Synthesis and biological evaluation of novel biaryl type α-noscapine congeners

Article abstract of DOI:10.1016/j.bmcl.2014.10.046

Natural α-noscapine, a known antitussive drug, is also now known to possess weak anticancer efficacy with relatively safe toxicity profile. In this study, we report synthesis and evaluation of novel biaryl type α-noscapine congeners designed by adding aryl unit to the tetrahydroisoquinoline part of natural α-noscapine core. Palladium catalyzed Suzuki cross coupling of 9-bromo α-noscapine with aryl boronic acids was employed using mild and inexpensive reagents to attain desired noscapinoids 5a-g in excellent yields. Screening anti-proliferative activity for new noscapinoids 5b-g, on human cancer cell lines resulted three compounds 5b, 5d and 5f as potent analogues, active against human breast epithelial (MCF-7), human cervix cancer (HeLa) and human lung adenocarcinoma epithelial (A549) cell lines.

Full text of DOI:10.1016/j.bmcl.2014.10.046

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5752–5757
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel biaryl type a-noscapine
congeners
d
a,b,
Naresh K. Manchukonda a, Pradeep K. Naik c, , Balasubramanian Sridhar , Srinivas Kantevari
⇑
⇑
a Organic Chemistry Division-II (CPC Division), CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India
b Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India
c Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, A Central University, Koni, Bilaspur 495 009, Chhattisgarh, India
d Laboratory of X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural
a-noscapine, a known antitussive drug, is also now known to possess weak anticancer efficacy
Received 4 August 2014
Revised 10 October 2014
Accepted 15 October 2014
Available online 22 October 2014
with relatively safe toxicity profile. In this study, we report synthesis and evaluation of novel biaryl type
a
a
-noscapine congeners designed by adding aryl unit to the tetrahydroisoquinoline part of natural
-noscapine core. Palladium catalyzed Suzuki cross coupling of 9-bromo -noscapine with aryl boronic
a
acids was employed using mild and inexpensive reagents to attain desired noscapinoids 5a–g in excellent
yields. Screening anti-proliferative activity for new noscapinoids 5b–g, on human cancer cell lines
resulted three compounds 5b, 5d and 5f as potent analogues, active against human breast epithelial
(MCF-7), human cervix cancer (HeLa) and human lung adenocarcinoma epithelial (A549) cell lines.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Noscapine
Suzuki reaction
Tubulin
Isoquinoline
Alkaloid
Noscapine, an opium alkaloid, non-narcotic, orally available,
safe antitussive agent1 was discovered as a tubulin-binding anti-
cancer agent that arrests cancer cells in mitosis and induce apop-
higher anticancer activity than natural
icant toxicity. Among them, 9-bromo
a
a
-noscapine without signif-
-noscapine 2c (EM011) is
recently introduced into clinical trials against non-Hodgkin’s
tosis.2 Unlike the other tubulin-targeting agents,
a-noscapine and
lymphoma and chronic lymphocytic leukemia.9 Similarly, 9-nitro
its derivatives do not alter the steady state monomer/polymer ratio
of tubulin.3 Rather they subtly attenuate microtubule dynamics
just enough to activate the mitotic checkpoints to stop the cell
cycle, particularly mitosis, leaving other processes dependent on
less dynamic microtubules relatively unperturbed.3 In addition,
noscapine has some other advantage properties as lead molecule:
(1) retains activity against paclitaxel-resistant cell lines (1A9/
PTX10, 1A9/PTX22)4 and epothilone-resistant cell line (1A9/A8);
(2) a favorable pharmacokinetics (clearance in 6–10 h);5 (3) a poor
substrate for drug-pumps (polyglycoprotein and MDR-related pro-
teins)6 that comprise a major cause of drug resistance; (4) free
from immunological and neurological toxicities.7
a
-noscapine 2e was active against T-cell lymphoma cells and
drug-resistant ovarian cancer.10 More recently our group synthe-
sized 9-amino
-noscapine (2g, Fig. 1)11 and was found to possess
improved tubulin binding activity.12 In continuation of our efforts13
to develop new congeners of -noscapine for improving the anti-
a
a
cancer activity profile, in this manuscript we describe synthesis of
a series of 9-aryl noscapinoids 5a–g using palladium catalyzed
Suzuki cross-coupling reaction conditions. All the new noscapi-
noids were screened for anti-proliferative activity against human
breast epithelial (MCF-7), human cervix cancer (HeLa) and human
lung adenocarcinoma epithelial (A549) cell lines.
It is known from the literature that natural
a-noscapine share
To enhance anticancer activity of noscapine, further efforts were
made to add various functional substituents on noscapine core. For
instance, 9-substituted analogues (9-bromo, 9-chloro, 9-iodo,
9-nitro, 9-iodo, 9-azido, 9-amino analogues, Fig. 1)8 exhibited
binding characteristics similar to Colchicine (I), having biaryl con-
figuration.14 However, due to toxic side effects, use of colchicine as
anticancer agent is limited.15 Later reports identified other natural
products II–VI (Fig. 2) with biaryl (red color) architecture as effec-
tive antimitotic agents affecting the tubulin-microtubule equilib-
rium.16 Jain et al.17 explored this phenomenon and developed
nitrovinyl biphenyl analogs as anti-mitotic agents and has shown
to inhibit tubulin polymerization. Inspired by this we rationalize
⇑
Corresponding authors. Tel.: +91 4027191437; fax: +91 4027198933 (S.K.); tel.:
+91 7752 260405, mobile: +91 09479268802 (P.K.N.).
E-mail addresses: pknaik1973@gmail.com (P.K. Naik), kantevari@yahoo.com,
kantevari@gmail.com (S. Kantevari).
to design novel biaryl type
a-noscapine congeners by hybridizing
http://dx.doi.org/10.1016/j.bmcl.2014.10.046
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

N. K. Manchukonda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5752–5757
5753
R
O
R
O
O
1: R =H
2a: R =F
3a: R =H
3b: R =F
3c: R =Cl
3d: R =Br
3e: R=I
N
H
N
H
O
CH3
2b: R =Cl
2c: R-Br
CH3
H
H
H3CO
H3CO
2d: R =I
O
O
2e: R =N3
2f: R =NO2
2g: R=NH2
OCH3
OCH3
O
H3CO
H3CO
Figure 1. Natural
a-noscapine and its congeners as potential chemotherapeutic cancer agents.
O
H3CO
O
O
CH3
N
CH3
CH3
H3CO
H3CO
CH3
H3CO
H3CO
N
H
HO
HO
OCH3
O
OCH3
I: Colchicine
O
O
III = Buflavin
II: Eupomatilone-6
H
O
O
H
H
R
O
RO
H
O
H3CO
H3CO
H3CO
O
OCH3
H3CO
OCH3
OCH3
H3CO
V: Schisanlignone-A, R = CH3
VI: Schisanlignone-B, R = H
IV: Steganacin: R = OCOCH3
Figure 2. Cytotoxic natural lignans with biaryl pharmacophore.
biaryl ring architecture in to the natural
evaluate them as anticancer agents. The design strategy adopted
a-noscapine skeleton to
B(OH)2
B(OH)2
B(OH)2
is depicted in Figure 3.
O
a-Noscapine 1 is structurally consisting of two major constitu-
4c
ents (isoquinoline and phthalide ring systems) connected with
sensitive C–C bond which is labile to strong acids and bases. There-
fore synthesis of noscapine analogues is always challenging.18 In
the present work we have optimized the reaction conditions for
Cl OEt
4b
4a
EtO
O
B(OH)2
B(OH)2
B(OH)2
B(OH)2
O
the synthesis of 9-aryl-
tive C–C bond. The starting material 9-bromo noscapine (2c)
required was synthesized from natural -noscapine 1 in excellent
a-noscapinods without affecting the sensi-
N
H
CH3
N
CF3
a
4d
4g
4e
4f
yield (90%) using bromine water in 48% aqueous HBr by modifying
the reaction conditions described in literature.8 It was fully charac-
terized by 1H & 13C NMR and mass (ESI & HRMS) spectral analysis
and is in agreement with literature.8b
Figure 4. Aryl boronic acids 4a–g used in the present study.
After synthesizing 9-bromo noscapine 2c in good yield, we next
examined palladium catalyzed Suzuki aryl coupling reaction with
various aryl boronic acids 4a–g (Fig. 4).
Initially, 9-bromo noscapine 2c was treated with phenyl boronic
acid 4a, 3.0 mol % Pd(PPh3)4, and potassium carbonate (2 equiv) as
base refluxing in toluene similar to the general Suzuki reaction con-
ditions reported in the literature19 (entry 1, Table 1). Disappoint-
ingly, no reaction took place and majority of bromo compound 2c
was recovered. Furthermore, changing the mole ratio of Pd(PPh3)4,
potassium carbonate and solvent to dimethoxy ethane (DME) did
not give any fruitful reaction (entries 2–4, Table 1). Even change
of base to cesium carbonate and reaction medium to DMF did not
help the progression of reaction (entries 5–6, Table 1). Further
taking the clues from the general Suzuki aryl cross coupling
reactions containing heterocyclic moieties, we next examined the
reaction in mixed solvent system ethanol:toluene (1:1, v/v) as reac-
tion medium at elevated temperatures. To our success, after series
of experiments, 9-bromo noscapine 2c was reacted with phenyl
boronic acid 4a, in the presence of 10 mol % of Pd(PPh3)4 and potas-
sium carbonate (2 equiv) ethanol:toluene (1:1, v/v) at 120 °C for
Variable substituent to tune
R
anticancer activity
Biaryl pharmacophore
O
N
O
CH3
H
Noscapine core
H
O
H3CO
OCH3
O
H3CO
Figure 3. Design strategy for new biaryl type a-noscapine congeners.

5754
N. K. Manchukonda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5752–5757
Table 1
Optimization of Suzuki reaction conditions
Br
O
O
O
O
N
H
B (OH)2
O
N
H
CH3
O
CH3
Catalyst
H
O
N
H
+
H3CO
H
+
CH3
H3CO
H
O
O
H3CO
OCH3
OCH3
O
O
H3CO
OCH3
H3CO
O
2c
H3CO
9-Phenylnoscapine (5a)
Noscapine (1)
α−
Entry
Catalyst
(mol %)
Solvent
Base (equiv)
Reaction time (h)
Temp (°C)
Products (%)a
5a
1
1
2
3
4
5
6
7
8
Pd(TPP)4
Pd(TPP)4
Pd(TPP)4
Pd(TPP)4
Pd(TPP)4
Pd(TPP)2Cl2
Pd(TPP)2Cl2
Pd(TPP)2Cl2
Pd(TPP)4
Pd(TPP)4
Pd(TPP)4
Pd(TPP)4
Pd(TPP)4
3
3
5
Toulene
DME
DME
DME
DME
DMF
DMF
DMF
EtOH/Toluene (1:1)
EtOH/Toluene (1:1)
EtOH/Toluene (1:1)
EtOH/Toluene (1:1)
EtOH/Toluene (1:1)
K2CO3 (2)
K2CO3 (2)
K2CO3 (2)
K2CO3 (3)
CsCO3 (2.0)
CsCO3 (2.0)
K2CO3 (3.0)
K2CO3 (3.0)
K2CO3 (2.0)
K2CO3 (2.0)
NaHCO3 (2.0)
NaHCO3 (2.0)
NaHCO3 (2.0)
12
12
24
24
12
12
12
24
24
48
24
48
48
110
100
100
100
120
130
130
130
120
120
120
120
120
<1
20
27
35
3
5
5
8
10
10
10
10
15
10
10
10
10
12
Sluggish reaction
Sluggish reaction
30
45
62
65
60
75
80
19
22
15
15
—
2
2
9
10
11
12
13
a
Isolated yields.
24 h to give 9-phenyl noscapine 5a in 62% yield. At this stage we
also observed formation of significant quantities (15%) of noscapine
1 through debromination of 2c (entry 9, Table 1). The reaction was
report from Porcu et al.21 demonstrated synthesis of 9-aryl nosca-
pines using palladium catalyzed Suzuki cross coupling reaction
using ‘Xphos’. In contrast, without using expensive ligand ‘Xphos’
in the Suzuki cross coupling reaction, we were able to attain the
product 5a in higher yield (80%). Here we also controlled the side
reaction and significantly reduced the formation of noscapine 1.
Having optimized Suzuki aryl coupling reaction conditions, we
best progressed when bromo-a-noscapine 2c was reacted with 4a
in ethanol:toluene (1:1, v/v) in the presence of 12 mol % of
Pd(PPh3)4 and NaHCO3 (2.0 equiv) as base, at 120 °C for 48 h (entry
13, Table 1) to give product 5a in 80% yield.
Here we noticed significant reduction in the debromination and
only 2% of noscapine 1 was isolated. The crude reaction mixture
was purified over silica gel column chromatography to give pure
next employed the coupling of 9-bromo-a-noscapine (2c) with
various aryl boronic acids. All the aryl boronic acids 4b–g reacted
well with bromonoscapine 2c to give biaryl type noscapine hybrids
5b–g (Fig. 5) with excellent yields. All the compounds 5a–g
obtained were fully characterized by 1H, 13C NMR and mass (ESI
9-phenyl
a
-noscapine 5a and was fully characterized by IR, 1H &
13C NMR and Mass (ESI and HRMS) spectra analysis.20 Recent
Cl
O
O
OEt
OEt
O
O
O
O
N
H
O
O
CH3
N
H
O
CH3
N
H
H3CO
H
CH3
H
O
H3CO
H
O
H3CO
OCH3
O
OCH3
N
H3CO
5a
O
5b
OCH3
5c
H3CO
O
H3CO
CF3
H
N
CH3
O
O
O
O
O
O
O
O
O
N
H
N
N
CH3
N
CH3
CH3
H
CH3
H
H
O
H
H3CO
H
O
H
O
H
O
H3CO
H3CO
H3CO
OCH3
OCH3
O
OCH3
OCH3
O
H3CO
O
O
H3CO
H3CO
5f
H3CO
5e
5g
5d
Figure 5. Novel biaryl type noscapine congeners 5a–g synthesized.

N. K. Manchukonda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5752–5757
5755
Figure 6. ORTEP diagram of 5c, showing the atom-labeling scheme. Displacement ellipsoids are drawn at the 30% probability level and H atoms are represented by circles of
arbitrary radii.22
and HR-MS) spectroscopic analysis.20 Single crystal X-ray analysis
unambiguously confirmed the structure of ethyl 4-((R)-5-((S)-4,
Table 2
IC50 values of noscapine derivatives 5a–g for various cancer cell typesa
Noscapine & analogues
HeLa (
l
M)
A549 (
l
M)
MCF-7 (lM)
5-dimethoxy-3-oxo-1,3-dihydro isobenzofuran-1-yl)-4-methoxy-
6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquin olin-9-yl)
benzoate 5c (Fig. 6).22
1
24.0 2.9
ND
62.9 4.6
ND
42.3 2.7
ND
5a24
5b
5c
5d
5e
5f
9.0 1.5
21.2 3.7
9.2 1.4
20.3 2.5
8.9 1.7
22.8 2.8
33.8 3.5
53.1 3.7
32.6 2.5
42.7 2.9
31.6 2.6
57.3 3.9
18.8 2.7
40.7 3.3
17.8 2.5
34.3 2.5
16.6 2.9
41.3 2.4
After successful synthesis of noscapinoids 5a–g, we next exam-
ined whether these new analogues will affect cancer cell prolifera-
tion.23 As a preliminary screen, all compounds including the lead
molecule, noscapine were evaluated for their anti-proliferative
activity in three human cancer cell lines; human breast epithelial
cell (MCF-7), human cervix cancer cells (HeLa) and human lung
adenocarcinoma epithelial cells (A549). The IC50 values for the test
compounds 5b–g are included in Table 2.24 Our results showed
that all the cancer cell types were more susceptible to compounds
5g
a
Cancer cells used in the assay namely, HeLa: human cervix cell line, A549:
human lung adenocarcinoma epithelial cell line and MCF7: human breast epithelial
cell line. Each value represents mean S.D. from three different experiments. ND,
not determined.
Figure 7. Representative figures show morphological evaluation of nuclei stained with DAPI in the absence and presence of the analogues (25
features of apoptotic cells such as condensed chromosomes, numerous fragmented micronuclei, and apoptotic bodies are evident (indicated by white head arrows) upon 72 h
of drug treatment. (Scale bar = 15 m).
lM each). Several typical
l

5756
N. K. Manchukonda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5752–5757
Joshi, H. C. Biochem. Pharmacol. 2006, 72, 415; (c) Aneja, R.; Vangapandu, S. N.;
Joshi, H. C. Bioorg. Med. Chem. 2006, 14, 8352.
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Yang, V. W.; Joshi, H. C. Int. J. Cancer 2012, 131, 1435.
10. Aneja, R.; Vangapandu, S. N.; Lopus, M.; Chandra, R.; Panda, D.; Joshi, H. C. Mol.
Pharmacol. 1801, 2006, 69.
11. (a) Manchukonda, N. K.; Sridhar, B.; Naik, P. K.; Joshi, H. C.; Kantevari, S. Bioorg.
Med. Chem. Lett. 2012, 22, 2983; (b) Naik, P. K.; Chatterji, B. P.; Vangapandu, S.
N.; Aneja, R.; Chandra, R.; Kanteveri, S.; Joshi, H. C. J. Comput. Aided Mol. Des.
2011, 25, 443.
12. Santoshi, S.; Naik, P. K. J. Comput. Aided Mol. Des. 2014, 28, 751.
13. Manchukonda, N. K.; Naik, P. K.; Santoshi, S.; Lopus, M.; Joseph, S.; Sridhar, B.;
Kantevari, S. PLoS ONE 2013, 21(8), e77970.
14. Naik, P. K.; Santoshi, S.; Rai, A.; Joshi, H. C. J. Mol. Graph. Model. 2011, 29, 947.
15. Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Med. Res. Rev. 2008, 28,
155.
16. Kumar, A. S.; Reddy, M. A.; Jain, N.; Kishor, C.; Murthy, T. R.; Ramesh, D.;
Supriya, B.; Addlagatta, A.; Kalivendi, S. V.; Sreedhar, B. Eur. J. Med. Chem. 2013,
60, 305.
5b–g in comparison to the lead compound, noscapine, with lower
IC50 value. Especially three compounds 5b, 5d and 5f possess
potent cytotoxic activity. The IC50 value amounted to 9.0
lM,
9.2 M and 8.9 M with 5b, 5d and 5f, respectively, for HeLa cells,
l
l
which reflects a pronounced anti-proliferative activity. Parentheti-
cally, it is worth mentioning that a similar low IC50 value of
18.8 lM, 17.8 lM and 16.6 lM was measured using 5b, 5d and
5f, respectively, for the MCF-7 cells. In contrast, modest anti-prolif-
erative activity for these compounds was noted against A549 cell
line. Thus this preliminary screen with the three chosen cell lines
revealed 5b, 5d and 5f as potent cytotoxic compounds compared
to noscapine. Besides the anti-proliferative effect, DAPI staining25
of the cells treated with noscapinoids showed condensed chroma-
tin along with numerous fragmented nuclei (shown by white
arrow heads), indicative of apoptotic cell death (Fig. 7).
In conclusion we have designed a series of novel biaryl type
noscapine analogues and synthesized using optimized Suzuki reac-
tion conditions in good yields. Evaluation of all the new derivatives
(5a–g) impacts their therapeutic potential for a variety of cancer
cell types, indicating a great potential for further preclinical and
clinical evaluation.
17. Jain, N.; Yada, D.; Shaik, T. B.; Vasantha, G.; Reddy, P. S.; Kalivendi, S. V.;
Sreedhar, B. ChemMedChem 2011, 6, 859.
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P. J. ChemMedChem 2012, 7, 2122; (b) DeBono, A. J.; Mistry, S. J.; Xie, J.;
Muthiah, D.; Phillips, J.; Ventura, S.; Callaghan, R.; Pouton, C. W.; Capuano, B.;
Scammells, P. J. ChemMedChem 2014, 9, 399.
19. (a) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457; (b) Torborg, C.; Beller, M.
Adv. Synth. Catal. 2009, 351, 3027.
20. General procedure for the preparation of 9-arylnoscapines 5a–g: To a solution of
9-bromonoscapine (200 mg, 0.41 mmol) in ethanol/toluene (1:1, v/v, 10 mL)
was added Pd(PPh3)4 (0.049 mmol), sodium bicarbonate (0.82 mmol) and 4a–g
(0.82 mmol), under nitrogen. Reaction mixture was heated at 120 °C for 48 h,
cooled to room temperature, solvents were removed under reduced pressure,
water (10 mL) was added, extracted with dichloromethane (3 Â 25 mL), and
combined organic portions were washed with water, dried over anhydrous
sodium sulphate and concentrated. Crude product was purified over silica gel
column chromatography eluted with 25% ethyl acetate in hexanes to give pure
compounds 5a–g as colorless solids.
Acknowledgments
Authors (N.K.M. and S.K.) are thankful to Dr. Lakshmi Kantham,
Director and Dr. V. J. Rao, Head, Crop Protection Chemicals Divi-
sion, IICT, Hyderabad for their continuous support and financial
assistance through ICMR – India project & CSIR-IICT-12th FYP
projects [ORIGIN, CSC 0108; DENOVA, CSC0205]. N.K.M. (SRF) is
thankful to CSIR for fellowship.
(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-methyl-9-phenyl-5,6,7,8-tetra
hydro-
[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one (5a): Yield: 80%;
25
m.p: 110 °C; [
a
]
À76 (c = 1, dichloromethane); 1H NMR (300 MHz, CDCl3):
D
d 7.48–7.20 (m, 5H), 7.02 (d, J = 8.3 Hz, 1H), 6.13 (d, J = 8.1 Hz, 1H), 6.00 (s, 1H),
5.92 (s, 1H), 5.56 (d, J = 3.7 Hz, 1H), 4.51 (d, J = 3.7 Hz, 1H), 4.13 (s, 6H), 3.90 (s,
3H), 2.56 (s, 3H), 2.30–2.09 (m, 2H), 1.78–1.55 (m, 2H); 13C NMR (75 MHz,
CDCl3): d 168.0, 152.2, 147.6, 145.9, 140.8, 139.5, 134.1, 133.7, 130.7, 129.8,
128.1,127.3, 120.4, 117.7, 116.4, 100.8, 81.9, 62.2, 61.0, 59.4, 56.8, 50.8, 46.7,
27.0; IR (KBr): 2942, 2900, 2791, 1751, 1605, 1497, 1437, 1380, 1310, 1270,
1034, 1006, 974, 791 cmÀ1; HRMS (ESI): m/z Calcd for C28H28NO7 (M+H)+,
490.18603; found: 490.18509.
Supplementary data
Supplementary data (complete experimental details, copies of
1H, 13C NMR and mass spectra (ESI and HR-MS) of synthesized
products 5a–g; single crystal X-ray diffraction data for compound
for 5c (CCDC 944746) can be found free of charge from the
Cambridge crystallographic data center via www.ccdc.cam.ac.uk/
data_ request/cif) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.046.
Ethyl 2-chloro-5-((R)-5-((S)-4,5-dimethoxy-3-oxo-1,3-dihydroiso benzofuran-1-
yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo
yl)benzoate (5b): Yield: 70%; m.p: 152 °C;
[4,5-g]isoquinolin-9-
À127.28 (c = 1,
25
[a
]
D
dichloromethane); 1H NMR (CDCl3, 300 MHz): d 7.64 (d, J = 2.0 Hz, 1H), 7.45
(d, J = 8.3 Hz, 1H), 7.34–7.27 (dd, J = 2.0, 8.1 Hz, 1H), 7.26 (s, 1H), 7.02 (d,
J = 8.1 Hz, 1H), 6.16–6.05 (d, J = 7.9 Hz, 1H), 6.00 (s, 1H), 5.92 (s, 1H), 5.45 (d,
J = 3.7 Hz, 1H), 4.49–4.34 (m, 3H), 4.10 (s, 6H), 3.91 (s, 3H), 2.72–2.53 (m, 4H),
2.26–2.09 (m, 2H), 1.66 (s, 1H), 1.42 (t, J = 7.1 Hz, 3H); 13C NMR (CDCl3,
75 MHz): 167.9, 165.6, 152.3, 147.6, 146.0, 140.8, 140.1, 133.8 133.7, 132.9,
132.5, 130.8, 130.5, 130.3, 120.4, 118.1, 117.7, 117.6, 114.1, 100.9, 81.8, 62.9,
61.6, 61.0, 59.5, 56.8, 50.5, 46.6, 26.9, 14.1; IR (KBr): 3418, 2922, 2853, 2795,
1756, 1633, 1597, 1498, 1363, 1274, 1159, 1036, 940, 813, 728, 506 cmÀ1; MS
(ESI): m/z 618 (M+Na)+; HRMS (ESI): m/z Calcd for C31H30NO9ClNa (M+Na)+,
618.1506; found: 618.1476.
References and notes
1. (a) Al-Yahya, M. A.; Hassan, M. M. A. Anal. Profiles Drug Subst. 1982, 11, 407; (b)
Karlsson, M. O.; Dahlström, B.; Eckernäs, S. A.; Johansson, M.; Alm, A. T. Eur. J.
Clin. Pharmacol. 1990, 39, 275.
2. (a) Ye, K.; Ke, Y.; Keshava, N.; Shanks, J.; Kapp, J. A.; Tekmal, R. R.; Petros, J.;
Joshi, H. C. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1601; (b) Tripathi, M.; Reddy, P.
L.; Rawat, D. S. Chem. Biol. Interface 2014, 4, 1.
3. (a) Jackson, T.; Chougule, M. B.; Ichite, N.; Patlolla, R. R.; Singh, M. Cancer
Chemother. Pharmacol. 2008, 63, 117; (b) Karna, P.; Rida, P. C. G.; Pannu, V.;
Gupta, K. K.; Dalton, W. B.; Joshi, H.; Yang, V. W.; Zhou, J.; Aneja, R. Cell Death
Differ. 2011, 18, 632; (c) Mishra, R. C.; Karna, P.; Gundala, S. R.; Pnnu, V.;
Stanton, R. A.; Guptha, K. K.; Robinson, M. H.; Lopus, M.; Wilson, L.; Henry, M.;
Aneja, R. Biochem. Pharmacol. 2011, 82, 110; (d) Aneja, R.; Miyagi, T.; Karna, P.;
Ezell, T.; Shukla, D.; Gupta, M. V.; Yates, C.; Chinni, S. R.; Zhau, H.; Chung, L. W.
K.; Joshi, H. C. Eur. J. Cancer 2010, 46, 1668; (e) Joshi, H. C.; Salil, A.; Bughani, U.;
Naik, P. K. Medicinal Plant Biotechnology, Arora, R., Ed., CAB e-Books, CABI, 2010;
(H ISBN 9781845936785).; (f) Mahmoudian, M.; Rahimi-Moghaddam, P. Recent
Pat. Anticancer Drug Discov. 2009, 4, 92.
4. (a) Zhou, J.; Gupta, K.; Yao, J.; Ye, K.; Panda, D.; Giannakakou, P.; Joshi, H. C. J.
Biol. Chem. 2002, 277, 39777; (b) Singh, H.; Singh, P.; Kumari, K.; Chandra, A.;
Dass, S. K.; Chandra, R. Curr. Drug Metab. 2013, 14, 351.
5. Dahlstrom, B.; Mellstrand, T.; Lofdahl, C. G.; Johansson, M. Eur. J. Clin.
Pharmacol. 1982, 22, 535.
6. Zhou, J.;Liu, M.;Aneja, R.;Chandra,R.;Lage,H.;Joshi,H.C.CancerRes.2006,66,445.
7. (a) Aneja, R.; Dhiman, N.; Idnani, J.; Awasthi, A.; Arora, S. K.; Chandra, R.; Joshi,
H. C. Cancer Chemother. Pharmacol. 2007, 60, 831; (b) Aneja, R.; Asress, S.;
Dhiman, N.; Awasthi, A.; Rida, P. C.; Arora, S. K.; Zhou, J.; Glass, J. D.; Joshi, H. C.
Int. J. Cancer 2010, 126, 256.
8. (a) Verma, A. K.; Bansal, S.; Singh, J.; Tiwari, R. K.; Sankar, K. V.; Tandon, V.;
Chandra, R. Bioorg. Med. Chem. 2006, 14, 6733; (b) Aneja, R.; Vangapandu, S. N.;
Lopus, M.; Viswesarappa, V. G.; Dhiman, N.; Verma, A.; Chandra, R.; Panda, D.;
Ethyl
4-((R)-5-((S)-4,5-dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-4-
methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-9-
yl)benzoate (5c): Yield: 67%; m.p: 143 °C;
25
[a
]
À140.52 (c = 1,
D
dichloromethane); 1H NMR (CDCl3, 300 MHz): d 8.09 (d, J = 8.4 Hz, 2H), 7.33
(d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.3 Hz, 1H), 6.16 (d, J = 8.1 Hz, 1H), 6.00 (d,
J = 1.3 Hz, 1H), 5.93 (d, J = 1.3 Hz, 1H), 5.54 (d, J = 4.1 Hz, 1H), 4.49 (d, J = 4.3 Hz,
1H), 4.40 (q, J = 7.1 Hz, 2H), 4.12 (s, 3H), 4.11 (s, 3H), 3.91 (s, 3H), 2.67–2.52 (m,
4H), 2.27–2.11 (m, 2H), 1.76–1.65 (m, 1H), 1.41 (t, J = 7.1 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d 167.9, 166.2, 152.3, 147.7, 146.0, 140.9, 140.0, 138.9, 133.7,
130.6, 130.0, 129.3, 120.5, 118.1, 117.7, 117.7, 115.4, 100.9, 81.8, 62.2, 61.1,
60.9, 59.5, 56.9, 56.7, 46.7, 27.0, 14.3; IR (KBr): 3413, 2915, 1767, 1697, 1616,
1498, 1464, 1443, 1381, 1306, 1262, 1165, 1088, 1034, 1012, 821, 621 cmÀ1
;
MS (ESI): m/z 584 (M+Na)+; HRMS (ESI): m/z Calcd for C31H31NO9Na (M+Na)+,
584.1896; found: 584.1881.
N-(3-((R)-5-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-4-
methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquin
olin-9-
À138.68 (c = 1,
25
yl)phenyl)acetamide (5d): Yield: 55%; m.p: 240 °C;
[
a
]
D
dichloromethane); 1H NMR (CDCl3, 300 MHz): d 7.68 (s, 1H), 7.46–7.28 (m,
2H), 7.25–7.12 (d, J = 8.3 Hz, 1H), 7.07–6.93 (d, J = 7 .2 Hz, 1H), 6.23–6.10 (d,
J = 8.3 Hz, 1H), 5.98 (s, 1H), 5.91 (s, 1H), 5.54 (d, J = 3.9 Hz, 1H), 4.50 (d,
J = 3.9 Hz, 1H), 4.10 (s, 6H), 3.94 (s, 3H), 2.54 (s, 4H), 2.27–2.10 (m, 5H), 1.77–
1.60 (m, 1H); 13C NMR (CDCl3, 75 MHz): d 168.4, 152.2, 147.3, 145.9, 140.6,
139.5, 138.2, 134.9, 133.6, 130.8, 128.5, 125.5, 121.3, 120.2, 118.3, 117.9, 117.6,

N. K. Manchukonda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5752–5757
5757
116.3, 100.8, 82.1, 62.1, 61.0, 59.4, 56.6, 56.4, 50.8, 46.7, 27.0, 24.4; IR (KBr):
3329, 2920, 2791, 1739, 1682, 1586, 1548, 1503, 1384, 1274, 1086, 1037,797,
620 cmÀ1; MS (ESI): m/z 569 (M+Na)+; HRMS (ESI): Calcd for C30H30N2O8Na
(M+Na)+, 569.1899; found: 569.1920.
atoms were located in difference Fourier maps and subsequently geometrically
optimized and allowed for as riding atoms, with C–H = 0.93–0.97 Å, with
U
iso(H) = 1.5Ueq(C) for methyl H or 1.2Ueq(C, N). The methyl groups were
allowed to rotate but not to tip. The absolute configuration of the procured
material was known in advance and was confirmed by unambiguous
refinement of the absolute structure parameter. In the absence of significant
anomalous scattering efforts, Friedel pairs were merged for 5c. Crystal data for
(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-methyl-9-(4-vinylphenyl)-5,6,7,8-
tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one
(5e):
25
Yield: 60%; m.p: 120 °C; [
a
]
D
À120.22 (c = 1, dichloromethane); 1H NMR
(CDCl3, 300 MHz): d 7.40 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 6.97 (d,
J = 8.1 Hz, 1H), 6.74–6.66 (dd, J = 10.8 Hz, 17.5 Hz, 1H) 6.10 (s, 1H), 5.98 (s, 1H),
5.91 (s, 1H), 5.74 (d, J = 17.5 Hz, 1H), 5.48 (s, 1H), 5.25 (d, J = 10.8 Hz, 1H), 4.47
(s, 1H), 4.10 (s, 6H), 3.90 (s, 3H), 2.66–2.54 (m, 4H), 2.27–2.13 (m, 2H), 1.77–
1.64 (m, 1H);13C NMR (75 MHz, CDCl3): d 157.9, 152.2, 147.7, 146.0, 143.6,
140.9, 139.6, 136.7, 133.7, 133.5, 130.7, 130.1, 126.0, 120.4, 117.8, 116.1, 114.2,
100.8, 81.9, 62.3, 61.1, 59.5, 56.9, 50.8, 46.6, 27.0, 23.2, 29.6 cmÀ1; MS (ESI): m/
z 538 (M+Na)+; HRMS (ESI): Calcd for C30H29NO7Na (M+Na)+, 538.1841; found:
538.1848.
5c:
C
31H31NO9, M = 561.57, colorless needle, 0.16 Â 0.08 Â 0.06 mm3,
orthorhombic, space group P212121 (No. 19), a = 11.0875(8), b = 15.3517(11),
c = 17.0221(12) Å, V = 2897.4(4) Å3, Z = 4, Dc = 1.287 g/cm3, F0 0 0 = 1184, CCD
Area Detector, MoK
27964 reflections collected, 2891 unique (Rint = 0.0196). Final GooF = 1.111,
R1 = 0.0402, wR2 = 0.1212, R indices based on 2737 reflections with I>2 (I)
(refinement on F2), 375 parameters, = 0.095 mmÀ1
restraints, CCDC
a radiation, k = 0.71073 Å, T = 294(2) K, 2hmax = 50.0°,
r
0
l
.
944746 contains supplementary Crystallographic data for the structure. (a)
Bruker (2001). SAINT (Version 6.28a) & SMART (Version 5.625) (b) Sheldrick G.
M. Acta Crystallogr. 2008, A64, 112. (c) Flack, K.; Bernardinelli, G. J. Appl. Cryst.
2000, i, 1143.
(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-methyl-9-(pyridin-3-yl)-5,6,7,8-
tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one
(5f):
25
Yield: 62%; mp: 193 °C; [
a
]
D
À124.25 (c = 1, dichloro methane); 1H NMR
23. In vitro cell proliferation assays (MTS assay): Cell culture reagents were obtained
from Sigma and Invitrogen. MCF-7, a human breast epithelial cancer cell line;
HeLa, a human cervix cell line and A549, a human lung cancer cell line were
obtained from the National Repository of Animal Cell Culture, National Centre
for Cell Sciences, Pune (NCCS), India. The cell lines were maintained in
Dulbecco’s Modification of Eagle’s Medium 1Â (DMEM) with 4.5 g/L glucose
(CDCl3, 500 MHz): d 8.52 (s, 1H), 8.43 (s, 1H) 7.56 (d, J = 7.6 Hz, 1H), 7.30 (t,
J = 6.6 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.12 (d, J = 7.6 Hz, 1H), 5.99 (s, 1H), 5.92
(s, 1H), 5.43 (d, J = 4.7 Hz, 1H), 4.43 (d, J = 4.7 Hz, 1H), 4.11 (s, 3H), 4.08 (s, 3H),
3.88 (s, 3H), 2.67–2.60 (m, 1H), 2.55 (s, 3H), 2.22–2.14 (m, 2H), 1.79–1.69 (m,
1H); 13C NMR (CDCl3, 75 MHz): 167.9, 152.3, 150.7, 148.36, 147.6, 146.4, 140.7,
140.2, 137.3, 133.7, 130.7, 130.3, 130.2, 123.1, 120.4, 118.2, 117.5, 100.9, 81.8,
62.2, 61.0, 59.4, 56.8, 50.6, 46.6, 26.8; IR (KBr): 3412, 2938, 1756, 1637, 1497,
1445, 1273, 1082, 1032, 943, 815, 714 cmÀ1; MS (ESI): m/z 513 (M+Na)+; HRMS
(ESI): Calcd for C27H26N2O7Na (M+Na)+, 513.1637; found: 513.1615.
and
L-glutamine (Sigma) supplemented with 10% fetal bovine serum
(Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Suspension cells
(MCF-7, HeLa and A549) were seeded into 96-well plates at a density of
5 Â 103 cells per well and were treated with increasing gradient concentrations
of noscapinoids, 5b–g for 72 h. Measurement of cell proliferation was
performed calorimetrically by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy
methoxyphenyl)-2-(4-sulpho phenyl)-2H-tetrazolium, inner salt (MTS) assay,
using the CellTiter 96 AQueous One Solution Reagent (Sigma). 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 was
then plotted to determine the IC50 value, which stands for the drug
concentration needed to prevent cell proliferation by 50%.
(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-methyl-9-(4-(trifluoromethyl)
phenyl)-
5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)iso benzofuran-1(3H)-one
25
(5g): Yield: 65%; mp: 135 °C; [
(CDCl3, 300 MHz)
a
]
D
À62.22 (c = 1, dichloromethane); 1H NMR
d
7.60–7.40 (m, 4H), 6.97 (d, J = 8.1 Hz, 1H), 6.05 (d,
J = 8.3 Hz, 1H), 6.02 (d, J = 1.3 Hz, 1H), 5.93 (d, J = 1.3 Hz, 1H), 5.44 (d,
J = 4.1 Hz, 1H), 4.46 (d, J = 4.3 Hz, 1H), 4.14 (s, 3H), 4.10 (s, 3H), 3.89 (s, 3H),
2.65–2.51 (m, 4H), 2.20–2.00 (m, 2H), 1.70–1.56 (m, 1H); 13C NMR (CDCl3,
75 MHz): d 167.9, 152.4, 147.5, 146.0, 140.5, 140.0, 134.9, 133.2, 128.7, 126.7,
126.6, 124.1, 120.5, 118.1, 117.2, 114.7, 100.9, 81.8, 62.2, 61.0, 59.5, 56.5, 50.7,
46.7, 29.6, 27.2; IR (KBr): 3413, 2948, 1765, 1614, 1497, 1422, 1237, 1158,
1120, 1039, 946, 806, 732, 701 cmÀ1; MS(ESI): m/z 580 (M+Na)+; HRMS (ESI):
Calcd for C29H26NO7F3Na (M+Na)+, 580.1559; found: 580.1568
24. Due to solubility issues, we could not report a conclusive anticancer activity for
5a.
25. DAPI staining: Nuclear morphology of cells treated with the noscapinoids was
evaluated by staining the cells with DAPI and imaging with fluorescence
microscopy. Briefly, MCF-7 cells (2 Â 103 cells) were grown on poly-L-lysine
coated cover slips in 6-well plates and were treated with the compounds at
21. Porcù, E.; Sipos, A.; Basso, G.; Hamel, E.; Bai, R.; Stempfer, V.; Udvardy, A.;
Bényei, A. C.; Schmidhammer, H.; Antus, S.; Viola, G. Eur. J. Med. Chem. 2014, 84,
476.
22. X-ray data for the compound 5c was collected at room temperature using a
25 lM for 72 h. After incubation, cover slips were fixed in cold methanol and
Bruker Smart Apex CCD diffractometer with graphite monochromated MoK
a
washed with PBS, stained with DAPI, and mounted on slides. Images were
captured using a BX60 fluorescence 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).
radiation (k = 0.71073 Å) with -scan method. Preliminary lattice parameters
x
and orientation matrices were obtained from four sets of frames. Integration
and scaling of intensity data were accomplished using SAINT program. The
structure was solved by direct methods using SHELXS97 and refinement was
carried out by full-matrix least-squares technique using SHELXL97. Anisotropic
displacement parameters were included for all non-hydrogen atoms. All H