A novel series of 4-methyl substituted pyrazole derivatives as potent glucagon receptor antagonists: Design, synthesis and evaluation of biological activities

Article abstract of DOI:10.1016/j.bmc.2018.02.036

A novel series of 4-methyl substituted pyrazole derivatives were designed, synthesized and biologically evaluated as potent glucagon receptor (GCGR) antagonists. In this study, compounds 9q, 9r, 19d and 19e showed high GCGR binding (IC₅₀ = 0.09 μM, 0.06 μM, 0.07 μM and 0.08 μM, respectively) and cyclic-adenosine monophosphate (cAMP) activities (IC₅₀ = 0.22 μM, 0.26 μM, 0.44 μM and 0.46 μM, respectively) in cell-based assays. Most importantly, the docking experiment demonstrated that compound 9r formed extensive hydrophobic interactions with the receptor binding pocket, making it justifiable to further investigate the potential of becoming a GCGR antagonist.

Full text of DOI:10.1016/j.bmc.2018.02.036

Accepted Manuscript
A novel series of 4-methyl substituted pyrazole derivatives as potent glucagon
receptor antagonists: Design, synthesis and evaluation of biological activities
Shuangjie Shu, Antao Dai, Jiang Wang, Bin Wang, Yang Feng, Jia Li, Xiaoqing
Cai, Dehua Yang, Dakota Ma, Ming-Wei Wang, Hong Liu
PII:
S0968-0896(18)30104-4
https://doi.org/10.1016/j.bmc.2018.02.036
BMC 14222
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 January 2018
19 February 2018
20 February 2018
Please cite this article as: Shu, S., Dai, A., Wang, J., Wang, B., Feng, Y., Li, J., Cai, X., Yang, D., Ma, D., Wang,
M-W., Liu, H., A novel series of 4-methyl substituted pyrazole derivatives as potent glucagon receptor antagonists:
Design, synthesis and evaluation of biological activities, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.02.036
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel series of 4-methyl substituted pyrazole derivatives as potent
glucagon receptor antagonists: Design, synthesis and evaluation of
biological activities
d
Shuangjie Shu a,b,c,†, Antao Dai d,†, Jiang Wang a,b,c,†, Bin Wang a,b,c, Yang Feng , Jia
Lia,b,c, Xiaoqing Cai d, Dehua Yang b,d, Dakota Ma d, Ming-Wei Wang b,d,e,* and Hong
Liu a,b,c,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203, China
bThe CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (CAS), 555 Zu Chong Zhi Road, Shanghai
201203, China
cUniversity of Chinese Academy of Sciences, 19A Yuquan Road, Beijing, 100049,
China
dThe National Center for Drug Screening, 189 Guo Shou Jing Road, Shanghai,
201203, China
eSchool of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203,
China
Author Contributions
†These authors contributed equally to this work.
Corresponding Author
*Ming-Wei Wang: E-mail, mwwang@simm.ac.cn.
*Hong Liu: E-mail, hliu@simm.ac.cn.
1

Graphical Abstract
Abstract
A novel series of 4-methyl substituted pyrazole derivatives were designed,
synthesized and biologically evaluated as potent glucagon receptor (GCGR)
antagonists. In this study, compounds 9q, 9r, 19d and 19e showed high GCGR
binding (IC50 = 0.09 μM, 0.06 μM, 0.07 μM and 0.08 μM, respectively) and
cyclic-adenosine monophosphate (cAMP) activities (IC50 = 0.22 μM, 0.26 μM, 0.44
μM and 0.46 μM, respectively) in cell-based assays. Most importantly, the docking
experiment demonstrated that compound 9r formed extensive hydrophobic
interactions with the receptor binding pocket, making it justifiable to further
investigate the potential of becoming a GCGR antagonist.
Keywords: pyrazole derivatives; glucagon receptor antagonist; docking study;
binding pocket
2

1. Introduction
Glucagon, a 29-amino acid peptide hormone, secreted by α-cells of the pancreatic
islets, was discovered by Kimball and Murlin in 1923.1-3 As a main hormone to
elevate the hepatic glucose production (HGP) by stimulating gluconeogenesis4 and
glycogenolysis,5 glucagon along with insulin maintains blood glucose homeostasis.6-8
It was reported that exogenous insulin may suppress the secretion of glucagon and
immunoneutralization of insulin increased glucagon release. 9-12 Thus, in type 1 and
type 2 diabetics, elevated levels of glucagon, insufficient glucagon suppression and
decreased insulin secretion all contribute to hyperglycemia.13,14
Type 2 diabetes mellitus (T2DM) is a chronic disorder accompanied by polydipsia,
polyphagia, polyuria and characterized by hyperglycemia. To reduce the risk of
late-stage complications and drug resistance, new therapies for diabetes are still in
need. Recently, several sodium-glucose cotransporter-2 (SGLT-2) inhibitors such as
canagliflozin, dapagliflozin and embagliflozin have been approved, but they may lead
to ketoacidosis.15 Given the importance of suppressing glucagon action in treating
T2DM, antagonism of glucagon receptor (GCGR) appears to be a promising approach
to glycemic control.16,17 GCGR is one of the 15 members of class B family of
G-protein coupled receptors (GPCRs). In 2013, the high resolution crystal structure of
seven-transmembrane (7-TM) helical domain of human GCGR was determined by
research groups led by Wang and Stevens, which is conducive to understanding the
molecular binding mode of the receptor.18 7-TM domain is the main part of GPCRs
that share similar signal transduction mechanisms.19 Compared with class A family of
3

GPCRs, GCGR has a large ligand binding pocket. The conformational states of the
full-length GCGR were revealed suggesting that glucagon binds to GCGR by a
conformational selection mechanism.20 A full-length GCGR structure was revealed
recently demonstrating how the extracellular domain interacts with the stalk to form a
compact β-sheet structure.21
To date, several GCGR antagonists with different chemical scaffolds were reported
to display efficacies in various bioassays.22-30 The biaryl containing compound BAY
27-9955 (Fig. 1) was the first GCGR antagonist to blunt elevation of HGP and plasma
glucose induced by exogenous glucagon.31 Clinical trial data of compounds MK-3577
32
33,34
and MK-0893
(Figure 1) exhibits that they are potent GCGR antagonists
capable of modulating glucose homeostasis.
Figure 1. Structures of BAY 27-9955, MK-3577 and MK-0893.
In this paper, a novel series of 4-methyl substituted pyrazole-containing derivatives
as GCGR antagonists was designed and synthesized. Most of compounds exhibited
good GCGR binding affinities and cyclic-adenosine monophosphate (cAMP)
responses. Our structural optimization was dedicated to improve both GCGR binding
and cAMP activities aiming at discovery of potent compounds for further
development.
4

2. Results and discussion
2.1. Design of the target compounds
Although the structure-activity relationship (SAR) of MK-0893 was explicit, it is
very limited to understand the binding mode of this compound with GCGR. Therefore,
structural modification of MK-0893 and docking studies were carried out to make the
binding information clearer. In 2016, the extra-helical binding site of MK-0893 for
GCGR was identified by a crystal complex structure.35 Unlike glucagon binding in the
7-TM, MK-0893 interacted with residues outside the 7-TM domain and formed
interactions in two binding pockets (Figure 2A) at allosteric sites. The β-alanine
moiety formed polar interactions with residues Lys349, Ser350, Arg346, Asn404 and
Lys405 including a water-mediated hydrogen bond with residues Ser350 and Leu399
between transmembrane 6 and transmembrane 7 (Figure 2B). The hydrophobic
moiety formed hydrophobic interactions with Leu329, Phe345, Leu352, Thr353 and
Lys349 between transmembrane 5 and transmembrane 6. As an important strategy of
lead compound optimization, introducing methyl group can modulate the
physicochemical, pharmacodynamic and pharmacokinetic properties. We
hypothesized that introducing methyl group on the pyrazole core may form potential
hydrophobic interactions with the binding pockets (Figure 3). Thus, a series of
4-methyl substituted pyrazole derivatives (9 and 19) was designed to evaluate the
influence of R1, R2 and R3 substituents on antagonistic activities.
5

Figure 2. MK-0893 allosteric binding sites of GCGR. (A) surface of interactions. (B)
interactions of MK-0893 with GCGR. All figures were prepared using PyMol
(http://www.pymol.org).
Figure 3. Design of 4-methyl substituted pyrazole derivatives 9 and 19.
2.2. Synthetic procedure of the target compounds
The desired compound 9 was synthesized according to the synthetic route shown in
Scheme 1. Esterification of compound 1 produced compound 2, which reacted with
tert-butyl carbazate and then reduced by sodium cyanoborohydride to give compound
3. Deprotection of compound 3 in the presence of trifluoroacetic acid afforded
hydrazine
4,
which
reacted
with
ethyl
3-(3,5-dichlorophenyl)-2-methyl-3-oxopropanoate 5 to provide compound 6.
Triflation of compound 6 with triflic anhydride generated compound 7, which was
6

subjected to a classic Suzuki coupling reaction to yield compound 8. Hydrolysis of
compound 8, condensation with β-alanine tert-butyl ester hydrochloride and
deprotection of tert-butyl ester obtained desired compound 9.
Scheme 1. Synthesis of the target compounds 9a-9sa
aReagents and conditions: (a) EtOH, H2SO4, reflux, 4 h; (b) cat. HOAc, toluene, 80 oC,
overnight; (c) NaBH3CN, p-toluenesulfonic acid, THF, rt, 3 h; (d) TFA, DCM, rt, 1 h;
(e) HOAc, reflux, 4 h; (f) triflic anhydride (Tf2O), TEA, THF, -78 oC to 0 oC, 1.5 h; (g)
o
R2-B(OH)2, Pd(PPh3)4, TEA, DME, 100 C, MW, 25 min; (h) NaOH, MeOH,
o
1,4-dioxane, 60 C, 1 h; (i) β-alanine tert-butyl ester hydrochloride, PyBOP, DIEA,
DMF, rt, overnight; (j) TFA, DCM, rt, 1 h.
The intermediate 12 and desired compound 19 were synthesized according to the
synthetic route shown in Scheme 2. Condensation of compound 10 with β-alanine
7

tert-butyl ester hydrochloride afforded compound 11, which was reduced by sodium
borohydride to provide intermediate 12. Esterification of compound 13 produced
compound 14, which reacted with ethyl propionate to give compound 15. Cyclization
of compound 15 and hydrazine hydrate in glacial acetic acid produced compound 16.
Nucleophilic reaction of compound 16 in the presence of TEA generated compound
17, which was subjected to Mitsunobu reaction with compound 12 to yield the main
product 18, the minor isomer is separated by chromatography. The desired compound
19 was generated by coupling reaction and deprotection of tert-butyl ester. 36
Scheme 2. Synthesis of key intermediate 12 and target compounds 19a-19da
aReagents and conditions: (a) β-alanine tert-butyl ester hydrochloride, PyBOP, DIEA,
DMF, rt, overnight; (b) NaBH4, DCM, rt, overnight; (c) EtOH, H2SO4, reflux,
o
overnight; (d) NaH, ethyl propionate, THF, 80 C, 24 h; (e) hydrazine hydrate (85%),
HOAc, 80 oC, overnight; (f) pyridine, Tf2O, THF, -78 oC to 0 oC, 2 h; (g) PPh3, DIAD,
8

DCM, rt, 2 h; (h) XPhos aminobiphenyl palladium chloride precatalyst, R3-B(OH)2,
XPhos, K2CO3, 17 h; (i) TFA, DCM, rt, 1 h.
The desired compound 19e was synthesized according to the synthetic route shown
in
Scheme
3.
Cyclization
of
hydrazine
4
with
ethyl
3-(3,4-dichlorophenyl)-2-methyl-3-oxopropanoate
20
gave
compound
21.
Nucleophilic reaction of compound 21 in the presence of TEA generated compound
22, which was subjected to a classic Suzuki coupling reaction to yield compound 23.
Hydrolysis of compound 23, condensation with β-alanine tert-butyl ester
hydrochloride and deprotection of tert-butyl ester obtained desired compound 19e.
Scheme 3. Synthesis of the target compounds 19ea
aReagents and conditions: (a) HOAc, reflux, 4 h; (b) Tf2O, TEA, THF, -78 oC to 0 oC,
1.5 h; (c) 6-Methoxy-2-naphthaleneboronic acid, Pd(PPh3)4, TEA, DME, 100 oC, MW,
o
25 min; (c) NaOH, MeOH, 1,4-dioxane, 60 C, 1 h; (e) β-alanine tert-butyl ester
hydrochloride, PyBOP, DIEA, DMF, rt, overnight; (f) TFA, DCM, rt, 1 h.
9

2.3. Biological evaluation
The antagonistic activities of all pyrazole derivatives were evaluated by a
competitive binding assay with rac-MK-0893 and MK-0893 as the positive control
using stably transfected CHO-K1 cells expressing human GCGR. The results are
reported as concentration for 50% inhibition (IC50) shown in Tables 1 and 2. SAR
analysis for these compounds is summarized below.
After introduction of methyl group on the pyrazole, we investigated R1 and R2
substitutions (Table 1). Compounds 9a, 9b and 9c with R1 as hydrogen at benzyl
position showed decreased activities compared with rac-MK-0893, while compound
9b with 2-naphthyl ring led to high binding activity (IC50 = 2.79 μM) among these
compounds. Compound 9d with methyl group at benzyl position substituted by
phenyl ring provided moderate binding activity (IC50 = 0.20 μM) and cAMP response
(IC50 = 0.80 μM). When compared with non-substitution on phenyl ring, compounds
9e and 9f with a methyl group on the phenyl ring exhibited high potencies. Between
them, methyl substitution at meta-position (9e) on the phenyl ring provided better
GCGR binding activity (IC50 = 0.1 μM). Sterically hindered moiety such as biphenyl
was subsequently introduced and showed good GCGR binding activities and cAMP
responses. Compound 9g with 2-biphenyl group showed moderate GCGR binding
activity (IC50 = 0.31 μM) and cAMP response (IC50 = 1.49 μM). Compound 9h with
3-biphenyl group showed equivalent GCGR binding activity (IC50 = 0.37 μM) and
cAMP response (IC50 = 0.45 μM). Various aryl rings were introduced to improve the
10

antagonistic activity. Thiophene substituted compound 9i displayed low binding
affinity (IC50 = 0.55 μM) and cAMP response (IC50 = 0.88 μM). Compounds with
fused rings such as quinoline (9j), benzofuran (9k), indole (9l) and 1-Me-indole (9m)
were subsequently synthesized. It was observed that (1) quinoline-substitution
presented decreased binding activity (IC50 = 0.50 μM) and cAMP response (IC50 =
2.03 μM); (2) benzofuran-substitution was more favorable than indole-substitution;
and (3) methyl group substituted on indole elevated the binding activity (IC50 = 0.28
μM) and cAMP response (IC50 = 2.31 μM) in comparison with compound 9l. In
addition, compared with these heterocyclic rings, naphthyl ring (compounds 9n and
9o) especially 2-naphthyl ring possessed good binding activity (IC50 = 0.11 μM) and
cAMP response (IC50 = 0.33 μM), while compound 9p with 6-ethoxy-substitution on
2-naphthyl ring showed good activities (GCGR biding activity, IC50 = 0.18 μM;
cAMP response, IC50 = 0.37 μM) as well. Compound 9q with a methoxy group
showed better GCGR binding activity (IC50 = 0.09 μM) and cAMP response (IC50 =
0.22 μM) compared with compound 9p. Furthermore, the resolution of compound 9q
yielded compounds 9r and 9s. Compound 9r bearing (S)-configured methyl group
showed equivalent GCGR binding activity (IC50 = 0.06 μM) and cAMP response
(IC50 = 0.26 μM) compared with (rac)-9q. The GCGR binding activity and cAMP
response of compound 9r were increased by approximately 10-fold and 5-fold,
respectively, compared with (R)-configuration (9s) (GCGR binding activity, IC50 =
0.63 μM; cAMP response, IC50 = 1.19 μM). This round of optimization indicated that
compounds with R1 as methyl at benzyl position gained equivalent activities.
11

Compounds with R2 as 6-methoxy-substitution on 2-naphthyl ring were more capable
of binding to GCGR and inducing better cAMP response activity.
Table 1. In vitro GCGR binding and functional cAMP activities of compounds 9a-9s.
GCGR bindinga cAMP responsea
Compound
R1
R2
IC50 (μM)
4.92 ± 1.65
2.79 ± 1.78
3.23 ± 1.79
0.20 ± 0.04
0.10 ± 0.01
0.39 ± 0.15
IC50 (μM)
27.34 ± 7.30
2.38 ± 0.49
7.53 ± 0.50
0.80 ± 0.17
0.45 ± 0.11
0.83 ± 0.20
H
H
4-Cl-Ph
naph-1-yl
6-MeO-naph-2-yl
Ph
9a
9b
9c
9d
9e
9f
H
Me
Me
Me
3-Me-Ph
4-Me-Ph
Me
Me
Me
Me
Me
biph-2-yl
biph-3-yl
0.31 ± 0.14
0.37 ± 0.20
0.55 ± 0.22
0.50 ± 0.10
0.30 ± 0.07
1.49 ± 0.45
0.45 ± 0.04
0.88 ± 0.21
2.03 ± 0.36
1.29 ± 0.82
9g
9h
9i
thioph-2-yl
quinoline-6-yl
benzofuran-5-yl
9j
9k
12

Me
Me
indole-5-yl
1-Me-indole-5-yl
naph-1-yl
3.90 ± 1.69
0.28 ± 0.02
0.57 ± 0.35
0.11 ± 0.01
0.18 ± 0.06
0.09 ± 0.04
0.06 ± 0.01
0.63 ± 0.37
0.05 ± 0.01
0.02 ± 0.01
5.52 ± 1.23
2.31 ± 0.60
17.06 ± 5.04
0.33 ± 0.15
0.37 ± 0.01
0.22 ± 0.05
0.26 ± 0.17
1.19 ± 0.29
0.10 ± 0.01
0.08 ± 0.001
9l
9m
Me
9n
Me
naph-2-yl
9o
Me
6-EtO-naph-2-yl
6-MeO-naph-2-yl
6-MeO-naph-2-yl
6-MeO-naph-2-yl
9p
Me
9q
9r
(S)-Me
(R)-Me
9s
rac-MK-0893
MK-0893
aActivities are reported as means ± SEM (N ≥ 3).
To fully understand the SAR of R3 group after making compounds with R1 as
methyl and with R2 as 6-MeO-naphthalene, multiple substituents were explored
(Table 2). All compounds in Table 2 are racemic, which is much better to explore the
SAR with racemic compounds 9a-9q in Table 1. Compound 19a with phenyl
substitution showed low binding activity (IC50 = 1.92 μM) and cAMP response (IC50
= 2.69 μM). Compound 19b (3-Me-Ph) exhibited better cAMP response (IC50 = 0.74
μM). According to the results of compounds 19a and 19b, we investigated the
electron-withdrawing substituent such as trifluoromethyl group on the phenyl ring at
13

the othor- and meta-positions. Compound 19c displayed low binding activity (IC50 =
0.23 μM) and cAMP response (IC50 = 1.70 μM). While compound 19d exhibited a
better binding activity (IC50 = 0.07 μM) than compound 19c. Furthermore, compound
19e containing 3,4-dichloro-substitution on the phenyl ring demonstrated good GCGR
binding activity (IC50 = 0.08 μM) and cAMP response (IC50 = 0.46 μM).
Table 2. In vitro GCGR binding and functional cAMP activities of compounds
19a-19e.
GCGR bindinga
IC50 (μM)
cAMP responsea
IC50 (μM)
Compound
R3
Ph
1.92 ± 0.94
2.75 ± 0.80
0.23 ± 0.07
0.07 ± 0.01
0.08 ± 0.01
0.05 ± 0.01
0.02 ± 0.01
2.69 ± 1.33
0.74 ± 0.16
1.70 ± 0.71
0.44 ± 0.26
0.46 ± 0.05
0.10 ± 0.01
0.08 ± 0.001
19a
19b
3-Me-Ph
2-CF3-Ph
3-CF3-Ph
3,4-Cl2-Ph
19c
19d
19e
rac-MK-0893
MK-0893
aActivities are reported as means ± SEM (N ≥ 3).
2.4. Docking studies
14

As compound 9r ((S)-isomer of compound 9q) exhibited both good GCGR binding
activity and cAMP response, it was docked into the GCGR binding domain to
investigate the binding modes and the interactions (Fig. 4). It was observed that
compound 9r occupied the P1 and P2 pockets and shown similar binding modes as
MK-0893 (Fig. 4A). The β-alanine moiety was in the P1 pocket forming hydrogen
bonds with residues Ser350, Arg346, Asn404, Lys349, Lys405 and a water-mediated
hydrogen bond with residues Ser350 and Leu399. Docking results showed that
6-MeO-naphthalene occupied P2 pocket and formed extensive hydrophobic
interactions with residues Leu329, Leu352 and Ala348 (Fig. 4B). Inspired by the
docking results, we proposed that addition of appropriate hindrance on the pyrazole
may modestly improve the antagonistic activity.
Fig. 4. Superposition of compounds 9r (yellow) with MK-0893 (green) (A). Docking
structures of compounds 9r (B). Some hydrogen atoms were omitted for clarity. All
figures were prepared using PyMol (http://www.pymol.org).
3. Conclusion
In this paper, we described the design, synthesis and biological evaluation of a
15

novel series of 4-methyl substituted pyrazole derivatives as potent GCGR antagonists.
SAR was summarized and the binding mode was studied. Among the derivatives,
compounds 9q, 9r, 19d and 19e showed high GCGR binding activities (IC50 = 0.09
μM, 0.06 μM, 0.07 μM and 0.08 μM, respectively) and cAMP responses (IC50 = 0.22
μM, 0.26 μM, 0.44 μM and 0.46 μM, respectively). These results indicate that
compounds 9q, 9r, 19d and 19e are useful leads for further investigations.
4. Experimental
4.1. Chemistry
All commercially available compounds and solvents were used without further
purification. The target products were characterized by their NMR and MS spectra.
Nuclear magnetic resonance spectra were recorded in deuterochloroform (CDCl3),
dimethylsulfoxid-d6 (DMSO-d6) or methonal-d4 (CD3OD) on a Brucker AMX-400 or
500 MHz instrument (TMS as IS). Chemical shifts were reported in parts per million
(ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are described as
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Low- and
high-resolution mass spectra (LRMS and HRMS) were measured on Finnigan MAT
95 spectrometer. All of the microwave-assistant reactions were performed in an
InitiatorTM EXP microwave system (Biotage, Inc.) at the specified temperature using
the standard mode of operation. All target compounds were confirmed with over 95%
purity which were determined by Agilent-1100 HPLC with binary pump, photodiode
array detector (DAD), using Agilent Extend-C18 column (4.6 × 150 mm, 5 µm),
16

CH3CN/H2O at 1 mL/min and calculated the peak areas at 254 nm. The enantiomeric
excess (ee) of compounds 9r and 9s were identified by Agilent-1100 HPLC with
photodiode array detector (DAD), and CHIRALPAK IE column (0.46 cm × 15 cm, 5
µm) was used with elution phase PE/EtOH/HAOc = 85/15/0.1 (v/v) at 1.0 mL/min.
The ee (%) values were calculated by peak areas at 254 nm.
Ethyl 4-acetyl benzoate (2). A solution of 4-acetylbenzoic acid (13.0 g, 79.2 mmol)
in ethanol (100 mL) was stirred in ice bath. Concentrated H2SO4 was slowly added
o
and the mixture was refluxed at 80 C for 3 h. Extracted with EtOAc, washed with
saturated brine, dried over anhydrous Na2SO4 and concentrated. The residue was
purified by chromatography to afford ethyl 4-acetylbenzoate as a white solid (13.6 g,
1
89.4%). H NMR (400 MHz, DMSO-d6) δ 8.06 (m, 4H), 4.35 (q, J = 7.1 Hz, 2H),
2.63 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H). MS (ESI, m/z): 191.1 [M-H]-.
tert-Butyl 2-{1-[4-(ethoxycarbonyl)-phenyl]ethyl}hydrazine carboxylate (3). A
solution of tert-butyl carbazate (14.0 g, 106.1 mmol) and compound 2 (13.6 g, 70.8
o
mmol) in toluene (120 mL) with catalytic HOAc was stirred at 80 C overnight.
tert-Butyl-2-{1-[4-(ethoxycarbonyl)phenyl]-ethylidene}hydrazine carboxylate was
collected by filtration as a crystalline solid (14.8 g, 68.3%). Without further
purification,
NaBH3CN
(3.3
g,
53.1
mmol)
and
tert-butyl
2-{1-[4-(ethoxycarbonyl)phenyl]ethylidene}-hydrazinecarboxylate (14.8 g, 48.3
mmol) were dissolved in THF (120 mL). A solution of p-toluenesulfonic acid (9.7 g,
56.5 mmol) in THF (50 mL) was slowly added for 3 h and the reaction was detected
by TLC. The mixture was extracted with EtOAc and washed with brine, dried over
17

anhydrous Na2SO4 and concentrated to give a white solid. The solid was redissolved
in DCM and washed with 1 N NaOH and the organic layer was washed with 1 N HCl
and brine, dried over Na2SO4 and concentrated to yield white solid. Flash column
chromatography petroleum ether/ethyl acetate = 4/1 to yield 13.6 g (91%) tert-butyl
1
2-{1-[4-(ethoxycarbonyl)-phenyl]ethyl}hydrazine carboxylate. H NMR (400 MHz,
DMSO-d6) δ 8.17 (s, 1H), 7.89 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.2 Hz, 2H), 4.73 (s,
1H), 4.30 (q, J = 7.1 Hz, 2H), 4.17 (s, 1H), 1.33 (d, J = 6.2 Hz, 9H), 1.30 (d, J = 7.1
Hz, 3H), 1.18 (d, J = 6.6 Hz, 3H). MS (ESI, m/z): 307.1 [M-H]-.
{1-[4-(Ethoxycarbonyl)phenyl]ethyl}hydrazinium-trifluoroacetate (4). The
tert-butyl 2-{1-[4-(ethoxycarbonyl)-phenyl]ethyl}hydrazine carboxylate (13.6 g, 44.1
mmol) was dissolved in DCM (50 mL) and treated with TFA (50 mL) at room
temperature for 1 h. The mixture was concentrated under reduced pressure without
further purification. MS (ESI, m/z): 209.1 [M+H]+.
Ethyl
3-(3,5-dichlorophenyl)-2-methyl-3-oxopropanoate
(5).
Ethyl
3,5-dichlorobenzoate (8.4 g, 38.3 mmol) and sodium hydride (1.1 g, 46.0 mmol) were
dissolved in 100 mL THF under N2. To the mixture was added 13.2 mL ethyl
propionate slowly at room temperature and refluxed overnight. The solvent was
removed under reduced pressure. The residue was extracted with ethyl acetate,
washed with water and brine and dried over Na2SO4. Flash column chromatography
gave 9.7 g (92%) ethyl 3-(3,5-dichlorophenyl)-2-methyl-3-oxopropanoate 5 as orange
1
solid. H NMR (400 MHz, DMSO-d6) δ 7.97 (t, J = 3.3 Hz, 3H), 4.80 (q, J = 6.9 Hz,
1H), 4.07 (q, J = 7.1 Hz, 2H), 1.31 (d, J = 6.9 Hz, 3H), 1.08 (t, J = 7.1 Hz, 3H). MS
18

(ESI, m/z): 274.0 [M-H]-.
Ethyl 4-(1-(3-(3,5-dichlorophenyl)-4-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)ethyl)benzoate (6). A solution of compound 4 and compound 5 (19.8 g, 72.0 mmol)
was refluxed in 300 mL HOAc for 4 h. The solvent was removed under reduced
pressure and the residue dissolved in ethyl acetate, washed with saturated NaHCO3,
brine and dried over Na2SO4. Flash column chromatography gave 8.1 g (30%) ethyl
4-(1-(3-(3,5-dichlorophenyl)-4-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)ethyl)benz
oate as pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 7.95 – 7.86
(m, 2H), 7.62 (d, J = 1.8 Hz, 2H), 7.55 – 7.48 (m, 1H), 7.38 (d, J = 8.3 Hz, 2H), 5.64
(q, J = 6.9 Hz, 1H), 4.29 (q, J = 7.0 Hz, 2H), 2.05 (s, 3H), 1.78 (d, J = 7.1 Hz, 3H),
1.29 (t, J = 7.1 Hz, 3H). MS (ESI, m/z): 420.0 [M+H]+.
Ethyl 4-(1-(3-(3,5-dichlorophenyl)-4-methyl-5-(((trifluoromethyl)sulfonyl)oxy)-
1H-pyrazol-1-yl)ethyl)benzoate (7). The solution of compound 6 (6.5 g, 15.5 mmol)
o
in THF (120 mL) at −78 C was added TEA (6.5 mL, 46.5 mmol). Triflic anhydride
(4.0 mL, 23.7 mmol) was added over half an hour. The reaction mixture was stirred
o
for 1 h at −78 C and moved to room temperature and detected by TLC. The reaction
was quenched by adding ethyl acetate and water. The organic layer was washed with
brine, dried over Na2SO4 and concentrated. The residue was purified by flash column
chromatography to provide ethyl 4-(1-(3-(3,5-dichlorophenyl)-4-methyl-5-
(((trifluoromethyl)sulfonyl)oxy)-1H-pyrazol-1-yl)ethyl)benzoate (6.0 g, 70.2%) as a
1
colorless oil. H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 8.1 Hz, 2H), 7.70 (d, J =
0.9 Hz, 2H), 7.66 (d, J = 2.2 Hz, 1H), 7.39 (d, J = 8.2 Hz, 2H), 5.66 (q, J = 6.6 Hz,
19

1H), 4.28 (q, J = 7.0 Hz, 2H), 2.15 (s, 3H), 1.86 (d, J = 6.8 Hz, 3H), 1.29 (t, J = 7.0
Hz, 3H). MS (ESI, m/z): 574.1 [M+Na]+.
Ethyl
4-(1-(3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-4-methyl-
1H-pyrazol-1-yl)ethyl)benzoate (8). Compound 7 (221.0 mg, 0.4 mmol),
(6-methoxynaphthalen-2-yl)boronic acid (101.2 mg, 0.5 mmol), and TEA (306 μL,
2.2 mmol) were dissolved in dimethoxyethane. Pd(PPh3)4 (46.3 mg, 40 μmol) was
o
added, and the mixture was deoxygenated before heated in microwave at 100 C for
25 min. The mixture was added EtOAc and saturated ammonium chloride, washed
with saturated brine, dried over Na2SO4 and concentrated. The residue was purified by
chromatography to give ethyl 4-(1-(3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen
-2-yl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzoate a colorless oil (175.0 mg, 78.0%). 1H
NMR (400 MHz, CD3OD) δ 7.94 – 7.77 (m, 4H), 7.72 (s, 3H), 7.60 (d, J = 1.8 Hz,
1H), 7.38 (s, 1H), 7.21 (ddd, J = 18.0, 10.7, 5.0 Hz, 4H), 5.53 (d, J = 4.1 Hz, 1H),
4.25 (dd, J = 5.6, 3.9 Hz, 2H), 3.88 (s, 3H), 2.09 (d, J = 2.0 Hz, 3H), 1.84 (d, J = 3.5
Hz, 3H), 1.26 (t, J = 8.0 Hz, 3H). MS (ESI, m/z): 557.1 [M-H]-.
3-(4-(1-(3-(3,5-Dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-4-methyl-1H-py
razol-1-yl)ethyl)benzamido)propanoic acid (9r). Compound 8 (175.0 mg, 0.3 mmol)
was dissolved in MeOH-dioxane (1:1). A solution of NaOH (0.7 g/15 mL) was added.
o
The mixture was heated to 60 C for 1 h. This was acidified with 2 N HCl, extracted
with EtOAc, washed with saturated brine, dried over Na2SO4 and concentrated to
produce a white solid (145 mg, 87.2%). This solid was suspended in 2 mL DMF,
followed by addition of DIEA (216 μL, 1.3 mmol), β-alanine tert-butyl ester
20

hydrochloride
(148.7
mg,
0.8
mmol).
A
solution
of
benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP)
(159.0 mg, 0.3 mmol) in DMF was added. After stirring at room temperature for 3 h,
46.4 mg PyBOP was added, and the reaction mixture was stirred overnight. After
o
addition of water (1 mL), the mixture was heated to 60 C for 30 min. Ethyl acetate
was added, and the organic layer was washed with 0.5 N HCl, 5% K2CO3 and brine.
The flash chromatography (SiO2, 25 % ethyl acetate in petroleum ether) gave 130 mg
(72.3%) of tert-butyl 3-(4-(1-(3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-
yl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzamido)propanoate as white solid. The solid
was then treated with TFA-DCM (1:2, 3 mL) for 1 hour. The solvent was removed
under reduced pressure. The flash chromatography (SiO2, 5% MeOH in DCM) gave
95.0
mg
(80%)
3-(4-(1-(3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-
1
yl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid 9r as white solid. H
NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 8.45 (t, J = 5.5 Hz, 1H), 7.93 (d, J = 8.5
Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.78 – 7.66 (m, 5H), 7.62 (t, J = 1.8 Hz, 1H), 7.41
(d, J = 2.1 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.27 – 7.21 (m, 1H), 7.14 (d, J = 8.4 Hz,
2H), 5.53 (q, J = 6.8 Hz, 1H), 3.91 (s, 3H), 3.43 – 3.38 (m, 2H), 2.47 (d, J = 7.1 Hz,
2H), 2.11 (s, 3H), 1.86 (d, J = 6.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 172.9,
165.9, 158.1, 145.5, 145.4, 143.1, 137.6, 134.4, 134.2, 133.5, 129.8, 129.2, 128.1,
127.5, 127.4, 126.7, 126.0, 125.3, 124.1, 119.4, 112.7, 105.9, 57.2, 55.4, 35.6, 33.8,
21.6, 9.8. LRMS (ESI, m/z): 600.1 [M-H]-. HRMS (ESI, m/z): calcd for
C33H29Cl2N3O4, 600.1457 [M-H]-; found 600.1459, purity: 98.4%; [α]20D = 1.18 (c = 1,
21

CH3OH).
3-(4-((5-(4-Chlorophenyl)-3-(3,5-dichlorophenyl)-4-methyl-1H-pyrazol-1-yl)me
thyl)benzamido)propanoic acid (9a). 3-(4-((5-(4-Chlorophenyl)-3-(3,5-
dichlorophenyl)-4-methyl-1H-pyrazol-1-yl)methyl)benzamido)propanoic acid (9a)
was prepared as white solid according to the preparation of compound 9r. 1H NMR
(400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.49 (t, J = 5.4 Hz, 1H), 7.73 (s, 1H), 7.72 –
7.68 (m, 3H), 7.63 (t, J = 1.9 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz,
2H), 7.04 (d, J = 8.3 Hz, 2H), 5.35 (s, 2H), 3.46 – 3.40 (m, 2H), 2.48 (d, J = 7.1 Hz,
13
2H), 2.11 (s, 3H). C NMR (125 MHz, DMSO-d6) δ 174.3, 164.9, 145.9, 141.8,
134.0, 137.1, 134.4, 131.6, 129.1, 127.1, 126.8, 126.6, 125.3, 113.1, 52.7, 37.4, 37.3,
9.8. LRMS (ESI, m/z): 540.0 [M-H]-. HRMS (ESI, m/z): calcd for C27H22Cl3N3O3,
540.0648 [M-H]-; found 540.0651, purity: 95.2%.
3-(4-((3-(3,5-Dichlorophenyl)-4-methyl-5-(naphthalen-1-yl)-1H-pyrazol-1-yl)m
ethyl)benzamido)propanoic acid (9b). 3-(4-((3-(3,5-Dichlorophenyl)-4-methyl-5-
(naphthalen-1-yl)-1H-pyrazol-1-yl)methyl)benzamido)propanoic acid (9b) was
1
prepared as white solid according to the preparation of compound 9r. H NMR (400
MHz, DMSO-d6) δ 12.22 (s, 1H), 8.43 (s, 1H), 8.07 (dd, J = 21.2, 8.2 Hz, 2H), 7.78
(d, J = 1.8 Hz, 2H), 7.67 – 7.54 (m, 5H), 7.51 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 6.8 Hz,
2H), 6.93 (d, J = 8.1 Hz, 2H), 5.28 (d, J = 16.0 Hz, 1H), 5.01 (d, J = 15.9 Hz, 1H),
13
3.40 (d, J = 5.8 Hz, 2H), 2.46 (d, J = 7.0 Hz, 2H), 1.97 (s, 3H). C NMR (125 MHz,
DMSO-d6) δ 172.9, 165.7, 145.8, 141.1, 140.1, 137.3, 134.4, 133.4, 133.2, 131.5,
129.9, 129.2, 128.6, 127.2, 127.2, 126.7, 126.5, 126.3, 125.5, 125.3, 124.7, 69.7, 52.7,
22

35.5, 33.7, 9.9. LRMS (ESI, m/z): 556.1 [M-H]-. HRMS (ESI, m/z): calcd for
C31H25Cl2N3O3, 556.1195 [M-H]-; found 556.1189, purity: 95.6%.
3-(4-((3-(3,5-Dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-4-methyl-1H-pyra
zol-1-yl)methyl)benzamido)propanoic acid (9c). 3-(4-((3-(3,5-Dichlorophenyl)-5-
(6-methoxynaphthalen-2-yl)-4-methyl-1H-pyrazol-1-yl)methyl)benzamido)propanoic
acid (9c) was prepared as white solid according to the preparation of compound 9r.
1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 8.45 (t, J = 5.5 Hz, 1H), 7.93 (d, J =
8.6 Hz, 1H), 7.85 (d, J = 9.1 Hz, 2H), 7.73 (d, J = 1.9 Hz, 2H), 7.70 (d, J = 8.3 Hz,
2H), 7.62 (t, J = 1.9 Hz, 1H), 7.43 – 7.32 (m, 2H), 7.23 (dd, J = 9.0, 2.5 Hz, 1H), 7.04
(d, J = 8.3 Hz, 2H), 5.39 (s, 2H), 3.90 (s, 3H), 3.41 (dd, J = 12.6, 7.0 Hz, 2H), 2.46 (d,
13
J = 7.1 Hz, 2H), 2.16 (s, 3H). C NMR (125 MHz, DMSO-d6) δ 174.5, 165.0, 158.2,
145.9, 143.2, 140.2, 137.4, 134.4, 134.1, 129.8, 129.1, 128.2, 127.4, 127.1, 126.8,
126.7, 125.3, 124.1, 119.5, 112.9, 106.0, 55.4, 52.8, 37.5, 37.3, 10.0. LRMS (ESI,
m/z): 586.0 [M-H]-. HRMS (ESI, m/z): calcd for C32H27Cl2N3O4, 586.1300 [M-H]-;
found 586.1315, purity: 96.2%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-phenyl-1H-pyrazol-1-yl)ethyl)benz
amido)propanoic acid (9d). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-phenyl-1H-
pyrazol-1-yl)ethyl)benzamido)propanoic acid (9d) was prepared as white solid
according to the preparation of compound 9r. 1H NMR (400 MHz, DMSO-d6) δ 13.17
– 10.49 (m, 1H), 8.44 (s, 1H), 7.77 – 7.66 (m, 5H), 7.61 (s, 1H), 7.50 (s, 5H), 7.31 –
7.20 (m, 3H), 7.13 (d, J = 8.0 Hz, 3H), 5.47 (d, J = 6.8 Hz, 1H), 3.42 (dd, J = 12.5,
6.6 Hz, 3H), 2.47 (d, J = 7.1 Hz, 3H), 2.08 (s, 4H), 1.84 (d, J = 6.9 Hz, 4H). 13C NMR
23

(125 MHz, DMSO-d6) δ 172.9, 165.9, 145.4, 145.3, 142.9, 137.5, 134.3, 133.5, 129.9,
129.2, 129.0, 128.9, 127.4, 126.7, 125.9, 125.3, 112.5, 57.1, 35.5, 33.8, 21.5, 9.7.
LRMS (ESI, m/z): 520.2 [M-H]-. HRMS (ESI, m/z): calcd for C28H25Cl2N3O3,
520.1196 [M-H]-; found 520.1188 purity: 95.5%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(m-tolyl)-1H-pyrazol-1-yl)ethyl)ben
zamido)propanoic acid (9e). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(m-tolyl)-
1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9e) was prepared as white solid
1
according to the preparation of compound 9r. H NMR (400 MHz, CD3OD) δ 12.15
(s, 1H), 8.44 (s, 1H), 7.72 (t, J = 5.6 Hz, 3H), 7.59 (s, 1H), 7.39 (t, J = 7.6 Hz, 1H),
7.30 (d, J = 7.6 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.5 Hz, 2H), 5.45 (q, J
= 6.8 Hz, 1H), 3.43 (dd, J = 12.7, 6.8 Hz, 2H), 2.47 (d, J = 7.1 Hz, 2H), 2.33 (s, 2H),
2.09 (d, J = 16.0 Hz, 2H), 1.83 (d, J = 6.9 Hz, 2H). 13C NMR (125 MHz, DMSO-d6) δ
172.9, 165.9, 145.5, 145.4, 143.0, 138.2, 137.6, 134.4, 133.5, 130.4, 129.6, 129.1,
128.8, 127.4, 127.0, 126.7, 125.9, 125.3, 112.4, 57.1, 35.6, 33.8, 21.6, 21.0, 9.7.
LRMS (ESI, m/z): 534.2 [M-H]-. HRMS (ESI, m/z): calcd for C29H28Cl2N3O3,
536.1502 [M+H]+; found 536.1510 purity: 94.9%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(p-tolyl)-1H-pyrazol-1-yl)ethyl)ben
zamido)propanoic acid (9f). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(p-tolyl)-
1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9f) was prepared as white solid
1
according to the preparation of compound 9r. H NMR (400 MHz, DMSO-d6) δ 8.47
(s, 1H), 7.80 – 7.66 (m, 4H), 7.60 (d, J = 1.8 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.14
(dd, J = 8.0, 2.7 Hz, 4H), 5.46 (q, J = 6.7 Hz, 1H), 3.42 (dd, J = 12.5, 6.7 Hz, 3H),
24

2.46 (d, J = 7.0 Hz, 2H), 2.37 (s, 3H), 2.12 – 1.99 (m, 3H), 1.81 (t, J = 13.7 Hz, 3H).
13C NMR (125 MHz, DMSO-d6) δ 172.8, 165.9, 145.4, 145.4, 142.9, 138.5, 137.5,
134.3, 133.4, 129.7, 129.5, 127.4, 126.6, 126.2, 125.9, 125.2, 112.4, 57.0, 35.5, 33.8,
21.5, 20.9, 9.7. LRMS (ESI, m/z): 534.2 [M-H]-. HRMS (ESI, m/z): calcd for
C29H27Cl2N3O3, 534.1351 [M-H]-; found 534.1358, purity: 95.5%.
3-(4-(1-(5-([1,1'-Biphenyl]-2-yl)-3-(3,5-dichlorophenyl)-4-methyl-1H-pyrazol-1-
yl)ethyl)benzamido)propanoic acid (9g). 3-(4-(1-(5-([1,1'-Biphenyl]-2-yl)-3-(3,5-
dichlorophenyl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9g) was
1
prepared as white solid according to the preparation of compound 9r. H NMR (400
MHz, DMSO-d6) δ 12.24 (s, 1H), 8.43 (t, J = 5.6 Hz, 1H), 7.68 – 7.55 (m, 9H), 7.35
(d, J = 7.3 Hz, 2H), 7.25 – 7.20 (m, 2H), 7.14 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 8.3 Hz,
2H), 4.90 (q, J = 7.1 Hz, 1H), 3.42 – 3.37 (m, 2H), 2.45 (t, J = 7.1 Hz, 2H), 2.08 (s,
3H), 1.13 (d, J = 6.9 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 173.4, 166.3, 145.6,
145.4, 142.5, 142.3, 140.3, 138.0, 134.8, 134.0, 132.2, 130.7, 130.5, 130.4, 129.1,
128.9, 128.8, 128.4, 128.3, 127.8, 127.7, 127.5, 127.1, 127.0, 126.2, 125.5, 125.3,
113.7, 57.4, 36.0, 34.3, 20.2, 10.2. LRMS (ESI, m/z): 596.1 [M-H]-. HRMS (ESI, m/z):
calcd for C34H29Cl2N3O3, 596.1513 [M-H]-; found 596.1514, purity: 99.2%.
3-(4-(1-(5-([1,1'-Biphenyl]-3-yl)-3-(3,5-dichlorophenyl)-4-methyl-1H-pyrazol-1-
yl)ethyl)benzamido)propanoic acid (9h). 3-(4-(1-(5-([1,1'-Biphenyl]-3-yl)-3-(3,5-
dichlorophenyl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9h) was
1
prepared as white solid according to the preparation of compound 9r. H NMR (400
MHz, DMSO-d6) δ 12.24 (s, 1H), 8.48 (t, J = 5.4 Hz, 1H), 7.81 – 7.71 (m, 5H), 7.65 –
25

7.52 (m, 4H), 7.41 (dq, J = 14.3, 7.2 Hz, 4H), 7.26 (d, J = 7.9 Hz, 1H), 7.16 (d, J =
8.3 Hz, 2H), 5.54 (q, J = 7.0 Hz, 1H), 3.43 (dd, J = 12.8, 6.6 Hz, 2H), 2.47 (m, 2H),
2.12 (s, 3H), 1.86 (d, J = 7.1 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 173.4, 166.3,
146.2, 145.9, 143.2, 141.1, 139.7, 138.0, 134.8, 133.9, 130.3, 130.0, 129.4, 129.3,
128.5, 128.3, 127.9, 127.7, 127.2, 127.2, 126.3, 125.8, 113.3, 57.9, 36.0, 34.3, 22.2,
10.2. LRMS (ESI, m/z): 596.1 [M-H]-. HRMS (ESI, m/z): calcd for C34H29Cl2N3O3,
596.1513 [M-H]-; found 596.1515, purity: 99.5%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(thiophen-2-yl)-1H-pyrazol-1-yl)et
hyl)benzamido)propanoic acid (9i). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-
(thiophen-2-yl)-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9i) was prepared as
1
white solid according to the preparation of compound 9r. H NMR (400 MHz,
DMSO-d6) δ 12.20 (s, 1H), 8.45 (s, 1H), 7.81 (dd, J = 5.1, 1.1 Hz, 1H), 7.78 – 7.68 (m,
4H), 7.62 (d, J = 1.8 Hz, 1H), 7.23 (dd, J = 5.1, 3.6 Hz, 1H), 7.16 (d, J = 8.3 Hz, 2H),
7.11 (dd, J = 3.5, 1.1 Hz, 1H), 5.63 (q, J = 6.9 Hz, 1H), 3.43 (dd, J = 12.7, 7.0 Hz,
2H), 2.47 (d, J = 7.1 Hz, 2H), 2.15 (d, J = 13.3 Hz, 3H), 1.84 (d, J = 6.9 Hz, 3H). 13C
NMR (125 MHz, DMSO-d6) δ 172.8, 165.8, 145.6, 145.3, 137.2, 135.6, 134.3, 133.5,
130.1, 129.3, 128.3, 127.9, 127.4, 126.8, 125.8, 125.4, 114.5, 57.3, 35.5, 33.8, 21.6,
9.8. LRMS (ESI, m/z): 526 [M-H]-. HRMS (ESI, m/z): calcd for C26H23Cl2N3O3S,
528.4480 [M+H]+; found 528.0745, purity: > 99.9%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(quinolin-6-yl)-1H-pyrazol-1-yl)eth
yl)benzamido)propanoic acid (9j). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-
(quinolin-6-yl)-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9j) was prepared as
26

1
white solid according to the preparation of compound 9r. H NMR (400 MHz,
DMSO-d6) δ 12.22 (s, 1H), 9.00 (dd, J = 4.3, 1.6 Hz, 1H), 8.46 (t, J = 5.7 Hz, 1H),
8.40 (d, J = 7.5 Hz, 1H), 8.16 – 8.06 (m, 1H), 7.94 (d, J = 1.3 Hz, 1H), 7.75 (d, J =
1.9 Hz, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.66 – 7.61 (m, 2H), 7.14 (d, J = 8.4 Hz, 2H),
5.57 (q, J = 6.9 Hz, 1H), 3.41 (dd, J = 12.6, 7.0 Hz, 2H), 2.47 (m, 2H), 2.13 (s, 3H),
13
1.87 (d, J = 6.9 Hz, 3H). C NMR (125 MHz, DMSO-d6) δ 172.9, 165.9, 151.6,
147.3, 145.6, 145.3, 142.2, 137.4, 136.4, 134.4, 133.5, 130.7, 129.9, 129.6, 127.8,
127.4, 127.3, 126.7, 125.9, 125.3, 122.2, 113.1, 57.4, 35.6, 33.8, 21.6, 9.7. LRMS
(ESI, m/z): 571.0 [M-H]-. HRMS (ESI, m/z): calcd for C31H26Cl2N4O3, 571.1309
[M-H]-; found 573.0453, purity: 95.7%.
3-(4-(1-(5-(Benzofuran-5-yl)-3-(3,5-dichlorophenyl)-4-methyl-1H-pyrazol-1-yl)
ethyl)benzamido)propanoic acid (9k). 3-(4-(1-(5-(Benzofuran-5-yl)-3-(3,5-
dichlorophenyl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9k) was
1
prepared as white solid according to the preparation of compound 9r. H NMR (400
MHz, DMSO-d6) δ 12.24 (s, 1H), 8.47 (t, J = 5.5 Hz, 1H), 8.11 (d, J = 2.1 Hz, 1H),
7.80 – 7.67 (m, 5H), 7.60 (dd, J = 7.1, 5.4 Hz, 2H), 7.14 (t, J = 9.5 Hz, 3H), 7.03 (d, J
= 2.2 Hz, 1H), 5.48 (q, J = 6.9 Hz, 1H), 3.42 (dd, J = 12.6, 7.0 Hz, 2H), 2.47 (m, 2H),
2.08 (s, 3H), 1.84 (d, J = 6.9 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 172.8, 165.9,
154.3, 147.1, 145.3, 145.3, 143.1, 137.6, 134.3, 133.4, 127.7, 127.3, 126.6, 126.1,
125.9, 125.2, 123.9, 123.0, 112.6, 111.8, 106.9, 57.0, 35.5, 33.8, 21.5, 9.7. LRMS
(ESI, m/z): 560.1 [M-H]-. HRMS (ESI, m/z): calcd for C30H25Cl2N3O4, 560.1149
[M-H]-; found 560.1159, purity: 95.9%.
27

3-(4-(1-(3-(3,5-Dichlorophenyl)-5-(1H-indol-5-yl)-4-methyl-1H-pyrazol-1-yl)et
hyl)benzamido)propanoic acid (9l). 3-(4-(1-(3-(3,5-Dichlorophenyl)-5-(1H-indol-
5-yl)-4-methyl-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9l) was prepared as
1
white solid according to the preparation of compound 9r. H NMR (400 MHz,
DMSO-d6) δ 11.35 (s, 1H), 8.48 (t, J = 5.3 Hz, 1H), 7.72 (t, J = 5.0 Hz, 4H), 7.60 (s,
1H), 7.51 (d, J = 8.4 Hz, 1H), 7.48 – 7.41 (m, 2H), 7.15 (d, J = 8.3 Hz, 2H), 6.93 (d, J
= 8.6 Hz, 1H), 6.49 (s, 1H), 5.51 (q, J = 6.5 Hz, 1H), 3.43 – 3.39 (m, 2H), 2.46 (m,
2H), 2.09 (s, 3H), 1.83 (d, J = 6.9 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 173.0,
165.9, 145.5, 145.2, 144.6, 137.8, 135.8, 134.3, 133.4, 127.7, 127.3, 126.5, 126.5,
126.0, 125.2, 122.6, 121.8, 119.5, 112.1, 111.8, 101.5, 56.7, 35.6, 334.0, 21.5, 9.9.
LRMS (ESI, m/z): 559.1 [M-H]-. HRMS (ESI, m/z): calcd for C30H26Cl2N4O3,
559.1309 [M-H]-; found 559.1306, purity: 96.4%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(1-methyl-1H-indol-5-yl)-1H-pyraz
ol-1-yl)ethyl)benzamido)propanoic acid (9m). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-
methyl-5-(1-methyl-1H-indol-5-yl)-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid
1
(9m) was prepared as white solid according to the preparation of compound 9r. H
NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.47 (t, J = 5.5 Hz, 1H), 7.73 (dt, J =
10.4, 5.2 Hz, 4H), 7.60 (t, J = 1.9 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.46 (s, 1H), 7.44
(d, J = 3.1 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 7.7 Hz, 1H), 6.48 (d, J = 3.0
Hz, 1H), 5.49 (q, J = 6.6 Hz, 1H), 3.84 (s, 3H), 3.42 (dd, J = 12.4, 6.9 Hz, 2H), 2.47
13
(m, 2H), 2.08 (s, 3H), 1.84 (d, J = 7.0 Hz, 3H). C NMR (125 MHz, DMSO-d6) δ
172.9, 165.9, 145.5, 145.2, 144.4, 137.8, 136.3, 134.3, 133.4, 130.8, 128.1, 127.3,
28

126.5, 126.0, 125.2, 122.6, 122.0, 119.6, 112.2, 110.2, 100.8, 56.8, 35.5, 33.8, 32.6,
21.5, 9.9. LRMS (ESI, m/z): 573.1 [M-H]-. HRMS (ESI, m/z): calcd for
C31H28Cl2N4O3, 573.1466 [M-H]-; found 573.1479, purity: > 99.9%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(naphthalen-1-yl)-1H-pyrazol-1-yl)
ethyl)benzamido)propanoic
acid
(9n).
3-(4-(1-(3-(3,5-Dichlorophenyl)-
4-methyl-5-(naphthalen-1-yl)-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9n)
was prepared as white solid according to the preparation of compound 9r.1H NMR
(400 MHz, DMSO-d6) δ 8.46 (t, J = 5.6 Hz, 1H), 8.34 (d, J = 5.8 Hz, 1H), 8.10 (t, J =
7.8 Hz, 3H), 8.00 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 1.9 Hz, 1H), 7.78 (d, J = 1.9 Hz,
1H), 7.72 (s, 1H), 7.70 (s, 1H), 7.67 – 7.60 (m, 4H), 7.57 (d, J = 7.2 Hz, 1H), 7.54 (s,
1H), 7.52 (s, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.20 (d, J = 8.4
Hz, 1H), 7.16 (d, J = 6.7 Hz, 1H), 7.10 (d, J = 8.3 Hz, 2H), 6.93 (d, J = 8.3 Hz, 2H),
5.28 (t, J = 7.0 Hz, 1H), 5.00 (q, J = 7.1 Hz, 1H), 3.49 – 3.24 (m, 4H), 2.48 – 2.41 (m,
2H), 1.96 (s, 3H), 1.92 (d, J = 6.8 Hz, 2H), 1.85 (d, J = 6.9 Hz, 2H), 1.78 (d, J = 6.9
Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 174.5, 165.1, 145.6, 145.0, 144.3, 141.0,
137.6, 134.5, 134.4, 134.1, 131.9, 131.5, 129.9, 129.9, 129.4, 128.8, 127.1, 126.7,
126.4, 126.2, 126.0, 125.3, 125.3, 124.4, 113.8, 57.6, 37.5, 37.3, 21.4, 9.7. LRMS
(ESI, m/z): 570.2 [M-H]-. HRMS (ESI, m/z): calcd for C32H27Cl2N3O3, 570.1351
[M-H]-; found 570.1357, purity: 97.3%.
3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-(naphthalen-2-yl)-1H-pyrazol-1-yl)
ethyl)benzamido)propanoic acid (9o). 3-(4-(1-(3-(3,5-Dichlorophenyl)-4-methyl-5-
(naphthalen-2-yl)-1H-pyrazol-1-yl)ethyl)benzamido)propanoic acid (9o) was prepared
29