Synthesis of novel andrographolide beckmann rearrangement derivatives and evaluation of their HK2-related anti-inflammatory activities

Article abstract of DOI:10.1016/j.ejmech.2019.04.022

Two series of andrographolide derivatives with introduction of amide moiety into ring A by Beckmann rearrangement were synthesized. In series 1, the ring A was converted to caprolactam, and an amide moiety was linked to C-19 of ring A in series 2. Among t

Full text of DOI:10.1016/j.ejmech.2019.04.022

Accepted Manuscript
Synthesis of novel andrographolide beckmann rearrangement derivatives and
evaluation of their HK2-related anti-inflammatory activities
Wang Wang, Yanli Wu, Kaiyin Yang, Canrong Wu, Ruotian Tang, Hua Li, Lixia Chen
PII:
S0223-5234(19)30328-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.022
EJMECH 11257
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 March 2019
Revised Date: 10 April 2019
Accepted Date: 10 April 2019
Please cite this article as: W. Wang, Y. Wu, K. Yang, C. Wu, R. Tang, H. Li, L. Chen, Synthesis of
novel andrographolide beckmann rearrangement derivatives and evaluation of their HK2-related anti-
inflammatory activities, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.04.022.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of novel andrographolide Beckmann rearrangement
derivatives and evaluation of their HK2-related
anti-inflammatory activities
1
,a
1,a
b
b
b
,a,b
Wang Wang, Yanli Wu, Kaiyin Yang, Canrong Wu, Ruotian Tang, Hua Li,*
,
a
Lixia Chen*
a
Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design &
Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang
110016, China
b
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation,
School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
1
These authors contributed equally to this work.
*
Corresponding author: Lixia Chen & Hua Li
E-mail: syzyclx@163.com (Lixia Chen)
li_hua@hust.edu.cn (Hua Li)

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ABSTRACT: Two series of andrographolide derivatives with introduction of amide
moiety into ring A by Beckmann rearrangement were synthesized. In series 1, the ring
A was converted to caprolactam, and an amide moiety was linked to C-19 of ring A in
series 2. Among them, compound 8h exhibited obvious inhibition on HK2 enzyme
activity (IC = 9.36 ± 0.08 µM) and LPS-induced NO production in RAW264.7 cells
5
0
(
IC = 22.38 ± 3.57 µM), and potent binding affinity with HK2 (Kd = 5.12 ± 0.82
50
µM). The preliminary structure-activity relationships (SARs) suggested that the
formation of caprolactam of ring A and esterification of C-19-hydroxyl could improve
the inhibitory effects on HK2 enzyme of andrographolide derivatives. Furthermore,
compound 8h significantly reduced the levels of IL-1β and IL-6, down-regulated the
expressions of iNOS and COX-2. Its anti-inflammatory effect was related to the
inhibition of both NF-κB pathway and glycolysis enzyme HK2. Since HK2 could be a
novel and effective target for anti-inflammation, compound 8h might be a new
anti-inflammatory agent targeting at HK2, or serve as a lead compound to design and
synthesize more HK2 inhibitors with better inflammatory effects.
Keywords: andrographolide, Beckmann rearrangement, anti-inflammatory activity,
HK2.

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1
. Introduction
Inflammation is a defensive response of the body to stimuli. In general, it is a
beneficial automatic defense response for human body. But persistent inflammation
could induce severe organ and tissue damage, which is harmful to health[1].
Therefore, safe and effective anti-inflammatory agents are highly desirable.
Andrographolide is a well-known ent-labdane diterpenoid isolated from
Andrographis paniculata, a traditional Chinese medicine. Its various bioactivities
including anti-bacterial[2], anti-hepatotoxic[3], anti-DENV (dengue virus)[4],
antitumor[5], hypoglycemic[6] and anti-hypertensive[7] activities has raised great
concern on andrographolide in recent years. In China and some other East Asian
countries, andrographolide has been widely used as the anti-inflammatory agent in
clinic for many years. It is reported that the anti-inflammation mechanisms of
andrographolide and some its derivatives were related to the inhibition of COX-2,
iNOS and NF-κB signal pathway[8-10]. We hope to synthesize some new
andrographolide derivatives with better anti-inflammatory activity and new
anti-inflammatory mechanism, which might lead to the discovery of novel potent
anti-inflammatory agents.
The fact that cancer cells mainly rely on glycolysis to meet the metabolic needs
of cell proliferation is named "Warburg effect"[11]. However, it has been recently
reported that glycolysis also played an important role in inflammation[12]. For
example, the inhibition of glycolysis in lipopolysaccharide (LPS)-induced
macrophages suppressed the expression of IL-1β[13, 14]. Hexokinase 2 (HK2)

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catalyzes the first rate-limiting glycolytic reaction, converting glucose to
glucose-6-phosphate (G6P) for energy production. It is a critical enzyme on the outer
mitochondrial membrane in shifting a cell from oxidative phosphorylation to
glycolytic metabolism[15-17]. HK2 has been reported to specifically express in
rheumatoid arthritis (RA) synovial lining and regulate fibroblast-like synoviocytes
(FLS) aggressive functions[18]. Therefore, HK2 was regarded as a new potential
anti-inflammatory target.
It is still a great challenge in modification of andrographolide into new series of
derivatives with appropriate structural diversity and improved bioactivity. The
previous derivatization of andrographolide has mainly focused on the positions of
several hydroxyl groups and double bonds[19-24], but the modification of double ring
skeleton is still very few[25]. Studies on the structure-activity relationships (SARs) of
andrographolide derivatives have revealed that introduction of nitrogen atom into the
structure could increase its basicity, and also affect its antitumor and antivirus
activities [26-29]. Based on these evidences, we designed and synthesized two series
of andrographolide derivatives with introduction of amide moiety into ring A by
Beckmann rearrangement. In series 1, the ring A was converted to caprolactam, and in
series 2, amide moiety was linked to C-19 of ring A. Their anti-inflammatory activity
and mechanism relating to HK2 were first investigated in this study.
2
. Results and discussion
2
.1 Chemistry

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The synthetic route of series 1 was shown in Scheme 1. At first, the hydroxyl group
at C-14 of andrographolide was removed by reacting with Al O in pyridine. Then the
2
3
dehydrated product 2 refluxed with tert-butyldimethylsilyl chloride (TBDMSCl) in
dichloromethane (DCM) to obtain C-19-hydroxyl protected product 3. The hydroxyl
group at C-3 of compound 3 was oxidized to carbonyl by pyridinium chlorochromate
(
PCC) and then was ammoniated by hydroxylamine hydrochloride (NH OH·HCl) to
2
yield compound 5. Beckmann rearrangement of ring A in compound 5 into
caprolactam structure was carried out with 4-toluene sulfochloride (p-TsCl) and
4
-dimethylamino-pyridin (DMAP) to furnish compound 6. Then the protective group
of compound 6 was removed by HCl gas to form the key intermediate 7. Previous
literatures have reported that esterification of hydroxyl groups at C-3 and C-19 of
andrographolide could enhance its bioactivities[20, 30], so we also adopted this
method to obtain compounds 8a-h.

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Scheme 1. Synthetic route of series 1. Reagents and conditions: (a) Al O , pyridine, reflux, 12 h;
2
3
(
b) TBDMSCl, TEA, DMAP, DCM, rt, 3 h; (c) PCC, DCM, 0 °C to rt, 2 h; (d) NH OH·HCl,
2
pyridine, 40 °C, 2 h; (e) p-TsCl, TEA, DMAP, DCM, rt, 3 h; (f) HCl(g)/ EtOAc, rt, 1 h; (g)
RCOOH, DCC, DMAP, DCM, rt, 2 h.
To investigate the influence of different positions of amide moiety in the ring A
on the anti-inflammatory activity, we designed and synthesized series 2 derivatives of
andrographolide. C-19-hydroxyl of compound
2
was oxidized by
N-bromosuccinimide (NBS) to obtain compound 9, which then was ammoniated by
NH OH·HCl to obtain ketoxime intermediate 10. Beckmann rearrangement of
2
compound 10 furnished the key intermediate 11, followed by the esterification of
compound 11 with acid molecules to yield the final products 12a-j.
Scheme 2. Synthetic route of series 2. Reagents and conditions: (h) TEMPO, TBAB, NBS,
NaHCO , DCM/H O, 0 °C to rt, 12 h.
3
2
2
.2 Biological evaluation

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2
.2.1 Inhibitory effects on HK2 activity
The recombinant HK2 was expressed and all synthesized compounds were tested
for their inhibitory activity on HK2. Compounds 8e, 8h and 12g showed inhibitory
effects against HK2 enzyme with IC values range from 4.80 to 26.08 µM (Table 1).
5
0
Compound 8h, with the most potent IC50 against HK2, was chosen for further
kinetic study. By using ATP and glucose as substrates, the steady-state enzyme kinetic
studies of compound 8h were performed for its inhibitory mode on HK2. As shown in
Fig. 1, while the maximal reaction rate (Vmax) values of both substrates were
decreased in dose-dependent manner for compound 8h (Fig. 1A and 1C, Table 2), the
kinetic constant (K ) values of both substrates remained little changes with or without
m
compound 8h treatment (Fig. 1B and 1D, Table 2). These results suggested that
compound 8h exhibited a non-competitive inhibition on HK2.
Fig. 1. In vitro enzyme inhibition assay. (A) ATP saturation profiles for HK2 at a range of

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concentrations of compound 8h. (B) Line weaver-Burk double-reciprocal representation of the
ATP saturation profiles for HK2 at a range of concentrations of compound 8h. (C) Glucose
saturation profiles for HK2 at a range of concentrations of compound 8h. (D) Line weaver-Burk
double-reciprocal representation of the glucose saturation profiles for HK2 at a range of
concentrations of compound 8h.
2
.2.2 Inhibitory effect on LPS-induced nitric oxide (NO) production and SAR study
As an important inflammatory mediator, NO is over produced and secreted out of
mouse macrophages in response to bacterial lipopolysaccharide (LPS) [31]. The
anti-inflammatory activity of the compounds was evaluated by detecting the secretion
level of NO in the medium of RAW264.7 cells after LPS stimulated. As showed in
Table 1, compounds 8d and 8h inhibited the secretion of NO with IC values range
5
0
from 22.38 to 33.51 µM.
The preliminary SARs showed that the oxidation of C-19-hydroxyl and
esterification of C-3-hydroxyl couldn't improve HK2 enzyme inhibitory activity of
andrographolide (i.e. compounds 12a-j). When C-3-hydroxyl was oxidized and
C-19-hydroxyl was esterified (i.e. compounds 8a-h), the derivatives exhibited better
HK2 enzyme inhibitory activity than series 2, showing that it is helpful to improve the
inhibitory effects on HK2 enzyme of andrographolide derivatives when the ring A was
converted to caprolactam and C-19-hydroxyl was esterified. But the effect of different
kinds of acid molecules on the inhibition of HK2 is not obvious. Therefore, the above
results would provide helpful information for the further structure modification of
andrographolide.
Table 1. The IC values of the inhibition against HK2 enzyme activity and NO production of all
5
0
a
compounds.

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Enzyme
NO
Enzyme
NO
Compound
7
Compound
IC (µM)
IC (µM)
IC (µM)
IC (µM)
5
0
50
50
50
>50
>50
>50
>50
12a
>50
>50
>50
>50
8
8
a
12b
b
>50
>50
12c
>50
>50
8
c
>50
>50
12d
>50
>50
8
d
>50
33.51 ± 8.62
>50
12e
>50
>50
8
e
4.80 ± 0.04
>50
12f
>50
>50
8
f
>50
12g
26.08 ± 0.21
>50
>50
8
8
1
g
h
1
>50
>50
12h
>50
9.36 ± 0.08 22.38 ± 3.57
12i
12j
>50
>50
>50
-
>50
>50
>50
Hydrocortisone
52.43 ± 2.74
Andrographolide
>50
14.42 ±1.39
a
The results were showed as means ± SD of at least three independent experiments
.
Table 2. Kinetic studies of enzyme inhibition by compound 8h on HK2 with ATP and glucose as
the substrates.
ATP
Glucose
C (comp. 8h, µM)
0
3.125
0.012
1.277
6.25
0.007
1.277
12.5
0.005
1.206
0
3.125
6.25
0.005
0.695
12.5
0.004
0.657
Vmax (OD/min)
Km (mM)
0.019
1.602
0.012
0.64
0.007
0.685
2
.2.3 Specific binding of compound 8h with HK2
The microscale thermophoresis method (MST) was used to assess protein-protein,
small molecule-protein, and nucleic acids-protein binding ability. Herein, compound
8
h, which showed strong inhibitory effect on both HK2 enzyme activity (IC : 9.36 ±
50
0
.08 µM) and NO production (IC : 22.38 ± 3.57 µM), was also selected to verify its
50
binding affinity with HK2. As shown in Fig. 2, compound 8h displayed a low
equilibrium dissociation constant (Kd) of 5.12 ± 0.82 µM, which meant a strong
binding affinity with HK2. Therefore, compound 8h was picked for the follow-up
study on its anti-inflammatory mechanism.

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Fig. 2. MST assay to measure the binding affinity of compound 8h with HK2. Compound 8h
displayed the equilibrium dissociation constant (Kd) of 5.12 ± 0.82 µM. Kd value was
automatically calculated by the curve fitting.
2
.2.4 Cell survival of RAW264.7
The cytotoxicity of compound 8h was measured by MTT to eliminate its influence
on the detection of NO production. The result showed that compound 8h had no
obvious effect on the cell survival at the concentration up to 100 µM (Fig. 3),
revealing that the anti-inflammatory activity of compound 8h is not produced by
cytotoxicity. In the further anti-inflammation study, the highest concentration of
compound 8h could be set below 100 µM.

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Fig. 3. NO inhibition rate curve of compound 8h and its cytotoxicity on RAW264.7 cells. (A)
Compound 8h inhibited LPS-induced NO production with IC50 values of 22.38 ± 3.57 µM. (B)
The RAW264.7 cell viability after exposure to different concentrations of compound 8h for 24 h.
The results were expressed as means ± SD of more than three independent experiments.
2
.2.5 Inhibitory effect on cytokines IL-1β and IL-6
When inflammation including acute and chronic inflammation occurs,
pro-inflammatory factors, such as IL-1β and IL-6, are over accumulated and
accelerate the development of inflammation in the local inflammatory area[32]. To
assess the anti-inflammatory activity of compound 8h, the levels of IL-1β and IL-6 in
the medium of macrophage cells after LPS induced were detected by ELISA. As
displayed in Fig. 4, apparently increased levels of IL-1β and IL-6 were observed in
LPS-stimulated cells, and then were both significantly down-regulated by compound
8
h in a dose-dependent manner.

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Fig. 4. The elevated levels of IL-1β and IL-6 were decreased by compound 8h. The production of
IL-1β (A) and IL-6 (B) in the medium of RAW264.7 cells after treatment with compound 8h for 3
#
##
h and LPS for 24 h. , p < 0.001, compared with control group. **, p < 0.01 and ***, p < 0.001,
compared with LPS-induced model group.
2
.2.6 Inhibitory effect on LPS-induced protein expression and NF-κB pathway
Inducible nitric oxide synthase enzymes (iNOS) are induced to high expression
in various inflammatory cells when exposed to microbial products, such as LPS or
pro-flammatory cytokines, such as IL-1 and tumor necrosis factor-α (TNF-α)[31]. NO
is synthesized by iNOS in activated murine macrophages. Besides, expression of
COX-2 is also up-regulated by pro-inflammatory cytokines such as IL-1 and TNF-α
in response to LPS[33]. In this study, iNOS and COX-2 expressions were detected by
western blotting. As shown in Fig. 5, the levels of iNOS and COX-2 were increased
after LPS activation, and then obviously reduced by compound 8h treatment in a
dose-dependent manner. Nuclear factor-κB (NF-κB) is a ubiquitously expressed
pro-inflammatory transcription factor that regulates the expression of genes related to
a number of physiological responses, including immune inflammatory responses,
acute-phase inflammatory responses, and oxidative stress responses[34]. The nuclear
translocation of NF-κB p65 is crucial for its function. As expected, NF-κB expression

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was decreased in macrophage cells after treated by compound 8h (Fig. 4). In addition,
the nuclear translocation of NF-κB p65 was also blocked by compound 8h (Fig. 4).
From the above results, compound 8h can be a potential anti-inflammatory agent by
inhibiting the expression of iNOS, COX-2, and NF-κB.
Fig. 5. The protein expressions of iNOS, COX-2, NF-κB and the nuclear translocation of NF-κB
p65 were inhibited by compound 8h. (A) Western blotting for iNOS and COX-2. (B) Western
blotting for NF-κB. (C) Immunofluorescence staining for NF-κB p65. RAW264.7 cells stained for
NF-κB p65 (green) and nuclei (DAPI, blue).
2
.2.7 Inhibitory effect on LPS-induced glycolysis
HK2 is an enzyme controlling the first step of glycolysis. The change in
metabolism toward glycolysis in LPS-induced macrophages is critical for enhanced
pro-inflammatory cytokines and mediators, such as IL-1β, NO, and

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prostaglandins[35]. A Seahorse Extracellular Flux Analyzer (EFA) was used to
measure metabolic phenotypes of cells. As opposed to other more traditional
metabolism assays, the advantages of Seahorse EFA are considerable, because assays
such as traditional glycolysis or beta-oxidation assay, require radioactively labeled
substrates and much larger numbers of cells [36]. In this study, the real-time
simultaneous measurement of the extracellular acidification rate (ECAR), an indicator
of glycolysis was performed to evaluate the effect of compound 8h on the
energy metabolism in macrophage RAW264.7 cells. As displayed in Fig. 6, LPS
enhanced the glycolysis but had no effect on the oxidative phosphorylation.
Compound 8h inhibited LPS-induced glycolysis in macrophages after treatment for 4
hours. This indicated that the anti-inflammatory effect of compound 8h was related to
its inhibition of glycolysis target HK2.
Fig. 6. The activation of glycolysis in RAW264.7 macrophages by LPS was blocked by
compound 8h (arrow, 50 µM) and LPS (black arrow, 1 µg/mL) for an extended period (> 6 h).
ECAR was monitoring. Data shown are mean ± SD, n=3 technical replicates.

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3
. Conclusion
Herein, two series of andrographolide derivatives with introduction of amide
moiety into ring A by Beckmann rearrangement were synthesized. Their inhibition on
HK2 enzyme activity and LPS-induced NO production in RAW264.7 cells were
tested. The structure-activity relationships (SARs) suggested that it is helpful to
improve the inhibitory effects on HK2 enzyme of andrographolide derivatives when
the ring A was converted to caprolactam and C-19-hydroxyl was esterified.
Compound 8h was measured for its binding affinity with HK2 through MST, and
exhibited a strong binding with HK2. Thus, the further anti-inflammatory
investigation of compound 8h revealed that it significantly reduced the levels of IL-1β
and IL-6, down-regulated the expressions of iNOS and COX-2. And its
anti-inflammatory effect was related to the inhibition of NF-κB pathway and the
suppression of glycolysis enzyme HK2. Since HK2 was suggested to be a novel and
effective target for anti-inflammation, compound 8h would be served as a lead
compound to design and synthesize more HK2 inhibitors with better
anti-inflammatory activity.
4
. Experimental
4
.1. Chemistry
All commercially available starting materials and solvents were reagent grade and
used without further purification. Reactions were monitored by thin-layer
chromatography (TLC) on 0.25 mm silica gel plates (GF ) and visualized under UV
2
54
1
13
light or by heating after spraying with anisaldehyde-H SO reagent. H NMR and C
2
4

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NMR spectra were recorded on a Bruker AV-600 spectrometer (Bruker Biospin,
Fallanden, Switzerland). Chemical shifts are stated relative to TMS and expressed in δ
values (ppm), with coupling constants reported in Hz. High resolution mass spectra
(HRMS) of all derivatives were recorded on a Waters Micromass Q-T of Micromass
spectrometer by electrospray ionization (ESI).
4
4
1
.2. Synthetic methods of all compounds
.2.1. Synthesis of compound 2
4-deoxy-11,12-didehydroandrographolide (compound 2). Andrographolide (3.0 g)
was dissolved in anhydrous pyridine (8 mL). Then Al O (600 mg) was added to the
2
3
solution. The mixture was stirred for 12 h at 115 °C. Upon completion, the soild was
filtered off and then pyridine was evaporated to dryness. The crude product was
redissolved in methylene chloride (DCM), washed with brine, dried over anhydrous
Na SO , filtered, evaporated, and purified by silica gel column chromatography (CC)
2
4
to obtain compound 2 as white solid (93% yield). HR-MS(ESI) m/z: 333.2062
+
1
[
M+H] (Calcd. for C H O , 333.2060); H-NMR (600 MHz, DMSO-d ): δ 7.64 (s,
20 29 4 6
1H, 14-H), 6.72 (dd, J = 15.8, 10.1 Hz, 1H, 11-H), 6.10 (d, J = 15.8 Hz, 1H, 12-H),
4.87 (s, 2H, 15-H), 4.71 (s, 1H, 17a-H), 4.40 (s, 1H, 17b-H), 4.12 (d, J = 10.7 Hz, 1H,
19a-H), 3.83 (d, J = 10.9 Hz, 1H, 19b-H), 3.23 – 3.17 (m, 1H, 3-H), 2.35 (dd, J = 9.5,
4.3 Hz, 1H, 9-H), 2.00 – 1.91 (m, 1H, 7-H), 1.76 – 1.66 (m, 2H, 2-H), 1.59 – 1.31 (m,
3H, 1-H, 6a-H), 1.21 – 1.14 (m, 1H, 5-H), 1.11 (dd, J = 13.4, 4.4 Hz, 1H, 6b-H), 1.07
(
s, 3H, 18-CH ), 0.74 (s, 3H, 20-CH ).
3
3
4
.2.2. Synthesis of compound 3

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1
9-tert-butyldimethylsilyl-14-deoxy-11,12-didehydroandrographolide (compound 3).
Compound 2 (3.3 g, 10 mmol) was dissolved in DCM (60 mL). Triethylamine (TEA,
.6 mL) was added to the solution. The mixture was put into an ice bath to cool down.
1
Then TBDMSCl (1.8 g, 11.94 mmol) and DMAP (catalytic amount) was added and
stirred for 3 h at 25 °C. Upon completion, the organic layers was washed with brine,
2
0% citric acid solution, and saturated sodium bicarbonate solution, respectively,
dried over anhydrous Na SO , and then filtered, evaporated, and purified by silica gel
2
4
CC to obtain compound 3 as white solid (91% yield). HR-MS(ESI) m/z: 447.2921
+
1
[
M+H] (Calcd. for C H O Si, 447.2925); H-NMR (600 MHz, CDCl ): δ 7.18 (s,
26 43 4 3
1H, 14-H), 6.92 (dd, J = 15.8, 10.1 Hz, 1H, 11-H), 6.14 (d, J = 15.8 Hz, 1H, 12-H),
4.81 (s, 2H, 15-H), 4.77 (s, 1H, 17a-H), 4.54 (s, 1H, 17b-H), 4.22 (d, J = 10.0 Hz, 1H,
19a-H), 3.41 (d, J = 10.0 Hz, 1H, 19b-H), 3.33 – 3.31 (m, 1H, 3-H), 2.32 (d, J = 10.1
Hz, 1H, 9-H), 2.05 (td, J = 13.3, 5.0 Hz, 1H, 7-H), 1.80 (d, J = 3.5 Hz, 2H, 2-H), 1.65
s, 2H), 1.50 (d, J = 13.6 Hz, 1H), 1.32 (s, 1H), 1.28 – 1.20 (m, 2H), 1.24 (s, 3H,
(
1
0
4
1
8-CH ), 1.14 (d, J = 3.4 Hz, 1H, 6b-H), 0.90 (s, 9H, t-Bu-CH ), 0.83 (s, 3H, 20-CH ),
3
3
3
.08 (d, J = 5.0 Hz, 6H, Si-CH3).
.2.3. Synthesis of compound 4
9-tert-butyldimethylsilyl-3-oxo-14-deoxy-11,12-didehydroandrographolide
compound 4). Pyridinium chlorochromate (PCC, 1 g, 3.98 mmol) was dissolved in
(
anhydrous DCM (10 mL). Then compound 3 (1 g, 2.24 mmol) was dissolved in
anhydrous DCM (10 mL) and added to the solution at 0 °C. The mixture was stirred
for about 2 h at room temperature. Upon completion, anhydrous ether (20 mL) was

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added. The solid was filtered off and the solvent was removed under reduced pressure.
The residue was redissolved in DCM and washed with brine, water and saturated
sodium bicarbonate solution, and dried over anhydrous Na SO , respectively, filtered,
2
4
evaporated, and purified by silica gel CC to obtain compound 4 as white solid (46%
+
yield). HR-MS(ESI) m/z: 445.2763 [M+H] (Calcd. for C H O Si, 445.2769);
26
41
4
1
H-NMR (600 MHz, CDCl ): δ 7.18 (s, 1H, 14-H), 7.04 – 6.88 (m, 1H, 11-H), 6.14 (d,
3
J = 15.8 Hz, 1H, 12-H), 4.85 (s, 1H, 17a-H), 4.83 (s, 2H, 15-H), 4.61 (s, 1H, 17b-H),
3
2
2
5
.86 (d, J = 9.9 Hz, 1H, 19a-H), 3.54 (d, J = 9.9 Hz, 1H, 19b-H), 2.60 (s, 1H), 2.53 –
.47 (m, 1H), 2.40 (d, J = 10.1 Hz, 1H), 2.30 (ddd, J = 15.1, 4.1, 3.0 Hz, 1H), 2.11 –
.02 (m, 1H), 1.85 – 1.78 (m, 1H), 1.77 – 1.72 (m, 1H), 1.60 (s, 2H), 1.47 (tt, J = 10.6,
.3 Hz, 1H), 1.15 (s, 3H, 18-CH ), 1.11 (s, 3H, 20-CH ), 0.85 (s, 9H, t-Bu-CH ), 0.00
3
3
3
(d, J = 3.8 Hz, 6H, Si-CH3).
4
.2.4. Synthesis of compound 5
9-tert-butyldimethylsilyl-3-oxime-14-deoxy-11,12-didehydroandrographolide
1
(compound 5). Compound 4 (0.1 g, 0.22 mmol) was dissolved in anhydrous pyridine
(5 mL). Then hydroxylamine hydrochloride (0.085 g, 1.22 mmol) was added to the
solution. The mixture was stirred for about 2 h at 40 °C. Upon completion, pyridine
was evaporated to dryness. The residue was redissolved in DCM and washed with 20%
citric acid solution, saturated sodium bicarbonate solution, water and brine, and dried
over anhydrous Na SO , respectively, filtered, and evaporated to obtain compound 5
2
4
as white solid (98% yield). This procedure was also applied to the preparation of
+
compound 10. HR-MS(ESI) m/z: 460.2870 [M+H] (Calcd. for C H NO Si,
26
42
4

ACCEPTED MANUSCRIPT
1
4
60.2878); H-NMR (600 MHz, CDCl ): δ 7.16 (s, 1H, 14-H), 6.95 (dd, J = 15.8, 10.1
3
Hz, 1H, 11-H), 6.11 (d, J = 15.8 Hz, 1H, 12-H), 4.82 (d, J = 5.5 Hz, 3H, 15-H, 17a-H),
4.58 (s, 1H, 17b-H), 3.75 (d, J = 10.0 Hz, 1H, 19a-H), 3.56 (d, J = 10.0 Hz, 1H,
19b-H), 3.29 (dt, J = 14.7, 3.4 Hz, 1H), 2.54 – 2.42 (m, 1H), 2.35 (d, J = 10.0 Hz, 1H),
2.12 – 1.96 (m, 1H), 1.94 – 1.79 (m, 2H), 1.64 (ddd, J = 13.0, 8.5, 4.6 Hz, 1H), 1.58
(td, J = 12.9, 4.0 Hz, 1H), 1.49 – 1.42 (m, 1H), 1.26 (s, 1H), 1.21 (s, 3H, 18-CH3),
1
.03 (s, 3H, 20-CH ), 0.85 (s, 9H, t-Bu-CH ), -0.01 (d, J = 6.4 Hz, 6H, Si-CH ).
3
3
3
4
.2.5. Synthesis of compound 6
(
1R,5aS,6R,9aS)-1-(((tert-butyldimethylsilyl)oxy)methyl)-1,5a-dimethyl-7-methylene-6
((E)-2-(2-oxo-2,5-dihydrofuran-3-yl)ethenyl)octahydro-1H-benzo[c]azepin-3(2H)-
-
one (compound 6). Compound 5 (0.8 g, 1.74 mmol) was dissolved in DCM (10 mL).
TEA (0.6 mL) was added to the sloution. The mixture was put in an ice bath to cool
down. Then p-TsCl (0.83 g, 4.35 mmol) and DMAP (catalytic amount) was added in
and stirred for 3 h at 25 °C. Upon completion, the organic layer was washed with 20%
citric acid solution, saturated sodium bicarbonate solution, water and brine, and dried
over anhydrous Na SO , respectively, filtered, evaporated, and purified by silica gel
2
4
CC to obtain compound 6 as light yellow solid (42% yield). This procedure was also
+
applied to the preparation of compound 11. HR-MS(ESI) m/z: 460.2871 [M+H]
1
(
Calcd. for C H NO Si, 460.2878); H-NMR (600 MHz, CDCl ): δ 7.04 (s, 1H,
2
6
42
4
3
15-H), 6.88 – 6.62 (m, 1H, 12-H), 5.98 (d, J = 15.8 Hz, 1H, 13-H), 4.69 (s, 3H, 16-H,
18a-H), 4.45 (s, 1H, 18b-H), 3.54 (d, J = 3.4 Hz, 2H, 20-H), 2.45 (d, J = 10.1 Hz, 1H),
2.38 – 2.25 (m, 3H), 2.00 – 1.87 (m, 1H), 1.76 – 1.65 (m, 2H), 1.63 – 1.56 (m, 1H),

ACCEPTED MANUSCRIPT
1
.50 (d, J = 8.5 Hz, 2H), 1.15 (s, 3H, 19-CH ), 0.87 (s, 3H, 21-CH ), 0.75 (s, 9H,
3
3
t-Bu-CH ), -0.10 (d, J = 1.6 Hz, 6H, Si-CH ).
3
3
4
.2.6. Synthesis of compound 7
(1R,5aS,6R,9aS)-1-(hydroxymethyl)-1,5a-dimethyl-7-methylene-6-((E)-2-(2-oxo-2,5-
dihydrofuran-3-yl)ethenyl)octahydro-1H-benzo[c]azepin-3(2H)-one (compound 7).
Compound 6 (0.5 g, 1.09 mmol) was dissloved in ethyl acetate solution of HCl and
stirred for about 1 h at room temperature, then ethyl acetate was evaporated to dryness.
Upon completion, ethyl acetate was added. The combined organic layers were washed
with brine, water and saturated sodium bicarbonate solution, and dried over
anhydrous Na SO , respectively, filtered, evaporated, and purified by silica gel CC to
2
4
obtain compound 7 as light yellow solid (96% yield). HR-MS(ESI) m/z: 346.2010
+
1
[
(
M+H] (Calcd. for C H NO , 346.2013); H-NMR (600 MHz, DMSO-d ): δ 7.67
20 28 4 6
s, 1H, 15-H), 6.72 (dd, J = 15.9, 10.2 Hz, 1H, 12-H), 6.58 (s, 1H, 4-NH), 6.15 (d, J =
5.9 Hz, 1H, 13-H), 4.90 (s, 2H, 16-H), 4.76 (s, 1H, 18a-H), 4.45 (s, 1H, 18b-H), 3.51
d, J = 10.9 Hz, 1H, 20a-H), 3.49 (d, J = 10.9 Hz, 1H, 20b-H), 2.72 (d, 1H, J = 10.0
Hz), 2.41 – 2.23 (m, 3H), 2.12 – 2.00 (m, 1H), 1.84 – 1.71 (m, 2H), 1.67 – 1.51 (m,
1
(
1
3
2
H), 1.50 – 1.40 (m, 1H), 1.20 (s, 3H, 19-CH ), 0.92 (s, 3H, 21-CH ). C-NMR (150
3 3
MHz, DMSO-d ): δ 175.2, 172.4, 149.2, 147.0, 134.5, 127.0, 121.7, 108.6, 70.2, 65.5,
6
6
0.1, 57.8, 53.1, 41.1, 36.0, 34.9, 30.9, 28.5, 25.2, 16.6.
4
.2.7. General procedure for the synthesis of compounds 8a-h
method 1: DCC (36 mg, 0.17 mmol) and acid molecules (0.20 mmol) were dissolved
in DCM (5 mL). After 20 minutes, compound 7 (60 mg, 0.17 mmol) and DMAP

ACCEPTED MANUSCRIPT
(catalytic amount) were added to the mixture. The mixture was stirred for about 2 h at
room temprature. Upon completion, the organic layer was washed with 20% citric
acid solution, saturated sodium bicarbonate solution, water and brine, respectively,
dried over anhydrous Na SO , filtered, evaporated, and purified by CC to afford
2
4
compounds 8a-d and compound 8h. This procedure was also applied to the
preparation of compounds 12a-d and compound 12h.
method 2: DCC (36 mg, 0.17 mmol) and acid molecules (0.20 mmol) were dissolved
in DCM (5 mL). After 20 minutes, compound 7 (60 mg, 0.17 mmol) and DMAP
(catalytic amount) were added to the mixture. The mixture was stirred for about 2 h at
room temprature. Upon completion, the organic layer was washed with 20% citric
acid solution, saturated sodium bicarbonate solution, water and brine, respectively,
dried over anhydrous Na SO , filtered, evaporated, and purified by CC to afford
2
4
products. Then the product was dissolved in ethyl acetate solution of HCl and stirred
for about 30 minutes at room temperature, then ethyl acetate was evaporated to
dryness. Upon completion, ethyl acetate was added. The combined organic layers
were washed with brine, water and saturated sodium bicarbonate solution, and dried
over anhydrous Na SO , respectively, filtered, evaporated, and purified by silica gel
2
4
CC to obtain compounds 8e-g. This procedure was also applied to the preparation of
compounds 12e-g and compounds 12i-j.
((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran
-3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl nicotinate (compound 8a).
+
White solid, 96% yield; HR-MS(ESI) m/z: 451.2223 [M+H] (Calcd. for C H N O ,
26
31
2
5

ACCEPTED MANUSCRIPT
1
4
4
51.2227); H-NMR (600 MHz, DMSO-d ): δ 9.19 (s, 1H, 2'-H), 8.84 (dd, 1H, J =
6
.8, 1.5 Hz, 6'-H), 8.36 (m, 1H, 4'-H), 7.70 (s, 1H, 15-H), 7.59 (dd, 1H, J = 7.8, 4.8
Hz, 5'-H), 7.29 (s, 1H, 4-NH), 6.73 (dd, 1H, J = 15.9, 10.2 Hz, 12-H), 6.17 (d, 1H, J
15.9 Hz, 13-H), 4.90 (s, 2H, 16-H), 4.80 (s, 1H, 18a-H), 4.72 (d, 1H, J = 11.0 Hz,
0a-H), 4.49 (s, 1H, 18b-H), 4.24 (d, 1H, J = 11.1 Hz, 20b-H), 2.78 (d, 1H, J = 10.0
Hz), 2.47 – 2.35 (m, 2H), 2.14 (dd, J = 17.7, 9.1 Hz, 1H), 1.93 – 1.75 (m, 1H), 1.72 –
=
2
1
1
1
7
.67 (m, 2H), 1.62 – 1.53 (m, 1H), 1.53 – 1.42 (m, 1H), 1.38 (s, 3H, 19-CH ), 1.15 –
3
1
3
.00 (m, 1H), 0.93 (s, 3H, 21-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.4, 172.4,
3
6
64.5, 153.7, 150.2, 148.7, 147.1, 137.1, 134.1, 127.0, 125.4, 123.9, 121.9, 108.9,
0.2, 68.2, 58.6, 57.7, 53.8, 41.2, 35.7, 34.6, 30.3, 28.21, 24.95, 16.5.
((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran
-3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl benzoate (compound 8b).
+
Light yellow solid, 90% yield; HR-MS(ESI) m/z: 450.2271 [M+H] (Calcd. for
1
C H NO , 450.2275); H-NMR (600 MHz, DMSO-d ): δ 8.02 (d, J = 7.4 Hz, 2H,
27
32
5
6
Ph-H), 7.74 – 7.61 (m, 2H, Ph-H, 15-H), 7.55 (t, J = 7.7 Hz, 2H, Ph-H), 7.19 (s, 1H,
-NH), 6.72 (dd, J = 15.9, 10.2 Hz, 1H, 12-H), 6.16 (d, J = 15.9 Hz, 1H, 13-H), 4.90
s, 2H, 16-H), 4.79 (s, 1H, 18a-H), 4.66 (d, J = 11.1 Hz, 1H, 20a-H), 4.48 (s, 1H,
4
(
1
1
1
8b-H), 4.21 (d, J = 11.2 Hz, 1H, 20b-H), 2.77 (d, J = 10.0 Hz, 1H), 2.44 – 2.36 (m,
H), 1.93 (dd, J = 19.7, 12.9 Hz, 2H), 1.74 – 1.69 (m, 2H), 1.66 (dd, J = 14.3, 9.7 Hz,
H), 1.59 (td, J = 12.9, 4.1 Hz, 2H), 1.36 (s, 3H, 19-CH ), 1.06 – 1.00 (m, 1H), 0.92
3
1
3
(
s, 3H, 21-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.4, 172.4, 165.5, 156.5, 149.2,
3
6
1
47.5, 134.5, 133.8, 129.9, 129.8, 129.1, 127.3, 122.3, 109.3, 70.2, 67.8, 58.5, 57.7,

ACCEPTED MANUSCRIPT
5
3.8, 41.2, 35.7, 34.7, 31.0, 28.3, 25.3, 16.4.
((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran
-3-yl) ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl 2-nitrobenzoate (compound
+
8
c). White solid, 95% yield; HR-MS(ESI) m/z: 495.2120 [M+H] (Calcd. for
1
C H N O , 495.2126); H-NMR (600 MHz, DMSO-d ): δ 8.05 (d, J = 7.6 Hz, 1H,
27
31
2
7
6
4
'-H), 7.96 (dd, J = 7.4, 1.2 Hz, 1H, 6'-H), 7.90 – 7.80 (m, 2H, 3', 5'-H), 7.69 (s, 1H,
5-H), 7.08 (s, 1H, 4-NH), 6.72 (dd, J = 15.8, 10.2 Hz, 1H, 12-H), 6.15 (d, J = 15.9
1
Hz, 1H, 13-H), 4.89 (s, 2H, 16-H), 4.77 (s, 1H, 18a-H), 4.65 (d, J = 11.1 Hz, 1H,
2
1
1
3
1
5
0a-H), 4.47 (s, 1H, 18b-H), 4.24 (d, J = 11.1 Hz, 1H, 20b-H), 2.75 (d, J = 10.0 Hz,
H), 2.44 – 2.33 (m, 2H), 1.87 (t, J = 10.0 Hz, 2H), 1.71 (d, J = 9.2 Hz, 1H), 1.66 –
.57 (m, 2H), 1.53 – 1.44 (m, 2H), 1.29 (s, 3H, 19-CH ), 1.07 – 0.99 (m, 1H), 0.90 (s,
3
13
H, 21-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.3, 172.4, 164.3, 148.7, 148.1,
3
6
47.1, 134.1, 133.4, 133.2, 130.1, 127.0, 125.5, 124.0, 121.9, 108.9, 70.2, 69.0, 58.3,
7.7, 53.7, 41.2, 35.6, 34.6, 30.9, 28.1, 25.3, 16.4.
((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran
-3-yl) ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl 4-nitrobenzoate (compound
+
8
d). Light yellow solid, 90% yield; HR-MS(ESI) m/z: 495.2121 [M+H] (Calcd. for
1
C H N O , 495.2126); H-NMR (600 MHz, DMSO-d ): δ 8.36 (d, J = 8.7 Hz, 2H, 3',
27
31
2
7
6
5
'-H), 8.27 (d, J = 8.6 Hz, 2H, 2', 6'-H), 7.69 (s, 1H, 15-H), 7.26 (s, 1H, 4-NH), 6.73
dd, J = 15.8, 10.2 Hz, 1H, 12-H), 6.17 (d, J = 15.9 Hz, 1H, 13-H), 4.90 (s, 2H, 16-H),
.79 (s, 1H, 18a-H), 4.73 (d, J = 11.1 Hz, 1H, 20a-H), 4.48 (s, 1H, 18b-H), 4.26 (d, J
11.1 Hz, 1H, 20b-H), 2.77 (d, J = 10.1 Hz, 1H), 2.47 – 2.35 (m, 2H), 1.93 (d, J =
(
4
=

ACCEPTED MANUSCRIPT
1
1
2
1
3
0.8 Hz, 2H), 1.79 (dd, J = 30.6, 12.2 Hz, 1H), 1.73 – 1.64 (m, 2H), 1.62 – 1.56 (m,
H), 1.53 – 1.45 (m, 1H), 1.38 (s, 3H, 19-CH ), 1.28 – 1.18 (m, 1H), 0.92 (s, 3H,
3
13
1-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.4, 172.4, 164.1, 150.3, 148.7, 147.1,
3
6
34.8, 134.1, 130.8, 127.0, 123.8, 121.9, 108.9, 70.2, 68.7, 58.5, 57.6, 53.7, 41.2, 35.6,
4.5, 31.0, 28.2, 25.3, 16.5.
(
R)-((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-
dihydrofuran-3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl 2-amino-3-
phenylpropanoate (compound 8e). Light yellow solid, 86% yield; HR-MS(ESI) m/z:
+
1
4
93.2694 [M+H] (Calcd. for C H N O , 493.2697); H-NMR (600 MHz,
29 37 2 5
DMSO-d ): δ 7.68 (s, 1H, 15-H), 7.31 – 7.23 (m, 2H, Ph-H), 7.19 (dd, J = 5.0, 2.8 Hz,
6
3
H, Ph-H), 7.01 (s, 1H, 4-NH), 6.70 (dd, J = 15.9, 10.2 Hz, 1H, 12-H), 6.15 (d, J =
1
5.8 Hz, 1H, 13-H), 4.89 (s, 2H, 16-H), 4.78 (s, 1H, 18a-H), 4.46 (s, 1H, 18b-H), 4.32
(d, J = 11.0 Hz, 1H, 20a-H), 3.92 (d, J = 11.0 Hz, 1H, 20b-H), 3.59 (t, J = 6.9 Hz, 1H,
2
'-H), 2.91 (dd, J = 13.4, 6.3 Hz, 1H), 2.78 – 2.71 (m, 2H), 2.39 – 2.33 (m, 2H), 2.17
2.07 (m, 1H), 1.84 (dd, J = 22.6, 7.6 Hz, 2H), 1.60 (dd, J = 13.3, 9.6 Hz, 1H), 1.49
–
1
3
(
qd, J = 12.9, 7.1 Hz, 2H), 1.18 (s, 3H, 19-CH ), 0.83 (s, 3H, 21-CH ). C-NMR (150
3 3
MHz, DMSO-d ): δ 175.2, 174.5, 172.4, 148.8, 147.1, 137.9, 134.1, 129.1, 128.1,
6
1
26.9, 126.2, 121.8, 108.9, 70.2, 67.3, 58.4, 57.4, 55.9, 53.3, 41.0, 40.3, 35.7, 34.4,
0.8, 28.2, 24.8, 16.6.
3
(
R)-((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-
dihydrofuran-3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl 2-amino-4-
methylpentanoate (compound 8f). Light yellow solid, 83% yield; HR-MS(ESI) m/z:

ACCEPTED MANUSCRIPT
+
1
4
59.2849 [M+H] (Calcd. for C H N O , 459.2853); H-NMR (600 MHz,
26 39 2 5
DMSO-d ): δ 7.68 (s, 1H, 15-H), 7.03 (s, 1H, 4-NH), 6.71 (dd, J = 15.9, 10.2 Hz, 1H,
6
1
2-H), 6.15 (d, J = 15.9 Hz, 1H, 13-H), 4.89 (s, 2H, 16-H), 4.78 (s, 1H, 18a-H), 4.47
s, 1H, 18b-H), 4.32 (d, J = 11.1 Hz, 1H, 20a-H), 3.99 (d, J = 11.1 Hz, 1H, 20b-H),
.76 (d, J = 10.1 Hz, 1H), 2.43 – 2.30 (m, 3H), 2.12 (dd, J = 13.0, 8.6 Hz, 1H), 1.87
d, J = 10.6 Hz, 2H), 1.71 (dt, J = 13.4, 6.6 Hz, 1H), 1.62 (dd, J = 13.0, 9.5 Hz, 1H),
.54 (dd, J = 13.3, 4.2 Hz, 1H), 1.50 – 1.38 (m, 3H), 1.33 – 1.27 (m, 1H), 1.24 (s, 3H,
(
2
(
1
1
1
5
1
3
9-CH ), 0.86 (9H, m, 21, 5', 5"-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.7,
3
3
6
75.2, 172.4, 148.9, 147.1, 134.1, 126.9, 121.8, 108.8, 70.2, 67.3, 58.4, 57.4, 53.2,
2.5, 43.4, 41.1, 35.7, 34.4, 30.8, 28.3, 24.9, 24.1, 22.8, 21.8, 16.6.
(S)-((1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-
dihydrofuran-3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl pyrrolidine-2-
carboxylate (compound 8g). White solid, 84% yield; HR-MS(ESI) m/z: 443.2538
+
1
[
(
M+H] (Calcd. for C H N O , 443.2540); H-NMR (600 MHz, DMSO-d ): δ 7.67
25 35 2 5 6
s, 1H, 15-H), 6.98 (s, 1H, 4-NH), 6.71 (dd, J = 15.9, 10.2 Hz, 1H, 12-H), 6.16 (d, J =
5.8 Hz, 1H, 13-H), 4.89 (s, 2H, 16-H), 4.78 (s, 1H, 18a-H), 4.46 (s, 1H, 18b-H), 4.35
d, J = 11.2 Hz, 1H, 20a-H), 4.02 (d, J = 11.2 Hz, 1H, 20b-H), 3.69 (dd, J = 8.4, 5.6
Hz, 1H, 2'-H), 2.87 (dt, J = 9.9, 6.8 Hz, 1H), 2.76 (dt, J = 10.0, 7.0 Hz, 2H), 2.42 –
1
(
2
1
1
1
.33 (m, 3H), 2.16 – 2.07 (m, 1H), 2.00 – 1.94 (m, 1H), 1.85 (d, J = 10.9 Hz, 2H),
.73 (dt, J = 12.7, 7.4 Hz, 1H), 1.67 – 1.58 (m, 3H), 1.50 – 1.42 (m, 1H), 1.23 (s, 3H,
13
9-CH ), 0.86 (s, 3H, 21-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.2, 174.3,
3
3
6
72.4, 148.8, 147.1, 134.1, 126.9, 121.8, 108.9, 70.2, 67.4, 59.2, 58.4, 57.5, 53.3, 46.2,

ACCEPTED MANUSCRIPT
4
1.1, 35.7, 34.5, 30.8, 29.4, 28.2, 24.9, 24.9, 16.4.
(1R,5aS,6R,9aS)-1,5a-dimethyl-7-methylene-3-oxo-6-((E)-2-(2-oxo-2,5-dihydrofuran
3-yl)ethenyl)decahydro-1H-benzo[c]azepin-1-yl)methyl 5-((R)-1,2-dithiolan-3-yl)
(
-
pentanoate (compound 8h). Light yellow solid, 98% yield; HR-MS(ESI) m/z:
+
1
5
34.2336 [M+H] (Calcd. for C H NO S , 534.2342); H-NMR (600 MHz,
28 40 5 2
DMSO-d ): δ 7.67 (s, 1H, 15-H), 6.94 (s, 1H, 4-NH), 6.71 (dd, J = 15.8, 10.2 Hz, 1H,
6
1
2-H), 6.15 (d, J = 15.8 Hz, 1H, 13-H), 4.89 (s, 2H, 16-H), 4.78 (s, 1H, 18a-H), 4.47
s, 1H, 18b-H), 4.35 (d, J = 11.2 Hz, 1H, 20a-H), 3.98 (d, J = 11.2 Hz, 1H, 20b-H),
.59 (td, J = 12.5, 6.2 Hz, 1H), 3.18 (dt, J = 12.1, 6.2 Hz, 1H), 3.10 (dt, J = 11.0, 6.8
Hz, 1H), 2.74 (d, J = 10.1 Hz, 1H), 2.43 – 2.35 (m, 4H), 2.34 – 2.30 (m, 2H), 2.18 –
(
3
2
1
1
1
3
4
1
.05 (m, 2H), 1.90 – 1.80 (m, 2H), 1.73 – 1.68 (m, 1H), 1.57 – 1.51 (m, 3H), 1.50 –
.43 (m, 2H), 1.36 (dt, J = 21.2, 10.5 Hz, 3H), 1.23 (3H, s, 19-CH ), 1.16 – 1.09 (m,
3
13
H), 0.86 (3H, s, 21-CH ). C-NMR (150 MHz, DMSO-d ): δ 175.2, 172.5, 172.4,
3
6
48.8, 147.0, 134.1, 127.0, 121.8, 108.8, 70.2, 66.8, 58.3, 57.7, 56.0, 56.0, 53.5, 41.1,
9.8, 38.0, 35.8, 34.6, 34.0, 33.3, 30.9, 28.3, 28.0, 25.0, 16.3.
.2.8. Synthesis of compound 9
9-oxo-14-deoxy-11,12-didehydroandrographolide (compound 9). Compound 2 (3.0
g, 9.30 mmol) was dissolved in DCM (50 mL). Then saturated sodium bicarbonate
solution (20 mL) was added to the solution. The mixture was put into an ice bath to
cool down. Then NBS (3.2 g, 17.98 mmol), TEMPO (catalytic amount) and TBAB
(catalytic amount) were added to the mixture cautiously. The mixture was stirred for
1
2 h at 0 °C to room temperature. Upon completion, the aqueous layer was extracted

ACCEPTED MANUSCRIPT
with DCM for several times, then the combined organic layers were washed with
water and brine, and dried over anhydrous Na SO , respectively, filtered, evaporated,
2
4
and purified by CC to afford compound 9 as light yellow solid (73% yield).
+
1
HR-MS(ESI) m/z: 331.1902 [M+H] (Calcd. for C H O , 331.1904); H-NMR (600
20
27
4
MHz, CDCl ): δ 7.19 (s, 1H, 14-H), 6.87 (dd, J = 15.8, 10.1 Hz, 1H, 11-H), 6.14 (d, J
3
=
15.8 Hz, 1H, 12-H), 4.87 – 4.79 (m, 3H, 15-H, 17a-H), 4.58 (s, 1H, 17b-H), 3.29 –
.19 (m, 1H, 3-H), 2.57 – 2.49 (m, 1H), 2.36 (dd, J = 15.4, 7.1 Hz, 2H), 2.14 – 1.99
m, 3H), 1.93 – 1.76 (m, 3H), 1.73 – 1.56 (m, 2H), 1.38 (dd, J = 13.3, 2.7 Hz, 1H),
3
(
1
.29 (s, 3H, 18-CH ), 1.23 – 1.16 (m, 2H), 0.81 (s, 3H, 20-CH ).
3 3
4
.2.9. 19-oxime-14-deoxy-11,12-didehydroandrographolide (compound 10). Light
+
yellow solid, 94% yield; HR-MS(ESI) m/z: 346.2010 [M+H] (Calcd. for C H NO ,
20
28
4
1
3
46.2013); H-NMR (600 MHz, CDCl ): δ 7.21 (s, 1H, 14-H), 6.87 (dd, J = 15.8, 10.0
3
Hz, 1H, 11-H), 6.13 (d, J = 15.8 Hz, 1H, 12-H), 4.88 – 4.82 (m, 3H, 15-H, 17a-H),
4.60 (s, 1H, 17b-H), 3.28 – 3.22 (m, 1H, 3-H), 2.57 – 2.51 (m, 1H), 2.40 (ddd, J =
15.1, 13.0, 4.2 Hz, 1H), 2.32 (d, J = 10.0 Hz, 1H), 2.25 (dt, J = 15.1, 3.0 Hz, 1H),
2.19 – 2.03 (m, 3H), 1.88 (tdd, J = 10.8, 7.8, 3.2 Hz, 2H), 1.79 – 1.64 (m, 2H), 1.61
(
dt, J = 13.7, 3.4 Hz, 1H), 1.52 (s, 3H, 18-CH ), 1.28 – 1.13 (m, 3H), 1.12 (s, 3H,
3
2
4
2
0-CH3).
.2.10.N-((1S,2R,4aS,5R,8aS)-2-hydroxy-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-
,5-dihydrofuran-3-yl)ethenyl)decahydronaphthalen-1-yl)formamide (compound 11).
+
Yellow solid, 47% yield; HR-MS(ESI) m/z: 346.2011 [M+H] (Calcd. for C H NO ,
20
28
4
1
3
46.2013); H-NMR (600 MHz, DMSO-d ):
δ
7.68 (s, 1H, 14-H), 6.74 (dd, J = 15.9,
6

ACCEPTED MANUSCRIPT
10.1 Hz, 1H, 11-H), 6.13 (d, J = 15.9 Hz, 1H, 12-H), 4.89 (s, 2H, 15-H), 4.82 (s, 1H,
17a-H), 4.49 (s, 1H, 17b-H), 3.16 (d, J = 10.8 Hz, 1H, 3-H), 2.46 – 2.38 (m, 2H), 2.14
– 2.04 (m, 1H), 1.89 – 1.82 (m, 1H), 1.72 – 1.65 (m, 1H), 1.58 – 1.49 (m, 1H), 1.44
1
3
(
td, J = 12.6, 4.3 Hz, 1H), 1.38 (s, 3H, 18-CH ), 0.96 (s, 3H, 20-CH ). C-NMR (150
3 3
MHz, DMSO-d ): δ 172.4, 147.9, 147.0, 133.5, 127.0, 122.5, 121.6, 109.1, 74.2, 70.2,
6
5
9.2, 50.0, 42.5, 38.3, 36.8, 34.8, 28.4, 23.4, 23.1, 13.4.
4
.2.11. Compounds 12a-j
(1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-
dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl nicotinate (compound 12a).
+
White solid, 98% yield; HR-MS(ESI) m/z: 451.2223 [M+H] (Calcd. for C H N O ,
26
31
2
5
1
4
8
7
1
4
51.2227); H-NMR (600 MHz, DMSO-d ):
δ
9.11 (dd, J = 2.1, 0.7 Hz, 1H, 2'-H),
6
.85 (dd, J = 4.8, 1.7 Hz, 1H, 4'-H), 8.36 – 8.22 (m, 1H, 6'-H), 7.71 (s, 1H, 14-H),
.65 – 7.60 (m, 1H, 5'-H), 6.77 (dd, J = 15.8, 10.1 Hz, 1H, 11-H), 6.19 (d, J = 15.8 Hz,
H, 12-H), 4.90 (d, J = 1.4 Hz, 2H, 15-H), 4.86 (dt, J = 8.2, 4.6 Hz, 2H, 3-H, 17a-H),
.53 (d, J = 1.5 Hz, 1H, 17b-H), 2.20 – 2.09 (m, 1H), 2.00 – 1.90 (m, 3H), 1.75 (dd, J
=
12.6, 2.5 Hz, 3H), 1.63 – 1.56 (m, 1H), 1.55 – 1.45 (m, 3H), 1.41 (s, 3H, 18-CH3),
1
3
1
1
7
.22 (s, 2H), 1.03 (s, 3H, 20-CH ). C-NMR (150 MHz, DMSO-d ): δ 172.4, 163.7,
3
6
54.1, 150.0, 147.5, 147.3, 136.8, 133.1, 126.9, 125.1, 124.1, 121.9, 121.4, 109.4,
7.2, 70.2, 58.8, 49.6, 40.2, 38.1, 35.9, 34.6, 24.9, 23.1, 23.0, 13.2.
(1S,2R,4aS,5R,8aS)-1-formamido-1,4a-dimethyl-6-methylene-5-((E)-2-(2-oxo-2,5-
dihydrofuran-3-yl)ethenyl)decahydronaphthalen-2-yl benzoate (compound 12b).
+
White solid, 98% yield; HR-MS(ESI) m/z: 450.2271 [M+H] (Calcd. for C H NO ,
27
32
5