One-pot three steps cascade synthesis of novel isoandrographolide analogues and their cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2017.07.035

An efficient one-pot synthesis of novel andrographolide analogues is reported from a naturally occurring and abundant andrographolide isolated from aerial parts of Andrographis paniculata. Reactions in the one-pot proceed through a cascade epoxide ring op

Full text of DOI:10.1016/j.ejmech.2017.07.035

Accepted Manuscript
One-pot three steps cascade synthesis of novel isoandrographolide analogues and
their cytotoxic activity
Teerapich Kasemsuk, Pawinee Piyachaturawat, Rada Bunthawong, Uthaiwan Sirion,
Kanoknetr Suksen, Apichart Suksamrarn, Rungnapha Saeeng
PII:
S0223-5234(17)30557-3
10.1016/j.ejmech.2017.07.035
EJMECH 9598
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 April 2017
Revised Date: 18 July 2017
Accepted Date: 20 July 2017
Please cite this article as: T. Kasemsuk, P. Piyachaturawat, R. Bunthawong, U. Sirion, K. Suksen,
A. Suksamrarn, R. Saeeng, One-pot three steps cascade synthesis of novel isoandrographolide
analogues and their cytotoxic activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.07.035.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
O
O
O
HO
O
AcO
O
O
R'
H2N
O
MeOH
O
R'
N
H
HO
HO
AcO
RO
AcO
RO
4a-e
5a-9e
Andrographolide

ACCEPTED MANUSCRIPT
One-pot three steps cascade synthesis of novel isoandrographolide
analogues and their cytotoxic activity
Teerapich Kasemsukb, Pawinee Piyachaturawatc, Rada Bunthawonga, Uthaiwan Siriona, Kanoknetr
Suksenc, Apichart Suksamrarnd and Rungnapha Saeenga*
Affiliation
a Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Burapha
University, Chonburi 20131, Thailand
b Department of Chemistry, Faculty of Science and Technology, Rambhai Barni Rajabhat
University, Chanthaburi 22000, Thailand
c Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
d Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240,
Thailand
Correspondence
Rungnapha Saeeng, Department of Chemistry and Center for Innovation in Chemistry, Faculty of
Science, Burapha University, Chonburi 20131, Thailand
E-mail: rungnaph@buu.ac.th Phone: +6681 6567524; fax: +6638 393494.
1

ACCEPTED MANUSCRIPT
Abstract
An efficient one-pot synthesis of novel andrographolide analogues is reported from a
naturally occurring and abundant andrographolide isolated from aerial parts of Andrographis
paniculata. Reactions in the one-pot proceed through a cascade epoxide ring opening by aniline
derivatives/intramolecular ring closing and oxa-conjugate addition-elimination reactions. This
methodology produces a new series of 17-amino-8-epi-isoandrographolide analogues in fair to
excellent yields with high stereoselectivity using an economic and environmental procedure without
base or catalyst at room temperature. Twenty-five analogues were obtained and cytotoxicity of all
new analogues were evaluated against six cancer cell lines to search for a new lead compound based
on andrographolide structure.
O
O
AcO
O
O
R'
H2N
O
MeOH
O
R'
N
H
AcO
RO
AcO
RO
4a-e
5a-9e
Keywords; epiandrographolide; cytotoxic activity; Andrographis paniculata
2

ACCEPTED MANUSCRIPT
1. Introduction
Natural products from medicinal herbs have long been the important sources of therapeutic
agents in drug discovery process. With diversity in their structures, the lead compound with
promising intrinsic therapeutic property has been employed as the template for chemical
transformation which is one of the common approaches to improve their therapeutic properties for
discovery of new drug in the pharmaceutical industry. Recently, more attention has been focused on
improving the biological activity by chemical modification of the labdane diterpenoid
andrographolide, a natural product occurring in the aerial parts of Andrographis paniculata Nees [1-
5]. Andrographolide is of interest because of its potential as a bioactive pharmacophore, capable of
energizing a wide range of biological activities including antibacterial, antihepatotoxic, anti-HIV,
anticancer, hypoglycemic and hypotensive activities [6-11]. Isolation and purification of
andrographolide from A. paniculata may be accomplished by simple chromatographic techniques
with 2% over all yields [12-13].
Modification of natural andrographolide into a library of new complex analogues with the
appropriate structural diversity to improve their bioactivity continues to represent a challenge in drug
discovery and drug development. Recently, new andrographolide analogues have been designed,
synthesized and evaluated for their biological activity [14-17]. Structural scaffold modification of
natural andrographolide has led to improved and diverse biological activities, especially with regard
to cytotoxic as well as anticancer activity [18-21].
The present study designed and synthesized andrographolide-based derivatives from the
perspective of their effectiveness as cytotoxic agents, specifically as anti-cancer agents. In an earlier
study we discovered some semi-synthetic compounds derived from the andrographolide framework
demonstrated higher bioactivity than the original natural product [22-24]. Andrographolide
modification at C-19 of andrographolide has indicated actively and the cytotoxic potential towards
cancer [25]. However, the modification of the core structure of ent-labdane diterpenoid has been
3

ACCEPTED MANUSCRIPT
rarely reported. Therefore, a series of epi-isoandrographolide were designed and synthesized by one-
pot procedure as novel structural type for searching new potential cytotoxic agents. This one pot
method is a suitable procedure for further drug development and an effective approach for scale-up,
minimize purification steps and reduce chemical waste are utilized.
In the present study, we explored the modification of andrographolide via tandem three steps
epoxide ring opening by aniline derivatives followed by intramolecular ring closing and oxa-
conjugate addition-elimination reactions leading to a series of 17-amino-8-epi-isoandrographolide
analogues.
2. Results and discussion
Chemistry
O
O
O
HO
O
AcO
O
AcO
O
a or b
c
d
O
HO
RO
AcO
RO
AcO
RO
2a (69%); R=Tr
2b (86%); R=TBS
3a (85%); R=Tr
4a (94%); R=Tr
3b
4b
(86%); R=TBS
(73%); R=TBS
O
O
O
O
AcO
O
HO
O
HO
O
HO
O
d
a
c
O
O
O
AcO
RO
HO
HO
HO
RO
HO
HO
4c (69%); R=TBDPS
4d
3c (81%); R=TBDPS
1
2c
(88% isomeric)
(69%); R=TIPS
3d
(72%); R=TIPS
O
O
AcO
O
AcO
O
d
c
O
AcO
AcO
AcO
AcO
4e
(68%)
3e
(84%)
Scheme 1. Reagents and Conditions; a) R3SiCl, pyridine, rt, b) TrCl, pyridine, reflux, for
2a; c) Ac2O, 145oC; d) m-CPBA,CH2Cl2/ MeOH
4

ACCEPTED MANUSCRIPT
C-8-epoxy-andrographolide analogues 4a-4e were synthesized from natural available
andrographolide as starting material for the one-pot synthesis of epi-isoandrographolide analogues
(Scheme 1). First, introducing of trityl and TBS groups at C-19 were carried out in the presence of
triphenylmethylchloride, tert-butyldimethylsilyl-chloride and pyridine followed by acetylation at
70oC and epoxidation with MCPBA to afford the desired compounds 4a and 4b in high yields.
Preparation of C-19 Silyl-precursors 4c and 4d, commenced with epoxidation followed by silylation
with TBDPSCl and TIPSCl in the presence of pyridine and acetylation to produce the designed
products in good yields. C-19 Silyl-precursors 4c and 4d can be prepared as per reaction sequence
4a and 4b. However, we found that in the preparation of C-19 TBDPS and TIPS-analogues,
epoxidation before silyl protection gave higher yield of designed compounds than initial C-19
protection.
5

ACCEPTED MANUSCRIPT
Table 1 Synthesis of 17-amino-8-epi-isoandrographolide analogues 5-9
O
O
AcO
O
O
R'
H2N
O
O
R'
MeOH
R= Tr, 5a-9a
5b 9b
R= TBS,
-
N
H
R= TBDPS, 5c-9c
R= TIPS, 5d-9d
AcO
RO
AcO
RO
5e 9e
-
R= Ac,
4a-4e
O
O
O
O
O
O
O
O
O
N
H
N
H
F
N
AcO
RO
AcO
RO
R= Tr,
R= TBS, 5b, 24 h, 86%
R= TBDPS, 5c, 4.5 h, 69%
5d,
AcO
RO
H
F
6a
7a
5a,
R= Tr, , 48 h, 53%
R= Tr, , 76 h, 61%
24 h, 51%
R= TBS, 6b, 48 h, 77%
R= TBS, 7b, 72 h, 79%
R= TBDPS,7c, 24 h, 80%
R= TBDPS, 6c, 48 h, 82%
6d,
7d,
48 h, 82%
R= TIPS,
72 h, 65%
R= Ac, 6e, 43 h, 87%
R= TIPS,
R= TIPS,
3 h, 65%
R= Ac, 5e, 1.5 h, 61%
R= Ac, 7e, 27 h, 82%
O
O
O
O
Br
OMe
OMe
O
O
N
H
N
AcO
RO
AcO
RO
H
8a
9a
R= Tr, , 127 h, 51%
R= Tr, , 1.5 h, 50%
R= TBS, 9b, 1 h, 67%
R= TBDPS, 9c, 2 h, 79%
R= TBS, 8b, 96 h, 77%
R= TBDPS, 8c, 24 h, 69%
R= TIPS,
R= Ac, 8e, 24 h, 63%
8d,
9d,
2 h, 73%
24 h, 66%
R= TIPS,
R= Ac, 9e, 2 h, 82%
A series of 17-amino-8-epi-isoandrographolide analogues were synthesized via tandem three
steps N-alkylation-epoxide ring opening reaction followed by intramolecular ring closing and oxa-
conjugate addition-elimination reactions. The reaction was first conducted with C-19-trityl
epoxyandrographolide analogue 4a and aniline in methanol without any base or catalyst to give the
cyclized product 5a in fair yield. In a previous study we found the introduction of halogenated and
dimethoxy-aniline to the andrographolide framework led to the enhancing of cytotoxic activity.6a
Therefore, anilines bearing halogenated groups (ortho-, meta-fluoro and para-bromo) and 3,4-
dimethoxy groups were investigated under the present condition to give the corresponding cyclized
product 17-amino-8-epi-isoandrographolide 6a-9a in moderate to good yields as shown in Table 1.
When 2-fluoro-, 3-fluro- and 4-bromo-aniline were employed as nucleophiles, the reactions
were performed more than one days to complete the reactions due to the decreased nucleophilicity of
aniline bearing electron withdrawing. Reactions of 3,4-dimethoxyaniline gave the corresponding
products 9a-9e in short reaction time. The preparation of new analogues by one pot reaction
6

ACCEPTED MANUSCRIPT
proceeded smoothly, selectively and efficiently at room temperature to give twenty-five analogues in
moderate to good yields proving the generality of this method. To our knowledge, this is the first
report of one pot cascade reactions of this sequential epoxide ring opening by aniline
derivatives/intramolecular ring closing and oxa-conjugate addition-elimination reactions. The cis-
stereochemistry at C-8 and C-12 of 5e was determined by NOESY correlations which were
supported by the observation between H-17/H-20, H-17/H-19, H-20/ ortho-Ar-H and H-12/ortho-Ar-
H as shown in Figure 1.
O
H
H
O
H
H
AcO
H3C
19
AcO
12
H
8
O
H
20
H
CH3
H
17
H
H
N
H
H
Figure 1. NOESY correlations of compound 5e.
7

ACCEPTED MANUSCRIPT
Biological activity
Table 2 Cytotoxic activities of 17-amino-8-epi-isoandrographolide analogues against cancer
cell lines.
IC50 (µM) a
Compounds
P-388
KB
HT 29
9.34
MCF-7
15.40
A 549
ASK
Andrographolide (1) 2.25
27.37
27.9
16.18
> 50
> 50
> 50
> 50
> 50
29.06±5.59
5.94±0.05
9.32±0.15
6.10±0.05
> 50
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
5.19±0.06
21.25±0.29
8.50±0.15
> 50
5.44±0.14
> 50
7.80±0.21
> 50
45.34±1.30 > 50
6.54±0.04
18.77±0.24
17.47±1.53 8.33±0.66
25.72±0.72 > 50
9.43±0.26
> 50
39.96±0.54 > 50
> 50
> 50
> 50
> 50
> 50
> 50
4.56±0.08
> 50
6.49±0.11
> 50
5.59±0.04
> 50
4.45±0.07
> 50
6.10±0.04
> 50
3.33±0.08
39.73±0.82
8.05±0.20
27.76±0.38
> 50
21.42±0.55 30.78±0.63
30.25±0.47 15.31±0.49
21.02±0.23
8.45±1.65
> 50
7.50±0.07
> 50
26.53±0.33
> 50
6.50±0.06
> 50
11.81±0.20
> 50
6.44±0.14
> 50
27.44±0.18
> 50
26.23±0.70 8.64±0.08
> 50 > 50
30.12±0.84 12.67±0.40
21.52±1.81
> 50
6.10±0.09
44.84±0.09
6.91±0.03
22.33±1.24
> 50
7.95±0.13
6.85±0.09
> 50
32.86±0.51
20.75±0.09
> 50
24.48±0.39
6.52±0.24
> 50
5.87±0.07
> 50
7.41±0.21
> 50
6.39±0.05
25.71±0.31
27.68±0.99 12.91±1.88
14.98±0.19
47.77±0.99
> 50
7.35±0.02
> 50
48.29±2.05 > 50
40.54±1.77 > 50
> 50
> 50
> 50
> 50
29.73±0.65
8.40±0.24
6.51±0.06
5.13±0.04
9.37±0.32
4.61±0.15
> 50
7.47±0.77
21.08±0.44
6.09±0.15
> 50
6.93±0.12
23.14±1.00
5.40±0.27
35.80±1.72
4.70±0.11
6.14±0.04
> 50
30.48±0.59 > 50
5.49±0.08
11.53±0.62
6.16±0.23
> 50
5.30±0.01
48.61±0.36
6.08±0.05
> 50
27.52±0.71 > 50
4.98±0.21
17.92±0.65
5.24±0.03
28.82±2.10 > 50
24.12±0.47 > 50
aCell lines used are P-388 (murine leukemia cell line); KB (human epidermoid carcinoma of the mouth); COL-
2 (human colon cancer); MCF-7 (human breast cancer); LU-1 (human lung cancer); and ASK (rat glioma).
Ellipticine (Ellipt) was used as a positive control. The results were expressed as IC50 values
8

ACCEPTED MANUSCRIPT
The cytotoxic activities of andrographolide, the parent compound 1 and a new series
of 17-amino-8-epi-isoandrographolide analogues were evaluated in vitro against seven
cancer cell lines; P-388 (mouse lymphoid neoplasma), KB (human epidermoid carcinoma in
the mouth), Col-2 (human colon cancer), MCF-7 (human breast cancer), Lu-1 (human lung
cancer) and ASK (rat glioma) using sulforhodamine B (SRB) assay. All tested analogues
were dissolved in DMSO (0.1%). Ellipticine, a potent anti-cancer agent was used as a
positive control. As shown in Table 2, results were expressed as IC50 values (drug
concentration causing 50% growth inhibition) in µM.
As shown in Table 2, an introduction of aniline to C-19 substituted analogues and
conversion of andrographolide scaffold to epi-isoandrographolide core structure provided
some analogues with greater cytotoxicity than that of the parent andrographolide.
Comparison of the cytotoxic activities of aniline analogues 5a-5e, compounds 5b and 5d
showed increasing cytotoxicity against almost all cancer cells than the natural
andrographolide. Interestingly, compound 5c showed selective cytotoxic activity to ASK
cancer cell. Among C-17-o-fluoroaniline analogues 6a-6e, compound 6b showed the
highest activities relative to other analogues on almost all cancer cell lines especially on
ASK cancer cells with an IC50 value 3.33 µM. 6b was 2 to 5-fold more active on various
cancer cells than 1. Changing the o-fluoro aniline to m-fluoro aniline derivatives, compound
7a-7e showed a trend in cytotoxic activity that was similar to that for 6a-6e. The effects of
substituent position of fluoro group on aniline ring did not alter activity. Compound 7e
showed the greatest activity in this group. Compound C-19-acetyl 8e bearing p-
bromoaniline at C-17 exhibited greater activities than other C-19 substituted analogues in
this group. Introduction of a more electron-donating group such as dimethoxyaniline to C-
17 position on the new scaffold led to a dramatic increase in cytotoxic activity of C-19 TBS
9b and C-19 TIPS 9d. Moreover, compound 9b showed the greatest activity on A 549
while compound 9d on HT 29 cancer cell and compound 9a showed selective cytotoxic
activity to ASK cancer cells.
3. Conclusions
In summary, a convenient and environmentally one pot, three steps cascade reactions
has been developed for modification of andrographolide scaffold to provide new 17-amino-
8-epi-isoandrographolide analogues to screen cytotoxic activity. The reaction proceed via
epoxide ring opening by aniline derivatives/intramolecular ring closing and oxa-conjugate
9

ACCEPTED MANUSCRIPT
addition-elimination reactions without any base or catalyst at room temperature. Twenty
five analogues were screened to study the SAR. Among the synthetic epi-
isoandrographolides, 6b exhibited the strongest activities relative to other analogues
especially on ASK cancer cells with an IC50 3.33 µM, while 5c and 9a showed selective
cytotoxic activity to ASK cancer cells.
4. Experimental section
General experimental
All chemicals were purchased from commercial sources and used without further
purification. Proton NMR spectra were recorded on a BRUKER AVANC (400 MHz). All
spectra were measured in CDCl3 solvent and chemical shifts are reported as δ values in parts
per million (ppm) relative to tetramethylsilane (δ 0.00) or CDCl3 (δ 7.26) as internal
standard. Data are reported as follows; chemical shift (multiplicity, integrate intensity or
assignment, coupling constants in Hz, assignment). Carbon NMR spectra were recorded on
a BRUKER AVANC (100 MHz). All spectra were measured in CDCl3 solvent and
chemical shifts are reported as δ values in parts per million (ppm) relative to CDCl3 (δ 77.0)
as internal standard. High-resolution mass spectra (HRMS) data were obtained with a
Finnigan MAT 95. Infrared spectra were determined on a PERKIN ELMER FT/IR-2000S
spectrophotometer and are reported in wave number (cm-1). Analytical thin-layer
chromatrography (tlc) was conducted on precoated tlc plates; silica gel 60F-254 [E. Merck,
Darmstadt, Germany]. Silica gel columns for open-column chromatrography utilized silica
gel 60 PF254 [E. Merck, Darmstadt, Germany]. Melting points were measured using a
Melting point apparatus (Griffin) and are uncorrected.
19-Tr-3,14-diacetyl-8,17-epoxy andrographolide (4a)
To a stirred solution of andrographolide (1) (2.05 mg, 4.91 mmol) in pyridine (2.5
mL) was added triphenylmethyl chloride (TrCl) (2.28 g, 8.18 mmol), then the reaction
mixture was heated to 70 oC. After the stirring was continued at 70 oC for 2.5 h, the reaction
mixture was quenched with cold NH4Cl and extracted with EtOAc (3×50 mL). The
combined organic layer was washed with brine (50 mL), dried over sodium sulfate
anhydrous, and then concentrated under reduced pressure. The residue was purified by
10

ACCEPTED MANUSCRIPT
column chromatography (50% EtOAc/n-hexane) to give 19-Tr-andrographolide (2a) in 69%
yield (1.47 g) as a white solid, Rf 0.45 (50% EtOAc/n-hexane).
To a stirred solution of 19-Tr-andrographolide (2a) (1.00 g, 1.69 mmol) in acetic
anhydride (10.0 mL) was refluxed to 145 oC. After the stirring was continued at 145 oC for
2.5 h, the reaction mixture was diluted with EtOAc (50 mL) and quenched with saturated
NaHCO3, washed with H2O, and extracted with EtOAc (3×50 mL). The combined organic
layer was washed with brine (50 mL), dried over sodium sulfate anhydrous, and then
concentrated under reduced pressure. The residue was purified by column chromatography
(30% EtOAc/n-hexane) to give 19-Tr-3,14-diacetyl-andrographolide (3a) in 73% yield (833
mg) as a white solid, Rf 0.54 (30% EtOAc/n-hexane).
To a stirred solution of 19-Tr-3,14-diacetyl andrographolide (3a) (501 mg, 0.74
mmol) in solution mixture of CH2Cl2/methanol (5:1) (20.0 mL) was added m-
chloroperoxybenzoic acid (m-CPBA) (307 mg, 1.78 mmol). After the stirring was continued
at room temperature for 24 h, the solvent was removed by evaporator. The reaction mixture
was diluted with EtOAc (50 mL) and quenched with saturated NaHCO3, washed with H2O,
and extracted with EtOAc (3×50 mL). The combined organic layer was washed with brine
(50 mL), dried over sodium sulfate anhydrous, and then concentrated under reduced
pressure. The residue was purified by column chromatography (30% EtOAc/n-hexane) to
give 19-Tr-3,14-diacetyl-8,17-epoxy andrographolide (4a) in 94% yield (481 mg) as a white
o
solid, Rf 0.65 (40% EtOAc/n-hexane). (4a): Mp. 83-85 C; IR (Neat): 2946, 1763, 1734,
1678, 1370, 1240, 1028, 750 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.52-7.47 (6H, m, PhH),
7.35-7.24 (6H, m, PhH), 7.23-7.22 (3H, m, PhH), 7.06 (1H, td, J = 7.0, 1.0 Hz, H-12), 5.82
(1H, brd, J = 6.0 Hz, H-14), 4.54 (1H, dd, J = 11.5, 4.0 Hz, H-3), 4.50 (1H, dd, J = 11.0, 6.0
Hz, H-15b), 4.20 (1H, dd, J = 11.0, 1.5 Hz, H-15a), 3.39 (1H, d, J = 10.0 Hz, H-19b), 3.16
(1H, d, J = 10.0 Hz, H-19a), 2.52 (1H, dd, J = 4.0, 2.0 Hz, H-17b), 2.47 (1H, d, J = 4.0 Hz,
H-17a), 2.12 (3H, s, COCH3), 2.03 (3H, s, COCH3), 2.01-1.77 (4H, m), 1.61-1.10 (8H, m),
1.23 (3H, s, H-18), 0.38 (3H, s, H-20); 13C NMR (100 MHz, CDCl3): δ 170.60 (2×C),
169.00, 150.96, 144.11 (3×C), 128.87 (6×C), 127.67 (6×C), 126.93 (3×C), 123.21, 86.95,
79.87, 71.59, 67.57, 62.22, 58.06, 55.09, 54.11, 49.84, 42.35, 39.46, 37.43, 35.97, 23.56,
23.00, 22.94, 22.45, 21.26, 20.87, 14.25; HRMS (ESI) m/z calcd for C43H48O8Na [M+Na]+
715.3247, found 715.3275.
11

ACCEPTED MANUSCRIPT
19-TBS-3,14-diacetyl-8,17-epoxy andrographolide (4b)
To a stirred solution of andrographolide (1) (504 mg, 1.44 mmol) in pyridine (5.0
mL) was added tert-butyldimethylsilyl chloride (TBDMSCl) (1.07 g, 7.10 mmol) at room
temperature. After the stirring was continued at room temperature for 1 h, the reaction
mixture was diluted with EtOAc (50 mL) and quenched with H2O (50 mL), and extracted
with EtOAc (3×50 mL). The combined organic layer was washed with brine (50 mL), dried
over sodium sulfate anhydrous, and then concentrated under reduced pressure. The residue
was purified by column chromatography (50% EtOAc/n-hexane) to give 19-TBS-
andrographolide (2b) in 88% yield (591 mg) as a white solid, Rf 0.42 (50% EtOAc/n-
hexane).
To a stirred solution of 19-TBS-andrographolide (2b) (591 mg, 1.27 mmol) in acetic
anhydride (10.0 mL) was refluxed to 145 oC. After the stirring was continued at 145 oC for 2
h, the reaction mixture was diluted with EtOAc (50 mL) and quenched with saturated
NaHCO3, washed with H2O, and extracted with EtOAc (3×50 mL). The combined organic
layer was washed with brine (50 mL), dried over sodium sulfate anhydrous, and then
concentrated under reduced pressure. The residue was purified by column chromatography
(40% EtOAc/n-hexane) to give 19-TBS-3,14-diacetyl andrographolide (3b) in 86% yield
(598 mg) as a white solid, Rf 0.61 (30% EtOAc/n-hexane).
To a stirred solution of 19-TBS-3,14-diacetyl andrographolide (3b) (598 mg, 1.09
mmol) in solution mixture of CH2Cl2/methanol (5:1) (6.0 mL) was added m-
chloroperoxybenzoic acid (m-CPBA) (553 mg, 3.20 mmol). After the stirring was continued
at room temperature for 19 h, the solvent was removed by evaporator. The reaction mixture
was diluted with EtOAc (50 mL) and quenched with saturated NaHCO3, washed with H2O,
and extracted with EtOAc (3×50 mL). The combined organic layer was washed with brine
(50 mL), dried over sodium sulfate anhydrous, and then concentrated under reduced
pressure. The residue was purified by column chromatography (22% EtOAc/n-hexane) to
give 19-TBS-3,14-diacetyl-8,17-epoxy andrographolide (4b) in 73% yield (451 mg) as a
o
white solid, Rf 0.18 (22% EtOAc/n-hexane). (4b): Mp. 114-116 C; IR (Neat): 2933, 2870,
1
1750, 1642, 1449, 1347, 1264, 1206, 1082, 1036, 892, 811 cm-1; H NMR (400 MHz,
CDCl3): δ 7.10 (1H, t, J = 7.0 Hz, H-12), 5.88 (1H, d, J = 5.0 Hz, H-14), 4.59-4.47 (1H, m,
H-3), 4.52 (1H, d, J = 11.0 Hz, H-15b), 4.22 (1H, d, J = 11.0 Hz, H-15a), 3.86 (1H, d, J =
12

ACCEPTED MANUSCRIPT
10.5 Hz, H-19b), 3.58 (1H, d, J = 10.5 Hz, H-19a), 2.62 (1H, brs, H-17b), 2.54 (1H, brs, H-
17a), 2.12 (3H, s, COCH3), 2.05 (3H, s, COCH3), 2.10-1.59 (8H, m), 1.43-1.36 (2H, m),
1.34-1.17 (2H, m), 0.97 (3H, s, H-18), 0.96 (3H, s, H-20), 0.89 (9H, s, SiC(CH3)3), 0.03
13
(6H, s, Si(CH3)2); C NMR (100 MHz, CDCl3): δ 170.68, 170.62, 169.10, 151.17, 123.07,
79.72, 71.61, 67.60, 63.98, 58.37, 55.06, 54.21, 49.90, 42.19, 39.66, 37.81, 36.33, 25.83
(3×C), 23.69, 23.30, 23.11, 22.83, 21.11, 20.88, 18.20, 14.51, -5.65, -5.74; HRMS (ESI) m/z
calcd for C30H48O8SiNa [M+Na]+ 587.3016, found 587.3047.
19-TBDPS-3,14-diacetyl-8,17-epoxy andrographolide (4c)
A mixture of 8,17-epoxy andrographolide (2c) (500 mg, 1.36 mmol) with tert-
butyldiphenylsilyl chloride (TBDPSCl) (1.12 mL, 4.31 mmol) was stirred at room
temperature for 1 h. The reaction mixture was quenched with NH4Cl, and extracted with
EtOAc. The combined organic layer was washed with brine, dried over sodium sulfate
anhydrous, and then concentrated under reduced pressure. The residue was purified by
column chromatography (60% EtOAc/n-hexane) to afford the corresponding product 3c in
81% yield (668 mg) as a white solid, Rf 0.36 (50% EtOAc/n-hexane).
To a stirred solution of 19-TBDPS-8,17-epoxy andrographolide (3c) (350 mg, 0.58
o
mmol) in acetic anhydride (2.0 mL) was heated to 145 C for 1h, the reaction mixture was
diluted with EtOAc (30 mL) and quenched with saturated NaHCO3, washed with H2O, and
extracted with EtOAc (3×30 mL). The combined organic layer was washed with brine (30
mL), dried over sodium sulfate anhydrous, and then concentrated under reduced pressure.
The residue was purified by column chromatography (30% EtOAc/n-hexane) to afford the
corresponding product 4c in 69% yield (275 mg) as a white solid, Rf 0.49 (30% EtOAc/n-
o
1
hexane). (4c): Mp. 92-94 C; IR (Neat): 2939, 1734, 1241, 1079, 749 cm-1; H NMR (400
MHz, CDCl3): δ 7.71-7.63 (4H, m, PhH), 7.47-7.34 (6H, m, PhH), 7.08 (1H, td, J = 7.0, 1.0
Hz, H-12), 5.86 (1H, brd, J = 5.5 Hz, H-14), 4.53 (1H, dd, J = 11.0, 4.0 Hz, H-3), 4.50 (1H,
dd, J = 11.5, 5.5 Hz, H-15b), 4.22 (1H, dd, J = 11.5, 1.5 Hz, H-15a), 3.84 (1H, d, J = 11.0
Hz, H-19b), 3.37 (1H, d, J = 11.0 Hz, H-19a), 2.60 (1H, d, J = 4.0 Hz, H-17b), 2.53 (1H, d,
J = 4.0 Hz, H-17a), 2.11 (3H, s, COCH3), 2.15-1.92 (5H, m), 1.90 (3H, s, COCH3), 1.84-
1.39 (5H, m), 1.31-1.17 (2H, m), 1.06 (9H, s, SiC(CH3)3), 1.04 (3H, s, H-18), 0.80 (3H, s,
H-20); 13C NMR (100 MHz, CDCl3): δ 170.61, 170.51, 169.01, 150.92, 135.82 (2×C),
135.74 (2×C), 133.44, 133.39, 129.67, 129.64, 127.62 (2×C), 127.59 (2×C), 123.19, 79.63,
13

ACCEPTED MANUSCRIPT
71.57, 67.57, 63.83, 58.20, 54.99, 54.13, 49.55, 42.82, 39.98, 37.57, 36.18, 26.92 (3×C),
23.58, 23.01, 22.81 (2×C), 21.06, 20.86, 19.25, 14.66; HRMS (ESI) m/z calcd for
C40H52O8SiNa [M+Na]+ 711.3329, found 711.3322.
19-TIPS-3,14-diacetyl-8,17-epoxy andrographolide (4d)
To a stirred solution of andrographolide (1) (1.50 g, 4.28 mmol) in solution mixture
of CH2Cl2/methanol (5:1) (30 mL) was added m-chloroperoxybenzoic acid (m-CPBA) (1.11
g, 6.43 mmol). After the stirring was continued at room temperature for 24 h, solvent was
removed by evaporator. The reaction mixture was diluted with EtOAc (50 mL) and
quenched with saturated NaHCO3, washed with H2O, and extracted with EtOAc (3×50 mL).
The combined organic layer was washed with brine (10 mL), dried over sodium sulfate
anhydrous, and then concentrated under reduced pressure. The residue was purified by
column chromatography (100% EtOAc) to give mixture of 8,17-epoxy andrographolide
isomers (2c) in 88% yield (1.38 g) as a white solid (major : minor ~ 18:1), Rf (major) 0.42
(100% EtOAc) and Rf (minor) 0.33 (100% EtOAc).
To a stirred solution of mixture of 8,17-epoxy andrographolide (2c) (500 mg, 1.36
mmol) in pyridine 1.0 mL was added triisopropyl chloride (TIPSCl) (0.90 mL, 4.20 mmol) at
room temperature. After the stirring was continued at room temperature for 5 h, the reaction
mixture was quenched with NH4Cl, and extracted with EtOAc. The combined organic layer
was washed with brine, dried over sodium sulfate anhydrous, and then concentrated under
reduced pressure. The residue was purified by column chromatography (60% EtOAc/n-
hexane) to afford the corresponding product 3d in 72% yield (514 mg) as a white solid, Rf
0.55 (45% EtOAc/n-hexane).
To a stirred solution of 19-TIPS-8,17-epoxy andrographolide (3d) (350 mg, 0.67
o
mmol) in acetic anhydride (2.0 mL) was heated to 145 C for 5.0 h. The reaction mixture
was diluted with EtOAc (30 mL) and quenched with saturated NaHCO3, washed with H2O,
and extracted with EtOAc (3×30 mL). The combined organic layer was washed with brine
(30 mL), dried over sodium sulfate anhydrous, and then concentrated under reduced
pressure. The residue was purified by column chromatography (40% EtOAc/n-hexane) to
afford the corresponding product 4d in 69% yield (280 mg) as a white solid, Rf 0.39 (20%
o
EtOAc/n-hexane). (4d): Mp. 104-106 C; IR (Neat): 2943, 1764, 1735, 1677, 1370, 1242,
1
1027, 883 cm-1; H NMR (400 MHz, CDCl3): δ 7.10 (1H, td, J = 7.0, 1.0 Hz, H-12), 5.88
14

ACCEPTED MANUSCRIPT
(1H, d, J = 5.5 Hz, H-14), 4.60-4.54 (1H, m, H-3), 4.51 (1H, dd, J = 11.0, 5.5 Hz, H-15b),
4.22 (1H, dd, J = 11.0, 2.0 Hz, H-15a), 3.95 (1H, d, J = 10.0 Hz, H-19b), 3.80 (1H, d, J =
10.0 Hz, H-19a), 2.63 (1H, d, J = 4.0 Hz, H-17b), 2.55 (1H, d, J = 4.0 Hz, H-17a), 2.12 (3H,
s, COCH3), 2.04 (3H, s, COCH3), 2.09-1.57 (9H, m), 1.45-1.05 (6H, m), 1.07 (18H, d, J =
6.0 Hz, Si(CH(CH3)2)3), 1.03 (3H, s, H-18), 0.96 (3H, s, H-20); 13C NMR (100 MHz,
CDCl3): δ 170.63, 170.56, 169.02, 151.03, 123.19, 79.74, 71.59, 67.59, 63.89, 58.28, 55.08,
54.20, 49.91, 42.78, 39.69, 37.74, 36.26, 23.67, 23.07, 22.98, 22.78, 21.18, 20.88, 18.09
(6×C), 14.86, 11.93 (3×C); HRMS (ESI) m/z calcd for C33H54O8SiNa [M+Na]+ 629.3486,
found 629.3446.
3,14,19-triacetyl-8,17-epoxy andrographolide (4e)
To a stirred solution of andrographolide (1) (1.00 g, 2.85 mmol) in acetic anhydride
o
o
(10.0 mL) was heated to 145 C. After the stirring was continued at 145 C for 3.5 h, the
reaction mixture was diluted with EtOAc (50 mL) and quenched with saturated NaHCO3,
washed with H2O, and extracted with EtOAc (3×50 mL). The combined organic layer was
washed with brine (50 mL), dried over sodium sulfate anhydrous, and then concentrated
under reduced pressure. The residue was purified by column chromatography (50%
EtOAc/n-hexane) to give 3,14,19-triacetyl andrographolide (3e) in 85% (1.15 g) as a white
solid, Rf 0.56 (50% EtOAc/n-hexane).
To a stirred solution of 3,14,19-triacetyl andrographolide (3e) (909 mg, 1.91 mmol)
in solution mixture of CH2Cl2/methanol (5:1) (10.0 mL) was added m-chloroperoxybenzoic
acid (m-CPBA) (660 mg, 3.82 mmol). After the stirring was continued at room temperature
for 24 h, the solvent was removed by evaporator. The reaction mixture was diluted with
EtOAc (50 mL) and quenched with saturated NaHCO3, washed with H2O, and extracted
with EtOAc (3×50 mL). The combined organic layer was washed with brine (50 mL), dried
over sodium sulfate anhydrous, and then concentrated under reduced pressure. The residue
was purified by column chromatography (40% EtOAc/n-hexane) to give 3,14,19-triacetyl-
8,17-epoxy andrographolide (4e) in 68% yield (641 mg) as a white solid, Rf 0.36 (40%
o
EtOAc/n-hexane). (4e): Mp. 170-172 C; IR (Neat): 2951, 1734, 1677, 1372, 1237, 1028,
749 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.08 (1H, td, J = 7.0, 1.5 Hz, H-12), 5.87 (1H, brd,
J = 5.0 Hz, H-14), 4.58 (1H, dd, J = 11.0, 5.0 Hz, H-3), 4.51 (1H, dd, J = 11.0, 6.0 Hz, H-
15b), 4.35 (1H, d, J = 11.5 Hz, H-19b), 4.23 (1H, dd, J = 11.0, 2.0 Hz, H-15a), 4.16 (1H, d,
15

ACCEPTED MANUSCRIPT
J = 11.5 Hz, H-19a), 2.62 (1H, dd, J = 4.0, 1.5 Hz, H-17b), 2.55 (1H, d, J = 4.0 Hz, H-17a),
2.11 (3H, s, COCH3), 2.06 (3H, s, COCH3), 2.04 (3H, s, COCH3), 2.01-1.90 (3H, m), 1.90-
1.79 (1H, m), 1.79-1.60 (3H, m), 1.47-1.41 (1H, m), 1.36-1.22 (4H, m), 1.04 (3H, s, H-18),
0.90 (3H, s, H-20); 13C NMR (100 MHz, CDCl3): δ 170.87, 170.60, 170.38, 169.02, 150.66,
123.35, 79.30, 71.61, 67.57, 64.62, 58.02, 54.78, 54.07, 49.80, 41.13, 39.57, 37.41, 35.95,
23.59, 23.01, 22.67, 22.16, 21.12, 21.08, 20.87, 14.72.
Synthesis of 17-amino-8-epi-12-epi-isoandrographolide analogues 5-9
General Procedure: To a stirred solution of 19-substituted-3,14-diacetyl-8,17-epoxy
andrographolide (4a-4e) (0.04-0.10 mmol) in methanol (1.0 mL) was added amine
compound (0.06-0.15 mmol) at room temperature. After the stirring was continued at room
temperature for 0.5 to 72 h, the reaction mixture was quenched carefully with cooled aq.
NH4Cl (10 mL), extracted with EtOAc (3×10 mL). The combined organic layer was washed
with water (10 mL) and brine (10 mL), dried over anhydrous Na2SO4 and then concentrated
under reduced pressure. The residue was purified by column chromatography (EtOAc/n-
hexane) to afford the corresponding products (5a-9e).
19-Tr-3-acetyl-17-(phenyl)amino-epi-isoandrographolide (5a)
According to the general procedure, 19-Tr-3,14-diacetyl-8,17-epoxy andrographolide (4a)
(51.9 mg, 0.075 mmol) with aniline (9.90 mg, 0.106 mmol) was stirred at room temperature
for 24 h. The residue was purified by column chromatography (30% EtOAc/n-hexane) to
afford the corresponding product 5a in 51% yield (27.7 mg) as a brown solid, Rf 0.31 (15%
o
EtOAc/n-hexane); Mp. 98-100 C; IR (Neat): 3361, 3093, 1794, 1732, 1603, 1402, 1246,
1
1067, 886, 708 cm-1; H NMR (400 MHz, CDCl3): δ 7.39 (6H, d, J = 8.0 Hz, PhH), 7.25-
7.13 (9H, m, PhH), 7.10-7.03 (3H, m, H-14, PhH), 6.60 (1H, t, J = 7.5 Hz, PhH), 6.40 (2H,
d, J = 7.5 Hz, PhH), 4.85 (1H, brs, NH), 4.69 (1H, d, J = 18.0 Hz, H-15b), 4.61 (1H, d, J =
18.0 Hz, H-15a), 4.43 (1H, dm, J = 11.0 Hz, H-3), 3.79 (1H, d, J = 10.0 Hz, H-12), 3.29
(1H, d, J = 10.0 Hz, H-19b), 3.04 (1H, d, J = 10.0 Hz, H-19a), 2.56 (1H, brs, H-17b), 2.46
(1H, brs, H-17a), 1.92 (3H, s, COCH3), 1.90-1.45 (8H, m), 1.10 (3H, s, H-18), 1.40-1.01
(3H, m), 0.60-0.50 (1H, m), 0.22 (3H, s, H-20); 13C NMR (100 MHz, CDCl3): δ 173.10,
170.61, 146.84, 146.16, 144.12 (3×C), 135.68, 129.24 (2×C), 128.83 (6×C), 127.61 (6×C),
126.85 (3×C), 117.18, 112.73 (2×C), 86.82, 80.20, 70.28, 62.13, 58.83, 54.81, 52.42, 51.85,
50.45, 42.30, 39.99, 35.99 (2×C), 26.31, 23.53, 22.88, 22.43, 21.28, 14.00; HRMS (ESI) m/z
calcd for C47H52NO6 [M+H]+ 726.3795, found 726.3789.
16

ACCEPTED MANUSCRIPT
19-Tr-3-acetyl-17-((2-fluoro)phenyl)amino-epi-isoandrographolide (6a)
According to the general procedure, 19-Tr-3,14-diacetyl-8,17-epoxy andrographolide (4a)
(51.3 mg, 0.074 mmol) with 2-fluoroaniline (10.5 mg, 0.094 mmol) was stirred at room
temperature for 48 h. The residue was purified by column chromatography (30% EtOAc/n-
hexane) to afford the corresponding product 6a in 53% yield (29.0 mg) as a yellow solid, Rf
o
0.50 (30% EtOAc/n-hexane); Mp. 112-114 C; IR (Neat): 3367, 3097, 1754, 1621, 1401,
1246, 1192, 1032, 708 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.39 (6H, d, J = 7.5 Hz, PhH),
7.25-7.11 (9H, m, PhH), 7.07 (1H, brs, H-14), 6.89 (1H, t, J = 10.0 Hz, PhH), 6.83 (1H, t, J
= 7.5 Hz, PhH), 6.56-6.48 (1H, m, PhH), 6.25 (1H, t, J = 7.5 Hz, PhH), 5.10 (1H, brs, NH),
4.69 (1H, d, J = 18.0 Hz, H-15b), 4.62 (1H, d, J = 18.0 Hz, H-15a), 4.43 (1H, dm, J = 11.0
Hz, H-3), 3.80 (1H, d, J = 10.0 Hz, H-12), 3.28 (1H, d, J = 10.0 Hz, H-19b), 3.04 (1H, d, J
= 10.0 Hz, H-19a), 2.55 (1H, brs, H-17b), 2.46 (1H, brs, H-17a), 1.92 (3H, s, COCH3), 1.91-
13
1.02 (11H, m), 1.09 (3H, s, H-18), 0.65-0.55 (1H, m), 0.22 (3H, s, H-20); C NMR (100
MHz, CDCl3): δ 172.94, 170.64, 151.39 (d, JC-F = 240 Hz), 146.04, 144.15 (3×C), 135.49,
135.32 (d, JC-F = 12.0 Hz), 128.86 (6×C), 127.62 (6×C), 126.86 (3×C), 124.47, 116.64 (d,
JC-F = 7.0 Hz), 114.59 (d, JC-F = 18.0 Hz), 112.24, 86.88, 80.25, 70.28, 62.17, 58.65, 54.94,
52.17, 51.91, 50.40, 42.36, 39.98, 36.11, 35.90, 26.34, 23.57, 22.88, 22.42, 21.25, 14.07;
HRMS (ESI) m/z calcd for C47H51FNO6 [M+H]+ 744.3700, found 744.3703.
19-Tr-3-acetyl-17-((3-fluoro)phenyl)amino-epi-isoandrographolide (7a)
According to the general procedure, 19-Tr-3,14-diacetyl-8,17-epoxy andrographolide (4a)
(52.4 mg, 0.076 mmol) with 3-fluoroaniline (10.45 mg, 0.094 mmol) was stirred at room
temperature for 76 h. The residue was purified by column chromatography (30% EtOAc/n-
hexane) to afford the corresponding product 7a in 61% yield (34.3 mg) as a white solid, Rf
0.50 (30% EtOAc/n-hexane); Mp. 96-98 oC; IR (Neat): 3339, 3119, 1754, 1621, 1404, 1247,
1
1152, 1032, 708 cm-1; H NMR (400 MHz, CDCl3): δ 7.40 (6H, d, J = 7.5 Hz, PhH), 7.26-
7.12 (9H, m, PhH), 7.08 (1H, brs, H-14), 6.98 (1H, dd, J = 15.0, 8.0 Hz, PhH), 6.28 (1H, t, J
= 8.0 Hz, PhH), 6.17 (1H, d, J = 8.0 Hz, PhH), 6.07 (1H, d, J = 11.5 Hz, PhH), 5.06 (1H,
brs, NH), 4.71 (1H, d, J = 18.0 Hz, H-15b), 4.63 (1H, d, J = 18.0 Hz, H-15a), 4.44 (1H, dm,
J =10.0 Hz, H-3), 3.75 (1H, brd, J = 7.0 Hz, H-12), 3.29 (1H, d, J = 9.5 Hz, H-19b), 3.05
(1H, d, J = 9.5 Hz, H-19a), 2.57 (1H, brs, H-17b), 2.48 (1H, brs, H-17a), 1.93 (3H, s,
COCH3), 1.99-1.03 (11H, m), 1.10 (3H, s, H-18), 0.59-0.48 (1H, m), 0.22 (3H, s, H-20); 13C
NMR (100 MHz, CDCl3): δ 172.90, 170.58, 164.05 (d, JC-F = 240 Hz), 148.72 (d, JC-F
=
17

ACCEPTED MANUSCRIPT
11.0 Hz), 146.13, 144.14 (3×C), 135.30, 130.33 (d, JC-F = 10.0 Hz), 128.85 (6×C), 127.62
(6×C), 126.87 (3×C), 108.57, 103.73 (d, JC-F = 20.0 Hz), 99.60 (d, JC-F = 25.0 Hz), 86.88,
80.15, 70.27, 62.17, 58.87, 54.88, 52.60, 52.00, 50.49, 42.34, 40.02, 36.04 (2×C), 26.26,
23.56, 22.89, 22.44, 21.26, 14.04; HRMS (ESI) m/z calcd for C47H51FNO6 [M+H]+
744.3700, found 744.3699.
19-Tr-3-acetyl-17-((4-Bromo)phenyl)amino-epi-isoandrographolide (8a)
According
to
the
general
procedure,
19-Tr-3,14-diacetyl-8,17-epoxy
andrographolide (4a) (51.0 mg, 0.074 mmol) with 4-bromoaniline (36.1 mg, 0.210 mmol)
was stirred at room temperature for 127 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 8a in 51%
yield (30.2 mg) as a white solid, Rf 0.34 (30% EtOAc/n-hexane); Mp. 98-101 oC; IR (Neat):
1
3372, 2932, 1753, 1595, 1490, 1245, 1070, 708, 632 cm-1; H NMR (400 MHz, CDCl3): δ
7.40 (6H, d, J = 7.5 Hz, PhH), 7.25-7.10 (11H, m, PhH), 7.06 (1H, s, H-14), 6.28 (2H, d, J =
8.0 Hz, PhH), 4.95 (1H, brs, NH), 4.69 (1H, d, J = 18.0 Hz, H-15b), 4.62 (1H, d, J = 18.0
Hz, H-15a), 4.45 (1H, dm, J = 10.0 Hz, H-3), 3.74 (1H, brd, J = 10.0 Hz, H-12), 3.29 (1H,
d, J = 10.0 Hz, H-19b), 3.05 (1H, d, J = 10.0 Hz, H-19a), 2.57 (1H, brs, H-17b), 2.48 (1H,
brs, H-17a), 2.05-1.02 (11H, m), 1.93 (3H, s, COCH3), 1.10 (3H, s, H-18), 0.60-0.48 (1H,
m), 0.22 (3H, s, H-20); 13C NMR (100 MHz, CDCl3): δ 172.88, 170.57, 146.11, 145.37,
144.10 (3×C), 135.24, 131.91 (2×C), 128.81 (6×C), 127.58 (6×C), 126.83 (3×C), 114.36
(2×C), 108.80, 86.85, 80.13, 70.23, 62.13, 58.86, 54.84, 52.57, 51.96, 50.46, 42.30, 39.98,
36.02 (2×C), 26.30, 23.51, 22.84, 22.40, 21.21, 14.00; HRMS (ESI) m/z calcd for
C47H51BrNO6 [M+H]+ 804.2900, found 804.2894.
19-Tr-3-acetyl-17-((3,4-dimethoxy)phenyl)amino-epi-isoandrographolide (9a)
According to the general procedure, 19-Tr-3,14-diacetyl-8,17-epoxy andrographolide (4a)
(50.7 mg, 0.073 mmol) with 3,4-dimethoxyaniline (20.4 mg, 0.133 mmol) was stirred at
room temperature for 1.5 h. The residue was purified by column chromatography (50%
EtOAc/n-hexane) to afford the corresponding product 9a in 50% yield (28.6 mg) as a yellow
o
solid, Rf 0.64 (50% EtOAc/n-hexane); Mp. 85-87 C; IR (Neat): 3367, 2924, 1749, 1614,
1513, 1446, 1238, 1027, 708 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.40 (6H, d, J = 7.5 Hz,
PhH), 7.25-7.12 (9H, m, PhH), 7.10 (1H, s, H-14), 6.61 (1H, d, J = 8.5 Hz, PhH), 6.09 (1H,
s, PhH), 5.84 (1H, d, J = 8.5 Hz, PhH), 4.70 (1H, d, J = 18.0 Hz, H-15b), 4.62 (1H, d, J =
18.0 Hz, H-15a), 4.50-4.40 (1H, m, H-3), 4.09-4.01 (1H, m, NH), 3.85-3.75 (1H, m, H-12),
18

ACCEPTED MANUSCRIPT
3.74 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.29 (1H, d, J = 9.5 Hz, H-19b), 3.04 (1H, d, J =
9.5 Hz, H-19a), 2.57 (1H, brs, H-17b), 2.46 (1H, brs, H-17a), 1.93 (3H, s, COCH3), 2.05-
13
1.00 (11H, m), 1.10 (3H, s, H-18), 0.63-0.53 (1H, m), 0.22 (3H, s, H-20); C NMR (100
MHz, CDCl3): δ 173.11, 170.61, 150.10, 146.16, 144.14 (3×C), 141.80, 141.38, 135.87,
128.85 (6×C), 127.61 (6×C), 126.86 (3×C), 113.30, 103.24, 98.87, 86.87, 80.21, 70.26,
62.17, 58.91, 56.71, 55.82, 54.88, 52.92, 51.70, 50.46, 42.34, 39.97, 36.08 (2×C), 26.49,
23.56, 22.88, 22.44, 21.25, 14.05; HRMS (ESI) m/z calcd for C49H56NO8 [M+H]+ 786.4006,
found 786.4004.
19-TBS-3-acetyl-17-(phenyl)amino-epi-isoandrographolide (5b)
According to the general procedure, 19-TBS-3,14-diacetyl-8,17-epoxy
andrographolide (4b) (23.6 mg, 0.042 mmol) with aniline (5.70 mg, 0.061 mmol) was
stirred at room temperature for 24 h. The residue was purified by column chromatography
(15% EtOAc/n-hexane) to afford the corresponding product 5b in 86% yield (21.6 mg) as a
yellow solid, Rf 0.31 (15% EtOAc/n-hexane); Mp. 124-126 oC; IR (Neat): 3376, 2954, 2857,
1
1749, 1732, 1651, 1603, 1513, 1471, 1248, 1060, 1030, 853, 838, 736 cm-1; H NMR (400
MHz, CDCl3): δ 7.19 (1H, d, J = 1.0 Hz, H-14), 7.14 (2H, t, J = 7.5 Hz, PhH), 6.68 (1H, t, J
= 7.5 Hz, PhH), 6.49 (2H, d, J = 7.5 Hz, PhH), 5.01 (1H, brd, J = 4.5 Hz, NH), 4.78 (1H, dt,
J = 18.0, 1.0 Hz, H-15b), 4.71 (1H, brd, J = 18.0 Hz, H-15a), 4.53 (1H, dd, J = 11.0, 5.5 Hz,
H-3), 3.96-3.89 (1H, m, H-12), 3.88 (1H, d, J = 10.5 Hz, H-19b), 3.58 (1H, d, J = 10.5 Hz,
H-19a), 2.76 (1H, brd, J = 3.0 Hz, H-17b), 2.63 (1H, d, J = 3.0 Hz, H-17a), 2.15 (1H, dt, J =
13.5, 3.0 Hz, H-11b), 2.04 (3H, s, COCH3), 1.94-1.61 (7H, m), 1.45-1.24 (2H, m), 1.18-1.12
(1H, m), 0.94 (3H, s, H-18), 0.90 (3H, s, H-20), 0.89 (9H, s, SiC(CH3)3), 0.77-0.68 (1H, m),
0.03 (6H, s, Si(CH3)2); 13C NMR (100 MHz, CDCl3): δ 173.15, 170.55, 146.94, 146.14,
135.84, 129.26 (2×C), 117.20, 112.82 (2×C), 80.12, 70.30, 64.03, 59.16, 54.93, 52.57,
52.10, 50.54, 42.30, 40.33, 36.46, 36.44, 26.59, 25.85 (3×C), 23.71, 23.11, 23.01, 21.21,
18.19, 14.28, -5.60, -5.70; HRMS (ESI) m/z calcd for C34H52NO6Si [M+H]+ 598.3564,
found 598.3569.
19-TBS-3-acetyl-17-((2-fluoro)phenyl)amino-epi-isoandrographolide (6b)
According to the general procedure, 19-TBS-3,14-diacetyl-8,17-epoxy
andrographolide (4b) (24.0 mg, 0.042 mmol) with 2-fluoroaniline (6.90 mg, 0.062 mmol)
was stirred at room temperature for 48 h. The residue was purified by column
19

ACCEPTED MANUSCRIPT
chromatography (25% EtOAc/n-hexane) to afford the corresponding product 6b in 77%
o
yield (20.0 mg) as a white solid, Rf 0.59 (25% EtOAc/n-hexane); Mp. 66-68 C; IR (Neat):
3372, 3119, 2956, 2857, 1755, 1620, 1526, 1402, 1249, 1093, 1062, 1030, 855, 837 cm-1;
1H NMR (400 MHz, CDCl3): δ 7.19 (1H, d, J = 1.5 Hz, H-14) 6.98 (1H, ddd, J = 12.0, 8.0,
1.5 Hz, PhH), 6.92 (1H, t, J = 8.0 Hz, PhH), 6.64-6.57 (1H, m, PhH), 6.36 (1H, td, J = 8.0,
1.5 Hz, PhH), 5.26 (1H, brs, NH), 4.79 (1H, dt, J = 18.0, 1.5 Hz, H-15b), 4.73 (1H, dt, J =
18.0, 1.5 Hz, H-15a), 4.54 (1H, dd, J = 10.0, 6.5 Hz, H-3), 3.96 (1H, brd, J = 10.0 Hz, H-
12), 3.88 (1H, d, J = 10.5 Hz, H-19b), 3.58 (1H, d, J = 10.5 Hz, H-19a), 2.76 (1H, brd, J =
3.5 Hz, H-17b), 2.63 (1H, d, J = 3.5 Hz, H-17a), 2.14 (1H, dt, J = 13.5, 3.5 Hz, H-11b), 2.04
(3H, s, COCH3), 1.95-1.68 (7H, m), 1.47-1.30 (2H, m), 1.19-1.13 (1H, m), 0.94 (3H, s, H-
18), 0.91 (3H, s, H-20), 0.89 (9H, s, SiC(CH3)3), 0.84-0.74 (1H, m), 0.03 (6H, s, Si(CH3)2);
13C NMR (100 MHz, CDCl3): δ 173.03, 170.60, 151.41 (d, JC-F = 240 Hz), 146.09, 135.55,
135.36 (d, JC-F = 10.0 Hz), 124.46 (d, JC-F = 3.0 Hz), 116.60 (d, JC-F = 6.0 Hz), 114.60 (d, JC-
= 18.0 Hz), 112.24 (d, JC-F = 3.0 Hz), 80.12, 70.34, 64.00, 59.00, 54.97, 52.25, 52.14,
F
50.51, 42.29, 40.29, 36.48, 36.33, 26.56, 25.84 (3×C), 23.71, 23.10, 22.98, 21.22, 18.19,
14.29, -5.63, -5.73; HRMS (ESI) m/z calcd for C34H51FNO6Si [M+H]+ 616.3470, found
616.3467.
19-TBS-3-acetyl-17-((3-fluoro)phenyl)amino-epi-isoandrographolide (7b)
According to the general procedure, 19-TBS-3,14-diacetyl-8,17-epoxy
andrographolide (4b) (22.3 mg, 0.039 mmol) with 3-fluoroaniline (6.40 mg, 0.058 mmol)
was stirred at room temperature for 72 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 7b in 79%
o
yield (19.0 mg) as a white solid, Rf 0.29 (30% EtOAc/n-hexane); Mp. 172-174 C; IR
1
(Neat): 3371, 2956, 2857, 1755, 1622, 1521, 1403, 1251, 1094, 1031, 837, 775 cm-1; H
NMR (400 MHz, CDCl3): δ 7.18 (1H, d, J = 1.5 Hz, H-14) 7.06 (1H, dd, J = 15.0, 8.0 Hz,
PhH), 6.36 (1H, td, J = 8.0, 2.0 Hz, PhH), 6.26 (1H, dd, J = 8.0, 2.0 Hz, PhH), 6.16 (1H, dt,
J = 11.5, 2.0 Hz, PhH), 5.22 (1H, brs, NH), 4.81 (1H, dt, J = 18.0, 1.5 Hz, H-15b), 4.73 (1H,
dt, J = 18.0, 1.5 Hz, H-15a), 4.55 (1H, dd, J = 10.5, 6.0 Hz, H-3), 3.92-3.87 (1H, m, H-12),
3.88 (1H, d, J = 10.5 Hz, H-19b), 3.57 (1H, d, J = 10.5 Hz, H-19a), 2.77 (1H, dm, J = 3.5
Hz, H-17b), 2.63 (1H, d, J = 3.5 Hz, H-17a), 2.13 (1H, dt, J = 13.5, 3.5 Hz, H-11b), 2.04
(3H, s, COCH3), 1.95-1.65 (7H, m), 1.45-1.29 (2H, m), 1.19-1.12 (1H, m), 0.94 (3H, s, H-
18), 0.90 (3H, s, H-20), 0.88 (9H, s, SiC(CH3)3), 0.76-0.66 (1H, m), 0.03 (6H, s, Si(CH3)2);
20

ACCEPTED MANUSCRIPT
13C NMR (100 MHz, CDCl3): δ 173.00, 170.58, 146.17, 135.38, 130.33 (d, JC-F = 10.0 Hz),
108.60, 103.69 (d, JC-F = 20.0 Hz), 99.61 (d, JC-F = 25.0 Hz), 80.04, 70.32, 64.04, 59.22,
54.92, 52.69, 52.20, 50.58, 42.28, 40.33, 36.46, 36.43, 26.48, 25.84 (3×C), 23.69, 23.10,
23.00, 21.23, 18.18, 14.26, -5.63, -5.73; HRMS (ESI) m/z calcd for C34H51FNO6Si [M+H]+
616.3470, found 616.3463.
19-TBS-3-acetyl-17-((4-Bromo)phenyl)amino-epi-isoandrographolide (8b)
According to the general procedure, 19-TBS-3,14-diacetyl-8,17-epoxy andrographolide (4b)
(23.4 mg, 0.041 mmol) with 4-bromoaniline (10.0 mg, 0.058 mmol) was stirred at room
temperature for 96 h. The residue was purified by column chromatography (15% EtOAc/n-
hexane) to afford the corresponding product 8b in 77% yield (21.3 mg) as a yellow solid, Rf
0.33 (15% EtOAc/n-hexane); Mp. 52-54 oC; IR (Neat): 3360, 3159, 3098, 2957, 2856, 1755,
1
1734, 1595, 1400, 1259, 1098, 1030, 837, 813 cm-1; H NMR (400 MHz, CDCl3): δ 7.21
(2H, d, J = 7.5 Hz, PhH), 7.16 (1H, brs, H-14) 6.37 (2H, d, J = 7.5 Hz, PhH), 5.13 (1H, brs,
NH), 4.79 (1H, d, J = 18.5 Hz, H-15b), 4.72 (1H, d, J = 18.5 Hz, H-15a), 4.57-4.51 (1H, m,
H-3), 3.87 (1H, d, J = 10.0 Hz, H-19b), 3.87-3.82 (1H, m, H-12), 3.57 (1H, d, J = 10.0 Hz,
H-19a), 2.76 (1H, brs, H-17b), 2.63 (1H, brs, H-17a), 2.13 (1H, dm, J = 13.5 Hz, H-11b),
2.04 (3H, s, COCH3), 1.93-1.67 (7H, m), 1.44-1.10 (3H, m), 0.94 (3H, s, H-18), 0.89-0.88
(12H, m, H-20, SiC(CH3)3), 0.80-0.70 (1H, m), 0.07 (6H, s, Si(CH3)2); 13C NMR (100 MHz,
CDCl3): δ 173.00, 170.57, 146.15, 145.99, 135.42, 131.97 (2×C), 114.44 (2×C), 108.84,
80.07, 70.31, 64.07, 59.23, 54.96, 52.74, 52.26, 50.60, 42.31, 40.36, 36.51 (2×C), 26.59,
25.86 (3×C), 23.71, 23.12, 23.02, 21.22, 18.20, 14.29, -5.7 (2×C); HRMS (ESI) m/z calcd
for C34H51BrNO6Si [M+H]+ 676.2669, found 676.2664.
19-TBS-3-acetyl-17-((3,4-dimethoxy)phenyl)amino-epi-isoandrographolide
(9b)According to the general procedure, 19-TBS-3,14-diacetyl-8,17-epoxy andrographolide
(4b) (21.0 mg, 0.037 mmol) with 3,4-dimethoxyaniline (8.30 mg, 0.054 mmol) was stirred
at room temperature for 1 h. The residue was purified by column chromatography (40%
EtOAc/n-hexane) to afford the corresponding product 9b in 67% yield (16.4 mg) as a
o
yellow solid, Rf 0.48 (40% EtOAc/n-hexane); Mp. 66-68 C; IR (Neat): 3373, 3124, 2955,
2857, 1751, 1616, 1516, 1403, 1261, 1248, 1093, 1030, 855, 837 cm-1; 1H NMR (400 MHz,
CDCl3): δ 7.21 (1H, brs, H-14) 6.69 (1H, d, J = 8.0 Hz, PhH), 6.18 (1H, s, PhH), 5.94 (1H,
d, J = 8.0 Hz, PhH), 4.79 (1H, d, J = 18.0 Hz, H-15b), 4.72 (1H, d, J = 18.0 Hz, H-15a),
4.60-4.51 (2H, m, H-3, NH), 3.91 (1H, d, J = 9.5 Hz, H-12), 3.87 (1H, d, J = 10.5 Hz, H-
21

ACCEPTED MANUSCRIPT
19b), 3.82 (3H, s, OCH3), 3.79 (3H, s, OCH3), 3.57 (1H, d, J = 10.5 Hz, H-19a), 2.77 (1H,
brs, H-17b), 2.62 (1H, brs, H-17a), 2.12 (1H, dm, J = 13.0 Hz, H-11b), 2.04 (3H, s,
COCH3), 1.93-1.65 (7H, m), 1.45-1.27 (2H, m), 1.19-1.12 (1H, m), 0.93 (3H, s, H-18), 0.89
(3H, s, H-20), 0.88 (9H, s, SiC(CH3)3), 0.80-0.70 (1H, m), 0.03 (6H, s, Si(CH3)2); 13C NMR
(100 MHz, CDCl3): δ 173.17, 170.56, 150.11, 146.19, 141.85, 141.38, 135.94, 113.33,
103.29, 98.91, 80.09, 70.29, 64.01, 59.23, 56.72, 55.82, 54.93, 53.03, 51.92, 50.54, 42.28,
40.27, 36.51, 36.47, 26.71, 25.83 (3×C), 23.70, 23.09, 22.99, 21.19, 18.17, 14.28, -5.65, -
5.75; HRMS (ESI) m/z calcd for C36H56NO8Si [M+H]+ 658.3775, found 658.3777.
19-TBDPS-3-acetyl-17-(phenyl)amino-epi-isoandrographolide (5c)
According to the general procedure, 19-TBDPS-3,14-diacetyl-8,17-epoxy
andrographolide (4c) (40.4 mg, 0.059 mmol) with aniline (18.2 mg, 0.195 mmol) was stirred
at room temperature for 4.5 h. The residue was purified by column chromatography (30%
EtOAc/n-hexane) to afford the corresponding product 5c in 69% yield (29.6 mg) as a white
o
solid, Rf 0.35 (30% EtOAc/n-hexane); Mp. 97-99 C; IR (Neat): 3367, 2936, 2857, 1753,
1600, 1508, 1371, 1245, 1077, 1027, 828, 760, 699, 501 cm-1; 1H NMR (400 MHz, CDCl3):
δ 7.69-7.63 (4H, m, PhH), 7.45-7.34 (6H, m, PhH), 7.18 (1H, brs, H-14), 7.13 (2H, t, J = 7.5
Hz, PhH), 6.67 (1H, t, J = 7.5 Hz, PhH), 6.48 (2H, d, J = 7.5 Hz, PhH), 4.77 (1H, brd, J =
18.0 Hz, H-15b), 4.70 (1H, brd, J = 18.0 Hz, H-15a), 4.50 (1H, dd, J = 12.0, 4.5 Hz, H-3),
3.90 (1H, brd, J = 10.0 Hz, H-12), 3.83 (1H, d, J = 10.5 Hz, H-19b), 3.71 (1H, d, J = 10.5
Hz, H-19a), 2.72 (1H, brd, J = 3.0 Hz, H-17b), 2.61 (1H, brd, J = 3.0 Hz, H-17a), 2.11-1.12
(11H, m), 1.88 (3H, s, COCH3), 1.05 (9H, s, SiC(CH3)3), 1.00 (3H, s, H-18), 0.73 (3H, s, H-
20), 0.73-0.64 (1H, m); 13C NMR (100 MHz, CDCl3): δ 173.08, 170.48, 146.97, 146.09,
135.84 (4×C), 135.76, 133.57, 133.44, 129.61 (2×C), 129.27 (2×C), 127.59 (2×C), 127.53
(2×C), 117.26, 112.85 (2×C), 80.04, 70.26, 63.91, 58.96, 54.86, 52.54, 51.96, 50.51, 42.89,
40.26, 36.29, 36.24, 26.96 (3×C), 26.51, 23.63, 22.88, 22.74, 21.06, 19.27, 14.49; HRMS
(ESI) m/z calcd for C44H56NO6Si [M+H]+ 722.3877, found 722.3873.
19-TBDPS-3-acetyl-17-((2-fluoro)phenyl)amino-epi-isoandrographolide (6c)
According to the general procedure, 19-TBDPS-3,14-diacetyl-8,17-epoxy
andrographolide (4c) (40.5 mg, 0.059 mmol) with 2-fluoroaniline (19.5 mg, 0.175 mmol)
was stirred at room temperature for 48 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 6c in 82%
o
yield (35.7 mg) as a white solid, Rf 0.39 (30% EtOAc/n-hexane); Mp. 83-85 C; IR (Neat):
22

ACCEPTED MANUSCRIPT
3373, 2930, 2857, 1753, 1620, 1519, 1368, 1260, 1081, 1029, 822, 753, 498 cm-1; 1H NMR
(400 MHz, CDCl3): δ 7.71-7.63 (4H, m, PhH), 7.46-7.34 (6H, m, PhH), 7.18 (1H, brs, H-
14), 6.98 (1H, t, J = 9.0 Hz, PhH), 6.91 (1H, t, J = 7.5 Hz, PhH), 6.65-6.57 (1H, m, PhH),
6.35 (1H, t, J = 8.5 Hz, PhH), 5.23 (1H, brs, NH), 4.78 (1H, brd, J = 18.0 Hz, H-15b), 4.72
(1H, brd, J = 18.0 Hz, H-15a), 4.52 (1H, dd, J = 11.5, 3.0 Hz, H-3), 3.93 (1H, brd, J = 8.5
Hz, H-12), 3.84 (1H, d, J = 10.5 Hz, H-19b), 3.72 (1H, d, J = 10.5 Hz, H-19a), 2.73 (1H,
brs, H-17b), 2.62 (1H, brs, H-17a), 2.12-1.95 (2H, m), 1.89 (3H, s, COCH3), 1.87-1.42 (7H,
m), 1.36-1.12 (2H, m), 1.06 (9H, s, SiC(CH3)3), 1.01 (3H, s, H-18), 0.75 (3H, s, H-20),
13
0.80-0.70 (1H, m); C NMR (100 MHz, CDCl3): δ 172.98, 170.56, 151.42 (d, JC-F = 239
Hz), 146.07, 135.84 (2×C), 135.75 (2×C), 135.53, 135.34 (d, JC-F = 11.0 Hz), 133.52,
133.39, 129.64, 129.61, 127.60 (2×C), 127.53 (2×C), 124.46, 116.64 (d, JC-F = 7.0 Hz),
114.61 (d, JC-F = 18.0 Hz), 112.23, 80.02, 70.32, 63.84, 58.81, 54.87, 52.22, 52.00, 50.50,
42.87, 40.20, 36.24, 36.15, 26.94 (3×C), 26.46, 23.61, 22.84, 22.73, 21.10, 19.26, 14.49;
HRMS (ESI) m/z calcd for C44H55FNO6Si [M+H]+ 740.3783, found 740.3782.
19-TBDPS-3-acetyl-17-((3-fluoro)phenyl)amino-epi-isoandrographolide (7c)
According to the general procedure, 19-TBDPS-3,14-diacetyl-8,17-epoxy
andrographolide (4c) (40.7 mg, 0.059 mmol) with 3-fluoroaniline (15.9 mg, 0.143 mmol)
was stirred at room temperature for 24 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 7c in 80%
o
yield (35.0 mg) as a white solid, Rf 0.32 (30% EtOAc/n-hexane); Mp. 85-87 C; IR (Neat):
1
3356, 2924, 2852, 1753, 1617, 1491, 1371, 1246, 1078, 1029, 825, 761, 702, 498 cm-1; H
NMR (400 MHz, CDCl3): δ 7.72-7.62 (4H, brs, PhH), 7.46-7.34 (6H, m, PhH), 7.18 (1H,
brs, H-14), 7.10-7.02 (1H, m, PhH), 6.36 (1H, td, J = 7.5, 1.0 Hz, PhH), 6.26 (1H, dd, J =
7.5, 1.0 Hz, PhH), 6.17 (1H, dt, J = 11.5, 1.0 Hz, PhH), 4.81 (1H, brd, J = 18.0 Hz, H-15b),
4.72 (1H, brd, J = 18.0 Hz, H-15a), 4.52 (1H, dd, J = 12.0, 4.5 Hz, H-3), 3.89-3.84 (1H, m,
H-12), 3.83 (1H, d, J = 10.5 Hz, H-19b), 3.71 (1H, d, J = 10.5 Hz, H-19a), 2.74 (1H, brd, J
= 3.0 Hz, H-17b), 2.63 (1H, d, J = 3.0 Hz, H-17a), 2.10-1.92 (2H, m), 1.89 (3H, s, COCH3),
1.87-1.41 (7H, m), 1.36-1.13 (2H, m), 1.06 (9H, s, SiC(CH3)3), 1.01 (3H, s, H-18), 0.74
13
(3H, s, H-20), 0.73-0.65 (1H, m); C NMR (100 MHz, CDCl3): δ 172.94, 170.53, 164.08
(d, JC-F = 241 Hz), 148.78 (d, JC-F = 11.0 Hz), 146.16, 135.84 (2×C), 135.75 (2×C), 135.35,
133.52, 133.38, 130.34 (d, JC-F = 10.0 Hz), 129.62 (2×C), 127.60 (2×C), 127.53 (2×C),
23

ACCEPTED MANUSCRIPT
108.62, 103.74 (d, JC-F = 22.0 Hz), 99.64 (d, J = 25.0 Hz), 79.96, 70.29, 63.88, 59.04, 54.83,
52.65, 52.06, 50.57, 42.87, 40.25, 36.29, 33.22, 26.95 (3×C), 26.40, 23.60, 22.86, 22.73,
21.07, 19.26, 14.47; HRMS (ESI) m/z calcd for C44H55FNO6Si [M+H]+ 740.3783, found
740.3777.
19-TBDPS-3-acetyl-17-((4-Bromo)phenyl)amino-epi-isoandrographolide (8c)
According to the general procedure, 19-TBDPS-3,14-diacetyl-8,17-epoxy
andrographolide (4c) (41.4 mg, 0.060 mmol) with 4-bromoaniline (15.0 mg, 0.087 mmol)
was stirred at room temperature for 24 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 8c in 69%
yield (33.2 mg) as a yellow solid, Rf 0.35 (30% EtOAc/n-hexane); Mp. 87-89 oC; IR (Neat):
1
3356, 2931, 2852, 1752, 1592, 1491, 1368, 1245, 1075, 1027, 819, 761, 704, 501 cm-1; H
NMR (400 MHz, CDCl3): δ 7.71-7.63 (4H, brs, PhH), 7.47-7.35 (6H, m, PhH), 7.21 (2H, d,
J = 8.5 Hz, PhH), 7.16 (1H, brs, H-14), 6.37 (2H, d, J = 8.5 Hz, PhH), 4.79 (1H, brd, J =
18.0 Hz, H-15b), 4.72 (1H, brd, J = 18.0 Hz, H-15a), 4.52 (1H, dd, J = 12.0, 4.5 Hz, H-3),
3.88-3.82 (1H, m, H-12), 3.83 (1H, d, J = 10.5 Hz, H-19b), 3.71 (1H, d, J = 10.5 Hz, H-
19a), 2.74 (1H, brd, J = 3.0 Hz, H-17b), 2.63 (1H, d, J = 3.0 Hz, H-17a), 2.09-1.97 (2H, m),
1.89 (3H, s, COCH3), 1.86-1.41 (7H, m), 1.35-1.12 (2H, m), 1.06 (9H, s, SiC(CH3)3), 1.00
(3H, s, H-18), 0.73 (3H, s, H-20), 0.72-0.65 (1H, m); 13C NMR (100 MHz, CDCl3): δ
172.97, 170.53, 146.16, 145.96, 135.84 (2×C), 135.76 (2×C), 135.36, 133.53, 133.38,
131.98 (2×C), 129.65 (2×C), 127.16 (2×C), 127.55 (2×C), 114.44 (2×C), 108.87, 79.96,
70.30, 63.88, 59.06, 54.84, 52.69, 52.11, 50.59, 42.87, 40.25, 36.31, 36.22, 26.96 (3×C),
26.47, 23.60, 22.88, 22.70, 21.10, 19.28, 14.48; HRMS (ESI) m/z calcd for C44H55BrNO6Si
[M+H]+ 800.2982, found 800.2977.
19-TBDPS-3-acetyl-17-((3,4-dimethoxy)phenyl)amino-epi-isoandrographolide
(9c)
According to the general procedure, 19-TBDPS-3,14-diacetyl-8,17-epoxy
andrographolide (4c) (40.2 mg, 0.058 mmol) with 3,4-dimethoxyaniline (13.9 mg, 0.091
mmol) was stirred at room temperature for 2 h. The residue was purified by column
chromatography (40% EtOAc/n-hexane) to afford the corresponding product 9c in 79%
yield (36.1 mg) as brown solid, Rf 0.50 (40% EtOAc/n-hexane); Mp. 107-109 oC; IR (Neat):
1
3366, 2935, 2852, 1751, 1515, 1460, 1365, 1240, 1077, 1029, 822, 752, 705, 502 cm-1; H
NMR (400 MHz, CDCl3): δ 7.70-7.64 (4H, brs, PhH), 7.46-7.34 (6H, m, PhH), 7.20 (1H,
24

ACCEPTED MANUSCRIPT
brs, H-14), 6.69 (1H, d, J = 8.5 Hz, PhH), 6.18 (1H, s, PhH), 5.93 (1H, d, J = 8.5 Hz, PhH),
4.78 (1H, brd, J = 18.0 Hz, H-15b), 4.72 (1H, brd, J = 18.0 Hz, H-15a), 4.51 (1H, brd, J =
11.0 Hz, H-3), 3.90 (1H, brd, J = 8.5 Hz, H-12), 3.83 (1H, d, J = 11.0 Hz, H-19b), 3.82 (3H,
s, OCH3), 3.79 (3H, s, OCH3), 3.71 (1H, d, J = 11.0 Hz, H-19a), 2.74 (1H, brs, H-17b), 2.61
(1H, brs, H-17a), 2.09-1.95 (2H, m), 1.89 (3H, s, COCH3), 1.85-1.12 (9H, m), 1.06 (9H, s,
SiC(CH3)3), 1.01 (3H, s, H-18), 0.96-0.83 (1H, m), 0.74 (3H, s, H-20); 13C NMR (100 MHz,
CDCl3): δ 173.14, 170.53, 150.10, 146.18, 141.82, 141.37, 135.90, 135.82 (2×C), 135.73
(2×C), 133.50, 133.36, 129.62 (2×C), 127.58 (2×C), 127.52 (2×C), 113.28, 103.24, 98.87,
79.98, 70.29, 63.84, 59.06, 56.70, 55.82, 54.81, 52.97, 51.78, 50.54, 42.84, 40.18, 36.31,
36.22, 26.93 (3×C), 26.60, 23.59, 22.86, 22.72, 21.08, 19.25, 14.47; HRMS (ESI) m/z calcd
for C46H60NO8Si [M+H]+ 782.4088, found 782.4083.
19-TIPS-3-acetyl-17-(phenyl)amino-epi-isoandrographolide (5d)
According to the general procedure, 19-TIPS-3,14-diacetyl-8,17-epoxy
andrographolide (4d) (49.9 mg, 0.082 mmol) with aniline (12.0 mg, 0.129 mmol) was
stirred at room temperature for 3 h. The residue was purified by column chromatography
(30% EtOAc/n-hexane) to afford the corresponding product 5d in 65% yield (34.2 mg) as a
o
white solid, Rf 0.35 (30% EtOAc/n-hexane); Mp. 87-89 C; IR (Neat): 3361, 2945, 2863,
1
1745, 1600, 1508, 1455, 1253, 1091, 1024, 878, 757, 495 cm-1; H NMR (400 MHz,
CDCl3): δ 7.19 (1H, brd, J = 1.0 Hz, H-14), 7.14 (2H, dd, J = 8.5, 7.5 Hz, PhH), 6.68 (1H, t,
J = 7.5 Hz, PhH), 6.49 (2H, d, J = 8.5 Hz, PhH), 5.01 (1H, brs, NH), 4.78 (1H, dt, J = 18.0,
1.5 Hz, H-15b), 4.72 (1H, dt, J = 18.0, 1.5 Hz, H-15a), 4.53 (1H, dd, J = 11.5, 4.5 Hz, H-3),
3.94 (1H, d, J = 10.5 Hz, H-19b), 3.95-3.89 (1H, m, H-12), 3.78 (1H, d, J = 10.5 Hz, H-
19a), 2.76 (1H, d, J = 3.5 Hz, H-17b), 2.63 (1H, d, J = 3.5 Hz, H-17a), 2.14 (1H, dt, J =
13.5, 3.0 Hz, H-11b), 2.03 (3H, s, COCH3), 1.98-1.69 (8H, m), 1.45-1.29 (2H, m), 1.06
(18H, brd, J = 4.0 Hz, 3×(SiCH(CH3)2)), 1.17-1.02 (3H, m, 3×(SiCH(CH3)2)), 0.99 (3H, s,
H-18), 0.89 (3H, s, H-20), 0.77-0.68 (1H, m); 13C NMR (100 MHz, CDCl3): δ 173.15,
170.58, 146.89, 146.18, 135.75, 129.25 (2×C), 117.19, 112.79 (2×C), 80.10, 70.30, 63.83,
59.08, 54.83, 52.52, 52.00, 50.55, 42.80, 40.27, 36.30, 36.27, 26.48, 23.64, 22.82 (2×C),
21.19, 18.08 (6×C), 14.58, 11.90 (3×C); HRMS (ESI) m/z calcd for C37H58NO6Si [M+H]+
640.4033, found 640.4028.
19-TIPS-3-acetyl-17-((2-fluoro)phenyl)amino-epi-isoandrographolide (6d)
25

ACCEPTED MANUSCRIPT
According to the general procedure, 19-TIPS-3,14-diacetyl-8,17-epoxy
andrographolide (4d) (50.2 mg, 0.083 mmol) with 2-fluoroaniline (13.7 mg, 0.123 mmol)
was stirred at room temperature for 72 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 6d in 65%
o
yield (35.4 mg) as a white solid, Rf 0.33 (30% EtOAc/n-hexane); Mp. 145-147 C; IR
(Neat): 3378, 2942, 2863, 1753, 1619, 1522, 1455, 1245, 1094, 1027, 853, 838, 758, 495
1
cm-1; H NMR (400 MHz, CDCl3): δ 7.18 (1H, brd, J = 1.0 Hz, H-14), 6.98 (1H, ddd, J =
12.0, 8.0, 1.0 Hz, PhH), 6.92 (1H, t, J = 8.0 Hz, PhH), 6.64-6.57 (1H, m, PhH), 6.36 (1H, td,
J = 8.0, 1.0 Hz, PhH), 5.25 (1H, brs, NH), 4.80 (1H, dt, J = 18.0, 1.5 Hz, H-15b), 4.73 (1H,
dt, J = 18.0, 1.5 Hz, H-15a), 4.55 (1H, dd, J = 12.0, 4.5 Hz, H-3), 3.94 (1H, d, J = 10.0 Hz,
H-19b), 3.98-3.93 (1H, m, H-12), 3.94 (1H, d, J = 10.0 Hz, H-19a), 2.76 (1H, d, J = 3.5 Hz,
H-17b), 2.64 (1H, d, J = 3.5 Hz, H-17a), 2.13 (1H, dt, J = 13.5, 3.0 Hz, H-11b), 2.03 (3H, s,
COCH3), 1.98-1.67 (8H, m), 1.48-1.29 (2H, m), 1.06 (18H, brd, J = 4.5 Hz,
3×(SiCH(CH3)2)), 1.12-1.03 (3H, m, 3×(SiCH(CH3)2)), 1.00 (3H, s, H-18), 0.90 (3H, s, H-
13
20), 0.83-0.73 (1H, m); C NMR (100 MHz, CDCl3): δ 173.01, 170.62, 151.39 (d, JC-F
=
238 Hz), 146.12, 135.50, 135.33 (d, JC-F = 11.0 Hz), 124.46 (d, JC-F = 3.0 Hz), 116.60 (d, JC-
F = 7.0 Hz), 114.59 (d, JC-F = 18.0 Hz), 112.22 (d, JC-F = 3.0 Hz), 80.11, 70.33, 63.81, 58.92,
54.90, 52.23, 52.07, 50.52, 42.82, 40.25, 36.33, 36.19, 26.47, 23.66, 22.82 (2×C), 21.20,
18.08 (6×C), 14.61, 11.91 (3×C); HRMS (ESI) m/z calcd for C37H57FNO6Si [M+H]+
658.3939, found 658.3939.
19-TIPS-3-acetyl-17-((3-fluoro)phenyl)amino-epi-isoandrographolide (7d)
According to the general procedure, 19-TIPS-3,14-diacetyl-8,17-epoxy
andrographolide (4d) (51.7 mg, 0.085 mmol) with 3-fluoroaniline (16.5 mg, 0.148 mmol)
was stirred at room temperature for 48 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 7d in 82%
o
yield (46.0 mg) as a white solid, Rf 0.39 (30% EtOAc/n-hexane); Mp. 147-149 C; IR
(Neat): 3361, 2942, 2866, 1753, 1620, 1495, 1370, 1245, 1097, 1030, 884, 837, 761, 495
1
cm-1; H NMR (400 MHz, CDCl3): δ 7.18 (1H, brd, J = 1.5 Hz, H-14), 7.10-7.02 (1H, m,
PhH), 6.35 (1H, td, J = 8.0, 1.5 Hz, PhH), 6.26 (1H, dd, J = 8.0, 1.5 Hz, PhH), 6.16 (1H, dt,
J = 11.5, 1.5 Hz, PhH), 5.21 (1H, brs, NH), 4.81 (1H, dt, J = 18.0, 1.5 Hz, H-15b), 4.73 (1H,
dt, J = 18.0, 1.5 Hz, H-15a), 4.55 (1H, dd, J = 12.0, 4.5 Hz, H-3), 3.94 (1H, d, J = 10.5 Hz,
H-19b), 3.88 (1H, brd, J = 10.0 Hz, H-12), 3.78 (1H, d, J = 10.5 Hz, H-19a), 2.76 (1H, d, J
26

ACCEPTED MANUSCRIPT
= 3.5 Hz, H-17b), 2.64 (1H, d, J = 3.5 Hz, H-17a), 2.12 (1H, dt, J = 13.5, 3.0 Hz, H-11b),
2.03 (3H, s, COCH3), 1.98-1.59 (8H, m), 1.46-1.27 (2H, m), 1.06 (18H, brd, J = 4.5 Hz,
3×(SiCH(CH3)2)), 1.12-1.03 (3H, m, 3×(SiCH(CH3)2)), 1.00 (3H, s, H-18), 0.89 (3H, s, H-
13
20), 0.76-0.66 (1H, m); C NMR (100 MHz, CDCl3): δ 172.98, 170.58, 164.04 (d, JC-F
=
241 Hz), 148.75 (d, JC-F = 10.0 Hz), 146.19, 135.33, 130.32 (d, JC-F = 10.0 Hz), 108.60,
103.70 (d, JC-F = 21.0 Hz), 99.61 (d, JC-F = 25.0 Hz), 80.04, 70.31, 63.85, 59.14, 54.86,
52.66, 52.13, 50.58, 42.81, 40.29, 36.33, 36.29, 26.40, 23.64, 22.83 (2×C), 21.19, 18.08
(6×C), 14.59, 11.91 (3×C); HRMS (ESI) m/z calcd for C37H57FNO6Si [M+H]+ 658.3939,
found 658.3934.
19-TIPS-3-acetyl-17-((4-Bromo)phenyl)amino-epi-isoandrographolide (8d)
According to the general procedure, 19-TIPS-3,14-diacetyl-8,17-epoxy
andrographolide (4d) (50.7 mg, 0.083 mmol) with 4-bromoaniline (21.5 mg, 0.125 mmol)
was stirred at room temperature for 24 h. The residue was purified by column
chromatography (40% EtOAc/n-hexane) to afford the corresponding product 8d in 66%
o
yield (39.6 mg) as a white solid, Rf 0.50 (40% EtOAc/n-hexane); Mp. 103-105 C; IR
(Neat): 3361, 2941, 2863, 1752, 1594, 1502, 1245, 1096, 1027, 881, 814, 762, 498 cm-1; 1H
NMR (400 MHz, CDCl3): δ 7.21 (2H, d, J = 8.5 Hz, PhH), 7.16 (1H, brs, H-14), 6.37 (2H,
d, J = 8.5 Hz, PhH), 5.13 (1H, brs, NH), 4.80 (1H, d, J = 18.0 Hz, H-15b), 4.72 (1H, d, J =
18.0 Hz, H-15a), 4.54 (1H, dd, J = 12.0, 4.5 Hz, H-3), 3.94 (1H, d, J = 10.5 Hz, H-19b),
3.86 (1H, brd, J = 10.0 Hz, H-12), 3.77 (1H, d, J = 10.5 Hz, H-19a), 2.76 (1H, d, J = 3.0 Hz,
H-17b), 2.64 (1H, d, J = 3.0 Hz, H-17a), 2.11 (1H, dt, J = 13.5, 3.0 Hz, H-11b), 2.02 (3H, s,
COCH3), 1.99-1.58 (8H, m), 1.45-1.29 (2H, m), 1.06 (18H, brd, J = 4.5 Hz,
3×(SiCH(CH3)2)), 1.18-1.03 (3H, m, 3×(SiCH(CH3)2)), 1.00 (3H, s, H-18), 0.88 (3H, s, H-
20), 0.76-0.66 (1H, m); 13C NMR (100 MHz, CDCl3): δ 172.98, 170.58, 146.19, 145.87,
135.27, 131.91 (2×C), 114.39 (2×C), 108.78, 80.01, 70.30, 63.81, 59.13, 54.81, 52.65,
52.12, 50.58, 42.76, 40.25, 36.30, 36.25, 26.43, 23.60, 22.80 (2×C), 21.17, 18.06 (6×C),
14.56, 11.87 (3×C); HRMS (ESI) m/z calcd for C37H57BrNO6Si [M+H]+ 718.3139, found
718.3133.
19-TIPS-3-acetyl-17-((3,4-dimethoxy)phenyl)amino-epi-isoandrographolide
(9d)
According to the general procedure, 19-TIPS-3,14-diacetyl-8,17-epoxy
andrographolide (4d) (47.8 mg, 0.079 mmol) with 3,4-dimethoxyaniline (19.2 mg, 0.125
27

ACCEPTED MANUSCRIPT
mmol) was stirred at room temperature for 2 h. The residue was purified by column
chromatography (30% EtOAc/n-hexane) to afford the corresponding product 9d in 73%
o
yield (40.2 mg) as a brown solid, Rf 0.17 (30% EtOAc/n-hexane); Mp. 117-119 C; IR
(Neat): 3356, 2936, 2863, 1748, 1513, 1454, 1239, 1096, 1027, 881, 822, 763, 495 cm-1; 1H
NMR (400 MHz, CDCl3): δ 7.21 (1H, brs, H-14), 6.69 (1H, d, J = 8.5 Hz, PhH), 6.18 (1H,
d, J = 2.5 Hz, PhH), 5.94 (1H, dd, J = 8.5, 2.5 Hz, PhH), 4.80 (1H, d, J = 18.0 Hz, H-15b),
4.72 (1H, d, J = 18.0 Hz, H-15a), 4.54 (1H, dd, J = 11.5, 4.5 Hz, H-3), 3.94 (1H, d, J = 10.5
Hz, H-19b), 3.94-3.88 (1H, m, H-12), 3.81 (1H, d, J = 10.5 Hz, H-19a), 3.82 (3H, s, OCH3),
3.79 (3H, s, OCH3), 2.77 (1H, d, J = 3.0 Hz, H-17b), 2.63 (1H, d, J = 3.0 Hz, H-17a), 2.11
(1H, dm, J = 13.5 Hz, H-11b), 2.03 (3H, s, COCH3), 1.98-1.60 (8H, m), 1.45-1.28 (2H, m),
1.06 (18H, brd, J = 4.5 Hz, 3×(SiCH(CH3)2)), 1.12-1.02 (3H, m, 3×(SiCH(CH3)2)), 1.00
(3H, s, H-18), 0.89 (3H, s, H-20), 0.80-0.70 (1H, m); 13C NMR (100 MHz, CDCl3): δ
173.20, 170.63, 150.10, 146.24, 141.84, 141.37, 135.93, 113.25, 103.23, 98.87, 80.11,
70.33, 63.85, 59.19, 56.71, 55.84, 54.89, 53.01, 51.87, 50.59, 42.83, 40.26, 36.40, 36.35,
26.65, 23.67, 22.88, 22.85, 21.21, 18.10 (6×C), 14.63, 11.93 (3×C); HRMS (ESI) m/z calcd
for C39H62NO8Si [M+H]+ 700.4245, found 700.4249.
3,19-Diacetyl-17-(phenyl)amino-epi-isoandrographolide (5e)
According to the general procedure, 3,14,19-triacetyl-8,17-epoxy andrographolide
(4e) (50.0 mg, 0.102 mmol) with aniline (14.1 mg, 0.151 mmol) was stirred at room
temperature for 1.5 h. The residue was purified by column chromatography (40% EtOAc/n-
hexane) to afford the corresponding product 5e in 61% yield (32.5 mg) as a yellow solid, Rf
o
0.38 (40% EtOAc/n-hexane); Mp. 149-151 C; IR (Neat): 3373, 2941, 2857, 1736, 1600,
1
1371, 1256, 1029, 752 cm-1; H NMR (400 MHz, CDCl3): δ 7.19 (1H, d, J = 1.5 Hz, H-
14), 7.14 (2H, dd, J = 8.5, 7.5 Hz, PhH), 6.68 (1H, t, J = 7.5 Hz, PhH), 6.49 (2H, dd, J =
8.5, 1.0 Hz, PhH), 4.97 (1H, brs, NH), 4.78 (1H, dt, J = 18.0, 1.5 Hz, H-15b), 4.71 (1H, d, J
= 18.0, 1.5 Hz, H-15a), 4.56 (1H, dd, J = 12.0, 4.5 Hz, H-3), 4.32 (1H, d, J = 11.5 Hz, H-
19b), 4.16 (1H, d, J = 11.5 Hz, H-19a), 3.92 (1H, brd, J = 10.0 Hz, H-12), 2.75 (1H, dd, J =
3.5, 1.5 Hz, H-17b), 2.64 (1H, d, J = 3.5 Hz, H-17a), 2.17 (1H, dt, J = 13.5, 2.0 Hz, H-11b),
2.05 (3H, s, COCH3), 2.04 (3H, s, COCH3), 1.98-1.77 (4H, m), 1.76-1.60 (4H, m), 1.49-
1.31 (2H, m), 1.00 (3H, s, H-18), 0.85 (3H, s, H-20), 0.78-0.67 (1H, m); 13C NMR (100
MHz, CDCl3): δ 173.13, 170.86, 170.35, 146.87, 146.17, 135.72, 129.27 (2×C), 117.24,
112.77 (2×C), 79.61, 70.31, 64.74, 58.72, 54.55, 52.49, 51.90, 50.41, 41.09, 40.14, 36.02,
28