Synthesis and biological evaluation of a novel class of β-carboline derivatives

Article abstract of DOI:10.1039/c4nj00262h

In this study, several novel β-carboline derivatives, 1-(4-hydroxy-3-methoxyphenyl)-β-carboline-3-carboxyl-Trp-Trp-AA-OBzl compounds, were designed and synthesized as potential anticancer agents. Their in vitro cytotoxic activities were evaluated using me

Full text of DOI:10.1039/c4nj00262h

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Synthesis and biological evaluation of a novel
class of b-carboline derivatives†
Cite this: New J. Chem., 2014,
38, 4155
Hao Chen, Pengchao Gao, Meng Zhang, Wei Liao and Jianwei Zhang*
In this study, several novel b-carboline derivatives, 1-(4-hydroxy-3-methoxyphenyl)-b-carboline-3-
carboxyl-Trp-Trp-AA-OBzl compounds, were designed and synthesized as potential anticancer agents.
Their in vitro cytotoxic activities were evaluated using methylthiazoltetrazolium (MTT) assay. The in vivo
anti-tumor activity of the newly synthesized b-carboline derivatives was determined in a S180 bearing
mouse model and some of the compounds demonstrated tumor growth inhibition similar to the positive
control, doxorubicin. The intercalation of the b-carboline derivatives synthesized with calf thymus (CT)
DNA was also studied.
Received (in Montpellier, France)
22nd February 2014,
Accepted 10th June 2014
DOI: 10.1039/c4nj00262h
www.rsc.org/njc
seems to be essential for many naturally occurring biologically
important indole alkaloids and represents an important lead struc-
Introduction
Cancer has long been one of the serious diseases threatening ture for the development of novel pharmacological agents.10–17
human health and continues to be a major health problem Some b-carboline alkaloids, such as harmine and its derivatives,
worldwide. Therefore, discovering new compounds with potent are highly cytotoxic against human tumor cell lines.18–23 In studies
anticancer activity is one important goal. Among the current with b-carboline, DNA intercalation and cytotoxicity showed a
anticancer chemotherapeutic agents, DNA-recognizing molecules, correlation.24–27 Thus, b-carbolines act through intercalation to
including intercalating agents, alkylating compounds and groove inhibit DNA topoisomerases I and II and cause DNA damage.
binders, are especially interesting. DNA intercalating agents
One of the commonly used approaches in the search for new
are important in clinical oncology, and several representative bioactive agents from natural products is chemical modifica-
compounds (anthracyclines, acridines, and anthraquinones) are tion of naturally occurring lead compounds. The b-carboline
routinely used.1 Intercalation results in conformational alteration framework is used as an important pharmacophore for the
of the double helix, and then changes the processes of DNA design of novel anti-tumor agents in this study. Previous
replication, transcription and repair. Thus the discovery of new studies on a variety of synthetic b-carboline derivatives have
DNA intercalating agents is considered as a promising approach demonstrated the influence of molecular planarity and sub-
toward anticancer drugs.
stitutions at positions 1, 3 and 9 of the b-carboline skeleton on
Polycyclic indolic compounds have attracted a great deal of anti-tumor activities.28 It has also been shown that substitu-
interest amongst synthetic chemists over the years, largely tions, especially at positions 1 and 3, could significantly reduce
because of the diverse biological activities demonstrated by the toxicity of b-carboline derivatives. Some studies demon-
this type of compounds.2 Given their rigid structures, polycyclic strated that b-carboline derivatives containing a substituted
indolic compounds are expected to show substantial selectivity phenyl group at position 1 possess anti-tumor activities.29
in their interactions with enzymes or receptors. However, Several compounds having 1-(4-hydroxy-3-methoxyphenyl)-b-
compounds bearing this ring system have displayed a diverse carbolines bearing the 2-substituted-1,3,4-oxadiazol-5-yl and
range of biological properties including antimalarial, anti-tumor, 5-substituted-1,2,4-triazol-3-yl moieties at C-3 were reported to
anti-HIV, anticonvulsive, hypnotic and anxiolytic, antiviral, and possess antitumoral activities.30
antimicrobial activities as well as inhibition of topoisomerase-II
It is well proved that Trp-Trp is biologically important either as
and cGMP-dependent processes.3–8 In addition, antiplasmodial a dipeptide or as a fragment of some peptides. Trp-Trp-OBz was
activity has also been reported.9 The structure of b-carboline used as a lead, and twenty tripeptide benzyl esters, Trp-Trp-AA-
OBz, were synthesized as DNA intercalators.31 In view of the
importance associated with the above cited moieties in their
potent anti-tumor activity, it was thought of considerable interest
to employ derivatives of Trp-Trp-AA-OBz for the generation of
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069,
People’s Republic of China. E-mail: jwzhang2006@163.com;
Fax: +86-10-8391-1533; Tel: +86-10-8391-1522
new 1-(4-hydroxy-3-methoxyphenyl)-3-carboxyl b-carbolines, which
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4nj00262h
are presented in Table 1. In addition, the use of tripeptides in
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Table 1 Structures of b-carboline derivatives synthesized
Compound no.
Chemical structure
Compound no.
Chemical structure
3
4
5
6
7
8
9
10
12
14
16
18
20
11
13
15
17
19
21
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b-carboline derivatives design was based upon the concept that double doublet at d 4.96–4.80 and 4.78–4.49 are in agreement
many amino acids with functional side chains are capable of with the a-methine proton of tryptophan. The signals at 4.96–
making base-specific contacts with more than one type of DNA 4.80, and 4.78–4.49 in the COSY spectrum indicated methylenes
1
bases,32 a series of amino acids containing either nonpolar, acidic, adjacent to methines. HNMR spectroscopic signals at d 4.80–
basic, or aromatic sides, and different functionalities with absolute 3.95 are in agreement with the a-methine proton of the AA in
stereochemistry were introduced into the b-carboline core. More- NH2-Trp-Trp-AA-OBzl.
over, amino acid conjugates might target the gastrointestinal
transporters involved in the absorption of amino acids and small
peptides resulting in improved oral bioavailability.33,34 The various
In vitro cytotoxicity
side chains of different amino acids allow the addition of amino The in vitro cytotoxicity of the b-carboline derivatives synthesized
acids to b-carboline to manipulate the pharmacokinetic profiles of above was evaluated in human colon cancer cells (HT-29), human
the compounds. In addition, various amino acids can be intro- lung adenocarcinoma cells (A549), chronic myeloid leukemia
duced to enhance the solubility. Therefore, in order to search for cells (K562) and human immature granulocyte leukemia cells
better DNA intercalating agents, b-carboline was used as the basic (HL-60) using MTT assay.22 Doxorubicin (adriamycin, ADM) was
system and a series of novel b-carboline derivatives bearing a used as a positive control. In brief, cells were exposed to 7–21 at
4-hydroxy-3-methoxyphenyl group at position 1 and Trp-Trp-AA- concentrations ranging from 1 mM to 400 mM for 48 h and cell
OBzl at position 3 were designed and synthesized and evaluated survival was then determined. IC50 is the concentration at which
for their anti-tumor activity in the present study.
50% of cells were killed. As shown in Table 2, the IC50 values of
ADM in K562, HT-29, HL60 and A549 cells were found to be 1.3,
8.1, 1.7 and 3.5 mM, respectively. 7–21 exhibited varied activities
in the four cell lines and they were found to be most effective in
K562 cells and least effective in HT-29 cells. In K562 cells, all
compounds demonstrated IC50 values of less than 90 mM, and 9,
Results and discussion
Chemistry
As shown in Scheme 2, the syntheses of b-carboline derivatives 12, 15, 18, 19 and 21 were found to be most effective with IC50
were carried out using a multi-step synthetic route. Firstly, 1-(4- values of less than 20 mM. The IC50 of 12 (IC50, 12.0 mM) is almost
hydroxy-3-methoxyphenyl)-tetrahydro-b-carboline-3-carboxylic acid equipotent to that of 21 (IC50, 14.3 mM), suggesting that the
(3) was synthesized via the Pictet–Spengler reaction of tryptophan methyl group at the b or g position of an amino acid residue
with vanillin in acetic acid. 3 obtained was composed of two does not cause changes in the cytotoxicity. In HL60 cells, all
isomers with 1S,3S-cis and 1R,3S-trans configurations.13,30,35 Then compounds showed IC50 values of less than 200 mM except for 16,
esterification of 3 with methanol in the presence of SOCl2 yielded and 20 was found to be most effective with an IC50 value of
methyl 1-(4-hydroxy-3-methoxyphenyl)-tetrahydro-b-carboline-3- 16.0 mM. In A549 cells, all but 13 demonstrated IC50 values less
carboxylate (4). Secondly, the intermediate methyl 1-(4-hydroxy- than 200 mM and 8 was found to be most potent with an IC50
3-methoxyphenyl)-b-carboline-3-carboxylate (5) was prepared by value of 52.0 mM. Compared to 9 (IC50, 56.4 mM), 19 (IC50,
the reaction of 4 with selenium dioxide in acetic acid.13,36 102.0 mM) exhibited a lower cytotoxicity in A549 cells suggesting
Subsequently, removal of the methyl group with NaOH resulted that the introduction of the additional methyl group in the Ser
in 1-(4-hydroxy-3-methoxyphenyl)-b-carboline-3-carboxylic acid (6). residue at the b position reduced the cytotoxicity. The results
Finally, tripeptide benzyl esters, NH2-Trp-Trp-AA-OBzl, from seem to suggest that the b-carboline derivatives synthesized
Scheme 1 were introduced into 6 by the DCC/HOBt/N-methyl- exhibited moderate cytotoxic activity.
morpholine (NMM) procedure to provide the respective b-carbo-
line derivatives, 1-(4-hydroxy-3-methoxyphenyl)-b-carboline-3-carboxyl-
Trp-Trp-AA-OBzl (7–21), as shown in Scheme 2 (chemical yield,
In vivo anti-tumor activity
54–79%). The chemical structures of all 19 (3–21) b-carboline deriva- The in vivo anti-tumor activity of fifteen b-carboline derivatives
tives synthesized in this study are provided in Table 1 and they were synthesized (7–21) was evaluated in mice bearing S180.37 The
confirmed by 1HNMR, 13CNMR, IR, and HR-ESIMS results. The mice were given a daily i.p. injection of 0.1 mmol kgÀ1 of 7–21
spectroscopic data are given in the ‘‘Experimental’’ section. The in 0.2 mL of normal saline (NS) for seven consecutive days with
1HNMR spectra of b-carboline derivatives 7–21 showed signals NS as the negative control. ADM at 0.1 mmol kgÀ1 (0.2 mL)
at d 8.74–8.68 for one proton corresponding to H-4 of the dissolved in 0.9% saline was used as a positive control. The
pyridine ring in the b-carboline skeleton. The two pairs of 1H tumor weights were found to be 0.45 g to 0.84 g compared to
Scheme 1 Preparation of NH2-Trp-Trp-AA-OBzl. AA = Tyr, Glu, Thr, Lys, Val, Ile, Ala, Asp, Pro, Gly, Trp, Phe, Ser, Met or Leu. Reagents and conditions:
(i) NMM, DCC, HOBt; (ii) 2 M NaOH; (iii) NH2-AA-OBzl, NMM, DCC, HOBt; (iv) hydrogen chloride in ethyl acetate (4 mol LÀ1).
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Scheme 2 Synthesis of 1-(4-hydroxy-3-methoxyphenyl)-b-carboline-3-carboxyl-Trp-Trp-AA-OBzl compounds. Compound 7 (AA = Tyr), 8 (AA = Glu),
9 (AA = Thr), 10 (AA = Lys), 11 (AA = Val), 12 (AA = Ile), 13 (AA = Ala), 14 (AA = Asp), 15 (AA = Pro), 16 (AA = Gly), 17 (AA = Trp), 18 (AA = Phe), 19 (AA = Ser),
20 (AA = Met), 21 (AA = Leu). Reagents and conditions: (i) AcOH; (ii) SOCl2 and MeOH; (iii) SeO2 and AcOH; (iv) 6 M NaOH; (v) NH2-Trp-Trp-AA-OBzl,
NMM, DCC, HOBt.
Table 2 IC50 values of 7–21 in K562, HT-29, HL-60 and A549 cell linesa
Table 3 Tumor inhibition % by 7–21 in S180 bearing micea
Compounds
K562
HT-29
HL60
A549
Dose
Tumor
weight (g)
Tumor
inhibition (%)
Compound
(mmol kgÀ1
)
7
8
9
34.0 Æ 3.1
35.0 Æ 1.2
17.3 Æ 3.5
43.5 Æ 0.9
43.0 Æ 1.2
12.0 Æ 1.6
25.1 Æ 0.9
30.2 Æ 1.1
13.7 Æ 2.1
86.3 Æ 1.9
36.4 Æ 1.5
15.3 Æ 1.9
18.6 Æ 2.6
66.0 Æ 2.3
14.3 Æ 1.2
1.3 Æ 0.1
4200
4200
4200
4200
4200
4200
4200
4200
4200
4200
4200
4200
4200
99.5 Æ 1.3
4200
8.1 Æ 0.6
50.0 Æ 1.7
28.5 Æ 0.9
45.0 Æ 2.5
39.0 Æ 0.9
60.0 Æ 1.5
84.0 Æ 3.1
134.0 Æ 2.5
37.5 Æ 1.4
51.0 Æ 2.4
4200
40.3 Æ 2.2
33.1 Æ 1.9
34.5 Æ 1.8
16.0 Æ 1.9
68.0 Æ 2.5
1.7 Æ 0.4
76.5 Æ 1.4
52.0 Æ 1.4
56.4 Æ 1.8
113.1 Æ 1.5
162.2 Æ 2.8
140.0 Æ 1.1
4200
NS
ADM
7
8
9
10
11
12
1.24 Æ 1.30
0.45 Æ 0.11
0.53 Æ 0.13b
0.68 Æ 0.18c
0.61 Æ 0.17b
0.66 Æ 0.13c
0.84 Æ 0.23c
0.59 Æ 0.14b
0.60 Æ 0.16b
0.45 Æ 0.11b
0.68 Æ 0.19c
0.62 Æ 0.16b
0.64 Æ 0.12c
0.73 Æ 0.16c
0.46 Æ 0.13b
0.62 Æ 0.12b
0.83 Æ 0.21c
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
61.1 Æ 6.9
57.3 Æ 10.4
45.5 Æ 14.8
51.1 Æ 13.8
46.6 Æ 10.3
31.9 Æ 18.6
52.4 Æ 11.7
51.9 Æ 13.0
63.4 Æ 8.7
45.1 Æ 15.6
50.4 Æ 12.9
48.1 Æ 9.5
41.2 Æ 13.5
62.9 Æ 10.7
50.0 Æ 9.5
33.0 Æ 16.9
10
11
12
13
14
15
16
17
18
19
20
21
ADM
77.0 Æ 0.8
98.0 Æ 1.7
175.0 Æ 0.8 13
63.4 Æ 2.4
79.0 Æ 1.5
14
15
102.0 Æ 2.1 16
92.1 Æ 1.9
17
114.0 Æ 0.7 18
3.5 Æ 0.6
19
20
21
a
Results shown are x Æ SD mM; n = 6.
%
a
ADM = positive control, NS = vehicle, n = 12, tumor weight is expressed
by x Æ SD g. Compared to NS p o 0.01, to 0.1 mmol kgÀ1 ADM p 4
b
%
1.24 g in the negative control group. The tumor inhibition % in
describing the anti-tumor effects was determined as
0.05. Compared to NS p o 0.01, to 0.1 mmol kgÀ1 ADM p o 0.05.
c
Tumor Inhibition % = (C À T)/C Â 100
tumor inhibition (63.4% and 62.9%, respectively) in mice
where T is the average tumor weight of the treated group and bearing S180. Compared to 12 (52.4%), 11 and 21 exhibited a
C the average tumor weight of the negative control group.
lower activity (31.9% and 33.0%) suggesting that the isopropyl
The tumor inhibition % of the compounds tested were group at the a or b position of an amino acid residue is likely
found to range from 31.9% (11) to 63.4% (14) compared to responsible for the reduced activity observed. The in vivo results
61.1% for 0.1 mmol kgÀ1 of ADM as shown in Table 3. The suggest that subtle changes in the size and shape of the alkyl
results suggested that the amino acid introduced in the Trp- side chains of neutral aliphatic amino acids of the b-carboline
Trp-AA-OBzl of the b-carboline derivatives synthesized affected derivatives could cause changes in the in vivo anti-tumor
their in vivo anti-tumor activities. 14 and 19 showed the highest activities, which may imply that the spatial orientation and
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Table 4 Tumor inhibition % by 14 at different doses in S180 bearing mice
Compounda Dose (mmol kgÀ1) Tumor weight (g) Tumor inhibition (%)
findings in surviving animals at the end of the experimental
period (7 days) revealed no apparent changes in any organs.
The most potent compound was examined for LD50 and neuro-
toxicity in the mice model. The results indicate that even when
the dose of 14 was up to 500 mg kgÀ1 the mice neither exhibited
neurotoxic behavior nor death occurred. The necropsy findings
in mice receiving 14 on the 7th day revealed that the adminis-
NS
ADM
14
14
14
1.24 Æ 1.30
0.45 Æ 0.11
0.45 Æ 0.11b
0.69 Æ 0.14c
0.83 Æ 0.11d
0.1
0.1
0.01
0.001
61.1 Æ 6.9
63.4 Æ 8.7
44.3 Æ 11.2
32.7 Æ 8.5
a
b
ADM = positive control, NS = vehicle, n = 12. Compared to NS and tration led to no apparent changes in any organs. These suggest
0.01 mmol kgÀ1 of 14 p o 0.01. Compared to NS and 0.001 mmol kgÀ1
that 14 is comparatively non-toxic and its LD50 values should be
d
c
of 14 p o 0.01, to 0.1 mmol kgÀ1 ADM p o 0.01. Compared to NS
more than 500 mg kgÀ1
.
p o 0.01, to 0.1 mmol kgÀ1 ADM p o 0.01.
Intercalation of b-carboline derivatives toward CT DNA
There is compelling evidence that cellular DNA is the common
target of many anti-tumor agents and the interaction of anti-
tumor agents with DNA has been widely studied to understand
the mechanisms of anti-tumor agents. Since two structural
elements b-carboline and Trp-Trp of the b-carboline derivatives
are synthesized, each has a planar polycyclic aromatic pharma-
cophore capable of stacking between DNA pairs at the intercala-
tion sites,40,41 the intercalation of the b-carboline derivatives
synthesized in this study with calf thymus (CT) DNA was studied
using 14. There are a number of techniques used to study the
interaction of small molecules with DNA. UV absorption and
fluorescence spectroscopy are well known as simple but sensitive
methods. The UV and fluorescence spectra and viscosity of 14 in
the presence of CT DNA were obtained and compared with those
in the absence of CT DNA.
The UV spectrum of 14 in PBS (pH 7.4, 2 mM) was recorded on a
Shimadzu 2550 spectrophotometer from 220 to 350 nm. Two (2)
mL of 14 was then titrated with 10 mL of CT DNA in PBS (pH 7.4)
solutions of 0, 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 mM, respectively. The
corresponding UV spectra of the mixtures were also determined.
As shown in Fig. 1, the absorption of 14 gradually decreased from
0.644 to 0.522 with the increase of the CT DNA concentration and
a hypochromic effect (18.9%) was induced. Most investigators
characteristics of the pendant group is important in DNA
recognition and binding.38 It was also noticed that the
in vitro efficacy did not match the in vivo efficacy. It is well
known that the in vitro efficacy not matching with the in vivo
efficacy is not a rare phenomenon. Perhaps the absorption,
delivery, distribution and metabolism are partly responsible for
the mentioned phenomenon in 7–21.
14, the most potent compound, was selected to further
explore the effect of dose on the in vivo anti-tumor activity. As
shown in Table 4, at 0.001, 0.01, and 0.1 mmol kgÀ1, 14 exhibited
tumor inhibitions of 32.7, 44.3 and 63.4%, respectively.
Toxicity of b-carboline derivatives in mice
During chemotherapy, an increase in the body weight is an
important parameter of health. To evaluate the preliminary
toxicity of 7–21 during the administration the body weights of
the mice were measured. The data are listed in Table 5 as
increased body weight from the start of treatment. 7–18 and 20
showed a weight gain similar to that of the vehicle-treated
group. Only two compounds (19 and 21) showed significantly
lower increases in the body weight.
It is reported that harmine and its derivatives exhibited
significant antitumor activities in mice bearing both Lewis lung
cancer and Sarcoma 180, while they exhibited remarkable
neurotoxic effects.39 All the tested compounds caused neither
obvious neurotoxic reaction including tremor, twitch, jumping,
tetanus and supination nor death at the tested dosage, except
for the partial contortion at the injection point. The necropsy
Table 5 Effect of 7–21 on the body weight increase of S180 mice
Compounda
Increased BW
Compounda
Increased BW
NS
ADM
7
8
9
10
11
12
13
6.03 Æ 2.75
6.64 Æ 2.54
6.16 Æ 3.65
5.64 Æ 3.16
6.76 Æ 2.78
6.96 Æ 1.68
7.05 Æ 2.65
6.42 Æ 2.82
5.93 Æ 3.07
14
15
16
17
18
19
20
21
5.90 Æ 3.04
8.78 Æ 2.32
7.23 Æ 2.99
6.11 Æ 1.69
7.07 Æ 2.92
4.61 Æ 2.98b
6.06 Æ 2.82
3.67 Æ 3.41b
a
Dose of ADM and 7–21: 0.1 mmol kgÀ1, NS = vehicle, n = 12, increased
Fig. 1 UV spectra of 14 (concentration 2 mM) in the absence and presence
of CT DNA (pH = 7.4) at concentrations of 2.0, 4.0, 6.0, 8.0, 10.0 and
32.0 mM, respectively.
b
BW = increased body weight is expressed by x Æ SD g. Compared to NS
%
p o 0.05.
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Fig. 3 Effect of 14 on the relative viscosity of CT DNA (200 mM).
Fig. 2 Fluorescence spectra of 14 (concentration 2 mM) in the absence
and presence of CT DNA (pH = 7.4) at concentrations of 1.0, 2.0, 4.0, 8.0,
16.0 and 32.0 mM, respectively.
Conclusions
In summary, novel b-carboline derivatives, 1-(4-hydroxy-3-
methoxyphenyl)-b-carboline-3-carboxyl-Trp-Trp-AA-OBzl 7–21,
were prepared in acceptable yields using the five-step route
described above. In vitro cytotoxicity assays against four human
carcinoma cell lines (HT-29, A549, K562 and HL-60) explored
the cell selective anti-proliferation for individual compounds.
The in vivo anti-tumor activity assays with S180 mice revealed
that 14 and 19 have the highest efficacy. The tumor inhibition %
of 14 and 19 at 0.1 mmol kgÀ1 (63.4% and 62.9%, respectively) is
equipotent to that (61.1%) of the positive control doxorubicin
ADM at 0.1 mmol kgÀ1. The UV and fluorescence spectra, as well
as the relative viscosity measurements of 14 with or without CT
DNA suggest that the binding of the b-carboline derivatives
synthesized to DNA was intercalative.
believed that the small molecule binding to the base pairs can
cause hypochromism. The classical intercalative binding has been
characterized by large changes in the absorbance (hypochromism
r 35%).42–44 This could be considered to be a direct evidence for
the intercalation of 14 with CT DNA.
To further confirm the intercalation of 14 with CT DNA, the
fluorescence spectrum of 14 in PBS (pH 7.4, 2 mM) was obtained
and compared with that in the presence of CT DNA in PBS (final
concentrations: 0, 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 mM, respec-
tively, pH 7.4) on a Shimadzu RF-5310PC spectrofluorometer
(emission wave-length 220–400 nm and excitation wavelength
310 nm).45 It was found that the fluorescence intensity of 14
gradually decreased with the increase of CT DNA concentration
as shown in Fig. 2. When the concentration of CT DNA was
increased to 32.0 mM the fluorescence intensity of 14 was
decreased by 42.2% as shown in Fig. 2. Fluorescence quenching
is a very common phenomenon. It refers to the process of
decrease in the fluorescence intensity samples. Many of the
molecular interactions can result in fluorescence quenching.46
Experimental
Syntheses
General. Unless otherwise stated, all reactions were per-
Fluorescence quenching is a result of intercalation of 14 with formed under a nitrogen atmosphere (1 bar). All reagents used
CT DNA. were purchased from Sigma Chemical Co (USA). Optical rota-
The most convincing evidence for DNA intercalation is tions were determined using a Schmidt + Haensch Polartromic
generally provided by viscosity measurements.47,48 Insertion D instrument (Germany). The IR spectra were recorded using
of ligands between adjacent nucleobase pairs leads to length- an Avatar 330, Nicolet, USA spectrometer. The 1H and 13C NMR
ening and stiffening of the double helix, i.e. to structural spectra were recorded at 300 MHz on a VXR-300 instrument or
changes that are reflected in an increase in DNA viscosity while at 500 MHz on a Bruker AM-500 instrument in CDCl3 or in
a non-classical intercalation or a groove mode would reduce the DMSO-d6 with tetramethylsilane as the internal standard and
DNA viscosity.48,49 To further clarify the interaction between chemical shifts are expressed in ppm (d). Chromatography was
the b-carboline derivatives synthesized and CT DNA, viscosity carried out using Qingdao silica gel H (Qingdao of China). TLC
measurements were carried out. The effect of the test com- analysis was carried out on silica gel F254. The purities (495%)
pound on the viscosity of CT DNA solution is shown in Fig. 3. It of the intermediates and the products were confirmed by TLC
is clear that as the concentration of 14 in the mixture of 14 and (Merck silica gel plates of type 60 F254, 0.25 mm layer thickness,
CT DNA increased the viscosity of the preparation increased, Germany) and HPLC (Waters, C18 column 4.6 Â 150 mm, USA).
which suggests that the binding resulted in the lengthening of MS was acquired on a Quattro Micro ZQ2000, Waters, USA
the DNA double helix. This is in consistent with the intercala- instrument, and m/z values are reported. High-resolution mass
tion of 14 toward CT DNA.
spectra were recorded using a microTOF-Q mass spectrometer.
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Methyl 1-(4-hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole- DMSO-d6): d 11.76 (1H, s, OH), 10.77 (2H, brs, N–H), 9.40 (1H, s,
3-carboxylate. To a cooled, rapidly stirring solution of 4 (1 g, N–H), 9.23 (1H, s, N–H), 8.70 (1H, s, Ar–H), 8.69 (1H, d, J = 5.4 Hz,
2.57 mmol) in acetic acid (100 mL) SeO2 (2.86 g, 25.8 mmol) was N–H), 8.49 (1H, d, J = 7.2 Hz, N–H), 8.39 (1H, d, J = 9.9 Hz, N–H),
added, and the resulting mixture was refluxed for 1 h. The pH of 8.34 (1H, d, J = 7.8 Hz, Ar–H), 7.69 (1H, d, J = 9.1 Hz, Ar–H), 7.61–
the reaction mixture was adjusted to 9 with ammonia and kept on 7.54 (4H, m, Ar–H), 7.41 (1H, dd, J1 = 9.1 Hz, J2 = 1.8 Hz, Ar–H),
ice. The precipitate was collected and purified by silica gel column 7.36–7.23 (8H, m, Ar–H), 7.16–7.15 (2H, m, Ar–H), 7.14–7.02 (6H,
chromatography (40 : 1 CH2Cl2–MeOH) to provide 5 as brown m, Ar–H), 6.83 (1H, t, J = 7.5 Hz, Ar–H), 6.66 (2H, d, J = 8.4 Hz,
powder (500 mg, 1.44 mmol, 56% yield). [a]2D5 À23.8 (c 0.1 in Ar–H), 5.05 (2H, dd, J = 15.3 Hz, CH2Ph), 4.84 (1H, dd, J1 = 12.6 Hz,
MeOH); IR (cmÀ1, KBr, neat): 3055, 1716, 740; 1HNMR (300 MHz, J2 = 7.8 Hz, CH), 4.71 (1H, dd, J1 = 13.2 Hz, J2 = 8.4 Hz, CH), 4.50
DMSO-d6): d 11.58 (1H, s, OH), 8.85 (1H, s, Ar–H), 8.72 (1H, s, (1H, dd, J1 = 14.4 Hz, J2 = 7.2 Hz, CH),3.85 (3H, s, OCH3), 3.27–3.09
Ar–H), 8.40 (1H, d, J = 8.1 Hz, Ar–H), 7.70 (1H, d, J = 8.4 Hz, Ar–H), (3H, m, CH2), 3.02–2.87 (3H, m, CH2); 13CNMR (75 MHz, DMSO-
7.60 (1H, dt, J1 = 8.1 Hz, J2 = 0.9 Hz, Ar–H), 7.46 (1H, d, J = 1.8 Hz, d6) d 172.0, 171.7, 171.6, 164.8, 156.6, 148.2, 148.1, 141.9, 141.5,
Ar–H), 7.44 (1H, dd, J1 = 8.1 Hz, J2 = 1.8 Hz, Ar–H), 7.33 (1H, t, J = 139.4, 136.6, 136.5, 136.2, 134.5, 130.5, 129.9, 129.1, 128.8, 128.4,
7.2 Hz, Ar–H), 7.04 (1H, d, J = 8.1 Hz, Ar–H), 3.93 (3H, s, OCH3), 128.3, 127.9, 127.8, 127.3, 124.2, 124.0, 122.3, 121.8, 121.7, 121.3,
3.91 (3H, s, OCH3); 13CNMR (125 MHz, DMSO-d6) d 166.6, 148.2, 120.6, 119.0, 118.9, 118.6, 116.2, 115.6, 113.2, 112.9, 112.7, 111.7,
148.1, 143.1, 141.8, 136.9, 134.9, 129.2, 129.1, 128.9, 122.4, 122.0, 110.5, 110.2, 66.4, 55.9, 55.4, 54.8, 53.9, 53.6, 36.6, 28.6, 28.3;
121.7, 120.8, 116.5, 116.1, 113.2, 112.9, 56.1, 52.5; ESIMS m/z 349 ESIMS m/z 958 (M À 1); HRMS calcd for: (C57H48N7O8 À 1), m/z
(M + 1); HRMS calcd for: (C20H15N2O4 À 1), m/z (347.1037); found, (958.3569); found, m/z (958.3620).
m/z (347.1003).
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic carboxyl-Trp-Trp-Glu(OBzl)-OBzl. HOBt (162 mg, 1.20 mmol),
acid. The solution of 5 (500 mg, 1.44 mmol) in NaOH (6 M) in DCC (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous
tetrahydrofuran (THF, 20 mL) was stirred at 0 1C until all the THF (10 mL) and NH2-Trp-Trp-Glu(OBzl)-OBzl (883 mg,
starting material was consumed as indicated by TLC (24 h). The 1.20 mmol) in anhydrous THF (5 mL). Yield: 70%. mp 139–
pH of the reaction mixture was adjusted to 2 with hydrogen 141 1C. [a]2D5 +58.3 (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3379,
chloride (2 M) and the mixture was subject to evaporation to 1736, 1658, 1519,740; 1HNMR (300 MHz, DMSO-d6): d 11.77 (1H,
remove solvent. The residue was washed with saturated NaCl to s, OH), 10.78 (2H, brs, N–H), 8.70 (1H, d, J = 8.1 Hz, N–H), 8.68
provide 6 as green powder (400 mg, 1.20 mmol, 83% yield). (1H, s, Ar–H), 8.71 (1H, d, J = 7.8 Hz, N–H), 8.44 (1H, d,
1HNMR (300 MHz, DMSO-d6): d 11.43 (1H, s, OH), 8.40 (1H, d, J = 7.8 Hz, N–H), 8.35 (1H, d, J = 7.8 Hz, Ar–H), 7.69 (1H, d,
J = 5.1 Hz, Ar–H), 8.24 (1H, d, J = 8.1 Hz, Ar–H), 8.04 (1H, d, J = J = 8.4 Hz, Ar–H), 7.60–7.53 (4H, m, Ar–H), 7.42–7.25 (14H, m,
5.1 Hz, Ar–H), 7.65 (1H, d, J = 8.1 Hz, Ar–H), 7.56–7.46 (2H, m, Ar–H), 7.17 (1H, d, J = 2.1 Hz, Ar–H), 7.15 (1H, d, J = 2.1 Hz,
Ar–H), 7.25 (1H, t, J = 7.2 Hz, Ar–H), 7.01 (1H, d, J = 7.2 Hz, Ar–H), Ar–H), 7.07–6.84 (5H, m, Ar–H), 5.15 (2H, s, CH2Ph), 5.04 (2H, s,
3.91 (3H, s, OCH3); 13CNMR (75 MHz, DMSO-d6) d 171.6, 147.9, CH2Ph), 4.80 (1H, dd, J1 = 12 Hz, J2 = 7.5 Hz, CH), 4.68 (1H, dd,
147.5, 136.9, 136.7, 131.5, 131.4, 126.6, 122.3, 121.8, 119.1, 118.4, J1 = 13.2 Hz, J2 = 8.7 Hz, CH), 4.44 (1H, dd, J1 = 13.2 Hz, J2 = 8.4 Hz,
115.5, 114.2, 111.7, 107.8, 107.7, 56.1.
CH), 3.83 (3H, s, OCH3), 3.27–3.10 (3H, m, CH2), 3.01 (1H, dd, J1 =
General synthetic procedure for preparing 1-(4-hydroxy-3- 15 Hz, J2 = 9 Hz, CH2), 2.54–2.37 (2H, m, CH2), 2.14–1.94 (2H, m,
methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxyl-Trp-Trp-AA-OBzl. CH2); 13CNMR (75 MHz, DMSO-d6) d 172.5, 172.3, 171.8, 171.7,
HOBt (1.2 eq.) and DCC (1.2 eq.) were added to a solution of 6 164.9, 148.2, 148.0, 141.8, 141.4, 139.4, 136.6, 136.5, 136.4, 136.3,
(1 eq.) in anhydrous THF at 0 1C. The reaction mixture was stirred 134.3, 129.9, 129.1, 128.9, 128.8, 128.5, 128.4, 128.3, 127.9, 127.8,
at 0 1C for 30 min. The solution of NH2-Trp-Trp-AA-OBzl (1.2 eq.) in 124.2, 123.9, 122.4, 121.8, 121.7, 121.3, 120.6, 119.0, 118.8, 118.7,
anhydrous THF was added and the pH of the reaction mixture was 116.2, 113.1, 112.8, 112.6, 111.7, 110.5, 110.2, 110.4, 110.2, 66.5,
adjusted to 9 with N-methylmorpholine. The reaction mixture 65.9, 55.9, 54.0, 53.7, 51.9, 28.6, 28.0, 26.5; ESIMS m/z 1015 (M);
obtained was kept at 0 1C for 2 h and then at room temperature HRMS calcd for: (C60H52N7O9 À 1), m/z (1014.3832); found, m/z
for 24 h. DCU formed was removed by filtration. The filtrate was (1014.3814).
subject to evaporation under reduced pressure and the residue was
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
dissolved in EtOAc (50 mL). The solution was washed successively carboxyl-Trp-Trp-Thr-OBzl. HOBt (162 mg, 1.20 mmol), DCC
with saturated NaHCO3, 5% KHSO4 and saturated NaCl, and the (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous THF
organic phase was collected and dried using Na2SO4. Following (10 mL) and NH2-Trp-Trp-Thr-OBzl (741 mg, 1.20 mmol) in
filtration and evaporation under reduced pressure, purification anhydrous THF (5 mL). Yield: 75%. mp 159–161 1C. [a]2D5 +46.4
of the residue by chromatography (100 : 1 CH2Cl2–MeOH) provided (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3356, 1658, 1519, 740;
7–21 as colorless powder in 54–79% yields.
1HNMR (300 MHz, DMSO-d6): d 11.82 (1H, s, OH), 10.83 (1H, brs,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxyl- N–H), 9.50 (1H, brs, N–H), 8.75 (1H, d, J = 8.1 Hz, N–H), 8.73 (1H,
Trp-Trp-Tyr-OBzl. HOBt (162 mg, 1.20 mmol), DCC (247 mg, s, Ar–H), 8.54 (1H, d, J = 8.1 Hz, N–H), 8.37 (1H, d, J = 7.8 Hz,
1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous THF (10 mL) Ar–H), 8.30 (1H, d, J = 8.1 Hz, N–H), 7.73 (1H, d, J = 8.1 Hz, Ar–H),
and NH2-Trp-Trp-Tyr-OBzl (815 mg, 1.20 mmol) in anhydrous THF 7.63–7.55 (4H, m, Ar–H), 7.46–7.30 (9H, m, Ar–H), 7.24 (1H, s,
(5 mL). Yield: 70%. mp 163–165 1C. [a]2D5 À10.7 (c 0.1 in MeOH); Ar–H), 7.19 (1H, s, Ar–H), 7.11–6.99 (4H, m, Ar–H), 6.87 (1H, t, J =
1
IR (cmÀ1, KBr, neat): 3356, 1658, 1519, 740; HNMR (300 MHz, 7.5 Hz, Ar–H), 5.19 (2H, s, CH2Ph), 5.13 (2H, d, J = 6 Hz, N–H),
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4.92–4.82 (2H, m, CH), 4.48 (1H, dd, J1 = 8.1 Hz, J2 = 3.3 Hz, CH),
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxyl-
4.26–4.21 (1H, m, CH), 3.86 (3H, s, OCH3), 3.35–3.16 (3H, m, Trp-Trp-Ile-OBzl. HOBt (162 mg, 1.20 mmol), DCC (247 mg,
CH2), 3.11–3.04 (1H, m, CH2), 1.15 (3H, d, J = 6.3 Hz,CH3); 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous THF (10 mL)
13CNMR (75 MHz, DMSO-d6) d 172.6, 171.6, 170.9, 164.9, 148.2, and NH2-Trp-Trp-Ile-OBzl (755 mg, 1.20 mmol) in anhydrous
141.9, 141.8, 141.5, 141.4, 139.4, 136.6, 136.5, 136.4, 136.3, 134.5, THF (5 mL). Yield: 64%. mp 144–146 1C. [a]2D5 À14.1 (c 0.1 in
134.3, 129.9, 129.1, 128.9, 128.4, 128.2, 128.0, 127.9, 127.8, 124.3, MeOH); IR (cmÀ1, KBr, neat): 3356, 1658, 1519, 748; 1HNMR
124.1, 123.9, 122.3, 121.8, 121.3, 120.6, 119.0, 118.9, 118.7, 116.2, (500 MHz, DMSO-d6): d 11.77 (1H, s, OH), 10.80 (1H, d, J = 10.5 Hz,
113.2, 112.8, 112.7, 111.7, 110.6, 110.2, 67.0, 66.9, 66.4, 58.7, N–H), 10.79 (1H, d, J = 8.4 Hz, N–H), 9.43 (1H, s, N–H), 8.72 (1H, d,
55.9, 53.8, 28.7, 28.2, 20.6; ESIMS m/z 897 (M), 896 (M À 1); J = 7.8 Hz, N–H), 8.71 (1H, s, Ar–H), 8.42 (1H, d, J = 8.3 Hz, N–H),
HRMS calcd for: (C52H46N7O8 À 1), m/z (896.3413); found, m/z 8.35 (1H, d, J = 7.9 Hz, Ar–H), 8.30 (1H, d, J = 7.9 Hz, N–H), 7.69
(896.3452).
(1H, d, J = 8.4 Hz, Ar–H), 7.63–7.51 (4H, m, Ar–H), 7.42–7.32 (9H,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- m, Ar–H), 7.21 (2H, d, J = 3.6 Hz, Ar–H), 7.03–6.96 (3H, m, Ar–H),
carboxyl-Trp-Trp-Lys(Nx-Z)-OBzl. HOBt (162 mg, 1.20 mmol), 6.93 (1H, t, J = 7.3 Hz, Ar–H), 6.87 (1H, t, J = 7.5 Hz, Ar–H), 5.17
DCC (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous (2H, dd, J1 = 29.3 Hz, J2 = 13.2 Hz, CH2Ph), 4.86–4.82 (1H, m, CH),
THF (10 mL) and NH2-Trp-Trp-Lys(No-Z)-OBzl (934 mg, 4.80–4.76 (1H, m, CH), 4.34 (1H, d, J = 14.1 Hz, CH), 3.87 (3H, s,
1.20 mmol) in anhydrous THF (5 mL). Yield: 79%. mp 124– OCH3), 3.26 (1H, dd, J1 = 15 Hz, J2 = 7.2 Hz, CH2), 3.17 (1H, dd, J1 =
126 1C. [a]2D5 +52.8 (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3290, 15 Hz, J2 = 4.5 Hz, CH2), 3.14 (1H, dd, J1 = 15 Hz, J2 = 4.5 Hz, CH2),
1
1662, 1512, 744; HNMR (300 MHz, DMSO-d6): d 11.80 (1H, s, 3.02 (1H, dd, J1 = 15 Hz, J2 = 8.8 Hz, CH2), 1.88–1.82 (1H, m, CH),
OH), 10.80 (1H, d, J = 8.7 Hz, N–H), 8.71 (1H, d, J = 9.1 Hz, N–H), 1.43–1.37 (1H, m, CH2), 1.26–1.16 (1H, m, CH2), 0.79 (3H, d, J =
8.69 (1H, s, Ar–H), 8.43 (1H, d, J = 7.2 Hz, N–H), 8.35 (1H, d, 2.75 Hz, CH3), 0.77 (3H, d, J = 2.65 Hz, CH3); 13CNMR (125 MHz,
J = 7.8 Hz, Ar–H), 7.70 (1H, d, J = 8.1 Hz, Ar–H), 7.60–7.54 (4H, m, DMSO-d6) d 172.2, 171.6, 171.5, 164.8, 148.2, 148.0, 141.8, 141.4,
Ar–H), 7.42–7.28 (14H, m, Ar–H), 7.17 (2H, d, J = 5.4 Hz, Ar–H), 139.4, 136.4, 136.3, 136.2, 134.3, 129.9, 129.1, 128.9, 128.6, 128.5,
7.08–6.90 (4H, m, Ar–H), 6.84 (1H, t, J = 7.5 Hz, Ar–H), 5.14 (2H, s, 127.9, 127.8, 124.0, 123.9, 123.8, 122.3, 121.8, 121.7, 121.3, 120.6,
CH2Ph), 4.99 (2H, s, CH2Ph), 4.82 (1H, m, CH), 4.72 (1H, m, CH), 118.9, 118.8, 118.6, 116.1, 113.1, 112.8, 112.6, 111.6, 110.4, 110.1,
4.33 (1H, t, J = 6.6 Hz, CH), 3.84 (3H, s, OCH3), 3.24–3.13 (2H, m, 66.4, 57.1, 55.9, 53.7, 53.4, 36.7, 28.6, 28.1, 25.2, 15.8, 11.6; ESIMS
CH2), 3.05–2.89 (4H, m, CH2), 1.84–1.67 (2H, m, CH2), 1.41–1.21 m/z 908 (MÀ1); HRMS calcd for: (C54H50N7O7 À 1), m/z (908.3766);
(4H, m, CH2); 13CNMR (75 MHz, DMSO-d6) d 172.2, 172.1, 171.5, found, m/z (908.3782).
164.9, 156.5, 148.2, 141.8, 141.5, 139.4, 137.7, 136.4, 136.3, 134.3,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
129.9, 129.1, 128.9, 128.8, 128.5, 128.3, 127.9, 127.8, 124.0, 123.8, carboxyl-Trp-Trp-Ala-OBzl. HOBt (150 mg, 1.11 mmol), DCC
122.3, 121.8, 121.3, 120.6, 118.9, 118.6, 116.2, 113.2, 112.8, 112.6, (228 mg, 1.11 mmol), 6 (308 mg, 0.92 mmol) in anhydrous THF
111.6, 110.5, 110.1, 103.5, 66.4, 55.6, 63.4, 55.9, 53.9, 53.4, 52.7, (10 mL) and NH2-Trp-Trp-Ala-OBzl (650 mg, 1.11 mmol) in
30.9, 29.5, 28.6, 28.2, 23.1; ESIMS m/z 1058 (M), 1057 (M À 1); anhydrous THF (5 mL). Yield: 69%. mp 149–151 1C. [a]2D5 À8.9
HRMS calcd for: (C62H57N8O9 À 1), m/z (1057.4254); found, m/z (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3371, 1666, 1527, 748;
(1057.4296).
1HNMR (300 MHz, DMSO-d6): d 11.78 (1H, s, OH), 10.81 (1H, brs,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxyl- N–H), 10.78 (1H, brs, N–H), 8.71 (1H, d, J = 6.3 Hz, N–H), 8.70
Trp-Trp-Val-OBzl. HOBt (162 mg, 1.20 mmol), DCC (247 mg, (1H, s, Ar–H), 8.57 (1H, d, J = 7.2 Hz, N–H), 8.44 (1H, d, J = 8.4 Hz,
1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous THF (10 mL) N–H), 8.37 (1H, d, J = 7.8 Hz, Ar–H), 7.69 (1H, d, J = 8.1 Hz, Ar–H),
and NH2-Trp-Trp-Val-OBzl (741 mg, 1.20 mmol) in anhydrous THF 7.61–7.55 (3H, m, Ar–H), 7.51 (1H, d, J = 1.5 Hz, Ar–H), 7.40–7.28
(5 mL). Yield: 68%. mp 136–138 1C. [a]2D5 +14.8 (c 0.1 in MeOH); (9H, m, Ar–H), 7.17 (1H, s, Ar–H), 7.15 (1H, s, Ar–H), 7.05–6.96
IR (cmÀ1, KBr, neat): 3387, 1666, 1519, 748; 1HNMR (300 MHz, (4H, m, Ar–H), 6.84 (1H, t, J = 7.5 Hz, Ar–H), 5.15 (2H, s, CH2Ph),
DMSO-d6): d 11.78 (1H, s, OH), 10.85 (1H, brs, N–H), 10.81 (1H, brs, 4.83 (1H, dd, J1 = 12 Hz, J2 = 7.2 Hz, CH), 4.69 (1H, dd, J1 = 12 Hz,
N–H), 8.71 (1H, d, J = 9.6 Hz, N–H), 8.69 (1H, s, Ar–H), 8.45 (1H, d, J = J2 = 4.8 Hz, CH), 4.47–4.38 (1H, m, CH),3.82 (3H, s, OCH3), 3.29–
8.1 Hz, N–H), 8.35 (1H, d, J = 7.8 Hz, Ar–H), 8.21 (1H, d, J = 8.1 Hz, 3.11 (3H, m, CH2), 2.99 (1H, dd, J1 = 15 Hz, J2 = 9.3 Hz, CH2), 1.34
N–H), 8.13 (1H, d, J = 7.8 Hz, N–H), 7.99 (1H, d, J = 8.1 Hz, N–H), (3H, d, J = 7.2 Hz, CH3); 13CNMR (75 MHz, DMSO-d6) d 172.8,
7.65 (1H, d, J = 8.4 Hz, Ar–H), 7.61–7.48 (3H, m, Ar–H), 7.48–7.27 172.0, 171.4, 164.8, 148.1, 148.0, 141.8, 141.4, 139.4, 136.5, 136.3,
(11H, m, Ar–H), 7.18–6.93 (5H, m, Ar–H), 6.82 (1H, t, J = 7.5 Hz, 134.3, 129.9, 129.1, 128.9, 128.5, 128.2, 127.9, 127.8, 124.2, 124.1,
Ar–H), 5.15 (2H, s, CH2Ph), 4.87–4.77 (1H, m, CH), 4.69–4.49 (1H, 123.8, 122.4, 121.8, 121.7, 121.3, 120.6, 119.0, 118.9, 118.6, 116.1,
m, CH), 4.27 (1H, dd, J1 = 9 Hz, J2 = 6.3 Hz, CH), 3.38 (3H, s, 113.1, 112.6, 111.6, 110.5, 110.4, 110.1, 66.4, 55.9, 53.9, 53.8,
OCH3), 3.30–2.80 (4H, m, CH2), 2.14–2.04 (1H, m, CH), 0.87 (3H, 53.4, 48.3, 28.6, 28.3, 17.3; ESIMS m/z 867 (M); HRMS calcd for:
d, J = 8.7 Hz, CH3), 0.84 (3H, d, J = 8.7 Hz, CH3); 13CNMR (75 MHz, (C51H44N7O7 À 1), m/z (866.3297); found, m/z (866.3036).
DMSO-d6) d 172.3, 171.6, 169.6, 164.7, 148.2, 141.8, 141.4, 139.4,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
136.4, 134.3, 129.9, 129.1, 128.9, 128.5, 128.0, 127.8, 123.8, 122.3, carboxyl-Trp-Trp-Asp(OBzl)-OBzl. HOBt (162 mg, 1.20 mmol),
121.7, 121.3, 120.6, 118.9, 118.6, 116.1, 113.1, 112.8, 111.6, 110.4, DCC (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous
110.0, 66.4, 58.1, 55.9, 53.7, 53.4, 30.4, 27.9, 22.9, 19.4, 18.8; ESIMS THF (10 mL) and NH2-Trp-Trp-Asp(OBzl)-OBzl (755 mg,
m/z 895 (M), 894 (M À 1); HRMS calcd for: (C53H48N7O7 À 1), m/z 1.20 mmol) in anhydrous THF (5 mL). Yield: 67%. mp 136–
(894.3609); found, m/z (894.3591).
138 1C. [a]2D5 +24.1 (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3387,
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1666, 1519, 748; HNMR (300 MHz, DMSO-d6): d 11.78 (1H, s, 127.9, 127.8, 124.1, 123.8, 122.3, 121.7, 121.3, 120.6, 119.0, 118.8,
OH), 10.80 (2H, brs, N–H), 8.71 (2H, d, J = 8.7 Hz, N–H), 8.70 (1H, 118.7, 116.1, 113.1, 112.8, 111.6, 110.4, 110.1, 66.4, 65.4, 56.0, 55.9,
s, Ar–H), 8.48 (1H, d, J = 8.1 Hz, N–H), 8.34 (1H, d, J = 7.8 Hz, 54.8, 53.9, 28.6, 28.3; ESIMS m/z 852 (M À 1), 853 (M); HRMS calcd
Ar–H), 7.69 (1H, d, J = 8.1 Hz, Ar–H), 7.61–7.53 (4H, m, Ar–H), for: (C50H44N7O7 + 1), m/z (854.3297); found, m/z (854.3394).
7.46–7.27 (14H, m, Ar–H), 7.18 (2H, d, J = 1.8 Hz, Ar–H), 7.07–
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
6.87 (5H, m, Ar–H), 5.11 (2H, s, CH2Ph), 5.08 (2H, s, CH2Ph), carboxyl-Trp-Trp-Trp-OBzl. HOBt (162 mg, 1.20 mmol), DCC
4.89–4.80 (2H, m, CH), 4.80 (1H, dd, J1 = 13.5 Hz, J2 = 8.7 Hz, CH), (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous
3.84 (3H, s, OCH3), 3.29–3.12 (3H, m, CH2), 3.04–2.89 (2H, m, THF (10 mL) and NH2-Trp-Trp-Trp-OBzl (843 mg, 1.20 mmol)
CH2), 2.78 (1H, dd, J1 = 16.8 Hz, J2 = 6.3 Hz, CH2); 13CNMR in anhydrous THF (5 mL). Yield: 59%. mp 158–160 1C. [a]D25 +11.3
(75 MHz, DMSO-d6) d 172.0, 171.7, 170.8, 170.3, 164.9, 148.2, (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3356, 1658, 1519, 740;
148.0, 141.8, 141.4, 139.4, 136.6, 136.5, 136.4, 136.3, 136.2, 136.1, 1HNMR (500 MHz, DMSO-d6): d 11.77 (1H, s, OH), 10.89 (1H, brs,
134.3, 129.9, 129.1, 128.9, 128.5, 128.4, 128.2, 127.9, 127.8, 124.2, N–H), 10.78 (2H, s, N–H), 9.42 (1H, s, N–H), 8.71 (1H, s, Ar–H),
123.9, 123.8, 122.3, 121.8, 121.7, 121.3, 120.6, 119.0, 118.8, 118.6, 8.69 (1H, d, J = 8 Hz, N–H), 8.60 (1H, d, J = 7 Hz, N–H), 8.43
116.2, 113.1, 112.9, 112.7, 111.7, 110.4, 110.3, 110.2, 66.8, 66.4, (1H, d, J = 8 Hz, N–H), 8.33 (1H, d, J = 8 Hz, Ar–H), 7.69 (1H, d,
55.9, 53.9, 53.7, 49.1, 36.2, 28.7, 28.3; ESIMS m/z 1002 (M + 1); J = 8 Hz, Ar–H), 7.61–7.51 (5H, m, Ar–H), 7.45–7.36 (2H, m, Ar–H),
HRMS calcd for: (C59H50N7O9 À 1), m/z (1000.3676); found, m/z 7.31–7.28 (6H, m, Ar–H), 7.21 (5H, m, Ar–H), 7.17–7.06 (5H, m,
(1000.3445).
Ar–H), 7.05–7.01 (1H, t, J = 7 Hz, Ar–H), 6.93 (1H, t, J = 7 Hz,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- Ar–H), 5.01 (2H, dd, J1 = 34 Hz, J2 = 13 Hz, CH2Ph), 4.87–4.83 (1H,
carboxyl-Trp-Trp-Pro-OBzl. HOBt (162 mg, 1.20 mmol), DCC m, CH), 4.77–4.73 (1H, m, CH), 4.66 (1H, dd, J1 = 14 Hz, J2 = 7 Hz,
(247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous THF CH), 3.85 (3H, s, OCH3), 3.25–3.21 (2H, m, CH2), 3.19–3.13 (3H,
(10 mL) and NH2-Trp-Trp-Pro-OBzl (736 mg, 1.20 mmol) in m, CH2), 3.02 (1H, dd, J1 = 14.5 Hz, J2 = 9 Hz, CH2); 13CNMR
anhydrous THF (5 mL). Yield: 54%. mp 137–139 1C. [a]2D5 À17.5 (75 MHz, DMSO-d6) d 172.2, 172.1, 171.6, 164.9, 148.2, 141.9,
(c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3425, 1635, 1519,1450,748; 141.5, 139.5, 136.6, 136.5, 136.4, 136.2, 134.5, 129.9, 129.1, 128.8,
1HNMR (500 MHz, DMSO-d6): d 11.79 (1H, s, OH), 10.85 (1H, s, 128.4, 128.2, 127.9, 127.8, 127.6, 124.1, 123.9, 121.8, 121.5, 121.3,
N–H), 8.78 (1H, d, J = 8.7 Hz, N–H), 8.74 (1H, s, Ar–H), 8.71 118.9, 118.6, 116.1, 111.9, 111.7, 110.5, 110.3, 109.7, 66.4, 56.0,
(1H, d, J = 8 Hz, N–H), 8.39 (1H, d, J = 7.5 Hz, Ar–H), 7.69 (1H, d, 54.0, 53.6, 28.6, 28.3, 27.7; ESIMS m/z 981 (M À 1), 982 (M); HRMS
J = 7.5 Hz, Ar–H), 7.61–7.53 (4H, m, Ar–H), 7.44–7.28 (9H, m, calcd for: (C59H51N8O7 + 1), m/z (983.3875); found, m/z (983.3944).
Ar–H), 7.19 (1H, s, Ar–H), 7.12 (1H, s, Ar–H), 7.04–6.95 (4H, m,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
Ar–H), 6.83 (1H, t, J = 7 Hz, Ar–H), 5.15 (2H, dd, J1 = 16.5 Hz, J2 = carboxyl-Trp-Trp-Phe-OBzl. HOBt (162 mg, 1.20 mmol), DCC
12.5 Hz, CH2Ph), 4.87 (2H, m, CH), 4.45 (1H, m, CH), 3.81 (3H, s, (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous THF
OCH3), 3.55 (1H, m, CH2), 3.29–3.20 (3H, m, CH2), 3.09–2.98 (2H, (10 mL) and NH2-Trp-Trp-Phe-OBzl (815 mg, 1.20 mmol) in
m, CH2), 2.17 (1H, m, CH2), 1.82 (3H, m, CH2); 13CNMR (125 MHz, anhydrous THF (5 mL). Yield: 70%. mp 139–141 1C. [a]D25 +10.2
DMSO-d6) d 172.1, 171.2, 170.6, 164.6, 148.2, 141.8, 141.4, 136.5, (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3387, 1666, 1519, 748;
136.3, 134.3, 129.9, 129.1, 128.9, 128.8, 128.5, 128.3, 128.2, 128.1, 1HNMR (300 MHz, DMSO-d6): d 11.78 (1H, s, OH), 10.79 (2H, brs,
127.6, 124.1, 122.4, 121.7, 121.4, 121.2, 120.6, 118.9, 118.8, 118.6, N–H), 8.71 (1H, d, J = 6 Hz, N–H), 8.70 (1H, s, Ar–H), 8.59 (1H, d,
118.4, 116.1, 113.1, 112.8, 112.6, 111.8, 111.6, 110.1, 109.9, 66.3, J = 7.2 Hz, N–H), 8.44 (1H, d, J = 8.4 Hz, N–H), 8.34 (1H, d,
59.1, 55.8, 53.6, 51.5, 47.0, 29.1, 28.7, 27.6, 24.9; ESIMS m/z 893 (M); J = 8.1 Hz, Ar–H), 7.69 (1H, d, J = 8.4 Hz, Ar–H), 7.61–7.53 (4H, m,
HRMS calcd for: (C53H46N7O7 À 1), m/z (892.2464); found, m/z Ar–H), 7.40 (1H, dd, J1 = 8.4 Hz, J2 = 2.1 Hz, Ar–H), 7.42–7.14
(892.3256).
(15H, m, Ar–H), 7.07–7.02 (4H, m, Ar–H), 6.83 (1H, t, J = 7.5 Hz,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- Ar–H), 5.05 (2H, dd, J = 12.6 Hz, CH2Ph), 4.84 (1H, dd, J1 = 12.3 Hz,
carboxyl-Trp-Trp-Gly-OBzl. HOBt (178 mg, 1.32 mmol), DCC J2 = 7.8 Hz, CH), 4.71 (1H, dd, J1 = 13.2 Hz, J2 = 8.4 Hz, CH), 4.59
(272 mg, 1.32 mmol), 6 (400 mg, 1.20 mmol) in anhydrous (1H, dd, J1 = 14.4 Hz, J2 = 7.2 Hz, CH), 3.84 (3H, s, OCH3), 3.37–
THF (10 mL) and NH2-Trp-Trp-Gly-OBzl (757 mg, 1.32 mmol) 2.94 (6H, m, CH2); 13CNMR (75 MHz, DMSO-d6) d 172.1, 171.6,
in anhydrous THF (5 mL). Yield: 61%. mp 140–142 1C. [a]2D5 164.8, 148.2, 148.0, 141.8, 141.4, 139.4, 137.3, 136.5, 136.4, 136.3,
À37.8 (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3340, 1658, 1519, 136.2, 136.1, 134.3, 129.9, 129.6, 129.1, 128.8, 128.7, 128.5, 128.4,
748; 1HNMR (300 MHz, DMSO-d6): d 11.76 (1H, s, OH), 10.78 127.9, 127.8, 127.0, 124.0, 122.3, 121.8, 121.7, 121.3, 120.6, 119.0,
(1H, d, J = 4.2 Hz, N–H), 8.72 (1H, d, J = 7.5 Hz, N–H), 8.71 (1H, s, 118.9, 118.6, 116.1, 113.1, 112.8, 111.6, 110.4, 110.2, 66.5, 55.9,
Ar–H), 8.51–8.42 (2H, m, N–H), 8.37 (1H, d, J = 7.8 Hz, Ar–H), 7.69 54.4, 53.8, 53.6, 37.2, 28.7, 28.3; ESIMS m/z 942 (M À 1), 944 (M + 1);
(1H, d, J = 9.1 Hz, Ar–H), 7.60–7.49 (4H, m, Ar–H), 7.41–7.27 (9H, HRMS calcd for: (C57H50N7O7 + 1), m/z (944.3766); found, m/z
m, Ar–H), 7.20 (1H, d, J = 7.5 Hz, Ar–H), 7.06–6.93 (4H, m, Ar–H), (944.3890).
6.86 (1H, t, J = 7.5 Hz, Ar–H), 5.16 (2H, s, CH2Ph), 4.86 (1H, dd,
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-
J1 = 12.3 Hz, J2 = 7.2 Hz, CH), 4.70 (1H, dd, J1 = 13.5 Hz, J2 = 8.4 Hz, carboxyl-Trp-Trp-Ser-OBzl. HOBt (162 mg, 1.20 mmol), DCC
CH), 3.95 (2H, dd, J1 = 10.5 Hz, J2 = 4.8 Hz, CH), 3.84 (3H, s, OCH3), (247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous
3.26–3.14 (3H, m, CH2), 3.02 (1H, dd, J1 = 14.7 Hz, J2 = 8.7 Hz,CH2); THF (10 mL) and NH2-Trp-Trp-Ser-OBzl (724 mg, 1.20 mmol) in
13CNMR (75 MHz, DMSO-d6) d 172.5, 171.6, 170.1, 164.8, 148.1, anhydrous THF (5 mL). Yield: 65%. mp 166–168 1C. [a]D25 À21.4
141.8, 141.4, 139.4, 136.4, 134.3, 129.9, 129.1, 128.9, 128.5, 128.4, (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3371, 1658, 1519,740;
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1HNMR (500 MHz, DMSO-d6): d 11.80 (1H, s, OH), 10.86 (1H, 4.82 (1H, dd, J1 = 12.3 Hz, J2 = 7.5 Hz, CH), 4.70 (1H, dd, J1 = 13.2
d, J = 9.5 Hz, N–H), 9.48 (1H, brs, N–H), 8.72 (1H, d, Hz, J2 = 8.4 Hz, CH), 4.42 (1H, dd, J1 = 13.2 Hz, J2 = 8.4 Hz, CH), 3.84
J = 8.0 Hz, N–H), 8.71 (1H, s, Ar–H), 8.53 (1H, d, J = 7.5 Hz, (3H, s, OCH3), 3.24–3.12 (3H, m, CH2), 3.15 (1H, dd, J1 = 17.4 Hz,
N–H), 8.46 (1H, d, J = 9.0 Hz, N–H), 8.37 (1H, d, J = 9.0 Hz, Ar–H), J2 = 7.2 Hz, CH2); 13CNMR (75 MHz, DMSO-d6) d 172.6, 172.2, 171.6,
7.70 (1H, d, J = 9.0 Hz, Ar–H), 7.59 (3H, t, J = 9.0 Hz, Ar–H), 7.53 164.9, 148.2, 148.0, 141.8, 141.4, 139.4, 136.6, 136.4, 136.3, 134.3,
(1H, d, J = 1.5 Hz, Ar–H), 7.40 (3H, d, J = 7.0 Hz, Ar–H), 7.36 (2H, t, 129.9, 129.1, 128.9, 128.6, 128.3, 127.9, 127.8, 124.2, 123.9, 123.8,
J = 7.5 Hz, Ar–H), 7.33–7.27 (4H, m, Ar–H), 7.20 (1H, s, Ar–H),7.16 122.3, 121.8, 121.7, 121.3, 120.6, 119.0, 118.9, 118.6, 116.1, 113.1,
(1H, s, Ar–H), 7.05 (1H, d, J = 9.0 Hz, Ar–H), 7.03–6.95 (2H, m, 112.8, 112.6, 111.7, 110.5, 110.1, 66.4, 55.9, 53.9, 53.6, 51.1, 40.77,
Ar–H), 6.93 (1H, t, J = 7.5 Hz, Ar–H), 6.84 (1H, t, J = 7.5 Hz, Ar–H), 28.6, 28.1, 24.6, 23.2, 21.9; ESIMS m/z 909 (M); HRMS calcd for:
5.19 (2H, s, CH2Ph), 4.87–4.77 (1H, m, CH), 4.69–4.65 (1H, m, CH), (C54H52N7O7 + 1), m/z (910.3922); found, m/z (910.3965).
4.51 (1H, dd, J1 = 10 Hz, J2 = 5 Hz, CH), 3.84 (3H, s, OCH3),
In vitro cytotoxicity
3.81–3.67 (2H, m, CH2), 3.30–3.16 (1H, m, CH2), 3.19–3.15 (2H, m,
CH2), 3.02 (1H, dd, J1 = 15 Hz, J2 = 9 Hz, CH2); 13CNMR (125 MHz, In vitro cytotoxicity of the compounds synthesized was deter-
DMSO-d6) d 172.2, 171.5, 170.8, 164.9, 148.2, 141.8, 139.4, 136.5, mined in HT-29, A549, K562 and HL-60 cell lines using the
136.4, 136.3, 134.3, 129.9, 129.1, 128.9, 128.8, 128.4, 128.1, 128.0, MTT assay according to the method described by Al-Allaf and
127.9, 127.8, 124.0, 123.8, 122.3, 121.8, 121.7, 121.2, 120.6, 119.0, Rashan.22 The cells were grown in DMEM or RPMI-1640 medium
118.9, 118.7, 116.2, 112.9, 112.6, 111.7, 111.6, 110.5, 110.4, 110.2, containing 10% (v/v) fetal bovine serum and 10 000 U mLÀ1
110.1, 66.4, 61.7, 56.0, 55.3, 53.9, 53.5, 28.6, 28.3; ESIMS m/z 883 penicillin and 100 mg mLÀ1 streptomycin in a humidified incu-
(M); HRMS calcd for: (C51H44N7O8 À 1), m/z (882.3257); found, m/z bator containing 5% CO2 at 37 1C for 4 h. 7–21 of different
(882.3246).
concentrations (ranging from 1 to 400 mM) in DMSO were
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- prepared. To each well of the 96-well plates, 25 mL of medium
carboxyl-Trp-Trp-Met-OBzl. HOBt (162 mg, 1.20 mmol), DCC containing test compounds of different concentrations of 7–21
(247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous were added. The final concentration of DMSO in the growth
THF (10 mL) and NH2-Trp-Trp-Met-OBzl (777 mg, 1.20 mmol) medium was 1% (v/v). After 48 h, 25 mL (final concentration,
in anhydrous THF (5 mL). Yield: 61%. mp 173–175 1C. 0.5 mg mLÀ1) of MTT was added to each well, and plates were
[a]2D5 À38.9 (c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3356, 1658, returned to the incubator for an additional 4 h. The culture
1519, 740; 1HNMR (500 MHz, DMSO-d6): d 11.81 (1H, s, OH), 10.83 medium was then removed. The resulting MTT–formazan product
(1H, d, J = 12 Hz, N–H), 8.72 (1H, d, J = 7.5 Hz, N–H), 8.70 (1H, s, was dissolved in 100 mL of DMSO, and the absorbance at 570 nm
Ar–H), 8.50 (1H, d, J = 7.5 Hz, N–H), 8.47 (1H, d, J = 8 Hz, N–H), was determined. IC50 is defined as the concentration that results
8.36 (1H, d, J = 8 Hz, Ar–H), 7.70 (1H, d, J = 8 Hz, Ar–H), 7.61–7.54 in 50% inhibition of tumor cell growth.
(4H, m, Ar–H), 7.42–7.28 (9H, m, Ar–H), 7.19 (2H, d, J = 4 Hz,
Ar–H), 7.08 (1H, d, J = 8.5 Hz, Ar–H), 7.03–7.01 (2H, m, Ar–H), 6.96
Determination of in vivo anti-tumor activity
(1H, t, J = 7.5 Hz, Ar–H), 6.87 (1H, t, J = 7.5 Hz, Ar–H), 5.17 (2H, dd, All animal experiments were conducted in accordance with China’s
J1 = 34 Hz, J2 = 13 Hz, CH2Ph), 4.84–4.80 (1H, m, CH), 4.70 (1H, dd, National Guide for the Care and Use of Laboratory Animals. The
J1 = 9 Hz, J2 = 5 Hz, CH), 4.53 (1H, dd, J1 = 9 Hz, J2 = 5 Hz, CH), 3.85 experimental procedures were approved by the Committee on
(3H, s, OCH3), 3.25 (1H, dd, J1 = 14.5 Hz, J2 = 4 Hz, CH2), 3.19–3.14 Animal Care and Usage, Capital Medical University, and all efforts
(2H, m, CH2), 3.03 (1H, dd, J1 = 15 Hz, J2 = 9 Hz, CH2), 2.50–2.46 were made to minimize animal suffering. This may be refined
(2H, m, CH2), 2.05–1.96 (2H, m, CH2); 13CNMR (125 MHz, DMSO- by selecting an injection site that will cause minimal pain and
d6) d 172.2, 171.9, 171.6, 148.2, 148.1, 141.5, 139.3, 136.4, 136.3, distress; using sharp needles of the narrowest possible gauge and
129.9, 129.1, 128.9, 128.6, 128.4, 127.9, 127.8, 124.0, 123.8, 122.3, catching mice by cupping in their home cage tunnel, instead of by
121.8, 121.7, 121.3, 120.6, 118.9, 118.8, 118.7, 116.2, 112.8, the tail. This method of capture is less aversive and induces less
112.7, 111.7, 110.5, 110.1, 66.6, 56.0, 53.9, 53.6, 51.7, 30.9, anxiety. Male ICR mice (10–12 week old), purchased from Peking
29.9, 28.6, 28.0, 15.0; ESIMS m/z 927 (M); HRMS calcd for: University Health Science Center, were maintained at 21 1C with a
(C53H48N7O7S À 1), m/z (926.3341); found, m/z (926.3370).
natural day–night cycle in a conventional animal colony. Food
1-(4-Hydroxy-3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- and water were supplied ad libitum. The breeding animals were
carboxyl-Trp-Trp-Leu-OBzl. HOBt (162 mg, 1.20 mmol), DCC maintained on solid floors with softwood shavings for bedding and
(247 mg, 1.20 mmol), 6 (334 mg, 1.00 mmol) in anhydrous nesting. All animals were allowed one week to adapt to their
THF (10 mL) and NH2-Trp-Trp-Leu-OBzl (755 mg, 1.20 mmol) environment before the treatment. S180 tumor cells passaged in
in anhydrous THF (5 mL). Yield: 75%. mp 156–158 1C. [a]2D5 +30.6 mouse abdomen were harvested once a week. Mice were inoculated
(c 0.1 in MeOH); IR (cmÀ1, KBr, neat): 3371, 1666, 1519, 748; with the harvested S180 tumor cells (1 Â 107 cells per mouse) by
1HNMR (300 MHz, DMSO-d6): d 11.78 (1H, s, OH), 10.81 (1H, s, subcutaneous injection. Twenty-four hours after inoculation,
N–H), 10.79 (1H, s, N–H), 9.45 (1H, s, N–H), 8.70 (1H, d, J = 9.1 Hz, 7–21 dissolved in 0.9% saline were injected by the intraperitoneal
N–H), 8.68 (1H, s, Ar–H), 8.44 (2H, d, J = 7.8 Hz, N–H), 8.34 (1H, d, (i.p.) route to mice (12 mice per group, 0.2 mL per mouse) at
J = 9.1 Hz, Ar–H), 7.69 (1H, d, J = 8.4 Hz, Ar–H), 7.60–7.53 (4H, m, 0.1 mmol kgÀ1 for seven consecutive days. ADM at 0.1 mmol kgÀ1
Ar–H), 7.46–7.26 (9H, m, Ar–H), 7.17 (2H, s, Ar–H), 7.07–6.95 (0.2 mL) dissolved in 0.9% saline was used as positive control and
(4H, m, Ar–H), 6.87 (1H, t, J = 7.5 Hz, Ar–H), 5.14 (2H, s, CH2Ph), 0.2 mL of 0.9% saline was used as negative control. All the tested
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Paper
14 M. Cain, R. W. Weber, F. Guzman, J. M. Cook, S. A. Barker,
K. C. Rice, J. N. Crawley, S. M. Paul and P. Skolnick, J. Med.
Chem., 1982, 25, 1081–1091.
15 T. J. Hagen, P. Skolnick and J. M. Cook, J. Med. Chem., 1987,
30, 750–753.
16 R. H. Dodd, C. Ouannes, L. P. Carvalho, A. Valin, P. Venault,
G. Chapouthier, J. Rossier and P. Potier, J. Med. Chem., 1985,
28, 824–828.
17 K. Patel, M. Gadewar, R. Tripathi, S. K. Prasad and
D. K. Patel, Asian Pac. J. Trop. Biomed., 2012, 2, 660–664.
18 T. Nakanishi, M. Suzuki, A. Mashiba, K. Ishikawa and
T. Yokotsuka, J. Org. Chem., 1998, 63, 4235–4239.
19 J. Jimenez, L. Riveron-Negrete and F. Abdullaev, Exp. Toxicol.
Pathol., 2008, 60, 381–389.
compounds did not cause obvious neurotoxic reaction including
tremor, twitch, jumping, tetanus and supination at the tested
dosage. The weights of animals were recorded every day. Most of
the compounds showed weight gain similar to that of the
vehicle-treated group. Only two compounds (19 and 21) showed
significantly lower increase in the body weight. It is observed
that only languishment was a significant indicator of distress
and the size of the tumor was the most significant predictor of
the animal’s condition. There was a strong correlation between
the incremental size of the tumor and further deterioration in
the animal’s condition. Twenty-four hours after the last admin-
istration, all mice were weighed, decapitated and dissected
immediately to obtain the weight of the tumor. The surgical
procedures were performed by personnel authorized by Beijing
Association on Laboratory Animal Care.
20 H. J. Guan, H. S. Chen and W. L. Peng, Eur. J. Med. Chem.,
2006, 41, 1167–1179.
21 J. Ishida, H. K. Wang, K. F. Bastow, C. Q. Hu and K. H. Lee,
Bioorg. Med. Chem. Lett., 1999, 9, 3319–3324.
22 T. A. K. Al-Allaf and L. J. Rashan, Eur. J. Med. Chem., 1998,
33, 817–820.
23 R. Cao, W. Peng, H. Chen, Y. Ma, X. Liu, X. Hou, H. Guan
and A. Xu, Biochem. Biophys. Res. Commun., 2005, 338,
1557–1563.
24 F. Pognan, J. M. Saucier, C. Paoletti, L. Kaczmarek,
P. Nantka-Namirski, M. Mordarski and W. Peczynska-
Czoch, Biochem. Pharmacol., 1992, 44, 2149–2155.
25 M. Laronze, M. Boisbrun, S. Leonce, B. Pfeiffer, P. Renard,
O. Lozach, L. Meijer, A. Lansiaux, C. Bailly, J. Sapi and
J. Y. Laronze, Bioorg. Med. Chem., 2005, 13, 2263–2283.
26 Y. Funayama, K. Nishio, K. Wakabayashi, M. Nagao,
K. Shimoi, T. Ohira, S. Hasegawa and N. Saijo, Mutat. Res.,
1996, 349, 183–191.
27 A. M. Sobhani, S. A. Ebrahimi and M. J. Mahmoudian,
J. Pharm. Pharm. Sci., 2002, 5, 19–23.
28 Y. Boursereau and I. Coldham, Bioorg. Med. Chem. Lett.,
2004, 14, 5841–5844.
Acknowledgements
This work was supported by the National Natural Scientific Foun-
dation of China (20642004), the Educational Council Foundation of
Beijing (KM200710025012), Academic Human Resources Develop-
ment in Institutions of Higher Learning under the Jurisdiction of
Beijing Municipality (PHR201007114), and Beijing Pharmacological
Society (2013BI-01).
References
1 G. Momekov, A. Bakalova and M. Karaivanova, Curr. Med.
Chem., 2005, 12, 2177–2191.
2 P. D. Bailey, P. J. Cochrane, A. H. Forster, K. M. Morgana
and D. P. J. Pearson, Tetrahedron Lett., 1999, 40, 4597–4600.
3 J. D. Winkler, A. T. Londregan and M. T. Hamann, Org. Lett.,
2006, 8, 2591–2594.
4 H. Guan, H. Chen, W. Peng, Y. Ma, R. Cao, X. Liu and
A. L. Xu, Eur. J. Med. Chem., 2006, 41, 1167–1179.
5 J. G. Tang, Y. H. Wang, R. R. Wang, Z. J. Dong, L. M. Yang,
Y. T. Zheng and J. K. Liu, Chem. Biodiversity, 2008, 5,
447–460.
29 P. Rivas, B. K. Cassels, A. Morello and Y. Repetto, Comp.
Biochem. Physiol., C: Comp. Pharmacol., 1999, 122, 27–31.
30 A. S. Formagio, L. T. D. Tonin, M. A. Foglio, C. Madjarof,
C. J. Ernesto, C. W. Ferreira, F. P. Cardoso and
M. H. Sarragiotto, Bioorg. Med. Chem., 2008, 16, 9660–9667.
31 X. Y. Zhang, Y. F. Yang, M. Zhao, L. Liu, M. Q. Zheng,
Y. J. Wang, J. H. Wu and S. Q. Peng, Eur. J. Med. Chem., 2011,
46, 3410–3419.
6 T. Kawasaki and K. Higuchi, Nat. Prod. Rep., 2005, 22,
761–793.
7 T. Herraiz and J. Galisteo, J. Agric. Food Chem., 2003, 51,
7156–7161.
32 C. A. Hastings and J. K. Barton, Biochemistry, 1999, 38,
10042–10051.
8 K. K. H. Ang, M. J. Holmes, T. Higa, M. T. Hamann and
U. K. Kara, Antimicrob. Agents Chemother., 2000, 44,
1645–1649.
9 J. Yu, T. Wang, X. X. Liu, J. Deschamps, J. F. Anderson,
X. Liao and J. M. Cook, J. Org. Chem., 2003, 68, 7565–7581.
10 R. S. Kusurkar, S. K. Goswami and S. M. Vyas, Tetrahedron
Lett., 2003, 44, 4761–4763.
33 H. Han, R. L. de Vrueh, J. K. Rhie, K. M. Covitz, P. L. Smith
and C. P. Lee, Pharm. Res., 1998, 15, 1154–1159.
34 M. E. Ganapathy, W. Huang, H. Wang, V. Ganapathy and
F. H. Leibach, Biochem. Biophys. Res. Commun., 1998, 246,
470–475.
35 W. Bi, Y. Bi, P. Xue, Y. Zhang, X. Gao, Z. B. Wang, M. Li,
M. B. Floch, N. Ngerebara, K. M. Gibson and L. R. Bi, Eur.
J. Med. Chem., 2011, 46, 1453–1462.
11 M. M. Airaksinen and I. Kari, Med. Biol., 1981, 59, 21–34.
12 G. M. Carbrera and A. M. Seldes, J. Nat. Prod., 1999, 62,
759–760.
36 M. Cain, O. Campos, F. Guzman and J. M. Cook, J. Am.
Chem. Soc., 1983, 105, 907–913.
13 K. P. Lippke, W. G. Schunack, W. Wenning and W. E. Mueller,
J. Med. Chem., 1983, 26, 499–503.
This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014
New J. Chem., 2014, 38, 4155--4166 | 4165

View Article Online
Paper
NJC
37 R. Cao, H. Chen, W. Peng, Y. Ma, X. Hou, H. Guan, X. Liu 43 S. S. Kalanur, U. Katrahalli and J. Seetharamappa,
and A. L. Xu, Eur. J. Med. Chem., 2005, 40, 991–1001. J. Electroanal. Chem., 2009, 636, 93–100.
38 J. S. Madalengoitia, J. J. Tepe, K. A. Werbovetz, E. K. Lehnert 44 Y. J. Guo, J. B. Chao and J. H. Pan, Spectrochim. Acta, Part A,
and T. L. Macdonald, Bioorg. Med. Chem., 1997, 5,
1807–1815.
39 R. H. Cao, Q. Chen, X. R. Hou, H. S. Chen, H. J. Guan, Y. Ma,
2007, 68, 231–236.
45 R. P. Queiroz, M. S. Castanheir, C. T. Lopes and K. Y. Cruz,
J. Photochem. Photobiol., A, 2007, 190, 45–52.
W. L. Peng and A. L. Xu, Bioorg. Med. Chem., 2004, 12, 46 G. W. Zhang, J. B. Guo, N. Zhao and J. R. Wang, Sens.
4613–4623.
Actuators, B, 2010, 144, 239–246.
40 L. H. Hurley, Nat. Rev. Cancer, 2002, 2, 188–200.
41 R. Martinez and L. Chacon-Garcia, Curr. Med. Chem., 2005,
12, 127–151.
47 M. Cusumano, M. L. DiPietro, A. Giannetto and
P. A. Vainiglia, J. Inorg. Biochem., 2005, 99, 560–565.
48 G. Cohen and H. Eisenberg, Biopolymers, 1966, 4, 429–440.
42 D. Suman and S. K. Gopinatha, J. Mol. Struct., 2008, 872, 49 D. Suh and J. B. Chaires, Bioorg. Med. Chem., 1995, 3,
56–63.
723–728.
4166 | New J. Chem., 2014, 38, 4155--4166
This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014