Phthalide Derivatives with Anticoagulation Activities from Angelica

Jul 11, 2016 - Their structures were elucidated using HRMS, NMR, and X-ray crystallographic data. Compound 1 is the first example of a phthalide trime...
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Phthalide Derivatives with Anticoagulation Activities from Angelica sinensis Lai-Bin Zhang, Jie-Li Lv,* and Jun-Wei Liu School of Pharmacy, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China S Supporting Information *

ABSTRACT: Two new phthalide derivatives, angesinenolides A and B (1 and 2), were isolated from the roots of Angelica sinensis. Their structures were elucidated using HRMS, NMR, and X-ray crystallographic data. Compound 1 is the first example of a phthalide trimer presumably formed through two [2+2] cycloaddition reactions. Compound 2 is a unique dimeric phthalide with a peroxy bridge between C-3a and C-6. Both phthalides were evaluated for in vitro anticoagulation activities. Compound 1 reduced the level of fibrinogen (FIB). Compound 2 significantly extended thrombin time and activated partial thromboplastin time, as well as markedly reduced the content of FIB.

1

H−1H COSY data exhibited the presence of a butylidene side chain [δH 5.28 (1H, t, J = 7.8 Hz, H-8″), 2.28 (2H, br dd, J = 15.2, 7.8 Hz, H2-9″), 1.45 (2H, overlapped, H2-10″), and 0.93 (3H, t, J = 7.4 Hz, H3-11″)], two propyl side chains [δH 1.51 (2H, overlapped, H2-9), 1.10 and 1.19 (each 1H, m, H2-10), and 0.83 (3H, t, J = 7.2 Hz, H3-11); δH 1.43 (2H, overlapped, H2-9′), 0.99 (2H, m, H2-10′), and 0.84 (3H, t, J = 7.0 Hz, H311′)], and two olefinic protons [δH 6.01 and 6.02 (each 1H, H6′ and H-7′)]. In the 13C NMR and DEPT spectra, 36 carbon signals were observed corresponding to three ester carbonyls [δC 169.9 (C-1), 169.3 (C-1′), and 168.6 (C-1″)], three methyls [δC 14.6 (C-11), 14.5 (C-11′), and 14.2 (C-11″)], two oxygenated sp3 tertiary carbons [δC 94.1 (C-3) and 93.8 (C3′)], 10 olefinic carbons, six sp3 methines, and 12 sp3 methylenes. The above information indicated that 1 is a trimeric phthalide. The structure of 1 was constructed on the basis of the 2D NMR data. All proton signals could be assigned to the attached carbons through an HSQC experiment (Table 1). Four protonbearing structural fragments as shown in bold in Figure 2a, corresponding to H3-11″/H2-10″/H2-9″/H-8″, H2-4″/H2-5″/ H-6″/H-7″/H-7/H-6/H2-5/H2-4, H3-11/H2-10/H2-9/H-8/H8′/H2-9′/H2-10′/H3-11′, and H2-4′/H2-5′/H-6′/H-7′, were evident in the 1H−1H COSY spectrum. The monomeric phthalide A unit was deduced from the HMBC correlations of H2-9″/C-11″, C-10″, C-8″, C-3″; H-8″/C-10″, C-9″, C-3″, C3a″; H2-4″/C-3a″, C-6″, C-7a″; and H-7″/C-7a″, C-3a″ (Figure 2a). In a similar fashion, the phthalide B and C units were deduced from the HMBC correlations. The 1H−1H COSY correlations of H-6″/H-6 and H-7″/H-7 revealed that units A

Angelica sinensis (Oliv.) Diels (Apiaceae) is a fragrant and perennial herb mainly cultivated in the Gansu, Yunnan, and Sichuan Provinces of western China. The dried root of A. sinensis, known as “Dang Gui”, was first recorded in ‘“Shen Nong Ben Cao Jing”’ in the Han Dynasty (A.D. 25−225).1,2 It has been commonly used to cure cardiovascular diseases and gynecological dysfunction in Asia.3 Phytochemical studies on A. sinensis have led to the isolation of phthalides, organic acids, and polysaccharides, showing neuroprotection as well as antiplatelet and immunomodulatory effects.4−7 Particularly, the phthalides from the roots of A. sinensis exhibited anticoagulation and antiplatelet aggregation activities in the hyperviscosity rat model.8 n-Butylidenephthalide also exhibited antiplatelet and antianginal activities.9−11 These results initiated an investigation into the anticoagulation-active phthalides of A. sinensis, which led to the isolation and characterization of two new phthalides (1 and 2). Compound 1 is the first example of a phthalide trimer. Compound 2 is a unique phthalide dimer with a peroxy bridge between C-3a and C-6. Herein, the isolation and structural elucidation of the new compounds and their anticoagulation activities are described. The 95% aqueous EtOH extract of the roots of A. sinensis was suspended in H2O and partitioned with CH2Cl2. The CH2Cl2soluble portion was subjected to silica gel and Sephadex LH-20 column chromatography, followed by semipreparative HPLC to afford the phthalide trimer (1) and dimer (2). Compound 1 was obtained as colorless crystals. Its molecular formula was determined as C36H42O6 by the [M + Na]+ ion at m/z 593.2862 (calcd for C36H42O6Na, 593.2874) in the HRESIMS and 13C NMR spectroscopic data, indicating 16 indices of hydrogen deficiency. The IR spectrum showed an absorption band for a five-membered α,β-unsaturated lactone carbonyl (1767 cm−1) group. The 1H NMR (Table 1) and © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 28, 2016

A

DOI: 10.1021/acs.jnatprod.6b00080 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H and 13C NMR Data (400 and 100 MHz, in Acetone-d6) for Compounds 1 and 2 (δ in ppm, J in Hz) 1 δH

position 1 2 3 3a 4 5 6 7 7a 8 9 10

2.35a 2.46a 1.68 m 1.91a 2.83a 3.31 t (8.1) 2.81a 1.51a 1.10 m 1.19 m 0.83 t (7.2)

2 δC

94.1 163.8 23.9

92.3 83.2 23.6

22.3 36.4 32.1 129.3 46.1 31.7 21.2

2.69 m

5′

2.36a

6′ 7′ 7a′ 8′

6.01a 6.02a 2.73a

131.6 117.2 126.4 45.5

9′

1.43a

31.6

10′ 11′ 1″ 2″ 3″ 3a″ 4″

0.99 m 0.84 t (7.0)

5″ 6″ 7″ 7a″ 8″ 9″ 10″ 11″ 1′-OCH3 a

5.28 t (7.8) 2.28 br dd (15.2, 7.8) 1.45a 0.93 t (7.4)

δC 166.3

11 1′ 2′ 3′ 3a′ 4′

2.44a 2.50a 1.78 m 1.92a 3.07 m 3.40 t (8.1)

δH

169.9

14.6 169.3 93.8 159.8 22.4 23.23

21.1 14.5 168.6

1.62a 1.79a 1.50a 2.17a 4.94 dt (4.7, 1.1) 7.30 d (6.1) 2.05a 1.17a 1.37a 1.17a 1.30a 0.87 t (7.3)

1.84a 2.21a 1.34a 1.99 m 2.93 m 7.81 d (7.3) 2.28a 2.46 ddd (15.8, 10.7, 4.8) 1.48a 1.58a 1.30a 0.86 t (7.2)

24.8 72.9 133.4 137.3 39.2 30.3

Figure 1. Structures of compounds 1 and 2.

21.3 14.3 165.0 210.5 59.5 28.7 17.4 34.0 153.8 134.5 42.2

Figure 2. (a) 1H−1H COSY and key HMBC correlations for 1. (b) Selected NOESY correlations for 1.

27.8

to C-6/C-7, from H2-5 to C-6/C-6″, from H-6 to C-5/C-7/C7″, and from H-7 to C-6″/C-7″/C-3a/C-7a further supported the above deduction. Similarly, phthalide unit C was deduced to be connected to the B unit via C-3/C-3′ and C-8/C-8′, on the basis of HMBC correlation of H2-9 and C-8/C-3/C-8′; of H-8 and C-3/C-3a/C-8′/C-3′; of H2-9′ and C-8′/C-3′/C-8; and of H-8′ and C-3′/C-3a′/C-8/C-3. The relative configuration of 1 was established on the basis of NOESY data (Figure 2b). The key NOESY correlations between H-6″ and H-7″/H-7 and between H-6 and H-7/H7″ implied the all-cis configuration of the cyclobutane ring connecting phthalide units A and B. The Z-configuration of the enolic double bond of unit A was confirmed by the NOESY correlations of H-8″ and H-4″. The NOESY correlations of H8/H-9′ and H-9/H-8′ implied an 8,8′-trans configuration. The configurations of the C-3 and C-3′ spiro centers were assigned by the NOESY correlations of H-8/H-4 and H-8′/H-4′. The relative configuration and structure of 1 was further confirmed by a single-crystal X-ray diffraction experiment performed with Cu Kα (λ = 1.541 78 Å) radiation (Figure 3). However, the crystal structure of 1 was found to exhibit centrosymmetric space group P121/c1, suggesting its crystallization as a racemate. Thus, the structure of angesinenolide A (1), the first trimeric phthalide, was defined as shown. Compound 2 was isolated as colorless crystals with a molecular formula of C25H32O7, as deduced from the 13C NMR and HRESIMS data (m/z 467.2045 [M + Na]+, calcd for

23.1 14.5

150.0 153.2 20.3 20.7 34.5 33.9 127.9 110.5 28.6 23.18 14.2 3.72 s

52.6

Signals overlapped.

and B were linked via C-6″/C-6 and C-7″/C-7, with the fourmembered ring presumably formed through a [2+2] cycloaddition reaction. The HMBC correlations from H2-5″ to C6″/C-6, from H-6″ to C-5″/C-7″/C-7a″/C-6/C-7, from H-7″ B

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and C-3. The aforementioned data suggested the absence of a five-membered α,β-unsaturated lactone moiety in the phthalide unit, contrary to its presence in a typical phthalide.12 Additionally, the presence of the peroxy bridge between C-3a and C-6 was inferred from the two oxygenated carbon signals at δC 72.9 (C-6) and 83.2 (C-3a) in the 13C NMR spectrum and the molecular formula with 10 indices of hydrogen deficiency. The HMBC correlations from H2-9 to C-8/C-6′, from H2-5′/ H-7′ to C-8, and from H2-4′/H-6′ to C-3 indicated that the monomeric phthalide units are connected through C-8/C-6′ and C-3/C-3a′. The NOESY correlations of H-8 and H-6′, H-9 and H-4′/H-5′, and H-8′ and H-4/H-5 (Figure 4b) confirmed the overall relative configuration shown in Figure 1. This was further confirmed by a single-crystal X-ray diffraction experiment performed with Cu Kα (λ = 1.541 78 Å) radiation (Figure 5). The crystal structure of 2 was found to exhibit

Figure 3. ORTEP drawing of compound 1.

467.2040). The IR spectrum indicated the presence of a carbonyl (1715 cm−1) and an α,β-unsaturated lactone (1778 cm−1) functionality. The 1H NMR data (Table 1) showed signals for two olefinic protons at δH 7.30 (1H, d, J = 6.1 Hz, H7) and 7.81 (1H, d, J = 7.3 Hz, H-7′), two methyls at δH 0.86 (3H, t, J = 7.2 Hz, H3-11′) and 0.87 (3H, t, J = 7.3 Hz, H3-11), and a methoxy group at δH 3.72 (3H, s, 1′-OCH3). The 13C NMR, DEPT, and HSQC spectra exhibited 25 carbon signals, comprising three carbonyls [δC 210.5 (C-3′), 166.3 (C-1), and 165.0 (C-1′)], two methyl [δC 14.5 (C-11′) and 14.3 (C-11)], one methoxy [δC 52.6 (1′-OCH3)], two oxygenated sp3 tertiary carbons [δC 92.3 (C-3) and 83.2 (C-3a)], one sp3 quaternary carbon [δC 59.5 (C-3a′)], four olefinic carbons, nine sp3 methylenes, and three sp3 methines. The above information suggested that compound 2 is a dimeric phthalide. Two phthalide units were deduced from analysis of the 1 H−1H COSY and HMBC spectra (Figure 4a). The HMBC

Figure 5. ORTEP drawing of compound 2.

centrosymmetric space group P1̅, suggesting crystallization as a racemate. Consequently, the structure of compound 2, angesinenolide B, was elucidated as shown. This is the first report of a phthalide dimer with a peroxy bridge. The phthalide derivatives were evaluated for their in vitro anticoagulation activities on the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB). As shown in Figures 6 and 7, compound 2 significantly extended APTT and TT, as well as markedly reduced the content of FIB compared with the DMSO control. However, 2 had no significant differences in PT. Compound 1 did not show any effects in the TT, APTT, and PT experiments, but reduced the level of FIB compared with the DMSO control. PT and APTT values reflect the activities of the extrinsic and intrinsic pathways of coagulation. TT and FIB are related to the third coagulation phase in plasma, and the prolongation of TT or reduced level of FIB indicates inhibition of thrombinmediated fibrin formation.13,14 In this study, the results implied that compound 2 exhibited an anticoagulant effect by acting on intrinsic coagulation pathways and preventing fibrin formation. Compound 1 exerted a potential anticoagulation effect just through hindering fibrin formation.

Figure 4. (a) 1H−1H COSY and key HMBC correlations for 2. (b) Selected NOESY correaltions for 2.

correlations of the methoxy protons (δH 3.72) with C-1′ (δC 165.0) indicated that the methoxy group was located at C-1′. The deshielding of C-3′ (δC 210.5) indicated that C-3′ was oxidized into a carbonyl group. The presence of a pentanoyl side chain was verified by the HMBC correlations from Me-11′ to C-9′ and C-10′ and from H-8′ to C-9′, C-10′, and C-3′, together with the 1H−1H COSY spin system of H2-8′/H2-9′/ H2-10′/H3-11′. The pentanoyl side chain was located at C-3a′ via the HMBC correlations from H2-4′ to C-3′, C-3a′, C-7a′,

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with an X-4 micromelting point apparatus. Optical rotations were measured on a polAAr 31 polarimeter. UV spectra were obtained on a Shimadzu UV-2550 instrument. IR spectra were measured on a Shimadzu IRPrestige-21 spectrometer with KBr pellets. C

DOI: 10.1021/acs.jnatprod.6b00080 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 6. Anticoagulant activity of compound 1 (C1) and compound 2 (C2) at different concentrations with respect to APTT, PT, and TT in vitro. The activity is expressed as the clotting time in seconds (s). The concentration “C” of C1 and C2 was 1 mM. DMSO was used as the control. Heparin sodium (0.5 μM) was used as the positive control. Values are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the control group. 2012. The sample was identified by Prof. Sui-Qing Chen from School of Pharmacy, Henan University of Traditional Chinese Medicine. A voucher specimen (AS201212) has been deposited at the Herbarium of the School of Pharmacy, Xinxiang Medical University. Extraction and Isolation. The air-dried and milled roots of A. sinensis (18 kg) were percolated with 95% EtOH (180 L) at room temperature. The filtrate was concentrated under vacuum to obtain a residue (2.1 kg), which was suspended in H2O (4 L) and extracted with CH2Cl2 (5 × 2 L). The CH2Cl2 extract (293 g) was subjected to CC over silica gel eluted with a gradient of petroleum ether−EtOAc (100:0, 50:1, 30:1, 15:1, 8:1, 4:1, 2:1, and 1:1) to give fractions 1−25. Fraction 9 (8.4 g) was separated by CC over silica gel eluted with petroleum ether−acetone (15:1, 10:1, and 8:1) to yield seven subfractions, 9.1−9.7. Subfraction 9.6 was subjected to Sephadex LH-20 CC eluted with CH2Cl2−MeOH (1:1) to afford compound 2 (12 mg). Fraction 20 (9.5 g) was separated by silica gel CC eluted with petroleum ether−acetone (3:1 and 2:1) to yield eight subfractions, 20.1−20.8. Subfraction 20.3 was subjected to Sephadex LH-20 CC eluted with CH2Cl2−MeOH (1:1) to give subfractions 20.3.1−20.3.3. Subfraction 20.3.2 was further isolated by silica gel CC eluted with CH2Cl2−acetone (20:1) and purified by semipreparative HPLC (MeOH−H2O, 82:18) to afford compound 1 (9.8 mg, 29 min). Angesinenolide A (1): colorless crystals (acetone); mp 154−155 °C; [α]25 D +6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 234 (4.02), 281 (4.27) nm; IR (KBr) νmax 2959, 2932, 1767, 1672, 1456, 1275, 1061, 1038, 957 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 593.2862 [M + Na]+ (calcd for C36H42O6Na, 593.2874). Angesinenolide B (2): colorless crystals (acetone); mp 139−140 °C; [α]25 D +35 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.32) nm; IR (KBr) νmax 2955, 1778, 1715, 1248, 1227, 1184, 1113, 1078, 1061 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 467.2045 [M + Na]+ (calcd for C25H32O7Na, 467.2040). X-ray Crystallographic Analysis. Crystal data for compounds 1 and 2 were collected on a Bruker APEX-II CCD diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.541 78 Å). Their structures were solved by direct methods using the SHELXS-97 program and refined with full-matrix least-squares calculations on F2 using the SHELXL-97 program. All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were positioned by geometric idealization. Crystallographic data for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre (deposition numbers CCDC 1436309 for 1 and CCDC 1436319 for 2). The data can be obtained free of charge from the CCDC via www. ccdc.cam.ac.uk. Crystal data of angesinenolide A (1): C36H42O6, M = 570.69, monoclinic, space group P121/c1, a = 12.9007(4) Å, b = 22.9463(7) Å, c = 11.5098(3) Å, α = 90°, β = 114.2340(10)°, γ = 90°, V = 3106.92(16) Å3, T = 296.15 K, Z = 4, F(000) = 1224, μ(Cu Kα) = 0.655 mm−1, 23 062 reflections collected, 5642 independent reflections

Figure 7. Effect of compound 1 (C1) and compound 2 (C2) at different concentrations on FIB in vitro. The activity is expressed as the content of FIB in accurate value plasma (mg·dL−1). The concentration “C” of C1 and C2 was 1 mM. DMSO was used as the control. Heparin sodium (0.5 μM) was used as the positive control. Values are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with the control group. X-ray data were collected with a Bruker APEX-II CCD diffractometer using graphite-monochromated Cu Kα radiation. NMR spectra were recorded on a Bruker AVANCE-III HD 400 spectrometer. Chemical shifts (δ) are expressed in ppm with reference to the residual solvent signals for acetone-d6 (δH 2.05 and δC 29.9). HRESIMS was carried out in the positive ion mode with a Bruker microOTOF-Q III spectrometer. Semipreparative HPLC was performed on a Shimadzu CTO-20A instrument and a YMC C18 column (150 × 10 mm, 5 μm, YMC Co., Kyoto, Japan), using a UV detector set at 210 nm. Column chromatography (CC) was performed on silica gel (200−300 mesh, Yantai Institute of Chemical Technology, Yantai, People’s Republic of China) and Sephadex LH-20 gel (GE Healthcare Amersham Biosciences, Uppsala, Sweden). TLC analysis was done on precoated silica gel GF254 plates (10−40 μm, Yantai Institute of Chemical Technology, Yantai, People’s Republic of China). The anticoagulant activity was measured on a platelet aggregation and blood coagulation factor analyzer (LG-PABER-I, Steellex, Beijing, People’s Republic of China). TT (No. 121148), PT (No. 105A275), APTT (No. 112183), and FIB (No. 136079; the accurate value plasma, No. 897066, content 268 mg/dL) kits were obtained from Sun Biochemical Co. Ltd. (Shanghai, People’s Republic of China). DMSO and heparin sodium were purchased from AMRESCO, LLC. (Solon, OH, USA) and Macklin Biochemical Co. Ltd. (Shanghai, People’s Republic of China), respectively. Plant Material. The roots of A. sinensis were collected in Min County, Gansu Province, People’s Republic of China, in December D

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(Rint = 0.0374), final R1 = 0.0423 and wR2 = 0.1173 [for I > 2σ(I)], R1 = 0.0484 and wR2 = 0.1226 (for all data). The goodness of fit on F2 was 1.040. Crystal data of angesinenolide B (2): C25H32O7, M = 444.50, triclinic, space group P1̅, a = 10.1553(17) Å, b = 10.4128(17) Å, c = 12.5099(19) Å, α = 80.379(10)°, β = 69.257(7)°, γ = 70.505(7)°, V = 1164.4(3) Å3, T = 293(2) K, Z = 2, F(000) = 476, μ(Cu Kα) = 0.755 mm−1, 9709 reflections collected, 4086 independent reflections (Rint = 0.0235), final R1 = 0.0470 and wR2 = 0.1364 [for I > 2σ(I)], R1 = 0.0502 and wR2 = 0.1445 (for all data). The goodness of fit on F2 was 1.060. Anticoagulation Activity Assay. Male New Zealand white rabbits (Animal Experimental Center of Zhengzhou University, Zhengzhou, China) weighing 2.0 ± 0.2 kg were used. The animal experimental procedures were performed in accordance with the permission of the animal ethical committee of Xinxiang Medical University. Fresh blood samples were acquired from the common carotid artery and mixed with 3.8% sodium citrate (blood/citrate: 9:1, v/v); then the mixture was centrifuged for 10 min at 3000 rpm to obtain plasma.15,16 The samples were dissolved in DMSO, and DMSO was used as a control group. Heparin sodium was used as positive control. The LGPABER-I coagulation analysis instrument was used to estimate the TT, PT, APTT, and FIB. TT was measured by incubating 50 μL of plasma with 10 μL of compound for 3 min at 37 °C. Then 50 μL of thrombin was added, and the clotting time was recorded. PT was measured by incubating 50 μL of plasma with 10 μL of compound for 5 min at 37 °C. Then 100 μL of warmed thromboplastin agent was added, and the clotting time was recorded. For the APTT assay, 50 μL of plasma was mixed with 10 μL of compound for 1 min at 37 °C. Then 50 μL of APTT assay reagent was added to the mixture and incubated for 5 min at 37 °C. Finally, 50 μL of warmed 25 mM CaCl2 was added, and the clotting time was recorded. FIB was measured by incubating 80 μL of accurate value plasma provided in the kit with 10 μL of sample solution for 3 min at 37 °C; then 40 μL of fibrinogen agent was added, and the clotting time was recorded. All the results were expressed as mean ± standard deviation, three times in four channels. Statistical significance was determined with Student’s t test. The P-values of less than 0.05 (P < 0.05) were considered significant.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00080.

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IR, MS, and NMR spectra of 1 and 2 (PDF) X-ray crystallographic data of 1 (CIF) X-ray crystallographic data of 2 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-373-3831652. Fax: +86-373-3029879. E-mail: [email protected] (J.-L. Lv). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81202916 and 31400305), the Project for Youth Backbone Teachers in the University of Henan Province (2013GGJS-129 and 2015GGJS-130), the Science and Technology Project of Henan Province (152102310113), and the Science and Technology Project of the Education Department of Henan Province (14A360012). E

DOI: 10.1021/acs.jnatprod.6b00080 J. Nat. Prod. XXXX, XXX, XXX−XXX