A new abietane norditerpenoid, 5-(6-isopropyl-2-methylnaphthalen-1-yl)pentan-2-one
(1), with a rare 4,5-seco-19-norabietane skeleton possessing a rearranged
angular methyl group at C-5, 2 new naturally occurring compounds,
19-norabieta-4,6,8,11,13-penttaen-3-one (2) and 14-hydroxy-7-oxo ester
(3), along with 10 known analogs (4−13) were isolated
from the resins of Populus euphratica. The new structures were elucidated
by spectroscopic analysis (one-/two-dimensional nuclear magnetic resonance and
high-reolution electrospray ionization mass spectrometry). Biological evaluation showed
that compounds 1 and 2 display low cytotoxicity against normal
cells and appreciable cytotoxicity in cancer cells, with 2 to be more
sensitive in HepG2 cells with a half-maximal inhibitory concentration value of 27.0
μM.
Populus euphratica (Salicaceae) is mainly distributed in western Inner
Mongolia, Gansu, Xinjiang and other places in China.1P. euphratica, also known as the desert polar, could survive in the desert
ecosystem characterized by salt stress, extreme temperatures, and drought.2P. euphratica Scbrenk, the resins from P. euphratica, has
been historically used as a traditional Chinese medicine and applied in Chinese folk remedy
for the treatment of swelling and pain in throat, tuberculosed adenitis, and toothache.3 Previous chemical studies on P. euphratica revealed that it contains
salicin derivatives, volatile oils, and some phenolic compounds.4-6 Besides, anti-inflammatory activity of the resin extract was also reported.1 As a part of our studies on plant resins, the title material was investigated,
resulting in the isolation of a new rearranged abietane norditerpenoid (1), 2 new
naturally occurring compounds (2 and 3), and 10 known but first
isolated abietane diterpenoids (4−13) from this species. Subsequently, the
cytotoxicity of compounds 1-3 in human cancer cells was assayed.
Compound 1, a colorless gum, has the molecular formula
C19H24O based on analysis of its high-resolution electrospray
ionization mass spectrometry (HRESIMS) m/z 269.1902 [M + H]+
(calculated for C19H24O, 269.1900), 13C nuclear magnetic
resonance (NMR), and distortion enhancement by polarization transfer (DEPT) spectra,
indicating 8 degrees of unsaturation. The 1H NMR data (Table 1) of 1 shows an ABX system
[δH 7.99 (d, J = 8.8 Hz), H-11, 7.59 (d, J = 1.8
Hz), H-14, 7.41 (dd, J = 8.8 Hz, 1.8 Hz), H-12], a double bond
[δH 7.25 (d, J = 8.1 Hz), H-6, 7.57 (d, J = 8.1
Hz), H-7], indicating the presence of one 1,2,3,4-tetrasubstituted and one
1,2,4-trisubstituted benzene rings. In addition, 4 methyl groups [δH 2.48 (s), 2.16 (s), 1.34 (d, J = 6.9 Hz, H3-17), and
1.33 (d, J = 6.9 Hz, H3-16)] are also observed. The 13C
NMR and DEPT spectra (Table 1)
display 19 carbons including 4 methyl, 3 methylene, 6 methine groups (5 olefinic and 1
aliphatic), and 6 carbons (1 keto-carbonyl and 5 olefinic). These data remind that
1 might be a diterpenoid derivative. Actually, the structural architecture of
1 was mainly assembled by using two-dimensional (2D) NMR. The
1H−1H COSY spectrum of 1 shows the correlations of
H2-1/H2-2/H2-3, H-6/H-7, H-11/H-12 and
H3-16/H-15/H3-17 (Figure 1). The heteronuclear multiple bond correlations (HMBCs) of H-6/C-8, C-10,
C-20, H-7/C-5, C-9, C-14, H-11/C-8, C-10, C-13 and H-12/C-9, C-14, C-15, H-14/C-9, C-15
indicate the presence of a substituted naphthalene ring. The HMBCs of H2-1/C-5,
C-9, C-10, H2-2, H2-3/C-4 (δC 208.9) and H3-18/C-3, C-4 indicate the substituted group
(4-ketone-1-pentane group) at C-10. An isopropyl group is connected to C-13 via C-15 supported
by the HMBCs of H3-16/C-13, C-15, C-17 and H3-17/C-13, C-15 (Figure 1). Thus, the structure of compound
1, named 5-(6-isopropyl-2-methylnaphthalen-1-yl)pentan-2-one, was identified.
Compound 1 is a rare 4,5-seco-19-norabietane skeleton
diterpenoid bearing a rearranged angular methyl group at C-5. To the best of our knowledge,
before only 3,4-dihydro-6-isopropyl-2-methyl-1-(4′-oxopentyl)naphthalene as a similar
structure was synthesized.7
1H (600 MHz) and 13C NMR (150 MHz) Data of 1-3
(δ in ppm, J in Hz, CD3OD).
No.
1
δC
No.
2
δC
No.
3
δC
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
1
3.04 (overlap)
28.0, t
1
2.62 (overlap); 2.24 (m)
33.3, t
1
2.29 (m); 1.63 (m)
37.2, t
2
1.91 (m)
24.1, t
2
2.73 (m); 2.62 (overlap)
34.2, t
2
1.77 (overlap)
18.3, t
3
2.58 (t, 7.0)
43.6, t
3
198.0, s
3
1.78 (overlap); 1.71 (m)
36.6, t
4
208.9, s
4
129.7, s
4
46.7, s
5
132.3, s
5
156.3, s
5
2.67 (dd, 14.0, 3.2)
43.5, d
6
7.25 (d, 8.4)
129.3, d
6
6.66 (d, 9.8)
123.7, d
6
2.76 (dd, 17.8, 14.0) 2.35 (dd, 17.8, 3.2)
37.9, t
7
7.57 (d, 8.4)
126.1, d
7
6.76 (d, 9.8)
133.5, d
7
205.3, s
8
132.9, s
8
130.8, s
8
114.8, s
9
130.9, s
9
142.9, s
9
153.8, s
10
134.9, s
10
38.8, s
10
37..6, s
11
7.99 (d, 8.8)
132.8, d
11
7.30 (d, 8.0)
123.9, d
11
6.77 (d, 8.0)
113.3, d
12
7.41 (dd, 8.8, 1.8)
125.9, d
12
7.15 (dd, 8.0, 1.9)
127.2, d
12
7.36 (d, 8.0)
133.6, d
13
145.0, s
13
147.5, s
13
134.9, s
14
7.59 (d, 1.8)
124.0, d
14
7.09 (d, 1.9)
126.5, d
14
160.9, s
15
3.04 (overlap)
34.0, d
15
2.89 (m)
33.7, d
15
3.32 (m)
26.2, d
16
1.34 (d, 6.9)
24.0, q
16
1.26 (d, 7.0)
24.0, q
16
1.22 (d, 6.9)
22.5, q
17
1.33 (d, 6.9)
24.0, q
17
1.25 (d, 7.0)
24.0, q
17
1.21 (d, 6.9)
22.3, q
18
2.16 (s)
30.2, q
18
1.90 (s)
10.7, q
18
1.33 (s)
16.7, q
19
19
19
177.9, s
20
2.48 (s)
20.2, q
20
1.39 (s)
30.1, q
20
1.24 (s)
22.3, q
21
3.66 (s)
52.4, q
Key COSY and HMBCs of 1.
Compound 2, obtained as a yellow gum, has a molecular formula
C19H22O deduced from its HRESIMS with a pseudo-molecular ion peak at
m/z 267.1741 [M + H]+ (calculated for
C19H22O, 267.1743), 13C NMR, and DEPT spectra. The
1H NMR spectrum of 2 (Table 1) shows signals assignable to 4 methyl groups
[δH 1.90 (s), 1.39 (s) 1.26 (d, J = 7.0 Hz, H3-16) , and
1.25 (d, J = 7.0 Hz, H3-17)] and 5 olefinic methines
[δH 7.30 (d, J = 8.0 Hz), 7.15 (dd, J = 8.0, 1.9
Hz), 7.09 (d, J = 1.9 Hz), 6.76 (d, J = 9.8 Hz), 6.66 (d,
J = 9.8 Hz)]. The 13C NMR and DEPT spectra (Table 1) display 19 carbons including
4 methyl, 2 methylene, 6 methine groups (5 olefinic and 1 aliphatic), and 7 carbons (1
keto-carbonyls, 5 olefinic, and 1 quaternary). Analyses of NMR data of 2 and
12 reveal that 2 is an analog of 12, based on the
close similarity of their NMR data with the exception of the carboncarbon single bond
(δC 27.0, C-6, δC 27.5, C-7) in 12 replacing the carboncarbon double bond
(δC 123.7, C-6, δC 133.5, C-7) in 2. This conclusion is supported by
1H-1H COSY correlation of H-6 (δH 6.66)/H-7 (δH 6.76) and HMBCs of H-6, H-7/C-8 (δC 130.8). Thus, the structure of 2 was determined as depicted (Figure 2).
Chemical structures of compounds 1 to 13.
Compound 3 was obtained as a pale yellow solid. Its molecular formula was
determined as C21H28O4 on the basis of its HRESIMS
m/z 345.2057 [M + H]+ (calcd for
C21H28O4, 345.2060), 13C NMR, and DEPT spectra.
The 1H NMR spectrum of 3 (Table 1) gives signals for 5 methyl groups
[δH 3.66 (s), 1.33 (s), 1.24 (s), 1.22 (d, J = 6.9 Hz,
H3-16) and 1.21 (d, J = 6.9 Hz, H3-17)] and two
olefinic methine [δH 7.36 (d, J = 8.0 Hz) and 6.77 (d, J = 8.0 Hz)].
The 13C NMR and DEPT spectra (Table 1) display 21 carbons including 5 methyl, 4 methylene, 4 methine groups (2
olefinic and 2 aliphatic), and 8 carbons (2 keto-carbonyls, 4 olefinic, and 2 quaternary).
Analyses of NMR data of 3 and 11 reveal that 3 is an
analog of 11. The difference is that an additional hydroxyl at C-14
(δC 160.9) in 3 is observed, which is supported by the HMBCs of H-11,
H-12/C-14 (δC 160.9). The structure of compound 3 was thus deduced.
Of note, both 2 and 3 were previously synthesized.8,9 However, they, as new naturally occurring products, were isolated from natural origins
for the first time. Their NMR data were also unambiguously assigned by 2D NMR experiments for
the first time. In addition, 10 known analogs were identified as 18-norabieta-8,11,13-4-ol
(4),10 6,8,11,13-abiteratrien-18-oic acid (5),11 19-nor-abieta-4(18),8,11,13-tetraen-7-one (6),12 abietane ester (7),13 dehydroabietic acid (8),14 7-oxodehydroabietic acid (9),11 7α-hydroxydehydroabietic acid (10),15 7-oxoabieta-8,11,13-trien-18-oate (11),16 4,4a,9,10-tetrahydro-1,4a-dimethyl-7-isopropyl-2(3H)-phenanthrone (12),17 and methyl-13-acetyl-podocarpa-8,11,13-trien-18-oate (13),18 respectively, by comparison their spectroscopic data with the literature. All the known
compounds were isolated from the resins of P. euphratica for the first
time.
In this study, compounds 1 and 2 were examined for their cytotoxic
activities against human cancer cells including Kyse30, HepG2, A549, BGC-823, and MDA-MB231.
The results showed that compounds 1 and 2 and 5-fluorouracil (5-FU)
significantly inhibit the growth of 5 human cancer cell lines in a dose-dependent manner
(Figure S1Figure S1). We found that the cytotoxicity of compound 2 is much better
than that of 5-FU in all cancer cell lines (Table S1). On the other hand, the IC50
values, which are higher in normal human umbilical vein endothelial cells than in the cancer
cells, suggesting that these compounds exert acceptable cytotoxicity in normal cells and high
selectivity in cancer cells.
Experimental
General
UV spectrum was recorded on a UV/VIS spectrophotometer. NMR spectra were recorded on a
Bruker Avance III 600 MHz spectrometer, with TMS as an internal standard. ESIMS and
HRESIMS were measured on an Agilent 6230 TOF MS spectrometer. C-18 silica gel (40-60
µm; Daiso Co., Japan), MCI gel CHP 20P (75-150 µm, Mitsubishi Chemical
Industries, Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia, Uppsala, Sweden), and
silica gel (200-300 mesh; Qingdao Marine Chemical Inc., China) were used for column
chromatography. Semipreparative HPLC was carried out using an Agilent 1200 liquid
chromatograph (Agilent Technologies, Santa Clara, CA, USA). The column used was a YMC-Pack
ODS-A 250 mm × 10 mm, i.d., 5 µm.
Plant Resins
The medicinal materials of P. diversifolia were obtained from Bayin,
Xinjiang Autonomous Region, in November, 2011, where the trees of P.
euphratica were grown. The material was secreted from P.
euphratica and a voucher specimen (CHYX0573) is deposited at School of
Pharmaceutical Sciences, Shenzhen University, China.
Extraction and Isolation
The dried resins (50 kg) were soaked with 95% EtOH (300 L × 3 × 24
h) to give a crude extract, which was extracted with EtOAc to afford an EtOAc soluble
extract (12.0 kg). This extract was divided into eight fractions (Fr.A-Fr.H) by using a
silica gel column eluted with petroleum ether–acetone (50:1, 35:1, 20:1, 15:1,
10:1, 7:1, 3:1, 1:1). Fr.A (640.0 g) was separated via MCI gel CHP 20P eluted with aqueous
MeOH (50%-100%) to provide nine portions (Fr.A.1-Fr.A.9). Among them, Fr.A.3
(30.0 g) was subjected to a RP-18 column eluted with aqueous MeOH (50%-100%)
to provide three portions (Fr.A.3.1-Fr.A.3.3). Fr.A.3.2 (26.0 g) was further separated via
MCI gel CHP 20P eluted with aqueous MeOH (50%-100%) to yield nine fractions
(Fr.A.3.2.1-Fr.A.3.2.9). Fr.A.3.2.6 (2.6 g) was passed through Sephadex LH-20 (MeOH) to
yield two fractions (Fr.A.3.2.6.1 and Fr.A.3.2.6.2). Fr.A.3.2.6.1 (1.1 g) was divided into
5 fractions (Fr.A.3.2.6.1.1- Fr.A.3.2.6.1.5) by using silica gel chromatography (petroleum
ether–acetone, 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 1:1). Fr.A.3.2.6.1.3 (245.7 mg)
was purified by semipreparative HPLC (aqueous MeOH, 90%) to give 4 portions.
Fr.A.3.2.6.1.3.1 (8.0 mg) was separated by RP-HPLC to produce compound 4 (1.2
mg, tR = 11.3 min, flow rate: 3 mL/min). Fr.A.3.2.8 (1.1 g) was submitted to
Sephadex LH-20 (MeOH) to provide three portions (Fr.A.3.2.8.1-Fr.A.3.2.8.3). Fr.A.3.2.8.3
(580.0 mg) was purified by semipreparative HPLC (aqueous MeOH, 90%) to give two
portions. Compound 5 (2.8 mg, tR = 17.0 min, flow rate: 3 mL/min) was purified from Fr.A.3.2.8.3.1 (3.2 mg) by
semipreparative HPLC (aqueous MeCN, 80%). Fr.A.7 (60.0 g) was separated via an
RP-18 column eluted with gradient aqueous MeOH (60%-100%) to yield 13
fractions (Fr.A.7.1-Fr.A.7.13). Fr.A.7.7 (1.2 g) was passed through Sephadex LH-20 (MeOH)
to give 4 portions. Fr.A.7.7.3 (191.0 mg) was subjected to preparative TLC developed with
petroleum ether–acetone (15:1) to afford eight portions
(Fr.A.7.7.3.1-Fr.A.7.7.3.8). From Fr.A.7.7.3.2 (44.7 mg), compound 6 (5.2 mg,
tR = 19.5 min, flow rate: 3 mL/min) was obtained by HPLC separation (aqueous
MeOH, 88%). Further purification of Fr.A.7.7.3.4 (26.7 mg) by semipreparative HPLC
eluted with aqueous MeOH (88%) afforded compound 3 (7.2 mg,
tR = 22.0 min, flow rate: 3 mL/min). Compound 1 (2.6 mg,
tR = 18.6 min) was obtained from Fr.A.7.7.3.6 (15.8 mg) by HPLC separation
(aqueous MeCN, 80%, flow rate: 3 mL/min). Fr.A.7.9 (393.0 mg) was submitted to
Sephadex LH-20 (MeOH) to yield two fractions (Fr.A.7.9.1 and Fr.A.7.9.2), of which,
Fr.A.7.9.2 (31.7 mg) was purified by semipreparative HPLC (aqueous MeCN, 93%) to
yield compound 7 (27.2 mg, tR = 17.1 min, flow rate: 3 mL/min). Fr.A.4 (180.0 g) was separated via RP-18
eluted with aqueous MeOH (65%-100%) to provide nine portions
(Fr.A.4.1-Fr.A.4.9). Compound 8 (3.0 g) was obtained from Fr.A.4.3 (10.1 g)
via MCI gel CHP 20P eluted with aqueous MeOH (50%-100%). Fr.A.4.4 (11.8 g)
was subjected to an RP-18 column washed with aqueous MeOH (60%-100%) to
provide 4 portions (Fr.A.4.4.1-Fr.A.4.4.4). Fr.A.4.4.1 (221.0 mg) was separated by
semipreparative HPLC (aqueous MeOH, 80%) to give 5 portions. Fr.A.4.4.1.5 (50.0 mg)
was further separated by HPLC (aqueous MeCN, 65%) to yield 9 (4.5 mg,
tR = 18.1 min, flow rate: 3 mL/min) and 10 (24.0 mg,
tR = 16.0 min). Separation of Fr.A.4.4.1.6 (11.0 mg) by HPLC (aqueous MeCN,
80%) afforded 11 (4.7 mg, tR = 17.9 min, flow rate: 3 mL/min). Fr.A.4.1 (4.8 g) was submitted to Sephadex
LH-20 (MeOH) to yield three fractions (Fr.A.4.1.1-Fr.A.4.1.3). Fr.A.4.1.3 (2.6 g) was
further divided into 4 parts (Fr.A.4.1.3.1-Fr.A.4.1.3.4) by a silica gel column washed
with increasing acetone in petroleum ether (100:1, 60:1, 30:1, 15:1, 5:1 1:1). HPLC
separation of Fr.A.4.1.3.3 (30 mg) by aqueous MeOH (82%) yielded 13
(5.3 mg, tR = 16.8 min, flow rate: 3 mL/min). Fr.A.5 (136.0 g) was subjected to an RP-18
column washed with aqueous MeOH (70%-100%) to provide nine portions
(Fr.A.5.1-Fr.A.5.9). Fr.A.5.2 (1.5 g) was passed through Sephadex LH-20 (MeOH) to yield 4
fractions (Fr.A.5.2.1-Fr.A.5.2.4). Further purification of Fr.A.5.2.2 (415.5 mg) by
semipreparative HPLC eluted with aqueous MeCN (72%) afforded compounds
2 (10.4 mg, tR = 24.7 min, flow rate: 3 mL/min) and 12 (4.2 mg,
tR = 25.6 min, flow rate: 3 mL/min).
HRESIMS m/z 269.1902 [M + H]+ (calculated for
C19H24O, 269.1900).
Biological Evaluation
Compounds 1 and 2 were evaluated for their inhibition on cancer
cell proliferation. In this assay, human cancer cells (Kyse30, HepG2, A549, BGC-823, and
MDA-MB231) derived from the stomach, liver, breast, lung, and esophagus were used. All
cells were exposed to various concentrations of compounds (0-80 µM) for 48 hours,
and cell viability was quantified by CCK-8 assay.
Footnotes
Acknowledgments
This work was supported by National Key Research and Development Program of China
(2017YFA0503900) and National Science Fund for Distinguished Young Scholars (81525026).
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research,
authorship, and/or publication of this article.
Funding
The author(s) disclosed no financial support for the research, authorship, and/or
publication of this article.
Supplemental Material
References
1.
GongX.LiZ.ZhangNet al. Anti-inflammatory activity and chemical composition of
resin extracts from Populus diversifolia Schrenk.
Modern Chin Med.
2018;12:1185-1189.
2.
YangQ.HanX-M.GuJ-K.LiuY-J.YangM-J.ZengQ-Y. Functional and structural profiles of GST gene family from
three Populus species reveal the sequence-function decoupling of
orthologous genes. New Phytol.
2019;221(2):1060-1073.doi:10.1111/nph.15430
3.
LiuYM. Pharmacognostic studies on Resina populi.
Zhong Yao Tong Bao.
1988;13(8):8-9.
4.
WeiW.RenaK.YangX-W. New salicin derivatives from the leaves of Populus
euphratica. J Asian Nat Prod Res.
2015;17(5):491-496.doi:10.1080/10286020.2015.1028920
5.
LuoJR.JiangHE.ZhaoYX.LiuYQ.QianJF. Volatiles from the heartwood of Populus
euphratica in ancient tomb. Nat Prod Res
Dev.
2007;19(5):819-821.
6.
LuoJ-R.JiangH-E.ZhaoY-X.ZhouJ.QianJ-F. Components of the heartwood of Populus
euphratica from an ancient tomb. Chem Nat
Compd.
2008;44(1):6-9.doi:10.1007/s10600-008-0003-2
7.
CambieRC.FranichRA.FullertonTJ. Chemistry of the Podocarpaceae. XXXI. Reactions of ring-A
epoxy derivatives of 18-Norabieta-8,11,13-triene and 12-Methoxy-18-norpodocarpa-8,1
1,13-triene. Aust J Chem.
1971;24(3):593-607.doi:10.1071/CH9710593
8.
CambieRC.FranichRA. Chemistry of the Podocarpaceae. XXIV. ring-A modifications
of Abieta- 8,11,13-trien-18-oic acid. Aust J Chem.
1970;23(1):93-105.doi:10.1071/CH9700093
LeeC.FangJM.ChengYS. Norditerpenes from Juniperus chinensis. Phytochemistry.
1995;39(2):391-394.doi:10.1016/0031-9422(94)00868-T
11.
LuY.HuaY. Abietane diterpenoids from Callicarpa
pedunculata. Nat Prod Res Dev.
2011;23(1):66-68.
12.
KinouchiY.OhtsuH.TokudaH.NishinoH.MatsunagaS.TanakaR. Potential antitumor-promoting diterpenoids from the stem
bark of Picea glehni. J Nat Prod.
2000;63(6):817-820.doi:10.1021/np0000217
13.
AlkhathlanHZ.Al-HazimiHM.Al-DhalaanFS.MousaAA. Three 2-(2-phenylethyl) chromones and two terpenes from
agarwood. Nat Prod Res.
2005;19(4):367-372.doi:10.1080/14786410412331280122
14.
van BeekTA.ClaassenFW.DoradoJ.GodejohannM.Sierra-AlvarezR.WijnbergJBPA. Fungal biotransformation products of dehydroabietic
acid. J Nat Prod.
2007;70(2):154-159.doi:10.1021/np060325e
15.
GuoPY.YangL.DaiJG.GuoSX. Diterpenoids from Aquilariae lignum
Resinatum (III). Chin Traditi Herb Drugs.
2015;46:486-489.
16.
ZaidiSF.AwaleS.KalauniSK.TezukaY.EsumiH.KadotaS. Diterpenes from "Pini Resina" and their preferential
cytotoxic activity under nutrient-deprived condition. Planta
Med.
2006;72(13):1231-1234.doi:10.1055/s-2006-947189
17.
KutneyJP.HanK. Studies with plant-cell cultures of the Chinese herbal
plant, Tripterygium wilfordii. Isolation and characterization of
diterpenes. Rec Trav Chim Pays Bas.
1996;115(1):77-93.doi:10.1002/recl.19961150115
18.
TanakaR.OhtsuH.MatsunagaS. Abietane diterpene acids and other constituents from the
leaves of Larix kaempferi. Phytochemistry.
1997;46(6):1051-1057.doi:10.1016/S0031-9422(97)84396-8
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