Discovery of novel negletein derivatives as potent anticancer agents for acute myeloid leukemia

Article abstract of DOI:10.1111/cbdd.13159

Baicalin and its aglycone baicalein derived from Scutellaria baicalensis exhibited potent anticancer effects in various types of cancer cell lines. However, the unfavorable pharmaceutical properties became the main obstacle for their potential clinical de

Full text of DOI:10.1111/cbdd.13159

DR HAIJUN CHEN (Orcid ID : 0000-0001-7945-2461)
Article type
: Research Article
Discovery of Novel Negletein Derivatives as Potent Anticancer Agents for Acute
Myeloid Leukemia
Jianlei Wu,†,§ Yingyu Chen,‡,§ Xuanping Liu,† Yu Gao,† Jianda Hu,‡,* Haijun Chen†,*
†College of Chemistry, Fuzhou University, Fuzhou, Fujian 350108, China
‡Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology,
Fujian Medical University Union Hospital, Fuzhou, Fujian 350000, China
§These authors contribute equally to this work.
*Corresponding Authors
(H. Chen) Phone: +86 591 22866234. Fax: +86 591 22866227.
E-mail: chenhaij@gmail.com.
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/cbdd.13159
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(J. Hu) Phone: +86 591 83357896. Fax: +86 591 83324116.
E-mail: drjiandahu@163.com.
ABSTRACT
Baicalin and its aglycone baicalein derived from Scutellaria baicalensis exhibited
potent anticancer effects in various types of cancer cell lines. However, the
unfavorable pharmaceutical properties became the main obstacle for their potential
clinical development. With the aim of development of novel anticancer agents based
on the skeleton of baicalin, a series of novel negletein derivatives were designed and
synthesized. Among them, compound 8 (FZU-02,006) with an N,N-dimethylamino
ethoxyl moitey at the C6 position exhibited significant enhanced antiproliferative
effect against HL-60 cells in vitro through regulating multi-signaling pathways. These
results revealed that compound 8 with the improved aqueous solubility (as HCl salt,
>1 mg/mL) and enhanced anti-leukemia potency might serve as a promising lead for
further development.
Keywords: negletein derivatives, aqueous solubility, multi-signaling pathways
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INTRODUCTION
Acute myeloid leukemia (AML) as one of the leading causes of cancer deaths is
characterized by rapid growth of abnormal white blood cells.1 For most of AML
patients, traditional targeted anticancer therapies might eventually result in drug
resistance due to compensatory cross-talk of various abnormal signal pathways.2-4
Natural products with diverse biological characteristics5, 6 have been explored as
invaluable sources for drug design with particular effectiveness in cancerous and
infectious diseases.7-9 Flavonoids, which are ubiquitously distributed in many dietary
plants, have received increasing attention because most of them display various
beneficial effects on human health.10 Previous studies have shown that many
flavonoids exhibit potent anticancer activity against several human cancer cell
lines.11, 12 Representative examples are baicalin (1, Figure 1) and baicalein (2).13
Accumulating evidence revealed that both of them induced growth inhibition and
apoptosis in multiple hematological cancer cell lines.13 However, like many
biologically active flavonoids such as naringenin and quercetin, the poor aqueous
solubility of baicalin or baicalein resulting in low in vivo bioavailability limits its
further clinical application.14 One potential solution to enhance aqueous solubility is
introduction of polar, preferably an ionizable functionality in the unstable metabolic
position.15-17 Recent studies indicated that negletein (7-O-methylated baicalein
derivative, 3) displayed similar bioactivities and higher metabolic stability.18, 19 Thus,
negletein can be used as a promising starting point for further structural
modification.
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Previous investigations indicated that a series of baicalein derivatives (4-6, Figure
1) with enhanced anticancer potency were synthesized by linking various functions
on the positions of 6 and 7.20, 21 In this work, we designed and synthesized a series of
negletein derivatives by incorporating nitrogen-containing fragments into the
unstable metabolic region on the positions of 5 and 6. Among them, compound 8
represented as a promising lead with enhanced anti-leukemia potency and improved
aqueous solubility (> 1 mg/mL).
EXPERIMENTAL SECTION
Experimental apparatus and reagents
All commercially available starting materials and solvents were reagent grade, and
used without further purification. Reactions were performed under a nitrogen
atmosphere in dry glassware with magnetic stirring. Preparative column
chromatography was performed using silica gel 60, particle size 0.063-0.200 mm
(70-230 mesh, Flash). Analytical TLC was carried out employing silica gel 60 F254
plates (Merck, Darmstadt). NMR spectra were recorded on a Bruker-400 (1H, 400
MHz; 13C, 101 MHz) spectrometer. 1H and 13C NMR spectra were recorded with TMS
as an internal reference. Chemical shifts were expressed in ppm, and J values were
given in Hz. High-resolution mass spectra (HRMS) were obtained from Thermo Fisher
Scientific Exactive Plus mass spectrometer. Purity of final compounds was
determined by analytical HPLC, which was carried out on a Shimadzu HPLC system.
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HPLC analysis conditions were as follows: Inertstil ODS2 C18 (150 mm × 4.6 mm, 5
μm) column; flow rate, 0.5 mL/min; UV detection at 210 and 254 nm; linear gradient
from 30% methanol in water to 100% methanol in water in 20 min followed by 10
min of the last-named solvent. All biologically evaluated compounds are >95% pure.
General Procedure for the Synthesis of Compounds 7, 14-17
Synthesis of compound 7 was shown as an example. To a solution of 3 (511 mg,
1.80 mmol), K2CO3 (481 mg, 4.80 mmol) and KI (30 mg, 0.18 mmol) in MeOH (30 mL)
was added 3-bromoprop-1-yne (257 mg, 2.20 mmol). The mixture was stirred at 70
oC for 3 h, and concentrated under reduced pressure. The residue was diluted with
EtOAc (50 mL), washed with 1N HCl (5 mL) followed by brine (50 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. Purification by flash column chromatography (CH2Cl2) furnished the
desired product 7 as a pale yellow solid (350 mg, 60%).
General Procedure for the Synthesis of Compounds 8-13
Synthesis of compound 8 was shown as an example. To a solution of 3 (57 mg,
0.20 mmol) and PPh3 (157 mg, 0.60 mmol) in THF (2 mL) was added
2-(dimethylamino) ethanol (90 mg, 1.00 mmol) and DIAD (101 mg, 0.50 mmol). The
mixture was stirred at r.t. for 2 h, and concentrated under reduced pressure. The
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residue was diluted with EtOAc (30 mL), washed with brine (30 mL). The organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. Purification by flash column chromatography (CH2Cl2:MeOH = 10/1)
furnished the desired product 8 as a yellow solid (62 mg, 87%).
General Procedure for the Synthesis of Compounds 18-21
Synthesis of compound 18 was shown as an example. To a solution of 7 (94 mg,
0.30 mmol), sodium ascorbate (36 mg, 0.18 mmol) and copper sulfate pentahydrate
(45 mg, 0.18 mmol) in DMSO/H2O (5/1, 2 mL) was added azidobenzene (214 mg,
1.80 mmol). The mixture was stirred at r.t. for 3 h. The reaction mixture was diluted
with EtOAc (20 mL) and extracted with H2O (20 mL). The organic layer was washed
with brine (20 mL), dried over anhydrous Na2SO4, and concentrated under reduced
pressure to give the crude product. This residue was purified by silica gel
chromatography (petroleum ether: EtOAc = 3/1) to provide desired product 18 (68
mg, 52%) as a yellow solid.
The detailed 1H NMR, 13C NMR, and HRMS data for all synthesized compounds are
shown in the Supporting Information.
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Cell Line and Cell Culture
The cell line HL-60 was obtained from the Cell Resource Center of Shanghai
Institute for Biological Sciences (Chinese Academy of Sciences, Shanghai, China).
Cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS),
100 U/mL penicillin G sodium and 100 μg/mL streptomycin sulfate. Cells were
maintained at 37 oC in a humidified and 5% CO2 incubator.22
Cell Proliferation Assay
Cells plated in 96-well plates were treated in triplicates with negletein or
negletein derivatives for 48 h at 37 oC. Cells were then incubated with 5 mg/mL MTT
(Sigma) for 4 h. The supernatants were removed and cells were pulsed with 100 μL
DMSO. The optical density (OD) was measured at 492/630 nm using a
spectrophotometer (STAT FAX-2100). The inhibitory rate on cell proliferation was
calculated as (1-ODtreated/ODcontrol) × 100%. The half inhibitory concentration (IC50)
values were obtained by the Logit method.
Mitochondrial Membrane Potential (∆Ψm) Assay
Changes of mitochondrial membrane potential (∆Ψm) was performed by JC-1
staining according to the manufacturer’s instruction (Beyotime, Shanghai, China).23
Briefly, HL-60 cells were cultured in 6-well plates and then incubated with compound
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8 in fresh culture medium for 24 h. Cells were harvested and resuspened in JC-1
staining solution for 20 min at 37 oC. After washed twice with 1X assay buffer, cells
were acquired by flow cytometer.
Apoptosis Assay
Briefly, 1.0 × 105/mL of HL-60 cells in RPMI 1640 medium with 10% fetal bovine
serum (FBS) were plated in 6-well plates. After 24 h of incubation with negletein or
negletein derivatives, cells were harvested and washed with PBS, and then stained
with Annexin V-FITC/7-AAD (Becton-Dickinson, NJ, USA) according to the
manufacturer's instructions. The early apoptotic cells were quantified by flow
cytometry.
Western Blotting
HL-60 cells were exposed to compound 8 at different concentrations for 24 h.
Total protein was extracted and western blotting was performed as described
previously.24 Primary antibodies against human caspase-9, caspase-3,
poly(ADP-ribose) polymerase (PARP), p-Akt (Ser473), p-p70S6K (Thr389) were
obtained from Cell Signaling Technology (Beverly, MA, USA). β-Actin and Bcl-2
antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,
USA).
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Statistical Analysis
Statistical significance was determined using student t-test. *Represents a p value
less than 0.05.
RESULTS AND DISCUSSION
Design
Extensive metabolic investigations revealed that baicalin with two hydroxyl groups
at the positions of C-5/C-6 and O-glucoside group at C-7 position did not possess
favorable metabolic stability. By contrast, negletein with methyl group at C7-position
displayed significantly enhanced metabolic stability.13 The skeleton of negletein with
two hydroxyl groups at 5, 6-position was allowed to improve the solubility via facile
introduction of functional fragments. Previous studies showed that introduction of
nitrogen-containing moiety at C-6 position based on the scaffold of 4 afforded
compound 6 may improve druglike properties and antiproliferative activities.21 In
addition, triazoles have been proved to possess various pharmacological properties
including anti-infective, anticancer, antiviral, and anti-hypertensive activities.25-27
Based on the above mentioned results and our initial studies, we directed our
chemical modification on C-5 and C-6 positions through introduction of different
kinds of moieties including various nitrogen-containing or triazole moieties (Figure
2).
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Chemistry
The synthetic routes to new negletein derivatives reported in this work are
outlined in Scheme 1. Negletein (3) was synthesized from baicalin (1) via our
developed simple two-steps/one-pot strategy.28 Esterification of 1 catalyzed by
catalytic H2SO4 in methanol followed by treatment with excess NaBH4 afforded 3 in
51% yield. Compounds 8-13 were synthesized from negletein via Mitsunobu coupling
with corresponding alcohols. Compounds 14-17 were obtained through nucleophilic
substitution of various organobromo compounds with negletein. Treatment of
negletein with 3-bromoprop-1-yne provided the key intermediate 7 in 60% yield. The
1,2,3-triazol derivatives 18-21 were easily prepared by click reaction of 7 with
various aryl azides29 in the presence of copper sulfate and sodium ascorbate.
Biology
The calculated lipophilicity (cLogP) and topological polar surface area (TPSA)
values of synthesized negletein derivatives are listed in Table 1. The result suggests
that most of these new compounds are conformed to the criteria of Lipinski’s “Rule
of Five” and may have good physicochemical properties. To explore the meaningful
SAR studies and examine how the substitutions on the key moieties affect biological
activities of newly synthesized negletein derivatives, the antiproliferative potency of
these compounds was evaluated on the proliferation of human promyelocytic
leukemia (HL-60) cells by using standard MTT assay. The activity is expressed as the
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concentration of drug inhibiting 50% cell growth (IC50) and the data are presented in
Tables 1.
Screening of 15 negletein derivatives showed that compound 8 had the strongest
antiproliferative activity with an IC50 value of 7.24 μM against HL-60 cells (Table 1).
Compounds 9 and 12 showed a slightly enhanced potency, indicating that
appropriate modifications on C-6 position might improve the antiproliferative
activity. Compounds 13 and 14 did not show any antiproliferative effect even at 40
µM, indicating that introduction of moieties into both C-5 and C-6 positions resulted
in a significant loss of antiproliferative activity. It was disappointed to find that
1,2,3-triazol derivatives 18-21 were inactive with a dramatic loss of antiproliferative
activity. Through our preliminary SAR studies, compound 8 with excellent aqueous
solubility (as HCl salt, aqueous solubility >1 mg/mL) exhibited promising
antiproliferative effect in a dose-dependent manner (Figure 3), and was subjected to
further biological characterization.
To determine whether compound 8 induces cell apoptosis in HL-60 cells, we next
used Annexin V-PE/7-AAD staining assay to investigate the role of compound 8 on
the induction of apoptosis. The result showed that compound 8 did induce cell
apoptosis in a dose-dependent manner (Figure 4). Apoptosis induces loss of
membrane asymmetry resulting in alterations in mitochondrial membrane potential
(∆Ψm). The ∆Ψm is obviously changed when treated with 10 μM of compound 8 for
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24 h as determined by flow cytometry analysis, further indicating that compound 8
induced HL-60 cells apoptosis (Figure 5).
To better understand the antiproliferative mechanism of compound 8, we
evaluated its effect on the cell cycle distribution of HL-60 cells. The cells were
treated with compound 8 for 24 h, stained with propidium iodide (PI) and analyzed
by flow cytometry. As shown in Figure 6, the incubation of HL-60 cells with 10 μM of
compound 8 for 24 h obviously increased the percentage of cells at G0/G1 phase
(from 54.7% to 72.7%). Meanwhile, at the same condition, the percentage of cells in
S phase was obviously decreased (from 28.0% to 13.7%). These results revealed that
compound 8 arrested the cell cycle at the G0/G1 stage, leading to the inhibition of
cell proliferation.
<Insert Figure 6 here>
To gain deeper insight into the mechanism of compound 8, we also investigated
its effect on the some key signal pathways by using western blot analysis. HL-60 cells
were treated with different concentrations of compound 8 for 24 h. Apoptosis as a
cell death program is regulated by a family of caspases. We found that treatment of
HL-60 cells with compound 8 resulted in the cleaved caspase-9 and proteolytic
cleavage of pro-caspase 3 to its active form (Figure 7). Meanwhile, an up-regulation
of cleaved poly (ADP-ribose) polymerase (PARP) was also observed. Activation of
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caspase-3 resulted in the cleaved PARP, yielding an 85 kDa fragment (cleaved PARP)
which is a marker of cells undergoing apoptosis.30 These results suggested that
compound 8 might induce cell death through caspase-dependent apoptosis.
<Insert Figure 7 here>
Constitutive Akt activation plays an important role in the progression of AML
patients. Akt activation indirectly stimulates the transcription of anti-apoptotic genes
including Bcl-2.24, 31 Our results showed that compound 8 suppressed the activation
of Akt in a dose-dependent way and the phosphorylation of p70S6K was also
down-regulated. Accordingly, the expression of anti-apoptotic protein Bcl-2 was also
blocked. Akt activation promoted cell cycle transition from the G1 to S phase via
phosphorylation of the downstream targets p70S6K. The above results revealed that
compound 8 arrested cell cycle at the G1 phase and reduced S phase cell population.
CONCLUSION
In conclusion, this SAR studies indicated that introduction of triazol groups at C-6
position resulted in loss of anticancer activities and C-5 hydroxyl moiety is essential
for potency. Compound 8 (FZU-02,006) with an N,N-dimethylamino ethoxyl moitey
at C-6 position exhibited enhanced anti-leukemia potency and improved excellent
aqueous solubility (>1 mg/mL). Further work demonstrated that this compound
might activate caspase-dependent apoptotic pathway and inhibit Akt signaling
pathway.
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ACKNOWLEDGEMENTS
This work was supported by Joint research project of health and education of Fujian
Province (No. WKJ2016-2-06), the National Natural Science Foundation of China
(Nos. 81402781 and 81571802), the Scientific Research Foundation for the Returned
Overseas Chinese Scholars, National and Fujian Provincial Key Clinical Specialty
Discipline Construction Program, Fujian Provincial Natural Science Foundation
(2015J0102), Fujian Provincial Medical Innovation Project (2015-CX-14), the Program
of New Century Excellent Talents in Fujian Province University (2016B032).
ASSOCIATED CONTENT
Supporting Information
Additional Supporting Information may be found online in the supporting
information tab for this article.
1H NMR, and 13C NMR data and spectra of all synthesized compounds (PDF)
CONFLICTS OF INTEREST
The authors declare no competing financial interest.
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THE LIST OF FIGURE LEGENDS
Figure 1. Chemical structures of baicalin (1), baicalein (2), negletein (3) and the
reported improved anticancer flavonoids (4-6) based on the skeleton of baicalein.
Figure 2. Drug design strategy for new negletein derivatives.
Figure 3. Effects of compound 8 on HL-60 cell proliferation after 48 h co-incubation.
Figure 4. The apoptotic cell percentages in HL-60 cells after incubation with
compound 8 at different concentrations for 24 h.
Figure 5. Changes of mitochondria membrane potential of HL-60 cells after treated
with compound 8 for 24 h.
Figure 6. Cell cycle analysis of HL-60 cells treated with compound 8 for 24 h. After
treatment with compound 8 for 24 h, the harvested cells were stained with PI and
analyzed by flow cytometry.
Figure 7. Western blot analysis of biochemical markers for apoptosis induction and
inhibition by compound 8 in HL-60 cell line. Cells were treated with compound 8 at
different concentrations. Levels of procaspase-3, cleaved Caspase-9, PARP, Bcl-2,
p-P70S6K, and p-Akt were probed by specific antibodies. Actin was used as the
loading control.
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Scheme 1. Synthesis of negletein derivatives
Reagents and conditions: (a) cat. H2SO4, MeOH, reflux, 2 h; NaBH4, r.t., 48 h, 51%; (b)
corresponding alcohols, DIAD, PPh3, r.t., 2 h, 9-87%; (c) organobromo compounds,
cat. KI, K2CO3, MeOH, 70 oC, 3 h, 17-60%; (d) 3-bromoprop-1-yne, cat. KI, K2CO3,
MeOH, 70 oC, 3 h, 60%; (e) N3-Ar, cat. CuSO45H2O, sodium ascorbate, DMF/H2O
(9/1), r.t., 3 h, 23-66%; (f) 1N HCl, NaNO2, NaN3, H2O, 0-5 oC, 1 h, 60-95%.
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Table 1. SARs of 5- and 6-Substituted Negletein Derivatives
IC50 (μM)
HL-60
No
R1
R2
TPSAb cLogPc
1
2
3
4
7
187.1 1.27
304.8 ± 2.4
234.6 ± 1.7
192.7 ± 1.9
13.8 ± 0.4
>40
90.9
79.9
79.9
68.9
3.19
2.76
4.77
3.12
H
H
H
H
H
8
72.1
72.1
72.1
72.1
2.79
3.05
3.23
3.62
7.24 ± 0.15
39.9 ± 0.9
>40
9
10
11
>40
12
13
H
81.4
82.9
2.42
2.20
34.3 ± 0.8
>40
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14
15
16
57.9
68.9
68.9
95.2
4.31
4.47
5.79
2.70
>40
>40
>40
H
H
17
H
H
>40
>40
18a
99.62 3.74
108.9 4.14
108.9 4.14
108.9 4.14
19a
20a
21a
H
H
H
>40
>40
>40
Doxorubicin (ADR)
Emodin
0.26 ± 0.01
24.3 ± 0.7
a The chemical structures of negletein derivatives were varied at aromatic group (Ar).
b TPSA: http://www.molinspiration.com/cgi-bin/properties.
c Average cLogP: http://146.107.217.178/lab/alogps/start.html.
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