'Click chemistry' synthesis of novel natural product-like caged xanthones bearing a 1,2,3-triazole moiety with improved druglike properties as orally active antitumor agents

Article abstract of DOI:10.3390/molecules22111834

DDO-6101, a natural-product-like caged xanthone discovered previously in our laboratory based on the pharmacophoric scaffold of the Garcinia natural product gambogic acid (GA), shows potent cytotoxicity in vitro, but poor efficacy in vivo due to its poor druglike properties. In order to improve the druglike properties and in vivo antitumor potency, a novel series of ten triazole-bearing caged xanthone derivatives of DDO-6101 has been efficiently synthesized by 'click chemistry' and evaluated for their in vitro antitumor activity and druglike properties. Most of the target compounds have sustained cytotoxicity against A549, HepG2, HCT116, and U2OS cancer cells and possess improved aqueous solubility, as well as permeability. Notably, these caged xanthones are also active towards taxol-resistant or cisplatin-resistant A549 cancer cells. Taking both the in vitro activities and druglike properties into consideration, compound 8g has been advanced into in vivo efficacy experiments. The results reveal that 8g (named as DDO-6318), both by intravenous or per os administration, are much more potent than the lead DDO-6101 in A549-transplanted mice models and it could be a promising antitumor candidate for further evaluation.

Full text of DOI:10.3390/molecules22111834

molecules
Article
‘Click Chemistry’ Synthesis of Novel Natural
Product-Like Caged Xanthones Bearing a
1,2,3-Triazole Moiety with Improved Druglike
Properties as Orally Active Antitumor Agents
Xiang Li 1,2, Yue Wu 1,3, Yanyan Wang 1,3, Qidong You 1,3,* and Xiaojin Zhang 1,4,
*
ID
1
Jiangsu Key Laboratory of Drug Design and Optimization, State Key Laboratory of Natural Medicines,
China Pharmaceutical University, Nanjing 210009, China; lixiang@cpu.edu.cn (X.L.);
wuyue_1994@163.com (Y.W.); emmawang_88@163.com (Y.W.)
Department of Pharmaceutical Engineering, China Pharmaceutical University, Nanjing 211198, China
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 21009, China
Department of Organic Chemistry, China Pharmaceutical University, Nanjing 211198, China
Correspondence: youqd@163.com (Q.Y.); zxj@cpu.edu.cn (X.Z.); Tel.: +86-25-8327-1351 (Q.Y.);
+86-139-1300-7140 (X.Z.)
2
3
4
*
Received: 12 October 2017; Accepted: 26 October 2017; Published: 27 October 2017
Abstract: DDO-6101, a natural-product-like caged xanthone discovered previously in our laboratory
based on the pharmacophoric scaffold of the Garcinia natural product gambogic acid (GA), shows
potent cytotoxicity in vitro, but poor efficacy in vivo due to its poor druglike properties. In order to
improve the druglike properties and in vivo antitumor potency, a novel series of ten triazole-bearing
caged xanthone derivatives of DDO-6101 has been efficiently synthesized by ‘click chemistry’ and
evaluated for their in vitro antitumor activity and druglike properties. Most of the target compounds
have sustained cytotoxicity against A549, HepG2, HCT116, and U2OS cancer cells and possess
improved aqueous solubility, as well as permeability. Notably, these caged xanthones are also active
towards taxol-resistant or cisplatin-resistant A549 cancer cells. Taking both the in vitro activities
and druglike properties into consideration, compound 8g has been advanced into in vivo efficacy
experiments. The results reveal that 8g (named as DDO-6318), both by intravenous or per os
administration, are much more potent than the lead DDO-6101 in A549-transplanted mice models
and it could be a promising antitumor candidate for further evaluation.
Keywords: caged xanthones; click chemistry; druglike; antitumor; natural-product-like
1. Introduction
Natural products (NPs) and natural-product-like compounds inspired by NP structures have
played an important role in new drug discovery [
than 70% of the anticancer drugs approved worldwide owe their origins to NPs [
(GA) (Figure 1), a naturally-occurring caged xanthone isolated from Garcinia plants, is a promising
antitumor agent in clinical study [ ]. Although great efforts has been put into revealing its antitumor
1
,2
]. Notably, in the case of anticancer drugs, more
1
]. Gambogic acid
3–5
mechanisms, the primary direct molecular target of GA is still debatable [6–12].
Intrigued by the unique structure and therapeutic promise of GA, a large library of its derivatives
and simplified caged xanthone analogues has been synthesized and evaluated in our laboratory to
explore the structure-activity relationship (SAR) [13–16], which allows us to understand that the intact
BCD ring containing the unique caged scaffold is the minimum pharmacophoric motif essential for its
activity. These studies have led to the identification of DDO-6101 (Figure 1), a natural-product-like
caged xanthone with a remarkably simple structure that retains the in vitro antitumor activity of
Molecules 2017, 22, 1834; doi:10.3390/molecules22111834
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1834
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GA [14]. Unfortunately, DDO-6101 showed poor efficacy in vivo on the tumor growth inhibition
in cancer cell-transplanted mice models [13]. This may be due to its poor druglike properties, such
as aqueous solubility and cell membrane permeability. Further medicinal chemistry research on
DDO-6101 have revealed that the modifications at the C1 site of the B ring and C15 site in the side
chain of the D ring are well-tolerated [17
groups can improve the druglike properties and enhance the in vivo antitumor potency [13
as shown in the structures of caged xanthone derivatives DDO-6306 and DDO-6337 (Figure 1) [17
,
18], and the introduction of hydrophilic heteroatom-containing
17 19],
18].
,
–
,
The main drawbacks of the two types of compounds lie in the unstable ester linker group, which
makes the molecules hardly able to survive in the presence of the ester hydrolases in plasma and the
acidic environment of the stomach when administered orally.
The copper-catalyzed 1-3-dipolar cycloaddition reaction that couples alkynes and azides forming
a 1,2,3-triazole ring, which is commonly known as ‘click chemistry’ due to its ease of use, has become
a powerful approach for the rapid construction of pharmacologically-active compounds [20,21].
The 1,2,3-triazole ring is chemically stable against acidic and basic hydrolysis and relatively resistant to
metabolic degradation. Moreover, it participates actively in hydrogen bond formation and is beneficial
to solubility in water. Herein we present a click chemistry-based linking strategy to couple hydrophilic
groups and caged xanthone pharmacophoric motifs to rapidly derive a new series of triazole-bearing
caged xanthones (Figure 1) with improved drug-like properties and oral antitumor activity in vivo.
Figure 1. Structures of GA, its simplified caged xanthone analogues and the newly-designed natural
product-like compounds.
2. Results and Discussion
2.1. Chemistry
The designed caged xanthane derivatives 8a
The starting material 1 was prepared according to our previously-reported procedure [14]. Heating 1
in DMF gave rise to the caged xanthone DDO-6101) via a Claisen rearrangement and Diels-Alder
addition cascade. The hydroxyl group at the C1 position is considered as a potential modification
site. Treatment of with 3-bromoprop-1-yne ( ) in the presence of potassium carbonate produced
the alkyne intermediate . Meanwhile, the substitution reaction between 6a 6j and sodium azide
provided the azide intermediates 7a 7j quantitatively, which were directly used for the following
reaction without further purification. The click reaction between alkyne and azide 7a 7j in the
presence of CuSO4 5H2O and sodium ascorbate led to the target triazole-bearing caged xanthone
derivatives 8a 8j in excellent yields. The chemical structures of these derivatives were characterized
using 1H-nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, and high-resolution mass
–8j and their synthetic routes are shown in Scheme 1.
3
(
3
4
5
–
–
5
–
·
–

Molecules 2017, 22, 1834
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spectrometry (HRMS). Representative compounds 8a
,
8c
,
8e
,
8g and 8h were further characterized by
13C-NMR. All of the target compounds yielded acceptable purity (>95%) by high-performance liquid
1
chromatography (HPLC) analysis. H-NMR, 13C-NMR, HRMS spectra for the target compounds are
shown in supplementary materials 1.
Scheme 1. The synthetic route of designed compounds. Reagents and conditions: (
a
) DMF, 120 ◦C,
c) NaN3 (1.5 equiv.),
2 h, 78.5%; (
K2CO3 (3 equiv.), H2O, 75 C, 15 h; (
b
)
4
(1.1 equi◦v.), K2CO3 (1.1 equiv.), DMF, r.t., overnight, 86.7%; (
d
)
7a–7j (2 equiv. relative to
5
), CuSO4 5H2O (0.01 equiv.), sodium
·
ascorbate (0.3 equiv.), t-BuOH/H2O (v:v = 1:1), r.t., overnight, 90.2–96.4%. For the detailed substitution
groups for 8a–8j see Table 1.
2.2. In Vitro Cytotoxic Effects
The antiproliferative activity of the ten synthesized caged xanthone derivatives, as well
as the lead compound DDO-6101 and GA were assessed with the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay, as previously reported [18], against six human cancer
cell lines, including the human lung carcinoma A549 cell line, taxol-resistant A549 cell line (A549/taxol),
cisplatin-resistant A549 cell line (A549/cisplatin), the human hepatocellular carcinoma HepG2 cell
line, the human colon carcinoma HCT-116 cell line, and the human glioblastoma U251 cell line.
The antiproliferative activities, expressed as IC50 values, are summarized in Table 1. DDO-6101 and
GA were used as positive controls for the in vitro assay.
As shown in Table 1, in general, most of the designed triazole-bearing caged xanthones 8a–8h
inhibited potent antiproliferative activities against A549, HepG2, HCT116, and U2OS cells, with IC50
values in the low micromolar range, which are comparable to those of DDO-6101 and natural product
GA. Interestingly, it was observed that these compounds showed comparable, or even better, cytotoxic
activity against A549/taxol and A549/cisplatin cell lines when compared to A549 cells, as well as
three other cancer cell lines. The results indicated that these xanthone derivatives with triazole- and
nitrogen-containing side chains at the C1 site function in cancer cells and multidrug-resistant ones, as well.
Different compounds showed different activities against the six cancer cell lines. 8f
8j showed potent cytotoxicity against A549 cells, with growth inhibition IC50 values of 1.07
0.31 0.02, 1.03 0.06, and 0.93 0.08 M, respectively, which were much more active than both
the DDO-6101 and GA. Moreover, the taxol-resistant and cisplatin-resistant A549 cell lines were both
significantly sensitive to 8g and 8h, with IC50 values ranging from 0.33 to 0.55 M. The HepG2 cell line
was sensitive to 8b and 8c, with IC50 values of 1.36 0.16 and 1.19 0.31 M, respectively, which were
,
8g
,
8h and
±
0.28,
±
±
±
µ
µ
±
±
µ

Molecules 2017, 22, 1834
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more potent than DDO-6101 and GA, while in the HCT116 cell lines 8g exerted the best cytotoxic
activity with an IC50 value of 0.28 0.03 M. Although many other compounds, such as 8c 8d 8h and
8j, possessed low micromolar activities against HCT116 cells, they were less active than DDO-6101
In the case of the U2OS cell line, it was generally less sensitive to this series of compounds as compared
to the five other cell lines, with IC50 values ranging from 0.73 to 9.51 M. However, eight compounds
8a 8h showed more potent cytotoxicity than DDO-6101 against U2OS, among which 8h was the most
±
µ
,
,
.
µ
–
active with an IC50 value of 0.73 ± 0.07 µM.
According to the in vitro data in Table 1, it was found that the introduction of the triazole moiety
with nitrogen-containing hydrophilic groups, especially the morpholino and 4-methylpiperizin-1-yl
groups as in 8g–8j, could improve the cytotoxic activity against most of the tested cancer cell lines.
Few differences in cytotoxicity were found between compounds with different carbon chain lengths
(n = 2 or 3). Among this series of target compounds, 8g showed the most potent inhibitory activities
in vitro towards A549, A549/taxol, A549/cisplatin, and HCT116 cells, with IC50 values of 0.31 ± 0.02
,
0.42 ± 0.05, 0.33
±
0.07, and 0.28 0.03, respectively, which was more active in vitro than the lead
±
DDO-6101. It was noteworthy that 8g displayed approximately eight-fold higher activity than
DDO-6101 in A549 cells.
Table 1. In vitro antiproliferative activity of the 1,2,3-triazole-bearing caged xanthone compounds.
IC50 (µM)
Cpd
8a
8b
8c
n
2
3
2
3
2
3
2
3
2
3
-NRR
A549
A549/Taxol A549/Cisplatin
HepG2
HCT116
U2OS
1.95 ± 0.40 1.22 ± 0.21
2.62 ± 0.20 1.30 ± 0.08
2.58 ± 0.39 1.29 ± 0.07
2.62 ± 0.04 1.70 ± 0.12
2.17 ± 0.12 2.03 ± 0.11
1.07 ± 0.28 1.29 ± 0.08
0.31 ± 0.02 0.42 ± 0.05
1.03 ± 0.06 0.55 ± 0.08
3.33 ± 0.49 2.12 ± 0.09
0.93 ± 0.08 0.82 ± 0.08
1.48 ± 0.10
1.66 ± 0.11
1.34 ± 0.09
0.89 ± 0.24
1.29 ± 0.48
1.32 ± 0.12
0.33 ± 0.07
0.36 ± 0.04
0.78 ± 0.08
1.29 ± 0.11
2.12 ± 0.41 1.64 ± 0.26 2.22 ± 0.09
1.36 ± 0.16 2.27 ± 0.05 2.73 ± 0.05
1.19 ± 0.31 0.98 ± 0.31 2.01 ± 0.20
4.48 ± 0.29 1.02 ± 0.03 3.05 ± 0.07
4.85 ± 0.39 2.14 ± 0.18 4.66 ± 0.03
6.30 ± 0.48 1.39 ± 0.04 2.07 ± 0.11
3.79 ± 0.43 0.28 ± 0.03 0.95 ± 0.21
3.07 ± 0.13 0.95 ± 0.12 0.73 ± 0.07
5.35 ± 0.23 6.15 ± 0.86 9.51 ± 1.52
2.91 ± 0.03 1.09 ± 0.21 5.86 ± 0.21
8d
8e
8f
8g
8h
8i
8j
DDO-6101
GA
—
—
2.59 ± 0.17 0.46 ± 0.03
2.02 ± 0.02 0.29 ± 0.04
2.11 ± 0.10
2.50 ± 0.06
2.04 ± 0.08 0.71 ± 0.10 5.35 ± 0.08
2.08 ± 0.07 0.34 ± 0.08 4.67 ± 0.11

Molecules 2017, 22, 1834
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2.3. Drug-Like Property Evaluation
In parallel with the in vitro antitumor activity testing, the physicochemical properties of the target
caged xanthone derivatives were theoretically predicted and experimentally determined in an effort
to identify potential druglike compounds prior to the time-consuming and costly development and
optimization of derivatives that may ultimately fail in efficacy experiments in vivo. The partition
coefficient (logD) was experimentally determined on a Gemini Profiler instrument (pION, Inc., Woburn,
MA, USA) by a potentiometric titration method [13]. The polar surface area (PSA), as well as intestinal
absorption level, were theoretically predicted by Accelrys Discovery Studio 4.0 (BIOVIA, San Diego,
CA, USA) using the ADMET protocol [17]. The aqueous solubility were experimentally measured
using HPLC at the maximum absorption wavelength of 290 nm based on the results of full-wave
scanning [18]. The permeability coefficient (Pe) was determined using a standard parallel artificial
membrane permeability assay (PAMPA) on a PAMPA Explorer instrument (pION) [17]. Ketoprofen
and propranolol were used as internal standards for permeability. These data are shown in Table 2 and
Figure 2.
Whether a drug can be well-absorbed intestinally after oral administration depends on the
intrinsic properties of the molecule. The polar surface area and distribution coefficient are the two
main aspects. Analysis of a plot of the logD versus PSA_2D of the caged xanthones 8a–8j (Table 2 and
Figure 2) showed that all of the compounds fell within the ellipse region of 95% (aborption_level = 0)
while GA fell outside the ellipse region of 95% (aborption_level = 1). These results suggested that the
xanthone derivatives are likely to have favorable intestinal absorption.
Meanwhile, compounds 8a
permeability, which are the two important properties reflecting the ability of molecules to penetrate to
the therapeutic target across the cell membrane and watery environment in vivo DDO-6101 and GA
–8j were set to determine experimentally their aqueous solubility and
.
were used as reference compounds for comparison. As shown in Table 2, all the xanthone derivatives
displayed aqueous solubility that was significantly improved over that of DDO-6101 and GA. This
may be attributed to the introduction of 1,2,3-triazole and hydrophilic groups, as well as the disruption
of intermolecular hydrogen bonds between C1-OH and the adjacent carbonyl group in the structure of
DDO-6101. Surprisingly, compounds with a chain of three carbon atom (e.g., 8b
slightly more solubility than the corresponding ones with a chain of two carbon atoms (e.g., 8a
8g 8i). Moreover, it was found that compounds 8g 8j were more soluble than that of 8a 8f. The results
,
8d
,
8f
,
8h
,
8j) showed
,
8c, 8e,
,
–
–
suggested that the introduction of hydrophilic groups, such as morpholino and 4-methylpiperazin-1-yl
groups, resulted in a significant increase in solubility.
Table 2. Druglike properties of the caged xanthone derivatives 8a–8j.
PSA_2D (Å2) a
Log D 7.4 b
S (mM) d
Cpd
Absorption_Level c
8a
8b
8c
8d
8e
8f
8g
8h
92.614
92.614
92.614
92.614
92.614
92.614
101.544
101.544
95.967
95.967
73.277
120.323
2.1
1.8
2.2
1.6
2.9
2.7
1.3
0.9
1.0
0.7
2.9
3.6
0
0
0
0
0
1
0
0
0
0
0
1
1.12
1.54
1.40
1.92
0.56
0.88
3.02
3.70
3.22
8i
8j
4.02
<0.005
<0.005
DDO-6101
GA
a Calculated value with Accelrys Discovery Studio 4.0; b Measured value of log D 7.4 on a Gemini Profiler instrument
(pION); c The data were predicted with Accelrys Discovery Studio 4.0. Absorption_level (0 = good, 1 = moderate);
d
The solubility in water.

Molecules 2017, 22, 1834
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Figure 2. Plot of log P versus PSA_2D.
In addition, the introduction of triazole moiety and hydrophilic groups to the C1 site of DDO-6101
also led to an improvement in permeability. As shown in Figure 3 (for the detailed data see
Supplementary Materials Table S1), most of the xanthone derivatives displayed improved permeability
than DDO-6101 (Pe = 18.0–23.6
decreased as the aqueous environment turned to be more acidic. Among the compounds tested,
×
10–6 cm/s) and GA (Pe = 0.9–2.3
×
10–6 cm/s). The permeability
8e and 8g showed preferable permeability under different pH from 5.0 to 7.4 with Pe values of
47.0–78.2 × 10–6 cm/s and 51.3–74.4
×
10–6 cm/s, respectively. This suggested that these xanthone
derivatives are likely to have favorable properties of absorption and distribution in vivo.
Figure 3. Experimental determination of membrane permeability of compounds 8a–8j at different
pH values.

Molecules 2017, 22, 1834
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2.4. In Vivo Antitumor Evaluations of Compound 8h
In the light of the in vitro antitumor activity and druglike properties, such as aqueous solubility
and permeability, we selected compound 8h to determine its in vivo efficacy. The lung cancer A549
transplanted mouse model was employed. DDO-6101 and antitumor drug 5-fluorouracil (5-FU) were
also tested for comparison. As shown in Table 3, the tumor growth inhibitory rates of DDO-6101 and
5-FU were 34.56% and 64.71%, respectively, at 20 mg/kg twice daily doses, over a period of two weeks.
8g displayed significant inhibitory effect on the growth of inoculated A549 in mice in a dose-dependent
manner, with 30.88%, 52.21% and 71.32% inhibition in tumor growth, at 5, 10 and 20 mg/kg twice
daily doses, respectively. No vascular irritation or weight loss was observed in any of these in vivo
tests. Compared with DDO-6101, the triazole-bearing derivative 8g was much more potent in vivo
on the inhibition of the growth of inoculated A549 in mice. This may be attributed to the increased
in vitro cytotoxicity and improved druglike properties, such as aqueous solubility and cell membrane
permeability of 8g compared to DDO-6101.
Table 3. The inhibitory effect of 8g with intravenous administration on the growth of A549
transplanted mice.
Body Weight (g)
Group
Dose (mg/kg)
Weight of Tumor (g) Inhibitory Rate (%)
Pre-Dose
After-Dose
control
8g
8g
0
5
10
20
20
20
20.95
±
±
±
±
±
±
1.22 29.25
±
±
±
±
±
±
3.37
2.81
2.66
2.78
1.90
2.62
1.36 ± 0.20
0.94 ± 0.24 a
0.65 ± 0.17 b
0.40 ± 0.14 b
0.89 ± 0.18 b
0.48 ± 0.17 b
0.0
20.80
21.40
20.67
21.13
20.25
a
1.53 28.53
1.81 28.08
1.30 27.73
1.22 27.69
1.27 24.43
30.88
52.21
71.32
34.56
64.71
8g
DDO-6101
5-FU
p < 0.05; b p < 0.01 vs. control.
Subsequently, 8g was set to determine further its in vivo efficacy with per os administration on
the growth of inoculated A549 in mice using DDO-6101 as a comparison control. As shown in Table 4,
8g displayed much more inhibitory potency on the growth of inoculated A549 in mice than DDO-6101
.
According to the data, 66.43% inhibition in tumor growth for 8g was observed at 50 mg/kg daily oral
dose, over a period of one week, while the inhibitory rate of DDO-6101 was 21.43%. Thus, compound
8g was identified as a new and orally-active natural-product-like caged xanthone and also a promising
antitumor agent for further clinical development.
Table 4. The inhibitory effect of 8g with per os administration on the growth of A549-transplanted mice.
Body Weight (g)
Group
Dose (mg/kg)
Weight of Tumor (g) Inhibitory Rate (%)
Pre-Dose
After-Dose
control
8g
DDO-6101
0
50
50
20.86
±
±
±
1.18 28.55
1.20 27.73
1.02 26.69
p < 0.05; b p < 0.01 vs. control.
±
±
±
2.97
2.78
1.90
1.40 ± 0.22
0.47 ± 0.12 a
1.10 ± 0.25 b
0.0
66.43
21.43
20.67
21.13
a
3. Materials and Methods
3.1. General Information
All reagents were purchased from commercial sources and, unless otherwise noted, were
used without further purification. Organic solutions were concentrated in a rotary evaporator
(Büchi Rotavapor, Büchi, Uster, Switzerland) below 45 ◦C (70 ◦C for DMF) under reduced pressure.
Reactions were monitored by thin-layer chromatography (TLC, Qingdao Haiyang Chemical Co., Ltd.,
Qingdao, China) on 0.25 mm silica gel plates (GF254) and visualized under UV light. Silica gel (60 Å,
300–400 mesh) was used for flash column chromatography. Melting points were determined with

Molecules 2017, 22, 1834
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a Melt-Temp II apparatus (Barnstead/Thermolyne Corp., Dubuque, IA, USA) and were reported
without correction. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR spectrometer (Thermo
1
Fisher Scientific, Waltham, MA, USA) using KBr film. The H-NMR and 13C-NMR spectra were
measured on a Bruker AV-300 instrument (Bruker, Billerica, MA, USA) using deuterated solvents with
tetramethylsilane (TMS) as an internal standard. High-resolution mass spectra (HRMS) were recorded
on a Water Q-Tof micro mass spectrometer (Waters, Milford, MA, USA). The purity is verified by the
HPLC study performed on an Amethyst C18-P (4.6
column using a mixture of solvent methanol or acetonitrile/water at the flow rate of 2 mL/min and
peak detection at 240 nm under UV.
×
150 mm, 5 µm, Merck, Darmstadt, Germany)
Cancer cell lines were obtained from Cell Bank of Shanghai, Chinese Academy of Sciences. Cells
were cultured in 90% RPMI 1640 medium (GIBCO, Invitrogen Corporation, New York, NY, USA)
supplemented with 10% fetal bovine serum (Sijiqing Biotechnology Co., Hangzhou, China), 100 U/mL
◦
benzyl penicillin, and 100
µ
g/mL streptomycin in a humidified environment with 5% CO2 at 37 C. All
the tested compounds were dissolved in DMSO at a concentration of 0.01 mol/L and stored at −4 ◦C.
3.2. Synthesis of Compound 3
Compound
1
. (5.0 g, 13.16 mmol) was dissolved in DMF (20 mL) and the reaction solution was stirred
◦
at 120 C under nitrogen for 2 h. The reaction mixture was then cooled to room temperature and
concentrated. The residue was purified by column chromatography (16:1 petroleum ether/ethyl
◦
acetate) to afford compound
3
(
DDO-6101) (3.925 g, 78.5%) as a yellow solid. m.p.: 130–132 C.
1H-NMR (300 MHz, CDCl3): 0.95 (s, 3H), 1.18–1.25 (m, 4H), 1.30 (s, 3H), 1.61 (s, 3H), 2.26 (dd,
J1 = 13.5 Hz, J2 = 4.5 Hz, 1H), 2.37 (d, J = 9.6 Hz, 1H), 2.54 (d, J = 7.8 Hz, 2H), 3.44 (dd, J1 = 6.9 Hz
,
J2 = 4.5 Hz, 1H), 4.32–4.36 (m, 1H), 6.43 (dd, J1 = 8.1 Hz, J2 = 0.9 Hz, 1H), 6.45 (dd, J1 = 8.1 Hz,
J2 = 0.9 Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 7.41 (d, J = 9.6 Hz, 1H), 12.00 (s, 1H); HRMS (ESI): calcd. for
C23H24O5 [M + H]+ 381.1697, found 381.1715; HPLC 99.5% (80% acetonitrile in water, tR: = 4.7 min).
3.3. Synthesis of Compound 5
To a stirring solution of compound
3 (3.8 g, 0.1 mol) in DMF (50 mL) was added K2CO3 (15.18 g,
0.11 mol) and 3-bromoprop-1-yne (12.83 mL, 0.11 mol). The reaction mixture was stirred at room
temperature overnight. A saturated ammonium chloride aqueous (200 mL) was added. The mixture
was extracted by ethyl acetate (100 mL
with brine (50 mL 3), dried over magnesium sulfate, and concentrated. The residue was purified by
column chromatography (5:1 petroleum ether/ethyl acetate) to afford compound (3.625 g, 86.7%) as
a white solid. m.p. 151–153 ◦C; 1H-NMR (300 MHz, CDCl3):
1.11 (s, 3H), 1.27–1.31 (m, 4H), 1.42 (s,
×
3). The ethyl acetate layer was partitioned, combined, washed
×
5
δ
3H), 1.72 (s, 3H), 2.33 (dd, J1 = 8.8 Hz, J2 = 4.5 Hz, 1H), 2.42 (d, J = 9.6 Hz, 1H), 2.54 (t, J = 2.3 Hz,1H),
2.63 (d, J = 7.7 Hz, 2H), 3.44–3.48 (m, 1H), 4.51 (t, J = 7.8 Hz, 1H), 4.86 (d, J = 2.3 Hz, 2H), 6.73–6.76 (m,
2H), 7.32 (d, J = 6.9 Hz, 1H), 7.41–7.47 (t, J = 8.4 Hz, 1H); IR (KBr, cm−1): 3253, 2993, 1738, 1672, 1613,
1600, 1573, 1467, 1109, 1061, 848, 799, 697; HRMS (ESI): calcd. for C26H27O5 [M + H]+ 419.1853, found
419.1864. HPLC purity: 97.7% (elution: 70% methanol in water, tR: 12.4 min).
3.4. Synthesis of Compounds 8a–8j
To a solution of corresponding reactant 6a
(1.656 g, 12 mmol) and sodium azide (0.390 g, 6 mmol). The reaction mixture was stirred at 75 ◦C for
10 h, then cooled to room temperature and extracted by dichloromethane (15 mL 3). The organic
layer was partitioned, combined, washed with bine (15 mL), dried over magnesium sulfate and
concentrated to afford crude product 7a 7j. The crude residue 7a 7j was then dissolved in a mixture
solvent of t-BuOH/H2O (v:v = 1:1, 15 mL). Compound (0.836 g, 2 mmol), sodium ascorbate (119 mg,
0.6 mmol), and CuSO4 5H2O (5 mg, 0.02 mmol) were added into the mixture. The reaction mixture
was stirring at room temperature under N2 protection overnight. Then water (200 mL) was added
and the mixture was extracted by dichloromethane (15 mL 3). The organic layer was partitioned,
–6j (4 mmol) in water (30 mL) was added K2CO3
×
–
–
5
·
×

Molecules 2017, 22, 1834
9 of 13
combined, washed with bine (15 mL), dried over magnesium sulfate and concentrated. The residue
was purified by column chromatography (20:1 dichloromethane/methanol) to afford the products
8a–8j.
Compound 8a. Yield 91.5%. Slight yellow solid. m.p. 156–158 ◦C; 1H-NMR (300 MHz, CDCl3):
δ 1.01
(s, 3H), 1.18–1.24 (m, 4H), 1.31 (s, 3H), 1.63 (s, 3H), 2.22–2.27 (m, 7H), 2.33 (d, J = 9.5 Hz, 1H), 2.54 (d,
J = 7.8 Hz, 2H), 2.73 (t, J = 6.5 Hz, 2H), 4.37–4.46 (m, 1H), 4.31–4.42 (m, 3H), 5.26 (s, 2H), 6.61–6.69 (m,
2H), 7.20–7.23 (m, 1H), 7.35 (t, J = 8.3 Hz, 1H), 7.95 (s, 1H); 13C-NMR (75 MHz, CDCl3):
δ 16.48, 25.03,
25.13, 28.48, 29.76, 30.38, 44.89, 46.21, 47.84, 47.99, 58.22, 63.35, 82.85, 83.91, 89.41, 105.73, 110.15, 110.55,
118.13, 123.04, 131.86, 134.15, 135.71, 136.02, 143.59, 159.09, 160.66, 175.09, 202.64; IR (KBr, cm−1): 3197,
3109, 2996, 1737, 1667, 1603, 1474, 1460, 1401, 1258, 1111, 1066, 846, 791, 669; HRMS (ESI): calcd. for
C30H37N4O5 [M + H]+ 533.2758, found 533.2774; HPLC purity: 100% (elution: 75% methanol in water,
tR: 4.63 min).
Compound 8b. Yeild 92.4%. Slight yellow solid. m.p. 133–136 ◦C; 1H-NMR (300 MHz, CDCl3):
δ
1.08 (s, 3H, C14-H), 1.25–1.29 (m, 4H), 1.37 (s, 3H), 1.69 (s, 3H, C20-H), 2.15–2.25 (m, 2H), 2.28–2.41 (m,
8H), 2.48–2.53 (m, 2H), 2.61 (d, J = 7.7 Hz, 2H), 3.43–3.47 (m, 1H), 4.45–4.49 (m, 3H), 5.31 (s, 2H), 6.71
(t, J = 7.8 Hz, 2H), 7.26–7.29 (m, 1H), 7.43 (t, J = 8.3 Hz, 1H), 7.98 (s, 1H); IR (KBr, cm−1): 3196, 3106,
2996, 1737, 1669, 1603, 1475, 1401, 1258, 1111, 1066, 846, 791, 669; HRMS (ESI): calcd. for C31H39N4O5
[M + H]+ 547.2915, found 547.2916; HPLC purity: 100% (elution: 75% methanol in water, tR: 4.47 min).
◦
Compound 8c. Yeild 90.2%. Slight yellow solid. m.p. 168–170 C; 1H-NMR (300 MHz, CDCl3):
δ
1.08 (s, 3H), 1.25–1.29 (m, 4H, C15-H), 1.37 (s, 3H), 1.70 (s, 3H), 1.78–1.81 (m, 4H), 2.33 (dd, J1 = 8.8 Hz
,
J2 = 4.5 Hz, 1H), 2.42 (d, J = 9.6 Hz, 1H), 2.60–2.62 (m, 6H), 3.04 (t, J = 6.7 Hz, 2H), 3.44–3.47 (m, 1H),
4.49–4.56 (m, 3H), 5.33 (s, 2H), 6.68–6.76 (m, 2H), 7.27–7.29 (m, 1H), 7.42 (t, J = 8.4 Hz, 1H), 8.05 (s, 1H);
13C-NMR (75 MHz, CDCl3):
δ 16.49, 23.02, 25.04, 25.13, 28.48, 28.50, 29.77, 46.22, 48.00, 48.66, 53.49,
54.77, 63.31, 82.85, 83.92, 89.45, 105.70, 110.15, 110.57, 118.12, 123.19, 131.89, 134.17, 135.69, 136.01,
143.65, 159.06, 160.67, 175.07, 202.67; IR (KBr, cm−1): 3196, 3110, 2996, 1737, 1668, 1603, 1474, 1458, 1400,
1258, 1110, 1066, 847, 791, 669; HRMS (ESI): calcd. for C32H39N4O5 [M + H]+ 559.2915, found 559.292;
HPLC purity: 95.1% (elution: 75% methanol in water, tR: 4.45 min).
Compound 8d. Yeild 96.4%. Slight yellow solid. m.p. 144–146 ◦C; 1H-NMR (300 MHz, CDCl3):
δ 1.10
(s, 3H), 1.26–1.33 (m, 4H), 1.39 (s, 3H), 1.72 (s, 3H), 1.83 (m, 4H), 2.14–2.23 (m, 2H), 2.33 (dd, J1 = 9.2 Hz,
J2 = 4.3 Hz, 1H), 2.42 (d, J = 9.6 Hz, 1H), 2.53–2.64 (m, 8H), 3.45–3.49 (m, 1H), 4.47–4.54 (m, 3H), 5.34
(s, 2H), 6.73 (t, J2 = 8.7 Hz, 2H), 7.29–7.31 (m, 1H), 7.44 (t, J = 8.4 Hz, 1H), 7.98 (s, 1H); IR (KBr, cm−1):
3196, 3110, 2996, 1736, 1670, 1604, 1474, 1458, 1401, 1263, 1110, 1066, 847, 791, 669; HRMS (ESI): calcd.
for C33H41N4O5 [M + H]+ 573.3071, found 573.3080; HPLC purity: 98.8% (elution: 75% methanol in
water, tR: 4.46 min).
◦
Compound 8e. Yeild 92.8%. Slight yellow solid. m.p. 173–176 C; 1H-NMR (300 MHz, CDCl3):
1.07 (s, 3H, C14-H), 1.28 (m, 4H), 1.36 (s, 3H), 1.42–1.44 (m, 2H), 1.56–1.60 (m, 4H), 1.69 (s, 3H), 2.31
(dd, J1 = 9.0 Hz J2 = 4.5 Hz, 1H), 2.39 (d, J = 9.6 Hz, 1H), 2.45 (m, 4H), 2.60 (d, J = 7.7 Hz, 2H), 2.80 (t,
J = 6.5 Hz, 2H), 3.42–3.46 (m, 1H), 4.46–4.50 (m, 3H), 5.32 (s, 2H), 6.67–6.75 (m, 2H), 7.27 (d, J = 6.8 Hz,
1H), 7.41 (t, J = 8.3 Hz, 1H), 8.07 (s, 1H); 13C-NMR (75 MHz, CDCl3):
16.45, 23.62, 25.04, 25.14, 25.28,
δ
,
δ
28.48, 28.50, 29.77, 46.21, 47.31, 47.99, 54.00, 57.66, 63.37, 82.85, 83.93, 89.45, 105.70, 110.16, 110.54, 118.12,
123.21, 131.80, 134.18, 135.67, 136.05, 143.56, 159.08, 160.65, 175.00, 202.69; IR (KBr, cm−1): 3200, 3111,
2996, 1738, 1668, 1603, 1476, 1458, 1400, 1259, 1116, 1064, 791, 669; HRMS (ESI): calcd. for C33H41N4O5
[M + H]+ 573.3071, found 573.3084; HPLC purity: 100% (elution: 75% methanol in water, tR: 4.69 min).
◦
Compound 8f. Yeild 93.6%. Slight yellow solid. m.p. 125–128 C; 1H-NMR (300 MHz, CDCl3):
δ
1.07 (s, 3H), 1.23–1.30 (m, 4H), 1.36 (s, 3H, C19-H), 1.42–1.44 (m, 2H), 1.57–1.61 (m, 4H), 1.68 (s, 3H),
2.10–2.15 (m, 2H), 2.29–2.40 (m, 8H), 2.60 (d, J = 7.7 Hz, 2H), 3.42–3.46 (m, 1H), 4.42–4.49 (m, 3H), 5.31
(s, 2H), 6.67–6.73 (m, 2H), 7.26–7.28 (m, 1H), 7.41 (t, J = 8.3 Hz, 1H), 7.93 (s, 1H); IR (KBr, cm−1): 3200,
3111, 2996, 1739, 1668, 1605, 1478, 1462, 1401, 1267, 1116, 1064, 878, 791, 669; HRMS (ESI): calcd. for

Molecules 2017, 22, 1834
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C34H43N4O5 [M + H]+ 587.3228, found 587.3228; HPLC purity: 98.5% (elution: 75% methanol in water,
tR: 4.45 min).
Compound 8g. Yeild 96.4%. Slight yellow solid. m.p. 175–178 ◦C; 1H-NMR (300 MHz, CDCl3):
δ 1.09
(s, 3H), 1.26–1.32 (m, 4H), 1.37 (s, 3H, C19-H), 1.70 (s, 3H), 2.32 (dd, J1 = 8.9 Hz, J2 = 4.5 Hz, 1H), 2.40 (d,
J = 9.5 Hz, 1H), 2.51 (t, 4H, J = 4.5 Hz), 3.72 (d, J = 7.6 Hz, 2H), 2.85 (t, J = 6.2 Hz, 2H), 3.44–3.48 (m, 1H),
3.72 (t, J = 4.7 Hz, 4H), 4.48–4.52 (m, 3H), 5.33 (s, 2H), 6.72 (t, J = 7.7 Hz, 2H), 7.27–7.29 (m, 1H), 7.43 (t,
J = 8.3 Hz, 1H), 8.14 (s, 1H); 13C-NMR (75 MHz, CDCl3):
δ 16.47, 25.03, 25.12, 28.48, 29.77, 30.37, 46.22,
46.93, 48.02, 52.97, 57.29, 63.44, 66.27, 82.85, 83.90, 89.49, 105.50, 110.10, 110.59, 118.15, 123.16, 131.90,
134.11, 135.71, 136.02, 143.79, 159.05, 160.70, 175.03, 202.61; IR (KBr, cm−1): 3200, 3111, 2993, 1737, 1668,
1603, 1474, 1457, 1401, 1258, 1109, 1066, 872, 846, 791, 689; HRMS (ESI): calcd. for 32H39N4O6 [M + H]+
575.2864, found 575.2892; HPLC purity: 95.8% (elution: 75% methanol in water, tR: 8.56 min).
Compound 8h. Yeild 91.8%. Slight yellow solid. m.p. 143–145 ◦C; 1H-NMR (300 MHz, CDCl3):
δ
1.09 (s, 3H), 1.26–1.30 (m, 4H), 1.38 (s, 3H), 1.71 (s, 3H), 2.10–2.17 (m, 2H), 2.30–2.44 (m, 8H), 2.60
(d, J = 7.7 Hz, 1H), 3.45–3.48 (m, 1H), 3.72 (t, J = 4.7 Hz, 4H), 4.46–4.53 (m, 3H), 5.33 (s, 2H), 6.72 (t,
J = 7.6 Hz, 2H), 7.27–7.30 (m, 1H), 7.44 (t, J = 8.4 Hz, 1H), 7.97 (s, 1H); 13C-NMR (75 MHz, CDCl3):
δ
16.48, 25.06, 25.12, 26.47, 28.49, 28.51, 29.77, 46.20, 47.65, 47.98, 53.00, 54.43, 63.41, 66.41, 82.87, 83.89,
89.44, 105.51, 110.05, 110.62, 118.15, 122.82, 131.88, 134.14, 135.77, 135.99, 143.73, 159.00, 160.68, 175.09,
202.62; IR (KBr, cm−1): 3201, 3112, 2963, 1737, 1668, 1604, 1477, 1457, 1400, 1267, 1115, 1064, 878, 844,
793, 696; HRMS (ESI): calcd. for C33H41N4O6 [M + H]+ 589.3021, found 589.3037; HPLC purity: 95.7%
(elution: 75% methanol in water, tR: 4.12 min).
Compound 8i. Yeild 90.6%. Slight yellow solid. m.p. 171–175 ◦C; 1H-NMR (300 MHz, CDCl3):
δ 1.07
(s, 3H), 1.25–1.30 (m, 4H), 1.37 (s, 3H), 1.69 (s, 3H), 2.30–2.62 (m, 15H), 2.85 (t, J = 6.4 Hz, 2H)), 3.43–3.47
(m, 1H), 4.46–4.50 (m, 3H), 5.32 (s, 2H), 6.68–6.75 (m, 2H), 7.26–7.29 (m, 1H), 7.42 (t, J = 8.4 Hz, 1H),
8.05 (s, 1H); IR (KBr, cm−1): 3210, 3113, 2967, 1738, 1672, 1602, 1475, 1457, 1400, 1109, 1064, 791, 685;
HRMS (ESI): calcd. for C33H42N5O5 [M + H]+ 588.3180 found 588.3189; HPLC purity: 95.9% (elution:
70% methanol in water, tR: 3.13 min).
◦
1
Compound 8j. Yeild 91.2%. Slight yellow solid. m.p. 126–129 C; H-NMR (300 MHz, CDCl3):
δ
1.10 (s, 3H), 1.24–1.33 (m, 4H), 1.39 (s, 3H), 1.71 (s, 3H), 2.05–2.15 (m, 2H), 2.31–2.48 (m, 15H), 2.63 (d,
J = 7.5 Hz, 1H), 3.45–3.49 (m, 1H), 4.44–4.51 (m, 3H), 5.34 (s, 2H), 6.70–6.76 (m, 2H), 7.28–7.31 (m, 1H),
7.44 (t, J = 8.3 Hz, 1H), 7.96 (s, 1H); IR (KBr, cm−1): 3103, 2973, 1741, 1683, 1616, 1605, 1478, 1462, 1399,
1262, 1110, 1013, 878, 792, 695; HRMS (ESI): calcd. for C34H44N5O5 [M + H]+ 602.3337 found 602.3346;
HPLC purity: 97.5% (elution: 75% methanol in water, tR: 4.39 min).
3.5. Cell Proliferation Assay
The inhibition of tumour cell growth was measured by a modified tetrazolium (MTT) salt assay.
Cells were cultured in a 96-well plate and treated with culture medium alone or different concentrations
of the compounds. The cells were incubated for 72 h, and then the MTT assay reagent was added. After
a 4 h incubation, the formazan product was quantitated at 570 nm. The IC50 values (the concentrations
that gave rise to 50% inhibition of cell viability) were calculated using GraphPad Prism 6 using a
variable slope (four parameters).
3.6. Determination of the Druglike Properties
A HPLC system with an ultraviolet (UV) detector was used to detect the solubility of the
compounds. The optimal wavelength was 290 nm. Ethanol/water (85:15) was an efficient eluent at a
flow rate of 0.5 mL/min. The compounds were stirred in deionized water overnight to maximize the
retention of the compounds in solution. Then, the solutions were filtered and the supernatant was
used as a fluid sample for HPLC detection. The permeability (Pe) of the compounds was tested with
the help of a standard PAMPA (pION). PAMPA was performed on a PAMPA Explorer instrument

Molecules 2017, 22, 1834
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(pION) with the PAMPA Explorer command software (Version 3.7.4.1). The compounds were diluted
to 10 mM with the system solution buffer (pH 7.4). Then, 150 L of the diluted compound solution
was transferred to a UV plate, and the UV spectrum was collected and used as a reference. Then, paint
the membrane with 5 L of gastrointestinal tract (GIT) lipid. The acceptor chamber was filled with
200 L of acceptor solution buffer, and the donor chamber was filled with 200 L of diluted compound
µ
µ
µ
µ
◦
solution. The PAMPA sandwich was assembled and left at 25 C for 4 h. The UV spectra (240–500 nm)
from the donor and the acceptor were collected. The permeability coefficient was calculated with the
PAMPA Explorer Command software (Version 3.7.4.1) based on the area under the curve (AUC) of the
reference plate, the donor plate, and the acceptor plate. The permeability coefficient of the co−m6pound
10 cm/s)
was tested for four times, and the data are shown as the average values. Ketoprofen (1.8
and propranolol (127.5 × 10−6 cm/s) were used as the standards in this assay.
×
3.7. In Vivo Tumor Growth Inhibition Assay
Kunming mice with body weights of 18–22 g were transplanted with lung cancer A549 cells according
to protocols of transplant tumor research, as previously reported [22]. Twenty-four hours after tumor
transplantation, animals were weighed and randomly divided into five groups. All test drugs were given
through injections 24 h after tumor transplantation. The treated groups were intravenously injected or
administered per os different doses of test compounds, respectively. The negative control group received
0.9% normal saline. Treatments were conducted at a frequency of one dose per two days for the intravenous
group or one dose per day for the per os group for two or one week, respectively. All animals were sacrificed
24 h after the final treatment. All data are presented as mean
evaluated by t-test.
±
SD and the statistical significance was
4. Conclusions
In summary, a click chemistry-based optimization of the C1-OH site of the lead compound
DDO-6101, utilizing a strategy that concerned both the in vitro activity and druglike property,
successfully led to the discovery of a novel series of natural-product-like triazole-bearing caged
xanthones with improved druglike properties as orally-active antitumor agents in vivo, represented by
compound 8g. It showed potent inhibitory activities in vitro towards A549, A549/taxol, A549/cisplatin,
and HCT116 cells, with IC50 values of 0.31
which was more active than the lead DDO-6101. Notably, 8g displayed 66.43% growth inhibition in the
±
0.02, 0.42
±
0.05, 0.33
±
0.07, and 0.28
±
0.03, respectively,
lung cancer cell (A549)-transplanted mice with per os administration. Thus, 8g (named as DDO-6318
was identified as a new and orally-active natural-product-like caged xanthone, and also a promising
antitumor agent for further clinical development.
)
1
Supplementary Materials: Supplementary Materials are available online.
for target compounds; Table S1: Data for Figure 3.
1
: H-NMR, 13C-NMR, HRMS spectra
Acknowledgments: We are thankful for the financial support of the National Natural Science Foundation of China
(No. 81603025, 81773571), the Natural Science Foundation of Jiangsu Province (BK20170088), the ‘Qing Lan’ Project
of Jiangsu Province, the National Major Science and Technology Project of China (2015ZX09101032, 2017ZX09302003,
2017ZX09101004-003-006), the Fundamental Research Funds for the Central Universities (2632017ZD03), and the
Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Author Contributions: Z.X. and Y.Q. conceived and designed the experiments; L.X., W.Y. and W.Y. performed the
experiments and analyzed the data; and L.X. and Z.X. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 8a–8j are available from the authors.

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