Second generation benzofuranone ring substituted noscapine analogs: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bcp.2011.03.029

Microtubules, composed of α/β tubulin heterodimers, represent a validated target for cancer chemotherapy. Thus, tubulin- and microtubule-binding antimitotic drugs such as taxanes and vincas are widely employed for the chemotherapeutic management of variou

Full text of DOI:10.1016/j.bcp.2011.03.029

Biochemical Pharmacology 82 (2011) 110–121
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Second generation benzofuranone ring substituted noscapine analogs:
Synthesis and biological evaluation
Ram Chandra Mishra a,1, Prasanthi Karna a,1, Sushma Reddy Gundala a,1, Vaishali Pannu a,
Richard A. Stanton a, Kamlesh Kumar Gupta b, M. Hope Robinson a, Manu Lopus d,
Leslie Wilson d, Maged Henary c, Ritu Aneja a,
*
a Department of Biology, Georgia State University, Atlanta, GA 30303, United States
b F.M. Kirby Neurobiology Center, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, United States
c Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States
d Department of Molecular, Cellular, and Developmental Biology, and the Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
Microtubules, composed of
a/b tubulin heterodimers, represent a validated target for cancer
Received 14 January 2011
Accepted 31 March 2011
Available online 8 April 2011
chemotherapy. Thus, tubulin- and microtubule-binding antimitotic drugs such as taxanes and vincas
are widely employed for the chemotherapeutic management of various malignancies. Although quite
successful in the clinic, these drugs are associated with severe toxicity and drug resistance problems.
Noscapinoids represent an emerging class of microtubule-modulating anticancer agents based upon the
parent molecule noscapine, a naturally occurring non-toxic cough-suppressant opium alkaloid. Here we
report in silico molecular modeling, chemical synthesis and biological evaluation of novel analogs
derived by modification at position-7 of the benzofuranone ring system of noscapine. The synthesized
analogs were evaluated for their tubulin polymerization activity and their biological activity was
examined by their antiproliferative potential using representative cancer cell lines from varying tissue-
origin [A549 (lung), CEM (lymphoma), MIA PaCa-2 (pancreatic), MCF-7 (breast) and PC-3 (prostate)].
Cell-cycle studies were performed to explore their ability to halt the cell-cycle and induce subsequent
apoptosis. The varying biological activity of these analogs that differ in the nature and bulk of substituent
at position-7 was rationalized utilizing predictive in silico molecular modeling.
Keywords:
Noscapine
Anticancer activity
Tubulin-binding
Cell cycle
ß 2011 Elsevier Inc. All rights reserved.
1. Introduction
Yet another emerging class of microtubule-modulating agents
is based upon noscapine, a non-sedative naturally occurring
Composed of
ubiquitous dynamic cytoskeletal polymers that have been long
recognized as validated pharmaceutical target in cancer
a
/
b
tubulin heterodimers, microtubules are
phthalideisoquinoline alkaloid from the opium poppy [3,4].
Noscapine, long-known innocuous cough-suppressant was
a
a
discovered for its tubulin-binding anticancer activity about a
decade ago [3]. Essentially, the identification and discovery of
noscapine was based upon its structural resemblance to commonly
known microtubule poisons such as colchicine [3]. Ever since,
noscapine has been successfully shown to inhibit various
neoplasms in vitro as well as in vivo such as leukemia and
lymphoma [3,5,6], melanoma [7], ovarian [8], gliomas [9], breast
[10], lung [11], and colon [12] cancer. Currently, noscapine is in
Phase I/II clinical trials for the treatment of multiple myeloma.
Our ongoing chemical synthetic efforts to improve the
therapeutic efficacy and pharmacological properties of noscapine
have yielded a battery of more potent first-generation noscapine
analogs, collectively referred to as noscapinoids [10,13–20].
Noscapinoids avoid the harsher effects of currently available
chemotherapeutic agents by leaving the total polymer mass of
tubulin unaffected [3,5,21]. Noscapine analogs have been exten-
sively shown to impede cell-cycle progression, inhibit cellular
chemotherapy [1,2]. Drugs that interfere with microtubule
dynamic stability are widely employed in the clinic to treat a
variety of cancers or are exploited as probes to gain insights into
microtubule structure and function. Three major classes of drugs
namely, taxanes, vinca alkaloids and colchicine analogs are well-
recognized and the positions they occupy on the cellular target,
tubulin, have been identified [1]. Traditionally, these three drug
classes are categorized into stabilizers and destabilizers; the
stabilizers predominantly causing overpolymerization of micro-
tubules into bundles and sheets and the destabilizers resulting in
depolymerization of microtubules into soluble tubulin.
* Corresponding author. Tel.: +1 404 413 5417; fax: +1 404 413 5301.
E-mail address: raneja@gsu.edu (R. Aneja).
1
All authors contributed equally.
0006-2952/$ – see front matter ß 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2011.03.029

R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
111
superimposing the drugs onto the colchicine molecule using Phase
Flexible Ligand Superpositioning program from Schrodinger
software [28] and then placing the resulting conformations into
their respective 3D protein models. UCSF Chimera was used to
determine hydrogen bonds and steric clashes of drugs docked to
protein [29,30]. Hydrophobic protein surface was made using
Tripos Sybyl (v. 8.1) to further visualize sites of potential steric
clashes.
2.2. Chemical synthesis
2.2.1. Synthesis of benzofuranone ring substituted noscapine analogs
1H NMR and 13C NMR spectra were measured in DMSO-d6 on
Bruker 400 NMR spectrometer. All proton NMR spectra were
recorded at 400 MHz and were referenced with residual DMSO
(2.50 ppm). Carbon NMR spectra were recorded at 100 MHz and
were referenced with 77.27 ppm (CDCl3) resonance of residual
chloroform. Abbreviations for signal coupling are as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet. High
resolution mass spectra were collected on Waters Q-TOF micro
mass spectrophotometer using 3-nitrobenzyl alcohol, in some
cases with addition of LiI as a matrix. All reactions were conducted
in oven-dried (125 8C) glassware under nitrogen atmosphere. All
common reagents and solvents were obtained from Sigma (St.
Louis, MO) and used without further purification unless otherwise
indicated. Solvents were dried by standard methods. The reactions
were monitored by thin layer chromatography (TLC) using silica
gel 60 F254 (Merck) pre-coated aluminum sheet. Flash chroma-
tography was carried out on standard grade silica gel (230–400
mesh).
Fig. 1. Molecular structure of noscapine ((S)-6,7-dimethoxy-3-((R)-4-methoxy-6-
methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-
1(3H)-one).
proliferation and induce apoptosis in a variety of cancer cells both
in vitro and in xenograft models of human cancers implanted in
nude mice [10,12,14,16,17,22,23]. From a synthetic perspective,
most of these first-generation analogs were generated by the
chemical manipulation of position-9 on the isoquinoline ring
system of noscapine (Fig. 1). The position-9 substituted (black-
arrowhead, Fig. 1) tubulin-binding analogs that displayed superior
anticancer activity included 9-nitronoscapine as well as haloge-
nated (fluoro, chloro, bromo, and iodo) analogs [13,19]. In
particular, the brominated analog of noscapine is most well-
studied because of its effectiveness against drug-resistant xeno-
graft tumors without any detectable toxicity [14,16,17,22,23]. It is
therefore no wonder that noscapine and its analogs have been
described as ‘kinder and gentler’ microtubule-modulating agents
that do not cause any apparent toxicity [24].
More recently, chemical modifications of position-7 (arrow,
Fig. 1) on the benzofuranone ring system of noscapine have been
reported [25,26]. The regioselective O-demethylation at position-7
yielded the O-alkylated derivatives including the hydroxy com-
pound which is ꢀ100-fold more potent than the parent noscapine
[25,26]. This strongly suggested that chemical maneuvering of the
benzofuranone ring had significant impact on the biological
activity of the parent molecule. Based upon this impetus, we
modeled noscapine in the colchicine binding site [27] to rationalize
the exceptionally enhanced biological activity of the 7-hydroxy
noscapine analog as well as aid future drug discovery by rational
drug design.
Here we report the chemical synthesis of second generation 7-
position benzofuranone noscapine analogs that differ in the steric
bulk of the substituent. The analogs were examined for their anti-
proliferative activity using representative cancer cell lines of lung,
myeloma, breast, pancreas and prostate. In silico molecular
modeling data were employed to rationalize and comprehend
the observed biological activity patterns of these analogs. This has
contributed to an enhanced understanding of structure-based drug
design to facilitate drug discovery and development of the
noscapinoid family, a novel class of tubulin-active, non-toxic
agents.
2.2.1.1. (S)-7-hydroxy-6-methoxy-3-((R)-4-methoxy-6-methyl-
5,6,7,8-tetrahydro[1,3]dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-
furan-1(3H)-one (2). Noscapine (2.0 g, 4.84 mmol), was dissolved
in anhydrous DMF (5.0 mL) followed by the addition of sodium
azide (0.63 g, 9.68 mmol) and sodium iodide (0.36 g, 2.42 mmol).
The mixture was stirred vigorously at 140 8C for 4 h. The mixture
was concentrated under reduced pressure to yield a dark residue
which was dissolved in EtOAc (50 mL). The insoluble material was
filtered through celite and the filtrate was diluted with EtOAc
(150 mL) followed by washing with water (2 ꢁ 25 mL) and brine
(2 ꢁ 25 mL). The organic layer was dried over sodium sulfate and
concentrated under reduced pressure to give crude product which
was crystallized from methanol. The product 2 was isolated as off-
white needles. (78% yield): mp 142–143 8C; 1H NMR (DMSO-d6,
400 MHz):
d 9.73 (s, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.47 (s, 1H), 6.01
(m, 2H), 5.81 (d, J = 8.0 Hz, 1H), 5.48 (d, J = 4.0 Hz, 1H), 4.24 (d,
J = 4.0 Hz, 1H), 3.96 (s, 3H), 3.79 (s, 3H), 2.48–2.34 (m, 2H) 2.43 (s,
3H), 2.31–2.18 (m, 1H), 1.95–1.83 (m, 1H): 13C NMR (CDCl3,
100 MHz):
d 174.6, 161.9, 151.6, 148.1, 140.7, 140.3, 133.9, 131.6,
116.8, 113.9, 111.8, 102.7, 102.4, 100.6, 80.9, 60.7, 59.3, 55.4, 48.2,
45.0, 25.76: HRMS: [M+H]+ [C21H21NO7+H]+, calcd: 400.1396,
found: 400.1382.
2.2.1.2. (S)-5-methoxy-1-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahy-
dro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-3-oxo-1,3-dihydroisobenzo-
furan-4-yl acetate (3). Compound
dissolved in anhydrous THF (5.0 mL) followed by the addition of
acetic anhydride (57 L, 0.6 mmol) and the dimethylamino
2 (0.2 g, 0.5 mmol), was
m
2. Materials and methods
pyridine (12 mg, 0.1 mmol). The mixture was stirred at ambient
temperature for 4 h, the reaction progress was monitored by TLC,
solvent was removed in vacuo and the residue thus obtained was
dissolved in EtOAc (25 mL) followed by washing with water
(2 ꢁ 25 mL). The organic layer was dried over sodium sulfate and
concentrated under reduced pressure to give crude product which
was separated over flash silica using methanol in chloroform as
2.1. In silico modeling studies
Crystal structure coordinates of the tubulin heterodimer-
colchicine model (PDB: 1sa0) [27] were used in this study.
Theoretical binding sites of noscapinoids were generated by

112
R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
eluent (1:99) to afford compound 3 which was crystallized using
methanol to yield pinkish crystals. (76% yield): mp 111–113 8C; 1H
(s, 3H), 2.73 (m, 3H), 2.45 (s, 3H), 1.94 (m, 1H); 13C NMR (DMSO-d6,
100 MHz): 167.2, 153.4, 152.5, 148.6, 144.3, 140.6, 137.2, 134.4,
d
NMR (DMSO-d6, 400 MHz):
d
7.40 (d, J = 8.4 Hz, 1H), 6.48 (s, 1H),
131.8, 124.6, 121.2, 120.5, 119.2, 116.9, 102.9, 101.3, 81.6, 79.7,
60.9, 59.7, 46.4, 35.9, 27.1: HRMS: [M+H]+ [C28H26N2O8+H]+, calcd:
519.1767, found: 519.1762.
6.40 (d, J = 8.4 Hz, 1H), 6.01 (s, 2H), 5.62 (d, J = 4.4 Hz, 1H), 4.25 (d,
J = 4.4 Hz, 1H), 3.95 (s, 3H), 3.81 (s, 3H), 2.67–2.42 (m, 3H), 2.42 (s,
3H) 2.32 (s, 3H), 1.95–1.85 (m, 1H); 13C NMR (DMSO-d6, 100 MHz):
d
168.5, 167.2, 151.7, 148.6, 140.5, 136.0, 134.3, 131.7, 121.4,
2.2.1.6. (S)-5-methoxy-1-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahy-
dro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-3-oxo-1,3-dihydroisobenzo-
furan-4-yl benzylcarbamate (7). Yellow solid. (78% yield): mp 122–
120.5, 119.5, 102.9, 101.3, 81.8, 60.8, 59.7, 57.1, 49.3, 45.9, 27.2,
20.6: HRMS: [M+H]+ [C23H22NO8+H]+, calcd: 442.1502, found:
442.1505.
123 8C; 1H NMR (DMSO-d6, 400 MHz):
d 8.39 (t, J = 5.2 Hz, 1H),
7.38–7.27 (m, 6H), 6.48 (s, 1H), 6.37 (d, J = 8.4 Hz, 1H), 6.01 (s, 2H),
5.60 (d, J = 4.4 Hz, 1H), 4.31–4.27 (m, 2H), 4.24 (d, J = 4.4 Hz, 1H),
3.95 (s, 3H), 3.81 (s, 3H), 2.72–2.48 (m, 2H), 2.44 (s, 3H), 2.32–2.28
(m, 1H), 1.91–1.86 (m, 1H); 13C NMR (DMSO-d6, 100 MHz):
d 158.6,
2.2.1.3. (S)-5-methoxy-1-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahy-
dro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-3-oxo-1,3-dihydroisobenzo-
furan-4-yl benzoate (4). Compound
2
(0.2 g, 0.5 mmol), was
dissolved in anhydrous THF (5 mL), potassium carbonate (0.1 g)
was added and the mixture was cooled over an ice bath (0–4 8C).
154.1, 152.5, 148.6, 140.6, 137.2, 134.4, 131.8, 121.2, 120.5, 119.2,
116.9, 102.9, 101.3, 81.6, 79.7, 60.9, 59.7, 46.4, 35.9, 27.1: HRMS:
[M+H]+ [C29H28N2O8+H]+, calcd: 533.1924, found: 533.1926.
Benzoyl chloride (76
ml, 0.65 mmol) was added drop-wise and
stirred vigorously at 0 8C then warmed to RT for overnight. Solvent
was removed in vacuo and the residue thus obtained was dissolved
in ethyl acetate (25 mL) and washed with water (2 ꢁ 25 mL). The
combined organic layers were dried over sodium sulfate and
concentrated under reduced pressure to give crude product which
was separated over flash silica using methanol in chloroform as
eluent (1:99) to obtain compound 4 which was crystallized with
methanol to yield dark yellow crystals. (92% yield): mp 152 8C; 1H
2.3. Cell lines and reagents
CEM (lymphoma), A549 (lung), PC-3 (prostate), MIA PaCa-2
(pancreatic), MCF-7 and MDA-MB-231 (breast) cancer cells were
purchased from ATCC. PC-3, A549, MDA-MB-231 and CEM were
cultured in RPMI-1640 medium supplemented with 10% Fetal
Bovine Serum (FBS) and 1% penicillin/streptomycin. MIA PaCa-2
and MCF-7 cells were cultured in DMEM supplemented with 10%
FBS and 1% penicillin/streptomycin. Primary human dermal
fibroblasts (HDF) from the dermis of normal human neonatal
foreskin were obtained from the Dermatology Department, Emory
University. MTT dye (Thiazolyl Blue Tetrazolium Bromide)
dimethyl sulfoxide (DMSO), propidium iodide and RNase were
purchased from Sigma (St. Louis, MO). Cells were cultured at 37 8C
with 5% CO2.
NMR (DMSO-d6, 400 MHz):
d 8.14 (m, 2H), 7.79 (m, 1H), 6.40 (d,
J = 8.4 Hz, 1H), 7.64 (m, 2H), 7.47 (d, J = 8.4 Hz, 1H), 6.50 (s, 1H),
6.44 (bs, 1H), 6.02 (s, 2H), 5.67 (d, J = 4.4 Hz, 1H), 4.29 (d, J = 4.4 Hz,
1H), 3.98 (s, 3H), 3.82 (s, 3H) 2.62 (m, 1H), 2.54 (m, 1H) 2.44 (s, 1H),
2.33 (m, 1H), 1.93 (m, 1H); 13C NMR (CDCl3, 100 MHz),
d 170.6,
166.7, 164.2, 152.1, 149.2, 140.4, 140.1, 136.9, 133.9, 132.7, 131.4,
130.5, 129.9, 128.9, 128.3, 121.5, 120.4, 118.5, 102.4, 100.9, 81.0,
61.2, 59.0, 56.9, 47.8, 45.0, 25.2.: HRMS: [M+H]+ [C28H25NO8+H]+,
calcd: 504.1658, found: 504.1668.
General synthesis procedure for carbamate derivatives 5–7:
Compound 2 (0.25 g, 0.626 mmol), was dissolved in anhydrous
dichloromethane (5 mL) followed by the addition of ethyl
2.3.1. Tubulin purification and polymerization assay
Microtubule proteins (MTP) consisting of ꢀ70% tubulin and
ꢀ30% microtubule-associated proteins (MAPs) were isolated from
bovine brain by three cycles of temperature-dependent polymeri-
zation and depolymerization. MAP-free tubulin (>99% pure) was
purified from MTP by phosphocellulose chromatography [31].
Purified tubulin was drop-frozen in liquid nitrogen, and stored at
ꢂ80 8C until use.
isocyanate (54
ml, 0.689 mmol) and the dimethylamino pyridine
(8 mg, 0.065 mmol). The mixture was stirred vigorously at ambient
temperature for 4 h. The mixture was condensed under reduced
pressure to dryness. The residue was dissolved in ethyl acetate
(25 mL) and washed with water (2 ꢁ 10 mL). The organic layer was
dried over sodium sulfate and concentrated under reduced
pressure to give crude product which was chromatographed over
flash silica using methanol in chloroform as eluent (2:98) to yield
carbamate derivatives 5–7.
The rate and extent of tubulin polymerization was monitored
using a light scattering assay at 350 nm as described previously
[32]. Briefly, phosphocellulose-purified MAP-free tubulin (12–
15
mM) was incubated with each compound at 0 8C for 10 min in
PEM buffer (80 mM PIPES, 3 mM MgCl2, and 1 mM EGTA, pH 6.8) in
a 96-well format. Following the addition of 1 mM GTP, assembly of
tubulin was initiated by transferring the sample containing plate to
Spectra Max Plus multi-well plate reader (Molecular Devices, USA).
which was temperature pre-adjusted at 35 8C.
2.2.1.4. (S)-5-methoxy-1-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahy-
dro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-3-oxo-1,3-dihydroisobenzo-
furan-4-yl ethylcarbamate (5). Yellow solid. (64% yield): mp 133–
1358C; 1H NMR (DMSO-d6, 400 MHz):
d 7.81 (t, J = 5.2 Hz, 1H), 7.34
(d, J = 8.4 Hz, 1H), 6.48 (s, 1H), 6.35 (d, J = 8.4 Hz, 1H), 6.00 (s, 2H),
5.58 (d, J = 4.4 Hz, 1H), 4.25 (d, J = 4.4 Hz, 1H), 3.95 (s, 3H), 3.79 (s,
3H), 3.09 (q, J = 7.2 Hz, 2H), 2.67–2.42 (m, 3H), 2.43 (s, 3H), 1.94–
1.92 (m, 1H), 1.09 (t, J = 7.2 Hz, 3H); 13C NMR (DMSO-d6,
2.3.2. Cytotoxicity assay
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay [16] was employed to evaluate the proliferative
capacity of cells. Essentially, MTT is a colorimetric assay, which
utilizes the colorless tetrazolium dye and converts it into a colored
formazan salt, which can be quantified by measuring absorbance at
100 MHz):
d 167.2, 153.4, 152.5, 148.6, 140.6, 137.2, 134.4,
131.8, 121.2, 120.5, 119.2, 116.9, 102.9, 101.3, 81.6, 79.7, 60.9, 59.7,
57.04, 45.89, 49.3, 35.9, 27.1, 15.3: HRMS: [M+H]+
[C24H26N2O8+H]+, calcd: 471.1767, found: 471.1761.
570 nm. Briefly, a 96-well format was used to seed 100
ml medium
containing cells at a density of 5 ꢁ 103 cells per well. After 24 h of
incubation, cells were treated with gradient concentration of the
test compounds, which were dissolved in DMSO. The final
concentration of DMSO in the culture medium was maintained
at 0.1%. After 48 h of drug incubation, the spent medium was
2.2.1.5. (S)-5-methoxy-1-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahy-
dro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-3-oxo-1,3-dihydroisobenzo-
furan-4-yl phenylcarbamate (6). Light yellow solid. (52% yield): mp
159–161 8C. 1H NMR (DMSO-d6, 400 MHz):
d 7.49–7.39 (m, 6H),
7.24 (t, J = 5.2 Hz, 1H), 6.48 (s, 1H), 6.35 (d, J = 8.4 Hz, 1H), 6.01 (s,
2H), 5.64 (d, J = 4.4 Hz, 1H), 4.32 (d, J = 4.4 Hz, 1H), 3.95 (s, 3H), 3.82
removed and the wells were washed twice with PBS. 100
ml of
fresh medium and 10 l of MTT (5 mg/ml in PBS) was added to the
m

R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
113
Fig. 2. (A) Colchicine. (B) Noscapine. (C) Flexible ligand superpositioning of noscapine (green carbons) on DAMA-colchicine (cyan carbons) from the colchicine-tubulin
complex as determined by Ravelli et al. [27]. Superpositioning was calculated based on best-fit geometry using the Schrodinger Phase Flexible Ligand Superpositioning
program [28]. Noscapine was found to share a similarity of 72.75% to the DAMA-colchicine conformation used.
wells and cells were incubated at 37 8C in dark for 4 h. The
formazan product was dissolved by adding 100 l of 100% DMSO
after removing the medium from each well. The absorbance was
measured at 570 nm using a Spectra Max Plus multi-well plate
reader (Molecular Devices, USA).
podophyllotoxin, and MTC [2-methoxy-5-(2,3,4-trimethoxyphe-
nyl)-2,4,6-cycloheptatrien-1-one], all of which are believed to bind
to the same region of the cellular target, tubulin [3]. The
identification of noscapine was based on its structural resemblance
with these drugs, such as a hydrophobic trimethoxyphenyl group
and other hydrophobic domains (like a lactone, tropolone, or other
aromatic rings) as well as small hydrophilic groups (like hydroxyl
m
2.3.3. Cell-cycle analysis
˚
Flow-cytometric evaluation of the cell-cycle status was
performed as described previously [14–17]. Briefly, control and
drug treated cells were centrifuged, washed 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
10 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
BD FACSCanto II flow-cytometer (BD Biosciences, Sparks, MD).
and amino groups) [3]. Since the 3.5 A crystal structure of tubulin
in complex with colchicine [27] clearly shows the binding
conformation of the drug, we first examined the structural
similarity of noscapine (Fig. 2B) to colchicine (Fig. 2A) using a
flexible ligand-superpositioning program [28]. The overlap of two
structures (Fig. 2C) yielded a geometric complementarity score of
72.75%, implying a strong structural similarity.
It has been recognized that traditional docking approaches have
several limitations when used with dynamic proteins such as
tubulin [34]. Thus, in order to model the binding of noscapine to
tubulin, we utilized the space-coordinates of colchicine in its
docked state to superimpose noscapine into the colchicine-binding
domain of 1SA0 PDB structure (Fig. 3A). Our modeling data show
2.3.4. Immunoblot analysis
Western blots were performed as described earlier [33]. Briefly,
proteins were resolved by polyacrylamide gel-electrophoresis and
transferred onto polyvinylidene difluoride membranes (Millipore).
The membranes were blocked in Tris-buffered saline containing
0.05% Tween-20 and 5% fat-free dry milk and incubated first with
primary antibodies against cleaved-PARP (Cell Signaling Inc.,
Beverly, MA) and survivin (Santa Cruz Biotechnology Inc., Santa
Cruz, CA) and then with horseradish peroxidase-conjugated
secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz,
noscapine in the same position as colchicine, within the b-subunit
near the intradimer interface (Fig. 3A). However, the methoxy
group at position-7 showed multiple clashes with the valine
residue 315 in the
b-subunit (Fig. 3B and C). This steric strain is
perhaps relieved upon O-demethylation at position-7 to yield a 7-
hydroxy-compound. This may possibly explain the significantly
increased activity of O-demethylated analogs that have been
reported [25,26].
CA).
b
-actin was from Sigma (St. Louis, MO). Specific proteins were
Given that replacing the methoxy group at position-7 with a
smaller, hydroxyl group increased efficacy by decreasing steric
hindrance, we rationalized that substituting an increasingly large-
sized group at this position should decrease drug activity. In order
to validate this predictive model, we synthesized noscapine
analogs by derivatizing position-7 on the benzofuranone ring
system of noscapine with larger functional groups.
visualized with enhanced chemiluminescence detection reagent
according to the manufacturer’s instructions (Pierce Biotechnology
Inc., Rockford, IL).
2.3.5. Caspase 3/7 activity assay
Control or lysates of PC-3 cells treated with 25
mM noscapine
derivatives were tested for caspase-3-like activity using Ac-DEVD-
7-amino-4-trifluoromethyl-coumarin, which detects the activities
of caspase-3 and caspase-7 according to manufacturer’s protocol
(Calbiochem, San Diego, CA). The results were evaluated using
VictorTM X5 multilabel reader (PerkinElmer, Inc., MA) and
expressed as relative fluorescence units.
When superimposed onto the colchicine-binding domain of our
tubulin docking model, these analogs showed an increase in the
number and magnitude of steric clashes at position-7 with an
increase in the size of the substituted subgroup (Fig. 4, Table 1).
This increase in clashes could decrease drug binding at the site and
decrease drug efficacy as well. These alterations would thus impact
the biological anticancer activity in cellular systems.
3. Results and discussion
3.2. Chemical synthesis of 7-position benzofuranone noscapine
analogs
3.1. In silico modeling studies
Noscapine was discovered through a semi-rational structural
screen of known microtubule poisons such as colchicine,
Sodium azide and sodium iodide in dimethylformamide (DMF)
were selectively used to cleave the methyl group at position-7 of

114
R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
Fig. 3. Model construction predicting noscapine bound in tubulin heterodimer. (A) Noscapine (green) is shown as a space-filling van der Waals model within the tubulin
heterodimer ( -subunit in blue, -subunit in yellow) in the colchicine-binding domain near the intra-dimer interface and the non-exchangeable GTP-site on -tubulin. (B)
b
a
a
Close-up of noscapine in the pocket reveals steric clashes (red lines) and H-bonds (blue lines) as predicted by UCSF Chimera software [29]. (C) Hydrophobic surface of binding
pocket created using Tripos Sybyl shows steric clash at 7-position of noscapine. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of the article.)
benzofuranone ring. As a result, we optimized an efficient method
to prepare compound 2 (7-hydroxy noscapine) that excluded the
use of Grignard reagent and simplified the work-up procedure to
obtain the reaction product. Briefly, noscapine was dissolved in
anhydrous DMF along with sodium azide and sodium iodide
followed by stirring at 140 8C for 4 h. The mixture was condensed
under reduced pressure and the residue was extracted in
ethylacetate followed by washing with water and brine to remove
excess salt. This synthetic method, offered a simple, economic and
easy work-up procedure compared to the one reported by
Anderson et al. [25]. The 7-hydroxy noscapine, 2 thus obtained,
served as a scaffold to synthesize various C-7-modified analogs of
noscapine.
Two strategies were followed for the synthesis of 7-substituted
noscapine derivatives as depicted in Scheme 1. Starting from the
key intermediate 2, in the first strategy, we performed acylation
reactions using acetic anhydride and benzoyl chloride in the
presence of a base to prepare compounds 3 and 4. Compound 3 is
the 7-acetyl derivative, which, in contrast to the almost inert
original methoxy derivative, has more polarized carbonyl func-
tionality. Compound 4, a benzoyl derivative, was prepared to
compare the effect of alkyl to aryl function in the same molecule.
It is well recognized that carbamate esters [30] are used to mask
free phenolic groups in cytotoxic cancer drugs [35]. Thus, in the
led to the formation of urea impurities thus increasing the
complexity of the purification process. Purification was accom-
plished by using repetitive flash chromatography. Another
challenge was the almost similar Rf of product and corresponding
starting materials on standard silica-coated TLC plates. We
resolved this problem by performing mass-spectrometric analysis
of the reaction mixture before the work-up step. Clearly, the mass
data also confirmed the presence of urea impurities in the reaction
mixture.
3.3. Biochemical characterization of novel benzofuranone analogs
3.3.1. Benzofuranone ring substituted noscapine analogs inhibit
tubulin polymerization in vitro
To determine the anti-tubulin activity of these benzofuranone
ring substituted noscapine analogs, their effects on tubulin
polymerization were examined in vitro. The effects of various
concentrations of all five noscapine analogs on the polymerization
of tubulin into microtubules are shown in Fig. 5. All five analogs
inhibited the light scattering signal in a concentration-dependent
manner, indicating that these benzofuranone ring substituted
noscapine analogs can bind to tubulin and inhibit microtubule
assembly.
second strategy,
a
series of carbamate esters of the key
3.4. Evaluation of antiproliferative activity and anti cell-cycle effects
intermediate, 2 were synthesized using readily available ethyl,
phenyl and benzyl isocyanates. These compounds were prepared
by the reaction of phenol derivative 2 with various isocyanates in
the presence of DMAP (4-N,N0-dimethylamino pyridine) in
anhydrous dichloromethane. The partial hydrolysis of isocyanates
3.4.1. Benzofuranone ring substituted noscapine analogs display
significant antiproliferative activity
The newly synthesized 7-position analogs of noscapine were
tested for their antiproliferative activity in various cancer cells

R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
115
Fig. 4. Predicted noscapinoid binding positions on tubulin. Panels on left show predicted binding position of noscapinoids based upon their superpositioning on DAMA-
colchicine from colchicine-tubulin crystal structure and then overlay into the protein [27]. Potential steric clashes (red lines) and H-bonds (blue lines) as predicted by UCSF
Chimera are shown. Larger substitutions at the 7-position show increased steric hindrances versus those of noscapine. Panels on right show noscapinoids in the hydrophobic
surface of the colchicine binding pocket created using Tripos Sybyl. Steric clashes can be seen in portions of the molecules shown outside of the pocket. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of the article.)

116
R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
Table 1
with compounds 5 and 6, with a few exceptions. Majority of the
cell lines were relatively resistant towards compound 6 (with IC50
values ranging from 50 to 250 M, Table 2). The significantly
Clashes and H-bonds.
m
Compound
Substituent
Average
clashes
Average
overlap
Average
H-bonds
higher IC50 values of compound 6 indicated that the bulk of the
substituent plays an important role in determining the biological
activity in cellular systems. Compound 7, as observed from Table 2,
showed relatively lower IC50 values when compared to compound
6 (Table 2). It is reasonable to speculate that the presence of a CH2
group between the nitrogen atom and aromatic ring system
provides flexibility to compound 7, which perhaps relates to its
higher activity. Comparison with noscapine demonstrated that 7-
position analogs, in particular, compounds 3–5 are significantly
better in antiproliferative activity (Table 2). Overall, the differences
in the IC50 value of these analogs for the cancer cell lines studied
can perhaps be attributed to the bulk of the functional group
introduced at position-7 of benzofuranone ring on the parent
molecule.
3
4
5
6
7
Acetyl
32.8
52.6
62.6
61.0
56.2
1.017
1.131
1.091
1.163
1.159
2.6
2.2
2.0
2.4
0.8
Benzoyl
Ethylcarbamato
Phenylcarbamato
Benzylcarbamato
using the MTT assay. Fig. 6 shows line plots of cell survival versus
gradient concentrations of various compounds to yield IC50 values
of each analog in different cell lines.
The IC50 value (drug concentration at which 50% inhibition of
cell proliferation occurs) of these synthesized compounds are
presented in Table 2. To appreciate the potency of these novel 7-
position substituted benzofurananone noscapine analogs, the IC50
values of the parent molecule noscapine for the various cell lines
under study are indicated in Table 2 (bottom-row).
3.4.2. Benzofuranone ring substituted noscapine analogs show
significant inter-line variation
Among the noscapine analogs tested, compound 3 (7-acetyl-
noscapine) was observed to be generally most-effective against
most cell lines used in the study (Fig. 6A, B, D, and E) except for
breast cancer cells (Fig. 6C). Pancreatic cancer MIA PaCa-2 cells
were particularly sensitive to compounds 3, 4 and 6 as compared
The expression level of oncogenes, tumor suppressor and key
molecules that regulate apoptosis, drug-resistance and angiogen-
esis can affect the sensitivity of tumor cells towards any given anti-
cancer agent [36,37]. It is recognizable that anti-tubulin agents like
paclitaxel, docetaxel offer superior anti-tumor outcomes in solid
malignancies such as breast and ovarian [38–40], while hemato-
logical malignancies are best managed by vinca alkaloids
(vinblastine, vincristine etc.) [41]. Differential sensitivity of cancer
cell lines was observed for each noscapine analog suggesting
significant inter-line variations (Fig. 7). Most cancer cells (lung,
lymphoma, pancreatic and prostate) significantly responded to
compound 3 and displayed much lower IC50 values in the range of
(IC50 in the range of 1–1.7
mM) to compounds 5 and 7 (IC50 49.0
and 24.5 respectively) (Fig. 6D and Table 2). Lung cancer A549 cells
also showed low IC50 for compounds 3–5 (Table 2) compared to
CEM, lymphoma cells, which were observed to be more sensitive
towards compound 5 (Fig. 6B). The IC50 values of the cell lines,
A549, CEM, MIA PaCa-2 and PC-3 were within 10
mM (Table 2) for
compounds 3–5. MCF-7, with higher IC50 values was found to be
resistant towards these analogs. Fig. 6F is a bar-graph representa-
tion depicting a comparison of the IC50 values of five noscapine
analogs towards each cancer cell used in the study. As the bulk of
the substituent increased in compound 4 (7-benzoyl-noscapine),
an increase in IC50s was evident, which was even more pronounced
1–15
mM (Fig. 7A). However, a very high IC50 (166.0 mM) was
observed for the breast cancer cell line, MCF-7 (Table 2, Fig. 7A),
suggesting a high degree of inter-line variability. Compound 4 with
a COPh (benzoyl) substituent, although quite effective against
lung, pancreatic and prostate cancer cells (IC50 values less than
Scheme 1. Synthesis of benzofuranone derivatives of noscapine 2–7.

R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
117
Fig. 5. Inhibition of tubulin assembly by noscapine analogs in vitro. Effects of various analogs on tubulin polymerization were assessed by monitoring the increase in light
scattering at 350 nm as described in Section 2. Each data point was obtained by subtracting the absorbance values of corresponding noscapine analogs in the absence of
tubulin. Data are representative of three independent experiments performed in triplicate (p < 0.05).
5
m
M) showed resistance towards lymphoma (IC50 49.0
breast cancer (IC50 34.0 M) cells (Fig. 7B). On the other hand,
compound 5 showed lower activity in CEM (1.7 M), A549
(5.6 M) and PC-3 (6.6 M) (Fig. 7C and Table 2. Interestingly,
pancreatic cancer cells were very sensitive to compound 6
m
M) and
(IC50 = 1
mM, Fig. 7D). These differences in cellular sensitivities
m
to the same compound may be due to altered expression of
b-
m
tubulin isotypes, or point mutations in tubulin resulting in
alterations of expression patterns of post-translational modifica-
tions of tubulin regulatory proteins, such as stathmin, microtubule
m
m
Fig. 6. Cancer cells of varying tissue origin are sensitive to the noscapine analogs. Cells were treated for 48 h with increasing gradient concentrations of various noscapine
analogs and the percentage of cell proliferation was measured using MTT assay. A, B, C, D and E represent the sensitivity profile the various cancer cells (A549, CEM, MCF-7,
MIA PaCa-2, PC-3) to the five 7-position substituted benzofuranone noscapine analogs. The plot of % cell survival versus noscapine analog concentrations for cancer cells in
them. F is a bar-graphical representation of IC50 values of noscapine analogs in various cancer cells used in our study. The values and error bars shown in all the graphs
represent average and standard deviations, respectively, of three independent experiments (p < 0.05).

118
R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
Table 2
In vitro cytotoxicity (IC50
,
mM) of noscapine analogs.
Compound
R
Cancer cell line, IC50
A549
(
m
M)
CEM
MCF-7
MIA PaCa-2
PC-3
3
4
5
6
7
–COMe
3.2
4.5
15.5
49.0
1.7
166.0
34.0
1.0
1.7
9.3
4.8
–COPh
–CONHEt
–CONHPh
–CONHBn
5.6
49.0
49.0
1.0
6.6
50.0
25.0
250.0
7.1
182.0
74.1
83.2
49.0
24.5
Noscapine
73.0
20.0
45.0
70.0
51.0
associated protein (MAP), tau and MAP4 [42,43]. These changes in
microtubule accessory proteins have been well recognized to affect
microtubule dynamicity and can perhaps contribute to the
development of drug resistance [44].
Interestingly, compounds 3–7 did not inhibit cell proliferation
in normal human dermal fibroblasts (HDFs) even at concentrations
48 h of treatment. Fig. 8(Ai–Ei) shows cell-cycle profiles upon
treatment with compounds 3–7 over various time points (0, 12, 24,
48 h) in a three-dimensional disposition. While 2N and 4N DNA
complements are representative of G1 and G2/M populations,
respectively, sub-G1 hypodiploid population is usually suggestive of
fragmented DNA and is a hallmark of apoptosis.
as high as 100
m
M. As shown in Suppl. Fig. 1, the proliferation
Treatment of MDA-MB-231 cells with compounds 3–7 showed
significant accumulation of cells in G2/M phase until 24 h of drug
exposure (Fig. 8Aii–Eii). The G2/M population however started to
decline beyond 24 h and thereafter a concomitant increase of sub-
G1 population was observed until 48 h. In case of compounds 3 and
5, as is evident from the bar graph; the G2/M population increased
considerably at 12 and 24 h (Fig. 8Aii and Cii). However, at 48 h the
G2/M population decreased significantly perhaps leading to
apoptotic cell death in case of compound 3 (Fig. 8Aii). Even after
48 h, compound 5 treated cells mostly remained arrested in G2/M
phase and the apoptotic index was lower compared to compound
3. Compounds 4, 6 and 7 also show similar pattern of G2/M arrest
over 24 h of treatment followed by a decline in the percent G2/M
cells and emergence of a hypodiploid population, indicative of
apoptosis (Fig. 8Bii, Dii and Eii).
capacity of these compounds were compared with the parent
compound noscapine and the most well-studied 9-position
substituted analog, 9-bromonoscapine (Suppl. Fig. 1).
3.4.3. Benzofuranone ring substituted noscapine analogs cause cell
cycle arrest followed by apoptosis
To gain further insights into the precise mechanisms responsible
forinhibitionof cellularproliferation, wenext examined the effect of
benzofuranone ring substituted analogs on cell cycle distributing
profiles of breast cancer MDA-MB-231 cells. The effect of
compounds 3–7 on mitotic index (percent G2/M cells) and apoptotic
index (percent sub-G1 cells) as a function of time in MDA-MB-231
cells was studied using fluorescence activated cell sorting (FACS)
analysis. All 5 compounds were used at 25
mM concentration over
Fig. 7. Benzofuranone ring substituted noscapine analogs exhibit significant inter-line variability. A, B, C, D and E are the plots of percent cell survival versus concentration of
noscapine analogs for cancer cells from varying tissue origins viz., lung (A549), lymphoma (CEM), breast (MCF-7), pancreas (MIA PaCa-2) and prostate (PC-3) used for
determination of IC50 values. F is a bar-graph representation of IC50 values of noscapine analogs in various cancer cells used in our study. The values and error bars shown in
the graphs represent average and standard deviations, respectively, of three independent experiments (p < 0.05).

R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
119
Fig. 8. Panels Ai–Ei depicts cell-cycle distribution of MDA-MB-231 cells in a three-dimensional disposition as determined by flow-cytometry at different time point upon
treatment with 25 M concentration of compounds 3–7. Results are representative of three experiments done in triplicate. The right panels (Aii–Eii) show bar-graph
m
representation of the percent G2/M and sub-G1 populations at different time points for compounds 3–7. Values and error bars shown in the graph represent mean and
standard deviation, respectively, of three independent experiments performed in triplicate.
Although staining of DNA with propidium iodide is extremely
useful to have an overview of the percent cell population in
various cell-cycle phases, however it has its limitations. It
cannot dissect out the differences between G2 and M phases as
both have 4N DNA amounts. Thus to explore further, we chose a
mitosis specific marker MPM-2, to distinctly identify which
phase the cells get stuck in. Our data revealed that all five
noscapine analogs appeared to induce strong G2 arrest
starting as early as 12 h (ꢀ65%) continuing till 24 h. In case of
compound 5, the arrest was maintained until 48 h.
Cell population corresponding to the G2/M peak was clearly
negative for MPM2, suggesting that the cells accumulated in G2
phase for a long time before succumbing to apoptosis (Suppl.
Fig. 2).
In addition, compounds 3–7 induced apoptotic cell death in PC-
3 cells which was associated with decreased expression of the anti-
apoptotic protein survivin (Suppl. Fig. 3A), an enhanced caspase-3
activity (Suppl. Fig. 3B), and cleavage of PARP (Poly (ADP-ribose)
Polymerase) (Suppl. Fig. 3A).
4. Discussion
Noscapine and its analogs, collectively referred to as the
noscapinoid family, typify a novel class of microtubule-modulating

120
R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
agents that evade the ‘harsher’ side-effects of currently available
tubulin-binding chemotherapeutics by preserving the total poly-
mer mass of tubulin [33]. This novel class of non-toxic microtu-
bule-modulating agents is based upon the parent molecule,
great potential for further preclinical evaluation of their therapeu-
tic efficacy in animals models. Based upon in silico cues and rational
drug-design, it is our ultimate goal to design, synthesize, and
develop a new class of novel ‘kinder and gentler’ noscapine analogs
that will target variant tubulins and thereby help reduce the
adverse side effects associated with their use. Owing to the
minimal toxicity of noscapine analogs, we envision that combina-
tion regimens of more toxic drugs with noscapinoids may present
an opportunity to reduce the dose-level of the toxic drugs to
improve disease-free survival and enhance the quality of life.
noscapine,
a relatively innocuous, non-sedative, isoquinoline
alkaloid from opium, known for its antitussive properties for
decades. Unlike the two major classes of tubulin-binding drugs,
which either overpolymerize and bundle microtubules (taxanes)
or depolymerize them and form paracrystals (vincas), noscapine
and its analogs do not exert gross affects on the microtubular
ultrastructure [33]. Thus, noscapine and its analogs do not impair
crucial microtubule functions and therefore cause minimal
toxicity, if any, and are best characterized as ‘kinder and gentler’
microtubule-modulating agents [24]. As the therapeutic efficacy is
based upon the potency and selectivity (non-toxicity to normal
cells) for clinical significance, noscapine derivatives can potentially
be exploited for therapeutic usage individually or in combination
with existing toxic anti-microtubule drugs.
Acknowledgements
This work was supported by grants to RA from the Department
of Defense and the National Cancer Institute at the National
Institutes of Health.
In silico molecular modeling efforts predicted the rational
design of novel second generation noscapine analogs substituted
at position-7 of the benzofuranone ring system, presented in this
study. Although each synthesized analog showed cytotoxicity
activity within a narrow range for most cell lines, significant inter
cell line variations were found to exist, in that a particular
compound exhibited differential sensitivity in cell lines from
varying tissue origin. These differences are not easy to compre-
hend, and can perhaps be attributable to the presence of varying
tubulin isotype expression and mutations in the tubulin gene in
different cell types [45–47]. It is also likely that altered expression
of survival and drug resistance mechanisms in cell lines from
different tissue types dictate cellular sensitivities [48].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bcp.2011.03.029.
References
[1] Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev
Cancer 2004;4:253–65.
[2] Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr
Med Chem Anticancer Agents 2005;5:65–71.
[3] Ye K, Ke Y, Keshava N, Shanks J, Kapp JA, Tekmal RR, et al. Opium alkaloid
noscapine is an antitumor agent that arrests metaphase and induces apoptosis
in dividing cells. Proc Natl Acad Sci USA 1998;95:1601–6.
[4] Checchi PM, Nettles JH, Zhou J, Snyder JP, Joshi HC. Microtubule-interacting
drugs for cancer treatment. Trends Pharmacol Sci 2003;24:361–5.
[5] Ke Y, Ye K, Grossniklaus HE, Archer DR, Joshi HC, Kapp JA. Noscapine inhibits
tumor growth with little toxicity to normal tissues or inhibition of immune
responses. Cancer Immunol Immunother 2000;49:217–25.
Successful chemotherapy relies on the strategic induction of
robust apoptosis in cancer cells while sparing normal cells. It is
noteworthy that these benzofuranone noscapine analogs did not
affect the viability of normal human fibroblasts at concentrations
[6] Sung B, Ahn KS, Aggarwal BB. Noscapine,
a benzylisoquinoline alkaloid,
as high as 100
mM. Essentially, chemotherapeutic agents induce
sensitizes leukemic cells to chemotherapeutic agents and cytokines by mod-
ulating the NF-kappaB signaling pathway. Cancer Res 2010;70:3259–68.
[7] Landen JW, Lang R, McMahon SJ, Rusan NM, Yvon AM, Adams AW, et al.
Noscapine alters microtubule dynamics in living cells and inhibits the pro-
gression of melanoma. Cancer Res 2002;62:4109–14.
[8] Zhou J, Gupta K, Yao J, Ye K, Panda D, Giannakakou P, et al. Paclitaxel-resistant
human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated
apoptosis in response to noscapine. J Biol Chem 2002;277:39777–85.
[9] Landen JW, Hau V, Wang M, Davis T, Ciliax B, Wainer BH, et al. Noscapine
crosses the blood-brain barrier and inhibits glioblastoma growth. Clin Cancer
Res 2004;10:5187–201.
[10] Aneja R, Lopus M, Zhou J, Vangapandu SN, Ghaleb A, Yao J, et al. Rational design
of the microtubule-targeting anti-breast cancer drug EM015. Cancer Res
2006;66:3782–91.
[11] Jackson T, Chougule MB, Ichite N, Patlolla RR, Singh M. Antitumor activity of
noscapine in human non-small cell lung cancer xenograft model. Cancer
Chemother Pharmacol 2008;63:117–26.
[12] Aneja R, Ghaleb AM, Zhou J, Yang VW, Joshi HC. p53 and p21 determine the
sensitivity of noscapine-induced apoptosis in colon cancer cells. Cancer Res
2007;67:3862–70.
[13] Aneja R, Vangapandu SN, Lopus M, Chandra R, Panda D, Joshi HC. Development
of a novel nitro-derivative of noscapine for the potential treatment of drug-
resistant ovarian cancer and T-cell lymphoma. Mol Pharmacol 2006;69:1801–
9.
[14] Aneja R, Zhou J, Zhou B, Chandra R, Joshi HC. Treatment of hormone-refractory
breast cancer: apoptosis and regression of human tumors implanted in mice.
Mol Cancer Ther 2006;5:2366–77.
[15] Karna P, Sharp SM, Yates C, Prakash S, Aneja R. EM011 activates a survivin-
dependent apoptotic program in human non-small cell lung cancer cells. Mol
Cancer 2009;8:93.
cell death by arresting cell cycle progression, upregulating the
expression of pro-apoptotic molecules while downregulating
survival signaling players that encumber apoptosis. The rate and
extent to which cell lines from various tissue types respond to a
particular test compound essentially depends on the status of
death-resisting anti-apoptosis molecules, as well as death-favoring
pro-apoptotic molecules in that cell type and how these molecules
are affected upon drug administration. For example, survivin, an
antiapoptotic protein of the inhibitor of apoptosis family that
blocks apoptosis by inhibiting caspases [49] has been shown to be a
player in dictating cellular sensitivity to 9-bromonoscapine [15].
Our data show that these compounds were able to alter survivin
levels as part of their anti-proliferative and pro-apoptotic program.
Examining the expression levels of survivin upon treatment with
these compounds revealed a decline in survivin, a property shared
in common with 9-bromonoscapine [15]. Even though a definite
relationship of the sensitivity of various cancer cells to these
analogs is cell-type dependent, it is apparent that tubulin presents
a potential target for these compounds. In addition, these second
generation benzofuranone ring substituted noscapine analogs
have been shown to spare the normal human dermal fibroblasts up
to a concentration as high as 100
mM. These data perhaps
demonstrate that the 7-position benzofuranone analogs may
prove to be significantly better than the founding compound,
noscapine.
[16] Aneja R, Miyagi T, Karna P, Ezell T, Shukla D, Vij Gupta M, et al. A novel
microtubule-modulating agent induces mitochondrially driven caspase-de-
pendent apoptosis via mitotic checkpoint activation in human prostate cancer
cells. Eur J Cancer 2010;46:1668–78.
In summary, we have provided the simplest methods for the
direct, and regioselective chemical manipulation of noscapine at
position-7 that yielded the benzofuranone analogs in high
quantitative yields. Taken together, like noscapine and its first
generation analogs, these second generation analogs indicate a
[17] Aneja R, Asress S, Dhiman N, Awasthi A, Rida PC, Arora SK, et al. Non-toxic
melanoma therapy by
2010;126:256–65.
a novel tubulin-binding agent. Int J Cancer
[18] Aneja R, Dhiman N, Idnani J, Awasthi A, Arora SK, Chandra R, et al. Preclinical
pharmacokinetics and bioavailability of noscapine, a tubulin-binding antican-
cer agent. Cancer Chemother Pharmacol 2007;60:831–9.

R.C. Mishra et al. / Biochemical Pharmacology 82 (2011) 110–121
121
[19] Aneja R, Vangapandu SN, Lopus M, Viswesarappa VG, Dhiman N, Verma A,
et al. Synthesis of microtubule-interfering halogenated noscapine analogs that
perturb mitosis in cancer cells followed by cell death. Biochem Pharmacol
2006;72:415–26.
[20] Aneja R, Vangapandu SN, Joshi HC. Synthesis and biological evaluation of a
cyclic ether fluorinated noscapine analog. Bioorg Med Chem 2006;14:8352–8.
[21] Zhou J, Gupta K, Aggarwal S, Aneja R, Chandra R, Panda D, et al. Brominated
derivatives of noscapine are potent microtubule-interfering agents that per-
turb mitosis and inhibit cell proliferation. Mol Pharmacol 2003;63:799–807.
[22] Aneja R, Zhou J, Vangapandu SN, Zhou B, Chandra R, Joshi HC. Drug-resistant T-
lymphoid tumors undergo apoptosis selectively in response to an antimicro-
tubule agent, EM011. Blood 2006;107:2486–92.
[23] Aneja R, Liu M, Yates C, Gao J, Dong X, Zhou B, et al. Multidrug resistance-
associated protein-overexpressing teniposide-resistant human lymphomas
undergo apoptosis by a tubulin-binding agent. Cancer Res 2008;68:1495–503.
[24] Heidemann S. Microtubules leukemia, and cough syrup. Blood
2006;107:2216a–7.
[25] Anderson JT, Ting AE, Boozer S, Brunden KR, Crumrine C, Danzig J, et al.
Identification of novel and improved antimitotic agents derived from nosca-
pine. J Med Chem 2005;48:7096–8.
multipolarity via centrosome amplification and declustering. Cell Death Differ
2011;18:632–44.
[34] Warren GL, Andrews CW, Capelli AM, Clarke B, LaLonde J, Lambert MH, et al. A
critical assessment of docking programs and scoring functions. J Med Chem
2006;49:5912–31.
[35] Ray S, Chaturvedi D. Application of organic carbamates in drug design. Part 1:
anticancer agents—recent reports. Drugs of the Future 2004;29.
[36] Efferth T, Sauerbrey A, Olbrich A, Gebhart E, Rauch P, Weber HO, et al.
Molecular modes of action of artesunate in tumor cell lines. Mol Pharmacol
2003;64:382–94.
[37] Anfosso L, Efferth T, Albini A, Pfeffer U. Microarray expression profiles of
angiogenesis-related genes predict tumor cell response to artemisinins. Phar-
macogenomics J 2006;6:269–78.
[38] Perez EA. Paclitaxel in Breast Cancer. Oncologist 1998;3:373–89.
[39] Katsumata N. Docetaxel: an alternative taxane in ovarian cancer. Br J Cancer
2003;89(Suppl. 3):S9–15.
[40] Crown J, O’Leary M, Ooi WS. Docetaxel and paclitaxel in the treatment of breast
cancer: a review of clinical experience. Oncologist 2004;9(Suppl. 2):24–32.
[41] Johnson IS, Armstrong JG, Gorman M, Burnett Jr JP. The Vinca alkaloids: a new
class of oncolytic agents. Cancer Res 1963;23:1390–427.
[26] Anderson JT, TingAE, Boozer S, Brunden KR, DanzigJ, Dent T, et al. Discovery of S-
phase arresting agents derived from noscapine. J Med Chem 2005;48:2756–8.
[27] Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, et al. Insight into
tubulin regulation from a complex with colchicine and a stathmin-like do-
main. Nature 2004;428:198–202.
[28] Dixon SL, Smondyrev AM, Knoll EH, Rao SN, Shaw DE, Friesner RA. PHASE a
new engine for pharmacophore perception, 3D QSAR model development, and
3D database screening: 1. Methodology and preliminary results. J Comput
Aided Mol Des 2006;20:647–71.
[29] Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al.
UCSF Chimera—a visualization system for exploratory research and analysis. J
Comput Chem 2004;25:1605–12.
[30] Bergenheim AT, Henriksson R. Pharmacokinetics and pharmacodynamics of
estramustine phosphate. Clin Pharmacokinet 1998;34:163–72.
[31] Miller HP, Wilson L. Preparation of microtubule protein and purified tubulin
from bovine brain by cycles of assembly and disassembly and phosphocellu-
lose chromatography. Methods Cell Biol 2010;95:3–15.
[32] Gaskin F, Cantor CR, Shelanski ML. Turbidimetric studies of the in vitro assembly
and disassembly of porcine neurotubules. J Mol Biol 1974;89:737–55.
[33] Karna P, Rida PC, Pannu V, Gupta KK, Dalton WB, Joshi H, et al. A novel
microtubule-modulating noscapinoid triggers apoptosis by inducing spindle
[42] Burkhart CA, Kavallaris M, Band Horwitz S. The role of beta-tubulin isotypes in
resistance to antimitotic drugs. Biochim Biophys Acta 2001;1471:O1–9.
[43] Cabral F, Abraham I, Gottesman MM. Revertants of a Chinese hamster ovary
cell mutant with an altered beta-tubulin: evidence that the altered tubulin
confers both colcemid resistance and temperature sensitivity on the cell. Mol
Cell Biol 1982;2:720–9.
[44] Liu B, Staren ED, Iwamura T, Appert HE, Howard JM. Mechanisms of taxotere-
related drug resistance in pancreatic carcinoma. J Surg Res 2001;99:179–86.
[45] Cowan NJ, Dudley L. Tubulin isotypes and the multigene tubulin families. Int
Rev Cytol 1983;85:147–73.
[46] Kavallaris M, Kuo DY, Burkhart CA, Regl DL, Norris MD, Haber M, et al. Taxol-
resistant epithelial ovarian tumors are associated with altered expression of
specific beta-tubulin isotypes. J Clin Invest 1997;100:1282–93.
[47] Huzil JT, Chen K, Kurgan L, Tuszynski JA. The roles of beta-tubulin mutations
and isotype expression in acquired drug resistance. Cancer Inform
2007;3:159–81.
[48] Kavallaris M, Tait AS, Walsh BJ, He L, Horwitz SB, Norris MD, et al. Multiple
microtubule alterations are associated with Vinca alkaloid resistance in
human leukemia cells. Cancer Res 2001;61:5803–9.
[49] Cheung CH, Cheng L, Chang KY, Chen HH, Chang JY. Investigations of survivin:
the past, present and future. Front Biosci 2011;16:952–61.