Design, synthesis, and biological evaluation of novel tetrahydroprotoberberine derivatives (THPBs) as proprotein convertase subtilisin/kexin type 9 (PCSK9) modulators for the treatment of hyperlipidemia.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulators may attenuate PCSK9-induced low-density lipoprotein receptor (LDLR) degradation in lysosome and promote the clearance of circulating low-density lipoprotein cholesterol (LDL-C). A novel series of tetrahydroprotoberberine derivatives (THPBs) were designed, synthesized, and evaluated as PCSK9 modulators for the treatment of hyperlipidemia. Among them, eight compounds exhibited excellent activities in downregulating hepatic PCSK9 expression better than berberine in HepG2 cells. In addition, five compounds 15, 18, 22, (R)-22, and (S)-22 showed better performance in the low-density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-LDL) uptake assay, compared with berberine at the same concentration. Compound 22, selected for in vivo evaluation, demonstrated significant reductions of total cholesterol (TC) and LDL-C in hyperlipidemic hamsters with a good pharmacokinetic profile. Further exploring of the lipid-lowering mechanism showed that compound 22 promoted hepatic LDLR expression in a dose-dependent manner in HepG2 cells. Additional results of human ether-à-go-go related gene (hERG) inhibition assay indicated the potential druggability for compound 22, which is a promising lead compound for the development of PCSK9 modulator for the treatment of hyperlipidemia.

Introduction

Cardiovascular diseases (CVDs) are a major cause of death throughout the world. Elevation of circulating low-density lipoprotein cholesterol (LDL-C) leads to an increased incidence of cardiovascular diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9), which originally found in patients with autosomal dominant hypercholesterolemia (ADH), could adjust the circulating LDL-C levels via inducing low-density lipoprotein receptor (LDLR) endocytosis and lysosomal degradation in hepatic cell3, 4. Loss-of-function mutations in PCSK9 in humans perform significantly lower LDL-C levels and CVDs morbidity, which indicates that PCSK9 may act as a therapeutic target for LDL-C-lowering.

Owing to the promise of its therapeutic potential, multiple PCSK9 monoclonal antibodies (mAbs) have been evaluated in clinical trials. Among them, two PCSK9 mAbs (alirocumab and evolocumab), have been approved by food and drug administration (FDA) for the treatment of coronary heart disease (CHD)6, 7. In addition, a small interfering RNA ALN-PCSSC, which inhibited the synthesis of PCSK9 also demonstrated lower LDL-C levels in patients administered in phase 3 clinical trial. However, both of these therapeutic drugs need to be administered by injection and costly, which may restrict their clinical promotion. Therefore, an orally available small molecule targets PCSK9 would be a highly desirable alternative therapeutic agent, based on its ease of administration and lower cost.

Small molecules target PCSK9 have been identified (Fig. 1). Compound 1 decreased PCSK9 protein expression in HepG2 cells with the IC50 value of 29.7 nmol/L. Compound 2 (PF-06446846) developed by Pfizer reduced secreted PCSK9 level with the IC50 value of 0.5 μmol/L10, 11, 12, 13. Natural products, such as moracin C (3), adenosine (4), and manglisin E (5), also exhibited potent down-regulation on PCSK9 mRNA expression in HepG2 cells, with IC50 values of 16.8, 18.46, and 3.15 μmol/L, respectively14, 15, 16.

Figure 1

Reported PCSK9 modulators.

Berberine (BBR, 6, Fig. 1), the main bioactive ingredient of Coptis chinensis, reduced serum total cholesterol (TC) by 29% and LDL-C by 25% in 32 hypercholesterolemic patients after 3 months of treatment. It was also identified reducing PCSK9 protein expression in HepG2 cells, and decreasing PCSK9 mRNA level with the IC50 value of 8.04 μmol/L. However, there are some factors that limit its wide prescription, such as its high oral dose of 1.0–1.5 g/day, poor bioavailability (F<1%) and high hERG channel inhibitory activity (IC50 = 3.1 μmol/L)17, 19, 20, 21, 22, 23, 24. Therefore, with the rapid rise in the number of patients with hyperlipidemia worldwide, discovering novel therapeutics to treat hyperlipidemia with better efficacy and fewer side effects has been a research focus in academia and industry.

It is known that tetrahydroprotoberberine deviratives (THPBs), which are the primary components of natural isoquinoline alkaloids, also exhibit a wide range of bioactivities25, 26, 27. Our group is dedicated to performing chemical modification of THPBs and constructing a library of small molecules with different scaffolds28, 29, 30, 31, 32, 33. Herein, we described a novel series of indole-containing THPB derivatives with anti-hyperlipidemia activity. We screened our in-house THPB library through PCSK9 expression in HepG2 cells. Fortunately, compound 7 featuring a novel scaffold, down-regulating PCSK9 expression assay at 5 μmol/L. In this study, based on the core structure of hit compound 7, a series of novel indole-containing THPBs as PCSK9 modulators were designed and synthesized, as well as the detailed structure–activity relationship (SAR) analysis, in vitro and in vivo biological evaluation, and pharmacokinetic studies.

Results and discussion

Chemistry

In order to obtain novel compounds to reduce the PCSK9 expression in HepG2 cells, we screened our in-house THPB library, and compound 7 showed biological activity (PCSK9 protein level 0.84 @5 μmol/L). To improve the potency, a series of novel indole-containing THPBs were designed based on the hit compound 7 (Scheme 1). Firstly, the oxacyclopentene of ring A was opened, we introduced various alkoxy groups at the 2- and 3-position on the ring A and maintained the ring D, and novel compounds 8‒13 were designed. Then, in order to reduce the potential hERG channel inhibition and improve the bioavailability, by introducing the methoxyl group at the 12-position of the ring D and investigating different substituents on the ring A, novel compounds 14‒26 were designed. In addition, chiral compounds (R)- and (S)-22 were also synthesized to explore the influence of the molecular configuration. Finally, introduction of the chlorine and bromine at the 13-position of the ring D, we obtained compounds 27 and 28.

Scheme 1

Design of indole-containing THPBs.

On the basis of the above design, indole-containing THPBs (7‒28) were synthesized through the route shown in Scheme 1. The procedures of Henry reaction was used to obtain the corresponding nitrostyrene 30. Reduction of the resulting nitrostyrenes with LiAlH4 produced the phenylethanamine intermediate 31. Then, phenylethanamine 31 and the substituted indole-3-acetic acid 32 were employed to generate amide 33, which was further cyclized under the presence of phosphoryl trichloride (POCl3) to give imine 34 in excellent yields, according to the procedures of the Bischler–Napieralski reaction. Reduction of the resulting imine 34 with sodium borohydride produced the key amine intermediate 35. Cyclization of amine 35 via the Pictect–Spengler reaction with formaldehyde resulted in the target products 7‒28 (Scheme 2).

Scheme 2

Synthesis of compounds 7‒28. Reagents and conditions: (a) CH3NO2, CH3COONH4, AcOH, 80 °C, 4 h; (b) LiAlH4, THF, 0‒65 °C, 4 h; (c) EDCI, Et3N, CH2Cl2, rt, 8 h; (d) POCl3, CH3CN, reflux; (e) NaBH4, methanol, rt, 8 h; (f) HCOOH, HCHO, 90 °C, 4 h.

Additionally, chiral compounds (R)- and (S)-22 were prepared according to the procedure outlined in Scheme 3. Asymmetric hydrogenation of imine 35p, catalyzed by a chiral Ru(II) complex (Noyori's catalyst)34, 35, 36 produced chiral amines (R)-35p, and (S)-35p, followed by cyclization with formaldehyde to give (R)- and (S)-22.

Scheme 3

Synthesis of chiral compounds (R)- and (S)-22. Reagents and conditions: (g) (S,S)-Noyori's catalyst, HCOONa, AgSbF6, La(OTf)3, CTAB, H2O, 40 °C, 12 h; (h) (R,R)-Noyori's catalyst, HCOONa, AgSbF6 , La(OTf)3, CTAB, H2O, 40 °C, 12 h; (i) HCOOH, 40% HCHO, CH3CN, 80 °C, 4 h.

Structure–activity relationship of indole-containing THPBs

The human hepatoma cell line HepG2 cells were used to investigate the PCSK9 expression. All indole-containing THPBs were screened to examine their ability to down-regulate PCSK9 expression in HepG2 cells. Structures of the indole-containing THPBs (compounds 7‒28) and their regulatory activity on PCSK9 protein expression are shown in Table 1 (data of PCSK9 protein level is expressed as a mean of the fold of vehicle). Initial screening was carried out of each compound at a concentration of 5 μmol/L using Western blot assay (Supporting Information Fig. S1).

Table 1

Effects of test compounds on PCSK9 expression in HepG2 cellsa.

Compd. (5 μmol/L)	R1	R2	R3	R4	PCSK9 protein level (compared to vehicle, mean ± SEM )
7	–OCH2O–		H	H	0.84±0.02*
8	OCH3	2-F-OBn	H	H	0.68±0.06*
9	2-F-OBn	OCH3	H	H	1.33±0.06*
10	3-F-OBn	OCH3	H	H	0.82±0.09
11	4-F-OBn	OCH3	H	H	0.81±0.07
12	3-Me-OBn	OCH3	H	H	0.93±0.03
13	4-Me-OBn	OCH3	H	H	0.69±0.17
14	-OCH2O-		OCH3	H	0.86±0.06
15	OCH3	OBn	OCH3	H	0.50±0.04∗∗
16	OCH3	2-F-OBn	OCH3	H	1.02±0.15
17	OCH3	3-F-OBn	OCH3	H	0.84±0.08
18	OCH3	4-F-OBn	OCH3	H	0.56±0.09*
19	OCH3	3-OMe-OBn	OCH3	H	1.04±0.16
20	OCH3	4-Me-OBn	OCH3	H	0.92±0.09
21	OCH3	OCH2CF3	OCH3	H	0.58±0.07*
22	OBn	OCH3	OCH3	H	0.24±0.02∗∗∗
(R)-22	OBn	OCH3	OCH3	H	0.21±0.01∗∗∗
(S)-22	OBn	OCH3	OCH3	H	0.51±0.03∗∗
23	2-F-OBn	OCH3	OCH3	H	0.87±0.06
24	3-F-OBn	OCH3	OCH3	H	0.48±0.02∗∗
25	3-Me-OBn	OCH3	OCH3	H	0.94±0.03
26	4-Me-OBn	OCH3	OCH3	H	0.95±0.08
27	OBn	OCH3	H	Cl	0.86±0.09
28	OBn	OCH3	H	Br	0.97±0.11
Berberine	–				0.71±0.05*
Vehicle	–				1.00±0.03

‒Not applicable.

Effects of test compounds on PCSK9 expression in HepG2 cells. Test compounds (5 μmol/L) were co-incubated with HepG2 cells for 24 h. Then the cells were collected and lysed in RIPA buffer containing protease inhibitor cocktail. Western blot was utilized to measure PCSK9 expression in HepG2 cells. The abundance of PCSK9 was quantified using imageJ software with normalization by signals of β-actin. Data are presented as mean ± SEM of three independent experiments. *P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; as compared to the vehicle group.

The SAR study was first focused on the effect of the side chains at the 2- and 3-position of the aromatic ring A (Table 1). The oxacyclopentene was opened, and a methoxyl or substituted-benzyloxy group was attached at the 2- and 3-position of the compound 7. The obtained compounds 8 and 13 showed higher activity on the down-regulation of PCSK9 expression than berberine and hit compound 7 (0.68 and 0.69 vs. 0.71 and 0.84, respectively). Compounds 9‒12 bearing 2-F-OBn, 3-F-OBn, 4-F-OBn, or 3-Me-OBn at the 2-position, respectively, reduced their activities on the down-regulation of PCSK9 (1.33, 0.82, 0.81, and 0.93).

Next, on the indole ring D of compound 7, the hydrogen atom at the 12-position was replaced with electron-donating methoxyl group. Compound 14 exhibited the similar biological activity with compound 7 (0.86 vs. 0.84). Then, retaining the methoxyl group at the 2-position, different substituted-benzyloxy group was introduced at the 3-position of the ring A. Compounds 15 and 18 displayed better biological activity than berberine on the down-regulation of PCSK9 (0.50 and 0.56 vs. 0.71). Replacement the substituted benzyloxy group to the trifluoroethoxyl group, compound 21 also exhibited good activity in down-regulating PCSK9 expression (0.58). Introduction of different substituted-benzyloxy group at the 2-position, compounds 22‒26 were obtained. Compound 22 displayed significant down-regulation of PCSK9 expression (0.24), which is much better than berberine. Therefore, we also synthesized the chiral compounds (R)- and (S)-22, and further evaluated their biological activities on the down-regulation of PCSK9. The R-configured enantiomer (R)-22 was more effective than the S-configured compound (S)-22 (0.21 vs. 0.51). Finally, by replacing the hydrogen to the chlorine and bromine at the 13-position, compounds 27 and 28 reduced their activities on the down-regulation of PCSK9 (0.86 and 0.97). We concluded that the methoxyl group at the 12-position played a significant role in down-regulating PCSK9 expression.

DiI-LDL uptake assays

Whether down-regulation of PCSK9 expression could promote LDL-C clearance in hepatic cells was determined by low-density lipoprotein, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI-LDL) uptake assay as previously described. Eight compounds 8, 15, 18, 21, 22, (R)-22, (S)-22, and 24 which exhibited better activity in decreasing hepatic cell PCSK9 expression than berberine, were co-incubated with HepG2 cells for the LDL-uptake assay. The results are summarized in Table 2. Firstly, berberine showed its LDL uptake rates with 1.38, compared to vehicle (DMSO). Subsequently, the above eight biological active compounds 8, 15, 18, 21, 22, (R)-22, (S)-22, and 24 were tested, and among them, five compounds 15, 18, 22, (R)-22 and (S)-22 displayed good LDL uptake rate (1.83, 1.78, 2.37, 2.19, and 1.83, respectively), which is much better than berberine (LDL uptake rate 1.38). The racemate 22 showed was slightly more potent than these two enantiomers (R)- and (S)-22, so the racemate 22 were used in the pharmacokinetic evaluation and in vivo biological assay. All selected compounds (<20 μmol/L) tested here showed no significant cytotoxicity in HepG2 cells (Supporting Information Table S1).

Table 2

Effects of test compounds on the regulation of LDL uptake by HepG2 cellsa.

Compd. (5 μmol/L)	DiI-LDL uptake rate (compared to vehicle, mean±SEM )
8	1.04±0.04
15	1.83±0.04∗∗
18	1.78±0.06∗∗
21	1.45±0.04∗∗
22	2.37±0.07∗∗∗
(R)-22	2.19±0.07∗∗∗
(S)-22	1.83±0.03∗∗
24	1.62±0.05∗∗
Berberine	1.38±0.03*
Vehicle	1.00±0.02

Effects of test compounds on the regulation of LDL uptake by HepG2 cells. HepG2 cells were treated with test compounds (5 μmol/L) for 24 h. Then the culture medium was changed to DMEM supplemented with 2% LPDS and 20 μg/mL DiI-LDL for another 4 h incubation. DiI-LDL uptaken by HepG2 cells was extracted by isopropanol and measured by microplate reader. Results are shown as mean ± SEM of three independent experiments. *P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; as compared to the vehicle group.

Selectivity evaluation

As a natural product, berberine and its derivatives exhibited various biological activities, including anti-depressant, anti-diabetes, and anti-inflammatory effects. Six compounds (8, 15, 18, 21, 22, and 24) which exhibited better activity in decreasing hepatic cell PCSK9 expression than berberine, were selected to evaluate the biological selectivity, such as β1 adrenergic receptor antagonist activity, dopamine D1 and D2 antagonist activity, and antagonistic activity on the serotonin 5-HT1B (Table 3). Among them, compound 22 exhibited good selectivity against β1 adrenergic receptor, dopamine D1 and D2 receptor (IC50 ≥ 100 μmol/L). Compound 22 showed moderate antagonistic activity on the serotonin 5-HT1B (IC50 = 13.43 μmol/L). The selectivity of compound 22 is better than berberine.

Table 3

Selectivity of six compounds on the typical biological targets of berberinea.

Compd.	IC50 (μmol/L)
	β1	D1	D2	5-HT1B
8	>100	5.56±0.08	17.87±0.86	47.16±1.22
15	>100	1.61±0.13	>100	40.83±0.80
18	∼100	1.71±0.10	>100	>100
21	∼100	2.35±0.07	∼50	>100
22	>100	∼100	>100	13.43±0.51
24	>100	>100	>100	15.70±0.59
Berberine	–	15.5b	17.1b	–

‒Not applicable.

Values are the average of 3 independent experiments. β1, beta-1 adrenergic receptor antagonist activity; D1, dopamine D1 antagonist activity; D2, dopamine D2 antagonist activity; 5-HT1B, serotonin 5-HT1B receptor antagonist activity.

Ref. 21.

Preliminary pharmacokinetic (PK) evaluation of compound 22

To explore the further druggability of the new identified indole-containing THPB derivatives, compound 22 was further evaluated for its pharmacokinetic properties in hamsters after oral administration and intravenous, respectively. As shown in Table 4, compound 22 given orally at 20 mg/kg displayed a half-life (t1/2) of 6.35 h, the maximum plasma concentration (Cmax) of 169 ng/mL, and an AUC value of 1005 ng/mL·h. Besides, compound 22 showed an oral bioavailability of 21.9%. Taken together, compared to the poor PK of the natural product berberine with low oral bioavailability (∼0%)22, 23, compound 22 exhibited significant improvements on overall PK properties.

Table 4

Pharmacokinetic parameters of compound 22 in hamstersa.

Compd.	Admin.	Cmax (ng/mL)	AUC0–∞ (ng/mL·h)	MRT (h)	t1/2 (h)	CL (mL/min/kg)	F (%)
22	p.o.	169	1005	10.3	6.35	–	21.9
	i.v.	–	2088	4.13	3.61	84.1

–Not determined.

Values are the average of three runs. Cmax, maximum concentration; AUC, area under the plasma concentration−time curve; MRT, mean residence time; t1/2, half-life; CL, clearance; F, oral bioavailability. Dose: p.o. at 20 mg/kg. Dose: i.v. at 10 mg/kg.

Oral efficacy evaluation of compound 22 in hyperlipidemic hamsters in vivo

In order to verify the hypolipidemic effect of compound 22 in vivo, high-fat diet (HFD)-induced hyperlipidemic hamster model was employed. Fenofibrate was used as the positive control during the in vivo studies. After 21 days treatment, compared to the HFD control group, HFD hamsters given compound 22 30 mg/kg by oral gavage showed 36.7% reduction in serum TC (Fig. 2A) and 41.4% reduction in serum LDL-C, respectively (Fig. 2B), which confirmed the hypolipidemic effect of compound 22 in vivo. The efficacy data showed that fenofibrate (p.o., 100 mg/kg) decreased TC and LDL-C in 71.3% and 79.4%, respectively, compared to hamsters in the HFD control group. Also, serum TC and LDL-C in the HFD control group increased 376% and 751%, respectively, as compared to the normal control group, which indicated that the high-fat-diet-induced hyperlipidemic hamster model was successful.

Figure 2

Hypolipidemic effect of compound 22 and fenofibrate in the hyperlipidemic hamsters. Hamsters involved in the experiment were divided into normal control group (n = 7) and HFD group (n = 21), and then the HFD group was switched into a high-fat diet (0.5% cholesterol) to induce hyperlipidemia. One week later, hamsters in the HFD group were divided into HFD control group and test groups (n = 7 per group). Test groups were given compound 22 at a daily dose of 30 mg/kg or fenofibrate at a daily dose of 100 mg/kg by oral gavage for 21 days. Blood samples of hamsters were collected at the end of experiment course. Serum TC levels (A) and LDL-C levels (B) of hamsters were measured by commercially available kits. Data are presented as mean ± SEM (##P < 0.01 as compared to the normal control group. P < 0.05; P < 0.01; P < 0.001; as compared to the HFD control group).

Effect of compound 22 on LDLR expression in hepatic cells

Mechanisms' studies demonstrated that PCSK9 could bind to the LDL receptor and target it for lysosomal degradation in hepatic cells. Lower PCSK9 may promote LDLR expression and increase LDL-C clearance. Since compound 22 decreases PCSK9 protein expression in HepG2 cells with an IC50 value of 1.34 μmol/L and PCSK9 mRNA expression with an IC50 value of 1.10 μmol/L (Supporting Information Figs. S2 and S3), for the purpose of exploring the lipid-lowering mechanism of compound 22, we tested the effect of compound 22 on LDLR expression in HepG2 cells.

HepG2 cells were treated with increasing concentrations of compound 22 (0.1, 0.5, 1, 2.5, and 5 μmol/L) for 24 h. Results demonstrated that compound 22 promotes hepatic cell LDLR expression in a dose-dependent manner with optimal 2.06-fold at 5 μmol/L compared to DMSO (Fig. 3), consistent with 2.37-fold LDL-uptake rate in DiI-LDL uptake assay (Table 2), which suggested that the increase of LDL-uptake in HepG2 cells mainly depended on the promotion of LDLR expression by compound 22.

Figure 3

Compound 22 promotes HepG2 cell LDLR expression in a dose-dependent manner. Total protein of HepG2 cells treated with increasing concentrations of compound 22 (0.1, 0.5, 1, 2.5, and 5 μmol/L) for 24 h was isolated by RIPA buffer. Western blot was utilized to measure LDLR expression in HepG2 cells. The protein abundance of LDLR was quantified using imageJ software with normalization by signals of β-actin. Results are shown as mean ± SEM of three independent experiments. P < 0.05; P < 0.01; as compared to the DMSO group.

hERG testing of compound 22

Blockade of the human hERG channel was a significant hurdle encountered in drug discovery. Because compound 22 was efficacious in vivo, it was subjected to hERG testing using a patch-clamp experiment. The result showed that the IC50 value of compound 22 on hERG inhibition was 7.99 μmol/L. Compared to berberine (IC50 = 3.1 μmol/L), compound 22 exhibited lower inhibitory activity on the hERG channel in vitro.

Conclusions

In this study, a novel series of indole-containing THPBs were designed, synthesized, and evaluated as PCSK9 modulators for the treatment of hyperlipidemia. SAR exploration led to the identification of a highly potent PCSK9 modulator 22 (IC50 = 1.34 μmol/L), which promoted 2.37-fold (5 μmol/L) LDL uptake in HepG2 cells. Notably, as a lead compound, compound 22 showed excellent in vivo hypolipidemic potency and a good pharmacokinetic profile, which is much better than berberine. To conclude, results we have earned suggest that compound 22 worths further investigation as an anti-hyperlipidemic agent.

Experimental

General metods of chemistry

Chemicals and solvents were purchased from commercial sources (Alfa, Acros, Sigma–Aldrich, and Shanghai Chemical Reagent Company) and used without further purification. Analytical thin layer chromatography (TLC) was HSGF 254 (0.15–0.2 mm thickness, YantaiHuiyou Company, China). Column chromatography was performed with CombiFlash Companion system (Teledyne Isco, Inc. Lincoln, NE, USA). Nuclear magnetic resonance spectra was recorded on a 400 MHz instrument (TMS as IS). Chemical shifts were reported in parts per million (ppm). Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low- and high-resolution mass spectra (MS and HR-MS) were given with electrospray ionization (ESI) produced by Q-TOF mass spectrometer. All target compounds were confirmed with over 95% purity (Supporting Information Table S2), which were determined by Agilent-1100 HPLC with binary pump, photodiode array detector (DAD), using Agilent Extend-C18 column (15 cm × 0.46 cm, 5 μm), CH3OH/H2O [0.1% triethylamine was added in CH3OH = 70/30 (v/v)] or CH3OH/H2O [0.1% triethylamine was added in CH3OH = 60/40 (v/v)] at 1.0 mL/min, and calculating the peak areas at 280 nm. Berberine was purchased from meilun biotechnology (Dalian, China).

General synthetic procedures for the target compounds 7‒28 (compound 7 as example)

(E)-5-(2-Nitrovinyl)benzo[d][1,3]dioxole (30a)

To a solution of benzo[d][1,3]dioxole-5-carbaldehyde (29a, 20 mmol) and ammonium acetate (3.08 g, 40 mmol) in nitromethane (20 mL) was added acetic acid (10 mL), and the resulting mixture was heated to 80 °C for 4 h. Then the mixture was concentrated, and the concentrate was added saturated NaHCO3 to obtained yellow solid, which was washed with isopropanol to get the intermediate (E)-5-(2-nitrovinyl)benzo[d][1,3]dioxole (30a) (3.67 g, 95%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 13.5 Hz, 1H), 7.51 (d, J = 13.5 Hz, 1H), 7.12 (d, J = 7.7 Hz, 1H), 7.04 (s, 1H), 6.91 (d, J = 7.9 Hz, 1H), 6.10 (s, 2H). ESI-MS m/z: 194 [M+H]+.

2-(Benzo[d][1,3]dioxol-5-yl)ethanamine (31a)

Lithium aluminium hydride (2.16 g, 57 mmol) was slowly added into dry tetrahydrofuran (50 mL) to form suspensions. A solution of 30a (3.67 g, 19 mmol) in tetrahydrofuran (50 mL) was added dropwise to suspensions at 0 °C, and the mixture was stirred for 4 h at 65 °C. The reaction mixture was cooled to room temperature (RT) and diluted with CH2Cl2 (150 mL), and then the reaction mixture was quenched with appropriate ice‒water and filtered. The filtrate was evaporated under reduced pressure to get the product (31a) (2.19 g, 71%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.76–6.71 (m, 1H), 6.69 (d, J = 4.5 Hz, 1H), 6.63 (dd, J = 7.9, 1.5 Hz, 1H), 5.91 (s, 2H), 2.90 (t, J = 6.8 Hz, 2H), 2.65 (t, J = 6.8 Hz, 2H), 1.40–1.13 (m, 2H). ESI-MS m/z: 166 [M+H]+.

N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-2-(1H-indol-3-yl)acetamide (33a)

2-(1H-Indol-3-yl)acetic acid (32a, 3.5 g, 19.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.8 g, 19.95 mmol), 1-hydroxybenzotriazole (3.23 g, 23.94 mmol), and triethylamine (2.01 g, 19.95 mmol) were dissolved in 50 mL of dichloromethane, and the mixture was stirred for 1 h at room temperature. Then a solution of 2-(benzo[d][1,3]dioxol-5-yl)ethanamine (31a) (13.3 mmol) in dichloromethane (20 mL) was added into the mixture and stirred for 8 h, water was added, the mixture was extracted with ethyl acetate, and the organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was purified by flash chromatography to give 33a (3.39 g, 79%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.28–7.19 (m, 1H), 7.17–7.10 (m, 1H), 7.06 (d, J = 2.1 Hz, 1H), 6.51 (d, J = 7.9 Hz, 1H), 6.42 (d, J = 1.6 Hz, 1H), 6.26 (dd, J = 7.9, 1.7 Hz, 1H), 5.88 (s, 2H), 5.76 (s, 1H), 3.71 (s, 2H), 3.36 (q, J = 6.7 Hz, 2H), 2.56 (t, J = 6.8 Hz, 2H). ESI-MS m/z: 323 [M+H]+.

3-((7,8-Dihydronaphtho[2,3-d][1,3]dioxol-5-yl)methyl)-1H-indole (35a)

N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-2-(1H-indol-3-yl)acetamide (33a) (3.39 g, 10.5 mmol) was dissolved in 20 mL of acetonitrile, and POCl3 (5.9 mL, 63.3 mmol) was added. The solution was heated to reflux under argon for 1 h. The solvents were evaporated under reduced pressure. The pH of the mixture was adjusted to alkalinity with the addition of saturated NaHCO3. The mixture was extracted with dichloromethane. The combined organic phase was evaporated under reduced pressure to get the crude product (34a) which was used in the next step without further purification. ESI-MS m/z: 305 [M+H]+. The intermediate 34a was dissolved in 50 mL of methanol, and NaBH4 (3.97 g, 105 mmol) was added in batches at 0 °C. The mixture was stirred for 2 h at room temperature. The reaction mixture was quenched with ammonium chloride and extracted with ethyl acetate. The organic layer was washed with brine, and the combined organic phase was evaporated under reduced pressure to get the crude product, which was purified by flash chromatography on silica gel to get intermediate 35a (1.19 g, 3.89 mmol, 37% over two steps). 1H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.18 (dd, J = 11.1, 4.0 Hz, 1H), 7.15–7.08 (m, 2H), 6.76 (s, 1H), 6.55 (s, 1H), 5.92 (d, J = 0.9 Hz, 2H), 4.32 (dd, J = 9.5, 3.8 Hz, 1H), 3.38 (dd, J = 14.8, 3.8 Hz, 2H), 3.27 (s, 1H), 3.13 (dd, J = 14.8, 9.6 Hz, 2H), 2.87–2.75 (m, 2H), 2.74–2.63 (m, 1H). ESI-MS m/z: 307 [M+H]+.

(5,6,14,14a-Tetrahydro-[1,3]dioxolo[4,5-g]indolo[3′,2′:4,5]pyrido[2,1-a]isoquino-lin-9(8H)-yl)methanol (7)

The intermediate 35a (0.5 g, 1.63 mmol), 0.5 mL of formaldehyde, and 0.1 mL of formic acid were dissolved in 30 mL of acetonitrile. The mixture was stirred for 4 h at 90 °C. Then the mixture was evaporated under reduced pressure to get the residue. With water added, the mixture was extracted with ethyl acetate, and the organic extracts were washed with brine, dried over Na2SO4, and concentrated. Then chromatographed on silica gel to give the target compound 7 (0.2 g). Yellow solid, m.p. = 258‒260 °C; Yield: 35%. 1H NMR (400 MHz, DMSO-d6) δ 7.45 (dd, J = 14.2, 7.8 Hz, 2H), 7.15–7.06 (m, 1H), 7.05–6.99 (m, 2H), 6.69 (s, 1H), 6.25 (t, J = 7.1 Hz, 1H), 5.96 (dd, J = 5.3, 0.9 Hz, 2H), 5.48–5.36 (m, 2H), 4.19 (d, J = 15.1 Hz, 1H), 3.69 (d, J = 15.1 Hz, 1H), 3.58 (dd, J = 10.4, 3.3 Hz, 1H), 3.37 (dd, J = 15.2, 2.7 Hz, 1H), 3.15–3.06 (m, 1H), 2.93 (m, 1H), 2.74–2.56 (m, 2H), 2.49–2.39 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 145.74, 145.45, 136.01, 133.11, 131.42, 127.49, 126.85, 120.63, 119.02, 117.61, 109.73, 108.05, 107.67, 106.03, 100.56, 65.29, 59.52, 51.13, 50.87, 29.41, 29.20; ESI-MS m/z: 349 [M+H]+. ESI-HR-MS Calcd. for C21H21N2O3+ [M+H]+ 349.1547, Found 349.1553. HPLC analysis: MeOH/H2O = 70:30 (v/v), 7.12 min, 98.44% purity.

(2-((2-Fluorobenzyl)oxy)-3-methoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (8)

Compound 8 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 180‒183 °C; Yield: 40%. 1H NMR (400 MHz, DMSO-d6) δ 7.60 (dd, J = 8.3, 6.8 Hz, 1H), 7.52–7.37 (m, 3H), 7.33–7.20 (m, 2H), 7.08 (dt, J = 29.0, 7.3 Hz, 3H), 6.74 (s, 1H), 6.27 (s, 1H), 5.43 (d, J = 7.2 Hz, 2H), 5.14 (q, J = 11.7 Hz, 2H), 4.20 (d, J = 12.1 Hz, 1H), 3.75 (s, 3H), 3.73–3.65 (m, 1H), 3.59 (m, 1H), 3.42 (d, J = 13.9 Hz, 1H), 3.15 (d, J = 7.8 Hz, 1H), 3.03–2.86 (m, 1H), 2.69 (d, J = 15.0 Hz, 2H), 2.45–2.26 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 160.45 (d, JC-F = 246.1 Hz), 147.64, 145.89, 136.01, 131.05 (d, JC-F = 4.0 Hz), 130.34 (d, JC-F = 8.2 Hz), 126.75, 124.48 (d, JC-F = 3.4 Hz), 124.16, 124.01, 120.67, 119.06, 117.57, 115.32 (d, JC-F = 21.1 Hz), 111.90, 111.75, 109.76, 107.64, 65.27, 64.44 (d, JC-F = 3.3 Hz), 59.23, 55.46, 51.08, 51.04, 28.90, 28.86; ESI-MS m/z: 459 [M+H]+; ESI-HR-MS Calcd. for C28H28FN2O3+ [M+H]+ 459.2078, Found 459.2090. MeOH/H2O = 70:30 (v/v), 15.04 min, >99% purity.

(3-((2-Fluorobenzyl)oxy)-2-methoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (9)

Compound 9 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 167‒169 °C; Yield: 38%. 1H NMR (400 MHz, DMSO-d6) δ 7.57 (td, J = 7.5, 1.4 Hz, 1H), 7.52–7.40 (m, 3H), 7.32–7.21 (m, 2H), 7.13–7.07 (m, 1H), 7.03 (dd, J = 10.4, 3.4 Hz, 2H), 6.86 (s, 1H), 6.26 (t, J = 7.1 Hz, 1H), 5.52–5.35 (m, 2H), 5.12–5.06 (m, 2H), 4.20 (d, J = 15.2 Hz, 1H), 3.79 (s, 3H), 3.70 (d, J = 15.1 Hz, 1H), 3.61 (dd, J = 10.3, 3.2 Hz, 1H), 3.46 (dd, J = 15.0, 2.7 Hz, 1H), 3.20–3.05 (m, 1H), 2.95 (m, 1H), 2.73–2.59 (m, 2H), 2.44 (d, J = 13.1 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 160.40 (d, J = 246.0 Hz), 147.57, 146.06, 136.02, 133.19, 131.02, 130.82 (d, JC-F = 4.1 Hz), 130.36 (d, JC-F = 8.1 Hz), 126.87, 126.43, 124.55 (d, JC-F = 3.4 Hz), 124.10 (d, JC-F = 14.6 Hz), 120.62, 119.02, 117.68, 115.37 (d, JC-F = 21.0 Hz), 113.43, 110.03, 109.76, 107.77, 65.30, 64.15 (d, JC-F = 3.4 Hz), 59.33, 55.87, 51.24, 51.08, 29.09, 28.97; ESI-MS m/z: 459 [M+H]+; ESI-HR-MS Calcd. for C28H28FN2O3 [M+H]+ 459.2078, Found 459.2090. MeOH/H2O = 70:30 (v/v), 12.75 min, 98.83% purity.

(3-((3-Fluorobenzyl)oxy)-2-methoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (10)

Compound 10 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 149‒151 °C; Yield: 43%. 1H NMR (400 MHz, DMSO-d6) δ 7.52–7.42 (m, 3H), 7.35–7.23 (m, 2H), 7.17 (td, J = 8.6, 2.2 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.02 (dd, J = 8.8, 6.2 Hz, 2H), 6.81 (s, 1H), 6.26 (t, J = 7.0 Hz, 1H), 5.50–5.37 (m, 2H), 5.14–5.03 (m, 2H), 4.20 (d, J = 15.1 Hz, 1H), 3.81 (s, 3H), 3.69 (d, J = 15.1 Hz, 1H), 3.61 (dd, J = 10.3, 3.0 Hz, 1H), 3.46 (dd, J = 15.0, 2.3 Hz, 1H), 3.19–3.07 (m, 1H), 3.00–2.90 (m, 1H), 2.64 (dd, J = 11.0, 7.7 Hz, 2H), 2.44 (d, J = 13.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 162.20 (d, JC-F = 243.4 Hz), 147.61, 145.99, 140.35 (d, JC-F = 7.5 Hz), 136.02, 133.17, 131.00, 130.45 (d, JC-F = 8.4 Hz), 126.86, 126.38, 123.54, 120.62, 119.01, 117.66, 114.52 (d, JC-F = 20.9 Hz), 114.18 (d, JC-F = 21.8 Hz), 113.57, 110.06, 109.75, 107.76, 69.13, 65.30, 59.32, 55.94, 51.23, 51.06, 29.08, 28.97; ESI-MS m/z: 459 [M+H]+; ESI-HR-MS Calcd. for C28H28FN2O3+ [M+H]+ 459.2078, Found 459.2081. MeOH/H2O = 70:30 (v/v), 13.63 min, 96.21% purity.

(3-((4-Fluorobenzyl)oxy)-2-methoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (11)

Compound 11 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 149‒151 °C; Yield: 43%. 1H NMR (400 MHz, DMSO-d6) δ 7.58‒7.41 (m, 4H), 7.23 (t, J = 8.9 Hz, 2H), 7.10 (t, J = 7.4 Hz, 1H), 7.06‒6.96 (m, 2H), 6.81 (s, 1H), 6.26 (t, J = 7.0 Hz, 1H), 5.43 (d, J = 6.7 Hz, 2H), 5.04 (s, 2H), 4.20 (d, J = 15.1 Hz, 1H), 3.80 (s, 3H), 3.69 (d, J = 15.1 Hz, 1H), 3.64‒3.57 (m, 1H), 3.52‒3.43 (m, 1H), 3.19‒3.08 (m, 1H), 2.94 (m, 1H), 2.68‒2.60 (m, 2H), 2.44 (d, J = 12.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 161.77 (d, JC-F = 243.5 Hz), 147.59, 146.12, 136.02, 133.58, 133.55, 133.18, 130.83, 130.03, 129.94, 126.87, 126.35, 119.01, 117.67, 115.33, 115.12, 113.49, 109.98, 109.76, 107.77, 69.22, 65.30, 59.33, 55.89, 51.24, 51.08, 29.09, 28.98; ESI-MS m/z: 459 [M+H]+; ESI-HR-MS Calcd. for C28H28FN2O3+ [M+H]+ 459.2078, Found 459.2090. MeOH/H2O = 70:30 (v/v), 12.73 min, 95.53% purity.

(2-Methoxy-3-((3-methylbenzyl)oxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (12)

Compound 12 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 152‒154 °C; Yield: 41%.1H NMR (400 MHz, DMSO-d6) δ 7.48 (dd, J = 7.8, 2.8 Hz, 2H), 7.27 (p, J = 7.5 Hz, 3H), 7.15 (d, J = 7.2 Hz, 1H), 7.11 (t, J = 7.3 Hz, 1H), 7.07–6.99 (m, 2H), 6.26 (t, J = 7.0 Hz, 1H), 5.43 (d, J = 7.7 Hz, 2H), 5.01 (s, 2H), 4.20 (d, J = 15.1 Hz, 1H), 3.80 (s, 3H), 3.70 (d, J = 15.5 Hz, 1H), 3.61 (d, J = 7.0 Hz, 1H), 3.46 (d, J = 14.5 Hz, 1H), 3.14 (d, J = 6.5 Hz, 1H), 3.02–2.87 (m, 1H), 2.65 (d, J = 13.2 Hz, 2H), 2.45 (d, J = 12.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 147.59, 146.32, 137.52, 137.21, 136.03, 133.17, 130.67, 128.45, 128.40, 128.32, 126.85, 126.32, 124.94, 120.64, 119.02, 117.68, 113.37, 109.97, 109.76, 107.76, 70.01, 65.30, 59.34, 55.89, 51.23, 51.08, 29.05, 28.98, 21.04; ESI-MS m/z: 455 [M+H]+; ESI-HR-MS Calcd. for C29H31N2O3+ [M+H]+ 455.2329, Found 455.2330. MeOH/H2O = 70:30 (v/v), 20.71 min, 98.07% purity.

(2-Methoxy-3-((4-methylbenzyl)oxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (13)

Compound 13 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 158‒160 °C; Yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 7.47 (dd, J = 7.8, 3.3 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 7.10 (t, J = 7.1 Hz, 1H), 7.02 (dd, J = 13.6, 5.8 Hz, 2H), 6.80 (s, 1H), 6.26 (t, J = 7.1 Hz, 1H), 5.43 (d, J = 6.6 Hz, 2H), 5.09–4.96 (m, 2H), 4.20 (d, J = 15.2 Hz, 1H), 3.79 (s, 3H), 3.69 (d, J = 15.0 Hz, 1H), 3.65–3.57 (m, 1H), 3.46 (dd, J = 15.0, 2.6 Hz, 1H), 3.18–3.08 (m, 1H), 3.00–2.85 (m, 1H), 2.70–2.59 (m, 2H), 2.44 (d, J = 12.9 Hz, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 147.58, 146.29, 137.02, 136.02, 134.28, 133.18, 130.62, 128.95, 127.90, 126.87, 126.34, 120.62, 119.01, 117.67, 113.37, 109.99, 109.76, 107.78, 69.80, 65.30, 59.34, 55.90, 51.24, 51.11, 29.09, 28.99, 20.82; ESI-MS m/z: 455 [M+H]+; ESI-HR-MS Calcd. for C29H31N2O3+ [M+H]+ 455.2329, Found 455.2342. MeOH/H2O = 70:30 (v/v), 19.28 min, >99% purity.

(12-Methoxy-5,6,14,14a-tetrahydro-[1,3]dioxolo[4,5-g]indolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (14)

Compound 14 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 169‒171 °C; Yield: 50%. 1H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 8.8 Hz, 1H), 7.01 (s, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.69 (s, 1H), 6.19 (t, J = 7.0 Hz, 1H), 5.96 (d, J = 1.6 Hz, 2H), 5.40–5.33 (m, 2H), 4.16 (d, J = 15.2 Hz, 1H), 3.76 (s, 3H), 3.66 (d, J = 14.1 Hz, 1H), 3.57 (d, J = 6.5 Hz, 1H), 3.36 (d, J = 15.6 Hz, 1H), 3.12 (d, J = 6.0 Hz, 1H), 3.02–2.86 (m, 1H), 2.66 (d, J = 15.8 Hz, 2H), 2.47–2.35 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 153.52, 145.73, 145.44, 133.59, 131.38, 131.09, 127.41, 127.21, 110.33, 110.01, 108.01, 107.43, 105.97, 100.54, 100.08, 65.37, 59.54, 55.31, 51.16, 50.86, 29.31, 29.23; ESI-MS m/z: 379 [M+H]+; ESI-HR-MS Calcd. for C22H23N2O4+ [M+H]+ 379.1652, Found 379.1661. MeOH/H2O = 70:30 (v/v), 5.63 min, 96.36% purity.

(2-(Benzyloxy)-3,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1 a]isoquinolin-9(8H)-yl)methanol (15)

Compound 15 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 182‒183 °C; Yield: 47%. 1H NMR (400 MHz, DMSO-d6) δ 7.52–7.47 (m, 2H), 7.45–7.40 (m, 2H), 7.38–7.32 (m, 2H), 7.05 (s, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 8.7, 2.4 Hz, 2H), 6.21 (t, J = 7.1 Hz, 1H), 5.42–5.32 (m, 2H), 5.16–5.03 (q, J = 11.8 Hz, 2H), 4.16 (d, J = 15.1 Hz, 1H), 3.79 (s, 3H), 3.76 (s, 3H), 3.64 (d, J = 14.9 Hz, 1H), 3.55 (dd, J = 12.8, 5.0 Hz, 1H), 3.32 (m, 1H), 3.18–3.05 (m, 1H), 2.95 (m, 1H), 2.71–2.57 (m, 2H), 2.40–2.25 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 153.57, 147.62, 146.07, 137.47, 133.77, 131.10, 130.17, 128.38, 127.95, 127.77, 127.23, 126.92, 111.89, 110.44, 110.10, 107.54, 99.87, 70.37, 65.39, 59.33, 55.52, 55.34, 54.94, 51.31, 51.20, 29.18, 29.00; ESI-MS m/z: 471 [M+H]+; ESI-HR-MS Calcd. for C29H31N2O4+ [M+H]+ 471.2278, Found 471.2286. MeOH/H2O = 70:30 (v/v), 10.10 min, 97.37% purity.

(2-((2-Fluorobenzyl)oxy)-3,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (16)

Compound 16 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 182‒183 °C; Yield: 47%. 1H NMR (400 MHz, DMSO-d6) δ 7.60 (t, J = 7.2 Hz, 1H), 7.44 (dd, J = 13.3, 6.2 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.27 (dd, J = 12.7, 6.0 Hz, 2H), 7.09 (s, 1H), 6.96 (d, J = 1.9 Hz, 1H), 6.86–6.66 (m, 2H), 6.22 (t, J = 7.0 Hz, 1H), 5.36 (t, J = 9.6 Hz, 2H), 5.14 (q, J = 11.6 Hz, 2H), 4.16 (d, J = 15.1 Hz, 1H), 3.78 (s, 3H), 3.74 (s, 3H), 3.65 (d, J = 15.0 Hz, 1H), 3.57 (d, J = 7.8 Hz, 1H), 3.40 (s, 1H), 3.20–3.07 (m, 1H), 2.97 (dd, J = 18.8, 7.2 Hz, 1H), 2.65 (dd, J = 21.8, 13.3 Hz, 2H), 2.43–2.31 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 160.48 (d, JC-F = 246.1 Hz), 153.56, 147.63, 145.92, 133.76, 131.11, 131.10, 131.07, 130.24, 127.29, 127.23, 124.50 (d, JC-F = 3.4 Hz), 124.16 (d, JC-F = 14.6 Hz), 115.44, 115.23, 111.94, 110.42, 110.09, 107.54, 99.88, 65.39, 64.60 (d, JC-F = 3.3 Hz), 59.30, 55.48, 55.34, 51.31, 51.15, 29.16, 29.03. ESI-MS m/z: 489 [M+H]+; ESI-HR-MS Calcd. for C29H30FN2O4+ [M+H]+ 489.2194, Found 489.2193. MeOH/H2O = 70:30 (v/v), 11.45 min, 95.77% purity.

(2-((3-Fluorobenzyl)oxy)-3,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (17)

Compound 17 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 176‒178 °C; Yield: 41%. 1H NMR (400 MHz, DMSO-d6) δ 7.45 (m, 1H), 7.39–7.27 (m, 3H), 7.18 (td, J = 8.4, 2.3 Hz, 1H), 7.05 (s, 1H), 6.93 (d, J = 2.4 Hz, 1H), 6.80–6.64 (m, 2H), 6.21 (t, J = 7.1 Hz, 1H), 5.44–5.31 (m, 2H), 5.20–5.05 (m, 2H), 4.16 (d, J = 15.1 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.64 (d, J = 15.0 Hz, 1H), 3.54 (dd, J = 10.1, 3.0 Hz, 1H), 3.31 (d, J = 2.6 Hz, 1H), 3.21‒3.07 (m, 1H), 3.00–2.89 (m, 1H), 2.81–2.57 (m, 2H), 2.36–2.28 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 162.21 (d, JC-F = 243.4 Hz), 153.56, 147.63, 145.72, 140.48 (d, JC-F = 7.4 Hz), 133.76, 131.08, 130.42 (d, JC-F = 8.2 Hz), 130.15, 128.17 (d, JC-F = 42.0 Hz), 127.19, 127.16, 123.72 (d, JC-F = 2.7 Hz), 114.60, 114.38, 112.00 (d, JC-F = 16.3 Hz), 110.45, 110.13, 107.47, 99.74, 69.44, 65.38, 59.31, 55.54, 55.31, 51.30, 51.20, 29.16, 28.99; ESI-MS m/z: 489 [M+H]+; ESI-HR-MS Calcd. for C29H30FN2O4+ [M+H]+ 489.2184, Found 489.2181. MeOH/H2O = 70:30 (v/v), 10.63 min, >99% purity.

(2-((4-Fluorobenzyl)oxy)-3,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (18)

Compound 18 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 151‒153 °C; Yield: 40%. 1H NMR (400 MHz, DMSO-d6) δ 7.53 (dd, J = 8.5, 5.7 Hz, 2H), 7.37 (d, J = 8.8 Hz, 1H), 7.24 (t, J = 8.9 Hz, 2H), 7.04 (s, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.74 (dd, J = 7.8, 3.1 Hz, 2H), 6.23 (t, J = 6.7 Hz, 1H), 5.37 (d, J = 7.5 Hz, 2H), 5.09 (q, J = 11.7 Hz, 2H), 4.17 (d, J = 14.8 Hz, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.61 (dd, J = 26.4, 16.6 Hz, 1H), 3.13 (d, J = 8.0 Hz, 1H), 2.99–2.93 (m, 1H), 2.69–2.63 (m, 2H), 2.35 (t, J = 11.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 161.78 (d, JC-F = 243.4 Hz), 153.57, 147.67, 145.92, 133.70, 133.67, 131.09, 130.19, 130.10, 127.17, 127.06, 115.28, 115.07, 112.07, 111.88, 110.47, 110.14, 107.48, 99.80, 69.70, 65.39, 59.33, 55.51, 55.33, 51.28, 51.16, 29.16, 29.00; ESI-MS m/z: 489 [M+H]+; ESI-HR-MS Calcd. for C29H30FN2O4+ [M+H]+ 489.2184, Found 489.2185. MeOH/H2O = 70:30 (v/v), 10.30 min, >99% purity.

(3,12-Dimethoxy-2-((3-methoxybenzyl)oxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (19)

Compound 19 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 85‒86 °C; Yield: 43%. 1H NMR (400 MHz, DMSO-d6) δ 7.51–7.20 (m, 2H), 7.21–7.00 (m, 3H), 7.01–6.85 (m, 2H), 6.83–6.66 (m, 2H), 6.20 (t, J = 6.9 Hz, 1H), 5.37 (d, J = 7.6 Hz, 2H), 5.09 (q, J = 12.1 Hz, 2H), 4.16 (d, J = 14.9 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.77 (s, 3H), 3.64 (d, J = 14.8 Hz, 1H), 3.54 (d, J = 8.2 Hz, 1H), 3.35 (d, J = 2.9 Hz, 1H), 3.13 (d, J = 9.5 Hz, 1H), 3.03‒2.84 (m, 1H), 2.64 (m, 2H), 2.33 (t, J = 12.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 159.32, 153.56, 147.62, 145.99, 139.07, 133.77, 131.10, 130.15, 129.48, 127.22, 126.94, 120.01, 113.36, 113.23, 111.94, 111.90, 110.43, 110.07, 107.50, 99.90, 70.19, 65.38, 59.33, 55.54, 55.37, 55.08, 51.31, 51.20, 29.16, 28.99; ESI-MS m/z: 501 [M+H]+; ESI-HR-MS Calcd. for C30H33N2O5+ [M+H]+ 501.2384, Found 501.2395. MeOH/H2O = 70:30 (v/v), 9.60 min, 95.32% purity.

(3,12-Dimethoxy-2-((4-methylbenzyl)oxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (20)

Compound 20 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 205‒207 °C; Yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 7.37 (dd, J = 8.4, 3.0 Hz, 3H), 7.22 (d, J = 7.8 Hz, 2H), 7.06 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 9.2, 2.8 Hz, 2H), 6.21 (t, J = 7.1 Hz, 1H), 5.37 (d, J = 6.6 Hz, 2H), 5.05 (q, J = 11.6 Hz, 2H), 4.16 (d, J = 15.1 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.64 (d, J = 15.1 Hz, 1H), 3.55 (dd, J = 10.5, 2.9 Hz, 1H), 3.38 (d, J = 2.9 Hz, 1H), 3.17–3.06 (m, 1H), 3.02–2.86 (m, 1H), 2.73–2.56 (m, 2H), 2.36 (d, J = 12.8 Hz, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 153.55, 147.58, 146.13, 137.00, 134.37, 133.77, 131.10, 130.15, 128.90, 128.12, 127.23, 126.81, 111.86, 111.77, 110.43, 110.08, 107.54, 99.91, 70.22, 65.38, 59.35, 55.50, 55.37, 51.32, 51.20, 29.16, 29.00, 20.84. ESI-MS m/z: 501 [M+H]+; ESI-HR-MS Calcd. for C30H33N2O4+ [M+H]+ 485.2435, Found 485.2432. MeOH/H2O = 70:30 (v/v), 16.71 min, >99% purity.

(3,12-Dimethoxy-2-(2,2,2-trifluoroethoxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (21)

Compound 21 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 180‒182 °C; Yield: 41%.1H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 8.8 Hz, 1H), 7.13 (s, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.80 (s, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.21 (t, J = 7.0 Hz, 1H), 5.37 (d, J = 6.4 Hz, 2H), 4.93‒4.59 (m, 2H), 4.17 (d, J = 15.1 Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H), 3.66 (d, J = 15.0 Hz, 1H), 3.56 (dd, J = 10.2, 2.8 Hz, 1H), 3.44 (dd, J = 15.0, 2.8 Hz, 1H), 3.19‒3.10 (m, 1H), 3.05‒2.90 (m, 1H), 2.72‒2.60 (m, 2H), 2.47‒2.35 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 153.56, 147.55, 144.68, 133.70, 131.09, 130.39, 128.95, 127.19, 124.14 (d, JC-F = 278.1 Hz), 113.02, 112.31, 110.42, 110.12, 107.48, 99.86, 66.10 (d, JC-F = 33.8 Hz), 65.38, 59.25, 55.62, 55.35, 51.28, 51.06, 29.10, 29.06; ESI-MS m/z: 463 [M+H]+; ESI-HR-MS Calcd. for C24H26F3N2O4+ [M+H]+ 463.1839, Found 463.1842. MeOH/H2O = 70:30 (v/v), 5.61 min, 95.21% purity.

(3-(Benzyloxy)-2,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (22)

Compound 22 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 145‒146 °C; Yield: 43%. 1H NMR (400 MHz, DMSO-d6) δ 7.49–7.44 (m, 2H), 7.41 (m, 2H), 7.38–7.32 (m, 1H), 7.06–6.95 (m, 2H), 6.82 (s, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.20 (t, J = 7.1 Hz, 1H), 5.43–5.36 (m, 2H), 5.06 (s, 2H), 4.17 (d, J = 15.2 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.66 (d, J = 15.1 Hz, 1H), 3.59 (dd, J = 10.5, 3.0 Hz, 1H), 3.43 (dd, J = 15.0, 2.7 Hz, 1H), 3.19‒3.04 (m, 1H), 2.94 (m, 1H), 2.70‒2.58 (m, 2H), 2.47‒2.37 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 153.54, 147.57, 146.26, 137.32, 133.74, 131.08, 130.74, 128.40, 127.80, 127.78, 127.24, 126.38, 113.40, 110.38, 110.10, 110.05, 107.56, 100.01, 69.92, 65.38, 59.39, 55.95, 55.37, 51.33, 51.13, 29.16, 28.99; ESI-MS m/z: 471 [M+H]+; ESI-HR-MS Calcd. for C29H31N2O4 [M+H]+ 471.2278, Found 471.2288. MeOH/H2O = 70:30 (v/v), 9.86 min, 96.68% purity.

(R)-(3-(Benzyloxy)-2,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol ((R)-22)

Compound 35p was prepared in a similar manner as described for compound 7. 6-(Benzyloxy)-7-methoxy-1-((5-methoxy-1H-indol-3-yl)methyl)-3,4-dihydroisoquinoline (35p, 2 mmol) dissolved in 10 mL of water, and (S,S)-Noyori's catalyst (0.04 mmol), AgSbF6 (0.06 mmol), La(OTf)3 (0.6 mmol), CTAB (2 mmol) and HCOONa (3 mmol) were added. The solution was heated to 40 °C under for 12 h. The reaction mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, and the combined organic phase was evaporated under reduced pressure to get the crude product, which was purified by flash chromatography on silica gel to get (R)-35p (441 g, 1.03 mmol, 51%). (R)-35p (428.5 mg, 1 mmol) was dissolved in 20 mL of acetonitrile, then excess of formaldehyde and 0.5 mL of formic acid were added into solution. The mixture was stirred for 2 h at 80 °C. The pH of the mixture was adjusted to alkalinity with the addition of saturated NaHCO3. The organic layer was separated and washed with water. The combined organic phase was evaporated under reduced pressure and then chromatographed on silica gel to give the target product (R)-22 (197 mg, 41%). Yellow solid, m.p. = 144‒145 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.46 (d, J = 7.7 Hz, 2H), 7.40 (dd, J = 14.8, 7.1 Hz, 2H), 7.34 (dd, J = 8.4, 4.8 Hz, 1H), 7.02–6.96 (m, 2H), 6.82 (s, 1H), 6.73 (dd, J = 8.8, 1.6 Hz, 1H), 6.22 (t, J = 6.9 Hz, 1H), 5.37 (d, J = 6.5 Hz, 2H), 5.06 (s, 2H), 4.17 (d, J = 15.1 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.62 (dd, J = 29.8, 12.5 Hz, 2H), 3.44 (d, J = 14.9 Hz, 1H), 3.16–3.08 (m, 1H), 2.94 (t, J = 12.0 Hz, 1H), 2.61 (dd, J = 24.1, 15.4 Hz, 2H), 2.47–2.38 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 153.55, 147.57, 146.26, 137.32, 133.74, 131.08, 130.73, 128.41, 127.80, 127.24, 126.38, 113.37, 110.39, 110.11, 110.04, 107.57, 100.00, 69.91, 65.38, 59.41, 55.95, 55.37, 51.33, 51.14, 29.18, 29.00; ESI-MS m/z: 471 [M+H]+. MeOH/H2O = 70:30 (v/v), 10.03 min, 95.06% purity.

(S)-(3-(Benzyloxy)-2,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]Pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol ((S)-22)

This compound was prepared by replacement of (S,S)-Noyori's catalyst with (R,R)-Noyori's catalyst using a similar synthetic procedure of (R)-22. Yellow solid, m.p. = 146‒147 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.46 (d, J = 7.0 Hz, 2H), 7.41 (dd, J = 10.0, 4.7 Hz, 2H), 7.35 (dd, J = 8.0, 5.1 Hz, 1H), 6.99 (dd, J = 7.1, 3.2 Hz, 2H), 6.81 (s, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.21 (t, J = 7.0 Hz, 1H), 5.37 (d, J = 6.6 Hz, 2H), 5.06 (s, 2H), 4.17 (d, J = 15.1 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.70–3.55 (m, 2H), 3.49–3.39 (m, 1H), 3.18–3.10 (m, 1H), 3.02–2.87 (m, 1H), 2.72–2.55 (m, 2H), 2.47–2.38 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 153.55, 147.57, 146.26, 137.32, 133.74, 131.08, 130.72, 128.41, 127.80, 127.24, 126.38, 113.37, 110.39, 110.11, 110.03, 107.57, 100.00, 69.91, 65.38, 59.40, 55.94, 55.37, 51.33, 51.14, 29.17, 28.99; ESI-MS m/z: 471 [M+H]+. MeOH/H2O = 70:30 (v/v), 9.87 min, 95.07% purity.

(3-((2-Fluorobenzyl)oxy)-2,12-dimethoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (23)

Compound 23 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 105‒106 °C; Yield: 38%. 1H NMR (400 MHz, DMSO-d6) δ 7.57 (td, J = 7.5, 1.4 Hz, 1H), 7.49–7.40 (m, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.30–7.23 (m, 2H), 6.99 (dd, J = 9.3, 3.1 Hz, 2H), 6.86 (s, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.21 (t, J = 7.1 Hz, 1H), 5.44–5.32 (m, 2H), 5.20–5.01 (m, 2H), 4.17 (d, J = 15.1 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.66 (d, J = 15.0 Hz, 1H), 3.59 (dd, J = 10.3, 3.0 Hz, 1H), 3.43 (dd, J = 14.9, 2.8 Hz, 1H), 3.20–3.09 (m, 1H), 3.02–2.89 (m, 1H), 2.73–2.57 (m, 2H), 2.47–2.35 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 160.41 (d, J = 245.9 Hz), 153.56, 147.56, 146.07, 133.75, 131.09, 131.04, 130.83 (d, JC-F = 4.1 Hz), 130.36 (d, JC-F = 8.2 Hz), 127.25, 126.46, 124.55 (d, JC-F = 3.4 Hz), 124.10 (d, JC-F = 14.7 Hz), 115.37 (d, JC-F = 21.0 Hz), 113.42, 110.40, 110.12, 110.06, 107.57, 100.00, 65.39, 64.14 (d, JC-F = 3.6 Hz), 59.41, 55.91, 55.37, 51.34, 51.13, 29.18, 28.97; ESI-MS m/z: 489 [M+H]+; ESI-HR-MS Calcd. for C29H30FN2O4+ [M+H]+ 489.2184, Found 489.2170. MeOH/H2O = 70:30 (v/v), 10.88 min, 95.00% purity.

(3-((3-Fluorobenzyl)oxy)-2,12-dimethoxy-5,6,14,14a tetrahydroindolo[3′,2′:4,5] pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (24)

Compound 24 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 98‒100 °C; Yield: 33%. 1H NMR (400 MHz, DMSO-d6) δ 7.50–7.39 (m, 1H), 7.38–7.25 (m, 3H), 7.17 (td, J = 8.6, 1.9 Hz, 1H), 7.00 (dd, J = 12.8, 3.5 Hz, 2H), 6.81 (s, 1H), 6.73 (dd, J = 8.8, 2.5 Hz, 1H), 6.21 (t, J = 7.1 Hz, 1H), 5.43–5.29 (m, 2H), 5.09 (s, 2H), 4.16 (d, J = 15.1 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.66 (d, J = 15.0 Hz, 1H), 3.59 (dd, J = 10.5, 3.3 Hz, 1H), 3.43 (dd, J = 15.0, 2.6 Hz, 1H), 3.21–3.05 (m, 1H), 3.18–3.07 (m, 1H), 3.00–2.87 (m, 1H), 2.71–2.57 (m, 2H), 2.48–2.36 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 162.20 (d, JC-F = 243.4 Hz), 153.55, 147.60, 146.00, 140.35 (d, JC-F = 7.4 Hz), 133.74, 131.05 (d, JC-F = 7.9 Hz), 130.46 (d, JC-F = 8.2 Hz), 128.11 (d, JC-F = 61.6 Hz), 127.25, 126.41, 123.54 (d, JC-F = 2.7 Hz), 114.53 (d, JC-F = 20.9 Hz), 114.20 (d, JC-F=21.8 Hz), 113.54, 110.40, 110.12, 110.09, 107.56, 100.00, 69.11, 65.39, 59.40, 55.98, 55.37, 51.33, 51.11, 29.16, 28.98; ESI-MS m/z: 489 [M+H]+; ESI-HR-MS Calcd. for C29H30FN2O4+ [M+H]+ 489.2184, Found 489.2174. MeOH/H2O = 70:30 (v/v), 10.72 min, 96.62% purity.

(2,12-Dimethoxy-3-((3-methylbenzyl)oxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (25)

Compound 25 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 96‒98 °C; Yield: 46%. 1H NMR (400 MHz, DMSO-d6) δ 7.37 (d, J = 8.8 Hz, 1H), 7.31–7.21 (m, 3H), 7.15 (d, J = 7.3 Hz, 1H), 7.01–6.97 (m, 2H), 6.81 (s, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.26 (t, J = 6.9 Hz, 1H), 5.46–5.31 (m, 2H), 5.12–4.92 (m, 2H), 4.18 (d, J = 15.2 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.70‒3.55 (m, 2H), 3.44 (d, J = 14.2 Hz, 1H), 3.13 (d, J = 7.5 Hz, 1H), 3.02‒2.85 (m, 1H), 2.65 (d, J = 14.2 Hz, 2H), 2.47–2.39 (m, 1H), 2.33 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 153.55, 147.57, 146.33, 137.53, 137.21, 133.74, 131.10, 130.63, 128.46, 128.42, 128.33, 127.22, 126.32, 124.96, 113.34, 110.42, 110.13, 109.99, 107.53, 100.00, 69.99, 65.37, 59.41, 55.93, 55.38, 51.32, 51.13, 29.14, 28.99, 21.05; ESI-MS m/z: 485 [M+H]+; ESI-HR-MS Calcd. for C30H33N2O4+ [M+H]+ 485.2435, Found 485.2424. MeOH/H2O = 70:30 (v/v), 15.58 min, 95.08% purity.

(2,12-Dimethoxy-3-((4-methylbenzyl)oxy)-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (26)

Compound 26 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 103‒105 °C; Yield: 43%. 1H NMR (400 MHz, DMSO-d6) δ 7.36 (dd, J = 17.5, 8.5 Hz, 3H), 7.26–7.13 (m, 2H), 7.07–6.95 (m, 2H), 6.80 (s, 1H), 6.73 (dd, J = 8.8, 2.5 Hz, 1H), 6.21 (t, J = 7.0 Hz, 1H), 5.36 (dd, J = 12.3, 10.8 Hz, 2H), 5.00 (s, 2H), 4.17 (d, J = 15.1 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.67 (d, J = 14.5 Hz, 1H), 3.59 (d, J = 9.8 Hz, 1H), 3.47–3.39 (m, 1H), 3.13 (d, J = 6.5 Hz, 1H), 2.94 (t, J = 11.5 Hz, 1H), 2.64 (d, J = 13.1 Hz, 2H), 2.47–2.37 (m, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 153.5, 147.57, 146.31, 137.02, 134.26, 131.09, 128.94, 127.90, 127.22, 126.32, 113.36, 110.39, 110.13, 110.03, 107.54, 100.01, 69.79, 65.38, 59.41, 55.94, 55.37, 51.31, 51.13, 29.13, 28.94. ESI-MS m/z: 485 [M+H]+; ESI-HR-MS Calcd. for C30H33N2O4+ [M+H]+ 485.2435, Found 485.2431. MeOH/H2O = 70:30 (v/v), 14.63 min, 97.92% purity.

(3-(Benzyloxy)-13-chloro-2-methoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (27)

Compound 27 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 150‒152 °C; Yield: 42%. 1H NMR (400 MHz, DMSO-d6) δ 7.49–7.44 (m, 3H), 7.40 (t, J = 7.3 Hz, 2H), 7.35 (d, J = 7.1 Hz, 1H), 7.10–7.00 (m, 2H), 6.95 (s, 1H), 6.82 (s, 1H), 6.39 (t, J = 7.1 Hz, 1H), 5.44 (d, J = 7.0 Hz, 2H), 5.06 (s, 2H), 4.20 (d, J = 15.4 Hz, 1H), 3.78 (s, 3H), 3.75‒3.68 (m, 2H), 3.63 (d, J = 10.0 Hz, 1H), 3.13 (dd, J = 6.6, 3.7 Hz, 1H), 2.98–2.86 (m, 1H), 2.76–2.58 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 147.55, 146.35, 137.31, 137.21, 134.68, 130.58, 128.40, 127.80, 127.76, 126.42, 124.13, 123.94, 121.39, 119.46, 113.54, 110.06, 109.04, 107.27, 69.94, 65.55, 59.09, 55.99, 51.01, 50.61, 30.87, 28.91; ESI-MS m/z:475 [M+H]+; ESI-HR-MS Calcd. for C28H28ClN2O3+ [M+H]+ 475.1783, Found 475.1794. MeOH/H2O = 60:40 (v/v), 6.31 min, 96.58% purity.

(3-(Benzyloxy)-13-bromo-2-methoxy-5,6,14,14a-tetrahydroindolo[3′,2′:4,5]pyrido[2,1-a]isoquinolin-9(8H)-yl)methanol (28)

Compound 28 was prepared in a similar manner as described for compound 7. Yellow solid, m.p. = 163‒165 °C; Yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 7.51 (d, J = 8.1 Hz, 1H), 7.49–7.38 (m, 4H), 7.38–7.30 (m, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.00 (t, J = 7.9 Hz, 1H), 6.94 (s, 1H), 6.83 (s, 1H), 6.40 (t, J = 6.9 Hz, 1H), 5.43 (d, J = 6.5 Hz, 2H), 5.06 (s, 2H), 4.19 (d, J = 15.3 Hz, 1H), 3.82 (s, 1H), 3.78 (s, 3H), 3.70 (d, J = 15.3 Hz, 1H), 3.61 (dd, J = 10.3, 2.7 Hz, 1H), 3.13 (dd, J = 6.4, 3.5 Hz, 1H), 3.00‒2.85 (m, 1H), 2.78‒2.57 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 147.53, 146.35 137.31, 137.08, 134.89, 130.62, 128.42, 127.81, 127.77, 126.42, 125.41, 122.75, 121.77, 113.53, 112.40, 110.01, 109.51, 107.89, 69.94, 65.50, 59.00, 55.95, 51.03, 50.59, 31.06, 28.90; ESI-MS m/z: 519 [M+H]+; ESI-HR-MS Calcd. for C28H28BrN2O3+ [M+H]+ 519.1278, Found 519.1281. MeOH/H2O = 60:40 (v/v), 6.26 min, 96.18% purity.

Pharmacology

Cell culture

The human hepatoma cell line HepG2 (ATCC® HB-8065™, Rockville, USA) cells were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, cat. SH30243.01, Logan, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco, cat. 10099141, Carlsbad, USA) at 37 °C in a 5% CO2 incubator. For the analysis of PCSK9 and LDLR expression or DiI-LDL uptake assay, the medium was replaced with DMEM supplemented with 2% (v/v) lipoprotein-deficient serum (LPDS) after cells were grown to confluence.

Western blot analysis of PCSK9 and LDLR in HepG2 cells

HepG2 cells plated in 12-well plate were treated with various test compounds or berberine (Meilun Biotechnology, cat. MB6000, Dalian, China) in the presence of 2% LPDS for 24 h. Then the cells were lysed in RIPA buffer (Beyotime Biotechnology, cat. P0013B, Shanghai, China) containing protease inhibitor cocktail (Merck Millipore, cat. 539134, Darmstadt, Germany). Total protein in cell lysates equivalent to 15 μg was separated by gel electrophoresis using 8% SDS-PAGE and then transferred to a PVDF membrane for protein blotting. Non-specific binding sites were blocked in 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad, cat. 170-6404, Hercules, USA) for 2 h. The membranes were incubated with antibodies specific for PCSK9 (Abcam, cat. ab181142, Cambridge, UK; 1:2000), LDLR (Proteintech, cat. 10785-1-AP, Wuhan, China; 1:2500) and β-actin (CST, cat. #3700, Danvers, MA, USA; 1:1000), respectively, at 4 °C overnight. After incubation with the primary antibodies, the membranes were washed in TBST (Tris-buffered saline, 0.1% Tween 20) 3 times and incubated with the secondary antibodies at room temperature for another 2 h. All bands were visualized with an ECL kit (Bio-Rad, cat. 170-5061, Hercules, USA) and chemiluminescence was detected on a ChemiDoc XRS (Bio-Rad). ImageJ (NIH, 1.50i, Rockville, MD, USA) was used for the quantification of band intensities.

DiI-LDL uptake assay

HepG2 cells plated in 24-well plate were used to implement DiI-LDL uptake assay. In general, after incubation with test compounds or berberine in the presence of 2% LPDS for 24 h, cell culture medium was changed to DMEM supplemented with 2% LPDS and 20 μg/mL DiI-LDL. Following another 4 h co-incubation with DiI-LDL, HepG2 cells were washed with PBS containing 0.4% albumin 2 times, and then PBS (phosphate buffered saline) 3 times. For the fluorescence quantification, 400 μL of isopropanol was added into each well of the 24-well plate, and the plate was gently shaken on a shaker for 20 min at room temperature. Then 200 μL of isopropanol in each well was used for the analysis with a SpectraMax M5e Microplate Reader (Molecular Devices, San Jose, USA; excitation wavelength of 520 nm, emission wavelength of 570 nm). The remaining cells were lysed in NaOH (0.5 mol/L) for protein determination. DiI-LDL was prepared as previously described.

Calcium mobilization assay

HEK293 cells stably expressing β1-AR/Gα16, D1/Gα16 or D2/Gα16 were seeded onto 96-well plates and incubated for 24 h. Cells were loaded with 2 μmol/L Fluo-4 AM in Hanks balanced salt solution (HBSS, containing KCl 5.4 mmol/L, Na2HPO4 0.3 mmol/L, KH2PO4 0.4 mmol/L, NaHCO3 4.2 mmol/L, CaCl2 1.3 mmol/L, MgCl2 0.5 mmol/L, Mg2SO4 0.6 mmol/L, NaCl 137 mmol/L, BSA 5 g/L, glucose 5.6 mmol/L, sulfinpyrazone 250 μmol/L, pH 7.4) at 37 °C for 45 min. The excess dye was removed and 50 μL HBSS containing test compounds were added. After incubation at room temperature for 10 min, 25 μL HBSS containing the respective agonists were dispensed into the well using a FlexStation III microplate reader (Molecular Devices, San Jose, CA, USA) and intracellular calcium change was recorded with an excitation wavelength of 485 nm and emission wavelength of 525 nm. The half maximal inhibitory concentrations (IC50) of compounds were determined with GraphPad Prism software by constructing their dose–response curves.

Whole-cell binding assay

CHO–K1 cells stably expressing 5-HT1B were seeded at a density of 3 × 104 cells/well into 96-well culture plates and incubated overnight at 37 °C in 5% CO2, and radioligand binding assay was carried out after seeding for 24 h. For homogeneous binding, the cells were incubated in binding buffer with constant concentration of 3H-GR125743 (1 nmol/L) or 3H-Mesulergine (1 nmol/L) and different concentrations of unlabeled compounds (1.28 nmol/L–100 μmol/L) at 4 °C overnight. Cells were washed three times with ice-cold PBS and lysed by 50 μL lysis buffer (PBS supplemented with 20 mmol/L Tris–HCl, 1% Triton X-100, pH 7.4). The plates were subsequently counted for radioactivity (counts per minute, CPM) in a scintillation counter (MicroBeta Plate Counter, PerkinElmer, Waltham, MA, USA) using a scintillation cocktail (PerkinElmer, cat. 1200-439, Waltham, MA, USA).

Pharmacokinetic profiles in male hamsters

Compound 22 was subjected to PK studies on male hamsters (n = 3, each group). Hamsters in oral administration group were given 20 mg/kg test compound (0.5% carboxymethylcellulose sodium) by oral gavage. Hamsters in intravenous administration group were given 10 mg/kg test compound (5% DMSO, 10% cremophor EL in 85% saline) by intravenous administration. Blood samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h after oral or intravenous administration. Serum samples were isolated by centrifuging for 10 min at 4000×g. The test compound concentrations in serum were measured by LC/MS/MS.

In vivo cholesterol-lowering assay

Male Syrian hamsters with body weights of 100–120 g were purchased from Beijing Vital River Laboratory Animal Technology and housed under controlled temperature (22±2 °C) and lighting (12 h light/dark cycle). All hamsters had free access to food and water. After 7 days of acclimatization, hamsters were divided into normal control group (n = 7) and HFD group (n = 21). Then the HFD group was switched into a high-fat diet (0.5% cholesterol; Research Diets, Inc. New Brunswick, NJ, USA) to induce hyperlipidemia. One week later, hamsters of the HFD group were divided into HFD control group and test groups randomly (n = 7 per group). Test groups were given a daily dose test compound 22 30 mg/kg or fenofibrate 100 mg/kg by oral gavage for 21 days. The control groups were given a vehicle (0.5% carboxymethylcellulose sodium).

At the end of the animal experiment, blood samples were collected from all the hamsters under anesthesia after 15 h of fasting (5 p.m.–8 a.m.). Serum was isolated by centrifuging for 10 min at 4000×g, and TC and LDL-C levels were measured using commercially available kits purchased from Maccura Biotechnology (Guangzhou, China).

The study was conducted in accordance with guidelines and ethics of Institutional Animal Care and Use Committee (IACUC), Shanghai Institute of Materia Medica, Chinese Academy of Sciences and the Ethics Committee of Shanghai Xuhui Central Hospital (China).

hERG testing using FluxOR™ thallium assay

Step 1: Growing cells. CHO-hERG-ZG cells are grown in 75 cm2 flask with complete medium with 100 μg/mL G418 and 100 μg/mL hygromycin B until 80%–90% confluency. Wash cells with PBS once. Incubate cells with 1 mL 0.25% trypsin until all cells are rounded and can be easily dislodged from the surface. Add 10 mL complete medium to stop trypsin activity. Disassociate cells by thoroughly and repetitively pipetting. Transfer them to 50 mL Falcon tube and spin down at 1000 rpm (Labofuge 400 Centrifuge, Thermo Fisher, Germany) for 5 min. Aspirate medium and resuspend cells using a small volume of complete medium, like 0.5 mL. Count cell density.

Step 2: Cell seeding. CHO-hERG-ZG cells are plated into 96-well plates and after plating, tap plates on sides to separate cells and let plates sit in the dark at RT for 30 min before incubation at 37 °C for 16‒18 h. Cells will reach 80% confluence. After overnight incubation, the media of cells are changed in loading buffer (old media is tapped out) and incubated in the dark at RT for 90 min. Remove the loading buffer and replace with assay buffer. Compounds were added to the cell plate. The cell plate is incubated with compound 22 for 20 min in the dark at RT. Load the cell plates on Functional Drug Screening System (FDSS). Fluorescent signals will be recorded every 2 s till 10 s. At 10 s, stimulus buffer will be added to cells. Then fluorescent signals will be recorded every second till 180 s on FDSS.

Real-time PCR

HepG2 cells were treated with the test compound in the presence of 2% LPDS for 24 h. The total RNA was isolated by Trizol (Invitrogen, cat. 10296010, Leawood, KA, USA) and reverse-transcribed to cDNA by reverse transcriptase (Promega, cat. M1705, Madison, USA). The expression of PCSK9 mRNA was assessed by real-time PCR using specific primers and SYBR® Green Supermix (Bio-Rad, cat. 1725124, Hercules, CA, USA). Primer sequences of PCSK9 gene are 5′-CCAAGCCTCTTCTTACTTCACC-3′ and 5′-GCATCGTTCTGCCATCACT-3′.

Statistical analysis

Data were analyzed with GraphPad Prism software. Results are presented as mean ± SEM. Nonlinear regression analysis was used to generate the dose–response curve and calculate a concentration of 50% inhibition (IC50) value. Differences between treatment groups were assessed by Student's t-test or one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.