Synthesis, cytotoxic activity and docking studies of new 4-aza-podophyllotoxin derivatives

Article abstract of DOI:10.1007/s00044-015-1375-z

The synthesized nine aza-podophyllotoxin derivatives (8a-f, 10, 12 and 14) have been evaluated for their cytotoxicity in a panel of tumor cancer cell lines (Zr-75-1, MCF7, KB, Gurav, DWD, Colo-205, A-549 and Hop62). Among them, 8a and 8b compounds show st

Full text of DOI:10.1007/s00044-015-1375-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1375-z
ORIGINAL RESEARCH
Synthesis, cytotoxic activity and docking studies of new
4-aza-podophyllotoxin derivatives
Islavathu Hatti1 Reddymasu Sreenivasulu1 Surender Singh Jadav2
•
•
•
Venkatesan Jayaprakash2 C. Ganesh Kumar3 Rudraraju Ramesh Raju1
•
•
Received: 5 September 2014 / Accepted: 25 April 2015
Ó Springer Science+Business Media New York 2015
Abstract The synthesized nine aza-podophyllotoxin
derivatives (8a–f, 10, 12 and 14) have been evaluated for
their cytotoxicity in a panel of tumor cancer cell lines (Zr-
75-1, MCF7, KB, Gurav, DWD, Colo-205, A-549 and
Hop62). Among them, 8a and 8b compounds show stron-
ger growth inhibition activity than the standard drug
etoposide. Further, molecular docking simulations were
carried out against human topoisomerase II, a putative
target for these classes of molecules.
mainstay in cancer chemotherapy for the near future.
Podophyllotoxin 1 is a naturally occurring aryl tetralin
lignan obtained from a number of plant species of the
podophyllum family (Lee et al., 1993; MacRae et al.,
1993) and is known as an antimicrotubule agent (Loike
et al., 1978).The biological activity of podophyllotoxin has
led to extensive structural modifications, resulting in sev-
eral clinically useful compounds. Several semisynthetic
derivatives of podophyllotoxin 1, namely etoposide 2
¨
¨
(Stahblin, 1973), teniposide 3 (Stahblin, 1970) and etopo-
phos 4 (Schacter, 1996; Witterland et al., 1996), are in
clinical use for the treatment of a variety of malignancies
including lung and testicular carcinoma, lymphoma, non-
lymphocytic leukemia (Belani et al., 1994; Hande, 1998)
shown in Fig. 1. The cytotoxic mechanism of these drugs is
the inhibition of topoisomerase II (Macdonald et al., 1991)
unlike the lead compound that inhibits mitosis. Podophyl-
lotoxin derivatives are widely used as anticancer agents,
but they still have several secondary effects such as poor
water solubility, development of drug resistance, metabolic
inactivation, and toxic effects (Kobayashi and Ratain,
1994). Because of the structural complexity of 1, arising
from the presence of four stereogenic carbons in ring C,
most of the SAR studies have been performed by deriva-
tization of the parent natural product rather than by de novo
chemical synthesis (Youngjae, 2005; Gordaliza et al.,
2000; Hitotsuyanagi et al., 1997, 2000).
Keywords Podophyllotoxin Á Knoevenagel
condensation Á Etoposide Á Cytotoxicity Á Docking studies
Introduction
Cancer is the second leading cause of mortality in devel-
oped countries, and cancer chemotherapy commonly in-
volves the use of cytotoxic agents that destroy rapidly
dividing cells. Within the past decade, advances in our
understanding of the cell cycle have presented new targets
that may allow for the development of more selective
chemotherapeutic agents—agents that target only cancer
cells. Despite this progress, cytotoxic agents will remain a
& Rudraraju Ramesh Raju
rrraju1@gmail.com
Heterocyclic compounds are widely distributed in nature
and are essential to life in various ways due to their dif-
ferent physiological activities. Pyrazole derivatives have
been found to possess a broad spectrum of biological ac-
tivities, which stimulated the research activity in this field.
Several pyrazole derivatives proved to have anticancer
activity (George et al., 2013). Recently, the research and
development of new pyrazoles for cancer therapy have
1
Department of Chemistry, Acharya Nagarjuna University,
Nagarjuna Nagar, Guntur 522510, Andhra Pradesh, India
2
Department of Pharmaceutical Sciences and Technology,
Birla Institute of Technology, Mesra, Ranchi 835 215,
Jharkhand, India
3
Centre for Medicinal Chemistry and Pharmacology,
CSIR-Indian Institute of Chemical Technology,
Tarnaka, Hyderabad 500 007, India
123

Med Chem Res
Fig. 1 Structures of
podophyllotoxin and semi
synthetic derivatives of
podophyllotoxin
H
H
O
O
H3C
O
O
O
HO
O
HO
S
OH
C
O
OH
O
OH
O
O
O
O
B
A
D
O
O
O
O
O
O
O
O
E
H3CO
OCH3
OCH3
H3CO
OCH3
H3CO
OCH3
OH
OH
Teniposide (3)
(1)
Podophyllotoxin
Etoposide (2)
H
O
O
H3C
O
O
HO
OH
O
O
O
O
H3CO
OCH3
OH
O
P
O
OH
Etopophos (4)
been one of the major focuses in anticancer drug design.
The indole scaffold is also found in a manifold of naturally
occurring plant-based alkaloids, and its derivatives have
displayed versatile pharmacological properties including
antitumor (Nguyen et al., 1990). Furthermore, indazole
(Showalter et al., 1998) and isoxazole (Dengler et al.,
1995) heterocyclic compounds also shown potent anti-
cancer activity on human cancer cell lines.
anticancer activity against a panel of human cancer cell
lines. The probable binding modes of present podophyllo-
toxin derivatives are explained through the molecular
docking simulations due to the history of the podophyllo-
toxins which behaves as DNA topoisomerase II inhibitors.
Results and discussion
The analogues of podophyllotoxins such as etoposide
and teniposide have fewer side effects. Further, various
scientists from worldwide have been paying great attention
to the synthesis of 4-aza-podophyllotoxin analogues and
observed very little side effects (Shi et al., 2011a, b; Kamal
et al., 2011, 2014). However, the podophyllotoxins are
known as the DNA topoisomerase II inhibitors with respect
to their mechanism of actions, having the greatest sig-
nificance in the use of several FDA-approved anticancer
drugs.
The synthesis of these aza-podophyllotoxin derivatives
(8a–f, 10, 12 and 14) was done by using the Knoevenagel
condensation as shown in Scheme 1. In this condensation
reaction, the substituted heterocyclic amines, tetronic acid
and substituted aromatic aldehydes were refluxed in etha-
nol. In all cases, the resulting aza-podophyllotoxins were
precipitated by cooling the reaction mixtures after the
completion of the reaction. Now, the precipitated com-
pounds were filtered and recrystallized from acetone to
afford the pure compounds. The structures of all synthetic
Some heterocyclic-fused podophyllotoxin analogues
potently induce apoptosis in cancerous Jurkat cells even
after a short 24-h exposure (Magedov et al., 2007). In the
view of pharmaceutical importance of heterocyclic-fused
4-aza-podophyllotoxin analogues and as continuation of
our research work toward the synthesis of potential anti-
cancer agents, here we report the synthesis of a series of
aza-podophyllotoxin derivatives with pyrazole, isoxazole,
indole, indazole and pyridine moieties as an inner ring part
of podophyllotoxins. The few podophyllotoxins are shown
in Fig. 1. All these congeners have been evaluated for their
1
compounds were confirmed by H NMR, 13C NMR and
mass spectral data.
The biological activities of this series of aza-podophyl-
lotoxin (8a–f, 10, 12 and 14) derivatives were evaluated by
an in vitro cytotoxicity test, which was carried out with a
panel of four tumor cell lines that comprise human breast
cancer (Zr-75-1, MCF7), human oral cancer (KB, Gurav and
DWD), human colon cancer (Colo-205) and human lung
cancer (A-549 and Hop62), using etoposide as reference
compound. The screening procedure was based on the
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Med Chem Res
H
N
O
O
NH
F
N
12
OCH3
NH2
Y
EtOH, reflux, 1 h
NH2
N
O
N
H
11
X
H
N
H
N
H3C
NH2
CHO
X
N
H
7
O
9
R
Y
O
O
R1
EtOH, reflux, 1 h
NH2
EtOH, reflux, 1 h
R
O
O
O
NH
6
5a-f
H3C
R1
OCH3
EtOH, reflux, 1 h
8a-f
OH
H3CO
N
8a; X= NH, Y= N, R= phenyl
R1= 3,4,5-trimethoxy
13
5a
; R1= 3,4,5-trimethoxy
10
5b; R1=4-hydroxy-3-methoxy
5c
8b; X= NH, Y= N, R= phenyl
R1= 4-hydroxy-3-methoxy
; R1= 3-hydroxy-4-methoxy
5d; R1= 3-nitro-4-methoxy
5e
H
8c
; X= NH, Y= N, R= phenyl
R1= 3-hydroxy-4-methoxy
8d
N
; R1= 4-flouro-3-methoxy
5f; R1= 2-flouro-4-methoxy
O
; X= NH, Y= N, R= phenyl
R1= 3-nitro-4-methoxy
8e
N
H3CO
O
; X= O, Y= N, R= 4-chloro-phenyl
R1= 4-flouro-3-methoxy
8f;X= O, Y= N, R= methyl
R1= 3-hydroxy-4-methoxy
H3CO
OCH3
OCH3
14
Scheme 1 Synthesis of various aza podophyllotoxin derivatives
standard SRB method (Skehan et al., 1990), and the GI50
values are given in Table 1. We have observed that all the
compounds are significantly cytotoxic with the concentra-
tion ranging from 0.11 to 2.98 lM and produced 50 % in-
hibition of cell growth (GI50). Where the drug etoposide
exhibited cytotoxic with GI50 concentration ranging from
0.13–3.08 lM. Among them, 8a and 8b compounds showed
a basically stronger inhibition than etoposide.
vanillin on six-membered ring makes it slightly lesser po-
tent (in case of Zr-75-1 and Hop62) than compound 8a.
The substitution of vanillin with isovanillin ring reduces
the potency of compound 8c when compared with 8b. The
presence of nitro and methoxy at the third and fourth po-
sition on the phenyl ring of six-membered nucleus slightly
increases the anticancer activity of compound 8d in colon
and lung cancer cell lines. However, the replacement of
pyrazole nucleus with five-membered ring, i.e., oxazole (8e
and 8f), pyrrole (10, 12) and six-membered ring, i.e.,
pyridine (14) is responsible for depleting them to moderate
actives. The compound 14 has the trimethoxy substituted
phenyl ring as compound 8a, the replacement of six-
membered ring with pyrazole made it least active. Among
the series, the compounds 8a and 8b exhibited potent cy-
totoxicities in vitro than etoposide, and we believed that
these compounds act as promising novel anticancer agents
in cancer chemotherapy. Further, the molecular docking
studies were carried out against the human topoisomerase
In the present study, a series of novel aza-podophyllo-
toxin derivatives 8a–f, 10, 12 and 14 have been designed,
synthesized and evaluated for their cytotoxic activity
against a panel of four types of human tumor cell lines. The
compounds 8a–8d comprises of the five-membered pyra-
zole ring along with phenyl group. The substituted phenyl
ring present on the adjacent six-membered ring to the
pyrazole makes them potent than the standard drug
etoposide. Almost all four cell lines were shown the sus-
ceptibility to compounds 8a and 8b (except Hop62 i.e., for
8b). The substitution of trimethoxy phenyl ring with
123

Med Chem Res
Hop62d
Table 1 Cytotoxic activity (GI50 lM) of compounds 8a–f, 10, 12 and 14
Compound
Zr-75-1a
MCF7a
KBb
Guravb
DWDb
Colo-205c
A-549d
8a
0.17
2.98
2.91
NA
0.13
0.18
2.76
2.20
2.27
2.43
2.71
NA
0.19
0.18
NA
0.15
0.14
2.09
NA
0.14
0.11
2.30
2.36
2.45
NA
0.11
0.12
NA
0.12
0.19
2.43
2.56
2.64
2.36
2.67
2.78
NA
2.01
NA
8b
8c
2.98
NA
8d
NA
0.19
2.37
NA
8e
2.47
2.31
2.26
2.39
2.15
0.20
NA
2.28
NA
NA
8f
2.22
NA
2.38
NA
10
2.07
NA
NA
2.90
NA
12
2.93
NA
2.12
2.54
0.62
2.95
NA
14
Etoposidee
2.78
2.11
NA
2.87
0.13
0.31
0.51
3.08
0.80
Growth inhibition of 50 % and the values are the mean of three determinations
NA not active
a
Breast cancer
b
Oral cancer
c
Colon cancer
d
Lung cancer
e
Etoposide
II-complexed DNA, in order to understand the approximate
binding modes of the present compounds.
dihydropyridyl ring plays a major role in determining the
activity of the isomers. Increasing the bulkiness of the
substitution on phenyl ring makes the S-isomer to loose
its activity.
Binding mode analysis
Analysis of interaction of 8a (R-isomer) with target
protein revealed the existence of two H-bonding interac-
tions and a p–p stacking interaction involving DNA
(DA12F and DG7C). The phenyl ring on pyrazole ring
established a hydrophobic interaction with Gln778A
(Fig. 2a, b). Similarly, analysis of 8b (R-isomer) with
target protein revealed the existence of p–p stacking in-
teraction involving DA12F similar to 8a, but here the
phenyl ring pyrazoline is involved (in 8a, it is phenyl ring
at dihydropyridyl). There is no H-bonding interaction too.
It is observed that R-isomers interact with the DNA rather
than the protein part. Analysis of 8b (S-isomer) revealed
that the isomer orients in a different way and it interacts
mainly with the protein part of the target and there is no p–
p stacking interaction DNA bases (Fig. 3a, b). In summary,
the R-isomers exhibited interaction with DNA (DA12F)
similar to etoposide and we supposed that these isomers be
the active isomers. The superpose of all ligands in active
site is shown in Fig. 4.
The complex of DNA with human topoisomerase II along
with etoposide (PDB: 3QX3) was employed; the chain A
along with DNA complex was selected for the present
molecular docking studies through the extra-precision
docking mode of GLIDE 5.0. The docking protocol was
˚
validated by redocking that resulted in 0.48 A RMSD.
The etoposide shows the H-bonding interactions with
Asp479 and DGF13 and hydrophobic interactions with
DF13F and DAF12 of DNA. Simulation study was carried
out for only two most potent molecules (8a and 8b) in
this series. Ligand preparation through LigPrep module in
Maestro 8.5 (Schrodinger LLC) provided four structures
for each molecule: two isomers (R and S) each with two
different protonation states (in pyrazole moiety). All the
four structures were docked on the target (PDB: 3QX3).
In case of 8a, only one structure with configuration of R
and protonation at 2 N of pyrazoline docked into the
target protein, while all the other three structures failed to
dock. Two structures of 8b were found to dock well
against the target protein: one with R configuration having
protonation at 1 N of pyrazole and one with S con-
figuration having protonation at 2 N of the pyrazole. Both
are having the docking score almost equal. This clearly
indicates that substitution on the phenyl ring on
Experimental
All the chemicals and reagents were obtained from Aldrich
(Sigma-Aldrich, St. Louis, MO, USA), Lancaster (Alfa
Aesar, Johnson Matthey Company, Ward Hill, MA, USA),
123

Med Chem Res
Fig. 3 a 2D Poseviwer plot of 8b. b Red line indicates DNA, and
curved line indicates protein; green thick residue indicates 8b and
others active-site residues (Color figure online)
trimethoxybenzaldehyde (5a) (196 mg, 1 mmol) and
3-phenyl-1H-pyrazol-5-amine (159 mg, 1 mmol). The re-
action mixture was reflux at ethanol temperature for 1 h.
The reaction mixture was allowed to cool to room tem-
perature. Now, the precipitated product was collected by
vacuum filtration, washed with ethanol (3 mL) and then
recrystallized from acetone to afford pure compound 8a as
in 390 mg, 93 % yield. Mp: 189–191 °C, 1H NMR
(200 MHz, DMSO-d6): d 3.61 (s, 3H, –OCH3), 3.62 (s, 6H,
(–OCH3)2), 4.83–5.02 (dd, 2H, J = 16.1, 15.4 Hz,
–CH2–O), 5.27 (s, 1H, –CH–), 6.43 (s, 2H, ArH), 7.35–7.58
(m, 5H, ArH), 8.72 (s, 1H, –OH), 10.32 (s, 1H, –NH); 13C
NMR (75 MHz, DMSO-d6): d 34.8(–CH–, C4), 55.5(–CH3,
Fig. 2 a 2D Poseviwer plot of 8a. b Red line indicates DNA, and
curved line indicates protein; green thick residue indicates 8a and
others active-site residues (Color figure online)
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. Chemical shifts (d) are re-
ported 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. Melting points were de-
termined with an electrothermal melting point apparatus
and are uncorrected.
00
00
00
C4 ), 59.7(–CH3, C3 , C5 ), 64.7(–CH2–, 0 C7), 96.1(–C–,
0
C10), 101.7(–C–, C11), 104.7(–C–, C2 , C6 ), 126.7(–CH–,
00
00
0
0
0
C2 , C6 ), 1270.09(–CH–, C4 ), 128.4(–CH–,C3 , C5 ),
0
129.3(–CH–, C1 ), 135.6(–C–, C010 ), 138.8(–C–, C3),
00
140.5(–C–, C5 ), 148.1(–C–, C3 ), 152.1(–C–, C9),
159.3(–C–, C12), 171.9(–C–, C5); MS (ESI): 420 [M ? H]?.
4-(4-hydroxy-3-methoxyphenyl)-3-phenyl-4,5,7,8-tetrahy-
dro-1H-furo[3,4-b] pyrazolo[4,3-e]pyridin-5-one (8b)
This compound 8b was prepared following the method
described for the preparation of the compound 8a, em-
ploying tetronic acid (100 mg, 1 mmol), 4-hydroxy-3-
3-phenyl-4-(3,4,5-trimethoxyphenyl)-4,5,7,8-tetrahydro-
1H-furo[3,4-b]pyrazolo[4,3-e] pyridin-5-one (8a) The
compound tetronic acid (100 mg, 1 mmol) was dissolved
in 4 mL of ethanol, followed by the addition of 3,4,5-
123

Med Chem Res
Fig. 4 Superpose: Red lines indicate DNA, and other curved lines indicate protein; red color stick indicates etoposide and green stick residues
indicate compounds (Color figure online)
methoxybenzaldehyde (5b) (152 mg, 1 mmol) and 3-phe-
nyl-1H-pyrazol-5-amine (159 mg, 1 mmol) to afford a
pure compound 8b as in 310 mg, 28 % yield. Mp:
–OH), 10.18 (s, 1H, –NH); 13C NMR (75 MHz, DMSO-
00
d6): d 34.4 (–CH–, C4), 55.3 (–CH3, O–CH3, C4 ), 64.6
0
00
(–CH2, C7), 97.2 (–C–, C10, C11), 1002.0 (–C–, C1 , C1 ),
1
0
0
0
304–306 °C, H NMR (200 MHz, DMSO-d6): d 3.58 (s,
111.4 (–CH–, C2 ), 114.9 (–CH–, C2 ), 118.2 (–CH–, C6 ),
0
0
0
3H, –OCH3), 4.73–4.85 (dd, 2H, J = 15.2, 15.2 Hz,
–CH2–O), 5.08 (s, 1H, –CH–), 6.40 (d, 1H, J = 9.3 Hz,
ArH), 6.52 (d, 1H, J = 8.4 Hz, ArH), 6.73 (s, 1H, ArH),
7.22–7.36 (m, 3H, ArH), 7.46 (d, 2H, J = 7.6 Hz, ArH),
12.55 (s, 1H, –NH); 13C NMR (75 MHz0,0 DMSO-d6): d
34.4 (–CH–, C4), 55.3 (–CH3, OCH3, C3 )0, 64.7 (–CH2,
126.1 (–CH–, C4 ), 127.8 (–CH–, C3 , C5 ), 128.5 (–CH–,
00
00
C6 ), 129.1 (–CH–, 00C5 ), 137.9 (–C–, C3), 138.3 (–C–,
00
C3 ), 145.9 (–C–, C4 ), 148.6 (–C–, C9), 158.4 (–C–, C12),
171.8(–C–, C5); MS (ESI): 376 [M ? H]?.
4-(4-methoxy-3-nitrophenyl)-3-phenyl-4,5,7,8-tetrahydro-
1H-furo[3,4-b]pyrazolo[4,3-e]pyridin-5-one (8d) This
compound 8d was prepared following the method de-
scribed for the preparation of the compound 8a, employing
tetronic acid (100 mg, 1 mmol), 4-methoxy-3-nitroben-
zaldehyde (5d) (181 mg, 1 mmol) and 3-phenyl-1H-pyra-
zol-5-amine (159 mg, 1 mmol) to afford a pure compound
00
C7), 96.8 (–C–, C10, C11), 102.30 (–C–, C1 , C1 ), 112.0
0
0
00
(–CH–, C1 ), 0115.1(–CH–, C6 ), 119.5(–CH–, C4 ),
0
126.5(–CH–, C5 ), 127.9 (–CH–, C3 ), 128.5 (–CH–, C2 ),
00
00
00
129.3 (–CH–, C6 ), 136.3(–CH–, C5 ), 138.5(–C–, C4 ),
00
144.5 (–C–, C3), 146.6 (–C–, C3 ), 148.3 (–C–, C9), 158.8
(–C–, C12), 172.0 (–C–, C5); MS (ESI): 376 [M ? H]?.
1
8d as in 380 mg, 94 % yield. Mp: 268–270 °C, H NMR
4-(3-hydroxy-4-methoxyphenyl)-3-phenyl-4,5,7,8-tetrahy-
dro-1H-furo[3,4-b] pyrazolo[4,3-e]pyridin-5-one (8c)
This compound 8c was prepared following the method
described for the preparation of the compound 8a, em-
ploying tetronic acid (100 mg, 1 mmol), 3-hydroxy-4-
methoxybenzaldehyde (5c) (152 mg, 1 mmol) and 3-phe-
nyl-1H-pyrazol-5-amine (159 mg, 1 mmol) to afford a
pure compound 8c as in 290 mg, 77 % yield. Mp:
(400 MHz, DMSO-d6): d 3.79 (s, 3H, –OCH3), 4.73–4.83
(dd, 2H, J = 15.5, 15.5 Hz, –CH2–O), 5.29 (s, 1H, –CH–),
7.04 (d, 1H, J = 8.6 Hz, ArH), 7.22 (t, 1H, ArH), 7.29 (t,
2H, ArH), 7.36–7.43 (m, 3H, ArH), 7.51 (d, 1H,
J = 2.5 Hz, ArH), 10.30 (s, 1H, –NH); 13C NMR
(75 MHz, DMSO-d6): d 33.8 (–CH–, C4), 56.3 (–CH3,
00
O–CH3, C4 ), 64.8 (–CH2, C7), 95.7 (–C–, C9), 101.0 (–C–,
0
0
0
C10), 113.7 (–CH–, C2 , C6 ), 123.7 (–CH–, C4 ), 126.4
0
1
0
00
00
304–306 °C, H NMR (400 MHz, DMSO-d6): d 3.70 (s,
(–CH–, C3 , C5 ),00128.1 (–CH–, C5 ), 128.4 (–CH–,C2 ),
0
00
3H, –OCH3), 4.88–5.02 (dd, 2H, J = 15.6, 16.3 Hz,
–CH2–O), 5.49 (s, 1H, –CH–), 6.41–6.43 (dd, 1H, J = 1.7,
1.7 Hz, ArH), 6.54 (d, 1H, J = 7.8 Hz, ArH), 7.05–7.07
(m, 4H, ArH), 7.83 (d, 1H, J = 7.8 Hz, ArH), 8.72 (s, 1H,
133.5 (–CH–, C6 ), 137.2 (–C–, C1 , 00C1 ), 138.2 (–C–,
00
C3 ), 138.8 (–C–, C3), 148.1 (–C–, C4 ), 150.3 (–C–,C9),
159.2 (–C–, C12), 171.7 (–C–, C5); MS (ESI): 405
[M ? H]?.
123

Med Chem Res
0
3-(4-chlorophenyl)-4-(4-fluoro-3-methoxyphenyl)-4,5,7,
8-tetrahydrofuro[30,40:5,6] pyrido[3,2-d]isoxazol-5-one
(8e) This compound 8e was prepared following the
method described for the preparation of the compound 8a,
employing tetronic acid (100 mg, 1 mmol), 4-fluoro-3-
methoxybenzaldehyde (5e) (154 mg, 1 mmol) and 3-(4-
chlorophenyl)isoxazol-5-amine (194 mg, 1 mmol) to af-
ford a pure compound 8e as in 385 mg, 93 % yield. Mp:
(–CH–, C10), 55.5 (–CH3, O–CH3, C3 ), 65.0 (–CH2, C7),
96.3 (–CH–,C3), 112.2 (–C–, 0C14,C15), 115.2 (–CH–, C4,
0
0
C5), 119.7 (–CH–, C2 , C5 , C6 ), 1036.00(–C–, C2, C11, C12),
0
145.0 (–C–, C1 ), 146.9 (–C–, C3 , C4 ), 157.7 (–C–, C13,
C16), 171.9(–C–, C9); MS (ESI): 363 [M ? H]?.
10-(2-fluoro-4-methoxyphenyl)-6,7,9,10-tetrahydro-1H-
furo[3,4-b]pyrazolo[3,4-f] quinolin-9-one (12) This
compound 12 was prepared following the method de-
scribed for the preparation of the compound 8a, employing
tetronic acid (100 mg, 1 mmol), 2-fluoro-4-methoxyben-
zaldehyde (5f) (154 mg, 1 mmol) and 1H-indazol-5-amine
(133 mg, 1 mmol) to afford a pure compound 12 as in
305 mg, 86 % yield. Mp: 298–300 °C, 1H NMR
(400 MHz, DMSO-d6): d 3.69 (s, 3H, –OCH3), 4.80–4.91
(dd, 2H, J = 15.2, 15.2 Hz, –CH2–O), 5.55 (s, 1H, –CH–),
6.56–6.64 (m, 2H, ArH), 6.98 (d, 1H, J = 9.3 Hz, ArH),
7.11 (t, 1H, ArH), 7.32 (d, 1H, J = 9.3 Hz, ArH), 7.60
(s, 1H, ArH), 9.96 (s, 1H, –NH); 13C NMR (75 MHz,
1
244–246 °C, H NMR (300 MHz, DMSO-d6): d 3.72 (s,
3H, –OCH3), 4.67–4.79 (dd, 2H, J = 16.6, 16.2 Hz,
–CH2–O), 4.92 (s, 1H, –CH–), 6.55–6.64 (m, 3H, ArH),
7.24 (d, 2H, J = 8.4 Hz, ArH), 7.39 (d, 2H, J = 8.4 Hz,
ArH), 8.32 (s br, 1H, ArH), 11.16 (s, 1H, –NH); 13C NMR
(75 MHz, DMSO-d6): d 34.7 (–CH–, C4), 55.6 (–CH3,
00
O–CH3, C3 ), 64.8 (–CH2,C7), 94.0 (–C–, C10), 100.8
00
00
(–C–, C11)0,0 113.5 (–CH–, C2 ), 115.4 (–CH–, C5 ), 115.7
0
0
(–CH–, C6 ), 120.0 (–CH–, C2 ), 126.9 (–CH–, C6 ), 128.6
0
00
0
0
(–C–, C5 ), 129.2 (–CH–, 0C3 ), 134.6 (–CH–, C1 ), 140.0
00
(–C–, C1 ), 146.2 (–C–, C4 ), 148.4 (–C–, C4 ), 151.6 (–C–,
0
0
DMSO-d6): d 31.2 (–CH–, C10), 55.3 (–CH3, O–CH3, C4 ),
00
C3 ), 157.2 (–C–, C12), 159.5 (–C–, C3), 162.0 (–C–, C9),
64.9 (–CH2, C7), 093.5 (–C–, C14, C15), 100.6 (–CH–, C3 ),
170.7 (–C–, C5); MS (ESI): 435 [M ? Na]?.
100.9 (–CH–, C5 ), 110.0 (–C–, C12), 110.5 (–CH–, C5),
0
4-(3-hydroxy-4-methoxyphenyl)-3-methyl-4,5,7,8-tetrahy-
drofuro[30,40:5,6]pyrido[3,2-d]isoxazol-5-one (8f) This
compound 8f was prepared following the method described
for the preparation of the compound 8a, employing tetronic
acid (100 mg, 1 mmol), 3-hydroxy-4-methoxybenzalde-
hyde (5c) (152 mg, 1 mmol) and 3-methylisoxazol-5-
2amine (98 mg, 1 mmol) to afford a pure compound 8f as
in 420 mg, 88 % yield. Mp: 205–207 °C, 1H NMR
(300 MHz, DMSO-d6): d 3.80 (s, 3H, –CH3), 3.86 (s, 3H,
–OCH3), 5.38–5.62 (m, 3H, –CH2–O, –CH–), 6.97–7.11
(m, 3H, ArH), 9.35 (s, 1H, –OH); 13C NMR (75 MHz,
DMSO-d6): d 12.7 (–CH3, C3), 37.7 (–CH–, C4), 55.5
112.6 (–C–, C11), 117.3 (–C–, C1 ), 129.4 (–CH–, C4),
0
131.0 (–CH–,C3), 137.3 (–CH–,C6 ), 157.8 (–C–, C13, C16),
0
0
158.9 (–C–, C4 ), 161.0 (–C–, C2 ), 171.9 (–C–, C9); MS
(ESI): 352 [M ? H]?.
2-methoxy-9-(3,4,5-trimethoxyphenyl)-5,6,8,9-tetrahy-
drofuro[3,4-b][1,5]naphthyridin-8-one (14) This com-
pound 14 was prepared following the method described for
the preparation of the compound 8a, employing tetronic
acid (100 mg, 1 mmol), 3,4,5-trimethoxybenzaldehyde
(5a) (196 mg, 1 mmol) and 6-methoxypyridin-3-amine
(124 mg, 1 mmol) to afford a pure compound 14 as in
340 mg, 88 % yield. Mp: 205–207 °C, 1H NMR
(400 MHz, DMSO-d6): d 3.85 (s, 6H, (–OCH3)2), 3.88 (s,
6H, (–OCH3)2), 4.65 (s, 2H, –CH2–O), 4.77 (s, 1H, –CH–),
7.88 (d, 1H, J = 6.0 Hz, ArH), 8.07–8.09 (m, 3H, ArH);
13C NMR (75 MHz, DMSO-d6): d 36.1 (–CH–, C9), 60.9
0
0
(–CH3, O–CH3, C4 ), 68.6 (–CH2, C7), 111.1 (–C0H–, C2 ,
0
0
C5 ), 116.9 (–CH–, C6 ), 1021.3 (–C–, C10, C11, C1 ), 121.9
0
(–C–, C3 ), 145.6 (–C–, C4 ), 149.2 (–C–, C12), 149.5 (–C–,
C9), 157.0 (–C–, C3), 170.1 (–C–, C5); MS (ESI): 314
[M - H]?.
0
0
(–CH3, O–CH3, C2), 61.2 (–CH3, C3 ), 65.1 (–CH3, C4 ),
0
0
0
10-(4-hydroxy-3-methoxyphenyl)-2-methyl-6,7,9,10-te-
trahydro-1H-furo[3,4-b]pyrrolo [2,3-f]quinolin-9-one
(10) This compound 10 was prepared following the
method described for the preparation of the compound 8a,
employing tetronic acid (100 mg, 1 mmol), 4-hydroxy-3-
methoxybenzaldehyde (5b) (152 mg, 1 mmol) and
2-methyl-1H-indol-5-amine(146 mg, 1 mmol) to afford a
pure compound 10 as in 310 mg, 85 % yield. Mp:
65.4 (–CH3, C5 ), 72.0 (–CH2, C6), 104.2 (–CH–, C2 , C6 ),
110.2 (–CH–, C3), 111.8 (–C–, C13), 111.9 (–CH–, C4),
0
0
136.8 (–C–, C1 ), 141.0 (–C–, C11), 141.8 (–C–, C4 ), 147.9
0
0
(–C–, C10), 157.5 (–C–, C3 , C5 ), 157.6 (–C–, C2), 170.7
(–C–, C12), 174.9 (–C–, C8); MS (ESI): 383 [M - H]?.
Procedure of the SRB assay
1
313–315 °C, H NMR (200 MHz, DMSO-d6): d 1.21 (s,
3H, –CH3), 3.80 (s, 3H, –OCH3), 4.95–5.12 (dd, 2H,
J = 15.69, 15.69 Hz, –CH2–O), 5.28 (s, 1H, –CH–), 5.59
(d, 1H, ArH), 5.71 (d, 1H, ArH), 6.21 (s, 1H, ArH), 7.00
(d, 1H, ArH), 7.92 (s, 1H, ArH), 9.48 (s, 1H, –OH); 13C
NMR (75 MHz, DMSO-d6): d 13.7 (–CH3, C2), 36.0
The synthesized compounds (8a–f, 10, 12 and 14) have
been evaluated for their in vitro cytotoxicity in human
cancer cell lines. A protocol of 48-h continuous drug ex-
posure has been used, and a sulforhodamine B (SRB)
protein assay has been used to estimate cell viability or
123

Med Chem Res
growth. The cell lines were grown in DMEM medium
containing 10 % fetal bovine serum and 2 mM L-glutamine
and were inoculated into 96-well microtiter plates in 90 lL
at plating densities depending on the doubling time of in-
dividual 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 lL of the drug dilutions were added to the appropriate
microtiter wells already containing 90 lL of cells, resulting
in the required final drug concentrations. For each com-
pound, four concentrations (0.1, 1, 10 and 100 lM) were
evaluated and done in triplicate wells. Plates were incu-
bated for further 48 h, and assay was terminated by the
addition of 50 lL of cold trichloroacetic acid (TCA; final
concentration, 10 % TCA) and incubated for 60 min at
4 °C. The plates were washed five times with tap water and
air-dried. 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 tem-
perature. The residual dye was removed by washing five
times with 1 % acetic acid. The plates were air-dried.
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
wavelengths. Percent growth was calculated on a plate by
plate basis for test wells relative to control wells. The
above determinations were repeated three times. Percent
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 calcu-
lated from [(Ti - Tz)/(C - Tz)] *100  50, which is the
drug concentration resulting in a 50 % reduction in the net
protein increase (as measured by SRB staining) in control
cells during the drug incubation, where Tz  optical
density at time zero, OD of control  C and OD of test
growth in the presence of drug  Ti.
studies were carried out by employing extra-precision
docking of Glide 5.0.
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