Chemistry of renieramycins. Part 8: Synthesis and cytotoxicity evaluation of renieramycin M-jorunnamycin A analogues

Article abstract of DOI:10.1016/j.bmc.2009.05.009

Twenty-four ester analogues of renieramycin M (1m) were prepared from jorunnamycin A (3a), which was easily transformed from marine natural 1m in three steps. These analogues, along with 1m itself, cyanojorumycin (2b), and jorunnamycins A (3a) and C (3b),

Full text of DOI:10.1016/j.bmc.2009.05.009

Bioorganic & Medicinal Chemistry 17 (2009) 4548–4558
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemistry of renieramycins. Part 8: Synthesis and cytotoxicity evaluation
of renieramycin M–jorunnamycin A analogues q
Kornvika Charupant a, Naomi Daikuhara b, Emi Saito b, Surattana Amnuoypol a, Khanit Suwanborirux a,
,
*
Takashi Owa c, Naoki Saito b,
*
a Department of Pharmacognosy and Pharmaceutical Botany, Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME),
Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
b Graduate School of Pharmaceutical Sciences, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
c Discovery Research Laboratories II, Integrative Oncology Area, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-four ester analogues of renieramycin M (1m) were prepared from jorunnamycin A (3a), which
was easily transformed from marine natural 1m in three steps. These analogues, along with 1m itself,
cyanojorumycin (2b), and jorunnamycins A (3a) and C (3b), were evaluated in vitro for cytotoxicity by
measuring IC50 values through the 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay using human HCT116 colon carcinoma and MDA-MB-435 breast carcinoma cell lines. Nitro-
gen-containing heterocyclic ester derivatives 9a–f showed similar in vitro cytotoxicity to 1m, whereas
the other derivatives were slightly less cytotoxic than 1m. 20-Pyridinecarboxylic acid ester derivative
(9c) exhibited a threefold increase in cytotoxicity relative to 1m.
Received 4 April 2009
Revised 1 May 2009
Accepted 2 May 2009
Available online 8 May 2009
Keywords:
Renieramycin
Marine natural product
Cytotoxicity
Ó 2009 Elsevier Ltd. All rights reserved.
Structure–activity relationship study
1. Introduction
edin), has a unique mechanism of action that is based on its
binding to the minor groove of DNA to interfere with cell division,
Bisisoquinolinequinone natural products and their reduced
forms have attracted considerable interest over the past 30 years
due to their potent biological properties (Fig. 1).2 The most bioac-
activated transcription, and DNA repair.3–6 The remarkable preclin-
ical and clinical results of ecteinascidin 743 have stimulated fur-
ther research of other antitumor agents, such as phthalascidin (Pt
650)7,8 and QAD9 (Fig. 2).
TM
tive member of this family, ecteinascidin 743 (Yondelis , trabect-
Following the discovery of renieramycins A–D (1a–d) from the
Mexican sponge Reniera sp. by Frincke and Faulkner in 1982,10 ten
marine natural renieramycins were isolated from marine sponges
belonging to genera Reniera,11 Xestospongia,12 Haliclona,13,14
Cribrochalina,15,16 and Neopetrosia.17 An additional related com-
pound, jorumycin (2a), which contains an acetyl ester instead of
an angelate ester, was isolated from the skin and the mucus of the
OCH3
HO
O
H
11
CH3
OCH3
O
O
NH
HO
4
1
H
CH3O
CH3
CH3
CH3
O
O
N
CH3
3
AcO
14
H
N 2
H
21
S
H
N
13
H
O
CH3O
N
CH3
H
X
22
NH
H
OCH3
OCH3
O
O
O
O
H
X
HO
H
CH3
HO
H
CH3
CH3
AcO
O
CH3O
HO
H
H
OCH3
CH3
CH3
saframycins
A: X = CN
S: X = OH
ecteinascidins
743: X = OH
770: X = CN
N
CH3
N
CH3
N
N
H
CH3O
H
O
H
H
CN
CN
Figure 1. Structures of representative isoquinoline natural products.
NH
O
N
N
O
O
q
See Ref. 1.
* Corresponding authors. Tel./fax: +66 2 218 8357 (K.S.), tel./fax: +81 42 495 8794
(N.S.).
E-mail addresses: skhanit@chula.ac.th (K. Suwanborirux), naoki@my-pharm
.ac.jp (N. Saito).
phthalascidin
Figure 2. Structures of synthetic analogues possessing high cytotoxicity.
QAD
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.009

K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
4549
OCH3
OCH3
three-step procedure for 1m, namely, hydrogenation, hydride
reduction, and air oxidation (Scheme 1).27
O
H
CH3
O
O
H
CH3
O
O
O
O
O
We first prepared the isomers of the ester side chain at C-22 of
1m, such as 4a and 4b. Acylation of 3a with DMAP and mixed anhy-
dride 5a29 at 45 °C for 7 h gave 4a in 38% yield. Treatment of 3a with
carbonate 5b30 in pyridine at ꢀ17 °C for 1 h afforded 4b in 81% yield.
Next, six aliphatic esters 6a–f were prepared from 3a with the anhy-
dride31 or the acid chloride in pyridine at -17 °C in 30–65%yields. Be-
cause commercially available racemic acid chloride was used to
prepare ester 6d, we obtained 6d as an inseparable 1:1 mixture of
epimers. Furthermore, five alicyclic compounds 7a–e were prepared
by esterifying 3a with acid chloride or carbonate30 following the
same procedure as above in 58–80% yields. Ester 7e was also ob-
tained as an inseparable 1:1 mixture of diastereomers. Finally, we
synthesized aromatic ester derivatives 8a–e (39–83% yields) and
nitrogen-containing heterocycles 9a–f (34–67% yields).
H
N
O
H
N
O
CH3
CH3
N
CH3
N
CH3
Y
Y
H
CH3O
CH3O
H
H
H
X
O
CH3
CH3
O
H
O
H
CH3
CH3
renieramycins
renieramycins
A (1a): X = H, Y = OH
C (1c): Y = OH
B (1b): X = H, Y = OC2H5
E (1e): X = OH, Y = H
F (1f): X = OH, Y = OCH3
M (1m) X = CN, Y = H
D (1d): Y = OC2H5
G (1g): Y = H
OCH3
OCH3
HO
O
H
CH3
O
CH3
OH
All the compounds were characterized by IR, mass, and 1H and
13C NMR measurements. All proton and carbon signals were as-
signed by 1H–1H, 1H–13C COSY, and a series of 1H detected two-
dimensional heteronuclear multiple-bond correlation (HMBC)
experiments. Signals of 16-CH3 and 17-OCH3 protons of aromatic
ester derivatives 8a–e and 9a–f were shifted upfield compared to
those of aliphatic, acyclic and cyclic derivatives, 4a,b, 6a–f, and
7a–f (Table 1). It was confirmed that both protons of quinone ring
E might be kept in the aromatic ring of the side chain.
O
O
O
O
H
H
CH3
CH3
N
CH3
N
CH3
N
N
O
O
H
CH3O
CH3O
H
H
H
X
O
OZ
CH3
O
H
jorumycin (2a): X = OH, Z = COCH3
CH3
cyanojorumycin (2b): X = CN, Z = COCH3
jorunnamycin A (3a): X = CN, Z = H
renieramycin H (1h)
jorunnamycin C (3b): X = CN, Z = COC2H5
(= cribrostatin 4)
3. Biology
Figure 3. Structures of marine natural renieramycins.
nudibranch Jorunna funebris (Fig. 3).18 The novel structures of renie-
ramycins, together with their remarkable biological activities and
lack of availability from natural sources, have made these com-
pounds extremely attractive and important synthetic targets. The
first total synthesis of renieramycin A (1a) was accomplished by
Fukuyama in 1990.19 To date, two total syntheses of renieramycin
G (1g),20,21 three total syntheses of renieramycin H (1h: cribrostatin
4),22–24 and one total synthesis of 2a have been published.21 How-
ever, the structure–activity relationships of renieramycins are rela-
tively unexplored because most synthetic approaches have focused
on their total synthesis.
As part of our search for new metabolites via the isolation and
characterization of biologically active compounds from Thai mar-
ine animals, we have reported the isolation and structure elucida-
tion of renieramycin M (1m) in gram scale from the Thai sponge
Xestospongia sp. by pretreatment with potassium cyanide.25,26
The availability of 1m has enabled us to prepare and evaluate
new members of this class of compounds. We succeeded in semi-
synthetically preparing 2a from 1m via deangeloylrenieramycin
M (3a: named jorunnamycin A),27 which was isolated from the
mantles and egg ribbons of the KCN-pretreated Thai nudibranch
J. funebris.28 Preliminary biological evaluation of 2a and jorunnam-
ycins A (3a) and C (3b) revealed growth inhibition at micromolar
concentrations when tested with two human carcinoma cell lines
(HCT-116 and QG 56). Therefore, this preliminary biological evalu-
ation established the basis for conducting new investigations of
this class of antitumor marine alkaloids with focus on the effect
of the variation of the ester side chain at C-22.
Twenty-four ester analogues of 1m, along with 1m itself, cyano-
jorumycin (2b), 3a, and 3b, were tested for cytotoxicity to repre-
sentative human tumor cell lines, HCT116 colon carcinoma and
MDA-MB-453 breast carcinoma cell lines, at doses ranging from
100 nM to 1.4 nM. The results are shown in Table 1. 4a and 4b,
which possessed isomeric groups at the side chain, showed three-
fold lower cytotoxicity than 1m. We are very interested in 1m ana-
logues that possessed elongated aliphatic ester groups in the side
chain, because both 2b and 3b exhibited high cytotoxicity similar
to 1m. However, 6b–e possessing long aliphatic alkyl groups at
the side chain and 6a with the formyl ester side chain showed sig-
nificantly decreased cytotoxicity. Furthermore, 7a–e and 8a–e,
which possessed, respectively, a variety of aliphatic rings and a
variety of monosubstituted phenyl groups, also showed decreased
cytotoxicity. In contrast, nitrogen-containing heterocyclic ester
derivatives (9a–f) had similar activity to 1m. It is noteworthy that
20-pyridinecarboxylic acid ester derivative (9c) was approximately
threefold more cytotoxic than 1m. Although a general structure–
activity relationship of the ester analogues of 1m with the antican-
cer effect could not be summarized from these data, these data
show that the side chain of the ester group appears to be some-
what relevant for the biological activity.
4. Conclusion
Twenty-four analogues of 1m were prepared from readily avail-
able 3a and were evaluated in vitro for cytotoxic activities against
both human HCT116 colon carcinoma and MDA-MB-435 breast
carcinoma cell lines. For good activity, the hydroxyl group at C-
22 has to be acylated by a small group, such as an acetyl or a pro-
pionyl group, whereas a formyl group dramatically decreased the
cytotoxicity. Ester analogues having an aliphatic ring or a substi-
tuted phenyl group were also slightly less cytotoxic than 1m. In
contrast, it is advantageous to introduce a nitrogen-containing het-
erocyclic ring, such as pyridine or quinoline. Interestingly, 9c and
2b displayed a low nanomolar cytotoxicity profile. This is a some-
what unexpected result and is currently under investigation.
In this paper, we report significant results gained from the
extension of our initial investigation and the results of cytotoxicity
evaluation of the ester analogues at C-22.
2. Chemistry
Synthesis of the ester analogues at C-22 of 1m was accom-
plished by acylation of readily available compound 3a, which
was prepared in 45–54% yield through our previously reported

4550
K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
OCH3
E
OCH3
17
18
O
H
CH3
O
O
CH3
1) H2, 20% Pd(OH)2/C
EtOAc
16
O
A
O
5
H19
12
N
15
2) AlH3, THF
3) O2
H
N
4
H
D
N
CH3
CH3
10
O
6
7
20CH3
14
H
11
21
CH3
3
C
B
N2
8
9
CH3O
H
13
CH3O
1
45-54% yield
H
H
23
O
27
O
CN
OH
O
CN
22
CH3
25
3a
1m
26
O
24
H
CH3
28
OCH3
O
H
CH3
O
O
O
H
N
O
CH3
CH3O
RCOX, base
N
CH3
H
H
CN
R
O
4a,b, 6a-f, 7a-e, 8a-e, 9a-f
H
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
H
6b
6a
4a
6d
4b
6c
6e
Cl
6f
7e
7d
7b
7c
7a
OCH3
NO2
OCH3
Br
8a
8d
8b
8e
8c
N
N
N
N
N
N
9f
9a
9e
9b
9c
9d
Scheme 1. Preparation of renieramycin M analogues.
We have witnessed in recent years a number of dramatic devel-
opmentsin thepreparation of syntheticanalogues and their antipro-
liferative activities.32–37 Second-generation synthetic analogues 10a
and zalypsis (10b) were evaluated by a Harvard University group38
and Pharma Mar,39 respectively ( Fig. 4). Our findings show that
the potent antitumor activity of our analogue 9c is promising and
further effort to explore the therapeutic potential of other reniera-
mycin analogues is under way.
(5.98
(62.5
l
l
L, 0.04 mmol) and 2,4,6-trichlorobenzoyl chloride
L, 0.4 mmol) at 0 °C for 1 h. A solution of 3a (18.0 mg,
0.0365 mmol) with DMAP (0.89 mg, 7.3 mmol) in CH2Cl2
(1.4 mL) was added to the above mixed anhydride solution 5a
at 0 °C over 5 min. The reaction mixture was stirred at 45 °C
for 7 h and then poured into water (20 mL) and extracted with
chloroform (20 mL ꢁ 3). The combined extracts were washed
with brine, dried, and concentrated in vacuo to give a residue
(105.7 mg). Chromatography on a silica gel column with hex-
ane–ethyl acetate (5:1) as eluent gave 4a (8.0 mg, 38.1%) as a
5. Experimental
pale yellow amorphous powder. ½a D22
ꢀ 61:7 (c 0.17, CHCl3); IR
ꢂ
(KBr) 2928, 2360, 1706, 1655, 1618, 1448, 1375, 1236, 1149,
Optical rotations were measured with a Horiba—SEPA polarim-
eter. IR spectra were obtained with a Shimadzu Prestige 21/IRA
Affinity-1 FT-IR spectrometer. 1H and 13C NMR spectra were re-
corded at 500 MHz and 125 MHz, respectively, on a JEOL–JNM–
LA 500 FT NMR spectrometer (ppm, J in hertz with TMS as internal
standard). Mass spectra were recorded on a JEOL JMS 700 instru-
ment with a direct inlet system operating at 70 eV.
1080, 957, 883, 731 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 6.48
;
(1H, qq, J = 7.2, 1.5 Hz, 30-H), 4.58 (1H, dd, J = 12.3, 3.5 Hz, 22-
Ha), 4.04–3.96 (4H, overlapped, 21-H, 1-H, 11-H, 22-Hb), 4.02
(3H, s, OCH3), 3.99 (3H, s, OCH3), 3.39 (1H, br d, J = 7.7 Hz, 13-
H), 3.10 (1H, ddd, J = 11.6, 3.0, 2.8 Hz, 3-H), 2.91 (1H, dd,
J = 16.9, 2.8 Hz, 4-Ha), 2.74 (1H, dd, J = 21.1, 7.7 Hz, 14-Ha),
2.31 (1H, d, J = 21.1 Hz, 14-Hb), 2.27 (3H, s, NCH3), 1.96 (3H, s,
16-CH3), 1.93 (3H, s, 6-CH3), 1.66 (3H, dq, J = 7.2, 1,2 Hz, 30-
CH3), 1.58 (3H, dq, J = 1.5, 1.2 Hz, 20-CH3), 1.32 (1H, ddd,
J = 16.9, 11.6, 2.4 Hz, 4-Hb); FABMS m/z 576 (MH+); HRFABMS
m/z 576.2348 (MH+, calcd for C31H34N3O8, 576.2346).
5.1. Tiglic acid ester 4a
A solution of mixed anhydride 5a29 was prepared by dissolv-
ing tiglic acid (40 mg, 0.4 mmol) in CH2Cl2 (0.6 mL) with DBU

K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
4551
Table 1
Cytotoxicity to two carcinoma cell lines along with selected 1H NMR spectral data of renieramycins and related analogues
Entry
Compound
R
Cytotoxicity IC50 SD nM
1H NMR (CDCl3) d
CH3
OCH3
HCT116a
MDA-MB-435a
6
16
7
17
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1m
3a
2b
3b
4a
4b
6a
6b
6c
6db
6e
6f
7a
7b
7c
7d
7eb
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
COC(CH3)@CHCH3 (Renieramycin M: Z-form
H (Jorunnamycin A)
COCH3 Cyanojorumycin
COCH2CH3 Jorunnamycin C
COC(CH3)@CHCH3 (E-form)
COCH@C(CH3)2
CHO
COCH2CH2CH3
COCH(CH3)2
9.4 1.6
1.3 ꢁ 102 8.9
3.2 0.47
11 0.37
34 0.33
27 0.31
38 1.9
34 1.4
22 1.5
33 0.89
1.0 ꢁ 102 2.4
16 0.70
93 5.2
33 1.1
33 2.2
49 2.0
43 0.74
25 1.3
26 1.7
34 0.89
26 1.5
3.8 0.54
2.9 ꢁ 102 4.8
1.4 0.071
4.2 0.087
11 0.33
11 0.31
38 1.6
12 0.41
10 0.12
12 0.31
40 0.63
12 0.47
30 0.51
13 0.72
13 0.70
29 0.26
27 3.1
8.6 0.42
8.9 0.76
11 0.37
9.4 0.81
13 0.50
4.4 0.12
3.5 0.11
1.4 0.031
4.1 0.095
3.8 0.052
4.2 0.12
1.90
1.93
1.94
1.95
1.93
1.92
1.95
1.95
1.94
1.94
1.95
1.95
1.93
1.94
1.94
1.94
1.93
1.97
1.97
1.95
1.97
2.00
2.00
1.99
1.96
2.02
2.03
2.02
1.94
1.93
1.96
1.95
1.96
1.95
1.96
1.95
1.94
1.94
1.95
1.96
1.93
1.95
1.95
1.95
1.95
1.69
1.72
1.48
1.69
1.76
1.76
1.74
1.69
1.16
1.31
1.42
3.99
3.98
3.99
4.01
3.99
4.00
4.01
4.01
4.00
4.01
4.01
4.01
4.02
4.00
4.00
4.01
4.00
4.03
4.03
4.00
4.02
4.05
4.04
4.04
4.01
4.05
4.06
4.03
4.02
4.03
4.01
4.01
4.02
4.01
4.02
4.02
4.02
4.01
4.02
4.03
4.02
4.02
4.02
4.02
4.02
3.67
3.71
3.62
3.77
3.78
3.76
3.77
3.85
3.65
3.44
3.28
COCH(CH3)CH2CH3
COCH2CH2CH2CH2CH3
COCH2CH2Cl
CO-Cyclopentyl
CO-1-Cyclopentenyl
CO-3-Cyclopentenyl
CO-1-Cyclohexenyl
CO-3-Cyclohexenyl
COC6H5
COC6H4OCH3-4
COC6H4OCH3-2
COC6H4NO2-4
COC6H4Br-4
CO-4-Pyridyl
CO-3-Pyridyl
CO-2-Pyridyl
CO-4-Quinolynyl
CO-3-Quinolynyl
CO-2-Qunolynyl
38 1.2
10 0.78
7.9 1.1
3.3 0.15
10 0.33
10 0.35
11 0.14
a
HCT116: human colon carcinoma; MDA-MB-435: human breast carcinoma.
An inseparable 1:1 mixture of diastereomers.
b
brine, dried, and concentrated in vacuo to give a residue (24.4 mg).
Chromatography on a silica gel column with hexane–ethyl acetate
(5:1) as eluent gave 4b (4.7 mg, 80.6%) as a pale yellow amorphous
OCH3
OCH3
HO
H
CH3
HO
H
CH3
CH3O
HO
AcO
O
H
H
powder. ½a 2D2
ꢂ
ꢀ 61:6 (c 0.4, CHCl3); IR (CHCl3) 2928, 2854, 2228,
OCH3
CH3
CH3O
CH3
N
CH3
N
CH3
1715, 1652, 1615, 1456, 1417, 1374, 1261, 801 cmꢀ1
;
1H NMR
N
N
H
H
O
(CDCl3, 500 MHz) d 5.30 (1H, br s, 20-H), 4.53 (1H, dd, J = 11.4,
3.1 Hz, 22-Ha), 4.06 (1H, d, J = 2.3 Hz, 21-H), 4.01 (3H, s, OCH3),
4.01 (1H, overlapped, 11-H), 4.00 (3H, s, OCH3), 4.00 (1H, over-
lapped, 1-H), 3.90 (1H, dd, J = 11.4, 3.4 Hz, 22-Hb), 3.37 (1H, ddd,
J = 7.5, 2.3, 0.5 Hz, 13-H), 3.10 (1H, ddd, J = 11.6, 3.1, 2.5 Hz, 3-H),
H
H
CN
OH
NH
NH
O
CF3
O
O
O
O
2.91 (1H, dd, J = 17.1, 2.5 Hz, 4-H
a), 2.73 (1H, dd, J = 21.0, 7.5 Hz,
Zalypsis (10b)
10a
14-H ), 2.30 (1H, d, J = 21.0 Hz, 14-Hb), 2.27 (3H, s, NCH3), 2.01
a
(3H, s, 40-H3), 1.95 (3H, s, 16-CH3), 1.92 (3H, s, 6-CH3), 1.81 (3H,
s, 30-CH3), 1.34 (1H, ddd, J = 17.1, 11.6, 2.4 Hz, 4-Hb), 0.82 (3H, t,
J = 7.3 Hz, 40-H3); 13C NMR (CDCl3, 125 MHz) d 185.9 (C-15),
185.5 (C-5), 182.5 (C-18), 181.0 (C-8), 165.4 (OCOR), 158.7 (C-30),
155.6 (C-7), 155.2 (C-17), 142.1 (C-20), 141.8 (C-10), 135.7 (C-9),
134.9 (C-19), 128.5 (C-6), 128.4 (C-16), 117.0 (CN), 114.8 (C-20),
62.1 (C-22), 61.1 (OCH3), 61.0 (OCH3), 58.8 (C-21), 56.1 (C-1),
54.6 (C-13), 54.4 (C-3), 54.4 (C-11), 41.5 (NCH3), 27.4 (30-CH3),
25.4 (C-4), 21.1 (C-14), 20.2 (C-40), 8.7 (16-CH3), 8.7 (6-CH3); EIMS
m/z (% intensity) 575 (M+, 8), 464 (5), 260 (9), 220 (100), 218 (24),
204 (10), 176 (6); HREIMS m/z 575.2268 (M+, calcd for C31H33N3O8,
575.2268).
Figure 4. Structures of second-generation synthetic analogues.
O
O
Cl
H
CH3
O
O
CH3
O
CH3
O
O
R
CH3
Cl
5a
Cl
5b: R = CH=C(CH3)2
5c: 3-cyclopentenyl
5d: 3-cyclohexenyl
5e: 2-pyridinyl
Figure 5. Structures of mixed anhydrides.
5.3. Formic acid ester 6a
5.2. 3,3-Dimethylacryloyl ester 4b
A CH2Cl2 solution of acetic formic anhydride31 (1.8 M, 66.7
lL,
Carbonate 5b30 (27.3 mg, 0.147 mmol) was added to a stirred
solution of 3a (5.0 mg, 0.01 mmol) in pyridine (0.2 mL) at ꢀ17 °C,
and the resulting solution was stirred at ꢀ17 °C for 1 h. The reac-
tion mixture was poured into water (20 mL) and extracted with
chloroform (20 mL ꢁ 3). The combined extracts were washed with
0.12 mmol) was added to a stirred solution of 3a (24.7 mg,
0.05 mmol) in pyridine (1.0 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at 0 °C for 24 h. The reaction mixture was diluted
with water (20 mL) and extracted with chloroform (20 mL ꢁ 3).
The combined extracts were washed with aqueous saturated NaH-

4552
K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
CO3 solution, dried, and concentrated in vacuo to give a residue
(27.5 mg). Chromatography on a silica gel column with hexane–
ethyl acetate (5:1) as eluent gave 6a (11.8 mg, 45.2%) as a pale yel-
2854, 2228, 1732, 1653, 1455, 1411, 1374, 1261, 1190, 1081, 956,
769 cmꢀ1 1H NMR (CDCl3, 500 MHz) d 4.30 (1H, dd, J = 11.6,
2.7 Hz, 22-Ha), 4.07 (1H, d, J = 2.1 Hz, 21-H), 4.06 (1H, dd,
J = 11.6, 3.6 Hz, 22-Hb), 4.04 (1H, overlapped, 11-H), 4.02 (3H, s,
OCH3), 4.01 (1H, br s, 1-H), 4.00 (3H, s, OCH3), 3.40 (1H, ddd,
J = 7.6, 2.1, 0.5 Hz, 13-H), 3.10 (1H, ddd, J = 11.5, 3.1, 2.8 Hz, 3-H),
;
low solid. ½a 2D2
ꢀ 92:2 (c 0.4, CHCl3); IR (CHCl3) 2944, 2852, 2229,
ꢂ
1724, 1654, 1619, 1451, 1374, 1310, 1234, 1079, 1021, 756 cmꢀ1
;
1H NMR (CDCl3, 500 MHz) d 7.80 (1H, s, HCO), 4.47 (1H, dd,
J = 11.3, 2.9 Hz, 22-Ha), 4.05 (1H, d, J = 2.3 Hz, 21-H), 4.02 (3H, s,
OCH3), 4.02 (1H, overlapped, 1-H), 4.01 (1H, overlapped, 11-H),
4.01 (3H, s, OCH3), 3.96 (1H, dd, J = 11.3, 4.3 Hz, 22-Hb), 3.38 (1H,
ddd, J = 7.6, 2.3, 1.7 Hz, 13-H), 3.12 (1H, ddd, J = 11.6, 3.1, 2.6 Hz,
2.91 (1H, dd, J = 17.4, 2.8 Hz, 4-H
a), 2.78 (1H, dd, J = 20.8, 7.6 Hz,
14-Ha
), 2.31 (1H, d, J = 20.8 Hz, 14-Hb), 2.30 (1H, overlapped, 20-
H), 2.29 (3H, s, NCH3), 1.94 (3H, s, 16-CH3), 1.94 (3H, s, 6-CH3),
1.35 (1H, ddd, J = 17.4, 11.5, 2.4 Hz, 4-Hb), 0.95 (3H, d, J = 7.0 Hz,
30-H3), 0.93 (3H, d, J = 7.0 Hz, 20-CH3); 13C NMR (CDCl3, 125 MHz)
d 186.3 (C-15), 185.4 (C-5), 182.5 (C-18), 181.0 (C-8), 176.0 (OCOR),
155.7 (C-7), 155.3 (C-17), 142.0 (C-20), 141.4 (C-10), 135.5 (C-9),
135.1 (C-19), 128.7 (C-6), 128.4 (C-16), 116.8 (CN), 63.3 (C-22),
61.1 (OCH3), 61.0 (OCH3), 58.8 (C-21), 56.2 (C-1), 54.6 (C-13),
54.4 (C-3), 54.2 (C-11), 41.5 (NCH3), 33.8 (C-20), 25.4 (C-4), 21.2
(C-14), 19.0 (C-30), 18.5 (20-CH3), 8.7 (16-CH3), 8.6 (6-CH3); EIMS
m/z (% intensity) 563 (M+, 7), 464 (4), 435 (3), 260 (15), 243 (5),
220 (100), 218 (20), 204 (9), 176 (5); HREIMS m/z 563.2273 (M+,
calcd for C30H33N3O8, 563.2268).
3-H), 2.94 (1H, dd, J = 17.2, 2.6 Hz, 4-H
a), 2.77 (1H, dd, J = 21.1,
7.6 Hz, 14-H ), 2.31 (3H, s, NCH3), 2.25 (1H, d, J = 21.1 Hz, 14-
a
Hb), 1.96 (3H, s, 16-CH3), 1.95 (3H, s, 6-CH3), 1.36 (1H, ddd,
J = 17.2, 11.6, 2.4 Hz, 4-Hb); 13C NMR (CDCl3, 125 MHz) d 186.2
(C-15), 185.3 (C-5), 182.5 (C-18), 181.0 (C-8), 159.8 (HCO), 155.5
(C-7), 155.2 (C-17), 142.2 (C-20), 142.1 (C-10), 135.0 (C-9), 134.9
(C-19), 128.8 (C-6), 128.7 (C-16), 116.8 (CN), 63.5 (C-22), 61.1
(OCH3), 61.1 (OCH3), 59.2 (C-21), 55.6 (C-1), 54.6 (C-13), 54.6 (C-
3), 54.3 (C-11), 41.5 (NCH3), 25.2 (C-4), 21.3 (C-14), 8.8 (6-CH3),
8.7 (16-CH3); EIMS m/z (% intensity) 521 (M+, 10), 495 (0.5), 462
(2), 435 (3), 260 (9), 243 (7), 220 (100), 218 (25), 204 (14), 176
(10); HREIMS m/z 521.1792 (M+, calcd for C27H27N3O8, 521.1798).
5.6. 20-(R/S)-Methylbutanoic Acid Ester 6d
5.4. Butyric acid ester 6b
( )-2-Methylbutyryl chloride (18.4 lL, 0.15 mmol) was added
to a stirred solution of 3a (14.8 mg, 0.03 mmol) and DMAP
(0.73 mg, 0.006 mmol) in dry CH2Cl2 (3 mL) at 0 °C, and the result-
ing solution was stirred at 0 °C for 3 h. The reaction mixture was
poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (19.6 mg). Chromatog-
raphy on a silica gel column with hexane–ethyl acetate (5:1) as
eluent gave an inseparable mixture of 6d (7.8 mg, 40.1%) as a pale
Butyric anhydride (24.0 lL, 0.147 mmol) was added to a stirred
solution of 3a (5.0 mg, 0.01 mmol) in pyridine (0.2 mL) at ꢀ17 °C,
and the resulting solution was stirred at ꢀ17 °C for 5 h. The reac-
tion mixture was poured into water (20 mL) and extracted with
chloroform (20 mL ꢁ 3). The combined extracts were washed with
brine, dried, and concentrated in vacuo to give a residue (12.2 mg).
Chromatography on a silica gel column with hexane–ethyl acetate
(5:1) as eluent gave 6b (3.7 mg, 64.8%) as a pale yellow amorphous
yellow amorphous powder. ½a D22
ꢀ 79:1 (c 0.7, CHCl3); IR (CHCl3)
ꢂ
powder. ½a 2D2
ꢂ
ꢀ 79:9 (c 0.4, CHCl3); IR (CHCl3) 2935, 2854, 2221,
3445, 3281, 2964, 2855, 2229, 1732, 1652, 1615, 1463, 1410,
1738, 1654, 1615, 1456, 1412, 1373, 1235, 757 cmꢀ1
;
1H NMR
1373, 1235, 1802 cmꢀ1 1H NMR (CDCl3, 500 MHz) one isomer d
;
(CDCl3, 500 MHz) d 4.39 (1H, dd, J = 11.5, 3.1 Hz, 22-Ha), 4.06
(1H, d, J = 2.4 Hz, 21-H), 4.02 (3H, s, OCH3), 4.01 (1H, overlapped,
11-H), 4.01 (3H, s, OCH3), 3.99 (1H, br s, 1-H), 3.93 (1H, dd,
J = 11.5, 3.6 Hz, 22-Hb), 3.38 (1H, br d, J = 7.3 Hz, 13-H), 3.10 (1H,
ddd, J = 11.6, 3.1, 2.7 Hz, 3-H), 2.93 (1H, dd, J = 17.4, 2.7 Hz, 4-
4.24 (1H, dd, J = 11.6, 2.7 Hz, 22-Ha), 4.12 (1H, dd, J = 11.6,
2.7 Hz, 22-Hb), 4.08 (1H, d, J = 2.7 Hz, 21-H), 4.03 (1H, br d,
J = 2.7 Hz, 11-H), 4.01 (3H, s, OCH3), 4.01 (3H, s, OCH3), 4.00 (1H,
overlapped, 1-H), 3.41 (1H, dd, J = 7.3, 1.2 Hz, 13-H), 3.11 (1H,
ddd, J = 11.4, 2.7, 2.5 Hz, 3-H), 2.90 (1H, dd, J = 17.1, 2.5 Hz, 4-
H
a
), 2.76 (1H, dd, J = 21.0, 7.3 Hz, 14-H
a
), 2.31 (1H, d, J = 21.0 Hz,
Ha), 2.78 (1H, dd, J = 21.0, 7.6 Hz, 14-Ha), 2.30 (1H, dd, J = 21.0,
14-Hb), 2.29 (3H, s, NCH3), 2.01 (2H, t, J = 7.3 Hz, 20-H2), 1.95 (3H,
s, 16-CH3), 1.95 (3H, s, 6-CH3), 1.42 (2H, sept, J = 7.3 Hz, 30-H2),
1.32 (1H, ddd, J = 17.4, 11.6, 2.4 Hz, 4-Hb), 0.82 (3H, t, J = 7.3 Hz,
40-H3); 13C NMR (CDCl3, 125 MHz) d 186.1 (C-15), 185.4 (C-5),
182.5 (C-18), 180.9 (C-8), 172.6 (OCOR), 155.6 (C-7), 155.2 (C-
17), 142.1 (C-20), 141.7 (C-10), 135.5 (C-9), 135.0 (C-19), 128.6
(C-6), 128.5 (C-16), 116.9 (CN), 63.3 (C-22), 61.1 (OCH3), 61.0
(OCH3), 58.9 (C-21), 56.0 (C-1), 54.6 (C-13), 54.4 (C-3), 54.3 (C-
11), 41.5 (NCH3), 36.0 (C-20), 25.4 (C-4), 21.2 (C-14), 18.3 (C-30),
13.6 (C-40), 8.8 (16-CH3), 8.6 (6-CH3); EIMS m/z (% intensity) 563
(M+, 7), 462 (3), 435 (4), 243 (13), 220 (100), 218 (23), 204 (11),
176 (6); HREIMS m/z 563.2271 (M+, calcd for C30H33N3O8,
563.2268).
1.2 Hz, 14-Hb), 2.29 (3H, s, NCH3), 2.09 (1H, m, 20-H), 1.94 (3H, s,
16-CH3), 1.94 (3H, s, 6-CH3), 1.35 (1H, ddd, J = 17.1, 11.4, 2.7 Hz,
4-Hb), 1.30 (2H, overlapped, 30-H2), 0.89 (3H, d, J = 7.0 Hz, 20-
CH3), 0.88 (3H, t, J = 7.2 Hz, 40-H3); other isomer d 4.27 (1H, dd,
J = 11.6, 2.7 Hz, 22-Ha), 4.12 (1H, dd, J = 11.6, 2.7 Hz, 22-Hb), 4.08
(1H, d, J = 2.7 Hz, 21-H), 4.03 (1H, br d, J = 2.4 Hz, 11-H), 4.01
(3H, s, OCH3), 4.01 (3H, s, OCH3), 4.00 (1H, overlapped, 1-H), 3.41
(1H, dd, J = 7.3, 1.2 Hz, 13-H), 3.11 (1H, ddd, J = 11.5, 3.1, 2.7 Hz,
3-H), 2.90 (1H, dd, J = 17.1, 2.4 Hz, 4-Ha), 2.78 (1H, dd, J = 21.0,
7.6 Hz, 14-H ), 2.30 (1H, dd, J = 21.0, 1.2 Hz, 14-Hb), 2.29 (3H, s,
a
NCH3), 2.09 (1H, m, 20-H), 1.94 (3H, s, 16-CH3), 1.94 (3H, s, 6-
CH3), 1.35 (1H, ddd, J = 17.1, 11.3, 2.7 Hz, 4-Hb), 1.27 (2H, m, 30-
H2), 0.91 (3H, d, J = 7.0 Hz, 20-CH3), 0.74 (3H, t, J = 7.2 Hz, 40-H3);
13C NMR (CDCl3, 125 MHz) one isomer d 186.2 (C-15), 185.4 (C-5),
182.5 (C-18), 180.9 (C-8), 175.4 (OCOR), 155.8 (C-7), 155.3 (C-
17), 142.0 (C-20), 141.3 (C-10), 135.5 (C-9), 135.2 (C-19), 128.6
(C-6), 128.3 (C-16), 116.8 (CN), 63.4 (C-22), 61.0 (OCH3), 61.0
(OCH3), 58.8 (C-21), 56.3 (C-1), 54.6 (C-13), 54.3 (C-3), 54.2 (C-
11), 41.5 (NCH3), 40.8 (C-20), 26.6 (C-30), 25.4 (C-4), 21.2 (C-14),
16.5 (20-CH3), 14.1 (C-40), 8.7 (16-CH3), 8.6 (6-CH3); other isomer
d 186.2 (C-15), 185.4 (C-5), 182.5 (C-18), 180.9 (C-8), 175.7 (OCOR),
155.8 (C-7), 155.3 (C-17), 142.0 (C-20), 141.3 (C-10), 135.5 (C-9),
135.1 (C-19), 128.6 (C-6), 128.3 (C-16), 116.8 (CN), 63.2 (C-22),
61.0 (OCH3), 61.0 (OCH3), 58.8 (C-21), 56.2 (C-1), 54.6 (C-13),
54.3 (C-3), 54.2 (C-11), 41.5 (NCH3), 40.8 (C-20), 26.2 (C-30), 25.4
5.5. 20-Methylpropanoic acid ester 6c
Isobutyric anhydride (21.9 lL, 0.735 mmol) was added to a stir-
red solution of 3a (24.7 mg, 0.05 mmol) in pyridine (0.9 mL) at
ꢀ17 °C, and the resulting solution was stirred at ꢀ17 °C for 5 h.
The reaction mixture was poured into water (20 mL) and extracted
with chloroform (20 mL ꢁ 3). The combined extracts were washed
with brine, dried, and concentrated in vacuo to give a residue
(199.5 mg). Chromatography on a silica gel column with hexane–
ethyl acetate (5:1) as eluent gave 6c (12.7 mg, 44.1%) as a pale yel-
low amorphous powder. ½a D22
ꢀ 67:0 (c 0.4, CHCl3); IR (CHCl3) 2963,
ꢂ

K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
4553
(C-4), 21.2 (C-14), 16.5 (20-CH3), 14.1 (C-40), 8.7 (16-CH3), 8.6 (6-
CH3); EIMS m/z (% intensity) 577 (M+, 10), 4642 (3), 435 (2), 260
(16), 243 (6), 220 (100), 218 (24), 204 (10), 176 (6); HREIMS m/z
577.2424 (M+, calcd for C31H35N3O8, 577.2424).
CH3); EIMS m/z (% intensity) 583 (M+, 3), 547 (5), 462 (3), 435
(1), 260 (7), 220 (100), 218 (24), 204 (10), 176 (6); HREIMS m/z
583.1728 (M+, calcd for C29H30ClN3O8, 583.1722).
5.9. Cyclopentanecarboxylic acid ester 7a
5.7. Hexanoic acid ester 6e
Cyclopentanecarbonyl chloride (35.7 lL, 0.294 mmol) was
Hexanoic anhydride (69.0
l
L, 0.298 mmol) was added to a stir-
added to a stirred solution of 3a (9.9 mg, 0.02 mmol) and DMAP
(0.24 mg, 0.002 mmol) in pyridine (0.4 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 1 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (41.5 mg). Chromatog-
raphy on a silica gel column with hexane–ethyl acetate (5:1) as
eluent gave 7a (9.8 mg, 78.8%) as a pale yellow amorphous powder.
red solution of 3a (9.9 mg, 0.02 mmol) and DMAP (0.24 mg,
0.002 mmol) in pyridine (0.4 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 1.5 h. The reaction mixture was
poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (68.8 mg). Chromatog-
raphy on a silica gel column with hexane–ethyl acetate (5:1) as
eluent gave 6e (7.0 mg, 58.4%) as a pale yellow amorphous powder.
½
a 2D2
ꢂ
ꢀ 66:6 (c 0.4, CHCl3); IR (CHCl3) 3444, 3277, 2960, 2855, 2228,
½
a 2D2
ꢂ
ꢀ 84:7 (c 0.5, CHCl3); IR (CHCl3) 2932, 2857, 2229, 1738, 1652,
1732, 1652, 1615, 1455, 1411, 1373, 1261, 802 cmꢀ1 1H NMR
;
1615, 1456, 1417, 1373, 1235, 803 cmꢀ1 1H NMR (CDCl3,
;
(CDCl3, 500 MHz) d 4.30 (1H, dd, J = 11.3, 2.7 Hz, 22-Ha), 4.08
(1H, d, J = 2.1 Hz, 21-H), 4.07 (1H, overlapped, 11-H), 4.02 (3H, s,
OCH3), 4.02 (3H, s, OCH3), 4.02 (1H, overlapped, 22-Hb), 3.99
(1H, br s, 1-H), 3.40 (1H, ddd, J = 7.3, 2.1, 0.6 Hz, 13-H), 3.10 (1H,
ddd, J = 11.6, 3.1, 2.5 Hz, 3-H), 2.91 (1H, dd, J = 17.1, 2.5 Hz, 4-
500 MHz) d 4.38 (1H, dd, J = 11.6, 3.1 Hz, 22-Ha), 4.06 (1H, d,
J = 2.0 Hz, 21-H), 4.02 (1H, overlapped, 11-H), 4.02 (3H, s, OCH3),
4.01 (3H, s, OCH3), 3.99 (1H, br s, 1-H), 3.91 (1H, dd, J = 11.6,
4.0 Hz, 22-Hb), 3.38 (1H, ddd, J = 7.3, 2.0, 0.6 Hz, 13-H), 3.10 (1H,
ddd, J = 11.6, 3.1, 2.7 Hz, 3-H), 2.93 (1H, dd, J = 17.1, 2.7 Hz, 4-
Ha), 2.78 (1H, dd, J = 20.8, 7.5 Hz, 14-Ha), 2.43 (1H, quintet,
H
a
), 2.76 (1H, dd, J = 20.7, 7.3 Hz, 14-H
a
), 2.31 (1H, d, J = 20.7 Hz,
J = 7.5 Hz, 10-H), 2.30 (1H, d, J = 20.8 Hz, 14-Hb), 2.29 (3H, s,
NCH3), 1.93 (3H, s, 16-CH3), 1.93 (3H, s, 6-CH3), 1.66 (2H, m),
1.65 (2H, m), 1.52 (1H, m), 1.50 (2H, m), 1.46 (1H, m), 1.35 (H,
ddd, J = 17.1, 11.6, 2.6 Hz, 4-Hb); 13C NMR (CDCl3, 125 MHz) d
186.2 (C-15), 185.4 (C-5), 182.5 (C-18), 180.9 (C-8), 175.7 (OCOR),
155.7 (C-7), 155.3 (C-17), 142.1 (C-20), 141.4 (C-10), 135.5 (C-9),
135.1 (C-19), 128.6 (C-6), 128.3 (C-16), 116.8 (CN), 63.3 (C-22),
61.1 (OCH3), 61.0 (OCH3), 58.8 (C-21), 56.3 (C-1), 54.6 (C-13),
54.4 (C-3), 54.2 (C-11), 43.8 (C-10), 41.5 (NCH3), 30.1 (C-20), 29.7
(C-50), 25.6 (C-30), 25.5 (C-40), 25.4 (C-4), 21.2 (C-14), 8.7 (16-
CH3), 8.6 (6-CH3); EIMS m/z (% intensity) 589 (M+, 9), 462 (4),
260 (11), 243 (47), 220 (100), 218 (24), 204 (12); HREIMS m/z
589.2427 (M+, calcd for C32H35N3O8, 589.2424).
14-Hb), 2.29 (3H, s, NCH3), 2.02 (2H, t, J = 8.3 Hz, 20-H2), 1.95 (3H,
s, 16-CH3), 1.95 (3H, s, 6-CH3), 1.40 (2H, sextet, J = 8.3 Hz, 30-H2),
1.31 (H, ddd, J = 17.1, 11.6, 2.4 Hz, 4-Hb), 1.23 (2H, m, 50-H2),
1.16 (2H, m, 40-H), 0.85 (3H, t, J = 7.3 Hz, 60-H3); 13C NMR (CDCl3,
125 MHz) d 186.1 (C-15), 185.4 (C-5), 182.5 (C-18), 180.9 (C-8),
172.8 (OCOR), 155.6 (C-7), 155.2 (C-17), 142.2 (C-20), 141.7 (C-
10), 135.5 (C-9), 135.0 (C-19), 128.6 (C-6), 128.5 (C-16), 116.9
(CN), 63.5 (C-22), 61.1 (OCH3), 61.0 (OCH3), 59.0 (C-21), 55.9 (C-
1), 54.6 (C-13), 54.4 (C-3), 54.3 (C-11), 41.5 (NCH3), 34.0 (C-20),
31.2 (C-40), 25.3 (C-4), 24.4 (C-30), 22.5 (C-50), 21.2 (C-14), 13.8
(C-60), 8.8 (16-CH3), 8.6 (6-CH3); EIMS m/z (% intensity) 591 (M+,
5), 464 (3), 435 (3), 260 (10), 243 (100), 220 (69), 204 (10); HREIMS
m/z 591.2578 (M+, calcd for C32H37N3O8, 591.2581).
5.10. 10-Cyclopentenecarboxylic acid ester 7b
5.8. 30-Chloropropanoic acid ester 6f
1-Cyclopentenecarbonyl chloride (57.6 mg, 0.440 mmol) was
added to a stirred solution of 3a (14.8 mg, 0.03 mmol) and DMAP
(0.37 mg, 0.003 mmol) in pyridine (0.6 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 2.5 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (66.4 mg). Chromatog-
raphy on a silica gel column with hexane–ethyl acetate (5:1) as
eluent gave 7b (11.9 mg, 60.5%) as a pale yellow amorphous pow-
3-Chloropropanoyl chloride (105.2 lL, 1.10 mmol) was added
to a stirred solution of 3a (37.0 mg, 0.075 mmol) and DMAP
(0.92 mg, 0.0075 mmol) in pyridine (1.5 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 3 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (170.7 mg). Chroma-
tography on a silica gel column with hexane–ethyl acetate (5:1)
as eluent gave 6f (13.4 mg, 29.8%) as a pale yellow amorphous
der. ½a 2D2
ꢂ
ꢀ 75:5 (c 0.4, CHCl3); IR (CHCl3) 3443, 3279, 2961, 2854,
powder. ½a 2D2
ꢂ
ꢀ 79:6 (c 0.7, CHCl3); IR (CHCl3) 3446, 2944, 2853,
2228, 1714, 1652, 1447, 1373, 1235, 801 cmꢀ1 1H NMR (CDCl3,
;
2229, 1737, 1654, 1449, 1417, 1374, 1311, 1235, 1150, 1080,
500 MHz) d 6.75 (1H, dt, J = 6.4, 2.1 Hz, 20-H), 4.55 (1H, dd,
J = 11.6, 2.7 Hz, 22-Ha), 4.05 (1H, d, J = 2.0 Hz, 21-H), 4.02 (1H,
overlapped, 1-H), 4.02 (3H, s, OCH3), 4.01 (1H, overlapped, 22-
Hb), 4.00 (3H, s, OCH3), 4.00 (1H, overlapped, 11-H), 3.38 (1H,
ddd, J = 7.6, 2.0, 0.6 Hz, 13-H), 3.10 (1H, ddd, J = 11.3, 3.1, 2.7 Hz,
966, 767 cmꢀ1 1H NMR (CDCl3, 500 MHz) d 4.39 (1H, dd, J = 11.6,
;
3.1 Hz, 22-Ha), 4.10 (1H, dd, J = 11.6, 3.7 Hz, 22-Hb), 4.07 (1H, d,
J = 3.1 Hz, 21-H), 4.03 (1H, overlapped, 11-H), 4.03 (3H, s, OCH3),
4.01 (3H, s, OCH3), 4.01 (1H, br s, 1-H), 3.53 (2H, m, 30-H2), 3.40
(1H, ddd, J = 7.4, 1.8, 0.5 Hz, 13-H), 3.11 (1H, ddd, J = 11.6, 3.1,
3-H), 2.91 (1H, dd, J = 17.4, 2.7 Hz, 4-H
a), 2.74 (1H, dd, J = 21.0,
2.5 Hz, 3-H), 2.92 (1H, dd, J = 17.4, 2.5 Hz, 4-H
a
), 2.76 (1H, dd,
7.6 Hz, 14-H
a
), 2.38 (2H, m, 30-H2), 2.31 (1H, d, J = 21.0 Hz, 14-
J = 21.0, 7.4 Hz, 14-H
a
), 2.53 (2H, m, 20-H2), 2.31 (1H, d,
Hb), 2.27 (3H, s, NCH3), 2.27 (2H, overlapped, 50-H2), 1.95 (3H, s,
16-CH3), 1.94 (3H, s, 6-CH3), 1.84 (2H, quintet, J = 7.6 Hz, 40-H2),
1.35 (H, ddd, J = 17.4, 11.3, 2.1 Hz, 4-Hb); 13C NMR (CDCl3,
125 MHz) d 186.0 (C-15), 185.5 (C-5), 182.5 (C-18), 181.0 (C-8),
164.1 (OCOR), 155.7 (C-7), 155.1 (C-17), 144.7 (C-20), 142.2 (C-
20), 141.8 (C-10), 135.8 (C-9), 135.5 (C-19), 134.8 (C-10), 128.5
(C-6), 128.4 (C-16), 117.0 (CN), 62.3 (C-22), 61.1 (OCH3), 61.0
(OCH3), 58.7 (C-21), 56.4 (C-1), 54.6 (C-13), 54.4 (C-3), 54.3 (C-
11), 43.0 (C-10), 41.5 (NCH3), 33.3 (C-30), 31.2 (C-50), 25.4 (C-4),
J = 21.0 Hz, 14-Hb), 2.30 (3H, s, NCH3), 1.96 (3H, s, 16-CH3), 1.95
(3H, s, 6-CH3), 1.37 (H, ddd, J = 17.4, 11.6, 2.6 Hz, 4-Hb); 13C NMR
(CDCl3, 125 MHz) d 186.3 (C-15), 185.3 (C-5), 182.4 (C-18), 181.0
(C-8), 169.5 (OCOR), 155.6 (C-7), 155.4 (C-17), 142.0 (C-20),
141.8 (C-10), 135.1 (C-9), 135.1 (C-19), 128.7 (C-6), 128.5 (C-16),
116.8 (CN), 63.8 (C-22), 61.1 (OCH3), 61.1 (OCH3), 58.8 (C-21),
55.9 (C-1), 54.6 (C-13), 54.4 (C-3), 54.2 (C-11), 41.5 (NCH3), 38.5
(C-30), 37.4 (C-20), 25.4 (C-4), 21.2 (C-14), 8.8 (16-CH3), 8.6 (6-

4554
K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
22.9 (C-40), 21.1 (C-14), 8.8 (16-CH3), 8.7 (6-CH3); EIMS m/z (%
intensity) 587 (M+, 10), 462 (4), 260 (10), 220 (100), 218 (24),
204 (10), 176 (6); HREIMS m/z 587.2267 (M+, calcd for
C32H33N3O8, 587.2268).
(C-30), 25.4 (C-4), 24.0 (C-60), 21.2 (C-50), 21.1 (C-14), 8.8 (16-
CH3), 8.8 (6-CH3); EIMS m/z (% intensity) 601 (M+, 10), 464 (6),
260 (12), 220 (100), 218 (27), 204 (12), 176 (8); HREIMS m/z
601.2427 (M+, calcd for C33H35N3O8, 601.2424).
5.11. 30-Cyclopentenecarboxylic acid ester 7c
5.13. (R/S)-30-Cyclohexenecarboxylic acid ester 7e
Carbonate 5c30 (87.4 mg, 0.440 mmol) was added to a stirred
solution of 3a (14.8 mg, 0.03 mmol) and DMAP (0.37 mg,
0.003 mmol) in pyridine (0.6 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 1 h. The reaction mixture was poured
into water (20 mL) and extracted with chloroform (20 mL ꢁ 3). The
combined extracts were washed with brine, dried, and concen-
trated in vacuo to give a residue (84.4 mg). Chromatography on a
silica gel column with hexane–ethyl acetate (5:1) as eluent gave
Carbonate 5d30 (31.2 mg, 0.147 mmol) was added to a stirred
solution of 3a (5.0 mg, 0.01 mmol) and DMAP (0.12 mg,
0.001 mmol) in pyridine (0.2 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 1 h. The reaction mixture was poured
into water (20 mL) and extracted with chloroform (20 mL ꢁ 3). The
combined extracts were washed with brine, dried, and concen-
trated in vacuo to give a residue (53.4 mg). Chromatography on a
silica gel column with hexane–ethyl acetate (5:1) as eluent gave
an inseparable mixture of 7e (5.0 mg, 80.4%) as a pale yellow amor-
7c (12.5 mg, 64.4%) as
ꢀ 82:5 (c 0.4, CHCl3); IR (CHCl3) 2942, 2855, 2228, 1732,
1652, 1615, 1456, 1410, 1374, 1261, 802 cmꢀ1 1H NMR (CDCl3,
a pale yellow amorphous powder.
½
a 2D2
ꢂ
phous powder. ½a D22
ꢂ
ꢀ 68:8 (c 0.4, CHCl3); IR (CHCl3) 2929, 2841,
;
2229, 1731, 1646, 1613, 1451, 1374, 1233, 798 cmꢀ1 1H NMR
;
500 MHz) d 5.53 (2H, m, 30-H and 40-H), 4.36 (1H, dd, J = 11.6,
2.7 Hz, 22-Ha), 4.08 (1H, d, J = 2.1 Hz, 21-H), 4.05 (1H, dd,
J = 11.6, 3.7 Hz, 22-Hb), 4.02 (1H, overlapped, 11-H), 4.02 (3H, s,
OCH3), 4.00 (3H, s, OCH3), 4.00 (1H, overlapped, 1-H), 3.40 (1H,
ddd, J = 7.4, 2.1, 0.6 Hz, 13-H), 3.10 (1H, ddd, J = 11.3, 3.1, 2.5 Hz,
(CDCl3, 500 MHz) one isomer d 5.78 (2H, br s, 30-H, 40-H), 4.42
(1H, dd, J = 11.6, 2.7 Hz, 22-Ha), 4.08 (1H, br s, 21-H), 4.07 (1H,
dd, J = 11.6, 3.1 Hz, 22-Hb), 4.02 (3H, s, OCH3), 4.01 (1H, over-
lapped, 11-H), 4.01 (3H, s, OCH3), 4.00 (1H, overlapped, 1-H),
3.40 (1H, ddd, J = 7.6, 1.8, 0.6 Hz, 13-H), 3.10 (1H, ddd, J = 11.6,
3-H), 2.91 (1H, dd, J = 17.3, 2.5 Hz, 4-H
J = 7.0 Hz, 10-H), 2.81 (2H, t, J = 7.0 Hz, 2.74 (1H, dd, J = 21.0,
7.4 Hz, 14-H
), 2.41 (2H, m, 30-H2), 2.31 (1H, d, J = 21.0 Hz, 14-
a
), 2.84 (1H, quintet,
3.1, 2.7 Hz, 3-H), 2.90 (1H, dd, J = 17.1, 2.7 Hz, 4-H
a), 2.77 (1H,
dd, J = 21.0, 7.6 Hz, 14-H ), 2.31 (1H, d, J = 21.0 Hz, 14-Hb), 2.28
a
a
(3H, s, NCH3), 2.26 (1H, m, 10-H), 2.04 (1H, m, 20-H), 2.01 (1H, m,
60-H), 1.95 (3H, s, 16-CH3), 1.94 (1H, m, 20-H), 1.93 (3H, s, 6-CH3),
1.87 (1H, m, 60-H), 1.35 (2H, m, 50-H2), 1.34 (H, ddd, J = 17.1,
11.6, 2.7 Hz, 4-Hb); other isomer d 5.60 (1H, m, 30-H), 5.51 (1H,
m, 40-H), 4.35 (1H, dd, J = 11.6, 2.7 Hz, 22-Ha), 4.13 (1H, dd,
J = 11.6, 3.1 Hz, 22-Hb), 4.08 (1H, d, J = 2.2 Hz, 21-H), 4.02 (3H, s,
OCH3), 4.01 (1H, overlapped, 11-H), 4.00 (3H, s, OCH3), 4.00 (1H,
overlapped, 1-H), 3.40 (1H, ddd, J = 7.6, 2.2, 0.6 Hz, 13-H), 3.10
(1H, ddd, J = 11.6, 3.1, 2.7 Hz, 3-H), 2.90 (1H, dd, J = 17.1, 2.7 Hz,
Hb), 2.28 (3H, s, NCH3), 1.95 (3H, s, 16-CH3), 1.94 (3H, s, 6-CH3),
1.35 (H, ddd, J = 17.3, 11.3, 2.7 Hz, 4-Hb); 13C NMR (CDCl3,
125 MHz) d 186.2 (C-15), 185.4 (C-5), 182.5 (C-18), 180.9 (C-8),
175.0 (OCOR), 155.7 (C-7), 155.2 (C-17), 142.1 (C-20), 141.6 (C-
10), 135.4 (C-9), 135.0 (C-19), 129.1 (C-30), 128.6 (C-40), 128.4 (C-
6), 128.4 (C-16), 116.8 (CN), 63.5 (C-22), 61.1 (OCH3), 61.0
(OCH3), 58.9 (C-21), 56.3 (C-1), 54.6 (C-13), 54.4 (C-3), 54.2 (C-
11), 41.6 (C-10), 41.5 (NCH3), 36.3 (C-20), 35.7 (C-50), 25.4 (C-4),
21.2 (C-14), 8.7 (16-CH3), 8.7 (6-CH3); EIMS m/z (% intensity) 587
(M+, 10), 462 (3), 260 (9), 220 (100), 218 (26), 204 (12), 176 (8);
HREIMS m/z 587.2267 (M+, calcd for C32H33N3O8, 587.2268).
4-Ha), 2.77 (1H, dd, J = 21.0, 7.6 Hz, 14-Ha), 2.31 (1H, d,
J = 21.0 Hz, 14-Hb), 2.28 (3H, s, NCH3), 2.26 (1H, m, 10-H), 2.04
(1H, m, 20-H), 2.01 (1H, m, 60-H), 1.95 (3H, s, 6-CH3), 1.94 (1H, m,
20-H), 1.93 (3H, s, 16-CH3), 1.87 (1H, m, 60-H), 1.35 (2H, m, 50-
H2), 1.34 (H, ddd, J = 17.1, 11.6, 2.7 Hz, 4-Hb); 13C NMR (CDCl3,
125 MHz) one isomer d 186.2 (C-15), 185.5 (C-5), 182.4 (C-18),
180.9 (C-8), 174.8 (OCOR), 155.8 (C-7), 155.3 (C-17), 142.1 (C-
20), 141.4 (C-10), 135.5 (C-9), 135.1 (C-19), 128.6 (C-6), 128.3 (C-
16), 126.9 (C-30), 124.8 (C-40), 116.8 (CN), 62.7 (C-22), 61.1
(OCH3), 61.0 (OCH3), 58.6 (C-21), 56.4 (C-1), 54.6 (C-13), 54.3 (C-
3), 54.2 (C-11), 41.5 (NCH3), 39.2 (C-10), 27.7 (C-60), 25.5 (C-4),
25.2 (C-50), 24.3 (C-20), 21.1 (C-14), 8.7 (16-CH3), 8.7 (6-CH3); other
isomer d 186.2 (C-15), 185.5 (C-5), 182.4 (C-18), 181.0 (C-8), 174.8
(OCOR), 155.8 (C-7), 155.3 (C-17), 142.1 (C-20), 141.4 (C-10), 135.5
(C-9), 135.1 (C-19), 128.6 (C-6), 128.4 (C-16), 126.6 (C-30), 124.6
(C-40), 116.8 (CN), 62.8 (C-22), 61.1 (OCH3), 61.0 (OCH3), 58.6 (C-
21), 56.4 (C-1), 54.6 (C-13), 54.3 (C-3), 54.2 (C-11), 41.5 (NCH3),
39.1 (C-10), 27.7 (C-60), 25.5 (C-4), 25.2 (C-50), 24.3 (C-20), 21.2 (C-
14), 8.7 (16-CH3), 8.6 (6-CH3); EIMS m/z (% intensity) 601 (M+, 9),
462 (3), 260 (11), 220 (100), 218 (22), 204 (10), 176 (5); HREIMS
m/z 601.2420 (M+, calcd for C33H35N3O8, 601.2424).
5.12. 10-Cyclohexenecarboxylic acid ester 7d
1-Cyclopentenecarbonyl chloride (63.7 mg, 0.440 mmol) was
added to a stirred solution of 3a (14.8 mg, 0.03 mmol) and DMAP
(0.37 mg, 0.003 mmol) in pyridine (0.6 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 2 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (31.3 mg). Chromatog-
raphy on a silica gel column with hexane–ethyl acetate (5:1) as
eluent gave 7d (11.9 mg, 58.4%) as a pale yellow amorphous pow-
der. ½a 2D2
ꢂ
ꢀ 60:9 (c 0.4, CHCl3); IR (CHCl3) 2935, 2856, 2228, 1714,
1652, 1615, 1456, 1261, 801 cmꢀ1
;
1H NMR (CDCl3, 500 MHz) d
6.62 (1H, dt, J = 6.4, 2.1 Hz, 20-H), 4.50 (1H, dd, J = 11.6, 2.7 Hz,
22-Ha), 4.04 (1H, br s, 21-H), 4.02 (1H, overlapped, 1-H), 4.02
(3H, s, OCH3), 4.01 (1H, overlapped, 22-Hb), 4.01 (3H, s, OCH3),
4.00 (1H, overlapped, 11-H), 3.38 (1H, ddd, J = 7.6, 1.8, 0.6 Hz, 13-
H), 3.09 (1H, ddd, J = 11.6, 3.1, 2.7 Hz, 3-H), 2.92 (1H, dd, J = 17.1,
2.7 Hz, 4-H
a), 2.75 (1H, dd, J = 21.0, 7.6 Hz, 14-Ha), 2.31 (1H, d,
5.14. Benzenecarboxylic acid ester 8a
J = 21.0 Hz, 14-Hb), 2.28 (3H, s, NCH3), 2.06 (2H, m, 30-H2), 1.95
(2H, overlapped, 60-H2), 1.95 (3H, s, 16-CH3), 1.94 (3H, s, 6-CH3),
1.94 (2H, overlapped, 50-H2), 1.51 (2H, m, 40-H2), 1.32 (H, ddd,
J = 17.1, 11.6, 2.7 Hz, 4-Hb); 13C NMR (CDCl3, 125 MHz) d 186.1
(C-15), 185.5 (C-5), 182.5 (C-18), 181.0 (C-8), 166.5 (OCOR),
155.7 (C-7), 155.1 (C-17), 142.2 (C-20), 141.7 (C-10), 140.6 (C-20),
135.6 (C-9), 134.8 (C-19), 129.6 (C-10), 128.4 (C-6), 128.3 (C-16),
116.9 (CN), 62.6 (C-22), 61.1 (OCH3), 61.0 (OCH3), 58.8 (C-21),
56.4 (C-1), 54.6 (C-13), 54.4 (C-3), 54.3 (C-11), 41.5 (NCH3), 25.7
Benzoyl chloride (72.8 lL, 0.63 mmol) was added to a stirred
solution of 3a (21.4 mg, 0.043 mmol) and DMAP (0.49 mg,
0.0043 mmol) in pyridine (0.8 mL) at ꢀ17 °C, and the resulting
solution was stirred at ꢀ17 °C for 1.5 h. The reaction mixture was
poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (103.7 mg). Chroma-
tography on a silica gel column with hexane–ethyl acetate (5:1)

K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
4555
as eluent gave 8a (10.2 mg, 39.4%) as a pale yellow amorphous
trated in vacuo to give a residue (122.4 mg). Chromatography on
a silica gel column with hexane–ethyl acetate (5:1) as eluent gave
powder. ½a 2D2
ꢀ 66:2 (c 0.2, CHCl3); IR (CHCl3) 3445, 2926, 2853,
ꢂ
2229, 1721, 1652, 1616, 1452, 1373, 1272, 1117, 956, 757 cmꢀ1
;
8c (14.3 mg, 54.9%) as
ꢀ 45:6 (c 0.11, CHCl3); IR (CHCl3) 3467, 2944, 2850, 2229,
1704, 1654, 1618, 1491, 1461, 1373, 1267, 1236, 1148, 759 cmꢀ1
a pale yellow amorphous powder.
1H NMR (CDCl3, 500 MHz) d 7.60 (2H, dd, J = 7.7, 1.2 Hz, 20-H, 60-
H), 7.50 (1H, dt, J = 7.7, 1.2 Hz, 40-H), 7.31 (2H, t, J = 7.7 Hz, 30-H,
50-H), 5.00 (1H, dd, J = 12.0, 3.1 Hz, 22-Ha), 4.12 (1H, br s, 21-H),
4.06 (1H, d, J = 12.0 Hz, 22-Hb), 4.06 (1H, overlapped, 1-H), 4.03
(3H, s, 7-OCH3), 3.97 (1H, br d, J = 2.3 Hz, 11-H), 3.67 (3H, s, 17-
OCH3), 3.44 (1H, ddd, J = 7.2, 1.8, 0.6 Hz, 13-H), 3.12 (1H, ddd,
½ ꢂ
a 2D7
;
1H NMR (CDCl3, 300 MHz) d 7.50 (1H, dd, J = 7.7, 1.6 Hz, 60-H),
7.41 (1H, dt, J = 7.7, 1.6 Hz, 40-H), 6.88 (1H, t, J = 7.7 Hz, 30-H),
6.85 (1H, d, J = 7.7 Hz, 30-H), 5.00 (1H, dd, J = 11.4, 2.1 Hz, 22-Ha),
4.26 (1H, br s, 21-H), 4.06 (2H, br s, 1-H, 11-H), 4.00 (3H, s, 7-
OCH3), 3.98 (1H, d, J = 11.4 Hz, 22-Hb), 3.80 (3H, s, OCH3), 3.62
(3H, s, 17-OCH3), 3.36 (1H, br, 13-H), 3.22 (1H, ddd, J = 11.5, 2.7,
J = 11.4, 2.4, 2.3 Hz, 3-H), 2.88 (1H, dd, J = 17.4, 2.4 Hz, 4-H
a),
2.74 (1H, dd, J = 21.1, 7.2 Hz, 14-H ), 2.40 (1H, d, J = 21.1 Hz, 14-
a
Hb), 2.25 (3H, s, NCH3), 1.97 (3H, s, 6-CH3), 1.69 (3H, s, 16-CH3),
1.27 (H, ddd, J = 17.4, 11.4, 2.4 Hz, 4-Hb); 13C NMR (CDCl3,
125 MHz) d 185.4 (C-15), 185.1 (C-5), 181.6 (C-18), 180.8 (C-8),
165.4 (OCOR), 155.7 (C-7), 154.7 (C-17), 142.0 (C-20), 141.7 (C-
10), 140.6 (C-20), 135.3 (C-9), 135.3 (C-19), 133.1 (C-40), 130.0 (C-
10), 129.2 (C-20, C-60), 129.0 (C-6), 128.6 (C-16), 128.4 (C-30, C-50),
116.6 (CN), 61.9 (C-22), 61.2 (OCH3), 60.9 (OCH3), 58.0 (C-21),
56.8 (C-1), 54.7 (C-13), 54.3 (C-3), 53.9 (C-11), 41.4 (NCH3), 25.5
(C-4), 21.8 (C-14), 9.0 (6-CH3), 9.0 (16-CH3); EIMS m/z (% intensity)
597 (M+, 10), 464 (3), 435 (3), 368 (5), 260 (13), 243 (19), 220 (100),
218 (25), 204 (11), 105 (24); HREIMS m/z 597.2115 (M+, calcd for
C33H31N3O8, 597.2111).
2.4 Hz, 3-H), 2.90 (1H, dd, J = 17.1, 2.4 Hz, 4-H
a), 2.76 (1H, dd,
J = 21.0, 7.6 Hz, 14-H ), 2.50 (1H, d, J = 21.0 Hz, 14-Hb), 2.35 (3H,
a
s, NCH3), 1.95 (3H, s, 6-CH3), 1.59 (H, ddd, J = 17.1, 11.5, 3.6 Hz,
4-Hb), 1.48 (3H, s, 16-CH3),; 13C NMR (CDCl3, 75 MHz) d 185.5
(C-15), 185.2 (C-5), 182.0 (C-18), 180.9 (C-8), 165.7 (OCOR),
158.2 (C-20), 155.7 (C-7), 154.4 (C-17), 142.1 (C-20), 141.8 (C-10),
135.4 (C-9), 135.4 (C-19), 133.9 (C-40), 132.6 (C-60), 128.6 (C-6),
128.4 (C-16), 120.2 (C-50), 118.5 (C-10), 116.8 (CN), 112.0 (C-30),
62.4 (C-22), 61.2 (7-OCH3), 60.9 (17-OCH3), 58.0 (C-21), 56.4 (C-
1), 55.6 (40-OCH3), 54.7 (C-13), 54.5 (C-3), 54.0 (C-11), 41.5
(NCH3), 25.3 (C-4), 21.2 (C-14), 9.0 (6-CH3), 8.6 (16-CH3); FABMS
m/z 628 (MH+); HRFABMS m/z 628.2298 (MH+, calcd for
C34H34N3O9, 628.2295).
5.15. 40-Methoxybenzenecarboxylic acid ester 8b
5.17. 40-Nitrobenzenecarboxylic acid ester 8d
4-Methoxybenzoyl chloride (20.2 lL, 0.15 mmol) was added to
a stirred solution of 3a (5.0 mg, 0.01 mmol) and DMAP (0.12 mg,
0.001 mmol) in pyridine (0.2 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 2.5 h. The reaction mixture was
poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (158.9 mg). Chroma-
tography on a silica gel column with hexane–ethyl acetate (5:1)
as eluent gave 8b (2.8 mg, 44.0%) as a pale yellow amorphous pow-
4-Nitrobenzoyl chloride (109.5 lL, 0.59 mmol) was added to a
stirred solution of 3a (20.4 mg, 0.04 mmol) and DMAP (0.49 mg,
0.004 mmol) in pyridine (0.8 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 1 h. The reaction mixture was poured
into water (20 mL) and extracted with chloroform (20 mL ꢁ 3). The
combined extracts were washed with brine, dried, and concen-
trated in vacuo to give a residue (104.1 mg). Chromatography on
a silica gel column with hexane–ethyl acetate (5:1) as eluent gave
der. ½a 2D7
ꢂ
ꢀ 51:3 (c 0.11, CHCl3); IR (CHCl3) 2927, 2854, 2229, 1715,
8d (13.0 mg, 48.6%) as
a pale yellow amorphous powder.
1652, 1512, 1455, 1372, 1258, 1167, 1027, 802, 768 cmꢀ1; 1H NMR
(CDCl3, 300 MHz) d 7.55 (2H, d, J = 8.8 Hz, 20-H, 60-H), 6.77 (2H, d,
J = 8.8 Hz, 30-H, 50-H), 4.97 (1H, dd, J = 12.1, 3.1 Hz, 22-Ha), 4.07
(1H, d, J = 2.0 Hz, 21-H), 4.03 (2H, d, overlapped, 22-Hb, 1-H),
4.03 (3H, s, 7-OCH3), 3.97 (1H, br s, 11-H), 3.81 (3H, s, OCH3),
3.71 (3H, s, 17-OCH3), 3.36 (1H, ddd, J = 7.2, 2.0, 0.6 Hz, 13-H),
3.06 (1H, ddd, J = 11.4, 2.7, 2.2 Hz, 3-H), 2.86 (1H, dd, J = 17.2,
½ ꢂ
a 3D0
ꢀ 316 (c 0.3, CHCl3); IR (CHCl3) 2929, 2853, 2229, 1727,
1654, 1613, 1528, 1449, 1374, 1348, 1273, 1149, 1016, 956 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) d 8.17 (2H, d, J = 8.5 Hz, 30-H, 50-H),
7.80 (2H, d, J = 8.5 Hz, 20-H, 60-H), 4.94 (1H, dd, J = 11.7, 2.4 Hz,
22-Ha), 4.19 (1H, d, J = 11.7 Hz, 22-Hb), 4.11 (2H, overlapped, 21-
H, 1-H), 4.02 (3H, s, 7-OCH3), 3.96 (1H, br s, 11-H), 3.77 (3H, s,
17-OCH3), 3.47 (1H, ddd, J = 7.3, 1.8, 0.6 Hz, 13-H), 3.14 (1H, ddd,
2.2 Hz, 4-H
a), 2.69 (1H, dd, J = 21.0, 7.6 Hz, 14-Ha), 2.34 (1H, d,
J = 11.1, 2.7, 2.0 Hz, 3-H), 2.91 (1H, dd, J = 17.3, 2.0 Hz, 4-H
a),
J = 21.0 Hz, 14-Hb), 2.19 (3H, s, NCH3), 1.97 (3H, s, 6-CH3), 1.72
(3H, s, 16-CH3), 1.30 (H, ddd, J = 17.2, 11.7, 2.2 Hz, 4-Hb); 13C
NMR (CDCl3, 75 MHz) d 185.6 (C-15), 185.3 (C-5), 182.0 (C-18),
180.9 (C-8), 165.9 (OCOR), 163.2 (C-40), 155.7 (C-7), 154.7 (C-17),
142.1 (C-20), 141.9 (C-10), 135.5 (C-9), 135.3 (C-19), 131.0 (C-20,
C-60), 128.5 (C-6), 128.2 (C-16), 121.4 (C-10), 116.9 (CN), 113.7
(C-30, C-50), 61.6 (C-22), 61.2 (7-OCH3), 60.7 (17-OCH3), 58.4 (C-
21), 56.9 (C-1), 55.4 (40-OCH3), 54.6 (C-13), 54.4 (C-3), 54.3 (C-
11), 41.5 (NCH3), 25.7 (C-4), 21.2 (C-14), 9.0 (6-CH3), 9.0 (16-
CH3); EIMS m/z (% intensity) 627 (M+, 12), 464 (7), 435 (2), 368
(10), 260 (17), 243 (20), 220 (100), 218 (25), 204 (11), 152 (27),
135 (56); HREIMS m/z 627.2220 (M+, calcd for C34H33N3O9,
627.2217).
2.73 (1H, dd, J = 21.2, 7.3 Hz, 14-H ), 2.33 (1H, d, J = 21.2 Hz, 14-
a
Hb), 2.26 (3H, s, NCH3), 1.97 (3H, s, 6-CH3), 1.69 (3H, s, 16-CH3),
1.27 (H, ddd, J = 17.3, 11.1, 2.0 Hz, 4-Hb); 13C NMR (CDCl3,
75 MHz) d 185.3 (C-15), 185.0 (C-5), 181.5 (C-18), 180.7 (C-8),
163.6 (OCOR), 155.5 (C-7), 154.6 (C-17), 150.4 (C-40), 142.0 (C-
20), 141.5 (C-10), 135.0 (C-9), 134.2 (C-19), 134.2 (C-10), 130.4
(C-20, C-60), 128.8 (C-6), 127.9 (C-16), 123.6 (C-30, C-50), 116.4
(CN), 63.3 (C-22), 61.3 (7-OCH3), 61.0 (17-OCH3), 58.0 (C-21),
56.5 (C-1), 54.6 (C-13), 54.4 (C-3), 54.0 (C-11), 41.4 (NCH3), 25.5
(C-4), 21.9 (C-14), 9.1 (6-CH3), 8.9 (16-CH3); FABMS m/z 643
(MH+); HRFABMS m/z 643.2037 (MH+, calcd for C33H31N4O10
,
643.2030).
5.18. 40-Bromobenzenecarboxylic Acid Ester 8e
5.16. 20-Methoxybenzenecarboxylic acid ester 8c
4-Bromobenzoyl chloride (64.5 mg, 0.44 mmol) was added to a
stirred solution of 3a (9.9 mg, 0.02 mmol) and DMAP (0.24 mg,
0.002 mmol) in pyridine (0.4 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 1 h. The reaction mixture was poured
into water (20 mL) and extracted with chloroform (20 mL ꢁ 3). The
combined extracts were washed with brine, dried, and concen-
trated in vacuo to give a residue (56.4 mg). Chromatography on a
2-Methoxybenzoyl chloride (91.9 lL, 0.62 mmol) was added to
a stirred solution of 3a (20.6 mg, 0.042 mmol) and DMAP (0.49 mg,
0.004 mmol) in pyridine (0.8 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 3 h. The reaction mixture was poured
into water (20 mL) and extracted with chloroform (20 mL ꢁ 3). The
combined extracts were washed with brine, dried, and concen-

4556
K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
silica gel column with hexane–ethyl acetate (5:1) as eluent gave 8e
powder. ½a 1D6
ꢀ 57:8 (c 0.3, CHCl3); IR (KBr) 2933, 2854, 2338, 2320,
ꢂ
(11.2 mg, 83.0%) as a pale yellow amorphous powder. ½a D30
ꢂ
ꢀ 85:9
1728, 1655, 1616, 1591, 1456, 1420, 1373, 1281, 1236, 1192, 1026,
957 cmꢀ1; 1H NMR (CDCl3, 500 MHz) d 8.83 (1H, m, 20-H), 8.74 (1H,
dd, J = 4.9, 1.6 Hz, 60-H), 7.93 (1H, dd, J = 7.9, 2.0 Hz, 40-H), 7.32 (1H,
ddd, J = 7.9, 4.9, 0.9 Hz, 50-H), 5.01 (1H, dd, J = 11.8, 2.9 Hz, 22-Ha),
4.15 (1H, dd, J = 11.8, 2.3 Hz, 22-Hb), 4.11 (2H, m, 1-H, 21-H), 4.04
(3H, s, 7-OCH3), 3.96 (1H, br s, 11-H), 3.77 (3H, s, 17-OCH3), 3.40
(1H, ddd, J = 7.6, 1.8, 0.6 Hz, 13-H), 3.10 (1H, ddd, J = 11.3, 3.1,
(c 0.19, CHCl3); IR (KBr) 2924, 2361, 2341, 1717, 1653, 1616,
1456, 1373, 1271, 1234, 1070, 1012, 957, 756 cmꢀ1 1H NMR
;
(CDCl3, 500 MHz) d 7.49 (2H, d, J = 9.5 Hz, 20-H, 60-H), 7.47 (2H, d,
J = 9.5 Hz, 30-H, 50-H), 5.02 (1H, dd, J = 11.3, 2.5 Hz, 22-Ha), 4.09
(1H, dd, J = 6.7, 2.5 Hz, 1-H), 4.07 (1H, br s, 21-H), 4.05 (1H, over-
lapped, 22-Hb), 4.05 (3H, s, 7-OCH3), 3.94 (1H, br d, J = 2.8 Hz,
11-H), 3.78 (3H, s, 17-OCH3), 3.38 (1H, ddd, J = 7.4, 1.8, 0.6 Hz,
13-H), 3.08 (1H, ddd, J = 11.3, 2.9, 2.6 Hz, 3-H), 2.89 (1H, dd,
2.4 Hz, 3-H), 2.92 (1H, dd, J = 17.2, 2.4 Hz, 4-Ha), 2.71 (1H, dd,
J = 21.1, 7.6 Hz, 14-H ), 2.35 (1H, d, J = 21.1 Hz, 14-Hb), 2.23 (3H,
a
J = 17.4, 2.6 Hz, 4-H
a
), 2.70 (1H, dd, J = 21.1, 7.4 Hz, 14-H
a
), 2.34
s, NCH3), 1.99 (3H, s, 6-CH3), 1.74 (3H, s, 16-CH3), 1.24 (H, ddd,
J = 17.2, 11.3, 2.8 Hz, 4-Hb); 13C NMR (CDCl3, 125 MHz) d 185.9
(C-15), 185.3 (C-5), 182.2 (C-18), 181.1 (C-8), 164.3 (OCOR),
155.6 (C-7), 155.0 (C-17), 153.6 (C-20), 150.5 (C-60), 142.1 (C-20),
142.0 (C-10), 136.9 (C-40), 135.2 (C-9), 134.6 (C-19), 128.9 (C-6),
128.3 (C-16), 125.1 (C-30), 123.4 (C-50), 116.8 (CN), 62.4 (C-22),
61.2 (OCH3), 60.9 (OCH3), 58.4 (C-21), 56.6 (C-1), 54.5 (C-13),
54.3 (C-3), 54.1 (C-11), 41.4 (NCH3), 25.5 (C-4), 21.0 (C-14), 8.9
(6-CH3), 8.7 (16-CH3); FABMS m/z 599 (MH+); HRFABMS m/z
599.2148 (MH+, calcd for C32H31N4O8, 599.2142).
(1H, d, J = 21.1 Hz, 14-Hb), 2.21 (3H, s, NCH3), 2.00 (3H, s, 6-CH3),
1.76 (3H, s, 16-CH3), 1.26 (H, ddd, J = 17.4, 11.3, 2.0 Hz, 4-Hb);
13C NMR (CDCl3, 125 MHz) d 185.8 (C-15), 185.5 (C-5), 182.2 (C-
18), 181.1 (C-8), 164.8 (OCOR), 155.7 (C-7), 155.0 (C-17), 142.1
(C-20), 142.0 (C-10), 135.4 (C-9), 134.5 (C-19), 131.9 (C-20, C-60),
130.9 (C-30, C-50), 128.7 (C-10), 128.5 (C-40), 128.1 (C-6), 128.0 (C-
16), 116.9 (CN), 62.0 (C-22), 61.2 (7-OCH3), 60.8 (17-OCH3), 58.3
(C-21), 56.8 (C-1), 54.6 (C-13), 54.3 (C-3), 54.1 (C-11), 41.4
(NCH3), 25.6 (C-4), 20.9 (C-14), 8.8 (6-CH3), 8.7 (16-CH3); FABMS
m/z 676 (MH+); HRFABMS m/z 676.1300 (MH+, calcd for
C33H31BrN3O10, 676.1294).
5.21. 20-Pyridinecarboxylic acid ester 9c
5.19. 40-Pyridinecarboxylic acid ester 9a
Carbonate 5e30 (60.0 mg, 0.31 mmol) was added to a stirred
solution of 3a (9.8 mg, 0.02 mmol) and triethylamine (0.28 lL,
4-Pyridinecarbonyl chloride-hydrochloride (52.3 mg, 0.29 mmol)
was added to a stirred solution of 3a (9.9 mg, 0.10 mmol) and trieth-
0.002 mmol) in pyridine (1.0 mL) at ꢀ17 °C, and the resulting solu-
tion was stirred at ꢀ17 °C for 3 h. The reaction mixture was poured
into water (20 mL) and extracted with chloroform (20 mL ꢁ 3). The
combined extracts were washed with brine, dried, and concen-
trated in vacuo to give a residue (14.9 mg). Chromatography on a
silica gel column with hexane–ethyl acetate (3:1) as eluent gave
ylamine (0.28
l
L, 0.002 mmol) in pyridine (1.0 mL) at ꢀ17 °C, and
the resulting solution was stirred at ꢀ17 °C for 3 h. The reaction
mixture was poured into water (20 mL) and extracted with chloro-
form (20 mL ꢁ 3). The combined extracts were washed with brine,
dried, and concentrated in vacuo to give a residue (79.2 mg). Chro-
matography on a silica gel column with hexane–ethyl acetate (3:1)
as eluent gave 9a (6.8 mg, 56.8%) as a pale yellow amorphous pow-
9c (4.8 mg, 40.1%) as
ꢀ 87:9 (c 0.3, CHCl3); IR (KBr) 2941, 2853, 2361, 2343, 1724,
1655, 1616, 1587, 1439, 1412, 1373, 1304, 1236, 1190, 1136,
1080, 1045, 1024, 957 cmꢀ1 1H NMR (CDCl3, 500 MHz) d 8.52
a pale yellow amorphous powder.
½ ꢂ
a 1D6
der. ½a 1D6
ꢂ
ꢀ 66:1 (c 0.5, CHCl3); IR (KBr) 2934, 2854, 2337, 2330,
;
1728, 1655, 1616, 1591, 1456, 1420, 1373, 1281, 1236, 1192,
(1H, ddd, J = 4.8, 1.8, 0.9 Hz, 60-H), 7.84 (1H, dt, J = 7.3, 1.2 Hz, 30-
H), 7.77 (1H, ddd, J = 8.3, 7.3, 1.8 Hz, 40-H), 7.43 (1H, ddd, J = 8.3,
4.8, 1.2 Hz, 50-H), 4.92 (1H, dd, J = 11.3, 3.1 Hz, 22-Ha), 4.25 (1H,
d, J = 2.0 Hz, 21-H), 4.15 (1H, ddd, J = 3.8, 3.1, 2.2 Hz, 1-H), 4.07
(1H, dd, J = 11.3, 3.8 Hz, 22-Hb), 4.01 (3H, s, 7-OCH3), 3.95 (1H, br
s, 11-H), 3.85 (3H, s, 17-OCH3), 3.38 (1H, ddd, J = 7.4, 2.0, 0.6 Hz,
13-H), 3.11 (1H, ddd, J = 11.6, 3.1, 2.6 Hz, 3-H), 2.87 (1H, dd,
1026, 957 cmꢀ1
; d 8.62 (2H, d,
1H NMR (CDCl3, 500 MHz)
J = 8.3 Hz, 20-H, 60-H), 7.44 (2H, d, J = 8.3 Hz, 30-H, 50-H), 4.99 (1H,
dd, J = 11.7, 2.9 Hz, 22-Ha), 4.17 (1H, dd, J = 11.7, 2.1 Hz, 22-Hb),
4.10 (2H, m, 1-H, 21-H), 4.04 (3H, s, 7-OCH3), 3.95 (1H, br d,
J = 2.2 Hz, 11-H), 3.76 (3H, s, 17-OCH3), 3.40 (1H, ddd, J = 7.6, 1.8,
0.6 Hz, 13-H), 3.10 (1H, ddd, J = 11.6, 3.1, 2.4 Hz, 3-H), 2.92 (1H,
dd, J = 17.4, 2.4 Hz, 4-H
a
), 2.71 (1H, dd, J = 20.9, 7.6 Hz, 14-H
a
),
J = 17.1, 2.6 Hz, 4-Ha), 2.68 (1H, dd, J = 21.0, 7.4 Hz, 14-Ha), 2.41
2.32 (1H, d, J = 20.9 Hz, 14-Hb), 2.23 (3H, s, NCH3), 2.00 (3H, s, 6-
CH3), 1.74 (3H, s, 16-CH3), 1.25 (H, ddd, J = 17.4, 11.6, 2.4 Hz, 4-
Hb); 13C NMR (CDCl3, 125 MHz) d 185.9 (C-15), 185.4 (C-5),
182.1 (C-18), 181.1 (C-8), 164.1 (OCOR), 155.6 (C-7), 154.9 (C-
17), 150.7 (C-20, C-60), 142.1 (C-20), 142.1 (C-10), 136.2 (C-40),
135.2 (C-9), 134.6 (C-19), 128.8 (C-6), 128.2 (C-16), 122.5 (C-30,
C-50), 116.8 (CN), 62.8 (C-22), 61.2 (OCH3), 60.9 (OCH3), 58.4 (C-
21), 56.6 (C-1), 54.5 (C-13), 54.3 (C-3), 54.1 (C-11), 41.4 (NCH3),
25.5 (C-4), 21.0 (C-14), 8.9 (6-CH3), 8.7 (16-CH3); FABMS m/z 599
(MH+); HRFABMS m/z 599.2133 (MH+, calcd for C32H31N4O8,
599.2142).
(1H, d, J = 21.0 Hz, 14-Hb), 2.23 (3H, s, NCH3), 1.96 (3H, s, 6-CH3),
1.69 (3H, s, 16-CH3), 1.63 (H, ddd, J = 17.1, 11.6, 2.5 Hz, 4-Hb);
13C NMR (CDCl3, 125 MHz) d 185.9 (C-15), 185.5 (C-5), 182.2 (C-
18), 181.2 (C-8), 164.7 (OCOR), 155.5 (C-7), 154.8 (C-17), 149.8
(C-60), 147.4 (C-20), 142.9 (C-10), 142.1 (C-20), 136.9 (C-40), 135.0
(C-9), 134.6 (C-19), 128.8 (C-6), 127.9 (C-16), 126.9 (C-50), 125.3
(C-30), 117.2 (CN), 62.3 (C-22), 61.1 (OCH3), 60.9 (OCH3), 58.9 (C-
21), 56.1 (C-1), 54.8 (C-3), 54.6 (C-13), 54.4 (C-11), 41.4 (NCH3),
25.5 (C-4), 20.9 (C-14), 8.9 (6-CH3), 8.7 (16-CH3); FABMS m/z 599
(MH+); HRFABMS m/z 599.2141 (MH+, calcd for C32H31N4O8,
599.2142).
5.20. 30-Pyridinecarboxylic acid ester 9b
5.22. 40-Quinolinecarboxylic acid ester 9d
3-Pyridinecarbonyl chloride-hydrochloride (67.4 mg, 0.38 mmol)
was added to a stirred solution of 3a (12.7 mg, 0.26 mmol) and tri-
4-Quinolinecarbonyl chloride (169.0 mg, 0.88 mmol) was added
to a stirred solution of 3a (30.0 mg, 0.06 mmol) and DMAP
(0.73 mg, 0.006 mmol) in pyridine (1.2 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 3 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloroform
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (170.4 mg). Chroma-
tography on a silica gel column with hexane–ethyl acetate (2:1)
as eluent gave 9d (26.1 mg, 67.1%) as a pale yellow amorphous
ethylamine (0.36
l
L, 0.026 mmol) in pyridine (1.0 mL) at ꢀ17 °C,
and the resulting solution was stirred at ꢀ17 °C for 3 h. The reac-
tion mixture was poured into water (20 mL) and extracted with
chloroform (20 mL ꢁ 3). The combined extracts were washed with
brine, dried, and concentrated in vacuo to give a residue (16.1 mg).
Chromatography on a silica gel column with hexane–ethyl acetate
(3:1) as eluent gave 9b (5.6 mg, 36.4%) as a pale yellow amorphous

K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
4557
powder. ½a 1D6
ꢂ
ꢀ 88:1 (c 0.6, CHCl3); IR (KBr) 2928, 2850, 2351, 1725,
1H NMR
(20 mL ꢁ 3). The combined extracts were washed with brine, dried,
and concentrated in vacuo to give a residue (30.8 mg). Chromatog-
raphy on a silica gel column with hexane–ethyl acetate (3:1) as
eluent gave 9f (9.0 mg, 69.4%) as a pale yellow amorphous powder.
1655, 1618, 1459, 1374, 1236, 1148, 1019, 768 cmꢀ1
;
(CDCl3, 500 MHz) d 8.90 (1H, d, J = 4.3 Hz, 20-H), 8.58 (1H, d,
J = 8.3 Hz, 50-H), 8.16 (1H, d, J = 8.3 Hz, 80-H), 7.77 (1H, t,
J = 8.3 Hz, 70-H), 7.57 (1H, t, J = 8.3 Hz, 60-H), 7.49 (1H, d,
J = 4.3 Hz, 20-H), 5.10 (1H, dd, J = 11.7, 2.9 Hz, 22-Ha), 4.30 (1H,
dd, J = 11.7, 2.9 Hz, 22-Hb), 4.17 (2H, m, 1-H, 21-H), 4.05 (3H, s,
7-OCH3), 3.97 (1H, br s, 11-H), 3.65 (3H, s, 17-OCH3), 3.40 (1H,
ddd, J = 7.4, 1.8, 0.6 Hz, 13-H), 3.16 (1H, ddd, J = 11.4, 3.1, 2.5 Hz,
½
a 1D6
ꢂ
ꢀ 133:7 (c 0.3, CHCl3); IR (KBr) 3649, 2943, 2847, 2359, 2343,
1724, 1654, 1616, 1560, 1506, 1458, 1412, 1373, 1312, 1294, 1281,
1236, 1211, 1190, 1138, 1109, 1080, 1047, 964, 842, 796 cmꢀ1 1H
;
NMR (CDCl3, 500 MHz) d 8.23 (1H, d, J = 8.4 Hz, 40-H), 7.95 (1H, dd,
J = 8.3, 1.2 Hz, 80-H), 7.88 (1H, d, J = 8.4 Hz, 30-H), 7.87 (1H, dd,
J = 8.3, 1.4 Hz, 50-H), 7.77 (1H, ddd, J = 8.3, 7.0, 1.4 Hz, 70-H), 7.67
(1H, ddd, J = 8.3, 7.0, 1.2 Hz, 60-H), 5.19 (1H, dd, J = 11.3, 2.8 Hz,
22-Ha), 4.24 (1H, d, J = 2.1 Hz, 21-H), 4.17 (1H, dt, J = 2.8, 2.5 Hz,
1-H), 4.04 (1H, dd, J = 11.3, 2.5 Hz, 22-Hb), 4.03 (3H, s, 7-OCH3),
3.91 (1H, br d, J = 2.0 Hz, 11-H), 3.38 (1H, ddd, J = 7.4, 2.1, 0.6 Hz,
13-H), 3.28 (3H, s, 17-OCH3), 3.13 (1H, dt, J = 11.4, 2.0 Hz, 3-H),
3-H), 2.95 (1H, dd, J = 17.7, 2.5 Hz, 4-H
7.4 Hz, 14-H ), 2.38 (1H, d, J = 21.0 Hz, 14-Hb), 2.22 (3H, s,
a), 2.67 (1H, dd, J = 21.0,
a
NCH3), 2.02 (3H, s, 6-CH3), 1.41 (H, ddd, J = 17.7, 11.4, 2.7 Hz, 4-
Hb), 1.16 (3H, s, 16-CH3); 13C NMR (CDCl3, 125 MHz) d 185.5 (C-
15), 185.4 (C-5), 182.2 (C-18), 181.1 (C-8), 165.1 (OCOR), 155.8
(C-7), 154.7 (C-17), 149.4 (C-20), 149.4 (C-80a), 149.1 (C-40a),
142.0 (C-20), 141.9 (C-10), 135.5 (C-9), 134.5 (C-19), 133.4 (C-40),
130.2 (C-70), 130.1 (C-80), 128.8 (C-6), 128.6 (C-60), 128.1 (C-16),
127.4 (C-40a), 125.4 (C-50), 122.0 (C-30), 116.8 (CN), 62.9 (C-22),
61.2 (OCH3), 60.7 (OCH3), 58.4 (C-21), 56.6 (C-1), 54.6 (C-13),
54.2 (C-3), 54.1 (C-11), 41.4 (NCH3), 25.7 (C-4), 21.0 (C-14), 8.9
(6-CH3), 7.8 (16-CH3); FABMS m/z 649 (MH+); HRFABMS m/z
649.2296 (MH+, calcd for C36H33N4O8, 649.2298).
2.95 (1H, dd, J = 17.4, 2.0 Hz, 4-H
a), 2.65 (1H, dd, J = 21.0, 7.4 Hz,
14-H ), 2.44 (1H, d, J = 21.0 Hz, 14-Hb), 2.18 (3H, s, NCH3), 2.08
a
(H, ddd, J = 17.4, 11.4, 2.4 Hz, 4-Hb), 2.02 (3H, s, 6-CH3), 1.42 (3H,
s, 16-CH3); 13C NMR (CDCl3, 125 MHz) d 185.9 (C-15), 185.6 (C-
5), 181.9 (C-18), 181.4 (C-8), 164.9 (OCOR), 155.5 (C-7), 154.1 (C-
17), 147.3 (C-40a), 147.3 (C-80a), 143.7 (C-10), 142.0 (C-20), 137.2
(C-40), 134.9 (C-9), 134.4 (C-19), 130.6 (C-70), 130.6 (C-80), 129.1
(C-20), 128.9 (C-60), 128.9 (C-6), 127.5 (C-50), 126.6 (C-16), 120.9
(C-30), 117.2 (CN), 63.6 (C-22), 61.1 (OCH3), 60.1 (OCH3), 58.7 (C-
21), 56.5 (C-1), 54.7 (C-3), 54.6 (C-13), 54.2 (C-11), 41.4 (NCH3),
25.6 (C-4), 20.9 (C-14), 8.9 (6-CH3), 8.4 (16-CH3); FABMS m/z 649
(MH+); HRFABMS m/z 649.2293 (MH+, calcd for C36H33N4O8,
649.2298).
5.23. 30-Quinolinecarboxylic acid ester 9e
3-Quinolinecarbonyl chloride (169 mg, 0.88 mmol) was added
to a stirred solution of 3a (10.8 mg, 0.022 mmol) and DMAP
(0.27 mg, 0.0022 mmol) in pyridine (1.0 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 6 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloro-
form (20 mL ꢁ 3). The combined extracts were washed with
6. Preparation of sample solutions and cell cultures
brine, dried, and concentrated in vacuo to give
a residue
Each sample was prepared as a 20 mM stock solution that was
dissolved in DMSO and added to the cells with less than 1% DMSO
in the final drug dilution with culture medium. HCT116 colon car-
cinoma and MDA-MB-453 breast carcinoma cell lines were cul-
tured in PMP11640 (Sigma) containing supplements: 10%
penicillin (100 U/mL) and streptomycin (100 U/mL). Cells were
incubated at 37 °C in a humidified atmosphere of 5% CO2 and
95% air.
(11.4 mg). Chromatography on a silica gel column with hexane–
ethyl acetate (2:1) as eluent gave 9e (4.9 mg, 34.4%) as a pale yel-
low amorphous powder. ½a D16
ꢀ 133:1 (c 0.3, CHCl3); IR (KBr)
ꢂ
3447, 2945, 2355, 2343, 2330, 1724, 1653, 1618, 1570, 1497,
1447, 1420, 1375, 1286, 1259, 1234, 1198, 1130, 1110, 1080,
1047, 1022, 966, 876, 841, 793 cmꢀ1 1H NMR (CDCl3, 500 MHz)
;
d 9.02 (1H, d, J = 2.1 Hz, 20-H), 8.50 (1H, d, J = 2.1 Hz, 40-H), 8.14
(1H, dd, J = 8.9, 1.2 Hz, 80-H), 7.87 (1H, dd, J = 5.5, 1.2 Hz, 50-H),
7.86 (1H, ddd, J = 8.9, 7.6, 1.2 Hz, 70-H), 7.65 (1H, ddd, J = 7.9,
7.6, 1.2 Hz, 60-H), 5.18 (1H, dd, J = 11.4, 2.1 Hz, 22-Ha), 4.16 (1H,
dd, J = 11.4, 2.1 Hz, 22-Hb), 4.13 (2H, m, 1-H, 21-H), 4.06 (3H, s,
7-OCH3), 3.95 (1H, br s, 11-H), 3.44 (3H, s, 17-OCH3), 3.40 (1H,
ddd, J = 7.4, 1.8, 0.6 Hz, 13-H), 3.12 (1H, ddd, J = 11.4, 3.1,
7. Cell growth inhibition assay (IC50
)
HCT116 colon carcinoma and MDA-MB-453 breast carcinoma
cell lines were, respectively seeded at 1500 cells per well and
3000 cells per well, in 96-well plates 24 h before the experiments.
The cell lines were treated with each sample at concentrations of
threefold dilution ranging from 100 nM to 0.41 nM and incubated
for 3 days in a cell culture incubator. MTT colorimetric assay was
performed to measure 50% cell growth inhibition (IC50) relative
to untreated control cells. MTT assay results were read in a TECAN
absorbance microplate reader by measuring absorbance at 540 nm.
All determinations were carried out in triplicate.
2.3 Hz, 3-H), 2.93 (1H, dd, J = 17.5, 2.3 Hz, 4-H
a), 2.69 (1H, dd,
J = 21.1, 7.6 Hz, 14-H ), 2.38 (1H, d, J = 21.1 Hz, 14-Hb), 2.20
a
(3H, s, NCH3), 2.03 (3H, s, 6-CH3), 1.33 (H, ddd, J = 17.5, 11.4,
2.6 Hz, 4-Hb), 1.31 (3H, s, 16-CH3); 13C NMR (CDCl3, 125 MHz) d
185.7 (C-15), 185.4 (C-5), 182.0 (C-18), 181.2 (C-8), 164.4 (OCOR),
155.6 (C-7), 154.8 (C-17), 149.2 (C-20), 149.2 (C-40a), 147.3 (C-80a),
142.1 (C-10), 141.9 (C-20), 139.1 (C-40), 135.4 (C-9), 134.5 (C-19),
132.4 (C-70), 129.4 (C-80), 129.3 (C-50), 128.1 (C-6), 127.9 (C-60),
126.5 (C-16), 121.9 (C-30), 116.9 (CN), 62.0 (C-22), 61.2 (OCH3),
60.4 (OCH3), 58.2 (C-21), 56.7 (C-1), 54.5 (C-13), 54.2 (C-3), 54.0
(C-11), 41.4 (NCH3), 25.7 (C-4), 20.9 (C-14), 8.9 (6-CH3), 8.0 (16-
CH3); FABMS m/z 649 (MH+); HRFABMS m/z 649.2295 (MH+, calcd
for C36H33N4O8, 649.2298).
Acknowledgments
K.C. was sponsored by the Thailand Research Fund (TRF) for the
2003 Royal Golden Jubilee (RGJ) Ph. D. Program Scholarship (PHD/
0157/2546). BNPME was supported by the Grant for Center of
Excellence from the Commission of Higher Education, Thailand.
This research was partially supported by the Japan Society for
the Promotion of Science (JSPS) AA Scientific Platform Program
and a grant from the High-Tech Research Center Project, the Min-
istry of Education, Culture, Sports, Science and Technology (MEXT),
Japan (S0801043). We are grateful to JSPS and the National Re-
5.24. 20-Quinolinecarboxylic acid ester 9f
2-Quinolinecarbonyl chloride (149 mg, 0.78 mmol) was added
to
a stirred solution of 3a (9.8 mg, 0.02 mmol) and DMAP
(0.24 mg, 0.0022 mmol) in pyridine (1.0 mL) at ꢀ17 °C, and the
resulting solution was stirred at ꢀ17 °C for 3 h. The reaction mix-
ture was poured into water (20 mL) and extracted with chloroform

4558
K. Charupant et al. / Bioorg. Med. Chem. 17 (2009) 4548–4558
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