α-Ketothioamide Derivatives: A Promising Tool to Interrogate Phosphoglycerate Dehydrogenase (PHGDH)

Article abstract of DOI:10.1021/acs.jmedchem.6b01166

Given the putative role of PHGDH in cancer, development of inhibitors is required to explore its function. In this context, we established and validated a straightforward enzymatic assay suitable for high-throughput screening and we identified inhibitors with similar chemical scaffolds. Through a convergent pharmacophore approach, we synthesized α-ketothioamides that exhibit interesting in vitro PHGDH inhibition and encouraging cellular results. These novel probes may be used to understand the emerging biology of this metabolic target.

Full text of DOI:10.1021/acs.jmedchem.6b01166

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Brief Article
#-Ketothioamide derivatives: a promising tool to
interrogate the phosphoglycerate dehydrogenase (PHGDH)
Séverine Ravez, Cyril Corbet, Quentin Spillier, Alice Dutu, Anita D Robin,
Edouard Mullarky, Lewis Clayton Cantley, Olivier Feron, and Raphaël Frédérick
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01166 • Publication Date (Web): 13 Jan 2017
Downloaded from http://pubs.acs.org on January 15, 2017
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αꢀKetothioamide derivatives: a promising tool to interrogate
phosphoglycerate dehydrogenase (PHGDH)
≠
≠
Séverine Ravez1 , Cyril Corbet2 , Quentin Spillier1,2, Alice Dutu1, Anita D. Robin3, Edouard Mullarky3, Lewis C. Cantley3,
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Olivier Feron2, Raphaël Frédérick1*
1 Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute (LDRI), Université Catholique de Louꢀ
vain, Bꢀ1200 Brussels, Belgium.
2 Pole of Pharmacology and Therapeutics (FATH), Institut de Recherche Expérimentale et Clinique (IREC), Université Caꢀ
tholique de Louvain, Bꢀ1200 Brussels, Belgium.
3 Meyer Cancer Center, Weill Cornell Medical College, New York, NY 10065. Department of Medicine, Weill Cornell Medꢀ
ical College, New York, NY 10065
KEYWORDS: PHGDH inhibitors, αꢀketothioamide, serine pathway, cancer metabolism
ABSTRACT: Given the putative role of PHGDH in cancer, development of inhibitors is required to explore its function. In this
context, we established and validated a straightforward enzymatic assay suitable for highꢀthroughput screening and we identified
inhibitors with similar chemical scaffolds. Through a convergent pharmacophore approach, we synthesized αꢀketothioamides that
exhibit interesting in vitro PHGDH inhibition and encouraging cellular results. These novel probes may be used to understand the
emerging biology of this metabolic target.
INTRODUCTION
large amount of glycolytic carbon was diverted into serine and
glycine biosynthesis due to genomic amplification of
PHGDH.4,5 In these two studies, RNAiꢀmediated suppression
of PHGDH caused a decrease in survival and growth of cancer
cells that harbored PHGDH amplification or PHGDH overexꢀ
pression but had no effect on lines lacking PHGDH. A recent
study also demonstrated the role of PHGDH in metastasis
formation and stem cell phenotype of breast cancer cells.6
Since 2011, several works extended the purported role of
PHGDH in a large panel of other cancers such as colon cancer,
glioma, lung cancer, thyroid cancer or leukemia.7ꢀ12
In this last decade, important efforts have been made by scienꢀ
tists to characterize the metabolic alterations in tumor cells
and it has been demonstrated that cancer cells exhibit an inꢀ
crease in the uptake of serine and glycine.1 These amino acids
provide essential precursors for the synthesis of proteins,
nucleic acids, and lipids that are crucial to cancer cell growth.2
Moreover, serine/glycine biosynthesis also affects cellular
antioxidative capacity, thus supporting tumor homeostasis.
Serine can be imported from the extracellular compartment via
amino acid transporters but it can also be synthesized by an
intracellular pathway that consists of three successive enzyꢀ
matic reactions (Fig. 1). Phosphoglycerate dehydrogenase
(PHGDH) catalyzes the first step of this pathway and produces
3ꢀphosphohydroxypyruvate (3ꢀPPyr) by NAD+ꢀcoupled oxidaꢀ
tion of 3ꢀphosphoglycerate (3ꢀPG). Subsequently, 3ꢀPPyr is
converted to phosphoserine by phosphoserine aminotransferꢀ
ase 1 (PSAT1) and then to serine by phosphoserine phosphaꢀ
tase (PSPH).
In 2011, Pollari and coworkers described the first clinical
results associated with serine biosynthetic elevation in cancer
cells.3 More precisely, they demonstrated that the three genes
involved in the serine biosynthesis were upregulated in the
highly metastatic MDAꢀMDꢀ231(SA) breast cancer cell line
compared to the parental cell line MDAꢀMDꢀ231. Subsequentꢀ
ly, the role of the first serine synthetic pathway (SSP) enzyme,
PHGDH, as a putative metabolic oncogene has emerged folꢀ
lowing two important publications nearꢀsimultaneously in
2011. Possemato et al. identified PHGDH as a metabolic gene
required for tumorigenesis via an in vivo shRNA screen while
Locasale et al. found that in some cancer cells, a relatively
Figure 1. Schematic representation of the SSP. SSP (repreꢀ
sented in blue) is a branching route of the glycolysis (repreꢀ
sented in red) that contributes to the production of proteins,
nucleic acids and lipids. In addition to catalyzing the oxidation
of 3ꢀPG, PHGDH catalyzes the reduction of αꢀKG to Dꢀ2HG
and stabilizes FOXM1.
The mechanism by which PHGDH supports tumorigenesis is
not yet fully understood. Indeed, it was suggested that serine
production is not the only major role for PHGDH in cancer
cells. This enzyme also can produce metabolites beyond serine
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that are essential for cell proliferation, invasion and tumorꢀ
Page 2 of 14
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igenicity of cancer cells.
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These reports strongly implicate PHGDH as an attractive drug
target in tumors that overexpress PHGDH or have PHGDH
gene amplification.13 Currently, only few specific inhibitors of
PHGDH have been described, severely limiting investigation
into this interesting novel cancer target (Fig. 2).14ꢀ16 To address
this need, we have initiated a study towards the discovery of
PHGDH inhibitors and herein, we describe this novel class of
inhibitors.
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Figure 2. Structure of reported PHGDH inhibitors.
RESULTS AND DISCUSSION
Enzymatic assay development. Prior to screening the comꢀ
pound library, we initially optimize a robust quantitative enꢀ
zymatic assay that is based on PHGDH activity. More preciseꢀ
ly, PHGDH oxidizes 3ꢀPG to 3ꢀPPyr with NAD+ as the elecꢀ
tron acceptor to yield NADH. The formation of 3ꢀPPyr is
therefore directly correlated with the NADH formation and the
enzymatic activity of PHGDH can be monitored by following
the fluorescence intensity (excitation wavelength 340 nm;
emission wavelength 460 nm). After optimization of the assay
(Fig. S1), we have undertaken the process of hit identification.
Figure 3. Process of hit identification. PHGDH inhibition
percentage at 100 µM is represented under each identified hit.
As highlighted in Fig. 3, the screening also highlighted several
inhibitors that present common structural groups. For examꢀ
ple, several compounds contain a nitrogen moiety such as
morpholine, piperazine or cyclohexamine that is often attached
to a thiocarbonyl (8ꢀ11,17). This observation is consistent with
the recent results of Pacold et al. that highlighted PHGDH
inhibitors containing a piperazineꢀ1ꢀcarbothioamide scaffold
(See compound 2).16 Three other compounds (12ꢀ14) showed
an acetophenone part attached to a bulky group such as aniꢀ
line, biphenylmethylamine or aminopyrimidine.
Primary screening. As described in Fig. 3, the primary
screening was carried out on a compound library of 336 moleꢀ
cules from a fragment library and an inꢀhouse compound
collection at high concentration (100 µM). Commercial fragꢀ
ments (4ꢀ7) were purchased from Prestwik Chemical™, and
most of them are derived from actual drugs following smart
fragmentation. The inꢀhouse collection was composed by
original molecules (8ꢀ18) showing a large structural diversity.
Of the 336 samples initially screened, 29 compounds (8.6%)
exhibited an inhibitory effect and among them, 15 molecules
(4.5%) decreased PHGDH activity by greater than 50%.
Among them, three molecules were quinone derivatives
(4,16,18). It was well known that quinones represent a class of
toxicological compounds which can promote a variety of side
effects in vivo, including acute cytotoxicity, immunotoxicity,
and carcinogenesis.17 For this reason, these three molecules
were not selected for the optimization process. The screening
also highlighted promising fragments like 6 that will be the
subject of further optimization studies.
PHGDH inhibitors design. Based on the primary screening,
we have merged the arylketo moiety of compounds 12ꢀ14 and
the highlighted thioamide template to design αꢀketothioamides
(Fig. 4). This operation of design which was undertaken is
sometimes referred to as convergent pharmacophore synthesis.
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O
S
or deplete medium. As depicted in Fig. 5B, 5D, extracellular
Cl
Cl
1
N
N
depletion of serine had low effect on proliferation of high
PHGDHꢀexpressing lines (SiHa and HLꢀ60ꢀshScr) while the
lack of serine considerably decreased the proliferation of the
cell line lacking PHGDH (MDAꢀMBꢀ231 and HLꢀ60ꢀ
shPHGDH). Given that the ability to proliferate in the absence
of extracellular serine is indicative of a high dependence for
serine synthesis, SiHa and HLꢀ60ꢀshScr cell lines should be
sensitive to αꢀketothioamides. Conversely, the identified inꢀ
hibitors should have no effect on MDAꢀMBꢀ231 and HLꢀ60ꢀ
shPHGDH cell lines.
H
NO2
O
Cl
2
12
8
3
4
5
6
59% at 100 ꢀM
96% at 100 ꢀM
O
N
O
N
O
O
N
N
NH
N
R
S
N
O2N
7
13
9
54% at 100 ꢀM
37% at 100 ꢀM
8
9
10
N
O
N
H
F3C
N
N
N
N
H
S
11
O2N
CF3
14
13% at 100 ꢀM
11
12
13
14
80% at 100 ꢀM
Figure 4. Process of convergent pharmacophore operation to
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design αꢀketothioamides.
Chemistry. To warrant a proof of concept, a limited set of αꢀ
ketothioamides was synthesized (Scheme 1). Firstly, the broꢀ
mination of appropriate acetophenone (1 eq) was carried out in
the presence of dibromine (1.2 eq) and TBAB in chloroform.
The resulting product was engaged in the WillgerodtꢀKindler
reaction without any further purification according to a reportꢀ
ed procedure.18 DMF, octasulfur (1.5 eq), and morpholine (3
eq) were introduced into the reaction mixture and the reaction
proceeded to completion at room temperature.
Scheme 1. Synthetic pathway of αꢀketothioamide derivatives.a
Figure 5. Ability of the selected cancer cell lines to proliferate
in serine replete or deplete medium. (A and C) Representative
immunobolot for PHGDH. (B and D) Cell growth extent (for 5
days) in either serineꢀreplete (+Ser) or serineꢀdeplete (ꢀSer)
medium. Data are shown as mean ± SEM (n ≥ 3).
O
O
O
O
Br
N
ii
i
R
R
R
S
19ꢀ35
19: R = H 20: R = 2ꢁF 23: R = 2ꢁCl 26: R = 2ꢁBr 29: R = 2ꢁI 32: R = 2ꢁNO2 35: R = 4ꢁPh
21 24 27 30
: R = 3ꢁI
33
: R = 3ꢁF
: R = 3ꢁCl
: R = 3ꢁBr
: R = 3ꢁNO2
34: R = 4ꢁNO2
22: R = 4ꢁF 25: R = 4ꢁCl 28: R = 4ꢁBr 31: R = 4ꢁI
Subsequently, the selected molecules were evaluated on the
following systems: (i) in MDAꢀMBꢀ231 lacking PHGDH, (ii)
in SiHa and HLꢀ60 expressing PHGDH in serineꢀreplete or
serineꢀdeplete medium and (iii) in HLꢀ60 with PHGDH
knockdown (shPHGDH) (Table 1). Assays in depleteꢀserine
medium were expected to enhance the de novo serine syntheꢀ
sis and increase the cell sensitivity toward the inhibitors.
a Reagents and conditions: (i) Br2, CHCl3, TBAB, rt for 2h; (ii)
S8, morpholine, DMF, rt.
Enzymatic evaluation. All the synthesized compounds (19ꢀ
35) were evaluated on PHGDH thanks to a coupledꢀenzyme
assay (Table 1). The compound 25 is the strict fusion between
hit compounds 12 and 8 identified during the primary screenꢀ
ing. Given the IC50 values of these three compounds on
PHGDH (8.7, 75.2 and 16.0 µM, respectively), we can valiꢀ
date the design based on the convergent pharmacophore synꢀ
thesis. In addition, within this concise series of αꢀ
ketothioamides, it appears that the paraꢀsubstitution in the aryl
moiety is more favorable than the substitution in the ortho or
metaꢀposition as illustrated by the fluoro (22: 68.9 µM), chloro
(25: 8.7 µM), and nitro (34: 35.1 µM) derivatives. However,
only the 4ꢀchloro derivative 25 exhibits a better inhibition than
the nonꢀsubstituted αꢀketothioamide 19.
As expected, all tested molecules had no effect on the MDAꢀ
MBꢀ231 and HLꢀ60ꢀshPHGDH cell lines sensitive to serine
removal. In contrast, the inhibitors (except compound 22)
exhibit interesting inhibition of SiHa and HLꢀ60 cell proliferaꢀ
tion in serineꢀreplete (+Ser) medium. For example, compound
25 shows IC50 values of 21.0 and 50.5 µM on SiHa and HLꢀ
60, respectively. According to plan, the amino acid depletion
(ꢀSer) enhanced the efficacy of the inhibitors by a factor of 3
for SiHa line and a factor 1.5 to 5.5 for the HLꢀ60 line. In
these conditions, compound 25 had an IC50 of 6.6 and 15.5 µM
on SiHa and HLꢀ60, respectively. Finally, the preliminary
SARs established were confirmed in the cellular assay. Indeed,
orthoꢀ and metaꢀchloro compounds (23 and 24) that show no
inhibition in the in vitro assay, also show no cell proliferation
inhibition on all systems (IC50 > 100 µM).
Cellular assays. To determine if the preliminary SARs can be
translated to cellular activities, the nonꢀsubstituted compound
(19), the 4ꢀsubstituted derivatives (22, 25, 28, 31 and 34) as
well as the 2ꢀ and 3ꢀchloro αꢀketothioamides (23 and 24) were
evaluated in several cellꢀbased assays (Table 1).
Mechanism of action of compound 19. To characterize the
mechanism by which αꢀketothioamides inhibit PHGDH, we
have undertaken competition and rapid dilution assays with
the leader of the series (19). Inhibition constants (Ki) for comꢀ
pound 19 with respect to 3ꢀPG and NAD+ were determined
(Fig. 6).
First of all, the proliferation ability of the three selected cancer
cell lines (MDAꢀMBꢀ231, human breast adenocarcinoma cells
(PHGDHꢀ); SiHa, human cervix carcinoma cells (PHGDH+);
HLꢀ60: human promyelocytic leukemia cells (PHGDH+); see
immunoblots in Fig. 5A, 5C) was evaluated in serine replete
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Table 1. PHGDH inhibition and cell proliferation inhibition results of compounds 19ꢀ35.
1
2
3
4
5
6
7
8
Cell proliferation inhibition (IC50, µM)a
PHGDH
inhibition
(IC50, µM) a
SiHa
HLꢀ60 shCTRL
IC50+Ser
Code
R
MDAꢀ
MBꢀ231
HLꢀ60
shPHGDH
IC50+Ser
/IC50ꢀSer
+ Ser
ꢀ Ser
+ Ser
ꢀ Ser
/IC50ꢀSer
12
8
19
75.2
16.0
30.9
> 200
> 200
68.9
> 200
> 200
8.7
160.7
> 200
130.7
> 200
> 200
177.7
> 200
88.1
> 100
> 100
> 100
nd
> 100
19.1
71.7
nd
57.5
5.6
7.9
nd
> 1.7
3.4
9,1
nd
nd
> 2.3
ꢀ
> 100
24.3
> 100
nd
83
> 1.2
3.5
> 5.5
nd
> 100
7.0
18.3
nd
> 100
> 100
nd
H
9
20 2ꢁF
21 3ꢁF
22 4ꢁF
23 2ꢁCl
24 3ꢁCl
25 4ꢁCl
26 2ꢁBr
27 3ꢁBr
28 4ꢁBr
29 2ꢁI
30 3ꢁI
31 4ꢁI
32 2ꢁNO2
33 3ꢁNO2
34 4ꢁNO2
35 4ꢁPh
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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49
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nd
nd
nd
nd
nd
nd
nd
> 100
> 100
> 100
> 100
nd
nd
> 100
nd
nd
> 100
nd
nd
> 100
nd
> 100
> 100
> 100
21.0
nd
nd
20.4
nd
nd
25.7
nd
nd
22.8
nd
43.3
> 100
> 100
6.6
nd
> 100
> 100
> 100
50.5
nd
64.4
> 100
> 100
15.5
nd
1.5
ꢀ
> 100
> 100
> 100
> 100
nd
nd
> 100
nd
nd
> 100
nd
nd
> 100
nd
ꢀ
ꢀ
3.2
nd
nd
2.6
nd
nd
2.8
nd
nd
3
3.3
nd
nd
7.9
nd
nd
9.3
nd
nd
7.6
nd
nd
nd
nd
37.2
nd
16.9
nd
2.2
nd
nd
nd
nd
19.6
nd
7.1
nd
2.8
nd
nd
nd
nd
35.1
> 200
17.6
nd
6.0
nd
2.9
nd
nd
a
All experiments to determine IC50 values were performed with at least triplicates at each compound dilution, and all IC50 values
were averaged when determined in two or more independent experiments. nd = not determined.
As highlighted in Fig. 6A, 6B, the αꢀketothioamide 19 inhibꢀ
its PHGDH in a noncompetitive mode of action with respect
to both substrates. The determined Ki values are 40 ± 2 ꢁM
and 27 ± 7 ꢁM for 3ꢀPG and NAD+, respectively. Moreover,
the rapid dilution experiment in Fig. 6C suggests that comꢀ
pound 19 may be a covalent inhibitor since enzymatic activiꢀ
ty is not recovered after dilution.
CONCLUSION
Recent studies implicate PHGDH as an attractive drug target
in tumors that overexpress PHGDH or amplify the PHGDH
gene. In the present work, we explored whether this enzyme
was druggable with small molecules using a screening apꢀ
proach. Initially, we established a straightforward fluoresꢀ
cenceꢀbased enzymatic assay that allowed us to screen
around 350 molecules. Structure analyses of these hits highꢀ
lighted common structural elements such as a thioamide or
acetophenone part. On these observations, a convergent
pharmacophore approach led to the synthesis of αꢀ
ketothioamides that appear as promising in vitro PHGDH
inhibitors. Moreover, cellular assays confirmed that the
identified inhibitors selectively abrogated the proliferation of
cancer cell with elevated PHGDH expression. These encourꢀ
aging results open the way to the development of a novel
series of PHGDH inhibitors around the αꢀketothioamide
scaffold.
EXPERIMENTAL SECTION
General chemistry. All reagents were purchased from chemꢀ
ical suppliers and used without purification. Thinꢀlayer
chromatography (TLC) was performed using silica gel 60
F254 plates, with observation under UV when necessary.
Melting points were recorded on an Electrothermal IA9000
Figure 6. Mechanism of action of compound 19. Inhibition
constants (Ki) were determined by titrating (A) 3ꢀPG and (B)
NAD+ at four concentrations of 19 (in nanomolar). (C)
Washout experiment of compound 19.
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1
melting point system. H NMR spectra were recorded an
AVANCE II 400 MHz Bruker spectrometer with CDCl3 or
DMSOꢀd6 as the solvent. 13C NMR spectra were recorded at
100 MHz. All coupling constants are measured in hertz (Hz),
and the chemical shifts (δH and δC) are quoted in parts per
million (ppm) relative to TMS (δ0), which was used as the
internal standard. Data are reported as follows: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, br = broad, m = multiplet), integration, and coupling
constant (Hz). Highꢀresolution mass spectroscopy (HRMS)
analyses were carried out on a LTQꢀOrbitrap XL hybrid
mass spectrometer (Thermo Fisher Scientific, Bremen, Gerꢀ
many). Data were acquired in positive ion mode using fullꢀ
scan MS with a mass range of 100ꢀ1000 m/z. The orbitrap
operated at 30000 resolutions (FWHM definition). All experꢀ
imental data were acquired using daily external calibration
prior to data acquisition. Appropriate tuning of the elecꢀ
trospray ion source was done. The following electrospray
inlet conditions were applied: flow rate, 100 µL minꢀ1; spray
voltage, 5 kV; sheath gas (N2) flow rate, 20 a.u.; auxiliary
gas (N2) flow rate, 10 a.u.; capillary temperature, 275 °C;
capillary voltage, 45 V; tube lens, 80V. Highꢀperformance
liquid chromatography (HPLC) analyses were performed on
a LC system using YMCꢀTriart Cꢀ18 (250 mm x 4.6 mm,
5ꢁm) column as the stationary phase. Mobile phase conꢀ
tained water/CH3CN (30:70, v/v) and maintained isocraticalꢀ
ly at the flow rate of 1 mL/min. The column temperature was
maintained at room temperature. The peaks were monitored
at the wavelength of 215 nm. The purity of all compounds
tested was greater than 95%, as determined by HPLC and 1H
NMR.
(C=S). HRMS (ESI+): m/z calcd for C12H13NO2S (M + H)+
1
2
3
4
5
6
7
8
236.0739, found 236.0737.
Compounds 20ꢀ35 were synthesized with the same condiꢀ
tions and procedure. The full descriptive paragraph of each
compound was described in the supplementary materials.
1ꢀ(2ꢀFluorophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (20).
Starting from the 1ꢀ(2ꢀfluorophenyl)ethanone (1.50 g, 11.10
mmol), the title compound 20 was obtained after recrystalliꢀ
zation in methanol as a yellow solid (39%).
9
1ꢀ(3ꢀFluorophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (21).
Starting from the 2ꢀbromoꢀ1ꢀ(3ꢀfluorophenyl)ethanone (0.50
g, 2.30 mmol), the title compound 21 was obtained after
recrystallization in methanol as a yellow solid (23%).
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12
13
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16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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1ꢀ(4ꢀFluorophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (22).
Starting from the 2ꢀbromoꢀ1ꢀ(4ꢀfluorophenyl)ethanone (0.50
g, 2.30 mmol), the title compound 22 was obtained after
recrystallization in methanol as a beige solid (41%).
1ꢀ(2ꢀChlorophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (23).
Starting from the 2ꢀbromoꢀ1ꢀ(2ꢀchlorophenyl)ethanone (0.50
g, 2.14 mmol), the title compound 23 was obtained after
recrystallization in acetonitrile as a yellow solid (36%).
1ꢀ(3ꢀChlorophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (24).
Starting from the 1ꢀ(3ꢀchlorophenyl)ethanone (2.00 g, 12.90
mmol), the title compound 24 was obtained after purification
by silica gel chromatography (cyclohexane/EtOAc, 8:2) as a
yellow solid (43%).
1ꢀ(4ꢀChlorophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (25).
Starting from the 2ꢀbromoꢀ1ꢀ(2ꢀchlorophenyl)ethanone (0.50
g, 2.14 mmol), the title compound 25 was obtained after
recrystallization in acetonitrile as a yellow solid (38%).
General procedure for the synthesis of αꢀketothioamide
analogs (19ꢀ35). To a stirred solution of ethanone derivative
(1 eq) in chloroform was added dropwise a solution of diꢀ
bromine (1.2 eq) in chloroform. After 2 hours, the solvent
was evaporated in vacuo to give a crude oil consisting mainꢀ
ly of 2ꢀbromoꢀ1ꢀ(substituted)ꢀethanone compound along
with trace amounts of 2,2ꢀdibromoꢀ1ꢀ(substituted)ꢀethanone
compound. The mixture was used without purification in the
next step. To the synthesized or commercial 2ꢀbromoꢀ1ꢀ
(substituted)ꢀethanone derivative were added in sequence
DMF, cyclooctasulfur (1.5 eq) and morpholine (3 eq). The
mixture was then stirred at room temperature. After compleꢀ
tion, the reaction mixture was quenched with distilled water
to give a precipitate which was further washed with distilled
water. The residue was recrystallized or purified by silica gel
chromatography if necessary.
1ꢀ(2ꢀBromophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (26).
Starting from the 2ꢀbromoꢀ1ꢀ(2ꢀbromophenyl)ꢀethanone
(0.50 g, 1.81 mmol), the title compound 26 was obtained
after recrystallization in ethanol as a white solid (52%).
1ꢀ(3ꢀBromophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (27).
Starting from the ꢀbromoꢀ1ꢀ(3ꢀbromophenyl)ꢀethanone (0.50
g, 1.81 mmol), the title compound 27 was obtained after
recrystallization in methanol as a white solid (26%).
1ꢀ(4ꢀBromophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (28).
Starting from the 2ꢀbromoꢀ1ꢀ(4ꢀbromophenyl)ꢀethanone
(0.50 g, 1.81 mmol), the title compound 28 was obtained
after recrystallization in cyclohexane as a white solid (19%).
1ꢀ(2ꢀIodophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
(29).
Starting from the 2ꢀbromoꢀ1ꢀ(2ꢀiodophenyl)ꢀethanone (1.00
g, 4.00 mmol), the title compound 29 was obtained after
purification by silica gel chromatography (cyclohexꢀ
ane/EtOAc, 8:2) as a yellow oil (47%).
1ꢀPhenylꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone (19). Brown
solid (73%). This compound was synthesized according to
the general procedure described above. Acetophenone (2.00
g, 16.60 mmol) and dibromine (1.01 mL, 19.90 mmol) were
mixed in chloroform (15 mL) to obtain the 2ꢀbromoꢀ1ꢀ
phenylꢀethanone and this intermediate was reacted in a seꢀ
cond time with morpholine (4.34 mL, 49.80 mmol) and
sulfur (0.79 g, 24.90 mmol) in DMF (10 mL). Methanol was
used for recrystallization to afford the title compound. Rf 0.2
1ꢀ(3ꢀIodophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
(30).
Starting from the 2ꢀbromoꢀ1ꢀ(3ꢀiodophenyl)ꢀethanone (1.00
g, 4.06 mmol), the title compound 30 was obtained after
purification by silica gel chromatography (cyclohexꢀ
ane/EtOAc, 8:2) as a yellow solid (51%).
1ꢀ(4ꢀIodophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
(31).
1
(cyclohexane/EtOAc 8:2). Mp: 110ꢀ112°C. H NMR (400
Starting from the 2ꢀbromoꢀ1ꢀ(4ꢀiodophenyl)ꢀethanone (1.00
g, 4.06 mmol), the title compound 31 was obtained after
purification by silica gel chromatography (cyclohexꢀ
ane/EtOAc, 8:2) as a yellow solid (63%).
MHz, CDCl3): δH (ppm) 3.59ꢀ3.62 (t, 2H, J = 4.8 Hz), 3.69ꢀ
3.71 (t, 2H, J = 4.8 Hz), 3.90ꢀ3.92 (t, 2H, J = 4.8 Hz), 4.33ꢀ
4.35 (t, 2H, J = 4.8 Hz), 7.48ꢀ7.52 (m, 2 ArH), 7.59ꢀ7.65 (m,
1 ArH), 7.99ꢀ8.01 (D, 2 ArH, J = 8.2 Hz). 13C NMR (100
MHz, CDCl3): δC (ppm) 47.13, 51.95, 66.40, 66.52, 128.99
(2C), 129.86 (2C), 133.26, 134.48, 187.90 (C=O), 195.70
1ꢀ(2ꢀNitrophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
Starting from the 2ꢀbromoꢀ1ꢀ(2ꢀnitrophenyl)ꢀethanone (0.50
(32).
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g, 2.05 mmol), the title compound 32 was obtained after
recrystallization in a mixture of cyclohexane/EtOAc (8:2) as
a yellow solid (26%).
Presto Blue assay (Life Technologies) according to manufacꢀ
turer’s instructions.
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Immunoblots. Protein extraction and western blot analysis
were carried out as reported elsewhere (Polet et al 2016
Oncotarget).
1ꢀ(3ꢀNitrophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
(33).
Starting from the 2ꢀbromoꢀ1ꢀ(3ꢀnitrophenyl)ꢀethanone (1.50
g, 6.17 mmol), the title compound 33 was obtained after
recrystallization in methanol as a yellow solid (73%).
Dilution experiment. Compound 19 (160 µM) or DMSO
control was incubated with the enzyme mix for 45min at
37oC. At the end of the incubation the enzyme drug mix was
diluted 10ꢀfold using assay buffer and concentrated back to
the original volume by centrifuging through a 10kD molecuꢀ
lar weight cutꢀoff filter. Subsequently enzyme activity was
measured via the PHGDH assay. An undiluted compound
(160 µM) condition was included as a positive control for
inhibition.
1ꢀ(4ꢀNitrophenyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
(34).
Starting from the 2ꢀbromoꢀ1ꢀ(4ꢀnitrophenyl)ꢀethanone (1.50
g, 6.17 mmol), the title compound 34 was obtained after
recrystallization in methanol as a yellow solid (47%).
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25
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1ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ2ꢀmorpholinoꢀ2ꢀthioxoethanone
(35). Starting from the 1ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ2ꢀ
bromoethanone (1.00 g, 3.64 mmol), the title compound 35
was obtained after purification by silica gel chromatography
(cyclohexane/EtOAc, 8:2) as a yellow solid (61%).
ASSOCIATED CONTENT
Primary PHGDH Screen. PHGDH inhibition assay was
performed in black polypropylene 96ꢀwell plates (Sigmaꢀ
Aldrich) using NAD (SigmaꢀAldrich) as cofactor and 3ꢀ
phosphoglycerate (SigmaꢀAldrich) as substrate. Tested comꢀ
pounds were dissolved in DMSO. The final concentration of
DMSO in assay solutions was 10 %, which was shown to
have no effect on PHGDH activity. The reaction volume per
well was 50 µL. Each assay was carried out in triplicate.
Briefly, a solution buffer (100 mM Tris HCl buffer (pH 8.8),
400 mM NaCl, 0.2 mM DTT, 1 mM NAD and 0.5 mM 3ꢀ
PG) was added at various concentrations of tested comꢀ
pounds. Then, an amount of 100 ng of PHGDH (human
recombinant protein, BPS Bioscience) is added to start the
reaction. Finally, the product formation, correlated with
NADH formation, is instantly monitored for 7 min at 25°C
using a SpectraMax spectrophotometer at an excitation
wavelength of 360 nm and emission wavelength of 460 nm.
1
Supporting Information. Synthetic details, H NMR, 13C NMR,
enzymatic assay optimization and molecular formula strings.
This material is available free of charge via the Internet at
http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
* Professor Raphaël Frédérick,
eꢀmail: raphael.frederick@uclouvain.be
phone: 32 (2)764 73 41, fax 32 (2)764 73 63.
Author Contributions
≠ SR and CC equally contributed to this work and share first
author position. The manuscript was written through contribuꢀ
tions of all authors. All authors have given approval to the final
version of the manuscript.
PHGDH assay. PHGDH activity was measured in 96ꢀwell
plates (96 ꢁL per well) at 37 °C by monitoring resorufin
fluorescence (Ex 550 nM/Em 580 nM) over time with a
FLUOstar Omega (BMG Labtech). PSAT1 was included to
prevent product inhibition of PHGDH. Assays were perꢀ
formed in PHGDH assay buffer (50 mM Tris, pH 8.5, and 1
mM EDTA). Substrates and enzyme concentrations were as
follows: 3ꢀPG, 240 ꢁM; NAD+, 120 ꢁM; glutamate, 30 mM;
resazurin, 0.1 mM; PHGDH, 12 ng/ꢁL; PSAT1, 20 ng/ꢁL,
diaphorase 0.18 µg/µL except for Ki measurements where
one of the PHGDH substrates was held constant at 3 mM
whereas the other substrate was titrated. For Ki measureꢀ
ments, drug and enzyme were preincubated for 30 min.
Initial rate plots were fit using Prism 6. Chemicals were
purchased from Sigma.
Notes
Disclosure of Potential Conflicts of Interest: A patent applicaꢀ
tion has been submitted based on the results described in this
manuscript. L.C.C. owns equity in, receives compensation
from, and serves on the Board of Directors and Scientific
Advisory Board of Agios Pharmaceuticals. Agios Pharmaꢀ
ceuticals is identifying metabolic pathways of cancer cells
and developing drugs to inhibit such enzymes in order to
disrupt tumour cell growth and survival. The authors declare
that they have no other conflicts of interest.
ACKNOWLEDGMENT
We are grateful to Laurenne Petit, Pr. Jacques Poupaert and
Romain Marteau for their participation in this project and to Dr.
MarieꢀFrance Hérent and Pr. Giullo Muccoli (UCL, BPBL) for
helpful discussions and assistance with HRMS. This work was
supported by a J. Maisin Foundation grant, the Fondatioun
Kriibskrank Kanner (Luxembourg), a Crédit de Recherche
(CDR) grant from the F.R.S.ꢀFNRS, and an Action de Recherꢀ
che Concertée (ARC 14/19ꢀ058) grant from the Fédération
WallonieꢀBruxelles. QS is a Télévie Research Fellow. L.C.C.
was supported by NIH grants P01 CA117969 and
P01CA120964.
Cell models and cytotoxicity assay. All cell lines were acꢀ
quired in the last 3 years from ATCC where they are regularꢀ
ly authenticated by short tandem profiling. Cells were stored
according to the supplier’s instructions and used within 6
months after resuscitation of frozen aliquots. SiHa cervix
carcinoma cells and MDAꢀMBꢀ231 breast cancer cells were
routinely cultured in DMEM supplemented with 10% FBS
and antibiotics. For the cytotoxicity assay, cells were seeded
at low density I 96ꢀwell plates in serine containing media.
Media were then aspirated and cells were incubated in fresh
serineꢀreplete orꢀdeplete media containing drugs or vehicle
(DMSO). Cells were grown for 5 days at 37°C with drug and
media changed daily before assaying cell viability using a
ABBREVIATIONS
3ꢀPG, 3ꢀphosphoglycerate; 3ꢀPPyr, 3ꢀphosphohydroxypyruvate;
NAD, nicotinamide adenine dinucleotide; PHGDH, 3ꢀ
phosphoglycerate dehydrogenase; PSA, molecular polar surface
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serine availability complements the inhibition of the glutamine
area; PSAT1, phosphoserine amintransferase 1; PSPH, phosꢀ
metabolism to block leukemia cell growth. Oncotarget 2016, 7,
1765ꢀ1776.
12 Fan, J.; Teng, X.; Liu, L.; Mattaini, K. R.; Looper, R. E.; Vander
Heiden, M. G.; Rabinowitz, J. D. Human phosphoglycerate dehyꢀ
drogenase produces the oncometabolite Dꢀ2ꢀhydroxyglutarate. ACS
Chem. Biol. 2015, 10, 510–516.
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7
8
phoserine phosphatase; TNBC, tripleꢀnegative breast cancer.
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Schematic representation of the SSP. SSP (represented in blue) is a branching route of the glycolysis
(represented in red) that contributes to the production of proteins, nucleic acids and lipids. In addition to
catalyzing the oxidation of 3ꢀPG, PHGDH catalyzes the reduction of αꢀKG to Dꢀ2HG and stabilizes FOXM1.
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Structure of reported PHGDH inhibitors.
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Process of hit identification. PHGDH inhibition percentage at 100 µM is represented under each identified hit.
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Process of convergent pharmacophore operation to design αꢀketothioamides.
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Ability of the selected cancer cell lines to proliferate in serine replete or deplete medium. (A and C)
Representative immunobolot for PHGDH. (B and D) Cell growth extent (for 5 days) in either serineꢀreplete
(+Ser) or serineꢀdeplete (ꢀSer) medium. Data are shown as mean ± SEM (n ≥ 3).
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Mechanism of action of compound 19. Inhibition constants (Ki) were determined by titrating (A) 3-PG and
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Synthetic pathway of αꢀketothioamide derivatives.
148x44mm (300 x 300 DPI)
ACS Paragon Plus Environment