Synthesis of novel ent-kaurane-type diterpenoid derivatives effective for highly aggressive tumor cells

Article abstract of DOI:10.3390/molecules23123216

We have designed and synthesized 6 ent-Kaurane-type diterpenoid derivatives containing α,β-unsaturated ketone moieties. In vitro, activity was evaluated against three human tumor cell lines and a rat myogenic cell line (HepG2, NSCLC-H292, SNU-1040, L6) by MTT assay. All the tested compounds exhibited comparable or higher activity than DDP and eriocalyxin B. Compounds 16, 17 and 18 are promising anti-tumor leads due to their cytotoxic potencies and higher selectivity, with SI values of 161.06, 47.80 and 128.20, respectively.

Full text of DOI:10.3390/molecules23123216

molecules
Article
Synthesis of Novel ent-Kaurane-Type Diterpenoid
Derivatives Effective for Highly Aggressive
Tumor Cells
Yu Hu 1, Xiao-Nian Li 2, Ze-Jin Ma 1, Pema-Tenzin Puno 2,*, Yong Zhao 1, Yan Zhao 1,
Ye-Zhi Xiao 1, Wei Zhang 1 and Jing-Ping Liu 1,
*
1
Department of Chemistry, Yunnan Normal University, Kunming 650092, China; xpcxyy@sina.com (Y.H.);
mazejinmzj@sina.com (Z.-J.M.); zhaooy@126.com (Y.Z.); zhaooyann@163.com (Y.Z.);
xyzlmm@sina.com (Y.-Z.X.); royzhangwei@sina.com (W.Z.)
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Kunming 650201, China; lixiaonian@mail.kib.ac.cn
2
*
Correspondence: punopematenzin@mail.kib.ac.cn (P.-T.P.); Liujp72@163.com (J.-P.L.);
Tel.: +86-871-65223616 (P.-T.P.); +86-871-65132144 (J.-P.L.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 November 2018; Accepted: 4 December 2018; Published: 5 December 2018
Abstract: We have designed and synthesized 6 ent-Kaurane-type diterpenoid derivatives containing
α
,
β-unsaturated ketone moieties. In vitro, activity was evaluated against three human tumor cell
lines and a rat myogenic cell line (HepG2, NSCLC-H292, SNU-1040, L6) by MTT assay. All the tested
compounds exhibited comparable or higher activity than DDP and eriocalyxin B. Compounds 16
17 and 18 are promising anti-tumor leads due to their cytotoxic potencies and higher selectivity,
with SI values of 161.06, 47.80 and 128.20, respectively.
,
Keywords: ent-kaurane-type diterpenoid derivatives; antitumor; synthesis
1. Introduction
Highly aggressive tumors are a major cause of death worldwide, accounting for 8.8 million deaths
each year, and are estimated to cause 9.6 million deaths in 2018. Globally, about 1 in 6 deaths is due
to cancer. Approximately 70% of deaths from cancer occur in low- and middle-income countries,
according to the World Health Organization (WHO) [1].
Plants are normally an important source of new drugs [
members have been promising leads due to their extensive pharmacological and physiological
effects, such as inhibition of hepatitis viral replication [ ] and bacterial infections of the lung or
gut [ ], as well as its antimalaria [ ], antiinflammatory [ ], and anti-tumor properties [ ]. Hitherto,
over 1200 ent-Kaurane-type diterpenoids with highly compact polycyclic ring systems have been
isolated from Isodon genus, notably the compounds oridonin [ ]. Natural Diterpenoid Isoferritin A
(IsoA) [ ], longikaurin [10], xerophilusins [11] (Figure 1, ), which have been studied for anti-tumor
activity. A common structural unit presented by these diterpenoid compounds is the functionalized
bridged C/D -unsaturated ketone moiety. Unfortunately, the use of these natural diterpenoids
2]. In recent years, Isodon genus
3
4
5
6
7
8
9
1–4
α,β
for anti-tumor was hampered by its moderate potency and complex oxygenated diterpenoid scaffold.
Therefore, it is very necessary to find compounds with simple structure and stronger activity.
Molecules 2018, 23, 3216; doi:10.3390/molecules23123216
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3216
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OH
OH
H
H
H
OH
O
OH
O
OH
O
OH
O
O
O
OH
O
O
O
H
H
OAc
H
OH
OH
OH
longikaurin A (3)
IsoA (2)
xerophilusins I (4)
oridonin (1)
Figure 1. Structures of ent-Kaurane-type diterpenoids.
In the preceding letter we reported the synthesis and biological evaluation of a series of
eriocalyxin B derivatives (Figure 2, ) and found that eriocalyxin B derivatives bearing one or two
-unsaturated ketone units are potent antitumor agents [12]. It is noteworthy that the 7-hydroxyl
5–7
α,β
group and the hydrogen bond between 6-hydroxyl group and 15-carbonyl group are not necessary for
antitumor activity.
12
O
O
13
11
20
O
O
16
H
O
1
17
H
H
8
14
9
2
3
O
15
10
O
O
O
5
7
H
O
4
H
O
H
OH
H
6
OH
O
H
OH
n=2-9
laxiflorin derivatives (7)
18
19
eriocalyxin B (5)
laxiflorin J (6)
Figure 2. Structures of eriocalyxin B, laxiflorin J and derivatives.
2. Results and Discussion
2.1. Chemistry
Herein we report the synthesis of ent-Kaurane-type diterpenoid derivatives containing two
α,β-unsaturated ketone moieties and their in vitro cytotoxic properties against three human tumor
cell lines. Our synthesis began with reacting isobutyraldehyde with methyl vinyl ketone (MVK) in the
presence of sulfuric acid which produced 4,4-dimethylcyclohex-2-enone (compound 8). Treatment of
commercially available 1,3-cyclohexan-dione with ethanol in a reflux of benzene in the presence of
p-toluene sulfonic acid resulted in 3-ethoxy-cyclohex-2-enone (compound
treated sequentially with NaH and diethyl carbonate to directly produce
-ketoester with NaH and allyl bromide produced compound 10 (81%). The resulting compound 10
9) (86%). Compound
9 was
β-ketoester. Treatment of
β
was reduced using NaBH4 followed by acidification to produce compound 11. Following the well-
established Ma protocol [13], the key intermediate 12 (52%) was obtained (Scheme 1).
Deprotonation of
8 with LiHMDS followed by a reaction with compound 12 led to compound
13 as the major product, along with isomer 14 in 82% combined yield (dr = 60:40). The structures
of 13 and 14 were confirmed by NMR spectral and X-ray crystallographic analysis (Figure 3). Next,
compound 13 was subjected to allylic oxidation with selenium dioxide (SeO2, CH2Cl2, t-BuOOH) and
IBX oxidation to form compounds 15 and 16, respectively. Compound 17 (or 18) was provided from
the diastereoisomer 14 under the same conditions as 15 (or 16) (Scheme 2).

Molecules 2018, 23, 3216
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H2SO4
O
benzene
reflux
O
CHO
92%
8
TsOH
ethanol
reflux
1. NaH
O
9
O
O
(CH3O)2CO
CO2C2H5
O
O
86%
O
2. Allyl bromide
81%
10
O
TBSOTf, Et3N
O
1.
THF, -60℃
1. NaBH4
2. 10% Pd(OAc)2, DMSO
2. HCl
84%
C2H5O2C
O2, 40℃
C2H5O2C
52%
11
12
Scheme 1. Synthesis of compound 8 and compound 12.
O
O
H
CO2C2H5
O
O
LiHMDS
13
THF, -78℃
O
82%
CO2C2H5
O
dr = 6:4
H
8
12
CO2C2H5
14
O
H
O
H
O
H
O
O
SeO2
t-BuOOH
O
IBX
O
OH
CH2Cl2
r.t.
EtOAc
CO2C2H5
O
O
O
O
15
13
16
O
H
O
O
O
H
O
OH
O
O
O
O
18
17
Scheme 2. Synthesis of cmopounds 13, 14, 15, 16, 17 and 18.

Molecules 2018, 23, 3216
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compound 14
compound 13
Figure 3. X-ray crystallographic analysis of compound 13 and 14.
2.2. Biological Evaluation
The cytotoxic properties of all newly synthesized compounds were evaluated in vitro against
three human tumor cell lines ((HepG2, NSCLC-H292, SNU-1040) and one rat myotubes cell line (L6)
by MTT assay. Cisplatin (DDP) and eriocalycin B (5) were used as reference drugs. The results are
summarized in Table 1. (IC50 value is defined as the concentrations corresponding to 50% growth
inhibition). Remarkably, all the tested compounds exhibited comparable or even higher activity than
their natural references eriocalyxin B and DDP. Although results are preliminary, we found a clear
structure-activity relationship: the
for compound 16 (IC50 = 0.33; 1.69; 2.44
-unsaturated ketone moieties, these had stronger in vitro potencies and cytotoxic activities to DDP.
To obtain insight into the cytotoxic potential of these new compounds on normal cells, the effect of
compounds 13 14 15 16 17 18 and DDP (cisplatin) were evaluated in human liver carcinoma cell
α
,
β-unsaturated ketone moiety is necessary for potency. Particularly
µ
M) and 18 (IC50 = 0.56; 1.35; 3.01 M), which display two
µ
α,β
,
,
,
,
,
(HepG2) and rat myotubes (L6) cells. All the tested analogues exhibited higher selectivity than DDP.
Especially, compound 16, which had more potency and selectivity than DDP and eriocalyxin B, with an
SI (selective index, IC50 of normal cells/IC50 of tumor cells) value of 161.06. Interestingly, the two
epimers 16, 18 showed no significant difference in their anticancer activities.
Table 1. In vitro cytotoxic activities of compounds 13–18 (IC50, µM) a,b
.
Cell Lines
Compounds
HepG2
NSCLC-H292
SNU-1040
L6
13
14
15
16
17
18
13.28
11.67
1.81
0.33
1.58
0.56
2.12
2.90
4.44
2.95
1.99
1.69
0.99
1.35
7.11
8.92
1.41
1.72
3.13
2.44
2.82
3.01
1.54
7.76
95.74
41.52
51.57
53.15
75.53
71.79
Eriocalyxin B
DDP
115.17
15.04
a: Cytotoxicity as IC50 values for each cell line, the concentration of compound that caused 50% reduction in
absorbance at 570 nm relative to untreated cells using the MTT assay; b: Human liver carcinoma cell line (HepG2),
non-small cell lung cancer cell line (NSCLC-H292), human colon cancer cell line (SNU-1040), normal skeletal muscle
of rat myotubes (L6).

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3. Materials and Methods
3.1. Chemistry
All regents were used as purchased from Energy Chemical (Shanghai, China), Tansoole (Shanghai,
China), Acros (Beijing, China) and Aldrich (Shanghai, China) without further purification. Anhydrous
CH2Cl2 was freshly distilled from CaH2, and THF was dried by Na. For reactions carried out under
Ar, at least three times. The course of reaction was monitored by TLC. 1H NMR and 13C NMR spectra
were recorded at ambient temperature with a Bruker Avance 300 (300 MHz for 1H and 75.5 MHz for
13C; Bruker, Germany) instrument in which TMS was used as internal standard for all measurements.
MS data were recorded by using a VG Auto spec-3000 spectrometer or a Finnigan MAT 90 instrument
(Bremen, Germany). IR spectra were measured as KBr pellets by using a Bio-Rad FTS-135 spectrometer
(California, CA, USA). All melting points are uncorrected. CC was performed by using silica gel
(100–200 mesh; Qingdao Marine Chemical, Shandong, China).
4,4-Dimethylcyclohex-2-enone (8): To a solution of isobutyraldehyde (3 g, 41.6 mmol) and methyl vinyl
ketone (2.91 g, 41.6 mmol) was added 1% mmol conc. H2SO4. The resulting mixture was stirred with a
Dean-Stark apparatus in refluxing benzene (20 mL) for 5 h. The reaction mixture was neutralized by
sat. NaHCO3 to pH = 7. Organic layer was separated and water was extracted with EtoAc (3
The organic phases were combined, dried over MgSO4, and concentrated on a rotary evaporator.
×
10 mL).
The residue was purified by flash chromatography (silica, ethyl acetate:petroleum = 1:10) to give
1
compound 8 (92%) as pale yellow liquid: H NMR (400 MHz, CDCl3)
δ = 6.67(d, J = 6 Hz, 1H), 5.82 (d,
J = 6 Hz, 1H), 2.45 (t, J = 6 Hz, 2H), 1.87 (t, J = 6 Hz, 2H), 1.17 (s, 6H) ppm; 13C NMR (100 M Hz, CDCl3)
δ
= 199.48, 159.79, 126.77, 36.03, 34.34, 32.77, 27.64 ppm; IR (KBr):
υ
= 3441.89, 2966.23, 2926.56,
max
1638.51 cm−1; MS (ESI): m/z: 125; HRMS (ESI) Calcd. for [(C8H12O) + H]+: 125.0966; found: 125.0961
[M + H]+.
3-Ethoxycyclohex-2-enone (9): To a solution of cyclohexane-1,3-dione (1.12 g, 10 mmol) in dry ethanol
was added 1% mmol TsOH. The resulting mixture was refluxed for overnight. Then the solvent
was removed in vacuo and the residue was purified by flash column chromatography (eluting with
1
EtOAc:petroleum = 1:20–1:10) to give compound
CDCl3)
J = 6.0 Hz, 2H), 0.80 (t, J = 6.9 Hz, 3H) ppm; 13C NMR (75.5 MHz, CDCl3)
9
(86%) as colorless liquid: H NMR (300 MHz,
δ
= 4.74 (s, 1H), 3.36 (q, J = 6.9 Hz, 2H), 1.85 (t, J = 6.0 Hz, 2H), 1.74 (t, J = 6.0 Hz, 2H), 1.11 (p,
= 197.496, 176.357, 101.367,
62.996, 35.635, 27.897, 20.201, 12.970 ppm; IR (KBr):υ
max = 2944.51, 1591.52 cm−1; MS (ESI): m/z: 141;
δ
HRMS (ESI) Calcd. for [(C8H12O2) + H]+: 141.0916; found: 141.0906 [M + H]+.
Ethyl 1-allyl-4-ethoxy-2-oxocyclohex-3-enecarboxylate (1◦0): NaH (0.6 g, 15 mmol, 60% in mineral oil)
was suspended in diethyl carbonate (10 mL) at 100 C. A solution of
carbonate (2 mL) was added to the suspension by syringe and stirring was continued for 5 h at 100 C.
9
(1.4 g, 10 mmol) in diethyl
◦
Upon cooling to ambient temperature, the reaction mixture was washed with brine (2
×
10mL) and
evaporated in vacuo without further purification. To a solution of crude product in dry THF was
added NaH (0.4 g, 10 mmol, 60% in mineral oil) at room temperature. After 30 min, the mixture was
added allyl bromide (1.2 g, 10 mmol) and refluxed for 3 h. The reaction mixture was poured into
ice-water and extracted with ether. The ether layer was washed with water and dried over Na2SO4.
1
Removal of solvent followed by CC gave compound 10 (81%) as colorless liquid: H NMR (300 MHz,
CDCl3)
2.74–2.32 (m, 5H), 1.97–1.91 (m, 1H), 1.19 (t, J = 6.9 Hz, 3H), 1.05 (t, J = 6.9 Hz, 3H) ppm; 13C NMR
(75.5 MHz, CDCl3) = 194.98, 176.59, 171.13, 133.50, 118.42, 101.65, 64.24, 60.98, 55.33, 38.38, 37.95,
26.10, 13.93 ppm; IR (KBr):
δ = 5.77 (m, 1H), 5.35 (s, 1H), 5.09 (m, 2H), 4.17 (q, J = 6.9 Hz, 2H), 3.92 (q, J = 6.9 Hz, 2H),
δ
υ
max = 2979.73, 2937.66, 1725.23, 1655.39, 1605.02, 1186.77 cm−1; MS (ESI):
m/z: 275; HRMS (ESI) Calcd. for [(C14H20O4) + Na]+: 275.1259; found: 275.1254 [M + Na]+.
Ethyl 1-allyl-4-oxocyclohex-2-enecarboxylate (11): To a solution of 10 (1 g, 3.97 mmol) in MeOH (5 mL) was
added CeCl3 7H2O (1.48 g, 3.97 mmol). After 10 min of stirring, the reaction mixture was added NaBH4
·

Molecules 2018, 23, 3216
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(0.3 g, 7.94 mmol) about for 30 min. Then the mixture was acidified to pH 6 by the dropwise addition
of concentrated HCl, and extracted with ethyl acetate and washed with brine. The combined organic
layers were dried over Na2SO4 and the solvent was removed under reduced pressure. Purification
1
of the residue by column chromatograph afford compound 11 (84%) as pale yellow liquid: H NMR
(300 MHz, CDCl3)
4.15 (q, J = 7.2 Hz, 2H), 2.52–2.34 (m, 5H), 1.98–1.93 (m, 1H), 1.24 (t, J = 7.2 Hz, 3H) ppm; 13C NMR
(75.5 MHz, CDCl3) = 198.498, 172.728, 150.539, 131.920, 129.227, 119.638, 61.435, 47.502, 42.884, 34.525,
δ = 6.88(d, J = 10.2 Hz, 1H), 5.97 (d, J = 10.2 Hz, 1H), 5.66 (m, 1H), 5.10 (m, 2H),
δ
30.238, 14.184 ppm; IR (KBr):υ
max = 2978.78, 2958.27, 1728.05, 1638.95, 1614.26 cm−1; MS (ESI): m/z:
209; HRMS (ESI) Calcd. for [(C12H16O3) + H]+: 209.1178; found: 209.1169 [M + H]+.
Ethyl 6-methylene-4-oxobicyclo[3.2.1]oct-2-ene-1-carboxylate (12): To a solution of 11 (6 g, 28.85 mmol,
◦
1.0 eq) in dry THF (200 mL) under Ar at –60 C was added Et3N (11.6 mL, 115.4 mmol, 4.0 eq).
The mixture was stirred for 20 min at this temperature, and then TBSOTf (19.11 g, 72.13 mmol, 2.5 eq)
was added dropwise. After full conversion was observed by TLC, the reaction mixture was quenched
with saturated NaHCO3 solution and extracted with EtOAc. The combined organic layers were dried
over Na2SO4 and the solvent was removed under reduced pressure. The residue was passed through a
short column of silica gel to provide the corresponding silyl enol ether as a colorless oil. The obtained
silyl enol ether was dissolved in DMSO (200 mL) and Pd(OAc)2 (650 mg, 2.89 mmol, 0.1 eq) was added,
and the resulting solution was stirred under oxygen atmosphere (balloon with O2) at 40 ◦C for 6 h.
The reaction was quenched with water, the mixture was extracted with Et2O, and the combined organic
layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure.
The residue was purified by flash column chromatography to afford compound 12 (40%) as pale yellow
1
liquid: H NMR (300 MHz, CDCl3)
δ = 7.43 (d, J = 9.9 Hz, 1H), 5.81 (d, J = 9.9 Hz, 1H), 5.26 (s, 1H),
5.05 (s, 1H), 4.19 (q, J = 7.2 Hz, 2H), 3.47 (br., 1H), 2.86 (d, J = 95.4 Hz, 1H), 2.52 (d, J = 95.4 Hz, 1H), 2.25
(m, 2H), 1.25 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (75.5 MHz, CDCl3)
= 197.515, 172.761, 152.359, 143.136,
= 2979.98, 1729.06,
δ
126.641, 113.126, 61.584, 57.960, 51.617, 43.207, 41.109, 14.120 ppm; IR (KBr):
υ
max
1685.40, 1280.90, 1222.04, 1193.09 cm−1; MS (ESI): m/z: 207; HRMS (ESI) Calcd. for [(C12H14O3) + H]+:
207.1021; found: 207.1015 [M + H]+.
3.1.1. General Method for Synthesis of 13 and 14
◦
To a stirred solution of
(2 eq, 1.3 M in THF) over a period of 5 min and stirring at
8
(1 eq) and 12 (1 eq) in dry THF under Ar at
−
78 C; was added LiHMDS
−
78 ◦C was continued for 45 min. The
resulting solution was warmed to room temperature, quenched with saturated NH4Cl and extracted
with Et2O. The combined organic layers were dried over Na2SO4 and the solvent was removed under
reduced pressure. The residue was purified by flash column chromatography to provide 13 and 14.
Compound 13: The compound is obtained as crystal; m.p.: 112.0–114.6 ◦C; 1H NMR (300 MHz,
CDCl3)
2H), 3.50 (m, 1H), 3.28 (s, 1H), 2.78 (s, 2H), 2.74–2.65 (m, 2H), 2.28 (m, 1H), 1.92–1.66 (m, 4H), 1.29 (t,
J = 6.0 Hz, 3H), 1.26 (s, 3H), 1.25 (s, 3H) ppm; 13C NMR (75.5 MHz, CDCl3)
= 208.93, 198.35, 174.52,
158.35, 145.59, 126.50, 109.91, 61.00, 58.73, 52.97, 46.11, 43.09, 38.80, 38.56, 36.41, 36.11, 33.70, 30.64,
δ = 6.56 (d, J = 6.0 Hz, 1H), 5.80 (d, J = 6.0 Hz, 1H), 5.10 (s, 1H), 5.01 (s, 1H), 4.15 (q, J = 6.0 Hz,
δ
24.95, 14.21 ppm; IR (KBr):
υ
= 2962.26, 2901.21, 1722.91, 1710.24, 1672.31, 1229.31, 1194.50 cm−1
;
max
MS (ESI): m/z: 353; HRMS (ESI) Calcd. for [(C20H26O4) + Na]+: 353.1729; found: 353.1724 [M + Na]+.
Compound 14: The compound is obtained as crystal; m.p.:103.2–105.8 ◦C; 1H NMR (300 MHz,
CDCl3)
2H), 3.30 (s, 1H), 2.65–2.39 (m, 6H),1.96 (m, 1H), 1.78–1.22 (m, 3H), 1.01 (t, J = 6.0 Hz, 3H), 0.88 (s, 6H)
ppm; 13C NMR (75.5 MHz, CDCl3)
= 206.74, 199.50, 175.14, 157.76, 146.00, 127.27, 109.41, 60.96, 59.66,
δ = 6.46(d, J = 6.0 Hz, 1H), 5.68 (d, J = 6.0 Hz, 1H), 5.04 (s, 1H), 4.88 (s,1H), 4.11 (q, J = 6.0 Hz,
δ
52.69, 45.89, 44.46, 43.35, 42.79, 37.86, 35.31, 34.03, 30.39, 24.74, 14.26 ppm; IR (KBr):
υ
= 2958.90,
max
1716.70, 1673.18, 1538.19, 1234.84, 1215.98 cm−1; MS (ESI): m/z: 353; HRMS (ESI) Calcd. for [(C20H26O4)
+ Na]+: 353.1729; found: 353.1732 [M + Na]+.

Molecules 2018, 23, 3216
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3.1.2. General Method for Synthesis of 15 and 17
To a stirred solution of 13 (1.0 eq) in DCM, SeO2 (0.9 eq) was added in one portion, and a solution
of t-BuOOH (5.5 M in decane, 3 eq) was added dropwise. The solution was vigorously stirred at room
temperature overnight, and then the mixture was filtered through a short pad of silica gel washed with
EtOAc. The filtrate was concentrated under vacuum and purified by flash column chromatography to
provide allylic alcohol 15.
1
Compound 15: The compound is obtained as pale yellow liquid; H NMR (300 MHz, CDCl3)
δ
= 6.52 (d, J = 6 Hz, 1H), 5.75 (d, J = 6 Hz, 1H), 5.40 (s, 1H), 5.30 (s, 1H), 4.58 (s, 1H), 4.16 (q,
J = 6 Hz, 2H), 3.32 (m, 2H), 2.57 (m, 3H), 2.17–1.69 (m, 5H), 1.21 (t, J = 6 Hz, 3H), 1.08 (s, 6H) ppm; 13
NMR (75.5 MHz, CDCl3) = 208.33, 198.08, 172.65, 158.66, 149.08, 126.53, 115.31, 79.98, 61.18, 59.47,
C
δ
56.68, 46.35, 37.91, 36.43, 35.20, 33.54, 32.64, 30.62, 25.07, 14.28 ppm; IR (KBr):υmax = 3451.12, 2961.46,
2927.59, 2871.68, 1714.49, 1669.43, 1280.80, 1223.47, 1199.50, 1059.84, 1037.64 cm−1; MS (ESI): m/z: 369;
HRMS (ESI) Calcd. for [(C20H26O5) + Na]+: 369.1678; found: 369.1677 [M + Na]+.
1
Compound 17: The compound is obtained as pale yellow liquid; H NMR (300 MHz, CDCl3)
δ
= 6.48 (d, J = 9 Hz, 1H), 5.68 (d, J = 9 Hz, 1H), 5.36 (s, 1H), 5.31 (s, 1H), 4.46 (s, 1H), 4.15 (q, J = 6 Hz,
2H), 3.37 (m, 1H), 2.45–2.39 (m, 5H), 1.98 (m, 2H), 1.78 (m, 1H), 1.68 (m, 1H), 1.24 (t, J = 6 Hz, 3H),
1.06 (s, 3H), 1.05 (s, 3H) ppm; 13C NMR (75.5 MHz, CDCl3)
= 206.21, 199.22, 173.03, 157.97, 150.47,
127.26, 114.27, 79.08, 61.06, 59.93, 57.98, 45.66, 43.62, 42.09, 37.51, 34.07, 31.78, 30.36, 24.80, 14.36 ppm;
δ
IR (KBr):υmax = 3374.71, 2985.31, 2963.32, 2945.34, 2928.06, 2873.40, 1726.97, 1705.28, 1670.93, 1633.80,
1616.71, 1228.42, 1203.67, 1085.27, 1045.22 cm−1; MS (ESI): m/z: 369; HRMS (ESI) Calcd. for [(C20H26O5)
+ Na]+: 369.1678; found: 369.1682 [M + Na]+.
3.1.3. General Method for Synthesis of 16 and 18
◦
To a solution of 15 (1.0 eq) in EtOAc was added IBX (3.0 eq). The mixture was heated at 70 C for
4 h and filtered through a short pad of silica gel washed with EtOAc. The solvent was removed under
reduced pressure and the residue was purified by flash column chromatography to provide 16.
1
Compound 16: The compound is obtained as pale yellow liquid; H NMR (300 MHz, CDCl3)
δ
= 6.57 (d, J = 9 Hz, 1H), 6.19 (s, 1H), 5.83 (d, J = 9 Hz, 1H), 5.59 (s, 1H), 4.27 (q, J = 6 Hz, 2H), 3.68 (m,
2H), 2.77–2.68 (m, 4H), 2.11 (d, 1H), 1.85 (m, 2H), 1.29 (t, J = 6 Hz, 3H), 1.16 (s, 6H) ppm; 13C NMR
(75.5 MHz, CDCl3) = 206.69, 198.72, 197.33, 168.85, 157.95, 142.16, 126.66, 120.53, 61.80, 61.16, 54.10,
δ
46.06, 39.07, 37.43, 35.08, 33.73, 32.93, 30.59, 25.18, 14.19ppm; IR (KBr):υmax= 3434.29, 2962.17, 2929.37,
2871.25, 1722.07, 1675.43, 1638.37, 1249.74, 1177.04, 1111.95 cm−1; MS (ESI): m/z: 367; HRMS (ESI)
Calcd. for [(C20H24O5) + Na]+: 367.1521; found: 367.1528 [M + Na]+.
1
Compound 18: The compound is obtained as pale yellow liquid; H NMR (600 MHz, CDCl3)
δ
= 6.58 (d, J = 9.9 Hz, 1H), 6.13 (s, 1H), 5.78 (d, J = 9.9 Hz, 1H), 5.58 (s, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.78
(s, 1H), 2.93 (d, J = 12.2 Hz, 1H), 2.66 (m, 3H), 2.45 (m, 1H), 2.13 (d, J = 17.2 Hz, 1H), 1.87 (m, 2H), 1.31
(t, J = 7.1 Hz, 3H), 1.18 (s, 3H), 1.14 (s, 3H) ppm; 13C NMR (150 MHz, CDCl3)
= 204.14, 200.19, 199.55,
169.71, 158.69, 142.90, 127.19, 119.93, 62.59, 61.80, 55.14, 45.85, 43.86, 40.88, 37.84, 34.18, 32.82, 30.26,
δ
24.81, 14.18 ppm; IR (KBr):
υ
= 3435.06, 2961.46, 2931.17, 2871.34, 1739.98, 1720.04, 1671.03, 1638.37,
max
1241.36, 1228.03, 1177.31, 1142.38 cm−1; MS (ESI): m/z: 367; HRMS (ESI) Calcd. for [(C20H24O5) + Na]+:
367.1521; found: 367.1515 [M + Na]+.
3.2. Biological Evaluation
Cells were purchased from the Wuxi Innovatbio Medicine Technology Co. LTD (Wuxi, China)
and were maintained at 37 ◦C under the atmosphere of 5% CO2. The cells of human non-small lung
cancer (H292) and colon cancer cell line (SNU-1040) cells were cultured in RPMI-medium and others
(rat myoblasts and HepG2) were cultured in dulbecco0s modified eagle medium (DMEM). 10% fetal
bovine serum (Tianhang Biotechnology Co., Ltd., Zhejiang, China) and 1% antibiotics (100 U/mL

Molecules 2018, 23, 3216
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penicillin and 100 mg/mL streptomycin) was supplemented. Cells are inoculated in 96-well plate and
cultured for 24 h. After attached to the culture bottle wall cells were divided cells into three groups,
(1) control group; (2) positive control group; (3) experiment group. The administration concentrations
of Cisplatin (DTT) and compounds were 0.1
µM–10
µM. After 24 h, 10% MTT was added and cells
were culture for 3.5 h. Then the culture medium was discard and 150
µL DMSO was added. Eventually,
after incubation for 10 min by shaking in the dark at room temperature, the absorbance was detected
at 490 nm using a microplate reader, and the difference was analyzed by prism 7 software.
4. Conclusions
In conclusion, we have designed and synthesized several compounds bearing two
α
,
β
-unsaturated ketone moiety. Their in vitro activities were evaluated against HepG2, NSCLC-H292,
SUM-1040 tumor cell lines and an L6 myoblast cell line, with IC50 values ranging from 0.33 to 115.17 M.
This study has revealed that compounds 16 17 and 18 are promising anti-tumor leads due to the
stronger cytotoxic potencies and higher selectivity they displayed than their natural counterparts.
µ
,
Author Contributions: Synthesis and experimental work, Y.H., Z.-J.M., Y.-Z.X. and W.Z.; X-ray crystallographic
analysis, X.-N.L.; Biological Evaluation, Y.Z. (Yong Zhao) and Y.Z. (Yan Zhao); Design and writing, P.-T.P. and
J.-P.L.
Funding: This work was supported by a grant from the National Natural Science Foundation of China (21462053),
the Science Foundation of Yunnan Province Office of Education (2014Z044), Program of the Undergraduate
Innovation (2018059).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 13–18 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).