Oxazole-Containing Diterpenoids from Cell Cultures of Salvia

ABSTRACT: Four new oxazole-containing diterpenoids, salvianans A−D (1−4), along with three known diterpenoids (5−7), were isolated from Salvia m...
0 downloads 0 Views 994KB Size
Download PDF
Article Cite This: J. Nat. Prod. 2017, 80, 3241−3246

pubs.acs.org/jnp

Oxazole-Containing Diterpenoids from Cell Cultures of Salvia miltiorrhiza and Their Anti-HIV‑1 Activities Dewu Zhang,†,‡,§ Jiamei Guo,†,§ Min Zhang,† Xiao Liu,† Mingyu Ba,† Xiaoyu Tao,†,‡ Liyan Yu,‡ Ying Guo,*,† and Jungui Dai*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, and ‡Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Four new oxazole-containing diterpenoids, salvianans A−D (1−4), along with three known diterpenoids (5−7), were isolated from Salvia miltiorrhiza cell cultures. The structures of the new compounds were elucidated using spectroscopic methods and single-crystal X-ray diffraction. The evaluation for their anti-HIV-1 activities revealed that 2 and 3 displayed inhibitory activities with IC50 values of 0.03 and 1.2 μM, respectively. The time of addition (TOA) assay and long terminal repeat (LTR) luciferase reporter assay results indicated that compound 2 was an HIV-1 transcription inhibitor and might be a lead compound of antiviral agents acting on HIV-1 transcription.

A

known compounds, salvianan (5),19 isosalviamine B (6),21 and neosalvianan (7).19 The isolates were evaluated for their antiHIV-1 activities, and compounds 2 and 3 showed inhibition effects on wild-type HIV-1 replication. Additional anti-HIV-1 assays identified 2 as an HIV-1 transcription inhibitor that could block HIV-1 replication at the submicromolar level. Herein, the isolation, structure identification, putative biosynthetic pathway, and anti-HIV-1 activities of compounds 1−7 are reported.

cquired immune deficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is still one of the most serious global public health threats. There were approximately 36.7 million people infected with HIV in 2015 worldwide.1 Currently, there are 26 antiretroviral agents that belong to the following four different drug classes: (1) viral entry inhibitors, (2) reverse transcriptase inhibitors, (3) integrase inhibitors, and (4) protease inhibitors.2 HIV transcription, which is driven by the long terminal repeat (LTR) promoter, occurs after viral integration. Viral DNA transcription results in the production of multiple viral RNA copies.3 To date, there are no available antiretroviral drugs acting on HIV transcription. Natural products have been demonstrated to be an important resource for anti-infective drugs.4 Salvia miltiorrhiza Bunge (Labiatae), known as “Danshen” in China, has been used to treat menstrual disorders, hepatitis, dysmenorrhea, and heart and cardiovascular diseases.5−8 More than 50 tanshinone derivates have been reported from the root of S. miltiorrhiza, some of which display diverse biological properties, such as antitumor,9−11 antioxidant,12−15 and antiplatelet aggregation16−18 activities. A few N-containing diterpenoids were also isolated from S. miltiorrhiza, some of which exhibited dose-dependent potent cytotoxicities against several tumor cell lines.19 A number of cytoxic diterpenoids were isolated from S. miltiorrhiza cell cultures.20 As part of a continuing investigation on biologically active diterpenoids from S. miltiorrhiza cell cultures, further systematic fractionation was performed and afforded four new oxazole-containing diterpenoids, salvianans A−D (1−4), together with three © 2017 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Salvianan A (1) was isolated as pale yellow lamellar crystals. Its molecular formula was established as C20H17NO2 via an HRESIMS ion at m/z 304.1318 [M + H]+ (calcd for C20H18NO2, 304.1332). The 1H NMR spectrum showed resonances of an AMX pattern for three protons at δH 10.15 (1H, d, 8.7 Hz, H1), 7.65 (1H, dd, 8.7, 6.9 Hz, H-2), and 7.48 (1H, d, 6.9 Hz, H3), a pair of ortho-aromatic protons at δH 8.03 (1H, d, 9.0 Hz, H-7) and 7.96 (1H, d, 9.0 Hz, H-6), the typical resonances of a methyl-substituted dihydrofuran moiety belonging to a tanshinone-type skeleton with signals at δH 5.00 (1H, t, 8.7 Hz, H-16a), 4.43 (1H, dd, 8.7, 7.2 Hz, H-16b), 4.08 (1H, m, H15), and 1.58 (3H, d, 6.6 Hz, H3-17), and two tertiary methyl signals at δH 2.79 (s, H3-18 and H3-21). The 1H and 13C NMR data (Table 1, Figures S3 and S4, Supporting Information) of 1 resembled those of isosalviamine B (6), which was isolated Received: July 30, 2017 Published: November 29, 2017 3241

DOI: 10.1021/acs.jnatprod.7b00659 J. Nat. Prod. 2017, 80, 3241−3246

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Spectroscopic Data (CDCl3) for 1−4 salvianan A (1)a position

δC, type

δH (J in Hz)

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

126.2, CH 126.2, CH 127.8, CH 134.1, C 131.3, C 121.5, CH 120.1, CH 115.7, C 122.7, C 130.0, C 132.1, C 146.1, C 110.7, C 154.8, C 36.5, CH 79.5, CH2

17 18 19 20 21 22 23 24 25 26 27

19.1, CH3 20.2, CH3

4.08 m 5.00 t (8.7) 4.43 dd (8.7, 7.2) 1.58 d (6.6) 2.79 s

160.4, C 14.7, CH3

2.79 s

10.15 d (8.7) 7.65 dd (8.7, 6.9) 7.48 d (6.9)

7.96 d (9.0) 8.03 d (9.0)

salvianan B (2)b δC, type 126.2, CH 126.4, CH 128.0, CH 134.3, C 131.4, C 122.0, CH 120.1, CH 116.3, C 123.3, C 129.8, C 130.9, C 145.6, C 110.9, C 155.8, C 36.4, CH 79.7, CH2 19.1, CH3 20.2, CH3

salvianan C (3)b

δH (J in Hz) 10.16 d (8.1) 7.66 dd (8.1, 7.2) 7.51 d (7.2)

8.01 d (9.0) 8.06 d (9.0)

4.13 m 5.04 t (8.7) 4.47 t (7.8) 1.60 d (6.6) 2.80 s

149.3, CH 8.23 s

δC, type 126.4, CH 126.3, CH 127.8, CH 134.1, C 131.3, C 121.6, CH 120.1, CH 115.8, C 123.0, C 130.0, C 132.1, C 146.4, C 110.9, C 155.0, C 36.4, CH 79.6, CH2 19.1, CH3 20.2, CH3 161.6 35.3, CH2 135.6, C 128.9, CH 128.7, CH 127.1, CH 128.7, CH 128.9, CH

δH (J in Hz) 10.21 d (8.4) 7.65 dd (8.4, 7.5) 7.49 d (7.5)

7.97 d (9.3) 8.02 d (9.3)

4.07 m 4.99 t (overlap) 4.42 dd (9.0, 9.0) 1.58 d (6.9) 2.79 s

salvianan D (4)b δC, type 30.4, CH2 19.6, CH2 38.7, CH2 34.6, C 144.6, C 124.3, CH 119.8, CH 117.4, C 125.4, C 132.1, C 130.2, C 145.5, C 109.4, C 155.6, C 36.4, CH 79.6, CH2 19.2, CH3 31.8, CH3 31.8, CH3 147.9, CH

δH (J in Hz) 3.73 t (6.3) 2.00 m 1.78 m

7.55 d (9.0) 7.89 d (9.0)

4.04 m 4.97 t (8.7) 4.40 t (7.8) 1.52 d (6.6) 1.39 s 1.39 s 8.08 s

4.44 s 7.47 7.37 7.31 7.37 7.47

a1 H NMR data were recorded at 300 MHz, and 13C NMR data were recorded at 100 MHz. NMR data were recorded at 150 MHz.

d (overlap) dd (7.5, 6.9) t (overlap) dd (7.5, 6.9) d (overlap) b1

H NMR data were recorded at 300 MHz, and 13C

from S. triguja,21 except that 1 displayed signals of an oxymethylene moiety (δH 5.00, H-16a, 4.43, H-16b; δC 79.5, C-16) and a methane function (δH 4.08, H-15; δC 36.5, C-15) instead of the Δ15(16) olefinic group in 8. The HMBC correlations (Figure 2) of H-1/C-3, C-5, and C-9; H-2/C-1, Figure 2. Selected HMBC correlations of 1.

C-3, C-4, and C-10; H-6/C-4, C-5, C-8, and C-10; and H3-18/ C-3, C-4, and C-5 indicated the presence of a methylnaphthalenyl group. The HMBC cross-peaks of H2-16/C-13, C-14, C-15, and C-17 and H3-17/C-13, C-15, and C-16 confirmed the location of the dihydrofuran moiety. Furthermore, the HMBC correlations of H3-21/C-20, together with the molecular formula and the chemical shifts of C-11, C-12, and C-20, established the location of the oxazole moiety. Accordingly, 1 was defined as a new diterpenoid with an oxazole unit. The structure and absolute configuration of 1 were further confirmed by X-ray diffraction analysis with Cu Kα radiation (Figure 3), and the absolute configuration of C-15 was assigned as R. The absolute configuration of C-15 and the presence of an oxazole ring for this type of compound were unambiguously determined by the single-crystal X-ray diffraction study. The negative Cotton effect at 291 nm observed in the electronic circular dichroism (ECD) spectrum (Figure S12, Supporting

Figure 1. Structures of 1−7. 3242

DOI: 10.1021/acs.jnatprod.7b00659 J. Nat. Prod. 2017, 80, 3241−3246

Journal of Natural Products

Article

Information) and specific rotation compared to those of 1 indicated the (15R) absolute configuration of salvianan C (3). Salvianan D (4) was obtained as a pale yellow powder. It showed an HR-ESIMS ion at m/z 308.1639 [M + H]+, in accordance with a molecular formula of C20H21NO2. The 1H and 13C NMR data of 4 resembled those of salvianan (5),19 except for the absence of the C-20 methyl group. The HMBC correlations from H-20 (δH 8.08) to C-11 and C-12 verified the above conclusion. The similar negative ECD Cotton effect around 306 nm (Figure S43, Supporting Information) and the specific rotation compared to those of 1 indicated the (15R) absolute configuration of salvianan D (4). Compounds 1−7 are characterized as unusual oxazolecontaining norditerpenoids, which might have a biosynthetic origin from dihydrotanshinone I/cryptotanshinone and various amino acids (Figure 4). The amino moiety of the starting amino acid (e.g., alanine, glycine, and phenylalanine) attacks the O-quinone group of the tanshinones via a nucleophilic substitution reaction to form an imino intermediate. The oxazole ring is formed through cyclization, and compounds 1− 7 are formed by reductive decarboxylation. Attack by the amino moiety at different positions (C-11 or C-12) results in the formation of the isomeric oxazoles 1−6 and 7, while different starting amino acids (alanine, glycine, and phenylalanine) lead to various substituents at C-20. The activities of 1−7 against wild-type HIV-1 replication were screened at a final concentration of 10 μM using a vesicular stomatitis virus glycoprotein (VSV-G)/HIV-1 replication assay (Table 2). Compounds 2 and 3 displayed

Figure 3. ORTEP drawing of 1.

Information) supported the (15R) configuration.22 Therefore, the structure of salvianan A was defined as shown in Figure 1. Salvianan B (2) was obtained as a pale yellow powder and possessed a molecular formula of C19H15NO2 via the HRESIMS ion peak at m/z 290.1167 [M + H]+ (calcd for C19H16NO2, 290.1176). Comparison of the 1H NMR data of 2 and 1 revealed a singlet at δH 8.23 (1H, s, H-20) in place of the tertiary methyl singlet at δH 2.79 (s, H3-21) in 1. Two signals including a methyl carbon (δC 14.7, C-21) and an oxygenated tertiary carbon (δC 160.4, C-20) in the 13C NMR spectrum of 1 were not observed in 2. Additionally, the 13C NMR spectrum of 2 contained a methine carbon (δC 149.3, C-20) that was not observed in the spectrum of 1. Thus, it was deduced that 2 was a derivative of 1 lacking the C-20 methyl group. The presence of the oxazole moiety was supported by the HMBC correlations from H-20 to C-11 and C-12. The similar negative Cotton effect around 292 nm (Figure S23, Supporting Information) and the specific rotation compared to those of 1 suggested the (15R) absolute configuration of salvianan B (2). Salvianan C (3) was isolated as a pale yellow powder. The molecular formula C26H21NO2 was deduced from the HRESIMS ion at m/z 380.1627 [M + H]+. The major difference between the 1H NMR spectra of 3 and 1 was that the C-20 methyl group in 1 was replaced by a benzyl moiety in 3. It was supported by the observation of a methylene group (δH 4.44, s, H2-21; δC 35.3, C-21) and a monosubstituted benzene moiety (δC 135.6, C-22; δC 128.9, C-23 and C-27, δH 7.47, overlapped, H-23 and H-27; δC 128.7, C-24 and C-26, δH 7.37, H-24 and H-26; and δC 127.1, C-25, δH 7.31, overlapped, H-25). The position of the benzyl moiety was confirmed by the HMBC cross-peak of H2-21 with C-20. The similar negative Cotton effect at 306 nm in the ECD spectrum (Figure S31, Supporting

Table 2. Inhibitory Effects of 1−7 on Wild-Type HIV-1 Replication; n = 2, x̅ ± s compound

inhibition (%) at 10 μM

1 2 3 4 5 6 7 EFV

2.82 ± 17.61 76.65 ± 0.45 62.09 ± 1.68 18.70 ± 1.04 18.30 ± 0.35 17.44 ± 9.36 11.18 ± 11.39 100.00 ± 2.60

IC50 (μM) 0.03 ± 0.01 1.2 ± 0.8

0.0007 ± 0.0002

inhibitory activities with IC50 values of 0.03 ± 0.01 and 1.2 ± 0.8 μM, respectively (Table 2). Compound 2 was also tested against HIV-1 replication by the VSV-G/HIV-1 assay and the

Figure 4. Putative biosynthesis routes toward the formation of 1−7. 3243

DOI: 10.1021/acs.jnatprod.7b00659 J. Nat. Prod. 2017, 80, 3241−3246

Journal of Natural Products

Article

reference compounds. This suggested that 2 functions at a step of HIV-1 replication postintegration. In a single-round HIV-1 replication model, HIV-1 undergoes major essential steps including reverse transcription, integration, transcription, and translation in a chronological manner. Because the TOA assay indicated that the action of 2 occurred after integration, a transient gene expression assay was performed to examine the direct effect of 2 on HIV-1 transcription and translation. In this assay, the plasmid pNL43.luc.R−E−, which was constructed from the HIV-1 gene backbone and carried a luciferase gene as a reporter, was transiently transfected into HEK 293T cells. The transfected cells treated with 2, but not AZT, EFV, or RAL, exhibited a dose-dependent luciferase activity reduction (Figure 5B). This indicated that 2 functions on either the HIV-1 transcription or translation step. A plasmid encoding Renilla luciferase was transfected into HEK 293T cells, and the Renilla luciferase activity of cell lysates was maintained when the cells were treated with 2 for 48 h at a final concentration of 1.0 μM (Figure 5C), which suggested that 2 had no influence on gene translation and more likely affected the transcription step. HIV-1 gene transcription is driven by the long terminal repeat, a functional promoter with a low basal transcriptional activity and a high transcription level facilitated by the virusencoded regulatory Tat protein.24 To further identify the mechanism of 2, HIV-1 LTR reporter assays were used to test its effect on basal and Tat-mediated HIV-1 gene transcription.25 As shown in Figure 6, reporter gene expression driven by the

same strain (NL4-3) live virus infection assay in MT2 cells (Table 3). Murine leukemia virus (MLV) is a retrovirus that Table 3. Effect of 2 on HIV-1 and MLV Replication; n = 2, x̅ ±s IC50 (μM) VSVG/HIV-1

HIV-1(NL4-3)

VSVG/MLV

compound

MT-2

MT-2

293T

2 EFV

0.03 ± 0.001 0.001 ± 0.0004

5.4 ± 2.6 0.004 ± 0.0005

0.15 ± 0.06 >10

can infect mice and cause cancer. Thus, an MLV-based pseudovirus was constructed for speculating the antiretroviral spectrum of 2. The results showed that 2 also inhibited MLV replication with an IC50 value of 0.15 ± 0.06 μM (Table 3). The time of addition (TOA) assay was performed to explore the anti-HIV-1 mechanism of 2 by comparing its profile with that of well-known HIV replication inhibitors.23 As shown in Figure 5A, the reference compounds, zidovudine (AZT,

Figure 6. Compound 2 inhibited basal and Tat-mediated LTR transcription activity in a dose-dependent fashion. (A) Basal LTRdirected luciferase expression. HEK 293T cells were cotransfected with pLTR-Luc and pSV40-Renilla and treated with the tested compound. Luciferase activity was measured 48 h post-transfection by using a dual-luciferase assay system. (B) Tat-induced LTR-directed luciferase expression. HEK 293T cells were cotransfected with pLTR-Luc, pcDNA3.1-Tat, and pSV40-Renilla plasmids and cultured in the presence of 2. Luciferase activity was measured 48 h post-transfection. n = 2.

Figure 5. Compound 2 inhibited HIV-1 transcription. (A) Time of addition study. The tested compounds (AZT, EFV, and RAL at final concentrations of 1 μM and 2 at a final concentration of 0.1 μM) were added at the indicated time points after infection. Luciferase activity was monitored at 48 h postinfection as a measure of viral replication. Infectivity (%) was calculated by comparison with the control. (B) Inhibition of luciferase expression encoded in pNL4-3.Luc.R−E−. HEK 293T cells were transfected with the pNL4-3.luc.R−E− for 4 h. Transfected cells were treated with the tested compounds (AZT, EFV, and RAL, 1 μM; 2, 0.001−1 μM) for an additional 20 h before measuring the luciferase activity. (C) Effect of 2 on Renilla luciferase expression. HEK 293T cells were transfected with pSV40-Renilla and cultured in the presence of 2 at indicated concentrations. Luciferase activity was measured 24 h post-transfection. n = 2.

LTR promoter, in the absence or presence of Tat, was suppressed by 2 in a dose-dependent manner. This result illustrated that 2 inhibited HIV-1 replication by blocking viral transcription. In summary, seven oxazole-containing diterpenoids including four new compounds were isolated and characterized from S. miltiorrhiza cell cultures. Compounds 2 and 3 displayed strong anti-HIV-1 activities. Notably, compound 2 showed potent inhibitory activity on HIV-1 replication by blocking viral gene transcription. To date, there are no available antiretroviral agents that act on HIV transcription. Thus, this research might provide a promising new lead compound for research and

nucleoside reverse transcriptase inhibitor), efavirenz (EFV, non-nucleoside reverse transcriptase inhibitor), and raltegravir (RAL, integrase inhibitor), exhibited a time of 50% failure (FT50) at 7.7, 10.1, and 12.0 h, respectively. However, compound 2 maintained its inhibitory activity up to 20 h postinfection, which was distinctly different from all the 3244

DOI: 10.1021/acs.jnatprod.7b00659 J. Nat. Prod. 2017, 80, 3241−3246

Journal of Natural Products

Article

967, 819, and 772 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 380.3 [M + H]+; HR-ESIMS m/z 380.1627 [M + H]+ (calcd for C26H22NO2, 380.1645). Salvianan D (4): pale yellow powder; [α]25D − 11 (c 0.2, CHCl3); ECD (CHCl3) λmax (Δε) 251 (−1.63), 306 (−1.14), 330 (−1.30), and 357 (−0.37) nm; UV (MeOH) λmax (log ε) 220 (0.86), 239 (1.04), 306 (0.23), 319 (0.23), and 334 (0.26) nm; IR νmax 2960, 2875, 1599, 1459, 1401, 1067, 957, and 820 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 308.4 [M + H]+; APCIMS m/z 308.3 [M + H]+, 306.3 [M − H]−; HR-ESIMS m/z 308.1639 [M + H]+ (calcd for C20H22NO2, 308.1645). X-ray Crystallographic Analysis of Salvianan A (1). Lamellar crystals of 1 were obtained in CH2Cl2−MeOH (10:1). A suitable crystal was mounted on a Rigaku MicroMax diffractometer using Cu Kα. The structure was solved by direct methods with SHELXS-97. All non-hydrogen atoms were refined anisotropically with anisotropic displacement parameters. All hydrogen atoms were fixed at calculated positions. The crystallographic data have been submitted to the Cambridge Crystallographic Data Center (CCDC 901834) for 1. C20H17NO2, M = 303.31, triclinic, space group P1, crystal dimensions 0.34 × 0.73 × 0.93 mm, Cu Kα radiation, a = 7.301(2) Å, b = 10.496(7) Å, c = 10.861(1) Å, α = 70.26(8)°, β = 77.00(6)°, γ = 81.658(10)°, V = 761.1(5) Å3, Z = 2, Dcalcd = 1.324 g·cm−3; 2863 reflections measured, 2833 independent reflections unique. Flack parameter = −0.0(2). The final values were R1 = 0.0455, wR2 = 0.1175(w = 1/σ|F|2), S = 1.042. Cell Culture, Plasmids, and Viruses. HEK 293T and MT-2 cells were grown in DMEM and RPMI 1640, respectively, both supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. pMX-Luc was constructed by replacing the GFP reporter gene of pMX-GFP with a firefly luciferase gene. The pLTR-Luc was obtained by subcloning the pNL4-3.Luc.R−E− 5′ LTR region (1−789 bp) into the pGL3-Basic vector (Promega, USA). The pcDNA3.1-Tat plasmid was generated by inserting the full-length HIV-1 Tat coding sequence into the pcDNA3.1 vector (Invitrogen, USA). VSV-G/HIV-1 pseudotyped viruses were prepared as described previously.26 VSV-G/MLV pseudotyped viruses were prepared by cotransfecting the VSV-G with pHIT60 and pMX-Luc into HEK 293T cells.27 The viral particles were harvested 48 h postinfection and filtered through a 0.45 μm filter. Antiretroviral Activity Assay with Pseudotyped Viruses.26 HEK 293T or MT-2 cells were plated in 24-well plates at a density of 6 × 104 cells per well on the day prior to infection. Compounds were added to the target cells 15 min ahead of VSV-G/HIV-1 or VSV-G/ MLV infection. Cells were lysed 48 h postinfection, and the substrate (Promega, cat. #E1501, USA) was added into the cell lysate. A Sirius luminometer (Berthold Detection System) was used to measure the luciferase activity according to its standard procedures. The same amount of DMSO was used as a solvent control for the tested compounds’ infectivity calculation. EFV was used as the reference control. Anti-HIV-1 Activity Assay through Multiple Rounds of HIV-1 Infection. MT-2 cells were seeded into a 384-well plate and infected with HIV-1 (NL4-3 strain) carrying a β-galactosidase (β-Gal) reporter gene at a multiplicity of infection (MOI) of 0.01. The compounds were dissolved in DMSO and added to the test wells. The β-Gal expression level was tested at 72 h postinfection with a FluorAce β-Gal reporter assay (Bio-Rad, USA). Cytotoxicity Assay.28 The tested compounds were added to HEK 293T or MT-2 cells and incubated for 48 h, and the same amount of solvent was added to the control. The cell viabilities were measured using the cell proliferation assay reagent (Promega, cat. #G111A, USA) according to the manufacturer’s instructions. Time of Addition Study.28 HEK 293T cells were seeded in 24well plates and infected with VSVG/HIV-1 followed by adding tested compounds at different time points. Compound 2 was added at a final concentration of 0.1 μM, and AZT, EFV, and RAL were used as

development on HIV-1 transcription inhibitors, which could serve as a novel anti-HIV-1 therapeutic strategy.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with a PerkinElmer model-343 polarimeter. IR spectra were measured on a Nicolet 5700 FT-IR microscope spectrometer (FTIR microscope transmission). The ECD data and UV spectra were obtained from a JASCO J-815 spectropolarimeter. ESIMS and HRESIMS data were recorded on an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS spectrometer. NMR spectroscopic data were acquired on Mercury-300, Mercury-Plus-400, and Bruker ARX-600 instruments. Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, China). Semipreparative HPLC was conducted on a Shimadzu HPLC instrument with a Shimadzu RID-10A detector. A Grace Allsphere silica column (10 mm × 250 mm, 5 μm) and Grace Adsorbosphere C18 column (10 mm × 250 mm, 5 μm) were used for HPLC separation. Analytical TLC was carried out on silica gel GF254 plates (Qingdao Marine Chemical Factory, Qingdao, China), and spots were visualized by UV light (254 nm) or stained with 10% H2SO4 in EtOH followed by heating. Tissue and Cell Culture of Salvia miltiorrhiza. The tissue and plant cell culture of S. miltiorrhiza were performed as described previously.20 Extraction and Isolation. The dried cell cultures of S. miltiorrhiza (1.2 kg) were extracted with 80% EtOH (2 L, 3 h × 3). After evaporating the solvent under reduced pressure, the resulting extract (370 g) was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. The petroleum ether sample (41 g) was initially chromatographed over silica gel CC (400 g, 200− 300 mesh), eluting with a gradient system of petroleum ether−acetone to obtain eight fractions. Fraction 1 (7.1 g) was subjected to silica gel column (210 g, 200−300 mesh) and eluted with petroleum ether− EtOAc (100:0−80:20, gradient system) to afford six fractions (1.1− 1.6). Fraction 1.2 (2.5 g) was treated similarly to yield seven fractions (1.2.1−1.2.7). Fraction 1.2.2 (230 mg) was separated using semipreparative HPLC (MeOH−H2O, 97:3) to give 6 (3 mg, tR = 12.9 min). Fraction 1.2.3 (730 mg) was purified using normal-phase HPLC (n-hexane−EtOAc, 100:0.5, v/v) to afford 1 (50 mg, tR = 25.9 min) and 5 (110 mg, tR = 33.4 min). The residue from the column was separated by reversed-phase HPLC (MeOH−H2O, 97:3) to yield 2 (14 mg, tR = 7.9 min) and 4 (5 mg, tR = 9.1 min). Fraction 1.2.4 was separated by RP-HPLC (MeOH−H2O, 97:3) to afford 3 (6 mg, tR = 13.1 min). Fraction 1.3 (1.3 g) was fractionated on silica gel CC (45 g, 200−300 mesh) by eluting with petroleum ether−acetone (100:0− 85:15, gradient system) to give four fractions (3.1−3.4). Fraction 3.2 (460 mg) was purified using semipreparative HPLC (n-hexane− EtOAc, 15:1) followed by Sephadex LH-20 CC (CHCl3−MeOH, 50:50) to afford 7 (3 mg, tR = 29.2 min). Salvianan A (1): pale yellow lamellar crystals (CH2Cl2−MeOH, 10:1); mp 203−204 °C; [α]25D −8 (c 0.2, CHCl3); ECD (CHCl3) λmax (Δε) 291 (−1.61), 351 (−0.32), and 368 (−0.36) nm; UV (MeOH) λmax (log ε) 223 (1.12), 252 (0.80), 289 (0.90), 335 (0.09), 351 (0.17), and 368 (0.21) nm; IR νmax 2971, 2940, 2876, 1600, 1586, 1463, 1423, 1290, 1117, 968, 817, and 783 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 304.22 [M + H]+; and HR-ESIMS m/z 304.1318 [M + H]+ (calcd for C20H18NO2, 304.1332). Salvianan B (2): pale yellow powder; [α]25D −7 (c 0.2, CHCl3); ECD (CHCl3) λmax (Δε) 268 (−0.42), 292 (−0.99), and 365 (−0.58) nm; UV (MeOH) λmax (log ε) 222 (1.04), 253 (0.71), 288 (0.80), 333 (0.08), 349 (0.15), and 366 (0.18) nm; IR νmax 2977, 2920, 2874, 1597, 1466, 1422, 1278, 1065, 968, 818, and 781 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 288.3 [M − H]−; APCIMS m/z 290.3 [M + H]+, 288.3 [M − H]−; HR-ESIMS m/z 290.1167 [M + H]+ (calcd for C19H16NO2, 290.1176). Salvianan C (3): pale yellow powder; [α]25D −11 (c 0.2, CHCl3); ECD (CHCl3) λmax (Δε) 275 (−1.01), 295 (−1.50), 332 (−0.57), and 365 (−0.57) nm; IR νmax 2960, 2923, 1600, 1573, 1465, 1423, 1115, 3245

DOI: 10.1021/acs.jnatprod.7b00659 J. Nat. Prod. 2017, 80, 3241−3246

Journal of Natural Products

Article

reference compounds with the final concentration of 1 μM. The luciferase activity of the cell lysate was monitored at 48 h postinfection. Transient Gene Expression Assay.25 The pNL4-3.luc.R−E− or pSV40-Renilla plasmid was transfected to HEK 293T cells seeded in a 24-well plate using the jetPRIME reagent (Polyplus, cat. #114-15, France) for 4 h. The test compounds were added to the wells, and viral gene transcription was determined by measuring the luciferase activity at 24 h post-transfection. HIV-1 LTR-Luciferase Reporter Assay.25 One hundred nanograms of pLTR-Luc and 2 ng of pSV40-Renilla with or without 70 ng of pcDNA3.1-Tat were transiently transfected into HEK 293T cells seeded in 24-well plates for 4 h. Compound 2 was added to the cell culture, and the luciferase activities of the cell lysates were measured 48 h post-transfection by using a dual-luciferase assay kit (Promega, cat. #E1910, USA) according to its standard procedures. The Renilla luciferase was used as a control reporter for normalizing transfection efficiencies.

■

(5) China Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China 2010 (Vol. I); China Medical Science Press: Beijing, 2010; p 71. (6) Xu, Y. Y.; Wan, R. Z.; Lin, Y. P.; Yang, L.; Chen, Y.; Liu, C. X. Asian J. Pharm. Pharmacokinet. 2007, 7, 99−130. (7) Chinese Materia Medica Committee. Chinese Materia Medica; Shanghai Science and Technology Press: Shanghai, 1999; pp 172−178. (8) Jiangsu New Medical College. Dictionary of Traditional Chinese Medicine; Shanghai Science & Technology: Shanghai, 1986; pp 478− 482. (9) Don, M. J.; Shen, C. C.; Syu, W. J.; Ding, Y. H.; Sun, C. M. Phytochemistry 2006, 67, 497−503. (10) Wang, X.; Bastow, K. F.; Sun, C. M.; Lin, Y. L.; Yu, H. J.; Don, M. J.; Wu, T. S.; Nakamura, S.; Lee, K. H. J. Med. Chem. 2004, 47, 5816−5819. (11) Haro, G.; Takenori, K.; Midori, O. I.; Hiroshi, K.; Zhao, W. J.; Chen, J.; Guo, Y. T. Tetrahedron Lett. 1988, 29, 4603−4606. (12) Wu, Y. J.; Hong, C. Y.; Lin, S. J.; Wu, P.; Shiao, M. S. Arterioscler., Thromb., Vasc. Biol. 1998, 18, 481−486. (13) Weng, X. C.; Gordon, M. H. J. Agric. Food Chem. 1992, 40, 1331−1332. (14) Huang, Y. S.; Zhang, J. T. Acta Pharm. Sin. (Yao Xue Xue Bao) 1992, 27, 96−100. (15) Zhang, K. Q.; Bao, Y. D.; Wu, P.; Rosen, R. T.; Ho, C. T. J. Agric. Food Chem. 1990, 38, 1184−1197. (16) Lin, H. C.; Ding, H. Y.; Chang, W. L. J. Nat. Prod. 2001, 64, 648−650. (17) Wang, N.; Luo, H. W.; Niwa, M.; Ji, J. Planta Med. 1989, 55, 390−391. (18) Lee, A. R.; Wu, W. L.; Chang, W. L.; Lin, H. C.; King, M. L. J. Nat. Prod. 1987, 50, 157−160. (19) Don, M. J.; Shen, C. C.; Lin, Y. L.; Syu, W. J.; Ding, Y. H.; Sun, C. M. J. Nat. Prod. 2005, 68, 1066−1070. (20) Zhang, D. W.; Liu, X.; Xie, D.; Chen, R.; Tao, X. Y.; Zou, J. H.; Dai, J. Chem. Pharm. Bull. 2013, 61, 576−580. (21) Lin, F. W.; Damu, A. G.; Wu, T. S. Heterocycles 2006, 68, 159− 165. (22) Berova, N.; Nakanishi, K.; Woody, R. W. Circular Dichroism: Principles and Applications; Wiley: New York, 1994; pp 413−442. (23) Daelemans, D.; Pauwels, R.; De Clercq, E.; Pannecouque, C. Nat. Protoc. 2011, 6, 925−933. (24) Jeeninga, R. E.; Hoogenkamp, M.; Armand-Ugon, M.; De Baar, M.; Verhoef, K.; Berkhout, B. E. N. J. Virol. 2000, 74, 3740−3751. (25) Wan, Z.; Chen, X. Retrovirology 2014, 11, 88. (26) Cao, Y. L.; Li, S. X.; Chen, H.; Guo, Y. Acta Pharm. Sin. (Yao Xue Xue Bao) 2009, 44, 355−361. (27) Zhang, R.; Bloch, N.; Nguyen, L. A.; Kim, B.; Landau, N. R. PLoS One 2014, 9, e89558. (28) Ma, X.; Li, L.; Zhu, T.; Ba, M.; Li, G.; Gu, Q.; Guo, Y.; Li, D. J. Nat. Prod. 2013, 76, 2298−2306.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00659. NMR, MS, IR, UV, and ECD spectra of 1−4 and the single-crystal X-ray diffraction analysis of compound 1 (PDF) X-ray crystallography data for 1 (CIF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel (Y. Guo): 86-10-63161716. Fax: 86-10-63161716. E-mail: [email protected]. *Tel (J. Dai): 86-10-63165195. Fax: 86-10-63017757. E-mail: [email protected]. ORCID

Dewu Zhang: 0000-0001-6289-0617 Liyan Yu: 0000-0002-8861-9806 Jungui Dai: 0000-0003-2989-9016 Author Contributions §

D. Zhang and J. Guo contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the CAMS Innovation Fund for Medical Sciences (Nos. 2016-I2M-3-012, 2016-I2M-2-002, 2016-I2M-3-014, and 2016-I2M-1-014), Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (No. BZ0150), the National Science and Technology Major Project (No. 2015ZX09102-023-004), the National Natural Basic Research Program of China (Nos. 81473256 and 81273561), the National Infrastructure of Microbial Resources (No. NIMR-2017-3), and the Science and Technology Program of Beijing (No. Z151100000115008).

■

REFERENCES

(1) World Health Organization Home Page. http://www.who.int/ hiv/en. (accessed July 5, 2017). (2) U.S. Food & Drug Administrationhome Home Page. https:// www.fda.gov/ForPatients/Illness/HIVAIDS/Treatment/ucm118915. htm (accessed July 5, 2017). (3) Wu, Y.; Marsh, J. W. Microbes Infect. 2003, 5, 1023−1027. (4) Guo, Z. Acta Pharm. Sin. B 2017, 7, 119−136. 3246

DOI: 10.1021/acs.jnatprod.7b00659 J. Nat. Prod. 2017, 80, 3241−3246