Design, synthesis, and biological evaluation of callophycin A and analogues as potential chemopreventive and anticancer agents

Article abstract of DOI:10.1016/j.bmc.2011.09.020

Callophycin A was originally isolated from the red aμgae Callophycus oppositifolius and shown to mediate anticancer and cytotoxic effects. In our collaborative effort to identify potential chemopreventive and anticancer agents with enhanced potency and se

Full text of DOI:10.1016/j.bmc.2011.09.020

Bioorganic & Medicinal Chemistry 19 (2011) 6182–6195
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of callophycin A and analogues
as potential chemopreventive and anticancer agents
Li Shen, Eun-Jung Park, Tamara P. Kondratyuk, Daniela Guendisch, Laura Marler, John M. Pezzuto,
⇑
Anthony D. Wright, Dianqing Sun
Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawai’i at Hilo, Hilo, HI 96720, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2011
Revised 6 September 2011
Accepted 10 September 2011
Available online 16 September 2011
Callophycin A was originally isolated from the red algae Callophycus oppositifolius and shown to mediate
anticancer and cytotoxic effects. In our collaborative effort to identify potential chemopreventive and
anticancer agents with enhanced potency and selectivity, we employed a tetrahydro-b-carboline-based
template inspired by callophycin A for production of a chemical library. Utilizing a parallel synthetic
approach, 50 various functionalized tetrahydro-b-carboline derivatives were prepared and assessed for
activities related to cancer chemoprevention and cancer treatment: induction of quinone reductase 1
Keywords:
Callophycin A
Tetrahydro-b-carboline
Anticancer agent
Chemoprevention
Aromatase
NFjB
Quinone reductase 1
iNOS
(QR1) and inhibition of aromatase, nitric oxide (NO) production, tumor necrosis factor (TNF)-
NF B activity, and MCF7 breast cancer cell proliferation. Biological results showed that the n-pentyl urea
S-isomer 6a was the strongest inducer of QR1 with an induction ratio (IR) value of 4.9 at 50 M [the con-
centration to double the activity (CD) = 3.8 M] and its corresponding R-isomer 6f had an IR value of 4.3
(CD = 0.2 M). The isobutyl carbamate derivative 3d with R stereochemistry demonstrated the most
potent inhibitory activity of NF B, with the half maximal inhibitory concentration (IC50) value of
4.8 M, and also showed over 60% inhibition at 50 M of NO production (IC50 = 2.8 M). The R-isomer
urea derivative 6j, having an appended adamantyl group, exhibited the most potent MCF7 cell prolifer-
ation inhibitory activity (IC50 = 14.7 M). The S-isomer 12a of callophycin A showed the most potent
activity in aromatase inhibition (IC50 = 10.5 M).
a-induced
j
l
l
l
j
l
l
l
l
l
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
and chemopreventive agents. To that end, we have undertaken syn-
thesis and biological evaluation of a chemical library of callophycin
A derivatives.11 In this paper, the synthetic efforts that focused on
the modifications of the 2- and 3-positions of callophycin A are
described. Subsequently, a series of N2-substituted tetrahydro-b-
carboline derivatives were synthesized and assessed in chemopre-
ventive and anticancer target-based bioassays: induction of quinone
reductase 1 (QR1) and inhibition of aromatase, nitric oxide (NO) pro-
duction by inducible nitric oxide synthases (iNOS) in lipopolysac-
charide (LPS)-stimulated RAW 264.7 murine macrophage cells,
Tetrahydro-b-carbolines have been featured as important tem-
plates for many naturally occurring and synthetic compounds,
which mediate a variety of biological activities.1–3 They have been
characterized as a new class of orally bioavailable compounds for
the potential treatment of malaria.4,5 Some were reported as po-
tential anti-thrombotic agents6,7 as well as human papillomavirus
antiinfective agents.8 More importantly for our research, several
tetrahydro-b-carboline derivatives have also been investigated as
anticancer agents.2,9
tumor necrosis factor (TNF)-a-induced NFjB activity, and MCF7 cell
Callophycin A (Fig. 1), a marine natural product displaying a tet-
rahydro-b-carboline scaffold, was first isolated from the red algae
Callophycus oppositifolius, and shown to have antiproliferative ef-
fects on various human cancer cell lines at low micromolar concen-
trations.10 Although no selectivity between tumor and normal
mammalian cells was noted, due to facile chemistry accessibility
and a variety of potential sites for chemical derivatization, callophy-
cin A offers a valuable chemical starting point for further investiga-
tion in an attempt to discover more selective and potent anticancer
proliferation.
More specifically, QR1 is categorized as a detoxifying enzyme
which is responsible for the reduction of electrophilic quinones
to their nontoxic hydroquinone forms.12 It is believed that QR1
elevation provides a reasonable biomarker for cancer chemopre-
vention.13 Aromatase catalyzes the conversion of androgen to
estrogen in the late stage of female sex hormone biosynthesis,
and it is found to be over expressed in breast cancer tissues.14,15
Clinically, nonsteroidal aromatase inhibitors such as letrozole
and anastrozole represent an important class of therapeutic agents
for the treatment of breast cancer and have shown their potential
as chemopreventive agents.16–18 NO elevation has been shown to
⇑
Corresponding author. Tel.: +1 808 933 2960; fax: +1 808 933 2974.
E-mail address: dianqing@hawaii.edu (D. Sun).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.020

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6183
isomer 1a or 1b21 with methanol in the presence of SOCl2 yielded
the enantiomeric methyl ester 2a or 2b in excellent yields, respec-
tively.22 The esters 2a–b were then subject to reaction with allyl or
isobutyl chloroformates to afford a series of carbamate derivatives
3a–d in high yields (79–100%).
COOH
4
5
3
*
N
2
N
H
1
8
9
callophycin A
Carboline sulfonamides 4a–f were obtained by reacting 2a or
2b with various sulfonyl chlorides in the presence of triethylamine
at room temperature. The reactions of 2a or 2b with 4-bro-
mobenzenesulfonyl chloride or p-toluenesulfonyl chloride pro-
ceeded smoothly to completion in 40 h, providing 4a–b and 4d–e
in good yields (78–100%). The reactions with methanesulfonyl
chloride occurred much faster and went to completion in 0.5 h to
give 4c and 4f in 100% and 93% yield, respectively. The N-alkylation
was carried out by the method reported by Cook’s group.23 A reac-
tion mixture of secondary amine 2a or 2b and various bromides in
acetonitrile was heated at reflux for 2–5 h in the presence of N,
N-diisopropylethylamine (DIPEA, Hunig’s base). Subsequent purifi-
cation by flash column chromatography on silica gel gave the alkyl-
ation products 5a–f in 62–84% yields. The urea derivatives 6a–j
were prepared by reaction of 2a or 2b with an array of substituted
isocyanates in dichloromethane at room temperature in >88%
yields.
OH
Figure 1. Chemical structure of callophycin A (MW: 322.36, calculated logP: 2.31).
be associated with the early stages of cancer development, and
inhibitors of NO production have been considered as cancer che-
mopreventive agents.19 In contrast, the role of NF
j
B in many cellu-
lar processes is well studied; it has been shown that NF B is
involved in inflammation, cell-cycle regulation, and apoptosis.20
Blocking of NF B can stop cell proliferation and ultimately lead
to apoptosis, thus, inhibitors of NF B have been investigated as
j
j
j
promising anticancer and cancer chemoprevention agents.20
Herein, we report our effort toward synthesis and biological
evaluation of callophycin A and analogues as potential chemopre-
ventive and anticancer agents; preliminary structure–activity
relationships (SARs) are also discussed.
2. Results and discussion
2.1. Chemistry
Compounds 3–5 were further hydrolyzed to the corresponding
free acids 7–9 in the presence of LiOH in aqueous THF at room tem-
perature. These hydrolysis reactions were quite clean based on
HPLC monitoring and products 7–9 were subsequently isolated in
39–84% yields. However, hydrolysis of carboline urea derivatives
6 in basic conditions led to the facile formation of intramolecular
cyclized hydantoin byproduct.24,25
Finally, the first total synthesis of callophycin A started with the
N-alkylation of 2a, b with 4-(benzyloxy)benzyl bromide.26 The
experiments were conducted under the same alkylation conditions
Design and synthesis of callophycin A-inspired tetrahydro-b-
carboline derivatives 2–12, including callophycin A itself, are
shown in Scheme 1. The design rationale of this focused carboline
library was to explore the chemical diversity at the 2 and 3 posi-
tions of the tetrahydro-b-carboline template and to improve the
poor solubility associated with 1a and 1b. Esterification of chiral
COOMe
*
COOH
*
i
NH
NH
N
H
N
1a
1b
2a
2b
H
(S)
(R)
(S)
(R)
iv
v
ii
iii
iv
COOMe
COOMe
O
COOMe
COOMe
COOMe
*
*
*
H
N
*
*
R1
R4
N
SO2R2
N
N
N
R3
N
N
H
N
H
O
N
H
N
H
O
N
H
4a-c (S)
4d-f (R)
6a-e (S)
6f-j (R)
10a (S)
5a-c (S)
5d-f (R)
3a-b (S)
3c-d (R)
10b (R)
OBn
R4
=
vii
vi
vi
6a f
,
vi
COOMe
COOH
O
COOH
COOH
*
*
*
*
6b g
,
R1
N
SO2R2
N
R3
N
N
N
N
H
O
N
H
N
H
6c h
,
H
11a (S)
11b (R)
9a-c (S)
9d-f (R)
8a-c (S)
7a-b (S)
7c-d (R)
)
8d-f (R
6d i
,
OH
COOH
vi
Br
R2
=
R3
=
4a d
,
5a d
,
R1
=
3a c
,
8a d
,
9a d
,
7a c
,
*
6e j
,
CH3
N
3b d
,
4b e
,
5b e
,
7d d
,
8b e
,
9b e
,
N
H
callophycin A
4c f
,
5c f
,
CH3
12a (S)
8c f
,
9c f
,
OH
12b
(R)
Scheme 1. Synthesis of callophycin A analogues 2–12. Reagents and conditions: (i) SOCl2, MeOH, rt; (ii) R1OCOCl, Et3N, CH2Cl2, rt; (iii) R2SO2Cl, Et3N, CH2Cl2, rt; (iv) R3Br or 4-
BnOBnBr, DIPEA, CH3CN, reflux; (v) R4NCO, CH2Cl2, rt; (vi) LiOH, THF/H2O (1:1), rt; (vii) 10% Pd/C, H2, MeOH, rt, 4.5 h.

6184
L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
as those of 5a–f, to give 10a, b in 58% and 61% yields, respectively,
followed by O-debenzylation (H2, Pd/C) to afford 11a, b.27 Enantio-
meric callophycin A (12a, b) were then obtained after ester hydro-
lysis under the same reaction conditions as in the preparation of
7–9. The mass spectrometric and NMR (1H, 13C NMR, HSQC, HMBC,
and COSY) spectroscopic data of 12a and 12b were in good agree-
ment with those of the reported natural callophycin A.10
the sulfonamide series (entries 11–15), approximately 20–40% of
partial racemization ocurred in the hydrolysis process due to in-
creased acidity originating from the electron withdrawing effect
of the sulfonamide group. The representative chiral HPLC chroma-
tographs are illustrated in Figure 3.
2.2. Biological activities
All compounds were characterized by 1H, 13C NMR, and mass
spectrometry.28 Purity was determined by reverse-phase C18 HPLC
with UV monitoring at 254 and 220 nm. Interestingly, the coupling
and splitting patterns of the 1H NMR spectra of carbamates 3 dif-
ferred from those of sulfonamides 4, alkylation products 5, and
urea derivatives 6 in that they were more complex and displayed
two sets of signals for most of the protons at room temperature
(24 °C). This phenomenon indicates that 3 exists as a mixture of
rotamers due to restricted rotation of the carbamate side chain.29
To confirm this deduction, temperature-dependent NMR studies
of 3d were undertaken. The 1H NMR spectra of 3d in DMSO-d6
were recorded at 24, 40, and 90 °C. For clarity, only four sets of pro-
ton signals of 3d are shown in Figure 2 to demonstrate the influ-
ence of temperature on the four sets of resonances shown. These
signals include: (i) 5.30–5.26 (Ha), (ii) 4.86–4.76 (Hb), (iii) 4.52–
4.36 (Hc), and (iv) 3.99–3.82 (Hd). As illustrated in Figure 2, all
these signals were doubled at 24 °C. When the sample temperature
was increased to 40 °C, the two sets of signals for Ha, Hb, and Hd
started to merge. At 90 °C, all signals coalesced. As rotamers were
only observed for the carbamates, it was concluded that the phe-
nomenon is possibly caused by an interaction, probably a weak
hydrogen bond, between the carbamate oxygen and Ha that re-
sulted in restricted rotation around the N–CO(O) bond, giving rise
to two distinct sets of NMR signals at normal room temperatures.
To evaluate the possible racemization in the final step of basic
hydrolysis, we performed chiral HPLC analysis for the targeted
compounds to determine their enantiomeric purity. For compari-
son, the ester derivatives were also evaluated. Ten pairs of enanti-
omers were randomly selected for analysis, and the chiral HPLC
analysis data are given in Table 1. In all cases, good to excellent
separations were achieved. As expected, no racemization occurred
for all the evaluated ester compounds (entries 1–8). Among the
free carboxylic acid derivatives analyzed, no racemization took
place and clean chiral HPLC profiles were obtained for the alkyl-
ation hydrolysis products (entries 17–20, Table 1). However, for
Biological evaluation of tetrahydro-b-carboline derivatives 1–
12 was undertaken in a series of bioassays related to cancer che-
moprevention and cancer treatment. An initial screen for activity
was performed at 50 lM. If a compound exhibited more than
50% inhibition (or 2.0 IR, for QR1) at this concentration, further
testing was then performed for the determination of an IC50 (or
CD) value. In the case of the evaluation of antiproliferative effects
using a sulforhodamine B (SRB) assay, compounds which exhibited
less than 50% survival are considered cytotoxic, and IC50 values
were determined. Results are summarized in Table 2.
2.2.1. QR1 induction activity
The effect of compounds 1–12 on QR1 induction was tested as de-
scribed in the experimentalsection. As shown in Table 2, the two iso-
mers of N2-unsubstituted b-carboline 2a and 2b showed the same
QR1 induction activity with an IR value of 1.1. Among the carbamate
derivatives 3a–d, allyl substituted R-isomer 3c demonstrated higher
activity (IR = 2.8, CD = 15.9 lM). There were no differences observed
between the two chiral isomers of 4-methylphenyl and methyl
substituted sulfonamide derivatives (4b and 4e, IR = 1.3; 4c and 4f,
IR = 1.6). In contrast, the 4-bromophenyl substituted R-isomer 4d
(IR = 2.1, CD = 29.7
tive and less cytotoxic than its corresponding S-isomer (4a, IR = 0.2,
36.4% survival at 50 M). For the alkylation products 5a–f and 10–
11, the S-isomers of benzyl 5a (IR = 1.6), cyclopropylmethyl substi-
tuted compound 5b (IR = 2.2, CD = 34.0 M), and compound 10a
lM, 83.7% survival at 50 lM) was much more ac-
l
l
(IR = 1.8) showed higher activity than their respective R-isomers
5d (IR = 1.2), 5e (IR = 1.8), and 10b (IR = 1.4). However, for the prop-
argyl alkylation R-isomer product 5f and compound 11b, higher IR
values (2.0 for 5f and 1.8 for 11b) were observed compared with
their corresponding S-isomers 5c (IR = 1.7) and 11a (IR = 1.3). It
was further noted that higher activities were observed for urea
derivatives with an alkyl chain. For example, compounds 6b and
6g with the isopropyl chain showed almost equal activity with IR
values of 2.9 and 2.7, respectively. More potent activity was ob-
served for the urea derivative with a longer alkyl chain, and the com-
pounds with an n-pentyl group proved to be the best inducers of
QR1: the R-isomer 6f had an IR value of 4.3 and a CD value of
COOMe
Ha
O
Hd
N
0.2
(CD = 3.8
significant cytotoxicity (12.7% and 7.8% survival at 50
l
M and its corresponding S-isomer 6a had an IR value of 4.9
M). Unfortunately, both compounds also demonstrated
M for 6f
Hd
l
N
H
Hc
O
Hb
l
and 6a, respectively). On the basis of these data, there appears to
be a linear correlation between QR1 induction and the length of alkyl
side chains. In general, most compounds with a free carboxylic acid
group showed lower QR1 induction activity than the corresponding
ester compounds. Higher activity was observed for the allyl substi-
tuted carbamate carboxylic acid 7a, with an IR value of 2.4, than
other derivatives 7b–d in this carbamate acid series. Overall, the
alkylation free acid products 9a–f, 12a, b were more active, with
IR values in the 1.5–2.4 range, compared with other acid compounds
8a–f.
90 °C
40 °C
24 °C
2.2.2. Nitrite inhibition
The ability of carboline compounds 1–12 to inhibit NO produc-
tion was assessed, results are given in Table 2. Stereoisomers of
carbamate derivatives 3a–d and sulfonamide derivatives 4a–f
showed different activities in the applied assay. In the case of car-
bamate derivatives, both allyl and isobutyl substituted R-isomers
Hc
Hd
Ha
Hb
Figure 2. 1H NMR spectra of 3d at 24, 40, and 90 °C.

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6185
Table 1
Chiral HPLC analysis of selected ester and targeted compoundsa
Entry
Compd
Config.
Mobile phases (hexanes: IPA)b
Enantiomeric purity (%)
tR (min)
1
2
3
4
5
6
7
8
2a
2b
4b
4e
6a
6f
S-
R-
S-
R-
S-
R-
S-
R-
S-
R-
S-
R-
S-
R-
S-
R-
S-
R-
S-
R-
80:20
80:20
60:40
60:40
80:20
80:20
70:30
70:30
80:20
80:20
60:40
60:40
50:50
50:50
60:40
60:40
60:40
60:40
40:60
40:60
100.0
100.0
100.0
100.0
96.0
89.3
99.3
97.8
92.4
13.72
13.06
9.94
22.03
15.84
11.19
18.46
9.11
6d
6i
9
7b
7d
8a
8d
8b
8e
8c
8f
9a
9d
12a
12b
7.67
8.36
10
11
12
13
14
15
16
17
18
19
20
98.9
72.4c
79.3c
78.5c
82.5c
62.7c
100.0
100.0
100.0
100.0
100.0
11.22
12.68
11.22
13.61
13.61
14.47
10.79
12.25
11.21
11.19
a
Chiral HPLC analysis was performed on a Lux 5
lm Amylose-2 analytical column (250 Â 4.6 mm, Phenomenex) at ambient temperature using isopropanol (IPA) in
hexanes as mobile phase at a flow rate of 1.0 mL/min. For entries 1–2, 6 lL of 0.5 mg/mL of sample in IPA was injected. For entries 3–20, 3 lL of 1 mg/mL of sample in IPA was
injected. UV absorbance was measured at 220 nm.
b
0.1% of formic acid was added in mobile phases except entries 1–2.
Approximately 20–40% of partial racemization in the sulfonamide series 8 was observed.
c
COOMe
O
A
(S)N
N
H
HN
6d
COOMe
O
(R)N
N
H
HN
6i
B
CO2H
(S)
N
N
H
9a
CO2H
(R)N
N
H
9d
Figure 3. Representative chiral HPLC chromatographs: (A) enantiomeric urea derivatives 6d, i; (B) enantiomeric alkylation derivatives 9a, d.

Table 2
Biological activities of compounds 1–12 evaluated in cancer chemopreventive and anticancer assaysa
Compd
R
Config.
QR1
% surv.c
Aromatase
Nitrite assay
% surv.c
NF
j
B
SRB assay
IRb
CDd
(l
M)
% inhib.e
IC50
(lM)
% inhib.e
IC50
(l
M)
% inhib.e
% surv.c
IC50
(
lM)
% surv.c
IC50 (lM)
1a
1b
2a
2b
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
7a
7b
7c
7d
8a
8b
8c
8d
8e
8f
9a
9b
9c
9d
9e
9f
10a
10b
—
—
—
—
S-
R-
S-
R-
S-
S-
R-
R-
S-
S-
S-
R-
R-
R-
S-
S-
S-
R-
R-
R-
S-
S-
S-
S-
S-
R-
R-
R-
R-
R-
S-
S-
R-
R-
S-
S-
S-
R-
R-
R-
S-
S-
S-
R-
R-
R-
S-
R-
S-
R-
S-
R-
1.1
0.8
1.1
1.1
1.9
1.7
2.8
1.1
0.2
1.3
1.6
2.1
1.3
1.6
1.6
2.2
1.7
1.2
1.8
2.0
4.9
2.9
0.5
1.1
0.9
4.3
2.7
1.5
1.0
0.4
2.4
1.8
1.9
1.5
1.3
1.4
1.1
1.3
1.2
1.9
2.4
2.4
2.2
1.8
1.8
1.6
1.8
1.4
1.3
1.8
1.7
1.5
99.2
90.7
124.9
115.0
83.4
88.3
77.3
78.8
36.4
61.0
85.8
83.7
85.8
86.3
100.5
68.6
89.6
96.8
81.8
77.2
7.8
61.2
59.6
71.5
78.1
12.7
77.0
51.7
71.8
30.6
82.9
94.2
93.8
87.5
97.8
88.8
96.7
88.3
93.8
80.6
105.2
90.7
91.9
107.5
101.6
94.6
72.8
85.4
91.7
98.6
88.3
91.2
20.7
34.9
18.3
14.3
54.6
41.5
35.1
31.7
36.7
41.7
30.5
35.5
28.2
40.2
31.5
47.7
24.4
38.4
37.9
31.7
À0.04
54.0
31.8
33.0
30.3
11.1
32.6
24.6
24.4
39.9
38.4
40.0
32.9
36.3
23.2
16.9
26.5
32.3
23.0
30.7
28.0
30.2
30.5
19.2
31.5
23.5
28.0
27.3
46.1
38.7
57.9
38.4
2.0
3.5
12.5
7.2
8.5
11.1
57.1
60.1
70.6
73.8
10.6
3.8
100.1
95.9
104.1
116.5
94.2
104.8
91.6
92.6
89.2
116.1
91.9
89.7
91.4
4.6
103.0
95.4
38.6
60.3
191.2
95.6
À72.5
58.3
58.4
33.3
59.8
50.7
91.6
32.8
6.0
38.9
22.1
Allyl
Isobutyl
Allyl
Isobutyl
4-Bromophenyl
4-Methylphenyl
Methyl
4-Bromophenyl
4-Methylphenyl
Methyl
44.8
64.5
70.4
76.7
69.5
72.0
À2.1
19.0
72.5
30.6
86.5
90.9
16.2
4.8
15.9
27.7
2.8
23.1
22.6
97.6
112.3
186.0
171.1
153.0
154.3
114.0
158.5
28.8
152.2
116.8
77.1
253.9
226.9
109.5
116.8
94.6
111.2
139.0
140.2
142.8
108.2
100.6
110.3
109.1
137.8
203.7
264.3
246.8
271.9
255.6
251.9
194.8
178.4
49.1
22.0
37.0
63.2
38.6
43.7
25.2
42.2
60.4
54.1
21.1
54.4
29.9
43.6
35.5
69.0
87.7
44.7
60.7
62.1
73.7
81.7
20.9
36.5
35.6
42.6
49.4
46.1
55.4
42.5
51.1
50.9
26.0
À2.3
59.5
33.7
50.2
41.8
48.4
À7.7
24.7
40.5
47.8
33.5
55.0
54.6
29.7
34.0
42.1
7.4
68.1
58.6
12.9
68.2
23.5
22.3
74.7
5.6
79.9
85.9
99.8
84.4
Benzyl
32.0
32.9
30.7
44.2
Cyclopropylmethyl
Propargyl
Benzyl
85.6
81.1
81.2
Cyclopropylmethyl
Propargyl
n-Pentyl
Isopropyl
Phenethyl
Cyclohexyl
1-Adamantyl
n-Pentyl
Isopropyl
Phenethyl
Cyclohexyl
1-Adamantyl
Allyl
Isobutyl
Allyl
Isobutyl
4-Bromophenyl
4-Methylphenyl
Methyl
4-Bromophenyl
4-Methylphenyl
Methyl
124.5
114.3
94.9
94.3
68.9
79.0
66.7
90.5
89.4
66.1
83.8
63.2
134.9
137.3
134.5
134.4
80.6
101.9
77.8
40.3
71.0
1.3
26.3
37.6
50.5
66.1
4.3
50.0
3.8
6.4
41.1
8.7
38.4
47.3
37.8
48.2
53.8
64.1
36.2
16.2
66.2
55.0
64.7
À6.3
À3.4
À4.0
1.5
50.0
7.0
6.4
56.2
2.9
5.0
26.5
16.9
25.4
35.8
36.9
41.1
13.4
10.2
15.9
21.2
13.3
0.2
8.6
11.0
41.6
11.7
27.7
18.4
14.7
5.3
9.9
26.5
107.9
104.6
101.5
97.4
77.0
96.5
96.2
91.2
99.8
88.7
72.0
49.7
46.1
85.5
94.2
102.6
102.3
93.1
119.4
134.6
126.5
108.9
129.8
87.9
93.9
86.8
75.5
87.3
Benzyl
8.1
27.6
18.7
6.9
À7.0
7.0
21.4
Cyclopropylmethyl
Propargyl
Benzyl
Cyclopropylmethyl
Propargyl
—
—
—
—
—
234.1
228.1
89.5
168.9
72.7
99.7
91.0
96.0
108.4
111.6
9.9
À10.3
16.4
58.0
75.9
62.1
63.7
16.0
27.8
113.4
106.6
99.4
96.7
94.7
100.3
85.2
71.1
39.2
29.4
38.8
37.5
11a
11b
57.4
10.5
0.23
122.0
102.4
132.9
12a(callophycin A)
12b(callophycin A)
Standard Controlf
90.3
94.7
—
0.01
22.1
4.9
0.056
a
b
c
d
e
f
Concentration for testing: 50
IR: induction ratio.
Percentage of cell survival in comparison with vehicle-treated controls.
CD is the concentration that doubles the activity. CD values were determined for compounds with IR > 2.
Percentage of inhibition in comparison with vehicle-treated controls.
lM.
IC50 or CD values of known bioactive compounds for the each assay; 4’-bromoflavone for QR1, naringenin for aromatase, N-tosyl-l-phenylalanine-chloromethyl ketone for NF
assay, and camptothecin for SRB assay.
j
B assay, L–NG-monomethyl arginine for nitrite

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6187
(3c and 3d) were more active than their corresponding S-isomers
(3a and 3b). For instance, compound 3d with an isobutyl substitu-
ent showed much lower micromolar inhibition of NO production
the R-isomer was inactive at 50
l
M. Most of the urea derivatives,
except 6b and 6g containing an isopropyl group, showed activities
with IC50 values in the 14.7–38.4 M range. The R-isomer 6j
having an adamantyl group being the most potently active
(IC50 = 14.7 M). Compounds 7–9 and 12, all displaying a carbox-
ylic acid group, showed no inhibition of MCF7 cell growth at
50 M presumably due to poor cell membrane penetration. Nota-
l
(IC50 = 2.8
50 M. For sulfonamide derivatives, S-isomers with a 4-bromophe-
nyl substituent (4a, IC50 = 23.1 M) and a 4-methylphenyl group
(4b, IC50 = 22.6 M) showed more potent activity than their corre-
sponding R-isomers (4d and 4e, both less than 10% inhibition at
50 M). Both methyl substituted isomers 4c (S) and 4f (R) exhib-
ited low inhibition. These data demonstrated that substituted phe-
nylsulfonamides with stereochemistry were more effective
lM) than the corresponding S-isomer 3b, 11.1% at
l
l
l
l
l
bly, compared with the previously reported antiproliferative activ-
ity of natural callophycin A,10 our synthetic sample didn’t show the
inhibitory activity in our MCF7 cell proliferation assay.
l
S
inhibitors of NO production than their R and less bulky methylsulf-
onamide analogues. In the alkylation series, clearly, the pair of chi-
ral isomers 5a (S) and 5d (R) with N-benzyl substitution showed
2.2.5. Aromatase inhibitory activity
For all compounds tested, the carbamate methyl ester 3a, urea
methyl ester 6b, and callophycin A (S) 12a showed more than
more potent inhibitory activities with IC50 = 32.0 and 32.9
respectively. The activity was maintained with further substitution
on the benzyl group (10–11, IC50 = 29.4–39.2 M). Nevertheless,
l
M,
50% inhibition at 50
12a being the most potent inhibitor (IC50 = 10.5
compounds were shown to be ineffective inhibitors of aromatase
at 50 M (Table 2).
l
M in an aromatase inhibitory assay, with
l
M); all the other
l
compounds 5b–c and 5e–f with alkyl and less bulky substituents,
l
such as cyclopropylmethyl and propargyl groups, showed less than
50% inhibition at 50 lM. In the cases of urea derivatives 6a–j, com-
3. Conclusion
pounds with a larger substituent showed enhanced activities. For
example, compounds 6d and 6i, each with a cyclohexyl group,
showed better inhibition of NO production (IC50 = 41.1 and
In summary, a focused chemical library of 50 tetrahydro-b-carb-
oline analogues inspired by callophycin A was designed, synthe-
sized, and tested in a series of bioassays related to cancer
chemoprevention and cancer treatment. This library yielded
five compounds considered to have significant activity in one or
more of the applied assays. The S-isomer of the carboline urea
derivative 6a bearing an n-pentyl group showed significant QR1
41.6
(6a–b, 6f–g, inhibition <50% at 50
was observed for adamantyl urea derivatives 6e and 6j, which
exhibited IC50 values of 13.4 and 11.7 M, respectively. Most com-
lM, respectively) than n-pentyl and isopropyl derivatives
lM). Further improved activity
l
pounds with free carboxylic acid functionality displayed negligible
inhibition, except that the 4-bromophenyl sulfonamide derivatives
8a and 8d demonstrated moderate inhibitions with IC50 values of
induction with an IR of 4.9 at 50
sponding R-isomer 6f has an IR value of 4.3 (CD = 0.2
isomer of the carbamate derivative 3d having an isobutyl group
was the most potent inhibitor of NF B, with an IC50 = 4.8 M. Good
inhibition of NO production was also observed for 3d
(IC50 = 2.8 M). The most potent inhibitor of MCF7 cell prolifera-
tion was the R-isomer of the urea derivative containing an ada-
mantyl group, 6j (IC50 = 14.7 M). The S-isomer 12a of
callophycin A demonstrated the most inhibitory activity of aroma-
tase (IC50 = 10.5 M). Based on these results, it is evident that these
lM (CD = 3.8
l
M), the corre-
lM). The R-
49.7 and 46.1 lM, respectively.
j
l
2.2.3. NF
Most of the R-isomers were slightly more active than their cor-
responding S-isomers in the NF B assay. The carbamate deriva-
jB inhibitory activity
l
j
tives 3b and 3d with an isobutyl substituent showed significant
inhibitory activity in this assay. The R-isomer 3d demonstrated
l
the most potent inhibitory activity with an IC50 of 4.8
activities were observed for the sulfonamide derivatives 4a–f
(inhibition <50% at 50 M). For the alkylation products, com-
pounds with a benzyl substituent (5a with IC50 = 30.7 M and 5d
with IC50 = 44.2 M) exhibited higher inhibition than compounds
with cyclopropylmethyl and propargyl groups (5b–c, 5e–f, 21.1–
54.1% inhibition at 50 M). Further substitution on the benzene
ring resulted in lower activities (10, 11, inhibition <50% at
50 M). Most urea derivatives (6b–c, 6e–h) exhibited good activi-
ties in the NF B inhibition assay. However, both isomers with a
cyclohexyl group (6d with 44.7% and 6i with 20.9% inhibition at
50 M) were significantly less potent than other urea derivatives
(6b–c, 6e–h with IC50 values in the 10.2–26.5 M range). All the
synthesized carboline derivatives with a free acid, except for alkyl-
ation products 9a and callophycin A, demonstrated weak NF
activity at 50 M. The benzyl substituted alkylation S-isomer 9a
showed inhibition of NF B activity with an IC50 of 21.4 M.
lM. Low
l
emerging callophycin A analogues will serve as important leads for
further synthesis and optimization. Future synthetic efforts will fo-
cus on optimization of the methyl ester group at the C2 position to
improve metabolic stability, and production of a series of N9
derivatives.
l
l
l
l
4. Experimental
4.1. Chemistry
l
j
l
All reagents and solvents obtained from commercial sources
were used without further purification. Reactions were monitored
either by thin-layer chromatography (TLC) or by analytical HPLC
employing a Shimadzu LC-20A series high performance liquid
chromatography (HPLC) system. TLC was performed using glass
plates pre-coated with silica gel (0.25 mm, 60-Å pore size, 230–
400 mesh, Sorbent Technologies, GA) impregnated with a fluores-
cent indicator (254 nm). TLC plates were visualized by exposure
to ultraviolet light (UV). Hydrogenation reactions were done using
domnick hunter NITROX UHP-60H hydrogen generator, USA. Flash
column chromatography on silica gel was performed using a Bio-
tage Isolera One system and a Biotage SNAP cartridge. Proton and
carbon nuclear magnetic resonance (1H and 13C NMR) spectra were
recorded employing a Bruker Avance DRX-400 spectrometer.
Chemical shifts were expressed in ppm, J values were in Hz. ESI
mass spectra in either positive or negative mode were recorded
l
jB
l
j
l
2.2.4. Inhibition of MCF7 cell proliferation
All carbamate derivatives (3a–d) and alkylation products (5a–f,
10–12) were inactive at 50 M in this assay. For sulfonamide deriv-
l
atives, S-isomers were more active than their corresponding R-iso-
mers. Compound 4a with a 4-bromophenyl substituent exhibited
good inhibition of MCF7 cell proliferation with an IC50 of
22.0
42.1
l
l
M, while the R-isomer (4d) demonstrated an IC50 of
M. The difference between the two isomers of the 4-methyl-
phenyl substituted compound 4b and 4e was larger: the S-isomer
inhibited the growth of MCF7 cells with an IC50 of 37.0 M, while
l

6188
L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
on a Varian 500-MS IT Mass Spectrometer. Compounds’ purity was
J = 15.6 Hz, 4b-H), 3.09–3.01 (1H, m, 4a
-H) ppm. 13C NMR
determined by analytical HPLC using a Gemini, 3
l
m, C18, 110Å
(DMSO-d6, 100 MHz): d = 172.0, 171.9, 156.1, 155.6, 136.7, 136.6,
133.6, 130.1, 130.0, 126.6, 121.6 (2C), 119.1, 118.2, 117.7, 117.6,
111.6, 104.7, 104.4, 66.4, 66.2, 53.6, 53.3, 52.9 (2C), 40.9, 40.7,
23.5, 23.2 ppm. ESI-MS: calcd for C17H17N2O4 [MÀH]À: 313.1,
found: 313.3. HPLC purity: 99.8(254 nm), tR: 6.82 min;
99.7(220 nm), tR: 6.82 min.
column (50 mm  4.6 mm, Phenomenex) and
a flow rate of
1.0 mL/min. Gradient conditions: solvent A (0.1% trifluoroacetic
acid in water) and solvent B (acetonitrile): 0–2.00 min 100% A,
2.00–7.00 min 0–100% B (linear gradient), 7.00–8.00 min 100% B,
UV detection at 254 and 220 nm.
4.1.2.2. (S)-2-Isobutyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]
4.1.1. General procedure for compounds 2a–b22
indole-2,3(9H)-dicarboxylate (3b).
Yellow viscous oil. Yield:
At 0 °C, thionyl chloride (2.1 mL, 24.1 mmol) was added drop-
wise to a solution of 1a or 1b (1 g, 98%, 4.6 mmol) in methanol
(80 mL). The reaction mixture was stirred at room temperature
for 3 days and completion of the reaction was monitored by HPLC.
The excess methanol and thionyl chloride were removed by evap-
oration. The residue was dissolved in dichloromethane and washed
successively with saturated Na2CO3 and NaCl solutions. The dried
dichloromethane layer was evaporated and purified by flash col-
umn chromatography on silica gel to provide 2a or 2b.
100%. 1H NMR (DMSO-d6, 400 MHz): a mixture of rotamers,
d = 10.91 and 10.87 (1H, s, 9-NH), 7.44 (1H, d, J = 7.7 Hz, 5-H),
7.30 (1H, d, J = 8.1 Hz, 8-H), 7.08–7.04 (1H, m, 7-H), 7.00–6.96
(1H, m, 6-H), 5.30 and 5.27 (1H, d, J = 5.3 and 5.4 Hz, 3-H), 4.84
and 4.78 (1H, d, J = 16.1 and 16.7 Hz, 1a-H), 4.50 and 4.38 (1H, d,
J = 16.2 and 16.6 Hz, 1b-H), 3.99-3.83 (2H, m, –CH2CH(CH3)2),
3.58 and 3.57 (3H, s, –COOCH3), 3.32 (1H, d, J = 16.0 Hz, 4b-H),
3.09–3.01 (1H, m, 4a-H), 2.01–1.86 (1H, m, –CH2CH(CH3)2), 0.96
and 0.90 (6H, d, J = 6.7 Hz; dd, J = 6.7 and 1.9 Hz, –CH2CH(CH3)2)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.1, 156.5, 156.1,
136.6 (2C), 130.2, 130.1, 126.6, 121.6 (2C), 119.1, 118.1, 111.6,
104.7, 104.3, 71.8 (2C), 53.5, 53.2, 52.9 (2C), 40.9, 28.0 (2C), 23.4,
4.1.1.1. (S)-Methyl 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
carboxylate (2a).
White powder. Yield: 91%. 1H NMR (DMSO-
d6, 400 MHz): d = 10.73 (1H, s, 9-NH), 7.38 (1H, d, J = 7.7 Hz, 5-H),
7.28 (1H, d, J = 7.9 Hz, 8-H), 7.02 (1H, t, J = 7.5 Hz, 7-H), 6.94 (1H,
t, J = 7.4 Hz, 6-H), 3.97 (2H, ABq, J = 15.9 Hz, 1-H), 3.74 (1H, dd,
J = 8.8 and 4.8 Hz, 3-H), 3.69 (3H, s, –COOCH3), 3.33 (1H, br s, 2-
NH), 2.94 (1H, dd, J = 14.9 and 4.6 Hz, 4b-H), 2.75 (1H, dd, J = 15.0
23.2, 19.3(2C), 19.2(2C) ppm. ESI-MS: calcd for
C18H21N2O4
[MÀH]À: 329.2, found: 329.4. HPLC purity: 98.4(254 nm), tR:
7.20 min; 98.4(220 nm), tR: 7.19 min.
4.1.2.3. (R)-2-Allyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]
and 8.8 Hz, 4a
-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 174.1,
indole-2,3(9H)-dicarboxylate (3c).
Yellow viscous oil. Yield:
a mixture of rotamers,
79%. 1H NMR (DMSO-d6, 400 MHz):
136.2, 133.9, 127.4, 120.9, 118.7, 117.7, 111.3, 106.0, 55.7, 52.1,
41.9, 25.5 ppm. ESI-MS: calcd for C13H15N2O2 [M+H]+: 231.1,
found: 231.2. HPLC purity: 99.5(254 nm), tR: 5.22 min; 99.5
(220 nm), tR: 5.22 min.
d = 10.92 and 10.87 (1H, s, 9-NH), 7.44 (1H, d, J = 7.7 Hz, 5-H),
7.31 (1H, d, J = 8.0 Hz, 8-H), 7.08–7.05 (1H, m, 7-H), 7.00–6.96
(1H, m, 6-H), 6.05–5.88 (1H, m, –CH2CH@CH2), 5.39–5.21 (3H, m,
3-H and –CH2CH@CH2), 4.86 and 4.81 (1H, d, J = 16.4 and
17.3 Hz, 1
(1H, d, J = 16.2 and 16.2 Hz, 1b-H), 3.58 (3H, s, –COOCH3), 3.33
(1H, d, J = 15.6 Hz, 4b-H), 3.11–2.98 (1H, m, 4
-H) ppm. 13C NMR
(DMSO-d6, 100 MHz): d = 172.0, 156.1, 155.6, 136.7, 136.6, 133.6,
130.1, 130.0, 126.6, 121.6, 119.1, 118.2, 117.7, 117.6, 111.6,
104.7, 104.4, 66.4, 66.2, 53.6, 53.3, 52.9 (2C), 40.6, 40.4, 23.5,
23.2 ppm. ESI-MS: calcd for C17H17N2O4 [MÀH]À: 313.1, found:
313.3. HPLC purity: 98.9(254 nm), tR: 6.83 min; 98.7(220 nm), tR:
6.84 min.
a-H), 4.68–4.60 (2H, m, –CH2CH@CH2), 4.51 and 4.39
4.1.1.2. (R)-Methyl 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3
-carboxylate (2b).
White powder. Yield: 90%. 1H NMR
a
(DMSO-d6, 400 MHz): d = 10.73 (1H, s, 9-NH), 7.38 (1H, d,
J = 7.7 Hz, 5-H), 7.27 (1H, d, J = 8.0 Hz, 8-H), 7.02 (1H, t, J = 7.5 Hz,
7-H), 6.94 (1H, t, J = 7.4 Hz, 6-H), 3.97 (2H, ABq, J = 15.9 Hz, 1-H),
3.74 (1H, dd, J = 8.7 and 4.8 Hz, 3-H), 3.68 (3H, s, –COOCH3), 3.34
(1H, br s, 2-NH), 2.94 (1H, dd, J = 15.0 and 4.5 Hz, 4b-H), 2.75
(1H, dd, J = 15.0 and 8.8 Hz, 4a
-H) ppm. 13C NMR (DMSO-d6,
100 MHz): d = 174.1, 136.2, 133.9, 127.4, 120.9, 118.7, 117.7,
111.3, 106.0, 55.7, 52.1, 41.9, 25.4 ppm. ESI-MS: calcd for
C
13H15N2O2 [M+H]+: 231.1, found: 231.2. HPLC purity:
4.1.2.4. (R)-2-Isobutyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]
indole-2,3(9H)-dicarboxylate (3d).
Yellow powder. Yield:
mixture of rotamers,
100(254 nm), tR: 5.21 min; 99.2(220 nm), tR: 5.21 min.
89%. 1H NMR (DMSO-d6, 400 MHz):
a
d = 10.91 and 10.87 (1H, s, 9-NH), 7.44 (1H, d, J = 7.8 Hz, 5-H),
7.30 (1H, d, J = 8.0 Hz, 8-H), 7.08–7.04 (1H, m, 7-H), 7.00–6.96
(1H, m, 6-H), 5.30 and 5.27 (1H, d, J = 5.3 and 5.4 Hz, 3-H), 4.84
and 4.78 (1H, d, J = 16.2 and 16.6 Hz, 1a-H), 4.50 and 4.38 (1H, d,
J = 16.2 and 16.5 Hz, 1b-H), 3.99–3.82 (2H, m, –CH2CH(CH3)2),
3.58 and 3.57 (3H, s, –COOCH3), 3.32 (1H, d, J = 16.0 Hz, 4b-H),
3.09–3.01 (1H, m, 4a-H), 2.01–1.86 (1H, m, –CH2CH(CH3)2), 0.95
and 0.90 (6H, d, J = 6.7 Hz; dd, J = 6.7 and 1.9 Hz, –CH2CH(CH3)2)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.0, 156.5, 156.1,
136.7 (2C), 130.3, 130.1, 126.6, 121.6 (2C), 119.1, 118.1, 111.6,
104.7, 104.3, 71.8 (2C), 53.5, 53.2, 52.9, 52.8, 40.9, 28.1, 28.0,
23.4, 23.2, 19.3(2C), 19.2(2C) ppm. ESI-MS: calcd for C18H21N2O4
[MÀH]À: 329.2, found: 329.3. HPLC purity: 97.4(254 nm), tR:
7.15 min; 97.9(220 nm), tR: 7.15 min
4.1.2. General procedure for compounds 3a–d
Triethylamine (29 lL, 0.21 mmol) was added to a solution of 2a
or 2b (0.17 mmol) in dichloromethane (2 mL), followed by the
addition of various chloroformates (0.21 mmol). The reaction
mixture was stirred at room temperature for 0.5–1.5 h. After the
reaction was complete (monitored by HPLC), the reaction mixture
was evaporated. The residue was purified by flash column
chromatography on silica gel to give products 3a–d.
4.1.2.1.
(S)-2-Allyl 3-methyl
3,4-dihydro-1H-pyrido[3,4-
b]indole-2,3(9H)-dicarboxylate (3a).
Yellow viscous oil.
Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): a mixture of rotamers,
d = 10.92 and 10.87 (1H, s, 9-NH), 7.44 (1H, d, J = 7.8 Hz, 5-H), 7.31
(1H, d, J = 8.0 Hz, 8-H), 7.09–7.05 (1H, m, 7-H), 7.00–6.96 (1H, m, 6-
H), 6.05-5.91 (1H, m, –CH2CH@CH2), 5.39–5.21 (3H, m, 3-H and –
4.1.3. General procedure for compounds 4a–f
CH2CH@CH2), 4.86 and 4.81 (1H, d, J = 16.4 and 17.4 Hz, 1a-H),
4.71–4.63 (2H, m, –CH2CH@CH2), 4.51 and 4.39 (1H, d, J = 16.2
Triethylamine (25.4 lL, 0.18 mmol) was added to a solution of
compound 2a or 2b (0.15 mmol) in dichloromethane (2 mL), fol-
and 16.6 Hz, 1b-H), 3.58 (3H, s, –COOCH3), 3.33 (1H, d,
lowed by the addition of substituted sulfonyl chlorides

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6189
(0.15 mmol). The reaction mixture was stirred at room tempera-
ture for 40–60 h. After the reaction was complete (monitored by
HPLC), the reaction mixture was evaporated. The residue was puri-
fied by flash column chromatography on silica gel to give products
4a–f.
For 4c and 4f: 1.5 equiv of methanesulfonyl chloride and trieth-
ylamine were used and the reaction was complete in 30 min based
on HPLC monitoring.
4.1.3.5. (R)-Methyl 2-tosyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (4e).
White powder. Yield: 99%. 1H
NMR (DMSO-d6, 400 MHz): d = 10.84 (1H, s, 9-NH), 7.74 (2H, d,
J = 8.3 Hz, MePh-H), 7.39 (2H, d, J = 8.1 Hz, MePh-H), 7.38 (1H, d,
J = 7.1 Hz, 5-H), 7.29 (1H, d, J = J = 8.0 Hz, 8-H), 7.07–7.03 (1H, m,
7-H), 6.97–6.93 (1H, m, 6-H), 5.18 (1H, dd, J = 6.4 and 1.0 Hz, 3-
H), 4.70 (1H, d, J = 15.1 Hz, 1
3.42 (3H, s, –COOCH3), 3.21 (1H, d, J = 15.9 Hz, 4b-H), 2.94 (1H,
dd, J = 15.8 and 6.5 Hz, 4
-H), 2.36 (3H, s, –PhCH3) ppm. 13C NMR
a-H), 4.47 (1H, d, J = 15.7 Hz, 1b-H),
a
4.1.3.1. (S)-Methyl 2-(4-bromophenylsulfonyl)-2,3,4,9-tetrahy-
(DMSO-d6, 100 MHz): d = 171.0, 144.0, 136.6, 136.5, 130.2, 129.2,
127.3, 126.6, 121.7, 119.2, 118.2, 111.6, 104.3, 54.2, 52.7, 41.0,
24.3, 21.4 ppm. ESI-MS: calcd for C20H19N2O4S [MÀH]À: 383.1,
found: 383.3. HPLC purity: 97.6(254 nm), tR: 7.11 min;
99.5(220 nm), tR: 7.11 min.
dro-1H-pyrido[3,4-b]indole-3-carboxylate (4a).
White floc.
Yield: 83%. 1H NMR (DMSO-d6, 400 MHz): d = 10.86 (1H, s, 9-NH),
7.84–7.79 (4H, m, BrPh-H), 7.39 (1H, d, J = 7.8 Hz, 5-H), 7.31–7.29
(1H, m, 8-H), 7.08–7.04 (1H, m, 7-H), 6.98–6.94 (1H, m, 6-H),
5.21 (1H, dd, J = 6.5 and 1.2 Hz, 3-H), 4.74 (1H, d, J = 15.4 Hz, 1
a-
H), 4.46 (1H, d, J = 15.6 Hz, 1b-H), 3.42 (3H, s, –COOCH3), 3.24
4.1.3.6. (R)-Methyl 2-(methylsulfonyl)-2,3,4,9-tetrahydro-1H-
(1H, d, J = 15.8 Hz, 4b-H), 2.97 (1H, dd, J = 15.9 and 6.5 Hz, 4
a
-H)
pyrido[3,4-b]indole-3-carboxylate (4f).
Pale yellow powder.
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 170.8, 138.7, 136.5,
132.9, 129.4, 129.0, 127.5, 126.5, 121.8, 119.2, 118.2, 111.7,
104.4, 54.4, 52.8, 41.2, 24.3 ppm. ESI-MS: calcd for C19H16BrN2O4S
[MÀH]À: 447.0, found: 447.2. HPLC purity: 99.5 (254 nm), tR:
7.24 min; 100(220 nm), tR: 7.24 min.
Yield: 93%. 1H NMR (DMSO-d6, 400 MHz): d = 10.94 (1H, s, 9-NH),
7.44 (1H, d, J = 7.8 Hz, 5-H), 7.32 (1H, d, J = 8.0 Hz, 8-H), 7.09–
7.05 (1H, m, 7-H), 7.01–6.97 (1H, m, 6-H), 5.06 (1H, dd, J = 6.4
and 1.4 Hz, 3-H), 4.70 (1H, d, J = 15.4 Hz, 1
J = 15.5 Hz, 1b-H), 3.60 (3H, s, –COOCH3), 3.30 (1H, d, J = 15.8 Hz,
4b-H), 3.14–3.06 (1H, m, 4
-H), 3.09 (3H, s, –SO2CH3) ppm. 13C
a-H), 4.56 (1H, d,
a
4.1.3.2. (S)-Methyl 2-tosyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylate (4b).
NMR (DMSO-d6, 400 MHz): d = 10.84 (1H, s, 9-NH), 7.74 (2H, d,
J = 8.3 Hz, MePh-H), 7.39 (2H, d, J = 8.1 Hz, MePh-H), 7.38 (1H, d,
J = 7.2 Hz, 5-H), 7.29 (1H, d, J = 8.0 Hz, 8-H), 7.07–7.03 (1H, m, 7-
H), 6.97–6.93 (1H, m, 6-H), 5.18 (1H, dd, J = 6.4 and 1.2 Hz, 3-H),
NMR (DMSO-d6, 100 MHz): d = 171.6, 136.6, 129.5, 126.7, 121.7,
White powder. Yield: 93%. 1H
119.2, 118.2, 111.6, 104.6, 54.4, 52.9, 40.8, 24.5 ppm. ESI-MS: calcd
for
C
14H15N2O4S [MÀH]À: 307.1, found: 307.2. HPLC purity:
95.4(254 nm), tR: 6.44 min; 97.7(220 nm), tR: 6.44 min.
4.1.4. General procedure for compounds 5a–f23
4.70 (1H, d, J = 15.4 Hz, 1
(3H, s, –COOCH3), 3.21 (1H, d, J = 15.9 Hz, 4b-H), 2.94 (1H, dd,
J = 15.8 and 6.5 Hz, 4
-H), 2.36 (3H, s, –PhCH3) ppm. 13C NMR
(DMSO-d6, 100 MHz): d = 171.0, 143.9, 136.7, 136.6, 130.2, 129.2,
127.3, 126.7, 121.7, 119.2, 118.1, 111.6, 104.4, 54.2, 52.6, 41.1,
24.3, 21.4 ppm. ESI-MS: calcd for C20H19N2O4S [MÀH]À: 383.1,
found: 383.3. HPLC purity: 99.4(254 nm), tR: 7.05 min;
99.9(220 nm), tR: 7.05 min.
a
-H), 4.47 (1H, d, J = 15.6 Hz, 1b-H), 3.42
Substituted bromides (0.12 mmol) and N,N-diisopropylethyl-
amine (DIPEA, 0.15 mmol) were added to the solution of 2
(0.1 mmol) in acetonitrile (5 mL). This mixture was heated under
reflux for 2–5 h. After the reaction was complete (monitored by
HPLC), the solvent was removed by evaporation. The residue was
dissolved in cold EtOAc (10 mL) and the mixture was then filtered
to remove the precipitate. The filtrate was concentrated in vacuo to
yield the yellow crude residue, which was further purified by flash
column chromatography on silica gel to give products 5a–f.
a
4.1.3.3. (S)-Methyl 2-(methylsulfonyl)-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylate (4c).
Light yellow pow-
4.1.4.1. (S)-Methyl 2-benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
der. Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): d = 10.94 (1H, s,
9-NH), 7.44 (1H, d, J = 7.8 Hz, 5-H), 7.32 (1H, d, J = 8.0 Hz, 8-H),
7.09–7.05 (1H, m, 7-H), 7.01–6.97 (1H, m, 6-H), 5.07 (1H, dd,
indole-3-carboxylate (5a).
Pale yellow powder. Yield: 62%.
1H NMR (DMSO-d6, 400 MHz): d = 10.66 (1H, s, 9-NH), 7.40–7.24
(7H, m, –CH2PhH, 5-H, and 8-H), 7.03–6.99 (1H, m, 7-H), 6.97–
J = 6.3 and 1.4 Hz, 3-H), 4.70 (1H, d, J = 15.7 Hz, 1
a-H), 4.56 (1H,
6.93 (1H, m, 6-H), 4.02–3.92 (4H, m, 3-H, –CH2Ph, 1a-H), 3.74
d, J = 15.5 Hz, 1b-H), 3.60 (3H, s, –COOCH3), 3.30 (1H, d,
(1H, d, J = 15.3 Hz, 1b-H), 3.60 (3H, s, –COOCH3), 3.10–3.00 (2H,
m, 4-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 173.3, 139.4,
136.4, 132.6, 129.0, 128.8, 127.6, 127.1, 121.0, 118.8, 117.8,
111.3, 104.8, 59.4, 58.7, 51.8, 45.8, 24.3 ppm. 13C NMR (DMSO-d6,
100 MHz): d = 173.3, 139.4, 136.4, 132.6, 129.0, 128.8, 127.6,
127.1, 121.0, 118.8, 117.8, 111.3, 104.8, 59.4, 58.7, 51.8, 45.8,
24.3 ppm. ESI-MS: calcd for C20H19N2O2 [MÀH]À: 319.1, found:
319.3. HPLC purity: 100(254 nm), tR: 5.74 min; 100(220 nm), tR:
5.74 min.
J = 15.8 Hz, 4b-H), 3.12–3.06 (1H, m, 4a-H), 3.09 (3H, s, –SO2CH3)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 171.6, 136.6, 129.5,
126.7, 121.7, 119.2, 118.2, 111.6, 104.6, 54.4, 52.9, 40.8,
24.5 ppm. ESI-MS: calcd for C14H15N2O4S [MÀH]À: 307.1, found:
307.3. HPLC purity: 99.3(254 nm), tR: 6.45 min; 99.3(220 nm), tR:
6.45 min.
4.1.3.4. (R)-Methyl 2-(4-bromophenylsulfonyl)-2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole-3-carboxylate
(4d).
White
powder. Yield: 78%. 1H NMR (DMSO-d6, 400 MHz): d = 10.86 (1H, s,
9-NH), 7.84–7.79 (4H, m, Ph-H), 7.39 (1H, d, J = 7.8 Hz, 5-H), 7.31–
7.29 (1H, m, 8-H), 7.08–7.04 (1H, m, 7-H), 6.98–6.94 (1H, m, 6-H),
4.1.4.2. (S)-Methyl 2-(cyclopropylmethyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (5b).
Brown viscous
oil. Yield: 72%. 1H NMR (DMSO-d6, 400 MHz): d = 10.75 (1H, s, 9-
NH), 7.37 (1H, d, J = 7.7 Hz, 5-H), 7.26 (1H, d, J = 8.0 Hz, 8-H),
7.03–6.99 (1H, m, 7-H), 6.95–6.92 (1H, m, 6-H), 4.09–3.99 (3H,
5.22 (1H, dd, J = 6.3 and 1.1 Hz, 3-H), 4.74 (1H, d, J = 15.5 Hz, 1
a-
H), 4.46 (1H, d, J = 15.6 Hz, 1b-H), 3.42 (3H, s, –COOCH3), 3.24 (1H,
d, J = 15.9 Hz, 4b-H), 2.97 (1H, dd, J = 15.8 and 6.5 Hz, 4
a
-H) ppm.
m, 3-H, 1-H), 3.54 (3H, s, –COOCH3), 3.05–2.96 (2H, m,
CH2CH(CH2)2), 2.72 (1H, dd, J = 12.6 and 6.3 Hz, 4b-H), 2.62 (1H,
dd, J = 12.6 and 6.9 Hz, 4 -H), 0.96–0.86 (1H, m, –CH2CH(CH2)2),
–
13C NMR (DMSO-d6, 100 MHz): d = 170.8, 138.7, 136.5, 132.9,
129.4, 129.0, 127.5, 126.5, 121.8, 119.2, 118.2, 111.7, 104.4, 54.4,
52.8, 41.2, 24.3 ppm. ESI-MS: calcd for C19H16BrN2O4S [MÀH]À:
447.0, found: 447.2. HPLC purity: 100(254 nm), tR: 7.19 min;
100(220 nm), tR: 7.19 min.
a
0.57–0.46 (2H, m, –CH2CH(CH2)2), 0.19–0.07 (2H, m,
–
CH2CH(CH2)2) ppm. 13C NMR (CDCl3, 100 MHz): d = 173.3, 136.2,
131.5, 127.1, 121.4, 119.3, 117.9, 110.7, 106.1, 59.4, 59.2, 51.6,

6190
L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
46.5, 23.8, 9.4, 4.2, 3.6 ppm. ESI-MS: calcd for C17H19N2O2 [MÀH]À:
283.1, found: 283.3. HPLC purity: 96.3(254 nm), tR: 5.50 min;
95.1(220 nm), tR: 5.50 min.
reaction mixture was evaporated. The residue was purified by flash
column chromatography on silica gel to give products 6a–j.
4.1.5.1. (S)-Methyl 2-(pentylcarbamoyl)-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylate (6a).
Light yellow pow-
4.1.4.3. (S)-Methyl 2-(prop-2-ynyl)-2,3,4,9-tetrahydro-1H-pyrido
der. Yield: 88%. 1H NMR (DMSO-d6, 400 MHz): d = 10.89 (1H, s, 9-
NH), 7.42 (1H, d, J = 7.8 Hz, 5-H), 7.30 (1H, d, J = 8.0 Hz, 8-H),
7.07–7.03 (1H, m, 7-H), 6.99–6.95 (1H, m, 6-H), 6.76 (1H, t,
J = 5.4 Hz, –CONH), 5.36 (1H, d, J = 5.0 Hz, 3-H), 4.75 (1H, d,
[3,4-b]indole-3-carboxylate (5c).
Light yellow powder.
Yield: 73%. 1H NMR (DMSO-d6, 400 MHz): d = 10.79 (1H, s, 9-NH),
7.38 (1H, d, J = 7.7 Hz, 5-H), 7.28 (1H, d, J = 8.0 Hz, 8-H), 7.05–
7.01 (1H, m, 7-H), 6.97–6.93 (1H, m, 6-H), 4.05–3.96 (3H, m, 3-H,
1-H), 3.72 (1H, dd, J = 16.7 and 2.4 Hz, –CH2C„CH), 3.66 (1H, dd,
J = 16.7 and 2.4 Hz, –CH2C„CH), 3.58 (3H, s, –COOCH3), 3.26 (1H,
t, J = 2.4 Hz, –CH2C„CH), 3.01 (2H, d, J = 4.6 Hz, 4-H) ppm. 13C
NMR (DMSO-d6, 100 MHz): d = 173.0, 136.4, 132.2, 126.9, 121.0,
118.8, 117.9, 111.4, 104.5, 80.6, 76.1, 58.6, 51.8, 45.9, 43.7,
24.7 ppm. ESI-MS: calcd for C16H15N2O2 [MÀH]À: 267.1, found:
267.2. HPLC purity: 93.8(254 nm), tR: 5.59 min; 92.9(220 nm), tR:
5.59 min.
J = 15.6 Hz, 1
COOCH3), 3.27 (1H, d, J = 15.6 Hz, 4b-H), 3.12–3.07 (2H, m, –CON-
HCH2), 2.96 (1H, dd, J = 15.6 and 6.3 Hz, 4 -H), 1.50–1.43 (2H, m,
a-H), 4.38 (1H, d, J = 15.7 Hz, 1b-H), 3.52 (3H, s, –
a
–CONHCH2CH2), 1.34–1.22 (4H, m, –CONHCH2CH2CH2CH2), 0.88
(3H, t, J = 7.0 Hz, –CONH(CH2)4CH3) ppm. 13C NMR (DMSO-d6,
100 MHz): d = 172.8, 158.5, 136.8, 130.9, 126.7, 121.4, 119.0,
118.1, 111.5, 105.0, 52.4(2C), 40.8, 29.9, 29.1, 23.7, 22.4,
14.4 ppm. ESI-MS: calcd for C19H24N3O3 [MÀH]À: 342.2, found:
342.3. HPLC purity: 97.2(254 nm), tR: 6.85 min; 97.5(220 nm), tR:
6.85 min.
4.1.4.4. (R)-Methyl 2-benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole-3-carboxylate (5d).
Pale yellow powder. Yield: 84%.
4.1.5.2. (S)-Methyl 2-(isopropylcarbamoyl)-2,3,4,9-tetrahydro-
1H NMR (DMSO-d6, 400 MHz): d = 10.66 (1H, s, 9-NH), 7.40–7.24
(7H, m, –CH2PhH, 5-H, and 8-H), 7.03–6.99 (1H, m, 7-H), 6.97–
1H-pyrido[3,4-b]indole-3-carboxylate (6b).
Yellow powder.
Yield: 99%. 1H NMR (DMSO-d6, 400 MHz): d = 10.87 (1H, s, 9-NH),
7.42 (1H, d, J = 7.8 Hz, 5-H), 7.30 (1H, d, J = 8.0 Hz, 8-H), 7.07–
7.03 (1H, m, 7-H), 6.99–6.95 (1H, m, 6-H), 6.48 (1H, d, J = 7.5 Hz,
6.93 (1H, m, 6-H), 4.02–3.92 (4H, m, 3-H, –CH2Ph, 1a-H), 3.74
(1H, d, J = 15.2 Hz, 1b-H), 3.60 (3H, s, –COOCH3), 3.10–3.00 (2H,
m, 4-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 173.3, 139.4,
136.4, 132.6, 129.0, 128.8, 127.6, 127.1, 121.0, 118.8, 117.8,
111.3, 104.8, 59.4, 58.7, 51.8, 45.8, 24.3 ppm. ESI-MS: calcd for
–CONH), 5.35 (1H, d, J = 5.0 Hz, 3-H), 4.80 (1H, d, J = 15.7 Hz, 1a-
H), 4.36 (1H, d, J = 15.8 Hz, 1b-H), 3.87–3.78 (1H, m, –CONHCH),
3.53 (3H, s, –COOCH3), 3.26 (1H, d, J = 15.6 Hz, 4b-H), 2.96 (1H,
C
20H19N2O2 [MÀH]À: 319.1, found: 319.3. HPLC purity:
dd, J = 15.6 and 6.3 Hz, 4a-H), 1.13 (3H, d, J = 6.6 Hz, –CON-
100(254 nm), tR: 5.75 min; 99.5(220 nm), tR: 5.74 min
HCH(CH3)2), 1.10 (3H, d, J = 6.5 Hz, –CONHCH(CH3)2) ppm. 13C
NMR (DMSO-d6, 100 MHz): d = 172.8, 158.0, 136.8, 131.0, 126.7,
121.4, 119.0, 118.1, 111.5, 104.9, 52.5, 52.4, 42.6, 23.7, 23.4,
23.3 ppm. ESI-MS: calcd for C17H20N3O3 [MÀH]À: 314.2, found:
314.2. HPLC purity: 94.8(254 nm), tR: 6.40 min; 97.1(220 nm), tR:
6.40 min.
4.1.4.5. (R)-Methyl 2-(cyclopropylmethyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (5e).
Light brown
solid. Yield: 69%. 1H NMR (DMSO-d6, 400 MHz): d = 10.76 (1H, s,
9-NH), 7.37 (1H, d, J = 7.7 Hz, 5-H), 7.26 (1H, d, J = 7.9 Hz, 8-H),
7.03–6.99 (1H, m, 7-H), 6.96–6.92 (1H, m, 6-H), 4.10–3.99 (3H,
m, 3-H, 1-H), 3.54 (3H, s, –COOCH3), 3.05–2.96 (2H, m,
CH2CH(CH2)2), 2.72 (1H, dd, J = 12.6 and 6.3 Hz, 4b-H), 2.62 (1H,
dd, J = 12.6 and 6.8 Hz, 4 -H), 0.96–0.85 (1H, m, –CH2CH(CH2)2),
0.57–0.46 (2H, m, –CH2CH(CH2)2), 0.19–0.07 (2H, m,
–
4.1.5.3. (S)-Methyl 2-(phenethylcarbamoyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (6c).
Yellow powder.
a
Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): d = 10.92 (1H, s, 9-
NH), 7.43 (1H, d, J = 7.7 Hz, 5-H), 7.32–7.19 (6H, m, -CH2PhH, 8-
H), 7.07–7.03 (1H, m, 7-H), 6.99–6.95 (1H, m, 6-H), 6.93 (1H, t,
J = 5.2 Hz, –CONH), 5.37 (1H, d, J = 5.8 Hz, 3-H), 4.73 (1H, d,
–
CH2CH(CH2)2) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 173.5,
136.4, 133.0, 127.1, 120.9, 118.7, 117.8, 111.3, 104.6, 59.7, 59.1,
51.6, 46.2, 24.5, 9.8, 4.5, 3.7 ppm. ESI-MS: calcd for C17H19N2O2
[MÀH]À: 283.1, found: 283.3. HPLC purity: 98.8(254 nm), tR:
5.44 min; 99.6(220 nm), tR: 5.44 min.
J = 15.5 Hz, 1a-H), 4.38 (1H, d, J = 15.8 Hz, 1b-H), 3.54 (3H, s, –
COOCH3), 3.32–3.26 (3H, m, –CONHCH2, 4b-H), 2.96 (1H, dd,
J = 15.5 and 6.3 Hz, 4a-H), 2.83–2.72 (2H, m, –CONHCH2CH2Ph)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.8, 158.4, 140.3,
136.8, 130.8, 129.2, 128.8, 126.7, 126.5, 121.4, 119.0, 118.1,
111.5, 105.0, 52.5, 52.4 (2C), 42.6, 36.3, 23.7 ppm. ESI-MS: calcd
4.1.4.6. (R)-Methyl 2-(prop-2-ynyl)-2,3,4,9-tetrahydro-1H-pyr-
ido[3,4-b]indole-3-carboxylate (5f).
Yellow powder. Yield:
84%. 1H NMR (DMSO-d6, 400 MHz): d = 10.79 (1H, s, 9-NH), 7.38
(1H, d, J = 7.7 Hz, 5-H), 7.28 (1H, d, J = 8.0 Hz, 8-H), 7.05–7.00
(1H, m, 7-H), 6.97–6.93 (1H, m, 6-H), 4.05–3.96 (3H, m, 3-H, 1-
H), 3.72 (1H, dd, J = 16.7 and 2.4 Hz, –CH2C„CH), 3.66 (1H, dd,
J = 16.7 and 2.5 Hz, –CH2C„CH), 3.58 (3H, s, –COOCH3), 3.26 (1H,
t, J = 2.4 Hz, –CH2C„CH), 3.01 (2H, d, J = 4.3 Hz, 4-H) ppm. 13C
NMR (DMSO-d6, 100 MHz): d = 173.0, 136.4, 132.2, 126.9, 121.0,
118.8, 117.9, 111.4, 104.5, 80.6, 76.1, 58.6, 51.8, 45.9, 43.7,
24.6 ppm. ESI-MS: calcd for C16H15N2O2 [MÀH]À: 267.1, found:
267.2. HPLC purity: 96.5(254 nm), tR: 5.59 min; 95.1(220 nm), tR:
5.58 min.
for
C
22H22N3O3 [MÀH]À: 376.2, found: 376.3. HPLC purity:
96.5(254 nm), tR: 6.88 min; 96.9(220 nm), tR: 6.88 min.
4.1.5.4. (S)-Methyl 2-(cyclohexylcarbamoyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (6d).
White powder.
Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): d = 10.86 (1H, s, 9-
NH), 7.42 (1H, d, J = 7.8 Hz, 5-H), 7.30 (1H, d, J = 8.0 Hz, 8-H),
7.07–7.03 (1H, m, 7-H), 6.98–6.95 (1H, m, 6-H), 6.47 (1H, d,
J = 7.7 Hz, –CONH), 5.34 (1H, dd, J = 6.0 and 1.1 Hz, 3-H), 4.80
(1H, d, J = 15.7 Hz, 1
s, –COOCH3), 3.50–3.44 (1H, m, –CONHCH), 3.26 (1H, d,
J = 15.6 Hz, 4b-H), 2.95 (1H, dd, J = 15.6 and 6.3 Hz, 4 -H), 1.81–
a-H), 4.36 (1H, d, J = 15.9 Hz, 1b-H), 3.52 (3H,
a
4.1.5. General procedure for compounds 6a–j
1.58 (5H, m, –CONHCH(CH2)5), 1.31–1.05 (5H, m, –CONHCH(CH2)5)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.8, 157.9, 136.8, 131.1,
126.7, 121.4, 119.0, 118.1, 111.5, 104.9, 52.4, 49.9, 33.5 (2C), 25.9,
25.6, 25.5, 23.7 ppm. ESI-MS: calcd for C20H24N3O3 [MÀH]À: 354.2,
The solutions of 2 (0.25 mmol) and a series of isocyanates in
dichloromethane (2 mL) were stirred at room temperature over-
night. After the reaction was complete (monitored by HPLC), the

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6191
found: 354.3. HPLC purity: 98.4(254 nm), tR: 6.84 min;
98.9(220 nm), tR: 6.84 min.
4.1.5.9. (R)-Methyl 2-(cyclohexylcarbamoyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (6i).
White powder.
Yield: 97%. 1H NMR (DMSO-d6, 400 MHz): d = 10.86 (1H, s, 9-NH),
7.42 (1H, d, J = 7.7 Hz, 5-H), 7.30 (1H, d, J = 8.0 Hz, 8-H), 7.07–
7.03 (1H, m, 7-H), 6.98–6.95 (1H, m, 6-H), 6.47 (1H, d, J = 7.6 Hz,
–CONH), 5.35 (1H, dd, J = 6.2 and 1.4 Hz, 3-H), 4.80 (1H, d,
4.1.5.5. (S)-Methyl 2-(1-adamantylcarbamoyl)-2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole-3-carboxylate
(6e).
Yellow
powder. Yield: 93%. 1H NMR (DMSO-d6, 400 MHz): d = 10.85 (1H,
s, 9-NH), 7.42 (1H, d, J = 7.8 Hz, 5-H), 7.29 (1H, d, J = 8.0 Hz, 8-H),
7.06–7.02 (1H, m, 7-H), 6.98–6.94 (1H, m, 6-H), 5.94 (1H, s, –
CONH), 5.31 (1H, dd, J = 6.3 and 1.5 Hz, 3-H), 4.81 (1H, d,
J = 15.8 Hz, 1
COOCH3), 3.51–3.42 (1H, m, –CONHCH), 3.26 (1H, d, J = 15.6 Hz,
4b-H), 2.95 (1H, dd, J = 15.6 and 6.2 Hz, 4 -H), 1.84–1.58 (5H, m,
a-H), 4.36 (1H, d, J = 15.8 Hz, 1b-H), 3.52 (3H, s, –
a
J = 15.6 Hz, 1
a
-H), 4.37 (1H, d, J = 15.7 Hz, 1b-H), 3.53 (3H, s, –
-CONHCH(CH2)5), 1.31–1.05 (5H, m, –CONHCH(CH2)5) ppm. 13C
NMR (DMSO-d6, 100 MHz): d = 172.8, 158.0, 136.8, 131.1, 126.8,
121.4, 119.0, 118.0, 111.5, 104.9, 52.5, 52.4, 50.0, 33.5 (2C), 25.9,
25.6, 25.5, 23.7 ppm. ESI-MS: calcd for C20H24N3O3 [MÀH]À:
354.2, found: 354.3. HPLC purity: 99.2(254 nm), tR: 6.85 min;
99.8(220 nm), tR: 6.84 min.
COOCH3), 3.25 (1H, d, J = 15.5 Hz, 4b-H), 2.95 (1H, dd, J = 15.6 and
6.3 Hz, 4a-H), 2.03–1.98 (9H, m, adamantyl-H), 1.66–1.60 (6H, m,
adamantyl-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.8,
157.6, 136.7, 131.3, 126.7, 121.3, 119.0, 118.0, 111.5, 104.9, 52.4,
51.3, 42.0, 40.9, 40.6, 36.7, 29.5, 23.7 ppm. ESI-MS: calcd for
C
24H28N3O3 [MÀH]À: 406.2, found: 406.3. HPLC purity:
96.5(254 nm), tR: 7.35 min; 97.9(220 nm), tR: 7.35 min.
4.1.5.10. (R)-Methyl 2-(1-adamantylcarbamoyl)-2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole-3-carboxylate
(6j).
Yellow
4.1.5.6. (R)-Methyl 2-(pentylcarbamoyl)-2,3,4,9-tetrahydro-1H-
powder. Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): d = 10.84
(1H, s, 9-NH), 7.41 (1H, d, J = 7.7 Hz, 5-H), 7.28 (1H, d, J = 8.0 Hz,
8-H), 7.06–7.02 (1H, m, 7-H), 6.98-6.94 (1H, m, 6-H), 5.92 (1H, s,
-CONH), 5.31 (1H, dd, J = 6.0 and 1.3 Hz, 3-H), 4.80 (1H, d,
pyrido[3,4-b]indole-3-carboxylate
(6f).
White
powder.
Yield: 89%. 1H NMR (DMSO-d6, 400 MHz): d = 10.90 (1H, s, 9-NH),
7.42 (1H, d, J = 7.7 Hz, 5-H), 7.30 (1H, d, J = 8.0 Hz, 8-H), 7.07–
7.03 (1H, m, 7-H), 6.99–6.95 (1H, m, 6-H), 6.77 (1H, t, J = 5.4 Hz,
J = 15.8 Hz, 1a-H), 4.37 (1H, d, J = 15.7 Hz, 1b-H), 3.53 (3H, s, –
–CONH), 5.35 (1H, d, J = 5.2 Hz, 3-H), 4.75 (1H, d, J = 15.7 Hz, 1a-
COOCH3), 3.25 (1H, d, J = 15.5 Hz, 4b-H), 2.95 (1H, dd, J = 15.6 and
H), 4.37 (1H, d, J = 15.7 Hz, 1b-H), 3.52 (3H, s, –COOCH3), 3.27
(1H, d, J = 15.6 Hz, 4b-H), 3.15–3.03 (2H, m, –CONHCH2), 2.96
(1H, dd, J = 15.7 and 6.2 Hz, 4a-H), 1.49–1.42 (2H, m, –CON-
HCH2CH2), 1.36–1.20 (4H, m, –CONHCH2CH2CH2CH2), 0.88 (3H, t,
J = 6.9 Hz, –CONH(CH2)4CH3) ppm. 13C NMR (DMSO-d6,
100 MHz): d = 172.8, 158.5, 136.8, 130.9, 126.7, 121.4, 119.0,
118.1, 111.5, 105.0, 52.5, 52.4, 40.7, 29.9, 29.1, 23.7, 22.4,
14.5 ppm. ESI-MS: calcd for C19H24N3O3 [MÀH]À: 342.2, found:
342.4. HPLC purity: 98.1(254 nm), tR: 6.85 min; 98.4(220 nm), tR:
6.85 min.
6.3 Hz, 4a-H), 2.03–1.98 (9H, m, adamantyl-H), 1.66–1.60 (6H, m,
adamantyl-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.9,
157.6, 136.7, 131.3, 126.7, 121.3, 119.0, 118.0, 111.5, 104.9, 52.4,
51.3, 42.0, 40.9, 40.6, 36.7, 29.5, 23.7 ppm. ESI-MS: calcd for
C
24H28N3O3 [MÀH]À: 406.2, found: 406.2. HPLC purity:
96.9(254 nm), tR: 7.35 min; 98.4(220 nm), tR: 7.36 min.
4.1.6. General procedure for compounds 7–9
A solution of an appropriate methyl ester 3, 4, or 5 (0.1 mmol)
and LiOH (0.3 mmol) in THF/H2O (1:1, 4 mL) was stirred at room
temperature until the completion of the reaction (monitored by
HPLC). THF was removed in vacuo and then the solution was care-
fully neutralized with glacial acetic acid, whereupon a heavy pre-
cipitate was formed. The product was collected by filtration,
washed with cold water, and dried.
4.1.5.7. (R)-Methyl 2-(isopropylcarbamoyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (6g).
Yellow powder.
Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): d = 10.87 (1H, s, 9-
NH), 7.42 (1H, d, J = 7.8 Hz, 5-H), 7.30 (1H, d, J = 8.0 Hz, 8-H),
7.07–7.03 (1H, m, 7-H), 6.99–6.95 (1H, m, 6-H), 6.48 (1H, d,
J = 7.5 Hz, –CONH), 5.35 (1H, d, J = 5.2 Hz, 3-H), 4.79 (1H, d,
4.1.6.1.
(S)-2-(Allyloxycarbonyl)-2,3,4,9-tetrahydro-1H-pyr-
light brown pow-
J = 15.7 Hz, 1
m, –CONHCH), 3.53 (3H, s, –COOCH3), 3.26 (1H, d, J = 15.6 Hz, 4b-
H), 2.96 (1H, dd, J = 15.6 and 6.3 Hz, 4 -H), 1.13 (3H, d, J = 6.6 Hz,
a-H), 4.36 (1H, d, J = 15.7 Hz, 1b-H), 3.89–3.77 (1H,
ido[3,4-b]indole-3-carboxylic acid (7a).
der. Yield: 55%. 1H NMR (DMSO-d6, 400 MHz): a mixture of
rotamers, d = 12.90 (1H, br, –COOH), 10.90 and 10.85 (1H, s, 9-
NH), 7.43 (1H, d, J = 7.7 Hz, 5-H), 7.31 (1H, d, J = 8.0 Hz, 8-H), 7.06
(1H, t, J = 7.2 Hz, 7-H), 6.98 (1H, t, J = 7.3 Hz, 6-H), 6.05-5.92 (1H,
m, -CH2CH=CH2), 5.39–5.17 (3H, m, 3-H and –CH2CH=CH2), 4.83
a
–CONHCH(CH3)2), 1.10 (3H, d, J = 6.5 Hz, –CONHCH(CH3)2) ppm.
13C NMR (DMSO-d6, 100 MHz): d = 172.8, 158.0, 136.8, 131.0,
126.7, 121.4, 119.0, 118.1, 111.5, 104.9, 52.4 (2C), 42.6, 23.7,
23.4, 23.3 ppm. ESI-MS: calcd for C17H20N3O3 [MÀH]À: 314.2,
found: 314.3. HPLC purity: 95.4(254 nm), tR: 6.42 min;
97.1(220 nm), tR: 6.42 min.
and 4.77 (1H, d, J = 16.2 and 16.3 Hz, 1
CH2CH=CH2), 4.52 and 4.41 (1H, d, J = 16.1 and 16.5 Hz, 1b-H),
3.30-3.17 (1H, m, 4b-H), 3.04–2.96 (1H, m, 4
-H) ppm. 13C NMR
a-H), 4.70–4.59 (2H, m, -
a
(DMSO-d6, 100 MHz): d = 173.1 (2C), 156.2, 155.8, 136.7, 136.6,
133.7, 130.4, 130.2, 126.7, 121.5 (2C), 119.1, 118.1, 117.7, 117.4,
111.5, 105.0, 104.7, 66.2, 66.1, 53.6, 53.3, 41.0, 40.8, 23.6,
23.3 ppm. ESI-MS: calcd for C16H15N2O4 [MÀH]À: 299.1, found:
299.2. HPLC purity: 96.0(254 nm), tR: 6.45 min; 97.9(220 nm), tR:
6.45 min.
4.1.5.8. (R)-Methyl 2-(phenethylcarbamoyl)-2,3,4,9-tetrahydro-
1H-pyrido[3,4-b]indole-3-carboxylate (6h).
Yellow powder.
Yield: 100%. 1H NMR (DMSO-d6, 400 MHz): d = 10.92 (1H, s, 9-
NH), 7.43 (1H, d, J = 7.7 Hz, 5-H), 7.32–7.19 (6H, m, –CH2PhH, 8-
H), 7.07–7.04 (1H, m, 7-H), 6.99–6.95 (1H, m, 6-H), 6.94 (1H, t,
J = 5.2 Hz, –CONH), 5.37 (1H, d, J = 5.5 Hz, 3-H), 4.73 (1H, d,
J = 15.6 Hz, 1
a
-H), 4.38 (1H, d, J = 15.7 Hz, 1b-H), 3.54 (3H, s, –
4.1.6.2 (S)-2-(Isobutoxycarbonyl)-2,3,4,9-tetrahydro-1H-pyr-
COOCH3), 3.34–3.26 (3H, m, –CONHCH2, 4b-H), 2.96 (1H, dd,
ido[3,4-b]indole-3-carboxylic acid (7b)
.
Pale orange pow-
J = 15.6 and 6.2 Hz, 4
a
-H), 2.83–2.72 (2H, m, –CONHCH2CH2Ph)
der. Yield: 58%. 1H NMR (DMSO-d6, 400 MHz): a mixture of
rotamers, d = 12.87 (1H, br, –COOH), 10.90 and 10.86 (1H, s, 9-
NH), 7.43 (1H, d, J = 7.7 Hz, 5-H), 7.30 (1H, dd, J = 7.9 and 2.7 Hz,
8-H), 7.08–7.04 (1H, m, 7-H), 6.98 (1H, t, J = 7.2 Hz, 6-H), 5.17
and 5.14 (1H, d, J = 5.3 and 5.4 Hz, 3-H), 4.81 and 4.75 (1H, d,
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.8, 158.5, 140.3,
136.8, 130.8, 129.2, 128.8, 126.7, 126.5, 121.4, 119.0, 118.1,
111.5, 105.0, 52.5, 52.4 (2C), 42.6, 36.3, 23.7 ppm. ESI-MS: calcd
for
C
22H22N3O3 [MÀH]À: 376.2, found: 376.4. HPLC purity:
96.5(254 nm), tR: 6.86 min; 98.1(220 nm), tR: 6.86 min.
J = 16.2 and 16.5 Hz, 1a-H), 4.51 and 4.40 (1H, d, J = 16.1 and

6192
L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
16.5 Hz, 1b-H), 3.98–3.82 (2H, m, –CH2CH(CH3)2), 3.32 (1H, d,
J = 16.0 Hz, 4b-H), 3.04–2.96 (1H, m, 4 -H), 1.99–1.89 (1H, m, –
d, J = 15.6 Hz, 4b-H), 2.89 (1H, dd, J = 15.6 and 6.5 Hz, 4a-H), 2.35
a
(3H, s, –PhCH3) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.1,
143.7, 137.0, 136.5, 130.2, 129.4, 127.3, 126.7, 121.7, 119.1,
118.1, 111.6, 104.6, 54.1, 41.0, 24.4, 21.4 ppm. ESI-MS: calcd for
CH2CH(CH3)2), 0.95 and 0.91 (6H, d, J = 6.7 and dd, J = 6.7 and
1.3 Hz, –CH2CH(CH3)2) ppm. 13C NMR (DMSO-d6, 100 MHz):
d = 173.2, 156.5, 156.2, 136.6 (2C), 130.5, 130.3, 126.7, 121.5,
119.1, 118.1, 111.5, 105.0, 104.6, 71.7, 71.6, 53.4, 53.1, 41.0, 28.1,
28.0, 23.5, 23.3, 19.4, 19.3, 19.2 ppm. ESI-MS: calcd for
C
19H17N2O4S [MÀH]À: 369.1, found: 369.3. HPLC purity:
99.6(254 nm), tR: 6.68 min; 100(220 nm), tR: 6.68 min.
C
17H19N2O4 [MÀH]À: 315.1, found: 315.3. HPLC purity:
4.1.6.7.
(S)-2-(Methylsulfonyl)-2,3,4,9-tetrahydro-1H-pyr-
98.1(254 nm), tR: 6.74 min; 99.1(220 nm), tR: 6.74 min.
ido[3,4-b]indole-3-carboxylic acid (8c).
White powder.
Yield: 70%. 1H NMR (DMSO-d6, 400 MHz): d = 13.04 (1H, br, –
COOH), 10.93 (1H, s, 9-NH), 7.44 (1H, d, J = 7.7 Hz, 5-H), 7.32 (1H,
d, J = 8.0 Hz, 8-H), 7.09-7.05 (1H, m, 7-H), 6.99 (1H, t, J = 7.2 Hz,,
6-H), 4.93 (1H, dd, J = 6.4 and 1.2 Hz, 3-H), 4.67 (1H, d,
4.1.6.3.
(R)-2-(Allyloxycarbonyl)-2,3,4,9-tetrahydro-1H-pyr-
Yellow viscous oil.
ido[3,4-b]indole-3-carboxylic acid (7c).
Yield: 58%. 1H NMR (DMSO-d6, 400 MHz): a mixture of rotamers,
d = 12.91 (1H, br, –COOH), 10.90 and 10.85 (1H, s, 9-NH), 7.43
(1H, d, J = 7.8 Hz, 5-H), 7.31 (1H, d, J = 8.0 Hz, 8-H), 7.08–7.04
(1H, m, 7-H), 7.00–6.96 (1H, m, 6-H), 6.05–5.92 (1H, m, –
CH2CH@CH2), 5.39–5.17 (3H, m, 3-H and –CH2CH@CH2), 4.83 and
J = 15.2 Hz, 1
m, 4b-H), 3.08 (3H, s, –SO2CH3), 3.03 (1H, dd, J = 15.8 and 6.5 Hz,
-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 172.7, 136.6,
a-H), 4.58 (1H, d, J = 15.4 Hz, 1b-H), 3.30–3.29 (1H,
4a
129.7, 126.8, 121.6, 119.1, 118.1, 111.6, 104.9, 54.4, 40.8, 39.3,
24.7 ppm. ESI-MS: calcd for C13H13N2O4S [MÀH]À: 293.0, found:
293.2. HPLC purity: 99.3(254 nm), tR: 6.02 min; 99.5(220 nm), tR:
6.02 min.
4.77 (1H, d, J = 15.9 and 16.3 Hz, 1
CH2CH@CH2), 4.52 and 4.41 (1H, d, J = 16.2 and 16.5 Hz, 1b-H),
3.35–3.30 (1H, m, 4b-H), 3.04–2.96 (1H, m, 4
-H) ppm. 13C NMR
a-H), 4.70–4.59 (2H, m, –
a
(DMSO-d6, 100 MHz): d = 173.1, 156.2, 155.8, 136.7, 136.6, 133.7,
130.3, 130.2, 126.7, 121.5 (2C), 119.1, 118.1, 117.7, 117.4, 111.5,
105.0, 66.2, 66.1, 53.6, 53.3, 41.0, 40.8, 23.6, 23.2 ppm. ESI-MS:
calcd for C16H15N2O4 [MÀH]À: 299.1, found: 299.2. HPLC purity:
96.3(254 nm), tR: 6.49 min; 99.6(220 nm), tR: 6.49 min.
4.1.6.8. (R)-2-(4-Bromophenylsulfonyl)-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylic acid (8d).
White powder.
Yield: 40%. 1H NMR (DMSO-d6, 400 MHz): d = 10.85 (1H, s, 9-NH),
7.80 (4H, s, BrPh-H), 7.39 (1H, d, J = 7.8 Hz, 5-H), 7.30 (1H, d,
J = 8.0 Hz, 8-H), 7.07–7.03 (1H, m, 7-H), 6.98–6.94 (1H, m, 6-H),
4.1.6.4. (R)-2-(Isobutoxycarbonyl)-2,3,4,9-tetrahydro-1H-pyrido
5.04 (1H, d, J = 5.4 Hz, 3-H), 4.72 (1H, d, J = 14.9 Hz, 1
(1H, d, J = 15.3 Hz, 1b-H), 3.24 (1H, d, J = 15.7 Hz, 4b-H), 2.93 (1H,
dd, J = 15.9 and 6.5 Hz,
-H) ppm. 13C NMR (DMSO-d6,
a-H), 4.53
[3,4-b]indole-3-carboxylic acid (7d).
White powder. Yield:
mixture of rotamers,
62%. 1H NMR (DMSO-d6, 400 MHz):
a
4a
d = 12.89 (1H, br, –COOH), 10.90 and 10.86 (1H, s, 9-NH), 7.43
(1H, d, J = 7.6 Hz, 5-H), 7.30 (1H, dd, J = 7.7 and 2.5 Hz, 8-H),
7.08–7.04 (1H, m, 7-H), 7.00–6.96 (1H, m, 6-H), 5.17 and 5.14
(1H, d, J = 5.2 and 5.2 Hz, 3-H), 4.81 and 4.75 (1H, d, J = 16.2 and
100 MHz): d = 171.9, 138.9, 136.5, 132.8, 129.3, 129.2, 127.3,
126.6, 121.7, 119.2, 118.1, 111.6, 104.6, 54.3, 41.2, 24.5 ppm. ESI-
MS: calcd for C18H14BrN2O4S [MÀH]À: 433.0, found: 433.2. HPLC
purity: 98.5(254 nm), tR: 6.88 min; 100(220 nm), tR: 6.88 min.
16.5 Hz, 1
3.98–3.82 (2H, m, –CH2CH(CH3)2), 3.35–3.30 (1H, m, 4b-H), 3.04–
2.95 (1H, m, 4 -H), 1.98–1.88 (1H, m, –CH2CH(CH3)2), 0.95 and
a-H), 4.51 and 4.40 (1H, d, J = 16.1 and 16.6 Hz, 1b-H),
4.1.6.9. (R)-2-Tosyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3
-carboxylic acid (8e).
a
White powder. Yield: 81%. 1H NMR
0.91 (6H, d, J = 6.7 and dd, J = 6.7 and 1.4 Hz, –CH2CH(CH3)2)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 173.2, 156.5, 156.2,
136.6, 130.5, 130.3, 126.7, 121.5(2C), 119.1, 118.1, 111.5, 105.0,
104.6, 71.7, 71.6, 53.5, 53.1, 41.0, 40.7, 28.1, 28.0, 23.5, 23.3,
19.4, 19.3, 19.2 ppm. ESI-MS: calcd for C17H19N2O4 [MÀH]À:
315.1, found: 315.3. HPLC purity: 98.9(254 nm), tR: 6.76 min;
99.6(220 nm), tR: 6.76 min.
(DMSO-d6, 400 MHz): d = 12.89 (1H, br, –COOH), 10.83 (1H, s, 9-
NH), 7.74 (2H, d, J = 8.3 Hz, MePh-H), 7.37 (3H, d, J = 8.2 Hz,
MePh-H and 5-H), 7.29 (1H, d, J = 8.0 Hz, 8-H), 7.06–7.02 (1H, m,
7-H), 6.97–6.93 (1H, m, 6-H), 5.03 (1H, dd, J = 6.2 and 0.9 Hz, 3-
H), 4.67 (1H, d, J = 15.5 Hz, 1
3.21 (1H, d, J = 15.6 Hz, 4b-H), 2.88 (1H, dd, J = 15.6 and 6.5 Hz,
-H), 2.35 (3H, s, –PhCH3) ppm. 13C NMR (DMSO-d6, 100 MHz):
a-H), 4.54 (1H, d, J = 15.6 Hz, 1b-H),
4a
d = 172.1, 143.7, 137.0, 136.5, 130.2, 129.4, 127.3, 126.7, 121.6,
4.1.6.5. (S)-2-(4-Bromophenylsulfonyl)-2,3,4,9-tetrahydro-1H-
119.1, 118.1, 111.6, 104.6, 54.1, 41.0, 24.4, 21.4 ppm. ESI-MS: calcd
pyrido[3,4-b]indole-3-carboxylic acid (8a).
White powder.
for
C
19H17N2O4S [MÀH]À: 369.1, found: 369.3. HPLC purity:
Yield: 61%. 1H NMR (DMSO-d6, 400 MHz): d = 12.94 (1H, br, –
COOH), 10.85 (1H, s, 9-NH), 7.80 (4H, s, BrPh-H), 7.39 (1H, d,
J = 7.8 Hz, 5-H), 7.30 (1H, d, J = 8.1 Hz, 8-H), 7.07-7.03 (1H, m, 7-
H), 6.98–6.94 (1H, m, 6-H), 5.04 (1H, d, J = 5.5 Hz, 3-H), 4.72 (1H,
99.1(254 nm), tR: 6.66 min; 99.1(220 nm), tR: 6.66 min.
4.1.6.10
.
(R)-2-(Methylsulfonyl)-2,3,4,9-tetrahydro-1H-pyr-
ido[3,4-b]indole-3-carboxylic acid (8f).
White powder.
d, J = 15.2 Hz, 1
a-H), 4.53 (1H, d, J = 15.5 Hz, 1b-H), 3.24 (1H, d,
Yield: 58%. 1H NMR (DMSO-d6, 400 MHz): d = 13.04 (1H, br, –
COOH), 10.93 (1H, s, 9-NH), 7.44 (1H, d, J = 7.8 Hz, 5-H), 7.32 (1H,
d, J = 8.0 Hz, 8-H), 7.09–7.05 (1H, m, 7-H), 7.00–6.96 (1H, m, 6-H),
J = 15.7 Hz, 4b-H), 2.93 (1H, dd, J = 15.7 and 6.5 Hz, 4
a-H) ppm.
13C NMR (DMSO-d6, 100 MHz): d = 171.9, 138.9, 136.5, 132.8,
129.4, 129.2, 127.3, 126.6, 121.7, 119.2, 118.1, 111.6, 104.6, 54.3,
41.2, 24.5 ppm. ESI-MS: calcd for C18H14BrN2O4S [MÀH]À: 433.0,
found: 433.2. HPLC purity: 99.8(254 nm), tR: 6.85 min;
99.3(220 nm), tR: 6.85 min.
4.93 (1H, dd, J = 6.3 and 1.3 Hz, 3-H), 4.67 (1H, d, J = 14.9 Hz, 1
a-
H), 4.57 (1H, d, J = 15.4 Hz, 1b-H), 3.34–3.28 (1H, m, 4b-H), 3.07
(3H, s, –SO2CH3), 3.04 (1H, dd, J = 15.8 and 6.5 Hz, 4a-H) ppm.
13C NMR (DMSO-d6, 100 MHz): d = 172.7, 136.6, 129.7, 126.8,
121.6, 119.1, 118.1, 111.6, 104.9, 54.4, 40.8, 39.3, 24.6 ppm. ESI-
MS: calcd for C13H13N2O4S [MÀH]À: 293.0, found: 293.2. HPLC pur-
ity: 97.4(254 nm), tR: 6.02 min; 99.4(220 nm), tR: 6.02 min.
4.1.6.6. (S)-2-Tosyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-
3-carboxylic acid (8b).
White powder. Yield: 84%. 1H NMR
(DMSO-d6, 400 MHz): d = 12.90 (1H, br, –COOH), 10.83 (1H, s, 9-
NH), 7.74 (2H, d, J = 8.3 Hz, MePh-H), 7.37 (3H, d, J = 8.0 Hz,
MePh-H and 5-H), 7.29 (1H, d, J = 8.0 Hz, 8-H), 7.06–7.02 (1H, m,
7-H), 6.95 (1H, t, J = 7.4 Hz, 6-H), 5.03 (1H, d, J = 5.6 Hz, 3-H), 4.67
4.1.6.11. (S)-2-Benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
-3-carboxylic acid (9a).
NMR (DMSO-d6, 400 MHz): d = 10.64 (1H, s, 9-NH), 7.40–7.27 (6H,
m, -CH2Ph-H and 5-H), 7.25 (1H, d, J = 7.8 Hz, 8-H), 7.03–6.99 (1H,
Light yellow powder. Yield: 39%. 1H
(1H, d, J = 15.5 Hz, 1a-H), 4.55 (1H, d, J = 15.6 Hz, 1b-H), 3.21 (1H,

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6193
m, 7-H), 6.96–6.92 (1H, m, 6-H), 4.04–3.94 (3H, m, 1
a-H and -
d = 173.9, 136.5, 132.2, 127.1, 120.9, 118.7, 117.8, 111.3, 105.1,
CH2Ph), 3.86 (1H, dd, J = 6.1 and 3.4 Hz, 3-H), 3.72 (1H, d,
J = 15.2 Hz, 1b-H), 3.08 (1H, dd, J = 15.6 and 2.5 Hz, 4b-H), 2.99
59.7, 59.2, 46.3, 24.2, 9.5, 4.5, 3.8 ppm. ESI-MS: calcd for
C
99.1(254 nm), tR: 5.35 min; 98.7(220 nm), tR: 5.35 min.
16H17N2O2 [MÀH]À: 269.1, found: 269.2. HPLC purity:
(1H, dd, J = 15.2 and 5.9 Hz, 4
a
-H) ppm. 13C NMR (DMSO-d6,
100 MHz): d = 174.7, 139.6, 136.4, 132.8, 129.0, 128.8, 127.5,
127.2, 120.9, 118.7, 117.8, 111.3, 105.2, 59.6, 58.7, 45.9, 24.4 ppm.
ESI-MS: calcd for C19H17N2O2 [MÀH]À: 305.1, found: 305.2. HPLC
purity: 99.6(254 nm), tR: 5.55 min; 99.1(220 nm), tR: 5.56 min.
4.1.6.16. (R)-2-(Prop-2-ynyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole-3-carboxylic acid (9f).
Light brown solid. Yield:
43%. 1H NMR (DMSO-d6, 400 MHz): d = 10.65 (1H, s, 9-NH), 7.32
(1H, d, J = 7.6 Hz, 5-H), 7.24 (1H, d, J = 7.9 Hz, 8-H), 6.98 (1H, t,
J = 7.5 Hz,, 7-H), 6.91 (1H, t, J = 7.2 Hz,, 6-H), 4.12 (1H, d,
4.1.6.12. (S)-2-(Cyclopropylmethyl)-2,3,4,9-tetrahydro-1H-pyr-
J = 14.7 Hz, 1a-H), 3.80–3.75 (2H, m, 1b-H and -CH2C„CH), 3.65
(1H, dd, J = 16.4 and 2.4 Hz, –CH2C„CH), 3.39 (1H, overlapped with
water peak, 3-H), 3.09 (1H, t, J = 2.1 Hz, –CH2C„CH), 3.00 (1H, dd,
ido[3,4-b]indole-3-carboxylic acid (9b).
Pale yellow powder.
Yield: 41%. 1H NMR (DMSO-d6, 400 MHz): d = 10.76 (1H, s, 9-NH),
7.38 (1H, d, J = 7.7 Hz, 5-H), 7.27 (1H, d, J = 8.0 Hz, 8-H), 7.04–
7.00 (1H, m, 7-H), 6.96–6.92 (1H, m, 6-H), 4.18 (1H, d, J = 14.9 Hz,
J = 14.9 and 5.2 Hz, 4b-H), 2.80 (1H, dd, J = 14.9 and 5.6 Hz, 4a-H)
ppm. 13C NMR (DMSO-d6, 100 MHz): d = 175.3, 136.4, 133.0,
127.4, 120.5, 118.5, 117.7, 111.2, 106.4, 82.1, 75.1, 59.9, 46.7,
43.5, 25.3 ppm. ESI-MS: calcd for C15H13N2O2 [MÀH]À: 253.1,
found: 253.2. HPLC purity: 98.9(254 nm), tR: 5.18 min;
98.8(220 nm), tR: 5.18 min.
1a
-H), 4.04 (1H, d, J = 15.2 Hz, 1b-H), 3.93 (1H, dd, J = 5.9 and
3.7 Hz, 3-H), 3.08 (1H, dd, J = 15.6 and 2.8 Hz, 4b-H), 2.97 (1H, dd,
J = 15.5 and 6.2 Hz, 4 -H), 2.81–2.68 (2H, m, –CH2CH(CH2)2),
0.98–0.91 (1H, m, –CH2CH(CH2)2), 0.58–0.49 (2H, m,
a
-
CH2CH(CH2)2), 0.20–0.11 (2H, m, –CH2CH(CH2)2) ppm. 13C NMR
(DMSO-d6, 100 MHz): d = 173.9, 136.5, 132.2, 127.1, 120.9, 118.7,
117.8, 111.3, 105.1, 59.7, 59.2, 46.3, 24.2, 9.5, 4.5, 3.8 ppm. ESI-
MS: calcd for C16H17N2O2 [MÀH]À: 269.1, found: 269.3. HPLC pur-
ity: 96.8(254 nm), tR: 5.29 min; 97.5(220 nm), tR: 5.29 min.
4.1.7. General procedure for compounds 10a, b
4-(Benzyloxy)benzyl bromide (1.48 mmol) and N,N-diisopro-
pylethylamine (DIPEA, 1.48 mmol) were added to the solution of
2 (0.74 mmol) in acetonitrile (40 mL). This mixture was heated un-
der reflux for 5 h. After the reaction was complete (monitored by
HPLC), the solvent was removed by evaporation. The residue was
dissolved in EtOAc and washed with water and saturated NaCl
solution. The organic layer was dried over anhydrous Na2SO4 and
then filtered. The filtrate was concentrated in vacuo to yield the
yellow crude residue, which was further purified by flash column
chromatography on silica gel to give products 10a and 10b.
4.1.6.13. (S)-2-(Prop-2-ynyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole-3-carboxylic acid (9c).
Brown viscous oil. Yield:
44%. 1H NMR (DMSO-d6, 400 MHz): d = 10.74 (1H, s, 9-NH), 7.35
(1H, d, J = 7.7 Hz, 5-H), 7.26 (1H, d, J = 7.9 Hz, 8-H), 7.02–6.98
(1H, m, 7-H), 6.95–6.91 (1H, m, 6-H), 4.08 (1H, d, J = 14.8 Hz, 1a-
H), 3.88 (1H, d, J = 14.8 Hz, 1b-H), 3.76 (1H, dd, J = 16.5 and
2.5 Hz, –CH2C„CH), 3.67–3.62 (2H, m, 3-H and –CH2C„CH), 3.17
(1H, t, J = 2.4 Hz, –CH2C„CH), 3.02 (1H, dd, J = 15.0 and 4.5 Hz,
4.1.7.1. (S)-Methyl 2-(4-(benzyloxy)benzyl)-2,3,4,9-tetrahydro-
4b-H), 2.88 (1H, dd, J = 15.1 and 5.8 Hz, 4a
-H) ppm. 13C NMR
1H-pyrido[3,4-b]indole-3-carboxylate (10a).
Yellow pow-
der. Yield: 58%. 1H NMR (DMSO-d6, 400 MHz): d = 10.66 (1H, s, 9-
NH), 7.47–7.45 (2H, m, Ar-H), 7.42–7.37 (3H, m, Ar-H), 7.35–7.24
(4H, m, Ar-H), 7.03–6.92 (4H, m, Ar-H), 5.10 (2H, s, –OCH2Ph),
(DMSO-d6, 100 MHz): d = 175.1, 136.4, 132.7, 127.2, 120.7, 118.6,
117.8, 111.3, 105.6, 81.5, 75.4, 60.2, 46.4, 43.6, 25.0 ppm. ESI-MS:
calcd for C15H13N2O2 [MÀH]À: 253.1, found: 253.2. HPLC purity:
94.2(254 nm), tR: 5.18 min; 92.1(220 nm), tR: 5.18 min.
3.99–3.95 (2H, m, 3-H and 1a-H), 3.89 (2H, ABq, J = 13.2 Hz, -
NCH2Ph), 3.72 (1H, d, J = 15.2 Hz, 1b-H), 3.59 (3H, s, –COOCH3),
3.09–2.98 (2H, m, 4-H) ppm. 13C NMR (DMSO-d6, 100 MHz):
d = 173.3, 158.0, 137.6, 136.4, 132.7, 131.4, 130.3, 128.9, 128.3,
128.1, 127.1, 120.9, 118.8, 117.8, 115.1, 111.3, 104.8, 69.7, 59.2,
58.1, 51.7, 45.7, 24.3 ppm. ESI-MS: calcd for C27H25N2O3 [MÀH]À:
425.2, found: 425.4. HPLC purity: 99.0(254 nm), tR: 6.15 min;
98.6(220 nm), tR: 6.15 min.
4.1.6.14.
(R)-2-Benzyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]
indole-3-carboxylic acid (9d).
Yellow powder. Yield: 52%.
1H NMR (DMSO-d6, 400 MHz): d = 10.66 (1H, s, 9-NH), 7.40–7.27
(6H, m, –CH2Ph-H and 5-H), 7.25 (1H, d, J = 7.8 Hz, 8-H), 7.03–
6.99 (1H, m, 7-H), 6.96–6.93 (1H, m, 6-H), 4.04–3.94 (3H, m, 1a-
H and –CH2Ph), 3.86 (1H, dd, J = 6.0 and 3.3 Hz, 3-H), 3.71 (1H, d,
J = 15.1 Hz, 1b-H), 3.08 (1H, dd, J = 14.9 and 2.3 Hz, 4b-H), 2.99
(1H, dd, J = 15.3 and 6.0 Hz, 4a
-H) ppm. 13C NMR (DMSO-d6,
4.1.7.2 . (R)-Methyl 2-(4-(benzyloxy)benzyl)-2,3,4,9-tetrahydro-
100 MHz): d = 174.7, 139.6, 136.4, 132.8, 129.0, 128.8, 127.5,
127.2, 120.9, 118.7, 117.8, 111.3, 105.2, 59.6, 58.7, 45.9,
24.4 ppm. 13C NMR (DMSO-d6, 100 MHz): d = 173.9, 136.5, 132.2,
127.1, 120.9, 118.7, 117.8, 111.3, 105.1, 59.7, 59.2, 46.3, 24.2, 9.5,
4.5, 3.8 ppm. ESI-MS: calcd for C19H17N2O2 [MÀH]À: 305.1, found:
305.3. HPLC purity: 100(254 nm), tR: 5.53 min; 99.4(220 nm), tR:
5.53 min.
1H-pyrido[3,4-b]indole-3-carboxylate (10b).
Yellow pow-
der. Yield: 61%. 1H NMR (DMSO-d6, 400 MHz): d = 10.67 (1H, s, 9-
NH), 7.46–7.45 (2H, m, Ar-H), 7.42–.37 (3H, m, Ar-H), 7.35–7.24
(4H, m, Ar-H), 7.03–6.92 (4H, m, Ar-H), 5.09 (2H, s, –OCH2Ph),
3.99–3.95 (2H, m, 3-H and 1a-H), 3.89 (2H, ABq, J = 13.2 Hz, -
NCH2Ph), 3.72 (1H, d, J = 15.3 Hz, 1b-H), 3.59 (3H, s, –COOCH3),
3.09–2.98 (2H, m, 4-H) ppm. 13C NMR (DMSO-d6, 100 MHz):
d = 173.3, 158.0, 137.6, 136.4, 132.7, 131.4, 130.3, 128.9, 128.3,
128.1, 127.1, 120.9, 118.8, 117.8, 115.1, 111.3, 104.8, 69.6, 59.2,
58.1, 51.7, 45.7, 24.3 ppm. ESI-MS: calcd for C27H25N2O3 [MÀH]À:
425.2, found: 425.5. HPLC purity: 98.6(254 nm), tR: 6.14 min;
96.5(220 nm), tR: 6.15 min.
4.1.6.15. (R)-2-(Cyclopropylmethyl)-2,3,4,9-tetrahydro-1H-pyr-
ido[3,4-b]indole-3-carboxylic acid (9e).
Yellow semisolid.
Yield: 42%. 1H NMR (DMSO-d6, 400 MHz): d = 10.68 (1H, s, 9-NH),
7.34 (1H, d, J = 7.6 Hz, 5-H), 7.24 (1H, d, J = 7.9 Hz, 8-H), 7.00–
6.96 (1H, m, 7-H), 6.94–6.90 (1H, m, 6-H), 4.24 (1H, d, J = 15.1 Hz,
4.1.8. General procedure for compounds 11a, b
1
a
-H), 3.90 (1H, d, J = 15.1 Hz, 1b-H), 3.66–3.65 (1H, m, 3-H),
3.04 (1H, dd, J = 15.1 and 3.7 Hz, 4b-H), 2.86 (1H, dd, J = 15.2 and
6.0 Hz, 4 -H), 2.70 (2H, d, J = 6.6 Hz, –CH2CH(CH2)2), 0.96–0.86
A mixture of 10a or 10b (150 mg, 0.35 mmol) and 10% Pd/C
(15 mg) in MeOH (10 mL) was stirred under an atmosphere of H2
(1.5 bar) at room temperature for 5 h. The reaction mixture was fil-
tered through a pad of celite. Solvent was removed by evaporation
a
(1H, m, -CH2CH(CH2)2), 0.53–0.44 (2H, m, –CH2CH(CH2)2), 0.16–
0.08 (2H, m, –CH2CH(CH2)2) ppm. 13C NMR (DMSO-d6, 100 MHz):

6194
L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
to give the crude product, which was further purified by flash col-
umn chromatography on silica gel (hexanes/ethyl acetate = 80:30)
to give 11a or 11b.
and 6.0 Hz, 4b-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 174.5,
156.9, 136.4, 132.7, 130.3, 129.3, 127.1, 120.9, 118.7, 117.8,
115.5, 111.3, 105.1, 59.2, 58.1, 45.7, 24.3 ppm. ESI-MS: calcd for
C
19H17N2O3 [MÀH]À: 321.1, found: 321.3. HPLC purity:
4.1.8.1. (S)-Methyl 2-(4-hydroxybenzyl)-2,3,4,9-tetrahydro-1H-
98.3(254 nm), tR: 5.37 min; 97.8(220 nm), tR: 5.37 min.
pyrido[3,4-b]indole-3-carboxylate (11a).
Yellow powder.
Yield: 43%. 1H NMR (DMSO-d6, 400 MHz): d = 10.66 (1H, s, 9-NH),
9.30 (1H, s, –OH), 7.38 (1H, d, J = 7.7 Hz, 5-H), 7.25 (1H, d,
J = 7.9 Hz, 8-H), 7.15 (2H, d, J = 8.4 Hz, Ph-H), 7.01 (1H, t,
J = 7.9 Hz, 7-H), 6.94 (1H, t, J = 7.2 Hz, 6-H), 6.73 (2H, d, J = 8.4 Hz,
4.2. Biology
4.2.1. NF
Human embryonic kidney cells 293 were used to monitor any
changes occurring along the NF B pathway. This cell line contains
chromosomal integration of a luciferase reporter construct regu-
lated by the NF B response element. Transcription factors can
bind to the response element when stimulated by certain agents,
allowing transcription of the luciferase gene. Following an incuba-
tion period of 6 h with TNFa and test compounds, cells were ana-
lyzed for luciferase activity using the Luc assay system from
jB luciferase assay
Ph-H), 3.97–3.94 (2H, m, 3-H and
1
a
-H), 3.83 (2H, ABq,
j
J = 13.1 Hz, –NCH2Ph), 3.71 (1H, d, J = 15.6 Hz, 1b-H), 3.59 (3H, s,
–COOCH3), 3.07–2.96 (2H, m, 4-H) ppm. 13C NMR (DMSO-d6,
100 MHz): d = 173.4, 156.9, 136.4, 132.8, 130.3, 129.3, 127.1,
120.9, 118.8, 117.8, 115.5, 111.3, 104.8, 59.1, 58.2, 51.7, 45.6,
24.3 ppm. ESI-MS: calcd for C20H19N2O3 [MÀH]À: 335.1, found:
335.3. HPLC purity: 98.1(254 nm), tR: 5.50 min; 98.2(220 nm), tR:
5.51 min.
j
Promega Corporation (Madison, WI). Results were expressed as
a percentage, relative to control (TNFa-treated) samples, and
dose-response curves were constructed for the determination of
IC50 values, which were generated from the results of five serial
dilutions of test compounds and were the mean of two different
experiments.
4.1.8.2. (R)-Methyl 2-(4-hydroxybenzyl)-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylate (11b).
Yellow semisolid.
Yield: 43%. 1H NMR (DMSO-d6, 400 MHz): d = 10.66 (1H, s, 9-NH),
9.31 (1H, s, –OH), 7.38 (1H, d, J = 7.7 Hz, 5-H), 7.25 (1H, d,
J = 8.0 Hz, 8-H), 7.15 (2H, d, J = 8.4 Hz, Ph-H), 7.01 (1H, t,
J = 7.9 Hz, 7-H), 6.94 (1H, t, J = 7.1 Hz, 6-H), 6.73 (2H, d, J = 8.4 Hz,
4.2.2. Measurement of the production of NO in LPS-stimulated
RAW 264.7 murine macrophage cells (nitrite assay)
Ph-H), 3.98–3.93 (2H, m, 3-H and
1a-H), 3.83 (2H, ABq,
J = 13.0 Hz, –NCH2Ph), 3.71 (1H, d, J = 15.3 Hz, 1b-H), 3.59 (3H, s,
–COOCH3), 3.07–2.97 (2H, m, 4-H) ppm. 13C NMR (DMSO-d6,
100 MHz): d = 173.4, 156.9, 136.4, 132.8, 130.2, 129.3, 127.1,
120.9, 118.8, 117.8, 115.5, 111.3, 104.7, 59.1, 58.2, 51.7, 45.6,
24.3 ppm. ESI-MS: calcd for C20H19N2O3 [MÀH]À: 335.1, found:
335.3. HPLC purity: 98.9(254 nm), tR: 5.50 min; 99.1(220 nm), tR:
5.50 min.
The level of NO in the cultured media was estimated by measur-
ing the level of nitrite due to the instability of NO and its subse-
quent conversion to nitrite. The nitrite assay was performed as
previously described.30 Briefly, RAW 264.7 cells were incubated
in 96-well culture plates at 37 °C, 5% CO2 in a humidified air incu-
bator for 24 h. Then cells were treated with serially diluted com-
pounds for 15 min, followed by treatment with or without LPS
(1 lg/mL) for an additional 20 h. After the incubation, nitrite re-
leased in the cultured media was measured using Griess reagent
[1:1 mixture (v/v) of 1% sulfanilamide in 5% H3PO4 and 0.1% N-
(1-naphthyl)ethylenediamine dihydrochloride solution], and
absorbance was measured at 540 nm. The concentration of nitrite
was calculated using a standard curve created with known concen-
trations of sodium nitrite. To evaluate the cytotoxic effects of com-
pounds with RAW 264.7 cells under the same experimental
condition, the SRB assay was performed.
4.1.9. General procedure for compounds 12a, b
A solution of 11a, or 11b (0.1 mmol) and LiOH (0.5 mmol) in
THF/H2O (1:1, 3 mL) was stirred at room temperature until
completion of the reaction (monitored by HPLC). THF was removed
in vacuo and then the solution was carefully neutralized with
glacial acetic acid, whereupon a heavy precipitate was formed.
The product was collected by filtration, washed with cold water,
and dried.
4.1.9.1. (S)-Callophycin A (12a).
Pale yellow powder. Yield:
4.2.3. Evaluation of antiproliferative effect of compounds in
MCF7 breast cancer cells (sulforhodamine B assay)
66%. 1H NMR (DMSO-d6, 400 MHz): d = 10.65 (1H, s, 9-NH), 9.33
(1H, br s, -OH), 7.38 (1H, d, J = 7.7 Hz, 5-H), 7.25 (1H, d, J = 7.8 Hz,
8-H), 7.16 (2H, d, J = 8.4 Hz, Ph-H), 7.01 (1H, t, J = 7.5 Hz, 7-H),
6.94 (1H, t, J = 7.3 Hz, 6-H), 6.73 (2H, d, J = 8.4 Hz, Ph-H), 4.00
The effect of compounds on MCF7 breast cancer cell prolifera-
tion was evaluated using the sulforhodamine B (SRB) cellular pro-
tein-staining method.31 In brief, MCF7 cells (1 Â 104 cells in 190
ll
(1H, d, J = 15.3 Hz, 1
a-H), 3.86 (2H, ABq, J = 13.0 Hz, -NCH2Ph),
of the complete media) were plated in 96-well plates containing
compounds and incubated at 37 °C, 5% CO2 in humidified air for
72 h. After the incubation, cells were fixed with 10% trichloroacetic
acid solution for 30 min and stained with 0.4% SRB in 1% acetic acid
solution for 30 min. After washing with 1% acetic acid solution,
protein-bound SRB was dissolved in 10 mM Tris buffer (pH 10.0)
and the absorbance was measured at 515 nm. The effect of com-
pounds on cell survival was demonstrated as% survival in compar-
ison with vehicle (DMSO)-treated control cells.
3.81 (1H, dd, J = 6.2 and 3.4 Hz, 3-H), 3.70 (1H, d, J = 15.1 Hz, 1b-
H), 3.06 (1H, dd, J = 15.3 and 2.3 Hz, 4a-H), 2.95 (1H, dd, J = 15.5
and 6.2 Hz, 4b-H) ppm. 13C NMR (DMSO-d6, 100 MHz): d = 174.5,
156.9, 136.4, 132.7, 130.3, 129.3, 127.1, 120.9, 118.7, 117.8,
115.5, 111.3, 105.1, 59.2, 58.1, 45.7, 24.3 ppm. ESI-MS: calcd for
C
19H17N2O3 [MÀH]À: 321.1, found: 321.3. HPLC purity:
96.3(254 nm), tR: 5.37 min; 99.0(220 nm), tR: 5.37 min.
4.1.9.2. (R)-Callophycin A (12b).
Pale yellow powder. Yield:
76%. 1H NMR (DMSO-d6, 400 MHz): d = 10.65 (1H, s, 9-NH), 9.31
(1H, br s, -OH), 7.38 (1H, d, J = 7.7 Hz, 5-H), 7.25 (1H, d, J = 7.9 Hz,
8-H), 7.16 (2H, d, J = 8.4 Hz, Ph-H), 7.01 (1H, t, J = 7.5 Hz, 7-H),
6.94 (1H, t, J = 7.4 Hz, 6-H), 6.73 (2H, d, J = 8.4 Hz, Ph-H), 4.00
4.2.4. Determination of QR1 activity in cell culture
QR1 was assessed using Hepa 1c1c7 murine hepatoma cells as
previously reported.32 QR1 was measured as a function of the
NADPH-dependent menadiol-mediated reduction of 3-(4,5-dim-
etylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue
formazan. Protein content was determined via crystal violet
staining of identical plates. Specific activity is defined as nmol of
(1H, d, J = 15.1 Hz, 1
3.82 (1H, dd, J = 6.0 and 3.3 Hz, 3-H), 3.71 (1H, d, J = 15.5 Hz, 1b-
H), 3.06 (1H, dd, J = 15.4 and 2.5 Hz, 4 -H), 2.96 (1H, dd, J = 15.3
a-H), 3.87 (2H, ABq, J = 13.1 Hz, –NCH2Ph),
a

L. Shen et al. / Bioorg. Med. Chem. 19 (2011) 6182–6195
6195
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of QR activity represents the specific enzyme activity of agent-trea-
ted cells compared with a DMSO-treated control. The concentra-
tion to double activity (CD) was determined through a dose-
response assay for active substances (IR > 2).
11. Part of this work has been presented at the following meeting. (Shen, L.; Park,
E.-J.; Kondratyuk, T. P.; Guendisch, D.; Marler, L.; Pezzuto, J. M.; Wright, A. D.;
Sun, D. 241st American Chemical Society National Meeting, Anaheim, CA, USA.
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4.2.5. Aromatase assay
Aromatase activity was assayed as previously reported33 with
the necessary modifications to perform the test in 384-well plates.
The test substance (3.5
lL at 20 lg/mL) was pre-incubated with
30
l
L of NADPH regenerating system (2.6 mM NADP+, 7.6 mM glu-
cose 6-phosphate, 0.8 U/mL glucose 6-phosphate dehydrogenase,
13.9 mM MgCl2, and 1 mg/mL albumin in 50 mM potassium phos-
phate buffer, pH 7.4) for 10 min. The enzyme and substrate mix-
ture [33
0.4 dibenzylfluorescein, and 4 mg/mL albumin in 50 mM
potassium phosphate, pH 7.4] was added and the plate was incu-
bated for 30 min at 37 °C before quenching with 25 L of 2 N NaOH
lL of 1 lM enzyme (165 lU CYP19, BD Biosciences),
l
M
l
solution. After termination of the reaction and shaking for 5 min,
the plate was further incubated for 2 h at 37 °C. This enhances
the ratio of signal to background. Fluorescence was measured
using a BioTek Synergy 2 plate reader at 485 nm (excitation) and
530 nm (emission). IC50 values and dose-response curves were
based on three independent experiments performed in duplicate
using five concentrations of tested substance.
20. Luqman, S.; Pezzuto, J. M. Phytother. Res. 2010, 24, 949.
21. S- and R-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acids were purchased
from Chem-Impex International, Inc., IL, USA.
22. Liu, J.; Jiang, X.; Zhao, M.; Zhang, X.; Zheng, M.; Peng, L.; Peng, S. J. Med. Chem.
2010, 53, 3106.
23. Kumpaty, H. J.; Van Linn, M. L.; Kabir, M. S.; Försterling, F. H.; Deschamps, J. R.;
Cook, J. M. J. Org. Chem. 2009, 74, 2771.
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Campa, J. G. J. Org. Chem. 1994, 59, 1583.
25. Representative characterization data of cyclization hydantoin byproduct of 6c:
Acknowledgments
O
We thank UH Hilo CoP for start-up funding. This work was also
supported in part by NCI P01 CA48112 (JMP) and NCRR INBRE pro-
gram P20 RR016467 (DS). We thank Annie Ma at Phenomenex for
the assistance with the chiral HPLC method development. We also
thank Dr. Cherie A. Motti of the Australian Institute of Marine Sci-
ences (AIMS), Queensland, Australia, for providing NMR spectra of
the isolated natural callophycin A.
N
N
O
N
H
1H NMR (DMSO-d6, 400 MHz): d = 11.03 (1H, s, 9-NH), 7.47 (1H, d, J = 7.8 Hz, 5-
H), 7.35 (1H, d, J = 8.1 Hz, 8-H), 7.30–7.27 (2H, m, –CH2CH2PhH), 7.22–7.19 (3H,
m, –CH2CH2PhH), 7.11–7.07 (1H, m, 7-H), 7.00 (1H, t, J = 7.3 Hz, 6-H), 4.89 (1H,
Supplementary data
Supplementary data (1H and 13C NMR spectra and HPLC chro-
matographs of compounds 2–12 and HSQC, HMBC, and COSY spec-
tra of 12b) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2011.09.020.
d, J = 16.2 Hz, 1
a-H), 4.41–4.34 (2H, m, 1b-H and 3-H), 371–3.66 (2H, m, –
NCH2CH2Ph), 3.19 (1H, dd, J = 14.9 and 5.2 Hz, 4b-H), 2.90 (2H, t, J = 7.3 Hz, –
NCH2CH2Ph), 2.60–2.53 (1H, m,
4a-H) ppm. ESI-MS: calcd for C21H20N3O2
[M+H]+: 346.2, found: 346.4. HPLC purity: 96.5 (254 nm), tR: 6.88 min;
96.9(220 nm), tR: 6.88 min.
26. Nieddu, G.; De Luca, L.; Giacomelli, G. Synthesis 2008, 2008, 3947.
27. According to the HPLC monitoring of the reaction mixture, about 30% of
byproduct 2a or 2b was observed due to the further N-debenzylation of the
corresponding product 11a or 11b. The byproduct can be easily removed by
flash column chromatography on silica gel.
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