Synthesis and biological evaluation of novel conjugates of podophyllotoxin and 5-FU as antineoplastic agents

Article abstract of DOI:10.1016/j.bmc.2009.03.009

A series of novel conjugates of podophyllotoxin and 5-FU were designed using association strategy and were synthesized by coupling 4′-demethylepipodophyllotoxin with 5-FU-N¹-alkyl amino acid ester. These derivatives have been evaluated for cyto

Full text of DOI:10.1016/j.bmc.2009.03.009

Bioorganic & Medicinal Chemistry 17 (2009) 3111–3117
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel conjugates of podophyllotoxin
and 5-FU as antineoplastic agents
Shi-Wu Chen a,b, , Rong Xiang a,c, Jian Liu b, Xuan Tian b,
*
*
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b Sates Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
c Lanzhou Hospital of Traditional Chinese Medicine Traumatocomium, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel conjugates of podophyllotoxin and 5-FU were designed using association strategy and
were synthesized by coupling 40-demethylepipodophyllotoxin with 5-FU-N1-alkyl amino acid ester.
These derivatives have been evaluated for cytotoxicity in vitro against tumour cell lines (HL-60, K562,
A-549 and AGS), and their octanol–water partition coefficients (logP) were also determined. As compared
with VP-16, most compounds showed superior water solubility, as well as more potent inhibitions
against these four tumour cell lines. Compound 21 showed interaction with calf thymus DNA, and it
was relatively resistant to metabolism by human plasma.
Received 15 January 2009
Revised 28 February 2009
Accepted 3 March 2009
Available online 12 March 2009
Keywords:
Podophyllotoxin
5-FU
Ó 2009 Elsevier Ltd. All rights reserved.
Antineoplastic agents
Calf thymus DNA
1. Introduction
showed stronger inhibitions to tumour or to TOP-II. In addition,
podophyllic aldehyde and its analogues were found to be of a
Anticancer drugs are rarely used singly to treat cancer, because
only a few tumours are sensitive enough to be cured by single
drugs. For a specific type of tumour, effective chemotherapy usu-
ally depends on suitable combinations.1 Many combinations,
which were named mutual drug, in clinical use consist of an anti-
metabolite with one or more other anticancer agents. A mutual
drug usually consists of two different synergistic drugs joined to-
gether. The two drugs may be connected directly or by means of
a linker.2
Podophyllotoxin (1) is an antimitotic natural product. Its
inhibitory activity on cell growth led to the development of
the clinically valuable anticancer agents, etoposide (2), tenipo-
side (3) and the water-soluble prodrug, etoposide phosphate
(4) (Fig. 1). The cytotoxic mechanism of these drugs is the inhi-
bition of topoisomerase II, unlike the lead compound that inhib-
its mitosis.3,4 Through extensive structure–activity relationship
studies, several potential drug candidates were synthesized such
as GL-331,5 TOP-53,6 NK6117 and F11782.8 Recently, more com-
plex and diverse analogues have been synthesized either to get
more potent compounds or to overcome drug resistance.9 4-N-
substituted 40-demethylepipodophyllotoxin derivatives, such as
4b-sulfonamide,10 (400-benzamide)-amino11 and carbamate,12
high selectivity against the HT-29 colon carcinoma.13 Further-
more, a number of multidrug approaches have been tried to en-
hance the tumour selectivity or to increase the aqueous
solubility.14
Our group has been aiming at the discovery of new antitu-
mour drugs of podophyllotoxin derivatives with improved bioac-
tivities, lower toxicity and without resistance effects in recent
years.15,16 In the present work, we reported the synthesis of a
series of conjugates of 1 together with 5-FU, in which the main
chemical differences are the length of the side chain (propyl, bu-
tyl or pentyl), and the difference of amino acids. Their cytotoxic
activities against four tumour cells (HL-60, K562, A-549 and
AGS) were evaluated. Also, the octanol–water partition coeffi-
cients (logP) were measured. Furthermore, metabolism stability
and interaction with calf thymus DNA (CT DNA) of 21 in vitro
were studied.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 7I–III and 8 is outlined in Scheme 1.
5-FU was protected with 1,1,1,3,3,3-hexamethyldisilazane
(HMDS)/chlorotrimethylsilane (TMSCl) and 6 was obtained in
excellent yield; then 6 reacted with 1,3-dibromo-propane (or 1,4-
dibromo-butane, or 1,5-dibromo-pentane) under 105–110 °C
* Corresponding authors. Tel./fax: +86 931 8915686 (S.-W.C.).
E-mail addresses: chenshwu@gmail.com (S.-W. Chen), xuant@lzu.edu.cn (X.
Tian).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.009

3112
S.-W. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3111–3117
R'
O
OH
O
5
4
11
3
2
O
HO
O
A
O
R'
R''
H
O
O
D
HO
B
C
Me
Etoposide
Teniposide
Etopophos
2
3
O
O
1
8
O
O
2'
H
6'
E
S
O
O
OH
OH
MeO
OMe
4'
Me
P
4
OMe
MeO
OMe
Podophyllotoxin
1
OR''
Figure 1. Podophyllotoxin (1), etoposide (2), teniposide (3) and etopophos (4).
resulting in the 7I–III
.
17 Compounds 8Ia–h, 8IId,g and 8IIId,g were pre-
carried out with a panel of four tumour cell lines that comprise hu-
man premyelocytic leukemia (HL-60), human erythromyeloblas-
toid leukemia (K562), human gastric cancer (AGS) and human
lung carcinoma (A-549), using VP-16 (1) and 5-FU (5) as reference
compounds. The screening procedure was based on the standard
MTT or SRB method. The IC50 values are shown in Table 1.
As shown in Table 1, 10–21 showed a basically stronger inhibi-
tion of these four cell lines than both VP-16 and 5-FU. In these four
tumour cell lines, the inhibition of these target compounds showed
decreased activity in the following: HL-60 ꢀ A-549 > K562 > AGS.
The large activity range of compounds 10–17 indicated that the
substituent of the amino acids markedly affected the activity pro-
files of this compound class, and some important SAR information
was revealed. As seen with compounds 12 and 17, inclusion of
moieties containing hydroxyl groups in the amino acid side chains
decreased the activity significantly. This decrease may be caused
by hydrogen bonding between the nitrogen or oxygen atoms and
the enzyme/DNA might cause the loss of activity, or the relatively
polar moieties might impede an important hydrophobic interac-
tion between the molecules and topo II/DNA. The different lengths
of alkyl linkages of target compounds did not affect obviously their
cytotoxicities (13 vs 18 vs 20, 16 vs 19 vs 21).
The octanol–water partition coefficients of 10–21 were also
determined and results are shown in Table 1. The logP of 10–21
is lower than VP-16, which means that their water solubility is bet-
ter than that of VP-16.
Ordinary, the analogues of 2 were considered as inhibitors of
TOP-II; however, other compounds, which showed potent inhibi-
tions against tumour cell lines, but not inhibitions to TOP-II were
also found.21 In order to study the interaction of 21 with CT DNA
in pH 7.2 PBS, the CD spectrum of CT DNA and its complexes with
pared in four steps from potassium salts of
L-a-amino acids as in
the previously reported method.16,18
Compounds 10–21 were synthesized from podophyllotoxin (1)
according to the literature methods (Scheme 2).19 Briefly, interme-
diate 4b-iodide 40-demethylepipodophyllotoxin (9) was prepared
stereoselectively from 1 through successive 40-demethylation, 4-
iodination with methanesulfonic acid/sodium iodide, and nucleo-
philic substitution with 8, afforded target compounds 10–21 by
one-pot reaction in the presence of triethylamine in refluxing
THF. The structure of all target compounds was identified by 1H
NMR, 13C NMR, IR and HRMS.
Biological activities of podophyllotoxin derivatives were af-
fected heavily by their configurations at positions C-4 and C-2,
compounds with the structure of C-4b substitute and trans lactone
C-ring generally show more potent biological activities. So it is
important to corroborate the relative configurations at C-4 and
C-2 of target compounds. While synthesis of the target compounds
10–21, the bulky C-1
stitute to be stereoselective in yielding the C-4b alkylamino iso-
mers as the major products. In some cases, C-4 -isomers were
a pendant aromatic ring E directed the sub-
a
also observed, but as minor products. The assignment of the con-
figuration at C-4 position for target compounds was based on J3,4
coupling constants20 or 1D NOE. When proton of C-4 was irradi-
1
ated in H NMR spectrum for 21, protons of C-3 and C-20,60 were
enhanced by 5.13% and 5.12%, respectively; however, proton of
C-2 was not enhanced. In addition, proton of C-2 was enhanced
by 4.56% while proton of C-1 was irradiated for 21, but protons
of C-3 and C-4 were not enhanced. Thus, we confirmed that H-4
is cis to H-3 and H-2 is trans to H-3 for 21.
2.2. Biological activities
21 at various concentrations: 0 (1), 10 (2), 20 (3) and 50 lg/mL (4)
were recorded from 200 to 320 nm at 5 °C on a Jasco-810 spec-
trometer, and the CD spectra of CT DNA in the presence and ab-
sence of 21 in pH 7.2 PBS are shown in Figure 2. The CD
The biological activities of this series of podophyllotoxin deriv-
atives were evaluated by an in vitro cytotoxicity test, which was
O
OSiMe3
F
F
F
HN
N
Br(CH2)nBr
HMDS/TMSCl
O
N
O
(CH2)nBr
HN
7I-III
O
N
H
5
Me3SiO
F
N
6
Ref. 16
R
III n = 5
I
n = 3
R = CH3
R = CH(OH)Me
R = CH(Me)CH2Me
R = CH2C6H5
II n = 4
N
O
O
a
c
e
g
b R = CHMe2
d R = CH2CHMe2
R = CH2CH2SCH3
h R = CH2(p-OH-C6H4)
H2N
n
NH
O
O
f
8
Ia-h, 8IId,g, 8IIId,g
Scheme 1. Synthesis of 7 and 8.

S.-W. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3111–3117
3113
F
R
O
O
N
O
OH
O
I
HN
n
NH
O
O
O
O
O
O
MeSO3H/NaI
CH2Cl2
O
8
O
Et3N/THF
O
O
O
O
MeO
OMe
MeO
OMe
MeO
OMe
OMe
OH
OH
9
10-21
1
Compd
10
n
3
3
3
3
R
Compd
14
n
3
3
3
3
R
Compd
18
n
4
4
5
5
R
Me
CH(Me)CH2Me
CH2CH2SMe
CH2C6H5
CH2CHMe2
CH2C6H5
11
CHMe2
15
19
12
CH(OH)Me
CH2CHMe2
16
20
CH2CHMe2
CH2C6H5
13
17
CH2(p-OH-C6H4)
21
Scheme 2. Synthesis of compounds 10–21.
Table 1
Biological evaluation of compounds 10–21
15
M)a
logP
1
2
3
4
Compound
Cytotoxic activities (IC50
,
l
K562b
AGSc
HL-60b
A-549c
10
5
10
11
12
13
14
15
16
17
18
19
20
21
5-FU
VP-16
19.5
13.5
30.8
9.5
5.1
8.8
5.8
28.6
5.3
9.2
6.0
40.4
22.3
5.80
23.2
0.13
0.24
0.99
0.31
26.6
0.73
0.42
0.31
0.04
65.3
2.75
2.59
1.92
6.95
0.01
0.18
0.29
0.48
21.7
<0.01
0.30
ꢁ0.07
0.34
ꢁ0.12
0.33
0.59
0.23
0.65
0.07
0.35
0.43
0.51
0.29
—
62.5
140.8
59.2
56.2
24.8
23.6
114.7
34.5
23.7
23.5
38.8
>100
50.8
0
0.53
<0.01
50.5
7.38
13.2
>100
>100
-5
0.69
a
The value is the average of triplicate.
MTT methods, drugs exposure was for 48 h.
SRB methods, drugs exposure was for 72 h.
200
220
240
260
280
300
320
b
c
Wavelength [nm]
1. 25µg/mL Calf Thymus DNA 2. 1+10µg/mL 21
3. 1+20µg/mL 21 4. 1+50µg/mL 21
spectrum of free CT DNA exhibits one positive peak at 272 nm due
to the base stacking and one negative band at 245 nm due to the
helicity, which is the characteristic of DNA in the right-handed B
form (Fig. 2, curve 1).22 Increasing the concentration of 21
Figure 2. CD spectra of calf thymus DNA.
While the 21 binds to the DNA, it also interacts with the ribose
and phosphate. Competition between water molecules and 21
induced water contents may be decreased in the micro sur-
rounding around the DNA. This would make the helicity of
DNA become stronger. Thus, the conformation of DNA is changed
from the B-form to a more compact C-form. These changes of
the CD spectrum of CT DNA revealed the interaction of CT
DNA with 21.
Further, metabolism stabilities of 21 were also investigated in
human plasma, mouse plasma and pH 7.4 PBS (1.0 lM) within
24 h at 37 °C, respectively. As shown in Figure 3, 21 was stable
within 24 h in human plasma and within 8 h in pH 7.4 PBS; how-
ever, its content was decreased dramatically in pH 7.4 PBS after
8 h, as well as in mouse plasma after 2 h, respectively. These re-
(c = 10, 20, 50 lg/mL, respectively) in above CT DNA solution, the
intensity of positive band decreases and negative band increases,
as well as a gradual blue shift of the negative band from 245 to
243 nm and a red shift from 272 to 276 nm of the positive band
were also observed (Fig. 2, curves 2, 3 and 4, respectively), sug-
gesting that DNA bound with 21 induces certain conformational
changes, DNA double helix in solution is in a more helical state
belonging to C form DNA.23 Double helix form DNA is not really
a rigid molecule, especially in solution, and water molecules are
important to the conformations of DNA. The major and minor
grooves are ‘coated’ by a unimolecular layer of water molecules
which interact with the exposed C@O, N and NH functions of ri-
bose and also extensively solvate the phosphate backbone.24

3114
S.-W. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3111–3117
layers were extracted with CHCl3 (20 mL). The combined organic
layers were washed with brine and dried over anhydrous Na2SO4,
filtered and concentrated to obtain yellow oil. Purification by
100
80
60
40
20
0
recyclization with isopropanol afforded 7I–III
.
4.2.1. 1-( -Bromopropyl)-5-FU (7I)
x
The yield 57%; mp: 112–113 °C; 1H NMR (300 MHz, CDCl3) d
9.81 (br, 1H, NH), 7.35 (d, J = 5.4 Hz, 1H, CH), 3.89 (t, J = 6.6 Hz,
2H, CH2), 3.44 (t, J = 6.0 Hz, 2H, CH2), 2.29–2.25 (m, 2H, CH2). MS
(EI): 252 ([M+2]+), 250 ([M]+), 185, 171 ([MꢁBr]+).
Human plasma
Mouse plasma
4.2.2. 1-(x-Bromobutyl)-5-FU (7II)
pH7.4 phosphate buffer
The yield 63%; mp: 129–130 °C; 1H NMR (300 MHz, CDCl3) d
9.81 (br, 1H, NH), 7.26 (d, J = 5.4 Hz, 1H, CH), 3.75 (t, J = 6.8 Hz,
2H, CH2), 3.43 (t, J = 6.3 Hz, 2H, CH2), 1.89–1.86 (m, 4H, 2CH2).
MS (EI): 266 ([M+2]+), 264 ([M]+), 185 ([MꢁBr]+).
0
5
10
15
Time (h)
20
25
4.2.3. 1-(x-Bromopentyl)-5-FU (7III)
Figure 3. The stability of compound 21.
Yield 72%; mp: 106–108 °C; 1H NMR (300 MHz, CDCl3) d 9.49
(br, 1H, NH), 7.25 (d, J = 6.3 Hz, 1H, CH), 3.73 (t, J = 7.5 Hz, 2H,
CH2), 3.41 (t, J = 6.3 Hz, 2H, CH2), 1.91–1.87 (m, 2H, CH2), 1.73–
1.69 (m, 2H, CH2), 1.52–1.48 (m, 2H, CH2). MS (EI): 280 ([M+2]+),
278 ([M]+), 199 ([MꢁBr]+).
sults revealed the following order of stability: human plasma > pH
7.4 phosphate buffer > mouse plasma.
3. Conclusion
4.3. General preparation of compounds 10–21
In summary, we believed that conjugate of podophyllotoxin and
5-FU is a promising direction in antineoplastic agents finding, be-
cause the target compounds showed more effective cytotoxicities
in vitro and superior water solubility than those of the reference
etoposide. In addition, the conformation of DNA is changed from
the B-form to the C-form in the presence of 21, and the result re-
vealed the interaction of CT DNA with 21. Furthermore, compound
21 is stable in human plasma, but not enough stable in mouse plas-
ma and pH 7.4 phosphate buffer solution.
To a solution of podophyllotoxin 1 (414 mg, 1 mmol) in dry
CH2Cl2 (10 mL), NaI (447 mg, 3 mmol) was added and stirred for
5 min. To this stirred suspension, MeSO3H (288 mg, 3 mmol) was
added dropwise with syringe at 0 °C and the stirring was continued
for another 5 h at room temperature. Nitrogen was bubbled
through the solution to drive off 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 intermediate
9 dissolved in dry THF (15 mL) was added the appropriate aryl-
amine 8 (3 mmol), followed by triethylamine (30 mg, 1.1 mmol).
The mixture was refluxed under nitrogen. While TLC analysis
showed that most of the starting material had been converted,
the reaction was stopped. The reaction mixture was diluted with
ethyl acetate and washed with water, 10% Na2S2O4 solution, dried
and purified via CC to afford compounds 10–21.
4. Experimental
4.1. Chemistry. General
Melting points were determined in Kofler apparatus and were
uncorrected. IR spectra were measured on a Nicolet 5DX-FT-IR
spectrometer on neat samples placed between KBr plates. 1H
NMR and 13C NMR spectra were recorded with a Varian Mercury-
300BB spectrometer with TMS as an internal standard, all chemical
shift values are reported as d ppm. Optical rotations were mea-
sured on a Perkin Elmer 341 polarimeter in a 1 dm cell at 23 °C.
Mass spectra were recorded on a Bruker Daltonics APEXII49e and
VGZAB-HS (70 eV) spectrometer with ESI and FAB source as ioniza-
tion, respectively. HPLC analyses were carried out on a Shimadzu
LC-10AT series HPLC system with SPD-M10AVP UV–vis detector.
Circular dichroism spectra were measured using a JASCO-810 spec-
trometer. All reactions were monitored by thin layer chromatogra-
phy (TLC) on silica gel GF254 (0.25 mm thick). CC was performed on
Silica Gel 60 (230–400 mesh, Qingdao Ocean Chemical Ltd., China).
Podophyllotoxin was isolated from a Chinese medicinal herb Podo-
phyllum emodi Wall var Chinesis Sprague, other starting materials
and regents were purchased commercially and used without fur-
ther purified, unless otherwise stated.
4.3.1. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L-alanyl]-4-
desoxy-podophyllotoxin (10)
The yield 46%; mp: 122–124 °C; ½a D23
ꢁ66 (c 0.3, acetone); IR
ꢃ
(KBr) 3436, 3229, 3070, 2915, 1772, 1698, 1613, 1482, 1371,
1231, 1114 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.28 (d, J = 5.4 Hz,
;
1H, H-600), 6.69 (s, 1H, H-5), 6.47 (s, 1H, H-8), 6.24 (s, 2H, H-20,60),
5.95 (s, 1H, H-13a), 5.94 (s, 1H, H-13b), 4.51 (d, J = 5.4 Hz, 1H, H-
1), 4.36–4.26 (m, 2H, H-3000), 4.20 (t, J = 6.0 Hz, 2H, H-11), 3.98 (d,
J = 3.9 Hz, 1H, H-4), 3.78 (t, J = 6.6 Hz, 2H, H-7000), 3.74 (s, 6H,
30,50-OMe), 3.42 (q, J = 7.2 Hz, 1H, H-2000), 3.30 (dd, J = 14.1, 5.4 Hz,
1H, H-2), 2.86–2.74 (m, 1H, H-3), 2.12–2.01 (m, 2H, H-4000), 1.41
(d, J = 6.3 Hz, 3H, Me); 13C NMR (75 MHz, CDCl3) (Table 2). HRMS
(ESI): 642.2095 for [M+H]+ (calcd 642.2094 for C31H33FN3O11).
4.3.2. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L-valinyl]-4-
desoxy-podophyllotoxin (11)
The yield 43%; mp: 122–124 °C; ½a D23
ꢁ69 (c 0.3, acetone); IR
ꢃ
(KBr) 3433, 2962, 1773, 1711, 1660, 1613, 1482, 1369, 1231,
1114 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.29 (d, J = 5.4 Hz, 1H, H-
;
4.2. General procedure of synthesis of 7
600), 6.67 (s, 1H, H-5), 6.47 (s, 1H, H-8), 6.23 (s, 2H, H-20,60), 5.95
(s, 2H, H-13), 5.50 (br, 1H, NH), 4.51 (d, J = 6.0 Hz, 1H, H-1),
4.32–4.28 (m, 2H, H-3000), 4.23 (t, J = 5.7 Hz, 1H, H-11a), 4.18 (t,
J = 6.0 Hz, 1H, H-11b), 3.96 (d, J = 3.6 Hz, 1H, H-4), 3.79 (t,
J = 8.1 Hz, 2H, H-7000), 3.74 (s, 6H, 30,50-OMe), 3.38 (dd, J = 13.5,
To dried 1,
x-dibromo alkane (20 mL) was added 2, 4-ditrim-
ethylsilyl 5-FU (5.5 g, 20 mmol), followed by the mixture that
was stirred for 3 h at 105 °C. The residue was cooled and diluted
with CHCl3 (20 mL), washed with water (2 ꢂ 15 mL), the aqueous

S.-W. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3111–3117
3115
Table 2
13C NMR data of compounds 10–21
F
6''
R
5''
4'''
6'''
1'''
O
N
O
O
4''
2'' NH
2'''
HN
7'''
3'''
5'''
5
8
O
10
6
O
O
4
1
3
2
11 O
13
12
9
7
O
1'
2'
6'
MeO5'
3O' Me
4'
OH
Compound
10
43.8
41.2
38.7
11
43.6
40.8
38.5
12
43.8
40.5
38.7
13
14
15
16
17
18
43.6
40.8
38.6
19
43.5
40.6
38.6
20
43.6
40.8
38.6
21
C-1
C-2
C-3
C-4
43.6
40.7
38.5
43.8
40.8
38.5
43.6
40.8
38.6
43.5
40.6
38.6
43.7
40.8
38.9
54.5
43.5
40.8
38.6
54.0
53.9
53.7
53.8
53.8
53.8
54.2
54.2
54.4
54.2
54.6
C-5
C-6
C-7
C-8
108.7
147.3
147.9
110.7
131.9
134.0
68.6
174.5
101.6
131.4
108.1
146.5
136.6
56.6
149.8
157.5
26.3
140.7
235.8
130.7
175.4
61.7
108.6
146.8
147.7
110.7
131.9
133.8
68.1
174.5
101.4
131.2
107.9
146.2
136.8
56.4
149.3
156.8
26.5
140.4
236.9
130.8
175.4
61.4
109.1
146.6
147.7
110.9
132.3
133.9
68.0
173.4
101.3
131.5
107.9
146.3
136.5
56.3
149.6
157.3
26.2
140.6
235.8
130.6
175.5
61.3
108.5
146.7
147.6
110.6
132.1
133.8
68.1
174.4
101.3
131.2
107.8
146.2
136.7
56.3
149.4
157.2
25.2
140.4
235.8
131.0
175.5
61.4
108.8
146.7
147.7
110.9
132.2
134.0
68.1
173.9
101.3
131.4
108.0
146.3
136.4
56.4
149.3
157.0
25.3
140.2
235.2
131.1
175.4
61.3
108.6
146.7
147.7
110.6
132.1
133.8
68.1
173.8
101.3
131.2
107.9
146.3
136.6
56.3
149.5
157.2
25.2
140.4
235.8
130.9
175.4
61.6
108.5
146.8
147.7
110.6
132.1
133.9
68.2
173.6
101.3
131.2
107.9
146.3
136.6
56.3
149.3
157.3
26.2
140.3
236.9
130.8
175.3
61.4
109.1
146.9
147.6
110.2
131.5
135.0
68.6
173.9
101.6
130.5
108.8
147.2
136.3
55.9
149.9
157.7
26.3
140.4
130.1
130.5
175.5
61.7
108.6
146.9
147.7
110.7
132.1
133.9
68.1
174.5
101.4
131.5
107.9
147.2
136.5
56.4
149.3
157.1
25.2
140.4
237.3
130.2
175.5
61.4
108.5
146.8
147.7
110.4
132.0
133.9
68.1
173.7
101.3
131.1
107.9
146.2
136.6
56.3
149.4
156.9
26.2
140.3
237.0
130.8
175.3
62.2
108.5
146.5
147.7
110.8
132.1
133.8
68.2
174.4
101.4
131.2
107.8
146.3
135.6
56.3
149.4
157.3
25.2
140.4
237.3
130.6
175.5
58.9
108.5
146.8
147.7
110.4
131.9
133.8
68.2
173.7
101.3
131.1
107.8
146.2
136.6
56.3
149.4
157.3
26.2
140.3
236.2
130.9
175.4
62.5
C-9
C-10
C-11
C-12
C-13
C-10
C-20,60
C-30,50
C-40
30,50-OMe
C-200
C-400
d, J(Hz)
C-500
d, J(Hz)
C-600
C-1000
C-2000
C-3000
C-4000
C-5000
C-6000
C-7000
55.9
28.1
58.5
28.0
64.8
27.9
58.4
27.9
63.9
28.1
58.7
27.8
62.1
27.7
62.7
27.9
63.8
28.0
63.8
30.8
64.8
28.4
22.8
28.1
48.7
42.6
25.0
22.5
64.4
28.4
22.7
27.9
25.4
48.2
42.7
25.0
22.6
22.4
25.2
48.2
46.3
19.5
46.2
42.7
22.4
22.2
45.7
69.1
20.0
46.1
42.6
24.9
22.3
22.2
46.3
38.7
25.8
15.4
11.5
46.0
32.7
30.8
15.5
46.0
136.6
129.1
128.7
127.1
40.0
45.7
156.4
132.3
129.8
115.3
29.8
48.7
130.8
129.0
128.5
126.9
39.9
133.8
129.0
128.6
126.9
39.9
R
22.3
5.4 Hz, 1H, H-2), 3.15 (d, J = 5.4 Hz, 1H, H-2000), 2.78–2.74 (m, 1H, H-
3), 2.07 (t, J = 6.0 Hz, 1H, b-H of valine), 1.58–1.54 (m, 2H, H-4000),
0.89 (d, J = 6.0 Hz, 3H, Me), 0.81 (d, J = 5.7 Hz, 3H, Me); 13C NMR
(75 MHz, CDCl3) (Table 2). HRMS (ESI): 670.2404 for [M+H]+ (calcd
670.2407 for C33H37FN3O11).
H-4000), 1.18 (d, J = 6.6 Hz, 3H, Me); 13C NMR (75 MHz, CDCl3) (Table
2). HRMS (ESI): 672.2191 for [M+H]+ (calcd 672.2199 for
C32H35FN3O12).
4.3.4. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L-leucyl]-4-
desoxy-podophyllotoxin (13)
4.3.3. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L
-threonyl]-4-
The yield 48%; mp: 110–112 °C; ½a D23
ꢁ65 (c 0.3, acetone); IR
ꢃ
desoxy-podophyllotoxin (12)
(KBr) 3436, 3223, 3072, 2957, 2844, 1773, 1715, 1699, 1660,
The yield 36%; mp: 119–121 °C; ½a D23
ꢃ
ꢁ68 (c 0.3, acetone); IR
1482, 1368, 1231, 1115 cmꢁ1 1H NMR (300 MHz, CDCl3) d 9.87
;
(KBr) 3441, 3219, 3069, 2918, 2845, 1770, 1698, 1612, 1481,
(br, 1H, NH), 7.29 (d, J = 5.4 Hz, 1H, H-600), 6.65 (s, 1H, H-5), 6.44
(s, 1H, H-8), 6.20 (s, 2H, H-20,60), 5.92 (s, 2H, H-13), 5.56 (br, 1H,
NH), 4.48 (d, J = 5.1 Hz, 1H, H-1), 4.34–4.26 (m, 2H, H-3000), 4.19
(t, J = 5.7 Hz, 1H, H-11a), 4.13 (t, J = 5.7 Hz, 1H, H-11b), 3.95 (d,
J = 3.0 Hz, 1H, H-4), 3.83–3.74 (m, 2H, H-7000), 3.70 (s, 6H, 30,50-
OMe), 3.37 (dd, J = 13.5, 5.4 Hz, 1H, H-2), 3.29 (t, J = 6.0 Hz, 1H,
H-2000), 2.82–2.78 (m, 1H, H-3), 1.82–1.78 (m, 2H, b-CH2 of leucine),
1371, 1231, 1112 cmꢁ1 1H NMR (300 MHz, CDCl3) d 9.77 (br, 1H,
;
NH), 7.35 (d, J = 5.1 Hz, 1H, H-600), 6.68 (s, 1H, H-5), 6.47 (s, 1H,
H-8), 6.22 (s, 2H, H-20,60), 5.93 (s, 2H, H-13), 5.51 (br, 1H, NH),
4.52 (d, J = 5.1 Hz, 1H, H-1), 4.31–4.28 (m, 3H, H-3000, b-H of threo-
nine), 4.17 (t, J = 6.0 Hz, 1H, H-11a), 4.12 (d, J = 3.0 Hz, 1H, H-4),
4.07 (t, J = 6.0 Hz, 1H, H-11b), 3.87–3.79 (m, 2H, H-7000), 3.74 (s,
6H, 30,50-OMe), 3.48 (dd, J = 14.1, 5.1 Hz, 1H, H-2), 3.28 (d,
J = 4.2 Hz, 1H, H-2000), 2.90–2.86 (m, 1H, H-3), 2.08–2.06 (m, 2H,
1.65–1.61 (m, 1H, CH,
0.87 (d, J = 5.4 Hz, 3H, Me), 0.79 (d, J = 5.4 Hz, 3H, Me); 13C NMR
c
-CH2 of leucine), 1.54–1.21 (m, 2H, H-4000),

3116
S.-W. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3111–3117
(75 MHz, CDCl3) (Table 2). HRMS (ESI): 684.2559 for [M+H]+ (calcd
684.2563 for C34H39FN3O11).
4.3.9. 40-O-Demethyl-4b-N-[(5-FU-N1-)-butyl
L-leucyl]-4-
desoxy-podophyllotoxin (18)
The yield 39%; mp: 115–117 °C; ½a D23
ꢁ62 (c 0.3, acetone); IR
ꢃ
4.3.5. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L
-isoleucyl]-4-
(KBr) 3438, 3222, 2957, 2843, 1772, 1711, 1698, 1613, 1511,
desoxy-podophyllotoxin (14)
1482, 1367, 1231, 1114 cmꢁ1 1H NMR (300 MHz, CDCl3) d 9.58
;
The yield 45%; mp: 118–120 °C; ½a D23
ꢃ
ꢁ81 (c 0.3, acetone); IR
(br, 1H, NH), 7.27 (d, J = 6.0 Hz, 1H, H-600), 6.66 (s, 1H, H-5),
6.47 (s, 1H, H-8), 6.24 (s, 2H, H-20,60), 5.95 (s, 2H, H-13), 5.45
(br, 1H, NH), 4.51(d, J = 5.4 Hz, 1H, H-1), 4.37 (t, J = 6.3 Hz, 2H,
H-11), 4.29–4.14 (m, 2H, H-3000), 3.95 (d, J = 3.6 Hz, 1H, H-4),
3.80 (t, J = 7.2 Hz, 2H, H-7000), 3.75 (s, 6H, 30,50-OMe), 3.39 (dd,
J = 13.5, 5.1 Hz, 1H, H-2), 3.36–3.28 (m, 1H, H-2000), 2.82–2.80
(m, 1H, H-3), 1.76–1.74 (m, 2H, b-H of leucine), 1.65–1.61 (m,
(KBr) 3439, 3229, 3072, 2963, 2840, 1773, 1714, 1700, 1663,
1613, 1481, 1369, 1231, 1114 cmꢁ1 1H NMR (300 MHz, CDCl3) d
;
7.28 (d, J = 5.1 Hz, 1H, H-600), 6.65 (s, 1H, H-5), 6.50 (s, 1H, H-8),
6.24 (s, 2H, H-20,60), 5.98 (s, 1H, H-13a), 5.96 (s, 1H, H-13b), 5.38
(br, 1H, NH), 4.55 (d, J = 5.7 Hz, 1H, H-1), 4.33–4.30 (m, 2H, H-
3000), 4.25 (t, J = 6.0 Hz, 1H, H-11a), 4.20 (t, J = 6.0 Hz, 1H, H-11b),
3.95 (d, J = 4.2 Hz, 1H, H-4), 3.82 (t, J = 7.8 Hz, 2H, H-7000), 3.75 (s,
6H, 30,50-OMe), 3.42 (dd, J = 13.8, 5.4 Hz, 1H, H-2), 3.29 (d,
J = 5.4 Hz, 1H, H-2000), 2.84–2.80 (m, 1H, H-3), 1.62–1.58 (m, 2H,
1H,
c
-H of leucine), 1.57–1.55 (m, 4H, H-4000,6000), 0.90 (d,
J = 5.4 Hz, 3H, Me), 0.83 (d, J = 6.0 Hz, 3H, Me); 13C NMR
(75 MHz, CDCl3) (Table 2). HRMS (ESI): 698.2732 for [M+H]+
(calcd 698.2720 for C35H41FN3O11).
H-4000), 1.41–1.19 (m, 3H, b,
c-H of isoleucine), 0.90 (d, J = 3.0 Hz,
3H, Me), 0.88 (t, J = 3.6 Hz, 3H, Me); 13C NMR (75 MHz, CDCl3) (Ta-
ble 2). HRMS (ESI): 684.2562 for [M+H]+ (calcd 684.2563 for
C34H39O11N3F).
4.3.10. 40-O-Demethyl-4b-N-[(5-FU-N1-)-butyl
L-phenylalanyl]-
4-desoxy- podophyllotoxin (19)
The yield 43%; mp: 111–113 °C; ½a D23
ꢁ68 (c 0.3, acetone); IR
ꢃ
4.3.6. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L
-methionyl]-4-
(KBr) 3442, 3184, 3063, 2920, 1773, 1715, 1698, 1609, 1506,
desoxy-podophyllotoxin (15)
1481, 1365, 1230, 1112 cmꢁ1 1H NMR (300 MHz, CDCl3) d 7.26–
;
The yield 54%; mp: 109–111 °C; ½a D23
ꢃ
ꢁ68 (c 0.3, acetone); IR
7.12 (m, 6H, H-600, Ar), 6.46 (s, 1H, H-5), 6.41 (s, 1H, H-8), 6.19 (s,
2H, H-20,60), 5.91 (s, 2H, H-13), 4.89 (br, 1H, NH), 4.41 (d,
J = 5.4 Hz, 1H), 4.25–4.22 (m, 2H, H-11), 4.09–4.05 (m, 2H, H-3000),
3.97 (d, J = 3.3 Hz, 1H, H-4), 3.71 (s, 6H, 30,50-OMe), 3.63–3.60 (m,
2H, H-7000), 3.49 (t, J = 6.6 Hz, 1H, H-2000), 3.13 (dd, J = 13.8, 5.7 Hz,
1H, H-2), 2.97 (d, J = 7.2 Hz, 1H, b-H of phenylalanine), 2.77–2.74
(m, 1H, H-3), 1.57–1.55 (m, 4H, H-4000,6000); 13C NMR (75 MHz,
CDCl3) (Table 2). HRMS (ESI): 732.2555 for [M+H]+ (calcd
732.2563 for C38H39FN3O11).
(KBr) 3438, 3229, 3069, 2915, 2840, 1773, 1715, 1698, 1660,
1612, 1481, 1369, 1231, 1113 cmꢁ1 1H NMR (300 MHz, CDCl3) d
;
7.31 (d, J = 5.4 Hz, 1H, H-600), 6.67 (s, 1H, H-5), 6.45 (s, 1H, H-8),
6.21 (s, 2H, H-20,60), 5.92 (s, 2H, H-13) 4.49 (d, J = 5.1 Hz, 1H, H-
1), 4.31 (d, J = 4.2 Hz, 1H, H-11a), 4.28 (d, J = 2.1 Hz, 1H, H-11b),
4.23–4.18 (m, 2H, H-3000), 4.01 (d, J = 3.6 Hz, 1H, H-4), 3.78 (t,
J = 6.3 Hz, 2H, H-7000), 3.71 (s, 6H, 30,50-OMe), 3.46 (t, J = 6.0 Hz,
1H, H-2000), 3.36 (dd, J = 13.8, 5.4 Hz, 1H, H-2), 2.84–2.80 (m, 1H,
H-3), 2.49(t, J = 6.9 Hz, 2H, SCH2), 2.05 (s, 3H, SMe), 2.11–2.08 (m,
2H, H-4000), 1.98–1.95 (m, 2H, CH2); 13C NMR (75 MHz, CDCl3) (Ta-
ble 2). HRMS (ESI): 702.2125 for [M+H]+ (calcd 702.2127 for
C33H37FN3O11S).
4.3.11. 40-O-Demethyl-4b-N-[(5-FU-N1-)-pentyl
L-leucyl]-4-
desoxy-podophyllotoxin (20)
The yield 35%; mp: 106–108 °C; ½a D23
ꢁ65 (c 0.3, acetone); IR
ꢃ
(KBr) 3429, 3214, 3070, 2954, 2870, 1773, 1715, 1697, 1613,
4.3.7. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L
-phenylalanyl]-
1481, 1365, 1230, 1114 cmꢁ1 1H NMR (300 MHz, CDCl3) d 9.56
;
4-desoxy- podophyllotoxin (16)
(br, 1H, NH), 7.36 (d, J = 5.1 Hz, 1H, H-600), 6.70 (s, 1H, H-5),
6.49 (s, 1H, H-8), 6.26 (s, 2H, H-20,60), 5.98 (s, 2H, H-11), 5.52
(br, 1H, NH), 4.54 (d, J = 6.0 Hz, 1H, H-1), 4.37 (t, J = 6.3 Hz, 2H,
H-3000), 4.21 (t, J = 5.4 Hz, 1H, H-11a), 4.11 (t, J = 5.4 Hz, 1H, H-
11b), 3.97 (d, J = 3.3 Hz, 1H, H-4), 3.76 (s, 6H, 30,50-OMe), 3.73
(t, J = 6.6 Hz, 2H, H-7000), 3.42 (dd, J = 14.1, 5.4 Hz, 1H, H-2),
3.29 (t, J = 6.0 Hz, 1H, H-2000), 2.84–2.81 (m, 1H, H-3), 1.75–1.72
The yield 54%; mp: 113–115 °C; ½a D23
ꢁ71 (c 0.3, acetone); IR
ꢃ
(KBr) 3440, 3211, 3066, 2918, 2844, 1772, 1714, 1697, 1666,
1607, 1510, 1481, 1369, 1230, 1112 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 9.58 (br, 1H, NH), 7.27–7.15 (m, 5H, Ar), 7.05 (d,
J = 5.1 Hz, 1H, H-600), 6.53 (s, 1H, H-5), 6.44 (s, 1H, H-8), 6.21 (s,
2H, H-20,60), 5.99 (s, 2H, H-13), 5.48 (br, 1H, NH), 4.44 (d,
J = 5.1 Hz, 1H, H-1), 4.27–4.24 (m, 2H, H-3000), 4.09 (t, J = 5.1 Hz,
2H, H-11), 4.01 (d, J = 3.3 Hz, 1H, H-4), 3.73 (s, 6H, 30,50-OMe),
3.67–3.48 (m, 3H, H-7000, H-2000), 3.15 (dd, J = 14.1, 5.1 Hz, 1H, H-
2), 3.08–2.98 (m, 2H, b-H of phenylalanine), 2.81–2.78 (m, 1H, H-
3), 1.87–1.84 (m, 2H, H-4000); 13C NMR (75 MHz, CDCl3) (Table 2).
HRMS (ESI): 740.2237 for [M+Na]+ (calcd 740.2226 for
C37H36FN3O11Na).
(m, 2H, b-H of leucine), 1.68–1.65 (m, 1H,
c-H of leucine),
1.65–1.62 (m, 4H, H-4000,6000), 1.25–1.23 (m, 2H, H-5000), 0.92 (d,
J = 6.0 Hz, 3H, Me), 0.87 (d, J = 6.0 Hz, 3H, Me); 13C NMR
(75 MHz, CDCl3) (Table 2). HRMS (ESI): 712.2883 for [M+H]+
(calcd 712.2876 for C36H43FN3O11).
4.3.12. 40-O-Demethyl-4b-N-[(5-FU-N1-)-pentyl
L-phenylalanyl]-
4-desoxy- podophyllotoxin (21)
4.3.8. 40-O-Demethyl-4b-N-[(5-FU-N1-)-propyl
L
-tyrosyl]-4-
The yield 24%; mp: 125–127 °C; ½a D23
ꢁ61 (c 0.3, acetone); IR
ꢃ
desoxy-podophyllotoxin (17)
(KBr) 3443, 3066, 2938, 2837, 1774, 1714, 1693, 1613, 1505,
The yield 27%; mp 124–126 °C; ½a D23
ꢃ
ꢁ56 (c 0.3, acetone); IR
1428, 1366, 1231, 1114 cmꢁ1 1H NMR (300 MHz, CDCl3) d 9.64
;
(KBr) 3394, 3070, 2912, 2841, 1771, 1698, 1665, 1612, 1514,
(br, 1H, NH), 7.28–7.12 (m, 6H, H-600, Ar), 6.47 (s, 1H), 6.42 (s,
1H), 6.20 (s, 2H, H-20,60), 5.93 (s, 2H, H-13), 5.49 (br s, 1H, NH),
4.43 (d, J = 5.4 Hz, 1H, H-1), 4.27(d, J = 9.0 Hz, 2H, H-11), 4.08–
4.05 (m, 2H, H-3000), 3.97 (d, J = 2.7 Hz, 1H, H-4), 3.72 (s, 6H, 30,50-
OMe), 3.64 (t, J = 7.2 Hz, 2H, H-7000), 3.49 (t, J = 6.0 Hz, 1H, H-2000),
3.14 (dd, J = 14.1, 5.7 Hz, 1H, H-2), 2.98 (d, J = 6.0 Hz, 2H, b-H of
phenylalanine), 2.78–2.75 (m, 1H, H-3), 1.63–1.60 (m, 4H, H-
4000,6000), 1.27–1.25 (m, 2H, H-5000); 13C NMR (75 MHz, CDCl3) (Table
2). HRMS (ESI): 746.2705 for [M+H]+ (calcd 746.2720 for
C39H41FN3O11).
1481, 1368, 1230, 1113 cmꢁ1 1H NMR (300 MHz, CD3COCD3) d
;
7.78 (d, J = 6.6 Hz, 1H, H-600), 7.04 (d, J = 8.1 Hz, 2H, Ar), 6.74 (d,
J = 8.1 Hz, 2H, Ar), 6.71 (s, 1H, H-5), 6.43 (s, 1H, H-8), 6.28 (s, 2H,
H-20,60), 5.95 (s, 1H, H-13a), 5.94 (s, 1H, H-13b), 4.43 (d,
J = 5.4 Hz, 1H, H-1), 4.37–4.28 (m, 2H, H-11), 4.16–4.10 (m, 3H,
H-3000, H-4), 3.76–3.69 (m, 2H, H-7000), 3.65 (s, 6H, 30,50-OMe),
3.43–3.40 (m, 1H, H-2000), 3.24 (dd, J = 14.1, 5.1 Hz, 1H, H-2),
2.95–2.92 (m, 2H, b-H of tyrosine), 2.89–2.86 (m, 1H, H-3), 1.99–
1.96 (m, 2H, H-4000); 13C NMR (75 MHz, CD3COCD3) (Table 2). HRMS
(ESI): 734.2367 for [M+H]+ (calcd 734.2356 for C37H37FN3O12).

S.-W. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3111–3117
3117
4.4. Octanol–water partition coefficients
Acknowledgement
The partition coefficient P was determined according to the
standard method25 and calculated with following equation:
P = (compound in 1-octanol)/(compound in water). The reported
P values are an average of three measurements.
This work was financially supported by the Interdisciplinary
Innovation Research Fund for Young Scholars, Lanzhou University
(LZU200526).
References and notes
4.5. Biology
1. Frei, E., III; Elias, A.; Wheeler, C.; Richardson, P.; Hryniuk, W. Clin. Cancer Res.
1998, 4, 2027.
2. Peters, G. J.; van der Wilt, C. L.; van Moorsel, C. J. A.; Kroep, J. R.; Bergman, A. M.;
Ackland, S. P. Pharmacol. Ther. 2000, 87, 227.
3. (a) Imbert, T. F. Biochimie 1998, 80, 207; (b) Canel, C.; Moraes, R. M.; Dayan, F.
E.; Ferreira, D. Phytochemistry 2000, 54, 115; (c) Gordaliza, M.; Garcia, P. A.;
Miguel del Corral, J. M.; Castro, M. A.; Gomez-Zurita, M. A. Toxicon 2004, 44,
431; (d) You, Y. Curr. Pharm. Design 2005, 11, 1695.
4.5.1. Cytotoxicity assays
Cytotoxicity assays are performed on human premyelocytic
leukemia (HL-60), human erythromyeloblastoid leukemia
(K562), human gastric cancer (AGS) and human lung carcinoma
(A-549). Cells (6000–10,000) in 100 lL culture medium per well
4. (a) Byl, J. A.; Cline, S. D.; Utsugi, T.; Kobunai, T.; Yamada, Y.; Osheroff, N.
Biochemistry 2001, 40, 712; (b) Srivastava, V.; Negi, A. S.; Kumaar, J. K.; Gupta,
M. M.; Khanuja, S. P. S. Bioorg. Med. Chem. 2005, 13, 5892.
5. (a) Lee, K. H. Med. Res. Rev. 1999, 19, 569; (b) Huang, T. S.; Lee, C. C.; Chao, Y.;
Shu, C. H.; Chen, L. T.; Chen, L. L.; Chen, M. H.; Yuan, C.; Whang, P.-J.
Pharmaceut. Res. 1999, 16, 997.
6. Mross, K.; Hutmann, A.; Herbst, K.; Hanauske, A. R.; Schilling, T.; Manegold, C.;
Burk, K.; Hossfeld, D. K. Cancer Chemother. Pharmacol. 1996, 38, 217.
7. Utsugi, T.; Shibata, J.; Sugimoto, Y.; Aoyagi, K.; Wierzba, K.; Kobunai, T.; Terada,
T.; Oh-hara, T.; Tsuruo, T.; Yamada, Y. Cancer Res. 1996, 56, 2809.
8. Barret, J. M.; Etievant, C.; Baudouin, C.; Skov, K.; Charveron, M.; Hill, B. T.
Anticancer Res. 2002, 22, 187.
were seeded into 96-well microtest plates (Falcon, CA). Cells
were treated in triplicate with gradient concentration of test
compounds and incubated at 37 °C for 48 h (or 72 h). For HL-
60 and K562 cell lines, the microculture tetrazolium [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT;
Sigma, St. Louis, MO] assay was performed to measure the cyto-
toxic effects. The growth inhibitory effect on AGS and A549 so-
lid tumour cell lines was measured by the Sulforhodamine B
(SRB; Sigma, St. Louis, MO) assay. The drug concentration re-
quired for 50% growth inhibition (IC50) of tumour cells was
determined from the dose–response curve.
9. Gordaliza, M. Clin. Transl. Oncol. 2007, 9, 767.
´
´
10. Guianvarc’h, D.; Duca, M.; Boukarim, C.; Kraus-Berthier, L.; Leonce, S.; Pierre,
A.; Pfeiffer, B.; Renard, P.; Arimondo, P. B.; Monneret, C.; Dauzonne, D. J. Med.
Chem. 2004, 47, 2365.
4.5.2. Circular dichroism (CD) studies of CT DNA
11. Xiao, Z.; Bastow, K. F.; Vance, J. R.; Sidwell, R. S.; Wang, H.-K.; Chen, M. S.; Shi,
Q.; Lee, K.-H. J. Med. Chem. 2004, 47, 5140.
12. Duca, M.; Guianvarc’h, D.; Meresse, P.; Bertounesque, E.; Dauzonne, D.; Kraus-
Berthier, L.; Thirot, S.; Leonce, S.; Pierre, A.; Pfeiffer, B.; Renard, P.; Arimondo, P.
B.; Monneret, C. J. Med. Chem. 2005, 48, 593.
CT DNA was dissolved and annealed in 10 mM PBS (pH 7.2). To
CT DNA in PBS (5 mL 50
lg/mL) was added compound 21 in PBS (0,
1, 2, 5 mL 100 g/mL, respectively), and diluted to 10 mL. Same
l
13. (a) Gordaliza, M.; Castro, M. A.; Miguel del Corral, J. M.; López-Vázquez, M. L.;
García, P. A.; García-Grávalos, M. D.; San Feliciano, A. Eur. J. Med. Chem. 2000,
35, 691; (b) Castro, M. A.; Miguel del Corral, J. M.; Gordaliza, M.; García, P. A.;
Gomez-Zurita, M. A.; García-Grávalos, M. D.; de la Iglesia-Vicente, J.; Gajate, C.;
An, F.; Mollinedo, F.; San Feliciano, A. J. Med. Chem. 2004, 47, 1214.
14. (a) Danieli, B.; Giardini, A.; Lesma, G.; Passarella, D.; Peretto, B.; Sacchetti, S.; Silvani,
A.; Pratesi, G.; Zumino, F. J. Org. Chem. 2006, 71, 2848; (b) Passarella, D.; Giardini, A.;
Peretto, B.; Fontana, G.; Sacchetti, A.; Silvani, A.; Ronchi, C.; Cappelletti, G.; Cartelli, D.;
Borlake, J.; Danieli, B. Bioorg. Med. Chem. 2008, 16, 6269.
concentration sample of 21 in PBS was also prepared. The above
mixture was incubated for 24 h in the dark at 37 °C. Circular
dichroism (CD) spectra were measured using a JASCO-810 spec-
trometer at 20 °C in quartz cells of 1 cm path length. Spectra of
CT DNA in PBS were recorded in the range 200–320 nm. The appro-
priate concentration samples of compound 21 in PBS were also
determined as blank.
15. (a) Tian, X.; Wang, Y. G.; Yang, M. G.; Chen, Y. Z. Life Sci. 1997, 60, 511; (b) Cui,
Y. J.; Tian, X. Curr. Sci. 1998, 75, 1383; (c) Tian, X.; Zhang, F. M.; Li, W. G. Life Sci.
2002, 70, 2433; (d) Zhang, F. M.; Tian, X. Acta Chim. Sinica 2002, 60, 720; (e) Jin,
Y.; Chen, S.-W.; Tian, X. Bioorg. Med. Chem. 2006, 14, 3062; (f) Zhang, F. M.; Yao,
X.-J.; Tian, X.; Tu, Y.-Q. Molecules 2006, 11, 849.
16. Chen, S.-W.; Tian, X.; Tu, Y.-Q. Bioorg. Med. Chem. Lett. 2004, 14, 5063.
17. U.S. Appl. 1965, July 22, 12; Chem. Abstr. 1967, 67, 91093e.
18. Zhao, R. L.; Fan, C. L.; Zhuo, R. X. Chem. J. Chin. Univ. 1989, 10, 605.
19. Kamal, A.; Laxman, N.; Ramesh, G. Bioorg. Med. Chem. Lett. 2000, 10, 2059.
20. (a) Lee, K. H.; Imakura, Y.; Haruna, M.; Beere, S. A.; Thurston, L. S.; Dai, H. J.;
Chen, C. H. J. Nat. Prod. 1989, 52, 606; (b) Hansen, H. F.; Jensen, R. B.;
Willumsen, A. M.; Lauritsen, N. N.; Ebbesen, P.; Nielsen, P. E.; Buchardt, O. Acta
Chem. Scand. 1993, 47, 1190.
21. (a) Xiao, Z.; Vance, J. R.; Bastow, K. F.; Brossi, A.; Vance, J. R.; Sidwell, R. S.;
Wang, H.-K.; Chen, M. S.; Shi, Q.; Lee, K.-H. Bioorg. Med. Chem. 2004, 12, 3363;
(b) Xiao, Z.; Han, S.; Bastow, K. F.; Lee, K. H. Bioorg. Med. Chem. Lett. 2004, 14,
1581; (c) Cho, S. J.; Tropsha, A.; Suffness, M.; Cheng, Y. C.; Lee, K. H. J. Med.
Chem. 1996, 39, 1383.
22. Nahadevan, S.; Palauiandavar, M. Inorg. Chem. 1998, 37, 3927.
23. Tanaka, K.; Okahata, Y. J. Am. Chem. Soc. 1996, 118, 10679.
24. Zhang, Z.; Huang, W.; Tang, J.; Wang, E.; Dong, S. Biophys. Chem. 2002, 97, 7.
25. Sosnovsky, G. Pure Appl. Chem. 1990, 62, 289.
4.5.3. Stability studies for compound 2126
For drug metabolism experiments, to 4.0 mL preincubated
human plasma, mouse plasma and pH 7.4 phosphate buffer,
respectively, was added test compound 21 (100
in acetonitrile. The mixture was incubated at 37 °C, and
100 L aliquots were taken at 0.5, 1, 2, 4, 6, 8, 12 and 24 h.
To precipitate plasma protein, 400
lg/mL, 1.0 mL)
l
lL acetonitrile (ꢁ20 °C) was
added, vortexed for 20 s and centrifuged at 10,000 rpm for
5 min. Supernatant was transferred to a glass vial and stored
at ꢁ20 °C immediately until HPLC analysis. HPLC analysis:
20 lL of solution obtained above was injected onto a Kromasil
100 A C18 column (350 mm ꢂ 4.6 mm, 5
lm), chromatographed
with acetonitrile–water as mobile phase (CH3CN/H2O = 45:55)
and flow rate is 1.0 mL/min. Compound 21 was detected with
SPD-M10AVP UV–vis detector at 278 nm and remaining time
is 10.7 min.
26. He, X.; Lu, W.; Jiang, X.; Cai, J.; Zhang, X.; Ding, J. Bioorg. Med. Chem. 2004, 12,
4003.