Fitoterapia 121 (2017) 206–211
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Picrotoxane sesquiterpenoids from the stems of Dendrobium nobile and their absolute configurations and angiogenesis effect
MARK
Chun-Wang Meng, Yu-Lin He, Cheng Peng⁎, Xing-Jie Ding, Li Guo, Liang Xiong⁎ School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Dendrobium nobile Sesquiterpenoids Picrotoxane Absolute configuration Angiogenesis effect Transgenic zebrafish
Five picrotoxane sesquiterpenoids belonging to the unusual dendrobine-type (1 and 4) and the picrotoxinin-type (2, 3, and 5) were isolated from the stems of Dendrobium nobile Lindl. Their structures were established by spectroscopic analyses and physical properties. Compound 1 was a new dendrobine analogue. Although the planar structure of 2 and 3 had been reported, their absolute configurations were first determined by singlecrystal X-ray diffraction and circular dichroism. Compound 2 exhibited angiogenesis effect against sunitinibinduced damage on intersegmental blood vessels in Tg (flk1: EGFP) and Tg (fli1: nEGFP) transgenic zebrafish at concentrations of 3.13, 6.25, 12.50, and 25.00 μM.
1. Introduction Picrotoxanes are a group of sesquiterpenes, sesquiterpene alkaloids, and nor-diterpenes with highly complex structures [1], only isolated from Menispermaceae [2], Coriariaceae [3], Picrodendraceae [4], Euphorbiaceae [5], and Orchidaceae families [6], and animals [7,8]. The picrotoxanes in Orchidaceae are mainly distributed in the genus Dendrobium, mostly with bicyclic or tricyclic ring system and up to 7 stereogenic centers. Particularly, dendrobine-type alkaloids are a class of characteristic picrotoxanes that are distributed only in the genus Dendrobium. To the best of our knowledge, although there are > 1500 species in Dendrobium, dendrobine-type alkaloids have been only found in D. nobile [9], D. findlayanum [10], D. friedricksianum [11], D. hildebrandii [11], D. moniliforme [12], and D. wardianum [13]. In our previous study, lignan, phenylpropanoid, and flavonoid glucosides were characterized from the n-BuOH soluble portion of the ethanolic extract of Dendrobium aurantiacum var. denneanum, but no picrotoxane sesquiterpenoids and alkaloids were discovered [14–16]. This phytochemical investigation was expected to obtain unusual picrotoxanes from D. nobile, one of the three major plant sources of the traditional Chinese medicine Dendrobii Caulis (Shihu in Chinese) that is rich in dendrobine (4) [17,18]. Most of picrotoxanes contain multiple chiral carbons, and the absolute configurations of many reported picrotoxanes have not been determined, such as dendronobilin C, D, K, L, and M [19,20], dendroside F and G [21], and dendronobiline A [22]. To establish the absolute
⁎
configurations of the new picrotoxane sesquiterpenoids isolated in this study (Fig. 1), NOESY, single-crystal X-ray diffraction, and circular dichroism (CD) experiments were conducted. Furthermore, the bioactivities of the isolates were also investigated in vivo and in vitro. Compound 2 exhibited significant angiogenesis effect against sunitinib-induced damage on intersegmental blood vessels in Tg (flk1: EGFP) and Tg (fli1: nEGFP) transgenic zebrafish. 2. Experimental 2.1. General Melting point was measured on a Büchi Melting Point M-565 (Buchi Corporation, Oldham, UK). Optical rotations were measured using a SGW®-2 automatic polarizer (Shanghai Precision Scientific Instrument Corporation, Shanghai, China). IR data were measured on Perkin-Elmer spectrum one FT-IR spectrometer (Perkin-Elmer, Waltham, USA) with KBr pellets. CD spectra were recorded on a J-815 CD spectrometer (JASCO Corporation, Tokyo, Japan). X-ray structure analyses were performed on a D8 Venture diffractometer (Bruker Corporation, Billerica, MA, USA). NMR spectra were obtained using an AVIIIHD-600 NMR spectrometer (Bruker Corporation, Billerica, MA, USA). Deuterated solvent peaks were used as NMR references. HRESIMS were measured using a Synapt G2 HDMS instrument (Waters Corporation Milford, MA, USA). Column chromatography was performed using silica gel (200–300 mesh; Yantai Institute of Chemical Technology,
Corresponding authors at: School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. E-mail addresses:
[email protected] (C. Peng),
[email protected] (L. Xiong).
http://dx.doi.org/10.1016/j.fitote.2017.07.017 Received 27 June 2017; Received in revised form 28 July 2017; Accepted 31 July 2017 Available online 01 August 2017 0367-326X/ © 2017 Published by Elsevier B.V.
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16
ROOC 17
H
11
HO
9 10
N 18 H
1 15O
3
O
9 1
HO
7
11
3
O
4
6
HO
7
H
14
13
12
1 R= H dendrine R= Me
O
O
N H
H
5
HO HO
1 2 3 4 5 6 7a 7b 8a 8b 9 10 11a
1b
4
3
δH
δC
δH
δC
– 3.42 d (3.0) 5.09 dd (6.0, 3.0) 2.27 m
51.6 68.7 78.2
– 3.56 brd (4.8) 4.25 brs
47.7 73.3 88.2
51.4 73.1 86.2
52.3
–
80.1
2.58 4.2) 2.14 6.0) 2.24 2.05 2.01 1.60 2.29 1.44 3.32
dd (6.0,
44.7
2.13 brd (3.0)
53.3
– 4.43 d (1.8) 4.66 brd (4.8) 2.34 dd (4.8, 4.2) 2.47 t (4.2)
dd (8.4,
44.8
1.95 m
46.6
2.96 m
46.3
m m m m m s m
33.4
25.4
52.3 21.2 20.4 179.9 37.8
2.02 m 1.78 m 1.89 m 1.25 m 2.68 m 1.13 s 3.56 dd (10.2, 9.0) 3.39 dd (10.2, 4.8) – 1.31 s 1.32 s
26.5
1.77 m 1.01 d (6.6) 1.03 d (7.2)
1.97 m 1.68 m 1.77 m 1.22 m 2.45 m 1.01 s 3.54 ddd (10.8, 7.2, 3.6) 3.18 (10.8, 6.0, 5.4) 1.74 m 0.82 d (6.6) 0.85 d (6.6) –
16b 17 18 OH-2 OH-4 OH-11
a b c
2.50 dd (15.6, 3.6) 2.46 dd (15.6, 6.0) 2.74 s
O
27.0 45.5 23.6 62.4
28.1 16.0 15.0 178.5
2.3. Extraction and isolation The stems of D. nobile (4 kg) were powdered and extracted with 95% EtOH (5 × 24 L) under reflux for 5 × 2 h. The EtOH extract was evaporated under reduced pressure to afford a black residue (640 g), which was suspended in water and partitioned sequentially with petroleum ether, ethyl acetate, and n-butanol. The n-butanol extract (180 g) was chromatographed over a macroporous adsorbent resin (D-101) column eluted with a gradient of increasing EtOH (0–90%) in H2O to afford six fractions (A–F). Fraction C (46 g) was subjected to a C18 silica gel column eluted with a gradient of increasing MeOH (5–100%) in H2O to give 11 fractions (C1–C11). Fraction C3 (1.7 g) was further separated by a Sephadex LH-20 column (30% MeOH in H2O) to yield two subfractions (C3-1 and C3-2). Subfraction C3-1 (0.8 g) was purified by repeated silica gel column chromatography, preparative TLC (CHCl2–MeOH, 10:1), and RP semipreparative HPLC (67% MeOH in H2O containing 0.2% diethylamine) successively to afford 1 (2.0 mg) and 4 (30.0 mg). Further purification of subfraction C3–2 (0.7 g) using silica gel column chromatography (CH2Cl2–MeOH, 50:1–5:1) and preparative TLC (CHCl2–Me2CO, 3:1), followed by semipreparative RP HPLC (50% MeOH in H2O) yielded 3 (12.0 mg) and 5 (22.0 mg). Compound 2 (8.0 mg) was crystallized in MeOH from fraction C7 by solvent evaporation. (−)-(1R,2S,3R,4S,5R,6S,9S,11R)-11-Carboxymethyldendrobine (1): white powder; [α]D25 − 40.1 (c 0.04, MeOH); IR (KBr) vmax 3446, 2948, 2854, 1776, 1603, 1458, 1384 cm− 1; CD (MeCN) 231.5 (Δε + 1.09) nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 322.2022 [M + H]+ (calcd for C18H28NO4, 322.2018) and 344.1834 [M + Na]+ (calcd for C18H27NO4Na, 344.1838). (+)-(1R,2S,3S,4R,5R,6S,9R)-2,4,11-Trihydroxypicrotoxane-3(15)lactone (2): colorless crystals (MeOH); m.p. 287–289 °C; [α]D25 + 14.2 (c 0.12, MeOH); IR (KBr) vmax 3324, 2958, 2873, 1781, 1463, 1290, 1043 cm− 1; CD (MeCN) 226 (Δε + 0.22) nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 307.1526 [M + Na]+ (calcd for C15H24O5Na, 307.1521) and 323.1266 [M + K]+ (calcd for C15H24O5K, 323.1261). (+)-(1R,2S,3R,4S,5R,6S,9R)-2,11,12-Trihydroxypicrotoxane-3(15)lactone (3): white powder; IR (KBr) vmax 3390, 2958, 2873, 1767, 1472, 1048 cm− 1; [α]D25 + 37.8 (c 0.06, MeOH); CD (MeCN) 226.5 (Δε + 0.78) nm; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 307.1522 [M + Na]+ (calcd for C15H24O5Na, 307.1521) and 323.1256 [M + K]+ (calcd for C15H24O5K, 323.1261).
54.7 47.6
28.2 46.7 22.3 63.5
69.9 30.4 30.1 181.9
178.4 33.9 5.80 d (4.8) 5.07 s 5.01 dd (5.4, 3.6)
Data (δ) were measured at 600 MHz for 1H and at 150 MHz for Data were measured in CD3OD. Data were measured in DMSO‑d6.
13
5
April 2013 and identified by Prof. Tingmo Zhang (Chengdu University of TCM, China). A voucher specimen (JCSH-20130420) was deposited at State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, Chengdu University of TCM.
3b
δC
60.7 31.8 72.7
H HO O
δH
31.7
H
OH
13
2
2c
11b 12 13 14 15 16a
O
O
Table 1 NMR data (δ) for compounds 1–3.a Position
Fig. 1. Structures of picrotoxane sesquiterpenoids (1–5) from D. nobile and dendrine.
H
OH
12 14
10
15O
2
H 5
HO
8
C, respectively.
Yantai, China), macroporous adsorbent resin (D-101; Anhui Sanxing Resin Technology Co., Ltd., Anhui, China), and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Semipreparative HPLC was performed on a 1220 Infinity LC instrument (Agilent Technologies Inc., CA, USA) equipped with an Ultimate XB-C18 (250 × 10 mm2) preparative column packed with C18 (5 μm). TLC was performed using glass plates precoated silica gel (GF254; Qingdao Marine Chemical Inc., Qingdao, China). Fluorescence images were obtained using a Leica M165-FC fluorescence stereomicroscopes (Leica Corporation, Wetzlar, Germany). Zebrafish embryos were cultured in MGC-100 constant temperature incubator (Shanghai Yiheng Instruments Co., Ltd., Shanghai, China).
2.4. X-ray crystallography data of 2
2.2. Plant material
C15H24O5, M = 284.34, orthorhombic, P212121, a = 9.1197(4) Å, b = 12.0474(5) Å, c = 12.7719(6) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1403.23(11) Å3, T = 170 (2) K, Z = 4, μ(Cu Kα) = 0.822 mm− 1, 12,725 reflections measured, 2572 independent
The stems of D. nobile were purchased from Sichuan WanAn Dendrobium Industrial Development Co., Ltd. (Chengdu, China) in 207
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C 16
B
11 9
+
-
E
F
A 6
2
10 3
14
(a)
Fig. 2. (a) Key NOESY correlations of 1; (b) Klyne's lactone sector rule of 1.
D
4
5
12
13
NOESY
(b) Klyne lactone sector district
reflections (Rint = 0.0211). The final R1 values were 0.0268 (I > 2σ(I)). The final wR(F2) values were 0.0693 (I > 2σ(I)). The final R1 values were 0.0273 (all data). The final wR(F2) values were 0.0697 (all data). The goodness of fit on F2 was 1.059. Flack parameter = − 0.01(4). CCDC number 1558355 for compound 2 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB21EZ, UK; fax: + 44 1223 336033; e-mail:
[email protected]).
embryos per group. The results are expressed as means ± standard error of the mean (SEM) from three independent experiments. Statistical significance of the data was determined using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests with GraphPad 5.0 Prism software (GraphPad, San Diego, CA, USA). 3. Results and discussion Compound 1 was isolated as a white powder. Its molecular formula was determined as C18H27NO4 with six degrees of unsaturation based on the quasi-molecular ions at 322.2022 [M + H]+ (calcd for C18H28NO4, 322.2018) and 344.1834 [M + Na]+ (calcd for C18H27NO4Na, 344.1838) in its HRESIMS. The IR spectrum of 1 exhibited strong absorptions for hydroxy (3446 cm− 1) and carbonyl (1776 cm− 1) functionalities. The 1H NMR spectrum of 1 exhibited characteristic signals attributed to four methyl groups (Table 1). In addition, the signals for three low-field methine protons at δH 5.09 (dd, J = 6.0, 3.0 Hz), 3.42 (d, J = 3.0 Hz), and 3.32 (m) in the 1H NMR spectrum suggested that they were connected to heteroatoms. The 13C NMR and DEPT spectra of 1 showed 18 carbon resonances that corresponded to the protonated units (4 × CH3, 3 × CH2, and 8 × CH) in addition to three quaternary carbons which were two carbonyl and an all‑carbon quaternary center. The above functionalities indicated that 1 was an analogue of dendrobine and dendrine (4) [9,24]. The main difference between dendrobine and 1 was that an additional carboxymethyl group was substituted at C-11 in 1. The planar structure was further confirmed by 2D NMR experiments. In particular, HMBC
2.5. Measurement of angiogenesis effect on zebrafish embryos The vascular green fluorescence transgenic Tg (flk1: EGFP) and Tg (fli1: nEGFP) zebrafish were supported by a Zebrafish experimental platform in Chengdu University of Traditional Chinese Medicine. Zebrafish husbandry and breeding were performed according to zebrafish book standard [23]. Adult zebrafish were maintained at 28.5 °C under a 14 h light/10 h dark cycle in fish water (pH 7.2–7.5 and conductivity 500–550 μs/cm). The embryos of Tg (flk1: EGFP) and Tg (fli1: nEGFP) zebrafish were obtained from spawning box with a partition and raised in cultivate water. The 10-hpf (10-hour-postfertilization) embryos were placed into 24-well plates (15 embryos per well) and treated with 1.56, 3.13, 6.25, 12.50 and 25.00 μM of compounds 1–5, respectively, then maintained at 28.5 °C in 1.5 mL cultivate water containing 1.6 μg/mL sunitinib to 48 hpf. At 48 hpf, fluorescence images were obtained using a Leica M165-FC fluorescence stereomicroscopes. All experiments were performed in triplicate using 15
Fig. 3. (a) Klyne's lactone sector rule of 2; (b) X-ray crystallographic structure of 2.
C B A
-
D
+E F
(a) Klyne lactone sector district
(b) X-ray crystallographic structure 208
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Fig. 4. Effect of compound 2 on the recovery of ISV angiogenesis in an injured Tg (flk1: EGFP) zebrafish model induced by sunitinib. (A–G) ISV of Tg (flk1: EGFP) zebrafish. (A) Untreated normal group (NOR). (B) Sunitinib-treated model group (SUB). (C) 1.56 μM, (D) 3.13 μM, (E) 6.25 μM, (F) 12.50 μM, and (G) 25.00 μM of 2 and 1.6 μg/mL of sunitinib. A1–G1 were 9.5fold images of A–G, respectively. (H) Data of ISV index of 2 in Tg (flk1: EGFP) transgenic zebrafish. ###p < 0.001 vs. NOR, ⁎⁎⁎p < 0.001 vs. SUB.
correlations of H2-16 with C-9, C-11, and C-17, together with 1H-1H COSY correlations between H2-16 and H-11 verified the substitution of a carboxymethyl group at C-11. NOESY correlations of H2-16 with H-9; of H-9 with H3-10; of H3-10 with H-2, H-6, and H-12; of H-2 with H-3, H-12, and H3-14; of H-6 with H-5, H-12, and H3-13; of H-3 with H3-14; and of H-5 with H3-13 (Fig. 2) indicated that H-2, H-3, isopropyl-4, H-5, H-6, H-9, H3-10, and carboxymethyl-11 were on the same orientation. Compound 1 is the free acid of dendrine, and the negative specific optical rotation {[α]D25 − 40.1 (c 0.04, MeOH)} of 1 was consistent with that of dendrine, a synthetic product with the same relative configuration as 1 [25]. Thus, the 1R,2S,3R,4S,5R,6S,9S,11R configuration was assigned for 1. This was supported by a positive Cotton effect at
231.5 nm in the CD spectrum on the basis of the Klyne's lactone sector rule [26] (Fig. 2). Therefore, compound 1 was determined to be (−)-(1R,2S,3R,4S,5R,6S,9S,11R)-11-carboxymethyldendrobine. Compound 2 was isolated as colorless crystals (MeOH) with a positive optical rotation. Its molecular formula, C15H24O5, was indicated by HRESIMS. The 1H NMR spectrum of 2 displayed resonances attributable to a tertiary and two secondary methyl groups, an oxymethylene group, two oxymethines, three exchangeable hydroxy protons, and several aliphatic methylenes and methines (Table 1). The 13C NMR and DEPT spectra of 2 revealed 15 carbon resonances corresponding to the above protonated units and three quaternary carbons (one ester carbonyl group, δC 178.5). The above spectroscopic data suggested that 2 Fig. 5. Effect of compound 2 on promoting the proliferation of ISV endothelial cells in an injured Tg (fli1: nEGFP) zebrafish model induced by sunitinib. (A–G) Fluorescence spots of endothelial cell nuclei in ISV of Tg (f fli1: nEGFP) zebrafish. (A) Untreated normal group (NOR). (B) Sunitinib-treated model group (SUB). (C) 1.56 μM, (D) 3.13 μM, (E) 6.25 μM, (F) 12.50 μM, and (G) 25.00 μM of 2 and 1.6 μg/mL of sunitinib. A1–G1 were 1.575-fold images of A–G, respectively.
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to repair sunitinib-induced ISV damage in Tg (fli1: nEGFP) zebrafish embryos via promoting the proliferation of ISV endothelial cells (Fig. 5D–G). The number of fluorescence spots in ISV increased in a dose-dependent manner.
had the same planar structure as dendronobilin B [19]. This deduction was further confirmed by 2D NMR data analysis. Since the absolute configuration of dendronobilin B has not been solved yet, a comprehensive analysis of ROESY, CD, and single-crystal X-ray diffraction was conducted. ROESY correlations of H-2 with H-3, of H-5 with H-6, and of both H-2 and H-6 with H3-10, together with the small coupling constants of J2,3 (≈ 0 Hz) and J5,6 (3.0 Hz) indicated that H-2, H-3, H-5, H6, and H3-10 were placed on the same face. In addition, ROESY correlations of H-2 and H-3 with H3-14, of H-5 and H-6 with H3-13, and of H2-11 with H3-10 further established the relative configuration of C-4 and C-9. The above spectroscopic and optical rotation data of 2 were consistent with those of dendronobilin B. However, the absolute configuration of dendronobilin B has not been reported yet. In the CD spectrum of 2, a positive Cotton effect at 226 nm revealed that 2 had 1R,2S,3S,4R,5R,6S,9R configuration by application of the Klyne's lactone sector rule (Fig. 3). This absolute configuration was further confirmed by a single-crystal X-ray diffraction experiment using Cu Kα radiation (Fig. 3). Therefore, compound 2 (dendronobilin B) was determined to be (+)-(1R,2S,3S,4R,5R,6S,9R)-2,4,11-trihydroxypicrotoxane-3(15)-lactone. Compound 3 showed the similar NMR data to those of 2 and had the same molecular formula as 2. Detailed comparison of the NMR data between 3 and 2 revealed that the hydroxy group at C-4 in 2 moved to C-12 in 3. In addition, the spectroscopic data of 3 matched with those of dendrodensiflorol [27]. However, the absolute configuration of dendrodensiflorol was also undetermined. What's more, its specific optical rotation was not provided. Thus, it was difficult to determine whether compound 3 was dendrodensiflorol. In the ROESY spectrum of 3, correlations of H3-10 with H-2, H-6, and H2-11, of H-2 with H3-14, of H-3 with H3-14, of H-5 with H-6 and H3-13, and of H-6 with H3-13 indicated that 3 had the same relative configuration as 2. Using the same lactone sector rule as described for 1 and 2, the 1R,2S,3R,4S,5R,6S,9R configuration of 3 was elucidated. Therefore, compound 3 was determined to be (+)-(1R,2S,3R,4S,5R,6S,9R)-2,11,12-trihydroxypicrotoxane-3(15)lactone. The known compounds were identified as dendrobine (4) [24] and findlayanin (5) [28] by comparing their spectroscopic data with those reported in literature. The isolates were assessed for their cytotoxic effects on HepG-2 and A-549 cancer cell lines, promoting effects on proliferation of LO2 cells, and protective effects against cobalt chloride-induced damage in PC12 cells. However, they were all inactive in the assays. Since the extract of D. nobile has been reported to possess effects on blood vessel [29–31], the angiogenesis effects against sunitinib-induced blood vessel loss in transgenic zebrafish of the isolates were investigated. Fortunately, compound 2 showed a significant angiogenesis effect. Since vascularspecific transgenic zebrafish with expression of green fluorescent protein (EGFP) in blood vessels and endothelial cell nuclei has been exceptionally useful for observation of angiogenesis [32,33], transgenic Tg (flk1: EGFP) and Tg (fli1: nEGFP) zebrafish were used in this study. As shown in Fig. 4A and B, 1.6 μg/mL of sunitinib caused a lack of physiological vessel formation as well as a loss of blood vessel in ISV (intersegmental vessel) of Tg (flk1: EGFP) zebrafish embryos. The ISV index decreased significantly to 8.0 ± 0.5 when compared to the untreated normal group (ISV index, 21.0 ± 0.87; Fig. 4H) (p < 0.001). However, co-treatment of zebrafish embryos with sunitinib (1.6 μg/mL) and 2 (3.13, 6.25, 12.50, and 25.00 μM, respectively) significantly attenuated the ISV damage induced by sunitinib (Fig. 4D–G). Quantitative analysis of ISV index showed a dose-dependent effect of 2 on the recovery of ISV angiogenesis (Fig. 4H). At a concentration of 25.00 μM, the ISV index (19.67 ± 0.93) was close to that of untreated normal group. The angiogenesis effect of 2 was further supported by another transgenic zebrafish Tg (fli1: nEGFP). By observing the number of fluorescent spots (Fig. 5A and B), it was found that sunitinib could inhibit the proliferation of endothelial cells in ISV. Similar to the Tg (flk1: EGFP) zebrafish assay, 3.13, 6.25, 12.50, and 25.00 μM of 2 were able
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