Synthesis and proteasome inhibition of lithocholic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.02.041

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among the

Full text of DOI:10.1016/j.bmcl.2011.02.041

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1926–1928
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and proteasome inhibition of lithocholic acid derivatives
Zhao Dang a, Andrew Lin a, Phong Ho a, Dominique Soroka a, Kuo-Hsiung Lee b,c, Li Huang a,
,
⇑
Chin-Ho Chen a,
⇑
a Surgical Science, Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
b Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at
the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of
potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13)
Received 14 January 2011
Revised 8 February 2011
Accepted 14 February 2011
Available online 17 February 2011
displayed the most potent proteasome inhibition with IC50 = 1.9
that these lithocholic acid derivatives are noncompetitive inhibitors of the proteasome.
Ó 2011 Elsevier Ltd. All rights reserved.
lM. Enzyme kinetic analysis indicates
Keywords:
Lithocholic acid
Proteasome
Proteasome inhibitor
The proteasome is an important protein complex that plays a
critical role in maintaining cellular homeostasis.1–3 The main func-
tion of the proteasome is intracellular degradation of damaged, un-
sources, the majority of these proteasome inhibitors have pep-
tide-related structures, interacting with the proteasome at the cat-
alytic site to competitively inhibit the proteolysis of substrate.
Examples of these compounds include MG132, CEP1612, PS341,
lactacystin, TMC-89A, and argyrin A.5 On the other hand, non-
peptide proteasome inhibitors are less common than the peptide-
related proteasome inhibitors. Two triterpene derivatives, celastrol
and withaferin A, and some green tea polyphenols, have been
reported with proteasome-inhibitory effects.6–9 We recently
reported that a series of triterpene 18b-glycyrrhetinic acid deriva-
tives had potent inhibitory activity on the 20S proteasome.10
Although the competitive proteasome inhibitors targeting the cat-
alytic sites are well documented, noncompetitive inhibitors are
less common and have generally not been well characterized11
though some quinolines were reported to be noncompetitive
inhibitors.12,13 In this paper, we report a class of novel proteasome
inhibitors that inhibit the proteasome in a noncompetitive manner.
Our previous study indicated that glycyrrhetinic acid can be
used as a scaffold to synthesize proteasome inhibitors through
esterification of its C-3 hydroxyl group.10 In an effort to search
for new scaffolds for the synthesis of proteasome inhibitors, sev-
eral natural products, including moronic acid, ursolic acid, olean-
olic acid, and lithocholic acid (LA), were evaluated as potential
scaffolds.10,14 Among these natural products, only LA inhibited
the chymotrypsin-like activity of the 20S proteasome with an
wanted, and misfolded proteins. The 20S proteasome has
cylindrical structure containing four rings stacked on top of each
other. The two outer rings each contain seven structural -sub-
a
a
units that do not have enzymatic activity. The two inner rings, each
with seven b-subunits, contain three major proteolytic activities: a
chymotrypsin-like (b5), a trypsin-like (b2), and a caspase-like (b1)
activity. These proteolytic activities allow the proteasome to cleave
unwanted proteins into 8–12 amino acid peptides. The chymotryp-
sin-like activity is believed to be the most important activity in
protein degradation and is thus the primary target of most protea-
some inhibitors.2,3
By regulating cellular protein levels, the proteasome is critical
for maintaining many important cellular functions, such as the cell
cycle, apoptosis, and immune response. Targeting proteasomal
proteolysis may lead to new treatments for a variety of clinical
conditions, such as cancers, inflammation, and neurodegenerative
diseases. One successful example is the proteasome inhibitor pep-
tide boronate, PS341 (Bortezomib), which was developed into an
anti-cancer drug for the treatment of multiple myeloma.4
In addition to bortezomib, a number of other proteasome inhib-
itors have been developed either as experimental tools or as poten-
tial drug candidates for clinical usage, especially for anti-cancer
therapy. Originating from both chemical synthesis and natural
IC50 of 18.1 lM (Table 1). Therefore, LA was used as a new scaffold
to further increase the potency of the proteasome inhibition. The
usage of LA as the scaffold is advantageous in that it is readily
available and less expensive than other triterpene natural prod-
ucts. The molecular size of LA is also smaller than other triterpenes.
⇑
Corresponding author. Tel.: +1 919 684 2952; fax: +1 919 684 3878 (L.H.); tel.:
+1 919 684 3819; fax: +1 919 684 3878 (C.-H.C.).
E-mail addresses: lihuang@duke.edu (L. Huang), chc@duke.edu (C.-H. Chen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.041

Z. Dang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1926–1928
1927
Table 1
The synthesis of LA C-3 ester derivatives was accomplished by
treating LA or a LA methyl ester with the corresponding dicarbox-
ylic acids or anhydrides under general esterification conditions
(Scheme 1). Compounds 3, 4, 6, 9, 11, and 12 were obtained by
treatment of LA with corresponding anhydrides in the presence
of 4-(dimethylamino)pyridine (DMAP), using microwave-assisted
heating. Treatment of LA, LA methyl ester, or LA ethyl ester with
corresponding dicarboxylic acid under microwave heating in the
presence of N,N0-dicyclohexylcarbodiimide (DCC)/DMAP yielded
the compounds 5, 7, 8, 10, 13–17.
The synthesized compounds were evaluated at various concen-
trations to determine their potencies against the chymotrypsin-
like activity of the proteasome by using a previously described
method.10 Two known proteasome inhibitors Ac-Leu-Leu-Met-
CHO (LLM-F) and lactacystin were included in the assay as controls
(Table 1).
Inhibition of proteasome activities by lithocholic acid and its derivatives
21
O
22
18
24
R2
12
H
23
17
11
19
H
16
1
2
15
H
H
7
3
R1O
6
Lithocholic acid and its derivatives
Compound
R1
R2
IC50 (lM)
a
Lithocholic
acid
1
2
H
OH
18.1 3.3
H
H
OCH3
NAb
Results of the proteasome assay indicated that with the excep-
tion of compound 8, C-3 esterifications of LA in general increased
the potency of inhibition of chymotrypsin-like proteasome activi-
ties by 2- to 10-fold when compared to unmodified LA. In addition,
compounds with C-3 aromatic or cyclic acetic acid moiety, such as
OC2H5 NAb
O
HOOC
3
OH
OH
10.3 2.1
O
4
11.5 2.3
5.8 0.9
10, 13, and 14, were in general more potent (IC50 = 1.9–3.8 lM)
than compounds without any ring system on their C-3 side chains.
However, compounds 11 and 12 with a cyclic carboxylic acid moi-
ety at C-3 were not as potent as other compounds with a ring sys-
tem on their C-3 side chains. Compound 8, with an unbranched
heptanoic acid C-3 side chain, did not inhibit the chymotrypsin-
like proteasome activity.
LA esterified at the C-24 position with methyl or ethyl groups
resulted in a loss of activity in proteasome inhibition, as seen in
compounds 1 and 2, suggesting that the carboxylic acid moiety
in the C-24 position is critical for inhibition of the chymotrypsin-
like proteasome activities. The negative impact on proteasome
inhibition by modifying the C-24 carboxylic acid was also evi-
denced by comparing compound 13 and its methylester derivative
15, which lost inhibitory activity after esterification at the C-24.
To determine whether the lithocholic acid derivatives could
also inhibit the other two main proteasomal activities, the most
potent lithocholic acid derivative compound 13 was tested against
the caspase-like and the trypsin-like activities of the proteasome.
Compound 13 inhibited the caspase-like and the trypsin-like activ-
HOOC
CH3
O
H3C
5
OH
HOOC
CH3
O
6
7
8
9
OH
OH
OH
OH
10.8 1.7
3.5 0.6
NAb
HOOC
HOOC
O
O
HOOC
O
O
O
HOOC
HOOC
6.9 1.5
10
11
OH
OH
2.2 0.3
8.5 1.4
O
H
H
COOH
ities of the proteasome at 6.5 and 9.1 lM, respectively (Table 1).
O
Most proteasome inhibitors, such as the anti-cancer drug bort-
ezomib, are competitive inhibitors that compete with substrates
for the catalytic site binding. Since the lithocholic acid derivatives
are not analogs of peptide substrates, it is possible that these com-
pounds are not competitive inhibitors of the proteasome. To test
this possibility, compound 10 was used in enzyme kinetic studies
to determine its mode of action.
12
OH
8.6 1.3
COOH
1.9 0.3 (6.5)d
(9.1)e
O
13
14
OH
OH
HOOC
HOOC
O
3.8 0.5
The chymotrypsin-like activity of the proteasome was deter-
mined using various concentrations of the substrate Suc-
Leu-Leu-Val-Tyr-AMC in the presence or absence of 2 and 4 lM
compound 10 (Fig. 1). The effects of compound 10 on the reaction
rates of the chymotrypsin-like activity of the proteasome were
analyzed using the Lineweaver–Burk plot (Fig. 1). Compound 10
O
15
16
OCH3
OCH3
NAb
NAb
HOOC
HOOC
O
O
OC2H5 NAb
5.2 0.8
HOOC
17
at 2 and 4 lM reduced the maximum reaction rate (Vmax) of the
LLM-Fc
proteasomal activity without affecting the Michaelis–Menten con-
stant (Km) of the substrate, suggesting that compound 10 inhibited
the proteasome in a noncompetitive manner. This data suggested
that lithocholic acid derivatives are allosteric inhibitors of the
proteasome.
Lactacystinc
5.6 0.9
a
The inhibition of chymotrypsin-like (ChT-L) activities of the 20S proteasome
was determined in the presence of various concentrations of the compounds as
previously described.10 IC50 is the concentration that inhibits the proteasomal
activity by 50%. The value of IC50 is expressed as mean standard deviation from
three independent assays.
In order to determine whether the lithocholic acid derivatives
b
can inhibit the proteasome in living cells, HeLa UbG76V-GFP cells
No inhibition.
c
were treated with compound 10 at 7.5 l
M for 24 h.15 In the pres-
LLM-F and lactacystin are known proteasome inhibitors.
IC50 against caspase-like activity of the proteasome.
IC50 against trypsin-like activity of the proteasome.
d
ence of proteasome inhibitors, HeLa UbG76V-GFP cells accumulate
UbG76V-GFP which emits green fluorescence under fluorescent
e

1928
Z. Dang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1926–1928
O
O
R2
R2
H
H
i or ii
H
H
H
H
R1O
HO
H
R1 = Acyl groups
2 = OH, OMe, or OEt
H
R2 = OH, OMe, or OEt
R
Reaction conditions: (i) dicarboxylic acid anhydride, DMAP, pyridine, microwave
heating, 120 °C, 2 hr; (ii) dicarboxylic acid, DCC, DMAP, pyridine, microwave
heating, 120 - 160 °C, 10 min - 2 hr.
Scheme 1. Synthesis of lithocholic acid derivatives. Reagents and conditions.
at C-24 resulted in the loss of inhibitory activity, shown by
compounds 1, 2, 15, 16, and 17.
Control (No inhibitor)
Compound 10 (2 uM)
Compound 10 (4 uM)
Bortezomib and most of the known proteasome inhibitors are
competitive inhibitors that interact at the catalytic sites. To our
knowledge, drug-like small molecule noncompetitive proteasome
inhibitors are relatively rare. Our data indicate that compound 10
is a noncompetitive proteasome inhibitor, which suggests that
the compound interacts with an allosteric site to inhibit the prote-
asomal activity. With further structural optimizations, this class of
compounds might have potential therapeutic applications and
could become a useful tool in dissecting the function of the
proteasome.
-1/Km
1/Vmax
1/S (1/mM)
Figure 1. Compound 10 is a noncompetitive inhibitor of the proteasome. Km is the
concentration of substrate (S) when the reaction rate (V) equals to 1/2 of the
maximum reaction rate (Vmax). The mean fluorescence units (mFU) are proportional
to the amount of cleaved substrates. Each data point in the figure represents the
average of two independent experiments.
Acknowledgments
HeLa UbG76V-GFP cells were kindly provided by Dr. Nico
P. Dantuma, Karolinska Institutet, Stockholm, Sweden. This work
was supported by the National Institutes of Health grant
GM084337 to CHC.
Supplementary data
Supplementary data (the method of proteasome assay, general
experimental procedures for preparation of compounds (Table 1
and Scheme 1), 1H and 13C NMR data, and high resolution mass
spectra) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmcl.2011.02.041.
References and notes
Figure 2. Compound 10 inhibited the proteasome in HeLa UbG76V-GFP cells. The
1. Voges, D.; Zwickl, P.; Baumeister, W. Annu. Rev. Biochem. 1999, 68, 1015.
2. Goldberg, A. L.; Dice, J. F. Annu. Rev. Biochem. 1974, 43, 835.
3. Goldberg, A. L.; St John, A. C. Annu. Rev. Biochem. 1976, 45, 747.
4. Kane, R. C.; Bross, P. F.; Farrell, A. T.; Pazdur, R. Oncologist 2003, 8, 508.
5. Huang, L.; Chen, C.-H. Curr. Med. Chem. 2009, 16, 931.
cells were cultured in the presence (A) or absence (B) of compound 10 at 7.5 lM for
24 h. Emission of green fluorescence from UbG76V-GFP was documented with a
Nikon fluorescence microscope system.
6. Yang, H.; Chen, D.; Cui, Q. C.; Yuan, X.; Dou, Q. P. Cancer Res. 2006, 66, 4758.
7. Walcott, S. E.; Heikkila, J. J. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2010,
156, 285.
8. Yang, H.; Shi, G.; Dou, Q. P. Mol. Pharmacol. 2007, 71, 426.
9. Stan, S. D.; Hahm, E. R.; Warin, R.; Singh, S. V. Cancer Res. 2008, 68, 7661.
10. Huang, L.; Yu, D.; Ho, P.; Qian, K.; Lee, K.-H.; Chen, C.-H. Bioorg. Med. Chem.
2008, 16, 6696.
11. Gaczynska, M.; Osmulski, P. A. Methods Enzymol. 2005, 398, 425.
12. Sprangers, R.; Li, X.; Mao, X.; Rubinstein, J. L.; Schimmer, A. D.; Kay, L. E.
Biochemistry 2008, 47, 6727.
13. Li, X.; Wood, T. E.; Sprangers, R.; Jansen, G.; Franke, N. E.; Mao, X., et al J. Natl.
Cancer Inst. 2010, 102, 1069.
microscope.16 Treatment of the HeLa UbG76V-GFP cells with com-
pound 10 resulted in accumulation of UbG76V-GFP (Fig. 2, panel
B). This result suggests that the lithocholic acid derivatives are able
to get into the cells and inhibit the proteasome.
In summary, we have identified LA derivatives modified with a
variety of acidic acyl substituents at the C-3 position exhibiting a
range of inhibitory activity against the proteasome. Compound
13, the most potent inhibitor with C-3 phenyl diacetic acid hemi-
ester side chain, inhibited the chymotrypsin-like activity of the
14. Huang, L.; Ho, P.; Chen, C.-H. FEBS Lett. 2007, 581, 4955.
15. Cell based proteasome assay: HeLa UbG76V-GFP cells were grown in DMEM with
10% (v/v) heat-inactivated fetal bovine serum (Sigma) at 37 °C with humidified
proteasome with an IC50 of 1.9 lM. Based on the data shown in
Table 1, the C-3 cyclic acetic acid moiety is critical for potent
proteasome inhibition. Compounds 10, 13, and 14, which possess
a C-3 cyclic acetic acid moiety, were all more potent than the
known proteasome inhibitors, LLM-F and lactacystin. In addition,
a C-24 free carboxylic acid was found to be important for the
proteasomal inhibitory activity. Esterification of the carboxylic acid
5% CO2. HeLa UbG76V-GFP cells (10,000 cells each well) in 100
ll of growth
medium were cultured in the presence of proteasome inhibitors in 96-well
plates (Corning-Costar) for 24 h. Accumulation of UbG76V-GFP in the cells was
documented by using a Nikon fluorescence microscope system.
16. Dantuma, N. P.; Lindsten, K.; Glas, R.; Jellne, M.; Masucci, M. G. Nat. Biotechnol.
2000, 18, 538.