Design, Synthesis, and Anti-SARS-CoV-2 Activity of Amodiaquine Analogs

Abstract

The pandemic of coronavirus disease 2019, caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a serious concern worldwide. Although some effective vaccines have been developed, only a few anti-SARS-CoV-2 drugs have been approved for their clinical use. In this study, we designed and synthesized new anti-SARS-CoV-2 drugs based on the chemical structure of amodiaquine, which is known as an antimalarial drug. Consequently, we have identified amodiaquine analogs functionalized with dialkylamino-pendant aminophenol moieties that possess a high level of anti-SARS-CoV-2 activity with a low level of toxicity.

Introduction

The coronavirus disease 2019 (COVID-19) pandemic, which began in the winter of 2019, has been characterized by severe pneumonia and related symptoms caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and it remains a serious threat to humanity worldwide.^1–3) In order to overcome this situation, the first approach has included an attempt at the development of various anti-SARS-CoV-2 drugs, and some drugs have been approved by the Food and Drug Administration in the U.S.A. as well as in several countries.^4–7)

One of the representative examples of anti-SARS-CoV-2 drugs is remdesivir, which is a nucleoside analog with broad-spectrum antiviral activity against Ebola virus and coronaviruses.^8–13) The inhibitory effect of remdesivir relies on the inhibition of the viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, which is one of the enzymes essential for viral replication.^14,15) In addition, molnupiravir (MPV) has been approved as an orally administrable agent for the treatment of COVID-19.^16–19) MPV (EIDD-2801) is a ribonucleoside-type prodrug of N-hydroxycytidine, which exhibits a similar inhibitory effect on SARS-CoV-2 RdRp and induces the mutation of viral genomes.

The second approach is the design and synthesis of new inhibitors of the main protease (Mpro) of SARS-CoV-2, such as nirmatrelvir (PF-07321332)^20,21) and ensitrelvir (S-217622),^22–24) as well as the repurposing of known viral protease inhibitors such as nelfinavir,²⁵⁾ which had been clinically used as an anti-human immunodeficiency virus drug, in combination with cepharanthine. These agents, however, have some potential drawbacks such as many contraindications with other drugs and the induction of drug resistance.^26–28) In addition to these anti-SARS-CoV-2 agents, various efforts have been made to develop new small-molecule drugs including peptide mimetics such as cyclic peptides,^29–36) vitamin K derivatives,³⁷⁾ and chemokine receptor CCR5 antagonists.³⁸⁾

Quinoline derivatives are known to be an important class of heterocyclic compounds that possess a wide range of biological properties. Typical quinoline-based drugs include chloroquine (CQ), hydroxychloroquine (HCQ), mefloquine (MQ), primaquine, and amodiaquine (ADQ), which have been known not only as antimalarial agents^39–44) but also as anticancer and antiviral agents^45–49) (Chart 1). In the initial stages of COVID-19, the anti-SARS-CoV-2 activity of these antimalarial agents^50–54) and the simultaneous use of MQ with nelfinavir were proposed.⁵⁵⁾ Although it has been reported that CQ, HCQ, MQ, and their analogs exhibit moderate to high antiviral activity against SARS-CoV-2 in vitro, possibly via the inhibition of endosome acidification,⁵⁶⁾ these drugs have not shown efficacy in human bodies. Also, there could be inevitable adverse effects due to the requirement of dosages that are higher than what is prescribed for their intended purpose.

Chart 1. Representative Approved Drugs with Quinoline Moieties

ADQ has been reported to exhibit antiviral activity against various RNA viruses, such as ebolavirus^57,58) and severe fever with thrombocytopenia syndrome virus (SFTSV),^59–61) and its anti-SARS-CoV-2 activity has recently been reported.^{50,53,62–64)} Furthermore, an in vivo study using SARS-CoV-2-infected hamsters has indicated that the infection is more effectively inhibited by ADQ than by chloroquine. In addition, ADQ and some ADQ derivatives have been reported to inhibit the production of interleukin-6 (IL-6) from J774.1 murine macrophages via the activation of Toll-like receptors (TLR3, TLR7, TLR8, and TLR9).⁶⁵⁾ However, the use of ADQ has been limited due to its severe side effects such as hepatotoxicity and agranulocytosis caused by its metabolites.^66,67)

Therefore, we have started designing and synthesizing more potent and less toxic anti-SARS-CoV-2 drug candidates based on ADQ scaffolds. As indicated in Chart 1, the structure of ADQ is composed of 3 parts, quinoline, aminophenol, and alkylamino moieties on the aminophenol units. The aminophenol moiety of ADQ metabolizes to afford reactive quinone imine species, which bind to cellular proteins to induce organ dysfunction.^68,69) In addition, the ethyl groups on the alkylamino moiety of ADQ undergo oxidative dealkylation to produce desethylamodiaquine,⁷⁰⁾ which is more cytotoxic to hepatic cells compared with the effect of ADQ itself.⁷¹⁾ In this manuscript, we describe the design and synthesis of more potent and less toxic ADQ derivatives for the development of novel anti-SARS-CoV-2 agents.

Results and Discussion

Design and Synthesis of ADQ Derivatives

We began the design and synthesis of compounds 1a–j, which possess a 4-(quinolin-4-ylamino)phenol scaffold (Chart 2) (the synthesis of 1a was previously reported in Ref. 65). Note that the synthesis and antiviral activity of only representative compounds are reported in this manuscript, although many antiviral drug candidates were synthesized by our research group. For example, the electron-withdrawing group such as halogens and the trifluoromethyl group were introduced into compounds 1b, 1c, 1d, 1e, 1g, 1h, and 1j to suppress the oxidation of their 4-aminophenol units in vivo, which affords reactive quinone imines.⁶⁹⁾ In addition, compounds 1h and 1j, bearing 7-trifluoromehtyl- and 7-iodoquinoline moieties, respectively, were synthesized to assess their effects.

Chart 2. Structures of the ADQ Derivatives Synthesized in This Work

Next, ADQ derivatives 2a–e bearing 2- or 3-aminophenol, 4-(N,N-dimethyl)aniline, and 4-mercaptophenol, were synthesized, because the change in the position of phenolic alcohol and its replacement with other functional groups may prevent the production of quinone imine species.⁷²⁾ Furthermore, we also designed 3a–d bearing various alkyl chains in alkylamino units. In addition, we synthesized 3e–i bearing 7-bromo and 7-iodoquinoline rings and 3j bearing non-substituted quinoline parts for comparison (Chart 2).

The synthesis of ADQ derivatives was carried out as shown in Charts 3–5. Initially, we synthesized compounds 1a–j from aminophenols 4a–g, as shown in Chart 3. The microwave-assisted condensation reactions⁷³⁾ of 4a–g with 4,7-dichloroquinoline 5a yielded HCl salts of 1a–g, respectively. In addition, 4e was reacted with 5b and 5c to afford 1h and 1i as HCl salts, respectively, and 4a was reacted with 5d to give 1j. In a similar manner, compounds 2a–e were synthesized from the corresponding anilines 6a–d and thiophenol 6e (Chart 3).

Chart 3. Synthesis of 1a–j and 2a–e

Chart 4. Synthesis of 3a–e

Chart 5. Synthesis of 3f–j

Next, ADQ derivatives bearing various alkylamino side chains were synthesized as shown in Charts 4 and 5. The Betti reactions⁷⁴⁾ of the HCl salts of the 7-chloroquinoline derivative 1a with secondary amines such as 7a–d gave 3a–d, respectively⁷⁵⁾ (Chart 4). In a similar manner, the 7-iodoquinoline derivative 1j was reacted with 7c to afford 3e.

Because the chemical yields in Chart 4 were not satisfactory, another synthetic route was examined as presented in Chart 5, in which 2-hydroxy-5-nitrobenzyl bromide 8 was reacted with secondary amines 7b and 7e to give 9a and 9b, respectively. The reduction of the nitro groups in 9a and 9b to amino groups by treatment with SnCl₂ and the successive reactions with 5c, 5d, and 5e afforded 3f–j (3f, 3g, and 3h were previously reported as anti-SFTSV agents in Refs. 59, 61).

Anti-SARS-CoV-2 Activity of the Synthesized Compounds

The antiviral activity of the synthesized compounds was evaluated in VeroE6 cells expressing TMPRSS2 (VeroE6/TMPRSS2), as established by Matsuyama et al.⁷⁶⁾ TMPRSS2 is a hydrolase that cleaves the S proteins of SARS-CoV-2 at the S1/S2 and S′2 sites involved in infection to the target cells, and VeroE6/TMPRSS2 cells are highly susceptible to SARS-CoV-2 infection. Therefore, this assay is considered as an effective method for finding selective anti-SARS-CoV-2 agents.^25,37,64,76)

The cells (2 × 10⁴ cells/well) were cultured in a 96-well plate at 37°C for 24 h and then infected or mock-infected with SARS-CoV-2 (WK-521 strain) at a multiplicity of infection (MOI) of 0.01 in the presence of various concentrations of ADQ derivatives. After incubation for 3 d, the number of viable cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. In the absence of compounds, the cells infected with SARS-CoV-2 were mostly killed after incubation for 3 d. The graphs in Fig. 1 show the cell viability of virus-infected (closed bars) and uninfected cells (open bars) in the presence of ADQ, MQ, 1a, 1b, 1c, 1d, 1e, 2a, 2b, 3e,⁶¹⁾ 3h,⁵⁹⁾ 3j, remdesivir, and nirmatrelvir at increasing concentrations (0, 0.16, 0.8, 4, 20, and 100 µM), from which the 50% effective concentration (EC₅₀) and 50% cytotoxic concentration (CC₅₀) of these compounds were calculated, as listed in Tables 1 and 2. For example, the EC₅₀ and CC₅₀ values of ADQ are approx. 6 µM and approx. 31 µM, respectively, as estimated from Fig. 1a, and these values for MQ were >8.8 and 8.8 µM (not listed in Table 1), respectively. These results suggest the high levels of cytotoxicity of these 2 compounds in VeroE6/TMPRSS2 cells.

Fig. 1. Anti-SARS-CoV-2 Activity of Representative ADQ Derivatives in VeroE6/TMPRSS2 Cells

The cells (2 × 104 cells) were cultured in a 96-well plate at 37°C for 24 h. Then, the cells were mock-infected (open bars) or infected (closed bars) with SARS-CoV-2 at an MOI of 0.01 in the presence of various concentrations of amodiaquine (a), mefloquine (b), 1a (c), 1b (d), 1c (e), 1d (f), 1e (g), 2a (h), 2b (i), 3e (j), 3h (k), 3j (l), remdesivir (m), and nirmatrelvir (n) and further incubated for 3 d. Then, the number of viable cells was determined using the MTT method. All data represent the mean ± standard deviation (S.D.) in a triplicate experiment.

Table 1. The EC₅₀ and CC₅₀ Values of 1a–j and 2a–e in VeroE6/TMPRSS2 Cells Determined by the MTT Method

Compound	X	R16	R17	R18	R19	EC50 (µM)	CC50 (µM)
Amodiaquine	NH	Cl	H		OH	5.6 ± 3.3	30.5 ± 6.6
1a (KUMB-0002)	NH	Cl	H	H	OH	>100	>100
1b (YMSA-0216)	NH	Cl	H	F	OH	>100	>100
1c (YMSA-0215)	NH	Cl	H	CI	OH	>100	>100
1d (YMSA-0214)	NH	Cl	H	Br	OH	>100	>100
1e (YMSA-0213)	NH	Cl	H	I	OH	19.6 ± 5.0	38.8 ± 20.0
1f (YMSA-0217)	NH	Cl	H	CH3	OH	>38.5	38.5
1g (YMSA-0364)	NH	Cl	H	CF3	OH	>43.2	43.2
1h (YMSA-0258)	NH	CF3	H	I	OH	>9.6	9.6
1i (YMSA-0269)	NH	H	H	I	OH	10.8 ± 2.3	37.5 ± 6.0
1j (YMSA-0264)	NH	I	H	H	OH	>33.6	33.6
2a (YMSA-0250)	NH	Cl	OH	H	H	>100	>100
2b (YMSA-0249)	NH	Cl	H	OH	H	>100	>100
2c (YMSA-0252)	NH	Cl	H	H	NMe2	>100	>100
2d (YMSA-0262)	NH	Cl	H	H	SO2NH2	>65.1	65.1
2e (YMSA-0362)	S	Cl	H	H	OH	>100	>100

Note that some compounds were used as HCl salts (see Experimental).

Table 2. The EC₅₀ and CC₅₀ Values of 3a–j, Remdesivir, and Nirmatrelvir in VeroE6/TMPRSS2 Cells Determined by the MTT Assay

Compound	R20	R21	EC50 (µM)	CC50 (µM)
3a (YMSA-0267)	CI		>14.4	14.4
3b (YMSA-0203)	CI		>9.1	9.1
3c (YMSA-0454)	CI		>8.2	8.2
3d (YMSA-0423)	CI		>28.1	28.1
3e (YMSA-0455)	I		4.1 ± 1.6	22.7 ± 3.0
3f (KUMB-0052)	Br		>70.5	70.5
3g (KUMB-0053)	I		>21.3	21.3
3h (KUMB-0090)	I		3.2 ± 1.7	>100
3i (KUMB-0101)	Br		3.0 ± 0.8	>100
3j (KUMB-0100)	H		3.2 ± 0.6	28.2 ± 1.2
Remdesivir			3.0 ± 1.8	>100
Nirmatrelvir			5.3 ± 4.3	>100

The EC₅₀ and CC₅₀ values of ADQ and the compounds synthesized in this work are listed in Tables 1 and 2. As shown in Table 1, a compound 1e exhibited moderate antiviral activity (EC₅₀ is approx. 20 µM) and low cytotoxicity (CC₅₀ is approx. 39 µM), while compounds 1a–d had weak antiviral activity and cytotoxicity in VeroE6/TMPRSS2 cells. On the other hand, alkylated compounds 1f and 1g exhibited relatively higher levels of cytotoxicity. A comparison of the CC₅₀ values of 1h, i with those of 1b–d suggests a reduction in cytotoxicity by introducing Cl and Br atoms on the aminophenol units. The very weak antiviral activity of compounds 2a–e suggests that hydroxyl groups are required at the p-position of the NH group in 1e for antiviral activity. The sigmoidal curves for the determination of the EC₅₀ and CC₅₀ values (listed in Tables 1 and 2) of ADQ, 1a, 1e, and 3h are presented in Supplementary Fig. S1.

Next, the EC₅₀ and CC₅₀ values of compounds 3a–j were determined and are summarized in Table 2. These values indicate that compounds bearing alkyl chains such as 3a, 3b, 3c, and 3d exhibited cytotoxicity in uninfected VeroE6/TMPRSS-2 cells. The introduction of Br and I atoms at the 7-position of quinoline rings in 3f and 3g instead of a Cl atom in ADQ negligibly improved antiviral activity and cytotoxicity (vs. EC₅₀ and CC₅₀ values of ADQ listed in Table 1). Among these ADQ analogs listed in Table 2, 3h and 3i displayed the most potent antiviral activity and the lower cytotoxicity. The EC₅₀ value of 3j, having no halogen at the 7-position of the quinoline part, was determined to be 3.2 µM and its CC₅₀ value was approx. 28 µM, suggesting that the introduction of iodide and bromide atoms at the 7-position of the quinoline ring reduces the toxicity of the antiviral agents (vs. 3b and 3j). It should be mentioned that the anti-SARS-CoV-2 activity and toxicity of these compounds are sensitive to the shape and/or hydrophobicity of alkyl groups on the side chains.

For comparison, the EC₅₀ values of remdesivir (RdRp inhibitor) and nirmatrelvir (Mpro inhibitor) were determined to be approx. 3.0 µM and approx. 5.3 µM, respectively, and their CC₅₀ values were >100 µM (Table 2) in the simultaneous assays (Figs. 1m, 1n). These data suggest that the anti-SARS-CoV-2 activity and cytotoxicity (safety) of compounds 3h and 3i are comparable to those of remdesivir and nirmatrelvir.

In order to support the aforementioned data, the EC₅₀ and CC₅₀ values of amodiaquine, 1a (KUMB-0002), 1e (YMSA-0213), and 3h (KUMB-0090) were evaluated by measuring the viral RNA level in the culture supernatants used for the incubation of the SARS-CoV-2-infected and -uninfected VeroE6/TMPRSS2 cells in the presence of these drugs for 56 h by reverse transcription real-time PCR (RT-PCR). As summarized in Supplementary Table S1, the EC₅₀ values of these representative compounds determined by these assays are slightly smaller than the EC₅₀ values determined by the MTT method and listed in Tables 1 and 2, suggesting that the evaluation of viral RNA level in the culture supernatant is more sensitive than the MTT method, although the MTT method is more convenient than the RT-PCR method for screening many drug candidates.

Mechanistic Study for the Antiviral Activity of ADQ Derivatives

The mechanism responsible for the inhibition of viral infection by ADQ derivatives was examined. In our preliminary experiments, the inhibition of Mpro and RdRp by some ADQ analogs including 3h was negligible (data not shown). Recent studies have reported that CQ and its derivatives exhibit antiviral activity against RNA viruses, such as ZIKA virus by inhibiting endocytosis,⁷⁷⁾ which is an important process for the uptake of viral particles. This background has prompted us to evaluate the inhibitory effect of ADQ, MQ, 1e, 2a, and 3h on endocytosis in A549 (human Caucasian lung carcinoma) cells, which were used as model cells, using ECGreen, an endosomal detection reagent. In the absence of inhibitors, bright green fluorescence was observed in the cytosol of A549 cells (Fig. 2a), which likely was due to the formation of endosomal particles. On the other hand, the number of endosomal particles was decreased in the presence of ADQ and MQ (Figs. 2b, 2c), suggesting that endocytosis was inhibited by these agents. In Figs. 2d–2f, the number of endosomal particles was obviously decreased in the presence of 1e and 3h, while weaker levels of inhibition were observed for 2a. These results suggest a relationship between their anti-SARS-CoV-2 activity and endocytosis inhibition. It is likely that dialkylaminomethyl groups on the aminophenol units of most of these compounds are protonated at neutral pH and hence neutralize the acidification of endocytic vesicles at the early stages of viral infection. It is indicated that the anti-SARS-CoV-2 activity and toxicity of quinoline-based drugs are sensitive to the shape and/or hydrophobicity of alkylamino groups on the side chains, and hence studies on their detailed structure–activity relationship are currently in progress.

Fig. 2 Fluorescence Microscopy Images of ECGreen in A549 Cells in the Absence and Presence of Compounds

(a) None, (b) ADQ, (c) Mefloquine, (d) 1e, (e) 2a, and (f) 3h at 100 µM of each compound.

Conclusion

In summary, we have reported herein on the design and synthesis of anti-SARS-CoV-2 drugs based on ADQ-type quinoline derivatives. Among the compounds synthesized in this study, compounds 1e, 1i, 3e, 3h, 3i, and 3g were found to exhibit high levels of antiviral activity and low levels of cytotoxicity. The EC₅₀ and CC₅₀ values of compounds 3h and 3i, which are the most potent compounds among the synthesized compounds, are comparable to the values of remdesivir and nirmatrelvir. It is likely that 3h and 3i inhibit endocytosis, which is involved in an initial step of the replication cycle of SARS-CoV-2 and other related viruses in the target cells, rather than other replication processes catalyzed by RdRp and Mpro. Besides, 3e (YMSA-0455) has been reported to be one of the moderate to potent inhibitors of IL-6 production from J774.1 murine macrophages via activation of TLR3 (by polyinosinic-polycytidylic acid), TLR7 (by imiquimod), and TLR9 (by cytosine-phosphate-guanosine oligodeoxynucleotide),⁶⁵⁾ suggesting the possibility of ADQ analogs as dual-functional drugs that have anti-SARS-CoV-2 and IL-6 production suppression activities. We believe that the present information would be useful for the design and synthesis of low molecular weight antiviral agents to fight against COVID-19 and other related diseases caused by viral infection.

Experimental

General Information

All reagents and solvents were of the highest commercial quality and were used without further purification unless otherwise noted. All aqueous solutions were prepared with deionized water. Remdesivir and nirmatrelvir were purchased from Selleck Chemicals (Houston, TX, U.S.A.). The ¹H-NMR spectra (300 and 400 MHz) and ¹³C-NMR (100 and 125 MHz) were recorded on a JEOL (Tokyo, Japan) Always 300 spectrometer and a JEOL Lamda 400 spectrometer, respectively. Tetramethylsilane (TMS) was used as an internal reference for the ¹H- and ¹³C-NMR spectroscopy measurements of samples in CDCl₃, CD₃OD, and dimethyl sulfoxide (DMSO)-d₆. IR spectra were obtained with a PerkinElmer (Waltham, MA, U.S.A.) FTIR Spectrum 100 (ATR). The electrospray ionization (ESI) mass spectra were recorded on a Sciex X500R QTOF (AB SCIEX, Framingham, MA, U.S.A.). Elemental analyses were performed on a PerkinElmer CHN 2400 series II CHNS/O analyzer. TLC and silica gel column chromatography were performed using Merck 5554 (silica gel) TLC plates and Fuji Silysia Chemical (Aichi, Japan) FL-100D, respectively.

Synthesis

4-((7-Chloroquinolin-4-yl)amino)phenol·HCl (1a·HCl, KUMB-0002·HCl) and 4-((7-chloroquinolin-4-yl)amino)-2-((methyl(pentyl)amino)methyl)phenol (3b, YMSA-0203) were synthesized according to Ref. 65.

4-((7-Chloroquinolin-4-yl)amino)-2-fluorophenol·HCl (1b·HCl, YMSA-0216·HCl)

A mixture of 4,7-dichloroquinoline (5a) (233 mg, 1.18 mmol) and 4-amino-2-fluorophenol (4b) (154 mg, 1.20 mmol) in EtOH (5 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 60 min. The reaction mixture was cooled down, and the resultant precipitates were isolated by filtration and washed with Et₂O. The obtained solid was recrystallized from Et₂O/MeOH to afford 1b·HCl (YMSA-0216·HCl) as pale yellow crystals (185 mg, 48%). Mp. 247–249°C (dec). IR (ATR): ν = 3023, 2921, 1608, 1586, 1523, 1428, 1376, 1360, 1307, 1286, 1259, 1234, 1112, 965, 869, 811, 758, 565 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.90 (br, 1H), 10.35 (s, 1H), 8.73 (d, J = 9.0 Hz, 1H), 8.50 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.89 (dd, J = 9.0, 2.1 Hz, 1H), 7.34 (dd, J = 9.0, 2.4 Hz, 1H), 7.15 (dd, J = 9.0, 8.4 Hz, 1H), 7.13 (s, J = 6.9 Hz, 1H), 6.73 (d, J = 6.9 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.3, 152.1, 149.7, 144.7 (d, J_CF = 12.5 Hz), 139.0, 138.3, 127.9, 127.8 (d, J_CF = 8.6 Hz), 119.2, 118.4, 115.7, 114.3 (d, J_CF = 20.2 Hz), 100.3 ppm; high-resolution MS (HRMS) (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₁³⁵ClFN₂O: 289.0538; Found 289.0537). Anal. Calcd (%) for C₁₅H₁₁Cl₂FN₂O: C 55.41, H 3.41, N 8.62. Found: C 55.24, H 3.27, N 8.44.

2-Chloro-4-((7-chloroquinolin-4-yl)amino)phenol·HCl (1c·HCl, YMSA-0215·HCl)

A mixture of 4,7-dichloroquinoline (5a) (109 mg, 0.55 mmol) and 4-amino-2-chlorophenol (4c) (72 mg, 0.5 mmol) in EtOH (4 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 60 min. The reaction mixture was cooled and concentrated under reduced pressure. The resulting solid was recrystallized from Et₂O/MeOH to afford 1c·HCl (YMSA-0215·HCl) as pale yellow crystals (91 mg, 48%). Mp. 258–261°C (dec). IR (ATR): ν = 3065, 3019, 2909, 1636, 1618, 1590, 1551, 1511, 1458, 1300, 1238, 1218, 1199, 1099, 901, 855, 820, 800, 770, 649 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.75 (d, J = 9.3 Hz, 1H), 8.50 (d, J = 6.9 Hz, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.88 (dd, J = 9.2, 1.9 Hz, 1H), 7.50 (d, J = 2.4 Hz, 1H), 7.27 (dd, J = 8.4, 2.7 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 6.9 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.3, 152.9, 143.5, 139.0, 138.4, 128.5, 127.4, 125.9, 120.3, 119.3, 117.4, 115.7, 100.3 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₁³⁵Cl₂N₂O: 305.0243; Found 305.0242). Anal. Calcd (%) for C₁₅H₁₀Cl₂N₂O: C 59.04, H 3.30, N 9.18. Found: C 59.36, H 3.17, N 9.16.

2-Bromo-4-((7-chloroquinolin-4-yl)amino)phenol·0.5HCl (1d·0.5HCl, YMSA-0214·0.5HCl)

A mixture of 4,7-dichloroquinoline (5a) (58 mg, 0.29 mmol) and 4-amino-2-bromophenol (4d) (51 mg, 0.27 mmol) in EtOH (3 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 1 h. The reaction mixture was cooled, and the precipitates were isolated by filtration and washed with Et₂O. The resultant powder was recrystallized from Et₂O/MeOH to afford 1d·0.5HCl (YMSA-0214·0.5HCl) as yellow crystals (105 mg, 95%). Mp. >285°C. IR (ATR): ν = 3078, 3018, 2817, 1635, 1615, 1589, 1548, 1509, 1456, 1413, 1366, 1296, 1238, 1217, 1198, 815, 798 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.90 (br, 1H), 10.76 (s, 1H), 8.72 (d, J = 6.9 Hz, 1H), 8.50 (d, J = 6.9 Hz, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.88 (dd, J = 8.7, 1.8 Hz, 1H), 7.62 (d, J = 2.4 Hz, 1H), 7.31 (dd, J = 8.4, 2.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 6.7 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.4, 153.9, 143.3, 139.0, 138.4, 130.3, 128.8, 127.4, 126.5, 126.0, 119.2, 117.0, 115.7, 109.7, 100.3 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₁⁷⁹Br³⁵ClN₂O: 348.9743; Found 348.9743). Anal. Calcd (%) for C₁₅H_10.5BrCl_1.5N₂O: C 48.98, H 2.88, N 7.62. Found: C 50.55, H 2.68, N 7.54.

4-((7-Chloroquinolin-4-yl)amino)-2-iodophenol·HCl (1e·HCl, YMSA-0213·HCl)

A mixture of 4,7-dichloroquinoline (5a) (167 mg, 0.84 mmol) and 4-amino-2-iodophenol (4e) (197 mg, 0.84 mmol) in EtOH (10 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 1 h. The reaction mixture was cooled, and the precipitates were isolated by filtration and washed with Et₂O. The resultant powder was recrystallized from MeCN/H₂O to afford 1e·HCl (YMSA-0213·HCl) as green crystals (214 mg, 58%). Mp. 238–242°C (dec). IR (ATR): ν = 3072, 2809, 1634, 1611, 1591, 1545, 1505, 1456, 1410, 1291, 1216, 1197, 827, 796, 667, 650, 543, 421 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.84 (brs, 1H), 8.71 (d, J = 9.0 Hz, 1H), 8.50 (d, J = 6.9 Hz, 1H), 8.07 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.77 (s, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 6.6 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 156.5, 155.3, 143.3, 139.0, 138.3, 135.9, 129.0, 127.3, 127.1, 126.0, 119.2, 115.7, 115.4, 100.2, 84.9 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₀³⁵ClIN₂O: 396.9605; Found 396.9615). Anal. Calcd (%) for C₁₅H₁₁Cl₂IN₂O: C 41.60, H 2.56, N 6.47. Found: C 41.34, H 2.28, N 6.33.

4-((7-Chloroquinolin-4-yl)amino)-2-methylphenol·HCl (1f·HCl, YMSA-0217·HCl)

A mixture of 4,7-dichloroquinoline (5a) (162 mg, 0.82 mmol) and 4-amino-2-methylphenol (4f) (102 mg, 0.83 mmol) in EtOH (7 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 60 min. The reaction mixture was cooled and the resultant precipitates were isolated by filtration and washed with Et₂O. The obtained solid was recrystallized from Et₂O/MeOH to afford 1f·HCl (YMSA-0217·HCl) as pale yellow crystals (105 mg, 40%). Mp. 228–232°C (dec). IR (ATR): ν = 2811, 1611, 1588, 1549, 1511, 1450, 1362, 1270, 1238, 1213, 1113, 1096, 890, 830, 805, 666, 651 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.89 (brs, 1H), 9.82 (s, 1H), 8.74 (d, J = 9.0 Hz, 1H), 8.42 (d, J = 6.9 Hz, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.81 (dd, J = 9.0, 1.8 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 7.06 (dd, J = 8.4, 2.7 Hz, 1H), 6.99 (s, J = 8.4 Hz, 1H), 6.62 (d, J = 7.5 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.4, 155.1, 143.1, 139.1, 138.3, 127.9, 127.5, 127.2, 125.9, 125.7, 123.3, 119.3, 115.6, 115.5, 100.1, 16.0 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₆H₁₄³⁵ClN₂O: 285.0795; Found 285.0796). Anal. Calcd (%) for C₁₆H₁₄Cl₂N₂O: C 59.83, H 4.39, N 8.72. Found: C 59.43, H 3.98, N 8.58.

4-((7-Chloroquinolin-4-yl)amino)-2-trifluoromethylphenol·HCl (1g·HCl, YMSA-0364·HCl)

A mixture of 4,7-dichloroquinoline (5a) (109 mg, 0.55 mmol) and 4-amino-2-trifluoromethylphenol (4g) (88 mg, 0.5 mmol) in EtOH (4 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 60 min. The reaction mixture was cooled, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/MeOH to afford 1g·HCl (YMSA-0364·HCl) as pale yellow crystals (95 mg, 56%). Mp. 224–247°C (dec). IR (ATR): ν = 3089, 2814, 1627, 1611, 1589, 1513, 1458, 1366, 1325, 1295, 1198, 1173, 1116, 1055, 903, 853, 810, 772, 651, 600, 537, 424 cm⁻¹; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ = 11.14 (s, 1H), 11.07 (s, 1H), 8.78 (d, J = 9.2 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 9.2, 2.0 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 8.6, 2.6 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 6.64 (d, J = 7.2 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.3, 155.2, 143.3, 138.9, 138.3, 131.4, 127.5, 127.3, 126.0, 124.3, 119.1, 118.3, 116.0, 115.7, 100.1 ppm; HRMS (ESI⁺): [M + H]⁺ (Calcd for C₁₆H₁₁³⁵ClF₃N₂O: 339.0507; Found 339.0509). Anal. Calcd (%) for C₁₆H₁₁Cl₂F₃N₂O: C 51.22, H 2.96, N 7.47. Found: C 51.19, H 2.39, N 7.57.

2-Iodo-4-((7-(trifluoromethyl)quinolin-4-yl)amino)phenol·HCl (1h·HCl, YMSA-0258·HCl)

A mixture of 4-chloro-7-trifluoromethylquinoline (5b) (88 mg, 0.38 mmol) and 4-amino-2-iodophenol (4e) (86 mg, 0.37 mmol) in EtOH (5 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 60 min. The reaction mixture was cooled, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/EtOH to afford 1h·HCl (YMSA-0258·HCl) as dark green crystals (102 mg, 57%). Mp. 227–230°C (dec). IR (ATR): ν = 3078, 2858, 2810, 1602, 1549, 1458, 1403, 1369, 1316, 1283, 1243, 1208, 1162, 1075, 1089, 1054, 899, 830, 811, 740, 684, 667, 545, 427 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.94 (d, J = 8.7 Hz, 1H), 8.60 (d, J = 6.9 Hz, 1H), 8.41 (brs, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 2.7 Hz, 1H), 7.34 (d, J = 8.6 Hz, 2.1 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 6.9 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 156.6, 155.3, 144.1, 137.9, 135.9, 129.0, 127.1, 125.9, 122.4, 119.2, 117.9, 115.5, 101.0, 85.0 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₆H₁₁F₃IN₂O: 430.9863; Found 430.9863). Anal. Calcd (%) for C₁₆H₁₁³⁵ClF₃IN₂O: C41.18, H 2.38, N 6.00. Found: C 41.55, H 2.21, N 5.93.

2-Iodo-4-(quinolin-4-ylamino)phenol·HCl (1i·HCl, YMSA-0269·HCl)

A mixture of 4-chloroquinoline (5c) (105 mg, 0.64 mmol) and 4-amino-2-iodophenol (4e) (151 mg, 0.64 mmol) in EtOH (5 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 1 h. The reaction mixture was cooled, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/MeOH to afford 1i·HCl (YMSA-0269·HCl) as green crystals (171 mg, 64%). Mp. 231–234°C (dec). IR (ATR): ν = 3059, 3006, 1619, 1595, 1541, 1494, 1453, 1415, 1292, 1199, 1147, 758, 668, 545, 511 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.90 (brs, 1H), 10.88 (brs, 1H), 8.78 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 8.03 (dd, J = 8.4, 7.5 Hz, 1H), 7.78 (dd, J = 7.8, 7.2 Hz, 1H), 7.31 (dd, J = 8.7, 2.1 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 6.9 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 156.4, 155.4, 142.5, 138.1, 136.0, 133.8, 129.3, 127.2, 126.9, 123.6, 120.1, 116.9, 115.4, 99.6, 84.9 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₂IN₂O: 362.9994; Found 362.9995). Anal. Calcd (%) for C₁₅H₁₂ClIN₂O: C 45.20, H 3.03, N 7.03. Found: C 45.55, H 3.02, N 6.72.

4-((7-Iodoquinolin-4-yl)amino)phenol (1j·HCl, YMSA-0264·HCl)

A mixture of 4-chloro-7-iodoquinoline (5d) (131 mg, 0.48 mmol) and 4-aminophenol (4a) (54 mg, 0.50 mmol) in EtOH (5 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 1 h. The reaction mixture was cooled, and the resultant precipitates were obtained by filtration, washed with Et₂O, and recrystallized from Et₂O/MeOH to afford 1j·HCl (YMSA-0264·HCl) as yellow crystals (100 mg, 53%). Mp. 263–266°C (dec). IR (ATR): ν = 3135, 2803, 1616, 1600, 1548, 1511, 1434, 1359, 1269, 1227, 1210, 1172, 879, 794, 767, 696, 670, 658, 546, 518, 439 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.48–8.40 (m, 3H), 8.09 (dd, J = 8.7 Hz, 1.5 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.63 (d, J = 6.9 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 157.0, 155.6, 142.8, 138.9, 135.3, 128.4, 127.7, 127.2, 124.9, 116.5, 116.1, 99.9 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₂IN₂O: 362.9989; Found 362.9988). Anal. Calcd (%) for C₁₅H₁₂ClIN₂O: C 45.20, H 3.03, N 7.03. Found: C 45.19, H 2.81, N 6.96.

2-((7-Chloroquinolin-4-yl)amino)phenol·HCl·0.5MeOH (2a·HCl·0.5MeOH, YMSA-0250·HCl·0.5MeOH)

A mixture of 4,7-dichloroquinoline (5a) (306 mg, 1.55 mmol) and 2-aminophenol (6a) (169 mg, 1.55 mmol) in EtOH (8 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 60 min. The reaction mixture was cooled, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/MeOH to afford 2a·HCl·0.5MeOH (YMSA-0250·HCl·0.5MeOH) as yellow-brown crystals (364 mg, 73%). Mp. 234–236°C (dec). IR (ATR): ν = 3031, 1616, 1585, 1542, 1509, 1458, 1441, 1361, 1208, 1097, 1058, 866, 810, 752, 648, 463, 416 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.81 (d, J = 9.3 Hz, 1H), 8.48 (d, J = 6.9 Hz, 1H), 8.15 (d, J = 2.1 Hz, 1H), 7.86 (dd, J = 9.3, 2.1 Hz, 1H), 7.35–7.21 (m, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.14 (dd, J = 8.6, 1.2 Hz, 1H), 6.98 (dt, J = 7.1, 1.2 Hz, 1H), 6.33 (d, J = 6.9 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.8, 153.3, 143.2, 139.4, 138.7, 130.0, 128.6, 127.6, 126.8, 123.8, 120.3, 119.6, 117.7, 116.1, 101.4 ppm; HRMS (FAB+): [M + H]⁺ (Calcd for C₁₅H₁₁³⁵ClN₂O: 271.0638; Found 271.0643). Anal. Calcd (%) for C_15.5H₁₃ClN₂O_1.5: C 57.60, H 4.05, N 8.66. Found: C 57.30, H 3.73, N 8.82.

3-((7-Chloroquinolin-4-yl)amino)phenol·HCl (2b·HCl, YMSA-0249·HCl)

A mixture of 4,7-dichloroquinoline (5a) (132 mg, 0.67 mmol) and 3-aminophenol (6b) (73 mg, 0.67 mmol) in EtOH (4 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 40 min. The reaction mixture was cooled down, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/MeOH to afford 2b·HCl (YMSA-0249·HCl) as pale yellow crystals (82 mg, 41%). Mp. 252–253°C (dec). IR (ATR): ν = 2811, 1611, 1588, 1549, 1511, 1450, 1362, 1270, 1238, 1213, 1113, 1096, 890, 830, 804, 666, 651 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 10.82 (brs, 1H), 9.33 (s, 1H), 8.71 (d, J = 9.0 Hz, 1H), 8.53 (d, J = 6.9 Hz, 1H), 8.09 (d, J = 1.8 Hz, 1H), 7.88 (dd, J = 9.0, 2.1 Hz, 1H), 7.37 (dd, J = 8.6, 8.4 Hz, 1H), 6.90–6.83 (m, 4H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 158.8, 154.9, 143.5, 139.1, 138.4, 137.8, 130.8, 127.4, 126.0, 119.3, 115.9, 115.6, 114.8, 112.4, 100.5 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₁³⁵ClN₂O: 271.0638; Found 271.0635). Anal. Calcd (%) for C₁₅H₁₂Cl₂N₂O: C 58.65, H 3.94, N 9.12. Found: C 58.29, H 3.86, N 8.94.

N¹-(7-Chloroquinolin-4-yl)-N⁴,N⁴-dimethylbenzene-1,4-diamine·HCl (2c·HCl, YMSA-0252·HCl)

A mixture of 4,7-dichloroquinoline (5a) (224 mg, 1.13 mmol) and N,N-dimethyl-p-phenylenediamine (6c) (158 mg, 1.16 mmol) in EtOH (7 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 40 min. The reaction mixture was cooled, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/MeOH to afford 2c·HCl (YMSA-0252) as orange crystals (222 mg, 59%). Mp. 240–244°C (dec). IR (ATR): ν = 2646, 1606, 1589, 1537, 1518, 1444, 1349, 1212, 1181, 1165, 1119, 1096, 895, 864, 815, 777, 666, 530, 508, 424 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.76 (d, J = 9.3 Hz, 1H), 8.42 (d, J = 6.9 Hz, 1H), 8.07 (d, J = 1.8 Hz, 1H), 7.81 (dd, J = 9.0, 2.1 Hz, 1H), 7.24 (dd, J = 8.7, 2.1 Hz, 1H), 6.85 (d, J = 9.3 Hz, 1H), 6.64 (d, J = 7.5 Hz, 4H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.3, 149.7, 142.9, 139.0, 128.2, 127.1, 126.4, 125.9, 124.9, 115.7, 112.9, 99.9 ppm. HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₇H₁₇³⁵ClN₃: 298.1106; Found 298.1103). Anal. Calcd (%) for C₁₇H₁₇Cl₂N₃: C 61.09, H 5.13, N 12.57. Found: C 61.12, H 5.15, N 12.55.

4-((7-Chloroquinolin-4-yl)amino)benzenesulfonamide·HCl (2d·HCl, YMSA-0262·HCl)

A mixture of 4,7-dichloroquinoline (5a) (211 mg, 1.06 mmol) and p-aminobenzenesulfonamide (6d) (184 mg, 1.07 mmol) in EtOH (10 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 40 min. The reaction mixture was cooled, and Et₂O was added. The resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from iPrOH to afford 2d·HCl (YMSA-0262·HCl) as yellow crystals (104 mg, 26%). Mp. 247–249°C (dec). IR (ATR): ν = 3284, 3055, 2754, 1611, 1582, 1533, 1445, 1366, 1323, 1212, 1162, 1096, 893, 817, 640, 571, 544, 520 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.90 (d, J = 9.0 Hz, 1H), 8.61 (d, J = 6.9 Hz, 1H), 8.19 (d, J = 2.1 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.92 (dd, J = 7.9, 1.8 Hz, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.51 (s, 1H), 7.70 (d, J = 7.2 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 154.4, 143.8, 142.3, 140.3, 139.2, 138.6, 127.6, 127.4, 126.4, 125.0, 119.4, 116.4, 101.0 ppm; HRMS (FAB⁺): [M + H]⁺ (Calcd for C₁₅H₁₃³⁵ClN₃O₂S: 334.0412; Found 334.0409). Anal. Calcd (%) for C₁₅H₁₃Cl₂N₃O₂S; C 48.66, H 3.54, N 11.35. Found: C 48.55, H 3.13, N 11.16.

4-((7-Chloroquinolin-4-yl)thio)phenol·HCl (2e·HCl, YMSA-0362·HCl)

A mixture of 4,7-dichloroquinoline (5a) (299 mg, 1.51 mmol) and 4-hydroxybenzenethiol (6e) (189 mg, 1.0 mmol) in EtOH (7 mL) was reacted in a microwave synthesizer at 50 W and 80°C for 1 h. The reaction mixture was cooled down, and the resultant precipitates were isolated by filtration, washed with Et₂O, and recrystallized from Et₂O/EtOH to afford 2e·HCl (YMSA-0362·HCl) as yellow crystals (162 mg, 33%). Mp. 200–201°C (dec). IR (ATR): ν = 3073, 2738, 1619, 1602, 1575, 1495, 1473, 1383, 1280, 1272, 1210, 1186, 1164, 1087, 986, 902, 888, 847, 836, 814, 692, 681, 533, 473 cm⁻¹; ¹H-NMR (300 MHz, DMSO-d₆/TMS): δ = 8.77 (d, J = 6.0 Hz, 1H), 8.38 (d, J = 9.0 Hz, 1H), 8.28 (d, J = 1.8 Hz, 1H), 7.91 (dd, J = 8.7, 2.1 Hz, 1H), 7.52 (d, J = 6.6 Hz, 2H), 7.05 (d, J = 6.6 Hz, 2H), 6.88 (d, J = 6.0 Hz, 1H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 160.5, 160.0, 145.7, 139.9, 137.9, 137.9, 129.4, 125.8, 123.2, 122.8, 121.1, 118.1, 116.2, 112.2 ppm; HRMS (FAB+): [M + H]⁺ (Calcd for C₁₅H₁₁³⁵ClN₂OS: 288.0244; Found 288.0241). Anal. Calcd (%) for C₁₅H₁₁Cl₂NOS: C 55.57, H 3.42, N 4.32. Found: C 55.30, H 3.10, N 4.32.

2-((Butyl(ethyl)amino)methyl)-4-((7-chloroquinolin-4-yl)-​amino)phenol (3a, YMSA-0267)^65,75)

A reaction mixture of 1a (57 mg, 0.20 mmol), N-ethylbutylamine (7a) (55 µL, 0.40 mmol), and 37% HCHO aqueous solution (42 µL, 0.52 mmol) in EtOH (2 mL) was refluxed for 15 h. The reaction mixture was concentrated under reduced pressure and the resultant residue was purified by silica gel column chromatography (hexanes/AcOEt = 1/1) to afford 3a (YMSA-0267) as a pale yellow powder (45 mg, 57%). The spectroscopic data of 3a are reported in Ref. 65.

4-((7-Chloroquinolin-4-yl)amino)-2-((isopentyl(methyl)-​amino)methyl)phenol (3c, YMSA-0454)^65,75)

A reaction mixture of 1a (65 mg, 0.24 mmol), N-methylisoamylamine (7c) (97 mg, 0.96 mmol), AcOH (40 µL), Et₃N (134 µL, 0.96 mmol), and 37% HCHO aqueous solution (101 µL, 1.25 mmol) in EtOH (4 mL) was refluxed for 19 h. The reaction mixture was concentrated under reduced pressure and the resultant residue was purified by silica gel column chromatography (AcOEt/MeOH = 1/0 to 4/1 containing 1% Et₃N) to afford 3c (YMSA-0455)⁶⁵⁾ as a pale yellow powder (60 mg, 65%). The spectroscopic data of 3c are reported in Ref. 65.

4-((7-Chloroquinolin-4-yl)amino)-2-((3-fluoropiperidin-1-yl)methyl)phenol (3d, YMSA-0423)^65,75)

A reaction mixture of 1a (31 mg, 0.12 mmol) in EtOH (1.2 mL), 3-fluoropiperidine HCl salt (7d)⁷⁸⁾ (64 mg, 0.46 mmol), and 37% HCHO aqueous solution (47 µL, 0.58 mmol) was refluxed for 24 h. The reaction mixture was concentrated under reduced pressure and the resultant residue was purified by silica gel column chromatography (hexanes/AcOEt = 5/1 to 0/1 containing 1% Et₃N) to afford 3e (YMSA-0423)⁶⁵⁾ as a yellow solid (11 mg, 24%). The spectroscopic data of 3d are reported in Ref. 67.

4-((7-Iodoquinolin-4-yl)amino)-2-((isopentyl(methyl)-amino)methyl)phenol (3e, YMSA-0455)^65,75)

A reaction mixture of 1j (44 mg, 0.12 mmol), N-methylisoamylamine (7c) (58 mg, 0.58 mmol), AcOH (5 drops), and 37% HCHO aqueous solution (50 µL, 0.62 mmol) in EtOH (2.0 mL) was refluxed for 15 h. The reaction mixture was concentrated under reduced pressure and the resultant residue was purified by silica gel column chromatography (hexanes/AcOEt = 1/1) to afford 3e (YMSA-0455)⁶⁵⁾ as a pale yellow powder (23 mg, 39%). The spectroscopic data of 3e are reported in Ref. 65.

2-((Methyl(pentyl)amino)methyl)-4-nitrophenol (9a)

A reaction mixture of 2-hydroxy-5-nitrobenzyl bromide (8) (101 mg, 0.433 mmol), N-methylpentylamine (7b) (64 µL, 0.48 mmol), and Et₃N (134 µL, 0.96 mmol) in THF (4.3 mL) was refluxed for 1 h. The reaction mixture was concentrated under reduced pressure and extracted with AcOEt from saturated NaHCO₃ aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 3/1) to afford 9a as a yellow oil (91 mg, 83%). IR (ATR): ν = 3039, 2957, 2931, 2925, 2856, 2163, 1592, 1554, 1465, 1428, 1415, 1276, 1249, 1232, 1165, 1125, 1083, 939, 907, 823, 791, 755, 717, 647, 619, 574, 546, 501, 456, 438, 424 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃/TMS): d = 8.07 (dd, J = 9.3, 3.0 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 6.84 (t, J = 9.3 Hz, 1H), 3.79 (s, 2H), 2.53 (t, J = 7.5 Hz, 2H), 2.32 (s, 3H), 1.60–1.56 (m, 2H), 1.32–1.26 (m, 4H), 0.91 (t, J = 6.9 Hz, 3H) ppm; ¹³C-NMR (100 MHz, CDCl₃/TMS) d = 165.2, 139.8, 125.3, 124.5, 121.6, 116.4, 60.7, 56.9, 41.9, 29.2, 26.3, 22.4, 13.9 ppm; HRMS (ESI⁺): [M + H]⁺ (Calcd for C₁₃H₂₁N₂O₃: 253.1547; Found 253.1546).

2-((Diethylamino)methyl)-4-nitrophenol (9b)

A reaction mixture of 2-hydroxy-5-nitrobenzyl bromide (8) (50 mg, 0.21 mmol), Et₂NH (7e) (24 µL, 0.23 mmol), and Et₃N (65 µL, 0.47 mmol) in THF (2.1 mL) was refluxed for 1 h. The reaction mixture was concentrated under reduced pressure and then extracted with AcOEt from saturated NaHCO₃ aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 1/1) to afford 9b as a yellow solid (28 mg, 58%). Mp. 149–150°C. IR (ATR): ν = 2974, 2876, 1734, 1671, 1587, 1519, 1479, 1389, 1330, 1280, 1193, 1181, 1162, 1108, 1084, 980, 924, 897, 832, 774, 751, 715, 656, 633, 523, 500, 471, 444, 408 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃/TMS): d = 8.08 (dd, J = 8.9, 2.7 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 6.81 (t, J = 9.0 Hz, 1H), 3.86 (s, 2H), 2.68 (q, J = 7.2 Hz, 4H), 1.15 (t, J = 7.2 Hz, 6H) ppm; ¹³C-NMR (100 MHz, CDCl₃/TMS) d = 165.8, 139.7, 125.3, 124.5, 121.7, 116.6, 56.5, 46.4, 11.0 ppm; HRMS (ESI⁺): [M + H]⁺ (Calcd for C₁₁H₁₇N₂O₃: 225.1239; found 225.1240).

4-((7-Bromoquinolin-4-yl)amino)-2-((diethylamino)-methyl)phenol (3f, KUMB-0052)⁶¹⁾

SnCl₂·2H₂O (70 mg, 0.31 mmol) was added to a solution of 9b (17 mg, 0.074 mmol) in EtOH (740 µL), and the reaction mixture was refluxed for 1 h. Then, 7-bromo-4-chloroquinoline (5e, Sigma-Aldrich) (20 mg, 0.082 mmol) was added, and the reaction mixture was allowed to react at reflux temperature for another 1 h. The reaction mixture was concentrated under reduced pressure and then extracted with AcOEt from 4 n NaOH aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (AcOEt) to afford 3f (KUMB-0052) as a pale yellow solid (21 mg, 72%). Mp. 194–196°C, IR (ATR): ν = 3237, 3052, 2972, 2933, 2829, 2160, 2032, 1610, 1565, 1540, 1492, 1453, 1418, 1389, 1368, 1327, 1288, 1249, 1215, 1196, 1168, 1106, 1077, 1051, 999, 955, 880, 842, 816, 781, 764, 719, 672, 632, 593, 575, 553, 503, 478, 443, 428 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.45 (d, J = 5.2 Hz, 1H), 8.18 (d, J = 2.4 Hz, 1H), 7.75 (d, J = 2.4 Hz, 1H), 7.56 (dd, J = 8.8, 2.4 Hz, 1H), 7.08 (dd, J = 8.4, 2.4 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 5.2 Hz, 1H), 6.57 (brs, 1H), 3.79 (s, 2H), 2.66 (q, J = 7.2 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H) ppm; ¹³C-NMR (100 MHz, CDCl₃/TMS): δ = 156.7, 151.8, 149.6, 149.5, 132.1, 129.9, 128.3, 125.6, 125.3, 123.4, 121.0, 117.7, 117.1, 101.4, 56.8, 50.9, 46.4, 11.2 ppm; HRMS (FAB+): [M + H]+ (Calcd for C₂₀H₂₃⁸¹BrN₃O: 400.1019; Found 400.1016). Anal. Calcd (%) for C₂₀H₂₂BrN₃O: C 60.01, H 5.54, N 10.28. Found: C 59.89, H 5.48, N 10.18.

2-((Diethylamino)methyl)-4-((7-iodoquinolin-4-yl)amino)-phenol (3g, KUMB-0053)⁶¹⁾

SnCl₂·2H₂O (58.9 mg, 0.261 mmol) was added to a solution of 9b (14 mg, 0.060 mmol) in EtOH (600 µL), and the reaction mixture was refluxed for 1 h. Then, 4-chloro-7-iodoquinoline (5d) (22 mg, 0.076 mmol) was added and the reaction mixture was allowed to react at reflux temperature for another 1 h. The reaction mixture was concentrated under reduced pressure and then extracted with AcOEt from 4 n NaOH aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (AcOEt to AcOEt/MeOH = 30/1) to afford 3g (KUMB-0053) as a yellow solid (24 mg, 89%). Mp. 190°C. IR (ATR): ν = 2972, 2821, 2160, 2035, 1603, 1570, 1493, 1446, 1418, 1387, 1367, 1328, 1289, 1254, 1193, 1167, 1106, 1169, 1050, 996, 879, 834, 813, 763, 654, 638, 609, 572, 550 cm⁻¹. ¹H-NMR (300 MHz, CDCl₃/TMS): δ = 8.44 (d, J = 5.2 Hz, 1H), 8.42 (d, J = 1.2 Hz, 1H), 7.72 (dd, J = 8.8, 1.2 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.08 (dd, J = 8.4, 2.4 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 5.2 Hz, 1H), 6.55 (brs, 1H), 3.78 (s, 2H), 2.65 (q, J = 7.2 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H) ppm; ¹³C-NMR (100 MHz, CDCl₃/TMS): δ = 156.7, 151.5, 149.8, 149.4, 138.8, 133.5, 129.8, 125.5, 125.3, 123.4, 120.8, 118.1, 117.2, 101.5, 95.3, 56.8, 46.4, 11.2 ppm; HRMS (FAB+): [M + H]+ (Calcd for C₂₀H₂₃IN₃O: 400.1019; Found 400.1016). Anal. Calcd (%) for C₂₀H₂₂IN₃O: C 53.70, H 4.96, N 9.39. Found: C 53.74, H 4.91, N 9.12.

4-((7-Iodoquinolin-4-yl)amino)-2-((methyl(pentyl)amino)-methyl)phenol·HCl (3h·HCl, KUMB-0090·HCl)⁵⁹⁾

SnCl₂·2H₂O (164 mg, 0.726 mmol) was added to a solution of 9a (44 mg, 0.18 mmol) in EtOH (2 mL), and the reaction mixture was refluxed for 1 h. Then, 4-chloro-7-iodoquinoline (5d) (79 mg, 0.27 mmol) was added, and the reaction mixture was refluxed for 1 h. The reaction mixture was concentrated under reduced pressure and extracted with AcOEt from 2 n NaOH aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 5/1 to 1/1) to afford 3h (KUMB-0090) as a pale yellow solid (55 mg, 67%).

To a solution of KUMB-0090 (3h) (10.22 mg, 0.0215 mmol) in EtOH (1 mL), 1n HCl aqueous solution (43 µL, 0.0430 mmol) and Et₂O (5.5 mL) were added. The resultant precipitates were isolated by filtration and washed with Et₂O to obtain 3h·HCl (KUMB-0090·HCl) as a yellow powder (6.5 mg, 59%). Mp. 171–173°C. IR (ATR): ν = 2926, 2857, 2614, 2162, 2040, 1623, 1602, 1565, 1543, 1496, 1448, 1408, 1328, 1276, 1255, 1206, 1168, 1109, 1089, 1071, 978, 929, 883, 831, 814, 800, 763, 728, 658, 551, 537, 523, 506, 477, 448, 439, 421 cm⁻¹; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ = 9.66 (s, 1H), 8.38 (d, J = 6.0 Hz, 1H), 8.34 (d, J = 8.8 Hz, 1H), 8.32 (d, J = 1.6 Hz, 1H), 7.87 (dd, J = 8.8, 1.6 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.27 (dd, J = 8.4, 2.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 6.72 (d, J = 5.6 Hz, 1H), 4.13 (s, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.60 (s, 3H), 1.72–1.66 (m, 2H), 1.29–1.27 (m, 4H), 0.88 (t, J = 6.8 Hz, 3H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 154.7, 154.0, 148.6, 146.2, 134.4, 133.2, 129.9, 128.4, 127.2, 124.3, 118.5, 117.5, 116.4, 100.6, 97.8, 55.1, 54.0, 28.2, 23.5, 21.6, 13.7 ppm; HRMS (ESI+): [M + H]+ (Calcd for C₂₂H₂₇IN₃O: 476.1193; Found 476.1194). Anal. Calcd (%) for C₂₂H₂₇ClIN₃O: C 51.63, H 5.32, N 8.21. Found: C 51.20, H 4.91, N 8.16.

4-((7-Bromoquinolin-4-yl)amino)-2-((methyl(pentyl)-amino)methyl)phenol·HCl (3i·HCl, KUMB-0101·HCl)

SnCl₂·2H₂O (122 mg, 0.54 mmol) was added to a solution of 9a (35 mg, 0.14 mmol) in EtOH (5 mL), and the reaction mixture was refluxed for 1.5 h. Then, 4-chloro-7-bromoquinoline (5e) (60 mg, 0.25 mmol) was added, and the reaction mixture was refluxed for 2 h. The reaction mixture was concentrated under reduced pressure and extracted with AcOEt from 1 n NaOH aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 2/1 to 0/1) to afford 3i (KUMB-0101) as a pale yellow solid (33 mg, 56%).

To a solution of KUMB-0101 (3i) (21.4 mg, 0.0500 mmol) in EtOH (1 mL), 1 n HCl aqueous solution (100 µL, 0.100 mmol) was added along with Et₂O (6 mL). The resultant precipitates were isolated by filtration and washed with Et₂O to obtain 3i·HCl (KUMB-0101·HCl) as a yellow powder (9.9 mg, 43% from 3i). Mp. 182–184°C. IR (ATR): ν = 2920, 1605, 1586, 1542, 1508, 1448, 1360, 1278, 1236, 1208, 1166, 1111, 1089, 1057, 894, 841, 818, 765, 653, 541, 498, 472, 419, 408 cm⁻¹; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ = 11.11 (s, 1H), 10.88 (s, 1H), 10.16 (s, 1H), 8.77 (d, J = 9.2 Hz, 1H), 8.49 (d, J = 7.2 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 9.2, 2.0 Hz, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.8, 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.84 (d, J = 7.2 Hz, 1H), 4.41–4.27 (m, 1H), 4.24–4.10 (m, 1H), 3.17–2.94 (m, 2H), 2.70 (s, 3H), 1.82–1.69 (m, 2H), 1.37–1.21 (m, 4H), 0.88 (t, J = 6.4 Hz, 3H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 156.0, 155.0, 143.0, 138.9, 130.2, 129.9, 127.8, 127.2, 125.7, 122.3, 117.5, 116.7, 115.8, 100.3, 54.9, 52.5, 28.1, 22.9, 21.6, 13.7 ppm; HRMS (ESI+): [M + H]+ (Calcd for C₂₂H₂₇⁷⁹BrN₃O: 428.1332; Found 428.1328). Anal. Calcd (%) for C₂₂H₂₇BrClN₃O·3H₂O: C 50.93, H 6.41, N 8.10. Found: C 50.25, H 5.93, N 7.98.

2-((Methyl(pentyl)amino)methyl)-4-(quinolin-4-ylamino)-phenol·HCl (3j·HCl, KUMB-0100·HCl)

SnCl₂·2H₂O (299 mg, 1.33 mmol) was added to a solution of 9a (83 mg, 0.33 mmol) in EtOH (5 mL), and the reaction mixture was refluxed for 2 h. Then, 4-chloroquinoline (5c) (109 mg, 0.67 mmol) was added, and the reaction mixture was refluxed for another 2 h. The reaction mixture was concentrated under reduced pressure and extracted with AcOEt from 1 n NaOH aqueous solution. The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/AcOEt = 2/1 to 0/1) to afford 3j (KUMB-0100) as a pale yellow solid (48 mg, 42%).

To a solution of KUMB-0100 (3j) (19.5 mg, 0.0557 mmol) in EtOH (1 mL), 1 n HCl aqueous solution (114 µL, 0.114 mmol) was added along with Et₂O (6 mL). The resultant precipitates were isolated by filtration and washed with Et₂O to obtain 3j·HCl (KUMB-0100·HCl) as a yellow powder (13.0 mg, 60% from 3j). Mp. 172–174°C. IR (ATR): ν = 2917, 1618, 1591, 1542, 1509, 1447, 1363, 1277, 1214, 1147, 761, 498, 474, 447, 421, 406 cm⁻¹; ¹H-NMR (400 MHz, DMSO-d₆/TMS): δ = 11.11 (s, 1H), 10.95 (s, 1H), 10.88 (s, 1H), 8.81 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 6.8 Hz, 1H), 8.11–7.99 (m, 2H), 7.80 (t, J = 7.2 Hz, 1H), 7.61 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.8, 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 7.2 Hz, 1H), 4.42–4.28 (m, 1H), 4.25–4.10 (m, 1H), 3.17–2.94 (m, 2H), 2.70 (s, 3H), 1.82–1.69 (m, 2H), 1.37–1.21 (m, 4H), 0.88 (t, J = 6.8 Hz, 3H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆/TMS): δ = 155.9, 155.1, 142.5, 138.0, 133.8, 130.3, 128.7, 128.0, 126.9, 123.4, 120.1, 117.5, 116.7, 99.7, 54.9, 52.5, 28.1, 22.9, 21.6, 13.7 ppm. HRMS (ESI+): [M + H]+ (Calcd for C₂₂H₂₈N₃O: 350.2227; Found 350.2228). Anal. Calcd (%) for C₂₂H₂₈ClN₃O·4H₂O: C 57.70, H 7.92, N 9.17. Found: C 57.12, H 7.30, N 8.84.

Evaluation of Anti-SARS-CoV-2 Activity of ADQ Derivatives

VeroE6/TMPRSS2 cells (2 × 10⁴ cells/well) were cultured in a 96-well plate and incubated at 37°C for 24 h. After incubation, the cells were infected with SARS-CoV-2 (WK-521 strain) at an MOI of 0.01 and then were further incubated in the presence of various concentrations (0, 0.16, 0.8, 4, 20, and 100 µM) of test compounds. After incubation for 3 d or 56 h, the number of viable cells was determined by the MTT method. The EC₅₀ and CC₅₀ values of each compound were determined. All experiments were conducted in triplicate. The % inhibition values were calculated based on the following equation: % inhibition = 100 × (the number of viable infected cells in the presence of compound at a certain concentration – the number of viable infected cells in the absence of compound)/(the number of viable uninfected cells in the absence of compound – the number of viable infected cells in the absence of compound). The viral RNA level in culture supernatants of infected VeroE6/TMPRSS2 cells was measured by real-time RT-PCR using PrimeScript One Step RT-PCR Kit (TaKaRa Bio, Kusatsu, Japan). The primer pair 5′-AAATTTTGGGGACCAGGAAC-3′ and 5′-TGGCAGCTGTGTAGGTCAAC-3′ and the probe 5′-FAM-ATGTCGCGCATTGGCATGGA-TAMRA-3′ were used for real-time PCR.³⁸⁾

Evaluation of Endocytosis Inhibition by ADQ Derivatives

A549 cells (1 × 10⁵ cells/dish) were incubated in 100 µM Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (100 µL) for 1 h in the presence of ADQ, MQ, 1e, 2a, and 3h, followed by incubation with 100 µL of ECGreen working solution (500 times dilution by DMEM containing 10% FBS) using the ECGreen-endocytosis detection kit (E298, Dojindo, Kumaoto, Japan). After incubation for 2 h, the cells were washed with phosphate-buffered saline and observed using an FV1000 confocal laser microscope (Olympus, Tokyo, Japan) (wavelengths used for excitation and emission were 405 and 425–525 nm, respectively).

Acknowledgments

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Nos. 22390005, 24659011, and 24640156 for S.A.). Support was also provided by the “Academic Frontiers” project for private universities (a matching fund study from MEXT), and the TUS (Tokyo University of Science) fund for strategic research areas, the funding from the Japan Agency for Medical Research and Development (AMED), Japan (20fk0108273h0001 for M.B. and 22fk0108555h0001 for S.A.), and the funding from the Shionogi Infectious Disease Research Promotion Foundation, Japan. This research was partially supported by the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under Grant Numbers: JP24ama121051 and JP24ama121053. We wish to thank Ms. Fukiko Hasegawa, Ms. Noriko Sawabe, Ms. Yayoi Yoshimura, Ms. Yuki Honda, and Ms. Hitomi Isoda (Faculty of Pharmaceutical Sciences, Tokyo University of Science and Research Equipment Center, Tokyo University of Science) for conducting MS spectrometry, NMR, and the elemental analyses, respectively. We wish to thank Dr. Yoshiki Yagi and Dr. Takuro Niwa (Nobelpharma Co., Ltd., Japan) for helpful discussions.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

References and Notes

• 1)  Di Gennaro F., Pizzol D., Marotta C., Antunes M., Racalbuto V., Veronese N., Smith L., Int. J. Environ. Res. Public Health, 17, 2690 (2020).
• 2)  Harapan H., Itoh N., Yufika A., Winardi W., Keam S., Te H., Megawati D., Hayati Z., Wagner A. L., Mudatsir M., J. Infect. Public Health, 13, 667–673 (2020).
• 3)  Helmy Y. A., Fawzy M., Elaswad A., Sobieh A., Kenney S. P., Shehata A. A., J. Clin. Med., 9, 1225 (2020).
• 4)  Toussi S. S., Hammond J. L., Gerstenberger B. S., Anderson A. S., Nat. Microbiol., 8, 771–786 (2023).
• 5)  Li G., Hilgenfeld R., Whitley R., De Clercq E., Nat. Rev. Drug Discov., 22, 449–475 (2023).
• 6)  Andrews H. S., Herman J. D., Gandhi R. T., Annu. Rev. Med., 75, 145–157 (2024).
• 7)  Morse J. S., Lalonde T., Xu S., Liu W. R., ChemBioChem, 21, 730–738 (2020).
• 8)  Chilamakuri R., Agarwal S., Cells, 10, 206 (2021).
• 9)  Saul S., Einav S., ACS Infect. Dis., 6, 2304–2318 (2020).
• 10)  Blair H. A., Drugs, 83, 1214–1237 (2023).
• 11)  Grein J., Ohmagari N., Shin D., et al., N. Engl. J. Med., 382, 2327–2336 (2020).
• 12)  Wang Y., Zhang D., Du G., et al., Lancet, 395, 1569–1578 (2020).
• 13)  Kokic G., Hillen H. S., Tegunov D., Dienemann C., Seitz F., Schmitzova J., Farnung L., Sievwert A., Höbartner C., Cramer P., Nat. Commun., 12, 279 (2021).
• 14)  Gao Y., Yan L., Huang Y., et al., Science, 368, 779–782 (2020).
• 15)  Bravo J. P. K., Dangerfield T. L., Taylor D. W., Johnson K. A., Mol. Cell, 81, 1548–1552.e4 (2021).
• 16)  Wahl A., Gralinski L. E., Johnson C. E., et al., Nature (London), 591, 451–457 (2021).
• 17)  Zarenezhad E., Marzi M., Med. Chem. Res., 31, 232–243 (2022).
• 18)  Alpizar S. A., Accini J., Anderson D. C., Eysa B., Medina-Piñón I., Ohmagari N., Ostrovskyy M. M., Aggrey-Amable A., Beck K., Byrne D., Grayson S., Hwang P. M. T., Lonchar J. D., Strizki J., Xu Y., Paschke A., De Anda C. S., Sears P. S., J. Infect., 87, 392–402 (2023).
• 19)  Kaore S., Atal S., Curr. Drug Res. Rev., 14, 203–214 (2022).
• 20)  Owen D. R., Allerton C. M. N., Anderson A. S., et al., Science, 374, 1586–1593 (2021).
• 21)  Hashemian S. M. R., Sheida A., Taghizadieh M., Memar M. Y., Hamblin M. R., Bannazadeh Baghi H., Nahand J. S., Asemi Z., Mirzaei H., Biomed. Pharmacother., 162, 114367 (2023).
• 22)  Yamato M., Kinoshita M., Miyazawa S., Seki M., Mizuno T., Sonoyama T., J. Infect. Chemother., 30, 946–950 (2024).
• 23)  Unoh Y., Uehara S., Nakahara K., et al., J. Med. Chem., 65, 6499–6512 (2022).
• 24)  Sasaki M., Tabata K., Kishimoto M., et al., Sci. Transl. Med., 15, eabq4064 (2023).
• 25)  Ohashi H., Watashi K., Saso W., Shionoya K., Iwanami S., Hirokawa T., Shirai T., Kanaya S., Ito Y., Kim K. S., Nomura T., Nishioka K., Ando S., Ejima K., Koizumi Y., Tanaka T., Aoki S., Kuramochi K., Suzuki T., Hashiguchi T., Maenaka K., Matano T., Muramatsu M., Saijo M., Aihara K., Iwami S., Takeda M., McKeating J. A., Wakita T., iScience, 24, 102367 (2021).
• 26)  Gandhi S., Klein J., Robertson A. J., et al., Nat. Commun., 13, 1547 (2022).
• 27)  Stevens L. J., Pruijssers A. J., Lee H. W., Gordon C. J., Tchesnokov E. P., Gribble J., George A. S., Hughes T. M., Lu X., Li J., Perry J. K., Porter D. P., Cihlar T., Sheahan T. P., Baric R. S., Götte M., Denison M. R., Sci. Transl. Med., 14, eabo0718 (2022).
• 28)  Szemiel A. M., Merits A., Orton R. J., MacLean O. A., Pinto R. M., Wickenhagen A., Lieber G., Turnbull M. L., Wang S., Furnon W., Suarez N. M., Mair D., da Silva Filipe A., PLoS Pathog., 17, e1009929 (2021).
• 29)  Hattori S., Higashi-Kuwata N., Hayashi H., et al., Nat. Commun., 12, 668 (2021).
• 30)  Kiso M., Yamayoshi S., Iida S., Furusawa Y., Hirata Y., Uraki R., Imai M., Suzuki T., Kawaoka Y., Nat. Commun., 14, 4231 (2023).
• 31)  Konno S., Kobayashi K., Senda M., et al., J. Med. Chem., 65, 2926–2939 (2022).
• 32)  Yamauchi Y., Konno S., Omura N., Yoshioka N., Hingst A., Gutschow M., Muller C. E., Taguchi A., Taniguchi A., Kawaguchi A., Hayashi Y., ACS Chem. Biol., 19, 1028–1034 (2024).
• 33)  Tsuji K., Kobayakawa T., Ishii T., Higashi-Kuwata N., Azuma C., Shinohara K., Miura Y., Yamamoto K., Nishimura S., Hattori S.-I., Bulut H., Mitsuya H., Tamamura H., Chem. Pharm. Bull., 71, 879–886 (2023).
• 34)  Tsuji K., Ishii T., Kobayakawa T., Higashi-Kuwata N., Shinohara K., Azuma C., Miura Y., Nakano H., Wada N., Hattori S.-I., Bulut H., Mitsuya H., Tamamura H., J. Med. Chem., 66, 13516–13529 (2023).
• 35)  Higashi-Kuwata N., Tsuji K., Hayashi H., et al., Nat. Commun., 14, 1076 (2023).
• 36)  Miura T., Malla T. R., Brewitz L., Tumber A., Salah E., Lee K. J., Terasaka N., Owen C. D., Strain-Damerell C., Lukacik P., Walsh M. A., Kawamura A., Schofield C. J., Katoh T., Suga H., Bull. Chem. Soc. Jpn., 97, uoae018 (2024).
• 37)  Homma T., Okamoto M., Koharazawa R., Hayakawa M., Fushimi T., Tode C., Hirota Y., Osakabe N., Baba M., Suhara Y., ACS Omega, 8, 42248–42263 (2023).
• 38)  Okamoto M., Toyama M., Baba M., Antiviral Res., 182, 104902 (2020).
• 39)  Hwang J. Y., Kawasuji T., Lowes D. J., Clark J. A., Connelly M. C., Zhu F., Guiguemde W. A., Sigal M. S., Wilson E. B., DeRisi J. L., Guy R. K., J. Med. Chem., 54, 7084–7093 (2011).
• 40)  Olliaro P., Nevill C., LeBras J., Ringwald P., Mussano P., Garner P., Brasseur P., Lancet, 348, 1196–1201 (1996).
• 41)  Gelb M. H., Curr. Opin. Chem. Biol., 11, 440–445 (2007).
• 42)  Uzor P. F., Evid. Based Complement. Alternat. Med., 2020, ID 8749083 (2020).
• 43)  Delarue-Cochin S., Paunescu E., Maes L., Mouray E., Sergheraert C., Grellier P., Melnyk P., Eur. J. Med. Chem., 43, 252–260 (2008).
• 44)  O’Neill P., Mukhtar A., Stocks P. A., Randle L. E., Hindley S., Ward S. A., Storr R. C., Bickley J. F., O’Neill I., Maggs J. L., Hughes R. H., Winstanley P. A., Bray P. G., Park K., J. Med. Chem., 46, 4933–4945 (2003).
• 45)  Afzal O., Kumar S., Haider M. R., Ali M. R., Kumar R., Jaggi M., Bawa S., Eur. J. Med. Chem., 97, 871–910 (2015).
• 46)  Zhang Y., Liao Z., Zhang L. J., Xiao H. T., Curr. Med. Res. Opin., 31, 1009–1013 (2015).
• 47)  Verbaanderd C., Maes H., Schaaf M. B., Sukhatme V. P., Pantziarka P., Sukhatme V., Agostinis P., Bouche G., Ecancermedicalscience, 11, 781 (2017).
• 48)  Kimura T., Takabatake Y., Takahashi A., Isaka Y., Cancer Res., 73, 3–7 (2013).
• 49)  Kaur R., Kumar K., Eur. J. Med. Chem., 215, 113220 (2021).
• 50)  Bocci G., Bradfute S. B., Ye C., Garcia M. J., Parvathareddy J., Reichard W., Surendranathan S., Bansal S., Bologa C. G., Perkins D. J., Jonsson C. B., Sklar L. A., Oprea T. I., ACS Pharmacol. Transl. Sci., 3, 1278–1292 (2020).
• 51)  Pastick K. A., Okafor E. C., Wang F., Lofgren S. M., Skipper C. P., Nicol M. R., Pullen M. F., Rajasingham R., McDonald E. G., Lee T. C., Schwartz I. S., Kelly L. E., Lother S. A., Mitjà O., Letang E., Abassi M., Boulware D. R., Open Forum Infect. Dis., 7, ofaa130 (2020).
• 52)  Seliem I. A., Panda S. S., Girgis A. S., Moatasim Y., Kandeil A., Mostafa A., Ali M. A., Nossier E. S., Rasslan F., Srour A. M., Sakhuja R., Ibrahim T. S., Abdel-Samii Z. K. M., Al-Mahmoudy A. M. M., Bioorg. Chem., 114, 105117 (2021).
• 53)  Zhao J., Zhang Y., Wang M., Liu Q., Lei X., Wu M., Guo S., Yi D., Li Q., Ma L., Liu Z., Guo F., Wang J., Li X., Wang Y., Cen S., ACS Infect. Dis., 7, 1535–1544 (2021).
• 54)  Gendrot M., Andreani J., Boxberger M., Jardot P., Fonta I., Le Bideau M., Duflot I., Mosnier J., Rolland C., Bogreau H., Hutter S., La Scola B., Pradines B., Travel Med. Infect. Dis., 37, 101873 (2020).
• 55)  Shionoya K., Yamasaki M., Iwanami S., Ito Y., Fukushi S., Ohashi H., Saso W., Tanaka T., Aoki S., Kuramochi K., Iwami S., Takahashi Y., Suzuki T., Muramatsu M., Matsuyama S., Takeda M., Wakita T., Watashi K., Front. Microbiol., 12, 651403 (2021).
• 56)  Shang C., Zhuang X., Zhang H., Li Y., Zhu Y., Lu J., Ge C., Cong J., Li T., Tian M., Jin N., Li X., Virol. J., 18, 46 (2021).
• 57)  Sakurai Y., Sakakibara N., Toyama M., Baba M., Davey R. A., Antiviral Res., 160, 175–182 (2018).
• 58)  DeWald L. E., Johson J. C., Gerhardt D. M., Torzewski L. M., Postnikova E., Honko A. N., Janosko K., Huzella L., Dowling W. E., Eakin A. E., Osborn B. L., Osborn B. L., Gahagen J., Tang L., Green C. E., Mirsalis J. C., Holbrook M. R., Jahrling P. B., Dayll J., Hensley L. E., Sci. Rep., 9, 20199 (2019).
• 59)  Baba M., Okamoto M., Toyama M., Sakakibara N., Shimojima M., Saijo M., Niwa T., Yagi Y., Antiviral Res., 210, 105479 (2023).
• 60)  Takayama-Ito M., Saijo M., Front. Microbiol., 11, 150 (2020).
• 61)  Baba M., Toyama M., Sakakibara N., Okamoto M., Arima N., Saijo M., Antivir. Chem. Chemother., 25, 83–89 (2017).
• 62)  Si L., Bai H., Rodas M., et al., Nat. Biomed. Eng., 5, 815–829 (2021).
• 63)  Seliem I., Panda S. S., Girgis A. S., Moatasim Y., Kandeil A., Mostafa A., Ali M. A., Nossier E. S., Rasslan F., Srour A. M., Sakhuja R., Ibrahim T. S., Abdel-Samii Z. K. M., Al-Mahmoudy A. M. M., Bioorg. Chem., 114, 105117 (2021).
• 64)  Baba M., Okamoto M., Toyama M., Aoki S., Tanaka T., PCT/JP2021/01862 (2021).
• 65)  Takenaka Y., Tanaka T., Otaki S., Kanbe A., Morita T., Yokoi K., Sekiguchi S., Nakamura K., Satoh H., Tojo T., Uchiumi F., Kitabatake K., Aoki S., Tsukimoto M., Biol. Pharm. Bull., 47, 2101–2118 (2024).
• 66)  Heidari R., Babaei H., Eghbal M. A., Res. Pharm. Sci., 9, 97–105 (2014).
• 67)  Neftel K. A., Woodtly W., Schmid M., Frick P. G., Fehr J., BMJ, 15, 721–723 (1986).
• 68)  Naisbitt D. J., Williams D. P., O’Neill P. M., Maggs J. L., Willock D. J., Pirmohamed M., Park B. K., Chem. Res. Toxicol., 11, 1586–1595 (1998).
• 69)  O’Neill P. M., Harrison A. C., Storr R. C., Hawley S. R., Ward S. A., Park B. K., J. Med. Chem., 37, 1362–1370 (1994).
• 70)  Tang Y., Wu Q., Beland F. A., Chen S., Fang J.-L., Toxicol. In Vitro, 62, 104669 (2020).
• 71)  Kalgutkar A. S., Soglia J. R., Expert Opin. Drug Metab. Toxicol., 1, 91–142 (2005).
• 72)  Fur N., Hochart G., Larchanche P.-E., Melnyk P., Eur. J. Med. Chem., 46, 3052–3057 (2011).
• 73)  Saari R., Törmä J.-C., Nevalainen T., Bioorg. Med. Chem., 19, 939–950 (2011).
• 74)  Phillips J. P., Barrall E. M., J. Org. Chem., 21, 692–694 (1956).
• 75)  The synthesis of 1a and 3b in this method was reported in Ref. 65 and 3a, 3c, 3d, and 3e were synthesized in different methods in the same paper.
• 76)  Matsuyama S., Nao N., Shirato K., Kawase M., Saito S., Takayama I., Nagata N., Sekizuka T., Katoh H., Kato F., Sakata M., Tahara M., Kutsuna S., Ohmagari N., Kuroda M., Suzuki T., Kageyama T., Takeda M., Proc. Natl. Acad. Sci. U.S.A., 117, 7001–7003 (2020).
• 77)  Delvecchio R., Higa L. M., Pezzuto P., Valadão A. L., Garcez P. P., Monteiro F. L., Loiola E. C., Dias A. A., Silva F.J. M., Aliota M. T., Caine E. A., Osorio J. E., Bellio M., O’Connor D. H., Rehen S., De Aguiar R. S., Savarino A., Campanati L., Tanuri A., Viruses, 8, 322 (2016).
• 78)  Senten K., Van der Veken P., De Meester I., Lambeir A.-M., Scharpe S., Haemers A., Augustyns K., J. Med. Chem., 47, 2906–2916 (2004).