Discovery and evolution of 12N-substituted aloperine derivatives as anti-SARS-CoV-2 agents through targeting late entry stage.

So far, there is still no specific drug against COVID-19. Taking compound 1 with anti-EBOV activity as the lead, fifty-four 12N-substituted aloperine derivatives were synthesized and evaluated for the anti-SARS-CoV-2 activities using pseudotyped virus model. Among them, 8a exhibited the most potential effects against both pseudotyped and authentic SARS-CoV-2, as well as SARS-CoV and MERS-CoV, indicating a broad-spectrum anti-coronavirus profile. The mechanism study disclosed that 8a might block a late stage of viral entry, mainly via inhibiting host cathepsin B activity rather than directly targeting cathepsin B protein. Also, 8a could significantly reduce the release of multiple inflammatory cytokines in a time- and dose-dependent manner, such as IL-6, IL-1β, IL-8 and MCP-1, the major contributors to cytokine storm. Therefore, 8a is a promising agent with the advantages of broad-spectrum anti-coronavirus and anti-cytokine effects, thus worthy of further investigation.

Introduction

Up to now, the Coronavirus Disease 2019 (COVID-19) epidemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still spreading around the world and has yet to be effectively controlled [1], [2], and SARS-CoV-2 is little known to us. Though several promising small molecule candidates had been used in clinic, such as RNA-dependent RNA polymerase inhibitor remdesivir [3], [4] and antimalarial hydroxychloroquine (Fig. 1 ) [5], [6], none of them showed satisfactory clinical effects [7], [8], [9], [10], [11], [12], [13]. In October 2020, remdesivir was approved by FDA for the treatment of COVID-19 patients in hospital [14]. However, with the emergence of the mutation of SARS-CoV-2 strains such as D614G and recurrent COVID-19 pneumonia, the treatment of COVID-19 is becoming more and more complicated and difficult [15], [16], [17], [18], [19]. Thus, it is of great significance to find broad-spectrum anti-SARS-CoV-2 drugs that are not affected by virus variation and drug resistance.

Fig. 1

Structures of remdesivir (left), hydroxychloroquine (center) and dexamethasone (right).

Cytokine storm is one of the leading causes of death in patients with severe and critically ill COVID-19 [20], [21], with significantly increased proinflammatory cytokine levels in patients, including interleukin (IL)-6, IL-1β, IL-2, IL-8, MCP-1 and tumour necrosis factor alpha (TNFα). However, both highly specific IL-6/IL-6R antibodies tocilizumab and sarilumab failed in the treatment of patients with severe or critically ill COVID-19 in phase III clinical trials. Meantime, dexamethasone (Fig. 1), a broad-spectrum immunosuppressive agent, effectively reduced mortality in critically ill patients [22], indicating the advantage of inhibiting multiple cytokines over inhibiting a single cytokine. Therefore, it is imperative to discover broad-spectrum anti-coronavirus and anti-cytokine agents to effectively prevent and control COVID-19 and cytokine storm syndrome.

In recent years, our team has been committed to the discovery of novel antiviral candidates from Tradition Chinese Medicine such as Sophora alopecuroides [23], [24], [25]. Earlier, our team found that 12N-3′,4′-dichlorobenzyl aloperine (1, Fig. 2 ) with a unique endocyclic skeleton had a broad-spectrum anti-filovirus effect against both Ebola virus (EBOV) and Marburg virus (MARV) with EC50 of 4.8 and 7.1 μM respectively, via targeting lysosome cathepsin B (Cat B) of host cells [23], [26], [27]. These results provoked us to further test its activity against SARS-CoV-2 using the newly constructed SARS-CoV-2 pseudotyped virus model in our lab [28], [29]. As anticipated, compound 1 exhibited a moderate activity against SARS-CoV-2 with an EC50 value of 28.2 μM and a selectivity index (SI) value of 5.2, showing an ideal lead for further structure modification and optimization for SARS-CoV-2.

Fig. 2

Chemical structures of aloperine and lead 1 as well as modification strategies.

Therefore, as showed in Fig. 2, taking compound 1 as the lead, several series of 12N-substituted aloperine derivatives were synthesized and evaluated for their anti-SARS-CoV-2 activity using pseudotyped virus model. Furthermore, the structure–activity relationship (SAR) analysis, anti-coronavirus and anti-cytokine effects, as well as pharmacokinetics (PK), safety profiles and primary mechanism exploration of key compounds were carried out in this study.

Results and discussion

Chemistry

A total of 54 12N-substituted aloperine derivatives, of which 39 were new, were prepared from commercially available aloperine with purity over 95%, which was purchased from Yanchi Dushun Biological and Chemical Co. Ltd. (Shanxi, China). The synthesis route of all the target compounds is displayed in Scheme 1 . Among all the target compounds, 12N-benzyl (2a–e), 12N-benzoyl (3a–c), 12N-sulfonyl (4a–c), 12N-alkyl (8a, 8b), 12N-carbamoylmethyl (11l, 11s) aloperine derivatives have been reported previously in our lab [23], [30], and the rest are newly synthesized ones.

Scheme 1

(a) R1XBr and R1XCl, MeCN or CH2Cl2, K2CO3, r.t., 2 h; (b) bromoacetyl bromide, CH2Cl2, Et3N, 0 °C to r.t., 2 h; (c) CH2Cl2, Et3N, r.t.; (d) ethyl bromoacetate, EtOH/NaHCO3, reflux, 8 h; (e) LiOH, H2O, reflux 1.5 h; (f) HOBt, DIEA, EDCI, CH2Cl2, 0 °C to r.t.; (g) SOCl2, reflux, 2 h; (h) K2CO3, CH2Cl2, r.t., 2 h; (i) LiAlH4, THF, r.t., 2 h; (j) R6NCO, CH2Cl2, Et3N, 0 °C to r.t., 1 h.

Various substituted amines (9a–s) were respectively acylated with bromoacetyl bromide to yield key intermediates 10a–s, which underwent an N-alkylation reaction with aloperine to get target compounds 11a–s in total yields of 11–53%. Another series of target compounds 14a–r was respectively gained through a condensation reaction of aloperine and the key intermediates 13a–r, which were prepared via an alkylation of commercially available different substituted amines 12a–r and ethyl bromoacetate, followed by an ester hydrolysis with LiOH as the base. The third series of products 15a–d were respectively obtained through a nucleophilic addition reaction between aloperine and isocyanates in equal molar ratio in 26–63% yields. All the final products were purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the gradient eluent.

Biological activity evaluation

SAR analysis for anti-SARS-CoV-2 activity

As reported earlier, the SARS-CoV-2 pseudovirus model was composed of plasmid pcDNA3.1.S2 expressing the key spike (S) protein and vesicular stomatitis virus (VSV) G pseudotyped virus (G*ΔG-VSV) that packages expression cassettes for firefly luciferase instead of VSV-G in the VSV genome [28]. The VSV G pseudovirus was then applied to identify their anti-VSV effects as a comparison, and inhibition of SARS-CoV-2 infection was detected as a decrease in luciferase signal. The structures, antivirus activity and cytotoxicity of all the 54 target compounds against SARS-CoV-2 and VSV were shown in Table 1 . The anti-SARS-CoV-2 potency of each compound was evaluated by the combination of its EC50 and SI values as the important evaluation indication, while referring to the EC50 of VSV.

Table 1

Structures and antiviral activities of all the target compounds against SARS-CoV-2.

No	R	R1	CC50(µM)a	EC50-SARS- CoV-2(µM)b	SIc	EC50-VSV (µM)
1	3′,4′-Cl2PhCH2	/	147.3	28.2	5.2	15.9
2a	PhCH2	/	>266.7	162.6	>1.6	68.9
2b	p-ClPhCH2	/	>266.7	115.5	>2.3	40.2
2c	o-CH3PhCH2	/	>266.7	97.0	>2.7	25.4
2d	m-OCH3PhCH2	/	>266.7	151.5	>1.8	15.9
2e	2′,4′-Cl2PhCH2	/	138.0	22.0	6.3	16.2
3a	p-ClPhCO	/	>266.7	235.6	−	214.4
3b	3′,4′-Cl2PhCO	/	>266.7	196.9	−	48.7
3c	3′,5′-Cl2PhCO	/	170.3	65.6	2.6	79.2
4a	p-ClPhSO2	/	>266.7	>266.7	−	267.6
4b	3′,4′-Cl2PhSO2	/	>266.7	136.8	>1.9	175.6
4c	3′,5′-Cl2PhSO2	/	>266.7	49.0	>5.4	38.5
8a	p-ClPh(CH2)2	/	>266.7	19.1	>14.0	25.9
8b	3′,5′-Cl2Ph(CH2)3	/	58.1	7.4	7.9	6.9
11a	p-ClPh	H	>266.7	103.2	>2.6	154.6
11b	p-BrPh	H	>266.7	62.8	>4.2	79.9
11c	p-IPh	H	124.4	29.5	4.2	47.8
11d	2′,4′-Cl2Ph	H	167.4	54.8	3.1	70.5
11e	2′,5′-Br2Ph	H	163.9	55.3	3.0	40.3
11f	2′-F-5′-BrPh	H	221.8	57.5	3.9	66.8
11g	2′-Cl-5′-BrPh	H	>266.7	90.3	>3.0	77.5
11h	2′-F-6′-CH3Ph	H	>266.7	>266.7	−	>266.7
11i	2′-Cl-4′-CH3Ph	H	>266.7	77.4	>3.4	87.9
11j	2′–CH3-4′-ClPh	H	>266.7	106.8	>2.5	75.1
11k	3′-Cl-5′-CF3Ph	H	57.1	10.4	5.5	17.5
11l	3′-Cl-5′-CF3OPh	H	62.8	14.7	4.3	21.8
11m	3′-CF3-4′-BrPh	H	38.3	4.0	9.6	6.0
11n	3′,4′-(CH3)2Ph	H	>266.7	70.8	>3.8	107.7
11o	3′,5′-(CF3)2Ph	H	71.0	17.0	4.2	21.9
11p		H	96.7	37.1	2.6	43.5
11q		H	>266.7	67.4	>4.0	119.8
11r	p-BrPh	CH3	>266.7	42.3	>6.3	152.9
11s		Ph	45.1	10.8	4.2	14.2
14a	Ph	H	>266.7	>266.7	−	>266.7
14b	p-FPh	H	>266.7	177.4	>1.5	>266.7
14c	m-FPh	H	>266.7	144.4	>1.8	>266.7
14d	p-ClPh	H	>266.7	61.3	>4.4	162.8
14e	p-BrPh	H	180.1	37.3	4.8	85.4
14f	p-IPh	H	106.8	9.4	11.4	50.2
14g	p-CH3Ph	H	>266.7	>266.7	−	>266.7
14h	m-CF3Ph	H	>266.7	69.2	>3.9	134.1
14i	m-CH3OPh	H	>266.7	>266.7	−	>266.7
14j	p-CF3OPh	H	117.6	19.3	6.1	47.6
14k	2′,4′-Cl2Ph	H	65.8	17.8	3.7	36.2
14l	3′,4′-Cl2Ph	H	63.0	15.8	4.0	35.9
14m	3′,5′-(CH3)2Ph	H	>266.7	>266.7	−	>266.7
14n	3′,5′-(CH3O)2Ph	H	>266.7	>266.7	−	>266.7
14o	3′,5′-(CF3)2Ph	H	112.7	37.6	3.0	48.3
14p		H	199.1	73.6	2.7	124.6
14q		H	224.1	87.2	2.6	108.3
14r	p-ClPh	CH3	191.8	21.1	9.1	85.6
15a	2′,4′-F2Ph	H	>266.7	195.4	>1.4	138.6
15b	3′,5′-(CH3)2Ph	H	>266.7	151.4	>1.8	94.9
15c	3′,5′-(CF3)2Ph	H	53.9	11.5	4.7	14.9
15d		H	>266.7	52.9	>5.0	106.7
CA-074	/	/	230.7	2.7	85.4	10.5

The concentration of 50% cellular toxicity in Huh 7 cells.

The concentration of compound that inhibited 50% of the virus level in Huh 7 cells.

The selectivity index-the ratio of CC50 to EC50.

SAR study was mainly focused on the influence of substituents on the 12-nitrogen atom, taking 1 with a 3, 4-dichlorobenzyl substituent as the lead. First, the methylene linker was retained, a series of 12N-benzyl aloperine derivatives 2a–e was synthesized and evaluated. Compounds 2a–d gave significantly decreased potency, while compound 2e with a 2, 4-dichlorobenzyl exhibited a moderate activity with EC50 of 22.0 μM, comparable potency to 1. Then, the mono- or dichlorophenyl was retained, and the replacement of methylene linker with carbonyl or sulfonyl yielded compounds 3a–c and 4a–c, which decreased activity in varying degrees. Furthermore, the extension of methylene to dimethylene and trimethylene gave compounds 8a and 8b, which exhibited promising potencies with EC50 values of 19.1 and 7.4 μM and SI values of over 14.0 and 7.9, respectively, indicating that the change of the linker might benefit the activity.

Next, methylene linker was replaced by carbamoylmethyl while a mono- or di-halophenyl element was retained, by which compounds 11a–g were invented and evaluated. As shown in Table 1, all of them displayed the declined activities to varying degrees, regardless of mono- or dihalophenyl substitution. Meanwhile, a halogen atom along with methyl, trifluoromethyl or trifluoromethoxy was introduced on the benzene ring to form compounds 11h–m, and most of them gave declined activities except that 11k and 11m gave slightly elevated activity. In addition, the introduction of dimethylphenyl, ditrifluoromethylphenyl, or bulky naphthyl, quinolyl and tertiary amine moiety in compounds 11n–s, caused a decrease in activity. Finally, the methylene linker was changed into aminoacetyl or carbamoyl linker to achieve compounds 14a–r and 15a–d, respectively. Most of them displayed comparable or decreased activity compared to the lead 1, except that compound 14f bearing the iodophenyl exhibited an elevated activity with the EC50 value of 9.4 μM and SI values of 11.4, indicating that iodine or chlorine atom on the phenyl ring might be beneficial for the activity.

In addition, as showed in Table 1, most of the target compounds also exhibited varying degrees of activity to VSV skeleton, therefore the antiviral activity against authentic SARS-CoV-2 should be conducted to exclude the active compounds through anti-VSV pathway for anti-SARS-CoV-2 effects.

Antiviral evaluation of compounds 2e, 8a and 8b against coronaviruses

The active compounds 2e, 8a and 8b bearing a common chlorobenzene were chosen to test their antiviral activities against authentic SARS-CoV-2 in Vero E6 cells in a BSL-3 lab. As displayed in Table 2 , compounds 2e and 8b had no activity against authentic SARS-CoV-2, indicating that the anti-SARS-CoV-2 activity in pseudovirus model might be from anti-VSV; while compound 8a displayed a moderate activity against authentic SARS-CoV-2 with an EC50 value of 39.1 µM and SI value of > 6.8, basically consistent with those in the pseudovirus model.

Table 2

Antiviral activities of part compounds against different coronaviruses.

No	CC50 (µM)a	EC50(µM)
		Authentic SARS-CoV-2b	Pseudo SARS-CoV-2c	Pseudo SARS-CoVc	Pseudo MERS-CoVc	Pseudo VSVc
2e	177.4	>3750	22.0	NTd	NTd	16.2
8a	>266.7	39.1	19.1	5.6	9.4	25.9
8b	109.6	>3750	7.4	NTd	NTd	6.9

CC50: the concentration of 50% cellular toxicity in Vero E6 cell;

EC50: the concentration of compound that inhibited 50% of the virus level in Vero E6 cell.

EC50: the concentration of compound that inhibited 50% of the virus level in Huh 7 cell.

NT: not tested.

To further evaluate the broad-spectrum anti-coronavirus effect of compound 8a, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudovirus models were applied herein [31]. As displayed in the Table 2, 8a showed better activity against SARS-CoV and MERS-CoV, indicating their broad-spectrum anti-coronavirus potential. As anticipated, 8a exhibited the higher anti-SARS-CoV and anti-MERS-CoV activity with EC50 values of 5.6 and 9.4 μM, respectively, and thus was selected as a representative compound for the next step.

Compound 8a blocks the late stage of SARS-CoV-2 entry

The SARS-CoV-2 pseudovirus model was virtually replication-defective VSV particles incorporated with the S protein of SARS-CoV-2, which could reflect only a few key steps in the entry into host cells. SARS-CoV-2 entry into host cells can be divided into several steps, including binding to angiotensin-converting enzyme 2 (ACE2) receptor on cell surface, entering host cell through endocytosis, and releasing the fuse peptide to fuse viral and lysosomal membranes [32], [33], [34], [35], [36]. Any step in the process of entry is blocked to prevent infections. Therefore, a single-cycle time-of-addition experiment was initially performed on our SARS-CoV-2 pseudovirus model to determine the specific blockage stage of compound 8a in SARS-CoV-2 entry, via adding 8a and pseudovirus at different time points and incubation temperature, as depicted in Fig. 3 A.

Fig. 3

Compounds 8a and CA-074 block SARS-CoV-2 entry into host cells. (A) Cartoon diagram of a single-cycle time-of-addition at different time and temperature. (B) Compound 8a and (C) CA-074 block SARS-CoV-2 infection at different entry stage. Error bars indicate standard deviations.

As shown in Fig. 3B, compound 8a had an inhibition rate of 27% on process a, but no inhibitory activity on process b and c, showing a weak or no inhibitory effect on the early stage of virus entry. Meanwhile, compound 8a showed a higher inhibition rate of 76% on process d, suggesting that compound 8a might affect membrane fusion process at late entry stage of SARS-CoV-2 infection, consistent with those of lead compound 1 against EBOV and MARV, mainly via inhibiting endosomal cysteine protease Cat B activity [23], [25].

Compound 8a inhibits Cat B activity in host cells

Considering the critical role of host Cat B and Cat L in membrane fusion of SARS-CoV-2 and similar mechanism of action of aloperine analogue 1, the inhibition activity of compound 8a on Cat B and Cat L enzyme in Huh 7 cells were evaluated respectively [37]. As displayed in Fig. 4 A and 4B, compound 8a significantly inhibited the Cat B activity with an IC50 value of 9.9 μM, but had no effect on Cat L. Furthermore, the affinity between 8a and recombinant Cat B was also measured to identify its direct binding with Cat B. However, as shown in Fig. 4C, compound 8a had a poor affinity with Cat B, indicating that 8a exerted a broad-spectrum anti-coronavirus effect, mainly via inhibiting host Cat B activity, rather than directly targeting Cat B protein.

Fig. 4

Effects of 8a on Cat B and Cat L. (A) Inhibitory activity of 8a Cat B in Huh 7 cells. (B) Inhibitory activity of 8a Cat L. (C) Affinity activity of 8a to recombinant Cat B.

In order to further confirm that Cat B is necessary for SARS-CoV-2 entry into the host cells, we selected a specific Cat B inhibitor CA-074 with a Ki value of 2–5 nM to test its anti-SARS-CoV-2 activity in pseudovirus model. As shown in Table 1, compound CA-074 exhibited a good potency with an EC50 value of 2.7 µM against SARS-CoV-2, and only moderate activity against VSV with an EC50 value of 10.5 µM, indicating that CA-074 could selectively inhibit SARS-CoV-2 activity. Then, the time-of-addition experiment of CA-074 was conducted, as depicted in Fig. 3C. Compound CA-074 significantly inhibited a, b and d stages with inhibitory rates of 87%, 77% and 93%, respectively, indicating a multi-target mechanism of action, in which the inhibition of Cat B in process d plays an important role. These results suggested that Cat B might be an ideal target for anti-SARS-CoV-2 drugs.

Inhibitory effects on cytokines of compound 8a

To further understand the inhibitory effects of 8a on the secretion of multiple proinflammatory factors, four cytokines including IL-6, IL-1β, IL-8, and MCP-1 were analyzed in phorbol 12-myristate 13-acetate-differentiated human monocytic cell line-1 (PMA-THP-1) triggered by lipopolysaccharide (LPS) plus nigericin (Nig) using ELISA assay [38]. Cells were primed with LPS (1 μg/mL) and then stimulated with Nig (5 μM) without or with 8a pre-treatment for different concentrations (5, 10 or 20 μM) and for different time (3 h, 6 h, 12 h or 24 h). As shown in Fig. 5 , compound 8a significantly reduced the releases of IL-6, IL-1β, IL-8 and MCP-1 in a dose-dependent and time-dependent manner, suggesting a broad-spectrum inhibiting activity of multiple cytokines.

Fig. 5

Compound 8a inhibits releases of cytokines. (A) IL-6, (B) IL-1β, (C) IL-8 and (D) MCP-1 in a concentration-dependent and time-dependent manner. Data were presented as the mean ± SEM, (###) p < 0.001 as compared to that of control group; (*) p < 0.05, (**) p < 0.01 (***) p < 0.01 as compared to that of LPS/Nig group. The experiment was repeated for 3 times.

PK and safety profiles of 8ain vivo

The in vivo PK behavior of compound 8a was evaluated in SD rat via oral administration at the dosage of 25 mg/kg. As showed in Table 3 , compound 8a gave the good in vivo PK parameters with a maximum plasma concentration (Cmax) value of 0.82 μM, a half-life (T1/2) of 2.5 h and an area under the curve (AUC) value of 4.47 μM·h, suggesting an ideal PK profile of compound 8a.

Table 3

The oral PK and safety profile of compound 8a.

No	Tmax (h)	Cmax (μM)	AUC0-t (μM·h)	AUC0-∞ (μM·h)	MRT (h)	t1/2 (h)	LD50 (mg/kg)
8a	2.50 ± 3.04	0.82 ± 0.32	4.47 ± 0.95	4.85 ± 0.26	4.59 ± 1.29	2.90 ± 1.69	812

AUC: Area under the concentration–time curve; Cmax: Maximum plasma concentration; MRT: Mean residence time; Tmax: peak time; T1/2: Half-life; LD50: 50% lethal dose.

Next, the acute toxicity assessment of 8a was performed in Kunming mice in a single oral administration at the dosage of 0, 250, 500 or 1000 mg·kg−1 respectively. The mice were closely monitored for 7 days. The results demonstrated that the oral median lethal dose (LD50) value of compound 8a was 812 mg/kg. At the end of the experiment, blood samples of 250 and 500 mg/kg groups were respectively taken for liver and kidney function measurement. As displayed in Fig. 6 , significant abnormality was not found in aminotransferase (AST) and alanine aminotransferase (ALT), as well as blood urea nitrogen (BUN) and serum creatinine (SCR) levels, indicating no toxicity to the liver or kidney function, and therefore a good oral safety profile of compound 8a.

Fig. 6

Effects of compound 8a on liver or kidney function in Kunming mice. (A) ALT and AST levels, (B) BUN and Scr levels in the treatment of 8a at the dosage of 250 or 500 mg/kg were tested. Data were presented as mean and sd values.

Conclusions

In summary, 54 aloperine derivatives with a unique endocyclic scaffold, of which 39 were new, were synthesized and evaluated for their anti-SARS-CoV-2 activities on a pseudotyped virus model. Among them, compound 8a with a 12N-p-chlorophenyl moiety exhibited the broad-spectrum anti-coronavirus effects against both pseudotyped and authentic SARS-CoV-2, as well as SARS-CoV and MERS-CoV, through targeting host cell components. It might block viral entry at a late stage by inhibiting host Cat B activity, which has the advantage of not being affected by virus mutation and drug resistance. Meanwhile, 8a could significantly reduce the levels of multiple inflammatory cytokines including IL-6, IL-1β, IL-8 and MCP-1 in a dose- and time-dependent manner. Furthermore, compound 8a showed a good PK and safety profile in vivo, suggesting an ideal drug-like feature. Therefore, aloperine derivative 8a has the advantage of broad-spectrum anti-coronavirus and anti-cytokine activity in the treatment of COVID-19, which is worth further investigation.

Experimental section

Apparatus, materials, and analysis reagents

All commercial solvents and reagents were used without any purification. All reactions were monitored by thin layer chromatography (TLC) and visualized under UV light at 254 nm or iodine fumigation. Melting points (mp) were obtained with a MP90 melting point apparatus (Mettler-Toledo, Greifensee, Switzerland). 1H and 13C NMR spectral data were recorded with a Bruker Avance 400, 500 or 600 spectrometers (Varian, San Francisco, USA) in DMSO‑d or CDCl3 using tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrometry (HRMS) was performed on an AutospecUitima-TOF mass spectrometer (Micromass UK Ltd., Manchester, U.K.). The flash column chromatograms were conducted on silica gel (200–300 mesh) using Combiflash Rf 200 (Teledyne, NE, USA).

General procedure for the synthesis of the reported compounds

The synthetic methods and structural identification of compounds 2a–e, 3a–c, 4a–c, 8a, 8b, 11l and 11s have been previously reported in our lab [23], [30].

General procedure for the synthesis of 12N-carbamoylmethyl aloperines (11a–s)

Bromoacetyl bromide (0.4 g, 2 mmol) was added dropwise to a mixture of the different amines 9a–s (2 mmol) and triethylamine (0.3 mL, 2 mmol) in anhydrous CH2Cl2 (30 mL) in ice bath, and the mixture was stirred for 30 min at 0 °C and stirred at r.t. for another 1.5 h until TLC indicated complete reaction. Then, triethylamine (0.3 mL, 2 mmol) and aloperine (0.5 g, 2 mmol) were added and the mixture was stirred overnight at r.t. till the TLC showed completion of the reaction. The mixture was then washed with water (20 mL) and brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by flash column chromatography with CH2Cl2/CH3OH to give the target compounds 11a–s.

12N-N′-p-Chlorophenylcarbamoylmethyl aloperine (11a)

Yield 48%; white solid; m.p. 169–171 °C. 1H NMR (500 MHz, CDCl3) δ 9.32 (s, 1H), 7.51–7.35 (m, 4H), 5.62 (d, J = 5.5 Hz, 1H), 3.67–3.49 (m, 1H), 2.95–2.87 (m, 2H), 2.79–2.71 (m, 1H), 2.68–2.61 (m, 2H), 2.58–2.50 (m, 1H), 2.47–2.41 (m, 1H), 2.34–2.24 (m, 2H), 2.12–2.13 (m, 1H), 2.08–1.97 (m, 2H), 1.93–1.85 (m, 2H), 1.80–1.73 (m, 1H), 1.73–1.66 (m, 1H), 1.63–1.50 (m, 2H), 1.50–1.41 (m, 2H), 1.40–1.35 (m, 1H), 1.34–1.23 (m, 2H); 13C NMR (126 MHz) δ 169.6, 137.0, 132.9, 132.1 (2), 129.2, 120.8 (2), 116.6, 64.1, 63.7, 57.3, 55.8, 55.2, 51.5, 35.6, 33.3, 31.4, 30.2, 26.0, 25.4, 24.6, 23.7. ESI-HRMS: calcd for C23H31ClN3O [M + H]+: 400.2150, found: 400.2152.

12N-N′-p-Bromophenylcarbamoylmethyl aloperine (11b)

Yield 47%; white solid; m.p. 168–170 °C. 1H NMR (500 MHz, CDCl3) δ 9.31 (s, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.30–7.25 (m, 2H), 5.62 (d, J = 5.5 Hz, 1H), 3.70–3.47 (m, 1H), 2.94–2.88 (m, 2H), 2.81–2.71 (m, 1H), 2.68–2.62 (m, 2H), 2.59–2.52 (m, 1H), 2.47–2.41 (m, 1H), 2.34–2.25 (m, 2H), 2.22–2.14 (m, 1H), 2.08–1.97 (m, 2H), 1.93–1.85 (m, 2H), 1.79–1.67 (m, 2H), 1.64–1.50 (m, 2H), 1.50–1.42 (m, 2H), 1.41–1.35 (m, 1H), 1.34–1.24 (m, 2H); 13C NMR (126 MHz) δ 169.6, 136.6, 133.0, 129.3 (3), 129.1, 120.5 (2), 64.2, 63.8, 57.4, 55.9, 55.3, 51.6, 35.7, 33.4, 31.5, 30.3, 26.1, 25.5, 24.7, 23.8. ESI-HRMS: calcd for C23H31BrN3O [M + H]+: 444.1645, found: 444.1646.

12N-N′-p-Iodophenylcarbamoylmethyl aloperine (11c)

Yield 35%; pink solid; m.p. 192–194 °C. 1H NMR (400 MHz, DMSO‑d) δ 11.21 (br, 1H), 7.79–7.37 (m, 4H), 5.78 (s, 1H), 4.57 (s, 1H), 4.44–4.10 (m, 1H), 3.73 (s, 1H), 3.60–3.31 (m, 5H), 3.30–3.08 (m, 3H), 2.78 (s, 1H), 2.45–2.23 (m, 3H), 2.21–2.03 (m, 2H), 1.85–1.53 (m, 6H), 1.45 (s, 1H); 13C NMR (151 MHz) δ 162.6, 138.0, 137.3 (2), 134.1, 128.0, 121.7 (2), 88.1, 63.0, 58.1, 54.6, 53.8, 52.6, 45.1, 32.6, 30.1, 27.3, 22.8, 22.5, 22.0, 20.7, 17.7. ESI-HRMS: calcd for C23H31IN3O [M + H]+, 492.1506, found: 492.1495.

12N-N′-(2,4-Dichlorophenyl)carbamoylmethyl aloperine (11d)

Yield 24%; brown solid; m.p. 71–73 °C. 1H NMR (500 MHz, CDCl3) δ 9.83 (s, 1H), 8.54–8.49 (m, 1H), 7.43–7.33 (m, 1H), 7.26–7.22 (m, 1H), 5.65 (s, 1H), 3.74–3.64 (m, 1H), 3.00–2.87 (m, 2H), 2.82–2.70 (m, 2H), 2.65 (s, 2H), 2.34–2.20 (m, 2H), 2.15–1.85 (m, 5H), 1.80–1.41 (m, 8H), 1.39–1.23 (m, 2H); 13C NMR (126 MHz) δ 169.8, 133.7, 132.6, 129.5, 129.0, 128.9, 128.1, 123.1, 121.5, 65.5, 63.8, 57.6, 56.0, 55.1, 52.1, 35.6, 33.4, 32.4, 29.9, 25.8, 25.2 (2), 23.5. ESI-HRMS: calcd for C23H30Cl2N3O [M + H]+, 434.1760, found: 434.1766.

12N-N′-(2,5-Dibromophenyl)carbamoylmethyl aloperine (11e)

Yield 20%; light brown solid; m.p. 79–81 °C. 1H NMR (500 MHz, CDCl3) δ 9.81 (s, 1H), 8.78 (d, J = 2.5 Hz, 1H), 7.40–7.36 (m, 1H), 7.13–7.06 (m, 1H), 5.65 (s, 1H), 3.75–3.67 (m, 1H), 2.98–2.88 (m, 2H), 2.82–2.71 (m, 2H), 2.63 (s, 3H), 2.36–2.18 (m, 3H), 2.09–2.02 (m, 2H), 1.91 (s, 1H), 1.78–1.65 (m, 4H), 1.60–1.40 (m, 4H), 1.34–1.23 (m, 2H); 13C NMR (101 MHz) δ 170.0, 137.2, 133.3, 132.3, 129.6, 127.8, 123.6, 122.2, 111.3, 65.2, 63.9, 57.7, 56.0, 55.1, 52.2, 35.7, 33.4, 32.6, 30.1, 25.7, 25.4, 25.3, 23.5. ESI-HRMS: calcd for C23H30Br2N3O [M + H]+, 522.0750, found: 522.0734.

12N-N′-(5-Bromo-2-fluorophenyl)carbamoylmethyl aloperine (11f)

Yield 11%; brown solid; m.p. 107–109 °C. 1H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.65–8.57 (m, 1H), 7.21–7.09 (m, 1H), 7.00–6.92 (m, 1H), 5.66 (s, 1H), 3.74–3.63 (m, 1H), 3.39–3.10 (m, 1H), 2.97–2.86 (m, 2H), 2.77–2.47 (m, 3H), 2.40–2.19 (m, 3H), 2.12–1.91 (m, 4H), 1.85–1.64 (m, 4H), 1.59–1.25 (m, 6H); 13C NMR (126 MHz) δ 169.4, 151.3, 132.6, 129.0, 127.5, 126.7, 123.7, 117.1, 116.1, 63.4, 57.2, 55.6 (2), 54.9, 51.4, 35.2, 33.1, 31.8, 29.5, 25.6, 24.9, 24.6, 23.2. ESI-HRMS: calcd for C23H30BrFN3O [M + H]+, 462.1551, found: 462.1557.

12N-N′-(5-Bromo-2-chlorophenyl)carbamoylmethyl aloperine (11g)

Yield 14%; brown solid; m.p. 75–77 °C. 1H NMR (500 MHz, CDCl3) δ 9.87 (s, 1H), 8.78 (d, J = 2.0 Hz, 1H), 7.25–7.21 (m, 1H), 7.19–7.14 (m, 1H), 5.65 (s, 1H), 3.76–3.69 (m, 1H), 2.97–2.88 (m, 2H), 2.82–2.74 (m, 1H), 2.63 (s, 3H), 2.35–2.22 (m, 2H), 2.11–2.01 (m, 3H), 1.92 (s, 1H), 1.78 (s, 2H), 1.71–1.60 (m, 3H), 1.58–1.41 (m, 4H), 1.36–1.25 (m, 2H); 13C NMR (126 MHz) δ 169.6, 135.6, 132.1, 129.9, 129.3, 127.0, 123.1, 121.2, 121.0, 65.1, 63.4, 57.3, 55.6, 54.7, 51.8, 35.3, 33.1, 32.1, 29.6 (2), 25.5, 24.9, 23.2. ESI-HRMS: calcd for C23H30BrClN3O [M + H]+, 478.1255, found: 478.1237.

12N-N′-(2-Fluoro-6-methylphenyl)carbamoylmethyl aloperine (11h)

Yield 15%; brown solid; m.p. 107–109 °C. 1H NMR (500 MHz, CDCl3) δ 9.92 (s, 1H), 7.14 (s, 1H), 7.06–6.87 (m, 2H), 5.67 (s, 1H), 3.80–3.64 (m, 1H), 3.58–3.35 (m, 3H), 3.30 (s, 1H), 3.19–3.05 (m, 1H), 2.98–2.67 (m, 3H), 2.64–2.46 (m, 2H), 2.38–2.23 (m, 5H), 2.17–1.91 (m, 4H), 1.85–1.72 (m, 3H), 1.65–1.48 (m, 3H), 1.32 (s, 1H); 13C NMR (126 MHz) δ 170.2, 158.9, 141.2, 138.2, 129.1, 128.0, 126.1, 123.3, 113.3, 61.9, 56.3, 55.9, 55.2, 51.7, 45.7, 35.6, 33.5, 32.2, 29.8, 25.7, 25.1, 24.0, 23.4, 18.6. ESI-HRMS: calcd for C24H33FN3O [M + H]+, 398.2602, found: 398.2602.

12N-N′-(2-Chloro-4-methylphenyl)carbamoylmethyl aloperine (11i)

Yield 25%; brown solid; m.p. 72–74 °C. 1H NMR (500 MHz, CDCl3) δ 9.73 (s, 1H), 8.44–8.33 (m, 1H), 7.21–7.15 (m, 1H), 7.12–7.04 (m, 1H), 5.69–5.60 (m, 1H), 3.75–3.66 (m, 1H), 3.03–2.85 (m, 2H), 2.64 (s, 4H), 2.35–2.21 (m, 5H), 2.12–1.99 (m, 3H), 1.91 (s, 1H), 1.81–1.60 (m, 5H), 1.56–1.38 (m, 4H), 1.35–1.23 (m, 2H); 13C NMR (126 MHz) δ 169.6, 134.5, 132.6, 132.4 129.5, 129.5, 128.5, 122.5, 120.7, 65.4, 63.8, 57.6, 55.9, 55.0, 52.1, 35.7, 33.5, 32.6, 30.0, 25.8, 25.4, 25.3, 23.5, 20.9. ESI-HRMS: calcd for C24H33ClN3O [M + H]+, 414.2307, found: 414.2309.

12N-N′-(4-Chloro-2-methylphenyl)carbamoylmethyl aloperine (11j)

Yield 23%; brown solid; m.p. 90–92 °C. 1H NMR (500 MHz, CDCl3) δ 9.21 (s, 1H), 8.25–8.15 (m, 1H), 7.22–7.10 (m, 2H), 5.64 (s, 1H), 3.77–3.63 (m, 1H), 2.99–2.87 (m, 2H), 2.82–2.59 (m, 4H), 2.48 (s, 1H), 2.33–2.21 (m, 5H), 2.12–2.01 (m, 3H), 1.92 (s, 1H), 1.81–1.68 (m, 3H), 1.62–1.39 (m, 5H), 1.34–1.23 (m, 2H); 13C NMR (126 MHz) δ 169.3, 134.9, 132.7, 130.3, 129.5, 129.0, 128.3, 127.1, 121.7, 64.9, 63.8, 57.5, 56.0, 55.1, 52.0, 35.7, 33.4, 32.0, 30.1, 26.1, 25.3, 25.0, 23.6, 18.1. ESI-HRMS: calcd for C24H33ClN3O [M + H]+, 414.2307, found: 414.2310.

12N-N′-(3-Chloro-5-trifluoromethylphenyl)carbamoylmethyl aloperine (11k)

Yield 53%; brown solid; m.p. 74–76 °C. 1H NMR (500 MHz, CDCl3) δ 9.58 (s, 1H), 7.90 (s, 1H), 7.65 (s, 1H), 7.33 (s, 1H), 5.66 (s, 1H), 3.71–3.48 (m, 1H), 3.00–2.86 (m, 2H), 2.83–2.74 (m, 1H), 2.71–2.40 (m, 4H), 2.39–2.19 (m, 3H), 2.13–1.92 (m, 4H), 1.81–1.30 (m, 9H); 13C NMR (126 MHz) δ 170.0, 139.7, 135.5, 132.8, 129.1, 124.3, 122.2, 120.9, 114.1, 63.6, 57.2, 55.7, 55.4, 51.2, 35.5, 33.3, 30.8, 30.2, 29.8, 25.9, 25.3, 24.2, 23.7 (2). ESI-HRMS: calcd for C24H30ClF3N3O [M + H]+, 468.2024, found: 468.2027.

12N-N′-(4-Bromo-3-trifluoromethylphenyl)carbamoylmethyl aloperine (11m).

Yield 34%; brown solid; m.p. 70–71 °C. 1H NMR (500 MHz, CDCl3) δ 9.52 (s, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.64 (s, 1H), 5.65 (d, J = 5.0 Hz, 1H), 3.64–3.59 (m, 1H), 2.97–2.89 (m, 2H), 2.83–2.76 (m, 1H), 2.71–2.61 (m, 2H), 2.56–2.51 (m, 1H), 2.47–2.42 (m, 1H), 2.37–2.29 (m, 2H), 2.25–2.18 (m, 1H), 2.09–1.99 (m, 2H), 1.97–1.89 (m, 2H), 1.79–1.70 (m, 2H), 1.66–1.43 (m, 4H), 1.41–1.27 (m, 3H); 13C NMR (126 MHz) δ 169.7, 137.1, 135.4, 132.8, 128.9, 123.1, 121.4, 118.2, 113.4, 63.5, 63.4, 57.0, 55.5, 55.1, 51.1, 35.4, 33.0, 30.7, 30.1, 29.6, 25.7, 25.1, 24.0, 23.6. ESI-HRMS: calcd for C24H30BrF3N3O [M + H]+, 512.1519, found: 512.1524.

12N-N′-(3,4-Dimethylphenyl)carbamoylmethyl aloperine (11n)

Yield 46%; white solid; m.p. 58–60 °C. 1H NMR (400 MHz, DMSO‑d) δ 9.40 (s, 1H), 7.39–7.23 (m, 2H), 7.05 (d, J = 7.6 Hz, 1H), 5.54 (s, 1H), 3.50–3.41 (m, 1H), 3.32 (s, 2H), 3.00–2.91 (m, 1H), 2.91–2.79 (m, 2H), 2.57 (s, 3H), 2.45–2.34 (m, 2H), 2.21–2.11 (m, 7H), 2.00–1.86 (m, 2H), 1.85–1.73 (m, 2H), 1.72–1.64 (m, 1H), 1.63–1.54 (m, 1H), 1.51–1.29 (m, 5H), 1.19–1.10 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 169.2, 137.4, 135.7, 133.2, 132.5, 130.1, 129.0, 120.7, 116.9, 64.2, 63.8, 57.3, 55.7, 55.1, 51.4, 35.7, 33.4, 31.4, 30.2, 26.0, 25.4, 24.6, 23.7, 20.0, 19.3. ESI-HRMS: calcd for C25H36N3O [M + H]+, 394.2853, found: 394.2840.

12N-N′-(3,5-Ditrifluoromethylphenyl)carbamoylmethyl aloperine (11o)

Yield 36%; white solid; m.p. 94–96 °C. 1H NMR (400 MHz, DMSO‑d 6) δ 10.24 (s, 1H), 8.34 (s, 2H), 7.76 (s, 1H), 5.53 (d, J = 5.2 Hz, 1H), 3.58–3.48 (m, 1H), 3.41–3.30 (m, 1H), 3.19–3.08 (m, 1H), 2.99 (s, 1H), 2.94–2.84 (m, 1H), 2.62–2.53 (m, 3H), 2.49–2.40 (m, 1H), 2.25–2.16 (m, 1H), 2.08–1.87 (m, 3H), 1.81 (s, 1H), 1.75–1.64 (m, 2H), 1.57 (s, 2H), 1.47–1.34 (m, 3H), 1.33–1.20 (m, 2H), 1.17–1.08 (m, 1H); 13C NMR (101 MHz, CDCl3) δ 170.3, 139.4, 133.2, 132.5 (2), 129.1, 123.3 (2), 119.0, 117.4 (2), 63.8, 63.4, 57.3, 55.6, 55.5, 51.2, 35.6, 33.3, 30.6, 30.4, 25.9, 25.4, 24.0, 23.9. ESI-HRMS: calcd for C25H30F6N3O [M + H]+: 502.2288, found: 502.2277.

12N-N′-(2-Naphthyl)carbamoylmethyl aloperine (11p)

Yield 39%; white solid; m.p. 64–66 °C. 1H NMR (400 MHz, CDCl3) δ 9.46 (s, 1H), 8.24 (s, 1H), 7.79 (t, J = 9.2 Hz, 3H), 7.52–7.42 (m, 2H), 7.39 (t, J = 7.6 Hz, 1H), 5.65 (d, J = 4.8 Hz, 1H), 3.74–3.63 (m, 1H), 3.04–2.91 (m, 2H), 2.87–2.77 (m, 1H), 2.76–2.64 (m, 2H), 2.60 (s, 1H), 2.38–2.28 (m, 2H), 2.27–2.18 (m, 1H), 2.13–1.98 (m, 2H), 1.97–1.89 (d, J = 8.0 Hz, 2H), 1.80–1.62 (m, 3H), 1.59–1.28 (m, 6H), 1.24 (t, J = 6.8 Hz, 1H); 13C NMR (101 MHz) δ 169.7, 135.4, 134.0, 133.1, 130.7, 129.1, 129.0, 127.7, 127.7, 126.6, 125.0, 119.5, 115.9, 64.1, 63.9, 57.4, 55.8, 55.3, 51.5, 35.7, 33.4, 31.4, 30.3, 26.1, 25.4, 24.6, 23.8. ESI-HRMS: calcd for C27H34N3O [M + H]+: 416.2696, found: 416.2696.

12N-N′-(2-Quinolyl)carbamoylmethyl aloperine (11q)

Yield 32%; white solid; m.p. 78–80 °C. 1H NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 8.43 (d, J = 8.8 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 7.2 Hz, 1H), 5.66 (s, 1H), 3.76–3.62 (m, 1H), 3.07–2.98 (m, 1H), 2.94 (s, 1H), 2.88–2.80 (m, 1H), 2.79–2.52 (m, 4H), 2.41–2.22 (m, 3H), 2.14–1.90 (m, 4H), 1.88–1.75 (m, 2H), 1.73–1.66 (m, 1H), 1.60–1.19 (m, 6H); 13C NMR (101 MHz) δ 170.8, 150.6, 147.0, 138.4, 133.3, 129.9, 128.9, 127.9, 127.6, 126.5, 125.2, 114.3, 64.2, 58.0 (2), 55.6, 55.4, 51.2, 35.7, 33.4, 31.5, 30.2, 25.7, 25.4, 24.6, 23.8. ESI-HRMS: calcd for C26H33N4O [M + H]+, 417.2649, found: 417.2638.

12N-N′-p-Bromophenyl-N′-methylcarbamoylmethyl aloperine hydrochloride (11r)

Yield 45%; white solid; m.p. 157–159 °C. 1H NMR (400 MHz, CDCl3) δ 10.80 (s, 1H), 7.60 (d, J = 7.2 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 5.61 (s, 1H), 3.37 (d, J = 5.6 Hz, 1H), 3.27–3.18 (m, 4H), 2.99–2.79 (m, 4H), 2.77–2.68 (m, 1H), 2.67–2.48 (m, 4H), 2.25–2.16 (m, 1H), 2.16–2.06 (m, 2H), 1.98–1.90 (m, 2H), 1.84–1.70 (m, 3H), 1.68–1.60 (m, 1H), 1.56–1.38 (m, 3H), 1.24 (s, 1H); 13C NMR (101 MHz) δ 169.4, 142.2, 135.4, 133.5 (2), 129.3 (2), 125.8, 122.2, 68.9, 62.5, 55.9, 54. 9, 53.0, 50.7, 37.5, 33.7, 32.9, 30.6, 27.3, 25.6, 23.5, 22.8, 22.5. ESI-HRMS: calcd for C24H33BrN3O ∙HCl [M-HCl + H]+, 458.1802, found: 458.1790.

General procedure for 12N-substituted aminoacetyl aloperines (14a–r)

To a mixture of the different substituted amines 12a–r (5 mmol) and NaHCO3 (0.5 g, 6 mmol) in anhydrous ethanol (50 mL), ethyl bromoacetate (1.1 g, 5.5 mmol) was added. The mixture was refluxed for 8 h, and then poured into water (30 mL) and extracted with ethyl acetate (50 mL). The organic layer was concentrated to give crude products, which was directly added to a solution of LiOH (5.5 mmol) in H2O (40 mL), and the mixture was refluxed for 1.5 h. After cooling, the pH of the mixture was acidified to 2 by 3 N HCl and extracted with CH2Cl2 (20 mL × 2). , the organic layer was concentrated to give crude products 13a–r for the next step directly.

To a solution of 13a–r (2 mmol) in anhydrous CH2Cl2 (30 mL) at 0 °C, N-hydroxy benzotrizol (HOBt, 2.7 mmol), diisopropylethylamine (5.2 mL) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 4 mmol) were added. The mixture was stirred for 30 min before the addition of aloperine (2.1 mmol), and was warmed to r.t. and stirred for 12 h. The mixture was washed with water and brine (30 mL), dried over anhydrous Na2SO4, filtered and then the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent to give the products 14a–r.

12N-N′-Phenylaminoacetyl aloperine (14a)

Yield 58%; white solid; m.p. 62–64 °C. 1H NMR (400 MHz, CDCl3) δ 7.19 (t, J = 7.6 Hz, 2H), 6.72 (t, J = 7.2 Hz, 1H), 6.64 (d, J = 8.0 Hz, 2H), 5.65 (s, 1H), 4.95 (s, 1H), 4.76 (s, 1H), 4.06–3.90 (m, 1H), 3.91–3.80 (m, 1H), 3.79–3.65 (m, 1H), 3.49–3.38 (m, 1H), 3.12–2.98 (m, 1H), 2.75 (d, J = 6.8 Hz, 2H), 2.65–2.54 (m, 1H), 2.45 (s, 1H), 2.40–2.32 (m, 1H), 2.31–2.21 (m, 1H), 2.17–2.06 (m, 1H), 1.97 (s, 1H), 1.92–1.76 (m, 5H), 1.73–1.62 (m, 2H), 1.51–1.39 (m, 1H), 1.16–1.01 (m, 2H); 13C NMR (101 MHz) δ 167.9, 147.6, 135.5, 129.4 (2), 128. 4, 117.5, 113.1 (2), 59.4, 59.2, 54.4, 46.9, 45.5, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.7, 24.1, 19.2. ESI-HRMS: calcd for C23H32N3O [M + H]+: 366.2540, found: 366.2540.

12N-N′-p-Fluorophenylaminoacetyl aloperine (14b)

Yield 42%; white solid; m.p. 64–66 °C. 1H NMR (600 MHz, CDCl3) δ 6.92–6.86 (m, 2H), 6.59–6.53 (m, 2H), 5.64 (d, J = 4.2 Hz, 1H), 4.82 (s, 1H), 4.75 (d, J = 4.8 Hz, 1H), 3.97–3.89 (m, 1H), 3.84–3.77 (m, 1H), 3.77–3.67 (m, 1H), 3.47–3.30 (m, 1H), 3.11–3.01 (m, 1H), 2.79–2.70 (m, 2H), 2.63–2.54 (m, 1H), 2.44 (s, 1H), 2.39–2.32 (m, 1H), 2.30–2.21 (m, 1H), 2.16–2.07 (m, 1H), 2.05–1.95 (m, 1H), 1.94–1.73 (m, 5H), 1.72–1.60 (m, 2H), 1.49–1.39 (m, 1H), 1.15–1.02 (m, 2H); 13C NMR (151 MHz) δ 167.9, 156.0, 144.2, 135.4, 128.5, 115.9, 115.7, 113.9, 113.8, 59.5, 59.3, 54.5, 46.9, 46.2, 40.6, 35.2, 31.6, 28.1, 26.3, 24.9, 24.7, 24.2, 19.3. ESI-HRMS: calcd for C23H31FN3O [M + H]+: 384.2446, found: 384.2447.

12N-N′-m-Fluorophenylaminoacetyl aloperine (14c)

Yield 40%; white solid; m.p. 64–66 °C. 1H NMR (600 MHz, CDCl3) δ 7.13–7.06 (m, 1H), 6.43–6.35 (m, 2H), 6.31–6.24 (m, 1H), 5.65 (d, J = 5.4 Hz, 1H), 5.10 (s, 1H), 4.75 (d, J = 4.8 Hz, 1H), 3.99–3.90 (m, 1H), 3.84–3.78 (m, 1H), 3.78–3.71 (m, 1H), 3.46–3.32 (m, 1H), 3.12–3.01 (m, 1H), 2.79–2.70 (m, 2H), 2.64–2.56 (m, 1H), 2.45 (s, 1H), 2.38–2.32 (m, 1H), 2.31–2.22 (m, 1H), 2.20–2.07 (m, 1H), 2.00–1.95 (m, 1H), 1.95–1.81 (m, 4H), 1.81–1.73 (m, 1H), 1.72–1.62 (m, 2H), 1.50–1.38 (m, 1H), 1.15–1.03 (m, 2H); 13C NMR (151 MHz) δ 167.4, 164.3, 149.4, 135.3, 130.4, 128.6, 109.1, 103.9, 99.6, 59.5, 59.4, 54.5, 46.9, 45.4, 40.6, 35.2, 31.6, 28.1, 26.3, 24.9, 24.7, 24.1, 19.3. ESI-HRMS: calcd for C23H31FN3O [M + H]+: 384.2446, found: 384.2449.

12N-N′-p-Chlorophenylaminoacetyl aloperine (14d)

Yield 45%; white solid; m.p. 73–75 °C. 1H NMR (400 MHz, CDCl3) δ 7.12 (d, J = 8.8 Hz, 2H), 6.54 (d, J = 8.4 Hz, 2H), 5.64 (d, J = 4.0 Hz, 1H), 4.99 (s, 1H), 4.74 (d, J = 4.4 Hz, 1H), 4.02–3.86 (m, 1H), 3.84–3.65 (m, 2H), 3.46–3.31 (m, 1H), 3.12–2.98 (m, 1H), 2.74 (d, J = 7.6 Hz, 2H), 2.65–2.54 (m, 1H), 2.43 (s, 1H), 2.38–2.21 (m, 2H), 2.18–2.04 (m, 1H), 1.97 (s, 1H), 1.93–1.74 (m, 5H), 1.72–1.61 (m, 2H), 1.51–1.35 (m, 1H), 1.15–0.99 (m, 2H); 13C NMR (101 MHz) δ 167.5, 146.1, 135.3, 129.2 (2), 128.5, 122.0, 114.1 (2), 59.4, 59.3, 54.4, 46.9, 45.5, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.6, 24.0, 19.2. ESI-HRMS: calcd for C23H31ClN3O [M + H]+: 400.2150, found: 400.2152.

12N-N′-p-Bromophenylaminoacetyl aloperine (14e)

Yield 43%; white solid; m.p. 73–75 °C. 1H NMR (600 MHz, DMSO‑d 6) δ 7.22–7.17 (m, 2H), 6.63 (d, J = 8.4 Hz, 2H), 5.79 (t, J = 4.8 Hz, 1H), 5.60 (d, J = 5.4 Hz, 1H), 4.54 (d, J = 5.4 Hz, 1H), 4.00–3.94 (m, 1H), 3.88–3.80 (m, 1H), 3.67–3.60 (m, 1H), 3.52–3.45 (m, 1H), 2.98–2.90 (m, 1H), 2.76–2.70 (m, 1H), 2.68–2.62 (m, 1H), 2.55–2.51 (m, 1H), 2.35–2.30 (m, 1H), 2.26 (s, 1H), 2.23–2.14 (m, 1H), 2.05–1.86 (m, 3H), 1.84–1.74 (m, 3H), 1.67–1.51 (m, 3H), 1.45–1.34 (m, 1H), 1.09–0.98 (m, 2H); 13C NMR (151 MHz, CDCl3) δ 167.5, 146.6, 135.3, 132.1 (2), 128.6, 114.6 (2), 109.1, 59.5, 59.4, 54.5, 46.9, 45.4, 40.6, 35.2, 31.6, 28.1, 26.3, 24.9, 24.7, 24.1, 19.3. ESI-HRMS: calcd for C23H31BrN3O [M + H]+: 444.1645, found: 444.1651.

12N-N′-p-Iodophenylaminoacetyl aloperine (14f)

Yield 46%; white solid; m.p. 85–87 °C. 1H NMR (600 MHz, CDCl3) δ 7.42 (d, J = 8.4 Hz, 2H), 6.41 (d, J = 8.4 Hz, 2H), 5.64 (d, J = 5.4 Hz, 1H), 5.07–4.97 (m, 1H), 4.74 (d, J = 6.6 Hz, 1H), 3.94–3.88 (m, 1H), 3.82–3.70 (m, 2H), 3.44–3.36 (m, 1H), 3.07–3.02 (m, 1H), 2.78–2.71 (m, 2H), 2.62–2.56 (m, 1H), 2.44 (s, 1H), 2.37–2.31 (m, 1H), 2.29–2.22 (m, 1H), 2.19–2.07 (m, 1H), 2.00–1.95 (m, 1H), 1.94–1.73 (m, 5H), 1.72–1.62 (m, 2H), 1.49–1.38 (m, 1H), 1.15–1.03 (m, 2H); 13C NMR (151 MHz) δ 167.4, 147.2, 137.9 (3), 135.3, 128.6, 115.3 (2), 59.5, 59.4, 54.5, 46.9, 45.3, 40.6, 35.2, 31.6, 28.1, 26.3, 24.9, 24.7, 24.1, 19.3. ESI-HRMS: calcd for C23H31IN3O [M + H]+: 492.1506, found: 492.1517.

12N-N′-p-Tolylaminoacetyl aloperine (14g).

Yield 48%; white solid; m.p. 59–61 °C. 1H NMR (400 MHz, CDCl3) δ 7.00 (d, J = 8.0 Hz, 2H), 6.56 (d, J = 8.4 Hz, 2H), 5.68–5.59 (m, 1H), 4.83–4.72 (m, 2H), 4.00–3.91 (m, 1H), 3.87–3.78 (m, 1H), 3.77–3.67 (m, 1H), 3.46–3.39 (m, 1H), 3.08–2.99 (m, 1H), 2.77–2.70 (m, 2H), 2.62–2.55 (m, 1H), 2.44 (s, 1H), 2.38–2.32 (m, 1H), 2.27–2.22 (m, 4H), 2.17–2.06 (m, 1H), 1.97 (s, 1H), 1.91–1.83 (m, 3H), 1.82–1.77 (m, 1H), 1.72–1.63 (m, 2H), 1.60 (s, 1H), 1.50–1.39 (m, 1H), 1.15–1.02 (m, 2H); 13C NMR (101 MHz) δ 168.1, 145.5, 135.5, 129.8 (2), 128.4, 126.7, 113.2 (2), 59.4, 59.2, 54.4, 46.9, 45.9, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.7, 24.1, 20.5, 19.2. ESI-HRMS: calcd for C24H34N3O [M + H]+: 380.2696, found: 380.2685.

12N-N′-m-Trifluoromethylphenylaminoacetyl aloperine (14h).

Yield 45%; white solid; m.p. 58–60 °C. 1H NMR (600 MHz, DMSO‑d) δ 7.26 (d, J = 8.4 Hz, 1H), 6.98–6.87 (m, 2H), 6.83 (d, J = 7.8 Hz, 1H), 6.14 (t, J = 4.8 Hz, 1H), 5.60 (d, J = 5.4 Hz, 1H), 4.60–4.51 (m, 1H), 4.10–4.02 (m, 1H), 3.96–3.88 (m, 1H), 3.71–3.64 (m, 1H), 3.54–3.46 (m, 1H), 2.97–2.89 (m, 1H), 2.75–2.69 (m, 1H), 2.69–2.62 (m, 1H), 2.56–2.51 (m, 1H), 2.36–2.29 (m, 1H), 2.28–2.16 (m, 2H), 2.04–1.96 (m, 1H), 1.95–1.88 (m, 2H), 1.84–1.74 (m, 3H), 1.68–1.58 (m, 2H), 1.58–1.50 (m, 1H), 1.45–1.33 (m, 1H), 1.09–0.97 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 167.2, 147.7, 135.3, 131.7, 129.7, 128.6, 124.5, 116.6, 113.8, 108.5, 59.4 (2), 54.4, 46.9, 45.1, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.7, 24.1, 19.2. ESI-HRMS: calcd for C24H31F3N3O [M + H]+: 434.2414, found: 434.2403.

12N-N′-m-Methoxyphenylaminoacetyl aloperine (14i)

Yield 50%; white solid; m.p. 60–62 °C. 1H NMR (400 MHz, CDCl3) δ 7.09 (t, J = 8.0 Hz, 1H), 6.27 (td, J = 8.0, 2.0 Hz, 2H), 6.17 (t, J = 2.0 Hz, 1H), 5.67–5.59 (m, 1H), 5.01–4.93 (m, 1H), 4.75 (d, J = 5.6 Hz, 1H), 3.99–3.91 (m, 1H), 3.86–3.79 (m, 1H), 3.78 (s, 3H), 3.76–3.68 (m, 1H), 3.45–3.37 (m, 1H), 3.08–3.01 (m, 1H), 2.77–2.71 (m, 2H), 2.62–2.56 (m, 1H), 2.47–2.40 (m, 1H), 2.39–2.32 (m, 1H), 2.31–2.22 (m, 1H), 2.16–2.06 (m, 1H), 2.00–1.94 (m, 1H), 1.93–1.83 (m, 3H), 1.82–1.65 (m, 3H), 1.65–1.60 (m, 1H), 1.50–1.40 (m, 1H), 1.14–1.02 (m, 2H); 13C NMR (101 MHz) δ 167.7, 160.9, 149.0, 135.4, 130.1, 128.5, 106.3, 102.5, 99.1, 59.4, 59.2, 55.2, 54.4, 46.9, 45.5, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.6, 24.1, 19.2. ESI-HRMS: calcd for C24H34N3O2 [M + H]+: 396.2646, found: 396.2634.

12N-N′-p-Trifluoromethoxyphenylaminoacetyl aloperine (14j)

Yield 35%; white solid; m.p. 57–59 °C. 1H NMR (600 MHz, CDCl3) δ 7.04 (d, J = 8.4 Hz, 2H), 6.61–6.54 (m, 2H), 5.65 (d, J = 4.2 Hz, 1H), 5.03 (s, 1H), 4.75 (d, J = 4.2 Hz, 1H), 4.00–3.91 (m, 1H), 3.85–3.79 (m, 1H), 3.78–3.66 (m, 1H), 3.45–3.33 (m, 1H), 3.13–3.00 (m, 1H), 2.74 (d, J = 7.8 Hz, 2H), 2.64–2.56 (m, 1H), 2.44 (s, 1H), 2.38–2.31 (m, 1H), 2.31–2.22 (m, 1H), 2.18–2.08 (m, 1H), 2.00–1.95 (m, 1H), 1.95–1.81 (m, 4H), 1.81–1.73 (m, 1H), 1.72–1.62 (m, 2H), 1.49–1.39 (m, 1H), 1.15–1.02 (m, 2H); 13C NMR (151 MHz) δ 167.5, 146.5, 140.7, 135.3, 128.6, 122.5 (2), 120.9, 113.3 (2), 59.5, 59.4, 54.5, 47.0, 45.7, 40.7, 35.2, 31.6, 28.1, 26.3, 24.9, 24.7, 24.1, 19.3. ESI-HRMS: calcd for C24H31F3N3O2 [M + H]+: 450.2363, found: 450.2363.

12N-N′-(2,4-Dichlorophenylamino)acetyl aloperine (14k).

Yield 46%; white solid; m.p. 79–81 °C. 1H NMR (600 MHz, CDCl3) δ 7.27 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 8.4, 2.4 Hz, 1H), 6.46 (d, J = 8.4 Hz, 1H), 5.71–5.62 (m, 2H), 4.77 (d, J = 5.4 Hz, 1H), 4.02–3.92 (m, 1H), 3.88–3.81 (m, 1H), 3.80–3.69 (m, 1H), 3.44–3.34 (m, 1H), 3.11–3.00 (m, 1H), 2.78–2.70 (m, 2H), 2.62–2.56 (m, 1H), 2.47 (s, 1H), 2.39–2.32 (m, 1H), 2.31–2.20 (m, 1H), 2.19–2.08 (m, 1H), 1.97 (s, 1H), 1.92–1.75 (m, 5H), 1.72–1.62 (m, 2H), 1.49–1.38 (m, 1H), 1.15–1.02 (m, 2H); 13C NMR (151 MHz) δ 167.0, 142.3, 135.2, 129.1, 128.6, 127.7, 121.4, 120.2, 111.8, 59.5 (2), 54.5, 47.0, 45.3, 40.7, 35.2, 31.6, 28.0, 26.3, 24.9, 24.7, 24.1, 19.3. ESI-HRMS: calcd for C23H30Cl2N3O [M + H]+: 434.1760, found: 434.1772.

12N-N′-(3,4-Dichlorophenylamino)acetyl aloperine (14l).

Yield 43%; white solid; m.p. 67–69 °C. 1H NMR (600 MHz, CDCl3) δ 7.20–7.16 (m, 1H), 6.64 (d, J = 3.0 Hz, 1H), 6.47 (dd, J = 9.0, 3.0 Hz, 1H), 5.65 (d, J = 4.8 Hz, 1H), 5.13–5.05 (m, 1H), 4.74 (d, J = 5.4 Hz, 1H), 3.95–3.87 (m, 1H), 3.81–3.72 (m, 2H), 3.43–3.35 (m, 1H), 3.08–3.02 (m, 1H), 2.78–2.70 (m, 2H), 2.64–2.56 (m, 1H), 2.43 (s, 1H), 2.37–2.32 (m, 1H), 2.31–2.24 (m, 1H), 2.19–2.07 (m, 1H), 2.05–1.95 (m, 1H), 1.95–1.81 (m, 4H), 1.81–1.74 (m, 1H), 1.71–1.60 (m, 2H), 1.49–1.38 (m, 1H), 1.15–1.02 (m, 2H); 13C NMR (151 MHz) δ 167.1, 147.0, 135.2, 133.0, 130.8, 128.7, 119.9, 113.7, 113.2, 59.5, 59.4, 54.4, 46.9, 45.2, 40.7, 35.2, 31.6, 28.0, 26.3, 24.9, 24.7, 24.1, 19.3. ESI-HRMS: calcd for C23H30Cl2N3O [M + H]+: 434.1760, found: 434.1767.

12N-N′-(3,5-Dimethylphenylamino)acetyl aloperine (14m).

Yield 54%; white solid; m.p. 64–66 °C. 1H NMR (400 MHz, CDCl3) δ 6.38 (s, 1H), 6.27 (s, 2H), 5.64 (d, J = 4.8 Hz, 1H), 4.84 (s, 1H), 4.75 (d, J = 4.8 Hz, 1H), 4.00–3.91 (m, 1H), 3.87–3.79 (m, 1H), 3.78–3.67 (m, 1H), 3.49–3.40 (m, 1H), 3.09–3.00 (m, 1H), 2.74 (d, J = 7.6 Hz, 2H), 2.63–2.54 (m, 1H), 2.44 (s, 1H), 2.38–2.31 (m, 1H), 2.25 (s, 7H), 2.16–2.06 (m, 1H), 1.97 (s, 1H), 1.92–1.83 (m, 3H), 1.82–1.74 (m, 2H), 1.72–1.62 (m, 2H), 1.47–1.39 (m, 1H), 1.15–1.02 (m, 2H); 13C NMR (101 MHz) δ 168.0, 147.8, 139.0 (2), 135.5, 128.4, 119.5, 111.1 (2), 59.4, 59.2, 54.4, 46.8, 45.7, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.7, 24.1, 21.6 (2), 19.2. ESI-HRMS: calcd for C25H36N3O [M + H]+: 394.2853, found: 394.2841.**

12N-N′-(3,5-Dimethoxyphenylamino)acetyl aloperine (14n).

Yield 39%; white solid; m.p. 61–63 °C. 1H NMR (400 MHz, CDCl3) δ 5.89 (s, 1H), 5.81 (s, 2H), 5.64 (d, J = 4.0 Hz, 1H), 4.98 (s, 1H), 4.74 (d, J = 4.8 Hz, 1H), 4.00–3.90 (m, 1H), 3.83 (d, J = 3.6 Hz, 1H), 3.76 (s, 6H), 3.45–3.34 (m, 1H), 3.11–2.99 (m, 1H), 2.74 (d, J = 8.0 Hz, 2H), 2.63–2.54 (m, 1H), 2.43 (s, 1H), 2.38–2.31 (m, 1H), 2.30–2.20 (m, 1H), 2.17–2.05 (m, 1H), 1.97 (s, 1H), 1.94–1.74 (m, 5H), 1.73–1.62 (m, 3H), 1.50–1.38 (m, 1H), 1.15–1.01 (m, 2H); 13C NMR (101 MHz) δ 167.7, 161.8 (2), 149.5, 135.4, 128.5, 92.0 (2), 89.8, 59.4, 59.2, 55.3 (2), 54.4, 46.9, 45.5, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.6, 24.1, 19.2. ESI-HRMS: calcd for C25H36N3O3 [M + H]+: 426.2751, found: 426.2739.

12N-N′-(3,5-Ditrifluoromethylphenylamino)acetyl aloperine (14o).

Yield 27%; white solid; m.p. 73–75 °C. 1H NMR (400 MHz, CDCl3) δ 7.16 (s, 1H), 6.94 (s, 2H), 5.67 (d, J = 5.2 Hz, 1H), 5.52 (s, 1H), 4.75 (d, J = 5.2 Hz, 1H), 4.04–3.95 (m, 1H), 3.90–3.73 (m, 2H), 3.48–3.39 (m, 1H), 3.10–3.01 (m, 1H), 2.75 (d, J = 7.6 Hz, 2H), 2.63–2.56 (m, 1H), 2.44 (s, 1H), 2.39–2.25 (m, 2H), 2.20–2.07 (m, 1H), 1.99 (s, 1H), 1.92–1.75 (m, 5H), 1.73–1.61 (m, 2H), 1.51–1.39 (m, 1H), 1.17–1.01 (m, 2H); 13C NMR (101 MHz) δ 166.5, 147.9, 135.1, 132.5 (2), 128.8, 123.7 (2), 112.1, 110.2 (2), 59.5, 59.4, 54.4, 46.9, 44.8, 40.7, 35.1, 31.5, 28.0, 26.3, 24.9, 24.7, 24.0, 19.2. ESI-HRMS: calcd for C25H30F6N3O [M + H]+: 502.2288, found: 502.2276.

12 N-N′-(1-Naphthylamino)acetyl aloperine (14p).

Yield 40%; white solid; m.p. 82–84 °C. 1H NMR (500 MHz, CDCl3) δ 8.06–7.97 (m, 1H), 7.83–7.75 (m, 1H), 7.50–7.42 (m, 2H), 7.34 (t, J = 7.5 Hz, 1H), 7.28–7.21 (m, 1H), 6.48 (d, J = 7.5 Hz, 1H), 5.96–5.85 (m, 1H), 5.67 (d, J = 5.0 Hz, 1H), 4.82 (d, J = 5.5 Hz, 1H), 4.20–4.09 (m, 1H), 4.04–3.96 (m, 1H), 3.84–3.74 (m, 1H), 3.57–3.46 (m, 1H), 3.15–3.03 (m, 1H), 2.81–2.69 (m, 2H), 2.66–2.54 (m, 1H), 2.50 (s, 1H), 2.43–2.37 (m, 1H), 2.33–2.22 (m, 1H), 2.20–2.10 (m, 1H), 2.02–1.62 (m, 8H), 1.51–1.37 (m, 1H), 1.17–1.03 (m, 2H); 13C NMR (126 MHz) δ 167.9, 142.9, 135.4, 134.4, 128.5 (2), 126.6, 126.0, 124.9, 123.5, 120.6, 117.3, 104.0, 59.4 (2), 54.4, 46.9, 45.5, 40.6, 35.1, 31.6, 28.0, 26.3, 24.9, 24.6, 24.0, 19.2. ESI-HRMS: calcd for C27H34N3O [M + H]+: 416.2696, found: 416.2688.

12N-N′-(2-Naphthylamino)acetyl aloperine (14q).

Yield 43%; white solid; m.p. 84–86 °C. 1H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 8.5 Hz, 1H), 7.63 (t, J = 8.5 Hz, 2H), 7.40–7.33 (m, 1H), 7.20 (t, J = 7.5 Hz, 1H), 6.99 (dd, J = 8.5, 2.0 Hz, 1H), 6.75–6.68 (m, 1H), 5.66 (d, J = 5.5 Hz, 1H), 5.23–5.16 (m, 1H), 4.79 (d, J = 5.5 Hz, 1H), 4.16–4.04 (m, 1H), 3.99–3.91 (m, 1H), 3.86–3.74 (m, 1H), 3.56–3.47 (m, 1H), 3.15–3.02 (m, 1H), 2.81–2.70 (m, 2H), 2.66–2.52 (m, 1H), 2.47 (s, 1H), 2.41–2.35 (m, 1H), 2.33–2.23 (m, 1H), 2.19–2.09 (m, 1H), 2.02–1.76 (m, 6H), 1.74–1.61 (m, 2H), 1.51–1.38 (m, 1H), 1.15–1.03 (m, 2H); 13C NMR (126 MHz) δ 167.7, 145.2, 135.4, 135.3, 129.1, 128.5, 127.8, 127.6, 126.4, 125.9, 122.0, 118.5, 104.0, 59.4, 59.3, 54.4, 46.9, 45.4, 40.6, 35.1, 31.5, 28.0, 26.3, 24.9, 24.7, 24.0, 19.2. ESI-HRMS: calcd for C27H34N3O [M + H]+: 416.2696, found: 416.2685.

12N-N′-Methyl-N’-p-chlorophenyl aminoacetylaloperine (14r).

Yield 38%; white solid; m.p. 54–56 °C. 1H NMR (600 MHz, CDCl3) δ 7.16–7.12 (m, 2H), 6.60 (d, J = 8.4 Hz, 2H), 5.62 (d, J = 3.6 Hz, 1H), 4.70 (s, 1H), 4.18–4.00 (m, 2H), 3.78–3.61 (m, 1H), 3.52–3.38 (m, 1H), 3.04 (s, 4H), 2.78–2.68 (m, 2H), 2.62–2.53 (m, 1H), 2.46–2.29 (m, 2H), 2.29–2.19 (m, 1H), 2.15–2.05 (m, 1H), 1.98–1.79 (m, 5H), 1.76–1.59 (m, 3H), 1.49–1.39 (m, 1H), 1.17–1.01 (m, 2H); 13C NMR (151 MHz) δ 168.3, 148.4, 135.7, 129.1 (3), 128.3, 122.1, 113.8, 59.6, 59.2, 54.9, 54.5, 47.0, 41.2, 40.0, 35.2, 31.5, 28.1, 26.3, 25.1, 24.9, 24.2, 19.4. ESI-HRMS: calcd for C24H33ClN3O [M + H]+: 414.2307, found: 414.2311.

General procedures for the synthesis of 12 N-carbamoyl aloperines (15a–d)

To a solution of aloperine (2 mmol) in anhydrous CH2Cl2 (30 mL) at 0 °C, after the addition of triethylamine (2.4 mmol), the different substituted isocyanates (2 mmol) were respectively added slowly. After 30 min, reaction was warmed to r.t. and stirred for 0.5 h. The gained mixture was concentrated to give a residue, which was purified by flash column chromatography on silica gel with CH2Cl2/CH3OH as the eluent to get the title compounds 15a–d.

12N-N′-(2,4-Difluorophenyl)carbamoyl aloperine (15a).

Yield 26%; white solid; m.p. 168–170 °C. 1H NMR (500 MHz, CDCl3) δ 8.15–8.05 (m, 1H), 6.88–6.79 (m, 2H), 6.45 (s, 1H), 5.64 (s, 1H), 4.48 (s, 1H), 3.80–3.69 (m, 1H), 3.63–3.52 (m, 1H), 3.15–3.04 (m, 1H), 2.82–2.66 (m, 2H), 2.6–2.53 (m, 2H), 2.33–2.25 (m, 2H), 2.18–2.12 (m, 1H), 2.03–1.80 (m, 5H), 1.72–1.63 (m, 3H), 1.52–1.37 (m, 1H), 1.17–1.02 (m, 2H). 13C NMR (126 MHz) δ 156.0, 154.8, 154.1, 135.9, 128.2, 124.4, 122.5, 111.2, 103.4, 59.9, 59.6, 54.4, 46.7, 40.3, 35.1, 33.4, 28.4, 26.3, 25.1, 24.3 (2), 19.5. ESI-HRMS: calcd for C22H28F2N3O [M + H]+: 388.2195, found: 388.2193.

12N-N′-(3,5-Dimethylphenyl)carbamoyl aloperine (15b).

Yield 63%; white solid; m.p. 106–108 °C. 1H NMR (600 MHz, CDCl3) δ 7.06 (s, 2H), 6.65 (s, 1H), 6.23 (s, 1H), 5.61 (d, J = 4.9 Hz, 1H), 4.49 (d, J = 4.8 Hz, 1H), 3.72 (dd, J = 13.4, 6.4 Hz, 1H), 3.58–3.53 (m, 1H), 3.07 (dd, J = 11.6, 3.6 Hz, 1H), 2.78 (d, J = 13.3 Hz, 1H), 2.74–2.69 (m, 1H), 2.58–2.55 (m, 2H), 2.31 (s, 1H), 2.27 (s, 6H), 2.24 (d, J = 11.2 Hz, 1H), 2.17–2.12 (m, 1H), 2.02–1.98 (m, 2H), 1.91 (d, J = 12.7 Hz, 1H), 1.87–1.79 (m, 2H), 1.71–1.66 (m, 3H), 1.48–1.40 (m, 1H), 1.12 (d, J = 13.2 Hz, 1H), 1.08 (d, J = 13.4 Hz, 1H); 13C NMR (151 MHz) δ 155.1, 139.2, 138.5 (2), 136.0, 127.6, 124.4, 117.2 (2), 59.5 (2), 54.2, 46.6, 40.0, 35.0, 33.3, 28.3, 26.1, 24.9, 24.3, 24.2, 21.4 (2), 19.3. ESI-HRMS: calcd for C24H34N3O [M + H]+: 380.2696, found: 380.2692.

12N-N′-(3,5-Ditrifluoromethylphenyl)carbamoyl aloperine (15c).

Yield 46%; white solid; m.p. 134–136 °C. 1H NMR (400 MHz, CDCl3) δ 7.95 (s, 2H), 7.48 (s, 1H), 6.66 (s, 1H), 5.66 (d, J = 4.4 Hz, 1H), 4.53 (s, 1H), 3.76–3.62 (m, 2H), 3.18–3.07 (m, 1H), 2.86–2.48 (m, 3H), 2.37–2.24 (m, 2H), 2.22–2.10 (m, 1H), 2.05–1.99 (m, 1H), 1.99–1.91 (m, 2H), 1.91–1.82 (m, 2H), 1.76–1.61 (m, 4H), 1.52–1.37 (m, 1H), 1.21–1.01 (m, 2H); 13C NMR (151 MHz, DMSO‑d) δ 154.1, 142.9, 135.8, 130.2 (2), 127.0, 123.4 (2), 118.9, 118.8, 113.6, 58.7, 58.3, 54.8, 53.6, 46.0, 34.4, 31.9, 27.6, 25.6, 24.4, 24.0, 23.4, 18.7. ESI-HRMS: calcd for C24H28F6N3O [M + H]+: 488.2131, found: 488.2122.

12N-N′-(1-Adamanty)carbamoyl aloperine (15d).

Yield 57%; white solid; m.p. 99–101 °C. 1H NMR (400 MHz, CDCl3) δ 5.55 (d, J = 4.8 Hz, 1H), 4.28 (d, J = 5.2 Hz, 1H), 4.09 (s, 1H), 3.64–3.52 (m, 1H), 3.41–3.29 (m, 1H), 3.08–2.98 (m, 1H), 2.83–2.63 (m, 2H), 2.59–2.48 (m, 1H), 2.28–2.16 (m, 2H), 2.14–2.01 (m, 5H), 1.99–1.96 (m, 5H), 1.95–1.92 (m, 1H), 1.91–1.76 (m, 3H), 1.71–1.68 (m, 2H), 1.68–1.57 (m, 9H), 1.44–1.38 (m, 1H), 1.18–1.03 (m, 2H); 13C NMR (101 MHz) δ 157.2, 136.8, 127.1, 59.9, 59.2, 54.4, 51.2, 46.8, 44.8, 42.8 (2), 42.7, 36.7 (2), 36.2, 35.1, 33.3, 29.8 (2), 29.7, 28.6, 26.1, 24.8, 24.7, 24.3, 19.7. ESI-HRMS: calcd for C26H40N3O [M + H]+: 410.3166, found: 410.3153.

Biological evaluation

Cell culture

HEK 293 T (American Type Culture Collection (ATCC), CRL-3216), Huh 7 (Japanese Collection of Research Bioresources [JCRB], 0403) and Vero E6 (ATCC, CRL-1586) cells were cultured in Dulbecco’s modified Eagle’s medium (HyClone, South Logan, UT). The medium was supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1% penicillin–streptomycin solution (Gibco, Carlsbad, CA, USA), 2% 4-(2-hydroxy ethyl)-1-piperazineethanesulfonic acid (Gibco, Carlsbad, CA, USA) at 37 °C with 5% CO2.

Pseudotyped virus

The monolayer 293 T cells were cotransfected with plasmid pcDNA3.1.S2 using lipofectamine 3000 (Invitrogen, Carlsbad, CA), and G*ΔG-VSV were added [28]. After incubated for 6 h, the medium was replaced with fresh medium and incubated for 24 h. The culture supernatants containing the pseudovirus were harvested, filtered (0.45 μM pore-size) and stored in 1 mL aliquots at –80 °C until use. 50% tissue culture infectious dose (TCID50) of pseudoviruses was determined as described previously [28]. All works involving pseudotyped viruses were performed in a BSL-2 facility at the National Institutes of Food and Drug Control (NIFDC), Beijing, China.

In vitro screening assay with pseudoviruses

For primary screening, serial dilutions (starting from 1:150) of each compound (30 mM) were mixed with 1.3 × 104 TCID50 of SARS-CoV-2 pseudovirus in 96-well Costar plates (Corning, Inc., Corning, NY) at 37 °C for 1 h, then mixed with Huh 7 cells (2 × 104 cells/well) and subsequently incubated for 24 h. The infectivity was determined by measuring the bioluminescence as described previously based on neutralization assay. The EC50 was determined from the dose–response curve, and cytotoxicity testing was performed to determine the CC50 of each test compound in the absence of pseudovirus.

Evaluation of key compounds with authentic SARS-CoV-2 in BSL-3

Serial dilutions of compounds 2e, 8a and 8b (30 mM) were mixed with 0.1 MOI authentic SARS-CoV-2 in 96-well Costar plates at 37 °C for 1 h, then Vero E6 cells (2 × 104 cells/well) were added and subsequently incubated for 3 days. EC50 of each compound were determined by CPE assay. Cytotoxicity testing was performed to determine the CC50 of each test compound in Vero E6 cells.

Time-of-addition experiment

The time of addition experiment of compounds 8a and CA-074 was performed to on SARS-CoV-2 pseudovirus. Huh 7 cells were prepackaged in 96-well plates 1 day in advance. Then 25 µM concentration of the compounds were added at during pseudovirus infection at 37 °C (a process), or at 0 °C (b process), and 1 h before pseudovirus infection (c process), and 1 h post infection (d process) at different temperature. After incubated at 37 °C for another 23 h, the inhibition rate was determined by measuring the bioluminescence as described previously [28].

Cathepsins activity assay

Cat B activity was assayed using the Cat B substrate N-benzyloxycarbonyl- l-arginyl-l-arginine-7-amido-4-methylcoumarin (Calbiochem) in cell lysates adjusted to pH of 5.0, as described previously [23]. Cat L activity was assayed in the same process as Cat B, but the Cat L substrate was N-benzyloxycarbonyl-l-phenyl alanyl-l-arginine-7-amido-4-methylcoumarin (Calbiochem).

PK study

Male SD rats were received 25 mg/kg with the compound 8a via oral administration. Nine blood samples were sequentially collected at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12 and 24 h after the dose, and immediately centrifuged to separate the plasma fractions at 10000 rpm for 10 min. The separated plasma samples were stored at –20 °C for assay. Non-compartmental PK analysis was performed on the serum concentration-versus-time profile of 8a derived from each SD rat with a nonlinear regression curve fitting program WinNonlin software, version 5.3.

Safety assessments

Kunming mice with weighting of 20 g ± 1.0 g were purchased from the Beijing HFK Biosciences Co. LTD. The mice were fed with a regular rodent diet and housed according to the guidelines of the CAMS & PUMC. After 3 days of accommodation, mice were randomly divided into 4 groups with 6 mice each, and compound 8a was given orally in a single-dosing experiment at 0, 250, 500 or 1000 mg•kg−1 (saline as control), respectively. The survival and body weight of the mice were closely monitored for 7 days. Seven days later, blood samples were collected and serum were isolated for determination of ALT, AST, BUN, and SCR levels.

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.