Design, synthesis, and biological evaluation of andrographolide derivatives as potent hepatoprotective agents

Article abstract of DOI:10.1111/cbdd.12246

Poor water solubility limits the clinical use of andrographolide and its derivatives. In an attempt to develop potent hepatoprotective drugs, a strategy was proposed to improve the aqueous solubility of andrographolide. Ten andrographolide derivatives were designed, synthesized, evaluated for aqueous solubility and in vivo hepatoprotective activity against CCl₄-induced liver injury in mice. As expected, the aqueous solubility of synthetic derivatives was effectively improved. All compounds demonstrated the effect of different degrees in improving the liver enzyme (ALT and AST) activity, especially the most promising compound 9d significantly improved liver enzyme activity, with high potency to be a new lead.

Full text of DOI:10.1111/cbdd.12246

Chem Biol Drug Des 2014; 83: 324–333
Research Article
Design, Synthesis, and Biological Evaluation of
Andrographolide Derivatives as Potent
Hepatoprotective Agents
Chunlei Tang1,2,†, Guolong Gu1,3,†, Bin Wang1,4
Xin Deng1, Xiaoyun Zhu1,5, Hai Qian1,* and
Wenlong Huang1,*
,
Liver is the largest internal organ in human body and is
the first stop for all nutrients, toxins, and drugs absorbed
by the digestive tract (1,2). This organ plays a major role in
metabolism and has a number of functions, including gly-
cogen storage, decomposition of red blood cells, plasma
protein synthesis, hormone production, detoxification, and
production of biochemicals necessary for digestion (3).
The liver also regulates various of biochemical reactions,
many of which are necessary for normal vital functions (4).
1State Key Laboratory of Natural Medicines, Centre of
Drug Discovery, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, China
2School of Pharmaceutical Science, Jiangnan University,
1800 Lihu Road, Wuxi 214122, China
3College of Pharmacy, Yancheng Teachers University,
Yancheng 224002, China
4R&D Center, Jiangsu Yabang Pharmaceutical Group,
Changzhou 213163, China
Hepatitis, both acute and chronic, is a common pathol-
ogy worldwide. It can lead to liver fibrosis and end-stage
cirrhosis, which contribute to life-threatening complica-
tions of portal hypertension, liver failure, and increased
incidence of hepatocellular carcinoma. The main etiology
of liver injury is represented by viral infections, excessive
alcohol consumption, toxins, and fatty liver (5). Natural
products, as a resource for the treatment of liver dis-
eases, have drawn attention from scientists and experts
worldwide in recent years (6), among which andrographo-
lide (1; Figure 1) is one of the potential agents (7,8). An-
drographolide, a bicyclic diterpenoid lactone, is the major
bioactive component of the medicinal plant of Androgra-
phis paniculata (9), and it is considered to protect the
liver against various hepatotoxic substances by increasing
the activity of hepatic microsomal enzyme (CYP1A2,
CYP2E1), maintaining the stability of phase II enzymatic
detoxification systems, and reducing oxidative stress and
anticholestasis (10–12). Despite the high potency of he-
patoprotective activity and its low toxicity, the poor aque-
ous solubility of andrographolide has been a concern as
it results in low dissolution and limited formulations and
has hindered the clinical use of andrographolide and its
derivatives (13–16). In this article, a series of derivatives
with improved aqueous solubility were designed and syn-
thesized and were screened in physicochemical studies
in vitro and the pharmacological evaluations in mice
model.
5Shanghai Institute of Pharmaceutical Industry, 1320 West
Beijing Road, Shanghai 200040, China
*Corresponding authors: Hai Qian, qianhai24@163.com;
Wenlong Huang, ydhuangwenlong@126.com
†These authors contributed equally to this work.
Poor water solubility limits the clinical use of androgra-
pholide and its derivatives. In an attempt to develop
potent hepatoprotective drugs,
a
strategy was
proposed to improve the aqueous solubility of androg-
rapholide. Ten andrographolide derivatives were
designed, synthesized, evaluated for aqueous solubility
and in vivo hepatoprotective activity against CCl4-
induced liver injury in mice. As expected, the aqueous
solubility of synthetic derivatives was effectively
improved. All compounds demonstrated the effect of
different degrees in improving the liver enzyme (ALT
and AST) activity, especially the most promising com-
pound 9d significantly improved liver enzyme activity,
with high potency to be a new lead.
Key words: andrographolide derivatives, aqueous solubility,
hepatoprotective activity, synthesis
Abbreviations: ALT, alanine transaminase; AST, aspartate
transaminase; DIPEA, N,N-diisopropylethylamine; DMAP, 4-
dimethylaminopyridine; EDCÁHCl, N-(3-Dimethylaminopropyl)-
N’-ethylcarbodiimide hydrochloride; PDC, pyridinium dichro-
mate; SAR, structure-activity relationships; TrCl, triphenylchlo-
romethane.
Methods and Materials
Received
2 July 2013, revised 2 September 2013 and
accepted for publication 4 October 2013
Chemistry
All chemical reagents were commercially available and
treated with standard methods before use. Solvents
324
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12246

Andro Derivatives as Potent Hepatoprotective Agents
The synthetic routes to the target compounds are outlined
in Schemes 1, 2, and 3. Andrographolide was dehydrated
with activated alumina in refluxing anhydrous pyridine to
give dehydroandrographolide 2. Compound 2 was treated
with triphenylchloromethane (TrCl) in the presence of
N-methylmorpholine in anhydrous dichloromethane at
room temperature to afford trityl ether 3.
Dehydroandrographolide (2)
1 (3.0 g, 8.6 mmol) was dissolved in anhydrous pyridine
(8 mL) and then activated alumina (0.6 g) was added. The
reaction mixture was stirred at 100–110 °C for 12 h. After
cooling, the whole solution was filtered, washed with chlo-
roform, and evaporated to obtain a residue purified by
column chromatography (PE/EtOAc 1:1 v/v). White solid,
88% yield. MS (ESI) m/z: 355.2 (M + Na)+.
Figure 1: Structure of andrographolide.
were dried and redistilled before use. Column chroma-
tography (CC): silica gel 60 (100–200 mesh). Thin-layer
chromatography (TLC): silica gel 60 F254 plates
(250 mm; Qingdao Ocean Chemical Company, Qingdao,
China). M.p.: capillary tube, uncorrected. IR spectra: Shi-
madzu FTIR-8400S spectrophotometer with KBr disks; in
19-O-trityldehydroandrographolide (3)
2 (2.0 g, 6.0 mmol) and N-Methylmorpholine (0.76 mL,
6.9 mmol) were dissolved in anhydrous dichloromethane
(25 mL) at room temperature. A solution of triphenylmethyl
chloride (1.70 g, 7.8 mmol) dissolved in dichloromethane
(10 mL) was then added dropwise over 20 min. The reac-
tion mixture was stirred for 12 h. Over this period, the
whole solution was diluted with dichloromethane (50 mL),
washed with saturated brine (30 mL 9 3), dried over
Na2SO4, and evaporated to obtain a residue purified by
recrystallization with EtOAc/PE. White solid, 83% yield. MS
(ESI) m/z: 575.3 (M + H)+.
cmÀ1
.
1H and 13C NMR spectra: Bruker ACF-300Q
apparatus at 300 MHz, in d6-DMSO unless otherwise
indicated, d in ppm rel. to Me4Si, J in Hz. Aqueous sol-
ubility and Mass spectrometry (MS): Hewlett–Packard
1100 LC/MSD spectrometer.
Synthetic procedures of target compounds
To improve the aqueous solubility of Andro, the nitrogen-
containing heterocyclic and carboxylic groups were
introduced to the andrographolide nucleus.
In Scheme 1, compound 3 was reacted with anhydrides in
the presence of 4-dimethylaminopyridine (DMAP) in anhy-
O
O
O
HO
O
O
O
a
b
c
H
H
H
HO
HO
HO
HO
TrO
HO
1
2
3
O
O
O
O
O
O
d
5a
5b
R=
COOH
H
H
RO
RO
HO
TrO
R=
COOH
5
4
Reagents and conditions: (a) Al2O3, pyridine, reflux; (b) TrCl, N-Methylmorpholine, CH2Cl2, rt; (c)
succinic or glutaric anhydride , DMAP, CH2Cl2, reflux; (d) HCOOH, CH2Cl2, rt;
Scheme 1: Synthesis of the target
compound 5a and 5b.
Chem Biol Drug Des 2014; 83: 324–333
325

Tang et al.
1
drous dichloromethane at room temperature to afford 4.
Then, compound 4 followed by hydrolysis of the trityl ether
with formic acid in dichloromethane obtained compound
5a and 5b.
1164.13, 1084.08; H-NMR (DMSO-d6, 300 MHz, ppm) d:
12.13 (brs, 1H, H-COCH2CH2COOH), 7.66 (s, 1H, H-14),
6.82–6.73 (dd, 1H, J = 10.1 Hz, 15.7 Hz, H-11), 6.18–
6.13 (d, 1H, J = 15.7 Hz, H-12), 4.89 (s, 2H, H-15), 4.74
(s, 1H, H-17a), 4.55–4.57 (dd, 1H, J = 4.4 Hz, 11.7 Hz,
H-3) 4.43 (s, 1H, H-17b), 4.13–4.09 (d, 1H, J = 11.8 Hz,
H-19a), 3.56–3.52 (d, 1H, J = 11.8 Hz, H-19b), 2.48–2.42
(t, 2H, H-COCH2CH2COOH), 2.38–2.34 (d, 1H, H-9),
2.02–1.95 (t, 2H, H-COCH2CH2COOH), 1.73–1.04 (m, 8H,
H-1, 2, 6, 7), 0.92 (s, 3H, H-18), 0.86 (s, 3H, H-20). 13C-
NMR (DMSO-d6, 75 MHz, ppm) d: 173.2, 171.9, 170.3,
148.6, 147.3, 134.2, 127.6, 122.0, 108.7, 70.7, 64.0,
60.4, 53.5, 41.5, 38.8, 38.0, 36.5, 29.2, 28.8, 24.2, 23.9,
22.7, 21.3, 15.2.
General procedures for the synthesis of 4a and 4b
3 (580 mg, 1.0 mmol), succinic anhydride or glutaric anhy-
dride (4 mmol), and 4-dimethylaminopyridine (DMAP)
(122 mg, 1 mmol) were dissolved in anhydrous dichlorom-
ethane (15 mL) at room temperature. The reaction mixture
was then heated to reflux for 24 h. After cooling, the
whole solution was diluted with dichloromethane (50 mL),
washed with water (100 mL 9 3) and saturated brine
(50 mL 9 3), dried over Na2SO4, and evaporated to obtain
crude product 4a and 4b, respectively.
3-O-monoglutaratedehydroandrographolide (5b)
Eluent from PE/EtOAc/AcOH 20:10:1, white powder, 73%
yield, mp 76–78 °C, MS (ESI) m/z: 469.2 (M + Na)+; IR
General procedures for the synthesis of compound
5a and 5b
(KBr, cmÀ1
) m: 3449.22, 3164.29, 1731.61, 1400.49,
1
Crude product 4a or 4b (1 mmol) was dissolved in dichlo-
romethane (10 mL) followed by addition of formic acid
(2 mL), and the reaction mixture was stirred under room
temperature for 1 h. Then, the whole solution was diluted
with dichloromethane (20 mL), washed with saturated
sodium bicarbonate (20 mL 9 3) and saturated brine
(20 mL 9 3), dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatogra-
phy.
1390.48, 1349.98, 1164.44, 1083.86; H-NMR (DMSO-d6,
300 MHz, ppm) d: 12.13 (brs, 1H, H-COCH2CH2COOH),
7.66 (s, 1H, H-14), 6.81–6.72 (dd, 1H, J = 10.2 Hz,
15.8 Hz, H-11), 6.18–6.13 (d, 1H, J = 15.8 Hz, H-12),
4.89 (s, 2H, H-15), 4.74 (s, 1H, H-17a), 4.55–4.57 (1H,
H-3), 4.43 (s, 1H, H-17b), 4.13–4.09 (d, 1H, J = 11.8 Hz,
H-19a), 3.56–3.52 (d, 1H, J = 11.8 Hz, H-19b), 2.42–2.38
(d, 1H, H-9), 2.35–2.30 (t, 2H, H-COCH2CH2CH2COOH),
2.27–2.22 (t, 2H, H-COCH2CH2CH2COOH), 1.78–1.73 (m,
2H, COCH2CH2CH2COOH), 2.20–1.04 (m, 8H, H-1, 2, 6,
7), 0.92 (3H, s, H-18), 0.86 (3H, s, H-20). 13C-NMR
(DMSO-d6, 75 MHz, ppm) d: 173.2, 171.9, 170.4, 148.6,
147.4, 134.2, 127.6, 122.0, 108.7, 70.6, 64.0, 60.4, 53.5,
41.5, 38.8, 37.9, 36.5, 29.2, 28.8, 24.2, 23.9, 22.7, 21.4,
19.7, 15.2.
3-O-monosuccinatedehydroandrographolide (5a)
Eluent from PE/EtOAc/AcOH 20:10:1, white powder, 82%
yield, mp 122–124 °C, MS (ESI) m/z: 431.2 (M À H)À; IR
(KBr, cmÀ1
) m: 3465.82, 3171.55, 1731.83, 1400.33,
O
O
O
O
O
O
b
c
a
H
H
H
O
O
HO
TrO
HO
TrO
7
6
3
O
O
O
O
O
N
N
9a
R=
d
H
9b
9c
9d
R=
R=
R=
H
O
O
N
N
O
O
R
Cl
O
O
OH
9 a-d
8
Reagents and conditions: (a) PDC, CH2Cl2, reflux; (b) HCOOH, CH2Cl2, rt; (c) Chloroacetyl chloride,
Et3N, CH2Cl2, rt; (d) Amine, DIPEA, THF, rt;
Scheme 2: Synthesis of the target
compound 9a–d.
326
Chem Biol Drug Des 2014; 83: 324–333

Andro Derivatives as Potent Hepatoprotective Agents
In Scheme 2, compound 3 was oxidized with pyridinium
dichromate (PDC) in refluxing anhydrous dichloromethane
to give oxide 6, compound 6 followed by hydrolysis of
the trityl ether with formic acid in dichloromethane
obtained compound 7, treatment of 7 with chloroacetyl
chloride in the presence of triethylamine in anhydrous di-
chloromethane to afford 8. Finally, 8 reacted with nitro-
gen-containing heterocyclic in the presence of N,N-
diisopropylethylamine in anhydrous THF to give the
desired product 9a–d.
rated brine (30 mL 9 3), dried over Na2SO4, and
concentrated in vacuo to afford crude product 8 as a
brown oil. The crude product can be used for the next
step without further purification.
General procedures for the synthesis of compound
9a–d
8
(540 mg, 1.23 mmol) and ethyldiisopropylamine
(643.2 mL, 3.69 mmol) were dissolved in anhydrous THF
(10 mL) and chilled to 0 °C. A solution of different amine
(3.69 mmol) dissolved in anhydrous THF (5 mL) was
added dropwise; the reaction mixture was allowed to
warm to room temperature and stirred for 3–8 h. The
whole solution was evaporated to obtain a residue, dis-
solved with EtOAc (80 mL), washed with water
(50 mL 9 3) and saturated brine (30 mL 9 3), dried over
Na2SO4, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography.
3-oxo-19-O-trityldehydehydroandrographolide (6)
3 (287 mg, 5.0 mmol) was dissolved in anhydrous dichlo-
romethane (80 mL) and then pyridinium dichromate (4.3 g,
10 mmol) was added at room temperature. The reaction
mixture was then heated to reflux for 48 h. After cooling,
the whole solution was filtered with a flash column chro-
matography, and the filtrate was diluted with dichlorome-
thane (80 mL), washed with diluted base (40 mL 9 3),
cold diluted acid (40 mL 9 3), and saturated brine
(40 mL 9 3), dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatogra-
phy (PE/EtOAc, 2:1) to afford 6 as a white solid. 64%
yield. MS (ESI) m/z: 573.3 (M + H)+.
3-oxo-19-O-(2-morpholinoacetyl)
dehydroandrographolide (9a)
Eluent from PE/EA 1:3, yellow solid, yield: 79%, mp 83–
85 °C, MS (ESI) m/z: 458.2 (M + Na)+; IR (KBr, cmÀ1) m:
3079.29, 1735.22, 1711.71, 1191.26, 1156.33, 1014.30,
996.41, 895.03; 1H-NMR (DMSO-d6, 300 MHz, ppm) d:
7.69 (s, 1H, H-14), 6.80 (dd, 1H, J = 10.1 Hz, 15.8 Hz,
H-11), 6.19 (d, 1H, J = 15.8 Hz, H-12), 4.90 (s, 2H, H-
15), 4.81 (s, 1H, H-17a), 4.51 (s, 1H, H-17b), 4.58 (d,
1H, J = 11.3 Hz, H-19a), 4.00 (d, 1H, J = 11.3 Hz, H-
19b), 3.55 (t, 4H, J = 4.5 Hz, H-CH2NCH2), 3.14 (s, 2H,
H-CH2CO), 2.55 (d, 1H, J = 13.8 Hz, H-9), 2.44 (t, 4H,
J = 4.5 Hz, H-CH2OCH2), 2.36–1.23 (m, 8H, H-1, 2, 6,
7), 1.08 (s, 3H, H-18), 1.02 (s, 3H, H-20); 13C-NMR
(DMSO-d6, 75 MHz, ppm) d: 212.2, 172.8, 170.0, 148.5,
147.5, 134.1, 127.5, 122.3, 109.4, 70.7, 66.5, 66.1,
59.8, 59.0, 55.3, 52.8, 52.0, 38.7, 38.6, 36.0, 35.4,
24.0, 21.0, 15.2.
3-oxodehydroandrographolide (7)
6 (580 mg, 1 mmol) was dissolved in dichloromethane
(10 mL) followed by addition of formic acid (2 mL), and
the reaction mixture was stirred under room temperature
for 1 h. Then, the whole solution was diluted with di-
chloromethane (20 mL), washed with saturated sodium
bicarbonate
(20 mL 9 3)
and
saturated
brine
(20 mL 9 3), dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatog-
raphy (PE/EtOAc, 2:1) to afford 7 as a white solid. 82%
yield. MS (ESI) m/z: 331.2 (M + H)+, 1H-NMR (DMSO-d6,
300 MHz, ppm) d: 7.68 (s, 1H, H-14), 7.32–7.29 (d, 2H,
J = 8.6 Hz, H-Ar), 6.84–6.75 (dd, 1H, J = 10.1 Hz,
15.8 Hz, H-11), 6.19–6.13 (d, 1H, J = 15.8 Hz, H-12),
4.89 (s, 2H, H-15), 4.79 (s, 1H, H-17a), 4.49 (s, 1H, H-
17b), 3.86–3.82 (d, 1H, J = 11.9 Hz, H-19a), 3.34–3.30
(d, 1H, J = 11.3 Hz, H-19b), 2.42–2.39 (d, 1H, H-9),
2.11-1.39 (m, 8H, H-1, 2, 6, 7), 1.04 (s, 3H, H-18),
1.02 (s, 3H, H-20).
3-oxo-19-O-(2-piperidinoacetyl)
dehydroandrographolide (9b)
Eluent from PE/EA 1:2, yellow solid, yield: 71%, mp 74–
76 °C, MS (ESI) m/z: 456.3 (M + Na)+; IR (KBr, cmÀ1) m:
1754.54, 1708.38, 1303.29, 1284.57, 1132.36, 1082.80,
958.82; 1H-NMR (DMSO-d6, 300 MHz, ppm) d: 7.68 (s,
1H, H-14), 6.85–6.76 (dd, 1H, J = 10.1 Hz, 15.8 Hz,
H-11), 6.19–6.14 (d, 1H, J = 15.8 Hz, H-12), 4.90 (s, 2H,
H-15), 4.82 (s, 1H, H-17a), 4.51 (s, 1H, H-17b), 4.57–4.53
(d, 1H, J = 11.4 Hz, H-19a), 3.99–3.95 (d, 1H,
J = 11.3 Hz, H-19b), 3.09 (s, 2H, H-CH2CO), 2.39 (m,
4H, H-CH2NCH2), 2.20–1.34 (m, 8H, H-1, 2, 6, 7), 1.49–
1.45 (t, 4H, H-CH2CH2OCH2CH2), 1.07 (s, 3H, H-18),
1.02 (s, 3H, H-20); 13C-NMR (DMSO-d6, 75 MHz, ppm) d:
212.2, 172.8, 170.4, 148.5, 147.6, 134.1, 127.5, 122.3,
109.4, 70.7, 66.1, 59.8, 59.7, 55,4, 53.6, 52.0, 38.7,
38.6, 36.1, 35.4, 25.9, 24.0, 21.0, 15.2.
3-oxo-19-O-(2-chloroacetyl)
dehydroandrographolide (8)
7 (660 mg, 2 mmol) and triethylamine (0.6 mL, 4.3 mmol)
were dissolved in anhydrous dichloromethane (10 mL) and
chilled to 0 °C.
A solution of chloroacetyl chloride
(0.33 mL, 4.0 mmol) dissolved in anhydrous dichlorome-
thane (5 mL) was added dropwise; the reaction mixture
was allowed to warm to room temperature and stirred for
1 h. Then, the whole solution was diluted with dichlorome-
thane (40 mL), washed with water (30 mL 9 3) and satu-
Chem Biol Drug Des 2014; 83: 324–333
327

Tang et al.
3-oxo-19-O-(2-pyrrolidinoacetyl)
dehydroandrographolide (9c)
1.99 (m, 4H, H-CH2CHOHCH2), 1.78–1.29 (m, 8H, H-1,
2, 6, 7), 1.07 (s, 3H, H-18), 1.02 (s, 3H, H-20); 13C-NMR
(CDCl3-d1, 75 MHz, ppm) d: 212.6, 172.2, 170.3, 147.0,
143.6, 135.1, 129.0, 121.8, 110.2, 69.6, 67.2, 66.3,
60.9, 59.0, 56.4, 52.2, 50.6, 39.1, 38.6, 36.2, 35.5, 24.0,
20.8, 15.3.
Eluent from PE/EA 1:3, yellow solid, yield: 65%, mp 68–
70 °C, MS (ESI) m/z: 442.2 (M + Na)+; IR (KBr, cmÀ1) m:
3166.76, 1752.02, 1708.00, 1398.69, 1184.13, 996.61,
895.03; 1H-NMR (DMSO-d6, 300 MHz, ppm) d: 7.69 (s,
1H, H-14), 6.85–6.76 (dd, 1H, J = 10.0 Hz, 15.8 Hz, H-
11), 6.19–6.14 (d, 1H, J = 15.8 Hz, H-12), 4.90 (s, 2H, H-
15), 4.82 (s, 1H, H-17a), 4.59–4.55 (d, 1H, J = 11.3 Hz,
H-19a), 4.51 (s, 1H, H-17b), 4.00–3.96 (d, 1H,
J = 11.3 Hz, H-19b), 3.23 (s, 2H, H-NCH2CO), 2.51 (m,
4H, H-CH2NCH2), 2.44–2.40 (d, 1H, H-9), 2.20–1.46 (m,
8H, H-1, 2, 6, 7), 1.67–1.64 (m, 4H, H-CH2CH2), 1.49–
1.45 (t, 4H, H- CH2CH2OCH2CH2), 1.07 (s, 3H, H-18),
1.02 (s, 3H, H-20); 13C-NMR (DMSO-d6, 75 MHz, ppm) d:
172.8, 170.3, 148.5, 147.6, 134.1, 127.5, 122.3, 109.5,
70.7, 66.2, 59.8, 56.0, 55.4, 53.4, 52.0, 38.8, 38.6, 36.0,
35.4, 24.0, 23.9, 20.9, 15.2.
In Scheme 3, compound 3 was acetylated with acetic
anhydride in the presence of 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCÁHCl) and 4-dimeth-
ylaminopyridine (DMAP) in anhydrous dichloromethane at
room temperature to afford 10, compound 10 followed by
hydrolysis of the trityl ether with formic acid in dichlorome-
thane obtained compound 11, treatment of 11 with chlo-
roacetyl chloride in the presence of triethylamine in
anhydrous dichloromethane to afford 12. Finally, 12
reacted with nitrogen-containing heterocyclic in the pres-
ence of N,N-diisopropylethylamine in anhydrous THF to
give the desired products 13a–d.
3-oxo-19-O-[2-(4-hydroxypiperidino)acetyl]
dehydroandrographolide (9d)
3-O-acetyl-19-O-trityldehydehydroandrographolide
(10)
Eluent from PE/EA/MeOH 5:15:1, yellow solid, yield: 72%,
mp 78–80 °C, MS (ESI) m/z: 472.2 (M + Na)+; IR (KBr,
cmÀ1) m: 3427.27, 1751.40, 1707.46, 1282.82, 1185.68,
1074.07, 996.61; 1H-NMR (DMSO-d6, 300 MHz, ppm) d:
7.69 (s, 1H, H-14), 6.85–6.76 (dd, 1H, J = 10.1 Hz,
15.8 Hz, H-11), 6.19–6.14 (d, 1H, J = 15.7 Hz, H-12),
4.90 (s, 2H, H-15), 4.82 (s, 1H, H-17a), 4.56–4.52 (d,
1H, J = 11.3 Hz, H-19a), 4.52 (s, 1H, H-17b), 3.99–3.95
(d, 1H, J = 11.3 Hz, H-19b), 3.44–3.36 (m, 1H,
H-CHOH), 3.09 (2H, s, H-CH2CO), 2.65–2.51 (m, 4H,
H-CH2NCH2), 2.45–2.41 (d, 1H, J = 12.4 Hz, H-9), 2.20–
3
(600 mg, 1.05 mmol), acetic anhydride (198.5 mL,
2.1 mmol), and DMAP (catalytic amount) were dissolved in
anhydrous dichloromethane (25 mL) and chilled to 0 °C. A
solution of EDC•HCl (400 mg, 2.1 mmol) dissolved in
anhydrous dichloromethane (15 mL) was added dropwise,
and the reaction mixture was stirred overnight allowed to
warm to room temperature. Then, the whole solution was
diluted with dichloromethane (40 mL), washed with water
(30 mL 9 3) and saturated brine (30 mL 9 3), dried over
Na2SO4, and concentrated in vacuo. The residue was
O
O
O
O
O
O
a
b
c
O
O
H
H
H
O
O
HO
TrO
HO
TrO
3
10
11
O
O
O
O
N
O
13a
R=
N
O
O
d
13b
13c
13d
R=
R=
R=
H
O
H
O
O
O
N
N
O
O
Cl
R
OH
12
13 a-d
Reagents and conditions: (a) Ac2O, EDC·HCl, DMAP, CH2Cl2, rt; (b) HCOOH, CH2Cl2, rt; (c)
Chloroacetyl chloride, Et3N, CH2Cl2, rt; (d) Amine, DIPEA, THF, rt;
Scheme 3: Synthesis of the target
compound 13a–d.
328
Chem Biol Drug Des 2014; 83: 324–333

Andro Derivatives as Potent Hepatoprotective Agents
purified by column chromatography (PE/EtOAc, 2:1) to
afford 10 as a white solid. 91% yield. MS (ESI) m/z: 617.3
(M + H)+.
due was purified by column chromatography (PE/EtOAc,
2:1) to afford 11 as a white solid. 87% yield. MS (ESI) m/z:
374.2 (M + H)+, 1H-NMR (DMSO-d6, 300 MHz, ppm) d:
7.68, (s, 1H, H-14), 6.81–6.72 (dd, 1H, J = 10.0 Hz,
15.6 Hz, H-11), 6.19–6.14 (d, 1H, J = 15.6 Hz, H-12),
4.89 (s, 2H, H-15), 4.77 (s, 1H, H-17a), 4.46 (s, 1H, H-
17b), 4.56–4.51 (dd, 1H, J = 4.3 Hz, 11.6 Hz, H-3), 4.32–
4.29 (d, 1H, J = 11.6 Hz, H-19a), 4.11–4.07 (d, 1H,
J = 11.6 Hz, H-19b), 2.01 (s, 3H, H-OCOCH3), 1.77–1.42
(m, 8H, H-1, 2, 6, 7), 0.96 (s, 3H, H-18), 0.80 (s, 3H, H-
20).
3-O-acetyl-dehydroandrographolide (11)
10 (620 mg, 1 mmol) was dissolved in dichloromethane
(10 mL) followed by the addition of formic acid (2 mL),
and the reaction mixture was stirred under room tempera-
ture for 1 h. Then, the whole solution was diluted with
dichloromethane (20 mL), washed with saturated sodium
bicarbonate (20 mL 9 3) and saturated brine (20 mL 9 3),
dried over Na2SO4, and concentrated in vacuo. The resi-
3-O-acetyl-19-O-(2-chloroacetyl)
dehydroandrographolide (12)
Following the procedure described for reduction of 8,
crude product of compound 12 was prepared from 11 as
a brown oil. The crude product can be used for the next
step without further purification.
General procedures for the synthesis of compound
13a–d
Following the procedure described for reduction of 9a–d,
compound 13a–d was prepared from 11 with different
amine.
3-O-acetyl-19-O-(2-morpholinoacetyl)
dehydroandrographolide (13a)
Eluent from PE/EA 1:3, yellow solid, yield: 72%, mp 114–
116 °C, MS (ESI) m/z: 502.2 (M + Na)+; IR (KBr, cmÀ1) m:
3174.31, 2371.68, 1746.9, 1400.59, 1121.92, 996.61,
Figure 2: The aqueous solubility of the compounds at
25 Æ 1 °C.
Table 1: Effect of andrographolide and its derivatives administration on the levels of ALT and AST in serum in the liver damaged mice
induced by CCl4
Group
Compound
ALT (IU/L)a
AST (IU/L)a
1
Normal
Model
Silymarin
Andrographolide (1)
2
5a
46.4 Æ 4.8
168.0 Æ 7.7
2
1380.0 Æ 124.6*
237.4 Æ 38.6†
589.7 Æ 71.6†
708.4 Æ 43.1†
516.9 Æ 62.1†,§1
807.7 Æ 64.3†
566.6 Æ 50.0†,§1
718.1 Æ 94.6†
684.2 Æ 49.0†
391.9 Æ 56.2†,‡2,§2
600.0 Æ 52.1†
812.4 Æ 46.3†
670.4 Æ 94.5†
487.6 Æ 48.1†,‡1,§2
2104.6 Æ 157.7*
545.7 Æ 51.9†
3
4
1012.9 Æ 83.1†
1275.2 Æ 86.3†
1006.1 Æ 96.8†
987.4 Æ 109.4†
942.5 Æ 89.7†,§1
1261.0 Æ 77.9†
1021.3 Æ 107.9†
778.3 Æ 50.2†,‡1,§2
842.3 Æ 74.5†,§2
1239.4 Æ 91.5†
1252.0 Æ 109.8
742.4 Æ 108.4†,‡1,§2
5
6
7
5b
9a
8
9
9b
9c
10
11
12
13
14
15
9d
13a
13b
13c
13d
aValues are expressed as mean Æ SEM (n = 8).
*Compared with control group P < 0.001.
†Compared with model group P < 0.001.
‡Compared with andrographolide group P < 0.05 (‡1), P < 0.01 (‡2).
§Compared with dehydroandrographolide (2) group P < 0.05 (§1), P < 0.01 (§2).
Chem Biol Drug Des 2014; 83: 324–333
329

Tang et al.
(A)
(B)
(C)
(D)
Figure 3: Histopathological
annlysis of liver tissue of mice. (A)
Liver section of normal group; (B)
Liver section of CCl4 control; (C)
Liver section of silymarin + CCl4
group; (D) Liver section of
9a + CCl4 group.
895.03; 1H-NMR (DMSO-d6, 300 MHz, ppm) d: 7.68 (s, 1H,
H-14), 6.81–6.72 (dd, 1H, J = 10.1 Hz, 15.8 Hz, H-11),
6.19–6.13 (d, 1H, J = 15.8 Hz, H-12), 4.89 (s, 2H, H-15),
4.77 (s, 1H, H-17a), 4.46 (s, 1H, H-17b), 4.57–4.51 (dd, 1H,
J = 4.5 Hz, 11.7 Hz, H-3), 4.37–4.33 (d, 1H, J = 11.3 Hz,
H-19a), 4.13–4.09 (d, 1H, J = 11.3 Hz, H-19b), 3.58–3.55
(t, 4H, J = 4.5 Hz, H-CH2OCH2), 3.22 (2H, s, H-CH2CO),
2.49–2.46 (t, 4H, J = 4.5 Hz, H-CH2NCH2), 2.42–3.37 (d,
1H, J = 13.7 Hz, H-9), 2.01 (s, 3H, H-CH3CO), 2.05–1.22
(m, 8H, H-1, 2, 6, 7), 0.96 (s, 3H, H-18), 0.80 (s, 3H, H-20);
13C-NMR (DMSO-d6, 75 MHz, ppm) d: 172.9, 170.3,
170.2, 149.0, 147.4, 134.2, 127.6, 122.0, 108.8, 79.6,
70.6, 66.6, 64.4, 60.5, 59.3, 53.7, 53.0, 41.5, 38.6, 38.0,
36.5, 24.2, 23.9, 22.8, 21.4, 15.2.
Figure 4: The relationship between aqueous solubility and in vivo
hepatoprotective activity.
3-O-acetyl-19-O-(2-piperidinoacetyl)
3-O-acetyl-19-O-(2-pyrrolidinoacetyl)
dehydroandrographolide (13b)
dehydroandrographolide (13c)
Eluent from PE/EA 1:2, yellow solid, yield: 63%, mp
143–145 °C, MS (ESI) m/z: 500.3 (M + H)+; IR (KBr,
cmÀ1) m: 3082.92, 1751.51, 1727.46, 1451.57, 1375.30,
1245.89, 1181.36, 1086.78; 1H-NMR (DMSO-d6,
300 MHz, ppm) d: 7.68 (s, 1H, H-14), 6.81–6.73 (dd,
1H, J = 10.1 Hz, 15.8 Hz, H-11), 6.19–6.14 (d, 1H,
J = 15.8, H-12), 4.89 (s, 2H, H-15), 4.77 (s, 1H, H-17a),
4.46 (s, 1H, H-17b), 4.57–4.51 (dd, 1H, J = 4.4 Hz,
11.4 Hz, H-3), 4.34–4.30 (d, 1H, J = 11.6 Hz, H-19a),
4.13–4.09 (d, 1H, J = 11.6 Hz, H-19b), 3.16 (s, 2H, H-
NCH2CO), 2.50–2.42(m, 4H, H-CH2NCH2), 2.01 (s, 3H,
H-CH3CO), 1.77–1.27 (m, 14H, H-1, 2, 6, 7, piperidine-
CH2CH2CH2), 0.96 (s, 3H, H-18), 0.80 (s, 3H, H-20);
13C-NMR (DMSO-d6, 75 MHz, ppm) d: 172.9, 170.6,
170.3, 149.0, 147.4, 134.2, 127.6, 122.0, 108.8, 79.6,
70.7, 66.6, 64.3, 60.5, 53.9, 41.5, 38.0, 36.6, 26.0,
24.3, 23.9, 22.8, 21.3, 15.2.
Eluent from PE/EA 1:3, yellow solid, yield: 63%, mp 125–
126 °C, MS (ESI) m/z: 486.2 (M + Na)+; IR (KBr, cmÀ1) m:
3080.01, 1752.43, 1729.55, 1400.10, 1245.27, 1182.67,
1085.64; 1H-NMR (DMSO-d6, 300 MHz, ppm) d: 7.68 (s,
1H, H-14), 6.81–6.72 (dd, 1H, J = 10.1 Hz, 15.8 Hz,
H-11), 6.19–6.13 (d, 1H, J = 15.8 Hz, H-12), 4.89 (s, 2H,
H-15), 4.77 (s, 1H, H-17a), 4.57–4.51 (dd, 1H, J = 4.6 Hz,
11.8 Hz, H-3), 4.46 (s, 1H, H-17b), 4.38–4.34 (d, 1H,
J = 11.3 Hz, H-19a), 4.11–4.07 (d, 1H, J = 11.3 Hz,
H-19b), 3.31 (s, 2H, H-CH2CO), 2.55–2.51 (t, 4H,
H-CH2NCH2), 2.42–2.38 (d, 1H, J = 13.6 Hz, H-9), 2.05
(s, 3H, H-CH3CO), 1.71–1.66 (m, 4H, H-pyrrolidine-
CH2CH2), 1.79–1.20 (m, 8H, H-1, 2, 6, 7), 0.96 (s, 3H,
H-18), 0.80 (s, 3H, H-20); 13C-NMR (DMSO-d6, 75 MHz,
ppm) d: 172.9, 170.5, 170.3, 149.0, 147.4, 134.2, 127.6,
122.0, 108.8, 79.6, 70.6, 64.3, 60.5, 56.3, 53.7, 53.6,
41.5, 38.6, 38.00, 36.5, 24.2, 23.9, 22.7, 21.3, 15.3.
330
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Andro Derivatives as Potent Hepatoprotective Agents
3-O-acetyl-19-O-[2-(4-hydroxypiperidino)acetyl]
dehydroandrographolide (13d)
the international rules considering animal experiments and
the internationally accepted ethical principles for laboratory
animal use and care.
Eluent from PE/EA/MeOH 5:15:1, yellow solid, yield: 73%,
mp 81–82 °C, MS (ESI) m/z: 516.3 (M + Na)+; IR (KBr,
cmÀ1) m: 3447.78, 1751.22, 1400.95, 1245.78, 1187.68,
1077.16, 1031.67; 1H-NMR (CDCl3-d1, 300 MHz, ppm) d:
7.18 (s, 1H, H-14), 6.96–6.88 (dd, 1H, J = 10.1 Hz,
15.8 Hz, H-11), 6.16–6.11 (d, 1H, J = 15.8 Hz, H-12),
4.82 (s, 2H, H-15), 4.81 (s, 1H, H-17a), 4.63–4.56 (1H,
H-3), 4.56 (s, 1H, H-17b), 4.42–4.38 (d, 1H, J = 11.3 Hz,
H-19a), 4.25–4.21 (d, 1H, J = 11.3 Hz, H-19b), 3.20 (s,
2H, H-NCH2CO), 2.85–2.81 (m, 1H, H-CHOH), 2.44–2.33
(m, 4H, H-CH2NCH2), 2.04 (s, 3H, H-CH3CO), 2.04–1.90
(m, 2H, H-CH2CHOHCH2), 1.82–1.13 (m, 10H, H-1, 2, 6,
7, CH2CHOHCH2), 1.02 (s, 3H, H-18), 0.89 (s, 3H, H-20);
13C-NMR (CDCl3-d1, 75 MHz, ppm) d: 172.2, 170.5,
170.4, 147.8, 143.2, 135.5, 129.2, 121.4, 109.3, 80.0,
69.6, 67.2, 64.9, 61.7, 59.4, 54.7, 50.8, 41.4, 38.6, 38.2,
36.7, 24.2, 23.9, 22.7, 21.2, 15.2.
CCl4-induced acute liver damage model in mice
and investigated compounds treatment
Mice were divided into 15 groups (n = 8). Group
1
(normal control) animals were administered a single dose
of water (25 mL/kg, p.o.) daily for 7 days and received
peanut oil (10 mL/kg, i.p.) on day 7. Group 2 (CCl4 con-
trol) received water (25 mL/kg, p.o.) once daily for
7 days and received 0.2% CCl4 in peanut oil (10 mL/kg,
i.p.) on day 7. Group 3 (positive control) animals received
standard drug silymarin (a drug commonly used in the
treatment for liver diseases, 100 mg/kg, p.o.) once daily
for 7 days. Groups 4–15 animals were administered 100
mg/kg of andrographolide or its derivatives once daily for
7 days, respectively. Group 3–15 received 0.2% CCl4 in
peanut oil (10 mL/kg, i.p.) after 2 h of administration of
the silymarin, andrographolide, and its derivatives on day
7 (20).
Aqueous solubility
Each target compound measuring 100 mg in accurate
weight was placed in a 50-mL beaker. Then, 10 mL water
was added and exposed to ultrasonic oscillation for 5 min
and stirred for 24 h in a 25 °C constant temperature water
bath. It was centrifuged at 12 000 9 g for 10 min, and
the supernatant was filtered through a microporous mem-
brane and injected to the HPLC system for determination
(17).
All animals were sacrificed 24 h after the treatment, and
blood samples were collected immediately, and the livers
were removed for the histopathological analysis.
Biochemical assays
The blood samples collected were centrifuged at 1500 9 g
for 10 min to obtain serum, which would be used for the
assessments of ALT and AST.
Biological evaluation
In this study, CCl4-induced acute liver injury in male Kun-
ming mice was introduced as an animal model to investi-
gate the protective effect of andrographolide and its
derivatives, and silymarin was the positive control (18,19).
Histopathological studies
Liver slices were fixed in 10% formalin and embedded in
paraffin wax. Sections of 5 micron thickness was made
using a microtome and stained with hematoxylin–eosin
and observed under microscope.
Drug and chemical agents
Compounds 5a, 5b, 9a–d, and 13a–d were synthesized
and identified in our laboratory. Andrographolide and
silymarin were purchased from Nanjing Zelang Technol-
ogy Co. Ltd. (Nanjing, China). CCl4 and peanut oil were
purchased from J & K Scientific Ltd. (Beijing China).
Assay kits for determination of aminotransferase (AST)
and alanine aminotransferase (ALT) were supplied by
Nanjing Jiancheng Biotechnology Institute (Nanjing,
China).
Statistical analysis
The results are presented as mean Æ SEM of the studied
group. Statistical analyses of the results were performed
using ANOVA with Tukey–Kramer multiple comparisons test.
Results and Discussion
Chemistry
In total, 10 compounds (5a, 5b, 9a–d, 13a–d) were
designed and synthesized based on chemical and physical
properties and structural features of Andro, and their syn-
thesis routes and chemical structures are shown in
Schemes 1–3, respectively. The details of the synthetic
procedures and structural characterizations (IR, 1H-NMR,
13C-NMR, mass spectroscopy) are described in synthetic
procedures of target compounds section.
Test animals
Male Kunming mice (20 Æ 2 g) were obtained from Nanj-
ing Qinglongshan animal center, Nanjing, China. The mice
were selected for the study and maintained at a controlled
temperature of 25 Æ 2 °C and constant humidity (50–
70%) under a 12-h light–dark cycle, with free access to
diet and water. The animal study was performed accord
Chem Biol Drug Des 2014; 83: 324–333
331

Tang et al.
1
Aqueous solubility
by ESI-MS, IR, H-NMR, and 13C-NMR and also evaluated
The solubility of these derivatives in water is shown in Fig-
ure 2. As expected, the aqueous solubility of synthetic
derivatives was effectively improved. Especially, com-
pounds 9d and 13d exhibited at least ten-fold of solubility
compared with Andro.
for their biological activity as hepatoprotective agents. The
results revealed that some of the derivatives exhibited
more potent hepatoprotective activity compared with an-
drographolide, especially compounds 9d and 13d. The
data analysis indicated no clear SAR for these com-
pounds. The aqueous solubility of andrographolide deriva-
tives was also measured, and result analysis indicated that
solubility is a significant factor for in vivo activity; suitable
aqueous solubility may benefit the absorption and bioavail-
ability of the agents, which indirectly improve the hepato-
protective activity in mice. In addition, andrographolide has
multiple biological activities and can act as multiple target
compound that has significant immunomodulatory effect
which may display indirectly hepatoprotective potency in
vivo, so further studies on biological evaluation, structure
activity relationship, and mechanism of this new class of
compounds are currently in progress, and the results will
be reported in due course.
Biological evaluation
Biochemical assays
All derivatives used in the study decreased the ALT and
AST levels in serum compared with the CCl4 model group,
especially 9d and 13d, among which compound 9d was
the most active derivative, its effect on decreasing the level
of ALT and AST was significantly higher than Andro, and
its effect on decreasing the level of ALT was no significant
difference with silymarin (Table 1).
Histopathological studies
The cellular architecture of the liver tissue of different group
of mice as studied by the histopathological analysis is pre-
sented in Figure 3. The liver tissues of untreated group (Fig-
ure 3A) showed normal architecture of hepatic cells with
clear cytoplasm and slightly dilated central veins, prominent
nucleus, and nucleolus. In CCl4-treated group (Figure 3B),
the liver showed that distorted architecture with markedly
congested central veins, extensive area of necrosis, and
hemorrhage nuclei was distorted, and some of the hepato-
cytes contain vacuolated cytoplasm. Figure 3C presents the
section of group treated with CCl4 and silymarin, slight
necrosis and neutrophil infiltration can be found, and section
of this group was nearly comparable to the control group. In
Figures 3D and 9d CCl4-treated group section showed
moderate degree of damage, and the liver damage was
greatly attenuated, just with local neutrophil infiltration and
necrosis. The histopathological observations supported the
result obtained on induction of hepatotoxicity by CCl4 and
reversal of the toxicity by silymarin and 9d as discernible
from the levels of serum ALT and AST.
Acknowledgments
This study was supported by the National Natural Science
Foundation of China (No. 30772647 and No. 81001363),
the Project Program of State Key Laboratory of Natural Med-
icines, China Pharmaceutical University (No. JKGZ201103),
Program Granted for Scientific Innovation Research of
College Graduate in Jiangsu Province (CXLX11_0785), and
Independent Research Funds for Youth of Jiangnan
University (JUSRP1039).
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