Discovery and kinetic evaluation of 6-substituted 4-benzylthio-1,3,5- triazin-2(1H)-ones as inhibitors of cathepsin B

Article abstract of DOI:10.1016/j.ejmech.2011.08.005

Cathepsin B is a lysosomal cysteine protease that has various physiological and pathophysiological functions. We present here the discovery of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as inhibitors of cathepsin B, starting from screening of a l

Full text of DOI:10.1016/j.ejmech.2011.08.005

European Journal of Medicinal Chemistry 46 (2011) 4648e4656
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery and kinetic evaluation of 6-substituted 4-benzylthio-1,3,
5-triazin-2(1H)-ones as inhibitors of cathepsin B
a
b
,
,
,
*
ꢀ a
ꢁ a
Izidor Sosic , Bojana Mirkovic , Samo Turk , Bogdan Stefane c, Janko Kos a d, Stanislav Gobec a
ꢀ
a
ꢀ
ꢀ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
b
ꢀ
ꢀ
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia
c EN-FIST Centre of Excellence, Dunajska 156, 1000 Ljubljana, Slovenia
d
ꢀ
Department of Biotechnology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Cathepsin B is a lysosomal cysteine protease that has various physiological and pathophysiological
functions. We present here the discovery of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as
inhibitors of cathepsin B, starting from screening of a library of variously 2,4,6-trisubstituted 1,3,5-
triazines and 1,3,5-triazin-2(1H)-ones on three different human cathepsins. The synthesis and enzy-
matic evaluation of a focused library of new 1,3,5-triazin-2(1H)-ones is also described. The detailed
kinetics analyses have shown that these compounds can act as reversible, partial mixed-type inhibitors
of cathepsin B, with Ki and Ki0 values in the low micromolar range. The inhibitory activities of selected
compounds were also assessed against two related cysteine proteases, cathepsin H and cathepsin L, to
estimate their selectivity; these compounds have a selective profile for catB and catL over catH.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 3 June 2011
Received in revised form
30 July 2011
Accepted 2 August 2011
Available online 9 August 2011
Keywords:
Cathepsin B
Cysteine protease
Noncovalent inhibitors
1,3,5-Triazine-2(1H)-ones
Partial mixed-type inhibition
1. Introduction
activity at neutral pH (typical of the environment of the membrane-
bound or extracellular catB), suggesting an extralysosomal as well
Cathepsin B (catB) (EC 3.4.22.1) is a lysosomal cysteine protease
that belongs to the papain family (C1) of clan CA of the cysteine
proteases [1]. It is unique among cathepsins as it exists in two
conformations, which are responsible for endopeptidase and
dipeptidyl carboxypeptidase activities [2]. In the exopeptidase
conformation access of the extended substrates into the active site
of catB is limited by a 21 amino acid insertion termed the occluding
loop (Ile105-Thr125) [3] that is held on to the body of the enzyme
by two salt bridges (Asp22-His110 and Asp224-Arg116). Addition-
ally, the loop provides two His residues (His110 and His111) that
bind the substrate’s C-terminal carboxylate, enabling the exopep-
tidase activity [4] that has a pH optimum around 5 [5], suitable for
lysosomal compartments. However, catB can also act as an endo-
peptidase since the occluding loop is flexible and can move away
from the active-site cleft [6]. It appears that the conformation of the
loop is pH dependent [3], with a prevalence of endopeptidase
as an extracellular role for cathepsin B endopeptidase activity. The
endopeptidase conformation of the occluding loop can also be
stabilized with inhibitors such as cystatin C [7] and chagasin [8].
As well as participating in protein turnover in lysosomes [2],
catB has some more specific physiological functions, such as bone
resorption [9], liberation of thyroid hormones from thyroglobulin
[10], antigen processing [11], and skin wound healing [12]. Alter-
ations in catB expression, protein levels, activity and localization
are associated with several disease states. Notably, catB has been
shown to be critically involved in tumor metastasis, angiogenesis
and progression [13]. These wide-ranging functions indicate that
catB is a promising druggable target in cancers, rheumatoid
arthritis and other important diseases.
Inhibitors of catB include endogenous inhibitors, such as the
cystatin superfamily of proteins, low molecular weight natural
inhibitors, and synthetic inhibitors. The majority of synthetic
inhibitors that have been described to date are peptidyl compounds
containing an electrophilic functionality, which reacts reversibly or
irreversibly with the catalytic cysteine in the active site of catB [14].
So far, none of these compounds have reached the clinical practice,
mostly due to poor bioavailability, off-target side effects and high
toxicity [15]. Therefore, due to the great number of possible
Abbreviations: Z, benzyloxycarbonyl; AMC, 7-amido-4-methylcoumarin; catB,
cathepsin B; EI, enzymeeinhibitor complex; ES, enzymeesubstrate complex; ESI,
enzymeesubstrateeinhibitor complex.
* Corresponding author. Tel.: þ386 1 476 9500; fax: þ386 1 425 8031.
E-mail address: stanislav.gobec@ffa.uni-lj.si (S. Gobec).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.005

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4649
therapeutic applications, inhibitors of catB still have potential in
drug design and development; thus a search for reversible, selec-
tive, and low molecular weight inhibitors should remain of great
interest in the future.
Recently, Mott and coworkers [16] described triazine nitriles as
promising reversible inhibitors of the catB-like protease from the
parasite Trypanosoma brucei (TbcatB). As it has been claimed that
the triazine nitrile scaffold is privileged in its nature toward
cysteine proteases [16,17], we screened our in-house library of
variously 2,4,6-trisubstituted-1,3,5-triazines (1e23) that were
recently synthesized [18] against three different human cathepsins:
B, H and L (Supplementary data, Tables S1, S2).
newly synthesized compounds were evaluated for inhibition of
catB, and to estimate their selectivity some selected compounds
additionally for inhibition of catH and catL. These data are pre-
sented in Tables 1 and 2, and the assay results clearly indicate
that these compounds have a selective profile for catB and catL
over catH.
From comparisons of the inhibitory activities of newly synthe-
sized compounds 23e38 in Table 1 with the compounds from
Table S1 (e.g. compounds 8e10, 12, 15) and Table S2 (e.g.
compounds 19e22), it can be postulated that the additional
hydrophobic benzylthio residue on position 6 of the 1,3,5-triazine
scaffold is essential for effective inhibition. When examining the
influence of the different substituents on the phenyl ring of the 6-
benzylthio moiety (Table 1, compounds 23e38), it appears that the
electronic nature and the position of the substituent do not have
important roles in the mode or potency of catB inhibition. For
instance, the inhibitory activities of compounds with an electron
donating methoxy group on the phenyl ring (23e25) were
comparable with compounds that have an electron withdrawing
nitro group (29e31). Placement of a chlorine atom on the phenyl
ring resulted in the most potent analogs (32e34), suggesting the
need for a large hydrophobic substituent on this part of the
molecule.
To further assess the importance for catB inhibition of the planar
phenyl ring of the 6-benzylthio substituent, this ring was replaced
with the methyl, ethyl, propyl, cyano, carboxyl, cyclopropyl, cyclo-
butyl, cyclopentyl, and cyclohexyl groups (compounds 39e47). Of
the analogs tested, compounds 41 and 45e47 retained comparable
potencies of catB inhibition to those of the 6-benzylthio substituted
derivatives 23e38. This thus shows that the nature of the hydro-
phobic residue does not have a decisive role in catB inhibition.
Although compounds 39, 40 and 42e44 did inhibit catB as mixed-
type inhibitors (see below), their inhibition was significantly
attenuated by up to two orders of magnitude, indicating that short
alkylthio, cyclopropylthio, eSCH2CN, and eSCH2COOH fragme-
nts are not well tolerated on position 6 of the 4-benzylthio-
1,3,5-triazin-2(1H)-one core. Only compounds 41 and 45e47,
which have larger hydrophobic substituents, can inhibit catB with
a Ki value in the low micromolar range.
All of these 1,3,5-triazines 1e23 were tested for relative inhibition
of catB, catH and catL according to the protocol described in
Materials and methods. As seen from Supplementary Tables S1 and
S2, all of these compounds were practically inactive against all
cathepsins at 50
mM, except for compound 23, which had a
6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-one core. Compound
23 showed high inhibitory activity against catB and catL, whereas it
showed no inhibition of catH. As compound 23 is unique in the series
(it has an additional hydrophobic substituent on position 6 of the
1,3,5-triazine scaffold) and because of our research interest in the
field of catB inhibitors [19], we synthesized new focused library of
similar compounds in order to explore and determine the structural
requirements for catB inhibition. Additionally, detailed kinetic anal-
ysis was performed for all catB inhibitors.
2. Chemistry
The 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones 23e47
were prepared similarly as in the previously described general
synthetic route (Scheme 1) [20]. Briefly, 4-benzylthio-6-mercapto-
1,3,5-triazin-2(1H)-one 22 was prepared from the appropriate
amidine S-(benzyl)isothiourea hydrochloride and ethoxycarbonyl
isothiocyanate, in the presence of 2 M NaOH in toluene at room
temperature. Furthermore, product 22 (Scheme 1) was readily
alkylated using various alkyl chlorides or bromides in a basic EtOH
solution at room temperature, which provided the 6-benzylthio
(23e38), 6-alkylthio (39e43), and 6-cycloalkylmethylthio deriva-
tives (44e47) in very good yields (71%e93%). Purification of these
derivatives was achieved by crystallization from CH3CN or EtOH.
It is not very surprising that the compounds do not exhibit
selectivity between catB and catL since these two enzymes are
structurally very similar [21]. However, this does not represent
crucial obstacle in their further optimization and research, because
recent studies have shown that these two cysteine proteases have
more important contributions to the proteolytic events during
tumor progression than catH [22]. Moreover, the latest trends in the
field of protease inhibitors implicate that the inhibition of multiple
proteases is likely to prove more effective than targeting an indi-
vidual protease within the protease network [23].
3. Results and discussion
3.1. Cathepsin B inhibitory activities
The ease of the chemistry in the second reaction step allowed
the evaluation of more than 20 different moieties attached to
position 6 of the 4-benzylthio-1,3,5-triazin-2(1H)-one core. These
SH
N
NH
EtO
NCS
HN
a
S
NH3+Cl-
O
O
N
S
Ph
22
X = Cl or Br
SH
N
S
N
R
S
R = substituted phenyl (for compounds 23-38)
alkyl (for compounds 39-41)
HN
HN
N
X
R
b
O
N
S
CN (for compound 42)
O
COOH (for compound 43)
Ph
Ph
22
23-47
cycloalkyl (for compounds 44-47)
Scheme 1. General synthesis of compounds 22e47. Reagents and conditions: (a) 2 M NaOH, toluene, H2O, 25 ꢀC, 1 h; (b) 2 M NaOH, EtOH, 25 ꢀC, 2 h.

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4650
Table 1
Inhibitory activities and Ki values of compounds with the substituted 6-benzylthio fragment on the 4-benzylthio-1,3,5-triazin-2(1H)-one core against cathepsins B, H and L.
Compound
R
Cathepsin B inhibition
Ki (
M)a Ki0
Cathepsin H inhibition (%)c, d
Cathepsin L inhibition (%)c, d
m
(m
M)a
bb
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
o-OMe
m-OMe
p-OMe
o-F
m-F
p-F
o-NO2
m-NO2
p-NO2
o-Cl
m-Cl
p-Cl
15.6 ꢁ 4.7
13.0 ꢁ 0.3
11.1 ꢁ 0.5
9.5 ꢁ 0.5
4.4 ꢁ 0.2
21.9 ꢁ 4.9
10.3 ꢁ 3.7
9.0 ꢁ 1.4
7.4 ꢁ 1.4
13.8 ꢁ 1.1
6.0 ꢁ 0.3
3.3 ꢁ 0.2
4.0 ꢁ 0.04
3.6 ꢁ 0.4
4.5 ꢁ 2.1
2.5 ꢁ 0.6
10.5 ꢁ 3.1
9.1 ꢁ 0.2
10.8 ꢁ 0.4
9.7 ꢁ 0.2
0.22 ꢁ 0.03
0.42 ꢁ 0.12
0.20 ꢁ 0.06
0.36 ꢁ 0.14
0.22 ꢁ 0.03
0.33 ꢁ 0.04
0.24 ꢁ 0.03
0.15 ꢁ 0.01
0.17 ꢁ 0.01
0.23 ꢁ 0.04
0.28 ꢁ 0.02
0.25 ꢁ 0.01
0.04 ꢁ 0.02
0.02 ꢁ 0.01
0.07 ꢁ 0.00
0.07 ꢁ 0.01
1.06
n.d.
n.d.
n.d.
4.18
n.d.
n.d.
3.92
n.d.
n.d.
n.d.
6.91
n.d.
n.d.
1.55
n.d.
61.33
n.d.
n.d.
n.d.
7.68
n.d.
n.d.
81.17
n.d.
n.d.
n.d.
85.23
n.d.
n.d.
44.94
n.d.
10.2 ꢁ 0.1
16.6 ꢁ 4.2
11.9 ꢁ 2.4
11.3 ꢁ 0.8
10.0 ꢁ 1.1
7.2 ꢁ 3.2
5.4 ꢁ 1.8
4.4 ꢁ 0.3
o-CN
m-CN
p-CN
40.7 ꢁ 7.3
46.7 ꢁ 0.4
24.3 ꢁ 2.1
23.0 ꢁ 5.8
o-F, p-CN
n.d. Not determined.
a
Ki values are an average of two independent determinations (performed in duplicate), with SDs given in the table.
b
c
b
is the factor by which the product formation changes when the inhibitor binds to the enzymeesubstrate (ES) complex.
Relative inhibition measurements represent averages of two independent determinations.
Inhibitory activities on catH and catL were determined at 50 mM of each compound (performed in duplicate).
d
3.2. Enzyme kinetics
described in Materials and methods. Compounds 23e38 and
compound 47 (Tables 1,2) inhibited catB in a reversible way and in
the low micromolar range as partial mixed-type inhibitors. Inter-
estingly, the same mechanism of action of catB inhibition was
observed for chiral cyclometallated complexes derived from N,N-
The kinetic parameters describing the inhibition of compounds
23e47 upon catB endopeptidase activity were determined using
the catB-specific substrate Z-Arg-Arg-AMC, according to a protocol
Table 2
Inhibitory activities and Ki values of the compounds with the 6-alkylthio and 6-cycloalkylmethylthio substituents on the 4-benzylthio-1,3,5-triazin-2(1H)-one core against
cathepsins B, H and L.
Compound
R
Cathepsin B inhibition
Ki (
M)a
Cathepsin H inhibition (%)c,d
Cathepsin L inhibition (%)c,d
m
Ki0
(
m
M)a
bb
39
40
41
42
43
44
Me
Et
Pr
CN
COOH
220.9 ꢁ 45.5
259.0 ꢁ 33.2
70.6 ꢁ 22.5
97.1 ꢁ 1.8
49.4 ꢁ 4.3
14.8 ꢁ 0.9
51.6 ꢁ 0.3
351.0 ꢁ 2.9
28.30 ꢁ 2.01
0
0
0
0
0
0
ꢂ4.06
ꢂ4.82
ꢂ1.81
4.94
6.89
10.02
ꢂ11.45
ꢂ8.66
9.33
ꢂ5.28
ꢂ2.36
42.02
207.7 ꢁ 33.2
560.0 ꢁ 77.0
211.5 ꢁ 35.08
45
46
34.13 ꢁ 6.60
23.03 ꢁ 3.01
10.13 ꢁ 1.14
8.95 ꢁ 1.05
0
0
1.57
37.39
9.96
10.55
47
11.9 ꢁ 4.4
3.3 ꢁ 0.2
0.12 ꢁ 0.01
10.17
76.24
n.d. Not determined.
a
Ki values are an average of two independent determinations (performed in duplicate), with SDs given in the table.
b
c
b
is the factor by which the product formation changes when the inhibitor binds to the ES complex.
Relative inhibition measurements represent averages of two independent determinations.
Inhibitory activities on catH and catL were determined at 50 mM of each compound (performed in duplicate).
d

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4651
dimethyl-1-phenethylamine with bridging bis(diphenylphosphine)
ferrocene ligand, described by Bincoletto and colleagues [24]. In the
case of mixed-type inhibition the substrate and the inhibitor bind
independently and reversibly to the enzyme to produce the ES, EI
and ESI complexes (Fig. 1). As for the partial mixed-type inhibition,
the formed ESI complex can also produce the product, but not as
[31]. As it is known [14,31] that covalent interactions with the
cysteine residue in the active site of the enzyme account for
a substantial portion of the binding energy of the inhibitor, it is very
promising that inhibitors 23e38 and 47 presented here, which are
without this electrophilic ‘warhead’ moiety, already show good
potencies and selectivities toward catB and also toward catL. The
subsequent introduction of an additional electrophilic ‘warhead’ on
an appropriate part of these molecules will most probably lead to
further increases in their potencies.
effectively as the ES complex. The parameter
the perturbation of product formation when inhibitor binds to the
ES complex and a low factor value denotes poor formation of the
b is used to describe
b
product from the ESI complex (Fig. 1). Representative Dixon plots
for compounds 24, 34, 40, and 43 are shown in Fig. 2.
In summary, our initial screening experiment was focused on
determining whether the 1,3,5-triazine scaffold truly represents
a privileged scaffold toward cysteine proteases. This screening
identified 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as
low micromolar and noncovalent inhibitors of catB and catL. These
compounds also show selectivity when evaluated against the
related cysteine protease, catH. Further studies will be focused on
additional exploration of the chemical space of these compounds,
to address as many noncovalent interactions with the enzyme as
possible. In a second phase, we will introduce the electrophilic
functionality into these compounds, thus attempting to further
improve the binding affinities of these molecules.
Furthermore, the kinetics analyses indicated that compounds
39e46 had slightly different mechanisms of action (mixed inhibi-
tors instead of partial mixed inhibitors) as their counterparts with
a planar phenyl ring or a cyclohexyl ring (Fig. 2). The equations to
which the data were fitted and that specify the mechanism of
action are disclosed in Materials and methods.
Since the fluorometric measurements with AMC and other fluo-
rophores are often biased by inner filter effects brought about by
molecules assayed, the compounds 23ꢂ47 were also tested for
absorption in the spectral range of the AMC substrates (350e500 nm)
at 50 mM in the assay buffer used for measuring catB activity. As seen
in Fig. 3, the tested compounds did not show significant absorption in
the spectral range of wavelengths of the AMC assay in this way
excluding the possibility of inner filter effects caused by the tested
compounds.
5. Experimental section
5.1. Enzymes and assay buffers
Human recombinant cathepsin B (EC 3.4.22.1) was prepared as
reported [32]. Human native cathepsin H (EC 3.4.22.16) was iso-
lated from human liver [33] and human recombinant cathepsin L
(EC 3.4.22.15) was expressed in Esherichia coli [34]. Assay buffers for
cathepsins B, H and L consisted of 100 mM phosphate buffer, pH 6.0,
100 mM phosphate buffer, pH 6.8, and 100 mM acetate buffer, pH
5.5, respectively. Each contained 0.1% PEG 8000 (SigmaeAldrich),
5 mM DTT and 1.5 mM EDTA. Prior to the assay each enzyme was
activated in the assay buffer for 5 min at 37 ꢀC.
3.3. Molecular docking of compound 34 in the active site of catB
It has been previously established [25e28] that the S10, S20 and
S2, S3 subsites of catB are likely to bind hydrophobic residues. We
predicted that hydrophobic moieties on positions 4 and 6 of 1,3,5-
triazin-2(1H)-one can bind to two of the hydrophobic subsites of
catB. The plausible binding mode of compound 34 was assessed by
molecular docking in the active site of catB (PDB entry: 1gmy) [27]
using FlexX incorporated into LeadIT 1.3 [29]. In the crystal struc-
ture the active site was defined as the area within 13 Å of the
catalytic Cys29. All water molecules and the co-crystallized ligand
were removed, and all OH and SH groups were defined as freely
rotatable. The inhibitor occupied the S10 and S20 subsites, as
5.2. Relative inhibition determination
The substrates Z-Arg-Arg-AMC (Calbiochem), Arg-AMC (Biomol),
Z-Phe-Arg-AMC (Bachem) were used to assess the activities of catB,
catH and catL, respectively. Five
AMC (5 M) or Z-Phe-Arg-AMC (1
compound (final concentration 50
a black microplate. The reaction was initiated by addition of 90
m
l of Z-Arg-Arg-AMC (5
M) and 5 l of the respective
M) were added into the wells of
mM), Arg-
anticipated, forming Van der Waals, hydrophobic and
interactions (Fig. 4).
p-stacking
m
m
m
m
ml
4. Conclusions
enzyme (final concentration for catB and catH was 5 nM and for catL
0.5 nM) in the assay buffer. Formation of the fluorescent degradation
products of the AMC substrates was continuously monitored at
460 nm ꢁ 10 nm with excitation at 380 nm ꢁ 20 nm, at 37 ꢀC. All
assay mixtures contained 5% (v/v) DMSO and 0.01% of Triton X-100,
to prevent false-positive inhibition due to the formation of
compound aggregates [35]. All measurements were performed in
duplicate. The relative inhibition of enzyme activities was calculated
Recently, research has been focused mostly on the discovery of
reversible covalent inhibitors of different cathepsins that have
a mildly electrophilic ‘warhead’, of which the nitrile-containing
functionalities are receiving the most attention. These ‘warheads’
combine strong, but reversible binding to the target protein
(through the formation of a thioimidate with the active site
cysteine), with low reactivities toward other cellular nucleophiles
according to the equation: Relative inhibition (%) ¼ 100 ꢃ (1 ꢂ ni
/no),
where ni and no denote the reaction velocity in the presence and
absence of inhibitor, respectively.
5.3. Determination of Ki values and the mechanism of inhibition
Inhibition constants were determined by measuring the initial
rate of hydrolysis at various substrate concentrations in the absence
or presence of different concentrations of inhibitors. Five
substrate Z-Arg-Arg-AMC at 60, 180 and 360 M and 5
respective inhibitor at 0, 10, 20, 30, 40, 50 and 70 M were added
into the wells of a black microplate. The reaction was initiated by
addition of 90 l enzyme (133 pM) in the assay buffer. Formation of
m
l of
m
m
ml of
Fig. 1. Representation of the mechanism of partial mixed-type inhibition. E is used to
denote the enzyme, S the substrate, Km the MichaeliseMenten constant, kcat the first-
order rate constant, P the product, I the inhibitor, Ki the inhibition constant,
perturbation parameter, and the kcat perturbation parameter.
a the Km
b
m

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4652
Fig. 2. Effect of compounds 24, 34, 40, 43 on catB activity. Compounds 24 (A) and 34 (B) are reversible, partial mixed-type inhibitors, whereas compounds 40 (C) and 43 (D) are
reversible, mixed-type inhibitors. Five l of substrate at 60, 180 and 360 M and 5 l of the respective inhibitor at 0, 10, 20, 30, 40, 50 and 70 M were added into the wells of a black
microplate. The reaction was initiated by addition of 90
m
m
m
m
m
l enzyme (133 pM). Formation of the fluorescent degradation product AMC was continuously monitored at 460 nm ꢁ 10 nm
with excitation at 380 nm ꢁ 20 nm at 37 ꢀC. All measurements were performed in duplicate and the Dixon plots presented here are representative of at least two independent
experiments.
the fluorescent degradation product AMC was continuously
monitored at 460 nm ꢁ 10 nm with excitation at 380 nm ꢁ 20 nm,
at 37 ꢀC. All assay mixtures contained 5% (v/v) DMSO. All
measurements were performed in duplicate. The resulting data
were analyzed by non-linear regression using the software
SigmaPlotÒ 11, Enzyme Kinetics ModuleÔ 1.3 and were fitted to the
equation: v ¼ Vmax/((Km/S) ꢃ (1 þ I/Ki) þ (1 þ I/( ꢃ Ki))), depicting
a
Fig. 4. Compound 34 (blue) modeled into the catB active site. Subsites S10 and S20 are
shown as semi-transparent surfaces. All of the pocket-forming amino-acid residues are
shown in yellow (S10, S20) and magenta (S1, S2 and S3) sticks, and are labeled. The
figure was prepared using PyMol [30]. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Absorption spectra of compounds 23ꢂ47. Data presented in the figure were
obtained by deduction of absorbance values acquired in the presence of DMSO which
was used as control.

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4653
the mixed type of inhibition; or equation
ꢃ Ki))/(1 þ I/( ꢃ Ki)))/(1þ(Km/S) ꢃ (1 þ I/Ki)/(1 þ I/(
depicting the partial mixed-type inhibition, where
reaction velocity, Vmax the maximal reaction velocity, Km the
MichaeliseMenten constant, S the substrate, I the inhibitor, Ki the
n
¼ Vmax ꢃ ((1þ
b
ꢃ I/
6 mL). The combined alkaline phases were acidified to pH 1 with
H2SO4, and in this way the precipitate was formed, which was then
filtered off and crystallized from CH3CN. Spectroscopic data
(without 13C NMR spectra) for this compound are described in
(a
a
a
ꢃ Ki))),
n
denotes
Ref. [25]. 13C NMR (100 MHz, DMSO-d6):
d 33.62, 127.55, 128.53,
inhibition constant,
a
the Km perturbation parameter, and
b
the
129.17, 136.14, 149.70, 168.15 (br s), 178.46 (br s).
Vmax (kcat) perturbation parameter.
5.6.2. General procedure for the synthesis of 6-alkylated derivatives
23e47
5.4. Measurements of absorption spectra
To asolutionofcompound 22(1.0mmol)inEtOH (8 mL)and NaOH
(2 M, 5 mL), the corresponding benzyl halide (forcompounds 23e38),
alkyl halide (for compounds 39e43) or cycloalkylmethyl halide (for
compounds 44e47) (1.1 mmol) was slowly added. The reaction
mixture was stirred for 2 h at room temperature. After the reaction
was complete, H2O (10 mL) was added, followed by the addition of
H2SO4 (2 M, 5 mL). The precipitate formed was filtered off and the
pure product obtained by crystallization from CH3CN or EtOH.
All compounds (23e47) were tested for absorption in the
spectral range of the AMC substrates. Five l of compound in DMSO
and 95 l of assay buffer (used for measuring catB activity) were
added to the wells of a black microplate. The concentration of each
compound was 50 M. The absorbance spectra were recorded in
m
m
m
the range from 350 to 500 nm in steps of 5 nm.
5.5. Materials and methods
5.6.2.1. 4-(Benzylthio)-6-(2-methoxybenzylthio)-1,3,5-triazin-2(1H)-
one (23). Crystallization from CH3CN gave 304 mg (82%) of white
crystals. Mp: 166.5e169.0 ꢀC; Rf: 0.68 (CH2Cl2/MeOH 5/1); 1H NMR
5.5.1. Chemistry
Reagents and solvents were obtained from commercial sources
(Fluka, SigmaeAldrich, Acros Organics, Alfa Aesar, Fluorochem).
Solvents were distilled before use, while the other chemicals were
used as received. Analytical TLC was performed on Merck silica gel
(60F254) pre-coated plates (0.25 mm). The compounds were visu-
alized under UV light and/or stained with the relevant reagent.
Column chromatography was performed on Merck silica gel 60
(mesh 70e230), using the indicated solvents. Yields refer to the
purified products, and they were not optimized. All of the melting
points were determined on a Reichert hot-stage apparatus, and are
uncorrected. 1H NMR spectra were recorded on a Bruker Avance 400
DPX spectrometer at 302 K, and are reported in ppm using tetra-
methylsilane or solvent as an internal standard (DMSO-d6 at
2.50 ppm, CDCl3 at 7.26 ppm). The coupling constants (J) are given in
Hz, and the splitting patterns are designated as: s, singlet; bs, broad
singlet; d, doublet; dd, double doublet; t, triplet; dt, double triplet;
td, triple doublet; and m, multiplet. 13C NMR spectra were recorded
on a Bruker Avance 400 DPX spectrometer at 302 K, and are reported
in ppm using solvent as an internal standard (DMSO-d6 at
39.43 ppm). Mass spectra data and high-resolution mass measure-
ments were performed on a VG-Analytical Autospec Q mass spec-
trometer. All computational procedures used for molecular docking
were performed on workstation, which has 4 dual core AMD
Opteron 2.0 GHz processors, 16 GB of RAM, 4320 GB hard drives in
RAID10 array and Nvidia GeForce 7900 graphic card and is running
64bit Fedora 7. The purity of all assayed compounds, as determined
by HPLC, was >95%. HPLC experiments were performed on an Agi-
(400 MHz, DMSO-d6): d 3.82 (s, 3H, CH3), 4.32 (s, 2H, SCH2), 4.38 (s,
2H, SCH2), 6.89 (td, J ¼ 7.5, 1.1 Hz, 1H, AreH), 7.02 (dd, J ¼ 8.1, 0.9 Hz,
1H, AreH), 7.23e7.37 (m, 5H, AreH), 7.38e7.45 (m, 2H, AreH), 12.91
(br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
d 28.97, 33.54,
55.48, 110.91, 120.25, 123.74, 127.32, 128.46, 128.92, 129.19, 130.29,
136.68, 149.84, 153.15 (br s), 157.10, 173.01 (br s); HRMS (ESI) m/z
calcd for C18H16N3O2S2 [M ꢂ H]ꢂ 370.0684, found 370.0692; purity
by HPLC: 100%, retention time: 22.71 min.
5.6.2.2. 4-(Benzylthio)-6-(3-methoxybenzylthio)-1,3,5-triazin-
2(1H)-one (24). Crystallization from CH3CN gave 271 mg (73%) of
white crystals. Mp: 125.5e127.5 ꢀC; Rf: 0.58 (CH2Cl2/MeOH 5/1); 1H
NMR (400 MHz, DMSO-d6):
d 3.73 (s, 3H, CH3), 4.34 (s, 2H, SCH2),
4.37 (s, 2H, SCH2), 6.83 (ddd, J ¼ 8.2, 2.5, 0.7 Hz, 1H, AreH),
6.94e7.01 (m, 2H, AreH), 7.19e7.36 (m, 4H, AreH), 7.36e7.42 (m,
2H, AreH), 12.98 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
d
33.60 (2C), 54.92, 112.90, 114.55, 121.09, 127.34, 128.46, 128.94,
129.55, 136.59, 138.07, 149.86, 152.89 (br s), 159.17, 173.12 (br s);
HRMS (ESI) m/z calcd for C18H18N3O2S2 [M þ H]þ 372.0840, found
372.0827; purity by HPLC: 100%, retention time: 21.82 min.
5.6.2.3. 4-(Benzylthio)-6-(4-methoxybenzylthio)-1,3,5-triazin-
2(1H)-one (25). Crystallization from EtOH gave 297 mg (80%) of
white crystals (prisms). Mp: 176.0e177.0 ꢀC; Rf: 0.75 (CH2Cl2/MeOH
5/1); 1H NMR (400 MHz, DMSO-d6):
d 3.73 (s, 3H, CH3), 4.32 (s, 2H,
SCH2), 4.38 (s, 2H, SCH2), 6.87 (dt, J ¼ 8.7, 3.0 Hz, 2H, AreH),
7.23e7.37 (m, 5H, AreH), 7.37e7.44 (m, 2H, AreH), 7.36e7.42 (m,
2H, AreH), 12.96 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
lent Eclipse C18 column (4.6 ꢃ 50 mm, 5
mm) with a flow rate of
1.0 mL/min, detection at 254 nm, and an eluent system of: A ¼ H2O
with 0.1% TFA; B ¼ MeOH. The following gradient was applied:
0e3 min, 40% B; 3e18 min, 40% B / 80% B; 18e23 min, 80% B;
23e30 min, 80% B / 40% B; run time ¼ 30 min; T ¼ 25 ꢀC.
d
33.21, 33.59, 55.00, 113.88, 127.34, 128.11, 128.47, 128.95, 130.21,
136.63, 149.31, 152.84 (br s), 158.54, 179.51 (br s); HRMS (ESI) m/z
calcd for C18H18N3O2S2 [M þ H]þ 372.0840, found 372.0838; purity
by HPLC: 99.76%, retention time: 21.74 min.
5.6. Experimental procedures
5.6.2.4. 4-(Benzylthio)-6-(2-fluorobenzylthio)-1,3,5-triazin-2(1H)-
one (26). Crystallization from CH3CN gave 316 mg (88%) of white
crystals (needles). Mp: 191.0e193.0 ꢀC; Rf: 0.63 (CH2Cl2/MeOH 5/1);
5.6.1. Procedure for the synthesis of 4-(benzylthio)-6-mercapto-
1,3,5-triazin-2(1H)-one (22) [25,26]
To a solution of S-(benzyl)isothiourea hydrochloride (203 mg,
1.00 mmol) in H2O (3 mL), toluene (5 mL) was added and the
1H NMR (400 MHz, DMSO-d6):
d 4.37 (s, 2H, SCH2), 4.39 (s, 2H,
SCH2), 7.13e7.43 (m, 8H, AreH), 7.50 (td, J ¼ 7.7, 1.5 Hz, 1H, AreH),
mixture stirred vigorously. After
5
min, ethoxycarbonyl iso-
13.03 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
d
27.31 and
3
thiocyanate (184 mg, 1.40 mmol) in toluene (2 mL) and NaOH (2 M,
4 mL) were simultaneously added over a period of 5 min. Additional
NaOH (2 M, 2 mL) was added after 15 min and then the reaction
mixture was stirred for 1 h at room temperature. The phases were
then separated and the organic layer washed with NaOH (2 M,
27.33 (1C, JF,C
¼
4.0 Hz), 33.58, 115.19 and 115.40 (1C,
2
2JF,C ¼ 21.3 Hz), 123.34 and 123.48 (1C, JF,C ¼ 14.7 Hz), 124.42 and
124.45 (1C, 3JF,C ¼ 4.2 Hz), 127.34, 128.45, 128.94, 129.66 and 129.74
3
4
(1C, JF,C ¼ 8.1 Hz), 131.27 and 131.31 (1C, JF,C ¼ 3.3 Hz), 136.48,
149.86, 152.92 (br s), 159.11 and 161.55 (1C, 1JF,C ¼ 245.8 Hz), 174.32

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I. Sosic et al. / European Journal of Medicinal Chemistry 46 (2011) 4648e4656
4654
(br s); HRMS (ESI) m/z calcd for C17H15N3OFS2 [M þ H]þ 360.0641,
5.6.2.10. 4-(Benzylthio)-6-(2-chlorobenzylthio)-1,3,5-triazin-2(1H)-
one (32). Crystallization from CH3CN gave 289 mg (77%) of white
crystals (needles). Mp: 158.0e160.0 ꢀC; Rf: 0.81 (CH2Cl2/MeOH 5/
found 360.0642; purity by HPLC: 100%, retention time: 22.17 min.
5.6.2.5. 4-(Benzylthio)-6-(3-fluorobenzylthio)-1,3,5-triazin-2(1H)-
one (27). Crystallization from EtOH gave 266 mg (74%) of white
crystals (needles). Mp: 165.0e167.0 ꢀC; Rf: 0.60 (CH2Cl2/MeOH 5/1);
1); 1H NMR (400 MHz, DMSO-d6):
d 4.37 (s, 2H, SCH2), 4.46 (s, 2H,
SCH2), 7.23e7.43 (m, 7H, AreH), 7.45e7.51 (m, 1H, AreH),
7.54e7.60 (m, 1H, AreH), 13.02 (br s, 1H, NHCO); 13C NMR
1H NMR (400 MHz, DMSO-d6):
d
4.37 (s, 2H, SCH2), 4.38 (s, 2H,
(100 MHz, DMSO-d6): d 31.82, 33.58, 127.33, 127.35, 128.46, 128.95,
SCH2), 7.05e7.14 (m, 1H, AreH), 7.21e7.43 (m, 8H, AreH), 13.03 (br
129.39, 129.48, 131.42, 133.28, 133.91, 136.50, 146.40, 153.08 (br s),
175.49 (br s); HRMS (ESI) m/z calcd for C17H15N3OS2Cl [M þ H]þ
376.0345, found 376.0342; purity by HPLC: 99.95%, retention time:
24.08 min.
s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
d 32.91 and 32.93 (1C,
4JF,C ¼ 2.9 Hz), 33.56, 114.06 and 114.27 (1C, 2JF,C ¼ 20.5 Hz), 115.61
2
and 115.83 (1C, JF,C
¼
22.0 Hz), 125.04 and 125.07 (1C,
4JF,C ¼ 2.9 Hz), 127.37, 128.48, 128.96, 130.33 and 130.42 (1C,
3JF,C ¼ 8.8 Hz), 136.54, 139.74 and 139.81 (1C, 3JF,C ¼ 8.1 Hz), 149.86,
152.94 (br s), 160.71 and 163.13 (1C, 1JF,C ¼ 242.1 Hz), 173.49 (br s);
HRMS (ESI) m/z calcd for C17H15N3OFS2 [M þ H]þ 360.0641, found
360.0651; purity by HPLC: 99.62%, retention time: 21.83 min.
5.6.2.11. 4-(Benzylthio)-6-(3-chlorobenzylthio)-1,3,5-triazin-2(1H)-
one (33). Crystallization from CH3CN gave 319 mg (85%) of white
crystals (needles). Mp: 159.0e160.0 ꢀC; Rf: 0.67 (CH2Cl2/MeOH 5/
1); 1H NMR (400 MHz, DMSO-d6):
d
4.37 (s, 4H, 2ꢃ SCH2), 7.23e7.42
(m, 8H, AreH), 7.47e7.51 (m, 1H, AreH), 12.96 (br s, 1H, NHCO); 13C
NMR (100 MHz, DMSO-d6): 32.87, 33.61, 127.26, 127.36, 127.64,
5.6.2.6. 4-(Benzylthio)-6-(4-fluorobenzylthio)-1,3,5-triazin-2(1H)-
one (28). Crystallization from CH3CN gave 284 mg (79%) of white
crystals (needles). Mp: 213.0e214.5 ꢀC; Rf: 0.72 (CH2Cl2/MeOH 5/1);
d
128.47, 128.80, 128.96, 130.26, 132.92, 136.52, 139.56, 147.66, 153.23
(br s), 175.68 (br s); HRMS (ESI) m/z calcd for C17H15N3OS2Cl
[M þ H]þ 376.0345, found 376.0352; purity by HPLC: 99.96%,
retention time: 23.61 min.
1H NMR (400 MHz, DMSO-d6):
d 4.36 (s, 2H, SCH2), 4.39 (s, 2H,
SCH2), 7.09e7.19 (symm m, 2H, AreH), 7.23e7.36 (m, 3H, AreH),
7.37e7.49 (m, 4H, AreH), 12.97 (br s, 1H, NHCO); 13C NMR
(100 MHz, DMSO-d6):
d
32.77, 33.61, 115.12 and 115.34 (1C,
5.6.2.12. 4-(Benzylthio)-6-(4-chlorobenzylthio)-1,3,5-triazin-2(1H)-
one (34). Crystallization from CH3CN gave 296 mg (79%) of white
crystals. Mp: 205.0e206.0 ꢀC; Rf: 0.80 (CH2Cl2/MeOH 5/1); 1H NMR
2JF,C ¼ 21.3 Hz), 127.34, 128.46, 128.96, 130.92 and 131.00 (1C,
3JF,C ¼ 8.1 Hz), 132.97 and 133.00 (1C, 4JF,C ¼ 2.9 Hz), 136.56, 147.84,
153.14 (br s), 160.14 and 162.57 (1C, 1JF,C ¼ 243.2 Hz), 175.64 (br s);
HRMS (ESI) m/z calcd for C17H15N3OFS2 [M þ H]þ 360.0641, found
360.0628; purity by HPLC: 100%, retention time: 21.88 min.
(400 MHz, DMSO-d6):
7.23e7.37 (m, 4H, AreH), 7.38e7.45 (m, 5H, AreH), 12.97 (br s, 1H,
NHCO); 13C NMR (100 MHz, DMSO-d6):
32.78, 33.59, 127.35,
d 4.35 (s, 2H, SCH2), 4.36 (s, 2H, SCH2),
d
128.38, 128.47, 128.95, 130.78, 131.94, 136.00, 136.54, 146.95, 153.22
(br s), 175.64 (br s); HRMS (ESI) m/z calcd for C17H15N3OS2Cl
[M þ H]þ 376.0345, found 376.0338; purity by HPLC: 100%, reten-
tion time: 23.96 min.
5.6.2.7. 4-(Benzylthio)-6-(2-nitrobenzylthio)-1,3,5-triazin-2(1H)-one
(29). Crystallization from CH3CN gave 336 mg (87%) of yellow
crystals. Mp: 175.0e176.5 ꢀC; Rf: 0.62 (CH2Cl2/MeOH 5/1); 1H NMR
(400 MHz, DMSO-d6):
d 4.37 (s, 2H, SCH2), 4.64 (s, 2H, SCH2),
7.23e7.36 (m, 3H, AreH), 7.37e7.44 (m, 2H, AreH), 7.53e7.62
(symm m, 1H, AreH), 7.67e7.75 (m, 2H, AreH), 8.06 (dd, J ¼ 7.7,
0.8 Hz, 1H, AreH), 13.02 (br s, 1H, NHCO); 13C NMR (100 MHz,
5.6.2.13. 4-(Benzylthio)-6-(2-cyanobenzylthio)-1,3,5-triazin-2(1H)-
one (35). Crystallization from EtOH gave 260 mg (71%) of white
crystals. Mp: 157.0e158.0 ꢀC; Rf: 0.67 (CH2Cl2/MeOH 5/1); 1H NMR
DMSO-d6):
d
30.90, 33.55, 124.98, 127.38, 128.47, 128.97, 129.11,
(400 MHz, DMSO-d6): d 4.36 (s, 2H, SCH2), 4.54 (s, 2H, SCH2),
132,26, 132.43, 133.97, 136.44, 147.97, 149.42, 152.83 (br s), 174.67
(br s); HRMS (ESI) m/z calcd for C17H15N4O3S2 [M þ H]þ 387.0586,
found 387.0574; purity by HPLC: 99.13%, retention time: 20.88 min.
7.21e7.40 (m, 5H, AreH), 7.44e7.53 (m, 1H, AreH), 7.65e7.71 (m,
2H, AreH), 7.84 (dt, J ¼ 7.5, 0.9 Hz, 1H, AreH), 13.05 (br s, 1H,
NHCO); 13C NMR (100 MHz, DMSO-d6):
d 32.13, 33.58,111.84,117.22,
127.37, 128.31, 128.46, 128.97, 130.35, 133.03, 133.31, 136.40, 140.18,
148.00, 152.96 (br s), 176.10 (br s); HRMS (ESI) m/z calcd for
C18H15N4OS2 [M þ H]þ 367.0687, found 367.0678; purity by HPLC:
99.60%, retention time: 18.80 min.
5.6.2.8. 4-(Benzylthio)-6-(3-nitrobenzylthio)-1,3,5-triazin-2(1H)-one
(30). Crystallization from EtOH gave 351 mg (91%) of yellow crys-
tals (prisms). Mp: 183.5e185.0 ꢀC; Rf: 0.70 (CH2Cl2/MeOH 5/1); 1H
NMR (400 MHz, DMSO-d6):
d 4.36 (s, 2H, SCH2), 4.50 (s, 2H, SCH2),
7.24e7.41 (m, 5H, AreH), 7.62 (dd, J ¼ 7.9, 7.9 Hz,1H, AreH), 7.87 (dt,
J ¼ 7.7, 1.1 Hz, 1H, AreH), 8.11 (ddd, J ¼ 8.2, 2.3, 0.9 Hz, 1H, AreH),
8.30 (dd, J ¼ 2.3, 2.3 Hz, 1H, AreH), 13.03 (br s, 1H, NHCO); 13C NMR
5.6.2.14. 4-(Benzylthio)-6-(3-cyanobenzylthio)-1,3,5-triazin-2(1H)-
one (36). Crystallization from CH3CN gave 286 mg (78%) of white
crystals. Mp: 142.0e143.0 ꢀC; Rf: 0.65 (CH2Cl2/MeOH 5/1); 1H
(100 MHz, DMSO-d6):
d
32.65, 33.60, 122.20, 123.64, 127.37, 128.47,
NMR (400 MHz, DMSO-d6): d 4.36 (s, 2H, SCH2), 4.41 (s, 2H, SCH2),
128.95, 129.86, 135.68, 136.44, 139.72, 147.58, 149.87, 153.04 (br s),
175.17 (br s); HRMS (ESI) m/z calcd for C17H15N4O3S2 [M þ H]þ
387.0586, found 387.0578; purity by HPLC: 100%, retention time:
20.23 min.
7.23e7.35 (m, 3H, AreH), 7.36e7.42 (m, 2H, AreH), 7.53 (dd,
J ¼ 7.7, 7.7 Hz, 1H, AreH), 7.70e7.78 (m, 2H, AreH), 7.87 (dd,
J ¼ 1.7, 1.7 Hz, 1H, AreH), 12.98 (br s, 1H, NHCO); 13C NMR
(100 MHz, DMSO-d6): d 32.62, 33.55, 111.28, 118.53, 127.36, 128.46,
128.94, 129.63, 131.04, 132.50, 133.88, 136.49, 138.96, 149.86,
152.93 (br s), 176.09 (br s); HRMS (ESI) m/z calcd for C18H15N4OS2
[M þ H]þ 367.0687, found 367.0695; purity by HPLC: 95.14%,
retention time: 18.69 min.
5.6.2.9. 4-(Benzylthio)-6-(4-nitrobenzylthio)-1,3,5-triazin-2(1H)-one
(31). Crystallization from CH3CN gave 282 mg (73%) of orange
crystals. Mp: 183.0e185.0 ꢀC; Rf: 0.54 (CH2Cl2/MeOH 5/1); 1H NMR
(400 MHz, DMSO-d6):
d 4.36 (s, 2H, SCH2), 4.49 (s, 2H, SCH2),
7.23e7.41 (m, 5H, AreH), 7.67 (d, J ¼ 8.7 Hz, 2H, AreH), 8.17 (d,
5.6.2.15. 4-(Benzylthio)-6-(4-cyanobenzylthio)-1,3,5-triazin-2(1H)-
one (37). Crystallization from CH3CN gave 322 mg (88%) of white
crystals (needles). Mp: 182.5e184.0 ꢀC; Rf: 0.61 (CH2Cl2/MeOH 5/1);
J ¼ 8.7 Hz, 2H, AreH), 13.04 (br s, 1H, NHCO); 13C NMR (100 MHz,
DMSO-d6):
d 32.78, 33.52, 123.48, 127.37, 128.46, 128.94, 130.11,
136.43, 145.33, 146.59, 149.86, 152.91 (br s), 175.88 (br s); HRMS
(ESI) m/z calcd for C17H15N4O3S2 [M þ H]þ 387.0586, found
387.0594; purity by HPLC: 95.31%, retention time: 20.43 min.
1H NMR (400 MHz, DMSO-d6):
d 4.35 (s, 2H, SCH2), 4.44 (s, 2H,
SCH2), 7.23e7.41 (m, 5H, AreH), 7.60 (d, J ¼ 8.5 Hz, 2H, AreH), 7.78
(d, J ¼ 8.5 Hz, 2H, AreH), 13.00 (br s,1H, NHCO); 13C NMR (100 MHz,

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I. Sosic et al. / European Journal of Medicinal Chemistry 46 (2011) 4648e4656
4655
DMSO-d6):
d
33.06, 33.54, 109.97, 118.64, 127.38, 128.47, 128.95,
5.6.2.21. 4-(Benzylthio)-6-(carboxymethylthio)-1,3,5-triazin-2(1H)-
129.67, 132.29, 136.43, 143.08, 147.57, 149.98 (br s), 177.86 (br s);
HRMS (ESI) m/z calcd for C18H15N4OS2 [M þ H]þ 367.0687, found
367.0698; purity by HPLC: 98.09%, retention time: 18.61 min.
one (43). Crystallization from CH3CN gave 250 mg (81%) of white
crystals (needles). Mp: 193.0e195.0 ꢀC; Rf: 0.09 (CH2Cl2/MeOH 5/
1); 1H NMR (400 MHz, DMSO-d6):
d
3.93 (s, 2H, SCH2), 4.38 (s, 2H,
SCH2), 7.24e7.37 (m, 3H, AreH), 7.38e7.44 (m, 2H, AreH), 12.96 (br
s, 2H, NHCO and COOH); 13C NMR (100 MHz, DMSO-d6):
32.75,
5.6.2.16. 4-(Benzylthio)-6-(2-fluoro-4-cyanobenzylthio)-1,3,5-
triazin-2(1H)-one (38). Crystallization from CH3CN gave 359 mg
(93%) of white crystals. Mp: 166.0e168.0 ꢀC; Rf: 0.54 (CH2Cl2/MeOH
d
33.61, 127.49, 128.59, 129.05, 136.52, 148.43, 153.15 (br s), 169.32,
176.00 (br s); HRMS (ESI) m/z calcd for C12H12N3O3S2 [M þ H]þ
310.0320, found 310.0329; purity by HPLC: 100%, retention time:
6.90 min.
5/1); 1H NMR (400 MHz, DMSO-d6):
d 4.35 (s, 2H, SCH2), 4.44 (s, 2H,
SCH2), 7.23e7.41 (m, 5H, AreH), 7.64e7.75 (m, 2H, AreH), 7.84 (dd,
J ¼ 9.9,1.1 Hz,1H, AreH),12.98 (br s,1H, NHCO); 13C NMR (100 MHz,
3
DMSO-d6):
d
3
27.07 and 27.10 (1C, JF,C ¼ 4.0 Hz), 33.54, 111.69 and
5.6.2.22. 4-(Benzylthio)-6-(cyclopropylmethylthio)-1,3,5-triazin-
2(1H)-one (44). Crystallization from CH3CN gave 253 mg (83%) of
white crystals. Mp: 139.0e140.0 ꢀC; Rf: 0.62 (CH2Cl2/MeOH 5/1); 1H
4
111.79 (1C, JF,C ¼ 9.9 Hz), 117.46 and 117.49 (1C, JF,C ¼ 2.9 Hz),
2
119.08 and 119.34 (1C, JF,C ¼ 25.3 Hz), 127.39, 128.06 and 128.31
2
4
(1C, JF,C ¼ 25.3 Hz), 128.47, 128.64 and 128.68 (1C, JF,C ¼ 3.6 Hz),
128.96, 132.35 and 132.40 (1C, 3JF,C ¼ 4.8 Hz), 136.39, 149.89, 153.07
(br s), 158.47 and 160.94 (1C, 3JF,C ¼ 249.1 Hz), 175.83 (br s); HRMS
(ESI) m/z calcd for C18H14N4OS2F [M þ H]þ 385.0593, found
385.0604; purity by HPLC: 95.07%, retention time: 19.96 min.
NMR (400 MHz, DMSO-d6): d 0.24e0.34 (m, 2H, cyclopropyl-H),
0.48e0.58 (m, 2H, cyclopropyl-H), 1.02e1.18 (m, 1H, cyclopropyl-H),
3.04 (d, J ¼ 7.2 Hz, 2H, SCH2CH), 4.37 (s, 2H, SCH2), 7.22e7.36 (m, 3H,
AreH), 7.38e7.45 (m, 2H, AreH), 12.92 (br s, 1H, NHCO); 13C NMR
(100 MHz, DMSO-d6):
d 5.63, 10.19, 33.64, 35.39, 127.31, 128.46,
128.90, 136.74, 149.88, 152.92 (br s), 176.22 (br s); HRMS (ESI) m/z
calcd for C14H16N3OS2 [M þ H]þ 306.0735, found 306.0375; purity
by HPLC: 100%, retention time: 20.36 min.
5.6.2.17. 4-(Benzylthio)-6-(ethylthio)-1,3,5-triazin-2(1H)-one
(39). Crystallization from CH3CN gave 201 mg (72%) of white
crystals (needles). Mp: 145.0e146.0 ꢀC; Rf: 0.62 (CH2Cl2/MeOH 5/
1); 1H NMR (400 MHz, DMSO-d6):
d
1.27 (t, J ¼ 7.3 Hz, 3H, SCH2CH3),
5.6.2.23. 4-(Benzylthio)-6-(cyclobutylmethylthio)-1,3,5-triazin-
2(1H)-one (45). Crystallization from CH3CN gave 230 mg (72%) of
white crystals (prisms). Mp: 129.0e130.5 ꢀC; Rf: 0.65 (CH2Cl2/
3.07 (q, J ¼ 7.3 Hz, 2H, SCH2CH3), 4.37 (s, 2H, SCH2), 7.23e7.37 (m,
3H, AreH), 7.38e7.42 (m, 2H, AreH), 12.92 (br s, 1H, NHCO); 13C
NMR (100 MHz, DMSO-d6):
d
14.23, 24.22, 33.56, 127.32, 128.47,
MeOH 5/1); 1H NMR (400 MHz, DMSO-d6):
d 1.60e1.88 (m, 4H,
128.90, 136.73, 149.61, 152.75 (br s), 177.76 (br s); HRMS (ESI) m/z
calcd for C12H14N3OS2 [M þ H]þ 280.0578, found 280.0571; purity
by HPLC: 98.21%, retention time: 17.74 min.
cyclobutyl-H), 1.94e2.07 (m, 2H, cyclobutyl-H), 2.48e2.65 (m, 1H,
cyclobutyl-H, overlaps with DMSO), 3.17 (d, J ¼ 7.4 Hz, 2H, SCH2CH),
4.38 (s, 2H, SCH2), 7.23e7.43 (m, 5H, AreH), 12.93 (br s, 1H, NHCO);
13C NMR (100 MHz, DMSO-d6):
d 17.28, 26.83, 33.56, 33.88, 35.45,
5.6.2.18. 4-(Benzylthio)-6-(propylthio)-1,3,5-triazin-2(1H)-one
(40). Crystallization from CH3CN gave 208 mg (71%) of white
crystals (needles). Mp: 118.0e119.0 ꢀC; Rf: 0.67 (CH2Cl2/MeOH 5/1);
127.30, 128.45, 128.85, 136.68, 149.50, 152.70 (br s), 172.80 (br s);
HRMS (ESI) m/z calcd for C15H18N3OS2 [M þ H]þ 320.0891, found
320.0888; purity by HPLC: 98.07%, retention time: 22.74 min.
1H NMR (400 MHz, DMSO-d6):
d
0.93 (t,
J
¼ 7.4 Hz, 3H,
SCH2CH2CH3), 1.58e1.70 (m, 2H, SCH2CH2CH3), 3.04 (t, J ¼ 6.9 Hz,
2H, SCH2CH2CH3), 4.37 (s, 2H, SCH2), 7.23e7.36 (m, 3H, AreH),
7.37e7.44 (m, 2H, AreH), 12.94 (br s, 1H, NHCO); 13C NMR
5.6.2.24. 4-(Benzylthio)-6-(cyclopentylmethylthio)-1,3,5-triazin-
2(1H)-one (46). Crystallization from CH3CN gave 253 mg (76%) of
white crystals. Mp: 135.0e136.5 ꢀC; Rf: 0.67 (CH2Cl2/MeOH 5/1); 1H
(100 MHz, DMSO-d6):
d
12.98, 22.00, 31.46, 33.55, 127.30, 128.46,
NMR (400 MHz, DMSO-d6): d 1.17e1.30 (m, 2H, cyclopentyl-H),
128.88, 136.72, 148.65, 153.22 (br s), 175.81 (br s); HRMS (ESI) m/z
calcd for C13H16N3OS2 [M þ H]þ 294.0735, found 294.0747; purity
by HPLC: 98.35%, retention time: 20.06 min.
1.40e1.82 (m, 6H, cyclopentyl-H), 2.03e2.22 (m, 1H, cyclopentyl-
H), 3.08 (d, J ¼ 7.2 Hz, 2H, SCH2CH), 4.36 (s, 2H, SCH2), 7.20e7.44 (m,
5H, AreH), 12.93 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
d
24.64, 31.54, 33.32, 33.36, 34.94, 127.20, 128.42, 128.83, 137.00,
5.6.2.19. 4-(Benzylthio)-6-(butylthio)-1,3,5-triazin-2(1H)-one
(41). Crystallization from CH3CN gave 261 mg (85%) of white
crystals (prisms). Mp: 101.0e102.0 ꢀC; Rf: 0.76 (CH2Cl2/MeOH 5/1);
149.43, 152.28 (br s), 175.62 (br s); HRMS (ESI) m/z calcd for
C16H20N3OS2 [M þ H]þ 334.1048, found 334.1040; purity by HPLC:
95.89%, retention time: 24.84 min.
1H NMR (400 MHz, DMSO-d6):
d
0.87 (t,
J
¼ 7.3 Hz, 3H,
SCH2CH2CH2CH3), 1.28e1.42 (m, 2H, SCH2CH2CH2CH3), 1.55e1.67
(m, SCH2CH2CH2CH3, 2H), 3.07 (t, J ¼ 7.3 Hz, 2H, SCH2CH2CH2CH3),
4.37 (s, 2H, SCH2), 7.23e7.37 (m, 3H, AreH), 7.38e7.45 (m, 2H,
AreH), 12.92 (br s, 1H, NHCO); 13C NMR (100 MHz, DMSO-d6):
5.6.2.25. 4-(Benzylthio)-6-(cyclohexylmethylthio)-1,3,5-triazin-
2(1H)-one (47). Crystallization from CH3CN gave 274 mg (79%) of
white crystals (needles). Mp: 144.0e145.0 ꢀC; Rf: 0.69 (CH2Cl2/
MeOH 5/1); 1H NMR (400 MHz, DMSO-d6):
d 0.88e1.05 (m, 2H,
d
13.45, 21.22, 29.31, 30.60, 33.57, 127.31, 128.46, 128.88, 136.67,
cyclohexyl-H), 1.05e1.26 (m, 3H, cyclohexyl-H), 1.49e1.79 (m, 6H,
cyclohexyl-H), 3.00 (d, J ¼ 6.7 Hz, 2H, SCH2CH), 4.38 (s, 2H, SCH2),
7.24e7.37 (m, 3H, AreH), 7.38e7.44 (m, 2H, AreH), 12.92 (br s, 1H,
149.85, 152.75 (br s), 177.88 (br s); HRMS (ESI) m/z calcd for
C14H18N3OS2 [M þ H]þ 308.0891, found 308.0881; purity by HPLC:
99.93%, retention time: 22.10 min.
NHCO); 13C NMR (100 MHz, DMSO-d6):
d 25.33, 25.66, 31.73, 33.57,
36.25, 36.88, 127.33, 128.47, 128.87, 136.65, 149.86, 152.77 (br s),
172.40 (br s); HRMS (ESI) m/z calcd for C17H22N3OS2 [M þ H]þ
348.1204, found 348.1200; purity by HPLC: 100%, retention time:
23.65 min.
5.6.2.20. 4-(Benzylthio)-6-(cyanomethylthio)-1,3,5-triazin-2(1H)-
one (42). Crystallization from EtOH gave 223 mg (77%) of white
crystals. Mp: 140.5e143.0 ꢀC; Rf: 0.55 (CH2Cl2/MeOH 5/1); 1H NMR
(400 MHz, DMSO-d6):
7.25e7.38 (m, 3H, AreH), 7.38e7.45 (m, 2H, AreH), 13.20 (br s, 1H,
NHCO); 13C NMR (100 MHz, DMSO-d6):
12.03, 33.88, 119.36,
d 4.18 (s, 2H, SCH2), 4.45 (s, 2H, SCH2),
Acknowledgments
d
127.89, 128.52, 129.02, 136.45, 148.32, 153.32 (br s), 175.34 (br s);
HRMS (ESI) m/z calcd for C12H11N4OS2 [M þ H]þ 291.0374, found
291.0363; purity by HPLC: 98.45%, retention time: 11.36 min.
This study was supported by the Slovenian Research Agency.
The authors thank Dr. Chris Berrie for critical reading of the
manuscript.

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4656
I. Sosic et al. / European Journal of Medicinal Chemistry 46 (2011) 4648e4656
C.P. Austin, C.J. Thomas, B.K. Shoichet, D.J. Maloney, J. Med. Chem. 53
(2010) 52e60.
Appendix. Supplementary data
[17] J.P. Mallari, A.A. Shelat, A. Kosinski, C.R. Caffrey, M. Connely, F. Zhu,
J.H. McKerrow, R.K. Guy, J. Med. Chem. 52 (2009) 6489e6493.
[18] I. Sosic, B. Stefane, A. Kovac, S. Turk, D. Blanot, S. Gobec, Heterocycles 81
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.08.005.
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(2010) 91e115.
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ꢀ
[19] B. Mirkovic, M. Renko, S. Turk, I. Sosic, Z. Jevnikar, N. Obermajer, D. Turk,
S. Gobec, J. Kos, ChemMedChem 6 (2011) 1351e1356.
References
[20] J. Goerdeler, J. Neuffer, Chem. Ber. 104 (1971) 1606.
ꢀ
[21] D. Turk, G. Guncar, Acta Cryst. D59 (2003) 203e213.
[1] N.D. Rawlings, A.J. Barrett, A. Bateman, Nucleic Acids Res. 38 (2010) 227e233.
[2] A.J. Barrett, H. Kirschke, Methods Enzymol. 80 (1981) 535e561.
[3] C. Illy, O. Quraishi, J. Wang, E. Purisima, T. Vernet, J.S. Mort, J. Biol. Chem. 272
(1997) 1197e1202.
[4] D. Musil, D. Zucic, D. Turk, R.A. Engh, I. Mayr, R. Huber, T. Popovic, V. Turk,
T. Towatari, N. Katunuma, W. Bode, EMBO J. 10 (1991) 2321e2330.
[5] P.C. Almeida, I.L. Nantes, J.R. Chagas, C.C. Rizzi, A. Faljoni-Alario, E. Carmona,
L. Juliano, H.B. Nader, I.L. Tersariol, Biol. Chem. 276 (2001) 944e951.
[6] D.K. Nägler, A.C. Storer, F.C. Portaro, E. Carmona, L. Juliano, R. Ménard,
Biochemistry 36 (1997) 12608e12615.
[7] A. Pavlova, J.C. Krupa, J.S. Mort, M. Abrahamson, I. Björk, FEBS Lett. 487 (2000)
156e160.
[8] I. Redzynia, A. Ljunggren, M. Abrahamson, J.S. Mort, J.C. Krupa, M. Jaskolski,
G. Bujacz, J. Biol. Chem. 283 (2008) 22815e22825.
[9] P.A. Hill, D.J. Buttle, S.J. Jones, A. Boyde, M. Murata, J.J. Reynolds, M.C. Meikle,
J. Cell. Biochem. 56 (1994) 118e130.
[10] S. Jordans, S. Jenko-Kokalj, N.M. Kühl, S. Tedelind, W. Sendt, D. Brömme,
D. Turk, K. Brix, BMC Biochem. 10 (2009) 23.
[11] T. Zhang, Y. Maekawa, J. Hanba, T. Dainichi, B.F. Nashed, H. Hisaeda, T. Sakai,
T. Asao, K. Himeno, R.A. Good, N. Katunuma, Immunology 100 (2000) 13e20.
[12] H. Büth, P. Luigi Buttigieg, R. Ostafe, M. Rehders, S.R. Dannenmann,
N. Schaschke, H.J. Stark, P. Boukamp, K. Brix, Eur. J. Cell Biol. 86 (2007)
747e761.
[13] M.M. Mohamed, B.F. Sloane, Nat. Rev. Cancer 6 (2006) 764e775.
[14] R. Frlan, S. Gobec, Curr. Med. Chem. 13 (2006) 2309e2327.
[15] B. Turk, Nat. Rev. Drug Discov. 5 (2006) 785e799.
[22] O. Vasiljeva, T. Reinheckel, C. Peters, D. Turk, V. Turk, B. Turk, Curr. Pharm. Des.
13 (2007) 387e403.
[23] S.D. Mason, J.A. Joyce, Trends Cell. Biol. 21 (2011) 228e237.
[24] C. Bincoletto, I.L.S. Tersariol, C.R. Oliveira, S. Dreher, D.M. Fausto, M.A. Soufen,
F.D. Nascimento, A.C.F. Caires, Bioorg. Med. Chem. 13 (2005) 3047e3055.
[25] D. Watanabe, A. Yamamoto, K. Tomoo, K. Matsumoto, M. Murata, K. Kitamura,
T. Ishida, J. Mol. Biol. 362 (2006) 979e993.
[26] K. Tomoo, Curr. Top. Med. Chem. 10 (2010) 696e707.
[27] P.D. Greenspan, C.L. Clark, R.A. Tomassi, S.D. Cowen, L.W. Mcquire, D.L. Farley,
J.H. Duzer, R.L. Goldberg, H. Zhou, Z. Du, J.J. Fitt, D.E. Coppa, Z. Fang,
W. Macchia, L. Zhu, M.P. Capparelli, R. Goldstein, A.M. Wigg, J. Doughty,
R.S. Bohacek, A.K. Knap, J. Med. Chem. 44 (2001) 4524e4534.
[28] P.D. Greenspan, C.L. Clark, S.D. Cowen, L.W. Mcquire, R.A. Tomassi, D.L. Farley,
E. Quadros, D.E. Coppa, Z. Du, Z. Fang, H. Zhou, J. Doughty, K.T. Toscano,
A.M. Wigg, S. Zhou, Bioorg. Med. Chem. Lett. 13 (2003) 4121e4124.
[29] M. Rarey, B. Kramer, T. Lengauer, G. Klebe, J. Mol. Biol. 261 (1996) 470e489.
[30] L.L.C. Schrödinger, The PyMOL Molecular Graphics System, Version 1.3r1
(2010).
[31] M. Frizler, M. Stirnberg, M.T. Sisay, M. Gütschow, Curr. Top. Med. Chem. 10
(2010) 294e322.
[32] R. Kuhelj, M. Dolinar, J. Pungercar, V. Turk, Eur. J. Biochem. 229 (2) (1995)
533e539.
ꢁ
[33] A. Schweiger, B. Stabuc, T. Popovic, V. Turk, J. Kos, J. Immunol. Methods 201
(1997) 165e172.
[34] M. Dolinar, D.B. Maganja, V. Turk, Biol. Chem. Hoppe-Seyler 376 (1995)
385e388.
[35] B.Y. Feng, B.K. Shoichet, Nat. Protoc. 1 (2006) 550e553.
[16] B.T. Mott, R.S. Ferreira, A. Simeonov, A. Jadhav, K.K. Ang, W. Leister,
M. Shen, J.T. Silveira, P.S. Doyle, M.R. Arkin, J.H. McKerrow, J. Inglese,