Synthesis of nigranoic acid and manwuweizic acid derivatives as HDAC inhibitors and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bioorg.2021.104728

As a successful anti-tumor drug target, the family of histone deacetylases (HDACs) is also a critical player in immune response, making the research of anti-inflammatory HDAC inhibitors an attractive new focus. In this report, triterpenoids nigranoic acid (NA) and manwuweizic acid (MA) were identified as HDAC inhibitors through docking-based virtual screening and enzymatic activity assay. A series of derivatives of NA and MA were synthesized and assessed for their biological effects. As a result, hydroxamic acid derivatives of NA and MA showed moderately increased activity for HDAC1/2/4/6 inhibition (the lowest IC₅₀ against HDAC1 is 1.14 μM), with no activity against HDAC8. In J774A.1 macrophage, compound 1–3, 13 and 17–19 demonstrated inhibitory activity against lactate dehydrogenase (LDH) and IL-1β production, without affecting cell viability. Compound 19 increased the histone acetylation level in J774A.1 cells, as well as inhibited IL-1β maturation and caspase-1 cleavage. These results indicated that compound 19 blocks the activation of NLRP3 inflammasome, probably related to HDAC inhibition. This work provided a natural scaffold for developing low-cytotoxic and anti-inflammatory HDAC inhibitors, as well as a class of tool molecules for studying the relationship between HDACs and NLRP3 activation.

Full text of DOI:10.1016/j.bioorg.2021.104728

Bioorganic Chemistry 109 (2021) 104728
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of nigranoic acid and manwuweizic acid derivatives as HDAC
inhibitors and anti-inflammatory agents
,
,
,
,*
Dong-Xuan Nia 1, Qi Wanga 1, Yi-Ming Lia 1, Yi-Man Cuia, Tian-Ze Shena, Xiao-Li Lia
,
Han-Dong Sunb, Xing-Jie Zhanga *, Ruihan Zhanga *, Wei-Lie Xiaoa
,
,
,*
a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan Research & Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091, PR China
b State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
As a successful anti-tumor drug target, the family of histone deacetylases (HDACs) is also a critical player in
immune response, making the research of anti-inflammatory HDAC inhibitors an attractive new focus. In this
report, triterpenoids nigranoic acid (NA) and manwuweizic acid (MA) were identified as HDAC inhibitors
through docking-based virtual screening and enzymatic activity assay. A series of derivatives of NA and MA were
synthesized and assessed for their biological effects. As a result, hydroxamic acid derivatives of NA and MA
Natural products modification
HDAC inhibition
Inflammasome
Triterpenoid
showed moderately increased activity for HDAC1/2/4/6 inhibition (the lowest IC50 against HDAC1 is 1.14 μM),
with no activity against HDAC8. In J774A.1 macrophage, compound 1–3, 13 and 17–19 demonstrated inhibitory
activity against lactate dehydrogenase (LDH) and IL-1β production, without affecting cell viability. Compound
19 increased the histone acetylation level in J774A.1 cells, as well as inhibited IL-1β maturation and caspase-1
cleavage. These results indicated that compound 19 blocks the activation of NLRP3 inflammasome, probably
related to HDAC inhibition. This work provided a natural scaffold for developing low-cytotoxic and anti-
inflammatory HDAC inhibitors, as well as a class of tool molecules for studying the relationship between
HDACs and NLRP3 activation.
1. Introduction
attention of pharmacologists and phytochemists, due to their notable
medicinal functions. The dried mature fruits of Schisandra chinensis and
Histone deacetylases (HDACs) are a family of enzymes for removing
the acetyl group on acetylated lysines. Besides histones, the substrates of
HDAC also include non-histone proteins like tubulin, Hsp90 etc [1].
There are 18 subtypes of HDAC, of which 11 subtypes are Zn2+ depen-
dent (HDAC 1–11). HDAC is a successful drug target for cancer therapy
(T cell lymphoma, multiple myeloma), and also involved in neurological
disorder, infection and inflammation [2]. The potential therapeutic ef-
fect of HDAC inhibitors in immune disorders has become a new focus in
recent years [3]. However, the relationship between HDAC inhibition
and anti-inflammation is complex. Previous studies led to mixed results,
depending on the experimental system, pathway under study and dosage
of compounds [4]. For instance, with the treatment of HDAC inhibitor
SAHA, LPS-induced IL-1β secretion was reduced in human peripheral
blood mononuclear cells, but increased in human dendritic cells [5].
Plants belonging to the family Schisandraceae have attracted the
S. sphenanthera have been used in traditional Chinese medicine to relieve
fatigue, protect the liver, strengthen the heart, reduce blood and treat
insomnia for over 2000 years [6]. The chemical components of Schi-
sandraceae often feature lanostane- and cycloartane-type triterpenoids
[7]. Nigranoic acid and manwuweizic acid, possessing cycloartane and
lanostane skeletons, respectively, are A-ring secocycloartene triterpe-
noid diacids isolated from several Schisandra species [8–12]. These two
representative compounds were known with various biological activ-
ities, including anti-HIV, anticancer, mental and intellectual functions
promotion, antiapoptotic and liver protection [13–21]. In this study, by
combining molecular docking-based virtual screening, enzyme inhibi-
tion assay, chemical modification and cell-based bioassays, we reported
a series of semi-synthesized derivatives of NA and MA as HDAC in-
hibitors and anti-inflammatory agents.
* Corresponding authors.
E-mail addresses: lixiaoli@ynu.edu.cn (X.-L. Li), zhangxj@ynu.edu.cn (X.-J. Zhang), zhangruihan@ynu.edu.cn (R. Zhang), xiaoweilie@ynu.edu.cn (W.-L. Xiao).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104728
Received 19 January 2021; Received in revised form 5 February 2021; Accepted 6 February 2021
Available online 16 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
2. Results
of the separated esters 1a and 2a gave pure samples of the corresponding
di-acids 1 (88% yield) and 2 (86% yield) (Scheme 1), whose structures
confirmed by comparison of their spectroscopic data with those previ-
ously reported [9].
2.1. Discovery of nigranoic acid as HDAC inhibitor
To identify potential HDAC inhibitors from our in-house natural
products library (containing 1068 compounds), virtual screening based
on molecular docking was performed using HDAC2 (PDB ID: 4LXZ) as
receptor [23] (Fig. 1A). Ten compounds were selected (as described in
experiment section) for in vitro inhibitory activity test against HDAC1/
2.3. Synthesis of the derivatives of nigranoic acid and manwuweizic acid
Diamides 3–18 derived from 1 and 2 were obtained by the reaction
of 1 or 2 with substituted amine or O-substituted hydroxylamine in
acetonitrile in the presence of triethylamine (TEA) and 2-(1H-benzo-
triazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU)
(Scheme 2). Most of the NMR data appeared as multiple, overlapping
signals, consistent with the relevant skeletal frameworks of the two se-
ries of derivatives, while expected signals and variations of shifts were
observed that corresponded to the various R groups of the relevant
amines or hydroxylamines (RH) (Figs. S1–16, supplementary material).
Treatment of 1a and 2a with hydroxylamine solution and potassium
hydroxide (KOH) in tetrahydrofuran (THF) and methanol afforded the
hydroxamic acid derivatives 19 and 20, respectively (Scheme 3). This
method was found to have selectivity in the production of 3-(N-
hydroxyamido)-27-carboxylic acid derivatives. For N-hydroxamido acid
formation, regiochemistry derivatives of the ester group at C-3 are
commonly produced. Though with excessive hydroxylamine solution,
the ester group at C-27, which has a “angelic acid methyl ester group”,
remains less reactive towards hydroxylamine than the “saturated car-
boxylic acid methyl ester” at the C-3 position, but susceptible to basic
hydrolysis. The molecular formula of compound 19 was established as
2/4/6/8. Under the compound concentration of 100 μM, NA (1) was
identified as active inhibitor against HDAC1/2/4/6 with the inhibition
rate >50%, but not for HDAC8. The interaction mode of 1 with HDAC2
was predicted by molecular docking (Fig. 1B). Compound 1 occupied the
same binding pocket of suberoylanilide hydroxamic acid (SAHA), which
is the first approved HDAC inhibitor. Tyr308, His146 and Arg275 were
involved in the hydrogen binding with 1. The C-27 carboxyl group of 1,
together with Asp269 and Asp181, were found to form polar interaction
with Zn2+ in the active center of HDAC2.
2.2. Improved separation of nigranoic acid and manwuweizic acid
Crude fractions containing NA (1) and MA (2) were isolated from the
stem of Schisandra sphenanthera, as previously reported [10]. Larger
quantities of the natural products, NA (1) and MA (2), were not able to
be separated nor taken to purity. The initial attempt to use the mixture in
reaction with O-methylhydroxylamine, with the expectation that the
desired derivatives (3 and 4) might be separated by fractional crystal-
lization. It is interesting that in practice the products cocrystallized
thereby making separation of these derivatives also difficult, although
good use was made of X-ray diffraction analysis to allow the molecular
structures of both to be confirmed (supplementary material). In this
case, the crude fractions were used for methylation reaction. Then the
products were purified with the silica gel column chromatography to
remove the unreacted large polar substances (87% yield). The methyl-
ated products 1a and 2a can be easily separated by C18 reversed phase
chromatographic column in preparative high-performance liquid chro-
matography (Prep-HPLC) (1a yield 44%, 2a yield 39%). Saponification
C
30H47NO4, on the basis of the high resolution (HR)-electrospray ion-
isation (ESI)-MS analysis which showed an adduct ion [M+Na]+ at m/z
508.3397 (Calcd 508.3397), indicating only one side N-hydroxamido
acid formation. Comparison of the 13C NMR of compound 19 and NA
showed a considerable upfield shift of the C-3 signal in the former. In
contrast, the signal position of the C-27 in the 13C NMR spectra of
compound 19 closely resembled that in the NA 13C NMR spectrum. This
gave good evidence of specific introduction of the hydroxamic acidifi-
cation at position C-3. The same method proved the N-hydroxamido acid
formation of compound 20 occurred at position C-3 (Figs. S19–20,
Fig. 1. Virtual screening of HDAC inhibitors from in-house natural products library.
2

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
Scheme 1. Improved Separation of Compounds 1 and 2.
compounds acids
R
yields compounds acids
R
yields
53%
49%
82%
71%
54%
57%
67%
56%
71%
3
4
1
2
1
2
1
2
1
2
11
12
13
14
15
16
17
18
1
2
1
2
1
2
1
2
57%
76%
66%
53%
63%
52%
55%
5
6
7
8
9
10
Scheme 2. General Synthesis of Compounds 3–18.
supplementary material).
2.4. HDAC inhibitory activity of the derivatives of nigranoic acid and
manwuweizic acid
Compared to 1 and 2, 19 and 20 possessed moderately increased
3

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
Scheme 3. General Synthesis of Compounds 19–20.
HDAC inhibitory activity, whereas other compounds have not shown
significant activity (IC50 > 50 M) (Table 1). The implementation of
histone fraction from J774A.1 cells was isolated and then performed by
a western blot assay. As shown in Fig. 3C and F, compound 19 increased
the histone acetylation level in a dose-dependent manner, indicating
that compound 19 inhibits HDAC activity in cell level, which was
consistent with the enzyme inhibition assay above. These results sug-
gested that compound 19 blocks NLRP3 inflammasome activation,
probably due to the inhibitory effects against HDAC.
μ
hydroxamic acid group, the most widely used zinc-biding group in
HDAC inhibitors, improved the interaction towards the zinc ion in
HDACs. (Fig. 2). The hydroxamate moiety of 19 formed zinc chelation
together with the histidine and aspartic acid residues in the catalytic
center of HDAC1/2/4/6. The shortest distance between the hydrox-
amate and Zn2+ is 1.9 Å for HDAC1, 1.8 Å for HDAC2, 2.3 Å for HDAC4
and 2.1 Å for HDAC6. The triterpenoid skeleton acted as the hydro-
phobic linker in HDAC inhibitors, fitted into the neck of HDAC pocket.
An extra hydrogen bond was formed by the carboxyl group in 19 with a
phenylalanine in HDAC1 (Phe205), HDAC4 (Phe168), HDAC6
(Phe680), but not in HDAC2. It is common for plant-derived HDAC in-
hibitors to possess a micromolar IC50 against HDACs1. Although weaker
than SAHA in HDAC inhibition, these compounds may still be valuable
as low-cytotoxicity HDAC inhibitors, for developing anti-inflammatory
agents.
3. Conclusion
In this work, we reported a series of semi-synthesized derivatives of
NA and MA, possessing HDAC inhibitory and anti-inflammatory activity.
The most potent compound 19 was identified to inhibit HDAC1/2/4/6,
but not HDAC8. The IC50 of compound 19 against HDACs ranges from
1.139 to 10.558 μM, weaker than the reference compounds SAHA and
TMP269. At the same time, compared to the anti-tumor HDAC in-
hibitors, 19 is low-cytotoxic against J774A.1 (CC50 > 20 M), making it
μ
potential for development of anti-inflammatory agent. Treatment with
compound 19 was found to increase the histone acetylation level in LPS
+ nigericin co-stimulated J774A.1 cells, as well as to reduce the
expression of IL-1β and caspase-1. These results indicated that com-
pound 19 inhibited the activity of HDACs and activation of NLRP3
inflammasome in inflammation cell model. However, further study is
expected to reveal the specific relationship between the inhibition of
HDAC and NLRP3 inflammasome activation.
2.5. Anti-inflammatory activity of the derivatives of nigranoic acid and
manwuweizic acid
Compounds 1–20 were tested for their anti-inflammatory activity in
murine macrophage J774A.1. summarized in Table 2, compounds 1–3,
13, 17–19 exhibited inhibitory activity against the production of LDH
and IL-1β, without reducing the cell viability. However, compound 20,
although possessing similar structure with 19, was cytotoxic towards
J774A.1 cells. The IC50 of LDH and IL-1β production of other compounds
4. Experimental section
were above 20 μM (data not shown). In western blot assays, compound
4.1. Chemistry materials and methods
19 was observed to inhibit IL-1β (Fig. 3A–B) maturation and caspase-1
cleavage (Fig. 3D–E).
Nigranoic acid (NA, 1) and manwuweizic acid (MA, 2) were isolated
as a mixture as previously described [11]. Other chemicals and solvents
used in synthesis were purchased from commercial supplies and were
further purified or dried when necessary. All reactions were monitored
by analytical thin layer chromatography (TLC) on silica gel pates GF 254
(Qingdao Haiyang Chemical, China). Column chromatography was
performed with silica gel (200–300 mesh). Preparative HPLC was car-
ried out on an Agilent 1260 Chromatograph equipped with a diode array
detector with a semi-preparative Agilent ZORBAX Extend-C18 reversed-
To verify their HDAC inhibitory effects in cell level, we analyzed the
acetylated-histone level of macrophages treated by compound 19. Total
Table 1
HDAC inhibitory activity.
Compound
IC50 (μM)
HDAC1
HDAC2
HDAC4
HDAC6
HDAC8
1
3.96
8.63
1.14
2.64
0.020
–
11.53
15.74
10.56
3.56
0.025
–
>50
>50
19.39
>50
–
3.35
6.31
2.23
1.08
0.014
–
>50
>50
>50
>50
0.210
–
phase column (9.4 × 250 mm, 5
μ
m). All solvents used in HPLC were of
2
1
chromatographic grade (Fisher Scientific, NJ, USA). H and 13C NMR
spectra were recorded on a Bruker AM-400 MHz spectrometer using
CDCl3, Pyridine‑d5 or Methanol‑d4 as solvents (TMS as internal stan-
dard), and the chemical shifts were reported in δ (ppm) and the coupling
constants (J) in Hz. HRESIMS data were obtained on an Agilent LC/MSD
19
20
SAHAa
TMP269b
0.289
a
b
SAHA was used as positive control in assays for HDAC1,2,6 and 8.
TMP269 was used as positive control in the assay for HDAC4.
4

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
Fig. 2. Binding modes of Compound 19 (in magenta sticks) in HDAC1 (A, B), HDAC2 (C, D), HDAC4 (E, F) and HDAC6 (G, H). Hydrogen bonds are shown in dash
line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
5

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
CH3CN-H2O (95:5) (5 mL/min) to give separated di-esters 1a and 2a as
oils. Some of them were used to get corresponding di-acids 1 and 2
through saponification reaction, and the rest to synthesize compounds
19 and 20.
Table 2
Anti-inflammatory effect of compounds in J774A.1 cells.
Compound
IC50 mean ± SD (
μ
M,n = 3b)
CC50 mean ± SD (μM,n = 3)
LDH
IL-1β
Dimethyl 3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-dioate (1a). col-
ourless oil. 755 mg, 44% yield; IR νmax (KBr) 2962, 1741, 1721, 1644,
1435, 1375, 1261, 1197, 1095, 1028, 866, 800 cmꢀ 1; 1H NMR (CDCl3,
400 MHz) δ 5.92 (1H, t, J = 7.2 Hz, H-24), 4.80 (1H, s, HA-28), 4.72 (1H,
s, HB-28), 3.73, 3.64 (6H, s, OCH3(A) and OCH3(B)), 1.88, 1.67 (6H, s,
CH3-26, CH3-29), 2.55–1.04 (24H, m, nigranoic acid scaffold), 0.95,
0.92 (6H, s, CH3-18, CH3-30), 0.89 (3H, d, J = 6.4 Hz, CH3-21), 0.72
(1H, d, J = 4.6 Hz, Hβ-19), 0.40 (1H, d, J = 4.4 Hz, Hα-19); 13C NMR
(CDCl3, 100 MHz) δ 174.5 (C, C-3), 168.7 (C, C-27), 149.6 (C, C-4),
144.3 (CH, C-24), 126.6 (C, C-25), 111.6 (CH2, C-28), 52.3 (CH, C-17),
51.6 (OC(A)H3), 51.3 (OC(B)H3), 49.1 (C, C-14), 47.8 (CH, C-8), 46.0 (CH,
C-5), 45.3 (C, C-13), 36.1 (CH, C-20), 36.0 (CH2, C-11), 35.8 (CH2, C-
12), 33.2 (CH2, C-15), 31.6 (CH2, C-2), 30.1 (CH2, C-19), 29.2 (CH2, C-
1), 28.2 (CH2, C-16), 27.9 (CH2, C-6), 27.2 (C, C-10), 27.1 (CH2, C-22),
26.8 (CH2, C-23), 25.1 (CH2, C-7), 21.5 (C, C-9), 20.8 (CH3, C-26), 19.9
(CH3, C-29), 19.4 (CH3, C-30), 18.3 (CH3, C-21), 18.2 (CH3, C-18);
HRMS-ESI: m/z calcd for C32H50NaO4 (M+Na)+ 521.3601, found
521.3599.
1
3.57 ± 0.41
7.48 ± 0.88
6.32 ± 0.91
5.97 ± 0.98
7.42 ± 0.84
15.98 ± 1.33
9.98 ± 1.01
>2.50
4.09 ± 0.51
9.32 ± 1.43
8.96 ± 1.36
3.01 ± 0.41
2.5 ± 0.22
14.88 ± 1.32
5.50 ± 0.51
>2.50
>20
2
>20
3
>20
13
>20
17
>20
18
>20
19
>20
20
2.90 ± 0.60
2.50 ± 0.80
>20
SAHA
>2.50
>2.50
MCC950a
0.31 ± 0.01
0.51 ± 0.02
a
Positive control.
b
The experiment was repeated three times.
TOF mass spectrometer. The IR spectra were recorded using a Thermo
Scientific Nicolet iS10 spectrometer. Melting points were determined on
a WRS-1 digital melting point instrument (Shanghai, China).
4.1.1. Procedures for the preparation of Di-esters 1a and 2a
Dimethyl 3,4-secocycloarta-4(28),8,24-(Z)-diene-3,-27-dioate (2a).
colourless oil. 664 mg, 39% yield; IR νmax (KBr) 2963, 1746, 1714, 1681,
1646, 1571, 1415, 1261, 1093, 1025, 800 cmꢀ 1; 1H NMR (CDCl3, 400
MHz) δ 5.93 (1H, t, J = 7.4 Hz, H-24), 4.89 (1H, s, HA-28), 4.67 (1H, s,
HB-28), 3.73, 3.65 (6H, s, OCH3(A) and OCH3(B)), 1.89, 1.75 (6H, s, CH3-
26, CH3-29), 2.55–1.08 (23H, m, manwuweizic acid scaffold), 0.95 (3H,
s, CH3-18), 0.94–0.89 (6H, overlap, CH3-30, CH3-21), 0.73 (3H, s, CH3-
19); 13C NMR (CDCl3, 100 MHz) δ 174.8 (C, C-3), 168.6 (C, C-27), 147.4
(C, C-4), 144.1 (CH, C-24), 139.1 (C, C-9), 129.4 (C, C-8), 126.4 (C, C-
25), 113.8 (CH2, C-28), 51.5 (OC(A)H3), 51.2 (OC(B)H3), 50.8 (C, C-14),
50.3 (CH, C-17), 46.8 (CH, C-5), 44.4 (C, C-13), 40.3 (C, C-10), 36.4 (CH,
To a solution of crude fractions (1 and 2, 1.60 g, 3.41 mmol) in DMF
(25 mL) was added K2CO3 (1.89 g, 13.6 mmol) and stirred for 1.5 h at
room temperature. To the mixture added CH3I (1.44 g, 10.2 mmol) and
stirred at room temperature was continued for 4 h. After the reaction
was complete, the reaction mixture was diluted with H2O (150 mL) and
extracted with EtOAc (3 × 60 mL). The combined organic layers were
washed with brine (100 mL). The organic phase was dried over anhy-
drous Na2SO4. Filtration and evaporation of the solvent at reduced
pressure gave a crude product, which was column chromatographed on
silica gel (petroleum ether-EtOAc, 5:1) and the combined, less polar
fractions (1.48 g, 87% yield) subjected to preparative HPLC with
Fig. 3. Compound 19 inhibited the activity of histone deacetylas and blocked NLRP3 inflammasome activation. (A) IL-1β expression in LPS + nigericin co-stimulated
J774A.1 cultured supernatant treated with Compound 19. (B) Caspase-1 expression in LPS + nigericin co-stimulated J774A.1 cultured supernatant treated with
Compound 19. (C) Histone acetylation levels in LPS + nigericin co-stimulated J774A.1 cultured supernatant treated with Compound 19. SAHA (100 nM) was used as
a positive control. (D)–(F) Gray analysis results. Data represent the mean values of three experiments (±SD). *P < 0.05,**P < 0.01, ***P < 0.001.
6

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
C-20), 35.9 (CH2, C-1), 32.5 (CH2, C-2), 31.1 (CH2, C-6), 31.0 (CH2, C-7),
29.4 (CH2, C-11), 28.0 (CH2, C-12), 26.7 (CH2, C-15), 25.9 (CH2, C-16),
25.2 (CH3, C-30), 24.0 (CH2, C-22), 23.0 (CH3, C-19), 22.3 (CH3, C-18),
21.7 (CH2, C-23), 20.7 (CH3, C-21), 18.6 (CH3, C-26), 15.9 (CH3, C-29);
HRMS-ESI: m/z calcd for C32H50NaO4 (M+Na)+ 521.3601, found
521.3597.
(CH2, C-7), 28.1 (CH2, C-12), 26.5 (CH2, C-15), 25.9 (CH2, C-16), 25.2
(CH3, C-30), 24.0 (CH2, C-22), 23.0 (CH3, C-19), 22.3 (CH3, C-18), 21.8
(CH2, C-23), 20.5 (CH3, C-21), 18.6 (CH3, C-26), 16.0 (CH3, C-29);
HRMS-ESI: m/z calcd for C32H52N2NaO4 (M+Na)+ 551.3819, found
551.3820.
N,N’-diallyl 3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide (5).
According to the above general procedures, derivative 5 was prepared
via synthesis of NA (1) with allylamine hydrochloride; white solid; 45
mg, 82% yield; mp 132–134 ◦C; IR νmax (KBr) 3439, 3309, 2924, 2878,
1652, 1617, 1531, 1382, 1146, 999 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ
4.1.2. Procedures for the synthesis of NA (1) and MA (2)
2 N aqueous NaOH (6 mL, 12 mmol) was added to a solution of di-
ester 1a (667 mg, 1.34 mmol) in THF-MeOH (1:1, 20 mL). The
mixture was stirred at room temperature for 6 h, acidified to pH 3 with 1
N HCl solution and extracted with EtOAc (3 × 50 mL). The combined
organic layers were washed with brine (80 mL) and dried over anhy-
drous Na2SO4. Evaporation of the solvent under reduced pressure and
column chromatography of the residue (CH2Cl2-MeOH, 15:1) gave NA
(1) (552 mg, 88% yield). Treatment of di-ester 2a (542 mg, 1.09 mmol)
under the same conditions and scale gave MA (2) (442 mg, 86% yield).
–
–
–
5.91–5.72 (2H, overlap, CH(A) CH and CH
CH ), 5.72–5.58 (2H,
2
–
2
(B)
overlap, CONH(A) and CONH(B)), 5.49 (1H, t, J = 7.5 Hz, H-24),
–
–
–
–
5.26–5.03 (4H, overlap, CH CH
and CH CH2(B)), 4.79 (1H, s, HA-
28), 4.71 (1H, s, HB-28), 3.928(–A3) .86 (2H, m, CH2(A) CH CH ),
–
–
–
2
–
3.86–3.75 (2H, m, CH2(B)–CH CH ), 1.88, 1.66 (6H, s, CH -26, CH -
–
29), 2.46–1.02 (24H, m, nigranoic a2cid scaffold), 0.92, 0.89 (6H, s, CH33-
18, CH3-30), 0.84 (3H, d, J = 6.4 Hz, CH3-21), 0.70 (1H, d, J = 4.5 Hz,
Hβ-19), 0.37 (1H, d, J = 4.5 Hz, Hα-19); 13C NMR (CDCl3, 100 MHz) δ
173.0 (C, C-3), 170.0 (C, C-27), 149.8 (C, C-4), 134.3 (CH, C-24), 134.2
3
4.1.3. Procedures for the synthesis of compound 3–18
–
–
–
H
–
To a solution of acid (1 or 2, 47 mg, 0.10 mmol) and TBTU (71 mg,
0.22 mmol) in anhydrous CH3CN (1 mL) was added TEA (40 mg, 0.40
mmol) and the mixture stirred for 30 min at room temperature. To the
mixture added substituted amine or O-substituted hydroxylamine (0.23
mmol) and stirred at room temperature was continued for 2 h. After the
reaction was complete, the reaction mixture was diluted with H2O (15
mL) and extracted with DCM (3 × 10 mL). The combined organic layers
were washed with brine (20 mL). The organic phase was dried over
anhydrous Na2SO4. Filtration and evaporation of the solvent at reduced
pressure gave a crude product, which was purified by column chroma-
tography (petroleum ether-EtOAc, 3:2) to give the pure corresponding
diamide (3–18).
(C(A)
H
CH ), 134.1 (C(B)
CH ), 131.4 (C, C-25), 116.5
2
2
–
–
–
(CH
C
H ), 116.1 (CH C(A)H2), 111.4 (CH2, C-28), 52.0 (CH, C-17),
2
–
49.0 (C(,AC) -14), 47.4 (CH, C-8), 45.9 (CH, C-5), 45.1 (C, C-13), 41.8
–
–
–
–
(C(A)H -CH CH ), 41.7 (C H -CH CH ), 36.2 (CH , C-11), 35.8 (CH,
2
2
2
C-20),235.5 (CH2, C-12), 33(.B8)(CH2, C-15), 32.9 (CH2, 2C-2), 29.9 (CH2, C-
19), 29.7 (CH2, C-1), 28.0 (CH2, C-16), 27.6 (CH2, C-6), 27.1 (C, C-10),
26.9 (CH2, C-22), 26.3 (CH2, C-23), 24.9 (CH2, C-7), 21.3 (C, C-9), 20.9
(CH3, C-26), 19.6 (CH3, C-29), 19.2 (CH3, C-30), 18.1 (CH3, C-21), 17.9
(CH3, C-18); HRMS-ESI: m/z calcd for C36H56N2NaO2 (M+Na)+
571.4234, found 571.4238.
N,N’-diallyl 3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide (6).
According to the above general procedures, derivative 6 was prepared
via synthesis of MA (2) with allylamine hydrochloride; white solid; 39
mg, 71% yield; mp 123–125 ◦C; IR νmax (KBr) 3432, 2925, 1640, 1546,
1401, 1323, 1267 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 5.91–5.74 (2H,
N,N’-dimethoxy 3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide
(3). According to the above general procedures, derivative 3 was pre-
pared via synthesis of NA (1) with O-methylhydroxylamine hydrochlo-
ride; white solid; 28 mg, 53% yield; mp 104–107 ◦C; IR νmax (KBr) 3443,
–
–
–
overlap, CH(A) CH and CH(B) CH ), 5.73–5.63 (2H, overlap,
–
2924, 2854, 1643, 1457, 1383, 1262, 1076, 1053 cmꢀ 1
;
1H NMR
CONH(A) and CONH(B2)), 5.48 (1H, t, J =27.4 Hz, H-24), 5.23–5.05 (4H,
–
–
–
–
(CDCl3, 400 MHz) δ 8.31, 8.25 (2H, s, CONH(A) and CONH(B)), 5.58 (1H,
t, J = 7.2 Hz, H-24), 4.81 (1H, s, HA-28), 4.73 (1H, s, HB-28), 3.82 (3H, s,
OCH3(A)), 3.73 (3H, s, OCH3(B)), 1.88, 1.67 (6H, s, CH3-26, CH3-29),
2.50–1.04 (24H, m, nigranoic acid scaffold), 0.94, 0.91 (6H, s, CH3-18,
CH3-30), 0.87 (3H, d, J = 6.4 Hz, CH3-21), 0.73 (1H, d, J = 4.5 Hz, Hβ-
19), 0.40 (1H, d, J = 4.5 Hz, Hα-19); 13C NMR (CDCl3, 100 MHz) δ 172.8
(C, C-3), 168.1 (C, C-27), 149.8 (C, C-4), 136.1 (CH, C-24), 128.5 (C, C-
25), 111.5 (CH2, C-28), 64.6 (OC(A)H3), 64.5 (OC(B)H3), 52.0 (CH, C-17),
48.9 (C, C-14), 47.4 (CH, C-8), 46.1 (CH, C-5), 45.1 (C, C-13), 36.1 (CH2,
C-11), 35.9 (CH, C-20), 35.5 (CH2, C-12), 33.0 (CH2, C-15), 29.7 (CH2,
C-2), 29.6 (CH2, C-19), 29.3 (CH2, C-1), 28.0 (CH2, C-16), 27.6 (CH2, C-
6), 27.1 (C, C-10), 27.9 (CH2, C-22), 26.5 (CH2, C-23), 24.9 (CH2, C-7),
21.3 (C, C-9), 20.5 (CH3, C-26), 19.7 (CH3, C-29), 19.3 (CH3, C-30), 18.1
(CH3, C-21), 17.9 (CH3, C-18); HRMS-ESI: m/z calcd for C32H52N2NaO4
(M+Na)+ 551.3819, found 551.3817.
overlap, CH CH
and CH CH2(B)), 4.87 (1H, s, HA-28), 4.66 (1H, s,
2(A)
–
–
–
HB-28), 3.98–3.86 (2H, m, CH2(A) CH CH ), 3.86–3.78 (2H, m, CH
2
3
2
–
–
–
CH CH ), 1.87, 1.73 (6H, s, CH -26, CH -29), 2.30–1.01 (23H, m,
(B)
2
3
manwuweizic acid scaffold), 0.93 (3H, s, CH3-18), 0.91–0.84 (6H,
overlap, CH3-30, CH3-21), 0.69 (3H, s, CH3-19); 13C NMR (CDCl3, 100
MHz) δ 173.3 (C, C-3), 170.0 (C, C-27), 147.5 (C, C-4), 138.7 (C, C-9),
–
–
–
–
134.4 (CH, C-24), 134.2 (C(A)
H
CH ), 134.1 (C
H CH ), 131.4 (C, C-
(B) 2
–
2
–
–
–
8), 129.5 (C, C-25), 116.5 (CH
C
(A)
H ), 116.2 (CH C(B)H2), 113.6
(CH2, C-28), 50.7 (C, C-14), 50.2 (CH, C-217), 46.9 (CH, C-5), 44.3 (C, C-
–
–
–
–
13), 41.8 (C(A)H -CH CH ), 41.7 (C H -CH CH ), 40.3 (C, C-10),
2
2
2
2
36.2 (CH2, C-1), 36.2 (CH, C-20), 33.2((BC) H2, C-2), 31.7 (CH2, C-6), 31.0
(CH2, C-7), 30.9 (CH2, C-11), 28.0 (CH2, C-12), 26.4 (CH2, C-15), 25.8
(CH2, C-16), 25.2 (CH3, C-30), 23.9 (CH2, C-22), 22.9 (CH3, C-19), 22.3
(CH3, C-18), 21.7 (CH2, C-23), 20.9 (CH3, C-21), 18.5 (CH3, C-26), 15.9
(CH3, C-29); HRMS-ESI: m/z calcd for C36H57N2O2 (M+H)+ 549.4415,
found 549.4413.
N,N’-dimethoxy
3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide
(4). According to the above general procedures, derivative 4 was pre-
pared via synthesis of MA (2) with O-methylhydroxylamine hydro-
chloride; white solid; 26 mg, 49% yield; mp 124–125 ◦C; IR νmax (KBr)
N,N’-diethoxy
3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide
(7). According to the above general procedures, derivative 7 was pre-
pared via synthesis of NA (1) with ethoxyamine hydrochloride; white
solid; 30 mg, 54% yield; mp 93–98 ◦C; IR νmax (KBr) 3440, 2924, 2854,
1640, 1495, 1458, 1402, 1324, 1263, 1039 cmꢀ 1; 1H NMR (CDCl3, 400
MHz) δ 8.30, 8.23 (2H, s, CONH(A) and CONH(B)), 5.56 (1H, t, J = 7.2 Hz,
H-24), 4.81 (1H, s, HA-28), 4.72 (1H, s, HB-28), 4.17–3.97 (2H, m, J =
6.4 Hz, OCH2(A)CH3), 3.96–3.77 (2H, m, J = 6.3 Hz, OCH2(B)CH3), 1.87,
1.67 (6H, s, CH3-26, CH3-29), 2.49–1.04 (30H, m, nigranoic acid scaf-
fold, OCH2CH3(A), OCH2CH3(B)), 0.93, 0.90 (6H, s, CH3-18, CH3-30),
0.86 (3H, d, J = 6.4 Hz, CH3-21), 0.72 (1H, d, J = 4.5 Hz, Hβ-19), 0.40
(1H, d, J = 4.5 Hz, Hα-19); 13C NMR (CDCl3, 100 MHz) δ 171.4 (C, C-3),
168.3 (C, C-27), 149.8 (C, C-4), 135.8 (CH, C-24), 128.5 (C, C-25), 111.4
(CH2, C-28), 72.3 (OC(A)H2CH3), 72.1 (OC(B)H2CH3), 52.0 (CH, C-17),
3440, 2923, 2853, 1640, 1402, 1323, 1263, 1038 cmꢀ 1
;
1H NMR
(CDCl3, 400 MHz) δ 8.66, 844 (2H, s, CONH(A) and CONH(B)), 5.58 (1H,
t, J = 7.1 Hz, H-24), 4.88 (1H, s, HA-28), 4.68 (1H, s, HB-28), 3.80 (3H, s,
OCH3(A)), 3.73 (3H, s, OCH3(B)), 1.87, 1.75 (6H, s, CH3-26, CH3-29),
2.33–1.01 (23H, m, manwuweizic acid scaffold), 0.94 (3H, s, CH3-18),
0.92–0.79 (6H, overlap, CH3-30, CH3-21), 0.72 (3H, s, CH3-19); 13C
NMR (CDCl3, 100 MHz) δ 171.7 (C, C-3), 168.2 (C, C-27), 147.4 (C, C-4),
139.1 (C, C-9), 136.0 (CH, C-24), 129.6 (C, C-8), 128.4 (C, C-25), 113.8
(CH2, C-28), 64.5 (OC(A)H3), 64.5 (OC(B)H3), 50.8 (C, C-14), 50.2 (CH, C-
17), 47.0 (CH, C-5), 44.4 (C, C-13), 40.5 (C, C-10), 36.3 (CH, C-20), 36.2
(CH2, C-11), 36.2 (CH2, C-1), 31.1 (CH2, C-2), 31.1 (CH2, C-6), 31.0
7

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
48.9 (C, C-14), 47.5 (CH, C-8), 46.1 (CH, C-5), 45.1 (C, C-13), 36.1 (CH2,
C-11), 35.9 (CH, C-20), 35.5 (CH2, C-12), 33.0 (CH2, C-15), 29.7 (CH2,
C-2), 29.7 (CH2, C-19), 29.7 (CH2, C-1), 28.0 (CH2, C-16), 27.7 (CH2, C-
6), 27.2 (C, C-10), 27.0 (CH2, C-22), 26.5 (CH2, C-23), 24.9 (CH2, C-7),
21.3 (C, C-9), 20.6 (CH3, C-26), 19.7 (CH3, C-29), 19.2 (CH3, C-30), 18.1
(CH3, C-21), 17.9 (CH3, C-18), 13.5(OCH2C(A)H3), 13.4(OCH2C(B)H3);
HRMS-ESI: m/z calcd for C34H56N2NaO4 (M+Na)+ 579.4132, found
579.4129.
(C, C-10), 36.3 (CH2, C-1), 36.3 (CH, C-20), 33.3 (CH2, C-2), 31.9 (CH2,
C-6), 31.1 (CH2, C-7), 31.0 (CH2, C-11), 28.0 (CH2, C-12), 26.4 (CH2, C-
15), 25.8 (CH2, C-16), 25.2 (CH3, C-30), 24.0 (CH2, C-22), 23.0 (CH3, C-
19), 23.0 (CH2C(A)H2CH3), 22.9 (CH2C(B)H2CH3), 22.4 (CH3, C-18), 21.8
(CH2, C-23), 20.9 (CH3, C-21), 18.6 (CH3, C-26), 15.9 (CH3, C-29), 11.5
(CH2CH2C(A)H3), 11.3 (CH2CH2C(B)H3); HRMS-ESI: m/z calcd for
C
36H60N2NaO2 (M+Na)+ 575.4547, found 575.4554.
N,N’-diisobutyl
3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide
N,N’-diethoxy 3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide (8).
According to the above general procedures, derivative 8 was prepared
via synthesis of MA (2) with ethoxyamine hydrochloride; white solid;
(11). According to the above general procedures, derivative 11 was
prepared via synthesis of NA (1) with isobutylamine; white solid. 41 mg,
71% yield; mp 119–122 ◦C; IR νmax (KBr) 3425, 2955, 2923, 2867, 1655,
1625, 1548, 1433, 1454, 1385, 1275, 1159, 1072, 1034 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz) δ 5.59 (1H, t, J = 5.2 Hz, CONH(A)), 5.56–5.49 (1H, t,
J = 5.6 Hz, CONH(B)), 5.44 (1H, t, J = 7.3 Hz, H-24), 4.80 (1H, s, HA-28),
4.71 (1H, s, HB-28), 3.20–3.07 [2H, m, J = 4.3, 2.0 Hz, CH2(A)CH
(CH3)2], 3.07–2.94 [2H, m, J = 6.3, 1.4 Hz, CH2(B)CH(CH3)2], 1.87, 1.66
(6H, s, CH3-26, CH3-29), 2.47–1.02 [26H, m, nigranoic acid scaffold,
CH2CH(A)(CH3)2, CH2CH(B)(CH3)2], 0.94–0.85 [18H, overlap, CH3-18,
CH3-30, CH2CH(CH3(A))2, CH2CH(CH3(B))2], 0.84 (3H, d, J = 6.6 Hz,
CH3-21), 0.70 (1H, d, J = 4.0 Hz, Hβ-19), 0.37 (1H, d, J = 4.4 Hz, Hα-19);
13C NMR (CDCl3, 100 MHz) δ 173.2 (C, C-3), 170.3 (C, C-27), 149.9 (C,
C-4), 133.2 (CH, C-24), 132.0 (C, C-25), 111.3 (CH2, C-28), 52.0 (CH, C-
17), 48.9 (C, C-14), 47.4 (CH, C-8), 46.7 [C(A)H2CH(CH3)2], 46.6
[C(B)H2CH(CH3)2], 45.9 (CH, C-5), 45.1 (C, C-13), 36.2 (CH2, C-11),
35.9 (CH, C-20), 35.5 (CH2, C-12), 34.0 (CH2, C-15), 32.9 (CH2, C-2),
30.1 (CH2, C-19), 29.7 (CH2, C-1), 28.5 [CH2C(A)H(CH3)2], 28.4
[CH2C(A)H(CH3)2], 28.0 (CH2, C-16), 27.6 (CH2, C-6), 27.1 (C, C-10),
26.9 (CH2, C-22), 26.4 (CH2, C-23), 24.9 (CH2, C-7), 21.3 (C, C-9), 20.9
(CH3, C-26), 20.1 [2C, CH2CH(C(A)H3)2], 20.0 [2C, CH2CH(C(A)H3)2],
19.6 (CH3, C-29), 19.2 (CH3, C-30), 18.1 (CH3, C-21), 17.9 (CH3, C-18);
HRMS-ESI: m/z calcd for C38H64N2NaO2 (M+Na)+ 603.4860, found
603.4856.
◦
32 mg, 57% yield; mp 115–120 C; IR νmax (KBr) 3445, 2924, 1640,
1401, 1052 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz) δ 8.35, 8.23 (2H, s,
CONH(A) and CONH(B)), 5.55 (1H, t, J = 7.2 Hz, H-24), 4.88 (1H, s, HA-
28), 4.68 (1H, s, HB-28), 4.09–3.79 (4H, overlap, OCH2(A)CH3 and OCH2
(B)CH3), 1.87, 1.75 (6H, s, CH3-26, CH3-29), 1.28 (3H, t, J = 6.8 Hz,
OCH2CH3(A)), 1.24 (3H, t, J = 7.0 Hz, OCH2CH3(B)), 2.33–1.05 (23H, m,
manwuweizic acid scaffold), 0.94 (3H, s, CH3-18), 0.92–0.86 (6H,
overlap, CH3-30, CH3-21), 0.71 (3H, s, CH3-19); 13C NMR (CDCl3, 100
MHz) δ 171.6 (C, C-3), 168.3 (C, C-27), 147.4 (C, C-4), 139.0 (C, C-9),
135.8 (CH, C-24), 129.5 (C, C-8), 128.6 (C, C-25), 113.7 (CH2, C-28),
72.3 (OC(A)H2CH3), 72.1 (OC(B)H2CH3), 50.7 (C, C-14), 50.2 (CH, C-17),
47.0 (CH, C-5), 44.3 (C, C-13), 40.4 (C, C-10), 36.2 (CH, C-20), 36.1
(CH2, C-11), 36.0 (CH2, C-1) 31.1 (CH2, C-2), 31.1 (CH2, C-6), 30.9 (CH2,
C-7), 28.0 (CH2, C-12), 26.5 (CH2, C-15), 25.8 (CH2, C-16), 25.2 (CH3, C-
30), 24.0 (CH2, C-22), 22.9 (CH3, C-19), 22.3 (CH3, C-18), 21.7 (CH2, C-
23), 20.6 (CH3, C-21), 18.5 (CH3, C-26), 15.9 (CH3, C-29); HRMS-ESI:
m/z calcd for C34H56N2NaO4 (M+Na)+ 579.4132, found 579.4132.
N,N’-dipropyl 3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide (9).
According to the above general procedures, derivative 9 was prepared
via synthesis of NA (1) with 1-propanamine; white solid; 37 mg, 67%
yield; mp 114–118 ◦C; IR νmax (KBr) 3442, 2923, 2853, 1652, 1545,
1457, 1382, 1233, 1151 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 5.53–5.30
(3H, overlap, CONH(A), CONH(B), H-24), 4.81 (1H, s, HA-28), 4.73 (1H, s,
HB-28), 3.35–3.21 (2H, m, J = 6.8, 2.6 Hz, CH2(A)CH2CH3), 3.21–3.11
(2H, m, J = 6.6 Hz, CH2(B)CH2CH3), 1.88, 1.68 (6H, s, CH3-26, CH3-29),
2.49–1.04 (28H, m, nigranoic acid scaffold, CH2CH2(A)CH3, CH2CH2
N,N’-diisobutyl
3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide
(12). According to the above general procedures, derivative 12 was
prepared via synthesis of MA (2) with isobutylamine; white solid. 33 mg,
57% yield; mp 103–105 ◦C; IR νmax (KBr) 3442, 2959, 2926, 1639, 1548,
1466, 1372, 1264, 1158, 1031 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz) δ
(B)CH3), 1.03–0.87 (12H, overlap, CH3-18, CH3-30, CH2CH2CH3(A)
,
5.54–5.35 (3H, overlap, CONH(A), CONH(B), H-24), 4.90 (1H, s, HA-28),
4.69 (1H, s, HB-28), 3.22–3.10 [2H, m, J = 4.1, 2.4 Hz, CH2(A)CH
(CH3)2], 3.10–3.01 [2H, m, J = 6.6 Hz, CH2(B)CH(CH3)2], 1.89, 1.76
(6H, s, CH3-26, CH3-29), 2.30–1.07 [25H, m, manwuweizic acid scaf-
fold, CH2CH(A)(CH3)2, CH2CH(B)(CH3)2], 1.01–0.79 [21H, overlap, CH3-
18, CH3-30, CH3-21, CH2CH(CH3(A))2, CH2CH(CH3(B))2], 0.72 (3H, s,
CH3-19); 13C NMR (CDCl3, 100 MHz) δ 173.4 (C, C-3), 170.3 (C, C-27),
147.7 (C, C-4), 138.8 (C, C-9), 133.4 (CH, C-24), 132.0 (C, C-8), 129.6
(C, C-25), 113.6 (CH2, C-28), 50.8 (C, C-14), 50.3 (CH, C-17), 47.0 (CH,
C-5), 46.8 [C(A)H2CH(CH3)2], 46.7 [C(B)H2CH(CH3)2], 44.4 (C, C-13),
40.4 (C, C-10), 36.3 (CH, C-20), 36.3 (CH2, C-21), 33.4 (CH2, C-2), 32.0
(CH2, C-6), 31.1 (CH2, C-7), 31.0 (CH2, C-11), 28.6 [CH2C(A)H(CH3)2],
28.5 [CH2C(A)H(CH3)2], 28.0 (CH2, C-12), 26.5 (CH2, C-15), 25.9 (CH2,
C-16), 25.2 (CH3, C-30), 24.0 (CH2, C-22), 23.0 (CH3, C-19), 22.4 (CH3,
C-18), 21.8 (CH2, C-23), 21.0 (CH3, C-21), 20.2 [2C, CH2CH(C(A)H3)2],
20.1 [2C, CH2CH(C(A)H3)2], 18.6 (CH3, C-26), 15.9 (CH3, C-29);
CH2CH2CH3(B)), 0.86 (3H, d, J = 6.5 Hz, CH3-21), 0.72 (1H, d, J = 4.2
Hz, Hβ-19), 0.37 (1H, d, J = 4.5 Hz, Hα-19); 13C NMR (CDCl3, 100 MHz)
δ 173.2 (C, C-3), 170.3 (C, C-27), 150.0 (C, C-4), 133.3 (CH, C-24), 132.0
(C, C-25), 111.3 (CH2, C-28), 52.0 (CH, C-17), 48.9 (C, C-14), 47.5 (CH,
C-8), 46.0 (CH, C-5), 45.1 (C, C-13), 41.1 (C(A)H2CH2CH3), 41.0
(C(B)H2CH2CH3), 36.3 (CH2, C-11), 35.9 (CH, C-20), 35.5 (CH2, C-12),
34.0 (CH2, C-15), 33.0 (CH2, C-2), 30.0 (CH2, C-19), 29.7 (CH2, C-1),
28.0 (CH2, C-16), 27.7 (CH2, C-6), 27.1 (C, C-10), 27.0 (CH2, C-22), 26.3
(CH2, C-23), 24.9 (CH2, C-7), 22.9 (CH2C(A)H2CH3), 22.8
(CH2C(B)H2CH3), 21.3 (C, C-9), 20.9 (CH3, C-26), 19.7 (CH3, C-29), 19.2
(CH3, C-30), 18.1 (CH3, C-21), 17.9 (CH3, C-18), 11.4 (CH2CH2C(A)H3),
11.3 (CH2CH2C(B)H3); HRMS-ESI: m/z calcd for C36H60N2NaO2
(M+Na)+ 575.4547, found 575.4546.
N,N’-dipropyl
3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide
(10). According to the above general procedures, derivative 10 was
prepared via synthesis of MA (2) with 1-propanamine; white solid. 31
mg, 56% yield; mp 108–111 ◦C; IR νmax (KBr) 3441, 2964, 2925, 1641,
1545, 1460, 1403, 1323, 1262, 1101, 1026, 802 cmꢀ 1; 1H NMR (CDCl3,
400 MHz) δ 5.53–5.32 (3H, overlap, CONH(A), CONH(B), H-24), 4.89
(1H, s, HA-28), 4.68 (1H, s, HB-28), 3.36–3.24 (2H, m, J = 7.2, 2.4 Hz,
CH2(A)CH2CH3), 3.23–3.09 (2H, m, J = 6.8 Hz, CH2(B)CH2CH3), 1.88,
1.76 (6H, s, CH3-26, CH3-29), 2.31–1.14 (27H, overlap, manwuweizic
acid scaffold, CH2CH2(A)CH3, CH2CH2(B)CH3), 0.99–0.86 (15H, overlap,
CH3-18, CH3-30, CH3-21, CH2CH2CH3(A), CH2CH2CH3(B)), 0.72 (3H, s,
CH3-19); 13C NMR (CDCl3, 100 MHz) δ 173.4 (C, C-3), 170.3 (C, C-27),
147.6 (C, C-4), 138.8 (C, C-9), 133.4 (CH, C-24), 132.0 (C, C-8), 129.6
(C, C-25), 113.7 (CH2, C-28), 50.7 (C, C-14), 50.2 (CH, C-17), 47.0 (CH,
C-5), 44.3 (C, C-13), 41.2 (C(A)H2CH2CH3), 41.0 (C(B)H2CH2CH3), 40.4
N,N’-dimethyl
3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide
(13). According to the above general procedures, derivative 13 was
prepared via synthesis of NA (1) with methylamine hydrochloride;
white solid. 38 mg, 76% yield; mp 177–179 ◦C; IR νmax (KBr) 3356,
2925, 2874, 1653, 1539, 1456, 1407, 1278, 1238, 1155 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz) δ 5.61–5.37 (3H, overlap, CONH(A), CONH(B), H-24),
4.80 (1H, s, HA-28), 4.72 (1H, s, HB-28), 2.87 (2H, d, J = 4.8 Hz,
CONHCH3(A)), 2.76 (2H, d, J = 4.5 Hz, CONHCH3(B)), 1.87, 1.67 (6H, s,
CH3-26, CH3-29), 2.47–1.04 (24H, m, nigranoic acid scaffold), 0.94,
0.91 (6H, s, CH3-18, CH3-30), 0.86 (3H, d, J = 6.4 Hz, CH3-21), 0.72
(1H, d, J = 3.3 Hz, Hβ-19), 0.38 (1H, d, J = 3.7 Hz, Hα-19); 13C NMR
(CDCl3, 100 MHz) δ 173.9 (C, C-3), 170.9 (C, C-27), 149.9 (C, C-4),
134.0 (CH, C-24), 131.6 (C, C-25), 111.3 (CH2, C-28), 52.0 (CH, C-17),
8

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
49.0 (C, C-14), 47.5 (CH, C-8), 46.0 (CH, C-5), 45.1 (C, C-13), 36.3 (CH2,
C-11), 35.9 (CH, C-20), 35.5 (CH2, C-12), 33.8 (CH2, C-15), 33.0 (CH2,
C-2), 29.9 (CH2, C-19), 29.7 (CH2, C-1), 28.0 (CH2, C-16), 27.7 (CH2, C-
6), 27.2 (C, C-10), 27.0 (CH2, C-22), 26.4 (CH2, C-23), 26.2 (CON-
HC(A)H3), 26.0 (CONHC(B)H3), 24.9 (CH2, C-7), 21.4 (C, C-9), 20.9 (CH3,
C-26), 19.7 (CH3, C-29), 19.3 (CH3, C-30), 18.1 (CH3, C-21), 17.9 (CH3,
C-18); HRMS-ESI: m/z calcd for C32H53N2O2 (M+H)+ 497.4102, found
497.4107.
31.0 (CH2, C-11), 28.0 (CH2, C-12), 26.3 (CH2, C-15), 25.8 (CH2, C-16),
25.2 (CH3, C-30), 24.0 (CH2, C-22), 23.0 (CH3, C-19), 22.3 (CH3, C-18),
21.8 (CH2, C-23), 20.8 (CH3, C-21), 18.6 (CH3, C-26), 15.9 (CH3, C-29),
14.9 (CH2C(A)H3), 14.9 (CH2C(B)H3); HRMS-ESI: m/z calcd for
C34H57N2O2 (M+H)+ 525.4415, found 525.4417.
N,N’-diallyloxy
3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide
(17). According to the above general procedures, derivative 17 was
prepared via synthesis of NA (1) with O-allylhydroxylamine hydro-
chloride; white solid. 30 mg, 52% yield; mp 110–112 ◦C; IR νmax (KBr)
3442, 2925, 1640, 1453, 1262, 1031, 929 cmꢀ 1; 1H NMR (CDCl3, 400
MHz) δ 8.16 (2H, s, CONH(A) and CONH(B)), 6.06–5.88 (2H, overlap,
N,N’-dimethyl
3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide
(14). According to the above general procedures, derivative 14 was
prepared via synthesis of MA (2) with methylamine hydrochloride;
white solid. 33 mg, 66% yield; mp 164–167 ◦C; IR νmax (KBr) 3423,
3320, 2963, 2926, 2881, 1654, 1633, 1560, 1467, 1408, 1323, 1274,
1160, 1026 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 5.60–5.43 (3H, overlap,
CONH(A), CONH(B), H-24), 4.88 (1H, s, HA-28), 4.67 (1H, s, HB-28), 2.86
(2H, d, J = 4.4 Hz, CONHCH3(A)), 2.78 (2H, d, J = 4.4 Hz, CONHCH3(B)),
1.87, 1.75 (6H, s, CH3-26, CH3-29), 2.26–1.03 (23H, m, manwuweizic
acid scaffold), 0.94 (3H, s, CH3-18), 0.92–0.84 (6H, overlap, CH3-21,
CH3-30), 0.71 (3H, s, CH3-19); 13C NMR (CDCl3, 100 MHz) δ 174.1 (C, C-
3), 170.6 (C, C-27), 147.6 (C, C-4), 138.8 (C, C-9), 134.0 (CH, C-24),
131.6 (C, C-8), 129.6 (C, C-25), 113.7 (CH2, C-28), 50.7 (C, C-14), 50.3
(CH, C-17), 47.0 (CH, C-5), 44.4 (C, C-13), 40.4 (C, C-10), 36.3 (CH2, C-
1), 36.2 (CH, C-20), 33.2 (CH2, C-2), 31.7 (CH2, C-6), 31.1 (CH2, C-7),
31.0 (CH2, C-11), 28.0 (CH2, C-12), 26.4 (CH2, C-15), 26.3 (CON-
HC(A)H3), 26.1 (CONHC(B)H3), 25.8 (CH2, C-16), 25.2 (CH3, C-30), 24.0
(CH2, C-22), 23.0 (CH3, C-19), 22.3 (CH3, C-18), 21.8 (CH2, C-23), 20.9
(CH3, C-21), 18.6 (CH3, C-26), 15.9 (CH3, C-29); HRMS-ESI: m/z calcd
for C32H53N2O2 (M+H)+ 497.4102, found 497.4100.
–
–
–
OCH2CH(A) CH and OCH CH
CH ), 5.57 (1H, t, J = 7.5 Hz, H-24),
2
–
2
2
(B)
–
–
–
5.41–5.26 (4H, overlap, OCH CH CH
and OCH CH CH2(B)), 4.82
–
(1H, s, HA-28), 4.74 (1H,2 s, HB-228()A,) 4.51–4.421 (2H, m, OCH2
–
–
–
–
–
(A)
–
CH CH ), 1.88, 1.68 (6H,
CH CH ), 4.41–4.28 (2H, m, OCH
2
2(B)
2
s, CH3-26, CH3-29), 2.50–1.05 (24H, m, nigranoic acid scaffold), 0.95,
0.92 (6H, s, CH3-18, CH3-30), 0.88 (3H, d, J = 6.0 Hz, CH3-21), 0.73
(1H, d, J = 3.2 Hz, Hβ-19), 0.41 (1H, d, J = 3.2 Hz, Hα-19); 13C NMR
(CDCl3, 100 MHz) δ 171.3 (C, C-3), 168.2 (C, C-27), 149.8 (C, C-4),
–
–
–
136.0 (CH, C-24), 132.2 (OCH2C(A)
H
CH ), 129.9 (OCH C
H CH ),
–
2
2
(B)
128.5 (C, C-25), 120.8 (OCH CH
C
H ), 120.7 (OCH CH C
(B)H22),
–
–
–
–
2
(A) 2 2
–
111.5 (CH2, C-28), 77.5 (overlap, OC(A)H CH CH ), 77.2 (overlap,
–
2
2
–
OC(B)H CH CH ), 52.1 (CH, C-17), 49.0 (C, C-14), 47.5 (CH, C-8), 46.1
–
(CH, C-25), 45.2 (2C, C-13), 36.2 (CH2, C-11), 35.9 (CH, C-20), 35.5 (CH2,
C-12), 33.0 (CH2, C-15), 33.0 (CH2, C-2), 29.8 (CH2, C-19), 29.3 (CH2, C-
1), 28.1 (CH2, C-16), 27.7 (CH2, C-6), 27.2 (C, C-10), 27.0 (CH2, C-22),
26.6 (CH2, C-23), 24.9 (CH2, C-7), 21.4 (C, C-9), 20.6 (CH3, C-26), 19.7
(CH3, C-29), 19.3 (CH3, C-30), 18.2 (CH3, C-21), 18.0 (CH3, C-18);
HRMS-ESI: m/z calcd for C36H56N2NaO4 (M+Na)+ 603.4132, found
603.4129.
N,N’-diethyl 3,4-secocycloarta-4(28),24-(Z)-diene-3,-27-diamide (15).
According to the above general procedures, derivative 15 was prepared
via synthesis of NA (1) with ethylamine hydrochloride; white solid. 28
mg, 53% yield; mp 140–141 ◦C; IR νmax (KBr) 3306, 3073, 2924, 2873,
1650, 1535, 1454, 1374, 1323, 1264, 1239, 1148, 890 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz) δ 5.54–5.42 (2H, overlap, CONH(A), H-24), 5.40–5.32
(1H, m, CONH(B)), 4.81 (1H, s, HA-28), 4.73 (1H, s, HB-28), 3.42–3.30
(2H, m, J = 6.6, 1,3 Hz, CH2(A)CH3), 3.29–3.20 (2H, m, J = 6.9, 1,5 Hz,
CH2(B)CH3), 1.88, 1.68 (6H, s, CH3-26, CH3-29), 2.47–1.22 (24H, m,
nigranoic acid scaffold), 1.18 (3H, t, J = 7.3 Hz, CH2CH3(A)), 1.11 (3H, t,
J = 7.2 Hz, CH2CH3(B)), 0.94, 0.91 (6H, s, CH3-18, CH3-30), 0.87 (3H, d,
J = 6.3 Hz, CH3-21), 0.72 (1H, d, J = 4.2 Hz, Hβ-19), 0.39 (1H, d, J = 4.4
Hz, Hα-19); 13C NMR (CDCl3, 100 MHz) δ 173.1 (C, C-3), 170.2 (C, C-
27), 150.0 (C, C-4), 133.5 (CH, C-24), 131.9 (C, C-25), 111.4 (CH2, C-
28), 52.0 (CH, C-17), 49.0 (C, C-14), 47.5 (CH, C-8), 46.0 (CH, C-5), 45.1
(C, C-13), 36.3 (CH2, C-11), 35.9 (CH, C-20), 35.5 (CH2, C-12), 34.3
(C(A)H2CH3), 34.2 (C(B)H2CH3), 34.0 (CH2, C-15), 33.0 (CH2, C-2), 30.0
(CH2, C-19), 29.7 (CH2, C-1), 28.1 (CH2, C-16), 27.7 (CH2, C-6), 27.2 (C,
C-10), 27.0 (CH2, C-22), 26.3 (CH2, C-23), 24.9 (CH2, C-7), 21.4 (C, C-9),
20.9 (CH3, C-26), 19.7 (CH3, C-29), 19.3 (CH3, C-30), 18.2 (CH3, C-21),
17.9 (CH3, C-18), 15.0 (CH2C(A)H3), 14.9 (CH2C(B)H3); HRMS-ESI: m/z
calcd for C34H57N2O2 (M+H)+ 525.4415, found 525.4412.
N,N’-diallyloxy 3,4-secocycloarta-4(28),8,24-(Z)-diene-3,-27-diamide
(18). According to the above general procedures, derivative 18 was
prepared via synthesis of MA (2) with O-allylhydroxylamine hydro-
chloride; white solid. 32 mg, 55% yield; mp 103–105 ◦C; IR νmax (KBr)
3440, 2923, 1640, 1402, 1324, 1029, 929 cmꢀ 1; 1H NMR (CDCl3, 400
MHz) δ 8.06 (2H, s, CONH(A) and CONH(B)), 6.12–5.81 (2H, overlap,
–
–
–
OCH2CH(A) CH and OCH CH
CH ), 5.57 (1H, t, J = 7.3 Hz, H-24),
–
2
2
(B)
2
–
–
–
5.43–5.22 (4H, overlap, OCH CH CH
and OCH CH CH2(B)), 4.89
–
(1H, s, HA-28), 4.67 (1H,2 s, HB-228()A,) 4.51–4.421 (2H, m, OCH2
–
–
–
–
–
(A)
–
CH CH ), 1.87, 1.75 (6H,
CH CH ), 4.41–4.26 (2H, m, OCH
2(B)
2
s, CH3-26, CH3-29), 2.31–1.04 (23H, m, manwuwei2zic acid scaffold),
0.95, 0.72 (6H, s, CH3-18, CH3-19), 0.93–0.84 (6H, overlap, CH3-21,
CH3-30); 13C NMR (CDCl3, 100 MHz) δ 171.6 (C, C-3), 168.2 (C, C-27),
147.5 (C, C-4), 139.1 (C, C-9), 136.0 (CH, C-24), 132.2 (OCH2
-
–
–
–
C(A)
H
CH ), 129.9 (OCH C
H CH ), 129.5 (C, C-8), 128.5 (C, C-25),
–
2
2
(B) 2
–
–
–
–
120.8 (OCH CH
C
(A)
H ), 120.8 (OCH CH C(B)H2), 113.8 (CH2, C-28),
2
(A) 2 2
–
–
–
–
77.5 (overlap, OC H CH CH ), 77.2 (overlap, OC H CH CH ),
2
2
(B)
2
2
50.8 (C, C-14), 50.3 (CH, C-17), 47.1 (CH, C-5), 44.4 (C, C-13), 40.5 (C,
C-10), 36.3 (CH2, C-1), 36.2 (CH, C-20), 29.8 (CH2, C-2), 29.3 (CH2, C-
6), 31.1 (CH2, C-7), 31.0 (CH2, C-11), 28.1 (CH2, C-12), 26.6 (CH2, C-
15), 25.9 (CH2, C-16), 25.3 (CH3, C-30), 24.0 (CH2, C-22), 23.0 (CH3, C-
19), 22.3 (CH3, C-18), 21.8 (CH2, C-23), 20.6 (CH3, C-21), 18.6 (CH3, C-
26), 16.0 (CH3, C-29); HRMS-ESI: m/z calcd for C36H56N2NaO4
(M+Na)+ 603.4132, found 603.4128.
N,N’-diethyl 3,4-secolanosta-4(28),8,24-(Z)-trien-3,-27-diamide (16).
According to the above general procedures, derivative 16 was prepared
via synthesis of MA (2) with ethylamine hydrochloride; white solid. 33
mg, 63% yield; mp 136–139 ◦C; IR νmax (KBr) 3425, 2963, 2924, 2853,
1642, 1402, 1323, 1262, 1097, 1030, 802 cmꢀ 1; 1H NMR (CDCl3, 400
MHz) δ 5.56–5.37 (3H, overlap, CONH(A), CONH(B), H-24), 4.88 (1H, s,
HA-28), 4.68 (1H, s, HB-28), 3.40–3.29 (2H, m, J = 7.1 Hz, CH2(A)CH3),
3.29–3.20 (2H, m, J = 7.2 Hz, CH2(B)CH3), 1.87, 1.75 (6H, s, CH3-26,
CH3-29), 2.30–1.19 (23H, m, manwuweizic acid scaffold), 1.17 (3H, t, J
= 7.1 Hz, CH2CH3(A)), 1.11 (3H, t, J = 7.2 Hz, CH2CH3(B)), 0.94, 0.71
4.1.4. Procedures for the synthesis of 19 and 20
To a solution of diesters (1a, 88 mg, 0.18 mmol) and hydroxylamine
solution (50 wt. % in Water, 0.46 mL, 7.2 mmol) in a mixed solvent
(THF-methanol, 1:1, 2 mL) was added KOH (81 mg, 1.44 mmol). The
mixture was stirred at room temperature for 3 h, acidified to pH 7 with 1
N HCl solution and extracted with EtOAc (3 × 10 mL). The combined
organic layers were washed with brine (20 mL). The organic phase was
dried over anhydrous Na2SO4. Filtration and evaporation of the solvent
at reduced pressure gave a crude product, which was purified by column
chromatography (DCM-methanol, 15:1) to give the pure compound 19.
Compound 20 was prepared from 2a (88 mg, 0.18 mmol) with the same
(6H, s, CH3-18, CH3-19), 0.92–0.84 (6H, overlap, CH3-21, CH3-30); 13
C
NMR (CDCl3, 100 MHz) δ 173.3 (C, C-3), 170.1 (C, C-27), 147.6 (C, C-4),
138.7 (C, C-9), 133.4 (CH, C-24), 131.9 (C, C-8), 129.7 (C, C-25), 113.6
(CH2, C-28), 50.7 (C, C-14), 50.3 (CH, C-17), 47.0 (CH, C-5), 44.4 (C, C-
13), 40.4 (C, C-10), 36.3 (CH2, C-1), 36.3 (CH, C-20), 34.3 (C(A)H2CH3),
34.1 (C(B)H2CH3), 33.3 (CH2, C-2), 31.8 (CH2, C-6), 31.1 (CH2, C-7),
9

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
procedure as that for 19.
reference compound for HDAC1, HDAC2, HDAC6 and HDAC8. TMP269
was used as the reference compound for HDAC4. For preliminary
screening, the inhibition rates at single compound concentration (100
3,4-secocycloarta-4(28),24-(Z)-diene-3-(hydroxyamino)-27-oic acid
(19). white solid. 33 mg, 38% yield; mp 116–121 ◦C; IR νmax (KBr) 3440,
3217, 2931, 1687, 1633, 1542, 1457, 1375, 1259, 1073, 993, 891 cmꢀ 1
;
μM) were tested in duplicates. For IC50 estimations, six concentrations
1H NMR (Pyridine‑d5, 400 MHz) δ 6.07 (1H, t, J = 6.9 Hz, H-24), 4.93
(1H, s, HA-28), 4.79 (1H, s, HB-28), 2.17, 1.70 (6H, s, CH3-26, CH3-29),
2.97–1.14 (24H, m, nigranoic acid scaffold), 0.99 (3H, d, J = 6.3 Hz,
CH3-21), 0.95, 0.85 (6H, s, CH3-18, CH3-30), 0.67 (1H, d, J = 4.0 Hz, Hβ-
19), 0.37 (1H, d, J = 4.0 Hz, Hα-19); 13C NMR (Pyridine‑d5, 100 MHz) δ
171.2 (C, C-3), 171.2 (C, C-27), 150.6 (C, overlap, C-4), 143.2 (CH, C-
24), 129.2 (C, C-25), 112.4 (CH2, C-28), 52.9 (CH, C-17), 49.6 (C, C-14),
48.2 (CH, C-8), 46.4 (CH, C-5), 45.8 (C, C-13), 36.9 (CH2, C-11), 36.9
(CH, C-20), 36.3 (CH2, C-12), 33.7 (CH2, C-15), 31.7 (CH2, C-2), 31.1
(CH2, C-19), 30.5 (CH2, C-1), 28.8 (CH2, C-16), 28.4 (CH2, C-6), 28.3 (C,
C-10), 27.7 (CH2, C-22), 27.5 (CH2, C-23), 25.7 (CH2, C-7), 22.0 (C, C-9),
22.0 (CH3, C-26), 20.5 (CH3, C-29), 19.9 (CH3, C-30), 18.9 (CH3, C-21),
18.7 (CH3, C-18); HRMS-ESI: m/z calcd for C30H47NNaO4 (M+Na)+
508.3397, found 508.3397.
were measured for each compound, with the starting point of 50 μM and
gradient five-fold dilution. The substrate solution was made by adding
trypsin and Ac-peptide substrate in Tris buffer. To perform the reaction,
firstly 15 μL of enzyme solution was transferred to assay plate with
compound solution, and incubated at room temperature for 15 min.
Then 10 L of substrate solution was added to each well and incubated at
μ
room temperature for 60 min. The plate was read by Synergy MX with
excitation at 355 nm and emission at 460 nm. The inhibition values were
obtained by using Equation (1): Inh%=(Max-Signal)/ (Max-Min)*100.
The IC50 values were obtained by fitting the data in GraphPad using
Equation (2): Y = Bottom + (Top-Bottom) / (1 + 10^ ((LogIC50-X)*Hill
Slope)), where Y is %inhibition and X is compound concentration.
4.4.2. NLRP3 inflammasome activation
3,4-secolanosta-4(28),8,24-(Z)-trien-3-(hydroxyamino)-27-oic
acid
Mouse mononuclear macrophages J774A.1 cells were placed in a six-
well plate at a density of 1 × 106/ml and cultured overnight. Then,
discarded all the supernatant in the six-well plate. The remaining part
was pre-stimulated with the opti-MEM (1 mL/well) which contains LPS
(200 ng/ml) for 3 h. After the stimulation was complete, discarded again
all the supernatant. Added the remaining part into the opti-MEM (500
◦
(20). white solid. 25 mg, 30% yield; mp 114 C; IR νmax (KBr) 3443,
2925, 1640, 1401, 1323, 1265 cmꢀ 1; 1H NMR (Pyridine‑d5, 400 MHz) δ
6.07 (1H, t, J = 6.9 Hz, H-24), 4.96 (1H, s, HA-28), 4.88 (1H, s, HB-28),
2.16, 1.79 (6H, s, CH3-26, CH3-29), 3.05–1.12 (23H, m, manwuweizic
acid scaffold), 1.03 (3H, d, J = 5.2 Hz, CH3-21), 0.98, 0.86, 0.76 (9H, s,
CH3-18, CH3-30, CH3-19); 13C NMR (Pyridine‑d5, 100 MHz) δ 171.5 (C,
C-3), 171.2 (C, C-27), 148.3 (C, C-4), 143.2 (CH, C-24), 139.2 (C, C-9),
130.7 (C, C-8), 129.2 (C, C-25), 114.7 (CH2, C-28), 51.5 (C, C-14), 51.1
(CH, C-17), 47.4 (CH, C-5), 45.0 (C, C-13), 41.2 (C, C-10), 37.2 (CH, C-
20), 36.9 (CH2, C-1), 34.7 (CH2, C-2), 31.8 (CH2, C-6), 31.8 (CH2, C-7),
29.5 (CH2, C-11), 28.8 (CH2, C-12), 27.5 (CH2, C-15), 26.6 (CH2, C-16),
25.8 (CH3, C-30), 24.8 (CH2, C-22), 23.9 (CH3, C-19), 23.0 (CH3, C-18),
22.5 (CH2, C-23), 22.0 (CH3, C-21), 19.4 (CH3, C-26), 16.7 (CH3, C-29);
HRMS-ESI: m/z calcd for C30H48NO4 (M+H)+ 486.3578, found
486.3578.
μ
L/well) containing either the synthesized compounds which need to be
tested or the positive control compound SAHA and pre-incubated for 0.5
h. Then Nig (500 L per well), the NLRP3 inflammasome stimulator, was
added at the final concentration of 10 M, and incubated with the
μ
μ
compound for 1 h [27]. After that, collected all the supernatant, a part of
it was used to directly measure the release of LDH (Promega, USA) and
IL-1β (mouse IL-1β/IL-1F2 DuoSet ELISA kit R&D Incorporation, USA)
[28]. The remaining supernatant was carried out by TCA for protein
concentration. The levels of IL-1β and Caspase-1 were detected by
Western Blot. Total histone was extracted by EpiQuik Total Histone
Extraction Kit. The histone acetylation level was analyzed by Western
Blot. The total protein lysate was used as an internal reference by Coo-
massie blue staining.
4.2. Virtual screening
To identify HDAC inhibitors from an in-house natural products li-
brary containing 1086 molecules, a structure-based virtual screening
was performed using FlexX molecular docking program [22]. The re-
ceptor model was built based on the crystal structure of HDAC2 (PDB ID:
4LXZ) [23], with the pocket composed of residues within 6.5 Å around
the reference ligand (SAHA) cocrystalized in 4LXZ. The coordination of
Zn2+ was set to spherical. Pharmacophore constraints were defined to
include the interaction between the ligand and Zn2+, His145 and
His146. The conformation optimization for ligands was performed with
Discovery Studio (Accelrys, San Diego, CA, USA). Docking parameters
remained as default. Top 100 compounds with lowest binding energy
were clustered to 30 clusters by Discovery Studio, and 0–2 compounds in
each cluster were selected by considering structure diversity and com-
pound amount. Finally, 10 compounds were chosen for experimental
validation.
4.4.3. Cytotoxicity assay
Cell viability studies induced by synthesized compounds were eval-
uated by MTT assay. J774A.1 macrophages were seeded in 96-well
plated at a density of 4 × 104 cells/well in complete medium and
incubated overnight. Each group consisted of 3 wells. Then the cells
were treated with synthesized compounds (2.5, 5, 10, 20 or 40 μM) for
48 h. MTT (5 mg/mL in PBS, 10% total volume) was added to each well
and the cells were further incubated for 4 h. The supernatant was
removed and the cells were lysed with 150 μL/well DMSO. The optical
density was measured at 540 (Measurement wavelength) and 630
(reference wavelength) nm on a microplate reader (Thermo Scientifc,
Waltham, MA, USA). The IC50 values were calculated using GraphPad
Prism 7.
4.4.4. Lactate dehydrogenase (LDH) assay
After inflammasome activation with the treatment of compounds,
cell culture supernatants were collected and analyzed by a LDH assay kit
(Promega) according the manufacturer’s instructions.
4.3. Molecular docking for binding mode analysis
To understand the interaction of active compounds with different
HDAC subtypes, the same molecular docking protocol was applied as for
virtual screening. The receptor models were built with following struc-
tures (PDB ID): 5ICN [24] for HDAC1, 4LXZ [23] for HDAC2, 2VQM
[25] for HDAC4, 5EDU [26] for HDAC6.
4.4.5. Statistical analysis
All values were expressed in the form of mean ± standard deviation,
and the analysis software was GraphPad Prism 7.0. One-way analysis of
variance (ANOVA) followed by Tukey post hoc test was used to analyze
the statistical significance among multiple groups. P-values < 0.05 were
considered to indicate statistical significance.
4.4. Biological study
4.4.1. HDAC inhibition assay
4.4.6. ELISA xxx
The in vitro HDAC inhibitory activity was tested by ChemPartner
Co., Ltd, shanghai, People’s Republic of China. SAHA was used as the
The supernatant of the cell culture was taken out and stored in the
10

D.-X. Ni et al.
Bioorganic Chemistry 109 (2021) 104728
refrigerator at ꢀ 70 ◦C. IL-1β ELISA kit (R&D DouSet Mouse IL-1β
inhibitor/IL-1F2) was used to detect the concentration of IL-1 in the
supernatant according to the instructions of the kit.
schisandra sphaerandra that inhibits HIV-1 reverse transcriptase, J. Nat. Prod. 59
(5) (1996) 525–527.
[10] J.S. Liu, M.F. Huang, Y. Tao, Anwuweizonic acid and manwuweizic acid, the
putative anticancer active principle of Schisandra propinqua, Can. J. Chem. 66 (3)
(1988) 414–415.
[11] C.Q. Liang, R.H. Luo, J.M. Yan, Y. Li, X.N. Li, Y.M. Shi, S.Z. Shang, Z.H. Gao, L.
M. Yang, Y.T. Zheng, W.L. Xiao, H.B. Zhang, H.D. Sun, Structure and bioactivity of
triterpenoids from the stems of Schisandra sphenanthera, Arch. Pharmacal Res. 37
(2) (2014) 168–174.
4.4.7. Biological experiment material
J774A.1 cells were purchased from Kunming Institute of Zoology.
SAHA and MCC950 purchased from sigma.
[12] M. Liu, J. Wan, Y.Q. Luo, X.N. Li, H.D. Sun, Triterpenoids from Schisandra
propinqua var. propinqua, Nat. Prod. Commun. 11 (7) (2016) 925–929.
[13] Y.G. Chen, G.W. Qin, L. Cao, Y. Leng, Y.Y. Xie, Triterpenoid acids from Schisandra
propinqua with cytotoxic effect on rat luteal cells and human decidual cells in
vitro, Fitoterapia 72 (4) (2001) 435–437.
Declaration of Competing Interest
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.
[14] W.L. Xiao, R.R. Tian, J.X. Pu, X. Li, L. Wu, Y. Lu, S.H. Li, R.T. Li, Y.T. Zheng, Q.
T. Zheng, Triterpenoids from Schisandra lancifolia with anti-HIV-1 activity, J. Nat.
Prod. 69 (2) (2006) 277–279.
[15] W. Wang, J. Liu, J. Han, Z. Xu, D. Guo, New triterpenoids from Kadsura heteroclita
and their cytotoxic activity, Planta Med. 72 (5) (2006) 450–457.
[16] X. Yang, S. Li, L. Wu, Y. Li, L. Feng, Y. Shen, J. Tian, J. Tang, N. Wang, Y. Liu,
Abiesatrines A-J: anti-inflammatory and antitumor triterpenoids from Abies
georgei Orr, Org. Biomol. Chem. 8 (11) (2010) 2609–2616.
Acknowledgments
This project was supported by the NSFC (81903541, 81860615 and
21762048), projects from Yunnan Province (2019FD127, 2018FY001,
2018FA048), the Project of Innovative Research Team of Yunnan
Province (202005AE160005), and Yunling Scholar grants (W.-L.X.), and
a grant from the Ministry of Education (IRT_17R94).
[17] R. Sun, H.C. Song, C.R. Wang, K.Z. Shen, Y.B. Xu, Y.X. Gao, Y.G. Chen, J.Y. Dong,
Compounds from Kadsura angustifolia with anti-HIV activity, Bioorg. Med. Chem.
Lett. 21 (3) (2011) 961–965.
[18] L. Zhu, B. Li, X. Liu, X. Meng, Hepatoprotective effects of triterpenoid isolated from
Schizandra chinensis against acute alcohol-induced liver injury in mice, Food Sci.
Technol. Res. 19 (6) (2013) 1003–1009.
[19] X.X. Yuan, L.P. Yang, Z.L. Yang, W.L. Xiao, H.D. Sun, G.S. Wu, H.R. Luo, Effect of
nigranoic acid on Ca2+ influx and its downstream signal mechanism in NGF-
differentiated PC12 cells, J. Ethnopharmacol. 153 (3) (2014) 725–731.
[20] T. Feng, Y. Liu, C. Li, Z. Li, Protective effects of nigranoic acid on cerebral
ischemia-reperfusion injury and its mechanism involving apoptotic signaling
pathway, Cell Biochem. Biophys. 71 (1) (2015) 345–351.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104728.
[21] H. Yu, R. Zeng, Y. Lin, X. Li, S. Tasneem, Z. Yang, Y.X. Qiu, B. Li, Y.H. Wang, X. Cai,
W. Wang, Kadsura heteroclita stem suppresses the onset and progression of
adjuvant-induced arthritis in rats, Phytomedicine 58 (2019), 152876.
[22] M. Rarey, B. Kramer, T. Lengauer, G. Klebe, A fast flexible docking method using
an incremental construction algorithm, J. Mol. Biol. 261 (3) (1996) 470–489.
[23] B.E.L. Lauffer, R. Mintzer, R. Fong, S. Mukund, C. Tam, I. Zilberleyb, B. Flicke,
A. Ritscher, G. Fedorowicz, R. Vallero, Histone deacetylase (HDAC) inhibitor
kinetic rate constants correlate with cellular histone acetylation but not
transcription and cell viability, J. Biol. Chem. 288 (37) (2013) 26926–26943.
[24] P.J. Watson, C.J. Millard, A.M. Riley, N.S. Robertson, L.C. Wright, H.Y. Godage, S.
M. Cowley, A.G. Jamieson, B.V.L. Potter, J.W.R. Schwabe, Insights into the
activation mechanism of class I HDAC complexes by inositol phosphates, Nat.
Commun. 7 (1) (2016) 11262.
References
[1] S. Tan, Z.P. Liu, Natural products as zinc-dependent histone deacetylase inhibitors,
ChemMedChem 10 (3) (2015) 441–450.
[2] K.J. Falkenberg, R.W. Johnstone, Histone deacetylases and their inhibitors in
cancer, neurological diseases and immune disorders, Nat. Rev. Drug Discov. 14 (3)
(2015) 219.
[3] E. Ballestar, T. Li, New insights into the epigenetics of inflammatory rheumatic
diseases, Nat. Rev. Rheumatol. 13 (10) (2017) 593–605.
[4] E.E. Hull, M.R. Montgomery, K.J. Leyva, HDAC inhibitors as epigenetic regulators
of the immune system: impacts on cancer therapy and inflammatory diseases,
BioMed Res. Int. 2016 (2016) 8797206.
[25] M.J. Bottomley, P. Lo Surdo, P. Di Giovine, A. Cirillo, R. Scarpelli, F. Ferrigno,
P. Jones, P. Neddermann, R. De Francesco, C. Steinkuhler, Structural and
functional analysis of the human HDAC4 catalytic domain reveals a regulatory
structural zinc-binding domain, J. Biol. Chem. 283 (39) (2008) 26694–26704.
[26] Y. Hai, D.W. Christianson, Histone deacetylase 6 structure and molecular basis of
catalysis and inhibition, Nat. Chem. Biol. 12 (9) (2016) 741–747.
[27] S. Chen, B. Sun, Negative regulation of NLRP3 inflammasome signaling, Protein
Cell 4 (4) (2013) 251–258.
[5] D. Stammler, T. Eigenbrod, S. Menz, J.S. Frick, K.A. Bode, Inhibition of histone
deacetylases permits lipopolysaccharide-mediated secretion of bioactive IL-1β via a
caspase-1–independent mechanism, J. Immunol. 195 (11) (2015) 5421–5431.
[6] Y.M. Shi, W.L. Xiao, J.X. Pu, H.D. Sun, Triterpenoids from the Schisandraceae
family: an update, Nat. Prod. Rep. 32 (3) (2015) 367–410.
[7] W.L. Xiao, R.T. Li, S.X. Huang, J.X. Pu, H.D. Sun, Triterpenoids from the
Schisandraceae family, Nat. Prod. Rep. 25 (5) (2008) 871–891.
[28] S.G. Lee, B. Kim, W. Choi, I. Lee, J. Choi, K. Song, Lack of association between pro-
inflammatory genotypes of the interleukin-1 (IL-1B -31 C/+ and IL-1RN *2/*2)
and gastric cancer/duodenal ulcer in Korean population, Cytokine 21 (4) (2003)
167–171.
[8] M. Kikuchi, A. Yoshikoshi, ChemInform abstract: nigranoic acid, a new a-seco-
cycloartene triterpenoid, Chemischer Informationsdienst 4 (49) (1973) 725–728.
[9] H.D. Sun, S.X. Qiu, L.Z. Lin, Z.Y. Wang, Z.W. Lin, T. Pengsuparp, J.M. Pezzuto, H.H.
S. Fong, G.A. Cordell, N.R. Farnsworth, nigranoic acid, a triterpenoid from
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