Glucoside derivatives of podophyllotoxin: Synthesis, physicochemical properties, and cytotoxicity

Article abstract of DOI:10.2147/DDDT.S215895

Background: Widespread concern of the side effects and the broad-spectrum anticancer property of podophyllotoxin as an antitumor agent highlight the need for the development of new podophyllotoxin derivatives. Although some per-butyrylated glucosides of podophyllotoxin and 4β-triazolyl-podophyllotoxin glycosides show good anticancer activity, the per-acetylated/free of podophyllotoxin glucosides and their per-acetylated are not well studied. Methods: A few glucoside derivatives of PPT were synthesized and evaluated for their in vitro cytotoxic activities against five human cancer cell lines, HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer), as well as the normal human pulmonary epithelial cell line (BEAS-2B). In addition, we investigated the structure–activity relationship and the physicochemical property–anticancer activity relationship of these compounds. Results: Compound 6b shows the highest cytotoxic potency against all five cancer cell lines tested, with IC₅₀ values ranging from 3.27±0.21 to 11.37±0.52 μM. We have also found that 6b displays higher selectivity than the etoposide except in the case of HL-60 cell line. The active compounds possess similar physicochemical properties: MSA > 900, %PSA < 20, ClogP > 2, MW > 700 Da, and RB > 10. Conclusion: We synthesized several glucoside derivatives of PPT and tested their cytotoxicity. Among them, compound 6b showed the highest cytotoxicity. Further studies including selectivity of active compounds have shown that the selectivity indexes of 6b are much greater than the etoposide except in the case of HL-60 cell line. The active compounds possessed similar physicochemical properties. This study indicates that active glucoside analogs of podophyllotoxin have potential as lead compounds for developing novel anticancer agents.

Full text of DOI:10.2147/DDDT.S215895

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O R I G I N A L R E S E A R C H
Glucoside Derivatives Of Podophyllotoxin: Synthesis,
Physicochemical Properties, And Cytotoxicity
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Cheng-Ting Zi1,2,
Liu Yang2,*
*
Background: Widespread concern of the side effects and the broad-spectrum anticancer
property of podophyllotoxin as an antitumor agent highlight the need for the development of
new podophyllotoxin derivatives. Although some per-butyrylated glucosides of podophyllo-
toxin and 4β-triazolyl-podophyllotoxin glycosides show good anticancer activity, the per-
acetylated/free of podophyllotoxin glucosides and their per-acetylated are not well studied.
Methods: A few glucoside derivatives of PPT were synthesized and evaluated for their in
vitro cytotoxic activities against five human cancer cell lines, HL-60 (leukemia), SMMC-
7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer),
as well as the normal human pulmonary epithelial cell line (BEAS-2B). In addition, we
investigated the structure–activity relationship and the physicochemical property–anticancer
activity relationship of these compounds.
Qing-Hua Kong2
Hong-Mei Li2
Xing-Zhi Yang2
Zhong-Tao Ding3
4
Zi-Hua Jiang
2
Jiang-Miao Hu
Jun Zhou2
1Key Laboratory of Pu-Er Tea Science,
Ministry of Education, College of Science,
Yunnan Agricultural University, Kunming,
650201, People’s Republic of China;
2State Key Laboratory of Phytochemistry
and Plant Resources in West China,
Kunming Institute of Botany, Chinese
Academy of Sciences, Kunming 650201,
People’s Republic of China; 3Key
Laboratory of Medicinal Chemistry for
Nature Resource, Ministry of Education,
School of Chemical Science and
Technology, Yunnan University, Kunming
650091, People’s Republic of China;
4Department of Chemistry, Lakehead
University, Thunder Bay ON P7B 5E1,
Canada
Results: Compound 6b shows the highest cytotoxic potency against all five cancer cell lines
tested, with IC50 values ranging from 3.27 0.21 to 11.37 0.52 μM. We have also found that
6b displays higher selectivity than the etoposide except in the case of HL-60 cell line. The
active compounds possess similar physicochemical properties: MSA > 900, %PSA < 20,
ClogP > 2, MW > 700 Da, and RB > 10.
Conclusion: We synthesized several glucoside derivatives of PPTand tested their cytotoxicity.
Among them, compound 6b showed the highest cytotoxicity. Further studies including selectiv-
ity of active compounds have shown that the selectivity indexes of 6b are much greater than the
etoposide except in the case of HL-60 cell line. The active compounds possessed similar
physicochemical properties. This study indicates that active glucoside analogs of podophyllo-
toxin have potential as lead compounds for developing novel anticancer agents.
Keywords: podophyllotoxin, glucoside, synthesis, cytotoxicity, physicochemical properties
*These authors contributed equally to
this work
Introduction
Correspondence: Zi-Hua Jiang
Department of Chemistry, Lakehead
University, 955 Oliver Road, Thunder Bay
ON P7B 5EI, Canada
Cancer is the second leading cause of death in the worldwide and remains one of
the most difficult diseases to combat.1 Developing new anticancer drugs and more
effective treatment strategies for cancer is of great importance in medicinal chem-
istry. Natural products with diverse structures and unique biological activities are
valuable sources for drug discovery. Close to 60% clinical drugs are either natural
products or structural analogs of natural products with improved pharmacological
activity.2–4 Podophyllotoxin (PPT, 1, Scheme 1), a well-known naturally occurring
aryltetralin lignan, is mainly isolated from the roots of the North American
Podophyllum peltatum Linnaeus, the Tibetan P. emodi Wall, or the Taiwanese
species Podophyllum peltatum.5 It shows strong cytotoxic activity against various
cancer cell lines and acts at the colchicine-binding site on tubulin.6
Tel +1 807 766 7171
Fax +1 807 346 7775
Email zjiang@lakeheadu.ca
Jiang-Miao Hu
State Key Laboratory of Phytochemistry
and Plant Resources in West China,
Kunming Institute of Botany, Chinese
Academy of Sciences, No. 132, Lanhei
Road, Kunming 650201, People’s Republic
of China
Tel +86 871 6522 3264
Fax +86 871 6522 3261
Email hujiangmiao@mail.kib.ac.cn
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Scheme 1 Structure of compounds 1–6: podophyllotoxin (1), etoposide (2), teniposide (3), etopophos (4), NK-611 (5), and podophyllotoxin glucosides (6).
Due to its high toxicity and poor water solubility, podo- derivatives, such as etopophos (4) and NK-611 (5) (Scheme
1), which reached clinical studies.15 The clinically useful
phyllotoxin has limited application as an anticancer drug.
Based on its potent anticancer activity, PPT has served as a
lead compound for the discovery and development of new
anticancer agents. For example, the two semisynthetic gluco-
sidic cyclic acetals of PPT, etoposide (2) and teniposide (3)
(Scheme 1), are in clinical use for the treatment of a variety of
cancers, including small-cell lung cancer, non-Hodgkin’s lym-
phoma, leukemia, Kaposi’s sarcoma, neuroblastoma, and soft-
tissue sarcoma.7–10 The mechanism of action for etoposide and
teniposide is different from that of PPT in that both etoposide
podophyllotoxin-derived glucosides 2–5 possess a 4,6-cyclic
acetal moiety and various other substitutions on the sugar
residue, suggesting the important role of substituents in mod-
ifying the biological activities of these podophyllotoxin
derivatives.
In recent years, we have been working on the structural
modification of podophyllotoxin and focused on glyco-
sides of podophyllotoxin (such as 6, Scheme 1) and 4β-
triazolyl-podophyllotoxin.16–19 Per-butyrylated glucosides
and teniposide block the DNA topoisomerase-II by stabilizing of podophyllotoxin16 as well as the glucosides of 4β-tria-
the enzyme–DNA complex.11–14 However, the therapeutic use zolyl-podophyllotoxin and their acylated analogues show
of etoposide and teniposide is often hindered by problems such good cytotoxicity.19,20 The glucosides of podophyllotoxin
as acquired drug resistance, myelosuppression, and their poor and their per-acetylated analogs are less well studied.21 In
water solubility. To overcome the problems of etoposide and this article, a few glucoside derivatives of PPT were
teniposide, further structure modifications of PPT have been synthesized (Table S1) and evaluated for their in vitro
carried out, which led to the synthesis of other PPT cytotoxic activity against five human cancer cell lines,
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HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung NMR data of compounds 6a – 6d are shown in Table 1. In
1
cancer), MCF-7 (breast cancer), and SW480 (colon can- the H-NMR spectra, the proton at C-4 of 4β-substituted
cer). To evaluate the selectivity of these compounds compounds appears as a doublet at 4.72–4.96 ppm, usually
between tumor cells and normal cells, their growth inhibi- with a coupling constant J3-4 < 4.0 Hz, indicating a cis-
tory effect was tested on normal human pulmonary epithe- relationship between C3-H and C4-H.31 The coupling con-
lial cell lines (BEAS-2B). In addition, the physicochemical stant of the anomeric proton of the glucose residue (J1”-2”
)
properties of these compounds were calculated and corre- is typically <4.0 Hz, which confirms that the glycosidic
lated with their anticancer activity.
linkage is fan α–linkage.
Results And Discussion
Chemical Synthesis
Evaluation Of Biological Activity
The per-butyrylated glucoside derivatives of podophyllo-
There have been several reports on constructing the glucosi- toxin 6e and 6f have been previously documented.16 Per-
dic linkages of podophyllotoxin according to known acetylated glucoside derivatives of podophyllotoxin (6a
literatures.22–25 The synthesis of glucoside derivatives of and 6b) and podophyllotoxin glucosides (6c and 6d)
podophyllotoxin 6a – 6d following a similar method is were tested for their cytotoxicity against five human can-
reported in the literature and is shown in Scheme 2. cer cell lines, including HL-60 (leukemia), SMMC-7721
1,2,3,4,6-Penta-O-acetyl-α/β-D-glucopyranose (mainly α- (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer),
form)26 was treated with ammonia solution (25%) in aceto- and SW480 (colon cancer). Etoposide (2) and cisplatin
nitrile to give 2,3,4,6-tetra-O-acetyl -α/β-D-glucopyranose were taken as control drugs, and their IC50 data are pre-
(8) as an anomeric mixture (α/β ratio = 6:1) in 46% yield.27,28 sented in Figure 1 and Table 2. Compounds 6c and 6d
Then, compound 8 was allowed to react with podophyllo- having a free glucose residue show weak activity (all
toxin (1) and 4ʹ-demethylepipodophyllotoxin (9)29 in the having IC50 > 40 μM), while peracetylated glucoside deri-
presence of trifluoroboran etherate (BF3•Et2O) at −78°C to vatives 6a and 6b show improved activity. Among these
give the per-acetylated glucoside derivatives of podophyllo- derivatives, compound 6b shows the highest cytotoxicity
toxin 6a and 6b in 58–62% yield.16 Compounds 6a and 6b against five cancer cells, with their IC50 values ranging
were treated with sodium methoxide in methanol at room from 3.27 0.21 to 11.37 0.52 μM, which is more potent
temperature for 2 hrs to yield podophyllotoxin glucosides 6c than the control drug etoposide against the MCF-7 and
and 6d in 78–80% yields.30
SW480 cell lines. In our previous study, we reported that
All the glucoside derivatives of PPT were character- the cytotoxic activity of 4β-triazolyl-podophyllotoxin deri-
1
ized by H and 13C-NMR, electrospray ionization mass vatives with a peracetylated glucose residue mostly shows
spectrometry (ESI-MS), and high-resolution mass spectro- weak activity.19 Furthermore, compound 6b with a
1
metry (HRESI-MS). The characteristic H-NMR and 13C- hydroxy group at the C-4ʹ position in the E ring is more
Scheme 2 Synthesis of glucoside derivatives of PPT 6a – 6d. Reagents and conditions: (A) Ac2O, sodium acetate, 100°C, 20 mins, ~99%; (B) NH3⋅ H2O, CH3CN, rt,
overnight, 46%; (C) BF3⋅ Et2O, CH2Cl2, −78°C to rt, 58–62%; (D) CH3ONa, CH3OH, 2 hrs, rt, 78–80%.
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1
Table 1 The Characteristic H-NMR And 13C-NMR Data Of Compounds 6a – 6d
Compound
1H-NMR
13C-NMR
4-Configuration
C4-H (ppm)
J3-4 (Hz)
C1”-H (ppm)
J1”-2” (Hz)
C-4 (ppm)
C-1” (ppm)
6a
4.76
4.72
4.78
4.77
4.96
2.7
3.8
3.4
3.4
7.4
5.22
5.35
5.01
4.99
–
3.2
3.2
3.6
3.6
–
75.7
75.7
75.3
75.3
72.6
95.7
95.7
99.4
99.4
–
β
β
β
β
α
6b
6c
6d
PPTa
Note: aData from Hartwell et al.38
active than compound 6a which has a methoxyl group at toward cancer cells with SI values in the range of 1.9–6.7 in
all cells tested. Compound 6b displays higher selectivity
than etoposide in four of the five cancer cell lines tested
except an HL-60 cell line. Among these derivatives, 6b
shows the highest potency (IC50 3.27 0.21 μM) and highest
selectivity (SI 6.7) in SW480 cell line, suggesting that 6b
may be a promising therapeutic agent for colon cancer.
the C-4ʹ position.
Cancer chemotherapy is often associated with low/non-
selectivity of cancer drugs which attack cancer cells as well
as normal cells, leading to serious side effects. In order to
test their selectivity, compounds 6a and 6b were tested for
their growth inhibitory effects on a normal human bronchial
epithelial cell line (BEAS-2B) (Table 2). The selectivity
index (SI) was expressed as the ratio of the IC50 value of
the compound in normal cell line over that in cancer cell
line. A larger SI value indicates that the drug displays
higher selectivity toward cancer cells over normal cells.32,33
The SI values of compound 6a, 6b and etoposide are pre-
Physicochemical Property–Cytotoxicity
Relationship
Values Of Partition Coefficient Of The Compounds
The logarithm of the octanol–water partition coefficient inves-
tigation (logP) is an important pharmaceutical parameter in
sented in Table 3. Compound 6b shows moderate selectivity evaluating solvency, absorption, and transport of drugs; the
Figure 1 Inhibitory effects of podophyllotoxin derivatives on cancer cells. (A–D) The inhibitory effects of compounds 6a – 6d on HL-60 (leukemia), SMMC-7721
(hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer) cells, as evaluated by the MTT assay.
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Table 2 Cytotoxicity Of Podophyllotoxin Derivatives 6a – 6f In Vitroa
Compound
IC50 (μM)
HL-60
SMMC-7721
A-549
MCF-7
SW480
BEAS-2B
6a
21.36 0.38
11.37 0.52
>40
14.50 0.56
8.41 0.48
>40
>40
36.55 0.78
9.18 0.49
>40
>40
30.60 0.54
21.78 0.36
NT
6b
10.74 0.37
>40
3.27 0.21
>40
6c
6d
>40
>40
>40
>40
>40
NT
6eb
>40
>40
>40
>40
>40
NT
6fb
16.87 0.32
0.31 0.24
1.17 0.34
16.82 0.12
8.12 0.72
6.43 0.57
16.04 0.73
11.92 0.12
9.24 0.36
39.13 0.52
32.82 0.44
15.56 0.52
38.72 0.92
17.11 0.67
13.42 0.44
NT
2
11.17 0.56
NT
Cisplatin
Note: aValues are means of three independent experiments; bExperimental data of compounds 6e and 16f from ref.16
Abbreviation: NT, not tested.
Table 3 Selectivity Of The Cytotoxicity Of Compounds 6a, 6b, And Etoposide To Cancer Cells As Compared With BEAS-2B
Normal Cells
Compound
Selectivity Index (SIa)
HL-60
SMMC-7721
A-549
MCF-7
SW480
6a
6b
2
1.4
2.1
2.6
1.4
–
0.8
2.4
0.3
–
1.9
2.0
0.9
6.7
0.7
36.0
Note: aSelectivity index (SI) = IC50 of the compound in BEAS-2B cell line/IC50 of the compound in cancer cell line.
preferred logP value is less than 5.11 Compounds etoposide (2) chemical properties of glucoside derivatives of podophyllo-
toxin 6a – 6d and 6e – 6f19 were calculated and compared
and the most potent compound 6b were measured for values of
logP. Solutes were equilibrated between octanol and water.
The concentration of compounds in octanol was determined
by the HPLC method.12,13 The logP values of compounds 2
and 6b were determined to be 1.44 and 1.78 at 30°C. As shown
in Table 4 (see supporting information for the details), com-
pound 6b expressed the logP value and was close to the
calculated value of 2.24.
with etoposide 2, which include molecular weight (MW),
molecular surface area (MSA), polar surface area (PSA),
relative polar surface area (%PSA), calculated partition
coefficient (ClogP), calculated distribution coefficient at
pH 7.4 (ClogD7.4), hydrogen bond donor (HD), hydrogen
bond acceptor (HA), and rotatable bond (RB) (Table 4).
Noteworthy is that almost all active compounds (having
IC50 < 40 μM) are relatively lipophilic (MSA > 900, %
PSA < 20, ClogP > 2), and since they have a higher
molecular weight (MW > 700 Da) and a larger number of
rotatable bonds (RB > 10), with the exception of compound
6e, they are placed at an advantage for further optimization.
By contrast, inactive compounds 6c and 6d (having a free
glucose residue) have %PSA values >22, ClogD7.4 <0, and
a smaller number of rotatable bonds (RB < 10). It is
obvious that derivatives with free glucose residues (6c and
6d) are relatively more polar, and this might account for the
general lack of activity for these compounds. This result
suggests that the peracetylated/perbutyrylated derivatives of
podophyllotoxin glucosides may, therefore, be more
Solubility
Poor water solubility is a common problem in developing
podophyllotoxin derivatives for therapeutic use. Compounds
with glucose residue are slightly soluble in water. The solubi-
lity of podophyllotoxin (1) and compounds 6b in aqueous at
temperature 25°C are reported 1 has a solubility of 2.2 mg/mL
in water, while 6b with a peracetylated glucoside residue has a
solubility of 1.7 mg/mL in water (see supporting information
for the details). The solubility values obtained for 6b become
unfairly soluble in water.
Physicochemical Property
The physicochemical properties of a drug can largely affect
the pharmacokinetics and efficacy of a drug. The physico- suitable for further optimization.
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Table 4 Physicochemical Properties Of Glucoside Derivatives Of Podophyllotoxin
Compound
Physicochemical Properties
MW
MSA
PSA
%PSAa
ClogPb
ClogD7.4c
H-Dd/H-Ae
RBf
logPg
6a
6b
6c
6d
6e
6f
745
731
576
562
857
843
588
1006
968
196.1
207.0
171.8
182.8
196.1
207.1
160.8
19.5
21.4
22.8
25.5
15.6
17.0
21.2
2.57
2.24
0.43
0.29
6.80
6.47
0.03
1.62
1.47
−0.15
−0.30
6.20
6.05
1.16
0/12
1/12
4/12
5/12
0/12
1/12
3/12
15
14
7
logPg
NT
755
2.14
NT
716
6
1257
1218
758
23
22
5
NT
NT
2
NT
Note: a%PSA: relative polar surface area = (PSA/MSA) × 100; bClogP: calculated partition coefficient; cClogD7.4: calculated distribution coefficient at pH 7.4; dHD: hydrogen
f
bond donor count; eHA: hydrogen bond acceptor count; RB: rotatable bond count; glogP: value for log octanol-water partition coefficients.
Abbreviations: MW, molecular weight; MSA, molecular surface area; PSA, polar surface area; NT, Not tested.
over calcium hydride. All reagents were commercially avail-
Chemical Stability Investigation
able and used without further purification unless indicated
otherwise. The melting points were measured by an X-4
melting point apparatus and were uncorrected. Optical rota-
tions were obtained with a Jasco P-1020 Automatic Digital
Polariscope MS data were obtained in the ESI mode on API
Qstar Pulsar instrument; HRMS data were obtained in the
ESI mode on LCMS-IT-TOF (Shimadzu, Kyoto, Japan); 1H-
NMR and 13C-NMR spectra were recorded on Bruker
AVANCE III 400 MHz, or 600 MHz (Bruker BioSpin
GmbH, Rheinstetten, Germany) instruments, using tetra-
methylsilane (TMS) as an internal standard: chemical shifts
(δ) are given in ppm, coupling constants (J) in Hz, and the
solvent signals were used as references (CDCl3: δC= 77.2
ppm; residual CHCl3 in CDCl3: δH= 7.26 ppm; CD3OD: δC=
49.0 ppm; residual CH3OH in CD3OD: δH= 4.78 ppm).
Column chromatography (CC): silica gel (200–300 mesh;
Qingdao Makall Group CO., LTD; Qingdao; China). All
reactions were monitored using thin-layer chromatography
(TLC) on silica gel plates.
The most potent compound 6b was selected for inves-
tigations of chemical stability in aqueous phase with
comparison of podophyllotoxin (1). The results indi-
cate that compound 6b exhibits better chemical stabi-
lity under the specific conditions (37°C, pH = 7.0,
Figure 2) (see supporting information for the details).
Obviously, 6b showed considerable stability with
podophyllotoxin.
Experimental
General
All cancer cells (HL-60, SMMC-7721, A-549, MCF-7, and
SW480) were obtained from a Shanghai cell bank in China.
D-glucose was purchased from Aladdin Chemical Co., Ltd
(Guangzhou, China); podophyllotoxin was obtained from
Chengdu Proifa Technology Development Co., Ltd
(Chengdu, China); boron trifluoride etherate was obtained
from J&K Chemical Technology Co., Ltd (Beijing China); 3-
(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) was purchased from Sigma-Aldrich (St. Louis,
MO, USA). Dichloromethane and acetonitrile were distilled
Chemistry
Synthesis Of 2,3,4,6-Tetra-O-Acetyl-α/β-D-
Glucopyranose (8)
D-glucose (1.8 g, 10 mmol) was suspended in acetic
anhydride (9.5 mL, 100 mmol) and anhydrous sodium
acetate (0.9 g, 11 mmol) was added, and the resulting
mixture was heated at 100°C for 20 mins. The reaction
was quenched (saturated aqueous sodium bicarbonate,
20 mL) and diluted with dichloromethane (30 mL); the
organic layer was washed with brine (3 × 30 mL) and
dried with sodium sulfate. The solvent was evaporated,
and the residue dried in vacuo to give the crude 1,2,3,4,6-
penta-O-acetyl-D-glucopyranose.
Figure 2 Chemical stability investigation of compounds 1 and 6b.
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The crude 1,2,3,4,6-penta-O-acetyl-D-glucopyranose (C=O), 169.3 (C=O), 169.2 (C=O), 153.4 (C-3ʹ, C-5ʹ),
was dissolved in acetonitrile (20 mL), and 25% ammonia 147.9 (C-6), 146.7 (C-7), 137.5 (C-4ʹ), 136.9 (C-1ʹ), 131.5
solution (0.4 mL, 20 mmol) was added dropwise slowly. (C-9), 128.4 (C-10), 109.7 (C-8), 106.8 (C-5), 105.4 (C-2ʹ,
The mixture was stirred at room temperature for 6 hrs. The C-6ʹ), 101.3 (OCH2O), 95.7 (C-1ʹ’), 75.7 (C-4), 72.3, 71.0,
solvent was evaporated, and the brown oily residue was 69.4, 68.2, 67.7 (C-11), 60.8 (4ʹ-OCH3), 60.7, 56.2 (3ʹ, 5ʹ-
passed a short pad of silica column (petroleum ether/ethyl OCH3), 44.9 (C-2), 44.2 (C-1), 38.1 (C-3), 20.7 (OCH3),
acetate 4:1, v/v) to afford the product 8 (1.6 g, 46% yield for 20.6 (OCH3), 20.6 (OCH3), 20.5 (OCH3); ESIMS: m/z 767
1
two steps). α/β ratio = 6:1. H-NMR (CDCl3, 400 MHz) δ [M + Na]+, HRESIMS: calcd for C36H40O17Na [M + Na]+
6.20 (d, 1/7H, J = 8.0 Hz, C1-Hβ), 5.54 (t, 1H, J = 9.6 Hz, 767.2285, found 767.2286.
C3-H), 5.47 (d, 6/7H, J = 3.2 Hz, C1-Hα), 5.08 (t, 1H, J =
4-O-(2ʹ’,3ʹ’,4ʹ’,6ʹ’-Tetra-O-Acetyl-α-D-
Glucopyranosyl)-4ʹ-Demethylepipodophyllotoxin
(6b)
9.6 Hz, C4-H), 4.91 (dd, 1H, J = 3.2 Hz, 10.0 Hz, C2-H),
4.27–4.23 (m, 2H, C6-CH2), 4.14–4.12 (m, 1H, C5-H),
2.10–2.00 (m, 12H, 4 × OCH3); 13C-NMR (CD3Cl, 400
MHz) δ 170.8 (C=O), 170.2 (C=O), 170.1 (C=O), 169.7
(C=O), 95.5 (C-1β), 90.1 (C-1α), 73.2 (C-5β), 72.1 (C-4β),
72.0 (C-2β), 71.9 (C-5α), 69.8 (C-4α), 68.4 (C-2α), 68.3 (C-
3β), 67.2 (C-3α), 61.9 (C-6), 20.7 (OCH3), 20.7 (OCH3),
20.6 (OCH3), 20.5 (OCH3); ESIMS: m/z 371 [M + Na]+.
White powder; yield 62%; mp 172–174 oC; 1H-NMR
(CDCl3, 400 MHz) δ 6.94 (s, 1H, C6-H), 6.55 (s, 1H,
C8-H), 6.45 (s, 2H, C2′, C6′-H), 5.97–5.95 (m, 2H,
OCH2O), 5.40 (t, 1H, J = 8.0 Hz), 5.35 (d, 1H, J = 3.2
Hz, C1”-H), 5.10–5.07 (m, 1H), 4.83–4.80 (m, 1H), 4.72
(d, 1H, J = 3.8 Hz, C4-H), 4.41 (d, 1H, J = 2.1 Hz, C1-H),
4.32–4.31 (m, 1H), 4.22–4.21 (m, 1H), 4.09–4.07 (m, 1H),
3.75 (s, 6H, C3ʹ, C5ʹ-OCH3), 3.67–3.64 (m, 1H), 3.45–3.42
(m, 1H, C3-H), 3.00–2.97 (m, 1H, C2-H), 2.15–2.09 (m,
12H, 4 × OCH3); 13C-NMR (CDCl3, 100 MHz) δ 177.7
(C-12), 170.5 (C=O), 170.0 (C=O), 169.3 (C=O), 169.2
(C=O), 152.3 (C-3ʹ, C-5ʹ), 148.0 (C-6), 146.8 (C-7), 140.4
(C-4ʹ), 140.4 (C-1ʹ), 131.1 (C-9), 128.4 (C-10), 109.8 (C-
8), 106.8 (C-5), 104.9 (C-2ʹ, C-6ʹ), 101.3 (OCH2O), 95.7
(C-1ʹ’), 75.7 (C-4), 71.0, 69.5, 68.2, 67.8, 67.5 (C-11),
60.7, 56.2 (3ʹ, 5ʹ-OCH3), 45.0 (C-2), 44.3 (C-1), 38.0 (C-
3), 20.7 (OCH3), 20.6 (OCH3), 20.6 (OCH3), 20.5
(OCH3); ESIMS: m/z 756 [M + Na]+, HRESIMS: calcd
for C35H38O17Na [M + Na]+ 756.2123, found 756.2126.
Synthesis Of 4ʹ-Demethylepipodophyllotoxin (9)
4ʹ-Demethylepipodophyllotoxin (9) was prepared accord-
ing to the literature.29
General Procedure For The Synthesis Of
Compounds 6a – 6b
To a mixture of 2,3,4,6-tetra-O-acetyl-α/β-D-glucopyra-
nose (0.2 mmol) and podophyllotoxin/4ʹ-demethylepipo-
dophyllotoxin (0.2 mmol) in dry CH2Cl2 (3 mL) was
o
added of BF3⋅ H2O (25 μL, 0.02 mmol) at −78 C, and
the resulting mixture was stirred for 1 hr. Then, triethyla-
mine (0.1 mL) was added to the mixture, and acetic acid
(0.1 mL) was added. The solvent was evaporated, and the
residue was purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate 2:1, v/v) to afford the major
product 6a or 6b as white powder.
General Procedure For The Synthesis Of
Compounds 6c – 6d
To a solution of 6a/6b (0.1 mmol) in methanol (1.5 mL)
was added sodium methoxide (0.03 mmol) at 0 oC, and the
resulting mixture was stirred for 2 hrs. The reaction was
slowly quenched (anhydrous Amberlite ion-exchange resin
IRA-400), and the resin was removed by filtration. The
filtrate was concentrated under vacuum, and the residue
was purified by flash chromatography on silica gel (chloro-
form/methanol 9:1, v/v) to afford compound 6c or 6d as
white powder.
4-O-(2ʹ’,3ʹ’,4ʹ’,6ʹ’-Tetra-O-Acetyl-α-D-
Glucopyranosyl)-Epipodophyllotoxin (6a)
White powder; yield 58%; mp 167–168 oC; 1H-NMR
(CDCl3, 400 MHz) δ 6.99 (s, 1H, C6-H), 6.55 (s, 1H, C8-
H), 6.23 (s, 2H, C2′, C6′-H), 6.00–5.98 (m, 2H, OCH2O),
5.33 (t, 1H, J = 8.0 Hz), 5.22 (d, 1H, J = 3.2 Hz, C1”-H),
5.08–5.02 (m, 2H), 4.76 (d, 1H, J = 2.7 Hz, C4-H), 4.66–
4.65 (m, 1H), 4.30 (d, 1H, J = 2.1 Hz, C1-H), 4.28–4.18 (m,
2H), 4.14–4.03 (m, 1H), 3.79 (s, 3H, C4ʹ-OCH3), 3.76 (s,
6H, C3ʹ, C5ʹ-OCH3), 3.45–3.42 (m, 1H, C3-H), 3.01–2.98 4-O-(α-D-Glucopyranosyl)-Epipodophyllotoxin (6c)
(m, 1H, C2-H), 2.14–2.04 (m, 12H, 4 × OCH3); 13C-NMR White powder; yield 80%; mp 190–191 oC; 1H-NMR
(CDCl3, 100 MHz) δ 177.8 (C-12), 170.6 (C=O), 170.0 (CDCl3, 600 MHz) δ 7.07 (s, 1H, C6-H), 6.51 (s, 2H,
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C2′, C6′-H), 6.48 (s, 1H, C8-H), 5.94–5.93 (m, 2H, cell culture plate and allowed to adhere for 12 hrs before
OCH2O), 5.01 (d, 1H, J = 3.6 Hz, C1”-H), 4.78 (d, 1H, J drug addition, while suspended cells were seeded just
= 3.4 Hz, C4-H), 4.47–4.45 (m, 1H), 4.42 (t, 1H, J = 9.6 before drug addition, both with an initial density of 1 ×
Hz), 4.36 (d, 1H, J = 2.2 Hz, C1-H), 3.77 (s, 6H, C3ʹ, C5ʹ- 105 cells/mL in 100 μL of the medium. Each tumor cell
OCH3), 3.76 (s, 3H, C4ʹ-OCH3), 3.69–3.63 (m, 2H), 3.55– line was exposed to the test compound at various concen-
3.53 (m, 1H), 3.41–3.38 (m, 2H), 3.36–3.33 (m, 1H), trations in triplicate for 48 hrs. After the incubation, MTT
3.24–3.21 (m, 1H, C3-H), 3.16–3.13 (m, 1H, C2-H); 13C- (100 μg) was added to each well, and the incubation
NMR (CDCl3, 150 MHz) δ 178.7 (C-12), 154.7 (C-3ʹ, C- continued for 4 hrs at 37°C. The cells were lysed with
5ʹ), 149.0 (C-6), 148.2 (C-7), 139.5 (C-4ʹ), 137.9 (C-1ʹ), SDS (200 μL) after removal of 100 μL of the medium. The
132.6 (C-9), 131.2 (C-10), 110.1 (C-8), 108.2 (C-5), 106.9 optical density of lysate was measured at 595 nm in a 96-
(C-2ʹ, C-6ʹ), 102.5 (OCH2O), 99.4 (C-1ʹ’), 75.3 (C-4), well microtiter plate reader (Bio-Rad 680). IC50 values
75.0, 74.3, 73.8, 71.1, 70.0 (C-11), 61.7, 61.1 (4ʹ-OCH3), were calculated by Reed and Muench’s method.35,36
56.6 (3ʹ, 5ʹ-OCH3), 46.6 (C-2), 45.4 (C-1), 38.7 (C-3);
ESIMS: m/z 575 [M - H]−, HRESIMS: calcd for
Calculated Molecular Physicochemical
Properties
All structures of podophyllotoxin derivatives were built
C28H32O13 [M - H]− 576.1843, found 576.1846.
4-O-(α-D-Glucopyranosyl)-4ʹ-
and energy minimized by the Tripos force field with 0.05
Demethylepipodophyllotoxin (6d)
White powder; yield 78%; mp 201–203 oC; 1H-NMR
kcal/(mol Å). The Gasteiger–Huchel method was used to
(CDCl3, 600 MHz) δ 7.07 (s, 1H, C6-H), 6.49 (s, 1H,
calculate charges. Energy minimization was performed by
C8-H), 6.47 (s, 2H, C2′, C6′-H), 5.93–5.92 (m, 2H,
the Powell method with 2000 iterations. Molecular surface
OCH2O), 4.99 (d, 1H, J = 3.6 Hz, C1”-H), 4.82–4.81 (m,
area (MSA), polar surface area (PSA), calculated partition
1H), 4.77 (d, 1H, J = 4.4 Hz, C4-H), 4.46–4.39 (m, 2H),
coefficient (ClogP), calculated solubility (ClogS), hydro-
4.33 (d, 1H, J = 2.2 Hz, C1-H), 3.78 (s, 6H, C3ʹ, C5ʹ-
gen bond donor (HD), hydrogen bond acceptor (HA) and
rotatable bond (RB) were obtained from MarvinSketch
version 5.3.8. (www.chemaxon.org).37
OCH3), 3.70–3.62 (m, 2H), 3.56–3.53 (m, 1H), 3.41–
3.34 (m, 3H), 3.36–3.33 (m, 1H), 3.26–3.23 (m, 1H, C3-
H), 3.20–3.18 (m, 1H, C2-H); 13C-NMR (CDCl3, 150
MHz) δ 178.8 (C-12), 149.5 (C-3ʹ, C-5ʹ), 148.9 (C-6), Conclusion
148.2 (C-7), 135.5 (C-4ʹ), 133.9 (C-1ʹ), 132.9 (C-9), In conclusion, we synthesized a few glucoside derivatives of
131.2 (C-10), 110.2 (C-8), 108.1 (C-5), 106.4 (C-2ʹ, C- podophyllotoxin and screened for cytotoxicity against a
6ʹ), 102.5 (OCH2O), 99.4 (C-1ʹ’), 75.3 (C-4), 75.0, 74.3, panel of five human cancer cell lines including HL-60 (leu-
73.8, 71.1, 69.9 (C-11), 61.7, 56.8 (3ʹ, 5ʹ-OCH3), 46.5 (C- kemia), SMMC-7721 (hepatoma), A-549 (lung cancer),
2), 45.6 (C-1), 38.7 (C-3); ESIMS: m/z 561 [M - H]−, MCF-7 (breast cancer), and SW480 (colon cancer).
HRESIMS: calcd for C27H30O13 [M - H]− 561.1686,
Derivatives having a free glucose residue show weak activity
(IC50 > 40 μM), while the peracetylated derivative 6b shows
the highest cytotoxic potency against all five cancer cell lines
tested, with IC50 values ranging from 3.27 0.21 to 11.37
0.52 μM. Compound 6b also displays moderate selectivity
toward cancer cells over normal human pulmonary epithelial
cells (BEAS-2B). The calculated physicochemical properties
of these PPT derivatives indicated that more lipophilic com-
pounds are generally more cytotoxic to cancer cells. Our
results suggest that some of these compounds have potential
as lead compounds for developing novel anticancer agents.
found 561.1684.
Cytotoxicity Assay
The following five human cancer lines were used in the
cytotoxicity assay: human myeloid leukemia (HL-60),
hepatocellular carcinoma (SMMC-7721), lung cancer (A-
549), breast cancer (MCF-7), and colon cancer (SW480).
All the cells were cultured in RMPI-1640 or DMEM
medium (Hyclone, Logan, UT, USA), supplemented with
10% FBS (Hyclone, USA) in 5% CO2 at 37°C. The
cytotoxicity assay was performed according to the MTT
[3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium Acknowledgment
bromide] method in 96-well microplates.34 Briefly, adher- We are grateful to the National Nature Science Foundation of
ent cells (100 μL) were seeded into each well of a 96-well China for financial support (No. 21602196); the Yunnan
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Disclosure
The authors declare no conflicts of interest in this work.
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