Design, synthesis and anticancer activities of halogenated Phenstatin analogs as microtubule destabilizing agent

Article abstract of DOI:10.1007/s00044-019-02299-4

A series of halogenated Phenstatin analogs were designed as microtubule destabilizing agent by docking study. It was synthesized within three steps starting from 2-chloro-5-iodobenzoic acid and substituted benzene. All the products were characterized by ¹H NMR and ¹³C NMR spectral analysis, and the stereochemical structure was also confirmed by a single crystal X-ray diffraction crystallographic analysis. The microtubule destabilizing activities were evaluated in vitro with human liver cancer Huh-7 cell line and human lung cancer A549 cell line. Some of the HPAs were achieved IC₅₀ about 5.0 μM against human liver cancer Huh-7 cells. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-019-02299-4

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MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02299-4
ORIGINAL RESEARCH
Design, synthesis and anticancer activities of halogenated
Phenstatin analogs as microtubule destabilizing agent
Shengquan Hu1 Wuji Sun1 Yeming Wang2 Hong Yan1
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Received: 30 November 2018 / Accepted: 24 January 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A series of halogenated Phenstatin analogs were designed as microtubule destabilizing agent by docking study. It was
synthesized within three steps starting from 2-chloro-5-iodobenzoic acid and substituted benzene. All the products were
characterized by 1H NMR and 13C NMR spectral analysis, and the stereochemical structure was also confirmed by a single
crystal X-ray diffraction crystallographic analysis. The microtubule destabilizing activities were evaluated in vitro with
human liver cancer Huh-7 cell line and human lung cancer A549 cell line. Some of the HPAs were achieved IC50 about 5.0
μM against human liver cancer Huh-7 cells.
Graphical Abstract
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Keywords Phenstatin analogs Microtubule destabilizing agent Docking Anticancer CCK-8
Introduction
tubulin polymerization (Lin et al. 1989; Pettit et al. 2003;
Kingston 2009). Combretastatins have special character-
istics that they can selectively target on the tumor blood
vessels and destroy the central area of the tumor (Li and
Tian 2016). Moreover, the CA-4 class of microtubule
destabilizing agent cannot be recognized by the drug efflux
transporter P-glycoprotein, which excessive expression in
tumor cells results in drug resistance (Singh and Kaur
2009). However, clinical studies have found that Com-
bretastatins have elevated blood pressure and other cardio-
vascular toxicities (Li and Tian 2016). Optimization
structure of lead compounds Combretastatins and synthesis
of new type Combretastatins derivatives with more efficient
and lower toxicity appear to be very important.
Combretastatins are natural antimitotic stilbenes derived
from the bark of Combretum caffrum Kuntze (Com-
bretaceae) (Pettit et al. 1982). The three most active mole-
cules Combretastatin A-1, A-2, and A-4 (CA-4) (Fig. 1)
have attracted a great attention as potent inhibitors of
* Hong Yan
hongyan@bjut.edu.cn
1
Beijing Key Laboratory of Environmental and Viral Oncology,
College of Life Science and Bio-engineering, Beijing University
of Technology, Beijing, China
The reported structural modifications of Combretastatins
have focused on trisubstituted benzene rings and double
2
Beijing Tide Pharmaceutical Co., Ltd, Beijing, China

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The activities of the HPAs (1 and 2) were evaluated
according to their binding energy (ΔG) and inhib constant
(Ki) by the virtual screening on Auto-Dock 4.0. The
synthesis of HPAs and anticancer activities evaluation were
described in details.
Materials and methods
Theoretical calculation
Fig. 1 Microtubule destabilizing agent structures
Initially, the virtual screening was utilized to identify the
potential inhibition of HPAs on tubulin polymerization. The
Tubulin-CA4 complex (PDB ID: 5lyj) (Gaspari et al. 2017)
was chosen as the study targets, owing to the ligand CA-4 is
similar structure to HPAs.
Molecular docking of compounds into the Tubulin-
Combretastatin A4 complex (PDB ID: 5lyj) was carried out
using the AutoDock software package as implemented
through the graphical user interface AutoDockTools (ADT
1.5.2). ADT was used to add polar hydrogens and gasteiger
charges. Ligands were prepared by drawing the 2D structure
in ChemDraw, and converted to PDB format using
Chem3D. Ligands were then energy minimized using Dis-
covery Studio Visualizer before using ADT to add polar
hydrogens and gasteiger charges. All bound water and
ligands were eliminated from the protein and the polar
hydrogen was added. The whole Tubulin-Combretastatin
A4 complex was defined as a receptor and the site sphere
was selected based on the ligand binding location of Ori-
ginal ligand, then the molecule of Original ligand CA-4 was
removed and new ligand was placed during the molecular
docking procedure. Types of interactions of the docked
protein with ligand were analyzed after the end of molecular
docking.
Scheme 1 Design strategy of HPAs
bond linker. The trisubstituted of CA-4, such as 2,3,4-tri-
methoxyphenyl (Álvarez et al. 2010), halogeno-
methoxyphenyl (Beale et al. 2010), and 3,4,5-trimethyl-
phenyl (Gaukroger et al. 2003) moieties, were demonstrated
equal or better activity. The pharmacophore model indicates
that other hydrogen bond acceptors could replace the tri-
methoxy group (Fig. 1) (Marrelli et al. 2011; Nguyen et al.
2005). The replacement of meta-methoxy groups with
halogen atoms (F, Cl, or Br) resulted in little difference in
growth inhibition of human cancer cell line comparison to
CA-4 (Pettit et al. 2005). Phenstatin (Fig. 1) was designed
from the CA-4 skeleton by replacement of the double bond
linker with a carbonyl moiety (Pettit et al. 1998). It is a
potent microtubule destabilizing agent that interacts with
colchicine binding site of tubulin and displays significant
anticancer and antimitotic activities comparable to those of
CA-4 (Pettit et al. 1998; 2000). 4,5-methylenedioxyphenyl
analog displaying the antiproliferative tubulin-destabilizing
activity at the same concentration range as CA-2 and CA-4
(Titov et al. 2011). A halogenated Phenstatin analogs
(HPAs), as an intermediate of Empagliflozin, was attracted
much attention for the very similar structure to that of some
reported microtubule destabilizing agents (Wang et al.
2014).
Materials and compound synthesis
Chemical reagents and solvents were obtained from com-
mercial sources. Commercial grade reagents were used
without further purification. When necessary, solvents were
dried or purified by standard methods. Melting points (m.p.)
were determined with an Electrothermal 9100 capillary
1
melting point apparatus and are uncorrected. H NMR and
13C NMR spectra were obtained using a Bruker 400
UltrashieldTM spectrometer at 400 and 100 MHz respec-
tively. These were analyzed using the Bruker TOPSPIN 2.1
program. Chemical shifts are reported in parts per million
relative to internal tetramethylsilane standard. Coupling
constants (J) are quoted to the nearest 0.1 Hz. The following
abbreviations are used: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet and br, broad. High resolution mass
spectra (HRMS) were obtained on a Thermo LTQ Orbitrap
In order to better study tubulin polymerization inhibition
of Phenstatin analogs, HPAs were designed based on the
importance of halogen in the Combretastatin (Scheme 1).

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XL (ThermoFisher, San Jose, CA, USA) spectrometer,
equipped with a quantropole detector. Analytic thin-layer
chromatography (TLC) was performed on the glass-backed
silica gel sheets (silica gel GF254). All compounds were
detected using UV light (254 or 365 nm).
Hz, 1H), 7.83(dd, J = 0.8, 6.8 Hz, 1H). 13C NMR (100
MHz, DMSO-d6): δ 13.58, 63.54, 92.07, 113.23, 120.67,
125.82, 129.30, 130.42, 131.31, 135.39, 136.20, 139.29,
143.17, 158.36, 192.09. HRMS (ESI) m/z (pos):386.96463
C15H13ClIO2 [M+H]+(calcd. 386.96488).
General procedure for the synthesis of HPAs (1a–1e)
(S)-(2-chloro-5-iodophenyl)(4-((tetrahydrofuran-3-yl)oxy)
phenyl)methanone (1d)
To a stirred suspension of 2-chloro-5-iodobenzoic acid
(5.65 g, 0.02 mol) in CH2Cl2(20 mL) was added oxalyl
chloride(1.80 mL, 0.021 mol) and DMF(0.02 mL). The
resulting mixture was stirred for 4 h at room temperature.
The mixture was concentrated, and the residual colorless
solid was dissolved in CH2Cl2(40 mL). To this solution
were added corresponding substituted benzene(0.02 mol)
and then AlCl3(2.75 g, 0.021 mol) portionwise so that the
temperature did not exceed 0 °C. After being stirred at 10–
20 °C for 3 h, the mixture was poured into ice water and
extracted with CH2Cl2 three times. The combined organic
layers were washed with 1 M HCl, water, and brine, then
dried over MgSO4 and concentrated. The residual solid was
crystallized from ethanol to give as white solid.
1
White solid, yield 81%, m.p. 110–111 °C. H NMR (400
MHz, DMSO-d6): δ 1.96–2.00(m, 1H), 2.24–2.31(m, 1H),
3.74–3.78(m, 1H), 3.81–3.88(m, 2H), 3.90–3.93(m, 1H),
5.14–5.16(m, 1H), 7.06(d, J = 6.8 Hz, 2H), 7.38(d, J = 6.8
Hz, 1H), 7.69(d, J = 6.8 Hz, 2H), 7.82(d, J = 0.8 Hz, 1H),
7.90(dd, J = 0.8, 6.8 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 32.36, 66.35, 72.11, 77.79, 92.82, 115.46,
128.24, 129.43, 131.58, 132.11, 136.55, 139.79, 140.38,
162.00, 191.10. HRMS (ESI) m/z (pos):428.97536
C17H15ClIO3 [M+H]+(calcd. 428.97544).
(R)-(2-chloro-5-iodophenyl)(4-((tetrahydrofuran-3-yl)oxy)
phenyl)methanone (1e)
According to this procedure the following compounds
were prepared.
1
White solid, yield 83%, m.p. 108–110 °C. H NMR (400
MHz, DMSO-d6): δ 1.96–1.99(m, 1H), 2.25–2.99(m, 1H),
3.74–3.80(m, 1H), 3.80–3.88(m, 2H), 3.90–3.93(m, 1H),
5.14–5.16(m, 1H), 7.06(d, J = 6.8 Hz, 2H), 7.38(d, J = 6.8
Hz, 1H), 7.69(d, J = 6.8 Hz, 2H), 7.82(d, J = 0.8 Hz, 1H),
7.90(dd, J = 0.8, 6.8 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 32.36, 66.35, 72.12, 77.80, 92.85, 115.48,
128.25, 129.42, 131.59, 132.12, 136.55, 139.81, 140.39,
162.02, 191.12. HRMS (ESI) m/z (pos):428.97536
C17H15ClIO3 [M+H]+(calcd. 428.97544)
(2-chloro-5-iodophenyl)(4-fluorophenyl)methanone (1a)
1
White solid, yield 92%, m.p. 69–70 °C. H NMR (400 MHz,
DMSO-d6): δ 7.38–7.40(m, 2H), 7.41(d, J = 8.0 Hz, 1H), 7.82
(dd, J = 2.4 Hz, 8.0 Hz, 2H), 7.90(d, J = 2.4 Hz, 1H), 7.92–
7.95(m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 116.19,
129.47, 131.65, 132.1, 132.65, 136.78, 139.69, 140.20,
164.54, 166.56, 191.36. HRMS (ESI) m/z (pos):382.91890
C13H7ClFIONa [M + Na]+(calcd. 382.91118).
Procedure for the synthesis of halogenated Phenstatin
analog (2-chloro-5-iodophenyl)(4-hydroxyphenyl)
methanone (1f)
(2-chloro-5-iodophenyl)(4-ethoxyphenyl)methanone (1b)
1
White solid, yield 72%, m.p. 103–104 °C. H NMR (400
MHz, DMSO-d6): δ 1.36(t, J = 5.6 Hz, 3H), 4.14(q, J = 5.6
Hz, 2H), 7.06(d, J = 6.8 Hz, 2H), 7.38(d, J = 6.8 Hz, 1H),
7.69(d, J = 6.8 Hz, 2H), 7.82(d, J = 0.8 Hz, 1H), 7.90(dd, J
= 0.8, 6.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ
14.31, 63.69, 92.74, 114.64, 128.04, 129.45, 131.54,
132.05, 136.53, 139.72, 140.43, 163.31, 191.03. HRMS
(ESI) m/z (pos):386.96463 C15H13ClIO2 [M+H]+(calcd.
386.96488).
To a solution of 1b (5.79 g, 0.015 mmol) in dichlor-
omethane(60 mL) at −20 °C was added boron tribromide
(1.65 mL, 0.023 mol) over 10 min at a rate that maintained
the reaction temperature below −10 °C under argon. After
addition, the mixture was slowly warmed to 0 °C and stirred
for 3 h. The reaction mixture was poured into 100 mL of ice
water and extracted with dichloromethane(2 × 100 mL). The
combined organic layers were washed with saturated
bicarbonate(80 mL), water(80 mL), and brine(80 mL) and
then dried over Na2SO4. The sample was concentrated and
purified by silica gel column chromatography to give pro-
(2-chloro-5-iodophenyl)(2-ethoxyphenyl)methanone (1c)
1
1
White solid, yield 18%, m.p. 109–110 °C. H NMR (400
duct 1f as a white solid, yield 87%, m.p. 181–182 °C. H
MHz, DMSO-d6): δ 0.81(t, J = 5.6 Hz,3H), 3.85(q, J = 5.6
Hz, 2H), 7.08–7.12(m, 2H), 7.30(d, J = 6.8 Hz, 1H), 7.59–
7.63(m, 1H), 7.66(d, J = 0.8 Hz, 1H), 7.72(dd, J = 0.8, 6.8
NMR (400 MHz, DMSO-d6): δ 6.88(d, J = 6.8 Hz, 2H),
7.37(d, J = 6.8 Hz, 1H), 7.58(d, J = 6.8 Hz, 2H), 7.78(d, J
= 0.8 Hz, 1H), 7.88(dd, J = 0.8, 6.8 Hz, 1H), 10.64(s, 1H).

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13C NMR (100 MHz, DMSO-d6): δ 115.70, 126.84, 129.44,
131.54, 132.40, 136.48, 139.59, 140.74, 163.20, 190.88.
HRMS (ESI) m/z (pos):356.91777 C13H7ClIO2 [M−H]
−(calcd. 356.91793)
3.71–3.77(m, 2H), 3.81–3.85(m,1H), 3.95(s, 2H), 4.93–
4.96(m, 1H), 6.84(d, J = 5.6 Hz, 2H), 7.10(d, J = 5.6 Hz,
2H), 7.22(d, J = 6.8 Hz, 1H), 7.58(dd, J = 0.8, 6.8 Hz, 1H),
7.65(d, J = 0.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ
32.37, 36.94, 66.29, 72.18, 76.89,92.73, 115.20, 129.66,
130.76, 131.21, 133.10, 136.57, 139.31, 141.27, 155.57.
General procedure for the synthesis of HPAs (2)
To a stirred solution of 1 (0.01 mol) in 30 mL of CH2Cl2
and 30 mL of CH3CN at 0 °C were added Et3SiH(4.79 mL,
0.03 mol), then BF3·Et2O(2.53 mL, 0.02 mol), and the
resulting mixture was warmed to room temperature and
stirred for 3–8 h. The mixture was poured into saturated
aqueous NaHCO3 and extracted with CH2Cl2 twice. The
combined organic layers were washed with brine, dried over
MgSO4, and concentrated. The residual solid was crystal-
lized from ethanol to give as white solid.
(R)-3-(4-(2-chloro-5-iodobenzyl)phenoxy)tetrahydrofuran
(2e)
1
White solid, yield 74%, m.p. 58–60 °C. H NMR (400
MHz, DMSO-d6): δ 1.90–1.95(m, 1H), 2.14–2.20(m, 1H),
3.70–3.76(m, 2H), 3.78–3.88(m, 2H), 3.94 (s, 2H), 4.94–
4.95 (m, 1H), 6.83(d, J = 6.8 Hz, 2H), 7.10(d, J = 6.8 Hz,
2H), 7.20(d, J = 6.8 Hz, 1H), 7.56(dd, J = 0.8, 6.8 Hz, 1H),
7.67(d, J = 0.8 Hz, 1H) 13C NMR (100 MHz, DMSO-d6): δ
32.37, 36.93, 66.30, 72.19, 76.91, 92.76, 115.22, 129.67,
130.78, 131.24, 133.10, 136.59, 139.33, 141.29, 155.59.
According to this procedure the following compounds
were prepared.
1-chloro-2-(4-fluorobenzyl)-4-iodobenzene (2a)
4-(2-chloro-5-iodobenzyl)phenol (2f)
1
1
White solid, yield 83%, m.p. 52–54 °C. H NMR (400
White solid, yield 75%, m.p. 101–102 °C. H NMR (400
MHz, DMSO-d6): δ 4.02(s, 2H), 7.10–7.13(m, 2H), 7.22–
7.25(m, 3H), 7.60(dd, J = 0.8, 6.8 Hz, 1H), 7.72(d, J = 0.8
Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 36.94, 92.85,
115.21(d, J = 68 Hz), 130.30(d, J = 24 Hz), 131.32,
133.16, 134.87, 136.82, 139.44, 140.77, 159.80, 161.73.
MHz, DMSO-d6): δ 3.90(s, 2H), 6.68(d, J = 5.6 Hz, 2H),
6.98(d, J = 5.6 Hz, 2H), 7.22(d, J = 6.8 Hz, 1H), 7.58(dd, J
= 0.8, 6.8 Hz, 1H), 7.62(d, J = 0.8 Hz, 1H), 9.22(s, 1H).
13C NMR (100 MHz, DMSO-d6): δ 37.01, 92.75, 115.24,
128.75, 129.57, 131.23, 133.06, 136.49, 139.27, 141.65,
155.76. HRMS (ESI) m/z (pos):342.93842 C13H9ClIO [M
−H]−(calcd. 342.93866)
1-chloro-2-(4-ethoxybenzyl)-4-iodobenzene (2b)
1
White solid, yield 87%, m.p. 61–63 °C. H NMR (400
X-ray crystal of compound (1d)
MHz, DMSO-d6): δ 1.30(t, J = 5.6 Hz, 3H), 3.94(s, 2H),
3.97(q, J = 5.6 Hz, 2H), 6.84(d, J = 5.6 Hz, 2H), 7.09(d, J
= 5.6 Hz, 2H), 7.22(d, J = 6.8 Hz, 1H), 7.58(dd, J = 0.8,
6.8 Hz, 1H), 7.65(d, J = 0.8 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 14.57, 36.95, 62.83, 92.76, 114.36, 129.59,
130.44, 131.25, 133.08, 136.57, 139.31, 141.41, 156.99.
To unambiguously assign the stereochemical structure of
compound 1d, a single crystal X-ray diffraction study was
performed. After many attempts, X-ray quality compound
1d crystals were obtained by slow evaporation of ethanol at
room temperature. The X-ray crystal structure of compound
1d confirmed the stereochemical assignment. And crystal-
lographic data for 1d has been deposited with Cambridge
Crystallographic Data Centre as supplementary number
CCDC 1444717. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK.
1-chloro-2-(2-ethoxybenzyl)-4-iodobenzene (2c)
1
White solid, yield 81%, m.p. 73–75 °C. H NMR (400
MHz, DMSO-d6): δ 1.30(t, J = 5.6 Hz, 3H), 3.95(s, 2H),
4.00(q, J = 5.6 Hz, 2H), 6.85–6.88(m, 1H), 6.95–6.97(m,
1H), 7.05–7.07(m, 1H), 7.19–7.23(m, 2H), 7.55–7.57(m,
2H). 13C NMR (100 MHz, DMSO-d6): δ 14.54, 63.08,
92.36, 111.63, 120.12, 126.41, 127.96, 130.12, 131.05,
133.30, 136.33, 139.50, 140.39, 156.24.
Cytotoxicity assay
For the evaluation of cytotoxicity, two different cancer cell
lines were used: human liver cancer Huh-7 cell line and
human lung cancer A549 cell line. Cancer cells were cul-
tured according to the supplier’s instructions. Cells were
seeded in 96-well plates at a density of 2–4 × 104 cells per
well and incubated overnight in 0.1 mL of media supple-
mented with 10 % Fetal Bovine Serum (Hyclone, USA) in a
(S)-3-(4-(2-chloro-5-iodobenzyl)phenoxy)tetrahydrofuran
(2d)
1
White solid, yield 79%, m.p. 60–62 °C. H NMR (400
MHz, DMSO-d6): δ 1.92–1.95(m, 1H), 2.15–2.19(m, 1H),