Design, synthesis and in vitro activity of phidianidine B derivatives as novel PTP1B inhibitors with specific selectivity

Article abstract of DOI:10.1016/j.bmcl.2015.12.097

A series of phidianidine B derivatives were synthesized by introducing various heterocyclic rings. Their inhibitory effects on PTP1B and other PTPs (TCPTP, SHP1, SHP2 and LAR) were evaluated. A majority of them displayed significant inhibitory potency and specific selectivity over PTP1B. The SAR and molecular docking analysis were also described.

Full text of DOI:10.1016/j.bmcl.2015.12.097

Accepted Manuscript
Design, synthesis and in vitro activity of phidianidine B derivatives as novel
PTP1B inhibitors with specific selectivity
Li Zhang, Cheng-Shi Jiang, Li-Xin Gao, Jing-Xu Gong, Zhong-Hua Wang, Jing-
Ya Li, Jia Li, Xu-Wen Li, Yue-Wei Guo
PII:
S0960-894X(15)30410-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.097
BMCL 23458
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 September 2015
23 December 2015
29 December 2015
Please cite this article as: Zhang, L., Jiang, C-S., Gao, L-X., Gong, J-X., Wang, Z-H., Li, J-Y., Li, J., Li, X-W., Guo,
Y-W., Design, synthesis and in vitro activity of phidianidine B derivatives as novel PTP1B inhibitors with specific
selectivity, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.097
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Design, synthesis and in vitro activity of phidianidine B derivatives as
novel PTP1B inhibitors with specific selectivity
Li Zhang a, ‡, Cheng-Shi Jiang a, b, ‡, Li-Xin Gaoa, Jing-Xu Gong a, Zhong-Hua Wang c, Jing-Ya Li
a, Jia Li a, Xu-Wen Li a, *, Yue-Wei Guoa, *
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR
China
b
School of Biological Science and Technology, University of Jinan, 336, Nan Xin Zhuang Xi
Road, Jinan, Shandong 250022, PR China
c
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Hai
Quan Road, Fengxian Dictrict, Shanghai, 201418, PR China
Abstract: A series of phidianidine B derivatives were synthesized by introducing
various heterocyclic rings. Their inhibitory effects on PTP1B and other PTPs (TCPTP,
SHP1, SHP2 and LAR) are evaluated. A majority of them displayed significant
inhibitory potency and specific selectivity over PTP1B. The SAR and molecular
docking analysis are also described.
Key words: Phidianidine; Oxadiazole derivative; PTP1B inhibitor; Specific
selectivity; Docking analysis; Function-Oriented Synthesis
* Authors for correspondence: ywguo@simm.ac.cn; xwli@simm.ac.cn
Tel & Fax: +86-21-50805813
‡Authors contributed equally

Protein tyrosine phosphatases (PTPs), a large family of signaling enzymes, act as
paramount regulatory components in numerous cell functions, such as growth,
mitogenesis, motility, cell–cellinteraction, metabolism, gene transcription, and
immune responses.1 Protein tyrosine phosphatases 1B (PTP1B), a member of PTPs
family, is a key negative regulator in both insulin and leptin signaling pathways,
thereby modulates both glucose and lipid metabolism.2 The independent studies from
Elchebly and Klaman, respectively, revealed that PTP1B knock-out mice processed
lower insulin and glucose levels with increased sensitivity to insulin, and resistance to
high-fat induced weight gain without any adverse effects,3 which enable PTP1B to
become a potential target for the treatment of type II diabetes and obesity. Great
efforts have been made for the developmentof PTP1B inhibitors as potential
therapeutics for type II diabetes. However, low selectivity over the other PTPs and
poor cell permeability are still two major challenges in the discovery of efficient
PTP1B inhibitors.4
Marine natural products are valuable resources and provide abundant lead
compounds to the development of new clinical drugs.5 Our group has long been
dedicated on the chemical and biological investigation of marine natural products. For
instance, phidianidines A and B (Figure 1), two indole alkaloids bearing an
uncommon 1,2,4-oxadiazole ring connected to the indole system, were isolated from
the marine opisthobranch mollusk Phidiana militaris in 2011.6 The subsequent vitro
assays showed that phidianidines A and B exhibited significant cytotoxicity against
tumor and nontumor mammalian cell lines after their total syntheses by Lindersley

and co-workers in 2012.7 Our further studies indicated that some of
phidianidine-based derivatives exhibited potential in vitro neuroprotective effects
against amyloid-β25–35(Aβ25–35)-, hydrogenperoxide (H2O2)-, and oxygen-glucose
deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells.8
The bioactivities of phidianidines toward diverse pharmacological targets could
be attributed to their unique structures. The indole fragment is a common but
important pharmacophore widely presented in numerous bioactive natural products or
drugs.9 The oxadiazole ring has been incorporated in drug discovery programs as an
essential element of pharmacophore in contribution of ligand binding.10 The
guanidine moiety, however, was proved to be not required for certain biological
activity according to Lindersley’s research.7b Based on the above observations and in
the courses of our continuous searching for novel effective PTP1B inhibitors,8 the
function-oriented synthesis (FOS) of phidianidine B derivatives aiming to simplify the
structure and synthetic routes while retain the activities were conducted.
In this paper, we prepared a series of phidanidine-based compounds (6a-6c,
7a-7x) by introducing heterocyclic rings to replace the guanidyl chain. Their PTP1B
inhibitory acitivity were evaluated and the structure-activity relationship (SAR) was
investigated. Selected compounds were subjected to specific selectivity studies
towards PTP1B over other PTPs (TCPTP, SHP1, SHP2 and LAR). A subsequent
docking study of compound 7o into the active site of PTP1B was perfomed to
rationalize the results.

Figure 1 Structure of phidianidine A and B
The preparation of phidianidin B derivatives was carried out following the
synthetic route shown in scheme 1. Synthesis was started from the commercially
available bromo aromatic aldehyde 1 (1a-1c, represented thiophenyl, phenyl, and
pyridyl, respectively). The reaction of 1 with hydroxylamine hydrochloride in the
presence of NaOAc gave oximes 2a-2c. Dehydration of
2
with
dichloro(p-cymene)ruthenium(II) dimer in CH3CN afforded nitriles 3.12 The treatment
of
3
with hydroxylamine hydrochloride and Et3N in MeOH provided
N-hydroxy-amidines 4a-4c. Esterification of 4 with 3-indoleacetic acid in the
presence of HATU and DIEA in CH2Cl2 gave rise to compounds 5a-5c.
Intramolecular cyclization of 5 in the presence of NaOAc in 30% EtOH gave bromo
oxadiazole analogues 6a-6c.13 The target compounds 7a-7x was obtained by the
coupling of 6a-6c with various substituted phenylboronic acids or pinacol esters
catalyed by bis(triphenylphosphine)palladium(II) dichloride in the presence of K2CO3
in mixture solvent of 1,4-dioxane and H2O.

Scheme
1
Reagent and conditions: (a) NH2OH·HCl, AcONa, r.t., 15 h; (b)
[Ru2(p-PriC6H4Me)2(µ-Cl)Cl]2, CH3CN, 80 oC, 3 h; (c) NH2OH·HCl, Et3N, MeOH, r.t., 15 h; (d)
3-indoleacetic acid, HATU, DIEA in CH2Cl2, r.t., 2 h; (e) AcONa in 30% EtOH, reflux, 12 h; f)
phenylboronic acid, Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane, H2O, r.t.
The inhibitory activities of the synthesized heterocyclic ring-substituted
phidianidine B derivatives 6a-6c, 7a-7x against PTP1B were measured using
p-nitrophenyl phosphate (pNPP) as a substrate. Oleanolic acid, a known PTP1B
inhibitor (IC50 = 3.02 0.22 µM), was used as the positive control. The results are
summarized in Table 1.
Table 1 Inhibitory activity of compounds 6a-6c and 7a-7x on PTP1Ba
Compd.
6a
X
R
IC50 (µM)
Compd.
X
R
IC50 (µM)
14.06 1.86
2-Me
24.44 1.78
7l
S
19.72 3.02
3- Me
15.78 1.01
6b
7m

NAb
NA
NA
6c
7a
7b
7c
7d
7e
7f
7n
7o
7p
7q
7r
7s
4-n-Pr
4-n-Bu
4-EtO
N
S
4-NH2
2-Me
8.80 0.91
15.90 2.03
18.36 1.49
NA
16.82 2.26
S
S
S
S
3- Me
4-Et
NA
4-n-PrO
13.14 1.32
4-O(CH2)3
CO2Me
12.51 0.60
8.96 0.63
17.26 1.77
13.73 1.28
NA
4-OH
2-Me
NA
4-n-Pr
4-n-Bu
4-EtO
4-n-PrO
N
N
N
N
N
N
26.12 3.06
21.67 3.28
9.66 0.88
31.49 1.82
9.66 1.02
3.02 0.22
7t
3-Me
7g
7h
7i
7u
7v
7w
7x
S
S
S
4-n-Bu
4-EtO
4-n-PrO
4-O(CH2)3
CO2Me
4-OH
21.93 2.97
NA
7j
4-NH2
Oleanoli
cacid
7k
a
all compounds herein showed no inhibitory activity on TCPTP, SHP-1, SHP-2 and LAR
(IC50>120 µM).
b “NA” means no activity (IC50>120 µM)
The bioassay results indicated that most of the target compounds displayed PTP1B

inhibitory activity with IC50 values in the range of 8.80–31.49 µM. Among them,
compounds 7f, 7o, 7v and 7x showed the most potent inhibitory effects with IC50
values of 8.96, 8.80, 9.66 and 9.66 µM, respectively, which is comparable with the
positive control oleanolic acid (IC50 = 3.02 µM). The primary SAR has been studied
regarding to the different functional groups on the side chain. Firstly, the
aforementioned results suggested that the PTP1B inhibitory activity was not
significantly affected by the aromatic rings (thiophene, benzene or pyridine) on the X
position of 6 or 7. A comparison of the activities across the position and
physicochemical properties of the substituents on the phenyl ring E indicated that
strong nucleophiles such as hydroxyl or amino was not critical for the activity against
PTP1B, since 7a, 7j, 7k, and 7s showed no or very low inhibition. The introduction of
carbonyl in ring E resulted in the disapear of the activity. The activity of 7b, 7c, 7l,
7m, 7t and 7u indicated that the substitution at 2 or 3-position lead to the decrease or
disappear of activity. The substitution at 4-position in the ring E played an important
role in the activity, the introduction of n-butyl group is beneficial for the improvement
of PTP1B inhibitory activity, as revealed from those of 7h, 7q, and 7x.
The selectivity bioassay was also evaluated by the investigation of bioactivities
against other PTPs. All the derivatives showed no activities against TCPTP, leukocyte
antigen-related phosphatase (LAR), Src homology phosphatase (SHP)-1 and SHP-2,
suggesting the highly specific selectivity of these molecules towards PTP1B.
In order to understand the inhibitory activity against PTP1B, the most bioactive
compound 7o has been selected to perform the molecular docking analysis. The X-ray

crystal structure of PTP1B with a resolution of 2.40 Å (Protein Data Bank, 1NL9) was
used for the docking studies. Figure 2 displayed the binding mode of compound 7o
with PTP1B. From this figure, it can be observed that the N-H in the ring B forms a
hydrogen bond (-NH…O=C-, 1.88 Å) with the carboxyl of Glu115, 4-site of the
nitrogen atom in ring C accepted another H-bond (-N…HN-, 2.84 Å) with the key
residue Ala217. Rings A and B form two π-σ bonds with Lys120. Moreover, rings C
and D form π-σ bonds with Ala217, Ile219 and Val49, respectively. Sulfur bond is
observed between ring A and sulfur atom of Cys215. According to this molecular
docking model, the studied phidianidin B analogue 7o was suggested to bind into the
catalytic site of PTP1B for the activity.
Figure 2. Docking result of compound 7o.
In summary, inspired by a function-oriented synthesis (FOS) point of view, a
series of phidianidine B derivatives with simplified structures and synthetic routes
were synthesized and identified as novel PTP1B inhibitors. Compound 7o exhibited
the best inhibitory activity with an IC50 value of 8.80 µM. The preliminary SAR study

provides a functional direction for the design of novel class of PTP1B inhibitors. The
selective inhibitory investigation of the active compounds against other PTPs showed
no inhibitory activities against TCPTP, LAR, SHP-1 or SHP-2, indicating their
specific selectivity against PTP1B. The mechanisms involved in the interaction
between the inhibitors and PTP1B were also predicted by docking analysis of the
represented compound 7o. Further FOS studies to improve pharmacological
performance, and further biological investigations to understand the interaction
mechanism for this class of PTP1B inhibitors should be conducted in the future.
Acknowledgements
This research work was financially supported by the National Marine “863”
Projects (Nos. 2013AA092902 and 2012AA092105), the Natural Science
Foundation of China (Nos. 81520108028, 81273430, 81302692 and 41476063),
SCTSM Project (Nos. 15431901000 and 14431901100), NSFC Shangdong
Joint Fund for Marine Science Research Centers (No. U1406402), the
SKLDR/SIMM Projects (SIMM1501ZZ-03, CASIMM0120152039).
Supplementary data
Supplementary data related to this article can be found, in the online version, at doi:#.

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Graphical Abstract
Function-Oriented Synthesis of phidianidine B derivatives were accomplished, their
specific inhibitory effects on PTP1B, the SAR and molecular docking analysis are
described.