Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues as potent cytotoxic and antimitotic agents

Article abstract of DOI:10.1016/j.bmc.2011.02.020

A series of novel conjugates of 4-aza-2,3-didehydropodophyllotoxins (11a-w) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated cytotoxicity against five human cancer cell lines (breast, oral, colon, lung and ovarian)

Full text of DOI:10.1016/j.bmc.2011.02.020

Bioorganic & Medicinal Chemistry 19 (2011) 2349–2358
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a new 4-aza-2,3-didehydropodophyllotoxin analogues
as potent cytotoxic and antimitotic agents
Ahmed Kamal a, , Paidakula Suresh a, Adla Mallareddy a, Banala Ashwini Kumar a, Papagari Venkat Reddy a,
⇑
Paidakula Raju a, Jaki R. Tamboli a, Thokhir B. Shaik b, Nishant Jain b, Shasi V. Kalivendi b,
⇑
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel conjugates of 4-aza-2,3-didehydropodophyllotoxins (11a–w) were synthesized by a
straightforward one-step multicomponent synthesis that demonstrated cytotoxicity against five human
cancer cell lines (breast, oral, colon, lung and ovarian). All the twenty three compounds (11a–w) have
been examined for the inhibition of tubulin polymerization. Among these compounds, 11a, 11k and
11p exhibited inhibition of polymerization tubulin comparable to podophyllotoxin apart from disruption
of microtubule organization within the cells. Flow cytometric analysis showed that these compounds
(11a, 11k and 11p) arrested the cell cycle in the G2/M phase of cell cycle leading to caspase-3 dependent
apoptotic cell death.
Received 22 December 2010
Revised 10 February 2011
Accepted 11 February 2011
Available online 24 February 2011
Keywords:
Multicomponent synthesis
Podophyllotoxin
Ó 2011 Elsevier Ltd. All rights reserved.
Anticancer activity
Tubulin polymerization activity
G2/M Cell cycle arrest
Immunohistochemistry
Caspase-3 activation
1. Introduction
topo-II,11 and induce apoptotic cell death through independent
mechanism which contributes to its cytotoxicity. This compound
Podophyllum peltatum L, also known as the American Mayapple,
is the source of the natural product podophyllotoxin (1).1 It has
cytotoxic properties and is known to inhibit polymerization inhibi-
tion. The biological activity2 of 1 has led to extensive structural
modifications resulting in several clinically useful compounds like
etoposide (2), teniposide (3) and the water-soluble prodrug, etopo-
phos (4).3–5 These compounds have shown to inhibit DNA topoiso-
merase-II (topo-II) by stabilizing the covalent topo-II DNA
cleavable complex6 and are used against a variety of cancers,
including germ-cell malignancies, small-cell lung cancer, non-
Hodgkin’s lymphoma, leukemia, kaposi’s sarcoma, neuroblastoma,
and soft tissue sarcoma.7–9 They have several limitations, such as
poor water solubility, development of drug resistance, metabolic
inactivation, and toxic effects.10 To overcome such problems,
extensive synthetic efforts have been carried out by a number of
researchers. This has led to the development of NK-611 (5), and
GL-331 (6). GL-331 contains a p-nitroanilino moiety at the 4b-
position instead of a glycoside of etoposide, which proved to be
more potent than etoposide. GL-331, is also an inhibitor of
has underwent phase II clinical trials for the treatment of various
cancers,12 however this did not proceed further. Recently, we have
been involved in the development of new synthetic procedures13
for the podophyllotoxin-based compounds and also in the design
and synthesis of new analogs of podophyllotoxin as potential
anticancer agents.14
Application of multicomponent reactions (MCRs) to the con-
struction of natural product-based libraries would be most benefi-
cial to this area of research. Such processes in which three or more
reactants are combined together in a single reaction flask to pro-
duce a product incorporating most of the atoms contained in the
starting materials have the advantages of the intrinsic atom econ-
omy, simpler procedures and equipment, time and energy savings,
as well as environmental friendliness.15–20 The structural complex-
ity of podophyllotoxin 1, arising from the presence of four stereo-
genic carbons in ring C (Fig 1). Most of the structure-activity
relationship studies have been performed by derivatization of par-
ent natural product rather than by de novo chemical synthesis.21 In
this connection, Itokawa and Takeya made an important contribu-
tion for the synthesis of 4-aza-podophyllotoxin (7a) and (7b)
derivatives via three steps of condensation, cyclization, and reduc-
tion. Cytotoxicity of these compounds is comparable to that of pod-
ophyllotoxin.22 The removal of the stereo centers at C-2 and C-3
solves the problem of epimerization at C-2 that has plagued the
⇑
Corresponding authors. Tel.: +91 40 27193157; fax: +91 40 27193189 (A.K.);
tel.: +91 04 27191814 (S.V.K.).
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), kalivendi@iict.res.in (S.V.
Kalivendi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.020

2350
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
H
R1
R2
R3
H
O
R1
OH
O
5
O
A
4
(2)
O
HO
Etoposide
R2
CH3
OH
3
B
D
O
C
2
1
O
O
O
Teniposide
3
( )
O
OH
H
O
S
E
O
P
O
OH Etopophos (4)
OH
H3CO
OCH3
OH
CH3
OCH3
1
( )
H3CO
OCH3
NK-611 (5)
H
OR3
NMe2
NMe2
NO2
H
RO
RO
N
HN
O
O
O
O
O
O
H3CO
OCH3
OCH3
H3CO
OCH3
OH
R = CH3 (7a)
7b
R = -CH2- (
)
6
GL 331 ( )
Figure 1.
clinical development of podophyllotoxin and its stereochemical
complex derivatives, because the rapidly formed in vivo cis-lactone
metabolite is significantly less potent.23 Tu et al. reported a multi-
component reaction leading to the formation of N-substituted
dihydropyridines, although no biological data for these compounds
were provided.24 Recently, Magedov et al. reported a multicompo-
nent reaction leading to the formation of didehydropyridopyrazole
analogues.25
In continuation of these efforts and our interest in the structural
modifications of the podophyllotoxin lignan, we would like to
report herein efficient access to the construction of a new novel
4-aza-2,3-didehydropodophyllotoxin derivatives with improved
cytotoxicity and inhibition of tubulin polymerization.
concentration of the compound that produced 50% inhibition of
cell growth (GI50) ranging from <0.1 to 2.9 M. It appears from
the data that 2-fluoro-4-methoxy substitutions in the 4-aryl pen-
dent ring enhances the cytotoxicity as seen in compounds 11l
and 11t for the breast cancer cell line (MCF-7). The presence of flu-
oro substituent adds the 4-position in the pendent aryl ring also
l
H
O
N
NH2
EtOH, reflux
1 h.
O
O
R
CHO
R
O
O
8
10
R1
R1
11
9
2. Chemistry
11a: R = 2,4 dimethoxy 5-pyrimidyl, R1 = 3,4,5-trimethoxy,
11b: R = 2,4 dimethoxy 5-pyrimidyl, R1 = 4-hydroxy-3-methoxy,
11c: R = 2,4 dimethoxy 5-pyrimidyl, R1 = 3-hydroxy-4-methoxy,
11d: R = 2,4 dimethoxy 5-pyrimidyl, R1 = 4-fluoro-3-methoxy,
11e: R = 5-indyl, R1 = 3,4,5-trimethoxy,
These new 4-aza-2,3-didehydropodophyllotoxin congeners
(11a–w) were synthesized by applying a multicomponent route
involving the condensation of substituted heteroaromatic amines,
tetronic acid, and substituted aromatic aldehydes that proceeds
smoothly in refluxing ethanol. In all cases, the resulting
4-aza-2,3-didehydropodophyllotoxins precipitate as the reaction
mixtures are allowed to cool to room temperature and are isolated
by simple filtration followed by recrystallized from ethanol to
afford the pure compounds (Scheme 1). All the synthesized
compounds were characterized by 1H NMR, 13C NMR, mass
spectral data and HRMS.
11f: R = 5-indyl, R1 = 4-hydroxy-3-methoxy,
11g: R = 5-indyl, R1 = 3-hydroxy-4-methoxy,
11h: R = 5-indyl, R1 = 4-fluoro-3-methoxy,
11i: R = 5-indyl, R1 = 3-nitro-4-methoxy,
11j: R = 2-methyl-5-indyl, R1 = 3,4,5-trimethoxy,
11k: R = 2-methyl-5-indyl, R1 = 4-fluoro-3-methoxy,
11l: R = 2-methyl-5-indyl, R1 = 2-fluoro-4-methoxy,
11m: R = 5-indazolyl, R1 = 4-hydroxy-3-methoxy,
11n: R = 5-indazoly, R1 = 3-hydroxy-4-methoxy,
11o: R = 5-indazolyl, R1 = 4-fluoro-3-methoxy,
11p: R = 6-benzthiazolyl, R1 = 3,4,5-trimethoxy,
11q: R = 2-methyl-6-benzthiazolyl, R1 = 3-nitro-4-methoxy,
11r: R = 2-mercapto-5-imidazolyl, R1 = 3-hydroxy-4-methoxy,
11s: R = 5-triazolyl, R1 = 3,4,5-trimethoxy,
3. Biological evaluation
The biological activities of this series of 4-aza-2,3-didehydrop-
odophyllotoxin derivatives were evaluated by an cytotoxicity as-
say, which was carried out in a panel of five human tumor cell
lines comprising (breast, oral, colon, lung and ovarian) by using
the sulforhodamine B (SRB) method. The results are summarized
in Table 1, and compared to etopside as well as adriamycin. It is ob-
served that all the compounds are significantly cytotoxic with the
11t: R = 5-triazolyl, R1 = 2-fluoro-4-methoxy,
11u: R = 3-(4-methoxyaryl)5-isoxazolyl, R1 = 3,4,5-trimethoxy,
11v: R = 3-(4-chloroaryl)5-isoxazolyl, R1 = 3-hydroxy-4-methoxy,
11w: R =2,3,4-trimethoxyaryl, R1 = 4-hydroxy-3-methoxy;
Scheme 1.

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
2351
Table 1
Cytotoxic activity (GI50
enhance the cytotoxicity in KB cell line as in case of 11h. It is also
observed that the change of A ring to hetero aryl or benzo hetero
fused system have not shown much difference in the cytotoxicity.
lM) of compounds 11a–2
Compound
Breast
MCF7
Oral
KB
Colo
Colo 205
Lung
A-549
Ovarian
A-2780
3.1. Inhibition of tubulin polymerization
11a
11b
11c
2.4
—
—
0.17
—
—
0.16
—
—
2.1
—
—
—
—
—
Since these 23 new compounds has structural resemblance to
podophyllotoxin, it has been considered of interest to investigate
their effect on tubulin polymerization. One of the possible explana-
tions of compounds showing anticancer activity and cell cycle ar-
rest is the inhibition of tubulin polymerization to functional
microtubules as it is observed with antimitotic agents such as pod-
ophyllotoxin. As tubulin subunits heterodimerize and self-
assemble to form microtubules in a time dependent manner, we
have investigated the progression of tubulin polymerization by
monitoring the increase in fluorescence emission at 460 nm
(excitation wavelength is 360 nm) in 384 well plate for 1 h at
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
—
—
2.3
2.9
2.1
—
2.2
2.0
—
2.2
0.17
—
2.0
—
2.2
—
—
—
2.1
—
—
2.9
—
—
2.7
—
0.3
0.13
—
2.6
2.4
—
2.3
2.2
—
—
2.1
—
—
—
2.3
2.4
—
—
—
—
—
—
—
3.08
<0.1
—
2.7
—
2.7
2.3
—
2.3
2.5
0.19
—
2.4
—
—
—
2.6
<0.1
2.0
—
—
2.4
2.3
—
—
—
2.3
—
—
—
—
—
—
—
1.3
<0.1
2.7
2.0
2.6
—
2.5
—
—
—
—
—
2.7
37 °C with and without the compounds at 3 lM concentration.
0.15
2.7
—
<0.1
—
Among the twenty three compounds examined, 11a, 11k and
11p inhibited tubulin polymerization to nearly 63.7%, 45.3%, and
49.4%, respectively compared to control and a similar pattern of
inhibition has been observed with the positive control, podophyl-
lotoxin (80.7%) (Fig. 2). From this data it is evident that these struc-
turally modified podophyllotoxin like molecules exhibit
cytotoxicity probably involving a different mechanism compare
to the typical podophyllotoxin ring system.
11s
11t
11u
11v
11w
Etoposide
ADR
—
—
—
0.13
<0.1
2.9
2.1
0.13
ADR = adriamycin is one of the control drug.
Figure 2. Effect of compounds on tubulin polymerization: Tubulin polymerization was monitored by the increase in fluorescence at 340 nm (excitation) and 460 nm
(emission) for 1 h at 37 °C. All the compounds were included at a final concentration of 3 M. Podophyllotoxin (Podo) was used as a positive control.
l

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
3.2. Immunohistochemistry of tubulin
treated A-549 cells with 11a, 11k and 11p along with the positive
control podophyllotoxin and examined for the activation of cas-
pase-3. Results indicate that there was nearly 2–3.5-fold induction
In order to substantiate the observed in vitro effects of the com-
pounds on the inhibition of tubulin polymerization to functional
microtubules, immunohistochemistry studies have been carried
out to examine the in situ effects of compounds 11a, 11k and
11p on cellular microtubules and their special arrangement. In this
study, untreated human lung cancer cells (A-549) displayed the
normal distribution of microtubules (Fig. 3). However, cells treated
in caspase-3 activity in cells treated with 5
compounds. Under similar conditions, podophyllotoxin (1
l
M concentration of
M) in-
l
duced caspase activity to nearly 4-fold as compared to controls as
shown in (Fig. 5).
4. Conclusion
with 5 lM concentration of compounds 11a, 11k and 11p and pod-
ophyllotoxin demonstrated disrupted microtubule organization as
seen in Fig. 3A–E, thus exemplifying the inhibition of tubulin
polymerization.
These new heterocyclic compounds (11a–w) are accessible by a
straightforward one-step multicomponent synthesis, which in-
volves simple isolation of the products by filtration and requires
no further purification. These compounds (11a–w) were evaluated
for their cytotoxic activity a five human cancer cell lines. All the
twenty three compounds (11a–w) have been examined for the
tubulin polymerization assay. Among these compounds 11a, 11k
and 11p exhibited inhibition of polymerization tubulin comparable
to podophyllotoxin apart from disruption of microtubule organiza-
tion within the cells. Further, flow cytometric analysis showed that
these 11a, 11k and 11p compounds arrested the cell cycle in the
G2/M phase of cell cycle leading to caspase-3 dependent apoptotic
cell death. These results provide an insight for future direction in
the development of such molecules.
3.3. Cell cycle analysis (flow cytometry)
Since the compounds 11a, 11k and 11p mediate their cytotoxic
effects by inhibiting the polymerization of tubulin, we have exam-
ined whether the same could be reflected in the pattern of cell cy-
cle arrest. Towards this, the cell cycle dependent DNA content was
determined by flow cytometry using propidium iodide staining
according to the standard protocol. The effect of compounds 11a,
11k and 11p (10 lM) along with DMSO control were examined
in A-549 lung cancer cells treated individually with these com-
pounds for 48 h. Podophyllotoxin was used as standard compari-
son as shown in (Fig. 4).
5. Experimental
3.4. Caspase-3 activation assay
All chemicals and reagents were obtained from Aldrich (Sigma–
Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Company, Ward Hill, MA, USA) and were used without further
purification. Reactions were monitored by TLC, performed on silica
gel glass plates containing 60 F-254, and visualization on TLC was
achieved by UV light or iodine indicator. 1H and 13C NMR spectra
were recorded on INOVA (400 MHz) or Gemini Varian-VXR-unity
(200 MHz) or Bruker UXNMR/XWIN-NMR (300 MHz) instruments.
Since in several instances cell cycle arrest at G2/M phase has
been shown to induce cellular apoptosis, we have examined
whether the cytotoxicity by 11a, 11k and 11p is by virtue of apop-
totic cell death. Cysteine aspartase group, namely, caspases play a
crucial role in the induction of apoptosis and amongst them cas-
pase-3 happens to be one of the effector caspase. Hence, we have
Figure 3. IHC analysis of tubulin polymerization (A) control untreated (B), (C), and (D) cells treated with compounds 11a, 11k, and 11p at 5
podophyllotoxin (1 M).
lM for 48 h, respectively, (E)
l

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
2353
Figure 4. FACS analysis of cell cycle distribution of A-549 cells (a) control untreated (b) Podophyllotoxin (Podo) was used at 1
lM concentration, (c–e) compounds 11a, 11p,
and 11k treated cells at 10 lM concentration.
Chemical shifts (d) are reported in ppm downfield from internal
TMS standard. ESI spectra were recorded on Micro mass, Quattro
LC using ESI+ software with capillary voltage 3.98 kV and ESI mode
positive ion trap detector. High-resolution mass spectra (HRMS)
were recorded on QSTAR XL Hybrid MS/MS mass spectrometer.
Melting points were determined with an electrothermal melting
point apparatus, and are uncorrected.
5.1. 2,4-Dimethoxy-5-(3,4,5-trimethoxyphenyl)-5,6,8,9-
tetrahydrofuro[30,40:5,6]pyrido[2,3-d]pyrimidin-6-one (11a)
The compound tetronic acid (102 mg, 1.02 mmol) was dissolved
in 4 mL of ethanol, followed by addition of 3,4,5-trimethoxybenz-
aldehyde (200 mg, 1.02 mmol) and 2,4-dimethoxypyrimidine-5-
amine (158 mg, 1.02 mmol). The reaction mixture was reflux at

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
5.4. 5-(4-Fluoro-3-methoxyphenyl)-2,4-dimethoxy-5,6,8,9-
tetrahydrofuro[30,40:5,6]pyrido[2,3-d]pyrimidin-6-one (11d)
This compound 11d was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (88 mg, 0.88 mmol) with 4-fluoro-3-methoxybenzal-
dehyde (200 mg, 0.88 mmol) and 2,4-dimethoxypyrimidine-5-
amine (137 mg, 0.88 mmol) to afford pure compound 11d as a
white solid in 470 mg, 97% yield. Mp: 267–270 °C, 1H NMR
(400 MHz, DMSO-d6): d 3.00 (s, 3H), 3.02 (s, 3H), 3.07 (s, 3H),
3.94– 4.12 (m, 3H), 5.77–5.84 (m, 1H), 6.15–6.29 (m, 2H), 9.78 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d 33.9, 53.8, 54.3, 55.8, 65.2,
93.7, 99.6, 113.2, 115.2, 115.5, 119.4, 141.1, 156.7, 157.9, 163.3,
168.7, 171.0; MS (ESI): 397 [M+Na]+; HRMS (ESI) calcd for
C
18H16N3O5FNa [M+Na]+ 396.0971; found: 396.0961.
Figure 5. Effect of compounds 11a, 11p, and 11k on caspase-3 activity: A-549 cells
were treated for 48 h with 5 mm concentration of compounds 11a, 11p and 11k.
Following termination of experiment, cellular caspase-3 activity was measured by
fluorimetric assay as described in the ‘Section 5’. Podophyllotoxin (Podo) was used
5.5. 10-(3,4,5-Trimethoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11e)
as
a positive control. Values indicated are the mean SD of two different
experiments performed in triplicates.
This compound 11e was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (102 mg, 1.02 mmol) with 3,4,5-trimethoxybenzalde-
hyde (200 mg, 1.02 mmol) and 1H-indol-5-amine (134 mg,
1.02 mmol) to afford pure compound 11e as a brown solid in
386 mg, 96% yield. Mp: 299–301 °C, 1H NMR (200 MHz, DMSO-
d6): d 3.61 (s, 3H), 3.69 (s, 6H), 4.83–5.06 (dd, 2H, J = 15.1,
15.1 Hz), 5.29 (s, 1H), 6.39 (br s,1H), 6.58 (s, 2H), 6.87 (d, 1H,
J = 9.0 Hz), 7.25 (t, 1H, J = 3.0, 2.2 Hz), 7.29 (d, 1H, J = 9.0 Hz), 9.92
(s, 1H); 13C NMR (75 MHz, DMSO-d6): d 55.7, 55.9, 59.7, 64.9,
94.7, 105.2, 109.9, 113.2, 117.3, 122.1, 129.2, 135.9, 141.9, 152.5,
157.7, 172.1. MS (ESI): 393 [M+H]+. HRMS (ESI) calcd for
ethanol temperature for 1 h. Then reaction mixture was allowed to
cool to room temperature, and the precipitated product was col-
lected by vacuum filtration and washed with ethanol (3 mL) then
recrystallized from ethanol to afford pure compound 11a as a
white solid in 390 mg, 92% yield. Mp: 299–300 °C, 1H NMR
(200 MHz, DMSO-d6): d 3.61 (s, 3H), 3.70 (s, 6H), 3.81 (s, 3H),
3.85 (s, 3H), 4.79–5.02 (m, 3H), 6.43 (s, 2H), 10.61 (s,1H); 13C
NMR (75 MHz, DMSO-d6): d 34.3, 53.8, 54.3, 55.7, 59.7, 65.2,
93.8, 99.6, 104.7, 136.1, 139.9, 152.4, 156.8, 163.2, 168.8, 171.1;
MS (ESI): 416 [M+H]+; HRMS (ESI) calcd for C20H22N3O7 [M+Na]+
416.1457; found: 416.1447.
C
22H20N2O5Na [M+Na]+ 415.1269; found: 415.1257.
5.6. 10-(4-Hydroxy-3-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11f)
5.2. 5-(4-Hydroxy-3-methoxyphenyl)-2,4-dimethoxy-5,6,8,9-
tetrahydrofuro[30,40:5,6]pyrido[2,3-d]pyrimidin-6-one (11b)
This compound 11f was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 4-hydroxy-3-methoxybenz-
aldehyde (200 mg, 1.27 mmol) and 1H-indol-5-amine (168 mg,
1.27 mmol) to afford pure compound 11f as a broun colour solid
in 440 mg, 96% yield. Mp: 301–303 °C, 1H NMR (200 MHz,
DMSO-d6): d 3.77 (s, 3H), 4.52– 5.03 (dd, 2H, J = 16.0, 15.3 Hz),
4,52 (s, 1H), 5.31 (s, 1H), 6.06 (d, 1H, J = 8.7 Hz), 6.40–6.49 (m,
2H), 6.56 (d, 1H, J = 8.7 Hz), 6.80–6.99 (dd, 1H, J = 2.1, 2.1 Hz),
6.79 (t, 1H, J = 2.1 Hz), 9.12 (s, 1H), 10.21(s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 56.0, 64.7, 68.8, 94.9, 100.1, 104.9, 110.8,
112.3, 114.9, 120.1, 122.2, 124.4, 125.2, 127.0, 132.7, 137.8,
144.5, 146.8, 157.4, 172.4; MS (ESI): 349 [M+H]+; HRMS (ESI) calcd
for C20H16N2O4Na [M+Na]+ 371.1007; found: 371.0995.
This compound 11b was prepared follwing the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 4-hydroxy-3-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 2,4-dimethoxypyrimi-
dine-5-amine (197 mg, 1.27 mmol) to afford pure compound 11b
as a white solid in 450 mg, 92% yield. Mp: 294–296 °C, 1H NMR
(200 MHz, DMSO-d6): d 3.77 (s, 3H), 3.84 (s, 3H), 3.90 (s, 3H),
4.82–5.03 (dd, 2H, J = 16.8, 16.8 Hz), 6.47–6.52 (dd, 1H, J = 2.1,
2.1 Hz), 6.66 (d, 1H, J = 8.0 Hz), 6.84 (d, 1H, J = 2.1 Hz), 10.64 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d 33.5, 53.8, 54.2, 55.4, 65.1,
94.3, 100.2, 111.8, 115.1, 119.4, 135.6, 145.0, 146.9, 156.6, 157.5,
163.0, 168.7, 171.1; MS (ESI): 372 [M+H]+; HRMS (ESI) calcd for
C20H16N2O4Na [M+Na]+ 371.1007; found: 371.0995.
5.3. 5-(3-Hydroxy-4-methoxyphenyl)-2,4-dimethoxy-5,6,8,9-
5.7. 10-(3-Hydroxy-4-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11g)
tetrahydrofuro[30,40:5,6]pyrido[2,3-d]pyrimidin-6-one (11c)
This compound 11c was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 3-hydroxy-4-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 2,4-dimethoxypyrimi-
dine-5-amine (197 mg, 1.27 mmol) to afford pure compound 11c
as white solid in 460 mg, 94% yield. Mp: 284–285 °C, 1H NMR
(400 MHz, DMSO-d6): d 3.71 (s, 3H), 3.77 (s, 3H), 3.85 (s, 3H),
4.65 (s, 1H), 4.76– 4.95 (dd, 2H, J = 15.9, 16.8 Hz), 9.10 (s, 1H),
10.60 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 33.4, 53.9, 54.3,
55.5, 65.1, 74.1, 82.8, 94.3, 100.4, 111.8, 114.7, 115.1, 117.9,
137.4, 146.2, 156.0, 157.2, 168.7; MS (ESI): 372 [M+H]+; HRMS
(ESI) calcd for C18H17N3O6Na [M+Na]+ 394.1015; found: 394.1000.
This compound 11g was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 3-hydroxy-4-methoxybenz-
aldehyde (200 mg, 1.27 mmol) and 1H-indol-5-amine (168 mg,
1.27 mmol) to afford pure compound 11g as a broun colour solid
in 447 mg, 97% yield. Mp: 297–299 °C, 1H NMR (200 MHz,
DMSO-d6): d 3.77 (s, 3H), 4.52– 5.03 (dd, 2H, J = 16.0, 15.3 Hz),
4,52 (s, 1H), 5.31 (s, 1H), 6.06 (d, 1H, J = 8.7 Hz), 6.40– 6.49 (m,
2H), 6.56 (d, 1H, J = 8.7 Hz), 6.80–6.99 (dd, 1H, J = 2.1, 2.1 Hz),
6.79 (t, 1H, J = 2.1 Hz), 9.12 (s, 1H), 10.21 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 37.8, 55.5, 64.7, 94.8, 99.9, 110.9, 111.2,
111.5, 113.8, 115.3, 118.3, 125.2, 127.0, 128.6, 132.7, 139.4,

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
2355
145.7, 145.9, 157.3, 172.3; MS (ESI): 349 [M+H]+; HRMS (ESI) calcd
for C20H16N2O4Na [M+Na]+ 371.1007; found: 371.0995.
15.5 Hz), 5.73 (s, 1H), 6.27 (s, 1H), 7.04–7.07 (m, 1H), 7.16 (d, 1H,
J = 8.6 Hz), 7.32–7.37 (dd, 1H, J = 7.7, 7.7 Hz), 7.55 (d, 1H,
J = 6.9 Hz), 7.61 (d, 1H, J = 8.6 Hz), 10.13 (s, 1H), 11.13 (s, 1H); 13C
NMR (75 MHz, DMSO-d6): d 13.2, 18.4, 38.0, 55.8, 64.7, 94.2,
98.0, 110.0, 112.1, 113.4, 115.2, 119.8, 127.9, 128.6, 132.8, 135.8,
143.1, 146.2, 157.5, 172.3; MS (ESI): 365 [M+H]+; HRMS (ESI) calcd
for C21H17N2O3FNa [M+Na]+ 387.1120; found: 387.1115.
5.8. 10-(4-Fluoro-3-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11h)
This compound 11h was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (88 mg, 0,88 mmol) with 4-fluoro-3-methoxybenzal-
dehyde (200 mg, 0.88 mmol) and 1H-indol-5-amine (116 mg,
0.88 mmol) to afford pure compound 11h as a white solid in
441 mg, 97% yield. Mp: 170–172 °C, 1H NMR (400 MHz, DMSO-
d6): d 3.76 (s, 3H), 4.71–4.87 (dd, 2H, J = 15.2, 15.2 Hz), 5.27 (s,
1H), 6.11 (br s, 1H), 6.51–6.56 (m, 1H), 6.72–6.77 (dd, 1H, J = 2.6,
2.6 Hz), 6.84–6.89 (m, 1H), 7.07–7.09 (m, 2H), 7.20 (d, 1H,
J = 8.9 Hz), 9.78 (s, 1H), 10.92 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): d 55.7, 55.9, 64.8, 94.3, 100.0, 111.2, 113.1, 115.1, 115.3,
119.9, 125.5, 126.9, 128.6, 132.7, 143.2, 146.1, 146.3, 148.1,
151.3, 157.7, 172.3; MS (ESI): 351 [M+H]+; HRMS (ESI) calcd for
5.12. 10-(2-Fluoro-4-methoxyphenyl)-2-methyl-6,7,9,10-
tetrahydro-1H-furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11l)
This compound 11l was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (129 mg, 1.29 mmol) with 2-fluoro-4-methoxybenz-
aldehyde (200 mg, 1.29 mmol) and 2-methyl-1H-indol-5-amine
(246 mg, 1.29 mmol) to afford pure compound 11l as a white solid
in 469 mg, 99% yield. Mp: 205–207 °C, 1H NMR (400 MHz, DMSO-
d6): d 2.80 (s, 3H), 4.30 (s, 3H), 5.22–5.32 (dd, 2H, J = 15.5, 15.5 Hz),
5.73 (s, 1H), 6.27 (s, 1H), 7.04–7.07 (m, 3H), 7.16 (d, 1H, J = 8.6 Hz),
7.61 (d, 1H, J = 8.6 Hz), 10.13 (s, 1H), 11.13 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 13.2, 18.4, 38.0, 55.8, 64.7, 94.2, 98.0,
110.0, 112.1, 113.4, 115.2, 119.8, 127.9, 128.6, 132.8, 135.8,
143.1, 146.2, 157.5, 172.3; MS (ESI): 365 [M+H]+; HRMS (ESI) calcd
for C21H17N2O3FNa [M+Na]+ 387.1120; found: 387.1134.
C
20H15N2O3FNa [M+Na]+ 373.0964; found: 373.0950.
5.9. 10-(4-Methoxy-3-nitrophenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11i)
This compound 11i was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (110 mg, 1.10 mmol) with 4-methoxy-3-nitrobenzal-
dehyde (200 mg, 1.10 mmol) and 1H-indol-5-amine (145 mg,
1.10 mmol) to afford pure compound 11i as a yellow solid in
400 mg, 96% yield. Mp: 280–281 °C, 1H NMR (400 MHz, DMSO-
d6): d 3.08 (s, 3H), 4.03–4.14 (dd, 2H, J = 16.1, 16.1 Hz), 4.62 (s,
1H), 5.34 (s, 1H), 6.05 (d, 1H, J = 8.7 Hz), 6.36–6.39 (m, 2H), 6.49
(d, 1H, J = 8.0 Hz), 6.72–6.74 (dd, 1H, J = 2.1, 1.4 Hz), 6.83 (d, 1H,
J = 2.1 Hz), 9.12 (s, 1H), 10.23 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): d 37.2, 56.4, 65.0, 93.7, 99.7, 111.3, 111.5, 112.3, 113.8,
123.5, 125.7, 126.7, 128.7, 132.7, 133.5, 138.6, 138.8, 150.2,
157.7, 172.2; MS (ESI): 429 [M+Na]+; HRMS (ESI) calcd for
5.13. 10-(4-Hydroxy-3-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrazolo[3,4-f]quinolin-9-one (11m)
This compound 11m was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 4-hydroxy-3-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 1H-indazol-5-amine
(168 mg, 1.27 mmol) to afford pure compound 11m as a broun so-
lid in 439 mg, 95% yield. Mp: 238–240 °C, 1H NMR (400 MHz,
DMSO-d6): d 3.73 (s, 3H), 4.77– 4.89 (dd, 2H, J = 14.7, 15.6 Hz),
5.27 (s, 1H), 6.49–6.58 (m, 2H), 6.91 (s, 1H), 7.02 (d, 1H,
J = 8.7 Hz), 7.34 (d, 1H, J = 8.7 Hz), 7.72 (s, 1H), 9.89 (s, 1H); 13C
NMR (75 MHz, DMSO-d6): d 37.8, 55.6, 64.9, 95.1, 109.8, 112.3,
113.9, 115.2, 117.3, 120.2, 122.2, 129.2, 132.2, 137.3, 137.5,
144.8, 147.0, 157.5, 172.3; MS (ESI): 350 [M+H]+; HRMS (ESI) calcd
for C19H15N3O4Na [M+Na]+ 372.0960; found: 372.0954.
C
20H15N3O5Na [M+Na]+ 400.0909; found: 400.0911.
5.10. 2-Methyl-10-(3,4,5-trimethoxyphenyl)-6,7,9,10-
tetrahydro-1H-furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11j)
This compound 11j was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (102 mg, 1.02 mmol) with 3,4,5-trimethoxybenzalde-
hyde (200 mg, 1.02 mmol) and 2-methyl-1H-indol-5-amine
(193 mg, 1.02 mmol) to afford pure compound 11j as a broun solid
in 409 mg, 98% yield. Mp: 280–281 °C, 1H NMR (400 MHz, DMSO-
d6): d 2.27 (s, 3H), 3.55 (s, 3H), 3.63 (s, 6H), 4.77–4.94 (dd, 2H,
J = 15.2, 15.2 Hz), 5.13 (s, 1H), 6.00 (br s, 1H), 6.48 (s, 2H), 6.69
(d, 1H, J = 8.9 Hz), 7.13 (d, 1H, J = 8.0 Hz), 9.83 (s, 1H), 10.85 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d 13.3, 38.5, 55.7, 59.7, 64.8,
94.4, 98.2, 105.2, 109.9, 110.1, 112.4, 128.0, 128.6, 132.8, 135.6,
135.7, 142.2, 152.3, 157.7, 172.3; MS (ESI): 401 [M+Na]+; HRMS
(ESI) calcd for C20H15N3O5Na [M+Na]+ 400.0909; found: 400.0911.
5.14. 10-(3-Hydroxy-4-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrazolo[3,4-f]quinolin-9-one (11n)
This compound 11n was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 3-hydroxy-4-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 1H-indazol-5-amine
(168 mg, 1.27 mmol) to afford pure compound 11n as a white solid
in 442 mg, 96% yield. Mp: 243–245 °C, 1H NMR (400 MHz, DMSO-
d6): d 3.73 (s, 3H), 4.77– 4.89 (dd, 2H, J = 14.7, 15.6 Hz), 5.27 (s, 1H),
6.49– 6.58 (m, 2H), 6.91 (s, 1H), 7.02 (s, 1H, J = 8.7 Hz), 7.34 (d, 1H,
J = 8.7 Hz), 7.72 (s, 1H), 9.89 (s, 1H); 13C NMR (75 MHz, DMSO-d6):
d 37.7, 55.5, 64.9, 95.0, 109.8, 111.6, 113.8, 115.3, 117.3, 118.4,
122.2, 129.2, 132.1, 137.3, 139.0, 146.1, 146.1, 157.4, 172.2; MS
(ESI): 350 [M+H]+; HRMS (ESI) calcd for C19H15N3O4Na [M+Na]+
372.0960; found: 372.0954.
5.11. 10-(4-Fluoro-3-methoxyphenyl)-2-methyl-6,7,9,10-
tetrahydro-1H-furo[3,4-b]pyrrolo[2,3-f]quinolin-9-one (11k)
This compound 11k was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (129 mg, 1.29 mmol) with 4-fluoro-3-methoxybenz-
aldehyde (200 mg, 1.29 mmol) and 2-methyl-1H-indol-5-amine
(246 mg, 1.29 mmol) to afford pure compound 11k as a white solid
in 461 mg, 97% yield. Mp: 198–199 °C, 1H NMR (400 MHz, DMSO-
d6): d 2.80 (s, 3H, –CH3), 4.30 (s, 3H), 5.22–5.32 (dd, 2H, J = 15.5,
5.15. 10-(4-Fluoro-3-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrazolo[3,4-f]quinolin-9-one (11o)
This compound 11o was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (88 mg, 0.88 mmol) with 4-fluoro-3-methoxybenzal-
dehyde (200 mg, 0.88 mmol) and 1H-indazol-5-amine (116 mg,

2356
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
0.88 mmol) to afford pure compound 11o as a white solid in
433 mg, 95% yield. Mp: 307–309 °C, 1H NMR (300 MHz, DMSO-
d6): d 3.79 (s, 3H), 4.85–5.01 (dd, 2H, J = 15.6, 15.6 Hz), 5.42 (s,
1H), 6.64–6.69 (m, 1H), 6.97–7.08 (m, 2H), 7.22–7.26 (dd, 1H,
J = 1.8, 1.8 Hz), 7.39 (d, 1H, J = 8.8 Hz), 7.87 (s, 1H), 10.01 (s,1H);
13C NMR (75 MHz, DMSO-d6): d 42.8, 60.8, 69.9, 99.5, 118.0,
118.4, 120.3, 120.5, 122.3, 124.9, 127.0, 134.2, 147.8, 147.9,
151.2, 153.6, 156.0, 162.7, 177.1; MS (ESI): 352 [M+H]+; HRMS
(ESI) calcd for C19H14N3O3FNa [M+Na]+ 374.0916; found: 374.0900.
tetronic acid (102 mg, 1.02 mmol) with 3,4,5-trimethoxybenzalde-
hyde (200 mg, 1.02 mmol) and 1H-benzo[d][1,2,3]triazol-5-amine
(136 mg, 1.02 mmol) to afford pure compound 11s as a white solid
in 378 mg, 94% yield. Mp: 324–325 °C, 1H NMR (400 MHz, DMSO-
d6): d 3.56 (s, 3H), 3.66 (s, 6H), 4.88–5.14 (dd, 2H, J = 15.6, 15.6 Hz),
5.27 (s, 1H), 6.59 (s, 2H), 7.12 (d, 1H, J = 8.5 Hz), 7.87 (d, 1H,
J = 8.5 Hz), 10.41 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 36.7,
55.7, 59.7, 65.1, 96.0, 104.8, 115.2, 118.3, 132.6, 135.3, 136.1,
140.2, 141.6, 152.6, 158.0, 171.8; MS (ESI): 395 [M+H]+;
HRMS (ESI) calcd for C20H18N4O5Na [M+Na]+ 417.1174; found:
417.1176.
5.16. 10-(3,4,5-Trimethoxyphenyl)-6,7,9,10-tetrahydrofuro[3,4-
b][1,3]thiazolo[5,4-f]quinolin-9-one (11p)
5.20. 10-(2-Fluoro-4-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b][1,2,3]triazolo[4,5-f]quinolin-9-one (11t)
This compound 11p was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (102 mg, 1.02 mmol) with 3,4,5-trimethoxybenzalde-
hyde (200 mg, 1.02 mmol) and benzo[d]thiazol-5-amine (153 mg,
1.02 mmol) to afford pure compound 11p as a broun solid in
393 mg, 94% yield. Mp: 309–310 °C, 1H NMR (400 MHz, DMSO-
d6): d 3.59 (s, 3H), 3.67 (s, 6H), 4.86–5.00 (dd, 2H, J = 16.1,
16.1 Hz), 5.09 (s, 1H), 6.48 (s, 2H), 7.22 (d, 1H, J = 8.7 Hz), 7.92 (d,
1H, J = 8.7 Hz), 9.06 (s, 1H), 10.33 (s, 1H); 13C NMR (300 MHz,
DMSO-d6): d 40.9, 55.7, 59.8, 64.9, 95.5, 105.9, 116.1, 116.2,
122.4, 134.0, 135.1, 136.2, 138.9, 149.4, 152.5, 153.7, 157.2,
171.6; MS (ESI): 411 [M+H]+; HRMS (ESI) calcd for C21H19N2O5
[M]+ 411.1014; found: 411.1021.
This compound 11t was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (88 mg, 0.88 mmol) with 2-fluoro-4-methoxybenzal-
dehyde (200 mg, 0.88 mmol) and 1H-benzo[d][1,2,3]triazol-5-
amine (118 mg, 0.88 mmol) to afford pure compound 11t as a
white solid in 439 mg, 96% yield. Mp: 308–310 °C, 1H NMR
(400 MHz, DMSO-d6): d 3.68 (s, 3H), 4.85–4.95 (dd, 2H, J = 15.6,
15.6 Hz), 5.06 (s, 1H), 6.55–6.61 (m, 2H), 7.08 (d, 1H, J = 8.7 Hz),
7.20 (br s, 1H), 7.69–7.71 (m, 1H), 10.29 (s, 1H); 13C NMR
(300 MHz, DMSO-d6): d 29.8, 55.3, 65.0, 94.6, 101.5, 101.8, 109.8,
111.6, 115.0, 118.2, 130.6, 130.9, 158.9, 159.3, 171.6; MS (ESI):
353 [M+H]+; HRMS (ESI) calcd for C18H13N4O3FNa [M+Na]+
375.0869; found: 375.0870.
5.17. 10-(3-Methoxy-4-nitrophenyl)-2-methyl-6,7,9,10-
tetrahydrofuro[3,4-b][1,3]thiazolo[5,4-f]quinolin-9-one (11q)
5.21. 3-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-4,5,7,8-
tetrahydrofuro[3’,4’:5,6]pyrido[3,2-d]isoxazol-5-one (11u)
This compound 11q was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (110 mg, 1.10 mmol) with 3-methoxy-4-nitrobenzal-
dehyde (200 mg, 1.10 mmol) and 2-methyl-benzo[d]thiazol-5-
amine (182 mg, 1.10 mmol) to afford pure compound 11q as a
yellow solid in 430 mg, 95% yield. Mp: 190–191 °C, 1H NMR
(400 MHz, DMSO-d6): d 2.71 (s, 3H), 3.83 (s, 3H), 4.80–4.96 (dd,
2H, J = 15.5, 16.4 Hz), 5.64 (s, 1H), 7.00–7.08 (m, 2H), 7.49–7.51
(dd, 1H, J = 2.5, 2.5 Hz), 7.66–7.71 (m, 2H), 8.08 (s, 1H), 10.11 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d 19.9, 35.9, 55.9, 56.4, 65.1,
95.2, 113.8, 114.4, 115.1, 121.3, 123.7, 129.6, 133.4, 134.9, 138.4,
150.4, 158.3, 167.7, 171.7; MS (ESI): 410 [M+H]+; HRMS (ESI) calcd
for C20H15N3O5NaS [M+Na]+ 432.0630; found: 432.0610.
This compound 11u was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (102 mg, 1.02 mmol) with 3,4,5-trimethoxybenzal-
dehyde (200 mg, 1.02 mmol) and 3-(4-methoxyphenyl)isoxazol-
5-amine (193 mg, 1.02 mmol) to afford pure compound 11u
as a white solid in 436 mg, 94% yield. Mp: 218–219 °C, 1H
NMR (400 MHz, DMSO-d6): d 3.54 (s, 3H), 3.59 (s, 6H), 3.75
(s, 3H), 4.89–5.04 (dd, 2H, J = 16.4, 16.4 Hz), 5.23 (s, 1H), 6.40
(s, 2H), 6.94 (d, 2H, J = 8.6 Hz), 7.52 (d, 2H, J = 8.6 Hz), 11.53
(s, 1H); 13C NMR (75 MHz, DMSO-d6): d 35.1, 55.6, 59.7, 64.9,
94.1, 100.8, 105.1, 127.2, 128.6, 129.3, 134.6, 136.0, 138.9,
152.3, 157.4, 159.7, 161.8, 170.7; MS (ESI): 451 [M+H]+; HRMS
5.18. 10-(3-Hydroxy-4-methoxyphenyl)-2-sulfanyl-6,7,9,10-
tetrahydro-1H-furo[3,4-b]imidazo[4,5-f]quinolin-9-one (11r)
(ESI) calcd for
473.1330.
C
24H22N2O7Na [M+Na]+ 473.1324; found:
This compound 11r was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 3-hydroxy-4-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 5-amine-1H-benzo[d]imid-
azole-2-thiol (210 mg, 1.27 mmol) to afford pure compound 11r as
a broun solid in 452 mg, 90% yield. Mp: 334–337 °C, 1H NMR
(400 MHz, DMSO-d6): d 4.00 (s, 3H), 7.07–7.17 (m, 4H), 7.49 (d,
1H, J = 8.9 Hz), 8.19–8.22 (dd, 1H, J = 2.2, 2.2 Hz), 8.40 (d, 1H,
J = 2.2 Hz), 8.69 (s, 1H), 12.58 (s, 1H), 12.63 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 55.6, 69.0, 103.9, 105.2, 111.3, 111.5,
117.1, 121.1, 122.8, 124.5, 133.5, 137.5, 145.9, 147.5, 148.3,
149.2, 162.5, 167.8, 173.5; MS (ESI): 381 [M+H]+; HRMS (ESI) calcd
for C19H13N3O4NaS [M+Na]+ 402.0524; found: 402.0537.
5.22. 3-(4-Chlorophenyl)-4-(3-hydroxy-4-methoxyphenyl)-
4,5,7,8-tetrahydrofuro[3’,4’:5,6]pyrido[3,2-d]isoxazol-5-one
(11v)
This compound 11v was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 3-hydroxy-4-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 3-(4-chlorophenyl)
isoxazol-5-amine (246 mg, 1.27 mmol) to afford pure compound
11v as a pale yellow solid in 459 mg, 85% yield. Mp: 245–246 °C,
1H NMR (300 MHz, DMSO-d6): d 3.72 (s, 3H), 4.67–4.79 (dd, 2H,
J = 16.6, 16.2 Hz), 4.92 (s, 1H), 6.55– 6.64 (m, 3H), 7.24 (d, 2H,
J = 8.4 Hz), 7.39 (d, 2H, J = 8.4 Hz), 8.32 (s, 1H), 11.16 (s, 1H); 13C
NMR (75 MHz, DMSO-d6): d 34.4, 55.4, 64.6, 94.1, 101.7, 111.4,
115.1, 118.4, 127.0, 128.3, 128.6, 129.0, 134.6, 136.1, 146.0,
146.3, 156.3, 159.4, 162.2, 167.1, 170.7; MS (ESI): 433 [M+Na]+;
HRMS (ESI) calcd for C21H15N2O5NaCl [M+Na]+ 433.0567; found:
433.0554.
5.19. 10-(3,4,5-Trimethoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b][1,2,3]triazolo[4,5-f]quinolin-9-one (11s)
This compound 11s was prepared following the method de-
scribed for the preparation of the compound 11a, employing

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2349–2358
2357
5.23. 9-(4-Hydroxy-3-methoxyphenyl)-5,6,7-trimethoxy-1,3,4,9-
tetrahydrofuro[3,4-b]quinolin-1-one (11w)
proceeds. Fluorescence emission at 460 nm (excitation wavelength
is 360 nm) was measured by using a Varioscan multimode plate
reader (Thermo scientific inc.). Podophyllotoxin was used as posi-
tive control in each assay.27,28
This compound 11w was prepared following the method de-
scribed for the preparation of the compound 11a, employing
tetronic acid (127 mg, 1.27 mmol) with 4-hydroxy-3-methoxy-
benzaldehyde (200 mg, 1.27 mmol) and 3,4,5-trimethoxybenzen-
amine (232 mg, 1.27 mmol) to afford pure compound 11w as a
yellow solid in 447 mg, 85% yield. Mp: 290–291 °C; 1H NMR
(400 MHz, DMSO-d6): d 3.38 (s, 3H), 3.63 (s, 3H), 3.70 (s, 3H),
3.78 (s, 3H), 4.75– 4.91 (m, 3H), 6.37–6.41(m, 2H), 6.60 (d, 1H,
J = 7.5 Hz), 6.76 (d, 1H, J = 1.5 Hz), 8.72 (s, 1H), 9.86 (s, 1H); 13C
NMR (75 MHz, DMSO-d6): d 34.7, 55.6, 59.9, 60.2, 64.8, 95.4,
96.2, 110.4, 112.0, 115.0, 119.7, 133.0, 137.4, 138.2, 144.6, 146.8,
151.7, 152.6, 157.4, 172.1; MS (ESI): 400 [M+H]+.
8. Immunohistochemistry and microscopy
A-549 cells were seeded on glass cover slips, incubated for 48 h
in the presence or absence of test compounds 11a, 11k and 11p
(5 lM). Following the termination of incubation, cells were fixed
with 3% paraformaldehyde, 0.02% glutaraldehyde in PBS and per-
meabilized by dipping the cells in 100% methanol (–20 °C). Later,
cover slips were blocked with 1% BSA in phosphate buffered saline
for 1 hr. followed by incubation with a primary anti tubulin (mouse
monoclonal) antibody and FITC conjugated secondary mouse anti
Ig G antibody. Photographs were taken using the confocal micro-
scope, equipped with FITC settings and the pictures were analyzed
for the integrity of microtubule network. In parallel experiments,
6. Procedure of the SRB-assay
The synthesized compounds (11a–w) have been evaluated for
their in vitro cytotoxicity in human cancer cell lines. A protocol
of 48 h continuous drug exposure has been used and a sulforhoda-
mine B (SRB) protein assay has been used to estimate cell viability
or growth. The cell lines were grown in DMEM medium containing
podophyllotoxin (1 l
M) is used as positive control.29
9. Analysis of cell cycle
Human lung cancer cell line (A-549) in 6 well plates were incu-
bated for 48 hrs in the presence or absence of test compounds 11a,
10% fetal bovine serum and 2 mM L-glutamine and were inoculated
into 96 well microtiter plates in 90 mL at plating densities depend-
ing on the doubling time of individual cell lines. The microtiter
plates were incubated at 37 °C, 5% CO2, 95% air, and 100% relative
humidity for 24 h prior to addition of experimental drugs. Aliquots
of 10 mL of the drug dilutions were added to the appropriate
microtiter wells already containing 90 mL of cells, sulting in the re-
quired final drug concentrations. For each compound four concen-
11k and 11p (10 lM) and podophyllotoxin (1 lM). Cells were har-
vested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 °C
for 30 min, ethanol was removed by centrifugation and cells were
stained with 250 lL of DNA staining solution [10 mg of propidium
iodide (PI), 0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100
were dissolved in 100 mL of sterile water] at room temperature for
30 min in the dark. The DNA contents of 20,000 events were mea-
sured by flow cytometer (BD CANTO II, San Jose, CA). Histograms
were analyzed using Summit Software.
trations (0.1, 1, 10 and 100 lM) were evaluated and each was done
in triplicate wells. Plates were incubated further for 48 h and assay
was terminated by the addition of 50 mL of cold trichloro acetic
acid (TCA) (final concentration, 10% TCA) and incubated for
60 min at 4 °C. The plates were washed five times with tap water
and airdried. Sulforhodamine B (SRB) solution (50 mL) at 0.4%
(w/v) in 1% acetic acid was added to each of the cells, and plates
were incubated for 20 min at room temperature. The residual
dye was removed by washing five times with 1% acetic acid. The
plates were airdried. Bound stain was subsequently eluted with
10 mM trizma base, and the absorbance was read on an ELISA plate
reader at a wavelength of 540 nm with 690 nm reference wave-
lengths. Percent growth was calculated on a plate by plate basis
for test wells relative to control wells. The above determinations
were repeated three times. Percentage growth was expressed as
the (ratio of average absorbance of the test well to the average
absorbance of the control wells) ꢀ 100. Growth inhibition of 50%
(GI50) was calculated from ½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ ꢀ 100 14 50, which
is the drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells dur-
ing the drug incubation. Where, Tz 1/4 Optical density at time zero,
OD of control 1/4 C, and OD of test growth in the presence of drug
1/4 Ti.26
10. Caspase assay
A-549 cells were plated in 6-well plates, grown to 60–80% con-
fluence, and treated with either no drug or test compound 11a, 11k
and 11p (5 lM). Podophyllotoxin (1 lM) was used as positive con-
trol. Following 24 h of incubation, cells were collected by scraping,
washed with PBS and briefly centrifuged to collect the pellet. Cells
were lysed in 200 lL of 1X lyses buffer by purging at least ten
times through an insulin syringe followed by incubation on ice
for 10–20 min. The lysate was centrifuged at 13,200 rpm for
20 min at 4 °C and the obtained clear supernatant was used for cas-
pase activity measurements employing AMC-conjugated substrate
for caspase-3 as described earlier.
Acknowledgment
One of the authors, P.S. is thankful to UGC, New Delhi for the
award of Senior Research Fellowship.
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