Shanpanootols G and H, Diterpenoids from the Rhizomes of Kaempferia pulchra Collected in Myanmar and Their Vpr Inhibitory Activities

Abstract

Two new trihydroxy derivative of Δ^8(14),15-isopimarane diterpenoids, shanpanootols G (1) and H (2), along with three known analogues were isolated from the ethyl acetate-soluble extract of Kaempferia pulchra rhizomes collected in Shan State of Myanmar. The structures of these compounds including their absolute configurations were elucidated by the combination of one dimensional (1D) and 2D-NMR spectroscopic methods, high resolution mass spectrometric technique, and the experimental and the calculated electronic circular dichroism (ECD) data. The isopimarane diterpenoids (1–5) were tested for their Viral protein R (Vpr) inhibitory activities against TREx-HeLa-Vpr cells. Shanpanootol H (2) and (1R,2S,5S,9R,10S,13R)-1,2-dihydroxypimara-8(14),15-dien-7-one (4) exhibited anti-Vpr activities at the 5 µM treated dose.

Introduction

Viral protein R (Vpr) is an accessary protein of human immune deficiency virus type-1 (HIV-1) and it is considered as a potential target for the development of anti-HIV drugs.^1–5) In the course of discovering bioactive compounds from medicinal plants of Myanmar,^6–11) the chloroform and ethyl acetate crude extracts of Kaempferia pulchra rhizomes were found to be potent Vpr inhibitors. K. pulchra is a perennial herb of the Zingiberaceae family. In our previous studies, thirty-one compounds including twenty-three unprecedented isopimarane diterpenoids, kaempulchraols A–W,^6,12–14) together with eight known compounds were isolated from the chloroform-soluble fraction of the K. pulchra rhizomes. Furthermore, six new isopimarane diterpenoids, shanpanootols A–F, together with two known analogues were obtained from the EtOAc-soluble fraction.¹⁵⁾ Some of the isolates from these active extracts exhibited the anti-Vpr activities.^6,15) The continuous work on the remaining fractions of the EtOAc-soluble extract afforded two new trihydroxy derivatives of Δ^8(14),15-isopimarane diterpenoids, shanpanootols G (1) and H (2). Herein, we report the isolation, structure elucidation, and Vpr inhibitory activities of the isolated isopimarane diterpenoids from the EtOAc extract of the K. pulchra rhizomes.

Results and Discussion

The EtOAc-soluble extract of K. pulchra inhibited the Vpr activity in TREx-HeLa-Vpr cells with the treated dose of 5 µg/mL. Thus, it was subjected to a series of chromatographic separations, which previously afforded six new isopimarane diterpenoids, named shanpanootols A–F, together with two known analogues.¹⁵⁾ The continuous work on the isolation of the remaining fractions recently furnished five analogues including two new compounds (1, 2) and three known ones (3–5) (Fig. 1).

Fig. 1. Chemical Structures of Compounds 1–5

Compound 1 was obtained as an amorphous powder, and its molecular formula was determined as C₂₀H₃₂O₃ via ¹³C-NMR and high resolution electrospray ionization (HRESI)MS data (m/z 343.2239 [M + Na]⁺). The IR spectrum of 1 showed absorption bands of hydroxy and olefinic groups at 3408 and 1636 cm⁻¹, respectively. The ¹H-NMR spectrum displayed resonances, due to four olefinic protons [δ_H 4.93, dd (J = 10.0, 1.2, H-16b), 4.95, dd (J = 17.8, 1.2 Hz, 16-a), 5.72, d (J = 1.8 Hz, H-14), 5.78, dd (J = 17.8, 10.0 Hz, H-15)], five methines where three were oxygenated ones [δ_H 1.87, d (J = 1.8 Hz, H-5), 2.77, td (J = 7.3, 1.8 Hz, H-9), 3.62, t (J = 3.2 Hz, H-1), 3.91, d (J = 3.2 Hz, H-7), 4.24, t (J = 2.7 Hz, H-6)], four methylenes, and four tertiary methyl singlets [δ_H 1.05 (H₃-19), 1.07 (H₃-20), 1.09 (H₃-17), 1.26 (H₃-18)] (Table 1). The ¹³C-NMR spectrum revealed 20 signals including four sp² carbons [δ_C 111.2 (C-16), 136.9 (C-8), 137.4 (C-14), 147.5 (C-15)], five methine carbons where three were oxygenated ones [δ_C 37.2 (C-9), 44.0 (C-5), 72.1 (C-6), 73.1 (C-1), 78.0 (C-7)], three sp³ quaternary carbons [δ_C 33.9 (C-4), 37.6 (C-13), 42.7 (C-10)], four methylenes [δ_C 17.3 (C-11), 25.8 (C-2), 33.5 (C-12), 35.7 (C-3)], and four tertiary methyls [δ_C 18.4 (C-20), 24.9 (C-18), 26.2 (C-17), 33.4 (C-19)] (Table 2). These NMR spectroscopic data were similar to those of a tetrahydroxylated Δ^8(14),15-isopimarane diterpenoid, roscorane C (5), isolated from K. roscoeana.¹⁶⁾ The significant difference is the disappearance of one oxygenated methine group [δ_H 3.99, ddd (J = 12.2, 4.3, 2.7 Hz), δ_C 66.7, C-2 of roscorane C (5)¹⁶⁾], suggesting 1 to be a trihydroxy derivative of Δ^8(14),15-isopimarane diterpenoid. The heteronuclear multiple bond connectivity (HMBC) correlations of H-1 to C-5/C-9/C-10/C-20, H-6 to C-8/C-10, and H-7 to C-5/C-8/C-9/C-14 (Fig. 2) revealed the attachment of hydroxy groups at C-1, C-6, and C-7, respectively. The relative configuration of 1 was assigned on the basis of a two dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) experiment. The presence of NOESY correlations (Fig. 3) between H-5α and H-6α/H-9α/H₃-19 and between H-1 and H₃-20, and the lack of NOESY correlation between H-7 and H-5α/H-9α suggested the α, β, and α orientations of OH-1, OH-6, and OH-7, respectively. Thus, the structure of 1 was elucidated as 1α, 6β, 7α -trihydroxy-isopimara-8(14),15-diene. The absolute configurations of 1 were determined on the basis of the comparison of the experimental and calculated electronic circular dichroism (ECD) data. The experimental ECD spectrum of 1 exhibited the negative Cotton effect (CE) at 210 nm, which was matched closely with the calculated ECD data for the model compound of 1 with 1S,5S,6S,7S,9S,10R,13R configurations, using the time-dependent density functional theory (TDDFT) method¹⁷⁾ (Fig. 4). Thus, the absolute configurations of 1 were elucidated as shown in Fig. 1 and 1 was named shanpanootol G.

Table 1. ¹H-NMR Spectroscopic Data (400 MHz, CDCl₃) of Compounds 1–3 (δ in ppm and J Values in (Hz) in Parentheses)

Position	1	2	3
1α	—	—	—
1β	3.62, t (3.2)	3.65, br s	3.69, m
2α	1.92, m	—	—
2β	1.56, m	3.87, m	3.93, m
3α	1.74, td (14.2, 3.6)	1.70, m	1.70, m
3β	1.17, dt (13.7, 3.6)	1.41, m	1.43, m
5α	1.87, d (1.8)	1.87, dd (13.3, 2.5)	1.47, m
6α	4.24, t (2.7)	1.75, m	2.00, m
6β	—	1.52, m	1.31, m
7α	—		3.99, m
7β	3.91, d (3.2)	4.15, t (2.7)	—
9α	2.77, td (7.3, 1.8)	2.74, m	2.33, t (7.9)
11α	1.63a), m	1.74, m	1.76, m
11β	1.63a), m	1.43a), m	1.50, m
12α	1.47, m	1.43a), m	1.45, m
12β	1.40, m	1.43a), m	1.59, m
14	5.72, d (1.8)	5.56, d (1.5)	5.65, br s
15	5.78, dd (17.8, 10.0)	5.78, dd (17.4, 10.5)	5.81, dd (17.4, 10.9)
16-a	4.95, dd (17.8, 1.2)	4.94, dd (17.4, 1.3)	4.94, dd (17.4, 1.3)
16-b	4.93, dd (10.0, 1.2)	4.90, dd (10.5, 1.3)	4.91, dd (10.9, 1.3)
17	1.09, s	1.03, s	1.07, s
18	1.26, s	0.88, s	0.91, s
19	1.05, s	0.94, s	0.97, s
20	1.07, s	0.76, s	0.82, s

a) Overlapping resonances within the same column.

Table 2. ¹³C-NMR Spectroscopic Data (400 MHz, CDCl₃) of Compounds 1–3, (δ in ppm)

Position	1	2	3
1	73.1	75.0	74.8
2	25.8	66.7	66.8
3	35.7	42.4	42.3
4	33.9	33.9	34.2
5	44.0	39.1	43.9
6	72.1	28.8	33.9
7	78.0	72.8	72.0
8	136.9	138.9	138.7
9	37.2	38.7	41.4
10	42.7	42.8	42.7
11	17.3	17.6	18.0
12	33.5	33.9	31.8
13	37.6	37.3	36.9
14	137.4	134.6	126.3
15	147.5	148.0	148.3
16	111.2	110.7	110.6
17	26.2	25.6	26.1
18	24.9	23.1	23.3
19	33.4	33.1	33.4
20	18.4	14.2	15.1

Fig. 2. COSY (Bold Lines) and Key HMBC (¹H→¹³C) (Arrows) Correlations in Compounds 1 and 2

Fig. 3. Selected NOESY Correlations (Arrows) in Compounds 1 and 2

Fig. 4. Experimental and Calculated Electronic Circular Dichroism (ECD) Data of 1 in MeOH

Compound 2 was obtained as an amorphous powder. The molecular formula, C₂₀H₃₀O₃ was deduced from the HRESIMS data (m/z 343.2229 [M + Na]⁺). The ¹H- and ¹³C-NMR spectroscopic data (Tables 1, 2) of 2 were similar to those of (1R,2S,5S,7S,9R,10S,13R)-1,2,7-trihydroxypimara-8(14),15-diene (3) isolated from K. pulchra in this study as well as K. marginata,¹⁸⁾ except for C-5, C-6, C-7, and C-9 positions (Tables 1, 2). These noticeable chemical shift differences were considered, because of the opposite orientation of the hydroxy group at C-7 in 2. This assumption was supported by the lack of the NOESY correlations (Fig. 3) of H-7 with H-5α (δ_H 1.87, dd, J = 13.3, 2.5 Hz) and H-9α (δ_H 2.74, m). Based on the aforementioned data, the relative configurations of 2 were determined as 1α, 2α, 7α-trihydroxy-isopimara-8(14),15-diene. Finally, the absolute configurations of 2 were determined by a calculated ECD experiment. The experimental ECD spectra of 2 matched closely with the theoretical ECD spectrum of a model of 2 with 1R,2S,5S,7R,9S,10S,13R configurations (Fig. 5). Thus, the absolute configurations of 2 were established as shown and 2 was named as shanpanootol H.

Fig. 5. Experimental and Calculated ECD Data of 2 in MeOH

The known compounds were identified as (1R,2S,5S,7S,9R,10S,13R)-1,2,7-trihydroxypimara-8(14),15-diene (3),¹⁸⁾ (1R,2S,5S,9R,10S,13R)-1,2-dihydroxypimara-8(14),15-dien-7-one (4),¹⁸⁾ and roscorane C (5)¹⁶⁾ by comparison of their observed and reported NMR data.

All the isolated compounds 1–5 were subjected for Vpr inhibitory activity against TREx-HeLa-Vpr cells according to the previously reported protocol.^6–8) All compounds could inhibit the Vpr activities at the 5 µM treated dose (Table S1, Fig. 6). Among the tested compounds, 2 and 4 were the most potent compounds [% Cell proliferation at 5 µM: 136.42 ± 5.38 (2), 135.25 ± 1.79 (4)] and their potencies were comparable to the effect of positive control, damnacanthal [% Cell proliferation at 5 µM: 143.94 ± 2.12]. The order of potency for other compounds at 5 µM are 5 (121.28 ± 2.76) > 1 (114.84 ± 8.28) > 3 (112.40 ± 2.34). All compounds were not cytotoxic or proliferative in the absence of tetracycline. Structure–activity relationship study revealed that the presence of the carbonyl group at C-7 (4) led to increase the activity, which is the new information for the anti-Vpr activity of the isopimara-8(14),15-diene scaffold.

Fig. 6. The Inhibitory Effects of the Ethyl Acetate Extract, the Isolated Compounds (1–5), and the Positive Control Damnacanthal against the Expression of Vpr in TREx-HeLa-Vpr Cells

Conclusion

The rhizome of K. pulchra native to Myanmar is a rich source of isopimarane diterpenoids. In the present study, two new trihydroxy derivatives of isopimara-8(14)-15-diene, shanpanootols G (1) and H (2), were isolated. Shanpanootol H (2) was found to be the most active compound. In our previous studies, compound containing the carbonyl group at C-14 led to decrease activity, whereas herein, we observed that the presence of the carbonyl group at C-7 could increase the activity.

Experimental

General Experimental Procedures

Optical rotations were recorded on a JASCO P2100 polarimeter. Circular dichroism (CD) measurements were carried out on a Jasco J-805 spectropolarimeter. Infrared spectra were recorded as KBr pellets on a JASCO FT/IR-460 Plus spectrometer. NMR spectra were recorded at 400 MHz (¹H-NMR) and 100 MHz (¹³C-NMR), respectively, on a JEOL ECX400 spectrometers. Chemical shift values were expressed in δ (ppm) and calibrated to the residual proton and carbon resonances of the deuterated chloroform (δ_H 7.26 ppm and δ_C 77.0 ppm). The HR-MS were recorded on a Shimadzu LCMS-IT-TOF spectrometer. Open column chromatography was performed with normal-phase silica gel (silica gel 60N, spherical, neutral, 40–50 µm, Kanto Chemical Co., Inc., Japan), Cosmosil 75C18-OPN (Nacalai Tesque Inc., Kyoto, Japan), and Wakosil 40C18 (30–50 µm) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). TLC was carried out on precoated silica gel 60F₂₅₄ and RP-18 F₂₅₄ plates (Merck, 0.25 or 0.50 mm thickness). The cell line TREx-HeLa-Vpr was maintained in our lab. Cell culture flasks and 48-well plates were from Corning Inc. (Corning, NY, U.S.A.). An SH-1200 Microplate Reader^® (Corona, Hitachinaka, Japan) was used to measure the absorbance.

Plant Material

The Kaempferia pulchra Ridl. rhizomes were collected from Pindaya Township, Shan State, Myanmar in September 2013 and identified by an authorized botanist in the Department of Botany, University of Yangon. A voucher specimen (TMPW 28301) was deposited at the Museum for Materia Medica, Analytical Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Japan.

Extraction and Isolation

The extraction and isolation of the previous works can be seen in the previous reports.^6,12–15) The rhizomes of K. pulchra (500 g) were extracted with CHCl₃ under sonication (1 L, 90 min, ×3) at 35 °C and the solvent was evaporated under reduced pressure to give 30 g of extract. The residue was then extracted with MeOH to give the MeOH extract (30 g). The MeOH extract was partitioned between EtOAc and water to give the EtOAc extract (15 g). In order to exclude the previous isopimarane diterpenoids from the CHCl₃ extract, the EtOAc extract (15 g) was chromatographed on a silica gel open column starting from an EtOAc–n-hexane (1 : 5) solvent system, and the percentages of EtOAc and MeOH were gradually increased. Two liters of solvent were eluted per fraction and a total of 24 fractions were obtained. On the basis of TLC profile, these fractions were pooled to give six main fractions [1: EtOAc–n-hexane (1 : 5) eluate, 7.33 g; 2: EtOAc–n-hexane (1 : 2) eluate, 0.95 g; 3: EtOAc–n-hexane (1 : 1) eluate, 1.22 g; 4: EtOAc–n-hexane (2 : 1) eluate, 1.24 g; 5: EtOAc–MeOH (20 : 1) eluate, 0.685 g; 6: EtOAc–MeOH (10 : 1) eluate, 0.254 g].

Fraction 2 (0.95 g) was rechromatographed on a Cosmosil 75C18-OPN open column with MeCN–H₂O (1 : 2, 1 : 1, 2 : 1, 200 mL/fraction) to give three subfractions [2-1: 50 mg; 2-2: 239 mg; 2-3: 500 mg]. In our previous report, shanpanootol D was obtained from the purification of subfr. 2-2.¹⁵⁾ In the present work, purification of subfr. 2-3 (500 mg) by a silica gel open column with CH₂Cl₂–EtOAc (3 : 1–1 : 1) followed by the normal-phased preparative TLC (CH₂Cl₂–EtOAc, 1 : 1) afforded shanpanootol G (1, 7.8 mg, Rf = 0.62), 3 (12.6 mg, Rf = 0.57), and 4 (25 mg, Rf = 0.71), respectively. Fraction 4 (1.24 g) was rechromatographed on a Wakosil 40C18 open column, eluted with MeCN–H₂O (1 : 2–2 : 1, 200 mL/fraction) to give six subfractions [4-1: 100 mg; 4-2: 147 mg; 4-3: 75 mg; 4-4: 100 mg, 4-5: 50 mg, 4-6: 200 mg]. In the previous report, purification of subfr. 4-1 and 4-3 afforded shanpanootols E and F.¹⁵⁾ Herein, purification of subfr. 4-5 (50 mg) by normal-phased preparative TLC using CHCl₃–MeOH (7 : 1) afforded shanpanootol H (2, 25 mg, Rf = 0.51) and roscorane C (5, 7.2 mg, Rf = 0.42).

Shanpanootol G (1)

Amorphous powder; [α]¹⁸_D −39.9 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 245 (2.13) nm; IR (KBr) ν_max 3408, 2953, 1636, 1459, 1210, 1042, 911 cm⁻¹; CD (c 3.12 × 10⁻⁴ M, MeOH) [medg]₂₀₃ −34.23, [medg]₂₁₀ −68.23; ¹H- and ¹³C-NMR data, see Tables 1, 2; HRESIMS m/z 343.2239 [M + Na]⁺ (Calcd for C₂₀H₃₂O₃Na 343.2244).

Shanpanootol H (2)

Amorphous powder; [α]¹⁸_D −36.0 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 229 (3.2) nm; IR (KBr) ν_max 3414, 2951, 1636, 1460, 1211, 1042, 913 cm⁻¹; CD (c 3.12 × 10⁻⁴ M, MeOH) [medg]₂₁₀ −151.40; ¹H- and ¹³C-NMR data, see Tables 1, 2; HRESIMS m/z 343.2229 [M + Na]⁺ (Calcd for C₂₀H₃₂O₃Na 343.2244).

ECD Calculation

The six conformers with low-energy of 1 and 2 were produced by the DFT/TDDFT calculations, Merck Molecular Force Field (MMFF94) using Winmostar program. All DFT/TDDFT calculations were conducted using the Gaussian 16 program. Firstly, each low-energy conformer obtained by MMFF calculations was subjected to geometry optimization by the DFT method at the B3LYP/6-31G(d) level. Each optimized conformer was then subjected to a frequency calculation at the B3LYP/6-31G(d) level in order to estimate the thermal free energy (ΔG) and check for the presence of imaginary frequencies. On the basis of the estimated thermal energy, the abundance ratio of each conformer was calculated by the Boltzmann distribution. Finally, four conformers of 1 and 2 (in total, occupying approximately 99%) were selected for ECD calculations. The ECD spectra of all conformers were calculated using the TDDFT method at the B3LYP/6-31G(d) level with PCM in methanol, and the weighted-average spectra were compared with the experimental ECD spectra recorded in methanol.

In Vitro Anti-Vpr Activity

The cell line used was TREx-HeLa-Vpr, which was established in our lab. The in vitro anti-Vpr activity of crude extract and isolated compounds was determined by the procedure as described previously.^6–8) Briefly, the established cell line, TREx-HeLa-Vpr (6000 cells/well, 150 µL), was seeded in 48-well plates and incubated in α-minimal essential medium (α-MEM, Wako), supplemented with 10% fetal bovine serum (FBS, Nichirei Bioscience), 1% antibiotic antimycotic solution (Sigma-Aldrich), 5 µg/mL of blasticidin (Invitrogen), and 50 µg/mL of zeocin, at 37 °C under a 5% CO₂ and 95% air atmosphere, for 24 h. Since the expression of Vpr is regulated by tetracycline, the tetracycline-treated cells were designed to express Vpr by the addition of 50 µL of tetracycline (10 µg/mL). After 24 h incubation, 50 µL portions of various samples at different concentrations (2.5, 5 µg/mL or µM) were added to the tetracycline-treated cells, and the wells without samples were used as controls. After 48 h incubation, 50 µL of 10% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) solution was added to the wells. After 3 h incubation, the absorbance at 570 nm was measured, and the cell viability was calculated from the mean values of data from three wells, by the following equation.

  

The inhibitory activity of the tested sample was obtained by comparing the number of viable cells treated with both tetracycline and sample to the number of viable cells treated with tetracycline without sample.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (JSPS KAKENHI Grants JP20H00490, JP20KK0173, JP19H04649, 19K23794, 20K07111).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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