Synthesis of andrographolide derivatives and their TNF-α and IL-6 expression inhibitory activities

Article abstract of DOI:10.1016/j.bmcl.2007.10.009

The synthesis of a series of andrographolide derivatives was described and their inhibitory effects on TNF-α and IL-6 secretion in mouse macrophages were also evaluated. Most of the tested compounds showed inhibitory effects, and the compounds with the structure of 12-hydroxy-14-dehydroandrographolide showed better inhibitory activity than the compounds with the structure of isoandrographolide.

Full text of DOI:10.1016/j.bmcl.2007.10.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6891–6894
Synthesis of andrographolide derivatives and their TNF-a
and IL-6 expression inhibitory activities
Jing Li,a Wenlong Huang,a Huibin Zhang,a,* Xinyang Wangb and Huiping Zhouc
aCenter of Drug Discovery, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
bMedical College of Nantong University, Nantong 226000, China
cDepartment of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA
Received 26 May 2007; revised 31 August 2007; accepted 3 October 2007
Available online 5 October 2007
Abstract—The synthesis of a series of andrographolide derivatives was described and their inhibitory effects on TNF-a and IL-6
secretion in mouse macrophages were also evaluated. Most of the tested compounds showed inhibitory effects, and the compounds
with the structure of 12-hydroxy-14-dehydroandrographolide showed better inhibitory activity than the compounds with the struc-
ture of isoandrographolide.
Ó 2007 Elsevier Ltd. All rights reserved.
Andrographolide 1 is a bicyclic diterpenoid lactone iso-
lated from leaves of Andrographis paniculata,1 which is
used extensively in the traditional systems of Chinese
medicine. Andrographolide was reported to possess a
wide spectrum of biological activities including antibac-
terial, antiinflammatory, antimalarial, immunomodula-
tion, antithrombotic, and hepatoprotective effect. It is
mainly used to treat pediatric pneumonia and respira-
tory tract infection in clinic.2–9
Recently, it has been reported that andrographolide can
decrease the expression of TNF-a, IL-1, IL-6, IL-8, IL-
12 at mRNA levels in a concentration-dependent man-
ner in murine macrophages via suppression of the
ERK1/2, PI3K-AKt signaling pathway.17–19 However,
the effect of the andrographolide derivatives on LPS-in-
duced TNF-a and IL-6 expression has not been exam-
ined so far. Herein, in this paper, we report the
synthesis of a series of andrographolide derivatives
and their potential inhibitory effects on LPS-induced
TNF-a and IL-6 release in mouse macrophages.
TNF-a and IL-6 are two major pro-inflammatory medi-
ators secreted by macrophages upon stimulation with
microbial infections such as LPS. These two pro-inflam-
matory cytokines have a wide array of functions. For
example, TNF-a can induce apoptosis and the secretion
of cytokines such as IL-1, IL-6, and IL-10; it can also
activate T cells and other inflammatory cells. However,
an overabundance of TNF-a and IL-6 is correlated with
the development of various diseases. It is reported that
TNF-a inhibitors can be used to treat many diseases
such as rheumatoid arthritis, diabetes, sepsis, Alzhei-
mer’s disease, tumor, and obesity,10–13 while the IL-6
inhibitors can be used in Alzheimer’s disease, psychiatric
disorders, cancer, diabetes, and depression.14–16
The 14-hydroxy of andrographolide is chemically unsta-
ble, it can be rearranged to form isoandrographolide,
which is much more stable than andrographolide. Isoan-
drographolide has similar biological activities to androg-
rapholide, such as antiinflammatory and anticancer
properties.20 In addition, the 14-hydroxy can also be rear-
ranged to form 12-hydroxy-14-dehydroandrographolide
easily. It has been reported that 12-hydroxy-14-dehydro-
andrographolide possessed similar biological activity of
anticancer asandrographolide.21 Therefore, isoandrogra-
pholide and 12-hydroxy-14-dehydroandrographolide
were chosen as the lead generation here, and their deriva-
tivesweresynthesized. The synthesis of these derivativesis
summarized in Schemes 1–3. All the prepared compounds
were characterized by spectroscopic tools.22
Keywords: TNF-a; IL-6; Inhibitors; Andrographolide; Derivatives;
Synthesis.
Andrographolide (1) was treated with cons. hydrochlo-
ric acid at room temperature to afford isoandrographo-
lide (2) in 70% yield (Scheme 1), 2 was followed by
*
Corresponding author. Tel.: +86 25 85230904; e-mail: zhanghb80@
163.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.009

6892
J. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6891–6894
O
O
O
HO
O
O
O
b
a
O
O
H
H
H
R1COO
HO
HO
HO
HO
1
2
3a-f
R2COO
c
R1
CH3
CH2CH3
R2
CH3
O
3a
3b
3c
3d
3e
3f
O
CH2CH3
CH2(CH2)3CH3
C6H5
CH2(CH2)3CH3
O
C6H5
H
H
C6H5
O
S
O
O
3g
O
O
CH2CH2COOH
CH2CH2COOH
Scheme 1. Reagents and conditions: (a) cons. HCl, rt; (b) for 3a–c, 3f: (R1CO)2O, CH2Cl2, reflux, for 3d and 3e: R1COCl, CH2Cl2, reflux; (c) 3g:
SOCl2, reflux.
O
O
O
O
O
O
O
O
a
b
c
O
O
O
O
H
H
H
H
HO
HO
HO
RCOO
2
RCOO
4
5h-j
3h-j
O
O
HO
R=CH3
3h
R=CH2CH3
R=CH2(CH2)3CH3
3i
3j
Scheme 2. Reagents and conditions: (a) TrCl, N-methylmorpholine, CH2Cl2, rt; (b) (R1CO)2O, DMAP, CH2Cl2, rt; (c) HCOOH, CH2Cl2, rt.
O
O
O
O
HO
HO
O
O
O
O
OH
OOCR
a
b
c
H
H
H
H
O
HO
HO
O
O
1
6
7
8a-h
O
O
O
O
R=CH3
9e
9f
R=
R=
9a
9b
9c
9d
O
NO2
R=CH2CH3
CH3
OOCR
d
R=CH2(CH2)3CH3
R=C6H5
9g
9h
O
O
R=
H
R=
O
O
HO
HO
O2N
9a-h
Scheme 3. Reagents and conditions: (a) (CH3)2C(OCH3)2, PPTS, CH2Cl2, reflux; (b) PDC, CH2Cl2, reflux; (c) for 8a–c: (RCO)2O, CH2Cl2, rt, for 8d:
RCOCl, CH2Cl2, rt, for 8e–h: RCOOH, DCC, DMAP, CH2Cl2, rt; (d) CH3COOH/H2O, rt.

J. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6891–6894
6893
Table 1. Mouse macrophage TNF-a inhibition assay data for
andrographolide and its derivatives
esterification with different acid anhydrides, acyl chlo-
rides, or SOCl2 to obtain 3a–g in good yields.
Normal (%)
LPS-induced TNF-a (%)
Treatment of 2 with triphenylchloromethane in the pres-
ence of N-methylmorpholine in CH2Cl2 at room temper-
ature afforded trityl ether 4 in 85% yield (Scheme 2).
Compound 4 was reacted with acetic anhydride, propi-
onic anhydride, or hexanoic anhydride in the presence
of 4-dimethylaminopyridine (DMAP) in CH2Cl2 at
room temperature to obtain corresponding esters 5h–j
in almost quantitative yield. Esters 5h–j were further
converted to 3h–j in 75% by hydrolysis of the trityl ether
with formic acid in CH2Cl2. To prepare 5h–j, it would
take much long time if DMAP is not added whether
the reaction was carried out at room temperature or at
refluxing temperature. The steric hindrance of the trityl
ether at C-19 was considered as a major factor.
Control
Andrographolide
100
100
63.94 2.60
80.03 2.13
82.78 2.21
35.66 2.04**
203.54 2.68
28.54 1.92**
81.74 2.09
61.82 2.08
44.40 3.23**
106.65 2.05
69.97 2.21
79.62 2.51
92.88 2.22
65.78 2.86
152.29 2.52
148.86 2.47
115.10 3.90
62.54 2.00
99.43 3.25
114.69 2.33
43.75 2.63**
293.69 2.77
36.45 2.23**
128.62 2.89
230.56 2.96
105.88 2.37
62.68 2.55
96.87 3.13
75.95 2.81
60.8 2.34
3a
3b
3c
3d
3e
3f
3g
3j
9a
9b
9c
9d
9e
9f
59.96 2.74
41.60 2.50**
61.03 1.82
94.36 2.80
9g
9h
Reaction of 1 with 2,2-dimethoxypropane in the pres-
ence of pyridinium p-toluenesulfonate (PPTs) in CH2Cl2
at 40 °C gave compound 6 in high yield (Scheme 3).
Treatment of 6 with pyridinium dichromate (PDC) in
refluxing CH2Cl2 obtained 7 in 50% yield. Compound
7 was followed by esterification with acid anhydrides,
acyl chlorides or with carboxylic acids in the presence
of dicyclohexylcarbodiimide (DCC) and DMAP to give
compounds 8a–h. For example, a mixture of 7, 4-nitro-
benzoic acid, DCC, and DMAP in CH2Cl2 was stirred
overnight at room temperature to obtain 8a in good
yield. Compounds 8a–h were treated with aq acetic acid
(acetic acid/water = 7:3) at room temperature for 30 min
and afforded 9a–h in almost quantitative yield.
Each value represents mean SD of four determinations. **P < 0.01
compared to andrographolide.
Table 2. Mouse macrophage IL-6 inhibition assay data for androg-
rapholide and its derivatives
Normal (%)
LPS-induced IL-6 (%)
Control
Andrographolide
100
100
56 2.20
49.21 3.67
72.87 2.52
60.53 2.25
31.39 3.67**
48.20 1.92
27.67 2.38**
55.02 2.69
79.66 2.12
69.80 3.11
95.10 2.95
82.56 2.14
82.93 2.67
81.97 3.51
58.56 2.80
127.68 2.83
96.05 1.60
70.14 2.36
3a
3b
3c
3d
3e
3f
150.15 3.13
110.25 2.51
85.25 1.57
107.19 3.52
85.71 2.81
114.12 2.77
207.64 3.09
101.81 2.50
44.73 2.11**
74.67 2.54
46.61 2.15**
37.53 2.65**
29.21 2.04**
32.1 2.93**
56.87 2.38
44.64 2.22**
The effects of andrographolide and its derivatives on
LPS-induced TNF-a and IL-6 expression in mouse
J774A.1 cells were examined using ELISA. Cells were
treated with 20 lM of andrographolide, its derivatives,
or vehicle control for 24 h. At the end of the treatment,
the culture media were collected and centrifuged at
14,000 rpm for 5 min. TNF-a and IL-6 levels in the med-
ia were determined by ELISA using mouse TNF-a and
mouse IL-6 ELISA MaxTM Set Deluxe Kits (Biolegend).
The total protein concentrations of the viable cell pellets
were determined using Bio-Rad protein assay reagents.
Total amounts of the TNF-a and IL-6 in the media were
normalized to the total protein amounts of the viable
cell pellets.
3g
3j
9a
9b
9c
9d
9e
9f
9g
9h
Each value represents mean SD of four determinations. **P < 0.01
compared to andrographolide.
The results (Tables 1 and 2) showed that andrographo-
lide and its derivatives inhibited LPS-induced TNF-a
and IL-6 expression to various degrees in mouse macro-
phages. Among these compounds, 3c, 3e, and 9f with the
% inhibition as 43.75, 36.45, and 41.60, respectively,
were more potent than andrographolide (62.54%) in
inhibiting LPS-induced TNF-a expression; 9a, 9d, 9e,
9f, and 9h with the % inhibition as 44.73, 37.53, 29.21,
32.1, and 44.64, respectively, were more potent than
andrographolide (56%) in inhibiting LPS-induced IL-6
expression. For the isoandrographolide derivatives 3a–
j, 3c having hexanoyl moieties at C-3 and C-19 showed
better inhibitory activity in both TNF-a and IL-6
expression compared to its mono-hexanoyl derivative
3j, indicating that both hydroxyls in isoandrographolide
need to be converted into esters for potent activity. But
for 3d and 3e, with di-benzoyl groups at C-3 and C-19 in
3d and mono-benzoyl group in 3e, the contradictive re-
sults were presented. The hydrophilic analog 3f, in
which carboxyl groups were introduced at C-3 and
C-19, displayed almost no inhibitory acitivity in LPS-in-
duced TNF-a and IL-6 expression, indicating that the
improvement of aqueous solubility of isoandrographo-
lide would lead to a decline in inhibitory effects, and
the introduction of cyclosulfurous substituent (3g) is
deleterious to the inhibitory activity of isoandrographo-
lide. For the derivatives of 12-hydroxy-14-dehydroan-
drographolide 9a–h, the compounds having aryl
moiety at 12-C (e.g., 9d, 9e, 9f) showed better inhibitory

6894
J. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6891–6894
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activities than the compounds having alkyl moiety at 12-
C (e.g., 9a, 9b, 9c) in both TNF-a and IL-6 expression,
suggesting that the electron-withdrawing group at 12-C
might be good for potent activity of 12-hydroxy-14-
dehydroandrographolide. However, the underlying
mechanisms by which andrographolide and its deriva-
tives inhibited LPS-induced TNF-a and IL-6 expression
remain unknown and are the focus of our current
research.
17. Lin, H. Q.; Ling, K.; Guo, J. S.; Zheng, T. W.; Bao, X. G.
Biol. Pharm. Bull. 2006, 29, 220.
18. Sheeja, K.; Kuttan, G. Integr. Cancer Ther. 2006, 5, 244.
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US6576662B2, 2003.
In conclusion, a series of andrographolide derivatives
have been synthesized and their inhibitory effects on
LPS-induced TNF-a and IL-6 expression in mouse mac-
rophages have been evaluated. The data analysis indi-
cated no clear SAR for these compounds, but the
compounds with the structure of 12-hydroxyl-14-dehy-
droandrographolide showed better inhibitory activity
than the compounds having the structure of isoandrog-
rapholide, suggesting the structure of 12-hydroxy-14-
dehydroandrographolide might be a good generation
for further optimization.
22. Analytical data for 3e: mp: 185–186 °C; IR (KBr, cmÀ1):
3554, 3105, 2938, 1755, 1708, 1470, 1295, 1124; 1H
NMR(CDCl3, 300 MHz) d: 7.97 (d, 2H, Ph), 7.65–7.45
(t, 3H, Ph), 7.27 (s, 1H, 14-H), 4.83 (s, 2H, 15-H), 4.70–
4.66 (m, 3H, 12-H, 3-H, 19-H), 4.44 (d, 1H, 19-H), 3.40
(br, 1H, 3-OH), 1.29 (s, 3H, 17-CH3), 1.13 (s, 3H, 18-
CH3), 1.04 (s, 3H, 20-CH3); EIMS: 477.2 [M+Na]+.
Analytical data for 9e: mp: 119–122 °C; IR (KBr, cmÀ1):
3409, 3111, 2950, 2873, 1729, 1625, 1545, 1344, 1279, 897;
1H NMR(CDCl3, 300 MHz) d: 9.25–9.14 (m, 4H, Ph),
7.50 (s, 1H, 14-H), 6.00 (t, 1H, J = 6.3 Hz, 12-H), 4.98 (s,
1H, 17-H), 4.91 (s, 2H, 15-H), 4.86 (s, 1H, 17-H), 4.18 (t,
1H, J = 7.2 Hz, 3-H), 3.43 (t, 1H, J = 9.9 Hz, 19-H), 3.30
(t, 1H, J = 9.9 Hz, 19-H), 1.23 (s, 3H, 18-CH3), 0.68 (s,
3H, 20-CH3); EIMS: 500.2 [M+H]+. Analytical data for
9f: mp: 176–178 °C; IR (KBr, cmÀ1): 3472, 2938, 2866,
1744, 1700, 1636, 1445, 1200, 1037, 828; 1H NMR(CDCl3,
300 MHz) d: 7.85 (d, 2H, J = 7.2 Hz, Ph), 7.41 (m, 3H, Ph,
14-H), 5.92 (t, 1H, J = 5.7 Hz, 12-H), 4.93 (s, 1H, 17-H),
4.84 (s, 3H, 15-H, 17-H), 4.18 (d, 1H, J = 11.4 Hz, 3-H),
3.49 (t, 1H, J = 7.5 Hz, 19-H), 3.31 (d, 1H, J = 10.8 Hz,
19-H), 2.38 (s, 3H, Ph–CH3), 1.23 (s, 3H, 18-CH3), 0.65 (s,
3H, 20-CH3); EIMS: 491.2 [M+Na]+.
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