Synthesis of 4β-carbamoyl epipodophyllotoxins as potential antitumour agents

Article abstract of DOI:10.1016/j.bmc.2011.03.030

A series of new 4β-carbamoyl epipodophyllotoxin analogues have been synthesized and evaluated for their anticancer activity against eleven cancer cell lines including Zr-75-1, MCF7, KB, Gurav, DWD, Colo 205, A-549, Hop62, PC3, SiHa and A-2780. Most of the

Full text of DOI:10.1016/j.bmc.2011.03.030

Bioorganic & Medicinal Chemistry 19 (2011) 2975–2979
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 4b-carbamoyl epipodophyllotoxins as potential antitumour agents
Ahmed Kamal a, , B. Ashwini Kumar a, Paidakula Suresh a, Aarti Juvekar b, Surekha Zingde b
⇑
a Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Khargar, Navi Mumbai 410 208, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 4b-carbamoyl epipodophyllotoxin analogues have been synthesized and evaluated for
their anticancer activity against eleven cancer cell lines including Zr-75-1, MCF7, KB, Gurav, DWD, Colo
205, A-549, Hop62, PC3, SiHa and A-2780. Most of the compounds exhibited better growth-inhibition
activities against tested cell lines than that of etoposide. Further, compounds 6g and 6i are also evaluated
for their DNA topoisomerase-II (topo-II) inhibition activity and they exhibited significant inhibition of
topo-II catalytic activity comparable to etoposide.
Received 29 January 2011
Revised 11 March 2011
Accepted 12 March 2011
Available online 21 March 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Podophyllotoxin
Epipodophyllotoxin
Carbamate
Topoisomerase II
Cytotoxicity
1. Introduction
development of drug resistance, metabolic inactivation, and toxic
effects.9 Therefore, the structure of etoposide has been extensively
Podophyllotoxin (Fig. 1) is a naturally occurring aryltetralin
lignan isolated from the roots of Podophyllum peltatum and Paeonia
emodi. Podophyllotoxin shows strong cytotoxic activity against
various cancer cell lines.1 It is effective in the treatment of Wilms
tumours, various genital tumours, non-Hodgkin’s, lymphomas
and lung cancer.2,3 However, the high toxicity and severe gastroin-
testinal side effect of podophyllotoxin has limited its application as
a drug in cancer chemotherapy. The biological activity of podo-
phyllotoxin has led to extensive structural modifications resulting
in several more potent and less toxic anticancer agents. The semi-
synthetic podophyllotoxin derivatives like etoposide and tenipo-
side are currently used in the chemotherapy for a variety of
malignancies including germ-cell malignancies, small-cell lung
cancer, non-Hodgkin’s lymphoma, leukemia, Kaposi’s sarcoma,
neuroblastoma, and soft tissue sarcoma.4–6
Podophyllotoxin inhibits the assembly of tubulin into microtu-
bules through interaction with protein at the colchicine binding
site, preventing the formation of the spindle. However, its semi-
synthetic derivatives showed different mechanisms of action.
Etoposide and congeners induce a premitotic blockade in late S
stage of the cell cycle because of the inhibition of DNA topoisomer-
ase II (top-II), an enzyme required for the unwinding of DNA during
replication.7,8
modified, thus increasing the information about its structure–
activity relationships. The most important modification is that of
the substituent in the 4b-position leading to potent inhibitors of
topoisomerase II. The replacement of C-4 sugar unit of etoposide
with a heteroatom (O, N, or S) linked moieties helped in overcom-
ing the problem of drug resistance to etoposide.10 Subsequently, a
large number of analogues have been reported exhibiting compa-
rable or better activity than etoposide and teniposide.11,12
In recent years, we have been involved in the development of
new methodologies, as well as synthesis of podophyllotoxin deriv-
atives with improved bioactivities and reduced toxicity.12,13 From
our previous studies as well as the investigations carried out in
the literature have shown that N-linked derivatives of podophyllo-
toxin possess lower bioavailability compared to O-linked congen-
ers. This finding has prompted us to design new carbamate
derivatives of podophyllotoxin that have promising anticancer
activity. In view of the available information on structure–activity
relationship studies and in continuation of our ongoing projects on
the design and development of structurally modified podophyllo-
toxin congeners we describe herein synthesis and structure–
activity relationships of a series of 4b-carbamoyl substituted
epipodophyllotoxin analogues.
Although etoposide is widely used in therapy, it presents sev-
eral limitations, such as moderate potency, poor water solubility,
2. Chemistry
The synthetic route for these new derivatives 6a–j is depicted in
Scheme 1. The podophyllotoxin (1) was first treated with BF3ꢀOEt2/
NaI followed by basic hydrolysis with barium carbonate to afford
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.030

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A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2975–2979
H
O
O
HO
R1
OH
O
R2
R3
R1
R2
O
O
O
O
O
CH3
OH
Etoposide (2)
Teniposide (3)
H
H
O
O
O
OH
S
H3CO
H3CO
OCH3
OCH3
OCH3
OR3
Podophyllotoxin (1)
Figure 1.
epipodophyllotoxin 4.13a Compound 4 was then converted into its
4b-activated derivative 5 by simple treatment with p-nitrophenyl-
chloroformate in pyridine. Introduction of the carbamate chains
was performed by reacting 5 with the appropriate amines in the
presence of triethylamine to afford compounds 6a–j in good yields.
In the earlier SAR studies on the podophyllotoxin based com-
pounds it has been observed that the O-linked derivatives of pod-
ophyllotoxin shows lower anticancer activity in comparison to the
N-linked congeners.11–13,15,16 However, in the present investiga-
tion these new class of 4b-carbamoyl podophyllotoxins possess po-
tent anticancer activity, that is comparable to previously reported
4b-N-sustituted podophyllotoxin derivatives.
3. Biological activity
In the literature, it has been observed that inversion of configura-
tion at 4-position to provide epipodophyllotoxin is essential for DNA
topo-II inhibition but there is no clarity about the role of 40-O-meth-
ylation or 40-O-demethylation. DNA topo-II is the pharmacological
target of clinical relevance for the podophyllotoxin lignans, there-
fore representative compounds 6g and 6i have been evaluated for
its inhibition. These compounds exhibit comparable in vitro inhibi-
tion of topo-II catalytic activity to etoposide and the results are illus-
trated in Figure 2. The topo-II-mediated relaxation assay performed
is in accordance to the previously reported procedure.17
Compounds 6a–j were evaluated for their anticancer activity in
selected human cancer cell lines, that is, breast (Zr-75-1, MCF7),
oral (KB, Gurav, DWD)), colon (Colo-205), lung (A-549, Hop62),
prostate (PC-3), cervix (SiHa) and ovarian (A-2780) origin by
employing the sulforhodamine B (SRB) assay method.14 The results
are summarized with standard drug etoposide and 5-fluorouracil
in Table 1 and reveal that all the new podophyllotoxin congeners
are significantly cytotoxic, with the concentration of the drug that
produces 50% inhibition of cell growth (GI50) ranging from <0.1 to
2.7 lM.
OH
OH
O
O
O
O
O
i, ii
O
O
O
H3CO
OCH3
OCH3
4
H3CO
OCH3
OCH3
1
iii
O
O
O
R2
R1
N
O
O
NO2
iv
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OCH3
OCH3
6a-j
5
6f: R1 = H, R2 = prop-2-yn-1-yl
6g: R1R2N = morpholinyl
6a: R1 = H, R2 = propyl
6b: R1 = H, R2 = isopropyl
6h: R1 = H, R2 = 4-trofluoromethylbenzyl
6i: R1 = H, R2 = 3-ethyl-1H-indole
6j: R1 = H, R2 = 2-dimethylaminoethyl
6c: R1 = H, R2 = cyclopropylmethyl
6d: R1 = CH3, R2 = CH3
6e: R1 = H, R2 = 2-methoxyethyl
Scheme 1. Reagents and conditions: (i) BF3ꢀOEt2/NaI, CH3CN, rt, 30 min; (ii) H2O–acetone, BaCO3, rt, 30 min; (iii) 4-nitrochloroformate, pyridine, 0 °C, 45 min; (iv) R1R2NH,
CH2Cl2, rt, 3–6 h.

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2975–2979
2977
Table 1
Anticancer activity data of compounds 6a–j
Compound
Breast
GI50 (lM)
Oral
Colon
Lung
Prostate
PC3
Cervix
SiHa
Ovarian
Zr-75-1
MCF7
KB
Gurav
DWD
Colo 205
A-549
Hop62
A-2780
6a
6b
6c
6d
6e
6f
6g
6h
0.19
0.15
0.19
0.12
<0.1
0.14
0.14
0.16
0.13
<0.1
0.2
2.0
0.19
2.3
2.1
0.18
2.4
0.17
0.16
0.18
0.17
0.14
0.14
0.14
0.19
0.12
0.15
0.5
2.0
<0.1
2.0
2.7
2.7
2.5
2.1
2.2
2.7
2.7
0.15
0.12
2.1
0.13
2.8
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
3.08
2.4
0.16
2.1
2.5
0.13
0.12
0.14
0.13
2.3
0.17
0.19
2.3
0.15
0.16
0.16
0.16
2.1
1.2
0.19
2.3
2.1
2.4
1.9
1.4
2.0
1.8
2.1
3.1
6.3
0.19
0.14
2.0
0.16
0.15
0.19
0.16
0.18
0.16
0.11
2.1
0.18
0.15
0.17
0.12
0.18
0.18
0.16
0.3
<0.1
<0.1
<0.1
<0.1
2.0
<0.1
<0.1
0.6
0.18
0.15
0.16
0.13
0.18
0.11
0.12
1.3
6i
6j
2.1
0.12
0.8
0.16
0.15
2.6
VP-16a
5FUb
2.3
2.8
5.2
3.1
6.1
4.1
12
3.5
a
VP-16 = Etoposide.
5FU = 5-Fluorouracil.
b
(1:1) and BaCO3 (2.17 mg, 11 mmol) were added successively.
After stirring for 30 min at room temperature, the solvent was
evaporated and the residue was treated with 10% Na2S2O3 solution
and extracted with ethyl acetate. The organic layer was dried over
anhydrous Na2SO4 and concentrated under vacuum to give the
crude product. This was purified by column chromatography
(CH2Cl2/CH3COCH3, 92:8) to afford 4, 3.52 g in 85% yield. Mp:
1
2 3 4 5 6 7 8 9 10
6g
148–152 °C, ½a 2D5
ꢁ
: ꢂ64.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d 2.77–
1
2
3 4
5
6
7 8
9
6i
2.80 (m, 1H), 3.21 (dd, 1H, J = 14.3, 5.2 Hz), 3.73 (s, 6H), 3.76 (s,
3H), 4.33–4.41 (m, 2H), 4.56 (d, 1H, J = 5.2 Hz), 4.81 (d, 1H,
J = 3.0 Hz), 5.96 and 5.99 (ABq, 2H, J = 1.5 Hz), 6.22 (s, 2H), 6.53
(s, 1H), 6.84 (s, 1H); IR(KBr) cmꢂ1: 3513, 2921, 2849, 1764, 1589,
1504, 1479; MS (FAB): 414 [M+].
Figure 2. Topoisomerase-II inhibition for compounds 6g and 6i. 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 10
10: 6g and 6i at concentrations 10, 50, 100, 200, 300, 500
lM. Lanes 4–
lM, and 1 mM.
4. Conclusion
5.1.2. Epipodophyllotoxin 4-(p-nitrophenyl)carbonate (5)
To a solution of p-nitrophenylchloroformate (2.5 g, 12.5 mmol)
in dry CH2Cl2 (10 mL) was added dry pyridine (1.2 mL). Instanta-
neously a white precipitate was formed. A solution of epipodo-
phyllotoxin (2.07 g, 5 mmol) in dry CH2Cl2 (10 mL) was added
drop wise under nitrogen atmosphere. The mixture was stirred
for 45 min at room temperature and washed with water and dried
over Na2SO4 and concentrated in vacuo. This residue was purified
by column chromatography using ethyl acetate/hexane (1:1) to af-
In summary, new 4b-carbamoyl substituted epipodophyllotoxin
analogues with different alkyl and aryl side chains have been pre-
pared. Interestingly these 4b-carbamoyl substituted epipodo-
phyllotoxin congeners exhibit promising anticancer activity. The
compounds of this series like etoposide also inhibit DNA topo-II
and have the potential for further development.
ford compound 5, 2.46 g in 85% yield. Mp: 115–120 °C, ½a D25
ꢁ
:
5. Experimental section
5.1. General methods
ꢂ122.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d 3.07–3.16 (m, 1H), 3.29
(dd, 1H, J = 14.8, 5.4 Hz), 3.76 (s, 6H), 3.80 (s, 3H), 4.13 (t, 1H,
J = 4.6 Hz), 4.44 (t, 1H, J = 8.5 Hz), 4.71 (d, 1H, J = 4.6 Hz), 6.03–
6.12 (m, 3H), 6.23 (s, 2H), 6.60 (s, 1H), 7.01 (s, 1H), 7.41 (d, 2H,
J = 9.3 Hz), 8.32 (d, 2H, J = 9.3 Hz); 13C NMR (CDCl3): d 36.8, 41.2,
43.8, 43.9, 56.3, 60.7, 66.9, 74.2, 101.9, 108.5, 109.6, 110.5, 121.6,
125.4, 126.3, 133.4, 134.3, 145.6, 147.6, 148.5, 149.6, 152.8,
155.2, 173.7; IR(KBr) cmꢂ1: 2911, 2836, 1766, 1720, 1590, 1506,
1485; MS (FAB): 580 [M+1]. Anal. Calcd for C29H25NO12: C, 60.10;
H, 4.35; N, 2.42. Found: C, 60.05; H, 4.39; N, 2.41.
The NMR spectra are recorded on Varian Gemini 200 MHz spec-
trometer, using TMS as an internal reference. IR spectra are
recorded on Perkin-Elmer model 683 or 1310 spectrometer with
sodium chloride optics. Mass spectra are recorded on CEC-21-
100B, Finnigan Mat 1210, or Micromass 7070 spectrometer operat-
ing at 70 eV using a direct inlet system. Optical rotations are
measured on Jasco Dip 360 digital polarimeter. Melting points
are determined 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 chromatog-
raphy.
5.1.3. Epipodophyllotoxin 4-[N-(n-propyl)]carbamate (6a)
To
phenyl)carbonate (5) (290 mg, 0.5 mmol) in dry CH2Cl2 (10 mL)
was added n-propylamine (50 L, 600 mol) and triethylamine
(85 L, 600 mol) at room temperature. The mixture was stirred
a stirred solution of epipodophyllotoxin 4-(p-nitro-
l
l
l
l
5.1.1. Epipodophyllotoxin (4)
for 3 h at room temperature and washed with cold saturated NaH-
CO3 and water. The extract was dried over anhydrous Na2SO4 and
concentrated in vacuo. This residue was purified by column chro-
matography using ethyl acetate/cyclohexane (1:1) to afford com-
To a solution of podophyllotoxin (1) (4.14 g, 10 mmol) in dry
CH3CN (100 mL), NaI (2.98 g, 20 mmol) was added and stirred for
5 minutes. To this stirred suspension, BF3ꢀOEt2 (2.5 mL, 20 mmol)
was added drop wise at 0 °C and stirring continued for another
30 min at room temperature. Then a mixture of H2O/Me2CO, v/v
pound 6a, 212 mg in 85% yield. Mp: 120–123 °C, ½a D25
ꢁ : ꢂ87.0 (c
1.0, CHCl3). 1H NMR (CDCl3): d 0.94–1.12 (m, 3H), 1.55–1.78 (m,

2978
A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2975–2979
2H), 2.92–2.99 (m, 1H), 3.13–3.53 (m, 3H), 3.73 (s, 6H), 3.77 (s, 3H),
3.94 (t, 1H, J = 10.2 Hz), 4.36 (t, 1H, J = 7.9 Hz), 4.60 (d, 1H,
J = 5.1 Hz), 4.73 (t, 1H, J = 5.8 Hz), 5.98–6.12 (m, 3H), 6.22 (s, 2H),
6.52 (s, 1H), 6.89 (s, 1H); 13C NMR (CDCl3): d 11.2, 23.1, 38.5,
40.9, 44.1, 44.5, 54.2, 56.8, 60.9, 68.8, 101.5, 108.2, 108.9, 111.0,
132.1, 131.5, 134.2, 136.8, 146.4, 147.8, 148.1, 155.4, 174.8; IR(KBr)
cmꢂ1: 3378, 2920, 2851, 1770, 1713, 1590, 1508, 1485; MS (FAB):
500 [M+1]. Anal. Calcd for C26H29NO9: C, 62.52; H, 5.85; N, 2.80.
Found: C, 62.47; H, 5.88; N, 2.82.
0.5 mmol) to afford compound 6e, 206 mg in 80% yield. Mp:
130–135 °C, ½a 2D5
ꢁ
: ꢂ86.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d 2.93–
2.97 (m, 1H), 3.13 (dd, 1H, J = 14.8, 5.4 Hz), 3.34 (s, 3H), 3.40–
3.56 (m, 4H), 3.73 (s, 6H), 3.77 (s, 3H), 3.97 (t, 1H, J = 10.1 Hz),
4.36 (t, 1H, J = 7.8 Hz), 4.61 (d, 1H, J = 4.6 Hz), 5.09 (t, 1H,
J = 5.4 Hz), 5.98–6.15 (m, 3H), 6.23 (s, 2H), 6.52 (s, 1H), 6.90 (s,
1H); 13C NMR (CDCl3): d 38.1, 40.8, 41.0, 43.9, 54.5, 56.9, 59.3,
61.3, 68.9, 70.7, 101.9, 108.6, 108.9, 110.9, 131.8, 132.3 , 134.6,
136.7, 146.9, 147.8, 147.8, 155.7, 174.9; IR(KBr) cmꢂ1: 3376,
2928, 2847, 1778, 1715, 1596, 1505, 1484; MS (FAB): 515 [M+].
Anal. Calcd for C26H29NO10: C, 60.58; H, 5.67; N, 2.72. Found: C,
60.61; H, 5.62; N, 2.76.
5.1.4. Epipodophyllotoxin 4-[N-(iso-propyl)]carbamate (6b)
This compound was prepared according to the method de-
scribed for 6a employing iso-propylamine (37 lL, 600 lmol) and
epipodophyllotoxin 4-(p-nitrophenyl)carbonate (5) (290 mg,
5.1.8. Epipodophyllotoxin 4-[N-(2-propynyl)]carbamate (6f)
0.5 mmol) to afford compound 6b, 200 mg in 80% yield. Mp:
This compound was prepared according to the method de-
scribed for 6a employing 2-propynylamine (41 lL, 600 lmol) and
epipodophyllotoxin 4-(p-nitrophenyl)carbonate (5) (290 mg,
110–115 °C, ½a 2D5
ꢁ
: ꢂ81.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d 1.19
(d, 6H, J = 6.8 Hz), 2.91–2.95 (m, 1H), 3.09 (dd, 1H, J = 13.6,
4.5 Hz), 3.73 (s, 6H), 3.77 (s, 3H), 3.79–3.86 (m, 1H), 3.93 (t, 1H,
J = 9.0 Hz), 4.37 (t, 1H, J = 9.0 Hz), 4.55 (d, 1H, J = 7.5 Hz), 4.60 (d,
1H, J = 5.2 Hz), 5.96–6.12 (m, 3H), 6.22 (s, 2H), 6.52 (s, 1H), 6.89
(s, 1H); 13C NMR (CDCl3): d 23.3, 38.6, 41.0, 44.2, 46.5, 54.1, 56.9,
61.0, 68.9, 101.6, 108.5, 108.9, 110.9, 131.6, 132.0, 134.4, 136.7,
146.6, 147.5, 148.3, 155.3, 174.6; IR(KBr) cmꢂ1: 3362, 2927,
2854, 1770, 1720, 1585, 1508, 1485; MS (FAB): 500 [M+1]. Anal.
Calcd for C26H29NO9: C, 62.52; H, 5.85; N, 2.80. Found: C, 62.55;
H, 5.79; N, 2.81.
0.5 mmol) to afford compound 6f, 186 mg in 75% yield. Mp: 163–
166 °C, ½a 2D5
ꢁ
: ꢂ90.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d 2.22 (s, 1H),
2.93–2.95 (m, 1H), 3.10 (dd, 1H, J = 14.3, 5.2 Hz), 3.73 (s, 6H),
3.77 (s, 3H), 3.90–4.10 (m, 3H), 4.36 (t, 1H, J = 9.0 Hz), 4.60 (d,
1H, J = 4.5 Hz), 4.99 (t, 1H, J = 5.2 Hz), 5.99–6.13 (m, 3H), 6.22 (s,
2H), 6.53 (s, 1H), 6.90 (s, 1H); 13C NMR (CDCl3): d 30.1, 38.1,
41.3, 43.5, 54.3, 56.8, 61.1, 69.1, 72.9, 80.3, 101.8, 108.5, 108.8,
110.3, 131.1, 132.2, 134.6, 136.8, 146.8, 147.2, 147.8, 155.8,
175.1; IR(KBr) cmꢂ1: 3392, 2932, 2843, 1776, 1715, 1598, 1507,
1484; MS (FAB): 495 [M+]. Anal. Calcd for C26H25NO9: C, 63.03;
H, 5.09; N, 2.83. Found: C, 62.96; H, 5.13; N, 2.81.
5.1.5. Epipodophyllotoxin 4-[N-(cyclopropylmethyl)]carbamate
(6c)
This compound was prepared according to the method de-
5.1.9. Epipodophyllotoxin 4-(morpholine)carbamate (6g)
scribed for 6a employing cyclopropylmethyl amine (52
lL,
This compound was prepared according to the method de-
600 mol) and epipodophyllotoxin 4-(p-nitrophenyl)carbonate
l
scribed for 6a employing morpholine (52 lL, 600 lmol) and epip-
(5) (290 mg, 0.5 mmol) to afford compound 6c, 204 mg in 80%
odophyllotoxin 4-(p-nitrophenyl)carbonate (5) (290 mg, 0.5 mmol)
yield. Mp: 134–137 °C, ½a D25
ꢁ
: ꢂ85.0 (c 1.0, CHCl3). 1H NMR (CDCl3):
to afford compound 6g, 237 mg in 90% yield. Mp: 155–158 °C, ½a D25
ꢁ
:
d 0.21–0.29 (m, 2H), 0.53–0.65 (m, 2H), 0.98–1.13 (m, 1H), 2.92–
2.98 (m, 1H), 3.07–3.45 (m, 3H), 3.73 (s, 6H), 3.77 (s, 3H), 3.96 (t,
1H, J = 10.5 Hz), 4.36 (t, 1H, J = 8.3 Hz), 4.6 (d, 1H, J = 4.5 Hz), 4.81
(t, 1H, J = 5.2 Hz), 5.97–6.14 (m, 3H), 6.22 (s, 2H), 6.52 (s, 1H),
6.90 (s, 1H); 13C NMR (CDCl3): d 3.5, 10.7, 38.5, 41.4, 43.6, 49.1,
54.2, 56.5, 60.9, 68.4, 101.5, 108.2, 108.8, 110.5, 131.6, 132.1,
ꢂ88.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d 2.90–2.96 (m, 1H), 3.06 (dd,
1H, J = 14.3, 5.2 Hz), 3.36–3.71 (m, 8H, morpholine), 3.73 (s, 6H),
3.77 (s, 3H), 3.88 (t, 1H, J = 10.5 Hz), 4.37–4.46 (m, 1H), 4.61 (d,
1H, J = 5.2 Hz), 5.99–6.17 (m, 3H), 6.22 (s, 2H), 6.53 (s, 1H), 6.89
(s, 1H); 13C NMR (CDCl3): d 38.9, 41.3, 43.5, 49.7, 54.3, 56.8, 60.9,
66.3, 68.4, 101.7, 108.5, 108.5, 110.6, 131.6, 131.7, 134.1, 136.8,
146.7, 147.6, 148.1, 155.9, 175.3; IR(KBr) cmꢂ1: 3357, 2946,
2841, 1775, 1715, 1596, 1505, 1488; MS (FAB): 527 [M+]. Anal.
Calcd for C27H29NO10: C, 61.47; H, 5.54; N, 2.66. Found: C, 61.41;
H, 5.60; N, 2.63.
134.6, 136.8, 146.4, 147.2, 147.7, 155.1, 174.3; IR(KBr) cmꢂ1
:
3387, 2927, 2845, 1780, 1723, 1592, 1510, 1487; MS (FAB): 511
[M+]. Anal. Calcd for C27H29NO9: C, 63.40; H, 5.71; N, 2.74. Found:
C, 63.34; H, 5.75; N, 2.79.
5.1.6. Epipodophyllotoxin 4-(N,N-dimethyl)carbamate (6d)
This compound was prepared according to the method de-
scribed for 6a employing N,N-dimethylamine hydrochloride
(49 mg, 0.6 mmol) and epipodophyllotoxin 4-(p-nitrophenyl)car-
bonate (5) (290 mg, 0.5 mmol) to afford compound 6d, 218 mg in
5.1.10. Epipodophyllotoxin 4-[N-(4-trifluoromethylbenzyl)]
carbamate (6h)
This compound was prepared according to the method de-
scribed for 6a employing 4-trifluoromethyl benzylamine (86
lL,
600 mol) and epipodophyllotoxin 4-(p-nitrophenyl)carbonate (5)
l
90% yield. Mp: 160–164 °C, ½a D25
ꢁ
: ꢂ101.0 (c 1.0, CHCl3). 1H NMR
(290 mg, 0.5 mmol) to afford compound 6h, 215 mg in 70% yield.
(CDCl3): d 2.92–3.0 (m, 7H), 3.10 (dd, 1H, J = 14.3, 4.9 Hz), 3.74 (s,
6H), 3.77 (s, 3H), 3.91 (t, 1H, J = 10.3 Hz), 4.36 (t, 1H, J = 8.8 Hz),
4.61 (d, 1H, J = 4.7 Hz), 5.98–6.12 (m, 3H), 6.23 (s, 2H), 6.53 (s,
1H), 6.91 (s, 1H); 13C NMR (CDCl3): d 37.8, 38.6, 41.6, 44.2, 54.5,
57.1, 61.0, 68.9, 101.8, 108.5, 108.9, 111.0, 131.7, 132.1, 134.5,
137.1, 146.8, 147.5, 147.8, 155.6, 174.9; IR(KBr) cmꢂ1: 2924,
1775, 1720, 1595, 1505, 1483; MS (FAB): 485 [M+]. Anal. Calcd
for C25H27NO9: C, 61.85; H, 5.61; N, 2.89. Found: C, 61.79; H,
5.66; N, 2.91.
Mp: 180–183 °C, ½a D25
ꢁ
: ꢂ65.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d
2.99–3.06 (m, 1H), 3.18 (dd, 1H, J = 14.4, 5.0 Hz), 3.74 (s, 6H), 3.8
(s, 3H), 4.03 (t, 1H, J = 10.1 Hz), 4.42– 4.54 (m, 3H), 4.63 (d, 1H,
J = 4.5 Hz), 6.01–6.21 (m, 3H), 6.26 (s, 2H), 6.55 (s, 1H), 6.93 (s,
1H), 7.39 (d, 2H, J = 7.6 Hz), 7.61 (d, 2H, J = 7.6 Hz); 13C NMR
(CDCl3): d 37.1, 41.5, 43.8, 44.7, 56.3, 60.6, 67.4, 69.1, 101.6,
108.4, 109.6, 110.1, 125.6, 125.6, 132.8, 134.5, 142.1, 147.5, 148.9,
152.7, 156.0, 174.2; IR(KBr) cmꢂ1: 3362, 2924, 1778, 1720, 1590,
1507, 1484; MS (FAB): 615 [M+]. Anal. Calcd for C31H28F3NO9: C,
60.49; H, 4.58; N, 2.28. Found: C, 60.54; H, 4.51; N 2.24.
5.1.7. Epipodophyllotoxin 4-[N-(2-methoxyethyl)]carbamate
(6e)
5.1.11. Epipodophyllotoxin 4-[N-2-(3-indolyl)ethyl]carbamate
(6i)
This compound was prepared according to the method de-
scribed for 6a employing 2-methoxyethylamine (51
and epipodophyllotoxin 4-(p-nitrophenyl)carbonate (5) (290 mg,
l
L, 600
l
mol)
This compound was prepared according to the method de-
scribed for 6a employing tryptylamine hydrochloride (118 mg,

A. Kamal et al. / Bioorg. Med. Chem. 19 (2011) 2975–2979
2979
0.6 mmol) and epipodophyllotoxin 4-(p-nitrophenyl)carbonate (5)
7. DNA Topoisomerase-II inhibition assay
(290 mg, 0.5 mmol) to afford compound 6i, 240 mg in 80% yield.
Mp: 170–175 °C, ½a D25
ꢁ
: ꢂ82.0 (c 1.0, CHCl3). 1H NMR (CDCl3): d
Topoisomerase-II activity was determined using kit (Topogen
Inc., USA, Cat No. 2000H). Reaction was assembled in micro centri-
2.98–3.14 (m, 4H), 3.58–3.62 (m, 2H), 3.73 (s, 6H), 3.77 (s, 3H),
3.85–3.88 (m, 1H), 4.33–4.36 (m, 1H), 4.60 (d, 1H, J = 4.5 Hz),
4.79 (t, 1H, J = 5.4 Hz), 5.98–6.12 (m, 3H), 6.23 (s, 2H), 6.51 (s,
1H), 6.88 (s, 1H), 7.00–7.25 (m, 3H), 7.32–7.62 (m, 2H); 13C
NMR (CDCl3): d 25.8, 38.2, 41.2, 41.4, 43.7, 54.3, 56.8, 60.8,
68.7, 101.8, 108.3, 108.8, 110.5, 111.7, 113.5, 119.5, 120.5,
121.7, 123.5, 127.8, 131.9, 132.3, 134.3, 136.8, 136.8, 146.6,
147.5, 148.1, 155.8, 175.3; IR(KBr) cmꢂ1: 3380, 2923, 2852,
1774, 1715, 1589, 1506, 1483; MS (FAB): 601 [M+1]. Anal. Calcd
for C33H32N2O9: C, 65.99; H, 5.37; N, 4.66. Found: C, 66.05; H,
5.32; N, 4.63.
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).
Acknowledgement
5.1.12. Epipodophyllotoxin 4-[2-(dimethylamino)ethyl]carba
mate (6j)
One of the author’s P.S. is thankful to UGC, New Delhi for the
award of senior research fellowship.
This compound was prepared according to the method de-
scribed for 6a employing N1,N1-dimethylethane-1,2-diamine
(53 mg, 0.6 mmol) and epipodophyllotoxin 4-(p-nitrophenyl)car-
bonate (5) (290 mg, 0.5 mmol) to afford compound 6j, 227 mg in
References and notes
86% yield. Mp: 172–174 °C, ½a D25
ꢁ
: ꢂ105.0 (c 1.0, CHCl3). 1H NMR
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(CDCl3): d 2.42–2.92 (m, 9H), 3.10 (dd, 1H, J = 14.3, 4.9 Hz), 3.32–
3.38 (m, 2H), 3.75 (s, 6H), 3.79 (s, 3H), 3.91 (t, 1H, J = 10.3 Hz),
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Tumour cells were grown in tissue culture flasks in growth
medium (RPMI-1640 with 2 mM glutamine, pH 7.4, 10% fetal calf
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37 °C in an atmosphere of 5% CO2 and 95% relative humidity in
a CO2 incubator. The cells at subconfluent stage were harvested
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containing 0.02% EDTA) and suspended in growth medium. Cells
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for 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 lay-
ering of 50 ll of 50% trichloroacetic acid. The plates were incu-
bated at 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.
100 ll 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 unbound SRB was quickly removed by washing the
wells five times with 1% acetic acid. Plates were air dried, tris-
buffer (100 ll of 0.01 M, pH 10.4) was added to all the wells
and plates were gently stirred for 5 minutes on a mechanical stir-
rer. The optical density was recorded on ELISA reader at 540 nm.
The cell growth at absence of any test material was considered
100% and in turn growth inhibition was calculated. IC50 values
were determined by regression analysis.
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