European Journal of Medicinal Chemistry 263 (2024) 115913

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European Journal of Medicinal Chemistry

journal homepage: www.elsevier.com/locate/ejmech

Research paper

Discovery of 4-hydroxyl pyrazole derivatives as potent

ferroptosis inhibitors
Danzhi Ying a, 1, Xin Shen b, 1, Shuqi Wang a, Junyi Chen b, Zhenying Wu a, Wenteng Chen a,
Fudi Wang b, Junxia Min b, **, Yongping Yu a, *
a
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China
b
The First Affiliated Hospital, The Second Affiliated Hospital, Institute of Translational Medicine, School of Public Health, Zhejiang University School of Medicine,
Hangzhou, 310058, China

A R T I C L E I N F O A B S T R A C T

Keywords: Ferroptosis, an iron-dependent form of regulated cell death, has been well recognized as a pathogenic mechanism
Ferroptosis inhibitor in driving many diseases, such as neurodegenerative disorders, ischemia-reperfusion (I/R) injury. Blocking
4-Hydroxyl pyrazole derivatives ferroptosis has been emerging as a feasible therapeutic strategy for the prevention and treatment of these dis­
Structure-activity relationship
eases. However, novel potent ferroptosis inhibitors remain to be developed for further clinical applications. In
Radical-trapping antioxidant
this study, we screened our in-house compound libraries by phenotypic assays and identified a 4-hydroxyl
pyrazole derivative HW-3 with good ferroptosis inhibitory activity (EC50 = 120.1 ± 3.5 nM). Based on the
structure of HW-3, a series of 4-hydroxyl pyrazole derivatives were further designed and synthesized. Among
these compounds, compound 25 could significantly inhibit RSL3-induced ferroptosis with an EC50 value of 8.6 ±
2.2 nM in HT-1080 cells, which was 3-fold more potent than the classical ferroptosis inhibitor ferrostatin-1 (Fer-
1) (EC50 = 23.4 ± 1.3 nM). The potent ferroptosis inhibitory activity of compound 25 was further validated in
multiple additional cell lines. Our mechanistic study revealed that compound 25 inhibited ferroptosis via
intrinsic radical-trapping antioxidative capacity. Taken together, the findings of our study demonstrate 4-hydrox­
yl pyrazole derivative 25 is a potent ferroptosis inhibitor, which holds a great therapeutic potential for further
development.

1. Introduction In the last decade, a plenty of ferroptosis inhibitors have been

Ferroptosis is an iron-dependent regulated cell death (RCD) driven by
excessive lipid peroxidation and cellular metabolism [1–3]. Morpholog­
ically, ferroptosis mainly manifests as obvious shrinkage of mitochondria
with increased membrane density and decreased cristae [4,5], which is
distinct from other forms of RCD such as apoptosis [6] and necroptosis
[7]. A major characteristic of ferroptosis is the accumulation of lipid
peroxides (LPO) that formed through iron-catalyzed polyunsaturated
fatty acids (PUFAs) autoxidation or enzyme-mediated process catalyzed
by iron-dependent lipoxygenases (LOXs) [8–11]. LPO are considered as
key inducers through their ability to crosslink ferroptosis-related proteins
and nucleic acids [12]. When cells fail to suppress the formation of LPO,
ferroptosis is triggered. It is thought as a promising strategy to inhibit
ferroptosis for the treatment of related diseases [13–15].
developed (Fig. 1). Ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) were
the first reported ferroptosis inhibitors discovered by high-throughput
screenings of compound library [1], and have been proved to act as
radical-trapping antioxidants (RTAs) to suppress the accumulation of
LPO for inhibiting ferroptosis [16–18]. CuATSM, a Cu2+ complex
recently used in clinical studies for the treatment of amyotrophic lateral
sclerosis (ALS), also showed anti-ferroptosis activity through quenching
the lipid radicals [19,20]. Additionally, a series of well-known RTAs [21,
22], such as α-TOH [23], phenothiazines [24–26] as well as nitroxides
[27,28], have been reported as ferroptosis inhibitors. Meanwhile, dex­
razoxane (DXZ), is the FDA-approved drug for preventing doxorubicin
(DOX)-induced cardiotoxicity in cancer patients [29]. Our previous
research indicated that DOX-induced cell death exhibited features of
typical ferroptotic cell death, and DXZ displayed protective effects

* Corresponding author.
** Corresponding author.
E-mail addresses: junxiamin@zju.edu.cn (J. Min), yyu@zju.edu.cn (Y. Yu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2023.115913
Received 4 September 2023; Received in revised form 11 October 2023; Accepted 24 October 2023
Available online 4 November 2023
0223-5234/© 2023 Elsevier Masson SAS. All rights reserved.
D. Ying et al.
against DOX-induced ferroptosis [30]. In addition, iron chelators
deferoxamine and deferiprone, have been proved to inhibit ferroptosis
as well [31,32]. Although a variety of ferroptosis inhibitors have been
developed so far, many of them suffer from poor activity, or unfavorable
pharmacokinetic properties, limiting their further clinical application.
Therefore, development of new ferroptosis inhibitors for treating
ferroptosis-related diseases is still highly desirable.
Herein, we discovered a new ferroptosis inhibitor HW-3 (EC50 =
120.1 ± 3.5 nM), a 4-hydroxy pyrazole derivative, through phenotypic
assays on 1 μM RSL3-induced HT-1080 cells ferroptosis model. Based on
the scaffold of HW-3, a series of derivatives were further designed,
synthesized, and evaluated for their ferroptosis inhibitory activity. Out
of them, compound 25 was found to be the most potent ferroptosis in­
hibitor (EC50 = 8.6 ± 2.2 nM), which was better than the classic fer­
roptosis inhibitor Fer-1 (EC50 = 23.4 ± 1.3 nM). The potent ferroptosis
inhibitory activity of compound 25 was further validated in multiple
additional cell lines. Further mechanism studies demonstrated com­
pound 25 effectively inhibited ferroptosis by intrinsic antioxidative
capacity.
2. Results and discussion
2.1. Chemistry
Synthetic routes for intermediates and the final products were
depicted in Scheme 1 and Scheme 2. Reaction of 2-bromoketone de­
rivatives with sodium azide provided phenacyl aizdes 1a-1j. Then
α-azido chalcones 2a-2w were obtained via Knoevengel condensation
reaction of phenacyl aizdes with corresponding aldehydes [33]. The
preparations of pyrazole derivatives were performed according to our
previous report [34]. To be precise, treatment of α-azido chalcones with
corresponding hydrazine and 1,8-diazabicyclo [5.4.0]undec-7-ene
(DBU) in acetonitrile/water afforded 4-hydroxy pyrazole derivatives
3–20, 23, 24, 27–31. Treatment of α-azido chalcone 2m with methyl­
hydrazine sulfate and sodium hydroxide in acetonitrile led to 4-amino
pyrazole derivative 21. Pyrazole derivative 22 was synthesized by
subjecting 2m to hydrazine hydrate and DBU in acetonitrile (Scheme 1).
Removing the methyl group of 23 and 24 afforded phenol derivatives 25
and 26, respectively. Hydrolysis of 31 under acid conditions afforded
the carboxyl derivative 32, followed by condensation with corre­
sponding amines to give amide derivatives 33, 34, 35a. Finally, the
tetrahydropyran group of 35a was easily removed under acid condi­
tions, providing the target compound 35 (Scheme 2).
Fig. 1. Chemical structures of representative ferroptosis inhibitors.
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European Journal of Medicinal Chemistry 263 (2024) 115913
2.2. Structure-activity relationship analysis for pyrazole derivatives
All the synthesized compounds were tested for their ferroptosis
inhibitory activity with the model of RSL3 (1 μM)-induced ferroptosis in
HT-1080 cells. The HT-1080 cell line was proved to show high sensi­
tivity to ferroptosis and widely used to evaluate the specificity of com­
pounds to ferroptosis [16,26,27]. We firstly explored the variety of R1
and R2 on the ferroptosis inhibitory activity. As shown in Table 1,
compound 3 (EC50 = 69.1 ± 2.7 nM) with phenyl ring on R1 and R2
exhibited better inhibitory activity than HW-3, while the typical fer­
roptosis inhibitor Fer-1 significantly rescued cells with an EC50 value of
23.4 ± 1.3 nM. The activity was slightly decreased when introducing
fluorine into the phenyl ring of R2 (4), while the phenyl ring bearing
chlorine (5) or bromine (6) led to an improved activity. The
electron-donating group methoxy (7) was also beneficial for the fer­
roptosis inhibition. Oppositely, substitution of the phenyl ring with
furanyl (8) or n-butyl (9) group was unfavorable, leading to a signifi­
cantly decreased activity. As for R1, 4-biphenyl substitution (10) was
more favorable than phenyl (3) group. Introduction of electron-donating
groups (11 and 12) into the phenyl ring was more beneficial than strong
electron-withdrawing groups (13 and 14). Of note, compound 15 with a
4-Cl substituted phenyl group at R1 exhibited significantly improved
potency (EC50 = 18.9 ± 0.9 nM), which was better than Fer-1. However,
the efficacy decreased no matter changing the position (16 and 17) or
increasing the number (18) of chlorine atom. Based on the structure of
compound 15, we next explored the influence of R3 and R4 on ferroptosis
inhibitory activity (Table 1). Substitution of R3 with methyl or phenyl
group was well-tolerated. However, the activity was notably decreased
when the hydroxyl of R4 was replaced by amino group or hydrogen
atom, demonstrating the necessity of hydroxy group at 4-position of the
pyrazole ring for ferroptosis inhibitory activity.
Inspired by the improved ferroptosis inhibition of compound 15, we
further optimized the R2 moiety to improve its efficacy (Table 2). Similar
to the above results, introduction of electron-donating group (23 and
24) into the phenyl ring was more favorable than those with electron-
withdrawing group (27 and 28). Of note, the efficacy of compound 25
bearing 4-hydroxy phenyl group at R2 was significantly improved (EC50
= 8.6 ± 2.2 nM), which was 3-fold more potent than Fer-1. Compound
26 with two hydroxyls on the phenyl ring exhibited slightly decreased
activity. Replacing the phenyl ring of R2 with naphthyl (29) or imidazole
group (30) resulted in an acceptable potency. Furthermore, it was not
beneficial for their ferroptosis inhibitory activity by introducing ester
(31), carboxy (32) or amide group (33–35) into the phenyl ring.
D. Ying et al. European Journal of Medicinal Chemistry 263 (2024) 115913

Scheme 1. Reagents and conditions: (a) NaN3, acetone, rt, 3 h, 75 %–91 %. (b) acetic acid, piperidine, ethanol, rt, 12–24 h, 38 %–91 %. (c) corresponding hydrazine,
DBU, CH3CN/H2O (10/1, v/v), rt, 3 h, 22 %–96 %. (d) methylhydrazine sulfate, NaOH, CH3CN, rt, 8 h, 86 %. (e) Hydrazine hydrate, DBU, CH3CN, rt, 3 h, 22 %.

Scheme 2. (A) BBr3, CH2Cl2, 0 ◦ C to rt, 3 h, 54%–65 %. (b) concentrated HCl, reflux, 24 h, 82 %. (c) corresponding amine, HATU, Et3N, DMF, rt, 6 h, 59 %–71 %. (d)
HCl/MeOH, rt, 12 h, 88 %.

2.3. Ferroptosis inhibitory activity of compound 25 in vitro
Among all tested compounds, the compound 25 was found to be the
most effective for inhibiting ferroptosis induced by RSL3, and thus was
chosen for further evaluation. Imidazole ketone erastin (IKE), derived
from Erastin, could effectively induce ferroptosis by inhibiting system
Xc− and preventing cystine import [35]. As shown in Fig. 2A, on
IKE-induced HT-1080 cells ferroptosis model, compound 25 also
showed stronger protection effect than Fer-1. To determine whether the
inhibitory effect of compound 25 was cell line specific, we tested its
activity on three additional cell lines: 786-O [36], BJ [37] and H9c2
[30], which all exhibited sensitivity to ferroptosis. As shown in Table 3,
Fig. 2C–E, consistent with the result in HT-1080 cells (Fig. 2B), the data
indicated that compound 25 significantly prevented 786-O, BJ and H9c2
cell lines from ferroptosis induced by RSL3 with EC50 values of 89.7 ±
7.2 nM, 11.3 ± 1.4 nM and 43.3 ± 6.2 nM, respectively, which were all
better than Fer-1 with EC50 values of 253.0 ± 20.1 nM, 38.2 ± 3.7 nM
and 68.6 ± 8.1 nM, respectively.
The inhibition activity of compound 25 against ferroptosis was also
validated by the cell morphologic observation. As shown in Fig. 2F, 786-
O cells became shrunken, membrane rupture and lost their normal
morphology after treatment with RSL3. Oppositely, the cells recovered
when mixed with Fer-1 (1 μM) or compound 25 (1 μM). The similar
changes were also observed when the concentration of compound 25
decreased to 0.0625 μM. However, unlike compound 25, there’re no
significant changes when treated with 0.0625 μM Fer-1. These results
further demonstrated that compound 25 had a better capacity of pro­
tecting HT-1080 cells from ferroptosis than Fer-1.

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D. Ying et al. European Journal of Medicinal Chemistry 263 (2024) 115913

Table 1 Table 2
Ferroptosis inhibitory activity of pyrazole derivatives in HT-1080 cells. Ferroptosis inhibitory activity of 4-hydroxyl pyrazole derivatives in HT-1080
cells.

Compound R1 R2 R3 R4 EC50 (nM)a

Compound R2 EC50 (nM)a
Fer-1 – – – – 23.4 ± 1.3
HW-3 4-NO2-phenyl 4-Me-phenyl H OH 120.1 ± Fer-1 – 23.4 ± 1.3
3.5 23 4-OMe-phenyl 23.1 ± 1.5
3 phenyl phenyl H OH 69.1 ± 2.7 24 3,4-di-OMe-phenyl 31.7 ± 5.1
4 phenyl 4-F-phenyl H OH 79.6 ± 2.3 25 4-OH-phenyl 8.6 ± 2.2
5 phenyl 4-Cl-phenyl H OH 30.8 ± 1.4 26 3,4-di–OH–phenyl 26.1 ± 4.3
6 phenyl 4-Br-phenyl H OH 22.6 ± 1.1 27 4-CF3-phenyl 187.2 ± 5.2
7 phenyl 4-OMe- H OH 24.8 ± 2.0 28 4-F-phenyl 52.7 ± 3.1
phenyl 29 1-naphthyl 44.8 ± 2.9
8 phenyl 2-furanyl H OH ＞1000 30 25.9 ± 1.2
9 phenyl n-butyl H OH 150.0 ±
5.8
10 4-biphenyl phenyl H OH 39.5 ± 2.3 31 51.1 ± 2.2
11 4-OMe-phenyl phenyl H OH 32.1 ± 1.7
12 3,4-OCH2O- phenyl H OH 27.4 ± 1.9
32b ＞1000
phenyl
13 4-CF3-phenyl phenyl H OH 79.6 ± 2.3
14 4-F-phenyl phenyl H OH 42.4 ± 1.3 33 81.9 ± 2.1
15 4-Cl-phenyl phenyl H OH 18.9 ± 0.9
16 2-Cl-phenyl phenyl H OH 94.9 ± 7.6
17 3-Cl-phenyl phenyl H OH 187.2 ±
5.2 34 44.8 ± 1.4
18 2,4-di-Cl-phenyl phenyl H OH 56.6 ± 3.3
19 4-Cl-phenyl phenyl Me OH 22.0 ± 0.8
20 4-Cl-phenyl phenyl phenyl OH 24.6 ± 1.2
21 4-Cl-phenyl phenyl Me NH2 720.5 ±
9.4
22 4-Cl-phenyl phenyl H H ＞1000 35 80.2 ± 5.6

a
Each compound was tested in three independent experiments; the values
were presented as the means ± SD.
2.4. Mechanistic study of compound 25 protecting cells from ferroptosis
a
Each compound was tested in three independent experiments; the values
were presented as the means ± SD. bCompound was tested in the form of hy­
drochloride salt.

It is reported that the classic ferroptosis inhibitor, Fer-1, act as a
radical-trapping antioxidant (RTA) [16,17]. To explore the mechanism
behind the anti-ferroptosis activity of 4-hydroxy pyrazole derivatives,
we firstly evaluated the antioxidative potency of compound 15, 21, 22,
25 by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) bleaching method,
which is widely used for the antioxidative capacity evaluation of the
ferroptosis inhibitors [38]. As shown in Fig. 3A and Table 4, compound
15, 25 were both capable of scavenging DPPH radicals in a
dose-dependent manner in vitro like Fer-1, in which compound 25 (EC50
= 3.94 μM) exhibited 1.6-fold more effective than Fer-1 (EC50 = 6.48
μM). However, compound 21 and 22, with amino and hydrogen at R4,
respectively, exhibited relatively poor antioxidative ability, suggesting
that the hydroxy at R4 plays a key role in exerting its antioxidative ca­
pacity, as well as ferroptosis inhibitory activity. Additionally, ABTS
radical cation scavenging capacity assay under cell-free condition was
performed to further validate their antioxidative capacity [39]. ABTS [2,
29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation
(ABTS•+) is a blue-colored cation with absorption maxima at wave­
lengths 415 nm, and the antioxidative capacity is determined by the
degree of blanching of ABTS•+. The results shown in Fig. 3B and Table 4
were similar with the results of DPPH assays, further proving that there’s
a significant correlation between the anti-ferroptosis activity and anti­
oxidative ability of 4-hydroxy pyrazole derivatives. In the ABTS assay,
compound 25 (EC50 = 6.3 μM) exhibited nearly 8-fold stronger anti­
oxidative capacity than Fer-1 (EC50 = 49.8 μM). Based on the above
results, we conclude that compound 25 holds stronger antioxidative
capacity than Fer-1.
Increasing level of lipid reactive oxygen species (ROS) is a major
hallmark of ferroptosis. We used BODIPY 581/591C11 (BODIPY-C11)
probe to detect levels of lipid ROS generation by flow cytometry. As
shown in Fig. 3C, RSL3 apparently induced the accumulation of lipid
ROS. Compound 25 exerted significantly decreased lipid peroxidation
compared with Fer-1 treatment. In addition, we measured the intracel­
lular ROS level with fluorescent probe 5-(and-6)-carboxy-2′,7′-dichlor­
odihydrofluorescein diacetate (DCFH-DA) by confocal laser scanning
microscopy (CLSM). The results demonstrated compound 25 could
effectively suppress the erastin-induced cytosolic ROS accumulation
(Fig. 3D).
Furthermore, iron imbalance is an essential initiator of ferroptosis
[1]. Thus, we evaluated the iron-chelating ability of compound 25 with
Fe (III) or Fe (II) by using UV–vis spectroscopy [40]. As displayed in
Fig. 3E, compound 25 had a characteristic absorption peak at ~261 nm
and no new peaks were observed when Fe (III) or Fe (II) was added to the
solution of compound 25. Moreover, it was observed that there were no
significant changes of UV–vis absorption at 261 nm in the presence of Fe
(III) or Fe (II) (Fig. 3F). This suggested that compound 25 had no ability
to chelate with Fe (III) or Fe (II). These results indicate that compound
25 mainly exerts anti-ferroptosis effect by its intrinsic antioxidative
capacity.
In conclusion, combining the results from both ferroptosis inhibitory
activity and antioxidative ability, a general SAR was summarized in
Fig. 4. The 4-hydroxy group is necessary for both anti-ferroptosis and
antioxidative activity. Substitution of 1-hydrogen with methyl or phenyl
group is well-tolerated. As for R1 or R2, furan or alkyl group is

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D. Ying et al. European Journal of Medicinal Chemistry 263 (2024) 115913

Fig. 2. The compound 25 shows more potent ferroptosis inhibitory activity than Fer-1 in cells.
(A) Inhibition effect of compound 25 against 2.5 μM IKE-induced ferroptosis in HT-1080 cells. (B–E) Inhibition effect of compound 25 against 1 μM RSL3-induced
ferroptosis in HT-1080 cells (B), 786-O cells (C), BJ cells (D) and H9c2 cells (E). (F) Morphology of 786-O cells treated with 1 μM RSL3 in the absence or presence of
Fer-1 or compound 25.

3. Conclusions
Table 3
Ferroptosis inhibitory activity of compound 25 and Fer-1 in different cell lines.
In this study, we firstly screen our in-house compound libraries by
No EC50 (nM) phenotypic assays and discovered a new ferroptosis inhibitor HW-3 with
HT-1080a HT-1080b 786-Ob BJb H9c2b 4-hydroxyl pyrazole scaffold. To obtain more potent compounds, we
25 48.3 ± 5.3 8.6 ± 2.2 89.7 ± 7.2 11.3 ± 1.4 43.3 ± 6.2 further designed and synthesized a series of derivatives based on the
Fer-1 75.2 ± 6.4 23.4 ± 1.3 253.0 ± 20.1 38.2 ± 3.7 68.6 ± 8.1 structure of HW-3. Among all tested compounds, compound 25 was
a identified as the most potent ferroptosis inhibitor in HT-1080 cells and
Cells were treated with 2.5 μM IKE in the presence of various concentrations
of compound 25 or Fer-1.
additional three cell lines. Notably, at the cellular level, its ferroptosis
b
Cells were treated with 1 μM RSL3 in the presence of various concentrations inhibitory activity was more potent than Fer-1. Furthermore, mecha­
of compound 25 or Fer-1. nistic studies revealed compound 25 mainly effectively inhibited fer­
roptosis by its intrinsic antioxidative capacity, which holds a great
unfavorable for ferroptosis inhibitory activity, while phenyl, naphtha­ therapeutic potential for further clinical development.
lene and imidazole are more favorable. With respect to substituents on
phenyl ring, chlorine, bromine or electron donating substituents im­
proves the anti-ferroptosis activity, while strong electron-withdrawing
group is not beneficial.

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D. Ying et al. European Journal of Medicinal Chemistry 263 (2024) 115913

Fig. 3. The compound 25 displays higher antioxidative activity than Fer-1. The cell-free antioxidant activity of Fer-1 and compound 15, 21, 22, 25 in various
concentrations analyzed by DPPH assay (A) and ABTS assay (B). (C) Flow cytometry analyses of lipid ROS levels in HT-1080 cells treated with 0.8 μM RSL3 in the
absence or presence of Fer-1 or compound 25. (D) The confocal image of DCFH-DA in HT-1080 cells treated with 5 μM Erastin in the absence or presence of Fer-1 or
compound 25. (E) UV–vis absorption spectra of compound 25 in the absence or presence of Fe (III) or Fe (II). (F) Quantification of UV–vis absorption peaks at 261 nm
for compound 25 in the absence or presence of Fe (III) or Fe (II). Ns, not significance.

4. Experimental section
Table 4
The ferroptosis inhibitory and antioxidant activity of pyrazole derivatives.
4.1. Chemistry

4.1.1. Materials and equipment

All reagents and solvents were obtained from commercial suppliers
and used without further purification unless otherwise noted. Reactions
were monitored by TLC carried out on silica gel 60 F254 plates. Melting
points were recorded on a Büchi B-540 melting point apparatus. 1H NMR
Compound EC50 and 13C NMR spectra were recorded at 500 MHz using a Bruker AVANCE
Ferroptosis inhibition DPPH clearance ABTS clearance III spectrometer in CDCl3 or dimethyl sulfoxide (DMSO)-d6 solution. For
1
(nM) (μM) (μM) H NMR, tetramethylsilane (TMS) served as the internal standard (δ =
Fer-1 23.4 ± 1.3 6.48 49.8
0), and data are reported as follows: chemical shift, integration, multi­
15 18.9 ± 0.9 4.97 111.8 plicity (s = singlet, d = doublet, t = triplet, q = quartet, and m =
21 720.5 ± 9.4 14.61 ＞200 multiplet), and coupling constant(s) in Hz. For 13C NMR, TMS (δ = 0) or
22 ＞1000 ＞100 ＞200 CDCl3 (δ = 77.26) was used as the internal standard and spectra were
25 8.6 ± 2.2 3.94 6.3
obtained with complete proton decoupling. HPLC purity analysis and
the HRMS of all final products were confirmed on an Agilent 1290 HPLC-
6224 time-of-flight mass spectrometer using a Phenomenex Luna 5 μm
C18, 100 Å, 150 × 4.60 mm, 5 μm column at a flow rate of 0.5 mL/min
using linear gradient buffer B in A (B, CH3OH containing 0.1 % formic
acid; A, H2O containing 0.1 % formic acid). Mobile phase B was
increased linearly from 5 to 95 % over 7 min and 95 % over the next 2

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D. Ying et al.
Fig. 4. Summary of the SAR of the pyrazole derivatives.
min after which the column was equilibrated to 5 % for 1 min. Purity of
all biologically tested compounds were determined by HPLC to be >95
%.
4.1.2. General procedures for the synthesis of 2a-2w
In a 100 mL round bottom flask, sodium azide (4 mmol) was added to
a solution of 2-bromoketone derivatives (2 mmol) in acetone (30 mL)
with vigorous stirring at room temperature. The mixture was stirred for
3 h and filtered under vacuum. The filtrate was concentrated to give
phenacyl azides without further purification. Aldehydes (2 mmol) were
added to a solution of phenacyl azides in ethanol (10 mL) at room
temperature. Then acetic acid (57 μL, 1 mmol) and piperidine (99 μL, 1
mmol) were added, and the mixture was stirred at room temperature for
12 h. Next, the reaction mixture was evaporated to dryness under
reduced pressure and the residue was extracted with dichloromethane/
water. The organic phase was separated, dried over anhydrous Na2SO4,
concentrated, and purified by silica-gel column chromatography using
petroleum ether/ethyl acetate (200/1 to 100/1) as eluent to yield
α-azido chalcones.
4.1.2.1. (Z)-2-Azido-1,3-diphenylprop-2-en-1-one (2a). Following gen­
eral procedure afforded 2a as a yellow solid (Yield: 80 %). 1H NMR (500
MHz, Chloroform-d) δ 7.85–7.75 (m, 4H), 7.66–7.58 (m, 1H), 7.53–7.48
(m, 2H), 7.42–7.32 (m, 3H), 6.47 (s, 1H). HRMS (ESI) m/z calcd for
C15H11N3O [M+Na]+: 272.0794. Found: 272.0799.
4.1.2.2. (Z)-2-Azido-3-(4-fluorophenyl)-1-phenylprop-2-en-1-one (2b).
Following general procedure afforded 2b as a yellow oil (Yield: 91 %).
1
H NMR (500 MHz, Chloroform-d) δ 7.85–7.76 (m, 4H), 7.64–7.59 (m,
1H), 7.50 (m, 2H), 7.10–7.04 (m, 2H), 6.42 (s, 1H). HRMS (ESI) m/z
calcd for C15H10FN3O [M+Na]+: 290.0706. Found: 290.0711.
4.1.2.3. (Z)-2-Azido-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (2c).
Following general procedure afforded 2c as a yellow solid (Yield: 66 %).
1
H NMR (500 MHz, Chloroform-d) δ 7.78 (dd, J = 15.9, 7.9 Hz, 4H),
7.65 (t, J = 7.2 Hz, 1H), 7.51 (t, J = 7.5 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H),
6.41 (s, 1H). HRMS (ESI) m/z calcd for C15H10ClN3O [M+Na]+:
306.0410. Found: 306.0400.
4.1.2.4. (Z)-2-Azido-3-(4-bromophenyl)-1-phenylprop-2-en-1-one (2d).
Following general procedure afforded 2d as a yellow solid (Yield: 65 %).
1
H NMR (500 MHz, Chloroform-d) δ 7.79 (dd, J = 8.2, 1.2 Hz, 2H), 7.68
(d, J = 8.6 Hz, 2H), 7.65–7.59 (m, 1H), 7.51 (d, J = 8.3 Hz, 4H), 6.37 (s,
1H). HRMS (ESI) m/z calcd for C15H10BrN3O [M+Na]+: 349.9899.
Found: 349.9901.
4.1.2.5. (Z)-2-Azido-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one
(2e). Following general procedure afforded 2e as a yellow oil (Yield: 91
%). 1H NMR (500 MHz, Chloroform-d) δ 7.78 (dd, J = 16.8, 7.6, 1.8 Hz,
4H), 7.61–7.57 (m, 1H), 7.51–7.47 (m, 2H), 6.93–6.90 (m, 2H), 6.46 (s,
1H), 3.85 (s, 3H). HRMS (ESI) m/z calcd for C16H13N3O2 [M+Na]+:
302.0900. Found: 302.0901.
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European Journal of Medicinal Chemistry 263 (2024) 115913
4.1.2.6. (Z)-2-Azido-3-(furan-2-yl)-1-phenylprop-2-en-1-one (2f).
Following general procedure afforded 2f as a yellow oil (Yield: 90 %). 1H
NMR (500 MHz, Chloroform-d) δ 7.75–7.71 (m, 2H), 7.62–7.57 (m, 1H),
7.52–7.46 (m, 3H), 7.25 (d, J = 3.6 Hz, 1H), 6.57 (dd, J = 3.9, 1.8 Hz,
1H), 6.47 (s, 1H). HRMS (ESI) m/z calcd for C13H9N3O2 [M+Na]+:
262.0587. Found: 262.0591.
4.1.2.7. (Z)-2-Azido-1-phenylhept-2-en-1-one (2g). Following general
procedure afforded 2g as a yellow oil (Yield: 88 %). 1H NMR (500 MHz,
Chloroform-d) δ 7.71–7.68 (m, 2H), 7.59–7.55 (m, 1H), 7.48–7.43 (m,
2H), 5.76 (t, J = 7.5 Hz, 1H), 2.32 (q, J = 7.4 Hz, 2H), 1.43–1.32 (m,
4H), 0.91 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for C13H15N3O
[M+Na]+: 252.1107. Found: 252.1114.
4.1.2.8. (Z)-1-([1,1′-biphenyl]-4-yl)-2-azido-3-phenylprop-2-en-1-one
(2h). Following general procedure afforded 2h as a yellow solid (Yield:
77 %). 1H NMR (500 MHz, Chloroform-d) δ 7.91–7.87 (m, 2H),
7.85–7.81 (m, 2H), 7.74–7.70 (m, 2H), 7.68–7.63 (m, 2H), 7.52–7.47
(m, 2H), 7.44–7.34 (m, 4H), 6.52 (s, 1H). HRMS (ESI) m/z calcd for
C21H15N3O [M+Na]+: 348.1113. Found: 348.1113.
4.1.2.9. (Z)-2-Azido-1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one
(2i). Following general procedure afforded 2i as a pale-yellow solid
(Yield: 80 %). 1H NMR (500 MHz, Chloroform-d) δ 7.86–7.82 (m, 2H),
7.82–7.78 (m, 2H), 7.42–7.37 (m, 2H), 7.36–7.32 (m, 1H), 7.01–6.95
(m, 2H), 6.41 (s, 1H), 3.90 (s, 3H). HRMS (ESI) m/z calcd for
C16H13N3O2 [M+Na]+: 302.0900. Found: 302.0896.
4.1.2.10. (Z)-2-Azido-1-(benzo[d] [1,3] dioxol-5-yl)-3-phenylprop-2-en-
1-one (2j). Following general procedure afforded 2j as a yellow solid
(Yield: 85 %). 1H NMR (500 MHz, Chloroform-d) δ 7.81 (d, J = 4.5 Hz,
2H), 7.46–7.32 (m, 5H), 6.89 (d, J = 8.4 Hz, 1H), 6.44 (s, 1H), 6.12 (s,
2H). HRMS (ESI) m/z calcd for C16H11N3O3 [M+Na]+: 316.0693.
Found: 316.0703.
4.1.2.11. (Z)-2-Azido-3-phenyl-1-(4-(trifluoromethyl) phenyl) prop-2-en-
1-one (2k). Following general procedure afforded 2k as a yellow solid
(Yield: 47 %). 1H NMR (500 MHz, Chloroform-d) δ 7.88 (d, J = 8.0 Hz,
2H), 7.85–7.80 (m, 2H), 7.77 (d, J = 8.1 Hz, 2H), 7.44–7.35 (m, 3H),
6.42 (s, 1H). HRMS (ESI) m/z calcd for C16H10F3N3O [M+Na]+:
340.0668. Found: 340.0665.
4.1.2.12. (Z)-2-Azido-1-(4-fluorophenyl)-3-phenylprop-2-en-1-one (2l).
Following general procedure afforded 2l as a yellow oil (Yield: 38 %). 1H
NMR (500 MHz, Chloroform-d) δ 7.87–7.77 (m, 4H), 7.42–7.34 (m, 3H),
7.22–7.15 (m, 2H), 6.42 (s, 1H). HRMS (ESI) m/z calcd for C15H10FN3O
[M+Na]+: 290.0700. Found: 290.0697.
4.1.2.13. (Z)-2-Azido-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one
(2m). Following general procedure afforded 2m as a pale-yellow solid
(Yield: 70 %). 1H NMR (500 MHz, Chloroform-d) δ 7.83–7.78 (m, 2H),
7.77–7.72 (m, 2H), 7.50–7.46 (m, 2H), 7.42–7.34 (m, 3H), 6.42 (s, 1H).
D. Ying et al.
HRMS (ESI) m/z calcd for C15H10ClN3O [M+Na]+: 306.0405. Found:
306.0400.
4.1.2.14. (Z)-2-Azido-1-(2-chlorophenyl)-3-phenylprop-2-en-1-one (2n).
Following general procedure afforded 2n as a yellow solid (Yield: 86 %).
1 364.0459. Found: 364.0459.
H NMR (500 MHz, Chloroform-d) δ 7.80–7.76 (m, 2H), 7.49–7.44 (m,
2H), 7.42–7.34 (m, 5H), 6.32 (s, 1H). HRMS (ESI) m/z calcd for
C15H10ClN3O [M+Na]+: 306.0405. Found: 306.0407.
4.1.2.15. (Z)-2-Azido-1-(3-chlorophenyl)-3-phenylprop-2-en-1-one (2o).
Following general procedure afforded 2o as a yellow oil (Yield: 80 %).
1 mmol) was stirred in acetonitrile/water (2 mL/0.2 mL) at room tem­
H NMR (500 MHz, Chloroform-d) δ 7.84–7.80 (m, 2H), 7.77 (t, J = 1.9
Hz, 1H), 7.66 (dt, J = 7.7, 1.3 Hz, 1H), 7.59 (dd, J = 8.1, 2.1, 1.1 Hz,
1H), 7.47–7.35 (m, 4H), 6.44 (s, 1H). HRMS (ESI) m/z calcd for
C15H10ClN3O [M+Na]+: 306.0405. Found: 306.0400.
4.1.2.16. (Z)-2-Azido-1-(2,4-dichlorophenyl)-3-phenylprop-2-en-1-one
(2p). Following general procedure afforded 2p as a yellow solid (Yield:
71 %). 1H NMR (500 MHz, Chloroform-d) δ 7.79 (dd, J = 7.5, 2.2 Hz,
2H), 7.51 (d, J = 1.7 Hz, 1H), 7.42–7.34 (m, 5H), 6.30 (s, 1H). HRMS
(ESI) m/z calcd for C15H9Cl2N3O [M+Na]+: 340.0015. Found:
340.0016.
4.1.2.17. (Z)-2-Azido-1-(4-chlorophenyl)-3-(4-(trifluoromethyl) phenyl)
prop-2-en-1-one (2q). Following general procedure afforded 2q as a
yellow solid (Yield: 68 %). 1H NMR (500 MHz, Chloroform-d) δ
7.93–7.88 (m, 2H), 7.79–7.74 (m, 2H), 7.64 (d, J = 8.2 Hz, 2H),
7.53–7.48 (m, 2H), 6.38 (s, 1H). HRMS (ESI) m/z calcd for
C16H9ClF3N3O [M+Na]+: 374.0278. Found: 374.0281.
4.1.2.18. (Z)-2-Azido-1-(4-chlorophenyl)-3-(4-fluorophenyl) prop-2-en-1-
one (2r). Following general procedure afforded 2r as a yellow solid
(Yield: 85 %). 1H NMR (500 MHz, Chloroform-d) δ 7.85–7.80 (m, 2H),
7.77–7.71 (m, 2H), 7.50–7.46 (m, 2H), 7.11–7.06 (m, 2H), 6.38 (s, 1H).
HRMS (ESI) m/z calcd for C16H9ClF3N3O [M+Na]+: 324.0316. Found:
324.0311.
4.1.2.19. (Z)-2-Azido-1-(4-chlorophenyl)-3-(4-methoxyphenyl) prop-2-
en-1-one (2s). Following general procedure afforded 2s as a yellow
solid (Yield: 48 %). 1H NMR (500 MHz, Chloroform-d) δ 7.82–7.77 (m,
2H), 7.74–7.68 (m, 2H), 7.50–7.45 (m, 2H), 6.94–6.90 (m, 2H), 6.41 (s,
1H), 3.85 (s, 3H). HRMS (ESI) m/z calcd for C16H12ClN3O2 [M+Na]+:
336.0510. Found: 336.0510.
4.1.2.20. (Z)-2-Azido-1-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl) prop-
2-en-1-one (2t). Following general procedure afforded 2t as a yellow
solid (Yield: 77 %). 1H NMR (500 MHz, Chloroform-d) δ 7.75–7.69 (m,
2H), 7.52 (d, J = 2.0 Hz, 1H), 7.50–7.45 (m, 2H), 7.33 (dd, J = 8.5, 2.0
Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.39 (s, 1H), 3.93 (s, 6H). HRMS (ESI)
m/z calcd for C17H14ClN3O3 [M+Na]+: 366.0616. Found: 366.0621.
4.1.2.21. (Z)-2-Azido-1-(4-chlorophenyl)-3-(naphthalen-1-yl) prop-2-en-
1-one (2u). Following general procedure afforded 2u as a yellow solid
(Yield: 47 %). 1H NMR (500 MHz, Chloroform-d) δ 8.30 (d, J = 7.3 Hz,
1H), 7.91–7.84 (m, 4H), 7.77–7.73 (m, 1H), 7.58–7.48 (m, 5H), 7.18 (s,
1H). HRMS (ESI) m/z calcd for C19H12ClN3O [M+Na]+: 356.0561.
Found: 356.0567.
4.1.2.22. (Z)-2-Azido-1-(4-chlorophenyl)-3-(1H-imidazole-5-yl) prop-2-
en-1-one (2v). Following general procedure afforded 2v as a yellow
solid (Yield: 74 %). 1H NMR (500 MHz, DMSO‑d6) δ 12.63 (d, J = 40.9
Hz, 1H), 8.00 (s, 1H), 7.81 (s, 1H), 7.77–7.72 (m, 2H), 7.68–7.60 (m,
2H), 6.51 (s, 1H). HRMS (ESI) m/z calcd for C12H8ClN5O [M+H]+:
274.0496. Found: 274.0493.
8
European Journal of Medicinal Chemistry 263 (2024) 115913
4.1.2.23. Methyl (Z)-4-(2-azido-3-(4-chlorophenyl)-3-oxoprop-1-en-1-yl)
benzoate (2w). Following general procedure afforded 2w as a yellow
solid (Yield: 40 %). 1H NMR (500 MHz, Chloroform-d) δ 8.07–8.02 (m,
2H), 7.88–7.84 (m, 2H), 7.79–7.75 (m, 2H), 7.52–7.48 (m, 2H), 6.40 (s,
1H), 3.94 (s, 3H). HRMS (ESI) m/z calcd for C17H12ClN3O3 [M+Na]+:
4.1.3. Synthesis of compounds 3–35
4.1.3.1. General procedures for the 4-hydroxyl pyrazole derivatives. A
mixture of vinyl azide 2 (0.5 mmol) and 80 % hydrazine hydrate (2.5
perature. Then DBU (0.5 mmol) was added and the mixture was stirred
for 5 h. The mixture was diluted with water (10 mL) and extracted three
times with ethyl acetate (10 mL). The combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, concentrated, and
purified by column chromatography using petroleum ether/ethyl ace­
tate (5/1 to 3/1) as eluent to afford the final compounds.
4.1.3.2. 3,5-Diphenyl-1H-pyrazol-4-ol (3). Following general procedure
afforded 3 as a white solid (Yield: 43 %). Mp 236–237 ◦ C. 1H NMR (500
MHz, DMSO‑d6) δ 12.89 (s, 1H), 8.33 (s, 1H), 7.93 (s, 4H), 7.45 (m, J =
7.5 Hz, 4H), 7.30 (m, J = 7.4 Hz, 2H). 13C NMR (125 MHz, Acetone-d6) δ
135.57, 128.44, 127.07, 125.87. HRMS (ESI) m/z calcd for C15H12N2O
[M+H]+: 237.1022. Found: 237.1025.
4.1.3.3. 3-([1,1′-biphenyl]-4-yl)-5-phenyl-1H-pyrazol-4-ol (4). Following
general procedure afforded 4 as a white solid (Yield: 52 %). Mp＞250 ◦ C.
1
H NMR (500 MHz, DMSO‑d6) δ 12.94 (s, 1H), 8.40 (s, 1H), 7.99 (d, J =
48.5 Hz, 4H), 7.75 (m, 4H), 7.47 (m, 4H), 7.34 (m, 2H). 13C NMR (125
MHz, DMSO‑d6) δ 135.98, 129.46, 129.02, 128.92, 127.90, 127.43,
127.20, 127.19, 126.97. HRMS (ESI) m/z calcd for C21H16N2O [M+H]+:
313.1335. Found: 313.1328.
4.1.3.4. 3-(4-methoxyphenyl)-5-phenyl-1H-pyrazol-4-ol (5). Following
general procedure afforded 5 as a yellow solid (Yield: 63 %). Mp
208–210 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.73 (s, 1H), 8.17 (s, 1H),
7.97–7.81 (m, 4H), 7.44 (t, J = 7.6 Hz, 2H), 7.30 (t, J = 7.3 Hz, 1H), 7.03
(d, J = 8.4 Hz, 2H), 3.80 (s, 3H)⋅13C NMR (125 MHz, Acetone-d6) δ
159.03, 134.79, 128.37, 127.19, 126.93, 125.82, 113.85, 54.67. HRMS
(ESI) m/z calcd for C16H14N2O2 [M+H]+: 267.1128. Found: 267.1133.
4.1.3.5. 3-(benzo[d] [1,3] dioxol-5-yl)-5-phenyl-1H-pyrazol-4-ol (6).
Following general procedure afforded 6 as a white solid (Yield: 59 %).
Mp 211–212 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.76 (s, 1H), 8.24 (s,
1H), 7.90 (s, 2H), 7.44 (s, 4H), 7.29 (m, 1H), 7.01 (d, J = 7.8 Hz, 1H),
6.05 (s, 2H). 13C NMR (125 MHz, Acetone-d6) δ 147.88, 146.81, 135.00,
128.43, 127.04, 125.83, 119.60, 108.24, 106.35, 101.12. HRMS (ESI)
m/z calcd for C16H12N2O3 [M+H]+: 281.0921. Found: 281.0921.
4.1.3.6. 5-Phenyl-3-(4-(trifluoromethyl) phenyl)-1H-pyrazol-4-ol (7).
Following general procedure afforded 7 as a white solid (Yield: 38 %).
Mp＞250 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 13.13 (s, 1H), 8.59 (s, 1H),
8.17 (d, J = 34.9 Hz, 2H), 7.99–7.74 (m, 4H), 7.47 (d, J = 7.8 Hz, 2H),
7.33 (m, J = 7.4 Hz, 1H). 13C NMR (125 MHz, Acetone-d6) δ 136.24,
128.62, 127.43, 126.11, 125.89, 125.30, 125.27. HRMS (ESI) m/z calcd
for C16H11F3N2O [M+H]+: 305.0896. Found: 305.0899.
4.1.3.7. 3-(4-fluorophenyl)-5-phenyl-1H-pyrazol-4-ol (8). Following
general procedure afforded 8 as a white solid (Yield: 64 %). Mp
236–237 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.89 (s, 1H), 8.34 (s, 1H),
7.93 (d, J = 31.7 Hz, 4H), 7.45 (m, J = 7.6 Hz, 2H), 7.35–7.21 (m, 3H).
13
C NMR (125 MHz, Acetone-d6) δ 135.31, 128.52, 127.87, 127.81,
127.19, 125.81, 115.22, 115.05. HRMS (ESI) m/z calcd for C15H11FN2O
[M+H]+: 255.0928. Found: 255.0931.
D. Ying et al.
4.1.3.8. 3-(4-chlorophenyl)-5-phenyl-1H-pyrazol-4-ol (9). Following
general procedure afforded 9 as a white solid (Yield: 78 %). Mp
230–231 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.97 (s, 1H), 8.43 (s, 1H),
7.93 (d, J = 35.4 Hz, 4H), 7.57–7.42 (m, 4H), 7.31 (t, J = 7.3 Hz, 1H).
13
C NMR (125 MHz, Acetone-d6) δ 135.61, 132.13, 128.52, 128.45,
127.37, 127.24, 125.83. HRMS (ESI) m/z calcd for C15H11ClN2O
[M+H]+: 271.0633. Found: 271.0633.
4.1.3.9. 3-(2-chlorophenyl)-5-phenyl-1H-pyrazol-4-ol (10). Following
general procedure afforded 10 as a white solid (Yield: 96 %). Mp
154–155 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.86 (d, J = 169.5 Hz,
1H), 8.41 (s, 1H), 7.92 (d, J = 29.1 Hz, 2H), 7.72 (s, 1H), 7.58–7.48 (m,
2H), 7.44 (s, 2H), 7.28 (t, J = 7.2 Hz, 1H). 13C NMR (125 MHz, Acetone-
d6) δ 136.79, 134.56, 134.08, 133.33, 129.23, 128.48, 127.10, 126.95,
125.41. HRMS (ESI) m/z calcd for C16H11ClN2O [M+H]+: 271.0633.
Found: 271.0636.
4.1.3.10. 3-(3-chlorophenyl)-5-phenyl-1H-pyrazol-4-ol (11). Following
general procedure afforded 11 as a white solid (Yield: 69 %). Mp
208–209 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 13.02 (s, 1H), 8.51 (s, 1H),
8.08–7.76 (m, 4H), 7.47 (m, J = 7.8 Hz, 3H), 7.39–7.28 (m, 2H). 13C
NMR (125 MHz, Acetone-d6) δ 135.84, 133.90, 130.12, 128.58, 127.34,
126.77, 125.86, 125.51, 124.12. HRMS (ESI) m/z calcd for C16H11ClN2O
[M+H]+: 271.0633. Found: 271.0633.
4.1.3.11. 3-(2,4-dichlorophenyl)-5-phenyl-1H-pyrazol-4-ol (12). Follow
ing general procedure afforded 12 as a white solid (Yield: 96 %). Mp
184–185 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.86 (d, J = 169.5 Hz,
1H), 8.41 (s, 1H), 7.92 (d, J = 29.1 Hz, 2H), 7.72 (s, 1H), 7.58–7.48 (m,
2H), 7.44 (s, 2H), 7.28 (t, J = 7.2 Hz, 1H). 13C NMR (125 MHz, Acetone-
d6) δ 136.79, 134.56, 134.08, 133.33, 129.23, 128.48, 127.10, 126.95,
125.41. HRMS (ESI) m/z calcd for C15H10Cl2N2O [M+H]+: 305.0243.
Found: 305.0250.
4.1.3.12. 5-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-ol (13). Following
general procedure afforded 13 as a white solid (Yield: 54 %). Mp
231–232 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.90 (s, 1H), 8.36 (s, 1H),
7.94 (d, J = 31.2 Hz, 4H), 7.45 (m, 2H), 7.30 (m, 3H). 13C NMR (125
MHz, Acetone-d6) δ 135.32, 128.51, 127.89, 127.82, 127.19, 125.83,
115.22, 115.05. HRMS (ESI) m/z calcd for C15H11FN2O [M+H]+:
255.0928. Found: 255.0930.
4.1.3.13. 5-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-ol (14). Following
general procedure afforded 14 as a pale-yellow solid (Yield: 79 %). Mp
240–241 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.95 (s, 1H), 8.41 (s, 1H),
7.93 (d, J = 26.6 Hz, 4H), 7.48 (d, J = 21.0 Hz, 4H), 7.32 (d, J = 6.5 Hz,
1H). 13C NMR (125 MHz, Acetone-d6) δ 135.59, 132.13, 131.11, 128.52,
128.45, 127.37, 127.24, 125.83. HRMS (ESI) m/z calcd for C15H11ClN2O
[M+H]+: 271.0633. Found: 271.0635.
4.1.3.14. 5-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-ol (15). Following
general procedure afforded 15 as a yellow solid (Yield: 80 %). Mp
222–223 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.98 (s, 1H), 8.43 (s, 1H),
7.90 (d, J = 48.8 Hz, 4H), 7.65 (d, J = 21.1 Hz, 2H), 7.46 (s, 2H), 7.32 (s,
1H). 13C NMR (125 MHz, Acetone-d6) δ 135.62, 131.44, 128.53, 127.68,
127.27, 125.84, 120.29. HRMS (ESI) m/z calcd for C15H11BrN2O
[M+H]+: 315.0128. Found: 315.0128.
4.1.3.15. 5-(4-methoxyphenyl)-3-phenyl-1H-pyrazol-4-ol (16). Following
general procedure afforded 16 as a yellow solid (Yield: 61 %). Mp
220–221 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.73 (s, 1H), 8.17 (s, 1H),
7.87 (s, 4H), 7.43 (s, 2H), 7.29 (t, J = 7.1 Hz, 1H), 7.02 (s, 2H), 3.79 (s,
3H). 13C NMR (125 MHz, Acetone-d6) δ 205.36, 159.02, 128.37, 127.19,
126.92, 125.81, 113.84, 54.67. HRMS (ESI) m/z calcd for C16H14N2O2
[M+H]+: 267.1128. Found: 267.1135.
9
European Journal of Medicinal Chemistry 263 (2024) 115913
4.1.3.16. 5-(Furan-2-yl)-3-phenyl-1H-pyrazol-4-ol (17). Following gen­
eral procedure afforded 17 as a black solid (Yield: 50 %). Mp
189–190 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.93 (s, 1H), 8.38 (s, 1H),
7.92 (s, 2H), 7.73 (s, 1H), 7.45 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.3 Hz,
1H), 6.77 (d, J = 3.1 Hz, 1H), 6.61 (s, 1H). 13C NMR (125 MHz, Acetone-
d6) δ 141.49, 135.04, 128.42, 127.07, 125.66, 111.19, 105.89. HRMS
(ESI) m/z calcd for C13H10N2O2 [M+H]+: 227.0815. Found: 227.0819.
4.1.3.17. 5-Butyl-3-phenyl-1H-pyrazol-4-ol (18). Following general pro
cedure afforded 18 as a yellow solid (Yield: 47 %). Mp 211–212 ◦ C. 1H
NMR (500 MHz, DMSO‑d6) δ 12.73 (s, 1H), 8.17 (s, 1H), 7.87 (s, 4H),
7.43 (s, 2H), 7.29 (t, J = 7.1 Hz, 1H), 7.02 (s, 2H), 3.79 (s, 3H). 13C NMR
(125 MHz, Acetone-d6) δ 205.36, 159.02, 128.37, 127.19, 126.92,
125.81, 113.84, 54.67. HRMS (ESI) m/z calcd for C13H16N2O [M+H]+:
217.1335. Found: 217.1344.
4.1.3.18. 3-(4-chlorophenyl)-1-methyl-5-phenyl-1H-pyrazol-4-ol (19). A
mixture of 2m (0.5 mmol), methylhydrazine sulfate (5 mmol) was stir­
red in acetonitrile/water (2 mL/0.2 mL) at room temperature. Then DBU
(1.0 mmol) was added and the mixture was stirred for 5 h. The mixture
was diluted with water (10 mL) and extracted three times with ethyl
acetate (10 mL). The combined organic extracts were washed with brine,
dried over anhydrous Na2SO4, concentrated, and purified by column
chromatography using petroleum ether/ethyl acetate (10/1 to 5/1) as
eluent to afford 19 as a yellow solid (Yield: 68 %). Mp 172–173 ◦ C. 1H
NMR (500 MHz, DMSO‑d6) δ 8.33 (s, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.54
(d, J = 7.8 Hz, 4H), 7.47 (d, J = 8.5 Hz, 3H), 3.76 (s, 3H). 13C NMR (125
MHz, Acetone-d6) δ 136.87, 136.37, 132.62, 131.64, 129.65, 129.23,
128.69, 128.30, 128.24, 127.31, 37.47. HRMS (ESI) m/z calcd for
C16H13ClN2O [M+H]+: 285.0789. Found: 285.0795.
4.1.3.19. 3-(4-chlorophenyl)-1,5-diphenyl-1H-pyrazol-4-ol (20). A mix
ture of 2m (0.5 mmol), phenyl hydrazine (5 mmol) was stirred in
acetonitrile/water (2 mL/0.2 mL) at room temperature. Then DBU (1.0
mmol) was added and the mixture was stirred for 5 h. The mixture was
diluted with water (10 mL) and extracted three times with ethyl acetate
(10 mL). The combined organic extracts were washed with brine, dried
over anhydrous Na2SO4, concentrated, and purified by column chro­
matography using petroleum ether/ethyl acetate (10/1 to 8/1) as eluent
to afford 20 as a yellow solid (Yield: 45 %). Mp 181–182 ◦ C. 1H NMR
(500 MHz, DMSO‑d6) δ 8.66 (s, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.52 (d, J
= 8.6 Hz, 2H), 7.42–7.24 (m, 10H). 13C NMR (125 MHz, DMSO‑d6) δ
140.53, 140.42, 138.12, 132.32, 131.97, 131.88, 129.95, 129.41,
129.33, 129.01, 128.95, 128.53, 128.14, 127.63, 125.00. HRMS (ESI)
m/z calcd for C21H15ClN2O [M+H]+: 347.0946. Found: 347.0950.
4.1.3.20. 3-(4-chlorophenyl)-1-methyl-5-phenyl-1H-pyrazol-4-amine
(21). A mixture of 2m (0.5 mmol) and methylhydrazine sulfate (0.5
mmol) was stirred in acetonitrile (2 mL) at room temperature. Then
NaOH (1.0 mmol) was added and the mixture was stirred for 8 h. The
mixture was diluted with water (10 mL) and extracted three times with
ethyl acetate (10 mL). The combined organic extracts were washed with
brine, dried over Na2SO4, concentrated, and purified by column chro­
matography using petroleum ether/ethyl acetate (5/1) as eluent to
afford 21 as a yellow solid (Yield: 86 %). Mp 103–104 ◦ C. 1H NMR (500
MHz, DMSO‑d6) δ 7.91–7.86 (m, 2H), 7.56 (t, J = 7.5 Hz, 2H), 7.51–7.43
(m, 5H), 3.80 (s, 2H), 3.73 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 138.72,
132.81, 132.06, 131.89, 129.43, 129.36, 129.25, 128.91, 128.57,
127.84, 124.42, 37.62. HRMS (ESI) m/z calcd for C16H14ClN3 [M+H]+:
284.0958. Found: 284.0954.
4.1.3.21. 3-(4-chlorophenyl)-5-phenyl-1H-pyrazole (22). A mixture of
vinyl azide 2m (0.5 mmol) and 80 % hydrazine hydrate (2.5 mmol) was
stirred in acetonitrile (2 mL) at room temperature. Then DBU (0.5
mmol) was added and the mixture was stirred for 5 h. The mixture was
D. Ying et al.
diluted with water (10 mL) and extracted three times with ethyl acetate
(10 mL). The combined organic extracts were washed with brine, dried
over anhydrous Na2SO4, concentrated, and purified by column chro­
matography using petroleum ether/ethyl acetate (5/1) as eluent to
afford 22 as a white solid (Yield: 22 %). Mp 215–216 ◦ C. 1H NMR (500
MHz, DMSO‑d6) δ 13.45 (s, 1H), 7.85 (d, J = 22.6 Hz, 4H), 7.48 (s, 4H),
7.36 (s, 1H), 7.23 (s, 1H). 13C NMR (125 MHz, Acetone-d6) δ 132.87,
128.87, 128.79, 128.01, 126.89, 125.31, 99.71. HRMS (ESI) m/z calcd
for C15H11ClN2 [M+H]+: 255.0684. Found: 255.0694.
4.1.3.22. 3-(4-chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazol-4-ol
(23). Following general procedure afforded 23 as a white solid (Yield:
54 %). Mp 239–240 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.78 (s, 1H),
8.24 (s, 1H), 7.89 (d, J = 70.6 Hz, 4H), 7.49 (d, J = 7.2 Hz, 2H), 7.02 (d,
J = 8.0 Hz, 2H), 3.79 (s, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 163.67,
136.45, 132.44, 132.27, 132.09, 131.91, 60.38. HRMS (ESI) m/z calcd
for C16H13ClN2O2 [M+H]+: 301.0738. Found: 301.0738.
4.1.3.23. 3-(4-chlorophenyl)-5-(3,4-di-methoxyphenyl)-1H-pyrazol-4-ol
(24). Following general procedure afforded 24 as a white solid (Yield:
65 %). Mp 221–222 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.84 (s, 1H),
8.35 (s, 1H), 7.98 (s, 2H), 7.49 (s, 4H), 7.04 (d, J = 7.7 Hz, 1H), 3.80 (d,
J = 12.9 Hz, 6H). 13C NMR (125 MHz, Acetone-d6) δ 149.49, 148.98,
135.04, 132.03, 128.42, 127.37, 118.48, 111.91, 109.74, 55.22, 55.16.
HRMS (ESI) m/z calcd for C17H15ClN2O3 [M+H]+: 331.0844. Found:
331.0846.
4.1.3.24. 3-(4-chlorophenyl)-5-(4-hydroxylphenyl)-1H-pyrazol-4-ol
(25). Under N2 atmosphere, 23 (0.5 mM) was stirred in dried CH2Cl2 (5
mL) at 0 ◦ C, then BBr3 in CH2Cl2 (2.5 mM, 1.0 M) was added slowly. The
reaction was allowed to continue at room temperature for another 3 h.
Then water (20 mL) was added and the mixture was extracted with ethyl
acetate (20 mL × 2). The combined organic extracts were washed with
saturated NaHCO3 (aq.) and brine. The organic phase was dried over
anhydrous Na2SO4, concentrated, and purified by column chromatog­
raphy using petroleum ether/ethyl acetate (3/1 to 2/1) as eluent to
afford compound 25 as a white solid (Yield: 54 %). Mp 240–241 ◦ C. 1H
NMR (500 MHz, DMSO‑d6) δ 12.73 (s, 1H), 9.57 (s, 1H), 8.20 (s, 1H),
7.95 (s, 2H), 7.69 (s, 2H), 7.49 (d, J = 7.8 Hz, 2H), 6.84 (d, J = 8.4 Hz,
2H). 13C NMR (125 MHz, DMSO‑d6) δ 157.14, 131.61, 128.91, 127.59,
127.52, 127.47, 127.35, 115.82. HRMS (ESI) m/z calcd for
C15H11ClN2O2 [M+H]+: 287.0582. Found: 287.0588.
4.1.3.25. 3-(4-chlorophenyl)-5-(3,4-di-hydroxylphenyl)-1H-pyrazol-4-ol
(26). Under N2 atmosphere, 24 (0.5 mM) was stirred in dried CH2Cl2 (5
mL) at 0 ◦ C, and then BBr3 in CH2Cl2 (2.5 mM, 1.0 M) was added slowly.
The reaction was allowed to continue at room temperature for another 3
h. Then water (20 mL) was added and the mixture was extracted with
ethyl acetate (20 mL × 2). The combined organic extracts were washed
with saturated NaHCO3 (aq.) and brine, dried over anhydrous Na2SO4,
concentrated, and purified by column chromatography using petroleum
ether/ethyl acetate (3/1 to 1/1) as eluent to afford 26 as a gray solid
(Yield: 65 %). Mp＞250 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.65 (s,
1H), 8.97 (s, 2H), 8.15 (s, 1H), 7.94 (s, 2H), 7.48 (d, J = 7.7 Hz, 2H),
7.32 (s, 1H), 7.11 (s, 1H), 6.83–6.75 (m, 1H). 13C NMR (125 MHz,
DMSO‑d6) δ 145.69, 145.31, 134.87, 131.58, 128.90, 117.46, 116.13,
113.84. HRMS (ESI) m/z calcd for C15H11ClN2O3 [M+H]+: 303.0531.
Found: 303.0529.
4.1.3.26. 3-(4-chlorophenyl)-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-
ol (27). Following general procedure afforded 27 as a white solid
(Yield: 57 %). Mp 247–248 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 13.20 (s,
1H), 8.70 (s, 1H), 8.23–7.77 (m, 6H), 7.54 (d, J = 20.8 Hz, 2H). 13C NMR
(125 MHz, Acetone-d6) δ 136.40, 132.55, 128.64, 127.44, 126.13,
10
European Journal of Medicinal Chemistry 263 (2024) 115913
125.37, 125.34. HRMS (ESI) m/z calcd for C16H10ClF3N2O [M+H]+:
339.0507. Found: 339.0506.
4.1.3.27. 3-(4-chlorophenyl)-5-(4-fluorophenyl)-1H-pyrazol-4-ol (28).
Following general procedure afforded 28 as a white solid (Yield: 67 %).
Mp＞250 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.97 (s, 1H), 8.45 (s, 1H),
7.94 (s, 4H), 7.52 (s, 2H), 7.30 (s, 2H)⋅13C NMR (125 MHz, Acetone-d6) δ
135.43, 132.28, 128.55, 127.91, 127.84, 127.34, 115.36, 115.18. HRMS
(ESI) m/z calcd for C15H10ClFN2O [M+H]+: 289.0538. Found:
289.0541.
4.1.3.28. 3-(4-chlorophenyl)-5-(naphthalen-1-yl)-1H-pyrazol-4-ol (29).
Following general procedure afforded 29 as a yellow solid (Yield: 62 %).
Mp 180–181 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.98 (d, J = 132.0 Hz,
1H), 8.42 (s, 1H), 8.03 (d, J = 29.3 Hz, 5H), 7.70–7.42 (m, 6H). 13C NMR
(125 MHz, Acetone-d6) δ 137.07, 133.93, 131.94, 131.85, 128.74,
128.46, 128.22, 128.16, 127.14, 126.38, 126.20, 126.07, 125.42. HRMS
(ESI) m/z calcd for C19H13ClN2O [M+H]+: 321.0789. Found: 321.0788.
4.1.3.29. 3-(4-chlorophenyl)-5-(1H-imidazole-5-yl)-1H-pyrazol-4-ol
(30). Following general procedure using dichloromethane/methanol
(100/1) as eluent afforded 30 as a yellow solid (Yield: 45 %). Mp＞
250 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 12.75 (s, 1H), 12.36 (s, 1H),
8.01–7.90 (m, 2H), 7.84 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.41 (s, 1H).
13
C NMR (125 MHz, DMSO‑d6) δ 140.50, 140.43, 136.12, 131.79,
131.75, 131.66, 131.60. HRMS (ESI) m/z calcd for C12H9ClN4O
[M+H]+: 261.0538. Found: 261.0528.
4.1.3.30. Methyl 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl) ben­
zoate (31). Following general procedure afforded 31 as a white solid
(Yield: 65 %). Mp 237–238 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 13.18 (s,
1H), 8.69 (s, 1H), 8.01 (m, 6H), 7.53 (s, 2H), 3.87 (s, 3H). 13C NMR (125
MHz, DMSO‑d6) δ 141.63, 136.82, 134.75, 133.87, 132.97, 132.61,
132.48, 132.08, 130.34, 130.10, 57.27. HRMS (ESI) m/z calcd for
C17H13ClN2O3 [M+H]+: 329.0687. Found: 329.0682.
4.1.3.31. 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl) benzoic acid
hydrochloride (32). In a 50 mL round bottom flask, methyl 4-(3-(4-
chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl) benzoate (31) (2 mmol) was
stirred in a solution of concentrated hydrochloride (20 mL) under reflux
condition for 24 h. The mixture was filtered under vacuum, washed with
water, and dried to yield the crude product, which was further purified
by treating with ethyl acetate to afford 32 as a white solid (Yield: 82 %).
Mp＞250 ◦ C. 1H NMR (500 MHz, DMSO‑d6) δ 13.06 (s, 2H), 8.66 (s, 1H),
8.03 (m, 4H), 7.94 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H). 13C NMR
(125 MHz, DMSO‑d6) δ 167.77, 136.75, 132.00, 130.13, 129.71,
129.09, 127.55, 125.56. HRMS (ESI) m/z calcd for C16H11ClN2O3
[M+H]+: 315.0531. Found: 315.0533.
4.1.3.32. 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl)-N-ethyl­
benzamide (33). In a 25 mL round bottom flask, 4-(3-(4-chlorophenyl)-
4-hydroxy-1H-pyrazol-5-yl) benzoic acid hydrochloride (32) (176 mg,
0.5 mmol) and HATU (285 mg, 0.75 mmol) was dissolved in dried DMF
(3 mL) for 10 min. Then ethylamine hydrochloride (49 mg, 0.6 mmol)
and triethylamine (139 μL, 1.0 mmol) were added after the mixture was
cooling to 0 ◦ C. The reaction was warmed to room temperature and
stirred for 3 h. Then water (10 mL) was added and the mixture was
extracted three times with ethyl acetate (10 mL). The combined organic
phases were washed with saturated NaHCO3 (aq.) and brine, dried over
anhydrous Na2SO4, concentrated, and purified by column chromatog­
raphy using petroleum ether/ethyl acetate (1/2) as eluent to afford 33 as
a pale-yellow solid (Yield: 71 %). Mp＞250 ◦ C. 1H NMR (500 MHz,
DMSO‑d6) δ 13.08 (s, 1H), 8.53 (d, J = 48.9 Hz, 2H), 7.95 (m, 6H), 7.52
D. Ying et al.
(s, 2H), 3.34–3.26 (m, 2H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz,
DMSO‑d6) δ 168.21, 136.46, 133.35, 131.88, 129.16, 128.98, 127.90,
127.05, 125.71, 124.93, 34.52, 15.34. HRMS (ESI) m/z calcd for
C18H16ClN3O2 [M+H]+: 342.1004. Found: 342.1000.
4.1.3.33. 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl)-N-(3-(dime­
thylamino)propyl)benza-mide (34). In a 25 mL round bottom flask, 4-(3-
(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl) benzoic acid hydrochlo­
ride (32) (176 mg, 0.5 mmol) and HATU (285 mg, 0.75 mmol) was
dissolved in dried DMF (3 mL) for 10 min. Then 3-dimethylaminopro­
pylamine (76 μL, 0.6 mmol) and triethylamine (83 μL, 0.6 mmol)
were added after cooling to 0 ◦ C. The reaction was warmed to room
temperature and stirred for 3 h. Then water (10 mL) was added and the
mixture was extracted three times with ethyl acetate (10 mL). The
combined organic phases were washed with saturated NaHCO3 (aq.) and
brine, dried over anhydrous Na2SO4, concentrated, and purified by
column chromatography using petroleum ether/ethyl acetate (1/3) as
eluent to afford 34 as a yellow solid (Yield: 67 %). Mp 240–241 ◦ C. 1H
NMR (500 MHz, DMSO‑d6) δ 12.95 (s, 1H), 8.51 (t, J = 5.3 Hz, 1H), 7.98
(dd, J = 22.7, 8.2 Hz, 4H), 7.88 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.3 Hz,
2H), 3.29 (q, J = 6.6 Hz, 2H), 2.26 (t, J = 7.0 Hz, 2H), 2.14 (s, 6H),
1.70–1.63 (m, 2H). 13C NMR (125 MHz, DMSO‑d6) δ 166.24, 133.06,
131.64, 128.93, 127.81, 127.40, 125.18, 57.47, 45.69, 38.27, 27.62.
HRMS (ESI) m/z calcd for C21H23ClN4O2 [M+H]+: 399.1582. Found:
399.1584.
4.1.3.34. 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl)-N-((tetrahy­
dro-2H-pyran-2-yl) oxy) benzamide (35a). In a 25 mL round bottom
flask, 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl) benzoic acid
hydrochloride (32) (176 mg, 0.5 mmol) and HATU (285 mg, 0.75 mmol)
was dissolved in dried DMF (3 mL) for 10 min. Then O-(tetrahydro-2H-
pyran-2-yl) hydroxylamine (70 mg, 0.6 mmol) and triethylamine (83 μL,
0.6 mmol) were added after cooling to 0 ◦ C. The reaction was warmed to
room temperature and stirred for 3 h. Then water (10 mL) was added
and the mixture was extracted three times with ethyl acetate (10 mL).
The combined organic phases were washed with saturated NaHCO3 (aq.)
and brine, dried over anhydrous Na2SO4, concentrated, and purified by
column chromatography using petroleum ether/ethyl acetate (1/1) as
eluent to afford 35a as a white solid (Yield: 59 %), Mp＞250 ◦ C. 1H NMR
(500 MHz, DMSO‑d6) δ 12.95 (s, 1H), 8.01 (d, J = 8.2 Hz, 2H),
7.99–7.94 (m, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H),
5.01 (s, 1H), 4.10–4.00 (m, 1H), 3.53 (m, 2H), 1.73 (m, 3H), 1.56 (m,
3H). 13C NMR (125 MHz, DMSO‑d6) δ 128.86, 127.81, 127.30, 125.01,
101.41, 61.98, 28.59, 25.29, 19.06. HRMS (ESI) m/z calcd for
C21H20ClN3O4 [M+H]+: 414.1215. Found: 414.1208.
4.1.3.35. 4-(3-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl)-N-hydrox­
ybenzamide (35). To a solution of hydrochloric in methanol, 4-(3-(4-
chlorophenyl)-4-hydroxy-1H-pyrazol-5-yl)-N-((tetrahydro-2H-pyran-2-
yl) oxy) benzamide (35a) was added and the reaction was stirred at
room temperature overnight. The mixture was concentrated. The res­
idue was washed with saturated NaHCO3 (aq.) and brine, dried over
anhydrous Na2SO4, concentrated, and purified by column chromatog­
raphy using petroleum ether/ethyl acetate (1/2) as eluent to afford 35 as
a white solid (Yield: 88 %), Mp＞250 ◦ C. 1H NMR (500 MHz, DMSO‑d6)
δ 11.37 (s, 1H), 8.81 (s, 3H), 8.04–7.97 (m, 4H), 7.85 (d, J = 8.5 Hz, 2H),
7.54–7.50 (m, 2H). 13C NMR (125 MHz, DMSO‑d6) δ 164.32, 136.44,
132.03, 131.47, 129.03, 127.70, 127.66, 125.61. HRMS (ESI) m/z calcd
for C16H12ClN3O3 [M+H]+: 330.0640. Found: 330.0645.
4.2. Biology
4.2.1. Cell lines and culture conditions
All cells were cultured in a humidified incubator at 37 ◦ C with 5 %
CO2. 786-O cell was cultured in RPMI-1640 (Gibco) media; HT-1080 and
11
European Journal of Medicinal Chemistry 263 (2024) 115913
H9c2 cells were cultured in DMEM (Gibco) media; BJ cell was cultured
in α-MEM (Gibco) media with 1 % 100 × nonessential amino acid so­
lution, 1 mM sodium pyruvate. All media were supplemented with 10 %
fetal bovine serum (Yeasen) and 1 % penicillin/streptomycin (Corning).
Cells were passaged by dissociation with 0.05 % trypsin - 0.2 % EDTA
(Gibco) every other day.
4.2.2. Cell viability assay
For cellular viability assays, cells were seeded in 96-well plates
(Corning) at 2000–5000 cells per well. Then 24 h after seeding, cells
were treated with compounds at the indicated concentrations for 24 h.
Cellular ATP levels were quantified using CellTiter-Glo (Promega).
Relative viability was normalized to the respective DMSO-treated con­
dition. For data presentation, the mean and standard deviation for the
three biological replicates of each data point in a representative exper­
iment were plotted in Prism 8 (Graphpad) and presented. Sigmoidal
nonlinear regression models were used to compute the regression fit
curves.
4.2.3. Morphological observation
For cell morphology observation, 5000 786-O cells were seeded per
well in 96-well plates. 1 μM RSL3 mixed with different concentration of
compound 25 and Fer-1 (1 μM，0.0625 μM) were added 24 h after
seeding. After one day of incubation, cells were observed by light
microscopy.
4.2.4. DPPH assay
DPPH solution (498 μL, 40.2 μM) in methanol was mixed with the
tested compound (2 μL of compound solution in DMSO). The final re­
action system contained 40 μM DPPH and different concentrations of
target compounds. The zero control (DMSO) and blank control (meth­
anol) were set up. After incubating at room temperature for 1 h, the
absorbance was measured at 517 nm, and the percentage scavenging
was calculated as follows: Scavenging % = [Ab (DMSO)-Ab (sample solu­
tion)]/[Ab (DMSO)-Ab (methanol)] × 100 %
4.2.5. ABTS assay
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid diammonium
salt (ABTS) was dissolved in double-distilled water to 14 mM. ABTS•+
was produced by reacting ABTS stock solution with potassium persulfate
(2.45 mM, final concentration) and allowing the mixture to incubate in
the dark at room temperature for 18 h. The solution of ABTS•+ was
diluted with sodium phosphate buffer (50 mM, pH 7.4) to 100 μM.
Tested compounds (1–100 μM, 50 μL) were added to 150 μL of 100 μM
ABTS•+ solution. The optical absorbance of ABTS•+ at 415 nm was
measured after incubated at 30 ◦ C for 6 min.
4.2.6. Measurement of lipid ROS
HT-1080 cells were cultured in a 6-well plate at the density of 4 ×
105/well overnight, which made the cells adherent. Cells were co-
treated with ferroptosis inducer (0.8 μM RSL3) and either vehicle
(DMSO) or a ferroptosis inhibitor (1 μM Fer-1 or 1 μM compound 25),
then incubated for 8 h. The medium was discarded and the cells were
washed twice with PBS. Treated cells were incubated with BODIPY lipid
probe 581/591C11 (Invitrogen, USA) at a final concentration of 5 μM at
37 ◦ C for 1 h. Following incubations, cells were harvested for FITC
fluorescence imaging and analysis to measure the intracellular lipid ROS
level.
4.2.7. Measurement of intracellular ROS
DCFH-DA, a total ROS assay kit (Solarbio, Beijing), was used to assess
the intracellular ROS generation. Briefly, HT-1080 cells were seeded on
glass coverslips at the density of 4 × 105/well overnight, which made
the cells adherent. Cells were co-treated with ferroptosis inducer (5 μM
Erastin) and either vehicle (DMSO) or a ferroptosis inhibitor (1 μM Fer-1
or 1 μM compound 25), then incubated for 24 h. The medium was
D. Ying et al.
discarded and the cells were washed twice with PBS. Treated cells were
incubated with DCFH-DA probe at a final concentration of 10 μM at
37 ◦ C for 20 min. Then, the medium was removed. Cells were washed
with PBS three times, and finally observed by CLSM.
4.2.8. Iron chelating assay
The chelating interaction of compounds with FeCl3 or FeSO4 was
determined using UV–vis spectrophotometer TU-1810. To investigate
the iron ion binding ability, the tested compounds were dissolved in
MeOH at a concentration of 20 mM, followed by mixing with FeCl3 or
FeSO4 at a concentration of 10 mM. Then, the absorbance value was
measured after dilution with MeOH/H2O.
4.2.9. Statistical analysis
Statistical analyses were performed by Prism 9 (GraphPad Software).
For statistical comparison, we performed two-tailed Student’s t-test to
analyze two groups with normally distributed data and one-way ANOVA
for multiple comparisons with Gaussian distribution. P < 0.05 was
considered significant (represented as × p < 0.05, **p < 0.01, ***p <
0.001, ****p < 0.0001).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This project was supported from the Zhejiang Provincial Natural
Science Foundation of China under Grant No. LY20H300001 (Yongping
Yu).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejmech.2023.115913.
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