Synthesis, cytotoxicity, and pharmacokinetic evaluations of niclosamide analogs for anti-SARS-CoV-2

Abstract

Niclosamide, an oral anthelmintic drug, could inhibit SARS-CoV-2 virus replication through autophagy induction, but high cytotoxicity and poor oral bioavailability limited its application. Twenty-three niclosamide analogs were designed and synthesized, of which compound 21 was found to exhibit the best anti-SARS-CoV-2 efficacy (EC₅₀ = 1.00 μM for 24 h), lower cytotoxicity (CC₅₀ = 4.73 μM for 48 h), better pharmacokinetic, and it was also well tolerated in the sub-acute toxicity study in mice. To further improve the pharmacokinetics of 21, three prodrugs have been synthesized. The pharmacokinetics of 24 indicates its potential for further research (AUC_last was 3-fold of compound 21). Western blot assay indicated that compound 21 could down-regulate SKP2 expression and increase BECN1 levels in Vero-E6 cells, indicating the antiviral mechanism of 21 was related to modulating the autophagy processes in host cells.

Graphical abstract

1. Introduction

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still evolving with the cases increasing globally. As of February 5, 2023, more than 754 million confirmed cases and over 6.8 million deaths have been reported according to the World Health Organization (WHO) [1]. Coronavirus was human-friendly until the highly pathogenic severe acute respiratory syndrome (SARS) broke out in 2003 [[2], [3], [4]]. After nine years of SARS, another highly pathogenic coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), emerged in Middle Eastern countries [5,6]. These two highly pathogenic coronaviruses have not spread worldwide, but SARS-CoV-2 has become a global pandemic due to its high transmissibility [7,8].

The tremendous success of antiviral agents in controlling SARS-CoV-2 patients since the beginning of the pandemic. Remdesivir, a prodrug of viral RNA-dependent RNA polymerase inhibitor, was the first approved drug for treatment of COVID-19 patients by FDA [9,10]. Molnupiravir was the second antiviral agent that received emergency use authorization by the U.S [11]. VV116 was also approved for the treatment of mild-to-moderate COVID-19 patients in China [12]. On the other hand, inhibiting the SARS-CoV-2 main protease is another important strategy to block viral replication. Paxlovid, a combination therapy using nirmatrelvir with ritonavir, has been demonstrated that significantly reduce the risk of hospital admission or death compared to the placebo group [13,14]. Last year, ensitrelvir was approved in Japan since its Phase III clinical trial reached the primary endpoint [15,16]. XIANNUOXIN, a combination therapy using simnotrelvir with ritonavir, approved for treatment of COVID-19 patients in China. Along with the likelihood that new coronavirus variants will appear in the future, it is still urgent to identify efficient antiviral inhibitors to control the current SARS-CoV-2 pandemic.

Drug repurposing is an efficient strategy to accelerate drug discovery and development. Niclosamide, an oral anthelmintic drug, has been approved for the treatment of tapeworm infection [[17], [18], [19], [20]]. Meanwhile, it also exhibits the ability to inhibit viral replication in a variety of viruses, including SARS [21], MERS-CoV [22], SARS-CoV-2 [23,24], Zika virus [25], and human adenovirus [26]. However, high cytotoxicity and poor bioavailability limit its further development and potential clinical applications. To improve antiviral activity and decrease the cytotoxicity of niclosamide, this study focused on the structure-activity relationships of niclosamide analogs by replacing groups at different moieties. And three prodrugs were also synthesized to improve the pharmacokinetics of lead compound. Furthermore, the mechanism of compound 21 against SARS-CoV-2 replication also has been explored. Western blot results indicated that 21 would down-regulate the level of SKP2 and increase the levels of BECN1.

2. Results and discussion

2.1. Chemistry

All designed compounds were prepared according to the corresponding methods and routes. To systematically evaluate the structure-activity relationship associated with niclosamide against SARS-CoV-2, we evaluated the effect of antiviral activity from substituted benzoic acid, linking groups, and substituted aniline, respectively (Fig. 1 ).

Fig. 1.

Synthesis of niclosamide analogs.

First, we focused our attention on the aniline moiety region of niclosamide. 1 was obtained by reduction the nitro of niclosamide, further ethanesulfonylation and acylation to obtain compounds 2 and 3, respectively. Compounds 4–6 were synthesized from 5-chloro-2-hydroxybenzoic acid and anilines substituted with electron-withdrawing groups (Cl, CF₃, OCF₃) on the ortho or para positions, in the presence of PCl₃. Second, 7–11 were synthesized to explore the effect of structural flexibility and bioisosteres for antiviral activity. 4-chlorophenol reacted with chlorosulfonic acid and then added 4-aminobenzotrifluoride into the reaction mixture to obtain compound 7.5-chlorosalicylic acid was esterified, hydrazinolysed to obtain compound 8b, and then compound 8b acylation with 4-(trifluoromethyl)benzoyl chloride to get compound 8c, from compound 8c can easily produce compound 8 via cyclization under thionyl chloride. Compound 8b directly reacted with trifluoro-p-tolunitrile to provide compound 10 (Scheme 1 ). 5-chlorosalicylamide reacted with 4-(trifluoromethyl)benzoyl chloride to obtain compound 9.5-chloro-2-hydroxybenzoic acid was reacted with oxalyl chloride and then the solution was added into 4-(trifluoromethyl)benzylamine in DCM to obtain compound 11.

Scheme 1.

Synthesis of niclosamide analogs and prodrugs of compound 21: a) oxalyl chloride, methanol, 95%; b) hydrazine hydrate, ethanol, 80 °C, 63% in two-step yield; c) oxalyl chloride, DCM, 0 °C; DIPEA, rt, 43.2%; d) thionyl chloride, reflux, 21.2%; e) K₂CO₃, n-butanol, reflux, 10%; f) anhydrous acetic, H₂SO₄; g) aluminum chloride, sodium chloride, 120 °C. h) toluene, PCl₃, reflux, 7%; i) N-(triphenylmethyl)-L-alanine, DMF, DCC, 12 h,64%; j) trifluoroacetic acid, DCM, hydrochloric acid.

After that, compounds 12–20 were synthesized to evaluate the effect of substitution on the benzoic acid moiety. Corresponding benzoic acid coupled with 4-aminobenzotrifluoride in the presence of PCl₃ to obtain compounds 12–19. Compound 8a underwent sequential acylation, rearrangement, and condensation processes to produce 20 (Scheme 1). We found that 4-chloro-5-fluoro-2-hydroxybenzoic acid perhaps has a favorable structure-activity relationship, so compounds 21–23 were synthesized for further investigation.

To improve the pharmacokinetic properties of compound 21, three prodrugs 24–26 were synthesized. Compound 21 was reacted with corresponding anhydride to obtain 24, 25. And compound 21 condensations with the corresponding protected amino acids and then deprotected to obtain compound 26 (Scheme 1).

2.2. Preliminary structure-activity relationship

In a biosafety level 3 laboratory, all newly synthesized niclosamide analogs were initially screened for antiviral efficacy against SARS-CoV-2. Remdesivir was used as the positive control, and DMSO was used as the negative control. Twenty-three niclosamide analogs were evaluated by detecting NP protein expression level at a concentration of 1 μM, quantified as the percentage of viral replication inhibition, of which six compounds (4, 12, 19, 21, 22, 23) demonstrating better anti-SARS-CoV-2 inhibitory activity compared to Remdesivir and niclosamide.

We focused on the aniline moiety of the niclosamide at the beginning (Table 1 ). Reduction of the nitro of niclosamide to amino, compound 1 loses the antiviral activity which may be caused by the electron-donating of amino. 2 (N-methanesulfonation) and 3 (N-aminoacylation) also lose potency in anti-SARS-CoV-2 assays. However, replacement of the nitro of niclosamide with CF₃, the antiviral activity of 6 was slightly improved. Removing the chlorine atom from compound 6 to obtain 4, and its antiviral activity was significantly enhanced. Then, replacing CF₃ with OCF₃ in compound 4 to obtain 5, the antiviral activity of 5 was weaker than compound 4. Based on the above analysis, we can conclude that the electron-withdrawing group has a positive effect on the antiviral activity than the electron-donating group, and also indicated that the introduction of trifluoromethyl and trifluoromethoxy group at the para positions of the aniline ring was beneficial for antiviral potency.

Table 1.

Preliminary structure-activity relationship.

Comp. No	Inhibition (%, 1 μM)	Comp. No	Inhibition (%, 1 μM)
NICa	39b	11	55
Remdesivir	<10	12	92
	99%c	13	<10
1	<10	14	<10
2	<10	15	50
3	<10	16	<10
4	95	17	50
5	55	18	<10
6	79	19	95
7	<10	20	50
8	<10	21	91
9	<10	22	87
10	<10	23	95

^aNIC, niclosamide.

^bTested at concentration of 2.5 μM.

^cTested at concentration of 5 μM.

Next, we turned our attention to the amide bond region. Bioisosterism is widely employed in the rational modification of lead compound, being used to increase activity and improve pharmacokinetic properties [27]. Several amide bioisostere analogs were synthesized and evaluated. Using sulfonamide, oxadiazoles, and 1,2,4-triazole to replace the amide bond of 4 to give compounds 7, 8, and 10, respectively (Fig. 1). All of those compounds lose antiviral activity. Compounds 9 and 11, inserting carbonyl and one carbon between the amide bond and aniline of compound 4, respectively, was synthesized to explore the effect of structural flexibility for antiviral activity. And their antiviral activity slightly decreased compared to compound 4. As a result, it appears to us that the amide bond seems to be the most effective for antiviral activity.

To investigate the benzoic acid moiety (Table 1), 5-fluoro replaced the 5-chloro on 2-hydroxybenzoicacid, the activity of 12 was similar to 4 and better than niclosamide. Removing the 5-chloro of compound 4, the antiviral activity of 13 disappeared. Replacement of the hydroxyl group of 4 with 2-methoxy and 2-cyano substituent (14 and 16) rendered the compounds inactive, respectively. Compound 15, 5-chloro-2-hydroxynicotinic acid replaced 5-chloro-2-hydroxybenzoic acid, and its antiviral activity was slightly diminished (50%, 1 μM). When electron-withdrawing nitro and acetyl were introduced to the 3-position of 4, respectively, 17 and 20 showed almost the same viral inhibition rate (50%, 1 μM) and were weaker than compound 4. Compared with 17 and 20, chlorine atom at 3 position directly caused compound 18 loses antiviral ability. On the other hand, when fluorine atom at 4 position of 4 gives compound 19, which can effectively inhibit viral replication (90%,1 μM).

Inspired by the antiviral potency of 19, more optimization was further pursued. By replacing trifluoromethyl of 19 with trifluoromethoxy, trifluoromethyl mercapto, or introducing a chlorine atom at the 3-position of 19 to obtain compounds 21–23, respectively. These three analogs (21–23) showed comparable levels of antiviral activity to compound 19.

2.3. Antiviral activity and cytotoxicity studies of active compound

In light of the above results, the antiviral activity (EC₅₀) and cytotoxicity (CC₅₀) of the active compounds (inhibition >85%) were further investigated (Table 2 ) (Fig. 2 ). From Table 2, we can conclude that all active compounds exhibited more potential antiviral activity than niclosamide (EC₅₀ = 4.63 μM). Among these compounds, compounds 19 and 21 exhibited equivalent antiviral activity (EC₅₀ = 1.00 μM, for 24 h). Niclosamide had high cytotoxicity against Vero-E6 (CC₅₀ = 1.96 μM, for 48 h), and the selectivity index between antiviral efficacy and cytotoxicity was 0.43. We are pleased that the cytotoxicity of compounds 19 and 21 were decreased, with CC₅₀ = 3.73 μM and CC₅₀ = 4.73 μM, respectively. In the following experiment, we will focus on the two lead compounds.

Table 2.

Antiviral activity and Cytotoxicity studies of active compounds.

Comp. No	Structure	EC50b (μM)	CC50c (μM)	Selectivity index
NICa		4.63	1.96	0.43
4		1.31	1.45	1.10
12		1.64	1.61	0.98
19		1.00	3.73	3.73
21		1.00	4.73	4.73
22		1.61	2.63	1.63
23		2.67	4.52	1.69

^aNIC, niclosamide.

^bVero-E6 cell incubation for 24 h.

^cVero-E6 cell incubation for 48 h.

Fig. 2.

The anti-SARS-CoV-2 activity and cytotoxicity of the niclosamide analogs 4 (A), 12 (B), 19 (C), 21 (D), 22 (E), 23 (F), niclosamide(G). As red line, the niclosamide analogs were screened for viral inhibitory activity in Vero-E6 cells. Vero-E6 cells were infected with SARS-CoV-2 at 0.01 multiplicity of infection (MOI) in the presence of different compounds, after incubation for 24 h and inhibition detected with IFA assay. As blue line, the cytotoxicity of niclosamide analogs was detected with serial dilution. The cell viability was tested by CCK8 assay after incubation for 48 h with different concentrations.

2.4. Active compound pharmacokinetic study (for screening)

Compounds 19 and 21 were progressed into pharmacokinetics studies in vivo where were administered orally to ICR (CD-1) mice at a dose of 30 mg/kg ( Table 3 ). 19 and 21 offered a 3.19-fold and 10.84-fold improvement in plasma exposure (AUC_last) compared to niclosamide, respectively. Compound 19 has similar maximum plasma concentrations to niclosamide (C_max = 452 ng/mL, C_max = 428 ng/mL), while the maximum plasma concentration of compound 21 is greatly improved compared to niclosamide (C_max = 2376 ng/mL). It is easily concluded that compound 21 was better than 19, indicating the potential for further research.

Table 3.

Active compound pharmacokinetic study for screening.

Comp. No	T1/2	Cmax	AUClastb	AUCINF_obs	MRTINF_obs
	(h)	(ng/mL)	(h*ng/mL)	(h*ng/mL)	(h)
NICa	1.99	428	478	522	2.81
19	3.10	452	1563	1573	5.45
21	3.33	2376	5547	5579	4.41

^aNIC, niclosamide.

^bMole correction has been applied when analyzing the data.

2.5. Determination of metabolic stability in liver microsomes

Drug-like properties, such as metabolic stability, help us to obtain the lead compound. The microsomal stability of compound 21 was significantly improved comparing with niclosamide ( Table 4 ). The half-life of niclosamide and 21 was 5.86 min and 22.6 min on the mouse species and 13.3 min and 120 min on the human species, respectively. It is worth noting that compound 21, the most promising derivative in this series, demonstrated adequate metabolic stability with a longer intrinsic half-life than niclosamide.

Table 4.

The metabolic stability of niclosamide and 21 in MLMs and HLMs.

Comp. No	Mouse t1/2 (min)	Human t1/2 (min)
NICa	5.86	13.3
21	22.6	120

^aNIC, niclosamide.

2.6. Sub-acute toxicity study (dose range toxicity study for ten days of 21)

Sub-acute toxicity study of compound 21 was conducted in male ICR mice weighing 18–20 g, 10 mice per dosage group. ICR mice were given 50 mg/kg, 100 mg/kg, and 200 mg/kg once a day for ten days and recorded symptoms. The animals fasted before dissection. No mortality occurred during the 10 days observation period. No significant changes in body weight, organ weight, or tissue histology were observed after animal sacrifice. This Sub-acute toxicity study indicated that compound 21 was well tolerated in ICR mice at an oral dosage up to 200 mg/kg.

2.7. Pharmacokinetic study of prodrug

Despite the significant improvement in antiviral activity, cytotoxicity, and microsomes liver stability of 21, its oral bioavailability still did not meet expectations. Several prodrugs were synthesized to further improve compound 21's pharmacokinetic characteristics. The application of prodrug approaches to successfully mitigate drug delivery issues of lead molecules is well-documented [28,29]. As shown in Table 5 , two esters were assessed the pharmacokinetics in mice, 24 and 25 were absorbed and rapidly hydrolyze to compound 21 after single-dose administration. 24 and 25 enhanced oral bioavailability in mice with F values of 39.75% and 37.61%, respectively, higher than compound 21 (F = 12.68%). As for amino acid prodrug 26, it barely transforms to parent compound 21, and almost retains prodrug form in mice plasma. The reason is that good stability makes prodrug 26 difficult to hydrolyze in plasma and liver microsomes. In summary, prodrug 24 improved the plasma exposures (3-fold) and oral bioavailability (F = 39.75%) compared to compound 21.

Table 5.

Pharmacokinetic study of prodrugs.

Comp. No	analyte	t1/2(h)	Cmax (ng/mL)	AUClasta (h*ng/mL)	AUCINF_obs (h*ng/mL)	MRTINF_obs(h)	F (%)
21 (po)	21	3.33	2376	5547	5579	4.41	12.68
21 (iv)	21	4.60		3646	3678	1.94
24	21	3.03	5175	17,393	17,551	5.52	39.75
	24	–	–	–	–	–
25	21	3.57	4771	16,458	16,726	6.03	37.61
	24	–	–	–	–	–
26	21	–	–	–	–	–	0
	26	2.92	3357	6668	6696	5.02

Oral bioavailability, F= (AUC_{last (po)}/30)*100/(AUC_{last (iv)}/2.5); po, Oral administration; iv, Intravenous injection; AUC_last, compound 21 or prodrug plasma exposure after dosing prodrug; a, Mole correction has been applied when analyzing the data.

2.8. Water solubility

Based on anti-SARS-CoV-2 activity and pharmacokinetic parameters (Table 2, Table 5), the most promising active compound 21 and prodrugs 24, 25 were tested for water solubility. As shown in Table 6 , the water solubility of niclosamide and 21 was 0.16 μg/mL and 0.10 μg/mL in water, respectively, while the water solubility of prodrugs 24 and 25 was 0.53 μg/mL and 0.71 μg/mL in water, respectively. Compared with niclosamide and 21, the solubility of prodrugs 24 and 25 increased slightly, which could be due to intramolecular hydrogen bonding limiting the solubility of niclosamide and 21 in water.

Table 6.

Solubility of active compound 21 and prodrugs 24, 25 in water.

Comp. No	Solubility in water (ug/mL)
NIC	0.16
21	0.10
24	0.53
25	0.71

2.9. Mechanisms of niclosamide analogs for anti-SARS-CoV-2

Autophagy is an essential cellular process affecting virus infections and Beclin 1 (BECN1) is one of the key regulators. SARS-CoV-2 can limit the signal of autophagy and block autophagy flux [30]. Nils C et al. reported that inhibition of S-phase kinase-associated protein 2 (SKP2) would slow down the process of BECN1 ubiquitination and reduce the degradation of BECN1 protein, thereby enhancing autophagic flux and inhibiting SARS-CoV-2 replication [22,30]. Niclosamide, an SKP2 inhibitor, has been demonstrated that increases the levels of the autophagy regulator BECN1, boosting autophagy in virus-infected cells. Inspired by this literature, we hypothesized that compound 21 may have the same antiviral mechanism as niclosamide. Therefore, western blot experiment was performed, monitoring the protein levels of SKP2 and BECN1 after addition of niclosamide and compound 21 in Vero-E6 cells. From Fig. 3 , western blot results indicated that the level of SKP2 is reduced after addition of niclosamide and 21 (A), the precise mechanism is unknown. As shown in Fig. 3 (B), the levels of BECN1 of niclosamide and 21 were higher than the control group at a concentration of 10 μM in Vero-E6 cells, and compound 21 also slightly increased BECN1 levels at concentrations of 3 μM, and 1 μM. This result indicated that compound 21 perhaps also prevents the replication of SARS-CoV-2 by changing SKP2 levels.

Fig. 3.

The levels of SKP2 and BECN1 after addition of niclosamide and 21.

3. Conclusion

In summary, we have synthesized a series of niclosamide analogs for anti-SARS-CoV-2 replication. Twenty-three niclosamide analogs were synthesized and evaluated for their anti-SARS-CoV-2 activity, cytotoxicity, metabolic stability, pharmacokinetics, and water solubility. The antiviral activity study revealed that compound 21 exhibited a better property for anti-SARS-CoV-2 (EC₅₀ = 1.00 μM), which was 4.6-fold than niclosamide (EC₅₀ = 4.63 μM), and the selectivity of cytotoxicity versus antiviral potency was improved from 0.43 to 4.73. Excepted that, three prodrugs of compound 21 were synthesized and evaluated pharmacokinetics in mice. Among these prodrugs, compound 24 was shown up as one of the most promising analogs which increased oral bioavailability in mice with F values of 39.75% and also improved the plasma exposure of 21 (3-fold). Accordingly, compound 24 is proposed as a better anti-SARS-CoV-2 agent than niclosamide, which is worth for advanced studies in the future.

4. Experimental

4.1. General chemistry information

All commercially available starting materials and solvents were reagent grade and were used without further purification. Preparative column chromatography was performed using silica gel 60, particle size 200−300 mesh. Analytical TLC was carried out by employing silica gel 60 HSGF254 plates. Molecular sieves were activated at 320 °C and cooled down to ambient temperature prior to use. ¹H NMR (500 MHz) and ¹³C NMR (126 MHz) spectra data were recorded on a Bruker 500 MHz spectrometer and H NMR (800 MHz) and ¹³C NMR (201 MHz) spectra data were recorded on a Bruker 800 MHz spectrometer. Data are reported as chemical shift with TMS as an internal standard, Chemical hifts were expressed in ppm, and J values were given in Hz, multiplicity (s = siglet, d = doublet, t = triplet, dd = doubledoublet, dt = doubletriplet, td = tribledoublet, m = multiplet). High-resolution mass spectra (HRMS) was measured by 1290–6545 UHPLC-QTOF.

4.1.1. N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide(1)

To a solution of niclosamide (400 mg, 1.22 mmol) in 15 mL of methanol was added hydrochloric acid (0.4 mL), Zinc dust (318 mg, 4.89 mmol) was added to the mixture under ice bath. The mixture was refluxed for 5 h, and TLC indicated that the raw material was consumed. Ethyl acetate (25 mL) was added to the solution, then filtered, and the filtrate was concentrated under vacuum. Residue was dissloved in ethyl acetate and extracted with sodium bicarbonate solution, and the organic phase dried by anhydrous sodium sulfate. Then the mixture was filtered and concentrated to obtain compound 1 (300 mg, 83%) as a pale white solid, ¹H NMR (800 MHz, DMSO‑d ₆) δ 12.25 (s, 1H), 10.39 (s, 1H), 8.01 (d, J = 2.7 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.71 (d, J = 2.5 Hz, 1H), 6.55 (dd, J = 8.7, 2.5 Hz, 1H), 5.38 (s, 2H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 163.84, 156.60, 147.61, 133.19, 128.86, 126.71, 126.18, 123.01, 122.61, 119.14, 118.78, 113.39, 112.80. HRMS (ESI): calcd for C₁₃H₁₁Cl₂N₂O₂, (M + H)⁺ 297.0197; found, 297.0193.

4.1.2. 5-Chloro-N-(2-chloro-4-(methylsulfonamido)phenyl)-2-hydroxybenzamide(2)

To a solution of compound 1 (50 mg, 0.17 mmol) and pyridine (32 mg, 0.40 mmol) in 4 mL of DCM was slowly added methane sulfonyl chloride (21 mg, 0.19 mmol) at 0 °C. The mixture was stirred at 0 °C for 5 h and the solvent was removed by a rotary evaporator. The residue was purified on flash column to provide the title compound 2 (50 mg, 80%) as a pale white solid. ¹H NMR (500 MHz, Methanol-d ₄) δ 8.31 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 2.8 Hz, 1H), 7.44–7.38 (m, 2H), 7.22 (dd, J = 8.9, 2.5 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 2.99 (s, 3H). ¹³C NMR (126 MHz, Methanol-d ₄) δ 163.91, 155.69, 135.45, 133.23, 131.64, 129.76, 125.26, 124.64, 123.92, 120.89, 119.39, 119.28, 118.25, 37.96. HRMS (ESI): calcd for C₁₃H₁₁Cl₂N₂O₂, (M + H)⁺ 374.9973; found, 374.9972.

4.1.3. N-(4-acetamido-2-chlorophenyl)-5-chloro-2-hydroxybenzamide(3)

To a solution of compound 1 (40 mg, 0.14 mmol) in 4 mL of DCM was added DIPEA (50 mg, 0.39 mmol). Acetic anhydride (30 mg, 0.27 mmol) was slowly added under ice-bath. The mixture was stirred at room temperature for 5 h and then concentrated. Residue was dissolved in ethyl acetate and washed with 1 M HCl solution, and the organic phase dried by anhydrous sodium sulfate. Then the mixture was filtered and concentrated to obtain compound 3 (33 mg, 75%) as a pale yellow solid. ¹H NMR (800 MHz, DMSO‑d ₆) δ 12.30 (s, 1H), 10.84 (s, 1H), 10.14 (s, 1H), 8.22 (d, J = 8.9 Hz, 1H), 7.98 (d, J = 2.8 Hz, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 8.8, 2.8 Hz, 1H), 7.43 (dd, J = 8.9, 2.4 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 2.05 (s, 3H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 168.54, 162.89, 155.98, 136.66, 133.36, 129.93, 129.49, 124.02, 123.64, 123.17, 119.42, 119.21, 119.19, 118.04, 23.97. HRMS (ESI): calcd for C₁₅H₁₂Cl₂N₂O₃, (M + H)⁺ 339.0303; found, 339.0300.

4.1.4. General procedure for prepare of niclosamide analogs

To a suspension of substituted benzoic acid (1 eq) and substituted aniline (1 eq) in 20 mL of anhydrous toluene was added PCl₃ (0.4 eq). Then the reaction mixture was refluxed at 110 °C for 5–10 h and the end up of reaction was determined by TLC. After the raw materials were consumed, cooling the reaction to room temperature. Ethyl acetate and sodium bicarbonate solution were added to the mixture and stirred for 20 min. Transfer the mixture to a separating funnel, separating, and the organic phase washed with brine. The organic phase was dried by Na₂SO₄, filtered, and concentrated. The residue was purified by column chromatography (PE/EA) to give the desired products.

4.1.5. 5-Chloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide(4)

Compound 4 (212 mg, 67%) was prepared as a white solid according to general procedure, starting from 5-chloro-2-hydroxybenzoic acid and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 11.58 (s, 1H), 10.65 (s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 2.7 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 165.08, 156.26, 141.87, 133.06, 128.65, 126.08, 126.06, 126.04, 124.33 (d, J = 271.0 Hz), 124.10 (q, J = 31.8 Hz), 122.81, 120.45, 120.40, 119.01. HRMS (ESI): calcd for C₁₄H₉ClF₃NO₂, (M + H)⁺ 316.0352; found, 316.0353.

4.1.6. 5-Chloro-2-hydroxy-N-(4-(trifluoromethoxy)phenyl)benzamide(5)

Compound 5 (180 mg, 53%) was prepared as a white solid according to general procedure, starting from 5-chloro-2-hydroxybenzoic acid and 4-(tifluoromethoxy)aniline. ¹H NMR (800 MHz, DMSO‑d ₆) δ 11.69 (s, 1H), 10.53 (s, 1H), 7.91 (d, J = 2.7 Hz, 1H), 7.84–7.81 (m, 2H), 7.47 (dd, J = 8.8, 2.7 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 165.04, 156.63, 144.28, 137.31, 133.06, 128.44, 122.73, 122.17, 121.60, 120.14 (q, J = 255.8 Hz), 119.86, 119.05. HRMS (ESI): calcd for C14H9ClF3NO3, (M + H)⁺ 332.0301; found, 332.0298.

4.1.7. 5-Chloro-N-(2-chloro-4-(trifluoromethyl)phenyl)-2-hydroxybenzamide(6)

Compound 6 (120 mg, 55%) was prepared as a white solid according to general procedure, starting from 5-chloro-2-hydroxybenzoic acid and 4-amino-3-chlorobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 12.41 (s, 1H), 11.16 (s, 1H), 8.72 (d, J = 8.7 Hz, 1H), 7.98 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 2.8 Hz, 1H), 7.77 (dd, J = 8.9, 2.2 Hz, 1H), 7.52 (dd, J = 8.7, 2.8 Hz, 1H), 7.08 (d, J = 8.7 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 163.07, 155.70, 139.28, 134.27, 130.48, 126.89, 125.70, 125.32 (q, J = 33.4 Hz), 124.14, 123.93 (d, J = 271.7 Hz), 123.51, 122.35, 120.04, 119.61. HRMS (ESI): calcd for C₁₄H₈Cl₂F₃NO₂, (M + H)⁺ 349.9962; found, 349.9954.

4.1.8. 5-Chloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzenesulfonamide(7)

4-Chlorophenol (500 mg, 5 mmol) was added into sulfurochloridic acid (3.3 mL) at 0 °C and stirred for 2.5 h. TLC indicated that the raw material was consumed. The reaction mixture was dropwise into ice-water under vigorously stirred. The aqueous phase was extracted with ethyl acetate and washed with brine, dried by Na₂SO₄, filtered, and concentrated. The residue was dissolved in DCM for next step. To a solution of 4-aminobenzotrifluoride (403 mg, 2.5 mmol) and pyridine (1.1 g, 15 mmol) in 20 mL of DCM was added the prepared sulfonyl chloride solution at 0 °C. The mixture was stirred for 12 h at 0 °C. 1 M HCl solution was added to the reaction system and extracted, the organic phase was washed with brine, dried by Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography to give the tittle compound 7 (160 mg, 18%) as a yellow solid. ¹H NMR (800 MHz, DMSO‑d ₆) δ 11.32 (s, 1H), 10.74 (s, 1H), 7.71 (d, J = 2.7 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.48 (dd, J = 8.8, 2.7 Hz, 1H), 7.27 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.8 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 154.49, 141.63, 134.80, 129.08, 126.44, 125.75, 124.28 (d, J = 271.6 Hz), 123.22 (d, J = 32.1 Hz), 121.97, 119.20, 118.08. HRMS (ESI): calcd for C₁₃H₉ClF₃NO₃S, (M + H)⁺ 352.0022; found, 352.0016.

4.1.9. 4-Chloro-2-(5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)phenol (8)

To a solution of 5-chloro-2-hydroxybenzoic acid (5.0 g, 29 mmol) and DMF (113 mg, 1.45 mmol) in 40 mL of DCM was dropwise added oxalyl chloride (5.52 g, 43.5 mmol) at 0 °C, stirred at room temperature for 4 h. Then the reaction mixture was slowly added into methanol at 0 °C, stirred the reaction for 30 min and concentrated to obtain crude compound 8a (5.12 g, 95%). ¹H NMR (500 MHz, CDCl₃) δ 10.63 (s,1H), 7.75 (d, J = 2.7 Hz, 1H), 7.33 (dd, J = 8.8, 2.7 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 3.89 (s, 3H).

To a solution of compound 8a in 15 mL of ethanol was added hydrazine hydrate (3.5 g, 58 mmol), the mixture was refluxed for 12 h, TLC indicated that the raw material was consumed. The solvent was removed by a rotary evaporator, and the residue was suspended in water, stirred for 1 h, and filtered to obtain a white solid 8b (3.3 g, 63% in two-step yield). ¹H NMR (500 MHz, DMSO‑d ₆) δ 12.45 (s, 1H), 10.10 (s, 1H), 7.86 (d, J = 2.7 Hz, 1H), 7.40 (dd, J = 8.8, 2.6 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 4.71 (s, 2H).

To a solution of 4-(trifluoromethyl)benzoic acid (1.9 g, 10 mmol) and DMF (40 mg, 0.5 mmol) in 20 mL of DCM was dropwise added oxalyl chloride (1.4 g, 11 mmol) at 0 °C, stirred at room temperature for 2 h. Then the prepared acyl-chloride solution was dropwise added to a solution of compound 8b and DIPEA (3.8 g, 30 mmol) in 15 mL of DCM at 0 °C. The mixture was stirred overnight at room temperature, and the solvent was removed by a rotary evaporator. The residue was dissolved in ethyl acetate and washed with saturated ammonium chloride solution and brine, dried by Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography to give the compound 8c (1.55 g, 43.2%). ¹H NMR (500 MHz, DMSO‑d ₆) δ 11.87 (s, 1H), 11.02 (s, 1H), 10.72 (s, 1H), 8.12 (d, J = 8.1 Hz, 2H), 7.94–7.91 (m, 3H), 7.51 (dd, J = 8.8, 2.7 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H).

Compound 8c (90 mg, 0.25 mmol) was dissolved in SOCl₂ and the mixture was refluxed for 5 h under nitrogen atmosphere. TLC indicated that the raw material was nearly consumed. The solvent was removed by a rotary evaporator and the residue was purified by column chromatography to give the tittle compound 8 (18 mg, 21.2%) as a yellow solid. ¹H NMR (800 MHz, DMSO‑d ₆) δ 10.69 (s, 1H), 8.34 (d, J = 8.1 Hz, 2H), 8.01 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 2.7 Hz, 1H), 7.54 (dd, J = 8.8, 2.7 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 162.82, 162.70, 155.42, 133.22, 131.65 (d, J = 32.2 Hz), 128.34, 127.63, 127.05, 126.46, 124.45, 123.11 (d, J = 5.1 Hz), 119.13, 111.17. HRMS (ESI): calcd for C₁₅H₈ClF₃N₂O₂, (M + H)⁺ 341.0304; found, 341.0299.

4.1.10. 5-Chloro-2-hydroxy-N-(4-(trifluoromethyl)benzoyl)benzamide(9)

To a solution of 4-(trifluoromethyl)benzoic acid (570 mg, 3 mmol) and DMF (12 mg, 0.15 mmol) in 8 mL of DCM was dropwise added oxalyl chloride (419 mg, 3.3 mmol) at 0 °C, stirred at room temperature for 1.5 h. Then the prepared acyl-chloride solution was dropwise added to a solution of 5-chlorosalicylamide and DBU (1.4 g, 9 mmol) in 15 mL of acetonitrile at 0 °C. The mixture was stirred for 4 h at room temperature, and the solvent was removed by a rotary evaporator. The residue was dissolved in ethyl acetate and extracted with 1 M HCl solution and brine. dried by Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography to give the tittle compound 9 (400 mg, 40%) as a white solid. ¹H NMR (500 MHz, DMSO‑d ₆) δ 11.93 (s, 2H), 8.10 (d, J = 7.9 Hz, 2H), 7.93 (d, J = 7.9 Hz, 2H), 7.74 (s, 1H), 7.64–7.25 (m, 1H), 6.99 (d, J = 8.9 Hz, 1H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 165.17, 164.71, 156.43, 138.01, 133.90, 132.80 (q, J = 32.0 Hz), 130.04, 129.34, 126.24, 124.24 (d, J = 272.7 Hz), 123.26, 121.65, 119.62. HRMS (ESI): calcd for C₁₅H₈ClF₃N₂O₂, (M + H)⁺ 344.0301; found, 344.0293.

4.1.11. 4-Chloro-2-(5-(4-(trifluoromethyl)phenyl)-4H-1,2,4-triazol-3-yl)phenol (10)

Compound 8b (186 mg, 1 mmol), 4-(trifluoromethyl)benzonitrile (205 mg, 1 mmol) and K₂CO₃ (276 mg, 2 mmol) were suspended in 5 mL of n-butanol. The mixture was refluxed for 3 h. Ethyl acetate (10 mL) was added to the mixture and extracted with acid water, the organic phase was washed with brine, dried by Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography to give the tittle compound 10 (34 mg, 10%).¹H NMR (500 MHz, DMSO‑d ₆) δ 14.35 (s, 1H), 11.40 (s, 1H), 8.30 (d, J = 8.1 Hz, 2H), 8.06 (s, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.41 (dd, J = 8.9, 2.6 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 158.88, 154.56, 152.45, 134.51, 131.35, 127.05, 126.57, 125.85, 123.10, 118.52, 114.12. HRMS (ESI): calcd for C₁₅H₉ClF₃N₃O, (M + H)⁺ 340.0464; found, 340.0461.

4.1.12. 5-Chloro-2-hydroxy-N-(4-(trifluoromethyl)benzyl)benzamide(11)

To a solution of 5-chloro-2-hydroxybenzoic acid (172 mg, 1 mmol) and DMF (4 mg, 0.05 mmol) in 4 mL of DCM was dropwise added oxalyl chloride (139 mg, 1.1 mmol) at 0 °C, stirred at room temperature for 1.5 h. Then the prepared acyl-chloride solution was dropwise added to a solution of 4-(trifluoromethyl)benzylamine (175 mg, 1 mmol) and DIPEA (380 mg, 3 mmol) in 10 mL of DCM at 0 °C. The mixture was stirred for 5 h at room temperature. The mixture was extracted with 1 M HCl solution and brine, dried by Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography to give the tittle compound 11 (107 mg, 32.5%). ¹H NMR (500 MHz, DMSO‑d ₆) δ 12.35 (s, 1H), 9.45 (t, J = 5.9 Hz, 1H), 7.97 (d, J = 2.7 Hz, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.46 (dd, J = 8.8, 2.6 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 4.59 (d, J = 5.8 Hz, 2H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 167.95, 158.82, 144.17, 133.88, 128.48, 128.03, 127.06 (d, J = 296.6 Hz), 125.76, 125.74, 122.94, 119.81, 117.43, 42.68. HRMS (ESI): calcd for C₁₅H₁₁ClF₃NO₂, (M + H)⁺ 330.0508; found, 330.0508.

4.1.13. 5-Fluoro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide(12)

Compound 12 (200 mg, 67%) was prepared as a white solid according to general procedure, starting from 5-fluorosalicylic acid and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 13.22 (s, 1H), 12.31 (s, 1H), 8.35 (d, J = 3.0 Hz, 1H), 8.11 (d, J = 3.0 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 161.29, 160.93, 144.22, 141.58, 138.84, 126.35 (q, J = 3.9 Hz), 124.28 (d, J = 271.3 Hz), 123.98 (q, J = 31.9 Hz), 120.59, 119.81, 112.20. HRMS (ESI): calcd for C₁₄H₉F₄NO₂, (M + H)⁺ 300.0647; found, 300.0649.

4.1.14. 2-Hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide(13)

Compound 13 (300 mg, 75%) was prepared as a white solid according to general procedure, starting from salicylic acid and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 11.50 (s, 1H), 10.63 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.91 (dd, J = 7.9, 1.7 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.45 (ddd, J = 8.5, 7.1, 1.7 Hz, 1H), 7.01 (dd, J = 8.3, 1.1 Hz, 1H), 6.98 (td, J = 7.5, 1.2 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 166.61, 157.82, 142.05, 133.75, 129.43, 124.37 (d, J = 271.5 Hz), 123.93 (d, J = 32.1 Hz), 120.49, 119.20, 118.25, 117.16. HRMS (ESI): calcd for C₁₄H₉F₄NO₂, (M + H)⁺ 282.0742; found, 282.0738.

4.1.15. 5-Chloro-2-methoxy-N-(4-(trifluoromethyl)phenyl)benzamide(14)

Compound 14 (218 mg, 69%) was prepared as a white solid according to general procedure, starting from 5-chloro-2-methoxybenzoic acid and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 10.55 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 2.8 Hz, 1H), 7.56 (dd, J = 8.9, 2.7 Hz, 1H), 7.22 (d, J = 8.9 Hz, 1H), 3.88 (s, 3H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 163.87, 155.31, 142.41, 131.52, 128.76, 126.69, 126.08 (d, J = 4.6 Hz), 124.37 (d, J = 270.9 Hz), 124.24, δ 123.71 (q, J = 32.0 Hz), 119.61, 114.06, 56.33. HRMS (ESI): calcd for C₁₅H₁₁ClF₃NO₂, (M + H)⁺ 330.0508; found, 330.0504.

4.1.16. 5-Chloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)nicotinamide(15)

Compound 15 (165 mg, 50%) was prepared as a white solid according to general procedure, starting from 5-Chloro-2-hydroxynicotinic acid and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 13.22 (s, 1H), 12.31 (s, 1H), 8.35 (d, J = 3.0 Hz, 1H), 8.11 (d, J = 3.0 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 161.29, 160.93, 144.22, 141.58, 138.84, 126.35 (q, J = 3.9 Hz), 124.28 (d, J = 271.3 Hz), 123.98 (q, J = 31.9 Hz), 120.59, 119.81, 112.20. HRMS (ESI): calcd for C₁₅H₁₁ClF₃NO₂, (M + H)⁺ 317.0304; found, 317.0299.

4.1.17. 5-Chloro-2-cyano-N-(4-(trifluoromethyl)phenyl)benzamide(16)

Compound 16 (70 mg, 21.5%) was prepared as a white solid according to general procedure, starting from 5-Chloro-2-cyanobenzoic acid and 4-aminobenzotrifluoride. ¹H NMR (500 MHz, DMSO‑d ₆) δ 10.53 (s, 1H), 8.34–8.28 (m, 1H), 8.00–7.96 (m, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 165.38, 157.96, 137.47, 137.04, 133.90, 130.84 (d, J = 250.9 Hz), 129.24, 128.68, 127.68 (d, J = 32.2 Hz), 125.65 (d, J = 3.9 Hz), 124.81, 122.98, 120.08. HRMS (ESI): calcd for C₁₅H₈ClF₃N₂O, (M + H)⁺ 325.0355; found, 325.0352.

4.1.18. 5-Chloro-2-hydroxy-3-nitro-N-(4-(trifluoromethyl)phenyl)benzamide(17)

Compound 17 (150 mg, 29.2%) was prepared as a yellow solid according to general procedure, starting from 5-chloro-2-hydroxy-3-nitrobenzoic acid and 4-aminobenzotrifluoride. ¹H NMR (500 MHz, DMSO‑d ₆) δ 11.73 (s, 1H), 8.17 (dd, J = 2.9, 1.3 Hz, 1H), 8.14 (d, J = 2.8 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 164.73, 153.49, 141.88, 139.21, 133.59, 127.67, 126.16 (d, J = 4.1 Hz), 124.94, 124.33 (d, J = 271.6 Hz), 124.18 (d, J = 31.7 Hz), 120.46, 119.68. HRMS (ESI): calcd for C₁₄H₈ClF₃N₂O₄, (M + H)⁺ 361.0203; found, 361.0199.

4.1.19. 3,5-Dichloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide(18)

Compound 18 (235 mg, 67.3%) was prepared as a white solid according to general procedure, starting from 3,5-dichloro-2-hydroxybenzoic acid and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 12.21 (s, 1H), 10.87 (s, 1H), 8.05 (d, J = 2.5 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 2.5 Hz, 1H), 7.77 (d, J = 8.7 Hz, 2H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 166.57, 154.13, 141.35, 133.03, 126.75, 126.06 (d, J = 4.8 Hz), 124.74 (d, J = 271.6 Hz), 124.68 (d, J = 31.6 Hz), 122.73, 122.53, 121.25, 119.75, HRMS (ESI): calcd for C₁₄H₈Cl₂F₃NO₂, (M + H)⁺ 349.9962; found, 349.9962.

4.1.20. 5-Chloro-4-fluoro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide(19)

Compound 19 (30 mg, 45.5%) was prepared as a white solid according to general procedure, starting from 5-chloro-4-fluoro-2-hydroxybenzoic acid and 4-aminobenzotrifluoride. ¹H NMR (500 MHz, DMSO‑d ₆) δ 12.08 (s, 1H), 10.61 (s, 1H), 8.08 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 10.7 Hz, 1H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 164.63, 159.47 (d, J = 251.7 Hz), 158.31 (d, J = 11.6 Hz), 141.76, 130.95, 126.06 (d, J = 3.9 Hz), 124.31 (d, J = 271.5 Hz), 124.18 (d, J = 32.1 Hz), 120.53, 116.83, 109.70 (d, J = 18.4 Hz), 105.48 (d, J = 23.4 Hz). HRMS (ESI): calcd for C₁₄H₈Cl₂F₃NO₂, (M + H)⁺ 334.0258; found, 334.0253.

4.1.21. 3-Acetyl-5-chloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide(20)

To a solution of compound 8a in 5 mL of anhydrous acetic anhydride was added sulphuric acid (100 μL), the mixture was stirred for 30 min. Ethyl acetate was added to the mixture and extracted with sodium bicarbonate solution, the organic phase was washed with brine, dried by Na₂SO₄, filtered and concentrated to obtain compound 20a. ¹H NMR (500 MHz, DMSO‑d ₆) δ 7.91 (dd, J = 2.7, 0.9 Hz, 1H), 7.76 (ddd, J = 8.7, 2.7, 1.0 Hz, 1H), 7.31 (dd, J = 8.6, 1.0 Hz, 1H), 3.82 (d, J = 0.9 Hz, 3H), 2.29 (d, J = 0.9 Hz, 3H).

Compound 20a, aluminum chloride (30 g), and sodium chloride (16 g) were added into a flask, the mixture was stirred for 3 h at 120 °C under nitrogen atmosphere. 1 M HCl solution was slowly added to the mixture under ice-bath and extracted with ethyl acetate (30 mL), the organic phase was washed with brine, filtered and concentrated. The residue was purified by column chromatography to give the tittle compound 20b (1.1 g, 40% in two steps). ¹H NMR (500 MHz, DMSO‑d ₆) δ 12.27 (s, 1H), 7.95 (d, J = 2.8 Hz, 1H), 7.86 (d, J = 3.0 Hz, 1H), 2.61 (s, 3H).

Compound 20 (105 mg, 7%) was prepared as a yellow solid according to general procedure, starting from compound 20b and 4-aminobenzotrifluoride. ¹H NMR (800 MHz, DMSO‑d ₆) δ 13.00 (s, 1H), 10.73 (s, 1H), 8.12 (d, J = 2.7 Hz, 1H), 8.05 (d, J = 2.7 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.7 Hz, 2H), 2.72 (s, 3H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 204.01, 164.44, 158.21, 142.42, 135.09, 133.88, 126.60 (d, J = 4.5 Hz), 125.41, 124.79 (d, J = 271.5 Hz), 124.60 (q, J = 31.8 Hz), 123.45, 123.05, 120.63, 28.87. HRMS (ESI): calcd for C₁₅H₁₁ClF₃NO₃, (M + H)⁺ 358.0458; found, 358.0459.

4.1.22. 5-Chloro-4-fluoro-2-hydroxy-N-(4-(trifluoromethoxy)phenyl)benzamide(21)

Compound 21 (2.0 g, 70.1%) as a white solid according to general procedure, starting from 5-chloro-4-fluoro-2-hydroxybenzoic acid and 4-(tifluoromethoxy)aniline. ¹H NMR (500 MHz, DMSO‑d ₆) δ 12.20 (s, 1H), 10.48 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.83–7.78 (m, 2H), 7.40–7.35 (m, 2H), 7.01 (d, J = 10.8 Hz, 1H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 164.64, 159.50 (d, J = 251.7 Hz), 158.71 (d, J = 11.6 Hz), 144.38, 137.14, 130.72, 122.29, 121.55, 120.12 (d, J = 255.7 Hz), 116.18, 109.67 (d, J = 18.2 Hz), 105.50 (d, J = 23.5 Hz). HRMS (ESI): calcd for C₁₄H₈ClF₄NO₃, (M + H)⁺ 350.0207; found, 350.0202.

4.1.23. 5-Chloro-4-fluoro-2-hydroxy-N-(4-((trifluoromethyl)thio)phenyl)benzamide(22)

Compound 22 (126 mg, 69.2%) as a white solid according to general procedure, starting from 5-chloro-4-fluoro-2-hydroxybenzoic acid and 4-(trifluoromethylthio)aniline. ¹H NMR (800 MHz, DMSO‑d ₆) δ 12.09 (s, 1H), 10.64 (s, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.90–7.85 (m, 2H), 7.74–7.70 (m, 2H), 7.00 (d, J = 10.8 Hz, 1H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 164.55, 159.45 (d, J = 251.9 Hz), 158.43 (d, J = 11.5 Hz), 141.20, 137.23, 130.93, 129.61 (q, J = 307.8 Hz), 121.43, 117.05, 116.87 (d, J = 2.8 Hz), 109.55 (d, J = 18.3 Hz), 105.49 (d, J = 23.3 Hz). HRMS (ESI): calcd for C₁₄H₈ClF₄NO₂S, (M + H)⁺ 365.9978; found, 365.9980.

4.1.24. 5-Chloro-N-(3-chloro-4-(trifluoromethyl)phenyl)-4-fluoro-2-hydroxybenzamide(23)

Compound 23 (126 g, 44.7%) as a white solid according to general procedure, starting from 5-chloro-4-fluoro-2-hydroxybenzoic acid and 3-chloro-4-(trifluoromethyl)phenyl]amine. ¹H NMR (500 MHz, DMSO‑d ₆) δ 11.92 (s, 1H), 10.69 (s, 1H), 8.13 (d, J = 1.8 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.88–7.80 (m, 2H), 7.01 (d, J = 10.8 Hz, 1H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 164.61, 159.47 (d, J = 252.0 Hz), 157.99 (d, J = 11.5 Hz), 142.93, 131.03, 128.56 (d, J = 5.2 Hz), 122.94 (d, J = 271.9 Hz), 121.97, 121.44 (d, J = 31.3 Hz), 118.49, 117.04, 109.70 (d, J = 18.3 Hz), 105.43 (d, J = 23.3 Hz). HRMS (ESI): calcd for C₁₄H₇Cl₂F₄NO₂, (M + H)⁺ 367.9868; found, 367.9869.

4.1.25. 4-Chloro-5-fluoro-2-((4-(trifluoromethoxy)phenyl)carbamoyl)phenyl isobutyrate (24)

To a solution of compound 21 (175 mg, 0.5 mmol) in 1 mL of isobutyric anhydride was added H₂SO₄ (100 μL). The mixture was stirred for 30 min at room temperature, and TLC indicated that the raw material was consumed. The mixture was slowly added to sodium bicarbonate solution and stirred for 15 min, filtered, the filter cake was washed with water 4 times and dried in vacuo to obtain compound 24 (180 mg, 86%) as a white solid. ¹H NMR (500 MHz, DMSO‑d ₆) δ 10.63 (s, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.79–7.76 (m, 2H), 7.57 (d, J = 9.8 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 2.75 (h, J = 7.0 Hz, 1H), 1.14 (d, J = 6.9 Hz, 7H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 174.01, 162.11, 157.93 (d, J = 251.2 Hz), 147.89, 147.80, 144.00, 137.97, 130.54, 127.56, 121.67, 121.08, 120.13 (d, J = 255.4 Hz), 116.73 (d, J = 18.0 Hz), 112.81 (d, J = 23.9 Hz), 33.27, 18.40. HRMS (ESI): calcd for C₁₈H₁₄ClF₄NO₄, (M + H)⁺ 420.0625; found, 420.0623.

4.1.26. 4-Chloro-5-fluoro-2-((4-(trifluoromethoxy)phenyl)carbamoyl)phenyl acetate (25)

Compound 25 was prepared by a procedure the same as that to prepare compound 24. Just change isobutyric anhydride to acetic anhydride. The title compound (162 mg, 82.5%) was obtained as a white solid. ¹H NMR (500 MHz, DMSO‑d ₆) δ 10.62 (s, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.81–7.75 (m, 2H), 7.55 (d, J = 9.7 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H), 2.21 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d ₆) δ 168.43, 162.19, 158.04 (d, J = 251.9 Hz), 148.11 (d, J = 10.5 Hz), 144.06, 137.94, 130.73, 127.03 (d, J = 3.8 Hz), 121.66, 121.34, 120.14 (d, J = 255.5 Hz), 116.72 (d, J = 17.6 Hz), 112.92 (d, J = 23.8 Hz). HRMS (ESI): calcd for C₁₆H₁₀ClF₄NO₄, (M + H)⁺ 392.0312; found, 392.0307.

4.1.27. 4-Chloro-5-fluoro-2-((4-(trifluoromethoxy)phenyl)carbamoyl)phenyl l-alaninate monohydrochloride (26)

To a solution of compound 21 (140 mg, 0.4 mmol), N-(triphenylmethyl)-L-alanine (132.4 mg, 0.4 mmol) and DMAP (5 mg, 0.04 mmol) in 4 mL of DMF were added DCC (82.4 mg, 0.4 mmol). The mixture was stirred for 12 h under nitrogen atmosphere, and filtered, ethyl acetate was added into the filtrate and washed with water, dried by Na₂SO₄, and the residue was purified by column chromatography to give the tittle compound 26a (170 mg, 64%) as a white solid. ¹H NMR (500 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.51–7.43 (m, 2H), 7.33–7.13 (m, 16H), 6.59 (d, J = 9.5 Hz, 1H), 3.68 (d, J = 7.4 Hz, 1H), 1.48 (d, J = 7.1 Hz, 3H).

To a solution of compound 26a (170 mg, 0.42 mmol) in 1.5 mL of DCM was added trifluoroacetic acid (0.4 mL), and the mixture was stirred for 30 min at room temperature. and the solvent was removed by a rotary evaporator. The residue was dissolved with ethyl acetate and extracted with sodium bicarbonate solution, the organic phase was washed with brine, filtered and concentrated. The residue was purified on flash column and gave a white compound and the compound was dissolved in 2 mL of methanol. Concentrated hydrochloric acid (0.6 mmol) was added, and the solvent was removed by a rotary evaporator. Toluene was added to the residue and removed by a rotary evaporator, repeating 3–4 times. to obtain title compound 26 (70 mg, 37%) as a white solid. ¹H NMR (800 MHz, DMSO‑d ₆) δ 12.75 (s, 1H), 10.36 (s, 1H), 9.10 (d, J = 6.6 Hz, 1H), 8.24 (d, J = 8.5 Hz, 1H), 7.74–7.70 (m, 2H), 7.33 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 10.7 Hz, 1H), 4.63 (p, J = 7.0 Hz, 1H), 1.45 (d, J = 7.1 Hz, 3H). ¹³C NMR (201 MHz, DMSO‑d ₆) δ 170.92, 166.06, 159.95 (d, J = 11.5 Hz), 159.64 (d, J = 252.2 Hz), 143.67, 138.06, 130.56, 121.67, 120.66, 120.13 (d, J = 255.5 Hz), 113.80, 109.45 (d, J = 18.4 Hz), 105.63 (d, J = 23.0 Hz), 49.74, 17.95. HRMS (ESI): calcd for C₁₇H₁₃ClF₄N₂O₄, (M + H)⁺ 449.0891; found, 449.0890.

4.2. Antiviral assay using SARS-CoV-2

Vero-E6 cells were seeded in 24-well plates (8 × 10⁴ cells per well) for antiviral assay. After one day of cultivation, the cells were incubated with compound diluents (2-fold) in different concentrations and infected with SARS-CoV-2 (MOI = 0.01) at 37 °C for 24 h. Then, the infected cells were fixed with cold (−20 °C) 5% acetone for IFA assay. The number of positive cells was calculated using ImageJ software. The antiviral activity of compounds was expressed as 50% effective concentration (EC₅₀) and calculated by GraphPad Prism software 8.0. Indirect immunofluorescence (IFA) assay. The cells were seeded on a Chamber Slide (Nalge Nunc). At time points of sample collection, the cells were fixed with cold (−20 °C) 5% acetone in methanol at room temperature for 10 min, washed three times with PBS and fixed with 3.7% formaldehyde for 24 h. For the detection of viral replication, the cells were incubated with rabbit antibody against NP protein (1:1000 dilution with PBS) for 1 h. After washing with PBS three times, the cells were incubated with FITC-conjugated goat anti-mouse IgG (1:125 dilution with PBS, Protein Tech Group) at room temperature for 1 h. Following PBS washing, the slides were mounted with 95% glycerol and analyzed under a Zeiss fluorescence microscope.

4.3. CCK8 cytotoxicity assay

Vero-E6 cells were plated in the 96-well plates at a density of 1 × 10⁴ cells per well for 48 h. Then the cells were incubated with the test articles at different concentrations (0.5–200 μM) for another 48 h (n = 3). A Cell Counting Kit 8 (CCK 8) purchased from Yeasen Biotech Co., Ltd. (Shanghai, China) was used for the cytotoxicity assay with 10 μl of CCK 8 being added to each well for 2 h. The absorbance was measured by an automatic microplate reader (Biotek, Winooski, VT, USA) at a wavelength of 450 nm. The half inhibitory concentration (CC₅₀) values for each compound were calculated by GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA).

4.4. Methods for pharmacokinetic study

The pharmacokinetic studies were operated at SIMM-Servier Joint Laboratory. ICR-1(CD-1) mice (N = 3 per group) fasted for 12 h before the administration of tested compounds. Each compound dissolved in DMSO/0.5%HMPC (5/95, v/v) was administered orally at 30 mg/kg and intravenously at 2.5 mg/kg, respectively. After dosing, blood samples were collected from the femoral vein of mice at a series of time points (0.25, 0.5, 1.00, 2.00, 4.00, 8.00, 24.00 h). Serum samples were obtained following general procedures and the concentrations of analytes in the supernatant were analyzed by LC-MS/MS system.

4.5. Methods for human liver microsome and mouse liver microsome studies

Microsomes in 0.1 M TRIS buffer pH 7.4 (final concentration 0.33 mg/mL), co-factor MgCl₂ (final concentration 5 mM), and tested compound (final concentration 1 μM, co-solvent (0.01% DMSO) and 0.005% Bovine serum albumin (BSA) were incubated at 37 °C for 10min. The reaction was started by the addition of NADPH (final concentration 1 mM). Aliquots were sampled at 0, 7, 17, 30, and 60 min respectively and methanol (cold at 4 °C) was added to terminate the reaction. After centrifugation (4000 rpm,5 min), samples were then analyzed by LC-MS/MS.

4.6. Methods for sub-acute toxicity study

ICR mice were male and weighted 18–20 g. Dose range toxicity studies for ten days were performed. ICR mice were assigned to four groups which contained one control group and three oral administration groups (ten ICR mice per group), the dosage of 21 were 50, 100, and 200 mg/kg, respectively. All animals were clinically observed once a day for at least ten days for toxic signs which included body weight, food intake, and behavior change. At the end of the experiment, samples of the heart, liver, spleen, lung, kidney, and administration site were collected.

4.7. Water solubility

Experiments were performed in singlicate. The solution of the tested compound was prepared at 0.5 mg/mL in 80% acetonitrile for a standard curve. The test compounds were suspended in water, vigorously vortexed then filtered with 0.22 μM microporous membrane, and the supernatant was recovered. 500 μL of supernatant was collected and then diluted 2 times with 80% acetonitrile, and analyzed by HPLC. Liquid chromatography was performed on an Agilent 1260 infinity II system with an Ascentis RP-Amide (Supelco) (5 μm, 250 mm × 4.6 mm) column. Aqueous concentration was determined by comparison of the peak intensity versus known concentrations.

4.8. Western blot assay

Culture medium was prepared with DMEM supplemented with 10% FBS and 1% P/S. 3 × 10⁵ Vero-E6 cells were seeded into a 6-well plate containing 2 mL of culture medium. The cells were cultured overnight at 37 °C, 5% CO₂ before being treated with test compounds in a series of concentrations for another 16 h. Cells were collected at the end of incubation and RIPA buffer was added to lyse the cells on ice for 20 min. The cells were then centrifuged at 14,000 rpm/4 °C for 15 min. The supernatant was collected and the total protein concentration of each sample was determined using BCA Protein Assay kit. The lysate was diluted to the same protein concentration with RIPA buffer after BCA assay. Samples of the same protein amount were mixed with 4x LDS sample buffer and boiled at 95 °C for 5min. The denatured samples were used for electrophoresis.

Western blot was performed according to the standard protocols. The same volume of protein was loaded onto 24-12% Bis-Tris gels separately. One gel is for SKP2 detection. Another is for Beclin-1 detection. The gels were run for 0.5 h at 80 V and 120 V for another 1 h. When the electrophoresis was completed, the iBlot™ 2 Gel Transfer Device was used to transfer SKP2/Beclin-1 and β-actin proteins at 20 V for 7 min. All the membranes were blocked in 5% milk in TBST buffer at room temperature for 1 h and then incubated with primary antibodies in TBST buffer containing 5% BSA at 4 °C overnight. After incubation with primary antibodies, membranes were washed with TBST, then incubated with the secondary antibody at room temperature for 1 h. Blots were visualized using the instrument Image Quant LAS-4000. The chemiluminescent signals from ECL Western blotting reagents were captured. The bands’ integrated intensity from 16-bit blot images was used for quantitation. The SKP2/Beclin-1 and the β-actin signal ratio of each sample were normalized to that of DMSO control as DMSO Control and then the normalized data of each compound were used for compound effect evaluation on SKP2/Beclin-1 protein.

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.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Data availability

Data will be made available on request.