Synthesis and cytotoxic activities of novel 4-methoxy-substituted and 5-methyl-substituted (3′S,4′S)-(-)-cis-khellactone derivatives that induce apoptosis via the intrinsic pathway

Article abstract of DOI:10.2147/DDDT.S131753

This study deals with the design and synthesis of a series of novel 4-methoxy-substituted and 5-methyl-substituted (3′S,4′S)-(-)-cis-khellactones. The newly synthesized compounds were characterized by¹H nuclear magnetic resonance (NMR),^13<

Full text of DOI:10.2147/DDDT.S131753

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O ꢀ ꢁ ꢂ ꢁ ꢃ ꢄ ꢅ ꢀ ꢆ ꢇ ꢆ ꢄ ꢀ ꢈ ꢉ
ꢇynthesis and cytotoxic activities of novel
-methoxy-substituted and 5-methyl-substituted
4
(
3′S,4′S)-(-)-cis-khellactone derivatives that
induce apoptosis via the intrinsic pathway
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
2
3 June 2017
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1
,
Jingrun ꢈhen *
Junjie ꢅiu *
Abstract:Thisstudydealswiththedesignandsynthesisofaseriesofnovel4-methoxy-substituted
and 5-methyl-substituted (3′S,4′S)-(-)-cis-khellactones. The newly synthesized compounds were
1
,
1
1
13
characterized by H nuclear magnetic resonance (NMR), C-NMR, mass spectrometry, and
elemental analysis. All the derivatives were subjected to in vitro cytotoxicity screening against
HEPG-2 (human liver carcinoma), SGC-7901 (human gastric carcinoma), and LS174T (human
colon carcinoma), by using the MTT assay. The results revealed that several of the 4-methoxy-
substituted compounds exhibited potent cytotoxicity. Among these, compound 12e showed the
Dongxiao ꢈui
1
ꢈhaoqun Yan
1
ꢅiqiang Meng
1
ꢅiqian ꢇun
1
ꢇhurong Ban
highest activity against cancer cells which 50% inhibitory concentration (IC ) values were in the
1
50
ꢀ
ui ꢂe
range of 6.1–9.2 μM with low toxicity on normal human hepatocyte. Preliminary investigation
of possible mechanisms of action of compound 12e against HEPG-2 cells indicated possible
induction of apoptosis, as determined by morphological observations and Annexin V/propidium
iodide (PI) double staining, in addition to apparent dissipation of mitochondrial membrane
potential (MMP), as measured by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine
iodide (JC-1) staining in combination with the activation of caspase-9 and caspase-3 by Western
blot analysis. Overall, the data suggest that compound 12e may be a promising potential anti-
cancer agent that could act primarily by inducing apoptosis through the mitochondria-mediated
intrinsic pathway in human hepatoma cells.
1
,2
Taigang ꢅiang
1
,2
Qingshan ꢅi
1
ꢅaboratory of Medicinal ꢈhemistry,
ꢇchool of Pharmaceutical ꢇcience,
ꢇhanxi Medical University, ꢈollege
of Traditional ꢈhinese Medicine,
ꢇhanxi University of Traditional
ꢈhinese Medicine,Taiyuan, ꢇhanxi,
People’s ꢀepublic of ꢈhina
2
*
These authors contributed equally
Keywords: 4-methoxy-substituted and 5-methyl-substituted (3′S,4′S)-(-)-cis-khellactones,
synthesis, cytotoxic activity, apoptosis
to this work
Abbreviations
AO, acridine orange; (DHQD)2-PYR, hydroquinidine-2,5-diphenyl-4,6-pyrimidinediyl
diether; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl
sulfoxide; EB, ethidium bromide; ECL, enhanced chemiluminescence; ee, enantio-
meric excess; ESI, electrospray ionization; FBS, fetal bovine serum; FITC, fluorescein
isothiocyanate; HPLC, high-performance liquid chromatography; HRP, horseradish
ꢈorrespondence: Taigang ꢅiang;
Qingshan ꢅi
peroxidase; IC , 50% inhibitory concentration; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-
ꢅaboratory of Medicinal ꢈhemistry,
ꢇchool of Pharmaceutical ꢇcience, ꢇhanxi
Medical University, ꢃo 56, Xinjian
ꢃan ꢀoad, Taiyuan 030001, ꢇhanxi,
People’s ꢀepublic of ꢈhina
Tel/fax +86 351 469 0322
50
tetraethyl-imidacarbocyanine iodide; MMP, mitochondrial membrane potential; NMR,
nuclear magnetic resonance; PE, petroleum ether; PI, propidium iodide; ppm, parts per
million; RIPA, radio-immunoprecipitation assay; SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel electrophoresis; TBS-T, tris-buffered saline plus Tween-20; TLC,
thin-layer chromatography; TMS, tetramethylsilane.
ꢆmail ltaigang@163.com;
sxlqs2012@163.com
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Introduction
a chiral catalyst of (DHQD) -PYR. Some of the synthesized
2
Cancer is now a leading cause of mortality worldwide derivatives exhibited considerable inhibition of human
1
and was responsible for 8.2 million deaths in 2012. In addi- cancer cells, and this finding prompted us to further explore
tion to surgery and radiation therapy, chemotherapy remains cis-khellactone derivatives with the 3′S,4′S configuration as
2
1
an important and widely used treatment option for many new potential anticancer agents. This study deals with the
2,3
human malignancies. Although considerable advances have design and synthesis of a new series of substituted (3′S,4′S)-
been made in chemotherapy over the past several decades, (-)-cis-khellactone. The derivatives were evaluated for
there is still an urgent need for novel chemotherapeutic agents their in vitro antitumor activity against three human cancer
with higher efficacy and lower toxicity. It is well known that cell lines (HEPG-2, SGC-7901, and LS174T). In addition,
a number of clinically useful chemotherapeutic agents such compound 12e, which exhibited the highest activity against
as taxol, topotecan, and irinotecan have been derived from the HEPG-2 cancer cell line, was further investigated in an
4
naturally occurring small organic molecules.
attempt to obtain insight into the primary mechanism of
Coumarins comprise a large class of natural compounds antitumor action of this class of agents.
found throughout the plant kingdom. Natural and synthetic
coumarin derivatives exhibit a wide range of pharmaco- Materials and methods
logical actions, such as antimicrobial, antiviral, anticancer, Materials
anti-inflammatory, antioxidant, anticoagulant, and central Melting points were measured on an XT-4 melting point
nervous system activities, and are therefore considered to be apparatus without correction. NMR spectra were generated
5–7
ideal lead compounds for drug development. Khellactone, on a BRUKER AVANCE-600 spectrometer and obtained as
1
a member of the coumarin family, possesses a pyrano ring CDCl or DMSO-d solutions (600 MHz for H and 150 MHZ
3
6
13
fused at positions seven and eight of the coumarin molecular for C). Chemical shifts are reported as δ values in ppm rela-
skeleton and is regarded as a biosynthetic pioneer found tive to TMS, and J values are expressed in Hertz. ESI mass
originally in plants with the angular-type pyranocoumarin. spectra were determined in an API QTRAP 3200 LC-MS spec-
Natural khellactone coumarins have been mainly isolated trometer. The elemental analyses were performed by using an
8–10
from Peucedanum species. In the past 2 decades, khel- Elementar Vario-III CHN analyzer and were within ±0.4% of
lactone and its derivatives have attracted great interest in theoretical values. Optical rotations were measured on Perkin-
medicinal chemistry owing to their wide range of biological Elmer 241 polarimeter in a 1-dm cell. The Chiralpak AS-H
1
1
effects including antiviral activity, inhibition of platelet chiral HPLC analysis by using Shimadzu LC-10A instrument
1
2
13
aggregation, calcium antagonist activity, and inhibition of was applied to detect the ee values of the compounds. TLC
1
4,15
P-glycoprotein.
In particular, attention has been focused analysis of reaction mixtures was carried out on silica gel
primarily on their anti-HIV activity, and some khellactone GF-254 (Qingdao Ocean Chemical Factory, Qingdao, China),
derivatives have been found to demonstrate extremely potent and the identification was performed under ultraviolet (UV)
16,17
inhibitory activity against HIV-1 replication.
It is notable light. Silica gel (200–300 mesh) used in column chroma-
that khellactone coumarins contain two chiral carbons of tography was provided by Tsingtao Marine Chemistry Co.
C-3′ and C-4′ (Figure 1), and previous studies showed that Ltd (Qingdao, China). Chiral catalyst (DHQD) -PYR was
2
3
′R- and 4′R-configured khellactone derivatives are critical procured from Sigma-Aldrich Co. (St Louis, MO, USA).
18–20
for anti-HIV activity.
Other commercial chemicals were purchased from Aladdin
In previous studies, we synthesized the amount of Chemicals Co. (Shanghai, China) and used without further
4
-methyl-(3′S,4′S)-(-)-cis-khellactone derivatives by using purification. Compounds 2, 3, 4, 6, and 7 were synthesized
18
according to previously reported procedure.
ꢁ
ꢂ
ꢁ
ꢂ
ꢇynthesis of compounds
ꢀ
ꢅ
ꢃ
ꢄ
ꢀ
ꢅ
ꢃ
ꢄ
ꢂeneral method for the preparation of substituted
seselins (8, 9)
2
2
2
ꢆ′
ꢆ
2
2
2
ꢆ′
ꢄ′
ꢆ
ꢄ′
ꢂ′
Compounds 8 and 9 were synthesized according to previous
ꢂ′
ꢃ′
ꢃ′
2
+
2
+
literature, andthemeltingpointsofthesetwocompoundswere
2
+
2
+
18
reportedtobe167°C–168°Cand143°C–144°C, respectively.
To a stirred solution of substituted 7-hydroxycoumarins 4,
ꢀ
′5ꢁꢂ′5ꢃFRQILJXUDWLRQ
ꢀ′6ꢁꢂ′6ꢃFRQILJXUDWLRQ
Figure 1 ꢇtructures of cis-khellactone with 3′R,4′R and 3′S,4′S configuration.
7 (10 mmol) and potassium carbonate (3.45 g, 25 mmol) in
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ꢇynthesis and cytotoxic activities
N,N-dimethylformamide (20 mL), potassium iodide (1.66 g, layer was dried with anhydrous sodium sulfate and evaporated
0 mmol) was added, followed by adding excess 3-chloro-3- in vacuo. The crude product was purified by column chro-
1
methyl-1-butyne (6 mL). The stirred reaction mixture was matography using the PE/ethyl acetate (6:1, v/v) to obtain
warmed at 70°C–80°C for 72 h. Then, the solution was fil- substituted (3′S,4′S)-(-)-cis-khellactone 10 and 11.
tered and the solvent was evaporated in vacuo. The residue
was directly added in 20 mL of N,N-diethylaniline, refluxed 4-Methoxy-(3′S,4′S)-(-)-cis-khellactone (10)
for 15 h. The mixture cooled down was diluted by 20 mL White solid; yield, 51%; m.p., 218°C–220°C; [α]D 20=-42.6
ethyl acetate, and successively washed using 10% aqueous (c 0.3, CHCl ). ee =86%, Chiral Pak AS-H column eluting
3
hydrochloric acid, water, and brine. The solvent was removed with hexane/isopropanol (90:10, v:v), flow rate =0.8 mL/min,
under reduced pressure. The crude product was further puri- wavelength =323 nm, t (minor) =26.21 min, tR
R
1
fied by silica gel column chromatography (PE/ethyl acetate: (major) =38.91 min; H-NMR (600 MHz, DMSO-d , ppm)
6
1
0/1) to obtain compounds 8 and 9.
δ 1.34 (s, 3H, C-2′-CH ), 1.36 (s, 3H, C-2′-CH ), 3.60 (br, 1H,
3
3
H-4′), 3.96(s, 3H, C-4-OCH ), 4.88(br, 1H, H-3′), 5.19(br, 2H,
3
4
-Methoxyseselin (8)
OH ×2), 5.72 (s, 1H, H-3), 6.73 (d, 1H, J =8.79 Hz, H-6), 7.57
1
3
Light yellow solid; yield, 41%; m.p., 162°C–164°C. (d, 1H, J =8.79 Hz, H-5). C-NMR (150 MHz, DMSO-d )
6
1
H-NMR (600 MHz, CDCl , ppm) δ 1.44 (s, 6H, C-2′-CH ), δ 20.99, 26.87, 56.78, 60.24, 71.18, 78.77, 87.13, 107.68,
3
3
3
.94 (s, 3H, C-4-OCH ), 5.53 (s, 1H, H-3), 5.68 (d, 1H, 111.65, 113.44, 123.07, 153.13, 156.08, 161.96, 166.34.
3
+
J =10.02 Hz, H-3′), 6.68 (d, 1H, J =8.68 Hz, H-6), 6.85 MS (ESI) m/z 314.8 ([M+Na] ). Anal. Calc for C H O : C,
15
16
6
(
d, 1H, J =10.02 Hz, H-4′), 7.51 (d, 1H, J =8.68 Hz, H-5). 61.64%; H, 5.52%. Found: C, 61.73%; H, 5.44%.
+
MS (ESI) m/z 280.9 ([M+Na] ). Anal. Calc for C H O : C,
1
5
14
4
6
9.76%; H, 5.46%. Found: C, 69.71%; H, 5.40%.
5-Methyl-(3′S,4′S)-(-)-cis-khellactone (11)
White solid; yield, 46%; m.p., 178°C–180°C; [α]D 20=-41.5
5
-Methylseselin (9)
(c 0.2, CHCl ). ee =87%, Chiral Pak AS-H column eluting
3
1
White solid; yield, 21%; m.p., 145°C–146°C. H-NMR with hexane/isopropanol (90:10, v:v), flow rate =0.8 mL/min,
(
(
(
600 MHz, CDCl , ppm) δ 1.46 (s, 6H, C-2′-CH ), 2.43 wavelength =323 nm, t (minor) =32.58 min, t (major) =
3
3
R
R
1
s, 3H, C-5-CH ), 5.66 (d, 1H, J =10.05 Hz, H-3′), 6.22 79.50 min; H-NMR (600 MHz, DMSO-d , ppm) δ 1.33
3
6
d, 1H, J =9.69 Hz, H-3), 6.58 (s, 1H, H-6), 6.86 (d, 1H, (s, 3H, C-2′-CH ), 1.35 (s, 3H, C-2′-CH ), 2.40 (s, 3H, C-5-
3
3
J =10.05 Hz, H-4′), 7.80 (d, 1H, J =9.69 Hz, H-4). MS (ESI) CH ), 3.58 (br m, 1H, H-4′), 4.86 (br m,1H, H-3′), 5.14(br, 2H,
3
+
m/z 265.0 ([M+Na] ). Anal. Calc for C H O : C, 74.36%; OH ×2), 6.24 (d, 1H, J =9.67 Hz, H-3), 6.61 (s, 1H, H-6), 8.05
15
14
3
1
3
H, 5.82%. Found: C, 74.40%; H, 5.89%.
(d, 1H, J =9.67 Hz, H-4). C-NMR (150 MHz, DMSO-d )
6
δ 17.80, 21.02, 26.88, 60.05, 71.28, 78.70, 109.62, 110.86,
ꢂeneral procedure for the preparation of
substituted (3′S,4′S)-(-)-cis-khellactone (10, 11)
111.04, 114.61, 137.20, 141.72, 154.49, 155.33, 160.20;
+
MS (ESI) m/z 277.0 ([M+H] ). Anal. Calc for C H O : C,
15
16
5
Potassium carbonate (105 mg, 0.75 mmol) was added to a 65.21%; H, 5.84%. Found: C, 65.14%; H, 5.89%.
solution of potassium ferricyanide (246 mg, 0.75 mmol) in
tert-butanol/water (1:1, v/v) (5 mL), and the mixture was ꢂeneral method for the preparation of substituted
stirred for 5 min. Potassium osmate (2 mg, 0.005 mmol) (±)-cis-khellactone (10′, 11′)
and (DHQD) -PYR (4.4 mg, 0.005 mmol) were then added, Substituted seselin (1 mmol) was added to a mixture solu-
2
and continuously stirred for 15 min. Then, the mixture was tion of N-methylmorpholine-N-oxide monohydrate (0.129 g,
cooled to 0°C and methane sulfonamide (24 mg, 0.25 mmol) 1.1 mmol) and osmium tetroxide (10 mg, 0.04 mmol) which
was added under stirring. When the color of solution were dissolved in tert-butanol/tetrahydrofuran/water (10/3/1,
turned from a light yellow to an orange, substituted seselin 10 mL); the mixture was stirred for 24 h at room tempera-
(0.25 mmol) was added, and the mixture was stirred at 0°C for ture. Then, saturated sodium hydrogen sulfite solution was
approximately 24 h. TLC analysis monitored the progress of added (80 mL) to keep stirring for another 2 h and was
reaction. After completion of the reaction as indicated on TLC, diluted with dichloromethane (80 mL). The organic layer
the contents such as dichloromethane (2.5 mL), sodium pyro- was dried by using sodium sulfate and then evaporated to
sulfite (1 g), and water (2.5 mL) were added. The mixture was dryness under reduced pressure. Purification by column chro-
continuously stirred for another 4 h at room temperature, and matography (PE/acetone: 5/1) obtained the substituted (±)-
then extracted with dichloromethane (3×15 mL). The organic cis-khellactone 10′ and 11′.
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4
-Methoxy-(±)-cis-khellactone (10′)
3.47 (s, 3H, OCH ), 3.95 (s, 3H, C-4-OCH ), 4.05
3
3
1
White solid; yield, 63%; m.p., 228°C–230°C. H-NMR (m, 4H, 2× CH ), 5.42 (d, 1H, J =4.85 Hz, H-3′), 5.53
2
(
600 MHz, DMSO-d , ppm) δ 1.34 (s, 3H, C-2′-CH ), 1.36 (s, 1H, H-3), 6.60 (d, 1H, J =4.85 Hz, H-4′), 6.79 (d, 1H,
6
3
1
3
(s, 3H, C-2′-CH ), 3.58 (br, 1H, H-4′), 3.97 (s, 3H, C-4-OCH ), J =8.89 Hz, H-6), 7.70 (d, 1H, J =8.89 Hz, H-5). C-NMR
3
3
4
.89(br, 1H, H-3′), 5.20(br, 2H, OH×2),5.72(s,1H,H-3),6.74 (150 MHz, CDCl ) δ 22.42, 24.91, 56.25, 59.41, 59.47, 61.44,
3
(
d, 1H, J =8.79 Hz, H-6), 7.58 (d, 1H, J =8.79 Hz, H-5). 69.32, 69.54, 70.54, 87.64, 105.89, 109.09, 113.88, 124.77,
+
MS (ESI) m/z 314.6 ([M+Na] ). Anal. Calc for C H O : C, 153.06, 156.85, 161.80, 166.46, 169.56, 169.69. MS (ESI)
6
15
16
6
+
1.64%; H, 5.52%. Found: C, 61.71%; H, 5.58%.
m/z 458.8 ([M+Na] ). Anal. Calc for C H O : C, 57.80%;
2
1
24 10
H, 5.54%. Found: C, 57.76%; H, 5.48%.
5
-Methyl-(±)-cis-khellactone (11′)
1
White solid; yield, 58%; m.p., 185°C–187°C; H-NMR 3′S,4′S-di-O-benzoyl-4-methoxy-(-)-cis-
(
(
600 MHz, DMSO-d , ppm) δ 1.33 (s, 3H, C-2′-CH ), 1.35 khellactone (12c)
6
3
s, 3H, C-2′-CH ), 2.40 (s, 3H, C-5-CH ), 3.57 (br m, 1H, White solid; yield, 42%; m.p., 242°C–244°C; [α]D 20=-3.3
3
3
1
H-4′), 4.85 (br m,1H, H-3′), 5.12 (br, 2H, OH ×2), 6.22 (d, 1H, (c 0.1, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.51
2
2
3
J=9.67Hz,H-3),6.60(s,1H,H-6),8.03(d,1H,J =9.67Hz,H-4). (s, 3H, C-2′-CH ), 1.64 (s, 3H, C-2′-CH ), 3.93 (s, 3H, C-4-
3
3
+
MS (ESI) m/z 277.0 ([M+H] ). Anal. Calc for C H O : C, OCH ), 5.50 (1H, s, H-3), 5.67 (d, 1H, J =4.92 Hz, H-3′), 6.88
15
16
5
3
6
5.21%; H, 5.84%. Found: C, 65.15%; H, 5.91%.
(d, 1H, J =8.92 Hz, H-6), 6.95 (d, 1H, J =4.92 Hz, H-4′), 7.33
(
m, 4H, Ar-H), 7.51 (m, 2H, Ar-H), 7.75 (d, 1H, J =8.92 Hz,
1
3
ꢂeneral procedure for the preparation of (3′S,4′S)-
-)-cis-khellactone derivatives (12a–g, 13a–g)
H-5), 7.87 (m, 4H, Ar-H). C-NMR (150 MHz, CDCl3)
(
δ 22.37, 25.61, 56.18, 61.24, 71.17, 77.43, 87.85, 106.84,
Excess acylating agent was added to a solution of substi- 109.08, 113.91, 124.75, 128.19, 128.31, 129.31, 129.78,
tuted (3′S,4′S)-(-)-cis-khellactone (0.2 mmol) in anhydrous 132.79, 133.22, 153.35, 157.09, 161.71, 165.25, 166.31.
+
pyridine (1 mL) and dichloromethane (2 mL) at 0°C, and MS (ESI) m/z 522.8 ([M+Na] ). Anal. Calc for C H O : C,
2
9
24
8
refluxed until completed by TLC monitor. The mixture was 69.59%; H, 4.83%. Found: C, 69.62%; H, 4.77%.
filtered, and the filtrate was evaporated in vacuo. Purifica-
tion by column chromatography (PE/acetone: 10/1) obtained 3′S,4′S-di-O-(2-methoxybenzoyl)-4-methoxy-(-)-cis-
target compounds.
khellactone (12d)
White solid; yield, 38%; m.p., 167°C–169°C; [α]D 20=-9.6 (c
1
3
′S,4′S-di-O-acetyl-4-methoxy-(-)-cis-
0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.52 (s, 3H,
2
2
3
khellactone (12a)
C-2′-CH ), 1.58 (s, 3H, C-2′-CH ), 3.70 (s, 3H, OCH ), 3.76
3
3
3
White solid; yield, 50%; m.p., 213°C–215°C; [α]D 20=+19.8 (s, 3H, OCH ), 3.91 (s, 3H, C-4-OCH ), 5.50 (s, 1H, H-3),
3
3
1
(
c 0.2, CH Cl ). H-NMR (600 MHz, DMSO-d , ppm) 5.65 (d, 1H, J =4.94 Hz, H-3′), 6.81 (d, 1H, J =8.89 Hz, H-6),
2
2
6
δ 1.40 (s, 3H, C-2′-CH ), 1.44 (s, 3H, C-2′-CH ), 2.10 (s, 6.86 (m, 4H, Ar-H), 6.92 (d, 1H, J =4.94 Hz, H-4′), 7.38
3
3
3
H, CH CO), 2.12 (s, 3H, CH CO), 3.95 (s, 3H, C-4-OCH ), (m, 2H, Ar-H), 7.69 (d, 1H, J =8.89 Hz, H-5), 7.69 (m, 2H,
3
3
3
1
3
5
.29 (d, 1H, J =4.87 Hz, H-3′), 5.55 (s, 1H, H-3), 6.51 Ar-H). C-NMR (150 MHz, CDCl ) δ 22.07, 25.67, 55.64,
3
(
d, 1H, J =4.87 Hz, H-4′), 6.78 (d, 1H, J =8.89 Hz, H-6), 7.69 55.68, 56.12, 60.93, 70.94, 77.53, 87.67, 107.22, 108.84,
13
(
d, 1H, J =8.89 Hz, H-5). C-NMR (150 MHz, DMSO-d ) 111.79, 113.80, 119.35, 119.92, 119.95, 120.64, 124.43,
6
δ 20.55, 20.59, 22.52, 24.89, 56.19, 60.99, 70.25, 77.36, 131.30, 132.02, 132.80, 133.57, 153.29, 157.05, 158.74,
8
7.63, 106.50, 109.01, 113.81, 124.43, 153.13, 156.95, 159.37, 161.79, 164.76, 165.20, 166.30. MS (ESI) m/z 582.7
61.96, 166.50, 169.92. MS (ESI) m/z 398.8 ([M+Na]+). ([M+Na] ). Anal. Calc for C H O : C, 66.42%; H, 5.03%.
+
1
3
1
28 10
Anal. Calc for C H O : C, 60.63%; H, 5.36%. Found: C, Found: C, 66.35%; H, 4.94%.
1
9
20
8
6
0.61%; H, 5.32%.
3
′S,4′S-di-O-(2-chlorobenzoyl)-4-methoxy-(-)-cis-
3
′S,4′S-di-O-methoxyacetyl-4-methoxy-(-)-cis-
khellactone (12e)
khellactone (12b)
White solid; yield, 48%; m.p., 221°C–222°C; [α]D 20=-33.3
1
White solid; yield, 43%; m.p.,116°C–118°C; [α]D 20=+6.5 (c 0.1, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.57
2
2
3
1
(
c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.42 (s, 3H, C-2′-CH ), 1.58 (s, 3H, C-2′-CH ), 3.95 (s, 3H, C-4-
2
2
3
3
3
(
s, 3H, C-2′-CH ), 1.43 (s, 3H, C-2′-CH ), 3.46 (s, 3H, OCH ), OCH ), 5.54 (s, 1H, H-3), 5.71 (d, 1H, J =4.92 Hz, H-3′),
3
3
3
3
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ꢇynthesis and cytotoxic activities
1
3
6
7
7
.84 (d, 1H, J =8.96 Hz, H-6), 6.93 (d, 1H, J =4.92 Hz, H-4′), (d, 1H, J =9.72 Hz, H-4). C-NMR (150 MHz, CDCl3)
.20 (m, 1H, Ar-H), 7.36 (m, 5H, Ar-H) 7.72 (m, 1H, Ar-H), δ 18.37, 20.57, 20.61, 22.54, 24.93, 60.87, 70.33, 77.34,
.73 (d, 1H, J =8.96 Hz, H-5), 7.86 (m, 1H, Ar-H). C-NMR 104.69, 111.73, 112.45, 115.27, 137.93, 140.18, 154.44,
150 MHz, CDCl ) δ 22.13, 25.86, 56.22, 61.99, 72.00, 77.42, 156.14, 159.86, 169.93. MS (ESI) m/z 382.7 ([M+Na]+).
13
(
3
8
1
1
7.81, 106.61, 109.05, 113.99, 124.89, 126.49, 126.62, Anal. Calc for C H O : C, 63.33%; H, 5.59%. Found: C,
19 20 7
29.65, 130.46, 130.83, 131.12, 132.05, 132.69, 133.01, 63.30%; H, 5.53%.
33.52, 153.31, 157.03, 161.75, 164.75, 165.18, 166.42. MS
+
(
ESI) m/z 592.6 ([M+Na] ). Anal. Calc for C H Cl O : C, 3′S,4′S-di-O-methoxyacetyl-5-methyl-(-)-cis-
29
22
2
8
6
1.17%; H, 3.89%. Found: C, 61.15%; H, 3.81%.
khellactone (13b)
White solid; yield, 43%; m.p., 117°C–118°C; [α]D 20=11.4
1
3
′S,4′S-di-O-(3-chlorobenzoyl)-4-methoxy-(-)-cis-
(c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.41
2
2
3
khellactone (12f)
(s, 3H, C-2′-CH ), 1.42 (s, 3H, C-2′-CH ), 2.45 (s, 3H,
3
3
White solid; yield, 43%; m.p., 200°C–201°C; [α]D 20=-9.6 C-5-CH ), 3.45 (3H, s, OCH ), 3.46 (3H, s, OCH ), 4.08
3
3
3
1
(
c 0.1, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.50 (4H, m, 2× CH ), 5.39 (1H, d, J =4.82 Hz, H-3′), 6.22
2
2
3
2
(
s, 3H, C-2′-CH ), 1.62 (s, 3H, C-2′-CH ), 3.94 (s, 3H, C-4- (1H, d, J =9.76 Hz, H-3), 6.57 (1H, d, J =4.82 Hz, H-4′),
3
3
13
CH O), 5.51 (s, 1H, H-3), 5.65 (d, 1H, J =4.94 Hz, H-3′), 6.89 6.65 (1H, s, H-6), 7.78 (1H, d, J =9.76 Hz, H-4). C-NMR
3
(
d, 1H, J =8.92 Hz, H-6), 6.91 (d, 1H, J =4.94 Hz, H-4′), 7.30 (150 MHz, CDCl ) δ 18.40, 22.37, 25.00, 59.40, 59.44, 61.32,
3
(
m, 2H, Ar-H), 7.50 (m, 2H, Ar-H), 7.77 (d, 1H, J =8.92 Hz, 69.35, 69.55, 70.68, 104.12, 111.78, 112.55, 115.32, 138.31,
1
3
H-5), 7.78 (m, 4H, Ar-H). C-NMR (150 MHz, CDCl3) 140.17, 154.41, 156.05, 159.65, 169.54, 169.71. MS (ESI)
+
δ 22.48, 25.36, 56.24, 61.67, 71.38, 77.29, 87.85, 106.23, m/z 442.8 ([M+Na] ). Anal. Calc for C H O : C, 59.99%;
2
1
24
9
1
1
1
09.19, 113.97, 124.99, 127.89, 127.99, 129.65, 129.70, H, 5.75%. Found: C, 59.94%; H, 5.81%.
29.77, 130.84, 131.30, 133.05, 133.44, 134.39, 134.57,
53.25, 156.99, 161.70, 164.15, 164.20, 166.33. MS (ESI) 3′S,4′S-di-O-benzoyl-5-methyl-(-)-cis-
+
m/z 592.1 ([M+Na] ). Anal. Calc for C H Cl O : C, 61.17%; khellactone (13c)
29
22
2
8
H, 3.89%. Found: C, 61.20%; H, 3.84%.
White solid; yield, 41%; m.p., 208°C–210°C; [α]D 20=-14.3
1
(
c 0.1, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.50
2
2
3
3
′S,4′S-di-O-(4-chlorobenzoyl)-4-methoxy-(-)-cis-
(s, 3H, C-2′-CH ), 1.64 (s, 3H, C-2′-CH ), 2.49 (s, 3H, C-5-
3
3
khellactone (12g)
CH ), 5.65 (d, 1H, J =4.93 Hz, H-3′), 6.18 (d, 1H, J =9.76 Hz,
3
White solid; yield, 46%; m.p., 273°C–275°C; [α]D 20=-22.4 H-3), 6.75 (s, 1H, H-6), 6.93 (d, 1H, J =4.93 Hz, H-4′), 7.33
1
(
c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.50 (m, 4H, Ar-H), 7.50 (m, 2H, Ar-H), 7.78 (d, 1H, J =9.76 Hz,
2
2
3
1
3
(
s, 3H, C-2′-CH ), 1.60 (s, 3H, C-2′-CH ), 3.93 (s, 3H, C-4- H-4), 7.88 (m, 4H, Ar-H). C-NMR (150 MHz, CDCl ) δ
3
3
3
OCH ), 5.50 (s, 1H, H-3), 5.64 (d, 1H, J =4.98 Hz, H-3′), 18.50, 22.36, 25.64, 61.06, 71.22, 77.38, 104.97, 111.75,
3
6
.88 (d, 1H, J =8.99 Hz, H-6), 6.89 (d, 1H, J =4.98 Hz, H-4′), 112.69, 115.36, 128.19, 128.31, 129.25, 129.76, 129.80,
.32 (m, 4H, Ar-H), 7.75 (d, 1H, J =8.99 Hz, H-5), 7.79 132.81, 133.24, 138.27, 139.99, 154.59, 156.22, 159.63,
7
13
+
(
m, 4H, Ar-H). C-NMR (150 MHz, CDCl ) δ 22.53, 25.35, 165.26, 165.30. MS (ESI) m/z 507.2 ([M+Na] ). Anal.
3
5
1
1
6.22, 61.66, 71.27, 77.36, 87.83, 106.40, 109.18, 113.93, Calc for C H O : C, 71.89%; H, 4.99%. Found: C,
29 24 7
24.89, 128.65, 128.78, 131.07, 139.44, 139.88, 153.29, 71.86%; H, 4.90%.
57.03, 161.66, 164.42, 164.49, 166.33. MS (ESI) m/z 592.3
+
([M+Na] ). Anal. Calc for C H Cl O : C, 61.17%; H, 3.89%. 3′S,4′S-di-O-(2-methoxybenzoyl)-5-methyl-(-)-cis-
29
22
2
8
Found: C, 61.13%; H, 3.82%.
khellactone (13d)
White solid; yield, 39%; m.p., 196°C–198°C; [α]D 20= -49.9
1
3
′S,4′S-di-O-acetyl-5-methyl-(-)-cis-khellactone (13a) (c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.51
2
2
3
White solid; yield, 48%; m.p., 202°C–204°C; [α]D 20=8.4 (s, 3H, C-2′-CH ), 1.58 (s, 3H, C-2′-CH ), 2.46 (s, 3H, C-5-
3
3
1
(
c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.39 CH ), 3.71 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 5.64 (d, 1H,
2
2
3
3
3
3
(
s, 3H, C-2′-CH ), 1.43 (s, 3H, C-2′-CH ), 2.09 (s, 3H, J =4.95 Hz, H-3′), 6.20 (d, 1H, J =9.76 Hz, H-3), 6.68 (s, 1H,
3
3
CH CO), 2.11 (s, 3H, CH CO), 2.45 (s, 3H, C-5-CH ), 5.27 H-6), 6.88 (d, 1H, J =4.95 Hz, H-4′), 6.88 (m, 4H, Ar-H); 7.37
3
3
3
(
d, 1H, J =4.74 Hz, H-3′), 6.24 (d, 1H, J =9.72 Hz, H-3), (m, 1H, Ar-H), 7.42 (m, 1H, Ar-H), 7.68 (m, 1H, Ar-H),
.49 (d, 1H, J =4.74 Hz, H-4′), 6.65 (s, 1H, H-6), 7.79 7.77 (d, 1H, J =9.76 Hz, H-4), 7.79 (m, 1H, Ar-H), C-NMR
13
6
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(
150 MHz, CDCl ) δ 18.45, 22.12, 25.75, 55.67, 55.73, 60.83, 7.32 (m, 4H, Ar-H), 7.78 (d, 1H, J =9.92 Hz, H-4), 7.79
3
1
3
7
1
1
1
1.04, 77.56, 105.44, 105.48, 111.61, 111.79, 111.85, 112.55, (m, 4H, Ar-H). C-NMR (150 MHz, CDCl ) δ 18.48, 22.54,
3
31.29, 132.06, 132.79, 132.84, 133.55, 133.61, 137.96, 25.39, 61.52, 71.35, 77.34, 104.60, 111.84, 112.75, 115.37,
39.94, 140.00, 154.62, 156.27, 158.77, 159.40, 159.67, 127.68, 128.16, 128.27, 128.65, 128.78, 131.07, 131.41,
59.73, 164.80, 165.28. MS (ESI) m/z 567.1 ([M+Na]+). 138.41, 139.44, 139.88, 139.99, 154.60, 156.19, 159.51,
+
Anal. Calc for C H O : C, 68.37%; H, 5.18%. Found: C, 164.43, 164.53; MS (ESI) m/z 576.7 ([M+Na] ). Anal. Calc
3
1
28
9
6
8.33%; H, 5.26%.
for C H Cl O : C, 62.94%; H, 4.01%. Found: C, 62.91%;
29 22 2 7
H, 4.03%.
3
′S,4′S-di-O-(2-chlorobenzoyl)-5-methyl-(-)-cis-
khellactone (13e)
ꢈell culture
White solid; yield, 46%; m.p., 188°C–190°C; [α]D 20=-45.1 HEPG-2 (human liver carcinoma), SGC-7901 (human
1
(
c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.57 gastric carcinoma), LS174T (human colon carcinoma),
2
2
3
(
s, 6H, 2× C-2′-CH ), 2.47 (s, 3H, C-5-CH ), 5.69 (d, 1H, and HL-7702 (normal human hepatocyte) cell lines were
3
3
J =4.86 Hz, H-3′), 6.23 (d, 1H, J =9.77 Hz, H-3), 6.70 (s, 1H, obtained from the Institute of Biochemistry and Cell Biology,
H-6), 6.91 (d, 1H, J =4.86 Hz, H-4′), 7.20 (m, 1H, Ar-H), 7.36 China Academy of Science. The cells were cultured in
(
m, 5H, Ar-H), 7.72 (m, 1H, Ar-H), 7.79 (d, 1H, J =9.77 Hz, DMEM, supplemented with 10% (v/v) heat-inactivated
1
3
H-4), 7.86 (m, 1H, Ar-H). C-NMR (150 MHz, CDCl3) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin
δ 18.46, 22.13, 25.92, 61.85, 72.09, 77.38, 104.79, 111.74, at 37°C in a humidified incubator containing 5% carbon
1
1
1
12.68, 115.41, 126.48, 126.62, 130.49, 130.83, 131.06, dioxide. Cells in logarithmic growth phase were employed
32.05, 132.69, 133.02, 133.51, 138.42, 140.07, 140.12, for further experiments.
54.60, 156.18, 159.62, 159.67, 164.75, 165.21; MS (ESI)
+
m/z 576.7 ([M+Na] ). Anal. Calc for C H Cl O : C, 62.94%; ꢁn vitro cytotoxic activity
H, 4.01%. Found: C, 62.91%; H, 4.09%.
29
22
2
7
The potential cytotoxic activity of all the target compounds
in vitro was assessed using the MTT assay. Briefly, cells were
4
3
′S,4′S-di-O-(3-chlorobenzoyl)-5-methyl-(-)-cis-
seeded in 96-well plates at a density of 2×10 cells/well. After
khellactone (13f)
overnight incubation, the cells were exposed to different con-
White solid; yield, 43%; m.p., 190°C–192°C; [α]D 20=-9.9 centrations of compounds for 48 h at 37°C in the atmosphere
1
(
c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.49 of 5% CO . Following 48 h of treatment, the cells of each
2
2
3
2
(
s, 3H, C-2′-CH ), 1.61 (s, 3H, C-2′-CH ), 2.50 (s, 3H, C-5- well were stained with 25 μL of MTT (5 mg/mL) and were
3
3
CH ), 5.64 (d, 1H, J =4.95 Hz, H-3′), 6.20 (d, 1H, J =9.42 Hz, incubated for a further 4 h at 37°C. Then, the media were
3
H-3), 6.75 (s, 1H, H-6), 6.89 (d, 1H, J =4.95 Hz, H-4′), 7.31 removed and shaken for 10 min by adding 150 μL DMSO
(
m, 2H, Ar-H), 7.49 (m, 2H, Ar-H), 7.77 (d, 1H, J =9.42 Hz, to all the wells. The absorbance values at 570 nm were
1
3
H-4), 7.78 (m, 4H, Ar-H). C-NMR (150 MHz, CDCl3) measured using a microplate reader (Model 680; Bio-Rad
δ 18.49, 22.51, 25.40, 61.59, 71.50, 77.30, 104.46, 111.89, Laboratories Inc., Hercules, CA, USA). The IC values were
50
1
1
1
12.77, 115.42, 127.89, 127.97, 128.16, 129.63, 129.76, calculated according to the equation for Boltzmann sigmoidal
30.12, 131.37, 133.03, 133.29, 133.43, 134.42, 134.59, concentration response curve using the nonlinear regression
38.51, 140.04, 154.58, 156.19, 159.58, 164.17, 164.26; fitting models (Prism Version 5; GraphPad Software, Inc.,
+
MS (ESI) m/z 576.6 ([M+Na] ). Anal. Calc for C H Cl O : La Jolla, CA, USA).
29
22
2
7
C, 62.94%; H, 4.01%. Found: C, 62.96%; H, 4.05%.
Morphological analysis by ꢄO/ꢆB double
3
′S,4′S-di-O-(4-chlorobenzoyl)-5-methyl-(-)-cis-
staining
khellactone (13g)
Morphological changes induced by compound 12e were
White solid; yield, 54%; m.p., 208°C–209°C; [α]D 20=-37.1 observed by fluorescence microscopy using AO/EB. In brief,
1
5
(
c 0.2, CH Cl ). H-NMR (600 MHz, CDCl , ppm) δ 1.49 HEPG-2 cells were seeded at a density of 1×10 cells/well in
2
2
3
(
s, 3H, C-2′-CH ), 1.59 (s, 3H, C-2′-CH ), 2.49 (s, 3H, C-5- six-well plates. After attachment, the cells were treated with
3
3
CH ), 5.62 (d, 1H, J =4.91 Hz, H-3′), 6.19 (d, 1H, J =9.92 Hz, compound 12e at 6.0, 12.0, and 24.0 μM for another 24 h;
3
H-3), 6.75 (s, 1H, H-6), 6.87 (d, 1H, J =4.91 Hz, H-4′), then, the mixture solution containing 100 mg/mL AO and
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ꢇynthesis and cytotoxic activities
EB was added for 10 min. The stained cells were washed ice-cold PBS. Whole-cell extracts were prepared using RIPA
twice in PBS and observed immediately under fluorescence lysis buffer, and protein concentrations were measured by
microscopy (IX-51; Olympus Corporation, Tokyo, Japan).
BCA Protein Assay kit. Proteins (40 μg) were resolved on
0% SDS-PAGE gels along with the equal amount of protein
molecular weight standards, followed by transferring onto
1
ꢄnnexinV-FꢁTꢈ/Pꢁ assay
5
HEPG-2 cells were seeded at a density of 1×10 cells/well in nitrocellulose membranes. The membranes were blocked in
six-well plates and grown in 5% CO at 37°C for 24 h. Then, TBS-T with 5% nonfat milk for 1.5 h and incubated overnight
2
cells were exposed to 6.0, 12.0, and 24.0 μM of compound with specific primary antibodies (for caspase-9, cleaved
1
2e for 48 h. After incubation, cells were collected and caspase-3, and β-actin) at 4°C, then incubated with second-
washed twice using cold PBS and resuspended in 200 μL of ary HRP-linked anti-rabbit antibody. Immunodetection was
6
binding buffer at 1×10 cells/mL. Subsequently, the samples carried out using ECL detection kit. The relative levels of
were incubated with 5 μL of Annexin V-FITC and PI in the each signaling event to control β-actin were determined by
dark for 15 min at room temperature and then analyzed by densimetric scanning.
flow cytometry within 1 h on an FACS Calibur (BD, Franklin
Lakes, NJ, USA). Each sample had at least 10,000 cells. The ꢇtatistical analysis
experiment was performed three times.
All experiments were repeated at least three times. The
results are presented as mean ± standard error of the mean.
We used Graph Pad software prism 5.1 for one-way analysis,
Measurement of MMP
Changes in the MMP were measured using JC-1 dye. followed by the Dunnett’s test. A value of P,0.05 denoted
Briefly, at the end of the treatment with compound 12e (6.0, statistical significance.
1
2.0, and 24.0 μM) for 48 h, HEPG-2 cells were collected,
Results and discussion
washed in ice-cold PBS, and incubated with 10 μg/mL JC-1
for 30 min at 37°C in the dark. After incubation, the stained ꢈhemistry
cells were transferred to a falcon tube and characterized by The title compounds derived in this study were obtained by
flow cytometry. The red/green fluorescence ratio of JC-1 applying the synthetic strategy described in Schemes 1 and 2.
was measured using fluorescent plate reader at 529 nm The condensation of 1,3-dihydroxybenzene (1) which is
emission (green)/590 nm emission (red). The relative red/ available by purchasing in the market with cyanoacetic acid
green fluorescence ratio in treated HEPG-2 cells was shown in the presence of zinc chloride/hydrogen chloride produces
graphically as a percentage of red/green fluorescence ratio 4-amino-7-hydroxycoumarin (2). Subsequent hydrolysis of
value of the control group (cells without treatment), which 2 in 50% sulfuric acid produced 4,7-dihydroxycoumarin (3).
were considered to have 100% MMP.
Then, the 4-hydroxyl group of 3 was selectively methylated
to produce 7-hydroxy-4-methoxycoumarin (4). To obtain
7-hydroxy-5-methylcoumarin (7), Wittig reaction and intra-
Western blot analysis
HEPG-2 cells were treated with different concentrations of molecular cyclization, which consisted of the reaction of 3,5-
compound 12e for 48 h, then collected and washed twice with dihydroxytoluene (5) with phosphorus oxychloride in excess
1
+ꢀ
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2
+
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ꢆ
Scheme 1 ꢇynthesis of substituted 7-hydroxycoumarins 4 and 7.
Notes: ꢀeagents and conditions: (i) cyanoacetic acid, zinc chloride/hydrogen chloride; (ii) 50% sulfuric acid; (iii) methanol/sulfuric acid; (iv) N,N-diethylaniline/phosphorus
oxychloride; (v) Ph P=ꢈꢉꢈOOꢆt.
3
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2
2
2
&
,
2
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ꢄ ꢂ ꢃ
ꢀ
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ꢂ
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&,
Scheme 2 ꢇynthesis of (3′S,4′S)-(-)-cis-khellactone derivatives (12–13).
Notes: ꢀeagents and conditions: (i) 3-chloro-3-methyl-1-butyne, potassium carbonate, potassium iodide in DMF, 70°ꢈ–80°ꢈ, 72 h; (ii) N,N-diethylaniline, reflux, 15 h;
iii) potassium ferricyanide, potassium carbonate, (DꢉQD) -PYꢀ, potassium osmate, methane-sulfonamide, in tert-butanol/water (v/v, 1:1) at 0°ꢈ, 24 h; (iv) various acyl
(
2
chlorides, anhydrous dichloromethane, pyridine, 0°ꢈ.
Abbreviations: (DꢉQD) -PYꢀ, hydroquinidine-2,5-diphenyl-4,6-pyrimidinediyl diether; DMF, N,N-dimethylformamide.
2
N,N-dimethylformamide, were used to derive the coumarin with various acyl chlorides in the presence of appropriate
nucleus from 2,4-dihydroxy-6-methylbenzaldehyde (6). pyridine, and dichloromethane used as solvent, adapting a
1
8
Compound 6 was further reacted with a triphenyl
previously reported procedure.
phosphonium ylide (Ph P = CHCOOEt) in dichloromethane
3
For the determination of ee, racemic substituted (±)-cis-
khellactones (10′ and 11′) were produced by oxidizing the
at room temperature and refluxed by heating in xylene with-
out purification to produce 7. In the presence of anhydrous
potassium carbonate and potassium iodide, the substituted
22
substituted seselins 8 and 9 (Scheme 3). Chiral HPLC
7
-hydroxycoumarins 4 and 7 were continuously reacted
with 3-chloro-3-methyl-1-butyne in N,N-dimethylforma-
mide and then thermal rearrangement occurred in boiling
diethylaniline to form substituted seselins (8, 9) accord-
R2
R1
O
R2
R1
O
i
1
8
ing to the procedures published previously. Similar to
the process used in the previous asymmetric preparation
of 4-methyl-(3′S,4′S)-(-)-cis-khellactone, the substituted
O
O
O
O
OH
OH
(
-)-cis-khellactones 10, 11 with the 3′S,4′S configuration
were successfully prepared by the reaction of osmium-
8 R = OCH , R = H
1 3 2
10′ R = OCH , R = H
1
3
2
9
R = H, R = CH
11′ R = H, R = CH
1 2 3
catalyzed asymmetric dihydroxylation of the seselins 8
1
2
3
2
1
and 9 in the presence of a chiral catalyst, (DHQD) -PYR.
Scheme 3 ꢇynthesis of substituted (±)-cis-khellactone (10′, 11′).
2
Note: ꢀeagents and conditions: (i) osmium tetroxide, N-methylmorpholine-N-
oxide monohydrate in tert-butanol/tetrahydrofuran/water (v/v, 10:3:1) at room
temperature, 24 h.
Target compounds 12a–g and 13a–g were synthesized by
reacting the unpurified forms of compounds 10 and 11
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ꢇynthesis and cytotoxic activities
analysis revealed that the reactions were highly stereose- ring at 3′ and 4′ positions (12d) led to a decrease in activity
lective with good ee (.86% ee). (DHQD) -PYR resulted compared to that of compound 12c. Conversely, significant
2
2
3
primarily in cis-diol with an S, S configuration.
activity was observed with 4-methoxy-substituted deriva-
tives having an electron-withdrawing C substituent on the
l
ꢁ
n vitro antitumor activity assays
benzoyl moiety at 3′ and 4′ positions (12e–g), and the
All the synthesized target compounds (12a-g and 13a-g) degree of inhibition ranged as follows: 2-Cl .4-Cl .3-Cl.
are novel and were screened for their preliminary antitumor Furthermore, the majority of the other compounds in this
activity against three human cancer cell lines (HEPG-2, group showed moderate activity. However, another group of
SGC-7901, and LS174T) by using the MTT assay with derivatives (13a–g) synthesized with 5-methyl substitution
doxorubicin as a positive reference compound. As shown did not show promising activity against the three cancer cell
in Table 1, the substituted (3′S,4′S)-(-)-cis-khellactones lines investigated. These results showed that the 4-methoxy-
1
0 and 11 did not show any cytotoxicity, while some substituted derivatives possessed greater activity than the
4
-methoxy-(3′S,4′S)-(-)-cis-khellactone derivatives exhib- 5-methyl-substituted derivatives and that the introduction
ited promising in vitro antitumor activity. Among the synthe- of a C substituent in the benzoyl moiety at 3′ and 4′ posi-
l
sized derivatives, compounds 12e, 12f, and 12g appeared to tions of 4-methoxy-(3′S,4′S)-(-)-cis-khellactone plays a
be the most potent. The IC values for compound 12e were key role in enhancing the activity. Furthermore, compound
5
0
6
.1, 9.2, and 8.0 μM in HEPG-2, SGC-7901, and LS174T 12e, which showed the highest activity against cancer cell
cells, respectively, while the corresponding values for com- lines, was chosen to investigate its cytotoxicity in HL-7702
pound 12f were 13.3, 21.3, and 23.6 μM. The IC values of cells, a normal human liver cell line. As shown in Figure S1,
5
0
compound 12g were 8.1, 29.6, and 16.7 μM, respectively, cell viability was greater than 90% after treatment with
in HEPG-2, SGC-7901, and LS174T cells. It is well known 100 μM compound 12e for 48 h. These results indicated
that various cancer cells have different responses to drug or that compound 12e is likely to be selectively toxic to liver
compound. Interestingly, all these three compounds tended cancer cell.
to be more sensitive in HEPG-2 cells compared to that in the
Morphological changes of ꢉꢆPꢂ-2 cell by
other two cancer cell lines. For HEPG-2 cells, the introduc-
tion of an electron-donating methoxy group in the benzene using ꢄO/ꢆB double-staining assay
To determine whether the cytotoxicity in HEPG-2 cells
caused by compound 12e was related to the induction of
Table 1 ꢈytotoxic activity of the synthesized compounds against
three human cancer cell lines
apoptosis, morphological observation with AO/EB stain-
ing was detected by fluorescence microscopy. As shown in
Compound
IC50 ± SD (μM)
Figure 2, green live cells with normal morphology were seen
in the control group, while the cells exposed to compound
HEPG-2
SGC-7901
LS174T
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
78.2±5.7
.100
83.3±5.1
.100
.100
1
2e showed apoptotic morphological changes such as cell
1
89.3±4.3
.100
2a
2b
2c
2d
2e
2f
59.1±6.9
66.8±5.5
31.3±7.0
.100
64.1±6.0
.100
shrinkage, chromatin condensation, cell nuclear fragmenta-
tion, and labeled orange color.
.100
51.8±4.5
53.3±8.1
9.2±2.0
21.3±4.0
29.6±5.2
45.0±6.6
.100
84.0±8.8
67.3±5.4
8.0±1.9
23.6±3.8
16.7±2.8
62.2±9.0
.100
ꢄnalysis of apoptosis by using the
ꢄnnexinV-FꢁTꢈ/Pꢁ method
To characterize the apoptosis induced by compound 12e, the
Annexin V/PI double-staining method was used to quantify
the apoptotic HEPG-2 cells following treatment with 6,
6.1±1.5
13.3±3.9
8.1±2.3
76.3±5.6
63.7±5.4
25.7±4.1
66.5±7.8
57.0±4.5
82.4±7.1
56.8±9.2
1.4±0.3
2g
3a
3b
3c
3d
3e
3f
73.9±3.7
.100
49.0±6.6
69.5±9.3
.100
1
2, and 24 μM of 12e. The percentage of early apoptotic
cells observed at these doses were 13.68%, 26.90%, and
30.72%, respectively, and this was statistically significant
compared to 5.91% observed with control cells (P,0.01).
Furthermore, total apoptotic cells at the three doses tested
were found to be 20.83%, 39.52%, and 43.88% respec-
tively, while the untreated control group had 8.75% of
68.9±5.3
.100
51.0±4.2
.100
3g
53.8±6.8
2.1±0.2
Doxorubicin
1.2±0.3
Note: Data represented the mean of three experiments in triplicate and are
expressed as mean ± ꢇD.
Abbreviation: ꢁꢈ , 50% inhibitory concentration.
50
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ꢈhen et al
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Figure 2 ꢆffects of compound 12e on the morphology of ꢉꢆPꢂ-2 cells stained by ꢄO/ꢆB.
Notes: ꢄfter being treated with compound 12e for 24 h, HEPG-2 cells were stained by AO/EB and morphologies were immediately observed using fluorescence microscopy.
Viable cells presented homogeneously stained green nuclei. ꢆarly apoptotic cells showed greenish yellow nuclei and late apoptotic cells indicated condensed orange nuclei.
Abbreviations: ꢄO, acridine orange; ꢆB, ethidium bromide.
total apoptotic cells (Figure 3). These results demonstrated the value associated with untreated control cells (Figure 4).
that compound 12e caused a dose-dependent induction of These findings suggest that apoptosis induced by compound
apoptosis in HEPG-2 cells.
12e in HEPG-2 cells is associated with depolarization of the
mitochondrial membrane.
ꢅoss of MMP
It is well known that altered mitochondrial function is linked
ꢈaspase activation induced by
to apoptosis. In particular, loss of MMP can lead to mito- compound 12e
chondrial dysfunction and has been regarded as an important During the process of apoptosis, mitochondrial disruption
24
factor in controlling the induction of apoptosis. To assess leads to the release of cytochrome c, forming apoptosomes
the involvement of disruption of MMP in the apoptotic with Apaf-1 and procaspase-9, and triggering caspase-9
action of compound 12e, the cationic lipophilic dye, JC-1, activation, which subsequently cleaves the effector
2
5
which readily penetrates the plasma membrane into cells and caspase-3. Western blotting analysis revealed that com-
accumulates in actively respiring mitochondria, was used to pound 12e markedly increased the levels of caspase-9 and
evaluate the disruption of MMP in HEPG-2 cells. Loss of the effector cleaved caspase-3 in a concentration-dependent
MMP was observed in HEPG-2 cells 48 h after treatment with manner (Figure 5). These data suggest that compound
6
, 12, and 24 μM 12e. The values observed at these doses 12e induces apoptosis of HEPG-2 cells via the intrinsic
decreased to 82.33%, 72.67%, and 55.36%, respectively, of pathway.
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ꢇynthesis and cytotoxic activities
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Figure 3 Flow cytometric analysis of compound 12e induced apoptosis in ꢉꢆPꢂ-2 cells using ꢄnnexin V/Pꢁ double-staining assay.
Notes: Treatment with compound 12e at 0 μM, 6 μM, 12 μM, and 24 μM for 48 h followed by staining with FITC-conjugated Annexin V and PI for flow cytometric analysis.
ꢆarly apoptotic cells are ꢄnnexin V positive but Pꢁ negative (low right quadrant); late apoptotic and necrotic cells are ꢄnnexin V and Pꢁ positive (upper right quadrant), and
the rate of total apoptosis was the sum of early and late apoptosis. (A) Representative flow cytometric plots. (B) Flow cytometric analysis result. Data are presented as
mean ± ꢇD (n=3), and the significance was established at **P,0.01 compared with the control group.
Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.
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Acknowledgments
This study was supported by Program for the Top Young
Academic Leaders of Higher Learning Institutions of Shanxi,
the Collaborative Innovation Center Project of Shanxi “Astra-
galus” Resource Industrialization and Industrial Internation-
alization (No HQXTCXZX2016-021), and Shanxi Natural
Science Foundation (No 2016011112). We would also like
to thank the support and help from all the teachers of School
of Pharmaceutical Science, Shanxi Medical University.
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Disclosure
The authors report no conflicts of interest in this work.
&RPSRXQGꢀꢁꢂH
Figure 4 ꢅoss of MMP induced by compound 12e in ꢉꢆPꢂ-2 cells.
Notes: ꢈells were treated with 0, 6, 12, and 24 μM 12e for 48 h and incubated
with MMP indicator JC-1. JC-1 dye-stained cells were analyzed by flow cytometry,
and the relative red/green fluorescence ratio in treated cells is expressed graphically
as a percentage of red/green fluorescence ratio value of the control cells. Each
value represents mean ± ꢇD (n=3). **P,0.01 compared with the control group.
Abbreviations: Jꢈ-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine
iodide; MMP, mitochondrial membrane potential.
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βꢅ$FWLQ
Figure 5 ꢆffect of compound 12e on the protein expression of cleaved caspase-3
and caspase-9 by Western blot analysis.
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Supplementary material
ꢀ
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Figure S1 ꢁn vitro cytotoxicity of compound 12e on ꢉꢅ-7702 normal human
hepatocyte.
Note: The toxicity of compound 12e to normal human liver cells was observed by
MTT assay at the concentration of 25, 50, 100, and 200 μM for 48 h.
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