Synthesis and structure-activity relationship of non-phosphorus-based fructose-1,6-bisphosphatase inhibitors: 2,5-Diphenyl-1,3,4-oxadiazoles

Article abstract of DOI:10.1016/j.ejmech.2014.06.011

With the aim of discovering a novel class of non-phosphorus-based fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of 2,5-diphenyl-1,3,4-oxadiazoles were synthesized based on the hit compound (1) resulting from a high-throughput screening (HTS). Structure-activity relationship (SAR) studies led to the identification of several compounds with comparable inhibitory activities to AMP, the natural allosteric inhibitor of FBPase. Notably, compound 22 and 27b, bearing a terminal carboxyl or 1H-tetrazole, demonstrated remarkable inhibition to gluconeogenesis (GNG). In addition, both inhibition and binding mode to the enzyme were investigated by enzymatic kinetics and in silico experiments for representative compounds 16 and 22.

Full text of DOI:10.1016/j.ejmech.2014.06.011

European Journal of Medicinal Chemistry 83 (2014) 15e25
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity relationship of
non-phosphorus-based fructose-1,6-bisphosphatase
inhibitors: 2,5-Diphenyl-1,3,4-oxadiazoles
,
,
c
,
,
Ben-Ren Liao a 1, Hai-Bing He a 1, Ling-Ling Yang b 1, Li-Xin Gao b, Liang Chang a,
Jie Tang a, Jing-Ya Li b *, Jia Li b *, Fan Yang a
,
,
, *
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, Department of Chemistry, East China Normal University,
Shanghai 200062, China
b National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China
c Chemistry and Chemical Engineering, Nantong University, Jiangsu 226019, China
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of discovering a novel class of non-phosphorus-based fructose-1,6-bisphosphatase (FBPase)
inhibitors, a series of 2,5-diphenyl-1,3,4-oxadiazoles were synthesized based on the hit compound (1)
resulting from a high-throughput screening (HTS). Structureeactivity relationship (SAR) studies led to
the identification of several compounds with comparable inhibitory activities to AMP, the natural allo-
steric inhibitor of FBPase. Notably, compound 22 and 27b, bearing a terminal carboxyl or 1H-tetrazole,
demonstrated remarkable inhibition to gluconeogenesis (GNG). In addition, both inhibition and binding
mode to the enzyme were investigated by enzymatic kinetics and in silico experiments for representative
compounds 16 and 22.
Received 7 February 2014
Received in revised form
3 June 2014
Accepted 7 June 2014
Available online 10 June 2014
Keywords:
Diabetes
Antidiabetic
© 2014 Published by Elsevier Masson SAS.
Fructose-1,6-bisphosphatase (FBPase)
Inhibitor
2,5-Diphenyl-1,3,4-oxadiazoles
1. Introduction
drug target, which enables inhibition of GNG from all GNG sub-
strates while avoiding direct effects on glycogenolysis, glycolysis,
Gluconeogenesis (GNG), a predominant factor of increased he-
patic glucose output [1e4], is a highly regulated process catalyzed
by several gluconeogenic enzymes such as fructose-1,6-
bisphosphatase (FBPase) which is one of the rate-limiting en-
zymes [5]. Comparing to the other two rate-limiting enzymes
(PEPCK and G6Pase) in multiple roles in GNG, FBPase functions only
within the GNG pathway by catalyzing the conversion of D-fruc-
tose-1,6-bisphosphate (FBP) to D-fructose-6-phosphate (F6P) [6].
Inhibition of FBPase would be expected as a reasonable way by
suppressing GNG to decrease plasma glucose. Actually, in insulin-
deficient and insulin-resistant animal models of diabetes, liver
FBPase activity is elevated, highlighting the importance of this
enzyme in the control of blood glucose [7]. Additionally, in view of
its position in the GNG pathway, FBPase shows more attraction as a
and the tricarboxylic acid cycle as well [8,9].
It has been clarified that the regulation of FBPase activity in-
volves changes in quaternary structure between the active (R) and
inactive (T) conformational states [10,11]. FBPase is naturally
inhibited by AMP by acting on the allosteric binding site (composed
of a hydrophilic phosphate binding site and a hydrophobic pocket),
inducing the enzymatic shifting from R to T conformation. More-
over, substrate analogue fructose-2,6-bisphosphate,
a potent
competitive inhibitor of FBPase, synergistically increases the
binding affinity of AMP [12e14]. For decades, significant effort has
been brought to develop small-molecular inhibitors against FBPase,
focusing on either substrate binding site [15] or AMP binding site
[16e25]. Although several series of compounds with FBPase
inhibitory activity were reported, such as anilinoquinazolines
[16,17], indole carboxylic acids [18], benzenesulfonamides [19,20],
sulfonylureas [22,23], tricyclic compounds [24] and achyrofurans
[25], few of them achieved such a success as CS-917, the prodrug of
MB05032 (Chart 1), which inhibits the enzyme by interacting with
the AMP binding site through the phosphate group [6,21,26e28].
* Corresponding authors.
E-mail addresses: jyli@mail.shcnc.ac.cn (J.-Y. Li), jli@mail.shcnc.ac.cn (J. Li),
fyang@chem.ecnu.edu.cn (F. Yang).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.06.011
0223-5234/© 2014 Published by Elsevier Masson SAS.

16
B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
formed by oxidation of the terminal alkenyl group of 21a with
K3Fe(CN)6 and K2OsO2(OH)4 [35] and 21a was generated conve-
niently from the condensation of 8 with hex-5-enoic acid. Com-
pound 22, which bears a terminal carboxyl group, was synthesized
by the reaction of 8 with pimelic acid in the presence of EDC and
HOBt in DCM.
The synthesis of the compounds containing at least one tertiary
amine unit as the hydrogen bond acceptor is displayed in Scheme 4.
The introduction of amine unit was carried out by replacement of
the terminal halogen with a secondary amine, including morpho-
lines (25a, 25c and 25i), piperidine (25b), 4-hydroxypiperidine
(25f), piperazine (25e) and N-methyl piperazines (25d and 25h).
However, due to the poor leaving property of chlorine, it was
necessary to previously replace the chlorine on 23 with iodine by
using NaI (step b) [36]. Acetylation of 25e with acetic anhydride
readily yielded 25g, whereas the ethyloic derivatization on the
piperazine ring of 25e was accomplished via a winding way which
was starting from 24 by coupling with tert-butyl-2-(piperazin-1-yl)
acetate 28 (step h) [37] and followed by deprotection in TFA/DCM
(step i) [38]. As the classical isostere of carboxyl, 5-substitued tet-
razole (27b) was synthesized from a cyano group (27a) with
Me3SiN3 at 120 ꢀC using NMP as the solvent under microwave
assistance. The intermediate 27a was obtained conveniently by
reacting 24 with Me3SiCN [39].
Chart 1. The previously reported inhibitors of FBPase.
Nevertheless, besides FBPase, AMP also modulates a number of
other enzymes controlling different biological functions [29]. Thus
it is of great attraction to develop new potent FBPase inhibitors
which are structurally distinct from AMP and likely bind to the
enzyme at least partly by a different mode.
With the intention of searching for new hits with FBPase
inhibitory activity, we carried out a high-throughput screening
(HTS) campaign of a lead-like library of small molecular com-
pounds, resulting in the identification of 1 endowed with a good
drug likeness according to Lipinski's rule [30,31]. Compound 1,
3. Results and discussion
inhibiting FBPase with the IC50 value of 15.45 mM at molecular level,
was characterized as 2,5-diphenyl-1,3,4-oxadiazoles structure type.
In previous report the preliminary SAR of derivatives of 1 by
modifying aromatic group A was disclosed, resulted in the discov-
3.1. Activities at molecular level
In vitro FBPase (recombinant human FBPase) inhibition assays
were carried out using a coupled spectrophotometric method re-
ported by Doris Rittmann [40], and AMP, the natural allosteric in-
hibitor to FBPase, served as a positive control in the experiments
ery of a three-fold more potent compound 2 (4.51 0.22 mM) [32].
Herein, we report further evolution of the novel non-phosphorus
FBPase inhibitors, mainly focused on the modification on the side
chain of compound 2 (Chart 1). Their effects on glucose output in
rat hepatocytes were explored for the compounds that showed
stronger potency at molecular level. Furthermore, both the inhibi-
tion and binding modes to the enzyme are discussed.
(IC50 ¼ 1.3
0.44 mM). On the basis of our previous work, 2,5-
diphenyl-1,3,4-oxadiazole scaffold with 4-methyl group on aro-
matic ring A was assayed as the optimum structure, thus the side
chain of the molecule would be the second key moiety to be
explored. At first, we attempted to replace the side chain (including
various terminal groups) with simple alkyl group without any
change of the amido bond which is usually considered to play a key
role in binding to enzymes via hydrogen bond interactions. When
the 3,4-dimethoxybenzyl unit was replaced with methyl group,
2. Chemistry
All the target compounds were obtained via an amidation re-
action of meta-amino substituted diphenyl-1,3,4-oxadiazole 8 with
kinds of acids, acyl chlorides or acetic anhydride. Compound 8 was
prepared via a classical procedure: dehydrated cyclization of
dihydrazide 6 with POCl3 in acetonitrile followed by the Raney-Ni-
catalyzed hydrogenation of the nitro to amino group on aromatic
ring B (Scheme 1) [33].
The reaction of 8 with acetic anhydride afforded 9 conveniently,
while 10e13 were prepared from the reactions of 8 with appro-
priate acyl chlorides (Scheme 2).
The synthesis of the compounds bearing hydroxyl groups at the
end of the side chain is illustrated in Scheme 3. Different pathways
were employed in the light of various lengths of the alkyl chains.
The reaction of 8 with 6-hydroxyhexanoic acid (16a) using EDC as
the condensation agent gave 16 directly. In contrast, due to the
instabilities of 4-hydroxybutanoic acid and 5-hydroxypentanoic
acid, compounds 14 and 15 were obtained from 8 via a relatively
complicated three-step procedure, including acylation with anhy-
dride, methylation with iodomethane and reduction of the terminal
ester group to hydroxyl group (step d, e and c). 17, 18, 19 and 20
were also prepared by the reduction of the corresponding terminal
ester group with NaBH4 in variable yields [34]. However, different
from 14a and 15a, the related ester intermediates 17ae20a were
synthesized by the reactions of 8 with the corresponding diacid
monoesters in one step. The vicinal diol group of compound 21 was
compound
9
kept
a
comparable inhibition potency
(IC50 ¼ 7.70 0.34
mM) to 2. However, along with the lengthening
or enlarging of the alkyl chain (Table 1, 10e13), inhibition potencies
of the corresponding compounds decrease. We speculated that a
group with strong hydrophobic property (a long or bulky alkyl)
would be disadvantageous to the binding with FBPase. On the other
hand, it also seemed disobedient that 2, bearing the largest side
chain, displayed the best activity comparing to the other com-
pounds in Table 1. It is suggested that the two methoxy groups on
the side aromatic ring in 2 contributed a lot to the interactions with
FBPase, probably acting as a hydrogen bonding acceptor, and thus it
can be inferred that a side chain composed of a small alkyl chain
having a hydrogen bonding acceptor would be beneficial to the
activity upon the enzyme.
Since a small alkyl chain with a hydrogen bonding acceptor was
considered to improve the inhibition against FBPase, linear alkyls
with terminal hydroxyl group (Table 2) or amino (Table 3) were
designed and synthesized for exploring. As outlined in Table 2,
most of the compounds with a hydroxyl group at the end of the
alkyl chain inhibited the enzyme significantly, and the potencies
varied with respect to the lengths of the alkyl chains. With the
increase of carbons from 4 (14) to 6 (16), the corresponding inhi-
bition potencies increased synchronously till about 12-fold more

B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
17
Scheme 1. Reagents and conditions: (a) Dioxane, 85% hydrazine hydrate, reflux, 6 h, (99%); (b) EDC, HOBt, 3-picoline, DCM, RT, 12 h, (65e75%); (c) POCl3, CH3CN, reflux, 8 h; (d)
Raney-Ni, H2, 1 atm, RT, overnight.
Scheme 2. Reagents and conditions: (a) 3-picoline, DCM, 0e8 ꢀC, 8 h; (b) 3-picoline, DCM, ꢁ5 ꢀC ~ 5 ꢀC, 3 h.
Scheme 3. Reagents and conditions: (a) EDC, HOBt, DCM, 3-picoline, RT, 16e30 h; (b) K3Fe(CN)6, K2CO3, K2OsO2(OH)4; (c) NaBH4, THF, MeOH, reflux; (d) Et3N, THF, RT; (e) MeI,
K2CO3, acetone, RT, 20 h; (f) Pimelic acid, EDC, HOBt, DCM, 3-picoline, RT.
Scheme 4. Reagents and conditions:(a) Appropriate chloroalkanoyl chloride, 3-picoline, DCM, 0 ꢀC ~ RT, overnight; (b) NaI, dried acetone, reflux, 12 h; (c) THF, RT, overnight; (d)
Ac2O, Et3N, 0 ꢀC ~ RT, 4 h; (e)Me3SiCN, Bu4NF, CH3CN, RT, 24 h; (f) Me3SiN3, NMP, 120 ꢀC, microwave, 30 min; (g) BrCH2COOBu-t, EtOH, 20 h; (h) K2CO3, THF, CH3CN, RT, 36 h; (i)
CF3COOH, DCM, 0 ꢀC ~ RT, 4 h.

18
B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
Table 1
example, 25i showed an improved two-fold activity than 25c.
0.55 M), replacement of
morpholine with 4-hydroxypiperidine (25f) led to approximate
IC50 values of compounds 11 ~ 13 against FBPase.
Comparing with 25c (IC50 ¼ 5.01
m
three-fold decrease of the activity. Acetylation of the terminal
piperazine 25e (IC50 ¼ 3.97 0.50
mM) yielded 25g which exhibited
a similar activity. On account of the enhanced water solubility
brought by carboxyl, an ethyloic group was incorporated to 25e,
affording 26b which displayed a half inhibitory activity. Another
strategy for applying the property of carboxyl is to introduce its
isosteres of which tetrazole (27b) is the most popular owing to a
similar pKa value and multiple hydrogen bond acceptors that
would be beneficial to the binding with FBPase. Fortunately, the
obtained compound 27b with a terminal tetrazole demonstrated an
Compd.
Y
FBPase, IC50 (m
M)a
2
4.51 0.22
7.70 0.34
27.15 0.63
>50
9
Me
Et
i-Pr
n-Bu
t-Bu
10
11
12
13
inhibitory activity (IC50 ¼ 2.98 0.35
mM) which is as good as those
of terminal hydroxyl or azacyclohexyl compounds.
33.80 0.55
>50
a
3.2. Inhibition of glucose output in rat hepatocytes
IC50 values were determined by regression analyses and expressed as
means SE of three replicates.
In order to further determine the inhibitory ability to FBPase of
the compounds which showed good potencies at molecular level,
potent (16, IC50 ¼ 1.32 0.21
mM) than that of the hit compound 1.
However, further lengthening the chain reduced the inhibitory
activity which was almost lost when the number of carbons added
up to 9 (19) and 10 (20). The intermediates 14a, 14b and 15, bearing
a hydrogen bonding acceptor of carboxyl or methyl carboxylate
group, also exhibited moderate inhibitions against FBPase. Despite
Table 3
IC50 values of compounds 25 ~ 27 against FBPase.
the poor potency of 14b (IC50 ¼ 42.90 3.22
mM), its better water
solubility inspired us to synthesize compound 22 with an opti-
mized length of side chain. Encouragingly, 22 demonstrated an 8-
fold more potent activity (IC50 ¼ 5.32
0.59 mM) than 14b.
Therefore, it was not surprising that the double hydroxyls
substituted compound 21 showed a better activity.
Compd.
n
Y
FBPase, IC50 (m
M)a
Azacyclohexanes, including piperidine, piperazine and mor-
pholine which usually furnish a structure with amphipathicity
(hydrophilicity & hydrophobicity) and potential hydrogen bonding
interactions, are widely incorporated into drug molecules. IC50
values of the compounds with a terminal azacyclohexane in replace
of hydroxyl are exhibited in Table 3. In contrast to piperidine (25b,
25a
25b
25c
25d
25e
25f
2
3
3
3
3
3
3
4.96 0.48
14.20 2.01
5.01 0.55
3.92 0.41
3.97 0.50
13.20 2.11
3.58 0.42
IC50 ¼ 14.20 2.01
mM), morpholines (25a, 25c and 25i), piperazine
(25e) or N-methyl piperazines (25d and 25h) which are composed
of two heteroatoms, inhibited FBPase more potently. Furthermore,
length of the alkyl chain also affected the activities obviously. For
Table 2
IC50 values of compounds 14 ~ 22 against FBPase.
25g
25h
25i
4
4
2.30 0.30
2.54 0.39
Compd.
n
Y
FBPase, IC50 (m
M)a
14
15
16
17
18
19
20
14a
14b
15a
21
2
3
4
5
6
7
8
1
1
2
2
4
OH
OH
OH
OH
OH
OH
OH
5.61 0.56
3.59 0.42
1.32 0.21
3.06 0.37
11.25 1.13
>50
26b
3
3
12
7.12 0.85
>50
eCOOMe
eCOOH
eCOOMe
eCHOHCH2OH
eCOOH
7.99 1.01
42.90 3.22
10.68 1.55
3.51 0.39
5.32 0.59
27b
2.98 0.35
22
a
a
IC50 values were determined by regression analyses and expressed as
means SE of three replicates.
IC50 values were determined by regression analyses and expressed as
means SE of three replicates.

B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
19
Table 4
Inhibition of glucose output in rat hepatocytes under FBPase inhibitors.a,b
the precipitate of 9 at 80
appeared when testing 25i, which might take the responsibility for
the dramatic rise of glucose concentration at 80 M and 160 M.
m
M and 160
m
M. Similar phenomenon
m
m
Compd.
10
mM (%)
20
m
M (%)
40 mM (%)
Fortunately, the inhibition effect against glucose output by com-
pound 22 and 27b was found to be concentration-dependent,
Metforminc
2
9
16
21
22
25e
25g
25h
25i
27b
75e87d
96.7
96.8
89.0
88.7
81.9
100
90.3
95
83.3
89.8
93.5
98.6
95.3
91.9
91.0
98.4
91.9
93.6
83.6
85.9
103.2
86.1
96.8
96.7
75.6
96.8
96.7
56.5
85.2
74.2
especially for 27b. At 160
production about 42% and 59% independently, and the corre-
sponding calculated IC50 values were 167.3 ( 0.11) M and 112.5
( 0.04) M respectively. Both of the two compounds demonstrated
mM, 22 and 27b suppressed the glucose
m
m
good water solubility as well as excellent membrane permeability,
which enable the compounds to be promising candidates for
development of drugs against T2DM.
a
The ratio of the produced glucose concentration under inhibitors into the blank
control (0.1% DMSO).
3.3. Characterization of the inhibitors on enzyme kinetics
b
Means of three parallel wells.
control, at 2 mM.
The ratio of the produced glucose concentration into the blank (0.1% DMSO).
c
For evaluating the inhibitory modality of the compounds,
compound 16 was selected for enzymatic kinetic study on FBPase.
As shown in Fig. 2(a), activity of the enzyme recovered gradually
after diluting the incubation mixture of FBPase and 16, and the
activity recovered about 90 percent just over 10 h. The result sug-
gests that inhibition of FBPase by 16 is a reversible reaction. Addi-
tionally, the relationship between Vmax and concentration or
between Km (calculated on Vmax by MichaeliseMenten equation)
and concentration was studied at five different concentrations
d
their effects on the glucose production in primary rat hepatocytes
were explored with the existence of sodium pyruvate (2 mmol/L) as
the GNG substrate [41]. Metformin was used as the control, because
it is recognized as the only marketed drug that acts, at least
partially, through inhibition of GNG [42]. As illustrated in Table 4,
five compounds (9, 22, 25h, 25i and 27b) inhibited the glucose
(0e4 mg/mL), as outlined in Fig. 2(b & c). Apparently, both of Vmax
and Km decreased with the increase of concentration of 16, indi-
cating that 16 might be an uncompetitive or mixed-type inhibitor
of FBPase.
production in hepatocytes effectively at 40
pound 22 and 25i, remarkable inhibition was obtained even at
10 M (81.9% and 83.3% respectively). Of all the compounds, 25h
showed the most potent inhibition of glucose output, up to 56.5% at
40 M. However, comparing to the blank control group (0.1%
DMSO), total proteins in the three parallel wells containing 25h at
40 M were significantly lower (68.7%, 65.0% and 73.4% of blank
group independently), which may be because the reduction of
glucose production by 25h is mainly associated with its cytotoxicity
to rat hepatocytes. Additionally, the decrease of total proteins was
not observed for the other compounds brought into the assays.
Therefore, for the sake of investigating detailed inhibition potencies
of the four compounds (9, 22, 25i and 27b), we carried out further
mM. Actually, for com-
m
m
3.4. In silico docking
m
In order to further clarify the inhibition modality of our novel
series to FBPase, 22, one of the compounds most potently sup-
pressing hepatocyte glucose output, was selected for a molecular
docking analysis using Ligandfit available with Discovery Studio 2.1.
Compound 22 may not bind at the FBPase active site since the
enzyme was still significantly inhibited even at a high dose of the
substrate, namely, there was no obvious competition between
compound 22 and the substrate. Instead, the preferred coordina-
tion mode of 22 with FBPase should be similar to the mode revealed
by Hebeisen [22]. As presented in Fig. 3, although interacts with the
assays at six concentrations (5e160
approximate 15% inhibition at 40 M, increasing the concentration
of 9 failed in improving the potency, which was well explained by
mM) [Fig. 1]. In spite of an
m
Fig. 1. Inhibition of glucose output in rat hepatocytes by 9, 22, 25i and 27b; Metformin (2 mM) was used as the control; Significances were expressed by asterisks.

20
B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
Fig. 2. Kinetic characterization of 16 on FBPase: (a) the reversible binding mode of 16 with enzyme was confirmed by the recovery activity of FBPase after dialysis by indicated
times; (b & c) effects on Vmax and Km values of FBPase by 16.
Fig. 3. Compounds 22 docked into FBPase (PDB id ¼ 2JJK). Molecular surface shows hydrophobic regions in white, hydrophilic regions in blue (left graph). Visualization was
performed with Discovery Studio 2.1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
enzyme partly through AMP binding sites, differently, compound
22 binds to two adjacent FBPase monomers simultaneously. The
aromatic ring A occupied an area near the AMP purine binding
domain on the left monomer through hydrophobic interacts with
Leu30 and Tyr113, while at the other end the carboxyl of the ligand
sit close to the phosphate recognition pocket (defined by Thr27,
Gly28, Glu29, Leu30, Lys112 and Tyr113) of the AMP binding site
through multiple hydrogen-bonding interactions with Thr27,
Tyr113 and Arg140. The 1,3,4-oxadiazole ring locates at the surface
region, engaged in hydrogen bonds with residues Gly28 and Thr31.
Aromatic ring B, located at the inter space of the two subunits, acts
as a linker without obvious interactions observed, which might
imply a possibility of replacement of this ring. The amido bond also
binds with Gly26 through a hydrogen bond. It is noted that the
distance between the amido and the terminal carboxyl is essential
to the binding with residues, by which the SAR of the side chain
length would be well explained.
hydroxyl on the side linear alkyl chain are beneficial to the activity
at molecular level. However, in consideration of cellular activities,
more hydrophilic carboxyl or 1H-tetrazole are preferred. Enzymatic
kinetic studies of 16 proved the inhibition modality to be reversible
mixed-type. In silico docking of 22 with FBPase revealed a possible
binding mode, together with enzymatic kinetic properties, which
make us to be aware of the suggesting allosteric inhibition to the
enzyme. These results may prompt further discovery of promising
candidates for new FBPase inhibitors, and thereby treating
diabetes.
5. Experimental section
5.1. Biological activity against fructose-1,6-diphosphatase (FBPase)
FBPase recombinant protein was expressed and purified ac-
cording to the previous report [41]. FBPase activity was measured in
a coupled spectrophotometric assay containing 5 mM MgCl2,
66.7 mM KCl, 66.7 mM MOPS pH 7.5, 0.25 mM NADPþ, yeast
glucose-6-phosphate-dehydrogenase (0.4 U/mL), yeast phospho-
glucoisomerase (0.7 U/mL) and 325 nmol/L FBPase. Fructose 6-
phosphate formed by the reaction of FBPase was converted to
glucose 6-phosphate and subsequently to 6-phosphogluconate by
coupling to phosphoglucoisomerase and glucose-6-dehydrogenase
and the concomitant formation of NADPH was detected at 340 nm.
4. Conclusion
In continuing the search for non-phosphorus-based inhibitors of
FBPase through targeted structural modifications of previously re-
ported analogues, 2,5-diphenyl-1,3,4-oxadiazoles with a long alkyl
side chain, were identified with moderate to good inhibitory ac-
tivities. In most cases, the tested compounds bearing a terminal
hydrogen-bonding acceptor showed potent inhibition against
FBPase at molecular level. Moreover, some representative com-
pounds were endowed with cellular activities. The highlighted two
compounds 22 and 27b were able to inhibit the glucose production
5.2. Effect on glucose output of primary hepatocytes
Primary hepatocytes were isolated from SpragueeDawley rats
which was fasted overnight as previously reported [43,44], then
suspended in DMEM containing 1 g/L glucose (Invitrogen™) and
10% FBS (Invitrogen™), and plated in 24-well plates (2 ꢂ 105 cells in
each well). After 4 h' attachment, the old medium was replaced
with fresh medium and incubated overnight. The medium was
in primary rat hepatocytes with an IC50 value of 167.3
mM and
112.5 M, respectively. These results indicated that the two com-
m
pounds are capable of inhibiting GNG likely by interacting with
FBPase. In regards to structure activity relationships, an electron-
donating group at 4-position on aromatic ring A and a terminal

B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
21
subsequently replaced with 500
DMEM supplemented with 20 mmol/L sodium lactate and
2 mmol/L sodium pyruvate, containing 0, 5, 10, 20, 40, 80 and
m
L glucose and phenol-red-free
chromatography (DCM/hexane ¼ 1/1) to give 7 as a white solid
(yield: 86%; mp 164e166 ꢀC). 1H NMR (400 MHz, CDCl3):
d
¼ 2.46 (s,
3H), 7.36 (d, J ¼ 6.8 Hz, 2H), 7.76 (s, 1H), 8.04 (d, J ¼ 7.3 Hz, 2H), 8.40
160
bation, 50
m
M relevant compounds with three parallels. After 4 h' incu-
L medium was collected and the glucose concentration
(d, J ¼ 7.3 Hz, 1H), 8.50 (d, J ¼ 7.1 Hz, 1H), 8.93 (s, 1H); 13C NMR
m
(100 MHz, CDCl3):
d
¼ 165.5, 162.4, 148.7, 142.9, 132.4, 130.4,
was measured by a colorimetric glucose assay kit (Fudan-Zhang-
jiang™, Shanghai, China).
129.9(2C), 127.1(2C), 125.9, 125.7, 121.6, 120.6, 21.7; HRMS (ESI):
Calcd for C15H12N3O3 [M þ H]þ, 282.0873; Found, 282.0869.
5.3. Characterization of the FBPase inhibitors
5.4.4. 3-(5-p-Tolyl-1,3,4-oxadiazol-2-yl)aniline (8)
After the pretreatment by ethanol, Raney-Ni (0.5 g) was sus-
pended in a mixed solution of methanol (20 mL) and THF (20 mL)
followed by the addition of 7 (1 g), the mixture was then hydro-
genated at 1atm overnight. The catalyst was removed by filtration
and the filtrate was evaporated under reduced pressure to give 8.
To characterize the inhibitor of FBPase, the assay was carried out
in a 50 mL system containing 5 mM MgCl2, 66.7 mM KCl, 66.7 mM
MOPS (pH 7.5), 0.25 mM NADPþ, yeast glucose-6-phosphate-de-
hydrogenase (0.4 U/mL), yeast phosphoglucose isomerase (0.7 U/
mL), 325 nmol/L FBPase, FBP(fructose-1,6-bisphosphate) in 2-fold
Yield: 83%; 1H NMR (400 MHz, CDCl3):
d
¼ 2.36 (s, 3H), 3.82 (s, 2H),
dilution from 400
mmol/L, and different concentrations of the in-
6.77 (s, 1H), 7.19e7.42 (m, 5H), 7.93 (d, J ¼ 7.6 Hz, 2H); HRMS (ESI):
hibitor, where Km is the Michaelis constant, v is the initial rate,
Vmax is the maximum rate, and [S] is the substrate concentration.
The Vmax and Km values of the FBPase in the presence and absence
of the inhibitor were derived from a non-linear regression fitting of
the curve in the plot of the initial rates and the substrate concen-
trations, using the MichaeliseMenton equation v ¼ Vmax$[S]/
(Kmþ[S]).
Calcd for C15H13N3NaO [M þ Na]þ, 274.0956; Found, 274.0999.
5.4.5. N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)acetamide(9)
To the solution of 8 (2 mmol) and 3-picoline (0.5 mL) in DCM
(30 mL) was added acetic anhydride (2.5 mmol) dropwise at 0 ꢀC,
then the temperature was raised to 8 ꢀC in 1 h. After stirring at 8 ꢀC
for 8 h, aqueous NaOH (5%, 20 mL) was added for hydrolyzing
excessive acetic anhydride for 1 h. After that, the DCM phase was
separated and washed with water, aqueous HCl (5%, 20 mL) and
saturated aqueous NaHCO3 (15 mL) successively. After dried over
anhydrous Na2SO4, the organic phase was evaporated in vacuo and
then purified by column chromatography (DCM/EtOAc ¼ 5/1) to
give 9 as a white solid. Yield: 91%; 1H NMR (500 MHz, DMSO-d6):
5.4. Chemistry
Microwave assisted synthesis was performed using a DISCOVER-
STM microwave reactor. 1H NMR spectra: were recorded at either
400 or 500 MHz on JEOL-400 or Bruker AM-500 instruments
respectively. Chemical shifts (
d
¼
H) are quoted in parts per million
d
¼ 2.09 (s, 3H), 2.41 (s, 3H), 7.45 (d, J ¼ 7.8 Hz, 2H), 7.54 (t,
(ppm) relative to TMS as internal standard. Signals were charac-
terized as s (singlet), dd (doublet of doublet), t (triplet), m (multi-
plet), brs (broad signal). 13C NMR spectra: were recorded at either
100 or 125 MHz on JEOL-400 or Bruker AM-500 instruments
J ¼ 8.0 Hz, 1H), 7.77e7.80 (m, 2H), 7.98 (d, J ¼ 7.8 Hz, 2H), 8.40 (s,
1H), 10.24 (s, 1H); HRMS (ESI): Calcd for C17H16N3O2 [M þ H]þ,
294.1243; Found, 294.1238.
respectively. Chemical shifts (
d
¼
C) are quoted in ppm relative to
5.4.6. N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)propionamide
(10)
TMS as internal standard. Degenerate peaks are suffixed by the
number of carbons. Mass spectra: High Resolution Mass Spec-
trometry (HRMS) data were obtained on a Micromass Tof II
spectrometer.
To the solution of 8 (2 mmol) and 3-picoline (0.5 mL) in DCM
(30 mL) was added propionyl chloride (2.5 mmol) dropwise
at ꢁ5 ꢀC, then the temperature was raised to 5 ꢀC in 1 h. After
stirring at 5 ꢀC for 3 h, aqueous NaOH (5%, 20 mL) was added for
hydrolyzing excessive propionyl chloride for 1 h. After that, the
DCM phase was separated and washed with water, aqueous HCl
(5%, 20 mL) and saturated aqueous NaHCO3 (15 mL) successively.
After dried over anhydrous Na2SO4, the organic phase was evapo-
rated in vacuo and then purified by column chromatography (DCM/
EtOAc ¼ 5/1) to give 10 as a white solid. Yield: 77%; 1H NMR
5.4.1. 3-Nitrobenzohydrazide (4)
To the solution of methyl 3-nitrobenzoate (3, 5 g) in dioxane
(50 mL) was added hydrazine hydrate aqueoussolution (85%,
10 mL), the mixture was heated to reflux for 6 h, and cooled to room
temperature. Evaporation of the solvent in vacuo give a yellow
solid, which was then washed with water (5 mL) and dried to afford
4 as a white solid. It was used in next step without any purification.
(400 MHz, CDCl3):
d
¼ 1.30 (t, J ¼ 7.6 Hz, 3H), 2.44e2.49 (m, 5H),
7.32(d, J ¼ 8.0 Hz, 2H), 7.47(t, J ¼ 8.0 Hz, 1H), 7.58(t, J ¼ 8.0 Hz, 1H),
7.86 (t, J ¼ 8.0 Hz, 1H), 8.01 (d, J ¼ 8.0 Hz, 2H), 8.26 (s, 1H); HRMS
(ESI): Calcd for C18H17N3O2, [M þ H]þ, 308.1394; Found, 308.1391.
5.4.2. N0-(4-methylbenzoyl)-3-nitrobenzohydrazide (6)
3-Nitrobenzohydrazide (4, 10 mmol), p-toluicacid (5, 10 mmol),
EDC (13 mmol) and HOBt (13 mmol) were suspended in anhydrous
DCM (25 mL). To this mixture, 3-picoline (14 mmol) was added.
After stirring at room temperature for 12 h, the reaction mixture
was filtered. The filter cake was washed with 5% HCl and water
successively, dried to give 6 as a white solid (yield: 67%). 1H NMR
5.4.7. N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)isobutyramide
(11)
A procedure similar to the preparation of 10 by replacing pro-
pionyl chloride with iso-butyryl chloride. 1H NMR (500 MHz,
(400 MHz, DMSO-d6):
d
¼ 2.37 (s, 3H), 7.32 (d, J ¼ 7.2 Hz, 2H),
CDCl3):
d
¼ 1.28 (d, J ¼ 6.9 Hz, 6H), 2.43 (s, 3H), 2.62 (q, J ¼ 6.9 Hz,
7.84e7.86 (m, 3H), 8.36 (d, J ¼ 7.6 Hz, 1H), 8.44 (d, J ¼ 8.0 Hz, 1H),
1H), 7.30 (d, J ¼ 7.8 Hz, 2H), 7.46 (t, J ¼ 8.0 Hz, 1H), 7.83e7.87 (m,
3H), 8.00 (d, J ¼ 7.8 Hz, 2H), 8.32 (s, 1H); HRMS (ESI): Calcd for
8.75 (s, 1H), 10.56 (s, 1H), 10.88 (s, 1H); HRMS (ESI): Calcd for
C
15H14N3O4 [M þ H]þ, 300.0979; Found, 300.0976.
C
19H20N3O2, [M þ H]þ, 322.1556; Found, 322.1551.
5.4.3. 2-(3-Nitrophenyl)-5-p-tolyl-1,3,4-oxadiazole (7)
5.4.8. N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)pentanamide
(12)
Dihydrazide 6 (10 mmol) was suspended in anhydrous aceto-
nitrile (20 mL). To this mixture phosphorusoxychloride (2 mL) was
added. After refluxing for 8 h, the reaction mixture was evaporated
in vacuo. The resultant residue was then purified by column
A procedure similar to the preparation of 10 by replacing pro-
pionyl chloride with valeryl chloride. 1H NMR (500 MHz, DMSO-
d6):
d
¼ 0.90 (t, J ¼ 6.8 Hz, 3H),1.30e1.37 (m, 2H), 1.57e1.63 (m, 2H),

22
B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
2.35 (t, J ¼ 7.5 Hz, 2H), 2.50 (s, 3H), 7.41 (d, J ¼ 7.7 Hz, 2H), 7.53 (t,
J ¼ 8.0 Hz,1H), 7.75 (d, J ¼ 7.5 Hz,1H), 7.80 (d, J ¼ 7.5 Hz,1H), 7.96 (d,
J ¼ 7.7 Hz, 2H), 8.43 (s,1H),10.18 (s, 1H); 13C NMR (100 MHz, CDCl3):
5.4.12. 5-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
pentanamide (15)
Compound 15 was prepared starting from 15a through a pro-
d
¼ 171.8,164.1,163.8,142.1, 140.2,130.0,129.9(2C),126.6(2C), 123.8,
cedure similar to that for the preparation of 14. White solid. 1H NMR
122.1, 121.1, 120.6, 116.6, 36.2, 27.2, 21.9, 21.2, 13.8; HRMS (ESI):
(400 MHz, DMSO-d6):
d
¼ 1.47(m, 2H), 1.65(m, 2H), 2.35 (t,
Calcd for C20H22N3O2, [M þ H]þ, 336.1712; Found, 336.1737.
J ¼ 7.3 Hz, 2H), 2.37 (s, 3H), 3.43(m, 2H), 4.42(t, J ¼ 5.2 Hz, 1H), 7.40
(d, J ¼ 8.0 Hz, 2H), 7.51 (t, J ¼ 7.8 Hz, 1H), 7.73 (d, J ¼ 7.8 Hz, 1H), 7.80
(d, J ¼ 7.8 Hz, 1H), 7.94 (d, J ¼ 8.0 Hz, 2H), 8.42 (s, 1H), 10.16 (s, 1H);
13C NMR (100 MHz, DMSO-d6):
d
¼ 171.7, 164.0, 163.7, 142.1, 140.2,
5.4.9. Methyl 4-oxo-4-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)
phenylamino)butanoate(14a)
129.9(2C), 129.8, 126.5(2C), 123.7, 122.1, 121.0, 120.5, 116.6, 60.4,
36.3, 32.0, 21.7, 21.1; HRMS (ESI): Calcd for C20H22N3O3 [M þ H]þ,
352.1656; Found, 352.1699.
In an ice-water bath, to a solution of 8 (1 g, 4 mmol) in THF
(15 mL) and Et3N (2 mL), a solution of succinic anhydride (0.5 g,
5 mmol) in THF (10 mL) was added over 20 min. After stirring at rt
overnight, the reaction mixture was poured into ice water (100 mL),
followed by adjusting the pH value to 3 by hydrochloric acid. After
stirring for 30 min, the precipitate was filtrated and dried to give
14b as a white solid (0.88 g, yield: 63%, mp 192e195 ꢀC), which was
used without any purification.
Anhydrous K2CO3 (1.5 g) was added to a solution of 14b (0.7 g,
2 mmol) and methyl iodide (1 mL) in acetone (15 mL), the resultant
mixture was then stirred at room temperature for 20 h. After
rotaryevaporation, the residue was dissolved in DCM (30 mL), and
then was washed by 5% NaOH、5%HCl and water successively. The
separated DCM phase was dried with anhydrous Na2SO4, then fil-
trated and evaporated to give 14a as a white solid (0.7 g, yield:
5.4.13. 6-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
hexanamide (16)
In an ice-water bath, to a mixture of 8 (0.25 g,1 mmol), EDC
(0.4 g), HOBt (0.3 g) and new-preprared 6-hydroxyhexanoic acid
(0.13 g) in DCM (10 mL) was added 3-picoline (0.3 mL), and after
stirring for 24 h, the reaction solution was diluted by DCM (20 mL)
and methanol (8 mL). The organic solution was washed by 5% HCl,
5% NaOH and saturated NaHCO3 successively, dried over anhydrous
Na2SO4, and then purified by column chromatography (DCM/
EtOAc ¼ 3/1) to give 16 as a white solid (0.21 g, yield: 57%). mp
128e130 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.31e1.36 (m, 2H),
1.41e1.46 (m, 2H), 1.59e1.62 (m, 2H), 2.31e2.35 (m, 5H),
3.39e3.41(m, 2H), 4.36(t, J ¼ 5.2 Hz,1H), 7.36 (d, J ¼ 7.9 Hz, 2H), 7.49
(t, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 7.8 Hz, 1H), 7.79 (d, J ¼ 7.8 Hz, 1H), 7.91
(d, J ¼ 7.9 Hz, 2H), 8.40 (s, 1H), 10.13(s, 1H); 13C NMR (125 MHz,
99%).1H NMR (400 MHz, CDCl3):
d
¼ 2.43 (s, 3H), 2.77e2.80 (m, 4H),
3.73 (s, 3H), 7.31 (d, J ¼ 8.2 Hz, 2H), 7.45 (t, J ¼ 8.0 Hz,1H), 7.83e7.85
(m, 2H), 8.00 (d, J ¼ 8.2 Hz, 2H), 8.27 (s, 1H), 8.45 (s, 1H); 13C NMR
DMSO-d6):
d
¼ 171.7, 164.0, 163.7, 142.1, 140.2, 129.9(2C), 129.8,
(100 MHz, CDCl3):
129.7(2C), 129.6, 126.8(2C), 124.2, 122.9, 122.1, 120.8, 117.8, 52.2,
31.7, 29.3, 21.7.
d
¼ 173.9, 165.2, 164.6, 163.0, 142.7, 139.1,
126.4(2C), 123.7, 122.0, 120.9, 120.5, 116.6, 60.6, 36.5, 32.3, 25.2,
25.0, 21.0; HRMS (ESI): Calcd for C21H24N3O3 [M þ H]þ, 366.1812;
Found, 366.1833.
5.4.14. 7-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
heptanamide (17)
5.4.10. 4-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
butanamide (14)
In an ice-water bath, to a mixture of 8 (0.25 g, 1 mmol), EDC
(0.4 g), HOBt (0.3 g) and monomethyl heptanedioate (17b, 0.15 g) in
DCM (10 mL) was added 3-picoline (0.3 mL), and after stirring for
24 h, the reaction solution was diluted by DCM (20 mL) and
methanol (8 mL). The organic solution was washed by 5% HCl, 5%
NaOH and saturated NaHCO3 successively, dried over anhydrous
Na2SO4, and then purified by column chromatography (DCM/
EtOAc ¼ 10/1) to give 17a as a white solid (0.25 g, yield: 61%).
17a was reduced by NaBH4 by a procedure similar to that for the
preparation of 14 to give 17 as a white solid. mp 125e128 ꢀC; 1H
NaBH4 (0.3 g) was suspended in a solution of 14a (0.9 g,
2.46 mmol) in THF (20 mL), the mixtured was then heated to reflux.
2 mL of methanol was added to the hot solution dropwise. After
1 h's reflux, the solution was then quenched by diluted hydrochloric
acid followed by the evaporation of THF. The resultant residue was
dissolved in DCM (30 mL), MeOH (10 mL) and water (50 mL), the
organic phase was collected and dried over anhydrous Na2SO4, and
then purified by column chromatography (DCM/MeOH ¼ 20/1) to
give 14 as a white solid (0.35 g, yield: 42%). 1H NMR (500 MHz,
CDCl3):
d
¼ 1.75e1.81(m, 2H), 2.40e2.43(m, 5H), 3.46e3.50(m, 2H),
NMR (500 MHz, DMSO-d6):
d
¼ 1.23e1.27(m, 2H), 1.30e1.35(m,
4.40(t, J ¼ 5.1 Hz, 1H), 7.43(d, J ¼ 7.9 Hz, 2H), 7.53(t, J ¼ 7.8 Hz, 1H),
7.76 (d, J ¼ 7.8 Hz,1H), 7.82 (d, J ¼ 7.7 Hz,1H), 7.98 (d, J ¼ 7.9 Hz, 2H),
2H), 1.60e1.63 (m, 2H), 2.34(t, J ¼ 7.3 Hz, 2H), 2.39 (s, 3H), 3.39 (t,
J ¼ 6.4 Hz, 2H), 7.41 (d, J ¼ 7.8 Hz, 2H), 3.52 (brs, 1H), 7.52 (t,
J ¼ 7.7 Hz,1H), 7.75 (d, J ¼ 7.7 Hz,1H), 7.80 (d, J ¼ 7.7 Hz,1H), 7.96 (d,
J ¼ 7.8 Hz, 2H), 8.43 (s, 1H), 10.15(s, 1H); 13C NMR (125 MHz, DMSO-
8.43 (s, 1H), 10.08 (s, 1H); 13C NMR (125 MHz, CDCl3):
d
¼ 171.6,
164.0, 163.7, 142.1, 140.1, 129.8(2C), 129.7, 126.4(2C), 123.6, 122.1,
120.9, 120.5, 116.6, 60.1, 33.1, 28.2, 21.0; HRMS (ESI): Calcd for
d6):
d
¼ 171.7, 164.0, 163.7, 142.2, 140.2, 129.9(2C), 129.8, 126.5(2C),
C
19H20N3O3 [M þ H]þ, 338.1499; Found, 338.1437.
123.7, 122.1, 121.0, 120.6, 116.6, 60.7, 36.4, 32.4, 28.6, 25.3, 25.0, 21.1;
HRMS (ESI): Calcd for C22H26N3O3 [M þ H]þ, 380.1969; Found,
380.1933.
5.4.11. Methyl 5-oxo-5-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)
phenylamino)pentanoate (15a)
Compound 15a was prepared starting from 8 and glutaric an-
hydride through a procedure similar to that for the preparation of
14a. White solid. White solid. 1H NMR (400 MHz, CDCl3):
5.4.15. 8-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
octanamide (18)
18 was prepared by a procedure similar to that for the prepa-
ration of 17, starting from 8 and monomethyl suberate(18b). 1H
d
¼ 2.04e2.11 (m, 2H), 2.39 (s, 3H), 2.45 (t, J ¼ 7.3 Hz, 2H), 2.54 (t,
NMR (500 MHz, DMSO-d6):
d
¼ 1.23e1.25 (m, 6H), 1.39e1.42 (m,
J ¼ 7.3 Hz, 2H), 3.65 (s, 3H), 7.26 (d, J ¼ 8.0 Hz, 2H), 7.41 (t, J ¼ 8.0 Hz,
1H), 7.79 (d, J ¼ 7.8 Hz,1H), 7.88 (d, J ¼ 7.8 Hz,1H), 7.93 (d, J ¼ 8.0 Hz,
2H), 1.60e1.62 (m, 2H), 2.34 (t, J ¼ 7.3 Hz, 2H), 2.40 (s, 3H),
3.35e3.37 (m, 2H), 4.37 (t, J ¼ 5.3 Hz, 1H), 7.43 (d, J ¼ 7.8 Hz, 2H),
7.53 (t, J ¼ 7.7 Hz, 1H), 7.76 (d, J ¼ 7.7 Hz, 1H), 7.80 (d, J ¼ 7.7 Hz, 1H),
7.97 (d, J ¼ 7.8 Hz, 2H), 8.44 (s, 1H), 10.19(s, 1H); 13C NMR (125 MHz,
2H), 8.33 (s, 1H), 8.81 (s, 1H); 13C NMR (100 MHz, CDCl3):
d
¼ 173.6,
171.2, 164.7, 164.1, 142.3, 139.1, 129.7(2C), 129.6, 126.8(2C), 124.2,
123.0, 122.1, 120.7, 117.9, 51.5, 36.1, 33.0, 29.5, 21.5.
DMSO-d6):
d
¼ 171.8, 164.1, 163.8, 142.2, 140.2, 130.0(2C), 129.9,

B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
23
126.6(2C), 123.7, 122.1, 121.1, 120.6, 116.6, 60.7, 36.5, 32.5, 28.7, 28.7,
5.4.20. 6-Oxo-6-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenylamino)
hexanoic acid (22)
25.4, 25.0, 21.2; HRMS (ESI): Calcd for C23H28N3O3 [M þ H]þ,
394.2125; Found, 394.2185.
In an ice-water bath, to a mixture of 8 (0.25 g, 1 mmol), pimelic
acid (1.6 g), EDC (0.3 g) and HOBt (0.25 g) in DCM (20 mL) was
added 3-picoline (0.3 mL), and after stirring for 24 h, the reaction
solution was flash washed by 5% HCl. Vigorous stirring of the
collected DCM phase with 20mLwater precipitated mass of white
solid. The following filtration and trituration with ether gave 22
5.4.16. 9-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
nonanamide (19)
19 was prepared by a procedure similar to that for the prepa-
ration of 17, starting from 8 and monomethyl azelate (19b). mp
111e113 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.23e1.28(m, 8H),
(70 mg, yield: 18%). 1H NMR (500 MHz, DMSO-d6):
(m, 2H), 1.63e1.78 (m, 4H), 2.35e2.64 (m, 7H), 7.52 (d, J ¼ 8.0 Hz,
2H), 7.65 (t, J ¼ 7.9 Hz, 1H), 7.84e7.94 (m, 2H), 8.07 (d, J ¼ 8.0 Hz,
2H), 8.55 (s, 1H), 10.30 (s, 1H), 12.17 (brs,1H); 13C NMR (125 MHz,
d
¼ 1.44e1.48
1.38e1.40(m, 2H), 1.59e1.62(m, 2H), 2.34 (t, J ¼ 7.3 Hz, 2H), 2.40(s,
3H), 3.35e3.37(m, 2H), 4.34(brs, 1H), 7.43 (d, J ¼ 7.8 Hz, 2H), 7.53 (t,
J ¼ 8.0 Hz,1H), 7.76 (d, J ¼ 7.7 Hz,1H), 7.80 (d, J ¼ 8.0 Hz,1H), 7.97 (d,
J ¼ 7.8 Hz, 2H),8.43 (s, 1H), 10.17 (s, 1H); 13C NMR (125 MHz, DMSO-
DMSO-d6):
d
¼ 174.5, 171.7, 164.0, 163.8, 142.2, 140.2, 130.0 (2C),
d6):
d
¼ 171.8, 164.1, 163.8, 142.2, 140.2, 130.0(2C), 129.9, 126.6(2C),
129.9, 126.6 (2C), 123.8, 122.1, 121.1, 120.6, 116.7, 36.3, 33.6, 28.2,
24.8, 24.3, 21.2; Calcd for C22H22N3O4 [MꢁH]þ, 392.1610; Found,
392.1637.
123.8,122.2,121.2,120.6,116.6, 60.7, 36.5, 32.5, 28.9, 28.9, 28.7, 25.5,
25.0, 21.2; HRMS (ESI): Calcd for C24H30N3O3 [M þ H]þ, 408.2282;
Found,408.2228.
5.4.21. 3-Chloro-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
propanamide (23a)
5.4.17. 10-Hydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
decanamide (20)
In an ice-water bath, to a solution of 8 (1 g, 3.98 mmol) and 3-
picoline (1 mL) in DCM (20 mL) was added 4-chlorobutyryl chlo-
ride (0.7 mL), then let the solution raise to rt naturally. After stirring
overnight, the mixture was diluted with DCM (25 mL) and washed
with 5% HCl, 5% NaOH and saturated NaHCO3 successively, dried
over anhydrous Na2SO4, and then purified by column chromatog-
raphy (DCM/EA ¼ 10/1) to give 23a as a white solid (0.87 g, yield:
20 was prepared by a procedure similar to that for the prepa-
ration of 17, starting from 8 and monomethyl sebacate (20b). mp
111e113 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.23e1.28 (m, 10H),
1.37e1.39(m, 2H), 1.59e1.61 (m, 2H), 2.32 (t, J ¼ 7.3 Hz, 2H), 2.41 (s,
3H), 3.35e3.37 (m, 2H), 4.31(t, J ¼ 5.3 Hz, 1H), 7.44 (d, J ¼ 7.8 Hz,
2H), 7.53 (t, J ¼ 7.7 Hz, 1H), 7.77 (d, J ¼ 7.7 Hz, 1H), 7.80 (d, J ¼ 7.7 Hz,
1H), 7.98 (d, J ¼ 7.8 Hz, 2H), 8.43 (s, 1H), 10.17(s, 1H); 13C NMR
57%). mp 106e108 ꢀC; 1H NMR (500 MHz, CDCl3):
d
¼ 2.21e2.26 (m,
(125 MHz, DMSO-d6):
d
¼ 171.3, 164.1, 163.80, 142.3, 140.2,
2H), 2.35 (s, 3H), 2.63 (t, J ¼ 7.1 Hz, 2H), 3.69 (t, J ¼ 6.1 Hz, 2H), 7.33
(d, J ¼ 8.0 Hz, 2H), 7.49 (t, J ¼ 8.0 Hz, 1H), 7.61 (s,1H), 7.82 (d,
J ¼ 8.0 Hz, 1H), 7.88 (d, J ¼ 8.0 Hz, 1H), 8.02 (d, J ¼ 8.0 Hz, 2H), 8.27
(s,1H).
130.0(2C), 129.9, 126.6(2C), 123.8, 122.2, 121.1, 120.6, 116.6, 60.7,
36.5, 32.5, 29.0, 28.9, 28.8, 28.7, 25.5, 25.0, 21.2; HRMS (ESI): Calcd
for C25H32N3O3 [M þ H]þ, 422.2438; Found, 422.2477.
5.4.18. N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)hex-5-enamide
(21a)
21a was prepared by a procedure similar to that for the prepa-
ration of 17a, starting from 8 and hex-5-enoic acid (21b). White
5.4.22. 3-Iodo-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
propanamide (24a)
The mixture of 23a (0.8 g,2 mmol), NaI (4.5 g, 28 mmol) and
anhydrous acetone (30 mL) was heated to reflux, after stirring for
12 h the reaction mixture was cooled and evaporated. The resultant
residue was dissolved in DCM (30 mL) and water (50 mL), and then
the organic phase was separated, dried over anhydrous Na2SO4,
filtrated, and then evaporated to give 24a as an off-white solid
(containing 9.5% 23a), which was used in the next step without
solid; 1H NMR (400 MHz, DMSO-d6):
d
¼ 1.83e1.89 (m, 2H), 2.13 (d,
J ¼ 6.6 Hz, 2H), 2.39e2.46 (m, 5H), 4.96e5.04 (m, 2H), 5.73e5.83
(m, 1H), 7. 29 (d, J ¼ 7.9 Hz, 2H), 7.43 (t, J ¼ 7.8 Hz, 1H), 7.81 (d,
J ¼ 7.8 Hz,1H), 7.81 (d, J ¼ 7.8 Hz,1H), 7. 97 (d, J ¼ 7.9 Hz, 2H), 8.34 (s,
1H); 13C NMR (100 MHz, DMSO-d6):
d
¼ 171.9, 164.8, 164.1, 142.4,
139.0, 137.7, 137.6, 129.7(2C), 126.9(2C), 124.3, 123.1, 122.3, 120.8,
118.0, 115.4, 36.7, 33.0, 24.5, 21.6.
purification. 1H NMR (500 MHz, CDCl3):
d
¼ 2.22e2.28 (m, 2H), 2.35
(s, 3H), 2.59 (t, J ¼ 7.0 Hz, 2H), 3.31 (t, J ¼ 6.6 Hz, 2H), 7.32 (d,
J ¼ 8.0 Hz, 2H), 7.48 (t, J ¼ 8.0 Hz,1H), 7.84e7.87 (m, 2H), 7.93 (s,1H),
8.01 (d, J ¼ 8.0 Hz, 2H), 8.31 (s,1H).
5.4.19. 5,6-Dihydroxy-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
hexanamide (21)
K3 [Fe(CN)6] (0.7 g), K2CO3 (0.3 g) and K2OsO2(OH)4 (0.05 g)
were dissolved in the solution composed of tertiarybutanol (3 mL)
and water (3 mL) at 0 ꢀC, followed by the adding of 21a
(0.2 g,0.57 mmol). After reacting at 0 ꢀC for 5 h, the reaction mixture
stirred at rt for 12 h. Sodium hydrosulfite (1 g) was added to the
mixture before the inner temperature was reduced to 0 ꢀC, and
after stirring at rt for 1 h, 20 mL water was added. The resultant
precipitation was filtered and dissolved in a mixed solution of DCM
and MeOH(1/1), after filtration, the filtrate was evaporated and
triturated with ether to give 21 as a white solid (0.19 g, yield: 84%).
5.4.23. 4-Morpholino-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
butanamide (25a)
To the solution of 24a (0.2 g, 0.4 mmol) in THF (10 mL) was
added morpholine (4 mmol), and then let the solution stir over-
night. After evaporation in vacuo, the resultant residue was dis-
solved in a solution composed of DCM (25 mL) and methanol
(5 mL), washed by 5% NaOH and water, dried over anhydrous
Na2SO4 and then purified by column chromatography (DCM/
MeOH ¼ 10/1) to give 25a as a white solid (87 mg, yield: 50%). 1H
mp 149e151 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.26e1.28 (m,
1H), 1.47e1.49 (m, 1H), 1.61e1.63 (m, 1H), 1.75e1.77 (m, 1H), 2.34 (t,
J ¼ 7.4 Hz, 2H), 2.35 (s, 3H), 3.24e3.29 (m, 2H), 3.41e3.44 (m, 1H),
4.45e4.49 (m, 2H), 7.38 (d, J ¼ 7.8 Hz, 2H), 7.50 (t, J ¼ 7.7 Hz, 1H),
7.75 (d, J ¼ 7.7 Hz, 1H), 7.79(d, J ¼ 7.7 Hz, 1H), 7.93 (d, J ¼ 7.8 Hz, 2H),
NMR (500 MHz, DMSO-d6):
d
¼ 1.75e1.80 (m, 2H), 2.31e2.42 (m,
11H), 3.54e3.56 (m, 4H), 7.45 (d, J ¼ 8.0 Hz, 2H), 7.54 (t, J ¼ 7.6 Hz,
1H), 7.77 (d, J ¼ 7.6 Hz, 1H), 7.81 (d, J ¼ 8.0 Hz, 2H), 7.99 (d,
J ¼ 8.0 Hz, 2H), 8.45 (s,1H),10.18 (s,1H); 13C NMR (125 MHz, DMSO-
8.41 (s, 1H), 10.16(s, 1H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 171.8,
d6):
d
¼ 171.7, 164.4, 164.1, 142.7, 140.3, 130.3(2C), 130.2, 126.9(2C),
164.0, 163.7, 142.1, 140.2, 129.9(2C), 129.8, 126.5(2C), 123.7, 122.1,
121.0, 120.5, 116.6, 70.9, 65.9, 36.7, 32.9, 21.5, 21.1; HRMS (ESI):
Calcd for C21H23N3NaO4 [M þ Na]þ, 404.1581; Found, 404.1510.
123.9, 122.5, 121.5, 120.7, 117.0, 66.6(2C), 57.9, 53.4(2C), 34.7, 22.0,
21.4; HRMS (ESI): Calcd for C23H27N4O3 [M þ H]þ, 407.2078; Found,
407.2033.

24
B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
5.4.24. 4-(Piperidin-1-yl)-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)
phenyl)butanamide (25b)
5.4.29. 5-(4-Acetylpiperazin-1-yl)-N-(3-(5-p-tolyl-1,3,4-oxadiazol-
2-yl)phenyl)pentanamide (25g)
25b was prepared by a procedure similar to that for the syn-
thesis of 25a with the replacement of mopholine with piperidine.
In an ice-water bath, 25e (50 mg) was suspended in DCM (5 mL),
then to the mixture was added triethylamine (40 mg) and acetic
anhydride (40 mg). After raising to rt, the reaction was allowed to
stir overnight. By evaporating and then recrystallizing in ethanol, a
white solid (21 mg) was obtained. mp 160e162 ꢀC; 1H NMR
1H NMR (500 MHz, CDCl3):
d
¼ 1.21e1.29 (m, 2H), 1.55e1.59 (m,
2H), 1.78e1.81 (m, 4H), 2.00e2.05 (m, 2H), 2.45(s, 3H), 2.61e2.66
(m, 8H), 7.33 (d, J ¼ 7.9 Hz, 2H), 7.47 (t, J ¼ 7.6 Hz, 1H), 7.86e7.88 (m,
2H), 8.02 (d, J ¼ 7.9 Hz, 2H), 8.33 (s, 1H), 9.94 (s, 1H); 13C NMR
(500 MHz, DMSO-d6):
d
¼ 1.49e1.51 (m, 2H),1.62e1.64 (m, 2H),1.97
(125 MHz, CDCl3):
d
¼ 171.6, 164.8, 164.2, 142.3, 139.4, 129.7(2C),
(s, 3H), 2.37e2.40 (m, 11H), 3.40e3.42 (m, 4H), 7.43 (d, J ¼ 6.7 Hz,
2H), 7.53 (t, J ¼ 7.8 Hz, 1H), 7.76 (d, J ¼ 7.1 Hz, 1H), 7.82 (d, J ¼ 7.9 Hz,
1H), 7.97 (d, J ¼ 6.8 Hz, 2H), 8.45 (s, 1H), 10.23 (s, 1H); 13C NMR
129.6, 126.9(2C), 124.4, 123.0, 122.2, 121.1, 117.9, 57.4, 54.1(2c), 35.6,
29.7, 24.9(2C), 23.7, 21.6, 21.5; HRMS (ESI): Calcd for C25H31N4O2
[M þ H]þ, 419.2447; Found, 419.2479.
(125 MHz, DMSO-d6):
d
¼ 171.6, 168.1, 164.0, 163.7, 142.2, 140.1,
130.0(2C), 129.9,126.5(2C),123.7,122.1, 121.0, 120.5,116.6, 57.2, 52.8
(2C), 52.3(2C), 40.5, 36.1, 25.4, 22.8, 21.1; HRMS (ESI): Calcd for
5.4.25. 5-Morpholino-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
pentanamide (25c)
C
26H32N5O3 [M þ H]þ, 462.2500; Found, 462.2542.
25c was prepared by a procedure similar to that for the syn-
thesis of 25b with the replacement of 24a with 24b. White solid;
5.4.30. 6-(4-Methylpiperazin-1-yl)-N-(3-(5-p-tolyl-1,3,4-
oxadiazol-2-yl)phenyl)hexanamide (25h)
25h was prepared by a procedure similar to that for the syn-
thesis of 25d with the replacement of 24c with 24b. White solid. mp
mp 140e142 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.46e1.51 (m,
2H), 1.57e1.62 (m, 2H), 2.33e2.44 (m, 11H), 3.56 (t, J ¼ 4.4 Hz, 4H),
7.40 (d, J ¼ 8.0 Hz, 2H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.73 (s, 1H), 7.78 (d,
132e134 ꢀC; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.27e1.30 (m, 2H),
J ¼ 7.6 Hz, 1H), 7.93 (d, J ¼ 8.0 Hz, 2H), 8.42 (s, 1H), 10.21 (s, 1H); 13
C
1.40e1.43 (m, 2H), 1.59e1.62 (m, 2H), 2.11 (s, 3H), 2.21e2.39 (m,
15H), 7.41 (d, J ¼ 8.0 Hz, 2H), 7.52 (t, J ¼ 7.6 Hz, 1H), 7.75 (d,
J ¼ 7.6 Hz,1H), 7.80 (d, J ¼ 7.6 Hz,1H), 7.96 (d, J ¼ 8.0 Hz, 2H), 8.43 (s,
NMR (125 MHz, DMSO-d6):
d
¼ 171.6, 164.0, 163.7, 142.2, 140.1,
130.0(2C), 129.9, 126.6(2C), 123.7, 122.1, 121.1, 120.5, 116.6, 65.6 (2C),
57.0, 52.9(2C), 36.1, 24.9, 22.7, 21.1; HRMS (ESI): Calcd for
1H), 10.21 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 171.7, 164.0,
C
24H29N4O3 [M þ H]þ, 421.2234; Found, 421.2265.
163.8, 142.2, 140.2, 130.0(2C), 129.9, 126.6(2C), 123.7, 122.1, 121.0,
120.6, 116.6, 57.7, 54.6(2C), 52.6(2C), 45.6, 36.4, 26.5, 26.1, 25.0, 21.1;
HRMS (ESI): Calcd for C26H34N5O2 [M þ H]þ, 448.2707; Found,
448.2747.
5.4.26. 5-(4-Methylpiperazin-1-yl)-N-(3-(5-p-tolyl-1,3,4-
oxadiazol-2-yl)phenyl)pentanamide (25d)
25d was prepared by a procedure similar to that for the syn-
thesis of 25c with the replacement of mopholine with N-methyl
piperazine. White solid; mp 182e184 ꢀC; 1H NMR (500 MHz,
5.4.31. 6-Morpholino-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
hexanamide (25i)
DMSO-d6):
d
¼ 1.45e1.49 (m, 2H), 1.58e1.63 (m, 2H), 2.16 (s, 3H),
25i was prepared by a procedure similar to that for the synthesis
of 25h with the replacement of N-methyl piperazine with mor-
pholine. Whitesolid; mp 135e137 ꢀC; 1H NMR (500 MHz, DMSO-d6):
2.28e2.40 (m, 15H), 7.44 (d, J ¼ 8.0 Hz, 2H), 7.53 (t, J ¼ 7.6 Hz, 1H),
7.77 (d, J ¼ 7.6 Hz,1H), 7.81 (d, J ¼ 7.6 Hz,1H), 7.98 (d, J ¼ 8.0 Hz, 2H),
8.44 (s, 1H), 10.21 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 171.7,
d
¼ 1.30e1.33 (m, 2H), 1.41e1.46 (m, 2H), 1.59e1.64 (m, 2H), 2.24 (t,
164.1, 163.8, 142.3, 140.2, 130.0(2C), 129.9, 126.6(2C), 123.7, 122.1,
121.1, 120.6, 116.6, 57.4, 54.5(2C), 52.4(2C), 45.5, 36.2, 25.8, 22.9,
21.1; HRMS (ESI): Calcd for C25H32N5O2 [M þ H]þ, 434.2551; Found,
434.2558.
J ¼ 7.3 Hz, 2H), 2.30e2.36 (m, 6H), 2.41 (s, 3H), 3.53 (t, J ¼ 4.5 Hz, 4H),
7.44 (d, J ¼ 8.0 Hz, 2H), 7.53 (t, J ¼ 7.6 Hz, 1H), 7.77 (d, J ¼ 7.6 Hz, 1H),
7.80 (d, J ¼ 7.6 Hz, 1H), 7.98 (d, J ¼ 8.0 Hz, 2H), 8.44 (s, 1H), 10.19 (s,
1H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 171.7, 164.1, 163.8, 142.3,
140.2, 130.0(2C), 129.9, 126.6(2C), 123.7, 122.1, 121.1, 120.6, 116.6,
66.2(2C), 58.2, 53.4(2C), 36.4, 26.5, 25.7, 25.0, 21.2; HRMS (ESI):
Calcd for C25H31N4O3 [M þ H]þ, 435.2391; Found, 435.2394.
5.4.27. 5-(Piperazin-1-yl)-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)
phenyl)pentanamide (25e)
25e was prepared by a procedure similar to that for the syn-
thesis of 25d with the replacement of N-methyl piperazine with
piperazine. White solid; 1H NMR (500 MHz, DMSO-
5.4.32. 2-(4-(5-Oxo-5-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)
phenylamino)pentyl)piperazin-1-yl)acetic acid (26b)
tert-Butyl bromoacetate (2.6 g) was added dropwise to a solu-
tion of piperazine (4 g) in EtOH (40 mL) at room temperature. After
stirring overnight, the solution was evaporated in vacuo with a hot
water bath (no higher than 40 ꢀC). After 30 mL ethanol was distilled
off, the residue was poured into 50 mL of water and extracted with
ether (20 mL). The separated ether phase was washed by saturated
brine, dried over anhydrous Na2SO4, and then evaporated to afford
28 as a yellow oil (1.8 g).
d6):
d
¼ 1.45e1.47 (m, 2H), 2.25e2.41 (m, 13H), 2.65e2.67 (m, 2H),
7.44 (d, J ¼ 8.0 Hz, 2H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.77 (d, J ¼ 7.6 Hz, 1H),
7.81 (d, J ¼ 7.6 Hz, 1H), 8.00 (d, J ¼ 8.0 Hz, 2H), 8.44 (s, 1H), 10.19 (s,
1H); 13C NMR (125 MHz, DMSO-d6):
d
¼ 171.7, 164.0, 163.7, 142.2,
140.1, 130.0(2C), 129.9, 126.5(2C), 122.1, 120.6, 116.6, 107.9, 58.3,
54.2(2C), 45.6(2C), 36.3, 25.6, 23.0, 21.1; HRMS (ESI): Calcd for
C
24H30N5O2 [M þ H]þ, 420.2394; Found, 420.2371.
To the solution of 24b (0.2 g, 0.4 mmol) in THF (8 mL) and
acetonitrile (8 mL) was added 28 (70 mg) and K2CO3 (0.2 g). After
stirring for 36 h at rt, DCM (10 mL) and methanol (5 mL) was added
to dilute the reacting mixture, then filtrate the mixture. The ob-
tained filtration was evaporated and purified by column chroma-
tography (DCM/EtOAc/MeOH ¼ 25/5/1) to give 26a (0.17 g),
containing a small amount of 28 but able to use without any further
purification.
5.4.28. 5-(4-Hydroxypiperidin-1-yl)-N-(3-(5-p-tolyl-1,3,4-
oxadiazol-2-yl)phenyl)pentanamide (25f)
25f was prepared by a procedure similar to that for the synthesis
of 25d with the replacement of N-methyl piperazine with 4-
hydroxypiperidine. White solid; 1H NMR (500 MHz, DMSO-d6):
d
¼ 1.44e1.78 (m, 10H), 2.37e2.42 (m, 7H), 2.83e2.88 (m, 2H),
3.52e3.57 (m,1H), 4.70 (brs, 1H), 7.46 (d, J ¼ 8.0 Hz, 2H), 7.55 (t,
J ¼ 7.6 Hz, 1H), 7.79 (d, J ¼ 7.6 Hz, 1H), 7.82 (d, J ¼ 7.6 Hz, 1H), 8.00(d,
J ¼ 8.0 Hz, 2H), 8.47 (s, 1H), 10.29 (s, 1H); HRMS (ESI): Calcd for
In an ice-water bath, a solution of 26a (0.17 g) in DCM (15 mL)
was added dropwise to the mixture of TFA (5 mL) and DCM (5 mL)
over 10 min. After raising to rt naturally, the reaction was stirred for
C
25H31N4O3 [M þ H]þ, 435.2391; Found, 435.2425.

B.-R. Liao et al. / European Journal of Medicinal Chemistry 83 (2014) 15e25
25
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4 h and then evaporated. Dissolving the resultant residue in 20 mL
water followed by the adjusting of pH to 7 with 10% HCl resulted in
a mass of precipitation. After filtrating, the obtained solid was then
recrystallized in ethanol to give 26b (90 mg) as a white solid. mp
166e167 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
¼ 1.42e1.44 (m, 2H),
1.57e1.60 (m, 2H), 2.25e2.50 (m, 15H), 2.83 (s, 2H), 7.40 (d,
J ¼ 7.2 Hz, 2H), 7.49 (t, J ¼ 7.4 Hz, 1H), 7.71 (d, J ¼ 7.4 Hz, 1H), 7.79 (d,
J ¼ 7.4 Hz, 1H), 7.94 (d, J ¼ 7.2 Hz, 2H), 8.41 (s,1H), 10.24 (s, 1H); 13
C
NMR (100 MHz, DMSO-d6):
d
¼ 171.6, 171.6, 164.0,163.7, 142.1, 140.1,
129.9(2C), 129.7, 126.5(2C), 123.6, 122.0, 120.9, 120.5, 116.6, 62.5,
57.5, 52.7(2C), 52.3(2C), 36.2, 25.9, 23.0, 21.1; HRMS (ESI): Calcd for
C
26H30N5O4 [MꢁH]þ, 476.2298; Found, 476.2271.
5.4.33. 5-Cyano-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)phenyl)
pentanamide (27a)
To a mixture of 24b (0.22 g) with acetonitrile (8 mL) was added
TMSCN (90 mg) and TBAF (0.25 g) successively at room tempera-
ture. After stirring overnight, the reacting solution was evaporated
and purified by column chromatography (DCM/EA ¼ 5/1) to afford
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27a as a light yellow solid. 1H NMR (400 MHz, CDCl3):
d
¼ 1.67e1.71
(m, 2H), 1.88e1.94 (m, 2H), 2.43e2.46 (m, 5H), 2.64 (t, J ¼ 6.9 Hz,
2H), 7.31 (d, J ¼ 7.9 Hz, 2H), 7.44 (t, J ¼ 7.8 Hz, 1H), 7.82 (d, J ¼ 7.8 Hz,
1H), 7.97 (d, J ¼ 7.8 Hz, 1H), 8.01 (d, J ¼ 7.9 Hz, 2H), 8.52 (s, 1H), 8.98
(s, 1H).
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5.4.34. 5-(1H-tetrazol-5-yl)-N-(3-(5-p-tolyl-1,3,4-oxadiazol-2-yl)
phenyl)pentanamide (27b)
27a (0.12g), TMSN3 (0.5mL)andTBAF(0.3g)wasdissolvedinNMP
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under 120 ꢀC for 30 min. After evaporated in vacuo, the resultant
residue was purified by column chromatography (DCM/MeOH ¼ 10/
1) to give a light yellow oil, which was then triturated with DCM to
give 27b as a white solid (62 mg, yield: 59%). mp 170e171 ꢀC; 1H NMR
(400 MHz, DMSO-d6):
d
¼ 1.66e1.71 (m, 2H), 1.77e1.81 (m, 2H),
2.40e2.42 (m, 5H), 2.94 (t, J ¼ 7.2 Hz, 2H), 7.45 (d, J ¼ 7.8 Hz, 2H), 7.55
(t, J¼ 7.9 Hz, 1H), 7.77e7.83(m, 2H), 7.99(d, J ¼ 7.8Hz,2H), 8.45(s,1H),
10.21 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d
¼ 171.3, 164.0, 163.7,
155.8,142.2,140.0,130.0(2C),129.9,126.5(2C),123.7,122.1,121.1,120.6,
116.6, 35.8, 26.6, 24.3, 22.5, 21.1. HRMS (ESI): Calcd for C21H22N7O2
[M þ H]þ, 404.1835; Found, 404.1865.
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Acknowledgments
This research was financially supported by Shanghai Science and
Technology
Council
(No
12142200800,
13142200900,
13DZ2290300), National Science and Technology Major Projects for
Major New Drugs Innovation and Development (2012ZX09304011
and 2013ZX09507002), National science Fund for Distinguished
Young Scholars (81125023). We also thank the Laboratory of Organic
Functional Molecules, Sino-French Institute of ECNU for support.
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.011.
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