Novel Aryl-Substituted Pyrimidones as Inhibitors of 3-Mercaptopyruvate Sulfurtransferase with Antiproliferative Efficacy in Colon Cancer

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Novel Aryl-Substituted Pyrimidones as Inhibitors of

3‑Mercaptopyruvate Sulfurtransferase with Antiproliferative

Eﬃcacy in Colon Cancer

Marina Bantzi, Fiona Augsburger, J é r é mie Loup, Yan Berset, So ﬁ a Vasilakaki, Vassilios Myrianthopoulos,

Emmanuel Mikros, Csaba Szabo,* and Christian G. Bochet *

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ABSTRACT: The enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is one of the

more recently identiﬁed mammalian sources of H₂S. A recent study identiﬁed several

novel 3-MST inhibitors with micromolar potency. Among those, (2-[(4-hydroxy-6-

methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one) or HMPSNE was found

to be the most potent and selective. We now took the central core of this compound and

modiﬁed the pyrimidone and the arylketone sides independently. A 63-compound library

was synthesized; compounds were tested for H₂S generation from recombinant 3-MST in

vitro. Active compounds were subsequently tested to elucidate their potency and

selectivity. Computer modeling studies have delineated some of the key structural features

necessary for binding to the 3-MST’s active site. Six novel 3-MST inhibitors were tested in

cell-based assays: they exerted inhibitory eﬀects in murine MC38 and CT26 colon cancer

cell proliferation; the antiproliferative eﬀect of the compound with the highest potency

and best cell-based activity (1b) was also conﬁrmed on the growth of MC38 tumors in

mice.

1. INTRODUCTION

Endogenously produced small molecules, including nitric oxide

(NO), carbon monoxide (CO), and hydrogen sulﬁde (H₂S),

are known as gasotransmitters, which inﬂuence many

important cellular functions in the body, including the

cardiovascular, nervous, and gastrointestinal systems. For

example, H₂S plays physiological roles in the regulation of

vascular tone, cell metabolism, and angiogenesis and

pathophysiological roles in cardiovascular disease, inﬂamma-

tion, and cancer.¹⁻³Three mammalian enzymes are respon-

sible for H₂S biosynthesis: cystathionine-β-synthase (CBS),

cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfur-

transferase (3-MST).¹⁻³The production of H₂S is increased in

various tumor cells, and this mediator supports cancer cell

growth, proliferation, and migration.⁴⁻⁶Depending on the

cancer cell type, one or more of these enzymes contribute to

H₂S production.⁴⁻⁶Human and murine colon cancer cells

express high levels of 3-MST, therefore pharmacological

inhibitors of 3-MST may have antitumor potential.⁶⁻⁹

Figure 1. (A−D) Novel 3-MST inhibitors identiﬁed by Hanaoka and

colleagues. Compound C (designated as compound 1a in the current

study) was used as a starting point for medicinal chemistry

modiﬁcations.¹⁰

pharmacological and potential translational standpoints due to

its selectivity for 3-MST. Molecular simulations sustained by

Recently, Hanaoka et al., using a high-throughput screening

of a 174,118 compound library via an HSip-1 ﬂuorescence

assay, identiﬁed four novel 3-MST inhibitors A−D (Figure 1)

with IC₅₀values in the micromolar range.¹⁰Among those,

inhibitor Calso known as (2-[(4-hydroxy-6-methylpyrimi-

din-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one) or

HMPSNE, which we refer to it as compound 1a in our

reportwas found to be the most interesting compound from

Received: February 9, 2021

Published: April 15, 2021

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the available crystallographic data of 1a in complex with 3-

MST conﬁrmed the pivotal importance of the interaction

formed between the inhibitor pyrimidone ring and the

sulfhydrated C248 sidechain of the enzyme.¹⁰However, a

number of additional interactions were identiﬁed as important

for stabilizing the inhibitor−enzyme complex, thus prompting

for further exploration of the structure−activity relationship

(SAR) landscape of lead compound 1a as a means to develop

analogues of potentially enhanced pharmacological interest. In

this direction, a systematic assessment of various 1a

substitution options was performed and characterization of

the resulting derivatives biological activity is performed. More

speciﬁcally, in this work, a collection of analogues of C was

synthesized by varying factors such as the electron density and

the H-bond donor pattern on the bicyclic part of 1a and

probing for the steric requirements on the pyrimidone part of

the lead compound.

The novel derivatives were assessed for their potency as 3-

MST inhibitors as well as for their selectivity over the related

CBS and CSE enzymes in vitro. Most importantly, a selected

group of the most potent derivatives was tested on the

proliferation and viability of two murine colon carcinoma cell

lines and a novel derivative exhibiting the highest in vitro

anticancer activity was also tested in vivo, in comparison with

the starting compound 1a. Molecular simulations were

undertaken alongside biological activity evaluations as a

means to rationalize the derived results and enable the design

and evaluation of new generations of 3-MST inhibitors.

2. RESULTS AND DISCUSSION

One of the common problems in screening large libraries is the

nonsystematic variation of structural features. In this respect,

the SAR landscape of any promising hit remains largely

unexplored, unless extended studies supported by synthesis of

novel derivatives are subsequently performed. Hence, in this

report the central core of the most promising currently known

inhibitor (lead compound 1a, Figure 2) of 3-MST was utilized

Figure 2. Optimization strategy of lead compound 1a. The two

extension vectors of the scaﬀold representing the underexplored

solvent-oriented (Ar, green vector) and pharmacophoric (R, red

vector) sides are depicted as colored spheres.

as a basis for performing a thorough subscaﬀold search in an

eﬀort to better comprehend SAR features responsible for

bioactivity and possibly suggest further potency-enhancing

modiﬁcations. In particular, a series of novel derivatives of the

aforementioned lead were developed by independently

modifying the pyrimidone (red vector “R”, Figure 2) and

arylketone (green vector “Ar”, Figure 2) sides of the scaﬀold by

introducing small substituents or their combinations.

The most extensive interventions were performed along the

Ar vector, which represents the least explored region, actually

positioned toward the solvent, with a series of aromatic

moieties attached (Figure 3A). On the other hand, based on

the already available SAR notions reported by Hanaoka et al.,

any bulky substitution motif on the pyrimidone region (R

vector) should be treated with great caution as it would likely

Figure 3. Diﬀerent variations in the 63 compounds of our library. (A)

Variations in the aromatic part of the molecule. (B) Variations in the

western part of the molecule. (C) Miscellaneous compounds.

result in steric repulsions, thus binding destabilization. Indeed,

this part of the scaﬀold is involved in the key pharmacophoric

interaction with C248, with the methyl moiety of 1a apparently

oﬀering optimal ﬁt.¹⁰Hence, to test this hypothesis, minor

variations in R such as hydrogen and methyl, triﬂuoromethyl,

and amino groups were introduced (Figure 3B) and, ﬁnally, to

achieve more eﬃcient SAR exploration, a small number of

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Figure 4. Screening for 3-MST inhibitors among 1a derivatives. The eﬀect of diﬀerent compounds on human recombinant 3-MST activity and H₂S

released by GYY4137 is measured with the ﬂuorescent probe AzMC. The reference compound 1a is shown in black and bold on all graphs. (A) 3-

MST activity after 1 h of preincubation with each compound at 100 μM concentration and 1 h of kinetic measurement at 37 °C with the substrate

3-MP. Data are represented in percentage of inhibition as a mean of three replicates. (B) Inset of compounds inhibiting the most in (A), classiﬁed

by their IC₅₀value calculated from the corresponding concentration response (see Figure 5) of three independent experiments, except if ﬂagged

with #, indicating that the value is from one experiment that could not be reproduced because of the instability of the molecule. (C) Eﬀect of

selected compounds on H₂S-induced ﬂuorescence represented in percentage of signal with GYY4137 or in the presence of DMSO only as mean

SEM of minimum three replicates. The compounds with value below 80% (shown in light blue) are potential H₂S scavengers.

uncategorized miscellaneous molecules were also synthesized

(Figure 3C).

The remaining compounds were subsequently tested to

elucidate their inhibitory potency, selectivity, and potential

interfering features. First, the eﬀect of the compounds on 3-

MST activity was determined by obtaining concentration−

response curves in vitro (Figure 5) with the resulting IC₅₀

values shown in Figure 4B. The derivatives 2a, 5a, and 5b were

found to be unstable and were not considered further.

To assess whether the concentration-dependent decrease of

the AzMC ﬂuorescent signal in the presence of the compounds

is, indeed, the result of inhibition of 3-MST activity (rather

than an interference with the detection method, either via

direct H₂S-scavenging or through quenching of the ﬂuorescent

signal), the eﬀect of the compounds on the AzMC signal was

assessed in response to a nonenzymatic H₂S source. GYY4137,

a slow-releasing H₂S donor,^11,12was used for this purpose.

Changes in this signal were observed with several compounds

at the highest concentration used in this study (300 μM)

(Figure 4C). When a compound inhibited the GYY4137/H₂S

signal by more than 20%, it was considered as a potential H₂S

scavenger or interferer with the assay.

Most of the compounds were prepared by simple alkylation

of the parent α-bromoketones and 2-mercapto-pyrimidin-

4(3H)-ones (Figure 2) or related nucleophiles (Figure 3) in

moderate to excellent yields (ranging from 21% for 7a up to

98% for 16a). Characterization by common spectroscopic

methods was straightforward, with the particularity of some of

the compounds showing very broad ¹³C NMR signals for the

pyrimidone core, presumably due to multiple tautomeric

forms.

Once compounds 1−33 were synthesized, the next step was

to test if the compounds similar to the lead 1a would inhibit

H₂S production from 3-MST by using the ﬂuorescent H₂S

sensor dye 7-azido-4-methylcoumarin (AzMC). Each com-

pound was incubated at 100 μM concentration with 3-MST

and the resulting AzMC signal was recorded. The results after

2 h of incubation are shown in Figure 4A. About half of the

compounds in our compound library inhibited more than 60%

of the 3-MST-induced signal, with compound 1a inhibition

being at 68%. The ﬁrst stage of our workﬂow enabled the

exclusion of compounds with no eﬀect or an eﬀect much lower

than the reference compound 1a. There were also some

compounds that increased (rather than decreased) the AzMC

signal; these compounds were also excluded from further

analysis.

Regarding the selectivity of the compounds, the eﬀect of the

compounds was evaluated against CBS and CSE, the two other

principal mammalian H₂S-producing enzymes. Compounds

were tested at 30 and 300 μM concentrations; the results for

CBS are shown in Figure 6A and for CSE in Figure 6B.

Unsurprisingly, all the compounds that were previously found

to have a H₂S-scavenging potential also produced concen-

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Figure 5. Concentration responses of 1a derivatives. Human recombinant 3-MST activity in the presence of serial dilutions of 1a derivatives

measured with the ﬂuorescent H₂S probe AzMC. Data are presented as mean SEM of minimum three independent experiments, except for

compounds 2a, 5a, and 5b, the data are from one experiment that could not be reproduced because of the instability of the molecule. The IC₅₀

value was calculated from each curve using the Graphpad Prism nonlinear ﬁtting curve function and is reported in Figure 4B.

tration-dependent inhibition of the CBS- and CSE-induced

H₂S signals. However, compounds 11b and 3a, which did not

exhibit any apparent H₂S-scavenging activity, exerted an

inhibition of CSE-derived H₂S production, and were, therefore,

not 3-MST-selective. Six of the compounds tested, namely, 1b,

2b, 13a, 13b, 21a, and 26a, showed no inhibition against either

CBS or CSE. The fact that all enzymatic or scavenging tests

were performed with the same probe and that the inhibitory

potency on 3-MST does not correlate with the inhibitory

potency on the other enzymes or with GYY4137 is viewed as

additional evidence to suggest that these six remaining

compounds are selective 3-MST inhibitors.

This second selection of compounds exhibited diverse

physicochemical properties (Table 1). The compounds were

subsequently further evaluated in two murine cancer cells

expressing 3-MST enzyme, the colon carcinoma cell lines

CT26 and MC38. First, we have tested if they are able to

inhibit H₂S generation in a cell-based environment. Com-

pound 1a was previously shown to inhibit 3-MST H₂S

production in CT26 cells.⁹In agreement with these prior

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Figure 6. Selectivity testing of potential 3-MST inhibitors. Eﬀect of the selected compounds on human full-length CBS and CSE activity, as

measured with the ﬂuorescent probe AzMC. The reference compound 1a is shown in black and bold on all graphs. The compounds are sorted by

increasing IC₅₀on all graphs. (A) CBS activity after 1 h of preincubation with each compound at 30 μM (top) and 300 μM (bottom)

concentrations and 2 h of kinetic measurement at 37 °C with cofactor PLP, substrates ^L-cysteine and L-homocysteine, and allosteric activator SAM.

Data are represented in percentage of control as mean SEM of minimum three replicates. (B) CSE activity after 1 h of preincubation with each

compound at 30 μM (top) and 300 μM (bottom) concentrations and 2 h of kinetic measurement at 37 °C with cofactor PLP and substrate ^L-

cysteine. Data are represented in percentage of control as mean SEM of minimum three replicates. The compounds with no signiﬁcant inhibition

of both CBS and CSE (shown in teal) are 3-MST-selective inhibitors.

a

Table 1. Predicted Physicochemical Properties of Nonscavenger and 3-MST-Selective Inhibitors

inhibitor

cLog P

cLog S

cCaco-2

cMDCK

cLog K_p

cHOA %

PSA

#in56

1a

1b

2b

13a

13b

21a

26a

2.72

2.32

2.44

1.31

0.96

0.49

1.44

−4.30

−4.02

−3.28

−3.03

−3.15

−3.48

564

451

487

168

137

47

380

291

375

117

95

−2.49

−2.50

−2.42

−3.87

−3.86

−4.91

−2.94

92.1

88.1

89.4

74.4

70.8

59.8

84.8

77.4

79.6

80.3

117.7

118.4

126.8

100.5

16

12

15

29

447

576

a

The calculated partition coeﬃcient (cLog P), water solubility (cLog S), apparent permeability in Caco-2 cells (cCaco-2), apparent permeability in

MDCK cells (cMDCK), skin permeability (cLog K_p), and percent human oral absorption (cHOA %); polar surface area (PSA); and the number of

atoms in 5- and 6-membered rings (#in56) of the derivatives were estimated by utilization of the QikProp algorithm.

ﬁndings, 300 μM of the novel 3-MST inhibitor compounds

markedly decreased the AzMC signal in CT26 cells, and in

MC38 cells as well (Figure 7).

Based on the functional role of endogenously produced H₂S

in cancer cells, the expected functional eﬀect of a 3-MST

inhibitor in a cancer cell would be an inhibition of cell

proliferation.⁴⁻⁹When assessing the eﬀect of the novel 3-MST

inhibitors on cell proliferation, signiﬁcant diﬀerences were

noted between the various compounds in terms of growth

impairment (Figure 7 and Table 2). Assessing the eﬀect of

these compounds on mitochondrial activity (ability of the cells

to convert the dye MTT), the same tendencies and rank-order

of compounds were noted as those established in the

proliferation assays, showing relatively minor inhibitory eﬀects

for compounds 13a, 13b, and 21a. The proliferative and MTT-

converting activity of CT26 cells was more substantially

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Figure 7. 3-MST inhibition in mouse colon cancer carcinoma cell lines. Eﬀective in situ inhibition and its eﬀect on cell growth and metabolism in

CT26 cells (left) and MC38 cells (right). H₂S production in living cells after 4 h of treatment with each compound at 300 μM concentration and 1

h of incubation with a ﬂuorescent probe is presented by the green channel images and the corresponding mean pixel intensity (MPI) in percentage

of control on the graphs. Both the cell conﬂuence after 48 h of growth (black) and MTT conversion by cells after 24 h of incubation (brown) with

serial dilutions of each compound are represented in percentage of control as mean SEM of n = 3 independent experiments.

inhibited by compounds 1b and 26a; nevertheless, these

compounds were, still, less eﬀective than the starting

compound 1a. Compound 2b was found to be equipotent

with 1a. Regarding the activity of the compounds in the second

cancer cell line (MC38), compounds 13a, 13b, 26a, and 21a

were without a signiﬁcant inhibitory eﬀect until 300 μM, while

MTT conversion slightly decreased with compound 21a only.

Compound 1b inhibited MC38 cell growth similar to

compound 1a. In contrast to the CT26 cells, MC38 cells

also express CBS enzyme (Figure 8), which is not aﬀected by

these compounds and which may explain the need for a higher

concentration to induce a functional eﬀect in these cells. This

ﬁnding can be seen as a further (indirect) conﬁrmation of the

3-MST-selectivity of the six new 3-MST inhibitors.

The eﬃcacy of 1b was next evaluated in vivo, in a murine

model of orthotopic transplantation of murine MC38 cells in

immunocompetent mice (Figure 9). In contrast to 1a, which,

at a dose of 20 mg/kg/day failed to aﬀect the growth of the

tumors, 1b exerted an inhibitory eﬀect on tumor growth in this

model (p < 0.05) (Figure 10). 3-MST inhibition also delayed

the experimental discontinuation time in this model

(euthanasia, according to our animal protocol, is required for

mice reaching tumor burden >1000 mm³; euthanasia rate on

day 32 was 75, 67, and 33% in the vehicle, 1a and 1b groups,

respectively).

Molecular simulations were also carried out in order to

rationalize the interaction between 3-MST and the series of

newly synthesized substituted pyrimidone analogues. Diﬀerent

levels of theory were utilized, aiming at reproducing the

experimentally assessed biological activities, thus facilitating

exploration of the SARs landscape of 3-MST inhibition. The

binding mode of 1a has been previously determined by

Hanaoka¹⁰(pdb code: 5WQK), providing the overall binding

geometry of pyrimidones in the active site of 3-MST. A key

observation regarding the aforementioned experimental model

is related to determination of the exact tautomeric state of the

ligand in its bioactive conformation. Indeed, on the basis of a

bidentate interaction involving the side chain of R188 and the

inhibitor N1 and carbonyl moieties, the dominant bioactive

tautomer could be unambiguously assigned as the one carrying

a hydrogen atom on N3.

As a ﬁrst step, a model utilizing docking calculations at the

molecular mechanics theory level was used. The highly

̈

eﬃcient induced-ﬁt docking algorithm (IFD, Schrodinger

LLC, New York, NY, USA, 2020) was implemented, allowing

for a more accurate simulation of the protein conformational

rearrangements induced by ligand binding. The top-ranked

interaction geometries obtained by IFD would be subsequently

used to perform MD simulations and calculate protein−ligand

interaction energies by utilizing an MM-GBSA approach. A

representative subset of the available pyrimidones was used,

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a

Table 2. Characterization of Nonscavenger and 3-MST-Selective Inhibitors

a

IC₅₀values are given according to each evaluation of the compounds, using recombinant 3-MST or murine colon cancer cell lines.

Figure 8. H₂S-producing enzyme expression in CT26 and MC38

cells. Representative western blot of CBS, 3-MST, and CSE in mouse

colon cancer CT26 and MC38 cell lysates. Detection of actin was

used as the internal control.

Figure 9. Evaluation of the eﬀect of 1b on the growth of the mouse

colon cancer carcinoma cell MC38 in vivo. Tumor size in C57Bl/6

mice was measured after the subcutaneous injection of 100,000 MC38

cells in the right ﬂank upon treatment with 20 mg/kg/day of

compound 1a, 1b, or vehicle via i.p. injection. Mice were distributed

in three groups at day 8 according to their tumor size to have a similar

mean tumor size and treated the next day for 2 weeks. Data are

presented as mean SEM, and Sidak’s multiple comparison tests are

shown per day vs vehicle (*p < 0.05).

comprised by analogues that span a broad range of bioactivities

so as to be compatible with regression statistics. Initial MD

calculations resulted in poses demonstrating extensive displace-

ment of the ligands with respect to their anticipated positions,

as these were assumed by the previously published crystallo-

graphic binding mode of 1a. A likely explanation for these

discrepancies could be poor description at the molecular

mechanics level for the interaction between the persulﬁdated

cysteine C248 and the pyrimidone ring, possibly attributable to

suboptimal force-ﬁeld parameterization involving a nontypical

and rather unusual for most biomolecule arrangement of

protein atoms such as C−S−S⁻. As a means to avoid major

displacement of the ligands, force constraints were applied on

the atoms of the pyrimidone core, with ﬂexibility being limited

to the rest of the ligand atoms as well as the surrounding

residues up to a radius of 5 Å. The application of core

constraints resulted in a signiﬁcant improvement of obtained

docking poses (Figure 10A), thus permitting construction of a

quantitative MM-GBSA model demonstrating a highly accept-

able correlation of experimental and predicted binding

aﬃnities with an r²value of 0.86 (Figure 10B). Two interesting

observations were derived from docking simulations. The ﬁrst,

arising from decomposition of the MM-GBSA energy into

individual terms, was that the main contributor discriminating

accounting for the 3-MST binding aﬃnity was the π−π

packing correction term. The second was that the hydroxyl

derivative 4a was predicted to be the most potent ligand, with

this observation based on the combination of docking with

protein hydration mapping. Indeed, utilization of the SZmap

algorithm (OpenEye Inc.) indicated that a putative low-energy

hydration site (shown in Figure 10A as a yellow sphere) might

bridge the aforementioned ligand with the side chain of Asp63,

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Figure 10. (A) Superposition of the lead compound 1a (green carbons) and derivatives 1b (magenta carbons) and 4a (yellow carbons) inside the

active site of 3-MST. Representative hydrogen bonds such as the bidentate interaction between the sidechain of Arg188 and the ligand are depicted

as red dashed lines. The putative hydration site predicted by SZmap that could bridge the ligand with Asp63 and thus provide an explanation for

the enhanced aﬃnity of the hydroxy derivative 4a is shown as a yellow opaque sphere; (B) plot of experimental versus predicted binding aﬃnities as

obtained by the MM-GBSA analysis of MD-derived docked poses for 3-MST; (C,D) depiction of the steric clash determined for the CF₃derivative

(1c) compared to its isosteric CH₃analogue (1a). An extensive steric clash is evident between the triﬂuoromethyl derivative (inset C) and the

protein (shown as an overlay of vdW surfaces in a grid and continuous representation for the ligand and the protein, respectively, and colored

according to the charge distribution), possibly explaining the diminished activity of the CF₃analogue. This is not the case for the methyl derivative

(inset D), where a reasonable ﬁt is observed, in accordance with the experimental results.

thus enabling enhancement of binding aﬃnity for that

derivative as a result of a rather unanticipated stabilizing

interaction facilitated by the thermodynamically stable water

molecule. Yet, the accuracy of this prediction could not be

conﬁrmed, as 4a was regarded as a potential H₂S scavenger

early in the workﬂow and was hence excluded from cell-based

evaluations.

While the methyl analogue retains to a good extent the

activity of the unsubstituted lead, the biological assessment of

the newly synthesized derivatives pointed toward the

diminished activity of the CF₃analogue 1c as a particularly

interesting SAR notion. This has drawn our attention, as the

triﬂuoro moiety is widely regarded as a bioisostere of the

methyl group. A structural explanation of this contradiction

was highly desired on the basis of the previously derived MM

model. However, a steric clash was the factor solely accounting

for this result at the MM level and that was clearly evident in

the major displacement observed in the case of the CF₃

analogueand to a lesser degree of the CH₃compound as

wellat poses resulting by the IFD calculations, albeit the core

constraints applied. This steric clash can be visualized in the

form of extensive overlaps between the respective protein and

ligand atoms (represented as spheres of corresponding van der

Waals radii), as is shown in Figure 10C,D.

direction and for enhancing the description of an interaction

previously described to be largely charge-assisted, high-level ab

initio calculations were performed. Based on this previous

report,¹⁰the stabilizing interaction between the persulﬁdated

cysteine anion and the carbon adjacent to pyrimidone carbonyl

that carries a positive partial charge greatly contributes to the

aﬃnity of HMPSNE for the enzyme. This interaction was

expected to be further strengthened by substituting the

pyrimidone ring with a CF₃group, as this substitution

increases the partial charge of the aforementioned carbon

atom. Yet, the opposite result was derived by ab initio

calculations of the interaction geometry between the cysteine

and the two isosteres and, interestingly, the trend was obtained

at two diﬀerent levels of theory, namely the density-functional

theory (DFT) level implementing the M06-2X functional and

̈

the second-order Moller−Plesset perturbation theory. In both

calculations, a correlation-consistent cc/PVDZ basis set was

utilized and the diﬀerence in interaction energy was −1.28 and

−1.78 kcal/mol in favor of the CF₃derivative, respectively.

Such a result advocates in favor of the notion that isosteric

replacements are to a great degree system-dependent and this

is likely the case for the 3-MST active site as well. In this

instance, calculations at diﬀerent levels of theory converge to

the hypothesis that the plasticity of the enzyme pocket is very

low, thus drastically restricting induced ﬁt eﬀects and rendering

small variations to the ligand bulk as critical activity

determinants, compensating for any potentially constructive

In this case, a gradual increase of steric overlap is recorded in

the order H < CH₃< CF₃, in agreement with the experimental

binding aﬃnities determined for the three analogues. To this

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contribution arising from charge-assisted interactions. In that

direction, each drug target has to be regarded as a distinct

biological system where the principles of isosterism should not

be generalized but be applied with consideration.

primary consequence of diﬀerences in direct potency, but,

more likely, in lipophilicity and cell uptake, as well as potential

diﬀerences in various pharmacological properties (e.g., cellular

residence timeas well as in vivo pharmacological properties

such as metabolism, elimination, and potential eﬀects of its

metabolites). Moreover, as with all pharmacological agents,

secondary and further oﬀ-target eﬀects on enzyme systems

unrelated to H₂S pathways also cannot be excluded.

Given the independent role of bacterial 3-MST (and its

product H₂S) in the regulation of bacterial growth, resistance,

and survival,²³⁻²⁵a further task will be to determine the

potential selectivity of the compounds characterized in the

current work against the bacterial 3-MST isoform.

Finally, in an eﬀort to identify additional trends that might

facilitate rational development of cell-active inhibitors, the

most promising derivatives in cell-based evaluations were

assessed in terms of their predicted physicochemical and

pharmacokinetic properties. Utilization of the QikProp

algorithm (Schrodinger LLC, New York, NY, USA, 2020)

aﬀorded preliminary data suggesting a well-anticipated and

potential correlation between cell-based potencies of 3-MST

inhibitors and factors related to their overall capacity to cross

biological barriers eﬃciently. Although the limited dataset

would not permit a thorough quantitative structure−property

relationship analysis, a strong trend was observed between

properties such as lipophilicity, membrane permeability, and

oral or transdermal absorption (Log P, Log S, Caco-2

permeability, MDCK permeability, and Log K_p) with the

cell-based activities of the studied inhibitors, whereas structural

features such as a low polar surface area and a high number of

atoms in 5- and 6-membered rings were shown to favor

activity. By inspecting the deviations in the results between the

cell-free and cell-based assays, it is clear that careful

consideration of such factors seems to be of pivotal importance

for the development of clinically relevant drug candidates

targeting 3-MST. (In this respect, one limitation of the current

study is that the cell-based inhibition of H₂S production by the

various compounds was only conﬁrmed at the highest

concentration tested (300 μM); potential diﬀerences in the

cell-based inhibitory potency of the various compounds on

H₂S generation at lower concentrations have not been

evaluated.)

The biological roles of 3-MST are multiple. This enzyme is

known to play a role in antioxidant and detoxiﬁcation

processes, and is also recognized as a source of H₂S,

polysulﬁdes, and reactive sulfur species. These actions are

relevant for the regulation of a variety of processes in the ﬁelds

of neurobiology, vascular biology, and immunology.¹³⁻²⁰The

current project focuses on the role of 3-MST in cancer, where

the H₂S produced has been implicated in the maintenance of

tumor cell bioenergetics and proliferation, in the maintenance

of tumor cells in the mesenchymal (as opposed to epithelial)

“state”, as well as in tumor angiogenesis.^6−9,21,22Inhibition of

these processes is expected to exert antitumor eﬀects (on its

own, or possibly in combination with anticancer agents with

diﬀerent molecular targets). The current results, indeed,

support this concept.

3. CONCLUSIONS

Taken together, the current report highlights some of the

molecular features that are useful for the design of 3-MST

inhibitors and demonstrates the anticancer eﬀect of pharmaco-

logical 3-MST inhibition in vitro and in vivo.

4. EXPERIMENTAL SECTION

4.1. Chemistry. 4.1.1. General. All reactions were carried out in

ﬂame-dried ﬂasks under an argon atmosphere. All reactions were

performed at room temperature (22−25 °C) and ambient pressure

(1013.25 hPa) unless otherwise indicated. Yields refer to chromato-

graphically and spectroscopically pure compounds. Unless otherwise

indicated, all reagents were obtained from commercial suppliers

(Chemie Brunschwig, Merck, Fluorochem, TCI) and were used

without further puriﬁcation. Deuterated solvents were obtained from

Cambridge Isotope Laboratories. Analytical thin layer chromatog-

raphy was carried out on alumina plates precoated with the silica gel

(Merck 60-F₂₅₄, 0.2 mm). The visualization was accomplished under

UV light (254 nm) and through coloring with KMnO₄(1.5 g of

KMnO₄, 10 g K₂CO₃, and 1.25 mL 10% NaOH in 200 mL water),

Seebach [10.0 g Ce(SO₄)₂·H₂O, 25.0 g molybdophosphoric acid

hydrate, 60.0 mL H₂SO₄concentrated, and 940 mL H₂O; the solution

was diluted 1:1 with H₂O], or ninhydrin (0.30 g ninhydrin, 3.0 mL

acetic acid, and 97 mL ethanol) staining solution, and the

development was achieved by a stream of hot air (350 °C). Flash

column chromatography was performed on silica gel (32−63, 60 Å)

from Chemie Brunschwig using a forced ﬂow of the eluent.

Concentration under reduced pressure was carried out by the rotary

evaporation method at 40 °C at an appropriate pressure. NMR

spectra were recorded on Bruker-DPX-400 (400 MHz) Bruker-DPX-

300 (300 MHz) NMR spectrometers. The residual solvent signal is

used as the internal standard: chloroform-d₁: 7.26 (¹H NMR), 77.16

(¹³C NMR); dimethyl sulfoxide-d₆: 2.50 (¹H NMR) and 39.52 (¹³C

NMR). The chemical shifts δ are reported in ppm and multiplicities

are classiﬁed using the following abbreviations: singlet (s), doublet

(d), triplet (t), quartet (q), multiplet (m), and combinations thereof.

Coupling constants, J, are given in Hertz (Hz), followed by the

integrated proton number and signal interpretation. All products’

purity has been identiﬁed as >95% by NMR analysis. High-resolution

ESI mass spectra were obtained on a Bruker FTMS 4.7T BioAPEXII

or a Waters SynaptG2-Si mass spectrometer. IR measurements were

recorded on a Bruker Tensor 27 equipped with a Specac MKII

Golden Gate Single-Reﬂection ATR system. Melting points were

Of the various derivatives synthesized in this study, 1b is

only approximately 2-fold more potent in terms of IC₅₀against

recombinant 3-MST than the existing lead 1a, while the other

compounds exhibit lower potency, accordingly, most of the

newly synthesized compounds are less eﬀective as antiprolifer-

ative agents in cancer cells than 1a; perhaps with the exception

of 2b, which (although less potent than 1a on recombinant 3-

MST) was a fairly active inhibitor of cancer cell proliferation.

The novel compound 1b (although more potent than 1a on

recombinant 3-MST) was comparable to 1a (but not more

eﬀective as an antiproliferative agent) in the in vitro

antiproliferative assays in cancer cells. However, compound

1b was more eﬀective than 1a in inhibiting tumor growth in

tumor-bearing mice in vivo. We hypothesize that this

diﬀerence in the observed in vivo eﬃcacy may not be the

measured on a Melting Point B-540 apparatus from Bu

uncorrected.

̈

chi and are

4.1.2. Synthesis of Compounds. 4-Acetamidophenol (33) was

purchased from Fluorochem (catalogue number 005037).

4.1.3. Preparation of Bromide Precursors. 5-Bromoacetyl

salicylamide (corresponding electrophile for 23a−23b) was originally

purchased from Merck (MDL number MFCD00792456). For the

synthesis of compounds 22a−22b, 30a, and 32, 2-chloro-3′,4′-

dihydroxyacetophenone (Merck, MDL number MFCD00002200),

chloroacetic acid (Acros Organics, catalogue number 220320010),

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and 1-naphthoyl chloride (Acros Organics, catalogue number

270380100) were used as the corresponding electrophiles.

4.1.4. Synthesis of the Precursors of Compounds 1a−2b, 4a−5b,

7a−9c, 11a−13c, 15a−27g, and 30a. The appropriate ketone (1.0

solution of N-hydroxysuccinimide (1.4 g, 12.0 mmol, 1.0 equiv) and

triethylamine (1.7 mL, 12.0 mmol, 1 equiv) in dry THF (20 mL) at 0

°C. The resulting suspension was stirred at 0 °C for 1 h and afterward

at room temperature for 4 h. The hydrobromide salt was removed by

ﬁltration and the solution of the newly formed activated acid was

added to a solution of 4-aminophenol (1.1 g, 10.0 mmol, 0.8 equiv) in

THF (10 mL). The reaction mixture was stirred at 0 °C for 2 h and

then all volatiles were removed under reduced pressure. The residue

was ﬁnally puriﬁed by ﬂash column chromatography (n-hexane/ethyl

acetate, 1:1 v/v) and the product was obtained as a white solid (2.11

g, 92%).

equiv) was dissolved in EtOAc (0.5 M) and then copper(II) bromide

(2.0 equiv) was added in one portion. The reaction mixture was

stirred for 2 days at room temperature or heated to 80 °C for 5 h.²⁶

Afterward, remaining precipitates were ﬁltered oﬀ and the ﬁltrate was

puriﬁed through a silica pad (eluent: n-hexane/ethyl acetate = 9:1 v/

v). All volatiles were removed under reduced pressure, the residue has

been dried under high vacuum for 6 h and has been directly used in

the next step without further puriﬁcation.

4.1.8. Synthesis of the Precursors of Compounds 28a and 28c.

(−)-cis-Myrtanylamine (0.2 mL, 1.0 mmol, 1.0 equiv), DMAP (24.4

4.1.5. Synthesis of the Precursors of Compounds 6a, 6g, and

10c. To a solution of the appropriate ketone (1.0 equiv) in Et₂O (0.7

mg, 0.2 mmol, 0.2 equiv), and triethylamine (0.2 mL, 1.2 mmol, 1.2

equiv) were dissolved in dry DCM (10 mL). The resulting mixture

was stirred at room temperature for 15 min. Bromoacetyl bromide

(0.1 mL, 1.2 mmol, 1.2 equiv) was added dropwise at 0 °C and the

solution was stirred at room temperature overnight. The reaction

mixture was quenched with water and extracted twice with EtOAc.

The combined organic layers were dried over Na₂SO₄and the

solvents were removed in vacuo. The residue was then puriﬁed by

ﬂash column chromatography (n-hexane/ethyl acetate, 10:1 → 3:1 v/

v) to yield the desired compound as a colorless oil (362 mg, quant.)

which has been dried under high vacuum for 6 h and has been directly

used in the next step.

M) at 0 °C, bromine (1.0 equiv) was added dropwise. The resulting

solution was stirred for 12 h at room temperature. Subsequently, the

reaction mixture was quenched with water and extracted twice with

Et₂O. The combined organic layers were successively washed with

saturated aqueous sodium hydrogen carbonate solution, saturated

aqueous sodium thiosulfate, brine, dried over Na₂SO₄, and the

solvents were removed under reduced pressure. The residue was then

puriﬁed by ﬂash column chromatography (n-hexane/ethyl acetate,

20:1 → 10:1 v/v) to yield the desired compound,²⁷which has been

dried under high vacuum for 6 h and has been directly used in the

next step without further puriﬁcation.

4.1.9. Synthesis of the Precursor of Compound 29c. 2-Bromo-2′-

nitroacetophenone (1.2 g, 5.0 mmol, 1.0 equiv) and urea (3.0 g, 50.0

4.1.6. Synthesis of the Precursor of Compounds 3a−3b. To a

suspension of 6-quinoline carboxylic acid (750.1 mg, 4.3 mmol, 1.0

mmol, 10.0 equiv) were dissolved in MeCN (50 mL) and the

resulting solution was heated to 80 °C for 3 days. Acetonitrile was

removed in vacuo and the crude was portioned between ethyl acetate

and water. The aqueous phase was extracted twice with EtOAc, the

combined organic layers were dried over Na₂SO₄, and the solvents

were removed in vacuo. The residue was then puriﬁed by ﬂash

column chromatography (n-hexane/ethyl acetate, 10:1 → 3:1 v/v) to

aﬀord the intermediate oxazole as a yellow solid (623 mg, 61%).²⁹In

the following step, oxazole (611.0 mg, 3.0 mmol, 1.0 equiv), DMAP

(72.7 mg, 0.6 mmol, 0.2 equiv), and triethylamine (0.5 mL, 3.6 mmol,

1.2 equiv) were dissolved in dry DCM (20 mL). The resulting

mixture was stirred at room temperature for 15 min. Bromoacetyl

bromide (0.3 mL, 3.6 mmol, 1.2 equiv) was added dropwise at 0 °C

and the solution was stirred at room temperature overnight. The

reaction mixture was quenched with water and extracted twice with

EtOAc. The combined organic layers were dried over Na₂SO₄and the

solvents were removed under reduced pressure. The crude product

was then puriﬁed by ﬂash column chromatography (n-hexane/ethyl

acetate, 10:1 → 3:1 v/v) to yield the desired bromide as a colorless oil

(362 mg, quant.), which has been dried under high vacuum for 6 h

and has been directly used in the next step.

equiv) in dichloromethane (DCM) (17 mL), oxalyl chloride (0.4 mL,

4.9 mmol, 1.2 equiv) and three drops of dimethyl formamide were

added. The reaction mixture was stirred at room temperature for 75

min and then all volatiles were removed under reduced pressure. The

solid residue was suspended in MeCN (17 mL), treated dropwise

with a 2 M solution of TMS-CHN₂in hexane (5.4 mL, 10.8 mmol,

2.5 equiv), and stirred for 2.5 h at room temperature. The reaction

mixture was cooled to 0 °C and a solution of HBr (33% in acetic acid,

3.6 mL, 21.7 mmol, 5.0 equiv) was added dropwise. The ice bath was

removed, and the reaction mixture was stirred for 1 h. Afterward, all

solvents were removed under reduced pressure and the crude mixture

was dried under high vacuum overnight. The solid residue was

subsequently mixed with heptane and reconcentrated twice (2 × 10

mL). The crude was then diluted in DCM (30 mL), washed with

saturated aqueous sodium hydrogen carbonate solution (20 mL), and

the aqueous layer was extracted with DCM (3 × 30 mL). The

combined organic layers were dried over Na₂SO₄and the solvents

were removed in vacuo. The residue was ﬁnally puriﬁed by ﬂash

column chromatography (heptane/ethyl acetate, 1:1 v/v) to yield the

desired compound as a white solid (340.9 mg, 1.4 mmol, 31% over

three steps), which has been dried under high vacuum for 8 h and has

been directly used in the next step.²⁸

4.1.10. Synthesis of the Precursor of Compound 31a.

Methanesulfonamide (300.0 mg, 3.2 mmol, 1.0 equiv) was dissolved

4.1.7. Synthesis of the Precursor of Compound 14a. Bromoacetyl

bromide (1.1 mL, 12.0 mmol, 1.0 equiv) was added dropwise to a

in dry toluene (18 mL) and then bromoacetyl bromide (1.1 mL, 12.6

mmol, 4.0 equiv) was added dropwise and the reaction mixture was

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reﬂuxed for 5 h. Afterward, the reaction mixture was cooled to room

temperature and stored in the fridge overnight. The desired product

was crystallized out of the toluene, ﬁltered under vacuum, and rinsed

with cold toluene (15 mL). The beige solid was collected and dried

under high vacuum overnight (548.8 mg, 2.5 mmol, 81%).³⁰

40.1, 22.6; HRMS (NSI positive, EtOAc): calcd for C₁₇H₁₅O₂N₂S,

[M + H]⁺311.0849; found, 311.0840; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 2920, 2361, 2163, 1989, 1646, 1573, 1509, 1459, 1385,

1358, 1291, 1263, 1229, 1213, 1176, 1162, 1089, 1026, 996, 949, 909,

892, 826, 794, 747, 710, 657, 640; mp 181−183 °C.

4.1.14.2. 2-((2-(Naphthalen-1-yl)-2-oxoethyl)thio)pyrimidin-

4(3H)-one (1b). Yield: 81%; ¹H NMR (400 MHz, DMSO-d₆): δ

[ppm] 12.90 (br, 1H), 8.33−8.37 (m, 1H), 8.19 (d, J = 7.21 Hz, 1H),

8.16 (d, J = 8.31 Hz, 1H), 8.00−8.03 (m, 1H), 7.64−7.67 (m, 1H),

7.58−7.63 (m, 3H), 6.05 (br, 1H), 4.82 (s, 2H); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 196.6, 175.9, 166.2, 160.7, 134.3, 133.2,

132.3, 129.2, 128.2, 127.8, 127.5, 126.3, 125.0, 124.5, 105.0, 40.2;

HRMS (NSI positive, EtOAc): calcd for C₁₆H₁₃O₂N₂S, [M + H]⁺

297.0692; found, 297.0687; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 3086, 2906, 1716, 1683, 1620, 1593, 1568, 1506, 1459, 1358,

1273, 1233, 1182, 1169, 1087, 1075, 1024, 990, 931, 863, 841, 802,

780, 768, 707, 642; mp 172−173 °C.

4.1.14.3. 2-((2-(Naphthalen-1-yl)-2-oxoethyl)thio)-6-

(triﬂuoromethyl)pyrimidin-4(3H)-one (1c). Yield: 66%; ¹H NMR

(400 MHz, DMSO-d₆): δ [ppm] 8.31−8.36 (m, 1H), 8.15−8.22 (m,

2H), 8.00−8.04 (m, 1H), 7.63−7.67 (m, 1H), 7.56−7.62 (m, 2H),

6.62 (s, 1H), 4.87 (s, 2H) (-NH is missing); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 196.6, 178.9, 140.5, 134.8, 133.9, 133.2, 129.9,

128.9 (2C), 128.7, 128.2, 127.0, 125.6 (2C), 125.2 (2C), 40.7;

HRMS (NSI positive, MeOH): calcd for C₁₇H₁₂O₂N₂F₃S, [M + H]⁺

365.0566; found, 365.0555; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 1674, 1548, 1476, 1417, 1278, 1228, 1142, 1087, 861, 803, 768,

711, 642; mp 166−170 °C.

4.1.11. Synthesis of the Corresponding Nucleophile for

Compounds 6g, 26g, and 27g. 2-Amino-4-(triﬂuoromethyl)benzoic

acid (1.0 g, 5.0 mmol, 1.0 equiv) and thiourea (457 mg, 6.0 mmol, 1.2

equiv) were heated at 220 °C under vigorous stirring in a DrySyn

heating block overnight (heat was homogenized using calcined sand).

The resulting malodorous slurry was cooled to room temperature and

was carefully poured into water. The suspension was ﬁltered and

washed several times with water to give the desired compound as a

yellow solid, which was used in the next step without further

puriﬁcation (887 mg, 72%).³¹

4.1.12. General Procedure for the Synthesis of Compounds 1a−

31a. To a suspension of the nucleophile (1.0 equiv) in MeOH (0.1

4.1.14.4. 6-Amino-2-((2-(naphthalen-1-yl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (1d). Yield: 88%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 11.48 (br, 1H), 8.38 (m, 1H), 8.22 (d, J =

6.4 Hz, 1H), 8.16 (d, J = 8.3 Hz, 1H), 8.02 (m, 1H), 7.65 (d, J = 7.9

Hz, 1H), 7.60 (m, 2H), 6.33 (br, 2H), 4.98 (br, 1H), 4.78 (s, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 197.7, 165.2, 163.9, 162.7,

M), KOH (1.0 equiv, 1 M solution in MeOH) was added dropwise.

The mixture was stirred for 15 min at room temperature and then the

electrophile (1.2 equiv) was added at once. The reaction mixture was

left to stir for 12 h at room temperature and afterward it was

quenched by addition of water (the same amount as MeOH). The

desired compound was recovered by ﬁltration, washed with Et₂O, and

dried under high vacuum.

134.7, 133.6, 132.8, 129.7, 128.7, 128.5, 128.0, 126.7, 125.5, 125.0,

81.6, 40.3; HRMS (NSI positive, MeOH): calcd for C₁₆H₁₄O₂N₃S,

[M + H]⁺312.0801; found, 312.0792; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 3536, 3429, 3314, 2920, 1615, 1574, 1507, 1445, 1384,

1291, 1228, 1167, 1091, 981, 950, 909, 805, 771; mp 191−193 °C.

4.1.14.5. 2-((4-Aminopyrimidin-2-yl)thio)-1-(naphthalen-1-yl)-

4.1.13. Synthesis of Compound 32. To a suspension of 2-amino-6-

methylpyrimidin-4(3H)-one (59.9 mg, 0.5 mmol, 1.0 equiv) and 4-

1

ethan-1-one (1e). Yield: 24%; H NMR (400 MHz, DMSO-d₆): δ

[ppm] 8.33 (m, 1H), 8.17 (dd, J = 7.2, 1.0 Hz, 1H), 8.13 (d, J = 8.2

Hz, 1H), 8.00 (m, 1H), 7.79 (d, J = 5.9 Hz, 1H), 7.66−7.56 (m, 3H),

6.89 (br, 2H), 6.12 (d, J = 5.7 Hz, 1H), 4.67 (s, 2H); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 198.3, 168.6, 163.0, 154.7, 135.2, 133.4,

132.2, 129.5, 128.4, 127.7, 127.5, 126.4, 125.3, 124.7, 101.4, 40.4;

HRMS (NSI positive, MeOH): calcd for C₁₆H₁₄ON₃S, [M + H]⁺

296.0852; found, 296.0843; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 3399, 3304, 3142, 3056, 3005, 2970, 2900, 1682, 1643, 1579,

1549, 1549, 1507, 1469, 1345, 1263, 1229, 1210, 1169, 1084, 1024,

985, 947, 935, 856, 798, 769, 716, 639; mp 111−112 °C.

dimethylaminopyridine (10.4 mg, 0.1 mmol, 0.2 equiv) in CHCl₃(2.6

mL), N,N-diisopropylethylamine (0.1 mL, 0.7 mmol, 1.5 equiv) and

1-naphthoyl chloride (101.0 mg, 0.5 mmol, 1.1 equiv) were added.

The reaction mixture was stirred under reﬂux for 20 h. The organic

layer was extracted twice with H₂O, dried over Na₂SO₄, and the

solvents were removed under reduced pressure. The crude mixture

was puriﬁed by ﬂash column chromatography (ethyl acetate/

dichloromethane, 9:1 v/v). Solvents were evaporated and the

compound was dried under high vacuum to furnish 33 (20.5 mg,

0.073 mmol, 15%) as a viscous transparent compound.

4.1.14. Analytical Characterization of the Final Compounds.

Some peaks are missing (as mentioned), because these types of

molecules could be present in the form of three tautomers (α-

pyrimidone, γ-pyrimidone, and hydroxypyrimidine), which do not

interconvert too quickly (intermolecular H transfer).

4.1.14.6. 2-((4,6-Diaminopyrimidin-2-yl)thio)-1-(naphthalen-1-

1

yl)ethan-1-one (1f). Yield: 63%; H NMR (400 MHz, DMSO-d₆):

δ [ppm] 8.36 (m, 1H), 8.21 (dd, J = 7.2, 1.0 Hz, 1H), 8.13 (d, J = 8.2

Hz, 1H), 8.01 (m, 1H), 7.63 (m, 1H), 7.59 (m, 2H), 6.02 (s, 4H),

5.14 (s, 1H), 4.64 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ

[ppm] 198.5, 166.9, 163.1, 134.8, 133.2, 132.0, 129.3, 128.2, 127.8,

127.3, 126.2, 125.2, 124.6, 78.9 (2C are missing); HRMS (NSI

positive, MeOH): calcd for C₁₆H₁₅ON₄S, [M + H]⁺311.0961; found,

311.0951; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3443, 3346,

3162, 3048, 1681, 1644, 1617, 1581, 1545, 1507, 1467, 1371, 1291,

1234, 1167, 1089, 976, 950, 929, 858, 812, 798, 766, 725, 591; mp

155−157 °C.

4.1.14.7. 6-Methyl-2-((2-(naphthalen-2-yl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (2a). Yield: 76%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.58 (br, 1H), 8.81 (s, 1H), 8.15 (d, J = 7.8

Hz, 1H), 8.03 (m, 3H), 7.69 (m, 1H), 7.64 (m, 1H), 5.95 (m, 1H),

4.87 (s, 2H), 1.93 (br, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ

[ppm] 193.6, 164.1, 164.1, 160.2, 135.1, 133.5, 132.1, 130.3, 129.6,

128.8, 128.3, 127.7, 127.0, 123.8, 107.2, 37.4, 22.9; HRMS (NSI

4.1.14.1. 6-Methyl-2-((2-(naphthalen-1-yl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (1a). Yield: 66%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.59 (br, 1H), 8.32 (m, 1H), 8.20 (d, J =

6.8 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.02 (m, 1H), 7.64 (m, 1H),

7.59 (m, 2H), 5.92 (br, 1H), 4.75 (s, 2H), 1.91 (s, 3H); ¹³C NMR

(100 MHz, DMSO-d₆): δ [ppm] 196.9, 163.7, 163.7, 161.2, 134.9,

133.2, 132.2, 129.2, 128.3, 127.6, 127.4, 126.3, 125.1, 124.5, 106.6,

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positive, MeOH): calcd for C₁₇H₁₄O₂N₂NaS, [M + Na]⁺333.0668;

found, 333.0736; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3059,

2924, 2832, 2743, 1652, 1625, 1575, 1538, 1508, 1460, 1437, 1385,

1360, 1293, 1270, 1240, 1218, 1182, 1162, 1132, 1029, 960, 934, 894,

864, 810, 767, 752, 745, 718, 685, 657, 640; mp 193−196 °C.

4.1.14.8. 2-((2-(Naphthalen-2-yl)-2-oxoethyl)thio)pyrimidin-

4(3H)-one (2b). Yield: 64%; ¹H NMR (400 MHz, DMSO-d₆): δ

[ppm] 12.88 (br, 1H), 8.81 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.03

(m, 3H), 7.76 (br, 1H), 7.70 (m, 1H), 7.65 (m, 1H), 6.09 (br, 1H),

4.97 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 193.1,

162.6, 154.4, 135.4, 133.2, 132.3, 130.6, 129.8, 129.1, 128.6, 127.9,

127.3, 123.9, 111.6, 38.3 (1C is missing); HRMS (TOF positive,

MeOH): calcd for C₁₆H₁₂O₂N₂NaS, [M + Na]⁺319.0512; found,

319.0582; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3057, 2918,

2680, 1656, 1625, 1567, 1542, 1469, 1435, 1386, 1353, 1283, 1244,

1226, 1208, 1144, 1123, 1063, 1019, 981, 939, 909, 855, 821, 780,

732, 708, 653, 628; mp 194−201 °C.

7.48 (dd, J = 10.3, 8.2 Hz, 1H), 6.09 (d, J = 5.3 Hz, 1H), 4.81 (s, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 195.7, 162.5, 160.1, 153.7,

131.5, 131.0, 129.5, 129.0, 127.3, 125.6, 123.1, 120.3, 109.2, 108.7,

40.3 (1C is missing); HRMS (TOF positive, MeOH): calcd for

C₁₆H₁₁O₂N₂NaFS, [M + Na]⁺337.0417; found, 337.0434; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3084, 3028, 3002, 2987, 2944,

2901, 2794, 2700, 2631, 1703, 1682, 1625, 1598, 1571, 1540, 1511,

1460, 1424, 1397, 1373, 1359, 1325, 1277, 1235, 1220, 1187, 1169,

1142, 1075, 1047, 1020, 989, 927, 899, 842, 788, 757, 744, 705, 654,

642, 615; mp 190−192 °C.

4.1.14.14. 2-((2-([1,1′-Biphenyl]-4-yl)-2-oxoethyl)thio)-6-methyl-

pyrimidin-4(3H)-one (6a). Yield: 21%; ¹H NMR (400 MHz, DMSO-

d₆): δ [ppm] 8.11 (d, J = 7.79 Hz, 2H), 7.85 (d, J = 7.89 Hz, 2H),

7.76 (d, J = 7.43 Hz, 2H), 7.48−7.55 (m, 2H), 7.42−7.47 (m, 1H),

5.93 (s, 1H), 4.75 (s, 2H), 1.99 (s, 3H) (1H is missing, −NH); ¹³C

NMR (100 MHz, DMSO-d₆): δ [ppm] 193.9, 164.3, 162.8, 145.2,

143.8, 139.3, 135.4, 129.6 (2C), 129.5, 128.9, 127.5, 127.5 (2C),

127.3 (2C), 106.8, 37.9, 23.4; HRMS (NSI positive, EtOH): calcd for

C₁₉H₁₇O₂N₂S, [M + H]⁺337.1005; found, 337.0998; FT-IR (golden

gate, 600−4000 cm⁻¹) ν_max: 2915, 1654, 1603, 1574, 1530, 1388,

1289, 1176, 991, 959, 830, 766, 700, 630; mp 162−170 °C.

4.1.14.15. 2-((2-([1,1′-Biphenyl]-4-yl)-2-oxoethyl)thio)-7-

4.1.14.9. 6-Methyl-2-((2-oxo-2-(quinolin-6-yl)ethyl)thio)-

pyrimidin-4(3H)-one (3a). Yield: 66%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.58 (br, 1H), 9.04 (d, J = 2.6 Hz, 1H),

8.89 (s, 1H), 8.59 (d, J = 8.2 Hz, 1H), 8.27 (dd, J = 1.6, 8.8 Hz, 1H),

8.13 (d, J = 8.8 Hz, 1H), 7.67 (dd, J = 4.2, 8.3 Hz, 1H), 5.92 (br, 1H),

4.86 (s, 2H), 1.90 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ

[ppm] 193.4, 153.0, 149.5, 137.7, 133.9, 130.6, 129.4, 127.4, 127.2,

37.4, 22.9 (5C are missing); HRMS (TOF positive, MeOH): calcd for

C₁₆H₁₃O₂N₃NaS, [M + Na]⁺334.0621; found, 334.0625; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3489, 3066, 2919, 2742, 2668,

1683, 1650, 1534, 1501, 1460, 1384, 1361, 1324, 1287, 1217, 1117,

1028, 999, 911, 875, 831, 770, 657; mp 189−192 °C.

1

(triﬂuoromethyl)quinazolin-4(3H)-one (6g). Yield: 84%; H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.04 (br, 1H), 8.19 (d, J = 8.34 Hz,

3H), 7.89 (d, J = 8.44 Hz, 2H), 7.73−7.82 (m, 2H), 7.62−7.69 (m,

1H), 7.40−7.58 (m, 3H), 7.36 (s, 1H), 4.92 (s, 2H); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 193.8, 160.9, 158.0, 148.5, 145.4, 139.3,

135.5, 134.3, 129.6 (4C), 129.0, 128.4, 127.5 (2C), 127.4 (2C),

125.7, 123.2, 123.0, 121.8, 38.1; HRMS (NSI positive, EtOH): calcd

for C₂₃H₁₆O₂N₂F₃S, [M + H]⁺441.0879; found, 441.0870; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 1686, 1670, 1582, 1465, 1376,

1347, 1318, 1175, 1123, 1061, 997, 844, 754, 695, 644; mp 209−216

°C.

4.1.14.10. 2-((2-Oxo-2-(quinolin-6-yl)ethyl)thio)pyrimidin-4(3H)-

1

one (3b). Yield: 66%; H NMR (400 MHz, DMSO-d₆): δ [ppm]

12.80 (br, 1H), 9.04 (dd, J = 1.5, 4.0 Hz, 1H), 8.87 (d, J = 1.2 Hz,

1H), 8.59 (d, J = 7.9 Hz, 1H), 8.26 (dd, J = 1.8, 8.8 Hz, 1H), 8.13 (d,

J = 8.8 Hz, 1H), 7.77 (br, 1H), 7.66 (dd, J = 4.2, 8.3 Hz, 1H), 6.10 (d,

J = 4.5 Hz, 1H), 4.98 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ

[ppm] 192.7, 153.1, 149.5, 137.9, 133.3, 130.8, 129.5, 127.4, 127.2,

122.5, 38.17 (4C are missing); HRMS (TOF positive, MeOH): calcd

for C₁₅H₁₁O₂N₃NaS, [M + Na]⁺320.0464; found, 320.0521; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3064, 2921, 1683, 1635, 1566,

1530, 1461, 1427, 1381, 1324, 1276, 1240, 1219, 1160, 1145, 1066,

1026, 941, 903, 888, 828, 773, 754, 710, 653; mp 201−206 °C.

4.1.14.11. 2-((2-(4-Hydroxynaphthalen-1-yl)-2-oxoethyl)thio)-6-

4.1.14.16. 6-Methyl-2-((2-oxo-2-phenylethyl)thio)pyrimidin-

4(3H)-one (7a). Yield: 65%; ¹H NMR (400 MHz, DMSO-d₆): δ

[ppm] 12.71 (br, 1H), 7.98−8.10 (m, 2H), 7.67 (d, J = 7.46 Hz, 1H),

7.51−7.61 (m, 2H), 5.92 (s, 1H), 4.73 (s, 2H), 1.93 (s, 3H); ¹³C

NMR (100 MHz, DMSO-d₆): δ [ppm] 197.9, 165.7, 159.7, 140.4,

136.0, 133.2, 128.5 (2C), 128.0 (2C), 107.2, 37.2, 22.8; HRMS (NSI

positive, MeOH): calcd for C₁₃H₁₃O₂N₂S, [M + H]⁺261.0692;

found, 261.0692; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 2915,

1638, 1578, 1212, 1177, 955, 848, 745, 682; mp 166−170 °C.

4.1.14.17. 2-((2-Oxo-2-phenylethyl)thio)-6-(triﬂuoromethyl)-

pyrimidin-4(3H)-one (7c). Yield: 73%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 13.46 (br, 1H), 8.01−8.03 (m, 1H), 8.00 (d,

J = 1.34 Hz, 1H), 7.66 (d, J = 7.34 Hz, 1H), 7.49−7.59 (m, 2H), 6.58

(s, 1H), 4.81 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm]

192.9, 163.5, 160.7, 152.6, 135.8, 133.52, 129.4, 128.7 (2C), 128.2

(2C), 118.8, 37.96; HRMS (NSI positive, MeOH): calcd for

C₁₃H₁₀O₂N₂F₃S, [M + H]⁺315.0410; found, 315.0406; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 2930, 1667, 1537, 1415, 1274,

1206, 1141, 1091, 988, 878, 857, 735, 710, 681; mp 178−182 °C.

4.1.14.18. 6-Methyl-2-((2-oxo-2-(p-tolyl)ethyl)thio)pyrimidin-

4(3H)-one (8a). Yield: 76%; ¹H NMR (400 MHz, DMSO-d₆): δ

[ppm] 12.68 (br, 1H), 7.95 (s, 1H), 7.93 (s, 1H), 7.37 (s, 1H), 7.35

(s, 1H), 5.91 (br, 1H), 4.71 (s, 2H), 2.39 (s, 3H), 1.96 (br, 3H); ¹³C

NMR (100 MHz, DMSO-d₆): δ [ppm] 193.7, 166.9, 154.2, 144.4,

134.1, 129.8 (2C), 128.9 (2C), 114.1, 110.7, 37.4, 23.1, 21.2; HRMS

(NSI positive, MeOH): calcd for C₁₄H₁₅O₂N₂S, [M + H]⁺275.0849;

found, 275.0847; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 2923,

1688, 1642, 1466, 1292, 1182, 1033, 960, 865, 811, 779, 661; mp

198−202 °C.

1

methylpyrimidin-4(3H)-one (4a). Yield: 80%; H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.64 (br. 1H), 11.18 (br, 1H), 8.66 (d, J = 8.56

Hz, 1H), 8.25 (d, J = 3.55 Hz, 1H), 8.23 (d, J = 3.18 Hz, 1H), 7.60

(td, J = 7.70, 1.34 Hz, 1H), 7.52 (td, J = 7.52, 0.98 Hz, 1H), 6.95 (d, J

= 8.07 Hz, 1H), 5.94 (s, 1H), 4.74 (s, 2H), 1.95 (s, 3H); ¹³C NMR

(100 MHz, DMSO-d₆): δ [ppm] 195.0, 158.4, 132.8 (2C), 132.4,

128.8, 125.9, 125.7 (2C), 125.1, 125.0, 122.8 (2C), 107.1 (2C), 23.6

(2C); HRMS (NSI positive, EtOH): calcd for C₁₇H₁₅O₃N₂S, [M +

H]⁺327.0798; found, 327.0791; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 3241, 2933, 1651, 1565, 1521, 1386, 1357, 1285, 1219,

1163, 1096, 965, 841, 757, 705, 655, 624; mp 225−229 °C.

4.1.14.12. 2-((2-(4-Fluoronaphthalen-1-yl)-2-oxoethyl)thio)-6-

1

methylpyrimidin-4(3H)-one (5a). Yield: 78%; H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.58 (br, 1H), 8.43 (d, J = 8.1 Hz, 1H), 8.29

(dd, J = 8.0, 5.6 Hz, 1H), 8.14 (m, 1H), 7.71 (m, 2H), 7.49 (dd, J =

0.3, 8.1 Hz, 1H), 5.95 (br, 1H), 4.74 (s, 2H), 1.89 (br, 3H); ¹³C

NMR (100 MHz, DMSO-d₆): δ [ppm] 196.6, 164.6, 161.7, 160.4,

132.0, 131.9, 129.9, 129.4, 127.8, 126.1, 123.5, 120.8, 109.2, 107.4,

40.6, 23.4 (1C is missing); HRMS (TOF positive, MeOH): calcd for

C₁₇H₁₃O₂N₂NaFS, [M + Na]⁺351.0574; found, 351.0580; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3000, 2916, 2888, 2740, 1747,

1673, 1646, 1575, 1538, 1507, 1462, 1423, 1385, 1360, 1321, 1288,

1266, 1240, 1216, 1173, 1166, 1144, 1086, 1049, 1022, 962, 914, 852,

838, 822, 809, 789, 766, 748, 703, 659, 614, 695; mp 206−209 °C.

4.1.14.13. 2-((2-(4-Fluoronaphthalen-1-yl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (5b). Yield: 57%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.88 (br, 1H), 8.48 (dd, J = 7.0, 1.5 Hz,

1H), 8.28 (dd, J = 8.1, 5.6 Hz, 1H), 8.14 (m, 1H), 7.78−7.66 (3H),

4.1.14.19. 2-((2-Oxo-2-(p-tolyl)ethyl)thio)-6-(triﬂuoromethyl)-

pyrimidin-4(3H)-one (8c). Yield: 79%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 13.46 (br, 1H), 7.92 (s, 1H), 7.90 (s, 1H),

7.37 (s, 1H), 7.35 (s, 1H), 6.58 (s, 1H), 4.78 (s, 2H), 2.40 (s, 3H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 190.3, 164.9, 163.5, 146.4,

144.5, 133.8, 129.8, 129.8 (2C), 128.8 (2C), 108.4, 38.4, 21.7; HRMS

(NSI positive, MeOH): calcd for C₁₄H₁₂O₂N₂F₃S, [M + H]⁺

329.0566; found, 329.0564; FT-IR (golden gate, 600−4000 cm⁻¹)

6232

https://doi.org/10.1021/acs.jmedchem.1c00260

J. Med. Chem. 2021, 64, 6221−6240

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

ν_max: 2929, 1684, 1608, 1548, 1416, 1362, 1318, 1292, 1236, 1180,

994, 862, 810, 711, 645; mp 199−204 °C.

(2C), 37.6, 24.7, 23.4 (2C are missing); HRMS (NSI positive,

EtOAc): calcd for C₁₅H₁₆O₃N₃S, [M + H]⁺318.0907; found,

318.0900; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3002, 2953,

1647, 1583, 1522, 1399, 1361, 1176, 1115, 1065, 1001, 843; mp 225−

228 °C.

4.1.14.20. 2-((2-(4-Ethylphenyl)-2-oxoethyl)thio)-6-methylpyri-

1

midin-4(3H)-one (9a). Yield: 73%; H NMR (400 MHz, DMSO-

d₆): δ [ppm] 12.67 (br, 1H), 7.97 (s, 1H), 7.95 (s, 1H), 7.40 (s, 1H),

7.38 (s, 1H), 5.92 (br, 1H), 4.72 (s, 2H), 2.69 (q, J = 7.58 Hz, 2H),

1.97 (br, 3H), 1.21 (t, J = 7.58 Hz, 3H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 194.2, 168.5, 163.0, 153.4, 150.4, 134.4, 129.0

(2C), 128.6 (2C), 37.9, 28.7, 23.6, 15.7 (1C is missing); HRMS (NSI

positive, MeOH): calcd for C₁₅H₁₇O₂N₂S, [M + H]⁺289.1005;

found, 289.1001; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 2970,

2888, 1693.9, 1652, 1541, 1458, 1241, 1217, 1178, 992, 958, 834, 735,

663; mp 174−178 °C.

4.1.14.27. N-(4-(2-((6-Oxo-1,6-dihydropyrimidin-2-yl)thio)-

1

acetyl)phenyl)acetamide (13b). Yield: 83%; H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.88 (br, 1H), 10.31 (s, 1H), 8.01 (s, 1H), 7.98

(s, 1H), 7.75 (s, 2H), 7.72 (s, 1H), 6.06 (br, 1H), 4.77 (s, 2H), 2.09

(s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 191.8, 169.5,

144.5, 130.6, 130.2 (2C), 118.7 (2C), 105.7, 38.3, 24.7 (3C are

missing); HRMS (NSI positive, EtOAc): calcd for C₁₄H₁₄O₃N₃S, [M

+ H]⁺304.0750; found, 304.0746; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 1650, 1598, 1523, 1464, 1299, 1260, 1174, 1069, 978,

826, 669; mp 220−226 °C.

4.1.14.21. 2-((2-(4-Ethylphenyl)-2-oxoethyl)thio)-6-

(triﬂuoromethyl)pyrimidin-4(3H)-one (9c). Yield: 66%; ¹H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.46 (br, 1H), 7.94 (s, 1H), 7.92

(s, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 6.59 (s, 1H), 4.79 (s, 2H), 2.70

(q, J = 7.54 Hz, 2H), 1.21 (t, J = 7.58 Hz, 3H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 192.8, 163.6, 160.6, 155.3, 150.5, 134.0, 130.0,

128.9 (2C), 128.6 (2C), 107.1, 38.4, 28.7, 15.6; HRMS (NSI positive,

MeOH): calcd for C₁₅H₁₄O₂N₂F₃S, [M + H]⁺343.0723; found,

343.0715; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 1676, 1602,

1551, 1475, 1415, 1278, 1179, 1141, 1091, 992, 863, 820, 711; mp

166−169 °C.

4.1.14.28. N-(4-(2-((6-Oxo-4-(triﬂuoromethyl)-1,6-dihydropyrimi-

1

din-2-yl)thio)acetyl)phenyl)acetamide (13c). Yield: 81%; H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.45 (br, 1H), 10.30 (s, 1H), 7.99

(s, 1H), 7.97 (s, 1H), 7.74 (s, 1H), 7.72 (s, 1H), 6.58 (s, 1H), 4.75 (s,

2H), 2.09 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 191.8,

169.5, 164.7, 144.5, 130.7, 130.1 (2C), 122.1, 118.6 (2C), 109.4, 38.1,

24.7 (2C are missing); HRMS (NSI positive, EtOAc): calcd for

C₁₅H₁₃O₃N₃F₃S, [M + H]⁺372.0624; found, 372.0616; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 1672, 1588, 1523, 1476, 1415,

1275, 1234, 1208, 1190, 1146, 1090, 991, 865, 832, 712; mp 241−248

°C.

4.1.14.22. 2-((2-Oxo-2-(4-(triﬂuoromethyl)phenyl)ethyl)thio)-6-

1

(triﬂuoromethyl)pyrimidin-4(3H)-one (10c). Yield: 56%; H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.54 (br, 1H), 8.20 (m, J = 7.98

Hz, 2H), 7.93 (m, J = 8.16 Hz, 2H), 6.55 (s, 1H), 4.82 (s, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ [ppm] 193.3, 165.5, 160.1, 151.7,

142.1, 136.4, 135.2, 129.5 (2C), 126.2 (2C), 126.0, 101.7, 38.5;

HRMS (NSI positive, EtOH): calcd for C₁₄H₉O₂N₂F₆S, [M + H]⁺

383.0283; found, 383.0278; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 2931, 1669, 1551, 1476, 1357, 1314, 1235, 1151, 1124, 1093,

1066, 991, 866, 832, 711, 671, 643; mp 201−206 °C.

4.1.14.29. N-(4-Hydroxyphenyl)-2-((4-methyl-6-oxo-1,6-dihydro-

1

pyrimidin-2-yl)thio)acetamide (14a). Yield: quant.; H NMR (400

MHz, DMSO-d₆): δ [ppm] 10.28 (s, 1H), 9.25 (br, 1H), 7.36 (s,

1H), 7.34 (s, 1H), 6.70 (s, 1H), 6.67 (s, 1H), 5.91 (s, 1H), 3.92 (s,

2H), 2.11 (s, 3H) (1H is missing −NH); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 166.6, 166.4, 164.1, 163.8, 153.9, 131.1, 121.3

(2C), 115.6 (2C), 106.9, 35.3, 23.6; HRMS (NSI positive, EtOH):

calcd for C₁₃H₁₄O₃N₃S, [M + H]⁺292.0750; found, 292.0747; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 2923, 1688, 1642, 1545, 1466,

1399, 1363, 1319, 1292, 1222, 1182, 1033, 960, 865, 811, 744, 661;

mp 223−228 °C.

4.1.14.30. 2-((2-(2-Methoxyphenyl)-2-oxoethyl)thio)-6-methyl-

pyrimidin-4(3H)-one (15a). Yield: 98%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.27 (br, 1H), 7.52−7.60 (m, 2H), 7.19 (d,

J = 8.19 Hz, 1H), 7.04 (td, J = 7.49, 0.92 Hz, 1H), 5.94 (s, 1H), 4.58

(s, 2H), 3.92 (s, 3H), 2.04 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆):

δ [ppm] 195.4, 164.6, 164.3, 159.6, 158.6, 134.6, 130.4, 127.3, 121.0,

112.9, 108.1, 56.4, 42.0, 23.6; HRMS (NSI positive, EtOH): calcd for

C₁₄H₁₅O₃N₂S, [M + H]⁺291.0798; found, 291.0792; FT-IR (golden

gate, 600−4000 cm⁻¹) ν_max: 3003, 2841, 1647, 1543, 1457, 1392,

1359, 1238, 1177, 1157, 1112, 962, 845, 756, 659, 634; mp 196−198

°C.

4.1.14.23. 2-((2-(4-Hydroxyphenyl)-2-oxoethyl)thio)-6-methyl-

pyrimidin-4(3H)-one (11a). Yield: 86%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.59 (br, 1H), 10.44 (s, 1H), 7.92−7.94 (m,

1H), 7.90−7.92 (m, 1H), 6.87−6.90 (m, 1H), 6.85−6.87 (m, 1H),

5.95 (br, 1H), 4.66 (s, 2H), 2.00 (s, 3H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 192.0, 164.9, 164.6, 162.8, 131.4 (2C), 128.0,

115.8 (2C), 107.2, 37.6, 23.6 (1C is missing); HRMS (NSI positive,

MeOH): calcd for C₁₃H₁₃O₃N₂S, [M + H]⁺277.0641; found,

277.0635; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3343, 2916,

1640, 1578, 1458, 1387, 1177, 1118, 956, 868, 816, 748; mp 228−233

°C.

4.1.14.24. 2-((2-(4-Hydroxyphenyl)-2-oxoethyl)thio)pyrimidin-

1

4(3H)-one (11b). Yield: 90%; H NMR (400 MHz, DMSO-d₆): δ

[ppm] 12.85 (br, 1H), 10.47 (s, 1H), 7.92 (s, 1H), 7.90 (s, 1H), 7.75

(br, 1H), 6.89 (s, 1H), 6.87 (s, 1H), 6.08 (br, 1H), 4.74 (s, 2H); ¹³C

NMR (100 MHz, DMSO-d₆): δ [ppm] 191.4, 163.0, 154.2, 131.4

(2C), 127.6, 115.8 (2C), 110.2, 38.2 (2C are missing); HRMS (NSI

positive, MeOH): calcd for C₁₂H₁₁O₃N₂S, [M + H]⁺263.0485;

found, 263.0478; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 1652,

1530, 1439, 1390, 1294, 1261, 1166, 980, 823, 647; mp 232−237 °C.

4.1.14.25. Methyl 4-(2-((4-methyl-6-oxo-1,6-dihydropyrimidin-2-

yl)thio)acetyl)benzoate (12a). Yield: 92%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.58 (br, 1H), 8.15 (m, J = 8.56 Hz, 2H), 8.10

(m, J = 8.56 Hz, 2H), 5.94 (s, 1H), 4.73 (s, 2H), 3.89 (s, 3H), 1.91 (s,

3H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 194.2, 166.0, 165.0,

140.3, 133.8, 129.9 (2C), 129.6, 129.0 (2C), 125.5, 108.9, 52.9, 37.9,

23.2; HRMS (NSI positive, EtOH): calcd for C₁₅H₁₅O₄N₂S, [M +

H]⁺319.0747; found, 319.0741; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 3232, 2919, 1714, 1648, 1384, 1280, 1174, 1106, 1032,

998, 963, 866, 830, 760, 693, 626; mp 185−189 °C.

4.1.14.31. 6-Methyl-2-((2-(2-nitrophenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (16a). Yield: 63%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.47 (br, 1H), 8.15 (d, J = 8.07 Hz, 1H),

7.84−7.93 (m, 2H), 7.74−7.82 (m, 1H), 6.00 (br, 1H), 4.58 (s, 2H),

2.10 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 196.4,

164.8, 156.6, 146.5, 135.0, 134.7, 132.5, 129.4, 124.7, 23.6 (3C are

missing); HRMS (NSI positive, EtOH): calcd for C₁₃H₁₂O₄N₃S, [M

+ H]⁺306.0543; found, 306.0538; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 2915, 1715, 1641, 1520, 1462, 1341, 1292, 1177, 1031,

998, 846, 738, 698, 638; mp 186−191 °C.

4.1.14.32. 2-((2-(2-Nitrophenyl)-2-oxoethyl)thio)-6-

1

(triﬂuoromethyl)pyrimidin-4(3H)-one (16c). Yield: 44%; H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.64 (br, 1H), 8.13−8.19 (m, 1H),

7.86−7.93 (m, 1H), 7.76−7.83 (m, 2H), 6.63 (s, 1H), 4.67 (s, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 195.9, 165.3, 146.5, 134.8,

134.7 (2C), 132.6 (2C), 129.0 (2C), 124.9 (2C), 37.5; HRMS (NSI

positive, EtOH): calcd for C₁₃H₉O₄N₃F₃S, [M + H]⁺360.0260;

found, 360.0252; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 1732,

1672, 1525, 1474, 1419, 1348, 1275, 1228, 1197, 1153, 1091, 877,

789, 737, 708, 622; mp 191−195 °C.

4.1.14.26. N-(4-(2-((4-Methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-

thio)acetyl)phenyl)acetamide (13a). Yield: 55%; ¹H NMR (400

MHz, DMSO-d₆): δ [ppm] 12.60 (br, 1H), 10.31 (s, 1H), 8.01 (s,

1H), 7.99 (s, 1H), 7.74 (s, 1H), 7.72 (s, 1H), 5.94 (br, 1H), 4.68 (s,

2H), 2.09 (s, 3H), 1.97 (br, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ

[ppm] 192.6, 169.5, 165.8, 163.3, 144.4, 131.1, 130.1 (2C), 118.7

4.1.14.33. 2-((2-(3-Fluoro-4-hydroxyphenyl)-2-oxoethyl)thio)-6-

methylpyrimidin-4(3H)-one (17a). Yield: 33%; ¹H NMR (400

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Article

MHz, DMSO-d₆): δ [ppm] 12.66 (br, 1H), 10.94 (s, 1H), 7.82 (dd, J

= 12.10, 2.08 Hz, 1H), 7.77 (dd, J = 8.50, 1.77 Hz, 1H), 7.07 (t, J =

8.56 Hz, 1H), 5.91 (br, 1H), 4.65 (s, 2H), 1.97 (br, 3H); ¹³C NMR

(100 MHz, DMSO-d₆): δ [ppm] 191.8, 167.7, 151.7, 150.2, 150.1,

126.1, 117.39, 117.36, 116.4, 116.2, 106.4, 36.9, 23.1; HRMS (NSI

positive, EtOAc): calcd for C₁₃H₁₂O₃N₂FS, [M + H]⁺295.0547;

found, 295.0541; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3224,

1666, 1634, 1591, 1566, 1525, 1384, 1308, 1238, 1200, 1165, 1109,

1005, 966, 830, 798, 655, 613; mp 218−223 °C.

4.1.14.34. 2-((2-(3-Fluoro-4-hydroxyphenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (17b). Yield: 42%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.84 (br, 1H), 11.08 (br, 1H), 7.81 (s, 1H),

7.78 (s, 2H), 7.08 (s, 1H), 6.10 (br, 1H), 4.75 (d, J = 12.23 Hz, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 190.5, 150.4, 127.4, 126.2,

ν_max: 3206, 1656, 1627, 1528, 1390, 1298, 1269, 981, 821, 646; mp

205−220 °C.

4.1.14.40. 2-((2-(3-Chloro-4-methoxyphenyl)-2-oxoethyl)thio)-6-

methylpyrimidin-4(3H)-one (20a). Yield: 85%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.69 (br, 1H), 8.10 (d, J = 2.08 Hz, 1H), 8.05

(dd, J = 8.68, 2.20 Hz, 1H), 7.31 (d, J = 8.80 Hz, 1H), 5.91 (br, 1H),

4.69 (s, 2H) 3.97 (s, 3H), 1.97 (br, 3H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 191.3, 167.0, 158.2, 129.8, 129.4, 129.2, 121.1,

112.3, 56.4, 36.7, 22.8 (3C are missing); HRMS (NSI positive,

MeOH): calcd for C₁₄H₁₄O₃N₂ClS, [M + H]⁺315.0408; found,

325.0405; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 1668, 1590,

1287, 1178, 1151, 1062, 1007, 961, 824, 696, 616; mp 207−212 °C.

4.1.14.41. 2-((2-(3-Chloro-4-methoxyphenyl)-2-oxoethyl)thio)-6-

1

(triﬂuoromethyl)pyrimidin-4(3H)-one (20c). Yield: 85%; H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.46 (br, 1H), 8.06 (d, J = 2.08 Hz,

1H), 8.02 (d, J = 8.68 Hz, 1H), 7.30 (d, J = 8.80 Hz, 1H), 6.58 (s,

1H), 4.75 (s, 2H), 3.97 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ

[ppm] 190.7, 158.5, 129.9, 129.4 (2C), 129.2, 121.3, 118.8, 112.5,

56.7, 37.5 (3C are missing); HRMS (NSI positive, MeOH): calcd for

C₁₄H₁₁O₃N₂ClF₃S, [M + H]⁺379.0126; found, 379.0120; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 1680, 1594, 1503, 1412, 1274,

1233, 1017, 994, 904, 710, 692; mp 132−138 °C.

4.1.14.42. 2-((2-(3,5-Dihydroxyphenyl)-2-oxoethyl)thio)-6-meth-

ylpyrimidin-4(3H)-one (21a). Yield: 74%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.57 (br, 1H), 9.67 (s, 2H), 6.85 (d, J = 2.2 Hz,

2H), 6.48 (t, J = 2.1 Hz, 1H), 5.95 (br, 1H), 4.63 (s, 2H), 2.03 (s,

3H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 193.3, 175.9, 161.0,

158.6, 158.6, 153.1, 138.0, 107.4, 106.2, 106.2, 103.7, 37.7, 23.2;

HRMS (NSI positive, EtOH): calcd for C₁₃H₁₃O₄N₂S, [M + H]⁺

293.0591; found, 293.0580; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 3260, 1661, 1638, 1588, 1531, 1350, 1297, 1203, 1168, 1152,

1048, 1007, 838; mp 210−212 °C.

4.1.14.43. 2-((2-(3,5-Dihydroxyphenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (21b). Yield: 56%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.73 (br, 1H), 9.67 (br, 2H), 7.78 (br, 1H),

6.84 (d, J = 1.5 Hz, 2H), 6.49 (m, 1H), 6.08 (br, 1H), 4.70 (s, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 192.9, 158.9, 158.9, 137.7,

107.8, 106.4, 106.4, 38.5 (4C are missing); HRMS (NSI positive,

MeOH): calcd for C₁₂H₁₁O₄N₂S, [M + H]⁺279.0434; found,

279.0437; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3236, 2929,

1667, 1636, 1592, 1552, 1522, 1461, 1447, 1390, 1350, 1291, 1262,

1157, 992, 894, 679; mp 221 °C.

117.5, 116.4, 116.2, 37.6 (4C are missing); HRMS (NSI positive,

EtOAc): calcd for C₁₂H₁₀O₃N₂FS, [M + H]⁺281.0391; found,

281.0382; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3208, 2163,

1853, 1655, 1602, 1564, 1441, 1264, 822, 724, 645, 617; mp 228−231

°C.

4.1.14.35. 2-((2-(3-Chloro-4-hydroxyphenyl)-2-oxoethyl)thio)-6-

methylpyrimidin-4(3H)-one (18a). Yield: 89%; ¹H NMR (300

MHz, DMSO-d₆): δ [ppm] 12.55 (br, 1H), 11.29 (br, 1H), 8.05

(d, J = 2.02 Hz, 1H), 7.87 (dd, J = 8.57, 2.06 Hz, 1H), 7.08 (d, J =

8.53 Hz, 1H), 5.96 (s, 1H), 4.65 (s, 2H), 1.98 (s, 3H); ¹³C NMR

(100 MHz, DMSO-d₆): δ [ppm] 191.7, 164.4, 158.3, 131.1 (2C),

130.9, 129.6 (2C), 129.0, 120.5, 116.8, 37.3, 23.4; HRMS (NSI

positive, EtOH): calcd for C₁₃H₁₂O₃N₂ClS, [M + H]⁺311.0252;

found, 311.0248; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3241,

2915, 1663, 1631, 1562, 1409, 1305, 1281, 1195, 1172, 1056, 1002,

967, 828, 799, 722, 693, 655, 618; mp 220−224 °C.

4.1.14.36. 2-((2-(3-Chloro-4-hydroxyphenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (18b). Yield: 53%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.86 (br, 1H), 11.31 (br, 1H), 8.02 (d, J =

2.08 Hz, 1H), 7.87 (dd, J = 8.56, 2.20 Hz, 1H), 7.70 (br, 1H), 7.08 (d,

J = 8.56 Hz, 1H), 6.06 (br, 1H), 4.74 (s, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 190.5, 158.0, 130.6, 129.2, 128.0, 120.1, 116.4,

37.6 (4C are missing); HRMS (NSI positive, EtOAc): calcd for

C₁₂H₁₀O₃N₂ClS, [M + H]⁺297.0095; found, 297.0091; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3219, 1655, 1532, 1425, 1307,

1275, 1195, 982, 865, 822, 696, 644, 615; mp 237−242 °C.

4.1.14.37. 2-((2-(3-Chloro-4-hydroxyphenyl)-2-oxoethyl)thio)-6-

(triﬂuoromethyl)pyrimidin-4(3H)-one (18c). Yield: quant.; ¹H

NMR (400 MHz, DMSO-d₆): δ [ppm] 13.46 (br, 1H), 11.27 (s,

1H), 8.00 (d, J = 2.20 Hz, 1H), 7.85 (dd, J = 8.56, 2.20 Hz, 1H), 7.07

(d, J = 8.56 Hz, 1H), 6.58 (s, 1H), 4.71 (s, 2H); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 190.9, 158.4, 131.0 (2C), 129.6 (2C),

128.7, 122.1, 120.5, 119.4, 116.8 (2C), 37.9; HRMS (NSI positive,

EtOH): calcd for C₁₃H₉O₃N₂ClF₃S, [M + H]⁺364.9969; found,

364.9965; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 2137, 1629,

1536, 1420, 1358, 1338, 1308, 1214, 1148, 997, 852, 817, 712, 694,

612; mp 210−213 °C.

4.1.14.38. 2-((2-(4-Hydroxy-3-iodophenyl)-2-oxoethyl)thio)-6-

methylpyrimidin-4(3H)-one (19a). Yield: 40%; ¹H NMR (400

MHz, DMSO-d₆): δ [ppm] 12.66 (br, 1H), 11.36 (br, 1H), 8.35

(d, J = 2.08 Hz, 1H), 7.92 (dd, J = 8.50, 2.14 Hz, 1H), 6.98 (d, J =

8.56 Hz, 1H), 5.90 (br, 1H), 4.63 (s, 2H), 1.99 (br, 3H); ¹³C NMR

(100 MHz, DMSO-d₆): δ [ppm] 164.7, 161.8, 161.6, 140.2, 131.1,

129.9, 115.0, 108.8, 85.2, 37.3, 23.7 (2C are missing); HRMS (NSI

positive, EtOAc): calcd for C₁₃H₁₂O₃N₂IS, [M + H]⁺402.9608;

found, 402.9598; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3216,

1658, 1301, 1275, 1194, 1171, 981, 846, 655; mp 215−219 °C.

4.1.14.39. 2-((2-(4-Hydroxy-3-iodophenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (19b). Yield: 41%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.87 (br, 1H), 11.39 (br, 1H), 8.33 (s, 1H),

7.86−7.99 (m, 1H), 7.72 (br, 1H), 6.92−7.04 (m, 1H), 6.06 (br, 1H),

4.74 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 190.7,

161.9, 140.2, 131.1, 129.4, 115.1, 112.1, 85.3, 38.1 (3C are missing);

HRMS (NSI positive, EtOAc): calcd for C₁₂H₁₀O₃N₂IS, [M + H]⁺

388.9451; found, 388.9444; FT-IR (golden gate, 600−4000 cm⁻¹)

4.1.14.44. 2-((2-(3,4-Dihydroxyphenyl)-2-oxoethyl)thio)-6-meth-

ylpyrimidin-4(3H)-one (22a). Yield: 24%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.55 (s, 1H), 9.76 (br, 2H), 7.45 (dd, J = 8.3,

2.2 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 5.93

(br, 1H), 4.63 (s, 2H), 2.03 (s, 3H); ¹³C NMR (100 MHz, DMSO-

d₆): δ [ppm] 191.5, 164.0, 151.1, 145.2, 127.7, 121.8, 115.2, 115.1,

37.2, 23.1 (3C are missing); HRMS (NSI positive, MeOH): calcd for

C₁₃H₁₂O₄N₂NaS, [M + Na]⁺315.0410; found, 315.0395; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3374, 1660, 1633, 1595, 1571,

1516, 1454, 1424, 1386, 1304, 1244, 1209, 1160, 1126, 1032, 960,

899, 811, 738; mp 217−218 °C.

4.1.14.45. 2-((2-(3,4-Dihydroxyphenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (22b). Yield: 40%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.76 (br, 1H), 9.69 (br, 2H), 7.78 (br, 1H),

7.45 (dd, J = 8.3, 2.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 6.84 (d, J =

8.3 Hz, 1H), 6.07 (br, 1H), 4.70 (s, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 190.9, 162.7, 154.1, 151.2, 145.3, 127.4, 121.8,

115.22, 115.15, 109.9, 37.8 (1C is missing); HRMS (NSI positive,

MeOH): calcd for C₁₂H₁₁O₄N₂S, [M + H]⁺279.0434; found,

279.0424; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3216, 1655,

1634, 1596, 1525, 1467, 1444, 1418, 1387, 1336, 1310, 1298, 1275,

1243, 1168, 1114, 1082, 1010, 982, 925, 870, 824, 791, 631; mp 238−

240 °C.

4.1.14.46. 2-Hydroxy-5-(2-((4-methyl-6-oxo-1,6-dihydropyrimi-

1

din-2-yl)thio)acetyl)benzamide (23a). Yield: 80%; H NMR (400

MHz, DMSO-d₆): δ [ppm] 13.87 (br, 1H), 12.67 (br, 1H), 8.73 (br,

1H), 8.65−8.68 (m, 1H), 8.04−8.14 (m, 2H), 7.02 (d, J = 8.80 Hz,

1H), 5.93 (br, 1H), 4.69 (s, 2H), 1.94 (br, 3H); ¹³C NMR (100

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MHz, DMSO-d₆): δ [ppm] 172.0 (2C), 165.8, 134.5 (2C), 130.3

(2C), 127.7, 118.3 (2C), 114.5, 37.4, 23.5 (1C is missing); HRMS

(NSI positive, EtOAc): calcd for C₁₄H₁₄O₄N₃S, [M + H]⁺320.0700;

found, 320.0695; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3440,

1593, 1561, 1192, 1028, 963, 865, 827, 735, 661; mp 248−255 °C.

4.1.14.47. 2-Hydroxy-5-(2-((6-oxo-1,6-dihydropyrimidin-2-yl)-

thio)acetyl)benzamide (23b). Yield: 75%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 13.88 (s, 1H), 12.89 (br, 1H), 8.74 (br, 1H),

8.13 (br, 1H), 8.07 (d, J = 2.08 Hz, 1H), 8.05 (d, J = 2.08 Hz, 1H),

7.71 (br, 1H), 7.02 (d, J = 8.68 Hz, 1H), 6.07 (br, 1H), 4.79 (s, 2H);

¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 190.8, 171.5, 165.5, 134.0,

129.9, 126.7, 118.0, 114.0, 37.8 (4C are missing); HRMS (NSI

positive, EtOAc): calcd for C₁₃H₁₂O₄N₃S, [M + H]⁺306.0543; found,

306.0541; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3443, 1661,

1497, 1369, 1281, 1185, 984, 831, 662, 609; mp 233−235 °C.

4.1.14.48. 2-((2-(4-Amino-3,5-dichlorophenyl)-2-oxoethyl)thio)-

6-methylpyrimidin-4(3H)-one (24a). Yield: 71%; ¹H NMR (400

MHz, DMSO-d₆): δ [ppm] 12.59 (br, 1H), 7.92 (s, 2H), 6.51 (s,

2H), 5.95 (br, 1H), 4.60 (s, 2H), 2.01 (s, 3H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 145.8, 128.8 (2C), 124.4, 117.3, 36.3, 23.1 (6C

are missing); HRMS (NSI positive, MeOH): calcd for

C₁₃H₁₂O₂N₃Cl₂S, [M + H]⁺344.0022; found, 344.0012; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 3447, 3343, 1647, 1545, 1326,

1274, 1179, 1133, 957, 846, 780, 658; mp 227−230 °C.

1500, 1437, 1313, 1239, 1174, 1105, 1026, 1006, 923, 893, 842, 799,

757, 617; mp 199−202 °C.

4.1.14.54. 2-((2-(Benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)thio)-7-

1

(triﬂuoromethyl)quinazolin-4(3H)-one (26g). Yield: 89%; H NMR

(300 MHz, CDCl₃): δ [ppm] 13.00 (br, 1H), 8.19 (d, J = 8.25 Hz,

1H), 7.77 (dd, J = 8.21, 1.70 Hz, 1H), 7.67 (dd, J = 8.34, 1.38 Hz,

1H), 7.57 (d, J = 1.56 Hz, 1H), 7.46 (s, 1H), 7.11 (d, J = 8.16 Hz,

1H), 6.17 (s, 2H), 4.83 (s, 2H); ¹³C NMR (100 MHz, CDCl₃): δ

[ppm] 192.0, 160.8, 157.9, 152.3, 148.6, 148.3, 134.8, 131.1, 128.4,

125.5, 123.2 (2C), 122.1, 121.8, 108.6, 108.3, 102.7, 38.1; HRMS

(NSI positive, EtOH): calcd for C₁₈H₁₂O₄N₂F₃S, [M + H]⁺

409.0464; found, 409.0456; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 1671, 1585, 1436, 1354, 1261, 1128, 1035, 962, 926, 899, 847,

787, 737, 695, 670, 643; mp 223−227 °C.

4.1.14.55. 6-Methyl-2-((2-oxo-2-(thiophen-2-yl)ethyl)thio)-

pyrimidin-4(3H)-one (27a). Yield: 93%; ¹H NMR (300 MHz,

CDCl₃): δ [ppm] 12.55 (br, 1H), 8.14 (dd, J = 3.85, 1.10 Hz,

1H), 8.06 (dd, J = 4.95, 1.01 Hz, 1H), 7.29 (dd, J = 4.91, 3.81 Hz,

1H), 5.96 (s, 1H), 4.66 (s, 2H), 1.95 (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): δ [ppm] 187.1, 164.5, 163.7, 160.8, 143.2, 135.7, 134.4,

129.2, 104.4, 37.5, 23.3; HRMS (NSI positive, EtOH): calcd for

C₁₁H₁₁O₂N₂S₂, [M + H]⁺267.0256; found, 267.0251; FT-IR (golden

gate, 600−4000 cm⁻¹) ν_max: 3102, 2918, 1674, 1634, 1461, 1410,

1179, 1061, 957, 850, 718, 657, 616; mp 195−201 °C.

4.1.14.56. 2-((2-Oxo-2-(thiophen-2-yl)ethyl)thio)-7-

1

4.1.14.49. 2-((2-(4-Amino-3,5-dichlorophenyl)-2-oxoethyl)thio)-

pyrimidin-4(3H)-one (24b). Yield: 68%; ¹H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.83 (br, 1H), 7.90 (s, 2H), 7.76 (br, 1H),

6.53 (s, 2H), 6.08 (br, 1H), 4.70 (s, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 145.9, 128.7 (2C), 124.0, 117.3, 37.3 (6C are

missing); HRMS (NSI positive, MeOH): calcd for C₁₂H₁₀O₂N₃Cl₂S,

[M + H]⁺329.9865; found, 329.9856; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 1673, 1608, 1571, 1541, 1452, 1312, 989, 851, 650; mp

197−207 °C.

(triﬂuoromethyl)quinazolin-4(3H)-one (27g). Yield: 76%; H NMR

(400 MHz, DMSO-d₆): δ [ppm] 13.02 (br, 1H), 8.24 (dd, J = 3.79,

1.10 Hz, 1H), 8.18 (d, J = 8.31 Hz, 1H), 8.10 (dd, J = 5.01, 1.10 Hz,

1H), 7.65 (dd, J = 8.31, 1.47 Hz, 1H), 4.81 (s, 2H) 7.30−7.37 (m,

2H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 187.1, 160.8, 157.7,

148.4, 143.2, 135.9, 134.6, 129.3, 128.4, 123.2, 123.0, 122.9, 121.83,

121.80, 37.8; HRMS (NSI positive, EtOH): calcd for

C₁₅H₁₀O₂N₂F₃S₂, [M + H]⁺371.0130; found, 371.0123; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 2904, 1669, 1580, 1464, 1415,

1382, 1324, 1259, 1208, 1167, 1059, 1043, 950, 903, 789, 720, 693,

617; mp 207−213 °C.

4.1.14.50. 2-((2-(3,5-Dichloro-4-hydroxyphenyl)-2-oxoethyl)-

thio)-6-methylpyrimidin-4(3H)-one (25a). Yield: 67%; ¹H NMR

(400 MHz, DMSO-d₆): δ [ppm] 12.67 (br, 1H), 11.29 (br, 1H), 8.04

(s, 2H), 5.90 (s, 1H), 4.64 (s, 2H), 1.97 (br, 3H); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 153.6, 129.0 (3C), 122.1, 36.6 (7C are

missing); HRMS (NSI positive, EtOAc): calcd for C₁₃H₁₁O₃N₂Cl₂S,

[M + H]⁺344.9862; found, 344.9856; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 2923, 1645, 1554, 1491, 1461, 1395, 1314, 1231, 1201,

1145, 1031, 852, 794, 664; mp 213−225 °C.

4.1.14.57. N-(((1S,2R,5S)-6,6-Dimethylbicyclo[3.1.1]heptan-2-yl)-

methyl)-2-((4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-

1

acetamide (28a). Yield: 60%; H NMR (400 MHz, DMSO-d₆): δ

[ppm] 8.11 (t, J = 5.64 Hz, 1H), 5.98 (s, 1H), 3.80 (s, 2H), 3.07 (d, J

= 6.05 Hz, 1H), 3.05 (d, J = 5.96 Hz, 1H), 2.23−2.33 (m, 1H), 2.14

(s, 3H), 2.10 (br, 1H), 1.85 (br, 2H), 1.83 (s, 2H), 1.80 (br, 1H),

1.34−1.47 (m, 1H), 1.12 (s, 3H), 0.98 (s, 3H), 0.82 (s, 1H), 0.79 (s,

1H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm] 173.6, 167.3, 164.5,

154.4, 107.2, 44.9, 43.3, 41.2, 41.1, 38.7, 34.4, 33.2, 28.2, 26.1, 23.6,

23.3, 19.6; HRMS (NSI positive, EtOH): calcd for C₁₇H₂₆O₂N₃S, [M

+ H]⁺336.1740; found, 336.1731; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 3286, 2903, 1650, 1581, 1470, 1432, 1397, 1361, 1244,

1178, 1032, 957, 851, 684, 619; mp 185−190 °C.

4.1.14.51. 2-((2-(3,5-Dichloro-4-hydroxyphenyl)-2-oxoethyl)-

1

thio)pyrimidin-4(3H)-one (25b). Yield: 80%; H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.85 (br, 1H), 8.02 (s, 2H), 7.77 (d, J = 5.38

Hz, 1H), 6.09 (br, 1H), 4.75 (s, 2H) (1H is missing); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 153.9, 129.1 (2C), 128.2, 122.3 (7C are

missing); HRMS (NSI positive, EtOAc): calcd for C₁₂H₉O₃N₂Cl₂S,

[M + H]⁺330.9705; found, 330.9703; FT-IR (golden gate, 600−4000

cm⁻¹) ν_max: 1657, 1516, 1401, 1313, 1278, 1174, 1037, 984, 828, 798,

646; mp 210−215 °C.

4.1.14.58. N-(((1S,2R,5S)-6,6-Dimethylbicyclo[3.1.1]heptan-2-yl)-

methyl)-2-((6-oxo-4-(triﬂuoromethyl)-1,6-dihydropyrimidin-2-yl)-

thio)acetamide (28c). Yield: 82%; ¹H NMR (300 MHz, DMSO-d₆):

δ [ppm] 8.20 (t, J = 5.46 Hz, 1H), 6.32 (s, 1H), 3.74 (s, 2H), 3.04 (t,

J = 5.87 Hz, 2H), 2.27 (q, J = 7.00 Hz, 1H), 2.02−2.16 (m, 1H),

1.73−1.88 (m, 5H), 1.33−1.44 (m, 1H), 1.12 (s, 3H), 0.97 (s, 3H),

0.80 (d, J = 9.45 Hz, 1H) (1H is missing −NH); ¹³C NMR (100

MHz, DMSO-d₆): δ [ppm] 167.9, 167.4, 151.9, 123.3, 119.7, 106.4,

44.9, 43.3, 41.2, 41.0, 38.7, 34.5, 33.1, 26.2, 26.1, 23.3, 19.5; HRMS

(NSI positive, EtOH): calcd for C₁₇H₂₃O₂N₃F₃S, [M + H]⁺

390.1458; found, 390.1446; FT-IR (golden gate, 600−4000 cm⁻¹)

ν_max: 2906, 1644, 1532, 1468, 1416, 1274, 1142, 1094, 854, 711, 617;

mp 148−151 °C.

4.1.14.52. 2-((2-(3,5-Dichloro-4-hydroxyphenyl)-2-oxoethyl)-

1

thio)-6-(triﬂuoromethyl)pyrimidin-4(3H)-one (25c). Yield: 66%; H

NMR (400 MHz, DMSO-d₆): δ [ppm] 13.45 (br, 1H), 11.29 (br,

1H), 8.00 (s, 2H), 6.58 (s, 1H), 4.71 (s, 2H); ¹³C NMR (100 MHz,

DMSO-d₆): δ [ppm] 190.2, 153.6, 128.9 (2C), 128.5, 122.1, 37.1 (6C

are missing); HRMS (NSI positive, MeOH): calcd for

C₁₃H₈O₃N₂Cl₂F₃S, [M + H]⁺398.9579; found, 398.9574; FT-IR

(golden gate, 600−4000 cm⁻¹) ν_max: 1660, 1552, 1313, 1283, 1202,

1150, 1099, 997, 866, 849, 796, 711; mp 241−250 °C.

4.1.14.53. 2-((2-(Benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)thio)-6-

methylpyrimidin-4(3H)-one (26a). Yield: 98%; ¹H NMR (300

MHz, DMSO-d₆): δ [ppm] 12.54 (br, 1H), 7.70 (dd, J = 8.21, 1.51

Hz, 1H), 7.48−7.54 (m, 1H), 7.07 (d, J = 8.25 Hz, 1H), 6.15 (s, 2H),

5.95 (br, 1H), 4.67 (s, 2H), 2.00 (s, 3H); ¹³C NMR (100 MHz,

CDCl₃): δ [ppm] 192.1, 165.0, 164.4, 163.7, 152.2, 148.3, 131.1,

125.3, 108.6, 108.2, 107.1, 102.6, 37.7, 23.6; HRMS (NSI positive,

EtOH): calcd for C₁₄H₁₃O₄N₂S, [M + H]⁺305.0591; found,

305.0586; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 1651, 1581,

4.1.14.59. N-(4-(2-Nitrophenyl)oxazol-2-yl)-2-((6-oxo-4-(triﬂuor-

omethyl)-1,6-dihydropyrimidin-2-yl)thio)acetamide (29c). Yield:

1

78%; H NMR (400 MHz, DMSO-d₆): δ [ppm] 11.82 (br, 1H),

8.26 (s, 1H), 7.86−7.92 (m, 1H), 7.70−7.81 (m, 2H), 7.58−7.64 (m,

1H), 6.49 (s, 1H), 4.14 (br, 2H) (1H is missing, −NH); ¹³C NMR

(100 MHz, CDCl₃): δ [ppm] 166.4, 165.1, 153.6, 151.9, 151.6, 148.6,

135.4, 133.6, 132.9, 132.4, 130.5, 130.1, 124.4, 124.3, 107.3, 35.1;

HRMS (NSI positive, EtOH): calcd for C₁₆H₁₁O₅N₅F₃S, [M + H]⁺

442.0428; found, 442.0416; FT-IR (golden gate, 600−4000 cm⁻¹)

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ν_max: 2155, 1674, 1528, 1476, 1272, 1225, 1146, 1091, 994, 936, 851,

709; mp 145−150 °C.

presence of 5 μM cofactor PLP. Substrates used for CBS activity were

2 mM ^L-cysteine and 2 mM L-homocysteine as well as 500 μM

allosteric activator SAM, while the substrate used with the CSE

enzyme was 2 mM ^L-cysteine. Speciﬁcity of the ﬂuorescence signal

was conﬁrmed using reference CBS inhibitor AOAA and CSE

inhibitor ^DL-propargylglycine,³⁴and the substrates’ background signal

was removed before data analysis. Compounds were also tested for

H₂S-scavenging activity at 300 μM using 50 mM Tris-HCl, pH 8 as

the reaction buﬀer. Fluorescence signal from 100 μM of H₂S-sensitive

ﬂuorescent probe AzMC was obtained in the presence of a

nonenzymatic source of H₂S using 5 mM GYY4137. After dispensing

all the reagents in a black 96-well plate, ﬂuorescence was immediately

measured in the kinetic mode for 2 h at 37 °C with an Inﬁnite 200

Pro reader (Tecan) as described above.

4.1.14.60. 2-((4-Methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-

acetic Acid (30a). Yield: 44%; ¹H NMR (400 MHz, CDCl₃): δ

[ppm] 12.60 (br, 2H), 5.99 (s, 1H), 3.93 (s, 2H), 2.14 (s, 3H); ¹³C

NMR (100 MHz, CDCl₃): δ [ppm] 169.5, 163.9, 161.5, 106.3, 32.3,

22.9 (1C is missing); HRMS (TOF positive, MeOH): calcd for

C₇H₉O₃N₂S, [M + H]⁺201.0328; found, 201.0332; FT-IR (golden

gate, 600−4000 cm⁻¹) ν_max: 2933, 1720, 1630, 1571, 1459, 1399,

1362, 1295, 1295, 1250, 1172, 969, 914, 872, 839, 780; mp 211−216

°C.

4.1.14.61. 2-((4-Methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio)-N-

1

(methylsulfonyl)acetamide (31a). Yield: 27%; H NMR (400 MHz,

DMSO-d₆): δ [ppm] 12.20 (br, 2H), 6.00 (br, 1H), 3.96 (s, 2H), 3.22

(s, 3H), 2.15 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ [ppm]

167.6, 164.6, 106.6, 40.9, 34.3, 23.1 (2C are missing); HRMS (TOF

positive, MeOH): calcd for C₈H₁₁N₃O₄NaS₂, [M + Na]⁺300.0083;

found, 300.0099; FT-IR (golden gate, 600−4000 cm⁻¹) ν_max: 3189,

3165, 3000, 2983, 2926, 2685, 1722, 1681, 1579, 1544, 1460, 1398,

1364, 1338, 1297, 1240, 1175, 1032, 971, 962, 902, 860, 838, 784,

700, 605; mp 192−198 °C.

4.2.3. Cell Growth Monitoring, Metabolic Activity Measurement,

and Enzyme Expression. Two murine colon carcinoma cell lines were

used in this study, the CT26 cell line purchased from ATCC

(American Type Culture Collection, Manassas, VA, USA) and the

MC38 cell line kindly given by Dr. Alena Donda from Department of

Oncology, UNIL (Lausanne, Switzerland). Both cell lines were

maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM)

containing high glucose, GlutaMAX supplement, and pyruvate

(Gibco, Thermo Fisher, Basel, Switzerland), supplemented with

10% fetal bovine serum (Hyclone, Pittsburgh, PA, USA), 100 units/

mL of penicillin, and 100 μg/mL of streptomycin in a humidiﬁed

incubator at 37 °C and a 5% CO₂atmosphere. Cell proliferation and

metabolic activity measurements were performed as previously

described.⁹Brieﬂy, each cell line was seeded in sterile TC-treated

ﬂat-bottom 96-well plates and treated after 1 h of adherence with

indicated compounds at various ﬁnal concentrations (3, 10, 30, 100,

and 300 μM) by keeping the ﬁnal DMSO concentration constant at

0.5%. The plates were then placed in a humidiﬁed incubator at 37 °C

and 5% CO₂atmosphere, either to record the conﬂuence every hour

by phase contrast scanning for 48 h using an IncuCyte Live Cell

Analysis device (20× objective) (Essen Bioscience, Hertfordshire,

UK) or to test mitochondrial activity after 24 h. The last one was

performed after replacing the medium by 50 μL/well of 2 mg/mL

MTT solution prepared in phosphate-buﬀered saline (PBS) 1×

(Gibco) and incubating the cells for three more hours. The formazan

produced by cells with active metabolism was solubilized in 150 μL/

well of DMSO and mixed by shaking in orbital way for 180 s using an

Inﬁnite 200 Pro reader (Tecan) before measuring the absorbance at

590 and 690 nm (background). For the 24 h incubation, cell density

used for seeding was at 10,000 cells/well, and for the 48 h incubation

5000 cells/well. Each assay was repeated three times in triplicate for

each compound. Enzyme expression was performed as previously

described.⁹The whole protein content was extracted from both CT26

cells and MC38 cells using RIPA Lysis and Extraction Buﬀer (Thermo

Scientiﬁc, Waltham, MA, USA) complemented with Halt Protease

and Phosphatase Inhibitor Cocktail (Thermo Scientiﬁc) just before

use. Protein concentrations were determined by measuring the

absorbance in Pierce Coomassie Plus (Bradford) Assay Reagent

(Thermo Scientiﬁc) using an Inﬁnite 200 Pro reader (Tecan,

4.1.14.62. N-(4-Methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-1-

1

naphthamide (32). Yield: 15%; H NMR (400 MHz, DMSO-d₆):

δ [ppm] 12.15 (br, 2H), 8.34 (m, 1H), 8.12 (d, J = 8.3 Hz, 1H), 8.03

(m, 1H), 7.86 (d, J = 7.0 Hz, 1H), 7.65−7.55 (3H), 5.99 (s, 1H), 2.19

(s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ [ppm] 170.4, 162.4, 160.9,

150.3, 133.9, 133.7, 130.1, 129.9, 128.8, 128.3, 127.1, 127.0, 124.7,

124.5, 108.9, 22.8; HRMS (NSI positive, MeOH): calcd for

C₁₆H₁₄O₂N₃, [M + H]⁺280.1081; found, 280.1072; FT-IR (golden

gate, 600−4000 cm⁻¹) ν_max: 3188, 1733, 1645, 1608, 1553, 1507,

1470, 1434, 1387, 1360, 1283, 1238, 1190, 1137, 1044, 967, 802, 773,

656, 617, 564, 541; mp 123−130 °C.

4.2. Enzymatic and Cell-Based Assays. 4.2.1. Chemicals. 7-

Azido-4-methylcoumarin (AzMC), ^L-homocysteine, thiazolyl blue

tetrazolium bromide (MTT), O-(carboxymethyl)hydroxylamine

hemihydrochloride (AOAA), ^DL-propargylglycine, S-(5′-adenosyl)-L-

methionine (SAM), and sodium mercaptopyruvate dehydrate (3-MP)

were purchased from Sigma-Aldrich Chemie Gmbh (Munich,

Germany). Methyl sulfoxide, 99.8+%, extra pure was obtained from

Fisher Scientiﬁc International, Inc. (Pittsburgh, PA, USA). ^L-cysteine

and pyridoxal-5′-phosphate (PLP) were purchased from AppliChem

GmbH (Darmstadt, Germany).

4.2.2. Enzymatic Assays and Counterscreens. Human recombi-

nant 3-MST was produced in an Escherichia coli expression system and

puriﬁed by GenScript (Piscataway, NJ, USA). A stock solution at 100

mM was prepared for each compound by diluting the powder in 100%

DMSO. The whole compound library was tested at a ﬁnal

concentration of 100 μM in black 96-well plates with 3 μg/well of

human recombinant 3-MST using 50 mM Tris-HCl, pH 8 as the

reaction buﬀer. After 1 h of incubation at 37 °C, the 3-MST substrate

3-MP and the H₂S-sensitive ﬂuorescent probe³²AzMC were added

for a ﬁnal concentration of 2 mM and 10 μM, respectively, in a total

volume of 200 μL/well. In this assay (which does not utilize an

additional sulfur acceptor, e.g., ^L-cysteine), 3-MP serves both as a

sulfur donor and as a sulfur acceptor.³³Fluorescence was immediately

measured in kinetic mode for 1 h at 37 °C with an Inﬁnite 200 Pro

̈

Mannedof, Switzerland). Samples were prepared for gel electro-

phoresis in Bolt LDS Sample Buﬀer (4X) (Invitrogen) and Bolt

Reducing Agent (10X) (Invitrogen, Thermo Scientiﬁc) according to

manufacturer’s instructions, loaded in Bolt 4−12% Bis-Tris Plus Gels

(Invitrogen, Thermo Scientiﬁc) and ran at constant 120 V. Proteins

were transferred to nitrocellulose membranes by dry transfer using the

iBlot 2 Device and Transfer Stacks (Invitrogen). Primary antibodies

were purchased from Abcam (Cambridge, UK): anti-CBS 1/500

(ab135626), anti-3-MST 1/500 (ab85377), and anti-CSE 1/500

(ab151769). The mouse anti-β-actin antibody 1/10,000 was obtained

from Sigma-Aldrich. The incubation was carried out overnight at 4 °C

under agitation, then the membranes were washed with TBST,

incubated for 1 h at RT with the secondary antibodies antirabbit IgG

or antimouse IgG and the HRP-linked antibody (CST, Danvers, MA,

USA) diluted 1/5000 in TBST/5% milk. The detection was

performed with Amersham ECL Prime Western Blotting Detection

Reagent (GE Healthcare, Pittsburgh, PA, USA) and the chemilumi-

̈

reader (Tecan; Mannedof, Switzerland), using excitation and emission

wavelengths of 365 and 450 nm, respectively. To evaluate the IC₅₀of

the compounds inhibiting at least 60% of 3-MST activity at 100 μM

concentration, the assay was repeated using each compound at various

ﬁnal concentrations (3, 10, 30, 100, and 300 μM) by keeping the ﬁnal

DMSO concentration constant at 0.3%. The assay was repeated three

times in triplicate for each compound. The potential IC₅₀of each

compound was calculated using GraphPad Prism (GraphPad Software

Inc.; San Diego, CA, USA) nonlinear ﬁtting curve function on data

points recorded at the slope of the sigmoidal ﬁtting curve generated

between min 20 and min 40 of the overall 60 min measurement.

Compounds with potential potent inhibition of 3-MST were tested

for selectivity in a similar assay using two other H₂S-producing

enzymes, CBS and CSE, at 0.5 and 2 μg/well, respectively, both in the

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nescence was detected with an Azure Imaging System 300 (Azure

Biosystems, Dublin, CA, USA).

4.4.3. MD Calculations. Molecular dynamics simulations were used

to investigate the dynamic aspect of binding between 3-MST and the

studied ligands. Each protein−ligand complex was simulated in an

explicit solvent with AMBER18⁴³for a total simulation time of 5 ns.

The Cys248 C−S−S⁻group was treated as a nonstandard amino acid.

Parameter ﬁles for the Cys248 C−S−S⁻and ligands were generated in

Antechamber, by using the partial charges derived by Gaussian and

modiﬁed in the Parmchk program. The tLEaP module was used to

generate the systems parameter ﬁles using the AMBER force ﬁeld

ff14SB⁴⁴and gaff 2⁴⁵for small molecules. The library ﬁle for Cys248

C−S−S⁻was manually modiﬁed to describe the bond connection of

the residue to the protein. The library ﬁle was then loaded into tLEaP

and topology and parameter ﬁles of the systems were generated.

Systems were solvated in a truncated octahedral water box and the

TIP3P water model was used. The system was neutralized by adding 6

Na⁺ions using the Joung and Cheatham ion parameters.⁴⁶The

systems were minimized in SANDER⁴⁷in three steps and heated up

to 310 K in three sequential steps. Finally, the system was equilibrated

under constant pressure for 50 ps without restraints, followed by the

MD simulations. The pmemd.cuda implementation was used for

heating, equilibration, and production time and a 10 Å nonbonded

cutoﬀ was applied. The SHAKE algorithm was used during the

production runs with a 2 fs time step and the particle mesh Ewald

method was used for long-range electrostatic interactions.⁴⁸

4.2.4. Detection of H₂S Production in Live Cells. H₂S generated in

living cells was measured using an AzMC probe similar to that

reported previously.⁹Each cell line was seeded at 60,000 cells/well in

a 8-well cell culture chamber and left adhering overnight in a

humidiﬁed incubator at 37 °C and 5% CO₂atmosphere. Treatment

with indicated compounds was performed at 300 μM concentration

for 4 h, then supernatant was replaced by 1 mM AzMC solution

prepared in HBSS 1× supplemented with glucose (Gibco). After 1

more hour of incubation, pictures were taken using an Olympus

CKX53 inverted microscope and the ﬂuorescence intensity was

calculated with ImageJ (NIH, Bethesda, MD, USA).

4.3. Tumor-Bearing Mice Model. The animal studies were

approved by the Swiss Federal Food Safety and Veterinary Oﬃce

(license no. 2020_19_FR). Female C57Bl/6 mice were maintained

on standard mouse chow and subcutaneously injected with 100,000

MC38 colon carcinoma cells in the right ﬂank.³⁵Health and tumor

growth were monitored every 2−3 days until day 8 at which mice

were distributed in three groups to reach similar patterns in all groups.

In addition to health and tumor growth monitoring, each group was

treated intraperitoneally for 2 weeks with 20 mg/kg/day of

compounds or vehicle (1a: n = 6; 1b: n = 6; vehicle: n = 4). The

tumor length (l) and width (w) were measured transcutaneously

4.4.4. QM-Level Complex Interaction Energy. As an eﬀort to

computationally predict and thus rationalize the impact of CF₃

substitution on the HMPSNE lead, high-level QM calculations were

performed. For comparison, a model system similar to the one

described earlier was utilized. Calculations were performed at two

diﬀerent levels of theory. The DFT method was used with the M06-

using calipers, and tumor volumes were calculated using the formula

l

2

V = w²×

.

4.4. Computations. 4.4.1. Solvent Mapping. Putative hydration

sites of importance for ligand binding on the studied enzyme were

identiﬁed using SZmap v1.2 (OpenEye Scientiﬁc Software Inc.).³⁶

The ligand structure with PDB code 4JGT of 3-MST cocrystallized

with the native substrate pyruvate was utilized to deﬁne the grid

center for calculations.

4.4.2. Induced Fit Docking. The selected compounds were

subjected to docking calculations using the crystal structure with

PDB id. 5WQK. The protein was prepared using the protein

preparation workﬂow, whereas ligands were subjected to preparation

̈

2X functional, whereas second-order Moller−Plesset perturbation

theory was utilized by the LMP2 method as implemented in Jaguar

̈

software (Schrodinger Release 2020-4: Jaguar, Schrodinger, LLC,

New York, NY, 2020). In both cases, the cc-PVDZ basis set was used.

The interaction energy between the two monomers of the complex

was determined by a single-point calculation, applying the counter-

poise method as implemented in Jaguar software.

̈

by the respective LigPrep module (Schrodinger Release 2020-4:

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00260.

̈

LigPrep, Schrodinger, LLC, New York, NY, 2020). Prior to docking

■

sı

grid generation, all cocrystallized water molecules were discarded. The

catalytic residue His74 interacting with the ligands was protonated at

Nε, based on its pK_avalue as calculated using the software. The

tautomeric form of the ligand pyrimidone carried a proton at N1, as

the alternative form at N3 would fail to sustain a key bidentate

hydrogen bond formed between the ligands and Arg188. The pK_a

values of the nitrogens of pyrimidone were calculated using Jaguar-

Molecular formula strings (CSV)

Homology (docking) model for 1a (PDB)

Homology (docking) model for 1b (PDB)

Homology (docking) model for 1c (PDB)

Homology (docking) model for 2a (PDB)

Homology (docking) model for 2b (PDB)

Homology (docking) model for 4a (PDB)

Homology (docking) model for 5a (PDB)

Homology (docking) model for 5b (PDB)

Homology (docking) model for 13a (PDB)

Homology (docking) model for 21a (PDB)

Homology (docking) model for 26a (PDB)

HPLC traces for lead compounds (PDF)

̈

v9.9 (Schrodinger Release 2020-4: Jaguar, Schrodinger, LLC, New

York, NY, 2020).³⁷IFD was performed for selected derivatives

spanning a wide range of biological activities by the use of the IFD

protocol (Schrodinger Suite 2020).³⁸⁻⁴⁰Unconstrained minimization

̈

resulted in poses of the reference ligand that were considerably

diﬀerent from the crystallographic geometry, thus suggesting the need

to apply external force constraints on the pyrimidone ring to

reproduce the experimental binding pose. Core constraints were

applied with a tolerance of 1 Å and residues were reﬁned within 5.0 Å

of the ligand, while all other parameters were kept default. The

Cys248 C−S−S⁻group was adjusted in its protonated analogue and

treated as such during the IFD calculations. The IFD poses of studied

compounds were rescored using the Prime v3.1 MM-GBSA method,

calculating binding energy using the equation ΔG = E_complex− (E_protein

+ E_ligand), where E denotes Coulombic and van der Waals energetic

contributions in protein−ligand binding and ligand desolvation

energies as well as strain energies derived by calculating the free

ligand and protein using the VSGB 2.0 implicit solvent model and the

OPLS2005 force ﬁeld.^41,42In this calculation, the terminal S atom of

the C−S−S⁻group in Cys248 was manually adjusted to its likely

anionic native form by carrying a partial charge of −0.76 and an atom

type “Thiolate anion” as calculated with Gaussian software at the

CCSD-T/AUG-CC-PVTZ level of theory.⁴³

AUTHOR INFORMATION

Corresponding Authors

■

Csaba Szabo − Chair of Pharmacology, Faculty of Science and

Medicine, University of Fribourg, 1700 Fribourg,

Switzerland; orcid.org/0000-0003-3110-4235;

Email: csaba.szabo@unifr.ch

Christian G. Bochet − Department of Chemistry, University of

Fribourg, 1700 Fribourg, Switzerland; orcid.org/0000-

0003-4267-0523; Email: christian.bochet@unifr.ch

6237

https://doi.org/10.1021/acs.jmedchem.1c00260

J. Med. Chem. 2021, 64, 6221−6240

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Authors

induced-ﬁt docking; IR, infrared; MD, molecular dynamics;

MM-GBSA, molecular mechanics, the generalized Bohr model

and solvent accessibility; MPI, mean pixel intensity; MTT,

thiazolyl blue tetrazolium bromide; NMR, nuclear magnetic

resonance; NO, nitric oxide; PBS, phosphate-buﬀered saline;

PDB, Protein Data Bank; PLP, pyridoxal-5′-phosphate; QM,

quantum mechanics; SAM, S-(5′-adenosyl)-^L-methionine;

SAR, speciﬁc absorption rate; SEM, standard error of the

mean; TC, tissue culture; THF, tetrahydrofuran; UV, ultra-

violet

Marina Bantzi − Department of Chemistry, University of

Fribourg, 1700 Fribourg, Switzerland; Chair of

Pharmacology, Faculty of Science and Medicine, University of

Fribourg, 1700 Fribourg, Switzerland

Fiona Augsburger − Chair of Pharmacology, Faculty of

Science and Medicine, University of Fribourg, 1700 Fribourg,

Switzerland

Jérémie Loup − Department of Chemistry, University of

Fribourg, 1700 Fribourg, Switzerland

Yan Berset − Department of Chemistry, University of Fribourg,

1700 Fribourg, Switzerland

Soﬁa Vasilakaki − Faculty of Pharmacy, Department of

Pharmaceutical Chemistry, University of Athens, 15772

Athens, Greece

Vassilios Myrianthopoulos − Faculty of Pharmacy,

Department of Pharmaceutical Chemistry, University of

Athens, 15772 Athens, Greece

Emmanuel Mikros − Faculty of Pharmacy, Department of

Pharmaceutical Chemistry, University of Athens, 15772

Athens, Greece; orcid.org/0000-0002-5984-5169

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00260

Author Contributions

M.B. and F.A. contributed equally to this work. M.B., F.A, J.L.,

Y.B, S.V., and V.M. designed, conducted, and performed

experiments and written parts of the methods and results

section of the manuscript. C.G.B., C.S., and E.M. designed

experiments and evaluated and interpreted the data and wrote

parts of the manuscript. The ﬁnal version of the manuscript

was written through contributions of all authors. C.G.B. and

C.S. ﬁnalized the manuscript. All authors have given approval

to the ﬁnal version of the manuscript.

Funding

C.G.B. and C.S. acknowledge the support of the Swiss

Krebsliga and the Novartis Foundation (Switzerland).

Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

The technical help of Drs. Nahzli Dilek and Vanessa Martins

during the performance of the in vivo experiments is

appreciated. CBS and CSE enzymes were kindly provided by

Dr. Tomas Majtan (Department of Pediatrics, University of

Colorado Anschutz Medical Campus, Aurora, CO, USA).

ABBREVIATIONS

■

3-MP, sodium mercaptopyruvate dehydrate; 3-MST, 3-

mercaptopyruvate sulfurtransferase; AzMC, 7-azido-4-methyl-

coumarin; AOAA, O-(carboxymethyl)hydroxylamine hemi-

hydrochloride; ATCC, American Type Culture Collection;

ATR, attenuated total reﬂectance; CBS, cystathionine-β-

synthase; CH₃, methyl group; CF₃, triﬂuoromethyl group;

CO, carbon monoxide; CO₂, carbon dioxide; CSE, cystathio-

nine-γ-lyase; DCM, dichloromethane; DFT, density functional

theory; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco’s

modiﬁed Eagle’s medium; DMSO, dimethyl sulfoxide; ES,

electrospray ionization; H₂S, hydrogen sulﬁde; HBSS, Hanks’

balanced salt solution; HMPSNE, 2-[(4-hydroxy-6-methylpyr-

imidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one; Hz,

Hertz; IC₅₀, half-maximal inhibitory concentration; IFD,

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Article Doi

Novel Aryl-Substituted Pyrimidones as Inhibitors of 3-Mercaptopyruvate Sulfurtransferase with Antiproliferative Efficacy in Colon Cancer

DOI: 10.1021/acs.jmedchem.1c00260

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