Carbamates of 4′-demethyl-4-deoxypodophyllotoxin: Synthesis, cytotoxicity and cell cycle effects

Article abstract of DOI:10.1016/j.bmcl.2011.10.024

In an attempt to generate compounds with superior bioactivity and reduced toxicity, 12 carbamates of 4′-demethyl-4-deoxypodophyllotoxin, N-(1-oxyl-4′-demethyl- 4-deoxypodophyllic)-α-amino acids amides, were synthesized and evaluated for antiproliferative activity and cell cycle effects. These synthesized compounds proved to be more hydrophilic, as well as improved or comparable in vitro cytotoxicities against four cell lines (A-549, HeLa, SiHa, and HL-60) compared with either parent DPT or anti-cancer drug VP-16. Furthermore, flow cytometric analysis exhibited that N-(1-oxyl-4′- demethyl-4-deoxypodophyllic)-d-α-methine amide (15f) induced cell cycle arrest in the G2/M phase in A-549 cells.

Full text of DOI:10.1016/j.bmcl.2011.10.024

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7355–7358
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Carbamates of 40-demethyl-4-deoxypodophyllotoxin: Synthesis, cytotoxicity
and cell cycle effects
Shi-Wu Chen a, , Yuan-Yu Gao a, Ni-Ni Zhou a, Jie Liu b, Wen-Ting Huang a, Ling Hui c, , Yan Jin b,
⇑
⇑
Yong-Xin Jin d,
⇑
a School of Pharmacy, State Key Laboratory of Applied Organic Chemistry, Key Lab of Preclinical Study for New Drugs of Gansu Province, Lanzhou University, Lanzhou 730000, China
b College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
c Experimental Center of Medicine and Key Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
d Department of Medicine, Second People’s Hospital of Gansu Province, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to generate compounds with superior bioactivity and reduced toxicity, 12 carbamates of
40-demethyl-4-deoxypodophyllotoxin, N-(1-oxyl-40-demethyl- 4-deoxypodophyllic)-
a-amino acids
Received 3 August 2011
Revised 26 September 2011
Accepted 7 October 2011
Available online 13 October 2011
amides, were synthesized and evaluated for antiproliferative activity and cell cycle effects. These synthe-
sized compounds proved to be more hydrophilic, as well as improved or comparable in vitro cytotoxicities
against four cell lines (A-549, HeLa, SiHa, and HL-60) compared with either parent DPT or anti-cancer drug
VP-16. Furthermore, flow cytometric analysis exhibited that N-(1-oxyl-40-demethyl-4-deoxypodophyl-
Keywords:
lic)-
D-
a
-methine amide (15f) induced cell cycle arrest in the G2/M phase in A-549 cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Podophyllotoxin
4-Deoxypodophyllotoxin
Amino acid
Antitumor activity
Cancer is a leading cause of human death and can be triggered
by environmental pollutants and genetic mutations. A number of
natural products, with diverse chemical structures, have been iso-
lated as anticancer agents until now.1 Several potential lead mole-
cules, such as podophyllotoxin (PPT, 1), camptothecin (2), taxol (3),
combretastatin A-4 (4), vincristine, vinblastine, etc., have been iso-
lated from plants and many of them have been modified to yield
analogues endowed with better activity, lower toxicity or higher
water-solubility. For example, etoposide (5, VP-16), teniposide
(6) and the water-soluble prodrug, etoposide phosphate (7) de-
rived from PPT are presently in clinical use for the treatment of
small cell lung cancer, testicular carcinoma, non-Hodgkin’s lym-
phoma, and Kaposi’s sarcoma.2
4-Deoxypodophyllotoxin (DPT, 8), an analogue of PPT, is a potent
antimitotic agent first isolated from Anthriscus sylvestris.3 It has been
reported that DPT exhibits antiproliferative and antitumor activi-
ties,4–8 anti-inflammatory,9,10 anti-viral activities11,12 and broad
insecticidal activity.13 The mechanism studies revealed that DPT
inhibits tubulin polymerization and induces cell cycle arrest at G2/
M, followed by apoptosis through multiple cellular processes,
involving the activation of ATM, upregulation of p53 and Bax, activa-
tion of caspase-3 and -7, and accumulation of PTEN resulting in the
inhibition of the Akt pathway.14,15 Besides, DPT inhibits migration
and MMP-9 via MAPK pathways in TNF-
-induced HASMC.16
a
Previously, it was reported that 40-demethyl-4-deoxypodo-
phyllotoxin (DDPT, 9, Fig. 1) exerted a comparable in vitro potency
with DPT. However, in vivo experiments revealed a substantial loss
of the antitumor activity of DDPT in the BDF1/3LL model.17 The re-
sults indicated that the free hydroxy group at the C-40 position in
DDPT was not favorable for antitumor activity. Lately, Ahn and
co-workers envisioned that transformation of the hydroxyl group
into bioreversible functionalities might improve the in vivo activity
of DDPT. Thus, they synthesized and evaluated in vivo antitumor
activity of many prodrugs of DDPT, including carbamates, carbon-
ate, esters (such as alkyl, carboxyl alkyl, unsaturated fatty acid es-
ters, and water-soluble amino acid esters). It was reported that the
esters showed increased in vivo antitumor activity despite the low-
er in vitro activity than DDPT.18–20
Inspired by previous work21–27 as well as the fact that amino
acids possess good water solubility, we here present the synthesis
of twelve new
a-amino acids carbamates derivatives of DDPT
(15a–l) and our measurements of their cytotoxic activity against
A-549, HeLa, SiHa, and HL-60 cell lines. Furthermore, the effect of
15f on A-549 cells was also investigated to reveal its actions on
the cell cycle.
Amino acids amides 12a–l were prepared as depicted in
Scheme 1. Compounds 11a–h were prepared by Boc-protected ami-
no acid 10 reacted with 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-
⇑
Corresponding authors. Tel./fax: +86 931 8915686.
E-mail address: chenshwu@gmail.com (S.-W. Chen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.024

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S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7355–7358
R1
R1
4
O
O
5
R1
R2
H
O
HO
O
O
R1
OH
H
R2
Me
Me
O
3
HO
O
Me
Etoposide
Teniposide
Etopophos
5
6
O
1
8
9
2
1
8
O
O
O
6'
2'
OMe
H
O
S
H
H
MeO
4'
O
OR2
OH
OH
P
7
Me
MeO
OMe
OR2
O
OH
O
AcO
O
MeO
MeO
N
Ph
NH
O
N
O
OMe
OH
O
O
OH
H
OMe
AcO
OBz
HO
OH
O
2
3
4
Figure 1. Structures of compounds 1–9.
in dichloromethane and afford compounds 12a–l, the later were
used directly for next reaction without further purified.
O
H
N
Boc
NH2
The synthetic route to the target compounds first involved gen-
eration of the intermediate DDPT, which was prepared from 1 as
described in previous publication (Scheme 2).27 Briefly, the inter-
mediate 9 is prepared through 40-demethylation of 1 with HBr
gas in CH2Cl2,30 and subsequent 4-deoxylation of 13 by hydrogen-
olysis with 10% Pd/C in HAc.31 Then treatment of 9 with 4-nitro-
phenyl chloroformate in the presence of pyridine provides the
intermediate 14 in high yield.32 Subsequently, introduction of the
O
O
i
R1
H
N
H2N
R2
iii
N
H
11a-h
O
Boc
OH
ii
R1
12a-l
R1
H
N
R2
10
Boc
N
R1
11i-l
H
Scheme 1. Synthesis of 12a–l. Reagents and conditions: (i) EEDQ, NH4HCO3, CHCl3,
rt; (ii) EDCI, DIEA, amine; (iii) TFA, CH2Cl2.
carbamate chains was performed by reacting 14 with
the appropriate -amino acid amide 12a–l in the presence of 4-
N,N-dimethylaminopyridine (DMAP) and triethylamine (Et3N),
affording -amino
N-(1-oxyl-40-demethyl-4-deoxypodophyllic)-
acids amides 15a–l with high or moderate yields. The structures
of the final products 15a–l were identified by IR, 1H NMR, 13C
NMR and HR-MS.
a-NH2 of
a
a
Table 1
Biological evaluation of compounds 15a–l
a
Compounds
Cytotoxicity (IC50
,
l
M)
logP
The biological activities of the DPT derivatives 15a–l were eval-
uated by an in vitro cytotoxicity test carried out with a panel of
four human tumor cell lines (A-549, lung carcinoma; HeLa, cervical
carcinoma; SiHa, cervical squamous cell carcinoma; and HL-60, hu-
man premyelocytic leukemia).33 The results expressed as IC50 val-
ues are summarized in Table 1.
As illustrated in Table 1, the DPT derivatives 15a–l were gener-
ally showed more potent or comparable activity in comparison
with DPT, DDPT and VP-16 in their in vitro cytotoxicity to these
four cell lines. These compounds were more effective in HL-60
cells, and had lower potency in A-549 cells, although the order of
potency varied in each cell line. In Table 1, we also show that dif-
A-549b
SiHab
HeLac
HL-60c
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
VP-16
DPT
DDPT
4.23
1.49
9.93
1.77
0.86
0.887
3.41
1.61
2.31
1.91
3.37
2.69
24.9
1.38
1.8
0.696
2.89
0.877
3.70
4.32
0.614
1.07
0.416
0.432
14.6
1.12
6.87
40.4
0.743
3.83
0.703
5.28
2.03
0.528
1.72
0.575
0.433
20.3
0.693
3.07
0.397
1.03
0.67
–0.09
0.84
1.26
1.36
0.28
0.26
1.35
1.33
1.45
2.57
1.95
2.06
0.69
3.16
2.92
1.02
0.711
0.0893
0.94
0.231
0.213
1.03
0.416
0.988
2.85
2.76
1.98
43.0
ferent substituent at the
pounds 15a–h have different effects on the activity of the
resultant compound. Notably, the compounds with -amino acids
incorporated appear to be more potent than those with -amino
a-carbon of the amino acids in com-
6.01
53.3
0.47
2.96
D
a
b
c
Data are the mean of three independent experiments.
MTT method with 48 h drug exposure.
L
acid (15e vs 15f, 15g vs 15h) on their anti-proliferative activity.
In addition, carbamates of deoxypodophyllotoxin with cyclopropyl
amine incorporated showed more potent anti-proliferative activity
in the in vitro assay than those of cyclopentyl amine except A-549
cell line (15i vs 15j, 15k vs 15l). In the previous reports, the ali-
phatic or aromatic carbamates of DDPT showed the lower cytotoxic
with approximately 10-fold drops in the in vitro activity relative to
that of DDPT. The authors considered the prodrugs of DDPT exhib-
ited less in vivo antitumor activity because they were stable in un-
CCK-8 method with 48 h drug exposure.
quinoline (EEDQ) under ammonium hydrogencarbonate in chloro-
form at room temperature.28 Boc-amino acid amides 11i–l were eas-
ily obtained through Boc protected L-valenine (or L-phenylalanine)
was reacted respectively with cyclopropylamine (or cyclopentyl-
amine) under 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide
hydrochloride (EDCI) in the presence of diisopropylethylamine
(DIEA) in dichloromethane.29 Then, the Boc protection group of com-
pounds 11a–l were easily removed using trifluoroacetic acid (TFA)
der cell culture conditions.18 These
DDPT showed more potent or comparable in vitro activity in
a-amino acids carbamates of

S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7355–7358
7357
OH
OH
O
O
O
O
O
O
O
O
O
ii
i
O
O
O
MeO
iii
OMe
MeO
OMe
MeO
OMe
OH
9
OH
13
OMe
1
O
O
O
O
O
O
O
O
iv
MeO
OMe
MeO
OMe
O
O
H
O
O
N
R2
N
H
O
O
R1
NO2
14
15a-l
1
R1
Compounds
15a
R2
H
H
H
H
H
H
Compounds
15g
R
R2
H
L-Me
L-CH2C6H5
D-CH2C6H5
L-CHMe2
15b
L-CHMe2
15h
H
15c
L-CH2CHMe2
15i
cyc-Pr
cyc-Pen
cyc-Pr
cyc-Pen
15d
L-CH(Me)CH2Me
L-CH2CH2SMe
D-CH2CH2SMe
15j
L-CHMe2
15e
15k
L-CH2C6H5
L-CH2C6H5
15f
15l
Scheme 2. Synthesis of 15a–l. Reagents and conditions: (i) HBr, acetone/H2O–BaCO3; (ii) H2, 10% Pd/C, HAc; (iii) 4-nitrophenyl chloroformate, pyridine, CH2Cl2; (iv) 12a–l,
DMAP/Et3N, CH2Cl2.
Figure 2. Effects of 15f on cell cycle progression. Values indicate the percentage of the cell population at the phase of the cell cycle. (A) Control A-549 cells; (B) A-549 cells
treated with 0.4 lM 15f at 12 h; (C) A-549 cells treated with 0.4 lM 15f at 24 h.
comparison with DPT, DDPT and VP-16, so they are a series of
amino acids carbamates derivatives, not prodrugs of DDPT.
a
-
The octanol–water partition coefficients of 15a–l were also
determined34 and results were shown in Table 1. In line with

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S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7355–7358
3. Masuda, T.; Oyama, Y.; Yonemori, S.; Takeda, Y.; Yamazaki, Y.; Mizuguchi, S.;
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sized carbamoyl derivatives 15a–l are significantly lower than
those of DPT and DDPT, which means that they are all more hydro-
philic than the parent compound. The result was in according with
the previously reported results,18 derivatives of DDPT formed by
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may improve the hydrophilic and solubility.
The effects of 15f on cell cycle progression were also deter-
mined by FACS analysis in propidium iodide-stained A-549 cells.
As shown in Figure 2, treatment with 15f (0.4 lM) led to a time-
dependent accumulation of cells in the G2/M phase with a con-
comitant decrease in the population of G1 phase cells. 27.7%
(Fig. 2B) and 58.1% (Fig. 2C) of the cells were in G2/M phase after
12 h and 24 h treatment with 15f, respectively, compared with
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than those identified in previous studies.14,15
In summary, the podophyllotoxin class of compounds is a
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Acknowledgments
This work was financially supported by the Fundamental Re-
search Funds for the Central Universities, Lanzhou University
(lzujbky-2012-136), The Open Funds of Key Lab of Preclinical Study
for New Drugs of Gansu Province (GSKFKT-00801).
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Supplementary data
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