Synthesis and biological evaluation of heterocyclic ring-substituted maslinic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.bmcl.2009.10.017

A series of maslinic acid derivatives have been synthesized by introducing various fused heterocyclic rings at C-2 and C-3 positions. Their inhibitory effects on PTP1B, TCPTP and related PTPs are evaluated. Most of the compounds exhibited a dramatic increase in inhibitory potency and selectivity, the two most potent PTP1B inhibitors 20 (IC₅₀ = 0.61 μM) and 29 (IC₅₀ = 0.64 μM) showed about 10-fold more potent than lead compound maslinic acid. More importantly, 29 possesses the best selectivity of 6.9-fold for PTP1B over TCPTP.

Full text of DOI:10.1016/j.bmcl.2009.10.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6618–6622
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of heterocyclic ring-substituted
maslinic acid derivatives as novel inhibitors of protein tyrosine phosphatase 1B
a,
Wen-Wei Qiu a, Qiang Shen b, Fan Yang a, Bo Wang a, Hui Zou a, Jing-Ya Li b, Jia Li b, , Jie Tang
*
*
a Institute of Medicinal Chemistry, Department of Chemistry, East China Normal University, Shanghai 200062, China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Science, Chinese Academy of Science, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of maslinic acid derivatives have been synthesized by introducing various fused heterocyclic
rings at C-2 and C-3 positions. Their inhibitory effects on PTP1B, TCPTP and related PTPs are evaluated.
Most of the compounds exhibited a dramatic increase in inhibitory potency and selectivity, the two most
potent PTP1B inhibitors 20 (IC50 = 0.61 lM) and 29 (IC50 = 0.64 lM) showed about 10-fold more potent
than lead compound maslinic acid. More importantly, 29 possesses the best selectivity of 6.9-fold for
Received 18 August 2009
Revised 23 September 2009
Accepted 5 October 2009
Available online 8 October 2009
PTP1B over TCPTP.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Maslinic acid
Protein tyrosine phosphatase 1B
Inhibitor
SAR
Diabetes
The protein tyrosine phosphatase (PTP) superfamily comprises
more than 100 enzymes.1 These protein tyrosine phosphatases
(PTPs) are expressed in insulin-sensitive tissues and can function
as negative modulators in insulin signal transduction by dephos-
phorylation of tyrosyl residues.2–5 The aberrant of PTP activity con-
tributes to several human pathologies, such as diabetes, obesity,
cancer and immune disorders.6–9 Protein tyrosine phosphatase
1B (PTP1B), one of the PTPs, is a key member in the down-regula-
tion of the insulin and leptin signaling pathway by dephosphoryl-
ating the insulin receptor (IR),10 insulin receptor substrates (IRS)11
and Janus kinase 2 (JAK2),12,13 which is downstream of leptin
receptor. Hereby, inhibition of PTP1B results in antidiabetes by
increasing insulin sensitivity14 and resistance to obesity.15 In re-
cent years PTP1B inhibitors are recognized as potential therapeu-
tics for treatment of type-2 diabetes and obesity.16,17 However
two major drawbacks prevent most of these inhibitors from further
development to clinical trials. Firstly, lack of sufficient cell perme-
ability due to the presence of highly negative charged residues
mimicking the phosphate group in IRS.18 Secondly, because of their
high conserved catalytic domain, the selectivity for PTP1B over the
most homogeneous T-cell protein tyrosine phosphatase (TCPTP) is
low.19
decades are natural products or their derivatives.20,21 In searching
for new types of PTP1B agonists, researchers have found pentacy-
clic triterpenoids including oleanolic acid (OA) are moderate PTP1B
inhibitors. Based on these results, some potent inhibitors with
good cell permeability were obtained by modifying with long
hydrophobic chains at C-3 and C-28 positions. However, these syn-
thetic compounds shared poor water solubility and had no obvious
selectivity between PTP1B and TCPTP.22,23
Maslinic acid (MA), a natural pentacyclic triterpene acid, which
presents in many plant species, especially in olive. It has various
pharmacological activities, such as anti-tumor,24–28 antioxi-
dant,29,30 anti-HIV,29 antimicrobial31 and anti-diabetic activities
by moderate to low inhibition to glycogen phosphorylase
(IC50 = 28 l
M).32,33 Recently we first screened the inhibitory effects
of MA (1), its isomers 3-epi-maslinic acid (2) and augustic acid
(3)32,34 on PTP1B. Among them MA exhibited good PTP1B inhibi-
tion (IC50 = 5.93 l
M). Considering the effect of hypoglycemic33
we postulate that the anti-diabetic pharmacological effect in KK-
Ay mice not only due to the glycogen phosphorylase inhibition
but also maybe mainly or partly caused by PTP1B inhibition. The
inhibitory activity data of TCPTP revealed MA has 3.3-fold selectiv-
ity for PTP1B over TCPTP (Table 1), therefore we speculate that
modification of C-2 and C-3 positions simultaneously may increase
the selectivity and MA is selected as a promising lead compound
for developing potent and high selective PTP1B inhibitors as ther-
apeutic agents for type-2 diabetes and obesity. It is well-known
that pentacyclic triterpenes including OA are too hydrophobic to
have reasonable water solubility and related pharmacokinetic
Natural products play a major role in drug discovery and nearly
half of the new drugs introduced into the market in the last two
* Corresponding authors. Tel.: +86 21 50801313x132; fax: +86 21 50801552
(J.L.); tel.: +86 21 62232764; fax: +86 21 62232100 (J.T.).
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), jtang@chem.ecnu.edu.cn (J. Tang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.017

W.-W. Qiu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6618–6622
6619
Table 1
of 12% after separation with column, which has a methylated
pyrimidine moiety. To avoid the undesired methylation, n-octyl-
amine was added. Indeed, compound 16 was successfully synthe-
sized in the presence of this MeI scavenger. Compounds 18 and
19 were obtained in a similar process as 16, the only difference
is the demethylation of C-28 methyl ester was smoothly carried
out in presence of LiI in anhydrous DMF without n-octylamine as
shown in Scheme 2.
Compound 20 was prepared by Fischer indole synthesis of 12
with phenylhydrazine.40 Bromination of 12 with pyridinium tri-
bromide in HOAc and DCM gave 21, which then condensed with
thiourea in refluxing EtOH to afford thiazole derivative 22.41,42 12
was formylated with HCOOEt in presence of MeONa to produce
23, which reacted with hydroxylamine hydrochloride in EtOH to
give isoxazole derivative 24.43 Pyrazole derivatives 25 and 26 were
synthesized by treatment of 23 with phenylhydrazine hydrochlo-
ride and hydrazine hydrochloride, respectively.44 Compound 27–
3045 were prepared by reacting 26 with acetyl chloride, hexanoyl
chloride, nicotinoyl chloride and isonicotinoyl chloride, respec-
tively, as shown in Scheme 3.
Inhibitory activity of maslinic acid derivatives against PTP1B and TCPTP
Compounds
IC50
(
l
M)
TCPTP/PTP1Bb
PTP1B
TCPTP
1
2
3
5.93 0.08
14.33 0.65
4.93 0.17
1.43 0.23
1.79 0.14
5.44 1.11
1.78 0.10
2.73 0.23
2.61 0.46
0.61 0.04
1.92 0.12
2.60 0.62
1.39 0.19
1.65 0.23
1.48 0.08
1.75 0.10
0.64 0.03
0.81 0.08
3.89 0.08
19.47 1.33
nda
nd
5.88 0.52
8.31 1.30
nd
5.51 0.77
8.19 0.31
6.50 0.65
1.60 0.21
6.44 0.55
8.44 0.54
3.80 0.04
5.99 0.35
4.93 0.15
5.56 0.55
4.39 0.31
3.62 0.12
6.24 0.19
3.3
9
4.1
4.6
11
15
16
18
19
20
22
24
25
26
27
28
29
30
31c
3.1
3.0
2.5
2.6
3.4
3.3
2.7
3.6
3.3
3.2
6.9
4.5
1.6
Results and discussion: Initial SAR analysis for modified OA ana-
logs with various side chains on C-3 and C-28 positions indicated a
strong preference for the hydrophobic group and these derivatives
have no obvious selectivity for PTP1B over TCPTP.23 So in this paper
we focus on improving water solubility, increasing inhibitory activ-
ity and selectivity between the two homogenous enzymes by intro-
ducing a series of heterocyclic rings at C-2 and C-3 positions. All the
synthetic maslinic acid derivatives were evaluated in the enzyme
inhibitionassay againstPTP1B by the methodof p-nitrophenyl phos-
phate using compound 31 as reference compound.46 Homogeneous
TCPTP inhibitory activities were investigated simultaneously by the
same method for further selectivity studying (Table 1). We also
tested the inhibitory activity of some synthetic derivatives on sev-
eral other critical PTPs, which negatively regulate insulin dephos-
phorylation, such as leukocyte antigen-related phosphatase (LAR),
src homology phosphatase-1 (SHP-1) and src homology phospha-
tase-2 (SHP-2) (Table 2). The assay results indicated that most of
the synthetic compounds exhibited more potent inhibition of PTP1B
than that of lead compound MA. All these compounds showed 2.5–
6.9-fold selectivity for PTP1B over TCPTP.
a
b
c
nd, not determined.
TCPTP/PTP1B, the ratio of IC50 of TCPTP and PTP1B.
Positive control.
properties. In this paper, a series of heterocyclic ring-substituted
maslinic acid derivatives at C-2 and C-3 have been synthesized
and biologically evaluated in order to find more potent and high
selective PTP1B inhibitors with improved water solubility.
COOH
COOH
HO
HO
HO
HO
(1)
maslinic acid
3-epi-maslinic acid
(2)
We tested the IC50 of MA (1) and its isomers 3-epi-maslinic acid
(2) and augustic acid (3) on PTP1B. The results indicated that the
stereo configuration of C-3 b-hydroxyl is important because the
COOH
C-3 epimer (2) with a-hydroxyl group has dramatically decreased
HO
HO
activity. However, the stereo configuration of C-2 has no significant
impact on PTP1B inhibition no matter the compound bears either
a-hydroxyl or b-hydroxyl group. For comparison, MA has 3.3-fold
(3)
augustic acid
selectivity for PTP1B over TCPTP. According to previous study,
modifying of C-3 and C-28 of triterpenoids couldn’t increase the
selectivity between the two most homogeneous PTPs.23 We postu-
late that modification on both C-2 and C-3 positions maybe helpful
to increase the selectivity. In this Letter, SAR analysis of A-ring
fused heterocyclic derivatives showed that six-member heterocy-
clic pyrazine-substituted and pyrimidine-substituted maslinic acid
derivatives could significantly increase PTP1B inhibitory potency
(9, 11, 16, 18 and 19) with the exception of N-methyl pyrimidinyl
derivative 15. Most of the five-member heterocyclics have higher
inhibitory potency on PTP1B than six-member heterocyclics do,
with the exception of thiazole-substituted derivative 22 and isox-
azole-substituted derivative 24. Among these five-member hetero-
A series of maslinic acid derivatives with heterocyclic rings
(pyrazine, pyrimidine, indole, thiazole, isoxazole and pyrazole) at
C-2 and C-3 positions were synthesized (Scheme 1–3). Benzylation
of OA followed by PCC oxidation35 afforded ketone 5. Stereoselec-
tive hydroxylation of 5 with mCPBA gave 2a-hydroxyl ketone 6.
Deprotection of 6 afforded 7, which was then treated with KOH
in MeOH to give diketone 8.32 As a common intermediate, 8 re-
acted with phenylenediamine to give quinoxaline derivative 9
and reacted with ethylene diamine to give 10. 10 underwent dehy-
dro-aromatization with MnO2 to produce pyrazine derivative 11 as
shown in Scheme 1.36,37
OA was oxidized with PCC to afford ketone 12, which reacted
with N,N-Dimethylformamide dimethyl acetal in presence of TEA
to afford enamine derivative 13.38 Condensation of 13 with guani-
dine hydrochloride in the presence of NaOEt produced 14. Demeth-
ylation of C-28 methyl ester with LiI in refluxing anhydrous DMF39
produced a complex, and compound 15 was obtained with a yield
cyclic compounds, 20 (IC50 = 0.61 lM) and 29 (IC50 = 0.64 lM) are
the best two PTP1B inhibitors, about 10-fold more potent than lead
compound MA. In addition, 29 is the most selective inhibitor (6.9-
fold) for PTP1B over TCPTP. Therefore, modifying the five-member
heterocyclics may provide the possibility to further improve inhib-
itory activity and selectivity.

6620
W.-W. Qiu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6618–6622
COOH
COOBn
COOBn
i
iii
ii
HO
HO
O
OA
4
5
COOBn
COOH
COOH
iv
HO
O
HO
v
O
O
O
6
8
7
vii
vi
COOH
COOH
N
N
viii
COOH
N
N
N
N
11
10
9
Scheme 1. Reagents and conditions: (i) BnCl, K2CO3, DMF, 100 °C, 3 h, (92%); (ii) PCC, DCM, rt, 12 h, (90%); (iii) mCPBA, concd H2SO4 (cat), MeOH-DCM, 10 °C, 20 h, (80%); (iv)
H2, Pd/C, MeOH, rt, 5 h, (98%); (v) NaOH, MeOH, rt, 12 h, (97%); (vi) phenylenediamine, EtOH, reflux, 3 h, (56%); (vii) ethylene diamine, EtOH, rt, 3 h, (95%); (viii) MnO2, KOH,
DMF, 120 °C, 6 h, (28%).
COOMe
COOMe
COOH
COOH
v
iii
N
N
N
N
H2N
N
O
N
H
N
N
13
14
15
16
vi
ii
iv
COOH
COOMe
COOH
H2N
N
O
R
N
12
17
v
i
ΟΑ
R = H
18
19
N
R = CH3
R
N
Scheme 2. Reagents and conditions: (i) PCC, DCM, rt, 12 h, (85%); (ii) N,N-dimethylformamide dimethyl acetal, TEA, toluene, reflux, 12 h, (78%); (iii) guanidine hydrochloride,
NaOEt, EtOH, reflux, 3 h, 70%; (iv) methanimidamide acetic acid or ethanimidamide hydrochloride, NaOEt, EtOH, reflux, 3 h, R = H, (78%); R = Me, (74%); (v) LiI, anhydrous
DMF, reflux, 48 h, 15 (12%); 18, R = H, (74%); 19, R = Me, (80%); (vi) LiI, n-octylamine, anhydrous DMF, reflux, (78%).
Someofsynthetic heterocyclic compounds werealsoevaluatedon
homogenous enzymes LAR, SHP-1 and SHP-2. No indication of inhibi-
tion on these PTPs was found. It has been reported that oleanolic acid
anditsderivatives(withthemodificationonC-28andC-3positionsof
OA) only have 2–10-folds selectivity for PTP1B over SHP-1.23 Com-
pared to these reported compounds, the heterocyclic ring-substi-
tuted maslinic acid derivatives developed by our group clearly have
better selectivity between the two homogenous enzymes.
In summary, we reported a series of novel maslinic acid analogs
with various fused heterocyclic rings on C-2 and C-3 positions.
These compounds have improved inhibitory activity on PTP1B
and especially selectivity for PTP1B over TCPTP. This Letter pro-
vides some important information for discovering specific potent
inhibitors of PTP1B by modifying naturally occurring triterpenoids.
A broader SAR research on A-ring fused heterocyclic analogues of
PTP1B inhibitors and the further evaluation in cell models of

W.-W. Qiu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6618–6622
6621
COOH
COO
H
COOH
Br
i
ii
N
H
O
O
21
iii
12
iv
20
COO
H
COOH
COOH
S
N
N
v
O
H2N
O
24
25
O
22
23
vi
vii
COO
H
N
N
COOH
viii
N
N
H
COOH
26
N
O
N
R
27
28
29
R =
R =
R =
CH3
O
O
CH3
COOH
O
O
O
N
HOOC
O
30
R =
31
N
O
Scheme 3. Reagents and conditions: (i) C6H5NHNH2, TFA, HOAc, 100 °C, 20 h, (55%); (ii) pyridinium tribromide, HOAc, DCM, 0 °C to rt, 1 h, (82%); (iii) thiourea, anhydrous
EtOH, reflux, 12 h, (63%); (iv) HCOOEt, MeONa, benzene, rt, 5 h, (94%); (v) hydroxylamine hydrochloride, EtOH/H2O, reflux, 1 h, (85%); (vi) phenylhydrazine hydrochloride,
EtOH/H2O, reflux, 1 h, (65%); (vii) hydrazine hydrochloride, EtOH/H2O, reflux, 1 h, (80%); (viii) RCOCl, pyridine, anhydrous DMF, rt, 3 h, for 27 (82%), 28 (85%), 29 (77%), 30
(80%).
Supplementary data
Table 2
Inhibitory activity of selected maslinic acid derivatives against related PTPs
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.017.
Compounds
IC50 (lM)
LAR
SHP-1
SHP-2
1
9
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
References and notes
11
18
22
24
25
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s), 0.92 (3H, s), 0.86 (3H, s), 0.84 (3H, s); 13C NMR (CDCl3, 125 MHz): d 183.2,
164.1, 164.0, 152.3, 152.1, 143.8, 139.4, 128.5, 126.5, 122.9, 122.2, 119.7, 53.1,
46.5, 46.0, 45.9, 41.8, 41.1, 39.3, 38.1, 36.1, 34.5, 33.9, 33.1, 32.4, 32.0, 31.4,
30.7, 27.7, 25.7, 24.7, 23.6, 23.3, 23.0, 19.4, 16.7, 15.2; ESI-HRMS (m/z): [M+H]+
calcd for C37H50N3O3, 584.3852; found 584.3843.
32. Wen, X.; Zhang, P.; Liu, J.; Zhang, L.; Wu, X.; Ni, P.; Sun, H. Bioorg. Med. Chem.
Lett. 2006, 16, 722.
46. Chen, Y. T.; Seto, C. T. J. Med. Chem. 2002, 45, 3946.