Discovery and preliminary SAR of 14-aryloxy-andrographolide derivatives as antibacterial agents with immunosuppressant activity

Article abstract of DOI:10.1039/c8ra01063c

Antibacterials (which restore gut flora balance) and immunosuppressants (which correct immune defects) are two important and effective therapeutic agents for the treatment of inflammatory bowel disease (IBD) in clinical use today. Since the structural skeleton of andrographolide, isolated from Andrographis paniculata, has become known as a natural antibiotic with anti-inflammation and heat-clearing and detoxifying properties, 14-aryloxy andrographolide derivatives have been designed, synthesized, and tested for their antibacterial effects on E. coli, S. aureus, and E. faecalis, which are related to IBD. It has been discovered in this study that the andrographolide skeleton is more selective against E. faecalis, the 14-aryloxy group with basicity is important for antibacterial functions, and the 14-(8′-quinolinyloxy) group is a good pharmacophore with antibacterial activity. In addition, we found that 7b1 and 8b1 are good and selective inhibitors of E. faecalis; two 14β-(8′-quinolinyloxy) andrographolide derivatives, 6b17 and 9b, exhibit good activity against E. coli, S. aureus, and E. faecalis. Likewise and importantly, further exploration of immunosuppressant activity for IBD shows that compound 7b1 is a selective inhibitor of the TNF-α/NF-κB signaling pathway, whereas 8b1 is selectively active against the TLR4/NF-κB signaling pathway; moreover, the compounds 6b17 and 9b are active in inhibiting the IL-6/STAT3, TLR4/NF-κB, and TNF-α/NF-κB signaling pathways. Based on these results, we have further focused on the development of dual function inhibitors of IBD as antibacterial and immunosuppressant agents by structural modification of andrographolide.

Full text of DOI:10.1039/c8ra01063c

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Discovery and preliminary SAR of 14-aryloxy-
andrographolide derivatives as antibacterial agents
Cite this: RSC Adv., 2018, 8, 9440
with immunosuppressant activity†
Feng Li,‡a Xiao-Min Li,‡b Dekuan Sheng,a Shao-Ru Chen,b Xin Nie,a Zhuyun Liu,§a
a
c
b
b
a
*
*
*
Decai Wang, Qi Zhao,
Yitao Wang, Ying Wang and Guo-Chun Zhou
Antibacterials (which restore gut flora balance) and immunosuppressants (which correct immune defects)
are two important and effective therapeutic agents for the treatment of inflammatory bowel disease (IBD) in
clinical use today. Since the structural skeleton of andrographolide, isolated from Andrographis paniculata,
has become known as a natural antibiotic with anti-inflammation and heat-clearing and detoxifying
properties, 14-aryloxy andrographolide derivatives have been designed, synthesized, and tested for their
antibacterial effects on E. coli, S. aureus, and E. faecalis, which are related to IBD. It has been discovered
in this study that the andrographolide skeleton is more selective against E. faecalis, the 14-aryloxy group
with basicity is important for antibacterial functions, and the 14-(80-quinolinyloxy) group is a good
pharmacophore with antibacterial activity. In addition, we found that 7b1 and 8b1 are good and selective
inhibitors of E. faecalis; two 14b-(80-quinolinyloxy) andrographolide derivatives, 6b17 and 9b, exhibit
good activity against E. coli, S. aureus, and E. faecalis. Likewise and importantly, further exploration of
immunosuppressant activity for IBD shows that compound 7b1 is a selective inhibitor of the TNF-a/NF-
kB signaling pathway, whereas 8b1 is selectively active against the TLR4/NF-kB signaling pathway;
moreover, the compounds 6b17 and 9b are active in inhibiting the IL-6/STAT3, TLR4/NF-kB, and TNF-a/
NF-kB signaling pathways. Based on these results, we have further focused on the development of dual
function inhibitors of IBD as antibacterial and immunosuppressant agents by structural modification of
andrographolide.
Received 3rd February 2018
Accepted 19th February 2018
DOI: 10.1039/c8ra01063c
rsc.li/rsc-advances
regulatory cytokines that are implicated in immune responses
related to IBD.5 Especially, overexpression of proinammatory
1. Introduction
Inammatory bowel disease (IBD)1 is an autoimmune disease
that is characterized by relapsing and remitting chronic
inammation of the gastrointestinal tract; it is believed to be
caused by unbalanced host-commensal microbiota and
a common immune defect in addition to genetic predisposi-
tion.2 Immune responses have been recognized to be related to
IBD pathogenesis,3 and a recent study demonstrated that the
innate immune system is a major determinant of the serum and
tissue proles of IBD,4 further explaining the existence of
cytokines, such as interleukin-6 (IL-6), toll-like receptor 4
(TLR4), and tumor necrosis factor-a (TNF-a), is crucial for IBD
onset and progression; these proinammatory cytokines, as
inammatory mediators, are connected with the activation of
nuclear factor of k light polypeptide gene enhancer in B-cells
(NF-kB) and signal transducer and activator of transcription 3
(STAT3).5 As NF-kB and STAT3 are known to play central roles in
regulating inammatory responses in patients with IBD, they
are recognized as important targets for therapeutic intervention
of IBD.6 Gastrointestinal microbiota are believed to be
commensal and mutualistic to humans and animals and to
have health benets for humans and animals in some aspects.7
Signicant imbalances of gastrointestinal ora are observed in
patients with IBD as compared to the case of healthy people; for
example, proteobacteria and actinobacteria appear to dominate
in people with ulcerative colitis (UC), whereas Enterococcus (E.)
faecium and several proteobacteria over-inhabit the gastroin-
testinal tracts of people with Crohn's disease (CD).8 Although
the etiology and pathogenesis of IBD are not fully understood,
E. coli and E. faecalis are recognized as pathogens of IBD.9 It is
controversial whether S. aureus infection is involved in early
aSchool of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211816, PR
China. E-mail: gczhou@njtech.edu.cn; dcwang@njtech.edu.cn; Tel: +86-25-58139415
bState Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao SAR,
PR China. E-mail: emilyywang@umac.mo
cFaculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa,
Macao SAR, PR China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra01063c
‡ These authors contributed equally to this work.
§ Current address: Department of Pharmaceutical and Chemical Engineering,
Taizhou Polytechnic College, Taizhou 225300, Jiangsu, PR China.
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lesions or established lesions of IBD; however, S. aureus infec- mechanisms and modes of action. On the other hand, some
tion occasionally may occur and complicate IBD during the evidence has been found that the anti-inammatory effects of
course of the disease.10 Metabolites from certain members of andrographolide are related to regulation of the immune
the gut ora may have causal contributions to disorders such as system19 and that andrographolide suppresses TLR4 expression
obesity and colon cancer by changing host signaling pathways.11 and NF-kB signaling in multiple myeloma cells.20 Multi-
In addition, the intrusion of gut ora components into other targeting andrographolide and its analogs have potential use
host compartments can lead to sepsis.11 Therefore, immuno- in the prevention and treatment of metabolic syndrome21 and
suppressants (which correct immune defects) and antibacte- stroke22 via the NF-kB signaling pathway. The andrographolide
rials (which restore ora balance) are two important and derivative isoandrographolide signicantly inhibited the
effective therapeutic agents to treat IBD in clinical use. release of NO and prostaglandin E2 and the production of
Considering this, we envisage that it is a meaningful explora- interleukin-1b (IL-1b) and IL-6 in lipopolysaccharide (LPS)-
tion to discover single compounds with dual functions of anti- stimulated J774A.1 macrophage cells in a dose-dependent
bacterial and immunosuppressant activities.
manner.23 Andrographolide analog AL-1 improved insulin
Andrographis paniculata [Burm. F.] Nees, an herb known as resistance by down-regulating the NF-kB signaling pathway.24
a “natural antibiotic”, is commonly used in China, India and Our previous report25 revealed that some analogs of androgra-
Southeast Asia for the treatment of a large variety of illnesses, pholide play a role in the attenuation of innate immunity; these
especially infectious diseases, by reducing inammation and were identied as potential immunomodulatory inhibitors of
“heat-clearing and detoxifying”. Andrographolide (1, Scheme 1), TLR signaling with distinct regulation of NF-kB family
a bicyclic diterpenoid lactone, is a major active component members. Furthermore, one compound effectively reduced LPS-
isolated from Andrographis paniculata [Burm. F.] Nees;12 its induced pulmonary injury in a mouse in vivo study; biochemical
derivatives,13 such as “Chuanhuning”,14,15 have been used in results showed that the nucleus translocation of phosphory-
China to treat bacterial and viral infections for many years. lated p65 and serum pro-inammatory cytokines decreased.
Numerous andrographolide derivatives have been designed and Based upon these facts, we are interested in developing dual-
synthesized in recent years.16 The antibacterial activities of function analogs of andrographolide as immunosuppressants
andrographolide and its derivatives are not potent and show to inhibit excessive immunity to “self” gastrointestinal micro-
only minimal or marginal direct inhibition of bacterial growth; biota and also as antibacterial agents to control infection by
however, it was discovered that andrographolide inhibits the specic “commensal/mutualistic” gut ora; such compounds
quorum sensing (QS) system17 of P. aeruginosa and that should be valuable in the treatment of IBD.
andrographolide derivatives block biolm formation18 of P.
In our search for dual-functional 14-aryloxy andrographolide
aeruginosa, indicating that andrographolide and its derivatives analogs inhibiting both bacterial growth and innate immunity,
can directly inhibit bacterial growth or/and infection by specic we launched a panel of screening platforms of these analogs to
Scheme 1 Reagents and conditions (andro ¼ andrographolide): (a) anhydrous DCM, 2,2-dimethoxypropane, PPTS, 40 ꢀC; (b) anhydrous THF; 3a
(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$H2O, 40 ꢀC. (d) anhydrous
THF; 3a or 3b (1.0 eq.), phenol (1.5 eq.), DIAD (1.5 eq.), PPh3 (1.5 eq.), 0 ꢀC to room temperature; (e) MeOH/H2O (4/1), TsOH$H2O, 20 ꢀC; (f) AcCl,
TEA, 0 ꢀC, 90% yield for 7b1 or TBSCl, TEA, rt, 89% yield for 7b2; (g) DCM, Dess–Martin periodinane, rt, 90% yield for 8b1 or 86% yield for 8b2; (h)
p-TSA, MeOH, 40 ꢀC, 8 h, 80% yield from 8b1 or DCM, TFA, ꢁ20 ꢀC, 20 min, 81% yield from 8b2.
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study their antibacterial activities against E. coli, S. aureus and treatment of digestive diseases caused by gastrointestinal
E. faecalis and preliminary structure–activity relationship (SAR) bacterial infection, antibacterial drugs with diverse and novel
studies as the rst step. Selected active anti-bacterial structural origins are required.28
compounds were explored as immunosuppressants to protect
On the basis of reported literature studies stating that
the host from abnormal immune response initiated by intes- andrographolide and some of its derivatives may function as
tinal commensal/mutualistic bacteria. In this paper, we antibiotics, we are pursuing the discovery of more potent and
describe the synthesis and evaluation of a series of 14-aryloxy specic andrographolide derivatives for the development of
andrographolide compounds against E. coli, S. aureus and E. chemotherapeutical agents against bacterial infection, espe-
faecalis as well as against the IL-6/STAT3, TLR4/NF-kB and TNF- cially for inhibiting gastrointestinal bacterial infection in the
a/NF-kB signaling pathways of innate immune response and development of IBD. The strategy in this study was to submit
their preliminary SAR. It was discovered that analogs of these synthesized compounds to a preliminary screening test
andrographolide are more sensitive to E. faecalis and that active against E. coli, S. aureus and E. faecalis (ciprooxacin was used
antibacterial compounds are greatly superior to andrographo- as a reference compound; see Table 1). Because these androg-
lide to attenuate innate immune response. Our results revealed rapholide analogs did not obviously function as bactericidal
that the andrographolide skeleton has immunosuppressant agents (data not shown), bacteriostatic screening was employed
and antibacterial properties and that these analogs of androg- with gradient concentrations and the percent growth was
rapholide can potentially be used to treat IBD.
plotted versus test concentration to afford the IC50 values.29 The
current antibacterial activity results are listed in Table 1.
In our study streamline, 5 series of 20 compounds bearing
14-(40-nitro)phenoxy groups were rst tested. It was found that
all of these compounds are more selective against E. faecalis
than against E. coli and S. aureus. 14b-3,19-Diol compounds
(6b1–6b5) are more active against E. faecalis than the corre-
sponding 14b-3,19-acetonylidene protected compounds (5b1–
5b5). Furthermore, 14b-(30-substituted 40-nitro)phenoxy-3,19-
diol compounds 6b4 and 6b5 are more sensitive to E. faecalis
than 14b-(20-substituted 40-nitro)phenoxy-3,19-diol compounds
6b1, 6b2 and 6b3. It was observed that the activity patterns of
the 14-(20-uro-40-nitro)phenoxy and 14-(20-carboxylate-40-nitro)
phenoxy derivatives are quite similar. The activity differences
between 14a-(20-substituted-40-nitro)phenoxy groups and 14b-
(20-substituted-40-nitro)phenoxy groups are variable, leading to
an order of 5a1 > 5b1, 5a2 > 5b2, 6b1 > 6a1 and 6b2 > 6a2 when
an electron-withdrawing group (EWG) is at the 20-position but
6b3 < 6a3 when an electron-donating group (EDG) is at the 20-
position. Overall, an EWG at the 20-position (5a1, 5a2, 6a1, 6a2,
6b1 and 6b2) was more favorable for antibacterial activity than
an EDG at the 20-position (5a3, 6a3 and 6b3). Compounds 6b4
and 6b5 with 14b-(30-substituted 40-nitro)phenoxy groups are
more active to E. faecalis than the corresponding compounds
6a4 and 6a5 with 14a-(30-substituted 40-nitro)phenoxy groups,
respectively.
2. Results and discussion
2.1. Synthesis
The title derivatives were synthesized according to our previ-
ously reported synthesis;25,26 the synthesis is outlined in Scheme
1 (ArO groups are depicted in Table 1). Accordingly, 14a-3,19-
acetonylidene-protected andrographolide (3a) was prepared by
the reaction of andrographolide (1) with 2,2-dimethoxypropane
catalyzed by PPTS, followed by a Mitsunobu reaction with acetic
acid, which inverted 14a-OH of 3a into 14b-OAc of 4. Full
hydrolysis of 4 by TsOH$H2O in MeOH/H2O (4/1) at 40 ꢀC
afforded 2 as the 14b-epimer of andrographolide; then, 3,19-
protection of 2 afforded 14b-3,19-acetonylidene andrographo-
lide (3b).
Mitsunobu reactions of 3a or 3b with varied aromatic
phenols were carried out in general from 0 ꢀC to room
temperature in anhydrous THF to afford 14b-aryloxy androgra-
pholide 5b (5b1–5b17) or 14a-aryloxy andrographolide 5a (5a1–
5a5, 5a8–5a9, 5a12, 5a15–5a17), respectively, in mild to
moderate isolated yield depending on the distinct properties of
the phenols. Deprotection of 3,19-acetonylidene from 5a or 5b
afforded the corresponding alcohol 6a (6a1–6a5, 6a8–6a9, 6a12,
6a15–6a17) or 6b (6b1–6b17). As we noted before,26 14a-aryloxy
derivatives are generally much less stable than their corre-
sponding 14b-isomers, and some 14a-aryloxy compounds were
not obtained or were obtained in very low yields. Furthermore,
mono-acetylation or mono-silylation of 6b17 produced 7b1 (R ¼
Ac) or 7b2 (R ¼ TBS), which was oxidized into ketone 8b1 or 8b2
by Dess–Martin periodinane (DMP); nally, deprotection of 8b1
or 8b2 afforded 3-keto-19-alcohol product 9b.
Then, 20-nitro compounds and 30-nitro compounds were
explored; it was discovered that the 14b-3,19-diol isomers of 14-
(20-nitro)phenoxy analog 6b6 and 14-(30-nitro)phenoxy analog
6b7 exhibited mild inhibitory activity to E. faecalis and were
more active than the corresponding 3,19-acetonylidene pro-
tected compounds 5b6 and 5b7. 14a- and 14b-(20-Carboxyl ester)
phenoxy-3,19-diol analogs of 6a8 and 6b8 possessed similar but
weak inhibitory activities to E. faecalis, indicating that 14-
stereochemistry is not the key to their activity; meanwhile, their
2.2. Antibacterial activity and SAR analysis
Numerous bacteriostatic and bactericidal agents have been 3,19-acetonylidene protected analogs 5a8 and 5b8 were almost
discovered and developed against bacterial growth and infec- inactive. We compared the 14-(20-carboxyl ester 40-nitro)phenoxy
tion; however, bacterial resistance to currently used antibacte- analogs 6a1 and 6b1 with the 14-(20-carboxyl ester)phenoxy
rial drugs is becoming a major problem in clinical practice, analogs 6a8 and 6b8; the results suggested that the 40-nitro
especially hospital-acquired infections by these drug-resistant group enhanced antibacterial activity against E. faecalis. A
bacteria.27 To meet the demand of clinical medication for the similar inhibition tendency was observed for 40-carboxyl ester
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Table 1 Antibacterial activities of the synthesized compounds against E. coli, S. aureus and E. faecalisa,b
IC50 (mM)c (inhibition rate (%)d)
IC50 (mM) (inhibition rate (%))
Cmpd
ArO
EC
SA
EF
Cmpd
ArO
EC
SA
EF
5a1
5b1
6a1
6b1
/
(14)
(20)
(10)
(40)
51.2 ꢂ 11.6
(56)
5a8
5b8
6a8
6b8
(32)
/
(21)
(21)
(20)
/
(17)
/
(33)
(22)
(22)
/
(17)
72.2 ꢂ 9.2
83.3 ꢂ 8.3
27.7 ꢂ 3.5
89.7 ꢂ 12.0
5a2
5b2
6a2
6b2
(20)
(11)
(67)
(42)
(20)
(26)
(65)
(25)
56.7 ꢂ 6.5
(56)
5a9
5b9
6a9
6b9
/
/
/
(10)
(15)
/
(45)
(42)
38.7 ꢂ 4.5
38.3 ꢂ 5.9
24.4 ꢂ 3.2
(17)
(40)
53.3 ꢂ 4.1
5a3
5b3
(21)
/
(18)
(16)
(60)
(19)
5b10
6b10
(20)
(39)
(22)
(29)
(46)
72.1 ꢂ 9.4
6a3
6b3
(31)
(9)
(17)
(44)
112.9 ꢂ 14.3
140.3 ꢂ 11.9
5b11
6b11
/
/
/
/
(39)
(17)
5a4
5b4
6a4
6b4
/
/
(46)
(73)
(44)
19.6 ꢂ 2.7
5a12
5b12
6a12
6b12
/
/
/
/
(49)
(52)
(19)
/
(60)
(15)
/
(28)
(24)
(14)
(13)
75.6 ꢂ 17.8
(12)
20.8 ꢂ 4.5
5a5
5b5
(12)
(18)
(10)
(32)
(73)
(34)
5b13
6b13
/
15
/
13
(17)
137.8 ꢂ 5.4
6a5
6b5
(11)
(23)
(10)
(37)
50.4 ꢂ 2.7
21.8 ꢂ 2.5
5b14
6b14
/
/
(47)
(50)
(35)
(18)
5b6
6b6
/
/
43
5a15
5b15
(13)
/
(11)
(33)
25.5 ꢂ 1.5
38.4 ꢂ 3.4
(28)
(12)
15.9 ꢂ 9.0
5b7
6b7
/
/
/
26
/
6a15
6b15
(14)
(14)
(13)
(41)
56.7 ꢂ 5.5
29.3 ꢂ 2.1
17.8 ꢂ 4.2
5a16
5b16
6a16
6b16
/
/
/
/
(14)
/
(22)
/
16.9 ꢂ 5.9
15.5 ꢂ 4.2
65.8 ꢂ 13.5
58.5 ꢂ 4.2
6b17
7b1
7b2
8b1
21.6 ꢂ 1.7
(60)
(24)
37.5 ꢂ 2.2
(58)
(23)
6.3 ꢂ 1.5
8.6 ꢂ 0.7
26.2 ꢂ 2.8
7.6 ꢂ 0.8
(34)
(42)
5a17
5b17
6a17
(44)
(47)
(70)
(35)
(27)
(66)
10.4 ꢂ 2.5
19.3 ꢂ 2.3
16.3 ꢂ 2.1
8b2
9b
Ciprooxacin
(25)
(28)
(71)
27.9 ꢂ 1.5
27.6 ꢂ 1.8
9.1 ꢂ 2.1
0.31 ꢂ 0.06
0.4 ꢂ 0.06
2.3 ꢂ 0.6
a
EC: E. coli; SA: S. aureus; EF: E. faecalis. b “/”represents no inhibition or lower than 10% inhibition rate at 250 mM of tested compound. c IC50 value
d
was generated when the inhibition rate at 250 mM of the tested compound was higher than 80%. The inhibition rate at 250 mM of the tested
compound is indicated in parentheses.
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phenoxy analogs, where the 40-carboxyl ester phenoxy-3,19-diol parallel to those of 6a16 and 6b16 and 14b-isomers 5a16 and
analogs of 6a9 and 6b9 exhibited weak activity against 6a16 were slightly more active than 14a-isomers 5b16 and 6b16,
E. faecalis, whereas 14a-isomer 6a9 was slightly more active respectively. These data imply that a positive charge of nitrogen
than 14b-isomer 6b9 against E. faecalis. However, the 40-carboxyl in the 14-aryloxy group under physiological conditions may play
ester phenoxy analogs 6a9 and 6b9 were more active against a role in antibacterial activity for the substitutions of phenoxy
E. faecalis than the 20-carboxyl ester phenoxy counterparts 6a8 groups by pyridinyloxy groups and of naphthyloxy groups by
and 6b8. Introduction of a cyano (CN) EWG at the 40-position quinolinyloxy groups.
did not improve the antibacterial activity, and only 14b-isomer
Continued exploration in the quinolinyloxy line disclosed
6b10 showed very weak activity against E. faecalis. As above, all that 14-(80-quinolinyloxy) derivatives exhibited novel antibacte-
of these analogs were very weakly active or inactive against rial activity proles. In contrast to the 14-(40-quinolinyloxy)
E. coli and S. aureus.
derivatives, the 14-(80-quinolinyloxy) derivatives were active
On the other hand, OMe as an EDG occupying the 20-, 30- or against all three bacteria; they also showed more selective
40-position afforded different results. 40-OMe derivatives 5b11 inhibition of E. faecalis than of E. coli or S. aureus, similar to the
and 6b11 showed very weak activity against E. faecalis and no other series of 14-aryloxy andrographolide derivatives in this
antibacterial activity against E. coli and S. aureus; it is possible work. Specically, 14a-3,19-acetonylidene compound 5a17
that 40-EDG substitution was not as benecial to antibacterial (clog P 6.77355) was more active against E. faecalis than 14a-
activity as 40-EWG substitution, according to the comparison of 3,19-diol compound 6a17 (clog P 4.73255), while 14b-3,19-ace-
40-OMe derivatives 5b11 and 6b11 with the 40-CO2Et and 40-CN tonylidene compound 5b17 (clog P 6.77355) was less active
derivatives 6a9, 6b9 and 6b10. 14b-(30-OMe) derivative 6b13 was against E. faecalis than 14b-3,19-diol compound 6b17 (clog P
very weakly active, while its corresponding 14b-(30-OMe-40-nitro) 4.73255); this activity pattern against E. faecalis is the same as
analog 6b5 possessed mild inhibitory activity toward E. faecalis, that of 14-(20-nitro-pyridinyl-3-oxy) derivatives 5a15, 5b15, 6a15
indicating that the 40-nitro group enhanced the inhibitory and 6b15. Importantly, 6b17 expressed good antibacterial
activity toward E. faecalis. 14a-20-OMe-3,19-diol derivative 6a12 activity against all three bacteria (Table 1) and was the most
was weakly active against E. faecalis and 6b12 (14b) showed mild potent inhibitor of E. faecalis in this paper (Table 1). These data
inhibitory activity toward E. faecalis, suggesting that the 14b- illustrate that the 14-(80-quinolinyloxy) compounds showed
isomer is more stereochemically active for antibacterial activity. superior results against all three bacteria compared to their
Meanwhile, the 40-nitro group negatively inuenced inhibitory corresponding 14-(40-quinolinyloxy) compounds. 19-Acetylated
activity toward E. faecalis; 6a12 and 6b12 were more active 7b1 (clog P 5.64055) and its 3-ketone 8b1 (clog P 5.37725) had
against E. faecalis than corresponding the 40-nitro analogs 6a3 almost no inhibitory activity toward E. coli and S. aureus but
and 6b3, respectively.
were still very active against E. faecalis. However, 19-silylated
Based on the above data, further exploration to introduce 7b2 (clog P 8.10955) had decreased inhibitory activity toward all
more complex aryl groups at the 14-position were conducted. three bacteria and possessed only mild activity against E. fae-
14b-(10-Naphthyloxy) compound 5b14 exhibited very weak calis, while the antibacterial activity of 3-ketone 8b2 (clog P
inhibitory activity only to E. faecalis, and 14b-(10-naphthyloxy) 7.85675) was very weak against all three bacteria. It is inter-
diol compound 6b14 expressed very weak activity against all esting that 3-keto-19-alcohol compound 9b (clog P 4.47495)
three bacteria. Interestingly, the introduction of a 20-nitro- regained the same antibacterial activity against all three
pyridinyl-3-oxy group at the 14-position increased the activity bacteria (Table 1) as 6b17, although its inhibitory activity
of all four derivatives 5a15, 5b15, 6a15 and 6b15 against E. toward E. faecalis was slightly less potent than that of 6b17. By
faecalis; 14a-3,19-acetonylidene analog 5a15 was the most combining these data, it is suggested that in addition to the
active, while 14a-3,19-diol analog 6a15 was the least active. importance of the andrographolide skeleton, the 14-(80-quino-
Moreover, 5b15 was more active than its corresponding 20-nitro linyloxy) group is essential to antibacterial activity; moreover,
phenoxy derivative 5b6, but 6b15 was less active than 20-nitro modications at the 3- or/and 19-positions are not ignorable,
phenoxy analog 6b6. Corresponding to the replacement of and 19-acetylation greatly reduced inhibitory activity to E. coli
phenoxy groups with pyridinyloxy groups, substitution of and S. aureus while transformation of 3-alcohols into 3-ketones
naphthyloxy by quinolinyloxy was subsequently studied. Unlike slightly inuenced the antibacterial activity.
14b-(10-naphthyloxy) analogs 5b14 and 6b14, four analogs of 14-
(40-quinolinyloxy) derivatives 5a16 (clog P 6.8988), 5b16 (clog P
6.8988), 6a16 (clog P 4.8578) and 6b16 (clog P 4.8578) expressed
2.3. Inhibition of innate immune response and SAR analysis
antibacterial activity against E. faecalis; however, none of these Considering that initiation of a dysregulated immune response
was active against E. coli, and two 14a-isomers, 5a16 and 6a16, within the intestinal mucosa by “self” gut ora and/or food is
were very weak inhibitors of S. aureus. The clog P values of 3,19- due to immune defects in IBD, reduction of over-immunity is an
protected compounds 5a16 and 5b16 were higher than 5.0 and effective treatment of IBD.3,30 TNF-a, the best studied NF-kB
higher than those of 6a16 and 6b16; 5a16 and 5b16 were more activator, is correlated with transformation of NF-kB by various
active against E. faecalis than the corresponding 3,19-diol stimuli from its inactive form to its active form. TLRs are
compounds 6a16 and 6b16, respectively. Unlike the activity involved in the regulation of innate and adaptive immunity,31
relationships between 5a15, 5b15, 6a15 and 6b15, the activity and LPS-activated TLR4 regulates the expression and nucleus
variation patterns against E. faecalis of 5a16 and 5b16 were translocation of NF-kB.32 IL-6 is a crucial pathogenic mediator
9444 | RSC Adv., 2018, 8, 9440–9456
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in IBD that functions by triggering cellular effects and functions
via STAT3 signaling. The proinammatory IL-6/STAT3-
dependent biological network is upregulated in active IBD
patients and is considered to be an important pathogenic factor
in IBD onset33 and progression.34 Based on these facts, immu-
nosuppressants, corticosteroids and anti-TNF-a antibodies
have become commonly used drugs in IBD treatment; they may
affect IBD progression by interfering with cellular oxidative
stress and cytokine production.35
3. Conclusion
Bacterial infections have long been regarded as a formidable
enemy of mankind. Bacterial pathogens dramatically affect the
normal function of host cells, including immune response, for
their own benet during infection; eventually, they destroy or
damage host cells. As reconstitution of gut ora balance and
correction of immune defects are two important strategies for
IBD treatment, we attempted to discover dual-functional
andrographolide analogs for use as antibacterial and immu-
nosuppressant agents that could synergize ultimate efficacy.
In this study, 14-aryloxy andrographolide derivatives were
designed, synthesized and tested against the growth of E. coli, S.
aureus and E. faecalis. Preliminary data suggest that the
andrographolide skeleton is primarily more selective against E.
faecalis; modications at the 3- or/and 19-positions are not
ignorable; the 14-aryloxy moiety strongly determines essential
efficacy against bacteria growth; 14a- and 14b-isomers exhibit
different antibacterial activities; and the substitution positions
at the phenoxy groups and the structural features of the
substitution groups may alter the inhibitory activities of the
compounds. The current data imply that nitrogen-containing
14-aryloxy arenes may play an enhanced role in antibacterial
activity upon the substitution of phenoxy groups by pyridinyloxy
groups and of naphthyloxy groups by quinolinyloxy groups,
suggesting the possibility that the nitrogen of the arene group is
charged under physiological conditions. Moreover, it was
revealed that the 14-(80-quinolinyloxy) group is an important
pharmacophore for antibacterial activity and that 14b-(80-qui-
nolinyloxy) andrographolide derivatives exhibit superb inhibi-
tory activity toward E. faecalis. Importantly, andrographolide
analogs with antibacterial activities also possess immunosup-
pressant activities against the IL-6/STAT3, TLR4/NF-kB and/or
TNF-a/NF-kB signaling pathways, enabling us to conclude that
dual functional andrographolide analogs can play synergistic
roles in IBD treatment.
We utilized luciferase reporters bearing the promoter
regions of TNF-a/NF-kB, TLR4/NF-kB, and IL-6/STAT3 (ref. 25)
to examine the activities of the antibacterial active androgra-
pholide analogs in the regulation of signaling pathways that
govern inammatory response (Table 2). It was noted that these
derivatives did not show obvious cytotoxicity to AD-293 cells;
DCB-3503 was used as a positive compound36 (Table 2). Our
previous study revealed that compound 6b3 is active against the
TNF-a/NF-kB and TLR4/NF-kB signaling pathways;25 however,
6b3 does not have antibacterial activity (Table 1). 14a-
Compounds 5a17 and 6a17 are active against the IL-6/STAT3
signaling pathway but do not inhibit the NF-kB signaling
pathways. 14b-Compounds 5b17 and 6b17 are active to
suppress the three signaling pathways (Table 2), suggesting that
3,19-acetonylidene does not obviously affect the inhibition of
these signaling pathways; although the feature of 3,19-aceto-
nylidene may contribute to the distinct activities of 5b17 and
6b17 against E. faecalis, 6b17 is also 3 times more active than
5b17 against E. faecalis. Compounds 7b1 and 8b1 possess
similar inhibitory activities to E. faecalis; however, 3-alcohol-19-
acetylated compound 7b1 shows selective anti-TNF-a/NF-kB
activity, while 3-keto-19-acetylated compound 8b1 is selectively
active against the TLR4/NF-kB signaling pathway. Like their
similarity in inhibitory activity against E. faecalis, E. coli and S.
aureus (Table 1), 3-keto-19-alcohol compound 9b1 suppresses
three signaling pathways in the same fashion as 6b17 (Table 2).
These interesting data suggest that dual-functional analogs of
andrographolide can behave as antibacterial and immunosup-
pressant agents and possibly have synergistic effects in IBD
treatment.
The results of this study suggest that the development of
andrographloide analogs as antibacterial and immunosup-
pressant agents is possible. Further discovery of more potent
Table 2 EC50 (half effective concentration) values of the antibacterially active compounds against the IL-6/STAT3, TLR4/NF-kB, and TNF-a/NF-
kB signaling pathways and cytotoxicities (CC50, half cytotoxic concentration) in AD-293 cells
EC50 (mM)
c
Entry
Cmpd
IL-6/STAT3a
TLR4/NF-kBa
TNF-a/NF-kBb
CC50 (mM)
1
2
3
4
5
6
7
8
9
10
1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6b3 (ref. 25)
5a17
5b17
6a17
6b17
7b1
8b1
9b
3.49 ꢂ 0.34
8.97 ꢂ 1.03
4.71 ꢂ 0.17
6.35 ꢂ 0.77
5.62 ꢂ 0.13
2.41 ꢂ 0.31
>10
>10
>10
2.85 ꢂ 0.07
>10
2.45 ꢂ 0.49
>10
2.00 ꢂ 0.28
4.91 ꢂ 0.02
>10
2.65 ꢂ 0.07
>10
>10
4.84 ꢂ 0.31
5.96 ꢂ 0.03
—
6.13 ꢂ 0.39
2.03 ꢂ 0.01
>10
1.83 ꢂ 0.39
d
DCB-3503 (ref. 36)
—
—
a
b
c
d
Treated for 16 h. Treated for 4 h. Treated for 24 h. Not applicable.
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structural cores, structural tuning of the 14-aryloxy substitu- 11.5 Hz, 1H), 2.41 (dd, J ¼ 16.5, 5.5 Hz, 1H), 2.26–2.14 (m, 2H),
tions and modications at other positions are underway on the 1.78–1.67 (m, 2H), 1.59 (d, J ¼ 10.8 Hz, 1H), 1.53–1.43 (m, 2H), 1.38
basis of these ndings.
(s, 3H), 1.33 (s, 3H), 1.08 (s, 3H), 1.00 (m, 2H), 0.94 (d, J ¼ 2.1 Hz,
1H), 0.91 (s, 3H), 0.87–0.83 (m, 1H); 13C NMR (101 MHz, C6D6)
d 167.90, 163.40, 160.05, 151.64, 148.46, 141.78, 128.31, 127.80,
124.20, 122.34, 114.01, 107.81, 99.48, 74.86, 73.23, 69.52, 64.21,
55.71, 51.99, 50.77, 38.37, 38.25, 37.61, 33.40, 26.17, 26.10, 25.89,
25.06, 24.41, 23.20, 16.70; ESI-HRMS: m/z 592.2523 [M + Na]+, calcd
for C31H39NNaO9, 592.2515.
4. Materials and methods
4.1. General information for chemistry
1H and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer at 400 and 100 MHz, respectively, in CD3Cl,
CD3OD, (CD3)2SO and C6D6 as indicated. Coupling constants (J)
are expressed in hertz (Hz). Chemical shis (d) of NMR are re-
ported in parts per million (ppm) units relative to the solvent.
High resolution ESI-MS spectra were recorded on an Applied
Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer.
Unless otherwise noted, materials were obtained from
commercial suppliers and were used without further purica-
tion. Melting points were measured using a YRT-3 melting point
apparatus (Shanghai, China) and were uncorrected.
(14a)-(20-Fluoro-40-nitro)phenoxy-3,19-acetonylidene androgra-
1
ꢀ
ꢀ
pholide (5a2). White solid; mp 147 C to 149 C; 47% yield; H
NMR (400 MHz, (CD3)2SO) d 8.26 (dd, J ¼ 10.9, 2.7 Hz, 1H), 8.16
(m, 1H), 7.48 (t, J ¼ 8.8 Hz, 1H), 6.97 (t, J ¼ 6.3 Hz, 1H), 6.07 (d, J
¼ 5.2 Hz, 1H), 4.83 (s, 1H), 4.74 (dd, J ¼ 11.1, 5.5 Hz, 1H), 4.59 (s,
1H), 4.45 (dd, J ¼ 11.1, 1.0 Hz, 1H), 3.82 (d, J ¼ 11.6 Hz, 1H), 3.36
(dd, J ¼ 9.2, 4.2 Hz, 1H), 3.07 (d, J ¼ 11.6 Hz, 1H), 2.47–2.36 (m,
2H), 2.33 (dd, J ¼ 10.3, 2.8 Hz, 1H), 2.03–1.91 (m, 2H), 1.89–1.77
(m, 1H), 1.69–1.55 (m, 2H), 1.54–1.45 (m, 1H), 1.28 (s, 3H), 1.25–
1.20 (m, 2H), 1.23 (s, 3H), 1.10 (s, 3H), 0.86–0.80 (m, 1H), 0.76 (s,
3H); 13C NMR (101 MHz, (CD3)2SO) d 168.64, 152.23, 151.34,
150.40, 150.30, 149.76, 147.60, 141.15, 141.07, 124.19, 121.28,
121.25, 115.75, 112.68, 112.45, 108.54, 98.15, 75.65, 73.01,
70.35, 62.74, 54.93, 51.35, 37.85, 37.14, 36.96, 33.83, 27.35,
25.73, 25.13, 24.96, 24.69, 22.59, 15.59; ESI-HRMS: m/z 552.2374
4.2. The preparation and characterization of 2, 3a, 3b, 4 and
5a1, 5b1, 6a1 and 6b1 were modied from our previous
papers25,26
4.2.1. Preparation of compounds 5a and 5b
General method for synthesis of 5a and 5b. Under N2 atmo- [M + Na]+, calcd for C29H36FNNaO7, 552.2368.
sphere and at 0 ꢀC, 0.5 g (1.28 mmol) 3a or 3b, assorted phenols
(14b)-(20-Fluoro-40-nitro)phenoxy-3,19-acetonylidene androgra-
1
ꢀ
ꢀ
(1.92 mmol) and 0.5 g PPh3 (1.92 mmol) were dissolved in pholide (5b2). White solid; mp 114 C to 117 C; 72% yield; H
8.0 mL anhydrous THF. To the above mixture, a solution of NMR (400 MHz, C6D6) d 7.57 (dd, J ¼ 10.6, 2.6 Hz, 1H), 7.47 (m,
0.39 g (1.92 mmol) diisopropyl azodiformate (DIAD) in 2.0 mL 1H), 7.24–7.19 (m, 1H), 5.76 (m, 1H), 4.84 (d, J ¼ 1.8 Hz, 1H),
anhydrous THF was added dropwise in 5 min, and the reaction 4.71 (d, J ¼ 5.1 Hz, 1H), 4.40 (d, J ¼ 1.7 Hz, 1H), 3.78 (d, J ¼
progress was monitored by TLC. Aer the reaction was 11.6 Hz, 1H), 3.64–3.51 (m, 2H), 3.44 (dd, J ¼ 7.0, 3.5 Hz, 1H),
complete, the reaction mixture was treated with ethyl acetate 3.06 (d, J ¼ 11.5 Hz, 1H), 2.27–2.09 (m, 3H), 1.80–1.61 (m, 2H),
and sol. sat. NaHCO3. The organic phase was washed with brine 1.56–1.44 (m, 2H), 1.40 (s, 3H), 1.34 (s, 3H), 1.27–1.35 (m, 2H),
and dried over anhydrous Na2SO4. The residue was ltered, 1.06 (s, 3H), 0.97 (td, J ¼ 14.7, 13.8, 5.4 Hz, 2H), 0.89 (s, 3H),
dried and subjected to silica gel chromatography using petro- 0.87–0.90 (m, 1H); 13C NMR (101 MHz, C6D6) d 167.99, 167.81,
leum ether and ethyl acetate to yield product 5b or 5a.
153.15, 151.64, 151.49, 150.64, 149.80, 149.70, 148.26, 142.41,
(14a)-(20-Carboxy methyl ester-40-nitro)phenoxy-3,19-acetonylidene 142.34, 124.26, 120.68, 115.14, 113.08, 112.85, 107.85, 99.62,
ꢀ
ꢀ
andrographolide (5a1). White solid; mp 77.3 C to 78.4 C; 51% 74.78, 73.52, 69.61, 64.32, 55.55, 50.74, 38.40, 38.35, 37.62,
yield; 1H NMR (400 MHz, C6D6) d 8.52 (d, J ¼ 2.9 Hz, 1H), 7.75 (dd, 33.33, 26.12, 25.93, 25.12, 24.35, 23.24, 16.78; ESI-HRMS: m/z
J ¼ 9.1, 2.9 Hz, 1H), 7.27–7.22 (m, 1H), 5.79 (d, J ¼ 9.1 Hz, 1H), 4.87 552.2374 [M + Na]+, calcd for C29H36FNNaO7, 552.2418.
(s, 1H), 4.81 (d, J ¼ 5.7 Hz, 1H), 4.61 (s, 1H), 3.79 (d, J ¼ 11.6 Hz,
(14a)-(20-Methoxy-40-nitro)phenoxy-3,19-acetonylidene androg-
1H), 3.73 (dd, J ¼ 10.7, 6.1 Hz, 1H), 3.66 (dd, J ¼ 10.7, 2.6 Hz, 1H), rapholide (5a3).25,26 Pale yellow solid; mp 121 ꢀC to 123 ꢀC; 46%
1
3.45 (dd, J ¼ 7.4, 3.3 Hz, 1H), 3.39 (s, 3H), 3.06 (d, J ¼ 11.6 Hz, 1H), yield; HNMR (400 MHz, C6D6) d 7.56 (dd, J ¼ 8.8, 2.4 Hz, 1H),
2.51 (m, 1H), 2.27–2.16 (m, 2H), 1.89–1.79 (m, 1H), 1.79–1.69 (m, 7.45 (d, J ¼ 2.5 Hz, 1H), 7.14–7.10 (m, 1H), 6.07–5.99 (m, 1H),
1H), 1.62–1.47 (m, 3H), 1.39 (s, 3H), 1.31 (s, 3H), 1.09 (s, 3H), 1.05– 4.90 (s, 1H), 4.82 (s, 1H), 4.61 (s, 1H), 3.85–3.76 (m, 2H), 3.71
0.96 (m, 2H), 0.94 (dd, J ¼ 6.4, 5.0 Hz, 1H), 0.89 (s, 3H), 0.86–0.82 (dd, J ¼ 10.8, 5.9 Hz, 1H), 3.46 (dd, J ¼ 7.5, 3.7 Hz, 1H), 3.11–3.03
(m, 1H); 13C NMR (101 MHz, (CD3)2SO) d 169.2, 164.5, 160.7, 151.6, (m, 4H), 2.41–2.24 (m, 1H), 2.22–2.05 (m, 2H), 1.85 (m, 1H),
148.1, 141.3, 129.3, 127.2, 124.8, 122.1, 116.0, 109.0, 98.6, 76.2, 1.79–1.67 (m, 1H), 1.62–1.52 (m, 1H), 1.62–1.52 (m, 1H), 1.52–
73.4, 70.9, 63.2, 55.3, 53.0, 51.8, 38.3, 37.6, 37.4, 34.4, 27.9, 26.2, 1.46 (m, 1H), 1.41 (s, 3H), 1.36 (s, 3H), 1.32 (m, 1H), 1.08 (s, 3H),
25.6, 25.2, 25.1, 23.0, 16.0; ESI-HRMS: m/z 592.2525 [M + Na]+, 1.04–0.89 (m, 3H), 0.81 (s, 3H); 13C NMR (101 MHz, C6D6)
calcd for C31H39NNaO9, 592.2523.
d 168.17, 151.11, 150.60, 147.17, 143.51, 124.80, 116.98, 115.61,
(14b)-(20-Carboxy methyl ester-40-nitro)phenoxy-3,19-acetonylidene 109.33, 107.40, 99.44, 75.17, 73.37, 69.90, 64.12, 55.82, 55.21,
andrographolide (5b1). White solid; mp 104 ꢀC to 106 ꢀC; 82% 51.25, 38.16, 38.04, 37.64, 34.06, 26.32, 26.01, 25.32, 25.20,
yield; 1H NMR (400 MHz, C6D6) d 8.57 (d, J ¼ 2.9 Hz, 1H), 7.74 (dd, 24.69, 23.16, 16.39; ESI-HRMS: m/z 564.2569 [M + Na]+, calcd for
J ¼ 9.1, 2.9 Hz, 1H), 7.25 (t, J ¼ 6.2 Hz, 1H), 5.71 (d, J ¼ 9.1 Hz, 1H),
4.86 (s, 1H), 4.80 (s, 1H), 4.42 (s, 1H), 3.78 (d, J ¼ 11.5 Hz, 1H), 3.62
C
30H39NNaO8, 564.2573.
(14b)-(20-Methoxy-40-nitro)phenoxy-3,19-acetonylidene androg-
(m, 2H), 3.43 (dd, J ¼ 7.1, 3.2 Hz, 1H), 3.35 (s, 3H), 3.06 (d, J ¼ rapholide (5b3).25,26 Pale yellow solid; mp 129 ꢀC to 131 ꢀC; 51%
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yield; 1H NMR (400 MHz, C6D6) d 7.56 (dd, J ¼ 8.8, 2.6 Hz, 1H), 98.16, 75.67, 71.68, 70.69, 62.73, 56.88, 54.95, 51.35, 37.92,
7.46 (d, J ¼ 2.6 Hz, 1H), 7.18 (d, J ¼ 1.5 Hz, 1H), 5.95 (d, J ¼ 37.14, 36.96, 33.87, 27.38, 25.73, 25.14, 24.97, 24.72, 22.58,
8.8 Hz, 1H), 4.90–4.86 (m, 1H), 4.85–4.80 (m, 1H), 4.39 (dd, J ¼ 15.60; ESI-HRMS: m/z 564.2569 [M
+
Na]+, calcd for
30H39NNaO8, 564.2573.
(14b)-(30-Methoxy-40-nitro)phenoxy-3,19-acetonylidene androg-
1.9, 1.0 Hz, 1H), 3.82–3.74 (m, 2H), 3.63–3.57 (m, 2H), 3.41 (dd, J
¼ 7.3, 3.6 Hz, 1H), 3.10–3.02 (m, 1H), 3.06 (s, 3H), 2.19–2.10 (m,
C
3H), 1.77–1.60 (m, 2H), 1.52–1.44 (m, 1H), 1.44–1.37 (m, 1H), rapholide (5b5). White solid; mp 169 ꢀC to 171 ꢀC; 75% yield; 1H
1.40 (s, 3H), 1.36 (s, 3H), 1.34–1.25 (m, 2H), 1.06 (s, 3H), 0.99 (m, NMR (400 MHz, C6D6) d 7.60 (d, J ¼ 9.0 Hz, 1H), 7.22 (m, 1H),
1H), 0.94–0.80 (m, 3H), 0.85 (s, 3H); 13C NMR (101 MHz, CDCl3) 5.98 (d, J ¼ 2.4 Hz, 1H), 5.54 (dd, J ¼ 9.0, 2.5 Hz, 1H), 4.87 (d, J ¼
d 168.12, 150.83, 150.47, 150.31, 148.06, 143.21, 128.02, 127.78, 0.9 Hz, 1H), 4.76 (d, J ¼ 5.4 Hz, 1H), 4.45 (s, 1H), 3.80–3.69 (m,
127.54, 124.91, 116.92, 114.96, 107.68, 107.11, 99.35, 74.78, 2H), 3.63 (m, 1H), 3.41 (m, 1H), 3.09 (s, 3H), 3.06 (m, 1H), 2.30–
72.86, 69.71, 64.04, 55.41, 55.12, 50.72, 38.16, 38.06, 37.43, 2.13 (m, 3H), 1.70 (m, 2H), 1.52 (t, J ¼ 8.6 Hz, 1H), 1.45–1.36 (m,
33.20, 26.08, 25.82, 25.55, 24.99, 24.32, 23.04, 16.44; ESI-HRMS: 1H), 1.38 (s, 3H), 1.35–1.28 (m, 2H), 1.32 (s, 3H), 1.06 (s, 3H),
m/z 564.2584 [M + Na]+, calcd for C30H39NNaO8, 564.2573.
1.04–0.96 (m, 1H), 0.95–0.90 (m, 1H), 0.98–0.82 (m, 1H), 0.87 (s,
(14a)-(30-Fluoro-40-nitro)phenoxy-3,19-acetonylidene androgra- 3H); 13C NMR (101 MHz, C6D6) d 168.23, 160.90, 155.43, 150.53,
1
ꢀ
ꢀ
pholide (5a4). White solid; mp 137 C to 139 C; 51% yield; H 148.07, 134.84, 127.80, 124.84, 107.95, 104.43, 101.58, 99.50,
NMR (400 MHz, C6D6) d 7.46 (m, 1H), 6.34–6.26 (m, 1H), 6.05 (t, 74.86, 71.75, 69.87, 64.15, 55.72, 55.64, 51.01, 38.39, 38.21,
J ¼ 7.9 Hz, 2H), 5.42 (s, 1H), 5.12 (m, 2H), 3.86 (d, J ¼ 11.5 Hz, 37.65, 33.57, 26.20, 25.92, 25.80, 25.08, 24.45, 23.16, 16.60; ESI-
1H), 3.69 (m, 2H), 3.50 (dd, J ¼ 7.0, 3.2 Hz, 1H), 3.09 (d, J ¼ HRMS: m/z 564.2569 [M + Na]+, calcd for C30H39NNaO8,
11.5 Hz, 1H), 2.32–2.22 (m, 2H), 2.03–1.91 (m, 2H), 1.86–1.64 564.2573.
(m, 4H), 1.43 (s, 3H), 1.41 (s, 3H), 1.50–1.32 (m, 2H), 1.11–1.06
(14b)-(20-Nitro)phenoxy-3,19-acetonylidene andrographolide (5b6).
(m, 2H), 1.04 (s, 3H), 1.01 (s, 3H); 13C NMR (101 MHz, C6D6) White solid; mp 146 ꢀC to 148 ꢀC; 72% yield; 1H NMR (400 MHz,
d 171.37, 163.02, 162.91, 158.53, 155.89, 147.16, 145.92, 133.57, C6D6) d 7.32–7.25 (m, 1H), 7.24–7.18 (m, 2H), 6.77–6.65 (m, 1H),
131.56, 131.49, 128.03, 127.79, 110.42, 110.39, 109.14, 104.87, 6.42–6.30 (m, 1H), 6.06–5.92 (m, 1H), 4.93–4.80 (m, 2H), 4.42–4.35
104.63, 99.58, 74.86, 73.48, 69.98, 64.29, 52.30, 51.40, 38.39, (m, 1H), 3.82 (d, J ¼ 11.5 Hz, 1H), 3.71–3.55 (m, 2H), 3.45 (dd, J ¼
38.28, 38.16, 33.88, 30.73, 26.12, 25.86, 25.14, 24.56, 23.45, 7.7, 3.3 Hz, 1H), 3.08 (d, J ¼ 11.5 Hz, 1H), 2.40–2.29 (m, 1H), 2.26–
16.99; ESI-HRMS: m/z 552.2365 [M + Na]+, calcd for C29H36- 2.11 (m, 2H), 1.86–1.67 (m, 2H), 1.62–1.47 (m, 3H), 1.42 (s, 3H),
FNNaO7, 552.2374.
1.36 (s, 3H), 1.39–1.31 (m, 1H), 1.12 (s, 3H), 1.05–0.91 (m, 3H), 0.87
(14b)-(30-Fluoro-40-nitro)phenoxy-3,19-acetonylidene androgra- (s, 3H); 13C NMR (101 MHz, C6D6) d 168.02, 168.02, 161.21, 151.64,
1
ꢀ
ꢀ
pholide (5b4). White solid; mp 184 C to 187 C; 52% yield; H 149.29, 149.29, 148.41, 141.55, 141.55, 133.24, 133.24, 128.28,
NMR (400 MHz, C6D6) d 7.51 (t, J ¼ 8.9 Hz, 1H), 7.20 (d, J ¼ 128.22, 128.16, 128.04, 127.92, 127.80, 127.68, 125.77, 124.31,
1.6 Hz, 1H), 5.89–5.77 (m, 2H), 4.86 (d, J ¼ 1.1 Hz, 1H), 4.54–4.48 121.71, 121.71, 115.78, 107.77, 99.27, 99.26, 99.26, 75.38, 73.27,
(m, 1H), 4.41 (d, J ¼ 0.7 Hz, 1H), 3.76 (d, J ¼ 11.5 Hz, 1H), 3.50 69.64, 64.11, 59.13, 55.72, 51.04, 38.39, 38.11, 37.63, 37.57, 33.61,
(d, J ¼ 3.8 Hz, 2H), 3.43 (dd, J ¼ 6.8, 3.5 Hz, 1H), 3.06 (d, J ¼ 26.66, 25.93, 25.22, 24.72, 23.19, 16.49; ESI-HRMS: m/z 534.2467
11.5 Hz, 1H), 2.23–2.06 (m, 3H), 1.76–1.63 (m, 2H), 1.48–1.35 [M + Na]+, calcd for C29H37NNaO7, 534.2468.
(m, 2H), 1.38 (s, 3H), 1.33 (s, 3H), 1.34–1.27 (m, 2H), 1.05 (d, J ¼
(14b)-(30-Nitro)phenoxy-3,19-acetonylidene andrographolide (5b7).
4.6 Hz, 3H), 1.04–0.95 (m, 1H), 0.95–0.87 (m, 2H), 0.89 (s, 3H); White solid; mp 163 ꢀC to 165 ꢀC; 32% yield; 1H NMR (400 MHz,
13C NMR (101 MHz, C6D6) d 167.85, 167.84, 161.50, 161.50, C6D6) d 7.51 (d, J ¼ 7.1 Hz, 1H), 7.31 (d, J ¼ 2.1 Hz, 1H), 7.20 (d, J ¼
161.40, 161.40, 158.59, 158.59, 155.95, 155.95, 151.01, 151.01, 6.8 Hz, 2H), 6.62 (t, J ¼ 8.2 Hz, 1H), 6.52 (dd, J ¼ 8.3, 1.8 Hz, 1H),
148.11, 148.11, 132.01, 131.94, 131.94, 124.20, 124.20, 111.06, 4.85 (s, 1H), 4.62 (d, J ¼ 5.1 Hz, 1H), 4.43 (s, 1H), 3.77 (d, J ¼
111.06, 111.03, 111.03, 107.94, 107.94, 104.20, 104.20, 103.96, 11.6 Hz, 1H), 3.64 (dd, J ¼ 10.8, 1.8 Hz, 1H), 3.58 (dd, J ¼ 10.8,
103.96, 99.72, 99.72, 74.57, 74.57, 72.19, 72.19, 69.41, 69.41, 5.5 Hz, 1H), 3.41 (dd, J ¼ 7.1, 3.5 Hz, 1H), 3.06 (d, J ¼ 11.5 Hz, 1H),
64.33, 64.33, 55.60, 55.60, 50.79, 50.79, 38.40, 38.40, 37.67, 2.24–2.05 (m, 3H), 1.68 (m, 2H), 1.52–1.41 (m, 2H), 1.39 (s, 3H),
37.67, 33.47, 33.47, 25.97, 25.97, 25.93, 25.93, 25.07, 25.07, 1.34 (s, 3H), 1.30 (m, 2H), 1.07 (s, 3H), 1.04–0.94 (m, 1H), 0.94–0.89
24.25, 24.25, 23.21, 23.21, 16.82, 16.82; ESI-HRMS: m/z 552.2415 (m, 1H), 0.87 (d, J ¼ 5.9 Hz, 1H), 0.84 (s, 3H); 13C NMR (101 MHz,
[M + Na]+, calcd for C29H36FNNaO7, 552.2374.
C6D6) d 168.13, 157.06, 150.18, 149.49, 147.85, 130.36, 128.16,
(14a)-(30-Methoxy-40-nitro)phenoxy-3,19-acetonylidene androg- 127.92, 127.80, 127.68, 124.91, 122.22, 116.85, 109.17, 107.97,
rapholide (5a5). White solid; mp 127 ꢀC to 129 ꢀC; 41% yield; 1H 99.46, 74.86, 71.83, 69.76, 64.15, 55.58, 50.96, 38.36, 38.22, 37.61,
NMR (400 MHz, (CD3)2SO) d 8.00 (d, J ¼ 9.1 Hz, 1H), 6.98 (dd, J ¼ 33.51, 26.20, 25.90, 25.79, 25.07, 24.38, 23.14, 16.58; ESI-HRMS: m/
10.0, 3.9 Hz, 1H), 6.86 (d, J ¼ 2.4 Hz, 1H), 6.74 (dd, J ¼ 9.1, z 534.2463 [M + Na]+, calcd for C29H37NNaO7, 534.2468.
2.5 Hz, 1H), 6.03 (d, J ¼ 5.2 Hz, 1H), 4.87 (s, 1H), 4.71 (dd, J ¼
(14a)-(20-Carboxy ethyl ester)phenoxy-3,19-acetonylidene
ꢀ
ꢀ
11.0, 5.5 Hz, 1H), 4.61 (s, 1H), 4.36 (dd, J ¼ 11.0, 0.9 Hz, 1H), andrographolide (5a8). White solid; mp 122 C to 124 C; 60%
3.93 (s, 3H), 3.83 (d, J ¼ 11.7 Hz, 1H), 3.39–3.35 (m, 1H), 3.08 (d, yield; 1H NMR (400 MHz, C6D6) d 7.78 (dd, J ¼ 7.7, 1.8 Hz, 1H),
J ¼ 11.6 Hz, 1H), 2.47–2.30 (m, 3H), 2.02–1.92 (m, 2H), 1.88–1.78 7.21 (t, J ¼ 6.0 Hz, 1H), 6.98–6.92 (m, 1H), 6.71 (t, J ¼ 7.6 Hz, 1H),
(m, 1H), 1.69–1.57 (m, 2H), 1.53–1.44 (m, 1H), 1.29 (s, 3H), 1.23 6.38 (d, J ¼ 8.2 Hz, 1H), 5.11 (s, 1H), 4.84 (s, 1H), 4.57 (s, 1H),
(s, 3H), 1.21–1.25 (m, 2H), 1.21–1.16 (m, 1H), 1.10 (s, 3H), 0.77 4.19–4.11 (m, 2H), 4.10–4.06 (m, 1H), 3.83–3.75 (m, 2H), 3.44
(s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 168.84, 161.72, 154.94, (dd, J ¼ 7.9, 3.7 Hz, 1H), 3.07 (d, J ¼ 11.5 Hz, 1H), 2.22–2.13
150.46, 147.60, 133.08, 127.88, 124.70, 108.46, 106.86, 101.33, (m, 3H), 1.84 (m, 1H), 1.78–1.67 (m, 1H), 1.60–1.51 (m, 1H),
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1.51–1.43 (m, 2H), 1.42 (s, 3H), 1.35 (s, 3H), 1.31 (d, J ¼ 2.5 Hz, 3.6 Hz, 1H), 3.06 (d, J ¼ 11.5 Hz, 1H), 2.23–2.04 (m, 3H), 1.74–
1H), 1.09 (s, 3H), 1.06 (t, J ¼ 7.1 Hz, 3H), 0.99–0.88 (m, 3H), 0.80 1.64 (m, 2H), 1.51–1.38 (m, 2H), 1.41 (s, 3H), 1.35 (s, 3H), 1.34–
(s, 3H); 13C NMR (101 MHz, C6D6) d 168.70, 165.60, 156.04, 1.24 (m, 2H), 1.07 (s, 1H), 0.98 (m, 1H), 0.97 (m, 1H), 0.92–0.84
149.51, 147.00, 132.85, 132.04, 125.51, 124.38, 122.50, 117.83, (m, 2H), 0.83 (s, 3H); 13C NMR (101 MHz, C6D6) d 168.06, 159.36,
109.13, 99.26, 75.48, 73.78, 70.37, 64.02, 60.96, 55.62, 51.54, 150.35, 148.00, 134.09, 124.74, 118.44, 115.68, 107.84, 106.04,
38.23, 38.07, 37.66, 34.21, 26.70, 26.05, 25.29, 25.24, 24.89, 99.53, 74.78, 71.45, 69.64, 64.18, 55.54, 50.88, 38.31, 38.25,
23.15, 16.22, 14.10; ESI-HRMS: m/z 561.2824 [M + Na]+ calcd for 37.60, 33.44, 26.12, 25.91, 25.72, 25.08, 24.33, 23.13, 16.59; ESI-
C
32H42NaO7, 561.2828.
(14b)-(20-Carboxy ethyl ester)phenoxy-3,19-acetonylidene androg- 514.2569.
HRMS: m/z 514.2566 [M + Na]+ calcd for C30H37NNaO5,
rapholide (5b8). White solid; mp 168 ꢀC to 170 ꢀC; 78% yield; 1H
(14b)-(40-Methoxy)phenoxy-3,19-acetonylidene andrographolide
1
ꢀ
ꢀ
NMR (400 MHz, C6D6) d 7.80 (dd, J ¼ 7.7, 1.8 Hz, 1H), 7.24–7.18 (5b11). White solid; mp 149 C to 151 C; 48% yield; H NMR
(m, 1H), 6.98–6.92 (m, 1H), 6.69 (m, 1H), 6.28 (d, J ¼ 8.2 Hz, 1H), (400 MHz, C6D6) d 7.20–7.17 (m, 1H), 6.69–6.62 (m, 2H), 6.57–
5.04 (d, J ¼ 5.4 Hz, 1H), 4.84 (d, J ¼ 1.1 Hz, 1H), 4.44 (s, 1H), 6.51 (m, 2H), 4.86–4.78 (m, 2H), 4.43 (q, J ¼ 1.4 Hz, 1H), 4.01
4.19–4.04 (m, 2H), 4.01 (dd, J ¼ 10.6, 1.4 Hz, 1H), 3.82 (d, J ¼ (dd, J ¼ 10.5, 1.8 Hz, 1H), 3.82 (d, J ¼ 11.5 Hz, 1H), 3.75–3.68 (m,
11.5 Hz, 1H), 3.68 (dd, J ¼ 10.6, 5.5 Hz, 1H), 3.42 (dd, J ¼ 7.6, 1H), 3.41 (m, 1H), 3.30 (s, 3H), 3.08 (d, J ¼ 11.5 Hz, 1H), 2.21–
3.7 Hz, 1H), 3.08 (d, J ¼ 11.5 Hz, 1H), 2.24–2.05 (m, 3H), 1.80– 2.05 (m, 3H), 1.82–1.64 (m, 2H), 1.55–1.43 (m, 2H), 1.41 (s, 3H),
1.65 (m, 2H), 1.59 (d, J ¼ 8.3 Hz, 1H), 1.53–1.43 (m, 1H), 1.41 (s, 1.38–1.29 (m, 2H), 1.37 (s, 3H), 1.08 (s, 3H), 1.02–0.90 (m, 2H),
3H), 1.40–1.31 (m, 2H), 1.37 (s, 3H), 1.11 (s, 3H), 1.04 (t, 3H), 0.89–0.79 (m, 1H), 0.83 (s, 3H); 13C NMR (101 MHz, C6D6)
1.00–0.88 (m, 3H), 0.85 (s, 3H); 13C NMR (101 MHz, C6D6) d 168.82, 155.47, 150.74, 149.23, 148.08, 126.05, 117.95, 117.95,
d 168.70, 165.65, 155.99, 149.74, 148.21, 132.91, 132.15, 128.16, 115.12, 115.12, 107.93, 99.28, 75.34, 72.90, 70.43, 64.10, 55.63,
127.92, 127.80, 127.68, 125.81, 123.92, 122.16, 116.61, 107.89, 55.06, 51.21, 38.34, 38.11, 37.64, 33.66, 26.57, 25.98, 25.63,
99.29, 75.26, 73.23, 70.29, 64.14, 60.97, 55.83, 50.97, 38.39, 25.24, 24.72, 23.18, 16.43; ESI-HRMS: m/z 519.2725 [M + Na]+,
38.15, 37.64, 33.49, 26.55, 25.91, 25.87, 25.20, 24.64, 23.22, calcd for C30H40NaO6, 519.2723.
16.51, 14.12; ESI-HRMS: m/z 561.2829 [M + Na]+, calcd for
32H42NaO7, 561.2828.
(14a)-(20-Methoxy)phenoxy-3,19-acetonylidene andrographolide
(5a12). White solid; mp 48.7 C to 52.6 C; 57% yield; H NMR
(14a)-(40-Carboxy ethyl ester)phenoxy-3,19-acetonylidene androg- (400 MHz, C6D6) d 6.81 (ddd, J ¼ 8.1, 6.9, 2.3 Hz, 1H), 6.69–6.61
1
ꢀ
ꢀ
C
rapholide (5a9). White solid; mp 67.8 ꢀC to 73.7 ꢀC; 30% yield; 1H (m, 2H), 6.48 (d, J ¼ 8.2 Hz, 1H), 5.15 (d, J ¼ 5.2 Hz, 1H), 4.82 (s,
NMR (400 MHz, C6D6) d 8.15–8.08 (m, 2H), 6.49 (d, J ¼ 8.7 Hz, 1H), 4.62 (s, 1H), 4.21 (dd, J ¼ 10.6, 1.6 Hz, 1H), 3.82 (d, J ¼
2H), 4.85 (s, 2H), 4.65 (s, 1H), 4.16 (q, J ¼ 7.1 Hz, 2H), 3.82–3.72 11.5 Hz, 1H), 3.75 (dd, J ¼ 10.6, 5.6 Hz, 1H), 3.46 (dd, J ¼ 7.7,
(m, 3H), 3.44 (dd, J ¼ 7.8, 3.8 Hz, 1H), 3.06 (d, J ¼ 11.6 Hz, 1H), 3.7 Hz, 1H), 3.28 (s, 3H), 3.08 (d, J ¼ 11.5 Hz, 1H), 2.25–2.12 (m,
2.39–2.28 (m, 1H), 2.21–2.12 (m, 2H), 1.86–1.69 (m, 2H), 1.54 (t, J 3H), 1.87 (m, 1H), 1.77–1.66 (m, 1H), 1.61–1.52 (m, 1H), 1.50–
¼ 10.1 Hz, 2H), 1.43–1.38 (m, 1H), 1.42 (s, 3H), 1.34 (s, 3H), 1.41 (m, 2H), 1.43 (s, 3H), 1.39 (s, 3H), 1.36–1.27 (m, 2H), 1.09 (s,
1.33–1.29 (m, 1H), 1.09 (s, 3H), 1.05 (t, J ¼ 7.1 Hz, 3H), 1.00–0.93 3H), 1.02–0.87 (m, 4H), 0.82 (s, 3H); 13C NMR (101 MHz,
(m, 2H), 0.85 (t, J ¼ 6.1 Hz, 2H), 0.75 (s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 169.8, 151.3, 149.8, 147.9, 145.9, 126.1, 124.1, 121.2,
(CD3)2SO) d 169.4, 165.7, 161.0, 150.8, 148.1, 131.9, 125.3, 123.7, 119.2, 113.1, 109.1, 98.6, 76.2, 73.3, 71.7, 63.2, 56.0, 55.3, 51.8,
115.8, 109.0, 98.6, 76.1, 71.8, 71.3, 63.2, 60.9, 55.4, 51.8, 38.3, 38.4, 37.6, 37.5, 34.4, 27.9, 26.2, 25.7, 25.2, 25.0, 23.1, 16.0; ESI-
37.6, 37.4, 34.3, 27.8, 26.2, 25.6, 25.4, 25.2, 23.1, 16.1, 14.7; ESI- HRMS: m/z 519.2719 [M + Na]+, calcd for C30H40NaO6, 519.2723.
HRMS: m/z 561.2822 [M + Na]+, calcd for C32H42NaO7, 561.2828.
(14b)-(20-Methoxy)phenoxy-3,19-acetonylidene andrographolide
1
(14b)-(40-Carboxy
ethyl
ester)phenoxyꢀ-3,19-acetoꢀnylidene (5b12). White solid; mp 130 C to 131 C; 60% yield; H NMR
ꢀ
ꢀ
andrographolide (5b9). White solid; mp 122 C to 125 C; 45% (400 MHz, C6D6) d 7.13–7.10 (m, 1H), 6.80 (m, 1H), 6.65 (m, 1H),
1
yield; H NMR (400 MHz, C6D6) d 8.14–8.09 (m, 2H), 7.23–7.18 6.58 (dd, J ¼ 7.9, 1.6 Hz, 1H), 6.47 (dd, J ¼ 8.1, 1.3 Hz, 1H), 5.18
(m, 1H), 6.45 (d, J ¼ 8.8 Hz, 2H), 4.86–4.78 (m, 2H), 4.44 (s, 1H), (d, J ¼ 5.4 Hz, 1H), 4.82 (d, J ¼ 1.2 Hz, 1H), 4.42 (s, 1H), 4.15 (dd,
4.16 (q, J ¼ 7.1 Hz, 2H), 3.80–3.71 (m, 2H), 3.68 (t, J ¼ 7.2 Hz, J ¼ 10.6, 1.5 Hz, 1H), 3.83 (d, J ¼ 11.5 Hz, 1H), 3.65 (dd, J ¼ 10.6,
1H), 3.38 (dd, J ¼ 7.4, 3.6 Hz, 1H), 3.05 (d, J ¼ 11.5 Hz, 1H), 2.16 5.5 Hz, 1H), 3.41 (dd, J ¼ 7.7, 3.7 Hz, 1H), 3.29 (s, 3H), 3.09 (d, J
(m, 3H), 1.75–1.63 (m, 2H), 1.52 (d, J ¼ 10.2 Hz, 1H), 1.46–1.36 ¼ 11.5 Hz, 1H), 2.26–2.04 (m, 3H), 1.83–1.62 (m, 2H), 1.55–1.44
(m, 1H), 1.37 (s, 3H), 1.35–1.26 (m, 2H), 1.33 (s, 3H), 1.06 (s, 3H), (m, 2H), 1.42 (s, 3H), 1.39 (s, 3H), 1.38–1.28 (m, 2H), 1.08 (s, 3H),
1.07–1.01 (m, 3H), 1.01–0.83 (m, 3H), 0.81 (s, 3H); 13C NMR (101 1.05–0.94 (m, 1H), 0.94–0.88 (m, 1H), 0.88–0.83 (m, 1H), 0.85 (s,
MHz, C6D6) d 168.29, 165.52, 160.29, 149.91, 147.98, 132.07 (2C), 3H); 13C NMR (101 MHz, C6D6) d 169.08, 151.78, 149.42, 148.27,
125.26, 124.92, 115.12 (2C), 107.85, 75.00, 71.48, 70.01, 64.14, 145.92, 128.15, 128.03, 127.91, 127.79, 127.67, 126.39, 124.00,
60.70, 55.66, 51.07, 38.37, 38.18, 37.64, 33.49, 26.32, 25.91, 121.11, 120.16, 112.54, 107.85, 99.30, 75.39, 73.44, 70.56, 64.14,
25.68, 25.11, 24.47, 23.17, 16.49, 14.21; ESI-HRMS: m/z 561.2825 55.72, 55.11, 51.10, 38.34, 38.12, 37.66, 33.56, 26.57, 26.00,
[M + Na]+, calcd for C32H42NaO7, 561.2828.
25.64, 25.29, 24.74, 23.24, 16.46; ESI-HRMS: m/z 519.2715 [M +
(14b)-(40-Cyano)phenoxy-3,19-acetonylidene andrographolide Na]+, calcd for C30H40NaO6, 519.2723.
1
ꢀ
ꢀ
(5b10). White solid; mp 177 C to 179 C; 42% yield; H NMR
(14b)-(30-Methoxy)phenoxy-3,19-acetonylidene andrographolide
1
ꢀ
ꢀ
(400 MHz, C6D6) d 7.22–7.18 (m, 1H), 6.97–6.91 (m, 2H), 6.12– (2b13). White solid; mp 168 C to 170 C; 58% yield; H NMR
6.07 (m, 2H), 4.85 (d, J ¼ 1.1 Hz, 1H), 4.66 (s, 1H), 4.42 (s, 1H), (400 MHz, C6D6) d 7.25–7.19 (m, 1H), 6.97 (t, J ¼ 8.2 Hz, 1H),
3.77 (d, J ¼ 11.5 Hz, 1H), 3.63–3.56 (m, 2H), 3.43 (dd, J ¼ 7.1, 6.43 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.38 (t, J ¼ 2.2 Hz, 1H), 6.19 (d,
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(14a)-(Quinolyl-40-oxy)-3,19-acetonylidene andrographolide (5a16).
J ¼ 8.2 Hz, 1H), 4.90 (s, 1H), 4.84 (d, J ¼ 0.9 Hz, 1H), 4.46 (s, 1H),
3.93 (d, J ¼ 10.6 Hz, 1H), 3.81 (d, J ¼ 11.5 Hz, 1H), 3.76–3.68 (m,
1H), 3.40 (dd, J ¼ 7.7, 3.7 Hz, 1H), 3.30 (s, 3H), 3.07 (d, J ¼
11.5 Hz, 1H), 2.34–2.25 (m, 1H), 2.15 (m, 2H), 1.79–1.65 (m, 2H),
1.58 (d, J ¼ 10.4 Hz, 1H), 1.51–1.42 (m, 1H), 1.41 (s, 3H), 1.40–
1.37 (m, 1H), 1.37–1.30 (m, 2H), 1.36 (s, 3H), 1.09 (s, 3H), 1.02–
0.92 (m, 2H), 0.88 (dd, J ¼ 12.9, 2.1 Hz, 1H), 0.81 (s, 3H); 13C
NMR (101 MHz, C6D6) d 168.61, 161.60, 158.18, 149.42, 147.97,
130.45, 125.84, 107.90, 107.58, 107.42, 102.64, 99.25, 75.36,
71.37, 70.39, 64.06, 55.70, 54.79, 51.24, 38.40, 38.10, 37.66,
33.61, 26.66, 25.95, 25.59, 25.20, 24.72, 23.16, 16.40; ESI-HRMS:
m/z 519.2717 [M + Na]+ calcd for C30H40NaO6, 519.2723.
1
ꢀ
ꢀ
White solid; mp 97.8 C to 102.9 C; 40% yield; H NMR (400
MHz, (CD3)2SO) d 8.79 (d, J ¼ 5.2 Hz, 1H), 8.07 (d, J ¼ 8.3 Hz,
1H), 7.99 (d, J ¼ 8.4 Hz, 1H), 7.79–7.74 (m, 1H), 7.57 (t, J ¼
7.6 Hz, 1H), 7.11–7.04 (m, 2H), 6.12 (d, J ¼ 6.9 Hz, 1H), 4.88–4.81
(m, 2H), 4.63 (s, 1H), 4.47–4.42 (m, 1H), 3.76 (d, J ¼ 11.7 Hz, 1H),
3.27 (dd, J ¼ 9.2, 3.9 Hz, 1H), 3.02 (d, J ¼ 11.5 Hz, 1H), 2.31 (d, J
¼ 11.7 Hz, 1H), 2.02 (s, 1H), 1.92 (s, 1H), 1.76–1.57 (m, 3H),
1.53–1.43 (m, 1H), 1.42–1.30 (m, 2H), 1.23 (s, 3H), 1.20 (s, 3H),
1.15–1.12 (m, 2H), 1.10 (s, 1H), 1.06–1.04 (m, 3H), 0.66 (d, J ¼
9.5 Hz, 3H), 0.65 (s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 169.4,
159.3, 151.9, 151.5, 149.5, 148.2, 132.1, 130.5, 129.2, 126.5,
124.9, 121.8, 109.0, 103.0, 98.6, 76.1, 72.1, 71.1, 63.1, 55.2, 51.8,
38.3, 37.5, 37.3, 34.3, 27.9, 26.2, 25.6, 25.5, 25.1, 23.0, 16.0; ESI-
HRMS: m/z 518.2902 [M + H]+, calcd for C32H40NO5, 518.2906.
(14b)-(Naphthyl-10-oxy)-3,19-acetonylidene andrographolide
1
ꢀ
ꢀ
(5b14). White solid; mp 158 C to 161 C; 46% yield; H NMR
(400 MHz, C6D6) d 8.16 (dd, J ¼ 6.2, 3.5 Hz, 1H), 7.59–7.54 (m,
1H), 7.39 (dd, J ¼ 11.0, 4.2 Hz, 1H), 7.30 (d, J ¼ 8.3 Hz, 1H), 7.26–
7.21 (m, 2H), 7.11–7.06 (m, 1H), 6.02 (d, J ¼ 7.6 Hz, 1H), 4.96 (d, J
¼ 5.6 Hz, 1H), 4.85 (s, 1H), 4.51 (s, 1H), 3.88 (d, J ¼ 7.2 Hz, 1H),
3.78 (dd, J ¼ 10.7, 5.6 Hz, 1H), 3.70 (d, J ¼ 11.5 Hz, 1H), 3.06 (dd,
J ¼ 7.6, 3.7 Hz, 1H), 2.98 (d, J ¼ 11.5 Hz, 1H), 2.24–2.03 (m, 3H),
1.74 (dd, J ¼ 20.3, 8.7 Hz, 2H), 1.40–1.33 (m, 1H), 1.32 (s, 3H),
1.31–1.24 (m, 1H), 1.28 (s, 3H), 1.12 (m, 1H), 0.98–0.85 (m, 1H),
0.92 (s, 3H), 0.84–0.73 (m, 1H), 0.71 (s, 3H), 0.70–0.60 (m, 2H);
13C NMR (101 MHz, C6D6) d 168.55, 152.54, 149.60, 147.88,
135.21, 127.02, 126.27, 126.02, 125.83, 125.54, 122.47, 121.65,
107.86, 105.47, 99.17, 74.98, 71.22, 70.42, 64.02, 56.55, 50.80,
38.39, 37.95, 37.74, 33.27, 26.47, 25.68, 25.07, 24.08, 23.17,
16.35, 14.06; ESI-HRMS: m/z 539.2768 [M + Na]+, calcd for
(14b)-(Quinolyl-40-oxy)-3,19-acetonylidene andrographolide
1
ꢀ
ꢀ
(5b16). White solid; mp 122 C to 124 C; 53% yield; H NMR
(400 MHz, C6D6) d 8.63 (d, J ¼ 5.1 Hz, 1H), 8.31–8.25 (m, 1H),
7.98 (dd, J ¼ 8.4, 1.4 Hz, 1H), 7.42 (m, 1H), 7.37–7.30 (m, 1H),
5.66–5.62 (m, 1H), 4.89–4.82 (m, 2H), 4.51 (d, J ¼ 1.7 Hz, 1H),
3.76–3.65 (m, 3H), 3.10 (dd, J ¼ 7.1, 3.5 Hz, 1H), 2.98 (d, J ¼
11.5 Hz, 1H), 2.23–2.05 (m, 3H), 1.80–1.63 (m, 2H), 1.34–1.24
(m, 2H), 1.30 (s, 3H), 1.28 (s, 3H), 1.14 (m, 1H), 1.00–0.91 (m,
1H), 0.90 (s, 3H), 0.76 (s, 3H), 0.74–0.59 (m, 3H); 13C NMR (101
MHz, C6D6) d 168.22, 158.91, 151.10, 150.54, 150.43, 147.82,
130.19, 129.97, 128.15, 127.91, 127.79, 127.67, 126.07, 125.40,
121.97, 121.27, 107.91, 100.92, 99.36, 74.50, 71.28, 69.95, 64.12,
56.67, 50.62, 38.42, 38.08, 37.75, 33.15, 26.10, 25.73, 25.63,
24.94, 23.67, 23.18, 16.53; ESI-HRMS: m/z 518.2900 [M + H]+,
calcd for C32H40NaO5, 518.2906.
C
33H40NaO5, 539.2773.
(14a)-(20-Nitro-pyridinyl-30-oxy)-3,19-acetonylidene androg-
ꢀ
ꢀ
(14a)-(Quinolyl-80-oxy)-3,19-acetonylidene andrographolide (5a17).
White solid; mp 143 ꢀC to 146 ꢀC; 42% yield; 1H NMR (400 MHz,
C6D6) d 8.64 (dd, J ¼ 4.1, 1.7 Hz, 1H), 7.46 (dd, J ¼ 8.4, 1.7 Hz,
1H), 7.27–7.22 (m, 1H), 7.08 (dd, J ¼ 8.2, 1.3 Hz, 1H), 6.99 (t, J ¼
7.8 Hz, 1H), 6.88 (m, 1H), 6.74 (dd, J ¼ 8.3, 4.1 Hz, 1H), 6.08 (d, J
¼ 5.6 Hz, 1H), 4.82 (s, 1H), 4.69 (s, 1H), 4.39 (dd, J ¼ 10.8, 1.6 Hz,
1H), 3.88 (dd, J ¼ 10.8, 5.6 Hz, 1H), 3.79 (d, J ¼ 11.5 Hz, 1H), 3.41
(dd, J ¼ 8.1, 3.9 Hz, 1H), 3.06 (d, J ¼ 11.5 Hz, 1H), 2.35 (m, 1H),
2.25–2.11 (m, 2H), 1.81 (m, 1H), 1.74–1.64 (m, 1H), 1.55–1.48
(m, 1H), 1.48–1.40 (m, 1H), 1.42 (s, 3H), 1.36 (s, 3H), 1.32–1.23
(m, 2H), 1.08 (s, 3H), 0.99–0.78 (m, 3H), 0.71 (s, 3H); 13C NMR
(101 MHz, C6D6) d 169.33, 153.10, 149.55, 149.07, 147.00,
142.46, 135.98, 130.11, 126.78, 126.52, 123.22, 121.41, 119.68,
109.42, 99.18, 75.65, 75.33, 70.92, 64.00, 55.65, 51.54, 38.19,
38.00, 37.68, 34.09, 26.83, 26.09, 25.63, 25.39, 25.04, 23.18,
16.12; ESI-HRMS: m/z 518.2901 [M + H]+, calcd for C32H40NO5,
518.2906.
(14b)-(Quinolyl-80-oxy)-3,19-acetonylidene andrographolide (5b17).
White solid; mp 146 ꢀC to 148 ꢀC; 87% yield; 1H NMR (400 MHz,
C6D6) d 8.67 (dd, J ¼ 4.1, 1.7 Hz, 1H), 7.49 (dd, J ¼ 8.3, 1.7 Hz,
1H), 7.30–7.24 (m, 1H), 7.08 (m, 1H), 7.01 (m, 1H), 6.75 (m, 2H),
5.97 (d, J ¼ 5.5 Hz, 1H), 4.84 (d, J ¼ 1.2 Hz, 1H), 4.54 (s, 1H), 4.25
(dd, J ¼ 10.7, 1.6 Hz, 1H), 3.84–3.75 (m, 2H), 3.28 (m, 1H), 3.06
(d, J ¼ 11.5 Hz, 1H), 2.37 (m, 1H), 2.30–2.21 (m, 1H), 2.21–2.13
(m, 1H), 1.74 (m, 1H), 1.69–1.58 (m, 2H), 1.39 (s, 3H), 1.36–1.30
(m, 1H), 1.34 (s, 3H), 1.29–1.19 (m, 2H), 1.04 (m, 3H), 1.01–0.92
rapholide (5a15). White solid; mp 174 C to 176 C; 53% yield;
1H NMR (400 MHz, (CD3)2SO) d 8.21 (dd, J ¼ 4.5, 1.0 Hz, 1H),
8.05–8.00 (m, 1H), 7.84 (m, 4.6 Hz, 1H), 6.94 (t, J ¼ 6.1 Hz, 1H),
6.07 (d, J ¼ 5.2 Hz, 1H), 4.81 (s, 1H), 4.73 (m, 1H), 4.50–4.43 (m,
2H), 3.83 (d, J ¼ 11.7 Hz, 1H), 3.38–3.34 (m, 1H), 3.08 (d, J ¼
11.6 Hz, 1H), 2.48–2.41 (m, 1H), 2.40–2.29 (m, 2H), 1.96 (m, 2H),
1.81 (m, 1H), 1.68–1.55 (m, 2H), 1.52–1.43 (m, 1H), 1.28 (m, 3H),
1.23 (s, 3H), 1.21–1.25 (m, 1H), 1.21–1.13 (m, 2H), 1.11 (s, 3H),
0.77 (s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 168.57, 151.44,
149.03, 147.50, 144.23, 140.41, 129.46, 126.43, 124.08, 108.52,
98.14, 75.70, 73.14, 70.22, 62.73, 54.90, 51.35, 37.86, 37.13,
36.90, 33.88, 27.41, 25.75, 25.15, 24.74, 24.72, 22.57, 15.57; ESI-
HRMS: m/z 535.2415 [M + Na]+, calcd for C28H36N2NaO7,
535.2420.
(14b)-(20-Nitro-pyridinyl-30-oxy)-3,19-acetonylidene androg-
ꢀ
ꢀ
rapholide (5b15). White solid; mp 104 C to 107 C; 42% yield;
1H NMR (400 MHz, C6D6) d 7.53 (d, J ¼ 4.6 Hz, 1H), 6.32 (m, 1H),
6.10 (m, 1H), 4.82 (s, 1H), 4.76 (s, 1H), 4.33 (s, 1H), 3.82 (d, J ¼
11.5 Hz, 1H), 3.51 (m, 2H), 3.44 (m, 1H), 3.07 (d, J ¼ 11.5 Hz,
1H), 2.30–2.12 (m, 3H), 1.87–1.66 (m, 2H), 1.63–1.47 (m, 3H),
1.42 (s, 3H), 1.39–1.31 (m, 1H), 1.36 (s, 3H), 1.10 (s, 3H), 1.04–
0.88 (m, 3H), 0.90 (s, 3H); 13C NMR (101 MHz, C6D6) d 167.65,
152.42, 151.04, 148.50, 143.65, 140.77, 127.47, 124.75, 123.67,
107.70, 99.31, 75.29, 73.91, 69.17, 64.12, 55.77, 51.06, 38.40,
38.13, 37.58, 33.67, 26.56, 26.05, 25.93, 25.21, 24.63, 23.19,
16.51; ESI-HRMS: m/z 535.2415 [M
28H36N2NaO7, 535.2420.
+
Na]+, calcd for
C
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(m, 1H), 0.84 (m, 2H), 0.75 (s, 3H); 13C NMR (101 MHz, C6D6) 112.45, 108.05, 78.36, 73.02, 70.34, 62.53, 55.12, 54.30, 42.18,
d 169.13, 153.03, 149.84, 149.10, 148.26, 142.17, 135.83, 130.10, 38.43, 37.36, 36.35, 27.77, 24.80, 23.84, 23.01, 14.52; ESI-HRMS:
126.66, 126.65, 122.68, 121.51, 117.63, 107.87, 99.13, 75.55, m/z 512.2054 [M + Na]+, calcd for C26H32FNNaO7, 512.2061.
74.63, 70.62, 64.03, 55.77, 51.17, 38.37, 37.99, 37.69, 33.57,
(14b)-(20-Fluoro-40-nitro)phenoxy-andrographolide (6b2). White
26.74, 25.99, 25.99, 25.31, 24.74, 23.30, 16.25; ESI-HRMS: m/z solid; mp 162 ꢀC to 165 ꢀC; 84% yield; 1H NMR (400 MHz,
518.2880 [M + H]+, calcd for C32H40NO5, 518.2906.
CD3OD) d 8.19–8.11 (m, 2H), 7.34 (t, J ¼ 8.4 Hz, 1H), 7.13–7.04
4.2.2. Preparation of compounds 6a and 6b
(m, 1H), 6.03–5.97 (m, 1H), 4.72 (dd, J ¼ 11.2, 5.5 Hz, 1H), 4.56
General method for synthesis of 6a and 6b. At room tempera- (s, 1H), 4.45 (dd, J ¼ 11.2, 1.4 Hz, 1H), 4.02 (d, J ¼ 11.1 Hz, 1H),
ture, 1.0 mmol 5a or 5b were dissolved in 15 mL methanol, and 3.32 (m, 1H), 3.21 (dd, J ¼ 11.8, 4.1 Hz, 1H), 2.52 (m, 1H), 2.45–
0.1 mmol TsOH$H2O was added. The reaction was stirred at rt 2.34 (m, 2H), 2.07–1.92 (m, 2H), 1.87–1.78 (m, 1H), 1.73–1.46
and was generally complete in 5 min. Aer the reaction was (m, 3H), 1.41–1.28 (m, 1H), 1.22 (m, 1H), 1.17 (s, 3H), 1.10–0.98
complete, the reaction mixture was treated with sol. sat. (m, 1H), 0.64 (s, 3H); 13C NMR (101 MHz, CD3OD) d 171.05,
NaHCO3 and extracted with ethyl acetate. The organic phase 154.37, 152.98, 151.89, 151.83, 151.73, 148.94, 143.42, 143.34,
was washed with brine and dried over anhydrous Na2SO4. The 126.12, 122.24, 122.20, 116.71, 113.79, 113.56, 108.54, 80.83,
solvent was removed in vacuo, and the residue was further 74.64, 72.22, 64.88, 57.72, 56.33, 43.63, 40.24, 38.93, 37.90,
puried using ash column chromatography eluting with 28.89, 26.76, 25.19, 23.39, 15.42; ESI-HRMS: m/z 512.2099 [M +
petroleum ether and ethyl acetate to afford 6a or 6b.
Na]+, calcd for C26H32FNNaO7, 512.2061.
(14a)-(20-Carboxy ethyl ester-40-nitro)phenoxy-andrographolide
(14a)-(20-Methoxy-40-nitro)phenoxy-andrographolide (6a3).25,26
1
1
ꢀ
ꢀ
(6a1). White solid; mp 82.6 C to 92.7 C; 97% yield; H NMR Yellow solid; mp 155 ꢀC to 159 ꢀC; 87% yield; H NMR (400 MHz,
(400 MHz, (CD3)2SO) d 8.51 (d, J ¼ 2.9 Hz, 1H), 8.44 (dd, J ¼ 9.2, CD3OD) d 7.95 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.92 (d, J ¼ 2.6 Hz, 1H),
3.0 Hz, 1H), 7.40 (d, J ¼ 9.3 Hz, 1H), 6.99–6.93 (m, 1H), 6.05 (d, J 7.16 (d, J ¼ 8.8 Hz, 1H), 7.12–7.03 (m, 1H), 5.87 (d, J ¼ 5.7 Hz,
¼ 5.4 Hz, 1H), 5.00 (d, J ¼ 4.9 Hz, 1H), 4.81–4.74 (m, 2H), 4.47 (s, 1H), 4.91 (s, 1H), 4.73 (dd, J ¼ 10.9, 5.8 Hz, 1H), 4.67 (s, 1H), 4.46
1H), 4.39 (dd, J ¼ 11.0, 1.5 Hz, 1H), 4.09 (dd, J ¼ 7.5, 2.9 Hz, 1H), (dd, J ¼ 10.9, 1.8 Hz, 1H), 4.10 (d, J ¼ 11.1 Hz, 1H), 3.98 (s, 3H),
3.79 (s, 3H), 3.79–3.74 (m, 1H), 3.24–3.11 (m, 2H), 2.48–2.44 (m, 3.38 (t, J ¼ 5.4 Hz, 1H), 2.58–2.40 (m, 3H), 2.09–1.95 (m, 2H),
1H), 2.38–2.27 (m, 2H), 1.96–1.86 (m, 2H), 1.70 (d, J ¼ 13.2 Hz, 1.90–1.83 (m, 1H), 1.80–1.60 (m, 3H), 1.44–1.32 (m, 1H), 1.32–
1H), 1.57–1.43 (m, 3H), 1.31 (m, 1H), 1.17–1.10 (m, 1H), 1.08– 1.17 (m, 2H), 1.22 (s, 3H), 0.66 (s, 3H); 13C NMR (101 MHz,
1.01 (m, 1H), 1.04 (s, 3H), 0.52 (s, 3H); 13C NMR (101 MHz, CD3OD) d 171.44, 153.11, 152.54, 151.94, 148.72, 144.43, 126.17,
(CD3)2SO) d 169.2, 164.5, 160.7, 151.7, 148.2, 141.3, 129.2, 127.2, 118.27, 116.57, 109.47, 108.44, 80.88, 74.75, 72.58, 64.91, 57.17,
124.6, 122.1, 116.0, 108.5, 78.8, 73.4, 70.9, 63.0, 55.5, 54.8, 53.1, 56.86, 56.29, 43.65, 39.84, 38.87, 38.15, 28.96, 26.36, 25.17,
42.7, 38.9, 37.8, 36.9, 28.2, 24.9, 24.3, 23.5, 14.9; ESI-HRMS: m/z 23.35, 15.43; ESI-HRMS: m/z 524.2253 [M + Na]+, calcd for
552.2203 [M + Na]+, calcd for C28H35NNaO9, 552.2210.
(14b)-(20-Carboxy ethyl ester-40-nitro)phenoxy-andrographolide
C
27H35NNaO8, 524.2260.
(14b)-(20-Methoxy-40-nitro)phenoxy-andrographolide (6b3).25,26
1
(6b1). White solid; mp 190 ꢀC to 192 ꢀC; 78% yield; H NMR Yellow solid; mp 151 ꢀC to 153 ꢀC; 90% yield; 1H NMR (400 MHz,
(400 MHz, (CD3)2SO) d 8.58 (d, J ¼ 2.9 Hz, 1H), 8.49 (dd, J ¼ 9.2, CDCl3) d 7.89 (m, 1H), 7.84–7.79 (m, 1H), 7.13–7.05 (m, 1H), 6.88
3.0 Hz, 1H), 7.44 (d, J ¼ 9.3 Hz, 1H), 7.02 (t, J ¼ 6.9 Hz, 1H), 6.15 (dd, J ¼ 9.2, 4.2 Hz, 1H), 5.71–5.65 (m, 1H), 4.83 (d, J ¼ 1.9 Hz,
(d, J ¼ 5.2 Hz, 1H), 4.99 (d, J ¼ 4.8 Hz, 1H), 4.83 (s, 1H), 4.77 (dd, 1H), 4.58 (dd, J ¼ 11.0, 5.8 Hz, 1H), 4.42 (dd, J ¼ 11.0, 1.8 Hz,
J ¼ 11.1, 5.5 Hz, 1H), 4.55 (s, 1H), 4.41 (d, J ¼ 11.0 Hz, 1H), 4.07 1H), 4.34 (s, 1H), 4.16–4.08 (m, 1H), 3.96 (s, 3H), 3.38 (dd, J ¼
(dd, J ¼ 7.5, 2.8 Hz, 1H), 3.82 (s, 3H), 3.75 (dd, J ¼ 11.0, 2.8 Hz, 11.6, 3.5 Hz, 1H), 3.28 (d, J ¼ 11.1 Hz, 1H), 2.58 (d, J ¼ 52.7 Hz,
1H), 3.20 (m, 1H), 3.02–2.95 (m, 1H), 2.44 (m, 1H), 2.46–2.30 (m, 2H), 2.44–2.36 (m, 2H), 2.32–2.21 (m, 1H), 1.97 (m, 1H), 1.89
2H), 1.90 (m, 2H), 1.71 (m, 1H), 1.55–1.38 (m, 2H), 1.38–1.24 (m, (dd, J ¼ 9.3, 2.7 Hz, 1H), 1.85–1.78 (m, 1H), 1.78–1.60 (m, 2H),
2H), 1.10–1.00 (m, 1H), 1.04 (s, 3H), 0.86 (m, 1H), 0.54 (s, 3H); 1.52 (m, 1H), 1.26–1.16 (m, 2H), 1.22 (s, 3H), 1.14–1.03 (m, 1H),
13C NMR (101 MHz, (CD3)2SO) d 168.69, 163.67, 160.48, 151.11, 0.61 (s, 3H); 13C NMR (101 MHz, CDCl3) d 168.99, 151.84,
147.59, 140.66, 129.03, 126.96, 124.68, 120.98, 115.50, 107.80, 151.03, 150.56, 147.09, 143.34, 124.42, 117.39, 116.00, 108.22,
78.25, 72.78, 70.46, 62.47, 55.62, 54.18, 52.51, 42.12, 38.67, 107.50, 80.35, 77.37, 77.26, 77.05, 76.73, 73.32, 70.60, 64.04,
37.35, 36.04, 27.71, 25.03, 23.86, 22.95, 14.54; ESI-HRMS: m/z 56.36, 55.75, 55.06, 42.82, 38.93, 37.65, 36.64, 28.16, 25.71,
552.2204 [M + Na]+, calcd for C28H35NNaO9, 552.2210.
(14a)-(20-Fluoro-40-nitro)phenoxy-andrographolide (6a2). White
solid; mp 172 ꢀC to 175 ꢀC; 87% yield; 1H NMR (400 MHz,
23.70, 22.77, 15.12; ESI-HRMS: m/z 524.2253 [M + Na]+, calcd for
C
27H35NNaO8, 524.2260.
(14a)-(30-Fluoro-40-nitro)phenoxy-andrographolide (6a4). White
(CD3)2SO) d 8.25 (dd, J ¼ 10.9, 2.7 Hz, 1H), 8.18–8.13 (m, 1H), solid; mp 191 ꢀC to 194 ꢀC; 86% yield; 1H NMR (400 MHz,
7.48 (t, J ¼ 8.7 Hz, 1H), 6.96 (t, J ¼ 6.3 Hz, 1H), 6.05 (d, J ¼ 5.3 Hz, (CD3)2SO) d 8.10 (t, J ¼ 9.1 Hz, 1H), 7.65 (s, 1H), 7.13 (dd, J ¼
1H), 5.03 (d, J ¼ 4.9 Hz, 1H), 4.79 (s, 1H), 4.74 (m, 1H), 4.54 (s, 13.4, 2.6 Hz, 1H), 6.90 (dd, J ¼ 9.3, 2.3 Hz, 1H), 5.11 (s, 1H), 5.04
1H), 4.44 (d, J ¼ 12.2 Hz, 1H), 4.11 (m, 1H), 3.78 (dd, J ¼ 11.0, (d, J ¼ 4.9 Hz, 1H), 4.93–4.86 (m, 1H), 4.89 (s, 3H), 4.11 (m, 1H),
2.9 Hz, 1H), 3.19 (m, 2H), 2.45 (s, 1H), 2.42–2.26 (m, 2H), 1.98– 3.82 (m, 1H), 3.24 (m, 2H), 2.31 (m, 1H), 2.03–1.91 (m, 2H), 1.89–
1.87 (m, 2H), 1.71 (m, 1H), 1.54 (m, 3H), 1.38–1.25 (m, 1H), 1.18– 1.59 (m, 6H), 1.40–1.27 (m, 1H), 1.26–1.11 (m, 2H), 1.05 (m, 3H),
1.10 (m, 2H), 1.06 (s, 3H), 0.54 (s, 3H); 13C NMR (101 MHz, 0.62 (s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 172.00, 163.60,
(CD3)2SO) d 168.64, 152.22, 151.45, 150.42, 150.32, 149.74, 163.49, 157.78, 155.17, 149.66, 146.99, 131.27, 130.46, 130.40,
147.67, 141.12, 141.04, 124.11, 121.27, 121.23, 115.72, 112.68, 128.16, 111.88, 111.86, 107.72, 105.08, 104.84, 78.29, 72.99,
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71.10, 62.58, 54.48, 51.23, 42.27, 38.45, 37.77, 36.50, 29.11, 1.31–1.18 (m, 2H), 1.24 (s, 3H), 1.10 (m, 1H), 0.58 (s, 3H); 13C
27.80, 23.99, 22.95, 14.77; ESI-HRMS: m/z 512.2055 [M + Na]+, NMR (101 MHz, CDCl3) d 168.74, 152.64, 149.38, 147.27, 141.14,
calcd for C26H32FNNaO7, 512.2061.
134.14, 126.28, 123.69, 122.33, 115.90, 108.07, 80.31, 77.37,
(14b)-(30-Fluoro-40-nitro)phenoxy-andrographolide (6b4). Yellow 77.25, 77.05, 76.73, 73.14, 70.30, 64.10, 55.80, 54.86, 42.80,
solid; mp 163 ꢀC to 166 ꢀC; 90% yield; 1H NMR (400 MHz, 38.89, 37.63, 36.54, 28.12, 25.81, 23.69, 22.63, 15.12; ESI-HRMS:
CD3OD) d 8.20 (t, J ¼ 9.0 Hz, 1H), 7.18–7.06 (m, 2H), 6.99 (m, 1H), m/z 494.2158 [M + Na]+, calcd for C26H33NNaO7, 494.2155.
5.97 (d, J ¼ 5.3 Hz, 1H), 4.70 (dd, J ¼ 11.1, 5.5 Hz, 1H), 4.57 (s,
(14b)-(30-Nitro)phenoxy-andrographolide (6b7). Yellow solid;
1H), 4.40 (dd, J ¼ 11.1, 1.3 Hz, 1H), 4.04 (d, J ¼ 11.1 Hz, 1H), 3.61 mp 144 ꢀC to 146 ꢀC; 93% yield; 1H NMR (400 MHz, CDCl3)
(q, J ¼ 7.0 Hz, 3H), 3.33 (s, 1H), 3.24 (m, 1H), 2.56 (m, 1H), 2.47– d 7.94 (m, 1H), 7.69 (t, J ¼ 2.3 Hz, 1H), 7.58–7.49 (m, 1H), 7.20
2.36 (m, 2H), 2.02 (m, 1H), 1.98–1.91 (m, 1H), 1.89–1.79 (m, 1H), (m, 1H), 7.17–7.11 (m, 1H), 5.62 (m, 1H), 4.87 (dd, J ¼ 2.5,
1.63 (m, 3H), 1.42–1.20 (m, 2H), 1.18 (s, 3H), 1.13–1.01 (m, 1H), 1.3 Hz, 1H), 4.72–4.63 (m, 1H), 4.45–4.35 (m, 2H), 4.16–4.08 (m,
0.66 (s, 3H); 13C NMR (101 MHz, CD3OD) d 171.07, 163.94, 1H), 3.41 (m, 1H), 3.33–3.25 (m, 1H), 2.51 (m, 1H), 2.46–2.38 (m,
163.83, 159.93, 157.31, 152.70, 148.90, 132.84, 132.77, 129.41, 1H), 2.37–2.23 (m, 1H), 2.29 (s, 3H), 2.01–1.93 (m, 1H), 1.93–1.87
126.15, 113.08, 113.05, 108.63, 106.14, 105.90, 80.82, 73.80, 72.22, (m, 1H), 1.83 (m, 1H), 1.74 (m, 1H), 1.62 (m, 2H), 1.34–1.18 (m,
64.90, 57.52, 56.34, 43.65, 40.25, 38.95, 38.09, 28.91, 26.76, 25.20, 2H), 1.24 (s, 3H), 1.18–1.06 (m, 1H), 0.60 (s, 3H); 13C NMR (101
23.39, 15.44; ESI-HRMS: m/z 512.2093 [M + Na]+, calcd for C26- MHz, CDCl3) d 168.90, 156.99, 151.67, 149.36, 146.91, 130.85,
H32FNNaO7, 512.2061.
124.19, 122.41, 117.26, 109.70, 108.28, 80.29, 77.38, 77.06,
(14a)-(30-methoxy-40-nitro)phenoxy-andrographolide (6a5). 76.74, 71.95, 70.39, 64.05, 55.77, 55.15, 42.80, 39.00, 37.68,
White solid; mp 131 ꢀC to 133 ꢀC; 81% yield; 1H NMR (400 MHz, 36.91, 28.05, 25.74, 23.68, 22.72, 15.10; ESI-HRMS: m/z 494.2156
(CD3)2SO) d 7.99 (d, J ¼ 9.1 Hz, 1H), 6.96 (t, J ¼ 6.8 Hz, 1H), 6.85 [M + Na]+, calcd for C26H33NNaO7, 494.2155.
(d, J ¼ 2.4 Hz, 1H), 6.73 (dd, J ¼ 9.1, 2.4 Hz, 1H), 6.01 (d, J ¼
(14a)-(20-Carboxy ethyl ester)phenoxy-andrographolide (6a8).
5.0 Hz, 1H), 5.04 (d, J ¼ 4.9 Hz, 1H), 4.83 (s, 1H), 4.71 (dd, J ¼ White solid; mp 157 ꢀC to 159 ꢀC; 96% yield; 1H NMR (400 MHz,
11.0, 5.4 Hz, 1H), 4.56 (s, 1H), 4.39–4.33 (m, 1H), 4.12 (dd, J ¼ C6D6) d 7.78 (dd, J ¼ 7.7, 1.8 Hz, 1H), 7.21 (t, J ¼ 6.0 Hz, 1H),
7.4, 2.9 Hz, 1H), 3.78 (dd, J ¼ 11.0, 2.8 Hz, 1H), 3.26–3.14 (m, 6.98–6.92 (m, 1H), 6.71 (t, J ¼ 7.6 Hz, 1H), 6.38 (d, J ¼ 8.2 Hz,
2H), 2.44 (dd, J ¼ 5.8, 3.0 Hz, 1H), 2.36–2.28 (m, 2H), 1.97–1.87 1H), 5.11 (s, 1H), 4.84 (s, 1H), 4.57 (s, 1H), 4.19–4.11 (m, 2H),
(m, 2H), 1.76–1.68 (m, 1H), 1.60–1.48 (m, 3H), 1.36–1.22 (m, 4.09 (dd, J ¼ 8.8, 4.0 Hz, 1H), 3.78 (dd, J ¼ 17.5, 8.6 Hz, 2H), 3.44
2H), 1.18–1.13 (m, 3H), 1.06 (s, 3H), 0.90–0.76 (m, 1H), 0.55 (s, (dd, J ¼ 7.9, 3.7 Hz, 1H), 3.07 (d, J ¼ 11.5 Hz, 1H), 2.22–2.13 (m,
3H); 13C NMR (101 MHz, CDCl3) d 168.7, 161.5, 155.8, 151.9, 3H), 1.89–1.80 (m, 1H), 1.78–1.67 (m, 1H), 1.60–1.51 (m, 1H),
146.6, 134.2, 128.7, 123.8, 109.1, 104.6, 101.7, 80.4, 71.8, 70.4, 1.51–1.43 (m, 2H), 1.42 (s, 3H), 1.35 (s, 3H), 1.31 (d, J ¼ 2.5 Hz,
64.1, 56.7, 55.8, 55.2, 42.8, 38.8, 37.7, 37.0, 28.1, 25.4, 23.6, 22.7, 1H), 1.09 (s, 3H), 1.06 (t, J ¼ 7.1 Hz, 3H), 0.99–0.88 (m, 3H), 0.80
15.2; ESI-HRMS: m/z 524.2252 [M
27H35NNaO8, 524.2260.
(14b)-(30-Methoxy-40-nitro)phenoxy-andrographolide
+
Na]+, calcd for (s, 3H); 13C NMR (101 MHz, C6D6) d 168.70, 165.60, 156.04,
C
149.51, 147.00, 132.85, 132.04, 125.51, 124.38, 122.50, 117.83,
(6b5). 109.13, 99.26, 75.48, 73.78, 70.37, 64.02, 60.96, 55.62, 51.54,
Yellow solid; mp 126 ꢀC to 129 ꢀC; 85% yield; 1H NMR (400 MHz, 38.23, 38.07, 37.66, 34.21, 26.70, 26.05, 25.29, 25.24, 24.89,
CD3OD) d 8.02 (d, J ¼ 9.1 Hz, 1H), 7.15 (t, J ¼ 6.6 Hz, 1H), 6.85 (d, 23.15, 16.22, 14.10; ESI-HRMS: m/z: 521.2509 [M + Na]+, calcd
J ¼ 2.5 Hz, 1H), 6.71 (dd, J ¼ 9.1, 2.5 Hz, 1H), 5.98 (d, J ¼ 5.4 Hz, for C29H38NaO7, 521.2515.
1H), 4.72 (dd, J ¼ 11.0, 5.5 Hz, 1H), 4.60 (s, 1H), 4.42 (dd, J ¼
(14b)-(20-Carboxy ethyl ester)phenoxy-andrographolide (6b8).
11.0, 1.3 Hz, 1H), 4.06 (d, J ¼ 11.1 Hz, 1H), 3.99 (s, 3H), 3.35 (d, J White solid; mp 181 ꢀC to 182 ꢀC; 88% yield; 1H NMR (400 MHz,
¼ 4.2 Hz, 1H), 3.25 (dd, J ¼ 11.8, 4.1 Hz, 1H), 2.60–2.52 (m, 1H), CDCl3) d 7.88 (dd, J ¼ 7.8, 1.7 Hz, 1H), 7.54–7.48 (m, 1H), 7.15 (t,
2.42 (m, 2H), 2.10–2.00 (m, 1H), 1.97 (m, 1H), 1.90–1.81 (m, 1H), J ¼ 7.6 Hz, 1H), 7.04 (t, J ¼ 7.0 Hz, 1H), 6.91 (d, J ¼ 8.3 Hz, 1H),
1.76–1.64 (m, 1H), 1.64–1.54 (m, 2H), 1.42–1.30 (m, 1H), 1.24 5.61 (d, J ¼ 5.3 Hz, 1H), 4.83 (d, J ¼ 1.9 Hz, 1H), 4.58 (dd, J ¼
(m, 1H), 1.20 (s, 3H), 1.15–1.05 (m, 1H), 0.68 (s, 3H); 13C NMR 10.7, 5.4 Hz, 1H), 4.52 (dd, J ¼ 10.7, 1.7 Hz, 1H), 4.34 (dd, J ¼
(101 MHz, CD3OD) d 171.33, 163.25, 156.92, 152.16, 148.93, 14.7, 7.6 Hz, 3H), 4.14 (d, J ¼ 11.1 Hz, 1H), 3.46–3.36 (m, 1H),
135.26, 129.27, 126.63, 108.56, 107.53, 102.64, 80.83, 73.31, 3.33–3.25 (m, 1H), 2.47–2.25 (m, 4H), 2.22–2.21 (m, 1H), 2.02–
72.52, 64.88, 57.55, 57.34, 56.33, 43.65, 40.24, 38.95, 38.03, 1.90 (m, 1H), 1.88–1.78 (m, 2H), 1.78–1.61 (m, 2H), 1.53–1.46
28.91, 26.71, 25.20, 23.38, 15.41; ESI-HRMS: m/z 524.2254 [M + (m, 1H), 1.36 (t, 3H), 1.32–1.16 (m, 2H), 1.24 (s, 3H), 1.10–0.99
Na]+, calcd for C27H35NNaO8, 524.2260.
(m, 1H), 0.56 (s, 3H); 13C NMR (101 MHz, CDCl3) d 169.45,
(14b)-(20-Nitro)phenoxy-andrographolide (6b6). Yellow solid; 165.73, 155.78, 150.94, 147.11, 133.38, 132.26, 124.95, 123.22,
mp 167 ꢀC to 168 ꢀC; 89% yield; 1H NMR (400 MHz, CDCl3) 122.60, 117.01, 108.19, 80.33, 77.36, 77.25, 77.04, 76.73, 73.39,
d 7.89 (dd, J ¼ 8.1, 1.6 Hz, 1H), 7.62–7.55 (m, 1H), 7.18 (t, J ¼ 70.98, 64.09, 61.16, 55.76, 54.90, 42.81, 38.81, 37.63, 36.55,
7.7 Hz, 1H), 7.13 (dd, J ¼ 10.1, 3.9 Hz, 1H), 6.92 (d, J ¼ 8.3 Hz, 28.12, 25.64, 23.68, 22.64, 15.07, 14.29 ESI-HRMS: m/z 521.2517
1H), 5.64 (d, J ¼ 5.6 Hz, 1H), 4.84 (d, J ¼ 15.2 Hz, 1H), 4.66 (dd, J [M + Na]+, calcd for C29H39NaO7, 521.2515.
¼ 10.9, 6.0 Hz, 1H), 4.44 (dd, J ¼ 10.9, 2.0 Hz, 1H), 4.31 (s, 1H),
(14a)-(40-Carboxy ethyl ester)phenoxy-andrographolide (6a9).
4.16–4.09 (m, 1H), 3.43 (dd, J ¼ 11.5, 4.6 Hz, 1H), 3.28 (d, J ¼ White solid; mp 183 ꢀC to 185 ꢀC; 84% yield; 1H NMR (400 MHz,
11.1 Hz, 1H), 2.56–2.45 (m, 1H), 2.38 (dd, J ¼ 13.2, 3.5 Hz, 1H), (CD3)2SO) d 8.00–7.91 (m, 2H), 7.09 (d, J ¼ 8.9 Hz, 2H), 6.93 (t, J
2.26 (m, 1H), 2.09 (s, 3H), 1.98 (t, J ¼ 12.3 Hz, 1H), 1.89 (d, J ¼ ¼ 6.2 Hz, 1H), 5.88 (d, J ¼ 5.5 Hz, 1H), 5.01 (d, J ¼ 4.7 Hz, 1H),
10.4 Hz, 1H), 1.85–1.78 (m, 1H), 1.78–1.63 (m, 2H), 1.59 (m, 1H), 4.82 (s, 1H), 4.73 (dd, J ¼ 10.9, 5.6 Hz, 1H), 4.58 (s, 1H), 4.33–
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4.26 (m, 3H), 4.10 (d, J ¼ 5.1 Hz, 1H), 3.77 (d, J ¼ 10.8 Hz, 1H), 2.21–2.11 (m, 1H), 1.94–1.81 (m, 2H), 1.70 (d, J ¼ 12.9 Hz, 1H),
3.24–3.13 (m, 2H), 2.43 (s, 1H), 2.37–2.27 (m, 2H), 1.96–1.87 (m, 1.62–1.51 (m, 2H), 1.50–1.43 (m, 1H), 1.30 (m, 1H), 1.15–1.07
2H), 1.71 (d, J ¼ 13.1 Hz, 1H), 1.58–1.45 (m, 3H), 1.38–1.24 (m, (m, 2H), 1.05 (s, 3H), 0.53 (s, 3H); 13C NMR (101 MHz, (CD3)2SO)
1H), 1.31 (t, J ¼ 7.1 Hz, 3H), 1.12 (dd, J ¼ 15.9, 12.0 Hz, 2H), 1.05 d 169.8, 151.2, 149.9, 148.0, 145.9, 126.0, 124.0, 121.2, 118.9,
(s, 3H), 0.52 (s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 169.4, 113.1, 108.6, 78.8, 73.3, 71.7, 63.0, 56.0, 55.5, 54.8, 42.7, 38.9,
165.7, 161.0, 150.9, 148.2, 131.9, 125.3, 123.7, 115.8, 108.5, 78.8, 37.9, 36.9, 28.3, 24.9, 24.4, 23.5, 15.0; ESI-HRMS: m/z 479.2406
71.8, 71.2, 63.0, 60.9, 55.6, 54.8, 42.7, 38.9, 37.9, 36.9, 28.3, 25.2, [M + Na]+, calcd for C27H36NaO6, 479.2410.
24.3, 23.5, 21.2, 15.0, 14.7, 14.5; ESI-HRMS: m/z 521.2507 [M +
(14b)-(20-Methoxy)phenoxy-andrographolide (6b12). White
Na]+, calcd for C29H38NaO7, 521.2515.
solid; mp 175 ꢀC to 178 ꢀC; 88% yield; 1H NMR (400 MHz,
(14b)-(40-Carboxy ethyl ester)phenoxy-andrographolide (6b9). CDCl3) d 7.13–7.02 (m, 1H), 7.01–6.83 (m, 4H), 5.56 (d, J ¼
White solid; mp 136 ꢀC to 139 ꢀC; 95% yield; 1H NMR (400 MHz, 5.4 Hz, 1H), 4.81 (d, J ¼ 1.8 Hz, 1H), 4.55–4.40 (m, 2H), 4.32 (s,
CD3OD) d 8.07–8.01 (m, 2H), 7.12 (t, J ¼ 7.3 Hz, 1H), 7.09–7.04 1H), 4.13 (t, J ¼ 8.9 Hz, 1H), 3.92–3.81 (m, 3H), 3.47–3.35 (m,
(m, 2H), 5.88 (d, J ¼ 5.4 Hz, 1H), 4.70 (dd, J ¼ 10.9, 5.5 Hz, 1H), 1H), 3.30 (d, J ¼ 11.1 Hz, 1H), 2.42–2.33 (m, 1H), 2.32–2.21 (m,
4.56 (s, 1H), 4.40–4.31 (m, 3H), 4.03 (d, J ¼ 11.1 Hz, 1H), 3.29 (s, 2H), 2.02–1.90 (m, 1H), 1.88–1.73 (m, 3H), 1.73–1.65 (m, 1H),
1H), 3.20 (dd, J ¼ 11.9, 4.1 Hz, 1H), 2.55–2.47 (m, 1H), 2.45–2.30 1.60–1.52 (m, 1H), 1.32–1.24 (m, 1H), 1.23 (s, 3H), 1.22–1.16 (m,
(m, 2H), 2.08–1.91 (m, 2H), 1.86–1.79 (m, 1H), 1.71–1.58 (m, 1H), 1.10 (td, J ¼ 13.2, 4.2 Hz, 1H), 0.59 (s, 1H); 13C NMR (101
1H), 1.58–1.48 (m, 2H), 1.42–1.27 (m, 4H), 1.24–1.14 (m, 1H), MHz, CDCl3) d 169.77, 151.35, 150.68, 147.23, 145.29, 125.46,
1.17 (s, 3H), 1.09–0.96 (m, 1H), 0.63 (s, 3H); 13C NMR (101 MHz, 124.33, 121.09, 119.97, 112.35, 108.12, 80.45, 73.42, 71.20,
CD3OD) d 171.50, 167.59, 162.09, 151.93, 148.95, 132.93, 126.89, 64.14, 55.80, 55.69, 55.03, 42.87, 38.88, 37.69, 36.64, 28.22,
125.30, 116.59, 108.53, 80.79, 72.88, 72.66, 64.90, 62.07, 57.61, 25.55, 23.73, 22.70, 15.10; ESI-HRMS: m/z 479.2400 [M + Na]+,
56.29, 43.62, 40.21, 38.95, 37.99, 28.87, 26.65, 25.18, 23.36, calcd for C27H36NaO6, 479.2410.
15.43, 14.68; ESI-HRMS: m/z 521.2509 [M + Na]+, calcd for
29H38NaO7, 521.2515.
(14b)-(30-Methoxy)phenoxy-andrographolide (6b13). White
C
solid; mp 156 ꢀC to 158 ꢀC; 92% yield; 1H NMR (400 MHz,
(14b)-(40-Cyano)phenoxy-andrographolide (6b10). White solid; (CD3)2SO) d 7.29–7.16 (m, 1H), 6.94 (dd, J ¼ 7.2, 6.0 Hz, 1H),
1
ꢀ
ꢀ
mp 182 C to 184 C; 76% yield; H NMR (400 MHz, CD3OD) 6.64–6.49 (m, 3H), 5.79 (d, J ¼ 5.3 Hz, 1H), 4.99 (d, J ¼ 4.9 Hz,
d 7.73–7.68 (m, 2H), 7.13–7.06 (m, 3H), 5.86 (d, J ¼ 5.5 Hz, 1H), 1H), 4.81 (s, 1H), 4.66 (dd, J ¼ 10.7, 5.4 Hz, 1H), 4.52 (s, 1H), 4.28
4.85 (s, 1H), 4.65 (dd, J ¼ 11.0, 5.5 Hz, 1H), 4.52 (s, 1H), 4.31 (dd, (dd, J ¼ 10.7, 1.1 Hz, 1H), 4.10 (dd, J ¼ 7.5, 2.8 Hz, 1H), 3.79–3.37
J ¼ 10.9, 1.4 Hz, 1H), 3.99 (d, J ¼ 11.1 Hz, 1H), 3.30–3.35 (m, 1H), (m, 1H), 3.74 (s, 3H), 3.19 (dd, J ¼ 10.8, 7.7 Hz, 1H), 3.05–2.98
3.18 (dd, J ¼ 11.8, 4.2 Hz, 1H), 2.53–2.45 (m, 1H), 2.41–2.28 (m, (m, 1H), 2.43–2.35 (m, 1H), 2.33–2.22 (m, 2H), 1.92 (t, J ¼
2H), 2.02–1.89 (m, 2H), 1.82–1.76 (m, 1H), 1.63 (m, 1H), 1.51 (m, 10.2 Hz, 2H), 1.70 (d, J ¼ 13.2 Hz, 1H), 1.47 (m, 3H), 1.36–1.21
2H), 1.36–1.24 (m, 1H), 1.20–1.15 (m, 1H), 1.14 (s, 3H), 1.06–0.97 (m, 1H), 1.11–1.05 (m, 1H), 1.02 (s, 3H), 0.94 (m, 1H), 0.55 (s,
(m, 1H), 0.60 (s, 3H); 13C NMR (101 MHz, CD3OD) d 171.30, 3H); 13C NMR (101 MHz, (CD3)2SO) d 169.09, 160.65, 157.69,
161.67, 152.20, 148.92, 135.62, 126.56, 119.68, 117.60, 108.51, 149.66, 147.70, 130.25, 125.73, 107.71, 107.55, 107.35, 102.01,
106.25, 80.79, 72.91, 72.40, 64.87, 57.60, 56.33, 43.62, 40.21, 78.24, 71.14, 71.04, 62.52, 55.66, 55.16, 54.21, 42.13, 38.68,
38.93, 38.04, 28.86, 26.67, 25.17, 23.35, 15.40; ESI-HRMS: m/z 37.41, 36.17, 27.75, 24.91, 23.88, 22.93, 14.56; ESI-HRMS: m/z
474.2252 [M + Na]+ calcd for C27H33NNaO5, 474.2256.
(14b)-(40-Methoxy)phenoxy-andrographolide (6b11). White
479.2400 [M + Na]+, calcd for C27H36NaO6, 479.2410.
(14b)-(Naphthyl-10-oxy)andrographolide (6b14). White solid;
solid; mp 178 ꢀC to 180 ꢀC; 75% yield; 1H NMR (400 MHz, mp 172 C to 175 C; 88% yield; H NMR (400 MHz, (CD3)2SO)
(CD3)2SO) d 6.97–6.88 (m, 4H), 6.87–6.82 (m, 1H), 5.66 (d, J ¼ d 8.07 (d, J ¼ 8.1 Hz, 1H), 7.92 (d, J ¼ 7.7 Hz, 1H), 7.62–7.41 (m,
5.4 Hz, 1H), 4.83–4.77 (m, 1H), 4.60 (dd, J ¼ 10.7, 5.4 Hz, 1H), 4H), 7.14 (t, J ¼ 7.2 Hz, 1H), 7.01 (d, J ¼ 7.5 Hz, 1H), 5.99 (d, J ¼
4.46–4.40 (m, 1H), 4.33 (dd, J ¼ 10.7, 1.4 Hz, 1H), 3.78 (d, J ¼ 5.2 Hz, 1H), 4.84 (s, 1H), 4.83–4.79 (m, 1H), 4.76 (d, J ¼ 4.9 Hz,
10.9 Hz, 1H), 3.72 (s, 3H), 3.21 (d, J ¼ 10.8 Hz, 1H), 3.09 (dd, J ¼ 1H), 4.62 (s, 1H), 4.44 (dd, J ¼ 10.8, 1.0 Hz, 1H), 4.05–4.00 (m,
11.2, 4.5 Hz, 1H), 2.30 (m, 1H), 2.21–2.12 (m, 2H), 1.97–1.83 (m, 1H), 3.63 (dd, J ¼ 11.0, 2.8 Hz, 1H), 3.07 (dd, J ¼ 10.8, 7.6 Hz,
2H), 1.71 (m, 1H), 1.61–1.22 (m, 4H), 1.14–0.91 (m, 2H), 1.05 (s, 1H), 2.45–2.33 (m, 2H), 2.33–2.22 (m, 2H), 1.96–1.86 (m, 2H),
3H), 0.55 (s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 169.24, 1.63–1.54 (m, 1H), 1.24 (m, 3H), 1.06 (m, 1H), 0.80 (s, 3H), 0.69
154.49, 150.27, 149.39, 147.73, 125.79, 117.95, 117.95, 114.82, (dd, J ¼ 12.6, 2.2 Hz, 1H), 0.58–0.47 (m, 1H), 0.43 (s, 3H); 13C
114.82, 107.75, 78.30, 72.48, 71.16, 62.55, 55.38, 55.36, 54.22, NMR (101 MHz, (CD3)2SO) d 169.13, 168.88, 151.95, 150.00,
42.16, 38.59, 37.38, 36.24, 27.74, 24.95, 23.87, 22.96, 14.58; ESI- 147.65, 134.31, 127.60, 126.69, 126.08, 125.97, 125.58, 124.93,
HRMS: m/z 479.2417 [M + Na]+, calcd for C27H36NaO6, 479.2410. 121.86, 121.66, 121.00, 107.65, 106.45, 99.49, 77.83, 71.21,
1
ꢀ
ꢀ
(14a)-(20-Methoxy)phenoxy-andrographolide (6a12). White 62.40, 56.28, 53.62, 48.13, 41.83, 38.72, 38.61, 37.39, 35.72,
1
ꢀ
ꢀ
solid; mp 155.2 C to 161.6 C; 92% yield; H NMR (400 MHz, 27.56, 24.92, 23.78, 22.59, 20.75, 20.75, 14.45; ESI-HRMS: m/z
(CD3)2SO) d 7.07–7.03 (m, 2H), 6.97 (d, J ¼ 7.5 Hz, 1H), 6.93–6.87 499.2462 [M + Na]+, calcd for C30H36NaO5, 499.2460.
(m, 1H), 6.82 (dd, J ¼ 6.8, 5.7 Hz, 1H), 5.58 (d, J ¼ 5.3 Hz, 1H),
(14a)-(20-Nitro-pyridinyl-30-oxy)andrographolide (6a15). White
5.04 (d, J ¼ 4.8 Hz, 1H), 4.79 (s, 1H), 4.57 (dd, J ¼ 10.7, 5.4 Hz, solid; mp 140 ꢀC to 142 ꢀC; 83% yield; 1H NMR (400 MHz,
1H), 4.52 (s, 1H), 4.36 (dd, J ¼ 10.7, 1.2 Hz, 1H), 4.12 (dd, J ¼ 7.4, (CD3)2SO) d 8.21 (dd, J ¼ 4.5, 1.1 Hz, 1H), 8.02 (dd, J ¼ 8.5,
2.7 Hz, 1H), 3.81–3.74 (m, 1H), 3.76 (s, 3H), 3.22 (dd, J ¼ 10.9, 1.0 Hz, 1H), 7.83 (dd, J ¼ 8.5, 4.6 Hz, 1H), 6.93 (t, J ¼ 6.0 Hz, 1H),
7.7 Hz, 1H), 3.16 (dd, J ¼ 10.4, 5.0 Hz, 1H), 2.36–2.26 (m, 2H), 6.06 (d, J ¼ 5.4 Hz, 1H), 5.02 (d, J ¼ 4.9 Hz, 1H), 4.77 (s, 1H), 4.72
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(dd, J ¼ 11.2, 5.6 Hz, 1H), 4.46 (dd, J ¼ 11.1, 1.3 Hz, 1H), 4.40 (s, d 8.89 (dd, J ¼ 4.1, 1.7 Hz, 1H), 8.39 (dd, J ¼ 8.4, 1.7 Hz, 1H), 7.69
1H), 4.12 (dd, J ¼ 7.4, 2.9 Hz, 1H), 3.79 (dd, J ¼ 11.0, 2.8 Hz, 1H), (dd, J ¼ 8.2, 0.9 Hz, 1H), 7.62–7.51 (m, 2H), 7.28 (dd, J ¼ 7.6,
3.26–3.13 (m, 2H), 2.47–2.41 (m, 1H), 2.36–2.25 (m, 2H), 1.91 1.0 Hz, 1H), 6.90 (t, J ¼ 6.3 Hz, 1H), 6.06 (d, J ¼ 5.3 Hz, 1H), 5.00
(m, 2H), 1.71 (m, 1H), 1.63–1.44 (m, 3H), 1.32 (m, 1H), 1.19–1.11 (d, J ¼ 4.9 Hz, 1H), 4.76–4.68 (m, 2H), 4.56–4.49 (m, 2H), 4.09
(m, 1H), 1.10–1.02 (m, 1H), 1.06 (s, 3H), 0.55 (s, 3H); 13C NMR (dd, J ¼ 7.5, 2.8 Hz, 1H), 3.71 (dd, J ¼ 11.0, 2.7 Hz, 1H), 3.19–3.00
(101 MHz, (CD3)2SO) d 168.58, 151.60, 149.00, 147.58, 144.24, (m, 2H), 2.27–2.18 (m, 2H), 2.17–2.07 (m, 1H), 1.81 (m, 2H), 1.64
140.35, 129.44, 126.35, 123.96, 108.04, 78.36, 73.08, 70.19, (m, 1H), 1.40 (m, 2H), 1.26–1.20 (m, 2H), 1.15 (m, 1H), 1.03 (m,
62.54, 55.07, 54.28, 42.19, 38.44, 37.30, 36.38, 27.77, 24.62, 1H), 1.00 (s, 3H), 0.36 (s, 3H); 13C NMR (101 MHz, (CD3)2SO)
23.82, 23.02, 14.51; ESI-HRMS: m/z 495.2102 [M + Na]+, calcd for d 169.9, 152.7, 150.1, 149.9, 147.8, 141.3, 136.7, 130.0, 127.2,
C25H32N2NaO7, 495.2107.
126.1, 122.9, 122.5, 116.3, 108.6, 78.7, 74.0, 71.8, 63.0, 55.4, 54.6,
(14b)-(20-Nitro-pyridinyl-30-oxy)andrographolide (6b15). White 42.6, 38.8, 37.8, 36.6, 28.2, 25.1, 24.3, 23.4, 14.8; ESI-HRMS: m/z
solid; mp 146 ꢀC to 149 ꢀC; 90% yield; 1H NMR (400 MHz, 478.2588 [M + H]+, calcd for C29H36NO5, 478.2593.
CD3OD) d 8.18 (dd, J ¼ 4.6, 1.2 Hz, 1H), 7.90 (dd, J ¼ 8.5, 1.1 Hz,
(14b)-(Quinolyl-80-oxy)andrographolide (6b17). White solid;
1
ꢀ
ꢀ
1H), 7.75 (dd, J ¼ 8.5, 4.6 Hz, 1H), 7.16 (td, J ¼ 7.3, 1.5 Hz, 1H), mp 134 C to 136 C; 76% yield; H NMR (400 MHz, CD3OD)
6.05 (d, J ¼ 5.5 Hz, 1H), 4.90 (s, 1H), 4.73 (dd, J ¼ 11.2, 5.6 Hz, d 8.88 (dd, J ¼ 4.3, 1.7 Hz, 1H), 8.40 (dd, J ¼ 8.3, 1.7 Hz, 1H),
1H), 4.54 (s, 1H), 4.47 (dd, J ¼ 11.2, 1.4 Hz, 1H), 4.07 (d, J ¼ 7.67–7.58 (m, 3H), 7.31–7.22 (m, 2H), 6.09 (d, J ¼ 5.5 Hz, 1H),
11.1 Hz, 1H), 3.34 (d, J ¼ 2.9 Hz, 1H), 3.28 (dd, J ¼ 11.9, 4.1 Hz, 4.89 (d, J ¼ 1.0 Hz, 1H), 4.83–4.79 (m, 1H), 4.62–4.55 (m, 2H),
1H), 2.54 (m, 1H), 2.47–2.33 (m, 2H), 2.04 (m, 1H), 1.95 (m, 1H), 3.91 (d, J ¼ 11.1 Hz, 1H), 3.63 (q, J ¼ 7.0 Hz, 1H), 3.21 (d, J ¼
1.90–1.82 (m, 1H), 1.77–1.53 (m, 3H), 1.41–1.26 (m, 2H), 1.21 (s, 10.3 Hz, 1H), 2.52 (dd, J ¼ 11.9, 4.2 Hz, 1H), 2.48–2.34 (m, 2H),
3H), 1.09–0.99 (m, 1H), 0.66 (s, 3H); 13C NMR (101 MHz, CD3OD) 2.32–2.21 (m, 1H), 2.06 (m, 2H), 1.77–1.68 (m, 1H), 1.48–1.34
d 170.88, 153.35, 151.17, 149.05, 145.93, 141.67, 130.25, 126.69, (m, 1H), 1.30–1.24 (m, 1H), 1.20 (m, 2H), 1.09 (m, 1H), 0.99
125.80, 108.52, 80.74, 74.60, 72.00, 64.95, 57.59, 56.03, 43.64, (s, 3H), 0.84 (m, 1H), 0.58 (m, 1H), 0.49 (s, 3H); 13C NMR (101
40.20, 38.89, 37.88, 28.93, 26.91, 25.20, 23.29, 15.47; ESI-HRMS: MHz, CD3OD) d 171.84, 153.59, 151.90, 150.48, 149.14, 141.29,
m/z 495.2101 [M + Na]+, calcd for C25H32N2NaO7, 495.2107.
138.32, 131.55, 128.29, 127.54, 123.50, 122.60, 112.92, 108.19,
(14a)-(Quinolyl-40-oxy)andrographolide (6a16). White solid; 80.50, 73.65, 73.10, 64.79, 57.59, 55.73, 43.35, 40.01, 38.80,
mp 102.6 ꢀC to 104.8 ꢀC; 83% yield; 1H NMR (400 MHz, 37.39, 28.65, 26.58, 25.10, 23.18, 15.28; ESI-HRMS: m/z 500.2407
(CD3)2SO) d 8.75 (d, J ¼ 5.2 Hz, 1H), 8.03 (d, J ¼ 7.5 Hz, 1H), 7.95 [M + Na]+, calcd for C29H35NNaO5, 500.2413.
(d, J ¼ 8.3 Hz, 1H), 7.77–7.71 (m, 1H), 7.53 (t, J ¼ 7.6 Hz, 1H),
4.2.3. Preparation of compound 7b1. To a solution of 0.48 g
7.05 (d, J ¼ 5.3 Hz, 1H), 7.01 (t, J ¼ 6.8 Hz, 1H), 6.08 (d, J ¼ of 6b17 (1.0 mmol) in 15 mL of dichloromethane, 0.15 g of
5.3 Hz, 1H), 4.96 (d, J ¼ 4.9 Hz, 1H), 4.84–4.76 (m, 2H), 4.55 (s, trimethylamine (1.5 mmol) in 2 mL of dichloromethane was
1H), 4.41 (d, J ¼ 11.0 Hz, 1H), 4.05 (dd, J ¼ 7.4, 2.8 Hz, 1H), 3.68 added; the mixture was then treated with 0.094 g of acetyl
(dd, J ¼ 11.0, 2.8 Hz, 1H), 3.13 (dd, J ¼ 11.1, 7.6 Hz, 1H), 3.10– chloride (1.2 mmol) in 2 mL of dichloromethane at 0 ꢀC for
3.02 (m, 1H), 2.43–2.34 (m, 1H), 2.25 (d, J ¼ 11.9 Hz, 1H), 1.94 (d, 30 min. Aer removing the volatile solvents by distillation, the
J ¼ 11.4 Hz, 1H), 1.89–1.83 (m, 1H), 1.63 (d, J ¼ 13.6 Hz, 1H), residue was dissolved in ethyl acetate and washed with satu-
1.49–1.41 (m, 1H), 1.40–1.35 (m, 2H), 0.98 (s, 3H), 0.85–0.67 (m, rated NaHCO3 solution and brine. The organic phase was dried
3H), 0.64–0.56 (m, 1H), 0.40 (s, 3H); 13C NMR (101 MHz, over anhydrous Na2SO4, ltered, and evaporated to dryness; the
(CD3)2SO) d 169.4, 159.3, 151.8, 151.6, 149.4, 148.3, 130.5, 129.2, residue was puried by silica gel column chromatography
126.5, 124.9, 121.8, 121.1, 108.5, 103.0, 78.8, 72.1, 71.1, 63.0, (petroleum ether/ethyl acetate 1/1) to afford 0.47 g of (14b)-
55.5, 54.7, 42.6, 38.9, 37.7, 36.8, 28.2, 25.3, 24.2, 23.5, 14.9; ESI- (quinolyl-80-oxy)-19-acetoxy andrographolide (7b1). 7b1: white
HRMS: m/z 478.2587 [M + H]+, calcd for C29H36NO5, 478.2593.
solid; mp 74 ꢀC to 77 ꢀC; 90% yield; 1H NMR (400 MHz, CD3Cl)
(14b)-(Quinolyl-40-oxy)andrographolide (6b16). White solid; d 9.11 (s, 1H), 8.42 (d, J ¼ 7.9 Hz, 1H), 7.64 (dd, J ¼ 23.1, 7.8 Hz,
1
ꢀ
ꢀ
mp 175 C to 177 C; 88% yield; H NMR (400 MHz, (CD3)2SO) 3H), 7.16 (q, J ¼ 7.0 Hz, 2H), 6.08 (s, 1H), 4.83 (s, 1H), 4.76–4.60
d 8.78 (d, J ¼ 5.2 Hz, 1H), 8.07 (d, J ¼ 7.6 Hz, 1H), 7.99 (d, J ¼ (m, 2H), 4.38 (s, 1H), 4.22 (d, J ¼ 11.6 Hz, 1H), 4.02 (d, J ¼
8.1 Hz, 1H), 7.81–7.74 (m, 1H), 7.21 (t, J ¼ 7.2 Hz, 1H), 7.07 (d, J 11.7 Hz, 1H), 3.00 (t, J ¼ 8.0 Hz, 1H), 2.48–2.33 (m, 2H), 2.28–
¼ 5.3 Hz, 1H), 6.13 (d, J ¼ 5.2 Hz, 1H), 4.84 (s, 1H), 4.78 (d, J ¼ 2.17 (m, 1H), 2.03 (s, 3H), 1.99–1.89 (m, 2H), 1.82 (s, 1H), 1.47–
4.8 Hz, 1H), 4.66 (s, 1H), 4.48 (d, J ¼ 11.1 Hz, 1H), 4.04–3.97 (m, 1.20 (m, 5H), 1.15 (dd, J ¼ 12.6, 2.7 Hz, 1H), 1.08 (s, 3H), 0.94 (q,
1H), 3.61 (dd, J ¼ 11.0, 2.9 Hz, 1H), 3.06 (dd, J ¼ 10.9, 7.6 Hz, J ¼ 11.7, 11.2 Hz, 1H), 0.47 (s, 3H); 13C NMR (101 MHz,
1H), 2.44 (d, J ¼ 8.6 Hz, 1H), 2.31–2.24 (m, 3H), 1.89 (d, J ¼ (CD3)2SO) d 170.8, 169.8, 152.7, 150.5, 149.9, 148.4, 140.7, 136.6,
9.3 Hz, 2H), 1.65–1.52 (m, 2H), 1.31–1.24 (m, 2H), 1.24–1.13 (m, 129.9, 127.2, 126.7, 122.6, 122.0, 113.3, 108.0, 76.6, 72.8, 71.7,
2H), 1.05–0.94 (m, 1H), 0.78 (s, 3H), 0.71–0.59 (m, 2H), 0.44 (s, 65.4, 56.2, 53.68, 4.76, 39.2, 37.9, 36.3, 27.7, 25.35, 25.1, 23.0,
3H); 13C NMR (101 MHz, (CD3)2SO) d 169.0, 166.5, 153.0, 147.9, 21.4, 14.2; ESI-HRMS: m/z 520.2691 [M + H]+, calcd for
147.7, 145.9, 135.4, 129.6, 124.5, 123.8, 121.2, 120.7, 108.4,
104.2, 78.6, 74.8, 70.6, 62.8, 56.7, 54.6, 42.5, 39.3, 37.9, 36.6,
C
31H38NO6, 520.2699.
4.2.4. Preparation of compound 7b2. At room temperature,
28.0, 25.7, 24.3, 23.3, 15.0; ESI-HRMS: m/z 478.2588 [M + H]+, to a solution of 0.4 g (0.84 mmol) 6b17 in 18.0 mL dry
calcd for C29H36NO5, 478.2593.
dichloromethane (DCM), 0.66 mL (5.0 mmol) triethylamine
(14a)-(Quinolyl-80-oxy)andrographolide (6a17). White solid; (TEA) and then 0.64 g (4.2 mmol) TBSCl in 2.0 mL dry DCM were
1
ꢀ
ꢀ
mp 151 C to 153 C; 79% yield; H NMR (400 MHz, (CD3)2SO) added carefully. Aer the reaction was complete in 40 min, the
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reaction mixture was treated with sol. sat. NH4Cl. The organic 169.7, 152.6, 150.1, 149.8, 147.9, 140.8, 136.6, 129.9, 127.2,
phase was extracted with ethyl acetate and washed with sol. sat. 126.6, 122.5, 122.2, 113.9, 108.9, 73.1, 71.7, 65.9, 55.4, 55.0, 53.5,
NaHCO3 and brine, then dried over anhydrous Na2SO4. The 38.9, 37.4, 36.9, 35.6, 26.8, 26.1, 26.0, 25.8, 24.9, 21.3, 18.2, 14.4,
residue was ltered and chromatographed on silica gel (petro- ꢁ5.3, ꢁ5.4; ESI-HRMS: m/z 590.3297 [M + H]+, calcd for
leum ether/ethyl acetate) to provide 0.44 g of (14b)-(quinolyl-80-
oxy)-19-(t-butyldimethylsilyloxy)andrographolide (7b2). 7b2:
C
35H48NO5Si, 590.3302.
4.2.6. Preparation of compound 9b from 8b1 or 8b2. 0.26 g
white solid; mp 149 ꢀC to 151 ꢀC; 89% yield; 1H NMR (400 MHz, (0.50 mmol) of 8b1 was dissolved in 5 mL methanol and then
ꢀ
(CD3)2SO) d 8.88 (dd, J ¼ 4.1, 1.7 Hz, 1H), 8.37 (dd, J ¼ 8.3, treated with 0.17 g (1.0 mmol) of p-TSA at 40 C for 8 h. The
1.6 Hz, 1H), 7.64 (d, J ¼ 7.5 Hz, 1H), 7.61–7.52 (m, 2H), 7.23 (d, mixture was diluted with ethyl acetate and washed with sat.
J ¼ 6.9 Hz, 1H), 7.06 (t, J ¼ 6.9 Hz, 1H), 6.06 (d, J ¼ 5.1 Hz, 1H), NaHCO3 solution and brine; the organic phase was dried over
4.77 (m, 2H), 4.54–4.45 (m, 2H), 4.22 (d, J ¼ 5.2 Hz, 1H), 3.58 (d, anhydrous Na2SO4, ltered, and then concentrated under
J ¼ 10.5 Hz, 1H), 3.47 (d, J ¼ 10.5 Hz, 1H), 2.36–2.14 (m, 4H), reduced pressure. The residue was puried by silica gel
2.00 (m, 1H), 1.87 (m, 1H), 1.62 (m, 1H), 1.48 (m, 1H), 1.22 (m, column chromatography to afford (14b)-(quinolyl-80-oxy)-3-keto
2H), 0.97 (m, 1H), 0.80 (s, 9H), 0.77 (s, 3H), 0.69–0.63 (m, 1H), andrographolide (9b) in 80% yield.
0.56 (m, 1H), 0.47 (s, 3H), ꢁ0.07 (d, J ¼ 2.3 Hz, 6H); 13C NMR
A solution of 0.20 g (0.34 mmol) 8b2 in 4.0 mL dry DCM was
ꢀ
(101 MHz, (CD3)2SO) d 169.27, 152.18, 150.02, 149.36, 148.25, cooled to ꢁ20 C and treated with 4.0 mL triuoroacetic acid
140.20, 136.03, 129.42, 126.68, 126.20, 122.06, 121.40, 112.65, (TFA) for 20 min. The reaction mixture was diluted with ethyl
107.18, 76.67, 72.24, 71.24, 64.12, 55.88, 53.68, 42.24, 38.75, acetate and carefully treated with sol. sat. NaHCO3. The organic
37.78, 36.08, 27.47, 25.60 (3C), 25.30, 24.88, 22.69, 17.73, 13.81, phase was washed with sol. sat. NaHCO3 and brine, then dried
ꢁ5.78, ꢁ5.81; ESI-HRMS: m/z 592.3453 [M + H]+, calcd for over anhydrous Na2SO4. The residue was ltered, dried and
C
35H50NO5Si, 592.3458.
puried by silica gel column chromatography (petroleum ether/
4.2.5. Preparation of compound 8b1 or 8b2. At room ethyl acetate) to afford (14b)-(quinolyl-80-oxy)-3-keto androgra-
temperature and under protection from light, 0.6 mmol of 7b1 pholide (9b) in 81% yield. 9b: white solid; mp 141 ꢀC to 143 ꢀC;
or 7b2 was dissolved in 8.0 mL dry DCM and treated with 0.50 g 1H NMR (400 MHz, (CD3)2SO) d 8.85 (dd, J ¼ 4.1, 1.7 Hz, 1H),
(1.17 mmol) Dess–Martin periodinane. The oxidation reaction 8.36 (dd, J ¼ 8.3, 1.7 Hz, 1H), 7.64 (d, J ¼ 7.5 Hz, 1H), 7.60–7.50
was complete in 3 h, and the mixture was diluted with 100 mL (m, 2H), 7.24 (d, J ¼ 6.8 Hz, 1H), 7.01 (dd, J ¼ 7.3, 6.1 Hz, 1H),
ethyl acetate and then treated with sol. sat. Na2S2O3. The 6.10 (d, J ¼ 5.3 Hz, 1H), 4.85 (s, 1H), 4.77 (dd, J ¼ 10.8, 5.5 Hz,
organic phase was washed with sol. sat. NaHCO3, brine and 1H), 4.56 (s, 1H), 4.49 (dd, J ¼ 10.8, 1.1 Hz, 1H), 4.43 (t, J ¼
distilled water. Aer drying over anhydrous Na2SO4, the solvent 5.4 Hz, 1H), 3.66 (dd, J ¼ 10.9, 5.7 Hz, 1H), 3.16 (dd, J ¼ 10.9,
was evaporated and the residue was puried by silica gel 5.2 Hz, 1H), 2.42 (m, 1H), 2.28 (m, 3H), 2.06 (m, 1H), 2.00–1.90
chromatography (petroleum ether/ethyl acetate) to afford (m, 1H), 1.67 (m, 1H), 1.62–1.47 (m, 2H), 1.34 (m, 1H), 1.25–1.18
(14b)-(quinolyl-80-oxy)-3-keto-19-acetoxy andrographolide (8b1) (m, 1H), 0.99–0.87 (m, 1H), 0.80 (s, 3H), 0.65 (s, 3H); 13C NMR
or (14b)-(quinolyl-80-oxy)-3-keto-19-(t-butyldimethyl silyloxy) (101 MHz, (CD3)2SO) d 212.64, 169.30, 152.13, 149.76, 149.45,
andrographolide (8b2).
147.42, 140.31, 136.18, 129.50, 126.72, 126.19, 122.12, 121.79,
8b1. White solid; mp 56 ꢀC to 59 ꢀC; 90% yield; 1H NMR (400 113.51, 108.43, 72.64, 71.29, 63.84, 55.38, 54.75, 53.68, 38.55,
MHz, (CD3)2SO) d 8.87 (d, J ¼ 4.2 Hz, 1H), 8.37 (d, J ¼ 8.3 Hz, 37.11, 36.98, 35.21, 25.32, 24.24, 20.10, 14.15; ESI-HRMS: m/z
1H), 7.71–7.47 (m, 3H), 7.25 (d, J ¼ 7.6 Hz, 1H), 7.02 (t, J ¼ 476.2431 [M + H]+, calcd for C29H34NO5, 476.2437.
7.2 Hz, 1H), 6.11 (d, J ¼ 5.3 Hz, 1H), 4.88 (s, 1H), 4.78 (dd, J ¼
4.2.7. Antibacterial evaluation. The tested compound or
10.9, 5.5 Hz, 1H), 4.60 (s, 1H), 4.50 (d, J ¼ 10.9 Hz, 1H), 4.35 (d, J ciprooxacin (positive drug) was dissolved in DMSO and diluted
¼ 11.3 Hz, 1H), 3.78 (d, J ¼ 11.3 Hz, 1H), 2.31 (q, J ¼ 9.3, 7.7 Hz, with LB liquid medium into a stock solution containing 20%
3H), 2.17–2.08 (m, 1H), 2.04–1.91 (m, 2H), 1.87 (s, 3H), 1.75–1.53 DMSO before use; the stock solution of the carrier was 20%
(m, 3H), 1.35 (s, 1H), 1.27–1.11 (m, 3H), 1.08–0.94 (m, 2H), 0.86 DMSO in LB liquid medium. All the experiments, including
(s, 3H); 13C NMR (101 MHz, (CD3)2SO) d 211.7, 170.5, 169.7, blank controls, were performed in triplicate.
152.6, 150.0, 149.9, 147.4, 140.8, 136.6, 129.9, 127.2, 126.7,
Bacteriostatic screening was performed by applying a modi-
122.5, 122.2, 113.9, 109.1, 73.1, 71.7, 65.8, 55.5, 54.9, 51.6, 38.9, ed protocol of the National Committee on Clinical Laboratory
37.2, 37.0, 35.0, 25.7, 24.7, 20.9, 20.7, 14.3; ESI-HRMS: m/z Standards (NCCLS).29 Briey, E. coli, S. aureus and E. faecalis
518.2536 [M + H]+, calcd for C31H36NO6, 518.25426.
cells (American Type Culture Collection, ATCC) were grown in
1
ꢀ
ꢀ
8b2. White solid; mp 125 C to 127 C; 86% yield; H NMR LB plate medium for one passage, then seeded in LB liquid
(400 MHz, (CD3)2SO) d 8.85 (dd, J ¼ 4.1, 1.7 Hz, 1H), 8.36 (dd, J ¼ medium and cultured at 37 ꢀC for 12 h. Aer being diluted 1000
8.4, 1.7 Hz, 1H), 7.64 (d, J ¼ 7.4 Hz, 1H), 7.59–7.50 (m, 2H), 7.24 times, 190 mL diluted cells were aliquoted to 96-well plates;
(d, J ¼ 6.8 Hz, 1H), 7.02 (t, J ¼ 6.8 Hz, 1H), 6.10 (d, J ¼ 5.3 Hz, then, 10 mL stock solution of carrier or a gradient amount of the
1H), 4.86 (s, 1H), 4.77 (dd, J ¼ 10.8, 5.5 Hz, 1H), 4.57 (s, 1H), tested compound or the positive compound, ciprooxacin, were
4.52–4.45 (m, 1H), 3.67 (d, J ¼ 10.1 Hz, 1H), 3.36 (d, J ¼ 10.3 Hz, added; the nal concentration of DMSO was 1% in all wells. The
1H), 2.28 (m, 4H), 2.09 (m, 1H), 1.97 (m, 1H), 1.76–1.68 (m, 1H), blank controls contained 1% DMSO and LB medꢀium but no
1.60 (m, 1H), 1.57–1.49 (m, 1H), 1.40–1.33 (m, 1H), 1.30–1.27 bacterial cells. The cultures were incubated at 37 C for 24 h,
(m, 1H), 0.97–0.90 (m, 1H), 0.79 (s, 3H), 0.75 (s, 9H), 0.63 (s, 3H), and the growth data were expressed as OD630; the IC50 values
ꢁ0.09 (d, J ¼ 1.7 Hz, 6H); 13C NMR (101 MHz, (CD3)2SO) d 212.4, were calculated based on growth data versus test concentration.
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4.2.8. Activity (EC50) against signaling pathways and cyto-
RSC Advances
experiments. All authors have read and approved the nal
manuscript.
toxicity (CC50) of testing compounds in AD-293 cells. Cells from
the AD-293 cell line, a derivative of the HEK293 cell line with
improved adherence and plaque formation properties, were
purchased from Stratagene (La Jolla, CA, USA). The protocols
were adopted and modied from our previous report.25 All
compounds were dissolved in DMSO at 10 mM as a stock
solution. The nal concentration of DMSO was 0.1% in the
culture medium. AD-293 cells bearing luciferase reporters with
promoter regions of IL-6/STAT3 and TNF-a/NF-kB in pGL4.20
vector (Promega) were maintained in DMEM high glucose
medium supplemented with 10% FBS and 1% penicillin
streptomycin in the presence of 1 mg mLꢁ1 puromycin. AD-293
cells overexpressing TLR4 and stably transfected with NF-kB
reporter were maintained in DMEM high glucose medium in
the presence of 1 mg mLꢁ1 puromycin and 10 mg mLꢁ1 blasti-
cidin. All cells were maintained in a humidied incubator at
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was supported in part by the Natural Science Foun-
dation of China (30973621) and Six Major Talents of Jiangsu
Province of China (2014). This work was partially supported by
the Macao Science and Technology Development Fund 041/
2014/A1, Research Fund of the University of Macao MYRG
2016-00105-ICMS-QRCM, and MYRG 2017-00116-ICMS-QRCM
to YW, and MYRG2015-00098-ICMS-QRCM to YTW.
ꢀ
37 C in 95% air and 5% CO2.
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tested compounds, the positive drug, DCB-3503 36 of a crypto-
pleurine analog, or the DMSO vehicle as a control were
dispensed appropriately into the partitioned 96-well plates,
which were then incubated for another 24 h. Then, the medium
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ꢀ
was discarded and the cells were incubated for 4 h at 37 C in
MTT solution (nal concentration 0.5 mg mLꢁ1). The solution
was then replaced with 100 mL DMSO to dissolve the violet
formazan crystals in the intact cells. Cell growth was assessed by
MTT according to the manufacturer's protocol. The absorbance
was measured at 570 nm as the reference wavelength. The
cytotoxicity as the CC50 value (concentration of 50% cell growth/
viability inhibition) was calculated based on the percentage of
cell viability data compared to the control group. Each
concentration was repeated 3 times independently.
Signaling pathway reporter assay. Reporter cells were treated
with 50 ng mLꢁ1 TNF-a to stimulate the NF-kB signaling
pathway, 1 mg mLꢁ1 LPS to stimulate the TLR4/NF-kB signaling
pathway, and 2.5 mg mLꢁ1 IL-6 to stimulate the IL-6/STAT3
signaling pathway. The medium was removed at the end of
the treatment, cell extracts were prepared, and the luciferase
activity was measured using a Luciferase assay kit (Promega)
according to the manufacturer's instructions. EC50 was
dened as the concentration of drug that inhibited stimulator-
triggered luciferase reporter activation by 50% aer continuous
drug exposure for 4 (TNF-a/NF-kB) or 16 (IL-6/STAT3 and TLR4/
NF-kB) hours. Each concentration was repeated 3 times
independently.
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Author contributions
GCZ, DW and YW conceived and supervised the project,
analyzed the data and wrote the paper. GCZ, FL, DS, XN, ZL and 10 (a) C. S. Pedamallu, A. S. Bhatt, S. Bullman, S. Fowler,
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