Xiaodong Dou1, Qi Sun2, Guofeng Xu1, Yameng Liu1, Caifang Zhang1, Bingding Wang1, Yangbin Lu2, Zheng Guo2, Lingyu Su1, Tongyu Huo1, Xinyi Zhao1, Chen Wang1, Zhongtian Yu2, Song Song1, Liangren Zhang1, Zhenming Liu1, Luhua Lai3, Ning Jiao4. 1. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China. 2. BNLMS, Peking-Tsinghua Center for Life Sciences at College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China. 3. BNLMS, Peking-Tsinghua Center for Life Sciences at College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China; Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China. Electronic address: lhlai@pku.edu.cn. 4. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China. Electronic address: jiaoning@pku.edu.cn.
Abstract
The COVID-19 posed a serious threat to human life and health, and SARS-CoV-2 Mpro has been considered as an attractive drug target for the treatment of COVID-19. Herein, we report 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 Mpro developed by in-house library screening and biological evaluation. Similarity search led to the identification of compound F8-S43 with the enzymatic IC50 value of 10.76 μM. Further structure-based drug design and synthetic optimization uncovered compounds F8-B6 and F8-B22 as novel non-peptidomimetic inhibitors of Mpro with IC50 values of 1.57 μM and 1.55 μM, respectively. Moreover, enzymatic kinetic assay and mass spectrometry demonstrated that F8-B6 was a reversible covalent inhibitor of Mpro. Besides, F8-B6 showed low cytotoxicity with CC50 values of more than 100 μM in Vero and MDCK cells. Overall, these novel SARS-CoV-2 Mpro non-peptidomimetic inhibitors provide a useful starting point for further structural optimization.
The COVID-19 posed a serious threat to human life and health, and SARS-CoV-2 Mpro has been considered as an attractive drug target for the treatment of COVID-19. Herein, we report 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 Mpro developed by in-house library screening and biological evaluation. Similarity search led to the identification of compound F8-S43 with the enzymatic IC50 value of 10.76 μM. Further structure-based drug design and synthetic optimization uncovered compounds F8-B6 and F8-B22 as novel non-peptidomimetic inhibitors of Mpro with IC50 values of 1.57 μM and 1.55 μM, respectively. Moreover, enzymatic kinetic assay and mass spectrometry demonstrated that F8-B6 was a reversible covalent inhibitor of Mpro. Besides, F8-B6 showed low cytotoxicity with CC50 values of more than 100 μM in Vero and MDCK cells. Overall, these novel SARS-CoV-2 Mpro non-peptidomimetic inhibitors provide a useful starting point for further structural optimization.
The outbreak of coronavirus infectious disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been recognized as a serious threat to human life and health [[1], [2], [3]]. As of March 27, 2021, the disease has caused over 480 million people infections with more than 6 million deaths globally [4]. SARS-CoV-2 is a positive-strand RNA enveloped beta-coronavirus, and similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), containing genome encodes non-structural proteins including main protease (Mpro, also known as 3-chymotrypsin-like protease, 3CLpro), papain-like protease (PLpro), helicase, and RNA-dependent RNA polymerase (RdRp) [5,6]. Among them, Mpro is a key enzyme in the viral life cycle, which is involved in the virus’ replication process, and results in the maturation of at least 12 non-structural proteins [[7], [8], [9]]. Furthermore, the highly conserved Mpro in coronavirus and the absence of closely related homologs in humans make Mpro an attractive target for the discovery of broad-spectrum antiviral drugs [10,11].To date, there is only one Mpro inhibitor on market, although a series of SARS-CoV-2 Mpro inhibitors have been identified (Fig. 1
) [10,12]. Among them, the organoselenium derivative 1 (Ebselen), was first disclosed as a covalent inhibitor of SARS-CoV-2 Mpro by screening the approved drugs and drug candidates [13]. It is reported that Ebselen could inhibit SARS-CoV-2 Mpro activity with an IC50 value of 0.67 μM, and SARS-CoV-2 in Vero cells with an EC50 value of 4.67 μM13. A variety of significant peptidomimetic inhibitors of SARS-CoV-2 Mpro have been reported, including compound 2 (N3), 3 (13b), 4 (11a), 5 (Boceprevir), 6 (GC-376), 7 (MI-09), and 8 (PF-07321332), which exhibited high SARS-CoV-2 Mpro inhibitory activity and SARS-CoV-2 inhibition at micromolar to sub-micromolar levels [[13], [14], [15], [16], [17], [18], [19], [20], [21]]. Furthermore, PF-07321332 was approved for mild and moderate symptoms caused by SARS-CoV-2 infection by US Food and Drug Administration (FDA), and PF-07321332 was combined with protease inhibitor and cytochrome P450 3A4 (CYP3A4) inactivator ritonavir to reduce its metabolism by CYP3A4 [21].
Fig. 1
Chemical structures of represented inhibitors of SARS-CoV-2 Mpro.
Chemical structures of represented inhibitors of SARS-CoV-2 Mpro.Although the peptidomimetic inhibitors are highly effective against SARS-CoV-2 Mpro, the stability of hydrolase limited the application of these inhibitors [[21], [22], [23], [24]]. In contrast, the non-peptidomimetic inhibitors are less developed [25,26]. Su et al. and Liu et al. simultaneously reported that compound 9 (Baicalein) was a non-peptidomimetic inhibitor of SARS-CoV-2 Mpro with the enzymatic inhibitory ability and antiviral activity both at micromolar levels [27,28]. Meanwhile, compound 10, an N-substituted isatin derivative, was identified as a potent non-peptidomimetic inhibitor of SARS-CoV-2 Mpro, with an IC50 value of 0.045 μM29. Furthermore, compound 11 (Masitinib), an orally bioavailable tyrosine kinase inhibitor, was revealed as a potent inhibitor of SARS-CoV-2 Mpro, with an IC50 value of 2.50 μM, through screening a library of 1900 clinically safe drugs [30]. Masitinib could block the replication of coronavirus in the lungs and nose of SARS-CoV-2 infected mice [30]. Moreover, Kneller and coworkers reported that compound 12 (HL-3-68) exhibited SARS-CoV-2 Mpro inhibitory activity, with an IC50 value of 0.29 μM, through X-ray/neutron crystallography guided drug design [31]. Additionally, compound 13 (Jun8-76-3A) was discovered as a non-peptidomimetic inhibitor of SARS-CoV-2 Mpro by the modification of known Mpro inhibitor ML188 [32]. Overall, the chemical diversity of the identified non-peptidomimetic inhibitors is still highly limited. Therefore, it is urgent to develop novel non-peptidomimetic inhibitors against SARS-CoV-2 Mpro for the broad-spectrum antiviral drug candidate discovery, especially the new scaffold inhibitors that were not reported in previous coronavirus studies.Herein, we report the identification of 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel non-peptidomimetic inhibitors of SARS-CoV-2 Mpro by screening our in-house library and subsequent similarity search. Interestingly, compound F8 was disclosed with the dissociation constant (KD) value of 27.7 μM, by surface plasmon resonance (SPR) assay, which also exhibited an IC50 value of 21.28 μM by enzymatic assay. Further two-dimensional similarity search based on the structure of compound F8 led to the identification of a series of 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel SARS-CoV-2 Mpro inhibitors. Among them, the most potent compound, F8–S43, exhibited the IC50 value of 10.76 μM against SARS-CoV-2 Mpro. Then, three rounds of optimization based on the structure-based drug design and synthetic modification discovered compounds F8–B6 and F8–B22 as non-peptidomimetic inhibitors of Mpro with IC50 values of 1.57 μM and 1.55 μM, respectively. Moreover, enzymatic kinetic and mass spectrometry studies demonstrated that F8–B6 was a reversible covalent inhibitor of Mpro. Besides, F8–B6 exhibited no obvious cytotoxicity in Vero and MDCK cells with CC50 values over 100 μM. Furthermore, the structure-activity relationship (SAR) of the newly identified scaffold was discussed, which provided useful guidance for further chemical optimization.
Results and discussion
Identification of novel Mpro inhibitors by screening in-house library
To discover novel SARS-CoV-2 Mpro inhibitors, especially the new chemical structures that were not previously reported in coronavirus study, an in-house library was screened by SPR assay (Fig. 2
A). As drawn in Fig. 2B, compound F8 showed the high possibility as a new inhibitor of SARS-CoV-2 Mpro, with the KD value of 27.7 μM. The further enzymatic assay demonstrated that compound F8 inhibited SARS-CoV-2 Mpro activity with an IC50 value of 21.28 μM (Fig. 2C). Similarity analysis of compound F8 showed that it is not similar to any of the previously reported SARS-CoV-2 Mpro inhibitors with the Tanimoto coefficient below 0.14 (Fig. 2D). All of these indicated that the identified non-peptidomimetic inhibitor F8 expanded the chemical space of SARS-CoV-2 Mpro inhibitors and provided a useful starting point for further structural optimization.
Fig. 2
The discovery of SARS-CoV-2 Mpro inhibitors. (A) Screen the in-house library through SPR assay. (B) The chemical structure and KD value of compound F8. (C) The dose-dependent curve of F8 against SARS-CoV-2 Mpro. (D) The similarity analysis of compound F8 with known SARS-CoV-2 Mpro inhibitors.
The discovery of SARS-CoV-2 Mpro inhibitors. (A) Screen the in-house library through SPR assay. (B) The chemical structure and KD value of compound F8. (C) The dose-dependent curve of F8 against SARS-CoV-2 Mpro. (D) The similarity analysis of compound F8 with known SARS-CoV-2 Mpro inhibitors.
Similarity search and preliminary SAR study
To investigate SAR and find more potent inhibitors of this new scaffold, a two-dimensional similarity search was performed based on the chemical structure of compound F8. A total of 70 compounds were captured and purchased from the ChemDiv and SPECS commercial databases. As exhibited in Table 1
, the replacement of the 3-carboxyl phenyl group of F8 with a 3-trifluoromethyl phenyl group (F8–S1), a 2-carboxyl phenyl group (F8–S2), a 2-nitro-4-methoxy phenyl group (F8–S3), a 4-nitrophenyl group (F8–S4), a 3-methyl-4-nitrophenyl group (F8–S5), or a 2,3-dimethyl-4-nitrophenyl (F8–S6), led to a decrease of potency, indicating that the 3-carboxyl phenyl group of F8 was essential for maintaining SARS-CoV-2 Mpro inhibitory activity. Furthermore, the replacement of the R1 group of F8 with a 10-methylacridin-9(10H)-one moiety (F8–S7 to F8–S16), or a poly-substituted pyridine ring (F8–S17 to F8–S20), the inhibitory ability of Mpro was decreased. Meanwhile, the replacement of the R1 group with a 1,2,5-oxadiazol-3-amine group (F8–S22), or a 1-methyl-1H-tetrazol-5-amine group (F8–S24), produced no significant effect on inhibitory potency. Besides, the introduction of the large hydrophobic substituents group (F8–S25, F8–S26, F8–S28 to F8–S39), was not conducive to the maintenance of inhibitory ability, which indicated that the R1 group of F8 was more suitable for small substituents. Notably, the replacement of a urea linker with a thiourea linker was likely to improve the inhibitory potency (F8–S40 to F8–S43). Among them, the most potent compound, F8–S43, displayed an IC50 value of 10.76 μM against SARS-CoV-2 Mpro (Fig. S1).
Table 1
The chemical structures and enzymatic activities of the 43 analogs of F8.
Compounds
R [1]
X
R [2]
Inhibition% (50 μM) a
IC50 ± SD (μM) a
Tideglusib
–
–
–
98.6
0.30 ± 0.02
F8
Image 2
O
Image 3
65.0
21.28 ± 0.89
F8–S1
Image 4
O
Image 5
25.5
N.T. b
F8–S2
Image 6
O
Image 7
41.7
N.T.
F8–S3
Image 8
O
Image 9
<20.0
N.T.
F8–S4
Image 10
O
Image 11
30.1
N.T.
F8–S5
Image 12
O
Image 13
42.9
N.T.
F8–S6
Image 14
O
Image 15
<20.0
N.T.
F8–S7
Image 16
O
Image 17
21.3
N.T.
F8–S8
Image 18
O
Image 19
26.8
N.T.
F8–S9
Image 20
O
Image 21
54.5
N.T.
F8–S10
Image 22
O
Image 23
55.4
N.T.
F8–S11
Image 24
O
Image 25
22.5
N.T.
F8–S12
Image 26
O
Image 27
<20.0
N.T.
F8–S13
Image 28
O
Image 29
<20
N.T.
F8–S14
Image 30
O
Image 31
30.0
N.T.
F8–S15
Image 32
O
Image 33
24.1
N.T.
F8–S16
Image 34
O
Image 35
26.0
N.T.
F8–S17
Image 36
O
Image 37
37.3
N.T.
F8–S18
Image 38
O
Image 39
36.2
N.T.
F8–S19
Image 40
O
Image 41
32.9
N.T.
F8–S20
Image 42
O
Image 43
38.3
N.T.
F8–S21
Image 44
O
Image 45
31.2
N.T.
F8–S22
Image 46
O
Image 47
70.9
25.02 ± 1.11
F8–S23
Image 48
O
Image 49
34.5
N.T.
F8–S24
Image 50
O
Image 51
86.0
17.50 ± 1.65
F8–S25
Image 52
O
Image 53
45.3
N.T.
F8–S26
Image 54
O
Image 55
22.4
N.T.
F8–S27
Image 56
O
Image 57
29.4
N.T.
F8–S28
Image 58
O
Image 59
44.6
N.T.
F8–S29
Image 60
O
Image 61
33.7
N.T.
F8–S30
Image 62
O
Image 63
35.1
N.T.
F8–S31
Image 64
O
Image 65
35.8
N.T.
F8–S32
Image 66
O
Image 67
24.2
N.T.
F8–S33
Image 68
O
Image 69
<20.0
N.T.
F8–S34
Image 70
O
Image 71
<20.0
N.T.
F8–S35
Image 72
O
Image 73
62.8
N.T.
F8–S36
Image 74
O
Image 75
23.3
N.T.
F8–S37
Image 76
O
Image 77
<20.0
N.T.
F8–S38
Image 78
O
Image 79
23.0
N.T.
F8–S39
Image 80
O
Image 81
38.3
N.T.
F8–S40
Image 82
S
Image 83
85.1
10.88 ± 0.16
F8–S41
Image 84
S
Image 85
51.4
N.T.
F8–S42
Image 86
S
Image 87
55.9
N.T.
F8–S43
Image 88
S
Image 89
95.0
10.76 ± 0.48
Data are presented as geometric mean values of at least two independent runs.
Not tested.
The chemical structures and enzymatic activities of the 43 analogs of F8.Data are presented as geometric mean values of at least two independent runs.Not tested.As shown in Table S1, the replacement of the thiourea linker of F8–S43 with a hydrazine linker (F8–S44 to F8–S48), an imine linker (F8–S49 to F8–S52), or a double bond linker (F8–S53 to F8–S65) led to the decrease of inhibitory potency, suggesting that the thiourea linker might be a good skeleton for these newly identified inhibitors. As for the right moiety of F8, the removal of the benzene ring resulted in the loss of potency (F8–S66 to F8–S70), indicating that the benzene ring of F8 was likely to play a hydrophobic role in the binding pocket of SARS-CoV-2 Mpro. As displayed in Table 2
, the replacement of the 4-nitrophenyl group of F8–S43 with a 4-bromobenzenyl group (F8–S43–S1), led to the remarkable loss of inhibitory activity, and the reduction of the volume of the R1 group was beneficial to the preservation of inhibitory activity (F8–S43–S2, IC50 = 8.08 μM; F8–S43–S3, IC50 = 9.69 μM). Notably, the replacement of the thiourea linker to urea or guanidine linker decreased the inhibitory potency (F8–S43–S4 to F8–S43–S6), indicating that the thiourea linker was very important for the maintenance and improvement of the potency. To study the SAR of the right moiety, the hydrophobic substituent of the left moiety and the thiourea linker were fixed according to the structure of F8–S43. As drawn in Table S2, the decrease of the hydrophobic volume of the right moiety was not conducive to the maintenance of activity (F8–S43–S11 to F8–S43–S32). All in all, these results indicate that the newly identified scaffold expanded the chemical diversity of SARS-CoV-2 Mpro inhibitors, which could serve as a starting point for subsequent structural optimization.
Table 2
The chemical structures and enzymatic activities of the 10 analogs of F8–S43.
Compounds
R [1]
X
R [2]
Inhibition% (50 μM) a
IC50 ± SD (μM) a
Tideglusib
–
–
–
98.6
0.30 ± 0.02
F8–S43
Image 91
S
Image 92
95.0
10.76 ± 0.48
F8–S43–S1
Image 93
S
Image 94
27.7
N.T. b
F8–S43–S2
NH2
S
Image 95
81.6
8.08 ± 0.38
F8–S43–S3
NH2
S
Image 96
81.1
9.69 ± 0.45
F8–S43–S4
NH2
O
Image 97
<20.0
N.T.
F8–S43–S5
Image 98
NH
Image 99
20.1
N.T.
F8–S43–S6
Image 100
NH
Image 101
28.7
N.T.
F8–S43–S7
Image 102
S
Image 103
28.4
N.T.
F8–S43–S8
Image 104
S
Image 105
34.2
N.T.
F8–S43–S9
Image 106
S
Image 107
30.2
N.T.
F8–S43–S10
Image 108
S
Image 109
<20.0
N.T.
Data are presented as geometric mean values of at least two independent runs.
Not tested.
The chemical structures and enzymatic activities of the 10 analogs of F8–S43.Data are presented as geometric mean values of at least two independent runs.Not tested.
Rational design and synthetic optimization
Structure-based design of newly identified scaffold
To guide the structural optimization of the newly identified scaffold, the representative compound F8–S43 was docked into the catalytic site of the SARS-CoV-2 Mpro (PDB ID, 7JU7) [30]. As shown in Fig. 3
A, compound F8–S43 was embedded into the catalytic site of SARS-CoV-2 Mpro and occupied the S1 and S2 sites, while the S1′, S3, and S4 sites were not filled with F8–S43. Furthermore, the 4-nitrophenyl moiety of F8–S43 was located at the S1 site and formed a hydrogen bond with His163, and the furan ring was deeply buried in the S2 site and had a π-π stacking interaction with the imidazole of His41 (Fig. 3B and Fig. S2). Meanwhile, the thiourea linker of F8–S43 formed two hydrogen bonds to the backbone carbonyl of Cys44, so the removal or replacement of the thiourea linker led to the loss of inhibitory ability. Besides, the right moiety of F8–S43 was located at the solvent-exposed area and formed a hydrogen bond with Ser46. As drawn in Fig. 3C, the removal of the dihydro-2H-pyrazol-2′-one moiety of F8–S43 had no impact on enzymatic inhibition in the aforementioned similarity search study, so the solvent-exposed region of this scaffold was structurally modified to improve the inhibitory potency of SARS-CoV-2 Mpro, followed by S1 and S2 pockets occupied moieties.
Fig. 3
Predicted binding mode of compound F8–S43. (A) The binding pattern of compound F8–S43 with SARS-CoV-2 Mpro in surface and 2D diagram (B). Hydrogen bonds are represented by yellow lines. Images depicting the proposed binding modes were generated using PyMOL software. (C) The design of newly identified SARS-CoV-2 Mpro inhibitors.
Predicted binding mode of compound F8–S43. (A) The binding pattern of compound F8–S43 with SARS-CoV-2 Mpro in surface and 2D diagram (B). Hydrogen bonds are represented by yellow lines. Images depicting the proposed binding modes were generated using PyMOL software. (C) The design of newly identified SARS-CoV-2 Mpro inhibitors.
Synthesis of target compounds
The target compounds F8-A1 to F8-A9 were synthesized as illustrated in Scheme 1
. Briefly, we synthesized the intermediates (15) from appropriate aryl boronic acid (14) with 5-bromofuran-2-carbaldehyde through Pd(PPh3)4 catalyzed Suzuki-Miyaura cross-coupling reaction [33]. Then, corresponding intermediates (15) reacted with substituted thiosemicarbazide to produce imine linkage (F8-A1 to F8-A9) through modification of published procedures or known methods [34,35].
Scheme 1
Synthetic Route of Compounds F8-A1 to F8-A9. Reagents and conditions: (a) 5-bromofuran-2-carbaldehyde, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) R1NH2CSNHNH2, MeOH, 50 °C, 4 h.
Synthetic Route of Compounds F8-A1 to F8-A9. Reagents and conditions: (a) 5-bromofuran-2-carbaldehyde, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) R1NH2CSNHNH2, MeOH, 50 °C, 4 h.The synthetic routes of the target compounds F8–B1 to F8–B13 were described in Scheme 2
. The building blocks (17 and 19) were prepared following previously described procedures [33]. Shortly, the starting material 5-iodofuran-2-carbaldehyde (16) or (5-formylfuran-2-yl)boronic acid (18) reacted with (4-carbamoylphenyl)boronic acid or substituted bromobenzene to form corresponding intermediates (17 and 19) through Suzuki-Miyaura cross-coupling reaction. Then, the aldehyde group of intermediates (17 and 19) condensed with the amino group of thiosemicarbazide to form the imine linkage giving the target compounds (F8–B1 to F8–B13).
Scheme 2
Synthetic Route of Compounds F8–B1 to F8–B13. Reagents and conditions: (a) (4-carbamoylphenyl)boronic acid, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h; (c) Substituted bromobenzene, Pd(PPh3)2Cl2, Na2CO3, MeCN/H2O, 90 °C, overnight; or substituted bromobenzene, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; or Substituted bromobenzene, Pd(PPh3)2Cl2, 2 M Na2CO3, DME/EtOH, 60 °C, overnight.
Synthetic Route of Compounds F8–B1 to F8–B13. Reagents and conditions: (a) (4-carbamoylphenyl)boronic acid, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h; (c) Substituted bromobenzene, Pd(PPh3)2Cl2, Na2CO3, MeCN/H2O, 90 °C, overnight; or substituted bromobenzene, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; or Substituted bromobenzene, Pd(PPh3)2Cl2, 2 M Na2CO3, DME/EtOH, 60 °C, overnight.As drawn in Scheme 3
, the synthetic methods of F8–B14 to F8–B21 were similar to the aforementioned synthesis of compounds F8–B1 to F8–B13. Simply, 5-bromofuran-2-carbaldehyde (20) reacted with corresponding aryl boronic acid to form intermediates (21), then the newly prepared intermediates condensed with thiosemicarbazide to give target compounds F8–B14 to F8–B21. Moreover, the intermediate (23), 5-benzylfuran-2-carbaldehyde, was synthesized by the starting material 5-(hydroxymethyl)furan-2-carbaldehyde (22) reacted with benzene through substitution reaction following previously described procedures [36]. Then, the intermediate (23) was condensed with thiosemicarbazide to give the target compound F8–B22.
Scheme 3
Synthetic Route of Compounds F8–B14 to F8–B22. Reagents and conditions: (a) Corresponding aryl boronic acid, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h; (c) benzene, trifluoromethanesulfonic acid, r.t.
Synthetic Route of Compounds F8–B14 to F8–B22. Reagents and conditions: (a) Corresponding aryl boronic acid, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h; (c) benzene, trifluoromethanesulfonic acid, r.t.As shown in Scheme 4
, the synthetic methods of F8–C1 to F8–C4 were similar to the aforesaid synthesis of compounds F8–B1 to F8–B13. Briefly, the appropriate aryl bromides (24) reacted with (4-carbamoylphenyl)boronic acid to form intermediates (25) through Suzuki-Miyaura cross-coupling reaction. Then, the corresponding intermediates condensed with thiosemicarbazide to give target compounds F8–C1 to F8–C4. All of the synthesized target compounds were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) experiments as single substances.
Scheme 4
Synthetic Route of Compounds F8–C1 to F8–C4. Reagents and conditions: (a) (4-carbamoylphenyl)boronic acid, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h.
Synthetic Route of Compounds F8–C1 to F8–C4. Reagents and conditions: (a) (4-carbamoylphenyl)boronic acid, Pd(PPh3)4, K2CO3, PhMe/EtOH/H2O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h.
Biological evaluation and SAR study
The synthesized target compounds were evaluated through aforesaid enzymatic assay, and the results were summarized in Table 3
. To investigate the SAR of the solvent-exposed region of the newly identified scaffold, the other regions were fixed with the same substituents. Notably, with the replacement of the 3-carboxyl phenyl group of F8-A1 with a 4-carboxyl phenyl group (F8-A2), the inhibitory potency was maintained. Furthermore, the introduction of hydrophobic substituents, including methyl, 4-chlorophenyl, 3-trifluoromethyl phenyl, and cyclohexyl amino groups (F8-A3 to F8-A9), led to decreased inhibitory ability. Thus, the R1 group of this scaffold was fused with a hydrogen atom, and the R2 group was further structural modified to enhance the inhibitory ability and explore explicit SAR.
Table 3
The chemical structures and enzymatic activities of F8-A1 to F8-A9.
Compounds
R [1]
R [2]
Inhibition% (50 μM) a
IC50 ± SD (μM) a
F8-A1 (F8–S40)
H
3-COOH
85.1
10.88 ± 0.16
F8-A2
H
4-COOH
89.4
17.53 ± 3.98
F8-A3
Image 111
3-COOH
22.2
N.T. b
F8-A4
Image 112
4-COOH
39.5
N.T.
F8-A5
Image 113
3-COOH
43.8
N.T.
F8-A6
Image 114
4-COOH
27.5
N.T.
F8-A7
Image 115
3-COOH
43.0
N.T.
F8-A8
Image 116
4-COOH
38.6
N.T.
F8-A9
Image 117
4-COOH
37.3
N.T.
Data are presented as geometric mean values of at least two independent runs.
Not tested.
The chemical structures and enzymatic activities of F8-A1 to F8-A9.Data are presented as geometric mean values of at least two independent runs.Not tested.As for the phenyl moiety of the newly identified scaffold (Table 4
), the replacement of 4-carboxyl phenyl group of F8-A2 with a 4-amide phenyl group (F8–B1), a 4-sulfonyl phenyl group (F8–B2), or a phenyl group (F8–B3), resulted in moderate improve in inhibitory activity. When the benzene ring had multiple substituents (F8–B4 to F8–B14), the polar substituents, including carboxyl group, hydroxyl group, and methoxy group, were favorable to maintain or promote the inhibitory ability. Among them, the 3-hydroxy-4-carboxyl phenyl group substituted compound F8–B6 exhibited good activity, with the IC50 value of 1.57 μM (Fig. S1). Whereas the replacement of the 4-carboxyl phenyl group of F8-A2 with a 3, 4-dichlorobenzyl group (F8–B15), led to the loss of inhibitory potency, which meant that the phenyl moiety of the newly identified scaffold was not suitable to be replaced by a completely hydrophobic substituent. Furthermore, this moiety was compatible with pyridine or substituted pyridine rings (F8–B16 to F8–B17). Intriguingly, the naphthalene ring substitution was not conducive to the preservation of inhibitory activity (F8–B18 to F8–B19), while the quinoline ring did not affect the potency (F8–B20 to F8–B21). Therefore, the aforementioned moiety might be more suitable for nitrogen heterocyclic replacement. Notably, the replacement of the 4-carboxyl phenyl group of F8-A2 with a benzyl group (F8–B22), led to the enhancement of potency, and the compound F8–B22 displayed an IC50 value of 1.55 μM (Fig. S1), which potency was 13-fold more than the initial compound F8.
Table 4
The chemical structures and enzymatic activities of F8–B1 to F8–B22.
Compounds
R [1]
Inhibition% (50 μM) a
IC50 ± SD (μM) a
F8-A2
Image 119
89.4
17.53 ± 3.98
F8–B1
Image 120
94.2
4.00 ± 0.25
F8–B2
Image 121
98.5
5.39 ± 0.14
F8–B3
Image 122
100.0
4.05 ± 0.26
F8–B4
Image 123
100.6
8.14 ± 1.14
F8–B5
Image 124
98.5
4.69 ± 0.34
F8–B6
Image 125
102.2
1.57 ± 0.08
F8–B7
Image 126
89.0
32.53 ± 2.33
F8–B8
Image 127
84.4
40.59 ± 1.06
F8–B9
Image 128
93.6
25.39 ± 2.05
F8–B10
Image 129
97.2
10.0 ± 0.50
F8–B11
Image 130
79.8
N.T. b
F8–B12
Image 131
114.8
9.09 ± 1.05
F8–B13
Image 132
106.6
4.14 ± 0.16
F8–B14
Image 133
102.5
5.81 ± 0.07
F8–B15
Image 134
49.8
N.T.
F8–B16
Image 135
95.4
7.09 ± 0.51
F8–B17
Image 136
97.4
8.76 ± 0.75
F8–B18
Image 137
73.2
N.T.
F8–B19
Image 138
54.1
N.T.
F8–B20
Image 139
92.3
10.40 ± 2.20
F8–B21
Image 140
88.9
10.30 ± 0.70
F8–B22
Image 141
100.1
1.55 ± 0.08
Data are presented as geometric mean values of at least two independent runs.
Not tested.
The chemical structures and enzymatic activities of F8–B1 to F8–B22.Data are presented as geometric mean values of at least two independent runs.Not tested.To investigate the furan ring moiety of the newly identified scaffold, the benzene ring moiety was designed to fix with the benzyl group, as the compound F8–B22 displayed an IC50 value of 1.55 μM. However, the designed compounds were difficult to synthesize, so the furan ring moiety was optimized with the 4-carboxyl phenyl group replacement. As drawn in Table 5
, the replacement of 2,5-position substituted furan ring of F8-A2 with a 2,4-position substituted furan ring (F8–C1), a 2,5-position substituted thiophene ring (F8–C2), or a 1,3-position substituted benzene ring (F8–C3), let to maintain or improve inhibitory potency. Whereas, the 1,4-position substituted benzene ring (F8–C4) was not conducive to the preservation of inhibitory activity, indicating that the furan ring moiety acted as an important connecting linker and prefer to three atomic lengths. It is noteworthy that the introduction of a substituent to the furan, thiophene, or benzene ring might be beneficial to the improvement of activity, however the synthesis was too difficult to achieve. Thus, we are trying to perform scaffold hopping based on this scaffold to improve the synthetic accessibility and inhibitory potency in the future.
Table 5
The chemical structures and enzymatic activities of F8–C1 to F8–C4.
Compounds
Ar
Inhibition% (50 μM) a
IC50 ± SD (μM) a
F8-A2
Image 143
89.4
17.53 ± 3.98
F8–C1
Image 144
90.9
24.56 ± 0.70
F8–C2
Image 145
95.1
5.78 ± 0.30
F8–C3
Image 146
99.2
5.44 ± 0.16
F8–C4
Image 147
64.0
N.T. b
Data are presented as geometric mean values of at least two independent runs.
Not tested.
The chemical structures and enzymatic activities of F8–C1 to F8–C4.Data are presented as geometric mean values of at least two independent runs.Not tested.Meanwhile, the mechanism of action of these newly identified inhibitors was investigated through enzymatic kinetic and mass spectrometry assays. As exhibited in Fig. 4
A and B, the Lineweaver-Burk plot with different F8–B6 concentrations yielded an intercept at the X-axis, indicating that F8–B6 is a non-competitive inhibitor of SARS-CoV-2 Mpro. Moreover, prolonged incubation of SARS-CoV-2 Mpro with F8–B6 exhibited a time-dependent increase of inhibition activity (Figs. S3A–3C). The addition of dithiothreitol (DTT) could reverse the inhibitory effect of F8–B6 (Fig. S3D). Further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that F8–B6 covalently bonded to SARS-CoV-2 Mpro (Fig. 4C), which was similar to the previously reported covalent inhibitor Ebselen [13], with more than one F8–B6 can be covalently bonded to the dimer of Mpro. Notably, thiacetazone moiety was identified as a covalent warhead to cystine in previous work [37,38], and this was consistent with the above SAR that the introduction of thiacetazone moiety led to the increase of inhibitory activity. However, the specific covalent mechanism still needed to be further studied. In addition, reversibility assay of F8–B6 to SARS-CoV-2 Mpro revealed that the ultrafiltration of inhibitor could recover enzymatic activity to a certain extent (Fig. 4D), which indicated that F8–B6 is a reversible inhibitor. Overall, the enzymatic kinetic and mass spectrometry studies demonstrated that F8–B6 is a reversible covalent inhibitor of SARS-CoV-2 Mpro. Interestingly, during the review process of this manuscript, Xu and coworkers reported that thiosemicarbazone is a promising scaffold for the inhibition of SARS-CoV-2 Mpro activity [39]. Besides, thiosemicarbazones are also well-known inhibitors of human Cathepsins. Pandey and coworkers reported that thiosemicarbazones derivatives are inhibitors of Cathepsin B, H, and L through a multi-target approach [40]. Notably, Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against [41,42]. Thus, the selectivity of compounds F8–B6 and F8–B22 against Cathepsin L was investigated through previously reported method [43], and the results demonstrated that compounds F8–B6 and F8–B22 exhibited moderate inhibition of Cathepsin L, with the IC50 values of 16.33 μM and 8.09 μM, respectively (Fig. S4).
Fig. 4
Michaelis-Menten kinetics analysis (A) and Lineweaver-Burk plot (B) of SARS-CoV-2 Mpro in presence or absence of F8–B6. (C) The liquid chromatograph-mass spectrometer of SARS-CoV-2 Mpro with F8–B6. (D) Reversibility assay of the inhibition ability of F8–B6 against SARS-CoV-2 Mpro.
Michaelis-Menten kinetics analysis (A) and Lineweaver-Burk plot (B) of SARS-CoV-2 Mpro in presence or absence of F8–B6. (C) The liquid chromatograph-mass spectrometer of SARS-CoV-2 Mpro with F8–B6. (D) Reversibility assay of the inhibition ability of F8–B6 against SARS-CoV-2 Mpro.Meanwhile, two compounds F8–B6 and F8–B22, with good SARS-CoV-2 Mpro inhibitory abilities, were further evaluated for their cytotoxic activities in Vero and MDCK cells by MTT assay. As drawn in Fig. 5
A and B, both compounds F8–B6 and F8–B22 exhibited low cytotoxicity, and F8–B6 showed the CC50 values more than 100 μM in Vero and MDCK cells, while F8–B22 displayed the CC50 values around 100 μM in Vero cells, and above 100 μM in MDCK cells.
Fig. 5
The cytotoxicity of compounds F8–B6 and F8–B22 in Vero (A) and MDCK cells (B).
The cytotoxicity of compounds F8–B6 and F8–B22 in Vero (A) and MDCK cells (B).
Conclusion
In the current work, a series of 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives were identified as non-peptidomimetic inhibitors of SARS-CoV-2 Mpro through screening an in-house library by SPR and enzymatic assays. Further similarity search led to the identification of compound F8–S43, which exhibited an IC50 value of 10.76 μM against SARS-CoV-2 Mpro. Then, three rounds of optimization based on the structure-based drug design and synthetic modification discovered compounds F8–B6 and F8–B22 as non-peptidomimetic inhibitors of Mpro with IC50 values of 1.57 μM and 1.55 μM, respectively. Moreover, enzymatic kinetic and mass spectrometry analysis demonstrated that F8–B6 was a reversible covalent inhibitor of Mpro. Besides, F8–B6 displayed no obvious cytotoxicity in Vero and MDCK cells. Taken together, this chemical series may serve as a good starting point for the further optimization of SARS-CoV-2 Mpro non-peptidomimetic inhibitors.
Experimental section
Chemistry
General method. Synthesis reagents and solvents were obtained from commercial suppliers and used without further purification. Characterizations of compounds are provided in the Supporting Information. 1H and 13C NMR spectra were recorded on Bruker (400 MHz) instruments, using dimethyl sulfoxide (DMSO‑d
6) as solvents. Chemical shifts are given in parts per million (ppm) downfield from tetramethylsilane (δ) as the internal standard in deuterated solvent and coupling constants (J) are in Hertz (Hz). Data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, ddd = doublet of doublet of doublet, dt = doublet of triplet, m = multiplet, bs = broad signal), and coupling constants. High-resolution mass spectra were recorded on a Bruker Apex IV FTMS mass spectrometer using electrospray ionization (ESI). All compounds tested in biological assays were >95% pure.
General procedure A
A schlenk tube was charged with 5-bromofuran-2-carbaldehyde (1 mmol), corresponding boronic acid (1.2 mmol), tetrakis(triphenylphosphine)palladium (0.05 mmol), and potassium carbonate (3 mmol). The vessel was evacuated and backfilled with argon. A mixed solvent of Toluene/EtOH/H2O (5/5/2 mL) was added. Then the schlenk tube was heated to 90 °C overnight. After cooling to room temperature, the reaction mixture was concentrated and diluted with water, filtered and the filtrate was adjusted to pH 2 with 2 N HCl. The precipitate was filtered and dried in vacuo to give the crude products without further purification.
General procedure B
A schlenk tube was charged with (5-formylfuran-2-yl)boronic acid (1.5 mmol), substituted bromobenzene (1 mmol), Pd(PPh3)2Cl2 (0.05 mmol), and sodium carbonate (2 mmol). The vessel was evacuated and backfilled with argon. A mixed solvent of MeCN/H2O (4/1.3 mL) was added. Then the schlenk tube was heated to 60 °C overnight. After cooling to room temperature, the reaction mixture was concentrated and diluted with water, filtered and the filtrate was adjusted to pH 2 with 2 N HCl. The precipitate was filtered and dried in vacuo to give the crude products without further purification.
General procedure C
A mixture of (5-formylfuran-2-yl)boronic acid (1.5 mmol), substituted bromobenzene (1 mmol), Pd(PPh3)2Cl2 (0.05 mmol) in DME/EtOH (3/3 mL), and 2 M aqueous Na2CO3 (3 mL, 6 mmol of Na2CO3) was flushed with nitrogen for 3 min and heated at 60 °C overnight under nitrogen atmosphere. The solvents were removed under reduced pressure, the residue was dissolved in water, the mixture obtained was filtered through Celite, and the filtrate was adjusted to pH 2 with 2 N HCl. The precipitate was filtered and dried in vacuo to give the crude products without further purification.
General procedure D
To a solution of the corresponding aldehyde (1.0 mmol) in MeOH (5.0 mL) was added thiosemicarbazide or substituted thiosemicarbazide (1.05 mmol). The mixture was heated to 50 °C and stirred for 4 h. The residue was recrystallized from ethanol, and the precipitate was filtered and dried in vacuo to give target compounds.
5-hydroxymethyl-2-furfuraldehyde (0.31 g, 2.45 mmol) was added to the mixture of TfOH (4 mL) and benzene (0.28 mL) in an ice bath. The reaction mixture was stirred at room temperature for 2 h. The mixture was poured into water (30 mL), and extracted with chloroform (3 × 30 mL). The combined extracts were washed with water, the saturated aqueous solution of NaHCO3, water again, and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was subjected to chromatographic separation on silica gel to give intermediate 5-(phenylmethyl)furan-2-carbaldehyde 0.1 g, yielding 21%. Then following the general procedure D, compound F8–B22 was obtained in 42% yield. Yellowish solid. 1H NMR (400 MHz, DMSO‑d
6) δ 11.38 (s, 1H), 8.17 (s, 1H), 7.89 (s, 1H), 7.55 (s, 1H), 7.37–7.19 (m, 5H), 6.88 (d, J = 3.6 Hz, 1H), 6.26 (d, J = 3.6 Hz, 1H), 4.02 (s, 2H). 13C NMR (100 MHz, DMSO‑d
6) δ 177.98, 157.32, 148.87, 138.02, 133.07, 129.08, 129.01, 127.03, 114.52, 109.71, 34.18. HRMS (ESI) [M − H]- calcd for C13H12N3OS−: 258.0707; found: 258.0699.
Tideglusib was purchased from Topscience Co. Ltd. SARS-CoV-2 Mpro fluorescent substrate Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH2 was synthesized by GL (Shanghai) Biochem Ltd. (Shanghai, China). Compounds used for SARS-CoV-2 Mpro inhibitors screening were purchased from ChemDiv () and SPECS () commercial databases.
Cloning, expression, and purification of SARS-CoV-2 Mpro
The full-length gene encoding SARS-CoV-2 Mpro was synthesized for Escherichia coli (E. coli) expression (Hienzyme Biotech). The expression and purification of SARS-CoV-2 Mpro were carried out using the reported protocol [29].
SPR assay
The SPR assay was used to analyze the interaction between SARS-CoV-2 Mpro with compounds on a Biacore 8 K instrument (GE Healthcare). SARS-CoV-2 Mpro was immobilized on a sensor chip (CM5) via Amine Coupling Kit (GE Healthcare, Buckinghamshire, UK) at levels of approximately 10,000 response units (RU). The first flow channel without immobilized protein was set as a reference, and the compounds were injected at the concentration of 50 μM for screening and 0.2 μM–100 μM for binding study in a period of 60 s. Dissociation was measured for 100–200 s at a flow rate of 30 μL/min using the following assay running buffer: 10 mM phosphate buffer containing 2.7 mM KCl, 137 mM NaCl, and 0.05% surfactant P20 (pH 7.5). All of the data were analyzed through Biacore evaluation software (8 K version 1.0), and the curve was fitted with a 1:1 kinetics binding model.
Enzymatic assay of SARS-CoV-2 Mpro
A fluorescent substrate Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH2 (GL Biochemistry Ltd) and assay buffer (40 mM PBS, 100 mM NaCl, 1 mM EDTA, 0.1% Triton 100, pH 7.3) was used for the inhibition assay. For the preliminary screening and IC50 measurements, 0.5 μM protease was incubated with inhibitor at room temperature for 30 min, and then the reaction was initiated by adding 20 μM substrate. The fluorescence signal generated by the cleavage of the substrate was monitored for 20 min at an emission wavelength of 460 nm with excitation at 360 nm using a plate reader (Synergy, Biotek). IC50 values were fitted with the Hill1 function of Origin 2018. For the enzymatic kinetic assay, 0.5 μM SARS-CoV-2 Mpro was pre-incubated with DMSO or F8–B6 for 3 h and different concentrations of the fluorescent substrate were added to initiate the reaction. Data was collected from three replicates and curve-fitted by Origin 2018. For the DTT assay, 0.5 μM SARS-CoV-2 Mpro was premixed with 400 mM DTT or H2O. Then, the protease solution was co-preincubated with DMSO or inhibitor at various concentrations for 30 min. Enzyme activity was tested and data was collected from three replicates. For time-dependent inhibitory measurement, various concentrations of inhibitors were pre-incubated with SARS-CoV-2 Mpro at a different time at room temperature before the addition of fluorescent substrate.
Mass spectrometry
2 μM protease was co-incubated with 15 μM F8–B6 on ice for 3 h. Then, the complex solution was analyzed by Quadrupole-TOF LC-MS/MS System (Vion, Waters). Raw data of mass signal was deconvoluted to obtain the total mass of the protein.
Enzymatic reversibility assay
SARS-CoV-2 Mpro (10 μM) was incubated with 100 μM F8–B6 for 180 min and divided into four parts which were ultra-filtrated for different times. In each time of ultrafiltration, an equal volume of buffer used in the enzyme activity assay was added into the protease solution and ultra-filtrated together for 5 min at 4 °C, 12,000 rpm. Protease left on the upper layer of Millipore was collected and diluted to the final concentration of 0.5 μM and enzyme activity was tested. All data were collected from three replicates to obtain average enzymatic activities and error bars.
Cathepsin L inhibition assay
The inhibition assay of Cathepsin L was performed as previously reported method [41,43]. Briefly, compounds F8–B6 and F8–B22 were tested using the commercial Cathepsin L Inhibitor Assay Kit (Abcam, Cat# ab197012). And the known Cathepsin L inhibitor FF-FMK was used as the positive compound.
Cell culture
Vero cells were cultured in (MEM, M&C Gene Technology, Beijing, China) and MDCK cells were cultured in Minimum Essential Medium Dulbecco's modified Eagle's medium (DMEM, M&C Gene Technology, Beijing, China), supplemented with 10% fetal bovine serum (FBS) (PAN Seratech, Aidenbach, Germany) and 100 U/mL penicillin/streptomycin (P/S), and maintained in a humidified atmosphere of 95% air and 5% CO2.
Cell viability assay
MTT assay was performed to examine the cell viability of the newly identified SARS-CoV-2 Mpro inhibitors. Vero and MDCK cells were seeded in 96-well plates. After 18 h incubation, SARS-CoV-2 Mpro inhibitors were added to each well. Corresponding vehicles were simultaneously added for blank well. Cell viability was determined 48 h after drug treatment.
Molecular docking
All of the chemical structures were processed using the LigPrep module in Schrödinger 10.2 software (Schrodinger, LLC, NY, USA) [46]. The OPLS3 force field was adopted to perform energy minimization. Default settings were used for all other parameters. The crystallographic structures of the Mpro in complex with inhibitors were retrieved from the RCSB Protein Data Bank (PDB) and crystal structures (PDB IDs: 7JUJ) were prepared by the Protein Preparation Wizard module in Schrödinger 10.2 software. Default settings were used for all parameters based on the OPLS3 force field. The molecular docking was performed using the Glide module with XP (extra precision) in Schrödinger 10.2 software with default settings for all other parameters [47,48].
Similarity search
The similarity search was performed using Pipeline Pilot 8.5 of Accelrys. The similarities were calculated through the Tanimoto coefficient based on the fingerprint ECFP_6 of each structure. Then, 70 analogs of compound F8 and 32 analogs of compound F8–S43 were captured and purchased for biological evaluation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Authors: Dafydd R Owen; Charlotte M N Allerton; Annaliesa S Anderson; Lisa Aschenbrenner; Melissa Avery; Simon Berritt; Britton Boras; Rhonda D Cardin; Anthony Carlo; Karen J Coffman; Alyssa Dantonio; Li Di; Heather Eng; RoseAnn Ferre; Ketan S Gajiwala; Scott A Gibson; Samantha E Greasley; Brett L Hurst; Eugene P Kadar; Amit S Kalgutkar; Jack C Lee; Jisun Lee; Wei Liu; Stephen W Mason; Stephen Noell; Jonathan J Novak; R Scott Obach; Kevin Ogilvie; Nandini C Patel; Martin Pettersson; Devendra K Rai; Matthew R Reese; Matthew F Sammons; Jean G Sathish; Ravi Shankar P Singh; Claire M Steppan; Al E Stewart; Jamison B Tuttle; Lawrence Updyke; Patrick R Verhoest; Liuqing Wei; Qingyi Yang; Yuao Zhu Journal: Science Date: 2021-11-02 Impact factor: 47.728
Authors: Naoya Kitamura; Michael Dominic Sacco; Chunlong Ma; Yanmei Hu; Julia Alma Townsend; Xiangzhi Meng; Fushun Zhang; Xiujun Zhang; Mandy Ba; Tommy Szeto; Adis Kukuljac; Michael Thomas Marty; David Schultz; Sara Cherry; Yan Xiang; Yu Chen; Jun Wang Journal: J Med Chem Date: 2021-04-23 Impact factor: 8.039
Authors: Sang Hoon Han; Christopher M Goins; Tarun Arya; Woo-Jin Shin; Joshua Maw; Alice Hooper; Dhiraj P Sonawane; Matthew R Porter; Breyanne E Bannister; Rachel D Crouch; A Abigail Lindsey; Gabriella Lakatos; Steven R Martinez; Joseph Alvarado; Wendell S Akers; Nancy S Wang; Jae U Jung; Jonathan D Macdonald; Shaun R Stauffer Journal: J Med Chem Date: 2021-08-04 Impact factor: 7.446
Fig. 1
Fig. 2
Fig. 3
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Fig. 4
Fig. 5