Sealed tube promoted coupling of camptothecin and norcantharidin acid ester and their preliminary biological activity evaluation in vitro

Article abstract of DOI:10.1007/s00044-017-2066-8

A facile synthetic method was developed for the novel acid-sensitive camptothecin norcantharidin acid ester derivatives 3 in sealed tube. This method offers several advantages including high yield and simple work procedure which can be extended for the synthesis of analogs. The new synthetic compounds 3 have shown better activity against several tumor cell lines in vitro test.

Full text of DOI:10.1007/s00044-017-2066-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2066-8
ORIGINAL RESEARCH
Sealed tube promoted coupling of camptothecin and
norcantharidin acid ester and their preliminary biological
activity evaluation in vitro
Xian H. Wang1 Fu H. Yang1 Chang K. Zhao1 Lei Gao1 Chan Li1
●
●
●
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Received: 21 June 2017 / Accepted: 5 September 2017
© Springer Science+Business Media, LLC 2017
Abstract A facile synthetic method was developed for the
novel acid-sensitive camptothecin norcantharidin acid ester
derivatives 3 in sealed tube. This method offers several
advantages including high yield and simple work procedure
which can be extended for the synthesis of analogs. The
new synthetic compounds 3 have shown better activity
against several tumor cell lines in vitro test.
EDCI
(3-dimethylaminopropyl)
monohydrochloride
4-dimethylaminopyridine
ethyl-carbodiimide
DMAP
●
●
●
Keywords Sealed tube Camptothecin Norcantharidin
Introduction
●
●
Coupling Anticancer Synthesis
In the last decade pharmaceutical industries have focused on
molecular targeting therapy and several drugs based on this
concept have been successfully used as anticancer therapy,
target therapy has been one of important strategies in new
drug discovery (Yadav and Khan 2013; Miao et al. 2013;
Gupta and Mahajan 2017). However, there are several
cancer genes or pathways operating within a given cancer.
At the same time, the drug resistance is still going to be a
problem for concern. Give these two things, the combina-
tion therapy will be needed for optimal therapeutic effect.
Camptothecin (CPT) was first isolated by Wall in 1966
from the Chinese tree Camptotheca acuminate. This isolated
compound has a pentacyclicer ring system with only one
asymmetrical cent in ring E with a 20(S)-configuration. This
pentacyclic ring system includes a pyrrol [3, 4-b] quinoline
moiety (rings A, B and C), a conjugated pyridine (ring D),
and a six-membered lactone (ring E) with an α-hydroxyl
group. However, the initial clinical studies showed that
camptothecin was not usable as an anticancer agent in vivo
due to its high toxicity and low bioavailability (Zhao et al.
2000; Cao et al. 1998; Vishnuvajjala and Garzon-Aburbeh
1990; Cao and Giovanella 2003; Li et al. 2007) (Fig. 1).
Cantharidin is a naturally occurring toxin which has been
isolated in the “Spanish fly” Cantharis vesicatoria, and then
found in many related Mylabris species. Despite potent
Abbreviations
CPT
TMS
MTT
Camptothecin
Tetramethylsilane
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2066-8) contains supplementary
material, which is available to authorized users.
* Chang K. Zhao
zck7103@hotmail.com
Xian H. Wang
xhwang32@hotmail.com
Fu H. Yang
1350324962@qq.com
Lei Gao
79725898@qq.com
Chan Li
911294175@qq.com
1
School of Pharmacy, Zunyi Medical University, No. 6 Xue Fu
West Road, Xin Pu New District, Zunyi City, Guizhou Province
563003, China

Med Chem Res
O
O
O
O
H
H
N
N
CPT
O
O
O
O
O
O
O
O
N
N
O
OH
O
O
R
O
Cantharidin
norcantharidin
Camptothecin
(I)
O
COOR
norcantharidin
Fig. 1 Structure of camptothecin, cantharidin, and norcantharidin
Fig. 2 Proposed molecule combining camptothecin with
norcantharidin
activity against different cancer cell lines, the nephrotoxi-
city of cantharidin has prevented it from entering main-
stream oncology. Norcantharidin, the demethylated analog
of cantharidin, owing a unique feature observed during
clinical trials was the stimulation of the bone marrow pro-
duction of white cells, which is in contrast to most other
anticancer drugs that readily induce myelo-suppression
(Hill et al. 2007; Essers et al. 2001; McCluskey et al. 2000).
In our present high throughout screen project to new
anticancer drugs, we proposed a new molecule that com-
bined CPT with norcantharidin in ester-bond way, which
was expected to have better activity against tumor cell lines.
Herein we reported the synthesis of this unique molecule
(Fig. 2).
condition of Pd/C (0.11 mg, 10%) for 2 days. The catalyst
was removed by filtration. The solvent was evaporated
completely under reduced pressure to give a white solid
1
(0.79 g, 72.2%). H NMR (DMSO-d6, 400 MHz): δ = 4.85
(s, 2H), 3.34 (d, J = 20 Hz, 2H), 1.65 (d, J = 8 Hz, 4H); 13
C
NMR (DMSO-d6, 100 MHz): δ = 173.35, 80.08, 51.14,
40.35, 40.14, 39.93, 39.72, 27.90.
Synthesis of 5,6-dibromo norcantharidin
To a suspension of 5-ene-norcantharidin (1, 5 g) in 20 ml of
CHCl3 was added drop-wise a solution of bromine (0.5 ml)
in chloroform (2.5 ml) at ambient temperature in 20 min.
The solid (8.3 g, 85.4%) was collected by filtration and
washed with carbon tetra-chloride for three times; mp
157–159 °C; Rf: 0.65 (petroleum ether: ethyl acetate = 2: 1);
Materials and methods
Melting points were determined by a Mettler Toledo FP62
melting point apparatus and are uncorrected. 1H NMR
spectra were recorded at 400 MHz on a Varian Unitynova
400 NMR spectrometer using tetramethylsilane as an
internal standard, and 13C NMR spectra were recorded at
100 MHz respectively. Mass spectra were run on a Waters
UPLC-MS instrument. UV spectra were determined by a
Lambda 25 spectrometer. HPLC spectra were run through a
Waters SunfireTM C18, 5 μm, 4.6 × 250 mm column on a
Waters apparatus (pump: e2695; detector: 2998). TLC
plates (GF 254) were bought from Branch Qingdao Haiyang
Chemical Plant. All the solvents and commercial materials
are bought from Sinopharm.
IR (KBr) νmax 1782, 1238, 1085, 972, 917 cm−1; H NMR
1
(DMSO-d6, 400 MHz): δ = 5.20 (d, J = 4 Hz, 1H), 5.02 (s,
1H), 4.51–4.57 (m, 2H), 3.87 (s, 2H); 13C NMR (DMSO-
d6, 100 MHz): δ = 171.82, 170.73, 86.98, 83.31, 52.44,
52.40, 48.96, 46.92.
A typical synthetic procedure for side chain
A suspension of norcantharidin (503.6 mg) in 5 ml of
methanol was refluxed for 3.5 h. After being cooled to room
temperature, the reaction mixture was then crystallized in
refrigerator for 12 h. The resulting solid was collected by
filtration (2a, 145.2 mg, 24.2%). 1H NMR (DMSO-d6, 400
MHz): δ = 12.51 (s, 1H), 6.48–6.43 (m, 2H), 5.08 (s, 2H),
3.54 (s, 3H), 2.73 (s, 2H); 13C NMR (DMSO-d6, 100 MHz):
δ = 172.64, 172.09, 136.71, 136.59, 80.00, 79.67, 51.48,
46.65, 45.94.
Experimental
Chemistry
1
Synthesis of side chain 2.
Synthesis of norcantharidin
Compound 2b (87%); a white solid; H NMR (CDCl3,
400 MHz): δ = 4.91 (d, J = 24 Hz, 2H), 4.11 (d, J = 8 Hz,
2H), 2.99 (q, J = 12 Hz, 3H), 1.81 (t, J = 4 Hz, 2H), 1.52
(d, J = 8 Hz, 2H), 1.21 (t, J = 8 Hz, 3H); 13C NMR (CDCl3,
100 MHz): δ = 176.45, 170.87, 78.59, 78.29, 77.03, 76.71,
61.16, 52.27, 28.97, 13.94.
5-ene-norcantharidin (1, 1.09 g) was dissolved in 20 ml of
tetrahydrofuran. The solution was hydrogenated in the

Med Chem Res
O
O
O
O
O
O
2a: R1=CH3, R2 = H
2b: R1= C2H5, R2 = H
2c: R1= Bn, R2 = H
2d: R1=CH3, R2 = Br
2e: R1= C2H5, R2 = Br
2f: R1= Bn, R2 = Br
O
R2
R2
COOH
O
R1OH
O
O
O
O
R1
O
Br
Br
1
O
O
O
O
2a-f
O
Scheme 1 Synthesis of side chain 2a–f (Scheme 1)
O
N
R2
R2
O
O
O
R2
R2
R1
O
O
O
O
O
OH
2a-f
N
O
O
N
O
O
O
3a-f
R1
O
N
OH
EDCI, DMAP, DCM,
sealed tube, 55oC
3a: R1=CH3, R2 = H
3b: R1= C2H5, R2 = H
3c: R1= Bn, R2 = H
3d: R1=CH3, R2 = Br
3e: R1= C2H5, R2 = Br
3f: R1= Bn, R2 = Br
Scheme 2 Synthesis of target compound 3a–f
Compound 2c (49.4%); a white solid; 1H NMR (DMSO-
d6, 400 MHz): δ = 12.30 (s, 1H), 7.35 (s, 5H), 4.97 (dd,
J = 44 Hz, 12 Hz, 2H), 4.71 (s, 2H), 3.05 (dd, J = 16 Hz,
12 Hz, 2H), 1.54 (dd, J = 20 Hz, 8 Hz, 4H); 13C NMR
(DMSO-d6, 100 MHz): δ = 172.35, 171.07, 136.06, 128.38,
128.06, 127.96, 126.64, 126.42, 77.93, 77.59, 65.64, 62.89,
52.00, 50.93, 28.52.
3.39 (t, J = 8 Hz, 1H); 13C NMR (DMSO-d6, 100 MHz): δ
= 171.48, 169.52, 135.75, 128.38, 128.36, 128.04, 128.01,
126.39, 86.11, 82.37, 66.15, 54.67, 53.88, 48.47, 46.77.
A typical synthesis procedure for compound 3a–f
(Scheme 2)
Compound 2d (86%); a white solid; mp > 240 °C; Rf:
0.41(ethyl acetate: methanol = 3: 1); IR (KBr) vmax 1730,
1693, 1435, 1351, 1273, 1227, 964, 932, 812, 797 cm−1; 1H
NMR (DMSO-d6, 400 MHz): δ = 12.61 (s, 1H), 4.78 (q,
J = 4 Hz, 2H), 4.70 (d, J = 4 Hz, 2H), 3.54 (s, 3H), 3.23 (s,
2H); 13C NMR (DMSO-d6, 100 MHz): δ = 170.76, 170.21,
86.79, 86.50, 54.31, 54.08, 51.68, 49.28, 48.25.
CPT (120 mg), norcantharidin mono methyl ester (2a,
157.2 mg), EDCI (320 mg) and DMAP (24 mg) was sus-
pended in CH2Cl2 (20 ml) and was stirred in a sealed tube at
55 °C for 3 d. CH2Cl2 (40 ml) was added to dilute the
reaction mixture. The mixture was then washed with H2O
(20 mL × 3), saturated Na2CO3 solution (20 ml) and brine
(40 ml) in sequence. The organic layer was dried over
MgSO4. The solvent was removed under reduced pressure,
and the residue was purified by flash column chromato-
graphy, eluting with CH2Cl2: CH3OH = 97:3) to afford the
title compound (3a, 108 mg, 60%) as a yellow solid. Rf =
Compound 2e (82%); a white solid; mp 164–165 °C; Rf:
0.65 (ethyl acetate: methanol = 3: 1); IR (KBr) vmax 1779,
1243, 1084, 969, 916 cm−1 1H NMR (DMSO-d6, 400
;
MHz): δ = 12.66 (s, 1H), 4.74 (d, J = 28 Hz, 2H), 4.42 (s,
1H), 4.00 (q, J = 8 Hz, 2H), 3.54 (t, J = 12 Hz, 1H), 3.35 (t,
J = 12 Hz, 1H), 3.21 (s, 1H), 1.15 (q, J = 4 Hz, 3H); 13C
NMR (DMSO-d6, 100 MHz): δ = 170.74, 169.63, 86.75,
86.46, 60.35, 54.31, 54.13, 49.21, 48.31, 13.83.
1
0.50 (CH2Cl2: CH3OH = 97: 3); H NMR (CDCl3, 400
MHz): δ = 8.39 (s, 1H), 8.20 (t, J = 8 Hz, 1H), 8.01 (s, 1H),
7.94 (d, J = 8 Hz, 1H), 7.83 (t, J = 8 Hz, 1H), 7.67 (d, J =
4 Hz, 1H), 7.26 (t, J = 28 Hz, 2H), 5.68 (d, J = 16 Hz, 1H),
5.42 (d, J = 16 Hz, 1H), 5.29 (d, J = 8 Hz, 2H), 4.98 (d, J
= 12 Hz, 1H), 4.89 (d, J = 8 Hz, 2H), 3.79 (s, 1H), 3.67 (t,
Compound 2f (81%); a white solid; mp 137–139 °C; Rf:
0.76 (ethyl acetate: methanol = 3: 1); IR (KBr) vmax 1785,
1
1615, 1238, 1086, 1007, 970, 919, 852 cm−1; H NMR
J = 28 Hz, 7H), 2.99 (s, 1H), 2.95 (s, 2H), 2.88 (s, 2H); 13
C
(DMSO-d6, 400 MHz): δ = 12.74 (br, 1H), 7.37 (s, 5H),
5.06 (t, J = 16 Hz, 1H), 4.95 (t, J = 4 Hz, 2H), 4.80 (d, J =
8 Hz, 1H), 4.43 (d, J = 16 Hz, 2H), 3.60 (d, J = 8 Hz, 1H),
NMR (CDCl3, 100 MHz): δ = 172.51, 172.42, 171.53,
170.34, 169.97, 167.27, 167.07, 157.28, 148.90, 148.81,
146.49, 146.38, 145.45, 145.30, 130.72, 129.55, 128.39,

Med Chem Res
128.36, 128.18, 128.10, 128.06, 120.32, 120.24, 95.89,
95.84, 80.60, 80.54, 80.46, 78.33, 77.97, 77.81, 67.18,
67.15, 52.28, 52.08, 51.26, 50.98, 50.86, 50.83, 50.00,
49.96, 39.04, 37.08, 36.42, 31.92, 29.16, 27.42, 26.74,
25.39, 22.69, 19.20, 14.17, 14.12, 7.55.
Compound 3d (48%); a yellow solid; Rf = 0.51 (CH2Cl2:
CH3OH = 97: 3); 1H NMR (CDCl3, 400 MHz): δ = 8.39 (s,
1H), 8.20 (t, J = 8 Hz, 1H), 7.93 (d, J = 8 Hz, 1H), 7.83 (t,
J = 8 Hz, 1H), 7.67 (t, J = 8 Hz, 1H), 7.23 (t, J = 8 Hz, 1H),
5.70 (dd, J = 4 Hz, 8 Hz, 1H), 5.4 (dt, J = 4 Hz,1H),
4.92–5.3 (m, 4H), 4.49 (dd, J = 4 Hz, 8 Hz, 1H), 4.32–4.36
(m, 1H), 3.64–3.90 (m, 4H), 3.09 (dd, J = 4 Hz, 4 Hz, 2H),
2.09–2.39 (m, 2H), 1.01 (dt, J = 8 Hz, 3H); 13C NMR
(CDCl3, 100 MHz): δ = 171.59, 170.27, 170.16, 169.29,
168.95, 167.23, 167.09, 166.97, 157.24, 152.06, 148.81,
146.74, 145.55, 145.30, 144.93, 131.26, 131.21, 130.80,
130.75, 129.55, 129.52, 128.44, 128.39, 128.34, 128.20,
128,12, 119.97, 119,63, 95.68, 95.52, 95.31, 88.76, 86.20,
85.96, 84.63, 67.31, 67.20, 67.11, 50.13, 53.14, 53.03,
52.99, 52.85, 50,59, 50.50, 50.23, 50.16, 50.11, 50.04,
49.60, 49.15, 49.12, 48.82, 45.07, 31.74, 31.63, 29.69,
7.71, 7.56.
Compound 3e (44.7%); a yellow solid; Rf = 0.47
(CH2Cl2: CH3OH = 97: 3); 1H NMR (CDCl3, 400 MHz): δ
= 8.40 (s, 1H), 8.21 (d, J = 8 Hz, 1H), 7.93 (d, J = 12 Hz,
1H), 7.83 (t, J = 8 Hz, 1H), 7.67 (t, J = 8 Hz,1H), 7.26 (t, J
= 8 Hz,1H), 5.70 (d, J = 16 Hz, 1H), 5.31 (d, J = 16 Hz,
1H), 5.31 (m, 4H), 4.98 (m, 2H), 4.45 (m, 1H), 4.27 (m,
1H), 4.14 (m, 1H), 3.83 (m, 1H), 3.00 (m, 1H), 2.31 (m,
1H), 2.17 (m, 1H), 2.03 (s, 1H); 13C NMR (CDCl3, 100
MHz): δ = 169.85, 169.76, 169.49, 169.04, 167.32,
157.24,152.02, 148.77, 146.74, 145.49, 145.17, 131.27,
129.54, 129.51, 128.37, 128.34, 128.19, 128.12, 119.88,
119.62, 95.79, 95.62, 95.43, 88.84, 86.22, 85.92, 84.71,
67.28, 67.16, 62.25, 62.10, 53.13, 52.91, 50.06, 49.76,
49.27, 49.03, 48.70, 31.92, 31.68, 31.60, 29.30, 22.69,
14.20, 14.14, 14.09, 7.72, 7.57.
Compound 3b (92.7%); a yellow solid; Rf = 0.47
(CH2Cl2: CH3OH = 97: 3); 1H NMR (CDCl3, 400 MHz): δ
= 8.37 (d, J = 8 Hz, 1H), 8.19 (t, J = 8 Hz, 1H), 7.92 (s,
1H), 7.82 (t, J = 8 Hz,1H), 7.65 (s, 1H), 7.27 (d, J = 36 Hz,
1H), 5.64 (d, J = 20 Hz, 1H), 5.42 (d, J = 16 Hz, 1H), 5.25
(t, J = 16 Hz, 2H), 5.00 (d, J = 16 Hz, 1H), 4.85 (s, 1H),
4.23 (m, 2H), 3.71 (t, J = 12 Hz, 1H), 3.03 (m, 1H), 2.30
(m, 1H), 2.11 (q, 2H), 1.77 (m, 5H), 0.99 (q, 1H), 0.83 (m,
3H); 13C NMR (CDCl3, 100 MHz): δ = 172.09, 172.01,
170.41, 170.12, 167.29, 167.10, 157.27, 157.25, 152.14,
152.06, 148.73, 146.47, 146.36, 145.51, 145.33, 131.20,
130.68, 129.53, 128.18, 128.12, 128.05, 120.26, 120.14,
95.88, 80.73, 80.59, 78.37, 78.20, 77.96, 76.39, 76.37,
67.15, 61.44, 61.28, 51.05, 50.95, 50.76, 50.70, 50.01,
49.95, 31.86, 29.68, 29.46, 29.15, 25.39, 22.68, 14.24,
14.18, 14.12, 7.55.
Compound 3c (10%); a yellow solid; Rf = 0.43 (CH2Cl2:
CH3OH = 97: 3); 1H NMR (CDCl3, 400 MHz): δ = 8.40 (s,
1H), 8.21 (dd, J = 4 Hz, 4 Hz, 1H), 7.95 (s, 1H), 7.93 (s,
1H), 7.83 (t, J = 8 Hz, 1H), 7.67 (t, J = 8 Hz, 1H), 7.39 (d, J
= 4 Hz, 1H), 7.33–7.36 (m, 1H), 7.21–7.26 (m, 4H), 5.68 (d,
J = 16 Hz, 1H), 5.39 (d, J = 16 Hz, 1H), 5.23–5.31 (m, 3H),
5.16–5.22 (dd, J = 4 Hz, 4 Hz, 1H), 5.12 (s, 1H), 4.98 (s,
1H), 4.93–5.01 (dd, J = 4 Hz, 4 Hz, 1H), 4.33 (dd, J = 8 Hz,
8 Hz, 1H), 3.81–3.86 (m, 1H), 1.51–1.67 (m, 4H), 1.03 (t, J
= 8 Hz, 2H), 0.93 (q, J = 8 Hz, 3H); 13C NMR (CDCl3, 100
MHz): δ = 171.86, 171.79, 170.37, 170.08, 167.32, 167.06,
157.29, 157.24, 152.17, 152.08, 148.80, 146.38, 145.33,
145.22, 135.58, 135.48, 130.71, 130.67, 129.67, 129.64,
120.27, 95.89, 95.85, 80.67, 80.64, 78.23, 77.96, 67.24,
67.09, 66.90, 66.69, 52.18, 51.08, 51.01, 50.75, 50,58,
50.01, 49.95, 31.92, 31.82, 29.05, 25.47, 25.37, 7.54.
Compound 3f (51.2%); a yellow solid; Rf = 0.46
1
(CH2Cl2: CH3OH = 97:3); H NMR (CDCl3, 400 MHz): δ
= 8.40 (s, 1H), 8.21(q, 1H), 7.94 (d, J = 8 Hz,1H), 7.67 (t,
J = 8 Hz, 1H), 7.36 (m, 6H), 5.69 (d, J = 16 Hz,1H), 5.40
(d, J = 20 Hz, 1H), 5.31 (s, 1H), 5.28 (d, J = 4 Hz, 1H),
Table 1 In vitro antitumor
HepG2
SW480
BGC803
PANC-1
activities (inhibition/%) of
Camptothecin (CPT) analogs
3a–g
Solventa
1.16
1.02
0.93
1.04
Cantharidin
CPT
78.08
74.19
53.31
72.64
37.45
67.45
52.45
38.81
75.04
71.04
71.05
55.22
61.05
76.04
62.55
60.57
75.33
73.95
71.62
60.63
70.06
70.01
66.09
66.64
77.21
73.88
39.49
24.33
37.48
70.75
36.98
32.32
Compound 3a
Compound 3b
Compound 3c
Compound 3d
Compound 3e
Compound 3f
Prelimary testing concentartion c = 50 μM
a
Test solvent DMSO

Med Chem Res
5.23 (t, J = 20 Hz, 1H), 5.13 (t, J = 16 Hz, 2H), 4.98 (m,
2H), 4.36 (m, 1H), 3.83 (m, 1H), 3.05 (m, 1H), 2.21 (m,
2H), 1.03 (d, J = 8 Hz, 2H), 0.94 (d, J = 8 Hz, 2H); 13C
NMR (CDCl3, 100 MHz): δ = 172.09, 172.01, 170.41,
170.12, 167.29, 167.10, 157.29, 157.25, 152.14, 152.06,
148.73, 146.47, 146.36, 145.51, 145.33, 131.20, 130.68,
129.53, 128.18, 128.12, 128.05, 120.26, 120.14, 95.88,
80.73, 80.59, 78.37, 78.20, 77.96, 76.39, 76.37, 67.15,
61.44, 61.28, 51.05, 50.76, 50.70, 50.01, 49.95, 31.86,
29.68, 29.46, 29.15, 25.39, 22.68, 14.24, 14.18, 14.12, 7.5.
most of the norcantharidin CPT compounds showed similar
activities against SW480 and BGC803 cell lines, and
compound 3b and 3d also showed potent activities against
Hep G2 and PANC-1 (Table 1).
Results and discussions
CPT and norcantharidin are both commercial available mate-
rials, which were thus chosen as starting materials to prepare
this target molecule (I). In the earlier literature (Cao 2008), a
general acid catalyzed process for the preparation of 20(S)-O-
linked ester. However, one of our reaction substrates nor-
cantharidin is high acid sensitive and subject to ring opening.
We then attempted to try the direct coupling of camp-
tothecin with norcantharidin or its acid in the presence of
EDCI and DMAP, DCM as a solvent under reflux condi-
tion. Neither can give any desired product (Scheme 3).
Based on the above result, it seems to be very difficult to
couple CPT with dicarboxylic acid directly in the normal
condition maybe because of the high polarity, low solubility,
low electrophilicity and space hindrance of the substrate.
Therefore we planed to modify the coupling substrate by
introducing a protecting group to decrease its polarity and
increase its electrophilicity. Thus, norcantharidin monoacid
monoester 2 was chosen as material to couple with CPT in
the condition of EDCI and DMAP under reflux for several
days, but no any reaction occurred. Finally, the target com-
pound 3 was smoothly obtained in a moderate yield when the
Proliferation inhibition assay
HepG2, SW480, BGC803, and PANC-1 cells were cultured
in RPMI 1640 or McCoy’s 5 A medium (Invitrogen)
respectively, supplemented with 10% heat-inactivated FBS
and 1% penicillin/streptomycin (Thermo Fisher Scientific).
All cell lines were maintained at 37 °C with 5% CO2. Cell
viability was evaluated by the MTT assay. Cells were see-
ded in 96-well plates and incubated for 24 h. A range of
concentrations of the test compounds were added and the
plates were incubated for 72 h before the addition of 10 μl
MTT (5 mg/ml)/well. After 4 h of incubation, the medium
was removed and 100 μl DMSO was added to each well.
The absorbance was measured using a SpectraMax M5
microplate reader at 550 nm.
A series of 20(S)-O-norcantharidin camptothecin deri-
vatives 3a–g were designed and synthesized to improve
antitumor activity and investigate the SARs. To our delight,
O
O
R2
R2
O
O
R2
R1
O
O
N
O
O
O
OH
N
R2
O
O
2
O
N
O
O
O
N
R1
OH
EDCI, DMAP, DCM,
sealed tube, 55oC
3
O
norcantharidin
O
O
O
N
O
O
N
O
O
O
N
O
O
HOOC
O
N
OH
or
O
O
COOH
HOOC
Scheme 3 The coupling of CPT with norcantharidin or its acid

Med Chem Res
Table 2 Direct coupling of CPT with norcantharidin monoester 2
References
Substrate R1
R2 Coupling
Solvent Temp. Time yield
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reagent
CPT + 2a CH3
CPT + 2b C2H5
CPT + 2c Bn
H
H
H
EDCI/DMAP DCM
50 °C 3 d
50 °C 3 d
50 °C 3 d
60%
93%
10%
48%
EDCI/DMAP DCM
EDCI/DMAP DCM
CPT + 2d CH3 Br EDCI/DMAP CHCl3 75 °C 5 d
CPT + 2e Et
CPT + 2f Bn
Br EDCI/DMAP DCM
Br EDCI/DMAP DCM
50 °C 24 h 45%
50 °C 24 h 51%
coupling reaction of CPT with compound 2 was set up in a
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A new acid-sensitive 20(S)-O-linked CPT norcantharidin
acid ester was firstly designed and synthesized via sealed
tube reaction from commercial available materials. This
method offers several advantages including high yield and
simple work procedure for the following similar synthesis.
This series of compounds have shown a strong activity
against several cancer cell lines. The further SAR study of
these derivatives is also ongoing.
Acknowledgements I am very grateful for the finacial support from
Joint fund project of Guizhou Provincial department of Science and
Technology (QianKeHe LH Zi [2014]7545), (QianKeHe LH Zi [2015]
7531), Science and Technology Department of Guizhou Province
(QKHSY [2017]2844) and (QKHSY [2015]3030). I am also thankful
for Dr. Yuqi He’s useful explanation on HPLC and LC/MS spectra
data.
Zhao H, Lee C, Sai P, Choe YH, Boro M, Pendri A, Guan S,
Greenwald RB (2000) 20-O acylcamptothecin derivatives: evi-
dence for lactone stabilization. J Org Chem 65:4601–4606
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.