Synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death.

feRitu Aneja a,*, Surya N. Vangapandu a, Manu Lopus b, Vijaya G. Viswesarappa a,
Neerupma Dhiman c, Akhilesh Verma c, Ramesh Chandra c,
Dulal Panda b, Harish C. Joshi a
a Laboratory for Drug Discovery and Research, Department of Cell Biology, Emory University School of Medicine,
615 Michael Street, Atlanta, GA 30322, USA
b School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
c BR Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
1. Introduction
Cellular microtubules are the major cytoskeletal components
in all eukaryotic cells. Microtubules are crucial for the
maintenance of cell shape, polarity, and intracellular trans-
port of vesicles and organelles [1,2]. Moreover, during cell
division, microtubules form the mitotic spindle, which is a key
machinery driving the alignment of replicated chromosomes
to the equatorial plane and mediating subsequent segregation
of chromosomes to the two daughter cells [3]. The critical role
that microtubules play in cell division makes them a very
suitable target for the development of chemotherapeutic
drugs against the rapidly dividing cancer cells [reviewed in
4,5]. Although the effectiveness of microtubule-targeting
drugs has been validated by the extensive use of several
vinca alkaloids and taxanes for the treatment of a wide variety
of human cancers, their clinical success has been limited by
the emergence of drug-resistance and associated toxicities
such as leucocytopenias, diarrhea, alopecia, and peripheral
neuropathies due to the blockage of axonal transport [6–8].
Keywords:
Cell cycle
Mitotic arrest
Anticancer
Tubulin-binding
a b s t r a c t
We have previously identified the naturally occurring non-toxic antitussive phthalideiso-
quinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces
apoptosis. Here we present high-yield efficient synthetic methods and an evaluation of
anticancer activity of halogenated noscapine analogs. Our results show that all analogs
display higher tubulin-binding activity than noscapine and inhibit proliferation of human
cancer cells (MCF-7, MDA-MB-231 and CEM). Surprisingly, the bromo-analog is �40-fold
more potent than noscapine in inhibiting cellular proliferation of MCF-7 cells. The ability of
these analogs to inhibit cellular proliferation is mediated by cell cycle arrest at the G2/M
phase, in that all analogs except 9-iodonoscapine, caused selective mitotic arrest with a
higher efficiency than noscapine followed by apoptotic cell death as shown by immuno-
fluorescence and quantitative FACS analyses. Furthermore, our results reveal the appear-
ance of numerous fragmented nuclei as evidenced by DAPI staining. Thus, our data indicate
a great potential of these compounds for studying microtubule-mediated processes and as
chemotherapeutic agents for the management of human cancers.Synthesis of microtubule-inter
analogs that perturb mitosis in
by cell deathring halogenated noscapine
cancer cells followed

This has prompted an ongoing worldwide search for novel
microtubule-targeting compounds that display favorable
toxicity profiles, have better therapeutic indices and improved
pharmacological characteristics.
Among the various antimitotic agents that perturb micro-
tubule dynamics, noscapinoids constitute an emerging class
of compounds receiving considerable attention [9–16]. A key
structure for the cytotoxic activity of this class of compounds
is the presence of two chiral centers forcing the two aromatic
rings to be non-coplanar and in half-chair conformation [17].
The lead compound, noscapine was discovered by our
laboratory as a microtubule-interfering agent that binds
accompanied by the appearance of numerous fragmented
nuclei at 72 h of drug exposure as shown by 40-6-diamidino-2-
phenylindole (DAPI) staining. The precise mechanism of action
of these compounds involved a selective arrest of cell cycle
progression at the G2/M phase in rapidly dividing cancer cells.
Interestingly, whereas noscapine-arrested cells have nearly
normal bipolar spindles [12], cells arrested by all halogenated
analogs of noscapine formed pronounced multipolar spindles
as revealed by immunofluorescence microscopy.
2. Materials and methods
416
ed
rity
0
5
0
5
5
etonstoichiometrically to tubulin and alters tubulin conformation
[18]. Like many other antimicrotubule agents, noscapine
suppresses the dynamics of microtubule assembly and blocks
cell cycle progression at mitosis, followed by apoptotic cell
death in a wide variety of cancer cell types [18–21]. Noscapine
inhibits the progression of murine lymphoma, melanoma, and
human breast tumors implanted in nude mice with little or no
toxicity to the kidney, heart, liver, bone marrow, spleen, or
small intestine and does not inhibit primary humoral immune
responses in mice [18,22–24]. The water solubility and
feasibility for oral administration also represent valuable
advantages of noscapine over many other antimicrotubule
drugs [25–27]. Recently, several noscapine analogs have been
reported by us and others that have much better therapeutic
indices and improved pharmacological profiles [9–16].
Here we describe selective and high-yield synthetic schemes
for the halogenation of noscapine and an evaluation of
anticancer activity of these halogenated analogs compared to
the parent compound, noscapine. Our results show that all
halogenated derivatives (viz. 9-fluoronoscapine (9-F-nos); 9-
chloronoscapine (9-Cl-nos); 9-bromonoscapine (9-Br-nos); 9-
iodonoscapine (9-I-nos)) have a higher binding affinity for
tubulin as compared to noscapine. With the exception of 9-
iodonoscapine, all analogs inhibited proliferation of cancer cells
more actively than noscapine. They displayed much lower IC50
values as compared to noscapine in the two human breast
cancer cell lines (MCF-7 and MDA-MB-231) and a T-cell
lymphoma line, CEM. Surprisingly, 9-Br-nos showed a �40-
fold higher cytotoxic activity (IC50 = 1.0 � 0.2 mM) in MCF-7 cells
as compared to the parent noscapine (IC50 = 39.6 � 2.2 mM). The
cellular proliferation of the hormone-refractory MDA-MB-231
cells was inhibited by 9-Br-nos with an IC50 that is �10–12-fold
lower (IC50 = 3.3 � 0.4 mM) than that of noscapine (IC50 =
36.3 � 1.8 mM). This inhibition of cellular proliferation was also
Table 1 – HPLC purity for halogenated analogs as determin
Compound Method 1
Retention time (min) HPLC pu
Nos 14.29 97.
9-F-nos 15.02 97.
9-Cl-nos 15.55 99.
9-Br-nos 15.27 98.
9-I-nos 16.75 97.
In method 1, the solvent systems used were 0.1% formic acid and ac
peak attributions are indicated as retention times in minutes.by using two different methods
Method 2
(%) Retention time (min) HPLC purity (%)
14.52 97.0
15.21 97.0
15.90 99.0
16.67 98.0
16.99 97.0
itrile, whereas, method 2 used 0.1% formic acid and methanol. The2.1. Synthesis of halogenated noscapine analogs
1H NMR and 13C NMR spectra were measured by 400 NMR
spectrometer in a CDCl3 solution and analyzed by INOVA.
Proton NMR spectra were recorded at 400 MHz and were
referenced with residual chloroform (7.27 ppm). Carbon NMR
spectra were recorded at 100 MHz and were referenced with
77.27 ppm resonance of residual chloroform. Abbreviations for
signal coupling are as follows: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet. Infrared spectra were recorded on
sodium chloride discs on Mattson Genesis II FT-IR. High
resolution mass spectra were collected on Thermo Finnigan
LTQ-FT Hybrid mass spectrophotometer using 3-nitrobenzyl
alcohol or with addition of LiI as a matrix. Melting points were
determined using a Thomas-Hoover melting point apparatus
and were uncorrected. All reactions were conducted with
oven-dried (125 8C) reaction vessels in dry argon. All common
reagents and solvents were obtained from Aldrich and were
dried using 4 A˚ molecular sieves. The reactions were mon-
itored by thin layer chromatography (TLC) using silica gel 60
F254 (Merck) on precoated aluminum sheets. Flash chromato-
graphy was carried out on standard grade silica gel (230–400
mesh). HPLC purity data in two different solvent systems and
the peak attributions were measured in Ultimate Plus, LC
Packings, Dionex, using C18 column as shown in Table 1.
2.2. (S)-3-((R)-9-Bromo-4-methoxy-6-methyl-5,6,7,8-
tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-
dimethoxyisobenzofuran-1(3H)-one (2)
To a flask containing noscapine (20 g, 48.4 mmol) was added
minimum amount of 48% hydrobromic acid solution (�40 ml)
to dissolve or make a suspension of the reactant. To the

417reaction mixture was added freshly prepared bromine water
(�250 ml) drop wise until an orange precipitate appeared. The
reaction mixture was then stirred at room temperature for 1 h
to attain completion, adjusted to pH 10 using ammonia
solution to afford solid precipitate. The solid precipitate was
recrystallized with ethanol to afford bromo-substituted
noscapine. Yield: 82%; mp 169–170 8C; IR: 2945 (m), 2800 (m),
1759 (s), 1612 (m), 1500 (s), 1443 (s), 1263 (s), 1091 (s), 933
(w) cm�1; 1H NMR (CDCl3, 400 MHz), d 7.04 (d, 1H, J = 7 Hz), 6.32
(d, 1H, J = 7 Hz), 6.03 (s, 2H), 5.51(d, 1H, J = 4 Hz), 4.55 (d, 1H,
J = 4 Hz), 4.10 (s, 3H), 3.98 (s, 3H), 3.89 (s, 3H), 2.52 (s, 3H), 2.8–
1.93 (m, 4H); 13C NMR (CDCl3, 100 MHz), d 167.5, 151.2, 150.5,
150.1, 148.3, 140.0, 135.8, 130.8, 120.3, 120.4, 120.1, 105.3, 100.9,
100.1, 87.8, 64.4, 56.1, 56.0, 55.8, 51.7, 41.2, 27.8; MS (FAB): m/z
(relative abundance, %), 494 (93.8), 492 (100), 300 (30.5), 298
(35.4); MALDI: m/z 491.37 (M+), 493.34; ESI/tandem mass
spectrometry: parent ion masses, 494, 492; daughter ion
masses (intensity, %), 433 (51), 431 (37), 300 (100), 298 (93.3);
HRMS (ESI): m/z Calcd. for C22H23BrNO7 (M + 1), 493.3211;
Found, 493.3215 (M + 1).
2.3. (S)-3-((R)-9-Fluoro-4-methoxy-6-methyl-5,6,7,8-
tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-
dimethoxyisobenzofuran-1(3H)-one (3)
To a solution of bromonoscapine (1 g, 2.42 mmol) in anhy-
drous THF (20 ml) was added an excess of Amberlyst-A 26
(fluorine, polymer-supported, 2.5 g, 10 mequiv. of dry resin,
the average capacity of the resin is 4 mequiv./g) and the
reaction mixture refluxed for 12 h. The resin was filtered off
and the solvent removed to afford the crude product which
was purified by flash column chromatography (ethyl acetate/
hexane = 4:1) to afford (S)-3-((R)-9-fluoro-4-methoxy-6-
methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-
6,7-dimethoxy-isobenzo-furan-1(3H)-one (3) as a light brown
crystals. The recovery of resin was achieved by washing with
1 M NaOH and then rinsing thoroughly with water until
neutrality to afford hydroxy-form of resin. It was then stirred
overnight with 1 M aqueous hydrofluoric acid (250 ml),
washed with acetone, ether and dried in a vacuum oven at
50 8C for 12 h to afford the regenerated Amberlyst-A 26
(fluorine, polymer-supported). Yield: 74%, light brown crystals;
mp 170.8–171.1 8C; 1H NMR (CDCl3, 400 MHz): d 7.11 (d, 1H,
J = 8.0 Hz), 6.99 (d, 1H, J = 8.0 Hz), 5.44 (s, 2H), 5.21 (d, 1H,
J = 4.1 Hz), 4.02 (d, 1H, J = 4.1 Hz), 3.95 (s, 3H), 3.78 (s, 3H), 3.64 (s,
3H), 2.65–2.62 (m, 2H), 2.51–2.47 (m, 2H), 2.30 (s, 3H); 13C NMR
(CDCl3, 100 MHz): d 167.5, 152.9, 148.4, 139.8, 134.5, 126.0, 121.8,
119.0, 108.8, 103.1, 93.8, 81.9, 64.8, 61.1, 59.7, 57.7, 55.0, 46.4,
45.8, 39.4, 20.7, 19.1; HRMS (ESI): m/z Calcd. for C22H23FNO7
(M + 1), 432.4192; Found, 432.4196 (M + 1).
2.4. (S)-3-((R)-9-Chloro-4-methoxy-6-methyl-5,6,7,8-
tetrahydro-[1,3]dioxolo[4,5-g]iso-quinolin-5-yl)-6,7-
dimethoxyisobenzofuran-1(3H)-one (4)
To a stirred solution of noscapine (5 g, 12.01 mmol) in
chloroform (200 ml), a solution of sulfuryl chloride (4.897 g,
36.28 mmol) in 100 ml chloroform was added drop wise over a
period of 1 h at 5–10 8C. The reaction mixture was allowed to
attain room temperature and stirring was continued for 10 h.The reaction progress was monitored using thin layer
chromatography (7% methanol in chloroform). The reaction
mixture was poured into 300 ml of water and extracted with
chloroform (2� 200 ml). The organic layer was washed with
brine, dried over anhydrous magnesium sulfate and the
solvent evaporated in vacuo to afford the crude product.
Purification of the crude product using flash chromatography
(silica gel, 230–400 mesh) with 7% methanol in chloroform as
an eluent afforded the desired product, (S)-3-((R)-9-chloro-4-
methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]iso-
quinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (4).
Yield: 90% (4.49 g), colorless needles; mp 169.0–169.1 8C; 1H
NMR (CDCl3, 400 MHz): d 7.14 (d, 1H, J = 8.26 Hz), 6.41 (d, 1H,
J = 8.26 Hz), 5.93 (s, 2H), 5.27 (d, 1H, J = 4.31 Hz), 4.20 (d, 1H,
J = 4.32 Hz), 3.99 (s, 3H), 3.87 (s, 3H), 3.83 (s, 3H), 2.79–2.65 (m,
2H), 2.54–2.46 (m, 2H), 2.35 (s, 3H); 13C NMR (CDCl3, 100 MHz): d
167.7, 152.4, 147.5, 139.3, 134.9, 126.1, 120.3, 118.4, 108.5, 102.3,
93.5, 81.9, 64.2, 61.8, 59.6, 57.7, 54.9, 46.1, 45.2, 39.8, 20.6, 18.6;
HRMS (ESI): m/z Calcd. for C22H23ClNO7 (M + 1), 448.11481;
Found, 448.11482 (M + 1).
2.5. (S)-3-((R)-9-Iodo-4-methoxy-6-methyl-5,6,7,8-
tetrahydro-[1,3]dioxolo[4,5-g]isoquino-lin-5-yl)-6,7-
dimethoxyisobenzofuran-1(3H)-one (5)
The iodination of noscapine was achieved using pyridine–
iodine chloride. Since this is not commercially available, we
first prepared the said reagent using the following procedure.
Iodine chloride (55 ml, 1 mol) was added to a solution of
potassium chloride (120 g, 1.6 mol) in water (350 ml). The
volume was then adjusted to 500 ml to give a 2 M solution. In
the event the iodine chloride was under or over chlorinated,
the solution was either filtered or the calculated quantity of
potassium iodide added. Over chlorination was more to be
avoided than under chlorination since iodine trichloride can
serve as a chlorinating agent. Alternatively, the solution of
potassium iododichloride was made as follows. A mixture of
potassium iodate (71 g, 0.33 mol), potassium chloride (40 g,
0.53 mol) and conc. hydrochloric acid (5 ml) in water (80 ml)
was stirred vigorously and treated simultaneously with
potassium iodide (111 g, 0.66 mol) in water (100 ml) and with
conc. hydrochloric acid (170 ml). The rate of addition of
hydrochloric acid and potassium iodide solutions were
regulated such that no chlorine was evolved. After addition
was completed, the volume was brought to 500 ml with water
to give a 2N solution of potassium iododichloride, which itself
is a very good iodinating agent. However, usage of reagent in
the aromatic iodination of noscapine resulted in hydrolysis
products due to the acidic nature of the reagent. This
prompted us to make basic iodinating reagent, pyridine–
iodine chloride and was prepared as follows. To a stirred
solution of pyridine (45 ml) in water (1 l) was added 2 M
solution of potassium iododichloride (250 ml). A cream colored
solid separated, the pH of the mixture was adjusted to 5.0 with
pyridine and the solid collected by filtration, washed with
water and air-dried to afford the pyridine–iodine chloride
reagent in 97.5% yield (117 g) that was crystallized from
benzene to afford light yellow solid.Iodination of noscapine was now carried out by addition of
pyridine–iodine chloride (1.46 g, 6 mmol) to a solution of

418noscapine (1 g, 2.42 mmol) in acetonitrile (20 ml) and the
resultant mixture was stirred at room temperature for 6 h and
then at 100 8C for 6 h. After cooling, excess ammonia was
added and filtered through celite pad to remove the black
nitrogen triiodide. The filtrate was made acidic with 1 M HCl
and filtered to collect the yellow solid, washed with water and
air-dried to afford (S)-3-((R)-9-iodo-4-methoxy-6-methyl-
5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-
dimethoxyisobenzofuran-1(3H)-one (5). Yield: 76%, mp 172.3–
172.6 8C; 1H NMR (CDCl3, 400 MHz): d 7.15 (d, 1H, J = 8.1 Hz), 7.01
(d, 1H, J = 8.1 Hz), 6.11 (s, 2H), 5.36 (d, 1H, J = 4.8 Hz), 4.25 (d, 1H,
J = 4.8 Hz), 3.85 (s, 3H), 3.74 (s, 3H), 3.72 (s, 3H), 2.78–2.72 (m, 2H),
2.55–2.50 (m, 2H), 2.32 (s, 3H); 13C NMR (CDCl3, 100 MHz): d
168.2, 155.1, 151.5, 148.3, 146.5, 143.1, 140.3, 120.4, 119.5, 113.3,
101.5, 85.9, 82.2, 61.8, 56.6, 55.7, 54.5, 54.1, 51.2, 39.8, 30.1, 18.8;
HRMS (ESI): m/z Calcd. for C22H23INO7 (M + 1), 540.3209; Found,
540.3227 (M + 1).
2.6. HPLC purity and peak attributions
2.6.1. Method 1
Ultimate Plus, LC Packings, Dionex, C18 column (pep Map 100,
3 mm, 100 A˚ particle size, i.d.: 1000 mm, length: 15 cm) with
solvent systems A (0.1% formic acid in water) and B
(acetonitrile), a gradient starting from 100% A and 0% B to
0% A and 100% B over 25 min at a flow of 40 ml/min (Table 1).
2.6.2. Method 2
Ultimate Plus, LC Packings, Dionex, C18 column (pep Map 100,
3 mm, 100 A˚ particle size, i.d.: 1000 mm, length: 15 cm) with
solvent systems A (0.1% formic acid in water) and B
(methanol), a gradient starting from 100% A and 0% B to 0%
A and 100% B over 25 min at a flow of 40 ml/min (Table 1).
2.6.3. Cell lines and chemicals
Cell culture reagents were obtained from Mediatech, Cellgro.
CEM, a human lymphoblastoid line was provided by Dr.
William T. Beck (Cancer Center, University of Illinois at
Chicago). MCF-7 cells were maintained in Dulbecco’s Mod-
ification of Eagle’s Medium 1� (DMEM) with 4.5 g/l glucose and
L-glutamine (Mediatech, Cellgro) supplemented with 10% fetal
bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin/
streptomycin (Mediatech, Cellgro). MDA-MB-231 and CEM cells
were grown in RPMI-1640 medium supplemented with 10%
fetal bovine serum, and 1% penicillin/streptomycin. Mamma-
lian brain microtubule proteins were isolated by two cycles of
polymerization and depolymerization and tubulin was sepa-
rated from the microtubule binding proteins by phosphocel-
lulose chromatography. The tubulin solution was stored at
�80 8C until use.
2.7. In vitro cell proliferation assays
2.7.1. Sulforhodamine B (SRB) assay
The cell proliferation assay was performed in 96-well plates
as described previously [12,28]. Adherent cells (MCF-7 and
MDA-MB-231) were seeded in 96-well plates at a density of
5 � 103 cells per well. They were treated with increasing
concentrations of the halogenated analogs the next day while
in log-phase growth. After 72 h of drug treatment, cells werefixed with 50% trichloroacetic acid and stained with 0.4%
sulforhodamine B dissolved in 1% acetic acid. After 30 min,
cells were then washed with 1% acetic acid to remove the
unbound dye. The protein-bound dye was extracted with
10 mM Tris base to determine the optical density at 564-nm
wavelength.
2.7.2. MTS assay
Suspension cells (CEM) were seeded into 96-well plates at a
density of 5 � 103 cells per well and were treated with
increasing concentrations of all halogenated analogs for
72 h. Measurement of cell proliferation was performed
colorimetrically by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox-
ymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner
salt (MTS) assay, using the CellTiter96 AQueous One
Solution Reagent (Promega, Madison, WI). Cells were
exposed to MTS for 3 h and absorbance was measured using
a microplate reader (Molecular Devices, Sunnyvale, CA) at an
optical density (OD) of 490 nm. The percentage of cell
survival as a function of drug concentration for both the
assays was then plotted to determine the IC50 value, which
stands for the drug concentration needed to prevent cell
proliferation by 50%.
2.7.3. 40-6-Diamidino-2-phenylindole (DAPI) staining
Cell morphology was evaluated by fluorescence microscopy
following DAPI staining (Vectashield, Vector Labs, Inc.,
Burlingame, CA). MDA-MB-231 cells were grown on poly-L-
lysine coated coverslips in six-well plates and were treated
with the halogenated analogs at 25 mM for 72 h. After
incubation, coverslips were fixed in cold methanol and
washed with PBS, stained with DAPI, and mounted on slides.
Images were captured using a BX60 microscope (Olympus,
Tokyo, Japan) with an 8-bit camera (Dage-MTI, Michigan City,
IN) and IP Lab software (Scanalytics, Fairfax, VA). Apoptotic
cells were identified by features characteristic of apoptosis
(e.g. nuclear condensation, formation of membrane blebs and
apoptotic bodies).
2.7.4. Tubulin-binding assay
Fluorescence titration for determining the tubulin-binding
parameters was performed as described previously [29]. In
brief, 9-F-nos, 9-Cl-nos, 9-Br-nos or 9-I-nos (0–100 mM) was
incubated with 2 mM tubulin in 25 mM PIPES, pH 6.8, 3 mM
MgSO4, and 1 mM EGTA for 45 min at 37 8C. The relative
intrinsic fluorescence intensity of tubulin was then monitored
in a JASCO FP-6500 spectrofluorometer (JASCO, Tokyo, Japan)
using a cuvette of 0.3-cm path length, and the excitation
wavelength was 295 nm. The fluorescence emission intensity
of noscapine and its derivatives at this excitation wavelength
was negligible. A 0.3-cm path-length cuvette was used to
minimize the inner filter effects caused by the absorbance of
these agents at higher concentration ranges. In addition, the
inner filter effects were corrected using a formula Fcorrected = -
Fobserved � antilog [(Aex + Aem)/2], where Aex is the absorbance
at the excitation wavelength and Aem is the absorbance at the
emission wavelength. The dissociation constant (Kd) was
determined by the formula: 1/B = Kd/[free ligand] + 1, where B
is the fractional occupancy and [free ligand] is the concentra-
tion of 9-F-nos, 9-Cl-nos, 9-Br-nos or 9-I-nos. The fractional

occupancy (B) was determined by the formula B = DF/DFmax,
where DF is the change in fluorescence intensity when tubulin
and its ligand are in equilibrium and DFmax is the value of
maximum fluorescence change when tubulin is completely
bound with its ligand. DFmax was calculated by plotting 1/DF
versus 1/[free ligand].
2.7.5. Cell cycle analysis
The flow cytometric evaluation of the cell cycle status was
performed as described previously [12]. Briefly, 2 � 106 cells
were centrifuged, washed twice with ice-cold PBS, and fixed in
70% ethanol. Tubes containing the cell pellets were stored at
4 8C for at least 24 h. Cells were then centrifuged at 1000 � g for
3. Results and discussion
Aromatic halogenation constitutes one of the most important
reactions in organic synthesis. Although, bromine and
chlorine are extensively used for carrying out electrophilic
aromatic substitution reactions in the presence of their
respective iron halides, their utility is limited because of the
practical difficulty in handling of these reagents in labora-
tories compared to N-bromo- (NBS) and N-chlorosuccinimide
(NCS). Thus, NBS and NCS have proven to be superior
halogenating reagents provided benzylic halogenation is
suppressed. For example, Schmid reported that benzene
and toluene gave nuclear brominated derivatives in good
41910 min and the supernatant was discarded. The pellets were
washed twice with 5 ml of PBS and then stained with 0.5 ml of
propidium iodide (0.1% in 0.6% Triton-X in PBS) and 0.5 ml of
RNase A (2 mg/ml) for 45 min in dark. Samples were then
analyzed on a FACSCalibur flow cytometer (Beckman Coulter
Inc., Fullerton, CA).
2.7.6. Immunofluorescence microscopy
Cells adhered to poly-L-lysine coated coverslips were treated
with noscapine and its halogenated analogs (9-F-nos, 9-Cl-
nos, 9-Br-nos, 9-I-nos for 0, 12, 24, 48 and 72 h). After
treatment, cells were fixed with cold (�20 8C) methanol for
5 min and then washed with phosphate-buffered saline (PBS)
for 5 min. Non-specific sites were blocked by incubating with
100 ml of 2% BSA in PBS at 37 8C for 15 min. A mouse
monoclonal antibody against a-tubulin (DM1A, Sigma) was
diluted 1:500 in 2% BSA/PBS (100 ml) and incubated with the
coverslips for 2 h at 37 8C. Cells were then washed with 2%
BSA/PBS for 10 min at room temperature before incubating
with a 1:200 dilution of a fluorescein-isothiocyanate (FITC)-
labeled goat anti-mouse IgG antibody (Jackson ImmunoRe-
search, Inc., West Grove, PA) at 37 8C for 1 h. Coverslips were
then rinsed with 2% BSA/PBS for 10 min and incubated with
propidium iodide (0.5 mg/ml) for 15 min at room temperature
before they were mounted with Aquamount (Lerner Labora-
tories, Pittsburgh, PA) containing 0.01% 1,4-diazobicy-
clo(2,2,2)octane (DABCO, Sigma). Cells were then examined
using confocal microscopy for microtubule morphology and
DNA fragmentation (at least 100 cells were examined per
condition). Propidium iodide staining of the nuclei was used to
visualize the multinucleated and micronucleated DNA in this
study.Scheme 1 – Semi-synthetic derivatives of noscapine. Reagents a
82%; compound 4: SO2Cl2, CHCl3, 90%; compound 5: Pyr-ICl, CHyields with NBS and AlCl3 without solvents under long reflux
using a large amount of the catalyst (>1 equiv.) [30]. However,
reactions using NBS in the presence of H2SO4, FeCl3, and ZnCl2
resulted in unsatisfactory yields (21–61%) together with the
polysubstituted products. In another report by Lambert et al.,
aromatic substituted derivatives were obtained in good yields
with NBS in 50% aqueous H2SO4 [31], however, this method
required considerably high acidic conditions which are not
suitable for acid labile compounds, such as noscapine. Thus,
there still exists a need to develop selective, reproducible and
efficient procedures for the halogenation of such labile
aromatic compounds that eliminate the limitations associated
with the above discussed synthetic methods and offer
quantitative yields of the desired compounds. Our lead
compound, noscapine consists of isoquinoline and benzofur-
anone ring systems joined by a labile C–C chiral bond and both
these ring systems contain several vulnerable methoxy
groups. Thus, achieving selective halogenation at C-9 position
without disruption and cleavage of these labile groups and C–C
bonds was challenging. After careful titration of many
conditions, we have been successful in developing simple,
selective, efficient, and reproducible synthetic procedures to
achieve halogenation at C-9 position, that are discussed
below.
First, we examined the bromination of noscapine with
bromine water in the presence of HBr (Scheme 1). 9-Br-nos, (2)
was prepared as described previously with minor modifica-
tions [12,32]. Noscapine (1) was dissolved in minimum amount
of 48% hydrobromic acid with continuous stirring followed by
the addition of freshly prepared bromine water over a period
of 1 h until the appearance of an orange precipitate. The
reaction mixture was then stirred at room temperature for 1 h
nd reaction conditions—(a) compound 2: Br -H O, 48% HBr,2 2
3CN, 71%. (b) F2, Amberlyst-A, THF, 74%.