Toward synthesis of third-generation spin-labeled podophyllotoxin derivatives using isocyanide multicomponent reactions

Article abstract of DOI:10.1016/j.ejmech.2014.01.038

Spin-labeled podophyllotoxins have elicited widespread interest due to their far superior antitumor activity compared to podophyllotoxin. To extend our prior studies in this research area, we synthesized a new generation of spin-labeled podophyllotoxin an

Full text of DOI:10.1016/j.ejmech.2014.01.038

European Journal of Medicinal Chemistry 75 (2014) 282e288
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Toward synthesis of third-generation spin-labeled podophyllotoxin
derivatives using isocyanide multicomponent reactions
,
,
Liang Kou a 1, Mei-Juan Wang a 1, Li-Ting Wang b, Xiao-Bo Zhao a, Xiang Nan a, Liu Yang d,
Ying-Qian Liu a
a School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
, Susan L. Morris-Natschke , Kuo-Hsiung Lee
,
e,
b
b,c,
*
**
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
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
e Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Spin-labeled podophyllotoxins have elicited widespread interest due to their far superior antitumor
activity compared to podophyllotoxin. To extend our prior studies in this research area, we synthesized a
new generation of spin-labeled podophyllotoxin analogs via isocyanide multicomponent reactions and
evaluated their cytotoxicity against four human cancer cell lines (A-549, DU-145, KB and KBvin). Most of
the compounds exhibited potent cytotoxic activity against all four cell lines, notably against the drug
Received 16 October 2013
Received in revised form
15 January 2014
Accepted 18 January 2014
Available online 29 January 2014
resistant KBvin cancer cell line. Among the new analogs, compounds 12e (IC50: 0.60e0.75
mM) and 12h
(IC50: 1.12e2.03 M) showed superior potency to etoposide (IC50: 2.03 to >20 M), a clinically available
m
m
Keywords:
Podophyllotoxin
C-4 position
anticancer drug. With a concise efficient synthesis and potent cytotoxic profiles, compounds 12e and 12h
merit further development as a new generation of epipodophyllotoxin-derived antitumor clinical trial
candidates.
Spin labeled
Ó 2014 Elsevier Masson SAS. All rights reserved.
Isocyanide multicomponent reactions
Cytotoxic activity
1. Introduction
environmentally benign procedures. From this standpoint, design-
or logic-based discovery utilizing IMCR could be advantageous.
In recent years, multicomponent reactions (MCRs) have played a
central role in the development of modern synthetic methodology
for drug discovery research. The applications in this field are quite
broad extending from initial lead structure identification to gen-
eration of large libraries of analogs. Combined with the power of in
silico screens, this methodology is an efficient tool for the modern
medicinal chemist. MCRs that involve isocyanides (IMCRs) are by
far the most versatile reactions in terms of scaffolds and number of
accessible compounds [1e3]. Many advantages over conventional
linear-type syntheses make IMCRs very popular in the medicinal
chemistry community: superior atom economy, low costs, high
variability, high bond forming efficiency, and simple
Consequently, the application of IMCR offers an efficient synthetic
route to construct diverse podophyllotoxin-based scaffolds for drug
discovery.
Podophyllotoxin (1, PPT), a naturally occurring lignin, exerts
cytotoxic activity by inhibition of microtubule assembly [4,5]. Its
two semisynthetic glucoconjugates, etoposide (2) and teniposide
(3), are novel DNA topoisomerase II inhibitors marketed in several
countries [6,7]. Some non-sugar substituted analogs, particularly
N-linked congeners, exhibit superior pharmacological properties to
etoposide, and consequently, several newer-generation clinical
candidates, including NPF (4) [8], GL-331 (5) [9], and TOP-53 (6)
[10], have emerged through C-4 modification as alternatives to
overcome the drawbacks of etoposide. In addition, another
important area of podophyllotoxin research involves the synthesis
and design of novel spin-labeled PPT derivatives by our group over
the past decades [11e20], providing potential drugs with beneficial
therapeutic profiles, e.g., improved activity or higher solubility,
better pharmacokinetics, or reduced side effects. Among the
highlights of this research, first generation spin-labeled podo-
phyllotoxin derivatives represented by GP-7 (7, Fig. 1) and GP-11 (8)
* Corresponding author. Natural Products Research Laboratories, UNC Eshelman
School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.
Tel.: þ1 919 962 0066; fax: þ1 919 966 3893.
** Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou
730000, PR China.
E-mail addresses: yqliu@lzu.edu.cn (Y.-Q. Liu), khlee@email.unc.edu, khlee@unc.
edu (K.-H. Lee).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.038

L. Kou et al. / European Journal of Medicinal Chemistry 75 (2014) 282e288
283
Fig. 1. Structures of podophyllotoxin derivatives.
are the most important, because of their high potency, low toxicity,
broad antitumor spectra, and better formulation/usage character-
istics. After them, second generation spin-labeled amino acid-
linked derivatives were investigated [21e23] and some of them
showed superior or comparable activity against particular cancer
types compared to 2, while their drug-resistance profiles were
significantly different from those of 1. Besides, some compounds
were effective at a lower dose than etoposide in drug-sensitive and
drug-resistant xenograft models, demonstrating their potential as
drug candidates for anticancer chemotherapy.
large-scale synthesis of this class of biologically active molecules
but also for synthesis of screening libraries for drug discovery or for
industrial production. To enrich the limited set of podophyllotoxin
derivatives typically employed in drug discovery, we recently
described an efficient synthesis of 4b-isocyanopodophyllotoxins by
dehydration of the corresponding formamide under both ultrasonic
and classical conditions [31]. Based on the success of the above
reaction, we subsequently developed a practical IMCR to synthesize
podophyllotoxin derivatives. Herein, we first report the use of Ugi
(Ugi-4CR) and Passirini (P-3CR) reactions as a versatile approach
towards the synthesis of third generation novel spin-labeled
podophyllotoxin series. To our knowledge, these examples are
also the first where a nitroxide participated in such IMCRs. Some of
the obtained compounds have been screened for cytotoxic activity
and exhibited considerable, yet selective potency against certain
cancer cell types. With a concise efficient synthesis and potent
cytotoxic profiles, compounds 12e, 12h and 13c merit further
development as new generation spin-labeled epipodophyllotoxin-
derived antitumor clinical trial candidates.
Although numerous syntheses and structure modifications of
podophyllotoxins have been performed, most changes at the C-4
position of PPT have been relatively simple, such as incorporation of
4b-substituted ethers, esters, and N-linked congeners [24e30].
Modification of this position with diverse structures via MCRs has
attracted less interest. Furthermore, typical synthetic strategies
often suffer from one or more limitations, including long reaction
times, tedious work-up procedures, or lack of general applicability
and stereoselectivity, which makes them unsuitable not only for

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L. Kou et al. / European Journal of Medicinal Chemistry 75 (2014) 282e288
2. Results and discussion
spin-labeled epipodophyllotoxin derivatives with an
carboxamide group.
The Ugi reaction (Scheme 2) involved four components: alde-
hyde/ketone, amine, stable nitroxide radical acid, -iso-
cyanopodophyllotoxin (11). This reaction mixture gave another
series of spin-labeled derivatives with a -acylamino carboxamide
a-acyloxy
2.1. Chemistry
4
b
Our synthetic routes are outlined in Schemes 1 and 2. Briefly, the
precursor 4
b-aminoepipodophyllotoxin (9) was prepared stereo-
a
selectively from 1 through azidation and catalytic hydrogenation
according to a previously reported method [32]. Next, compound 9
was converted to 4b-formamido-4-deoxypodophyllotoxin (10) in
group. All newly synthesized compounds were purified by column
chromatography and their structures were characterized by HRMS,
ESR and IR spectroscopic analysis.
excellent yield by reaction with ethyl formate under both classical
and ultrasonic conditions. Subsequently, dehydration of 10 with p-
toluenesulfonyl chloride in pyridine furnished the key intermediate
2.2. Cytotoxicity
4b-isocyanopodophyllotoxin (11) in good yield [31]. Finally, two
Target compounds 12aej and 13ael were evaluated for in vitro
cytotoxicity against four human tumor cell lines, A549 (non-small
cell lung cancer), DU145 (prostate cancer cell line), KB (nasopha-
ryngeal carcinoma), and KBvin [multi-drug resistant (MDR) KB
subline selected using vincristine], using a sulforhodamine B
colorimetric (SRB) assay with triplicate experiments [33].
novel series of target compounds 12aej and 13ael were obtained
via Passerini and Ugi reactions in moderate to good yields,
respectively. The Passirini reaction (Scheme 1) involved three
components: aldehyde/ketone, stable nitroxyl radical acid, and 4b-
isocyanopodophyllotoxin (11). The reaction gave a series of novel
OH
NH2
NHCHO
O
O
HN3 BF ·Et O
/
O
O
O
O
3
2
HCO2C2H5/CH2Cl2
u.s. reflux, 1h
O
O
O
O
O
Pd/H2 EtOAc
O
OCH3
H3CO
OCH3
H3CO
OCH
H3CO
3
OCH3
OCH3
OCH3
1
9
10
R
1 R2
R3
O
O
NC
O
O
P-TsCl/pyridine
O
O
NH
O
R1 R2
R3 OH
O
O
u.s. r.t., 2h
O
O
O
MeOH, reflux, 5 h.
Passirini reaction
O
H3CO
OCH3
H3CO
OCH3
OCH3
12a-j
OCH3
11
O
O
R3 OH
R1 R2
COOH
COOH
COOH
O
CHO
12a
12d
12b
12c
12f
CH3(CH2)2CHO
CH2CHO
N
O
N
N
O
O
O
COOH
COOH
COOH
COOH
CHO
12e
12h
N
O
N
O
N
O
H3CO
COOH
COOH
O
CHO
CHO
O
O
12g
12j
12i
CH3(CH2)2CHO
N
O
N
O
N
O
COOH
CH3(CH2)2CHO
N
O
Scheme 1. Synthesis of target compounds 12aej via the Passerini reaction.

L. Kou et al. / European Journal of Medicinal Chemistry 75 (2014) 282e288
285
Scheme 2. Synthesis of target compounds 13ael via the Ugi reaction.
Compound 2 was included as a positive control and the results are
summarized in Table 1.
Table 1
In vitro cytotoxicity of compounds 12aej and 13aec against four human tumor cell
As illustrated in Table 1, except for compounds 12b and 13del,
most of the tested compounds exhibited moderate to potent
cytotoxicity against the four human tumor cell lines. Remarkably,
these compounds retained similar levels of cytotoxicity against the
drug resistant KBvin and KB tumor cell lines, while 2 lost its ac-
lines.a
Compd
IC50
(
mM)
A549
DU145
KB
KBvin
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
13a
13b
13c
2
2.38 ꢀ 0.27
>20
2.39 ꢀ 0.26
>20
2.32 ꢀ 0.12
>20
2.55 ꢀ 0.55
>20
tivity completely (IC50 > 20 vs 3.88 mM). This result agrees with
8.86 ꢀ 0.43
9.52 ꢀ 0.82
0.75 ꢀ 0.02
7.68 ꢀ 0.37
7.38 ꢀ 0.04
1.46 ꢀ 0.17
8.82 ꢀ 0.26
9.05 ꢀ 0.26
8.98 ꢀ 1.21
8.40 ꢀ 0.304
6.10 ꢀ 0.192
2.58 ꢀ 0.252
7.07 ꢀ 0.44
8.74 ꢀ 0.77
0.63 ꢀ 0.09
6.82 ꢀ 0.16
6.55 ꢀ 0.12
1.37 ꢀ 0.08
7.13 ꢀ 0.07
7.27 ꢀ 0.05
10.95 ꢀ 1.20
9.78 ꢀ 2.20
6.08 ꢀ 0.138
2.03 ꢀ 0.121
7.12 ꢀ 0.45
9.24 ꢀ 1.57
0.60 ꢀ 0.02
6.53 ꢀ 0.06
7.12 ꢀ 0.22
1.12 ꢀ 0.13
6.58 ꢀ 0.14
6.88 ꢀ 0.24
9.35 ꢀ 2.01
8.49 ꢀ 0.72
5.94 ꢀ 0.37
3.88 ꢀ 0.199
8.88 ꢀ 0.37
13.68 ꢀ 1.96
0.73 ꢀ 0.06
7.71 ꢀ 0.19
7.35 ꢀ 0.36
2.03 ꢀ 0.38
7.96 ꢀ 0.14
8.35 ꢀ 0.06
8.83 ꢀ 1.33
7.47 ꢀ 1.15
6.45 ꢀ 0.396
>20
our prior observation that C4-amino substitution of epi-
podophyllotoxin is good for overcoming drug-resistance [34e36].
Among the tested compounds, compounds 12e and 12h showed
superior activity with IC50 values of 0.60e1.46
mM compared to
those of 2 (IC50 2.03e3.88
mM) against A549, DU-145, and KB tu-
mor cell lines.
Compounds 12e and 12h have a methoxy and methylenedioxy
substituted phenyl ring, respectively, in their -acyloxy carbox-
a
amide side chain, while 12b has an unsubstituted phenyl ring in
the same position. This minor structural difference is notable as
12b was completely inactive against all four tumor cell lines. Two
a
The results are average of three independent experiments. Compounds 13del
M).
additional compounds with an aromatic ring in the
a-acyloxy
did not show significant inhibition (IC50 > 20
m

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L. Kou et al. / European Journal of Medicinal Chemistry 75 (2014) 282e288
carboxamide side chain, 12g with a furan ring and 12f with a
benzyl group, displayed moderate potency (IC50 6.53e7.71 M).
Therefore, future structural modifications will focus on intro-
4.2. Synthesis of key intermediate 4
b-isocyanopodophyllotoxin (11)
m
The key intermediate 4 -isocyanopodophyllotoxin (11) used for
b
ducing various substituted aromatic rings in the
boxamide side chain.
a
-acyloxy car-
the experiments was prepared by our previous procedure [31].
Yield 67%; m.p. 115e117 ꢁC; IR (KBr, cmꢂ1) 2130 (NC), 1779
(lactone), 1588, 1506 and 1485 (aromatic C]C), 930 (OCH2O). 1H
Among compounds 12a, 12c, and 12d with alkyl groups in the
a
-acyloxy carboxamide side chain, cytotoxic activity generally
NMR (400 MHz, CDCl3) d 6.91 (s, 1H, H-5), 6.56 (s, 1H, H-8), 6.26 (s,
decreased as the size of the substituents increased. For example,
against the A549 cancer cell, the IC50 value of 12a (two methyl
groups) was 2.38
group) and 12d (cyclohexane) were 8.86 and 9.52
2H, H-20,60), 6.03 (dd, J ¼ 7.9, 1.1 Hz, 2H, OCH2O), 4.99 (d, J ¼ 4.4 Hz,
1H, H-4), 4.68 (d, J ¼ 5.1 Hz,1H, H-1), 4.39 (m, 2H, H-11), 3.81 (s, 3H,
40-OCH3), 3.75 (s, 6H, 30,50-OCH3), 3.20 (q, 1H, H-2), 2.96 (m, 1H, H-
m
M, while the IC50 values of 12c (H and propyl
M, respec-
m
3). 13C NMR: (CDC13, 400 MHz)
d: 173.01, 160.01, 152.78, 149.27,
tively, indicating that the size of substituent groups might
critical.
Compounds 12c, 12j and 12i differ only in the identity of the
nitroxide. The three compounds exhibited similar potency; thus,
ring size and degree of unsaturation had no effect, which was
consistent with the literature [19].
147.95, 137.64, 134.32, 131.59, 125.13, 110.53, 108.71, 108.33, 101.95,
67.69, 60.73, 56.35, 53.53, 43.53, 41.37, 35.24. MS (EI) m/z: 424
(M þ 1); HRMS (m/z) calcd for C23H21NO7: 441.1656 [M þ NH4]þ,
Found: 441.1652 [M þ NH4]þ.
4.3. General procedure for synthesis of 20aej
Surprisingly, in the
(13aec) compounds displayed cytotoxicity, albeit only moderate,
against the four human tumor cell lines. It is not clear why most
acylamino carboxamide analogs were inactive, while correspond-
ing -acyloxy carboxamide derivatives (e.g., 13h vs 12e, 13d vs 12a)
were active. However, a general relationship between substituent
size and activity appears to be present and merits further
investigation.
a-acylamino carboxamide series, only three
To a solution of aldehyde/ketone (0.13 mmol) in MeOH (10 mL)
was added stable nitroxide radical acid (0.13 mmol) and 4b-iso-
a
-
cyanopodophyllotoxin (11) (0.10 mmol) at room temperature. The
reaction mixture was subsequently heated at reflux for 5 h. Upon
completion of the reaction (TLC monitoring), the mixture was
cooled to room temperature and solvent was evaporated. The res-
idue was purified by chromatography on silica gel using EtOAc/
petroleum ether as eluant to give 12aej.
a
3. Conclusion
4.3.1. Compound 12a
Yield 36%; m.p: 83e85 ꢁC; IR (KBr) cmꢂ1: 3431 (NeH),1776,1717
(C]O), 1589, 1506, 1484, 1465 (Ar), 1383 (NeO), 931 (OCH2O); ESR
In summary, efficient isocyanide multicomponent reactions
have been developed to prepare newer-generation structurally
diverse spin-labeled derivatives of epipodophyllotoxin. This
approach is a valuable tool in design and synthesis of new podo-
phyllotoxin analogs with advantages of simplicity, atom-economy,
and good yields. The cytotoxic results showed that most of the
new compounds exhibited moderate to potent cytotoxic activity
against A-549, DU-145, KB and KBvin, and overcame acquired drug
resistance in the latter cell line. Among them, compounds 12e and
12h were the most promising derivatives and were selected as lead
molecules for further development. Additional systematic struc-
tural modifications will be carried out to further clarify these initial
interesting findings. Future applications could also involve devel-
opment of versatile biologically significant podophyllotoxin frag-
ments into MCR products, chemical libraries created via MCRs, and
heterocycles built from isocyanides.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.9 ꢃ 10ꢂ4
,
D
Ho ¼ 2.66 ꢃ 10ꢂ4; MS (EI) (m/z): 667 [M þ 2H]þ; HRMS (ESI) (m/z)
for C35H41N2O11 [M þ Na]þ: calc. 688.2710, found 688.2723.
4.3.2. Compound 12b
Yield 52%; m.p: 138e139 ꢁC; IR (KBr) cmꢂ1: 3433 (NeH), 1778,
1723 (C]O), 1589, 1504, 1485 (Ar), 1385 (NeO), 935 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.8 ꢃ 10ꢂ4
,
D
Ho ¼ 2.61 ꢃ10ꢂ4; MS (EI) (m/z): 736 [M þ Na]þ; HRMS (ESI) (m/z)
for C39H41N2O11 [M þ Na]þ: calc. 736.2710, found 736.2752.
4.3.3. Compound 12c
Yield 79%; m.p: 113e115 ꢁC; IR (KBr) cmꢂ1: 3437 (NeH), 1778,
1720 (C]O), 1588, 1506, 1484, 1466 (Ar), 1383 (NeO), 932 (OCH2O);
ESR (1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.7 ꢃ 10ꢂ4
,
D
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 680 [M þ H]þ; HRMS (ESI) (m/z)
4. Experimental section
for C36H43N2O11 [M þ H]þ: calc. 680.2867, found 680.2864.
4.1. Chemistry
4.3.4. Compound 12d
Yield 66%; m.p: 119e121 ꢁC; IR (KBr) cmꢂ1: 3438 (NeH), 1776,
1718 (C]O), 1588, 1505, 1484 (Ar), 1382 (NeO), 932 (OCH2O); ESR
Melting points were determined on a Kofler apparatus and are
uncorrected. IR spectra were measured on a Nicolet 380 FT-IR
spectrometer on neat samples placed between KBr plates. Mass
spectra were recorded on a Bruker Daltonics APEXII49e spec-
trometer with ESI ionization source. Electron spin resonance (ESR)
spectra were obtained on a Bruker A300 X-band EPR spectrometer.
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 start-
ing material for preparation of all new derivatives. The precursor
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.9 ꢃ 10ꢂ4
,
D
Ho ¼ 2.66 ꢃ 10ꢂ4; MS (EI) (m/z): 706 [M þ H]þ; HRMS (ESI)(m/z)
for C38H45N2O11 [M þ H]þ: calc. 706.3023, found 706.3021.
4.3.5. Compound 12e
Yield 48%; m.p: 95e97 ꢁC; IR (KBr) cmꢂ1: 3431 (NeH),1774,1718
(C]O), 1610, 1508, 1458 (Ar), 1364 (NeO), 932 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.7 ꢃ 10ꢂ4
,
D
Ho ¼ 2.61 ꢃ 10ꢂ4; MS (EI) (m/z): 744 [M þ H]þ; HRMS (ESI) (m/z)
for C40H43N2O12 [M þ H]þ: calc. 744.2816, found 744.2815.
4b-aminoepipodophyllotoxin (9) was synthesized by our previ-
ously reported procedures, and its structure was confirmed by
direct comparison with an authentic sample and previously re-
ported spectroscopic data [32].
4.3.6. Compound 12f
Yield 64%; m.p: 105e107 ꢁC; IR (KBr) cmꢂ1: 3428 (NeH), 1775,
1719 (C]O), 1588, 1506, 1456 (Ar), 1384 (NeO), 931 (OCH2O); ESR

L. Kou et al. / European Journal of Medicinal Chemistry 75 (2014) 282e288
287
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.9 ꢃ 10ꢂ4
,
4.4.4. Compound 13d
D
Ho ¼ 2.61 ꢃ 10ꢂ4; MS (EI) (m/z): 728 [M þ H]þ; HRMS (ESI) (m/z)
Yield 50%; m.p: 115e117 ꢁC; IR (KBr) cmꢂ1: 3433 (NeH), 1772
(C]O), 1590, 1506, 1483 (Ar), 1370 (NeO), 934 (OCH2O); ESR
for C40H43N2O11 [M þ H]þ: calc. 728.2867, found 728.2864.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.9 ꢃ 10ꢂ4
,
D
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 708 [Mþ2H]þ; HRMS (ESI) (m/z)
4.3.7. Compound 12g
for C38H48N3O10 [M þ K]þ: calc. 745.3340, found 745.3342.
Yield 62%; m.p: 119e121 ꢁC; IR (KBr) cmꢂ1: 3438 (NeH), 1776,
1719 (C]O), 1589, 1506, 1466 (Ar), 1383 (NeO), 930 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.9 ꢃ 10ꢂ4
,
4.4.5. Compound 13e
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 705 [M þ 2H]þ; HRMS (ESI) (m/z)
Yield 42%; m.p: 115e116 ꢁC; IR (KBr) cmꢂ1: 3429 (NeH), 1590,
1507, 1483 (Ar), 1363 (NeO), 934 (OCH2O); ESR (1 ꢃ 10ꢂ5 mol/L in
D
for C37H39N2O12 [M þ Na]þ: calc. 726.2503, found 726.2511.
EtOH): go ¼ 2.0058, AN ¼ 14.7 ꢃ 10ꢂ4
,
D
Ho ¼ 2.61 ꢃ 10ꢂ4; MS (EI)
(m/z): 72 1[M þ H]þ; HRMS (ESI) (m/z) for C39H50N3O10 [M þ H]þ:
4.3.8. Compound 12h
Yield 59%; m.p: 110e112 ꢁC; IR (KBr) cmꢂ1: 3423 (NeH), 1774,
1718 (C]O), 1589, 1507, 1458 (Ar), 1363 (NeO), 931 (OCH2O); ESR
calc. 721.3496, found 721.3493.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.7 ꢃ 10ꢂ4
,
4.4.6. Compound 13f
D
Ho ¼ 2.61 ꢃ 10ꢂ4; MS (EI) (m/z): 758 [M þ H]þ; HRMS (ESI) (m/z)
Yield 68%; m.p: 86e88 ꢁC; IR (KBr) cmꢂ1: 3434 (NeH), 1772 (C]
O), 1592, 1507, 1466 (Ar), 1368 (NeO), 935 (OCH2O); ESR
for C40H41N2O13 [M þ H]þ: calc. 758.2609, found 758.2611.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.9 ꢃ 10ꢂ4
,
4.3.9. Compound 12i
D
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 722 [Mþ2H]þ; HRMS (ESI) (m/z)
Yield 85%; m.p: 110e112 ꢁC; IR (KBr) cmꢂ1: 3436 (NeH), 1778,
1736 (C]O), 1588, 1506, 1465 (Ar), 1364 (NeO), 933 (OCH2O); ESR
for C39H50N3O10 [M þ Na]þ: calc. 743.3496, found 743.3492.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.9 ꢃ 10ꢂ4
,
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 697 [Mþ2H]þ; HRMS (ESI) (m/z)
4.4.7. Compound 13g
D
Yield 84%; m.p: 147e149 ꢁC; IR (KBr) cmꢂ1: 3426 (NeH), 1775
(C]O), 1507, 1458 (Ar), 1368 (NeO), 932 (OCH2O); ESR
for C37H47N2O11 [M þ Na]þ: calc. 718.3180, found 718.3185.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.8 ꢃ 10ꢂ4
,
4.3.10. Compound 12j
D
Ho ¼ 2.66 ꢃ 10ꢂ4; MS (EI) (m/z): 755 [M þ H]þ; HRMS (ESI) (m/z)
Yield 76%; m.p: 106e108 ꢁC; IR (KBr) cmꢂ1: 3437 (NeH), 1778,
1718 (C]O), 1588, 1506, 1466 (Ar), 1362 (NeO), 934 (OCH2O); ESR
for C42H48N3O10 [M þ H]þ: calc. 755.3340, found 755.3342.
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.7 ꢃ 10ꢂ4
,
D
Ho ¼ 2.67 ꢃ 10ꢂ4; MS (EI) (m/z): 694 [M þ H]þ; HRMS (ESI) (m/z)
4.4.8. Compound 13h
for C37H45N2O11 [M þ H]þ: calc. 694.3023, found 694.3014.
Yield 60%; m.p: 95e96 ꢁC; IR (KBr) cmꢂ1: 3392 (NeH), 1779,
1717 (C]O), 1609, 1508, 1451 (Ar), 1382 (NeO), 930 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.7 ꢃ 10ꢂ4
,
4.4. General procedure for synthesis of 13ael
D
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 807 [M þ Na]þ; HRMS (ESI) (m/z)
for C43H50N3O11 [M þ Na]þ: calc. 807.3445, found 807.3443.
Aldehyde/ketone (0.23 mmol), stable nitroxide radical acid
(0.23 mmol), and 4 -isocyanopodophyllotoxin (11) (0.18 mmol)
b
were added sequentially to a solution of amine (0.23 mmol) in
MeOH (10 mL) at room temperature. The reaction mixture was
heated at reflux for 5 h. Upon completion of the reaction (TLC
monitoring), the mixture cooled to room temperature and solvent
was evaporated. The crude reaction mixture was purified by col-
umn chromatography (EtOAc/petroleum ether) to give the target
product 13ael.
4.4.9. Compound 13i
Yield 76%; m.p: 106e109 ꢁC; IR (KBr) cmꢂ1: 3426 (NeH), 1768
(C]O), 1591, 1506 (Ar), 1374 (NeO), 934 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.7 ꢃ 10ꢂ4
,
D
C
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 746 [M]þ; HRMS (ESI) (m/z) for
41H52N3O10 [M]þ: calc. 746.3653, found 746.3655.
4.4.10. Compound 13j
4.4.1. Compound 13a
Yield 45%; m.p: 110e112 ꢁC; IR (KBr) cmꢂ1: 3447 (NeH), 1772
(C]O), 1590, 1507, 1466 (Ar), 1375 (NeO), 932 (OCH2O); ESR
Yield 54%; m.p: 120e123 ꢁC; IR (KBr) cmꢂ1: 3448 (NeH), 1772
(C]O), 1592, 1507, 1465 (Ar), 1377 (NeO), 934 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.9 ꢃ 10ꢂ4
,
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0058, AN ¼ 14.8 ꢃ 10ꢂ4
,
D
Ho ¼ 2.66 ꢃ 10ꢂ4; MS (EI) (m/z): 736 [Mþ2H]þ; HRMS (ESI) (m/z)
D
Ho ¼ 2.66 ꢃ 10ꢂ4; MS (EI) (m/z): 762 [Mþ2H]þ; HRMS (ESI) (m/z)
for C40H52N3O10 [M þ Na]þ: calc. 757.3653, found 757.3652.
for C40H54N3O10 [M þ Na]þ: calc. 783.3809, found 783.3806.
4.4.11. Compound 13k
4.4.2. Compound 13b
Yield 39%; m.p: 86e89 ꢁC; IR (KBr) cmꢂ1: 3432 (NeH), 1772 (C]
O), 1591, 1507, 1465 (Ar), 1378 (NeO), 935 (OCH2O); ESR
Yield 47%; m.p: 103e105 ꢁC; IR (KBr) cmꢂ1: 3421 (NeH), 1775,
1718 (C]O), 1591, 1507, 1465 (Ar), 1358 (NeO), 931 (OCH2O); ESR
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.8 ꢃ 10ꢂ4
,
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.8 ꢃ 10ꢂ4
,
Ho ¼ 2.61 ꢃ 10ꢂ4; MS (EI) (m/z): 736 [Mþ2H]þ; HRMS (ESI) (m/z)
D
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 755 [M þ H]þ; HRMS (ESI) (m/z)
D
for C40H52N3O10 [M þ Na]þ: calc. 757.3653, found 757.3652.
for C42H48N3O10 [M þ H]þ: calc. 755.3340, found 755.3343.
4.4.3. Compound 13c
4.4.12. Compound 13l
Yield 42%; m.p: 157e159 ꢁC; IR (KBr) cmꢂ1: 3423 (NeH), 1777,
1735 (C]O), 1592, 1505, 1458 (Ar), 1368 (NeO), 932 (OCH2O); ESR
Yield 46%; m.p: 92e94 ꢁC; IR (KBr) cmꢂ1: 3356 (NeH), 1778 (C]
O), 1585, 1451 (Ar), 1381 (NeO), 933 (OCH2O); ESR (1 ꢃ 10ꢂ5 mol/L
(1 ꢃ 10ꢂ5 mol/L in EtOH): go ¼ 2.0057, AN ¼ 14.8 ꢃ 10ꢂ4
,
in EtOH): go ¼ 2.0057, AN ¼ 14.9 ꢃ 10ꢂ4
,
D
Ho ¼ 2.66 ꢃ 10ꢂ4; MS (EI)
D
Ho ¼ 2.65 ꢃ 10ꢂ4; MS (EI) (m/z): 805 [M þ H]þ; HRMS (ESI) (m/z)
(m/z): 738 [Mþ2H]þ; HRMS (ESI) (m/z) for C40H54N3O10 [Mþ2H]þ:
for C46H50N3O10 [M þ H]þ: calc. 805.3496, found 805.3497.
calc. 738.3809, found 738.3811.

288
L. Kou et al. / European Journal of Medicinal Chemistry 75 (2014) 282e288
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(SRB) colorimetric assay as previously described [33]. In brief, the
cells (3e5 ꢃ 103 cells/well) were seeded in 96-well plates filled
with RPMI-1640 medium supplemented with 10% fetal bovine
serum (FBS) containing various concentrations of samples, and
incubated for 72 h. At the end of the exposure period, the attached
cells were fixed with cold 50% trichloroacetic acid for 30 min fol-
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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.
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Acknowledgments
This work was supported financially by the National Natural
Science Foundation of China (30800720, 31371975); Partial
financial support was supplied by Xinjiang Production & Con-
struction Corps Key Laboratory of Protection and Utilization of
Biological Resources in Tarim Basin (BYRB1306); the Fundamental
Research Funds for the Central Universities (lzujbky-2013-69) and
the Young Scholars Science Foundation of Lanzhou Jiaotong Uni-
versity (2011011). Partial support was also supplied by NIH grant
CA177584 from the National Cancer Institute awarded to K.H. Lee.
Thanks are also due to the support of Taiwan Department of
Health Cancer Research Center of Excellence (DOH-100-TD-C-111-
005).
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