Synthesis of 4β-N-polyaromatic substituted podophyllotoxins: DNA topoisomerase inhibition, anticancer and apoptosis-inducing activities

Article abstract of DOI:10.1016/j.bmc.2010.10.042

A new class of 4β-N-polyaromatic substituted podophyllotoxin congeners have been synthesized and evaluated for their DNA topoisomerase-II (topo-II) inhibition as well as anticancer potential in some human cancer cell lines. The ease of synthesis and inter

Full text of DOI:10.1016/j.bmc.2010.10.042

Bioorganic & Medicinal Chemistry 18 (2010) 8493–8500
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 4b-N-polyaromatic substituted podophyllotoxins: DNA
topoisomerase inhibition, anticancer and apoptosis-inducing activities
Ahmed Kamal a, , B. Ashwini Kumar a, Paidakula Suresh a, Satyam Kumar Agrawal b, Gousia Chashoo b,
⇑
Shashank K. Singh b, A. K. Saxena b
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of 4b-N-polyaromatic substituted podophyllotoxin congeners have been synthesized and
evaluated for their DNA topoisomerase-II (topo-II) inhibition as well as anticancer potential in some
human cancer cell lines. The ease of synthesis and interesting biological activities make the present series
of polyaromatic-podophyllotoxin congeners as a promising new structure for the development of new
anticancer agents based on podophyllotoxin scaffold.
Received 25 August 2010
Revised 14 October 2010
Accepted 15 October 2010
Available online 10 November 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Podophyllotoxin
Polyarylamines
DNA topoisomerase inhibition
Etoposide
Anticancer activity
Apoptosis
1. Introduction
which exhibit improved topoisomerase II inhibition. The replace-
ment of the C-4 sugar moiety of etoposide and teniposide with a
Podophyllotoxin (1), a well-known naturally occurring aryltetr-
alin lignan, extracted from the roots of Podophyllotoxin peltatum,
whose medicinal properties particularly cytotoxicity have been
well-known for centuries.1–3 Attempts to use 1 for the treatment
of cancer were mostly unsuccessful due to the severe side effects,
hence this cyclolignan has been employed as a lead compound
for the design and development of anticancer agents. This led to
the synthesis of a number of semisynthetic derivatives that are
in clinical use for the treatment of a variety of malignancies. The
prominent ones are etoposide (2), teniposide (3), and etopophos
(4), interestingly these differ substantially in their mechanism of
action compared to the parent compound podophyllotoxin. Etopo-
side and other analogs inhibit DNA topo-II, while podophyllotoxin
inhibit the assembly in the microtubulin.4,5 In view of the develop-
ment of drug resistance by cancer cells as well as side effects asso-
ciated with the use of these agents in clinic (which include
myelosuppression, eutropenia, and nausea), the search for new
effective anticancer drugs with 1 as a lead compound still remains
as an intense area of research.
non-sugar substituent has improved the therapeutic value of
etoposide.6 The C-4 non-sugar substituent can be linked through
O-, S- or N-linkage. In general, the O-linked (ethers, esters) and
S-linked (thioethers) compounds are less active in comparison to
the N-linked congeners7–10 like NPF (7) and GL-331 (8). Prior
molecular area-oriented structure–activity relationship (SAR)
studies11 and the composite pharmacophore model proposed by
MacDonald et al.12 designated the C-4 molecular area of 1 analogs
as a variable region. The comparative molecular field analysis
(CoMFA) models generated by Lee and co-workers13,14 further
demonstrated that bulky substituents at C-4 might be favorable
for DNA topo-II inhibition. These postulates are compatible with
the excellent activity profiles of NK 611 (5), TOP-53 (6), and
GL-331.15 In addition, both GL-331 and TOP-53 showed good
topoisomerase II inhibitory and antitumor activity, while the
drug-resistance profiles are significantly different from those of
1. This investigation thus suggest the important role played by var-
ious substitutions at C-4 in the activity profile of such analogs and
the feasibility of optimizing the activity through rational C-4 mod-
ification in this class of compounds.16
The recent synthetic studies on podophyllotoxin have been fo-
cused on the synthesis of the C-4 non-sugar substituted analogs,
Polycyclic aromatic hydrocarbons have been considered as one
of the most prevalent antitumor agents. Becker and co-workers,
explained an exploratory synthetic and biological evaluation of
unique polycyclic aromatic compounds such as chrysene (9) and
1-pyrene methylamine derivatives (10).17–19 These compounds
⇑
Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.042

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A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
possess a broad spectrum of chemotherapeutic activity against
both murine and human tumors. Interestingly, some of them like
crisnatol (11) and 502U83 (12) were chosen for clinical develop-
ment, as shown in Figure 1. Based on the information available
on the structure–activity relationship studies and in continuation
of our ongoing project on the design and development of structur-
ally modified podophyllotoxin analogs,16,20 we became interested
in the synthesis of different polycyclic aromatic hydrocarbons
linked to the epipodophyllotoxin ring system.21 Hence, in the pres-
ent work we report the synthesis of a series of 4b-N-polyaryl
substituted podophyllotoxin congeners, and their cytotoxic activi-
ties against certain tumor cell lines (502713, HCT-15, HEP-2, IMR-
32, A-549, DU-145, and PC-3). Furthermore, DNA topo-II inhibition
and cell cycle analysis was evaluated for some representative com-
pounds of this series.
these were reacted in the crude form for the next step to yield the
final products. The synthetic strategy developed for the prepara-
tion of these new 4b-N-polyaryl substituted podophyllotoxin cong-
eners is shown in Scheme 2. Two of the amino substituted
precursors, that is, benzophenone amine 16 and its mustard 19
were obtained from 4-fluoro-40-nitrobenzophenone 15, which in
turn was obtained by the reaction of fluorobenzene 13 and 4-nitro-
benzoyl chloride 14 as shown in Scheme 1. All the synthesized
compounds were characterized by 1H NMR, 13C NMR, IR, and mass
spectral data.
3. Biological activity
Compounds 20a–f and 21a–e were evaluated for their antican-
cer activity in selected human cancer cell lines, that is, colon
(502713, HCT-15), liver (HEP-2), neuroblastoma (IMR-32), lung
(A-549), and prostate (DU-145, PC-3) origin by employing the sul-
forhodamine B (SRB) assay method.23 The results are summarized
in Table 1 and compared to etoposide and 5-fluorouracil. Most of
these new compounds exhibited significant anticancer activity
compared to etoposide and camptothecin as well as 5-fluorouracil.
DNA topo-II is the pharmacological target of clinical relevance
for most of the podophyllotoxin lignans;24 therefore, some of the
representative compounds ( 20c, 20d, and 21d) were evaluated
for its inhibition. All these compounds exhibited comparable
2. Chemistry
These new 4b-polyarylamino podophyllotoxin congeners were
synthesized by coupling of various substituted polyarylamine
compounds with podophyllotoxin by employing BF3ꢀOEt2/NaI
reagent system.22 The reaction proceeded via the formation of
4b-iodopodophyllotoxin or 4b-iodo-40-O-demethylpodophyllotox-
in as intermediates. Since these iodo intermediates are highly reac-
tive and susceptible to nucleophilic attack in presence of moisture,
H
O
R1
OH
R1
R2
R3
H
O
O
O
O
HO
R2
CH3
O
Etoposide
OH
(2)
O
O
O
O
Teniposide
(3)
OH
H
S
O
O
H3CO
OH
OCH3
Etopophos (4)
NK-611 (5)
P
CH3
OH
OCH3
OH
H3CO
OCH3
OR3
(1)
Podophyllotoxin
NMe2
NMe2
H
R
N
HN
N
HN
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OH
OH
R = F
(NPF) (7)
TOP 53 (6)
R = NO2 (GL 331) (8)
OH
NH R OH
NH
CH3
OH
HCl
OH
CH3
NH
CH3
CH3SO3H
OCH2CH2OH
OH
Chrycene
(10)
(9)
1-Pyrenemethylamine
(12)
Crisnatol (11)
502U83
Figure 1.

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
8495
O
O
i
F
+
NO2
Cl
O2N
F
13
14
15
iii
ii
O
O
OH
H2N
F
O2N
N
17
16
OH
iv
O
O
ii
Cl
Cl
O2N
N
H2N
N
18
19
Cl
Cl
Scheme 1. Reagents and conditions: (i) AlCl3, CH2Cl2, 30 min, rt. (ii) SnCl2ꢀ2H2O, MeOH, reflux, 3 h. (iii) Diethanolamine, DMSO, reflux, 5 h. (iv) (a) MsCl, TEA, CH2Cl2, rt, 4 h.
(b) NaCl, DMF, reflux, 15 min.
inhibition of DNA topo-II catalytic activity comparable to etoposide
and the results are illustrated in Figure 2.
Further it was considered of interest to evaluate the DNA frag-
mentation aspect for 20d. This compound at 0.5, 1, 5, and 10 lM
concentrations induced DNA fragmentation in leukemia (MOLT-
4) cells after 24 h incubation as shown in Figure 3. In order to
understand the mode of action of 20d, its cell cycle effects were
corded on Perkin–Elmer model 683 or 1310 spectrometer with so-
dium chloride optics. Mass spectra are recorded on CEC-21-100B,
Finnigan Mat 1210, or Micromass 7070 spectrometer operating
at 70 eV using a direct inlet system. Optical rotations are measured
on Jasco Dip 360 digital polarimeter. Melting points are deter-
mined on an electrothermal melting point apparatus and are
uncorrected. TLC is performed with E. Merck precoated silica gel
plates (60F-254) with iodine as a developing agent. Acme, India
silica gel (100–200 mesh) is used for column chromatography.
examined. Thus 20d was tested at 0.5, 1, 5, and 10
tions by treatment with leukemia (MOLT-4) cells and compared to
camptothecin (5 M). This study indicated that it blocks the G1
lM concentra-
l
phase of cell cycle and there was an increase in sub-G1 cell popu-
5.1.1. 4-Fluoro-40-nitrobenzophenone (15)
lation indicating apoptosis as seen from Figure 4.
To a solution of fluorobenzene (13) (2.71 mL, 20 mmol) in dry
CH2Cl2 was added 4-nitrobenzoyl chloride (14) (4.44 g, 24 mmol)
and stirred for 5 min. To this stirred solution AlCl3 (5.32 g,
40 mmol) was added and stirred for 30 min. This reaction mixture
was poured in ice cold water. This solution was filtered, extracted
with ethyl acetate, and dried over anhydrous Na2SO4. This solvent
was removed under vacuum and the crude product was subjected
to column chromatography using ethyl acetate/hexane (2:3)
affords 14, 3.70 g in 75% yield. 1H NMR (200 MHz, DMSO-d6 +
CDCl3): d 7.26 (t, 2H, J = 8.2 Hz), 7.82–7.98 (m, 4H), 8.38 (d, 2H,
J = 8.2 Hz).
4. Conclusion
In summary, new 4b-N-polyaromatic-podophyllotoxin congeners
havingdifferentpolyarylsubstitutionsincludingabenzophenoneand
its mustard moieties were prepared and evaluated against some hu-
man cancer cell lines. Almost all the compounds exhibited significant
in vitro anticancer activity at micromolar (lM) concentration. These
new podophyllotoxins also inhibited DNA topo-II and interestingly
the cell cycle studies revealed that these compounds possess apopto-
sis-inducing activity. The insights obtained from these investigations
are considered useful for the development of newer structures based
on the podophyllotoxin lignans.
5.1.2. 4-Amino-40-fluorobenzophenone (16)
The compound 15 (735 mg, 3 mmol) dissolved in methanol
(40 mL) and added SnCl2ꢀ2H2O (3.38 g, 15 mmol) was refluxed for
3 h. The reaction mixture was cooled and the methanol was evap-
orated under vacuum, and the residue was carefully adjusted to pH
8 with saturated NaHCO3 solution and then extracted with ethyl
acetate (2 ꢁ 30 mL). The combined organic phase was washed with
brine (15 mL), dried over anhydrous Na2SO4, and evaporated under
vacuum. This crude product was subjected to column chromatog-
raphy using ethyl acetate/hexane (1:1) affords 16, 516 mg in 80%
5. Experimental section
5.1. General methods
The NMR spectra are recorded on Varian Gemini 200 MHz
spectrometer, using TMS as an internal reference. IR spectra are re-

8496
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
I
I
OH
O
O
O
O
O
ii
O
O
i
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
OH
1
iii
iii
R
R
HN
HN
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OCH3
OH
21 a-f
20 a-f
O
R
=
,
,
,
F
,
,
e
a
d
b
c
O
Cl
.
N
f
Cl
Scheme 2. Reagents and conditions: (i) BF3ꢀOEt2/NaI, CH3CN, rt, 30 min. (ii) BF3ꢀOEt2/NaI, CH2Cl2, rt, 5 h. (iii) R-NH2, THF, BaCO3, rt, 8 h.
Table 1
Anticancer activity data of compounds 20a–f and 21a–e
Compound
IC50 (lM)
Colon
Liver
Neuroblastoma
IMR-32
Lung
Prostate
502713
HCT-15
HEP-2
A-549
DU-145
PC-3
20a
20b
20c
20d
20e
20f
21a
21b
21c
21d
21e
Etoposide
Camptothecin
5FUa
9.4
5.5
0.002
0.1
4.1
0.1
—
0.001
0.002
0.1
6.3
6.7
3
—
6.7
5.5
—
0.003
19
6.3
2.3
10
0.2
0.001
1.3
6.3
0.08
18
8.8
7.1
21
6.6
—
2.2
—
3.7
18
2.7
2.2
0.0003
0.3
3.7
0.02
4.8
7.9
4.3
0.0001
0.1
6.7
2.6
—
0.5
—
0.0003
6.0
2.6
0.03
12
0.09
0.0001
0.002
6.2
0.9
8.2
0.0001
0.0001
0.1
0.08
0.9
0.0009
0.04
17
3.08
8.3
0.002
7.1
0.9
8.6
1
0.0002
0.01
1.3
0.9
0.7
0.3
1
1.7
0.0003
0.2
3.08
0.03
2.4
1.4
0.1
nd
4.6
0.067
3.6
nd = not determined.
a
5FU = 5-Fluorouracil is one of the control drug.
yield. 1H NMR (200 MHz, DMSO-d6 + CDCl3): d 5.34 (br s, 2H), 6.65
(d, 2H, J = 9.0 Hz), 7.16 (t, 2H, J = 9.0 Hz), 7.58 (d, 2H, J = 9.0 Hz),
7.69–7.78 (m, 2H).
5.1.3. 4-[Di(2-hydroxyethyl)amino]-40-nitrorobenzophenone (17)
The compound 15 (2.45 g, 10 mmol) dissolved in DMSO (25 mL)
and added diethanolamine (3.15 g, 15 mmol) was refluxed for 5 h.

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
8497
The reaction mixture was cooled and extracted with ethyl acetate
and ice cold water. The combined organic phase was washed with
brine (15 mL), dried over anhydrous Na2SO4, and evaporated under
vacuum. This crude product was subjected to column chromatog-
raphy using ethyl acetate/hexane (8:1) affords 17, 2.64 g in 80%
yield. 1H NMR (200 MHz, DMSO-d6 + CDCl3): d 3.65–3.88 (m, 4H),
3.90–4.46 (m, 4H), 6.65 (d, 2H, J = 8.2 Hz), 7.65 (d, 2H, J = 8.2 Hz),
7.82 (d, 2H, J = 8.2 Hz), 8.35 (d, 2H, J = 8.2 Hz).
1 2 3 4
5
6
7
8
9
20c
20d
1 2 3 4 5 6 7 8 9 10
5.1.4. 4-[Di(2-chloroyethyl)amino]-40-nitrorobenzophenone (18)
The compound 17 (1.65 g, 5 mmol) dissolved in dry CH2Cl2
(20 mL) was added triethylamine (2.09 mL, 15 mmol) at 0 °C and
stirred for 5 min. To this reaction mixture mesyl chloride
(0.77 mL, 10 mmol) was added drop wise and stirred for 4 h at
room temperature, dichloromethane was removed under vacuum
and extracted with ethyl acetate. The combined organic phase
was dried over anhydrous Na2SO4 and evaporated under vacuum.
This crude product was subjected to next step without any further
purification. The above crude compound was dissolved in DMF was
added NaCl (1.45 g, 25 mmol) and heated to reflux for 15 min. The
reaction mixture was cooled and extracted with ethyl acetate,
dried over anhydrous Na2SO4, and evaporated under vacuum. This
crude product was subjected to column chromatography using
ethyl acetate/hexane (9:1) as eluent affords 18, 1.28 g in 70% yield.
1H NMR (200 MHz, DMSO-d6 + CDCl3): d 3.74 (t, 4H, J = 6.7 Hz),
3.91 (t, 4H, J = 6.0 Hz), 6.80 (d, 2H, J = 9.0 Hz), 7.74 (d, 2H,
J = 8.2 Hz), 7.87 (d, 2H, J = 8.2 Hz), 8.35 (d, 2H, J = 8.2 Hz).
1
2 3 4 5 6 7 8 9 10 21d
Figure 2. Topoisomerase-II inhibition for compounds 20c, 20d, and 21d. Lane 1:
contain supercoiled PBR322 DNA in the absence of topoisomerase-II. Lane 2: CCC
DNA related by topoisomerase-II. Lane 3: inhibitory effect of etoposide (VP-16)
10
500
l
l
M. Lanes 4–10: 20c, 20d, and 21d at concentrations 10, 50, 100, 200, 300,
M, and 1 mM.
5.1.5. 4-Amino-40-[di(2-chloroyethyl)amino]benzophenone (19)
The compound 18 (1.10 g, 3 mmol) dissolved in methanol
(30 mL) and added SnCl2ꢀ2H2O (3.38 g, 15 mmol) was refluxed for
1.5 h. The reaction mixture was cooled and the methanol was
evaporated under vacuum, and the residue was carefully adjusted
to pH 8 with saturated NaHCO3 solution and then extracted with
ethyl acetate (2 ꢁ 30 mL). The combined organic phase was
washed with brine (15 mL), dried over anhydrous Na2SO4, and
evaporated under vacuum. This crude product was subjected to
Figure 3. DNA fragmentation assay for compound 20d. Lane 1: untreated cells.
Lane 2: camptothecin (5 M). Lanes 3–6: 20d at concentration 0.5, 1, 5, and 10 M.
l
l
Figure 4. DNA cell cycle analysis for compound 20d.

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A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
column chromatography using ethyl acetate/hexane (9:1) affords
19, 809 mg in 80% yield. 1H NMR (200 MHz, DMSO-d6 + CDCl3): d
3.36 (t, 4H, J = 6.6 Hz), 3.50 (t, 4H, J = 6.5 Hz), 6.27–6.40 (m, 4H),
7.20–7.37 (m, 4H).
Calcd for C38H35NO7: C, 74.38; H, 5.09; N, 2.28. Found: C, 74.21;
H, 5.15; N, 2.31.
5.1.9. 4b-(600-Chrycenylamino)-4-desoxypodophyllotoxin (20d)
This compound was prepared by method described for 20a
employing 6-chrycenylamine (296 mg, 1.2 mmol) and podophyllo-
toxin (414 mg, 1 mmol) affords 20d, 456 mg, in 71% yield. Mp:
5.1.6. 4b-(100-Anthrylamino)-4-desoxypodophyllotoxin (20a)
To a solution of podophyllotoxin (414 mg, 1 mmol) in dry aceto-
nitrile (10 mL), sodium iodide (298 mg, 2 mmol) was added and
stirred for 5 min to this stirred suspension BF3ꢀOEt2 (0.13 mL,
2 mmol) was added dropwise at 0 °C and the stirring was contin-
ued for another 0.5 h at room temperature. This solution was then
evaporated in vacuo and used for the next reaction without further
purification. To the crude product, anhydrous barium carbonate
(395 mg, 2 mmol) and 1-anthraceneamine (231 mg, 1.2 mmol) in
10 mL of dry THF under nitrogen was added and stirred for 8 h at
room temperature. The reaction mixture was filtered, diluted with
ethyl acetate and washed with water, 10% aqueous sodium thiosul-
phate solution, dried, and purified via column chromatography
using ethyl acetate/hexane (2:3) as eluent affords 20a, 530 mg in
157–160 °C,
½
a 2D5
ꢂ
:
ꢃ48.0 (c = 1.0, CHCl3). 1H NMR (200 MHz,
CDCl3): d 2.98–3.30 (m, 2H), 3.82 (s, 6H), 3.83 (s, 3H), 4.07 (t, 1H,
J = 9.5 Hz), 4.61 (t, 2H, J = 7.1 Hz), 4.72–4.82 (m, 2H), 5.21–5.28
(m, 1H), 6.01 (d, 2H, J = 2.3 Hz), 6.38 (s, 2H), 6.57 (s, 1H), 6.86 (s,
1H), 7.40–9.01 (m, 11H); 13C NMR (75 MHz, CDCl3): d 38.6, 42.2,
43.7, 52.5, 56.3, 60.6, 69.0, 101.4, 108.6, 109.2, 110.0, 111.3,
111.4, 118.4, 119.1, 122.4, 123.3, 123.5, 126.9, 129.2, 124.4,
129.9, 131.1, 131.4, 131.8, 134.0, 134.2, 135.1, 142.4, 147.8,
148.3, 152.6, 174.5; IR (KBr) cmꢃ1: 3409, 2906, 1774, 1598, 1503,
1483; MS (FAB): 639 [M+]. Anal. Calcd for C40H37NO7: C, 74.10;
H, 5.20; N, 2.19. Found: C, 74.21; H, 5.16; N, 2.17.
90% yield. Mp: 195–200 °C, ½a D25
ꢂ
: –39.0 (c = 1.0, CHCl3). 1H NMR
d 2.87–3.10 (m, 1H), 3.30–3.43 (dd, 1H,
5.1.10. 4b-[400-(400-Fluorobenzoyl)anilino]-4-desoxypodophyllot-
oxin (20e)
(200 MHz, CDCl3):
J = 13.6, 4.5 Hz), 3.78 (s, 6H), 3.80 (s, 3H), 3.97 (t, 1H, J = 9.0 Hz),
4.43 (t, 1H, J = 8.0 Hz), 4.67 (d, 2H, J = 4.5 Hz), 4.95 (br s, 1H),
5.97 (d, 2H, J = 3.0 Hz), 6.33 (s, 2H), 6.43 (d, 1H, J = 7.5 Hz), 6.61
(s, 1H), 6.80 (s, 1H), 7.28–7.37 (m, 1H), 7.43–7.78 (m, 3H),
7.94–8.12 (m, 2H), 8.24 (s, 1H), 8.35 (s, 1H); 13C NMR (75 MHz,
CDCl3): d 38.7, 42.3, 43.7, 52.5, 56.4, 60.7, 69.0, 101.5, 108.7,
109.3, 110.0, 118.4, 118.8, 122.9, 125.4, 125.5, 125.8, 126.9,
127.8, 128.2, 130.6, 131.0, 131.8, 132.2, 132.5, 135.2, 142.4,
147.7, 148.4, 152.7, 174.6; IR (KBr) cmꢃ1: 3409, 2903, 2834,
1774, 1586, 1503, 1481; MS (FAB): 589 [M+]. Anal. Calcd for
This compound was prepared by method described for 20a
employing 4-amino-40-fluorobenzophenone (258 mg, 1.2 mmol)
and podophyllotoxin (414 mg, 1 mmol) affords 20e, 458 mg, in
75% yield. Mp: 106–110 °C, ½a D25
ꢂ
: ꢃ106.0 (c = 1.0, CHCl3). 1H NMR
(200 MHz, CDCl3): d 2.99–3.02 (m, 2H), 3.75 (s, 6H), 3.78 (s, 3H),
4.40–4.50 (m, 2H), 4.58–4.63 (m, 1H), 4.80–4.89 (m, 1H), 5.95
and 5.98 (ABq, 2H, J = 1.5 Hz), 6.25 (s, 2H), 6.55–6.67 (m, 3H),
6.78 (s, 1H), 7.12–7.65 (m, 2H), 7.72–7.89 (m, 4H); 13C NMR
(75 MHz, CDCl3): d 38.7, 42.3, 43.6, 52.5, 56.4, 60.7, 69.0, 101.5,
108.8, 109.3, 110.0, 114.2, 115.6, 117.2, 118.3, 118.9, 131.1,
131.7, 132.5, 135.1, 144.2, 145.2, 146.2, 147.8, 148.4, 152.7,
164.5, 174.6, 194.2; IR (KBr) cmꢃ1: 3348, 2923, 1772, 1641, 1596,
1504, 1481; MS (FAB): 611 [M+]. Anal. Calcd for C35H30FNO8: C,
68.73; H, 4.94; N, 2.29. Found: C, 68.75; H, 4.91; N, 2.27.
C36H31NO7: C, 73.33; H, 5.30; N, 2.38. Found: C, 73.29; H, 5.28;
N, 2.35.
5.1.7. 4b-(100-Fluorenylamino)-4-desoxypodophyllotoxin (20b)
This compound was prepared by method described for 20a
employing 1-fluorenylamine (220 mg, 1.2 mmol) and podophyllo-
toxin (414 mg, 1 mmol) affords 20b, 432 mg in 75% yield. Mp:
5.1.11. 4b-(400-{400-[Di(200-chloroethyl)amino]benzoyl}anilino)-4-
desoxypodophyllotoxin (20f)
209–212 °C, ½a 2D5
ꢂ
: ꢃ129.0 (c = 1.0, CHCl3). 1H NMR (200 MHz,
This compound was prepared by method described for 20a
CDCl3): d 2.89–3.07 (m, 1H), 3.20–3.49 (dd, J = 13.6, 4.5 Hz), 3.77
(s, 6H), 3.82 (s, 3H), 3.84 (s, 2H), 3.92 (br s, 1H), 4.07 (t, 1H,
J = 9.0 Hz), 4.44 (t, 1H, J = 8.3 Hz), 4.63 (d, 1H, J = 4.5 Hz),
4.76–4.80 (m, 1H), 5.98 (d, 2H, J = 3.0 Hz), 6.34 (s, 2H), 6.55 (s,
1H), 6.58 (dd, 1H, J = 8.3, 2.2 Hz), 6.75–6.79 (m, 1H), 6.81 (s, 1H),
7.10–7.70 (m, 5H); 13C NMR (75 MHz, CDCl3): d 36.9, 38.6, 42.4,
43.8, 52.4, 56.4, 60.7, 69.0, 101.4, 108.8, 109.3, 109.9, 111.2,
118.2, 118.3, 119.9, 122.9, 125.0, 126.6, 126.8, 127.0, 131.1,
131.8, 133.7, 135.1, 140.7, 142.3, 143.6, 147.8, 148.3, 152.8,
174.5; IR (KBr) cmꢃ1: 3364, 2906, 2834, 1774, 1615, 1585, 1503,
1457; MS (FAB): 577 [M+]. Anal. Calcd for C35H31NO7: C. 72.78;
H, 5.41; N, 2.42. Found: C, 72.75; H, 5.39; N, 2.43.
employing
4-amino-40-[di(2-chloroethyl)amino]benzophenone
(404 mg, 1.2 mmol) and podophyllotoxin (414 mg, 1 mmol) affords
20f, 476 mg, in 65% yield. Mp: 186–190 °C, ½a D25
ꢂ : ꢃ110.0 (c = 1.0,
CHCl3). 1H NMR (200 MHz, CDCl3):
d 2.97–3.13 (m, 2H),
3.64–3.91 (m, 17H), 3.99–4.10 (m, 1H), 4.26–4.48 (m, 2H),
4.63–4.70 (m, 1H), 4.81–4.90 (m, 1H), 5.99 (d, 2H, J = 6.8 Hz),
6.33 (s, 2H), 6.55 (s, 1H), 6.57–6.74 (m, 4H), 6.80 (s, 1H),
7.66–7.80 (m, 4H); 13C NMR (75 MHz, CDCl3): 38.6, 42.2, 43.2,
43.7, 52.5, 53.2, 56.3, 60.6, 69.0, 101.5, 108.7, 109.2, 110.0, 111.3,
113.6, 116.9, 118.4, 128.1, 131.1, 131.2, 131.8, 133.4, 135.2, 146.2,
147.3, 147.8, 148.4, 152.7, 153.4, 174.6, 193.2; IR (KBr) cmꢃ1
:
3380, 2924, 2854, 1773, 1727, 1596, 1507, 1480; MS (FAB): 732
[M+]. Anal. Calcd for C39H38Cl2N2O8: C, 63.85; H, 5.22; N, 3.82.
Found: C, 63.81; H, 5.19; N, 3.85.
5.1.8. 4b-(100-Pyrenylamino)-4-desoxypodophyllotoxin (20c)
This compound was prepared by method described for 20a
employing 1-pyrenylamine (265 mg, 1.2 mmol) and podophyllo-
toxin (414 mg, 1 mmol) affords 20c, 413 mg, in 67% yield. Mp:
5.1.12. 40-O-Demethyl-4b-(100-anthrylamino)-4-desoxypodophy-
llotoxin (21a)
190–193 °C, ½a 2D5
ꢂ
: ꢃ122.0 (c = 1.0, CHCl3). 1H NMR (200 MHz,
To a solution of podophyllotoxin (414 mg, 1 mmol) in dry
CH2Cl2 (10 mL), sodium iodide (298 mg, 2 mmol) was added and
stirred for 5 min to this stirred suspension BF3ꢀOEt2 (0.13 mL,
2 mmol) was added dropwise at 0 °C and the stirring was contin-
ued for another 8 h at room temperature. Nitrogen was bubbled
through the solution to drive of the excess hydrogen iodide. This
solution was then evaporated in vacuo and used for the next reac-
tion without further purification. To the above crude product,
anhydrous barium carbonate (395 mg, 2 mmol) and 1-anthracene-
amine (231 mg, 1.2 mmol) in 10 mL of dry THF under nitrogen was
CDCl3): d 2.97–3.19 (m, 1H), 3.34–3.67 (dd, 1H, J = 14.1, 5.3 Hz),
3.82 (s, 6H), 3.84 (s, 3H), 4.02 (t, 1H, J = 10.1 Hz), 4.50 (t, 1H,
J = 8.0 Hz), 4.70 (d, 1H, J = 4.7 Hz), 4.86–4.92 (m, 1H), 5.11–5.23
(m, 1H), 6.01 (s, 2H), 6.37 (s, 2H), 6.62 (s, 1H), 6.83 (s, 1H),
7.12–8.10 (m, 9H); 13C NMR (75 MHz, CDCl3): d 38.6, 42.2, 43.8,
52.4, 56.3, 60.6, 69.1, 101.4, 108.9, 109.4, 110.1, 110.8, 114.1,
118.3, 122.2, 125.0, 125.9, 125.9, 127.8, 129.12, 131.2, 131.0,
134.2, 135.2, 142.4, 147.8, 148.4, 152.8, 174.7; IR (KBr) cmꢃ1
3394, 2924, 1770, 1615, 1505, 1483; MS (FAB): 613 [M+]. Anal.
:

A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
8499
added and stirred for 8 h at room temperature. The reaction mix-
ture was filtered, diluted with ethyl acetate and washed with
water, 10% aqueous sodium thiosulphate solution, dried and puri-
fied via column chromatography using ethyl acetate/hexane (1:1)
147.7, 148.3, 152.7, 174.6; IR (KBr) cmꢃ1: 3394, 2923, 1768,
1615, 1503, 1482; MS (FAB): 625 [M+]. Anal. Calcd for
C39H35NO7: C, 74.87; H, 4.99; N, 2.24. Found: C, 74.79; H, 5.03;
N, 2.23.
as eluent affords 21a, 374 mg in 65% yield. Mp: 180–182 °C, ½a D25
ꢂ
:
ꢃ59.0 (c = 1.0, CHCl3). 1H NMR (200 MHz, CDCl3): d 2.97–3.11 (m,
1H), 3.39–3.68 (dd, 1H, J = 13.6, 4.5 Hz), 3.83 (s, 6H), 3.97 (t, 1H,
J = 9.1 Hz), 4.46 (t, 1H, J = 8.3 Hz), 4.73–4.80 (m, 2H), 5.45 (br s,
1H), 5.98 (d, 2H, J = 1.5 Hz), 6.40 (s, 2H), 6.47 (d, 1H, J = 7.5 Hz),
6.63 (s, 1H), 6.82 (s, 1H), 7.35–7.40 (m, 1H), 7.48–7.69 (m, 3H),
7.97–8.12 (m, 2H), 8.24 (s, 1H), 8.35 (s, 1H); 13C NMR (75 MHz,
CDCl3): d 38.7, 42.3, 43.81, 52.5, 56.4, 69.1, 101.6, 108.7, 109.3,
110.0, 118.4, 118.8, 123.0, 125.3, 125.6, 125.8, 126.9, 127.7,
128.1, 130.7, 131.1, 131.8, 132.2, 132.5, 134.4, 142.6, 148.1,
148.4, 152.7, 175.3; IR (KBr) cmꢃ1: 3416, 2924, 2852, 1773, 1576,
1481; MS (FAB): 575 [M+]. Anal. Calcd for C35H29NO7: C, 73.03;
H, 5.08; N, 2.43. Found: C, 72.99; H, 5.11; N, 2.41.
5.1.16. 40-O-Demethyl-4b-[400-(400-fluorobenzoyl)anilino]-4-des-
oxypodophyllotoxin (21e)
This compound was prepared by method described for 21a
employing 4-amino-40-fluorobenzophenone (258 mg, 1.2 mmol)
and podophyllotoxin (414 mg, 1 mmol) affords 21e, 388 mg in
65% yield. Mp: 162–165 °C, ½a D25
ꢂ
: ꢃ129.0 (c = 1.0, CHCl3). 1H NMR
(200 MHz, CDCl3): d 2.68–3.02 (m, 2H), 3.79 (s, 6H), 4.36–4.45
(m, 1H), 4.52–4.63 (m, 2H), 4.79–4.88 (m, 1H), 5.35 (br s, 1H),
5.95 and 5.98 (ABq, 2H, J = 1.5 Hz), 6.28 (s, 2H), 6.51 (s, 1H), 6.57
(d, 2H, J = 8.6 Hz), 6.76 (s, 1H), 7.13–7.46 (m, 2H), 7.68–7.79 (m,
4H); 13C NMR (75 MHz, CDCl3): d 38.7, 42.3, 43.6, 52.5, 60.7,
69.0, 101.4, 108.7, 109.3, 110.0, 114.2, 115.7, 117.2, 118.3, 118.9,
131.0, 131.7, 132.5, 135.1, 144.2, 145.2, 146.4, 147.6, 148.3,
152.7, 164.5, 174.5, 194.2; IR (KBr) cmꢃ1: 3402, 2924, 1775,
1610, 1503, 1481; MS (FAB): 597 [M+]. Anal. Calcd for C34H28FNO8:
C, 68.34; H, 4.72; N, 3.18. Found: C, 68.29; H, 4.75; N, 3.16.
5.1.13. 40-O-Demethyl-4b-(100-fluorenylamino)-4-desoxypodo-
phyllotoxin (21b)
This compound was prepared by method described for 21a
employing 1-fluorenylamine (220 mg, 1.2 mmol) and podophyllo-
toxin (414 mg, 1 mmol) affords 21b, 354 mg, in 63% yield. Mp:
5.1.17. 40-O-Demethyl-4b-(400-{400-[di(200-chloroethyl)amino]-
benzoyl}anilino)-4-desoxy podophyllotoxin (21f)
250–252 °C, ½a 2D5
ꢂ
: ꢃ105.0 (c = 1.0, CHCl3). 1H NMR (200 MHz,
CDCl3): d 3.01–3.30 (m, 2H), 3.74 (s, 6H), 3.79 (s, 2H), 3.88–4.23
(m, 1H), 4.34 (t, 1H, J = 7.8 Hz), 4.52 (d, 1H, J = 5.2 Hz), 4.86–4.98
(m, 1H), 5.96 (s, 2H), 6.28 (s, 2H), 6.5 (s, 1H), 6.67–6.78 (m, 1H),
6.81 (s, 1H), 6.86–6.98 (m, 1H), 7.06–7.58 (m, 5H); 13C NMR
(75 MHz, CDCl3): d 36.9, 38.7, 42.2, 43.6, 52.5, 60.6, 68.9, 101.4,
108.8, 109.2, 109.9, 111.2, 118.3, 118.5, 119.9, 122.9, 125.0,
126.6, 126.8, 127.0, 131.1, 131.9, 133.8, 135.1, 140.8, 142.3,
143.7, 147.8, 148.3, 152.8, 174.4; IR (KBr) cmꢃ1: 3349, 2925,
2854, 1758, 1610, 1515, 1458; MS (FAB): 563 [M+]. Anal. Calcd
for C34H29NO7: C, 72.46; H, 5.19; N, 2.49. Found: C, 72.45; H,
5.21; N, 2.45.
This compound was prepared by method described for 21a
employing
4-amino-40-[di(2-chloroethyl)amino]benzophenone
(404 mg, 1.2 mmol) and podophyl-lotoxin (414 mg, 1 mmol) af-
fords 21f, 366 mg in 51% yield. Mp: 173–175 °C, ½a D25
ꢂ : ꢃ124.0
(c = 1.0, CHCl3). 1H NMR (200 MHz, CDCl3): d 2.69–3.12 (m, 2H),
3.65–3.88 (m, 14H), 3.99 (t, 1H, J = 10.5 Hz), 4.48–4.56 (m, 2H),
4.62 (d, 1H, J = 4.5 Hz), 4.82–4.98 (m, 1H), 5.34 (br s, 1H), 5.98 (d,
2H, J = 6.8 Hz), 6.33 (s, 2H), 6.55 (s, 1H), 6.57–6.74 (m, 4H), 6.8 (s,
1H), 7.66–7.80 (m, 4H); 13C NMR (75 MHz, CDCl3): d 38.7, 42.3,
43.2, 43.7, 52.5, 53.6, 60.7, 69.0, 101.5, 108.7, 109.2, 109.9, 111.5,
113.6, 117.3, 118.4, 118.8, 127.9, 131.1, 131.2, 131.7, 133.5,
135.1, 144.8, 146,2, 147.3, 147.8, 148.4, 152.7, 153.3, 174.6,
195.1; IR (KBr) cmꢃ1: 3395, 2920, 1772, 1598, 1507, 1481; MS
(FAB): 718 [M+]. Anal. Calcd for C38H36Cl2N2O8: C, 63.42; H, 5.04;
N, 3.89. Found: C, 63.38; H, 4.99; N, 3.91.
5.1.14. 40-O-Demethyl-4b-(100-pyrenylamino)-4-desoxypodophy-
llotoxin (21c)
This compound was prepared by method described for 21a
employing 1-pyrenylamine (265 mg, 1.2 mmol) and podophyllo-
toxin (414 mg, 1 mmol) affords 21c, 313 mg, in 55% yield. Mp:
a 2D5
ꢂ
:
ꢃ76.0 (c = 1.0, CHCl3). 1H NMR (200 MHz,
6. Procedure of the SRB-assay
148–153 °C,
½
CDCl3): d 2.89–3.13 (m, 1H), 3.29–3.67 (dd, 1H, J = 13.6, 4.5 Hz),
3.83 (s, 6H), 3.98 (t, 1H, J = 10.5 Hz), 4.44 (t, 1H, J = 8.3 Hz),
4.55–4.60 (m, 1H), 4.66 (d, 1H, J = 5.2 Hz), 5.07–5.25 (m, 1H),
5.34 (br s, 1H), 5.97 (s, 2H), 6.35 (s, 2H), 6.59 (s, 1H), 6.8 (s, 1H),
7.18–7.67 (m, 1H), 7.90–8.29 (m, 8H); 13C NMR (75 MHz, CDCl3):
d 38.6, 42.2, 43.8, 52.5, 60.6, 69.1, 101.4, 108.8, 109.4, 110.1,
110.8, 114.2, 118.3, 122.1, 125.1, 125.9, 125.9, 127.7, 131.0, 131.8,
Tumor cells were grown in tissue culture flasks in growth med-
ium (RPMI-1640 with 2 mM glutamine, pH 7.4, 10% fetal calf ser-
um, 100 lg/mL streptomycin, and 100 units/mL penicillin) at
37 °C in an atmosphere of 5% CO2 and 95% relative humidity in a
CO2 incubator. The cells at subconfluent stage were harvested from
the flask by treatment with trypsin (0.05% trypsin in PBS contain-
ing 0.02% EDTA) and suspended in growth medium. Cells with
more than 97% viability (trypan blue exclusion) were used for
134.1, 135.3, 142.2, 147.8, 148.3, 152.8, 174.6; IR (KBr) cmꢃ1
:
3381, 2920, 1775, 1603, 1510, 1483; MS (FAB): 599 [M+]. Anal.
Calcd for C37H33NO7: C, 74.11; H, 4.87; N, 2.34. Found: C, 74.03;
H, 4.91; N, 2.29.
determination of cytotoxicity. An aliquot of 100 ll of cells were
transferred to a well of 96-well tissue culture plate. The cells were
allowed to grow for 24 h at 37 °C in a CO2 incubator as stated
above. Test materials (100 ll) were then added to the wells and
cells were further allowed to grow for another 48 h. Suitable blanks
and positive controls were also included. Each test was done in
triplicate. The cell growth was stopped by gently layering of
5.1.15. 40-O-Demethyl-4b-(100-chrycenylamino)-4-desoxypodo-
phyllotoxin (21d)
This compound was prepared by method described for 21a
employing 6-chrycenylamine (296 mg, 1.2 mmol) and podophyllo-
toxin (414 mg, 1 mmol) affords 21d, 327 mg, in 52% yield. Mp:
50 ll of 50% trichloroacetic acid. The plates were incubated at
158–160 °C,
½
a 2D5
ꢂ
:
ꢃ39.0 (c = 1.0, CHCl3). 1H NMR (200 MHz,
4 °C for an hour to fix the cells attached to the bottom of the wells.
Liquids of all the wells were gently pipette out and discarded. The
plates were washed five times with distilled water to remove TCA,
growth medium, etc. and were air-dried. Hundred microliters of
SRB solution (0.4% in 1% acetic acid) was added to each well and
the plates were incubated at room temperature for 30 min. The un-
bound SRB was quickly removed by washing the wells five times
CDCl3): d 2.99–3.34 (m, 2H), 3.84 (s, 6H), 4.08 (t, 1H, J = 10.2 Hz),
4.59–4.63 (m, 1H), 4.74–4.89 (m, 2H), 5.21–5.69 (m, 1H), 5.98 (s,
2H), 6.34 (s, 2H), 6.51 (s, 1H), 6.83 (s, 1H), 7.40–9.01 (m, 11H);
13C NMR (75 MHz, CDCl3): d 38.6, 42.3, 43.7, 52.4, 60.6, 69.0,
101.5, 108.7, 109.3, 110.0, 111.2, 111.3, 118.4, 119.3, 122.3,
123.2, 123.5, 131.1, 131.3, 131.7, 134.1, 134.3, 135.1, 142.3,

8500
A. Kamal et al. / Bioorg. Med. Chem. 18 (2010) 8493–8500
with 1% acetic acid. Plates were air dried, tris-buffer (100 ll of
Acknowledgment
0.01 M, pH 10.4) was added to all the wells and plates were gently
stirred for 5 min on a mechanical stirrer. The optical density was
recorded on ELISA reader at 540 nm. The cell growth in absence
of any test material was considered 100% and in turn growth inhi-
bition was calculated. IC50 values were determined by regression
analysis.
One of the authors P.S. is thankful to UGC, New Delhi for the
award of Senior Research Fellowship.
References and notes
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Mechanisn of Interaction of DNA Topoisomerase II with Chemotherapeutic Agents.
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Topoisomerase-II activity was determined using kit (Topogen
Inc., USA, Cat no. 2000H). Reaction was assembled in micro centri-
fuge tube that contains super coiled DNA 250 ng/
ase-II (4 units) in assay buffer (A 0.1 volume and B 1 volume). In
each reaction 2 l sample was added then volume was made up
to 20 l with water and then incubated at 37 °C. Reaction was ter-
minated by addition of 2 l of 10% SDS. Each sample tube was trea-
ll & Topoisomer-
l
l
l
ted with proteinase K and extracted once with chloroform: isoamyl
alcohol (24:1). Products were resolved by 1% agarose gel electro-
phoresis in TAE buffer (40 mM tris-acetate, pH 8.0, and 1 mM
EDTA) and stained with 0.5 lg/mL ethidium bromide (EtBr).
8. DNA gel electrophoresis
DNA fragmentation was determined by electrophoresis of ex-
tracted genomic DNA from leukemia cell line (MOLT-4). Briefly,
exponentially growing cells (2 ꢁ 106 cells/ mL) in 6-well plate were
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Shibata, J.; Sugimoto, Y.; Yamada, Y.; Kobunai, T.; Takeda, S.; Minami, Y.;
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Arifuddin, M.; Dastidar, S. G.; Kondapi, A. K.; Rajkumar, M. Bioorg. Med. Chem.
2005, 13, 6218; (c) Kamal, A.; Laxman, E.; Khanna, G. B. R.; Reddy, P. S. M. M.;
Rehana, T.; Arifuddin, M.; Neelima, K.; Kondapi, A. K.; Dastidar, S. G. Bioorg.
Med. Chem. 2004, 12, 4197; (d) Kamal, A.; Kumar, B. A.; Arifuddin, M.; Dastidar,
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073375, 2004.; (g) Kumar. B. A. Ph.D. Thesis, Osmania University, 2006.; (h)
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treated with compound 20d in 0.5, 1, 5, and 10 lM concentrations
for 24 h. Cells were harvested, washed with PBS, pellets were
dissolved in lysis buffer (10 mM EDTA, 50 mM Tris pH 8.0, 0.5%
w/v SDS, and proteinase K (0.5 mg/mL) and incubated at 50 °C
for 1 h. Finally the DNA obtained was heated rapidly to 70 °C, sup-
plemented with loading dye and immediately resolved on to 1.5%
agarose gel at 50 V for 2–3 h.
9. Flow-cytometric analysis of phase distribution of nuclear
DNA
17. Bair, K. W.; Tuttle, R. L.; Cory, M.; McKee, D. D. J. Med. Chem. 1990, 33,
2385.
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Med. Chem. 1991, 34, 1983.
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Laxminarayana, B.; Gayatri, N. L. Tetrahedron Lett. 1997, 38, 6871; (c) Kamal, A.;
Gayatri, N. L.; Rao, N. V. Bioorg. Med. Chem. Lett. 1998, 8, 3097; (d) Kamal, A.;
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Effect of compound 20d on DNA content by cell cycle phase dis-
tribution was assessed using Molt-4 cells by incubating the cells
(1 ꢁ 106 mL/well) with 20d (0.5, 1, 5, and 10
lM) for 24 h. The cells
were then washed twice with ice-cold PBS, harvested, fixed with
ice cold PBS in 70% ethanol, and stored at ꢃ20 °C for 30 min. After
fixation, these cells were incubated with RNase A (0.1 mg/mL) at
37 °C for 30 min, stained with propidium iodide (50 lg/mL) for
30 min on ice in dark, and then measured for DNA content using
BD-LSR flow cytometer (Becton Dickinson, USA) equipped with
electronic doublet discrimination capability using blue (488 nm)
excitation from Argon laser. Data were collected in list mode on
10,000 events for FL2-A versus FL2-W.
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