Phytochemical and biological studies on rare and endangered plants endemic to China. Part XIV. Structurally diverse terpenoids from the twigs and needles of the endangered plant Picea brachytyla

Article abstract of DOI:10.1016/j.phytochem.2019.112161

A phytochemical investigation on the MeOH extract of the twigs and needles of the endangered plant Picea brachytyla led to the isolation and characterization of thirty-eight structurally diverse terpenoids. Seven of these molecules are previously undescribed, including three abietane-type (brachytylins A–C) and one labdane-type (brachytylin D) diterpenoids, an unseparated C-24 epimeric mixture of cycloartane-type triterpenoids (brachytylins E/F, ratio: 1:1), and a rare rearranged 12(1 → 6)-abeo-megastigmane glycoside (brachytylins G). Their structures and absolute configurations were determined by extensive spectroscopic (e.g., detailed 2D NMR and ECD) methods and/or X-ray diffraction analyses. All the isolates were evaluated for their inhibitory activities against the adenosine triphosphate (ATP)-citrate lyase (ACL) and the Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2). Among them, abiesadine J showed inhibitory effect against ACL, displaying an IC₅₀ value of 17 μM. 3S,23R-Dihydroxycycloart-24-en-26-oic acid exhibited inhibitory effect on SHP2, with an IC₅₀ value of 19 μM. Meanwhile, 3R*,23S*-dihydroxycycloart-24-en-26-oic acid was found to have inhibitory effects against both ACL and SHP2, with IC₅₀ values of 16 and 12 μM, respectively.

Full text of DOI:10.1016/j.phytochem.2019.112161

Phytochemistry 169 (2020) 112161
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phytochemical and biological studies on rare and endangered plants
endemic to China. Part XIV. Structurally diverse terpenoids from the
twigs and needles of the endangered plant Picea brachytyla
a
a
b
c
a,d
b
b
Wei Jiang , Juan Xiong , Yi Zang , Junmin Li , Ezzat E.A. Osman , Jing-Ya Li , Yu-Bo Zhou ,
b,∗∗
a,c,∗
Jia Li , Jin-Feng Hu
a
Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, PR China
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, PR China
Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation, Taizhou University, Taizhou, 318000, Zhejiang, PR China
Laboratory of Medicinal Chemistry, Theodor Bilharz Research Institute, Giza, 12411, Egypt
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Picea brachytyla (Franch.) E.Pritz.
Pinaceae
Endangered plant
Terpenoids
Brachytylins
A phytochemical investigation on the MeOH extract of the twigs and needles of the endangered plant Picea
brachytyla led to the isolation and characterization of thirty-eight structurally diverse terpenoids. Seven of these
molecules are previously undescribed, including three abietane-type (brachytylins A–C) and one labdane-type
(brachytylin D) diterpenoids, an unseparated C-24 epimeric mixture of cycloartane-type triterpenoids (bra-
chytylins E/F, ratio: 1:1), and a rare rearranged 12(1 → 6)-abeo-megastigmane glycoside (brachytylins G). Their
structures and absolute configurations were determined by extensive spectroscopic (e.g., detailed 2D NMR and
ECD) methods and/or X-ray diffraction analyses. All the isolates were evaluated for their inhibitory activities
against the adenosine triphosphate (ATP)-citrate lyase (ACL) and the Src homology-2 domain containing protein
tyrosine phosphatase-2 (SHP2). Among them, abiesadine J showed inhibitory effect against ACL, displaying an
IC50 value of 17 μM. 3S,23R-Dihydroxycycloart-24-en-26-oic acid exhibited inhibitory effect on SHP2, with an
IC50 value of 19 μM. Meanwhile, 3R*,23S*-dihydroxycycloart-24-en-26-oic acid was found to have inhibitory
effects against both ACL and SHP2, with IC50 values of 16 and 12 μM, respectively.
ATP citrate lyase (ACL)
SHP2 tyrosine phosphatase
1. Introduction
change impacts. There are 39 Pinaceae species recorded in the first
volume of the China Plant Red Data Book (CPRDB) published in 1992
In general, plants from the Pinaceae family are trees (rarely shrubs),
(Fu and Jin, 1992). The second volume of the CPRDB is currently under
preparation and will contain more than six hundreds of additional en-
dangered plants (López-Pujol et al., 2006), including increased numbers
of Pinaceae species. Recently, several statistical surveys unveiled that
the rare and endangered plants (REPs) could serve as better sources for
drug discovery than other botanic sources (Zhu et al., 2011; Ibrahim
et al., 2013). A pioneering phylogenetic study of the terrestrial plants
showed that nature-derived drugs are mainly produced by specific
drug-productive plant families, and most REPs species are in drug-
producing families (Ibrahim et al., 2013). Therefore, there is a tre-
mendous need to prioritize protection and utilization of these REPs
species at extinction risk. Since 2013, a special program has been
launched to systematically identify bioactive/novel natural products
from REPs native to China (Xiong et al., 2018), and more attention has
which can grow up to 2–100 m tall. Based on the morphology and the
microscopical anatomy of the cones, pollen, seeds, wood, and leaves,
this family is now divided into 11 genera (The Plant List, 2013): Abies,
Cathaya, Cedrus, Keteleeria, Larix, Picea, Pinus, Pseudolarix, Pseudotsuga,
Tsuga, and Nothotsuga. The last one was not listed in an earlier paper, in
which the family was described to comprise 10 genera only (Hu et al.,
2
016a; Zheng and Fu, 1978). In fact, Nothotsuga contains only one
species endemic to China, N. longibracteata. It is now regarded as a
genus of coniferous trees in the family Pinaceae (The Plant List, 2013;
Qiu et al., 2013). It is noteworthy to point out that Pinaceae is among
the major plant families that produce high numbers of approved drugs
Zhu et al., 2011). However, the threats to the Pinaceae species have
increased under anthropogenic activities and other massive ecological
(
∗
Corresponding author. Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, PR China.
Corresponding author.
∗∗
E-mail addresses: jli@simm.ac.cn (J. Li), jfhu@fudan.edu.cn (J.-F. Hu).
https://doi.org/10.1016/j.phytochem.2019.112161
Received 13 May 2019; Received in revised form 25 September 2019; Accepted 28 September 2019
0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Table 1
1
a
H NMR data (δ in ppm, J in Hz) for compounds 1–3 and 7 .
No.
1b
2b
3c
No.
7d
1
1.59 (m) (H-1ax
)
1.63 (ddd, 12.6, 12.4, 4.2) (H-1ax
)
1.63 (m) (H-1ax
2.18 (br d, 13.0) (H-1eq
1.77 (m) (H-2eq
1.81 (m) (H-2ax
1.45 (m)
)
1
2
3
4
5
6
7
/
2
.38 (br d, 12.9) (H-1eq
)
)
2.20 (br d, 12.4) (H-1eq
)
)
6.16 (s)
/
6.16 (s)
/
2
1.81 (br d, 13.6) (H-2eq
1.77 (m) (H-2eq
1.81 (m) (H-2ax
1.49 (m)
)
)
)
)
1
.87 (qt-like, 13.6, 4.0) (H-2ax
1.58 (m)
.43 (m)
)
3
1
1.43 (m)
2.41 (dd, 2.9, 2.2)
5.96 (dd, 9.7, 2.2)
/
5
6
2.31 (dd, 13.5, 4.4)
2.75 (dd, 18.1, 4.4) (H-6eq
2.40 (dd, 3.0, 2.7)
5.93 (dd, 10.0, 2.7)
2.05 (m)
1.84 (m)
)
2
/
/
.69 (dd, 18.1, 13.5) (H-6ax
)
8
9
1.02 (m)
7
9
6.56 (dd, 9.7, 2.9)
/
7.14 (d, 8.1)
7.24 (dd, 8.1, 1.9)
7.09 (d, 1.9)
/
1.52 (s)
1.53 (s)
3.94 (d, 11.0)
3.88 (d, 11.0)
1.09 (s)
6.54 (dd, 10.0, 3.0)
/
3.81 (dd, 11.6, 5.5)
1.11 (d, 6.0)
2.08 (s)
1.32 (s)
2.08 (s)
4.28 (d, 7.8)
3.11 (dd, 8.0, 7.8)
3.35 (dd, 8.0, 7.0)
3.26 (m)
3.24 (m)
3.87 (br d, 11.8)
3.67 (dd, 11.8, 5.1)
10
11
12
13
1′
2′
3′
4′
5′
6′
1
1
1
1
1
1
1
1
1
2
2
3
1
2
4
5
6
7
8a
8b
9
0
′
7.52 (d, 8.3)
8.15 (dd, 8.3, 1.6)
8.54 (d, 1.6)
/
7.10 (d, 8.0)
7.07 (dd, 8.0, 2.0)
6.93 (d, 2.0)
2.86 (sept, 7.0)
1.24 (d, 7.0)
1.24 (d, 7.0)
3.94 (d, 11.0)
3.86 (d, 11.0)
1.07 (s)
1.08 (s)
2.63 (m)
2.63 (m)
/
/
2.64 (s)
3.48 (d, 11.0)
3.21 (d, 11.0)
0.98 (s)
1.30 (s)
1.09 (s)
2.63 (m)
2.63 (m)
3.09 (s)
/
/
/
′
OMe
a
Assignments were made by a combination of 1D and 2D NMR experiments.
b
c
Measured in CDCl
Measured in CDCl
3
(600 MHz).
(400 MHz).
3
d
3
Measured in CD OD (400 MHz).
been paid to the REPs species in the family Pinaceae (Hu et al., 2016a,
017). A convictive example for the scientific community is that two
rare sesquiterpenoids featuring a 6/6/5 ring system with an oxaspir-
olactone motif, beshanzuenones C and D, were obtained from the cri-
tically endangered Pinaceae plant Abies beshanzuensis (Hu et al.,
reported spectroscopic data and physicochemical properties, the known
structures were identified to be metaglyptin B (8) (Tu et al., 2019), 7-
oxo-dehydroabietinol (9) (Tanaka et al., 1997), 7α,18-dihydroxy-de-
hydroabietanol (10) (Ohmoto et al., 1987), 7β,18-dihydroxy-dehy-
droabietanol (11) (Ohmoto et al., 1987), 18-succinoyloxyabieta-
8,11,13-triene (12) (Raldugin et al., 2005), abiesadine J (13) (Yang
et al., 2010), abiesadine Q (14) (Yang et al., 2010), 12β-hydroxyabieta-
7,13-abietadien-18-oic acid (15) (Bleif et al., 2011), 12α-methoxy-
abieta-7,13-dien-18-oic acid (16) (Wu et al., 2010), abietic acid (17)
(Karlberg et al., 1988), 7-oxo-dehydroabietic acid (18) (Ayer and
Macaulay, 1987), abieta-8,11,13,15-tetraen-18-oic acid (19) (Tanaka
et al., 1997), 7α-methoxy-dehydroabietic acid (20) (Wu et al., 2010),
dehydroabietic acid (21) (van Beek et al., 2007), 15-hydroxy-dehy-
droabietic acid (22) (Cheung et al., 1993), 15-methoxy-dehydroabietic
acid (23) (Wang et al., 2010), 13S-hydroxy-9-oxo-9,10-seco-abiet-
8(14)-en-18,10α-olide (24) (Tanaka et al., 2004), karamatsuic acid (25)
(Ohtsu et al., 1999), 8(14)-podocarpen-13-oxo-18-oic acid (26)
(Cheung et al., 1993), isopimaric acid (27) (Ryu et al., 2010), cis-
abienol (28) (Hieda et al., 1983), labd-13(E)-ene-8α,15-diol (29)
(Forster et al., 1985), 8-hydroxy-14,15-dinor-11(Z)-labden-13-one (30)
(Hlubucek et al., 1974), (+)-3β-hydroxymanool (31) (Munesada et al.,
1992), 3S,23R-dihydroxycycloart-24-en-26-oic acid (32) (Anjaneyulu
et al., 1999), 3S*,23S*-dihydroxycycloart-24-en-26-oic acid (33)
(Anjaneyulu et al., 1999), 3R*,23S*-dihydroxycycloart-24-en-26-oic
acid (= 23-hydroxyisomangiferolic acid A, 34) (Nguyen et al., 2017),
2
2
016b). The unique structures, the precious source, and the inhibiting
activities of beshanzuenones against the protein tyrosine phosphatase
B (PTP1B) have soon attracted the attention of scientists for their total
1
syntheses and also provided valuable clues for producing new synthetic
analogues with extended biological activity on the Src homology-2
domain containing protein tyrosine phosphatase-2 (SHP2) (Davis et al.,
2018).
Among the 39 Pinaceae species in the first volume of the CPRDB (Fu
and Jin, 1992), six belong to the genus Picea (P. aurantiaca, P. brachy-
tyla, P. montigena, P. neoveitchii, P. obovate, and P. smithiana). As an
endemic species to Qinling Mountains, Picea. brachytyla (Franch.)
E.Pritz. has been protected as a ‘vulnerable’ plant in the CPRDB (Fu and
Jin, 1992). The mountains geographically provide a special natural
boundary between North and South China, and support a huge variety
of plant species, some of which could be found nowhere else on Earth.
P. brachytyla has never been phytochemically or pharmacologically
investigated. In the course of our continuous interest in identifying
bioactive compounds from REPs endemic to China (Xiong et al., 2018),
the chemical constituents of the twigs and needles of this plant have
been studied. As a result, a total of 38 structurally diverse terpenoids
were isolated and characterized, including seven previously un-
described ones (1–7). Reported herein are their isolation, structure
determination, and biological evaluations.
3R*,23R*-dihydroxycycloart-24-en-26-oic
acid
(=
23-hydro-
xyisomangiferolic acid B, 35) (Nguyen et al., 2017), 6S,9S-dihydroxy-
megastigma-4-en-9-O-β-D-glucopyranoside (36) (Wang et al., 2009),
dilatanone (37) (Park et al., 2018), and 4-[4-hydroxy-6-(hydro-
xymethyl)-2,6-dimethyl-1-cyclohex-1-en-1-yl]butan-2-one (38) (Miyase
et al., 1987), respectively. Metaglyptin B (8) was just recently reported
from Metasequoia glyptostroboides (Tu et al., 2019), but the absolute
configuration remaining unknown. In this study, its absolute config-
uration was unequivocally determined as (4R,5R,10S) by comparison of
2. Results and discussion
The water-suspended 90% MeOH extract of the twigs and needles of
P. brachytyla (10 kg) was fractionated successively with petroleum
ether, EtOAc, and n-BuOH. Further chromatographic separation of the
EtOAc and n-BuOH fractions afforded seven previously undescribed
its ECD data (Δε2 +0.50, Δε
11
227
+0.47) with those analogues reported
in literature (Machumi et al., 2010).
(
1–7) and 31 known (8–38) terpenoids. By comparing the observed and
2

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Table 2
1965), allowing the assignment of a 5R,10S configuration for 1. The
structure with the absolute configuration of 1 was finally established by
a Ga-Ka X-ray crystallographic analysis (Fig. 5). Accordingly, com-
pound 1, named brachytylin A, was characterized as (4R,5R,10S)-7,15-
dioxo-17-nor-abieta-8,11,13-trien-18-ol. Biogenetically, compound 1,
featuring an acetyl group at C-13, would be generated by an oxidative
fission of the double bond between C-15 and C-17.
The molecular formula of brachytylin B (2) was established as
25 34 5
C H O based on C NMR data and a quasi-molecular ion at m/z
13.2337 [M – H] in its negative mode HRESIMS. Like compound 1,
13
a
C NMR data (δ in ppm, 150 MHz) for compounds 1–4 and 7 .
1b
2b
3b
4b
7c
No.
No.
1
2
3
4
5
6
7
8
9
37.3
18.1
34.7
37.8
42.1
35.8
198.4
130.9
160.6
38.3
124.4
132.9
135.2
128.0
197.3
35.5
18.2
34.9
36.1
34.9
18.3
35.5
37.5
38.2
27.2
78.7
38.8
55.0
19.9
43.8
74.2
61.8
38.7
23.1
133.4
131.2
133.3
114.1
19.9
24.4
28.2
15.4
15.5
1
2
3
4
5
6
7
8
169.2
128.9
188.7
128.9
169.1
48.5
33.6
33.2
74.8
19.8
19.9
25.7
19.9
102.0
75.2
78.2
71.8
77.9
62.9
45.6
45.6
1
3
128.6
128.5
132.5
146.4
37.5
121.6
125.2
143.4
124.1
76.6
28.0
27.8
72.5
18.2
20.7
172.3
28.6
29.0
175.5
50.7
128.5
128.5
132.6
145.4
36.1
121.7
125.7
146.3
124.7
33.6
24.0
24.0
72.4
18.2
20.8
172.2
28.3
29.0
174.1
-
4
signals assignable to the 1,2,4-trisubstitued aromatic C ring [δ
H
7.14 (d,
9
J = 8.1 Hz, H-11), 7.24 (dd, J = 8.1, 1.9 Hz, H-12), and 7.09 (d,
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
0
1
2
3
4
5
6
7
8
9
0
′
10
11
12
13
1′
2′
3′
4′
5′
6′
J = 1.9 Hz, H-14)] could be readily distinguished in its 1H NMR spec-
trum (Table 1), implying an abieta-8,11,13-triene scaffold for 2. The H
1
13
and C NMR data of 2 were comparable to those of abiesadine J (13), a
known dehydroabietanol congener with a succinoyloxy substituent at
C-18 previously isolated from the conifer Abies georgei (Yang et al.,
6
,7
26.7
70.8
17.3
23.7
2010). Unlike 13, a Δ double bond presented in the structure of 2,
which was evidenced from the olefinic protons resonated at δ 5.96
dd, J = 9.7, 2.2 Hz, H-6) and 6.56 (dd, J = 9.7, 2.9 Hz, H-7), and the
H
(
key HMBC correlations of H-6/C-8, H-7/C-5, and H-7/C-9 (Fig. 4). The
relative configuration of 2 was determined (as depicted in Fig. 6) based
′
′
′
on the key NOE correlations of H-2ax with H
3 3
-19 and H -20, and of H-5
with H -18. The absolute configuration of 2 was established by analysis
2
OMe
of its ECD data. The Cotton effects (Δε217 –5.1, Δε258 +1.3) shown in its
ECD spectrum (Fig. 7) were opposite to those of chlorabietin L (Xiong
et al., 2016), an ent-abieta-6,8,11,13-tetraene diterpenoid from Chlor-
anthus oldhamii. This suggested 2 is a normal abietane diterpenoid, in
which C-10 has an S configuration and Me-20 is β-oriented (as depicted
in Fig. 1). Thus, the structure of 2 was defined as (4R,5R,10S)-15-
methoxy-18-succinoyloxy-abieta-6,8,11,13-tetraene.
a
Assignments were made by a combination of 1D and 2D NMR experiments.
b
c
In CDCl
3
.
In CD OD.
3
Compound 1 was isolated as colorless needles (from CH
hexane), and the molecular formula was determined to be C19
based on the protonated ion at m/z 301.1800 [M + H]+ (calcd for
2 2
Cl /n-
H O
24 3
-
With a deprotonated ion at m/z 383.2227 [M – H] in its HRESIMS,
C
1
19
H
25
O
3
, 301.1798) in its positive mode HRESIMS. The IR spectrum of
brachytylin C (3) was found to have the molecular formula of
-
1
showed absorption bands (νmax) attributable to hydroxy (3446 cm ),
24 32 4
C H O , with the loss of a methoxy group when compared with that
−1
−1
1
13
conjugated carbonyl (1690, 1676 cm ), and aryl (1594 cm ) func-
tional groups. The presence of the latter two was consistent with the
maximum absorption (λmax) at 233, 257 (sh), and 295 nm in its UV
of 2. Consistent with this, the H and C NMR spectroscopic data of 3
were superimposable on those of 2, except for the absence of the signals
assigned to the methoxy group at C-15. The planar structure of 3 was
further verified by the key HMBC correlations as shown in Fig. 4. Its
relative configuration was also congruent with that of 2 based on the
1
spectrum (Ayer and Macaulay, 1987). Its H NMR spectrum displayed
signals of three methyl singlets [δ
H
0.98 (s, H
-17)], a hydroxymethylene [δ 3.21 and 3.48 (ABq, J = 11.0 Hz,
-18)], and a typical AMX system for three aromatic protons at δ
3 3
-19), 1.30 (s, H -20), 2.64
(
s, H
3
H
3 3 3 2
NOE correlations of H -20/H-1eq, H -20/H-2ax, H-2ax/H -19, H -18/H-
H
2
H
5, and H-5/H-1ax (see Supporting Information). Similar Cotton effects in
the ECD spectra (Fig. 7) of 3 (Δε217 –3.6, Δε258 +1.3) and 2 (Δε217 –5.1,
Δε258 +1.3) suggested that they have the same absolute configurations
(5R,10S) at C-5 and C-10. Compound 3 was thus identified as
(4R,5R,10S)-18-succinoyloxy-abieta- 6,8,11,13-tetraene.
7
(
.52 (d, J = 8.3 Hz, H-11), 8.15 (dd, J = 8.3, 1.6 Hz, H-12), and 8.54
d, J = 1.6 Hz, H-14) (Table 1). Analysis of the 13C and HSQC NMR
spectra of 1 revealed the presence of 19 carbon signals comprising three
methyls, five methylenes (one oxygenated at δc 70.8, C-18), four me-
thines [three olefinic at δc 124.4 (C-11), 128.0 (C-14), 132.9 (C-12)],
five quaternary carbons [three olefinic at δc 130.9 (C-8), 135.2 (C-13),
The positive mode HREIMS of brachytylin D (4) gave a sodium-
adduct ion at m/z 329.2454 [M + Na]+, indicating a molecular for-
1
60.6 (C-9)], and two keto-carbonyl carbons (δ
C
198.4, 197.3)
34 2
mula of C20H O with four degrees of unsaturation. In accordance with
13
(
Table 2). The above data showed general features similar to those of 7-
the molecular formula, the C NMR spectrum of 4 displayed 20 carbon
signals (Table 2) comprising five methyls, six methylenes [one olefinic
oxo-dehydroabietinol (9) (Tanaka et al., 1997), a co-occurring known
abietane-type diterpenoid featuring the aromatic C ring with an AMX
spin system. The most notable difference was that the isopropyl group
at 114.1 (C-15)], five methines [one oxygenated at δ
olefinic at δ 133.4 (C-12) and 133.3 (C-14)], one oxygen-bearing ter-
tiary carbon at δ 74.2 (C-8), and three quaternary carbons [one ole-
finic at δ 131.2 (C-13)]. Its H NMR spectrum indicated the existence
6.88 (dd, J = 17.2,
C
78.7 (C-3), two
C
in 9 was replaced by an acetyl group [δ
5), 26.7 (C-17)] in 1. This, in conjunction with its molecular formula,
suggested that compound 1 is a C-17 norabietene derivative. The 2D
H
: 2.64 (s, H
3
-17), δ
C
: 197.3 (C-
C
1
1
C
of four olefinic protons, among which three [δ
H
1
1
structure of 1 was then confirmed by H– H COSY (see Supporting
Information) and HMBC (Fig. 4) data. The large proton-proton coupling
constant (13.5 Hz) between H-5 and H-6ax (Table 1) was indicative of a
trans diaxial relationship between these two protons. The observed NOE
10.8 Hz, H-13), 5.23 (d, J = 17.2 Hz, H-14a), 5.13 (d, J = 10.8 Hz, H-
14b)] were characteristic for a terminal double bond. Meanwhile, sig-
nals for an oxymethine proton resonated at δ
4.7 Hz, H-3) and five methyl singlets at δ 0.78 (s, H
20), 1.00 (s, H -18), 1.21 (s, H -17), and 1.81 (s, H -16) were observed
H
3.24 (dd, J = 11.5,
H
3
-19), 0.86 (s, H -
3
correlations of H
H-6eq implied that H
8, H-5, and H-6eq were all on the opposite side of the molecule. The
ECD spectrum of 1 displayed a positive Cotton effect at 325 nm (Δε
1.26), which was in good accordance with those of reported
5R,10S)-7-oxo-abieta-8,11,13-trienes (Cambie et al., 1964; Snatzke,
3
-19 with H
3
-20 and H-6ax, and of H
2
-18 with H-5 and
3
3
3
-20, H
3
-19, and H-6ax were cofacial, whereas H
2
-
(Table 1). The above data revealed that 4 is a bicyclic labdane-type
diterpenoid similar to cis-abienol (28) (Hieda et al., 1983). A major
difference between the two molecules was the appearance of an addi-
tional secondary hydroxy group in 4, which was concluded to be at C-3
3
1
+
(
according to the key HMBC correlations from H
3 3
-18 and H -19 to C-3
(
Fig. 4). The H-3 was axial as it exhibited a large coupling constant
3

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Fig. 1. Diterpenoids from Picea brachytyla.
(
11.5 Hz) with the vicinal proton H-2ax, implying an equatorial or-
Attempts to separate the mixture by employing different chromato-
ientation for 3-OH. The relative configuration of the trans-decalin core
of 4 was further confirmed by the clear NOE correlations of H-3/H-5, H-
graphic methods especially HPLC, but failed (for details, see Experi-
mental section). In the 1H NMR spectrum of the mixture, signals at δ
H
5
/H-9, H-3/H
peak between H
of 8-OH. Like 28, the clear NOE correlation between H-12 and H
Fig. 6) was indicative of a 12Z-configuration for 4. Therefore, com-
pound 4 was ascertained as labda-12Z,14-dien-3,8-diol.
3
-18, and H
3
-19/H
-17 demonstrated the equatorial orientation
-16
3
-20 (Fig. 6). An intense NOE cross-
0.34 and 0.55 (d, J = 4.0 Hz, H
methylene group. Meanwhile, signals for five methyls [δ
28), 0.88 (d, J = 6.0 Hz, H -21), 0.89 (s, H -30), and 0.97(s, H
-29)], two oxymethine protons [δ 3.30 (dd, J = 10.8, 4.8 Hz, H-3)
and 4.38 (m, H-24)], and a pair of olefinic protons from a terminal
6.37 (1H, br s) and 5.91/5.89 (each 0.5 H, br s)]
2
-19) were typical for a cyclopropane
0.81 (s, H
-18 and
3
-20 and H
3
H
3
-
3
3
3
3
(
H
3
H
Compounds 5 and 6 were obtained as a pair of unseparated isomers
methylene group [δ
H
(
(
in a ratio of 1:1, Fig. 2), and both have the same molecular formula
were observed. These data suggested that both compounds 5 and 6
1
3
13
C
30
H
48
O
4
) based on their HRESIMS and C NMR data (Table 3).
were cycloartane-type triterpenoids. Correspondingly, the C NMR
4

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Fig. 2. Triterpenoids from Picea brachytyla.
data showed two sets of carbon resonances and each contained 30
signals (Table 1), including five methyls, twelve methylenes, six me-
thines, six quaternary carbons, and one carboxyl carbon. Among them,
the carbon resonances arising from the side chain (C-17 and C-21~C-
32, especially for the stereochemistry at C-23 (23R), was undoubtedly
established by the X-ray crystallography analysis (Fig. 2). Correspond-
ingly, compound 33, the 23-epimer of 32, could be concluded to pos-
1
13
sess a (3S,23S) configuration. A direct comparison of the H and
C
2
6, see Table 3) appeared in duplicate. The aforementioned NMR data
NMR spectroscopic data of these two C-23 epimeric isomers (Table 3)
revealed that appreciable differences could be found around C-23,
especially for the coupling pattern of H-23 [appeared as br dd (J = 9.4,
8.0 Hz) for 23R-isomer, but ddd (J = 9.6, 9.3, 4.6 Hz) for the 23S-
isomer]. Compounds 34 and 35, another pair of C-23 epimers featuring
a 3ax-OH group, were previously isolated from Trigona minor (Nguyen
et al., 2017), but their stereochemistry at C-23 are open until this study.
As described above, the configuration of 23-OH was readily assigned as
“23S” in 34, whereas “23R” in 35 based on their 1D NMR data
(Table 3).
for each component were comparable to those of 3S,23R-dihydrox-
ycycloart-24E-en-26-oic acid (32) (Anjaneyulu et al., 1999), and var-
iation was observed only in the nature of the side chain. The difference
2
4
between 32 and 5/6 is that the Δ group in 32 was replaced by a
2
5(27)
terminal Δ
in 5/6, which was evidenced from the 1D NMR data
and HMBC correlations (Fig. 4). Meanwhile, the secondary hydroxy
group at C-23 in 32 was shifted to C-24 in 5/6 as deduced from the
HMBC correlations between this oxymethine carbon (δ
C
72.8/72.1, C-
24) and H -27. A further comparison between the NMR data of com-
2
pounds 5 and 6 (Table 3) revealed that they are C-24 epimeric isomers.
Owing to the limited sample (1.4 mg in total), the absolute configura-
tion at C-24 of 5 and 6 could not be defined. The relative configurations
for the other chiral centers (i.e., C-3, C-5, C-8, C-9, C-10, C-13, C-14,
and C-20) were determined to be the same as those of 32 by inter-
pretation of the NOESY data (Fig. 5) and proton-proton coupling con-
stants (Table 3). In particular, 3-OH was determined to be equatorially
oriented from the large J value (10.8 Hz) between H-3 and H-2ax and
30 7
The molecular formula C19H O of brachytylin G (7) was de-
+
termined by its HRESIMS (m/z 371.2056 [M + H] , calcd for
371.2064) and 13C NMR data (Table 2). The 1H and C NMR spectra
showed characteristic signals for a β-glucose moiety, with the anomeric
13
proton resonated at δ
carbons at δ
The remaining 13 carbon signals comprised four methyls (δ
19.9, and 25.7), two methylenes (δ 33.6 and 33.2), an oxymethine (δ
74.8), two olefinic methines (duplicate at δ 128.9), three quaternary
carbons (δ 169.2, 169.1, and 48.5), and one carbonyl group (δ 188.7).
H
4.28 (J = 7.8 Hz, H-1′) and six oxygen-bearing
102.0, 75.2, 78.2, 71.8, 77.9, and 62.9 (Tables 1 and 2).
19.8, 19.9,
C
C
C
C
the NOE correlations of H-3 with H
3
-28 and H-5. Thus, 5 and 6 were
C
defined as a pair of C-24 epimers of 3β,24ξ-dihydroxycycloart-25-en-
C
C
26-oic acid.
These data were strongly reminiscent of the co-occurring megastigmane
glycoside, 6S,9S-dihydroxymegastigma-4-ene-9-O-β-D-glucopyranoside
(36) (Wang et al., 2009). Unlike the regular megastigmane skeleton (e.
g., compounds 36–38) with a geminal dimethyl group at C-1, Me-12 in
the structure of 7 was shifted to the neighboring C-6 via a Wagner-
Meerwein rearrangement (1,2-alkyl shift) (Hanson, 1991). This was
Along with compounds 5 and 6, four related known cycloartane-
type triterpenoids (32–35) were isolated and purified from the title
plant. Compounds 32 and 33 were first reported from the Indian plant
Mangifera indica, with the stereochemistry at C-23 being unknown
(
Anjaneyulu et al., 1999). In this study, the absolute configuration of
Fig. 3. Megastigmene-type derivatives from Picea brachytyla.
5

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Fig. 4. Observed key HMBC correlations of 1–7.
Fig. 5. ORTEP drawing of 1.
Fig. 7. Experimental ECD curves of 1–3 in MeOH.
Fig. 6. Key NOE correlations of 2, 4, and 5/6.
6

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Table 3
1
13
a
H and C NMR data (δ in ppm) for compounds 5/6 and 32–35 .
No. 5/6b 32c
33c
(J in Hz)d
34c
(J in Hz)d
35c
(J in Hz)d
δ
e
δ
H
(J in Hz)d
δ
e
δ
δ
e
δ
δ
C
e
δ
H
(J in Hz)d
δ
e
δ
H
C
C
H
C
H
C
1
2
1.81 (m)
.25 (m)
1.76 (m)
.57 (m)
3.30 (dd, 10.8, 4.8) 78.9
32.9
2.05 (m)
1.03 (m)
1.69 (m)
1.63 (m)
3.22 (dd, 10.6, 4.2)
/
1.88 (m)
1.63 (m)
0.84 (m)
1.32 (m)
1.11 (m)
1.54 (m)
/
33.3
31.0
2.02 (m)
1.00 (m)
1.68 (m)
1.63 (m)
3.21 (dd, 10.3, 3.8)
/
1.88 (m)
1.63 (m)
0.84 (m)
1.32 (m)
1.12 (m)
1.56 (m)
/
33.2
31.0
1.88 (m)
1.00 (m)
1.95 (m)
1.61 (m)
3.39 (br s)
/
1.88 (m)
1.50 (m)
0.80 (m)
1.33 (m)
1.15 (m)
1.57 (m)
/
28.7
29.6
1.86 (m)
1.02 (m)
1.93 (m)
1.62 (m)
3.39 (br s)
/
1.86 (m)
1.55 (m)
0.81 (m)
1.35 (m)
1.13 (m)
1.58 (m)
/
28.7
29.6
1
30.4
1
3
4
5
6
79.5
41.6
49.6
22.3
79.5
41.6
49.6
22.3
77.7
40.6
42.6
22.2
77.7
40.6
42.2
22.2
/
40.5
47.1
21.1
1.31 (m)
1.60 (m)
0
.80 (m)
1.34 (m)
.10 (m)
7
26.0
29.2
29.5
26.9
26.9
1
8
9
1
1
1.50 (m)
/
/
48.0
20.0
26.1
26.4
49.9
21.2
27.4
27.2
49.5
21.1
27.4
27.2
49.6
21.0
27.8
27.4
49.6
21.0
27.8
27.4
0
1
/
/
/
/
2.00 (m)
2.05 (m)
1.17 (m)
1.69 (m)
1.36 (m)
/
2.02 (m)
1.19 (m)
1.68 (m)
1.41 (m)
/
2.04 (m)
1.16 (m)
1.66 (m)
1.66 (m)
/
2.04 (m)
1.17 (m)
1.69 (m)
1.69 (m)
/
1
.11 (m)
1
2
1.62 (m)
32.0
33.6
34.1
34.2
33.6
1
/
/
.49 (m)
13
14
15
16
45.3
48.8
35.5
28.1
46.7
50.1
36.7
27.5
46.5
50.1
36.6
27.5
46.5
50.2
36.6
29.5
46.6
50.2
36.6
29.2
/
/
/
/
1.30 (m)
1.90 (m)
1.31 (m)
1.99 (m)
1.73 (m)
1.61 (m)
1.05 (s)
0.58 (d, 3.8)
0.37 (d, 3.8)
1.35 (m)
0.99 (d, 6.0)
1.68 (m)
1.68 (m)
1.31 (m)
1.99 (m)
1.87 (m)
1.59 (m)
0.98 (s)
0.56 (d, 3.6)
0.35 (d, 3.6)
1.43 (m)
0.96 (d, 7.0)
1.68 (m)
1.68 (m)
1.32 (m)
1.92 (m)
1.31 (m)
1.65 (m)
0.98 (s)
0.52 (d, 3.5)
0.37 (d, 3.5)
1.32 (m)
0.97 (d, 6.0)
1.66 (m)
1.40 (m)
1.32 (m)
1.92 (m)
1.31 (m)
1.69 (m)
1.05 (s)
0.54 (d, 4.2)
0.38 (d, 4.2)
1.32 (m)
0.99 (d, 6.0)
1.66 (m)
1.51 (m)
1
.31 (m)
17
18
19
1.60 (m)
0.97 (s)
52.2/52.1
18.0
54.1
18.7
30.9
54.2
19.9
30.8
54.2
18.5
30.7
54.1
18.7
30.7
0.55 (d, 4.0)
29.9
0
.34 (d, 4.0)
2
2
2
0
1
2
1.57 (m)
0.88 (d, 6.0)
1.44 (m)
32.2/32.1
18.3/18.2
35.9/35.7
34.3
18.7
44.1
34.6
19.8
44.9
34.6
19.7
44.9
34.3
18.6
44.1
1
.44 (m)
23
24
25
26
27
1.75 (m)
4.38 (m)
/
/
33.0/32.9
72.8/72.1
141.5/141.2
168.2
4.54 (br dd, 9.4, 8.0) 66.7
6.69 (d, 8.0)
4.53 (ddd, 9.6, 9.3, 4.6) 67.6
146.4 6.56 (d, 9.3) 145.0 6.56 (d, 9.4)
129.6
171.5
4.53 (ddd, 9.6, 9.4, 4.6) 67.6
4.54 (br dd, 9.2, 8.0) 66.7
145.1 6.69 (d, 8.0)
146.4
127.7
171.5
12.7
/
/
127.7
171.5
12.7
/
/
/
/
129.6
171.5
13.2
/
/
6.37 (br s)
127.2/126.9 1.83 (s)
1.87 (s)
13.1
1.87 (s)
1.83 (s)
5
.91/5.89 (br s)
28
29
30
0.81 (s)
0.97 (s)
0.89 (s)
14.0
25.4
19.3
0.81 (s)
0.95 (s)
0.94 (s)
14.7
26.1
19.8
0.80 (s)
0.94 (s)
0.94 (s)
14.7
26.4
18.5
0.92 (s)
0.88 (s)
0.96 (s)
26.6
21.9
19.9
0.92 (s)
0.89 (s)
0.95 (s)
26.6
21.9
19.8
a
Assignments were made by a combination of 1D and 2D NMR experiments.
b
c
In CDCl
3
.
In CD OD.
3
d
e
Measured at 400 MHz.
Measured at 600 MHz.
confirmed by the key HMBC correlations from H
3
-12 to C-1, C-5, C-6,
effectively (DeFronzo et al., 2015). Type 2 DM is the most common type
of diabetes, accounting for approximately 90% of all DM cases (Yang
et al., 2012). Recently, quite a few new enzyme-based targets related to
type 2 DM have attracted great attentions (Chu et al., 2010; Dorenkamp
et al., 2018; Li et al., 2019; Sun et al., 2019). Among them, the ade-
nosine triphosphate (ATP)-citrate lyase (ACL) is a strategic enzymeαα
linking both the glycolytic and lipidic metabolism (Granchi, 2018). The
ACL serves as an epigenetic regulator to promote nephropathy in obe-
sity and type 2 DM (Deb et al., 2017; Paton, 2017). Meanwhile, the Src
homology-2 domain containing protein tyrosine phosphatase-2 (SHP2)
is essential for receptor tyrosine kinase signaling and mitogen-activated
protein kinase (MAPK) pathway activation. DM conditions exposure has
been documented to be related with the upregulation of the expression
of the SHP2 tyrosine phosphatase. Therefore, SHP2 is also regarded as a
new target for improving monocyte function in Type 2 DM (Dorenkamp
et al., 2018). Natural products have always played significant roles in
the discovery of new chemical entities (NCEs) and drugs (Newman and
Cragg, 2016). In this study, all the isolates were evaluated for their
inhibitory activity against ACL using BMS 303141 as the positive con-
trol. Compounds 13, 17, 27, and 34 showed moderate ACL inhibitory
activities, with IC50 values ranging from 16 to 29 μM (Table 4). The
1
,4
and C-7 (Fig. 4). In addition, the long conjugated system of Δ -dien-3-
one and the linkage position of the glucose were all verified by the
HMBC correlations as depicted in Fig. 4. The stereochemistry of C-9 was
characterized as R* by comparing its chemical shift (δ
of related megastigmane glycosides reported in literature (9R: δ
75.0; 9S: δ ~77.5) (Matsunami et al., 2010). The identification of the
C
74.8) with those
C
~
C
D-glucose moiety in the mass-limited 7 could be determined by analogy
with the co-occurring 36 based on their similar NMR data and on a
biogenetic consideration. In this study, acid hydrolysis of 36 gave a
monosaccharide, which was identified to be D-glucose by direct com-
parison of its HPLC profile and optical rotation datum with those of an
authentic sample (see Experimental section). Thus, the structure of 7
was elucidated as shown in Fig. 3. Such a rearranged 12(1 → 6)-abeo-
megastigmane scaffold was rarely encountered in nature. Only balsa-
mitone possessing such a backbone was previously obtained from the
Turkish plant Tanacetum balsamita almost 45 years ago (Bohlmann
et al., 1975).
Diabetes mellitus (DM) is a chronic metabolic disorder that occurs
when there are raised levels of blood glucose ascribed to the body not
producing any or enough hormone insulin and/or using insulin
7

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
Table 4
a Sunfire C18 column (5 μm, 250 × 10 mm) and/or a TSK-Gel column
(5 μm, 250 × 4.6 mm). Column chromatography (CC) was carried out
using MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Industries,
Japan), silica gel (100–200 and 200–300 mesh, Ji-Yi-Da Silysia
Chemical Ltd., Qingdao, PR China), and Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Sweden). TLC analysis was conducted on
precoated silica gel GF 254 plates (0.25 mm thick, Kang-Bi-Nuo Silysia
Chemical Ltd., Yantai, PR China). Spots were visualized using UV light
(254 nm) and by spraying with vanillin in sulfuric acid.
Inhibitory activities of indicated compounds against ACL and SHP2.
a
Compound
IC50 (μM)
ACL
SHP2
13
17
27
32
34
17 ±
28 ±
29 ±
> 50
16 ±
2
5
3
> 50
> 50
> 50
19 ±
12 ±
N.T.e
b
b
1
1
2
c
BMS 303141
0.4 ± 0.1
d
N.T.e
4.2. Plant material
Na
3
VO
4
0.9 ± 0.1
a
The values (μM) indicate 50% ACL or SHP2 inhibitory effects. These data
The twigs and needles of Picea brachytyla (Franch.) E.Pritz.
(Pinaceae) were collected from Taibai Mountains (33°88′ N, 107°41′ E),
Shaanxi Province of PR China, in July 2017 (wet season). The plant was
collected and identified by Mr. Gen-Lu Bai (Mei county botanist,
Shaanxi Province). A voucher specimen (20170701) is deposited in the
Department of Natural Product Chemistry, School of Pharmacy, Fudan
University, PR China.
are expressed as the mean ± SEM of triplicate experiments.
b
In this study, abietic acid (17) and isopimaric acid (27) showed comparable
inhibitory effects against SHP2, when compared with literature data [17 (IC50):
0
.32 ± 0.13; 27 (IC50): 0.37 ± 0.13 mM (Hjortness et al., 2018)].
c
Positive control for the ACL assay.
Positive control for the SHP2 assay.
N.T.: Not tested.
d
e
4
.3. Extraction and isolation
inhibitory effects against SHP2 were examined using DiFMUP as the
substrate and Na VO as the positive control. Compounds 32 and 34
exhibited inhibitory activities against SHP2, with IC50 values of 19 and
2 μM, respectively (Table 4). The rest of the isolates were found to be
inactive (IC50 > 50 μM).
3
4
The powdered twigs and needles of P. brachytyla (10 kg) were ex-
tracted four times with 90% MeOH (4 × 10 L) at room temperature.
The MeOH extracts were combined and evaporated in vacuum to yield
a brown residue (1 kg, semi-dry). The crude extract was suspended in
1
2
H O (5 L) and partitioned successively with petroleum ether (PE),
3
. Concluding remarks
EtOAc, and n-BuOH. The entire EtOAc-soluble fraction (330 g) was
subjected to column chromatography (CC) over silica gel [PE-EtOAc
gradient (10:1–0:1, v/v) to EtOAc-MeOH gradient (20:1–0:1, v/v)],
affording ten fractions (Fr.1−Fr.10) based on TLC analysis. Fraction 4
Terpenoids have been often encountered from the endangered
plants in the family Pinaceae (Handa et al., 2013; Hu et al., 2016a,
016b; 2017; Yang et al., 2010; Zhao et al., 2015). This is the first
2
(
10.9 g) was separated over silica gel (PE-Acetone, from 30:1 to 1:1, v/
phytochemical investigation on the endangered coniferous plant Picea
brachytyla, the Part XIV in our series of phytochemical and biological
studies on REPs endemic to China (Xiong et al., 2018). From the MeOH
extract of the twigs and needles of the title plant, a total of thirty-eight
structurally diverse terpenoids were isolated and characterized, in-
cluding seven previously undescribed ones. The absolute configurations
of compounds 1 (abietane-type diterpenoid) and 32 (cycloartane-type
triterpenoid) were finally confirmed based on the X-ray diffraction
analyses, which could help to substantiate the absolute configurations
of related compounds isolated in this study. Compound 7 features a rare
v) to give eight subfractions, Fr.4.1–4.8. Subfraction Fr.4.3 (1.0 g) was
preseparated by gel permeation chromatography on Sephadex LH-20
(
in MeOH), followed by semi-preparative RP-HPLC [MeOH–H
2
O (con-
taining 0.05% TFA, v/v), 85:15], to afford compounds 15 (1.5 mg,
t
t
R
= 8.9 min), 19 (1.1 mg,
= 38.7 min).
R
t = 14.1 min), and 12 (4.4 mg,
R
Fraction 5 (13.7 g) was fractionated on an MCI column with a
stepwise gradient elution of MeOH–H O (50:50–100:0, v/v), and nine
2
fractions (Fr. 5.1–5.9) were collected. Fr. 5.8 (1.2 g) was applied to a
silica gel CC (stepwise PE-EtOAc, 20:1–1:1, v/v) to yield five fractions
12(1 → 6)-abeo-megastigmane scaffold. Compounds 13, 17, 27, and 34
(
Fr. 5.8.1–5.8.5). Fr.5.8.1 was separated via Sephadex LH-20 eluted in
MeOH and semi-preparative HPLC [MeOH–H O (containing 0.05%
TFA, v/v), 80:20] to yield compounds 22 (2.7 mg, t = 9.9 min), 23
2.6 mg, t = 15.6 min), 18 (2.9 mg, t = 22.9 min), and 16 (1.5 mg,
= 28.5 min). Fr. 5.9 (1.9 g) was loaded onto silica gel CC eluted with
showed moderate inhibitory effects on ACL, whereas compounds 32
and 34 exhibited significant inhibitory activities on SHP2. The above
bioactive terpenoids may provide useful clues for discovery and de-
velopment of new therapeutic or preventive agents for treatment of
metabolic disorders (e.g., Type 2 DM) and other ACL or SHP2 related
diseases. Moreover, the identification of new molecules from en-
dangered plants reveals the importance in conservation efforts to pre-
vent species diversity loss in the control of emerging targets.
2
R
(
R
R
t
R
PE-EtOAc (40:1–5:1, v/v) in a stepwise gradient of increasing polarity
to afford five subfractions (Fr.5.9.1–5.9.5). Compound 27 (10.0 mg)
was crystallized from fraction Fr.5.9.2 (300 mg), and the residue was
further purified via a combination of silica gel CC (PE-CH
and semi-preparative HPLC [MeOH–H O (containing 0.05% TFA, v/v),
92:8] to afford compounds 21 (2.0 mg, t = 14.8 min), 28 (7.8 mg,
= 18.9 min), and 17 (2.9 mg, t = 21.1 min).
Fraction 6 (23.7 g) was subjected to an MCI gel column eluted with
gradients of MeOH–H O (30:70–100:0, v/v) to obtain eight fractions
(Fr. 6.1–6.8). Subsequent separation of Fr. 6.4 (1.07 g) by CC over
polyamide (MeOH–H O, 30:70–100:0, v/v) to give three subfractions
2 2
Cl 1:1, v/v)
2
4. Experimental
R
t
R
R
4.1. General experimental procedures
2
Optical rotations were measured on a Rudolf Autopol IV automatic
polarimeter. UV spectra were recorded on a Hitachi U-2900E spectro-
photometer. ECD spectra were recorded on a JASCO-810 CD spectro-
meter. IR measurements were performed on a Nicolet Is5 FT-IR spec-
2
(Fr. 6.4.1–6.4.3). After further purification by Sephadex LH-20 (in
MeOH), subfraction Fr. 6.4.1 (0.87 g) gave four portions
(Fr.6.4.1.1–6.4.1.4). Fr. 6.4.1.2 was chromatographed on silica gel CC
trometer. X-ray data were collected on
a Bruker D8 Venture
diffractometer. NMR spectra were acquired on a Bruker Avance III
and further purified by semi-preparative HPLC [MeOH–H
taining 0.05% TFA, v/v), 75:25] to provide compounds 24 (4.1 mg,
= 10.6 min), 4 (2.0 mg, t = 20.7 min), 30 (5.0 mg, t = 36.5 min),
and 31 (1.9 mg, t = 38.5 min). In a similar way, compounds 1 (4.0 mg,
= 10.2 min) and 26 (7.8 mg, t = 15.2 min) [HPLC mobile phase:
2
O (con-
400 MHz or 600 MHz spectrometer, and the chemical shifts (δ) are
given in ppm with reference to the solvent signals. HRESIMS data were
recorded on an AB Sciex Triple TOF 5600 spectrometer. Semi-pre-
parative HPLC was conducted on a Waters e2695 system equipped with
t
R
R
R
R
t
R
R
8

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
MeOH–H
2
O (containing 0.05% TFA, v/v), 70:30] were obtained from
(calcd for C24
31 4
H O , 383.2228, Δ = −0.2 ppm).
Fr. 6.4.1.3 (100 mg). Fr. 6.5 was also processed in the same way as that
described for Fr. 6.4, and the resultant Fr. 6.5.3 (260 mg) was subjected
4.3.4. Brachytylin D (4)
20
to semi-preparative HPLC (MeOH–H
1 (0.9 mg, t = 18.4 min), 29 (4.1 mg, t
= 29.7 min). Fr. 6.6 (910 mg) was repeatedly separated via Sephadex
2
O, 80:20, v/v) to give compounds
Colorless oil; [α]
D 3
+4.1 (c 0.08, CHCl ); UV (MeOH) λmax (logε)
1
R
R
= 26.6 min), and 10 (2.6 mg,
234 (3.73) nm; IR (KBr) νmax 3450, 2950, 1640, 1460, 1445, 1115,
−1 1
t
R
3
1085, 990, 940, 900 cm ; H NMR (δ in ppm, J in Hz, in CDCl ): 1.10
LH-20 (in MeOH) to afford five fractions (Fr. 6.6.1–6.6.5), and sub-
fraction Fr. 6.6.3 (0.15 g) was further separated using silica gel CC
eluted with PE-acetone (20:1–1:1, v/v) to obtain six fractions (Fr.
(ddd, J = 12.6, 12.4, 4.2 Hz, H-1ax), 1.72 (m, H-1eq), 1.87 (m, H-2a),
1.66 (m, H-2b), 3.24 (dd, J = 11.5, 4.7 Hz, H-3), 0.94 (dd, J = 12.0,
2.0 Hz, H-5), 1.67 (m, H-6a), 1.32 (m, H-6b), 1.87 (m, H-7a), 1.44 (m,
H-7b), 1.30 (m, H-9), 2.44 (m, H-11a), 2.21 (m, H-11b), 5.48 (dd,
J = 8.0, 8.0 Hz, H-12), 6.88 (dd, J = 17.2, 10.8 Hz, H-13), 5.23 (d,
J = 17.2 Hz, H-14a), 5.13 (d, J = 10.8 Hz, H-14b), 1.81 (3H, s, H-16),
1.21 (3H, s, H-17), 1.00 (3H, s, H-18), 0.78 (3H, s, H-19), 0.86 (3H, s,
H-20). For 13C NMR data, see Table 2; HRESIMS [M + Na] m/z
6
.6.3.1−Fr. 6.6.3.6), all of which were purified by semi-preparative
HPLC. Consequently, compound 8 (2.2 mg, t = 10.6 min, MeOH–H O,
8:12, v/v) from Fr. 6.6.3.1, compounds 20 (1.5 mg, t = 17.5 min) and
3 (3.1 mg, t = 19.2 min) [MeOH–H O (containing 0.05% TFA),
0:20, v/v] from Fr. 6.6.3.2, compounds 32 (2.1 mg, t = 18.7 min), 33
= 11.8 min), 34 (1.5 mg, = 20.3 min), 35 (1.0 mg,
= 19.5 min), and 3 (1.0 mg, t = 24.2 min) [MeCN–H O (containing
.05% TFA), 60:40, v/v] from Fr. 6.6.3.3, and compounds 14 (2.2 mg,
= 18.8 min) and the mixture of 5/6 (1.4 mg, = 21.4 min)
MeOH–H O, 80:20, v/v) from Fr. 6.6.3.4, were obtained. The mixture
R
2
8
1
8
R
R
2
+
R
(
3.4 mg,
t
R
t
R
34 2
329.2454 (calcd for C20H O , 329.2451, Δ = +0.8 ppm).
t
R
R
2
0
4.3.5. Brachytylins E and F (5 and 6)2
Amorphous, white powder; [α]
0
t
R
t
R
+12.8 (c 0.14, MeOH); UV
max max
(log ε) 204 (3.66) nm; IR (KBr) ν 3400, 3030, 2950,
2870, 1680, 1630, 1450 cm
D
(
2
(MeOH) λ
−
1
1
13
of 5/6 showed a single spot on TLC in various developing solvent sys-
tems (e. g., PE: EtOAc 1:1, CHCl : MeOH 9:1, PE: acetone 2:1). Further
; H and C NMR data, see Table 3;
-
3
HRESIMS [M – H] m/z 471.3483 (calcd for C30H47O , 471.3480,
4
chromatographic separations of this mixture on HPLC using different
columns (e. g., Waters Sunfire C18, Waters Xbrige, and Cosmosil cho-
Δ = +0.7 ppm).
lester) with the mobile phase of either MeOH/H
not achieved. Compounds 9 (2.0 mg, t = 20.1 min; MeCN–H
v/v) and 25 (1.4 mg, t = 23.5 min) [MeOH–H O (containing 0.05%
TFA), 80:20, v/v] were isolated from Fr. 6.6.5. Fr. 6.7 (600 mg) was
purified over Sephadex LH-20 gel CC and semi-preparative HPLC
2
O or MeCN/H
2
O were
4.3.6. Brachytylin G (7)
20
R
2
O, 75:25,
Yellow gum; [α] −4.1 (c 0.09, MeOH); UV (MeOH) λ
D
max
(log ε)
max
3446, 2924, 1656, 1619, 1380,
R
2
241 (3.99) nm; IR (KBr)
ν
−
1
1
13
1075 cm ; H and C NMR data, see Tables 1 and 2; HRESIMS [M +
+
H] m/z 371.2056 (calcd for C19H31O , 371.2064, Δ = +2.3 ppm).
7
[
2
MeOH–H
(2.3 mg, t
Fraction 8 (23.7 g) was subjected to a MCI gel column eluted with
gradients of MeOH–H O (30:70–100:0, v/v) to obtain five fractions (Fr.
.1–8.5). Fr. 8.2 (11.6 g) was further separated on silica gel CC eluted
with CH Cl –MeOH (30:1–1:1, v/v) to obtain ten fractions (Fr.
.2.1−Fr. 8.2.10). Fr. 8.2.3 (910 mg) was purified using Sephadex LH-
0 gel CC and then semi-preparative HPLC (MeOH–H O, 45:55, v/v) to
= 10.9 min) and 38 (2.3 mg,
2
O (containing 0.05% TFA, v/v), 86:14] to obtain compound
R
= 14.2 min).
4.3.7. Metaglyptin B (8)
2
0
Amorphous, white powder; [α] +9.3 (c 0.14, CHCl ); UV (MeCN)
D
3
−3
2
λ
λ
max
(log ε) 203 (3.73), 265 (2.22) nm; ECD (c 1.0 × 10 M, MeCN)
8
(Δϵ) 211 (+0.50), 221 (+0.47) nm; IR (KBr) ν
max
3447, 2975,
max
2
2
2927, 2853, 2361, 2337, 1738, 1682, 1463, 1383, 1255, 1164,
−1
1
13
8
2
1070 cm
; H and C NMR data, see ref. (Tu et al., 2019); HRESIMS
[M+Na]+ m/z 339.2294 (calcd for C21H32O Na, 339.2295,
2
2
obtain compounds 37 (3.4 mg,
= 12.3 min).
The n-BuOH-soluble fraction was subjected to CC over silica gel
eluted with CH Cl –MeOH (15:1–1:1) to afford eight fractions. The
third fraction was resubmitted to Sephadex LH-20 and semipreparative
HPLC (MeOH–H O, 30:70, v/v), yielding compounds 36 (7.2 mg,
= 17.8 min) and 7 (2.0 mg, t = 20.6 min).
t
R
Δ = +0.0 ppm).
t
R
4.3.8. 3S,23R-dihydroxycycloart-24-en-26-oic acid (32)
Amorphous, white powder; [α] +38.0 (c 0.10, MeOH); H and 13C
2
0
1
2
2
D
–
NMR data, see Table 3; ESIMS [M – H] m/z 470.8.
2
t
R
R
4.3.9. 3S*,23S*-dihydroxycycloart-24-en-26-oic acid (33)
Amorphous, white powder; [α]
NMR data, see Table 3; ESIMS [M – H] m/z 471.0.
2
0
+48.0 (c 0.10, MeOH); H and 13C
1
D
–
4
.3.1. Brachytylin A (1) 2
0
Colorless needles; [α]
D
+2.2 (c 0.12, MeOH); UV (MeOH) λmax (log
ε) 233 (3.75), 257 (3.36, sh), 295 (2.70) nm; IR (KBr) νmax 3446, 2965,
2
2
4.3.10. 3R*,23S*-dihydroxycycloart-24-en-26-oic acid (34)
−1
Amorphous, white powder; [α] +36.0 (c 0.15, MeOH); H and 13C
20
1
923, 1690, 1676, 1594, 1412, 1384, 1212, 1182, 1075 cm ; ECD (c
D
−3
–
.0 × 10 M, MeOH) λmax (Δε) 215 (+2.19), 258 (−2.54), 325
NMR data, see Table 3; ESIMS [M – H] m/z 470.8.
1
13
(
+1.26) nm; H and C NMR data, see Tables 1 and 2; HRESIMS [M +
+
H] m/z 301.1800 (calcd for C19
H
25
O
3
, 301.1798, Δ = +0.6 ppm).
4.3.11. 3R*,23R*-dihydroxycycloart-24-en-26-oic acid (35)
Amorphous, white powder; [α]
NMR data, see Table 3; ESIMS [M – H] m/z 471.2.
2
0
+12.9 (c 0.08, MeOH); H and 13C
1
D
–
4
.3.2. Brachytylin B (2)
2
0
Yellow oil; [α]
D
−20.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
2
1
λ
1
4
19 (4.40), 265 (3.93) nm; IR (KBr) νmax 3444, 2970, 2928, 2855, 1736,
4.3.12. X-ray crystallographic analysis of 1 and 32
−
1
−3
716, 1679, 1467, 1385, 1185 cm ; ECD (c 2.4 × 10 M, MeOH)
max (Δε) 217 (−5.1), 258 (+1.3) nm; H and 13C NMR data, see Tables
X-ray crystallographic data for 1: C19H24O , colorless crystals ob-
tained from a solution of CH Cl /n-hexane (1:10, v/v), M = 300.38,
2 2
Trigonal, Space group P3 , a = 20.1158(2) Å, b = 20.1158(2) Å,
3
1
and 2; HRESIMS [M – H]- m/z 413.2337 (calcd for C25
13.2333, Δ = +0.8 ppm).
33 5
H O ,
2
3
c = 6.77244(9) Å, α = β = 90.00°, γ = 120.00°, V = 2373.28(6) Å ,
3
Z = 6, Dcalcd = 1.261 Mg/m , μ (Ga Kα) = 1.34139 Å, crystal size
3
4
.3.3. Brachytylin C (3)
0.12 × 0.05 × 0.03 mm , F (000) = 972, 33480 reflections collected,
2
0
Yellow oil; [α]
D
−4.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 218
5994
[I > 2σ(I)], wR
wR = 0.2479 (all data), goodness of fit = 1.050, Flack para-
independent
reflections
(Rint = 0.0578),
1
R = 0.0891
= 0.0899 (all data),
(
3.73), 265 (3.28) nm; IR (KBr) νmax 3439, 2955, 2923, 2843, 1736,
2
= 0.2464 [I > 2σ(I)],
R
1
−
1
1
2
716, 1651, 1606, 1469, 1383, 1260, 1171 cm
;
ECD (c
2
.6 × 10 M, MeOH) λmax (Δε) 217 (−3.6), 258 (+1.3) nm; 1H and
−3
meter = 0.11(9).
1
3
-
C NMR data, see Tables 1 and 2; HRESIMS [M – H] m/z 383.2227
48 4
X-ray crystallographic data for 32: C30H O , colorless crystals
9

W. Jiang, et al.
Phytochemistry 169 (2020) 112161
obtained from MeOH, M = 472.68, monoclinic, space group P
1
2
1
1,
4.7. SHP2 inhibitory activity assay
a = 7.1450(2) Å, b = 11.3122(3) Å, c = 17.2791(5) Å, α = 90.00°,
3
β = 100.64°, γ = 90.00°, V = 1372.59(7) Å , Z = 2, Dcalcd = 1.144 Mg/
Using the 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP;
Invitrogen, Carlsbad, CA) as the substrate, reaction buffer contained
3
3
m , μ (Ga Kα) = 1.34139 Å, crystal size 0.1 × 0.03 × 0.01 mm , F
(
000) = 520, 19,450 reflections collected, 5197 independent reflec-
tions (Rint = 0.0531), = 0.0391 [I > 2σ(I)], wR = 0.0952
= 0.0452 (all data), wR = 0.1006 (all data), goodness
60 mM Hepes (pH 7.0), 75 mM NaCl, 75 mM KCl, 0.05% Tween 20,
R
1
2
5 mM DTT, 1 mM EDTA, 25 μM DiFMUP, 0.1 μM GST-SHP2, and dif-
ferent concentrations of test compounds or DMSO in a total reaction
volume of 50 μL in black 384-well plates. Reaction was initiated by
addition of DiFMUP, and the incubation time was 30 min at 37 °C.
DiFMUP fluorescence signal was measured at an excitation of 358 nm
and an emission of 455 nm with a plate reader (PerkinElmer EnVision).
Na VO was used as the positive control (Dorenkamp et al., 2018; Wang
[
I > 2σ(I)], R
1
2
of fit = 1.051, Flack parameter = −0.04(11).
The crystal structures of compounds 1 and 32 were solved by direct
methods using SHELXT. Refinements were performed with SHELXL-
2
2
015 using full-matrix least-squares calculations on F , with anisotropic
displacement parameters for all the non-hydrogen atoms. The hydrogen
atom positions were geometrically idealized and allowed to ride on
their parent atoms. CCDC-1912386 (1) and CCDC-1910919 (32), con-
taining the supplementary crystallographic data, can be obtained free of
charge from the Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif.
3
4
et al., 2017).
Declaration of competing interest
The authors confirm that this article content has no conflict of in-
terest.
4.4. Acid hydrolysis of compound 36
Acknowledgements
3
Compound 36 (3.0 mg) was hydrolyzed with 2 M aqueous CF COOH
(
TFA). After refluxing at 105 °C for 6 h, it was cooled to room tem-
perature, and the solution was extracted with CHCl . The aqueous
phase was concentrated to yield glucose (0.8 mg), which was identified
by HPLC-ELSD using a TSK-Gel column (mobile phase: H O; flow rate:
mL/min; t = 10.1 min), with the authentic sample of D-glucose
Batch # 088K00391, Sigma-Aldrich) as a standard. The D-configura-
This work was supported by NSFC grants (Nos. 21937002,
81773599, 21772025), a STCSM grant (No. 17430742200), and
3
National Science and Technology Major Project for New Drug Research
and Development of MOST (2019ZX09735002-005). WJ thanks Dr.
Jerome M. Fox and his colleague Michael K. Hjortness (Department of
Chemical and Biological Engineering, University of Colorado, USA) for
sharing their valuable biological information of abietic acid and iso-
pimaric acid against the protein tyrosine phosphatase SHP2. Their
prompt replies via emails are highly appreciated.
2
1
(
R
20
tion was confirmed based on its optical rotation value of [α]
(
D
+55.6
2
0
c 0.08, H
2
O) [ref. [α]
D
2
+80.0 (c 0.1, H O)] (Hu et al., 2017).
4.5. Preparation of acetate 37a
Appendix A. Supplementary data
To a solution of 37 (1.5 mg) in 1 mL of dry pyridine was added six
drops of (CH CO) O. This mixture was stirred at room temperature
overnight and concentrated under reduced pressure to give a residue,
which was purified by semi-preparative HPLC (MeOH–H O, 60:40, v/v)
to furnish an acetate 37a (1.9 mg). Compound 37a, [α]
3
2
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.112161.
2
2
0
D
−20.2 (c
.10, MeOH); ECD (c 3.5 × 10 M, MeOH) λmax (Δϵ) 228 (+3.3), 255
References
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and deplete the unconsumed ATP within 60 min at room temperature.
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