Synthesis and anticancer activity of heteroaromatic linked 4β-amido podophyllotoxins as apoptotic inducing agents

Article abstract of DOI:10.1016/j.bmcl.2012.10.099

A series of different heteroaromatic linked 4β-amidopodophyllotoxin conjugates (16a-i, 17a-i and 18a-d) were synthesized and evaluated for anticancer activity against five human cancer cell lines. Among the series, one of the compound 17g showed significant antiproliferative activity in A549 (lung cancer) cell line. Flow cytometric analysis showed that 17g arrested the cell cycle in the G2/M phase leading to caspase-3 dependent apoptotic cell death. Further, Hoechst 33258 staining and DNA fragmentation assay also suggests that 17g induces cell death by apoptosis.

Full text of DOI:10.1016/j.bmcl.2012.10.099

Bioorganic & Medicinal Chemistry Letters 23 (2013) 273–280
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer activity of heteroaromatic linked 4b-amido
podophyllotoxins as apoptotic inducing agents
Ahmed Kamal a, , Jaki R. Tamboli a, M. V. P. S. Vishnuvardhan b, S. F. Adil c, V. Lakshma Nayak b,
⇑
S. Ramakrishna b
a Division of Organic Chemistry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
c Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of different heteroaromatic linked 4b-amidopodophyllotoxin conjugates ( 16a–i, 17a–i and
18a–d) were synthesized and evaluated for anticancer activity against five human cancer cell lines.
Among the series, one of the compound 17g showed significant antiproliferative activity in A549 (lung
cancer) cell line. Flow cytometric analysis showed that 17g arrested the cell cycle in the G2/M phase
leading to caspase-3 dependent apoptotic cell death. Further, Hoechst 33258 staining and DNA fragmen-
tation assay also suggests that 17g induces cell death by apoptosis.
Received 21 September 2012
Revised 19 October 2012
Accepted 23 October 2012
Available online 1 November 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Podophyllotoxin
Anticancer activity
Cell cycle analysis
Caspase-3
Apoptosis
Podophyllotoxin (1) is a naturally occurring aryltetralin lignan
obtained from a number of plant species of the Podophyllum fam-
ily.1,2 Podophyllotoxin as well as its congeners and derivatives ex-
hibit pronounced biological activity mainly as strong antiviral
agents and as antineoplastic drugs.3 However, its use as a lead
agent in the development of new anticancer drugs has led to the
discovery of semisynthetic podophyllotoxin derivatives like etopo-
side (VP-16, 2a) and teniposide (VM-26, 2b)4–6 that are currently
used in the chemotherapy for various types of cancer, including
small cell lung cancer, testicular carcinoma, lymphoma, kaposi’s
sarcoma, neuroblastoma, and soft tissue sarcoma.7–9 Their clinical
efficacy is due to its ability to inhibit the enzyme DNA topoisomer-
ase-II (topo-II) by stabilizing a cleavable enzyme–DNA-complex in
which the DNA is cleaved and covalently linked to enzyme.10 In
view of the development of drug resistance by cancer cells as well
as side effects associated with the use of these agents in clinic,
including myelosuppression, neutropenia, and nausea, the search
for new effective anticancer drugs based on podophyllotoxin scaf-
fold as a lead still remains an intense area of research.11,12
4b-position instead of a glycoside of etoposide, which proved to
be more potent than etoposide. GL-331, is also an inhibitor of
topo-II and induces apoptotic cell death16 through independent
mechanism which contributes to its cytotoxicity. This compound
has underwent phase II clinical trials for the treatment of various
cancers.17 A study on molecular-area oriented chemical modifica-
tions of podophyllotoxin has revealed certain structural features
that are critical for the topo-II inhibition.18 The comparative molec-
ular field analysis18,19 model further demonstrate that bulky
substituents at C-4 might be favorable for topo-II inhibition. In
the past few years, a variety of compounds have been prepared
and evaluated for their biological activity.20,21
Over the past two decades, pyrazole-containing compounds
have received considerable attention owing to their diverse
chemotherapeutic potentials including versatile antineoplastic
activities. Literature survey revealed that some pyrazoles have
been implemented as antileukemic,22–24 antitumor25–28 and anti-
proliferative29,30 agents beside their capability to exert remarkable
anticancer effects through inhibiting different types of enzymes
that play important roles in cell division.31–33 Pyrazolo[1,5-
a]pyrimidine derivatives have been reported as inhibitors of tyro-
sine kinase and cyclin-dependent kinases (CDK),34 and many of
them are known to block proliferation of various cancer cell lines.35
Some selective CDK inhibitors have been described in the litera-
ture, and some of them that are undergoing clinical trials, like
roscovitine (5),36 and SCH-727965 (6),37 as shown in Figure 1.
To overcome such problems, extensive synthetic efforts have
been carried out by a number of researchers. This has led to the
development of the N-linked congeners,13–15 for example, GL-331
(3a) and NPF (3b). GL-331 contains a p-nitroanilino moiety at the
⇑
Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.099

274
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
H
O
R
O
O
O
HO
OH
R
O
OH
O
O
O
O
NH
OH
O
O
O
O
O
O
H3CO
OCH3
O
OH
H3CO
OCH3
OCH3
2a
H3CO
R = NO2
OCH3
R = CH3; Etoposide
1
2b
Teniposide
R =
GL-331 3
NPF
S
R = F
4
-O
H
N
N+
NH
N
N
HO
N
N
N
N
N
N
HN
Et
SCH-727965
6
OH
5
Figure 1. Chemical structures of podophyllotoxin (1), etoposide (2a), teniposide (2b), GL-331(3), NPF (4), roscovitine (5), SCH-727965 (6).
O
O
O
R1
R2
O
N
R1
R2
OCH3
CH3
OCH3
R1
R2
iv
i
O
O
R3
R3
9a-i
O
7a-i
8a-i
R3
vi
v
ii
HN
N
R1
R2
OCH3
N
OH
O
R1
R2
O
N
13a-i
N
R3
N
N
10a-i
R1
R2
OCH3
R3
vii
O
11a-d
iii
HN
R3
OH
R1
R2
O
14a-i
R3
N
7a,10a,14a: R1 = R2 = R3 = H
N
N
7b,10b,14b: R1 = H; R2 = NO2; R3 = H
7c,10c,14c: R1 = H; R2 = CH3; R3 = H
7d,10d,14d: R1 = R3 = H; R2 = OCH3
7e,10e,14e: R1 = R2 = OCH3; R3 = H
7f,10f,14f: R1 = R2 = R3 = OCH3
7g,10g,14g: R1 = R2 = Cl; R3 = H
7h,10h,14h: R1 = R2 = F; R3 = H
7i,10i,14i: R1 = R3 = H; R2 = F
R1
R2
OH
O
R3
12a-d
12a: R1 = H; R2 = NO2; R3 = H
12b: R1 = R3 = H; R2 = OCH3
12c: R1 = R2 = OCH3; R3 = H
12d: R1 = R2 = R3 = OCH3
Scheme 1. Reagents and conditions: (i) Dimethyl oxalate, NaOMe, THF, rt, 8 h; (ii) 3-amino-5-phenyl-1H-pyrazole, cat. HCl, ethanol, reflux, 2 h; (iii) 2 N NaOH, MeOH, reflux,
2 h; (iv) NH2OHꢀHCl, CH3OH, reflux, 3 h; (v) 1 N NaOH, THF, 60 °C, 1 h; (vi) NH2NH2ꢀH2O, acetic acid, reflux, 1 h; (vii) hydroxylammonium chloride, 2 N NaOH, MeOH, 80 °C 1 h.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
275
NH2
O
O
O
O
H3CO
OCH3
OCH3
i, 12a-d
15
i, 10a-i
C6H5
R1
R1
i, 14a-i
R2
R3
R1
R2
R3
N
N
H
N
O
R2
N
N
N
R3
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
OCH3
OCH3
OCH3
18a-d
16a-i
17a-i
16a,17a: R1 = R2 = R3 = H
18a: R1 = H; R2 = NO2; R3 = H
18b: R1 = R3 = H; R2 = OCH3
18c: R1 = R2 = OCH3; R3 = H
18d: R1 = R2 = R3 = OCH3
16b,17b: R1 = H; R2 = NO2; R3 = H
16c,17c: R1 = H; R2 = CH3; R3 = H
16d,17d: R1 = R3 = H; R2 = OCH3
16e,17e: R1 = R2 = OCH3; R3 = H
16f,17f: R1 = R2 = R3 = OCH3
16g,17g: R1 = R2 = Cl; R3 = H
16h,17h: R1 = R2 = F; R3 = H
16i,17i: R1 = R3 = H; R2 = F
Scheme 2. Reagents and conditions: (i) EDCI/HOBt, CH2Cl2, 0 °C-rt, 8 h.
Table 1
Anticancer activity of compounds 16a–i, 17a–i and 18a–d expressed in
4b-amidopodophyllotoxin congeners as outlined in Schemes 1 and
2. A variety of substituted acetophenones 7a–i were oxalylated by
dimethyl oxalate in the presence of sodium methoxide, to give b-
diketoester 8a–i. Treatment of the aryl b-diketoester with hydroxyl
ammonium chloride in refluxing methanol yielded isoxazole ester
9a–i which was then hydrolyzed to give 5-phenyl isoxazole-3-car-
boxylic acid 10a–i. On the other side, aryl diketo ester 8a–i was
readily converted into pyrazole ester 13a–i by coupling with hydra-
zine in the presence of acetic acid which was then hydrolyzed into
5-phenyl-1H-pyrazole-3-carboxylic acids39 14a–i. Thus the treat-
ment of the b-diketoester 8a–i with 3-amino-5-phenyl-1H-pyra-
zole affords the cyclodehydrated methyl 2,7-diphenyl-
pyrazolo[1,5-a]pyrimidine-5-carboxylate 11a–d which was then
hydrolyzed to 2,7-diphenylpyrazolo[1,5-a]pyrimidine-5-carboxylic
acid40 12a–d. 4b-Amino podophyllotoxin (15) has been synthesized
as described in our previously reported procedure.41,42 This upon
treatment with different heteroaromatic carboxylic acids on amide
bond formation with 10a–i, 14a–i and 12a–d using EDCI/HOBt af-
ford the required conjugates 16a–i, 17a–i and 18a–d in good yields,
respectively,47,48 as shown in Scheme 2.
l
M
Compd
A549a
HT-29b
ACHNc
B-16d
HELAe
16a
16b
16c
16d
16e
16f
16g
16h
16i
17a
17b
17c
17d
17e
17f
17g
13.6
17.9
24.7
38.1
23.1
19.8
31.07
12.7
20.6
28.9
19.4
22.3
5.3
39.5
15.2
2.1
15.8
21.2
10.6
11.2
17.3
9.5
19.5
22.6
27.9
32.1
38.4
25.1
11.2
14.1
19.9
26.5
20.6
26.5
8.4
36.3
16.8
7.1
10.8
26.3
10.9
5.7
17.5
26.4
28.5
30.3
23.1
34.2
16.8
16.4
15.6
30.2
23.5
25.8
11.6
31.6
27.1
15.9
32.5
18.3
17.7
17.3
19.3
13.8
7.61
2.3
16.3
16.8
20.3
22.5
12.7
23.4
22.7
27.5
23.5
16.3
NA
16.6
9.5
45.3
18.3
9.5
25.6
26.1
18.4
18.3
10.7
12.7
1.39
2.5
5.9
7.5
5.26
11.2
13.3
18.4
12.6
21.2
16.6
7.5
14.6
16.9.
9.4
25.1
11.5
9.3
14.9
15.2
12.1
8.9
17h
17i
18a
18b
18c
18d
14.5
9.2
1.81
1.02
14.4
13.7
1.52
2.8
These compounds have been tested for thier cytotoxicity
against a different set of human cancer cell lines A-549 (lung), B-
16 (melanoma), HeLa (cervical), HT-29 (colon) and ACHN (renal)
using MTT assay43 and the values obtained were compared to a
standard drug like etoposide as shown in Table 1. The screening re-
sults suggest that compound 17g exhibits significant activity in
Etoposide
Podophyllotoxin
2.8
3.6
a
b
c
Lung cancer.
Colon cancer.
Renal cancer.
Melanoma cancer.
Cervical cancer.
d
e
A549 with IC50 in (2.1
lM) and good activity in other cell lines like
HT-29 (7.1 M), B-16 (9.3
l
l
M) and HeLa (9.5 M), where as other
l
Encouraged by these observations and in continuation of our
conjugates showed promising moderate activity in different cancer
cell lines. Therefore, it was considered of interest to examine the
effect of 17g on cell cycle progression by FACS analysis.44 A549
research work towards the development of new molecules based
on podophyllotoxins,38 in the present investigation, we have
designed and synthesized three types of heteroaromatic linked
cells were treated with the compounds 17g at 1 and 3 lm along

276
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
Figure 2. FACS analysis of compounds 17g, podophyllotoxin and etoposide in A-549 lung cancer cell line.
Table 2
Cell cycle distribution of A549 cell line with podo, 17g and etoposide
Compd
Control
Sub-G1
G1 phase
S phase
G2/M phase
1.08
6.77
2.05
1.83
1.25
8.30
85.33
63.35
27.71
78.31
62.32
31.49
3.69
2.27
8.96
3.67
4.10
2.42
9.89
27.68
61.28
16.24
32.33
57.86
17g (1
17g (3
l
l
M)
M)
Podo (1 lM)
Etoposide (1
Etoposide (3
l
l
M)
M)
Figure 4. Effect of compounds 17g and etoposide on caspase-3 activity: A-549 cells
were treated for 48 h with 1 and 3 M concentrations of compounds 17g. Etoposide
is used as positive control. Values indicate the mean SD of two different
l
a
experiments performed in triplicates.
Figure 3. DNA fragmentation of compounds 17g and etoposide in A-549 lung
cancer cells. Lane-1: marker (50 bp), Lane-2: C-1, untreated control DNA (1
Lane-3: C-2, untreated control DNA (5 l), Lane-4: 17g at 3 M, and Lane-5: etop
(etoposide) at 3 M. Loaded DNA for 17g, and etoposide is 5 l.
ll),
with the standard etoposide 2a (1 and 3 lM) and incubated for
48 h. The data obtained infers that compounds show more
l
l
l
l

A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
277
Figure 5. Hoechst staining in A-549 lung cancer cell line, (A) A-549 control cells, (B) 17g at 1 lM, (C) 17g at 3 lM, (D) etoposide-3 lM.
accumulation of cells in G2/M phase compared with untreated
control with (17g) 27.6% at 1 M, 61.28% at 3 M and (control)
9.89% while standard showed 32.33% at 1 M and 57.86% at
M resulting G2/M arrest as shown in Figure 2. To confirm the
exhibit promising anticancer activity at micromolar (
tration. Among the series one compound 17g exhibited significant
l
M) concen-
l
l
l
anticancer activity (IC50, 2.1 M) against A-549 cancer cell line and
l
3
l
flow cytometric analysis of this compound shows arrest of cell cy-
cle in the G2/M phase. Hoechst 33258 staining and DNA fragmen-
tation assay reveals that this compound induces cell death by
apoptosis. Further, activation of caspase-3 also suggested that this
compound produces apoptotic cell death. Hence from this data it
can be deduced that coupling of a heteroaromatic carboxylic acid
moieties to 4b-aminopodophyllotoxin scaffold through an amide
functionality has shown promising anticancer activity and could
be considered as a lead to be taken up for further structural mod-
ifications of such podophyllotoxin conjugates.
effect of 17g on the induction of apoptosis, fragmentation of chro-
mosomal DNA was analyzed45 (Fig. 3).
The DNA fragmentation analysis reveals that compounds 17g
induced a discrete ladder pattern in A-549 cell line at 3 lM after
48 h of incubation thereby showing significant fragmentation.
Etoposide also exhibits DNA fragmentation, however, no fragmen-
tation was observed in untreated cells. Chromatin condensation
and fragmented nuclei are known as the classic characteristics of
apoptosis.46 To investigate the apoptotic inducing effect of the
potent compound (17g) by Hoechst staining (H 33258) method
in A-549 cancer cell line (Fig. 5), cells were treated with 17g at 1
Acknowledgment
and 3 lM concentrations for 24 h wherein etoposide was used as
the standard. Manual field quantification of apoptotic cells based
on cytoplasmic condensation, presence of apoptotic bodies, nucle-
ar fragmentation and relative fluorescence of the test compounds
(17g and etoposide) revealed that there was significant increase
in the percentage of apoptotic cells. It is well known49–51 that the
cell cycle arrest at G2/M phase is shown to induce cellular apopto-
sis (see Table 2).
Hence it was considered of interest to examine whether the
cytotoxicity of 17g and etoposide is by virtue of apoptotic cell
death. Cystein aspartase group namely caspase plays a crucial role
in the induction of apoptosis and amongst them caspase-3 happens
to be one of the effector caspase. This prompted us to treat A549
cells with compounds 17g and etoposide to examine the activation
of caspases. The results indicate that there is nearly fourfold
J.R.T. is thankful to DST (India) for the award of research
fellowship.
References and notes
1. Kaye, S. B. Curr. Opin. Oncol. 1998, 10, S15.
2. MacRae, W. D.; Hudson, J. B.; Towers, B. H. N. Planta Med. 1993, 55, 531.
3. Canel, C.; Moraes, R. M.; Dayan, F. E.; Ferreira, D. Phytochemistry 2000, 54, 115.
4. Stahelin, H. Eur. J. Cancer 1973, 9, 215.
5. Stahelin, H. Eur. J. Cancer 1970, 6, 303.
6. (b) Schacter, L. Semin. Oncol. 1996, 23, 1; (b) Witterland, H. A.; Koks, H. C.;
Beijinen, H. J. Pharm. World Sci. 1996, 18, 63.
7. Belani, P. C.; Doyle, A. L.; Aisner, J. Cancer Chemother. Pharmacol. 1994, 34, S118.
8. Hande, R. K. Eur. J. Cancer 1998, 34, 1514.
9. Pommier, Y.; Fesen, R. M.; Goldwasser, F. In Cancer Chemotherapy and
Biotherapy: Principles and Practice; Chabner, A. B., Longo, L. D., Eds.;
Lippincott-Raven: Philadelphia, PA, 1996; p 435.
10. Macdonald, T. L.; Lehnert, E. K.; Loper, J. T.; Chow, K. C.; Ross, W. E. In DNA
Topoisomerases in Cancer; Potmesil, M., Kohn, K. W., Eds.; Oxford University:
New York, 1991; p 199. and references cited therein.
11. You, Y. J. Curr. Pharm. Des. 2005, 11, 1695.
12. Gordaliza, M.; Castro, M. A.; Corral, J. M. M.; San Feliciano, A. Curr. Pharm. Des.
1811, 2000, 6.
13. Lee, K. H.; Beers, S. A.; Mori, M.; Wang, Z. Q.; Kuo, Y. H.; Li, L.; Liu, S. Y.; Cheng, J.
Y.; Han, F. S.; Cheng, Y. C. J. Med. Chem. 1990, 33, 1364.
14. Wang, Z. Q.; Kuo, Y. H.; Schnur, D.; Bower, J. P.; Liu, S. Y.; Han, F. S.; Chang, J. Y. J.
Med. Chem. 1990, 33, 2660.
15. (a) Zhou, X. M.; Wang, Z. Q.; Chang, J. Y.; Chen, H. X.; Cheng, Y. C.; Lee, K. H. J.
Med. Chem. 1991, 34, 3346; (b) Xiao, Z.; Xiao, Y.-D.; Golbraikh, A.; Tropsha, A.;
Lee, K. H. J. Med. Chem. 2002, 45, 2294; (c) VanVilet, D. S.; Tachibana, Y.;
Bastow, K. F.; Huang, E. S.; Lee, K. H. J. Med. Chem. 2001, 44, 1422.
induction in caspase-3 activity in cells treated with 1
tration and six- to sevenfold induction with 3 M concentration
by 17g compound. Interestingly under similar conditions, etopo-
side (3 M) also induced the caspase activity by nearly sixfold as
lM concen-
l
l
compared to control as shown in Figure 4. Therefore activation of
caspase-3 by 17g indicates that they are capable of inducing apop-
tosis in A-549 cells.
In summary, twenty two conjugates of heteroaromatic linked
4b-amido podophyllotoxins have been synthesized and evaluated
for their anticancer potential against five human cancer cell lines
(A-549, HeLa, MCF-7, HT-29 and ACHN). Some of these compounds

278
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
16. (a) Chen, Y.; Su, H. Y.; Wang, H. C.; Wu, M. J.; Chen, C. J.; Tseng, H. S. Anticancer
Res. 2005, 25, 4203; (b) Huang, S. T.; Shu, H. C.; Lee, C. C.; Chen, T. L.; Whang-
Peng J. Apoptosis 2000, 5, 79; (c) Huang, S. T.; Lee, C. C.; Chao, Y.; Shu, H. C.;
Chen, T. L.; Chen, L. L.; Chen, H. M.; Yuan, C. C.; Whang-Peng, J. Pharm. Res.
1999, 16, 997.
17. Lee, H. K.; Wang, K. H. J. Food Drug Anal. 1995, 3, 209.
18. Xiao, Z.; Xiao, Y. D.; Golbraikh, A.; Tropsha, A.; Lee, K. H. J. Med. Chem. 2002, 45,
2294.
19. Cho, S. J.; Tropsha, A.; Suffness, M.; Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1996,
39, 1383.
20. Pan, J. L.; Wang, Y. G.; Chen, Y. Z. Curr. Sci. 1997, 72, 268.
21. Zhou, X. M.; Wang, Z.; Chen, H. X.; Cheng, Y. C.; Lee, K. H. Pharm. Res. 1993, 10,
214.
22. Daidone, G.; Maggio, B.; Raffa, D.; Plescia, S.; Schillaci, D.; Raimondi, M. V. Il
Farmaco 2004, 59, 413.
23. Chou, L. C.; Huang, L. J.; Yang, J. S.; Lee, F. Y.; Teng, C. M.; Kuo, S. C. Bioorg. Med.
Chem. 2007, 15, 1732.
24. Manetti, F.; Brullo, C.; Magnani, M.; Mosci, F.; Chelli, B.; Crespan, E.; Schenone,
S.; Naldini, A.; Bruno, O.; Trincavelli, M. L.; Maga, G.; Carraro, F.; Martini, C.;
Bondavalli, F.; Botta, M. J. Med. Chem. 2008, 51, 1252.
25. Li, J.; Zhao, Y. F.; Zhao, X. L.; Yuan, X. Y.; Gong, P. Arch. Pharm. Chem. Life Sci.
2006, 339, 593.
26. Xia, Y.; Dong, Z. W.; Zhao, B. X.; Ge, X.; Meng, N.; Shin, D. S.; Miao, J. Y. Bioorg.
Med. Chem. 2007, 15, 6893.
27. Xia, Y.; Fan, C. D.; Zhao, B. X.; Zhao, J.; Shin, D. S.; Miao, J. Y. Eur. J. Med. Chem.
2008, 43, 2347.
28. Farag, A. M.; Mayhoub, A. S.; Barakat, S. E.; Bayomi, A. H. Bioorg. Med. Chem.
2008, 16, 881.
29. Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa, P.; Mosti, L.;
Menozzi, G.; Carraro, F.; Naldini, A.; Bernini, C.; Manettic, F.; Botta, M. Bioorg.
Med. Chem. Lett. 2004, 14, 2511.
30. Daidone, G.; Raffa, D.; Maggio, B.; Raimondi, M. V.; Plescia, F.; Schillaci, D. Eur. J.
Med. Chem. 2004, 39, 219.
31. Warshakoon, N. C.; Wu, S.; Boyer, A.; Kawamoto, R.; Renock, S.; Xu, K.; Pokross,
M.; Evdokimov, A. G.; Zhou, S.; Winter, C.; Walter, R.; Mekel, M. Bioorg. Med.
Chem. Lett. 2006, 16, 5687.
32. Huang, S.; Lin, R.; Yu, Y.; Lu, Y.; Connolly, P. J.; Chiu, G.; Li, S.; Emanuel, S. L.;
Middleton, S. A. Bioorg. Med. Chem. Lett. 2007, 17, 1243.
33. Zhu, G.-D.; Gong, J.; Gandhi, V. B.; Woods, K.; Luo, Y.; Liu, X.; Guan, R.;
Klinghofer, V.; Johnson, E. F.; Stoll, V. S.; Mamo, M.; Li, Q.; Rosenberg, S. H.;
Giranda, V. L. Bioorg. Med. Chem. 2007, 15, 2441.
1H NMR (300 MHz, CDCl3): d 2.71–2.88 (m, 1H), 3.01 (dd, 1H, J = 3.7, 13.5 Hz),
3.75 (s, 6H), 3.78 (s, 3H), 3.84–3.94(m,1H), 4.38–4.57 (m, 2H), 5.40 (dd, 1H,
J = 3.0, 7.7 Hz), 6.0 (d, 2H, J = 6.7 Hz), 6.23 (s, 2H), 6.48 (d, 1H, J = 15), 6.84 (s,
1H), 6.95 (s, 1H), 7.51 (m, 3H), 7.81 (dd, 2H, J = 2.2, 7.7 Hz); 13C NMR (300 MHz,
CDCl3): d 37.4, 41.7, 43.6, 46, 48.4, 56, 61.1, 68.9, 98.8, 101.7, 106.4, 108, 109.1,
109.9, 125.9, 126.4, 127.8, 129.3, 131.1, 132.3, 134.6, 137.1, 147.6, 152.7, 158.1,
158.7, 159.4, 174; MS (ESI): 585 [M+H]+; HRMS (ESI) Calcd for C32H29O9N2
[M+H]+ 585.1873. Found: 585.1865. 4b-5-(4-Nitrophenyl)isoxazole-3-amido
podophyllotoxin (16b): The compound 16b was prepared according to the
general procedure. Yield 88%; mp: 164–165 °C; ½a D25
ꢂ
ꢃ69.0 (c 0.5 in CHCl3), 1
H
NMR (300 MHz, CDCl3): d 2.99–3.12 (m, 2H), 3.73 (s, 6H), 3.80 (s, 3H), 4.12 (dd,
1H, J = 7.1, 14 Hz), 4.44–4.63 (m, 2H), 5.46 (d, 1H, J = 10.7 Hz), 5.96 (d, 2H,
J = 6.7 Hz), 6.30 (s, 2H), 6.51 (s, 1H), 6.83 (s, 1H), 7.19 (s, 1H), 7.80 (d, 2H,
J = 8.6 Hz), 8.26 (d, 2H, J = 8.7 Hz) 13C NMR (300 MHz, CDCl3): d 37.4, 41.8, 43.7,
47.7, 56.1, 60.9, 69.1, 101.6, 102.9, 103.4, 108, 109.1, 110, 124.2, 128.6, 130.9,
132.2, 134.7, 137.1, 145.7, 147.5, 148.3, 152.5, 152.9, 153.7, 161.8, 174.7; MS
(ESI): 652 [M+Na]+; HRMS (ESI) Calcd for C32H27O11N3Na [M+Na]+ 652.1556.
Found: 652.1543. 4b-5-p-Tolylisoxazole-3-amido podophyllotoxin (16c): The
compound 16c was prepared according to the general procedure. Yield 83%;
mp: 143–144 °C; ½a D25
ꢂ
ꢃ30.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.44
(s, 3H), 3.02–3.10 (m, 2H), 3.76 (s, 6H), 3.81 (s, 3H), 3.89–3.99 (m, 1H), 4.45–
4.58 (m, 2H), 5.42 (d, 1H, J = 9.8 Hz), 6.00 (d, 2H, J = 3.7 Hz), 6.28 (s, 2H), 6.45 (s,
1H), 6.84 (s, 1H), 6.93 (s, 1H), 7.22–7.36 (m, 3H), 7.71 (d, 2H, J = 8.3 Hz); 13C
NMR (300 MHz, CDCl3): d 20.6, 37.2, 41.7, 43.8, 47.7, 55.9, 60.7, 69, 101.4, 103,
107.4, 108.3, 109.7, 124.9, 125.2, 128.1, 129.3, 131.9, 134.5, 136.7, 140, 145.6,
146.3, 147.1, 147.7, 152.2, 161.9, 174.4; MS (ESI): 598 [M+H]+. 4b-5-(4-
Methoxyphenyl)isoxazole-3-amido podophyllotoxin (16d):The compound 16d
was prepared according to the general procedure. Yield 87%; mp: 161–162 °C;
½
a 2D5
ꢂ
ꢃ67.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.94–3.04 (m, 1H),
3.08 (dd, 1H, J = 4.5, 14.3 Hz), 3.73 (s, 6H), 3.77 (s, 3H), 3.89 (s, 3H), 4.10 (dd,
1H, J = 7.5, 14 Hz), 4.35–4.49 (m, 2H), 5.38 (dd, 1H, J = 4.5, 7.5 Hz), 6.01 (d, 2H,
J = 2.2 Hz), 6.17 (s, 2H), 6.28 (s, 1H), 6.79 (s, 1H), 6.84 (s, 1H), 7.03 (d, 2H,
J = 9.0 Hz), 7.76 (d, 2H, J = 9.0 Hz); 13C NMR (300 MHz, CDCl3): d 37.3, 41.3,
43.5, 48.6, 55.4, 55.7, 61.1, 69, 97.7, 101.7, 108, 109, 109.7, 114.7, 118.9, 127.5,
127.8, 132.1, 134.6, 137.2, 147.4, 148.5, 152.5, 158, 158.7, 161.8, 171.8, 174.1;
MS (ESI): 637 [M+Na]+. 4b-5-(3,4-Dimethoxyphenyl)isoxazole-3-amido
podophyllotoxin(16e): The compound 16e was prepared according to the
general procedure. Yield 72%; mp: 141–142 °C; ½a D25
ꢂ
ꢃ96.0 (c 0.5 in CHCl3), 1
H
NMR (500 MHz, CDCl3): d 2.78–2.95 (m, 2H), 3.68 (s, 6H), 3.73 (s, 3H), 3.76–
3.85 (m, 1H), 3.87 (s, 3H), 3.89 (s, 3H), 4.36–4.48 (m, 2H), 5.40 (dd, 1H, J = 3.9,
7.9 Hz), 5.98 (d, 2H, J = 6.5 Hz), 6.19 (t, 2H, J = 4.3 Hz), 6.27 (s, 1H), 6.43 (s, 1H),
6.69–6.91 (m, 2H), 7.02 (d, 2H, J = 3.5 Hz); 13C NMR (500 MHz, CDCl3): d 36.9,
41.1, 43, 48, 55.3, 55.4, 55.5, 60.2, 68, 97.6, 101.3, 107.3, 107.5, 108.4, 109, 110,
110.9, 118.4, 119, 125.7, 127.2, 131.4, 134, 146.8, 147.8, 149, 150.9, 151.9,
157.5, 158.1, 173.7; MS (ESI): 667 [M+Na]+; HRMS (ESI) Calcd for
34. Shenone, S.; Bruno, O.; Bondavalli, F.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa,
P.; Donnini, S.; Santoro, A.; Ziche, M.; Manetti, F.; Botta, M. Eur. J. Med. Chem.
2004, 39, 939.
35. Krystof, V.; Moravcova, D.; Paprskarova, M.; Barbier, P.; Peyrot, V.; Hlobikova,
A.; Havlicek, L.; Stramd, M. Eur. J. Med. Chem 2006, 41, 1405.
36. McClue, S. J.; Blake, D.; Clarke, R.; Cowan, A.; Cummings, L.; Fischer, P. M.;
MacKenzie, M.; Melville, J.; Stewart, K.; Wang, S.; Zhelev, N.; Zheleva, D.; Lane,
D. P. Int. J. Cancer 2002, 102, 463.
C
34H32O11N2Na
[M+Na]+
667.1903.
Found:
667.1885.
4b-5-(3,4,5-
Trimethoxyphenyl)isoxazole-3-amido podophyllotoxin (16f): The compound 16d
was prepared according to the general procedure. Yield 77%; mp: 152–153 °C;
½
a 2D5
ꢂ
ꢃ112.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.68–2.90 (m, 2H),
37. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.-C.; Barbosa, S. A.; Hunt, J. T.;
Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B.-C.; Zhao, R.; Bednarz,
M. S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.; Roongta, U.; Kamath, A.;
Marathe, P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y.
F.; Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
38. Kamal, A.; Suresh, P.; Ramaiah, M. J.; reddy, A. M.; Imthiajali, S.; Pushpavalli, S.
N. C. V. L.; Lavanya, A. A.; Pal-Bhadra, M. Bioorg. Med. Chem 2012, 20, 2054.
39. Zeng, L. F.; Zhang, H. S.; Yun-Hua Wang, Y. H.; Sanchez, T.; Zheng, Y. T.;
Neamati, N.; Long, Y. Q. Bioorg. Med. Chem. Lett. 2008, 18, 4521.
40. Kamal, A.; Tamboli, J. R.; Ramaiah, M. J.; Adil, S. F.; Rao, G. K.; Viswanath, A.;
reddy, A. M.; Pushpavalli, S. N. C. V. L.; Pal-Bhadra, M. Chem. Med. Chem. 2012, 7,
1453.
41. Kamal, A.; Laxman, N.; Ramesh, G. Bioorg. Med. Chem. Lett. 2000, 10, 2059.
42. Kamal, A.; Kumar, B. A.; Arifuddin, M.; Dastidar, S. G. Bioorg. Med. Chem. 2003,
11, 5135.
43. Ren, J.; Wu, L.; Xin, W. Q.; Chen, X.; Hu, K. Bioorg. Med. Chem. Lett. 2012, 22,
4778.
44. Kamal, A.; Ramakrishna, G.; Raju, P.; Rao, A. V. S.; Viswanath, A.; Nayak, V. L.;
Ramakrishna, S. Eur. J. Med. Chem. 2011, 46, 2427.
45. Kamal, A.; Reddy, A. M.; Suresh, P.; Nayak, V. L.; Shetti, V. C. R. N. C.; Rajesh, V.
C. R. N. C. R.; Rao, N. S.; Tamboli, J. R.; Shaik, T. B.; Vishnuvardhan, M. V. P. S.;
Ramakrishna, S. Eur. J. Med. Chem. 2012, 47, 530.
46. Shankar, R.; Chakravarti, B.; Singh, U. S.; Ansari, M. I.; Deshpande, S.; Dwivedi,
S. K. D.; Bid, H. K.; Konwar, R.; Kharkwal, G.; Chandra, V.; Dwivedi, A.; Hajela, K.
Bioorg. Med. Chem. 2009, 17, 3847.
3.65 (s, 6H), 3.72 (s, 3H), 3.80 (s, 6H), 3.82 (s, 3H), 4.03 (dd, 1H, J = 4.5, 14 Hz),
4.33–4.46 (m, 2H), 5.35 (dd, 1H, J = 3.9, 7.3 Hz), 5.93 (d, 2H, J = 2.4 Hz), 6.16 (d,
2H, J = 7.7 Hz), 6.26 (s, 1H), 6.82 (d, 2H, J = 9.7 Hz), 7.15 (d, 2H, J = 8.5 Hz); 13C
NMR (500 MHz, CDCl3): d 37.7, 41.8, 43.7, 45.2, 47.8, 56.2, 60.7, 61, 69.1, 101.4,
101.6, 103, 103.4, 108.1, 109.2, 110.1, 124.3, 128.6, 132.1, 134.8, 145.8, 147.6,
148.4, 152.6, 153.8, 159.8, 161.9, 170.3, 174.6; MS (ESI): 675 [M+H]+; HRMS
(ESI) Calcd for C35H35O12N2 [M+H]+ 675.2160. Found: 675.2182. 4b-5-(3,4-
Dichlorophenyl)isoxa-zole-3-amido podophyllotoxin (16g): The compound 16g
was prepared according to the general procedure. Yield 76%; mp: 188–189 °C;
½
a 2D5
ꢂ
ꢃ63.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.92–3.12 (m, 2H),
3.73 (s, 6H), 3.77 (s, 3H), 3.80–3.98 (m, 1H), 4.39–4.58 (m, 2H), 5.39 (dd, 1H,
J = 2.0, 6.4 Hz), 6.0 (d, 2H, J = 6.4 Hz), 6.23 (s, 2H), 6.46 (d, 1H, J = 14 Hz), 6.81 (t,
1H, J = 4.1 Hz), 6.99 (s, 1H), 7.56–7.71 (m, 2H), 7.92 (S, 1H); 13C NMR (300 MHz,
CDCl3): d 37.3, 41.5, 43.6, 48.4, 56, 60.6, 68.7, 98.9, 101.7, 107.9, 109, 109.9,
116.4, 116.7, 122.6, 127.7, 128, 128.1, 132, 143.5, 137.1, 147.5, 148.5, 152.5,
158.2, 158.5, 162.5, 165.8, 174; MS (ESI): 653 [M+H]+. 4b-5-(3,4-
Difluorophenyl)isoxazole-3-amido podophyllotoxin (16h): The compound 16h
was prepared according to the general procedure. Yield 79%; mp: 139–140 °C;
½
a 2D5
ꢂ
ꢃ77.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 3.00 (dd, 1H, J = 4.5,
14.3 Hz), 3.04 (m, 1H), 3.76 (s, 6H), 3.82 (s, 3H), 3.94 (t, 1H, J = 9.8 Hz), 4.50 (dd,
1H, J = 6.7, 9.0 Hz), 4.62–4.68 (m, 1H), 5.44 (dd, 1H, J = 4.5, 7.5 Hz), 6.0 (dd, 2H,
J = 1.5, 6.0 Hz), 6.31 (s, 2H), 6.55 (s, 1H), 6.72 (d, 1H, J = 3.0 Hz), 6.97–7.11 (m,
2H), 7.14 (d, 1H, J = 3.7 Hz), 7.91–8.01 (m, 1H); 13C NMR (500 MHz, CDCl3): d
37.3, 41.7, 43.7, 48.4, 56.2, 60.7, 68.8, 101.8, 102.7, 105.1, 105.6, 108.1, 109,
110.2, 127.8, 128.6, 128.9, 132.3, 134.5, 134.6, 137.2, 147.7, 148.5, 152.6, 155.6,
158.4, 158.6, 161.4, 174.1; MS (ESI): 621 [M+H]+; HRMS (ESI) Calcd for
47. General procedure for the synthesis of compounds 16a–i, 17a–i and 18a–d: To a
solution of compound 15 (1.0 mmol) in dichloromethane (20 ml) was added
EDCIꢀHCl (1.2 mmol) and HOBt (0.1 mmol). Then added corresponding
hetroaromatic acids 10a–i, 14a–i and 12a–d (1.0 mmol) and the reaction
mixture was stirred at room temperature for 10 h. The reaction was monitored
by TLC. After completion of reaction, water was added to reaction mixture and
extracted with dichloromethane (2 ꢁ 30 ml). The solvent was evaporated
under reduced pressure to afford the crude product which was further purified
by column chromatography on silica gel using ethyl acetate and hexane as
solvent system to obtain the pure products as solids. 4b-5-Phenylisoxazole-3-
amido podophyllotoxin (16a): The compound 16a was prepared according to
C
32H27O9N2F2
[M+H]+
621.1679.
Found:
621.1676.
4b-5-(4-
Fluorophenyl)isoxazole-3-amido podophyllotoxin (16i): The compound 16i was
prepared according to the general procedure. Yield 78%; mp: 150–151 °C; ½a D25
ꢂ
ꢃ86.0 (c 0.5 in CHCl3), 1H NMR (500 MHz, CDCl3): d 2.93–3.08 (m, 2H), 3.73 (s,
6H), 3.76 (s, 3H), 3.80–3.92 (m,1H), 4.38–4.50 (m, 2H), 5.39 (dd, 1H, J = 2.8,
6.9 Hz), 5.9 (d, 2H, J = 3.9 Hz), 6.18 (s, 2H), 6.34 (s, 1H), 6.87 (d, 2H, J = 14 Hz),
7.16–7.34 (m, 2H), 7.83 (dd, 2H, J = 5.2, 6.7 Hz); 13C NMR (500 MHz, CDCl3): d
37.3, 41.6, 43.8, 48.5, 56.1, 60.7, 68.7, 99, 101.8, 108, 109, 109.8, 116.5, 116.8,
127.8, 128.1, 128.2, 132.2, 134.6, 137.2, 147.6, 148.6, 152.6, 158.2, 158.6, 165.9,
174.1; MS (ESI): 603 [M+H]+. 4b-5-Phenyl-1H-pyrazole-3-amido podophyllotoxin
(17a): The compound 17a was prepared according to the general procedure.
the general procedure. Yield 89%; mp: 155–156 °C; ½a D25
ꢃ43.0 (c 0.5 in CHCl3),
ꢂ

A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
279
Yield 80%; mp: 145–146 °C; ½a D25
ꢂ
ꢃ53.0 (c 0.5 in CHCl3), 1H NMR (300 MHz,
110.1, 123.6, 126.6, 128.4, 128.8, 129.6, 130.6, 131.7, 132.5, 134.6, 136.4, 137.4,
144.6, 147.2, 147.7, 148.6, 149.1, 149.2, 152.6, 157.7, 162.8, 173.9; MS (ESI):
755 [M]+. 4b-7-(4-Methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine-5-amido
podophyllotoxin (18b): The compound 18b was prepared according to the
CDCl3): d 2.79–2.94 (m, 2H), 3.67 (s, 6H), 3.75 (s, 3H), 3.84–3.98 (dd, 1H,
J = 10.1, 13.7), 4.26–4.57 (m, 2H), 5.30 (t, 1H, J = 6.9), 5.84 (s, 2H), 6.13 (s, 1H),
6.27–6.52 (m, 1H), 6.72 (s, 1H), 6.88–7.07 (m, 1H), 7.30–7.44 (m, 4H), 7.58 (dd,
2H, J = 7.7, 16 Hz); 13C NMR (300 MHz, CDCl3): d 37.4, 41.7, 43.6, 47.7, 56.1,
56.2, 60.7, 69, 101.5, 103.5, 108, 108.3, 109, 109.9, 125.4, 128.5, 129, 129.1,
132, 134.8, 145.5, 146.3, 147.4, 148.2, 152.5, 162, 174.7; MS (ESI): 584 [M+H]+;
HRMS (ESI) Calcd for C32H30O8N3 [M+H]+ 584.2027. Found: 584.2025. 4b-5-(4-
Nitrophenyl)-1H-pyrazole-3-amido podophyllotoxin (17b): The compound 17b
was prepared according to the general procedure. Yield 84%; mp: 203–204 °C;
general procedure. Yield 85%; mp: 179–180 °C; ½a D25
ꢂ
ꢃ63.0 (c 0.5 in CHCl3), 1
H
NMR (300 MHz, CDCl3): d 3.08 (dd, 1H, J = 4.0, 14.1 Hz), 3.10–3.16 (m, 1H), 3.78
(s, 6H), 3.83 (s, 3H), 3.88 (s, 3H), 3.90–3.98 (m, 1H), 4.51 (dd, 1H, J = 6.0, 9.0 Hz),
4.70 (d, 1H, J = 4.0 Hz), 5.48 (dd, 1H, J = 4.0, 8.0 Hz), 6.0 (d, 2H, J = 3.0 Hz), 6.36
(s, 2H), 6.61 (s, 1H), 6.86 (s, 1H), 7.07 (s, 1H), 7.12 (d, 2H, J = 9.0 Hz), 7.39–7.51
(m,3H), 8.05 (dd, 3H, J = 7.0, 13 Hz), 8.35 (d, 2H, J = 9.0 Hz); 13C NMR (300 MHz,
CDCl3): d 37.5, 41.9, 43.8, 48.3, 55.4, 56.2, 60.7, 68.8, 94.2, 101.6, 103.8, 108.3,
109.2, 110.1, 114, 122.6, 126.3, 126.6, 128.6, 128.7, 129.3, 131.4, 132.3, 132.4,
134.7, 137.3, 146.9, 147.7, 148.5, 149.5, 152.6, 157.1, 162.2, 163.5, 174; MS
(ESI): 741 [M+H]+; HRMS (ESI) Calcd for C42H37O9N4 [M+H]+ 741.2555. Found:
741.2553. 4b-7-(3,4-Dimethoxyphenyl)-2-phenyl pyrazo lo[1,5-a]pyrimidine-5-
amido podophyllotoxin (18c): The compound 18c was prepared according to the
½
a 2D5
ꢂ
ꢃ74.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.90–3.05 (m, 2H),
3.72 (s, 3H), 3.74 (s, 6H), 3.89 (t, 1H, J = 9.8 Hz), 4.31–4.63 (m, 2H), 5.48 (dd, 1H,
J = 4.7, 7.9 Hz), 5.97 (s, 2H), 6.29 (s, 2H), 6.52 (s, 1H), 6.85 (s, 1H), 7.73–7.86 (m,
1H), 8.0 (d, 2H, J = 8.6 Hz), 8.25 (d, 2H, J = 8.3 Hz); MS (ESI): 629 [M+H]+. 4b-5-
p-Tolyl-1H-pyrazole-3-amido podophyllotoxin (17c): The compound 17c was
prepared according to the general procedure. Yield 80%; mp: 171–172 °C; ½a D25
ꢂ
ꢃ38.0 (c 0.5 in CHCl3), 1H NMR (400 MHz, CDCl3): d 2.41 (s, 3H), 2.70–2.96 (m,
2H), 3.68 (s, 6H), 3.77 (s, 3H), 3.91–4.03 (m, 1H), 4.28–4.45 (m, 2H), 5.18–5.29
(m, 1H), 5.83 (d, 2H, J = 13.9 Hz), 6.13 (s, 2H), 6.27 (s, 1H), 6.68–7.10 (m, 2H),
7.20 (d, 2H, J = 7.3 Hz), 7.42 (d, 2H, J = 5.8 Hz); 13C NMR (300 MHz, CDCl3): d
20.9, 37, 41.6, 43.6, 47.7, 56.1, 60.6, 68.9, 101.5, 103.1, 108, 108.8, 109.9, 125.2,
125.4, 128.3, 129.8, 132.1, 134.7, 137, 139.2, 145.4, 146.4, 147.3, 148.1, 152.4,
162.1, 174.7; MS (ESI): 598 [M+H]+; HRMS (ESI) Calcd for C33H32O8N3 [M+H]+
598.2183. Found: 598.2182.4b-5-(4-Methoxyphenyl)-1H-pyrazole-3-amido
podophyllotoxin (17d): The compound 17d was prepared according to the
general procedure. Yield 80%; mp: 167–168 °C; ½a D25
ꢂ
ꢃ40.0 (c 0.5 in CHCl3), 1
H
NMR (300 MHz, CDCl3): d 2.91 (dd, 1H, J = 4.5, 14.3 Hz), 2.96–3.05 (m, 1H), 3.70
(s, 6H), 3.73 (s, 3H), 3.94 (s, 3H), 3.95 (s, 3H), 4.03 (dd, 1H, J = 7.5, 15 Hz), 4.36–
4.58(m, 2H), 5.35 (dd, 1H, J = 4.5, 7.5 Hz), 5.93 (s, 2H), 6.22 (s, 2H), 6.48 (s, 1H),
6.77 (s, 1H), 6.98 (t, 2H, J = 3.7 Hz), 7.28–7.42 (m, 3H), 7.74 (s,1H), 7.82–8.0 (m,
4H); MS (ESI): 771 [M+H]+; HRMS (ESI) Calcd for C43H39O10N4 [M+H]+
771.2660.
Found:
771.2680.
4b-2-Phenyl-7-(3,4,5-trimethoxyphenyl)
pyrazolo[1,5-a]pyrimidine-5-amido podophyllotoxin (18d): The compound 18d
was prepared according to the general procedure. Yield 76%; mp: 183–184 °C;
general procedure. Yield 81%; mp: 144–145 °C; ½a D25
ꢂ
ꢃ64.0 (c 0.5 in CHCl3), 1
H
½
a 2D5
ꢂ
ꢃ118.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 3.08–3.14 (m, 2H),
NMR (500 MHz, CDCl3): d 2.78–2.91 (m, 2H), 3.68 (s, 9H), 3.74 (s, 3H), 3.95 (t,
1H, J = 7.9 Hz), 4.29–4.41 (m, 2H), 5.24 (br s, 1H), 5.84 (d, 2H, J = 9.7 Hz), 6.13 (s,
2H), 6.28 (s, 1H), 6.73 (s, 1H), 6.86–7.02 (m, 3H), 7.45 (d, 2H, J = 6.9 Hz); 13C
NMR (300 MHz, CDCl3): d 37.4, 41.7, 43.7, 47.8, 55.3, 56.2, 60.7, 69, 101.6,
102.7, 108.1, 109, 110, 114.6, 121, 126.9, 128.6, 132.2, 134.8, 137.1, 145.3,
146.6, 147.5, 148.3, 152.5, 160.2, 162.1, 174.6; MS (ESI): 614 [M+H]+; HRMS
(ESI) Calcd for C33H32O9N3 [M+H]+ 614.2133. Found: 614.2127. 4b-5-(3,4-
Dimethoxyphenyl)-1H-pyrazole-3-amido podophyllotoxin (17e): The compound
17e was prepared according to the general procedure. Yield 84%; mp: 178–
3.79 (s, 6H), 3.83 (s, 3H), 3.90–3.96 (m, 1H), 3.99 (s, 9H), 4.48–4.57 (m, 1H),
4.72 (d, 1H, J = 4.5 Hz), 5.48 (dd, 1H, J = 3.7, 7.5 Hz), 6.01 (d, 2H, J = 2.2 Hz), 6.35
(s, 2H), 6.62 (s, 1H), 6.85 (s, 1H), 7.11 (s, 1H), 7.27 (s, 1H), 7.41–7.52 (m,2H),
7.60 (s, 2H), 7.84 (s, 1H), 7.99–8.12 (m, 2H); MS (ESI): 801 [M+H]+; HRMS (ESI)
Calcd for C44H41O11N4 [M+H]+ 801.2764. Found: 801.2759
48. Evaluation of in vitro anti-cancer activity: The cytotoxic activity of the
compounds was determined using MTT assay. 1 ꢁ 10ꢃ4 cells/well were
seeded in 200 ll DMEM, supplemented with 10% FBS in each well of 96-well
micro culture plates and incubated for 24 h at 37 °C in a CO2 incubator.
Compounds, diluted to the desired concentrations in culture medium, were
added to the wells with respective vehicle control. After 48 h of incubation,
10 ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
(5 mg/ml) was added to eachwell and the plates were further incubated for 4 h.
Then thesupernatant from each well was carefully removed, formazoncrystals
179 °C; ½a 2D5
ꢂ
ꢃ89.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.74–2.98 (m,
2H), 3.69 (s, 6H), 3.75 (s, 3H), 3.79–3.84 (m,1H), 3.86 (s, 3H), 3.88 (s, 3H), 4.30–
4.52 (m, 2H), 5.33 (d, 1H, J = 8.1 Hz), 5.93 (d, 2H, J = 6.7 Hz), 6.21 (t, 2H, J = 5.4),
6.30 (s, 1H), 6.47 (s, 1H), 6.72–6.93 (m, 2H), 7.06–7.19 (m, 2H); MS (ESI): 666
[M+Na]+; HRMS (ESI) Calcd for C34H33O10N3Na [M+Na]+ 666.2063. Found:
666.2089. 4b-5-(3,4,5-Trimethoxyphenyl)-1H-pyrazole-3-amido podophyllotoxin
(17f): The compound 17f was prepared according to the general procedure.
were dissolved in 100 ll of DMSO and absorbance at 540 nm wavelength was
recorded. Cell cycle analysis: To determine the effect of compound on the stages
Yield 77%; mp: 165–166 °C; ½a D25
ꢂ
ꢃ105.0 (c 0.5 in CHCl3), 1H NMR (300 MHz,
of cell cycle, A549 cells (1 ꢁ 10ꢃ6) were seeded in six-well plates and treated
CDCl3): d 2.79–2.96 (m, 2H), 3.67 (s, 6H), 3.75 (s, 3H), 3.82 (s,6H), 3.83 (s,
3H),4.09 (dd, 1H, J = 6.7, 14.3 Hz), 4.32–4.48 (m, 2H), 5.33 (d, 1H, J = 6.7 Hz),
5.91 (d, 2H, J = 4.7 Hz), 6.13–6.20 (m, 2H), 6.29 (s, 1H), 6.77 (d, 2H, J = 3.7 Hz),
6.90 (s, 1H), 7.12 (d, 1H, J = 7.5 Hz); 13C NMR (300 MHz, CDCl3): d 37.8, 41.6,
43.6, 47.7, 56, 60.3, 60.6, 60.8, 69, 101.5, 102.9, 103.2, 108, 108.9, 109.8, 124.2,
128.4, 128.8, 132, 134.7, 137, 138.6, 145.7, 145.9, 147.4, 148.2, 152.4, 153.5,
161.8, 174.7; MS (ESI): 674 [M+H]+; HRMS (ESI) Calcd for C35H36O11N3 [M+H]+
674.2344. Found: 674.2346. 4b-5-(3,4-Dichlorophenyl)-1H-pyrazole-3-amido
podophyllotoxin (17g): The compound 17g was prepared according to the
with compound 17g at 1 and 3 lM for 48 h. After 48 h treatments, both floating
and trypsinized adherent cells were collected and fixed with 70% ethanol. After
fixation cells were washed with PBS and stained with 50 mg/mL propidium
iodide in hypotonic lysis buffer (0.1% sodium citrate, 0.1% Triton X-100)
containing DNase-free RNase-A for 20 min. Stained cells were analyzed using
fluorescence-activated cell sortercaliber (Becton Dickinson). DNA fragmentation
assay: Cells were seeded (1 ꢁ 10ꢃ6) in six-well plates. After incubation of 48 h
cells were treated with compounds 17g at 1 and 3 lM. After 48 h of treatment,
cells were collected and centrifuged at 2500 rpm for 5 min at 4 °C. Pellet was
collected and washed with phosphate buffered saline (PBS). Then added 100
general procedure. Yield 80%; mp: 180–181 °C; ½a D25
ꢂ
ꢃ56.0 (c 0.5 in CHCl3), 1
H
ll
NMR (300 MHz, CDCl3): d 2.93–3.09 (m, 2H), 3.72 (s, 6H), 3.80 (s, 3H), 4.13 (dd,
1H, J = 6.7, 14.3 Hz), 4.43–4.60 (m, 2H), 5.44 (dd, 1H, J = 3.0, 6.7 Hz), 5.96 (d, 2H,
J = 3.0 Hz), 6.29 (s, 2H), 6.49 (s, 1H), 6.81 (s, 1H), 7.05 (s, 1H), 7.41–7.52 (m, 2H),
7.72 (d, 1H, J = 2.2 Hz); 13C NMR (300 MHz, CDCl3): d 37.3, 41.8, 43.7, 47.9, 56.1,
60.8, 69.1, 101.7, 103.9, 108.1, 109.1, 110, 124.8, 127.2, 128.4, 129, 131.1,
132.2, 133, 133.3, 134.8, 137.1, 144, 145.2, 147.6, 148.3, 152.5, 161.5, 174.8;
of lysis buffer centrifuged at 3000 rpm for 5 min at 4 °C and collected
supernant. And added 10 ml of 10% SDS and 10 ml of (50 mg/ml) RNase-A
and incubated for 2 h at 56 °C. After incubation proteinase K (25 mg/ml) was
added and further incubated at 37 °C for 2 h. Then added 65 ml of 10 M
ammonium acetate and 500 ml of ice cold ethanol and mixed well. And these
samples were incubated at 80 °C for 1 h. After incubation samples were
centrifuged at12000 rpm for 20 min at 4 °C. After centrifuge pellet was washed
with 80% ethanol and air dried for 10 min at room temperature. Dissolved the
pellet in 50 ml of TE buffer and DNA laddering was determined by using 2%
agarose gel electrophoresis inTE Buffer. Caspase-3: To determine the caspase-3
activity of the active compound 17g AFC conjugated Ac-DEVD substrate was
used. Lung cancer cells (A-549) were seeded in six well plates with the
confluence of per (2.5 ꢁ 10ꢃ6) well and are treated with the compounds at 1
MS
(ESI):
652
[M]+.4b-5-(3,4-Difluorophenyl)-1H-pyrazole-3-amido
podophyllotoxin (17h): The compound 17h was prepared according to the
general procedure. Yield 77%; mp: 158–159 °C; ½a D25
ꢂ
ꢃ82.0 (c 0.5 in CHCl3), 1
H
NMR (300 MHz, CDCl3): d 2.92–3.04 (m, 2H), 3.74 (s, 6H), 3.77 (s, 3H), 3.91 (t,
1H, J = 8.9 Hz), 4.41–4.55 (m, 2H), 5.40 (dd, 1H, J = 3.7, 7.5 Hz), 5.96 (d, 2H,
J = 5.3 Hz), 6.24 (s, 2H), 6.48 (s, 1H), 6.72 (s, 1H), 6.82 (s, 1H), 6.92–7.14 (m, 3H),
7.63–7.74 (m, 1H); MS (ESI): 620 [M+H]+; HRMS (ESI) Calcd for C32H28O8N3F2
[M+H]+ 620.1832. Found: 620.1830. 4b-5-(4-Fluorophenyl)-1H-pyrazole-3-
amido podophyllotoxin (17i): The compound 17i was prepared according to
and 3 lM concentration along with standard etoposide. After incubation for
48 h cell were washed with PBS and then cells were scraped in to the PBS and
centrifuged at 2000 rpm for 10 min at 4 °C. Pellet was resuspended in 80 ml of
lysis buffer, pellet was passed through insulin syringe followed by incubation
of suspension on ice for 20–30 min centrifuged the lysate at 13,200 rpm for
20 min at 4 °C and transferred the supernatant to fresh tubes. In a 96 well black
polystyrene plate, 50 ml of 2X assay buffer, 50 ml cell lysate and 2 ml of
caspase-3substrate were taken. The reaction was allowed to take place for 1 h.
The fluorescence generated by the release of the fluorogenic group AFC on
cleavage by caspase-3 was measured by excitation at 400 nm and emission at
505 nm for every 5 min over 1 h. Protein was estimated by Bradford’s method
and normalized accordingly. Morphological analysis for apoptosis with Hoechst
staining: Cells were seeded at a density of 10,000 cells over 18-mmcoverslips
and incubated for 24 h. Then, the medium was replaced, and cells were treated
the general procedure. Yield 81%; mp: 170–171 °C; ½a D25
ꢃ58.0 (c 0.5 in CHCl3),
ꢂ
1H NMR (300 MHz, CDCl3): d 2.94–3.10 (m, 2H), 3.73 (s, 6H), 3.81 (s, 3H), 3.91–
4.02 (m,1H), 4.41–4.60 (m, 2H), 5.41 (dd, 1H, J = 2.6, 7.1 Hz), 5.95 (d, 2H,
J = 3.5 Hz), 6.28 (s, 2H), 6.47 (s, 1H), 6.81 (s, 1H), 6.01 (s, 1H), 7.13 (t, 2H,
J = 8.4 Hz), 7.57 (dd, 2H, J = 5.2, 8.6 Hz); 13C NMR (300 MHz, CDCl3): d 37.4,
41.8, 43.7, 47.8, 56.1, 60.7, 69.1, 101.6, 103.4, 108.1, 109.1, 110, 116.1, 116.4,
124.9, 127.4, 127.5, 128.6, 132.3, 134.7, 137.1, 147.6, 148.4, 152.5, 161.4, 161.7,
174.6; MS (ESI): 602 [M+H]+; HRMS (ESI) Calcd for C32H29O8N3F [M+H]+
602.1935. Found: 602.1933. 4b-7-(4-Nitrophenyl)-2-phenylpyrazolo[1,5-
a]pyrimidine-5–amido podophyllotoxin (18a): The compound 18a was
prepared according to the general procedure. Yield 84%; mp: 174–175 °C;
½
a 2D5
ꢂ
ꢃ62.0 (c 0.5 in CHCl3), 1H NMR (300 MHz, CDCl3): d 2.99 (dd, 1H, J = 14.3,
5.2 Hz), 3.03–3.14 (m, 1H), 3.77 (s, 6H), 3.79 (s, 3H), 3.87 (t, 1H, J = 9.0 Hz), 4.49
(dd, 1H, J = 6.7, 9.0 Hz), 4.66 (d, 1H, J = 4.5 Hz), 5.44 (dd, 1H, J = 4.5, 8.3 Hz), 6.01
(d, 2H, J = 1.5 Hz), 6.28 (s, 2H), 6.58 (s, 1H), 6.82 (s, 1H), 7.13 (s, 1H), 7.37–7.50
(m, 3H), 7.86 (s,1H), 7.94 (d, 3H, J = 8.3 Hz), 8.47 (s, 3H); 13C NMR (300 MHz,
CDCl3): d 37.4, 41.8, 43.8, 48, 56.2, 60.6, 68.7, 95.2, 101.6, 105.4, 108.3, 109.1,
with compound 17g at 1 and 3 lM for 24 h. Cells treated with vehicle (0.001%
DMSO) were included as controls for all experiments. An overnight treatment,
Hoechst 33342 (Sigma–Aldrich) were added to medium at a concentration of
0.5 mg/mL containing 4% paraformaldehyde. After incubation for 30 min at
37 °C, cells from each dish were captured from randomly selected fields under

280
A. Kamal et al. / Bioorg. Med. Chem. Lett. 23 (2013) 273–280
fluorescent microscope (Leica, Germany) to qualitatively determine the 50. Iyer, S.; Chaplin, D. J.; Rosenthal, D. S.; Boulares, A. M.; Li, L.; Smulson, M. E.
proportion of viable and apoptotic cells based on their relative fluorescence
and nuclear fragmentation.
Cancer Res. 1998, 58, 4510.
51. Zhu, H.; Zhang, J.; Xue, N.; Hu, Y.; Yang, B.; He, Q. Invest. New Drugs 2010, 28,
49. Browne, L. J.; Gude, C.; Rodriguez, H.; Steele, R. E.; Bhatnager, A. J. Med. Chem.
1991, 34, 725.
493.