 1H), 3.66−3.56 (m, 2H), 3.16 (t, J = 11.1 Hz, 1H), 2.89−2.76 (m, 2H), 1.92 (s, 3H), 1.90−1.82 (m, 2H), 1.33−1.20 (m, 2H), 1.13 (d, J = 6.8 Hz, 6H). HRMS (ESI): calcd for C23H30N3O2 [M + H]+ 380.226, found 380.2335.

13C

NMR (75 MHz, DMSO-d6) δ 171.65, 168.01, 147.79, 146.17, 137.51, 130.50,

127.21, 126.56, 126.03, 125.66, 114.22, 112.27, 47.78, 44.21, 33.01, 32.11, 31.45, 23.90, 21.28. Purity: 96.16% by HPLC (MeOH/H2O = 90:10) 4'-isopropyl-4-(piperidin-4-ylamino)-[1,1'-biphenyl]-3-carboxamide (79) Intermediate 25 (1.67 g, 7.0 mmol) was dissolved into 5 mL DMSO, 4-amino-1-Boc-piperidine (6.3 g, 31.5 mmol) and DIPEA (11 ml, 63 mmol) were added. The mixture was heated at 120 °C for 24 h and


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then poured into 60 mL water and extracted with 60 mL EA for two times. The EA layer was combined and dried over anhydrous Na2SO4. After concentration, the residue was dissolved into 60 mL ethanol, 6 mL 1M NaOH solution and 6 mL 30% H2O2 were added. Then the mixture was stirred at 30 °C overnight. After removing ethanol under reduced pressure, the residue was dissolved into 60 mL EA and washed with water (60 mL × 2) and brine (60 mL × 1). After concentration, the residue was dissolved into 120 mL DCM and TFA (3.9 mL, 52.6 mmol) was added, the mixture was stirred at room temperature for 48 h. After completely reacted, the mixture was concentrated and the residue was dispersed into 50 mL water. Then adjusted the PH value to 9−10, light yellow solid precipitated and filtered to afford compound 79. Light yellow solid, 1.3 g, yield: 55.1%. mp 216−218 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.13 (d, J = 7.9 Hz, 1H), 7.93 (s, 1H), 7.78 (d, J = 2.2 Hz, 1H), 7.48−7.45 (m, 3H), 7.17 (d, J = 8.0 Hz, 2H), 7.09 (s, 1H), 6.70 (d, J = 8.8 Hz, 1H), 3.44−3.37 (m, 2H), 2.88−2.75 (m, 3H), 2.57−2.50 (t, J = 11.2 Hz, 2H), 1.85−1.81 (m, 2H), 1.24−1.17 (m, 2H), 1.13 (d, J = 6.9 Hz, 6H). HRMS (ESI): calcd for C21H28N3O [M + H]+ 338.2154, found 338.2232. Purity: 96.43% by HPLC (MeOH 100%). General procedures for the synthesis of compounds 80−86 Compound 79 was dissolved into 5 mL DMF, various Boc-protected amino acids (1.5 eq), BOP (1.5 eq), DIPEA (3 eq) were added. The mixture was stirred at room temperature for 5 h. Then the mixture was concentrated and re-dissolved into 20 mL DCM, washed with water (20 mL × 2) and brine (20 mL × 1). The DCM layer was dried over anhydrous Na2SO4, and then purified by silica gel chromatography (PE/EA, 5:1～2:1) to afford a white solid. The white product was dissolved into 20 mL DCM, TFA was added and the mixture was stirred at room temperature for 36 h. The mixture was dispersed into 20 mL


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saturated Na2CO3 solution and 20 mL EA. After stirring for a while, the EA layer was separated and washed with water (20 mL × 3) and brine (20 mL × 1). The EA layer was dried over anhydrous Na2SO4, and then purified by silica gel chromatography (DCM/MeOH, 50:1～30:1) to afford compounds 80−86. 4-((1-(2-aminoacetyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide

(80).

Compound 80 was prepared from compound 79 (120 mg, 0.36 mmol) and Boc-glycine (95 mg, 0.54 mmol) according to the general procedures. White solid, 50 mg, yield: 35.2%. mp 110−112 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.28 (d, J = 7.2 Hz, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.57 (d, J = 8.0 Hz, 3H), 7.27 (d, J = 8.2 Hz, 3H), 6.87 (d, J = 8.8 Hz, 1H), 4.17−4.12 (m, 1H), 3.69−3.64 (m, 2H), 3.50−3.44 (m, 2H), 3.23−3.15 (m, 1H), 3.03−2.96 (m, 1H), 2.94−2.85 (m, 1H), 1.99−1.96 (m, 2H), 1.41−1.31 (m, 2H), 1.22 (d, J = 6.7 Hz, 6H). HRMS (ESI): calcd for C23H31N4O2 [M + H]+ 395.2369, found 395.2446. Purity: 97.83% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N). 4-((1-(2-aminopropanoyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide

(81).

Compound 81 was prepared from compound 79 (120 mg, 0.36 mmol) and N-Boc-DL-alanine (102 mg, 0.54 mmol) according to the general procedures. Light yellow solid, 63 mg, yield: 42.9%. mp 106−108 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.94 (s, 1H), 7.53 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.18 (s, 2H), 6.70 (d, J = 8.8 Hz, 1H), 5.81 (brs, 2H), 4.17 (brs, 1H), 3.81−3.60 (m, 3H), 3.22−3.07 (m, 2H), 2.90−2.81 (m, 1H), 2.19 (brs, 2H), 2.00−1.97 (m, 2H), 1.50 (brs, 2H), 1.21−1.19 (m, 9H). HRMS (ESI): calcd for C24H33N4O2 [M + H]+ 409.2525, found 409.2603. Purity: 96.34% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N). 4-((1-(3-aminopropanoyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide

(82).

Compound 82 was prepared from compound 79 (120 mg, 0.36 mmol) and Boc-beta-alanine (102 mg,


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0.54 mmol) according to the general procedures. Light yellow solid, 30 mg, yield: 20.4%. mp 84−86 °C. 1H

NMR (300 MHz, DMSO-d6) δ 8.28 (d, J = 8.1 Hz, 1H), 8.07 (s, 1H), 7.89 (s, 1H), 7.57 (d, J = 7.7 Hz,

3H), 7.27 (d, J = 8.0 Hz, 3H), 6.87 (d, J = 8.8 Hz, 1H), 4.18−4.13 (m, 1H), 3.78−3.68 (m, 2H), 3.28−3.21 (m, 2H), 3.01−2.92 (m, 1H), 2.87 (t, J = 6.4 Hz, 2H), 2.59−2.55 (m, 2H), 1.97 (brs, 2H), 1.44−1.25 (m, 2H), 1.22 (d, J = 6.7 Hz, 6H). HRMS (ESI): calcd for C24H33N4O2 [M + H]+ 409.2525, found 409.2600. Purity: 96.70% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N). 4-((1-(2-amino-3-methylbutanoyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamid e (83). Compound 83 was prepared from compound 79 (120 mg, 0.36 mmol) and N-Boc-DL-valine (117 mg, 0.54 mmol) according to the general procedures. White solid, 58 mg, yield: 36.9%. mp 94−96 °C. 1H

NMR (300 MHz, Chloroform-d) δ 8.07−8.00 (m, 1H), 7.62 (s, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.46 (d,

J = 8.0 Hz, 2H), 7.28 (s, 2H), 6.81 (d, J = 8.7 Hz, 1H), 5.99 (brs, 2H), 4.32−4.27 (m, 1H), 3.86−3.82 (m, 1H), 3.71−3.66 (m, 2H), 3.38−3.13 (m, 2H), 3.01−2.91 (m, 1H), 2.42 (brs, 2H), 2.09 (brs, 2H), 1.97−1.88 (m, 1H), 1.68−1.50 (m, 2H), 1.31−1.27 (m, 6H), 1.04−0.90 (m, 6H). HRMS (ESI): calcd for C26H37N4O2 [M + H]+ 437.2838, found 437.2917. Purity: 97.88% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N). (S)-4-((1-(2-amino-4-methylpentanoyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carbox amide (84). Compound 84 was prepared from compound 79 (120 mg, 0.36 mmol) and Boc-L-leucine (125 mg, 0.54 mmol) according to the general procedures. White solid, 48 mg, yield: 29.6%. mp 95−97 °C. 1H NMR (300 MHz, Chloroform-d) δ 8.07−8.02 (m, 1H), 7.61 (s, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.28 (s, 2H), 6.81 (d, J = 8.7 Hz, 1H), 5.94 (brs, 2H), 4.28−4.24 (m, 1H), 3.90−3.83 (m, 1H), 3.70 (brs, 1H), 3.36−3.13 (m, 2H), 3.00−2.91 (m, 1H), 2.32 (brs, 2H), 2.16−2.07 (m, 2H), 1.90



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(brs, 1H), 1.68−1.53 (m, 2H), 1.48−1.35 (m, 2H), 1.31−1.27 (m, 6H), 0.98−0.85 (m, 6H). HRMS (ESI): calcd for C27H39N4O2 [M + H]+ 451.3002, found 451.3074. Purity: 97.67% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N). 4-((1-((2S,3S)-2-amino-3-methylpentanoyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-car boxamide (85). Compound 85 was prepared from compound 79 (120 mg, 0.36 mmol) and Boc-L-isoleucine (125 mg, 0.54 mmol) according to the general procedures. White solid, 40 mg, yield: 24.7%. mp 102−104 °C. 1H NMR (300 MHz, Chloroform-d) δ 8.07−8.00 (m, 1H), 7.61−7.56 (m, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.28 (s, 2H), 6.80 (d, J = 8.7 Hz, 1H), 5.95 (brs, 2H), 4.28 (brs, 1H), 3.87−3.84 (m, 1H), 3.72−3.68 (m, 2H), 3.38−3.14 (m, 2H), 3.00−2.91 (m, 1H), 2.29 (brs, 2H), 2.09 (brs, 2H), 1.65−1.55 (m, 3H), 1.29 (d, J = 6.9 Hz, 6H), 1.19−1.11 (m, 2H), 1.00−0.85 (m, 6H). HRMS (ESI): calcd for C27H39N4O2 [M + H]+ 451.3000, found 451.3071. Purity: 97.03% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N). 4-((1-(6-aminohexanoyl)piperidin-4-yl)amino)-4'-isopropyl-[1,1'-biphenyl]-3-carboxamide

(86).

Compound 86 was prepared from compound 79 (665 mg, 1.97 mmol) and Boc-6-aminohexanoic acid (683 mg, 2.96 mmol) according to the general procedures. Light yellow solid, 631 mg, yield: 71.1%. mp﹥250 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.27 (d, J = 7.7 Hz, 1H), 8.08 (s, 1H), 7.91 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.20 (brs, 1H), 6.87 (d, J = 8.9 Hz, 1H), 6.30 (brs, 2H), 4.15−4.12 (m, 1H), 3.80−3.67 (m, 2H), 3.27−3.19 (m, 2H), 2.96−2.91 (m, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.33 (s, 2H), 1.97 (brs, 2H), 1.50−1.48 (m, 3H), 1.32 (s, 3H), 1.23 (d, J = 7.0 Hz, 6H). HRMS (ESI): calcd for C27H39N4O2 [M + H]+ 451.2995, found 451.3067. Purity: 95.78% by HPLC (MeOH/H2O = 90:10, 1‰ Et3N).


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ASSOCIATED CONTENT Supporting Information The detailed scoring criteria for DAI (Table S1); Representative images to show the general status of the UC mice at day 7 (Figure S1); 1H, 13C NMR and HRMS (ESI) spectrums of compounds 42−86.

AUTHOR INFORMATION Corresponding Authors * Dr. Xiao-li Xu, Tel/Fax: +86 025 83271216. E-mail address: [email protected] * Prof. Qi-Dong You, Tel/Fax: +86 025 83271351. E-mail address: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the project 81573346, 81502990, 81773639 and 81872737 of National Natural

Science

Foundation

of

China;

2015ZX09101032,

2017ZX09302003

and

2018ZX09711002-003-006 of the National Major Science and Technology Project of China (Innovation and Development of New Drugs); BK 20150691 of the Natural Science Foundation of Jiangsu Province of China; A project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions; CX16B-004HH co-funded by China Pharmaceutical University and Huahai Pharmaceutical Co., Ltd.

ABBREVIATIONS USED



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Hsp90, Heat-shock protein 90; Grp94, glucose regulated protein 94; Trap1, tumor necrosis factor receptor-associated protein-1; ER, endoplasmic reticulum; POAG, primary open-angle glaucoma; UC, ulcerative colitis; SAR, structure-activity relationship; TLRs, Toll-like receptors; IGFs, insulin-like growth factors; FP, fluorescence polarization; CPMG, Carr-Purcell-Meiboom-Gill; MD, molecular dynamic; Hsp70, Heat-shock protein 70; DAI, Disease activity index; PAMPs, pathogen-associated molecular patterns; DIPEA, N,N-Diisopropylethylamine; TFA, trifluoroacetic acid; BGG, bovine gamma-globulin.

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Table of Contents Graphic


Figure 1. Structures and selectivity profiles of the reported Grp94-selective inhibitors. 157x169mm (300 x 300 DPI)


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Figure 2. Structural basis of the Grp94-selective inhibitory activity of PU-class compounds. (A) Compound 2 adopts “Binding mode A” to bind with Hsp90α (PDB: 3O0I). Phe138 is shown as a pink stick. (B) Compound 2 adopts “Binding mode B” to bind with Grp94 (PDB: 3O2F). Phe199 is shown as a gray stick. (C) ATP binding pocket of Apo-Grp94N (PDB: 1YT2). Phe199 is shown in blue stick. (D) Superimposition of the 2:Grp94 complex and Apo-Grp94N. The “Phe199 shift” effect is indicated by a small magenta arrow, and the conformational change in Grp94 (H5 helix rearrangement) is indicated by a large magenta arrow. 119x105mm (300 x 300 DPI)



Figure 3. Design strategies for Grp94-selective inhibitors. (A) Benzamide Hsp90 inhibitor 11 adopts “Binding mode A” to bind with Grp94 (PDB: 4NH9). Compound 11 is shown as a pink stick. (B) Superposition of the 11:Grp94 complex and 2:Grp94 complex. Compound 2 is shown as a yellow stick, the meta position to the carbamoyl group in 11 is indicated by a red arrow, and the “Phe199 shift” is indicated by a magenta arrow. (C) Predicted binding mode of compound 42. Compound 42 and the residues in the Grp94 active site are shown as green and gray sticks, respectively. Conserved water molecules are shown as red spheres, and Hbonds are indicated by magenta dashed lines. (D) Superimposition of the 42:Grp94 complex and 2:Grp94 complex. 170x107mm (300 x 300 DPI)


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Figure 4. Predicted binding mode of compound 54. The Protein structure of Grp94 was extracted from PDB: 3O2F. Active site of Grp94 is surfaced in the hydrophobic state, compound 54 and the surrounded residues are shown as green and gray sticks, respectively. Conserved water molecules are shown as red spheres, and H-bonds are indicated by magenta dashed lines. 170x88mm (300 x 300 DPI)



Figure 5. Selectivity of compound 54 to Grp94 and Hsp90α. (A) The inhibition curves of compound 54 to inhibit Grp94 and Hsp90α. The mP values for negative control and blank control in the determination of Hsp90α are 428 and 318, respectively. The results are shown as mean ± SD, n = 3. (B) BLI dose−response curves reflecting the direct binding of 54 to Grp94. (C) BLI dose−response curves reflecting the direct binding of 54 to Hsp90α. (D) NMR-based CPMG determination of direct binding of 54 to Grp94. NMR spectra of 54 at different concentrations of Grp94N are shown in different colors. 139x224mm (300 x 300 DPI)


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Figure 6. MD simulation to analyze the structural basis of Grp94-selective inhibition. (A) ATP binding pocket of Apo-Grp94N (PDB: 1YT2), Phe199 is shown in blue stick. (B) MD simulation results (20 ns) of the compound 2 removed Grp94N (PDB: 3O2F), Phe199 is shown in gray stick. (C) Superimposition of A and B. (D) ATP binding pocket of compound 2 bound Grp94N (PDB: 3O2F), Phe199 and compound 2 are shown in green and yellow sticks, respectively. (E) MD simulation results (20 ns) of the 54:Grp94N docking complex, Phe199 and compound 54 are shown in pink and navy blue sticks, respectively. (F) Superimposition of D and E. 170x114mm (300 x 300 DPI)



Figure 7. Western blot analysis of some biomarkers in 54 treated panc-1 cells. (A) Integrin α2 and integrin αL levels in cell surface protein extracts after treatment with 54 at indicated concentrations for 36 h. (B) Hsp70 and Akt levels in total protein extracts after treatment with 54 at indicated concentrations for 36 h. AT13387 (AT) is used as the reference. β-actin is used as the control for protein loading. 140x31mm (300 x 300 DPI)


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Figure 8. Anti-inflammatory efficacy of compound 54 in DSS-induced UC mice (n = 8). (A) Body weight changes of the mice during treatment. (B) DAI score changes during treatment. (C) Colon length of each group at day 7. (D) Representative H&E images of the colon samples. Scale bar, 200 μm. (E) TNFα and IL-6 levels in the serum and colonic tissues of each group of mice (n = 5, 5 samples were analyzed). *p < 0.05; **p < 0.01; ***p < 0.001, compared with the “Disease Control” group. (F) Western blot analysis of the biomarkers in colonic tissues. β-actin is used as the control for protein loading.



Figure 9. Subacute toxicity evaluation of compound 54 in healthy C57BL/6 mice (n = 8). The mice were treated with vehicle or three different dosages of compound 54. (A) Body weight change of each group during the treatment. (B) H&E staining analysis for the different organs of the mice in each group. Scale bar, 50 μm. 119x158mm (300 x 300 DPI)


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Table of Contents Graphic 77x55mm (300 x 300 DPI)


Discovery of a Potent Grp94 Selective Inhibitor with Anti-inflammatory

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