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Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities
Journal Pre-proofs Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities Changshan Niu, Sheng Liu, Yong Li, Yunbao Liu, Shuanggang Ma, Fei Liu, Li Li, Jing Qu, Shishan Yu PII: DOI: Reference:
S0045-2068(19)31609-8 https://doi.org/10.1016/j.bioorg.2019.103502 YBIOO 103502
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Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
25 September 2019 12 November 2019 8 December 2019
Please cite this article as: C. Niu, S. Liu, Y. Li, Y. Liu, S. Ma, F. Liu, L. Li, J. Qu, S. Yu, Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103502
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Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities Changshan Niu#, Sheng Liu#, Yong Li, Yunbao Liu, Shuanggang Ma, Fei Liu, Li Li, Jing Qu*, Shishan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People's Republic of China.
To
whom
correspondence
should
be
addressed.
Email:
[email protected]
(Jing
Qu);
[email protected] (Shishan Yu); Telephone: +8610666574; Fax: +86-10-6516757. #These
authors contributed equally to this work.
Abstract Seven new diterpenoids, including four ent-kaurane-type pierisentkaurans B–E (1–4), one 4,5-secoent-kaurane-type pierisentkauran F (5), two leucothane-type 3β,7α,14β-trihydroxy-leucoth-10(20),15dien-5-one (6) and 10α,16α-dihydroxy-leucoth-5-one (7), along with three known diterpenoids entkaurane-type 16α-dihydroxy-6-oxo-ent-kauran-18-oic-acid (8), kalmane-type rhodomollein XXIII (9), and grayanane-type pierisformosoid J (10), were isolated from the roots of Pieris formosa. Their structures with absolute configurations were determined by a series of spectroscopic methods and electronic circular dichroism (ECD) calculations. Compounds 2 and 7 displayed weak analgesic activity at a dose of 5.0 mg/kg (i.p.) compared to the vehicle tests (p < 0.05) in an acetic acid-induced writhing 1
test. At a dose of 0.5 mg/mL, compounds 3 and 7 showed antifeedant activity against Plutella xylostella larvae with inhibition ratios of 27.1% and 52.5%, respectively.
Keywords Pieris formosa; Diterpenoids; Grayanane; Ent-kaurane; Leucothane; Kalmane; Anagesic; Antifeedant
1.
Introduction Pain is defined as an “unpleasant sensory and emotional experience associated with actual or potential
tissue damage.” [1]. With global estimates suggesting that 1 in 5 adults suffer from pain and that another 1 in 10 adults are diagnosed with chronic pain each year [2], pain is an enormous problem. It can be a chronic disease, a barrier to cancer treatment, or occur concurrently with other diseases and conditions such as depression, post-traumatic stress disorder, traumatic brain injury [1]. Although, scientists already developed different kinds of pain medicines in the past years. Current pain management options are still inadequate. Some analgesics, such as the opioids, exhibited severe side effects and addiction potential. Given the ever-growing demand for safe and efficacious pain management options, the search for new 2
analgesics from natural sources remains extremely relevant. P. formosa is a well-known poisonous, evergreen shrub that is widely distributed in southwest China. Accidental consumption of its leaves by poultry leads to dyspnea, motion imbalance, and eventually coma. As a Traditional Chinese Medicine (TCM), the juice of fresh leaves is used as an insecticide and for the treatment of tinea and scabies [3]. Previous studies of P. formosa led to the discovery of numerous grayanane diterpenoids, some of which exhibited potent analgesic and antifeedant activities [4−10]. As part of our ongoing efforts to characterize bioactive molecules from Traditional Chinese Medicine (TCM), seven new (1–7) and three known diterpenoids (8–10), were isolated from the roots of P. formosa (Fig.1). The analgesic and antifeedant activities of the isolated compounds were assayed. Compounds 2 and 7 displayed weak analgesic activity at a dose of 5.0 mg/kg (i.p.) when compared to the vehicletreated controls (p < 0.05) in an acetic acid-induced writhing test. At a dose of 0.5 mg/mL, compounds 3 and 7 showed antifeedant activity against Plutella xylostella larvae with inhibition ratios of 27.1% and 52.5%, respectively. Herein, we report on the isolation, structure elucidation, and biological activities. 2.
Experimental
2.1 General Experimental Procedures Optical rotations were measured on an AUTOPOL V polarimeter. IR spectra were recorded using a Nicolet 5700 FT-IR microscope (FT-IR microscope transmission). 1D and 2D NMR spectra were obtained using an INOVA-500 or a Bruker-600 spectrometer. HRESIMS data were recorded on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed using a Shimadzu LC-6AD instrument equipped with RID-20A and SPD-20A detectors, using a YMC-Pack RPC18 column (250 × 10 mm, 5 µm) or a COSMOSIL packed column (250 × 10 mm, 5 µm). Column chromatography was performed using polyamide (30-60 mesh, Changzhou Changfeng Chemical Factory, China), macroporous resin D101 (Qingdao Marine Chemical Factory, China), MCI (Mitsubishi Chemical Corporation), and ODS (50 μm, Merck, Germany). TLC was carried out with glass precoated Si gel GF254 plates (Qingdao Marine Chemical Factory, China). Spots were visualized by spraying with 10% H2SO4 in EtOH-H2O (95:5, v/v) followed by heating. 2.2 Plant material Roots of P. formosa (120 kg) were collected in Chuxiong Yi Autonomous Prefecture in Yunnan 3
Province, People’s Republic of China, in June 2014 and were identified by Prof. Lin Ma (Department of Natural Products Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences). A voucher specimen (ID-s-2747) was deposited in the herbarium at the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences. 2.3 Extraction and Isolation The isolation procedures were the same as in our previous report [7–10]. The roots of P. formosa (120 kg) were extracted with 95% EtOH (150 L × 3 h × 3). The evaporation of the solvent under reduced pressure yielded a black residue (5.0 kg). The residue was mixed with diatomite (1:2) and then successively Soxhlet extracted using a gradient of petroleum ether, CH2Cl2, EtOAc and CH3OH. The CH2Cl2 portion and the EtOAc portion were then applied to a macroporous resin D101 column (10 kg) and eluted with a gradient of 30% EtOH-H2O, 50% EtOH-H2O and 70% EtOH-H2O, to yield six fractions (D1–D3, and E1–E3). Fractions D2 (57 g) was separated through a polyamide column and eluted with H2O-EtOH (100:0, 30:70, 50:50, 70:30, v/v), to yield four fractions (D2a–D2d). Fraction D2a (4 g) was separated via an MCI column and eluted with a gradient of H2O-MeOH (1:9– 9:1, v/v), led to the four subfractions D2a1–D2a4. Subfraction D2a4 (0.8 g) was segmented by semipreparative HPLC with MeCN-H2O (23:77, v/v, 4.0 ml/min), to yield four subfractions (D2a4a– D2a4d). Subfraction D2a4d (0.18 g) was subjected to semipreparative HPLC eluted with MeCN-H2O (14:86, v/v, 4.0 ml/min), to yield 3 (35.0 mg, tR = 99.0 min), 6 (1.2 mg, tR = 104.0 min) and 9 (1.8 mg, tR = 102.0 min). Fraction D2b (8 g) was separated through an ODS column and eluted with 10% CH3OHH2O, 30% CH3OH-H2O, 50% CH3OH-H2O, and 70% CH3OH-H2O, successively, to yield four fractions (D2b1–D2b4). Fraction D2b2 (1.8 g) was further purified by MCI column using H2O-MeOH (1:9–9:1, v/v) as the mobile phase and got four subfractions (D2b2a–D2b2d). The purification of D2b2a (180 mg) by using semipreparative HPLC equipped with a YMC-Pack RP-C18 column (250 × 10 mm, 5 µm) and eluted with MeCN-H2O (18:82, v/v, 4.0 ml/min), led to the isolation of 4 (5.0 mg, tR = 110.0 min) and 5 (8.0 mg, tR = 101.0 min). Purification of D2b2c (230 mg) with semipreparative HPLC eluted with MeCNH2O (23:77, v/v, 4.0 ml/min), led to the isolation of 1 (3.0 mg, tR = 60.0 min), 7 (50.0 mg, tR = 84.0 min) and 8 (6.0 mg, tR = 115.0 min). Fraction E2a (15 g) was separated via an MCI column and eluted with a gradient of H2O-MeOH (1:9–9:1, v/v), gave five subfractions E2a1–E2a5. Subfraction E2a3 (1.3 g) was purified by semipreparative HPLC equipped a COSMOSIL packed column (250 × 10 mm, 5 µm) with 4
MeCN-H2O (18:82, v/v, 5.0 ml/min) as the mobile phase, to yield 2 (10.0 mg, tR = 54.0 min) and 10 (3.0 mg, tR = 56.0 min). Pierisentkauran B (1): Colorless, amorphous powder; [α] 20 D +61.8 (c 0.04, MeOH); IR νmax 3422, 2929, 1716, 1671, 1414, 1088 cm-1; HRESIMS m/z 373.1990 [M + Na]+ (calcd for 373.1985, C20H30O5Na); 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1 and 2. Pierisentkauran C (2): Colorless, amorphous powder; [α] 20 D -55.7 (c 0.30, MeOH); IR νmax 3412, 2936, 1714, 1459, 1202, 1153 cm-1; HRESIMS m/z 365.2329 [M + H]+ (calcd for 365.2323, C21H33O5); 1H
NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz), see Tables 1 and 2. Pierisentkauran D (3): Colorless, amorphous powder; [α] 20 D +2.9 (c 0.13, MeOH); IR νmax 3413,
2942, 1716, 1464, 1373, 1232, 1048, 550 cm-1; HRESIMS m/z 365.2323 [M + H]+ (calcd for 365.2323, C21H33O5); 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1 and 2. Pierisentkauran E (4): Colorless, amorphous powder; [α] 20 D -2.8 (c 0.11, MeOH); HRESIMS m/z 345.2395 [M + Na]+ (calcd for 345.2400, C20H34O3Na); 1H NMR (C5D5N, 500 MHz) and
13C
NMR
(C5D5N, 125 MHz), see Tables 1 and 2. Pierisentkauran F (5): Colorless, amorphous powder; [α] 20 D +41.3 (c 0.08, MeOH); IR νmax 3383, 2937, 1649, 1377, 1227, 1140, 1101, 1035, 929 cm-1; HRESIMS m/z 359.2201 [M + Na]+ (calcd for 359.2193, C20H32O4Na); 1H NMR (C5D5N, 500 MHz) and
13C
NMR (C5D5N, 125 MHz); 1H NMR
(CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Tables 1 and 2. 3β,7α,14β-trihydroxy-leucoth-10(20),15-dien-5-one (6): Colorless, amorphous powder; [α] 20 D +37.3 (c 0.15, MeOH); IR νmax 3394, 2936, 1737, 1373, 1251, 1049 cm-1; HRESIMS m/z 333.2062 [M + H]+ (calcd for 333.2060, C20H29O4); 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 2 and 3. 10α,16α-dihydroxy-leucoth-5-one (7): Colorless, amorphous powder; [α] 20 D -44.1 (c 0.21, MeOH); IR νmax 3358, 2935, 2869, 1700, 1444, 1372, 1116, 1080 cm-1; HRESIMS m/z 343.2252 [M + Na]+ (calcd for 343.2244, C20H32O3Na); 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz), see Tables 2 and 3. 2.4 Analgesic assay
5
The acetic acid-induced writhing model was employed for the analgesic assay as reported previously [11]. Groups of 8 Kunming mice were used as controls and test mice. The mice were given an intraperitoneal injection of 1% v/v acetic acid solution 15 minutes after the administration of the test compounds (vehicle controls were received 0.9 % NaCl, 5 ml/kg; positive controls were received ibuprofen 20.0 mg/kg or morphine 1.0, 0.5 and 0.2 mg/kg, respectively). The mice were placed individually in glass boxes. The numbers of writhes produced in these mice were counted for 20 minutes. For scoring purposes, a writhe was indicated by a stretching of the abdomen with a simultaneous stretching of at least one hind limb. 2.5 Antifeedant assay A lobular dish method was used for the antifeedant tests. The P. xylostella larvae were raised in the laboratory of Institute of Plant Protection, Chinese Academy of Agricultural Sciences under a controlled photoperiod (14 : 10 h light : dark), temperature (25 ± 2 °C), and relative humidity (75 ± 5%). P. xylostella larvae (third instar) were starved for 4 h prior to the test. Air drying leaf discs were cut from oilseed rape, using a borer (4 cm in diameter). Leaf discs were submerged in 0.05% Triton water-solution containing the test compounds (0.5 mg/mL, blank control was 0.05% Triton water-solution) for 5 s. After air drying, the treated leaf discs were set in the petri dish with filter paper at the bottom. Every ten larvae were divided into a group and then placed at the center of the leaf discs. Each test repeated three times. After feeding for 72 h, the areas of the leaf discs consumed were measured. The antifeedant index (%) was calculated as follow: 100 * (C - T)/C. 2.6 Computations The quantum-chemical calculations of 1 and 5 were performed using the Gaussian 09 program package. A system conformational analysis was employed via the MOE software using the MMFF94 force field. The obtained conformers were further optimized and checked as the true minima of potential energy surface by the density functional theory method at the B3LYP/6-31G(d) basis set level. Conductor-like polarizable continuum model (CPCM) was adopted to consider solvent effects using the dielectric constant of MeOH. The calculated ECD spectra were generated by Boltzmann weighting of each conformer’s population contribution with a bind-width of 0.35 eV for 1 and 0.3 eV for 5. 2.7 Animals 6
All animal care and experimental procedures were in accordance with the current guidelines of the National Institutes of Health (NIH). Animal experiments were approved by the Ethics Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. 3.
Results and Discussions Pierisentkanuran F (5) was obtained as a colorless powder, with a molecular formula of C20H32O4,
based on the quasi-molecular ion at m/z 359.2201 [M + Na]+ (calcd for C20H32O4Na, 359.2193) in the HRESIMS, indicating five degrees of unsaturation. The 1H NMR (Table 1) combined with HSQC spectra (Fig. 2) of 5 indicated resonances for four methyl singlets (δH 1.55, 1.36, 1.32, and 1.27). The 13C NMR (Table 2) and DEPT spectra showed a total of 20 carbon resonances which could be assigned to be six quaternary carbons (one keto carbonyl at δC 201.4; one oxygenated olefinic at δC 147.3; two oxygenated at δC 78.5 and 69.4), three methines (one olefinic at δC 128.8), seven methylenes, as well as four methyls. The 1H-1H COSY and HSQC spectra (Fig. 2) revealed the presence of three spin systems: C(2)H2–C(3)H2, C(9)H–C(11)H2 and C(12)H2–C(13)H–C(14)H2. One carbonyl and one double bond account for two degrees of unsaturation and the remaining three degrees of unsaturation indicated the presence of a tricyclic ring system in 5. The aforementioned structural features of 5 are similar to those of rhodomicranone E [12], except for two main differences. The first difference is that the oxygenated methylene at C-19 (δC 70.3) in rhodomicranone E was reduced to a methyl group (C-19, δC 29.3) in 5, which could be confirmed by the cross-peaks from H3-18 to C-19 and from H3-19 to C-18 in the HMBC spectrum; the second difference is that the two methylenes at C-6 and C-7 in rhodomicranone E were replaced by a double bond, which together with the carbonyl group formed an α-hydroxyl-α,βunsaturated ketone fragment in 5. The key HMBC correlations from OH-7 to C-5 (δC 201.4), C-6 (δC 147.3), and C-7 (δC 128.8) and from H-7 to C-5 (δC 201.4) proved the deduction. The NOESY cross peaks between H-9/H-15b and H-15b/CH3-17 demonstrated that H-9, H-15b and CH3-17 possessed an β-orientation. However, the NOESY cross peak between CH3-20/H-14a revealed the α-orientation of these groups. Finally, the absolute configuration of 5 was elucidated by comparing the experimental with calculated electronic circular dichroism (ECD) spectra. Two enantiomers 5a (8S,9S,10R,13S,16S) and 5b (8R,9R,10S,13R,16R) of 5 were calculated using a time-dependent density functional theory (TDDFT) at B3LYP/6-31G(d) level with MeOH as solvent. The results showed that the calculated ECD spectrum
7
of 5b matches very well with the experimental ECD (Fig. 3). Thus, compound 5 was finally identified as shown in Fig. 1. Compound 1 had a molecular formula of C20H30O5, as deduced from the HRESIMS ion at m/z 373.1990 [M + Na]+ (calcd for C20H30O5Na, 373.1985), requiring six degrees of unsaturation. Comparison of the 1H and
13C
NMR data of 1 and the known compound 16α-dihydroxy-6-oxo-ent-
kauran-18-oic-acid (8) revealed that these two compounds are structural analogues, with the only difference lying in an extra hydroxyl group at C-7 in 1. The extra oxygen atom in the molecular formula of 1 and the critical HMBC correlations from H-7 (δC 84.4, C-7) to C-6, C-14 and C-15 verified that the extra hydroxyl group attached to C-7. The NOESY correlations between H-7/H-9, H-9/H-5, H-9/CH317, and H-5/CH3-19 proved that H-5, H-7, H-9, H3-17 and CH3-19 are β-orientation. On the contrary, the NOESY correlation between H-14b/CH3-20 demonstrated that H-14b and CH3-20 have α-orientation The absolute configuration of 1 was illuminated by comparing the experimental ECD spectrum of 1 with the calculated ECD spectrum of two enantiomers 1a (4S,5R,7R,8S,9S,10R,13S,16S) and 1b (4R,5S,7S,8R,9R,10S,13R,16R) by using TDDFT method at B3LYP/6-31G(d) level. The result showed that (Fig. 4) the calculated ECD spectrum of 1b agreed well with the experimental ECD spectrum of 1. Thus, compound 1 was determined as shown in Fig. 1 and named pierisentkauran B. Compound 2 gave a molecular formula of C21H32O5, based on the HRESIMS ion at m/z 365.2329 [M + H]+ (calcd for C21H31O5, 365.2323), with six degrees of unsaturation. Analysis of the 1H and 13C NMR data of 2 with those of 1 illustrated they are structural analogues with only two main differences. The spin system C(9)H-C(11)H verified in the 1H-1H COSY spectrum together with the HMBC correlations (Fig. 2) from OH-11 to C-12 and from H-7 (δC 56.3, C-7) to C-5, C-6, C-9, C-14, and C-15 revealed that the hydroxyl located at C-11 in 2 instead of C-7 in 1. Moreover, the HMBC correlation from CH3-21 to C-18 certified that an extra methoxyl group connected to C-18. The NOESY correlations between H5/H-9, H-9/H-11, and H-11/CH3-17 discovered that these groups are β-orientation; NOESY correlations between CH3-21/CH3-20 and CH3-20/OH-11 proved the α-orientation of CH3-20 and CH3-21. Thus, compound 2 was determined as shown in Fig. 1 and named pierisentkauran C. Compound 3 showed the same molecular formula of C21H32O5 as 2, based on the HRESIMS ion at m/z 365.2323 [M + H]+ (calcd for C21H31O5, 365.2323). Detailed comparison of the NMR data between 2 and 3 uncovered that the only difference between these two compounds is that the hydroxyl group at 8
C-11 in 2 was moved to C-1 in 3. The two spin coupling systems C(1)H(OH)-C(2)H2-C(3)H2 and C(11)H2-C(12)H2 detected in 1H-1H COSY and HSQC spectrum (Fig. 2) confirmed that the hydroxyl group linked to C-1. Cross peaks between H-1/H-5 and OH-1/CH3-20 in the NOESY spectrum verified OH-1 is α-orientated. Thus, compound 3 was determined as shown in Fig. 1 and named pierisentkauran D. The molecular formula of 4 was assigned as C20H34O3 by the Na+ liganded molecular ion at m/z 345.2395 (calcd for C20H34O3Na, 345.2400) in the HRESIMS data, with six degrees of unsaturation. Careful analysis of the 1H and 13C NMR spectroscopic data of 4 (Tables 2 and 3) showed a close structural resemblance to a known compound 16α-hydroxy-ent-kaurane13, expect for two extra hydroxy groups substituted at C-3 and C-7 in 4, respectively, which confirmed by the spin coupling systems C(2)H2C(3)H and C(6)H2-C(7)H detected in 1H-1H COSY and HSQC spectrum and the key HMBC correlations (Fig. 2) from gem-dimethyl CH3-18(19) and H-5 to C-3, from H-5 to C-7, and from H-7 to C-9. The NOESY correlations between H-15a/H-9 and H-15a/H-7 demonstrated that OH-7 is α-orientation. Similarly, OH-3 has β-orientation deduced by NOESY correlation between H-3/CH3-18. Thus, compound 4 was determined as shown in Fig. 1 and named pierisentkauran E. The HRESIMS of compound 6 indicated a molecular formula of C20H28O4, which indicated seven degrees of unsaturation. Analysis of the NMR data clearly indicated that 6 is related closely to a known leucothane-type diterpenoid, rhodomicranol F [13]. Except for the difference that 6 has an endocyclic double bond at Δ15,16 instead of an exocyclic double bond at Δ16,17 and an additional hydroxyl at C-7. The long spin coupling system of C(3)H-C(2)H2-C(1)H-C(6)H-C(7) identified in 1H-1H COSY spectrum and in conjunction with correlations from H-7 to C-15 and from CH3-17 to C-14, C-15, and C-16 in HMBC spectrum confirmed the above assignment (Fig. 2). The β-orientation of H-7 was established via the critical NOESY correlations of H-7/H-1 and H-7/H-9. Thus, compound 6 was defined as 3β,7α,14βtrihydroxy-leucoth-10(20),15-dien-5-one as shown in Fig. 1. The molecular formula of 7 was determined to be C20H32O3 based on the HRESIMS data, with five degrees of unsaturation, two less than 6. The NMR data of 7 (Tables 1 and 3) resembled those of 6. The main differences were that the two double bonds at Δ16,17 and Δ10,20 in 6 were hydrolyzed in 7; meantime, 7 lost the hydroxyls at C-3 and C-7. HMBC correlations from CH3-20 to C-1, C-9, and C-10 (δC 75.0) and from CH3-17 to C-15 (δC 58.3) and C-16 (δC 77.8) combined with the spin systems of C(3)H2-C(2)H2 9
and C(1)H-C(6)H-C(7)H2 established in 1H-1H COSY spectrum proved this deduction. The NOESY correlations of H-1/CH3-20, H-9/CH3-17, and H-9/CH3-20 revealed these groups are β-orientation. Thus, compound 7 was defined as 10α,16α-dihydroxy-leucoth-5-one as shown in Fig. 1. The three known compounds were defined as 16α-dihydroxy-6-oxo-ent-kauran-18-oic-acid (8) [14], rhodomollein XXIII (9) [15], pierisformosoid J (10) [9], by comparison of their experimental and reported NMR data. To date, more than one hundred diterpenoids were identified from the genus Pieris and most of which are grayanane diterpenoids [16–18]. In the viewpoint of biosynthesis, grayanane diterpenoids derive from ent-kaurane-type diterpenoids through A-nor-B-homo rearrangement [19], in other words, ent-kauranetype was assumed to be the biogenetic precursor of grayanane-type. However, ent-kaurane-type diterpenoids were rarely discovered in genus Pieris, until now there are only two ent-kaurane-type diterpenoids pierosides D [20] and E [21] and one 4,5-seco-ent-kaurane pierisformoside G [22] were identified from P. formosa. The discovery of ent-kaurane-type diterpenoids 1–5 further supported the hypothesis of the bioprecursor of grayanane. Grayanane-type undergone ring rearrangements to form leucothane-type (A-homo-B-nor) [23] and kalmane-type (B-homo-C-nor) [24] (Fig. 5). Previous acetic acid-induced writhing test showed that the 95% ethanol extract of the roots of P. formosa exhibited potent analgesic activity. In order to further illustrate the active components and the structure-activity relationships (SARs), the analgesic activities of compounds 2 and 5–10 were tested in an acetic acid-induced writhing test. Ibuprofen (ib) and morphine were used as positive control drugs. As shown in Fig. 6, compounds 2 and 7 displayed promising analgesic activity compared to the vehicle tests (p < 0.05). As far as we know, 2 is the first report of ent-kaurane diterpenoids showed analgesic activity. Comparison with 1, 3 and 8, the main difference of 2 in structural is the OH-11, which indicated the crucial role to the activity. Since the juice of fresh leaves of P. formosa was used as an insecticide in TCM for a long time, compounds 3 and 7 were tested for their antifeeding activity against P. xylostella larvae using the lobular dish method with 0.05% triton water-solution as the negative control. At a dose of 0.5 mg/mL, 3 and 7 showed antifeeding activity with the inhibition ratios of 27.1% and 52.5%, respectively. 4. Conclusions 10
In summary, ten diterpenoids with diverse carbon skeletons, including four new ent-kaurane-type pierisentkaurans B–E (1–4), one new 4,5-seco-ent-kaurane-type pierisentkauran F (5), two new leucothane-type 3β,7α,14β-trihydroxy-leucoth-10(20),15-dien-5-one (6) and 10α,16α-dihydroxyleucoth-5-one (7), and three knowns ent-kaurane-type 16α-dihydroxy-6-oxo-ent-kauran-18-oic-acid (8), kalmane-type rhodomollein XXIII (9), and grayanane-type pierisformosoid J (10), were isolated from the roots of P. formosa. From the viewpoint of biosynthesis, the discovery of 1–5 further supported the hypothesis that ent-kaurane-type was the bioprecursor of grayanane diterpenoids. Acetic acid-induced writhing test showed that compounds 2 and 7 displayed weak analgesic activity at a dose of 5.0 mg/kg compared to the vehicle tests (p < 0.05). Compounds 3 and 7 showed antifeedant activities at 0.5 mg/mL against P. xylostella larvae with inhibition ratios of 27.1% and 52.5%, respectively. Through the systematic study of P. formosa not only enriched the diversity of grayanane diterpenoids, but also in some degree clarified the chemical material foundation of P. formosa used as analgesic and antifeedant in Traditional Chinese Medicine (TCM). Conflict of interest The authors declare no competing financial interest Acknowledgments This work was supported by the CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-1-010), the Natural Science Foundation of China (No. 21732008 and No. 81673314), and the National Megaproject for Innovative Drugs (No. 2018ZX09711001-008). The authors are grateful to the Department of Instrumental Analysis at our institute for the spectroscopic measurements and Li Cui from Institute of Plant Protection, Chinese Academy of Agricultural Sciences for the antifeedant activity screening. Supporting information In the Supporting Information, 1D/2D NMR, IR, and HRESIMS spectra for seven new compounds (1–7) were attached.
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Legends for Figures and Tables Fig. 1 The structures of compounds 1–10. Fig. 2 Key 2D NMR correlations of 1, 2, 4, 5, and 6 Fig. 3 Experimental and calculated ECD spectra for 5. Fig. 4 Experimental and calculated ECD spectra for 1. Fig. 5 Biogenetic relationships of different scaffolds and pure compounds isolated from roots of P. formosa. Fig. 6 Analgesic activities of the purified compounds from the roots of P. formosa in an acetic acidinduced writhing test. Ibuprofen (ib) and morphine (morph) were used as control drugs. Each bar represents the mean SEM of the values obtained from 8 mice. *p < 0.05, ***p < 0.001, significant differences between the test compound or control drug groups and vehicle group (veh) (one-way 13
ANOVA followed by the Bonferroni test). Table 1 1H NMR data of compounds 1–5 recorded in Pyridine-d5 Table 2 13C NMR data of compounds 1–7 recorded in Pyridine-d5. Table 3 1H NMR data of compounds 6 and 7 recorded in Pyridine-d5 Graph 1 Graphic abstract .
14
H 20
12
11
14
9 2 3
1
10
8
H7
5
4
O 18
OH
OH
R
O
O
19
18 21
3
19
4 18
20
H 2
1 6
5
O
H 6
HO O
11
8
14
OH
15
13
OH
17
O
O
OH
OH 5
12
20 9
HO
3
18
OH 7
1 5
10
HO
13
H
6
19
H
H
5
4
H
H
4
OH
1
3
OH
H
HO H H
H
10 9 7
19
2
H
2 R1 = H, R2 = OH 3 R1 = OH, R2 = H
1 R = OH 8R=H
HO
O
H
H OH
OH H
15
6
H
16
H
H
R2 R1
17
7
OH
14 8
17
16 15
OH
9
Fig. 1 The structures of compounds 1–10.
15
OH
11
H
H
HO
3
1 5
10
OH 6
H 13 15
HO
HO 10
H
14
8
17
OH OH
Fig. 2 Key 2D NMR correlations of 1, 2, 4, 5, and 6
16
Fig. 3 Experimental and calculated ECD spectra for 5.
17
Fig. 4 Experimental and calculated ECD spectra for 1.
18
Fig. 5 Biogenetic relationships of different scaffolds and pure compounds isolated from roots of P. formosa.
19
Fig. 6 Analgesic activities of the purified compounds from the roots of P. formosa in an acetic acidinduced writhing test. Ibuprofen (ib) and morphine (morph) were used as control drugs. Each bar represents the mean SEM of the values obtained from 8 mice. *p < 0.05, ***p < 0.001, significant differences between the test compound or control drug groups and vehicle group (veh) (one-way ANOVA followed by the Bonferroni test).
20
Table 1 1H NMR data of compounds 1–5 recorded in Pyridine-d5 1b
no. 1
2
3
5
2a
a 1.83, dt (13.2, 3.0)
a 1.83, dt (13.5, 2.5)
b 1.20, dd (12.6, 3.6)
b 1.45, overlap
a 2.11, overlap
a 2.03, m
b 1.50, overlap
3b 3.86, m
4a
5a
a 1.86, dt (13.0, 3.5)
a 2.00, overlap
b 1.08, overlap
b 1.61, d (4.0)
a 2.35, m
a 1.98, overlap
a 1.74, m
b 1.50, overlap
b 1.88, m
b 1.90, m
b 1.50, m
a 2.48, dt (13.2, 3.0)
a 2.13, overlap
a 2.09, dt (13.8, 3.6)
3.57, dd (11.5, 4.5)
a 1.71, overlap
b 1.04, dt (13.8, 3.6)
b 1.21, dt (12.5, 4.0)
b 1.38, overlap
3.90, s
2.54, s
2.56, s
6
b 1.63, m 2.05, d (13.0) a 2.21, dd (11.0, 4.5) b 1.94, d (11.0)
7
4.25, s
a 2.73, d (12.5)
a 2.76, d (12.6)
b 2.65, d (12.5)
b 2.50, d (12.6)
4.07, t (3.0)
6.29, s
9
2.15, d (9.0)
2.11, d (6.5)
1.97, overlap
1.77, d (6.0)
2.28, d (8.5)
11
a 1.61, m
4.71, m
a 3.45, m
a 1.64, overlap
a 1.69, overlap
b 1.95, overlap
b 1.60, overlap
b 1.59, d (5.0)
b 1.59, overlap 12
a 1.92, m
a 2.46, d (10.0)
a 1.74, m
a 1.93, overlap
a 1.67, overlap
b 1.48, overlap
b 2.17, d (11.0)
b 1.66, overlap
b 1.78, overlap
b 1.46, m
13
2.20, t (3.0)
2.26, s
2.15, overlap
2.25, t (3.0)
2.21, overlap
14
a 2.29, dd (11.4, 4.8)
a 2.24, overlap
a 2.22, dd (11.4, 4.2)
a 1.69, m
a 2.51, dd (11.5, 4.0)
b 1.69, d (11.4)
b 2.03, m
b 2.13, d (12.6)
b 1.67, m
b 1.83, d (11.5)
a 2.57, d (14.4)
a 1.99, overlap
a 2.04, d (13.8)
a 2.69, d (14.0)
a 2.23, d (14.0)
b 2.07, d (16.2)
b 1.99, overlap
b 1.98, d (14.4)
b 1.99, d (13.5)
b 1.80, d (14.5)
1.60, s
1.59, s
1.59, s
1.62, s
1.55, s
1.11, s
1.36, s
15
17 18 19
1.29, s
1.48, s
1.40, s
1.31, s
1.32, s
20
1.15, s
1.73, s
1.65, s
1.14, s
1.27, s
3.68, s
3.70, s
21 1-OH
6.16, d (5.4)
4-OH
7.53, s
6-OH
10.07, s
11-OH
6.15, d (4.5)
16-OH
5.65, s
aRecorded
5.45, s
at 500 MHz. bRceorded at 600 MHz.
21
7.58, s
Table 2 13C NMR data of compounds 1–7 recorded in Pyridine-d5. no.
1a
2a
3b
1
41.4, CH2
42.7, CH2
80.4, CH
39.4, CH2
39.2, CH2
43.1, CH
56.7, CH
2
19.3, CH2
19.6, CH2
30.4, CH2
28.5, CH2
20.2, CH2
32.9, CH2
21.0, CH2
3
38.6, CH2
38.8, CH2
36.6, CH2
78.4, CH
45.3, CH2
77.5, CH
40.2, CH2
4
43.5, C
43.4, C
42.5, C
39.1, C
69.4, C
50.8, C
44.6, C
5
59.9, CH
66.8, CH
66.5, CH
45.5, CH
201.4, C
214.3, C
215.6, C
6
222.5, C
208.7, C
208.5, C
37.1, CH
147.3, C
54.0, CH
45.4, CH
7
84.4, CH
56.3, CH2
56.7, CH2
77.0, CH
128.8, CH
73.1, CH
40.3, CH2
8
54.6, C
48.8, C
49.0, C
50.0, C
44.8, C
57.8, C
44.5 C
9
50.2, CH
61.1, CH
57.8, CH
51.7, CH
42.8, CH
43.1, CH
54.8, CH
10
47.1, C
46.2, C
50.1, C
39.3, C
49.7, C
150.8, C
75.0, C
11
18.1, CH2
67.7, CH
20.1, CH2
18.3, CH2
18.3, CH2
21.6, CH2
18.8, CH2
12
26.7, CH2
37.0, CH2
26.6, CH2
28.5, CH2
26.6, CH2
21.8, CH2
26.9, CH2
13
48.7, CH
48.2, CH
49.0, CH
49.3, CH
48.5, CH
55.3, CH
49.5, CH
14
35.1, CH2
37.9, CH2
38.8, CH2
27.8, CH2
39.4, CH2
82.9, CH
36.9, CH2
15
54.6, CH2
59.0, CH2
58.8, CH2
55.4, CH2
58.5, CH2
127.7, CH
58.3, CH2
16
77.5, C
78.5, C
78.1, C
77.8, C
78.5, C
140.1, C
77.8, C
17
24.9, CH3
24.9, CH3
24.8, CH3
25.1, CH3
24.9, CH3
15.8, CH3
25.0, CH3
18
176.4, C
176.3, C
175.9, C
16.7, CH3
30.4, CH3
24.1, CH3
24.9, CH3
19
27.3, CH3
29.1, CH3
28.2, CH3
28.8, CH3
29.6, CH3
21.8, CH3
26.1, CH3
20
17.3, CH3
20.2, CH3
15.8, CH3
18.1, CH3
25.1, CH3
106.4, CH2
21.0, CH3
51.3, CH3
51.4, CH3
21 aRecorded
at 125 MHz.
bRceorded
4a
at 150 MHz.
22
5a
6a
7b
Table 3 1H NMR data of compounds 6 and 7 recorded in Pyridine-d5 no.
6a
7b
1
2.98, dt (12.6, 2.5)
1.65, m
2
a 2.39, dt (13.8, 2.4)
a 2.57, dd (13.5, 3.5)
b 2.24, dt (13.2, 3.6)
b 1.88, dd (13.5, 3.5)
4.16, t (3.0)
a 1.73, dt (13.5, 3.6)
3
b 1.50, dd (13.0, 3.5) 6
3.24, dd (12.6, 9.6)
2.67, dt (12.5, 3.0)
7
4.62, d (9.6)
a 2.18, dd (13.5, 3.0) b 1.84, overlap
9
2.01, d (6.6)
1.70, dd (9.0, 3.5)
11
a 2.18, m
a 2.32, dd (14.5, 6.0)
b 1.60, m
b 1.63, m
a 2.09, m
a 1.82, overlap
b 1.68, m
b 1.70, overlap
13
2.65, t (3.0)
2.22, overlap
14
4.51, brs
a 2.09, overlap
12
b 1.84, d (11.0) 15
5.47, m
a 2.12, d (14.0) b 1.77, overlap
17
1.75, d (1.8)
1.56, s
18
1.17, s
1.10, s
19
1.46, s
1.11, s
20
a 5.07, s
1.54, s
b 5.00, overlap 10-OH
5.37, s
16-OH
5.43, s
aRecorded
at 500 MHz. bRceorded at 600 MHz.
23
Graph 1 Graphic abstract
Pieris formosa
24
Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities Changshan Niu#, Sheng Liu#, Yong Li, Yunbao Liu, Shuanggang Ma, Fei Liu, Li Li, Jing Qu*, Shishan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People's Republic of China.
Conflict of interest The authors declare no competing financial interest.
25
Highlights
1. Seven new diterpenoids with diverse carbon skeletons were isolated from the roots of Pieris formosa. 2. Compounds 2 and 7 displayed weak analgesic activity. 3. Compounds 3 and 7 showed antifeedant activity against Plutella xylostella larvae. 4. In the view of biosynthesis, the discovery of compounds 1–5 further supported the bio-origin of grayanane-type diterpenoids.
26
S0045-2068(19)31609-8 https://doi.org/10.1016/j.bioorg.2019.103502 YBIOO 103502
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
25 September 2019 12 November 2019 8 December 2019
Please cite this article as: C. Niu, S. Liu, Y. Li, Y. Liu, S. Ma, F. Liu, L. Li, J. Qu, S. Yu, Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103502
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© 2019 Published by Elsevier Inc.
Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities Changshan Niu#, Sheng Liu#, Yong Li, Yunbao Liu, Shuanggang Ma, Fei Liu, Li Li, Jing Qu*, Shishan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People's Republic of China.
To
whom
correspondence
should
be
addressed.
Email:
[email protected]
(Jing
Qu);
[email protected] (Shishan Yu); Telephone: +8610666574; Fax: +86-10-6516757. #These
authors contributed equally to this work.
Abstract Seven new diterpenoids, including four ent-kaurane-type pierisentkaurans B–E (1–4), one 4,5-secoent-kaurane-type pierisentkauran F (5), two leucothane-type 3β,7α,14β-trihydroxy-leucoth-10(20),15dien-5-one (6) and 10α,16α-dihydroxy-leucoth-5-one (7), along with three known diterpenoids entkaurane-type 16α-dihydroxy-6-oxo-ent-kauran-18-oic-acid (8), kalmane-type rhodomollein XXIII (9), and grayanane-type pierisformosoid J (10), were isolated from the roots of Pieris formosa. Their structures with absolute configurations were determined by a series of spectroscopic methods and electronic circular dichroism (ECD) calculations. Compounds 2 and 7 displayed weak analgesic activity at a dose of 5.0 mg/kg (i.p.) compared to the vehicle tests (p < 0.05) in an acetic acid-induced writhing 1
test. At a dose of 0.5 mg/mL, compounds 3 and 7 showed antifeedant activity against Plutella xylostella larvae with inhibition ratios of 27.1% and 52.5%, respectively.
Keywords Pieris formosa; Diterpenoids; Grayanane; Ent-kaurane; Leucothane; Kalmane; Anagesic; Antifeedant
1.
Introduction Pain is defined as an “unpleasant sensory and emotional experience associated with actual or potential
tissue damage.” [1]. With global estimates suggesting that 1 in 5 adults suffer from pain and that another 1 in 10 adults are diagnosed with chronic pain each year [2], pain is an enormous problem. It can be a chronic disease, a barrier to cancer treatment, or occur concurrently with other diseases and conditions such as depression, post-traumatic stress disorder, traumatic brain injury [1]. Although, scientists already developed different kinds of pain medicines in the past years. Current pain management options are still inadequate. Some analgesics, such as the opioids, exhibited severe side effects and addiction potential. Given the ever-growing demand for safe and efficacious pain management options, the search for new 2
analgesics from natural sources remains extremely relevant. P. formosa is a well-known poisonous, evergreen shrub that is widely distributed in southwest China. Accidental consumption of its leaves by poultry leads to dyspnea, motion imbalance, and eventually coma. As a Traditional Chinese Medicine (TCM), the juice of fresh leaves is used as an insecticide and for the treatment of tinea and scabies [3]. Previous studies of P. formosa led to the discovery of numerous grayanane diterpenoids, some of which exhibited potent analgesic and antifeedant activities [4−10]. As part of our ongoing efforts to characterize bioactive molecules from Traditional Chinese Medicine (TCM), seven new (1–7) and three known diterpenoids (8–10), were isolated from the roots of P. formosa (Fig.1). The analgesic and antifeedant activities of the isolated compounds were assayed. Compounds 2 and 7 displayed weak analgesic activity at a dose of 5.0 mg/kg (i.p.) when compared to the vehicletreated controls (p < 0.05) in an acetic acid-induced writhing test. At a dose of 0.5 mg/mL, compounds 3 and 7 showed antifeedant activity against Plutella xylostella larvae with inhibition ratios of 27.1% and 52.5%, respectively. Herein, we report on the isolation, structure elucidation, and biological activities. 2.
Experimental
2.1 General Experimental Procedures Optical rotations were measured on an AUTOPOL V polarimeter. IR spectra were recorded using a Nicolet 5700 FT-IR microscope (FT-IR microscope transmission). 1D and 2D NMR spectra were obtained using an INOVA-500 or a Bruker-600 spectrometer. HRESIMS data were recorded on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed using a Shimadzu LC-6AD instrument equipped with RID-20A and SPD-20A detectors, using a YMC-Pack RPC18 column (250 × 10 mm, 5 µm) or a COSMOSIL packed column (250 × 10 mm, 5 µm). Column chromatography was performed using polyamide (30-60 mesh, Changzhou Changfeng Chemical Factory, China), macroporous resin D101 (Qingdao Marine Chemical Factory, China), MCI (Mitsubishi Chemical Corporation), and ODS (50 μm, Merck, Germany). TLC was carried out with glass precoated Si gel GF254 plates (Qingdao Marine Chemical Factory, China). Spots were visualized by spraying with 10% H2SO4 in EtOH-H2O (95:5, v/v) followed by heating. 2.2 Plant material Roots of P. formosa (120 kg) were collected in Chuxiong Yi Autonomous Prefecture in Yunnan 3
Province, People’s Republic of China, in June 2014 and were identified by Prof. Lin Ma (Department of Natural Products Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences). A voucher specimen (ID-s-2747) was deposited in the herbarium at the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences. 2.3 Extraction and Isolation The isolation procedures were the same as in our previous report [7–10]. The roots of P. formosa (120 kg) were extracted with 95% EtOH (150 L × 3 h × 3). The evaporation of the solvent under reduced pressure yielded a black residue (5.0 kg). The residue was mixed with diatomite (1:2) and then successively Soxhlet extracted using a gradient of petroleum ether, CH2Cl2, EtOAc and CH3OH. The CH2Cl2 portion and the EtOAc portion were then applied to a macroporous resin D101 column (10 kg) and eluted with a gradient of 30% EtOH-H2O, 50% EtOH-H2O and 70% EtOH-H2O, to yield six fractions (D1–D3, and E1–E3). Fractions D2 (57 g) was separated through a polyamide column and eluted with H2O-EtOH (100:0, 30:70, 50:50, 70:30, v/v), to yield four fractions (D2a–D2d). Fraction D2a (4 g) was separated via an MCI column and eluted with a gradient of H2O-MeOH (1:9– 9:1, v/v), led to the four subfractions D2a1–D2a4. Subfraction D2a4 (0.8 g) was segmented by semipreparative HPLC with MeCN-H2O (23:77, v/v, 4.0 ml/min), to yield four subfractions (D2a4a– D2a4d). Subfraction D2a4d (0.18 g) was subjected to semipreparative HPLC eluted with MeCN-H2O (14:86, v/v, 4.0 ml/min), to yield 3 (35.0 mg, tR = 99.0 min), 6 (1.2 mg, tR = 104.0 min) and 9 (1.8 mg, tR = 102.0 min). Fraction D2b (8 g) was separated through an ODS column and eluted with 10% CH3OHH2O, 30% CH3OH-H2O, 50% CH3OH-H2O, and 70% CH3OH-H2O, successively, to yield four fractions (D2b1–D2b4). Fraction D2b2 (1.8 g) was further purified by MCI column using H2O-MeOH (1:9–9:1, v/v) as the mobile phase and got four subfractions (D2b2a–D2b2d). The purification of D2b2a (180 mg) by using semipreparative HPLC equipped with a YMC-Pack RP-C18 column (250 × 10 mm, 5 µm) and eluted with MeCN-H2O (18:82, v/v, 4.0 ml/min), led to the isolation of 4 (5.0 mg, tR = 110.0 min) and 5 (8.0 mg, tR = 101.0 min). Purification of D2b2c (230 mg) with semipreparative HPLC eluted with MeCNH2O (23:77, v/v, 4.0 ml/min), led to the isolation of 1 (3.0 mg, tR = 60.0 min), 7 (50.0 mg, tR = 84.0 min) and 8 (6.0 mg, tR = 115.0 min). Fraction E2a (15 g) was separated via an MCI column and eluted with a gradient of H2O-MeOH (1:9–9:1, v/v), gave five subfractions E2a1–E2a5. Subfraction E2a3 (1.3 g) was purified by semipreparative HPLC equipped a COSMOSIL packed column (250 × 10 mm, 5 µm) with 4
MeCN-H2O (18:82, v/v, 5.0 ml/min) as the mobile phase, to yield 2 (10.0 mg, tR = 54.0 min) and 10 (3.0 mg, tR = 56.0 min). Pierisentkauran B (1): Colorless, amorphous powder; [α] 20 D +61.8 (c 0.04, MeOH); IR νmax 3422, 2929, 1716, 1671, 1414, 1088 cm-1; HRESIMS m/z 373.1990 [M + Na]+ (calcd for 373.1985, C20H30O5Na); 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1 and 2. Pierisentkauran C (2): Colorless, amorphous powder; [α] 20 D -55.7 (c 0.30, MeOH); IR νmax 3412, 2936, 1714, 1459, 1202, 1153 cm-1; HRESIMS m/z 365.2329 [M + H]+ (calcd for 365.2323, C21H33O5); 1H
NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz), see Tables 1 and 2. Pierisentkauran D (3): Colorless, amorphous powder; [α] 20 D +2.9 (c 0.13, MeOH); IR νmax 3413,
2942, 1716, 1464, 1373, 1232, 1048, 550 cm-1; HRESIMS m/z 365.2323 [M + H]+ (calcd for 365.2323, C21H33O5); 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 1 and 2. Pierisentkauran E (4): Colorless, amorphous powder; [α] 20 D -2.8 (c 0.11, MeOH); HRESIMS m/z 345.2395 [M + Na]+ (calcd for 345.2400, C20H34O3Na); 1H NMR (C5D5N, 500 MHz) and
13C
NMR
(C5D5N, 125 MHz), see Tables 1 and 2. Pierisentkauran F (5): Colorless, amorphous powder; [α] 20 D +41.3 (c 0.08, MeOH); IR νmax 3383, 2937, 1649, 1377, 1227, 1140, 1101, 1035, 929 cm-1; HRESIMS m/z 359.2201 [M + Na]+ (calcd for 359.2193, C20H32O4Na); 1H NMR (C5D5N, 500 MHz) and
13C
NMR (C5D5N, 125 MHz); 1H NMR
(CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Tables 1 and 2. 3β,7α,14β-trihydroxy-leucoth-10(20),15-dien-5-one (6): Colorless, amorphous powder; [α] 20 D +37.3 (c 0.15, MeOH); IR νmax 3394, 2936, 1737, 1373, 1251, 1049 cm-1; HRESIMS m/z 333.2062 [M + H]+ (calcd for 333.2060, C20H29O4); 1H NMR (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz), see Tables 2 and 3. 10α,16α-dihydroxy-leucoth-5-one (7): Colorless, amorphous powder; [α] 20 D -44.1 (c 0.21, MeOH); IR νmax 3358, 2935, 2869, 1700, 1444, 1372, 1116, 1080 cm-1; HRESIMS m/z 343.2252 [M + Na]+ (calcd for 343.2244, C20H32O3Na); 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz), see Tables 2 and 3. 2.4 Analgesic assay
5
The acetic acid-induced writhing model was employed for the analgesic assay as reported previously [11]. Groups of 8 Kunming mice were used as controls and test mice. The mice were given an intraperitoneal injection of 1% v/v acetic acid solution 15 minutes after the administration of the test compounds (vehicle controls were received 0.9 % NaCl, 5 ml/kg; positive controls were received ibuprofen 20.0 mg/kg or morphine 1.0, 0.5 and 0.2 mg/kg, respectively). The mice were placed individually in glass boxes. The numbers of writhes produced in these mice were counted for 20 minutes. For scoring purposes, a writhe was indicated by a stretching of the abdomen with a simultaneous stretching of at least one hind limb. 2.5 Antifeedant assay A lobular dish method was used for the antifeedant tests. The P. xylostella larvae were raised in the laboratory of Institute of Plant Protection, Chinese Academy of Agricultural Sciences under a controlled photoperiod (14 : 10 h light : dark), temperature (25 ± 2 °C), and relative humidity (75 ± 5%). P. xylostella larvae (third instar) were starved for 4 h prior to the test. Air drying leaf discs were cut from oilseed rape, using a borer (4 cm in diameter). Leaf discs were submerged in 0.05% Triton water-solution containing the test compounds (0.5 mg/mL, blank control was 0.05% Triton water-solution) for 5 s. After air drying, the treated leaf discs were set in the petri dish with filter paper at the bottom. Every ten larvae were divided into a group and then placed at the center of the leaf discs. Each test repeated three times. After feeding for 72 h, the areas of the leaf discs consumed were measured. The antifeedant index (%) was calculated as follow: 100 * (C - T)/C. 2.6 Computations The quantum-chemical calculations of 1 and 5 were performed using the Gaussian 09 program package. A system conformational analysis was employed via the MOE software using the MMFF94 force field. The obtained conformers were further optimized and checked as the true minima of potential energy surface by the density functional theory method at the B3LYP/6-31G(d) basis set level. Conductor-like polarizable continuum model (CPCM) was adopted to consider solvent effects using the dielectric constant of MeOH. The calculated ECD spectra were generated by Boltzmann weighting of each conformer’s population contribution with a bind-width of 0.35 eV for 1 and 0.3 eV for 5. 2.7 Animals 6
All animal care and experimental procedures were in accordance with the current guidelines of the National Institutes of Health (NIH). Animal experiments were approved by the Ethics Committee of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. 3.
Results and Discussions Pierisentkanuran F (5) was obtained as a colorless powder, with a molecular formula of C20H32O4,
based on the quasi-molecular ion at m/z 359.2201 [M + Na]+ (calcd for C20H32O4Na, 359.2193) in the HRESIMS, indicating five degrees of unsaturation. The 1H NMR (Table 1) combined with HSQC spectra (Fig. 2) of 5 indicated resonances for four methyl singlets (δH 1.55, 1.36, 1.32, and 1.27). The 13C NMR (Table 2) and DEPT spectra showed a total of 20 carbon resonances which could be assigned to be six quaternary carbons (one keto carbonyl at δC 201.4; one oxygenated olefinic at δC 147.3; two oxygenated at δC 78.5 and 69.4), three methines (one olefinic at δC 128.8), seven methylenes, as well as four methyls. The 1H-1H COSY and HSQC spectra (Fig. 2) revealed the presence of three spin systems: C(2)H2–C(3)H2, C(9)H–C(11)H2 and C(12)H2–C(13)H–C(14)H2. One carbonyl and one double bond account for two degrees of unsaturation and the remaining three degrees of unsaturation indicated the presence of a tricyclic ring system in 5. The aforementioned structural features of 5 are similar to those of rhodomicranone E [12], except for two main differences. The first difference is that the oxygenated methylene at C-19 (δC 70.3) in rhodomicranone E was reduced to a methyl group (C-19, δC 29.3) in 5, which could be confirmed by the cross-peaks from H3-18 to C-19 and from H3-19 to C-18 in the HMBC spectrum; the second difference is that the two methylenes at C-6 and C-7 in rhodomicranone E were replaced by a double bond, which together with the carbonyl group formed an α-hydroxyl-α,βunsaturated ketone fragment in 5. The key HMBC correlations from OH-7 to C-5 (δC 201.4), C-6 (δC 147.3), and C-7 (δC 128.8) and from H-7 to C-5 (δC 201.4) proved the deduction. The NOESY cross peaks between H-9/H-15b and H-15b/CH3-17 demonstrated that H-9, H-15b and CH3-17 possessed an β-orientation. However, the NOESY cross peak between CH3-20/H-14a revealed the α-orientation of these groups. Finally, the absolute configuration of 5 was elucidated by comparing the experimental with calculated electronic circular dichroism (ECD) spectra. Two enantiomers 5a (8S,9S,10R,13S,16S) and 5b (8R,9R,10S,13R,16R) of 5 were calculated using a time-dependent density functional theory (TDDFT) at B3LYP/6-31G(d) level with MeOH as solvent. The results showed that the calculated ECD spectrum
7
of 5b matches very well with the experimental ECD (Fig. 3). Thus, compound 5 was finally identified as shown in Fig. 1. Compound 1 had a molecular formula of C20H30O5, as deduced from the HRESIMS ion at m/z 373.1990 [M + Na]+ (calcd for C20H30O5Na, 373.1985), requiring six degrees of unsaturation. Comparison of the 1H and
13C
NMR data of 1 and the known compound 16α-dihydroxy-6-oxo-ent-
kauran-18-oic-acid (8) revealed that these two compounds are structural analogues, with the only difference lying in an extra hydroxyl group at C-7 in 1. The extra oxygen atom in the molecular formula of 1 and the critical HMBC correlations from H-7 (δC 84.4, C-7) to C-6, C-14 and C-15 verified that the extra hydroxyl group attached to C-7. The NOESY correlations between H-7/H-9, H-9/H-5, H-9/CH317, and H-5/CH3-19 proved that H-5, H-7, H-9, H3-17 and CH3-19 are β-orientation. On the contrary, the NOESY correlation between H-14b/CH3-20 demonstrated that H-14b and CH3-20 have α-orientation The absolute configuration of 1 was illuminated by comparing the experimental ECD spectrum of 1 with the calculated ECD spectrum of two enantiomers 1a (4S,5R,7R,8S,9S,10R,13S,16S) and 1b (4R,5S,7S,8R,9R,10S,13R,16R) by using TDDFT method at B3LYP/6-31G(d) level. The result showed that (Fig. 4) the calculated ECD spectrum of 1b agreed well with the experimental ECD spectrum of 1. Thus, compound 1 was determined as shown in Fig. 1 and named pierisentkauran B. Compound 2 gave a molecular formula of C21H32O5, based on the HRESIMS ion at m/z 365.2329 [M + H]+ (calcd for C21H31O5, 365.2323), with six degrees of unsaturation. Analysis of the 1H and 13C NMR data of 2 with those of 1 illustrated they are structural analogues with only two main differences. The spin system C(9)H-C(11)H verified in the 1H-1H COSY spectrum together with the HMBC correlations (Fig. 2) from OH-11 to C-12 and from H-7 (δC 56.3, C-7) to C-5, C-6, C-9, C-14, and C-15 revealed that the hydroxyl located at C-11 in 2 instead of C-7 in 1. Moreover, the HMBC correlation from CH3-21 to C-18 certified that an extra methoxyl group connected to C-18. The NOESY correlations between H5/H-9, H-9/H-11, and H-11/CH3-17 discovered that these groups are β-orientation; NOESY correlations between CH3-21/CH3-20 and CH3-20/OH-11 proved the α-orientation of CH3-20 and CH3-21. Thus, compound 2 was determined as shown in Fig. 1 and named pierisentkauran C. Compound 3 showed the same molecular formula of C21H32O5 as 2, based on the HRESIMS ion at m/z 365.2323 [M + H]+ (calcd for C21H31O5, 365.2323). Detailed comparison of the NMR data between 2 and 3 uncovered that the only difference between these two compounds is that the hydroxyl group at 8
C-11 in 2 was moved to C-1 in 3. The two spin coupling systems C(1)H(OH)-C(2)H2-C(3)H2 and C(11)H2-C(12)H2 detected in 1H-1H COSY and HSQC spectrum (Fig. 2) confirmed that the hydroxyl group linked to C-1. Cross peaks between H-1/H-5 and OH-1/CH3-20 in the NOESY spectrum verified OH-1 is α-orientated. Thus, compound 3 was determined as shown in Fig. 1 and named pierisentkauran D. The molecular formula of 4 was assigned as C20H34O3 by the Na+ liganded molecular ion at m/z 345.2395 (calcd for C20H34O3Na, 345.2400) in the HRESIMS data, with six degrees of unsaturation. Careful analysis of the 1H and 13C NMR spectroscopic data of 4 (Tables 2 and 3) showed a close structural resemblance to a known compound 16α-hydroxy-ent-kaurane13, expect for two extra hydroxy groups substituted at C-3 and C-7 in 4, respectively, which confirmed by the spin coupling systems C(2)H2C(3)H and C(6)H2-C(7)H detected in 1H-1H COSY and HSQC spectrum and the key HMBC correlations (Fig. 2) from gem-dimethyl CH3-18(19) and H-5 to C-3, from H-5 to C-7, and from H-7 to C-9. The NOESY correlations between H-15a/H-9 and H-15a/H-7 demonstrated that OH-7 is α-orientation. Similarly, OH-3 has β-orientation deduced by NOESY correlation between H-3/CH3-18. Thus, compound 4 was determined as shown in Fig. 1 and named pierisentkauran E. The HRESIMS of compound 6 indicated a molecular formula of C20H28O4, which indicated seven degrees of unsaturation. Analysis of the NMR data clearly indicated that 6 is related closely to a known leucothane-type diterpenoid, rhodomicranol F [13]. Except for the difference that 6 has an endocyclic double bond at Δ15,16 instead of an exocyclic double bond at Δ16,17 and an additional hydroxyl at C-7. The long spin coupling system of C(3)H-C(2)H2-C(1)H-C(6)H-C(7) identified in 1H-1H COSY spectrum and in conjunction with correlations from H-7 to C-15 and from CH3-17 to C-14, C-15, and C-16 in HMBC spectrum confirmed the above assignment (Fig. 2). The β-orientation of H-7 was established via the critical NOESY correlations of H-7/H-1 and H-7/H-9. Thus, compound 6 was defined as 3β,7α,14βtrihydroxy-leucoth-10(20),15-dien-5-one as shown in Fig. 1. The molecular formula of 7 was determined to be C20H32O3 based on the HRESIMS data, with five degrees of unsaturation, two less than 6. The NMR data of 7 (Tables 1 and 3) resembled those of 6. The main differences were that the two double bonds at Δ16,17 and Δ10,20 in 6 were hydrolyzed in 7; meantime, 7 lost the hydroxyls at C-3 and C-7. HMBC correlations from CH3-20 to C-1, C-9, and C-10 (δC 75.0) and from CH3-17 to C-15 (δC 58.3) and C-16 (δC 77.8) combined with the spin systems of C(3)H2-C(2)H2 9
and C(1)H-C(6)H-C(7)H2 established in 1H-1H COSY spectrum proved this deduction. The NOESY correlations of H-1/CH3-20, H-9/CH3-17, and H-9/CH3-20 revealed these groups are β-orientation. Thus, compound 7 was defined as 10α,16α-dihydroxy-leucoth-5-one as shown in Fig. 1. The three known compounds were defined as 16α-dihydroxy-6-oxo-ent-kauran-18-oic-acid (8) [14], rhodomollein XXIII (9) [15], pierisformosoid J (10) [9], by comparison of their experimental and reported NMR data. To date, more than one hundred diterpenoids were identified from the genus Pieris and most of which are grayanane diterpenoids [16–18]. In the viewpoint of biosynthesis, grayanane diterpenoids derive from ent-kaurane-type diterpenoids through A-nor-B-homo rearrangement [19], in other words, ent-kauranetype was assumed to be the biogenetic precursor of grayanane-type. However, ent-kaurane-type diterpenoids were rarely discovered in genus Pieris, until now there are only two ent-kaurane-type diterpenoids pierosides D [20] and E [21] and one 4,5-seco-ent-kaurane pierisformoside G [22] were identified from P. formosa. The discovery of ent-kaurane-type diterpenoids 1–5 further supported the hypothesis of the bioprecursor of grayanane. Grayanane-type undergone ring rearrangements to form leucothane-type (A-homo-B-nor) [23] and kalmane-type (B-homo-C-nor) [24] (Fig. 5). Previous acetic acid-induced writhing test showed that the 95% ethanol extract of the roots of P. formosa exhibited potent analgesic activity. In order to further illustrate the active components and the structure-activity relationships (SARs), the analgesic activities of compounds 2 and 5–10 were tested in an acetic acid-induced writhing test. Ibuprofen (ib) and morphine were used as positive control drugs. As shown in Fig. 6, compounds 2 and 7 displayed promising analgesic activity compared to the vehicle tests (p < 0.05). As far as we know, 2 is the first report of ent-kaurane diterpenoids showed analgesic activity. Comparison with 1, 3 and 8, the main difference of 2 in structural is the OH-11, which indicated the crucial role to the activity. Since the juice of fresh leaves of P. formosa was used as an insecticide in TCM for a long time, compounds 3 and 7 were tested for their antifeeding activity against P. xylostella larvae using the lobular dish method with 0.05% triton water-solution as the negative control. At a dose of 0.5 mg/mL, 3 and 7 showed antifeeding activity with the inhibition ratios of 27.1% and 52.5%, respectively. 4. Conclusions 10
In summary, ten diterpenoids with diverse carbon skeletons, including four new ent-kaurane-type pierisentkaurans B–E (1–4), one new 4,5-seco-ent-kaurane-type pierisentkauran F (5), two new leucothane-type 3β,7α,14β-trihydroxy-leucoth-10(20),15-dien-5-one (6) and 10α,16α-dihydroxyleucoth-5-one (7), and three knowns ent-kaurane-type 16α-dihydroxy-6-oxo-ent-kauran-18-oic-acid (8), kalmane-type rhodomollein XXIII (9), and grayanane-type pierisformosoid J (10), were isolated from the roots of P. formosa. From the viewpoint of biosynthesis, the discovery of 1–5 further supported the hypothesis that ent-kaurane-type was the bioprecursor of grayanane diterpenoids. Acetic acid-induced writhing test showed that compounds 2 and 7 displayed weak analgesic activity at a dose of 5.0 mg/kg compared to the vehicle tests (p < 0.05). Compounds 3 and 7 showed antifeedant activities at 0.5 mg/mL against P. xylostella larvae with inhibition ratios of 27.1% and 52.5%, respectively. Through the systematic study of P. formosa not only enriched the diversity of grayanane diterpenoids, but also in some degree clarified the chemical material foundation of P. formosa used as analgesic and antifeedant in Traditional Chinese Medicine (TCM). Conflict of interest The authors declare no competing financial interest Acknowledgments This work was supported by the CAMS Innovation Fund for Medical Sciences (No. 2016-I2M-1-010), the Natural Science Foundation of China (No. 21732008 and No. 81673314), and the National Megaproject for Innovative Drugs (No. 2018ZX09711001-008). The authors are grateful to the Department of Instrumental Analysis at our institute for the spectroscopic measurements and Li Cui from Institute of Plant Protection, Chinese Academy of Agricultural Sciences for the antifeedant activity screening. Supporting information In the Supporting Information, 1D/2D NMR, IR, and HRESIMS spectra for seven new compounds (1–7) were attached.
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Agric. Biol. Chem. 41 (1977) 2109–2110. (20) T. Kaiya, J. Sakakibara, Occurrence of pieroside C, a grayanotox-9-ene derivative, in Pieris japonica, Chem. Pharm. Bull. 33 (1985) 4637−4639. (21) J. Sakakibara, N. Shirai, T. Kaiya, Diterpene glycosides from Pieris japonica, Phytochemistry 20 (1981) 1744−1745. (22) L.Q. Wang, G.W. Qin, S.N. Chen, C.J. Li, Three diterpene glucosides and a diphenylamine derivative from Pieris Formosa, Fitoterapia 72 (2001) 779−787. (23) T. Kaiya, N. Shirai, J. Sakakibara, One-step synthesis of the diterpenoid leucothol D from grayanotoxin-II, J. Chem. Soc., Chem. Commun. 9 (1979) 431–432. (24) J.W. Burke, R.W. Doskotch, C.Z. Ni, J. Clardy, Kalmanol, a pharmacologically active diterpenoid with a new ring skeleton from Kalmia angustifolia L, J. Am. Chem. Soc. 111 (1989) 5831–583.
Legends for Figures and Tables Fig. 1 The structures of compounds 1–10. Fig. 2 Key 2D NMR correlations of 1, 2, 4, 5, and 6 Fig. 3 Experimental and calculated ECD spectra for 5. Fig. 4 Experimental and calculated ECD spectra for 1. Fig. 5 Biogenetic relationships of different scaffolds and pure compounds isolated from roots of P. formosa. Fig. 6 Analgesic activities of the purified compounds from the roots of P. formosa in an acetic acidinduced writhing test. Ibuprofen (ib) and morphine (morph) were used as control drugs. Each bar represents the mean SEM of the values obtained from 8 mice. *p < 0.05, ***p < 0.001, significant differences between the test compound or control drug groups and vehicle group (veh) (one-way 13
ANOVA followed by the Bonferroni test). Table 1 1H NMR data of compounds 1–5 recorded in Pyridine-d5 Table 2 13C NMR data of compounds 1–7 recorded in Pyridine-d5. Table 3 1H NMR data of compounds 6 and 7 recorded in Pyridine-d5 Graph 1 Graphic abstract .
14
H 20
12
11
14
9 2 3
1
10
8
H7
5
4
O 18
OH
OH
R
O
O
19
18 21
3
19
4 18
20
H 2
1 6
5
O
H 6
HO O
11
8
14
OH
15
13
OH
17
O
O
OH
OH 5
12
20 9
HO
3
18
OH 7
1 5
10
HO
13
H
6
19
H
H
5
4
H
H
4
OH
1
3
OH
H
HO H H
H
10 9 7
19
2
H
2 R1 = H, R2 = OH 3 R1 = OH, R2 = H
1 R = OH 8R=H
HO
O
H
H OH
OH H
15
6
H
16
H
H
R2 R1
17
7
OH
14 8
17
16 15
OH
9
Fig. 1 The structures of compounds 1–10.
15
OH
11
H
H
HO
3
1 5
10
OH 6
H 13 15
HO
HO 10
H
14
8
17
OH OH
Fig. 2 Key 2D NMR correlations of 1, 2, 4, 5, and 6
16
Fig. 3 Experimental and calculated ECD spectra for 5.
17
Fig. 4 Experimental and calculated ECD spectra for 1.
18
Fig. 5 Biogenetic relationships of different scaffolds and pure compounds isolated from roots of P. formosa.
19
Fig. 6 Analgesic activities of the purified compounds from the roots of P. formosa in an acetic acidinduced writhing test. Ibuprofen (ib) and morphine (morph) were used as control drugs. Each bar represents the mean SEM of the values obtained from 8 mice. *p < 0.05, ***p < 0.001, significant differences between the test compound or control drug groups and vehicle group (veh) (one-way ANOVA followed by the Bonferroni test).
20
Table 1 1H NMR data of compounds 1–5 recorded in Pyridine-d5 1b
no. 1
2
3
5
2a
a 1.83, dt (13.2, 3.0)
a 1.83, dt (13.5, 2.5)
b 1.20, dd (12.6, 3.6)
b 1.45, overlap
a 2.11, overlap
a 2.03, m
b 1.50, overlap
3b 3.86, m
4a
5a
a 1.86, dt (13.0, 3.5)
a 2.00, overlap
b 1.08, overlap
b 1.61, d (4.0)
a 2.35, m
a 1.98, overlap
a 1.74, m
b 1.50, overlap
b 1.88, m
b 1.90, m
b 1.50, m
a 2.48, dt (13.2, 3.0)
a 2.13, overlap
a 2.09, dt (13.8, 3.6)
3.57, dd (11.5, 4.5)
a 1.71, overlap
b 1.04, dt (13.8, 3.6)
b 1.21, dt (12.5, 4.0)
b 1.38, overlap
3.90, s
2.54, s
2.56, s
6
b 1.63, m 2.05, d (13.0) a 2.21, dd (11.0, 4.5) b 1.94, d (11.0)
7
4.25, s
a 2.73, d (12.5)
a 2.76, d (12.6)
b 2.65, d (12.5)
b 2.50, d (12.6)
4.07, t (3.0)
6.29, s
9
2.15, d (9.0)
2.11, d (6.5)
1.97, overlap
1.77, d (6.0)
2.28, d (8.5)
11
a 1.61, m
4.71, m
a 3.45, m
a 1.64, overlap
a 1.69, overlap
b 1.95, overlap
b 1.60, overlap
b 1.59, d (5.0)
b 1.59, overlap 12
a 1.92, m
a 2.46, d (10.0)
a 1.74, m
a 1.93, overlap
a 1.67, overlap
b 1.48, overlap
b 2.17, d (11.0)
b 1.66, overlap
b 1.78, overlap
b 1.46, m
13
2.20, t (3.0)
2.26, s
2.15, overlap
2.25, t (3.0)
2.21, overlap
14
a 2.29, dd (11.4, 4.8)
a 2.24, overlap
a 2.22, dd (11.4, 4.2)
a 1.69, m
a 2.51, dd (11.5, 4.0)
b 1.69, d (11.4)
b 2.03, m
b 2.13, d (12.6)
b 1.67, m
b 1.83, d (11.5)
a 2.57, d (14.4)
a 1.99, overlap
a 2.04, d (13.8)
a 2.69, d (14.0)
a 2.23, d (14.0)
b 2.07, d (16.2)
b 1.99, overlap
b 1.98, d (14.4)
b 1.99, d (13.5)
b 1.80, d (14.5)
1.60, s
1.59, s
1.59, s
1.62, s
1.55, s
1.11, s
1.36, s
15
17 18 19
1.29, s
1.48, s
1.40, s
1.31, s
1.32, s
20
1.15, s
1.73, s
1.65, s
1.14, s
1.27, s
3.68, s
3.70, s
21 1-OH
6.16, d (5.4)
4-OH
7.53, s
6-OH
10.07, s
11-OH
6.15, d (4.5)
16-OH
5.65, s
aRecorded
5.45, s
at 500 MHz. bRceorded at 600 MHz.
21
7.58, s
Table 2 13C NMR data of compounds 1–7 recorded in Pyridine-d5. no.
1a
2a
3b
1
41.4, CH2
42.7, CH2
80.4, CH
39.4, CH2
39.2, CH2
43.1, CH
56.7, CH
2
19.3, CH2
19.6, CH2
30.4, CH2
28.5, CH2
20.2, CH2
32.9, CH2
21.0, CH2
3
38.6, CH2
38.8, CH2
36.6, CH2
78.4, CH
45.3, CH2
77.5, CH
40.2, CH2
4
43.5, C
43.4, C
42.5, C
39.1, C
69.4, C
50.8, C
44.6, C
5
59.9, CH
66.8, CH
66.5, CH
45.5, CH
201.4, C
214.3, C
215.6, C
6
222.5, C
208.7, C
208.5, C
37.1, CH
147.3, C
54.0, CH
45.4, CH
7
84.4, CH
56.3, CH2
56.7, CH2
77.0, CH
128.8, CH
73.1, CH
40.3, CH2
8
54.6, C
48.8, C
49.0, C
50.0, C
44.8, C
57.8, C
44.5 C
9
50.2, CH
61.1, CH
57.8, CH
51.7, CH
42.8, CH
43.1, CH
54.8, CH
10
47.1, C
46.2, C
50.1, C
39.3, C
49.7, C
150.8, C
75.0, C
11
18.1, CH2
67.7, CH
20.1, CH2
18.3, CH2
18.3, CH2
21.6, CH2
18.8, CH2
12
26.7, CH2
37.0, CH2
26.6, CH2
28.5, CH2
26.6, CH2
21.8, CH2
26.9, CH2
13
48.7, CH
48.2, CH
49.0, CH
49.3, CH
48.5, CH
55.3, CH
49.5, CH
14
35.1, CH2
37.9, CH2
38.8, CH2
27.8, CH2
39.4, CH2
82.9, CH
36.9, CH2
15
54.6, CH2
59.0, CH2
58.8, CH2
55.4, CH2
58.5, CH2
127.7, CH
58.3, CH2
16
77.5, C
78.5, C
78.1, C
77.8, C
78.5, C
140.1, C
77.8, C
17
24.9, CH3
24.9, CH3
24.8, CH3
25.1, CH3
24.9, CH3
15.8, CH3
25.0, CH3
18
176.4, C
176.3, C
175.9, C
16.7, CH3
30.4, CH3
24.1, CH3
24.9, CH3
19
27.3, CH3
29.1, CH3
28.2, CH3
28.8, CH3
29.6, CH3
21.8, CH3
26.1, CH3
20
17.3, CH3
20.2, CH3
15.8, CH3
18.1, CH3
25.1, CH3
106.4, CH2
21.0, CH3
51.3, CH3
51.4, CH3
21 aRecorded
at 125 MHz.
bRceorded
4a
at 150 MHz.
22
5a
6a
7b
Table 3 1H NMR data of compounds 6 and 7 recorded in Pyridine-d5 no.
6a
7b
1
2.98, dt (12.6, 2.5)
1.65, m
2
a 2.39, dt (13.8, 2.4)
a 2.57, dd (13.5, 3.5)
b 2.24, dt (13.2, 3.6)
b 1.88, dd (13.5, 3.5)
4.16, t (3.0)
a 1.73, dt (13.5, 3.6)
3
b 1.50, dd (13.0, 3.5) 6
3.24, dd (12.6, 9.6)
2.67, dt (12.5, 3.0)
7
4.62, d (9.6)
a 2.18, dd (13.5, 3.0) b 1.84, overlap
9
2.01, d (6.6)
1.70, dd (9.0, 3.5)
11
a 2.18, m
a 2.32, dd (14.5, 6.0)
b 1.60, m
b 1.63, m
a 2.09, m
a 1.82, overlap
b 1.68, m
b 1.70, overlap
13
2.65, t (3.0)
2.22, overlap
14
4.51, brs
a 2.09, overlap
12
b 1.84, d (11.0) 15
5.47, m
a 2.12, d (14.0) b 1.77, overlap
17
1.75, d (1.8)
1.56, s
18
1.17, s
1.10, s
19
1.46, s
1.11, s
20
a 5.07, s
1.54, s
b 5.00, overlap 10-OH
5.37, s
16-OH
5.43, s
aRecorded
at 500 MHz. bRceorded at 600 MHz.
23
Graph 1 Graphic abstract
Pieris formosa
24
Diterpenoids with diverse carbon skeletons from the roots of Pieris formosa and their analgesic and antifeedant activities Changshan Niu#, Sheng Liu#, Yong Li, Yunbao Liu, Shuanggang Ma, Fei Liu, Li Li, Jing Qu*, Shishan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, People's Republic of China.
Conflict of interest The authors declare no competing financial interest.
25
Highlights
1. Seven new diterpenoids with diverse carbon skeletons were isolated from the roots of Pieris formosa. 2. Compounds 2 and 7 displayed weak analgesic activity. 3. Compounds 3 and 7 showed antifeedant activity against Plutella xylostella larvae. 4. In the view of biosynthesis, the discovery of compounds 1–5 further supported the bio-origin of grayanane-type diterpenoids.
26