Preparation and α-glucosidase inhibition of andrographolide derivatives

Article abstract of DOI:10.1007/s00044-020-02612-6

Series of novel analogs, which were primarily modified on its lactone moiety, was synthesized based on Andrographolide (1), a natural product sesquiterpene inhibitor of α-glucosidase. Among new analogs, 14-deoxy-11,12-didehydro-15-(4-ethoxybenzylidene)andrographolide (3h) was determined to have the greatest potential of α-glucosidase inhibitor through the calculation of IC₅₀ value of 160 ± 5.1 μM, a significant improvement compared to the clinical dose of Acarbose, which showed the IC₅₀ value of 390 ± 8.1 μM. In addition, 14-deoxy-11,12-didehydro-3,19-(2′-hydroxybenzylidene)-15-(2-hydroxybenzylidene) andrographolide (7), a 15-benzylidene derivative of 14-deoxy-11,12-didehydroandrographolide containing a 1,3-dioxane moiety at C(3) and C(19), also displayed good inhibition with IC₅₀ 260 ± 13 μM. These results are promising avenues in the subsequent optimization of antidiabetic drugs.

Full text of DOI:10.1007/s00044-020-02612-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02612-6
ORIGINAL RESEARCH
Preparation and α-glucosidase inhibition of andrographolide
derivatives
1
Minh Huy Ly1,2 Tuyen Ngoc Truong1 Tuoi Thi Hong Do
●
●
Received: 11 May 2020 / Accepted: 1 August 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Series of novel analogs, which were primarily modified on its lactone moiety, was synthesized based on Andrographolide
(1), a natural product sesquiterpene inhibitor of α-glucosidase. Among new analogs, 14-deoxy-11,12-didehydro-15-(4-
ethoxybenzylidene)andrographolide (3h) was determined to have the greatest potential of α-glucosidase inhibitor through
the calculation of IC50 value of 160 5.1 μM, a significant improvement compared to the clinical dose of Acarbose, which
showed the IC50 value of 390 8.1 μM. In addition, 14-deoxy-11,12-didehydro-3,19-(2′-hydroxybenzylidene)-15-(2-
hydroxybenzylidene) andrographolide (7), a 15-benzylidene derivative of 14-deoxy-11,12-didehydroandrographolide
containing a 1,3-dioxane moiety at C(3) and C(19), also displayed good inhibition with IC50 260 13 μM. These results are
promising avenues in the subsequent optimization of antidiabetic drugs.
●
●
Keywords Andrographolide derivatives α-glucosidase inhibitors Diabetes
Introduction
(Fig. 1) is currently the only α-glucosidase inhibitor clini-
cally used to support type 2 diabetes patients (Rosak and
Mertes 2012).
Diabetes mellitus is one of the most widespread and pro-
blematic metabolic disorders globally. Maintaining optimal
blood glucose is one of the treatment objectives in type 2
diabetes therapy. Toward this end, α-glucosidases (amylase,
sucrase-isomaltase, and maltase-glucoamylase), found on
the luminal surface of enterocytes, contribute to increase
postprandial blood glucose and have emerged as potential
targets to diminish gastrointestinal glucose absorption
(Priebe et al. 2018). Therefore, there has been a high interest
in the optimization of α-glucosidase inhibitors. Acarbose
Andrographolide (Fig. 1), the major component in
Andrographis paniculate, is well known as a com-
plementary medicine to treatments of infections or inflam-
mations (Chao and Lin 2010). Andrographolide and its
derivatives possess several attractive biological activities,
such as antibacterial (Jiang et al. 2009), antiviral properties
(Wang et al. 2010; Uttekar Mayur et al. 2012), tumor
growth inhibition (Sirion et al. 2012; Wei et al. 2013), liver
protection and immunity booster (Kumar et al. 2004).
Regarding α-glucosidase inhibition, some of its derivatives
possess improved potency compared to acarbose, making
them potential candidates toward novel antidiabetic drugs
(Xu et al. 2007).
Previous structure–activity relationship (SAR) studies
have shown that the lactone moiety and the conjugated
double bond Δ12(13) played essential roles in biological
activity (Xu et al. 2010; Jitendra et al. 2012). Recent reports
indicate that introducing a silyl ether or triphenylmethyl
ether group into C(19) increased toxicity against cancer
cells (Jiang et al. 2011). In other studies, synthetic 14-
deoxy-11,12-didehydroandrographolide (2, Fig. 1) and its
derivatives exhibited high stability in both acidic and basic
medium (Wei et al. 2013). In terms of α-glucosidase inhi-
bition, derivatives of C(15)-isopropylidene and benzylidene
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02612-6) contains supplementary
material, which is available to authorized users.
* Minh Huy Ly
huyh2002@USherbrooke.ca
* Tuyen Ngoc Truong
truongtuyen@ump.edu.vn
1
Faculty of Pharmacy, University of Medicine and Pharmacy at Ho
Chi Minh city, 41 Dinh Tien Hoang, District 1, Ho Chi Minh city
700000, Vietnam
2
Université de Sherbrooke, Faculté des Sciences, 2500, boul. de
l’Université Sherbrooke, Sherbrooke, QC J1K 2R1, Canada

Medicinal Chemistry Research
Fig. 1 Structures of acarbose,
andrographolide (1) with
numbering and its derivatives
Scheme 1 Preparation of some derivatives of 15-isopropylidene and benzylidene-14-deoxy-11,12-didehydroandrographolide (3b–3j). Reagents
and conditions: (a) H2SO4, pyridine, 90 °C, 91%; (b) K2CO3 in MeOH, rt; (c) CH3COOH/pyrrolidine in MeOH, 50–60 °C
(3, Fig. 1) of 14-deoxy-11,12-didehydroandrographolide (2)
exhibited more potent inhibition against α-glucosidase than
andrographolide, with % inhibition determined at 100 μM
up to 100% (for compound 3a) (Xu et al. 2007). Together,
existing studies point to three groups as crucial for biolo-
gical activity: C(15), C(3), and C(19) (Wei et al. 2013; Xu
et al. 2010; Nanduri et al. 2004). Due to their potential
inhibition, many efforts have been made to develop pro-
cedures for the semisynthesis of these derivatives.
mixture of CH3COOH/pyrrolidine in MeOH. In the present
study, we report new syntheses of andrographolide deriva-
tives that enable further investigation of its SAR on the
inhibition of α-glucosidase.
Results and discussion
Chemistry
We recently reported a novel approach to the semi-
synthesis of andrographolide derivatives based on two
simple steps: dehydration and vinylogous aldol condensa-
tion (Ly Minh et al. 2019). Notably, in the preparation of
Firstly (Scheme 1), the conjugated dehydration of andro-
grapholide was conducted to prepare the intermediate 2.
Subsequently, synthesis of derivatives of 15-isopropylidene
and benzylidene-14-deoxy-11,12-didehydroandrographolide
(3b–3j) was reached via a reaction of 2 with different ben-
zaldehydes and ketones under suitable conditions.
14-deoxy-11,12-didehydro-andrographolide
(2),
con-
centrated sulfuric acid in pyridine was utilized as an alter-
native condition for Al2O3 in xylene (Kumar et al. 2004). In
the vinylogous aldol condensation, two new catalysts were
successfully implemented: (a) K2CO3 in MeOH and (b) a
Overall, nine derivatives of 14-deoxy-11,12-didehy-
droandrographolide (3b–3j) were successfully synthesized

Medicinal Chemistry Research
Scheme 2 Preparation of 3,19-
diacetyl-15-(4-clorobenzylidene)-
14-deoxy-11,12-
didehydroandrographolide (5).
Reagents and conditions: (a)
Ac2O, pyridine, ZnCl2, rt, 93%;
(b) 4-chlorobenzaldehyde,
CH3COOH/pyrrolidine in MeOH,
50–60 °C, 78%
Scheme 3 Preparation of dioxane and oxirane derivatives (6a–6c, 7, 8)
of andrographolide (1) and 14-deoxy-11,12-didehydroandrographolide
(2). Reagents and conditions: (a) R1COR2, pyridinium chloride,
CHCl3, rt; (b) salicylaldehyde, pyridinium chloride, CHCl3, rt, 78%;
(c) m-CBPA, 10 °C, CHCl3, 41%
by aldol-like condensation with different catalysts. As
reported in our previous study (Ly Minh et al. 2019),
K2CO3 in MeOH was favorable for the preparation of
3c–3j, while the mixture of CH3COOH and pyrrolidine in
MeOH was used to prepare 3b. In addition, the acetylated
form of 15-(4-chlorobenzylidene)-14-deoxy-11,12-didehy-
droandrographolide (5) was prepared following Scheme 2.
The structures of 2, 3b–3j, and 5 were elucidated based
on their IR, NMR, and HRMS spectra. According to pre-
vious studies, the stereoisomer of double bond Δ11(12) was
assumed to be E based on the coupling constant JH-11,H-12
(15.6 Hz) and the relative geometry of the new exocyclic
double bond of the lactone has been identified as Z isomer
due to the interaction between H(14) and the vinylic proton
in NOE spectrum (Xu et al. 2007). In order to obtain more
insight into the SAR study at C(3), C(19), and the double
bond Δ8(17), dioxane and oxirane derivatives of andro-
grapholide (1) and 14-deoxy-11,12-didehydroandro-
grapholide (2), respectively were prepared (Scheme 3) in
good yields.
Evaluation of α-glucosidase inhibition activity
With these derivatives in hand, their inhibition of α-gluco-
sidase was investigated as described previously (Xu et al.
2007). Briefly, compounds dissolved in DMSO as stock
solutions (1 mM) were diluted in buffer (pH 6.8) to obtain
100-μM samples. These samples were incubated in the
presence of α-glucosidase for 20 min, then the enzyme
substrate p-NPG (p-nitrophenyl-α-D-glucopyranoside) was

Medicinal Chemistry Research
Table 1 α-Glucosidase inhibition activity of andrographolide analogs
α-Glucosidase inhibition activitya (IC50 μM)
Weak inhibition by 3i suggests that rigid groups, such as
conjugated aryl groups, are favorable for enzyme–inhibitor
interactions. In particular, 3h, with one ethoxy group
(–OC2H5) at the para position, shows the most potent
inhibition; this result is also accordant with a previous 3D
SAR analysis which stated that the introducing of the
hydrophobic group in particular positions, such as C(3), C
(15), and C(19) of phenyl ring could enhance the activity
(Jitendra et al. 2012). Interestingly, the transformation from
3c to 7, in which the addition of one more hydroxyl group
activated the non-active compound (3c), indicated that the
1,3-dioxane ring at C(3) and C(19) with proper hydrogen
donating groups might contribute to the promotion of
inhibition activity.
Compound
α-Glucosidase
1
9.47 6.27%
N/Ab
2
3b
3c
3d
3e
3f
3g
3h
3i
3j
4
N/A
N/A
N/A
N/A
N/A
N/A
36.6 3.63% (160 5.1)
5.84 0.09%
N/A
N/A
Conclusion
5
N/A
6a
6b
6c
7
N/A
In summary, 17 derivatives of andrographolide were synthe-
sized in medium-to-good yields. Furthermore, selected com-
pounds were evaluated for in vitro α-glucosidase inhibitions.
Compound structures were characterized by IR, NMR, and
HRMS spectra. Among newly synthesized compounds, 3h
and 7 exhibit good α-glucosidase inhibition, which will be
useful for further development of this class as anti-diabetes
drugs. Particularly, 14-deoxy-11,12-didehydro-15-(4-ethox-
ybenzylidene) andrographolide (3h) is the most potent inhi-
bitor of α-glucosidase, with an IC50 value of 160 5.1 μM.
This is about 2.5 times more potent than the clinically used
acarbose (IC50 390 8.1 μM) in the same assay. Moreover,
good inhibition of 14-deoxy-11,12-didehydro-3,19-(2′-hydro-
xybenzylidene)-15-(2-hydroxybenzylidene) andrographolide
(7, IC50 260 13 μM), shows an interesting SAR.
N/A
N/A
23.3 0.41% (260 13)
N/A
8
Acarbose was taken as positive control. The inhibition percentage of 1-
mM acarbose was 75.3 0.23% (IC50 = 390 8.1 μM).
N/A not active at 100 μM.
a% Inhibition determined at 100-μM concentration of test compound.
added and incubated for another 20 min, using acarbose as a
reference. The reaction was quenched using Na2CO3 0.2 M
(pH 9.8) and % inhibition was evaluated based on p-NPG
absorbance at 405 nm. The results are reported in Table 1.
Compounds showing more than 20% inhibition at 100 μM
were tested in full concentration–response curves to deter-
mine their IC50.
In vitro α-glucosidase inhibition of the above derivatives
showed that 1, 3h, 3i and 7 inhibit α-glucosidase, while the
others showed no inhibition at 100 μM (Table 1). In parti-
cular, the compounds 3h and 7 gave the most promising
results.
Andrographolide and acarbose inhibited α-glucosidase
by 9.47 6.27% at 100 μM and 75.3 0.23% at 1 mM.
Among the newly tested derivatives, 3h and 7 emerge as the
most potent inhibitors at 100 μM. Their IC50 values were
determined using full concentration–response curves. IC50
values of 3h and 7 were found to be 160 5.1 μM and
260 13 μM, respectively. These IC50 values are lower than
that of acarbose (IC50 = 390 8.1 μM) in the same assay.
Moreover, based on the structures of the new active
compounds against α-glucosidase at 100 μM (3h, 3i, and 7),
the addition of lipophilic groups and the removal of
hydrogen acceptors enhance inhibition of α-glucosidase.
Materials and methods
Materials
All the reactions were monitored by TLC analysis (pre-
coated silica gel plates with fluorescent indicator UV 254,
0.2 nm) and visualized with 254- and 365-nm UV light.
Andrographolide was extracted by the procedure of
Faculty of Pharmacy, University of Medicine and Phar-
macy at Ho Chi Minh city. Other chemicals were pur-
chased from Sigma-Aldrich, Merck (Germany), Spain,
China. NMR spectra were recorded on Bruker Avance 500
NMR Spectrometer (Ha Noi, Vietnam). High-resolution
mass spectrometry was carried out on 1100 series LC/MS/
MS Trap Agilent and LC–MS Shimadzu. IR spectra were
recorded as ATR method on an IRAFFINITY-1S Shi-
madzu Spectrometer.

Medicinal Chemistry Research
Specific chemical transformation
deoxy-11,12-didehydroandrographolide
(2)
(332 mg,
1 mmol) was dissolved and the reaction was slightly heated
up to 50–60 °C. After 24 h, the reaction was quenched with
distilled water (20 mL) and saturated NaCl solution (20 mL)
then cooled down to afford a crude product as a yellow
solid. It was then dissolved into ethyl acetate (30 mL). The
organic phase was washed with water successively and
NaHCO3 10% solution (20 mL × 2), then removed. The
crude product was purified by silica chromatography using
an eluent chloroform–acetone = 5:1 to afford corresponding
products 3.
Procedure for preparation of 14-deoxy-11,12-didehy-
droandrographolide (2). Sulfuric acid 98% (0.25 mL) was
added into pyridine (10 mL) followed by the addition of
andrographolide (3.15 g, 9 mmol). The mixture was stirred
at 85–95 °C for 5–6 h. After the completion of reaction by
TLC analysis, a yellow-brown crude product was obtained
by slowly adding HCl 2-M solution (70 mL) into the reac-
tion mixture and then cooling down. The crude product was
purified by silica gel chromatography using an eluent
chloroform–acetone = 5:1 to afford colorless corresponding
product 2. Yield 90.4%, mp. 209–210 °C. IR (ATR), ν
(cm−1): 3294 (νO–H), 2933–2850 (νC–H), 1735 (νC=O), 1637
*Catalyst solution: a mixture of MeOH–CH3COOH–pyrrolidine
=25:4:1 by volume. 14-Deoxy-11,12-didehydro-15-(4-nitrobenzyli-
dene)andrographolide (3b). Yield 46%, mp. 199–201 °C. IR
(ATR), ν (cm−1): 3366 (νO–H), 3082 (νAr–H), 2926–2849(νC–
H), 1751 (νC=O), 1591 (νC=C), 1518 (νN–O), 1337 (νC–N),
1
(νC=C), 1350 (νC–Oester). H-NMR (500 MHz, DMSO-d6, δ
ppm): 7.63 (s, 1H, H-14), 6.74 (dd, 1H, J = 15.5, 10.0 Hz,
H-11), 6.12 (d, 1H, J = 15.5 Hz, H-12), 5.03 (d, 1H, J =
5.0 Hz, OH-3), 4.88 (s, 2H, H-15), 4.73 (s, 1H, H-17a), 4.42
(s, 1H, H-17b), 4.14 (dd, 1H, J = 7.5, 2.5 Hz, OH-19), 3.86
(d, 1H, J = 11.0 Hz, H-19a), 3.28 (dd, 1H, J = 11.0, 7.5 Hz,
H-19b), 3.22 (dt, 1H, J = 10.5, 5.0 Hz, H-3), 2.36 (d, 2H, J =
10.0 Hz, H-9, H-7a), 1.98 (dd, 1H, J = 13.0, 9.0 Hz, H-7b),
1.72 (d, 1H, J = 11.0 Hz, H-1a), 1.58 (m, 2H, H-6), 1.42 (td,
1H, J = 8.0, 4.0 Hz, H-1b), 1.33 (d, 1H, J = 13.5 Hz, H-2a),
1.18 (d, 1H, J = 12.5 Hz, H-5), 1.13 (dd, 1H, J = 8.0,
4.0 Hz, H-2b), 1.09 (s, 3H, H-20), 0.76 (s, 3H, H-18). 13C-
NMR (125 MHz, MeOD, δ ppm): 174.8, 150.1, 146.7,
136.5, 129.6, 122.5, 109.1, 81.2, 71.6, 65.0, 62.8, 55.8,
43.8, 39.6, 39.5, 37.8, 28.9, 24.4, 23.3, 16.3. HRMS m/z:
[M + Na]+ 355.1876 (calcd 355.1887).
1
1107 (νC–Oester), 750 (νAr–H). H-NMR (500 MHz, MeOD-
d4, δ ppm): 8.28 (d, 2H, J = 9.0 Hz, Ar–H-3′, Ar–H-5′),
8.00 (d, 1H, J = 9.0 Hz, Ar–H-2′, Ar–H-6′), 7.51 (s, 1H,
H-14), 7.03 (dd, 1H, J = 16.0, 10.0 Hz, H-11), 6.32 (m, 2H,
H-12, H-21), 4.81 (s, 1H, H-17a) 4.53 (d, 1H, J = 1.5 Hz,
H-17b), 4.16 (d, 1H, J = 11.5 Hz, H-3), 3.43 (d, 1H, J =
11.5 Hz, H-19a), 3.41 (d, 1H, J = 11.0 Hz, H-19b), 1.25 (s,
1H, H-20), 0.91 (s, 3H, H-18). 13C-NMR (125 MHz,
DMSO-d6, δ ppm): 168.1, 149.9, 148.7, 146.4, 139.8,
138.4, 136.6, 130.8, 130.2, 127.7, 124.0, 123.9, 121.2,
110.1, 108.23, 78.6, 62.6, 60.8, 53.7, 42.4, 38.5, 38.0, 36.2,
27.6, 23.1, 23.0, 15.4. HRMS m/z: [M − H]− 464.1878
(calcd 464.2073).
14-Deoxy-11,12-didehydro-15-(2-hydroxybenzylidene)
andrographolide (3c). A mixture of Z and E isomers (7:3),
yield 51%, mp. 175–176 °C. IR (ATR), ν (cm−1): 3321
(νO–H), 2986–2928 (νC–H), 1732 (νC=O), 1645–1601 (νC=C),
1259 (νC–Oester), 1030 (νC–O), 750 (νAr–H). 1H-NMR
(500 MHz, DMSO-d6, δ ppm): 10.15 (s, 0.7H. Ar–OH),
10.03 (s, 0.3H, Ar–OH), 8.03 (s, 0.3H, H-14), 7.90 (dd,
0.7H, J = 8.5, 2.0 Hz, Ar–H-6′), 7.78 (s, 0.7H, H-14), 7.46
(dd, 0.3H, J = 8.0, 1.5 Hz, Ar–H-6′), 7.19 (m, 1H, Ar–H-
4′), 6.90 (m, 2H, Ar–H3′, Ar–H-5′), 6.87 (s, 0.3H, H-21),
6.80 (dd, 1H, J = 15.5, 10.0 Hz, H-11), 6.57 (s, 0.7H, H-
21), 6.26 (d, 0.3H, J = 16.0 Hz, H-12), 6.23 (d, 0.7H, J =
16.0 Hz, H-12), 5.03 (d, 1H, J = 5.0 Hz, OH-3), 4.75 (s, 1H,
H-17a), 4.45 (s, 1H, H-17b), 4.14 (dd, 1H, J = 7.5, 3.0 Hz,
OH-19), 3.86 (d, 1H, J = 11.0 Hz, H-19a), 3.23 (dt, 1H, J =
10.5, 5.0 Hz, H-3), 2.43 (d, 1H, J = 10.0 Hz, H-9), 1.10 (s,
3H, H-20), 0.79 (s, 3H, H-18). 13C-NMR (125 MHz,
DMSO-d6, δ ppm): 167.9, 156.1, 148.8, 147.9, 146.8,
137.8, 136.3, 132.2, 128.8, 125.1, 121.5, 119.6, 115.6,
110.6, 108.3, 78.9, 62.7, 60.9, 53.7, 42.4, 38.5, 38.0, 36.2,
27.6, 23.2, 23.0, 15.5. HRMS m/z: [M − H]− 435.2173
(calcd 435.2173).
General procedure for preparation of 15-alkylidene and
benzylidene derivatives of 14-deoxy-11,12-didehydroan-
drographolide (3b–3j) and 15-(4-chlorobenzylidene)-3,19-
diacetyl-14-deoxy-11,12-didehydroandrographolide (5)
General procedure for catalyst K2CO3 in MeOH. To a
solution of 14-Deoxy-11,12-didehydro-andrographolide (2)
(332 mg, 1 mmol) and K2CO3 (100 mg, 0.72 mmol) in
methanol (10 mL) was slowly added benzaldehyde or
ketone derivatives (5 mmol). The mixture was stirred at
room temperature for 1–3 h. After the completion of reac-
tion by TLC analysis, the reaction was quenched with dis-
tilled water (20 mL) and saturated NaCl solution (20 mL)
then cooled down to afford a crude product as a yellow
solid, which was then dissolved into ethyl acetate (30 mL).
The organic phase was washed with water successively then
removed. The crude product was purified by silica chro-
matography using an eluent chloroform–acetone = 5:1 to
afford corresponding products 3.
General procedure for catalyst acetic acid-pyrrolidine in
MeOH. To a 100 mL round bottom flask was added the
catalyst solution* (10 mL) and a benzaldehyde or ketone
derivative (5 mmol). The mixture was stirred at room tem-
perature until it turned intense yellow or red. Then, 14-
14-Deoxy-11,12-didehydro-15-(4-hydroxybenzylidene)
andrographolide (3d). Yield 50%, mp. 267–268 °C. IR

Medicinal Chemistry Research
(ATR), ν (cm−1): 3451 (νO–H), 3084 (νAr–H), 2924–2849
(νC–H), 1740 (νC=O), 1607 (νC=C), 1173 (νC–Oester), 903
(νAr–H). 1H-NMR (500 MHz, DMSO-d6, δ ppm): 7.67
(s, 1H, H-14), 7.61 (d, 2H, J = 8.5 Hz, Ar–H-2′, Ar–H-6′),
6.82 (d, 2H, J = 9.0 Hz, Ar–H-3′, Ar–H-5′), 6.78 (dd, 1H,
J = 15.5, 10.0 Hz, H-11), 6.24 (d, 1H, J = 16.0 Hz, H-12),
6.23 (s, 1H, H-21), 5.03 (d, 1H, J = 5.0 Hz, OH-3), 4.75 (s,
1H, H-17a), 4.44 (s, 1H, H-17b), 4.14 (dd, 1H, J = 7.5,
3.0 Hz, OH-19), 3.85 (dd, 1H, J = 10.5, 2.5 Hz, H-19a),
2.42 (d, 1H, J = 10.5 Hz, H-9), 1.10 (s, 3H, H-20), 0.78 (s,
3H, H-18). 13C-NMR (125 MHz, DMSO-d6, δ ppm): 168.7,
158.5, 148.9, 145.4, 137.2, 135.8, 132.2, 124.44, 124.37,
121.4, 116.0, 113.6, 108.1, 78.6, 62.6, 60.8, 53.7, 42.4,
38.4, 38.0, 36.2, 27.6, 23.1, 23.0, 15.4. HRMS m/z: [M −
H]− 435.2067 (calcd 435.2173).
14-Deoxy-11,12-didehydro-15-(3,4-dimethoxybenzylidene)
andrographolide (3g). Yield 48%, mp. 133–134 °C. IR
(ATR), ν (cm−1): 3377 (νO–H), 2930–2849 (νC–H), 1746
(νC=O), 1643 (νC=C), 1263 (νC–Oester), 1022 (νC–O), 887
(νAr–H). 1H-NMR (500 MHz, DMSO-d6, δ ppm): 7.71 (s, 1H,
H-14), 7.36 (m, 2H, Ar–H-2′, Ar-H-6′), 7.05 (d, 1H, J =
9.0 Hz, Ar–H-5′), 6.79 (dd, 1H, J = 16.0, 10.0 Hz, H-11),
6.27 (s, 1H, H-21), 6.24 (d, 1H, J = 15.5 Hz, H-12), 5.04 (d,
1H, J = 5.0 Hz, OH-3), 4.75 (s, 1H, H-17a), 4.45 (s, 1H, H-
17b), 4.14 (dd, 1H, J = 7.5, 2.5 Hz, OH-19), 3.85 (d, 1H, J =
11.0 Hz, H-19a), 3.81 (s, 3H, Ar–OCH3), 3.79 (s, 3H,
Ar–OCH3), 3.22 (m, 2H, H-3, H-19b), 2.42 (m, 2H, H-9, H-
7a), 1.10 (s, 3H, H-20), 0.79 (s, 3H, H-18). 13C-NMR
(125 MHz, DMSO-d6, δ ppm): 168.7, 149.8, 148.9, 148.7,
146.1, 137.1, 136.2, 126.1, 124.9, 124.1, 121.4, 113.4, 113.3,
112.0, 108.2, 78.6, 62.7, 60.8, 55.6, 55.5, 53.7, 42.4, 38.5,
38.0, 36.2, 27.6, 23.1, 23.0, 15.5. HRMS m/z: [M − H]−
479.2401 (calcd 479.2435), [M + Na]+ 503.2707 (calcd
503.2411).
14-Deoxy-11,12-didehydro-15-(3,4-methylenediox-
ybenzylidene)andrographolide (3e). Yield 60%, mp.
202–203 °C. IR (ATR), ν (cm−1): 3402 (νO–H), 2943–2845
(νC–H), 1732 (νC=O), 1699 (νC=C), 1263 (νC–Oester), 1082
(νC–O), 804 (νAr–H). 1H-NMR (500 MHz, MeOD-d4, δ ppm):
7.46 (s, 1H, H-14), 7.14 (d, 1H, J = 8.0 Hz, Ar–H-6′), 7.08
(s, 1H, Ar-H-2′), 6.88 (dd, 1H, J = 16.0, 10.0 Hz, H-11),
6.82 (d, 1H, J = 8.0 Hz, Ar-H-5′), 6.19 (d, 1H, J = 16.0 Hz,
H-12), 6.01 (s, 2H, O–CH2-O), 5.88 (s, 1H, H-21), 4.80 (s,
1H, H-17a), 4.54 (s, 1H, H-17b), 4.22 (d, 1H, J = 11.0 Hz,
H-19a), 3.49 (dd, 1H, J = 11.5, 4.5 Hz, H-3), 3.36 (d, 1H,
J = 11.0 Hz, H-19b), 1.27 (s, 1H, H-20), 0.84 (s, 3H, H-18).
13C-NMR (125 MHz, DMSO-d6, δ ppm): 169.0, 149.4,
148.5, 148.3, 146.7, 137.6, 136.9, 128.0, 126.2, 125.7,
121.9, 113.5, 109.6, 109.4, 108.7, 102.1, 79.1, 63.2, 61.3,
54.2, 42.9, 39.0, 38.5, 36.7, 28.1, 23.7, 23.5, 16.0. HRMS
m/z: [M + Na]+ 487.1732 (calcd 487.2097).
14-Deoxy-11,12-didehydro-15-(3-hydroxy-4-methox-
ybenzylidene)andrographolide (3f). Yield 52%, mp.
159–160 °C. IR (ATR), ν (cm−1): 3362 (νO–H), 3080 (νAr–H),
2918–2849 (νC–H), 1740 (νC=O), 1609 (νC=C), 1258 (νC–
Oester), 1130 (νC–O), 885 (νAr–H). 1H-NMR (500 MHz,
DMSO-d6, δ ppm): 9.28 (s, 1H, Ar–OH), 7.66 (s, 1H, H-
14), 7.33 (d, 1H, J = 2.0 Hz, H-2′), 7.11 (dd, 1H, J = 8.5,
2.0 Hz, H-6′), 6.98 (d, 1H, J = 8.5 Hz, H-5′), 6.78 (dd, 1H,
J = 16.0, 10.0 Hz, H-11), 6.25 (d, 1H, J = 16.0 Hz, H-12),
6.19 (s, 1H, H-21), 5.03 (d, 1H, J = 5.0 Hz, OH-3), 4.75 (s,
1H, H-17a), 4.44 (s, 1H, H-17b), 4.14 (dd, 1H, J = 7.5,
3.0 Hz, OH-19), 3.85 (d, 1H, J = 11.0 Hz, H-19a), 3.81 (s,
3H, Ar–OCH3), 3.28–3.22 (m, 2H, H-19b, H-3), 1.10 (s,
3H, H-20), 0.78 (s, 3H, H-18). 13C-NMR (125 MHz,
DMSO-d6, δ ppm): 168.7, 148.9, 148.8, 146.6, 145.8,
137.2, 136.0, 126.2, 124.6, 123.1, 121.4, 116.4, 113.6,
112.2, 108.1, 78.6, 62.6, 60.8, 55.5, 53.7, 42.4, 38.4, 38.0,
36.2, 27.6, 23.1, 23.0, 15.4. HRMS m/z: [M − H]−
465.2138 (calcd 465.2279), [M + H]+ 467.1835 (calcd
467.2435), [M + Na]+ 489.2098 (calcd 489.2255), [M]−
466.2150 (calcd 466.2357).
14-Deoxy-11,12-didehydro-15-(4-ethoxybenzylidene)
andrographolide (3h). A mixture of Z and E isomers (2:1),
yield 53%, mp. 200–201 °C. IR (ATR), ν (cm−1): 3281
(νO–H), 3076 (νAr–H), 2972–2849 (νC–H), 1744 (νC=O), 1603
1
(νC=C), 1248 (νC–Oester), 1034 (νC–O), 829 (νAr–H). H-NMR
(500 MHz, MeOD-d4, δ ppm): 7.91 (s, 1H, H-14), 7.82 (s,
0.5H, H-14), 7.74 (d, 2H, J = 8.5 Hz, Ar–H-2′, Ar–H-6′),
7.45 (d, 1H, J = 9.0 Hz, Ar–H-2′, Ar–H-6′), 7.03 (dd, 0.5H,
J = 16.0, 10.0 Hz, H-11), 6.96 (d, 3H, J = 8.5 Hz, Ar–H-3′,
Ar–H-5′), 6.92 (dd, 1H, J = 16.0, 10.0 Hz, H-11), 6.71 (s,
0.5H, H-21), 6.32 (d, 0.5H, J = 16.0 Hz, H-12), 6.27 (d, 1H,
J= 15.5 Hz, H-12), 6.15 (s, 1H, H-21), 4.79 (s, 1.5H, H-
17a), 4.54 (s, 1.5H, H-17b), 4.16 (d, 1,5H, J = 11.0 Hz, H-
3), 4.11 (m, 3H, Ar–O–CH2CH3), 3.41 (d, 3H, J = 11.5 Hz,
H-19a, H-19b), 1.42 (t, 4.5H, J = 7.0 Hz, Ar–OCH2–CH3),
1.25 (s, 4.5H, H-20), 0.89 (s, 4.5H, H-18). 13C-NMR
(125 MHz, DMSO-d6, δ ppm): 168.6, 159.1, 148.8, 145.9,
137.1, 136.1, 131.9, 130.9, 124.8, 121.4, 115.0, 113.0,
108.1, 78.6, 63.2, 62.6, 60.8, 53.7, 42.4, 38.5, 38.0, 36.2,
27.6, 23.1, 23.0, 15.4, 14.5. HRMS m/z: [M + Na]+
487.2563 (calcd 487.2462), [M + K]+ 503.2048 (calcd
503.2048).
14-Deoxy-11,12-didehydro-15-(4-methylcyclohex-
ylidene)andrographolide (3i). Yield 55%, mp. 175–176 °C.
IR (ATR), ν (cm−1): 3285 (νO–H), 2926–2851 (νC–H), 1742
1
(νC=O), 1643 (νC=C), 1259 (νC–Oester). H-NMR (500 MHz,
MeOD-d4, δ ppm): 7.67 (s, 1H, H-14), 6.88 (dd, 1H, J =
15.5, 10.0 Hz, H-11), 6.22 (d, 1H, J = 16.0 Hz, H-12), 4.78
(s, 1H, H-17a), 4.53 (s, 1H, H-17b), 4.15 (d, 1H, J =
11.0 Hz, H-3), 3.40 (d, 2H, J = 11.0 Hz, H-19a,b), 3.03 (d,
1H, J = 14.0 Hz, cyclo-H), 2.76 (d, 1H, J = 14.0 Hz,
cyclo-H), 2.47 (d, 1H, J = 13.5 Hz, H-7a), 2.42 (d, 1H, J =
10.0 Hz, H-9), 2.18 (td, 1H, J = 13.5, 3.0 Hz, cyclo-H),
2.10 (td, 2H, J = 13.0, 3.0 Hz, cyclo-H), 1.94 (t, 2H, J =

Medicinal Chemistry Research
15.0 Hz, cyclo-H), 1.71 (m, 2H, cyclo-H), 1.25 (s, 3H, H-
20), 0.97 (d, 3H, J = 6.5 Hz, cyclo-CH3), 0.87 (s, 3H, H-
18). 13C-NMR (125 MHz, DMSO-d6, δ ppm): 168.7, 148.9,
142.1, 135.7, 132.2, 129.9, 126.0, 121.3, 108.1, 89.1, 78.6,
62.6, 60.7, 53.7, 42.4, 38.4, 37.9, 36.2, 35.6, 34.9, 34.1,
31.5, 27.6, 23.1, 23.0, 21.5, 15.4. HRMS m/z: [M + Na]+
449.2558 (calcd 449.2670), [M + K]+ 465.2137 (calcd
465.2409).
reached 7–8. The precipitate formed was filtered and rinsed
with 20 mL cool ethanol 96% solution. The crude product
was purified by silica chromatography using an eluent
petroleum ether–chloroform–acetone = 4:5:1 to afford the
pure product 4. Yield 93%, mp. 136–137 °C. IR (ATR), ν
(cm−1): 2986–2934 (νC–H), 1748–1726 (νC=O), 1643 (νC=C),
1246 (νC–Oester). 1H-NMR (500 MHz, DMSO-d6, δ ppm):
7.67 (s, 1H, H-14), 6.76 (dd, 1H, J = 16.0, 10.0 Hz, H-11),
6.16 (d, 1H, J = 16.0 Hz, H-12), 4.89 (s, 2H, H-15), 4.77 (s,
1H, H-17a), 4.53 (dd, 1H, J = 12.0, 4.0 Hz, H-3), 4.46 (s,
1H, H-17b), 4.35 (d, 1H, J = 11.5 Hz, H-19a), 4.02 (d, 1H,
J = 12.0 Hz, H-19b), 2.01 (s, 3H, OCOCH3), 1.99 (s, 3H,
OCOCH3), 1.09 (s, 3H, H-20), 0.76 (s, 3H, H-18). 13C-
NMR (125 MHz, DMSO-d6, δ ppm): 172.4, 170.2, 169.8,
148.4, 146.9, 133.7, 127.1, 121.5, 108.3, 79.2, 70.2, 63.7,
60.0, 53.2, 41.0, 38.1, 37.5, 36.0, 23.7, 23.3, 22.2,
20.8, 20.7, 14.8. HRMS m/z: [M − H]− 415.2077
(calcd 415.2122), [M + Na]+ 439.2080 (calcd 439.2098),
[M + K]+ 455.1846 (calcd 455.1837).
14-Deoxy-11,12-didehydro-15-(2,4-dimethoxybenzylidene)
andrographolide (3j). Yield 66%, mp. 144–145 °C. IR
(ATR), ν (cm−1): 3360 (νO–H), 3076 (νAr–H), 2928– 2849 (νC–
H), 1744 (νC=O), 1603 (νC=C), 1267 (νC–Oester), 1030 (νC–O),
889 (νAr–H). 1H-NMR (500 MHz, DMSO-d6, δ ppm): 7.92 (d,
1H, J = 8.5 Hz, Ar–H-6′), 7.74 (s, 1H, H-14), 6.77 (dd, 1H, J
= 16.0, 10.0 Hz, H-11), 6.68 (dd, 1H, J = 9.0, 2.5 Hz, Ar–H-
5′), 6.63 (m, 1H, Ar–H-3′), 6.48 (s, 1H, H-21), 6.23 (d, 1H, J
= 16.0 Hz, H-12), 5.04 (d, 1H, J = 5.0 Hz, OH-3), 4.75 (s,
1H, H-17a), 4.44 (s, 1H, H-17b), 4.14 (dd, 1H,
J = 7.5, 2.5 Hz, OH-19), 3.86 (s, 3H, Ar–OCH3), 3.83 (d, 1H,
J = 10.0 Hz, H-19a), 3.82 (s, 3H, Ar–OCH3), 2.40 (m, 2H, H-
9, H-7a), 1.10 (s, 3H, H-20), 0.79 (s, 3H, H-18). 13C-NMR
(125 MHz, DMSO-d6, δ ppm): 168.7, 161.6, 158.7, 148.9,
145.9, 137.5, 131.6, 130.5, 128.3, 124.3, 121.5, 114.7, 108.1,
106.7, 98.2, 79.1, 78.6, 62.7, 55.8, 55.4, 53.7, 42.4, 38.5, 38.0,
36.2, 27.6, 23.1, 23.0, 15.5. HRMS m/z: [M − H]− 479.2358
(calcd 479.2435), [M + Na]+ 503.2497 (calcd 503.2411).
3,19-Diacetyl-15-(4-clorobenzylidene)-14-deoxy-11,12-
didehydroandrographolide (5). Yield 78%, mp.
158–159 °C. IR (ATR), ν (cm−1): ν (cm−1): 3082 (νO–H),
2922–2853 (νC–H), 1734 (νC=O), 1674 (νC=C), 1236 (νC–Oester),
General procedure for preparation of 3,19-iso-
propylideneandrographolide (6a–6c) and 14-deoxy-11,12-
didehydro-3,19-(2′-hydroxybenzylidene)-15-(2-hydro-
xybenzylidene) andrographolide (7). In 100 mL round bot-
tom flask, andrographolide (350 mg, 1 mmol) was dispersed
into a solution of chloroform (20 mL) and a benzaldehyde
derivative (5 mmol). To the stirred suspension was added
pyridinium chloride at 40– 50 °C for 1–3 h. After the com-
pletion of reaction by TLC analysis, the solution was dried
by sufficient Na2SO4 and filtered. The solvent was removed
to obtain a crude product which was then purified by silica
chromatography using an eluent chloroform–acetone = 5:1.
3,19-(3-Hydroxy-4-methoxybenzylidene) andrographolide
(6a). Yield 66%, mp. 239–240 °C. IR (ATR), ν (cm−1): 3402
(νO–H), 3048 (νAr–H), 2967–2837 (νC–H), 1717(νC=O), 1661
1
841 (νAr–H), 770 (νC–Cl). H-NMR (500 MHz, DMSO-d6, δ
ppm): 7.77 (d, 2H, J = 8.5 Hz, Ar–H-2′, Ar–H-6′), 7.76 (s,
1H, H-14), 7.51 (d, 2H, J = 8.5 Hz, Ar–H-3′, Ar–H-5′), 6.85
(dd, 1H, J = 16.0, 10.0 Hz, H-11), 6.35 (s, 1H, H-21), 6.31
(d, 1H, J = 16.0 Hz, H-12), 4.79 (s, 1H, H-17a), 4.56 (dd,
1H, J = 12.0, 4.5 Hz, H-3), 4.48 (s, 1H, H-17b), 4.36 (d, 1H,
J = 11.5 Hz, H-19a), 4.03 (d, 1H, J = 12.0 Hz, H-19b), 2.01
(s, 3H, OCOCH3), 1.99 (s, 3H, OCOCH3), 0.98 (s, 3H, H-
20), 0.83 (s, 3H, H-18). 13C-NMR (125 MHz, DMSO-d6, δ
ppm): 170.2, 169.8, 168.3, 148.3, 147.8, 137.1, 136.7, 133.3,
132.2, 131.7, 130.9, 129.0, 126.4, 121.6, 111.4, 108.5, 79.1,
63.7, 60.2, 53.2, 41.0, 38.4, 37.5, 36.0, 23.7, 23.3, 22.2, 20.8,
14.9. HRMS m/z: [M − H]− 537.1948 (calcd 537.2045), [M
+ Na]+ 561.2007 (calcd 561.2021).
Procedure for preparation of 3,19-diacetyl-14-deoxy-
11,12-didehydroandrographolide (4). To A stirred suspen-
sion of zinc chloride (150 mg) in acetic anhydride (20 mL,
0.21 mol) was added 14-deoxy-11,12-didehydroandro-
grapholide (3.32 g, 10 mmol) at 0 °C in 6 h. After the
completion of reaction by TLC analysis, the reaction mix-
ture was quenched with cool water (50 mL). To the strongly
stirred aqueous solution was slowly added sodium bicar-
bonate until no longer effervescence and solution pH
1
(νC=C), 1279–1231 (νC–Oester), 1026 (νC–O), 795 (νAr–H). H-
NMR (500 MHz, MeOD-d4, δ ppm): 6.97 (s, 1H, Ar–H-2′),
6.91 (m, 2H, Ar–H-5′, Ar–H-6′), 6.90 (m, 1H, H-12), 5.75 (s,
1H, H-21), 5.05 (d, 1H, J = 6.0 Hz, H-14), 4.94 (s, 1H, H-
17a), 4.73 (s, 1H, H-17b), 4.50 (dd, 1H, J = 10.0, 6.0 Hz, H-
15a), 4.34 (d, J = 11.5, H-19a), 4.19 (dd, 1H, J = 10.0,
1.5 Hz, H-15b), 3.86 (s, 3H, C4′–OCH3), 3.66 (dd, J = 13.0,
5.0 Hz, H-3), 3.58 (d, 1H, J = 11.5 Hz, H-19b), 1.48 (s, 3H,
H-20), 0.93 (s, 3H, H-18). 13C-NMR (125 MHz, DMSO-d6,
δ ppm): 169.9, 147.7, 147.2, 146.1, 146.0, 132.2, 129.0,
117.2, 113.6, 111.4, 108.7, 94.2, 79.7, 74.3, 68.3, 64.5, 55.6,
55.1, 53.6, 38.4, 37.0, 36.3, 35.2, 25.5, 23.9, 22.3, 21.5, 14.8.
HRMS m/z: [M − H]− 483.2166 (calcd 483.2383).
3,19-(4-Dimethylaminobenzylidene)
andrographolide
(6b). Yield 52%, mp. 201–202 °C. IR (ATR), ν (cm−1):
3146 (νO–H), 2978–2849 (νC–H), 1724 (νC=O), 1668 (νC=C),
1
1223 (νC–Oester), 1198 (νC–N), 1026 (νC–O), 810 (νAr–H). H-
NMR (500 MHz, MeOD-d4, δ ppm): 7.32 (d, 2H, J =
9.0 Hz, Ar–H-2′, Ar–H-6′), 6.89 (t, 1H, J = 6.5 Hz, H-12),

Medicinal Chemistry Research
6.76 (d, 2H, J = 9.0 Hz, Ar–H-3′, Ar–H-5′), 5.77 (s, 1H, H-
21), 5.05 (d, 1H, J = 6.0 Hz, H-14), 4.94 (s, 1H, H-17a),
4.73 (s, 1H, H-17b), 4.50 (dd, 1H, J = 6.0, 10.0 Hz, H-15a),
4.19 (dd, 1H, J = 2.0, 6.0 Hz, H-15b), 4.34 (d, 1H, J =
11.5 Hz, H-19a), 3.66 (dd, 1H, J = 4.5, 12.5 Hz, H-3), 3.56
(d, 1H, J = 11.5 Hz, H-19b), 2.95 (s, 6H, –N(CH3)2), 1.49
(s, 3H, H-20), 0.93 (s, 3H, H-18). 13C-NMR (125 MHz,
DMSO-d6, δ ppm): 169.9, 150.5, 147.3, 146.1, 129.0,
127.3, 127.0, 111.4, 108.7, 94.6, 79.7, 74.3, 68.3, 64.5,
55.1, 53.6, 38.4, 37.0, 36.3, 35.2, 25.6, 23.9, 22.4, 21.5,
14.8. HRMS m/z: [M − H]− 480.2685 (calcd 480.2750),
[M + H]+ 482.2917 (calcd 482.2907), [M + Na]+ 504.2458
(calcd 504.2726), [M + K]+ 520.2520 (calcd 520.2466).
3,19-(2,4-Dimethoxybenzylidene) andrographolide (6c).
Yield 50%, mp. 204–205 °C. IR (ATR), ν (cm−1): 3414
(νO–H), 2959–2920 (νC–H), 1759 (νC=O), 1676 (νC=C), 1207
579.2061 (calcd 579.2151), [M + Na]+ 563.2195 (calcd
563.2411).
Procedure for preparation of 3,19-diacetyl-14-deoxy-
11,12-didehydro-8,17-epoxyandrographolide (8). In an
erlenmeyer flask, compound 4 (416 mg, 1 mmol) was dis-
solved into a solution of m-CPBA (525 mg, 3 mmol) in
CHCl3 (10 mL) at room temperature. The reaction mixture
was stirred at 10–15 °C for 48 h. After the completion of
reaction by TLC analysis, the reaction was quenched by
adding of Na2SO3 and stirring for 10–15 min to completely
reduce the remaining m-CBPA. The solution was then fil-
tered and rinsed with NaHCO3 10% solution (3 × 20 mL). A
crude product was obtained by removing the organic sol-
vent and then purified by silica chromatography using an
eluent petroleum ether–chloroform–acetone = 4:5:1 to
afford the pure product 8. Yield 41%, mp. 161–162 °C. IR
(ATR), ν (cm−1): 2920–2851 (νC–H), 1753–1726 (νC=O),
1244 (νC–Oester), 1034 (νC–O), 752 (νAr–H). 1H-NMR
(500 MHz, DMSO-d6, δ ppm): 7.65 (s, 1H, H-14), 6.33
(dd, 1H, J = 15.5, 10.0 Hz, H-11), 6.11 (d, 1H, J = 15.5 Hz,
H-12), 4.87 (d, 2H, J = 1.5 Hz, H-15), 4.53 (dd, 1H, J =
12.0, 4.5 Hz, H-3), 4.36 (d, 1H, J = 12.0 Hz, H-19a), 4.08
(d, 1H, J = 11.5 Hz, H-19b), 2.54 (d, 1H, J = 4.5 Hz, H-
17a), 2.52 (d, 1H, J = 4.0 Hz, H-17b), 2.02 (s, 3H,
OCOCH3), 2.00 (s, 3H, OCOCH3), 0.98 (s, 3H, H-20), 0.94
(s, 3H, H-18). 13C-NMR (125 MHz, DMSO-d6, δ ppm):
172.3, 170.2, 169.8, 146.9, 129.9, 127.0, 123.6, 79.1, 70.2,
63.7, 57.9, 57.7, 52.7, 49.5, 40.9, 38.5, 37.2, 35.2, 23.3,
23.2, 21.5, 20.8, 20.7, 14.9. HRMS m/z: [M − H]−
493.2382 (calcd 493.1994), [M + Na]+ 477.2200 (calcd
477.2047).
1
(νC–Oester), 1121 (νC–O), 793 (νAr–H). H-NMR (500 MHz,
MeOD-d4, δ ppm): 7.49 (d, 1H, J = 8.5 Hz, Ar–H-6′), 6.89
(td, 1H, J = 6.5, 1.5 Hz, H-12), 6.54 (m, 2H, Ar–H-3′,
Ar–H-5′), 6.13 (s, 1H, H-21), 5.05 (d, 1H, J = 6.0 Hz, H-
14), 4.94 (s, 1H, H-17a), 4.73 (s, 1H, H-17b), 4.50 (dd, 1H,
J = 10.0, 6.0 Hz, H-15), 4.33 (d, 1H, J = 11.0 Hz, H-19a),
4.19 (dd, 1H, J = 10.0, 2.0 Hz, H-15), 3.83 (s, 3H, C-
2′–OCH3)–3.81 (s, 3H, C-4′–OCH3), 3.65 (dd, 1H, J =
12.5, 4.5 Hz, H-3), 3.56 (d, 1H, J = 11.0 Hz, H-19b), 1.49
(s, 3H, H-20), 0.93 (s, 3H, H-18). 13C-NMR (125 MHz,
DMSO-d6, δ ppm): 169.9, 160.6, 157.3, 147.2, 146.1,
129.0, 128.0, 119.9, 108.7, 104.7, 97.9, 89.3, 79.8, 74.3,
68.6, 64.5, 55.5, 55.2, 55.1, 53.6, 38.4, 37.0, 36.3, 35.2,
25.6, 23.9, 22.3, 21.4, 14.9. HRMS m/z: [M + Na]+
521.2508 (calcd 521.2516).
14-deoxy-11,12-didehydro-3,19-(2′-hydroxybenzylidene)-
15-(2-hydroxybenzylidene) andrographolide (7). A mixture
of Z and E isomers (1:1), yield 78%, mp. 158–159 °C. IR
(ATR), ν (cm−1): 3354 (νO–H), 3080 (νAr–H), 2918–2851 (νC–
Biological assay
General procedure for α-glucosidase inhibition assay
H), 1732 (νC=O), 1645–1587 (νC=C), 1254 (νC–Oester
)
1092–1065 (νC–O), 752 (νAr–H). 1H-NMR (500 MHz, DMSO-
d6, δ ppm): 10.17 (s, 1H, Ar-OH), 10.05 (s, 1H, Ar-OH), 9.36
(s, 2H, Ar-2′-OH), 8.07 (s, 1H, H-14), 7.91 (dd, 1H, J = 8.5,
1.0 Hz, Ar-H-6), 7.81 (s, 1H, H-14), 7.47 (d, 1H, J = 7.0 Hz,
Ar–H-6), 7.40 (m, 2H, Hz, Ar–H-6′), 7.19 (m, 2H, Ar–H-4′),
7.12 (td, 2H, J = 8.0, 1.5 Hz, Ar–H-5′), 6.91–6.85 (m, 5H,
Ar–H-3, Ar–H-4, Ar–H-5, H-3′, H-21), 6.79–6.77 (m, 4H,
Ar–H-4, Ar–H-3′, H-11), 6.58 (s, 1H, H-21), 6.29 (d, 1H, J
= 15.5 Hz, H-12), 6.28 (d, 1H, J = 15.5 Hz, H-12), 6.05 (s,
2H, H-21′), 4.79 (s, 2H, H-17a), 4.50 (s, 2H, H-17b), 4.19 (d,
2H, J = 11.0 Hz, H-19a), 3.56 (m, 2H, H-3, H-19b), 1.40 (s,
6H, H-20), 0.93 (s, 6H, H-18). 13C-NMR (125 MHz, DMSO-
d6, δ ppm): 168.8, 160.7, 156.1, 148.5, 147.9, 146.8, 137.7,
136.4, 132.3, 130.3, 129.7, 128.7, 125.3, 121.7, 120.3, 119.6,
117.2, 115.6, 111.5, 110.7, 108.9, 107.4, 89.9, 80.1, 68.7,
60.6, 52.9, 38.4, 36.5, 29.8, 25.5, 21.6, 21.3, 15.7. HRMS
m/z: [M − H]− 539.2268 (calcd 539.2435), [M + H]+
p-Nitrophenyl-α-D-glucopyranoside (p-NPG), the substrate,
and α-glucosidase (Saccharomyces cerevisiae) were pur-
chased from Sigma-Aldrich.
The inhibition assays were evaluated at 37 °C in
K2HPO4/KH2PO4 buffer with pH = 6.8. The reaction mix-
ture comprised of an enzyme 0.2 U/mL solution (40 μL),
inhibitors or acarbose solution with various concentrations
(50 μL) and p-NPG 1 mM solution (40 μL). Acarbose was
tested as a positive control. Inhibitors were dissolved into
aqueous DMSO 10% solution, whereas both enzyme and
substrate were dissolved into the buffer solution. After
incubation of the enzyme solution in the presence of the
inhibitor for 20 min at room temperature, the enzymatic
reaction was started by the addition of a substrate (p-NPG).
The mixture was incubated at room temperature for 20 min,
then the reaction was quenched by the addition of Na2CO3
0.2 M solution (130 μL). The absorbance at 405 nm

Medicinal Chemistry Research
(Ainhibitor) was measured immediately and taken as relative
inhibition capacity for an inhibitor. All the experiment was
carried out with blank sample (without an inhibitor) to
obtain Ablank in triplicate.
The inhibition percentage was calculated by the follow-
ing equation:
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¼ ½ðAblank À AinhibitorÞ=AblankŠ  100:
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starch. In: Starch in food, 2nd edn. Elsevier, pp. 805–826. https://
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Data availability
Copies of HRMS, IR, H-NMR, and 13C-NMR spectra of
6c and 7 are available online.
1
Acknowledgements We gratefully thank the Faculty of Pharmacy,
University of Medicine and Pharmacy at Ho Chi Minh city, Institute of
drug quality control Ho Chi Minh city, Vietnam Academy of Science
and Technology for their support of this research. We also gratefully
thank professor Éric Marsault, Faculty of Medicine and Health Sci-
ences, Université de Sherbrooke, for his comments and corrections.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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(2010) Design, synthesis and antibacterial activity of novel
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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