Synthesis and biological evaluation of new 4β-anilino-4′-O- demethyl-4-desoxypodophyllotoxin derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2010.11.016

A series of new 4β-anilino-4′-O-demethyl-4-desoxypodophyllotoxin derivatives were prepared and evaluated for their cytotoxicities against four human cancer cell lines including KB, KB/VCR, A549 and 95D. Most compounds showed better growth-inhibition activities against tested cell lines than that of etoposide (VP-16). Preliminary structure-activity relationships (SARs) were concluded and it indicated that the side chains substituted at 4β position of podophyllotoxin significantly influenced the cytotoxic activity, especially for the drug resistance profile. In vivo studies of compound 26c on highly metastatic human lung cancer xenograft in nude mice showed that it can significantly inhibit tumor growth with administrating by oral route.

Full text of DOI:10.1016/j.ejmech.2010.11.016

European Journal of Medicinal Chemistry 46 (2011) 285e296
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new 4b
-anilino-40-O-demethyl-
4-desoxypodophyllotoxin derivatives as potential antitumor agents
Li Wang a, Fenyan Yang a, Xiaochun Yang b, Xianghong Guan a, Chunqi Hu a, Tao Liu a, Qiaojun He b,
,
Bo Yang b, Yongzhou Hu a
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
A series of new 4b
-anilino-40-O-demethyl-4-desoxypodophyllotoxin derivatives were prepared and
evaluated for their cytotoxicities against four human cancer cell lines including KB, KB/VCR, A549 and
95D. Most compounds showed better growth-inhibition activities against tested cell lines than that of
etoposide (VP-16). Preliminary structureeactivity relationships (SARs) were concluded and it indicated
Article history:
Received 28 July 2010
Received in revised form
5 November 2010
Accepted 11 November 2010
Available online 19 November 2010
that the side chains substituted at 4b position of podophyllotoxin significantly influenced the cytotoxic
activity, especially for the drug resistance profile. In vivo studies of compound 26c on highly metastatic
human lung cancer xenograft in nude mice showed that it can significantly inhibit tumor growth with
administrating by oral route.
Keywords:
4b
-Anilino-40-O-demethyl-4-
desoxypodophyllotoxin derivative
Cytotoxicity
Ó 2010 Elsevier Masson SAS. All rights reserved.
Drug resistance
Tumor growth inhibition
1. Introduction
drug-resistance and poor water-solubility, which block their further
application [6]. Thus, it is valuable for medicinal chemists to over-
Etoposide (VP-16, 1) (Fig. 1) and teniposide (VM-26, 2), first
synthesized in the 1960s, are two semi-synthetic glycoside deriva-
tives of podophyllotoxin (PDT, 3) [1]. In 1983 and 1992,1 and 2 were
officially approved respectively for clinical use against various types
of cancers including breast cancer, testicular cancer, small-cell lung
cancer, lymphoma, Kaposi’s sarcoma and childhood leukemia [2,3].
The mechanism of VP-16 was elucidated eleven years later after its
introduction in medical practice. Being a topoisomerase II inhibitor,
VP-16 stabilizes the double-stranded DNA cleavage normally cata-
lyzed by topoisomerase II and inhibits faithful relegation of DNA
breaks. The break of double-strand DNA subsequently triggers the
desired antitumor effects of the drug [4]. Recent researches reveal
that the antitumor activity of VP-16 is attributed primarily to its
come drug-resistance and develop more active, less toxic podo-
phyllotoxin derivatives. After the approval of VP-16, NK-611 (4) [7],
GL-331 (5) [8], TOP-53 (6) [9], etopophos (7) [10] and tafluposide (8)
[11] were emerged through C4 modification as alternatives to over-
come the drawbacks of VP-16. The excellent activity profiles of these
agents including improved water solubility, topoisomerase II
inhibitory activity, drug resistance and antitumor spectra suggested
the important role of C4 substitution pattern. Molecular area-
oriented chemical modification of podophyllotoxin revealed
structural features critical for the topoisomerase II inhibition: (1)
b-D-glucopyranose is not essential, the 4b configuration is funda-
mental with various substitution accommodated at C4; (2) the free
40-hydroxy is crucial, but 40-ester pro-drugs are allowed; (3) the
inhibition of topo II
a
, whereas the carcinogenic effect has been
trans-lactone D ring with 2a, 3b configuration is very important; (4)
attributed to the isoform [5].
b
the methylenedioxy cycle (ring A) is optimal; and (5) the free rota-
tion of ring E is required. This study also showed that the C4 is an
optimal position tolerable to significant structural modification [12].
The later discovered comparative molecular field analysis (CoMFA)
model further demonstrated that bulky substituents at C4 might be
favorable for topo-II inhibition [13].
Although being used extensively in clinic, VP-16 and VM-26
exhibited several toxic side effects such as bone-marrowdepression,
increased risk of secondary acute myelogenous leukemia, acquired
In previous studies, we reported that 4
phyllotoxin derivatives were potent cytotoxic agents against some
b-anilino substituted podo-
* Corresponding author. Tel./fax: þ86 571 88208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.016

286
L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
Fig. 1. Structures of etoposide (VP-16, 1), teniposide (VM-26, 2), podophyllotoxin (PDT, 3), NK-611 (4), GL-331 (5), TOP-53 (6), etopophos (7), tafluposide (8) and 9.
human cancer cell lines and drug-resistant cancer cell lines
Compound 24 was prepared from 1 with the same procedure as
described above. Deprotection of the amine group of 24 afforded
the hydrochloride salt 25, which was reacted with the corre-
sponding aromatic carboxylic acids to obtain the target compounds
of class III 26ael (Scheme 2).
[14]. One representative compound, 4b-(N-[3-(piperidin-1-ylmethyl)
phenyl]amino)-40-O-demethyl-4-desoxypodophyllotoxin (9), strongly
inhibited the growth of the tested drug-sensitive cancer cells with IC50
values ranging from 0.4 mM (Glima, U251 cell line) to 14.5 mM (lung
cancer, A549 cell line), which also inhibited drug-resistant cancer cells
with at least 3-fold more potent activity than that of VP-16. Besides,
compound 9 was effective in the treatment of cancer at a lower dose
than that of VP-16 in drug-sensitive xenograft and drug-resistant
xenograft models, demonstrating that it is a potential drug candidate
for anticancer chemotherapy [14]. Based on the structural features of
compound 9 and previously obtained biological results, we designed
2.2. Pharmacology
2.2.1. In vitro cytotoxic activities
All the synthesized compounds were tested for their cytotoxic
activities against four human cancer cell lines that comprised of KB,
A549, 95D and drug-resistant cell line KB/VCR. VP-16 and 9 were
chosen as the positive controls. IC50 values were obtained using the
standard MTT assay. The results are summarized in Table 1.
As shown in Table 1, most synthesized compounds exhibited
potent cytotoxic activities than that of VP-16. The RFs of most
compounds against KB cell lines were lower than that of VP-16. The
results implied that the introduction of aniline group at C4 position
contributed to overcome drug-resistance in the tested compounds.
This is in agreement with observation in other literature reports on
podophyllotoxin derivatives which suggest that C4-amino substi-
tution is good for overcoming drug-resistance [20].
and synthesized three classes of podophyllotoxin derivatives. A 4
anilino moiety was included to retain the drug-resistance profiles;
second basic group (compounds 22aem) or amide groups
b-
a
(compounds 26ael) were introduced to optimize activity profiles; and
hydroxyl groupswereintroduced tomodulatewatersolubilityof target
compounds (compounds 23aed). Preliminary structureeactivity
relationships (SARs) of synthesized podophyllotoxin derivatives were
derived from the in vitro results. Compounds 23a, 26c and 26e were
chosen for in vivo studies.
2. Results and discussion
The biological results of compounds in class I indicated that the
alkyl amino group on the end of 4b-side chain was important for in
2.1. Chemistry
vitro cytotoxic activity. The IC50 values of compounds 9, 22d and 22j
demonstrated that the length of the alkyl linker between the benzene
ring and tertiary amino influenced the cytotoxic activity obviously. In
general, the compounds with a CH2CH2 linker (i.e. 22d and 22j)
showed better activities than the molecule with a CH2 linker (9).
Compounds with pyrrolidine 22a, morpholine 22b or N,N-diethyl-
amine group 22c on the end of 4b-side chain displayed similar or
more potent activities than that of compound 9. Among them, the
pyrrolidineanalogue22a exhibitedthemost potent cytotoxic activity,
The synthetic routes of targetpodophyllotoxinderivatives22aem,
23aed and 26ael are outlined in Schemes 1 and 2. Aryl aldehydes
10aeb were efficiently converted into dibromoalkene derivatives
11aeb by Wittig Reaction, which were then treated with secondary
amines to afford intermediates 12aej [15]. The obtained aryl amides
12aej were further reduced to the corresponding amines 14aem first
by borane and then by Pd/C under hydrogen atmosphere [16]. On the
other hand, treatment of p-nitrophenol 15 with 2-chloroethanol in
a potassium hydroxide solution at an elevated temperature gave 2-(4-
nitrophenoxy)ethanol 17. Reaction of 1-(bromomethyl)-4-nitroben-
zene 20 with different glycols yielded the benzyloxy alcohols 20aec.
Reduction of the nitro groups of 17 and 20aec using Pd/C under
hydrogen atmosphere led to 18 and 21aec. Finally, the target mole-
cules of class I 22aem and class II 23aed were prepared utilizing our
recently developed synthetic protocol (Scheme 1) [17e19].
with IC50 ranging from 0.036 mM to 1.57 mM against the tested cancer
cell lines. Transfer of the alkyl amino group from 4-position of aniline
ring on the side chain (22a, 22b and 22c) to 3-position (22g, 22h and
22i) made a slightly decreased activity. Besides, compounds 22kem,
which possess amide groups on the end of 4b-side chain, maintained
fairly the same cell growth inhibitionpotencyagainst KB, KB/VCR and
A549 cell lines compared to the activities of compounds 22a, 22d and
22i, but greatly reduced the inhibition of 95D cell lines.

L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
287
Scheme 1. Synthesis of 22aem, 23aed and 24. Reagents and conditions: (a) CBr4, PPh3, CH2Cl2, 0 ꢀC; (b) amine, H2O, rt, 1 h (method 1); amine, THF, H2O, KOH, rt, 12 h (method 2);
(c) BF3$Et2O, NaBH4, THF, reflux; (d) H2, Pd/C, MeOH; (e) KOH, H2O; (f) ClCH2CH2OH, H2O; (g) H2, Pd/C, EtOAc; (h) glycol, KOH, 80 ꢀC, 3 h; (i) NaI, TMSCl, CH3CN, 0 ꢀC, 1 h; (j) BaCO3,
Et3N, CH3CN, 14aem, rt, 15e18 h; (k) BaCO3, Et3N, CH3CN, 18 or 21aec, rt, 15e18 h; (l) BaCO3, Et3N, CH3CN, t-butyl 4-aminophenylcarbamate, rt, 15e18 h.

288
L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
Scheme 2. Synthesis of 26ael. Reagents and conditions: (a) concentrated HCl/EtOAc, rt, 5 h; (b) EDCI, HOBt, Et3N, carboxylic acids, CH2Cl2, 0 ꢀC-rt.
Compounds containing the hydrophilic tails in class II were
designed to improve water-solubility. As illustrated in Table 1, the
length of alkoxy side chain on the end of 4 -side chain was found to
rates of 10 mg/kg 23a and 10 mg/kg 26e groups were 37.5% and
9.2%, respectively.
b
influence the cytotoxic activity. Compound 23a with a short chain
showed the most potent cytotoxic activity, as the number of atoms
between the benzene ring and the terminal hydroxyl group
increased (23b, 23c and 23d), the activities decreased except that of
compound 23b against 95D tumor cell lines.
Compounds 26ael in class III, which possess the amide moieties
on para-position of anilino ring, displayed comparable or slightly
weaker cell growth inhibition against KB and A549 cell lines in
comparison with that of VP-16, but a cytotoxic potency superior to
that of VP-16 on KB/VCR and 95D cell lines, except the activities of
compounds 26k and 26l. Among them, compound with fluorine
2.2.3. Physicochemical characteristics
Compounds 23aed were designed to improve the water solu-
bility, based on the structural features of compound 9, by intro-
ducing the hydroxyl groups. In order to determine the water-lipid
coefficient, calculated values of c Log P by ChemDraw 9.0 software
were reported. In addition, based on the method initiated by Baker
et al. [21], retention behavior of compounds 9 and 23aed was
measured by RP-HPLC to indicate the hydrophobicity parameter of
drugs. The retention index (RI) values of tested compounds were
calculated by an equation based on the retention time of the alkan-
2-ones used. The results are summarized in Table 3.
atom 26b or acetoxy group 26c on the benzene ring of 4
b
-side chain
As shown in Table 3, both the calculated c Log P value and
retention index value of compound 9 were higher than those of
compounds 23aed, indicating that introducing the hydroxyl
groups improved the water solubility.
showed the most potent activities. It is interesting that two di-
substituted derivatives 26k and 26l showed decreased activities
compared with those of mono-disubstituted compounds 26d and
26j as well as VP-16.
In an overall view, the activities of class II derivatives were
superiortothoseofclass IIIcompounds, but inferiortothose ofclassI
compounds. This result revealed that the 4b-aniline substitution
3. Conclusions
In summary, a series of new 4b
-anilino-40-O-demethyl-4-des-
was beneficial for the cytotoxic activity, especially for overcoming
acquired drug resistance. The activity correlated well with the
oxypodophyllotoxin derivatives have synthesized and evaluated for
their biological activities. Most of synthesized compounds exhibi-
ted potent cytotoxic activities against the tested cancer cell lines
including MDR cancer cell lines in vitro. Preliminary structur-
eeactivity relationships were put forward based on the biological
results. Compound 26c was a promising agent which can be orally
administered, and at 50 mg/kg it significantly inhibited high
metastatic human lung cancer growth in vivo. Additional work is
underway to perform the pre-clinical evaluation of compound 26c.
alkalinity of the substituents at the tail of the 4
Compounds of class I with alkyl amino groups on the end of 4
b
-aniline side chain.
-side
b
chain possessed the most potent inhibition activity, compounds in
class II with the hydroxyl groups ranked the second and compounds
in class IIIbearing the substituted amide moieties showed the lowest
activities among all these three classes of synthesized compounds.
2.2.2. In vivo antitumor effects
Based on the cytotoxic activities of the tested compounds in
vitro, compounds 23a, 26c and 26e were chosen to investigate their
growth inhibitory activities against the xenograft model of a highly
metastatic human lung cancer 95D in nude mice. Compound 26c
(50 mg/kg) was administered intragastrically two or three times
a week for 20 days and compounds 23a (10 mg/kg) and 26e
(10 mg/kg) were administered intramuscularly every other day for
20 days. The results of experimental therapeutic efficacies of 23a,
26c and 26e are shown in Table 2 and Fig. 2.
4. Experimental protocols
4.1. Chemistry
Melting points were taken on a BÜCHI B-540 apparatus and are
uncorrected. IR spectra were recorded on a Brüker VECTOR-22
spectrophotometer. Optical rotations were measured with a JASCO
P-1010 polarimeter. NMR spectra were obtained using a Brüker
Advance DMX 500-MHz spectrometer with TMS as the internal
standard. All chemical shifts are reported in parts per million
(ppm). Mass spectra were recorded on a Fourier Transform Ion
Tumor weights of 95D in 50 mg/kg 26c group reduced signifi-
cantly (P < 0.05), and the inhibition rate was 57.3%. The inhibition

L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
289
Table 1
In vitro cytotoxic activities of synthesized compounds and VP-16, 9 against four human cancer cell lines.
RFb
a
Compound
R(n1, n2)
IC50
(
m
M)
KB
KB/VCR
A549
95D
VP-16
9
e
e
4.61
0.97
83.4
3.44
2.56
0.50
20.2
0.20
18.09
3.55
22a
22b
22c
22d
22e
0.21
0.78
0.45
4.21
2.42
1.57
1.27
1.53
19.1
3.95
0.24
1.94
0.40
13.4
19.0
0.036
11.5
9.84
17.2
17.1
7.48
1.63
3.4
4.54
1.63
22f
0.44
2.20
0.49
10.0
5
22g
22h
22i
22j
0.39
0.48
0.77
0.43
1.85
2.09
3.87
1.78
3.91
5.10
7.49
2.75
1.17
14.8
14.6
7.40
4.74
4.35
5.03
4.14
22k
22l
1.52
0.40
0.42
2.20
2.33
27.2
15.9
5.20
4.78
>50
25.8
>50
1.45
5.83
64.76
22m
23a
23b
23c
23d
n1 ¼ 0, n2 ¼ 2
n1 ¼ 1, n2 ¼ 2
n1 ¼ 1, n2 ¼ 3
n1 ¼ 1, n2 ¼ 4
2.88
3.12
6.00
6.60
6.51
7.39
5.08
9.55
4.54
5.11
7.45
8.5
4.13
0.55
12.04
20.30
2.26
2.37
0.85
1.45
(continued on next page)

290
L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
Table 1 (continued )
RFb
a
Compound
R(n1, n2)
IC50
(
m
M)
KB
KB/VCR
9.28
A549
3.41
95D
0.44
26a
26b
26c
26d
26e
6.76
1.37
1.82
4.44
<0.27
0.54
2.91
1.91
>50
6.88
5.31
8.48
13.7
3.70
1.56
2.68
11.0
3.31
1.08
0.42
3.41
1.15
26f
6.69
5.30
8.43
0.41
0.79
26g
26h
26i
26j
6.53
>50
8.75
22.1
2.80
3.08
5.72
13.06
>50
>50
18.9
19.5
1.25
1.23
1.66
6.56
0.43
<0.06
0.65
0.59
26k
>50
>50
>50
>50
>50
15.7
19.6
NT
26l
14.21
<0.28
a
Cells were exposed with various concentrations of compounds and VP-16 for 72 h.
Resistance factor that was calculated as the ratio of the IC50 value of the MDR cells to that of the corresponding drug-sensitive parental cells.
b
Cyclotron Resonance Mass Spectrometer. RP-HPLC analyses were
carried out on an Agilent 1200 system with DAD detector.
butanediol (5 mL), and the resulting mixture heated at about
80 ꢀC for 20 h until the raw product disappeared. After cooling to
room temperature, the mixture was diluted with water and
extracted with dichloromethane (10 mL ꢁ 3). Combined organic
layers were dried over Na2SO4 and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
using PE/EtOAc (3:1, v/v) as eluent to afford 20c. Yellow oil
4.1.1. Synthesis of key intermediates (14aem, 18 and 21aeb)
Compounds 14aem, 18 and 21aeb were synthesized according
to the known methods [15e18].
4.1.2. 4-[(4-nitrobenzyl)oxy]butan-1-ol (20c)
Potassium hydroxide (145 mg, 2.6 mmol) was added to
a solution of 4-nitrobenzyl bromide (512 mg, 2.4 mmol) in 1,4-
(535 mg, 83.1%). 1H NMR (500 MHz, CDCl3)
d
8.19 (d, J ¼ 9.0 Hz,
2H), 7.49 (d, J ¼ 9.0 Hz, 2H), 4.61 (s, 2H), 3.66 (m, 2H), 3.56 (m,
2H), 1.73 (m, 2H), 1.68 (m, 2H).
Table 2
Inhibitory effects of 23a, 26c and 26e on the tumor growth of 95D in nude mice.
Group
Average body weight (g)
Tumor volume (mm3)
Tumor weight (g)
RTV
T/C (%)
Inhibition rate (%)
D1
D20
D1
D20
Control
23a
26c
17.5 ꢂ 0.8
17.6 ꢂ 0.4
18.2 ꢂ 1.0
17.4 ꢂ 0.5
21.6 ꢂ 0.6
153 ꢂ 26
166 ꢂ 18
165 ꢂ 37
168 ꢂ 27
1370 ꢂ 225
663 ꢂ 112*
527 ꢂ 21*
910 ꢂ 80
1.97 ꢂ 0.31
1.23 ꢂ 0.18
0.84 ꢂ 0.10*
1.79 ꢂ 0.26
9.3 ꢂ 1.5
4.2 ꢂ 1.0*
4.3 ꢂ 0.6*
5.8 ꢂ 0.8
e
e
17.0 ꢂ 0.4***
16.8 ꢂ 0.5***
20.2 ꢂ 0.7
45.4
46.3
62.4
37.5
57.3
9.2
26e
D1: first day treated with drug. *P < 0.05, ***P < 0.001, compared with control on the same day. Significant difference was calculated by Student’s t test.

L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
291
Fig. 2. (a) Effects of compounds 23a, 26c and 26e on the tumor volume of transplanted tumor (95D) in nude mice ðx ꢂ sÞ; (b) Effects of compounds 23a, 26c and 26e on the relative
tumor volume (RTV) of transplanted tumor (95D) in nude mice ðx ꢂ sÞ; (c) Effects of compounds 23a, 26c and 26e on the body weight of transplanted tumor (95D) in nude mice
ðx ꢂ sÞ.
4.1.3. 4-[(4-aminobenzyl)oxy]butan-1-ol (21c)
146.48, 142.82, 134.15, 133.77, 131.93, 129.90, 129.68, 125.78, 112.69,
109.95,109.10,107.98,101.56, 69.09, 57.24, 56.52, 53.86, 52.63, 45.84,
43.42, 41.91, 38.66, 31.29, 23.40; HR-MS (ESIþ) calculated for
Compound 20c (300 mg, 1.3 mmol) was dissolved in methanol
(15 mL) in a pressure bottle, 5% Pd/C (30 mg) was added and the
reaction mixture was stirred at room temperature under hydrogen
for 12 h. When hydrogen uptake ceased, the catalyst was removed
by filtration and the solvents evaporated. The resulting crude
residue was purified by flash column chromatography on silica gel
using PE/EtOAc (1:2, v/v) as eluent to afford 21c. Yellow oil (162 mg,
C33H36N2O7 [M þ 1]þ: 573.2556, found: m/z ¼ 573.2549 [M þ 1]þ
.
4.1.6. 4
b-(N-[4-(2-morpholin-4-ylethyl)phenyl]amino)-
40-O-demethyl-4-desoxypodophyllotoxin (22b)
Yellow solid (62.3%), mp 117.9e118.9 ꢀC; IR(KBr, cmꢃ1) 3387,
62.3%). 1H NMR (500 MHz, CDCl3)
J ¼ 8.5 Hz, 2H), 4.37 (s, 2H), 3.57 (t, J ¼ 6.0 Hz, 2H), 3.46 (t, J ¼ 6.0 Hz,
2H), 1.67 (q, J ¼ 6.0 Hz, 2H), 1.61 (q, J ¼ 6.0 Hz, 2H).
d
7.11 (d, J ¼ 8.5 Hz, 2H), 6.63 (d,
2936, 1771, 1614, 1517, 1482, 1227, 1114, 1036; [
a
]
25e50.4 (c ¼ 0.1,
D
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.04 (d, J ¼ 9.0 Hz, 2H, 300,500-H),
6.76 (s, 1H, 5-H), 6.52 (s, 1H, H-8), 6.47 (d, J ¼ 9.0 Hz, 2H, 200,600-H),
6.33 (s, 2H, 20,60-H), 5.94 and 5.96 (AB q, J ¼ 1.5 Hz, 2H, eOCH2Oe),
4.64 (m, 1H, 1-H), 4.58 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.35 (t, J ¼ 8.0 Hz, 1H,
11-H), 3.98 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.81 (br, 1H, 4-NH), 3.79 (s, 6H,
30,50-OCH3), 3.74 (m, 4H, morphaline-H), 3.16 (m, 1H, 3-H), 3.13 (dd,
J ¼ 5.0, 14.0 Hz, 1H, 2-H), 3.07 (m, 1H, 3-H), 2.72 (m, 2H,
NeCH2eCH2-Ph), 2.59 (m, 2H, NeCH2eCH2-Ph), 2.56 (m, 4H,
4.1.4. General procedure for the synthesis of 22, 23 and 24
A solution of TMSCl (0.76 mL) in dry acetonitrile (15 mL) was
added dropwise to a mixture of VP-16 (0.88 g, 1.5 mmol) and
sodium iodide (0.9 g, 6.0 mmol) in dry acetonitrile at 0 ꢀC and
stirred for 1 h. Dried barium carbonate (0.9 g, 4.5 mmol) was added
to the mixture. After stirring for 10 min, the pH was adjusted to 8
using anhydrous triethylamine. Then the appropriate amine
(1.8 mmol) was added and the reaction mixture was stirred at room
temperature for 15e18 h. The mixture was filtered; the filtrate was
evaporated, washed with 20% sodium thiosulfate solution and
extracted with ethyl acetate (30 mL ꢁ 3). Combined organic layers
were dried over Na2SO4 and concentrated in vacuo. The crude
product was purified by flash column chromatography on silica gel
using PE/EtOAc/Et3N (100:100e200:1, v/v/v) to give 22, 23 and 24.
morphaline-H); 13C NMR (125 MHz, CDCl3)
d 175.04, 148.43, 147.77,
146.70, 146.00, 134.41, 132.10, 131.00, 130.82, 129.94, 129.03, 112.62,
110.11, 109.29, 108.27, 101.68, 69.16, 67.00, 61.24, 60.55, 56.72, 56.28,
53.82, 52.98, 43.64, 42.12, 38.92, 32.30; HR-MS (ESIþ) calculated for
C33H36N2O8 [M þ 1]þ: 589.2505, found: m/z ¼ 589.2510 [M þ 1]þ
.
4.1.7. 4
40-O-demethyl-4-desoxypodophyllotoxin (22c)
Yellowewhite solid (70.8%), mp 113.1e113.9 ꢀC; IR(KBr, cmꢃ1
b-(N-[4-[2-(diethylamino)ethyl]phenyl]amino)-
)
3387, 2969, 2935, 2840, 1771, 1614, 1518, 1482, 1464, 1227, 1115,
4.1.5. 4
b
-(N-[4-(2-pyrrolidin-1-ylethyl)phenyl]amino)-
1036; [
a
]
25e39.9 (c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d 7.05
D
40-O-demethyl-4-desoxypodophyllotoxin (22a)
(d, J ¼ 8.0 Hz, 2H, 300,500-H), 6.76 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.48
(d, J ¼ 8.0 Hz, 2H, 200,600-H), 6.33 (s, 2H, 20,60-H), 5.94 and 5.96 (AB q,
J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.64 (m, 1H, 1-H), 4.58 (d, J ¼ 5.0 Hz, 1H,
4-H), 4.35 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.95 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.91
(br, 1H, NH), 3.79 (s, 6H, 30,50-OCH3), 3.14 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-
H), 3.00 (m, 1H, 3-H), 2.95 (m, 6H, NeCH2eCH2-Ph, Ne(CH2CH3)2),
Yellow solid (58.3%), mp 135.2e136.7 ꢀC; IR(KBr, cmꢃ1) 3487,
2942, 1771, 1614, 1518, 1482, 1458, 1227, 1116, 1036; [a]
25e73.1
D
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.07 (d, J ¼ 8.0 Hz, 2H,
300,500-H), 6.75 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.49 (d, J ¼ 8.0 Hz,
2H, 200,600-H), 6.33 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q, J ¼ 1.0 Hz,
2H, eOCH2Oe), 4.65 (m,1H,1-H), 4.59 (d, J ¼ 4.5 Hz,1H, 4-H), 4.34 (t,
J ¼ 8.0 Hz, 1H, 11-H), 4.00 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.84 (br, 1H, NH),
3.79 (s, 6H, 30,50-OCH3), 3.11 (m,1H, 2-H), 3.06 (m, 4H, NeCH2eCH2-
Ph), 2.99 (m, 1H, 3-H), 2.09 (m, 4H, pyrrolidine-H), 1.36 (t, J ¼ 7.5 Hz,
2.86 (m, 2H, NeCH2eCH2-Ph), 1.25 (t, J ¼ 7.5 Hz, 6H, eCH2CH3); 13
C
NMR (125 MHz, CDCl3)
d 175.00, 148.33, 147.66, 146.58, 146.31,
134.20, 131.98, 130.70, 130.04, 127.70, 112.65, 110.00, 109.23, 108.04,
101.63, 69.06, 56.71, 53.76, 52.75, 46.35, 43.51, 41.99, 38.79, 30.45,
9.77; HR-MS (ESIþ) calculated for C33H38N2O7 [M þ 1]þ: 575.2713,
4H, pyrrolidine-H); 13C NMR (125 MHz, CDCl3)
d 174.82, 146.58,
found: m/z ¼ 575.2720 [M þ 1]þ
.
4.1.8. 4b
-(N-[4-(2-piperidin-1-ylethyl)phenyl]amino)-40-O-
Table 3
Calculated c Log P and retention index (RI) of compounds 9 and 23aed.
demethyl-4-desoxypodophyllotoxin (22d)
Compd.
Calculated c Log P
RI
Yellow solid (68.2%), mp 132.3e133.3 ꢀC; IR(KBr, cmꢃ1) 3387,
2936, 1775, 1613, 1517, 1481, 1230, 1114, 1036; [
a
]
25e63.7 (c ¼ 0.1,
9
3.505
2.267
2.116
2.470
2.415
590.4
476.8
472.4
514.6
572.2
D
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.12 (d, J ¼ 9.0 Hz, 2H, 300,500-H),
23a
23b
23c
23d
6.68 (d, J ¼ 9.0 Hz, 2H, 200,600-H), 6.62 (s, 1H, 5-H), 6.57 (s, 1H, 8-H),
6.34 (s, 2H, 20,60-H), 5.97 (s, 2H, eOCH2Oe), 5.14 (m, 1H, 1-H), 4.66
(d, J ¼ 5.0 Hz, 1H, 4-H), 4.28 (t, J ¼ 8.0 Hz, 1H, 11-H), 4.12 (t,

292
L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
J ¼ 8.0 Hz, 1H, 11-H), 3.80 (br, 1H, NH), 3.79 (s, 6H, 30,50-OCH3), 3.04
(dd, J ¼ 5.0, 14.5 Hz, 1H, 2-H), 2.98 (m, 1H, 3-H), 2.89 (m,
4H, NeCH2eCH2-Ph), 2.79 (m, 4H, piperidine-H), 1.81 (m, 6H,
(m,1H, 3-H), 3.12(dd, J ¼ 5.0,14.0 Hz,1H, 2-H), 3.07 (m,1H, 3-H), 2.80
(m, 2H, NeCH2eCH2-Ph), 2.68 (m, 2H, NeCH2eCH2-Ph), 2.63 (m, 4H,
morphaline-H); 13C NMR (125 MHz, CDCl3)
d 175.04, 148.42, 147.82,
piperidine-H); 13C NMR (125 MHz, CDCl3)
d
173.93, 147.68, 147.35,
147.76, 134.42, 132.13, 130.86, 129.89, 118.95, 113.12, 110.27, 110.10,
109.36, 108.29, 101.70, 69.15, 66.63, 60.66, 56.72, 53.62, 52.65, 46.02,
43.64, 42.12, 38.93, 33.13; HR-MS (ESIþ) calculated for C33H36N2O8
147.24, 146.02, 133.63, 132.08, 129.89, 129.33, 128.07, 113.96, 111.16,
109.43, 108.34, 107.38, 107.14, 101.00, 68.50, 59.71, 56.01, 55.77,
55.45, 53.28, 43.40, 43.05, 42.93, 38.32, 34.87, 23.88, 22.86; HR-MS
(ESIþ) calculated for C34H38N2O7 [M þ 1]þ: 587.2713, found:
[M þ 1]þ: 589.2505, found: m/z ¼ 573.2515 [M þ 1]þ
.
m/z ¼ 587.2720 [M þ 1]þ
.
4.1.13. 4
demethyl-4-desoxypodophyllotoxin (22i)
b
-(N-[3-[2-(diethylamino)ethyl]phenyl]amino)-40-O-
4.1.9. 4
b
-(N-[4-[2-(dimethylamino)ethyl]phenyl]amino)-40-O-
Yellow solid (54.1%), mp 134.2e135.8 ꢀC; IR(KBr, cmꢃ1) 3381,
demethyl-4-desoxypodophyllotoxin (22e)
2944, 1772, 1614, 1518, 1482, 1227, 1115, 1036; [
a
]
25e105.7 (c ¼ 0.1,
D
Yellow solid (36.7%), mp 101.1e101.8 ꢀC; IR(KBr, cmꢃ1) 3387,
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.13 (t, J ¼ 8.5 Hz, 1H, 500-H),
2966, 2934, 1612, 1774, 1515, 1482, 1229, 1115, 1036; [
a
]
25e27.3
6.75 (s, 1H, 5-H), 6.58 (m, 2H, 400-H and 600-H), 6.52 (s, 1H, 8-H), 6.43
(s, 1H, 200-H), 6.34 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q, J ¼ 1.5 Hz, 2H,
eOCH2Oe), 4.72 (m, 1H, 1-H), 4.59 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.40 (t,
J ¼ 8.0 Hz, 1H, 11-H), 4.00 (br, 1H, NH), 3.93 (t, J ¼ 8.0 Hz, 1H, 11-H),
3.80 (s, 6H, 30,50-OCH3), 3.13 (dd, J ¼ 6.5, 14.0 Hz, 1H, 2-H), 3.08 (m,
1H, 3-H), 3.00 (m, 8H, NeCH2eCH2-Ph, Ne(CH2CH3)2), 1.38 (t,
J ¼ 7.5 Hz, 6H, eCH2CH3); HR-MS (ESIþ) calculated for C33H38N2O7
D
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
7.11 (d, J ¼ 8.5 Hz, 2H,
d
300,500-H), 6.66 (d, J ¼ 8.5 Hz, 2H, 200,600-H), 6.64 (s, 1H, 5-H), 6.56 (s,
1H, 8-H), 6.33 (s, 2H, 20,60-H), 5.97 (s, 2H, eOCH2Oe), 5.13 (m,1H,1-
H), 4.66 (d, J ¼ 4.5 Hz, 1H, 4-H), 4.31 (t, J ¼ 9.0 Hz, 1H, 11-H), 3.88 (t,
J ¼ 9.0 Hz, 1H, 11-H), 3.80 (s, 6H, 30,50-OCH3), 3.40 (br, 1H, NH), 3.02
(dd, J ¼ 5.0, 15.0 Hz, 1H, 2-H), 2.96 (m, 1H, 3-H), 2.93 (m, 2H,
NeCHe(CH3)2),
2.92
(m,
2H,
NeCH2eCH2-Ph),
2.87
[M þ 1]þ: 575.2713, found: m/z ¼ 575.2719 [M þ 1]þ
.
(m, 2H, NeCH2eCH2-Ph), 1.28 (m, 12H, NeCHe(CH3)2); HR-MS
(ESIþ) calculated for C31H34N2O7 [M þ 1]þ: 547.2400, found:
4.1.14. 4
demethyl-4-desoxypodophyllotoxin (22j)
Yellow solid (51.4%), mp 216.8e222 ꢀC; IR(KBr, cmꢃ1) 3387,
b
-(N-[3-(2-piperidin-1-ylethyl)phenyl]amino)-40-O-
m/z ¼ 547.2408 [M þ 1]þ
.
4.1.10. 4
b
-(N-[4-[2-(diisopropylamino)ethyl]phenyl]amino)-40-O-
2942, 2840, 1771, 1614, 1518, 1482, 1227, 1116, 1036; [a]
25e68.9
D
demethyl-4-desoxypodophyllotoxin (22f)
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.11 (t, J ¼ 7.5 Hz, 1H,
Light yellow solid (77.4%), mp 127.8e132.8 ꢀC; IR(KBr, cmꢃ1
)
500-H), 6.76 (s,1H, 5-H), 6.58 (d, J ¼ 7.0 Hz,1H, 400-H), 6.54 (m,1H, 200-
H), 6.52 (s, 1H, 8-H), 6.41 (s, 1H, 600-H), 6.34 (s, 2H, 20,60-H), 5.95 and
5.97 (AB q, J ¼ 0.5 Hz, 2H, eOCH2Oe), 4.70 (m, 1H, 1-H), 4.58 (d,
J ¼ 5.0 Hz, 1H, 4-H), 4.40 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.97 (br, 1H, NH),
3.92 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.79 (s, 6H, 30,50-OCH3), 3.13 (dd,
J ¼ 5.0, 14.0 Hz, 1H, 2-H), 3.05 (m, 1H, 3-H), 3.03 (m, 4H, piperidine-
H), 2.97 (m, 4H, NeCH2eCH2-Ph), 1.34 (m, 6H, piperidine-H); HR-
MS (ESIþ) calculated for C34H38N2O7 [M þ 1]þ: 587.2713, found: m/
3387, 2936, 2777, 1772, 1614,1517, 1482, 1227, 1116, 1036; [a]
25e57.9
D
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.05 (d, J ¼ 8.0 Hz, 2H,
300,500-H), 6.76 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.49 (d, J ¼ 8.0 Hz, 2H,
200,600-H), 6.33(s, 2H, 20,60-H), 5.95 and 5.96 (AB q, J ¼ 1.5 Hz, 2H,
eOCH2Oe), 4.64 (m, 1H, 1-H), 4.58 (d, J ¼ 4.5 Hz, 1H, 4-H), 4.34 (t,
J ¼ 8.0 Hz, 1H, 11-H), 3.96 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.81 (br, 1H, NH),
3.79 (s, 6H, 30,50-OCH3), 3.12 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-H), 3.06 (m,
1H, 3-H), 2.84 (m, 2H, NeCH2eCH2-Ph), 2.79 (m, 2H, NeCH2eCH2-
Ph), 2.52 (s, 6H, NeCH3); HR-MS (ESIþ) calculated for C35H42N2O7
z ¼ 587.2705 [M þ 1]þ
.
[M þ 1]þ: 603.3026, found: m/z ¼ 603.3032 [M þ 1]þ
.
4.1.15. 4b
-(N-[4-(2-oxo-2-pyrrolidin-1-ylethyl)phenyl]amino)-40-
O-demethyl-4-desoxypodophyllotoxin (22k)
4.1.11. 4
b
-(N-[3-(2-pyrrolidin-1-ylethyl)phenyl]amino)-40-O-
Yellow solid (34.2%), mp 75.8e80.4 ꢀC; IR(KBr, cmꢃ1) 3376,
2969, 2879, 1771, 1618, 1518, 1482, 1453, 1227, 1114, 1036;
demethyl-4-desoxypodophyllotoxin (22g)
Yellow solid (53.8%), mp 116.9e117.2 ꢀC; IR(KBr, cmꢃ1) 3387,
[
a
]
25e46.0 (c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d 7.12 (d,
D
2942, 2840, 1772, 1605, 1590, 1482, 1226, 1115, 1036; [R]2D5e55.5
J ¼ 10.0 Hz,, 2H, 300,500-H), 6.76 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.49 (d,
J ¼ 11.0 Hz, 2H, 200,600-H), 6.33 (s, 2H, 20,60-H), 5.95 and 5.96 (AB q,
J ¼ 1.5 Hz, 2H, eOCH2Oe), 4.65 (m, 1H, 1-H), 4.59 (d, J ¼ 4.5 Hz, 1H,
4-H), 4.35 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.98 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.81
(br,1H, NH), 3.79 (s, 6H, 30,50-OCH3), 3.55 (s, 2H, NCOeCH2-Ph), 3.44
(m, 4H, pyrrolidine-H), 3.13 (dd, J ¼ 3.5, 14.0 Hz, 1H, 2-H), 2.97 (m,
1H, 3-H),1.91 (m, 2H, pyrrolidine-H),1.81 (m, 2H, piperidine-H); 13C
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.12 (t, J ¼ 7.5 Hz,
8.5 Hz,1H, 500-H), 6.76 (s,1H, 5-H), 6.59 (d, J ¼ 7.0 Hz,1H, 400-H), 6.52
(m, 2H, 600-H and 8-H), 6.41 (d, 7.5 Hz, 1H, 200-H), 6.33(s, 2H, 20,60-H),
5.95 and 5.97 (AB q, J ¼ 1.5 Hz, 2H, eOCH2Oe), 4.70 (m, 1H, 1-H),
4.59 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.39 (t, J ¼ 7.5 Hz, 1H, 11-H), 3.93 (t,
J ¼ 7.5 Hz, 1H, 11-H), 3.82 (br, 1H, NH), 3.79 (s, 6H, 30,50-OCH3), 3.12
(dd, J ¼ 5.0,14.0 Hz,1H, 2-H), 3.11 (m, 2H, NeCH2eCH2-Ph), 3.03 (m,
1H, 3-H), 3.00 (m, 2H, NeCH2eCH2-Ph), 2.04 (m, 4H, pyrrolidine-
H), 1.34 (t, J ¼ 7.5 Hz, 4H, pyrrolidine-H); HR-MS (ESIþ) calculated
for C33H36N2O7 [M þ 1]þ: 573.2556, found: m/z ¼ 573.2550
NMR (125 MHz, CDCl3)
d 174.47, 169.61, 147.05, 146.49, 146.03,
145.93, 133.54, 133.32, 131.34, 130.18, 129.65, 126.13, 124.07, 111.95,
108.65, 107.42, 104.77, 101.00, 70.49, 68.52, 56.01, 55.98, 46.37,
45.44, 45.08, 42.91, 42.03, 41.35, 40.64, 38.20, 25.65, 23.85; HR-MS
(ESIþ) calculated for C33H34N2O8 [M þ 1]þ: 587.2349, found: m/
[M þ 1]þ
.
z ¼ 587.2345 [M þ 1]þ
.
4.1.12. 4b
-(N-[3-(2-morpholin-4-ylethyl)phenyl]amino)-40-O-
demethyl-4-desoxypodophyllotoxin (22h)
4.1.16. 4b
-(N-[4-(2-oxo-2-piperidin-1-ylethyl)phenyl]amino)-40-O-
Yellow solid (57.8%), mp 97.1e98.3 ꢀC; IR(KBr, cmꢃ1) 3381, 2936,
demethyl-4-desoxypodophyllotoxin (22l)
1771, 1614, 1518, 1481, 1227, 1116, 1036; [
a
]
25e85.5 (c ¼ 0.1, CHCl3);
Yellow solid (38.8%), mp 103.8e104.2 ꢀC; IR(KBr, cmꢃ1) 3373,
D
1H NMR (500 MHz, CDCl3)
d
7.12 (t, J ¼ 7.5 Hz, 1H, 500-H), 6.77 (s, 1H,
2936, 1772, 1615, 1518, 1482, 1227, 1115, 1036; [
a
]
25e72.1 (c ¼ 0.1,
D
5-H), 6.62 (d, J ¼ 8.5 Hz,1H, 400-H), 6.52 (s,1H, 8-H), 6.43 (s,1H, 200-H),
6.38 (m, 1H, 600-H), 6.34 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q,
J ¼ 1.5 Hz, 2H, eOCH2Oe), 4.68 (m,1H,1-H), 4.58 (d, J ¼ 5.0 Hz,1H, 4-
H), 4.35 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.96 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.87 (br,
1H, 4-NH), 3.81 (m, 4H, morphaline-H), 3.79(s, 6H, 30,50-OCH3), 3.16
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.08 (d, J ¼ 8.5 Hz,, 2H, 300,500-
H), 6.76 (s, 1H, 5-H), 6.50 (d, J ¼ 8.0 Hz, 2H, 200,600-H), 6.52 (s, 1H,
8-H), 6.33 (s, 2H, 20,60-H), 5.94 and 5.96 (AB q, J ¼ 1.0 Hz,
2H, eOCH2Oe), 4.66 (m, 1H, 1-H), 4.58 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.34
(t, J ¼ 8.0 Hz, 1H, 11-H), 3.97 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.87 (br, 1H,

L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
293
NH), 3.79 (s, 6H, 30,50-OCH3), 3.62 (s, 2H, NCOeCH2-Ph), 3.55 (m,
2H, piperidine-H), 3.40 (m, 2H, piperidine-H), 3.14 (dd, J ¼ 5.0,
14.5 Hz, 1H, 2-H), 3.08 (m, 1H, 3-H), 1.58 (m, 2H, piperidine-H),
1.51 (m, 2H, piperidine-H), 1.38 (m, 2H, piperidine-H); HR-MS
(ESIþ) calculated for C34H36N2O8 [M þ 1]þ: 601.2505, found:
4.1.21. 4b
-(N-[4-[(4-hydroxybutoxy)methyl]phenyl]amino)-40-O-
demethyl-4-desoxypodophyllotoxin (23d)
Yellow solid (58.1%), mp 88.2e90 ꢀC; [
a
]
25e72.9 (c ¼ 0.1, CHCl3);
D
1H NMR (500 MHz, CDCl3)
d
7.18 (d, J ¼ 8.5 Hz, 2H, 300,500-H), 6.76 (s,
1H, 5-H), 6.52 (d, J ¼ 8.0 Hz, 2H, 200,600-H), 6.53 (s, 1H, 8-H), 6.34 (s,
2H, 20,60-H), 5.95 and 5.97 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.67 (m,
1H, 1-H), 4.59 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.42 (s, 2H, Ph-CH2eO), 4.35
(t, J ¼ 8.0 Hz, 1H, 11-H), 3.77 (s, 6H, 30,50-OCH3), 3.68 (m, 2H, eOCH2
CH2CH2CH2OH), 3.62 (t, J ¼ 5.5 Hz, 2H, eOCH2CH2CH2CH2OH), 3.50
(t, J ¼ 5.5 Hz, 2H, eOCH2CH2CH2CH2OH), 3.12 (dd, J ¼ 5.0, 13.5 Hz,
1H, 2-H), 2.98 (m, 1H, 3-H), 1.69 (m, 2H, eOCH2CH2CH2CH2OH);
HR-MS (ESIþ) calculated for C32H35NO9 [M þ 1]þ: 578.2345, found:
m/z ¼ 601.2498 [M þ 1]þ
.
4.1.17. 4b
-(N-[3-(2-oxo-2-(diethylamino)ethyl)phenyl]amino)-40-
O-demethyl-4-desoxypodophyllotoxin (22m)
Yellow solid (59.6%), mp 78.9e82.3 ꢀC; IR(KBr, cmꢃ1) 3387, 2973,
2935, 1772, 1605, 1517, 1483, 1481, 1227, 1114, 1036; [a]
25e60.3
D
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.12 (t, J ¼ 7.5 Hz, 1H,
500-H), 6.76 (s,1H, 5-H), 6.62 (d, J ¼ 7.5 Hz,1H, 400-H), 6.54 (m,1H, 600-
H), 6.50 (s,1H, 8-H), 6.42 (m,1H, 200-H), 6.33 (s,1H, 20-H), 6.29 (s,1H,
60-H)5.95 and 5.97 (AB q, J ¼ 2.0 Hz, 2H, eOCH2Oe), 4.68 (m, 1H, 1-
H), 4.58 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.33 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.92 (t,
J ¼ 8.0 Hz, 1H, 11-H), 3.85 (br, 1H, NH), 3.79 (s, 6H, 30,50-OCH3), 3.62
(s, 2H, NCOeCH2-Ph), 3.36 (m, 2H, Ne(CH2CH3)2), 3.28 (m, 2H, Ne
(CH2CH3)2), 3.10 (dd, J ¼ 5.5, 14.5 Hz, 1H, 2-H), 3.06 (m, 1H, 3-H),
1.36 (t, J ¼ 7.0 Hz, 3H, eCH2CH3), 1.24 (t, J ¼ 7.0 Hz, 3H, eCH2CH3);
HR-MS (ESIþ) calculated for C33H36N2O8 [M þ 1]þ: 589.2505,
m/z ¼ 578.2351 [M þ 1]þ
.
4.1.22. 4b
-(N-[4-(tert-butyl-(4-aminophenyl)carbamate)])-40-O-
demethyl-4-desoxypodophyllotoxin (24)
Yellow solid (85.7%), mp 136.1e138.6 ꢀC; 1H NMR (500 MHz,
CDCl3)
d 7.20e7.21 (m, 2H), 6.74 (s, 1H), 6.51 (s, 1H), 6.48e6.50 (d,
J ¼ 9.0 Hz, 2H), 6.36 (ws, 1H), 6.33 (s, 2H), 5.95 and 5.97 (d,
J ¼ 10.5 Hz, 2H), 5.47 (ws, 1H), 4.61 (m, 1H), 4.57e4.58 (d, J ¼ 5.0 Hz,
1H), 4.34e4.37 (t, J ¼ 8.0 Hz, 1H), 3.96e4.00 (m, 1H), 3.79 (s, 6H),
3.13e3.16 (dd, J ¼ 5.0, 14.5 Hz, 1H), 2.94e3.01 (m, 1H), 1.52 (s, 9H).
found: m/z ¼ 589.2512 [M þ 1]þ
.
4.1.18. 4
b
-(N-[4-(2-hydroxyethoxy)phenyl]amino)-40-O-demethyl-
4.1.23. 4b
-(N-[4-aminophenyl]amino)-40-O-demethyl-4-
4-desoxypodophyllotoxin (23a)
desoxypodophyllotoxin hydrochloride (25)
Yellow solid (84%), mp 126.1e127.3 ꢀC; [R]D25e100.3 (c ¼ 0.1, CHCl3);
Compound 24 (2.14 g, 3.63 mmol) was dissolved in saturated
HCl/EtOAc (50 mL) and then stirred at room temperature for 5 h.
The mixture was filtered, the filtration cake was dried overnight to
obtain the desired product 25, yellow solid (95.7%), mp
1H NMR (500 MHz, CDCl3)
d
6.83 (d, J ¼ 7.0 Hz, 2H, 300,500-H), 6.76 (s,1H,
5-H), 6.52 (s,1H, 8-H), 6.49 (d, J ¼ 7.0 Hz, 2H, 200,600-H), 6.33 (s, 2H, 20,60-
H), 5.95 and 5.96 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.58 (m, 2H, 4-H,1-
H), 4.34 (t, J ¼ 8.0 Hz,1H,11-H), 4.02 (m, 3H,11-H, eOCH2CH2OH), 3.92
(t, J ¼ 4.5 Hz, 2H, eOCH2CH2OH), 3.79 (s, 6H, 30,50-OCH3), 3.14 (dd,
J ¼ 5.0,14.0 Hz,1H, 2-H), 2.96 (m,1H, 3-H); 13C NMR (125 MHz, CDCl3)
183.3e186.3 ꢀC; 1H NMR (500 MHz, DMSO)
d 9.93 (s, 1H), 7.11e7.13
(m, 2H), 6.76e6.77 (m, 3H), 6.54 (s, 1H), 6.25 (m, 2H), 6.33 (s, 2H),
5.96 and 5.99 (d, J ¼ 10.5 Hz, 2H), 4.85 (m, 1H), 4.50e4.51 (d,
J ¼ 5.0 Hz, 1H), 4.33e4.35 (m, 2H), 3.64 (s, 6H), 3.27e3.28 (dd,
J ¼ 5.0, 14.5 Hz, 1H), 2.96e3.03 (m, 1H).
d
175.01, 151.52, 148.16, 147.54, 146.48, 142.17, 134.12, 131.81, 130.96,
130.70, 116.17, 113.38, 109.92, 109.05, 108.24, 101.51, 70.13, 69.01, 61.59,
56.50, 53.37, 43.42, 41.91, 38.79; HR-MS (ESIþ) calculated for
C29H29NO9 [M þ 1]þ: 536.1876, found: m/z ¼ 536.1875 [M þ 1]þ
.
4.1.24. General procedure for the synthesis of 26
To a solution of compound 25 (60 mg, 0.1 mmol), the corre-
sponding acid (0.1 mmol) and HOBt (15 mg, 0.11 mmol) in dry
dichloromethane, triethylamine and EDC$HCl (21 mg, 0.11 mmol)
were added at 0 ꢀC, this mixture was stirred at room temperature
until the starting material disappeared. The reaction mixture was
diluted with water and extracted with dichloromethane
(10 mL ꢁ 3). Combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The crude product was purified by flash
column chromatography on silica gel using PE/EtOAc (1:1, v/v) to
give 26.
4.1.19. 4b
-(N-[4-[(2-hydroxyethoxy)methyl]phenyl]amino)-40-O-
demethyl-4-desoxypodophyllotoxin (23b)
Yellow solid (61%), mp 73.6e74.9 ꢀC; [
a]
25e57.4 (c ¼ 0.1, CHCl3);
D
1H NMR (500 MHz, CDCl3)
d
7.20 (d, J ¼ 8.5 Hz, 2H, 300,500-H), 6.76 (s,
1H, 5-H), 6.52 (m, 3H, 200,600,8-H), 6.33 (s, 2H, 20,60-H), 5.96 (m, 2H,
eOCH2Oe), 4.66 (m, 1H, 1-H), 4.60 (d, J ¼ 5.5 Hz, 1H, 4-H), 4.47 (s,
2H, Ph-CH2eO), 4.38 (m, 1H, 11-H), 3.97 (t, J ¼ 4.5 Hz, 2H,
eOCH2CH2OH), 3.80 (s, 6H, 30,50-OCH3), 3.75 (t, J ¼ 6.0 Hz, 2H,
eOCH2CH2CH2OH), 3.65 (t, J ¼ 6.0 Hz, 2H, eOCH2CH2CH2OH), 3.12
(dd, J ¼ 5.0, 13.5 Hz, 1H, 2-H), 3.00 (m, 1H, 3-H), 1.85 (t, J ¼ 6.0 Hz,
2H, eOCH2CH2CH2OH); 13C NMR (125 MHz, CDCl3)
d
175.33,149.09,
4.1.25. 4b
-[N-(4000-methoxy-phenyl-1000-carbonyl)-400-
148.55, 147.88, 147.47, 146.78, 134.40, 133.17, 132.21, 130.88, 130.17,
129.93, 128.59, 127.92, 115.31, 112.45, 110.21, 109.45, 108.37, 101.91,
74.87, 72.78, 69.37, 66.72, 62.08, 58.71, 56.75, 44.01, 42.22, 37.80;
HR-MS (ESIþ) calculated for C30H31NO9 [M þ 1]þ: 550.2032, found:
m/z ¼ 550.2025 [M þ 1]þ.
aminoanilino]-40-O-demethyl-4-desoxypodophyllotoxin (26a)
Yellow solid (54.5%), mp 143.8e145.7 ꢀC; IR(KBr, cmꢃ1) 3384,
3379, 3060, 3036, 2960, 2932, 2875, 1771, 1728, 1647, 1607, 1515,
1482, 1464, 1253, 1228, 1115, 1036; [
a
]
25e125.7 (c ¼ 0.1, CHCl3); 1
H
D
NMR (500 MHz, CDCl3)
d
7.82 (d, J ¼ 8.5 Hz, 2H, 2000,6000-H), 7.65 (s,
1H, eCONHe), 7.44 (d, J ¼ 8.0 Hz, 2H, 300,500-H), 6.96 (d, J ¼ 8.5 Hz,
3000,5000-H), 6.76 (s, 1H, 5-H), 6.55 (d, J ¼ 9.0 Hz, 2H, 200,600-H), 6.52
(s, 1H, 8-H), 6.34 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q, J ¼ 1.0 Hz,
2H, eOCH2Oe), 4.69 (m, 1H, 1-H), 4.61 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.39
(t, J ¼ 8.0 Hz, 1H, 11-H), 4.02 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.91 (s, 3H,
eOCH3), 3.83 (s, 6H, 30,50-OCH3), 3.16 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-H),
4.1.20. 4b
-(N-[4-[(3-hydroxypropoxy)methyl]phenyl]amino)-40-O-
demethyl-4-desoxypodophyllotoxin (23c)
Yellow solid (64%), mp 101.4e103.7 ꢀC; [
a
]
D
25e92.5 (c ¼ 0.1,
CHCl3); 1H NMR (500 MHz, CDCl3)
d
6.83 (d, J ¼ 7.0 Hz, 2H, 300,500-H),
6.76 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.49 (d, J ¼ 7.0 Hz, 2H, 200,600-H),
6.33 (s, 2H, 20,60-H), 5.95 and 5.96 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe),
4.58 (m, 2H, 4-H, 1-H), 4.34 (t, J ¼ 8.0 Hz, 1H, 11-H), 4.02 (m, 3H, 11-
H, eOCH2CH2OH), 3.92 (t, J ¼ 4.5 Hz, 2H, eOCH2CH2OH), 3.79 (s, 6H,
30,50-OCH3), 3.14 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-H), 2.96 (m, 1H, 3-H);
HR-MS (ESIþ) calculated for C31H33NO9 [M þ 1]þ: 564.2189, found:
m/z ¼ 564.2195 [M þ 1]þ.
3.04 (m, 1H, 3-H); 13C NMR (125 MHz, CDCl3)
d 175.04, 162.46,
148.33, 147.68, 146.56, 144.70, 134.18, 132.54, 131.99, 131.02, 130.75,
129.34, 128.95, 127.25, 122.81, 114.02, 112.64, 110.01, 109.27, 108.03,
101.65, 69.08, 68.28, 56.60, 55.58, 52.97, 43.53, 42.03, 38.83; HR-MS
(ESIþ) calculated for C35H32N2O9 [M þ 1]þ: 625.2141, found: m/
z ¼ 625.2135 [M þ 1]þ
.

294
L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
4.1.26. 4
b
-[N-(4000-fluoro-phenyl-1000-carbonyl)-400-aminoanilino]-
4.1.30. 4b
-[N-(2000-nitro-phenyl-1000-ethylcarbonyl)-400-
40-O-demethyl-4-desoxypodophyllotoxin (26b)
aminoanilino]-40-O-demethyl-4-desoxypodophyllotoxin (26f)
Brown solid (31.9%), mp 158.3e159.2 ꢀC; [R]D25e133.3 (c ¼ 0.1,
Yellow solid (57%), mp 157.2e158.9 ꢀC; IR(KBr, cmꢃ1) 3373, 2960,
2930, 1770, 1726, 1643, 1604, 1515, 1482, 1227, 1160, 1115, 1038;
CHCl3); 1H NMR (500 MHz, CDCl3)
d
8.15 (d, J ¼ 9.0 Hz, 2H, 3000,6000-H),
[
a
]
25e77.5 (c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.86 (m, 2H,
7.41 (d, J ¼ 9.0 Hz, 2H, 4000,5000-H), 6.91 (s, 1H, eCONHe), 6.73 (s, 1H, 5-
H), 6.53 (s,1H, 8-H), 6.48 (d, J ¼ 8.5 Hz, 2H, 200,600-H), 6.33 (s, 2H, 20,60-
H), 5.95 and 5.97 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.62 (m, 1H, 1-H),
4.59 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.34 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.95 (t,
J ¼ 8.0 Hz, 11-H), 3.79 (s, 6H, 30,50-OCH3), 3.15 (t, J ¼ 7.5 Hz, 2H,
eNHCOCH2CH2-Ph), 3.11 (dd, J ¼ 5.0,14.0 Hz,1H, 2-H), 2.99 (m,1H, 3-
H), 2.66 (t, J ¼ 7.5 Hz, 2H, eNHCOCH2CH2-Ph); HR-MS (ESIþ) calcu-
lated for C36H33N3O10 [M þ 1]þ: 668.2199, found: m/z ¼ 668.2190
[M þ 1]þ.
D
2000,6000-H), 7.66 (s, 1H, eCONHe), 7.44 (d, J ¼ 8.5 Hz, 2H, 300,500-H), 7.14
(t, J ¼ 8.5 Hz, 3000,5000-H), 6.76 (s,1H, 5-H), 6.55 (d, J ¼ 9.0 Hz, 2H, 200,600-
H), 6.52 (s, 1H, 8-H), 6.34 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q,
J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.66 (m,1H,1-H), 4.59 (d, J ¼ 5.5 Hz,1H, 4-
H), 4.36 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.98 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.85 (br,
1H, eCONH), 3.79 (s, 6H, 30,50-OCH3), 3.13 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-
H), 3.00 (m, 1H, 3-H); HR-MS (ESIþ) calculated for C34H29FN2O8
[M þ 1]þ: 613.1941, found: m/z ¼ 613.1935 [M þ 1]þ
.
4.1.27. 4
b
-[N-(4000-acetyloxyl-phenyl-1000-carbonyl)-400-
4.1.31. 4b
-[N-(phenyl-1000-vinylcarbonyl)-400-aminoanilino]-40-O-
aminoanilino]-40-O-demethyl-4-desoxypodophyllotoxin (26c)
Yellow solid (28.1%), mp 182.2e184.5 ꢀC; IR(KBr, cmꢃ1) 3420,
2909, 1759, 1651, 1605, 1516, 1482, 1371, 1311, 1227, 1199, 1162, 1037;
demethyl-4-desoxypodophyllotoxin (26g)
Yellow solid (29.8%), mp 153.7e155.2 ꢀC; [
a
]
D
25e144.0 (c ¼ 0.1,
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.73 (m, 1H, eCH]CH), 7.54 (d,
[
a
]
25e105.6 (c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.87 (d,
J ¼ 5.0 Hz, 2H, 2000,6000-H), 7.47 (d, J ¼ 5.5 Hz, 2H, 300,500-H), 7.39 (m, 3H,
3000,4000,5000-H), 7.15(s,1H, eCH]CH), 6.77(s,1H, 5-H), 6.57 (s,1H, 8-H),
6.54 (m, J ¼ 4.0 Hz, 2H, 200,600-H), 6.35 (s, 2H, 20,60-H), 5.97 and 5.98
(AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.66 (m,1H,1-H), 4.60 (d, J ¼ 4.5 Hz,
1H, 4-H), 4.37 (t, J ¼ 8.0 Hz, 1H, 11-H), 4.00 (t, J ¼ 8.0 Hz, 11-H), 3.80
(s, 6H, 30,50-OCH3), 3.13 (dd, J ¼ 5.0, 14.0 Hz,1H, 2-H), 3.01 (m, 1H, 3-
D
J ¼ 8.5 Hz, 2H, 2000,6000-H), 7.71 (s, 1H, eCONHe), 7.44 (d, J ¼ 8.5 Hz,
2H, 300,500-H), 7.20 (d, J ¼ 8.5 Hz, 3000,5000-H), 6.76 (s, 1H, 5-H), 6.55 (d,
J ¼ 9.0 Hz, 2H, 200,600-H), 6.53 (s, 1H, 8-H), 6.34 (s, 2H, 20,60-H), 5.95
and 5.97 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.65 (m, 1H, 1-H), 4.58 (d,
J ¼ 4.5 Hz, 1H, 4-H), 4.36 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.98 (t, J ¼ 8.0 Hz,
1H, 11-H), 3.85 (br, 1H, eCONH), 3.79 (s, 6H, 30,50-OCH3), 3.12 (dd,
J ¼ 5.0, 14.0 Hz, 1H, 2-H), 2.98 (m, 1H, 3-H). 2.33 (s, 3H, eCOCH3);
H); 13C NMR (125 MHz, CDCl3)
d 175.19,164.11,148.46,147.80,146.69,
144.79, 141.98, 134.99, 134.30, 131.18, 130.88, 129.10, 128.14, 122.48,
121.20, 112.73, 109.39, 108.16, 101.77, 72.06, 69.19, 65.84, 56.73,
53.03, 43.66, 42.13, 38.92; HR-MS (ESIþ) calculated for C36H32N2O8
[M þ 1]þ: 621.2192, found: m/z ¼ 621.2187 [M þ 1]þ.
13C NMR (125 MHz, CDCl3)
d 174.9, 169.1, 164.9, 153.2, 148.3, 147.6,
146.5, 144.8, 134.2, 132.6, 131.9, 130.7, 130.6, 128.9, 128.5, 122.8,
121.9, 112.5, 109.9, 109.2, 108.0, 101.5, 68.9, 56.4, 52.8, 41.9, 38.7,
21.1; HR-MS (ESIþ) calculated for C36H32N2O10 [M þ 1]þ: 653.2091,
found: m/z ¼ 653.2085 [M þ 1]þ
.
4.1.32. 4b
-[N-(4000-methoxy-phenyl-1000-ethylcarbonyl)-400-
aminoanilino]-40-O-demethyl-4-desoxypodophyllotoxin (26h)
4.1.28. 4
b
-[N-(4000-nitro-phenyl-1000-carbonyl)-400-aminoanilino]-40-
Greyewhite solid (71.8%), mp 149.4e151.2 ꢀC; IR(KBr, cmꢃ1
)
O-demethyl-4-desoxypodophyllotoxin (26d)
3469, 2935, 2838, 1771, 1646, 1611, 1514, 1482, 1228, 1114, 1036;
Orange solid (43.6%), mp 230.1e231.7 ꢀC; IR(KBr, cmꢃ1) 3413,
2964, 2848, 1772, 1642, 1602, 1515, 1481, 1229, 1107, 1038;
[
a
]
25e95.5 (c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.2400(m, 2H,
D
2000,6000-H), 7.16 (d, J ¼ 8.5 Hz, 2H, 300,500-H), 6.84 (m, 3H, 300,4 ,500-H),
6.74 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.48 (d, J ¼ 8.0 Hz, 2H, 200,600-H),
6.33 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe),
4.62 (m, 1H, 1-H), 4.59 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.34 (t, J ¼ 8.0 Hz, 1H,
11-H), 3.96 (t, J ¼ 8.0 Hz,11-H), 3.79 (m, 9H, 30,50-OCH3, eOCH3), 3.11
(dd, J ¼ 5.0,14.0 Hz,1H, 2-H), 3.00 (m,1H, 3-H), 2.98 (t, J ¼ 7.5 Hz, 2H,
eNHCOeCH2CH2-Ph), 2.59 (t, J ¼ 7.5 Hz, 2H, eNHCOeCH2CH2-Ph);
HR-MS (ESIþ) calculated for C37H36N2O9 [M þ 1]þ: 653.2454, found:
[
a
]
25e97.1 (c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d 8.34 (d,
D
J ¼ 8.5 Hz, 2H, 2000,6000-H), 8.03 (d, J ¼ 8.5 Hz, 2H, 3000,5000-H), 7.70 (s,
1H, eCONHe), 7.47 (d, J ¼ 8.0 Hz, 2H, 300,500-H), 6.77 (s, 1H, 5-H),
6.58 (d, J ¼ 9.0 Hz, 2H, 200,600-H), 6.54 (s, 1H, 8-H), 6.34 (s, 2H, 20,60-
H), 5.97 and 5.98 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.68 (m, 1H, 1-H),
4.61 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.37 (t, J ¼ 8.0 Hz, 1H, 11-H), 3.99 (t,
J ¼ 8.0 Hz, 11-H), 3.87 (br, 1H, eCONHe), 3.79 (s, 6H, 30,50-OCH3),
3.13 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-H), 3.00 (m, 1H, 3-H); HR-MS (ESIþ)
calculated for C34H29N3O10 [M þ 1]þ: 640.1886, found: m/
z ¼ 640.1892 [M þ 1]þ.
m/z ¼ 653.2460 [M þ 1]þ
.
4.1.33. 4b
-[N-(4000-bromo-phenyl-1000-carbonyl)-400-aminoanilino]-
40-O-demethyl-4-desoxypodophyllotoxin (26i)
4.1.29. 4
b
-[N-(phenyl-1000-ethylcarbonyl)-400-aminoanilino]-40-O-
Purple solid (46.8%), mp 160.9e162.6 ꢀC; [
a
]
25e46.9 (c ¼ 0.1,
D
demethyl-4-desoxypodophyllotoxin (26e)
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.74 (d, J ¼ 8.5 Hz, 2H, 2000,6000-
Yellow solid (60.6%), mp 156.8e158.5 ꢀC; IR(KBr, cmꢃ1) 3473,
H), 7.62 (d, J ¼ 8.5 Hz, 2H, 3000,5000-H), 7.45 (d, J ¼ 9.0 Hz, 2H, 300,500-H),
6.77 (s, 2H, eCONHe), 6.56 (d, J ¼ 8.5 Hz, 2H, 200,600-H), 6.34 (s, 2H,
20,60-H), 5.97 and 5.99 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.67 (m, 1H,
1-H), 4.61 (m, 1H, 4-H), 4.40 (m, 1H, 11-Ha), 3.96 (m, 1H, 11-Hb),
3.80 (s, 6H, 30,50-OCH3), 3.14 (m, 1H, 2-H), 3.00 (m, 1H, 3-H); 13C
2907, 1771, 1642, 1603, 1515, 1482, 1227, 1114, 1037; [R]2D5e84.27
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
8.15 (d, J ¼ 9.0 Hz, 2H,
3000,6000-H), 7.41 (d, J ¼ 9.0 Hz, 2H, 4000,5000-H), 6.91 (s, 1H, eCONHe),
6.73 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.48 (d, J ¼ 8.5 Hz, 2H, 200,600-H),
6.33 (s, 2H, 20,60-H), 5.95 and 5.97 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe),
4.62 (m, 1H, 1-H), 4.59 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.34 (t, J ¼ 8.0 Hz, 1H,
11-H), 3.95 (t, J ¼ 8.0 Hz, 11-H), 3.79 (s, 6H, 30,50-OCH3), 3.15 (t,
J ¼ 7.5 Hz, 2H, eNHCOCH2CH2-Ph), 3.11 (dd, J ¼ 5.0,14.0 Hz,1H, 2-H),
2.99 (m, 1H, 3-H), 2.66 (t, J ¼ 7.5 Hz, 2H, eNHCOCH2CH2-Ph); 13C
NMR (125 MHz, CDCl3)
d 175.23, 165.07, 148.53, 147.86, 146.76,
145.13, 144.00, 134.37, 134.15, 132.18, 131.24, 130.89, 129.13, 128.95,
126.59, 123.68, 122.72, 115.73, 112.76, 108.22, 105.46, 101.84, 69.23,
58.74, 56.78, 53.09, 43.71, 40.34, 38.98; HR-MS (ESIþ) calculated for
C34H29BrN2O8 [M þ 1]þ: 674.1087, found: m/z ¼ 674.1082 [M þ 1]þ.
NMR (125 MHz, CDCl3)
d 174.96, 170.37, 148.22, 147.56, 146.48,
144.52, 140.80, 134.11, 131.86, 130.65, 128.94, 128.61, 128.43, 126.33,
122.49, 122.17, 115.38, 112.41, 109.90, 109.13, 107.98, 101.53, 68.96,
56.50, 52.82, 43.42, 41.89, 39.20, 38.69, 31.72; HR-MS (ESIþ) calcu-
lated for C36H34N2O8 [M þ 1]þ: 623.2349, found: m/z ¼ 623.2355
[M þ 1]þ.
4.1.34. 4b
-[N-(4000-chlorophenyl-2000-carbonyl)-400-aminoanilino]-40-
O-demethyl-4-desoxypodophyllotoxin (26j)
Purple solid (14.3%), mp 165.6e167.2 ꢀC; [
a]
D
25e63.1 (c ¼ 0.1,
CHCl3); 1H NMR (500 MHz, CDCl3)
d
7.82 (d, J ¼ 8.5 Hz, 2H, 2000,6000-H),
7.64 (d, J ¼ 8.5 Hz, 2H, 3000,5000-H), 7.44 (d, J ¼ 9.0 Hz, 2H, 300,500-H), 6.76

L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
295
(m, 1H, 5-H, eCONHe), 6.55 (d, J ¼ 8.5 Hz, 2H, 200,600-H), 6.34 (s, 2H,
20,60-H), 5.95 and 5.96 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.67 (m, 1H,
1-H), 4.60 (m, 1H, 4-H), 4.39 (m, 1H, 11-Ha), 3.99 (m, 1H, 11-Hb), 3.80
(s, 6H, 30,50-OCH3), 3.14 (m, 1H, 2-H), 3.00 (m, 1H, 3-H); 13C NMR
Rodent Laboratory Animal Resource, Shanghai Branch, China).
Treatments were initiated when tumors reached a mean group size
of about 100e200 mm3. Nude mices were administered intra-
gastrically with 26c (50 mg/kg, 5 mg/mL in 0.9% sterile sodium
chloride solution) two or three times a week for 20 days.
Compounds 23a (10 mg/kg, 2 mg/mL in 0.9% sterile sodium chlo-
ride solution) and 26e (10 mg/kg, 2 mg/mL in 0.9% sterile sodium
chloride solution) were administered intramuscularly once every
day for 20 days. Mice weight and tumor volume were recorded
every other day until animals were sacrificed.
All experiments were approved by an internal ethical committee
and in accordance with the institutional guidelines. Tumor volume
(mm3) was measured with calipers and calculated as (W2 ꢁ L)/2,
where W is the width and L is the length. The T/C% value was
determined by calculating relative tumor volume (RTV) as T/C% ¼
100 ꢁ (mean RTV of treated group)/(mean RTV of control group).
The tumor growth inhibition rate was calculated by using the
formula IR (%) ¼ (1 ꢃ TWt/TWc) ꢁ 100, where TWt and TWc were
the mean tumor weight of treatment and control groups.
(125 MHz, CDCl3)
d 175.25, 164.98, 148.57, 147.90, 146.80, 145.17,
144.03, 138.19, 134.42, 133.67, 132.21, 130.86, 129.31, 128.81, 123.11,
122.75, 115.77, 112.80, 110.22, 109.46, 108.26, 101.86, 69.26, 60.75,
58.76, 56.81, 53.12, 43.74, 42.23, 39.00; HR-MS (ESIþ) calculated for
C34H29ClN2O8 [M þ 1]þ: 629.1646, found: m/z ¼ 629.1650 [M þ 1]þ.
4.1.35. 4
b
-[N-(4000-nitro-phenyl-1000-carbonyl)-400-aminoanilino]-
[N-(40000-nitro-phenyl-10000-carbonyl)]-40-O-demethyl-4-
desoxypodophyllotoxin (26k)
According to the procedure used to prepare 26a, reaction of 25
(60 mg, 0.1 mmol) with 4-nitrobenzoic acid (33 mg, 0.2 mmꢃo1l)
provided 26k, red solid (37.7%), mp 217e220 ꢀC; IR(KBr, cm
)
3471, 3110, 2940, 2907, 1774, 1749, 1643, 1601, 1515, 1482, 1348,
1
1265, 1129, 1037; [
CDCl3)
a
]
D
25e72.7 (c ¼ 0.1, CHCl3); H NMR (500 MHz,
d
8.32 (m, 8H, 2000,3000,5000,6000,20000,30000,50000,60000-H), 8.03 (d,
J ¼ 8.5 Hz, 2H, 300,500-H), 7.78(s,1H, eCONHe), 7.48 (d, J ¼ 8.0 Hz, 2H,
200,600-H), 6.78 (s, 1H, 5-H), 6.58 (d, J ¼ 8.5 Hz, 2H, 20,60-H), 6.57 (s,
1H, 8-H), 5.98 and 5.99 (AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.69 (m,
1H, 1-H), 4.67 (d, J ¼ 5.0 Hz, 1H, 4-H), 4.41 (t, J ¼ 8.0 Hz, 1H, 11-H),
4.00 (t, J ¼ 8.0 Hz, 11-H), 3.71 (s, 6H, 30,50-OCH3), 3.19 (dd, J ¼ 5.0,
14.0 Hz, 1H, 2-H), 3.00 (m, 1H, 3-H); HR-MS (ESIþ) calculated for
4.3. Retention index
4.3.1. Chromatographic conditions
The RP-HPLC analysis was performed on an Agilent 1200 Series
instrumentfittedwith an Eclipse PlusC18 (5
m
m, 4.6 mm ꢁ 250 mm).
C41H32N4O18 [M þ 1]þ: 789.1999, found: m/z ¼ 789.1995 [M þ 1]þ
.
The optimum separation in HPLC was carried out with a mobile
phase composed of 1% HOAc: acetonitrile (45:55, v/v) at a flow-rate
4.1.36. 4b
-[N-(4000-chloro-phenyl-1000-carbonyl)-400-aminoanilino]-
of 1.0 mL/min. The volume of sample injected was 20 mL, the column
[N-(40000-chloro-phenyl-10000-carbonyl)]-40-O-demethyl-4-
temperature was set at 25 ꢀC, and the elution was detected at
254 nm, according to the UV absorption of the sample.
desoxypodophyllotoxin (26l)
According to the procedure used to prepare 26a, reaction of 25
(60 mg, 0.1 mmol) with 4-chlorobenzoic acid (31 mg, 0.2 mmol)
4.3.2. RI evaluation
provided 26l, brown solid (17.4%), mp 143.1e145 ꢀC; [ 25e62.8
a]
D
Each of the compounds 9 and 23aed was dissolved in acetoni-
trile (0.1 mg/mL) and then injected into the HPLC apparatus. The
retention time of these five compounds was recorded. The capacity
factors (k0x) and RI values of the samples were calculated using the
equations as follows:
(c ¼ 0.1, CHCl3); 1H NMR (500 MHz, CDCl3)
d
8.13 (d, J ¼ 9.0 Hz, 2H,
2000,6000-H), 7.81 (m, 2H, 20000,60000-H), 7.44 (m, 8H, 200, 300, 500, 600, 3000, 5000,
3
0000, 50000-H), 6.78 (s, 1H, 5-H), 6.57 (m, 3H, 20,60, 8-H), 5.98 and 6.00
(AB q, J ¼ 1.0 Hz, 2H, eOCH2Oe), 4.68 (m, 2H, 4-H, 1-H), 4.43 (t,
J ¼ 8.0 Hz, 1H, 11-Ha), 4.00 (t, J ¼ 8.0 Hz, 11-Hb), 3.70 (s, 6H, 30,50-
OCH3), 3.19 (dd, J ¼ 5.0, 14.0 Hz, 1H, 2-H), 3.02 (m, 1H, 3-H); HR-MS
(ESIþ) calculated for C41H32ClN2O9 [M þ 1]þ: 768.1455, found:
m/z ¼ 768.1452 [M þ 1]þ.
kx0
¼
ðtx ꢃ t0Þ=t0
À
ÁꢀÀ
Á
RI ¼ 100N þ 100 lg k0x ꢃ lg kn0
lg k0nþ1 ꢃ lg k0n
4.2. Pharmacology
where x ¼ compounds 9 or 23aed measured, n ¼ alkan-2-one
eluting immediately before x, n þ 1 ¼ alkan-2-one eluting imme-
diately after x and N ¼ carbon number of alkan-2-one. The k0 values
were calculated using the retention time of NaNO3 as a dead time.
4.2.1. Cytotoxicity assay
The cytotoxic activity in vitro against several cancer cell lines
was measured by MTT assay. The cancer cell lines included oral
squamous cell carcinoma (KB), vincristine (VCR)-selected MDR-
positive KB/VCR, human non-small cell lung cancer (A549) and
highly metastatic human lung cancer (95D). MTT solution
(2.5 mg/mL in RPIM-1640, Sigma, St. Louis, MO) was added
Acknowledgements
This study was financially supported by the National Key Tech
Project for Major Creation of New drugs (No. 2009ZX09501-003)
and the Fundamental Research Funds for the Central Universities
(No. KYJD038).
(20.0
mL/well), and the plates were incubated for an additional 4 h
at 37 ꢀC. The purple formazan crystals were dissolved in 100.0
m
L
DMSO. After 5 min, the plates were read on a Multiskan Spectrum
(Thermo Electron Corporation, Marietta, Ohio) at 570 nm. Assays
were performed in triplicate on three independent experiments.
The concentration of drug inhibit for 50% of cells (IC50) was
calculated using the software of Dose-Effect Analysis with
Microcomputers.
References
[1] D. Guianvarc’h, M. Duca, C. Boukarim, L. Kraus-Berthier, S. Leonce, A. Pierre,
B. Pfeiffer, P. Renard, P.B. Arimondo, C. Monneret, D. Dauzonne, J. Med. Chem.
47 (2004) 2365e2374.
[2] E.L. Baldwin, N. Osheroff, Curr. Med. Chem.: Anti-Cancer Agents 5 (2005)
363e372.
[3] A. Kamal, B.A. Kumar, M. Arifuddin, Tetrahedron Lett. 44 (2003) 8457e8459.
[4] J.L. Nitiss, Nat. Rev. Cancer 9 (2009) 338e350.
[5] J. Yang, A. Bogni, E.G. Schuetz, M. Ratain, M.E. Dolan, H. McLeod, L. Gong,
C. Thorn, M.V. Relling, T.E. Klein, R.B. Altman, Pharmacogenet. Genom. 19
(2009) 552e553.
4.2.2. Antitumor activity in 95D bearing mice
95D highly metastatic human lung cancer model was estab-
lished by injection of a highly metastatic human lung cancer 95D
cells (1 ꢁ107 cells per animal, subcutaneously into the armpit of the
nude mice) to 4- to 5-week-old BALB/C female nude mice (National
[6] Y.J. You, Curr. Pharm. Des. 11 (2005) 1695e1717.

296
L. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 285e296
[7] I. Rassmann, T. Schilling, H. Schrodel, A. KaeserFrohlich, K. Burk, J. Rastetter,
A.R. Hanauske, Eur. J. Cancer 31A (1995) 111.
[14] C.Q. Hu, D.Q. Xu, W.T. Du, S.J. Qian, L. Wang, J.S. Lou, Q.J. He, B. Yang, Y.Z. Hu,
Mol. Biosyst. 6 (2010) 410e420.
[8] T.S. Huang, C.H. Shu, W.K. Yang, J. WhangPeng, Cancer Res. 57 (1997)
2974e2978.
[15] D.H. Huh, J.S. Jeong, H.B. Lee, H. Ryu, Y.G. Kim, Tetrahedron 58 (2002)
9925e9932.
[9] T. Utsugi, J. Shibata, Y. Sugimoto, K. Aoyagi, K. Wierzba, T. Kobunai, T. Terada,
T. OhHara, T. Tsuruo, Y. Yamada, Cancer Res. 56 (1996) 2809e2814.
[10] S.Z. Fields, L.N. Igwemezie, S. Kaul, L.P. Schacter, R.J. Schilder, P.P. Litam,
B.S. Himpler, C. Mcaleer, J. Wright, R.H. Barbhaiya, C.J. Langer, P. Odwyer, Clin.
Cancer Res. 1 (1995) 105e111.
[16] R.J. Gentile, R.J. Murray, J.E. MacDonald, W.C. Shakespeare, WO 9505363 A1.
[17] Q.Y. Liu, H.T. Xi, Y. Jiang, X.Q. Sun, Huaxue Gongye Yu Gongcheng 21 (2004)
425e428.
[18] M.R. Pitts, J.R. Harrison, C.J. Moody, J. Chem. Soc., Perkin Trans. 1 (2001)
955e977.
[11] C. Etievant, A. Kruczynski, J.M. Barret, D. Perrin, B.T. Hill, Biochem. Pharmacol.
65 (2003) 755e763.
[12] R.M. Moreas, F.E. Dayan, C. Canel, Stud. Nat. Prod. Chem. 26 (2002) 149e182.
[13] S.J. Cho, A. Tropsha, M. Suffness, Y.C. Cheng, K.H. Lee, J. Med. Chem. 39 (1996)
1383e1395.
[19] Y.Z. Hu, W.T. Du, Q.J. He, B. Yang, X.C. Yang, CN 101074233.
[20] Z.Y. Xiao, K.F. Bastow, J.R. Vance, R.S. Sidwell, H.K. Wang, M.S. Chen, Q. Shi,
K.H. Lee, J. Med. Chem. 47 (2004) 5140e5148.
[21] J.K. Baker, R.E. Skelton, T.N. Riley, J.R. Bagley, J. Chromatogr. Sci. (1980)
153e158.