Pentacyclic triterpenes. Part 2: Synthesis and biological evaluation of maslinic acid derivatives as glycogen phosphorylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.10.014

The synthesis of a series of maslinic acid derivatives is described and their effect on rabbit muscle glycogen phosphorylase a evaluated. Within this series of compounds, 15 (IC₅₀ = 7 μM) is the most potent GPa inhibitor. SAR of the maslinic acid derivatives are discussed.

Full text of DOI:10.1016/j.bmcl.2005.10.014

Bioorganic & Medicinal Chemistry Letters 16 (2006) 722–726
Pentacyclic triterpenes. Part 2: Synthesis and biological
evaluation of maslinic acid derivatives as glycogen
phosphorylase inhibitors
Xiaoan Wen,a Pu Zhang,a Jun Liu,b Luyong Zhang,b Xiaoming Wu,a
Peizhou Nia and Hongbin Suna,*
aDepartment of Medicinal Chemistry, College of Pharmacy, China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009, China
bJiangsu Center for Drug Screening, China Pharmaceutical University, 1 Shennonglu, Nanjing 210038, China
Received 22 August 2005; revised 26 September 2005; accepted 6 October 2005
Available online 21 October 2005
Abstract—The synthesis of a series of maslinic acid derivatives is described and their effect on rabbit muscle glycogen phosphorylase
a evaluated. Within this series of compounds, 15 (IC50 = 7 lM) is the most potent GPa inhibitor. SAR of the maslinic acid deriv-
atives are discussed.
Ó 2005 Elsevier Ltd. All rights reserved.
Energy metabolism, which mainly involves fat metab-
olism, glucose metabolism, and protein metabolism, is
one of the basic characteristics of biological processes.
Multiple abnormalities in energy metabolism, especial-
ly abnormalities in glucose metabolism, result in a
variety of severe diseases. As part of our project
aimed at pharmacological interference with glucose
metabolism, we have interest in developing glycogen
phosphorylase inhibitors as therapeutic agents. Glyco-
gen phosphorylase (GP) is the enzyme responsible for
glycogen breakdown to produce glucose and related
metabolites for energy supply.1 Due to its key role
of GP in modulation of glycogen metabolism, phar-
macological inhibition of GP has been regarded as
an effective therapeutic approach for treating diseases
caused by abnormalities in glycogen metabolism, such
as type 2 diabetes,2 myocardial ischemia,3 and tu-
mors.4 Several structural classes of GP inhibitors have
been reported, whose binding sites identified in GP
include the catalytic site, the purine inhibitor site (also
known as I-site), the allosteric site, the glycogen stor-
age site, a novel allosteric inhibitor site, and the newly
discovered benzimidazole-binding site.5
Maslinic acid (1), which is an abundant constituent of
olive fruit, has recently drawn much interest due to its
anti-tumor,6 anti-HIV,7 antioxidation,8 and antiobesity
activities.9 More recently, we have first reported that 1
and related triterpenes represent a new class of GP
inhibitors.10 This discovery afforded pentacyclic triter-
penes as novel lead compounds in searching for potent
and low-toxic GP inhibitors as therapeutic agents for
type 2 diabetes and other diseases. In our previous
studies, 1 exhibited moderate inhibition against both
rabbit muscle GP and rat liver GP.10 Moreover, 1
effectively inhibited the increase of fasted plasma glu-
cose of diabetic mice induced by adrenaline.10 In an ef-
fort to clarify SAR of the triterpene class of GP
inhibitors, the present study has mainly focused on
structural modifications at C-28, C-2, and C-3 posi-
tions of 1. Herein, we describe the synthesis, biological
evaluation, and SAR of maslinic acid derivatives as a
new class of GP inhibitors.
COOH
Keywords: Glycogen phosphorylase; Inhibitor; Maslinic acid; Deriva-
tives; Synthesis; SAR.
HO
*
Corresponding author. Tel.: +86 25 85327950; fax: +86 25
83271335; e-mail: hbsun2000@yahoo.com
HO
1
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.014

X. Wen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 722–726
723
The syntheses of maslinic acid derivatives are summa-
rized in Schemes 1–4. Maslinic acid (1) was synthesized
based on a hydroboration-oxidation reaction as de-
scribed previously.10 On the other hand, Takayama
and co-workers reported the preparation of corosolic
acid via ketone-hydroxylation strategy,11 and based on
this methodology, we employed modified procedures
to carry out the preparation of 1 and related compounds
(Scheme 1). It was found that protection of the carboxyl
groups as benzyl esters was very convenient and neces-
sary, since process manipulation on the triterpene acids
was very tedious and low-yielding. Thus, esterification
of 2 with benzyl chloride followed by an oxidation reac-
tion with PCC afforded ketone 4 in high yields.10 Stereo-
selective hydroxylation of 4 with mCPBA catalyzed by
H2SO4 in MeOH-CH2Cl2 at 0 °C gave 2a-hydroxyl ke-
tone 5 in 80% yield. Reduction of 5 with NaBH4 in
THF at 0 °C gave 2a,3b-diol 6 (77%) as the major prod-
uct, together with 2a,3a-diol 7 (18%) as a minor prod-
uct. The relative stereochemistry at C-2 and C-3 of 6
and 7 was determined by 1H NMR coupling data
(J = 9.5 Hz in response to trans-coupling between
H-3a and H-2b in 6; J = 2.6 Hz in response to cis-cou-
pling between H-3b and H-2b in 7). Hydrogenolysis of
6 or 7 over palladium/carbon in THF furnished maslinic
acid (1) or 3-epi-maslinic acid (8) in quantitative yield,
respectively. It should be mentioned that 5 was unstable
at basic conditions, for example, treatment of 5 with
KOH in MeOH-DMF at room temperature gave
a,b-unsaturated ketone 9 in almost quantitative yield.
COOR1
COOH
HO
HO
HO
HO
i
1
10 R1 = -CH3
CH2
11 R1
=
N
N
12 R1 = -CH2COOC2H5
ii
13 R1 = -CH2COOH
Scheme 2. Reagents: (i) R1X, K2CO3, DMF; (ii) THF-4 N NaOH.
(R1X = CH3I, N-chloromethylpyrazole or ethyl bromoacetate,
respectively).
hydrolysis of 12 with 4 N NaOH aqueous solution in
THF without effecting the ester bond of C-28.
Reaction of 1 with 1,2-dibromoethane or 1,4-dibromo-
butane in the presence of potassium carbonate in
DMF at room temperature gave compounds 14 and
15 in high yields, respectively (Scheme 3). Treatment
of 14 in refluxing aqueous ethanol in the presence of
Et3N afforded16 in 78% yield. Nitrogen-containing
derivatives 17–22 were synthesized through reactions
of 14 or 15 with corresponding amines. For example, a
mixture of 14, diethylamine, and potassium carbonate
in DMF was stirred overnight at room temperature to
give 17 in 82% yield. In the same way, compounds
18–22 were prepared in moderate to good yields.13
Treatment of 1 with iodomethane in the presence of
potassium carbonate in DMF at room temperature
afforded methyl ester 10 in 95% (Scheme 2). In a similar
fashion, maslinic acid esters 11 and 12 were obtained in
good yields upon treating 1 with N-chloromethylpyraz-
ole12 and ethyl bromoacetate, respectively. Ester 12
was further converted to 13 in quantitative yield by
We examined how steric hindrance at C-2 affected
stereoselectivity of the reduction of the C-3 carbony
group by adding bulky substituents on 2a-hydroxyl
group of 5. In this regard, acetate 23 and silyl ether 24
H
H
H
iii
ii
COOBn
i
COOBn
COOH
O
HO
4
HO
3
2
H
H
H
COOBn
COOBn
iv
HO
HO
COOBn
HO
HO
HO
O
(minor)
v
(major)
7
6
5
v
vi
H
H
H
COOH
COOBn
COOH
HO
HO
HO
HO
HO
O
8
1
9
Scheme 1. Reagents and conditions: (i) K2CO3, BnCl, DMF, 50 °C; (ii) PCC, CH2Cl2; (iii) mCPBA, concd H2SO (cat), MeOH-CH2Cl2, 0 °C;
4
(iv) NaBH4, THF, 0 °C; (v) H2, Pd/C, THF, rt; (vi) KOH, MeOH-DMF.

724
X. Wen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 722–726
COO(CH2)nBr
HO
HO
COOH
i
HO
HO
14 n=2
15 n=4
1
16 n=2 R2= -OH
17 n=2 R2= -N(C2H5)2
COO(CH2)nR2
HO
HO
18 n=2 R2=
ii
N
19 n=2 R2=
N
O
20 n=4 R2= -N(C2H5)2
21 n=4 R2=
22 n=4 R2=
N
N
O
Scheme 3. Reagents and conditions: (i) BrCH2CH2Br or BrCH2(CH2)2CH2Br, K2CO3, DMF, rt; (ii) for 16: Et3N, aqueous ethanol, reflux; for 17–22:
amine, K2CO3, DMF, rt.
H
H
COOBn
COOBn
R3O
O
i
HO
O
23 R3=Ac
5
24 R3=TBDMS
COOBn
COOBn
R3O
HO
R3O
HO
ii
27 R3=Ac
25 R3=Ac
26 R3=TBDMS
28 R3=TBDMS
Scheme 4. Reagents and conditions: (i) for 23: Ac2O, pyridine, rt; for 24: TBDMSCl, imidazole, DMF, rt; (ii) NaBH4, THF, 0 °C.
were prepared in high yields using the regular methods,
respectively (Scheme 4). Our preliminary results showed
that reduction of acetate 23 with NaBH4 resulted in a
similar stereoselectivity compared with reduction of 5,
furnishing 2a,3b-isomer 25 (75%) as the major product,
together with 2a,3a-isomer 27 (14%) as a minor product.
Even with the very bulky silyl ether 24, the stereoselec-
tivity of reduction was not significantly distinguished
compared with that of reduction of 5, and as a result,
2a,3b-isomer26 (74%) was obtained as the major prod-
uct, together with 2a,3a-isomer28 (19%).
Our initial effort for lead optimization was focused on
improving water solubility of the triterpenoids since it
is well known that pentacyclic triterpenoids including
1 have very poor water solubility. Unfortunately, the
incorporation of hydrophilic groups at C-28 side chain
resulted in a significant decrease of potency (e.g., 16,
17, and 21). SAR analysis for C-28 side chains indicated
a strong preference for the hydrophobic groups (12, 14,
and 15) over hydrophilic groups (13, 16, and 20). One
hypothesis for this phenomenon is that the triterpenoids
might bind to the inhibitor site (I-site) of GP, which is a
hydrophobic binding pocket.5 As an effort to identify
the possible binding sites in GP for these triterpenoids,
we performed molecular modeling studies, and the
docking results showed a high probability of interaction
between these triterpenoids and the inhibitor site of
GP.15 Furthermore, our previous studies showed that
GP inhibition by 1 was synergistic with glucose,10b
which was supportive of the above hypothesis since
the I-site inhibitors were reported to be more potent in
the presence of high glucose concentrations.16 Certainly,
in order to unanimously determine the GP-binding site
for these triterpene inhibitors, an X-ray crystallographic
study is needed.
The synthesized maslinic acid derivatives were evaluated
in the enzyme inhibition assay against rabbit muscle
glycogen phosphorylase a which shared considerable
sequence similarity with human liver GPa and was com-
mercially available. As described previously,14 the activ-
ity of rabbit muscle GPa was measured through
detecting the release of phosphate from glucose-1-phos-
phate in the direction of glycogen synthesis. The assay
results showed that most of the newly synthesized com-
pounds exhibited inhibitory activity against rabbit mus-
cle GPa with IC50 values in the range of 7–1707 lM
(Table 1).

X. Wen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 722–726
Table 1. Rabbit muscle GPa inhibition assay results for maslinic acid
derivatives
725
References and notes
a
1. Kurukulasuriya, R.; Link, J. T.; Madar, D. J.; Pei, Z.;
Richards, S. J.; Rohde, J. J.; Souers, A. J.; Szczepankie-
wicz, B. G. Curr. Med. Chem. 2003, 10, 123.
Compound
RMGPa IC50 (lM)
1
5
28
29
ndb
2. (a) Oikonomakos, N. G. Curr. Protein Pept. Sci. 2002, 3,
´
6
561; (b) Somsak, L.; Nagy, V.; Hadady, Z.; Docsa, T.;
7
66
Gergely, P. Curr. Pharm. Des. 2003, 9, 1177.
3. Tracey, W. R.; Treadway, J. L.; Magee, W. P.; Sutt, J. C.;
McPherson, R. K.; Levy, C. B.; Wilder, D. E.; Yu, L. J.;
Chen, Y.; Shanker, R. M.; Mutchler, A. K.; Smith, David
M.; Flynn, A. H.; Knight, D. R. Am. J. Physiol. Heart
Circ. Physiol. 2004, 286, H1177.
4. (a) Schnier, J. B.; Nishi, K.; Monks, A.; Gorin, F. A.;
Bradbury, E. M. Biochem. Biophys. Res. Commun. 2003,
309, 126; (b) Geschwind, J.-F.; Georgiades, C. S.; Ko, Y.
H.; Pedersen, P. L. Expert Rev. Anticancer Ther. 2004, 3,
449; (c) Laszlo, G. B.; Vay Liang, W. G.; Wai-Nang, P. L.
Pancreas 2003, 4, 368.
5. (a) Lu, Z. J.; Bohn, J.; Bergeron, R.; Deng, Q. L.;
Ellsworth, K. P.; Geissler, W. M.; Harris, G.; McCann,
P. E.; Mckeever, B.; Myers, R. W.; Saperstein, R.;
Willoughby, C. A.; Yao, J.; Chapman, K. Bioorg. Med.
Chem. Lett. 2003, 13, 4125; (b) Wright, S. W.; Rath, V.
L.; Genereux, P. E.; Hageman, D. L.; Levy, C. B.;
McClure, L. D.; McCoid, S. C.; McPherson, R. K.;
Schelhorn, T. M.; Wilder, D. E.; Zavadoski, W. J.;
Gibbs, E. M.; Treadway, J. L. Bioorg. Med. Chem.
Lett. 2005, 15, 459; (c) Treadway, J. L.; Mendys, P.;
Hoover, D. J. Expert Opin. Invest. Drugs 2001, 10, 439,
and references therein; (d) Chrysina, E. D.; Kosmopo-
lou, M. N.; Tiraidis, C.; Kardarakis, R.; Bischler, N.;
Leonidas, D. D.; Hadady, Z.; Somsak, L.; Docsa, T.;
Gergely, P.; Oikonomakos, N. G. Protein Sci. 2005, 14,
873.
8
144
30
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Caffeine
393
66
19
1651
50
7
153
51
31
43
62
580
121
29
ndb
80
1707
63
580
114
a Values are means of three experiments.
b nd, not determined.
The effect of C-28 side-chain size could be clarified by a
comparison of 14 and 15. Compound 15 (IC50 = 7 lM)
is much more potent than 14 (IC50 = 50 lM). The only
structural difference between 14 and 15 is the length of
carbon side chain linked to the C-28 carboxyl, indicating
that the size of C-28 hydrophobic side chains affects the
enzyme inhibitory activity.
6. (a) Taniguchi, S.; Imayoshi, Y.; Kobayashi, E.; Takama-
tsu, Y.; Ito, H.; Hatano, T.; Sakagami, H.; Tokuda, H.;
Nishino, H.; Sugita, D.; Shimura, S.; Yoshida, T. Phyto-
chemistry 2002, 59, 315; (b) Noriyasu, K.; Shinohara, G.
Toshiyuki, I.U.S. Patent Application: US 2003153538,
2002.
7. Xu, H. X.; Zeng, F. Q.; Wan, M.; Sim, K. Y. J. Nat. Prod.
1996, 59, 643.
8. Montilla, M. P.; Agil, A.; Navarro, M. C.; Jimenez, M. I.;
Garcia-Granados, A.; Parra, A.; Cabo, M. M. Planta
Med. 2003, 69, 472.
9. Shinohara, H.; Shinohara, G.; Noriyasu, K. PCT Patent:
WO 03011267, 2003.
Data analysis indicated no clear SAR for compounds
based on structural modifications at C-2 and C-3 posi-
tions of 1. In addition, no specific stereochemistry pre-
ferred by the enzyme could be inferred.
10. (a) Sun, H. B; Wen, X. A.; Liu, J.; Zhang, L. Y.; Wang, S.
Z.; Ni, P. Z. Chinese Patent Application: CN
200510038094.2, 2005; (b) Wen, X. A.; Sun, H. B.; Liu,
J.; Wu, G. Z.; Zhang, L. Y.; Wu, X. M.; Ni, P. Z. Bioorg.
Chem. Med. Lett. 2005, 15, 4944.
11. Takayama H.; Kitajima, M.; Ishizuka, T.; Seo S. U.S.
Patent Application: US2005020681 A1, 2005.
12. (a) Dvoretzky, I.; Richtern, G. H. J. Org. Chem. 1950, 15,
1285; (b) Machin, P. J.; Hurst, D. N.; Bradshaw, R. M.;
Blaber, L. C.; Burden, D. T.; Melaranget, R. A. J. Med.
Chem. 1984, 27, 503.
In summary, a series of maslinic acid derivatives have
been synthesized and evaluated as novel GP inhibi-
tors. Within this series of compounds, 15 was the
most potent GPa inhibitor (IC50 = 7 lM). A clear
preference for hydrophobic groups at C-28 is evident
for enzyme inhibition, which raises a challenge for
lead optimization based on 1 and related triterpenoids
since good water solubility is usually preferred for
drug design. A broader SAR study on triterpene class
of GP inhibitors is ongoing in our laboratory and will
be reported in due time.
13. Analytical data for 15: IR (KBr, cmÀ1) 3572, 3427, 2947,
1726, 1464, 1383, 1258, 1159, 1051, 1036; 1H NMR (CDCl3,
300 MHz): d 0.73, 0.83, 0.90, 0.93, 0.98, 1.03, 1.13 (each, 3H,
s), 2.85 (1H, dd, J = 4.2, 13.7 Hz, H-18), 2.99 (1H, d,
J = 9.5 Hz, H-3a), 3.43 (2H, t, J = 6.7 Hz, -CH2Br), 3.70
(1H, m, H-2b), 4.05 (2H, t, J = 6.2 Hz, –COOCH2–), 5.29
(1H, t, J = 3.5 Hz, H-12); EIMS: 629 [M+Na]+. Analytical
data for 20: IR (KBr, cmÀ1) 3570, 3429, 2945, 1724, 1464,
1383, 1259, 1161, 1051; 1H NMR (CDCl3, 300 MHz): d
0.74, 0.83, 0.90, 0.92, 0.98, 1.03, 1.13 (each, 3H, s), 1.04 (6H,
Acknowledgments
This work was supported by Jiangsu Natural Science
Foundation (Grant BK2005101) and Jiangsu Outstand-
ing Researcher Grant.

726
X. Wen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 722–726
t, J = 7.3 Hz, –N(CH2CH3)2), 2.48 (2H,
–CH2N(CH2CH3)2), 2.55 (4H, q,
–N(CH2CH3)2), 2.89 (1H, dd, J = 4.0, 14.2 Hz, H-18),
2.98 (1H, d, J = 9.4 Hz, H-3a), 3.69 (1H, m, H-2b), 4.02
(2H, m, –COOCH2–), 5.28 (1H, br s, J = 3.5 Hz, H-12);
EIMS: 600 [M+H]+.
t J = 6.9,
J = 7.1 Hz,
14. Engers, H. D.; Shechosky, S.; Madsen, N. B. Can. J.
Biochem. 1970, 48, 746.
15. Manuscript in preparation.
16. (a) Kasvinsky, P. J.; Shechosky, S.; Fletterick, R. J.
J. Biol. Chem. 1978, 253, 9102; (b) Ercan-Fang, N.;
Nuttall, F. Q. J. Pharmcol. Exp. Ther. 1997, 280, 1312.