Design and Synthesis of Biotinylated Bivalent Carboline Derivatives as Potent Antitumor Agents

Article abstract of DOI:10.1021/acs.joc.0c01067

Compound 6, a novel β-carboline comprising two 1-methyl-9H-β-carboline-3-carboxylic acids and a biotin moiety conjugated together using tris(2-Aminoethyl)amine, was synthesized and tested for its cytotoxicity toward MCF-7 and HepG2 cell lines and antitumor potency in an S180 tumor-bearing mouse model. Compound 6 was delivered via biotin receptor-mediated endocytosis and exerted its therapeutic effects by intercalation binding with DNA. In vivo antitumor evaluations of 6 revealed that it is efficacious and exhibits low systemic toxicity.

Full text of DOI:10.1021/acs.joc.0c01067

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Article
Design and Synthesis of Biotinylated Bivalent
Carboline Derivatives as Potent Anti-tumor Agents
Xueyuan Chen, Yi Zheng, Songlin Song, Ying Liu, Yi Wang, Yong
Huang, Xiaoyi Zhang, Meng Zhang, Ming Zhao, Yuji Wang, and Li Li
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01067 • Publication Date (Web): 18 Aug 2020
Downloaded from pubs.acs.org on August 19, 2020
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Design and Synthesis of Biotinylated Bivalent Carboline
Derivatives as Potent Anti-tumor Agents
Xueyuan Chen, Yi Zheng, Songlin Song, Ying Liu, Yi Wang, Yong Huang, Xiaoyi
Zhang, Meng Zhang, Ming Zhao, Yuji Wang,* Li Li*
Beijing Area Major Laboratory of Peptide and Small Molecular Drugs; Engineering Research Center of
Ministry of Education of China; Beijing Laboratory of Biomedical Materials; School of Pharmaceutical
Sciences, Capital Medical University, Beijing 100069, P.R. China
* E-mail: wangyuji@ccmu.edu.cn (Y. Wang).
* E-mail: renulee@ccmu.edu.cn (L. Li).
Supporting Information
O
OH
2
N
N
H
O
NH2
O
N
N
H
5
6
N
N
H
HN
O
NH
S
N
N
H
HN
O
NH
N
N
H
O
N
N
H
HN
O
N
N
H
ABSTRACT: Compound 6, a novel -carboline comprising two 1-methyl-9H--carboline-3-carboxylic
acids and a biotin moiety conjugated together using tris(2-aminoethyl)amine, was synthesized and tested for
its cytotoxicity towards MCF-7 and HepG2 cell lines, and anti-tumor potency in an S180 tumor bearing
mouse model. Compound 6 was delivered via biotin receptor-mediated endocytosis, and exerted its
therapeutic effects by intercalation binding with DNA. In vivo anti-tumor evaluations of 6 found it to be
efficacious, and of low systemic toxicity.
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INTRODUCTION
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Peganum harmala. L is a perennial, herbaceous plant which has been used in Chinese folk medicines
to treat alimentary tract cancers for hundreds of years.1 -Carboline alkaloids isolated from Peganum
harmala can induce apoptosis and inhibit tumor cell proliferation, and have recently attracted attention for
their potential anti-tumor activities.2-4 -Carboline alkaloids exert their anti-tumor effects via multiple
mechanisms, including DNA intercalation and inhibition of topoisomerases I and II; cyclin-dependent kinase
(CDK); and IkK kinase complex (IkK).5 As DNA intercalators, their planar aromatic moieties insert between
adjacent base pairs, which decreases the DNA helical twist and length of the DNA strand, and disrupts
important processes such as DNA replication, transcription and repair.6 Recently, many DNA intercalators
have been used as potential lead compounds due to their inhibitory effect on the growth of cancer cells.7,8
Also, bivalent ligands formed by the tethering of two DNA intercalating agents by an appropriately designed
spacer have proved more effective inhibitors than their corresponding monomers by increasing the number
of binding sites and improving the interactions of ligands with their corresponding receptors.9-11 We recently
reported a bivalent ligand based on isoquinoline-3-carboxylic acid exhibited improved anti-tumor activity,
compared to isoquinoline-3-carboxylic acid alone.12
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-carboline alkaloid derivatives are interesting synthetic targets due to their medicinal importance. 13 In
synthetic organic chemistry we generally seek to develop novel synthetic route that can make new
compounds. However, when chemistry addresses disease problems, the chemistry must be considered in the
context of biological system, such as cellular system, or even whole animal.14 There is a great demand for
expanding the availability of -carboline-containing synthetic building blocks to promote medicinal
chemistry research.15 When -carboline derivatives are used as anti-tumor agents, the lack of tumor
specificity leads to relatively high dose administration. Therefore, the synthetic strategies varied accordingly.
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One possible solution to this problem is the conjugation of therapeutic agents with targeting ligands whose
receptors are over-expressed on the surface of cancer cells. Receptor-mediated internalization would result in
their selective delivery at high doses.16 Biotin has attracted much attention as a potential targeting ligand due
to its relatively simple chemical structure and low molecular weight, and the very high demands of cancer
cells for biotin compared to normal cells. Receptors for biotin such as avidin and its bacterial counterpart
streptavidin are present on the membranes of cancer cells, and biotin can form stable complexes with both.
Consequently, biotin or biotin-conjugates should be assimilated by cancer cells through receptor-mediated
endocytosis at higher rates than normal cells. 17,18
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We previously reported that the conjugation of 1-methyl-9H-β-carboline-3-carboxylic acid 2 (Scheme 1)
with amino acid benzylesters can increase the in vitro anti-proliferation and in vivo anti-tumor activities of
2.19 The conjugation of two of 1-methyl-9H-β-carboline-3-carboxylic acid units with a biotin moiety in the
same molecule was suggested as a possible strategy to reduce the in vivo treatment dose and improve the
therapeutic index of 2. To test this idea, we constructed a new bivalent ligand 6 bearing a biotin moiety
based
on
1-methyl-9H-β-carboline-3-carboxylic
acid.
Compound
6
comprises
two
1-methyl-9H-β-carboline-3- carboxylic acid pharmacophores; biotin; and the tris(2-aminoethyl) amine
(TREN) linker. This concept was supported by the therapeutic effect of compound 6 in vitro and in vivo
bioactivity evaluations.
RESULTS AND DISCUSSION
Scheme 1 depicts our synthesis of 6. Compound 1, obtained by Pictet-Spengler reaction of tryptophan and
acetaldehyde, was hydrolyzed in the presence of sodium hydroxide to give compound 2 in 91% yield.
Mono-N-Boc-protected TREN 3,20 obtained from the reaction of TREN with di-tert-butyldicarbonate in 74%
yield, was condensed with compound 2 in 1:2 stoichiometry by DMTMM (4-(4,6-dimethoxy-1,3,5-triazin
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-2-yl)-4-methyl morpholinium chloride) to give the compound 4 in 14% yield. Compound 4 was treated with
TFA to remove the Boc protecting group, giving compound 5 in 29% yield. The conjugation of compound 5
with biotin-NHS ester afforded the desired compound 6 in 29% yield.
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Scheme 1. Synthesis of Compound 6
L-Trp
O
NH
O
O
O
O
N
C
O
N
OH
O
OCH3
H
NaOH
91%
N
N
N
N
DMTMM
N
H
HN
N
H
H
DMF/CH3OH
14%
1
2
N
NH2
N
(Boc)2O
74%
NH
N
NH2
N
C
H
NH2
4
O
NH2
NH2
O
3
29%
O
N
H
TFA
N
NH
S
HN
NH2
O
O
NH
O
N
N
Biotin-NHS
N
O
H
N
N
HN
N
H
H
DMF
29%
HN
N
N
N
N
H
H
6
5
UV-visible absorption titration studies were used to study the binding behavior of compound 6 with DNA,
figure S1. Upon the addition of calf thymus DNA (CT-DNA) to a solution of compound 6 in Tris-HCl buffer
at pH 7.4, the characteristic absorption peaks corresponding to compound 6 (3.0 × 10-5 mol/L) were seen to
gradually decrease in intensity, accompanied by a bathochromic shift of about 19 nm at the absorption
maximum (267 nm). This hypochromic effect and bathochromic shift are thought to arise from intercalating
binding between compounds 6 and CT-DNA.8 The binding constant Kb between compound 6 and CT-DNA
was estimated to be 9.7  104 M-1according to the Scatchard equation.21 As the CT-DNA and compound 6
have an overlapping UV-vis absorption band, the corresponding CT-DNA was added to the reference
cuvette to compensate for the control absorbance.
DNA-antitumor agent intercalation binding plays an important role in cell proliferation,7 and is able to
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induce DNA conformational changes. It is known that the CD (circular dichroism) spectra of the B-form of
DNA has a positive band at about 275 nm due to base stacking, and a negative band of similar magnitude at
about 245 nm due to base right-hand helixity.22 In order to verify if compound 6 could induce DNA
conformational changes, the CD spectrum of CT-DNA in Tris-HCl buffer was recorded. It was found that
the negative band intensities of CT-DNA (100 µM) gradually decreased with increasing concentrations of
compound 6 (0, 10, 20 µM) at 245 nm, while only subtle differences were seen to the band at 275 nm
(Figure S2). The decrease in band intensity at 245 nm may arise from the insertion of compound 6 into the
DNA, resulting in the uncoupling and elongation of DNA double helix, and a decrease in the DNA helicity.
23 Thus, the results of the CD spectrum analysis together with the UV-visible spectra verify the intercalative
mode of binding of compound 6 into CT-DNA helix.
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The binding specificity of biotinylated 6 to avidin was investigated in an avidin-HABA competitive
binding assay.24 HABA (2-(4’-hydroxyazobenzene) benzoic acid) is an orange dye, capable of binding to
avidin in the same pocket used to bind biotin. HABA has a maximum UV absorbance at 350 nm; and the
non-covalent complex of avidin/HABA displays a new UV-visible absorbance band at 500 nm. The intensity
of this characteristic absorbance reduces upon addition of biotin, since biotin has much a stronger affinity for
avidin compared with HABA, and thus easily displaces the HABA from the avidin/HABA complex. This
displacement can be quantitatively monitored by the absorbance decrease at 500 nm. The avidin/HABA
complex was titrated with biotinylated 6, and the data are shown in the overlaid UV-vis spectra (Figure 1).
Upon addition of biotinylated 6, the absorbance at 500 nm decreased and the shade of orange color of
complex faded with an increase in the molar ratio of biotinylated compound 6 to avidin, suggesting the
avidin/HABA complex is displaced by biotinylated 6. There was no further absorbance decrease until the
molar ratio of biotinylated 6 to avidin reached four, where upon the color of the complex remained constant
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(Figure S3). Thus, the binding stoichiometry of biotinylated 6 to avidin is four, which is the same
stoichiometry as unmodified biotin (biotin: avidin = 4:1) and consistent with literature reports.25 These
results constitute evidence that 6 can form stable complex with avidin, and therefore 6 is able to selectively
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target the biotin receptors over-expressed on the cancer cells membrane surface.
0.30
0.25
1.4
0.20
1.2
0.15
1.0
0.10
0.8
0.05
0
2
4
6
8
10
a
k
0.6
0.4
0.2
0.0
[6]/[avidin]
(a) Avidin-HABA complex
(b-k) Compex of Avidin with 6
300
400
500
600
700
Wavelength / nm
Figure 1. UV/vis spectra of the avidin-HABA complex upon additions of biotinylated 6([6]/[avidin] = 0
~ 10) in buffer solution (pH = 7.4). Inserted: Absorption changes of avidin-HABA complex at 500 nm upon
the addition of 6.
To evaluate the anti-tumor potency of compound 6, its in vitro cytotoxic activity against a selection of
different cancer cell lines was ascertained using the MTT assay.26 The biotin receptor-expressing cell lines
MCF-7 and HepG2 were treated with varying concentrations of compound 6 for 72 h. Doxorubicin (DOX)
was used as positive control (IC50 = 0.16 ± 0.02 µM for MCF-7 and 0.58 ± 0.06 µM for HepG2 at 48 h),
compounds 2 and 5 were used as references. Due to the low aqueous solubility of 2, it was found to be
inactive above 100 μM. Cell viability graphs (Figure 2A) show that the tested compounds reduced MCF-7
and HepG2 viability at 72 h after seeding, in a concentration-dependent manner (P < 0.05). For instance, the
cell viability of compound 6 at 155 μM was ca. 45% in HepG2 cells over 72 h, while the cell viability of
compound 5 was ca 62% at the same concentration. It is evident that the compound 6 displays higher
cytotoxicity compared with the compound 5 against the biotin receptor-positive cancer cell lines tested
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(MCF-7 and HepG2 cells), within a concentration range of 40 to 225 μM, as shown in Figure 2A. The
MCF-7 cell line was more sensitive to compound 6 than the HepG2 cell line, showing that compound 6 was
a more potent inhibitor of the growth of this cell line. However, biotin free compound 5 and biotinylated
compound 6 separately exhibited no significantly different cytotoxicity against the biotin receptor-negative
cancer line HCT-116 (P > 0.05) within all tested concentration ranges. These results demonstrate that
biotinylated compound 6 accomplished the selective delivery of 1-methyl-9H-β-carboline-3-carboxylic acid
pharmacophores into biotin receptor-positive cancer cells over biotin receptor-negative cells.
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To further verify the role of biotin in receptor-mediated endocytosis, the biotin competitive MTT assay
was performed. When HepG2 cells were pre-incubated with an excess of biotin (100 μM) for 24 h, the biotin
receptors of HepG2 cells became occupied. The reduced in vitro cytotoxic activity of compound 6 against
biotin-treated HepG2 cells compared to the biotin-untreated HepG2 cells at all tested concentrations
indicated the competitive binding of biotinylated 6 with the biotin receptors of HepG2 cells was prevented or
reduced. Moreover, the difference in cytotoxic activity of compound 5 against biotin-treated and
biotin-untreated HepG2 cells was not significantly (P > 0.05), as shown in Figure 2B. The results of the
27
competitive MTT assay constitute evidence that the biotinylated compound 6 could be targeted to the
surface of cancer cells via biotin receptor-mediated endocytosis, accumulate with a higher dose, and exhibit
more potent activity, compared to compound 5.
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A
Compound 5
Compound 6
100
80
60
40
20
0
P<0.01
P>0.05
P>0.05
P>0.05
P>0.05
P>0.05
P>0.05
P<0.01
P<0.05
P<0.05
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P<0.01
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P<0.01
40 85 155 170 200 225 40 85 155 170 200 225
40 85 155 170 200 225
HCT-116
HepG2
MCF-7
Concentration(μM)
B
Compound 5
100
100μM Biotin/Compound 5
Compound 6
P<0.01
P<0.01
P<0.01
P>0.05
P>0.05
P>0.05
100μM Biotin/Compound 6
80
60
40
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P<0.01
P<0.01
P>0.05
P<0.01
P>0.05
P>0.05
40
85
155
170
200
225
Concentration(μM)
Figure 2. (A) Cell viability for biotin receptor-positive MCF-7, HepG2 cell lines, and biotin
receptor-positive cancer line HCT-116. (B) Cell viability of HepG2 cell without and with pretreatment with
excess of biotin (100 μM).
Apoptosis-induced cell death can alter cell nuclear morphology, which can be visualized by confocal laser
scanning microscopy.28 To examine the morphology of the HepG2 cells, they were stained blue with
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4'-6-diamidino-2-phenylindole (DAPI). It was observed that the morphology of the nuclei of the cells treated
with compound 6 became abnormal, containing condensed and irregular patterns of chromatin distribution,
after 24 h of exposure (Figure 3). With a further increase in the concentration of compound 6 to 225 μM, the
number of apoptotic cells with condensed nuclei increases. The similar phenomenon was observed for
positive control DOX, which also reported the nuclear accumulation of cancer cell following exposure
to DOX.29 On the other hand, cells treated with negative control NS (normal saline) and biotin free 5 showed
no or fewer nuclear morphological changes.
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Control
5 (150 µM)
5 (225 µM)
6 (225 µM)
DOX (0.5 µM)
6 (150 µM)
Figure 3. Confocal microscopy of HepG2 cells treated with indicated concentration of 5, 6 and DOX. The
non-treated and treated cells were stained with DAPI and observed by confocal microscopy. Scale bar = 75
m.
The percentage of apoptotic HepG2 cells was determined by monitoring the translocation of
phosphatidylserine from the inner face of the plasma membrane to the cell surface using the Annexin
V-FITC apoptosis detection kit. HepG2 cells treated with 6 (0, 150 μM and 225 μM, respectively) were
evaluated for apoptosis by Annexin-V FITC/PI double staining followed by flow cytometry, doxorubicin
(DOX, 0.5 μM) was used as positive control. HepG2 cells, which were harvested in the absence of
compound 6, were ~100% viable (negatively stained, lower left quadrant of the cytogram). When HepG2
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cells were treated with 150 μM compound 6, the percentage of early apoptotic cells (annexin-V positive and
propidium iodide negative, lower right quadrant) and cells in the late stages of apoptosis (annexin-V and
propidium iodide positive, upper right quadrants) increased to 12.6% in Figure 4. Upon increasing
concentration of 6 to 225 μM, the percentage of apoptotic cells further increased to 17.1%. However, the
percentage of necrotic cells (upper left) did not increase with the increasing concentration of 6. These results
indicate that HepG2 cells underwent the early and late stages of apoptosis after treatment with 6. The
increased percentage of apoptotic cells of 6 is consistent with the increased anti-proliferative activity of 6
compare to 5.
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Control
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5 (225 µM)
PI
6 (225 µM)
DOX (0.5 µM)
6 (150 µM)
14.6%
12.4%
18.5%
54.5%
Annexin V-FITC
Figure 4. HepG2 cells were treated with the indicated concentration of 5, 6 and DOX for 24 h, and stained
with FITC-Annexin V and PI, followed by flow cytometry analysis.
The therapeutic potential of 6 in vivo was evaluated using the S180 tumor bearing mouse model.12
Twenty-four hours after implantation, the mice were randomized into groups of 5 with 10 in each group.
Normal saline (NS, 10 mL/kg) and doxorubicin (DOX, 2 μmol/kg) were used as a negative and positive
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control, respectively. Test agents were administered every day at the indicated doses for 10 days. All
animals were sacrificed on the twelfth day. The activity was represented by tumor weight. The tumor
weights of the mice receiving DOX at dose of 2 μmol/kg (0.62 ± 0.17 g); compound 2 at dose of 8
μmol/kg (1.31 ± 0.26 g); compound 5 at dose of 2 μmol/kg (1.09 ± 0.32 g); and compound 6 at dose of
0.2 μmol/kg (0.93 ± 0.20 g), were significantly lower than those of the mice receiving NS (2.09 ± 0.47
g), table S1. This shows that DOX and the test agents all display anti-tumor activity at the treatment
doses. As shown in Figure 5, the in vivo anti-tumor activity of compound 5 at dose of 2 μmol/kg was
comparable to that compound 2 at dose of 8 μmol/kg. This implies that in vivo the effective dose of
compound 5 was four times lower than that of compound 2. The improved anti-tumor potency of 5
compared to 2 could be due to an increased local concentration of pharmacophore associated with 5,
due to the bivalent approach. To examine the role of biotin in the enhancement of drug uptake at the
tumor, the pharmacokinetics of compounds 5 and 6 were evaluated in S180 tumor bearing mice.30 Test
compounds were introduced by a single tail vein injection. Dose ratio of compound 5 to 6 in
pharmacokinetic study was the same to the dose ratio in vivo evaluation. Plasma levels of tested
compounds were monitored over time using an LC-MS/MS system. As shown in Figure S4, compound
6 displayed a similar pharmacokinetic profile to that of biotin free compound 5. These results indicated
that the therapeutic effectiveness of the mice administered compound 6 at lower dose compared with
those administered compound 5 was caused by biotin mediated uptake. The biotin receptor-mediated 6
maintained the higher pharmacophore accumulation to reach the tumor site. Thus, 6 exerted an
equivalent therapeutic effect at a dose ten times lower than that of biotin free bivalent ligand 5. It is
noteworthy that 6 exhibited better anti-tumor activity than 2 at 1/40 of the dose of compound 2 (P <
0.01).
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)
)
S
)
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N
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C
o
C
Figure 5. Tumor weights of S180 mice treated with the indicated doses of test agents, n =10.
The body weights of mice receiving test agents were monitored for 10 consecutive days. In Figure S4, the
body weight gain of the mice receiving DOX was suppressed throughout the 10 days. Conversely, mice
treated with compounds 2, 5 and 6 achieved similar levels of weight gain as compared with the NS treatment
group (body weights consistently increased from ~ 22 g to ~ 32 g). The organ to body weight ratio of liver
and spleen were significantly decreased in the DOX group in Figure S5. In contrast, the organ to body
weight ratios of treatment with compound 6 were not significantly different with that of NS. These results
suggest that compound 6 did not affect body weights, organ to body weight ratio to mice at the treatment
dose, which suggests it was not toxic at that dose.31
CONCLUSION
In summary, compound 6, a novel anti-tumor agent comprising two 1-methyl-9H-β-carboline-3-
carboxylic acids as double pharmacophores, biotin as a targeting ligand, and TREN as the bridging moiety,
has been designed, synthesized, and tested in vitro against biotin receptor-positive MCF-7 and HepG2 cell
lines, and in vivo in an S180 tumor bearing mouse model. In vivo evaluations of 6 found it to be efficacious
at 1/40 of the dose of compound 2, and of low systemic toxicity. Its superior performance compared with
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reference compound 2 is thought to arise from the preferential delivery of 6 to cancer cells due to the
over-expression of biotin receptors on the cancer cell surface, with which the biotin moiety of 6 can interact.
After endocytosis, compound 6 exerts its therapeutic effects by intercalation binding with the DNA.
Accordingly, compound 6 is proposed as a new anti-tumor lead compound.
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EXPERIMENTAL SECTION
General Information. All coupling and deprotection reactions were carried out under anhydrous conditions
and under a positive pressure of nitrogen, unless otherwise stated. 1-Methyl-9H-β-carboline-3-carboxylate
(1),19 1-methyl-9H-β-carboline-3-carboxylic acid (2),19 mono-N-Boc-protected TREN (3),20 and biotin
32
NHS ester were synthesized as previously described.
Avidin from egg white (avidin), and HABA
(2-(4’-hydroxyazobenzene) benzoic acid) were purchased from Sigma (Shanghai,China). All pH values were
obtained using pH paper, unless otherwise stated. ESI-MS experiments were performed on a Quattro micro
APITM and Bruker micrOTOF-Q TM, and Fourier transform ion cyclotron resonance (FT-ICR, 9.4T solariX,
Bruker, US) with dual ion source of ESI/matrix-assisted laser desorption ionization ESI-MS, respectively. 1H
NMR spectra were recorded on a Bruker Avance-500 MHz. 13C NMR spectra were recorded on a Bruker
Avance-125 MHz and 75 MHz. HPLC was performed on a Shimadzu LC30AD equipped with UV and
fluorescence detectors. Phenomenex Luna Omega C18 100A 1.6 μm  50  2.1 mm, Cloumn Number: 144,
eluting with 80% water containing 0.1% FA (formic acid) + 20% acetonitrile containing 0.1% FA. Statistical
analyses of all the biological data were carried out using use of analysis of variance (ANOVA). P-values <
0.01 were considered statistically significant, P-values > 0.05 were considered not differ significantly.
Tert-butyl 2-(bis(2-(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamido)ethyl)amino)ethyl)carbamate(4).
DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) (0.80 g, 2.92 mmol) and
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NMM (N-methylmorpholine) (2 mL) were added dropwise to a solution of 2 (0.66 g, 2.92 mmol) and
mono-N-Boc-protected TREN (3) (0.30 g, 1.22 mmol) in DMF (30 mL) at room temperature, with stirring,
until the pH of the resulting mixture was about 9, the pH value was checked by pH paper. Then solution was
diluted with methanol (2 mL) and allowed to stir at room temperature. After 18h, the solvent was removed in
vacuo and the residue was dissolved in ethyl acetate. After removal of solvent the crude product was purified
by column chromatography using silica gel (EtOAc:MeOH = 8:2). The product 4 (0.11 g ， 14%) was
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1
obtained as a white solid. TLC (EtOAc:i-Pro:NH3 = 15:1:0.1), Rf = 0.3. Mp: 252-254C. H NMR (300
MHz, DMSO-d6): δ/ppm = 12.11 (s, 2H), 8.66-8.62 (m, 4H), 8.29 (d, J = 7.5 Hz, 2H), 7.61-7.51 (m, 4H),
7.25 (t, J = 7.5 Hz, 2H), 6.67 (s, 1H), 3.48-3.46 (m, 4H), 3.10-3.08 (m, 2H), 2.72-2.76 (m, 10H), 2.64-2.66
(m, 2H), 1.31-1.23 (m, 9H). 13C{1H} NMR (75MHz, DMSO-d6) δ/ppm = 165.3, 141.3, 139.5, 136.3, 128.5,
-
127.8, 122.4, 121.8, 120.2, 112.7, 112.2, 63.5, 54.1, 37.7, 28.6, 20.8. ESI-TOF (m/z): Calc for C37H41N8O4 ,
([M-H]-1): 661.3251, found: 661.3358.
N,N'-(((2-Aminoethyl)azanediyl)bis(ethane-2,1-diyl))bis(1-methyl-9H-pyrido[3,4-b]indole-3-carboxami
de) (5).
Compound 4 (0.33 g, 0.50 mmol) was dissolved in TFA (4 mL). The solution was agitated for 6 h. The
.
TFA was evaporated off under reduced pressure at room temperature. The residue was dissolved in NH3 H2O
(0.3-0.4 mL) to get a pH of about 7. After removal of the solvent, the crude product was purified by column
chromatography using silica gel and CH2Cl2: MeOH: NH3 (10: 1: 0.2). The product 5 (0.08 g, 29%) was
1
obtained as a pale yellow solid. TLC (CH2Cl2: MeOH: NH3 =10: 1: 0.2), Rf = 0.29. Mp: 242-243C. H
NMR (500 MHz, DMSO-d6): δ/ppm =11.73 (s, 2H), 8.64 (t, J = 5.0 Hz, 2H), 8.63 (s, 2H), 8.26 (d, J = 10.0
Hz, 2H), 7.54-7.50 (m, 4H), 7.23-7.25 (m, 2H), 3.49 (d, J = 5.0 Hz, 4H), 2.73 - 2.76 (m, 4H), 2.66-2.70 (m,
8H), 2.61-2.62 (m, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ/ppm = 165.1, 141.1, 139.5, 136.1, 128.6,
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127.7, 122.4, 121.8, 120.2, 112.5, 112.1, 58.2, 53.9, 37.6, 20.7. HR-MS (FT-MS) (m/z): Calc for
5
6
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-
C32H33N8O2 , ([M-H]-1): 561.2726, found: 561.2756.
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N,N'-(((2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)azanediyl)bis(ethane-
2,1-diyl))bis(1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide) (6).
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Compound 5 (0.16 g, 0.29 mmol) was added to a solution of biotin-NHS ester (0.12 g, 0.35 mmol) in
dry DMF (4 mL). The reaction mixture was stirred at room temperature for 17 h. The solvent was then
removed in vacuo, and the crude product was purified by column chromatography using silica gel (CH2Cl2:
MeOH: NH3=10: 1: 0.1). Product 6 (0.06g, 29%) was obtained as a white solid. TLC (CH2Cl2: MeOH: NH3
1
=10: 1: 0.1), Rf = 0.29. Mp: 204-205C. H NMR (500 MHz, DMSO-d6): δ/ppm = 11.76 (s, 2H), 8.59-8.62
(m, 4H), 8.26-8.25 (m, 2H), 7.63 (t, J = 5.0 Hz, 1H), 7.51-7.56 (m, 4H), 7.22-7.26 (m, 2H), 6.36 (s, 1H),
6.30 (s, 1H), 4.21 (t, J = 5.0 Hz, 1H), 3.99 (t, J = 5.0 Hz, 1H), 3.46-3.48 (m, 4H), 3.22-3.23 (m, 2H), 3.18 (d,
J = 5.0 Hz, 1H), 2.87-2.91 (m, 1H), 2.76 (t, J = 5.0 Hz, 4H), 2.70-2.73 (m, 6H), 2.64-2.67 (m, 2H), 2.54 (s,
1H), 2.03 (t, J = 7.5 Hz, 2H), 1.33-1.53 (m, 4H), 1.04-1.22 (m, 2H). 13C{1H} NMR (125 MHz, DMSO-d6)
δ/ppm = 172.4, 165.4, 163.2, 141.2, 141.2, 139.6, 136.2, 128.6, 127.8, 122.4, 121.9, 120.3, 112.6, 112.2,
61.5, 59.7, 55.8, 54.2, 53.9, 49.1, 37.8, 37.7, 35.7, 28.7, 28.5, 25.7, 20.7. HR-MS (FT-MS) (m/z): Calc for
C42H49N10O4S+ , ([M+H]+1): 789.3659, found: 789.3631.
UV-visible absorption titrations.
UV-visible absorption titrations were performed using a UV–Vis spectrophotometer (UV-2550,
Shimadzu) at 25 °C using 1 cm path length quartz cuvette. The CT-DNA stock solution was prepared by
dissolving CT-DNA in Tris buffer (pH = 7.40, C = 0.1 mol/L). If the ratio of absorbance at 260 and 280 nm,
A260/A280, was greater than 1.8, the CD-DNA solution was considered to be sufficiently free from protein.
The concentration of CT-DNA stock solution was determined by Lambert-Beer law using the absorption
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coefficient at 260 nm (6600 M-1•cm-1). The stock solution was stored at 4 ºC and used within 4 days.
The binding constant Kb between compound 6 and CT-DNA was estimated as 5.0  104 M-1 according
to the Scatchard equation.21
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[DNA]/(εa - εf)= [DNA]/(εb - εf) +1/K(εb - εf)
Where [DNA] is the concentration of CT-DNA in base pairs; and εa , εf and εb correspond to the apparent
extinction coefficient for Aobsd/[6], the extinction coefficient for the free compound, and the extinction
coefficient for the compound in the fully bound form, respectively. The binding constant Kb was obtained
from the slope and intercept of the plot of [DNA]/(εa - εf) versus [DNA].
Circular dichroism (CD) studies.
Circular dichroism (CD) spectra were recorded with Jasco J-810 spectrometer at 25 °C using a 1 mm
quartz sample cell. Wavelength scans were acquired from 200 nm to 400 nm at 1 nm bandwidth, with a
scanning speed of 100 nm/min. All CD experiments were carried out in Tris-HCl buffer (pH = 7.40, C = 0.1
mol/L) upon addition of 0, 10, 20 µmol/L of compound 6 onto 100 µmol/L of CT-DNA, respectively.
Biotin binding: HABA assay. 24
The binding specificity of 6 towards avidin was investigated by HABA (2-(4’-hydroxyazobenzene)
benzoic acid) assay in Tris-HCl buffer solution (pH = 7.40, C = 0.1 mol/L, 1% DMSO).The HABA assay
was used to determine the extent of biotinylation. Avidin solution (3.710-6 mol/L, 9.0 mL), and HABA
solution (2.0 10-3 mol/L, 0.5 mL) were combined to afford a stock solution (Cavidin= 3.5  10-6 mol/L, CHABA
= 1.0  10-4 mol/L). Different concentrations of compound 6 of (0 ~ 3.5  10-5 mol/L) were added into the
stock solution. The final value was 1.0 mL. HABA dye is bound to avidin and displays a new UV-visible
absorbance at 500 nm. This characteristic absorbance was reduced upon addition of 6. The corresponding 6
was added to the reference cuvette to compensate for the control absorbance.
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In vitro antiproliferation assay.
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The cytotoxicities of compounds 5 and 6 against biotin receptor-positive cancer cell lines MCF-7
(human breast adenocarcinoma), HepG2 (human hepatocellular liver carcinoma), and biotin
receptor-negative cancer cell lines HCT-116 (human colorectal cancer) were separately studied using the
MTT assay.26 A suspension of cells (100 µL of 5 × 105 cells/mL) was seeded in 96-well plates and incubated
at 37ºC in a humidified atmosphere of 5% CO2 for 4 h. The medium was replaced by medium containing
different concentrations of test samples in PBS containing 5‰ DMSO and tests were in triplicates.
Incubation was carried out at 37 ºC in an incubator supplied with 5% CO2 for 72 h. Following incubation, 25
µL of 5 mg/mL of MTT were added to each well and the plate was put back in the incubator for another 4 h
at 37 ºC. Then culture medium was removed. The resulting MTT-formazan product was dissolved in 100 µL
DMSO. The amount of formazan was determined by measuring the optical density at 570 nm. The cytotoxic
activities were observed to occur in a dose-dependent manner in the cells. The activity was expressed as cell
viability, which is the percentage of living cells in the total cells.
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Biotin competitive MTT assay 27
A suspension of HepG2 cells (100 µL of 2 × 103 cells/mL) was seeded in 96-well plates and incubated
at 37ºC for 4 h in a humidified atmosphere of 5% CO2, until the cells were fully attached. 100 μM Biotin
were pre-incubated into HepG2 cells for 24 h. After the medium was removed, and washed three times with
PBS, the different concentrations of compounds were added to the medium and incubated for another 72 h.
The cytotoxicities were determinted according to MTT assays.
Confocal microscopy imaging: Identification of apoptotic cells by DAPI Staining.
HepG2 cells (2 × 105 cells/mL) were seeded in confocal dish with pore size of 20 mm and were
cultured in an incubator at 37 °C under an atmosphere of 5% CO2 for 4 h. The cells were then washed with
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PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1 % Triton X-100. The cells were incubated
for 10 min in the dark with DAPI (10 µg/mL). Cells were washed again with PBS and finally sealed with
50% glycerin. The stained cells were viewed by laser scanning confocal microscope (Leica TCS SP8,
Germany).
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Apoptosis assay.
Apoptosis of HepG2 cells was detected using the Annexin V-FITC Apoptosis Detection Kit. HepG2 cells, in
log phase of growth, were seeded at a density of 2  105 cells/mL in a 6-well cell culture plate. The cells
were cultured in an incubator at 37 °C under an atmosphere of 5% CO2. After the cells were attached, the
original medium in the cell plate was discarded, and different concentrations of the compound in medium
were added. The cells were collected by adding EDTA-free trypsin after 24 hours culture, and the cells were
washed three times with PBS (centrifuge at 2000 rpm for 5 min) to collect 5 × 105 cells. The cells were
resuspended in 500 μL of 1X Binding Buffer. After addition 5 µL of annexin V-FITC and 5 µL of propidium
iodide (PI, optional), the cells were incubated at room temperature for 5 min in the dark. The assay was
performed using a flow cytometer (LSR Fortessa). Fluorescence was measured with an excitation
wavelength of 488 nm through FL-1 (525 nm) and FL-3 filters (620 nm).
In vivo anti-tumor study.
All studies described herein were performed under a review protocol approved by the ethics committee of
Capital Medical University. The committee was assured that the welfare of the animals was maintained in
accordance to the requirements of the animal welfare act and according to the guide for care and use of
laboratory animals. The in vivo antitumor potency was determined using ICR mice (Swiss, 6-8 weeks old,
20.00 ± 2.00 g weight) inoculated with S180 sarcoma. S180 ascites tumor cells were used to form solid tumors
after subcutaneous injection. For initiation of subcutaneous tumors, the cells were obtained in their anascitic
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form from the tumor-bearing mice, which were serially transplanted once per week. Subcutaneous tumors
were implanted by injecting 0.2 mL of 0.9% saline containing 1.2  107 viable tumor cells under the skin on
the right armpit. 24 hours after implantation, the mice were randomly divided into five experimental groups
with 10 in each group, and treated with normal saline (NS, 10 mL/kg), DOX (2 μmol/kg), compound 2 (8
μmol/kg), compound 5 (2 μmol/kg) and compound 6 (0.2 μmol/kg) for 10 consecutive days. The weights of
the mice were recorded every day. Twenty-four hours after the last administration, all mice were weighed,
sacrificed (diethyl ether anesthesia), and immediately dissected. The tumor, heart, liver, spleen, kidney, and
brain of each mouse were weighed.
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Pharmacokinetics
For plasma: 10 µL standard samples in duplicate, samples in triplicate, and mouse plasma samples were
mixed with 60 µL acetonitrile containing IS (200 ng/mL of tolbutamide, 50 ng/mL of propranolol and 500
ng/mL of Dic ) in EP tubes. After the mixture was vortexed for 1 min, then centrifuged for 10 min (13000
rpm, 4℃), transfer 50 µL supernatant to a 96-well plate with 150 µL pure water, shake for 10 min and finally
inject 10 µL into LC-MS/MS system.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
NMR, MS, UV and CD spectra; the effect of compound 6 on the body weight and organ-to-body weight ratio of
S180 mice (file type, i.e., PDF).
AUTHOR INFORMATION
Corresponding Authors
* E-mail: wangyuji@ccmu.edu.cn (Y. Wang).
* E-mail: renulee@ccmu.edu.cn (L. Li).
ORCID
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Y. Wang: 0000-0003-3842-7627
L. Li: 0000-0002-2244-3967
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ACKNOWLEDGMENT
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This work was supported by Beijing Natural Science Foundation (2172018), Great Wall Scholar Project of Beijing
Municipal Education Commission (CIT&TCD20180332), and 863 Programme 2015AA020902.
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