Synthesis and biological evaluation of novel spin labeled 18β-glycyrrhetinic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.10.041

Eighteen novel spin-labeled 18β-glycyrrhetinic acid (GA) derivatives were designed, synthesized, and evaluated for cytotoxicity against four human tumor cell lines (A-549, DU-145, KB and KBvin). Most of the derivatives showed more significant cytotoxicity than that of the parent compound GA. The best compound, 6j, with a tryptophan amino moiety and piperidine nitroxyl radical showed GI₅₀ values of 13.7-15.0 μM, and was fivefold more potent than GA. In a mechanism of action study, compound 7a was confirmed as a 20S proteasome inhibitor in both in vitro and cell-based assays. These findings support further optimization efforts based on 18β-GA as a lead compound to develop potential anticancer drug candidates.

Full text of DOI:10.1016/j.bmcl.2012.10.041

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7530–7533
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel spin labeled 18b-glycyrrhetinic
acid derivatives
Yingqian Liu a,d, Keduo Qian a, Chih-Ya Wang a, Chin-Ho Chen b, Xiaoming Yang a, Kuo-Hsiung Lee a,c,
⇑
a Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
b Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan, ROC
d School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eighteen novel spin-labeled 18b-glycyrrhetinic acid (GA) derivatives were designed, synthesized, and
evaluated for cytotoxicity against four human tumor cell lines (A-549, DU-145, KB and KBvin). Most of
the derivatives showed more significant cytotoxicity than that of the parent compound GA. The best com-
pound, 6j, with a tryptophan amino moiety and piperidine nitroxyl radical showed GI50 values of 13.7–
Received 30 July 2012
Revised 3 October 2012
Accepted 8 October 2012
Available online 16 October 2012
15.0 lM, and was fivefold more potent than GA. In a mechanism of action study, compound 7a was con-
firmed as a 20S proteasome inhibitor in both in vitro and cell-based assays. These findings support fur-
ther optimization efforts based on 18b-GA as a lead compound to develop potential anticancer drug
candidates.
Keywords:
Spin-labeled
18b-Glycyrrhetinic acid
Nitroxide
Ó 2012 Elsevier Ltd. All rights reserved.
Cytotoxicity
20S proteasome inhibitors
Cancer is currently one of the most severe health problems
worldwide. Because natural products exhibit significant biological
properties and often have a broad safety window during adminis-
tration, the development of natural product-derived derivatives to
treat various types of cancer has received increasing interest.1–4
Glycyrrhetinic acid (1, GA), an active triterpenoid metabolite of
glycyrrhizin present abundantly in licorice roots, shows diverse
biological activities, including antiviral, anti-allergic, anti-inflam-
matory, and anti-ulcer effects.5,6 In addition, GA and its derivatives
also display promising antitumor activities, such as inhibition of
tumorigenesis and induction of apoptosis in various cancer cells,
including hepatoma, leukemia, breast cancer, and gastric cancer
cells, at high concentrations. It was discovered that GA acts directly
on mitochondria to induce apoptosis through increased mitochon-
drial swelling, loss of mitochondrial membrane potential, and re-
lease of cytochrome c.7–10 GA was also reported to potentiate the
apoptotic effects of trichostatin A, a histone deacetylase inhibitor,
in ovarian cancer cells by increasing the activation of the cas-
pase-8-dependent pathway, as well as the activation of the mito-
However, because GA’s cytotoxicity is usually only weak to mod-
erate, many researchers have tried to enhance the potency of GA by
various derivatizations.8–10,13,14 In our recent search for potential
antitumor drugs derived from natural products, we found that
incorporation of a stable nitroxyl radical into antitumor molecules
could increase their activity and decrease their toxicity compared
with parent compounds, as well as impart some lipophilicity. In
addition, reports have shown that conjugation of amino acids and
natural products can provide improved bioactivity;15,16 an approach
that was also successful in our prior studies where amino acid moi-
eties effectively enhanced the potency of antitumor molecules.17,18
The above information together with our previous studies of GA
derivatives as potential anticancer compounds19,20 prompted us to
introduce a nitroxyl functionality and amino acid segments into
the design of novel spin labeled GA derivatives, in order to improve
inhibitory effects against human tumor cell lines. All newly synthe-
sized compounds (4a–c, 6a–j and 7a–e) were tested for cytotoxic
activity against a panel of human tumor cell lines, including
A549 (non-small cell lung cancer), DU145 (prostate cancer cell
line), KB (nasopharyngeal carcinoma) and KB-VIN (MDR KB subline
selected using vincristine). We report herein the design, synthesis,
and in vitro cytotoxicity screening of these novel spin-labeled GA
derivatives, as well as a mechanistic investigation of proteasome
inhibitory effect.
chondria-mediated cell death.11 GA was found to be
a
proteasome inhibitor, and other studies have revealed that GA
and its derivatives can target peroxysome proliferator-activated
receptors (PPARs) and act on the tumor cell environment, angio-
genesis, inflammation, and immune cell functions.12
Scheme 1 outlines the synthetic route to compounds 4a–c.
Compound 1 was first reacted with acetic anhydride in pyridine
to convert into its 3-O-acetate 2, which was then treated with
⇑
Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966 3893.
E-mail address: khlee@unc.edu (K.-H. Lee).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.041

Y. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7530–7533
7531
COOH
COOH
COCl
H
H
H
O
H
O
H
O
H
Ac2O/pyridine
r.t. overnight
(CO)2Cl2/CH2Cl2
NH2R/Et3N
CH2Cl2, r.t., overnight
0°C
r.t., 2h
AcO
H
H
H
HO
AcO
3
2
1
CONHR
H
O
H
R1=Ac
R =
R =
4b
4a
R1=Ac
R =
N
4N NaOH
THF/MeOH
overnight
H
N
O
O
R1O
4a-c
4c
R1=H
N
O
Scheme 1. Syntheses of compounds 4a–c.
oxalyl chloride to give the 30-acyl chloride 3. This key intermediate
was then condensed with the appropriate piperidine (pyrroline)
nitroxyl amines in the presence of triethylamine at room temper-
ature overnight to give amides 4a–b. Saponification of the C-3
acetyl protection of compound 4a using 4 N NaOH in MeOH and
THF mixed solvent yielded the corresponding compound 4c.
The syntheses of compounds 6a–j and 7a–e were conducted
according to the procedures in Scheme 2. Compound 1 was reacted
with appropriate amino acid methyl esters in the presence of
HOBt/EDCI/Et3N in DMF at room temperature overnight, which
afforded the corresponding intermediate amides. Subsequently,
the saponification of the methyl ester of the intermediate amides
was carried out with 4 N NaOH in MeOH/THF which gave the cor-
responding carboxylic acid derivatives 5a–j. Reaction of 5a–j with
piperidine nitroxyl amine in the presence of HOBt/EDCI in DMF led
to target compounds 6a–j in good yields. Compounds 7a–e were
obtained with similar methods using pyrroline nitroxyl amine un-
der HOBt/EDCI/Et3N in yields of 67% to 82%.
Target compounds 4a–c, 6a–j and 7a–e were evaluated for cyto-
toxic activity against four human tumor cell lines, A549, DU-145,
KB and KBvin, using a sulforhodamine B colorimetric (SRB) assay
in parallel with 1.21 The results are summarized in Table 1. Gener-
ally, the introduction of a piperidine or pyrroline nitroxyl radical
into the C-30 side chains of 1 increased the cytotoxic effects of 1
by 2- to 4-fold. Compound 4a, with a C-3 acetyl moiety, exhibited
slightly improved antitumor activity compared with 4c, with a free
C-3 hydroxy group. The best compound in this series, 4a, showed
cytotoxicity against four human tumor cell lines with GI50 values
of 14.6–18.7
lM, which were fourfold better than those of 1
(GI50: 61.2–64.9
l
M). This finding indicated that both the 3-O-
acetyl group and 30-nitroxyl radical group might be important
for increasing the tumor cell growth inhibition of GA derivatives.
Regarding the influences of the nitroxyl radical functionality
and amino acid segments, it was first obvious that intermediates
5a–j with only various free amino acids at C-30 showed no signif-
icant cytotoxicity (GI50 >70 lM). However, incorporation of a
R2
O
H
N
C
N
N
O
H
H
O
O
R2
7a-e
H
HO
O
7a
7b
H
H
CH3
7c CH2Ph
H2N
7d
7e
CH(CH3)2
(CH2)2SCH3
N
, EDCI/HOBt/Et3N
DMF, r.t., overnight
R2
O
C
R2
O
C
COOH
H
N
OH
N
O
H
N
H
i. amino acid methyl ester
EDCI/HOBt/Et3N, DMF
N
H
H2N
N O
, EDCI/HOBt/Et3N
O
H
H
O
H
O
O
O
H
ii. 4N NaOH
THF/MeOH
DMF, r.t., overnight
H
H
H
HO
1
HO
HO
R2
6a-j
H
R2
5a-j
5f
5a
5b
H
CH2OH
6a
6f CH2OH
H
CH3
CH2Ph
5g CH2PhOH
5h D-CH2OH
6g
6h
6b CH3
CH2PhOH
5c
6c CH2Ph
D-CH2OH
5i
5j
6d
5d CH(CH3)2
5e
CH2CH(CH3)2
CH(CH3)2
6i CH2CH(CH3)2
6e (CH2)2SCH3
(CH2)2SCH3
6j
N
H
N
H
Scheme 2. Syntheses of compounds 6a–j and 7a–e.

7532
Y. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7530–7533
Table 1
of 13.7–15.0 lM, and thus, was fivefold more potent than the par-
ent compound 1.
To investigate whether the chiral center introduced in the ami-
no acids has any influence on the cytotoxic activity, compound 6f
Growth inhibition of spin labeled 18b-GA derivatives against four human tumor cell
linesa
Entry
GI50
(l
M)
KB
containing an
diastereomer 6h were prepared. The
more active than the -serine 6h, which showed no significant
L-serine side chain and its corresponding
D-serine
A549
DU145
KBvin
L-serine substituted 6f was
4a
4b
4c
6a
6b
6c
6d
6e
6f
18.7 1.21
27.3 2.29
24.8 1.90
>70
14.9 0.768
32.5 2.46
24.6 1.02
>70
14.6 0.415
22.1 0.500
20.6 0.606
>70
15.2 0.235
20.6 0.886
19.2 0.887
>70
D
cytotoxicity, suggesting that the stereochemistry in the amino acid
side chain might be important for the derivatives’ growth inhibi-
tion effects.
>70
>70
>70
>70
19.4 0.909
34.2 1.88
23.3 0.304
44.0 0.057
18.3 0.373
>70
19.6 1.60
15.0 0.689
46.7 1.90
46.1 0.653
19.0 1.13
34.5 0.187
41.5 1.83
61.2 2.33
19.3 0.292
28.9 0.921
21.7 0.402
45.5 0.666
17.4 0.619
>70
22.0 0.546
15.0 0.363
46.2 0.697
45.2 1.27
22.5 0.606
39.5 1.05
43.2 1.61
64.9 0.505
14.6 0.448
17.5 0.927
16.9 0.501
39.9 0.618
15.3 0.469
>70
16.0 0.368
14.2 0.670
45.5 1.04
41.3 0.346
17.8 0.193
30.7 0.480
38.4 1.15
61.2 0.118
14.9 0.471
18.6 0.931
19.2 0.497
47.6 1.06
19.5 1.33
>70
17.0 0.377
13.7 1.25
46.9 0.230
44.2 0.280
16.6 0.591
27.3 0.338
38.5 0.956
62.3 1.41
Proteasome hydrolyzes various unwanted cell cycle regulators,
transcription factors, and antigenic proteins. It is a promising tar-
get for the development of therapeutic treatments for various dis-
orders, such as neurodegenerative diseases, cancers, and
inflammatory diseases. Because proteasome is so critically related
to the development of numerous major human diseases, various
proteasome inhibitors have been designed and synthesized,22–24
resulting in novel classes of anticancer drugs or clinical trial candi-
dates, such as bortezomib and salinosporamide A. In our previous
studies, we reported that many triterpenoids, such as betulinic,
oleanolic, ursolic, moronic, and glycyrrhetinic acids, as well as their
derivatives, can regulate proteasome activities.20,25 Among these
compounds, 1 and its derivatives are interesting structural pheno-
types for the identification of novel anti-proliferative agents and
20S proteasome inhibitors.
6g
6h
6i
6j
7a
7b
7c
7d
7e
1
a
The results are averaged from three independent experiments. Compounds 5a–
5g did not show significant inhibition (GI50 >70 lM).
Therefore, in the current study, we also evaluated the protea-
some regulating activity of newly synthesized spin-labeled GA
derivative 7a, using in vitro 20S proteasome and cell-based assays
in HeLa UbG76V-GFP cells,26 with lactacystin as positive control.
Compound 7a was selected due to its availability in quantity, and
should represent the mechanism of other novel spin-labeled GA
derivatives due to their structural similarity. In the in vitro assay
for inhibition against the chymotrypsin-like activity of 20S protea-
Table 2
Inhibition of the chymotrypsin-like activity of 20S proteasome
Compound
IC50 M)
7a
1
Lactacystin
11.4
(
l
16.9
22.3
piperidine (6a–j) or pyrroline (7a–e) nitroxyl radical at the termi-
nus of the C-30 side chains potentiated the cytotoxic effects against
the tested human tumor cell lines. The difference in the GI50 values
paralleled the size and lipophilic character of the substituents at
some, 7a inhibited proteasome activity with an IC50 of 16.9 lM,
slightly better than that of the parent compound 1 (Table 2). This
effect was confirmed in the cell-based luminescent proteasome
inhibition assay using HeLa UbG76V-GFP cells, which light up when
proteasome is inhibited (Fig. 1).
In conclusion, three series of novel spin-labeled derivatives of
18b-GA were designed and synthesized, and the in vitro cytotoxic-
ity was evaluated against four human tumor cell lines using an SRB
assay. Most of the new compounds with nitroxyl radical function-
alities displayed better activity compared with 1. Among the new
derivatives, 6j with a tryptophan amino moiety and piperidine
nitroxyl radical showed the greatest cytotoxicity, five-fold more
potent than 1. These results suggested that the incorporation of
the
a-carbon of the C-30 amino acid chain in these derivatives.
Similar results were seen in our previous study.17,18 Specifically,
glycine (6a and 7a) and alanine (6b and 7b) amino substituents
showed little to weak potentiation of the cytotoxicity, while com-
pounds 6c and 7c with a phenylalanine amino moiety showed sig-
nificant growth inhibitory activity with GI50 values of 14.6–19.4
and 16.6–22.5
increased the cytotoxicity, with GI50 values of 15.3–19.5 and 16.0–
22.0 M, respectively. The best compound 6j with a tryptophan
lM, respectively. Tyrosine (6g) and leucine (6i) also
l
amino moiety and piperidine nitroxyl radical exhibited GI50 values
O
C
H
N
N
H
O
N
H
O
O
H
HO
7a (YQL-30)
H
Figure 1. The cell-based proteasome inhibition assay. HeLa UbG76V-GFP cells light up when the proteasome is inhibited. (Left: HeLa UbG76V-GFP without compounds).

Y. Liu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7530–7533
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mechanistic investigation, it was found that 7a showed protea-
some inhibition activity in both in vitro and cell-based assays.
Although the anti-proteasome activity is in a concentration range
similar to that of the anti-proliferative inhibitory activity against
cancer cells, further studies are required to determine whether
the proteasome is the major anticancer target of 7a. Further opti-
mization efforts based on GA are ongoing.
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Supplementary data
Supplementary data associated with this article can be found,
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