Synthesis and discovery of andrographolide derivatives as non-steroidal farnesoid X receptor (FXR) antagonists

Article abstract of DOI:10.1039/c3ra46715e

Based upon the discovery of the natural compound andrographolide (1) as a non-steroidal farnesoid X receptor (FXR) antagonist, a series of andrographolide derivatives were designed and synthesized accordingly. Our primary SAR studies demonstrated that 14-phenoxy andrographolide scaffold is an excellent structural pharmacophore for FXR antagonists. Remarkably, 14β-compounds of 12b, 12f and 10g were found to be the most potent FXR antagonists in this work. Structural docking discovered that the phenoxy substitution at the 14-position and the modification at 3,19-positions altered the putative binding positions of small FXR ligands, resulting in their FXR antagonistic activity discrepancy. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ra46715e

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PAPER
Synthesis and discovery of andrographolide
derivatives as non-steroidal farnesoid X receptor
Cite this: RSC Adv., 2014, 4, 13533
(FXR) antagonists†
Zhuyun Liu,‡a Wai-Kit Law,b Decai Wang,a Xin Nie,a Dekuan Sheng,a Genrui Song,a
c
c
c
b
a
*
*
Kai Guo, Ping Wei, Pingkai Ouyang, Chi-Wai Wong and Guo-Chun Zhou
Based upon the discovery of the natural compound andrographolide (1) as a non-steroidal farnesoid X
receptor (FXR) antagonist, a series of andrographolide derivatives were designed and synthesized
accordingly. Our primary SAR studies demonstrated that 14-phenoxy andrographolide scaffold is an
excellent structural pharmacophore for FXR antagonists. Remarkably, 14b-compounds of 12b, 12f and
10g were found to be the most potent FXR antagonists in this work. Structural docking discovered that
the phenoxy substitution at the 14-position and the modification at 3,19-positions altered the putative
binding positions of small FXR ligands, resulting in their FXR antagonistic activity discrepancy.
Received 15th November 2013
Accepted 28th January 2014
DOI: 10.1039/c3ra46715e
www.rsc.org/advances
acids are high.1–5 It is reported that FXR has an active role in
regulating cholesterol, lipoprotein, and glucose metabolism
and its association with liver disease,22,23 liver regeneration24
Introduction
Nuclear receptors are classied as ligand-activated transcrip-
tion factors that regulate the expression of specic target genes
and are involved in several physiological functions including
reproduction, development, and metabolism.1 Farnesoid X
receptor (FXR), also known as bile acid receptor (BAR) or
NR1H4, is a nuclear receptor that is encoded by the NR1H4 gene
in humans and expressed at high levels in the liver, intestine
and other cholesterol-rich tissues.1,2 Cholic acid (CA), cheno-
deoxycholic acid (CDCA), and other bile acids are natural
ligands2–5 for FXR (Fig. 1).
Aer activated by bile acid ligand/s,2–5 FXR translocates to
the cell nucleus, where FXR recruits retinoid X receptor (RXR) to
form a heterodimer and then binds to hormone response
elements on DNA, which up- or down-regulates the expression
of certain genes.1,2 The down-regulation of CYP7A1 (ref. 21)
(cholesterol 7a-hydroxylase) of the rate-limiting enzyme in bile
acid synthesis from cholesterol is one of the primary functions
of FXR activation, resulting in a negative feedback in which
synthesis of bile acids is inhibited if the cellular levels of bile
and tumorigenesis,25 suggesting that FXR represents an attrac-
tive pharmacological target.26,27
Currently, most studies are focused on FXR agonists
(Fig. 1).6–11 Because it is possible that FXR over-regulation cau-
ses some diseases and an unnatural FXR agonist diverts the
normal pathway to result in potentially undesirable side
effects,25–28 it is necessary to develop FXR antagonists as FXR
modulators.
The known FXR antagonists12–20 (Fig. 1) are mainly derived
from a steroidal scaffold and a nite number of other structural
skeletons have been reported in recent years. Thus, seeking
potent, structurally diverse and non-steroidal FXR antagonists
should be an important step forward in this eld. The utiliza-
tion of natural products as a source of structural or functional
diversity for the design and synthesis of novel molecules has
been an important aspect of new drug design.29,30 So as to
increase structural diversity of FXR antagonists, we launched a
project initiated by the high-throughput screening of the
natural compound libraries from a series of Traditional Chinese
Medicines (TCMs) based on FXR luciferase activity assay.31
Andrographolide (1, Fig. 1),32,33 a diterpene ester from Androg-
raphis panniculata (Burm.f.) Nees, was identied as a weak FXR
antagonist (IC50 ¼ 9.7 mM, Fig. 2).
aSchool of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, P. R.
China. E-mail: gczhou@njtech.edu.cn; Tel: +86-25-58139415
bNeuMed Pharmaceuticals Limited, Shatin, N. T., Hong Kong, P. R. China. E-mail:
wongcw@neumedpharma.com.hk; Tel: +852-67487218
cCollege of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China
† Electronic supplementary information (ESI) available: Table S1, 1H NMR and
13C NMR spectra of compounds 5–13. CCDC 960845. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c3ra46715e
Andrographolide (1) is the representative active ingredient
of Andrographis panniculata (Burm.f.) Nees and it works as a
broad therapeutic agent.34,35 Although 1 has a molecular
weight of 350 Da, 3 hydrogen bond donors, 5 hydrogen bond
acceptors and 2.1186 of the calculated Log P (Table S1†, entry
23), which conforms to the “Rule of Five”, its poor water-
solubility and also relatively low lipo-solubility, weak potency
‡ Current address: Department of Pharmaceutical and Chemical Engineering,
Taizhou Polytechnic College, Taizhou 225300, Jiangsu, PR China.
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Fig. 1 Natural and synthetic known ligands for FXR and general structures of andrographolide and their derivatives.
and inadequate therapeutic efficacy restrict its further appli- Herein, we describe the synthesis and SAR studies of 14-ary-
cation. To improve its physiochemical properties and phar- loxy andrographolide derivatives (3 and 4, Fig. 1), leading to
maceutical features, numerous andrographolide derivatives the identication of a series of 14-phenoxy andrographolide
have been reported from time to time, especially preparation derivatives as more potent FXR antagonists than the lead
of 14-acyloxy andrographolide derivatives36–40 (2, Fig. 1). andrographolide (1).41
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acetylated 3,9-diol analogue
7
in antagonizing FXR. By
Results and discussion
comparison of 14b-andrographolide (8) with its modied
derivatives (6, 7, 9), it was discovered that 3,19-isopropylidene
modication alone (9) almost did not change the antagonistic
activity and 14-acetylation modication alone (7) decreased the
antagonistic activity, but simultaneous modications (6) of
3,19-isopropylidene and 14-acetylation enhanced somewhat the
antagonistic activity. It was inferred from these preliminary
data (Fig. 2 and Table 1, entries 23–28) that the optimal
combination of 3,19- and 14-modications including 14a- or
14b-conguration is possibly helpful for the FXR antagonistic
activity.
In our preliminary study, andrographolide (1) was found to be a
weak FXR antagonist (IC50 ¼ 9.7 mM) in the concentration-
dependent mode (Fig. 2),31 this result spurred us on to discover
more potent FXR antagonists on the basis of andrographolide
derivatives. As 14-acyloxy andrographolide esters (2, Fig. 1) were
effective in some reported studies for other assays,36–38 we were
interested in investigating 14-modied andrographolide deriv-
atives as FXR novel antagonists.
In an initial exploration step, some simple derivatives of
andrographolide were studied (Scheme 1). Starting from 1, 3,19-
acetonylidene protection was conducted in DCM and catalyzed
by PPTS yielding 5 as reported.36–38 Inversion of 14a-OH of 5 into
14b-OAc of 6 was achieved by Mitsunobu reaction under normal
conditions. 14-Epimeric andrographolide (8) or its 14b-acety-
lated derivative (7) was generated in MeOH/H2O (4/1) with
TsOH$H2O by partial (at 20 ꢀC) or complete (40 ꢀC) hydrolysis of
6, respectively. Finally, 14b-3,19-acetonylidene derivative (9) was
made by the same preparation conditions as 5.
Fig. 2 The FXR antagonistic activities of compounds 1 and 5–9.
Control (100%) is the activity of FXR in the presence of 25.0 mM CDCA.
Considering that aryloxy ether is chemically more stable
than the acyloxy ester, we are interested in 14-aryloxy ether
analogues of andrographolide (Fig. 1) in our exploration.
Incrementing the structural diversity is the important superi-
ority of 14-aryloxy ether by the utilizing of varied aromatic
skeletons (e.g. phenyl, naphthyl, quinolinyl, pyridinyl, etc.) and
the incorporation of different substituents into aromatic scaf-
folds. In this work, 14-phenoxy andrographolide derivatives
(Ar ¼ Ph, Fig. 1, series 3 and 4) were rstly studied.
Scheme 1 Reagents and conditions: (a) anhydrous DCM, 2,2-dime-
thoxypropane, PPTS, 40 ꢀC; (b) anhydrous THF; 5 (1.0 eq.), anhydrous
HOAc (1.5 eq.), DIAD (1.5 eq.), PPh3 (1.5 eq.), 0 ꢀC to room temperature;
(c) MeOH/H2O (4/1), TsOH$H2O, 20 ꢀC; (d) MeOH/H2O (4/1),
TsOH$H2O, 40 ꢀC.
FXR antagonistic results of the synthesized compounds were
measured by the luciferase reporter assay31 and shown in
Table 1 (entries 23–28). The antagonistic potencies at 5.0 mM are
depicted in Fig. 2. Compared to 1, 3,19-isopropylidene
andrographolide (5) slightly improved the FXR antagonistic
activity. 14b-Epimeric andrographolide (8) was a much weaker
FXR antagonist than 1 and 14b-acetylated andrographolide (7)
became inactive to FXR. Unlike the relationship between 1 and
5, 3,19-isopropylidene 14b-andrographolide (9) was indeed a
Scheme 2 Reagents and conditions: (a) anhydrous THF; 5 or 9 (1.0
eq.), phenol (1.5 eq.), DIAD (1.5 eq.), PPh3 (1.5 eq.), 0 ꢀC to room
temperature; (b) MeOH/H2O (4/1), TsOH$H2O, 20 ꢀC.
As shown in Scheme 2, 14a-compound 5 or its 14b-epimer 9
weaker FXR antagonist than 8. 14b-Acetylated 3,19-iso- were used as starting materials for the synthesis of 14b- or 14a-
propylidene andrographolide (6) is more active than 14b- phenoxy andrographolide derivatives, respectively. The key
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synthetic approach to 14-phenoxy andrographolide derivatives the 14-(40-nitrophenoxy) group is an important pharmacophore
is the Mitsunobu reaction. Diverse phenols were used to (10a < 10b and 12a < 12b). In addition, it seems that the 3,19-
prepare two series of 14b-phenoxy (10) and 14a-phenoxy (11) acetonylidene group decreases the FXR antagonistic activity (10a
ꢀ
compounds. The reaction was performed in general from 0 C < 12a and 10b < 12b) for these compounds.
to room temperature in anhydrous THF and afforded 10 or 11 in
mild to moderate isolated yield depending on the distinct
phenol property. Removal of the protective group of 3,19-ace-
Table 1 FXR antagonistic activity of synthesized compounds 1, 5–9,
10–13
tonylidene from 10 or 11 by TsOH$H2O in MeOH/H2O (4/1) at
b
c
20 ꢀC, the series of compounds 12 or 13 were obtained in high
yields, respectively. It is worthwhile noting that a-isomers of 11
and 13 were generally less stable than their corresponding
b-isomers of 10 and 12.
Entry
Cmpda
R
14-Isomer
IC50
NA
CC50
1
2
3
4
5
6
7
8
10a
12a
10b
12b
10c
12c
10d
12d
10e
12e
10f
H
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
a
a
a
a
a
a
b
b
b
b
—
H
5.7
8.6
0.55
>15.0
>15.0
>15.0
—
40-NO2
40-NO2
In order to conrm the stereochemistry of C-14 in these
series, the single crystal of 10b was cropped from petroleum
ether/ethyl acetate (5/4) and its crystal structure was determined
as shown in Fig. 3a by X-ray crystallography, which is in accor-
dance with our expected structure. The crystal packing diagram
(Fig. 3b) of 10b demonstrated that the molecule 10b is stabilized
and linked by the intermolecular hydrogen bonds of C(9)–
H(b)/O(40-NO2) and C(14)–H(a)/O(lactone)–C(15).
30-NO2
NA
NA
3.0
NA
2.9
3.5
2.4
0.98
2.0
11.9
6.3
NA
3.9
NA
15.1
16.0
2.5
30-NO2
—
20-NO2
8.5
20-NO2
—
9
20-CO2Et
20-CO2Et
20-OCH3
12.3
>15.0
>15.0
>15.0
>15.0
>15.0
>15.0
—
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
12f
20-OCH3
10g
12g
10h
12h
10i
20-OCH3-40-NO2
20-OCH3-40-NO2
20-CH3-40-NO2
20-CH3-40-NO2
20-F-40-NO2
20-F-40-NO2
40-NO2
>15.0
—
12i
11b
13b
11g
13g
>15.0
>15.0
12.0
>15.0
>15.0
>15.0
>15.0
—
40-NO2
20-OCH3-40-NO2
20-OCH3-40-NO2
7.1
9.7
9.0
9.3
1, Andrographolide
5
6
7
8
9
NA
>15.0
>15.0
—
—
a
See Scheme 2. b FXR-transfected 293T cells were agonized by 25.0 mM
Fig. 3 Molecular and crystal structures (a) and crystal packing diagram
(b) of 10b. ESI.†
CDCA in all experiments and the vehicle control was set as 100%, “NA”
means “not active”. The 293T cells were used to test compound's
c
cellular toxicity, “—” represents “not detected”.
The calculated data of “Lipinski's Rule of Five” of 1 and these
synthesized compounds 5–9 and 10–13 are included in Table
S1.† Even some of these compounds do not conform very well to
Aer the above, 30- and 20-nitrophenoxy andrographolide
the “Rule of Five”; they have the potential to be optimized and derivatives 10c and 12c, 10d and 12d, respectively, were
developed into drug-like candidates in the future. FXR antago- designed to investigate the effects of different nitro positions on
nistic activity and the cellular toxicity of 1 and these synthesized the FXR antagonistic activity in comparison to 40-nitrophenoxy
compounds 5–9, and 10–13 are listed in Table 1.
analogues 10b and 12b. Among these, 10c and 12c of 30-nitro-
At rst, 14b-phenoxy andrographolide derivative 10a bearing phenoxy analogues, and 12d of the 20-nitrophenoxy analogue
the 3,19-acetonylidene group was found to be inactive (Table 1, did not express FXR antagonistic activity (Table 1, entries 5, 6
entry 1); however, its hydrolyzed product of 3,19-diol 12a (Table and 8); nevertheless, 10d of the 20-nitrophenoxy analogue with
1, entry 2) exhibited as a moderate FXR antagonist (IC50 ¼ 5.7 3,19-acetonylidene (Table 1, entry 7) showed a good FXR
mM), is better than our lead 1. Then, we moved to incorporate antagonistic activity (IC50 ¼ 3.0 mM) but it possessed an obvious
substituted groups into the phenyl ring at the 14-position to cellular toxicity (CC50 ¼ 8.5 mM). Based upon these results, we
investigate the structure–activity relationship (SAR). 40-Nitro- further explored other substitution at the 20-position of the
phenoxy compounds of 10b with 3,19-acetonylidene group and 14-phenoxy group. Compound 10e with an electron-with-
12b of 3,19-diol derivative were designed and tested. Compound drawing group of 20-carboxylic ester (Table 1, entry 9) exhibited
10b (IC50 ¼ 8.6 mM; Table 1, entry 3) is less potent than 12a but it similar FXR antagonistic activity (IC50 ¼ 2.9 mM) and cellular
is much more active than its corresponding counterpart 10a as a toxicity (CC50 ¼ 12.3 mM) to its 20-nitro counterpart 10d. 20-
FXR antagonist; notably, the IC50 value of 12b reached 0.55 mM Carboxylic ester 3,19-diol compound 12e (Table 1, entry 10),
(Table 1, entry 4) in the FXR antagonistic activity, indicating that which is different from its inactive 20-nitro-3,19-diol counterpart
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12d, acted as a good FXR antagonist (IC50 ¼ 3.5 mM) but did not corresponding mother 3,19-diol analogues 12b and 12f, suggest-
show any obvious cellular toxicity. Interestingly, an electron- ing that the “integration” of 20-methoxy and 40-nitro groups affects
donating group of 20-methoxy-substituted compound 10f with the binding ability of these small molecules to FXR. More
3,19-acetonylidene exhibited similar FXR antagonistic activity importantly, these data reveal that there are synergistic effects
(IC50 ¼ 2.4 mM, Table 1, entry 11) to 10d and 10e containing between substitutions at 14-phenoxy and 3,19-modications on
20-electron-withdrawing substitutions. Moreover, 20-methoxy- the antagonistic activities to the receptor FXR.
3,19-diol 12f was very active (IC50 ¼ 0.98 mM, Table 1, entry 12)
As part of exploring the effects of 14-phenoxy substitutions on
and its relatively low cellular toxicity was detected. Summarized the FXR antagonistic activity, substituents of the methyl group
from the above-mentioned results, it is concluded that the as a weak electron-donating group and uoro group as an elec-
mono substitution at 20-position is very exible to the electron- tron-withdrawing group were introduced at the 20-position into
donating group and the electron-withdrawing group for the FXR the 14-(40-nitro)-phenoxy group, respectively. The assay data
antagonistic activity, especially for 10 series compounds with disclosed that 20-methyl-40-nitro derivative 10h was a moderate
the 3,19-acetonylidene group.
FXR antagonist (IC50 ¼ 6.3 mM, Table 1, entry 15) and its diol
Inspired by these promising results of 12b and 12f bearing 40- derivative 12h lost its FXR antagonistic activity (Table 1, entry
nitro and 20-methoxy groups at 14-phenoxy, respectively, 16); meanwhile, introduction of 20-uoro at 14-(40-nitro)-phenoxy
20-methoxy-40-nitro-andrographolide derivatives 10g and 12g were made 10i bearing 3,19-acetonylidene (IC50 ¼ 3.9 mM, Table 1,
logically designed (Fig. 4) and tested for their FXR antagonistic entry 17) slightly more active than its 20-methyl counterpart 10h
activity (Table 1 and Fig. 4). We envisioned that the “integration” but its diol analogues 12i (Table 1, entry 18) had a lack of FXR
of 20-methoxy and 40-nitro groups should retain or increase the antagonistic activity as the 20-methyl counterpart diol 12h.
antagonistic activity. Compared to its mother analogues 10b (40-
Taking into consideration that 1 is a 14a-isomer, we subse-
nitro) and 10f (20-methoxy) with single substitution at 14-phenoxy, quently investigated 14a-(40-nitro-phenoxy) and 14a-(20-
the dual substituted 20-OMe-40-NO2 derivative 10g with 3,19-ace- methoxy-40-nitro-phenoxy) andrographolide analogues (Scheme
tonylidene (IC50 ¼ 2.0 mM, Fig. 4 and Table 1, entry 13) was a more 2 and Table 1, entries 19–22) since their corresponding 14b-
potent FXR antagonist than 10b (IC50 ¼ 8.6 mM) and 10f (IC50
¼
isomers 12b and 12g are very potent FXR antagonists. Contrary
2.4 mM) as expected. However, it was surprising that 3,19-diol 12g to their 14b-isomers of 10b and 12b, two 14a-(40-nitro)-phenoxy
(IC50 ¼ 11.9 mM) with dual substitutions at 14-phenoxy by 20- analogues of 11b (IC50 ¼ 15.1 mM, Table 1, entry 19) and 13b
methoxy group and 40-nitro group (Fig. 4 and Table 1, entry 14) (IC50 ¼ 16.0 mM, Table 1, entry 20) were unfortunately weaker
showed a much weaker FXR antagonistic activity than 10g and its FXR antagonists than 1 (IC50 ¼ 9.7 mM). 11g (IC50 ¼ 2.5 mM,
Table 1, entry 21) of 3,19-acetonylidene-14a-(20-methoxy-40-
nitro)-phenoxy derivative showed a similar FXR antagonistic
activity to its 14b-counterpart 10g (IC50 ¼ 2.0 mM) but 11g
exhibited a cellular toxicity (CC50 ¼ 12.0 mM); meanwhile, its
corresponding diol 13g (IC50 ¼ 7.1 mM, Table 1, entry 22) was a
moderate FXR antagonist. Thus, these data together with their
decreased stability suggest that at the current stage, 14a-isomer
analogues should not be considered further as FXR antagonists.
To decipher the structural basis for the above SARs, we
compared the putative binding positions of compounds 1, 5, 6,
10b, 12b, 10g and 12g by means of molecular docking. The
docking model was generated from the chain A of 3DCT42 from
the same view using Discovery Studio 3.5 and the modelling
results are shown in Fig. 5 and 6.
As shown in Fig. 5, even FXR antagonistic activities of 1, 5 and 6
are similar, the putative binding positions of 1 (Fig. 5a), 5 (Fig. 5b)
and 6 (Fig. 5c) in NS3 appear to be quite different, in which their
orientation of decalin rings are distinctive, the bindings of lactone
rings in 1 and 5 are similar but different from that in 6. The
putative binding position of 1 can be transformed into 5's by
making a rotation of 180ꢀ along the bond of C9–C11 or C11–C12 or
6's by a rotation of 90ꢀ clockwise and then 180ꢀ along the bond of
C9–C11 or C11–C12. These simulation results suggest that the
spatial volume of the binding pocket in NS3 is large enough for
different occupation modes which are dependent on the interac-
tion between a specic ligand with the binding site. This means
that optimal structural tuning will benet binding potency.
Further, the predictive binding of the most potent compound
Fig. 4 Design of 10g and 12g from 12b and 12f.
of 3,19-diol 12b is in good agreement with the large spatial
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with aromatic rings of Trp454 and Phe284, leading to the tight
binding between 12b and FXR (Fig. 6b). However, due to the
relatively large and rigid 6-membered ring formed by 3,19-ace-
tonylidene protection as indicated in Fig. 6a, compound 10b,
whose binding position is close to 5 but not 1 or 6, was obstructed
by the putative “gate” established by 4 amino acid residues of
Met290, His294, Met328 and Arg331, which lets compound 10b
not fully enter the binding pocket, resulting in the loss of
hydrogen bond and p–p interaction in the binding of 10b to FXR.
These molecular binding positions of 10b and 12b are in good
agreement with their FXR antagonistic activities (10b < 12b).
Moreover, by comparison with the predictive binding positions of
10b and 12b, the orientations of 14b-(40-nitro)-phenoxy moiety
and the lactone ring of 10b are reciprocally exchanged, suggest-
ing the importance of their orientations for binding.
Since compounds 10g and 12g expressed big differences in
their FXR antagonistic activities (10g > 12g) and their SAR is
totally distinct from that of 10b and 12b (10b < 12b), we were
interested in understanding how 20-methoxy group affects the
binding positions of 10g and 12g. Interestingly and notably, the
predictive binding position of 10g (Fig. 6c) bearing 3,19-aceto-
nylidene protective group is different from that of its corre-
sponding counterpart 10b (Fig. 6a) but is quite similar to that of
the diol 12b (Fig. 6b); meanwhile, the predictive binding posi-
tion of the diol 12g (Fig. 6d) is not similar to that of the diol 12b
(Fig. 6b) but is almost identical to that of 3,19-acetonylidene
protected compound 10b (Fig. 6a), these observations gave a
good explanation for the above-mentioned FXR antagonistic
relationship of 10b < 12b vs. 10g > 12g (Fig. 6 and Table 1,
entries 1, 2, 13 and 14). Except for one more hydrogen bond in
the binding pose of 12b as shown in Fig. 6b, the interaction of
10g with FXR (Fig. 6c), which is stabilized by one hydrogen bond
and three p–p interactions, is similar to 12b. Because 3,19-
acetonylidene protected molecule 10g was partially blocked by
the “gate” (Fig. 6c), the binding pose of 10g is much more at
than 12b in order to reach the same binding site as 12b. These
results demonstrated that the 20-methoxy group plays an
important role in the binding of these small molecules to FXR.
These modelling results are interesting in that the putative
binding positions of 10b (14b) and 12g (14b), and the putative
orientations of decalin rings of 12b (14b) and 10g (14b) are
similar to those of 5 (14a) but not of 6 (14b), respectively.
Conclusions
In summary, starting from the readily available natural
compound of andrographolide (1), a series of andrographolide
derivatives by the introduction of various substituted phenoxy
groups to andrographolide at 14-position were straightforwardly
synthesized with inversion of the 14-conguration by the Mit-
sunobu reaction. Our primary SAR studies revealed that 14-
phenoxy andrographolide scaffold is an excellent structural
moiety for FXR antagonists. Structural tuning of the substitu-
tions at 14-phenoxy, modications at 3,19-positions and
Fig. 5 Three-dimensional (3D) interaction diagrams of predicted
binding positions of 1 (a), 5 (b) and 6 (c) in FXR active sites. Critical
amino acid residues of the binding pocket are labelled. The green dash
line denotes hydrogen bond between ligand and amino acid residue
atoms of FXR protein.
volume binding pocket (Fig. 6b), in which there are two hydrogen 14-congurations could be an efficient way to discover 14-phe-
bonds between the 40-nitro group and the hydroxyl group of the noxy andrographolide derivatives as effective FXR antagonists
Thr 288 residue and three p–p interactions of the 40-nitro group with low cellular toxicity. Remarkably, 14b-compounds of 12b,
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Fig. 6 Three-dimensional (3D) interaction diagrams of predicted binding positions of 10b (a), 12b (b), 10g (c) and 12g (d) in FXR active site. Critical
amino acid residues of the binding pocket are labelled. The green dash line denotes hydrogen bonding between ligand and amino acid residue
atoms of FXR protein, while the orange line denotes p–p interaction from the ligand to amino acid aromatic ring residue of the FXR protein. The
inset (in red rectangle) of the hydrogen bond in 12g (d) was viewed from a different visual angle.
12f and 10g were found to be the most potent FXR antagonists in Melting points were measured using an YRT-3 melting point
this work. Lesser stability and relatively weaker potency with apparatus (Shanghai, China) and were uncorrected. 1H and 13
C
somewhat cellular toxicity of a-isomers restricted their applica- NMR spectra were recorded on a Bruker AV-400 spectrometer at
tion in our current data. The structural docking unveiled that the 400 and 100 MHz, respectively, in CDCl3, CD3OD, DMSO-d6, etc.
structural features of a small FXR ligand affects its binding as indicated. Coupling constants (J) are expressed in Hertz (Hz).
position, resulting in its FXR antagonistic discrepancy. It should Chemical shis (d) of NMR are reported in parts per million
be mentioned that these compounds have the potential to be (ppm) units relative to the solvent. The low or high resolution of
optimized and developed into drug-like candidates in the future ESIMS (in the positive ion or negative ion acquisition mode) was
even compounds 6, 10b–h, 11b, 11g and 12h have too high recorded on an Agilent 1200 HPLC-MSD mass spectrometer or
C Log P values (>5.0) to obey the Lipinski rule of 5. Taken Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spec-
together, the data strongly supports that andrographolide trometer, respectively.
backbone combined with phenoxy substitution at the 14-posi-
Preparation of 3,19-acetonylidene andrographolide (5)36–38
tion provides an excellent scaffold for FXR antagonists.
To the solution of 10 g (28.5 mmol) of andrographolide (1) and
24 ml (196 mmol) of 2,2-dimethoxypropane in 20.0 ml of
anhydrous dichloromethane, 0.72 g (2.9 mmol) of PPTS was
Experimental
Materials and equipment
ꢀ
added and the reaction mixture was heated at 40 C. The reac-
Unless otherwise noted, materials were obtained from tion was monitored by TLC and then treated with ethyl acetate
commercial suppliers and used without further purication. and sat. NaHCO3 aer the reaction was complete. The organic
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phase was washed with brine, dried over anhydrous Na2SO4,
and then ltered organic solution was evaporated to dryness.
The residue was puried by silica gel column chromatography
(petroleum ether/ethyl acetate 1/1) to afford a white solid 10.05 g
(90.2%); mp 187–192 ꢀC. 1H NMR (400 MHz, DMSO-d6) d 6.63 (t,
J ¼ 6.4 Hz, 1H), 5.74 (d, J ¼ 6.0 Hz, 1H), 4.93 (t, J ¼ 5.8 Hz, 1H),
4.86 (s, 1H), 4.69 (s, 1H), 4.41 (dd, J ¼ 10.0, 6.0 Hz, 1H), 4.04 (dd,
J ¼ 9.8, 1.8 Hz, 1H), 3.89 (d, J ¼ 11.6 Hz, 1H), 3.42 (dd, J ¼ 9.2,
3.6 Hz, 1H), 3.12 (d, J ¼ 11.6 Hz, 1H), 2.54–2.50 (m, 1H), 2.40–
1.88 (m, 3H), 1.78–1.63 (m. 3H), 1.34 (s, 3H), 1.26 (s, 3H), 1.34–
1.15 (m, 3H), 1.14 (s, 3H), 0.88 (s, 3H).
Preparation of 14b-andrographolide (8)
0.5 mmol of compound 6 was dissolved in 4 ml of methanol and
then treated with 0.05 mmol of TsOH at 40–50 ꢀC for 4 h.
Diluted by ethyl acetate and washed with sat. NaHCO3, brine,
the organic phase was dried over anhydrous Na2SO4, ltered,
evaporated by a rotavap to dryness. Compound 8 was puried by
silica gel column chromatography (dichloromethane–ethyl
acetate 1 : 2) (white solid, 80%, mp 200–202 C). H NMR (400
MHz, DMSO-d6) d 6.61 (ddd, J ¼ 7.9, 6.1, 1.8 Hz, 1H), 5.64 (d, J ¼
6.1 Hz, 1H), 5.06 (d, J ¼ 4.8 Hz, 1H), 4.94 (t, J ¼ 6.1 Hz, 1H), 4.79
(d, J ¼ 1.8 Hz, 1H), 4.45–4.36 (m, 2H), 4.12 (dd, J ¼ 7.6, 2.8 Hz,
1H), 4.02 (dd, J ¼ 9.9, 2.2 Hz, 1H), 3.84 (dd, J ¼ 10.9, 2.9 Hz, 1H),
3.24 (ddd, J ¼ 14.8, 9.9, 6.1 Hz, 2H), 2.63–2.53 (m, 1H), 2.43–2.26
(m, 2H), 1.92 (t, J ¼ 12.2 Hz, 2H), 1.79–1.59 (m, 4H), 1.43–1.15
(m, 3H), 1.08 (s, 3H), 0.66 (s, 3H); 13C NMR (101 MHz, C6D6) d
175.2, 153.3, 151.8, 134.5, 113.0, 83.8, 79.6, 70.2, 68.0, 60.7, 59.8,
47.6, 44.1, 42.9, 41.8, 33.2, 29.8, 29.3, 28.4, 20.2; ESI-HRMS: m/z
373.1981 [M + Na]+, calcd for C20H30NaO5, 373.1991.
1
ꢀ
Preparation of 3,19-acetonylidene-14b-acetoxy-
andrographolide (6)
Under N2 atmosphere, 1.0 mmol of compound 5, 1.5 mmol of
PPh3, 1.5 mmol of acetic acid were dissolved in 10.0 ml of
anhydrous THF. The solution was cooled to 0 ꢀC and then
treated with 1.5 mmol of DIAD in 2.0 ml of anhydrous THF. The
reaction was stirred overnight at room temperature aer being
stirred at 0 ꢀC for 1 h. Aer distilling off the volatile solvents, the
residue was dissolved in ethyl acetate and washed with brine
about 5 times and dried over anhydrous Na2SO4. The ltered
organic solution was evaporated to dryness and the residue was
puried by silica gel column chromatography (petroleum ether–
ethyl acetate 1 : 1) to give 6 (61%) as a white solid; mp 140–142
ꢀC. 1H NMR (400 MHz, C6D6) d 7.00 (dd, J ¼ 7.1, 1.8 Hz, 1H), 5.93
(dd, J ¼ 5.6, 2.5 Hz, 1H), 4.89–4.84 (m, 1H), 4.56 (dd, J ¼ 11.3, 6.2
Hz, 1H), 4.41 (d, J ¼ 1.7 Hz, 1H), 4.22 (dd, J ¼ 11.3, 1.9 Hz, 1H),
3.94 (d, J ¼ 11.6 Hz, 1H), 3.49 (dd, J ¼ 8.4, 3.9 Hz, 1H), 3.16 (d,
J ¼ 11.6 Hz, 1H), 2.55–2.31 (m, 3H), 2.10 (s, 3H), 1.98 (dd, J ¼
12.2, 6.4 Hz, 2H), 1.91–1.83 (m, 1H), 1.83–1.64 (m, 3H), 1.39 (s,
3H), 1.35 (s, 3H), 1.28 (tdd, J ¼ 12.5, 8.2, 4.6 Hz, 3H), 1.18 (s, 3H),
0.94 (s, 3H); 13C NMR (101 MHz, C6D6) d 168.7, 161.3, 152.0,
146.9, 142.5, 126.4, 123.9, 115.3, 108.3, 80.3, 77.3, 71.8, 70.3,
64.0, 55.7, 55.2, 42.8, 39.0, 37.7, 36.9, 28.1, 25.8, 23.7, 22.7, 15.1;
ESI-HRMS: m/z 455.2397 [M + Na]+, calcd for C25H36NaO6,
455.2410.
Preparation of 3,19-acetonylidene-14b-andrographolide (9)
The same procedure was used as the preparation of compound
5. The compound 9 was puried by silica gel column with
petroleum ether/ethyl acetate 10/7 as a white solid (90%), mp
167–168 ꢀC. 1H NMR (400 MHz, CDCl3) d 6.95 (ddd, J ¼ 8.0, 6.5,
1.8 Hz, 1H), 5.08 (t, J ¼ 6.5 Hz, 1H), 4.87 (t, J ¼ 1.5 Hz, 1H), 4.47
(dd, J ¼ 10.5, 6.1 Hz, 1H), 4.43 (q, J ¼ 1.3 Hz, 1H), 4.25 (dd, J ¼
10.5, 2.1 Hz, 1H), 3.96 (d, J ¼ 11.6 Hz, 1H), 3.50 (dd, J ¼ 8.6, 3.6
Hz, 1H), 3.18 (d, J ¼ 11.6 Hz, 1H), 2.72–2.61 (m, 1H), 2.54–2.44
(m, 1H), 2.41 (dt, J ¼ 13.3, 2.9 Hz, 1H), 2.07 (d, J ¼ 6.8 Hz, 1H),
2.05–1.89 (m, 3H), 1.85–1.70 (m, 3H), 1.41 (s, 3H), 1.37 (s, 3H),
1.36–1.24 (m, 3H), 1.21 (s, 3H), 0.96 (s, 3H); 13C NMR (101 MHz,
DMSO) d 169.8, 147.8, 146.3, 129.2, 108.1, 98.2, 75.8, 74.2, 64.8,
62.8, 55.2, 51.5, 38.2, 37.2, 37.1, 33.8, 27.4, 25.8, 25.2, 24.8, 24.6,
22.7, 15.8; ESI-HRMS: m/z 413.2302 [M + Na]+, calcd for
C
23H34NaO5, 413.2304.
Preparation of the series compounds 10a–10i, 11b and 11g
The same procedure was used as the preparation of 3,19-ace-
tonylidene 14b-acetoxy andrographolide (6). Generally, the
purication was conducted by silica gel column chromatog-
raphy with petroleum ether–ethyl acetate from 3 : 1 to 1 : 1.
Preparation of 14b-acetoxy-andrographolide (7)
0.5 mmol of compound 6 was dissolved in 4 ml of methanol and
then treated with 0.05 mmol of TsOH at 20 ꢀC for 30 min.
Diluted by ethyl acetate and washed with sat. NaHCO3, brine,
the organic phase was dried over anhydrous Na2SO4, ltered,
evaporated by a rotavap to dryness. Compound 7 was puried by
3,19-Acetonylidene-14b-phenoxy-andrographolide (10a)
silica gel column chromatography (petroleum ether–ethyl From 5 in 51% yield, white solid, mp 153–155 ꢀC. 1H NMR (400
acetate 10 : 7) (white solid, 93%, mp 163–165 ꢀC). 1H NMR (400 MHz, CDCl3) d (ppm) 7.39–7.28 (m, 2H), 7.13 (td, J ¼ 7.4, 1.8 Hz,
MHz, CDCl3) d 6.98 (dd, J ¼ 7.0, 1.8 Hz, 1H), 5.91 (dt, J ¼ 6.0, 1.8 1H), 7.05 (tt, J ¼ 7.5, 1.0 Hz, 1H), 6.88–6.80 (m, 2H), 5.53 (d, J ¼
Hz, 1H), 4.85 (q, J ¼ 1.3 Hz, 1H), 4.54 (dd, J ¼ 11.3, 6.1 Hz, 1H), 5.7 Hz, 1H), 4.87 (d, J ¼ 1.6 Hz, 1H), 4.60 (dd, J ¼ 10.7, 5.8 Hz,
4.36 (d, J ¼ 1.7 Hz, 1H), 4.20 (ddd, J ¼ 25.9, 11.0, 1.8 Hz, 2H), 1H), 4.46 (s, 1H), 4.40 (dd, J ¼ 10.7, 2.0 Hz, 1H), 3.91 (d, J ¼ 11.6
3.46 (dt, J ¼ 10.2, 4.6 Hz, 1H), 3.36–3.26 (m, 1H), 3.00–2.89 (m, Hz, 1H), 3.42 (dd, J ¼ 8.3, 3.9 Hz, 1H), 3.14 (d, J ¼ 11.5 Hz, 1H),
2H), 2.53–2.26 (m, 3H), 2.11 (s, 3H), 1.96 (td, J ¼ 12.5, 5.0 Hz, 2.57–2.46 (m, 1H), 2.45–2.26 (m, 2H), 1.95 (dd, J ¼ 27.6, 12.1 Hz,
1H), 1.89–1.75 (m, 4H), 1.75–1.68 (m, 1H), 1.36–1.12 (m, 6H), 2H), 1.88–1.77 (m, 1H), 1.75–1.67 (m, 1H), 1.64 (dd, J ¼ 11.8, 6.0
0.66 (s, 3H); 13C NMR (101 MHz, CDCl3) d 170.5, 169.1, 150.6, Hz, 1H), 1.51 (dd, J ¼ 8.0, 5.4 Hz, 1H), 1.37 (s, 3H), 1.34 (s, 3H),
146.8, 123.9, 108.4, 80.4, 71.7, 68.1, 64.1, 55.7, 55.2, 42.9, 39.0, 1.31–1.19 (m, 3H), 1.17 (s, 3H), 0.87 (s, 3H); 13C NMR (101 MHz,
37.7, 36.9, 28.2, 28.2, 25.6, 23.7, 22.7, 20.8, 15.2; ESI-HRMS: m/z C6D6) d 168.8, 157.0, 149.6, 148.1, 130.1, 126.0, 122.2, 115.9,
415.2067 [M + Na]+, calcd for C22H32NaO6, 415.2097.
108.0, 99.4, 75.4, 71.5, 70.5, 64.2, 55.8, 51.3, 38.5, 38.3, 37.8,
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33.7, 26.7, 26.1, 25.7, 25.3, 24.8, 23.3, 16.6; ESI-HRMS: m/z 6.98–6.92 (m, 1H), 6.69 (td, J ¼ 7.6, 0.9 Hz, 1H), 6.28 (d, J ¼ 8.2
489.2658 [M + Na]+, calcd for C29H38NaO5, 489.2617.
Hz, 1H), 5.04 (d, J ¼ 5.4 Hz, 1H), 4.87–4.81 (m, 1H), 4.44 (s, 1H),
4.12 (qq, J ¼ 10.9, 7.1 Hz, 2H), 4.01 (dd, J ¼ 10.6, 1.4 Hz, 1H),
3.82 (d, J ¼ 11.5 Hz, 1H), 3.68 (dd, J ¼ 10.6, 5.5 Hz, 1H), 3.42 (dd,
J ¼ 7.6, 3.7 Hz, 1H), 3.08 (d, J ¼ 11.5 Hz, 1H), 2.24–2.05 (m, 3H),
1.73 (dq, J ¼ 13.4, 6.8 Hz, 2H), 1.59 (d, J ¼ 8.3 Hz, 1H), 1.53–1.43
(m, 1H), 1.43–1.31 (m, 2H), 1.41 (s, 3H), 1.37 (s, 3H), 1.11 (s, 3H),
1.04 (t, J ¼ 7.1 Hz, 3H), 1.01–0.89 (m, 3H), 0.85 (s, 3H); 13C NMR
(101 MHz, C6D6) d 168.7, 165.7, 156.0, 149.7, 148.2, 132.9, 132.2,
127.8, 125.8, 123.9, 122.2, 116.6, 107.9, 99.3, 75.3, 73.2, 70.3,
64.1, 61.0, 55.8, 51.0, 38.4, 38.2, 37.6, 33.5, 26.6, 25.91, 25.87,
25.2, 24.6, 23.2, 16.5, 14.1; ESI-HRMS: m/z 561.2829 [M + Na]+,
calcd for C32H42NaO7, 561.2828.
3,19-Acetonylidene-14b-(40-nitro-phenoxy)-andrographolide
(10b)
From 5 in 52% yield, white solid, mp 179–181 ꢀC. 1H NMR (400
MHz, CDCl3) d 8.31–8.22 (m, 2H), 7.19 (ddd, J ¼ 8.0, 6.7, 1.7 Hz,
1H), 6.97–6.88 (m, 2H), 5.69–5.62 (m, 1H), 4.89 (dd, J ¼ 1.8, 1.0
Hz, 1H), 4.67 (dd, J ¼ 10.9, 5.8 Hz, 1H), 4.45 (d, J ¼ 1.5 Hz, 1H),
4.37 (dd, J ¼ 10.9, 1.9 Hz, 1H), 3.88 (d, J ¼ 11.6 Hz, 1H), 3.44 (dd,
J ¼ 7.8, 3.8 Hz, 1H), 3.14 (d, J ¼ 11.5 Hz, 1H), 2.54 (ddd, J ¼ 15.9,
7.8, 2.5 Hz, 1H), 2.47–2.31 (m, 2H), 2.04–1.89 (m, 2H), 1.85–1.75
(m, 1H), 1.75–1.68 (m, 1H), 1.68–1.59 (m, 1H), 1.51 (td, J ¼ 8.0,
4.2 Hz, 1H), 1.35 (s, 3H), 1.33 (s, 3H), 1.31–1.21 (m, 3H), 1.16 (s,
3H), 0.89 (s, 3H); 13C NMR (101 MHz, CDCl3) d 168.68, 161.28,
151.98, 147.27, 142.53, 126.35, 123.93, 115.16, 108.26, 99.37,
75.42, 71.78, 70.26, 64.05, 55.82, 51.66, 38.50, 38.10, 37.58,
33.85, 26.47, 26.01, 25.94, 25.10, 24.43, 23.17, 16.44; ESI-HRMS:
m/z 534.2473 [M + Na]+, calcd for C29H37NNaO7, 534.2468.
3,19-Acetonylidene-14b-(20-methoxy-phenoxy)-
andrographolide (10f)
From 5 in 60% yield, white solid, mp 130–131 ꢀC. 1H NMR (400
MHz, C6D6) d 7.13–7.10 (m, 1H), 6.80 (td, J ¼ 7.8, 1.7 Hz, 1H),
6.65 (td, J ¼ 7.7, 1.5 Hz, 1H), 6.58 (dd, J ¼ 7.9, 1.6 Hz, 1H), 6.47
(dd, J ¼ 8.1, 1.3 Hz, 1H), 5.18 (d, J ¼ 5.4 Hz, 1H), 4.82 (d, J ¼ 1.2
Hz, 1H), 4.42 (s, 1H), 4.15 (dd, J ¼ 10.6, 1.5 Hz, 1H), 3.83 (d, J ¼
11.5 Hz, 1H), 3.65 (dd, J ¼ 10.6, 5.5 Hz, 1H), 3.41 (dd, J ¼ 7.7, 3.7
Hz, 1H), 3.29 (s, 3H), 3.09 (d, J ¼ 11.5 Hz, 1H), 2.26–2.04 (m, 3H),
1.83–1.62 (m, 2H), 1.54–1.28 (m, 10H), 1.08 (s, 3H), 1.05–0.82
(m, 6H); 13C NMR (101 MHz, C6D6) d 169.08, 151.78, 149.42,
148.27, 145.92, 128.15, 128.03, 127.91, 127.79, 127.67, 126.39,
124.00, 121.11, 120.16, 112.54, 107.85, 99.30, 75.39, 73.44,
70.56, 64.14, 55.72, 55.11, 51.10, 38.34, 38.12, 37.66, 33.56,
26.57, 26.00, 25.64, 25.29, 24.74, 23.24, 16.46; ESI-HRMS: m/z
519.2715 [M + Na]+, calcd for C30H40NaO6, 519.2723.
3,19-Acetonylidene-14b-(30-nitro-phenoxy)-andrographolide
(10c)
From 5 in 32% yield, white solid, mp 163–165 ꢀC. 1H NMR (400
MHz, C6D6) d 7.51 (d, J ¼ 7.1 Hz, 1H), 7.31 (d, J ¼ 2.1 Hz, 1H),
7.23–7.16 (m, 1H), 6.62 (t, J ¼ 8.2 Hz, 1H), 6.52 (dd, J ¼ 8.3, 1.8
Hz, 1H), 4.85 (s, 1H), 4.62 (d, J ¼ 5.1 Hz, 1H), 4.43 (s, 1H), 3.77 (d,
J ¼ 11.6 Hz, 1H), 3.67–3.54 (m, 2H), 3.41 (dd, J ¼ 7.1, 3.5 Hz, 1H),
3.06 (d, J ¼ 11.5 Hz, 1H), 2.12 (ddd, J ¼ 22.8, 17.7, 9.7 Hz, 3H),
1.68 (dt, J ¼ 13.0, 6.6 Hz, 2H), 1.53–1.36 (m, 2H), 1.39 (s, 3H),
1.35–1.25 (m, 2H), 1.34 (s, 3H), 1.07 (s, 3H), 0.99 (td, J ¼ 13.0, 3.8
Hz, 1H), 0.94–0.80 (m, 2H), 0.84 (s, 3H); 13C NMR (101 MHz,
C6D6) d 168.1, 157.1, 150.2, 149.5, 147.8, 130.4, 124.9, 122.2,
116.8, 109.2, 108.0, 99.5, 74.9, 71.8, 69.8, 64.2, 55.6, 51.0, 38.4,
38.2, 37.6, 33.5, 26.2, 25.9, 25.8, 25.1, 24.4, 23.1, 16.6; ESI-HRMS:
m/z 534.2463 [M + Na]+, calcd for C29H37NNaO7, 534.2468.
3,19-Acetonylidene-14b-(20-methoxy-40-nitro-phenoxy)-
andrographolide (10g)
1
ꢀ
From 5 in 51% yield, pale yellowish solid, mp 129–131 C. H
NMR (400 MHz, C6D6) d 7.56 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.46 (d, J ¼
2.6 Hz, 1H), 7.20–7.15 (m, 1H), 5.95 (d, J ¼ 8.8 Hz, 1H), 4.88 (d, J
¼ 5.5 Hz, 1H), 4.83 (d, J ¼ 1.5 Hz, 1H), 4.41–4.37 (m, 1H), 3.82–
3.74 (m, 2H), 3.60 (dd, J ¼ 10.8, 5.7 Hz, 1H), 3.41 (dd, J ¼ 7.3, 3.6
Hz, 1H), 3.08 (s, 1H), 3.05 (s, 3H), 2.19–2.10 (m, 3H), 1.77–1.60
(m, 2H), 1.52–1.44 (m, 1H), 1.44–1.36 (m, 1H), 1.40 (s, 3H), 1.36
(s, 3H), 1.34–1.25 (m, 2H), 1.06 (s, 3H), 1.03–0.93 (m, 1H), 0.94–
0.80 (m, 3H), 0.85 (s, 3H); 13C NMR (101 MHz, C6D6) d 168.1,
150.8, 150.5, 150.3, 148.1, 143.2, 124.9, 116.9, 115.0, 107.7,
107.1, 99.4, 74.8, 72.9, 69.7, 64.0, 55.4, 55.1, 50.7, 38.2, 38.1,
37.4, 33.2, 26.1, 25.8, 25.6, 25.0, 24.3, 23.0, 16.4; ESI-HRMS: m/z
564.2584 [M + Na]+, calcd for C30H39NNaO8, 564.2573.
3,19-Acetonylidene-14b-(20-nitro-phenoxy)-andrographolide
(10d)
1
ꢀ
From 5 in 73% yield, white solid, mp 146–148 C. H NMR (400
MHz, C6D6) d 7.27 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.21 (ddd, J ¼ 8.1, 6.3,
1.9 Hz, 1H), 6.77–6.65 (m, 1H), 6.34 (td, J ¼ 7.8, 1.1 Hz, 1H), 6.06–
5.92 (m, 1H), 4.93–4.80 (m, 2H), 4.38 (t, J ¼ 2.8 Hz, 1H), 3.82 (d, J ¼
11.5 Hz, 1H), 3.60 (qd, J ¼ 10.8, 4.2 Hz, 2H), 3.45 (dd, J ¼ 7.7, 3.3 Hz,
1H), 3.08 (d, J ¼ 11.5 Hz, 1H), 2.40–2.29 (m, 1H), 2.26–2.11 (m, 2H),
1.86–1.67 (m, 2H), 1.62–1.47 (m, 3H), 1.42 (s, 3H), 1.36 (s, 3H), 1.36–
1.31 (m, 1H), 1.12 (s, 3H), 1.05–0.91 (m, 3H), 0.87 (s, 3H); 13C NMR
(101 MHz, C6D6) d 168.0, 151.6, 149.3, 148.4, 141.6, 133.2, 127.7,
125.8, 124.3, 121.7, 115.8, 107.8, 99.3, 75.4, 73.3, 69.6, 64.1, 55.7,
51.0, 38.4, 38.1, 37.6, 33.6, 26.7, 25.9, 25.2, 24.7, 23.2, 16.5; ESI-
HRMS: m/z 534.2467 [M + Na]+, calcd for C29H37NNaO7, 534.2468.
3,19-Acetonylidene-14b-(20-methyl-40-nitro-phenoxy)-
andrographolide (10h)
From 5 in 61% yield, white solid, mp 168–170 ꢀC. 1H NMR (400
MHz, C6D6) d 8.13 (d, J ¼ 7.7 Hz, 2H), 7.19 (td, J ¼ 7.2, 1.7 Hz,
1H), 6.79–6.70 (m, 1H), 5.67 (d, J ¼ 5.6 Hz, 1H), 4.91–4.84 (m,
1H), 4.67 (dd, J ¼ 10.9, 5.7 Hz, 1H), 4.43 (d, J ¼ 1.8 Hz, 1H), 4.35
(dd, J ¼ 11.0, 1.7 Hz, 1H), 3.87 (d, J ¼ 11.6 Hz, 1H), 3.43 (dd, J ¼
Ethyl 3,19-acetonylidene-14b-(20-carboxy-phenoxy)-
andrographolide (10e)
From 5 in 78% yield, white solid, mp 168–170 ꢀC. 1H NMR (400 7.7, 3.7 Hz, 1H), 3.13 (d, J ¼ 11.6 Hz, 1H), 2.51 (ddd, J ¼ 16.2, 7.4,
MHz, C6D6) d 7.80 (dd, J ¼ 7.7, 1.8 Hz, 1H), 7.24–7.18 (m, 1H), 2.6 Hz, 1H), 2.45–2.34 (m, 2H), 2.27 (s, 3H), 1.96 (ddd, J ¼ 27.0,
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12.4, 6.9 Hz, 2H), 1.83–1.66 (m, 2H), 1.65–1.44 (m, 3H), 1.34 (s,
Preparation of the series compounds 12a–12i, 13b and 13g
3H), 1.33 (s, 3H), 1.31–1.17 (m, 3H), 1.15 (s, 3H), 0.89 (s, 3H); 13
C
The same procedure was used as the preparation of compound
7. Generally, the purication was conducted by silica gel
column chromatography with petroleum ether–ethyl acetate
from 2 : 1 to 1 : 1.
NMR (101 MHz, C6D6) d 162.6, 153.7, 145.2, 142.7, 136.9, 123.1,
121.4, 119.4, 117.9, 105.4, 102.5, 94.2, 69.1, 66.5, 64.3, 58.8, 50.5,
45.4, 32.9, 32.2, 27.9, 20.5, 19.6, 18.7, 17.7, 11.4, 10.5; ESI-
HRMS: m/z 548.2636 [M + Na]+, calcd for C30H39NNaO7,
548.2624.
14b-Phenoxy-andrographolide (12a)
3,19-Acetonylidene-14b-(20-uoro-40-nitro-phenoxy)-
1
ꢀ
From 10a in 86% yield, white solid, mp 164–166 C. H NMR
(400 MHz, CDCl3) d 7.40–7.29 (m, 2H), 7.14–7.01 (m, 2H), 6.89–
6.79 (m, 2H), 5.55–5.48 (m, 1H), 4.85 (dd, J ¼ 1.9, 1.0 Hz, 1H),
4.60 (dd, J ¼ 10.7, 5.9 Hz, 1H), 4.44–4.36 (m, 2H), 4.17–4.08 (m,
1H), 3.40 (dd, J ¼ 11.5, 4.3 Hz, 1H), 3.29 (d, J ¼ 10.9 Hz, 1H),
2.54–2.44 (m, 1H), 2.44–2.36 (m, 1H), 2.33–2.23 (m, 1H), 2.16 (d,
J ¼ 26.3 Hz, 3H), 1.97 (td, J ¼ 12.7, 10.7, 6.2 Hz, 1H), 1.92–1.85
(m, 1H), 1.85–1.70 (m, 2H), 1.70–1.55 (m, 2H), 1.33–1.16 (m,
5H), 1.11 (td, J ¼ 13.5, 3.7 Hz, 1H), 0.58 (s, 3H); 13C NMR (101
MHz, C6D6) d 169.5, 156.4, 150.8, 147.1, 130.0, 125.0, 122.3,
115.7, 108.2, 80.4, 71.3, 71.0, 64.1, 55.9, 55.1, 42.8, 39.0, 37.7,
36.8, 28.1, 25.6, 23.7, 22.7, 15.1; HRMS (ESI): m/z 449.2342 [M +
Na]+, calcd for C26H34NaO5, 449.2304.
andrographolide (10i)
From 5 in 72% yield, white solid, mp 114–117 ꢀC. 1H NMR (400
MHz, C6D6) d 7.56 (dd, J ¼ 10.6, 2.6 Hz, 1H), 7.47 (ddd, J ¼ 9.1,
2.7, 1.5 Hz, 1H), 7.23–7.18 (m, 1H), 5.75 (td, J ¼ 8.4, 3.3 Hz, 1H),
4.84 (d, J ¼ 1.8 Hz, 1H), 4.70 (d, J ¼ 5.1 Hz, 1H), 4.39 (d, J ¼ 1.7
Hz, 1H), 3.77 (d, J ¼ 11.6 Hz, 1H), 3.63–3.51 (m, 2H), 3.43 (dd, J
¼ 7.0, 3.5 Hz, 1H), 3.05 (d, J ¼ 11.5 Hz, 1H), 2.26–2.08 (m, 3H),
1.79–1.60 (m, 2H), 1.55–1.43 (m, 2H), 1.39 (s, 3H), 1.34 (s, 3H),
1.36–1.27 (m, 2H), 1.05 (s, 3H), 1.01–0.85 (m, 3H), 0.89 (s, 3H);
13C NMR (101 MHz, C6D6) d 167.9, 167.7, 153.1, 151.5, 151.4,
150.6, 149.7, 149.6, 148.17, 142.3, 142.3, 124.2, 120.6, 115.0,
113.0, 112.8, 107.8, 99.5, 74.7, 73.4, 69.5, 64.2, 55.5, 50.7, 38.3,
38.3, 37.5, 33.2, 26.0, 25.8 (2C), 25.0, 24.2, 23.1, 16.7; ESI-HRMS:
m/z 552.2418 [M + Na]+, calcd for C29H36FNNaO7, 552.2374.
14b-(40-Nitro-phenoxy)-andrographolide (12b)
1
ꢀ
3,19-Acetonylidene-14a-(40-nitro-phenoxy)-andrographolide
(11b)
From 10b in 89% yield, white solid, mp 184–186 C. H NMR
(400 MHz, CDCl3) d 8.32–8.23 (m, 2H), 7.18–7.11 (m, 1H), 6.93
(d, J ¼ 9.2 Hz, 2H), 5.63 (d, J ¼ 5.7 Hz, 1H), 4.87 (s, 1H), 4.67 (dd,
J ¼ 10.9, 5.8 Hz, 1H), 4.43–4.32 (m, 2H), 4.13 (d, J ¼ 11.2 Hz, 1H),
3.42 (dd, J ¼ 11.6, 4.3 Hz, 1H), 3.29 (d, J ¼ 11.0 Hz, 1H), 2.50 (dd,
J ¼ 7.0, 2.7 Hz, 1H), 2.46–2.37 (m, 1H), 2.32 (dd, J ¼ 10.7, 7.1 Hz,
1H), 2.05 (s, 3H), 1.97 (dd, J ¼ 12.7, 5.2 Hz, 1H), 1.90 (d, J ¼ 10.5
Hz, 1H), 1.87–1.70 (m, 2H), 1.70–1.63 (m, 1H), 1.60 (dd, J ¼ 13.0,
3.5 Hz, 1H), 1.34–1.18 (m, 5H), 1.13 (td, J ¼ 13.2, 3.8 Hz, 1H),
0.60 (s, 3H); 13C NMR (101 MHz, CDCl3) d 168.7, 161.3, 152.0,
146.9, 142.5, 126.4, 123.9, 115.3, 108.3, 80.3, 77.3, 71.8, 70.3,
64.0, 55.7, 55.2, 42.8, 39.0, 37.7, 36.9, 28.1, 25.8, 23.7, 22.7, 15.1;
HRMS (ESI): m/z 494.2148 [M + Na]+, calcd for C26H33NNaO7,
494.2155.
From 9 in 56% yield, white solid, mp 149–152 ꢀC. 1H NMR (400
MHz, Benzene-d6) d 7.80–7.74 (m, 2H), 6.39 (dd, J ¼ 9.5, 2.4 Hz,
2H), 6.11 (dt, J ¼ 4.7, 1.9 Hz, 1H), 5.49 (s, 1H), 5.20 (dt, J ¼ 10.7,
1.8 Hz, 1H), 5.17–5.13 (m, 1H), 3.86 (d, J ¼ 11.5 Hz, 1H), 3.68
(dd, J ¼ 3.3, 2.0 Hz, 2H), 3.50 (dd, J ¼ 7.1, 3.4 Hz, 1H), 3.09 (d, J ¼
11.5 Hz, 1H), 2.36–2.20 (m, 2H), 2.09–2.03 (m, 1H), 2.02–1.92
(m, 1H), 1.85 (dd, J ¼ 14.6, 10.6 Hz, 1H), 1.81–1.63 (m, 3H), 1.51
(dd, J ¼ 12.6, 6.7 Hz, 1H), 1.43 (s, 3H), 1.41 (s, 3H), 1.33 (ddt, J ¼
6.9, 5.1, 2.9 Hz, 1H), 1.12–0.97 (m, 2H), 1.03 (s, 3H), 1.02 (s, 3H);
13C NMR (101 MHz, C6D6) d 171.5, 162.4, 147.2, 145.7, 142.2,
134.0, 125.9, 115.0, 109.1, 99.5, 75.0, 73.0, 69.9, 64.3, 52.3, 51.4,
38.4, 38.3, 38.2, 33.9, 30.9, 26.2, 25.8, 25.2, 24.6, 23.4, 17.0; ESI-
HRMS: m/z 534.2500 [M + Na]+, calcd for C29H37NNaO7,
534.2468.
14b-(30-nitro-phenoxy)-andrographolide (12c)
1
ꢀ
From 10c in 92% yield, white solid, mp 144–146 C. H NMR
(400 MHz, CDCl3) d 7.94 (ddd, J ¼ 8.2, 2.0, 0.9 Hz, 1H), 7.69 (t, J
¼ 2.3 Hz, 1H), 7.54 (t, J ¼ 8.2 Hz, 1H), 7.20 (ddd, J ¼ 8.3, 2.6, 0.9
3,19-Acetonylidene-14a-(20-methoxy-40-nitro-phenoxy)-
andrographolide (11g)
From 9 in 50% yield, white solid, mp 121–123 ꢀC. 1H NMR (400 Hz, 1H), 7.14 (td, J ¼ 7.1, 1.7 Hz, 1H), 5.62 (dt, J ¼ 5.6, 1.7 Hz,
MHz, C6D6) d 7.56 (dd, J ¼ 8.8, 2.4 Hz, 1H), 7.45 (d, J ¼ 2.5 Hz, 1H), 4.87 (q, J ¼ 1.3 Hz, 1H), 4.67 (dd, J ¼ 10.9, 5.7 Hz, 1H),
1H), 7.14–7.10 (m, 1H), 6.07–5.99 (m, 1H), 4.90 (s, 1H), 4.82 (s, 4.45–4.35 (m, 2H), 4.12 (d, J ¼ 11.2 Hz, 1H), 3.41 (ddd, J ¼ 11.7,
1H), 4.61 (s, 1H), 3.81 (dd, J ¼ 13.3, 6.7 Hz, 2H), 3.71 (dd, J ¼ 4.5, 1.3 Hz, 1H), 3.29 (dd, J ¼ 11.2, 1.3 Hz, 1H), 2.51 (ddd, J ¼
10.8, 5.9 Hz, 1H), 3.46 (dd, J ¼ 7.5, 3.7 Hz, 1H), 3.07 (t, J ¼ 6.6 Hz, 16.1, 7.2, 3.0 Hz, 1H), 2.46–2.38 (m, 1H), 2.37–2.23 (m, 4H), 1.98
4H), 2.41–2.24 (m, 1H), 2.22–2.05 (m, 2H), 1.85 (td, J ¼ 13.6, 6.4 (td, J ¼ 12.6, 4.9 Hz, 1H), 1.93–1.87 (m, 1H), 1.87–1.79 (m, 1H),
Hz, 1H), 1.79–1.67 (m, 1H), 1.62–1.52 (m, 1H), 1.52–1.28 (m, 1.79–1.69 (m, 1H), 1.62 (ddt, J ¼ 24.2, 13.1, 3.8 Hz, 2H), 1.34–
9H), 1.06 (d, J ¼ 16.4 Hz, 3H), 1.04–0.89 (m, 3H), 0.84–0.74 (m, 1.18 (m, 5H), 1.12 (td, J ¼ 13.2, 4.1 Hz, 1H), 0.60 (s, 3H); 13C
3H); 13C NMR (101 MHz, C6D6) d 168.17, 151.11, 150.60, 147.17, NMR (101 MHz, CDCl3) d 168.9, 157.0, 151.7, 149.4, 146.9,
143.51, 124.80, 116.98, 115.61, 109.33, 107.40, 99.44, 75.17, 130.9, 124.2, 122.4, 117.3, 109.7, 108.3, 80.3, 71.9, 70.4, 64.1,
73.37, 69.90, 64.12, 55.82, 55.21, 51.25, 38.16, 38.04, 37.64, 55.8, 55.2, 42.8, 39.00, 37.7, 36.9, 28.1, 25.7, 23.7, 22.7, 15.1;
34.06, 26.32, 26.01, 25.32, 25.20, 24.69, 23.16, 16.39; ESI-HRMS: HRMS (ESI): m/z 494.2156 [M + Na]+, calcd for C26H33NNaO7,
m/z 564.2569 [M + Na]+, calcd for C30H39NNaO8, 564.2573.
494.2155.
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14b-(20-nitro-phenoxy)-andrographolide (12d)
1H), 1.68–1.60 (m, 1H), 1.57 (dd, J ¼ 12.9, 3.3 Hz, 1H), 1.34–1.15
(m, 5H), 1.08 (td, J ¼ 13.4, 3.6 Hz, 1H), 0.59 (s, 3H); 13C NMR
(101 MHz, CDCl3) d 169.0, 151.8, 151.0, 150.6, 147.1, 143.3,
124.4, 117.4, 116.0, 108.2, 107.5, 80.4, 73.3, 70.6, 64.0, 56.4, 55.7,
55.1, 42.8, 38.9, 37.6, 36.6, 28.2, 25.7, 23.7, 22.8, 15.1; HRMS
(ESI): m/z 524.2254 [M + Na]+, calcd for C27H35NNaO8, 524.2260.
1
ꢀ
From 10d in 86% yield, white solid, mp 167–168 C. H NMR
(400 MHz, CDCl3) d 7.89 (dd, J ¼ 8.1, 1.6 Hz, 1H), 7.62–7.55 (m,
1H), 7.18 (t, J ¼ 7.7 Hz, 1H), 7.15–7.10 (m, 1H), 6.92 (d, J ¼ 8.3
Hz, 1H), 5.64 (d, J ¼ 5.6 Hz, 1H), 4.83 (s, 1H), 4.66 (dd, J ¼ 10.9,
6.0 Hz, 1H), 4.44 (dd, J ¼ 10.9, 2.0 Hz, 1H), 4.31 (s, 1H), 4.13 (d, J
¼ 11.2 Hz, 1H), 3.43 (dd, J ¼ 11.5, 4.6 Hz, 1H), 3.28 (d, J ¼ 11.1
Hz, 1H), 2.56–2.45 (m, 1H), 2.38 (dd, J ¼ 13.2, 3.5 Hz, 1H), 2.26
(ddd, J ¼ 16.5, 11.2, 7.8 Hz, 1H), 2.09 (s, 3H), 1.98 (t, J ¼ 12.3 Hz,
1H), 1.89 (d, J ¼ 10.4 Hz, 1H), 1.85–1.78 (m, 1H), 1.78–1.63 (m,
2H), 1.59 (dt, J ¼ 13.0, 3.3 Hz, 1H), 1.24 (s, 5H), 1.10 (td, J ¼ 13.2,
3.9 Hz, 1H), 0.58 (s, 3H); 13C NMR (101 MHz, CDCl3) d 168.7,
152.6, 149.4, 147.3, 141.1, 134.1, 126.3, 123.7, 122.3, 115.9,
108.1, 80.3, 73.1, 70.3, 64.1, 55.8, 54.9, 42.8, 38.9, 37.6, 36.5,
28.1, 25.8, 23.7, 22.6, 15.1; HRMS (ESI): m/z 494.2158 [M + Na]+,
calcd for C26H33NNaO7, 494.2155.
14b-(20-methyl-40-nitro-phenoxy)-andrographolide (12h)
1
ꢀ
From 10h in 87% yield, white solid, mp 115–117 C. H NMR
(400 MHz, CDCl3) d 8.13 (d, J ¼ 7.7 Hz, 2H), 7.19 (td, J ¼ 7.2, 1.7
Hz, 1H), 6.79–6.70 (m, 1H), 5.67 (d, J ¼ 5.6 Hz, 1H), 4.91–4.84
(m, 1H), 4.67 (dd, J ¼ 10.9, 5.7 Hz, 1H), 4.43 (d, J ¼ 1.8 Hz, 1H),
4.35 (dd, J ¼ 11.0, 1.7 Hz, 1H), 3.87 (d, J ¼ 11.6 Hz, 1H), 3.43 (dd,
J ¼ 7.7, 3.7 Hz, 1H), 3.13 (d, J ¼ 11.6 Hz, 1H), 2.51 (ddd, J ¼ 16.2,
7.4, 2.6 Hz, 1H), 2.45–2.34 (m, 2H), 2.27 (s, 3H), 1.96 (ddd, J ¼
27.0, 12.4, 6.9 Hz, 2H), 1.83–1.66 (m, 2H), 1.65–1.44 (m, 3H),
1.33 (d, J ¼ 6.2 Hz, 6H), 1.31–1.17 (m, 3H), 1.15 (s, 3H), 0.89 (s,
3H); 13C NMR (101 MHz, CDCl3) d 168.7, 152.6, 149.3, 147.2,
141.0, 134.1, 126.2, 123.6, 122.3, 115.8, 108.0, 80.3, 73.1, 70.3,
64.1, 55.7, 54.8, 42.7, 38.8, 37.6, 36.5, 28.1, 25.8, 23.6, 22.6, 15.1;
HRMS (ESI): m/z 508.2305 [M + Na]+, calcd for C27H35NNaO7,
508.2311.
Ethyl 14b-(20-carboxy-phenoxy)-andrographolide (12e)
1
ꢀ
From 10e in 88% yield, white solid, mp 181–182 C. H NMR
(400 MHz, CDCl3) d 7.88 (dd, J ¼ 7.8, 1.7 Hz, 1H), 7.56–7.46 (m,
1H), 7.20–7.11 (m, 1H), 7.04 (ddd, J ¼ 7.7, 6.0, 1.6 Hz, 1H), 6.91
(d, J ¼ 8.3 Hz, 1H), 5.61 (d, J ¼ 5.3 Hz, 1H), 4.83 (d, J ¼ 1.9 Hz,
1H), 4.63–4.48 (m, 2H), 4.41–4.26 (m, 3H), 4.14 (d, J ¼ 11.1 Hz,
1H), 3.41 (dt, J ¼ 11.7, 2.8 Hz, 1H), 3.29 (d, J ¼ 10.9 Hz, 1H),
2.47–2.25 (m, 4H), 2.17 (ddd, J ¼ 16.4, 11.2, 8.2 Hz, 1H), 1.96 (td,
J ¼ 12.6, 5.2 Hz, 1H), 1.88–1.78 (m, 2H), 1.78–1.61 (m, 2H), 1.36
(t, J ¼ 7.1 Hz, 3H), 1.32–1.16 (m, 5H), 1.04 (td, J ¼ 13.3, 3.9 Hz,
1H), 0.56 (s, 3H); 13C NMR (101 MHz, CDCl3) d 169.5, 165.7,
155.8, 150.9, 147.1, 133.4, 132.3, 124.9, 123.2, 122.6, 117.0,
108.2, 80.3, 73.4, 71.0, 64.1, 61.2, 55.8, 54.9, 42.8, 38.8, 37.6,
36.6, 28.1, 25.6, 23.7, 22.6, 15.1, 14.3; HRMS (ESI): m/z 521.2517
[M + Na]+, calcd for C29H39NaO7, 521.2515.
14b-(20-uoro-40-nitro-phenoxy)-andrographolide (12i)
1
From 10i in 91% yield, white solid, mp 162–165 ꢀC. H NMR
(400 MHz, CD3OD) d 8.20–8.12 (m, 2H), 7.35 (t, J ¼ 8.4 Hz, 1H),
7.14–7.05 (m, 1H), 6.04–5.98 (m, 1H), 4.91 (s, 1H), 4.73 (dd, J ¼
11.2, 5.5 Hz, 1H), 4.57 (s, 1H), 4.46 (dd, J ¼ 11.2, 1.4 Hz, 1H),
4.03 (d, J ¼ 11.1 Hz, 1H), 3.33 (d, J ¼ 1.2 Hz, 1H), 3.22 (dd, J ¼
11.8, 4.1 Hz, 1H), 2.53 (ddd, J ¼ 15.3, 8.0, 3.2 Hz, 1H), 2.46–2.35
(m, 2H), 2.08–1.93 (m, 2H), 1.84 (ddt, J ¼ 12.5, 4.6, 2.3 Hz, 1H),
1.74–1.47 (m, 3H), 1.34 (qd, J ¼ 12.8, 4.1 Hz, 1H), 1.23 (dd, J ¼
12.9, 2.3 Hz, 1H), 1.18 (s, 3H), 1.11–0.99 (m, 1H), 0.65 (s, 3H);
13C NMR (101 MHz, CD3OD) d 171.0, 154.4, 153.0, 151.9, 151.8,
151.7, 148.9, 143.4, 143.3, 126.1, 122.2, 122.2, 116.7, 113.8,
113.6, 108.5, 80.8, 74.6, 72.2, 64.9, 57.7, 56.3, 43.6, 40.2, 38.9,
37.9, 28.9, 26.8, 25.2, 23.4, 15.4; HRMS (ESI): m/z 512.2099 [M +
Na]+, calcd for C26H32FNNaO7, 512.2061.
14b-(20-methoxy-phenoxy)-andrographolide (12f)
1
From 10f in 93% yield, white solid, mp 176–178 ꢀC. H NMR
(400 MHz, CDCl3) d 7.13–7.02 (m, 1H), 7.01–6.83 (m, 4H), 5.58 (t,
J ¼ 13.3 Hz, 1H), 4.82 (t, J ¼ 7.3 Hz, 1H), 4.55–4.40 (m, 2H), 4.32
(dd, J ¼ 7.6, 6.7 Hz, 1H), 4.13 (t, J ¼ 8.9 Hz, 1H), 3.92–3.81 (m,
3H), 3.47–3.35 (m, 1H), 3.30 (d, J ¼ 11.1 Hz, 1H), 2.42–2.33 (m,
1H), 2.32–2.21 (m, 2H), 2.02–1.66 (m, 5H), 1.56 (dt, J ¼ 13.3, 3.6
Hz, 1H), 1.31–1.02 (m, 6H), 0.61 (dd, J ¼ 18.6, 2.5 Hz, 3H); 13C
NMR (101 MHz, CDCl3) d 169.77, 151.35, 150.68, 147.23, 145.29,
125.46, 124.33, 121.09, 119.97, 112.35, 108.12, 80.45, 73.42,
71.20, 64.14, 55.80, 55.69, 55.03, 42.87, 38.88, 37.69, 36.64,
28.22, 25.55, 23.73, 22.70, 15.10; HRMS (ESI): m/z 479.2408 [M +
Na]+, calcd for C27H36NaO6, 479.2410.
14a-(40-nitro-phenoxy)-andrographolide (13b)
1
ꢀ
From 11b in 82% yield, white solid, mp 163–166 C. H NMR
(400 MHz, C6D6) d 8.18–8.13 (m, 2H), 7.25 (m, 1H), 6.91–6.86 (m,
2H), 5.21 (d, J ¼ 10.4 Hz, 1H), 5.00 (d, J ¼ 6.9 Hz, 2H), 4.84–4.79
(m, 2H), 4.18 (d, J ¼ 11.1 Hz, 1H), 3.54–3.46 (m, 1H), 3.32 (d, J ¼
11.1 Hz, 1H), 2.48 (s, 1H), 2.43–2.35 (m, 1H), 2.13–2.02 (m, 1H),
1.96–1.76 (m, 6H), 1.43–1.14 (m, 6H), 1.25 (s, 3H), 1.23 (s, 3H),
0.66 (s, 3H); 13C NMR (101 MHz, C6D6) d 171.54, 162.43, 147.22,
145.74, 142.21, 134.02, 125.95, 115.01, 109.07, 99.53, 74.96,
72.97, 69.92, 64.28, 52.29, 51.37, 38.36, 38.27, 38.16, 33.92,
30.89, 26.20, 25.85, 25.16, 24.63, 23.45, 16.98; HRMS (ESI): m/z
494.2185 [M + Na]+, calcd for C29H37NNaO7, 494.2155.
14b-(20-methoxy-40-nitro-phenoxy)-andrographolide (12g)
From 10g in 87% yield, pale yellowish solid, mp 151–153 ꢀC. 1H
NMR (400 MHz, CDCl3) d 8.13 (d, J ¼ 7.1 Hz, 2H), 7.16 (q, J ¼ 7.0,
6.5 Hz, 1H), 6.75 (d, J ¼ 9.6 Hz, 1H), 5.64 (d, J ¼ 5.4 Hz, 1H), 4.86
(s, 1H), 4.67 (dd, J ¼ 10.9, 5.7 Hz, 1H), 4.39–4.32 (m, 2H), 4.12 (d,
J ¼ 11.1 Hz, 1H), 3.39 (dd, J ¼ 11.7, 4.3 Hz, 1H), 3.29 (d, J ¼ 11.2
14a-(20-methoxy-40-nitro-phenoxy)-andrographolide (13g)
1
ꢀ
Hz, 1H), 2.54–2.38 (m, 2H), 2.31 (d, J ¼ 32.5 Hz, 5H), 2.11 (s, 3H), From 13g in 79% yield, white solid, mp 155–159 C. H NMR
1.97 (dt, J ¼ 13.0, 6.4 Hz, 1H), 1.92–1.77 (m, 2H), 1.78–1.68 (m, (400 MHz, CD3OD) d 7.93 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.90 (d, J ¼ 2.6
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Hz, 1H), 7.14 (d, J ¼ 8.8 Hz, 1H), 7.05 (dd, J ¼ 9.7, 3.6 Hz, 1H), was set to 1.0 ꢂ 107 as the run terminates. For clustering the
5.85 (d, J ¼ 5.7 Hz, 1H), 4.89 (s, 1H), 4.71 (dd, J ¼ 10.9, 5.8 Hz, conformations, the root mean square deviation tolerance was
1H), 4.65 (s, 1H), 4.44 (dd, J ¼ 10.9, 1.8 Hz, 1H), 4.08 (d, J ¼ 11.1 2.0. Two hundred independent docking runs were carried out
Hz, 1H), 3.96 (s, 3H), 3.36 (t, J ¼ 5.4 Hz, 2H), 2.56–2.38 (m, 3H), for every ligand. ALL other parameters were set to default.
2.08–1.93 (m, 2H), 1.85 (dd, J ¼ 12.8, 2.5 Hz, 1H), 1.78–1.58 (m, Analysis of the results was performed using AutoDockTools and
3H), 1.36 (ddd, J ¼ 25.0, 12.5, 4.0 Hz, 1H), 1.30–1.15 (m, 5H), the docked structures were viewed using Discovery Studio 3.5
0.64 (s, 3H); 13C NMR (101 MHz, CD3OD) d 171.45, 153.12, Client.
152.55, 151.94, 148.73, 144.44, 126.18, 118.28, 116.58, 109.47,
108.45, 80.89, 74.76, 72.59, 64.92, 57.18, 56.87, 56.30, 43.66,
Acknowledgements
39.85, 38.88, 38.16, 28.97, 26.37, 25.18, 23.36, 15.44; HRMS
(ESI): m/z: 524.2253 [M + Na]+, calcd for C27H35NNaO8, 524.2260.
Authors thank Dr Qian Li for his help of the molecular docking.
General information for assay
Notes and references
Every compound was dissolved in DMSO and diluted with PBS
into a stock solution before use. The nal concentration of
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13544 | RSC Adv., 2014, 4, 13533–13545
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