Identification and Structure–Activity Relationship (SAR) of potent and selective oxadiazole-based agonists of sphingosine-1-phosphate receptor (S1P1)

Article abstract of DOI:10.1016/j.bioorg.2018.09.008

Agonism of S1P₁ receptor has been proven to be responsible for peripheral blood lymphopenia and elicts the identification of various S1P₁ modulators. In this paper we described a series of oxadiazole-based S1P₁ direct-acting agonists disubstituted on terminal benzene ring, with high potency for S1P₁ receptor and favorable selectivity against S1P₃ receptor. In addition, two representative agents named 16-3b and 16-3g demonstrated impressive efficacy in lymphocyte reduction along with reduced effect on heart rate when orally administered. Furthermore, these compounds have been shown to possess desired pharmacokinetic (PK) and physicochemical profiles. The binding mode between 16-3b and the activated S1P₁ model was also studied.

Full text of DOI:10.1016/j.bioorg.2018.09.008

Bioorganic Chemistry 82 (2019) 41–57
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification and Structure–Activity Relationship (SAR) of potent and
selective oxadiazole-based agonists of sphingosine-1-phosphate receptor
(
S1P )
1
a
a
a
b
a
b
a
Tianqi Liu , Jing Jin , Yonghui Chen , Qiumu Xi , Jinping Hu , Wenqiang Jia , Xiaoguang Chen ,
a
a,b,⁎
a,b
Yan Li , Xiaojian Wang
, Dali Yin
a
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of
Medical Sciences, Beijing 100050, PR China
b
Department of Medicinal Chemistry, Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Peking Union
Medical College and Chinese Academy of Medical Sciences, Beijing 100050, PR China
A R T I C L E I N F O
A B S T R A C T
Agonism of S1P
Keywords:
1
receptor has been proven to be responsible for peripheral blood lymphopenia and elicts the
S1P
1
agonist
identification of various S1P modulators. In this paper we described a series of oxadiazole-based S1P direct-
1
1
Oxadiazole
acting agonists disubstituted on terminal benzene ring, with high potency for S1P
1
receptor and favorable se-
SAR
lectivity against S1P receptor. In addition, two representative agents named 16-3b and 16-3g demonstrated
3
Lymphocyte
Cascade modification
impressive efficacy in lymphocyte reduction along with reduced effect on heart rate when orally administered.
Furthermore, these compounds have been shown to possess desired pharmacokinetic (PK) and physicochemical
profiles. The binding mode between 16-3b and the activated S1P model was also studied.
1
1
. Introduction
FTY720 can be phosphorylated to (S)-FTY720-P, which exhibits affinity
for 4 of the 5 S1P receptors (S1P1,3,4,5) [18–21]. During the clinical trial
of FTY720, several researches reveal that there exist some potential side
Sphingolipids are important plasma-membrane lipids, where
sphingosine-1 phosphate (S1P, Fig. 1) is the endogenous ligand for a
family of five G-protein coupled receptors known as S1PR1-5, regulating
a variety of physiological processes including cell differentiation, vas-
cular stabilization, inflammation, endothelium integrity and angio-
effects which are related to the activation of S1P receptor in rodent,
3
such as the risk of cardiovascular effects [22,23]. On the other hand, the
overlong half-life of FTY720 raises some concerns about the prognosis
after the drug administration is ceased. As a result, more potent S1P -
3
genesis [1–5]. In recent studies, S1P
1
receptor has become an attractive
sparing S1P
1
direct-acting agonists with improved pharmacokinetic
drug target, involved in occurrence and development of many diseases,
profile remain to be the focus in drug discovery.
especially for immune-mediated diseases [6,7]. Notably, sustained ac-
Despite structural diversity of S1P agonists, the key features can be
1
tivation of S1P
1
receptor by the synthetic modulators instead of S1P
generally divided into three parts: polar head, aromatic region, and
results in the internalization and degradation of S1P receptors, thereby
1
lipophilic tail. In our previous study, highly predictive 3D QSAR
lymphocytes are sequestrated in the lymph nodes and secondary lym-
phoid organs, restricting the auto immune reactivity, which is me-
chanistically identified as “functional antagonism” [8–12].
pharmacophore model of S1P agonists has been constructed, which
1
indicated that properly rigid hydrophobic region was favorable for
S1P /S1P receptor selectivity [24]. Moreover, in 2012 Stevens’s group
disclosed the crystal structure of the S1P receptor fused to T4-lysozyme
(S1P
1
3
Along with the research for the mechanism of S1P
1
receptor toward
1
lymphocyte migration, a great deal of effort has been devoted to the
discovery of potent S1P agonists, including FTY720 (2), BAF312, ACT-
2800, ONO-4641 (3), RPC1063 (4), etc. [13–16]. In 2010, Fingolimod
1
-T4L) in complex with an antagonist ML056, which suggested that
1
binding pocket appeared to be amphiphilic and significant π-π stacking
was observed between Phe125 and aromatic heterocyclic region [25].
Inspired by the valuable results above, we intended to employ di-
phenyl ether scaffold to maintain the rigidity of hydrophobic tail, which
1
(
FTY720, Fig. 1) is the first oral drug approved for the treatment of
relapsing remitting multiple sclerosis (RRMS) [17]. As a pro-drug,
⁎
Corresponding author at: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Peking Union Medical
College and Chinese Academy of Medical Sciences, Beijing 100050, PR China.
E-mail address: wangxiaojian@imm.ac.cn (X. Wang).
https://doi.org/10.1016/j.bioorg.2018.09.008
Received 5 July 2018; Received in revised form 31 August 2018; Accepted 7 September 2018
Available online 11 September 2018
0045-2068/ © 2018 Elsevier Inc. All rights reserved.

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
Fig. 1. Structures of sphingosine, Fingolimod (FTY720), and their phosphates.
Fig. 2. Design of oxadiazole-based S1P modulators.
1
is derived from CS2100 (5, Fig. 2), a potent S1P
1
agonist with 5000-fold
from aryl halides (9) by using isopropylmagnesium chloride and 1-
formylpiperidine [28]. Initially, aromatic aldehydes (10) were con-
verted to acetals (11) under the catalysis of PTSA to protect the formyl
group in advance [29]. Subsequent addition reaction was performed
between 11 and hydroxylamine hydrochloride to provide N'-hydro-
xybenzimidamides (12) [30]. Intermediates 12 were then coupled
with different substituted benzoic acids, which were commercially
available or prepared according to literature in two steps, employing
HOBt and EDCI·HCl as activating reagents to afford 1,2,4-oxadiazoles
13 [31]. After deprotection of 13 with hydrochloric acid, the corre-
sponding aldehydes 14 were then reacted with amino carboxylic ester
or alkamine through reductive amination, using either sodium cya-
noborohydride or sodium triacetoxyborohydride as the reducing agent
to give corresponding intermediates 15 or alkamine end-products
selectivity against S1P receptor reported by Nakamura et al. [26]
3
Meanwhile, in order to further decrease the electricity of aromatic re-
gion, the oxadiazole group derived from CYM5442 and CS-2100 was
also adopted as part of the linker to enhance its interaction with S1P
1
receptor [27]. As illustrated in Fig. 2, selective S1P
synthesized on the basis of rational design. After several rounds of
cascade modification, highly potent oxadiazole-based S1P agonists
1
modulators were
1
(
structures 8) were identified, where terminal phenyl ring was dis-
ubstituted, represented by compound 16-3b and 16-3g. In addition to
the exploration of structure–activity relationship (SAR) in vitro, effects
on lymphocyte reduction and heart rate, pharmacokinetic and physi-
cochemical profiles, in silico docking results were fully evaluated and
analyzed in detail.
[
32,33]. Finally, hydrolysis of amino carboxylic esters 15 with lithium
2
. Results and discussion
hydroxide followed by hydrochloric acid treatment afforded the de-
sired hydrochloride of oxadiazole-based derivatives 16. Besides,
wittig reaction of 14a with Methyl (triphenylphosphoranylidene)
acetate was carried out to prepare substituted α, β-unsaturated ester
2.1. Chemistry
The synthetic pathway utilized in the preparation of oxadiazole-
17 [34]. Reduction of 17 was performed with Pd/C and H , sub-
2
based derivatives is outlined in Scheme 1. Polysubstituted aromatic
sequent hydrolysis of the reduction product furnished phenylpro-
aldehydes (10) were either commercially available or easily prepared
pionic acid derivative 16-1l.
42

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
Scheme 1. Synthesis of 1,2,4-oxadiazole derivatives. Reagents and Conditions: (a) N-Formylpiperidine, i-PrMgCl, THF, 0 °C, 2 h; (b) Ethylene glycol, PTSA, Toluene,
reflux; (c) NH OH-HCl, NaHCO , MeOH, reflux; (d) Substituted benzoic acid, HOBt, EDCI, K CO , DMF, 110 °C; (e) 2 mol/L HCl, Acetone, 55 °C; (f) Amino acid ester
2
3
2
3
(
hydrochloride) or alkamine, DIPEA, AcOH, Sodium cyanoborohydride, DCM + MeOH; (g) 0.5 mol/L LiOH, MeOH, Acidification; (h) Methyl (triphenylpho-
sphoranylidene)acetate, DMF; (i) H
plementary Material.
2
, Pd/C, EA. For the structure of compounds 13a∼13 l, 14a∼14 l, 15-1a∼15-3n and 16-1a∼16-3n see Tables 1–3 and Sup-
2
.2. Biological evaluation
model would be discussed in later docking studies. Compounds 16-
1
q∼16-1s showed almost equipotent to S1P receptor, while the so-
1
All compounds were evaluated for affinity and selectivity against
lubility of 16-1q appeared to be much better than 16-1r. 3-cyano-4-
isopropoxy was then determined to be the optimized piece for the SAR
exploration of the rest scaffold.
the S1P receptor in an HTRF-IP1 functional assay [35,36]. As illu-
1
strated in Table 1, phenoxy on the 4-position of terminal benzene was
selected as the lipophilic tail, various polar groups were screened for
As described in Table 2, with terminal phenyl ring disubstituted
with isopropoxy and nitrile group, a number of compounds listed below
human S1P
1
and S1P receptor agonism at first. Among the selected
3
groups, glycine (16-1a), pyrrolidine-3-carboxylic acid (16-1d), sarco-
exhibited satisfied EC50 (< 10 nM) and selectivity at S1P
1
receptor,
receptor
sine (16-1f), aspartic acid (16-1j) and 2-(piperidin-4-yl) acetic acid (16-
demonstrating that with the suitable lipophilic tail, the S1P
1
1
k) exhibited moderate nanomolar S1P
1
agonistic activities in the same
can accommodate various moieties in the polar head. Meanwhile, in
order of magnitude. However, in the case of pyrrolidine-3-carboxylic
acid functioned as the polar head, a relative loss in S1P /S1P se-
lectivity was observed. Additionally, proline and 2-amino-2-methyl-
comparison with other derivatives, compounds 16-2f and 16-2i were
1
3
relative less potent on S1P receptor. The former might due to the ab-
1
sence of carboxyl or alcoholic hydroxyl group in polar motif, which
propanoic acid displayed poor potency for S1P
1
receptor, which in-
resulted in scarcely any electrostatic interaction vs S1P receptor. In
1
dicated that steric hindrance and high rigidity in polar head are
disadvantageous for affinity. To keep proper rigidity and O/W partition
coefficient, glycine was chosen for next investigation.
terms of the later, the inappropriate length and rigidity of 4-aminocy-
clohexane-1-carboxylic acid should be accounted for the decrease of
potency on S1P receptor. On the basis of the SAR analysis, five ap-
1
Thus, we extended our studies to compounds wherein glycine was
maintained as the polar head, evaluating the influence of substitution
on the benzene ring of lipophilic tail. It was found that replacing the
phenoxy by bulk group led to no measurable agonist activity against
proving polar head groups were selected for subsequent optimization,
including glycine, sarcosine, pyrrolidine-3-carboxylic acid, aspartic
acid and 2-(piperidin-4-yl) acetic acid.
Since the polar head has been investigated in detail, a particular
attention was focused on exploring substitution at the phenyl ring of
aromatic region. As shown with examples in Table 3, the disubstituted
pattern of terminal benzene ring with 3-cyano-4-isopropoxy was
maintained, when fluorine was introduced in the aromatic ring, most
S1P receptor, exemplified by phenyl or tertiary butyl monosubstituted
1
compounds 16-1m and 16-1n. Interestingly, potency was regained
when terminal phenyl ring was disubstituted with isopropoxy and other
specific groups (16-1q∼16-1s), which proved to be important factors
contributing to favorable S1P
1
agonistic activity. Compared the obvious
compounds displayed fairly high potency for S1P receptor, especially
1
difference of S1P
1
agonistic activity between 16-1p and 16-1q, this was
for compound 16-3b with an EC50 of 0.46 nM. Besides, it was worth
might due to the electric distinction of aromatic region as well as the
necessity of proper steric effect on terminal benzene ring, further ana-
noting that removing halogen at the phenyl ring furnished compounds
with considerable S1P
1
agonist activities and good S1P selectivity over
1
lysis of binding mode between compounds and the activated S1P
1
S1P receptor, which suggested that halogen is not indispensable for the
3
43

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
Table 1
SAR of design and optimization in polar head and lipophilic tail.a
No
R
1
R
2
R
3
EC50 (nM)
hS1P
1
hS1P
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6-1a
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenoxy
phenyl
H
Glycine
39.5
111
204
59.2
415
52.9
> 10,000
4610
6-1b
6-1c
6-1d
6-1e
6-1f
6-1g
6-1h
6-1i
H
Alanine
H
3-Aminopropanoic acid
pyrrolidine-3-carboxylic acid
Proline
> 10,000
671
H
H
> 10,000
> 10,000
> 10,000
2919
H
Sarcosine
H
Azetidine-3-carboxylic acid
2-Amino-2-methylpropanoic acid
Piperidine-4-carboxylic acid
Aspartic acid
> 10,000
368
H
H
472
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
6-1j
H
18.3
6-1k
6-1l
H
2-(Piperidin-4-yl) acetic acid
Acetic acid
18.2
H
99.8
6-1m
6-1n
6-1o
6-1p
6-1q
6-1r
6-1s
6-1t
H
Glycine
> 10,000
> 10,000
824
tert-butyl
isopropyl
isopropoxy
isopropoxy
isopropoxy
isopropoxy
isopropoxy
H
Glycine
H
Glycine
H
Glycine
> 10,000
26.2
cyano
Glycine
trifluoromethyl
chlorine
methyl
Glycine
17.1
Glycine
26.2
Glycine
53.6
a
EC50 is the mean of three experimental determinations.
interaction between S1P
1
receptor and agonists. Remarkably, com-
size of lipophilic tail left the affinity and selectivity for S1P receptor
1
pound 16-3g was very efficacious as well, similar in potency to com-
pound 16-3b with an EC50 of 0.47 nM, which spurred interest in further
research.
largely unchanged. Nevertheless, the substitution of aspartic acid and 2-
(piperidin-4-yl) acetic acid led to slight reduction in potency, which
confirmed that the S1P binding pocket applied restriction on the shape
1
In the last series, we intended to replace isopropoxy by cyclopentyl
to evaluate the influence of rigidity of hydrophobic region, while
keeping the cyano group to retain its electric and steric function on
aromatic region. Apparently, under this circumstance, increasing the
and size of agonists to some extent, supporting our initial assumption
about the significance of proper rigidity in S1P
selectivity.
1
agonistic activity and
In general, the cascade optimization afforded a relatively elaborate
SAR study, coming up with several compounds which performed
Table 2
SAR of design and optimization in polar head.a
Table 3
a
SAR of design and optimization in aromatic region, lipophilic tail and polar head.
No
R
EC50 (nM)
No
R
1
R
2
R
3
EC50 (nM)
hS1P
1
hS1P
3
hS1P
1
hS1P
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6-2a
Sarcosine
6.2
4.8
1.71
1.1
2.9
70
> 10,000
> 10,000
4737
6-2b
6-2c
6-2d
6-2e
6-2f
6-2g
6-2h
6-2i
Azetidine-3-carboxylic acid
Aspartic acid
16-3a
16-3b
16-3c
16-3d
16-3e
16-3f
16-3g
16-3h
16-3i
16-3j
16-3k
16-3l
16-3m
16-3n
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
F
F
F
F
Glycine
1.37
0.46
1.2
33
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
1940
Sarcosine
Pyrrolidine-3-carboxylic acid
2-(Piperidin-4-yl) acetic acid
Pyrrolidin-2-one
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
Aspartic acid
2-(Piperidin-4-yl) acetic acid
Glycine
H
H
H
H
H
H
H
H
H
H
4.7
1.3
0.47
2.5
21
3-(Methylamino)propanoic acid
Piperidine-3-carboxylic acid
4-Aminocyclohexane-1-carboxylic acid
Serine
4.1
1.7
121
1.46
8.3
3.4
6.6
1.0
1.0
Sarcosine
Pyrrolidine-3-carboxylic acid
Aspartic acid
6-2j
2-(Piperidin-4-yl) acetic acid
Glycine
6-2k
6-2l
Homoserine
8.7
6.4
3.6
31
Glutamic acid
Sarcosine
5-2m
5-2n
5-2o
2-Aminopropane-1,3-diol
2-Aminoethan-1-ol
Pyrrolidine-3-carboxylic acid
Aspartic acid
1-Aminopropan-2-ol
2-(Piperidin-4-yl) acetic acid
123
> 10,000
a
a
EC50 is the mean of three experimental determinations.
EC50 is the mean of three experimental determinations.
44

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
Table 4
lipophilicity efficiency (LLE) calculated by both the activity and lipo-
philicity of compound, was basically recommended over 6 to maintain
proper druggability for oral drugs. Therefore, LogD at pH 7.4 and LLE
were also measured to evaluate the lipophilicity and efficiency com-
prehensively on a Gemini Profiler instrument (pION) by potentiometric
titration method. To our delight, the whole compounds exhibited log D
Lymphocyte count, plasma concentrations of compounds 16-2d, 16-2e, 16-3b,
1
6-3g and 16-3l after 3 mg/kg oral administration to SD rats.a
No
LC reduction (%)
Plasma concentration (ng/mL)
4
h
8 h 12 h
24 h
4 h
8 h
12 h
24 h
(
pH 7.4) < 3 and LLE > 6, which provided satisfying balance of S1P
1
RPC1063 −5
−30 −80 −35 23.98
15.2
4.38
424
13.36
1.83
2.4
potency and druglike properties, indicating promising prospect of the
1
1
1
1
1
6-2d
6-2e
6-3b
6-3g
6-3l
−54 −38 −38 131
97.24
673.8
BLQ
11.25
BLQ
2.34
BLQ
−27 +36 −36 154
251.98
5.28
following modification.
−74 −82 −91 −47 44.46
−52 −90 −83 −64 153.2
11.59
35.19
9.98
It is well established from the profiles above that compound 16-3b
and 16-3g are promising in pharmacodynamics and pharmacokinetics
studies. In consequence, they were further tested for effect on heart rate
at dose of 10 mg/kg oral administration to rats. As summarized in
Fig. 4, compared with marketed drug FTY720, neither of these two
compounds had obvious influence on heart rate, let alone induced
bradycardia. Additionally, the apparent decrease in the risk of cardio-
vascular side effect appeared to be driven by improvement in selectivity
9.14
−23 −73 53
7
54.54
3.04
a
Data are expressed as mean ± SD of three animals.
superior in selective S1P
excellent functional potency for S1P
profile for S1P receptor were advanced to mice models for evaluating
1
receptor agonism. Hence, 5 compounds with
1
receptor and desirable selectivity
3
against S1P
3
receptor, which confirmed the importance of developing
/S1P modulators.
their ability in lymphocytes reduction, including 16-2d, 16-2e, 16-3b,
selective S1P
1
3
1
6-3g and 16-3l.
As outlined in Table 4, blood lymphocyte reduction along with
plasma exposure was measured at multiple time points, after oral ad-
ministration of the compounds discussed above at dose of 3 mg/kg. As
expected, in comparison with positive control RPC1063, most com-
pounds displayed delightful levels of lymphopenia in vivo efficacy,
especially for compound 16-3b and 16-3g, which provided around 90%
decrease of lymphocyte counts. In addition, the largest lymphocyte
reduction occurred at 8–12 h, which was generally consistent with the
agonist activity trends observed before (Fig. 3). Overall, the lymphocyte
reduction usually recovered to baseline 24 h after administration. In the
meantime, the plasma concentration almost vanished thoroughly, in-
dicating the relative rapid onset of function as well as complete re-
covery of lymphopenia.
2.3. Molecular docking
To rationalize the structure activity relationships (SARs) in this
study, compound 16-3b and 16-3g were evaluated by docking studies
in order to better understand the ligand-protein interactions. The active
state of S1P model was induced by Gaussian accelerated molecular
1
dynamics, which was used as receptor for docking study. The binding
mode analysis was then performed by Schrödinger and PyMOL [37]. As
is evident from Fig. 5, the docking result of 16-3b in binding pocket was
presented in 3D diagram (Fig. 5a). The charged carboxyl group in polar
head strongly contacted with Arg120 via salt bridge and established an
H-bond with Tyr29, while α-amino group linked to Glu121 by elec-
tronic interaction as well. Phenyl moiety in lipophilic tail was adjacent
to Phe125, a π-π stacking interaction was found for both compounds,
which was enhanced by electron-withdrawing effect of nitrile group
To gain insight into the metabolic feature in vivo, pharmacokinetics
studies in mice were subsequently assessed at dose of 3 mg/kg, with
RPC1063 as the positive control. It was of note that compound 16-2e
exhibited rather high blood concentration (Cmax) and area under the
curve (AUC(0-t)) in oral administration, which was likely due to lower
plasma clearance. On the other hand, other compounds possessed
moderate Cmax, differences in Cmax and clearance might be ascribed to
plasma protein binding and tissue distribution. Besides, as depicted in
Table 5, all these compounds showed relatively short t1/2, which
achieved our previous goal of properly curtailing t1/2 to improve
and proved to be obligatory for S1P agonist activity. In addition, iso-
1
propoxy group and nitrile group of terminal benzene ring were involved
in hydrophobic interaction with some lipophilic amino acid residues,
such as Phe210, Trp269, Leu272, Phe273 and Leu276. Besides, com-
paring the binding mode of 16-3b and 16-3g (Fig. 5a), they behaved
similar in the cavity of S1P receptor, where all the significant contacts
1
mentioned above could be observed. Moreover, in terms of the docking
result between compound 16-3g and 16-3i (Fig. 5b), nearly every bond
length of important contacts attached to 16-3g was shorter than that of
prognosis. In light of outstanding S1P agonist activity and lympho-
1
penia of compound 16-3b and 16-3g, they were further progressed to
intravenous administration, where 16-3g was observed for favorable
oral bioavailability of 50.31%.
1
3
6-3i, which further proved the reasonability of high efficiency in 16-
g. Hence, the computational assay depicted a general binding mode
Moreover, it is recognized that both pharmacological activity and
druggability are essential elements in drug discovery. Ligand
along with key interactions, which elucidated high S1P
1
potency of 16-
3
b and 16-3g primarily.
1
00
3
. Conclusion
9
0
0
0
0
0
0
0
0
0
8
7
6
5
4
3
2
1
In summary, a series of potent oxadiazole-based S1P
agonists possessing specific disubstitution on terminal benzene ring was
identified with favorable selectivity against S1P receptor. Taking
multiple pharmacological parameters into consideration, cascade and
direct-acting
1
Control
RPC1063
3
1
6-2d
6-2e
1
targeted structural modification led to elaborate SAR study of S1P
1
16-3b
selective modulators. Moreover, most compounds demonstrated im-
pressive efficacy in vitro, while two representative compounds 16-3b
and 16-3g (EC50 < 1 nM) displayed satisfied lymphopenia activities in
vivo and exhibited reduced effect on heart rate. Apart from the im-
provement in pharmacokinetics properties, the two compounds also
possessed desired physicochemical features. As a consequence, further
optimization and evaluation of this scaffold is ongoing to provide more
1
6-3g
6-3l
1
0
Control RPC1063 1 6-2d
1 6-2e 1 6-3b 1 6-3g
1 6-3l
Fig. 3. Maximum LC reduction of compounds 16-2d, 16-2e, 16-3b, 16-3g and
6-3l in SD rats.
potent and selective S1P
files.
1
agonists with superior pharmacokinetic pro-
1
45

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
Table 5
Pharmacokinetic parameters after 3 mg/kg oral administration in SD rats and physicochemical profiles of compounds 16-2d, 16-2e, 16-3b, 16-3g and 16-3l.a,b
No
C
max (ng/ml)
Tmax (h)
T
1/2 (h)
AUC0-t (ng·h/mL)
CLz (ml/(h·kg))
Vz (ml/kg)
log D (pH 7.4)
LLEc
RPC1063
26.4
3.5
1
5.95
1.75
2.66
1.71
1.98
1.73
306.87
3998.89
9434.1
388.133
1053.88
367.98
9709.98
1612.01
329.46
77503.5
2981.2
–
–
1
1
1
1
1
6-2d
6-2e
6-3b
6-3g
6-3l
895
1.79
1.38
1.76
1.49
2.19
7.17
7.16
7.58
7.84
6.25
1419
86.32
232.8
74.32
1.4
1.8
2.8
2.2
1261.34
20719.15
8144.34
19620.8
8373.71
2946.96
8381.94
a
b
c
Data are expressed as mean ± SD of three animals.
Data are expressed as mean of three measurements.
LLE = pIC50 − ClogP.
Control
FTY720
16-3b
16-3g
performed on Biotage Isolera one. All the solvents and chemicals were
obtained from commercial sources and used without further purifica-
tion.
5
4
4
3
3
2
2
40
90
40
90
40
90
40
4
.1.2. General procedure for the synthesis of 2-halogen-4-
formylbenzonitrile (10a∼b)
To a solution of 4-halogen-2-chlorobenzonitrile (23.10 mmol) in dry
THF under Argon at 0 °C was added isopropyl magnesium chloride
(30.03 mmol, 2 M solution in ether) dropwise during 20 min, and the
reaction mixture was stirred for 2 h, keeping the temperature around
0 °C. Then 1-formyl piperidine (30.03 mmol) was added dropwise at
0 °C and stirred for additional 2 h at 0 °C. The reaction mixture was
0
h
2 h
4 h
8 h
Time (hr)
1 2 h
2 4 h
quenched with saturated NH Cl solution and extracted with ethyl
4
Fig. 4. Effect on heart rate after 10 mg/kg oral administration of compound 16-
b and 16-3g to SD rats.
acetate. The combined organic layers were washed with brine, dried
3
over Na
2
4
SO , then filtered and concentrated. The residue was purified
by silica gel flash column chromatography (PE/EtOAc = 3:1) to afford
4
. Experimental
compound 10a and 10b.
4
.1. Chemistry
4
.1.2.1. 2-Chloro-4-formylbenzonitrile (10a). Yield:65.9%; White solid;
4.1.1. General experimental information
1
Mp: 114–115 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.07 (s, 1H),
All reagents and solvents were obtained from commercial sources
8
.23–8.22 (m, 2H), 8.02 (d, J = 7.6 Hz, 1H); MS (ESI) m/z 166.0 (M
and used without further purification. Melting points were determined
on Yanaco MP-J3 microscope melting point apparatus. NMR spectra
were recorded on Mercury-400, Mercury-500, and Mercury-600 spec-
trometers. Chemical shifts are referenced to the residual solvent peak
and reported in ppm (δ scale) and all coupling constant (J) values are
given in Hz. ESI-HRMS data were measured on Thermo Exactive
Orbitrap plus spectrometer. Flash column chromatography was
+
+
H) .
4
.1.2.2. 2-Fluoro-4-formylbenzonitrile (10b). Yield: 53.1%; White solid;
1
Mp: 87–88 °C; H NMR (400 MHz, DMSO‑d ) δ 10.07 (s, 1H), 8.20 (dd,
6
J = 8.0, 6.4 Hz, 1H), 7.99 (dd, J = 9.2, 1.2 Hz, 1H), 7.94 (dd, J = 7.8,
.2 Hz, 1H); MS (ESI) m/z 150.0 (M+H)+
1
.
Fig. 5. Docking poses of compound 16-3b, 16-3g and 16-3i within S1P
1
binding pocket. (5-1) 3D binding modes of compound 16-3b (orange sticks) and 16-3g
green sticks). Ionic bonds are reported as dotted red lines, π-π stacking is reported as dotted blue lines. (5-2) 3D binding modes of compound 16-3i (A) and 16-3g
B). Ionic bonds are reported as dotted red lines, π-π stacking is reported as dotted yellow lines.
(
(
46

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
4
.1.3. General procedure for the synthesis of 4-(1,3-dioxolan-2-yl)
with brine, dried over Na
2
SO
4
, filtered and concentrated. The residue
benzonitrile (11a∼c)
was purified by silica gel flash column chromatography (PE/
In a round flask was placed 4-formylbenzonitrile (15.10 mmol),
EtOAc = 3:1) to afford compound 13a∼l.
ethane-1,2-diol
(60.39 mmol)
and
p-toluenesulfonic
acid
(
1.2 mmol) as catalyst. The mixture was dissolved in dry toluene
and stirred for 18 h under reflux, with the continuous removal of
O in a Dean–Stark apparatus. The solvent was removed by dis-
tillation, the residue was redissolved in EtOAc (15 mL) and then
neutralized with saturated aqueous NaHCO solution. The aqueous
phase was extracted with EtOAc. The combined organic layers were
4.1.5.1. 3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-5-(4-phenoxyphenyl)-
1
1,2,4-oxadiazole (13a). Yield: 64.7%; White solid; Mp: 79–81 °C;
H
H
2
NMR (500 MHz, DMSO‑d ) δ 8.20 (d, J = 6.8 Hz, 2H), 8.04 (d,
6
J = 6.0 Hz, 1H), 7.72 (s, 1H), 7.62 (d, J = 6.0 Hz, 1H), 7.50 (t,
3
J = 6.2 Hz, 2H), 7.29 (t, J = 6.0 Hz, 1H), 7.21–7.18 (m, 4H), 5.87 (s,
1H), 4.12–3.97 (m, 4H); MS (ESI) m/z 421.1 (M+H)+
.
washed with brine, dried over Na
2
SO , then filtered and con-
4
centrated. The residue was purified by silica gel flash column
4
.1.5.2. 5-([1,1′-Biphenyl]-4-yl)-3-(2-chloro-4-(1,3-dioxolan-2-yl)
chromatography (PE/Acetone/DCM = 24:4:3) to afford compound
phenyl)-1,2,4-oxadiazole (13b). Yield: 61.8%; White solid; Mp:
1
1a∼c.
1
1
8
2
7
4
34–137 °C; H NMR (500 MHz, DMSO‑d
6
) δ 8.30 (d, J = 8.0 Hz, 2H),
.11 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 7.5 Hz,
H), 7.76 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H),
.49 (t, J = 7.5 Hz, 1H), 5.91 (s, 1H), 4.12–4.03 (m, 4H); MS (ESI) m/z
4
.1.3.1. 2-Chloro-4-(1,3-dioxolan-2-yl)benzonitrile (11a). Yield: 74.2%;
1
White solid; Mp: 60–61 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.02 (d,
J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 5.86 (s, 1H),
05.1 (M+H)+
.
+
4
.09–3.94 (m, 4H); MS (ESI) m/z 210.0 (M+H)
.
4
.1.5.3. 5-(4-(tert-Butyl)phenyl)-3-(2-chloro-4-(1,3-dioxolan-2-yl)
4
.1.3.2. 4-(1,3-Dioxolan-2-yl)-2-fluorobenzonitrile (11b). Yield: 78.4%;
phenyl)-1,2,4-oxadiazole (13c). Yield: 62.2%; White solid; Mp:
1
Colorless oil; H NMR (400 MHz, DMSO‑d
6
) δ 7.96 (dd, J = 8.0, 6.4 Hz,
1
7
8
5
7–79 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.11 (d, J = 8.4 Hz, 2H),
1
4
H), 7.54 (dd, J = 10.0, 1.2 Hz, 1H), 7.48–7.46 (m, 1H), 5.86 (s, 1H),
+
.05 (d, J = 8.0 Hz, 1H), 7.72–7.67 (m, 3H), 7.62 (d, J = 8.0 Hz, 1H),
.08–3.94 (m, 4H); MS (ESI) m/z 194.1 (M+H)
.
.87 (s, 1H), 4.12–3.96 (m, 4H), 1.33 (s, 9H); MS (ESI) m/z 385.1 (M
+
+
H)
.
4
.1.3.3. 4-(1,3-Dioxolan-2-yl)benzonitrile (11c). Yield: 72.5%; White
1
solid; Mp: 32–35 °C;
H
NMR (400 MHz, DMSO‑d ) δ 7.87 (d,
6
4
.1.5.4. 3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-5-(4-isopropylphenyl)-
J = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 5.83 (s, 1H), 4.08–3.94 (m,
1
1
,2,4-oxadiazole (13d). Yield: 47.5%; White solid; Mp: 53–55 °C;
H
+
4
H); MS (ESI) m/z 176.1 (M+H)
.
NMR (500 MHz, DMSO‑d
6
) δ 8.11 (d, J = 8.0 Hz, 2H), 8.05 (d,
J = 8.0 Hz, 1H), 7.72 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.54 (d,
J = 8.0 Hz, 2H), 5.87 (s, 1H), 4.10–3.98 (m, 4H), 3.02 (hept,
4
.1.4. General procedure for the synthesis of 4-(1,3-dioxolan-2-yl)-N′-
hydroxybenzimidamide (12a∼c).
J = 7.0 Hz, 1H), 1.25 (d, J = 7.0 Hz, 6H); MS (ESI) m/z 371.1 (M
To a solution of 4-(1,3-dioxolan-2-yl) benzonitrile (10.97 mmol) in
MeOH was added hydroxyamine hydrochloride (38.40 mmol) and
+
+
H)
.
NaHCO
3
(43.89 mmol) and heated to reflux for 5 h. The reaction
4
.1.5.5. 3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-5-(4-
mixture was then cooled to rt, then filtered and concentrated. The
filtrate was diluted with EtOAc, washed with brine, dried over
isopropoxyphenyl)-1,2,4-oxadiazole (13e). Yield: 42.3%; White solid;
1
Mp: 88–90 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.12–8.09 (m, 2H),
Na
2
SO , filtered and concentrated. The residue was purified by silica
4
8
1
1
.03 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 1.2 Hz, 1H), 7.62 (dd, J = 7.8,
gel flash column chromatography (DCM / MeOH = 20:1) to afford
.4 Hz, 1H), 7.19–7.15 (m, 2H), 5.87 (s, 1H), 4.79 (hept, J = 6.0 Hz,
compound 12a∼c.
H), 4.11–3.98 (m, 4H), 1.32 (d, J = 6.4 Hz, 6H); MS (ESI) m/z 387.1
+
(
M+H)
.
4
.1.4.1. 2-Chloro-4-(1,3-dioxolan-2-yl)-N′-hydroxybenzimidamide
1
(
12a). Yield: 79.1%; White solid; Mp: 110–113 °C; H NMR (400 MHz,
4
.1.5.6. 5-(3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-1,2,4-oxadiazol-5-
DMSO‑d
6
) δ 9.50 (s, 1H), 7.50 (s, 1H), 7.45–7.39 (m, 2H), 5.84 (s, 2H),
+
yl)-2-isopropoxybenzonitri-le (13f). Yield: 87.0%; White solid; Mp:
5
.77 (s, 1H), 4.09–3.92 (m, 4H); MS (ESI) m/z 243.0 (M+H)
.
1
1
53–155 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.51 (d, J = 2.4 Hz, 1H),
8
.40 (dd, J = 9.0, 2.2 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.72 (d,
4
.1.4.2. 4-(1,3-Dioxolan-2-yl)-2-fluoro-N′-hydroxybenzimidamide
1
J = 1.2 Hz, 1H), 7.62 (dd, J = 8.0, 1.2 Hz, 1H), 7.55 (d, J = 9.2 Hz,
(
12b). Yield: 76.9%; White solid; Mp: 87–88 °C; H NMR (400 MHz,
1
H), 5.87 (s, 1H), 4.98 (hept, J = 6.0 Hz, 1H), 4.13–3.96 (m, 4H), 1.38
DMSO‑d
6
) δ 9.66 (s, 1H), 7.54–7.50 (m, 1H), 7.28–7.24 (m, 2H), 5.83
+
+
(d, J = 6.0 Hz, 6H); MS (ESI) m/z 412.1 (M+H)
.
(
s, 2H), 5.77 (s, 1H), 4.08–3.92 (m, 4H); MS (ESI) m/z 227.1 (M+H) .
4
8
9
.1.4.3. 4-(1,3-Dioxolan-2-yl)-N′-hydroxybenzimidamide (12c). Yield:
4.1.5.7. 3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-5-(4-isopropoxy-3-
1
1.2%; White solid; Mp: 135–136 °C; H NMR (400 MHz, DMSO‑d
6
) δ
(trifluoromethyl)phenyl)-1,2,4-oxadiazole (13g). Yield: 72.3%; White
1
.66 (s, 1H), 7.69–7.67 (m, 2H), 7.43–7.41 (m, 2H), 5.82 (s, 2H), 5.74
solid; Mp: 131–134 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.39 (dd,
+
(
s, 1H), 4.07–3.93 (m, 4H); MS (ESI) m/z 209.1 (M+H)
.
J = 8.8, 2.4 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.06 (d, J = 8.0 Hz,
1
H), 7.72 (d, J = 1.6 Hz, 1H), 7.62 (dd, J = 8.4, 1.6 Hz, 1H), 7.58 (d,
4
.1.5. General procedure for the synthesis of 3-(4-(1,3-dioxolan-2-yl)
J = 8.8 Hz, 1H), 5.87 (s, 1H), 4.97 (hept, J = 6.0 Hz, 1H), 4.12–3.96
+
phenyl)-5-(substituted phenyl)-1,2,4-oxadiazole (13a∼l).
(m, 4H), 1.35 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 455.1 (M+H)
.
A
solution of substituted benzoic acid (8.61 mmol), 1-
Hydroxybenzotriazole (7.83 mmol), 1-Ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride (7.83 mmol) and CO
4.1.5.8. 3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-5-(3-chloro-4-
K
2
3
isopropoxyphenyl)-1,2,4-oxadiazole (13h). Yield: 71.7%; White solid;
1
(
11.74 mmol) in DMF was stirred at rt for 30 min. 4-(1,3-dioxolan-2-yl)-
Mp: 130–133 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.18 (dd, J = 2.4,
N′-hydroxybenzimidamide (7.83 mmol) was then added to the reaction
mixture at rt and the resulting slurry was stirred under Argon at 110 °C
for additional 2 h. The reaction mixture was then cooled to rt, then
filtered and concentrated. The filtrate was diluted with EtOAc, washed
0.8 Hz, 1H), 8.11 (ddd, J = 8.7, 2.2, 0.7 Hz, 1H), 8.05 (d, J = 8.0 Hz,
1H), 7.72 (m, 1H), 7.63–7.61 (m, 1H), 7.44 (d, J = 8.4 Hz, 1H), 5.87 (s,
1H), 4.88 (hept, J = 6.1 Hz, 1H), 4.13–3.98 (m, 4H), 1.36 (d,
J = 6.0 Hz, 6H); MS (ESI) m/z 421.1 (M+H)+
.
47

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
4
.1.5.9. 3-(2-Chloro-4-(1,3-dioxolan-2-yl)phenyl)-5-(4-isopropoxy-3-
4.1.6.5. 3-Chloro-4-(5-(4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
1
methylphenyl)-1,2,4-oxadiazole (13i). Yield: 64.9%; White solid; Mp:
benzaldehyde (14e). Yield: 73.2%; White solid; Mp: 107–109 °C;
H
1
7
8
1
4
4
1–74 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.03 (d, J = 8.0 Hz, 1H),
NMR (400 MHz, DMSO‑d ) δ 10.10 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H),
6
.00–7.97 (m, 2H), 7.71 (d, J = 1.6 Hz, 1H), 7.61 (dd, J = 8.0, 1.6 Hz,
8.20 (d, J = 1.2 Hz, 1H), 8.14–8.10 (m, 2H), 8.06 (dd, J = 7.8, 1.4 Hz,
H), 7.20 (d, J = 8.4 Hz, 1H), 5.87 (s, 1H), 4.78 (hept, J = 6.1 Hz, 1H),
1H), 7.20–7.16 (m, 2H), 4.80 (hept, J = 6.1 Hz, 1H), 1.32 (d,
.12–3.96 (m, 4H), 2.23 (s, 3H), 1.33 (d, J = 6.0 Hz, 6H); MS (ESI) m/z
J = 6.0 Hz, 6H); MS (ESI) m/z 343.1 (M+H)+
.
+
01.1 (M+H)
.
4
.1.6.6. 5-(3-(2-Chloro-4-formylphenyl)-1,2,4-oxadiazol-5-yl)-2-
4
.1.5.10. 5-(3-(4-(1,3-Dioxolan-2-yl)-2-fluorophenyl)-1,2,4-oxadiazol-5-
isopropoxybenzonitrile (14f). Yield: 76.9%; White solid; Mp:
1
yl)-2-isopropoxybenzonitrile (13j). Yield: 81.7%; White solid; Mp:
177–179 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.10 (s, 1H), 8.53 (d,
1
1
8
1
4
48–150 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.51 (d, J = 2.4 Hz, 1H),
J = 2.4 Hz, 1H), 8.41 (dd, J = 9.0, 2.2 Hz, 1H), 8.25 (d, J = 8.0 Hz,
1H), 8.21 (d, J = 1.2 Hz, 1H), 8.08–8.05 (m, 1H), 7.56 (d, J = 9.2 Hz,
1H), 4.98 (hept, J = 5.9 Hz, 1H), 1.39 (d, J = 6.0 Hz, 6H); MS (ESI) m/z
.41 (dd, J = 9.2, 2.4 Hz, 1H), 8.18–8.14 (m, 1H), 7.56 (d, J = 9.2 Hz,
H), 7.53–7.49 (m, 2H), 5.87 (s, 1H), 4.98 (hept, J = 5.9 Hz, 1H),
368.1 (M+H)+
.
.12–3.96 (m, 4H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 396.1 (M
+
+
H)
.
4
.1.6.7. 3-Chloro-4-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-1,2,4-
oxadiazol-3-yl)benzaldehyde (14g). Yield: 70.9%; White solid; Mp:
4
.1.5.11. 5-(3-(4-(1,3-Dioxolan-2-yl)phenyl)-1,2,4-oxadiazol-5-yl)-2-
1
1
21–124 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.10 (d, J = 2.0 Hz,
isopropoxybenzonitrile (13k). Yield: 59.7%; White solid; Mp:
1
1H), 8.41 (d, J = 8.8 Hz, 1H), 8.31 (m, 1H), 8.26 (dd, J = 7.8, 1.4 Hz,
1
19–120 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.46 (s, 1H), 8.37 (d,
1
H), 8.20 (m, 1H), 8.07–8.05 (m, 1H), 7.59 (d, J = 8.8 Hz, 1H), 4.98
J = 8.8 Hz, 1H), 8.09 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.52
(
hept, J = 6.0 Hz, 1H), 1.36 (dd, J = 6.0, 1.6 Hz, 6H); MS (ESI) m/z
(
(
d, J = 9.2 Hz, 1H), 5.82 (s, 1H), 4.96 (hept, J = 6.0 Hz, 1H), 4.10–3.95
+
m, 4H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 378.1 (M+H)+
.
411.1 (M+H)
.
4
.1.6.8. 3-Chloro-4-(5-(3-chloro-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
1
4
.1.5.12. 5-(3-(4-(1,3-Dioxolan-2-yl)phenyl)-1,2,4-oxadiazol-5-yl)-2-
yl)benzaldehyde (14h). Yield: 86.8%; White solid; Mp: 136–138 °C;
H
(
cyclopentyloxy)benzonitrile (13l). Yield: 72.9%; White solid; Mp:
1
NMR (400 MHz, DMSO‑d
6
) δ 10.10 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H),
1
8
7
47–148 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d, J = 2.4 Hz, 1H),
8
1
.21–8.19 (m, 2H), 8.12 (dd, J = 8.8, 2.4 Hz, 1H), 8.06 (dd, J = 8.0,
.6 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 4.89 (hept, J = 5.9 Hz, 1H), 1.36
.41 (dd, J = 9.0, 2.2 Hz, 1H), 8.12–8.10 (m, 2H), 7.67–7.65 (m, 2H),
.52 (d, J = 8.8 Hz, 1H), 5.84 (s, 1H), 5.18–5.14 (m, 1H), 4.12–3.96
(
d, J = 6.0 Hz, 6H); MS (ESI) m/z 377.0 (M+H)+
.
(
m, 4H), 2.06–1.99 (m, 2H), 1.82–1.63 (m, 6H); MS (ESI) m/z 404.2 (M
+
+
H)
.
4
.1.6.9. 3-Chloro-4-(5-(4-isopropoxy-3-methylphenyl)-1,2,4-oxadiazol-3-
1
yl)benzaldehyde (14i). Yield: 83.1%; White solid; Mp: 107–109 °C;
H
4
.1.6. General procedure for the synthesis of 4-(5-(substituted phenyl)-
,2,4-oxadiazol-3-yl)benzaldehyde (14a∼l)
NMR (400 MHz, DMSO‑d
6
) δ 10.09 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H),
1
8
2
1
.19 (d, J = 1.6 Hz, 1H), 8.05 (dd, J = 8.0, 1.6 Hz, 1H), 8.02–7.98 (m,
H), 7.22 (d, J = 8.4 Hz, 1H), 4.78 (hept, J = 6.1 Hz, 1H), 2.23 (s, 3H),
To a stirred suspension of 3-(4-(1,3-dioxolan-2-yl)phenyl)-5-(sub-
stituted phenyl)-1,2,4-oxadiazole (4.75 mmol) in acetone was added
.34 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 357.1 (M+H)+
.
2
N aqueous hydrochloric acid (38.00 mmol), and the resulting mixture
was stirred at 55 °C for 5 h. The reaction mixture was cooled to room
temperature, the precipitated solid was filtered and then dried in vacuo
to afford compound 14a∼l.
4
.1.6.10. 5-(3-(2-Fluoro-4-formylphenyl)-1,2,4-oxadiazol-5-yl)-2-
isopropoxybenzonitrile (14j). Yield: 63.2%; White solid; Mp:
1
1
1
84–185 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.09 (d, J = 1.2 Hz,
H), 8.51 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 8.8, 2.4 Hz, 1H), 8.36–8.32
4
.1.6.1. 3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
(
m, 1H), 7.99–7.94 (m, 2H), 7.56 (d, J = 9.2 Hz, 1H), 4.98 (hept,
1
benzaldehyde (14a). Yield: 94.4%; White solid; Mp: 191–193 °C;
H
J = 6.1 Hz, 1H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 352.1 (M
NMR (400 MHz, DMSO‑d
6
) δ 10.10 (s, 1H), 8.25–8.20 (m, 4H), 8.06 (d,
+
+
H)
.
J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H),
+
7
.22–7.19 (m, 4H); MS (ESI) m/z 377.1 (M+H)
.
4
.1.6.11. 5-(3-(4-Formylphenyl)-1,2,4-oxadiazol-5-yl)-2-
isopropoxybenzonitrile (14k). Yield: 82.6%; White solid; Mp:
4
.1.6.2. 4-(5-([1,1′-Biphenyl]-4-yl)-1,2,4-oxadiazol-3-yl)-3-
1
1
60–161 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.11 (s, 1H), 8.48 (d,
chlorobenzaldehyde (14b). Yield: 94.8%; White solid; Mp: 193–195 °C;
J = 2.4 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 8.28 (d, J = 8.4 Hz,
1
H NMR (500 MHz, DMSO‑d ) δ 10.11 (s, 1H), 8.30–8.27 (m, 3H), 8.22
6
2
H), 8.10 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 9.2 Hz, 1H), 4.97 (hept,
(
s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.81 (d,
J = 6.0 Hz, 1H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 334.1 (M
J = 8.0 Hz, 2H), 7.54 (t, J = 7.5 Hz, 2H), 7.47 (t, J = 7.5 Hz, 1H); MS
+
+
H)
.
(
ESI) m/z 361.1 (M+H)+
.
4
.1.6.12. 2-(Cyclopentyloxy)-5-(3-(4-formylphenyl)-1,2,4-oxadiazol-5-
1
4
.1.6.3. 4-(5-(4-(tert-Butyl)phenyl)-1,2,4-oxadiazol-3-yl)-3-
yl)benzonitrile (14l). Yield: 64.4%; White solid; Mp: 160–161 °C;
H
chlorobenzaldehyde (14c). Yield: 88.0%; White solid; Mp: 132–135 °C;
NMR (400 MHz, DMSO‑d ) δ 10.12 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H),
6
1
H NMR (500 MHz, DMSO‑d
6
) δ 10.10 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H),
8.42 (dd, J = 8.8, 2.4 Hz, 1H), 8.32–8.29 (m, 2H), 8.14–8.10 (m, 2H),
8
.21 (s, 1H), 8.13 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.70 (d,
7.53 (d, J = 9.2 Hz, 1H), 5.19–5.15 (m, 1H), 2.07–1.98 (m, 2H),
+
1.83–1.63 (m, 6H); MS (ESI) m/z 360.1 (M+H)+
.
J = 8.5 Hz, 2H), 1.34 (s, 9H); MS (ESI) m/z 341.1 (M+H)
.
4
.1.6.4. 3-Chloro-4-(5-(4-isopropylphenyl)-1,2,4-oxadiazol-3-yl)
benzaldehyde (14d). Yield: 89.0%; White solid; Mp: 119–121 °C;
NMR (400 MHz, DMSO‑d ) δ 10.11 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H),
4.1.7. General procedure for the synthesis of (4-(5-(substituted phenyl)-
1,2,4-oxadiazol-3-yl)benzyl) carboxylic ester or alkamine (15-1a∼15-3n)
To a solution of 4-(5-(substituted phenyl)-1,2,4-oxadiazol-3-yl)ben-
zaldehyde (0.53 mmol)in 10 mL DCM was added methyl glycinate hy-
drochloride (0.80 mmol), acetic acid (2.12 mmol), and N,N-diisopropy-
lethylamine (0.80 mmol), and 10 mL MeOH. After the reaction was stirred
1
H
6
8
.22 (d, J = 1.6 Hz, 1H), 8.14 (dt, J = 8.4, 1.9 Hz, 2H), 8.07 (dd,
J = 7.8, 1.4 Hz, 1H), 7.58–7.55 (m, 2H), 3.04 (hept, J = 6.9 Hz, 1H),
+
1
.27 (d, J = 6.8 Hz, 6H); MS (ESI) m/z 327.1 (M+H)
.
48

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
1
for 3 h, sodium cyanoborohydride (0.53 mmol) was added. The reaction
solid; Mp: 96–98 °C;
H
NMR (400 MHz, DMSO‑d ) δ 8.19 (d,
6
mixture was quenched with saturated aqueous NaHCO
3
solution and
J = 8.8 Hz, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.50 (t,
J = 7.4 Hz, 3H), 7.28 (t, J = 7.4 Hz, 1H), 7.21–7.18 (m, 4H), 3.70 (d,
J = 6.8 Hz, 2H), 3.64 (s, 3H), 2.78 (t, J = 7.4 Hz, 1H), 1.28 (s, 6H); MS
extracted with DCM. The combined organic layers were washed with
brine, dried over Na
2
SO , then filtered and concentrated. The residue was
4
purified by silica gel flash column chromatography (DCM:MeOH = 20:1)
(ESI) m/z 478.2 (M+H)+
.
to afford compound 15-1a∼15-3n (except for 15-1l).
4
.1.7.9. Ethyl 1-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
4
.1.7.1. Methyl (3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
benzyl)piperidine-4-carboxylate (15-1i). Yield: 68.9%; White solid; Mp:
1
1
benzyl)glycinate (15-1a). Yield: 80.5%; White solid; Mp: 62–65 °C;
H
93–95 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.21 (d, J = 8.4 Hz, 2H),
NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.4 Hz, 2H), 7.98 (d,
J = 7.6 Hz, 1H), 7.57 (s, 1H), 7.41 (q, J = 8.5 Hz, 3H), 7.22 (t,
7.96 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.51 (t, J = 7.8 Hz, 3H), 7.30 (t,
J = 7.4 Hz, 1H), 7.23–7.20 (m, 3H), 5.77 (s, 1H), 4.08 (q, J = 7.1 Hz,
2H), 3.57 (s, 2H), 2.78 (d, J = 11.2 Hz, 2H), 2.36–2.30 (m, 1H), 2.08 (t,
J = 11.2 Hz, 2H), 1.83 (d, J = 12.8 Hz, 2H), 1.66–1.56 (m, 2H), 1.19 (t,
J = 7.4 Hz, 1H), 7.11 (d, J = 8.0 Hz, 4H), 3.88 (s, 2H), 3.75 (s, 3H),
+
3
.44 (s, 2H), 1.92 (s, 1H); MS (ESI) m/z 450.1 (M+H)
.
J = 7.0 Hz, 3H); MS (ESI) m/z 518.2 (M+H)+
.
4
.1.7.2. Methyl (3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
1
benzyl)alaninate (15-1b). Yield: 74.1%; White solid; Mp: 77–79 °C;
H
4.1.7.10. Dimethyl (3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-
NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.8 Hz, 2H), 7.97 (d,
J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.41 (q, J = 8.0 Hz, 3H), 7.22 (t,
J = 7.4 Hz, 1H), 7.10 (d, J = 8.4 Hz, 4H), 3.90 (d, J = 13.6 Hz, 1H),
yl)benzyl)aspartate (15-1j). Yield: 74.9%; White solid; Mp: 62–63 °C;
1
H NMR (400 MHz, DMSO‑d ) δ 8.19 (d, J = 8.4 Hz, 2H), 7.93 (d,
6
J = 8.0 Hz, 1H), 7.63 (s, 1H), 7.49 (q, J = 8.0 Hz, 3H), 7.28 (t,
J = 7.4 Hz, 1H), 7.21–7.18 (m, 4H), 3.90 (dd, J = 15.0, 6.0 Hz, 1H),
3.73 (dd, J = 15.0, 6.0 Hz, 1H), 3.64 (s, 3H), 3.61 (s, 3H), 3.58–3.54
3
.75 (m, 3H), 3.71 (s, 1H), 3.40 (q, J = 6.9 Hz, 1H), 2.22 (s, 1H), 1.36
+
(
d, J = 7.2 Hz, 3H); MS (ESI) m/z 464.1 (M+H)
.
(
m, 1H), 3.02–2.96 (m, 1H), 2.73 (dd, J = 16.0, 6.4 Hz, 1H), 2.64 (dd,
+
4
.1.7.3. Methyl 3-((3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-
J = 16.0, 7.2 Hz, 1H); MS (ESI) m/z 522.1 (M+H)
.
yl)benzyl)amino)propanoate (15-1c). Yield: 79.6%; White solid; Mp:
1
6
0–63 °C; H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.4 Hz, 2H),
.97 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.44–7.36 (m, 3H), 7.22 (t,
4.1.7.11. Methyl 2-(1-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-
7
3-yl)benzyl)piperidin-4-yl)acetate (15-1k). Yield: 69.3%; White solid;
1
J = 7.4 Hz, 1H), 7.11 (d, J = 8.0 Hz, 4H), 3.87 (s, 2H), 3.71 (s, 3H),
Mp: 109–112 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.18 (d, J = 8.4 Hz,
2
.91 (t, J = 6.4 Hz, 2H), 2.56 (t, J = 6.4 Hz, 2H); MS (ESI) m/z 464.1
2H), 7.94 (d, J = 7.6 Hz, 1H), 7.59 (s, 1H), 7.51–7.46 (m, 3H), 7.28 (t,
+
(
M+H)
.
J = 7.4 Hz, 1H), 7.20–7.18 (m, 4H), 3.58 (s, 3H), 3.54 (s, 2H), 2.78 (d,
J = 11.2 Hz, 2H), 2.24 (d, J = 6.4 Hz, 2H), 1.98 (t, J = 11.4 Hz, 2H),
+
4
.1.7.4. Methyl 1-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-
1.70–1.60 (m, 3H), 1.27–1.18 (m, 2H); MS (ESI) m/z 518.2 (M+H)
.
yl)benzyl)pyrrolidine-3-carboxylate (15-1d). Yield: 70.8%; White solid;
1
Mp: 78–79 °C; H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.4 Hz,
4.1.7.12. Methyl
(4-(5-([1,1′-biphenyl]-4-yl)-1,2,4-oxadiazol-3-yl)-3-
2
H), 7.94 (d, J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.44–7.36 (m, 3H), 7.22 (t,
chlorobenzyl)glycinate (15-1m). Yield: 72.5%; White solid; Mp:
1
J = 7.6 Hz, 1H), 7.10 (d, J = 8.0 Hz, 4H), 3.70 (s, 3H), 3.68–3.63 (m,
88–90 °C; H NMR (500 MHz, DMSO‑d
6
) δ 8.27 (d, J = 8.5 Hz, 2H),
2
2
H), 3.10–3.02 (m, 1H), 2.88 (t, J = 8.6 Hz, 1H), 2.72–2.68 (m, 2H),
7.98 (dd, J = 8.0, 2.0 Hz, 3H), 7.80 (d, J = 7.5 Hz, 2H), 7.67 (s, 1H),
7.56–7.50 (m, 3H), 7.46 (t, J = 7.2 Hz, 1H), 3.84 (s, 2H), 3.64 (s, 3H),
.58 (q, J = 8.0 Hz, 1H), 2.16–2.10 (m, 2H); MS (ESI) m/z 490.2 (M
+
3.37 (s, 2H), 2.77 (s, 1H); MS (ESI) m/z 434.1 (M+H)+
.
+
H)
.
4
.1.7.5. Methyl (3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
4.1.7.13. Methyl
(4-(5-(4-(tert-butyl)phenyl)-1,2,4-oxadiazol-3-yl)-3-
1
benzyl)prolinate (15-1e). Yield: 73.2%; White solid; Mp: 52–54 °C;
H
chlorobenzyl)glycinate (15-1n). Yield: 57.7%; White solid; Mp:
1
NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.4 Hz, 2H), 7.95 (d,
J = 8.0 Hz, 1H), 7.56 (s, 1H), 7.41 (q, J = 8.0 Hz, 3H), 7.22 (t,
J = 7.4 Hz, 1H), 7.11 (d, J = 8.0 Hz, 4H), 3.99 (d, J = 13.2 Hz, 1H),
79–82 °C; H NMR (500 MHz, DMSO‑d
6
) δ 8.11 (d, J = 8.0 Hz, 2H),
7.95 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.65 (s, 1H), 7.50 (d,
J = 8.0 Hz, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.37 (s, 2H), 2.76 (s, 1H),
3
3
2
.70 (s, 3H), 3.59 (d, J = 13.4 Hz, 1H), 3.33 (t, J = 7.4 Hz, 1H),
.08–3.03 (m, 1H), 2.43 (q, J = 8.3 Hz, 1H), 2.22–2.12 (m, 1H),
1.34 (s, 9H); MS (ESI) m/z 414.2 (M+H)+
.
.04–1.89 (m, 2H), 1.86–1.82 (m, 1H); MS (ESI) m/z 490.2 (M+H)+
.
4.1.7.14. Methyl (3-chloro-4-(5-(4-isopropylphenyl)-1,2,4-oxadiazol-3-yl)
1
benzyl)glycinate (15-1o). Yield: 70.7%; White solid; Mp: 68–70 °C;
H
4
.1.7.6. Ethyl N-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
NMR (400 MHz, DMSO‑d ) δ 8.12–8.10 (m, 2H), 7.95 (d, J = 8.0 Hz,
6
benzyl)-N-methylglycinate (15-1f). Yield: 57.5%; White solid; Mp:
1H), 7.66 (d, J = 1.2 Hz, 1H), 7.56–7.54 (m, 2H), 7.50 (dd, J = 8.0,
1.2 Hz, 1H), 3.83 (s, 2H), 3.63 (s, 3H), 3.37 (s, 2H), 3.03 (hept,
J = 6.9 Hz, 1H), 2.78 (s, 1H), 1.26 (d, J = 6.8 Hz, 6H); MS (ESI) m/z
1
5
2
7
2
8–60 °C; H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.4 Hz,
H), 7.96 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.42 (t, J = 7.6 Hz, 3H),
.22 (t, J = 7.4 Hz, 1H), 7.11 (d, J = 8.4 Hz, 4H), 4.20 (q, J = 7.1 Hz,
H), 3.77 (s, 2H), 3.32 (s, 2H), 2.43 (s, 3H), 1.30 (t, J = 7.0 Hz, 3H); MS
400.1 (M+H)+
.
(
ESI) m/z 478.2 (M+H)+
.
4.1.7.15. Methyl (3-chloro-4-(5-(4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
yl)benzyl)glycinate (15-1p). Yield: 86.6%; White solid; Mp: 59–60 °C;
1
4
.1.7.7. Methyl 1-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-
H NMR (400 MHz, DMSO‑d ) δ 8.11 (d, J = 8.8 Hz, 2H), 7.93 (d,
6
yl)benzyl)azetidine-3-carboxylate (15-1g). Yield: 73.5%; White solid;
J = 8.0 Hz, 1H), 7.65 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.18–7.16 (m,
1
Mp: 73–76 °C;
H
NMR (400 MHz, Chloroform-d)
δ
8.17 (d,
2H), 4.80 (hept, J = 6.1 Hz, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.36 (s, 2H),
+
J = 8.4 Hz, 2H), 7.95 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.42 (t,
J = 7.8 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H),
2.76 (s, 1H), 1.32 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 416.1 (M+H)
.
7
3
.10 (d, J = 8.4 Hz, 4H), 3.72 (s, 3H), 3.66 (s, 2H), 3.58–3.54 (m, 2H),
4.1.7.16. Methyl
(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
+
.36 (m, 3H); MS (ESI) m/z 476.1 (M+H)
.
oxadiazol-3-yl)benzyl)glycinate (15-1q). Yield: 88.3%; White solid;
1
Mp: 107–110 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d, J = 2.4 Hz,
4
.1.7.8. Methyl 2-((3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-
1H), 8.39 (dd, J = 9.2, 2.4 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.65 (d,
J = 1.2 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.50 (dd, J = 8.0, 1.6 Hz,
yl)benzyl)amino)-2-methylpropanoate (15-1h). Yield: 71.6%; White
49

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
1
2
H), 4.98 (hept, J = 6.1 Hz, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.37 (s, 2H),
69.1%; White solid; Mp: 130–132 °C; 1H NMR (400 MHz, DMSO‑d
) δ
6
.77 (s, 1H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 441.1 (M+H)+
.
8.49 (d, J = 2.0 Hz, 1H), 8.39 (dd, J = 8.8, 2.4 Hz, 1H), 7.95 (d,
J = 8.0 Hz, 1H), 7.59 (s, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.48 (d,
J = 8.0 Hz, 1H), 4.98 (hept, J = 6.1 Hz, 1H), 3.58 (s, 3H), 3.54 (s,
2H), 2.78 (d, J = 11.2 Hz, 2H), 2.25 (d, J = 6.8 Hz, 2H), 1.98 (t,
J = 11.0 Hz, 2H), 1.72–1.61 (m, 3H), 1.38 (d, J = 6.0 Hz, 6H),
4
.1.7.17. Methyl (3-chloro-4-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-
1
,2,4-oxadiazol-3-yl)benzyl)glycinate (15-1r). Yield: 73.5%; White
1
solid; Mp: 88–90 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.39 (dd,
J = 8.8, 2.0 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.96 (d, J = 7.6 Hz,
H), 7.66 (d, J = 1.6 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.50 (dd,
1.28–1.18 (m, 2H); MS (ESI) m/z 509.2 (M+H)+
.
1
J = 8.0, 1.6 Hz, 1H), 4.98 (hept, J = 6.0 Hz, 1H), 3.83 (s, 2H), 3.63 (s,
4
.1.7.25. Methyl 4-((3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
3
4
H), 3.37 (s, 2H), 2.77 (s, 1H), 1.35 (d, J = 6.0 Hz, 6H); MS (ESI) m/z
oxadiazol-3-yl)benzyl)-amino)butanoate (15-2f). Yield: 58.2%; White
+
1
84.1 (M+H)
.
solid; Mp: 115–116 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.62 (d,
J = 2.0 Hz, 1H), 8.51 (dd, J = 9.2, 2.0 Hz, 1H), 8.07 (d, J = 8.0 Hz,
4
.1.7.18. Methyl
(3-chloro-4-(5-(3-chloro-4-isopropoxyphenyl)-1,2,4-
1
5
2
6
H), 7.77 (s, 1H), 7.68 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H),
.10 (hept, J = 5.9 Hz, 1H), 3.89 (s, 2H), 3.71 (s, 3H), 3.45 (s, 1H),
.63–2.61 (m, 4H), 1.82 (quint, J = 7.0 Hz, 2H), 1.51 (d, J = 6.0 Hz,
oxadiazol-3-yl)benzyl)glycinate (15-1s). Yield: 82.5%; White solid; Mp:
1
8
3–86 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.16 (d, J = 2.0 Hz, 1H),
H); MS (ESI) m/z 469.2 (M+H)+
.
8
.09 (dd, J = 8.6, 2.2 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.64 (d,
J = 1.2 Hz, 1H), 7.48 (dd, J = 8.0, 1.6 Hz, 1H), 7.43 (d, J = 9.2 Hz,
1
2
H), 4.87 (hept, J = 6.1 Hz, 1H), 3.82 (s, 2H), 3.63 (s, 3H), 3.36 (s, 2H),
4.1.7.26. Methyl
3-((3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
.76 (s, 1H), 1.36 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 450.1 (M+H)+
.
oxadiazol-3-yl)benzyl)-(methyl)amino)propanoate (15-2g). Yield: 54.0%;
1
White solid; Mp: 80–81 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.48 (d,
4
.1.7.19. Methyl
(3-chloro-4-(5-(4-isopropoxy-3-methylphenyl)-1,2,4-
J = 2.4 Hz, 1H), 8.38 (dd, J = 8.8, 2.4 Hz, 1H), 7.95 (d, J = 8.0 Hz,
H), 7.58 (d, J = 1.2 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.45 (dd,
J = 8.0, 1.2 Hz, 1H), 4.97 (hept, J = 6.1 Hz, 1H), 3.60 (s, 3H), 3.57 (s,
oxadiazol-3-yl)benzyl)glycinate (15-1t). Yield: 77.7%; White solid; Mp:
1
1
8
3–85 °C; H NMR (400 MHz, DMSO‑d
6
) δ 7.99–7.96 (m, 2H), 7.93 (d,
J = 8.0 Hz, 1H), 7.64 (d, J = 1.6 Hz, 1H), 7.48 (dd, J = 8.0, 1.6 Hz,
2
H), 2.66 (t, J = 6.8 Hz, 2H), 2.53 (t, J = 6.6 Hz, 2H), 2.17 (s, 3H), 1.38
1
3
6
H), 7.19 (d, J = 8.8 Hz, 1H), 4.77 (hept, J = 6.1 Hz, 1H), 3.82 (s, 2H),
d, J = 6.0 Hz, 6H); MS (ESI) m/z 469.2 (M+H)+
(
.
.63 (s, 3H), 3.36 (s, 2H), 2.76 (s, 1H), 2.22 (s, 3H), 1.33 (d, J = 6.4 Hz,
+
H); MS (ESI) m/z 430.2 (M+H)
.
4
.1.7.27. Methyl 1-(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
oxadiazol-3-yl)benzyl)-piperidine-3-carboxylate (15-2h). Yield: 54.0%;
4
.1.7.20. Ethyl
N-(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
1
White solid; Mp: 102–103 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.51
oxadiazol-3-yl)benzyl)-N-methylglycinate (15-2a). Yield: 62.5%; White
(
d, J = 2.4 Hz, 1H), 8.40 (dd, J = 9.2, 2.4 Hz, 1H), 7.97 (d, J = 8.0 Hz,
1
solid; Mp: 68–69 °C;
H
NMR (400 MHz, DMSO‑d ) δ 8.50 (d,
6
1
H), 7.61 (d, J = 1.2 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 7.48 (dd,
J = 2.4 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.97 (d, J = 8.0 Hz,
H), 7.64 (m, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.51–7.49 (m, 1H), 4.98
hept, J = 6.1 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.75 (s, 2H), 3.36 (s,
J = 8.0, 1.6 Hz, 1H), 4.98 (hept, J = 6.0 Hz, 1H), 3.63–3.53 (m, 5H),
1
2
.77–2.74 (m, 1H), 2.67–2.56 (m, 2H), 2.34–2.15 (m, 2H), 1.80–1.66
(
(
m, 2H), 1.53–1.48 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z
2
H), 2.30 (s, 3H), 1.38 (d, J = 6.0 Hz, 6H), 1.21 (t, J = 7.0 Hz, 3H); MS
4
95.2 (M+H)+
.
(
ESI) m/z 469.2 (M+H)+
.
4
.1.7.28. Methyl
4-((3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-
4
.1.7.21. Methyl 1-(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
1
,2,4-oxadiazol-3-yl)benzyl)-amino)cyclohexane-1-carboxylate
(15-
oxadiazol-3-yl)benzyl)-azetidin-e-3-carboxylate (15-2b). Yield: 72.8%;
1
1
2i). Yield: 69.0%; White solid; Mp: 126–127 °C;
H
NMR
White solid; Mp: 110–111 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49
(
400 MHz, DMSO‑d ) δ 8.50 (d, J = 2.0 Hz, 1H), 8.39 (dd, J = 9.0,
6
(
d, J = 2.0 Hz, 1H), 8.39 (dd, J = 9.2, 2.0 Hz, 1H), 7.95 (d, J = 8.0 Hz,
2
.2 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 1.2 Hz, 1H), 7.55
1
H), 7.57–7.54 (m, 2H), 7.45 (d, J = 7.6 Hz, 1H), 4.98 (hept,
(
d, J = 9.2 Hz, 1H), 7.50 (d, J = 8.0, 1.2 Hz, 1H), 4.98 (hept,
J = 6.0 Hz, 1H), 3.66 (s, 2H), 3.64 (s, 3H), 3.47 (t, J = 7.0 Hz, 2H),
J = 6.0 Hz, 1H), 3.76 (s, 2H), 3.58 (m, 3H), 2.32 (d, J = 6.8 Hz,
2H), 2.23 (tt, J = 12.2, 3.4 Hz, 1H), 1.91–1.82 (m, 4H), 1.38 (d,
J = 6.0 Hz, 6H), 1.36–1.26 (m, 2H), 0.96–0.86 (m, 2H); MS (ESI) m/z
3
.39–3.34 (m, 1H), 3.27 (t, J = 6.6 Hz, 2H), 1.38 (d, J = 6.0 Hz, 6H);
+
MS (ESI) m/z 467.1 (M+H)
.
5
09.2 (M+H)+
.
4
.1.7.22. Dimethyl (3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
oxadiazol-3-yl)benzyl)-aspartate (15-2c). Yield: 78.1%; White solid;
1
4
.1.7.29. Methyl
(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
Mp: 89–90 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d, J = 2.4 Hz,
oxadiazol-3-yl)benzyl)-serinate (15-2j). Yield: 64.2%; White solid; Mp:
1
H), 8.39 (dd, J = 8.8, 2.4 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.63 (d,
J = 0.8 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.47 (dd, J = 8.0, 1.2 Hz,
1
1
8
09–110 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (d, J = 2.4 Hz, 1H),
.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.66 (d,
1
H), 4.98 (hept, J = 6.1 Hz, 1H), 3.93–3.72 (m, 2H), 3.64 (s, 3H), 3.61
J = 0.8 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.50 (dd, J = 8.0, 1.2 Hz,
(
s, 3H), 3.57 (s, 1H), 3.00 (s, 1H), 2.76–2.61 (m, 2H), 1.38 (d,
+
1
H), 4.97 (hept, J = 6.1 Hz, 1H), 4.88 (t, J = 5.8 Hz, 1H), 3.92–3.71
J = 6.0 Hz, 6H); MS (ESI) m/z 513.2 (M+H)
.
(
m, 2H), 3.63–3.60 (m, 5H), 3.30 (t, J = 5.2 Hz, 1H), 1.38 (d,
+
J = 6.0 Hz, 6H); MS (ESI) m/z 471.1 (M+H)
.
4
.1.7.23. Methyl
1-(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
oxadiazol-3-yl)benzyl)-pyrrolidine-3-carboxylate (15-2d). Yield: 69.1%;
1
White solid; Mp: 111–113 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (d,
4.1.7.30. 5-(3-(2-chloro-4-(((2-oxotetrahydrofuran-3-yl)amino)methyl)
J = 2.4 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H),
phenyl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
(15-2k). Yield:
) δ
1
7
1
3
.62 (d, J = 1.6 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.49 (dd, J = 8.0,
62.5%; White solid; Mp: 134–136 °C; H NMR (400 MHz, DMSO‑d
6
.6 Hz, 1H), 4.98 (h, J = 6.1 Hz, 1H), 3.73–3.64 (m, 2H), 3.61 (s, 3H),
8.49 (d, J = 2.4 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.96 (d,
J = 7.6 Hz, 1H), 7.69 (d, J = 1.2 Hz, 1H), 7.56–7.52 (m, 2H), 4.97
(hept, J = 5.9 Hz, 1H), 4.32 (td, J = 8.5, 2.7 Hz, 1H), 4.18–4.12 (m,
.11–3.03 (m, 1H), 2.76–2.67 (m, 2H), 2.56 (t, J = 6.8 Hz, 2H), 2.05–1.95
+
(
m, 2H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 481.2 (M+H) .
1
H), 4.00–3.89 (m, 2H), 3.58–3.53 (m, 1H), 3.03–3.01 (m, 1H),
4
.1.7.24. Methyl
2-(1-(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-
(15-2e). Yield:
2.44–2.37 (m, 1H), 2.03–1.95 (m, 1H), 1.38 (d, J = 6.0 Hz, 6H); MS
1
,2,4-oxadiazol-3-yl)benzyl)-piperidin-4-yl)acetate
(ESI) m/z 453.1 (M+H)+
.
50

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
1
4
.1.7.31. Dimethyl
(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
87–89 °C; H NMR (400 MHz, DMSO‑d ) δ 8.49 (s, 1H), 8.39 (d,
6
oxadiazol-3-yl)benzyl)-glutamate (15-2l). Yield: 67.3%; White solid; Mp:
J = 8.0 Hz, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.37
(t, J = 11.2 Hz, 2H), 5.00–4.94 (m, 1H), 3.92 (dd, J = 15, 2.2 Hz, 1H),
3.75 (dd, J = 15.8, 3.4 Hz, 1H), 3.64–3.56 (m, 7H), 2.99 (s, 1H), 2.76–2.61
1
9
1–93 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d, J = 2.0 Hz, 1H), 8.39
(
dd, J = 9.0, 2.2 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 1.6 Hz,
+
1
H), 7.55 (d, J = 9.2 Hz, 1H), 7.47 (dd, J = 8.0, 1.6 Hz, 1H), 4.98 (hept,
(m, 2H), 1.38 (d, J = 5.9 Hz, 6H); MS (ESI) m/z 497.2 (M+H) .
J = 6.0 Hz, 1H), 3.88–3.84 (m, 1H), 3.66–3.61 (m, 4H), 3.57 (s, 3H),
3.25–3.19 (m, 1H), 2.81–2.75 (m, 1H), 2.47–2.42 (m, 2H), 1.92–1.74 (m,
4.1.7.38. Methyl 2-(1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
+
2H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 527.2 (M+H) .
3-yl)-3-fluorobenzyl)-piperidin-4-yl)acetate (15-3d). Yield: 53.4%; White
1
solid; Mp: 110–111 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (s, 1H),
4
.1.7.32. 5-(3-(2-Chloro-4-(((1,3-dihydroxypropan-2-yl)amino)methyl)
8.39 (d, J = 8.8 Hz, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 9.2 Hz,
1H), 7.36 (d, J = 9.6 Hz, 2H), 5.00–4.94 (m, 1H), 3.58 (s, 3H), 3.54 (s,
2H), 2.78 (d, J = 10.0 Hz, 2H), 2.25 (d, J = 6.4 Hz, 2H), 1.98 (t,
J = 11.2 Hz, 2H), 1.64–1.61 (m, 3H), 1.38 (d, J = 6.0 Hz, 6H),
phenyl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile hydrochloride (15-
1
2
m). Yield: 63.5%; White solid; Mp: 134–136 °C; H NMR (400 MHz,
DMSO‑d
6
) δ 8.49 (d, J = 2.4 Hz, 1H), 8.38 (dd, J = 9.0, 2.2 Hz, 1H),
.94 (d, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.53 (t, J = 8.0 Hz, 2H), 4.97
+
7
1.28–1.19 (m, 2H); MS (ESI) m/z 493.2 (M+H) .
(
hept, J = 6.0 Hz, 1H), 4.47 (s, 2H), 3.88 (s, 2H), 3.42 (s, 2H), 3.38 (s,
1
3
2
H), 2.54 (q, J = 5.6 Hz, 1H), 1.38 (d, J = 6.0 Hz, 6H); C NMR
4.1.7.39. Methyl (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
(
151 MHz, DMSO‑d
6
) δ 173.21, 167.06, 162.56, 147.10, 134.63,
yl)benzyl)glycinate (15-3e). Yield: 78.7%; White solid; Mp:
1
1
1
33.82, 131.92, 131.47, 129.89, 126.97, 123.26, 115.80, 115.24,
122–124 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (m, 1H), 8.39 (dt,
14.91, 102.50, 72.56, 61.10, 60.33, 49.67, 21.49; HRMS (ESI) m/z
J = 9.2, 1.7 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.56–7.53 (m, 3H), 4.97
+
calcd. For C22
H
24ClN
4
O
4
(M+H) 443.1481, found 443.1480.
(hept, J = 5.9 Hz, 1H), 3.81 (s, 2H), 3.63 (s, 3H), 3.36 (s, 2H), 2.73 (s,
+
1
H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 407.2 (M+H)
.
4
.1.7.33. 5-(3-(2-Chloro-4-(((2-hydroxyethyl)amino)methyl)phenyl)-
1
,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
hydrochloride
(15-
4.1.7.40. Ethyl N-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
1
2
n). Yield: 70.2%; White solid; Mp: 132–135 °C; H NMR (400 MHz,
yl)benzyl)-N-methyl-glycinate (15-3f). Yield: 68.8%; White solid; Mp:
1
DMSO‑d
6
) δ 8.52 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H),
93–94 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.47 (d, J = 2.0 Hz, 1H),
8
.01 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.62–7.59 (m, 1H), 7.56 (d,
8.38 (dd, J = 9.0, 1.8 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.53 (t,
J = 7.8 Hz, 3H), 4.97 (hept, J = 5.8 Hz, 1H), 4.10 (q, J = 7.1 Hz,
2H), 3.73 (s, 2H), 3.33 (s, 2H), 2.28 (s, 3H), 1.38 (d, J = 6.0 Hz, 6H),
J = 9.2 Hz, 1H), 5.76 (s, 1H), 4.98 (hept, J = 6.0 Hz, 1H), 4.86 (s, 1H),
4
6
1
1
.02 (s, 2H), 3.57 (s, 2H), 2.77 (t, J = 5.6 Hz, 2H), 1.38 (d, J = 6.0 Hz,
13
1.20 (t, J = 7.0 Hz, 3H); MS (ESI) m/z 435.2 (M+H)+
.
H); C NMR (101 MHz, DMSO‑d
6
) δ 173.32, 166.92, 162.59, 142.74,
34.64, 133.85, 132.01, 131.66, 130.88, 127.96, 124.23, 115.74,
15.22, 114.93, 102.50, 72.57, 58.75, 50.54, 50.07, 21.48; HRMS
4.1.7.41. Methyl 1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
+
(
ESI) m/z calcd. For
C
21
H
4
22ClN O
3
(M+H)
413.1375, found
3-yl)benzyl)pyrrolidine-3-carboxylate (15-3g). Yield: 68.8%; White
1
4
13.1376.
solid; Mp: 94–95 °C;
H
NMR (400 MHz, DMSO‑d ) δ 8.48 (d,
6
J = 2.0 Hz, 1H), 8.39 (dd, J = 8.8, 2.4 Hz, 1H), 8.04–8.02 (m, 2H),
7.56–7.51 (m, 3H), 4.97 (hept, J = 6.0 Hz, 1H), 3.70–3.63 (m, 2H),
3.61 (s, 3H), 3.10–3.02 (m, 1H), 2.75 (t, J = 8.6 Hz, 1H), 2.68–2.64 (m,
1H), 2.56–2.53 (m, 2H), 2.07–1.94 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H);
4
.1.7.34. 5-(3-(2-Chloro-4-(((2-hydroxypropyl)amino)methyl)phenyl)-
1
,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile
hydrochloride
(15-
1
2
o). Yield: 58.4%; White solid; Mp: 172–175 °C; H NMR (400 MHz,
DMSO‑d
6
) δ 8.51 (d, J = 2.0 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H),
MS (ESI) m/z 447.2 (M+H)+
.
8
.01 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.63 (d, J = 7.2 Hz, 1H), 7.56 (d,
J = 9.2 Hz, 1H), 4.98 (hept, J = 6.1 Hz, 2H), 4.05–4.00 (m, 2H),
.47–3.44 (m, 2H), 2.90 (s, 1H), 1.38 (d, J = 6.0 Hz, 6H), 1.10 (d,
4.1.7.42. Dimethyl (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
3
3-yl)benzyl)aspartate (15-3h). Yield: 71.3%; White solid; Mp:
1
3
1
J = 6.4 Hz, 3H); C NMR (101 MHz, DMSO‑d
6
) δ 173.30, 166.91,
75–76 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (d, J = 2.0 Hz, 1H),
1
1
2
4
62.58, 143.05, 134.63, 133.84, 131.98, 131.63, 130.92, 127.97,
8.39 (dd, J = 9.0, 2.2 Hz, 1H), 8.03–8.01 (m, 2H), 7.56–7.50 (m, 3H),
4.97 (hept, J = 6.1 Hz, 1H), 3.91–3.71 (m, 2H), 3.64 (s, 3H), 3.60 (s,
3H), 3.57 (m, 1H), 2.87 (s, 1H), 2.76–2.61 (m, 2H), 1.38 (d, J = 6.0 Hz,
24.19, 115.74, 115.22, 114.92, 102.49, 72.56, 63.52, 54.27, 48.00,
1.48, 15.55; HRMS (ESI) m/z calcd. For C22
H
24ClN
4
O
3
(M+H)+
27.1531, found 427.1531.
6H); MS (ESI) m/z 479.2 (M+H)+
.
4
.1.7.35. Methyl (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
4.1.7.43. Methyl 2-(1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
yl)-3-fluorobenzyl)-glycinate (15-3a). Yield: 92.1%; White solid; Mp:
3-yl)benzyl)piperidin-4-yl)acetate (15-3i). Yield: 74.8%; White solid; Mp:
1
1
1
50–151 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d, J = 2.2 Hz, 1H),
122–123 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (d, J = 2.4 Hz, 1H),
8
.39 (dd, J = 8.8, 2.4 Hz, 1H), 8.05 (t, J = 7.6 Hz, 1H), 7.55 (d,
8.39 (dd, J = 9.0, 2.2 Hz, 1H), 8.04–8.02 (m, 2H), 7.55 (d, J = 9.2 Hz,
1H), 7.50 (d, J = 8.0 Hz, 1H), 4.97 (hept, J = 6.1 Hz, 1H), 3.58 (s, 3H),
3.52 (s, 2H), 2.78 (d, J = 11.6 Hz, 2H), 2.24 (d, J = 6.8 Hz, 2H),
J = 9.2 Hz, 1H), 7.43–7.38 (m, 2H), 4.98 (hept, J = 6.1 Hz, 1H), 3.84
(
s, 2H), 3.63 (s, 3H), 3.37 (s, 2H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/
+
z 425.1 (M+H)
.
1.99–1.93 (m, 2H), 1.63–1.60 (m, 3H), 1.38 (d, J = 6.0 Hz, 6H),
+
1
.27–1.17 (m, 2H); MS (ESI) m/z 475.2 (M+H) .
4
.1.7.36. Ethyl N-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
yl)-3-fluorobenzyl)-N-methylglycinate (15-3b). Yield: 58.9%; White
4.1.7.44. Methyl (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
1
solid; Mp: 101–102 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.48 (d,
yl)-3-fluorobenzyl)-glycinate (15-3j). Yield: 92.1%; White solid; Mp:
1
J = 2.4 Hz, 1H), 8.38 (dd, J = 8.8, 2.4 Hz, 1H), 8.06 (t, J = 7.8 Hz,
150–151 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d, J = 2.2 Hz, 1H),
1
H), 7.54 (d, J = 9.2 Hz, 1H), 7.40–7.37 (m, 2H), 4.97 (hept,
8.39 (dd, J = 8.8, 2.4 Hz, 1H), 8.05 (t, J = 7.6 Hz, 1H), 7.55 (d,
J = 9.2 Hz, 1H), 7.43–7.38 (m, 2H), 4.98 (hept, J = 6.1 Hz, 1H), 3.84
(s, 2H), 3.63 (s, 3H), 3.37 (s, 2H), 1.38 (d, J = 6.0 Hz, 6H); MS (ESI) m/
J = 6.1 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.75 (s, 2H), 3.36 (s, 2H),
2
.30 (s, 3H), 1.38 (d, J = 6.0 Hz, 6H), 1.21 (t, J = 7.0 Hz, 3H); MS (ESI)
+
z 425.1 (M+H)+
.
m/z 453.2 (M+H)
.
4
.1.7.37. Dimethyl (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
4.1.7.45. Ethyl N-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
yl)-3-fluorobenzyl)-aspartate (15-3c). Yield: 73.7%; White solid; Mp:
yl)-3-fluorobenzyl)-N-methylglycinate (15-3k). Yield: 58.9%; White
51

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
1
solid; Mp: 101–102 °C; H NMR (400 MHz, DMSO‑d
J = 2.4 Hz, 1H), 8.38 (dd, J = 8.8, 2.4 Hz, 1H), 8.06 (t, J = 7.8 Hz,
H), 7.54 (d, J = 9.2 Hz, 1H), 7.40–7.37 (m, 2H), 4.97 (hept,
6
) δ 8.48 (d,
4.1.8.3. 3-((3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
benzyl)amino)propanoic acid hydrochloride (16-1c). Yield: 68.4%; White
1
1
solid; Mp: 218–219 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.64 (s, 1H),
J = 6.1 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.75 (s, 2H), 3.36 (s, 2H),
9.32 (s, 2H), 8.20 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.94 (s,
1H), 7.72 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.29 (t,
J = 7.4 Hz, 1H), 7.20 (t, J = 7.6 Hz, 4H), 4.29 (s, 2H), 3.16 (t,
2
.30 (s, 3H), 1.38 (d, J = 6.0 Hz, 6H), 1.21 (t, J = 7.0 Hz, 3H); MS (ESI)
+
m/z 453.2 (M+H)
.
1
3
J = 7.2 Hz, 2H), 2.74 (t, J = 7.2 Hz, 2H);
C NMR (101 MHz,
4
.1.7.46. Methyl 1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
DMSO‑d ) δ 174.61, 171.55, 166.74, 161.53, 154.69, 136.86, 132.23,
6
3
-yl)-3-fluorobenzyl)-pyrrolidine-3-carboxylate (15-3l). Yield: 66.7%;
132.15, 131.88, 130.45, 130.42, 129.26, 125.80, 125.06, 120.21,
1
White solid; Mp: 96–98 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (d,
118.11, 117.45, 48.77, 42.25, 30.28; HRMS (ESI) m/z calcd. For
J = 2.0 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 8.06 (t, J = 7.6 Hz, 1H),
.55 (d, J = 9.2 Hz, 1H), 7.39 (m, 1H), 7.37 (m, 1H), 4.98 (p,
C
24
H
21ClN
3
O
4
(M+H)+ 450.1215, found 450.1206.
7
J = 6.1 Hz, 1H), 3.73–3.65 (m, 2H), 3.61 (s, 3H), 3.11–3.03 (m, 1H),
4.1.8.4. 1-(3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
2
.77–2.66 (m, 2H), 2.56 (t, J = 7.0 Hz, 2H), 2.08–1.95 (m, 2H), 1.38 (d,
benzyl)pyrrolidine-3-carboxylic acid hydrochloride (16-1d). Yield:
+
1
J = 6.0 Hz, 6H); MS (ESI) m/z 465.2 (M+H)
.
59.7%; White solid; Mp: 228–231 °C; H NMR (400 MHz, DMSO‑d
6
) δ
1
2.87 (s, 1H), 11.71 (s, 1H), 8.20 (d, J = 8.4 Hz, 2H), 8.07–8.03 (m,
4
.1.7.47. Dimethyl (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-
2H), 7.80 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.28 (t,
yl)-3-fluorobenzyl)-aspartate (15-3m). Yield: 73.7%; White solid; Mp:
J = 7.4 Hz, 1H), 7.20 (t, J = 7.2 Hz, 4H), 4.47 (s, 2H), 3.34 (m, 5H),
1
13
8
7–89 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (s, 1H), 8.39 (d,
2.33–2.08 (m, 2H); C NMR (101 MHz, DMSO‑d
6
) δ 174.62, 173.05,
J = 8.0 Hz, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.37
166.74, 161.53, 154.70, 136.25, 132.76, 132.30, 131.99, 130.46,
(
t, J = 11.2 Hz, 2H), 5.00–4.94 (m, 1H), 3.92 (dd, J = 15, 2.2 Hz, 1H),
130.43, 129.75, 126.14, 125.06, 120.21, 118.13, 117.46, 55.40,
3
.75 (dd, J = 15.8, 3.4 Hz, 1H), 3.64–3.56 (m, 7H), 2.99 (s, 1H), 2.76–2.61
53.71, 52.59, 40.74, 26.64; HRMS (ESI) m/z calcd. For C26
H
23ClN
O
3 4
+
(M+H)+ 476.1372, found 476.1363.
(m, 2H), 1.38 (d, J = 5.9 Hz, 6H); MS (ESI) m/z 497.2 (M+H) .
4
.1.7.48. Methyl 2-(1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
4.1.8.5. (3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)benzyl)
3-yl)-3-fluorobenzyl)-piperidin-4-yl)acetate (15-3n). Yield: 53.4%; White
proline hydrochloride (16-1e). Yield: 54.1%; White solid; Mp:
1
1
solid; Mp: 110–111 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (s, 1H), 8.39
200–202 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.05 (s, 1H), 8.19 (d,
(
d, J = 8.8 Hz, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 9.2 Hz, 1H),
J = 8.8 Hz, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.82 (s, 1H), 7.65 (d,
J = 8.0 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H),
7.21–7.18 (m, 4H), 4.35 (d, J = 13.2 Hz, 1H), 4.14 (d, J = 13.6 Hz,
7
2
2
.36 (d, J = 9.6 Hz, 2H), 5.00–4.94 (m, 1H), 3.58 (s, 3H), 3.54 (s, 2H),
.78 (d, J = 10.0 Hz, 2H), 2.25 (d, J = 6.4 Hz, 2H), 1.98 (t, J = 11.2 Hz,
H), 1.64–1.61 (m, 3H), 1.38 (d, J = 6.0 Hz, 6H), 1.28–1.19 (m, 2H); MS
1H), 3.94 (s, 1H), 3.30 (s, 1H), 2.95 (s, 1H), 2.35–2.30 (m, 1H),
+
3
(ESI) m/z 493.2 (M+H) .
2.01–1.92 (m, 2H), 1.85–1.82 (m, 1H); C NMR (151 MHz, DMSO‑d
6
) δ
1
74.59, 166.77, 161.54, 154.68, 132.21, 132.13, 131.92, 130.46,
4
.1.8. General procedure for the synthesis of (3-chloro-4-(5-(4-
130.43, 129.26, 125.63, 125.08, 120.23, 118.12, 117.46, 65.39,
phenoxyphenyl)-1,2,4-oxadiazol-3-yl)benzyl) carboxylic acid (16-
56.35, 54.11, 28.36, 22.43; HRMS (ESI) m/z calcd. For C26
H
3 4
23ClN O
1
a∼16-3n)
(M+H)+ 476.1372, found 476.1368.
To a solution of (4-(5-(substituted phenyl)-1,2,4-oxadiazol-3-yl)
benzyl) carboxylic ester (0.43 mmol) in 15 mL MeOH was added 0.5 N
aqueous lithium hydroxide solution (3.41 mmol) and heated to 45 °C for
4.1.8.6. N-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
benzyl)-N-methylglycine Hydrochloride (16-1f). Yield: 76.3%; White
1
3
h. The reaction mixture was cooled to rt and then added 2 N aqueous
solid; Mp: 189–191 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.28 (s, 1H),
hydrochloric acid to adjust the PH to 2–3. The solvent was evaporated
and 25 mL H O was added. The precipitated solid was filtered, washed
with ether, and then dried in vacuo to afford compound 16-1a∼16-3n.
8.20 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.62 (d,
2
J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H),
1
3
7.22–7.18 (m, 4H), 4.09 (s, 2H), 3.67 (s, 2H), 2.54 (s, 3H); C NMR
(
151 MHz, DMSO‑d ) δ 174.52, 169.74, 166.85, 161.50, 154.69,
6
4
.1.8.1. (3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)benzyl)
139.86, 132.16, 131.91, 131.82, 130.44, 130.40, 129.01, 125.19,
glycine hydrochloride (16-1a). Yield: 75.3%; White solid; Mp:
125.05, 120.20, 118.11, 117.49, 58.39, 56.18, 41.02; HRMS (ESI) m/
1
(M+H)+ 450.1215, found 450.1206.
2
20–223 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.77 (s, 2H), 8.19 (d,
z calcd. For C24
H
21ClN
O
3 4
J = 8.4 Hz, 2H), 8.05 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.68 (d,
J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H),
4.1.8.7. 1-(3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
1
3
7
.20 (t, J = 7.4 Hz, 4H), 4.24 (s, 2H), 3.82 (s, 2H); C NMR (101 MHz,
benzyl)azetidine-3-carboxylic acid hydrochloride (16-1g). Yield: 63.4%;
1
DMSO‑d
6
) δ 174.61, 167.88, 166.75, 161.53, 154.68, 136.52, 132.46,
White solid; Mp: 183–185 °C; H NMR (400 MHz, DMSO‑d
6
) δ 13.13 (s,
1
1
32.09, 131.86, 130.46, 130.42, 129.45, 125.87, 125.07, 120.22,
1H), 11.49 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.05 (d, J = 8.0 Hz, 1H),
7.92 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.29 (t,
J = 7.4 Hz, 1H), 7.20 (t, J = 7.2 Hz, 4H), 4.49 (s, 2H), 4.20 (d,
18.12, 117.45, 48.69, 46.58; HRMS (ESI) m/z calcd. For
+
C
23
H
19ClN
3
O
4
(M+H) 436.1059, found 436.1057.
1
3
J = 8.4 Hz, 4H), 3.68–3.62 (m, 1H); C NMR (101 MHz, DMSO‑d ) δ
6
4
.1.8.2. 3-((3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
174.61, 171.52, 166.69, 161.53, 154.68, 135.33, 132.36, 132.31,
132.08, 130.45, 130.42, 129.30, 126.18, 125.06, 120.21, 118.11,
117.44, 55.61, 54.60, 32.04; HRMS (ESI) m/z calcd. For
benzyl)amino)propanoic acid hydrochloride (16-1b). Yield: 69.5%; White
1
solid; Mp: 232–235 °C; H NMR (400 MHz, DMSO‑d
6
) δ 9.78 (s, 2H),
8
.20 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H), 7.72 (d,
C
25
H21ClN
3
O
4
(M+H)+ 462.1215, found 462.1206.
J = 8.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.20
(
t, J = 7.8 Hz, 4H), 4.29 (q, J = 13.5 Hz, 2H), 4.06 (q, J = 7.1 Hz, 1H),
4.1.8.8. 2-((3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
1
3
1
1
1
5
.54 (d, J = 7.2 Hz, 3H); C NMR (101 MHz, DMSO‑d
6
) δ 174.62,
benzyl)amino)-2-methylpropanoic acid hydrochloride (16-1h). Yield:
1
70.80, 166.75, 161.53, 154.69, 136.74, 132.40, 132.08, 131.86,
30.46, 130.43, 129.41, 125.83, 125.07, 120.22, 118.12, 117.46,
59.4%; White solid; Mp: 230–233 °C; H NMR (400 MHz, DMSO‑d
6
) δ
9.74 (s, 2H), 8.20 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.93 (s,
1H), 7.72 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.29 (t,
J = 7.4 Hz, 1H), 7.20 (t, J = 7.8 Hz, 4H), 4.25 (s, 2H), 1.61 (s, 6H);
4.54, 47.37, 14.59; HRMS (ESI) m/z calcd. For C24
H21ClN
3
O (M
4
+
+
H) 450.1215, found 450.1206.
52

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
1
3
Mp: 212–215 °C; 1H NMR (500 MHz, DMSO‑d
) δ 8.12 (d, J = 8.0 Hz,
C NMR (151 MHz, DMSO‑d
6
) δ 174.64, 172.48, 166.76, 161.54,
6
1
1
54.68, 137.13, 132.46, 132.00, 131.86, 130.45, 129.53, 125.81,
2H), 8.02 (d, J = 8.0 Hz, 1H), 7.78 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H),
13
25.07, 120.21, 118.13, 117.45, 61.77, 45.34, 21.72; HRMS (ESI) m/
7.60 (d, J = 8.0 Hz, 1H), 4.06 (s, 2H), 3.47 (s, 2H), 1.35 (s, 9H);
C
+
z calcd. For C25
H
23ClN
3
O
4
(M+H) 464.1372, found 464.1364.
NMR (151 MHz, DMSO‑d ) δ 174.99, 169.04, 166.81, 156.70, 132.10,
6
1
31.87, 131.76, 131.71, 128.77, 127.88, 126.53, 125.35, 120.44,
4
.1.8.9. 1-(3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
49.34, 47.69, 35.05, 30.76; HRMS (ESI) m/z calcd. For C21
H
23ClN
O
3 3
benzyl)piperidine-4-carboxylic acid hydrochloride (16-1i). Yield: 48.3%;
(M+H)+ 400.1422, found 400.1427.
1
White solid; Mp: 233–235 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.14 (s,
1
7
3
H), 8.19 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H),
4.1.8.15. (3-Chloro-4-(5-(4-isopropylphenyl)-1,2,4-oxadiazol-3-yl)
.50 (t, J = 7.6 Hz, 3H), 7.28 (t, J = 7.4 Hz, 1H), 7.21–7.18 (m, 4H),
.55 (s, 2H), 2.76 (d, J = 11.2 Hz, 2H), 2.22 (m, 1H), 2.05 (t,
benzyl)glycine hydrochloride (16-1o). Yield: 64.2%; White solid; Mp:
1
117–119 °C; H NMR (400 MHz, DMSO‑d
6
) δ 11.14 (s, 1H), 8.11 (d,
J = 11.4 Hz, 2H), 1.80 (d, J = 12.8 Hz, 2H), 1.59 (t, J = 11.8 Hz,
J = 8.4 Hz, 2H), 8.06 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 1.2 Hz, 1H), 7.72
1
3
2
1
1
2
H);
C
NMR (101 MHz, DMSO‑d ) δ 174.63, 174.59, 166.73,
6
(dd, J = 8.2, 1.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 4.27 (s, 2H), 3.87 (s,
1
3
61.54, 154.68, 134.45, 133.66, 132.28, 131.92, 130.73, 130.45,
2H), 3.03 (hept, J = 6.8 Hz, 1H), 1.26 (d, J = 6.8 Hz, 6H); C NMR
30.42, 126.39, 125.06, 120.21, 118.12, 117.44, 57.51, 50.73, 37.94,
(151 MHz, DMSO‑d ) δ 175.07, 167.85, 166.75, 154.51, 136.52,
6
+
4.99; HRMS (ESI) m/z calcd; For C27
H25ClN
3
O
4
(M+H) 490.1528,
132.49, 132.09, 131.85, 129.47, 128.14, 127.64, 125.87, 120.76,
found 490.1524.
48.68, 46.58, 33.61, 23.44; HRMS (ESI) m/z calcd. For C20
H
3 3
21ClN O
+
(
M+H) 386.1266, found 386.1269.
4
.1.8.10. (3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)benzyl)
aspartic acid hydrochloride (16-1j). Yield: 48.3%; White solid; Mp:
4.1.8.16. (3-Chloro-4-(5-(4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
1
2
12–214 °C; H NMR (400 MHz, DMSO‑d
6
) δ 11.20 (s, 2H), 8.19 (d,
benzyl)glycine hydrochloride (16-1p). Yield: 65.6%; White solid; Mp:
1
J = 8.4 Hz, 2H), 8.01 (d, J = 7.6 Hz, 1H), 7.89 (s, 1H), 7.68 (d,
204–207 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.13 (s, 1H), 8.10 (d,
J = 8.0 Hz, 1H), 7.49 (t, J = 7.8 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1H),
J = 8.8 Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.63 (d,
J = 8.0 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 4.80 (hept, J = 6.1 Hz,
7
.19 (dd, J = 8.4, 4.8 Hz, 4H), 4.22 (q, J = 12.9 Hz, 2H), 3.98 (t,
13
13
J = 6.0 Hz, 1H), 3.38 (q, J = 7.1 Hz, 1H), 2.97–2.85 (m, 2H); C NMR
1H), 4.15 (s, 2H), 3.64 (s, 2H), 1.32 (d, J = 6.0 Hz, 6H); C NMR
(
151 MHz, DMSO‑d
6
) δ 174.54, 171.22, 171.04, 166.86, 161.51,
(151 MHz, DMSO‑d ) δ 174.91, 167.95, 166.65, 161.64, 136.54,
6
1
1
54.71, 139.84, 132.02, 131.70, 131.61, 130.46, 130.42, 128.63,
132.39, 132.09, 131.84, 130.08, 129.39, 126.00, 116.24, 114.95,
25.09, 125.06, 120.22, 118.12, 117.51, 55.71, 48.80, 35.40; HRMS
69.95, 48.73, 46.66, 21.66; HRMS (ESI) m/z calcd. For C20
H
3 4
21ClN O
+
(M+H)+ 402.1215, found 402.1216.
(
ESI) m/z calcd. For
C
25
H
3
21ClN O
6
(M+H)
494.1113, found
4
94.1104.
4
.1.8.17. (3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
4
.1.8.11. 2-(1-(3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
3-yl)benzyl)glycine hydrochloride (16-1q). Yield: 62.3%; White solid;
1
benzyl)piperidin-4-yl)acetic acid hydrochloride (16-1k). Yield: 61.4%;
Mp: 198–200 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.06 (s, 1H), 8.50
1
White solid; Mp: 234–236 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.25
(m, 1H), 8.40 (m, 1H), 8.04 (m, 1H), 7.83 (s, 1H), 7.65–7.57 (m, 2H),
13
(
s, 1H), 10.84 (s, 1H), 8.20–8.04 (m, 4H), 7.79 (m, 2H), 7.49 (m, 2H),
4.98 (s, 1H), 4.13 (s, 2H), 3.61 (s, 2H), 1.39 (m, 6H); C NMR
7
2
.40–6.97 (m, 5H), 4.47–4.36 (m, 2H), 3.14 (s, 1H), 2.97–2.95 (m, 3H),
(151 MHz, DMSO‑d ) δ 173.46, 167.98, 166.75, 162.63, 136.73,
6
1
3
.41 (s, 1H), 2.19 (m, 2H), 2.05–1.39 (m, 5H); C NMR (101 MHz,
) δ 174.62, 172.97, 166.73, 161.53, 154.68, 134.62, 133.61,
134.66, 133.89, 132.44, 132.10, 131.91, 129.41, 125.65, 115.68,
DMSO‑d
6
115.22, 114.96, 102.51, 72.59, 48.75, 46.69, 21.49; HRMS (ESI) m/z
1
1
32.26, 131.89, 130.67, 130.45, 130.42, 126.33, 125.06, 120.21,
calcd. For C21
H
20ClN
4
O
4
(M+H)+ 427.1168, found 427.1166.
18.12, 117.44, 57.63, 51.42, 30.18, 28.27; HRMS (ESI) m/z calcd.
+
For C28
H
27ClN
3
O
4
(M+H) 504.1685, found 504.1686.
4.1.8.18. (3-Chloro-4-(5-(4-isopropoxy-3-(trifluoromethyl)phenyl)-1,2,4-
oxadiazol-3-yl)benzyl)-glycine hydrochloride (16-1r). Yield: 68.3%;
1
4
.1.8.12. 3-(3-Chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl)
White solid; Mp: 202–205 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.39
phenyl)propanoic acid (16-1l). Yield: 63.5%; White solid; Mp:
(d, J = 8.4 Hz, 1H), 8.30 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H),
1
1
37–139 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.22 (s, 1H), 8.18 (d,
7.60–7.54 (m, 2H), 5.01–4.95 (m, 1H), 3.94 (s, 2H), 3.23 (s, 2H), 1.35
1
3
J = 8.4 Hz, 2H), 7.90 (d, J = 8.0 Hz, 1H), 7.59 (s, 1H), 7.49 (t,
J = 7.8 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H),
(d, J = 5.6 Hz, 6H); C NMR (101 MHz, CF COOD-d) δ 178.57, 172.27,
3
167.72, 164.20, 137.62, 136.14, 134.96, 134.61, 131.02, 130.56 (q,
J = 5.4 Hz), 128.46, 124.88 (q, J = 272.6 Hz), 124.03 (q, J = 32.3 Hz),
116.82, 114.44, 75.48, 53.74, 49.42, 22.28; HRMS (ESI) m/z calcd. For
7
.20–7.18 (m, 4H), 2.92 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.4 Hz, 2H);
1
3
C NMR (151 MHz, DMSO‑d ) δ 174.36, 173.47, 166.99, 161.43,
6
1
1
54.72, 146.29, 131.92, 131.59, 130.57, 130.43, 130.36, 127.67,
C
21
H20ClF
3
N
3
O
4
(M+H)+ 470.1089, found 470.1091.
25.01, 123.17, 120.18, 118.09, 117.57, 34.44, 29.73; HRMS (ESI)
+
m/z calcd. For C23
H18ClN
2
O
4
(M+H) 421.0950, found 421.0942.
4.1.8.19. (3-Chloro-4-(5-(3-chloro-4-isopropoxyphenyl)-1,2,4-oxadiazol-
3
-yl)benzyl)glycine hydrochloride (16-1s). Yield: 64.5%; White solid;
1
4
.1.8.13. (4-(5-([1,1′-biphenyl]-4-yl)-1,2,4-oxadiazol-3-yl)-3-
Mp: 217–220 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.16 (d, J = 2.4 Hz,
chlorobenzyl)glycine hydrochloride (16-1m). Yield: 64.7%; White solid;
1H), 8.09 (dd, J = 8.6, 2.2 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.80 (d,
J = 1.6 Hz, 1H), 7.61 (dd, J = 8.0, 1.6 Hz, 1H), 7.44 (d, J = 9.2 Hz,
1H), 4.88 (hept, J = 5.9 Hz, 1H), 4.09 (s, 2H), 3.49 (s, 2H), 1.36 (d,
1
Mp: 248–250 °C; H NMR (500 MHz, DMSO‑d
6
) δ 9.81 (s, 1H), 8.28 (d,
J = 8.5 Hz, 2H), 8.10 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.93
1
3
(
s, 1H), 7.81 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 8.0 Hz, 1H), 7.55 (t,
J = 6.4 Hz, 6H); C NMR (151 MHz, DMSO‑d ) δ 173.87, 168.06,
6
1
3
J = 7.5 Hz, 2H), 7.48–7.46 (m, 1H), 4.29 (s, 2H), 3.91 (s, 2H); C NMR
166.72, 156.93, 136.84, 132.38, 132.08, 131.90, 129.49, 129.35,
(
151 MHz, DMSO‑d
6
) δ 174.86, 167.83,166.85, 144.84, 138.52, 136.46,
128.69, 125.72, 122.99, 115.82, 115.35, 71.81, 48.78, 46.80, 21.63;
1
1
32.53, 132.12, 131.91, 129.51, 129.20, 128.65, 127.76, 127.00,
HRMS (ESI) m/z calcd. For C20
H
20
l2
C N
3
O
4
(M+H)+ 436.0825, found
25.85, 121.93, 48.68, 46.52; HRMS (ESI) m/z calcd. For
436.0827.
+
C
23
H
19ClN
3
O
3
(M+H) 420.1109, found 420.1107.
4
.1.8.20. (3-Chloro-4-(5-(4-isopropoxy-3-methylphenyl)-1,2,4-oxadiazol-
4
.1.8.14. (4-(5-(4-(tert-Butyl)phenyl)-1,2,4-oxadiazol-3-yl)-3-
3-yl)benzyl)glycine hydrochloride (16-1t). Yield: 61.2%; White solid;
chlorobenzyl)glycine hydrochloride (16-1n). Yield: 71.4%; White solid;
Mp: 212–214 °C; 1H NMR (400 MHz, DMSO‑d
) δ 8.00–7.97 (m, 3H),
6
53

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
7
.77 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 4.78
J = 6.0 Hz, 6H); 13C NMR (101 MHz, DMSO‑d
) δ 173.48, 172.99,
6
(
hept, J = 5.9 Hz, 1H), 4.05 (s, 2H), 3.41 (s, 2H), 2.23 (s, 3H), 1.33 (d,
166.74, 162.64, 134.70, 134.67, 133.86, 133.65, 132.27, 131.94,
1
3
J = 6.0 Hz, 6H); C NMR (151 MHz, DMSO‑d
6
) δ 175.05, 168.31,
130.68,126.13, 115.66, 115.22, 114.97, 102.51, 72.60, 57.68, 51.48,
1
1
1
66.62, 159.89, 137.24, 132.14, 132.08, 131.85, 130.14, 129.15,
30.17, 28.29, 21.49; HRMS (ESI) m/z calcd. For C26
H28ClN
4
4
O (M
+
27.91, 127.81, 125.87, 114.46, 112.89, 70.10, 48.92, 47.08, 21.84,
+H) 495.1794, found 495.1794.
+
6.04; HRMS (ESI) m/z calcd. For C21
H
23ClN
3
O
4
(M+H) 416.1372,
found 416.1369.
4.1.8.26. 5-(3-(2-Chloro-4-((2-oxopyrrolidin-1-yl)methyl)phenyl)-1,2,4-
oxadiazol-5-yl)-2-isopropoxybenzonitrile hydrochloride (16-2f). Yield:
1
4
.1.8.21. N-(3-chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
60.5%; White solid; Mp: 107–109 °C; H NMR (400 MHz, DMSO‑d
6
) δ
oxadiazol-3-yl)benzyl)-N-methyl-glycine Hydrochloride (16-2a). Yield:
8.50 (d, J = 2.0 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.98 (d,
J = 8.0 Hz, 1H), 7.57–7.55 (m, 2H), 7.41 (d, J = 8.4 Hz, 1H), 4.98
(hept, J = 6.1 Hz, 1H), 4.48 (s, 2H), 3.32–3.28 (m, 2H), 2.33 (t,
J = 8.2 Hz, 2H), 1.97 (quint, J = 7.5 Hz, 2H), 1.38 (d, J = 6.0 Hz,
1
6
4.1%; White solid; Mp: 152–155 °C; H NMR (400 MHz, DMSO‑d
6
) δ
8
.51 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H), 8.08 (d,
J = 8.0 Hz, 1H), 7.90 (d, J = 1.2 Hz, 1H), 7.70 (dd, J = 8.2, 1.4 Hz,
1
3
1
3
H), 7.57 (d, J = 9.2 Hz, 1H), 4.98 (hept, J = 6.1 Hz, 1H), 4.34 (s, 2H),
6H); C NMR (101 MHz, DMSO‑d ) δ 174.38, 173.31, 166.88, 162.58,
6
1
3
.97 (s, 2H), 2.73 (s, 3H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (101 MHz,
) δ 173.45, 168.08, 166.75, 162.63, 136.40, 134.66, 133.86,
142.54, 134.63, 133.82, 132.28, 132.01, 129.70, 126.72, 124.10,
DMSO‑d
6
115.73, 115.21, 114.92, 102.49, 72.55, 46.35, 44.73, 30.10, 21.47,
+
1
1
33.08, 132.27, 132.00, 130.10, 125.88, 115.67, 115.22, 114.97,
17.47; HRMS (ESI) m/z calcd. For C23
H
4
22ClN O
3
(M+H) 437.1375,
02.51, 72.59, 57.84, 55.06, 40.67, 21.48; HRMS (ESI) m/z calcd.
found 437.1376.
+
For C22
H
22ClN
4
O
4
(M+H) 441.1324, found 441.1326.
4
.1.8.27. 3-((3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
4
.1.8.22. 1-(3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
oxadiazol-3-yl)benzyl)(methyl)-amino)propanoic acid hydrochloride (16-
1
oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid hydrochloride (16-
2g). Yield: 62.8%; White solid; Mp: 194–196 °C; H NMR (400 MHz,
1
2
b). Yield: 69.3%; White solid; Mp: 145–147 °C; H NMR (400 MHz,
DMSO‑d ) δ 8.52 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 9.2, 2.0 Hz, 1H),
6
DMSO‑d
6
) δ 8.52 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H),
8.09 (d, J = 8.0 Hz, 1H), 8.02 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.57 (d,
J = 9.2 Hz, 1H), 4.98 (hept, J = 6.0 Hz, 1H), 4.41 (s, 2H), 3.30–3.28
(m, 2H), 2.87 (t, J = 7.6 Hz, 2H), 2.67 (s, 3H), 1.38 (d, J = 6.0 Hz, 6H);
8
7
4
6
1
1
.06 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.68 (dd, J = 7.8, 2.0 Hz, 1H),
.57 (d, J = 9.2 Hz, 1H), 4.98 (hept, J = 6.2 Hz, 1H), 4.38 (s, 2H),
.11–4.09 (m, 4H), 3.59 (quint, J = 8.2 Hz, 1H), 1.38 (d, J = 6.0 Hz,
1
3
C NMR (101 MHz, DMSO‑d ) δ 173.46, 171.60, 166.71, 162.63,
6
1
3
H); C NMR (101 MHz, DMSO‑d
6
) δ 173.33, 172.72, 166.79, 162.59,
135.46, 134.65, 133.87, 133.29, 132.32, 132.01, 130.32, 126.06,
39.00, 134.61, 133.81, 132.21, 131.92, 131.41, 128.42, 124.98,
115.66, 115.21, 114.96, 102.51, 72.59, 57.24, 50.59, 39.02, 28.68,
+
15.68, 115.20, 114.92, 102.49, 72.57, 58.05, 55.46, 32.73, 21.48;
21.48; HRMS (ESI) m/z calcd. For C23
H
4
24ClN O
4
(M+H) 455.1481,
+
HRMS (ESI) m/z calcd. For C23
H
22ClN
4
O
4
(M+H) 453.1324, found
found 455.1484.
4
53.1326.
4
.1.8.28. 1-(3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
4
.1.8.23. (3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
oxadiazol-3-yl)benzyl)piperidine-3-carboxylic acid hydrochloride (16-
1
3
-yl)benzyl)aspartic acid hydrochloride (16-2c). Yield: 67.2%; White
2h). Yield: 55.1%; White solid; Mp: 212–215 °C; H NMR (400 MHz,
1
solid; Mp: 165–167 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.14 (s, 1H),
DMSO‑d ) δ 12.92 (s, 1H), 11.00 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H), 8.40
6
8
.50 (d, J = 2.0 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 7.99 (d,
(dd, J = 9.0, 2.2 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.77
J = 8.0 Hz, 1H), 7.75 (d, J = 1.2 Hz, 1H), 7.58–7.54 (m, 2H), 4.97
(m, 1H), 7.57 (d, J = 9.2 Hz, 1H), 4.98 (hept, J = 5.9 Hz, 1H), 4.40 (s,
(
hept, J = 6.0 Hz, 1H), 4.09–3.93 (m, 2H), 3.62 (t, J = 6.6 Hz, 1H),
2H), 3.47 (s 2H), 2.92 (s, 3H), 2.01 (s, 1H), 1.84 (s, 2H), 1.47 (s, 1H),
1
3
13
2
.74–2.59 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (101 MHz,
) δ 173.28, 172.35, 171.69, 166.91, 162.59, 142.27, 134.63,
1.38 (d, J = 6.0 Hz, 6H); C NMR (101 MHz, DMSO‑d
6
) δ 173.46,
DMSO‑d
6
173.03, 166.75, 162.64, 134.66, 133.86, 133.29, 132.29, 131.98,
130.41, 125.96, 115.67, 115.22, 114.96, 102.51, 72.59, 58.05, 51.98,
51.39, 38.48, 24.94, 21.65, 21.49; HRMS (ESI) m/z calcd. For
1
1
33.82, 132.04, 131.67, 130.94, 127.94, 124.32, 115.74, 115.24,
14.92, 102.50, 72.59, 56.25, 49.03, 36.51, 21.51; HRMS (ESI) m/z
+
(M+H)+ 481.1637, found 481.1640.
calcd. For C23
H
22ClN
4
O
6
(M+H) 485.1222, found 485.1226.
C
25
4 4
H26ClN O
4
.1.8.24. 1-(3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
4.1.8.29. 4-((3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
oxadiazol-3-yl)benzyl)pyrrolidine-3-carboxylic acid hydrochloride (16-
oxadiazol-3-yl)benzyl)amino)-cyclohexane-1-carboxylic acid hydrochloride
1
1
2
d). Yield: 58.3%; White solid; Mp: 212–214 °C; H NMR (400 MHz,
(16-2i). Yield: 71.3%; White solid; Mp: 149–151 °C;
H NMR
DMSO‑d
H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 8.08–8.01 (m, 2H), 7.82 (d,
J = 7.6 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H), 4.98 (hept, J = 5.9 Hz,
H), 4.50 (s, 2H), 3.44–3.36 (m, 5H), 2.22 (s, 2H), 1.38 (d,
6
) δ 12.90 (s, 1H), 11.76–11.40 (m, 1H), 8.50 (d, J = 2.0 Hz,
(400 MHz, DMSO‑d ) δ 12.06 (s, 1H), 9.28 (s, 2H), 8.51 (d,
6
1
J = 2.0 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H), 8.07 (d, J = 8.0 Hz,
1H), 7.99 (d, J = 1.6 Hz, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H), 7.57 (d,
J = 9.2 Hz, 1H), 4.98 (hept, J = 6.1 Hz, 1H), 4.24 (s, 2H), 2.79 (d,
J = 6.4 Hz, 2H), 2.15 (tt, J = 12.2, 3.3 Hz, 1H), 1.94–1.85 (m, 4H),
1
1
3
J = 6.0 Hz, 6H); C NMR (101 MHz, DMSO‑d
6
) δ 173.40, 173.00,
1
3
1
1
5
66.69, 162.62, 136.04, 134.62, 133.81, 132.89, 132.32, 132.03,
1.38 (d, J = 6.0 Hz, 6H), 1.35–1.24 (m, 3H), 1.04–0.94 (m, 2H);
C
29.81, 125.98, 115.63, 115.20, 114.94, 102.49, 72.60, 55.37, 53.73,
NMR (101 MHz, DMSO‑d ) δ 176.52, 173.44, 166.77, 162.63, 136.99,
6
2.18, 40.70, 26.60, 21.49; HRMS (ESI) m/z calcd. For C24
H
24ClN
4
O
4
134.66, 133.88, 132.45, 131.80, 129.45, 125.52, 115.69, 115.22,
114.96, 102.51, 72.59, 52.37, 49.29, 41.98, 33.88, 29.14, 27.93,
+
(
M+H) 467.1481, found 467.1487.
2
6.72, 25.24, 21.49; HRMS (ESI) m/z calcd. For C26
H
28ClN
4
4
O (M
+
4
.1.8.25. 2-(1-(3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-
+H) 485.1794, found 495.1779.
oxadiazol-3-yl)benzyl)piperidin-4-yl)acetic acid hydrochloride (16-
1
2
e). Yield: 58.3%; White solid; Mp: 205–207 °C; H NMR (400 MHz,
4.1.8.30. (3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
DMSO‑d
6
) δ 12.25 (s, 1H), 10.43 (s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.40
dd, J = 9.0, 2.2 Hz, 1H), 8.11–8.08 (m, 1H), 8.00 (s, 1H), 7.77–7.75
m, 1H), 7.57 (d, J = 9.2 Hz, 1H), 4.99 (hept, J = 6.1 Hz, 1H),
.37–4.36 (m, 2H), 3.41 (m, 2H), 3.02–2.93 (m, 2H), 2.20 (d,
3-yl)benzyl)serine hydro-chloride (16-2j). Yield: 61.9%; White solid;
1
(
(
Mp: 219–222 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (s, 1H), 8.39
(d, J = 9.6 Hz, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.64–7.54
4
(m, 2H), 4.98 (m, 1H), 4.17–4.07 (m, 2H), 3.84–3.75 (m, 2H), 3.58 (s,
J = 6.8 Hz, 2H), 1.87–1.84 (m, 3H), 1.58–1.48 (m, 2H), 1.38 (d,
1H), 1.39–1.38 (m, 6H); 13C NMR (151 MHz, DMSO‑d
) δ 173.44,
6
54

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
1
1
6
69.36, 166.82, 162.65, 137.80, 134.68, 133.89, 132.35, 132.01,
31.78, 129.32, 125.35, 115.72, 115.25, 114.98, 102.53, 72.63,
4.1.8.36. 2-(1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-
3-fluorobenzyl)piperidin-4-yl)acetic acid hydrochloride (16-3d). Yield:
1
1.26, 59.10, 48.27, 21.51; HRMS (ESI) m/z calcd. For C22
H22ClN
4
O
5
65.9%; White solid; Mp: 199–201 °C; H NMR (400 MHz, DMSO‑d
6
) δ
+
(
M+H) 457.1273, found 457.1277.
12.22 (s, 1H), 11.06–10.91 (m, 1H), 8.50 (d, J = 2.4 Hz, 1H), 8.40 (dd,
J = 9.0, 2.2 Hz, 1H), 8.18 (t, J = 7.6 Hz, 1H), 7.85 (d, J = 11.2 Hz, 1H),
7.66 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 9.2 Hz, 1H), 4.98 (hept,
J = 6.1 Hz, 1H), 4.37–4.36 (m, 2H), 3.36 (s, 2H), 2.96 (q,
J = 11.3 Hz, 2H), 2.19 (d, J = 6.4 Hz, 2H), 1.90–1.83 (m, 3H),
4
.1.8.31. (3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
3
-yl)benzyl)homoserine hydrochloride (16-2k). Yield: 59.0%; White
1
solid; Mp: 189–192 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.51 (s, 1H),
1
3
8
.40 (d, J = 8.8 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.55 (t,
1.64–1.55 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (151 MHz,
J = 7.2 Hz, 2H), 4.98 (hept, J = 6.0 Hz, 1H), 3.99–3.77 (m, 2H),
DMSO‑d ) δ 173.47, 172.98, 164.70 (d, J = 5.6 Hz), 162.60, 159.42 (d,
6
3
1
.60–3.49 (m, 2H), 3.59–3.50 (m, 2H), 3.24 (t, J = 6.4 Hz, 1H),
J = 257.0 Hz), 135.80, 134.61, 133.82, 130.83, 128.14, 119.82 (d,
J = 21.4 Hz), 115.66, 115.19, 115.04, 114.93, 102.47, 72.59, 57.78,
1
3
.83–1.68 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (151 MHz,
) δ 174.49, 173.31, 167.00, 162.60, 144.36, 134.68, 133.88,
DMSO‑d
6
51.43, 30.20, 28.27, 21.48; HRMS (ESI) m/z calcd. For C26
H
28FN
4
O (M
4
+
1
1
31.97, 131.62, 130.33, 127.40, 123.85, 115.80, 115.25, 114.95,
+H) 479.2089, found 479.2087.
02.51, 72.58, 58.20, 58.14, 49.57, 35.06, 21.50; HRMS (ESI) m/z
+
calcd. For C23
H
4
24ClN O
5
(M+H) 471.1430, found 471.1430.
4.1.8.37. (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
benzyl)glycine hydrochloride (16-3e). Yield: 70.6%; White solid; Mp:
1
4
.1.8.32. (3-Chloro-4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-
212–215 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49 (d, J = 2.0 Hz, 1H),
3
-yl)benzyl)glutamic acid hydrochloride (16-2l). Yield: 57.6%; White
8.39 (dd, J = 8.8, 2.4 Hz, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.70 (d,
J = 8.4 Hz, 2H), 7.56 (d, J = 9.2 Hz, 1H), 4.98 (hept, J = 5.9 Hz,
1
solid; Mp: 147–149 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d,
1
3
J = 2.0 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H), 7.97 (d, J = 8.0 Hz,
1H), 4.18 (s, 2H), 3.66 (s, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR
1
3
1
H), 7.70 (s, 1H), 7.57–7.53 (m, 2H), 4.98 (hept, J = 6.0 Hz, 1H),
.99–3.78 (m, 2H), 3.22 (t, J = 6.6 Hz, 1H), 2.43–2.33 (m, 2H),
(101 MHz, DMSO‑d ) δ 173.94, 167.97, 167.85, 162.54, 135.53,
6
134.58, 133.79, 131.04, 127.30, 126.43, 115.87, 115.23, 114.91,
1
3
.92–1.79 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (101 MHz,
102.45, 72.58, 49.44, 46.77, 21.51; HRMS (ESI) m/z calcd. For
DMSO‑d
6
) δ 174.96, 173.30, 166.91, 162.58, 142.17, 134.63, 133.82,
C
21
H
21
N
4
O
4
(M+H)+ 393.1557, found 393.1555.
1
1
32.17, 131.84, 129.93, 126.91, 124.10, 115.75, 115.23, 114.91,
02.50, 72.57, 58.94, 44.28, 28.84, 22.67, 21.49; HRMS (ESI) m/z
4.1.8.38. N-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
+
calcd. For C24
H
4
24ClN O
6
(M+H) 499.1379, found 499.1375.
benzyl)-N-methylglycine hydrochloride (16-3f). Yield: 75.5%; White
1
solid; Mp: 199–210 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.50 (d,
4
.1.8.33. (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-3-
J = 2.4 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H), 8.16 (d, J = 8.4 Hz,
2H), 7.79 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 9.2 Hz, 1H), 4.98 (hept,
J = 6.1 Hz, 1H), 4.45 (s, 2H), 4.07 (s, 2H), 2.79 (s, 3H), 1.38 (d,
fluorobenzyl)glycine hydrochloride (16-3a). Yield: 72.5%; White solid;
1
Mp: 212–214 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.51 (d, J = 2.4 Hz,
1
3
1
H), 8.39 (dd, J = 8.8, 2.0 Hz, 1H), 8.15 (t, J = 7.8 Hz, 1H), 7.62 (d,
J = 6.0 Hz, 6H); C NMR (101 MHz, DMSO‑d ) δ 173.98, 167.79,
6
J = 11.2 Hz, 1H), 7.55 (t, J = 9.4 Hz, 2H), 4.98 (hept, J = 6.1 Hz, 1H),
167.29, 162.55, 134.57, 133.76, 133.62, 132.28, 127.46, 127.01,
115.83, 115.21, 114.92, 102.45, 72.58, 58.18, 54.31, 40.29, 21.50;
1
3
4
.18 (s, 2H), 3.67 (s, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (151 MHz,
DMSO‑d ) δ 173.49, 167.93, 164.74 (d, J = 5.4 Hz), 162.61, 159.44 (d,
J = 256.5 Hz), 137.80 (d, J = 8.0 Hz), 134.64, 133.88, 130.80, 126.86,
HRMS (ESI) m/z calcd. For C22
H
23
N
4
O
4
(M+H)+ 407.1714, found
6
407.1712.
1
18.54 (d, J = 22.0 Hz), 115.70, 115.21, 114.94, 114.56 (d,
J = 12.4 Hz), 102.48, 72.59, 48.83, 46.59, 21.49; HRMS (ESI) m/z
4.1.8.39. 1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
+
calcd. For C21
H
20
4
N O
4
(M+H) 411.1463, found 411.1461.
benzyl)pyrrolidine-3-carboxylic acid hydrochloride (16-3g). Yield: 75.5%;
1
White solid; Mp: 187–189 °C; H NMR (400 MHz, DMSO‑d
6
) δ 12.04 (s,
4
.1.8.34. N-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-3-
0H), 8.50 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 9.0, 2.2 Hz, 1H), 8.13 (d,
J = 8.4 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 9.2 Hz, 1H), 4.98
(hept, J = 6.0 Hz, 1H), 4.34 (s, 2H), 3.26–3.16 (m, 5H), 2.29–2.10 (m,
fluorobenzyl)-N-methylglycine hydrochloride (16-3b). Yield: 61.4%;
1
White solid; Mp: 208–211 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.49
1
3
(
d, J = 2.0 Hz, 1H), 8.39 (dd, J = 9.0, 2.2 Hz, 1H), 8.08 (t, J = 7.8 Hz,
2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (101 MHz, DMSO‑d ) δ 173.94,
6
1
H), 7.55 (d, J = 9.2 Hz, 1H), 7.42 (t, J = 9.4 Hz, 2H), 4.97 (hept,
173.54, 167.86, 162.54, 136.25, 134.58, 133.79, 131.11, 127.40,
J = 5.9 Hz, 1H), 3.84 (s, 2H), 3.36 (s, 2H), 2.35 (s, 3H), 1.38 (d,
126.41, 115.88, 115.22, 114.91, 102.46, 72.56, 56.65, 54.15, 52.80,
1
3
+
J = 6.0 Hz, 6H); C NMR (151 MHz, DMSO‑d
6
) δ 173.44, 169.21,
40.79, 26.65, 21.49; HRMS (ESI) m/z calcd. For C22
H
23
N
4
4
O (M+H)
1
1
1
4
4
64.83 (d, J = 5.4 Hz), 162.60, 159.64 (d, J = 256.7 Hz), 139.9,
433.1870, found 433.1873.
34.64, 133.86, 130.79, 126.81, 118.40 (d, J = 21.4 Hz), 115.73,
15.22, 114.93, 114.19 (d, J = 11.6 Hz), 102.49, 72.59, 58.38, 55.82,
4.1.8.40. (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
0.96, 21.49; HRMS (ESI) m/z calcd. For C22
H
22FN
4
O
4
(M+H)+
benzyl)aspartic acid hydrochloride (16-3h). Yield: 67.5%; White solid;
1
15.1620, found 415.1615.
Mp: 216–219 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.51 (s, 2H), 8.48 (d,
J = 2.4 Hz, 1H), 8.38 (dd, J = 9.0, 2.2 Hz, 1H), 8.07 (d, J = 8.4 Hz,
2H), 7.64 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 9.2 Hz, 1H), 4.97 (hept,
J = 6.0 Hz, 1H), 4.15–4.01 (m, 2H), 3.69 (dd, J = 7.6, 5.2 Hz, 1H),
4
.1.8.35. (4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)-3-
fluorobenzyl)aspartic acid hydrochloride (16-3c). Yield: 68.8%; White solid;
1
13
Mp: 223–225 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.48 (s, 1H), 8.48 (d,
2.77–2.62 (m, 2H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (101 MHz,
J = 2.4 Hz, 1H), 8.37 (dd, J = 9.0, 2.2 Hz, 1H), 8.08 (t, J = 7.8 Hz, 1H),
DMSO‑d ) δ 173.89, 171.26, 171.20, 167.96, 162.53, 138.74, 134.59,
6
7
.56–7.52 (m, 2H), 7.46 (d, J = 8.0 Hz, 1H), 4.97 (hept, J = 5.9 Hz, 1H),
.14–3.98 (m, 2H), 3.68 (t, J = 6.4 Hz, 1H), 2.77–2.63 (m, 2H), 1.38 (d,
133.82, 130.18, 127.21, 125.71, 115.93, 115.24, 114.90, 102.46,
4
72.56, 55.72, 49.44, 35.76, 21.50; HRMS (ESI) m/z calcd. For
13
(M+H)+ 451.1612, found 451.1607.
J = 6.0 Hz, 6H); C NMR (151 MHz, DMSO‑d
6
) δ 173.29, 172.28, 171.65,
C
23
H
23
N
O
4 6
1
64.90 (d, J = 5.6 Hz), 162.55, 159.70 (d, J = 256.1 Hz), 143.45 (d,
J = 5.3 Hz), 134.58, 133.79, 130.48, 125.31, 116.90 (d, J = 21.6 Hz),
4.1.8.41. 2-(1-(4-(5-(3-cyano-4-isopropoxyphenyl)-1,2,4-oxadiazol-3-yl)
1
4
4
15.74, 115.21, 114.87, 113.24 (d, J = 12.5 Hz), 102.47, 72.58, 56.18,
benzyl)piperidin-4-yl)acetic acid hydrochloride (16-3i). Yield: 69.2%;
(M+H)+
White solid; Mp: 252–256 °C; H NMR (400 MHz, DMSO‑d
1
) δ 12.24
9.17, 36.37, 21.50; HRMS (ESI) m/z calcd. For C23
H22FN O
4 6
6
69.1518, found 469.1514.
(s, 1H), 10.78 (s, 1H), 8.50 (d, J = 2.3 Hz, 1H), 8.40 (dd, J = 9.0,
55

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
+
2
.3 Hz, 1H), 8.14 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.57 (d,
J = 9.3 Hz, 1H), 4.98 (p, J = 6.1 Hz, 1H), 4.35 (d, J = 4.1 Hz, 2H), 3.31
s, 2H), 3.04–2.89 (m, 2H), 2.18 (d, J = 6.6 Hz, 2H), 1.86 (t,
J = 15.1 Hz, 3H), 1.58 (q, J = 13.0, 12.2 Hz, 2H), 1.38 (d,
30.29, 28.31, 23.53; HRMS (ESI) m/z calcd. For C28
H
31
N
4
4
O (M+H)
487.2340, found 487.2335.
(
4
.1.9. Synthesis of methyl 3-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-
1
3
J = 6.0 Hz, 6H); C NMR (101 MHz, DMSO‑d
6
) δ 173.92, 172.96,
oxadiazol-3-yl)phenyl)acrylate (17)
1
1
2
67.79, 162.52, 134.53, 133.72, 133.45, 132.38, 127.30, 126.80,
To a solution of 3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-
yl)benzaldehyde (200 mg, 0.53 mmol) under Argon in DMF was added
Methyl (triphenylphosphoranylidene)acetate (212.96 mg, 0.64 mmol),
and stirred at rt for 3 h. The reaction mixture was quenched with water
and extracted with ethyl acetate. The combined organic layers were
15.81, 115.19, 114.89, 102.43, 72.55, 58.43, 51.30, 30.25, 28.26,
+
1.48; HRMS (ESI) m/z calcd. For C26
H
29
N
4
O
4
(M+H) 461.2183,
found 461.2182.
4
.1.8.42. (4-(5-(3-cyano-4-(cyclopentyloxy)phenyl)-1,2,4-oxadiazol-3-yl)
washed with brine, dried over Na
2
SO , then filtered and concentrated.
4
benzyl)glycine hydrochloride (16-3j). Yield: 71.3%; White solid; Mp:
The residue was purified by silica gel flash column chromatography
1
2
8
09–211 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.37 (s, 1H), 8.48 (s, 1H),
(
PE/EtOAc = 3:1) to afford compound 17 (160 mg, 69.7% yield) as
.39 (dd, J = 9.2, 2.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 2H), 7.70 (d,
1
white solid. Mp: 124–126 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.20 (d,
J = 8.0 Hz, 2H), 7.52 (d, J = 9.2 Hz, 1H), 5.17–5.14 (m, 1H), 4.19 (s,
J = 8.4 Hz, 2H), 8.12 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.93 (d,
J = 8.0 Hz, 1H), 7.73 (d, J = 16.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H),
1
3
2
H), 3.69 (s, 2H), 2.06–1.97 (m, 2H), 1.80–1.64 (m, 6H); C NMR
(
151 MHz, DMSO‑d
6
) δ 174.00, 167.90, 167.85, 162.60, 135.32,
7
3
.29 (t, J = 7.6 Hz, 1H), 7.21–7.18 (m, 4H), 6.89 (d, J = 16.0 Hz, 1H),
1
8
34.50, 133.73, 131.08, 127.31, 126.49, 115.93, 115.13, 102.49,
.76 (s, 3H); MS (ESI) m/z 433.1 (M+H)+
.
1.74, 49.43, 46.57, 32.20, 23.53; HRMS (ESI) m/z calcd. For
+
C
23
H
23
N
4
O
4
(M+H) 419.1714, found 419.1771.
4
.1.10. Synthesis of methyl 3-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-
oxadiazol-3-yl)phenyl)propanoate (15-1l)
4
.1.8.43. N-(4-(5-(3-cyano-4-(cyclopentyloxy)phenyl)-1,2,4-oxadiazol-
To a solution of methyl 3-(3-chloro-4-(5-(4-phenoxyphenyl)-1,2,4-
oxadiazol-3-yl)phenyl)acrylate (150 mg, 0.35 mmol) in ethyl acetate
3
-yl)benzyl)-N-methylglycine hydrochloride (16-3k). Yield: 70.9%; White
1
solid; Mp: 193–196 °C; H NMR (400 MHz, DMSO‑d
6
) δ 8.45 (s, 1H),
was added Pd/C (15 mg) in batches. The mixture was stirred under H
2
8
2
2
.37 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 6.8 Hz, 2H), 7.58 (d, J = 6.4 Hz,
H), 7.50 (d, J = 8.4 Hz, 1H), 5.14 (s, 1H), 3.91 (s, 2H), 3.42 (s, 2H),
at rt for 4 h, then filtered via celite and concentrated. The filtrate was
1
3
diluted with EtOAc, washed with brine, dried over Na SO , filtered and
.41 (s, 3H), 2.00 (m, 2H), 1.80–1.65 (m, 6H); C NMR (151 MHz,
) δ 173.95, 168.61, 167.90, 162.59, 134.49, 133.71, 131.52,
2
4
concentrated. The residue was purified by silica gel flash column
DMSO‑d
6
chromatography (PE/EtOAc = 3:1) to afford compound 15–1l (119 mg,
1
4
4
31.35, 127.36, 126.30, 115.12, 115.12, 102.49, 81.72, 58.83, 55.55,
1
(M+H)+
52.1% yield) as white solid. Mp: 88–90 °C;
H NMR (400 MHz,
0.77, 32.19, 23.52; HRMS (ESI) m/z calcd. For C24
H
25
N
O
4 4
Chloroform-d) δ 8.17 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.0 Hz, 1H),
.44–7.41 (m, 3H), 7.24–7.20 (m, 2H), 7.10 (d, J = 8.4 Hz, 4H), 3.69
33.1870, found 433.1868.
7
(
s, 3H), 3.01 (t, J = 7.6 Hz, 2H), 2.68 (t, J = 7.6 Hz, 2H), MS (ESI) m/z
4
.1.8.44. 1-(4-(5-(3-cyano-4-(cyclopentyloxy)phenyl)-1,2,4-oxadiazol-3-
4
35.1 (M+H)+
.
yl)benzyl)pyrrolidine-3-carboxylic acid hydrochloride (16-3l). Yield:
1
6
1
2
4.1%; White solid; Mp: 229–232 °C; H NMR (400 MHz, DMSO‑d
6
) δ
2.94–11.36 (m, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.41 (dd, J = 9.0,
.2 Hz, 1H), 8.15 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.54 (d,
4
.2. Biological evaluation
.2.1. IP functional assay
J = 9.2 Hz, 1H), 5.18–5.15 (m, 1H), 4.46 (s, 2H), 3.35 (s, 5H),
4
1
1
3
2
.33–2.16 (m, 2H), 2.06–1.97 (m, 2H), 1.82–1.61 (m, 6H); C NMR
The Homogeneous Time Resolved Fluorescence-IP One (HTRF-IP1)
(
151 MHz, DMSO‑d
6
) δ 173.97, 173.09, 167.79, 162.59, 134.95,
assay was performed by IP-One Tb kit purchased from Cisbio (Cisbio
Bioassays). The CHO-S1P and CHO-S1P cells were prepared in sus-
1
8
34.47, 133.69, 131.46, 127.43, 126.72, 115.89, 115.11, 102.48,
1
3
1.73, 56.21, 53.69, 52.63, 40.66, 32.19, 26.55, 23.52; HRMS (ESI)
pension in stimulation buffer containing LiCl and 7 μl of suspension was
+
m/z calcd. For C26
H
27
N
4
O
4
(M+H) 459.2027, found 459.2024.
4
distributed in 384-well plates with density of 7 × 10 cells/well. Then,
7
μl different concentrations of test agonists were added and the cells
4
.1.8.45. (4-(5-(3-cyano-4-(cyclopentyloxy)phenyl)-1,2,4-oxadiazol-3-yl)
were incubated at 37 °C with 5% CO for 2 h. Meanwhile, a standard
2
benzyl)aspartic acid hydrochloride (16-3m). Yield: 64.1%; White solid;
curve was also built according to the protocol. Subsequently, 3 μl IP1-
d2 and Ab-Cryp diluted by Lysis Buffer were added into each well in
succession and incubated at room temperature for 1 h at 25 °C. Plates
were then read in EnVision reader (PE Company), ratio of 665 nm/
1
Mp: 229–232 °C; H NMR (400 MHz, DMSO‑d
6
) δ 10.56 (s, 2H), 8.48 (d,
J = 2.4 Hz, 1H), 8.38 (dd, J = 9.0, 2.2 Hz, 1H), 8.08 (d, J = 8.0 Hz,
2
1
2
H), 7.64 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 9.2 Hz, 1H), 5.17–5.14 (m,
H), 4.15–4.01 (m, 2H), 3.73–3.69 (m, 1H), 2.78–2.63 (m, 2H),
6
15 nm fluorescence was calculated and corrected by standard curve.
1
3
.06–1.97 (m, 2H), 1.81–1.63 (m, 6H);
C NMR (151 MHz,
EC50 values are calculated by Graph pad software 5.0.
DMSO‑d
6
) δ 173.99, 170.88, 169.99, 167.90, 162.61, 136.51, 134.52,
1
5
33.77, 130.88, 127.28, 126.26, 115.96, 115.13, 102.50, 81.74, 64.92,
4.2.2. Blood lymphocyte reduction assay
5.26, 49.34, 34.59, 32.19, 23.52, 15.18; HRMS (ESI) m/z calcd. For
+
Male Sprague-Dawley rats (200–220 g) were dosed through in-
C
25
H
25
N
4
O
6
(M+H) 477.1769, found 477.1766.
tragastric administration with test agonists (as a solution in double
distilled water), positive control and vehicle. Blood was drawn at the
time 0 h, 4 h, 8 h, 12 h and 48 h after administration via tail vein and
peripheral blood lymphocyte counts were assessed using MEK-7222K
hematology analyzer.
4
.1.8.46. 2-(1-(4-(5-(3-cyano-4-(cyclopentyloxy)phenyl)-1,2,4-
oxadiazol-3-yl)benzyl)piperidin-4-yl)acetic acid hydrochloride (16-
1
3
n). Yield: 65.0%; White solid; Mp: 244–246 °C; H NMR (400 MHz,
DMSO‑d
6
) δ 12.17 (s, 1H), 10.58–10.43 (m, 1H), 8.47 (d, J = 2.4 Hz,
1
H), 8.37 (dd, J = 9.0, 2.2 Hz, 1H), 8.12 (d, J = 8.0 Hz, 2H), 7.77 (d,
J = 8.0 Hz, 2H), 7.50 (d, J = 9.2 Hz, 1H), 5.15–5.11 (m, 1H), 4.32–4.31
4.2.3. Heart rate assay
(
m, 2H), 3.32 (s, 2H), 2.98–2.88 (m, 2H), 2.15 (d, J = 6.8 Hz, 2H),
Male SD rats (200–220 g) were dosed orally with 10 mg/kg of test
agonists, positive control and vehicle. The heart rate at the time 0 h,
2 h, 4 h, 8 h, 12 h and 24 h after administration were assessed using
intelligent non-invasive blood pressure measurement meter.
1
3
2
.03–1.94 (m, 2H), 1.83–1.45 (m, 11H); C NMR (151 MHz, DMSO‑d )
6
δ 174.01, 173.00, 167.84, 162.61, 134.51, 133.73, 133.39, 132.38,
1
27.36, 126.87, 115.92, 115.14, 102.50, 81.74, 58.49, 51.42, 32.19,
56

T. Liu et al.
Bioorganic Chemistry 82 (2019) 41–57
4
.2.4. In vivo pharmacokinetic assay
[17] R. Albert, K. Hinterding, V. Brinkmann, D. Guerini, C. Muller-Hartwieg, H. Knecht,
C. Simeon, M. Streiff, T. Wagner, K. Welzenbach, F. Zecri, M. Zollinger, N. Cooke,
E. Francotte, Novel immunomodulator FTY720 is phosphorylated in rats and hu-
mans to form a single stereoisomer. identification, chemical proof, and biological
characterization of the biologically active species and its enantiomer, J. Med. Chem.
Blood samples of male SD rats were obtained at 0.08, 0.25, 0.5, 1, 2,
, 6, 8, 12, 24, 36 and 48 h postdose. The blood was prepared by
4
centrifugation and the plasma samples were analyzed using liquid
chromatography coupled to mass spectrometry (LC-MS/MS) after pro-
tein precipitation. Data fitting and pharmacokinetic parameter esti-
mates were carried out using WinNonLin Software.
4
8 (2005) 5373–5377.
[
[
[
18] J. Chun, V. Brinkmann, A mechanistically novel, first oral therapy for multiple
sclerosis: the development of fingolimod (FTY720, Gilenya), Discov. Med. 12
(
2011) 213–228.
19] V. Brinkmann, A. Billich, T. Baumruker, P. Heining, R. Schmouder, G. Francis,
S. Aradhye, P. Burtin, Fingolimod (FTY720): discovery and development of an oral
drug to treat multiple sclerosis, Nat. Rev. Drug Discov. 9 (2010) 883.
20] A. Billich, F. Bornancin, P. Dévay, D. Mechtcheriakova, N. Urtz, T. Baumruker,
Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases, J.
Biol. Chem. 278 (2003) 47408–47415.
Acknowledgments
This work was financially supported by National Natural Science
Foundation of China (NSFC no. 81473096 and 81602949), Beijing
Natural Science Foundation (no. 7172140) and CAMS Collaborative
Innovation Project (no. 2017-I2M-3-011).
[21] R. Albert, K. Hinterding, V. Brinkmann, D. Guerini, C. Müller-Hartwieg, H. Knecht,
C. Simeon, M. Streiff, T. Wagner, K. Welzenbach, F. Zécri, M. Zollinger, N. Cooke,
E. Francotte, Novel immunomodulator FTY720 is phosphorylated in rats and hu-
mans to form a single stereoisomer. identification, chemical proof, and biological
characterization of the biologically active species and its enantiomer, J. Med. Chem.
4
8 (2005) 5373–5377.
Appendix A. Supplementary material
[
[
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