Synthesis, in vitro anti-inflammatory and cytotoxic evaluation, and mechanism of action studies of 1-benzoyl-β-carboline and 1-benzoyl-3-carboxy-β-carboline derivatives

Article abstract of DOI:10.1016/j.bmc.2011.01.034

In the present study, various 1-substituted and 1,3-disubstituted β-carboline derivatives were synthesized by a modified single-step Pictet-Spengler reaction. The compounds were examined for cytotoxicity and anti-inflammatory activity, as measured by the

Full text of DOI:10.1016/j.bmc.2011.01.034

Bioorganic & Medicinal Chemistry 19 (2011) 1674–1682
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, in vitro anti-inflammatory and cytotoxic evaluation,
and mechanism of action studies of 1-benzoyl-b-carboline
and 1-benzoyl-3-carboxy-b-carboline derivatives
Mei-Lin Yang a, Ping-Chung Kuo b, , Tsong-Long Hwang c, , Wen-Fei Chiou d, Keduo Qian e, Chin-Yu Lai e,
a,f,
Kuo-Hsiung Lee e,f, , Tian-Shung Wu
⇑
⇑
a Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, ROC
b Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, ROC
c Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan, ROC
d National Research Institute of Chinese Medicine, Taipei 105, Taiwan, ROC
e Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
f Chinese Medicine Research and Development Center and Department of Pharmacy, China Medical University and Hospital , Taichung 401, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, various 1-substituted and 1,3-disubstituted b-carboline derivatives were synthesized
by a modified single-step Pictet–Spengler reaction. The compounds were examined for cytotoxicity and
anti-inflammatory activity, as measured by the inhibition of prostaglandin E2 (PGE2) production and nitric
oxide (NO) production. While only two compounds (28 and 31) showed marginal cytotoxicity against four
human cancer cell lines, most of the tested compounds exhibited potent inhibitory activity of both NO and
PGE2 production. Moreover, compounds 6 and 16 significantly reduced the expression of inducible nitric
oxide synthase (iNOS) and cyclooxygenase 2 (COX2), suggesting that b-carboline analogs can inhibit NO
and PGE2 production at the translational level. In addition, several of the b-carboline derivatives (1, 2, 4–
Received 22 November 2010
Revised 6 January 2011
Accepted 16 January 2011
Available online 22 January 2011
Keywords:
b-Carboline derivatives
Anti-inflammatory activity
Nitric oxide production inhibition
8, 11, 13, 22, 25, 27, 31, and 41–43) displayed significant inhibitory activity of superoxide anion (O2ꢀꢁ
generation or elastase release compared to the reference compound, with 6 being the most potent. N-For-
myl- -methionyl-phenylalanine (FMLP)-induced phosphorylation of c-Jun N-terminal kinase (JNK) and
)
L
protein kinase B (AKT) were also inhibited by 6, suggesting that it suppresses human neutrophil functions
by inhibiting the activation of JNK and AKT signaling pathways. Therefore, the synthetic 1-benzoyl-3-car-
boxy b-carboline analogs may have great potential to be developed as anti-inflammatory agents.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
eases, such as rheumatoid arthritis, ischemia, reperfusion injury,
chronic obstructive pulmonary disease, and asthma.4,5 In response
Sustained nitric oxide (NO) release by inducible nitric oxide
synthase (iNOS) and prostaglandin E2 (PGE2) production by cyclo-
oxygenase 2 (COX2) have been implicated as mediators of inflam-
mation and are induced by bacterial lipopolysaccharide (LPS) or
immunological stimuli.1,2 It has been reported that excess produc-
tion of NO and PGE2 by macrophages and other cells exposed to
endotoxins may contribute to septic shock, cerebral injury,
myocardical ischemia, diabetes, arteriosclerosis, and other local
or systemic inflammatory disorders.2,3 Thus, inhibition of NO syn-
thesis and PGE2 production stands as an important therapeutic
goal. On the other hand, overwhelming activation of neutrophils
is known to play important roles in the pathogenesis of various dis-
to diverse stimuli, activated neutrophils secrete a series of cytotox-
ins, such as superoxide anion (Oꢀ2ꢁ), a precursor of other reactive
oxygen species, and elastase, a granule protease. Therefore, it is
crucial to retain Oꢀ2ꢁ production and elastase release in physiologi-
cal conditions, while potentiating these functions in infected
tissues and organs. Currently, only a few agents are available in
clinical practice that can directly modulate neutrophil proinflam-
matory responses.
Natural and synthetic products containing a b-carboline phar-
macophore exhibit a wide range of important bioactivities, partic-
ularly on the central nervous system.6,7 Due to their unique rigid
heterocyclic skeleton, many b-carbolines bind with high affinity
to benzodiazepine,8 serotonin,6 and dopamine9 receptor sites and
inhibit monoamine oxidase A.10 The reported biological effects of
this class of compounds include sedative,11 antithrombotic,12
anti-HIV,13 and DNA-targeting properties,14 as well as suppression
of CDK,15,16 topoisomerase,17 and IrK.18 For example, flazin19 and
⇑
Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.H.L.);
tel.: +886 6 2757575x65333; fax: +886 6 2740552 (T.S.W).
E-mail addresses: khlee@unc.edu (K.-H. Lee), tswu@mail.ncku.edu.tw (T.-S. Wu).
These authors had equal contributions as the first author.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.034

M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
1675
flazinamide20 possess anti-HIV activity, while dichotomine
C
Table 1
Yields of differently substituted b-carbolines 1–44
exhibits inhibitory effects against the release of b-hexosaminidase,
TNF-a
, and IL-4 from RBL-2H3 cells.21 With appropriate substitu-
Products, yield
ents at the 3-position, b-carboline derivatives also showed cyto-
R
R
R
R
1 = R2 = R3 = H
1, 4%
2, 6%
3, 5%
4, 8%
5, 3%
6, 5%
7, 4%
8, 4%
9, trace
10, trace
11, 2%
12, trace
13, 2%
40, 5%
43, 55%
44, 50%
14, 35%
15, 20%
16, 18%
17, 22%
18, 20%
19, 18%
20, 20%
21, 15%
22, 15%
23, trace
24, 10%
25, 10%
26, trace
41, 13%
27, 45%
28, 35%
29, trace
30, trace
31, 35%
32, trace
33, trace
34, trace
35, trace
36, trace
37, trace
38, trace
39, trace
42, 30%
toxic activity against human tumor cell lines.22–24
1 = CH3, R2 = R3 = H
2 = CH3, R1 = R3 = H
3 = CH3, R1 = R2 = H
As a continuing study of bioactive 1-substituted b-carbolines, we
synthesized more novel 1-substituted and 1,3-disubstituted b-carb-
oline derivatives, using the previously reported single-step Pictet–
Spengler reaction.25 We report the synthesis, cytotoxic and anti-
inflammatory evaluation, as well as the structure–activity relation-
ship (SAR) analysis of these derivatives in the present paper.
R1 = Br, R2 = R3 = H
R
R
R
R
R
R
R
R
2 = Br, R1 = R3 = H
3 = Br, R1 = R2 = H
1 = OCH3, R2 = R3 = H
2 = OCH3, R1 = R3 = H
3 = OCH3, R1 = R2 = H
1 = NO2, R2 = R3 = H
2 = NO2, R1 = R3 = H
3 = NO2, R1 = R2 = H
2. Chemistry
The synthetic methods used to obtain compounds 1–39 by sin-
gle-step Pictet–Spengler reaction of L-tryptophan with different
functionalized phenylglyoxal derivatives were described in our
previous report as shown in Figure 1.25 The yields of the synthetic
b-carboline compounds are listed in Table 1. The 1-benzoyl-3-car-
boxy-b-carboline products 1–8 and 11–13 were obtained in 2–6%
yields, while 9 and 10 were present in trace amounts, insufficient
to be isolated and characterized. These compounds displayed very
strong affinity for silica gel, considering that chromatography elut-
ing with highly polar solvent systems, such as mixtures of EtOAc
and MeOH, were still barely able to elute and purify these
compounds.
The decarboxylated 1-benzoyl-b-carboline analogs 14–22 and
24–25 were obtained in moderate yields (ꢂ20%), while 23 and
26 were available in only trace amounts. Generally, these com-
pounds could be purified by chromatography with medium polar
solvent systems. Compounds 27, 28, and 31, were obtained by
reduction of the ketone group in yields of 45%, 35%, and 35%,
respectively.
3. Results and discussion
The novel 1-substituted and 1,3-disubstituted b-carboline
derivatives (1–2, 4–8, 11, 13–22, 24–25, 27–28, 31, and 40–44)
were first tested against four different human cancer cell lines:
A549 (lung), DU145 (prostate), KB (nasopharyngeal), and KB-VIN
(its vincristine-resistant subline) using paclitaxel as a positive con-
trol (Table 2). Overall, the newly obtained b-carboline derivatives
did not show strong cytotoxicity. Except for 27, 28, and 31, the
new derivatives did not exhibit cytotoxicity against any of the four
tested cell lines at concentrations less than 10 lg/mL. Comparing
the potency of active 28 and 31 with inactive 2/15 and 5/18,
respectively, we found that a 3-carboxylic acid and 10-ketone were
detrimental, while a 10-hydroxy moiety was necessary for the com-
pounds to exhibit cytotoxicity. The three active compounds dif-
fered structurally only in the substituents on the pendant phenyl
ring. Compound 28 with a para-methyl group showed the best
In addition, as shown in Figure 2, pyruvic aldehyde was con-
densed with L-tryptophan or L-tryptophanylamide to yield b-carbo-
line derivatives 40–42 and 43, respectively. Moreover, compound 44
was obtained by coupling between 4-methoxyphenylglyoxal and
tryptophan methyl ester. While compounds 40–42 were obtained
insimilaryields tothose previously reported,25 changingthecarbox-
ylic acid group to amide and ester functionalities significantly im-
proved the yields in the modified Pictet–Spengler condensation
reactions, as was seen with 43 and 44. Not only were b-carbolines
43 and 44 produced in higher yields, but the condensation reactions
afforded only one product, due to the difference of the 3-substituent
in the b-carboline basic skeleton.
activity (GI50
: 2.68–7.68 lg/mL, 9.29–26.6 lM). Interestingly,
these three compounds showed better activity against KB-VIN than
KB.
The synthetic b-carboline derivatives were then examined for
inhibition of PGE2 production, which was stimulated by lipopoly-
saccharide in interferon-
c (LPS/IFN-c)-stimulated macrophages.
The data are displayed in Table 3. Among the 1-benzoyl-3-car-
boxy-b-carboline derivatives (1–8, 11, and 13), compounds 2, 3,
5, and 6, with methyl and bromide at the para- and meta-positions
O
NH2
H
TsOH
CO2H
N
O
MeOH
H
R1
R3
100°C , 2 h
R2
l-tryptophan
5
8
4
CO2H
3
6
7
N
N
N
N
H
N
H
N
H
HO
1
O
O
R3
R1
R3
R1
R3
R1
R2
R2
R2
1-13
14-26
27-39
Figure 1. Synthesis of differently substituted b-carbolines 1–39.

1676
M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
O
NH2
CO2H
TsOH
H
MeOH
100 °C , 2 h
N
H
O
CO2H
N
N
N
N
H
N
N
H
HO
H
O
O
40
41
42
O
NH2
HCl
CONH2
H
O
MeOH
N
CONH2
N
N
H
O
H
100 °C , 2 h
O
43
CO2CH3
NH2
HCl
H
MeOH
N
N
O
CO2CH3
100 °C , 2 h
H
N
H
H3CO
O
OCH3
44
Figure 2. Synthesis of b-carboline analogs 40–44.
Table 2
Table 3
Cytotoxicity of compounds against human tumor cell linesa,b
Effects of synthetic compounds on LPS/IFN-b-induced PGE2 production in RAW 264.7
macrophages
Compound
KB
KB-VIN
A549
DU145
Basal
LPS/IFN-
0–12.9 pg/mL
113–259 pg/mL
27
28
31
Paclitaxel
>40
20.6
15.9
0.00487
22.56
9.29
14.5
>1.0
>40
26.6
19.0
0.00558
>40
13.8
13.3
0.00555
c
% of inhibition
% of inhibition
% of inhibition
10
90
—
—
100
—
—
Concentration
1
lM
3
lM
lM
a
1
2
3
4
5
6
7
8
11
13
14
15
16
17
18
19
20
21
22
24
25
27
28
31
34
40
41
42
43
44
0
12
Mean GI50 (lM), from two or more independent tests.
Compounds 1, 2, 4–8, 11, 13–22, 24, 25, and 40–44 were inactive (GI50 >40 lM)
b
100
98
0
98
100
0
28
0
0
100
100
82
100
100
78
98
92
97
39
99
100
44
100
98
37
95
71
100
95
0
against all four cell lines.
of the 1-benzoyl ring exhibited the most significant inhibition of
PGE2 synthesis with inhibitory percentages of 100%, 98%, 98%,
and 100%, respectively, at 1
compound 1 and ortho-substituted analogs 4 and 7 were not active
at this concentration, although they were active at 3 and 10 M.
Moreover, the electron-releasing methoxy and electron-withdraw-
ing nitro substituents in 8 and 11/13 did not contribute to PGE2
inhibition. Based on these results, we concluded that m- or p-
substituted weak electron-donating or -withdrawing groups in
the 1-benzoyl ring could improve the inhibitory activity on PGE2
production. In addition, the 3-methyl ester derivative 44 also
96
—
lM. In comparison, the unsubstituted
100
100
82
100
—
82
—
—
89
95
98
100
96
0
0
l
25
97
0
98
97
0
0
0
0
0
0
15
0
0
0
0
0
0
98
8
showed a high inhibition percentage of 98% at 1 lM. Similarly,
among the 1-benzoyl substituted b-carboline derivatives 14–26,
only the m-methyl-, p-bromo-, and m-bromo-benzoyl substituted
compounds 16, 18, and 19 exhibited high inhibition (97%, 98%,
and 97%, respectively) of PGE2 synthesis in LPS/IFN-
cells at 1 M. The (9H-b-carbolin-1-yl)-phenyl-methanol deriva-
tives 27, 28, 31, and 34 did not show significant inhibition at any
tested concentration, suggesting that the 1-carbonyl functionality
played an important role in the inhibitory activity of the deriva-
tives on PGE2 production.
29
21
0
0
0
—
34
19
0
59
0
95
—
42
c-stimulated
l
0
95
100
21
Aminoguanidine
The inhibitory effects of the synthetic analogs on the generation
of NO were further evaluated in LPS/IFN-c stimulated RAW 264.7
‘—’: Not determined.

M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
1677
Table 4
Effects of synthetic compounds on LPS/IFN-
macrophages
c-induced NO production in RAW 264.7
Compound
EC50
M)
CC50
cytotoxic concn
(
l
M)
Therapeutic index (T.I.)
(CC50/EC50)
(
l
1
2
3
4
5
6
7
8
11
13
14
15
16
17
18
19
20
21
22
24
25
27
28
31
34
40
41
42
43
44
3.65
0.50
0.47
2.58
0.53
0.57
6.96
0.23
2.94
2.88
2.67
1.26
0.20
2.12
0.30
1.16
6.97
4.59
2.96
0.66
1.06
0.31
4.94
0.74
2.08
0.25
6.65
NA
564
155
35
34
>2000
20
51
75
187
5
4
49
17.7
16.4
62300
10.7
29.4
524
43.1
15.0
12.8
131
52.8
23.7
51500
149
51.4
53.8
83.4
16.3
3.86
21.6
574
42
Figure 3. Western blot analysis of synthetic compounds 6 and 16 on iNOS and
COX2 expressions in LPS/IFNc-induced RAW 264.7 macrophages.
120
>2000
492
44
8
18
6
6
20
186
>1010
95
764
>1010
>1010
ND
21
Table 5
Effects of synthetic compounds on superoxide anion generation and elastase release
by human neutrophils in response to FMLP/cytochalasin B
a
Compound
IC50
(lM)
Superoxide anion generation
Elastase release
NT
1
2
4
5
6
7
8
11
17.7 0.89
6.27 1.00
5.36 0.62
2.28 0.33
2.05 0.21
5.33 0.53
16.1 0.95
6.65 1.62
17.6 0.50
>30
13.4 0.54
3.13 1.24
14.83 1.01
1.99 0.10
2.78 0.15
3.07 1.71
10.4 1.11
6.21 0.82
13.2 1.22
>30
70.1
1590
NT
NT
0.28
19.8
1.95
358
0.92
0.51
22.6
4
16
Aminoguanidine
(iNOS inhibitor)
13
14
NA: not active; ND: not determined; NT: not toxic.
15
>30
>30
16
>30
>30
17
18
19
20
21
22
24
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
macrophages, according to the method reported in literature,26 and
the data are summarized in Table 4. Among the tested compounds,
the 1-benzoyl-3-carboxy-b-carbolines 2, 3, 5, 6, 8, 40, 43, and 44, 1-
benzoyl-b-carbolines 16, 18, and 24, and (9H-b-carbolin-1-yl)-phe-
nyl-methanol derivatives 27 and 31 showed significant NO produc-
>30
13.2 1.22b
>30
tion inhibitory activity with EC50 values of <1
16 with an m-methylbenzoyl group displayed the strongest NO
inhibition activity with an EC50 value of 0.20 M, which was
two-order better than that of the iNOS inhibitor aminoguanidine.
Although the corresponding o-methylbenzoyl compound 17 was
lM. The b-carboline
25
27
28
31
40
41
42
43
16.6 1.70
7.88 0.33
>30
8.84 0.82
>30
5.03 1.33
9.40 4.81
14.5 0.71
>30
38.3 5.38
1.12 0.20
—
>30
>30
14.17 2.00
>30
>30
>30
5.82 0.58
>30
32.0 6.49
1.92 0.22
l
less potent (EC50: 2.12 lM) than 16, it had a higher therapeutic in-
dex due to low cytotoxicity. Overall, these 1-methylbenzoyl-b-
carbolines may have potential to be developed as new iNOS inhib-
itors. While the 1,3-disubstituted b-carbolines 43 and 44 with 3-
amide and -ester substitutions still possessed significant NO inhi-
bition activity, they also showed some increased cytotoxicity,
which resulted in lower therapeutic indexes. Thus, 3-amide and -
ester substituted compounds may not merit development as new
anti-inflammatory lead compounds.
44
Indomethacin
LY294002c
Results are presented as mean SEM (n = 3).
a
Concentration necessary for 50% inhibition.
Compound 22 induced elastase release in the pretreatment of cytochalasin B.
LY294002, a phosphatidylinositol-3-kinase inhibitor, was used as a positive
b
c
control for superoxide anion generation and elastase release.
Following the initial screening, the mechanisms of action of the
selected synthetic compounds 6 and 16 on iNOS and COX2 expres-
sion in LPS/IFN-c-stimulated RAW264.7 macrophages were then
investigated using western blot analysis. The results are displayed
in Figure 3. These two compounds significantly reduced the protein
carboxy-b-carboline derivatives 1, 2, 4–8, 11, and 13 inhibited O2ꢀꢁ
generation and elastase release in FMLP/cytochalasin B-activated
human neutrophils in a concentration-dependent manner. Com-
pound 6 demonstrated the most significant inhibition towards O2ꢀꢁ
generation and compound 5 towards elastase release with IC50 val-
expression of iNOS and COX2 at 18 h after LPS/IFN-c stimulation,
which suggested that the synthetic b-carboline analogs inhibited
NO and PGE2 production at the translational level or the upstream
pathway of transcription.
ues of 2.05 0.21 and 1.99 0.10
the reference compound LY29400227,28 (IC50 1.12 0.20 and
1.92 0.22
M towards Oꢀ2ꢁ generation and elastase release, respec-
lM, respectively, compared with
l
In addition, the b-carboline derivatives were also assayed for
tively). In contrast, the decarboxylated 1-benzoyl-b-carboline ana-
logs 14–25 were inactive, except for 22 (elastase) and 25 (Oꢀ2ꢁ). The
(9H-b-carbolin-1-yl)-phenyl-methanol derivatives 27 and 31 also
significantly inhibited Oꢀ2ꢁ generation, but were inactive or less
inhibition of Oꢀ2ꢁ generation and elastase release by human neutro-
phils in response to N-formyl-L-methionyl-phenylalanine (FMLP)/
cytochalasin B. The data are displayed in Table 5. The 1-benzoyl-3-

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M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
active against elastase release. In addition, the b-carboline analogs
41–43 were generally effective against Oꢀ2ꢁ generation, but less
active against elastase release. Compounds 40 and 44 were inactive
in both anti-inflammatory assays.
HRFABMS were measured on a JEOL JMS-700 mass spectrometer.
Elemental analyses were performed on an Elementar Vario EL III
analyzer. The identities of the reported compounds have been
unambiguously proven by their spectroscopic data, and the purity
assessed by analytical HPLC performed on a Shimadzu LC-10ATVP
series pumping system equipped with a Shimadzu SPD-6AV
UV–vis spectrophotometric detector at 254 nm, and a Luna C-18
Generally speaking, the formation of Oꢀ2ꢁ in neutrophils can be
inhibited by modulating cellular signaling pathways, but also by
direct radical scavenging. However, at a concentration of 250 lM,
the tested synthetic compounds (1–13) did not significantly
scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals in cell-
free systems, indicating that the inhibition of Oꢀ2ꢁ generation by
b-carboline derivatives 1–13 is mediated by modulating cellular
signaling pathways (data not shown). FMLP is known to activate
phospholipase C and phosphatidylinositol-3-kinase (PI3K), and it
also activates MAPKs. Activation of these signal transduction path-
ways is known to cause various physiological responses.29 Upon
activation of PI3K, protein kinase B (AKT) is recruited to the plasma
membrane, where it undergoes phosphorylation and activation.
Activation of c-Jun N-terminal kinase (JNK) and PI3K/AKT has been
shown to contribute to human neutrophil functions, such as respi-
ratory burst and degranulation.30–32 Therefore, the effects of 6 on
phosphorylation of JNK and AKT in FMLP-stimulated human neu-
trophils were further determined by western blot analysis, and
the results are displayed in Figure 4. FMLP-induced phosphoryla-
tions of JNK and AKT were clearly inhibited by 6, suggesting that
6 suppressed human neutrophil functions by inhibiting the activa-
tion of JNK and AKT signaling pathways.
packed column (4.6 ꢃ 250 mm, 5
lm) with an isocratic elution of
mixing solvent of methanol and water (60:40). The purity of all
compounds, as determined by HPLC, was better than 98.0%.
5.2. General synthetic procedure
To a stirred suspension of L-tryptophan (0.174 g, 0.854 mmol,
1.3 equiv) and p-toluenesulfonic acid monohydrate (0.125 g,
0.657 mmol, 1.0 equiv), phenylglyoxal monohydrate (0.1 g, 0.657
mmol, 1.0 equiv) was added. The resulting solution was stirred at
50 °C for 2 h, and the phenylglyoxal was shown by HPLC to be com-
pletely consumed. The reaction mixture was poured into water,
and the precipitate was filtered and purified by silica gel column
chromatography eluted with a gradient of n-hexane and EtOAc to
afford 1, 14, and 27. Compounds 2–13, 15–26, 28–39, and 40–44
were prepared with similar procedures.
5.2.1. 1-Benzoyl-9H-b-carboline-3-carboxylic acid (1)
Yellow powder (EtOAc–MeOH), mp 281–283 °C. 1H NMR and
13C NMR: see Ref. 25.
4. Conclusions
5.2.2. 1-(4-Methylbenzoyl)-9H-b-carboline-3-carboxylic acid (2)
Yellow powder (EtOAc–MeOH), mp 259–260 °C. 1H NMR and
13C NMR: see Ref. 25.
In summary, we designed and synthesized new 1-substituted
and 1,3-disubstituted b-carbolines, which were evaluated for cyto-
toxicity and anti-inflammatory activity. The results demonstrated
that, among the synthetic b-carbolines tested, most of the syn-
thetic derivatives exhibited significant NO inhibition activity, and
were more effective than the iNOS inhibitor aminoguanidine. The
most active compound 16 displayed the strongest NO inhibition
5.2.3. 1-(3-Methylbenzoyl)-9H-b-carboline-3-carboxylic acid (3)
Yellow powder (EtOAc–MeOH), mp 280–282 °C. 1H NMR
(DMSO-d6, 300 MHz): d 2.40 (3H, s, CH3), 7.28 (1H, t, J = 7.5 Hz,
H-6), 7.42 (2H, m, H-40 and -50), 7.56 (1H, t, J = 7.5 Hz, H-7), 7.76
(1H, d, J = 8.1 Hz, H-8), 8.20 (1H, br s, H-20), 8.30 (1H, m, H-60),
8.31 (1H, d, J = 7.8 Hz, H-5), 8.93 (1H, s, H-4), 11.90 (1H, br s,
D2O exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 21.4,
113.2, 119.0, 120.4, 121.1, 122.0, 128.2, 128.9, 129.2, 130.9,
131.8, 133.3, 134.9, 136.0, 137.5, 137.8, 142.2, 166.0, 199.7. FABMS
m/z 331 [M+H]+ (12), 316 (14), 287 (18), 167 (16), 154 (100), 136
(79). HRFABMS m/z 331.1081 [M+H]+ (calcd for C20H15N2O3,
331.1083).
activity with an EC50 value of 0.20 lM. 1-Benzoyl-3-carboxy deriv-
atives 1–13 also inhibited Oꢀ2ꢁ generation and elastase release in
FMLP/cytochalasin B-activated human neutrophils, compared with
the reference compound LY294002. In addition, the synthetic
compounds showed low cytotoxicity. Therefore, this group of
b-carbolines has the potential to be developed as new anti-inflam-
matory drugs.
5. Experimental
5.2.4. 1-(2-Methylbenzoyl)-9H-b-carboline-3-carboxylic acid (4)
Yellow powder (EtOAc–MeOH), mp 286–287 °C. 1H NMR
(DMSO-d6, 300 MHz): d 2.37 (3H, s, CH3), 7.31 (1H, t, J = 7.5 Hz,
H-6), 7.37 (2H, m, H-30 and -50), 7.47 (1H, t, J = 7.5 Hz, H-7), 7.65
(2H, m, H-40 and -60), 7.86 (1H, d, J = 8.2 Hz, H-8), 8.47 (1H, d,
J = 7.8 Hz, H-5), 9.14 (1H, s, H-4), 12.45 (1H, br s, D2O exchange-
able, NH), 12.88 (1H, br s, D2O exchangeable, CO2H). 13C NMR
(DMSO-d6, 75 MHz): d 20.4, 113.6, 120.6, 121.0, 121.2, 122.4,
125.2, 129.6, 130.7, 130.7, 130.9, 131.8, 135.8, 136.6, 136.9,
137.2, 138.2, 142.4, 166.5, 198.0. EIMS m/z 330 [M]+ (35), 302
(91), 283 (16), 258 (100), 257 (50), 256 (95), 255 (70), 166 (15),
140 (21), 91 (62). HREIMS m/z 330.1002 [M]+ (calcd for
5.1. General analytical and spectroscopic methods
Melting points were measured on a Yanaco MP-S3 micro melt-
ing point apparatus and are uncorrected. IR spectra were obtained
with a Shimadzu FT-IR DR-8011 spectrophotometer. 1H and 13C
NMR spectra were obtained on the Bruker Avance-300 NMR spec-
trometer, with tetramethylsilane (TMS) as internal standard and
the chemical shifts were reported in d values (ppm). EI and HREIMS
spectra were recorded on a VG 70–250 S spectrometer. FAB and
C20H14N2O3, 330.1004).
5.2.5. 1-(4-Bromobenzoyl)-9H-b-carboline-3-carboxylic acid (5)
Yellow powder (EtOAc–MeOH), mp 290–291 °C. 1H NMR and
13C NMR: see Ref. 25.
5.2.6. 1-(3-Bromobenzoyl)-9H-b-carboline-3-carboxylic acid (6)
Yellow powder (EtOAc–MeOH), mp 282–283 °C. 1H NMR
(DMSO-d6, 300 MHz): d 7.37 (1H, t, J = 7.4 Hz, H-6), 7.57 (1H, t,
Figure 4. Western blot analysis of synthetic compound
phosphorylations in FMLP-induced human neutrophils.
6 on JNK and AKT

M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
1679
J = 7.8 Hz, H-50), 7.66 (1H, t, J = 7.4 Hz, H-7), 7.86 (1H, d, J = 8.5 Hz,
H-8), 7.89 (1H, br d, J = 9.4 Hz, H-40), 8.37 (1H, d, J = 7.7 Hz, H-60),
8.48 (1H, d, J = 7.8 Hz, H-5), 8.68 (1H, br s, H-20), 9.18 (1H, s, H-
4), 12.43 (1H, br s, D2O exchangeable, NH). 13C NMR (DMSO-d6,
75 MHz): d 113.5, 120.6, 121.0, 121.2, 121.5, 122.5, 129.6, 130.2,
130.5, 131.9, 134.1, 135.2, 135.4, 136.5, 136.9, 139.1, 142.3,
166.5, 191.0. EIMS m/z 396 [M+2]+ (95), 394 [M]+ (94), 349 (53),
322 (51), 315 (52), 271 (81), 241 (100). HREIMS m/z 393.9949
[M]+ (calcd for C19H11N2O3Br, 393.9953).
166.1, 194.2. FABMS m/z 362 [M+H]+ (2), 307 (20), 289 (11).
HRFABMS m/z 361.0699 [M]+ (calcd for C19H11N3O5, 361.0699).
5.2.12. (9H-b-Carbolin-1-yl)-phenyl-methanone (14)
Yellow needles (EtOAc), mp 135–138 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.13. (9H-b-Carbolin-1-yl)-p-tolyl-methanone (15)
Yellow needles (EtOAc), mp 158–161 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.7. 1-(2-Bromobenzoyl)-9H-b-carboline-3-carboxylic acid (7)
Yellow powder (EtOAc–MeOH), mp 281–283 °C. 1H NMR
(DMSO-d6, 300 MHz): d 7.39 (1H, t, J = 7.5 Hz, H-6), 7.47–7.57
(2H, m, H-50 and -7), 7.67 (2H, m, H-30 and -40), 7.75 (1H, d,
J = 6.5 Hz, H-60), 7.87 (1H, d, J = 8.0 Hz, H-8), 8.48 (1H, d,
J = 8.1 Hz, H-5), 9.15 (1H, s, H-4), 12.54 (1H, br s, D2O exchange-
able, NH), 12.90 (1H, br s, D2O exchangeable, CO2H). 13C NMR
(DMSO-d6, 75 MHz): d 113.6, 119.6, 120.6, 121.4, 121.5, 122.6,
127.3, 129.8, 130.7, 131.8, 131.9, 132.8, 134.6, 136.5, 137.2,
140.4, 142.5, 166.3, 196.4; EIMS m/z 394 [M]+ (1), 393 (2), 315
(100), 271 (16), 242 (24). HREIMS m/z 393.9951 [M]+ (calcd for
5.2.14. (9H-b-Carbolin-1-yl)-m-tolyl-methanone (16)
Yellow needles (EtOAc), mp 142–144 °C. 1H NMR (DMSO-d6,
300 MHz): d 2.41 (3H, s, CH3), 7.31 (1H, t, J = 7.5 Hz, H-6),
7.44 (2H, m, H-40 and -50), 7.60 (1H, t, J = 7.5 Hz, H-7), 7.81
(1H, d, J = 8.2 Hz, H-8), 7.96 (2H, m, H-20 and -60), 8.32 (1H,
d, J = 7.8 Hz, H-5), 8.44 (1H, d, J = 4.9 Hz, H-4), 8.52 (1H, d,
J = 4.9 Hz, H-3), 12.04 (1H, br s, D2O exchangeable, NH). 13C
NMR (DMSO-d6, 75 MHz): d 21.2, 113.1, 119.0, 120.2, 120.4,
122.0, 128.0, 128.3, 129.1, 131.1, 131.1, 133.0, 135.9, 136.6,
137.3, 137.3, 137.8, 141.8, 194.3. EIMS m/z 286 [M]+ (100),
285 (98), 271 (27), 258 (68), 257 (86), 243 (12), 167 (15),
140 (32). HREIMS m/z 286.1108 [M]+ (calcd for C19H14N2O,
286.1106).
C19H11N2O3Br, 393.9953).
5.2.8. 1-(4-Methoxybenzoyl)-9H-b-carboline-3-carboxylic acid
(8)
Yellow powder (EtOAc–MeOH), mp 264–266 °C. 1H NMR and
5.2.15. (9H-b-Carbolin-1-yl)-o-tolyl-methanone (17)
13C NMR: see Ref. 25.
Yellow needles (EtOAc), mp 150–151 °C. 1H NMR (DMSO-d6,
300 MHz): d 2.27 (3H, s, CH3), 7.28–7.35 (3H, m, H-6, -30 and -50),
7.43 (1H, t, J = 7.4 Hz, H-40), 7.49 (1H, d, J = 7.5 Hz, H-60), 7.62
(1H, d, J = 7.5 Hz, H-7), 7.83 (1H, d, J = 8.2 Hz, H-8), 8.32 (1H, d,
J = 7.8 Hz, H-5), 8.43 (2H, s, H-3 and -4), 12.15 (1H, br s, D2O
exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 19.9, 113.3,
119.4, 120.2, 120.5, 122.1, 125.1, 129.2, 129.3, 130.0, 130.5,
131.3, 135.5, 136.0, 136.4, 137.8, 139.4, 142.0, 199.1. EIMS m/z
286 [M]+ (79), 285 (100), 271 (29), 258 (60), 257 (61), 243 (14),
167 (9), 140 (18), 119 (29), 91 (48). HREIMS m/z 286.1109 [M]+
(calcd for C19H14N2O, 286.1106).
5.2.9. 1-(3-Methoxybenzoyl)-9H-b-carboline-3-carboxylic acid
(9)
Yellow powder (EtOAc–MeOH), mp 282–284 °C. 1H NMR
(DMSO-d6, 300 MHz): d 3.87 (3H, s, OCH3), 7.26 (1H, dd, J = 8.1,
2.6 Hz, H-40), 7.37 (1H, t, J = 7.6 Hz, H-6), 7.51 (1H, t, J = 8.0 Hz,
H-50), 7.65 (1H, t, J = 7.4 Hz, H-7), 7.85 (1H, d, J = 8.1 Hz, H-8),
7.94 (1H, d, J = 7.6 Hz, H-60), 8.27 (1H, br s, H-20), 8.48 (1H, d,
J = 8.4 Hz, H-5), 9.17 (1H, s, H-4), 12.41 (1H, br s, D2O exchange-
able, NH), 13.03 (1H, br s, D2O exchangeable, CO2H). 13C NMR
(DMSO-d6, 75 MHz): d 55.4, 113.5, 116.7, 119.3, 120.7, 120.7,
121.2, 122.4, 123.5, 129.4, 129.5, 131.7, 135.8, 136.3, 136.9,
138.2, 142.2, 158.9, 166.6, 191.9. FABMS m/z 347 [M+H]+ (7), 313
(7), 191 (10), 154 (26). HRFABMS m/z 347.1031 [M+H]+ (calcd for
5.2.16. (4-Bromophenyl)-(9H-b-carbolin-1-yl)-methanone (18)
Yellow needles (EtOAc), mp 194–196 °C. 1H NMR and 13C NMR:
see Ref. 25.
C20H15N2O4, 347.1032).
5.2.17. (3-Bromophenyl)-(9H-b-carbolin-1-yl)-methanone (19)
Yellow needles (EtOAc), mp 148–150 °C. 1H NMR (DMSO-d6,
300 MHz): d 7.32 (1H, t, J = 7.5 Hz, H-6), 7.55 (1H, t, J = 7.8 Hz,
H-50), 7.61 (1H, t, J = 7.6 Hz, H-7), 7.82 (1H, d, J = 8.3 Hz, H-40),
7.86 (1H, d, J = 8.2 Hz, H-8), 8.16 (1H, d, J = 7.9 Hz, H-60), 8.33
(1H, d, J = 7.2 Hz, H-5), 8.34 (1H, d, J = 1.4 Hz, H-20), 8.48 (1H, d,
J = 4.9 Hz, H-4), 8.55 (1H, d, J = 4.9 Hz, H-3), 12.10 (1H, br s, D2O
exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 113.2, 119.5,
120.2, 120.5, 121.2, 122.1, 129.2, 129.8, 130.4, 131.4, 133.3,
134.9, 135.9, 136.1, 137.5, 139.8, 141.9, 192.5. EIMS m/z 352
[M+2]+ (87), 351 [M+1]+ (94), 350 [M]+ (88), 349 (79), 324 (56),
323 (53), 322 (57), 321 (43), 271 (56), 243 (100), 242 (53). HREIMS
m/z 350.0058 [M]+ (calcd for C18H11N2OBr, 350.0055).
5.2.10. 1-(4-Nitrobenzoyl)-9H-b-carboline-3-carboxylic acid (11)
Yellow powder (EtOAc–MeOH), mp 279–280 °C. 1H NMR
(DMSO-d6, 300 MHz): d 7.38 (1H, t, J = 7.5 Hz, H-6), 7.66 (1H, t,
J = 7.5 Hz, H-7), 7.87 (1H, d, J = 8.3 Hz, H-8), 8.39 (2H, d,
J = 8.7 Hz, H-20 and -60), 8.49 (1H, d, J = 8.2 Hz, H-5), 8.53 (2H, d,
J = 8.7 Hz, H-30 and -50), 9.20 (1H, s, H-4), 12.49 (1H, br s, D2O
exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 113.7, 120.7,
121.4, 121.5, 122.6, 123.2, 129.8, 132.1, 132.7, 134.9, 136.8,
137.0, 142.5, 142.7, 149.7, 166.6, 192.0. EIMS m/z 361 [M]+ (33),
331 (14), 316 (22), 287 (16), 270 (22), 214 (19), 194 (19), 184
(14), 168 (18), 167 (18), 140 (18), 120 (20), 105 (100), 77 (62).
HREIMS m/z 361.0699 [M]+ (calcd for C19H11N3O5, 361.0699).
5.2.11. 1-(2-Nitrobenzoyl)-9H-b-carboline-3-carboxylic acid (13)
Yellow powder (EtOAc–MeOH), mp 280–281 °C. 1H NMR
(DMSO-d6, 300 MHz): d 7.39 (1H, t, J = 7.4 Hz, H-6), 7.68 (1H, t,
J = 7.7 Hz, H-7), 7.84 (1H, d, J = 7.5 Hz, H-60), 7.87 (1H, d,
J = 6.7 Hz, H-8), 7.88 (1H, t, J = 8.0 Hz, H-50), 7.98 (1H, t, J = 7.4 Hz,
H-40), 8.25 (1H, d, J = 8.3 Hz, H-30), 8.47 (1H, d, J = 7.8 Hz, H-5),
9.11 (1H, s, H-4), 12.55 (1H, br s, D2O exchangeable, NH), 12.81
(1H, br s, D2O exchangeable, CO2H). 13C NMR (DMSO-d6,
75 MHz): d 113.7, 120.5, 121.4, 121.5, 122.6, 123.8, 129.9, 130.2,
131.8, 132.0, 134.2, 134.8, 134.8, 135.7, 137.0, 142.6, 148.3,
5.2.18. (2-Bromophenyl)-(9H-b-carbolin-1-yl)-methanone (20)
Yellow plate crystals (EtOAc), mp 209–211 °C. 1H NMR
(DMSO-d6, 300 MHz): d 7.31 (1H, t, J = 7.5 Hz, H-6), 7.41–7.55
(3H, m, H-30, -40, and -50), 7.61 (1H, t, J = 7.4 Hz, H-7), 7.69 (1H, d,
J = 7.8 Hz, H-60), 7.82 (1H, d, J = 8.2 Hz, H-8), 8.29 (1H, d,
J = 7.8 Hz, H-5), 8.40 (2H, br s, H-3 and -4), 12.17 (1H, br s, D2O
exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 113.3, 119.1,
119.9, 120.1, 120.8, 122.1, 127.5, 129.5, 129.6, 131.4, 131.5,
132.5, 135.3, 135.5, 138.1, 141.5, 142.1, 197.1. EIMS m/z 352
[M+2]+ (13), 351 [M+1]+ (7), 350 [M]+ (13), 324 (7), 323 (7), 322

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M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
(8), 321 (6), 271 (100), 243 (63), 242 (48), 136 (49). HREIMS m/z
352.0211). Anal. Calcd for C18H13N2OBr: C, 61.21; H, 3.17; N, 7.93.
Found: C, 61.12; H, 3.79; N, 7.75.
350.0056 [M]+ (calcd for C18H11N2OBr, 350.0055).
5.2.19. (9H-b-Carbolin-1-yl)-(4-methoxyphenyl)-methanone (21)
Yellow needles (EtOAc), mp 185–187 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.26. (9H-b-Carbolin-1-yl)-(4-methoxyphenyl)-methanol (34)
Yellow powder (EtOAc), mp 145–147 °C. 1H NMR (DMSO-d6,
300 MHz): d 3.66 (3H, s, OCH3), 6.08 (1H, d, J = 3.9 Hz, H-14),
6.41 (1H, d, J = 3.9 Hz, OH-14), 6.82 (2H, d, J = 8.6 Hz, H-30 and
-50), 7.19 (1H, dd, J = 7.6, 7.4 Hz, H-6), 7.48 (2H, d, J = 8.5 Hz, H-20
and -60), 7.49 (1H, dd, J = 7.8, 7.4 Hz, H-7), 7.71 (1H, d, J = 7.8 Hz,
H-8), 7.96 (1H, d, J = 5.2 Hz, H-4), 8.17 (1H, d, J = 7.6 Hz, H-5),
8.21 (1H, d, J = 5.2 Hz, H-3), 11.22 (1H, br s, D2O exchangeable,
NH). 13C NMR (DMSO-d6, 75 MHz): d 55.2, 75.6, 112.7, 113.6,
113.7, 119.3, 120.6, 121.5, 127.7, 128.1, 128.8, 132.3, 136.0,
137.0, 140.8, 148.0, 158.5. EIMS m/z 304 [M]+ (100), 285 (98),
271 (76), 255 (72), 242 (30), 197 (12), 168 (46), 140 (30),
121 (60). HREIMS m/z 304.1215 [M]+ (calcd for C19H16N2O2,
304.1212).
5.2.20. (9H-b-Carbolin-1-yl)-(3-methoxyphenyl)-methanone (22)
Yellow plate crystals (EtOAc), mp 192–194 °C. 1H NMR
(DMSO-d6, 300 MHz): d 3.93 (3H, s, OCH3), 7.20 (1H, dd, J = 7.2,
1.9 Hz, H-20), 7.29 (1H, t, J = 7.5 Hz, H-6), 7.45 (1H, t, J = 8.0 Hz, H-
50), 7.58 (1H, t, J = 7.5 Hz, H-7), 7.68 (1H, d, J = 1.9 Hz, H-20), 7.73
(1H, d, J = 7.5 Hz, H-60), 7.79 (1H, d, J = 8.2 Hz, H-8), 8.27 (1H, d,
J = 7.8 Hz, H-5), 8.39 (1H, d, J = 4.8 Hz, H-4), 8.49 (1H, d, J = 4.8 Hz,
H-3), 12.01 (1H, br s, D2O exchangeable, NH). 13C NMR (DMSO-d6,
75 MHz): d 55.7, 113.3, 116.3, 118.2, 119.3, 120.5, 120.7, 122.2,
123.7, 129.5, 129.6, 131.5, 136.2, 136.7, 137.6, 139.1, 142.0, 159.1,
194.0. EIMS m/z 302 [M]+ (99), 301 (100), 287 (31), 274 (48), 273
(82), 271 (25), 259 (21), 258 (21), 244 (29), 243 (26), 230 (16).
HREIMS m/z 302.1052 [M]+ (calcd for C19H14N2O2, 302.1055).
5.2.27. 1-Acetyl-9H-b-carboline-3-carboxylic acid (40)
Yellow powder (EtOAc–MeOH), mp 292 °C (dec). 1H NMR and
13C NMR: see Ref. 25.
5.2.21. (9H-b-Carbolin-1-yl)-(4-nitrophenyl)-methanone (24)
Yellow needles (EtOAc), mp 216–217 °C. 1H NMR (DMSO-d6,
300 MHz): d 7.34 (1H, t, J = 7.6 Hz, H-6), 7.63 (1H, t, J = 7.5 Hz,
H-7), 7.84 (1H, d, J = 8.2 Hz, H-8), 8.33 (2H, d, J = 8.7 Hz, H-20 and
-60), 8.34 (1H, m, H-5), 8.40 (2H, d, J = 8.7 Hz, H-30 and -50), 8.50
(1H, d, J = 4.9 Hz, H-4), 8.54 (1H, d, J = 4.9 Hz, H-3), 12.18 (1H, br
s, D2O exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 113.3,
119.8, 120.2, 120.7, 122.1, 123.1, 129.4, 131.6, 132.0, 135.5,
136.1, 137.7, 142.0, 143.4, 149.3, 193.2. EIMS m/z 317 [M]+ (100),
289 (46), 270 (39), 243 (62), 167 (17), 140 (23). HREIMS m/z
317.0800 [M]+ (calcd for C18H11N3O3, 317.0800).
5.2.28. 1-(9H-b-Carbolin-1-yl)-ethanone (41)
Yellow needles (EtOAc), mp 207–209 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.29. 1-(9H-b-Carbolin-1-yl)-ethanol (42)
Yellow powder (EtOAc), mp 168–170 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.30. 1-Acetyl-9H-b-carboline-3-carboxylic acid amide (43)
Yellow powder (EtOAc–MeOH), mp 275–277 °C. 1H NMR
(DMSO-d6, 300 MHz): d 2.89 (3H, s, CH3), 7.33 (1H, dd, J = 7.8,
7.5 Hz, H-6), 7.61 (1H, dd, J = 8.1, 7.5 Hz, H-7), 7.64 (1H, br s, D2O
exchangeable, NH), 7.83 (1H, d, J = 8.2 Hz, H-8), 8.20 (1H, br s, D2O
exchangeable, NH), 8.42 (1H, d, J = 7.8 Hz, H-5), 9.10 (1H, s, H-4),
12.14 (1H, br s, D2O exchangeable, NH). 13C NMR (DMSO-d6,
75 MHz): d 26.2, 113.5, 118.2, 120.5, 121.0, 122.4, 129.4, 132.1,
134.1, 135.0, 139.1, 142.5, 166.5, 201.4. EIMS m/z 253 [M]+ (100),
236 (18), 208 (26), 194 (14), 182 (8). HREIMS m/z 253.0849 [M]+
(calcd for C14H11N3O2, 253.0851).
5.2.22. (9H-b-Carbolin-1-yl)-(3-nitrophenyl)-methanone (25)
Yellow plate crystals (EtOAc), mp 215–217 °C. 1H NMR
(DMSO-d6, 300 MHz): d 7.34 (1H, t, J = 7.4 Hz, H-6), 7.63 (1H, t,
J = 7.6 Hz, H-7), 7.84 (1H, d, J = 7.7 Hz, H-8), 7.88 (1H, t, J = 8.0 Hz,
H-50), 8.35 (1H, d, J = 7.8 Hz, H-5), 8.50 (1H, dd, J = 9.0, 1.3 Hz,
H-60), 8.51 (1H, d, J = 4.5 Hz, H-4), 8.56 (1H, d, J = 4.5 Hz, H-3),
8.61 (1H, d, J = 7.6 Hz, H-40), 9.01 (1H, br s, H-20), 12.17 (1H, br s,
D2O exchangeable, NH). 13C NMR (DMSO-d6, 75 MHz): d 113.4,
119.8, 120.3, 120.7, 122.2, 125.7, 126.7, 129.4, 130.0, 131.7,
135.6, 136.3, 137.2, 137.7, 139.0, 142.1, 147.6, 191.8. FABMS m/z
341 [M+Na]+ (34), 318 [M+H]+ (66). HRFABMS m/z 318.0878
[M+H]+ (calcd for C18H12N3O3, 318.0879).
5.2.31. 1-(4-Methoxybenzoyl)-9H-b-carboline-3-carboxylic acid
methyl ester (44)
Yellow powder (EtOAc–MeOH), mp 181–183 °C. 1H NMR
(DMSO-d6, 300 MHz): d 3.89 (3H, s, OCH3-40), 3.95 (3H, s, OCH3-
15), 7.13 (2H, d, J = 8.9 Hz, H-30 and -50), 7.36 (1H, dd, J = 7.6,
7.5 Hz, H-6), 7.64 (1H, dd, J = 7.9, 7.6 Hz, H-7), 7.82 (1H, d,
J = 7.9 Hz, H-8), 8.48 (1H, d, J = 7.5 Hz, H-5), 8.51 (2H, d, J = 8.9 Hz,
H-20 and -60), 9.15 (1H, s, H-4), 12.37 (1H, br s, D2O exchangeable,
NH). 13C NMR (DMSO-d6, 75 MHz): d 52.5, 55.8, 113.5, 113.8,
120.5, 120.7, 121.1, 122.4, 129.4, 129.5, 131.4, 134.0, 135.1, 136.8,
142.2, 163.4, 165.6, 190.5. EIMS m/z 360 [M]+ (100), 359 (71), 345
(15), 331 (32), 299 (28), 274 (22), 229 (16), 135 (51). HREIMS m/z
360.1110 [M]+ (calcd for C21H16N2O4, 360.1110). Anal. Calcd for
5.2.23. (9H-b-Carbolin-1-yl)-phenyl-methanol (27)
Yellow powder (EtOAc), mp 141–144 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.24. (9H-b-Carbolin-1-yl)-p-tolyl-methanol (28)
Yellow powder (EtOAc), mp 184–185 °C. 1H NMR and 13C NMR:
see Ref. 25.
5.2.25. (4-Bromophenyl)-(9H-b-carbolin-1-yl)-methanol (31)
Yellow powder (EtOAc), mp 150–151 °C. 1H NMR (DMSO-d6,
300 MHz): d 6.14 (1H, d, J = 4.1 Hz, H-14), 6.62 (1H, d, J = 4.1 Hz,
OH-14), 7.20 (1H, dd, J = 7.6, 7.5 Hz, H-6), 7.47 (2H, d, J = 8.5 Hz,
H-20 and -60), 7.51 (1H, dd, J = 8.0, 7.5 Hz, H-7), 7.55 (2H, d,
J = 8.5 Hz, H-30 and -50), 7.71 (1H, d, J = 8.0 Hz, H-8), 7.99 (1H, d,
J = 5.3 Hz, H-4), 8.17 (1H, d, J = 7.6 Hz, H-5), 8.23 (1H, d, J = 5.3 Hz,
H-3), 11.27 (1H, br s, D2O exchangeable, NH). 13C NMR (DMSO-d6,
75 MHz): d 75.2, 112.7, 114.0, 119.4, 120.3, 120.6, 121.6, 128.2,
128.7, 129.0, 131.1, 132.4, 137.1, 140.9, 143.3, 147.1. EIMS m/z 354
[M+2]+ (34), 352 [M]+ (35), 335 (24), 333 (22), 255 (100), 197 (15),
168 (30). HREIMS m/z 352.0211 [M]+ (calcd for C18H13N2OBr,
C21H16N2O4: C, 69.99; H, 4.48; N, 7.77. Found: C, 69.82; H, 4.48; N,
7.74.
5.3. Chemicals and antibodies
Prior to use, the synthetic compounds were dissolved in di-
methyl sulfoxide (DMSO) to make stock solutions. LPS (Escherichia
coli serotype 0111:B4), FMLP, cytochalasin B, ferricytochrome c,
penicillin, streptomycin, sulfanilamide, naphthylenediamine,
EDTA, Triton X-100, phenylmethylsulfonyl fluoride, Tris–HCl,
leupeptin, aprotinin, and NP-40 were obtained from Sigma (St.

M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
1681
Louis, MO, USA). IFN-
c
was obtained from Genzyme (Cambridge,
5.8. Cell viability
MA). Dulbecco’s Modified Eagle’s Medium (DMEM) and Hank’s
balanced salt solution (HBSS) were purchased from Gibco (Grand
Island, NY).
Cell respiration, an indicator of cell viability, was determined by
the mitochondrial-dependent reduction of 3-(3,4-dimethylthiazol-
2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan. The
extent of the reduction of MTT to formazan within cells was quan-
tified by measuring the optical density at 550 nm.
5.4. Cell growth inhibition assay
All stock cultures were grown in T-25 flasks. Freshly trypsinized
cell suspensions were seeded in 96-well microtiter plates with
compounds added from DMSO diluted stock. The plates were incu-
bated for an additional 72 h after attachment and drug addition,
and the assay was terminated by 10% trichloroacetic acid. Then,
0.4% sulforhodamine B dye in 1% HOAc was added to stain the cells
for 10 min. Unbound dye was removed by repeated washing with
1% HOAc, and the plates were air-dried. Bound stain was subse-
quently solved with 10 mM Trizma base and the absorbance read
at 515 nm. Growth inhibition of 50% (GI50) was calculated as the
drug concentration that caused a 50% reduction in the net protein
increase in control cells during the drug incubation. The mean GI50
is the concentration of agent that reduces cell growth by 50% under
the experimental conditions and is the average from at least three
independent determinations. Variation between replicates was no
more than 5% of the mean. The following human tumor cell lines
were used in the assay: A549 (non-small cell lung cancer),
DU145 (prostate cancer), KB (nasopharyngeal carcinoma), and
KB-VIN (vincristine-resistant KB subline). All cell lines were ob-
tained from the Lineberger Cancer Center (UNC-CH) or from ATCC
(Rockville, MD). Cells propagated in RPMI-1640 supplemented
5.9. iNOS and COX2 expression
After incubation, cells were lysed in ice-cold RIPA buffer
(25 mM Tris–HCl, pH 7.2; 0.1% SDS; 0.1% Triton X-100; 1% sodium
deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM sodium orthovan-
adate; 1 mM phenylmethanesulfonyl fluoride; 10
lg/mL aprotinin,
and 5 g/mL leupeptin). The protein concentration was deter-
l
mined using Bio-Rad protein assay kit and separated by 7.5%
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS–PAGE). After transferring onto nitrocellulose membrane, blot
was first incubated with antibodies for iNOS and COX2 (Chemicon,
Temecula, CA), respectively, and then incubated with horseradish
peroxidase (HRP)-conjugated anti-IgG. Immunoreactive bands
were visualized by an enhanced chemiluminescence system
(Amersham Biosciences Buckinghamshire, UK). Thereafter, the
same membrane was stripped and re-probed with mouse anti-
tubulin antibody.
a-
5.10. Preparation of human neutrophils
with 10% FBS, penicillin (100 IU/mL), streptomycin (1
l
g/mL),
Blood was taken from healthy human donors (20–30 years old)
by venipuncture, using a protocol approved by the institutional re-
view board at Chang Gung Memorial Hospital. Neutrophils were
isolated with a standard method of dextran sedimentation prior
to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis
of erythrocytes. Purified neutrophils that contained >98% viable
cells, as determined by the trypan blue exclusion method, were
re-suspended in a calcium (Ca2+)-free HBSS buffer at pH 7.4, and
were maintained at 4 °C before use.
and amphotericin B (0.25 g/mL), and were cultured at 37 °C in a
l
humidified atmosphere of 95% air/5% CO2.
5.5. Cell culture
RAW 264.7 macrophages (ATCC TIB-71) were cultured in DMEM
containing 10% heat-inactivated fetal calf serum, 100 U/mL penicil-
lin and 100 lg/mL streptomycin and grown at 37 °C with 5% CO2 in
fully humidified air. Cells used for experiments were between pas-
sages 9 and 20.
5.11. Measurement of Oꢀ2ꢁ generation
The Oꢀ2ꢁ generation assay was based on the SOD-inhibitable
reduction of ferricytochrome c.33 In brief, after supplementation
with 0.5 mg/mL ferricytochrome c and 1 mM Ca2+, neutrophils
(6 ꢃ 105 cells/mL) were equilibrated at 37 °C for 2 min and incu-
bated with drugs or an equal volume of vehicle (0.1% DMSO) for
5 min. Cells were activated with 100 nM FMLP during the preincu-
5.6. Measurement of PGE2
Assessment of PGE2 synthesis was performed using a commer-
cially available PGE2 competitive enzyme immunoassay kit
(Cayman, MI). Supernatant from the culture medium and the
standards were put into different wells on the goat anti-mouse
IgG-coated microtiter plate provided in the kit. Mouse anti-PGE2
antibody and peroxidase-conjugated PGE2 were added to each
well, and the plate was incubated at room temperature with shak-
ing for 1 h. The wells were drained and washed, and 3,30,5,50-tetra-
methylbenzidine/hydrogen peroxide solution was then added. The
plate was incubated at room temperature with shaking, and the
reaction was stopped after 30 min through the addition of H2SO4.
The absorbance of the content of each well was then measured
at a wavelength of 450 nm.
bation of 1 lg/mL cytochalasin B (FMLP/cytochalasin B) for 3 min.
Changes in the absorbance with a reduction in ferricytochrome c at
550 nm were continuously monitored in a double-beam, six-cell
positioner spectrophotometer with constant stirring (Hitachi
U-3010, Tokyo, Japan).
5.12. Measurement of elastase release
Degranulation of azurophilic granules was determined by
elastase release as described previously.30 Experiments were
performed using MeO-Suc-Ala-Ala-Pro-Val-pnitroanilide as the
elastase substrate. Briefly, after supplementation with MeO-Suc-
5.7. Nitrite assay
Ala-Ala-Pro-Val-p-nitroanilide (100
mL) were equilibrated at 37 °C for 2 min and incubated with drugs
l
M), neutrophils (6 ꢃ 105/
LPS (1 lg/mL) plus IFN-c (50 U/mL) was added to fresh culture
medium for 18 h to induce nitrite formation, an indicator of NO
synthesis. Nitrite concentration in the absence or presence of the
synthetic compounds was measured by adding Griess reagent
(1% sulfanilamide and 0.1% naphthylenediamine in 5% phosphoric
acid). The optical density at 550 nm (OD550) was measured with
a microplate reader.
or an equal volume of vehicle (0.1% DMSO) for 5 min. Cells were
activated by 100 nM FMLP and 0.5 lg/mL cytochalasin B, and
changes in absorbance at 405 nm were continuously monitored
to assay elastase release. The results were expressed as the percent
of elastase release in the FMLP/cytochalasin B-activated, drug-free
control system.

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M.-L. Yang et al. / Bioorg. Med. Chem. 19 (2011) 1674–1682
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Acknowledgments
The authors are thankful for the financial support from the
National Science Council, Taiwan, Republic of China, awarded to
T. S. Wu and for partial support from NIH Grant CA 17625 awarded
to K.-H. Lee. Authors are also grateful to the National Center for
High-performance Computing, Taiwan, Republic of China, for com-
puter time and facilities.
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