Discovery, Optimization, and Target Identification of Novel Potent

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Discovery, Optimization and Target Identification of Novel Potent Broad-Spectrum Antiviral Inhibitors Yiqing Yang, Lin Cao, Hongying Gao, Yue Wu, Yaxin Wang, Fang Fang, Tianlong Lan, Zhiyong Lou, and Yu Rao J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00091 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Journal of Medicinal Chemistry

Discovery, Optimization and Target Identification of Novel Potent Broad-Spectrum Antiviral Inhibitors Yiqing Yang†, §, #, Lin Cao‡, #, Hongying Gao†, §, #, Yue Wu†, Yaxin Wang⊥, Fang Fang†, Tianlong Lan†, Zhiyong Lou&, *, Yu Rao†, * †MOE

Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, MOE Key

Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, P.R. China. §Tsinghua

University-Peking University Joint Center for Life Sciences, Beijing 100084, P.R.

China. ‡College

of Life Sciences, Nankai University, Tianjin 300071, P.R. China.

⊥College

&School

of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei 050024, P.R. China.

of Medicine and Collaborative Innovation Center of Biotherapy, Tsinghua University,

Beijing 100084, P.R. China.

ABSTRACT: Viral Infections are increasing and probably long-lasting global risks. In this study, a chemical library was exploited by phenotypic screening to discover new antiviral inhibitors. After optimizations from hit to lead, a novel potent small molecule (RYL-634) was identified, showing excellent broad-spectrum inhibition activity against various pathogenic viruses, including

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hepatitis c virus (HCV), dengue virus (DENV), zika virus (ZIKV), Chikungunya virus (CHIKV), enterovirus 71 (EV71), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV) and others. The mechanism of action and potential targets of RYL-634 were further explored by the combination of activity-based protein profiling (ABPP) and other techniques. Finally, human dihydroorotate dehydrogenase (HsDHODH) was validated as the major target of RYL-634. We did not observe any mutant resistance under our pressure selections with RYL-634, and it had strong synergistic effect with some FDA-approved drugs. Hence, there is great potential for developing new broad-spectrum antivirals based on RYL-634.

INTRODUCTION Virus infection seriously threatens global health. For example, HCV, which can be cured now, still annually infects over 185 million people and causes up to 500,000 deaths1; the infection numbers of two other flaviviruses, DENV and ZIKV, are approximately 390 million around the world and 4 million in Brazil alone, respectively2-3. Since neither specific antiviral drugs nor vaccines are available for newly emerging viruses, including DENV and ZIKV, related diseases result in heavy social burdens4-5. Although the research community has made great efforts to discover therapeutic small molecules for coping with such emergencies6-8, novel potent molecules have been rarely reported. The majority of reported effective agents in the literature are nucleotides or known antivirals. To fight against future viral epidemics, which may emerge from known, currently unknown or neglected viruses, broad-spectrum antiviral agents are particularly needed. According to the mechanisms of action, antiviral reagents can be divided into virus-targeting antivirals (VTAs) and host-targeting antivirals (HTAs)9-11. Although VTAs are more virusspecific, they can relatively easily generate resistant mutants12-14. The allure of HTAs is their


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broad-spectrum antiviral activity. This activity is particularly attractive for some viral emergencies, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), the influenza virus, the Ebola virus and the recent ZIKV outbreaks because a broad-spectrum agent allows for immediate treatment without a lead time being required to develop a specific therapy15. HTAs could also provide therapeutic opportunities for niche indication in cases when the patient population is not large enough to provide an economic incentive for the development of virusspecific drugs but when the medical need is acute15. Although the discovery of HTAs has attracted much attention13, 15-16, ribavirin is the only approved host-targeting antiviral small molecule drug, and new HTAs are much needed13, 17. In the process of drug discovery, compounds discovered from phenotypic assays on the cellular level may be more likely to be efficacious than target-based approaches in the following preclinical or clinical studies18. There has been a resurgence of phenotypic drug discovery in recent years, which evades the incompletely understood complexity of diseases and does not rely on knowledge of a specific target or its role in a disease19. However, the mechanisms behind biologically active molecules, which are usually first-in-class drugs from phenotypic screens, urgently need to be revealed. Although various methods for targets identification, such as ABPP2021,

reverse docking22, genome-wide RNAi or CRISPR-Cas923, proteome microarray24 and so on25-

26,

have been developed to solve this problem, target identification is still a challenge.

Here, we report the discovery of RYL-634 (compound 1, Figure 1) via a phenotypic screen, which is a novel potent broad-spectrum and host-targeting antiviral small molecule against HCV, DENV, ZIKV, CHIKV, EV71, HIV, Middle East respiratory syndrome coronavirus (MERS-CoV), RSV, severe fever with thrombocytopenia syndrome virus (SFTSV) and the influenza virus. Its mechanism of action and a target study are also described here.


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RESULTS AND DISCUSSION Discovery and optimization of RYL-634 as a novel antiviral agent. In our previous project of developing antimalarial PfNDH2 inhibitors, we constructed a mini focused library consisting of ~200 biaryl-substituted quinolones27. Quinolone is a privileged structure in medicinal chemistry that behaves many biological activities, but this kind of biaryl-substituted quinolone was never reported as antiviral. Considering the current antiviral status mentioned above, we initiated an antiviral drug discovery study via screening the library in our anti-HCV assays. First, the compounds were screened by a high-content fluorescence microscope with a green fluorescent protein (GFP)-fused HCV to rapidly find hit compounds (Figure 1A). Then, the hits were quantitatively validated by RT-PCR or the luciferase reporter method. Once the EC50 values were precisely calculated, the structure-activity relationship (SAR) was summarized and instructed us to design and synthesize new molecules for hit optimization. Serendipitously, some active compounds were found from this mini library which exhibited the advantage of privileged structure in drug discovery. Compound RYL-552S (compound 2, Figure 1B and Figure S1) stood out in the screening (EC50 = 0.39 μM) (Table 1) and was used as the hit compound for the next optimization process. To synthesize the target compounds, various 1-biarylpropan-1-ones were constructed via Suzuki cross-coupling. These intermediates were then condensed and cyclized with compound 64 under acidic condition to generate the target compounds (Scheme 1, Table 1). The EC50 value dropped to 1.56 μM for the similar analogue RYL-552 (compound 3) when the sulfur atom was merely replaced by the oxygen atom. It indicated that the -SCF3 group may play an important role in the biological activity. Therefore, some alternatives to -SCF3 were introduced into the molecules. (Figure 1B, Table 1). We first used -OCH2CF3 to probe the possible modifications (compound 5, Table 1). The insertion of methylene


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between the oxygen and -CF3 made it more potent than compound 3. Further methylation and hydroxylation on the methylene improved the potency (compound 6), but it decreased largely when only hydroxy group was kept on the methylene (compound 7). It seemed that some hydrophobic groups like methyl groups would be good for the activity (compare compound 3 with 5, 6 and 7). To verify the above hypothesis, we oxidized the sulfur atom of RYL-552S (3) to generate more polar molecules like sulfoxide, sulfone and sulfoximines (compounds 8-11). All these modifications interfered the anti-HCV activity (compare compound 2 with 8-11) which indicated that the better potency of RYL-552s compared with RYL-552 resulted from the hydrophobic property of the sulfur atom. Thus, compound 4 (Figure 1B) with substitution by a hydrophobic t-butyl group was synthesized and was found to inhibit viral infection with similar potency. Obviously, the -CF3 group was not essential for keeping the antiviral activity and it could be replaced by hydrophobic groups. We also tried to simplify the substitution with -OCHF2, -SF5, -Cl and -OMe (compounds 12-15) as -SCF3 alternatives. These smaller functional groups didn’t give good results possibly due to the unsuitable distance or orientation to the residues in the target. Similarly, the introduction of constrained 6-member rings morpholine and piperidine gave bad results too (compounds 16 and 17). When the -F and -O- groups was used to replace the -H on benzene ring and the methylene between the di-aryl respectively, we found that compounds 3 and 18 (Table 2) behaved nearly same potency. With the -F and -O- groups remained, these 1-biarylpropan-1-one intermediates can be synthesized more easily via metal-free nucleophilic aromatic substitution reaction (Scheme 1, A). Then some hydrophobic alkyl groups were introduced on the nitrogen atom of the aniline, which led to the discovery of the most potent molecule RYL-634 (Figure 1B, Table 2, compound 1). In our test, the significant antiviral activity of RYL-634 could be observed by fluorescence microscope in a dose-dependent manner (Figure 1C). It had EC50


5


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values of approximately 5 nM from both RT-PCR and luciferase method with PSI-7977 (sofosbuvir, EC50 = 0.17 μM) as the positive control (Figure 1D and 1E). The cell viability was also monitored at the same time (Table 1-2), and the CC50 value of RYL-634 was higher than 2.5 μM. The high selectivity index (SI = CC50/EC50) suggested that the excellent potency of RYL-634 was not due to cytotoxicity. During this round optimizing process, we found again that constrained rings were worse than ring-opened substitutions (compare compound 1 with 19, 20 and 25 in Table 2). The size of substitutions was also important so that modifications with di-ethyl gave the best inhibition (compound 1). If the ethyl group was replaced by smaller methyl or larger propyl (compounds 22-24 and 21), the inhibition significantly became weak.


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Figure 1. Discovery and optimization of novel antiviral small molecules. (A) The workflow for novel antiviral drug discovery in this project. Each compound was screened at 10 μM with the anti-HCV assay, the intensity of the fused-GFP was monitored quickly under a fluorescence microscope. Only compounds with over 50% fluorescence decay were diluted to different concentrations and re-evaluated by qRT-PCR or luciferase reporter method. The calculated IC50 values then guided the structure-activity relationship (SAR) study. (B) RLY-634 was identified after hit optimization. (C) The antiviral activity of RYL-634 was observed by fluorescence microscope. The diagrams in the first line were GFP signals. The diagrams in the second line were stained nucleus. The scale bar is 100 m. (D) The antiviral activity of RYL-634 was confirmed by RT-PCR and luciferase reporter. (E) The antiviral activity of RYL-634 was compared with PSI-7977 (sofosbuvir) by luciferase reporter. Each experiment was repeated three times. Error bars stand for SD.


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Scheme 1. The synthetic routes for target molecules.

F

N F

O O

64

R

NH2

O

77

O

SCF3 HO

O

Pd(dppf)2Cl2.DCM KOAc, DMF

O

Br O

Pd(dppf)2Cl2.DCM, K3PO4, Toluene O

49

When R=-SCF3 Pd(dppf)2Cl2.DCM, K3PO4, Toluene

54

63

NH2 F

AlCl3

F

O CF3

55

N CF3

58

SCF3

O

Cl

O S

S

OMe

59

60

O

53 O

CF3

S

NH CF3

57 O

N

N

61

62 O

O NH2

HO F

R Alkylation reagent O

K2CO3, MeCN

O

K2CO3, DMF

65

CF3

52

56

Cl

OH

SF5

F

51 F

50 CF3

S

R=: O

CF3

OH

O

F

R

Br

B O

35-47

O

F

SCF3

K2CO3, DMF

R

O

O

76

O

O B B

R

48

1.1 NaN3, DMF 1.2 PPh3, THF O

O

NH2

EDCI, DMAP, HOBt Et3N, DMF

O

Br

O

O

HO

SCF3

R

1, 4-25 and 29-34 SCF3

H N

N H

TfOH, n-BuOH

49-63 and 67-80 O

O

F

66 R=:

SCF3 HO K2CO3, DMF O

H N

N

68

69

N

N

70

71

SCF3

NH

O F

67

N

N

N

72

73

74

N

75

O

O

R=:

Alkylation reagent OH

K2CO3, Acetone

O

R

N

78

O

N

N

79

80


8

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Table 1. The anti-HCV activity of compounds with -SCF3 alternatives. F

O R

N H

Compound

R

Activity (EC50, μM)

Toxicity (CC50, μM)

SI

2

OCF3

1.56±0.15

>10

>6

3

SCF3

0.36±0.02

>10

>27

0.29±0.02

>5

>17

0.84±0.04

>5

>6

0.11±0.01

>2.5

>23

3.49±0.21

>10

>3

0.76±0.11

>5

>7

1.76±0.29

>2.5

>2

1.42±0.31

>10

>7

5.39±0.36

>10

>2

1.01±0.11

>5

>5

4 5

O

CF3

OH

6

CF3

OH

7

CF3 O S

8 9 10 11 12

O O

O

S S

S

CF3

O CF3 NH CF3

N CF3

O

F F

13

SF5

0.73±0.07

>5

>7

14

Cl

4.40±0.42

>10

>2

15

OMe

2.17±0.37

>10

>5

O

3.65±0.11

>10

>3

1.92±0.07

>10

>5

16 17

N

N


9


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Table 2. The anti-HCV activity of compounds with hydrophobic alternatives. F

O R

N H O F

Compound

R

18 1 19 20 21 22

SCF3

N

N

N

N

N

23 24 25

N

H N NH N



SI

0.36±0.01

>10

>27

0.006±0.007

>2.5

>416

0.10±0.01

>10

>100

0.035±0.01

>10

>285

0.066±0.01

>10

>151

0.24±0.05

>10

>42

1.05±0.12

>10

>10

0.17±0.02

>10

>58

2.57±0.15

>10

>4

The antiviral profile of RYL-634. With RYL-634 in hand, its antiviral potential was further exploited. In current antiviral therapies, drug combinations or cocktails have been utilized more commonly to avoid or lower the chance of developing drug-resistant mutations28-29. The combination effects of RYL-634 with some FDA-approved antiviral drugs were tested on HCV (Figure 2A). We treated HCVcc-infected cells with various concentrations and with RYL-634 either alone or in combination. The results revealed a concentration-dependent inhibition with all


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of them alone and with any two in combination (Figure 2A). The data were further analyzed using a mathematic model, MacSynergy II (Figure S2), and revealed that RYL-634-boceprevir, RYL634-cyclosporin A, RYL-634-ribavirin and RYL-634-sofosbuvir pairs have strong synergy (CI > 50), whereas the synergistic antiviral effect of RYL-634 with IFN-α, daclatasvir and telaprevir is under moderate (50 > CI >10), according to the suggested criteria. Next, we investigated the working step of RYL-634 in the HCV lifecycle. First, we observed that both HCVcc and HCV subgenomic replicon (HCVrep) are potently inhibited by RYL-634 treatment; however, HCV pseudo-typed virus (HCVpp) activity was not affected in the presence of RYL-634 at a high concentration (Figure 2B). This meant that RYL-634 inhibited HCV at the step of viral replication. Next, we assessed the step that RYL-634 worked on via time-of-addition analysis (Figure 2C). Huh7.5.1 cells were infected with HCVcc at 4 °C for 2 h (T = -2 h). After removing unbound viruses, cells were incubated at 37 °C (T = 0 h), and compounds were added to the infected cells at different time points. We selected the anti-CD81 antibody as a control to represent typical working HCV entry inhibitors. HCV inhibiting effects of the anti-CD81 antibody decreased at the time of the temperature shift. The growth curve of HCV in the presence of RYL-634 indicated that RYL-634 acts on the replication. Further study revealed that RYL-634 is a potent compound against other RNA viruses (Figure 2D). It showed excellent potency against DENV (EC50 = 7 nM), ZIKV (EC50 = 20 nM), EV71 (EC50 = 4 nM) and HIV (EC50 = 13 nM), and obvious inhibition against CHIKV, RSV, SFTSV, MERS-CoV and influenza virus at sub-micromolar concentrations. Thus far, there have been no effective inhibitors discovered for many of these viruses. Although the recent ZIKV outbreak has resulted in some projects for anti-ZIKV and anti-DENV drug discovery, RYL-634 inhibited these viruses as well as, if not better than, the best molecules from those projects7, 30-31. The broad-spectrum antiviral activity indicated that RYL-634 may be a host-


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targeting molecule. If RYL-634 was a viral-targeting molecule, viral strains with resistance against RYL-634 might be generated under the sustained pressure of high concentrations. However, any mutant resistant strain was never observed after 8 rounds of screening in our pressure selections with boceprevir as the control32.

Figure 2. The antiviral profile of RYL-634. (A) The combination of RYL-634 with FDA-approved drugs (IFNα, ribavirin, cyclosporin A, telaprevir, boceprevir, daclatasvir and sofosbuvir). The combination indexes were shown in Figure S2. (B) Inhibitory activities of RYL-634 on HCVcc, HCVpp and HCVrep. (C) The antiviral time course of RYL-634. (D) The broad-spectrum antiviral activity of RYL-634. SI, selectivity index. Each experiment was repeated three times. Error bars stand for SD.

Target identification by ABPP. To explore the potential targets of RLY-634 and understand the mechanism of action at the molecular level, we next focused on target identification. ABPP has


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become a powerful method for target identification20. In a typical ABPP procedure (Figure 3A), a probe with a crosslinker and alkyne tag is first designed and synthesized33. Then, the probe is incubated with live cells in vivo or in cell lysate in vitro to bind with target proteins. Once the covalent binding is triggered by UV, the probe-labeled sample reacts with azide-tagged rhodamine and biotin (Figure 3B) via click chemistry. The results are monitored by in-gel fluorescence and LC-MS/MS34. ABPP may be quite suitable for the target identification of RYL-634 because our SAR study has indicated that some hydrophobic functional groups are essential for its biological activity (Figure 1B, Table 1-2). As illustrated in Scheme 2, the terminal alkyne-containing diazirine photocrosslinker could be used as a hydrophobic functional group to replace the alkyl chain in RYL-634. Compound 87 was used as the positive probe (EC50 = 8 nM on HCV). When a methylation reaction occurred on the oxygen atom of compound 87, the quinolone ring became a quinoline. This change made compound 88 lose the most potency and serve as an ideal negative control (EC50 > 5 μM on HCV), which also indicated that the quinolone scaffold is vital for the antiviral activity. In the following ABPP studies, we firstly observed the labeling position via ingel fluorescence scanning for different probe concentrations (Figure 3C and S3). Three experimental groups were set up for each concentration in which the positive probe, negative probe and competitor (RYL-634) were studied. After optimizations, the labeling process can be efficiently conducted with a probe concentration between 1.0 and 10.0 μM. In the first pull-down experiment, 833 proteins were identified that bound with the positive probe more than the negative or DMSO control (Figure 3D and Table S1). If a threshold (positive/negative ratio of >3 and positive/DMSO of >5) was set to sort these proteins, the amount could be narrowed down to 290 proteins over it (Figure 3D and Table S1). Interestingly, only human proteins were found in the proteomic data, despite the presence of viral components in the pull-down experiment. Both this


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point and the broad-spectrum antiviral property described above (Figure 2A) strongly suggested that RYL-634 is a host-targeting antiviral molecule. Five proteins (DOCK1, SIAE, DHODH, UTRO and MLC1) occupied the highest region of the P/N ratio in the first experiment (Figure 3D and 3E). However, these candidates were treated with caution because the protein scores in the hazardous group were very low, indicating that their signal may be caused by the stochastic nature of data-dependent acquisition by LC-MS/MS35. In contrast, the abundance of proteins in the conservative group is always high; however, the selectivity (P/N ratio) may not be good. Proteins in that group may be originally abundant in the biological system, and their non-specific binding with the probes may be strong. Then, we further conducted several replicates of the experiment to eliminate random signals and to further enrich the target proteins. In this case, 78 proteins appeared in all three replicates, and another 302 proteins appeared in two of the three replicates (Figure 3F). For the sake of insurance, all the proteins that appeared in at least two replicates were considered in the following study.


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Figure 3. Target identification by activity-based protein profiling (ABPP). (A) The workflow for ABPP. (B) The structures of rhodamine and biotin with azide tag. (C) The in-gel fluorescence of ABPP. Left, in-gel fluorescence scanning. Right, the corresponding Commassie brilliant blue staining of the same gel which indicated the same sample loading. Neg, negative probe. Pos, positive probe. Com, competitive (positive and RYL-634). For the enlarged version, see Figure S3. (D) The detected proteins by LC-MS/MS in the first experiment. (E) Protein distribution in a coordinate system of P/N (positive probe to negative probe) ratio and protein scores. (F). The Venn diagram of three experiments.

Bioinformatic analysis of the potential targets. We performed a bioinformatic analysis next. First, the subcellular distributions of the candidates were concluded from the gene ontology analysis (Figure 4A and Table S1). Most of them were distributed in cytosol (29%), in the nucleus


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(29%) and in the mitochondrion (15%). Some others also dispersed in the extracellular matrix (7%), the endoplasmic reticulum (ER, 6%), the Golgi apparatus (4%), on the cell surface (3%), the proteasome (3%), the ribosome (2%) and the endosome (2%). In the following analysis of the biological process, we found that these candidates were involved in many biological processes, and the representative ones are summarized in Figure 4B, especially the small molecule metabolic process (Table S1). To our delight, some of the enriched proteins participated in the viral process, the viral life cycle and viral transcription, which might be related with the broad-spectrum antiviral activity of RYL-634 (Figure 4B). They correlated with the entire viral life cycle, including viral entry, molecular metabolism, transcription and translation. (Table S2, Figure 4C and 4D)36-37. More importantly, some of them, such as HSP90, HSPA5, IMPDH2, DHODH, lipid-related enzymes and PDI, have been purposed to be or validated as broad-spectrum antiviral targets37-38. RYL-634 had a high chance to act on them. Scheme 2. Design and synthesis of clickable and photoreactive probes. O

O

H N

NH2 EtI, K2CO3, MeCN

O

O F

66

F 1. NaBH4, MeOH, -30oC, 2h 2. I2, imidazole, PPh3, DCM

O HO

81 F

N

O 67

F

N K2CO3, MeCN F

I

OH

O

OH

84

83 F

O

O

O 64

NH2

TfOH, n-BuOH F 1.1 NH3 in MeOH 1.2 NH2OSO3H 1.3 Et3N, I2

O

Dess-Martin, DCM

N

N H F

OH

O 85

F F

O F

O N

N N N

O F

87

Postive probe, EC50=8 nM on HCV

O

O 86

MeI, K2CO3. DMF

N

N H

N

N H

N N

88

Negative probe, EC50>5 M on HCV


16

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Figure 4. Bioinformatic analysis of the potential target proteins. (A) Cellular component analysis of the potential target proteins. (B) Biological process analysis of the potential target proteins. (C) Replication cycle and polyprotein organization of flaviviruses. (D) Some potential target proteins in different viral infection stages were selected by searching literatures. See Table S1 and S2 for details.

Target enrichment by SAR-guided selection in reverse docking. The 3D structures for the most enriched proteins have been provided (Figure S4), which inspired us to also probe the structurebased methods. Reverse docking (Figure 5A) with its low-cost and high throughput is proving to be a useful tool for drug repositioning and target identification39-40. Therefore, some programs or databases, such as TarFishDock41, have been developed. Different scoring functions (DOCK, GOLD, AutoDock, Glide, etc.) have been utilized in reverse docking for protein ranking; however, reverse docking for protein ranking can be strongly influenced by the different scoring functions42.


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Some specific factors should be introduced into the manual selection stage to filter more possible targets from the candidate pool. Since the SAR for RYL-634 has been extensively studied as described above (Figure 1B, Table 1-2) and because SAR represents a unique scaffold feature for bioactive compounds, we envisioned that the consistency between SAR and reverse docking can be used as a filter to specifically enrich the target candidates. In this workflow (Figure 5A), the conformation of RYL-634 with the lowest energy was calculated first; meanwhile, a protein library was constructed from PDTD. The top 120 binding models with Glide scores below -8.0 were further sorted according to SAR. This meant that the binding model should excellently explain the SAR but should not conflict with it. Finally, 20 proteins were enriched by this SAR-guided selection (Figure 5B and Figure S5). We noticed that dihydroorotate dehydrogenase (DHODH) appeared again in the enrichment. Furthermore, its binding model had the best consistency with the SAR. In the binding model (Figure 5C and Figure 5D), the carbonyl group and the fluorine atom on the quinolone ring formed a hydrogen bonding network with R136 and Q47 of DHODH. Accordingly, the potency abated if the fluorine atom was moved to other positions on the quinolone ring (Table 3, compounds 3, 26-28) or if a methyl group was installed on the oxygen atom, which would disrupt these hydrogen bonds (the positive probe 87 vs. negative probe 88). The right benzene ring of RYL-634 was vised by F62 and Y38 via π-π stacking so that the potency was mostly lost when the benzene ring was replaced by some saturated groups (Table 3, compounds 29-31). At the end of RYL-634, the nitrogen atom and the diethyl group contributed to a hydrogen bond with Y38 and hydrophobic interactions around the Leu residues, respectively. This can explain why the diethyl-substituted amine group could be used to replace other hydrophobic groups and give the best activity (Figure 1B, Table 1-2). Since the above described π-π stacking, hydrogen bonding and hydrophobic interactions with DHODH in the model, keeping


18

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a one-atom length, such as an oxygen atom, on the linker between di-aryl was reasonable (Figure 5D). Regardless of the abstraction of an atom or insertion of more atoms, the tandem interactions were disarranged, and the potency decreased (Table 3, compounds 32-34). A similar binding style of the positive probe (Scheme 2, compound 87) with DHODH was also observed from the molecular docking (Figure 5E) by which the low abundance of DHODH in LC-MS/MS may be elucidated (Figure 3E). In the light of the high protein expression level of DHODH in cells (Figure S6), the low abundance may be caused by the low reaction efficiency of the probe with the protein. In detail, the additional terminal alkyne-containing diazirine photocrosslinker in the positive probe (Scheme 2) brought about more hydrophobic interactions with several Leu residues compared with RYL-634 (Figure 5E). The C-H bonds from the Leu residues had a lower reactivity with the carbene intermediate than the polar protic O-H or N-H bonds43; however, the diazirine group was fixed at an orientation toward the solvent exposure by these hydrophobic interactions. The diazirine could rearrange into a diazo compound, and this binding position could make it form an unstable intermediate, which rapidly reacted with nucleophiles including water44. Thus, three clues to DHODH have been collected. First, DHODH was identified by LC-MS/MS in the pull-down experiment. Second, the bioinformatic analysis showed that DHODH is closely related with the viral life cycle (Figure 4D). DHODH supplies pyrimidine for viral replication, and we observed that RYL-634 extensively inhibits the viral replication stage (Figure 2D). Finally, it was enriched again by the SAR-guided selection in the reverse docking (Figure 5B). All these data encouraged us to focus on DHODH in the following study.


19


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Figure 5. Target enrichment by SAR-guided selection in reverse docking. (A) The workflow for reverse docking. (B) The selected proteins after SAR-guided filtration. (C) The binding model of RYL-634 with HsDHODH. (D) The binding model of RYL-634 with HsDHODH was consistent with its SAR very well. (E) The binding model of positive probe 87 with HsDHODH.


20

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Table 3. The anti-HCV activity of compounds with other modifications. Compound

Structure F



SI

0.36±0.02

>10

>27

2.48±0.2

>10

>4

0.97±0.1

>5

>5

1.85±0.32

>5

>3

28.12±1.34

>100

>4

25.56±1.94

>100

>4

16.65±0.9

>100

>6

2.33±0.13

>10

>4

1.59±0.17

>10

>6

11.05±0.88

>50

>5

O

3

SCF3

N H

O

26

F SCF3

N H O

27 F

SCF3

N H O

28 F

F

SCF3

N H

O

29

N H O

N O

F

O

30 N H

N

O

F

O

31 N H

N

O F

O

32

N H SCF3

F

33

O

N H

O SCF3

F

34

O

N H

SCF3 H N O


21


Page 22 of 62

DHODH was validated as a target of RYL-634. DHODH is a flavin-dependent mitochondrial enzyme that catalyzes the oxidation of the intermediate, dihydroorotate, to orotate with coenzyme Q as a cofactor in the de novo biosynthesis of pyrimidine (Figure 6A)45. Both de novo and salvage pathways provide precursors for further synthesis of RNA, DNA, glycoproteins and phospholipids. The inhibition of DHODH has been validated as a promising therapeutic strategy for viral infection, cancer, arthritis and immunosuppression45. To verify if RYL-634 is an inhibitor of DHODH, a chemical rescue experiment was designed. In the hypothesis, the de novo pathway was repressed by RYL-634 by inhibiting DHODH, and the downstream supply of intermediates for viral replication was blocked; then, the viral replication would be rescued if the intermediate is compensated from the salvage pathway. The uridine, a metabolite in the pyrimidine salvage pathway (Figure 6A), was supplemented to the viral infection system, and the antiviral activity of RYL-634 was most significantly reversed (Figure 6B). In the presence of 0.5 mM uridine, the EC50 value of RYL-634 on HCV declined near 200-fold (Figure 6C). This phenomenon was as same as previously reported cases for other DHODH inhibitors46-47. Then, the recombinant DHODH enzyme was purified. In a DARTS study (Figure 6D), a protein might be less susceptible to proteolysis when it is ligand-bound than when it is ligand-free48. RYL-634 protected DHODH from the degradation of pronase in a dose-dependent manner, while GAPDH was mostly degraded. Obviously, RYL-634 can bind with DHODH very well. We also conducted enzymatic activity assays to reveal the functional inhibition of RYL-634 to DHODH. The inhibition of RYL-634 to DHODH was stronger 4-fold than teriflunomide, the only DHODH-targeted immunomodulatory drug approved by the FDA for multiple sclerosis (Figure 6E)49. Accordingly, the positive probe (compound 87) had a better inhibitory effect than the negative probe (compound 88). Although DHODH had been purposed as antiviral target, its inhibitors had been rarely well studied for the


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broad-spectrum antiviral activity. Teriflunomide was purposed to be used as antiviral by targeting DHODH very recently from a genome-wide siRNA screen50, but its antiviral activity of micromolar level was not good enough, while RYL-634 had better antiviral activity, corresponding to its stronger inhibition to DHODH. DHODH was a conserved protein between different species such as human, monkey, dog and mouse (Figure S7), that could explain why the antiviral activity assays from host cells of different species were all good (Figure 2E). All the above results made us believe that the enzymatic activity of RYL-634 can mostly explain its cellular antiviral activity and DHODH should be a major antiviral target of RYL-634. Noteworthy, these biaryl-substituted quinolone compounds have been mainly used as antimalarial PfNDH2 inhibitors in previous studies27,51. Their inhibition on DHODH have never been reported. What’s more, there are no NDH2 or homologous proteins in both human cells and in the virus. RYL-634, a HsDHODH inhibitor, has a completely different mechanism of action with the previous compounds. Although DHODH could be viewed as a major target of RYL-634, these results did not exclude the possibility that besides inhibiting DHODH, it may also exert its antiviral activities through other mechanisms. In parallel, we also attempted to validate other potential targets during this process; however, we only confirmed that RYL-634 has no activity on purified enzymes, including HSP90, PRPS1 and IMPDH2.


23


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Figure 6. DHODH was validated as a target of RYL-634. (A) The de novo and salvage pathways for pyrimidine biosynthesis. (B) The antiviral activity of RYL-634 was reversed by supplementing uridine. (C) The EC50 value of RYL-634 significantly increased in the presence of uridine. (D) DHODH was protected by RYL-634 in the study of DARTS. (E) The inhibition of DHODH activity by teriflunomide, RYL-634, positive probe and negative probe. Each experiment was repeated three times. Error bars stand for SD.

CONCLUSIONS In summary, RYL-634, a novel potent broad-spectrum antiviral small molecule, has been identified by phenotypic screening and validated as a DHODH inhibitor. A detailed structureactivity-relationship (SAR) of these compounds has been discussed. Although some viral infections, such as HCV and HIV, can be controlled by medications, there are still no good choices for many others, such as DENV, ZIKV, CHIKV, and SFTSV. Moreover, viruses usually generate resistances to drugs. The known broad-spectrum antivirals including some DHODH inhibitors usually are not as potent as RYL-634 in cellular assays, or else show serious cytotoxicity which extremely limited their clinical utilization36, 52. For example, brequinar, a strong DHODH inhibitor,


24

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had gained a lot attention in previous but failed in the clinical study due to its side effect and poor solubility53-54. Moreover, the DENV resistance to brequinar via enhancement of polymerase activity was reported55. Most of the DHODH inhibitors including the only FDA-approved teriflunomide behaved very weak antiviral activity that couldn’t be competent for a drug56-57. Some of the DHODH inhibitors contained “soft groups” like labile esters that easily metabolized in vivo and presumably led to poor pharmacokinetic stability46, 58-59. The potent broad-spectrum antiviral activity of RYL-634 with only weak cytotoxicity that does not show detectable resistance in antiHCV assays and its combination with other drugs provide new insights on the development of antivirals that are likely to avoid drug resistance. Considering the importance of pharmacokinetic properties which might lead to failure of previous DHODH inhibitor in vivo46, tremendous efforts for lead optimization to give more drug-like analogues of RYL-634 in the following pre-clinical study are essential. Target identification is a time- and budget-consuming process, especially for some promiscuous molecules. In ABPP, the sensitivity is limited for compounds that exhibit a low binding affinity toward their target or for targets expressed at low levels. In these cases, the target protein can be lost during washing steps, or its binding may be obscured by the presence of highly abundant and non-specific binding proteins60. In the reverse docking, which is limited by the volume of the PDB database, protein preparation and scoring functions, the bias and false positives are always observed. Therefore, combining multiple methods for target identification is helpful for overcoming the corresponding limitations and making the process more direct. DHODH has been validated as an antiviral target in previous studies13,

37, 61-64.

In addition, it can be used as a

therapeutic target for cancer, rheumatoid arthritis, sclerosis, malarial and immunosuppression45, 6566.

RYL-634, a new hit compound for human DHODH, could be used in the development of


25


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therapeutic agents for these conditions. Especially for malarial, this kind of compounds are likely to inhibit PfDHODH too, because the HsDHODH and PfDHODH are near homologues59, 67-68. Since these compounds were originally used as antimalarial PfNDH2 inhibitors27, 51, there is great chance to develop dual inhibitors of PfNDH2 and PfDHODH based on RYL-552 and RYL-634. This work provided some important information for the further mechansim study and potential clinical utilizations of RYL-634 in the future. EXPERIMENTAL SECTION Chemistry. All commercial chemical materials (Energy, Bide, Macklin, J&K Chemical Co. Ltd.) were used without further purification. All solvents were analytical grade. The 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer in CDCl3, CD3OD, or DMSO-d6 using tetramethylsilane (TMS) or solvent peak as a standard. All 13C NMR spectra were recorded with complete proton decoupling. Low resolution mass spectral analyses were performed with a Waters AQUITY UPLCTM/MS. Analytical thin-layer chromatography (TLC) was performed on silica gel F254 plates from Yantai Chemical Industry Research Institute, and flash column chromatography was performed on silica gel (200 − 300 mesh) from Qingdao Haiyang Chemical Co. Ltd. A BUCHI Rotavapor R-3 was used to remove solvents by evaporation. The purities of all the final tested compounds was more than 95%, which were confirmed by NMR or UPLC. Compounds 2, 3 and 26 – 28 were prepared according to the previously reported procedures27. All the other final compounds (compounds 1, 4 – 25, 29 – 34 and 85) were synthesized according to the following synthetic procedure for compound 1, when the corresponding intermediates were replaced. The details for preparing the intermediates were shown in Supporting Information.


26

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2-(4-(4-(Diethylamino)phenoxy)-3-fluorophenyl)-5-fluoro-3-methylquinolin-4(1H)-one (1).To a 10 ml round bottom flask was added 560 mg compound 68 (2 mmol, 1,.0 eq), 400 mg 64 (1.0 eq), 170 μL TfOH (1.0 eq) and 3 mL n-BuOH as solvent. The mixture was refluxed at 130 oC overnight. Compound 1 was obtained after the purification by column chromatography (EtOAc: hexane = 1: 1.5, isolated yield = 25%). To a 10 ml round bottom flask was added 560 mg compound 68 (2 mmol, 1,.0 eq), 400 mg 64 (1.0 eq), 170 μL TfOH (1.0 eq) and 3 mL n-BuOH as solvent. The mixture was refluxed at 130 oC overnight. Compound 1 was obtained after the purification by column chromatography (EtOAc: hexane = 1: 1.5, isolated yield = 25%). 1H-NMR (400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.48 (dd, J = 12.92 Hz, J = 6.92 Hz, 1H), 7.34 (d, J = 8.40 Hz, 1H), 7.29 (d, J = 11.04 Hz, 1H), 6.97 – 6.87 (m, 4H), 6.80 (d, J = 8.28 Hz, 2H), 3.33 (q, J = 7.08 Hz, 4H), 1.98 (s, 3H), 1.13 (t, J = 7.08 Hz, 3H). 13C-NMR (100 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 178.64, 163.09, 160.50, 154.48, 152.00, 148.62, 148.51, 147.93, 146.34, 146.15, 142.39, 142.35, 132.65, 132.55, 129.52, 129.45, 125.91, 125.88, 118.89, 118.28, 118.01, 117.82, 114.51, 114.40, 114.31, 109.81, 109.60, 45.48, 12.68, 12.49. LC-MS: calcd for C26H25F2N2O2 [M+H]+: 435.18, found 435.23. 2-(4-(4-(Tert-butyl)benzyl)phenyl)-5-fluoro-3-methylquinolin-4(1H)-one (4). 1H-NMR (400 MHz, CDCl3, ppm): 11.54 (s, 1H), 7.77 (d, J = 8.44 Hz, 1H), 7.48 – 7.42 (m, 1H), 7.26 (d, J = 8.08 Hz, 2H), 7.03 (d, J = 8.16 Hz, 2H), 6.99 (d, J = 8.44 Hz, 2H), 6.80 (dd, J = 11.64Hz, J = 7.96 Hz, 1H), 3.83 (s, 2H), 1.82 (s, 2H), 1.29 (s, 9H).

13C-NMR

(100 MHz, CDCl3, ppm): 177.59,

162.56, 159.97, 149.19, 148.14, 142.60, 142.08, 142.04, 137.29, 134.82, 132.60, 131.41, 131.31, 128.85, 128.80, 128.71, 125.51, 117.55, 114.71, 114.01, 113.92, 108.88, 108.67, 41.16, 34.49, 31.51, 12.43. LC-MS: calcd for C27H27FNO [M+H]+: 400.20, found 400.23.


27


Page 28 of 62

5-Fluoro-3-methyl-2-(4-(4-(2,2,2-trifluoroethoxy)benzyl)phenyl)quinolin-4(1H)-one (5). 1HNMR (400 MHz, CDCl3, ppm): 11.78 (s, 1H), 7.81 (d, J = 8.28 Hz, 1H), 7.46 – 7.43 (m, 1H), 7.22 (d, J = 7.56 Hz, 2H), 7.00 (d, J = 8.08 Hz, 2H), 6.92 (d, J = 7.48 Hz, 2H), 6.78 (d, J = 7.84 Hz, 2H), 4.26 (q, J = 7.96 Hz, 2H), 3.79 (s, 2H), 1.76 (s, 3H). 13C-NMR (100 MHz, CDCl3, ppm): 177.54, 162.46, 159.87, 156.02, 148.22, 142.52, 142.09, 142.05, 134.45, 132.62, 131.47, 131.36, 130.29, 128.91, 124.6, 122.10, 117.45, 115.11, 114.82, 113.94, 113.85, 108.88, 108.67, 66.53, 66.17, 65.82, 65.47, 40.69, 12.40. LC-MS: calcd for C25H20F4NO2 [M+H]+: 442.14, found 442.22. 5-Fluoro-3-methyl-2-(4-(4-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)phenyl)quinolin4(1H)-on (6). 1H-NMR (400 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 7.51 (d, J = 7.84 Hz, 2H), 7.46 – 7.42 (m, 1H), 7.36 – 7.29 (m, 5H), 7.19 (d, J = 7.80 Hz, 2H), 6.89 – 6.83 (m, 1H), 4.00 (s, 2H), 1.95 (s, 3H), 1.71 (s, 3H). 13C-NMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 177.41, 161.87, 159.28, 148.48, 142.50, 141.20, 141.16, 140.14, 137.34, 131.99, 131.35, 131.25, 129.81, 128.55, 128.34, 127.95, 126.97, 126.07, 124.13, 116.94, 113.40, 113.18, 113.09, 108.53, 108.32, 73.86, 73.57, 73.28, 73.00, 40.60, 22.25, 11.30. LC-MS: calcd for C26H22F4NO2 [M+H]+: 456.15, found 456.22. 5-Fluoro-3-methyl-2-(4-(4-(2,2,2-trifluoro-1-hydroxyethyl)benzyl)phenyl)quinolin-4(1H)one (7). 1H-NMR (400 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 7.48 – 7.33 (m, 6H), 7.30 (d, J = 7.96 Hz, 2H), 7.20 (d, J = 7.88 Hz, 2H), 4.96 (q, J = 6.92 Hz, 1H), 4.01 (s, 2H), 1.94 (s, 3H). 13CNMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 179.25, 163.72, 161.14, 150.36, 144.36, 143.06, 143.02, 142.95, 134.91, 133.88, 133.23, 133.12, 130.40, 130.22, 130.07, 129.15, 127.66, 124.86, 118.76, 115.29, 115.24, 115.01, 114.92, 110.39, 110.17, 73.58, 73.27, 72.96, 72.64, 42.54, 13.07. LC-MS: calcd for C25H20F4NO2 [M+H]+: 441.14, found 441.21.


28

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5-Fluoro-3-methyl-2-(4-(4-((trifluoromethyl)sulfinyl)benzyl)phenyl)quinolin-4(1H)-one (8). 1H-NMR

(400 MHz, CDCl3, ppm): 11.46 (s, 1H), 7.69 (m, 3H), 7.52 – 7.40 (m 1H), 7.35 – 7.27

(m, 4H), 7.04 (d, J = 8.24 Hz, 2H), 6.80 (t, J = 9.60 Hz, 1H), 3.98 (s, 2H), 1.84 (s, 3H). 13C-NMR (100 MHz, CDCl3, ppm): 177.50, 162.58, 160.01, 147.72, 146.89, 141.98, 140.84, 133.49, 133.23, 131.61, 130.24, 129.23, 128.89, 126.41, 124.77, 123.09, 122.04, 117.48, 114.53, 113.93, 108.98, 108.77, 41.51, 12.44. LC-MS: calcd for C24H18F4NO2S [M+H]+: 460.09, found 460.12. 5-Fluoro-3-methyl-2-(4-(4-((trifluoromethyl)sulfonyl)benzyl)phenyl)quinolin-4(1H)-one (9). 1H-NMR

(400 MHz, d6-DMSO, ppm): 11.55 (s, 1H), 8.10 (d, J = 8.24 Hz, 2H), 7.78 (d, J = 8.36

Hz, 2H), 7.54 – 7.50 (m, 5H), 7.36 (d, J = 8.44 Hz, 1H), 6.93 (dd, J = 11.96 Hz, J = 7.88 Hz, 1H), 4.27 (s, 2H), 1.82 (s, 3H). 13C-NMR (100 MHz, d6-DMSO, ppm): 175.42, 161.73, 159.29, 152.00, 146.61, 141.80, 140.98, 132.92, 131.67, 131.56, 131.10, 129.32, 129.08, 127.20, 121.10, 117.88, 116.07, 114.09, 114.05, 113.01, 108.34, 108.14, 40.57, 12.02. LC-MS: calcd for C24H18F4NO3S [M+H]+: 476.09, found 476.19. 5-Fluoro-3-methyl-2-(4-(4-(S-(trifluoromethyl)sulfonimidoyl)benzyl)phenyl)quinolin-4(1H)one (10). 1H-NMR (400 MHz, CDCl3:d4-MeOD = 0.2:0.3, ppm): 8.03 (d, J = 7.96 Hz, 2H), 7.52 – 7.47 (m, 3H), 7.43 (d, J = 7.80 Hz, 2H), 7.36 (d, J = 7.68 Hz, 2H), 7.30 (d, J = 8.40 Hz, 1H), 6.89 (dd, J = 11.72 Hz, J = 8.16 Hz, 1H), 4.19 (s, 2H), 1.97 (s, 3H). 13C-NMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 179.16, 163.61, 161.03, 150.65, 149.77, 142.90, 142.40, 134.34, 133.09, 132.98, 131.95, 131.15, 131.05, 130.43, 130.38, 123.93, 120.62, 118.72, 115.03, 114.98, 114.84, 110.28, 110.07, 42.57, 13.02. LC-MS: calcd for C24H19F4N2O2S [M+H]+: 475.10, found 475.26.


29


Page 30 of 62

5-Fluoro-3-methyl-2-(4-(4-(N-methyl-S(trifluoromethyl)sulfonimidoyl)benzyl)phenyl)quinolin-4(1H)-one (11). 1H-NMR (400 MHz, CDCl3, ppm): 11.77 (s, 1H), 7.88 (d, J = 7.80 Hz, 2H), 7.76 (d, J = 8.28 Hz, 1H), 7.46 (dd, J = 12.72 Hz, J = 7.52 Hz, 1H), 7.30 (m, 4H), 6.97 (d, J = 7.56 Hz, 2H), 6.78 (dd, J = 11.20 Hz, J = 8.08 Hz, 1H), 3.97 (s, 2H), 3.05 (s, 3H), 1.82 (s, 3H). 13C-NMR (100 MHz, CDCl3, ppm): 177.59, 162.54, 159.96, 148.67, 147.90, 142.07, 140.59, 133.27, 131.69, 131.58, 130.52, 130.05, 129.96, 129.30, 128.89, 123.63, 120.25, 117.50, 114.73, 113.97, 113.88, 109.03, 108.82, 41.57, 29.59, 12.55. LC-MS: calcd for C25H21F4N2O2S [M+H]+: 489.12, found 489.21. 2-(4-(4-(Difluoromethoxy)benzyl)phenyl)-5-fluoro-3-methylquinolin-4(1H)-one

(12).

1H-

NMR (400 MHz, CDCl3:d4-MeOD = 0.2: 0.3, ppm): 7.47 (dd, J = 13.12 Hz, J = 7.84 Hz, 1H), 7.39 (d, J = 7.56 Hz, 2H), 7.34 – 7.29 (m, 3H), 7.20 (d, J = 7.84 Hz, 2H), 7.03 (d, J = 7.92 Hz, 2H), 6.89 (dd, J = 11.52 Hz, J = 8.28 Hz, 1H), 6.59 (t J = 74.12 Hz, 1H), 4.03 (s, 2H), 1.97 (s, 3H). 13C-NMR

(100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 178.67, 163.11, 160.52, 150.56, 149.47,

143.54, 142.37, 138.45, 133.32, 132.51, 132.41, 130.80, 129.67, 129.56, 120.13, 119.46, 118.20, 116.90, 114.47, 114.42, 114.33, 109.72, 109.51, 41.43, 12.49. LC-MS: calcd for C24H19F3NO2 [M+H]+: 410.13, found 410.36. 5-Fluoro-3-methyl-2-(4-(4-(pentafluorosulfanyl)benzyl)phenyl)quinolin-4(1H)-one (13). 1HNMR (400 MHz, d6-DMSO, ppm): 11.67 (s, 1H), 7.83 (d, J = 8.60 Hz, 2H), 7.56 – 7.43 (m, 7H), 7.39 (d, J = 8.44 Hz, 1H), 6.93 (dd, J = 11.92 Hz, J = 7.96 Hz, 1H), 4.14 (s, 2H), 1.83 (s, 3H). 13CNMR (100 MHz, d6-DMSO, ppm): 175.33, 161.73, 159.16, 151.32, 151.16, 151.00, 147.14, 145.95, 141.83, 141.76, 132.68, 131.83, 131.72, 129.69, 129.28, 128.97, 126.12, 126.08, 126.03, 116.18, 114.25, 114.21, 113.00, 112.91, 108.60, 108.39, 40.18, 12.11. LC-MS: calcd for C23H18F6NOS [M+H]+: 470.09, found 470.16.


30

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2-(4-(4-Chlorobenzyl)phenyl)-5-fluoro-3-methylquinolin-4(1H)-one

(14).

1H-NMR

(400

MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 7.50 – 7.46 (m, 1H), 7.39 (d, J = 8.08 Hz, 2H), 7.33 (d, J = 8.04 Hz, 2H), 7.09 (d, J = 8.56 Hz, 1H), 7.25 (d, J = 8.36 Hz, 2H), 7.16 (d, J = 8.32 Hz, 2H), 6.91 (dd, J = 11.80 Hz, J = 7.96 Hz, 1H), 4.03 (s, 2H), 1.97 (s, 3H). 13C-NMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 178.77, 163.30, 160.72, 149.63, 143.53, 142.59, 140.05, 133.53, 132.86, 132.65, 132.54, 131.01, 129.81, 129.69, 129.29, 118.29, 114.56, 114.52, 109.18, 109.60, 41.58, 12.48. LC-MS: calcd for C23H18FNO [M+H]+: 378.10, found 378.19. 5-Fluoro-2-(4-(4-methoxybenzyl)phenyl)-3-methylquinolin-4(1H)-one (15).

1H-NMR

(400

MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 7.45 – 7.42 (m, 1H), 7.33 (d, J = 8.04 Hz, 2H), 7.28 (d, J = 7.88 Hz, 2H), 7.09 (d, J = 8.44 Hz, 2H), 6.89 – 6.80 (m, 3H), 3.95 (m, 2H), 3.76 (s, 3H), 1.95 (s, 3H). 13C-NMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 177.37, 161.87, 159.29, 157.58, 148.16, 142.98, 141.06, 132.06, 131.84, 131.17, 131.06, 129.26, 128.36, 128.19, 116.99, 113.40, 113.19, 113.10, 108.45, 108.24, 54.51, 40.17, 11.41. LC-MS: calcd for C24H21FNO2 [M+H]+: 374.15, found 374.23. 5-Fluoro-3-methyl-2-(4-(4-morpholinobenzyl)phenyl)quinolin-4(1H)-one (16). 1H-NMR (400 MHz, CDCl3, ppm): 10.80 (s, 1H), 7.58 (d, J = 8.20 Hz, 1H), 7.46 –7.41 (m, 1H), 7.26 (d, J = 7.84 Hz, 2H), 7.03 (d, J = 8.44 Hz, 2H), 7.01 (d, J = 8.76 Hz, 2H), 6.83 – 6.78 (m, 3H), 3.81 – 3.79 (m, 6H), 3.07 (t, J = 4.64 Hz, 4H), 1.83 (s, 3H). 13C-NMR (100 MHz, d6-DMSO, ppm): 177.58, 162.69, 160.10, 149.83, 147.62, 143.16, 141.86, 132.65, 131.82, 131.56, 131.45, 129.79, 128.81, 117.69, 115.93, 114.19, 113.99, 113.90, 109.02, 108.81, 67.01, 49.53, 40.81, 12.40. LC-MS: calcd for C27H26FN2O2 [M+H]+: 429.19, found 429.23.


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5-Fluoro-3-methyl-2-(4-(4-(piperidin-1-yl)benzyl)phenyl)quinolin-4(1H)-one (17). 1H-NMR (400 MHz, CDCl3: d4-MeOD = 0.2: 0.3, ppm): 7.51 – 7.45 (m, 1H), 7.36 – 7.32 (m, 5H), 7.07 (d, J = 8.64 Hz, 2H), 6.92 – 6.90 (m, 3H), 3.95 (s, 2H), 3.07 (m, 4H), 1.97 (s, 3H), 1.70 (m, 4H), 1.55 (m, 2H). 13C-NMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 178.71, 163.16, 160.58, 151.47, 149.73, 144.47, 142.37, 133.03, 132.94, 132.51, 132.41, 130.12, 129.67, 129.41, 118.18, 114.45, 109.72, 109.51, 52.21, 41.43, 26.37, 24.73, 12.55. LC-MS: calcd for C28H28FN2O [M+H]+: 427.27, found 427.35. 5-Fluoro-2-(3-fluoro-4-(4-((trifluoromethyl)thio)phenoxy)phenyl)-3-methylquinolin-4(1H)one (18). 1H-NMR (400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.63 (d, J = 8.20 Hz, 2H), 7.53 – 7.46 (m, 1H), 7.40 – 7.25 (m, 4H), 7.04 (d, J = 8.32 Hz,1H), 6.91 (dd, J = 8.80 Hz, J = 11.68 Hz, 1H), 2.01 (s, 3H). 13C-NMR (100 MHz, CD3OD: CDCl3=0.2: 0.3, ppm): 178.51, 163.02, 160.43, 160.13, 155.78, 153.28, 147.18, 144.25, 144.14, 142.33, 139.05, 132.99, 132.92, 132.74, 132.64, 131.70, 128.65, 126.52, 126.48, 123.17, 118.78, 118.58, 118.43, 118.27, 114.48, 114.37, 114.28, 109.88, 109.67, 12.43. LC-MS: calcd for C24H15F5NO2S [M+H]+: 464.07, found 464.13. 5-Fluoro-2-(3-fluoro-4-(4-(piperidin-1-yl)phenoxy)phenyl)-3-methylquinolin-4(1H)-one (19). 1H-NMR

(400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.48 – 7.45 (m, 1H), 7.33 (d, J = 8.80 Hz,

1H), 7.29 (d, J = 11.28 Hz,1H), 7.14 (d, J = 8.16 Hz, 1H), 7.01 – 6.86 (m, 6H), 3.10 – 3.07 (m, 4H), 1.98 (s, 3H), 1.72 – 1.67 (m, 4H), 1.58 – 1.56 (m, 2H).

13C-NMR

(100 MHz, CD3OD:

CDCl3=0.2: 0.3, ppm): 178.53, 154.69, 152.21, 149.99, 149.64, 147.57, 147.47, 142.22, 132.55, 132.44, 130.17, 130.10, 125.89, 125.85, 120.24, 119.70, 119.23, 118.21, 118.10, 117.91, 114.41, 114.36, 114.24, 109.74, 109.53, 53.32, 26.30, 24.52, 12.44. LC-MS: calcd for C27H25F2N2O2 [M+H]+: 447.18, found 447.15.


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5-Fluoro-2-(3-fluoro-4-(4-(pyrrolidin-1-yl)phenoxy)phenyl)-3-methylquinolin-4(1H)-one (20). 1H-NMR (400 MHz, d6-DMSO, ppm): 11.61 (b, 1H), 7.61 – 7.53 (m, 2H), 7.38 (d, J = 8.24 Hz, 1H). 7.27 (d, J = 8.08 Hz, 1H), 7.00 – 6.91 (m, 4H), 6.57 (d, J = 7.92 Hz, 2H), 3.21 (s, 4H), 1.94 (s, 4H), 1.85 (s, 3H). 13C-NMR (100 MHz, d6-DMSO, ppm): 175.51, 161.77, 159.20, 152.95, 150.51, 147.41, 147.06, 145.32, 144.65, 141.75, 131.80, 131.69, 129.10, 129.03, 125.94, 118.03, 117.79, 117.96, 116.32, 114.11, 113.03, 112.94, 108.46, 108.26, 47.60, 25.02, 12.01. LC-MS: calcd for C26H23F2N2O2 [M+H]+: 433.16, found 433.24. 2-(4-(4-(Ethyl(propyl)amino)phenoxy)-3-fluorophenyl)-5-fluoro-3-methylquinolin-4(1H)one (21). 1H-NMR (400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.52 – 7.46 (m, 1H), 7.32 (d, J = 8.80 Hz, 1H), 7.29 (d, J = 11.28 Hz,1H), 7.13 (d, J = 8.16 Hz, 1H), 6.99 – 6.88 (m, 4H), 6.68 (d, J = 8.80 Hz, 2H), 3.35 (q, J = 6.96 Hz, 2H), 3.20 (t, J = 7.44 Hz, 2H), 1.99 (s, 3H), 1.60 (m, 2H), 1.13 (t, J = 6.96 Hz, 3H), 0.93 (J = 7.44 Hz, 3H). 13C-NMR (100 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 178.70, 163.12, 160.53, 154.46, 151.99, 148.76, 148.65, 147.98, 146.41, 146.02, 142.37, 132.70, 132.59, 129.46, 129.39, 125.93, 125.89, 121.29, 118.82, 118.31, 118.01, 117.82, 114.49, 114.34, 114.09, 109.85, 109.64, 53.32, 46.04, 21.20, 12.50, 12.45, 11.70. LC-MS: calcd for C27H27F2N2O2 [M+H]+: 449.20, found 449.16. 2-(4-(4-(Ethyl(methyl)amino)phenoxy)-3-fluorophenyl)-5-fluoro-3-methylquinolin-4(1H)one (22). 1H-NMR (400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.52 – 7.46 (m, 1H), 7.33 (d, J = 8.80 Hz, 1H), 7.29 (d, J = 11.28 Hz,1H), 7.13 (d, J = 8.16 Hz, 1H), 6.97 – 6.88 (m, 4H), 6.75 (d, J = 8.80 Hz, 2H), 3.36 (q, J = 7.08 Hz, 1H), 2.88 (s, 3H), 1.99 (s, 3H), 1.11 (t, J = 7.08 Hz, 3H). 13C-NMR

(100 MHz, CD3OD: CDCl3=0.2: 0.3, ppm): 178.63, 163.09, 160.50, 154.54, 152.06,

148.47, 148.36, 147.83, 147.49, 146.90, 142.33, 132.63, 132.53, 129.66, 129.59, 125.89, 125.86,


33


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121.03, 119.03, 118.28, 118.03, 117.83, 114.86, 114.48, 114.43, 114.31, 109.80, 109.58, 48.05, 38.23, 12,47, 11.26. LC-MS: calcd for C25H23F2N2O2 [M+H]+: 421.16, found 421.19. 2-(4-(4-(Dimethylamino)phenoxy)-3-fluorophenyl)-5-fluoro-3-methylquinolin-4(1H)-one (23). 1H-NMR (400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.52 – 7.46 (m, 1H), 7.32 (d, J = 8.80 Hz, 1H), 7.29 (d, J = 11.28 Hz,1H), 7.13 (d, J = 8.16 Hz, 1H), 6.97 – 6.88 (m, 4H), 6.78 (d, J = 8.80 Hz, 2H), 2.92 (s, 6H), 1.99 (s, 3H). 13C-NMR (100 MHz, CD3OD: CDCl3=0.2: 0.3, ppm): 178.74, 163.13, 160.63, 154.62, 152.12, 148.98, 148.38, 148.27, 147.79, 147.39, 142.37, 132.63, 132.53, 129.69, 125.89, 125.86, 120.86, 119.13, 118.28, 118.05, 117.86, 114.98, 114.41, 109.80, 109.58, 41.51, 12.45. LC-MS: calcd for C24H21F2N2O2 [M+H]+: 407.15, found 407.23. 2-(4-(4-(Ethylamino)phenoxy)-3-fluorophenyl)-5-fluoro-3-methylquinolin-4(1H)-one 1H-NMR

(24).

(400 MHz, CD3OD: CDCl3 = 0.2: 0.3, ppm): 7.48 - 7.43 (m, 1H), 7.30 – 7.25 (m, 2H),

7.09 (d, J = 8.44 Hz, 1H), 6.94 – 6.84 (m, 4H), 6.64 (d, J = 8.40 Hz, 2H), 3.11 (q, J = 7.04 Hz, 2H), 1.97 (s, 3H), 1.23 (t, J = 6.88 Hz, 3H). 13C-NMR (100 MHz, CD3OD: CDCl3=0.2: 0.3, ppm): 178.32, 162.82, 160.23, 154.14, 151.66, 148.15, 148.05, 147.25, 147.01, 146.30, 142.01, 132.27, 132.16, 129.33, 129.27, 125.55, 125.51, 120.96, 118.58, 118.06, 117.72, 117.52, 114.49, 114.10, 114.05, 109.53, 109.31, 39.32, 14.73, 12.33. LC-MS: calcd for C24H21F2N2O2 [M+H]+: 407.15, found 407.11. 5-Fluoro-2-(3-fluoro-4-(4-(piperazin-1-yl)phenoxy)phenyl)-3-methylquinolin-4(1H)-one (25). Finally, a salt with 4-methylbenzenesulfonate was obtained. 1H-NMR (400 MHz, d6-DMSO, ppm): 11.65 (s, 1H), 7.66 (d, J = 11.4 Hz, 1H). 7.62 – 7.52 (m, 1H), 7.49 (d, J = 7.68 Hz, 2H), 7.40 (d, J = 8.36 Hz, 1H), 7.35 (d, J = 8.28 Hz, 1H), 7.15 – 7.52 (m, 7H), 6.95 (dd, J = 8.80 Hz, J = 11.68 Hz, 1H), 3.30 – 7.52 (m, 8H), 2.28 (s, 3H), 1.86 (s, 3H). 13C-NMR (100 MHz, d6-DMSO,


34

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ppm): 174.95, 161.23, 158.70, 153.01, 150.55, 148.57, 146.56, 145.06, 144.93, 144.66, 141.22, 137.29, 131.34, 131.23, 129.78, 129.70, 127.61, 125.66, 124.97, 119.21, 118.82, 117.54, 117.33, 115.82, 113.62, 112.51, 107.99, 107.77, 45.72, 42.46, 20.26, 11.52. LC-MS: calcd for C26H24F2N3O2 [M+H]+: 448.18, found 448.21. 5-Fluoro-3-methyl-2-(4-(3-morpholinopropoxy)phenyl)quinolin-4(1H)-one (29).

1H-NMR

(400 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 7.55 – 7.49 (m, 1H), 7.43 (d, J = 8.64 Hz, 2H), 7.35 (d, J = 8.48 Hz, 1H), 7.06 (d, J = 8.64 Hz, 2H), 6.93 (dd, J = 11.88 Hz, J = 7.72 Hz, 1H), 4.13 (t, J = 6.12 Hz, 3H), 3.75 (t, J = 4.56 Hz, 4H), 2.61 (t, J = 7.44 Hz, 2H), 2.55 (m, 4H), 2.07 – 2.02 (m, 5H). 13C-NMR (100 MHz, CDCl3: d4-MeOD = 0.2:0.3, ppm): 178.76, 163.21, 160.83, 160.63, 149.66, 142.48, 142.44, 132.53, 132.43, 130.89, 127.71, 118.24, 115.18, 114.52, 114.47, 114.36, 109.74, 109.52, 67.30, 66.88, 56.30, 54.31, 26.77, 12.55. LC-MS: calcd for C23H26FN2O3 [M+H]+: 397.18, found 397.25. 2-(4-(2-(Diethylamino)ethoxy)phenyl)-5-fluoro-3-methylquinolin-4(1H)-one (30).

1H-NMR

(400 MHz, CDCl3, ppm): 7.79 (d, J = 8.40 Hz, 1H), 7.37 (m, 1H), 7.21 (d, J = 8.32 Hz, 2H), 6.76 (m, 1H), 6.66 (d, J = 8.28 Hz, 2H), 4.06 (t, J = 5.04 Hz, 2H), 3.03 (t, J = 5.04 Hz, 2H), 2.79 (q, J = 6.96 Hz, 4H). 1.84 (s, 3H), 1.13 (t, J = 6.96 Hz, 6H). 13C-NMR (100 MHz, CDCl3, ppm): 177.48, 162.32, 159.74, 158.83, 147.74, 142.05, 142.01, 131.29, 131.18, 130.39, 127.24, 117.23, 114.78, 113.98, 113.80, 108.70, 108.48, 65.06, 52.90, 47.76, 12.42, 10.49. LC-MS: calcd for C22H26FN2O2 [M+H]+: 369.19, found 369.23. 5-Fluoro-3-methyl-2-(4-(2-(piperidin-1-yl)ethoxy)phenyl)quinolin-4(1H)-one (31). 1H-NMR (400 MHz, CDCl3, ppm): 7.70 (d, J = 8.36 Hz, 1H), 7.40 (dd, J = 13.12 Hz, J = 7.96 Hz, 1H), 7.23 (d, J = 8.36 Hz, 1H), 6.80 (dd, J = 11.68 Hz, J = 8.08 Hz, 1H), 6.68 (d, J = 8.24 Hz, 2H), 4.05 (t,


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J = 5.26 Hz, 2H), 2.84 (t, J = 5.26 Hz, 2H), 2.60 (m, 4H), 1.84 (s, 3H), 1.66 – 1.63 (m, 4H), 1.49 – 1.47 (m, 2H). 13C-NMR (100 MHz, CDCl3, ppm): 177.36, 162.30, 159.70, 159.08, 148.08, 141.96, 131.15, 131.06, 130.18, 127.04, 117.26, 114.74, 113.97, 113.78, 113.69, 108.62, 108.40, 65.32, 57.51, 54.87, 25.34, 23.73, 12.35. LC-MS: calcd for C23H26FN2O2 [M+H]+: 381.19, found 381.32. 5-Fluoro-3-methyl-2-(4'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)quinolin-4(1H)-one 1H-NMR

(32).

(400 MHz, d6-DMSO, ppm): 11.69 (s, 1H), 7.95 (m, 4H), 7.85 (d, J = 8.08 Hz, 2H), 7.69

(d, J = 8.04 Hz, 2H), 7.56 (m, 1H), 7.42 (d, J = 7.76 Hz, 1H), 6.96 (dd, J = 11.88 Hz, J = 8.04 Hz, 1H), 1.89 (s, 3H). 13C-NMR (100 MHz, d6-DMSO, ppm): 175.95, 162.27, 159.69, 146.80, 142.72, 142.37, 142.32, 140.00, 137.28, 135.06, 132.24, 132.13, 131.64, 130.24, 128.82, 128.59, 127.59, 122.97, 116.71, 114.65, 114.61, 113.55, 113.46, 108.91, 108.71, 12.54. LC-MS: calcd for C23H16F4NOS [M+H]+: 430.18, found 430.24. 5-Fluoro-3-methyl-2-(4-((4-((trifluoromethyl)thio)phenoxy)methyl)phenyl)quinolin-4(1H)one (33). 1H-NMR (400 MHz, d6-DMSO, ppm): 11.62 (s, 1H), 7.66 (m, 4H), 7.58 (d, J = 7.96 Hz, 2H), 7.52 (m, 1H), 7.39 (d, J = 8.40 Hz, 1H), 7.20 (d, J = 8.56 Hz, 2H), 6.94 (dd, J = 11.88 Hz, J = 7.96 Hz, 1H), 5.30 (s, 2H), 1.4 (s, 3H). 13C-NMR (100 MHz, d6-DMSO, ppm): 175.49, 161.79, 160.84, 159.22, 146.56, 141.85, 141.81, 138.31, 137.88, 134.32, 131.73, 131.62, 131.16, 129.16, 128.10, 127.86, 116.35, 114.14, 114.10, 113.58, 113.06, 112.97, 108.41, 108.21, 69.04, 12.02. LCMS: calcd for C24H18F4NO2S [M+H]+: 460.09, found 460.21. N-(4-(5-fluoro-3-methyl-4-oxo-1,4-dihydroquinolin-2-yl)benzyl)-4((trifluoromethyl)thio)benzamide (34). 1H-NMR (400 MHz, d6-DMSO, ppm): 11.58 (s, 1H), 9.37 (t, J = 5.88 Hz, 1H), 8.03 (d, J = 8.20 Hz, 2H), 7.84 (d, J = 8.12 Hz, 2H), 7.56 – 7.50 (m, 5H),


36

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7.38 (d, J = 8.44 Hz, 1H), 6.93 (dd, J = 12.00 Hz, J = 7.92 Hz, 1H), 4.59 (d, J = 5.92 Hz, 2H), 1.83 (s, 3H). 13C-NMR (100 MHz, d6-DMSO, ppm): 175.46, 165.34, 161.77, 159.20, 146.79, 141.83, 141.78, 140.90, 136.85, 135.88, 133.19, 131.67, 131.57, 131.02, 128.94, 128.67, 127.96, 127.32, 126.33, 116.06, 114.11, 114.06, 113.02, 112.93, 108.36, 108.15, 42.55, 11.99. LC-MS: calcd for C25H19F4N2O2S [M+H]+: 487.10, found 487.27. 2-(4-(4-(Ethyl(3-hydroxyhept-6-yn-1-yl)amino)phenoxy)-3-fluorophenyl)-5-fluoro-3methylquinolin-4(1H)-one (85). 1H-NMR (400 MHz, CDCl3, ppm): 10.83 (s, 1H), 7.51 (d, J = 8.40 Hz, 1H), 7.38 (m, 1H), 7.10 (dd, J = 10.92 Hz, J = 1.6 Hz, 1H), 6.95 (d, J = 8.40 Hz, 1H), 6.78 – 6.63 (m, 6H), 3.85 – 3.73 (m, 1H), 3.42 (t, J = 6.72 Hz, 2H), 3.34 (q, J = 6.84 Hz, 2H), 2.35 – 2.29 (m, 2H), 1.96 (m, 1H), 1.85 (s, 3H), 1.67 – 1.52 (m, 4H), 1.01 (t, J = 6.84 Hz, 3H). 13CNMR (100 MHz, CDCl3 ppm): 177.49, 162.80, 160.01, 154.10, 147.41, 146.97, 146.30, 145.51, 141.87, 132.49, 131.49, 129.68, 125.40, 120.22, 118.99, 117.88, 116.10, 114.28, 113.80, 109.30, 84.18, 70.30, 69.07, 49.04, 46.64, 36.20, 34.16, 15.06, 12.34, 12.12. LC-MS: calcd for C31H31F2N2O3 [M+H]+: 517.22, found 517.18. 2-(4-(4-(Ethyl(3-oxohept-6-yn-1-yl)amino)phenoxy)-3-fluorophenyl)-5-fluoro-3methylquinolin-4(1H)-one (86). A mixture of 160 mg compound 85 (0.3 mmol, 1.0 eq) and 150 mg Dess-Martin reagent (1.2 eq) in 5 ml DCM was stirred at room temperature for 2 hours. Then wash with brine and extract with EtOAc. Compound 86 was obtained after column chromatography (Hexane: EtOAc = 1: 1, isolated yield = 50%). It should be used in the next step immediately because β-elimination made it seriously decomposed. 1H-NMR (400 MHz, CDCl3, ppm): 10.93 (s, 1H), 7.60 (m, 1H), 7.38 (m, 1H), 7.49 – 7.41 (m, 1H), 7.19 (d, J = 10.8 Hz, 1H), 7.03 (d, J = 8.12 Hz, 1H), 6.88 – 6.79 (m, 3H), 6.75 – 6.67 (m, 1H), 6.61 (d, J = 8.48 Hz, 2H), 3.55 (t, J = 6.72 Hz, 2H), 3.32 (q, J = 6.84 Hz, 2H), 2.75 – 2.64 (m, 4H), 2.24 (t, J = 5.86 Hz, 2H),


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1.95 (s, 3H), 1.90 (s, 3H), 1.12 (t, J = 6.84 Hz, 3H). 13C-NMR (100 MHz, CDCl3 ppm): 207.91, 177.41, 162.35, 159.77, 153.60, 151.13, 147.12, 147.01, 146.14, 144.70, 141.95, 131.65, 131.54, 129.25, 129.19, 125.36, 120.70, 118.05, 117.58, 117.37, 114.83, 113.79, 109.03, 108.83, 82.97, 69.03, 45.81, 45.41, 42.09, 40.65, 12.98, 12.34. LC-MS: calcd for C31H29F2N2O3 [M+H]+: 515.21, found 515.26. It should be noted that the intermediate 86 must be used in the next step as soon as possible, because the carbon-nitrogen bond near the newly generated carbonyl group could break spontaneously via β-elimination. 2-(4-(4-((2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)(ethyl)amino)phenoxy)-3fluorophenyl)-5-fluoro-3-methylquinolin-4(1H)-one (87). 70 mg compound 86 (0.14 mmol, 1.0eq) was added into 2 ml 7 N ammonia solution in methanol at 0 oC. The hydroxylamine-Osulfonic acid (20 mg, 1.2 eq) was added after 2 hours and the mixture was stirred at room temperature for 16 hours. The excess ammonia was removed under reduced pressure, then the solid was suspended in methanol and filtered. The filtration was concentrated to 1ml and cooled at ice bath, followed by adding Et3N (2.0 eq, 40 μL) and I2 (0.9 eq, 32 mg) subsequently. The solution was washed with brine and extracted with EtOAc. Compound 87 was obtained after column chromatography (Hexane: EtOAc = 1: 1, isolated yield= 11%). 1H-NMR (400 MHz, CDCl3, ppm): 10.21 (s, 1H), 7.55 – 7.43 (m, 2H), 7.21 (d, J = 10.88 Hz, 1H), 7.04 (d, J = 8.32 Hz, 1H), 6.95 – 6.80 (m, 3H), 6.75 (t, J = 8.32 Hz,1H), 6.60 (d, J = 8.92 Hz, 2H), 3.30 (q, J = 7.00 Hz, 2H), 3.12 (t, J = 7.52 Hz, 2H), 2.05 – 1.96 (m, 3H), 1.91 (s, 3H), 1.71 – 1.60 (m, 4H), 1.11 (t, J = 6.96 Hz, 3H). 13C-NMR (100 MHz, CDCl3, ppm): 177.49, 162.38, 159.80, 153.59, 151.07, 146.98, 146.00, 144.74, 141.90, 131.54, 129.08, 125.33, 120.82, 117.92, 117.64, 117.52, 117.33, 114.83, 113.82, 109.05, 108.84, 82.78, 69.47, 45.55, 45.21, 32.49, 30.72, 29.81, 27.14, 22.81, 14.25, 13.43, 12.35, 12.30. LC-MS: calcd for C31H29F2N4O2 [M+H]+: 527.22, found 527.26.


38

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N-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-N-ethyl-4-(2-fluoro-4-(5-fluoro-4-methoxy3-methylquinolin-2-yl)phenoxy)aniline (88). A mixture of 22 mg compound 87 (0.04 mmol, 1.0 eq), 25 μL MeI (10.0 eq) and 27 mg K2CO3 (5.0 eq) in 1 ml DMF was stirred at room temperature for 6 hours. Then wash with brine and extract with EtOAc. Compound 88 was obtained after column chromatography (Hexane: EtOAc = 5: 1, isolated yield= 30%). 1H-NMR (400 MHz, CDCl3, ppm): 7.90 (d, J = 8.52 Hz, 1H), 7.60 – 7.52 (m, 1H), 7.41 (dd, J = 11.40 Hz, J = 1.88 Hz, 1H), 7.18 (m, 1H), 7.02 (m, 4H), 6.65 (d, J = 9.04 Hz, 2H), 3.31 (q, J = 7.00 Hz, 2H), 3.13 (t, J = 7.52 Hz, 2H), 2.41 (s, 3H), 2.05 – 1.96 (m, 3H), 1.91 (s, 3H), 1.71 – 1.60 (m, 4H), 1.11 (t, J = 6.96 Hz, 3H).

13C-NMR

(100 MHz, CDCl3, ppm): 155.73, 147.11, 144.59, 143.72, 128.77, 128.67,

125.99, 125.95, 125.30, 122.53, 120.44, 119.06, 117.90, 117.70, 114.12, 111.75, 111.53, 82.84, 69.42, 61.91, 45.74, 45.34, 32.60, 32.07, 30.78, 29.85, 29.81, 29.51, 22.84, 14.28, 13.49, 13.44, 12.36. LC-MS: calcd for C32H30F2N4O2 [M+H]+: 541.23 found 541.20. Biological materials. Huh7.5.1 cells were gifted by Apath L.L.C. Other cells were purchased from the American Type Culture Collection (ATCC) (Manassas, USA). The HCV, DENV, EV71 assays were constructed by ourselves. The ZIKV, MERS-CoV, RSV, HIV, SFTSV, CHIKV and influenza assays were gifted or conducted by Wuhan institute of virology or Peking union medical college. The WST-1 reagent for cell viability assays was purchased from Roche. The avidin agarose used in ABPP was purchased from Thermo (20219). The mini protease inhibitor cocktail and pronase used in DARTS were purchased from Roche. The antibodies of DHODH and GAPDH were purchased from CST. Recombinant GAPDH protein was purchased from Cloud-Clone Corp. Wuhan (RPB932Hu02). Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (catalog number 10099-141) (GIBCO,


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USA), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L l-glutamine were used for the cell culture. HCVrep, HCVcc and HCVpp were constructed according to a previously described method with modifications69. Based on the pJFH-1 plasmid gifted by Apath L.L.C., a humanized Renilla luciferase reporter gene was inserted between amino acids 399 and 400 of NS5A in the HCV JFH-1 genome containing four adaptive mutations (core-K78E, E2-G451R, NS3-M260K and NS5AT462I). These plasmids harboring HCV genomes were made through digestion with the XbaI restriction enzyme and used as a template for RNA transcription. The transcripts were prepared in vitro using the Ambion MEGAscript Kits, and then 10 μg RNA was mixed with 400 μL Huh7.5.1 cells at a concentration of 1 × 107 cells/mL. After electroporation, Huh7.5.1 cells containing viral transcripts were seeded in 10 cm dishes. The cells were cultured and passaged every 3 days until they showed significant apoptosis, and the supernatant was collected and filtered to obtain a stock solution of hRluc-JFH-1 virus. HCV JFH-1 virus with an EGFP reporter gene is an infectious HCV monocistronic reporter virus constructed by inserting an EGFP reporter gene between amino acids 399 and 400 of NS5A of the HCV JFH-1 genome. The stable replicon-containing cells were selected and maintained in medium containing 1 mg/mL G418 geneticin (Invitrogen, USA). Pseudotyped viral particles of HCV were generated by co-transfection of the JFH1 full-length envelope-expressing plasmid (pCDN3.1-JFH1-cE1E2) together with the pNL4-3R-E-luciferase backbone construct into HEK 293T cells. At 48 h post-transfection, the culture supernatant was harvested, clarified, filtered through 0.45 μm filters (Millipore, USA) and tested for luciferase activity to standardize the viral input in the subsequent inhibition analysis. Phenotypic screening. Huh 7.5.1 cells were seeded at 3 × 104 cells per well in 96-well microtiter plates. After overnight culture at 37 °C in 5% CO2, Huh 7.5.1 cells were treated with a gradient of


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concentrations of different compounds ranging from 0.0004 to 0.05 μM. After 2 h, Huh 7.5.1 cells were infected with HCV-GFP at a multiplicity of infection (MOI) of 1. At 48 h post infection (hpi), the level of GFP expression was monitored using an epifluorescence microscope (OLYMPUS DP973) and data analysis using CellSens standard software70-71. Antiviral activity assays. Cells (Huh7.5.1) or cells containing HCV (JFH-1) subgenomic replicon were seeded in 96-well plates at a density of 1 ×104 cells per well and cultured at 37 °C overnight. The initial concentration of the compounds was 30 μM or 6 μM, and serial dilutions were made. For HCVcc and HCVpp the serially diluted compounds were mixed with a certain titer of the virus and added to different cells. For the inhibition of HCVrep, the serially diluted compounds were mixed with the stable Huh7.5.1 cell line containing a subgenomic replicon, followed by 2 days of culture at 37 °C. The viral amounts were detected by luminescence or qRT-PCR. EC50 is the concentration of the compound at which the luminescence or HCV RNA levels is reduced by 50%. For the time-of-addition experiments, Huh7.5.1 cells were infected with HCVcc at 4 °C for 2 h or 6 h (T = -2 h or -6 h). After removing unbound viruses, cells were incubated at 37 °C (T = 0 h), and compounds were added to the infected cells at different time points. We selected the antiCD81 antibody as a control to represent typical working HCV entry inhibitors. HCV inhibiting effects of the anti-CD81 antibody decreased at the time of the temperature shift. The EC50 values were calculated using GraphPad Prism 5.0 software. For DENV and ZIKV, a similar procedure was used: The Huh7.5.1 cells were infected by DENV II or ZIKV (0.1 MOI). After 3 days, the viral RNA was detected by qRT-PCR. For EV71, RD cells were seeded at 1×104 per well in 96-well microtiter plates. After overnight culture, EV71(FY)-Luc at a multiplicity of infection (MOI) of 1 was used to infect the RD cells, immediately followed by treatment with various concentrations of different compounds ranging


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from 0.05 to 20 nM. At 24 hpi, the viral amounts were detected by luminescence or qRT-PCR. EC50 is the concentration of the compound at which the luminescence or RNA levels is reduced by 50%. For MERS-CoV, Caco-2 cells were seeded at 1×104 per well in 24-well microtiter plates. After overnight culture, MERS at a multiplicity of infection (MOI) of 0.005 was used to infect the Caco2 cells, and after 1 h, they were treated with 0.05 to 20 µM concentrations of the compounds. At 18 hpi, the viral amounts were detected by virus plaque formation assay. For RSV, Vero cells were seeded at 2×104 per well in 96-well microtiter plates. After 18 h of culture, RSV at 2000TCID50/ml was used to infect the Vero cells, immediately followed by treatment with various concentrations of different compounds ranging from 0.01 to 20 µM. At 57 days, the viral amounts were detected by a virus plaque formation assay. For SFTSV and CHIKV, the Vero cells were also used with 0.01 multiplicity of infection (MOI) in the assay, and the virus titers were tested at 4 days and 36 hours, respectively, after infections. For the anti-HIV activity assay by the pseudo-typed virus, VSVG plasmid and env-deficient HIV vector (pNL4-3.luc.R-E-) were co-transfected into HEK 293T cells using the Ca3(PO4)2 method. Sixteen hours post-transfection, the cells were washed with PBS, and fresh media was added. The supernatant containing pseudotyped virions was collected 48 h post-transfection. The harvested virus solution was quantified using p24 concentrations, which were detected by ELISA (ZeptoMetrix, Cat.: 0801111) and diluted to 0.2 ng p24/ml. HEK 293T cells were seeded on a 24well plate one day prior to infection. The compounds were added into the target cells 15 min prior to infection. For each well, 0.5 mL of pseudo-virus was added (0.2 ng p24/ml). Forty-eight hours post-infection, infected cells were lysed in Cell Lysis Reagent (Promega). The luciferase activity


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of the cell lysate was measured by a Sirius luminometer (Berthold Detection System) according to the manufacturer's instructions. For the influenza virus, MDCK cells were seeded at 1×105 per well in 96-well microtiter plates. After overnight culture, A/PR/08/34(H1N1)-Gaussia Luciferase at a multiplicity of infection (MOI) of 0.1 was used to infect the MDCK cells, immediately followed by treatment with various concentrations of different compounds ranging from 0.05 to 20 µM. At 24 hpi, the viral amounts were detected by luminescence or qRT-PCR. EC50 is the concentration of the compound at which the luminescence or viral RNA levels is reduced by 50%. Cell viability assays. Cells were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured at 37 °C overnight. The cells were incubated with the serially diluted compounds for 48 h. The cell viability was determined in 96-well tissue culture plates using the cell proliferation reagent, WST-1 (Roche, Swiss), and the absorbance (OD450/OD630) was measured to detect the cytotoxicity of the compounds according to the manufacturer’s protocol. Target identification by ABPP. This procedure was similar to previous methods with further optimizations33, 72. The Huh7.5.1 cells in three culture plates (10 cm) were infected with HCV for 2 days and isolated by trypsinization. The cell plates were suspended in PBS buffer containing an EDTA-free protease inhibitor cocktail (cOmpleteTM, Roche) to prepare the cell lysate by sonication (40% W, 5 s on, 5 s off, 8 min). The supernatant was collected by centrifugation at 15000 rpm at 4 °C for 30 min, and the protein concentration was determined by the BCA method and was adjusted to approximately 1.5 mg/ml. For the in-gel fluorescence, we added 50 μl supernatant and probes (1:100) in the DMSO stocks to each 1.5 ml tube. The mixture was incubated at room temperature for 1 hour and irradiated


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under UV light (UVP CL-1000, 350 nM, 10000 mJ/cm2) for 10 min. Then, 3 µl of freshly prepared premixed click chemistry reaction cocktail (1 μl, 50 mM CuSO4; 0.5 μl 10 mM TBTA; 0.5 μl 10 mM Rodamine-N3; 1 μl 50 mM TCEP) was added and gently mixed. After incubation at room temperature for 1 hour, 500 μl of prechilled acetone was added, and the mixture was kept at -20 °C for 30 min to precipitate the proteins. The precipitated proteins were collected at 15000 rpm at 4 °C over 10 min. The supernatant was discarded, and the pellet was washed 2 times with 200 μL of prechilled methanol. SDS loading buffer was added and heated at 95 °C for 10 min. The proteins were separated by SDS-PAGE (12% gel) and visualized by in-gel fluorescence scanning (BIO RAD ChemiDocTM XRS+, Qdots565). Finally, the gel was stained with Coomassie brilliant blue to confirm the equal loading amount of the cell lysates. For the pull-down and LC-MS/MS analysis, 1 ml of the supernatant was used. After UV irradiation, the click chemistry reaction cocktail (23 µl, 50 mM CuSO4; 10 μl 10 mM TBTA; 12 μl 10 mM Biotin-N3; 23 μl 50 mM TCEP) was incubated at room temperature for 1 hour. Then, each sample was transferred to a 15 ml conical centrifuge; 4 ml of methanol, 1 ml of chloroform and 3 ml of water were added; then, the sample was vortexed. The top and bottom layers were removed to leave behind the precipitated proteins. They were transferred into a 1.5 ml tube with 600 μl of methanol. We added 150 μl of chloroform and 600 μl of water and vortexed again to collect the precipitated proteins. The proteins were washed with 600 μl of methanol and dissolved in 8.5 ml of 0.2% SDS in PBS. We added 100 μl of avidin beads (Pierce, product number 20219) that were prewashed three times according to the manufacturer’s instructions and incubated at room temperature for 1 hour on an end-over-end rotator. The sample was centrifuged at 3400 rpm at room temperature for 2 min to pellet beads. Then, the beads were washed with 1% SDS, 6 M urea and PBS, respectively. The beads were mixed with SDS loading buffer, heated for 10 min at


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95 °C, and separated by SDS-PAGE. Then, quantitative analysis was performed by LC-MS/MS with TMT labeling or iBAQ in the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University. Bioinformatic analysis. The bioinformatic analysis of the cellular component, biological process and protein-protein interaction network was performed in STRING (https://string-db.org/)73. The Venn diagram was generated by VENNY 2.1, Oliveros, J.C. (2007-2015) Venny, which is an interactive

tool

for

comparing

lists

with

Venn's

diagrams

(http://bioinfogp.cnb.csic.es/tools/venny/index.html). Reverse docking. The PDB files were downloaded from PDTD74. Docking was performed by Schrödinger (Release 2017-1). Water, ions and other HETATM records, which were not related to the binding, were all removed from the PDB files. The conformation of RYL-634 with the lowest energy and proteins were prepared by LigPrep and Protein Preparation Wizard, respectively. In the docking, the precision was set to XP (extra precision), and the descriptor information was written in the result. The results were browsed by the XP Visualizer, and the proteins were ranked by Glide scores. Expression and purification of HsDHODH. Endogenous human DHODH contains a 29 residue N-terminal mitochondrial signal peptide. A truncated DHODH lacking the N-terminal signal peptide (Δ29DHODH) was expressed and purified according to a previously reported method with further optimization47. In brief, the cDNA was cloned into pET21a with NdeI and EcoRI as the restriction sites to encode Δ29DHODH with a C-terminal His6 tag. The constructed plasmid was transformed into E. coli BL21 (DE3) cells. The single colony was grown in a 37 °C shaker for 12−15 h in 10 ml of 2× YT medium containing 100 μg/ml ampicillin and transformed into 1 L 2×


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YT medium. Expression of the recombinant protein was induced by 0.1 mM IPTG (isopropyl βD-thiogalactopyranoside) and 100 μl of flavin mononucleotide when the culture reached an OD600 of 0.7. Cultivation was continued at 25 °C for 20 h before the cells were harvested by centrifugation at 4000 rpm and 4 °C for 20 min. Recombinant human Δ29DHODH was purified as follows: cell pellets were resuspended in ~100 ml buffer A containing HEPES (50 mM, pH 7.7), NaCl (300 mM), glycerol (10% v/v) and Triton X-100 (0.5% v/v) and lysed by sonication (35% amplitude, 4.0 s pulse, 9.0 s pause, 15 min). The sample was centrifuged at 2,300 g and 4 °C for 30 min, and the supernatant was passed through a 0.45 μm syringe filter. The sample was loaded onto a 1 ml HisTrap HP column (GE Healthcare Life Sciences).and gradually washed with buffer A containing 20 mM to 500 mM of imidazole. Subsequently, a SuperDex200 size exclusion column (GE Healthcare) was used to purify the DHODH again. The eluate was collected and analyzed by SDS-PAGE. Then, the protein was concentrated by ultrafiltration with a 10-kDa molecular mass cut-off (Millipore) by buffer B containing HEPES (50 mM, pH 7.7), KCl (150 mM) and glycerol (10% v/v). Finally, the enzyme was aliquoted, flash-frozen with liquid nitrogen, and stored at −80 °C. It should be noted that the purity of recombinant DHODH was vital for the enzymatic assays. When the enzyme was only purified through a HisTrap HP column, the IC50 of RLY-634 was 0.24 μM. Subsequently, a SuperDex200 size exclusion column (GE Healthcare) was used to purify the DHODH again after the HisTrap HP column. The purity of DHODH was improved from 50% to over 85%. As a result, the IC50 of RLY-634 decreased to 60 nM, while there was no obvious fluctuation for teriflunomide. The impurities in the protein buffer could disturb the enzymatic activity of RYL-634. Targets validation by DARTS. A previous DARTS procedure for purified DHODH was modified according to the pronase bulletin (Roche, 0111.11743791001, version 7)48. In brief, the


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aliquoted DHODH were diluted to 20 μg/ml with TNC buffer (50 mM Tris·HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl2) and treated with different concentrations of RYL-634 or DMSO. After incubation for 1 h at room temperature, pronase was added into the above mixture for a further 15 min at 37 °C. Reactions were quenched by adding the protease inhibitor cocktail. Then, SDSPAGE and a western blot were performed with a specific anti-DHODH antibody. Enzymatic activity assays of HsDHODH. The DHODH inhibition assays were carried out by using a previously reported DCIP assay method with further optimization47. The purified HsDHODH was diluted into a final concentration of 10 nM with an assay buffer containing 50 mM HEPES at pH 7.7, 150 mM KCl and Triton X-100 (0.1% v/v). Then, UQ0 and DCIP were added into the assay buffer to final concentrations of 100 and 120 μM, respectively. The mixture was transferred into a 96-well plate and incubated for 5 min at room temperature. Inhibitor compounds were prepared as 10 mM stock solutions in DMSO and further diluted by the assay buffer to prepare working stocks. In the following step, dihydroorotate was added to a final concentration of 500 μM to initiate the reaction. The reaction was monitored by measuring the decrease of DCIP according to its absorption at 600 nm for each 30 s over a period of 6 min. Inhibition studies were performed in this assay with additional variable amounts of compounds. Teriflunomide was also measured as the positive control. The percent inhibition relative to the no inhibitor control was calculated from (1 − Vi/V0) × 100. For the determination of the IC50 values, eight to nine different concentrations were applied. Each inhibitor concentration point was tested in triplicate. IC50 values were calculated using the sigmoidal fitting option of the Origin 8.0 program. ASSOCIATED CONTENT


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Supporting Information. The following files are available free of charge. The result of phenotypic screening (Figure S1), the combination indexes for RYL-634 with other FDA-approved drugs, the details of ABPP, bioinformatic analysis, reverse docking, and sequence alignment of DHODH (Figure S2-S7, Table S1-S2), the SMILES spreadsheet, the coordinates of docking, the synthesis and spectrums of compounds. AUTHOR INFORMATION Corresponding Author *Yu Rao, [email protected] *Zhiyong Lou, [email protected] Author Contributions #These

authors contributed equally. The manuscript was written through contributions of all

authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We would like to thank Dr. Honglin Li, Dr. Pengfei Tu, Dr. Chu Wang and Dr. Qidong You for their kind help in ABPP study and enzymatic activity assays. This work was supported by the National Natural Science Foundation of China (81811530340, 81773567, 81622042, 81573277


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and 31770309), Tsinghua University-Peking University Joint Center for Life Sciences and China Postdoctoral Science Foundation (2017M620806). ABBREVIATIONS HIV, human immunodeficiency virus; HCV, hepatitis C virus; DENV, dengue virus; ZIKV, Zika virus; CHIKV, Chikungunya virus; EV71, enterovirus 71; RSV, respiratory syncytial virus; ABPP, activity-based protein profiling; HsDHODH, human dihydroorotate dehydrogenase; FDA, Food and Drug Administration; VTAs, virus-targeting antivirals; HTAs, host-targeting antivirals; SARS-CoV, severe acute respiratory syndrome coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; SFTSV, severe fever with thrombocytopenia syndrome virus; GFP, green fluorescent protein; SAR, structure-activity relationship; SD, standard deviation; SI, selectivity index; CI, combination index; DMSO, dimethyl sulfoxide; DOCK1, dedicator of cytokinesis protein 1; SIAE, sialate O-acetylesterase; UTRO, utrophin; MLC1, megalencephalic leukoencephalopathy with subcortical cysts 1; HSP90, heat shock protein 90; HSPA5 or GRP78, 78 kDa glucose-regulated protein; IMPDH2, inosine-5'-monophosphate dehydrogenase 2; PDI, protein disulfide-isomerase; DARTS, drug affinity responsive target stability; PfNDH2, NADHubiquinone oxidoreductase of Plasmodium falciparum; PRPS1, phosphoribosyl pyrophosphate synthetase 1; MOI, multiplicity of infection. REFERENCES (1) Hill, A.; Cooke, G. Hepatitis C can be cured globally, but at what cost? Science 2014, 345 (6193), 141-142. (2) Guzman, M. G.; Harris, E. Dengue. Lancet 2015, 385 (9966), 453-465.


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(3) Solomon, T.; Baylis, M.; Brown, D. Zika virus and neurological disease—approaches to the unknown. Lancet Infect. Dis. 2016, 16 (4), 402-404. (4) Low J.G., Gatsinga R., Vasudevan S.G., Sampath A. Dengue antiviral development: a continuing journey in dengue and zika: control and antiviral treatment strategies. Adv. Exp. Med. Biol. 2018,1062, 319-332. (5) Castro, M. C.; Wilson, M. E.; Bloom, D. E. Disease and economic burdens of dengue. Lancet Infect. Dis. 2017, 17 (3), 70-78. (6) Aldrich, C. C. The known unknowns of emerging viruses. ACS Infect. Dis. 2016, 2 (5), 310311. (7) Lu, L.; Liu, Q.; Zhu, Y.; Chan, K.-H.; Qin, L.; Li, Y.; Wang, Q.; Chan, J. F.-W.; Du, L.; Yu, F.; Ma, C.; Ye, S.; Yuen, K.-Y.; Zhang, R.; Jiang, S. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor. Nat. Commun. 2014, 5, 3067. (8) Shan, C.; Xie, X.; Muruato, Antonio E.; Rossi, Shannan L.; Roundy, Christopher M.; Azar, Sasha R.; Yang, Y.; Tesh, Robert B.; Bourne, N.; Barrett, Alan D.; Vasilakis, N.; Weaver, Scott C.; Shi, P.-Y. An infectious cDNA clone of zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors. Cell Host Microbe 2016, 19 (6), 891-900. (9) Lou, Z.; Sun, Y.; Rao, Z. Current progress in antiviral strategies. Trends Pharmacol. Sci. 2014, 35 (2), 86-102. (10) Kaufmann, S. H. E.; Dorhoi, A.; Hotchkiss, R. S.; Bartenschlager, R. Host-directed therapies for bacterial and viral infections. Nat. Rev. Drug Discov. 2018, 17, 35-56.


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(11) Martinez, J. P.; Sasse, F.; Brönstrup, M.; Diez, J.; Meyerhans, A. Antiviral drug discovery: broad-spectrum drugs from nature. Nat. Prod. Rep. 2015, 32 (1), 29-48. (12) Bright, R. A.; Shay, D. K.; Shu, B.; Cox, N. J.; Klimov, A. I. Adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states. JAMA 2006, 295 (8), 891-894. (13) Hoffmann, H.-H.; Kunz, A.; Simon, V. A.; Palese, P.; Shaw, M. L. Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (14), 5777-5782. (14) Sun, J.-H.; O’Boyle Ii, D. R.; Fridell, R. A.; Langley, D. R.; Wang, C.; Roberts, S. B.; Nower, P.; Johnson, B. M.; Moulin, F.; Nophsker, M. J.; Wang, Y.-K.; Liu, M.; Rigat, K.; Tu, Y.; Hewawasam, P.; Kadow, J.; Meanwell, N. A.; Cockett, M.; Lemm, J. A.; Kramer, M.; Belema, M.; Gao, M. Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A. Nature 2015, 527 (7577), 245-248. (15) Harvey, R.; Brown, K.; Zhang, Q.; Gartland, M.; Walton, L.; Talarico, C.; Lawrence, W.; Selleseth, D.; Coffield, N.; Leary, J.; Moniri, K.; Singer, S.; Strum, J.; Gudmundsson, K.; Biron, K.; Romines, K. R.; Sethna, P. GSK983: a novel compound with broad-spectrum antiviral activity. Antiviral Res. 2009, 82 (1), 1-11. (16) Wolf, M. C.; Freiberg, A. N.; Zhang, T.; Akyol-Ataman, Z.; Grock, A.; Hong, P. W.; Li, J.; Watson, N. F.; Fang, A. Q.; Aguilar, H. C.; Porotto, M.; Honko, A. N.; Damoiseaux, R.; Miller, J. P.; Woodson, S. E.; Chantasirivisal, S.; Fontanes, V.; Negrete, O. A.; Krogstad, P.; Dasgupta, A.; Moscona, A.; Hensley, L. E.; Whelan, S. P.; Faull, K. F.; Holbrook, M. R.; Jung, M. E.; Lee,


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