The optimization of xanthine derivatives leading to HBK001 hydrochloride as a potent dual ligand targeting DPP-IV and GPR119

Article abstract of DOI:10.1016/j.ejmech.2019.112017

A series of xanthine compounds derived from the previous hit 20i with modification on the terminal side chain was discovered through ring formation strategy. Systematic optimization of the compounds with rigid heterocycles in the hydrophobic side chain led to the new lead compound HBK001 (21h) with the improved DPP-IV inhibition and moderate GPR119 agonism activity in vitro. As a continuing work to further study the PK and PD profiles, 21h and its hydrochloride (22) were synthesized on grams scale and evaluated on the ADME/T and oral glucose tolerance test (OGTT) in ICR mice. Compound 22 showed the improved bioavailability and blood glucose-lowering effect in vivo compared to its free base 21h probably attributed to its improved solubility and permeability. The preliminary toxicity studies on compound 22 exhibited that the result of mini-Ames was negative and the preliminary acute toxicity LD₅₀ in mice was above 1.5 g/kg, while it showed moderate inhibition on hERG channel with IC₅₀ 4.9 μM maybe due to its high lipophilicity. These findings will be useful for the future drug design for more potent and safer dual ligand targeting DPP-IV and GPR119 for the treatment of diabetes.

Full text of DOI:10.1016/j.ejmech.2019.112017

Journal Pre-proof
The optimization of xanthine derivatives leading to HBK001 hydrochloride as a potent
dual ligand targeting DPP-IV and GPR119
Gang Li, Bingxu Meng, Baokun Yuan, Yi Huan, Tian Zhou, Qian Jiang, Lei Lei, Li
Sheng, Weiping Wang, Ningbo Gong, Yang Lu, Chen Ma, Yan Li, Zhufang Shen,
Haihong Huang
PII:
S0223-5234(19)31175-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.112017
EJMECH 112017
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 October 2019
Revised Date: 27 December 2019
Accepted Date: 27 December 2019
Please cite this article as: G. Li, B. Meng, B. Yuan, Y. Huan, T. Zhou, Q. Jiang, L. Lei, L. Sheng, W.
Wang, N. Gong, Y. Lu, C. Ma, Y. Li, Z. Shen, H. Huang, The optimization of xanthine derivatives leading
to HBK001 hydrochloride as a potent dual ligand targeting DPP-IV and GPR119, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.112017.
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Graphical Abstract
The optimization of xanthine derivatives leading to HBK001 hydrochloride as a
potent dual ligand targeting DPP-IV and GPR119
1
1
Gang Li , Bingxu Meng , Baokun Yuan, Yi Huan, Tian Zhou, Qian Jiang, Lei Lei, Li Sheng,
Weiping Wang, Ningbo Gong, Yang Lu, Chen Ma, Yan Li, Zhufang Shen*, Haihong Huang*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Beijing Key
Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia
Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing
1
00050, P. R. China
*
Corresponding author. E-mail addresses: shenzhf@imm.ac.cn (Z. Shen), joyce@imm.ac.cn (H. Huang).

The optimization of xanthine derivatives leading to HBK001 hydrochloride as a
potent dual ligand targeting DPP-IV and GPR119
1
1
Gang Li , Bingxu Meng , Baokun Yuan, Yi Huan, Tian Zhou, Qian Jiang, Lei Lei, Li Sheng,
Weiping Wang, Ningbo Gong, Yang Lu, Chen Ma, Yan Li, Zhufang Shen*, Haihong Huang*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Beijing Key
Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia
Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing
1
00050, P. R. China
Abstract
A series of xanthine compounds derived from the previous hit 20i with modification
on the terminal side chain was discovered through ring formation strategy. Systematic
optimization of the compounds with rigid heterocycles in the hydrophobic side chain
led to the new lead compound HBK001 (21h) with the improved DPP-IV inhibition
and moderate GPR119 agonism activity in vitro. As a continuing work to further study
the PK and PD profiles, 21h and its hydrochloride (22) were synthesized on grams
scale and evaluated on the ADME/T and oral glucose tolerance test (OGTT) in ICR
mice. Compound 22 showed the improved bioavailability and blood glucose-lowering
effect in vivo compared to its free base 21h probably attributed to its improved
solubility and permeability. The preliminary toxicity studies on compound 22
exhibited that the result of mini-Ames was negative and the preliminary acute toxicity
LD50 in mice was above 1.5 g/kg, while it showed moderate inhibition on hERG
channel with IC 4.9 µM maybe due to its high lipophilicity. These findings will be
5
0
useful for the future drug design for more potent and safer dual ligand targeting
DPP-IV and GPR119 for the treatment of diabetes.
Key words
DPP-IV; GPR119; xanthine compounds; HBK001; druggability;
*
Corresponding author. E-mail addresses: shenzhf@imm.ac.cn (Z. Shen), joyce@imm.ac.cn (H. Huang).
1
These authors contributed equally to this article.
1

1
. Introduction
Type 2 diabetes mellitus (T2DM) is a progressive chronic disease characterized
by insulin resistance and defective insulin secretion from the pancreas [1]. During the
development of T2DM, pancreatic β-cell function gradually declines resulted in a
severe deduction of insulin secretion [2]. Glucagon like peptide-1 (GLP-1) is one of
the major incretins released from intestinal enteroendocrine L cells and plays an
important role in nutrient metabolism and glucose homeostasis [3, 4]. As GLP-1 is
rapidly degraded by dipeptidyl peptidase IV (DPP-IV), DPP-IV inhibitors can prolong
the half-life of active GLP-1, which promotes glucose-stimulated insulin secretion
and decreases glucose [5].
Recently, along with the successful commercial launch of GLP-1 analogues and
DPP-IV inhibitors, intensive interest in the development of T2DM therapeutics has
been stimulated on the targets which enhance insulin secretion in a glucose-dependent
manner in order to minimize the risk of hypoglycemia [6]. On the other hand,
G-protein-coupled receptor 119 (GPR119) has been emerged as one of the most
exciting targets for the treatment of T2DM. GPR119 agonists mediate a unique dual
elevation of both insulin and GLP-1, followed by a reduction in blood glucose level
[
7]. Representative launched DPP-IV inhibitors such as Sitagliptin, Linagliptin,
Tenegliptin and GPR119 agonists under research such as AR231453, MBX-2982 are
depicted in Fig. 1.
Fig. 1. Structures of representative DPP-IV inhibitors and GPR119 agonists
2

Therefore, based on the biological effects of GLP-1, the synergistic effects
between DPP-IV inhibition and GPR119 agonism may bring advantages in blood
glucose control. Indeed, in animal experiments, combining a DPP-IV inhibitor with a
GPR119 agonist significantly increased the plasma active GLP-1 levels and improved
glucose clearance [8, 9]. Inspired by the promising results, the proof of concept hit 20i
with jointed DPP-IV inhibition and GPR119 agonism activity was discovered by our
group through systematic structural modification based on the pharmacophore merged
strategy [10]. The multi-target ligands with the lower risk of drug-drug interactions
and simplified clinical development could offer a new therapeutic option in the
treatment of type 2 diabetes [11-14].
In order to further improve the dual DPP-IV and GPR119 activities, the amide in
the hydrophobic side chain of 20i was converted to various rigid heterocycles through
ring formation strategy (Fig. 2). Among them, compound HBK001 (21h) with
oxadiazole side chain can both inhibit DPP-IV and activate GPR119 ex and in vivo.
Furthermore, HBK001 (21h) can improve islet morphology, increase β-cell
proliferation and up-regulate genes involved in improved β-cell function [15].
Fig. 2. Structure optimization through the ring formation strategy
Herein, we present the synthesis, SARs and ADME/T of HBK001 (21h) and its
derivatives. All the compounds exhibited good DPP-IV inhibitory activities and 21h
showed better DPP-IV inhibition and comparative GPR119 agonism activity than the
hit 20i. Further druggability results revealed that HBK001 hydrochloride (22) showed
3

improved bioavailability and glucose tolerance compared to its free base. The further
modification based on the identified compound 22 is ongoing for the discovery of
dual ligand drug candidate targeting DPP-IV and GPR119 for the treatment type 2
diabetes.
2
. Chemistry
The target xanthine derivatives can be conveniently afforded by reacting
xanthine intermediate 20 with piperidyl/piperazinyl intermediates through
retrosynthetic analysis (Scheme 1). The 1,3-dibromopropane was used as the key
precursor reagent to construct the linker between the xanthine and
piperidyl/piperazinyl scaffolds. A series of five/six-member heterocycles, derived
from the privileged scaffold of DPP-IV inhibitors and GPR119 agonists, was involved
in the terminal of the aromatic side chain.
Scheme 1. Retrosynthetic analysis of the target compounds
The synthesis of key intermediates 6a-g, 16a-f and 19a-f containing heterocycles
is outlined in Scheme 2-4. The xanthine intermediate 20 is synthesized according to
our previous work [10]. The general synthetic procedures of the target compounds
2
1a-s and a hydrochloride 22 are illustrated in Scheme 5.
2
.1 Synthesis of the piperidyl intermediates 6a-g
The N-Cbz piperidine-4-carboxylic acid 1a was treated with oxalyl chloride
followed by refluxing with N'-hydroxybenzimidamide 2 in toluene and deprotection
of Cbz to afford the oxadiazole intermediate 6a [16]. The triazole piperidyl 6b was
obtained through the click reaction between the azide 1b and phenylacetylene 3 in the
presence of CuI and DIPEA [17]. The thiazole 6c was formed by the heterocyclization
4

of N-Boc piperidine-4-thioformamide 1c and α-bromoacetophenone 4 followed by the
deprotection of Boc group. The intermediate 1e, prepared from the amidation of 1a
and 1d, reacted with AcONH in the solvent of AcOH to produce imidazole 6d while
4
the treatment of 1e with POCl in the presence of pyridine generated oxazole 6e [18].
3
The phenylacetylene 3 was treated with 1f in the presence of potassium
peroxymonosulfate to form isoxazole 6f. Tetrazole 6g was obtained by the amidation
of N-Boc protected piperidine-4-carboxylic acid 1g and aniline 5 followed by
heterocyclization and deprotection [19].
Scheme 2. Synthesis of the intermediates 6a-g. Reagents and conditions: i. Oxalyl chloride, DMF,
DCM, r.t., 3h; ii. Toluene, reflux, 4h; iii. TMSI, DCM, 0°C - r.t., 4h; iv. CuI, DIPEA, DCM, r.t.,
2
h; v. TFA, DCM, r.t., 2h; vi. NaHCO , ethanol, reflux, 3h; vii. CDI, DIPEA, DCM, 0°C - r.t., 12h;
3
viii. AcONH , AcOH, reflux, 5h; ix. POCl , pyridine, r.t., 2h; x. KCl, HKO S, H O, 0°C - r.t., 12h;
4
3
6
2
xi. EDCI, HOBt, TEA, acetonitrile, r.t., 5h; xii. TMS-N , Ph P, DIAD, r.t. 6 days then reflux 1h;
3
3
xiii. 7N HCl-EA, r.t., 1h.
2
.2 Synthesis of the piperazinyl intermediates 16a-f
The N'-hydroxybenzimidamide 2 was treated with trichloroacetic anhydride
followed by the substitution with piperazine to form the oxadiazole intermediate 16a
20]. The treatment of benzimidamide 8 with (trichloromethyl)sulfenyl chloride in the
[
presence of NaOH generated intermediate 9. The thiadiazole 16b was formed by the
5

substitution of 9 followed by the deprotection of Boc group [21]. Reacting 10 with
Boc-piperazine produced intermediate 11 [22]. The triazole 16c was obtained by the
heterocyclization of 11 and hydrazine monohydrate followed by the deprotection [23].
The methyl 3-oxobutanedithioate 12 was treated with Boc-piperazine followed by the
heterocyclization with phenylhydrazine and deprotection of Boc to afford the pyrazole
1
6d. The thiazole 16e was formed by the heterocyclization of 14 and
2
-bromoacetophenone followed by the deprotection [24]. The intermediate 15 was
prepared from the substitution of cyanogen bromide followed by the addition of
hydroxylamine. The treatment of 15 with benzoic acid and CDI under refluxing
condition followed by the deprotection afforded intermediate 16f [25].
Scheme 3. Synthesis of the intermediates 16a-f. Reagents and conditions: i. Trichloroacetic
anhydride, toluene, reflux, 10h; ii. Anhydrous piperazine, DMF, r.t., 6h. iii.
(Trichloromethyl)sulfenyl chloride, NaOH, DCM, 0°C - r.t., 3h; iv. 1-Boc-piperazine, TEA, DMF,
r.t., 12h; v. TFA, DCM, r.t., 2h. vi. 1-Boc-piperazine, DCM, 0°C - r.t., 2h; vii. Hydrazine
monohydrate, CHCl , reflux, 4h; viii. Phenylhydrazine, AcOH, 4A molecular sieve, dry ethanol,
3
reflux, 15h; ix. 2-Bromoacetophenone, NaHCO , ethanol, reflux, 4h. x. Cyanogen bromide,
3
NaHCO DCM/H O, r.t., 2h; xi. 50% NH -OH aqueous solution, EtOH, reflux, 6h; xii. Benzoic
3,
2
2
acid, CDI, DMF, 120°C, 8h.
2
.3 Synthesis of the pyrimidyl intermediates 19a-f
The reactions of commercially available starting material 17a-f with
6

Boc-piperazine 13 in the presence of K CO produced 18a-f [26]. Intermediates 19a-f
2
3
were obtained by the N-Boc deprotection of 18a-f.
Scheme 4. Synthesis of the intermediates 19a-f. Reagents and conditions: i. TEA, ethanol, reflux,
6
h; ii. TFA, DCM, r.t., 3h.
2
.4 General synthesis of the target compounds 21a-s and 22
Finally, the nucleophilic substitution of xanthine intermediate 20 [10] with the
corresponding intermediates 6a-g, 16a-f and 19a-f in the presence of DIPEA,
followed by deprotection of Boc group, provided the target compounds 21a-s
respectively. The HBK001 hydrochloride (22) was formed with a solution of
HCl/EtOH via salification of its free base 21h.
Scheme 5. General synthesis of the target compounds 21a-s and 22. Reagents and conditions: i.
DIPEA, DMF, 80°C, 10h; ii. TFA, DCM, r.t., 2h; iii. HCl/EtOH, r.t., 3h.
2
.5 Single-crystal X-ray structure of compound 21h
The configuration of 21h was confirmed by an X-ray crystal structure (CCDC
943980, Fig. 3). The X-ray analysis data demonstrates the exclusive formation of
1
(R)-3-aminopiperidine at C-8. No intramolecular hydrogen bond was found and the
hydrophobic phenyl oxadiazole side chain was linear as expected, which was in
accordance with the pharmacophore feature for the GPR119 agonist [7].
7

Fig. 3. X-ray crystallographic structure of compound 21h
3
. Results and discussion
The target compounds were evaluated in vitro for their inhibition of DPP-IV,
DPP-8/9 and agonism activities of GPR119. The inhibition against DPP-IV as IC50
values and the agonism to GPR119 as EC values of the selected compounds were
5
0
summarized in Table 1-2 with sitagliptin and AR231453 as the positive controls. The
relative activity percentages of the corresponding reference compounds are
normalized to the activity of AR231453 at the same concentration, and the EC value
5
0
refers to the concentration of the indicated compound reaching half potency of
GPR119 agonism itself [10].
3
.1 Hit optimization through ring formation strategy
Our previous work revealed that the linker length of 3 methylene units and
(R)-3-aminopiperidine were the crucial constituents for the activities [10]. Hence, our
continuous systematic modification was focused on the terminal side chain through
ring formation strategy based on our previous hit 20i. The amide group was directly
converted to various five-member heterocycles 21a-g (Table 1). To our delight, the
compound 21a-g with rigid heterocycles retained good potency of DPP-IV with IC50
values ranged from 0.044 µM to 0.27 µM. In the meanwhile, the compounds 21c-e
which have the same heteroatom position exhibited good GPR119 agonism above 60%
activity of AR231453 at 1 µM. In particular, compound 21b and 21d with the triazole
and imidazole units demonstrated much better DPP-IV inhibitory activities than 20i
(
DPP-IV, IC = 0.22 µM) [10], which offered 5-fold improvement in potency. The
50
results indicated that the introduction of additional conformational rigidity led to the
improved DPP-IV inhibitory activities. Specifically, not only did compound 21d
8

display potent DPP-IV inhibition (IC = 0.044 µM), it also exhibited good GPR119
5
0
agonism activity (EC = 0.6 µM). Meanwhile, these compounds showed excellent
5
0
DPP-IV selectivity over DPP-8/9, which is one key requirement due to the safety
concern.
Inspired by the improved DPP-IV inhibition from 0.22 µM (20i) to 0.044 µM
(21d) through the ring formation strategy, other versatile five-member heterocycles
with the piperazine linker were conducted (21h-m). It turned out that the installation
of polar nitrogen atom led to the improved activities against DPP-IV exemplified by
compound 21a with piperidine linker (IC = 0.23 µM) versus 21h with piperazine
5
0
linker (IC =0.04 µM), and similarly compound 21c (IC = 0.19 µM) versus 21l
5
0
50
(
IC = 0.059 µM). Especially, compound 21h and 21l also showed the good potency
50
of GPR119 compared to the positive control AR231453 in vitro. When the oxygen
atom in the oxadiazole in compound 21h was replaced by sulfur and nitrogen atom,
the corresponding compounds 21i and 21j decreased DPP-IV potency to 0.36 µM and
0
.076 µM, respectively. Among them, the GPR119 agonism of compound 21j was
disappeared. In addition, the position exchange of oxygen and nitrogen in the
oxadiazole (21m) resulted in the decreased DPP-IV inhibitory activity and maintained
GPR119 activity compared to compound 21h. The introduction of
1
-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine unit which selected from the side
chain of Teneligliptin afforded the desirable DPP-IV potency, but lost GPR119
agonism maybe due to the loss of structural linearity (21k). To our delight, the
compounds 21h and 21i could maintain comparable potency toward GPR119 (EC =
5
0
1
.40 and 1.93 µM), especially, compound 21h displayed very potent DPP-IV
inhibitory activity.
Table 1. Activity of the target compounds with five-member heterocycles against DPP-IV,
DPP-8/9 and GPR119
9

DPP-IV
GPR119
DPP-8/9
Compds.
Ar
X
a
b
c
d
%
Inhibition
IC50
%Activity
EC50
(10 µM)
(10 µM)
(µM)
(1 µM)
(µM)
2
1a
CH
CH
CH
CH
CH
CH
91.8
98.8
79.5
89.2
97.3
89.2
0.23
-
55
-
21b
0.047
0.19
0
47
83
65
80
49
-
2
1c
2.2
5.1
3.3
3.3
3.13
0.6
0.83
-
21d
0.044
0.12
2
1e
2
1f
0.19
21g
CH
89.0
0.27
4.6
53
-
2
1h
N
N
N
97.8
86.5
94.5
0.04
0.36
0
58
81
38
1.40
1.93
-
2
1i
1.6
7.3
2
1j
0.076
2
1k
N
N
82.9
86.3
0.083
0.059
15.4
0
42
56
-
2
1l
0.65
1
0

21m
N
87.1
0.68
-
89
1.45
e
2
0i
93.6
99.0
-
0.22
0.0084
-
36.8
81.5
-
0.95
-
Sitagliptin
AR231453
-
-
100
0.64
a
b
Maximum % inhibition against DPP-IV. Concentration resulting in 50% maximum inhibition of
c
DPP-IV. Relative activity percentage was normalized to the activation fold of AR231453.
d
e
Concentration resulting in 50% maximum activation activity of GPR119. The data were cited
from our previous result [10].
Subsequently, we concentrated our efforts on the further modification of GPR119
pharmacophore. Attracted by the GPR119 agonist MBX-2982 in phase II clinical
stage, its substituted pyrimidine unit was chosen as the terminal side chain to form the
new target compounds 21n-s (Table 2). It revealed that all the xanthine compounds
containing pyrimidine could maintain GPR119 agonism above 60% activity at 1 µM
with EC values ranged from 1.41 µM to 4.02 µM. Among them, compound 21r
5
0
(EC50 = 1.41 µM) with fluorine substituted pyrimidine side chain exhibited better
potency than 21o (EC50 = 3.99 µM) containing ethyl pyrimidine side chain like
MBX-2982. The bulky hydrophobic propyl pyrimidine compound 21p showed better
activity than compounds 21n and 21o toward GPR119 (EC = 1.85 µM vs 2.56 µM,
5
0
3
.99 µM). The compounds with electron-donating group (21n and 21q) displayed
better GPR119 agonism than the compound with strong electron-withdrawing group
21s) (EC50 = 2.56 µM, 2.26 µM vs 4.02 µM). In addition, the substitution of
(
N-benzyl amide with pyrimidine could maintain the DPP-IV inhibitory activity with
IC values ranged from 0.35 µM to 0.74 µM compared to the previous hit 20i (IC =
5
0
50
0
.22 µM). Although the target compounds with pyrimidine side chain possessed
moderate to good GPR119 agonism activity compared to the compounds containing
five-member heterocycles, that these compounds decreased DPP-IV inhibitor activity
to some extent was unanticipated.
Table 2. Activity of the target compounds with six-member heterocycles against DPP-IV and
GPR119
1
1

DPP-IV
GPR119
Compds.
R
a
b
c
d
%
Inhibition IC50
%Activity EC50
(
10 µM)
91.2
86.3
89.2
90.5
93.4
88.0
91.7
-
(µM)
(1 µM)
(µM)
CH3
21n
0.66
0.61
0.59
0.43
0.35
0.74
0.018
-
60
2.56
3.99
1.85
2.26
1.41
4.02
-
CH CH
21o
3
2
85
CH CH CH
2
2
1p
1q
3
2
2
84
CH O
3
79
F
2
1r
1s
78
CF3
2
90
Sitagliptin
AR231453
-
100
0.64
a
b
Maximum % inhibition against DPP-IV. Concentration resulting in 50% maximum inhibition of
c
DPP-IV. Relative activity percentage was normalized to the activation fold of AR231453.
d
Concentration resulting in 50% maximum activation activity of GPR119.
3
.2 Lead identification through preliminary druggability evaluation
To identify the metabolic and toxic liabilities of the novel xanthine series,
compounds 21b, 21d, 21h and 21l exhibited improved DPP-IV inhibitory activities
IC = 0.04-0.059 µM) and compound 21m showed potent GPR119 agonism (89%
(
5
0
activity; EC50 = 1.45 µM), were evaluated for their hepatocyte stability and
cytotoxicity in vitro (Table 3). As shown in Table 3, the selected compounds with
five-member heterocycles displayed good hepatocyte stability in human, but low to
medium stability in mouse or rat. The Vero cells were often used in the cytotoxicity
experiment in vitro to evaluate the preliminary toxicity. Their IC s against Vero cell
5
0
line were substantially different. Unfortunately, compound 21l with potent dual
activities and relatively good hepatocyte stability exhibited obvious toxicity (IC =
5
0
4
.3 µg/mL). Based on the comprehensive analysis of other xanthine compounds, 21h
which possessed favorable human hepatocyte stability and noncytotoxicity was
identified as a promising lead for the treatment of diabetes. Indeed, as it was reported
1
2

that compound 21h exerted good activities in ICR mice as well as the diabetic db/db
and KKAy mice. Moreover, 21h can increase first-phase insulin secretion in KKAy
mice, suggesting a direct effect on islet β-cells via GPR119 activation [15].
Table 3. Hepatocyte stability and cytotoxicity of selected xanthine compounds
a
Vero
IC50
Substrate remaining (%)
Compds.
Mouse
Rat
Human
(µg/mL)
2
2
2
1b
1d
1h
21.0
69.4
32.0
68.3
24.2
19.7
21.7
23.8
39.6
25.0
68.1
84.0
101.2
91.5
82.0
25.9
35.2
46.6
4.3
2
1l
21m
8.7
a
Substrate concentrations were determined in incubations after 30 min and normalized to
concentrations at time zero.
3
.3 Potential candidate confirmation through salt formation
As shown in Table 4, compound 21h as a free base displayed a relatively low
bioavailability (12.2%). It prompted us to improve the bioavailability by forming its
hydrochloride 22. Following a single oral and an intravenous administration of 22,
this compound exhibited high plasma exposure (AUC0−∞ = 656 ng·h/mL), high
maximum plasma concentration (Cmax
t1/2 = 1.08 h) after oral administration dosed at 30 mg/kg (free base content) in rat.
The oral bioavailability was increased from 31.8% to 42.4% compared to its free base
1h at the same dose (30 mg/kg). In addition, compound 22 showed slower tmax and
longer MRT(0-∞) in rat after oral administration. These results indicated that compound
= 226 ng/ml) and medium elimination half-life
(
2
2
2 with superior PK properties may enhance the in vivo efficacy in animal models.
Table 4. Mouse and Rat Pharmacokinetic Properties of 21h and 22
2
1h
22
Parameters
Units
a
b
Mouse (PO ) Mouse (IV ) Rat (PO)
Rat (PO)
30
Rat (IV)
3
Dose
mg/kg
h
20
2
30
c
t1/2
1.06
0.42
213
308
318
1.64
-
1.11
1.17
192
468
502
1.08
1.33
226
0.63
-
d
tmax
h
e
Cmax
ng/mL
h*ng/mL
h*ng/mL
850
257
261
416
147
167
AUC(0-t)
AUC(0-∞)
623
f
656
1
3

g
MRT(0-∞)
h
1.22
0.76
2.11
31.8
2.22
42.4
0.73
h
F
%
12.2
a
b
c
d
Per os. Intravenous injection. Plasma elimination half-life. Time of maximum plasma
e
f
g
concentration. Maximum plasma concentration. Plasma exposure. Mean residence time.
h
Bioavailability.
To confirm the pharmacokinetic results of 22 and 21h, the euglycemic ICR mice
was used to explore the glucose-lowering effect (Fig. 4A). In ICR mice, a single oral
administration of 22 (30 mg/kg, free base content) significantly reduced blood glucose
level at 30 minutes after oral glucose loading. Impressively, 22 could achieve a much
better in vivo effect compared to its free base at the same dose (30 mg/kg) (Fig. 4A).
The oral glucose tolerance test (OGTT) is the reference method assessing glucose
tolerance in the body. It reflects glucose utilization and insulin sensitivity after oral
intake of glucose. To further evaluate the anti-diabetic effect of 22, we performed
OGTT in ICR mice with 22 and positive controls (Fig. 4B). The area under the curve
of OGTT (AUCOGTT) of 22-treated group was significantly decreased by 26.8% at the
dose of 30 mg/kg, which was almost equivalent to the AUC of the linagliptin-treated
group (26%, 2 mg/kg) and sitagliptin-treated group (27.3%, 10 mg/kg), better than the
AUC of APD597-treated group (19.9%, 10 mg/kg) as a positive control of GRP119
agonist (Fig. 4B). These findings indicated that compound 22 could achieve
comparative glucose-lowering effect in vivo. Thus far, all the above results supported
compound 22 to be worth of further investigation on druggability.
Fig. 4. Oral glucose tolerance test in ICR mice. (A) Blood glucose level and area under the curve
of glucose tolerance test (AUCOGTT) measurements after single dose treatment of 21h and 22. (B)
1
4

Blood glucose level and AUCOGTT measurements after single dose treatment of 22 and positive
controls. All experiments were performed independently. Data are shown as mean ± SEM of every
group, n = 10. *P < 0.05, **P < 0.01, ***P < 0.001, vs vehicle group.
Inspired by the promising PK and PD profiles aforementioned, selected
ADME/T studies were conducted on compound 22. As presented in Table 5,
compound 22 showed high permeability and moderate metabolic stability. This
compound 22 exhibited very high plasma protein binding (PPB, >96%) in five species,
possibly due to its high lipophilicity. The ClogP value of 21 (free base form of 22) is
3
.41, while the values of linagliptin and AR231453 are 1.91 and 2.21, respectively,
calculated with ChemDraw Professional 16.0.
As depicted in Table 6, compound 22 displayed high IC value against Vero cell,
5
0
indicating a lack of toxicity to renal cell. A preliminary in vivo tolerability study was
carried out in mice with a single dose at 1.5 g/kg. All animals survived after oral
administration followed by a 7-day observation. Notably, the result of bacterial
reverse mutation assay in Salmonella typhimurium tester strains TA98 and TA100 was
negative, which suggests low potential for genotoxicity. Maybe attributed its high
lipophilicity and aromatic character of linear side chain being curial for GPR119
agonism activity, compound 22 showed moderate inhibition of the hERG channel in a
manual patch clamp assay (IC = 4.9 µM). It precludes that it could be a challenge to
5
0
balance the physiological properties and biological activities to afford a promising
candidate with more potent anti-diabetic activity and lower toxicity targeting DPP-IV
and GPR119 at the same time.
Table 5. In vitro ADME data for compound 22
a
c
Papp
Mouse Liver Microsome
Plasma Protein Binding (%)
Compd
-6
b
(
×10 cm/s) substrate remaining (%) Mouse Rat Dog Monkey Human
2
2
19.1±1.1
49.3%
98.5
96.1 96.3
96.0
97.2
a
b
Apparent permeability coefficient. Substrate concentrations were determined in incubations with
c
NADPH after 30 min and normalized to concentrations at time zero. Binding at 5 h, starting
concentration 5 µM.
Table 6. Preliminary toxicity study of compound 22
1
5

a
+
Cytotoxicity
Acute Toxicity Study
(LD )
hERG K inhibition
b
Compd
Mini-Ames
Vero (µg/mL)
IC (µM)
5
0
50
2
2
>64
> 1.5 g/kg
negative
4.9
a
No. of animals that survived/total no. of animals after 7 days.
b
Genetic safety assay using two Salmonella strains: TA98 and TA100.
4
. Conclusion
In summary, we have synthesized and characterized a series of xanthine
compounds derived from our previous hit 20i modification on the terminal
hydrophobic side chain through ring formation strategy. Systematic optimization led
to the identification of compound HBK001 hydrochloride 22, which proved to be a
dual modulator targeting DPP-IV and GPR119 with efficient in vivo glucose-lowering
effect and favorable PK properties. The results of preliminary toxicity investigation
exhibited that this compound 22 displayed the moderate inhibition of the hERG
channel possibly due to its high lipophilicity. The efforts to maintain the activity but
reduce the hERG inhibition through increasing the hydrophilic or the rigidity of the
entire molecule are ongoing in order to develop the more promising candidates as
anti-diabetic agents with dual mechanism targeting both DPP-IV and GPR119.
5
5
5
. Experimental
.1. Chemistry
.1.1. General experimental information
Melting points were determined on Yanaco MP-J3 microscope melting point
apparatus which is uncorrected. NMR spectra were recorded on Varian-400 and
Mercury-500 spectrometer. ESI-HRMS data were measured on Thermo Exactive
Orbitrap plus spectrometer. Chemical shifts are referenced to the residual solvent peak
and reported in ppm (d scale) and all coupling constant (J) values are given in Hz.
The following multiplicity abbreviations are used: (s) singlet, (d) doublet, (t) triplet,
(
q) quartet, (m) multiplet, and (br) broad. TLC was carried out on silica gel plates
GF254) with visualization of components by UV light (254 nm) or exposure to I2.
(
1
6

Column chromatography was carried out on silica gel (200-300 mesh). Petroleum
ether (PE), extra pure, boiling range 60-90°C. All the solvents and chemicals were
obtained from commercial sources and used without further purification.
5
.1.2.
-((2-oxo-2-phenylethyl)carbamoyl)piperidine-1-carboxylate (1e)
,1'-Carbonyldiimidazole (1.42 g, 8.74 mmol) was added to 1a (2.00 g, 7.60
mmol) in CH Cl (15 mL) cooled with an ice bath. The mixture was stirred at room
Preparation
of
benzyl
4
1
2
2
temperature under argon for 3 h. 2-Aminoacetophenone hydrochloride 1d (1.37 g,
.98 mmol) and DIPEA (1.39 mL, 7.98 mmol) were then added to the reaction
7
mixture. The mixture was stirred at room temperature for additional 9 h, then
evaporated under reduced pressure. The residue was purified by silica gel flash
column chromatography (CH Cl /EtOAc = 40:1, V/V) to afford the compound 1e
2
2
1
(
1.50 g, 51.9% yield) as a light-brown oil. H NMR (400 MHz, CDCl ) δ: 7.97 (m,
3
2
H), 7.63 (m, 1H), 7.51 (m, 2H), 7.34 (m, 5H), 6.60 (brs, 1H), 5.14 (s, 2H), 4.76 (d, J
=
4.0 Hz, 2H), 4.24 (m, 2H), 2.88 (m, 2H), 2.42 (m, 1H), 1.88 (m, 2H), 1.73 (m, 2H).
+
MS (ESI) m/z 381.19 [M+H] .
5
5
.1.3. Preparation of the piperidyl intermediates 6a-g
.1.3.1. Preparation of 3-phenyl-5-(piperidin-4-yl)-1,2,4-oxadiazole (6a)
To a solution of 1a (1.50 g, 5.70 mmol) and DMF (0.03 mL) in anhydrous
dichloromethane (5 mL) was added oxalyl chloride (0.68 mL, 7.98 mmol). The
mixture was stirred at room temperature for 3 h, then concentrated. The residue was
dissolved in toluene (4 mL). A solution of 2 (0.29 g, 3.17 mmol) in toluene (4 mL)
was added to the solution at 0 °C, then the mixture was heated to reflux for 4 h. After
evaporated under reduced pressure, the residue was purified by silica gel flash
column chromatography (PE/EtOAc = 9:1, V/V) to give a yellow oil (0.89 g, 77.4%
1
yield). H NMR (500 MHz, CDCl ) δ 7.95 (m, 2H), 7.35 (m, 3H), 7.22 (m, 5H), 5.04
3
1
3
(
s, 2H), 4.06 (brs, 2H), 2.99 (m, 3H), 1.99 (m, 2H), 1.79 (m, 2H). C NMR (125 MHz,
1
7

CDCl ) δ 180.7, 167.9, 154.8, 136.3, 130.8, 128.5, 128.2, 127.7, 127.6, 127.1, 126.5,
3
6
6.9, 42.7, 33.9, 28.7.
Iodotrimethylsilane (0.84 mL, 5.88 mmol) was added to the above yellow oil
(
0.89 g, 2.45 mmol) in CH Cl (6 mL) cooled with an ice bath. The mixture was
2 2
stirred at room temperature for 4 h and quenched with methanol (1 mL), followed by
the addition of 2N HCl aqueous solution (15mL) and ethyl ether (10 mL). The organic
phase was separated and the aqueous phase (15 mL) was basified with K CO to pH
2
3
8
-9 and extracted three times with CH Cl (3×15 mL). The combined organic phases
2 2
were washed with brine, dried over Na SO , filtered and evaporated in vacuo to afford
2
4
1
the title compound 6a (0.49 g, 87.2% yield) as a yellow solid. M.p.: 85-87°C. H
NMR (400 MHz, CDCl ) δ: 8.08 (m, 2H), 7.49 (m, 3H), 3.23 (m, 2H), 3.15 (m, 1H),
3
13
2
.82 (m, 2H), 2.54 (brs, 1H), 2.15 (m, 2H), 1.92 (m, 2H). C NMR (100 MHz, CDCl )
3
δ 181.9, 168.3, 131.2, 128.9, 127.5, 127.1, 45.7, 34.9, 30.3. MS (ESI) m/z 230.15
+
[
M+H] .
5
.1.3.2. Preparation of 4-(4-phenyl-1H-1,2,3-triazol-1-yl)piperidine (6b)
To a solution of 1b (0.23 g, 1.0 mmol), DIPEA (0.65 g, 5.0 mmol) and CuI (0.057
g, 0.03 mmol) in methanol (15 mL) was added phenylacetylene 3 (0.68 mL, 7.98
mmol). The mixture was stirred at room temperature for 2 h, then concentrated. The
residue was diluted with EA (50 mL) and H O (10 mL). The organic phase was
2
separated and washed with 10% citric acid (10 mL) and brine (10 mL), dried over
Na SO , filtered and evaporated in vacuo to give a light-yellow solid (0.28 g, 80.3%
2
4
1
yield). H NMR (400 MHz, CDCl ) δ: 7.82 (m, 2H), 7.77 (s, 1H), 7.43 (m, 2H), 7.34
3
(m, 1H), 4.66 (m, 1H), 4.30 (m, 2H), 2.96 (m, 2H), 2.24 (m, 2H), 2.01 (m, 2H), 1.49
+
(
s, 9H). MS (ESI) m/z 329.25 [M+H] .
Trifluoroacetic acid (0.7 mL) was added to the above light-yellow solid dissolved
in CH Cl (2 mL). The solution was stirred at room temperature for 2 h. The mixture
2
2
was diluted with H O (15 mL) and ethyl ether (15 mL). The organic phase was
2
separated and the aqueous phase (15 mL) was basified with K CO to pH 8-9 and
2
3
1
8

extracted with CH Cl (5×20 mL). The combined organic phases were washed with
2
2
brine (10 mL), dried over Na SO , filtered and evaporated in vacuo to afford the title
2
4
1
compound 6b (170 mg, 89.0% yield) as an off-white solid. M.p.: 90-92°C. H NMR
(
400 MHz, CDCl ) δ: 7.83 (m, 2H), 7.78 (s, 1H), 7.43 (m, 2H), 7.32 (m, 1H), 4.62 (m,
3
1
H), 3.28 (m, 2H), 2.82 (m, 2H), 2.24 (m, 2H), 1.99 (m, 2H), 1.57 (brs, 1H). MS (ESI)
+
m/z 229.15 [M+H] .
5
.1.3.3. Preparation of 4-phenyl-2-(piperidin-4-yl)thiazole (6c)
To a solution of 1c (0.30 g, 1.23 mmol) and NaHCO (0.21 g, 2.46 mmol) in
3
ethanol (5 mL) was added α-bromoacetophenone (0.26 g, 1.29 mmol). The mixture
was heated to reflux for 3 h. After evaporated under reduced pressure, the residue was
purified by silica gel flash column chromatography (CH Cl /EtOAc = 40:1, V/V) to
2
2
1
give a light-brown oil (0.40 g, 93.2% yield). H NMR (400 MHz, CDCl ) δ: 7.90 (m,
3
2
H), 7.42 (m, 2H), 7.38 (s, 1H), 7.34 (m, 1H), 4.21 (m, 2H), 3.33 (m, 1H), 2.93 (m,
+
2
H), 2.17 (m, 2H), 1.77 (m, 2H), 1.48 (s, 9H). MS (ESI) m/z 345.18 [M+H] .
Trifluoroacetic acid (1.0 mL) was added to the above light-brown oil (0.39 g,
.15 mmol) dissolved in CH Cl (3 mL). The solution was stirred at room temperature
1
2
2
for 2 h. The mixture was diluted with H O (15 mL) and ethyl ether (10 mL). The
2
organic phase was separated and the aqueous phase (15 mL) was basified with K CO
2
3
to pH 8-9 and extracted with CH Cl (3×20 mL). The combined organic phases were
2
2
washed with brine, dried over Na SO , filtered and evaporated in vacuo to afford the
2
4
1
title compound 6c (0.24 g, 85.7% yield) as an off-white solid. M.p.: 76-78°C. H
NMR (400 MHz, CDCl ) δ: 7.88 (m, 2H), 7.41 (m, 2H), 7.35 (s, 1H), 7.30 (m, 1H),
3
1
3
3
.20 (m, 3H), 2.80 (m, 2H), 2.17 (m, 2H), 1.73-1.84 (m, 3H). C NMR (100 MHz,
CDCl ) δ 175.8, 155.0, 134.9, 128.8, 128.1, 126.5, 111.7, 46.5, 41.3, 34.0. MS (ESI)
3
+
m/z 245.13 [M+H] .
5
.1.3.4. Preparation of 4-(4-phenyl-1H-imidazol-2-yl)piperidine (6d)
To a solution of 1e (0.73 g, 1.92 mmol) in acetic acid (5 mL) was added
1
9

ammonium acetate (2.07 g, 26.86 mmol). The mixture was heated to reflux for 5 h.
After cooling to room temperature, the reaction mixture was poured into water (20
mL) and extracted with CH Cl (3×20 mL). The combined organic phases were
2
2
washed with brine, dried over Na SO , then filtered. After evaporated under reduced
2
4
pressure, the residue was purified by silica gel flash column chromatography
1
(
PE/EtOAc = 1:1, V/V) to give a light-yellow oil (0.66 g, 83.7% yield). H NMR (400
MHz, CDCl ) δ: 7.67 (m, 2H), 7.38 (m, 7H), 7.23 (m, 2H), 5.13 (s, 2H), 4.28 (m, 2H),
3
3
.04 (m, 1H), 2.93 (m, 2H), 2.05 (m, 2H), 1.73 (m, 2H).
Iodotrimethylsilane (0.60 mL, 4.15 mmol) was added to the above light-yellow
oil (0.63 g, 1.73 mmol) in anhydrous dichloromethane (10 mL) at room temperature
under argon. The mixture was stirred for 4 h and quenched with methanol (1 mL),
followed by the addition of 2N HCl aqueous solution (15mL) and ethyl ether (10 mL).
The organic phase was separated and the aqueous phase (15 mL) was basified with
K CO to pH 8-9 and extracted with CH Cl (3×15 mL). The combined organic
2
3
2
2
phases were washed with brine, dried over Na SO , filtered and evaporated in vacuo
2
4
to afford the title compound 6d (0.44 g, 99.3% yield) as an off-white solid. M.p.:
1
9
3
2
2-94°C. H NMR (400 MHz, CDCl ) δ: 7.68 (m, 2H), 7.35 (m, 2H), 7.21 (m, 2H),
3
.18 (m, 2H), 2.94 (m, 1H), 2.72 (m, 2H), 2.02 (m, 2H), 1.76 (m, 2H). MS (ESI) m/z
+
28.97 [M+H] .
5
.1.3.5. Preparation of 5-phenyl-2-(piperidin-4-yl)oxazole (6e)
To a solution of 1e (0.80 g, 2.10 mmol) in pyridine (5 mL) was added phosphorus
oxychloride (1.0 mL, 10.5 mmol). The mixture was stirred at room temperature for 2
h and then slowly poured into saturated NaHCO solution (20 mL) cooled by ice bath.
3
The aqueous phase was extracted with ethyl acetate (3×20 mL) and the combined
organic phases were dried over Na SO , filtered and evaporated in vacuo. The
2
4
resulting residue was purified by silica gel flash column chromatography (PE/EtOAc
1
=
1:1, V/V) to give a light-yellow oil (0.66 g, 86.8% yield). H NMR (400 MHz,
CDCl ) δ: 7.60 (m, 2H), 7.37 (m, 8H), 7.24 (s, 1H), 5.15 (s, 2H), 4.20 (m, 2H), 3.07
3
2
0

+
(
m, 2H), 2.11 (m, 2H), 1.05 (m, 3H). MS (ESI) m/z 363.17 [M+H] .
Iodotrimethylsilane (0.66 mL, 4.64 mmol) was added to the above light-yellow
oil (0.60 g, 1.93 mmol) in anhydrous dichloromethane (10 mL) at room temperature
under argon. The mixture was stirred for 4 h and quenched with methanol (1 mL),
followed by the addition of 2N HCl aqueous solution (15mL) and ethyl ether (10 mL).
The organic phase was separated and the aqueous phase (15 mL) was basified with
K CO to pH 8-9 and extracted with CH Cl (3×15 mL). The combined organic
2
3
2
2
phases were washed with brine (15 mL), dried over Na SO , filtered and evaporated
2
4
in vacuo to afford the title compound 6e (0.43 g, 96.4% yield) as an off-white solid.
1
M.p.: 62-64°C. H NMR (400 MHz, CDCl ) δ: 7.61 (m, 2H), 7.40 (m, 2H), 7.30 (m,
3
1
1
1
H), 7.22 (s, 1H), 3.19 (m, 2H), 3.00 (m, 1H), 2.77 (m, 2H), 2.10 (m, 2H), 1.96 (brs,
1
3
H), 1.84 (m, 2H). C NMR (100 MHz, CDCl ) δ 166.7, 150.9, 129.0, 128.3, 128.3,
3
+
24.1, 121.7, 46.0, 36.2, 30.8. MS (ESI) m/z 229.13 [M+H] .
5
.1.3.6. Preparation of 5-phenyl-3-(piperidin-4-yl)isoxazole (6f)
To a solution of 1f (2.13 g, 9.33 mmol), phenylacetylene 3 (0.68 mL, 7.98 mmol)
and KCl (0.70 g, 9.33 mmol) in water (20 mL) was added HKO S (8.57 g, 13.95
6
mmol). The mixture was stirred at room temperature overnight and then extracted
with CH Cl (3×30 mL). The combined organic phases were washed with brine, dried
2
2
over Na SO , filtered and evaporated under reduce pressure. The resulting residue
2
4
was purified by silica gel flash column chromatography (CH Cl /EtOAc = 40:1, V/V)
2
2
1
to give a colorless oil (1.16 g, 37.9% yield). H NMR (400 MHz, CDCl ) δ: 7.76 (m,
3
2
2
1
H), 7.46 (m, 3H), 6.38 (s, 1H), 4.18 (m, 2H), 2.94 (m, 3H), 1.98 (m, 2H), 1.72 (m,
13
H), 1.48 (s, 9H). C NMR (100 MHz, CDCl ) δ 170.0, 167.5, 154.9, 130.2, 129.1,
3
+
27.7, 125.9, 97.7, 79.8, 43.7, 34.4, 31.0, 28.6. MS (ESI) m/z 351.17 [M+H] .
Trifluoroacetic acid (3.0 mL) was added to the above colorless oil (1.10 g, 3.55
mmol) dissolved in CH Cl (10 mL). The solution was stirred at room temperature for
2
2
2
h. The mixture was diluted with H O (20 mL) and ethyl ether (20 mL). The organic
2
phase was separated and the aqueous phase (20 mL) was basified with K CO to pH
2
3
2
1

8
-9 and extracted with CH Cl (3×30 mL). The combined organic phases were
2 2
washed with brine (20 mL), dried over Na SO , filtered and evaporated in vacuo to
2
4
afford the title compound 6f (0.65 g, 85.0% yield) as an off-white solid. M.p.:
1
9
6-99°C. H NMR (400 MHz, CDCl ) δ: 7.82 (m, 2H), 7.50 (m, 3H), 6.98 (s, 1H),
3
3
.83 (brs, 1H), 3.05 (m, 2H), 2.86 (m, 1H), 2.66 (m, 2H), 1.87 (m, 2H), 1.59 (m, 2H).
1
3
C NMR (100 MHz, CDCl ) δ 169.9, 168.0, 130.2, 129.1, 127.7, 125.9, 97.7, 46.2,
3
+
3
4.4, 31.9. MS (ESI) m/z 229.13 [M+H] .
5
.1.3.7. Preparation of 4-(1-phenyl-1H-tetrazol-5-yl)piperidine hydrochloride
(6g)
To a solution of 1g (1.00 g, 4.37 mmol) in acetonitrile (10 mL) was added EDCI
0.84 g, 44 mmol), HOBt (0.59 g, 4.37 mmol), triethylamine (0.68 mL, 4.81 mmol)
(
and aniline 5 (0.44 g, 4.37 mmol) in turn. The mixture was stirred at room
temperature for 5 h and then diluted with CH Cl (50 mL) and saturated NaHCO
3
2
2
solution (10 mL). The organic phase was separated and washed with brine (20 mL),
dried over Na SO , filtered and evaporated in vacuo to give an off-white solid (0.83 g,
2
4
1
6
7
2.5% yield). H NMR (400 MHz, CDCl ) δ 7.52 (m, 3H), 7.31 (t, J = 7.9 Hz, 2H),
3
.10 (t, J = 7.4 Hz, 1H), 4.18 (m, 2H), 2.76 (m, 2H), 2.38 (m, 1H), 1.87 (m, 2H), 1.75
(m, 2H), 1.47 (s, 9H).
To a solution of the above off-white solid (0.40 g, 1.32 mmol),
triphenylphosphine (0.80 g, 2.63 mmol) and TMS-N (0.35 mL, 2.63 mmol) in
3
anhydrous THF (10 mL), DIAD (0.52 mL, 2.63 mmol) was added dropwise cooled
with an ice bath. The mixture was stirred at room temperature for 6 days, and then
heated to reflux for 1 h. After cooling to room temperature, 7 N HCl-EA solution (10
mL) was added to the mixture and stirred at room temperature for additional 1 h. The
mixture was diluted with ethyl acetate (10 mL) and then filtered. The residue was
washed with ethyl acetate to afford the title compound 6g (0.30 g, 86.0%) as an
1
off-white solid. M.p.:110-112°C. H NMR (400 MHz, DMSO-d ) δ: 9.29 (brs, 1H),
6
9
.16 (brs, 1H), 8.35 (brs, 1H), 7.68 (m, 5H), 3.29 (m, 3H), 2.96 (m, 2H), 2.02 (m, 4H).
2
2

+
MS (ESI) m/z 230.14 [M+H] .
5
.1.4. Preparation of 3-phenyl-5-(trichloromethyl)-1,2,4-oxadiazole (7)
To a solution of 2 (2.50 g, 18.36 mmol) in toluene (20 mL) was added
trichloroacetic anhydride (4.30 mL, 23.50 mmol) dropwise and the mixture was
heated to reflux for 10 h. After evaporated under reduced pressure, the residue was
diluted with ethyl acetate (30 mL). The organic phase was washed with brine (15 mL),
dried over Na SO , filtered and evaporated in vacuo. The residue was purified by
2
4
silica gel flash column chromatography (PE/CH Cl = 100:1) to give a colorless oil
2
2
1
(
4.39 g, 90.7% yield). H NMR (400 MHz, CDCl ) δ: 8.13 (m, 2H), 7.53 (m, 3H).
3
5
.1.5. 5-Chloro-3-phenyl-1,2,4-thiadiazole (9)
To a solution of 8 (0.50 g, 3.19 mmol) and (trichloromethyl)sulfenyl chloride (10
mL) in CH Cl (10 mL) cooled by ice bath was added NaOH (0.64 g, 15.96 mmol)
2
2
and the mixture was stirred for 1 h, then stirred at room temperature for additional 2 h.
The mixture was diluted with CH Cl (30 mL). The combined organic phases were
2
2
washed with brine, dried over Na SO , filtered and evaporated in vacuo to give a
2
4
light-yellow oil. The product was used to the next step without further purification.
5
.1.6.
-(benzoylcarbamothioyl)piperazine-1-carboxylate (11)
To a solution of 1-Boc-piperazine (0.50 g, 2.68 mmol) in anhydrous CH Cl (10
Preparation
of
tert-butyl
4
2
2
mL), a solution of 10 (0.38 mL, 2.68 mmol) in CH Cl (5 mL) was added dropwise
2
2
cooled by ice bath. The mixture was stirred at room temperature for 2 h. After
evaporated under reduced pressure, the resulting residue was washed with the mixed
solvent (6 mL, diethyl ether/hexane = 1:2, V/V) to give an off-white solid (0.85 g,
1
9
2
0.6%). M.p.: 142-145 °C. H NMR (400 MHz, CDCl ) δ: 8.49 (brs, 1H), 7.83 (m,
3
H), 7.60 (m, 1H), 7.50 (m, 2H), 4.18 (m, 2H), 3.64 (m, 6H), 1.48 (s, 9H). MS (ESI)
+
m/z 372.13 [M+Na] .
2
3

5
.1.7. Preparation of tert-butyl 4-carbamothioylpiperazine-1-carboxylate (14)
Intermediate 11 (0.66 g, 1.86 mmol) was dissolved in 50% hydrazine hydrate
aqueous solution (3 mL). The mixture was stirred at room temperature for 4 h, then
diluted with CH Cl (30 mL). The organic phase was washed with 10% citric acid
2
2
(
2×30 mL) and brine (25 mL), dried over Na SO , filtered and evaporated under
2 4
reduced pressure. The resulting residue was washed with diethyl ether (20 mL) to give
1
an off-white solid (0.38 g, 81.1% yield). H NMR (400 MHz, CDCl ) δ: 5.82 (brs, 2H),
3
+
3
.84 (m, 4H), 3.55 (m, 4H), 1.47 (s, 9H). MS (ESI) m/z 246.13 [M+H] .
5
.1.8.
Preparation
of
tert-butyl
(Z)-4-(N'-hydroxycarbamimidoyl)piperazine-1-carboxylate (15)
To a solution of 1-Boc-piperazine 13 (0.50 g, 2.68 mmol) and NaHCO aqueous
3
solution (1.10 g in 20 mL H O) in CH Cl (10mL), a solution of cyanogen bromide
2
2
2
(
0.34 g, 3.22 mmol) in CH Cl (10 mL) was added dropwise cooled by ice bath. The
2 2
mixture was stirred at room temperature overnight, then diluted with the saturated
NaHCO solution (10 mL) and extracted with CH Cl (3×15 mL). The combined
3
2
2
organic phases were washed with brine (20 mL), dried over Na SO , filtered and
2
4
1
evaporated in vacuo to afford a white solid (0.54 g, 95.4% yield). H NMR (400 MHz,
CDCl ) δ 3.53-3.50 (m, 4H), 3.20-3.18 (m, 4H), 1.46 (s, 9H).
3
5
0% hydroxylamine aqueous solution (0.15 mL, 2.45 mmol) was added to the
solution of the above white solid (0.50 g, 2.37 mmol) in ethanol (5 mL). The mixture
was heated to reflux for 6 h, then concentrated. The residue was dissolved in toluene
(10 mL) and evaporated in vacuo to afford an off-white solid (0.53 g, 91.5% yield).
1
M.p.: 164-166 °C. H NMR (500 MHz, CDCl ) δ 4.50 (s, 1H), 3.57-3.43 (m, 4H),
3
3
.13 (m, 3H), 3.00-2.93 (m, 1H), 1.55-1.47 (m, 9H).
5
5
.1.9. Preparation of the piperazinyl intermediates 16a-f
.1.9.1. Preparation of 3-phenyl-5-(piperazin-1-yl)-1,2,4-oxadiazole (16a)
2
4

A solution of anhydrous piperazine (2.8 g, 33.32 mmol) in DMF (20 mL) was
added slowly to the mixture of 7 (4.39 g, 16.66 mmol) and DMF (15 mL). The
mixture was stirred at room temperature for 6 h. After evaporated under reduced
pressure, the residue was diluted with EA (200 mL) and washed with brine (100
mL×2), dried over Na SO , filtered and evaporated under reduced pressure. The
2
4
residue was purified by silica gel flash column chromatography (CH Cl /MeOH =
2
2
1
00:1, V/V) to afford the title compound 16a (2.35 g, 61.2% yield) as a light-yellow
1
solid. M.p.: 65-67°C. H NMR (400 MHz, CDCl ) δ: 7.99 (m, 2H), 7.44 (m, 3H), 3.73
3
1
3
(
m, 4H), 3.04 (m, 4H), 2.16 (brs, 1H). C NMR (100 MHz, CDCl ) δ 171.2, 168.8,
3
+
1
5
1
30.8, 128.7, 127.9, 127.3, 47.0, 45.5. MS (ESI) m/z 231.13 [M+H] .
.1.9.2. Preparation of 3-phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole (16b)
Triethylamine (1.8 mL, 12.77 mmol) was added to a solution of 9 and
-Boc-piperazine (0.60 g, 3.19 mmol) in DMF (4 mL). The mixture was stirred at
room temperature overnight, then concentrated. The residue was diluted with water
10 mL) and extracted with ethyl acetate (3×20 mL). The combined organic phases
were washed with brine (20 mL), dried over Na SO , filtered. After evaporated under
(
2
4
reduced pressure, the residue was purified by silica gel flash column chromatography
(PE/ EtOAc = 10:1, V/V) to give a light-yellow solid (0.62 g, 55.6% yield over two
1
steps). H NMR (400 MHz, CDCl ) δ: 8.19 (m, 2H), 7.42 (m, 3H), 3.60 (m, 8H), 1.49
3
+
(
s, 9H). MS (ESI) m/z 347.17 [M+H] .
Trifluoroacetic acid (5.0 mL) was added to the above light-yellow solid (0.61 g,
1
.76 mmol) dissolved in CH Cl (5 mL). The solution was stirred at room temperature
2 2
for 2 h. The mixture was diluted with H O (15 mL) and ethyl ether (15 mL). The
2
organic phase was separated and the aqueous phase (15 mL) was basified with K CO
2
3
to pH 8-9 and extracted with CH Cl (4×20 mL). The combined organic phases were
2
2
washed with brine, dried over Na SO , filtered and evaporated in vacuo to afford the
2
4
1
title compound 16b (0.34 g, 79.2% yield) as an off-white solid. M.p.:78-79°C. H
NMR (400 MHz, CDCl ) δ: 8.19 (m, 2H), 7.42 (m, 3H), 3.60 (m, 4H), 3.03 (m, 4H),
3
2
5

+
2
.08 (brs, 1H). MS (ESI) m/z 247.11 [M+H] .
5
.1.9.3. Preparation of 1-(5-phenyl-4H-1,2,4-triazol-3-yl)piperazine (16c)
Hydrazine monohydrate (Hydrazine, 64%) (0.59 g, 12 mmol) was added to a
solution of 11 (0.85 g, 2.3 mmol) in chloroform (20 mL). The mixture was heated to
reflux for 4 h, then concentrated. The residue was diluted with water (30 mL) and
extracted with ethyl acetate (3×30 mL). The combined organic phases were washed
with brine, dried over Na SO , filtered. After evaporated under reduced pressure, the
2
4
residue was purified by silica gel flash column chromatography (PE/ EtOAc = 2:1,
1
V/V) to give an off-white solid (0.46 g, 61.3% yield). M.p.:176-178 °C. H NMR
(400 MHz, CDCl ) δ: 8.81 (brs, 1H), 7.89 (m, 2H), 7.41 (m, 3H), 3.52 (m, 4H), 3.45
3
13
(
m, 4H), 1.48 (s, 9H). C NMR (125 MHz, CDCl ) δ 163.0, 157.5, 154.8, 129.8,
3
+
1
29.2, 128.8, 126.3, 80.2, 46.5, 43.1, 28.5. MS (ESI) m/z 330.24 [M+H] .
Trifluoroacetic acid (2.0 mL) was added to the above off-white solid (0.46 g, 1.40
mmol) dissolved in CH Cl (5 mL). The solution was stirred at room temperature for
2
2
2
h. The mixture was diluted with H O (15 mL) and ethyl ether (15 mL). The organic
2
phase was separated and the aqueous phase (15 mL) was basified with K CO to pH
2
3
8
-9 and extracted with CH Cl (4×30 mL). The combined organic phases were
2 2
washed with brine, dried over Na SO , filtered and evaporated in vacuo to afford the
2
4
1
title compound 16c (0.26 g, 81.3% yield) as an off-white solid. M.p.: 160-161°C. H
NMR (400 MHz, CDCl ) δ: 7.91 (m, 2H), 7.39 (m, 3H), 5.14 (brs, 1H), 3.42 (m, 4H),
3
13
2
.96 (m, 4H). C NMR (100 MHz, CDCl ) δ 162.6, 158.3, 129.9, 129.7, 128.8, 126.4,
3
+
4
7.6, 45.3. MS (ESI) m/z 230.14 [M+H] .
5
.1.9.4. Preparation of 1-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine (16d)
1
2 (0.95 g, 6.40 mmol) was added to a solution of 1-Boc-piperazine (1.43 g, 7.68
mmol) in anhydrous ethanol (30 mL). The mixture was heated to reflux for 3 h,
followed by addition of phenylhydrazine (0.75 mL, 7.68 mmol), acetic acid (5 drops)
and 4Å molecular sieve (1.28 g). The mixture was heated to reflux for additional 12 h,
2
6

then concentrated. The mixture was diluted with CH Cl (90 mL), washed
2
2
subsequently with 1 mol/L HCl (10 mL), water (10 mL), saturated NaHCO solution
3
(
10 mL) and brine (10 mL), dried over Na SO , filtered and evaporated in vacuo. The
2 4
residue was purified by silica gel flash column chromatography (PE/ EtOAc = 9:1,
1
V/V) to give a white solid (1.77 g, 80.7% yield). M.p.: 92-95°C. H NMR (400 MHz,
CDCl ) δ: 7.77 (m, 2H), 7.40 (m, 2H), 7.26 (m, 1H), 5.68 (s, 1H), 3.45 (m, 4H), 2.81
3
(brs, 4H), 2.27 (s, 3H), 1.45 (s, 9H).
Trifluoroacetic acid (4 mL) was added to the above white solid (1.14 g, 3.33
mmol) dissolved in CH Cl (12 mL). The solution was stirred at room temperature for
2
2
1
.5 h. The mixture was diluted with H O (15 mL) and ethyl ether (15 mL). The
2
organic phase was separated and the aqueous phase (15 mL) was basified with K CO
2
3
to pH 8-9 and extracted with ethyl acetate (3×40 mL). The combined organic phases
were washed with brine (15 mL), dried over Na SO , filtered and evaporated in vacuo
2
4
to afford the title compound 16d (0.79 g, 97.8% yield) as an off-white solid. M.p.:
1
1
8
03-105°C. H NMR (400 MHz, DMSO-d ) δ: 7.76 (d, J = 8.0 Hz, 2H), 7.45 (t, J =
6
.0 Hz, 2H), 7.26 (t, J = 7.6 Hz, 1H), 5.77 (s, 1H), 2.72 (brs, 4H), 2.70 (brs, 4H), 2.50
1
3
(
s, 3H). C NMR (100 MHz, CDCl ) δ 152.7, 149.0, 140.4, 129.0, 126.4, 122.8, 94.2,
3
+
5
2.3, 45.7, 14.2. MS (ESI) m/z 243.17 [M+H] .
5
.1.9.5. Preparation of 4-phenyl-2-(piperazin-1-yl)thiazole (16e)
To a solution of 14 (0.38 g, 1.55 mmol), NaHCO (0.26 g, 3.10 mmol) in ethanol
3
(5 mL) was added 2-bromoacetophenone (0.33 g, 1.64 mmol). The mixture was
heated to reflux for 4 h, then evaporated in vacuo. The residue was purified by silica
gel flash column chromatography (PE/ CH Cl = 1:1, V/V) to give a light-yellow oil
2
2
1
(
0.43 g, 80.0% yield). H NMR (400 MHz, CDCl ) δ: 7.83 (m, 2H), 7.39 (m, 2H),
3
7
.30 (m, 1H), 6.78 (s, 1H), 3.59 (m, 8H), 1.49 (s, 9H).
Trifluoroacetic acid (1 mL) was added to the above light-yellow oil (0.38 g, 1.10
mmol) dissolved in CH Cl (3 mL). The solution was stirred at room temperature for
2
2
2
h. The mixture was diluted with H O (10 mL) and ethyl ether (10 mL). The organic
2
2
7

phase was separated and the aqueous phase (10 mL) was basified with K CO to pH
2
3
8
-9 and extracted with CH Cl (3×15 mL). The combined organic phases were
2 2
washed with brine (15 mL), dried over Na SO , filtered and evaporated in vacuo to
2
4
1
afford the title compound 16e (0.24 g, 87.4% yield) as a light-yellow oil. H NMR
(
400 MHz, CDCl ) δ: 7.82 (m, 2H), 7.37 (m, 2H), 7.28 (m, 1H), 6.80 (s, 1H), 3.64 (m,
3
+
4
H), 3.11 (m, 4H). MS (ESI) m/z 246.13 [M+H] .
5
.1.9.6. Preparation of 5-phenyl-3-(piperazin-1-yl)-1,2,4-oxadiazole (16f)
To a solution of benzoic acid (0.50 g, 4.09 mmol) and 15 (0.83 g, 3.41 mmol) in
anhydrous DMF (5 mL) was added CDI (0.66 g, 4.09 mmol). The mixture was stirred
at room temperature for 3 h, followed by the addition of CDI (0.66 g, 4.09 mmol).
The reaction mixture was heated to 120°C for additional 8 h. The reaction was
quenched by water (15mL) and extracted with ethyl acetate (3×15 mL). The organic
layer was washed with brine (20 mL) and dried over Na SO , filtered and evaporated
2
4
in vacuo. The residue was purified by silica gel flash column chromatography
(
CH Cl /CH OH = 50:1, V/V) to give a white solid (0.84 g, 62.2% yield). M.p.:
2 2 3
1
9
2-95°C. H NMR (500 MHz, CDCl ) δ 8.08-8.06 (m, 2H), 7.58-7.48 (m, 3H),
3
3
.56-3.55 (m, 4H), 3.52 (brs, 4H), 1.49 (s, 9H).
Trifluoroacetic acid (1 mL) was added to the above white solid (0.20 g, 0.61
mmol) dissolved in CH Cl (3 mL). The solution was stirred at room temperature for
2
2
2
h. The mixture was diluted with H O (10 mL) and ethyl ether (10 mL). The organic
2
phase was separated and the aqueous phase (10 mL) was basified with K CO to pH
2
3
8
-9 and extracted with CH Cl (3×15 mL). The combined organic phases were
2 2
washed with brine, dried over Na SO , filtered and evaporated in vacuo to afford the
2
4
1
title compound 16f (0.11 g, 79.7% yield) as a light-yellow oil. H NMR (400 MHz,
CDCl ) δ 8.08-8.06 (m, 2H), 7.58-7.48 (m, 3H), 3.56-3.53 (m, 4H), 3.03-3.01 (m, 4H),
3
1
3
2
1
.23 (brs, 1H). C NMR (100 MHz, CDCl ) δ 174.5, 171.0, 132.5, 129.0, 128.0,
3
+
24.9, 47.2, 45.5. MS (ESI): m/z 231.12 [M+H]
2
8