Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy

Article abstract of DOI:10.1016/j.bioorg.2021.105159

Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC₅₀ values of 5.96 ± 0.61 μM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.

Full text of DOI:10.1016/j.bioorg.2021.105159

Bioorganic Chemistry 115 (2021) 105159
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of novel inhibitors of human phosphoglycerate dehydrogenase
by activity-directed combinatorial chemical synthesis strategy
,
,
,
Xia Zhoua 1, Yuping Tana 1, Kun Goua 1, Lei Taoa, Yuan Luoa, Yue Zhoua, Zeping Zuoa,
Qingxiang Suna *, Youfu Luoa *, Yinglan Zhaoa
,
,
,b,*
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, and Collaborative Innovation Center for
Biotherapy, Chengdu 610041, China
b West China School of Pharmacy, Sichuan University, Chengdu 610041, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a
crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first,
rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we
identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based
on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this
hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values
Serine synthesis
3-phosphoglycerate dehydrogenase
Inhibitor
Combinatorial chemical synthesis
of 5.96 ± 0.61 μM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and
ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle ar-
rest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator
to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.
1. Introduction
Cells can obtain serine not only from exogenous sources through
amino acid transporters [5] but also from a branch of glycolysis through
Metabolic reprogramming is one of the most significant features in
the development of cancers [1]. Cancer support growth and prolifera-
tion by altering their glucose uptake, aerobic glycolysis, and folate-
dependent one-carbon units (1C) metabolism [2]. Otto Warburg
pointed out that the rate of aerobic glycolysis of proliferating cancer
cells was higher than that of normal cells, which mainly relied on
oxidative phosphorylation of glucose-fuel mitochondria to generate
energy [3]. This abnormal metabolic pattern was called “the Warburg
effect,” and the elevated glucose consumption is still considered a
marker of cancer cells. Serine is the predominant source of 1C carried by
tetrahydrofolate, which provides methyl groups to modify lipids, nucleic
acids, and proteins [4]. Serine metabolism is often dysregulated in
cancer, which is essential for the occurrence and development of
cancers.
the classical serine synthesis pathway (SSP) (Fig. 1) [6]. The cells
transfer the glycolysis intermediate 3-phosphoglyceride (3-PG) into the
serine synthesis pathway and eventually converts serine to glycine and
generates a one-carbon unit, which enable cancer cells proliferation by
supporting purine and pyrimidine biosynthesis [7]. Firstly, 3-phospho-
glycerate dehydrogenase (PHGDH) catalyzes glycolytic intermediate 3-
PG transformed into 3-phosphohydroxy pyruvate (3-PPyr), with nico-
tinamide adenine dinucleotide (NAD+) as a cofactor. Subsequently, 3-
PPyr is converted to 3-phosphoserine (3-Pser) and alpha-ketoglutarate
(αKG) by phosphoserine aminotransferase (PSAT) with glutamate as
the nitrogen donor, and then to serine by the action of phosphoserine
phosphatase (PSPH). Finally, serine hydroxymethyltransferase (SHMT)
catalyzes serine and tetrahydrofolate (THF) into glycine and 5,10-
methylene-THF.
Abbreviations: SSP, serine synthesis pathway; 1C, one-carbon units; 3-PG, 3-phosphoglyceride; PHGDH, 3-phosphoglycerate dehydrogenase; 3-PPyr, 3-phos-
phohydroxy pyruvate; NAD+, nicotinamide adenine dinucleotide;
αKG, alpha-ketoglutarate; PSAT, phosphoserine aminotransferase; SHMT, serine hydrox-
ymethyltransferase; THF, tetrahydrofolate; D-2-HG, D-2-Hydroxyglutarate; ADS, activity-directed synthesis.
* Corresponding authors at: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, and
Collaborative Innovation Center for Biotherapy, Chengdu 610041, China (Y. Zhao).
E-mail addresses: sunqingxiang@hotmail.com (Q. Sun), luo_youfu@scu.edu.cn (Y. Luo), zhaoyinglan@scu.edu.cn (Y. Zhao).
1
These authors made equal contribution to this work.
https://doi.org/10.1016/j.bioorg.2021.105159
Received 5 April 2021; Received in revised form 4 July 2021; Accepted 6 July 2021
Available online 8 July 2021
0045-2068/© 2021 Published by Elsevier Inc.

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
PHGDH is the first and only rate-limiting enzyme in the SSP, which is
includes design, synthesis, separation, purification, and biological
evaluation, which is time-consuming, uneconomical, and less environ-
mentally friendly [26]. Although it is still widely used drug candidates’
discovery, these shortcomings limit its use on a broader scale. The
emergence of activity-directed synthesis (ADS), a novel discovery
approach in which bioactive molecules and related synthesis appear
simultaneously, has expanded the diversity of small molecule screening
collections [27]. The integration of synthesis and assay significantly
reduce material requirements and achieve rapid analysis. Combinatorial
chemistry strategy can combine different building blocks with a one-pot
reaction to obtain structurally diverse libraries of compounds [28].
Herein we report an activity-directed combinatorial chemical synthesis
strategy to obtain novel PHGDH inhibitors as promising anticancer drug
candidates.
considered one of the ways for cancer cells proliferation and stress
resistance [8]. Focal amplifications of the gene encoding PHGDH have
been identified, and the mRNA and protein levels of PHGDH were
significantly increased, particularly in breast cancers and melanomas
[9–11]. Notably, PHGDH overexpressed in 40% of melanoma and 70%
of estrogen receptor-negative and triple-negative breast cancer [12]. In
breast cancer, melanoma, glioma, colon cancer, cervical cancer,
pancreatic cancer, non-small cell lung cancer (NSCLCs), and ovarian
cancer, the amplification and overexpression of PHGDH were associated
with poor prognosis and short overall survival rate [13–17]. In most
cases, increased PHGDH levels promote cancer cell proliferation, which
is inhibited when PHGDH is knocked out or mutated at specific sites.
PHGDH has been further demonstrated to use
α-ketoglutarate as a
substrate to produce cancer metabolite D-2-Hydroxyglutarate (D-2-HG),
which is essential for the proliferation, invasion, and tumorigenicity of
cancer cells [18]. Together these findings suggest that PHGDH is an
attractive antitumor target, and the development of PHGDH inhibitors
will open a new way for cancer treatment.
2. Results and discussion
2.1. Identification of H-G6 as a PHGDH inhibitor.
In recent years, several compounds have been reported to inhibit
PHGDH, which can be divided into two categories (Fig. 2). The first
category is allosteric inhibitors, such as CBR-5884 (1) [19], NCT-503 (2)
To identify small molecules inhibiting human PHGDH, we screened
an in-house library of 2680 compounds by using an in vitro enzymatic
activity assay (Fig. 3A). Initially, compounds were tested at a single
[20], α-ketothioamide derivatives (3) [21], and PKUMDL-WQ-2201 (4)
concentration of 5 μM to inhibit PHGDH, and we selected 70 compounds
[22], which display micromolar potency and do not depend on the level
of intracellular cofactors. The second category is orthosteric inhibitors,
all of which are indole derivatives. Astra Zeneca discovered a series of
indole-2-carboxamide compounds (1) that binds the NAD+ pocket of
PHGDH and inhibit its activity with low nanomolar affinities [23]. Due
to structural similarity, compound (6) of RAZE is likely to share the same
binding mode as compound (1) [24]. Boehringer Ingelheim [25]
discovered another indoleamine compound, BI-4916 (8), a prodrug of
the cofactor nicotinamide adenine dinucleotide (NADH/NAD+)-
competitive PHGDH inhibitor BI-4924 (7), which combines with NAD+
competitive pocket and inhibits PHGDH activity at nanomolar concen-
tration. Nevertheless, the chemical structure types of PHGDH inhibitors
are quite limited, and most of them have moderate cell activity. There is
a pressing need to develop inhibitors with new scaffold structure and
antitumor activity.
with an inhibition ratio of over 80% for diaphrorase counter-screening
analysis to exclude false-positive results. 48 compounds were obtained
and we verified compounds’ activity and got the hit compound H-G6
which exhibited a significant inhibition against PHGDH activity with the
inhibition ratio>50% at 1 μM, and IC50 value of 4.57 ± 0.29 μM.
2.2. Optimization of hit compound H-G6 through the activity-directed
combinatorial chemical synthesis strategy
To prepare a series of H-G6 analogs rapidly and economically, we
used the activity-directed combinatorial chemical synthesis strategy to
advance structure optimization (Fig. 4A). We selected 10 types I sub-
stituent building blocks and 10 types II internal substituent building
blocks for the micro-one-pot aldol condensation experiment (Fig. 4C) by
purchasing or synthesizing (The chemical synthesis method of the in-
termediates is shown in Scheme 1). All reactions were carried out in PCR
The traditional modification iteration of the hit compound (s)
Fig. 1. The serine synthesis pathway and downstream anabolic reactions of serine.
2

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Fig. 2. The chemical structures of reported PHGDH inhibitors.
Fig. 3. Identification of a novel PHGDH inhibitor H-G6 from the chemical library screen. (A) The workflow for the identification of novel PHGDH inhibitors in an in-
house library. (B) A random screen of 2680 compounds identified compounds with the inhibition ratio>80% at 5
hibition curve of the hit compound H-G6.
μM. (C) Chemical structure and enzymatic in-
tubes at 50
× 60) crude products were obtained by micro-one-pot synthesis, and the
PHGDH enzymatic activity at a single-point concentration of 5 M was
μ
L scale and heated by qPCR instrument. Eventually, 480 (8
with meta or para electron-donating groups and hydrophobic groups at
moiety II exhibited strong inhibition potency.
μ
determined. The building block had no inhibitory effect on PHGDH at
this concentration. Finally, we inferred the preliminary structure ꢀ ac-
tivity relationship (SAR) from 480 crude products obtained from the
microscale combination screening. The compounds with para-
substituted benzene ring in moiety I exhibited better inhibitory activity,
and –COOCH3 > halogens > no substitutions > –COOH. Compounds
2.3. Biological activity assay of the optimized compounds
The products with high PHGDH inhibitory activity were screened
from 480 one-pot reactions for routine synthesis, structure confirmation,
and activity determination. Firstly, we repeated the micro-synthesis by
the traditional method and then separated and purified the reaction
3

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Fig. 4. Structural optimization of H-G6 through the activity-directed combinatorial chemical synthesis strategy. (A) The workflow for structural optimization by
combinatorial chemical synthesis. (B, C) Micro-one-pot aldol condensation and building blocks for moieties I and II for the combinatorial chemical.
4

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Scheme 1. Synthetic route for building blocks. Reagents and conditions: (a) NaNO2, HCl, SnCl2 ▪HCl, ꢀ 10 ℃; (b) CH3CO2H, 100 ℃, overnight; (c) (pph)3pdCl2,
CH3COOK, dioxane : H2O = 3 : 1, N2, 90 ℃, overnight; (d) DMSO (micro-one-pot synthesis) or EtOH (traditional synthesis), CH3CO2H, 55 ℃, 3 h.
liquid to obtain two main products. One is the aldol reaction adduct;
other is the bis-pyrazolone derivative (Scheme 2). As we used activity-
directed synthesis, the reaction products may be PHGDH inhibitors.
The enzymatic activity assay revealed that all these compounds dis-
played potent inhibitory activity against PHGDH. The enzymatic
inhibitory activities of monomer adducts’ and their corresponding bis-
pyrazolone derivatives were in the same order of magnitude. In order
to exclude non-specific binding, we selected compounds H-G6, b36, and
bis-b36 for false positive and non-specific binding exclusion experi-
ments, because they have three representative structures. The result
showed that all the compounds did not inhibit the activity of diaphorase,
and the addition of Triton X-100 did not weaken the inhibition activity
of the compounds against PHGDH (Figure S1), confirming that these
compounds are inhibitors of PHGDH. However, bis-pyrazolone de-
rivatives had higher molecular weight, lower ligand efficiency and poor
water solubility. Since all of the optimized compounds exhibited
considerable PHGDH inhibition activity in extracellular enzyme assay,
we evaluated their antiproliferative activity against a panel of human
cancer cell lines including human breast cancer cells MDA-MB-468 and
Hs578T, and human ovarian cancer cells A2780 and SKOV3 after 96 h
treatment. The results showed that compounds H-G6, b16, b36, and bis-
ovarian cells (A2780 and SKOV3) with IC50 value of 2.69–18.76 μM than
that of breast cancer cells,suggesting the possible important role of
PHGDH in the pathogenesis of ovarian cancer (Table 2). The difference
sensitive of cancer cells to compounds may be the difference of gene
expression in cancer cells. Thus, the mechanism by which ovarian cancer
cells sensitive to PHGDH inhibitor is worthy further study. In addition,
we measured the toxicity of these compounds on normal cells and the
results showed that high concentration (40 μM) of compounds b36, bis-
b36 and H-G6 didn’t significantly inhibit the proliferation of MCF-10A,
suggesting that these compounds are nontoxic to normal cell and spe-
cifically inhibit the cancer cells proliferation. In summary, these com-
pounds are more selective for tumor cells than normal cells.
Absorption, distribution, metabolism, elimination, and toxicity
(ADMET) are very important in current drug discovery and develop-
ment. In silico assessment of ADMET predictions of these active com-
¨
pounds were carried out using Qikprop (Schrodinger Small-Molecule
Drug Discovery Suite) (Table S1). Compound b36 exhibited more “drug-
like” e“ properties and was selected for further study.
2.5. Formate supplementation rescue experiment
b36 displayed dominant antiproliferative activity at 20
μM against
cancer cells (Table 1).
Formic acid (FA) is a crucial one-carbon metabolite of serine, which
generates 10-formyl tetrahydrofuran in cells and participates in the
synthesis of purine nucleotides [29]. As the inhibition of PHGDH ac-
tivity reduced the serine and pyrimidine nucleotide synthesis vital for
cancer cell proliferation, supplementation of exogenous FA in cancer
cells could rescue the proliferation inhibitory effects of PHGDH in-
hibitors. MDA-MB-468 cells were treated with compound b36 with or
without formate supplementation, the result indicated that the addition
of 1 mM formate recovered the proliferation inhibition of MDA-MB-468
induced by b36, especially 5 days after b36 treatment (Fig. 5).
2.4. Cell proliferation inhibitory assay of compounds
As compounds H-G6, b16, b36, and bis-b36 displayed dominant
antiproliferative activity at 20
μM against cancer cells, we further
investigated their antiproliferative activities in various concentrations
after 96 h exposure using the MTT assay. The results showed that they
inhibited the breast cancer cells proliferation with IC50 value of
12.97–33 μM. Notably, compounds exhibited stronger inhibition against
5

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Scheme 2. Traditional methods synthesized the optimized compounds.
2.6. Cell metabolism studies
because serine synthesis and metabolism can provide a carbon unit for
the synthesis of purine and pyrimidine. It has been reported that
siPHGDH can increase the accumulation of the DNA damage marker
histone H2AX on Ser139 (generally referred to as γH2AX) [32]. We
hypothesize that the same effect can be achieved by inhibiting PHGDH
with our compounds. Western blot experiment showed that b36 mark-
edly increased the level of γH2AX in MDA-MB-468 cells in a dose-
dependent manner (Fig. 8A). We then found that the accumulation of
γH2AX foci was also significantly increased in MDA-MB-468 cells after
b36 treatment (Fig. 8B and 8C). These data indicated that the inhibition
of PHGDH in tumor cells accumulate DNA strand breaks.
As b36 inhibited PHGDH activity, we then determined whether b36
inhibited de novo biosynthesis of serine synthesis in cancer cells. The
level of serine and its downstream glycine in MDA-MB-468 cells after
b36 treatment was determined by using a targeted reversed-phase
gradient UPLC-MS/MS assay method for quantitative/monitoring of
intracellular amino acids established by the precolumn derivation of 6-
aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQTag Ultra)
[30]. The results showed that b36 significantly decreased the serine and
glycine level in a concentration-dependent manner, suggesting that
PHGDH inhibition by b36 inhibited the de novo synthesis of serine
(Fig. 6).
2.8. Cell cycle arrest analysis
A series of biological processes such as cell proliferation, growth, and
differentiation are regulated by the cell cycle [33]. As the inhibition of
PHGDH activity suppressed the formation of purine nucleotides required
for DNA synthesis, we speculated that b36 treatment may resulted in cell
cycle arrest. Thus, we investigated the effect of b36 on the cell cycle of
2.7. DNA damage studies
Inhibition of serine synthesis reduces the synthesis of purine nucle-
otides, which are the main components of DNA. The DNA synthesis
ability of MDA-MB-468 cells treated with b36 was determined by 5-
Ethynyl-20-deoxyuridine (EdU) incorporation. EdU is a thymidine
nucleoside analog that can replace thymidine in cell proliferation. Based
on the specific reaction of EdU with fluorescent dyes and azido biotin,
the synthesis of DNA in cells is detected, which reflect the inhibition of
cell proliferation [31]. The results showed that b36 significantly
inhibited the production of intracellular DNA in MDA-MB-468 cell in a
concentration-dependent manner, suggesting DNA synthesis was
MDA-MB-468 by flow cytometry. The results showed that b36 at 40 μM
induced cell cycle arrest of the MDA-MB-468 cell at S-phase, which is
consistent with DNA synthesis inhibition (Fig. 9).
2.9. Analysis of compounds inhibition modality
We sought to deeply characterize the mechanism by which com-
pounds inhibit PHGDH, and look for the pockets where they bind to the
enzyme. We selected compounds H-G6, b36, and bis-b36 to perform an
enzyme kinetics study to explore the mode of inhibition, because they
have three representative structures [33]. Competition studies showed
damaged (Fig. 7). For example, 20 μM of b36 treatment resulted in 70%
inhibition of DNA production, which is consistent with the PHGDH in-
hibition effect of b36.
We have confirmed that compound b36 can inhibit DNA synthesis,
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X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
MDA-MB-468, Hs578T, A2780, and SKOV3 cell lines, but not normal
cells (MCF-10A). Mechanistically, compound b36 is an allosteric in-
hibitor of PHGDH, reduces intracellular serine synthesis, blocks DNA
replication, leads to S phase arrest, activates DNA damage accumula-
tion, and finally causes cell death. Collectively, our data demonstrated
that b36 is a promising PHGDH inhibitor and might provide a rationale
for the development of PHGDH inhibitors for cancer therapy.
Table 1
The PHGDH inhibitory activity and preliminary cancer cell proliferation
inhibitory activity of the active compounds.
Compound
Inhibitory
activity
Antiproliferative activity
(Inhibition rate at 20
μ
M) b
(IC50
,
μ
M) a
PHGDH
MDA-MB-
468
Hs578T
A2780
SKOV3
c
4. Experimental section
H-G6
b16
4.57 ± 0.29
6.39 ± 0.04
4.69 ± 0.12
3.41 ± 0.18
5.38 ± 0.51
3.24 ± 0.05
10.01 ± 0.34
4.36 ± 0.34
5.96 ± 0.61
7.04 ± 0.45
4.52 ± 0.53
5.95 ± 0.56
3.12 ± 0.30
13.29 ± 1.99
8.9 ± 0.17
88%
97%
40%
11%
39%
29%
20%
5%
50%
12%
6%
10%
13%
9%
5%
–
75%
85%
–
9%
6%
9%
–
4%
–
10%
94%
68%
–
-
80%
31%
6%
3%
8%
5%
2%
74%
29%
2%
7%
5%
–
b20
4.1. General procedures
bis-b20
b28
3%
4%
9%
4%
89%
66%
–
All reagents and solvents were purchased from commercial sources
and were used without further purification. All the chemical reactions
were monitored by thin-layer chromatography (TLC) on precoated silica
gel 60 F254 plates (0.25 mm, Qingdao Haiyang Inc.), and components
were visualized by ultraviolet light (254 nm). All the NMR spectra were
recorded on Bruker Avance (Varian Unity Inova) spectrometer in CDCl3
or DMSO‑d6 with TMS as internal standard. 1H NMR and 13C NMR
spectra were recorded respectively at 400 MHz and 100 MHz, analyzed
by MestReNova Software. Coupling constants were reported as Hz with
multiplicity denoted as s (singlet), d (doublet), t (triplet), q(quartet), m
(multiplet), and br (broad). High-resolution mass spectrometry (HRMS)
was acquired on a Waters Q-TOF Premier mass spectrometer.
bis-b28
b35
bis-b35
b36
49%
68%
–
bis-b36
b52
b56
19%
9%
5%
–
bis-b56
c37
26%
19%
–
–
bis-c37
e31
–
–
–
20.09 ± 1.10
12.84 ± 2.21
6.27 ± 0.34
4.58 ± 0.17
4.58 ± 0.35
4.77 ± 0.04
9.33 ± 0.37
6.12 ± 1.01
3.95 ± 0.29
4.79 ± 0.71
–
bis-e31
e38
5%
8%
11%
–
17%
12%
5%
–
38%
16%
15%
7%
30%
2%
29%
–
bis-e38
e42
–
–
bis-e42
e43
–
4.2. Compound library
13%
21%
–
–
13%
–
29%
28%
0%
3%
bis-e43
e59
9%
12%
–
–
1%
The in-house small molecular library used for the screening assay
consists of 2636 commercially available pure compounds with structural
diversity (Source: MedChem Express). They are stored at concentrations
of 10 mM in 100% dimethyl sulfoxide (DMSO) with the purities of >
95%. The hit H-G6 from the screening assay were purchased from a local
agent, confirmed by 1H NMR and HRMS before use.
bis-e59
–
a
Each compound was tested in triplicate; the data are presented as the mean
± SD.
b
c
Cell proliferation inhibition at a single concentration.
Compounds had no proliferation inhibition.
4.3. Implementation of combinatorial chemistry one-pot reaction
Table 2
IC50 a value (
μM) of compounds against cancer and normal cell lines.
All reactions were carried out in PCR tubes at 50
μL scale. The re-
Compound
MDA-MB-
468
Hs578T
A2780
SKOV3
MCF-10A
actants were dissolved in DMSO to a final concentration of 40 mM (1.0
equiv), and 0.3 μL glacial acetic acid (2.0 equiv) was added as a catalyst.
H-G6
b16
27.24 ±
0.84
33.55 ±
2.93
2.69 ±
3.22
4.14 ±
3.92
>40.00
The reaction mixtures were heated in a real-time PCR detection systems
(Bio-Rad, CFX96) at 55 ℃ for 3 h,followed by cooing to room
temperature.
12.97 ±
1.37
> 40.00
5.60 ±
0.28
6.38 ±
0.90
38.95 ±
0.91
b36
31.13 ±
0.53
24.00 ±
0.39
8.99 ±
0.19
18.76 ±
5.10
>40.00
4.4. Chemistry
bis-b36
31.59 ±
0.45
22.42 ±
0.58
9.9 ±
2.01
8.462 ±
3.32
>40.00
4.4.1. General procedure for the synthesis of building blocks for moieties I (I
b-h)
a
Each compound was tested in triplicate; the data are presented as the mean
± SD.
Aniline (1.0 equiv) was dissolved in conc. HCl and cooled to ꢀ 10 ℃,
then an aqueous sodium nitrite solution (1.5 equiv) was added and
stirred cold for 40 min. The newly prepared tin dichloride (3.0 equiv)
hydrochloric acid solution was added to the reaction solution slowly and
stired at ꢀ 10 ℃ for 2–3 h [34]. At the end of the reaction, the precipitate
is formed and filtered, washed with water and ethyl acetate, then dried
in a vacuum, and used for the next step without further purification.
The phenylhydrazine (1.0 equiv) obtained in the previous step is
dissolved in an appropriate amount of glacial acetic acid and added
ethyl acetoacetate (1.5 equiv) in one portion. The mixture was stirred
and heated to reflux for 12 h [34]. The solution was cooled to room
temperature and a precipitate was formed, followed by washing with a
small amount of organic solvent and dried in a vaccum. The residue was
purified by chromatography on Si gel column with DCM-MeOH.
that all the compounds inhibited PHGDH in a non-competitive mode
with respect to both 3-PG and NAD+, as evidenced by dose-dependent
decrease in Vmax and little effects on Km for the substrate 3-PG and
the cofactor NAD (Fig. 10), suggesting that compounds might be binding
to an allosteric pocket.
3. Conclusion
In summary, we identified a potential PHGDH inhibitor H-G6 from
the screening of an in-house small molecule library by in vitro enzymatic
activity assay. To avoid the disadvantages of traditional synthesis, which
is time-consuming and costly, we used the activity-directed combina-
torial chemical synthesis strategy to optimize the lead compound. We
designed and synthesized a series of pyrazolone derivatives as novel
4.4.2. General procedure for the synthesis of building blocks for moieties II
(II 5 ~ 12)
PHGDH inhibitors with IC50 values ranging from 3.12 to 20.09
Compound b36 showed potent activities with the IC50 value of 5.96 ±
0.61 M and exhibited significant antiproliferative activities against
μM.
A solution of the corresponding Aryl halides (1.0 equiv) and 5-
formyl-2-furanboronic acid (1.5 mmol) in 1,4- dioxane: H2O = 3 : 1
μ
7

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Fig. 5. Formate rescues the proliferation inhibition of MDA-MB-468 induced by b36. MDA-MB-468 cells were treated with b36 and formate (1 mM), followed by
MTT assay. Statistical analysis of MDA-MB-468 cells proliferation after 5 days of treatment. The data are shown as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 6. Compound b36 inhibits serine synthesis in MDA-MB-468 cells. (A) The level of seine in MDA-MB-468 cells after various concentration of b36 treatment. (B)
The level of glycine in MDA-MB-468 cells after various concentration of b36 treatment. *P < 0.05.
were prepared with bis (triphenylphosphine) palladium (II) chloride
(0.1 equiv and potassium acetate (3.0 equiv). The mixture was stirred
under nitrogen and heated to 90 ℃ for 12 h [34]. Then the solution was
diluted with H2O and extracted with DCM for 3 times. The combined
organic phase was washed with brine and dried over anhydrous Na2SO4,
filtered and concentrated, then purified by chromatography on Si gel
column with PE-EA.
acid (2.0 equiv) in ethanol were stirred at 55 ◦C for 3–5 h. After cooling
to room temperature, the products were precipitated, filtered, and
collected, then purified by column chromatography using PE-EA or
MeOH-DCM as an eluent.
4.4.4. 5-Methyl-4-((5-nitrothiophen-2-yl)methylene)-2-phenyl-2,4-
dihydro-3H-pyrazol-3-one (b16)
I-e was synthesized from I-d by reflux esterification in anhydrous
methanol and catalytic concentrated sulfuric acid. After heating to
reflux with stirring for 10 h, the reaction mixture was cooled to room
temperature and concentrated under reduced pressure. Finally, the
crude products were purified by chromatography on Si gel column with
DCM-MeOH.
Solid, yield 76%; 1H NMR (400 MHz, DMSO) δ 8.26 (s, 1H), 8.24 (d,
J = 4.4 Hz, 1H), 8.09 (d, J = 4.4 Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.47
(t, J = 8.0 Hz, 2H), 7.24 (t, J = 7.4 Hz, 1H), 2.36 (s, 3H); 13C NMR (101
MHz, DMSO) δ 162.02, 156.80, 151.29, 141.02, 138.18, 137.53, 129.47,
129.34, 126.35, 125.42, 118.65, 13.22; HRMS [ESI+]: calcd for
313.0521, C15H11N3O3S, [M + H]+, found, 314.0598. Purity ≥ 95.0%.
4.4.3. General procedure for the synthesis of building blocks for the
optimized compounds
4.4.5. 5-Methyl-4-(4-nitrobenzylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-
3-one (b20)
Building blocks for moieties I (1.0 equiv), II (1.2 equiv), and acetic
Solid, yield 21%; 1H NMR (400 MHz, DMSO) δ 8.70 (d, J = 8.0 Hz,
8

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Fig. 7. Compound b36 inhibits DNA syn-
thesis in MDA-MB-468 cells. (A) MDA-MB-
468 cells were treated with various concen-
trations of b36 (5–20
μM) for 48 h and
labeled by EdU (red fluorescence) and
Hoechst (blue fluorescence), followed by
ArrayScan VTI HCS reader detection. (B) The
statistical results of DNA damage assay in
MDA-MB-468 cells. The data are shown as
means ± SD. **P < 0.01; ****P < 0.0001.
(For interpretation of the references to colour
in this figure legend, the reader is referred to
the web version of this article.)
Fig. 8. Compound b36 induces DNA damage in MDA-MB-468 cells. (A) Immunoblotting of γH2AX after MDA-MB-468 cells were treated with compound b36 for 48
h. GAPDH as loading control. (B, C) Representative images (B) and quantification (C) of γ-H2AX foci (red) in MDA-MB-468 cells treated with compound b36 for 48 h.
Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, blue). The data are shown as means ± SD. ***P < 0.001; ****P < 0.0001. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
2H), 8.37 (d, J = 8.0 Hz, 2H), 7.97 (s, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.45
(t, J = 7.4 Hz, 2H), 7.22 (s, 1H), 2.37 (s, 3H); 13C NMR (101 MHz,
DMSO) δ 161.48, 152.15, 149.37, 145.31, 138.91, 138.32, 134.56,
129.96, 129.36, 125.33, 123.84, 118.87, 13.51; HRMS [ESI + ]: calcd
for 307.0957, C17H13N3O3, [M + H]+, found, 308.1031. Purity ≥ 95.0%.
4.4.6. 4,4′-((4-Nitrophenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-
5-ol) (bis-b20)
Solid, yield 53%; 1H NMR (400 MHz, DMSO) δ 8.13 (d, J = 8.8 Hz,
2H), 7.79 (d, J = 7.8 Hz, 4H), 7.55 (d, J = 8.8 Hz, 2H), 7.38 (t, J = 7.9
Hz, 4H), 7.16 (t, J = 7.4 Hz, 2H), 4.96 (s, 1H), 2.27 (s, 6H); 13C NMR
9

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
Fig. 9. Compound b36 induces MDA-MB-468 cells arrest at S-phase. Cell cycle analysis of MDA-MB-468 cells treated with different concentrations of b36 (5–40
for 48 h. The statistical results of cell cycle assay in MDA-MB-468 cells. The data are shown as means ± SD.
μM)
Fig. 10. Compounds exhibits non-competitive inhibition with respect to both 3-PG and NAD+. Data are average of three experiments and error bars represent
standard deviations.
(101 MHz, DMSO) δ 157.57, 151.73, 146.61, 146.27, 138.36, 129.27,
129.06, 125.66, 123.76, 120.80, 103.80, 40.65, 40.44, 40.23, 40.02,
39.81, 39.60, 39.40, 34.03, 12.35; HRMS [ESI+]: calcd for 481.1750,
4.4.7. 5-Methyl-4-(3-nitrobenzylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-
3-one (b28)
Solid, yield 27%; 1H NMR (400 MHz, DMSO) δ 9.60 (s, 1H), 8.84 (d,
J = 7.8 Hz, 1H), 8.43 (dd, J = 7.9, 1.8 Hz, 1H), 8.02 (s, 1H), 7.93 – 7.82
C
27H23N5O4, [M + H]+, found,482.1825. Purity ≥ 95.0%.
(m, 3H), 7.49 – 7.41 (m, 2H), 7.22 (t, J = 7.4 Hz, 1H), 2.37 (s, 3H); 13
C
NMR (101 MHz, DMSO) δ 161.69, 152.17, 148.24, 145.80, 139.80,
138.35, 134.51, 130.56, 129.35, 129.24, 127.81, 127.28, 125.32,
10

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
118.92, 13.51; HRMS [ESI + ]: calcd for 307.0957, C17H13N3O3, [M +
H]+, found, 308.1032. Purity ≥ 95.0%.
4.4.14. 5-Methyl-2-phenyl-4-(quinolin-3-ylmethylene)-2,4-dihydro-3H-
pyrazol-3-one (b56)
Solid, yield 17%, 1H NMR (400 MHz, DMSO) δ 9.75 (s, 1H), 9.67 (d,
J = 2.0 Hz, 1H), 8.13–8.08 (m, 3H), 7.75–7.71 (m, 3H), 7.73 (t, J = 7.6
4.4.8. 4,4′-((3-Nitrophenyl)methylene)bis(3-methyl-1-phenyl-1H-pyrazol-
5-ol) (bis-b28)
Hz, 1H), 7.51 – 7.42 (m, 2H), 7.24 (d, J = 7.4 Hz, 1H), 2.41 (s, 3H); 13
C
Solid, yield 48%; 1H NMR (400 MHz, DMSO) δ 8.13 (s, 1H), 8.08 (d,
J = 8.1 Hz, 1H), 7.75 (d, J = 7.9 Hz, 5H), 7.60 (t, J = 7.9 Hz, 1H), 7.44 (t,
J = 7.9 Hz, 4H), 7.24 (t, J = 7.4 Hz, 2H), 5.12 (s, 1H), 2.35 (s, 6H).1H
NMR (400 MHz, DMSO) δ 8.13 – 8.00 (m, 2H), 7.73 (t, J = 7.9 Hz, 5H),
7.60 (t, J = 7.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 4H), 7.25 (d, J = 7.4 Hz, 2H),
5.11 (s, 1H), 2.34 (s, 6H); 13C NMR (101 MHz, DMSO) δ 148.23, 146.70,
145.60, 138.05, 134.83, 130.10, 129.36, 125.90, 122.24, 121.56,
120.93, 104.15, 33.54, 12.24.; HRMS [ESI + ]: calcd for 481.1750,
NMR (101 MHz, DMSO) δ 192.76, 161.97, 153.92, 152.08, 150.24,
149.26, 148.84, 146.43, 144.88, 141.49, 140.90, 138.48, 133.18,
132.85, 130.41, 130.20, 129.49, 129.36, 129.33, 129.14, 129.05,
128.90, 128.76, 128.40, 128.31, 128.10, 127.93, 127.25, 127.14,
126.95, 126.73, 125.26, 120.57, 118.93, 13.53, 12.67; HRMS [ESI + ]:
calcd for 313.1215, C20H15N3O, [M + H]+, found, 312.1292. Purity ≥
95.0%.
C
27H23N5O4, [M + H]+, found, 482.1821. Purity ≥ 95.0%.
4.4.15. 4,4′-(quinolin-3-ylmethylene)bis(3-methyl-1-phenyl-1H-pyrazol-5-
ol) (bis-b56)
4.4.9. 4-(4-Bromobenzylidene)-5-methyl-2-phenyl-2,4-dihydro-3H-
pyrazol-3-one (b35)
Solid, yield 36%, 1H NMR (400 MHz, DMSO) δ 8.82 (d, J = 2.1 Hz,
1H), 8.13 (s, 1H), 7.94 (dd, J = 16.1, 8.2 Hz, 2H), 7.79 (d, J = 8.2 Hz,
4H), 7.67 (t, J = 7.6 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 8.0 Hz,
4H), 7.18 (t, J = 7.4 Hz, 2H), 5.13 (s, 1H), 2.33 (s, 6H); 13C NMR (101
MHz, DMSO) δ 157.81, 152.14, 146.50, 146.40, 139.26, 137.21, 132.79,
129.10, 129.02, 128.90, 128.40, 127.95, 126.94, 124.98, 120.47,
103.20, 32.36, 12.76;HRMS [ESI + ]: calcd for 487.2008, C30H25N5O2,
[M + H]+, found, 488.2079. Purity ≥ 95.0%.
Solid, yield 28%; 1H NMR (400 MHz, DMSO) δ 8.53 (d, J = 8.6 Hz,
2H), 7.89 (d, J = 7.7 Hz, 2H), 7.86 – 7.75 (m, 3H), 7.47 – 7.41 (m, 2H),
7.21 (t, J = 7.4 Hz, 1H), 2.35 (s, 3H); 13C NMR (101 MHz, DMSO) δ
161.88, 152.25, 147.26, 138.52, 135.81, 132.51, 132.23, 129.32,
127.65, 125.18, 120.57, 118.87, 13.55; HRMS [ESI+]: calcd for
340.0211, C17H13BrN2O, [M + H]+, found, 341.0290. Purity ≥ 95.0%.
4.4.10. 4,4′-((4-Bromophenyl)methylene)bis(3-methyl-1-phenyl-1H-
pyrazol-5-ol) (bis-b35)
4.4.16. 2-(4-Chlorophenyl)-4-(3-hydroxy-4-methoxybenzylidene)-5-
methyl-2,4-dihydro-3H-pyrazol-3-one (c37)
Solid, yield 56%; 1H NMR (400 MHz, DMSO) δ, 7.75 (d, J = 7.7 Hz,
4H), 7.50 – 7.34 (m, 6H), 7.26 – 7.15 (m, 4H), 4.86 (s, 1H), 2.28 (s, 6H);
13C NMR (101 MHz, DMSO) δ 157.61, 146.64, 142.88, 138.37, 131.32,
130.05, 129.27, 125.61, 120.76, 119.32, 104.34, 33.42, 12.31; HRMS
[ESI+]: calcd for 514.1004, C27H23BrN4O2, [M + H]+, found, 515.1082.
Purity ≥ 95.0%.
Solid, 37%, 1H NMR (400 MHz, DMSO) δ 9.44 (s, 1H), 8.43 (s, 1H),
8.03 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 8.9 Hz, 2H), 7.68 (s, 1H), 7.49 (d, J
= 8.9 Hz, 2H), 7.13 (d, J = 8.5 Hz, 1H), 3.91 (s, 3H), 2.32 (s, 3H). 13
C
NMR (101 MHz, DMSO) δ 162.34, 153.64, 152.72, 149.67, 146.59,
137.73, 129.88, 129.21, 128.52, 126.87, 123.55, 120.44, 120.06,
112.04, 56.29, 13.63.HRMS [ESI
+
]: calcd for 342.0771,
C
18H15ClN2O3, [M + Na]+, found, 365.0667. Purity ≥ 95.0%.
4.4.11. 5-Methyl-4-(naphthalen-1-ylmethylene)-2-phenyl-2,4-dihydro-3H-
pyrazol-3-one (b36)
4.4.17. 1-(4-Chlorophenyl)-3-((1-(4-chlorophenyl)-5-hydroxy-3-methyl-
1H-pyrazol-4-yl)(3-hydroxy-4-methoxyphenyl)methyl)-4-methyl-1H-
pyrazol-5-ol (bis-c37)
Solid, yield 23%; 1H NMR (400 MHz, DMSO) δ 8.65 (d, J = 7.1 Hz, 3/
5H), 8.54 (s, 1H), 8.33 (d, J = 11.5 Hz, 1H), 8.18 – 7.81 (m, 13/5H),
7.70–7.63 (m, 24/5H), 7.50–7.40 (m, 2H), 7.25–7.17 (m, 1H), 2.48 (s,
3H);13C NMR (101 MHz, DMSO) δ 163.19, 161.77, 152.16, 148.96,
145.21, 144.32, 138.64, 138.39, 133.47, 133.33, 131.96, 131.89,
131.34, 131.27, 130.36, 130.07, 129.77, 129.49, 129.37, 129.29,
129.23, 128.52, 128.35, 128.03, 127.32, 126.93, 125.65, 125.45,
125.16, 125.06, 124.96, 124.54, 118.64, 118.39, 40.64, 40.43, 40.22,
40.01, 39.81, 39.60, 39.39, 17.05, 13.62; HRMS [ESI+]: calcd for
312.1263, C21H16N2O, [M + H]+, found, 313.1332. Purity ≥ 95.0%.
Solid, yield 41%, 1H NMR (400 MHz, DMSO) δ 8.68 (s, 1H), 7.86 (d,
J = 8.9 Hz, 4H), 7.42 (d, J = 8.9 Hz, 4H), 6.82 – 6.70 (m, 2H), 6.60 (d, J
= 8.3 Hz, 1H), 4.65 (s, 1H), 3.68 (s, 3H), 2.21 (s, 6H). 13C NMR (101
MHz, DMSO) δ 147.39, 146.65, 146.42, 134.92, 129.90, 129.37, 129.24,
122.16, 118.06, 115.25, 112.61, 100.00, 56.17, 55.39, 32.76, 12.11.
HRMS [ESI + ]: calcd for 550.1175, C28H24Cl2N4O4, [M + Na]+, found,
573.1028. Purity ≥ 95.0%.
4.4.18. Methyl 4-(3-methyl-4-(4-methylbenzylidene)-5-oxo-4,5-dihydro-
1H-pyrazol-1-yl)benzoate (e31)
4.4.12. 4,4′-(Naphthalen-1-ylmethylene)bis(3-methyl-1-phenyl-1H-
pyrazol-5-ol) (bis-b36)
Solid, yield 28%, 1H NMR (400 MHz, DMSO) δ 8.53 (d, J = 8.2 Hz,
2H), 8.12 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 9.0 Hz, 2H), 7.85 (s, 1H), 7.41
(d, J = 8.1 Hz, 2H), 3.86 (s, 3H), 2.43 (s, 3H), 2.37 (s, 3H) ; 13C NMR
(101 MHz, DMSO) δ 166.21, 162.50, 153.38, 149.50, 145.02, 142.42,
134.61, 130.87, 130.76, 129.88, 125.67, 125.33, 117.69, 52.44, 22.02,
13.64.HRMS [ESI + ]: calcd for 334.1317, C20H18N2O3, [M + Na]+,
found, 357.1212. Purity ≥ 95.0%.
Solid, yield 47%; 1H NMR (400 MHz, DMSO) δ 8.05–8.04 (m, 1H),
7.94–7.92 (m, 1H), 7.81– 7.69 (m, 6H), 7.54 – 7.40 (m, 7H), 7.23–0.21
(d, J = 6.7 Hz, 2H), 5.60 (s, 1H), 2.22 (s, 6H); 13C NMR (101 MHz,
DMSO) δ 146.13, 137.89, 137.36, 134.17, 131.25, 129.36, 129.27,
127.51, 126.42, 126.27, 125.68, 124.01, 120.83, 12.50, 0.58.HRMS
[ESI + ]: calcd for 486.2056, C31H26N4O2, [M + H]+, found, 487.2127.
Purity ≥ 95.0%.
4.4.19. Dimethyl 4,4′-((p-tolylmethylene)bis(5-hydroxy-3-methyl-1H-
pyrazole-4,1-diyl))dibenzoate (bis-e31)
4.4.13. 5-Methyl-2-phenyl-4-(2,4,6-tribromo-3-hydroxybenzylidene)-2,4-
dihydro-3H-pyrazol-3-one (b52)
Solid, yield 42%, 1H NMR (400 MHz, DMSO) δ 8.00 (dd, J = 21.8,
8.9 Hz, 8H), 7.16 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 4.76 (s,
1H), 3.85 (d, J = 7.5 Hz, 6H), 2.23 (d, J = 8.4 Hz, 9H); 13C NMR (101
MHz, DMSO) δ 166.33, 158.41, 148.40, 143.20, 134.90, 130.60, 129.02,
127.56, 125.07, 118.99, 104.45, 52.41, 33.80, 21.00, 12.84.HRMS [ESI
+ ]: calcd for 566.2165, C32H30N4O6, [M + Na]+, found, 589.2053.
Purity ≥ 95.0%.
Solid, yield 73%; 1H NMR (400 MHz, DMSO) δ 7.96 (d, J = 11.1 Hz,
1H), 7.93 – 7.69 (m, 3H), 7.44 (dt, J = 25.3, 8.0 Hz, 2H), 7.21 (dt, J =
20.4, 7.4 Hz, 1H), 2.38 (s, 1H), 1.84 (s, 2H); 13C NMR (101 MHz, DMSO)
δ 160.46, 151.45, 150.57, 144.24, 138.04, 135.37, 134.83, 131.22,
129.42, 125.28, 118.28, 113.19, 112.95, 111.27, 13.37; HRMS [ESI+]:
calcd for 511.8371, C17H11Br3N2O2, [M + H]+, found, 512.3079. Purity
≥ 95.0%.
11

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
4.4.20. Methyl 4-(4-(3-methoxybenzylidene)-3-methyl-5-oxo-4,5-dihydro-
1H-pyrazol-1-yl)benzoate (e38)
10H). 13C NMR (101 MHz, DMSO) δ 166.20, 162.33, 157.79, 153.40,
149.89, 142.38, 134.45, 130.78, 130.06, 126.45, 125.98, 125.37,
121.38, 120.13, 117.70, 52.47, 13.64.HRMS [ESI+]: calcd for 336.1110,
Solid, yield 35%, 1H NMR (400 MHz, DMSO) δ 8.42 (s, 1H), 8.16 –
8.00 (m, 5H), 7.85 (s, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.23 (dd, J = 8.2, 2.5
Hz, 1H), 3.85 (d, J = 2.4 Hz, 6H), 2.36 (s, 3H); 13C NMR (101 MHz,
DMSO) δ 166.20, 162.39, 159.57, 153.40, 149.52, 142.33, 134.54,
130.80, 130.18, 127.37, 126.87, 125.46, 120.34, 118.23, 117.81, 55.82,
52.49, 13.66.HRMS [ESI + ]: calcd for 350.1267, C20H18N2O4, [M +
H]+, found,351.1351. Purity ≥ 95.0%.
C
19H16N2O4, [M + Na]+, found, 359.1004. Purity ≥ 95.0%.
4.4.27. Dimethyl 4,4′-(((3-hydroxyphenyl)methylene)bis(5-hydroxy-3-
methyl-1H-pyrazole-4,1-diyl))dibenzoate (bis-e59)
Solid, yield 47%; 1H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.07 (d,
J = 8.8 Hz, 4H), 7.96 (d, J = 8.9 Hz, 4H), 6.99 (t, J = 7.8 Hz, 1H), 6.73 (s,
1H), 6.68 (d, J = 7.8 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 4.67 (s, 1H), 3.84
(s, 6H), 2.22 (s, 6H); 13C NMR (101 MHz, DMSO) δ 166.37, 158.39,
157.53, 148.46, 146.13, 143.60, 130.56, 129.23, 124.80, 118.86,
118.43, 114.72, 112.95, 103.97, 52.40, 34.18, 13.00. HRMS [ESI+]:
calcd for 568.1958, C31H28N4O7, [M + Na]+, found, 591.1854. Purity ≥
95.0%.
4.4.21. Dimethyl 4,4′-(((3-methoxyphenyl)methylene)bis(5-hydroxy-3-
methyl-1H-pyrazole-4,1-diyl))dibenzoate (bis-e38)
Solid, yield 51%, 1H NMR (400 MHz, DMSO) δ 7.99 (s, 8H), 7.18 (t, J
= 7.9 Hz, 1H), 6.89 – 6.81 (m, 2H), 6.74 (d, J = 7.9 Hz, 1H), 4.83 (s, 1H),
3.84 (s, 6H), 3.68 (s, 3H), 2.28 (s, 6H); 13C NMR (101 MHz, DMSO) δ
166.25, 159.61, 148.46, 142.56, 130.67, 129.59, 125.59, 120.08,
119.34, 114.27, 110.70, 104.66, 55.31, 52.47, 33.92, 12.61.HRMS [ESI
+ ]: calcd for 582.2114, C32H30N4O7, [M + Na]+, found, 605.2004.
Purity ≥ 95.0%.
4.5. PHGDH enzymatic activity assay
PHGDH activity was tested in 96-well plates at room temperature by
measuring NADH production medicated by PHGDH through the
diaphorase-mediated reduction of resazurin. Hydrazine sulfate was
included to prevent product inhibition of PHGDH. PHGDH assay buffer
contained 30 mM Tris 8.0 and 1 mM EDTA. Substrates and enzyme
concentrations were as follows: 200 nM PHGDH 1–314, mixtures (0.1
4.4.22. Methyl 4-(4-(4-fluorobenzylidene)-3-methyl-5-oxo-4,5-dihydro-
1H-pyrazol-1-yl)benzoate (e42)
Solid, yield 29%, 1H NMR (400 MHz, DMSO) δ 8.71 (dd, J = 8.8, 5.8
Hz, 2H), 8.07 (dd, J = 26.6, 8.8 Hz, 4H), 7.91 (s, 1H), 7.44 (t, J = 8.8 Hz,
2H), 3.85 (s, 3H), 2.37 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 166.84,
166.72, 164.28, 162.15, 151.60, 145.99, 142.07, 136.64, 136.55,
130.55, 129.28, 129.25, 126.73, 126.71, 125.88, 117.72, 117.41,
116.24, 116.02, 52.04, 52.02, 13.34; HRMS [ESI + ]: calcd for
338.1067, C19H15FN2O3, [M + H] +, found, 339.1145. Purity ≥ 95.0%.
mM resazurin; 0.001 U/μL diaphorase; 1 mM hydrazine sulfate; 20 μM
NAD+; 0.1 mM 3-PG). Compounds and PHGDH were preincubated for 2
h, and then the mixtures were added. The 96-well plate was shaken
evenly and incubated in dark for 1 h. The resulting fluorescence was
measured using a Synergy H1 Hybrid Multi-Mode Reader (BioTek) at
Ex/Em = 550/590 nm. PHGDH inhibition data were calculated by the
following formula: inhibition rate = 1 ꢀ (Fcompound ꢀ FNo-3PG)/ (FDMSO
ꢀ FNo-3PG). Fcompound was resorufin fluorescence of compound well;
FDMSO was resorufin fluorescence of DMSO well, and FNo-3PG was
resorufin fluorescence of non-substrate well. IC50 values were calculated
by Graph-Pad Prism 7.0 software using XY modeling.
4.4.23. Dimethyl 4,4′-(((4-fluorophenyl)methylene)bis(5-hydr Qoxy-3-
methyl-1H-pyrazole-4,1-diyl))dibenzoate (bis-e42)
Solid, yield 43%, 1H NMR (400 MHz, DMSO) δ 8.02 (d, J = 8.9 Hz,
4H), 7.96 (d, J = 8.9 Hz, 4H), 7.28 (dd, J = 8.4, 5.7 Hz, 2H), 7.09 (t, J =
8.8 Hz, 2H), 4.93 (s, 1H), 3.85 (s, 6H), 2.31 (s, 6H); 13C NMR (101 MHz,
CDCl3) δ 166.72, 162.76, 160.32, 147.98, 141.10, 136.03, 130.51,
128.73, 128.65, 126.71, 119.67, 117.85, 115.29, 115.08, 106.25, 52.23,
33.07, 11.96; HRMS [ESI + ]: calcd for 570.1915, C31H27FN4O6, [M +
Na]+, found, 593.1811. Purity ≥ 95.0%.
For inhibition mode measurements, compounds and enzyme were
pre-incubated for 2 h. Then, we added different concentrations of the
substrate mixed solution, and read the fluorescence value every two
minutes at room temperature for a total of 20 min, finally, used Prism to
fit the initial rate plots.
4.4.24. Methyl 4-(3-methyl-5-oxo-4-(3-(trifluoromethoxy)benzylidene)-
4,5-dihydro-1H-pyrazol-1-yl)benzoate (e43)
4.6. Cell culture
Solid, yield 32%, 1H NMR (400 MHz, DMSO) δ 8.66 (d, J = 7.9 Hz, 7/
10H), 8.14 – 7.80 (m, 63/10H), 7.76–7.71 (m,1H), 7.59–7.54 (m, 2H),
3.85 (s, 3H), 2.36 (s, 21/10H), 2.05 (s, 9/10H); 13C NMR (101 MHz,
DMSO) δ 166.15, 161.68, 152.62, 142.07, 140.06, 134.92, 133.38,
130.79, 130.53, 129.03, 127.54, 125.57, 121.37, 118.97, 117.67,
117.51, 52.47, 13.31; HRMS [ESI+]: calcd for 404.0984,C20H15F3N2O4,
[M + H]+, found, 405.1055. Purity ≥ 95.0%.
The cell lines, including human breast cancer cell line MDA-MB-468
and Hs578T, human ovarian cancer cell line A2780 and SKOV3, and
normal cell line MCF-10A were obtained from American Type Culture
Collection (ATCC; Manassas, VA, USA). The cells were cultured in
Dubelco’s Modified Eagle Medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and grown in a CO2 incubator at 37 ℃ with a
humidified atmosphere of 5% CO2 and 95% air according to ATCC
guidelines.
4.4.25. Dimethyl 4,4′-(((3-(trifluoromethoxy)phenyl)methylene)bis(5-
hydroxy-3-methyl-1H-pyrazole-4,1-diyl))dibenzoate (bis-e43)
Solid, yield 49%; 1H NMR (400 MHz, DMSO) δ 7.98 (dd, J = 27.8,
8.9 Hz, 8H), 7.75 (s, 1H), 7.43 – 7.23 (m, 3H), 5.09 (s, 1H), 3.85 (s, 6H),
2.25 (s, 6H); 13C NMR (101 MHz, DMSO) δ 166.22, 158.59, 147.71,
146.32, 142.35, 134.89, 130.67, 128.52, 127.22, 125.74, 122.05,
120.10, 119.50, 103.64, 100.00, 52.47, 28.95, 12.22; HRMS [ESI+]:
calcd for 636.1832, C32H27F3N4O7, [M + Na]+, found, 659.1731. Purity
≥ 95.0%.
4.7. Cell viability assay
MDA-MB-468, Hs578T, A2780, and SKOV3 cells were seeded (2–10
× 103 cells per well) into 96-well plates. After incubation for 24 h, the
cells were treated with serially diluted compounds for 96 h. Cell viability
was determined by using MTT assay. Briefly, 20 μL of MTT reagent (5
mg/mL) was added to each of the wells and then incubated for 2–4 h.
The absorbance was measured at 570 nm using a Microplate Reader
3550-UV (ThermoFisher Scientific).
4.4.26. Methyl 4-(4-(3-hydroxybenzylidene)-3-methyl-5-oxo-4,5-dihydro-
1H-pyrazol-1-yl)benzoate (e59)
In the formate supplementation experiment, the appropriate media
was added to cells, which included vehicle (DMSO), or b36 and formate
(1 mM). Each experiment was repeated three times. The IC50 value of
compounds was defined as the concentration of the compounds required
to inhibit cell proliferation by 50% relative to the untreated controls, as
Solid, yield 28%;1H NMR (400 MHz, DMSO) δ 9.87 (d, J = 39.4 Hz,
1H), 8.22 – 8.00 (m, 23/5H), 7.92 (t, J = 8.4 Hz, 7/5H), 7.78 (s, 7/10H),
7.38 (t, J = 7.9 Hz, 13/10H), 7.06 (dd, J = 8.1, 2.3 Hz, 7/10H), 6.97 (t, J
= 7.8 Hz, 3/10H), 3.84 (d, J = 10.5 Hz, 3H), 2.36 (s, 23/10H), 2.18 (s, 7/
12

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
determined by non-linear curve fitting of the experimental data and
calculated using GraphPad Prism 7.0 software.
Hoechst 33342 at room temperature for 10 min, followed by observation
under a fluorescence microscope (Thermo Fisher Scientific).
4.8. In silico ADMET prediction
4.11. Cell cycle assay
To explore the pharmacokinetics of the best active compounds, we
MDA-MB-468 cells were seeded (2 × 105 cells per well) in a 6-well
plate and treated with compound b36 or vehicle (DMSO) for 48 h.
After incubation, the cells were harvested and washed with ice-cold PBS.
Cell cycle progression was analyzed using propidine iodide (PI) (50 mg/
L, RNase free) staining. Then stained cells were analyzed using an Attune
NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed
using the FlowJo software.
¨
used the Qikprop program (Schrodinger, 2018–1) to predict their
ADMET properties. All the compounds were prepared from LigPrep and
calculated by their pharmacokinetic properties with Qikprop. Finally,
we obtained physically significant descriptors and pharmaceutically
relevant properties, including octanol/water partitioning coefficient,
aqueous solubility, intestinal wall permeability, plasma protein binding,
and others.
4.9. Serine and glycine level detection
4.12. Western blot
MDA-MB-468 cells (2.0 × 105) were seeded in a 6-well plate and
treated with compound b36 or vehicle (DMSO) for 48 h. Subsequently,
the treated cells were lysed and boiled for 10 min. Proteins were sepa-
rated by SDS-PAGE, transferred to a PVDF membrane, and detected with
relevant antibodies against γH2AXor GAPDH.
4.9.1. Sample preparation and derivatization
MDA-MB-468 cells were seeded (2 × 105 cells per well) in 6-well
plates and allowed to attach for 24 h, then treated with compound
b36 or vehicle (DMSO) for 48 h. The media was removed by aspiration,
and the cells were washed twice with PBS. Metabolome extraction was
performed by adding 500 μL of ice-cold extraction solvent (methanol:
water = 1:1) and was repeatedly freezed-thawed 3 times by liquid ni-
trogen, then centrifuged (20 min, 20000 rpm, 4 ◦C). Finally, the su-
pernatant was collected and dried with a nitrogen blowing instrument
and dissolved in methanol: water (1:1) for derivatization. The sample’
derivatization was carried out strictly according to the instruction of the
AccQTag Ultra reagent pack (waters). The resulting supernatant was
frozen on dry ice and kept at ꢀ 80 ◦C until LC-MS/MS analysis.
4.13. Immunofluorescence and quantification
MDA-MB-468 cells (3 × 105 cells in a six-well plate containing
coverslips) were treated with DMSO or compound b36 for 48 h, and then
cells were fixed in pre-cold 4% paraformaldehyde for 20 min, washed
three times in 1 × PBS, and then extracted with 0.1% Triton X-100 PBS
solution for 5 min, then blocked in PBS containing 1.5% bovine serum
albumin (BSA) for 20 min at room temperature, and then incubated
sequentially with primary antibody against phospho-H2AX (1:1000
dilution) at 4 ◦C overnight. Cells were washed three times with 1 × PBS
and incubated with Alexa Fluor 568-conjugated secondary antibody
(Thermo Fisher Scientific) at room temperature for 1 h. The cells were
4.9.2. UPLC-MS/MS analyses
The chromatographic separation was achieved by injecting 2 μL of
sample on a reversed-phase gradient chromatography column (ACQ-
UITY UPLC BEH amide, 2.1 mm × 100 mm, 1.7 μm, Waters). Mobile
phase A consisted of 0.1% formic acid in water (v/v), and mobile Phase
B was 0.1% formic acid in acetonitrile (v/v). The column compartment
then stained with DAPI (1 μg/mL) and the confocal images were ac-
◦
was maintained at 25 C, and the autosampler sample tray was set to
quired using an Olympus SpinSR10 laser scanning microscope. Cells that
displayed 20 or more discrete bright dots were considered positive for
γ-H2AX foci which were quantified using Fiji. Foci pictures of each in-
dividual experiment were obtained with the same exposure parameters.
Author contributions
4 ◦C. The linear gradient elution was performed at 0.4 mL/min, and the
mobile phase gradient (%B) was as follows: 0 min 3%, 1 min 3%, 2 min
13%, 5.5 min 15%, 6.5 min 60% for 1 min, and finally returning to 3% B
(1.3 min) for re-equilibration. The weak and the intense washes were
90:10 water/acetonitrile (v/v) and 100% 10:90 water/acetonitrile. The
mass spectrometer was via electrospray ionization (ESI) in positive ion
mode and used nitrogen as the dissolvent gas and argon as the collision
gas. All mobile phase was introduced into the generic source, and the
conditions were: capillary voltage, 1.5 kV; source offset, 50 V; des-
olvation temperature, 500 ◦C; source temperature, 150 ◦C, desolvation
gas flow, 1000 L/h; cone gas flow, 150 L/h; nebulizer gas, 7.0 bar;
collision gas, 0.15 mL/min. The raw data were processed by the Tar-
getLynx application package within MassLynx software (Waters), and
the mean smoothed, and peak integration was performed using the
ApexTrak algorithm. Further statistical analysis was performed on the
resulting calculated concentrations using Graph-Pad Prism 7.0.
YLZ, YFL, and QXS designed the experiment scheme. XZ did the
synthesis. The high-throughput screening was done by XZ, TPT, LT, and
YZ; XZ, KG, YPT and YL performed the assays. XZ and KG were
responsible for drafting the manuscript and drawing the figures of this
article. YLZ and YFL edited and revised the manuscript. All authors
reviewed and approved the final version of the manuscript.
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.
4.10. EdU incorporation assay
Acknowledgements
The MDA-MB-468 cells were seeded (6000 per well) into 96-well
plates and left to adhere overnight. The cells were treated with
different concentrations of b36 in DMSO as a vehicle (<0.1%) for 48 h.
This work was supported by the Project of National S&T Major
Project (2018ZX09201018), National Natural Sciences Foundation of
China (81773198) and the National Key R&D Program of China
(2016YFC1303200).
The cells were then incubated with 50 μM EdU for 2 h, followed by
washed twice with PBS and fixed using 4% paraformaldehyde (PFA) for
30 min. Next, the cells were incubated with glycine for 5 min to remove
the residual paraformaldehyde, washed with PBS to neutralize PFA, and
incubated with 0.5% Triton X-100 in PBS. The cells were incubated for
30 min with 1 × Apollo® dye staining reaction in the dark and washed
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105159.
with PBS 2–5 times. At last, the cells were incubated with 5 μg/mL
13

X. Zhou et al.
Bioorganic Chemistry 115 (2021) 105159
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