Antiproliferative Dimeric Aporphinoid Alkaloids from the Roots of

Article Cite This: J. Nat. Prod. 2017, 80, 2893-2904

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Antiproliferative Dimeric Aporphinoid Alkaloids from the Roots of Thalictrum cultratum Da-Hong Li,†,‡,§ Jian-Yong Li,†,‡,§ Chun-Mei Xue,†,‡ Tong Han,†,‡ Chun-Mei Sai,†,‡ Kai-Bo Wang,‡ Jin-Cai Lu,‡ Yong-Kui Jing,† Hui-Ming Hua,†,‡ and Zhan-Lin Li*,†,‡ †

Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and ‡School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China S Supporting Information *

ABSTRACT: Inspired by the intriguing structures and bioactivities of dimeric alkaloids, 11 new thalifaberine-type aporphinebenzylisoquinoline alkaloids, thalicultratines A−K, a tetrahydroprotoberberine-aporphine alkaloid, thalicultratine L, and five known ones were isolated from the roots of Thalictrum cultratum. Their structures were defined on the basis of NMR and HRESIMS data. The antiproliferative activities of compounds 1−17 were evaluated against human leukemia HL-60 and prostate cancer PC-3 cells. Most alkaloids showed potent cytotoxicity against selected cancer cells. Preliminary SARs are discussed. The most active new compound (3), with an IC50 value of 1.06 μM against HL-60 cells, was selected for mechanism of action studies. The results revealed that compound 3 induced apoptosis and arrested the HL-60 cell cycle at the S phase with the loss of mitochondria membrane potential. The nuclear morphological Hoechst 33258 staining assay was also carried out, and the results confirmed apoptosis.

P

tetrandrine in regulating tumor cellular functions were also reported, including the inhibition of angiogenesis, proliferation, migration, and invasion; the induction of autophagy and apoptosis; the reversal of multidrug resistance (MDR); and the enhancement of radiation sensitization.14−20 Novel structures found from Thalictrum are sure to greatly enrich the diversity of the compound library of dimeric alkaloids. Inspired by the intriguing structures and diverse bioactivities of the dimeric alkaloids,21−29 investigation of the alkaloids of Thalictrum cultratum Wall led to the isolation of the new thalicultratines A−L (1−3, 7−10, 12−16) and the known thalifaronine (4),3 thalifaberine (5),6 thalifabatine (6),6 dehydrothalifaberine (11),6 and thalibealine (17).10 Herein, the isolation and structural elucidation of the new alkaloids as well as their antiproliferative activities toward human leukemia HL-60 and prostate cancer PC-3 cells are described. Compound 3 was selected for further cellular mechanism studies in the HL-60 cell line. The cell cycle effect, apoptosis-

lants in the genus Thalictrum L. (Ranunculaceae) comprise about 200 species and are widely distributed in Asia, Europe, Africa, and North and South America. There are 67 species throughout China, the majority of which originate from southwest China.1 Approximately 30 species have been used traditionally as folk medicines, especially in Inner Mongolia and Tibet regions.2 These species display diverse biological effects, such as antitumor, anti-inflammatory, antihypertension, antifungal, antisilicosis, and antiplatelet aggregation activities, and are used for the treatment of cancer, esoenteritis, dysentery, arthritis, jaundice, and gingivitis in clinical applications.3 The main active components from the genus are isoquinoline alkaloids, including simple isoquinoline, benzylisoquinoline, aporphinoid, protoberberine, morphine, pavine, and phenanthrene alkaloids and their dimers.4−12 The dimeric alkaloids isolated from Thalictrum include bisbenzylisoquinoline, aporphine-benzylisoquinoline, protoberberine-benzylisoquinoline, aporphine-protoberberine, and pavine-aporphine types. For example, tetrandrine is a well-known natural bisbenzylisoquinoline alkaloid, which was recently found to inhibit Ebola virus infection in vitro and in vivo.13 The important roles of © 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 3, 2017 Published: November 13, 2017 2893

DOI: 10.1021/acs.jnatprod.7b00387 J. Nat. Prod. 2017, 80, 2893−2904

Journal of Natural Products

Article

Chart 1

correlations of H-11 (δH 8.02) to C-1a (δC 128.1), C-7a (123.4), C-8 (144.9), C-9 (141.5), and C-10 (152.0), of H-3 (δH 6.62) to C-1 (δC 144.8), C-1b (127.2), C-2 (152.1), and C4 (δC 29.0), of N-methyl (δH 2.35) to C-5 (δC 53.2) and C-6a (62.2), and from the O-methyl protons at δH 3.90, 3.89, 3.80, and 3.71 to C-10, C-2, C-9, and C-1, respectively. The structure of fragment A was deduced by the HMBC cross-peaks from the proton signal at δH 7.00 (2H, d, J = 8.4 Hz) to C-α (δC 40.3) and C-11′ or C-13′ (115.1), the signal at δH 6.78 (2H, d, J = 8.4 Hz) to C-10′ (or C-14′) and C-12′ (δC 157.4), H-8′ at δH 5.55 to C-1′ (δC 65.3), C-4′a (130.5), C-6′ (134.2), and C-7′ (150.0), and the N-methyl at δH 2.63 to C-1′ (δC 65.3) and C3′ (45.1), as well as from the O-methyl signals at δH 3.84 and 3.51 to C-6′ and C-7′, respectively. The locations of the Omethyl groups were defined by the NOE associations of H-3/2OMe, 1-OMe/H-11/10-OMe, and 7′-OMe/H-8′. The ether linkage between C-8 of the aporphine moiety and C-12′ of the benzylisoquinoline moiety was defined by their deshielded chemical shifts and supported by the NOE correlations of 9OMe/H-11′, H-13′. The (6aS, 1′S) absolute configuration was defined by the electronic circular dichroism (ECD) spectrum, which showed positive and negative Cotton effects at 238 and 281 nm, respectively (Figure S1, Supporting Information), identical to that of (6aS,1′S)-thalifaberine (5).6 Therefore, the structure of thalicultratine A (1) was defined as shown. Compound 2, thalicultratine B, was obtained as a yellowish, amorphous powder. Its molecular formula was deduced as C42H50N2O9 from a positive HRESIMS ion at m/z 727.3587 [M + H]+ (calcd 727.3589), indicative of two additional Omethyl groups compared to 1. The 1H and 13C NMR data (Table 1) of 2 were similar to those of 1, except for the absence

inducing ability, and influence on the mitochondria membrane potential in HL-60 cells were assessed. The nuclear morphological evaluation was also carried out using the Hoechst 33258 staining assay to confirm apoptosis.

■

RESULTS AND DISCUSSION Compound 1, thalicultratine A, was obtained as a yellowish amorphous powder. It showed a positive reaction to the Dragendorff reagent (BiI3·KI), indicating 1 as an alkaloid. Its molecular formula C40H46N2O8, with 19 indices of hydrogen deficiency, was defined based on a protonated molecular ion at m/z 683.3325 [M + H]+ (calcd 683.3327) in the HRESIMS. The 13C NMR data of 1 showed 40 resonances (Table 1) comprising 24 aromatic carbons and 16 aliphatic carbons, including two N-methyl groups at δC 43.7 (6-NMe) and 41.4 (2′-NMe) and six aromatic O-methyl groups at δC 61.1 (9OMe), 61.0 (6′-OMe), 60.5 (1-OMe), 56.1 (10-OMe), 55.9 (2OMe), and 55.6 (7′-OMe). The above data revealed that 1 was a dimeric benzylisoquinoline alkaloid. The 1H NMR data of 1 exhibited a deshielded aromatic proton signal at δH 8.02 (1H, s) characteristic of H-11 of an aporphine moiety, an AA′BB′ spin system at δH 7.00 (2H, d, J = 8.4 Hz) and 6.78 (2H, d, J = 8.4 Hz), and a shielded aromatic proton signal at δH 5.55 (1H, s) characteristic of H-8′ of a benzylisoquinoline moiety. Additionally, six aromatic O-methyls at δH 3.90, 3.89, 3.84, 3.80, 3.71, and 3.51 and two N-methyl proton resonances at δH 2.63 and 2.35 were observed. On the basis of the above data, 1 was deduced as a thalifabenine-type aporphine-benzylisoquinoline alkaloid.4,5 The 2D structure of 1 was elucidated by analyses of the HMBC and NOESY data as shown in Figures 1 and 2. The structure of fragment B was assigned based on the long-range 2894



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Table 1. 1H NMR and 13C NMR Data for Compounds 1−3 in CDCl3 1a position 1 1a 1b 2 3 3a 4 5 6-NMe 6a 7 7a 8 9 10 11 11a 1′ 2′-NMe 3′ 4′ 4′a 5′ 6′ 7′ 8′ 8′a α 9′ 10′ 11′ 12′ 13′ 14′ 1-OMe 2-OMe 3-OMe 9-OMe 10-OMe 5′-OMe 6′-OMe 7′-OMe −OCH2O− a1

δH (J in Hz)

6.62, s 3.16, m, 2.67, m 3.02, m, 2.49, m 2.35, s 2.90, m 3.23, m, 2.06, m

8.02, s 3.82, m 2.63, s 3.35, m, 3.02, m 2.90, m

5.55, s 3.46, m, 2.79, m 7.00, d (8.4) 6.78, d (8.4) 6.78, d, (8.4) 7.00, d (8.4) 3.71, s 3.89, s

2b,c δC 144.8 128.1 127.2 152.1 111.2 128.9 29.0 53.2 43.7 62.2 26.8 123.4 144.9 141.5 152.0 109.6 126.6 65.3 41.4 45.1 29.8 130.5 146.7 134.2 150.0 103.6 113.0 40.3 131.7 131.2 115.1 157.4 115.1 131.2 60.5 55.9

3.80, s 3.90, s

61.1 56.1

3.84, s 3.51, s

61.0 55.6

H NMR (600 MHz). MHz).

b1

δH (J in Hz)

2.82, m, 2.74, m 2.95, m, 2.31, m 2.27, s 2.82, m 3.20, m, 1.98, m

7.84, s 3.60, m 2.43, s 3.07, m, 2.74, m 2.68, m, 2.50, m

5.80, s 3.07, m, 2.68, m 6.93, d (8.4) 6.73, d (8.4) 6.73, d (8.4) 6.93, d (8.4) 3.74, s 3.90, s 3.83, s 3.74, s 3.87, s 3.78, s 3.76, s 3.49, s

3a,c δC 149.6 127.8 123.0 145.1 150.1 131.0 23.5 52.7 43.7 62.3 26.4 122.1 144.6 141.0 151.8 109.0 122.8 65.0 42.3 45.9 19.6 132.5 150.6 140.1 150.6 107.0 119.6 39.9 133.0 130.7 114.7 157.0 114.7 133.0 60.8 60.9 60.3 60.7 56.0 60.3 60.6 55.5

H NMR (400 MHz).

δH (J in Hz)

2.82, m, 2.75, m 2.97, m, 2.32, m 2.29, s 2.82, m 3.22, m, 1.99, m

7.86, s 3.61, s 2.46, s 3.14, m, 2.75, m 2.68, m, 2.43, m

5.64, s 3.07, m, 2.68, m 6.94, d (8.4) 6.75, d (8.4) 6.75, d (8.4) 6.94, d (8.4) 3.76, s 3.92, s 3.85, s 3.76, s 3.89, s

3.55, s 5.87, s c13

δC 149.5 127.8 123.0 145.1 150.1 131.0 23.5

Figure 1. Key HMBC correlations of compound 1.

52.7 43.7 62.3 26.5 122.0 144.6 141.0 151.7 108.9 122.9 64.8 42.2 44.9

Figure 2. Key NOESY correlations of compound 1.

the O-methyl signal at δH 3.83 and H2-4 at δH 2.82, 2.74 to C-3 (δC 150.1) and from the O-methyl signal at δH 3.78 and H2-4′ at δH 2.68, 2.50 to C-5′ (δC 150.6). The absolute configuration of 2 was determined as (6aS, 1′S) by the similarity of its ECD spectrum to that of (6aS,1′S)-thalifaberine (5).6 Compound 3, thalicultratine C, was obtained as a yellowish, amorphous powder with the molecular formula C41H46N2O9, as suggested by the HRESIMS data. The 1H and 13C NMR spectra of 3 exhibited similar signals to 2 except for a methylenedioxy group at δH 5.87 (2H, s) in 3 instead of the two O-methyl groups in 2. Analyses of the NOESY, HSQC, and HMBC (Figure S3, Supporting Information) data unambiguously confirmed the above deduction. The HMBC spectrum of 3 revealed that the protons at δH 5.87 (2H, s) correlated with C-5′ (δC 145.6) and C-6′ (132.8), and H2-4′ at δH 2.68 and 2.43 with C-5′, suggesting that the methylenedioxy group was located at C-5′ and C-6′. The (6aS, 1′S) absolute configuration was again defined by the Cotton effects in the ECD spectrum at λmax (Δε): 241 (+21.07), 280 (−3.93) nm (Figure S3). Compound 7, thalicultratine D, was obtained as a yellowish, amorphous powder. The HRESIMS data showed an [M + H]+ ion at m/z 665.3228 (calcd 665.3221), establishing the molecular formula as C40H44N2O7, i.e., 2 amu less than that of thalifaronine (4).3 The 1H and 13C NMR data (Table 2) showed similarity with those of 4, except for the one aromatic proton at δH 6.60 in 7 replacing the three aliphatic protons in 4 and two additional sp2 carbons at δC 142.6 (C-6a) and 94.6 (C7) characteristic of dehydrothalifaberine (11),5 suggesting that 7 is a dehydro derivative of 4. Compound 7 was unambiguously defined as the 6a,7-dehydro derivative of 4, based on the HMBC cross-peaks from H-7 (δH 6.60) to C-1b (δC 119.0), C6a (142.6), and C-8 (140.9) and from H-11 (δH 9.09) to C-1a (δC 124.9), C-7a (120.9), C-9 (141.9), and C-10 (149.0) as well as the long-range couplings of the O-methyl protons at δH 4.01, 4.00, 3.92, 3.88, 3.80, and 3.32 to C-2 (δC 151.0), C-10 (149.0), C-1 (145.0), C-9 (141.9), C-6′ (148.7), and C-7′ (147.0), respectively (Figure 3). The (1′S) absolute configuration was defined by the ECD spectrum [λmax (Δε) nm: 197

18.6 131.1 145.6 132.8 141.1 106.5 109.0 40.0 132.9 130.7 114.6 156.9 114.6 130.7 60.8 60.8 60.2 60.8 56.0

56.0 101.2

C NMR (150

of an aromatic proton and the presence of two more O-methyl groups. Analyses of the HSQC and HMBC (Figure S2, Supporting Information) data assigned the location of the Omethyl groups at C-3 and C-5′ via the HMBC cross-peaks from 2895



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Table 2. 1H NMR and 13C NMR Data for Compounds 7−10 in CDCl3a 7 position 1 1a 1b 2 3 3a 4 5 6-NMe 6a 7 7a 8 9 10 11 11a 1′ 2′-NMe 3′ 4′ 4′a 5′ 6′ 7′ 8′ 8′a α 9′ 10′ 11′ 12′ 13′ 14′ 1-OMe 2-OMe 3-OMe 9-OMe 10-OMe 5′-OMe 6′-OMe 7′-OMe −OCH2O− a1

δH (J in Hz)

8 δC

6.85, d (8.6) 6.94, d (8.6) 3.92, s 4.01, s

145.0 124.9 119.0 151.0 111.3 125.2 31.3 50.4 40.4 142.6 94.6 120.9 140.9 141.9 149.0 107.1 121.1 65.4 40.6 45.2 22.8 122.2 111.1 148.7 147.0 111.3 123.3 40.4 129.9 131.4 115.7 158.2 115.7 131.4 60.2 56.5

3.88, s 4.00, s 3.80, s 3.32, s

6.99, s 3.29, m, 3.21, m 3.29, m 2.87, s 6.60, s

9.09, s 4.02, m 2.77, s 3.54, m, 3.21, m 3.00, m, 2.92, m 6.56, s

5.67, s 3.76, m, 3.00, m 6.94, d (8.6) 6.85, d (8.6)

δH (J in Hz)

9 δC

61.1 55.9

6.84, d (8.6) 6.97, d (8.6) 3.99, s 4.07, s 3.94, s 3.88, s 4.03, s

150.5 121.1 121.0 146.0 148.1 121.7 24.2 49.9 40.5 142.4 96.2 124.1 141.1 141.5 149.3 106.4 121.7 65.3 41.7 45.1 18.5 131.0 146.6 133.9 149.6 103.6 112.1 40.3 132.2 131.0 115.3 157.7 115.3 131.0 60.6 61.4 60.9 61.1 56.0

56.0 55.5

3.80, s 3.41, s

61.0 55.5

3.20, m 3.25, m 2.87, s 6.72, s

9.00, s 3.75, m 2.57, s 3.29, m, 2.96, m 2.77, m, 2.64, m

5.49, s 3.35, m, 2.71, m 6.97, d (8.6) 6.84, d (8.6)

δH (J in Hz)

3.21, m 3.25, m 2.88, s 6.74, s

8.99, s 3.66, m 2.52, s 3.14, m, 2.77, m 2.77, m, 2.54, m


10 δC 150.5 121.1 121.0 146.0 148.1 121.7 24.3 49.9 40.5 142.4 96.3 124.2 141.1 141.6 149.4 106.4 121.8 65.3 42.4 46.1 19.8 132.1 150.8 140.4 150.7 107.2 119.5 40.1 132.7 131.0 115.3 157.6 115.3 131.0 60.6 61.4 60.9 61.1 56.1 60.5 60.9 55.6

δH (J in Hz)

3.33, m, 3.20, m 3.25, m 2.88, s 6.72, s

9.00, s 3.78, m 2.60, s 3.33, m, 2.98, m 2.80, m, 2.63, m

5.54, s 3.33, m, 2.71, m 6.94, d (8.6) 6.84, d (8.6) 6.84, d (8.6) 6.94, d (8.6) 3.99, s 4.08, s 3.94, s 3.89, s 4.03, s

3.46, s 5.94, d (1.2) 5.93, d (1.2)

δC 150.5 121.2 121.1 146.1 148.2 121.7 24.3 49.9 40.5 142.4 96.3 124.2 141.1 141.6 149.4 106.4 121.8 65.3 41.7 44.7 18.1 132.5 146.0 133.6 141.5 107.0 107.5 40.4 133.5 131.1 115.4 157.8 115.4 131.1 60.7 61.4 60.9 61.1 56.1

56.1 101.7

H NMR (600 MHz). 13C NMR (150 MHz).

thalifaberine (11).6 Therefore, the structure of 7 was determined as shown. Compound 8, thalicultratine E, was obtained as a yellowish, amorphous powder. The HRESIMS showed an [M + H]+ ion at m/z 711.3276 (calcd 711.3276), indicating the molecular formula as C41H46N2O9. Comparison of the 1H and 13C NMR data of 8 (Table 2) with those of 7 and thalifabatine (6)5 suggested 8 as the 6a,7-dehydro derivative of 6. The analyses of the HMBC and NOESY (Figure S5, Supporting Information) data unambiguously confirmed the above conclusion. The (1′S) absolute configuration of 8 was determined by the ECD spectrum [λmax (Δε) nm: 193 (−25.84), 230 (+11.28), 264


(−21.18), 213 (+13.02), 234 (+13.60), 286 (+1.75)] (Figure S4, Supporting Information), identical to that of S-dehydro2896



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Table 3. 1H NMR and 13C NMR Data for Compounds 12−15 in CDCl3 12a,c position 1 1a 1b 2 3 3a 4 5 6-NMe 6a 7 7a 8 9 10 11 11a 1′ 2′-NMe 3′ 4′ 4′a 5′ 6′ 7′ 8′ 8′a α 9′ 10′ 11′ 12′ 13′ 14′ 1-OMe 2-OMe 3-OMe 9-OMe 10-OMe 5′-OMe 6′-OMe 7′-OMe a1

δH (J in Hz)

2.99, m, 2.77, m 2.99, m, 2.34, m 2.29, s 2.82, m 3.20, m, 2.00, m

7.89, s 4.72, d (8.6) 3.31, s 3.73, m 3.20, m, 2.90, m

6.09, s 3.95, m, 2.77, m 7,11, d (8.5) 6.83, d (8.5) 6.83, d (8.5) 7.11, d (8.5) 3.79, s 3.95, s 3.88, s 3.77, s 3.91, s 3.87, s 3.79, s 3.48, s

H NMR (600 MHz).

b1

13b,c δC 149.7 122.9 128.2 145.3 150.4 130.6 23.8 52.8 43.9 62.5 26.7 122.2 144.6 141.0 151.9 109.4 123.3 78.1 52.9 62.6 21.3 116.4 150.7 141.2 152.2 106.6 128.7 37.3 131.3 130.3 115.7 157.8 115.7 130.3 60.9 61.1 60.6 61.0 56.3 60.5 60.9 55.9

H NMR (400 MHz).

14a,c

δH (J in Hz)

2.95, m, 2.75, m 2.95, m, 2.30, m 2.32, s 2.80, m 3.20, m, 2.01, m

7.89, s 4.42, d (11.6) 3.36, s 3.76, m 3.21, m, 2.88, m


δC

δH (J in Hz)

149.7 122.9 128.2 145.3 150.4 130.5 23.8 52.8 43.9 62.5 26.7 122.2 144.6 141.0 151.9 109.4 123.3 78.1 55.5 59.0 21.3 115.3 150.7 141.2 152.2 106.6 128.7 37.8 131.3 130.4 115.7 157.8 115.7 130.4 60.9 61.1 60.6 61.1 56.3 60.5 60.9 55.9

15b,c δC

6.83, d (8.2) 7.15, d (8.2) 3.79, s 3.95, s 3.88, s 3.77, s 3.92, s

149.8 122.9 128.2 145.3 150.4 131.3 23.7 52.9 43.9 62.5 26.7 122.2 144.6 141.1 152.0 109.4 123.3 78.3 52.2 63.1 21.2 110.5 147.1 135.1 150.9 102.8 128.9 36.8 131.0 130.3 115.7 157.8 115.7 130.3 61.1 61.1 60.5 61.0 56.3

3.80, s 3.47, s

60.9 55.7

2.84, m, 2.78, m 3.00, m, 2.35, m 2.29, s 2.84, m 3.20, m, 2.01, m

7.89, s 4.75, d (8.6) 3.31, s 3.15, m 3.20, m, 2.88, m

5.92, s 3.98, m, 2.78, m 7,15, d (8.2) 6.83, d (8.2)

δH (J in Hz)

δC

6.77, d (8.2) 6.97, d (8.2) 3.79, s 3.95, s 3.88, s 3.79, s 3.92, s

149.8 123.0 128.2 145.3 150.4 131.3 23.7 52.9 43.9 62.5 26.7 122.2 144.7 141.1 152.0 109.3 123.3 78.8 55.4 58.9 21.7 107.9 147.6 135.3 150.4 103.6 128.5 37.8 131.0 131.7 115.2 157.8 115.2 131.7 61.1 61.1 60.5 61.0 56.3

3.83, s 3.40, s

60.9 55.6

2.83, m, 2.78, m 3.00, m, 2.36, m 2.31, s 2.83, m 3.21, m, 2.01, m

7.88, s 4.40, d (11.6) 3.28, s 3.00, m 3.11, m, 2.92, m

5.25, s 3.61, m, 2.78, m 6.97, d (8.2) 6.77, d (8.2)

c13

C NMR (100 MHz).

Compound 10, thalicultratine G, was obtained as a yellowish, amorphous powder with the molecular formula C41H44N2O9, as suggested by the HRESIMS ion at m/z 709.3119 [M + H]+ (calcd 709.3120). The deshielded proton signal at δH 8.99 (1H, s, H-11), the characteristic signal of 6a,7-dehydroaporphine, suggested that 10 was the 6a,7-dehydro derivative of 3, which was confirmed by the long-range couplings of H2-4′ δH 2.80 (1H, m), 2.63 (1H, m) to C-5′ (δC 146.0) and of the methylenedioxy protons at δH 5.94 (1H, d, J = 1.2 Hz), 5.93 (1H, d, J = 1.2 Hz) to C-5′ and C-6′ (δC 133.6). The (1′S) absolute configuration was defined by the ECD spectrum [λmax (Δε) nm: 196 (−5.98), 209 (+1.34), 231 (+30.58), 281 (+4.90), 333 (+0.94)] (Figure S7, Supporting Information), identical to that of 7.

(+5.22)] (Figure S5, Supporting Information), identical to that of 7. Compound 9, thalicultratine F, was obtained as a yellowish, amorphous powder. Its molecular formula was deduced as C42H48N2O9 from the HRESIMS ion at m/z 725.3432 [M + H]+ (calcd 725.3433). The 1H and 13C NMR spectra showed similar signals to those of 8 except for the presence of an additional O-methyl group, which was located at C-5′ by the HMBC cross-peak between the O-methyl signal at δH 3.81 and C-5′ (δC 150.8). Therefore, 9 was defined as the 6a,7-dehydro derivative of 2. The (1′S) absolute configuration was defined by the ECD spectrum [λmax (Δε) nm: 190 (−20.21), 213 (+15.99), 233 (+23.43), 283 (+9.51), 331 (+2.00)] (Figure S6, Supporting Information), identical to that of 7. 2897



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(Figure S11, Supporting Information).6 Thus, the (2′S) absolute configuration was assigned. Compound 16, thalicultratine L, was obtained as a yellowish, amorphous powder. The HRESIMS data showed an [M + H]+ ion at m/z 737.3098 (calcd 737.3069), suggesting the molecular formula as C42H44N2O10. The 1H NMR data (Table 4) exhibited six aromatic proton singlets at δH 8.09,

Compound 12, thalicultratine H, was obtained as a yellowish, amorphous powder. The HRESIMS data showed an [M + H]+ ion at m/z 743.3553 (calcd 743.3538), which was suggestive of a molecular formula of C42H50N2O10 with one additional oxygen atom compared to 2.6 Comparison of the NMR data of 12 (Table 3) with those of 2 showed significant downfield shifts of carbon and proton signals at C-1′, C-3′, and 2′-NMe, suggesting that 12 is the 2′-N-oxide derivative of 2.7−9 The H1′ resonance at δH 4.72 (1H, d, J = 8.6 Hz) showed an NOE correlation with 2′-NMe at δH 3.31, proving that 2′-NMe and H-1′ were cofacial. The (6aS, 1′S) absolute configuration was defined by the ECD spectrum [λmax (Δε) nm: 204 (−16.68), 245 (+39.69)] (Figure S8, Supporting Information), identical to that of 5.6 Thus, the absolute configuration of N-2′ is R. Compound 13, thalicultratine I, was obtained as a yellowish, amorphous powder. The HRESIMS data showed the same molecular formula, C42H50N2O10, as 12 by an [M + H]+ ion at m/z 743.3545 (calcd 743.3538). Comparison of the 1H and 13C NMR data (Table 3) with those of 12 showed similar signals except for the shielded chemical shifts of H-1′ at δH 4.42 (1H, d, J = 11.6 Hz) and H-8′ at 5.44 (1H, s), as well as the shielded signal of C-3′ (δC 59.0) and the deshielded signal of 2′-NMe (δC 55.5), indicating that 13 and 12 possess different absolute configurations at N-2′. No coupling of 2′-NMe with H-1′ was observed in the NOESY spectrum, confirming the 2′β-N-oxide orientation. The (6aS, 1′S) absolute configuration was determined by the ECD spectrum [Δε (λmax) nm: 205 (−16.53), 243 (+45.10)] (Figure S9, Supporting Information), identical to that of 5.6 Therefore, the absolute configuration of N-2′ is S. Compound 14, thalicultratine J, was obtained as a yellowish, amorphous powder. Its molecular formula was deduced as C41H48N2O10 from an ion at m/z 729.3393 [M + H]+ (calcd 729.3382). The 1H and 13C NMR data indicated the absence of the 5′-O-methyl group compared to 12. Key HMBC crosspeaks of compound 14 are shown in Figure 4. The almost

Table 4. 1H NMR and 13C NMR Data for Compound 16 in CDCl3a position 1 1a 1b 2 3 3a 4 5 6-NMe 6a 7 7a 8 9 10 11 11a 1′ 2′ 3′ 4′ 4′a a1

δH (J in Hz)

2.92, m, 2.80, m 3.13, m, 2.43, m 2.50, s 3.00, m 2.43, m 6.41, s

8.09, s 7.17, s

6.70, s

δC

position

δH (J in Hz)

δC

149.6 122.3 123.0 145.4 150.1 122.7 23.5 52.9 43.8 62.5 33.6 129.1 115.1 146.9 147.7 112.7 126.7 108.3 150.3 148.5 110.5 128.4

5′ 6′ 7′ 8′ 8′a 9′ 10′ 11′ 12′ 12′a 13′ 13′a 13′b 1-OMe 2-OMe 3-OMe 10-OMe 2′-OMe 3′-OMe 10′-OMe 11′-OMe

4.35, m, 2.92, m 4.35, m, 2.92, m

28.2 40.0

7.81, s

7.03, s

3.76, 3.94, 3.87, 4.06, 3.90, 3.89, 4.03, 3.92,

s s s s s s s s

161.4 120.8 105.8 152.7 146.2 142.7 126.9 95.9 136.3 122.7 60.7 61.1 60.4 56.7 56.1 56.4 56.4 61.2 126.3

H NMR (600 MHz). 13C NMR (100 MHz).

7.81, 7.17, 7.03, 6.70, and 6.41, eight O-methyl signals at δH 4.06, 4.03, 3.94, 3.92, 3.90, 3.89, 3.87, and 3.76, and an Nmethyl signal at δH 2.50. The characteristic H-11 signal of an aporphine moiety resonated at δH 8.09 (1H, s). The 13C NMR data showed 42 resonances (Table 4) comprising 27 sp2 carbons including a carbonyl carbon at δC 161.4 and 15 aliphatic carbons, indicating that 16 possessed a different skeleton compared to the above structures. Its 1H and 13C NMR data showed similarity to those of thalibealine (17),10 suggesting that 16 might be a derivative of an aporphineprotoberberine type dimer. The aporphine moiety was established by the similar NMR resonances to those of 17, with the long-range couplings of H-11 (δH 8.09) to C-1a (δC 122.3), C-7a (129.1), C-8 (115.1), C-9 (146.9), and C-10 (147.7), of H2-4 (δH 2.92, 2.80) to C-1b (δC 123.0) and C-3 (150.1), of H-8 (δH 6.41) to C-7 (δC 33.6), C-9, C-10, and C11a (126.7), and of 6-NMe (δH 2.50) to C-5 (δC 52.9) and C6a (62.5), as well as from the O-methyl protons at δH 4.06, 3.94, 3.87, and 3.76 to C-10, C-2 (δC 145.4), C-3, and C-1 (149.6), respectively (Figure 5). Comparison of the 1H and 13C NMR data of 16 with those of 1710 showed an additional aromatic proton at δH 7.03, two olefinic carbons at δC 95.9 and 136.3, and a carbonyl carbon at δC 161.4 in the protoberberine moiety, which was suggestive of 16 as a derivative of 17 formed via oxidation and dehydrogenation. The long-range couplings of H-9′ at δH 7.81 to C-8′ (δC 161.4), C-8′a (120.8), C-10′ (152.7), C-11′ (146.2), C-12′ (142.7), and C-12′a (126.9) and


identical 1H and 13C NMR data of 14 to 12 assigned the same relative configuration. The (6aS, 1′S) absolute configuration was defined by the similarity of its ECD spectrum [λmax (Δε) nm: 240 (+36.72), 281 (−5.01)] (Figure S10, Supporting Information) to that of 5.6 Thus, the absolute configuration of N-2′ is R. Compound 15, thalicultratine K, was obtained as a yellowish, amorphous powder. The HRESIMS data showed an [M + H]+ ion at m/z 729.3385 (calcd 729.3382), establishing the same molecular formula of C41H48N2O10 as 14. Comparing the 1H and 13C NMR data of 15 with those of 14, the differences of H1′ at δH 4.40 (1H, d, J = 11.6 Hz), H-8′ at 5.25 (1H, s), 2′-NMe (δC 55.4), and C-3′ (58.9) suggested 15 as an N-2′ epimer of 14. The (6aS, 1′S) absolute configuration was defined by the ECD spectrum [λmax (Δε) nm: 240 (+44.50), 281 (−5.85)] 2898



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Generally, thalifabenine-type aporphine-benzylisoquinoline alkaloids 1−6 were more active than their 6a,7-unsaturated derivatives 7−11. For example, compound 4 showed stronger antiproliferative activities with IC50 values of 1.44 and 2.72 μM against HL-60 and PC-3 cells, respectively, than compound 7, with an IC50 of 4.09 and 7.86 μM. Similarly, the efficacy of compounds 2, 5, and 6 was stronger than those of 9, 11, and 8. However, in the PC-3 cell line, compounds 3 and 10 were the exceptions, as 10 showed a smaller IC50 value of 3.29 μM. The 2′-N-oxides 12 and 13 were also not as potent as compound 2. The 2′-N-oxides of 6, 14, and 15 exhibited lower antiproliferative activities than 6. Based on this, the oxidation of 2′-N adversely affected the antiproliferative activities of these dimeric aporphinoid alkaloids. Furthermore, the 2′β-N-oxides 13 (IC50 4.21 and 8.35 μM against HL-60 and PC-3 cells, respectively) and 15 (IC50 2.86 and 12.66 μM, respectively) were more potent than the 2′α-N-oxides 12 (IC50 5.05 and 13.40 μM, respectively) and 14 (IC50 4.57 and 18.66 μM, respectively). The dimeric alkaloids 16 and 17 also showed cytotoxicity against PC-3 (IC50 3.72 μM) and HL-60 (IC50 1.77 μM) cells, respectively. The new compound 3 was selected for intensive apoptosis-related mechanism studies. Cellular proliferation and the series of events leading to cell division and replication are generally linked to cell cycle progression. Therefore, to better understand the antiproliferative mechanisms, the effects of compound 3 on the cell cycle distribution of HL-60 cells were evaluated. The cells were treated with different concentrations (0.06, 0.3, and 1.5 μM) of compound 3 for 24 h, then stained with propidium iodide (PI) and analyzed by flow cytometry. As shown in Figure 6, compound 3 was found to arrest cells at the S phase, increasing from 25.75% of cells treated with the negative control to 42.51% and 48.45% of cells treated with 0.3 and 1.5 μM compound 3, respectively, accompanied by decreased cell numbers in the G2/M phase (from 30.93% of the vehicle to 21.47% and 18.53% of cells treated with 0.3 and 1.5 μM compound 3, respectively) and the G1/S phase (from 43.32% of the vehicle to 36.01% and 33.02% of cells treated with 0.3 and 1.5 μM compound 3, respectively). These revealed that compound 3 arrested the HL-60 cell cycle at the S stage, leading to the inhibition of cell proliferation. To confirm the apoptosis-inducing effects of compound 3, a nuclear morphological evaluation study was performed by fluorescence microscopy following staining with Hoechst 33258. It is well known that apoptotic cells are characterized by specific morphological features, such as cell shrinkage and chromatin condensation. After the treatment with 0.06, 0.3, and 1.5 μM compound 3 for 24 h, the morphological analyses (Figure 7) showed that the control cells were uniformly stained with Hoechst 33258 and showed normally blue, homogeneous, and round nuclei, without morphological changes. HL-60 cells exposed to 0.06, 0.3, and 1.5 μM compound 3 for 24 h presented remarkable morphological changes, such as cell shrinkage and chromatin condensation. These results supported the proapoptotic effects of compound 3, especially at the concentrations of 0.06 and 0.3 μM, while late apoptosis was observed at a concentration of 1.5 μM. To shed more light on the apoptosis-inducing effects of this series of dimeric alkaloids, compound 3, with a methylenedioxy group, was analyzed for the effects on apoptosis in HL-60 cells. One of the hallmarks of apoptosis observed in the early stages of an apoptotic process is the disruption of membrane asymmetry and the translocation of phosphatidylserine (PS)


of H-13′ at δH 7.03 to C-8′a, C-12′, and C-13′a (δC 136.3) confirmed the carbonyl group at C-8′ and the additional Δ13′(13′a) double bond. The correlations from the O-methyl protons at δH 4.03, 3.92, 3.90, and 3.89 to C-10′, C-11′, C-2′ (δC 150.3), and C-3′ (148.5), respectively, confirmed the location of these O-methyl groups. Consequently, the above information assigned the protoberberine moiety. The NOE correlation of H-8 to 11′-OMe (δH 3.92) indicated that the two moieties were linked by an ether bond between C-9 and C-12′. The (6aS) absolute configuration was determined by the ECD spectrum [λmax (Δε) nm: 218 (−9.53), 242 (+22.50), 276 (−3.16), 300 (−3.20)] (Figure S12, Supporting Information) similar to the spectrum of S-preocoteine.11,12 The antiproliferative activities of compounds 1−17 were evaluated against human leukemia HL-60 and prostate cancer PC-3 cells (Table 5). These dimeric aporphinoid alkaloids Table 5. Inhibitory Effects of Compounds 1−17 on HL-60 and PC-3 Cells (IC50 ± SD in μM) compound

HL-60

PC-3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 5-Fu camptothecin

2.10 ± 0.13 1.16 ± 0.10 1.08 ± 0.09 1.44 ± 0.13 1.58 ± 0.14 1.01 ± 0.10 4.09 ± 0.25 4.28 ± 0.36 2.12 ± 0.14 1.77 ± 0.12 2.41 ± 0.13 5.05 ± 0.40 4.21 ± 0.31 4.57 ± 0.32 2.86 ± 0.18 10.34 ± 0.47 1.77 ± 0.12 2.80 ± 0.14 0.92 ± 0.08

5.85 ± 0.19 6.05 ± 0.30 5.59 ± 0.24 2.72 ± 0.11 7.27 ± 0.40 2.47 ± 0.14 7.86 ± 0.27 7.10 ± 0.31 6.01 ± 0.36 3.29 ± 0.18 2.56 ± 0.12 13.40 ± 0.37 8.35 ± 0.50 18.66 ± 0.57 12.66 ± 0.45 3.72 ± 0.23 11.64 ± 0.35 22.02 ± 1.08 6.96 ± 0.41

showed superior growth inhibitory activities against HL-60 cells with IC50 values ranging from 1.01 to 5.05 μM, except for compound 16, with an IC50 value of 10.34 μM. The IC50 values against PC-3 cells ranged from 2.47 to 18.66 μM, and the antiproliferative potency of all the compounds was higher than that of 5-fluorouracil (5-Fu, IC50 22.02 μM). Compound 6 showed the most promising antiproliferative activity against selected human cancer cells. For the HL-60 cell line, the IC50 value of 1.01 μM was approximately 2-fold stronger than 5-Fu and as potent as camptothecin. For the PC-3 cell line, the IC50 value of 6 was 2.47 μM, approximately 9-fold stronger than 5Fu and 3-fold stronger than camptothecin. 2899



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Figure 6. Cell cycle effects of compound 3 in HL-60 cells.

the cells were treated with different concentrations (0.06, 0.3, and 1.5 μM) of compound 3 for 24 h, stained with annexin VAPC/7-AAD, and analyzed by flow cytometry. As shown in Figure 8, compound 3 was a potent dose-dependent apoptosis inducer. Specifically, when compared with the control (4.25%, early and late apoptosis), treatment with compound 3 increased the apoptosis rates, which gradually increased from 16.75% to 29.06% and 56.95% after treatment with 0.06, 0.3, and 1.5 μM compound 3, respectively. The results showed that compound 3 caused a remarkable increase of the cellular apoptosis in a concentration-dependent manner. Moreover, time-dependent apoptosis was also observed using the annexin V-FITC/PI double-staining assay. HL-60 cells were treated with a 1.5 μM solution of compound 3 for 24, 48, and 72 h, respectively, then stained with annexin V-FITC/PI and analyzed by flow cytometry (Figure 9). Specifically, when compared with the control (5.33%, early and late apoptosis), treatment with compound 3 increased the apoptosis rates, which gradually increased from 23.71% to 46.47%, and 63.12% after treatment for 24, 48, and 72 h, respectively. Compound 3 also caused apoptosis of HL-60 cells in a time-dependent manner. Mitochondria play a crucial role in the induction and control of apoptosis, and the loss of mitochondrial membrane potential is an important characteristic of mitochondrial dysfunction. To investigate the mitochondria-related effects of compound 3, the fluorescent probe JC-1 was used to detect the changes in mitochondrial membrane potentials. HL-60 cells were treated with different concentrations (0.06, 0.3, and 1.5 μM) of compound 3 for 24 h, and collapsed mitochondria were

Figure 7. Morphological changes in HL-60 cells caused by compound 3.

from the inner to the outer cell membrane. The externalized PS can be detected and quantified by annexin V-APC, a fluorescently labeled conjugate that binds to PS with high affinity. In the late stages of apoptosis, the cell membrane loses its integrity, allowing the penetration of 7-AAD, which intercalates with DNA, through the cell membrane. Therefore, 2900



Article

Figure 8. Compound 3 induced apoptosis in a concentrationdependent manner in HL-60 cells.

Figure 10. Compound 3 induced mitochondrial depolarization in HL60 cells.

are rare N-oxide dimeric alkaloids. The antiproliferative activities were evaluated against HL-60 and PC-3 cancer cells. Compound 3 with an IC50 value of 1.06 μM against HL-60 cells showed the most potent cytotoxicity and was selected for further mechanistic studies. The results revealed that compound 3 induced apoptosis and arrested the HL-60 cell cycle at the S phase through mitochondria-related intrinsic pathways, which deserve further investigation.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a PerkinElmer 241MC polarimeter. The UV spectra were recorded on a Shimadzu UV-2201 spectrometer. NMR spectra were recorded on Bruker AV-400 or AV-600 NMR spectrometers, with tetramethylsilane as the internal standard. IR spectra were acquired on a Bruker IFS-55 spectrometer (KBr tablets). ECD spectra were obtained on a Bio-Logic MOS-450 spectrometer. HRESIMS was measured on a Bruker micrOTOF-Q mass spectrometer. ESIMS was conducted on an Agilent 1100 mass spectrometer. Semipreparative HPLC was performed on a YMC ODS-A column (250 × 20 mm i.d., 5 μm) equipped with an LC-6AD pump and a Shimadzu SPD-20A UV− vis detector (Shimadzu Co., Ltd., Japan). Silica gel (Qingdao Haiyang Chemical Group Co.; 200−300 mesh), neutral alumina (100−200 mesh, Sinopharm Chemical Reagent Co. Ltd., Shanghai, People’s Republic of China), ODS (50 μm, YMC Co. Ltd., Kyoto, Japan), 001×7 ion-exchange resin (Dianli Resin Co. Ltd., Langfang, People’s Republic of China), and Sephadex LH-20 (GE Healthcare, Sweden) were used for column chromatography (CC). Plant Material. The roots of T. cultratum Wall were purchased from Anguo Traditional Chinese Medicinal Materials Trading Center, Hebei Province, People’s Republic of China. The sample was identified by Professor Jin-Cai Lu (School of Traditional Chinese Materia

Figure 9. Compound 3 induced apoptosis in a time-dependent manner in HL-60 cells.

monitored by flow cytometry. As shown in Figure 10, compound 3 induced a concentration-dependent increase (from 0.58% of control to 24.06%, 33.51%, and 59.1% at 0.06, 0.3, and 1.5 μM compound 3, respectively) in the proportion of cells with depolarized mitochondria. In summary, to enrich the diversity of the compound library of dimeric alkaloids, 12 new and five known alkaloids were obtained from T. cultratum Wall, and the structures were defined by NMR spectroscopic analyses. Thalicultratines H−K 2901



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(4.08) nm; IR (KBr) νmax 2921, 2851, 1607, 1494, 1463, 1392, 1273, 1196, 1118, 838 cm−1; ECD (MeOH) λmax (Δε) 190 (−20.21), 213 (+15.99), 233 (+23.43), 283 (+9.51), 331 (+2.00) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 725.3432 [M + H]+ (calcd for C42H49N2O9, 725.3433). Thalicultratine G (10): yellowish, amorphous powder; [α]20D +40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 255 (4.29), 336 (4.08) nm; IR (KBr) νmax 2921, 2850, 1608, 1503, 1462, 1392, 1273, 1214, 1131, 1068, 831 cm−1; ECD (MeOH) λmax (Δε) 196 (−5.98), 209 (+1.34), 231 (+30.58), 281 (+4.90), 333 (+0.94) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 709.3119 [M + H]+ (calcd for C41H45N2O9, 709.3120). Thalicultratine H (12): yellowish, amorphous powder; [α]20D +95 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 280 (4.29), 310 (4.08 sh) nm; IR (KBr) νmax 2920, 2851, 1462, 1384, 1121, 839 cm−1; ECD (MeOH) λmax (Δε) 204 (−16.68), 245 (+39.69) nm; 1H and 13C NMR data, see Table 3; HRESIMS m/z 743.3553 [M + H]+ (calcd for C42H51N2O10, 743.3538). Thalicultratine I (13): yellowish, amorphous powder; [α]20D +72 (c 0.1, MeOH); IR (KBr) νmax 2921, 2850, 1606, 1503, 1462, 1384, 1219, 1121, 1052, 842 cm−1; ECD (MeOH) λmax (Δε) 205 (−16.53), 243 (+45.10) nm; 1H and 13C NMR data, see Table 3; HRESIMS m/z 743.3545 [M + H]+ (calcd for C42H51N2O10, 743.3538). Thalicultratine J (14): yellowish, amorphous powder; [α]20D +45 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 282 (4.26), 310 (3.93 sh) nm; IR (KBr) νmax 2921, 2851, 1609, 1505, 1463, 1384, 1219, 1121, 841 cm−1; ECD (MeOH) λmax (Δε) 240 (+36.72), 281 (−5.01) nm; 1H and 13C NMR data, see Table 3; HRESIMS m/z 729.3393 [M + H]+ (calcd for C41H49N2O10, 729.3382). Thalicultratine K (15): yellowish, amorphous powder; [α]20D +32 (c 0.1, MeOH); IR (KBr) νmax 2921, 2851, 1608, 1505, 1462, 1384, 1219, 1122, 1051, 839 cm−1; ECD (MeOH) λmax (Δε) 240 (+44.50), 281 (−5.85) nm; 1H and 13C NMR data, see Table 3; HRESIMS m/z 729.3385 [M + H]+ (calcd for C41H49N2O10, 729.3382). Thalicultrtine (16): yellowish, amorphous powder; [α]20D −92 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 226 (4.64), 265 (4.56), 316 (3.96), 336 (4.03) nm; IR (KBr) νmax 2920, 2851, 1640, 1599, 1511, 1464, 1384, 1271, 1116 cm−1; ECD (MeOH) λmax (Δε) 218 (−9.53), 242 (+22.50), 276 (−3.16), 300 (−3.20) nm; 1H and 13C NMR data, see Table 4; HRESIMS m/z 737.3098 [M + H]+ (calcd for C42H45N2O10, 737.3069). Growth Inhibitory Assays. Compounds 1−17 were evaluated for antiproliferative activity against human leukemia HL-60 cell line (America Type Culture Collection) by using the trypan blue method and human prostate cancer PC-3 cells (America Type Culture Collection) using the MTT assay.30−32 The cells were cultured in RPMI-1640 medium (Gibco, New York, NY, USA), which was supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin, 1 mM glutamine, and 10% heat-inactivated fetal bovine serum (FBS, Gibco), then cultured at 37 °C in a humidified atmosphere with 5% CO2. Two-milliliter cell suspensions (4.5 × 104 cells/mL) were seeded in 24-well plates and incubated with the compounds in a concentration gradient (dissolved in DMSO and diluted with absolute EtOH) for 72 h, followed by the addition of trypan blue to stain the cells, and the total cell number was counted using a hematocytometer. The MTT assay was performed in 96-well plates. PC-3 cells in log phase were added to each well and treated with various concentrations of the samples in triplicates at 37 °C in a humidified atmosphere of 5% CO2. The cultures were treated with MTT solution (5 mg/mL) for an additional 4 h. DMSO was added to each well, and the OD value of each well was measured using a microplate reader (Bio-Rad 550) at the wavelength of 490 nm. The growth inhibition rates of the tested compounds were described as IC50 values, based on the average values of three parallel experiments. In the experiments, the negative reference was 0.1% DMSO. 5-Fu and camptothecin were used as positive controls. Effects on Cell Cycle. HL-60 cells were incubated in 10% FBS supplemented culture medium at 37 °C for 24 h. Exponentially growing cells were incubated with different concentrations of compound 3 in triplicate. Untreated cells (control) or cells treated

Medica, Shenyang Pharmaceutical University). A voucher sample (TC20110314) was deposited in Shenyang Pharmaceutical University. Extraction and Isolation. The air-dried roots of T. cultratum Wall (30 kg) were extracted with 95% EtOH (3 × 300 L, 2 h each) under reflux. The extracts were concentrated in vacuo to give a residue, which was suspended in H2O (2 L) and adjusted to pH 3 with 5% HCl. The acidic aqueous phase was filtered off. The filtrate was loaded on ion-exchange resin (001×7), eluted with 20% EtOH until the eluate approached colorless to give the nonalkaloid parts, and then eluted with 2% NaOH in 65% EtOH solution (5-fold of retention volume) to afford the crude total alkaloids. The alkaloid-containing solution was acidified to pH 5 with 5% HCl and partitioned with CH2Cl2 (3 × 2 L) to afford the CH2Cl2 extract (142 g). The CH2Cl2-soluble portion was subjected to neutral Al2O3 CC and eluted with petroleum ether (60−90 °C)−acetone (100:10, 100:20, 100:30, 100:50, v/v) to yield five fractions (Fr.1−Fr.5). Fr.3 was separated by silica gel CC using a gradient of acetone in petroleum ether (60−90 °C) to afford five subfractions, Fr.3A−Fr.3E. Fr.3E was subjected to ODS CC with MeOH−H2O (60:40, 70:30, 75:25, v/v) to give three subfractions, 3E-1, 3E-2, and 3E-3. The 60% MeOH eluting fraction 3E-1 was subjected to neutral Al2O3 CC to provide two subfractions, Fr.3E1-1 and Fr.3E1-2, which were separated by preparative HPLC to afford compounds 2 (525.1 mg), 4 (80.5 mg), 5 (155.8 mg), 9 (38.7 mg), 11 (62.0 mg), 12 (13.0 mg), and 13 (14.6 mg). The 70% and 75% MeOH eluting fractions 3E-2 and 3E-3 were purified by semipreparative HPLC to yield 3 (19.7 mg) and 10 (15.3 mg). Fr.4 was subjected to ODS CC with MeOH−H2O (30:70, 35:65, 45:55, v/v) to give four subfractions, Fr.4A−4D. Fr.4A was separated by macroporous resin D101 CC and then purified by semipreparative HPLC to afford 7 (22.7 mg). Compound 17 (22.7 mg) was obtained from Fr.4B by semipreparative HPLC. Fr.4C was separated by Sephadex LH-20 (MeOH) and semipreparative HPLC, successively, to afford 16 (23.5 mg). Fr.5 was subjected to neutral Al2O3 CC and silica gel CC in succession to yield three subfractions, Fr.5A1−5A3, of which Fr.5A3 was subjected to ODS CC with MeOH−H2O (60:40, 65:35, v/v) and then purified by semipreparative HPLC to obtain 1 (20.7 mg), 6 (167.5 mg), 14 (13.6 mg), 15 (12.7 mg), and 8 (50.4 mg). Thalicultratine A (1): yellowish, amorphous powder; [α]20D +67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 278 (4.26), 310 (3.96 sh) nm; IR (KBr) νmax 3430, 2920, 2850, 1608, 1504, 1462, 1384, 1217, 1120, 838 cm−1; ECD (MeOH) λmax (Δε) 238 (+21.55), 281 (−2.65) nm; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 683.3325 [M + H]+ (calcd for C40H47N2O8, 683.3327). Thalicultratine B (2): yellowish, amorphous powder; [α]20D +61 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 281 (4.33), 310 (3.98 sh) nm; IR (KBr) νmax 2933, 2850, 2787, 1606, 1503, 1461, 1340, 1216, 1120, 847 cm−1; ECD (MeOH) λmax (Δε) 241 (+22.57), 282 (−2.53) nm; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 727.3587 [M + H]+ (calcd for C42H51N2O9, 727.3589). Thalicultratine C (3): yellowish, amorphous powder; [α]20D +67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 280 (4.30), 310 (4.03 sh) nm; IR (KBr) νmax 2921, 2850, 1608, 1504, 1462, 1340, 1216, 1122, 849 cm−1; ECD (MeOH) λmax (Δε) 241 (+21.07), 280 (−3.93) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 711.3279 [M + H]+ (calcd for C41H47N2O9, 711.3276). Thalicultratine D (7): yellowish, amorphous powder; [α]20D +42 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 263 (4.56), 332 (4.06) nm; IR (KBr) νmax 2921, 2851, 1610, 1504, 1463, 1384, 1220, 1124, 838 cm−1; ECD (MeOH) λmax (Δε) 197 (−21.18), 213 (+13.02), 234 (+13.60), 286 (+1.75) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 665.3228 [M + H]+ (calcd for C40H45N2O7, 665.3221). Thalicultratine E (8): yellowish, amorphous powder; [α]20D +48 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 258 (4.32), 269 (4.52), 332 (4.06) nm; IR (KBr) νmax 2922, 2850, 1608, 1503, 1463, 1392, 1273, 1216, 1121, 834 cm−1; ECD (MeOH) λmax (Δε) 193 (−25.84), 230 (+11.28), 264 (+5.22), 333 (+0.01) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 711.3276 [M + H]+ (calcd for C41H47N2O9, 711.3276). Thalicultratine F (9): yellowish, amorphous powder; [α]20D +64 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 257 (4.32), 271 (4.56), 336 2902


Journal of Natural Products with the solvent (DMSO) were included. After 24 h of treatment, cells were centrifuged and fixed in 70% EtOH at 4 °C overnight and subsequently resuspended in phosphate-buffered saline (PBS) containing 100 μL of RNase A and 400 μL of PI. Cellular DNA was measured using a flow cytometer (FACS Calibur Becton-Dickinson) for the cell cycle distribution analysis.33 Hoechst Staining. The nuclear morphological changes and apoptotic effects induced by compound 3 were detected using Hoechst 33258 staining. In this experiment, 1 × 108 HL-60 cells were seeded in culture plates and treated with compound 3 for 24 h. Cells were collected and mounted on a slide, fixed for 30 min with 4% paraformaldehyde, and washed three times with PBS. Cells were stained with Hoechst 33258 (2 mg/mL in PBS) for 10 min at room temperature in the dark. The cells were subsequently washed with PBS and observed with a fluorescence microscope.36,37 Analysis of Cellular Apoptosis. The annexin V-APC/7-AAD cytometry assay was carried out to detect the cell population in the early and late apoptosis stage. HL-60 cells were seeded in six-well plates to grow overnight and treated with or without 3 at indicated concentrations in triplicate for a 24 h time period. Cells were washed twice in PBS and resuspended in annexin V binding buffer. Annexin VAPC was added, and the mixture was incubated in the dark at 25 °C for 15 min. 7-AAD was added just prior to acquisition. Apoptosis was analyzed by annexin V-APC and 7-AAD double staining by flow cytometry. Cells undergoing early and late apoptosis were detected by fluorescence from only annexin V-APC and annexin V-APC/7-AAD, respectively, according to the manufacturer’s instructions.34 Additionally, time-dependent apoptosis was analyzed with the annexin VFITC/propidium iodide test. HL-60 cells were seeded in six-well plates to grow overnight and treated with or without 3 at 1.5 μM in triplicate for 24, 48, and 72 h, respectively. Cells were washed twice in PBS and resuspended in annexin V binding buffer. Annexin V-FITC was added, and the mixture was incubated under dark conditions at 25 °C for 15 min. PI was added just prior to acquisition. Apoptosis was analyzed using annexin V-FITC and PI double staining by flow cytometry. Mitochondrial Membrane Potential Loss. HL-60 cells were incubated in triplicate and treated with compound 3 or vehicle for 24 h. The cells were washed with PBS and stained with JC-1 dye (Keygen, KGA601) under dark conditions according to the manufacturer’s instructions. After incubation, the cells were washed twice with PBS, and the percentage of cells with collapsed or healthy mitochondrial membrane potentials was monitored by flow cytometry analysis (FACS Calibur Becton-Dickinson).35

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Mrs. W. Li and Mr. Y. Sha (Analytical Test Center, Shenyang Pharmaceutical University) for acquiring the NMR data. The work was financially supported by the National Natural Science Foundation of China (Grant No. 81172958).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00387. 1 H NMR, 13 C NMR, HSQC, NOESY, HMBC, HRESIMS, UV, and ECD spectra of 1−3, 7−10, and 12−16 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-24-23986465. E-mail: [email protected] (Z. L. Li). ORCID

Kai-Bo Wang: 0000-0002-2934-9906 Hui-Ming Hua: 0000-0002-0258-3647 Zhan-Lin Li: 0000-0001-5306-3396 Author Contributions §

D. H. Li and J. Y. Li contributed equally.

Notes

The authors declare no competing financial interest. 2903



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Antiproliferative Dimeric Aporphinoid Alkaloids from the Roots of

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