Structure–activity relationships (SARs) of α- ketothioamides as inhibitors of phosphoglycerate dehydrogenase (PHGDH)

Article abstract of DOI:10.3390/ph13020020

For many years now, targeting deregulation within cancer cells’ metabolism has appeared as a promising strategy for the development of more specific and efficient cancer treatments. Recently, numerous reports highlighted the crucial role of the serine synthetic pathway, and particularly of the phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the pathway, to sustain cancer progression. Yet, because of very weak potencies usually in cell-based settings, the inhibitors reported so far failed to lay ground on the potential of this approach. In this paper, we report a structure–activity relationship study of a series of α-ketothioamides that we have recently identified. Interestingly, this study led to a deeper understanding of the structure–activity relationship (SAR) in this series and to the identification of new PHGDH inhibitors. The activity of the more potent compounds was confirmed by cellular thermal shift assays and in cell-based experiments. We hope that this research will eventually provide a new entry point, based on this promising chemical scaffold, for the development of therapeutic agents targeting PHGDH.

Full text of DOI:10.3390/ph13020020

ASrttirclue cture–Activity Relationships (SARs) of α‐
Ketothioamides as Inhibitors of Phosphoglycerate
Dehydrogenase (PHGDH)
1,2,†
3,†
4
2
1,2
Quentin Spillier , Séverine Ravez , Judith Unterlass , Cyril Corbet , Charline Degavre
,
2
1,
Olivier Feron and Raphaël Frédérick *
1
Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute (LDRI), Université
Catholique de Louvain, 1200 Brussels, Belgium; Quentin.Spillier@nyulangone.org (Q.S.);
charline.degavre@student.uclouvain.be (C.D.)
2
Pole of Pharmacology and Therapeutics (FATH), Institut de Recherche Expérimentale et Clinique (IREC),
Université Catholique de Louvain, 1200 Brussels, Belgium; cyril.corbet@uclouvain.be (C.C.);
olivier.feron@uclouvain.be (O.F.)
UMR‐S1172—JPArc—Centre de Recherche Jean‐Pierre AUBERT Neurosciences et Cancer, Université de
Lille, Inserm, CHU Lille, 59000 Lille, France; severine.ravez@univ‐lille.fr
Science for Life Laboratory, Department of Oncology‐Pathology, Karolinska Institutet, 171 21 Solna,
Sweden; judith_unterlass@web.de
3
4
* Correspondence: raphael.frederick@uclouvain.be; Tel.: +32‐2‐764‐73‐41; Fax: +32‐2‐764‐73‐63
† These authors equally contributed to the work and share the first authorship position.
Received: 3 December 2019; Accepted: 14 January 2020; Published: 22 January 2020
Abstract: For many years now, targeting deregulation within cancer cells’ metabolism has appeared
as a promising strategy for the development of more specific and efficient cancer treatments.
Recently, numerous reports highlighted the crucial role of the serine synthetic pathway, and
particularly of the phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the pathway, to
sustain cancer progression. Yet, because of very weak potencies usually in cell‐based settings, the
inhibitors reported so far failed to lay ground on the potential of this approach. In this paper, we
report a structure–activity relationship study of a series of α‐ketothioamides that we have recently
identified. Interestingly, this study led to a deeper understanding of the structure–activity
relationship (SAR) in this series and to the identification of new PHGDH inhibitors. The activity of
the more potent compounds was confirmed by cellular thermal shift assays and in cell‐based
experiments. We hope that this research will eventually provide a new entry point, based on this
promising chemical scaffold, for the development of therapeutic agents targeting PHGDH.
Keywords: PHGDH; α‐ketothioamides; serine synthesis pathway inhibitors
1. Introduction
The ability of cancer cells to reprogram their metabolism was demonstrated in numerous studies
over the past two decades [1–3]. These metabolic modifications are used by cancer cells in order to
proliferate in harmful environments (hypoxic zones, deficiencies in the external supply of glucose
and amino acids) by producing certain metabolites and antioxidants more efficiently. It quickly
became apparent that the development of treatments specifically targeting these deregulated
metabolic pathways would reduce tumor development while significantly limiting the side effects
associated with conventional non‐targeted therapies [4].
In this purpose, among the many pathways studied, the serine synthesis pathway appeared to
be a promising target [5,6]. Indeed, this pathway plays a central role in one‐carbon metabolism,
methylation processes, and allows the cells to produce the building blocks required for their
Pharmaceuticals 2020, 13, 20; doi:10.3390/ph13020020
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proliferation [7]. Serine can also be used to produce glutathione and therefore offers resistance against
the oxidative stress associated with intense proliferation [8]. In addition, the three enzymes
constituting this pathway (phosphoglycerate dehydrogenase (PHGDH), PSAT‐1, and PSPH) are
overexpressed in many cancer types (breast cancer, melanoma, glioma) and often associated with
poor prognosis in the patient [9].
Phosphoglycerate dehydrogenase (PHGDH) is the first enzyme to catalyze the rate‐limiting step
of the serine synthesis pathway. Recently, its study highlighted key roles in diverting glycolytic flow
to the serine pathway leading to a tumor invasion process. PHGDH is overexpressed and/or
genomically amplified in more than 16% of cancer lines [10]. Moreover, the overexpression of this
protein appears to lead to greater metastatic invasion in animal models and could also be correlated
with greater aggressiveness and lower survival rates [11,12]. Finally, it was demonstrated that
inhibition or silencing of this enzyme can selectively impact tumor growth while sparing healthy cells
[13]. Therefore, in recent years, there has been an increased interest in the development of small
molecule PHGDH inhibitors.
Since 2015, several chemical series were identified to inhibit PHGDH enzymatic activity (Figure
+
1). Two of them are NAD ‐competitive and thus interact with the Rossmann fold (BI‐4924 and
compound 18). These compounds exhibit PHGDH inhibition in the submicromolar range in isolated
enzyme assays but have poor cellular efficacy notably because of the competition with a high
+
intracellular NAD concentration [14,15]. The other series of inhibitors (CBR‐5884, NCT‐503,
disulfiram, Azacoccone, PKUMLD‐WQ‐220, Ixocarpolactone) have an allosteric mode of action, and
+
although less effective than the NAD ‐competitive compounds, they are usually characterized by a
better inhibition profile on cancer cell lines in in vitro and in vivo xenograft models. However, for
most of these allosteric inhibitors, their precise sites of action are yet to be determined or have been
concluded from docking studies so far [16–21].
Despite the interest in targeting PHGDH and the increasing number of reported inhibitors, the
weak in cellulo and in vivo efficacy of the inhibitors hamper the understanding of the role PHGDH
plays in cancer progression. To date, the developed compounds were mainly used to study the serine
synthesis pathway in restoring the sensitivity of cancer cells to conventional chemotherapy
(bortezomib, doxorubicin, sorafenib) [22–24]. The development of more effective PHGDH inhibitors
to investigate the serine synthesis pathway in cancer biology hence remains necessary.
Figure 1. Representative phosphoglycerate dehydrogenase (PHGDH) inhibitors and their IC50. (A)
Allosteric inhibitors. (B) NAD competitive inhibitors.

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In 2017, our group identified a series of PHGDH inhibitors based on an α‐ketothioamide motif
and endowed with micromolar inhibitory potency (Figure 1A, compounds 1 and 2). [25] Although
these compounds remain only moderately active on the isolated target, they are characterized by an
excellent ligand efficiency (LE) of around 0.34. Moreover, these molecules demonstrated their ability
to reduce the proliferation of cancer cell lines overexpressing PHGDH (SiHa, HL‐60) without
affecting proliferation in cell lines with little or no expression of this enzyme (MDA‐MB‐231, HL‐60
shPHGDH). The study also identified these molecules as irreversible and non‐competitive PHGDH
inhibitors [25]. Preliminary structure–activity relationships (SARs) were undertaken and revealed
that substituents, notably halogens, introduced in the para position enhanced PHGDH inhibition
whereas ortho‐ or meta‐ substitutions led to a decrease of the PHGDH inhibitory potency. Since α‐
ketothioamides proved to be useful as PHGDH inhibitors, we put significant effort to explore the
SARs around this series.
2. Results and Discussion
Compound 1 is characterized by an aryl group (part A) and a morpholine moiety (part C) that
are connected by an α‐ketothioamide linker (part B) (Figure 1). In this manuscript, the SAR study was
investigated by: (1) modulation of the aromatic moiety or by insertion of alkyl groups on part A; (2)
introduction of different amino moieties on part C; and (3) variation of the nature of the linker (part
B). As a result, a library of 39 compounds was designed, synthesized, and evaluated in vitro using a
PHGDH coupled assay at a single compound concentration of 150 μM.
2.1. Chemistry
The α‐ketothioamides 1–21 were obtained using a two‐step procedure involving the
bromination of the corresponding acetone (1 equiv.) derivatives when necessary in the presence of
bromine (1.2 equiv.) and tetra‐n‐butylammonium bromide (TBAB) in chloroform (Scheme 1). The
resulting product or the commercially available brominated compound was engaged in the
Willgerodt–Kindler reaction without any further purification according to a reported procedure. [25]
DMF, octasulfur (1.5 equiv.), and appropriated amine (3 equiv.) were introduced into the reaction
mixture and the reaction proceeded to completion at room temperature.
a
a
Scheme 1. Synthetic pathway of α‐ketothioamide derivatives 1–21, 38, and 39
.
Reagents and
conditions: (i) Br2, CHCl3, TBAB, r.t., for 2 h; (ii) S8, appropriated amino group, DMF, r.t., 8%–73%. R
represents aryl or alkyl moieties and R’ and R’’ represent different amino group as detailed in § 2.3
SAR’s Investigation.
For the modification of the central linker, syntheses of compounds are outlined in Scheme 2.
Compounds 22, 24, 28, and 30 were synthesized by nucleophilic acyl substitution. In the first step,
different carboxylic acids were acylated with oxalyl chloride to obtain the corresponding acid
chlorides. These chlorides then reacted with a morpholine molecule, or 4‐amino‐morpholine in the
case of compound 30, to obtain the desired compounds. Benzaldehyde or acetophenone were used
in the Willgerodt–Kindler reaction described above to lead to the synthesis of compounds 23 and 25.
The compound 26 was obtained thanks to an SN2‐type reaction between morpholine and bromo‐
acetophenone and the alcohol 27 was obtained after the NaBH4 reduction of 24. The reaction of
commercially available ethyl morpholine‐4‐carboxylate with POCl3 afforded the corresponding acyl
chloride, which was converted to compound 29 by reaction with aniline. The reaction of 4‐
aminomorpholine and benzaldehyde furnished the desired methylethanimine derivative 31. The
reaction of morpholine with isothiocyanatobenzene or isocyanatobenzene afforded the

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hydrazinecarbothioamide 32 and the urea derivative 33, respectively, with good yield (>76%).
Compound 34 was synthesized by a two‐step reaction. Firstly, CS2 and morpholine were used in
order to produce the corresponding morpholinium salt. Then, this salt was reacted with
(thiocyanatomethyl)benzene in acetonitrile to afford the desired derivative 34. Coupling between the
commercially available alcohol and morpholine‐4‐carbonyl chloride furnished the desired compound
35. Finally, the reaction of morpholine or 4‐amino‐morpholine with a benzenesulfonyl chloride
generated compounds 36 and 37.
22
24
28
30
38
: X = -, Y = - , Z = H, 87%
: X = CO, Y = - , Z = H, 99%
: X = CH2, Y = - , Z = H, 78%
: X = -, Y = NH2 , Z = H, 10%
: X = -, Y = - , Z = Cl, 65%
X
Y
X
OH
N
i
O
S
O
O
Z
O
O
ii
X
N
23
25
: X = - , 62%
: X = CH2 , 39%
XH
O
O
O
O
Br
iii
iv
N
N
26:
27:
34%
52%
OH
O
O
N
O
O
O
O
O
H
N
N
O
N
N
v
29
31
: 53%
: 78%
O
O
N
vi
O
H
N
H
N
NCX
32
33
: X = S, 80%
: X = O, 76%
vii
N
N
X
S
O
O
S
H
N
viii
34
35
: 42%
: 89%
O
HO
O
ix
x
H
N
N
NH2
O
HO
O
S
O
S
O
36
37
39
: X = - , Y = H, 85%
: X = NH , Y = H, 83%
: X = NH , Y = Cl, 74%
N
Cl
X
Y
O
O
a
a
Scheme 2. Synthetic pathway of compounds 22–39 . Reagents and conditions: (i) COCl2, DIPEA,
appropriated amine, CH2Cl2, r.t., 10%–99%; (ii) S8, morpholine, DMF, 39%–62%; (iii) morpholine,
K2CO3, MeCN, r.t., 34%; (iv) NaBH4, MeOH, r.t., 52%; (v) (1) POCl3, MeCN, 50 °C, (2) aniline, DIPEA,
0 °C to r.t., 53%; (vi) 4‐amino‐morpholine, MgSO4, CH2Cl2, r.t., 78%; (vii) 4‐amino‐morpholine,
CH2Cl2, r.t., 76%–80%; (viii) (1) CS2, Et2O, r.t., (2) (thiocyanatomethyl)benzene, ACN, r.t., 42%; (ix)
morpholine‐4‐carbonyl chloride, DIPEA, CH2Cl2, r.t., 89%. (x) Appropriated amine, Et3N, r.t., 83%–
85%.

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2.2. PHGDH Biochemical Assay Optimization
Prior to inhibitor evaluation, we undertook the optimization of our biochemical assay in order
to achieve reliable results. Our initial PHGDH assay contained solely the dehydrogenase and its
substrates; the enzyme activity was quantified by directly measuring NADH fluorescence, hence
yielded a low fluorescent signal (Figure 2B).
Figure 2. PHGDH coupled biochemical assay system. (A) Schematic representation of the coupled
system. Continuous measurement of PHGDH activity for initial PHGDH assay (B), for
PHGDH/PSAT1 coupled assay (C), and for present fully coupled assay (D). (3‐PG, 3‐
phosphoglycerate; 3‐PPyr, 3‐phosphopyruvate; 3‐PSer, 3‐phosphoserine).
PHGDH activity was monitored by measuring a continuous increase of fluorescence (Ex 544
nm/Em 590 nm) in a coupled‐enzyme system including three enzymes: PHGDH, PSAT1, and
diaphorase (Figure 2A). The addition of PSAT1 promotes accumulation of NADH by the PHGDH
reaction and generates a significant increase in the fluorescent signal (Figure 2C). Moreover, by
coupling PHGDH to diaphorase, which utilizes NADH to produce the fluorescent molecule resorufin
from resazurin, the assay red‐shifted into a spectral region that reduced interference from
compounds, as well as greatly increased.
In optimized conditions, Michaelis–Menten constants were estimated for substrates of PHGDH
+
+
(3‐PG and NAD ) and substrates of PSAT1 (3‐PSer and α‐KG). K
m
values for 3‐PG, NAD , 3‐PSer, and
α‐KG were determined to be 294.3, 30.4, 25.1, and 35 μM, respectively (Table 1).
Table 1. Michaelis–Menten enzyme kinetics for PHGDH and PSAT1. All experiments were performed
with at least triplicates and K values were determined in two or more independent experiments.
m
PHGDH
3‐PG
PSAT1
3‐PSer
NAD+
α‐KG
Experimental K
m
294.3 ± 12.4 μM 30.4 ± 4.3 μM 25.1 ± 5.3 μM 35 ± 4.2 μM
22 μM [26] 5 μM [27] 800 μM [28]
Km
from literature 260 μM [26]
The optimized fully coupled assay provides robust measurement of PHGDH activity,
suggesting it was amenable to evaluation of our inhibitors.
2.3. SARs Investigation

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Although a structural variety has been introduced at part A of the α‐ketothioamide scaffold in
our previous work, the substitution by alkyl groups has not been previously studied in detail. Hence,
we began our PHGDH SAR investigations with a focus on part A. As depicted in Table 2, only aryl
groups led to PHGDH inhibition (1, 2, and 4) whereas the introduction of alkyl groups in this position
resulted in inactive derivatives. Moreover, substitutions on the aryl moiety are only tolerated in the
para position with the chlorinated derivative 2 being the most potent.
Table 2. Results of PHGDH inhibition for compounds 1–11. All experiments were performed with at
least triplicates and IC50 values were determined in two or more independent experiments. *
Compounds previously published in Ravez et al. [25].
A
B
C
O
O
N
S
Part A
Phenyl *
4‐Chlorophenyl *
3‐Chlorophenyl *
PHGDH IC50 (μM)
111.1 [87.3–131.2]
20.3 [15.6–26.4]
>150
1
2
3
4
5
6
7
8
9
2,4‐Dichlorophenyl 77 [65.3–98.6]
Cyclohexyl
4‐Pyridine
>150
>150
3,4‐Dichlorophenyl >150
2‐Naphtyl
Ethyl
>150
>150
>150
>150
10 Adamantyl
11 t‐Butyl
To expand the SARs, the introduction of different amino moieties on part C was undertaken
keeping fixed parts A and B (Table 3). Except for the 4‐methylpiperidine analog (14), the introduction
of a bulky group in part C of the molecule abolished the inhibitory potency.
Table 3. Results of PHGDH inhibition for compounds 12–21. All experiments were performed with
at least triplicates and IC50 values were determined in two or more independent experiments.
Part C
PHGDH IC50 (μM)
12 Pyrrolidine
>150
13 4,4‐Difluoropiperidine >150
14 4‐Methylpiperidine
15 Piperidine
16 Diethylamine
17 Thiomorpholine
85.1 [37.8–101.2]
>150
>150
>150
>150
18 N‐methylpiperazine
19
20
>150
>150

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21
>150
Subsequently, we shifted our SAR analysis to the linker portion B (Table 4). This feature of the
pharmacophore proved essential to activity as the replacement led to significant loss of PHGDH
activity (IC50 > 150 μM). However, two modifications to this region of the molecule tolerated such
shortening of the linkage from the thiocarbonyl (23, IC50 = 106 μM) or modification by
sulfonohydrazide (37, IC50 = 92 μM).
Table 4. Results of PHGDH inhibition for compounds 22–37. All experiments were performed with
at least triplicates and IC50 values were determined in two or more independent experiments.
Part B
PHGDH IC50 (μM)
22
23
>150
106 [63.2–178.8]
>150
24
25
26
>150
>150
27
>150
28
29
>150
>150
30
31
32
>150
>150
>150
N
33
34
>150
>150
35
>150
36
37
>150
92.1 [58.4–145.2]
Based on the modification of the linker portion, two 4‐chloroderivatives carrying a thiocarbonyl
(38) or a sulfonohydrazide (39) instead of a ketothioamide linker were designed and synthesized
(Scheme 2). As expected, substitution of the para position by a chlorine atom resulted in an
improvement in PHGDH inhibition (Figure 3B). The most promising compounds (1, 2, 23, 38, 37, and
39) were validate in a counter screen against PSAT1 and the diaphorase. Based on the preliminary
study reported earlier and the present work, the following SARs can be highlighted (Figure 3A).

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Figure 3. Overview of the structure−activity relationships. Color code: green > yellow > red, for
decreasing PHGDH activity (A). Influence of the para‐chloro substitution on the potency of PHGDH
inhibition (B). Under brackets: standard deviation in μM.
2.4. PHGDH Engagement Investigated by Cellular Thermal Shift Assay (CETSA)
Next, in order to confirm that the most potent inhibitors target PHGDH in more complex
biological models, we measured their ability to stabilize PHGDH in cell lysates using the cellular
thermal shift assay (CETSA). In this assay, HL‐60 (leukemia cancer cells) lysates were incubated with
DMSO or with 80 μM of the inhibitor. The lysate was then heated to a pre‐determined temperature,
at which most of PHGDH was denaturated (60 °C). The fraction of non‐denatured PHGDH was
quantified by Western blot and allowed to identify the compounds that bind and stabilize PHGDH.
The percentages of stabilization compared to the DMSO control and non‐denatured PHGDH (heated
to 37 °C) of the six most potent PHGDH inhibitors are depicted in Figure 4.
Figure 4. Cellular thermal shift assay (CETSA) for the most promising inhibitors in HL‐60 cells. (A)
Schematic overview of CETSA experiment. (B) Quantification of thermostable PHGDH normalized
to the respective control at 37 °C. Results represent the average of two independent experiments.
While in untreated cells PHGDH was denaturated at 60 °C, the addition of compounds 1, 23,
and 37 leads to a small stabilization of the protein (1.2%, 0.8%, and 1.1%, respectively) thus proving

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the interaction between these compounds and PHGDH in cell lysates. More interestingly, the para‐
chlorinated derivatives (2, 38, and 39) present a greater stabilization of the protein compared to the
non‐halogenated compounds (2.8%, 3.1%, and 3.2% respectively); hence, this is in agreement with
increased PHGDH inhibition in the enzyme activity assay.
2.5. Cell‐Based Evaluation
We assessed the ability of para‐chlorinated derivatives, 38 and 39, to inhibit tumor proliferation.
To this end, breast cancer cells overexpressing PHGDH (BT‐20), breast cancer cells that do not express
PHGDH (MDA‐MB‐231) and healthy fibroblast cells as control (BJ‐5ta) were treated with compounds
1, 2, 38, 39 and the reference inhibitor NCT‐503, used for comparison purpose. The IC50 values
determined are depicted on Table 5.
Table 5. Cell proliferation inhibition data (IC50). 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. * Values previously published in Ravez et al.
[25].
Tumor Cell Lines
PHGDH‐Dependent
BT‐20
67.8 μM
PHGDH‐Independent
MDA‐MB‐231
>100 μM *
Normal Cell Line
BJ‐5ta
>100 μM
1
2
21.3 μM
>100 μM *
>100 μM
38
42.2 μM
>100 μM
>100 μM
39
>100 μM
>100 μM
>100 μM
NCT‐503
6.52 μM
>100 μM
>100 μM
Except for compound 39, which was deprived of any inhibition of tumor proliferation up to 100
μM probably due to poor membrane permeability, compounds 1, 2, and 38 selectively inhibit BT‐20
growth without affecting healthy BJ‐5ta or the PHGDH‐independent tumor cell line, MDA‐MB‐231.
Interestingly, these inhibitions were similar to the one observed with NCT‐503. Again, these results
demonstrate the potential of this compound class as a starting point for the design of new therapeutic
agents targeting PHGDH.
3. Conclusions
The development of new PHGDH inhibitors is emerging as a promising strategy to develop new
targeted therapy. We previously reported the discovery of compound 1, a PHGDH inhibitor with
robust cancer cell inhibition. Here, we have detailed the SARs investigation around this α‐
ketothioamide motif. Replacement of the aryl and modulation of the amino groups were not tolerated
in terms of PHGDH inhibition. However, modification of the ketothioamide linker by a thiocarbonyl
or by a sulfonohydrazide led to the identification of novel inhibitors. As expected, the addition of a
chlorine atom in para position on the most promising molecules resulted in the discovery of novel
micromolar range inhibitors (38 and 39). Target engagement was confirmed in cellular thermal shift
assay (CETSA) and the anticancer potential was validated in cell‐based experiments. Interestingly,
the identification of the sulfonohydrazide motif as a new series of inhibitors of this enzyme offers
interesting prospects in the development of future PHGDH inhibitors, this function being reported
in several works as non‐toxic [29,30]. It should be noted that recent mass spectrometry studies suggest
that some of the inhibitors discussed here would interact at the c‐terminal end of PHGDH. This will
be detailed in a forthcoming publication.
4. Materials and Methods
4.1. General Chemistry

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All reagents were purchased from chemical suppliers and used without purification. Thin‐layer
chromatography (TLC) was performed using silica gel 60 F254 plates, with observation under UV
when necessary. Melting points were recorded on an Electrothermal IA9000 melting point system.
1
H NMR spectra were recorded on an AVANCE II 400 MHz Bruker spectrometer with CDCl3 or
13
DMSO‐d6 as the solvent. C NMR spectra were recorded at 100 MHz. All coupling constants were
measured in hertz (Hz), and the chemical shifts (δH and δC) were quoted in parts per million (ppm)
relative to TMS (δ0), which was used as the internal standard. Data were reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet),
integration, and coupling constant (Hz). High‐resolution mass spectroscopy (HRMS) analyses were
carried out on an LTQ‐Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, Bremen,
Germany). Data were acquired in positive ion mode using full‐scan MS with a mass range of 100–
1000 m/z. The orbitrap resolution was set at 30,000 (fwhm definition). All experimental data were
acquired using daily external calibration prior to data acquisition. Appropriate tuning of the
electrospray ion source was done. The following electrospray inlet conditions were applied: flow rate,
100 μL/min; spray voltage, 5 kV; sheath gas (N2) flow rate, 20 arbitrary unit (au); auxiliary gas (N2)
flow rate, 10 au; capillary temperature, 275 °C; capillary voltage, 45 V; tube lens, 80 V. High‐
performance liquid chromatography (HPLC) analyses were performed on an LC system using a
YMC‐Triart C‐18 (250 mm × 4.6 mm, 5 μm) column as the stationary phase. Mobile phase contained
water/CH3CN (30:70, v/v) and was maintained isocratically at the flow rate of 1 mL/min. The column
temperature was maintained at room temperature. The peaks were monitored at a wavelength of 215
1
nm. The purity of all compounds tested was greater than 95%, as determined by HPLC and H NMR.
Compounds 1–11 were synthesized according to General Procedure I.
4.1.1. General Procedure I
To a stirred solution of ethanone derivative (1 equiv.) in chloroform was added dropwise a
solution of dibromine (1.2 equiv.) in chloroform. After 2 h, the solvent was evaporated in vacuo to
give a crude oil consisting mainly of 2‐bromo‐1‐(substituted)‐ethanone compound along with trace
amounts of 2,2‐dibromo‐1‐(substituted)‐ethanone compound. The mixture was used without
purification in the next step. To the synthesized or commercial 2‐bromo‐1‐(substituted)‐ethanone
derivative were added, in sequence, DMF, cyclooctasulfur (1.5 equiv.), and morpholine (3 equiv.).
The mixture was then stirred at room temperature. After completion, 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.
2‐Morpholino‐1‐phenyl‐2‐thioxoethan‐1‐one (1). Acetophenone (2.00 g, 16.60 mmol) and dibromine
(1.01 mL, 19.90 mmol) were mixed in chloroform (15 mL) to obtain the 2‐bromo‐1‐phenyl‐ethanone,
and this intermediate was reacted in a second 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
1
compound (2.85 g, 73%). Rf 0.2 (cyclohexane/EtOAc: 8/2). Mp: 110–112 °C. H NMR (400 MHz, CDCl3):
δH (ppm) 3.59–3.62 (t, 2H, J = 4.8 Hz), 3.69–3.71 (t, 2H, J = 4.8 Hz), 3.90–3.92 (t, 2H, J = 4.8 Hz), 4.33–
13
4.35 (t, 2H, J = 4.8 Hz), 7.48–7.52 (m, 2 ArH), 7.59–7.65 (m, 1 ArH), 7.99–8.01 (d, 2 ArH, J = 8.2 Hz).
C
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 (C=S). HRMS (ESI ): m/z calcd for C12H13NO2S [M + H] 236.0739, found 236.0737.
1‐(4‐Chlorophenyl)‐2‐morpholino‐2‐thioxoethan‐1‐one (2). This compound was synthesized
according to General Procedure I using 2‐bromo‐1‐(2‐chlorophenyl)ethanone (0.50 g, 2.14 mmol),
morpholine (0.56 mL, 6.42 mmol), and sulfur (0.10 g, 3.21 mmol) in DMF (10 mL). Acetonitrile was
used for recrystallization to afford the title compound as a yellow solid (0.22 g, 38%). Rf 0.2
1
(cyclohexane/EtOAc: 8/2). Mp: 135–137 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.58–3.71 (m, 4H),
3.89–3.92 (t, 2H, J = 4.8 Hz), 4.31–4.33 (t, 2H, J = 4.8 Hz), 7.46–7.48 (d, 2 ArH, J = 8.8 Hz), 7.93–7.95 (d,
13
2 ArH, J = 8.6 Hz). C NMR (100 MHz, CDCl3): δC (ppm) 47.18, 51.97, 66.39, 66.53, 129.36 (2C), 131.22

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+
+
(2C), 131.74, 141.06, 186.45 (C=O), 194.94 (C=S). HRMS (ESI ): m/z calcd for C12H13ClNO2S [M + H]
270.0350, found 270.0350.
1‐(3‐Chlorophenyl)‐2‐morpholino‐2‐thioxoethan‐1‐one (3). This compound was synthesized
according to General Procedure I. 1‐(3‐Chlorophenyl)ethanone (2.00 g, 12.90 mmol) and dibromine
(0.78 mL, 15.50 mmol) were mixed in chloroform (15 mL) to obtain the 2‐bromo‐1‐(3‐chlorophenyl)‐
ethanone and this intermediate was reacted in a second time with morpholine (3.38 mL, 38.80 mmol)
and sulfur (0.62 g, 19.40 mmol) in DMF (10 mL). The residue was purified by silica gel
chromatography (cyclohexane/EtOAc: 8/2) and the obtained oil was collected by filtration with
diethyl ether to give the title compound as a yellow solid (1.50 g, 43%). Rf 0.2 (cyclohexane/EtOAc:
1
8/2). Mp: 92–94 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.59–3.61 (t, 2H, J = 4.8 Hz), 3.70–3.72 (t,
2H, J = 4.8 Hz), 3.90–3.92 (t, 2H, J = 4.8 Hz), 4.31–4.33 (t, 2H,J = 4.8 Hz), 7.42–7.46 (dd, 1 ArH, J = 7.8
Hz), 7.57–7.59 (ddd, 1 ArH, J = 8.0 and 1.0 Hz), 7.85–7.87 (ddd, 1 ArH, J = 7.8 and 1.4 Hz), 7.96–7.97
13
(dd, 1 ArH, J = 1.8 Hz). C NMR (100 MHz, CDCl3): δC (ppm) 47.21, 52.00, 66.39, 66.52, 127.98, 129.61,
+
130.28, 134.33, 135.05, 135.30, 186.07 (C=O), 194.60 (C=S). HRMS (ESI ): m/z calcd for C12H12ClNO2S
+
[M + H] 270.0277, found 270.0278.
1‐(2,4‐Dichlorophenyl)‐2‐morpholino‐2‐thioxoethan‐1‐one (4). This compound was synthesized
according to General Procedure I using 2‐bromo‐1‐(2,4‐dichlorophenyl)‐ethanone (1.00 g, 3.76 mmol),
morpholine (0.99 mL, 11.28 mmol), and sulfur (0.18 g, 5.64 mmol) in DMF (10 mL). Acetonitrile was
used for recrystallization to afford the title compound as a yellow solid (0.31 g, 28%). Rf 0.4
1
(cyclohexane/EtOAc: 8/2). Mp: 116–118 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.75–3.77 (t, 2H, J
= 4.8 Hz), 3.77–3.79 (t, 2H, J = 4.8 Hz), 3.89–3.91 (t, 2H, J = 4.8 Hz), 4.10–4.12 (t, 2H, J = 4.8 Hz), 7.30
13
(dd, 1 ArH, J = 2.6 and 8.4 Hz), 7.38 (d, 1 ArH, J = 2.6 Hz), 7.80 (d, 1 ArH, J = 8.4 Hz). C NMR (100
MHz, CDCl3): δC (ppm) 47.68, 52.11, 65.95, 66.03, 127.83, 130.49 (2C), 133.08, 133.56, 139.55, 183.73
+
+
(C=O), 195.19 (C=S). HRMS (ESI ): m/z calcd for C12H12Cl2NO2S [M + H] 303.99603, found 303.99607.
1‐Cyclohexyl‐2‐morpholino‐2‐thioxoethan‐1‐one (5). This compound was synthesized according to
General Procedure I using 1‐cyclohexylethanone (2.00 g, 15.86 mmol) and dibromine (0.96 mL, 19.03
mmol) mixed in chloroform (20 mL) to obtain the 2‐bromo‐1‐cyclohexylethanone and this
intermediate was reacted in a second time with morpholine (4.18 mL, 47.58 mmol) and sulfur (0.76 g,
23.79 mmol) in DMF (10 mL). The residue was purified by silica gel chromatography
(Cyclohexane/EtOAc: 8/2) to give the title compound as a brown solid (0.58 g, 15%). Rf 0.3
1
(Cyclohexane/EtOAc: 8/2). Mp: 57–59 °C. H NMR (400 MHz, CDCl3): δH (ppm) 1.18–1.37 (m, 5H),
1.69–1.99 (m, 5H), 3.22 (m, 1H), 3.61–3.62 (m, 2H), 3.73–3.74 (t, 2H, J = 4.8 Hz), 3.82–3.84 (t, 2H, J = 4.8
13
Hz), 4.19–4.21 (t, 2H, J = 4.8 Hz). C NMR (100 MHz, CDCl3): δC (ppm) 25.52 (2C), 25.71, 28.10 (2C),
+
47.28, 47.49, 52.07, 66.31, 66.60, 197.85 (C=O, 201.05 (C=S). HRMS (ESI ): m/z calcd for C12H20NO2S [M
+
+ H] 242.1209, found 242.1208.
2‐Morpholino‐1‐(pyridin‐4‐yl)‐2‐thioxoethan‐1‐one (6). This compound was synthesized according
to General Procedure I using 2‐bromo‐1‐(pyridine‐4‐yl)ethanone (0.50 g, 1.79 mmol), morpholine
(0.47 mL, 5.37 mmol), and sulfur (0.08 g, 2.68 mmol) in DMF (10 mL). The residue was purified by
silica gel chromatography (CH2Cl2/EtOAc: 5/5) to give the title compound as a yellow solid (0.15 g,
1
36%). Rf 0.3 (CH2Cl2/EtOAc: 5/5). Mp: 96–98 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.60–3.63 (m,
2H), 3.71–3.74 (m, 2H), 3.91–3.93 (m, 2H), 4.31–4.34 (m, 2H), 7.77–7.79 (m, 2 ArH), 8.83–8.85 (m, 2
13
ArH). C NMR (100 MHz, CDCl3): δC (ppm) 45.30, 50.05, 64.40, 64.56, 120.26 (2C), 137.86, 149.10 (2C),
+
+
183.37 (C=O), 191.58 (C=S). HRMS (ESI ): m/z calcd for C11H13N2O2S [M + H] 237.06922, found
237.06923.
1‐(3,4‐Dichlorophenyl)‐2‐morpholino‐2‐thioxoethan‐1‐one (7). This compound was synthesized
according to General Procedure I using 2‐bromo‐1‐(3,4‐dichlorophenyl)ethan‐1‐one (1.00 g, 3.73
mmol), morpholine (0.96 mL, 11.2 mmol), and sulfur (0.18 g, 5.60 mmol) in DMF (15 mL). The residue
was purified by silica gel chromatography (Cyclohexane/EtOAc: 3/2) to give the title compound as a

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1
yellow solid (0.32 g, 28%). Rf 0.6 (Cyclohexane/EtOAc: 3/2). Mp: 134–136 °C. H NMR (400 MHz,
CDCl3): δH (ppm) 3.60–3.61 (m, 2H), 3.71–3.72 (m, 2H), 3.90–3.93 (m, 2H), 4.31–4.33 (m, 2H), 7.55–7.60
13
(m, 1 ArH), 7.79–7.84 (m, 1 ArH), 8.07–8.09 (m, 1 ArH). C NMR (100 MHz, CDCl3): δC (ppm) 44.87,
49.63, 63.99, 64.15, 126.38, 128.69, 129.11, 130.76, 131.36, 136.75, 182.57 (C=O), 191.61 (C=S). HRMS
+
+
(ESI ): m/z calcd for C12H12Cl2NO2S (M + H) 303.99603, found 303.99549.
2‐Morpholino‐1‐(naphthalen‐2‐yl)‐2‐thioxoethan‐1‐one (8). This compound was synthesized
according to General Procedure I using 2‐bromo‐1‐(naphthalene‐2‐yl) ethanone (0.50 g, 2.01 mmol),
morpholine (0.53 mL, 6.04 mmol), and sulfur (0.09 g, 3.01 mmol) in DMF (10 mL). Cyclohexane was
used for recrystallization to afford the title compound as a yellow solid (0.31 g, 28%). Rf 0.2
1
(Cyclohexane/EtOAc: 8/2). Mp: 152–154 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.63–3.65 (m, 2H),
3.69–3.71 (m, 2H), 3.93–3.96 (m, 2H), 4.38–4.41 (m, 2H), 7.55–7.66 (m, 2 ArH), 7.88–7.97 (m, 3 ArH),
13
8.03–8.05 (dd, 1 ArH, J = 2.1 and 8.8 Hz), 8.52 (s, 1 ArH). C NMR (100 MHz, CDCl3): δC (ppm) 46.92,
51.70, 66.14, 66.25, 124.02, 126.87, 127.63, 128.67, 129.06, 129.54, 130.28, 132.11, 132.22, 135.88, 187.75
+
+
(C=O), 195.51 (C=S). HRMS (ESI ): m/z calcd for C16H15NO2S (M + Na) 308.07157, found 308.07146.
1‐Morpholino‐1‐thioxobutan‐2‐one (9). This compound was synthesized according to General
Procedure I using 1‐bromobutan‐2‐one (0.34 mL, 3.33 mmol), morpholine (0.88 mL, 10.0 mmol), and
sulfur (0.15 g, 4.99 mmol) in DMF (10 mL). The residue was purified by silica gel chromatography
(Hexane/EtOAc: 7/3) to give the title compound as a yellow solid (0.08 g, 8%). Rf 0.5
1
(Cyclohexane/EtOAc: 3/2). H NMR (400 MHz, CDCl3): δH (ppm) 1.19 (t, 3H, J = 5.1 Hz), 2.88–2.94 (q,
2H, J = 5 Hz), 3.59–3.62 (m, 2H), 3.74–3.76 (m, 2H), 3.82–3.85 (m, 2H), 4.17–4.20 (m, 2H). 13C NMR
(100 MHz, CDCl3): δC (ppm) 7.41, 33.52, 46.94, 51.45, 65.92, 66.21, 197.42 (C+O), 199.14 (C=S) HRMS
+
+
(ESI ): m/z calcd for C8H14NO2S [M + H] 188.07398, found 188.07384.
1‐((1r,3r,5r,7r)‐Adamantan‐2‐yl)‐2‐morpholino‐2‐thioxoethan‐1‐one (10). This compound was
synthesized according to General Procedure I using 2‐bromo‐1‐((1 r,3r,5r,7r)‐Adamantan‐2‐yl)‐10‐
ethanone (1 g, 3.90 mmol), morpholine (1.02 mL, 11.70 mmol), and sulfur (0.18 g, 5.90 mmol) in DMF
(10 mL). The residue was purified by silica gel chromatography (cyclohexane/EtOAc: 8/2) to give the
1
title compound as yellow oil (0.08 g, 8%). Rf 0.3 (hexane/EtOAc 8:2). H NMR (400 MHz, CDCl3): δΗ
(ppm) 1.69–1.77 (m, 6H), 2.02–2.05 (m, 9H), 3.51 (m, 2H), 3.73–3.75 (t, 2H, J = 4.8 Hz), 3.80–3.83 (t, 2H,
13
J = 4.8 Hz), 4.18 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 27.96 (3C), 36.30 (3C), 39.79 (3C),
+
45.03, 46.41, 52.39, 66.30, 66.36, 196.66 (C=O), 205.54 (C=S). HRMS (ESI ): m/z calcd for C16H23NO2S [M
+
+ H] 294.1522, found 294.1523.
3,3‐Dimethyl‐1‐morpholino‐1‐thioxobutan‐2‐one (11). This compound was synthesized according to
General Procedure I using 1‐bromo‐3,3‐dimethylbutan‐2‐one (2.0 g, 11.2 mmol), morpholine (2.96
mL, 33.7 mmol), and sulfur (0.54 g, 16.8 mmol) in DMF (10 mL). The residue was purified by silica
gel chromatography (Cyclohexane/EtOAc: 8/2) to give the title compound as orange oil (0.35 g, 14%).
1
Rf 0.2 (Cyclohexane/EtOAc: 8/2). H NMR (400 MHz, CDCl3): δH (ppm) 1.34 (s, 9H), 3.50–3.53 (m, 2H),
13
3.74–3.76 (m, 2H), 3.81–3.83 (m, 2H), 4.18 (broad, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 26.16
+
(3C), 40.52, 44.11, 50.03, 64.01 (2C), 194.65 (C=O), 204.21 (C=S). HRMS (ESI ): m/z calcd for C10H18NO2S
+
[M + H] 216.10528, found 216.10524.
Compounds 12–21 were synthesized according to General Procedure II.
4.1.2. General Procedure II
To commercial 2‐bromo‐1‐phenylethanone were added, in sequence, DMF, cyclooctasulfur (1.5
equiv.), and amine derivative (3 equiv.). The mixture was then stirred at room temperature. After
completion, 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.

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1‐Phenyl‐2‐(pyrrolidin‐1‐yl)‐2‐thioxoethan‐1‐one (12). This compound was synthesized according to
General Procedure II using 2‐bromo‐1‐phenylethanone (0.5 g, 2.52 mmol), pyrrolidine (0.62 mL, 7.56
mmol), and sulfur (0.12 g, 3.78 mmol) in DMF (10 mL). The residue was purified by silica gel
chromatography (Cyclohexane/EtOAc: 8/2) to give the title compound as a yellow solid (0.12 g, 22%).
1
Rf 0.4 (Cyclohexane/EtOAc: 8/2). Mp: 57–59 °C. H NMR (400 MHz, CDCl3): δH (ppm) 1.61 (m, 4H),
13
3.53–3.56 (m, 2H), 3.94–3.98 (m, 2H), 7.46–7.48 (m, 2H), 7.50–7.62 (m, 1H), 7.99–8.01 (m, 2H). C NMR
(100 MHz, CDCl3): δC (ppm) 22.97, 25.24, 50.22, 50.40, 127.97 (2C), 129.24 (2C), 131.96, 133.37, 187.92
+
+
(C=O), 191.87 (C=S). HRMS (ESI ): m/z calcd for C12H14NOS [M + H] 220.07906, found 220.07877.
2‐(4,4‐Difluoropiperidin‐1‐yl)‐1‐phenyl‐2‐thioxoethan‐1‐one (13). This compound was synthesized
according to General Procedure II using 2‐bromo‐1‐phenylethanone (0.5 g, 2.52 mmol), 4,4‐
difluoropiperidine HCl (1.19 g, 7.56 mmol), and sulfur (0.12 g, 3.78 mmol) in DMF (10 mL).
Cyclohexane was used for recrystallization to afford the title compound as a white solid (0.12 g, 18%).
1
Rf 0.8 (Cyclohexane/EtOAc: 3/2). Mp: 117–119 °C. H NMR (400 MHz, CDCl3): δH (ppm) 1.42–1.58 (m,
2H), 2.08–2.25 (m, 2H), 3.68 (m, 2H), 4.44 (m, 2H), 7.48–7.52 (m, 2H), 7.61–7.65 (m, 1H), 7.98–8.00 (m,
13
2H). C NMR (100 MHz, CDCl3): δC (ppm) 31.25 (t), 32.32 (t), 41.73 (t), 46.22 (t), 118.84, and 121.26
+
(C‐F), 127.18 (2C), 128.02 (2C), 131.23, 132.77, 186.10 (C=O), 194.87 (C=S). HRMS (ESI ): m/z calcd for
+
C13H14NOSF2 [M + H] 270.07587, found 270.07568.
2‐(4‐Methylpiperidin‐1‐yl)‐1‐phenyl‐2‐thioxoethan‐1‐one (14). This compound was synthesized
according to General Procedure II using 2‐bromo‐1‐phenylethanone (1.0 g, 5.05 mmol), 4‐
methylpiperidine (3.82 g, 15.15 mmol), and sulfur (0.24 g, 7.57 mmol) in DMF (10 mL). The residue
was purified by silica gel chromatography (Hexane/EtOAc: 9/1) to give the title compound as a
1
yellow solid (0.47 g, 38%). Rf 0.3 (Hexane/EtOAc: 7/3). Mp: 71–73 °C. H NMR (400 MHz, CDCl3): δH
(ppm) 1.00 (s, 3H), 1.15–1.25 (m, 1H), 1.30 (m, 1H), 1.78–1.92 (m, 3H), 3.08–3.14 (m, 1H) 1.15–1.20 (m,
3H), 3.26–3.32 (m, 1H), 3.74–3.76 (m, 1H), 5.39 (m, 1H), 7.47–7.49 (m, 2H), 7.59 (m, 1H), 7.98 (m, 2H).
13
C NMR (100 MHz, CDCl3): δC (ppm) 18.86, 28.42, 30.95, 31.97, 45.03, 49.83, 126.48 (2C), 127.41 (2C),
+
+
131.01, 131.82, 185.65 (C=O), 192.08 (C=S). HRMS (ESI ): m/z calcd for C14H17NOSNa (M + Na)
270.09231, found 270.09219.
1‐Phenyl‐2‐(piperidin‐1‐yl)‐2‐thioxoethan‐1‐one (15). This compound was synthesized according to
General Procedure II using 2‐bromo‐1‐phenylethanone (2.0 g, 10.01 mmol), piperidine (2.99 mL, 30.30
mmol), and sulfur (0.48 g, 15.10 mmol) in DMF (10 mL). The residue was purified by silica gel
chromatography (Cyclohexane/EtOAc: 8/2) to give the title compound as a yellow solid (0.47 g, 20%).
1
Rf 0.6 (Cyclohexane/EtOAc: 3/2). Mp: 67–69 °C. H NMR (400 MHz, CDCl3): δH (ppm) 1.56–1.63 (m,
2H), 1.77–1.84 (m, 4H), 3.53–3.55 (m, 2H), 4.24–4.27 (m, 2H), 7.47–7.50 (m, 2H), 7.58–7.62 (m, 1H), 7.98–
13
8.00 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 23.79, 25.13, 26.20, 47.88, 52.79, 52.79, 128.63 (2C),
129.52 (2C), 133.14, 133.97, 187.76 (C=O), 194.03 (C=S). 18.86, 28.42, 30.95, 31.97, 45.03, 49.83, 126.48
+
(2C), 127.41 (2C), 131.01, 131.82, 185.65 (C=O), 192.08 (C=S). HRMS (ESI ): m/z calcd for C13H15NOSNa
+
(M + Na) 256.07666, found 256.07661.
N,N‐Diethyl‐2‐oxo‐2‐phenylethanethioamide (16). This compound was synthesized according to
General Procedure II using 2‐bromo‐1‐phenylethanone (1.0 g, 5.05 mmol), diethylamine (1.56 mL,
15.15 mmol), and sulfur (0.24 g, 7.57 mmol) in DMF (10 mL). The residue was purified by silica gel
chromatography (Hexane/EtOAc: 7/3) to give the title compound as brown oil (0.09 g, 8%). Rf 0.3
1
(Hexane/EtOAc: 7/3). H NMR (400 MHz, CDCl3): δH (ppm) 1.15–1.20 (m, 3H), 1.26–1.34 (m, 3H), 3.37–
13
3.42 (m, 2H), 3.94–3.99 (m, 2H), 7.36–7.40 (m, 2H), 7.48–7.52 (m, 1H), 7.87–7.89 (m, 2H). C NMR (100
MHz, CDCl3): δC (ppm) 11.29, 13.63, 44.55, 47.99, 128.80 (2C), 129.90 (2C), 133.51, 134.09, 187.51 (C=O),
+
+
195.58 (C=S). HRMS (ESI ): m/z calcd for C12H16NOS [M + H] 222.09471, found 222.09456.
1‐Phenyl‐2‐thiomorpholino‐2‐thioxoethan‐1‐one (17). This compound was synthesized according to
General Procedure II using 2‐bromo‐1‐phenylethanone (1.0 g, 5.05 mmol), thiomorpholine (1.52 mL,
15.15 mmol), and sulfur (0.24 g, 7.57 mmol) in DMF (10 mL). The residue was purified by silica gel

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chromatography (Hexane/EtOAc: 7/3) to give the title compound as a yellow solid (0.40 g, 43%). Rf
1
0.7 (Cyclohexane/EtOAc: 3/2). Mp: 127–129 °C. H NMR (400 MHz, CDCl3): δH (ppm) 2.69 (m, 2H),
2.88–2.90 (t, 2H, J = 5.1 Hz), 3.83–3.86 (t, 2H, J = 5.1 Hz), 4.59 (m, 2H), 7.47–7.51 (m, 2H), 7.59–7.63 (m,
13
1H), 7.97–7.99 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 27.31, 28.25, 49.71, 54.64, 128.96 (2C),
+
129.90 (2C), 133.23, 134.44, 187.73 (C=O), 196.06 (C=S). HRMS (ESI ): m/z calcd for C12H14NOS2 [M +
+
H] 252.05113, found 252.05117.
2‐(4‐Methylpiperazin‐1‐yl)‐1‐phenyl‐2‐thioxoethan‐1‐one (18). This compound was synthesized
according to General Procedure II using 2‐bromo‐1‐phenylethanone (2 g, 10.10 mmol), N‐
methylpiperazine (3.37 mL, 30.30 mmol), and sulfur (0.48 g, 15.15 mmol) in DMF (10 mL). The residue
was purified by silica gel chromatography (Cyclohexane/EtOAc: 8/2) to give the title compound as
1
brown solid (0.58 g, 23%). Rf 0.2 (Cyclohexane/EtOAc: 8/2). Mp: 93–95 °C H NMR (400 MHz, CDCl3):
δH (ppm) 2.34 (s, 3H, CH3), 2.41–2.44 (m, 2H), 2.62–2.64 (m, 2H), 3.58–3.60 (m, 2H), 4.31–4.34 (m, 2H),
13
7.47–7.51 (m, 2H), 7.59–7.63 (m, 1H), 7.98–8.00 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 45.53,
46.61, 51.31, 53.95, 54.57, 128.78 (2C), 129.71 (2C), 133.18, 134.21, 187.80 (C=O), 195.17 (C=S). HRMS
+
+
(ESI ): m/z calcd for C13H17N2OS [M + H] 249.10561, found 249.10561.
2‐(4‐(2‐Oxo‐2‐phenylethyl)piperazin‐1‐yl)‐1‐phenyl‐2‐thioxoethan‐1‐one (19). This compound was
synthesized according to General Procedure I using 2‐bromo‐1‐phenylethanone (1.00 g, 5.05 mmol),
piperazine (1.30 g, 15.15 mmol), and sulfur (0.24 g, 7.57 mmol) in DMF (10 mL). The residue was
purified by silica gel chromatography (hexane/EtOAc: 9/1) to give the title compound as orange oil
1
(0.40 g, 22%). Rf 0.2 (cyclohexane/EtOAc: 8/2). H NMR (400 MHz, CDCl3): δΗ (ppm) 2.67–2.70 (t, 2H,
J = 4.8 Hz), 2.87–2.90 (t, 2H, J = 4.8 Hz), 3.66–3.69 (t, 2H, J = 4.8 Hz), 4.40–4.43 (t, 2H, J = 4.8 Hz), 7.42–
+
7.53 (m, 4 ArH), 7.58–7.64 (m, 2 ArH), 7.94–8.15 (m, 4 ArH). HRMS (ESI ): m/z calcd for C20H20N2O2S
+
[M + H] 353.1318, found 353.1318.
1‐Phenyl‐2‐(4‐phenylpiperazin‐1‐yl)‐2‐thioxoethan‐1‐one (20). This compound was synthesized
according to General Procedure II using 2‐bromo‐1‐phenylethanone (1.0 g, 5.05 mmol), 1‐phenyl‐
piperazine (2.31 mL, 15.15 mmol), and sulfur (0.24 g, 7.57 mmol) in DMF (10 mL). The residue was
purified by silica gel chromatography (Cyclohexane/EtOAc: 8/2) to give the title compound as a
1
yellow solid (0.20 g, 13%). Rf 0.4 (Cyclohexane/EtOAc: 8/2). Mp: 89–91 °C. H NMR (400 MHz, CDCl3):
δH (ppm) 3.20–3.22 (m, 2H), 3.40–3.43 (m, 2H), 3.73–3.76 (m, 2H), 4.46–4.48 (m, 2H), 6.92–6.96 (m,
13
3H), 7.28–7.31 (m, 2H), 7.48–7.52 (m, 2H), 7.60–7.64 (m, 1H), 8.00–8.02 (m, 2H). C NMR (100 MHz,
CDCl3): δC (ppm) 45.06, 47.45, 48.10, 49.66, 115.28 (2C), 119.55, 126.78, 127.30 (2C), 127.74 (2C), 128.24
+
(2C), 131.65, 132.77, 148.47, 186.30 (C=O), 193.88 (C=S). HRMS (ESI ): m/z calcd for C18H19N2OS [M +
+
H] 311.12181, found 311.12068.
2‐(4‐Benzhydrylpiperazin‐1‐yl)‐1‐phenyl‐2‐thioxoethan‐1‐one (21). This compound was synthesized
according to General Procedure II using 2‐bromo‐1‐phenylethanone (1.0 g, 5.05 mmol), 1‐
(diphenylmethyl)piperazine (3.82 g, 15.15 mmol) and sulfur (0.24 g, 7.57 mmol) in DMF (10 mL).
Cyclohexane was used for recrystallization to afford the title compound as a white solid (0.60 g, 30%).
1
Rf 0.5 (Hexane/EtOAc: 8/2). Mp: 130–132 °C. H NMR (400 MHz, CDCl3): δH (ppm) 2.40 (m, 2H), 2.62
(m, 2H), 3.57 (m, 2H), 4.29 (m, 3H), 7.17–7.25 (m, 6H), 7.39–7.47 (m, 6H), 7.56–7.59 (m, 1H), 7.95–7.96
13
(m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 45.65, 49.77, 50.33, 50.41, 74.22, 126.09 (2C), 126.41
(4C), 127.44 (4C), 127.55 (2C), 128.48 (2C), 131.99, 132.94, 140.26 (2C), 186.66 (C=O), 193.69 (C=S).
+
+
HRMS (ESI ): m/z calcd for C25H25N2OS [M + H] 401.16821, found 401.16826.
4.1.3. Synthesis of Compounds 22–39
Morpholino(phenyl)methanone (22). To a stirring solution of benzoic acid (2.50 g, 20.47 mmol, 1
equiv.) in CH2Cl2 (50 mL) were added catalytic amount of DMF and oxalyl chloride (2.60 g, 20.47
mmol, 1 equiv.). The reaction mixture was stirred at room temperature until the generation of gasses
was stopped (HCl), cooled to 0 °C, and a solution of morpholine (2.67 g, 30.7 mmol, 1.5 equiv.) and

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DIPEA (5.29 g, 40.94 mmol, 2 equiv.) was slowly added. The mixture was stirred at room temperature
for 6 h. The reaction was quenched by adding water (50 mL) following by extraction with CH2Cl2 (75
mL). The combined organic layer were washed with brine and dried over Na2SO4. The solvent was
removed in vacuo, and the residue was purified by silica gel chromatography (Cyclohexane/EtOAc:
2/3) to give the title compound as a white solid (17.81 mmol, 3.40 g, 87%). Rf 0.45 (Cyclohexane/EtOAc:
1
3/2). Mp: 62–63 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.41–3.50 (m, 4H), 3.64–3.79 (m, 4H), 7.39–
13
7.44 (m, 5H). C NMR (100 MHz, CDCl3): δC (ppm) 26.84, 42.60, 48.19, 66.74, 127.20 (2C), 128.67 (2C),
+
+
129.99, 135.24, 170.32 (C=O). HRMS (ESI ): m/z calcd for C11H14NO2 [M + H] 192.1024, found 192.1014.
Morpholino(phenyl)methanethione (23). Benzaldehyde (1.00 g, 9.42 mmol, 1 equiv.), morpholine
(2.05 g, 23.5 mmol, 2.5 equiv.), sulfur (0.30 g, 9.42 mmol, 1 equiv.), and para‐toluene sulfonic acid (0.05
g, 0.26 mmol, 0.028 equiv.) were mixed together and the solution stirred at 85 °C for 12 h. The brown
solid formed was recovered with 50 mL of CH2Cl2. The solution was then filtered and the organic
layer was extracted 3 times with water (80 mL). The organic layer was evaporated under vacuo and
the desired product was recrystallized in EtOH to give the title compound as a yellow solid (5.84
1
mmol, 1.21 g, 62%). Rf 0.7 (Cyclohexane/EtOAc: 2/3). Mp: 128–129 °C. H NMR (400 MHz, CDCl3): δH
(ppm) 3.59–3.62 (m, 2H), 3.64–3.66 (m, 2H), 3.88–3.91 (m, 2H), 4.44–4.46 (m, 2H), 7.27–7.30 (m, 2H),
13
7.34–7.39 (m, 3H). C NMR (100 MHz, CDCl3): δC (ppm) 47.32, 50.29, 64.33, 64.54, 123.57 (2C), 126.45
+
+
(2C), 126.68, 140.13, 198.90 (C=S). HRMS (ESI ): m/z calcd for C11H14NOS [M + H] 208.0796, found
208.0791.
1‐Morpholino‐2‐phenylethane‐1,2‐dione (24). To a solution of 2‐oxo‐2‐phenylacetic acid (2.50 g, 16.6
mmol, 1 equiv.) in CH2Cl2 were added catalytic amount of DMF and oxalyl chloride (2.11 g, 16.6
mmol, 1 equiv.). The reaction was stirred at room temperature until the generation of gasses (HCl)
was stopped, cooled to 0 °C and a solution of morpholine (2.17 g, 24.9 mmol, 1.5 equiv.) with DIPEA
(4.29 g, 33.2 mmol, 2 equiv.) was slowly added. The mixture was stirred at room temperature for 14
h, quenched by adding water and extracted with CH2Cl2. The combined organic layer were washed
with brine and dried over Na2SO4. The solvents were removed in vacuo, and the resulting crude
product was purified by silica gel chromatography (Cyclohexane/EtOAc: 3/2) to give the title
compound as a white solid (16.4 mmol, 3.60 g, 99%). Rf 0.35 (Cyclohexane/EtOAc: 3/2). Mp: 50–52 °C.
1
H NMR (400 MHz, CDCl3): δH (ppm) 3.38–3.40 (m, 2H), 3.65–3.68, (m, 2H), 3.80 (m, 4H), 7.51–7.55
13
(m, 2H), 7.65–7.69 (m, 1H), 7.96–7.98 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 41.64, 46.28,
66.70, 66.77, 129.14 (2C), 129.71 (2C), 133.06, 134.99, 165.48 (C=O), 191.19 (C=O). HRMS (ESI ): m/z
calcd for C12H14NO3 [M + H] 220.0973, found 220.0968.
+
+
1‐Morpholino‐2‐phenylethane‐1‐thione (25). To a solution of acetophenone (1.50 g, 12.48 mmol, 1
equiv.) and sulfur (0.60 g, 18.73 mmol, 1.5 equiv.) in DMF (20 mL), morpholine (3.26 g, 12.48 mmol,
3 equiv.) was slowly added and the reaction was stirred at reflux for 10 h. After the completion of the
reaction, water was added on the mixture and the solution was extracted with EtOAc (150 mL). The
organic layers were dried and evaporated under vacuo to afford the crude mixture. The residue was
purified by silica gel chromatography (Cyclohexane/EtOAc: 2/3) to give the title compound as a
1
yellow solid (4.87 mmol, 1.08 g, 39%). Rf 0.67 (EtOAc/Cyclohexane: 3/2). Mp: 66–67 °C. H NMR (400
MHz, (CD3)2SO): δH (ppm) 3.34 (s, 2H), 3.40–3.42 (m, 2H), 3.63–3.66 (m, 2H), 3.69–3.72 (m, 2H), 4.22–
13
4.25 (m, 2H), 7.23–7.28 (m, 1H), 7.32–7.35 (m, 4H). C NMR (100 MHz, (CD3)2SO): δC (ppm) 48.66,
+
49.40, 50.22, 65.23, 65.33, 126.27, 127.53 (2C), 128.19 (2C), 135.95, 198.39 (C=S). HRMS (ESI ): m/z calcd
+
for C12H16NOS [M + H] 222.0952, found 222.0941.
2‐Morpholino‐1‐phenylethan‐1‐one (26). 2‐bromo‐1‐phenylethanone (1.00 g, 5.02 mmol, 1 equiv.) and
morpholine (1.71 g, 19.59 mmol, 3.9 equiv.) were mixed in a solution of K2CO3 (3.12 g, 22.59 mmol,
4.5 equiv.) in acetonitrile and stirred for 3 h. Then the solution was filtered to remove K2CO3 and
evaporated under reduced pressure. The resulting crude product was purified by silica gel
chromatography (Cyclohexane/EtOAc: 3/2) to give the title compound as red oil (1.71 mmol, 0.35 g,
1
34%). Rf 0.4 (Cyclohexane/EtOAc: 3/2). H NMR (400 MHz, CDCl3): δH (ppm) 261–2.63 (m, 4H), 3.78–

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13
3.80 (m, 4H), 3.83 (s, 2H), 7.45–7.49 (m, 2H), 7.56–7.60 (m, 1H), 7.99–8.01 (m, 2H). C NMR (100 MHz,
CDCl3): δC (ppm) 53.91 (2C), 64.72, 66.84 (2C), 128.09 (2C), 128.61 (2C), 133.37, 135.95, 196.10 (C=O).
+
+
HRMS (ESI ): m/z calcd for C11H16NO2 [M + H] 206.11810, found 206.11425. Data are in agreement
with the literature [12].
2‐Hydroxy‐1‐morpholino‐2‐phenylethan‐1‐one (27). To a solution of 1‐morpholino‐2‐phenylethane‐
1,2‐dione (0.25 g, 1.14 mmol, 1 equiv.) in 10 mL of MeOH, NaBH4 (0.11 g, 2.85 mmol, 2.5 equiv.) was
added and stirred during 5 h. The reaction mixture was then evaporated, diluted in water, and
extracted with CH2Cl2. The organic layer was evaporated and purified by silica gel chromatography
(Cyclohexane/EtOAc: 8/2) to give the title compound as a white solid (0.59 mmol, 0.13 g, 52%). Rf 0.53
1
(Cyclohexane/EtOAc: 8/2). Mp: 85–87 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.02–3.09 (m, 1H),
3.13–3.18 (m, 1H), 3.44–3.49 (m, 1H), 3.52–3.63 (m, 2H), 3.67–3.71 (m, 1H), 3.76–3.81 (m, 1H), 4.73 (s,
13
1H, OH), 5.19 (s, 1H), 7.29–7.40 (m, 5H). C NMR (100 MHz, CDCl3): δC (ppm) 43.15, 45.31, 65.81,
+
66.56, 71.54, 127.39 (2C), 128.74, 129.21 (2C), 139.13, 171.03 (C=O). HRMS (ESI ): m/z calcd for
+
C12H16NO3 [M + H] 222.1130, found 222.1125.
1‐Morpholino‐2‐phenylethan‐1‐one (28). To a solution of 2‐phenylacetic‐acid (0.50 g, 3.70 mmol, 1
equiv.) in CH2Cl2 were added catalytic amounts of DMF and oxalyl chloride (0.47 g, 3.7 mmol, 1
equiv.). The reaction was stirred at room temperature until the generation of gasses (HCl) was
stopped. The resulting solution was cooled to 0 °C and a solution of morpholine (0.48 g, 5.55 mmol,
1.5 equiv.) and DIPEA (0.96 g, 7.40 mmol, 2 equiv.) was slowly added. The mixture was stirred at
room temperature for 14 h, quenched by adding water (100 mL), and the mixture was extracted with
CH2Cl2 (150 mL). The combined organic layers were washed with brine and dried over Na2SO4. The
residue was purified by silica gel chromatography (Cyclohexane/EtOAc: 2/3) to give the title
compound as a white solid (2.90 mmol, 0.59 g, 78%). Rf 0.29 (Cyclohexane/EtOAc: 2/3). Mp: 60–62 °C.
1
H NMR (400 MHz, CDCl3): δH (ppm) 3.42–3.45 (m, 2H), 3.47–3.49 (m, 2H), 3.65 (s, 4H), 3.74 (s, 2H),
13
7.23–7.27 (m, 3H), 7.31–7.35 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 39.10, 40.36, 44.75, 64.69,
65.03, 125.23, 126.75 (2C), 127.06 (2C), 133.02. HRMS (ESI ): m/z calcd for C12H16NO2 [M + H] 206.1181,
+
+
found 206.1176.
N‐Phenylmorpholine‐4‐carboxamide (29). To a MeCN solution of ethyl morpholine‐4‐carboxylate
(0.40 g, 2.50 mmol, 1 equiv.) were added POCl3 (1.15 g, 7.50 mmol, 3 equiv.) under Ar. The reaction
mixture was stirred at reflux and monitored by TLC. After completion, the solution was diluted in
water and extracted with CH2Cl2. The organic layer was evaporated, diluted in dry CH2Cl2, cooled to
0 °C, and a solution of aniline (0.35 g, 3.75 mmol, 1.5 equiv.) and DIPEA (0.65 g, 3.75 mmol, 2 equiv.)
in CH2Cl2 was slowly added. The reaction mixture was stirred at room temperature for 50 h. The
reaction was quenched by adding water and the mixture was extracted with CH2Cl2. The combined
organic layers were washed with brine and dried over Na2SO4. The resulting crude product was
purified by silica gel chromatography (Cyclohexane/EtOAc: 8/2) to give the title compound as a green
1
solid (1.32 mmol, 0.27 g, 53%). Rf 0.14 (Cyclohexane/EtOAc: 3/2). Mp: 156–158 °C. H NMR (400 MHz,
CDCl3): δH (ppm) 3.48–3.50 (m, 4H), 3.74–3.77 (m, 4H), 6.69 (s, 1H, NH), 7.04–7.08 (m, 1H), 7.28–7.37
13
(m, 4H). C NMR (100 MHz, CDCl3): δC (ppm) 44.26 (2C), 66.51 (2C), 120.14 (2C), 123.40, 128.95 (2C),
+
+
138.71, 155.19 (C=O). HRMS (ESI ): m/z calcd for C11H15N2O2 [M + H] 207.1133, found 207.1128.
N‐Morpholinobenzamide (30). To a solution of benzoic acid (2.50 g, 20.47 mmol, 1 equiv.) in CH2Cl2,
a catalytic amount of DMF and oxalyl chloride were added (2.60 g, 20.47 mmol, 1 equiv.) and the
mixture was stirred at room temperature until the generation of gasses (HCl) was stopped. The
reaction was cooled to 0 °C before adding a solution of morpholin‐4‐amine (3.13 g, 30.7 mmol, 1.5
equiv.) and DIPEA (4.53 g, 40.94 mmol, 2 equiv.) and the reaction was stirred at room temperature
for 6 h. The solution was quenched by water (30 mL) and the mixture was extracted with CH2Cl2 (100
mL). The combined organic layers were washed with brine and dried over Na2SO4. The solvent was
removed in vacuo, and the resulting crude product was purified by silica gel chromatography
(Cyclohexane/EtOAc: 3/2) to give the title compound as yellow oil (2.05 mmol, 0.42 g, 10%). Rf 0.25

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1
(Cycloxane/EtOAc: 3/2). H NMR (400 MHz, CDCl3): δH (ppm) 2.96–2.97 (m, 4H), 3.87–3.89 (m, 4H),
13
6.84 (s, 1H), 7.40–7.56 (m, 2H), 7.52–7.54 (m, 1H), 7.74–7.75 (m, 2H). C NMR (100 MHz, CDCl3): δC
+
(ppm) 56.12 (2C), 66.44 (2C), 127.05, 128.69 (2C), 128.69 (2C), 144.76, 151.61(C=O). HRMS (ESI ): m/z
+
calcd for C11H15N2O2 [M + H] 207.11335, found 207.11221.
(E)‐N‐Morpholino‐1‐phenylmethanimine (31). To a solution of MgSO4 (2.27 g, 18.86 mmol, 2 equiv.)
in CH2Cl2 was added benzaldehyde (0.96 mL, 9.43 mmol, 1 equiv.) under Ar and the solution was
stirred overnight at room temperature. The solution was filtered to remove MgSO4 and evaporated
under reduced pressure. The resulting crude product was purified by silica gel chromatography
(CH2Cl2) to give the title compound as a white solid (7.35 mmol, 1.39 g, 78%). Rf 0.2 (CH2Cl2). Mp: 86–
1
88 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.17–319 (m, 4H), 3.88–3.91 (m, 4H), 7.26–730 (m, 1H),
13
7.33–7.37 (m, 2H), 7.60–7.61 (m, 3H). C NMR (100 MHz, CDCl3): δC (ppm) 51.88 (2C), 66.50 (2C),
+
+
126.23 (2C), 128.40, 128.58 (2C), 135.92, 136.31(C=N). HRMS (ESI ): m/z calcd for C11H15N2O [M + H]
191.11844, found 191.11733.
1‐Morpholino‐3‐phenylthiourea (32). To a solution of isothiocyanatobenzene (1.00 g, 7.40 mmol, 1
equiv.) in MeOH, 4‐amino‐morpholin (0.91 g, 8.9 mmol, 1.2 equiv.) was slowly added and the
reaction was monitored with TLC until complete disappearance of the starting material and the
formation of a white solid. The solution was then filtered and residue recovered was recrystallized
in MeOH to afford the desired product as a white solid (5.92 mmol, 1.40 g, 80%). Rf 0.35
1
(Cyclohexane/EtOAc: 3/2). Mp: 160–161 °C. H NMR (400 MHz, CDCl3): δH (ppm) 2.99–3.02 (m, 4H),
13
3.74–3.76 (m, 4H), 7.54–7.59 (m, 2H) 7.62–7.66 (m, 1H), 7.75–7.78 (m, 2H). C NMR (100 MHz, CDCl3):
+
δC (ppm) 45.54 (2C), 65.74 (2C), 127.45 (2C), 128.80 (2C), 132.64, 134.60, 157.40 (C=S). HRMS (ESI ):
+
m/z calcd for C11H16N3OS [M + H] 238.1114, found 238.1009.
1‐Morpholino‐3‐phenylurea (33). To a stirring solution of phenylisocyanate (1.00 g, 8.4 mmol, 1
equiv.) in CH2Cl2, 4‐amino‐morpholin (1.03 g, 10.1 mmol, 1.2 equiv.) was slowly added and the
reaction was monitored with TLC until complete disappearance of the starting material. A white solid
appeared in the mixture. The solution was filtered and the residue was purified by silica gel
chromatography (EtOAc) to give the title compound as a white solid (6.64 mmol, 1.47 g, 79%). Rf 0.4
1
(EtOAc). Mp: 147–148 °C. H NMR (400 MHz, CDCl3): δH (ppm) 2.67–3.05 (m, 4H), 3.71–3.91 (m, 4H),
13
5.53 (s, 1H), 7.04–7.08 (m, 1H), 7.29–7.33 (m, 2H), 7.47–7.49 (m, 2H), 8.05 (s, 1H). C NMR (100 MHz,
CDCl3): δC (ppm) 56.81 (2C), 66.75 (2C), 119.21 (2C), 123.26, 129.02 (2C), 138.07, 154.75 (C=O). HRMS
+
+
(ESI ): m/z calcd for C11H14NO2 [M + H] 222.1242, found 222.1231.
Benzyl morpholine‐4‐carbodithioate (34). To a solution of CS2 (0.5 g, 6.6 mmol, 1 equiv.) in diethyl
ether, morpholine (1.13 mL, 13.2 mmol, 2 equiv.) was slowly added. The resulting mixture was stirred
for 1 h at room temperature. The precipitated salt was recovered by filtration and dried over vacuum.
The morpholinium salt (0.5 g, 2.0 mmol, 1 equiv.) was then mixed with (thiocyanatomethyl)benzene
(0.3 g, 2.0 mmol, 1 equiv.) in acetonitrile and stirred at room temperature during 2 h. The solution
was evaporated under vacuum, the resulting powder was diluted in 15 mL of water and extracted
with EtOAc. The combined organic layers were washed with brine and dried over Na2SO4. The
solvents were removed in vacuo, and the resulting crude product was purified by silica gel
chromatography (Cyclohexane/EtOAc: 3/2) to give the title compound as a white solid (0.8 mmol, 0.2
1
g, 42%). Rf 0.7 (Cyclohexane/EtOAc: 3/2). Mp: 70–71 °C. H NMR (400 MHz, CDCl3): δH (ppm) 3.38–
3.40 (m, 2H), 3.65–3.68, (m, 2H), 3.80 (m, 4H), 3.76 (bs, 4H), 3.93–4.34 (m, 4H), 4.58 (s, 2H), 7.26–7.34
13
(m, 3H), 7.38–7.39 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 42.03 (2C), 66.43 (2C), 127.66, 128.66
+
+
(2C), 129.41 (2C), 135.69, 197.20 (C=S). HRMS (ESI ): m/z calcd for C12H16NOS2 [M + H] 254.06733,
found 254.06602.
(S)‐N‐(2‐Hydroxy‐1‐phenylethyl)morpholine‐4‐carboxamide (35). To
a
solution of (S)‐(+)‐
2phenylglycinol (1.00 g, 7.29 mmol, 1.5 equiv.) in anhydrous CH2Cl2 under Ar, DIPEA (1.71 mL, 9.71
mmol, 2 equiv.) and 4‐morpholinylcarbonyl chloride (0.57 mL, 4.86 mmol, 1 equiv.) were added at 0

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°C. The resulting solution was stirred at room temperature for 12 h. The solvent was removed in
vacuo, and the resulting crude product was purified by silica gel chromatography (CH2Cl2/MeOH:
9/1) to give the title compound as a yellow solid (4.32 mmol, 1.62 g, 89%). Rf 0.2 (EtOAc). Mp: 90–91
1
°C. H NMR (400 MHz, CDCl3): δH (ppm) 3.10–3.21 (m, 4H), 3.45–3.54 (m, 4H), 3.57–3.67 (m, 2H), 4.31
13
(s, 1H), 4.76–4.81 (m, 1H), 5.83 (d, 1H, J = 6.2 Hz), 7.20–7.25 (m, 3H), 7.29–7.32 (m, 2H). C NMR (100
MHz, CDCl3): δC (ppm) 43.89 (2C), 57.11, 66.02, 66.38 (2C), 126.58 (2C), 127.43, 128.54 (2C), 140.67,
+
+
158.06 (C=O). HRMS (ESI ): m/z calcd for C13H19N2O3 [M + H] 251.13957, found 251.13835.
4‐(Phenylsulfonyl)morpholine (36). To a solution of benzenesulfonyl chloride (1.65 g, 9.60 mmol, 1.2
equiv.) in CH2Cl2, morpholine (1.00 g, 11.5 mmol, 1 equiv.) and Et3N (0.97 mL, 9.60 mmol, 1.2 equiv.)
were added and the reaction was stirred during 3 h. A white solid slowly appeared in the mixture.
The residue was filtered and recrystallized in EtOH to afford the desired compound as a white solid
1
(8.16 mmol, 1.85 g, 85%). Rf 0.27 (Cyclohexane/EtOAc: 3/2). Mp: 107–109 °C. H NMR (400 MHz,
CDCl3): δH (ppm) 2.99–3.02 (m, 4H), 3.74–3.76 (m, 4H), 7.54–7.59 (m, 2H), 7.62–7.66 (m, 1H), 7.75–7.78
13
(m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 45.63 (2C), 65.74 (2C), 127.50 (2C), 128.80 (2C), 132.75,
+
+
134.70. HRMS (ESI ): m/z calcd for C10H14NO3S [M + H] 228.0694, found 228.0688.
N‐Morpholinobenzenesulfonamide (37). To a solution of benzenesulfonyl chloride (1.65 g, 9.60 mmol,
1.2 equiv.) in CH2Cl2 was added 4‐amino‐morpholine (1.17 g, 11.5 mmol, 1 equiv.) and Et3N (0.97 mL,
9.60 mmol, 1.2 equiv.). The reaction was stirred for 3 h and followed by TLC. After complete
disappearance of the starting material, 100 mL of water was added on the mixture and the solution
was extracted with CH2Cl2 (75 mL). The organic layers were dried and evaporated under vacuum.
The solid recovered was recrystallized in EtOH to afford the desired compound as a white solid (7.97
1
mmol, 1.93 g, 83%). Rf 0.29 (Cyclohexane/EtOAc: 3/2). Mp: 110–112 °C. H NMR (400 MHz, CDCl3):
δH (ppm) 2.60–2.63 (m, 4H), 3.60–3.62 (m, 4H), 5.34 (s, 1H, NH), 7.51–7.55 (m, 2H), 7.60–7.64 (m, 1H),
13
7.97–7.99 (m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 56.74 (2C), 66.64 (2C), 128.16 (2C), 128.89
+
+
(2C), 133.21, 138.61. HRMS (ESI ): m/z calcd for C10H15N2O3S [M + H] 243.0803, found 243.0798.
(4‐Chlorophenyl)(morpholino)methanethione (38). 4‐chloro‐benzaldehyde (1.00 g, 7.10 mmol, 1
equiv.), morpholine (1.86 g, 21.30 mmol, 3.0 equiv.), and sulfur (0.34 g, 10.70 mmol, 1 equiv.) were
mixed in DMF and the solution stirred at 85 °C for 12 h. The solution was filtered, H2O (100 mL) was
added and the organic layer was extracted 3 times with EtOAc. The organic layer was evaporated
under vacuo, the residue was purified by silica gel chromatography (Cyclohexane/EtOAc: 4/1) to give
a yellow powder. This powder was recrystallized in EtOH to give the title compound as a yellow
1
solid (4.59 mmol, 1.11 g, 65%). Rf 0.5 (Cyclohexane/EtOAc: 3/2). Mp: 140–142 °C. H NMR (400 MHz,
CDCl3): δH (ppm) 3.60–3.65 (m, 4H), 3.87–3.89 (m, 2H), 4.41–4.43 (m, 2H), 7.23–7.25 (m, 2H), 7.33–7.35
13
(m, 2H). C NMR (100 MHz, CDCl3): δC (ppm) 49.55, 52.53, 66.46, 66.62, 126.97 (2C), 128.95 (2C),
+
+
134.93, 140.60, 199.54 (C=S). HRMS (ESI ): m/z calcd for C11H13ClNOS [M + H] 242.04064, found
242.03942.
4‐Chloro‐N‐morpholinobenzenesulfonamide (39). To a solution of 4‐chlorobenzenesulfonyl chloride
(1.24 g, 5.87 mmol, 1.2 equiv.) in CH2Cl2 was added 4‐amino‐morpholine (0.5 g, 4.89 mmol, 1 equiv.)
and Et3N (0.82 mL, 5.87 mmol, 1.2 equiv.). The reaction was stirred for 12 h. After complete
disappearance of the starting material, 100 mL of water was added on the mixture and the solution
was extracted with CH2Cl2 (75 mL). The organic layers were dried and evaporated under vacuum.
The solid recovered was recrystallized in EtOH to afford the desired compound as a white solid (4.34
1
mmol, 1.20 g, 74%). Rf 0.55 (Cyclohexane/EtOAc: 3/2). Mp: 161–163 °C. H NMR (400 MHz, CDCl3):
δH (ppm) 2.64–2.66 (m, 4H), 3.61–3.64 (m, 4H), 5.34 (s, 1H, NH), 7.50–7.52 (m, 2H), 7.90–7.92 (m, 2H).
13
C NMR (100 MHz, CDCl3): δC (ppm) 54.65 (2C), 64.46 (2C), 127.04 (2C), 127.44 (2C), 134.95, 137.64.
+
+
HRMS (ESI ): m/z calcd for C10H15NO3S [M + H] 277.0414, found 277.0408.
4.2. WT PHGDH and PSAT‐1 Purification

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pET28a human PHGDH and pET28a human PSAT‐1 were transformed into BL21 Escherichia coli.
A single colony was grown to an OD600 0.6 in 1 L of Terrific broth. Protein expression was induced
with 1 mM isopropyl thiogalactopyranoside (IPTG). The culture was chilled on ice for 30 min,
cultured for 18 h at 24 °C, and pelleted (6000× g, 20 min). Pellets were resuspended in 60 mL of lysis
buffer (50 mM Tris, pH 8.5, 10 mM MgCl2, 300 mM NaCl, 10% glycerol, 5 mM imidazole) and
sonicated, and cell debris were pelleted by centrifugation (20,000× g, 30 min). The supernatant was
TM
collected and purified using Akta purifier on HisTrap FF column (GE Healthcare). After column
equilibration with wash buffer (50 mM Tris, pH 8.5, 10 mM MgCl2, 300 mM NaCl, 10% glycerol, and
30 mM imidazole), bound proteins were eluted with elution buffer (50 mM Tris, pH 8.5, 10 mM
MgCl2, 250 mM NaCl, 10% glycerol, 250 mM imidazole) and collected (1 mL fractions). Fraction
protein content was measured via a Bradford assay. The most concentrated fractions were pooled
and dialyzed overnight into 4 L of dialysis buffer (50 mM Tris, pH 8.5, 10 mM MgCl2, 250 mM NaCl,
20% glycerol, 1 mM TCEP). Protein purity was assessed via SDS/PAGE and Coomassie staining.
4.3. PHGDH Assay
The enzymatic assay was adapted from a previously described procedure [25]. In brief, the
reactions were performed in PHGDH assay buffer (50 mM Tris, pH 8.5, and 1 mM EDTA) following
resorufin fluorescence emission (Ex 550 nm/Em 580 nm) over time. Substrate and enzyme
+
concentrations were as follows: 240 μM 3‐PG; 120 μM NAD ; 30 mM glutamate; 0.1 mM resazurin;
12 ng/μL human PHGDH; 20 ng/μL human PSAT1; 0.18 μg/μL diaphorase. Inhibitors concentration
varied from 0.03 μM to 1000 μM. The final concentration of DMSO in the assay mixture was set to
5%.
4.4. Target Engagement in Cell Lysates
Target engagement in cell lysates was determined by the cellular thermal shift assay (CETSA)
using cell lysates. Therefore, HL‐60 cells were harvested by centrifugation, washed in PBS, and
resuspended in TBS‐T to a density of 1.67 × 106 cells/mL. Cells were lysed with three freeze‐thaw
cycles (3 min/3 min at −80 °C/37 °C). The lysate was clarified by centrifugation at 17,000× g for 20 min
at 4 °C. The supernatant was aliquoted into PCR tubes (60 mL/tube), and 0.5 mL DMSO (0.8% v/v
final) or 83 mM compound was added to the supernatant and mixture incubated for 30 min at room
temperature. The lysates were then heated at 60 °C (or 37 °C for DMSO reference sample) for 3 min
in an Applied Biosystems Veriti Thermal Cycler, followed by cooling for 3 min at 4 °C. In order to
pellet protein aggregates, the samples were centrifuged at 20,000× g for 20 min at 4 °C. Forty five
milliliters of clarified supernatant was mixed with 15 mL 4 × Laemmli buffer and heated for 10 min
at 95 °C. Fifteen milliliters of the mixture was separated by SDS‐PAGE on a Mini‐PROTEAN TGX or
Criterion TGX gel (4%–15%) and transferred onto nitrocellulose membrane using a Trans‐Blot Turbo
Transfer System (BioRad, Hercules, USA). Membranes were blocked in 1:1 Odyssey blocking buffer
(PBS)/PBS for 1 h at room temperature and incubated with primary antibody at 4 °C overnight.
Primary antibodies for PHGDH (HPA021241, Sigma Aldrich (Overijse, Belgium), rabbit antibody),
PSAT (ab96136, abcam, rabbit antibody) and Vinculin (H‐10) (sc‐25336, Santa Cruz, goat antibody),
were diluted 1:1000 (PSAT and PHGDH) or 1:2000 (Vinculin) in 1:1 Odyssey blocking buffer
(PBS)/PBS. Secondary antibody incubation was performed for 1 h at room temperature with anti‐
rabbit or anti‐goat antibody (IRDye 800 CW) at 1:10,000 dilution in 1:1 Odyssey blocking buffer
(PBS)/PBS. Protein bands were visualized with an Odyssey Fc Imager and analyzed with Image
Studio Software (LiCor Biosciences, Lincoln, USA).
4.5. Cell Models and Cytotoxicity Assay
BT‐20, MDA‐MB‐468 breast cancer cells, and BJ‐5ta were purchased from ATCC and cultured in
DMEM medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS) and 1%
penicillin/streptomycin. For the cytotoxicity assay, cells were seeded at low density in 96‐well plates
in serine containing media following a previously reported procedure [25]. Media was then aspirated,

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and cells were incubated in fresh serine‐replete or ‐deplete media containing compounds at indicated
concentrations or vehicle (DMSO). Cells were grown for 5 days at 37 °C with drug and media changed
daily before assaying cell viability using a Presto Blue assay (Life Technologies) according to the
manufacturer’s instructions.
Author Contributions: The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript.
Funding: 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 Recherche Concertée
(ARC 14/19‐058) grant from the Fédération Wallonie‐Bruxelles. Q.S. is Télévie Research Fellows.
Conflicts of Interest: The authors declare that they have no conflicts of interest.
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