Synthesis and cytotoxic activity of novel derivatives of 4′-demethylepipodophyllotoxin

Article abstract of DOI:10.1016/j.bmcl.2004.07.094

The IC₅₀ values of 9g (n = 5, R = CH₂Ph), which is the most potent inhibitor against HL-60 and A-549 cells, are 0.04 and <0.01 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2004.07.094

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5063–5066
Synthesis and cytotoxic activity of novel derivatives of
40-demethylepipodophyllotoxin
Shi-Wu Chen, Xuan Tian* and Yong-Qiang Tu*
State Key Laboratory of Applied Organic Chemistry and Department of Chemistry, Lanzhou University, Lanzhou 730000, PR China
Received 7 July 2004; accepted 30 July 2004
Available online 1 September 2004
Abstract—Nine novel 4b-N-substituted-5-FU-40-demethylepipodophyllotoxin derivatives were synthesized and evaluated as poten-
tial antitumor agents. All of the target compounds showed more significant cytotoxic activity against HL-60 and A-549 in vitro than
VP-16 and 5-FU. Among them, 4b-N-substituted-phenylalanine 5-Fu pentyl ester-40-demethylepipodophyllotoxin (9g) was found
to exhibit most potent cytotoxic activity against HL-60 and A-549 cell (IC50 is 0.04 and <0.01lM, respectively).
Ó 2004 Elsevier Ltd. All rights reserved.
Podophyllotoxin (1) and its derivatives exhibit pro-
nounced biological activity as antiviral agents and
antineoplastic drugs.1,2 The podophyllotoxin derivatives
etoposide (VP-16, 2a), etopophos (etoposide phosphate,
2b), and teniposide (VM-26, 2c) are currently used in the
chemotherapy for various types of cancer, including
small cell lung cancer, testicular carcinoma, lymphoma,
and KaposiÕs sarcoma.3–5 NK611 (2d), as well as GL331
and TOP53 are presently under clinical trials.1,6 In addi-
tion, podophyllic aldehyde and its analogues were found
to be a highly selectivity against the HT-29 colon carci-
noma.7,8 Interestingly, these semisynthetic derivatives
and the parent compound, podophyllotoxin, showed
differentmechanisms of action. 9 Podophyllotoxin inhib-
its the assembly of tubulin into microtubules through
interaction with protein at the colchicine binding site,
preventing the formation of the spindle.1,9 While etopo-
side and congeners induce a premitotic blockade in late
S stage of the cell cycle because of the inhibition of
DNA topoisomerase II(TopII), an enzyme required for
the unwinding of DNA during replication. Etoposide
binds to and stabilizes the DNA–protein complex pre-
venting religation of the double-stranded breaks.1,9
However, due to the typical adverse effects, such as ane-
mia, hair loss and severe gastrointestinal disturbances,
the application of them has been limited to a certain
extent.10 Therefore, being continued research on podo-
phyllotoxin is currently focused on structure optimiza-
pharmacological profiles and broader therapeutic
scope.2,11
Antimetabolite 5-fluorourcial (5-FU, 3) is one of the
major anticancer agents used clinically for the treatment
of stomach, colorectal, head, and neck cancers.12 How-
ever, response rate and duration have limited its efficacy,
although some 5-FU dosing schedules such as continu-
ous infusion have increased the response rate compared
to bolus and injection.13 One main strategy of potentiat-
ing the antitumor activity of 5-FU is chemical and bio-
chemical modulations by combination with cytotoxic or
noncytotoxic agents (Fig. 1).14
As mechanism of action is concerned, 5-FU also belongs
to DNA-interacting agent.14 According to the principle
of drug design, combination of 40-demethyl-
epipodophyllotoxin and 5-FU may overcome some
faults and lead to decreased repair of DNA damage or
increased DNA adduct formation in cancer cells. Struc-
ture–activity relationship (SAR) studies suggested that
the structural features essential for the antineoplastic
activity include 40-demethylation, 4-epimerization, 4-
substituted, and the trans-fused lactone ring.15 And it
was found that C4b-N-substituted (including 4b-aniline
or alkylamino,16–18 4b-amido or sulphonamido19 and
amino acid20,21) congeners of podophyllotoxin resulted
in inhibitors, which have biological activity comparable
or superior to podophyllotoxin.
tion
to
generate
derivatives
with
superior
Based on the above, as well as amino acids with better
biological activity and good water solubility in the human
body, we combined 40-demethylepipodophyllotoxin and
Keywords: Podophyllotoxin; 5-FU; Amino acid; Antitumor drugs.
*
Corresponding authors. Tel.: +86 931 8912410; fax: +86 931
8912582; e-mail addresses: tuyq@lzu.edu.cn; xuant@lzu.edu.cn
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.094

5064
S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5063–5066
B
OH
A
5
8
R1
O
11
10
9
6
7
O
O
O
4
3
HO
O
12
O
No
2a
2b
R1
R2
R3
R2
O
4
5
F
2
O
O
1
CH3
CH3
OH
OH
1'
NH
O
O
3
6'
5'
OMe
2'
MeO
OH PO(ONa)2
6
N
H
O
2
1
O
2c
2d
OH
OH
OH
3'
4'
OMe
S
CH3
NMe2
5-Fluorouracil 3
MeO
OMe
3
R
Podophyllotoxin 1
Figure 1. (A) Structure of podophyllotoxin and related compounds. (B) Structure of 5-fluorouracil.
5-FU with natural L-amino acid, and hoped to improve
their anticancer activity and decrease their cytotoxicity
for normal cells. Here, we report the synthesis of novel
4b-N-substituted-5-FU-40-demethylepipodophyllotoxin
and evaluation of cytotoxicity against human leukemic
(HL-60) and human lung carcinoma (A-549) in vitro.
The preliminary results showed these compounds pos-
sessed more significant cytotoxicity against HL-60 and
A-549 than both VP-16 and 5-FU.
amine in methylene chloride yielded L-a-amino acid
5-fluorouracil–propyl (butyl or pentyl) esters 7a–i. As
described in Scheme 2, 4b-N-substituted-amino acid
5-FU propyl (butyl or pentyl) ester––40-demethylepip-
odophyllotoxins 9a–i24 were synthesized by direct nucle-
ophilic substituted (SN1) of 1.3 equivalentappropriaet
7a–i with 4b-bromo-40-demethyl-epipodophyllotoxin 8,
which prepared according to literature,25 in the presence
of triethylamine in refluxing THF.
As shown in Scheme 1, compounds 7a–i were synthe-
sized in three steps from potassium salts of L-amino
acids 4a–c.22 Compounds 4a–c were treated initially
with ethyl acetoacetate to afford 5a–c, in which the
a-amino group of amino acid were protected. N-pro-
tected L-a-amino acid 5-fluorouracil–propyl (butyl or
pentyl) esters 6a–i were prepared through combination
5a–c with 1-[x-bromo-propyl (butyl or pentyl)]-5-fluor-
ouracil23 under catalyzed by KI in N,N-dimethylforma-
mide (DMF). Subsequent deprotection of compounds
6a–i with methanolic HCl and basification with triethyl-
While synthesis of the target compounds, the bulky C-1
a pendant aromatic ring E directed the substitution to
be stereoselective in yielding the C-4b alkylamino iso-
mers as the main products. In some cases, C-4a isomers
were also observed, butas minor producst. The C-4
b
alkylamino isomers were purified by column chromato-
graphy. The assignment of the configuration at C-4 posi-
tion for compounds 9a–i was based on J3,4 coupling
constants. The C-4b substituted compounds have
J3,4 ꢀ 4.0Hz due to a cis relationship between H-3 and
H-4. The C-4a substituted compounds, however, have
R
CH3
R
O
O
CHCOOK
C
NH
CH3CCH2COC2H5
NH2CHCOOK
HC
+
5a~c
C
O
4a~c
H5C2O
F
N (CH2)nBr
(n = 3,4,5)
O
N
H
O
O
O
F
HN
R
F
CH3
HN
R
O
N(CH2)nOOCCHNHC
O
N(CH2)nOOCCHNH2
CH
O
OC2H5
6a~i
7a~i
compds 6, 7
_____________________________________
n
R=CH2Ph R=CH(Me)CH2Me R=CH2SMe
4a, 5a R=CH2Ph
_____________________________________
4b, 5b R=CH(Me)CH2Me
4c, 5c R=CH2SMe
3
4
5
6a,7a
6d,7d
6g,7g
6b,7b
6e,7e
6h,7h
6c,7c
6f,7f
6i, 7i
_____________________________________
Scheme 1. Synthesis of L-amino acid 5-fluorouracil esters 7a–i.

S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5063–5066
5065
F
R
Br
O
O
N
O
O
HN
n
N
H
F
O
O
O
R
O
O
O
N
O
O
H2N
n
+
N
H
O
O
O
O
MeO
OMe
MeO
OMe
OH
OH
8
9a~i
7a~i
Scheme 2. Synthesis of compounds 9a–i (R and n, see the Table 1).
J3,4 P 10.0Hz because of H-3 is trans to H-4.26,27 The
relative stereochemistry at C-4 position was also demon-
strated by the observation of NOE between H-3 and
Acknowledgements
This work was financially supported by NSFGS
(ZGS033-A43-013) and NSFC (No. 20021001); We also
thank National Center for Drug Screening of China for
help in performing the anticancer activity in vitro.
1
H-4. While H-3 was irradiated in H NMR spectrum,
the H-4 was enhanced 4–5%, however, H-2 was not en-
hanced. Thus we ensured that H-3 is cis to H-4 and H-2
is trans to H-3.
Cytotoxicities of all the target compounds against two
tumor cell lines HL-60 and A-549 were tested in vitro
following procedure of the methods described by Ber-
geron.28 The results of these assays were used to obtain
the corresponding inhibition rates, from which IC50
(lM) values were calculated. As shown in Table 1, com-
pounds 9a–i are more active than both VP-16 and 5-FU.
Compound 9g is the most potent against HL-60 and
A-549 cells.
References and notes
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In conclusion, the strategy to synthesize novel deriva-
tives of podophyllotoxin through combination of
antimetabolite 5-FU and TOP-II inhibitor 40-
demethylepipodophyllotoxin with L-amino acid may be
successful. The target compounds 9a–i showed more
effective cytotoxic activity than both VP-16 and 5-FU.
The IC50 values of compounds 9a–i againstHL-60 and
A-549 were less than those of VP-16 and 5-FU. Among
them, Compound 9g was the most potent cytotoxic
7. Gordaliza, M.; Castro, M. A.; Miguel del Corral, J. M.;
´
´
´
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´
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values 0.04lM againstHL-60 and less
50
than 0.01lM against A-549, respectively. Further inves-
tigation of this series of compounds is underway.
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Table 1. Cytotoxic activity of 9a–i in vitro (IC50, lM)
Compd
n
R
HL-60a
A-549b
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Fryer, J. B. J. Clin. Oncol. 1989, 7, 425.
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S. J.; Cheng, H. H.; Cheng, Y. C.; Gurwith, M.; Lee, K.
H. J. Med. Chem. 1999, 42, 2441.
9a
9b
3
3
3
4
4
4
5
5
5
CH2Ph
0.31
0.24
0.99
0.42
0.53
0.05
0.04
0.30
0.04
2.75
65.3
0.48
0.18
0.29
0.30
0.74
1.87
<0.01
0.53
0.23
7.38
50.5
CH(Me)CH2Me
CH2SMe
CH2Ph
9c
9d
9e
CH(Me)CH2Me
CH2SMe
CH2Ph
9f
9g
9h
9i
CH(Me)CH2Me
CH2SMe
VP-16
5-FU
17. Zhu, X. K.; Guan, J.; Xiao, Z.; Cosentino, L. M.; Lee, K.
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a MTT methods, drug exposure was for 48h.
b SRB methods, drug exposure was for 72h.

5066
S.-W. Chen et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5063–5066
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1989, 10, 605.
OCHaCH2CH2CH2CH25-FU), 4.24 (s, 1H, OCHb-
CH2CH2CH2CH25-FU), 4.06 (m, 2H, H-11), 3.97 (d, 1H,
H-4, J = 2.7Hz), 3.72 (s, 6H, OMe-30, 50), 3.64 (t, 2H, CH2-
N-5Fu, J = 7.2Hz), 3.49 (t, 1H, CHCH2Ph, J = 6.0Hz),
3.14 (dd, 1H, H-2, J = 14.1, 5.7Hz), 2.98 (d, 2H, CH2Ph,
J = 6.0Hz), 2.76 (m, 1H, H-3), 1.61 (m, 4H,
CH2CH2CH2CH2CH25-FU), 1.25 (m, 2H, CH2CH2CH2-
CH2CH2 5-FU);13 C NMR (CDCl3, 75M) d 175.4, 173.7,
157.3, 149.4, 147.7, 146.8, 146.2, 140.3, 136.6, 133.8, 131.9,
131.1, 130.9, 129.0, 128.8, 128.5, 126.9, 110.4, 108.5, 107.8,
101.3, 68.2, 64.4, 62.4, 56.3, 54.6, 48.6, 43.5, 40.6, 39.9,
38.6, 28.3, 27.9, 22.6; HRMS (ESI): 746.2705 for [M+H]+
(calcd 746.2720 for C39H41O11N3F).
23. U. S. Appl. 1965, July 22, 12. Chem. Abstr. 1967, 67,
91093e.
24. To a solution of 8 (3mmol) in 15mL dry THF was added
30mg triethylamine and a solution of 7 (10mmol) in dry
THF (5mL). The mixture was refluxed under Ar. While
TLC analysis showed that most of the starting material had
been converted, the reaction was stopped. Then the
mixture was filtered and evaporated under reduced pres-
sure. The residue was purified by silica gel column
chromatography to afford compound 9. For example, 4b-
N-substituted-phenylalanine 5-Fu-pentyl ester-40-demeth-
25. Kuhn, M.; Keller-Juslen, G.; Wartburg, A. Helv. Chim.
Acta 1969, 52, 944.
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R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, M.
R. J. Natl. Cancer Inst. 1990, 82, 1113.
25
ylepipodophyllotoxin 9g: yield 23.8%; mp 125–127°C; ½aꢁD
ꢂ61 (c 0.3, acetone); IR (KBr) 3443, 3066, 2938, 2837,
1774, 1714, 1693, 1613, 1505, 1428, 1366, 1231, 1114cmꢂ1
;
1H NMR (CDCl3, 300M) d 9.64 (br, 1H, NH), 7.28–7.12
(m, 6H, H-6 of 5-Fu, CH2Ph), 6.47 (s, 1H, H-5), 6.42 (s,
1H, H-8), 6.20 (s, 2H, H-20, 60), 5.93 (s, 2H, OCH2O), 5.49
(br, 1H, 40-OH), 4.43 (d, 1H, H-1, J = 5.4Hz), 4.27(s, 1H,