Synthesis and in?vitro anticancer activities of biotinylated derivatives of glaucocalyxin A and oridonin

Article abstract of DOI:10.1080/10286020.2020.1760851

Fourteen glaucocalyxin A biotinylated derivatives, one glaucocalyxin C biotinylated derivative, and two oridonin biotinylated derivatives were designed and synthesized. Their structures were confirmed from ¹H NMR, ¹³C NMR and HRMS data. The derivatives were evaluated for cytotoxic activities against lung (A549), cervical cancer cell line HeLa derivative (KB), multidrug-resistant KB subline (KB-VIN), triple-negative breast (MDA-MB-231), and estrogen receptor-positive breast (MCF-7) cancer cell lines. (Figure presented.).

Full text of DOI:10.1080/10286020.2020.1760851

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis and in vitro anticancer activities of
biotinylated derivatives of glaucocalyxin A and
oridonin
Xiao-Lei Huang, Jing-Lei Chen, Xian-Lun Li, Lei Zhao, Ya-Dong Cui, Jiang-Yun
Liu, Susan L. Morris-Natschke, Goto Masuo, Yung-Yi Cheng, Kuo-Hsiung Lee,
Dao-Feng Chen & Jian Zhang
To cite this article: Xiao-Lei Huang, Jing-Lei Chen, Xian-Lun Li, Lei Zhao, Ya-Dong Cui, Jiang-
Yun Liu, Susan L. Morris-Natschke, Goto Masuo, Yung-Yi Cheng, Kuo-Hsiung Lee, Dao-
Feng Chen & Jian Zhang (2020): Synthesis and in vitro anticancer activities of biotinylated
derivatives of glaucocalyxin A and oridonin, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2020.1760851
To link to this article: https://doi.org/10.1080/10286020.2020.1760851
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1760851
Synthesis and in vitro anticancer activities of biotinylated
derivatives of glaucocalyxin A and oridonin
a
a
a
a
a
ꢀ
ꢀ
Xiao-Lei Huang , Jing-Lei Chen , Xian-Lun Li , Lei Zhao , Ya-Dong Cui ,
Jiang-Yun Liua, Susan L. Morris-Natschkeb, Goto Masuob, Yung-Yi Chengb,
Kuo-Hsiung Leeb,c, Dao-Feng Chend and Jian Zhanga
b
aCollege of Pharmaceutical Science, Soochow University, Suzhou 215123, China; Natural Products
Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
c
North Carolina 27599, United States; Chinese Medicine Research and Development Center, China
d
Medical University and Hospital, Taichung 20277, Taiwan; Department of Pharmacognosy, School of
Pharmacy, Fudan University, Shanghai 201203, China
ABSTRACT
ARTICLE HISTORY
Received 9 January 2020
Accepted 22 April 2020
Fourteen glaucocalyxin A biotinylated derivatives, one glaucoca-
lyxin C biotinylated derivative, and two oridonin biotinylated
derivatives were designed and synthesized. Their structures were
1
confirmed from H NMR, 13C NMR and HRMS data. The derivatives
KEYWORDS
Glaucocalyxin A; oridonin;
glaucocalyxin C; biotin;
anticancer activity
were evaluated for cytotoxic activities against lung (A549), cervical
cancer cell line HeLa derivative (KB), multidrug-resistant KB sub-
line (KB-VIN), triple-negative breast (MDA-MB-231), and estrogen
receptor-positive breast (MCF-7) cancer cell lines.
CONTACT Jian Zhang
215123, China; Dao-Feng Chen
Fudan University, Shanghai 201203, China; Kuo-Hsiung Lee
jianzhang@suda.edu.cn
College of Pharmaceutical Science, Soochow University, Suzhou
dfchen@shmu.edu.cn Department of Pharmacognosy, School of Pharmacy,
khlee@unc.edu Natural Products Research
Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599,
United States
ꢀ
These authors contribute equally to this work.
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1760851.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
X.-L. HUANG ET AL.
1. Introduction
Glaucocalyxin A (GLA), glaucocalyxin B (GLB) and glaucocalyxin C (GLC) are ent-
kaurane type diterpenoids isolated and purified from the aerial parts of Rabdosia
japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara by us [1]. During the last decade,
we performed several studies on GLA and GLB [2–7]. GLA showed various biological
activities, such as cytotoxic effects, as well as inhibition of platelet activation and
thrombus formation [2–6]. GLA showed strong cytotoxic effects against HL60, Jurkat,
BRL, SK-BR-3, SW480, Bel-7402, SGC-7901, HUT-78 cancer cells [2, 3]. Mannich
base-type derivatives of GLA were prepared and evaluated for cytotoxic activities
against six tumour cell lines [5]. GLA nanosuspensions, prepared with precipitation-
combined ultrasonication, showed higher antitumor efficacy against HepG2 cells and
H22 bearing mice than free GLA [6]. GLA and GLB strongly decreased lipopolysac-
charides (LPS) induced NO generation in BV-2 microglia cells; furthermore, GLB
exerted anti-inflammatory effects mainly by targeting p38 mitogen-activated protein
kinase, nuclear factor-jB and the heme oxygenase (HO)-1 signaling pathway [7].
Studies by other researchers on GLA revealed the following findings: GLA c-cyclodex-
trin clathrate improved the water solubility and anticancer activity of GLA; GLA
induced G2/M cell cycle arrest and apoptosis through the PI3K/Akt pathway in
human bladder cancer cells; GLA reversed EMT and TGF-b1-induced EMT by inhib-
iting TGF-b1/Smad2/3 signaling pathway in osteosarcoma [8–10]. Oridonin is a
related highly oxygenated 7, 20-epoxy-ent-kaurane type diterpenoid, and its deriva-
tives also exhibited strong antitumor activities [11–14]. However, the mechanisms
underlying GLA and oridonin induced cytotoxicities remain unclear.
Biotin (vitamin H or B-7) promotes growth at the cellular level and is found in
substantially higher levels in tumors than in normal tissues [15]. Also, biotin recep-
tors are over expressed in many cancer cells, such as L1210 FR, A549, and MCF-7
[16, 17]. Consequently, biotin receptors could be explored for targeted delivery of
biotinylated derivatives of cytotoxic drugs to cancer cells and the biotin-avidin label-
ing method could be used to study the anti-tumor mechanism.
In this study, we designed and synthesized biotinylated derivatives of GLA, GLC
and oridonin in order to find the biomarkers which could be used to study the anti-
tumor mechanism. The cytotoxic activities of these derivatives against lung (A549),
cervical cancer cell line HeLa derivative (KB), multidrug-resistant KB subline (KB-
VIN), triple-negative breast (MDA-MB-231), and estrogen receptor-positive breast
(MCF-7) cancer cell lines were evaluated. In addition, the stabilities of biotinylated
GLA in trichloromethane (CHCl3), methanol (MeOH) and dimethyl sulfoxide
(DMSO) were analysed by high performance liquid chromatography (HPLC).
2. Results and discussion
2.1. Synthesis of biotin derivatives
Following literature methods [18, 19], biotin was linked to one and two units of 6-
aminocaproic acid as well as to various aminobenzoic acids to give six biotin
extended-chain reactants (1-4c, Figure 1). Then, GLA-14-biotin derivatives (5, 7, 9,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 1. Synthesis of biotin derivatives 1-4c. Reagent and conditions: (A) DMF, NHS, DCC, DMAP
at room temperature, overnight. (B) 6-Aminocaproic acid, DMF, Triethylamine, overnight. (C) a:
Tributylamine, DMF, Isobutyl chloroformate, at room temperature; b: 3-Aminobenzoic acid or 4-
Aminobenzoic acid or 4-Amino-3-methylbenzoic acid or 4-Amino-3-fluorobenzoic acid, 0 ꢁC, 2 h.
Figure 2. Synthesis of GLA biotin derivatives 5-14c. Reagent and conditions: (A) GLA, DMF, biotin
or compound 1 or 2, EDCI, DMAP, at room temperature, 24 h; (B) GLA, DMF, compound 3, EDCI,
DMAP, at room temperature, 24 h; (C) GLA, DMF, compound 4a or 4 b or 4c, EDCI, DMAP, at room
temperature, 24 h.
11, 13a-c, Figure 2) and GLA-7-biotin derivatives (6, 8, 10, 12, 14a-c, Figure 2), as
well as oridonin-7-biotin, oridonin-14-biotin and GLC-14-biotin derivative (15-17,
Figure 3) were obtained by esterification between the kaurene diterpenoid and biotin
or 1-4c. The products were purified by extraction and column chromatography; their
structures were confirmed from NMR and HRMS data (supplemental material).

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X.-L. HUANG ET AL.
Figure 3. Synthesis of oridonin biotin derivatives and GLC biotin derivative. Reagent and condi-
tions: (A) oridonin, DMF, biotin, EDCI, DMAP, at room temperature, 24 h; (B) GLC, DMF, compound
4a, EDCI, DMAP, at room temperature, 24 h.
Among the 14 new biotin-extended-chain-linked GLA biotin markers, the products
of the reactions of GLA with biotin or 6-(biotinylamido)caproic acid were obtained
in significantly higher yields than those of the reactions between GLA and biotinyla-
mido benzoic acids. This phenomenon might be explained by steric hindrance. The
esterification reactions between GLA and the (biotinylamido)benzoic acids were diffi-
cult and, thus, the yields of the target products were relatively low.
Esterification could occur at either the 7- or 14-hydroxyl group of GLA, and the
GLA-14-biotin derivatives were generally obtained in higher yields compared with the
GLA-7-biotin derivatives. Hydrogen bonding between the 7-hydroxyl and the 15-car-
bonyl group of GLA might hinder the esterification between the hydroxyl group of
the diterpenoid and the carboxylic acid of the biotin reactants. Similarly, the yield of
the oridonin-14-biotin derivative (15) was significantly higher than that of the orido-
nin-7-biotin derivative (16). In this reaction, LC-MS revealed two additional products;
these possible di-esterified compounds were formed in only small amounts and were
not isolated. The reaction between GLC and 4-(biotinylamido)benzoic acid led to a
complex mixture; only the main product (17) from esterification of the 14-hydroxyl
group was isolated.
2.2. The stability of biotinylated GLA
GLA-7-biotin (6) and GLA-14-biotin (5) were most stable in DMSO, slightly less sta-
ble in MeOH, and least stable in CHCl3. GLA-14-biotin (5) was more stable than
GLA-7-biotin (6) in these solvents, as shown in Figure 4. GLA-biotin was stable in
DMSO and MeOH, presumably due to strong hydrogen bond between the compound
and the solvent. On the other hand, the lower stability in CHCl3 might result from
acyl group transfer. GLA-7-biotin and GLA-14-biotin could interconvert to reach
equilibrium. We speculated that the 7-hydroxyl group, at a b-position to the carbonyl
group at C-15 of GLA-biotin, might show an inductive effect in the Lewis acid
CHCl3. Similar phenomena occurred between oridonin-7-biotin (16) and oridonin-
14-biotin (15). However, the GLC-14-biotin derivative (17) was stable in all three sol-
vents. The only structural difference between GLA and GLC is the presence of a

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 4. Time course for compounds 5 and 6 in different solutions. (A) compound 5 in different
solutions (B) compound 6 in different solutions.
carbonyl group at C-15 in GLA rather than a hydroxyl group at the same position
in GLC.
2.3. The anticancer activities of biotinylated derivatives in vitro
The target compounds were evaluated for their activities against A549, MCF-7, KB,
MDA-MB-231, KB-VIN cancer cell lines [20]. As shown in Table 1, the cytotoxic
potencies of GLA-biotinylated derivatives 5 and 6 were similar. Among the five tested
human cancer cell lines, MDA-MB-231 was the most sensitive. All IC50 values of the
remaining tested compounds (7 – 10, 13 b, 13c, 14a, and 14 b) were greater than
10 lM. Although compounds 5 and 6 were less potent than GLA, they could be used
as biomarkers for studying the anti-tumor mechanism of GLA especially in MDA-
MB-231 cells using a biotin-avidin labeling method.
The methylenecyclopentanone moiety in GLA is certainly an essential cytotoxic
pharmacophore. The biotin esters attached to hydroxyl groups at positions 7 or 14
would likely hinder binding of methylenecyclopentanone to corresponding protein
receptors; thus, the cytotoxic potencies of all tested GLA-biotinylated derivatives were
lower than that of GLA itself.
3. Experimental
3.1. General experimental procedures
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian UNITY
INOVA - 600 with tetramethylsilane (TMS) as an internal standard. High resolution
electrospray ionisation mass (HR-ESI-MS) spectra were recorded on Agilent 6520 B
Q-TOF spectrometer (Agilent Technologies, Santa Clara, CA, USA). Silica gel (200-
300 mesh, Qingdao Marine Chemical Co., Qingdao, China) and RP-C18 (50-70 lm,
Daiso, Japan) were used for column chromatography.
3.2. General procedure for the synthesis of biotin derivatives
A solution of Biotin (488 mg, 2.0 mmol, 1.0 equiv) in N, N-dimethylformamide
(DMF, 20 ml) was added N-hydroxysuccinimide (NHS, 276 mg, 2.4 mmol, 1.2 equiv)

6
X.-L. HUANG ET AL.
Table 1. The anticancer activities of biotinylated derivatives in vitro.
tumor cells, IC50, lM
Compound
A549
KB
KB-VIN
MDA-MB-231
MCF-7
GLA
5
6
2.063 0.350
10.267 0.041
8.705 1.322
4.582 0.993
11.991 1.647
10.480 1.647
2.807 1.162
13.494 1.689
10.196 0.110
1.417 0.252
6.077 0.420
6.074 0.217
4.490 1.516
10.993 0.146
10.196 0.705
7
8
9
10
14a
13b
14b
13c
taxol
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.924 0.063
1.372 0.016
1.241 0.028
7.246 0.913
8.231 1.006
and dicyclohexylcarbodiimide (DCC, 824 mg, 4.0 mmol, 2.0 equiv) and 4-dimethyla-
minopyridine (DMAP, 610 mg, 5.0 mmol, 2.5 equiv) at room temperature. The solu-
tion was stirred overnight, and then the solvent was removed under reduced
pressure. The residue was dissolved in isopropanol (40 ml) with ultrasonic. The solu-
tion was cool to 4 ꢁC and stood for 1 h. The resulted mixture was filtered to afford
intermediate a. Intermediate a (341 mg, 1.0 mmol, 1.0 equiv) and 6-aminocaproic acid
(157 mg, 1.2 mmol, 1.2 equiv) were dissolved in 20 ml DMF. Triethylamine (0.3 ml,
2.16 mmol, 2.16 equiv) was added. The solution was stirred overnight, and then the
solvent was removed under reduced pressure. The residue was dissolved in water
(25 ml) and formic acid (2 ml) with ultrasonic. The solution was cooled to 4 ꢁC. The
resulted mixture was filtered to afford compound 1. Intermediate b was obtained
according to the similar procedure of preparing intermediate a staring from 1.
Compound 2 was obtained according to the similar procedure of preparing com-
pound 1 staring from intermediate b. To a solution of biotin (400 mg, 2.0 mmol, 1.0
equiv) and tributylamine (0.64 ml, 2.7 mmol, 1.3 equiv) in DMF (40 ml) was added
isobutyl chloroformate (0.32 ml, 2.5 mmol, 1.2 equiv) at room temperature. The reac-
tion mixture was stirred 10 min, and then 3-aminobenzoic acid (548 mg, 4.0 mmol,
2.0 equiv) in 40 ml DMF was added slowly to the mixture at 0 ꢁC and left to stir for
2 h. The solvent was removed under reduced pressure. The residue was dissolved in
warm ethanol solution (50%, 36 ml). The pH of the mixture was adjusted to 2.0 by
hydrochloric acid. The mixture was cooled to 0 ꢁC and stood for 12 h. The resulted
mixture was filtered to give compound 3. Compounds 4a, 4 b and 4c were obtained
according to the similar procedure of preparing 3 starting from 4-aminobenzoic acid,
4-amino-3-methylbenzoic acid and 4-amino-3-fluorobenzoic acid. The biotin deriva-
tives were all white solids. Compound 1, 270 mg, yield 75.6%. Compound 2, 340 mg,
yield 72.2%. Compound 3, 465 mg, yield 62.4%. Compound 4a, 483 mg, yield 64.9%.
Compound 4 b, 600 mg, yield 77.6%. Compound 4c, 465 mg, yield 62.4%.
Compound 1, a white powder, 270 mg, yield 75.6%, HRESIMS: m/z 358.1791
1
[M þ H]þ (calcd for C16H28N3O4S, 358.1756). H NMR (600 MHz, DMSO-d6) d: 7.71
(1 H, s), 6.39 (1 H, s), 6.33 (1 H, s), 4.29 (1 H, t, J ¼ 6.4 Hz), 4.10-4.11 (1 H, m), 3.06-
3.08 (1 H, m), 3.00 (2 H, dd, J ¼ 12.5, 6.3 Hz), 2.81 (1 H, dd, J ¼ 12.4, 4.9 Hz), 2.56
(1 H, d, J ¼ 12.4 Hz), 2.18 (2 H, t, J ¼ 7.3 Hz), 2.03 (2 H, t, J ¼ 7.3 Hz), 1.66-1.22 (12 H,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
m). 13C NMR (150 MHz, DMSO-d6) d: 174.9, 172.2, 163.1, 61.5, 59.6, 55.9, 38.7, 35.6,
34.1, 29.3, 28.7, 28.5, 26.4, 25.8, 24.7.
3.3. General procedure for the synthesis of GLA-biotin derivatives
A solution of glaucocalyxin A (332 mg, 1.0 mmol, 1.0 equiv) in DMF (20 ml) was
added biotin (293 mg, 1.2 mmol, 1.2 equiv) and 1-ethyl-3(3-dimethylpropylamine)
carbodiimide (EDCI, 383 mg, 2.0 mmol, 2.0 equiv) and DMAP (122 mg, 1.0 mmol, 1.0
equiv) at room temperature. The reaction mixture was stirred at room temperature
for 24 h. The reaction mixture was then diluted with water and extracted with ethyl
acetate. The extract was washed with saturated NaCl (aqueous) solution, dried over
anhydrous Na2SO4, filtered, and evaporated to give a white solid residue. The crude
residue was purified by silica gel column, eluted with EtOAc/MeOH (12:1, v/v) to
afford a mixture. Then the crude residue was further purified by preparative reversed
phase liquid chromatography, elution with MeOH-H2O (70:30, v/v) at 3 ml/min to
afford the desired compounds 5 and 6. Compounds 7, 8, 9, 10, 11, 12, 13a, 14a,
13 b, 14 b,13c and 14c were obtained according to the similar procedure of preparing
5 and 6 starting from 1, 2, 3, 4a, 4 b and 4c. GLA-biotin derivatives were all white
solids. Compound 5, 280 mg, yield 50.2%. Compound 6, 101 mg, yield 18.1%.
Compound 7, 251 mg, yield 18.0%. Compound 8, 121 mg, yield 37.4%. Compound 9,
110 mg, yield 13.4%. Compound 10, 105 mg, yield 14.0%. Compound 11, 94 mg, yield
13.9%. Compound 12, 30 mg, yield 4.4%. Compound 13a, 102 mg, yield 15.1%.
Compound 14a, 65 mg, yield 9.6%. Compound 13 b, 169 mg, yield 21.0%. Compound
14 b, 45 mg, yield 6.5%. Compound 13c, 96 mg, yield 13.8%. Compound 14c, 64 mg,
yield 9.2%.
Compound 5: a white powder, 280 mg, yield 50.2%. HRESIMS: m/z 559.2843
1
[M þ H]þ (calcd for C30H43N2O6S, 559.2842). H NMR (600 MHz, DMSO-d6) d: 6.40
(1 H, s), 6.35 (1 H, s), 5.96 (1 H, s), 5.85 (1 H, s), 5.46 (1 H, s), 4.41 (1 H, d,
J ¼ 4.3 Hz), 4.31 (1 H, t, J ¼ 5.8 Hz), 4.10-4.13 (2 H, m), 3.93-3.95 (1 H, m), 3.08-3.10
(1 H, m), 3.03 (1 H, s), 2.83 (1 H, dd, J ¼ 12.4, 5.1 Hz), 2.58 (1 H, d, J ¼ 12.4 Hz), 2.47-
2.49 (1 H, m), 2.41-2.43 (1 H, m), 1.12 (3 H, s), 1.01 (3 H, s), 0.96 (3 H, s). 13C NMR
(150 MHz, DMSO-d6) d: 216.1, 205.5, 172.6, 162.7, 147.0, 116.3, 74.2, 70.9, 61.3, 61.0,
59.2, 55.4, 53.9, 50.6, 48.6, 46.1, 43.7, 39.8, 38.4, 37.3, 33.8, 33.4, 31.9, 28.9, 28.0, 26.9,
24.0, 20.7, 17.8, 17.5.
3.4. General procedure for the synthesis of oridonin-biotin derivatives
Compounds 15 and 16 were obtained according to the similar procedure of preparing
5 and 6 starting from oridonin. Compound 15, a white solid, 212 mg, yield 35.9%.
Compound 16, a white solid, 78 mg, yield 13.2%.
Compound 15: a white powder, 212 mg, yield 35.9%. HRESIMS: m/z 591.2715
1
[M þ H]þ (calcd for C30H43N2O8S, 591.2695). H NMR (600 MHz, DMSO-d6) d: 6.38
(1 H, s), 6.33 (1 H, s), 5.98 (1 H, s), 5.88 (1 H, d, J ¼ 10.3 Hz), 5.82 (1 H, s), 5.82 (1 H,
s), 5.59 (1 H, s), 4.38 (1 H, d, J ¼ 4.9 Hz), 4.11 (1 H, t, J ¼ 4.9 Hz), 4.06 (1 H, d,
J ¼ 10.2 Hz), 3.82 (1 H, d, J ¼ 10.2 Hz), 3.50 (1 H, dd, J ¼ 10.2, 7.0 Hz), 3.05-3.07 (1 H,

8
X.-L. HUANG ET AL.
m), 2.95 (1 H, d, J ¼ 9.7 Hz), 2.81 (1 H, dd, J ¼ 12.5, 5.1 Hz), 2.56 (1 H, d, J ¼ 12.4 Hz),
0.98 (3 H, s), 0.97 (3 H, s). 13C NMR (150 MHz, DMSO-d6) d: 207.6, 172.0, 163.2,
151.3, 119.8, 96.2, 74.5, 73.6, 72.0, 62.9, 62.2, 61.5, 59.6, 59.5, 55.8, 54.3, 41.8, 40.9,
40.3, 38.8, 34.2, 33.8, 33.2, 30.7, 29.8, 28.4 ꢂ 2, 24.5, 22.1, 20.2.
3.5. General procedure for the synthesis of GLC-biotin derivative
Compound 17 was obtained according to the similar procedure of preparing 5 and 6
starting from GLC and 4a. 7-substitued compound was not found in this reaction.
1
Compound 17: a white powder, 51 mg, yield 8.9%. H NMR (400 MHz, DMSO-d6)
d: 10.2 (1 H, s), 7.94 (2 H, d, J ¼ 8.7 Hz), 7.68 (2 H, d, J ¼ 8.7 Hz), 6.42 (1 H, s), 6.35
(1 H, s), 5.20 (1 H, d, J ¼ 7.2 Hz), 5.00 (1 H, s), 4.88 (1 H, s), 4.51 (1 H, s), 4.41 (1 H,
s), 4.34 (1 H, s), 4.28 (1 H, t, J ¼ 7.4 Hz), 4.10-4.12 (1 H, m), 3.07-3.09 (1 H, m), 2.81
(1 H, dd, J ¼ 12.4, 5.0 Hz), 2.60-2.62 (1 H, m), 2.31-2.33 (2 H, m), 1.01 (3 H, s), 0.92
(6 H, s). 13C NMR (100 MHz, DMSO-d6) d: 216.8, 171.8, 165.2, 162.8, 157.4, 143.6,
130.6 ꢂ 2, 124.5, 118.2 ꢂ 2, 106.2, 75.7, 73.7, 71.2, 61.1, 59.2, 55.5, 54.1, 50.7, 48.8,
47.1, 45.9, 40.5, 38.5, 37.8, 36.3, 33.5, 32.8, 28.3, 28.1, 27.6, 25.0, 20.6, 18.6, 17.6.
3.6. The stability of biotinylated GLA
HPLC analyses were performed on an Agilent 1260 HPLC system equipped with a
photodiode array detector (DAD). A 100-5C18 column (250 mm ꢂ 4.6 mm, 5 lm,
Kromasil) was used. The mobile phase was methanol and water (65: 35, v/v). The
flow rate was 1 ml/min. Monitoring was performed at 230 nm and the injection vol-
ume was 20 ll. The stabilities of compounds 5 and 6 in different solvents (DMSO,
MeOH, CHCl3) were studied at room temperature (25 ꢁC). At different time intervals,
the remaining compound was measured according to the above-mentioned
HPLC method.
3.7. Cytotoxic activity assay
Cytotoxic activity was determined by the sulforhodamine B (SRB) colorimetric assay.
In brief, the cells (4-11 ꢂ 103 cells/well) were seeded in 96-well plates filled with
RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) containing
various concentrations of samples, and incubated for 72 h. At the end of the exposure
period, the attached cells were fixed with cold 50% trichloroacetic acid for 30 min fol-
lowed by staining with 0.04% SRB (Sigma Chemical Co.) for 30 min. The bound SRB
was solubilized in 10 mM Tris-base and the absorbance was measured at 515 nm on a
Microplate Reader ELx800 (Bio-Tek Instruments, Winooski, VT) with a Gen5 soft-
ware. All results were representative of three or more experiments and IC50 is
expressed as the average with standard deviation.
Disclosure statement
No potential conflict of interest was reported by the author(s).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
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Funding
This study was funded by the National Natural Science Foundation of China (grant number
81202882); the Social Development Project in Jiangsu Province of China (grant number
BE2018322); the Priority Academic Program Development of the Jiangsu Higher Education
Institutes, China (PAPD). Partial support was received from National Institutes of Health,
NIH (grant number CA177584) awarded to Kuo-Hsiung Lee.
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