Synthesis and biological evaluation of pseudolaric acid B derivatives as potential immunosuppressive agents

Article abstract of DOI:10.1080/10286020.2015.1030400

Pseudolaric acid B (PB) derivatives with immunosuppressive activity were found by our group. In order to find potential immunosuppressive agents with high efficacy and low toxicity, a series of novel PB derivatives were synthesized and evaluated on their immunosuppressive activities. Most of the synthesized compounds were tested in vitro on murine T and B proliferation. In particular, compound 11 exhibited excellent inhibitory activity toward murine T cells (up to 19-fold enhancement compared to that of mycophenolatemofetil) and little cytotoxicity toward normal murine spleen cells. These experimental data demonstrated that some of these PB derivatives have great potential for future immunosuppressive studies.

Full text of DOI:10.1080/10286020.2015.1030400

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Synthesis and biological evaluation
of pseudolaric acid B derivatives as
potential immunosuppressive agents
Shou-Qiang Chenab, Jie Wangab, Chuan Zhaoa, Qiang-Wen Sunab,
Yi-Teng Wangb, Ting Aib, Tan Lib, Ying Gaob, Huo Wangac & Hong
Chenb
a Tianjin Key Laboratory on Technologies Enabling Development of
Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin
Medical University, Tianjin 300070, China
b Tianjin Key Laboratory for Biomarkers of Occupational and
Environmental Hazard, Logistics University of Chinese People's
Armed Police Forces, Tianjin 300162, China
Click for updates
c The Affiliated Hospital of Logistics University of Chinese People's
Armed Police Forces, Tianjin 300162, China
Published online: 21 Apr 2015.
To cite this article: Shou-Qiang Chen, Jie Wang, Chuan Zhao, Qiang-Wen Sun, Yi-Teng Wang,
Ting Ai, Tan Li, Ying Gao, Huo Wang & Hong Chen (2015): Synthesis and biological evaluation of
pseudolaric acid B derivatives as potential immunosuppressive agents, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2015.1030400
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1030400
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Journal of Asian Natural Products Research, 2015
http://dx.doi.org/10.1080/10286020.2015.1030400
Synthesis and biological evaluation of pseudolaric acid B derivatives as
potential immunosuppressive agents
Shou-Qiang Chenab, Jie Wangab, Chuan Zhaoa, Qiang-Wen Sunab, Yi-Teng Wangb,
Ting Aib, Tan Lib, Ying Gaob, Huo Wangac* and Hong Chenb*
aTianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and
Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China; bTianjin Key
Laboratory for Biomarkers of Occupational and Environmental Hazard, Logistics University of
Chinese People’s Armed Police Forces, Tianjin 300162, China; cThe Affiliated Hospital of Logistics
University of Chinese People’s Armed Police Forces, Tianjin 300162, China
(Received 10 December 2014; final version received 10 March 2015)
Pseudolaric acid B (PB) derivatives with immunosuppressive activity were found by
our group. In order to find potential immunosuppressive agents with high efficacy and
low toxicity, a series of novel PB derivatives were synthesized and evaluated on their
immunosuppressive activities. Most of the synthesized compounds were tested in vitro
on murine T and B proliferation. In particular, compound 11 exhibited excellent
inhibitory activity toward murine T cells (up to 19-fold enhancement compared to that
of mycophenolatemofetil) and little cytotoxicity toward normal murine spleen cells.
These experimental data demonstrated that some of these PB derivatives have great
potential for future immunosuppressive studies.
Keywords: pseudolaric acid B derivatives; immunosuppressive activity; cytotoxicity
Ca2þ-sensitive T-cell signal transduction
1. Introduction
Immunosuppressant is an important class
of clinical drugs for an array of medical
processes, including transplant rejection
and treatment of autoimmune diseases such
as systemic lupus erythematosus, rheuma-
toid arthritis, and psoriasis [1]. The major
immunosuppressive drugs in clinical use
today are cyclosporin A (CsA), sirolimus
(rapamycin), tacrolimus (FK506), and
mycophenolatemofetil (MMF) [2]. In
clinical, most compounds influence both
the innate and adaptive immune system,
and interact with pathways of lymphocyte
activation and proliferation [3]. It is well
known that T-lymphocyte plays an import-
ant role in transplant rejection and
autoimmune diseases. CsA and FK506
are calcineur inhibitors, which exert their
immunosuppressive effects through the
pathway, thereby preventing the activation
of specific transcription factors involved
in lymphokine gene expression [4].
Sirolimus is an inhibitor of the mammalian
target of rapamycin while MMF is an anti-
metabolite.
Although immunosuppressive drugs
have been successfully used for organ
transplantation and treatment of auto-
immune diseases in clinic, their side effects
cannot be neglected, such as secondary
infection, inducing cancer, kidney toxicity,
and liver toxicity [5–7]. Furthermore,
chronic rejection is not satisfactorily
controlled with current immunosuppres-
sants. Therefore, there is a clinical need for
new therapeutic agents capable of mod-
ulating immune responses with high
efficacy and low toxicity [7–10].
*Corresponding authors. Email: wanghuo56@126.com; chenhongtian06@163.com
q 2015 Taylor & Francis

2
S.-Q. Chen et al.
It is known that a majority of drugs in
has a variety of biological activities, such as
anti-fertile, antitumor, and anti-angiogenic
properties [13,14]. PB served as the antic-
ancer drug lead and a series of structurally
modified PB derivatives showed significant
and specific inhibition on HMEC-1 cell
migration in vitro anticancer tests [15]. The
7, 8-carbon–carbon double bond, 13, 14,
15, 16-conjugated double bonds, 4-acetoxy
and 19-methyl ester of PB are the essential
groups of PB for its antitumor activity,
which has been confirmed by Chen et al.
[16,17]. The in vitro antifungal activities of
structurally diversified analogues of pseu-
dolaric acids tested against the major
pathogenic fungus have led to the establish-
ment of a very clear structure–activity
relationship of pseudolaric acids deriva-
tives [18–20]. In addition, recent reports
clinical use derive from natural products,
including drugs for the treatment of cancer
and infections as well as immunosuppres-
sants [11]. Some traditional Chinese
medicines have been used for centuries in
China to treat various immune system
disorders, and represent a valuable resource
to find new immunosuppressive agents.
In view of the facts mentioned above, we
turned our attention to the natural product
pseudolaric acid B (PB; shown in Figure 1),
which was isolated from the root and trunk
bark of Pseudolarix kaempferi Gordon
(Pinaceae). The barks of this tree, known as
“Tu-Jin-Pi” in traditional Chinese herbal
medicine, have been used to treat skin
diseases caused by fungal infections as
early as the seventeenth century [12]. PB
O
20
O
8
9
OH
O
O
O
H
N
1
N
N
O
N
H
O
19
O
10
O
7
O
O
4
O
N
N
2
N
O
13
6
15
3
5
N
H
N
18
O
11
O
HO
16
17
14
H
N
N
H
O
12
O
O
pseudolaric acid B
PB
cyclosporin A
CsA
O
O
HO
O
H
O
OH
O
N
O
O
H
O
O
O
O
O
O
O
H
O
OH
O
N
OH
OH
O
O
HO
O
tacrolimus
FK506
sirolimus (rapamycin)
OH
O
O
N
O
O
O
O
mycophenolate mofetil
MMF
Figure 1. Structures of PB, CsA, sirolimus, FK506, and MMF.

Journal of Asian Natural Products Research
3
showed that PB can suppress human T
lymphocytes activation [21], indicating
that it might have immunosuppressive
activity. PB contains an unique backbone
of a polyhydroazulene with a trans-
substitution pattern at the junction sites
[22]. In order to investigate PB derivatives
for possible application as immunosup-
pressants, 12 PB derivatives were syn-
thesized and their immunosuppressive
activities were evaluated with murine T
and B lymphocytesassays, and their cyto-
toxicities were tested using MTT method.
Most of the synthetic compounds exhibited
potent immunosuppressive activities.
methanol by NaBH4 at 08C for 1 h to
generate the alcohols. And then in the
presence of 1-hydroxy-1H-benzotriazole
(HOBt), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI)
and triethylamine (Et3N), the alcohols
from the first step were reacted with PB in
anhydrous CH2Cl2 to get the compounds
5–8. The second derivatization was the
synthesis of PB amides compounds. In the
presence of HOBt, EDCI, and Et3N, a
series of different aromatic amines were
reacted with PB in anhydrous CH2Cl2 to
get the compounds 9–12. The third
derivatization was the synthesis of PB
amides in which the acetyl group at the 4th
position was hydrolyzed. The selective
hydrolysis of the acetyl group was
achieved by stirring the methanol solution
of PB in the presence of CH3ONa. And
then the standard amide coupling con-
ditions were applied to get the compounds
13–16.
2. Results and discussion
In this study, 12 novel PB derivatives were
synthesized to screen for immunosuppres-
sive activity. The route that enabled the
syntheses of the PB derivatives 5–16 is
outlined in Scheme 1. The syntheses of
these derivatives utilized three structural
derivatizations. The first derivatization
was the syntheses of the PB esters
compounds. Four kinds of thiophenealde-
hydes were firstly reduced in anhydrous
The inhibitory activity on ConA-
induced murine T and LPS-induced murine
B cell proliferation of all the synthesized
compounds was tested with MMF as the
control. To exclude the possibility that the
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
c
b
b
HN
OH
O
HO
HN
HO
O
OH
R3
R
3-NH2
H
R
2-NH2
H
H
H
R2
O
13-16
O
4
9-12
3
a
b
R1CH2-OH
2
R1-CHO
1
O
O
O
O
O
O
R1
O
H
5-8
O
R1 R2
9
R3
15
5
13
16
7
8
14
6
10
12
11
N
S
O
O
O
O
O
S
O
S
S
O
O
Scheme 1. The synthesis route of target compounds. Conditions and reagents: (a) NaBH4,
anhydrous CH3OH, 08C, 84–93% yield; (b) HOBt, EDCI, Et3N, anhydrous CH2Cl2, rt, 8 h, 85–93%
yield; (c) CH3ONa, anhydrous CH3OH, rt, 93–97% yield.

4
S.-Q. Chen et al.
observed inhibitory activity originates
from the toxic effects, we evaluated the
cytotoxicity of these compounds on murine
spleen cells with MTT assay with MMF as
the control in vitro. The pharmacological
results of these compounds are summar-
ized in Table 1. The data showed that most
of these compounds were low toxic to
normal cells. As can be seen from the
immunosuppressive activity of all the
synthesized compounds (Table 1), the
activities of PB amides are better than PB
esters against T and B cells, except for the
PB amides in which the 4-acetyl group has
been hydrolyzed.
compounds 10 (IC50 13.49 mmol l21),
adding one more carbon between the
nitrogen and the benzene ring compared
to 9 significantly improved the activity
against T cells. Notably, compound 11
displayed the most outstanding immuno-
suppressive effects in vitro with IC50
values of 0.35 and 11.12 mmol l21 against
T and B cells, respectively. Its activity
against T cells is 19-fold more potent than
MMF. This result indicated that the
introduction of electron-donating group
(ZOCH3) on the phenyl ring are beneficial
for the activity. Compound 12, installed
with two electron-donating groups on the
R3-position and R4-position of phenyl ring
showed decreased activity (IC50
3.25 mmol l21) compared to compound
11. This result indicated that the position
of substituent on benzene ring significantly
influenced the activities. Derivatives 13–
16, without the acetyl group generally
exhibit poor activities against both T and
B cells.
Compounds 5 and 6 containing the
thiophene motif showed inferior activities
while compounds 7 and 8 showed
moderate activities with IC50 values of
10.12 and 15.25 mmol l21 respectively,
compared to PB with an IC50 value of
10.12 mmol l21 against T cell. Although
the amide derivative 9 showed very low
activity (IC50 41.73 mmol l21) against T
cells, amides 10–12 exhibit higher inhibi-
tory activities against T cells than PB. For
For the assays with B cells, all the
compounds (5–16) except 8 (IC50 16.15
mmol l21) and 11 (IC50 11.12 mmol l21
)
showed poor activities. Therefore, it was
difficult to get a clear structure–activity
relationship on the immunosuppressive
activity of B lymphocytes.
Table 1. In vitro inhibitory effects and
cytotoxicities of novel PB derivatives (5–16).
a
IC50 (1026 mol 121
)
In summary, a series of PB derivatives
have been synthesized and tested for
immunosuppressive activities. We found
that the amide derivatives are the most
potent species, especially for compound
11, which has the potential to be developed
into an immunosuppressive drug. The
further study of the structure–activity
relationship is ongoing in our group.
Compounds T Cell B Cell Cytotoxicity
5
6
7
8
9
40.26
53.34
10.12
15.25
41.73
13.49
0.35
3.25
6.85
.100 .100
.100 .100
.100 .100
.100
.100
.100
16.15
.100
.100
11.12
.100
.100
36.56
.100
.100
.100
13.61
32.07
.100
22.23
.100
.100
35.56
.100
.10
10
11
12
13
14
15
16
PB
MMFb
3. Experimental
10.12
6.71
40.35
11.83
3.1 General experimental procedures
41.00
Melting points (uncorrected) were deter-
mined onaXT4MP apparatus(TaikeCorp.,
Note: IC50: concentration that causes a 50% reduction
of cell growth. Values are the mean of two
independent experiments.
1
Beijing, China). H NMR and 13C NMR
a IC50 . 100 was defined as inactive.
b Mycophenolatemofetil.
spectra were taken on Bruker Advance
2B/400 (Bruker Technologies, Bremen,

Journal of Asian Natural Products Research
5
Germany) spectrometer (400 MHz) in
CDCl3, using TMS an internal standard
(chemical shifts in d). HR-ESI-MS were
recorded using an Agilent-6210LC/TOF-
MS spectrometer (Agilent Technologies,
Waldbronn, Germany). PB was extracted
from the “Tu-Jin-Pi” by our group. All
chemicals used were purchased from J&K
Chemical (Beijing, China). The reaction
was monitored by thin layer chromatog-
raphy on E. Merck (Darmstadt, Germany)
precoated silica GF254 plates.
J ¼ 5.2 Hz, H-40), 7.17–7.21 (2H, m, H-
8, 15), 7.11 (1H, d, J ¼ 3.2 Hz, H-60), 6.99
(1H, dd, J ¼ 5.1, 3.5 Hz, H-50), 6.53 (1H,
dd, J ¼ 15.1, 11.5 Hz, H-14), 5.87 (1H, d,
J ¼ 15.1 Hz, H-13), 5.35 (2H, s, H-10),
3.72 (3H, s, H3-19), 3.29 (1H, d,
J ¼ 5.0 Hz, H-3), 3.07 (1H, dd, J ¼ 14.3,
5.6 Hz, H-5a), 2.89 (1H, dd, J ¼ 15.6,
6.3 Hz, H-6a), 2.75 (1H, dd, J ¼ 15.0,
8.8 Hz, H-9a), 2.58–2.63 (1H, m, H-9b),
2.14–2.18 (1H, m, H-6b), 2.12 (3H, s, H3-
OAC), 1.97 (3H, s, H3-17), 1.69–1.89
(5H, m), 1.58 (3H, s, H3-12); 13C NMR
(100 MHz, CDCl3) d 172.8 (C-18), 169.3
(C-20), 167.9 (ZCOCH3), 167.8 (C-19),
143.8 (C-13), 141.6 (C-8), 138.2 (C-20),
137.2 (C-15), 134.5 (C-7), 128.3 (C-16),
127.9 (C-40), 126.8 (C-50), 126.7 (C-60),
121.7 (C-14), 90.1 (C-4), 83.7 (C-11), 60.8
(C-10), 55.3 (C-10), 51.9 (ZCOCH3), 49.3
(C-3), 33.3 (C-1), 30.7 (C-5), 28.5 (C-12),
27.8 (C-9), 24.3 (C-2), 21.7 (ZCOCH3),
20.2 (C-6), 12.9 (C-17); HR-ESI-MS: m/z
551.1703 [M þ Na]þ (calcd for
C28H32O8SNa, 551.1716).
3.2 Synthesis of pseudolaricesters
compounds 5–8
2-Thiophenecarboxaldehyde (0.224 g,
2.0 mmol) (as shown in Scheme 1) was
dissolved with anhydrous CH3OH (10 ml),
and then the mixture was moved into an ice
bath at 08C. NaBH4 (0.152 g, 4.0 mmol)
was added to the solution slowly and stirred
at 08C for 1 h. After the reaction was
accomplished, some water was added to the
mixture, and 5% HCl was added to adjusted
pH 7. The resulting mixture was extracted
with EtOAc, washed with water and brine,
and the organic phase was evaporated
under vacuum. The crude product 2 was
isolated as a solid (0.179 g, 80% yield). The
corresponding alcohols were added to a
solution of PB (0.432 g, 1 mmol), HOBt
(0.136 g, 1.2 mmol), EDCI (0.229 g,
1.2 mmol) and Et3N (0.1 ml) in anhydrous
CH2Cl2 (10 ml) at 08C. The reaction
mixture was stirred at room temperature
for 10 h, then washed with 5% HCl
(3 £ 15 ml), saturated NaHCO3 (20 ml),
H2O (20 ml) and dried with anhydrous
Na2SO4. The crude was purified by
chromatography on silica gel using pet-
roleum ether/ethyl acetate (2:1) as the
eluent and afforded 5–8 (0.376 g, 87%
yield as a brown oil).
3.2.2 Compound 6
White powder; m.p. 139–1408C; 1H NMR
(400 MHz, CDCl3) d 7.16–7.21 (2H, m,
H-8, 15), 6.91 (1H, d, J ¼ 3.4 Hz, H-50),
6.65 (1H, d, J ¼ 3.4 Hz, H-60), 6.52 (1H,
dd, J ¼ 15.1, 11.5 Hz, H-14), 5.86 (1H, d,
J ¼ 15.1 Hz, H-13), 5.27 (2H, s, H-10),
3.72 (3H, s, H3-19), 3.29 (1H, d,
J ¼ 5.1 Hz, H-3), 3.07 (1H, dd, J ¼ 13.9,
6.0 Hz, H-5a), 2.72–2.92 (4H, m, H-6a,
9a, 70), 2.58–2.63 (1H, m, H-9b), 2.14–
2.18 (1H, m, H-6b), 2.12 (3H, s, H3-OAc),
1.96 (3H, s, H3-17), 1.80 (5H, m), 1.58
(3H, s, H3-12), 1.30 (3H, t, J ¼ 7.5 Hz, H-
80); 13C NMR (100 MHz, CDCl3) d 172.8
(C-20), 169.4 (C-18), 168.0 (ZCOCH3),
167.9 (C-19), 149.3 (C-13), 143.6 (C-20),
141.7 (C-8), 137.1 (C-15), 135.4 (C-40),
134.5 (C-7), 128.5 (C-16), 127.9 (C-50),
122.9 (C-60), 121.8 (C-14), 90.2 (C-4),
83.7 (C-11), 61.2 (C-10), 55.3 (C-10), 51.9
(ZOCH3), 49.4 (C-3), 33.3 (C-1), 30.7 (C-
3.2.1 Compound 5
White powder; m.p. 142–1438C; 1H NMR
(400 MHz, CDCl3) d 7.32 (1H, d,

6
S.-Q. Chen et al.
5), 28.5 (C-12), 27.8 (C-9), 24.3 (C-2),
23.5 (C-70), 21.7 (ZCOCH3), 20.2 (C-6),
15.8 (C-80), 12.9 (C-17); HR-ESI-MS: m/z
579.2016 [M þ Na]þ (calcd for
C30H36O8SNa, 579.2029).
(1H, dd, J ¼ 15.0, 8.8 Hz, H-9a), 2.58–
2.63 (1H, m, H-9b), 2.15–2.18 (1H, m, H-
6b), 2.13 (3H, s, H3-OAC), 1.99 (3H, s,
H3-17), 1.70–1.87 (5H, m), 1.59 (3H, s,
H3-12); 13C NMR (100 MHz, CDCl3) d
172.8 (C-20), 169.4 (C-18), 168.0
(ZCOCH3), 167.9 (C-19), 143.6 (C-13),
141.7 (C-8), 136.9 (C-40), 136.3 (C-15),
134.5 (C-7), 134.2 (C-30), 128.6 (C-60, C-
80), 128.1 (C-50, C-90), 126.6 (C-70), 123.4
(C-16), 121.8 (C-14, C-20), 90.2 (C-4),
83.7 (C-11), 65.4 (C-10), 55.3 (C-10), 52.0
(ZOCH3), 49.3 (C-3), 33.3 (C-1), 30.7 (C-
5), 28.5 (C-12), 27.8 (C-9), 24.3 (C-2),
21.8 (ZCOCH3), 20.2 (C-6), 12.9 (C-17);
HR-ESI-MS: m/z 571.2310 [M þ Na]þ
(calcd for C32H36O8Na, 571.2308).
3.2.3 Compound 7
White powder; m.p. 144–1458C; 1H NMR
(400 MHz, CDCl3) d 7.36 (1H, d,
J ¼ 1.5 Hz, H-60), 7.19–7.22 (2H, m, H-
8, 15), 6.46–6.58 (2H, m, H-14, 30), 6.38
(1H, dd, J ¼ 3.2, 2.0 Hz, H-70), 6.28 (1H,
d, J ¼ 3.2 Hz, H-80), 6.24 (1H, m, H-20),
5.88 (1H, d, J ¼ 15.1 Hz, H-13), 4.79 (2H,
d, J ¼ 7.4 Hz, H-10), 3.72 (3H, s, H3-19),
3.30 (1H, d, J ¼ 5.2 Hz, H-3), 3.08 (1H,
dd, J ¼ 14.2, 5.6 Hz, H-5a), 2.90 (1H, dd,
J ¼ 15.6, 6.3 Hz, H-6a), 2.75 (1H, dd,
J ¼ 15.0, 8.8 Hz, H-9a), 2.58–2.64 (1H,
m, H-9b), 2.18 (1H, overlap, H-6b), 2.12
(3H, s, H3-OAc), 1.98 (3H, s, H3-17),
1.72–1.87 (5H, m), 1.59 (3H, s, H3-12);
13C NMR (100 MHz, CDCl3) d 172.9 (C-
20), 169.4 (C-18), 168.1 (ZCOCH3),
167.9 (C-19), 151.8 (C-40), 143.6 (C-13),
142.4 (C-60), 141.7 (C-8), 136.9 (C-15),
134.5 (C-7), 128.5 (C-16), 122.1 (C-20),
121.9 (C-30), 121.8 (C-14), 111.4 (C-70),
108.9 (C-80), 90.1 (C-4), 83.7 (C-11), 64.9
(C-10), 55.2 (C-10), 52.1 (ZOCH3), 49.3
(C-3), 33.3 (C-1), 30.7 (C-5), 28.5 (C-12),
27.8 (C-9), 24.3 (C-2), 21.8 (ZCOCH3),
20.1 (C-6), 12.9 (C-17); HR-ESI-MS: m/z
561.2061 [M þ Na]þ (calcd for
C30H34O9Na, 561.2101).
3.3 Synthesis of pseudolaricamides
compounds 9–12
To a solution of PB (0.432 g, 1 mmol) in
anhydrous CH2Cl2 (10 ml) were added
corresponding amines (as shown in
Scheme 1), HOBt (0.136 g, 1.2 mmol),
EDCI (0.229 g, 1.2 mmol), and Et3N
(0.1 ml) at 08C. The reaction mixture was
stirred at room temperature for 10 h, then
washed with 5% HCl (3 £ 15 ml), satu-
rated NaHCO3 (20 ml), H2O (20 ml), and
dried with anhydrous Na2SO4. The crude
product was purified by column chroma-
tography on silica gel using petroleum
ether/ethyl acetate (3:2) as the eluent to
afford 9–12 (0.367 g, 85% yield as a
brown oil).
3.2.4 Compound 8
3.3.1 Compound 9
White powder; m.p. 139–1408C; 1H NMR
(400 MHz, CDCl3) d 7.20–7.41 (7H, m,
H-8, 15, 50, 60, 70, 80, 90), 6.68 (1H, d,
J ¼ 15.9 Hz, H-30), 6.55 (1H, dd, J ¼ 15.1,
11.5 Hz, H-14), 6.33 (1H, dt, J ¼ 15.9,
6.4 Hz, H-20), 5.88 (1H, d, J ¼ 15.1 Hz, H-
13), 4.82 (2H, d, J ¼ 6.4 Hz, H-10), 3.72
(3H, s, H3-19), 3.30 (1H, d, J ¼ 5.1 Hz, H-
3), 3.08 (1H, dd, J ¼ 14.1, 6.0 Hz, H-5a),
2.90 (1H, dd, J ¼ 15.5, 6.3 Hz, H-6a), 2.75
White powder; m.p. 142–1438C; 1H NMR
(500MHz, CDCl3) d 7.26–7.35 (5H, m, H-
30,40, 50, 60, 70), 7.18–7.20 (1H, m, H-8),
6.97 (1H, d, J ¼ 11.3Hz, H-15), 6.50 (1H,
dd, J ¼ 15.0, 11.4Hz, H-14), 6.07 (1H, t,
J ¼ 5.3 Hz, N-H), 5.80 (1H, d, J ¼ 15.0Hz,
H-13), 4.52 (2H, d, J ¼ 6.0 Hz, H-10), 3.71
(3H, s, H3-19), 3.28 (1H, d, J ¼ 5.6 Hz, H-
3), 3.07 (1H, dd, J ¼ 14.1, 6.1 Hz, H-5a),
2.88 (1H, dd, J ¼ 15.5, 6.2 Hz, H-6a), 2.73

Journal of Asian Natural Products Research
7
(1H, dd, J ¼ 15.0, 8.8 Hz, H-9a), 2.58–2.61
(1H, m, H-9b), 2.13–2.16 (1H, m, H-6b),
2.12 (3H, s, H3-OAc), 1.98 (3H, s, H3-17),
8, 70), 6.90 (1H, d, J ¼ 11.2 Hz, H-15),
6.74–6.80 (3H, m, H-40, 60, 80), 6.47 (1H,
dd, J ¼ 15.0, 11.0 Hz, H-14), 5.78 (1H, d,
J ¼ 15.1 Hz, H-13), 3.79 (3H, s, H-90), 3.72
(3H, s, H3-19), 3.59 (2H, q, J ¼ 4.8 Hz,
H-10), 3.28 (1H, d, J ¼ 5.5 Hz, H-3), 3.07
(1H, dd, J ¼ 14.0, 6.0 Hz, H-5a), 2.89 (1H,
dd, J ¼ 16.0, 6.5 Hz, H-6a), 2.83 (2H, t,
J ¼ 5.2 Hz, H-20), 2.74(1H, dd, J ¼ 15.0,
9.0 Hz, H-9a), 2.58–2.62 (1H, m, H-9b),
2.14–2.17 (1H, m, H-6b), 2.12 (1H, s, H3-
OAc), 1.90 (3H, s, H3-17), 1.71–1.82 (5H,
m), 1.57 (3H, s, H3-12); 13C NMR
(100 MHz, CDCl3) d 172.9 (C-20, C-18),
169.4 (ZCOCH3), 168.0 (C-19), 159.8 (C-
50), 142.2 (C-13), 141.7 (C-8), 140.5 (C-30),
134.5 (C-15), 132.6 (C-7), 129.7 (C-70, C-
16), 121.4 (C-14), 121.1 (C-80), 114.5 (C-
40), 112.0 (C-60), 90.1 (C-4), 83.7 (C-11),
55.2 (C-90, C-10), 52.0 (ZOCH3), 49.4 (C-
3), 40.9 (C-10), 35.7 (C-20), 33.3 (C-1), 30.7
(C-5), 28.6 (C-12), 27.8 (C-9), 24.3 (C-2),
21.8 (ZCOCH3), 20.1 (C-6), 13.1 (C-17);
1.68–1.84 (5H, m), 1.57 (3H, s, H3-12); 13
C
NMR (100MHz, CDCl3) d 172.9 (C-20),
169.4 (C-18), 168.4 (ZCOCH3), 168.0 (C-
19), 142.3 (C-13), 141.8 (C-8), 138.2 (C-
15), 134.4 (C-7), 132.8 (C-20), 130.7 (C-40,
C-60), 128.7 (C-30, C-70), 127.9 (C-50), 127.6
(C-16), 121.4 (C-14), 90.1 (C-4), 83.7 (C-
11), 55.2 (C-10), 52.0 (ZOCH3), 49.3 (C-3),
43.9 (C-10), 33.2 (C-1), 30.7 (C-5), 28.6 (C-
12), 27.8 (C-9), 24.3 (C-2), 21.8
(ZCOCH3), 20.1 (C-6), 13.2 (C-17); HR-
ESI-MS m/z: 522.2480 [M þ H]þ (calcd for
C30H36NO7, 522.2486).
3.3.2 Compound 10
White powder; m.p. 139–1408C; 1H NMR
(400MHz, CDCl3) d 7.30 (2H, m, H-50, 70),
7.17–7.23 (4H, m, H-8, 40, 60, 80), 6.89 (1H,
d, J ¼ 11.5 Hz, H-15), 6.46 (1H, dd,
J ¼ 15.1, 11.5 Hz, H-14), 5.84 (1H, t,
J ¼ 5.5 Hz, N-H), 5.77 (1H, d,
J ¼ 15.0Hz, H-13), 3.70 (3H, s, H3-19),
3.57 (2H, q, J ¼ 7.0 Hz, H-10), 3.27 (1H, d,
J ¼ 5.5 Hz, H-3), 3.05 (1H, dd, J ¼ 15.0,
6.5 Hz, H-5a), 2.83–2.90 (3H, m, H-6a, 20),
2.72(1H, dd, J ¼ 15.5, 9.5 Hz, H-9a), 2.57–
2.61 (1H, m, H-9b), 2.14 (1H, overlap, H-
6b), 2.11 (3H, s, H3-OAc), 1.87 (3H, s, H3-
17), 1.70–1.81 (5H, m), 1.56 (3H, s, H3-12);
13C NMR (100MHz, CDCl3) d 173.0 (C-
20), 169.4 (C-18), 168.4 (ZCOCH3), 168.0
(C-19), 142.2 (C-13), 141.7 (C-8), 138.9 (C-
15), 134.5 (C-7), 132.6 (C-30), 131.6 (C-50,
70) 130.8 (C-40, 80), 128.7 (C-16), 126.6 (C-
60), 121.4 (C-14), 90.1 (C-4), 83.6 (C-11),
55.2 (C-10), 52.0 (ZOCH3), 49.4 (C-3),
41.0 (C-10), 35.6 (C-20), 33.3 (C-1), 30.7 (C-
5), 28.6 (C-12), 27.8 (C-9), 24.3 (C-2), 21.8
(ZCOCH3), 20.1 (C-6), 13.1 (C-17); HR-
ESI-MS m/z: 536.2652 [M þ H]þ (calcd for
C31H38NO7, 536.2702).
þ
HR-ESI-MS m/z: 566.2751 [M þ H]
(calcd for C32H40NO8, 566.2754).
3.3.4 Compound 12
White powder; m.p. 144–1458C; 1H NMR
(400 MHz, CDCl3) d 7.19–7.21 (1H, m,
H-8), 6.91 (1H, d, J ¼ 11.5 Hz, H-15),
6.82 (1H, d, J ¼ 8.0 Hz, H-70), 6.72–6.75
(2H, m, H-40, 80), 6.48 (1H, dd, J ¼ 15.0,
11.3 Hz, H-14), 5.79 (1H, d, J ¼ 15.0 Hz,
H-13), 3.87 (6H, s, H3-90, H3-100), 3.72
(3H, s, H3-19), 3.57 (2H, m, H-10), 3.29
(1H, d, J ¼ 6.0 Hz, H-3), 3.07 (1H, dd,
J ¼ 14.0, 6.0 Hz, H-5a), 2.89 (1H, dd,
J ¼ 15.5, 6.5 Hz, H-6a), 2.80 (2H, t,
J ¼ 7.0 Hz, H-20), 2.74 (1H, dd, J ¼ 15.0,
8.5 Hz, H-9a), 2.58–2.63 (1H, m, H-9b),
2.14–2.16 (1H, m, H-6b), 2.12 (3H, s, H3-
OAc), 1.90 (3H, s, H3-17), 1.71–1.82 (5H,
m), 1.58 (3H, s, H3-12); 13C NMR
(100 MHz, CDCl3) d 173.0 (C-20), 169.4
(C-18), 168.4 (ZCOCH3), 168.0 (C-19),
149.1 (C-50), 147.7 (C-60), 142.2 (C-13),
141.7 (C-8), 134.5 (C-15), 132.6 (C-7),
131.4 (C-30), 130.7 (C-16), 121.4 (C-14),
3.3.3 Compound 11
White powder; m.p. 145–1468C; 1H NMR
(400 MHz, CDCl3) d 7.19–7.25 (2H, m, H-

8
S.-Q. Chen et al.
120.6 (C-80), 111.9 (C-70), 111.3 (C-40),
90.1 (C-4), 83.7 (C-11), 55.9 (C-90), 55.2
(C-100, C-10), 52.0 (ZOCH3), 49.3 (C-3),
41.1 (C-10), 35.2 (C-20), 33.3 (C-1), 30.7
(C-5), 28.6 (C-12), 27.8 (C-9), 24.3 (C-2),
22.8 (ZCOCH3), 20.1 (C-6), 13.1 (C-17);
HR-ESI-MS: m/z 596.2866 [M þ H]þ
(calcd for C33H42NO9, 596.2860).
3), 35.1 (C-5), 33.4 (C-1), 28.4 (C-12),
27.3 (C-9), 24.4 (C-2), 19.8 (C-6), 13.0
(C-17); HR-ESI-MS: m/z 553.2032
[M þ H]þ (calcd for C29H33N2O7S,
553.2008).
3.4.2 Compound 14
White powder; m.p. 145–1468C; 1H NMR
(400 MHz, CDCl3) d 7.20–7.23 (1H, m,
H-8), 7.17 (1H, d, J ¼ 5.2 Hz, H-50), 6.94–
6.98 (2H, m, H-15, 60), 6.84 (1H, d,
J ¼ 3.2 Hz, H-70), 6.48 (1H, dd, J ¼ 15.0,
11.4 Hz, H-14), 6.10 (1H, t, J ¼ 5.7 Hz, N-
H), 5.81 (1H, d, J ¼ 15.0 Hz, H-13), 3.70
(3H, s, H3-19), 3.60 (2H, q, J ¼ 6.4 Hz, H-
10), 3.08 (1H, t, J ¼ 6.4 Hz, H-20), 2.83
(1H, dd, J ¼ 14.6, 5.7Hz, H-5a), 2.58–
2.65 (3H, m, 6a), 2.24 (1H, d, J ¼ 6.2 Hz,
H-3), 2.08–2.17 (3H, m, 9a, 9b, H-6b),
1.92 (3H, s, H3-17), 1.64–1.89 (4H, m),
1.53 (3H, s, H3-12); 13C NMR (100 MHz,
CDCl3) d 174.5 (C-20), 168.7 (C-18),
168.3 (C-19), 143.3 (C-13), 142.8 (C-8),
141.2 (C-30), 134.3 (C-15), 133.2 (C-7),
130.2 (C-70), 127.1 (C-16), 125.4 (C-60),
124.0 (C-50), 121.1 (C-14), 83.6 (C-11),
80.0 (C-4), 55.1 (C-10), 54.0 (C-3), 51.9
(ZOCH3), 41.1 (C-10), 35.2 (C-5), 33.5 (C-
1), 29.8 (C-20), 28.6 (C-12), 27.3 (C-9),
24.5 (C-2), 19.9 (C-6), 13.0 (C-17); HR-
ESI-MS: m/z 500.2112 [M þ H]þ (calcd
for C27H34NO6S, 500.2107).
3.4 Synthesis of pseudolaricamides
without 4-acetyl 13–16
The CH3ONa (0.065 g, 1.2 mmol) was
added to a solution of PB (0.432 g,
1 mmol) in anhydrous CH3OH (10 ml) at
08C slowly. Ten minutes later, it was
moved to room temperature for 4 h. Then,
the pH value of the mixture was adjusted
to 6 with 5% HCl, extracted with EtOAc
(3 £ 15 ml), and washed with saturated
NaCl. The combined organic layers were
dried with anhydrous Na2SO4 and the
solvents were evaporated in vacuo. The
crude product 4 was isolated as a solid
(0.397 g, 92% yield). Then, compounds
13–16 were synthesised via the method b
(as shown in Scheme 1).
3.4.1 Compound 13
White powder; m.p. 142–1438C; 1H NMR
(400 MHz, CDCl3) d 7.49 (1H, d,
J ¼ 9.2 Hz, H-70), 7.27–7.29 (1H, m, H-
40), 7.21–7.23 (1H, m, H-8), 6.70–6.97
(2H, m, H-15, 60), 6.48 (1H, dd, J ¼ 15.0,
11.1 Hz, H-14), 5.42 (1H, d, J ¼ 15.0 Hz,
H-13), 3.85 (3H, s, H-100), 3.72 (3H, s, H3-
19), 2.86 (1H, dd, J ¼ 15.1, 5.8 Hz, H-5a),
2.62 (3H, m, H-3, 6a, 9a), 2.43 (1H, s, N-
H), 2.13 (3H, s, H3-17), 2.05–2.09 (1H, m,
H-6b), 1.59–2.01 (5H, m), 1.37 (3H, s, H3-
12); 13C NMR (100 MHz, CDCl3) d 174.1
(C-10), 168.2 (C-20), 167.2 (C-18), 157.9
(C-19), 156.9 (C-50), 144.9 (C-13), 142.7
(C-8), 141.7 (C-80), 135.8 (C-15), 134.2
(C-7), 133.0 (C-30), 129.5 (C-16), 120.9
(C-14), 120.8 (C-70), 115.3 (C-60), 104.4
(C-40), 83.3 (C-11), 80.4 (C-4), 55.8 (C-
10), 55.0 (C-100), 53.7 (ZOCH3), 52.0 (C-
3.4.3 Compound 15
White powder; m.p. 140–1418C; 1H NMR
(400 MHz, CDCl3) d 7.44 (1H, brs, N-H),
7.29 (1H, d, J ¼ 2.0 Hz, H-20), 7.22–7.24
(1H, m, H-8), 7.03 (1H, d, J ¼ 11.3 Hz, H-
15), 6.82 (1H, d, J ¼ 8.4 Hz, H-50), 6.75
(1H, d, J ¼ 8.4 Hz, H-60), 6.53 (1H, dd,
J ¼ 15.0, 11.1 Hz, H-14), 5.96 (2H, s, 70),
5.87 (1H, d, J ¼ 15.0 Hz, H-13), 3.72 (3H,
s, H3-19), 2.88 (1H, dd, J ¼ 15.0, 5.9 Hz,
H-5a), 2.50–2.70 (3H, m, H-3, 6a, 9a),
2.20 (1H, m, H-9b), 2.09–2.16 (1H, m, H-
6b), 2.07 (3H, s, H3-17), 1.68–2.00 (5H,
m), 1.55 (3H, s, H3-12); 13C NMR

Journal of Asian Natural Products Research
9
(100 MHz, CDCl3) d 174.2 (C-20), 168.2
(C-18), 166.7 (C-19), 147.9 (C-30), 144.5
(C-40), 143.4 (C-13), 142.7 (C-8), 134.2
(C-15), 133.4 (C-7), 132.1 (C-10), 131.0
(C-16), 121.2 (C-60), 113.5 (C-14), 108.1
(C-50), 103.2 (C-20), 101.3 (C-70), 83.5 (C-
11), 80.4 (C-4), 55.1 (C-10), 54.1 (C-3),
51.9 (ZOCH3), 35.2 (C-5), 33.4 (C-1),
28.6 (C-12), 27.3 (C-9), 24.5 (C-2), 19.8
(C-6), 13.3 (C-17); HR-ESI-MS: m/z
plemented with 2 mmol l21 L-glutamine,
10% (v/v) heat-inactivated fetal bovine
serum, and 100 U ml21 penicillin–strepto-
mycin. T cells were purified from the
auricular and mandibular lymphnodes
using immunomagnetic negative selection
as described by Wei et al. [23]. The purity
of T cells was consistently more than 95%
as assessed by flow cytometric antibody
(BD Biosciences, New Jersey, USA).
510.2176
C28H32NO8, 510.2128).
[M þ H]þ
(calcd
for
3.5.2 Cytotoxicity assay
The cytotoxicity of PB and its derivatives
on T cells was assessed by MTT assay as
described previously [23]. The optical
density (OD) values were recorded at
570 nm wavelength on a microplate reader
(Model 680, Bio-Rad, California, USA).
Cell viability was calculated according to
the following formula: viability rate (%)
¼ OD treated/OD control £ 100% [21].
3.4.4 Compound 16
White powder; m.p. 141–1428C; 1H NMR
(400 MHz, CDCl3) d 7.21–7.24 (1H, m,
H-8), 7.00 (1H, d, J ¼ 11.3 Hz, H-15),
6.92 (1H, t, J ¼ 4.6 Hz, N-H), 6.50 (1H,
dd, J ¼ 15.0, 11.4 Hz, H-14), 5.80 (1H, d,
J ¼ 15.0 Hz, H-13), 3.72 (3H, s, H3-19),
3.59 (3H, m, H-30, 50), 3.46 (1H, q,
J ¼ 5.2 Hz, H-10), 2.85 (1H, dd, J ¼ 14.9,
5.9 Hz, H-5a), 2.55–2.70 (3H, m, H-3, 6a,
9a), 2.24 (1H, m, H-9b), 2.11 (2H, m, H-
20), 1.95 (3H, s, H3-17), 1.69–1.88 (5H,
m), 1.54 (3H, s, H3-12), 1.17 (6H, s, H-60,
70); 13C NMR (100 MHz, CDCl3) d 174.3
(C-20), 168.3 (C-18,C-19), 142.8 (C-13),
142.7 (C-8), 134.2 (C-15), 132.7 (C-7),
130.5 (C-16), 121.3 (C-14), 83.5 (C-11),
80.3 (C-4), 72.0 (C-50), 68.3 (C-30), 55.0
(C-10), 54.1 (C-3), 51.9 (ZOCH3), 39.8
(C-10), 35.2 (C-5), 33.4 (C-1), 28.9 (C-20),
28.7 (C-12), 27.3 (C-9), 24.5 (C-2), 22.2
(C-60, 70), 19.8 (C-6), 13.0 (C-17); HR-
ESI-MS: m/z 490.2867 [M þ H]þ (calcd
for C27H40NO7, 490.2805).
3.5.3 T and B cell proliferation assay
Splenocytes (5 £ 105/well) were cultured
with 5 mg ml21 of ConA/LPS in triplicate
for 48 h. Cell proliferation was determined
by the WST-1 assay, a colorimetric assay
for the quantification of cell proliferation,
based on the cleavage of the tetrazolium
salt WST-1 by mitochondrial dehydrogen-
ases in viable cells [24]. At the end of
incubation, WST-1 (1:10) was added to
each well and the absorption of samples
was measured at 450 nm. The inhibitory
rate was calculated as follows: inhibitory
rate (%) ¼ [(OD control–OD treated)/OD
control] £100%.
3.5 Immunoactivity
Disclosure statement
3.5.1 Cell preparation
No potential conflict of interest was reported by
the authors.
Mice were killed by cervical dislocation,
and their spleens were removed asepti-
cally. Cell debris and clumps were
removed, and erythrocytes were lysed
with ammonium chloride buffer solution.
Mononuclear cell suspensions were resus-
pended in RPMI 1640 medium sup-
Funding
This work was financially supported by the
Special Financial Grant from China Postdoc-
toral Science Foundation [grant number
201104796]; Tianjin Municipal Science and

10
S.-Q. Chen et al.
Technology Commission [grant number
11JCYBJC14600]; and National Nature
Science Foundation of China (NSFC) for
Young Scientists Fund [grant number
81202843].
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