Synthesis and biological evaluation of innovative thiourea derivatives as PHGDH inhibitors

Article abstract of DOI:10.1007/s11696-020-01188-0

In order to discover novel compounds with inhibitory activity against 3-phosphoglycerate dehydrogenase (PHGDH), a series of thiourea derivatives were designed and synthesized based on the structural modification of compound 5d. Compound 5d emerged from the visual database of ChemDiv of 200,000 small molecules by docking score ranking. Inhibition experiments on PHGDH activity of newly synthesized compounds were performed in vitro. Compounds with more than 30percent inhibitory rate at 25?μM on PHGDH were screened for IC₅₀ measurement. Anti-proliferative activity of 4a, 5a, 6e, 6n against A2780, MDA-MB-468, MDA-MB-231 and HEK293T in vitro was evaluated. The results showed that the compound 4a displayed the best inhibitory activity on PHGDH among the newly synthesized compounds, and the compounds 4a, 5a, 6n had a better proliferation inhibition effect on human A2780 cell line than NCT-503 reported previously. In addition, 2D interaction diagrams revealed potential action modes of active compounds with PHGDH.

Full text of DOI:10.1007/s11696-020-01188-0

Chemical Papers
https://doi.org/10.1007/s11696-020-01188-0
ORIGINAL PAPER
Synthesis and biological evaluation of innovative thiourea derivatives
as PHGDH inhibitors
Jiawei Xiang · Lei Tao · Yue Zhou · Yuping Tan1,2 · Zicheng Li1 · Yinglan Zhao · Qingxiang Sun · Youfu Luo
1
2
1,2
1
2
2
Received: 22 October 2019 / Accepted: 7 May 2020
©
Institute of Chemistry, Slovak Academy of Sciences 2020
Abstract
In order to discover novel compounds with inhibitory activity against 3-phosphoglycerate dehydrogenase (PHGDH), a series
of thiourea derivatives were designed and synthesized based on the structural modiꢀcation of compound 5d. Compound 5d
emerged from the visual database of ChemDiv of 200,000 small molecules by docking score ranking. Inhibition experiments
on PHGDH activity of newly synthesized compounds were performed in vitro. Compounds with more than 30% inhibitory
rate at 25 μM on PHGDH were screened for IC measurement. Anti-proliferative activity of 4a, 5a, 6e, 6n against A2780,
5
0
MDA-MB-468, MDA-MB-231 and HEK293T in vitro was evaluated. The results showed that the compound 4a displayed
the best inhibitory activity on PHGDH among the newly synthesized compounds, and the compounds 4a, 5a, 6n had a bet-
ter proliferation inhibition eꢁect on human A2780 cell line than NCT-503 reported previously. In addition, 2D interaction
diagrams revealed potential action modes of active compounds with PHGDH.
Keywords 3-Phosphoglycerate dehydrogenase (PHGDH) · Thiourea derivatives · Virtual screening · Proliferation
inhibition eꢁect · 2D interaction diagrams
Introduction
that PHGDH is highly expressed in a range of tumors. It is
also related to tumor cell growth and low survival rate of
tumor patients (Jia et al. 2016; Jing et al. 2015; Mattaini
et al. 2015).
The phosphoglycerate dehydrogenase (PHGDH) is the ꢀrst
and important branching enzyme in the glycolytic-serine
biosynthetic pathway (Pacold et al. 2016; Ravez et al. 2017;
Reid et al. 2018). PHGDH oxidizes glycolysis intermediate
In previous studies, PHGDH has been conꢀrmed as a
promising target in cancer by target validation, performed
in vitro and in vivo knockdown models (Gromova et al.
2015; Ou et al. 2015; Vandekeere et al. 2018). So far, there
have been few reports of PHGDH small molecule inhibitors.
There were only three major PHGDH inhibitors published
in peer-reviewed, namely CBR-5884 (IC =33± 12 μM),
3
-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate
(
p-Pyr), and ꢀnally serine is synthesized through a series
of enzymes (Sullivan et al. 2019; Truong et al. 2017; Wein-
stabl et al. 2019). Serine plays an important role in the syn-
thesis of proteins and other biomolecules required for cell
proliferation, such as nucleotides, phosphatidylserine and
sphingosine (Ducker et al. 2017). Recent studies have found
5
0
NCT-503 (IC =2.5± 0.6 μM) and PKUMDL-WQ-2101
5
0
(IC =34.8± 3.6 μM) (Pacold et al. 2016; Wang et al. 2017).
50
CBR-5884 and NCT-503 were obtained by high-throughput
screening (HTS), and the third type was PHGDH allosteric
inhibitor discovered by virtual screening (Mullarky et al.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-020-01188-0) contains
supplementary material, which is available to authorized users.
2
019; Reid et al. 2018). These three inhibitors had simi-
lar potency in vitro biochemical assays. They had moderate
selectivity for PHGDH and inhibited PHGDH activity in
a noncompetitive manner (Ravez et al. 2017; Wang et al.
2017).
*
Zicheng Li
sculzc@scu.edu.cn
1
2
School of Chemical Engineering, Sichuan University,
Chengdu 610065, Sichuan, People’s Republic of China
In order to further search for PHGDH small molecule
inhibitors with other novel structures, a virtual screening was
conducted by Schrodinger_Suites_2018-1, a virtual docking
State Key Laboratory of Biotherapy and Cancer Center,
West China Hospital, West China Medical School,
Chengdu 610041, Sichuan, People’s Republic of China
Vol.:(0
1
123456789)
3

Chemical Papers
software. Compound 5d emerged from the visual database
of ChemDiv of 200,000 small molecules. The molecular
docking procedure was performed by using glide docking
program of Schrodinger_Suites_2018-1. The 200,000 small
molecule databases were used to dock using standard preci-
sion. We ranked the docking scores of all molecules. NCT-
diꢁerent piperazine or piperidine structures again to obtain
compounds 4a, 5a–c.
Moreover, anti-proliferative activity of 4a, 5a, 6e and
6n against A2780, MDA-MB-468, MDA-MB-231 and
HEK293T in vitro was evaluated. To understand the poten-
tial action modes of molecules and protein, 2D interaction
diagrams were generated by Schrodinger_Suites_2018-1.
5
03 was used as the control. The number of compounds
with docking score above 8.0 was about 65 (NCT-503 was
.124). We considered synthesis diꢂculty and availability of
raw materials and then selected two molecules 116 and 5d
docking scores were 8.806 and 8.987, respectively)(Fig. 1).
9
Experimental
Chemistry
(
Partial reason for selecting compound 5d was structural sim-
ilarity with NCT-503. This increased our conꢀdence that
compound 5d had great activity. The two molecules were
then synthesized, and their inhibitory activity on PHGDH
was measured (116, IC =174 μM; 5d, IC =56 μM). Due
Materials and instruments
5
0
50
All the reagents were purchased from commercial suppli-
ers without further puriꢀcation unless otherwise speciꢀed.
Melting points were determined with a capillary method and
are uncorrected. IR spectra were recorded on a Spectrum
to the better activity against PHGDH, Compound 5d was
selected as a lead compound.
Due to the structural similarity, the piperazine structure of
NCT-503 and the SAR of piperazine-1-thiourea compounds
were referred (Pacold et al. 2016; Rohde et al. 2018). We
synthesized a series of 5d derivatives and discussed struc-
ture–activity relationships of newly synthesized molecules.
Firstly, to explore the eꢁect of diꢁerent piperidine or
piperazine structures on the activity of compound 5d, we
modiꢀed the piperidine structure of 5d to obtain compounds
1
13
Two Li10014 spectrometer. H NMR and C NMR spectra
were recorded on a Bruker Avance 400 MHz spectrometer.
NMR spectra were recorded in DMSO-d or CDCl solu-
6
3
1
13
tions at room temperature (20± 2 °C). H and C chemical
shifts were quoted in parts per million downꢀeld from TMS.
ESI–MS spectra were recorded on a Bruker Esquire 3000
instrument. High-resolution mass spectra (HRMS) were
obtained on a MicrOTOF-Q II mass spectrometer with an
ESI source (Waters, Manchester). Compound NCT-503 was
purchased from MCE. Other newly synthesized compounds
4
b, 5e, 5f and 6o. The inhibition rates of compounds 4b, 5d,
e, 5f and 6o at 25 μM were 20%, 30%, 23%, 24% and 31%,
5
respectively. We found that not only 6o maximized the activ-
ity of 5d maximally, but the piperazine structure of 6o was
also more easily synthesized than the piperidine structure
of 5d. So, compound 6o was selected as the starting point
for our next optimization. Then, we maintained the pipera-
zine structure of compound 6o and changed the aromatic
structure on the other side of the thiocarbonyl group. We
used diꢁerent substituted benzene rings, aromatic heterocy-
clic rings and cycloalkyl groups as R groups to explore the
eꢁect of them on the activity of the compounds. A series of
compounds 6a–n were obtained. The results showed that
aromatic heterocyclic rings had better activity than other
groups, and 3-(oxazol-5-yl) phenyl was the best active of
all R groups. Compound 6n showed the best inhibition rate
of 47% at 25 μM. Finally, compound 6n was replaced with
1
13
were characterized by IR, H NMR, C NMR spectra and
mass spectra.
Synthesis of compound 116
Compound 116 was prepared according to literature methods
[
Supporting Information] (Won et al. 2003).
General procedure for the preparation
of intermediates 2
Add compound 1 (1 mmol) to a solution of di(1H-imidzazol-
1-yl)methanethione (196 mg, 1.1 mmol) in 5 ml THF. The
Fig. 1 Structures of PHGDH inhibitors NCT-503, 116 and 5d
1
3

Chemical Papers
resulting reaction mixture was heated at 45 °C for 0.5–1.5 h.
The reaction was then cooled to room temperature to give a
mixture containing intermediate 2.
in vacuo. After that, the resulting residue was puriꢀed via
phase column chromatography (ethyl acetate/petroleum
ether=1:5–1:15) to give product 4, 5 or 6, respectively.
The speciꢀc mass of piperazine or piperidine derivatives
required for the reaction was: 1-(4-(triꢃuoromethyl)benzyl)
piperazine (244 mg, 1 mmol), 4-(4-ꢃuorophenyl)piperi-
dine (179 mg, 1 mmol), 1-(3-(triꢃuoromethyl)phenyl)pip-
erazine (230 mg, 1 mmol), methyl 2-(piperidin-4-yl)acetate
(157 mg, 1 mmol), 4-(3-chlorophenyl)piperidine (196 mg,
1 mmol).
The speciꢀc mass of compound 1 required for the reac-
tion was: 3-chloro-4-ꢃuoroaniline (146 mg, 1 mmol), thia-
zol-2-amine (100 mg, 1 mmol), 4-chloroaniline (128 mg,
1
1
mmol), 4-ꢃuoroaniline (111 mg, 1 mmol), 1,3-dimethyl-
H-pyrazol-5-amine (111 mg, 1 mmol), 3-chloroaniline
(
128 mg, 1 mmol), 3,4-dichloroaniline (162 mg, 1 mmol),
4
,4-dimethylcyclohexan-1-amine (127 mg, 1 mmol), 4-bro-
moaniline (172 mg, 1 mmol), naphthalen-1-amine (143 mg,
mmol), 4-(trifluoromethyl)aniline (161 mg, 1 mmol),
1,1′-biphenyl]-4-amine (169 mg, 1 mmol), 4-isopropy-
1
N‑(3‑(oxazol‑5‑yl)phenyl)‑4‑(3‑(triꢀuoromethyl)phenyl)pip‑
erazine‑1‑carbothio‑amide (4a) Yellow solid, yield: 65%;
[
−
1
laniline (135 mg, 1 mmol), 3-(oxazol-5-yl)aniline (160 mg,
mp: 124.9–125.1 °C; IR (KBr, cm ): 3283, 3101, 2922,
1
1
1
mmol), 1-methyl-3-phenyl-1H-pyrazol-5-amine (173 mg,
2839, 1615, 1540, 1497, 1449, 1321, 787, 714, 691; H NMR
mmol).
(400 MHz, DMSO-d ): δ 9.53 (s, 1H), 8.44 (s, 1H), 7.70 (s,
6
1
H), 7.67 (s, 1H), 7.51–7.38 (m, 3H), 7.36 (s, 1H), 7.21 (s,
Synthesis of compound 3
2H), 7.09 (d, J=7.6 Hz, 1H), 4.16–4.02 (m, 4H), 3.48–3.35
13
(m, 4H); C NMR (100 MHz, DMSO-d ): δ 181.88, 152.29,
6
Piperazine (27.61 g, 320 mmol) was dissolved in THF
151.08, 150.82, 142.22, 130.50, 130.43 (q, J = 31.0 Hz),
129.32, 127.74, 126.03, 124.92 (q, J =272.5 Hz), 122.46,
121.35, 120.60, 118.95, 115.10 (q, J=4.0 Hz), 111.25 (q,
J=4.0 Hz), 47.96, 47.42. HRMS Calcd. for C H F N OS
(
300 ml) by heating. 1-(Bromomethyl)-4-(triꢃuoromethyl)
benzene (9.56 g, 40 mmol) was added dropwise to a solution
of piperazine in THF under reꢃux. After being stirred over-
night under reꢃux, the reaction mixture was cooled to room
temperature, and the THF was removed by evaporation. The
resulting residue was washed with aq. K CO , extracted with
2
1
19
3
4
+
[M+H] 433.1305, found 433.1311.
N‑(1‑methyl‑3‑phenyl‑1H‑pyrazol‑5‑yl)‑4‑(3‑(triꢀuoromethyl)
phenyl)piperazine‑1‑carbothioamide (4b) White solid, yield:
2
3
ethyl acetate, washed with sat. NaCl, dried with Na SO and
2
4
−
1
evaporated to dryness under vacuum to yield 3 (O’Dowd
60%; mp: 176.4–177.5 ℃; IR (KBr, cm ): 3122, 2931,
et al. 2017).
1610, 1509, 1476, 1443, 1363, 1307, 833, 767, 721, 697;
1
H NMR (400 MHz, CDCl ): δ 7.77–7.69 (m, 2H), 7.37
3
1
‑(4‑(trifluoromethyl)benzyl)piperazine (3) Colorless
(s, 1H), 7.36–7.32 (m, 3H), 7.30–7.23 (m, 1H), 7.12 (d,
1
liquid, yield: 86%; H NMR (400 MHz, DMSO): δ 7.67
J=7.8 Hz, 1H), 6.99 (s, 1H), 6.93 (d, J=8.4 Hz, 1H), 6.37
(
d, J=8.0 Hz, 2H), 7.52 (d, J=8.0 Hz, 2H), 3.51 (s, 2H),
(s, 1H), 3.99–3.88 (m, 4H), 3.69 (s, 3H), 3.27–3.16 (m, 4H);
1
3
13
2
.76–2.62 (m, 4H), 2.29 (s, 4H); C NMR (100 MHz,
C NMR (100 MHz, DMSO-d ): δ 181.98, 151.03, 148.25,
6
DMSO): δ 143.92, 129.84, 127.99 (q, J=31.6 Hz), 125.43
140.34, 134.04, 130.51, 130.44 (q, J = 31.1 Hz) 129.06,
127.76, 125.10, 124.92 (q, J = 272.6 Hz), 118.97, 115.16
(q, J=4.0 Hz), 111.27 (q, J=3.6 Hz), 100.39, 48.06, 47.33,
(
q, J=3.8 Hz), 124.85 (q, J=271.8 Hz), 62.58, 54.58, 46.07.
+
HRMS Calcd. for C H F N [M + H] 245.1260, found
1
2
15
3
2
+
2
45.1259.
36.14. HRMS Calcd. for C H F N S [M+H] 446.1621,
2
2
22
3
5
found 446.1619.
General procedure for the preparation
of compounds 4, 5, 6
4‑(4‑fluorophenyl)‑N‑(3‑(oxazol‑5‑yl)phenyl)piperi‑
dine‑1‑carbothioamide (5a) Yellow solid, yield: 65%;
−
1
The mixture containing intermediate 2 was transferred to
another vial containing a solution comprised of piperazine
or piperidine derivatives (1 mmol), THF (3 ml), and trieth-
ylamine (0.14 ml, 1 mmol). An additional aliquot of THF
mp: 78.3–79.7 ℃; IR (KBr, cm ): 2923, 2853, 1618,
1
1532, 1508, 1439, 1323, 832, 790, 770, 714, 688; H NMR
(400 MHz, DMSO-d ): δ 9.41 (s, 1H), 8.45 (s, 1H), 7.73–
6
7.61 (m, 2H), 7.47 (d, J=7.8 Hz, 1H), 7.40 (t, J=7.8 Hz,
1H), 7.37–7.27 (m, 3H), 7.13 (t, J = 9.0 Hz, 2H), 4.92
(d, J = 13.2 Hz, 2H), 3.15 (t, J = 12.0 Hz, 2H), 2.90 (dd,
J=16.4, 8.0 Hz, 1H), 1.91–1.80 (m, 2H), 1.65 (qd, J=12.8,
(
2 ml) was used to complete the transfer. The resulting reac-
tion mixture was heated with stirring at 75 °C for 1.5–2.5 h.
Then, the reaction mixture was concentrated and diluted
with water and DCM. The layers were separated and the
aqueous layer was reextracted with DCM. The combined
organic layers were dried with Na SO and concentrated
1
3
3.8 Hz, 2H); C NMR (100 MHz, DMSO-d ): δ 181.41,
6
161.25 (d, J=241.8 Hz), 152.27, 150.87, 142.49, 142.00 (d,
J=2.9 Hz), 129.26, 129.00 (d, J=7.9 Hz), 127.67, 125.83,
2
4
1
3

Chemical Papers
1
4
3
22.41, 121.08, 120.33, 115.55 (d, J = 20.9 Hz), 49.20,
4‑(3‑chlorophenyl)‑N‑(1‑methyl‑3‑phenyl‑1H‑pyrazol‑5‑yl)
piperidine‑1‑carbo‑thioamide (5e) White solid, yield: 55%;
+
1.34, 33.36. HRMS Calcd. for C H FN OS [M + H]
2
1
20
3
−
1
82.1384, found 382.1389.
mp: 141.2–142.9 ℃; IR (KBr, cm ): 3198, 3063, 2932,
2
856, 1566, 1591, 1498, 1476, 1439, 1319, 1301, 879, 831,
1
4
‑(3‑Chlorophenyl)‑N‑(3‑(oxazol‑5‑yl)phenyl)piperi‑
771, 724, 699; H NMR (400 MHz, CDCl ): δ 7.77–7.69 (m,
3
dine‑1‑carbothioamide (5b) Light green solid, yield: 60%;
2H), 7.36 (t, J=7.6 Hz, 2H), 7.28 (t, J=4.8 Hz, 1H), 7.25–
7.18 (m, 2H), 7.14 (s, 1H), 7.05–6.99 (m, 1H), 6.36 (s, 1H),
4.73 (d, J=12.8 Hz, 2H), 3.72 (s, 3H), 3.15–2.95 (m, 2H),
2.72 (tt, J=12.0, 3.6 Hz, 1H), 1.88 (d, J=12.0 Hz, 2H), 1.67
−
1
mp: 146.2–147.8 ℃; IR (KBr, cm ): 3233, 3088, 2933,
2
1
851, 1618, 1594, 1576, 1535, 1503, 1473, 1442, 1407,
1
322, 900, 825, 797, 733, 692; H NMR (400 MHz, DMSO-
1
3
d ): δ 9.41 (s, 1H), 8.44 (s, 1H), 7.67 (d, J= 1.8 Hz, 1H),
(qd, J = 13.0, 3.8 Hz, 2H); C NMR (100 MHz, CDCl ):
6
3
7
1
4
.66 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz,
H), 7.38 – 7.31 (m, 3H), 7.27 (dd, J = 9.2, 4.6 Hz, 2H),
.92 (d, J = 13.2 Hz, 2H), 3.15 (t, J = 12.0 Hz, 2H), 2.92
δ 181.88, 149.65, 146.42, 139.08, 134.43, 133.30, 129.96,
128.70, 127.84, 126.99, 126.89, 125.25, 124.94, 99.62,
49.47, 41.88, 36.03, 32.60. HRMS Calcd. for C H ClN S
2
2
23
4
+
(
(
dd, J=13.8, 10.4 Hz, 1H), 1.88 (d, J=11.4 Hz, 2H), 1.67
[M+H] 411.1405, found 411.1408.
13
qd, J=12.8, 3.8 Hz, 2H); C NMR (100 MHz, DMSO-d ):
6
δ 181.43, 152.26, 150.88, 148.43, 142.48, 133.61, 130.78,
Methyl 2‑(1‑((1‑methyl‑3‑phenyl‑1H‑pyrazol‑5‑yl)carba‑
mothioyl)piperidin‑4‑yl)acetate (5f) Yellow solid, yield:
1
1
29.26, 127.68, 127.27, 126.73, 126.00, 125.86, 122.41,
−
1
21.12, 120.35, 49.12, 41.79, 32.98. HRMS Calcd. for
45%; mp: 85.3–86.7 ℃; IR (KBr, cm ): 2931, 1734, 1628,
+
1
C H ClN OS [M+H] 398.1089, found 398.1093.
1568, 1524, 1442, 1364, 1311, 766, 726, 697, 670; H NMR
2
1
20
3
(
400 MHz, CDCl ): δ 7.76–7.66 (m, 2H), 7.36 (t, J=7.6 Hz,
3
Methyl 2‑(1‑((3‑(oxazol‑5‑yl)phenyl)carbamothioyl)piperi‑
din‑4‑yl)acetate (5c)Methyl 2‑(1‑((3‑(oxazol‑5‑yl)phenyl)
carbamothioyl)piperidin‑4‑yl)acetate (5c) White solid,
2H), 7.31 (s, 1H), 7.30–7.24 (m, 1H), 6.31 (s, 1H), 4.55
(d, J=12.4 Hz, 2H), 3.67 (s, 3H), 3.66 (s, 3H), 3.06–2.88
(m, 2H), 2.23–2.16 (m, 2H), 2.03–1.95 (m, 1H), 1.73 (d,
−
1
13
yield: 50%; mp: 87.5–88.9 ℃; IR (KBr, cm ):2926, 1730,
J=12.8 Hz, 2H), 1.25–1.15 (m, 2H); C NMR (100 MHz,
1
1
634, 1529, 1442, 1321,1250, 910, 828, 755; H NMR
CDCl ): δ 181.58, 172.58, 149.54, 139.13, 133.33, 128.64,
3
(
400 MHz, DMSO-d ): δ 9.33 (s, 1H), 8.44 (s, 1H), 7.64 (d,
127.75, 125.23, 99.63, 51.64, 48.98, 40.16, 35.95, 32.51,
6
+
J=5.6 Hz, 2H), 7.46 (d, J=7.8 Hz, 1H), 7.38 (t, J=7.8 Hz,
H), 7.29 (d, J=8.2 Hz, 1H), 4.72 (d, J=13.2 Hz, 2H), 3.61
s, 3H), 3.10 (t, J=11.8 Hz, 2H), 2.32 (d, J=7.0 Hz, 2H),
31.38. HRMS Calcd. for C H N O S [M+H] 373.1693,
1
9
24
4
2
1
found 373.1701.
(
2
2
.02 (ddd, J=16.6, 8.4, 4.6 Hz, 1H), 1.73 (d, J=11.0 Hz,
N‑(3‑chloro‑4‑ꢀuorophenyl)‑4‑(4‑(triꢀuoromethyl)benzyl)
piperazine‑1‑carbo‑thioamide (6a) White solid, yield: 71%;
1
3
H), 1.30–1.17 (m, 2H); C NMR (100 MHz, DMSO-d ):
6
−
1
δ 181.14, 172.76, 152.22, 150.88, 142.46, 129.23, 127.64,
mp: 164.1–165.3 ℃; IR (KBr, cm ):3246, 3038, 2925,
1
25.84, 122.36, 121.12, 120.31, 51.70, 48.61, 40.14, 32.87,
2888, 2806, 1620, 1532, 1503, 1458, 1425, 1319, 881, 845,
+
1
3
1.71. HRMS Calcd. for C H N O S [M+H] 360.1377,
729; H NMR (400 MHz, CDCl ): δ 7.59 (d, J=8.0 Hz, 2H),
1
8
21
3
3
3
found 360.1379.
7.46 (d, J=7.8 Hz, 2H), 7.24 (s, 1H), 7.16–7.00 (m, 3H),
13
3
.88 (s, 4H), 3.61 (s, 2H), 2.54 (s, 4H); C NMR (100 MHz,
4
‑(4‑ꢀuorophenyl)‑N‑(1‑methyl‑3‑phenyl‑1H‑pyrazol‑5‑yl)
DMSO-d ): δ 181.67, 154.61 (d, J = 243.9 Hz), 143.35,
6
piperidine‑1‑carboth‑ioamide (5d) White solid, yield:
138.75 (d, J = 3.2 Hz), 129.96, 128.22 (q, J = 31.7 Hz),
127.65, 126.33 (d, J = 7.1 Hz), 125.58 (q, J = 3.7 Hz),
124.83 (q, J = 271.9 Hz), 118.72 (d, J = 18.6 Hz), 116.42
(d, J = 21.8 Hz), 61.30, 52.66, 48.48. HRMS Calcd. for
−
1
6
1%; mp: 178.2–179.8 ℃; IR (KBr, cm ): 2931, 1606,
1
564, 1509, 1476, 1443, 1363, 1307, 833, 766, 725, 697;
1
H NMR (400 MHz, CDCl ): δ 7.80–7.73 (m, 2H), 7.37
3
+
(
t, J=7.6 Hz, 2H), 7.28 (t, J=7.4 Hz, 1H), 7.17–7.10 (m,
C H ClF N S [M+H] 432.0919, found 432.0920.
1
9
18
4
3
2
H), 7.08 (s, 1H), 7.04–6.95 (m, 2H), 6.39 (s, 1H), 4.78 (d,
J=12.8 Hz,2H), 3.78 (s, 3H), 3.15 (td, J=13.2,2.4 Hz, 2H),
N‑(thiazol‑2‑yl)‑4‑(4‑(trifluoromethyl)benzyl)pipera‑
zine‑1‑carbothioamide (6b) Yellow solid, yield: 66%; mp:
2
1
.78 (tt, J = 11.8, 3.6 Hz, 1H), 1.92 (d, J = 12.4 Hz, 2H),
1
3
−1
.73 (ddd, J=25.8, 12.8, 3.8 Hz, 2H); C NMR (100 MHz,
200.1–201.4 °C; IR (KBr, cm ):3126, 3030, 2980, 2936,
CDCl ): δ 182.18, 161.62 (d, J=245.0 Hz), 149.79, 140.04
2857, 2802, 1620, 1526, 1475, 1457, 1365, 788, 756, 695;
3
1
(
m), 138.86, 133.34, 128.62, 128.09 (d, J=7.8 Hz), 127.76,
H NMR (400 MHz, DMSO-d ): δ 12.47 (s, 1H), 7.70 (d,
6
1
25.28, 115.46 (d, J=21.2 Hz), 99.35, 49.71, 41.54, 36.03,
J=8.2 Hz, 2H), 7.57 (d, J=8.0 Hz, 2H), 7.30 (d, J=4.6 Hz,
+
3
2.99. HRMS Calcd. for C H FN S [M+H] 395.1700,
1H), 6.72 (d, J = 4.6 Hz, 1H), 4.05 (s, 4H), 3.61 (s, 2H),
2
2
23
4
1
3
found 395.1707.
2.41 (s, 4H); C NMR (100 MHz, DMSO-d ): δ 184.20,
6
1
70.46, 143.51, 129.93, 128.17 (q, J=31.8 Hz), 125.56 (q,
J=3.7 Hz), 124.83 (q, J=271.8 Hz), 123.69 (br), 108.31,
1
3

Chemical Papers
+
6
3
1.49, 52.99. HRMS Calcd. for C H F N S [M + H]
J=271.8 Hz), 124.20, 123.71, 61.30, 52.68, 48.59. HRMS
1
6
17
3
4 2
+
87.0920, found 387.0926.
Calcd. for C H ClF N S [M + H] 414.1013, found
1
9
19
3
3
4
14.1017.
N‑(4‑fluorophenyl)‑4‑(4‑(trifluoromethyl)benzyl)pipera‑
zine‑1‑carbothioamide (6c) White solid, yield: 65%; mp:
N‑(3,4‑dichlorophenyl)‑4‑(4‑(triꢀuoromethyl)benzyl)piper‑
azine‑1‑carbothio‑amide (6g) White solid, yield: 67%; mp:
−
1
1
1
70.2–171.8 ℃; IR (KBr, cm ):3250, 2919, 2816, 1617,
1
−1
529, 1425, 1394, 1327, 833, 724, 687; H NMR (400 MHz,
155.8–156.9 ℃; IR (KBr, cm ):3242, 2922, 2815, 1621,
DMSO-d ): δ 9.28 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.57
1578, 1525, 1471, 1421, 1377, 1327, 873, 826, 722, 675;
6
1
(
d, J=8.0 Hz, 2H), 7.27 (dd, J =9.0, 5.2 Hz, 2H), 7.11 (t,
H NMR (400 MHz, DMSO-d ): δ 9.45 (s, 1H), 7.71 (d,
6
J = 8.8 Hz, 2H), 3.96 – 3.85 (m, 4H), 3.63 (s, 2H), 2.48–
J=8.2 Hz, 2H), 7.61 (d, J=2.4 Hz, 1H), 7.57 (d, J=7.8 Hz,
1
3
2
1
1
1
.41 (m, 4H); C NMR (100 MHz, DMSO-d ): δ 181.99,
2H), 7.53 (d, J=8.8 Hz, 1H), 7.32 (dd, J=8.8, 2.4 Hz, 1H),
6
1
3
59.63 (d, J = 241.0 Hz), 143.40, 137.80 (d, J = 2.8 Hz),
29.96, 128.21 (q, J = 31.7 Hz), 128.06 (d, J = 8.3 Hz),
25.58 (q, J = 3.6 Hz), 124.84 (q, J = 271.9 Hz), 115.04
3.91 (s, 4H), 3.63 (s, 2H), 2.48–2.44 (m, 4H); C NMR
(100 MHz, DMSO-d ): δ 181.36, 143.33, 141.77, 130.47,
6
130.16, 129.98, 128.21 (q, J = 31.7 Hz), 126.63, 126.23,
125.60 (q, J = 3.4 Hz), 125.29, 124.82 (q, J = 272.1 Hz),
61.27, 52.65, 48.61. HRMS Calcd. for C H Cl F N S
(
d, J = 22.4 Hz), 61.33, 52.70, 48.37. HRMS Calcd. for
+
C H F N S [M+H] 398.1309, found 398.1312.
1
9
19
4
3
19 18
2
3
3
+
[
M+H] 448.0624, found 448.0627.
N‑(4‑chlorophenyl)‑4‑(4‑(trifluoromethyl)benzyl)pipera‑
zine‑1‑carbothioamide (6d) Yellow solid, yield: 64%; mp:
N‑(4,4‑dimethylcyclohexyl)‑4‑(4‑(trifluoromethyl)benzyl)
piperazine‑1‑carbothio‑amide (6h) White solid, yield:
−
1
1
1
6
68.9–169.4 ℃; IR (KBr, cm ):3255, 2950, 2907, 2814,
−
1
620, 1589, 1527, 1490, 1424, 1392, 1328, 835, 723, 699,
72%; mp: 132.1–133.8 ℃; IR (KBr, cm ):3346, 2942,
1
79; H NMR (400 MHz, DMSO-d ): δ 9.35 (s, 1H), 7.71
2865, 2807, 2764, 1623, 1537, 1454, 1414, 1348, 1323,
6
1
(
d, J=8.2 Hz, 2H), 7.57 (d, J=8.0 Hz, 2H), 7.36 – 7.27 (m,
883, 838, 800, 704, 673; H NMR (400 MHz, DMSO-
4
H), 3.96–3.84 (m, 4H), 3.63 (s, 2H), 2.49–2.38 (m, 4H);
d ): δ 7.69 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H),
6
1
3
C NMR (100 MHz, DMSO-d ): δ 181.71, 143.38, 140.52,
7.25 (d, J = 7.8 Hz, 1H), 4.20–4.07 (m, 1H), 3.82–3.72
(m, 4H), 3.59 (s, 2H), 2.42–2.31 (m, 4H), 1.75–1.60 (m,
2H), 1.46 (td, J = 13.2, 3.2 Hz, 2H), 1.35 (d, J = 12.6 Hz,
2H), 1.19 (td, J = 13.4, 3.4 Hz, 2H), 0.90 (d, J = 8.0 Hz,
6
1
1
5
4
29.96, 128.63, 128.31, 128.21 (q, J = 31.6 Hz), 127.20,
25.58 (q, J = 3.7 Hz), 124.83 (q, J = 272.0 Hz), 61.31,
+
2.70, 48.51. HRMS Calcd. for C H ClF N S [M + H]
1
9
19
3
3
1
3
14.1013, found 414.1018.
6H).; C NMR (100 MHz, DMSO-d ): δ 180.81, 143.43,
6
1
29.93, 128.17 (q, J = 31.7 Hz), 125.55 (q, J = 3.8 Hz),
N‑(1,3‑dimethyl‑1H‑pyrazol‑5‑yl)‑4‑(4‑(triꢀuoromethyl)ben‑
zyl)piperazine‑1‑carbothioamide (6e) Yellow solid, yield:
124.83 (q, J=271.9 Hz), 61.41, 55.09, 52.72, 47.62, 38.39,
32.79, 29.81, 28.08, 24.45. HRMS Calcd. for C H F N S
2
1
30
3
3
−
1
+
5
2
7
2
4
6%; mp: 101.2–102.1 ℃; IR (KBr, cm ): 3223, 2926,
[M+H] 414.2186, found 414.2190.
816, 1621, 1571, 1491, 1442, 1418, 1370, 1326, 850, 820,
1
93, 729; H NMR (400 MHz, CDCl ): δ 7.59 (d, J=8.0 Hz,
N‑(4‑bromophenyl)‑4‑(4‑(trifluoromethyl)benzyl)pipera‑
zine‑1‑carbothioamide (6i) White solid, yield: 65%; mp:
3
H), 7.45 (d, J=8.0 Hz, 2H), 5.81 (s, 1H), 3.99 – 3.83 (m,
H), 3.59 (s, 2H), 3.56 (s, 3H), 2.52 (d,J=4.2 Hz, 4H), 2.18
−
1
160.1–161.9 ℃; IR (KBr, cm ):3257, 2940, 2804, 1628,
1
3
1
(
s, 3H); C NMR (100 MHz, CDCl ): δ 182.38, 146.85,
1586, 1525, 1485, 1420, 1324, 819, 712, 672, 649; H NMR
3
1
41.78, 138.59, 129.59 (q, J = 32.2 Hz), 129.19, 125.34
q, J = 3.8 Hz), 124.20 (q, J = 272.0 Hz), 101.98, 61.92,
2.42, 48.76, 35.27, 14.04. HRMS Calcd. for C H F N S
(400 MHz, DMSO-d ): δ 9.35 (s, 1H), 7.71 (d, J=8.2 Hz,
6
(
2H), 7.57 (d, J=8.0 Hz, 2H), 7.50–7.40 (m, 2H), 7.26 (d,
5
J=8.8 Hz, 2H), 3.96–3.84 (m, 4H), 3.63 (s, 2H), 2.49–2.40
1
8
22
3
5
+
13
[
M+H] 398.1621, found 398.1623.
(m, 4H); C NMR (100 MHz, DMSO-d ): δ 181.64, 143.32,
6
1
40.95, 131.24, 129.98, 128.21 (q, J = 31.6 Hz), 127.52,
N‑(3‑chlorophenyl)‑4‑(4‑(trifluoromethyl)benzyl)pipera‑
zine‑1‑carbothioamide (6f) Yellow solid, yield: 66%; mp:
125.57 (q, J = 3.7 Hz), 124.82 (q, J = 272.0 Hz), 116.78,
61.31, 52.68, 48.51. HRMS Calcd. for C H BrF N S
1
9
19
3
3
−
1
+
1
1
35.1–136.4 ℃; IR (KBr, cm ):2920, 1645, 1525, 1457,
[M+H] 458.0508, found 458.0521.
1
427, 1393, 1319, 844, 727, 673; H NMR (400 MHz,
DMSO-d ): δ 9.40 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.57
N‑(naphthalen‑1‑yl)‑4‑(4‑(trifluoromethyl)benzyl)piper‑
azine‑1‑carbothio‑amide (6j) Pink solid, yield: 61%; mp:
6
(
d, J=8.0 Hz, 2H), 7.41 (t, J=1.8 Hz, 1H), 7.34–7.23 (m,
−
1
2
H), 7.14 (dd, J=7.6, 1.8 Hz, 1H), 3.99–3.84 (m, 4H), 3.63
139.9–140.8 ℃; IR (KBr, cm ): 3250,2907, 2810, 1620,
1
3
1
(
s, 2H), 2.49–2.43 (m, 4H); C NMR (100 MHz, DMSO-
1598, 1514, 1416, 1392, 1327, 842, 798, 774, 694; H
d ): δ 181.55, 143.36, 143.09, 132.55, 129.97, 128.21 (q,
NMR (400 MHz, DMSO-d ): δ 9.48 (s, 1H), 7.95–7.87 (m,
6
6
J = 31.7 Hz), 125.58 (q, J = 3.8 Hz) 124.89, 124.83 (q,
1H), 7.85–7.77 (m, 2H), 7.72 (d, J=8.2 Hz, 2H), 7.59 (d,
1
3

Chemical Papers
−
1
J=8.0 Hz, 2H), 7.53–7.44 (m, 3H), 7.29 (d, J=7.2 Hz, 1H),
mp: 137.2–139.1 ℃; IR (KBr, cm ): 2918, 2810, 1619,
1
4
.08–3.93 (m, 4H), 3.66 (s, 2H), 2.52 (d, J=4.8 Hz, 4H);
1593, 1532, 1467, 1426, 1325, 847, 822, 791, 723, 687; H
1
3
C NMR (100 MHz, DMSO-d ): δ 183.03, 143.43, 137.80,
NMR (400 MHz, DMSO-d ): δ 9.43 (s, 1H), 8.44 (s, 1H),
6
6
1
1
34.27, 131.14, 130.00, 128.40, 128.22 (q, J = 32.1 Hz),
7.72 (d, J=8.2 Hz, 2H), 7.66 (s, 2H), 7.58 (d, J=8.0 Hz,
26.89, 126.42, 126.31, 126.27, 125.98, 125.59 (q,
2H), 7.47 (d, J=7.8 Hz, 1H), 7.40 (t, J=7.8 Hz, 1H), 7.31
J=3.8 Hz), 124.85 (q, J=272.0 Hz), 124.17, 61.39, 52.81,
(d, J=8.2 Hz, 1H), 3.93 (m, 4H), 3.65 (s, 2H), 2.52–2.48
+
13
4
8.44. HRMS Calcd. for C H F N S [M+H] 430.1560,
(m, 4H); C NMR (100 MHz, CDCl ): δ 183.13, 150.87,
2
3
22
3
3
3
found 430.1570.
150.63, 141.75, 140.71, 129.75, 129.70 (q, J = 32.3 Hz),
1
29.17, 128.65, 125.35 (q, J = 3.8 Hz), 124.15 (q,
4
‑(4‑(triꢀuoromethyl)benzyl)‑N‑(4‑(triꢀuoromethyl)phenyl)
J=272.0 Hz), 123.13, 122.08, 121.00, 118.81, 62.02, 52.39,
+
piperazine‑1‑carbothioamide (6k) Yellow solid, yield: 64%;
49.42. HRMS Calcd. for C H F N OS[M+H] 447.1461,
22
21
3
4
−
1
mp: 131.2–132.6 ℃; IR (KBr, cm ): 3236, 2953, 2918,
found 447.1460.White solid, yield: 67%; mp: 137.2–139.1
−
1
2
7
7
812, 1618, 1595, 1531, 1479, 1431, 1399, 1323, 843,
℃; IR (KBr, cm ): 2918, 2810, 1619, 1593, 1532, 1467,
1
1
41, 703; H NMR (400 MHz, DMSO-d ): δ 9.57 (s, 1H),
1426, 1325, 847, 822, 791, 723, 687; H NMR (400 MHz,
6
.71 (d, J=8.2 Hz, 2H), 7.63 (d, J=8.6 Hz, 2H), 7.58 (d,
DMSO-d ): δ 9.43 (s, 1H), 8.44 (s, 1H), 7.72 (d, J=8.2 Hz,
6
J=8.0 Hz, 2H), 7.53 (d, J=8.6 Hz, 2H), 4.00–3.85 (m, 4H),
2H), 7.66 (s, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.47 (d,
1
3
3
.64 (s, 2H), 2.47 (d, J=5.0 Hz, 4H); C NMR (100 MHz,
J=7.8 Hz, 1H), 7.40 (t, J=7.8 Hz, 1H), 7.31 (d, J=8.2 Hz,
1
3
DMSO-d ): δ 181.56, 145.42, 143.35, 129.97, 128.22 (q,
1H), 3.93 (m, 4H), 3.65 (s, 2H), 2.52–2.48 (m, 4H);
C
6
J=31.6 Hz), 125.55 (m), 124.93 (q, J=271.3 Hz), 124.83
NMR (100 MHz, CDCl ): δ 183.13, 150.87, 150.63, 141.75,
3
(
q, J = 272.0 Hz), 124.51, 124.09 (q, J= 32.0 Hz), 61.27,
140.71, 129.75, 129.70 (q, J = 32.3 Hz), 129.17, 128.65,
125.35 (q, J = 3.8 Hz), 124.15 (q, J = 272.0 Hz), 123.13,
122.08, 121.00, 118.81, 62.02, 52.39, 49.42. HRMS Calcd.
+
5
4
2.69, 48.76. HRMS Calcd. for C H F N S [M + H]
20 19 6 3
48.1277, found 448.1281.
+
for C H F N OS[M+H] 447.1461, found 447.1460.
2
2
21
3
4
N‑([1,1′‑biphenyl]‑4‑yl)‑4‑(4‑(triꢀuoromethyl)benzyl)pipera‑
zine‑1‑carbothio‑amide (6l) Yellow solid, yield: 66%; mp:
N‑(1‑methyl‑3‑phenyl‑1H‑pyrazol‑5‑yl)‑4‑(4‑(triꢀuoromethyl)
benzyl)piperazine‑1‑carbothioamide (6o) Light yellow solid,
−
1
1
1
33.2–134.9 ℃; IR (KBr, cm ): 2929, 1742, 1646, 1539,
1
−1
455, 1422, 1330, 838, 763, 727, 693; H NMR (400 MHz,
yield: 62%; mp: 187.4–188.1 °C; IR (KBr, cm ): 3217,
DMSO-d ): δ 9.38 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.65
3020, 2924, 2821, 1622, 1559, 1528, 1447, 1327, 855, 819,
6
1
(
d, J=7.2 Hz, 2H), 7.59 (dd, J =8.2, 5.0 Hz, 4H), 7.45 (t,
763, 699; H NMR (400 MHz, DMSO-d ): δ 9.31 (s, 1H),
6
J=7.8 Hz, 2H), 7.38 (d, J=8.6 Hz, 2H), 7.34 (t, J=7.4 Hz,
7.76 (d, J=7.2 Hz, 2H), 7.72 (d, J=8.2 Hz, 2H), 7.58 (d,
J=8.0 Hz, 2H), 7.38 (t, J=7.6 Hz, 2H), 7.27 (t, J=7.4 Hz,
1H), 6.52 (s, 1H), 3.95 (s, 4H), 3.65 (s, 2H), 3.62 (s, 3H),
1
H), 3.92 (s, 4H), 3.64 (s, 2H), 2.47 (d, J = 5.0 Hz, 4H);
1
3
C NMR (100 MHz, DMSO-d ): δ 181.76, 143.40, 141.00,
6
1
3
1
1
1
40.25, 136.30, 129.97, 129.38, 128.21 (q, J = 31.6 Hz),
2.47 (d, J=4.8 Hz, 4H); C NMR (100 MHz, DMSO-d ):
6
27.57, 126.83, 126.65, 125.74, 125.59 (q, J = 3.8 Hz),
δ 181.83, 148.20, 143.35, 140.40, 134.04, 129.98, 129.05,
24.84 (q, J=271.9 Hz), 61.34, 52.74, 48.55. HRMS Calcd.
128.23 (q, J = 31.7 Hz), 127.74, 125.60 (q, J = 3.8 Hz),
+
for C H F N S [M+H] 456.1716, found 456.1718.
125.08, 124.84 (q, J=272.0 Hz), 100.33, 61.26, 52.63, 48.62,
2
5
24
3
3
+
3
6.08. HRMS Calcd. for C H F N S [M+H] 460.1778,
2
3
24
3
5
N‑(4‑isopropylphenyl)‑4‑(4‑(triꢀuoromethyl)benzyl)pipera‑
zine‑1‑carbothio‑amide (6m) Yellow solid, yield: 69%; mp:
found 460.1782.
−
1
1
1
66.1–168.5 ℃; IR (KBr, cm ): 2961, 2808, 1621, 1588,
1
529, 1462, 1420, 1395, 1325, 843, 741, 703; H NMR
Biological assays
(
400 MHz, DMSO-d ): δ 9.21 (s, 1H), 7.71 (d, J=8.2 Hz,
6
2
H), 7.57 (d, J=8.0 Hz, 2H), 7.21–7.10 (m, 4H), 3.96–3.83
General
(
m, 4H), 3.63 (s, 2H), 2.85 (dq, J=13.8, 7.0 Hz, 1H), 2.48–
13
2
.40 (m, 4H), 1.19 (d, J=7.0 Hz, 6H); C NMR (100 MHz,
A2780 (human ovarian cancer cell lines), MDA-MB-468,
MDA-MB-231 (human breast cancer cell lines) and
HEK293T (human embryonic kidney cell lines) were pur-
chased from American Type Culture Collection (ATCC).
Cells were maintained in RPMI-1640 media or DMEM
media (Gibco) supplemented with 10% fetal bovine serum
DMSO-d ): δ 181.86, 144.87, 143.41, 139.17, 129.96,
6
1
28.19 (q, J=31.6 Hz), 126.23, 126.19, 125.77, 125.58 (q,
J=3.4 Hz), 124.84 (q, J=271.9 Hz), 61.34, 52.73, 48.37,
+
3
4
3.39, 24.43. HRMS Calcd. for C H F N S [M + H]
22 26 3 3
22.1873, found 422.1877.
−1
(
Hyclone), 100 U ml penicillin (Sigma-Aldrich, USA) and
−
1
N‑(3‑(oxazol‑5‑yl)phenyl)‑4‑(4‑(triꢀuoromethyl)benzyl)pip‑
erazine‑1‑carbothio‑amide (6n) White solid, yield: 67%;
100 μg ml streptomycin (Sigma-Aldrich, USA) at 37 ℃ in
a humidiꢀed atmosphere containing 5% CO . All tumor cell
2
1
3

Chemical Papers
lines were maintained according to the ATCC procedures.
All enzyme assay reagents were purchased from Ronghai
Huakang Biotechnology.
MTT assay
The anti-proliferative activity of selected PHGDH- active
compounds (4a, 5a, 6e, 6n and NCT-503) against cancer
cell lines was evaluated by MTT assay. Brieꢃy, cell lines
were seeded in 96-well plate at 2000 cells per well and then
treated with compounds for 72 h. Ten microliters of MTT
(10 mg/ml) was added and incubated for another 2.5 h at
37 °C. Then, the supernatant ꢃuid was removed and 150 μl/
well DMSO was added for 15–20 min. The absorbance (OD)
of each well was measured at 450 nm by SpectraMAX M5
microplate spectrophotometer (Molecular Devices, CA,
USA). The inhibition rates were calculated and IC values
Enzyme assays
The inhibitory activity of all the synthesized compounds
(
4a–b, 5a–f, 6a–o) against PHGDH was evaluated. The
experimental schematic is illustrated in Fig. 2 (Wang et al.
017). Brieꢃy, 1 μl of test compounds and 50 μl of PHGDH
ꢀnal concentration was 200 nM) were added to the opaque
2
(
9
6-well plate, followed by incubation for 1 h. Then, 49 μl
of mixtures (0.1 mM resazurin; 0.001 U/μl diaphorase;
5
0
+
1
mM hydrazine sulfate; 20 μM NAD ; 0.1 mM 3-PG; the
were calculated by Graph-Pad Prism 7 software (San Diego,
USA) using XY modeling (Nguyen et al. 2018).
above were the ꢀnal concentration) was added to each well.
3
-PG is not in the blank control group. Mixtures were fur-
ther incubated for 1 h. The resulting ꢃuorescence was read
using a ViewLux uHTS Microplate Imager (PerkinElmer).
PHGDH inhibition data were calculated by using the fol-
Molecular docking and 2D simulation
The molecular docking procedure was performed by using
glide docking program of Maestro. A crystal structure of the
catalytic subunit of PHGDH (amino acids (aa) 3-314) with
lowing formula: inhibition rate=1 − (F
− F
)/
No-3PG
compound
was resoruꢀn ꢃuorescence of
(
F
− F
). F
DMSO
DMSO
No-3PG
compound
+
compound well; F
was resoruꢀn ꢃuorescence of DMSO
cofactor, NAD , and substrate analogue, L-malate (1), has
well; and F
was resoruꢀn ꢃuorescence of nonsubstrate
been reported (PDB: 2G76) (Kujundzic et al. 2019). The
No-3PG
+
well. IC values were calculated by Graph-Pad Prism 7 soft-
active site was close to NAD in this structure (Kujundzic
50
ware (San Diego, USA) using XY modeling. NCT-503 was
et al. 2019). Therefore, the crystal structure of 2G76 was
selected to perform molecular docking. Brieꢃy, the structure
2G76 was downloaded and optimized into receptor protein.
Then, receptor grid was generated. The square area centered
used as positive control.
To exclude false positive molecules which inhibited
diaphorase, exclusion experiments were performed on the
active molecules screened. One microliter (1 mM) of test
compounds, positive control (DMSO) and negative control
+
on NAD was set as the docking pocket; 200,000 small mol-
ecule database was then used to dock using standard preci-
sion. Finally, 2D interaction diagrams of docking molecules
were simulated based on the data of docking.
(
no NADH) was added to the opaque 96-well plates, respec-
tively. Then, 50 μl of diaphorase (0.001 U/μl, the ꢀnal con-
centration) was added followed by incubation for 1 h. After
that, 49 μl of mixtures (0.1 mM resazurin; 10 μM NADH)
was added to each well and incubated for 30 min. The data
were analyzed as described above.
Fig. 2 Experimental schematic of PHGDH activity assay in vitro
1
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Chemical Papers
Results and discussion
(Zhang et al. 2018). The synthesis of compounds 4a–b,
5
a–f and 6a–o involved the formation of thiourea bonds
Chemistry
(Hemdan and El-Bordany 2016). One-pot method was
applied in this synthesis process. Firstly, aromatic amine
and TCDI formed active intermediate. The intermediate
was then reacted with the piperazine or piperidine deriva-
tive added later to form the expected product. The average
yield of entire reaction process was 62%.
The synthetic routes of compounds 4a–b, 5a–f and 6a–o
are depicted in Scheme 1. Structures of all synthesized
compounds are shown in Tables 1 and 2. Compound 116
was obtained by one-step reaction, amide condensation
Scheme 1 Conditions: a di(1H-imidazol-1-yl)methanethione, THF, 45 °C, 0.5–1.5 h; b piperazine or piperidine derivatives, Et N, THF, heat to
3
7
5 °C, 1.5–2.5 h, 45–72%; c 1-(bromomethyl)-4-(triꢃuoromethyl)benzene, THF, reꢃux, overnight, 86%
Table 1 Structure of compounds 5a−f
Comp
R
R1
Comp
R
R1
5
a
5b
5
5
c
e
5d
5f
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Chemical Papers
Table 2 Structure of compounds 4a–b and 6a–o
Comp
R
Comp
R
4
6
a
a
4b
6b
6d
6f
6
6
c
e
6
g
6h
6j
6
i
6k
6l
6m
6n
6o
Biology
enzyme activity system was 16 µM. It diꢁers from previ-
ously reported data by less than an order of magnitude.
Therefore, we thought it was reasonable. Compound 5d
from virtual screening displayed better inhibitory activity
against PHGDH than 116; Furthermore, six other deriva-
tives 4a, 5a–b, 6e, 6n–o from 5d showed better activity at
Enzyme assays
The inhibitory activity of synthesized compounds against
PHGDH was evaluated. The results are shown in Table 3.
2
5 μM. However, the other compounds showed low or no
Structure–activity relationship analysis It can be seen from
Table 3 that the IC value of NCT-503 measured by our
activity against PHGDH. Among the six derivatives of bet-
ter activity, 4a showed the best inhibitory activity toward
5
0
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Chemical Papers
Table 3 Inhibitory activity of compounds against PHGDH in vitro
Compound
Structure
No.
Inh%
IC50
(μM)
10 μM
26
25 μM
30
5
d
56
ND
ND
ND
60
5
e
10
9
23
24
20
31
16
5
f
4
b
19
10
7
6
o
a
6
ND
6
b
14
8
30
15
54
6
c
ND
6
d
8
16
38
ND
36
6
e
15
6
f
19
13
25
20
ND
ND
6
g
6
h
15
14
17
22
ND
ND
6
i
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Chemical Papers
Table 3 (continued)
Compound
Inh%
IC50
(μM)
Structure
No.
10 μM
25 μM
6
j
9
9
ND
ND
6k
9
12
26
6
l
12
ND
6
m
8
21
8
14
47
65
ND
27
6
n
4
a
18
5a
30
49
25
5
b
18
8
32
24
51
5
c
ND
NCT-503
43
67
16
1
16
ND
ND
174
a
Inh% values represent the inhibition rates of compounds at 10 μM or 25 μM. Values are the mean of three repeated experiments. ND not
detected
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Chemical Papers
Table 4 IC50 values of compounds against human cancer cell lines
A2780 and an IC value of 7.52 μM against MDA-
50
in vitro
MB-468. It indicated 6n had a good cellular potency
against PHGDH high-expression cell lines. In addi-
tion, these compounds had relatively higher IC values
Compound no. IC50 (μM)
5
0
A2780 MDA-MB-468 MDA-MB-231 HEK293T
against PHGDH low-expression cell line MDA-MB-231
and human embryonic kidney cell line HEK293T. This
indicated that they had good selectivity and low toxicity.
4
5
6
6
a
a
e
15.58
7.73 13.05
27.86 31.06
4.74
22.18 33.95
9.91
63.42
107.1
123.4
30.13
>160
40.53
55.22
34.63
17.34
60.24
n
7.52
Interaction prediction We docked the active molecules
4a, 5a, 6e and 6n, predicted and analyzed the 2D diagram
of their interactions with the surrounding amino acid resi-
dues (Fig. 3). In summary, oxazole formed pi–pi conjuga-
tion with residues close to it. This moiety was indispensa-
ble for maintaining the interactions, which was consistent
with structure–activity analysis above. In addition, hydro-
gen on the thiourea formed hydrogen bond with surround-
ing residues. Other moieties such as thiocarbonyl and
substituted piperazine were essential to keep the suitable
conꢀguration that made the molecule go into the groove.
NCT-503
PHGDH with IC50 value 18 μM, while 5a and 6n had the
similar inhibition activity with IC about 25 μM. In addi-
5
0
tion, compounds 5b, 6b, 6e displayed IC which was lower
5
0
than 55 μM. The preliminary SAR showed that R group
of heterocycle was crucial for maintaining PHGDH activ-
ity, while those with R group of phenyl, naphthyl or alkyl
displayed low inhibitory activity. Generally, compounds
with 3-(oxazol-5-yl) phenyl displayed better activity than
compounds with other R group, such as 4a, 5a–b and 6n.
Among the compounds containing diꢁerent piperazine or
piperidine structures, those with ꢃuorine substituents at
the end commonly had better PHGDH inhibitory activity.
Finally, compounds with low IC were screened to perform
Conclusions
We have designed and synthesized a novel series of thio-
urea derivatives, according to the structural characteristics
of PHGDH inhibitor 5d. These newly synthesized com-
5
0
cell proliferation inhibition experiment.
1
13
Anti‑proliferative activity against human cancer cells lines
in vitro Knockdown of PHGDH is selectively toxic toward
PHGDH-dependent cell lines and minimally toxic toward
PHGDH-independent cell lines. Therefore, treatment of
PHGDH-independent cell lines and PHGDH-dependent cell
lines in dose–response assays with PHGDH inhibitors could
demonstrate that PHGDH inhibitors had a good inhibitory
activity against PHGDH (Pacold et al. 2016).
pounds were characterized by IR, MS, H NMR and
C
NMR. Six other derivatives from 5d showed better activity
toward PHGDH. Compound 4a displayed the best inhibi-
tory activity on PHGDH with IC of 18 μM. The anti-
5
0
proliferative activity of four compounds against four cell
lines (A2780, MDA-MB-468, MDA-MB-231 and 293 T)
was evaluated by MTT assay, and compounds with good
anti-proliferative activity and low toxicity were discov-
ered. Finally, 2D interaction diagrams revealed potential
action modes of active compounds with PHGDH. From the
previous discussion of SAR, we obtained that the enzyme
activity of compound was better when the R group was
3-(oxazol-5-yl) phenyl, 1-methyl-3-phenyl-1h-pyrazol-5-yl
or 1, 3-dimethyl-1h-pyrazol-5-yl. In addition, we found
that heterocyclic groups played a key role in the interac-
tion between molecules and proteins. Based on the infor-
mation above, we concluded that compounds were more
likely to be active when the R group was heterocyclic.
Therefore, this research provided supporting information
for further design and synthesis of PHGDH inhibitors.
MDA-MB-468 and A2780 were PHGDH high-expression
cell lines and PHGDH-dependent. However, MDA-MB-231
was PHGDH low-expression cell lines and PHGDH-inde-
pendent. We could judge whether a compound acted on
PHGDH by activity diꢁerence toward cell lines (MDA-
MB-468, A2780, MDA-MB-231). Human embryonic kidney
cell line HEK293T was used to assess the toxicity to normal
human cell. We further evaluated the anti-proliferative activ-
ity of compounds 4a, 5a, 6e and 6n, which could inhibit the
PHGDH activity. The results are shown in Table 4.
From Table 4, we can see that 6n, which contains
3
-(oxazol-5-yl) phenyl, was the most active compound in
the MTT assay, with an IC value of 4.74 μM against
5
0
Acknowledgements We thank State Key Laboratory of Biother-
1
13
apy, West China Hospital, Sichuan University for H NMR,
C
NMR and HRMS determination. We thank Engineering Teaching
Experiment Center, School of Chemical Engineering for IR deter-
mination. This work was supported by National S&T Major project
(
2018ZX09201018), Project of the National Keypoint Research and
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3

Chemical Papers
Fig. 3 2D interaction diagrams
of 4a, 5a, 6e, 6n. a–d were
interaction simulations of 4a,
5
a, 6e, 6n with surrounding
amino acid residues, respec-
tively. Arrows represent hydro-
gen bonds, and round headlines
represent pi–pi conjugation
Invention Program of China Ministry of Science and Technology
Compliance with ethical standards
(
MOST-2016YFC1303200) and National Natural Science Foundation
of China (81773198).
Conflict of interest On behalf of all authors, the corresponding author
states that there is no conꢃict of interest.
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3

Chemical Papers
fate. Nat Chem Biol 12:452–458. https://doi.org/10.1038/nchem
bio.2070
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