Design, synthesis and potent cytotoxic activity of novel podophyllotoxin derivatives

Article abstract of DOI:10.1016/j.bmc.2013.01.069

Twenty new acyl thiourea derivatives of podophyllotoxin and 4′-demethylepipodophyllotoxin were prepared and screened for their cytotoxicity against four human tumor cell lines, A-549, DU-145, KB, and KBvin. With IC₅₀ values of 0.098-1.13 μM, co

Full text of DOI:10.1016/j.bmc.2013.01.069

Bioorganic & Medicinal Chemistry 21 (2013) 2363–2369
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and potent cytotoxic activity of novel
podophyllotoxin derivatives
b
d
a
Wen-Qun Li a, , Xu-Li Wang a, , Keduo Qian b, Ying-Qian Liu a, , Chih-Ya Wang , Liu Yang , Jin Tian ,
⇑
Susan L. Morris-Natschke b, Xing-Wen Zhou a, Kuo-Hsiung Lee b,c,
⇑
a School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
d Environmental and Municipal Engineering School, Lanzhou Jiaotong University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Twenty new acyl thiourea derivatives of podophyllotoxin and 40-demethylepipodophyllotoxin were pre-
pared and screened for their cytotoxicity against four human tumor cell lines, A-549, DU-145, KB, and
Article history:
Received 8 November 2012
Revised 18 January 2013
Accepted 28 January 2013
Available online 17 February 2013
KBvin. With IC50 values of 0.098–1.13
activity than the control etoposide. Most importantly, 13b and 13o exhibited promising cytotoxicity
against the drug resistant tumor cell line KBvin with IC50 values of 0.098 and 0.13 M, respectively, while
lM, compounds 13b, 13c, and 13o displayed much better cytotoxic
l
etoposide lost activity completely. Structure–activity relationship (SAR) correlations of the new deriva-
tives have been established. Compounds 13b and 13o merit further development as a new generation
of epipodophyllotoxin-derived antitumor clinical trial candidates.
Keywords:
Podophyllotoxin
Acyl thiourea
Synthesis
Ó 2013 Elsevier Ltd. All rights reserved.
Cytotoxic activity
1. Introduction
epipodophyllotoxin system led to two clinical anticancer drugs,
etoposide (3) and teniposide (4), as well as a water-soluble prodrug
Topoisomerases are enzymes that control the changes in DNA
structure by catalyzing the breaking and rejoining of the phospho-
diester backbone of DNA strands during the normal cell cycle.
Topoisomerases are classified into two categories. Monomeric type
I enzymes catalyze the formation of DNA single-strand breaks,
while multimeric type II enzymes engender DNA double-strand
breaks. Topoisomerase-II is highly involved in DNA replication,
transcription, chromosome condensation, and de-condensation,
and therefore, represents an important cellular target for clinical
anticancer drugs, such as the epipodophyllotoxins (etoposide and
teniposide), the anthracyclines (daunorubicin and doxorubicin),
and the aminoacridines.
Podophyllotoxin (1) is a naturally occurring aryltetralin lignan
isolated from various plant species within the Podophyllum family.
Its powerful cytotoxic properties have been attributed to its ability
to bind to tubulin during mitosis and thus inhibit microtubule
assembly.1,2 During early chemical modification studies, 40-
demethylepipodophyllotoxin (2) was obtained by 40-O-demethyla-
of 3, etopophos (5) (Fig. 1).3–5 The structural modification also led
to a change in the mechanism of action of these ligands, while 1
acts as antimicrotubule agent, 3 and 4 act as topoisomerase-II
inhibitors.6,7 Their clinical success and intriguing mechanism of ac-
tion have stimulated great interest in further exploration of epip-
odophyllotoxin derivatives with better antitumor activity.
Some nonsugar substituted analogues, particularly N-linked
congeners, such as NPF (6)8 and GL-331(7),9 and C-linked congen-
ers, such as TOP-53 (8) (Fig. 1),10 were then found to exhibit supe-
rior pharmacological properties compared to 3. Among them,
compound 7 was brought into phase II clinical trials.9 Overall,
these variants displayed improved water solubility and cytotoxic
activity, as well as drug resistant and antitumor profiles, suggest-
ing the possibility of further optimizing epipodophyllotoxins
through rational C-4 modifications. Both the composite pharmaco-
phore model and comparative molecular field analysis also con-
firmed that the C-4 molecular area could accommodate
considerable structural diversity.11
tion and stereo transformation of
Investigation of semisynthetic glucoconjugates based on the
a
to b at the C-4 position.
Based on critical modeling clues, we have successfully prepared
several potent epipodophyllotoxin derivatives during the past few
years.2,12–17 In our continuing work in this research area, we dis-
covered that thiourea was a good ligand for metal ions, which play
important roles in tumorigenesis. Moreover, thiourea derivatives
possessed good inhibitory activity against protein tyrosine kinases
⇑
Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.-H.L.).
E-mail addresses: khlee@email.unc.edu (K.-H. Lee), yqliu@lzu.edu.cn (Y.-Q. Liu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.069

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W.-Q. Li et al. / Bioorg. Med. Chem. 21 (2013) 2363–2369
O
R1
O
O
HO
O
HO
O
O
OH
O
O
OH
O
O
O
O
O
O
O
O
H3CO
OCH3
OR2
OCH3
H3CO
R1
R2
H
OH
OCH3
H3CO
3.
4.
Etoposide
Teniposide
CH3
OCH3
2. 4'-Demethylepipodophyllotoxin
1.Podophyllotoxin
H
O
S
Etopophos
P OH
OH
5.
CH3
N
H
HN
F
HN
NO2
O
O
O
O
O
O
O
O
O
O
O
O
OCH3
H3CO
H3CO
OCH3
OCH3
H3CO
OH
OH
OH
6. NPF
8. TOP-53
7. GL331
Figure 1. Structures of podophyllotoxin derivatives.
(PTKs), which also are critical in many aspects of tumorigene-
sis.18,19 Thus, incorporation of a thiourea group could possibly
potentiate biochemical or pharmacological properties of a bioac-
tive original molecule.20,21 Therefore, it was intriguing to introduce
thiourea groups into podophyllotoxins (1 and 1a) via coupling
reactions, in order to investigate the impact on the derivatives’
cytotoxicity against human tumor cell lines. Herein, this paper re-
ports the design, synthesis, and cytotoxic activity of the newly syn-
thesized acyl thiourea epipodophyllotoxin derivatives 13a–o and
14a–e.
including A549 (non-small cell lung cancer), DU145 (prostate can-
cer cell line), KB (nasopharyngeal carcinoma), and KBvin [multi-
drug resistant (MDR) KB subline selected using vincristine], using
a sulforhodamine B colorimetric (SRB) assay with triplicate exper-
iments.24 Compound 3 was included as control and the results are
summarized in Table 1.
Except for compounds 13j–l, 13n, and 14b, most of the new
compounds showed moderate to potent cytotoxic activity against
the four human tumor cell lines. Among them, compounds 13b–e
and 13o showed superior activity with IC50 values of 0.099–
1.44 lM compared to that of 3 (IC50 2.03–3.88 lM) against A549,
DU-145, and KB tumor cell lines. Most importantly, these com-
pounds retained their significant cytotoxicity (IC50 0.098–
2. Results and discussion
2.1. Chemistry
2.01
lM) against the drug resistant KBvin tumor cell line, while 3
lost its activity completely (IC50 >20
lM). This result is in agree-
ment with our prior observation that C4-amino substitution of
epipodophyllotoxin is good for overcoming drug-resistance.25
The 40-O-methylated derivatives (13c, 13e, and 13m–13o) were
more potent than their corresponding 40-hydroxyl analogues (14a–
14e). While 13c and 13o showed significant cytotoxicity with IC50
The synthetic route to target compounds 13a–o and 14a–e is
depicted in Scheme 1. The starting materials, 1 and 1a, were first
converted to 4b-chloroamido podophyllotoxins (9 and 10) in excel-
lent yields by reaction with ClCH2CN in the presence of 60% w/w
methanesulfonate/aluminium oxide (MsOH/Al2O3). Subsequently,
the chloroacetyl group was cleaved with thiourea in the presence
of acetic acid (HOAc) to furnish the key intermediates 4b-aminop-
odophyllotoxins (11 and 12).22 Intermediates 11 and 12 were then
coupled with the appropriate acylisothiocyanates in dry acetoni-
trile to provide a series of acyl thiourea derivatives 13a–o and
14a–e in 56–95% yields.23 All newly synthesized compounds were
purified by column chromatography and their structures were
characterized by 1H NMR, 13C NMR and HRMS analyses.
values of 0.81–1.13 and 0.099–0.13
lM, respectively, their corre-
sponding 14a and 14e displayed only marginal activity with IC50
values of 8.96–12.00 and 6.55–11.75
demethylation of 13e (IC50 1.16–2.01
l
M, respectively. The 40-O-
lM) led to a complete loss
of cytotoxicity in the corresponding 14b. These results highlight
the critical role of the 40-O-methyl functionality in acyl thiourea
substituted epipodophyllotoxin derivatives.
Within the 40-O-methylated series (13a–o), cyclohexyl (13b),
phenyl (13c), and naphthyl (13o) R2 groups within the acyl thio-
urea side chains led to significantly enhanced cytotoxicity, as these
derivatives displayed IC50 values of 0.098–0.32, 0.81–1.13, and
2.2. Cytotoxicity and SAR
Target compounds 13a–o and 14a–e were evaluated for their
in vitro cytotoxicity against a panel of human tumor cell lines,
0.099–0.13
lM, respectively. Notably, annular aliphatic substi-
tuted 13b, with a cyclohexyl R2 group, exhibited better activity

W.-Q. Li et al. / Bioorg. Med. Chem. 21 (2013) 2363–2369
2365
O
Cl
NH2
HN
OH
O
O
O
O
O
O
NCCH2Cl
thiourea
AcOH
O
O
O
O
O
O
MsOH/Al2O3
OCH3
OCH3
OCH3
H3CO
H3CO
H3CO
OR1
OR1
1
OR1
1. R1=CH3
9
10
11. R1=CH3
. R =CH3
1
1
1
1a
. R =H
. R =H
12
. R =H
S
O
R2
CH3
HN
N
H
CH3
O
R2
N
14a
14b
13a
13b
13f
13k
13l
R2 NCS
O
O
Cl
N
Cl
O
NO2
CH3CN
13g
O
Br
O
S
S
13h
13i
13j
13m
13c
13d
14c
14d
14e
NO2
Cl
F
H3CO
OCH3
O
OR1
13n
13o
N
N
1
13a-o.
R =CH3
Cl
14a-e. R1=H
13e
Scheme 1. Synthesis of 4b-amino-4-desoxypodophyllotoxin (13a–o) and 4b-amino 40-demethyl-desoxypodophyllotoxin (14a–e).
Table 1
thiourea side chains play an important role in the derivatives’ cyto-
toxic activity.
In vitro cytotoxicity of 13a–o and 14a–e against four human tumor cell lines with
etoposide (3) as control
The introduction of heterocyclic groups into the acyl thiourea
side chains of 13i–n resulted in dramatically decreased cytotoxic-
ity. For instance, compounds with furan (13n), thiophene (13m),
and pyridine-containing groups (13i–13l), all exhibited marginal
or no activity against the four human tumor cell lines. This finding
further confirmed that the electron density and distribution of the
substituents within the acyl thiourea side chain is important to-
wards the compounds’ activity.
Compd
IC50
(
l
M)
A549
DU-145
KB
KBvin
13a
13b
13c
13d
13e
13f
13g
13h
13i
13.12 0.246
0.19 0.010
1.13 0.050
1.37 0.050
1.62 0.039
11.31 0.291
2.06 0.095
10.31 0.431
15.47 2.261
>20
12.30 0.176
0.17 0.007
0.97 0.089
1.15 0.059
1.16 0.047
10.52 0.561
1.57 0.094
9.82 0.091
11.40 0.146
>20
13.43 0.121
0.32 0.014
0.90 0.059
1.44 0.049
1.20 0.040
10.48 0.389
1.33 0.075
9.27 0.395
14.20 0.503
>20
13.63 0.664
0.098 0.005
0.81 0.101
1.38 0.051
2.01 0.045
12.25 0.461
1.65 0.071
9.86 0.270
16.84 0.811
>20
3. Conclusion
13j
In summary, new acyl thiourea derivatives of epipodophyllo-
toxin were designed and synthesized successfully, and their cyto-
toxic activity was evaluated against four human tumor cell lines.
Results showed that the C4-acyl thiourea compound retained cyto-
toxicity against the drug resistant KBvin tumor cell line, while eto-
poside (3) lost its activity completely. The best compounds 13b,
13c, and 13o exhibited significant cytotoxicity against all four hu-
13k
13l
>20
>20
>20
>20
>20
>20
>20
>20
13m
13n
13o
14a
14b
14c
14d
14e
3
12.29 0.333
>20
0.11 0.002
12.00 0.214
>20
13.74 1.000
9.82 0.624
11.75 1.317
2.58 0.252
11.19 0.173
>20
0.099 0.004
9.88 1.070
>20
8.49 0.160
7.34 0.303
6.55 0.073
2.03 0.121
11.56 0.306
>20
0.12 0.000
8.96 0.490
>20
8.74 0.508
8.40 0.502
7.23 0.312
3.88 0.199
12.51 0.338
>20
0.13 0.007
9.93 0.966
>20
9.46 0.207
9.24 0.501
10.29 1.267
>20
man tumor cell lines with IC50 values of 0.098–1.13 lM, 20-fold
better than that of the control 3. The SAR study found that the size,
electron density, and distribution of the substituents within the
acyl thiourea side chain are critical to the derivatives’ activity.
With a concise synthesis and potent cytotoxic profiles, the new
acyl thiourea epipodophyllotoxins, especially 13b and 13o, are
promising new candidates for further development as anticancer
agents. Studies on the enzymatic interaction of these new acyl
thiourea epipodophyllotoxins with the topoisomerase-II target
are currently ongoing.
than compound 13a, with a methyl R2 group, which showed only
marginal IC50 values of 12.30–13.63 M. This observation indicates
that the size of the acyl substituent is critical. Compounds with
electron-withdrawing substituents on the phenyl ring (13d–e,
13g) showed similar or slightly reduced activity (IC50 1.15–
l
2.06 lM) compared with non-substituted 13c. An electron-donat-
ing group was detrimental to cytotoxicity, as compound 13f
(para-methylphenyl) had drastically reduced IC50 values of
4. Experimental section
4.1. Chemistry
10.48–12.25
group from the para-position (13g) to the meta-position (13h) also
decreased the cytotoxicity remarkably (IC50 9.27–10.31 M), sug-
gesting that the electron distribution and substituents on benzoyl
lM. The transfer of a electron-withdrawing nitro
l
Melting points were taken on a Kofler melting point apparatus
and are uncorrected. High resolution mass spectroscopic analyses

2366
W.-Q. Li et al. / Bioorg. Med. Chem. 21 (2013) 2363–2369
were performed on ZAB-HS and BrukerDaltonics APEXII49e instru-
ments. 1H and 13C NMR spectra were recorded at 400 MHz and
100 MHz on a Bruker AM-400 spectrometer using TMS as reference
(Bruker Company, USA). Podophyllotoxin (1) was isolated from the
Chinese medicinal herb Juniperus sabina Linnaeus, and served as
the starting material for the preparation of all new derivatives.
(d, 1H, 1-H), 5.82 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.97 and 6.00
(ABq, 2H, –OCH2O–), 6.30 (s, 2H, 20,60-H), 6.54 (s, 1H, 8-H), 6.80
(s, 1H, 5-H), 8.96 (s, 1H, –CSNHCO–), 10.72 (d, 1H, J = 8.0 Hz, 4-
NH); 13C NMR (100 MHz, CDCl3): 24.4, 37.4, 42.1, 43.7, 53.9, 56.2,
60.7, 68.6, 101.7, 108.1, 109.0, 110.2, 127.6, 132.3, 134.6, 137.2,
147.7, 148.7, 152.6, 171.2, 173.9, 180.2. HRMS m/z calcd for
C
25H26N2O8S: 515.1483 [M+H]+, found: 515.1487 [M+H]+.
4.1.1. General synthetic procedure for the key intermediates 4b-
amino-4-desoxypodophyllotoxin (11) and 4b-amino-40-
demethyl-4-desoxypodophyllotoxin (12)
4.1.2.2. 4b-(3-Cyclohexanecarbonyl-thioureido)-4-deoxypodo-
phyllotoxin (13b).
Yield: 78%; mp: 153–157 °C; 1H NMR
To a stirred mixture of podophyllotoxin (4 mmol) and ClCH2CN
(10 mL), a homogeneous mixture of MsOH/Al2O3 (60 mass %, 1 g)
was added, and the mixture was irradiated by an ultrasonic gener-
ator in a water bath at 60 °C for 30 min. The solvent was evapo-
rated under reduced pressure and the residue was poured onto
ice water and extracted with EtOAc (3 ꢀ 50 mL). Combined organic
extracts were dried over anhydrous Na2SO4, the mixture was fil-
tered, and the filtrate was evaporated under reduced pressure to
give the crude product, which was purified by flash column chro-
matography (CHCl3/MeOH, 9.5:0.5) to give the pure intermediate
9 in 90% yield. Subsequently, a solution of amide 9 (5 mmol) and
thiourea (0.46 g, 6 mmol) in HOAc (50 mL) was sonicated at 80 °C
for 4 h, and then the solution was neutralized with aq 20% NaHCO3
and extracted with CHCl3 (3 ꢀ 30 mL). The combined organice ex-
tracts were washed with brine (30 mL) and dried (MgSO4), filtered,
and concentrated in vacuo. The residue was purified by column
chromatography to give compound 11 as a white solid in 82% yield.
Analogously, amide 10 and subsequently amine 12 were prepared
starting from the podophyllotoxin derivative 1a in 93% and 85%
yields, respectively.
(400 MHz, CDCl3): d 1.23–1.93 (m, 10H, 200,300,400,500,600-H), 2.17–
2.21 (m, 1H, 100-H), 2.90 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.06–3.08
(m, 1H, 3-H), 3.76 (s, 6H, 30,50-OCH3), 3.81 (s, 3H, 40-OCH3), 3.94–
3.99 (m, 1H, 11a-H), 4.50–4.54 (m, 1H, 11b-H), 4.63 (d, 1H, 1-H),
5.82 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.98 and 6.00 (ABq, 2H, –
OCH2O–), 6.30 (s, 2H, 20,60-H), 6.53 (s, 1H, 8-H), 6.82 (s, 1H, 5-H),
8.66 (s, 1H, –CSNHCO–), 10.80 (d, 1H, J = 8.0 Hz, 4-NH). 13C NMR
(100 MHz, CDCl3): 25.2, 25.3, 26.9, 28.8, 28.9, 37.5, 42.1, 43.7,
45.8, 53.9, 56.2, 60.7, 68.7, 101.7, 108.1, 109.1, 110.1, 127.6,
132.3, 134.6, 137.2, 147.7, 148.7, 152.6, 173.9, 177.1, 180.6. HRMS
m/z calcd for C30H34N2O8S: 583.2109 [M+H]+, found: 583.2120
[M+H]+.
4.1.2.3. 4b-(3-Benzoyl-thioureido)-4-deoxypodophyllotoxin
(13c).
Yield: 83%; mp: 162–165 °C; 1H NMR (400 MHz,
CDCl3): d 2.96 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.09–3.14 (m, 1H, 3-
H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 4.00–4.06 (t,
1H, 11a-H), 4.53–4.57 (m, 1H, 11b-H), 4.67 (d, 1H, 1-H), 5.89 (dd,
J = 7.6, 4.8 Hz, 1H, 4-H), 5.97 and 6.00 (ABq, 2H, –OCH2O–), 6.33
(s, 2H, 20,60-H), 6.55 (s, 1H, 8-H), 6.87 (s, 1H, 5-H), 7.54 (t, 2H,
300,500-H), 7.66 (t, 1H, 400-H), 7.83 (d, 2H, 200,600-H), 9.11 (s, 1H, –
CSNHCO–), 10.98 (d, 1H, J = 8.0 Hz, 4-NH). 13C NMR (100 MHz,
CDCl3): 37.6, 42.2, 43.8, 54.1, 56.2, 56.3, 60.4, 60.7, 60.8, 68.7,
101.7, 108.2, 109.2, 110.2, 127.5, 127.7, 129.3, 131.2, 132.3,
134.1, 134.6, 137.3, 147.8, 148.8, 152.7, 167.0, 171.1, 173.9,
180.5. HRMS m/z calcd for C30H28N2O8S: 599.1459 [M+Na]+, found:
599.1474 [M+Na]+.
4.1.1.1. 4b-Amino-4-desoxypodophyllotoxin (11).
MS (FAB)
m/z 414 (M+1); 1H NMR (400 MHz, CDCl3): d 6.84 (s, 1H, H-5),
6.47 (s, 1H, H-8), 6.36 (s, 2H, H-20,60), 5.97 (s, 2H, OCH2O), 4.58
(d, J = 5.1 Hz, lH, H-I), 4.32 (m, 2H, H-11), 4.25 (d, J = 4.0 Hz, 1H,
H-4), 3.80 (s, 3H, 40-OCH3), 3.72 (s, 6H, 30,50-OCH3), 3.32 (q, 1H,
H-2), 2.92–2.63 (m, 1H, H-3), 1.84 (d, 2H, 4-NH2); 13C NMR
(CDC13, 100 MHz): d 37.9, 40.2, 43.7, 48.9, 56.4, 68.1, 101.3,
107.9, 108.6, 110.2, 131.1, 131.2, 133.9, 134.1, 146.3, 147.2,
147.6, 175.4. HRMS m/z calcd for C22H23NO7: 414.1366 [M+H]+,
found: 414.1365 [M+H]+.
4.1.2.4. 4b-[3-(4-Fluorobenzoyl)-thioureido]-4-deoxypodo-
phyllotoxin (13d).
Yield: 86%; mp: 161–164 °C; 1H NMR
(400 MHz, DMSO-d6): d 3.07–3.16 (m, 1H, 3-H), 3.21 (dd, J = 14.8,
5.2 Hz, 1H, 2-H), 3.61 (s, 3H, 40-OCH3), 3.66 (s, 6H, 30,50-OCH3),
4.1.1.2.
(12).
4b-Amino-40-demethyl-4-desoxypodophyllotoxin
MS (EI) m/z 400 (M+1); 1H NMR (400 MHz, CDCl3): d
3.99–4.04 (m, 1H, 11a-H), 4.41–4.45 (m, 1H, 11b-H), 4.62 (d, 1H,
1-H), 5.86 (dd, J = 8.0, 4.4 Hz, 1H, 4-H), 6.02 and 6.03 (ABq, 2H, –
OCH2O–), 6.31 (s, 2H, 20,60-H), 6.58 (s, 1H, 8-H), 6.87 (s, 1H, 5-H),
7.33 (t, 2H, 300,500-H), 7.98–8.01 (m, 2H, 200,600-H), 10.92 (d, 1H,
J = 8.0 Hz, 4-NH), 11.54 (s, 1H, –CSNHCO–); 13C NMR (100 MHz,
DMSO-d6): 36.7, 40.6, 42.5, 52.6, 55.3, 59.4, 67.8, 101.0, 107.7,
108.4, 109.2, 114.8, 115.0, 127.9, 128.2, 128.3, 131.1, 131.2,
131.9, 135.0, 135.9, 146.3, 147.3, 151.5, 163.1, 165.6, 166.3,
6.81 (s, 1H, H-5), 6.50 (s, lH, H-8), 6.30 (s, 2H, H-20,60), 5.98 and
5.95 (2s, 2H, OCH2O), 4.56 (d, J = 5.2 Hz, 1H, H-l), 4.30 (d,
J = 10 Hz, 2H, H-11), 4.18 (d, J = 4.0 Hz, 1H, H-4), 3.78 (s, 6H,
30,50-OMe), 3.15 (dd, J = 5.2, 14 Hz, 1H, H-2), 2.80 (m, 1H, H-3);
13C NMR (CDC13, 100 MHz): d 38.2, 40.2, 43.7, 48.9, 56.4, 68.1,
101.3, 107.9, 108.6, 110.2, 131.1, 131.2, 133.9, 134.1, 146.3,
147.3, 147.6, 175.4. HRMS m/z calcd for C21H21NO7: 400.1822
[M+H]+, found: 400.1825 [M+H]+.
173.5, 180.2. HRMS m/z calcd for
C30H27FN2O8S: 617.1364
[M+Na]+, found: 617.1374 [M+Na]+.
4.1.2. General synthetic procedure for compounds 13a–o and
14a–e
4.1.2.5. 4b-[3-(4-Chlorobenzoyl)-thioureido]-4-deoxypodo-
phyllotoxin (13e).
Yield: 85%; mp: 170–173 °C; 1H NMR
The key intermediates 11 and 12 (1 mmol) were added to a
solution of 1.5 mmol of an acyl isothiocyanate in dry acetonitrile
(10 mL). The reaction mixture was stirred for 2 h at rt and then
concentrated. The residue was purified by chromatography on sil-
ica gel using EtOAc–petroleum ether to give 13a–o and 14a–e.
(400 MHz, CDCl3): d 2.96 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.10–3.15
(m, 1H, 3-H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 4.02
(t, 1H, 11a-H), 4.53–4.57 (m, 1H, 11b-H), 4.67 (d, 1H, 1-H), 5.88
(dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.99 and 6.01 (ABq, 2H, –OCH2O–),
6.32 (s, 2H, 20,60-H), 6.56 (s, 1H, 8-H), 6.86 (s, 1H, 5-H), 7.52 (d,
J = 8.8 Hz, 2H, 300,500-H), 7.78 (d, J = 8.8 Hz, 2H, 200,600-H), 9.07 (s,
1H, –CSNHCO–), 10.91 (d, 1H, J = 7.6 Hz, 4-NH); 13C NMR
(100 MHz, CDCl3): 37.5, 42.2, 43.8, 54.2, 56.2, 60.8, 68.7, 101.7,
108.2, 109.1, 110.2, 127.6, 128.9, 129.6, 132.3, 134.6, 137.3,
140.6, 147.8, 148.8, 152.7, 166.0, 173.8, 180.3. HRMS m/z calcd
4.1.2.1. 4b-(3-Acetyl-thioureido)-4-deoxypodophyllotoxin
(13a).
Yield: 75%; mp: 168–172 °C; 1H NMR (40 0 MHz,
CDCl3): d 2.17 (s, 3H, COCH3), 2.28 (dd, J = 14.4, 5.2 Hz, 1H, 2-H),
3.04–3.12 (m, 1H, 3-H), 3.76 (s, 6H, 30,50-OCH3), 3.81 (s, 3H, 40-
OCH3), 3.93–3.99 (m, 1H, 11a-H), 4.50–4.54 (m, 1H, 11b-H), 4.64

W.-Q. Li et al. / Bioorg. Med. Chem. 21 (2013) 2363–2369
2367
for C30H27ClN2O8S: 628.1515 [M+NH4]+, found: 628.1510
[M+NH4]+.
(m, 1H, 3-H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 3.99–
4.04 (m, 1H, 11 -H), 4.52–4.57 (m, 1H, 11b-H), 4.63 (d, 1H, 1-H),
a
5.87 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.98 and 6.01 (ABq, 2H, –
OCH2O–), 6.32 (s, 2H, 20,60-H), 6.57 (s, 1H, 8-H), 6.85 (s, 1H, 5-H),
7.51 (d, J = 8.4 Hz, 1H, 500-H), 8.10 (dd, J = 8.0, 2.4 Hz, 1H, 600-H),
8.88 (d, 1H, J = 2.4 Hz, 200-H), 9.20 (s, 1H, –CSNHCO–), 10.77 (d,
1H, J = 8.0 Hz, 4-NH); 13C NMR (100 MHz, CDCl3): 37.5, 42.2, 43.8,
54.3, 56.3, 60.8, 68.6, 101.8, 108.3, 109.1, 110.3, 124.9, 126.3,
127.5, 132.4, 134.5, 137.4, 137.7, 147.8, 148.9, 152.7, 156.7,
164.6, 173.8, 179.9. HRMS m/z calcd for C29H26ClN3O8S: 634.1021
[M+Na]+, found: 634.1027 [M+Na]+.
4.1.2.6. 4b-[3-(4-Methylbenzoyl)-thioureido]-4-deoxypodo-
phyllotoxin (13f).
Yield: 78%; mp: 162–165 °C; 1H NMR
(400 MHz, CDCl3): d 2.45 (s, 3H, 400-CH3), 2.96 (dd, J = 14.4,
5.2 Hz, 1H, 2-H), 3.10–3.14 (m, 1H, 3-H), 3.77 (s, 6H, 30,50-OCH3),
3.82 (s, 3H, 40-OCH3), 4.01–4.06 (m, 1H, 11
a-H), 4.53–4.57 (m,
1H, 11b-H), 4.66 (d, 1H, 1-H), 5.89 (dd, J = 8.0, 4.4 Hz, 1H, 4-H),
5.97 and 6.00 (ABq, 2H, –OCH2O–), 6.32 (s, 2H, 20,60-H), 6.55 (s,
1H, 8-H), 6.87 (s, 1H, 5-H), 7.33 (d, J = 8.0 Hz, 2H, 300,500-H), 7.72
(d, J = 8.4 Hz, 2H, 200,600-H), 9.07 (s, 1H, –CSNHCO–), 11.03 (d, 1H,
J = 8.0 Hz, 4-NH); 13C NMR (100 MHz, CDCl3): 21.7, 37.6, 42.2,
43.8, 54.1, 56.2, 60.7, 68.7, 101.7, 108.2, 109.2, 110.2, 127.5,
127.8, 128.3, 130.0, 132.3, 134.6, 137.3, 145.1, 147.8, 148.8,
152.7, 167.0, 173.9, 180.6. HRMS m/z calcd for C31H30N2O8S:
613.1615 [M+Na]+, found: 613.1622 [M+Na]+.
4.1.2.11. 4b-[3-(2-Chloronicotinoyl)-thioureido]-4-deoxypodo-
phyllotoxin (13k).
Yield: 88%; mp: 183–186 °C; 1H NMR
(400 MHz, CDCl3): d 2.94 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.09–3.12
(m, 1H, 3-H), 3.76 (s, 6H, 30,50-OCH3), 3.80 (s, 3H, 40-OCH3), 3.98–
4.03 (m, 1H, 11a-H), 4.50–4.55 (m, 1H, 11b-H), 4.66 (d, 1H, 1-H),
5.85 (dd, J = 7.6, 4.8 Hz, 1H, 4-H), 5.99 and 6.01 (ABq, 2H, –
OCH2O–), 6.30 (s, 2H, 20,60-H), 6.55 (s, 1H, 8-H), 6.87 (s, 1H, 5-H),
7.41 (dd, J = 7.6, 4.8 Hz, 1H, 500-H), 8.07 (dd, J = 7.6, 2.0 Hz, 1H, 600-
H), 8.56 (dd, 1H, J = 4.8, 2.0 Hz, 400-H), 9.76 (s, 1H, –CSNHCO–),
10.64 (d, 1H, J = 8.0 Hz, 4-NH); 13C NMR (100 MHz, CDCl3): 37.5,
42.2, 43.7, 54.1, 56.2, 60.7, 68.6, 101.7, 108.1, 109.1, 110.2, 122.9,
127.4, 128.4, 132.4, 134.5, 137.2, 139.8, 147.2, 147.8, 148.8,
152.6, 152.7, 164.7, 173.8, 179.7. HRMS m/z calcd for
4.1.2.7. 4b-[3-(4-Nitrobenzoyl)-thioureido]-4-deoxypodophyllo-
toxin (13g).
Yield: 56%; mp: 171–174 °C; 1H NMR (400 MHz,
CDCl3): d 2.95 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.10–3.19 (m, 1H, 3-H),
3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 3.99–4.04 (m, 1H,
11a-H), 4.53–4.57 (m, 1H, 11b-H), 4.68 (d, 1H, 1-H), 5.87 (dd,
J = 8.0, 4.8 Hz, 1H, 4-H), 5.99 and 6.01 (ABq, 2H, –OCH2O–), 6.31
(s, 2H, 20,60-H), 6.57 (s, 1H, 8-H), 6.85 (s, 1H, 5-H), 8.03 (d,
J = 8.4 Hz, 2H, 200,600-H), 8.38 (d, J = 8.8 Hz, 2H, 300,500-H), 9.25 (s,
1H, –CSNHCO–), 10.79 (d, 1H, J = 8.0 Hz, 4-NH). 13C NMR
(100 MHz, CDCl3): 37.5, 42.2, 43.8, 54.3, 56.3, 60.8, 68.6, 101.7,
108.2, 109.0, 110.3, 124.3, 127.4, 128.8, 132.4, 134.5, 136.7,
137.3, 147.8, 148.9, 150.8, 152.7, 165.1, 173.8, 179.9. HRMS m/z
C
29H26ClN3O8S: 634.1021 [M+Na]+, found: 634.1041 [M+Na]+.
4.1.2.12. 4b-{3-(5-Bromonicotinoyl)-thioureido]-4-deoxypodo-
phyllotoxin (13l).
Yield: 68%; mp: 173–176 °C; 1H NMR
(400 MHz, CDCl3): d 2.94 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.12–3.15
calcd for
C
30H27N3O10S: 644.1309 [M+Na]+, found: 644.1326
(m, 1H, 3-H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 3.99–
[M+Na]+.
4.04 (m, 1H, 11a-H), 4.53–4.57 (m, 1H, 11b-H), 4.68 (d, 1H, 1-H),
5.86 (dd, J = 7.6, 4.8 Hz, 1H, 4-H), 5.99 and 6.01 (ABq, 2H, –
OCH2O–), 6.31 (s, 2H, 20,60-H), 6.57 (s, 1H, 8-H), 6.85 (s, 1H, 5-H),
8.29 (s, 1H, 400-H), 8.92 (s, 1H, 600-H), 8.99 (s, 1H, 200-H), 9.27 (s,
1H, –CSNHCO–), 10.74 (d, 1H, J = 8.0 Hz, 4-NH); 13C NMR
(100 MHz, CDCl3): 37.5, 42.2, 43.7, 54.3, 56.3, 60.8, 68.6, 101.8,
108.2, 109.0, 110.3, 127.4, 132.4, 134.5, 137.3, 137.8, 146.4,
147.8, 148.9, 152.7, 155.4, 164.2, 173.8, 179.8. HRMS m/z calcd
for C29H26BrN3O8S: 678.0516 [M+Na]+, found: 678.0529 [M+Na]+.
4.1.2.8. 4b-[3-3(3-Nitrobenzoyl)-thioureido]-4-deoxypodo-
phyllotoxin (13h).
Yield: 63%; mp: 168–172 °C; 1H NMR
(400 MHz, CDCl3): d 2.96 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.10–3.18
(m, 1H, 3-H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 4.00–
4.05 (m, 1H, 11a-H), 4.54–4.68 (m, 1H, 11b-H), 4.69 (d, 1H, 1-H),
5.88 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.99 and 6.01 (ABq, 2H, –
OCH2O–), 6.32 (s, 2H, 20,60-H), 6.57 (s, 1H, 8-H), 6.86 (s, 1H, 5-H),
7.78 (t, J = 8.0 Hz, 1H, 500-H), 8.18 (d, J = 8.0 Hz, 1H, 600-H), 8.51 (d,
J = 8.0 Hz, 1H, 400-H), 8.72 (s, 1H, 200-H), 9.28 (s, 1H, –CSNHCO–),
10.82 (d, 1H, J = 8.0 Hz, 4-NH). 13C NMR (100 MHz, CDCl3): 37.5,
42.2, 43.8, 54.3, 56.2, 60.8, 68.6, 101.7, 108.2, 109.0, 110.3, 122.7,
127.4, 128.2, 130.6, 132.4, 133.0, 133.1, 134.5, 137.3, 147.8,
148.5, 148.9, 152.7, 164.8, 173.8, 179.9. HRMS m/z calcd for
4.1.2.13. 4b-[3-(2-Thiophenecarbonyl)-thioureido]-4-deoxypod-
ophyllotoxin (13m).
Yield: 92%; mp: 170–173 °C; 1H NMR
(400 MHz, CDCl3): d 2.94 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.08–3.12
(m, 1H, 3-H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 3.99–
4.04 (m, 1H, 11a-H), 4.52–4.56 (m, 1H, 11b-H), 4.65 (d, 1H, 1-H),
C
30H27N3O10S: 644.1309 [M+Na]+, found: 644.1317 [M+Na]+.
5.87 (dd, J = 7.6, 4.8 Hz, 1H, 4-H), 5.97 and 6.00 (ABq, 2H, –
OCH2O–), 6.32 (s, 2H, 20,60-H), 6.54 (s, 1H, 8-H), 6.84 (s, 1H, 5-H),
7.19 (dd, J = 5.2, 4.0 Hz, 1H, 400-H), 7.71–7.74 (m, 2H, 300,500-H),
8.98 (s, 1H, –CSNHCO–), 10.81 (d, 1H, J = 8.0 Hz, 4-NH); 13C NMR
(100 MHz, CDCl3): 37.6, 42.2, 43.8, 54.1, 56.3, 60.7, 68.7, 101.7,
108.3, 109.1, 110.1, 127.6, 128.6, 130.8, 132.3, 134.6, 134.7,
135.6, 137.3, 147.8, 148.8, 152.7, 161.3, 173.8, 180.1. HRMS m/z
4.1.2.9.
toxin (13i).
4b-(3-Nicotinoyl-thioureido)-4-deoxypodophyllo-
Yield: 80%; mp: 173–176 °C; 1H NMR (400 MHz,
CDCl3): d 2.95 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.07–3.16 (m, 1H, 3-
H), 3.75 (s, 6H, 30,50-OCH3), 3.80 (s, 3H, 40-OCH3), 3.98–4.03 (m,
1H, 11a-H), 4.50–4.52 (m, 1H, 11b-H), 4.66 (d, 1H, 1-H), 5.86 (dd,
J = 8.0, 4.8 Hz, 1H, 4-H), 5.97 and 5.99 (ABq, 2H, –OCH2O–), 6.30
(s, 2H, 20,60-H), 6.54 (s, 1H, 8-H), 6.84 (s, 1H, 5-H), 7.46 (dd,
J = 8.0, 4.8 Hz, 1H, 600-H), 8.13–8.15 (m, 1H, 500-H), 8.83 (dd,
J = 4.8, 1.2 Hz, 1H, 400-H), 9.09 (d, 1H, J = 2.0 Hz, 200-H), 9.58 (s, 1H,
–CSNHCO–), 10.87 (d, 1H, J = 8.0 Hz, 4-NH). 13C NMR (100 MHz,
CDCl3): 37.5, 42.1, 43.7, 54.1, 56.2, 60.3, 60.7, 68.6, 101.7, 108.1,
109.0, 110.2, 123.7, 127.4, 127.5, 132.3, 134.5, 135.3, 137.2,
147.7, 148.7, 148.8, 152.6, 154.2, 165.8, 173.8, 180.2. HRMS m/z
calcd for C29H27N3O8S: 578.1592 [M+H]+, found: 578.1606 [M+H]+.
calcd for
C
28H26N2O8S2: 600.1469[M+NH4]+, found: 600.1480
[M+NH4]+.
4.1.2.14. 4b-[3-(2-Furoyl)-thioureido]-4-deoxypodophyllotoxin
(13n).
Yield: 89%; mp: 158–161 °C; 1H NMR (400 MHz,
CDCl3): d 2.93 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.09–3.12 (m, 1H, 3-
H), 3.77 (s, 6H, 30,50-OCH3), 3.82 (s, 3H, 40-OCH3), 3.99–4.04 (m,
1H, 11a-H), 4.53–4.57 (m, 1H, 11b-H), 4.65 (d, 1H, 1-H), 5.87 (dd,
J = 7.6, 4.8 Hz, 1H, 4-H), 5.97 and 6.00 (ABq, 2H, –OCH2O–), 6.31
(s, 2H, 20,60-H), 6.54 (s, 1H, 8-H), 6.64 (dd, J = 3.6, 1.2 Hz, 1H, 400-
H), 6.85 (s, 1H, 5-H), 7.32 (d, J = 3.6 Hz, 1H, 500-H), 7.63 (d,
J = 1.2 Hz, 1H, 300-H), 9.19 (s, 1H, –CSNHCO–), 10.73 (d, 1H,
J = 8.0 Hz, 4-NH); 13C NMR (100 MHz, CDCl3): 37.5, 42.2, 43.7,
4.1.2.10. 4b-[3-(4-Chloronicotinoyl)-thioureido]-4-deoxypodo-
phyllotoxin(13j).
(400 MHz, CDCl3): d 2.94 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.12–3.15
Yield: 84%; mp: 172–175 °C; 1H NMR

2368
W.-Q. Li et al. / Bioorg. Med. Chem. 21 (2013) 2363–2369
54.0, 56.2, 60.7, 68.7, 101.7, 108.1, 109.1, 110.1, 113.5, 119.3, 127.7,
132.3, 134.6, 137.2, 144.6, 146.6, 147.7, 148.8, 152.6, 156.8, 173.9,
180.0. HRMS m/z calcd for C28H26N2O9S: 567.1432 [M+H]+, found:
567.1448 [M+H]+.
4.1.2.19. 4b-[3-(Thiophene-2-carbonyl)-thioureido]-40-demeth-
yl-4-deoxypodophyllotoxin (14d).
Yield: 93%; mp: 194–
197 °C; 1H NMR (400 MHz, CDCl3): d 2.93 (dd, J = 14.4, 4.8 Hz,
1H, 2-H), 3.09–3.10 (m, 1H, 3-H), 3.80 (s, 6H, 30,50-OCH3), 3.99–
4.04 (m, 1H, 11a-H), 4.50–4.54 (m, 1H, 11b-H), 4.64 (d, 1H, 1-H),
4.1.2.15. 4b-[3-(2-Naphthoyl)-thioureido]-4-deoxypodophyllo-
toxin (13o).
5.49 (s, 1H, 40-OH), 5.87 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.97 and
6.00 (ABq, 2H, –OCH2O–), 6.33 (s, 2H, 20,60-H), 6.54 (s, 1H, 8-H),
6.85 (s, 1H, 5-H), 7.19 (dd, J = 4.8, 4.0 Hz, 1H, 400-H), 7.71–7.74
(m, 2H, 300,500-H), 8.97 (s, 1H, –CSNHCO–), 10.81 (d, 1H, J = 8.0 Hz,
4-NH); 13C NMR (100 MHz, CDCl3): 37.5, 42.3, 43.6, 54.1, 56.4,
56.5, 60.4, 68.7, 101.7, 107.9, 109.1, 110.2, 127.6, 128.6, 130.1,
130.9, 132.5, 134.2, 134.7, 135.6, 146.5, 147.7, 148.8, 171.1,
Yield: 77%; mp: 156–159 °C; 1H NMR
(400 MHz, CDCl3): d 3.00 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.10–3.20
(m, 1H, 3-H), 3.78 (s, 6H, 30,50-OCH3), 3.83 (s, 3H, 40-OCH3), 4.05–
4.10 (m, 1H, 11a-H), 4.56–4.60 (m, 1H, 11b-H), 4.69 (d, 1H, 1-H),
5.93 (dd, J = 8.0, 4.4 Hz, 1H, 4-H), 5.98 and 6.01 (ABq, 2H,
–OCH2O–), 6.34 (s, 2H, 20,60-H), 6.57 (s, 1H, 8-H), 6.90 (s, 1H,
5-H), 7.63–7.68 (m, 2H, 500,600-H), 7.84 (dd, J = 8.4, 1.2 Hz, 1H, 300-
H), 7.93 (d, J = 8.0 Hz, 1H, 200-H), 7.99 (d, J = 8.4 Hz, 2H, 400,700-H),
8.39 (s, 1H, 800-H), 9.27 (s, 1H, –CSNHCO–), 11.07 (d, 1H,
J = 8.0 Hz, 4-NH). 13C NMR (100 MHz, CDCl3): 37.6, 42.2, 43.8,
54.2, 56.3, 60.8, 68.7, 101.7, 108.3, 109.2, 110.2, 122.8, 127.6,
127.8, 127.9, 128.3, 129.0, 129.2, 129.3, 129.5, 132.3, 132.4,
134.6, 135.7, 137.4, 147.8, 148.8, 152.7, 167.2, 173.9. HRMS m/z
173.9, 180.1. HRMS m/z calcd for
C27H24N2O8S2: 591.0866
[M+Na]+, found: 591.0876 [M+Na]+.
4.1.2.20.
4b-[3-(2-Naphthoyl)-thioureido]-40-demethyl-4-
deoxypodophyllotoxin (14e).
Yield: 87%; mp: 178–181 °C;
1H NMR (400 MHz, CDCl3): d 2.98 (dd, J = 14.4, 4.8 Hz, 1H, 2-H),
3.09–3.18 (m, 1H, 3-H), 3.80 (s, 6H, 30,50-OCH3), 3.04–4.10 (m,
calcd for
C
34H30N2O8S: 649.1615 [M+Na]+, found: 649.1629
1H, 11a-H), 4.53–4.57 (m, 1H, 11b-H), 4.67 (d, 1H, 1-H), 5.49 (s,
[M+Na]+.
1H, 40-OH), 5.92 (dd, J = 8.0, 4.4 Hz, 1H, 4-H), 5.98 and 6.00 (ABq,
2H, –OCH2O–), 6.35 (s, 2H, 20,60-H), 6.56 (s, 1H, 8-H), 6.89 (s, 1H,
5-H), 7.62–7.67 (m, 2H, 500, 600-H), 7.84 (dd, J = 8.4, 1.6 Hz, 1H, 300-
H), 7.92 (d, J = 7.6 Hz, 1H, 200-H), 7.98 (d, J = 8.4 Hz, 2H, 400,700-H),
8.38 (s, 1H, 800-H), 9.28 (s, 1H, –CSNHCO–), 11.07 (d, 1H,
J = 8.0 Hz, 4-NH); 13C NMR (100 MHz, CDCl3): 37.5, 42.3, 43.6,
54.2, 56.4, 56.5, 60.4, 68.7, 101.7, 107.9, 109.1, 110.2, 122.8,
127.6, 127.7, 127.9, 128.3, 129.0, 129.2, 129.3, 129.4, 130.1,
132.3, 132.5, 134.2, 135.7, 146.5, 147.7, 148.8, 167.2, 171.1,
173.9, 180.5. HRMS m/z calcd for C33H28N2O8S: 635.1459[M+Na]+,
found: 635.1477 [M+Na]+.
4.1.2.16. 4b-(3-Benzoyl-thioureido)-40-demethyl-4-deoxypodo-
phyllotoxin (14a).
Yield: 84%; mp: 177–180 °C; 1H NMR
(400 MHz, CDCl3): d 2.95 (dd, J = 14.4, 5.2 Hz, 1H, 2-H), 3.09–3.14
(m, 1H, 3-H), 3.80 (s, 6H, 30,50-OCH3), 4.00–4.05 (m, 1H, 11
-H),
a
4.51–4.55 (m, 1H, 11b-H), 4.66 (d, 1H, 1-H), 5.49 (s, 1H, 40-OH),
5.90 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.97 and 6.00 (ABq, 2H, –
OCH2O–), 6.34 (s, 2H, 20,60-H), 6.55 (s, 1H, 8-H), 6.87 (s, 1H, 5-H),
7.54 (t, 2H, 300,500-H), 7.66 (t, 1H, 400-H), 7.84 (d, 2H, 200,600-H), 9.11
(s, 1H, –CSNHCO–), 10.99 (d, 1H, J = 8.0 Hz, 4-NH). 13C NMR
(100 MHz, CDCl3): 37.5, 42.3, 43.6, 54.1, 56.4, 56.5, 68.7, 101.7,
107.9, 109.1, 110.2, 127.5, 127.7, 129.3, 130.1, 131.2, 132.5, 134.0,
134.2, 146.5, 147.7, 148.8, 167.0, 173.9, 180.5. HRMS m/z calcd for
4.2. Antiproliferative activity assay
C
29H26N2O8S: 585.1302 [M+Na]+, found: 585.1323 [M+Na]+.
Antiproliferative activity was determined by the sulforhoda-
mine B (SRB) colorimetric assay as previously described.21 In brief,
the cells (3–5 ꢀ 103 cells/well) were seeded in 96-well plates filled
with RPMI-1640 medium supplemented with 10% fetal bovine ser-
um (FBS) containing various concentrations of samples, and incu-
bated for 72 h. At the end of the exposure period, the attached
cells were fixed with cold 50% trichloroacetic acid for 30 min fol-
lowed by staining with 0.04% SRB (Sigma Chemical Co.) for
30 min. The bound SRB was solubilized in 10 mM Tris-base and
the absorbance was measured at 515 nm on a Microplate Reader
ELx800 (Bio-Tek Instruments, Winooski, VT) with a Gen5 software.
All results were representative of three or more experiments.
4.1.2.17.
4b-[3-(4-Chlorobenzoyl)-thioureido]-40-demethyl-4-
deoxypodophyllotoxin (14b).
Yield: 92%; mp:180–183 °C;
1H NMR (400 MHz, CDCl3): d 2.94 (dd, J = 14.4, 4.8 Hz, 1H, 2-H),
3.07–3.15 (m, 1H, 3-H), 3.80 (s, 6H, 30,50-OCH3), 3.99–4.04 (m,
1H, 11a-H), 4.51–4.55 (m, 1H, 11b-H), 4.66 (d, 1H, 1-H), 5.49 (s,
1H, 40-OH), 5.89 (dd, J = 8.0, 4.8 Hz, 1H, 4-H), 5.97 and 6.00 (ABq,
2H, –OCH2O–), 6.33 (s, 2H, 20,60-H), 6.56 (s, 1H, 8-H), 6.85 (s, 1H,
5-H), 7.52 (d, J = 8.4 Hz, 2H, 300,500-H), 7.78 (d, J = 8.4 Hz, 2H, 200,600-
H), 9.07 (s, 1H, –CSNHCO–), 10.90 (d, 1H, J = 8.0 Hz, 4-NH). 13C
NMR (100 MHz, CDCl3): 37.5, 42.3, 43.6, 54.2, 56.4, 56.5, 60.4,
68.6, 101.7, 107.9, 109.1, 110.2, 127.6, 128.9, 129.6, 129.7, 130.1,
132.5, 134.2, 140.6, 146.5, 147.7, 148.8, 166.0, 171.1, 173.9,
180.3. HRMS m/z calcd for C29H25ClN2O8S: 619.0912 [M+Na]+,
found: 619.0944 [M+Na]+.
Acknowledgments
This work was supported financially by the National Natural
Science Foundation of China (30800720); the Post-Doctor Research
Foundation (20090450142); the Fundamental Research Funds for
the Central Universities (860120); and the Young Scholars Science
Foundation of Lanzhou Jiaotong University (2011011). Thanks are
also due to the support of Taiwan Department of Health Cancer Re-
search Center of Excellence (DOH-100-TD-C-111–005).
4.1.2.18. 4b-[3-(2-Furoyl)-thioureido]-40-demethyl-4-deoxypod-
ophyllotoxin (14c).
(400 MHz, CDCl3): d 2.92 (dd, J = 14.4, 4.8 Hz, 1H, 2-H), 3.07–3.12
(m, 1H, 3-H), 3.80 (s, 6H, 30,50-OCH3), 3.98–4.03 (m, 1H, 11
-H),
Yield: 86%; mp: 180–184 °C;1H NMR
a
4.50–4.54 (m, 1H, 11b-H), 4.64 (d, 1H, 1-H), 5.47 (s, 1H, 40-OH),
5.87 (dd, J = 8.8, 4.8 Hz, 1H, 4-H), 5.96 and 5.99 (ABq, 2H, –
OCH2O–), 6.33 (s, 2H, 20,60-H), 6.54 (s, 1H, 8-H), 6.64 (dd, J = 3.6,
1.2 Hz, 1H, 400-H), 6.84 (s, 1H, 5-H), 7.32 (d, J = 3.6 Hz, 1H, 500-H),
7.63 (d, J = 1.2 Hz, 1H, 300-H), 9.19 (s, 1H, –CSNHCO–), 10.72 (d,
1H, J = 8.0 Hz, 4-NH). 13C NMR (100 MHz, CDCl3): 37.5, 42.3, 43.6,
54.0, 56.4, 56.5, 60.4, 68.7, 101.6, 107.9, 109.1, 110.1, 113.5,
119.2, 127.7, 130.1, 132.5, 134.2, 144.6, 146.5, 146.6, 147.7,
148.8, 156.8, 171.1, 173.9, 180.0. HRMS m/z calcd for C27H24N2O9S:
575.1095 [M+Na]+, found: 575.1114 [M+Na]+.
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