Structural elucidation, bio-inspired synthesis, and biological activities of cyclic diarylpropanes from Horsfieldia kingii

Article abstract of DOI:10.1016/j.tet.2020.131494

Bioactivity-guided phytochemical investigation on 70% aqueous acetone extracts of the twigs and leaves of Horsfieldia kingii led to the isolation of two novel cyclic diarylpropanes (1 and 2) bearing a 2,3-dihydro-1H-indene core, one new diarylpropane (3), six known diarylpropanes (4–9), one flavanol (10), and seven lignans (11–17). Their structures were determined by extensive spectroscopic analysis, electronic circular dichroism calculations, and X-ray diffraction crystallography. Moreover, a biomimetic synthesis of 1 and 2 were accomplished in four steps. The in vitro nitric oxide production inhibition tests of these compounds revealed that compounds (±)-2, (+)-2, (?)-2, and 10 were potential with IC₅₀ values lower than 10 μM. Compound 2 could inhibit iNOS expression in LPS-induced RAW264.7 cells at a series of non-cytotoxic concentrations (<20 μM). Furthermore, the bioassay results also suggested the primary SARs of 1-phenyl-2,3-dihydro-1H-indene based scaffold.

Full text of DOI:10.1016/j.tet.2020.131494

Tetrahedron 76 (2020) 131494
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Structural elucidation, bio-inspired synthesis, and biological activities
of cyclic diarylpropanes from Horsfieldia kingii
,
,
,
, *
Rui Zhan a 1, Dashan Li b 1, Yuan-Lie Liu a 1, Xiao-Yan Xie b, Lei Chen b, Li-Dong Shao b
,
Wen-Jing Wang b, Ye-Gao Chen a
,
**
a School of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming, 650050, PR China
b School of Chinese Materia Medica, Yunnan University of Chinese Medicine, Kunming, 650500, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioactivity-guided phytochemical investigation on 70% aqueous acetone extracts of the twigs and leaves
of Horsfieldia kingii led to the isolation of two novel cyclic diarylpropanes (1 and 2) bearing a 2,3-dihydro-
1H-indene core, one new diarylpropane (3), six known diarylpropanes (4e9), one flavanol (10), and
seven lignans (11e17). Their structures were determined by extensive spectroscopic analysis, electronic
circular dichroism calculations, and X-ray diffraction crystallography. Moreover, a biomimetic synthesis
of 1 and 2 were accomplished in four steps. The in vitro nitric oxide production inhibition tests of these
compounds revealed that compounds ( )-2, (þ)-2, (ꢀ)-2, and 10 were potential with IC50 values lower
Received 19 March 2020
Received in revised form
28 July 2020
Accepted 8 August 2020
Available online 23 August 2020
Keywords:
than 10
cytotoxic concentrations (<20
1-phenyl-2,3-dihydro-1H-indene based scaffold.
m
M. Compound 2 could inhibit iNOS expression in LPS-induced RAW264.7 cells at a series of non-
Cyclic diarylpropane
Horsfieldia kingii
Anti-inflammation
Bio-inspired synthesis
m
M). Furthermore, the bioassay results also suggested the primary SARs of
© 2020 Elsevier Ltd. All rights reserved.
1. Introduction
resulted in seven DAPs without cytotoxicity against many cancer
cell lines [10e12,14]. Given that the folk use of Horsfieldia genus
depended on its anti-inflammatory effects, and invoked by our
continuous interests of exploring the fundamental substances of
these ethno-pharmacological resources, the anti-inflammatory
activity guided phytochemical investigations were carried out in
this work. As a result, three new DAPs, horsfielenides C-E (1e3)
along with 14 known ones (4e17) were isolated from the active
fractions. To our interest, compounds 1 and 2 possess a 2,3-
dihydro-1H-indene core representing a new type of natural diary-
lpropane. Herein, the isolation, structural elucidation, bio-inspired
synthesis of 1 and 2, NO production inhibitory activity evaluation of
1e17, and inhibitory effect of 2 on iNOS expression in LPS-induced
RAW264.7 cells are reported.
Horsfieldia is an important genus of Myristicaceae, accounting
for ~11% of species in the family [1]. Species of Horsfieldia have been
used as folk medicines to treat kinds of inflammation related dis-
eases, such as pains and infections, for a long history in South Asia
[2e5]. Previous studies on the chemical components of Horsfieldia
have proved the genus to be plenty of flavonoids [6e14] along with
less other compounds [3,15e17]. Among the reported flavonoids,
diarylpropanes (DAPs) [10e14] or called reduced chalcones usually
possess a linear C6eC3eC6 skeleton representing a rare class of
flavonoids and taking a comparable percentage. However, com-
pounds isolated from Horsfieldia genus were poorly documented
with their bioactivity profiles, especially the relative anti-
inflammatory activity of the genus for folk use.
H. kingii is an importantly economic and ethnopharmacological
plant in South Asia, and has been cultivated in subtropical area of
Yunnan. Previous investigations on the non-oil parts of H. Kingii
2. Results and discussion
A 70% aqueous acetone extracts of the air-dried and powdered
twigs and leaves of H. kingii were partitioned between EtOAc and
H2O. The resulting EtOAc portion was then subjected to column
chromatography (silica gel) eluting with petroleum ether/acetone
to obtain fractions A-D. Fractions C and D with apparent NO pro-
duction inhibitory activities (Table S1, supplementary material)
were further performed on column chromatography (silica gel, MCI
* Corresponding author.
** Corresponding author.
E-mail addresses: shaolidong@ynutcm.edu.cn (L.-D. Shao), ygchen48@126.com
(Y.-G. Chen).
1
These authors contributed equally.
https://doi.org/10.1016/j.tet.2020.131494
0040-4020/© 2020 Elsevier Ltd. All rights reserved.

2
R. Zhan et al. / Tetrahedron 76 (2020) 131494
CHP-20 gel, Sephadex LH-20, and preparative TLC), leading to the
isolation of two new cyclic diarylpropanes, horsfielenides C-D
(1e2), a new diarylpropane, horsfielenide E (3), and 14 known
compounds (4e17) (Fig. 1).
7.05) to C-400
(dC 159.5), along with HREIMS data (m/z 256.1100,
[M]þ, cacld 256.1099, molecular formula: C16H16O3), together
proved 2 to be a 400-methoxy analogue of 1. Similarly, 2 was resolved
to (þ)-2 and (ꢀ)-2 by chiral HPLC (Fig. S22). Further comparing
their experimental spectra with those of 1 (Fig. S1), the absolute
configurations of (þ)-2 and (ꢀ)-2 were assigned as 3S and 3R,
respectively.
Horsfielenide C (1) was obtained as a brown oil. The molecular
formula of 1 was assigned as C15H14O3 by HREIMS (m/z 242.0949,
[M]þ, cacld 242.0943) with 9ꢁ of unsaturation. The 1H NMR data
(Table 1) suggested a symmetric benzene ring [dH 6.94 (2H, d,
J ¼ 8.6 Hz, H-200, 600), 6.70 (2H, d, J ¼ 8.6 Hz, H-300, 500)] and two
singlet aromatic protons [dH 6.69 (1H, s, H-20), 6.31 (1H, s, H-50)]. 13C
NMR spectra (Table 1) showed 15 carbon signals, including 12 ar-
omatic carbons (three were substituted with hydroxy groups,
dC ¼ 156.4, 145.4, and 144.8, respectively), and other three sp3-
hybridized carbons which belonged to one methine (dC 51.9) and
two methylenes (dC 32.2 and 38.4). The 1He1H COSY spectrum
disclosed cross signals for two fragments, CH2(1)ꢀCH2(2)ꢀCH(3)
and CH(200)ꢀCH(300) (Fig. 2). The HMBC correlations from H-1 (dH
2.81, 2.70) to C-2’ (dC 112.3), and from H-2’ (dH 6.69)/H-5’ (dH 6.31)
Compound 3, a colorless oil with a molecular formula of
C
17H18O3 as deduced by HREIMS (m/z 270.1253, [M]þ, calcd
270.1256), exhibited a typical diarylpropane-type 13C NMR spectra
with 12 aromatic carbons and three sp3-hybridized methylenes
(Table 1) [12]. By further analyzing the 1He1H COSY, HSQC, and
HMBC spectrum, compound 3 was determined as 1-(30,40-methyl-
enedioxyphenyl)-3-(400-methoxyphenyl)propane,
and
named
horsfielenide E.
Other compounds isolated were elucidated as virolane (4) [7], 1-
(20-hydroxy-40-methoxyphenyl)-3-(300, 400-methylenediox-
yphenyl)-propan-2-ol (5) [19], 1-(20-hydroxy-40-methoxyphenyl)-
3-(400-hydroxy-300-methoxyphenyl)-propane (6) [20], horsfieleni-
dine A (7) [13], virolanol B (8) [21], virolanol C (9) [21], (þ)-catechin
(10) [22], (ꢀ)-kobusin (11) [23], (þ)-eudesmin (12) [23], (þ)-phil-
lygenin (13) [24], 30-desmethylarctigenin (14) [25], (ꢀ)-hinokinin
(15) [26], matairesinol (16) [27], and 30,40-de-O-methyl-
enehinokinin (17) [28].
to C-3’
dihydroxyphenyl moiety was linked to C-1. In addition, the HMBC
correlations from H-3 (dH 4.05) to C-100 dC 138.5)/C-200
dC 129.9),
and from H-200 dH 6.94) to C-400(dC 156.4) suggested that the 4-
(dC 145.4)/C-4’ (dC 144.8) indicated that the 3,4-
(
(
(
hydroxyphenyl group was placed at C-3. Moreover, the HMBC
correlations from H-3 to C-50 (dC 112.6, d)/C-60 (dC 139.9, s) revealed
the CeC linkage of C-3 to C-60, which altogether permitted com-
pound 1 to be a unique cyclic diarylpropane with a 2,3-dihydro-1H-
indene core. Compound 1 was detected as a racemic mixture by
chiral HPLC (Fig. S11). Being decisive in the absolute configuration
determination for natural prodcuts especially the new ones before
biological evaluation is necessary [18], ECD calculation was used to
establish the absolute configurations of (þ)-1 and (ꢀ)-1. The tested
ECD sprctra of (þ)-1 and (ꢀ)-1 were in good agreement with those
computational calculations of 3S and 3R (Fig. 3), indicating 3S and
3R configurations for (þ)-1 and (ꢀ)-1, respectively.
Horsfielenides C and D (1e2) are the first examples of natural
cyclic diarylpropanes with 2,3-dihydro-1H-indene core. Biogenet-
ically, 1 and 2 were likely derived from chalcone (Scheme 1). To be
detailed, reduction of the corresponding chalcone might give three
intermediates including i (totally reduced), ii (partially reduced),
and iii (partially reduced). Subsequently, diarylpropane i was likely
oxidized to form diarylpropanyl cation iv [29], which was perhaps
also generated by diarylpropanol ii and diarylpropene iii under
acidic conditions. The key transformation would be the Friedel-
Crafts cyclization of iv to give compounds 1 and 2.
The spectroscopic characterization of horsfielenide
D
(2)
Based on the hypothetic biosynthesis of 1 and 2, a total synthetic
route was subsequently developed (Scheme 2). Initially, aldehyde
18 was transformed to aldehyde 19 by homologation using the
Wittig/hydrolysis sequence [30] in 90% yield. Next, the proposed
implied that it was an analogue of 1. The presence of OMe [dH 3.75
(3H, s, H-700)] signal in 1D NMR spectra, similar HMBC correlations
from H-20 (dH 6.68) to C-30 (dC 145.2)/C-40 (dC 145.0), and H-200
(dH
Fig. 1. Structures of compounds 1e17 from H. kingii.

R. Zhan et al. / Tetrahedron 76 (2020) 131494
3
Table 1
1H and13C NMR data for 1e3 (
d in ppm and J in Hz).
1a
2a
3b
No.
dH (J in Hz)
dC (mult.)
dH (J in Hz)
dC (mult.)
dH (J in Hz)
2.58 (t, 7.8)c
dC (mult.)
1
2
2.81 m;
2.70 m
2.40 m
32.2 t
38.4 t
2.84 (ddd, 15.0, 8.5, 3.5)
2.75 m
2.46 m
32.2 t
35.2 t
38.4 t
1.91 m
33.5 t
1.84 m
4.05 (t, 8.1)
1.89 (dq, 12.4, 8.6)
4.11 (t, 8.1)
c
3
51.9 d
136.8 s
112.3 d
145.4 s
144.8 s
112.6 d
139.9 s
e
51.9 d
136.3 s
111.9 d
145.2 s
145.0 s
112.5 d
139.5 s
e
2.60 (t, 7.7)
e
34.5 t
10
20
30
40
50
60
70
100
200
300
400
500
600
700
e
e
136.3 s
109.0 d
144.7 s
145.6 s
108.2 d
121.2 d
100.8 t
134.4 s
129.4 d
113.9 d
157.9 s
113.9 d
129.4 d
55.4 q
6.69 s
e
6.68 s
e
6.71 s
e
e
e
e
6.31 s
e
6.29 s
e
6.75 (d, 7.9)
6.65 (d, 7.9)
5.93 s
e
e
e
138.5 s
129.9 d
116.2 d
156.4 s
116.2 d
129.9 d
e
e
139.4 s
129.8 d
114.7 d
159.5 s
114.7 d
129.8 d
55.6 q
e
6.94 (d, 8.6)
6.70 (d, 8.6)
e
7.05 (d, 8.6)
6.82 (d, 8.6)
e
7.12 (d, 8.6)
6.86 (d, 8.5)
e
6.70 (d, 8.6)
6.94 (d, 8.6)
-
6.82 (d, 8.6)
7.05 (d, 8.6)
3.75 s
6.86 (d, 8.5)
7.12 (d, 8.6)
3.81 s
a
b
c
Data were recorded in CD3OD, 1H NMR (500 MHz),13C NMR (125 MHz).
Data were recorded in CD3Cl, 1H NMR (500 MHz),13C NMR (125 MHz).
Partial overlapped signals.
precursor diarylpropane 3 and diarylpropene 21 were obtained by
Wittig/catalytic hydrogenation sequence and Wittig reaction in 80%
and 88% yield, respectively.
With compounds 3 and 21 in hand, the biomimetic cyclization
was explored. Given benzylic CeH bond is sensitive to oxidants,
compound 3 was chosen as substrate to investigate the desired
cyclization by forming the benzylic cation in situ using DDQ [31],
Fe(II)/DDQ [32], and Fe(II)/(tBuO)2 [33] (Table S2). As a result, dia-
rylpropane 3 was oxidized by DDQ to form dihydrochalcones 3a/3b
and chalcones 3c/3d as inseparable mixtures, respectively (entries
1e3, Table S2). A catalytic amount of FeCl2 (0.2 equiv) significantly
accelerated the conversion of substrate 3, but not the desired
cyclization (entry 4, Table S2). Moreover, no reaction was detected
using (tBuO)2 as an oxidant (entries 5e6, Table S2). All the obser-
vations suggested that the benzylic cation (like diarylpropanic
cation iv in Scheme 1) generated in situ by DDQ could not be
trapped by either of the aromatic rings of 3 but DDQ itself [34].
To carry on the synthesis, various acid-promoted conditions
were screened using diarylpropene 21 as substrate (Table 2). Initial
attempts using trifluoromethanesulfonic acid (TfOH) [35] gave
desired cyclized product 22 albeit in low yields (<15%, entries 1e3).
Other protic acids like sulfuric acid and hydrochloric acid resulted
in decomposion of the starting material 21 (data not shown). Silver
salts [36] (AgBF4, AgOTf, and AgPF6, entries 4e6) also promoted the
Fig. 2. Key 1He1H COSY and key HMBC correlations for 1e3.
Fig. 3. Experimental and calculated ECD spectra of 1.
Scheme 1. Plausible biogenetic pathway for 1 and 2.

4
R. Zhan et al. / Tetrahedron 76 (2020) 131494
Scheme 2. Total synthesis of 1 and 2. Conditions: a) MeOCH2PPh3Cl, LiHMDS, THF, ꢀ78 ꢁC e RT, 2 h; then 2 N HCl, reflux, 3 h, 90%. b) 20, LiHMDS, THF, ꢀ78 ꢁC e RT, 2 h; then Pd/C,
H2 (balloon), EA/MeOH (5 : 1), RT, 6 h, 80%. c) 20, LiHMDS, THF, ꢀ78 ꢁC e RT, 2 h, 88%. d) BBr3, DCM, ꢀ40 ꢁC, 0.5 h, 83%. e) BBr3, DCM, 0 ꢁC, 4 h, 65%.
Table 2
Acid-promoted cyclization of 21.a
c
Entry
Conditions
Time (h)
Product (yield%)
1
2
3
4
5
6
7
8
9
TfOH (0.1 equiv), DCM, ꢀ20 ꢁC
12
3
3
6
4
22 (trace)
22 (10)
22 (15)
22 (17)
22 (30)
22 (10)
22 (16)
TfOH (0.1 equiv), DCM, 0 ꢁC - RT
TfOH (0.1 equiv), DCM, reflux
AgBF4 (0.1 equiv), DCM, sealed, 60 ꢁC
AgOTf (0.1 equiv), DCM, sealed, 60 ꢁC
AgPF6 (0.1 equiv), DCM, sealed, 60 ꢁC
AuCl$Me2S (0.1 equiv), 1,4-dioxane, sealed, 100 ꢁC
Cu(OTf)2 (0.1 equiv), DCM, RT
b
4
12
12
16
10
12
8
b
d
NR
Cu(OTf)2 (0.1 equiv), DCM, 60 ꢁC
22 (33)
22 (35)
22 (45)
22 (55)
10
11
12
Cu(OTf)2 (0.2 equiv), DCM, 60 ꢁC
Cu(OTf)2 (0.1 equiv), DCM, O2 (1 atm), 60 ꢁC
Cu(OTf)2 (0.1 equiv), DCM, O2, sealed, 60 ꢁC
a
All the reactions were performed on 0.1 mmol scale.
the starting materials were recovered.
isolated yield.
no reaction.
b
c
d
Table 3
In vitro NO production inhibitory activities of compounds 1e17 and 22.
cyclization, of which AgOTf could give higher yield of 30%. More-
over, a harsh condition using AuCl$Me2S [37] resulted in low yield
of 22 (entry 7). In addition, we found that cyclization could be also
catalyzed by Cu(OTf)2 [38] in boiling DCM (60 ꢁC) to give 22 in 33%
yield although no reaction was observed at room temperature
(entries 8 and 9). The commonly used solvents like DCE, MeCN,
toluene, etc. were also screened using Cu(OTf)2 as catalyst, but all
the tested solvents gave lower yield than DCM (data not shown).
Higher catalyst loading did not increase the yield but accelerated
the reaction rate (entry 10). Pleasingly, the satisfactory yield of 22
was obtained by filling reaction system with one atm of oxygen in a
sealed tube (entries 11 and 12). Finally, ( )-1 and ( )-2 were pre-
pared by removal of dioxymethylene and methyl of 22 using BBr3 in
DCM, which could further support the structural elucidations of 1
and 2 on the basis of X-ray structure of 22 (Scheme 2).
Compd.
)-1
IC50
(
m
M) a
Compd.
IC50
(m
M) a
(
> 40
> 40
> 40
4.40 0.69
4.76 0.31
6.41 0.21
> 40
28.78 2.12
30.34 4.78
30.43 5.22
24.52 2.13
29.16 2.42
9
10
11
12
13
14
15
16
17
22
L-NMMA
e
> 40
8.86 0.35
> 40
> 40
> 40
> 40
> 40
> 40
> 40
(þ)-1
(ꢀ)-1
(
)-2
(þ)-2
(ꢀ)-2
3
4
5
6
7
8
> 40
13.82 0.81
e
a
Inhibition of NO production, L-NMMA (NG-Monomethyl-
acetate salt) was used as positive control.
L-arginine, mono-
After completion of the synthesis, all isolated compounds (1e17)
and synthetic compound 22 were tested against NO production in
RAW264.7 macrophages. As shown in Table 3, compounds 2, 4e8,
and 10 could dose-dependently inhibit NO production in LPS-
stimulated RAW264.7 macrophages without cytotoxicity
cells, and secondly the binding affinity to target proteins. Moreover,
the similar IC50 values of ( )-2, (þ)-2, and (ꢀ)-2 might be resulted
from a non-selective inhibition mode of these compounds towards
the target. As it is well known, inducible NO synthase (iNOS) plays
an important role in NO amount in inflammation [39]. We docked
(þ)-2 and (ꢀ)-2 into iNOS to provide the possibility for validating
this non-selective inhibition. As shown in Fig. S2, both (þ)-2 (A) and
(ꢀ)-2 (B) could bind to iNOS protein and occupy its active pocket
nearby the Heme-Fe by interacting with residues Tyr341, Gly365,
Trp366, Glu371, and Heme-Fe itself [39,40]. However, the detailed
mechanism deserves to be elucidated in future. Thus, the primary
structure-activity relationships (SARs) of this scaffold are
concluded based on these observations as: hydroxy groups at C30/
(CC50 > 40
showed strong inhibitory activities with IC50 values of 4.40 0.69,
4.76 0.31, and 6.41 0.21 M, respectively, which were more
potent than that of catechin (10, IC50 ¼ 8.86 0.35 M) and positive
M). Interestingly, compound
mM). To be specified, compounds ( )-2, (þ)-2, and (ꢀ)-2
m
m
control L-NMMA (IC50 ¼ 13.82 0.81
m
1 with three free phenolic hydroxy groups (more polar) and com-
pound 22 with no free phenolic hydroxy group (less polar) were
both inactive in inhibition of NO production (IC50 > 40 mM), indi-
cating that the free phenolic hydroxy groups in 1-phenyl-2,3-
dihydro-1H-indene scaffold (1, 2, and 22) might account for firstly
and most importantly the permeability of these compounds into

R. Zhan et al. / Tetrahedron 76 (2020) 131494
5
C40 and methoxy group at C400 are favorable; C3 stereocenter is
tolerable with both 3S and 3R.
China), and spots were visualized by heating silica gel plates
sprayed with 10% H2SO4 in EtOH. Preparative TLC (Si gel 60 GF254
,
To further evaluate the anti-inflammatory potency of compound
2, the inhibitory effect of 2 on iNOS expression in LPS-induced
RAW264.7 cells were performed. As a result, 2 showed dose-
dependent inhibitory effects on iNOS expression in LPS-induced
RAW264.7 macrophages compared to control (Fig. 4), indicating
that compound 2 inhibited LPS-induced NO production likely by
down-regulating iNOS protein expression.
Qingdao Marine Chemical, Inc., Qingdao, China). All reactions were
carried out under an atmosphere of argon in dry and freshly
distilled solvents under anhydrous conditions, unless otherwise
noted, monitoring by TLC (Si gel GF254), and spraying phospho-
molybdic acid (10% in EtOH) followed by heating.
4.2. Plant material
The twigs and leaves of H. kingii were collected from the Guanlei
town, Mengla County of the Yunnan Province in China in November
2016. The samples were identified by Mr. Shishun Zhou, a botanist
of Xishuangbanna Tropical Botanical Garden, Chinese Academy of
Sciences. The voucher specimen (No. 16112110) was deposited in
Yunnan Normal Univesity.
3. Conclusion
In summary, an ethnopharmacological plant H. kingii was
investigated on its bioactive components. A total of 17 compounds
were isolated from the bioactive fractions of H. kingii extracts.
Among these compounds, 2, 4e8, and 10 showed inhibitory effects
on NO production in LPS-stimulated RAW264.7 macrophages
without cytotoxicity. Moreover, a four-step synthetic route to 1 and
2 was developed based on biogenetic hypothesis. Most importantly,
4.3. Extraction and isolation
The twigs and leaves (5.0 Kg) of H. kingii were extracted for four
times (two days for each time) with 70% aqueous acetone (40 L) at
room temperature, and then were filtered followed by concentra-
tion in vacuum to remove acetone. The residue (~10 L) was
extracted with EtOAc (5 L ꢂ 6) and the EtOAc layer was concen-
trated in vacuum to yield EtOAc portions (280 g). The EtOAc portion
was separated by column chromatography (silica gel, petroleum
ether/acetone, 20:1 to 1:1) to yield fractions A-D based on the TLC
analysis. Fraction C (83 g) was subjected to column chromatog-
raphy on Lichroprep RP-18 (MeOHeH2O, 50:50 to 95:5) to provide
four subfractions C1eC4. After purification by repeated column
chromatography (silica gel, CHCl3/acetone, 50:1e10:1 gradient
system, and Sephadex LH-20, CHCl3/MeOH, 3:2), subfraction C1
afforded compounds 2 (22 mg), 6 (14 mg), 14 (7 mg), and 16
(17 mg), repectively. Moreover, C2 was then purified by repeated
column chromatography of Sephadex LH-20 (CHCl3/MeOH, 3:2)
and silica gel (CHCl3/acetone, 30:1 to 10:1) to give compound 4
(25 mg), 13 (18 mg), and compound 16 (12 mg), repectively. Com-
pounds 3 (11 mg), 11 (7 mg), 12 (9 mg), 13 (157 mg), and 15 (16 mg)
were separated from C4 by column chromatography of RP-18
(MeOH/H2O, 50:50 to 100:0), MCI gel (MeOH/H2O, 80:20 to
100:0), and Sephadex LH-20 (CHCl3/MeOH, 3:2), repectively.
Fraction D (42 g) was subjected to column chromatography of
Lichroprep RP-18 (MeOH/H2O, 30:80 to 95:5) to obtain three sub-
fractions D1-D3. From D1, compound 10 (40 mg) was isolated by
column chromatography of Sephadex LH-20 (CHCl3/MeOH, 3:2). D2
was separated by column chromatography of Sephadex LH-20
(CHCl3/MeOH, 3:2), silica gel (CHCl3/acetone, 30:1 to 5:1), and sil-
ica gel (petroleum ether/ethyl acetate, 20:1 to 10:1) to yield 7
(8 mg), 8 (13 mg), and 9 (15 mg), respectively. Compounds 1
(17 mg), 5 (22 mg), and 17 (11 mg) were isolated from D3 by
Sephadex LH-20 (CHCl3/MeOH, 3:2) and preparative TLC (CH2Cl2/
acetone, 4:1, for two times).
compound 2 was the most potential one (IC50 ¼ 4.40 0.69
mM)
representing a promising anti-inflammatory lead. Our present
findings provide the scientific basis to further support the folk
treatments of Horsfieldia species.
4. Experimental
4.1. General experimental procedures
Optical rotations were measured with an AUTOPOL VI polar-
imeter (Rudolph, USA). UV data were obtained on a Shimadzu UV-
2401A spectrophotometer (Shimadzu, Kyoto, Japan). An IR Affinity-
1S spectrophotometer was used for scanning IR spectroscopy with
KBr pellets (Shimadzu, Kyoto, Japan). ECD spectra were taken with a
Chirascan instrument (Applied photophysics, England). 1D and 2D
NMR spectra were recorded on ADVANCE III 400 MHz, AM-
500 MHz, and ADVANCE III 600 MHz spectrometers (Bruker,
German). Unless otherwise specified, chemical shifts (d) were
expressed in ppm with reference to solvent signals (CDCl3:
dC ¼ 77.16 ppm; CD3OD: dC ¼ 49.00 ppm; residual CHCl3 in CDCl3:
dH ¼ 7.26 ppm; residual CH3OH in CD3OD: dH ¼ 3.31 ppm). HREIMS
(70 eV) were measured on a VG Auto Spec-3000 spectrometer (VG
PRIMA, England). Column chromatography was performed with
silica gel (100e200 mesh) (Qingdao Marine Chemical, Inc., Qing-
dao, People’s Republic of China), MCI gel CHP 20P (75e150 mm;
Mitsubishi Chemical Corporation, Tokyo), Sephadex LH-20 (Amer-
sham Biosciences AB, Uppsala, Sweden), Lichroprep RP-18
(40e63 mm, Merck, Darmstadt, Germany). Fractions were exam-
ined by TLC (Si gel GF254) (Qingdao Marine Chemical, Inc., Qingdao,
Compound 1 was separated using a chiral stationary phase
[Agilent 1260 HPLC with Ultimate Cellu-Amy-DR (10 ꢂ 250 mm)
column (MeOH/H2O 96:4, v/v, 2 mL/min)] respectively to provide
(þ)-1 (6.8 mg, tR ¼ 7.5 min), and (ꢀ)-1 (6.6 mg, tR ¼ 8.0 min).
Similarly, compound 2 was resolved [Agilent 1260 HPLC with Ul-
timate Cellu-Amy-DR (10 ꢂ 250 mm) column (MeOH/H2O 95:5, v/v,
2 mL/min)] to provide (þ)-2 (10.4 mg, tR ¼ 9.6 min) and (ꢀ)-2
(10.5 mg, tR ¼ 10.6 min), respectively.
4.3.1. Horsfielenide C (1)
Fig. 4. Effects of compound 2 on LPS-induced iNOS expression in RAW264.7 cells. Cells
Brown oil; UV (MeOH) lmax (log ε) 202 (3.91), 219 (3.66), 283
(3.22) nm; IR (KBr) nmax 3441, 2972, 1632, 1512, 1237, 836 cmꢀ1; 1H
and 13C NMR: see Table 1; HREIMS [M]þ m/z 242.0949 (calcd for
were pretreated with 2 (5, 10, and 20 mM) for 1 h, then co-incubated with LPS (1 mg/
mL) for another 12 h. Values represent the mean SD of three independent experi-
ments. (# vs Control, * vs LPS, ***/###P < 0.001).

6
R. Zhan et al. / Tetrahedron 76 (2020) 131494
25
C15H14O3, 242.0943). (þ)-Horsfielenide C: [
a
]
þ70.9 (c 0.15,
mixture was passed a short pad of Celite® to remove Pd/C, the filter
D
MeOH). (ꢀ)-Horsfielenide C: [
a
]
2D5-72.2 (c 0.15, MeOH).
cake was washed by ethyl acetate (10 mL ꢂ 3), the filtrates were
then combined and evaporated under vacuum to give slightly yel-
low oil, which was purified by column chromatography (silica gel,
petroleum ether/ethyl acetate ¼ 20:1) to yield 3 (1.3 g, 80%) as a
colorless oil. IR (KBr) nmax 2929, 1612, 1513, 1489, 1244, 1039, 939,
4.3.2. Horsfielenide D (2)
Brown oil; UV (MeOH) lmax (log ε) 203 (3.81), 221 (3.43), 293
(2.99) nm; IR (KBr) nmax 3429, 2922, 1604, 1510, 1462, 1026,
842 cmꢀ1; 1H and 13C NMR: see Table 1; HREIMS [M]þ m/z 256.1100
810 cmꢀ1. 1H NMR (500 MHz, CDCl3)
d
7.10 (d, J ¼ 8.6 Hz, 2H, H-200,
(calcd for C16H16O3, 256.1099). (þ)-Horsfielenide D: [
a
]
25þ18.6 (c
H-600), 6.84 (d, J ¼ 8.6 Hz, 2H, H-300, H-500), 6.73 (d, J ¼ 7.9 Hz, 1H, H-
50), 6.68 (d, J ¼ 1.5 Hz, 1H, H-20), 6.63 (dd, J ¼ 7.9, 1.6 Hz, 1H, H-60),
5.92 (s, 2H, H-70), 3.80 (s, 3H, H-700), 2.57 (dd, J ¼ 15.7, 9.1 Hz, 4H, H-
D
0.20, MeOH). (ꢀ)-Horsfielenide D: [
a
]2D5e20.1 (c 0.20, MeOH).
4.3.3. Horsfielenide E (3)
1, H-3), 1.89 (m, 2H, H-2); 13C NMR (125 MHz, CDCl3) 157.9 (C-400),
d
Colorless oil; UV (MeOH) lmax (log ε) 209 (2.01), 230 (2.21), 285
(1.78) nm; IR (KBr) nmax 2935, 1612, 1513, 1489, 1244, 1039, 937,
810 cmꢀ1; 1H and 13C NMR: see Table 1; HREIMS [M]þ m/z 270.1253
(calcd for C17H18O3, 270.1256).
147.7 (C-40), 145.6 (C-30), 136.4 (C-10), 134.5 (C-30), 129.4 (C-200, C-
600), 121.3 (C-60), 113.9 (C-300, C-500), 109.0 (C-20), 108.2 (C-50), 100.8
(C-70), 55.4 (C-700), 35.2 (C-1), 34.5 (C-3), 33.6 (C-2). HREIMS m/z:
[M]þ calcd. for C17H18O3 270.1256, found 270.1253.
4.4. Total synthesis of 1 and 2
4.4.3. Synthesis of diarylpropene 21
To a suspension of 20 (2.95 g, 6.4 mmol) in THF (20 mL) was
added dropwise of LiHMDS (1 M in THF, 6.4 mL, 6.4 mmol) at 0 ꢁC,
the resulting mixture was stirred at 0 ꢁC for 15 min to give a clear
orange solution before the reaction was moved to ꢀ78 ꢁC cold bath.
To this mixture, aldehyde 19 (1.0 g, 6.1 mmol) in THF (10 mL) was
added, and the resulting mixture was warmed to room temperature
and stirred for 2 h. After consumption of the starting materials, the
reaction was quenched by adding of water (10 mL) at 0 ꢁC. The
resulting mixture was portioned by ethyl acetate (50 mL) and water
(50 mL) was added, the organic layer was washed by water
(50 mL ꢂ 3) and brine (50 mL ꢂ 2), and the aqueous layer was
extracted with ethyl acetate (50 mL). The combined organic layer
was dried over Na2SO4, and evaporated under vacuum to give an
orange oil which was purified by column chromatography (silica
gel, petroleum ether/ethyl acetate ¼ 20:1) to yield 21 (1.4 g, 88%) as
a slightly yellow oil. IR (KBr) nmax 2895, 1606, 1508, 1487, 1246, 1176,
1037, 929, 804 cmꢀ1. 1H NMR (400 MHz, CDCl3) E-isomer displayed
4.4.1. Synthesis of aldehyde 19
To a suspension of (methoxymethyl)triphenylphosphonium
chloride (7.2 g, 21.0 mmol) in THF (40 mL) was added dropwise of
LiHMDS (1 M in THF, 21.0 mL, 21.0 mmol) at 0 ꢁC, the resulting
mixture was stirred at 0 ꢁC for 30 min to give a clear red solution
before the reaction was moved to ꢀ78 ꢁC cold bath. To this mixture,
aldehyde 18 (3.0 g, 20.0 mmol) in THF (15 mL) was added, and the
resulting mixture was warmed to room temperature and stirred for
2 h. After consumption of the starting materials, the reaction was
quenched by adding of 2 N HCl (60 mL) at 0 ꢁC. The resulting
mixture was heated to reflux for 4 h, then was cooled at 0 ꢁC, and
NaHCO3 (~10 g) was added portionwise to the mixture. Ethyl ace-
tate (100 mL) and water (100 mL) was added, the organic layer was
washed by water (100 mL ꢂ 3) and brine (100 mL ꢂ 2), and the
aqueous layer was extracted with ethyl acetate (100 mL). The
combined organic layer was dried over Na2SO4, and evaporated
under vacuum to give crude product which was purified by column
chromatography (silica gel, petroleum ether/ethyl acetate ¼ 10:1)
to yield aldehyde 19 (2.95 g, 90%) as a slightly yellow oil. IR (KBr)
d
7.31 (d, J ¼ 8.7 Hz, 2H, H-200, H-600), 6.85 (d, J ¼ 8.7 Hz, 2H, H-300, H-
500), 6.77 (d, J ¼ 7.9 Hz,1H, H-50), 6.75 (d, J ¼ 1.2 Hz,1H, H-20), 6.71 (d,
J ¼ 7.8 Hz, 1H, H-60), 6.40 (d, J ¼ 15.7 Hz, 1H, H-3), 6.24e6.14 (m, 1H,
H-2), 5.93 (s, 2H, H-70), 3.81 (s, 3H, H-700), 3.45 (d, J ¼ 6.8 Hz, 2H, H-
nmax 2897, 2827, 2725, 1722, 1489, 1246, 1039, 925, 810 cmꢀ1 1H
.
NMR (500 MHz, CDCl3)
d
9.70 (d, J ¼ 1.0 Hz, 1H, H-2), 6.80 (d,
1); 13C NMR (100 MHz, CDCl3) E-isomer displayed 159.0 (C-400),
d
J ¼ 7.9 Hz, 1H, H-50), 6.69 (s, 1H, H-20), 6.66 (d, J ¼ 7.9 Hz, 1H, H-60),
147.8 (C-40), 146.0 (C-30), 134.4 (C-10), 130.5 (C-30), 130.4 (C-3), 127.3
(C-200, C-600), 127.3 (C-2), 121.5 (C-60), 114.0 (C-300, C-500), 109.3 (C-20),
108.3 (C-50), 100.9 (C-70), 55.4 (C-700), 39.1 (C-1). HREIMS m/z: [M]þ
calcd. for C17H16O3 268.1099, found 268.1100.
5.96 (s, 2H, H-70), 3.59 (d, J ¼ 1.0 Hz, 2H, H-1); 13C NMR (125 MHz,
CDCl3) d
199.4 (C-2), 148.4 (C-40), 147.2 (C-30), 125.4 (C-10), 122.9 (C-
60), 110.0 (C-20), 108.9 (C-50), 101.3 (C-70), 50.3 (C-1). HREIMS m/z:
[M]þ calcd. for C9H8O3 164.0473, found 164.0473.
4.4.4. Synthesis of cyclic diarylpropane 22
4.4.2. Synthesis of horsfielenide E (3)
To an oven-dried tube was charged with diarylpropene 21
(27.0 mg, 0.1 mmol) and dry DCM (4.0 mL) was added Cu(OTf)2
(4.0 mg, 0.01 mmol). The reaction tube was filled with oxygen and
sealed. The sealed reaction mixture was stirred at 60 ꢁC for 8 h.
Then the mixture was cooled to room temperature and was added
saturated brine (5.0 mL) followed by dilution with DCM (10 mL).
The organic layer was collected and washed with water (10 mL ꢂ 2)
and dried over anhydrous Na2SO4. After removal of the solvent, the
residue was purified by flash column chromatography (silica gel,
petroleum ether/ethyl acetate ¼ 80:1) to yield 22 (14.7 mg, 55%) as
a white powder which was further crystalized to give colorless
needles. mp 143e145 ꢁC [petroleum ether/ethyl acetate (3:2, v/v)].
IR (KBr) nmax 2927, 1632, 1512, 1474, 1248, 1037, 938, 831 cmꢀ1. 1H
To a suspension of 20 (2.95 g, 6.4 mmol) in THF (20 mL) was
added dropwise of LiHMDS (1 M in THF, 6.4 mL, 6.4 mmol) at 0 ꢁC,
the resulting mixture was stirred at 0 ꢁC for 15 min to give a clear
orange solution before the reaction was moved to ꢀ78 ꢁC cold bath.
To this mixture, aldehyde 19 (1.0 g, 6.1 mmol) in THF (10 mL) was
added, and the resulting mixture was warmed to room temperature
and stirred for 2 h. After consumption of the starting materials, the
reaction was quenched by adding of water (10 mL) at 0 ꢁC. The
resulting mixture was portioned by ethyl acetate (50 mL) and water
(50 mL) was added, the organic layer was washed by water
(50 mL ꢂ 3) and brine (50 mL ꢂ 2), and the aqueous layer was
extracted with ethyl acetate (50 mL). The combined organic layer
was dried over Na2SO4, and evaporated under vacuum to give an
orange oil which was passed through a short pad of silica gel
eluting with petroleum ether/ethyl acetate ¼ 20:1 (200 mL) to yield
crude diarylpropene 21 (~1.5 g). This crude product was dissolved
in ethyl acetate/MeOH (5:1, 60 mL), Pd/C (150 mg, 5% Pd) was
added. The resulting dark suspension was connected to a H2
balloon, and stirred at room temperature for 6 h. Then the reaction
NMR (600 MHz, CDCl3)
d
7.09 (d, J ¼ 8.6 Hz, 2H, H-200, H-600), 6.85 (d,
J ¼ 8.6 Hz, 2H, H-300, H-500), 6.74 (s, 1H, H-20), 6.40 (s, 1H, H-50), 5.90
(dd, J ¼ 11.1, 1.3 Hz, 2H, H-70), 4.18 (t, J ¼ 8.1 Hz, 1H, H-3), 3.80 (s, 3H,
H-700), 2.92 (ddd, J ¼ 15.3, 8.5, 3.5 Hz, 1H, H-1a), 2.86e2.78 (m, 1H,
H-1b), 2.59e2.51 (m, 1H, H-2a), 2.00 (ddd, J ¼ 17.2, 12.5, 8.6 Hz, 1H,
H-2b). 13C NMR (150 MHz, CDCl3)
d
158.2 (C-400), 146.7 (C-30), 146.6
(C-40), 140.2 (C-60), 137.8 (C-100), 137.1 (C-10), 129.0 (C-200, C-600), 114.0

R. Zhan et al. / Tetrahedron 76 (2020) 131494
7
(C-300, C-500), 105.6 (C-50), 104.9 (C-20), 101.0 (C-70), 55.4 (C-700), 50.7
(C-3), 37.4 (C-2), 31.8 (C-1). HREIMS m/z: [M]þ calcd. for C17H16O3
268.1099, found 268.1097.
1 h followed by the treatment with LPS (1
m
g/mL). The detailed
procedures were recorded on the manual of Beyotime’s Griess so-
lution kits. The absorbance at 540 nm was measured in a microplate
reader (Thermo Fisher Scientific, USA). The purity of all tested
compounds was proved on HPLC (Table S4).
4.4.5. Synthesis of horsfielenide D (2)
To a solution of 22 (20.0 mg, 0.074 mmol) in DCM (4 mL) was
added dropwise of BBr3 (1 M in DCM, 148
m
L, 0.15 mmol) at ꢀ40 ꢁC,
4.7. Western blot analysis
the resulting mixture was stirred at ꢀ40 ꢁC for 30 min. The reaction
was quenched by adding of water (1 mL) at ꢀ40 ꢁC. DCM (10 mL)
and water (10 mL) was added, the organic layer was washed by
water (10 mL ꢂ 3) and brine (10 mL ꢂ 2), and the aqueous layer was
extracted with DCM (10 mL). The combined organic layer was dried
over Na2SO4, and evaporated under vacuum to give crude product
which was purified by column chromatography (silica gel, petro-
leum ether/ethyl acetate ¼ 3:1) to yield compound 2 (15.7 mg, 83%)
RAW264.7 cells were pretreated with compound 2 (5, 10, and
20 mM) for 1 h, and then were co-stimulated with LPS (1 mg/mL) for
12 h in a 5% CO2 containing incubator at 37 ꢁC. The cells were
collected on ice, washed twice with ice-cold PBS, and suspended in
120 mL of the lysis buffer containing 1 mM PMSF and 1:25 complete
PI. After incubation of lysates on ice for 0.5 h, the mixtures were
subjected to centrifugation (14000 g) at 4 ꢁC for 5 min to give the
cytosolic fractions. The nuclear and cytoplasmic protein samples
were extracted using Nuclear and Cytoplasmic Protein Extraction
kit (Beyotime). The western blot method was according to the
literature [42].
as a brown oil. IR (KBr) nmax 3420, 2927, 1604, 1510, 1026, 839 cmꢀ1
.
1H NMR (600 MHz, CD3OD)
d
7.04 (d, J ¼ 8.5 Hz, 2H, H-200, H-600),
6.82 (d, J ¼ 8.6 Hz, 2H, H-300, H-500), 6.68 (s, 1H, H-20), 6.30 (s, 1H, H-
50), 3.75 (s, 3H, H-700), 2.84 (m, 1H, H-1a), 2.75 (m, 1H, H-1b), 2.45
(m, 1H, H-2a), 1.89 (dq, J ¼ 12.0, 8.6 Hz, 1H, H-2b). 13C NMR
(150 MHz, CD3OD) d
159.5 (C-400), 145.2 (C-30), 144.9 (C-40), 139.5 (C-
Declaration of Competing Interest
60), 139.4 (C-100), 136.3 (C-10), 129.8 (C-200, C-600), 114.7 (C-300, C-500),
112.5 (C-50), 111.9 (C-20), 55.6 (C-700), 51.9 (C-3), 38.4 (C-2), 32.2 (C-
1). HREIMS m/z: [M]þ calcd. for C16H16O3 256.1099, found 256.1100.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.4.6. Synthesis of horsfielenide C (1)
To a solution of 22 (20.0 mg, 0.074 mmol) in DCM (4 mL) was
Acknowledgments
added dropwise of BBr3 (1 M in DCM, 296 m
L, 0.30 mmol) at 0 ꢁC,
the resulting mixture was stirred at 0 ꢁC for 4 h. The reaction was
quenched by adding of water (1 mL) at 0 ꢁC. DCM (10 mL) and water
(10 mL) was added, the organic layer was washed by water
(10 mL ꢂ 3) and brine (10 mL ꢂ 2), and the aqueous layer was
extracted with DCM (10 mL). The combined organic layer was dried
over Na2SO4, and evaporated under vacuum to give crude product
which was purified by column chromatography (silica gel, petro-
leum ether/ethyl acetate ¼ 1:2) to yield compound 1 (11.6 mg, 65%)
as a brown oil. IR (KBr) nmax 3440, 2927, 1632, 1612, 1510, 1237, 1174,
The authors thank Prof. Philip J. Proteau for the helpful sug-
gestions on revision of the manuscript. This research was finan-
cially supported by the National Natural Science Foundation of
China (Nos. 31460086, 31860097, and 81960631), the Science and
Technology Program of Yunnan Province (L.-D. S), the Top Young
Talent of Ten Thousand Talents Program of Yunnan Province (L.-D. S
and R. Z), the Start-up fund of Yunnan University of Chinese Med-
icine (2019YZG03), and the Discipline Funding of School of Chinese
Materia Medica, Yunnan University of Chinese Medicine
(2019ZY021).
839 cmꢀ1. 1H NMR (400 MHz, CD3OD)
d
6.95 (d, J ¼ 8.5 Hz, 2H, H-200,
H-600), 6.69 (d, J ¼ 8.6 Hz, 2H, H-300, H-500), 6.67 (s, 1H, H-20), 6.30 (s,
1H, H-50), 4.08 (t, J ¼ 8.3 Hz, 1H, H-3), 2.83 (ddd, J ¼ 15.0, 8.6, 3.6 Hz,
1H, H-1a), 2.74 (m, 1H, H-1b), 2.44 (dtd, J ¼ 12.0, 7.7, 3.6 Hz, 1H, H-
2a), 1.87 (dq, J ¼ 12.4, 8.6 Hz, 1H, H-2b). 13C NMR (100 MHz, CD3OD)
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2020.131494.
d
156.6 (C-400), 145.1 (C-30), 144.9 (C-40), 139.7 (C-60), 138.3 (C-100),
136.4 (C-10), 129.8 (C-200, C-600), 116.1 (C-300, C-500), 112.5 (C-50), 111.9
(C-20), 51.9 (C-3), 38.4 (C-2), 32.2 (C-1). HREIMS m/z: [M]þ calcd. for
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