Design, synthesis and SAR exploration of tri-substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction

Article abstract of DOI:10.1016/j.bmc.2014.07.043

Recent target validation studies have shown that inhibition of the protein interaction between annexin A2 and the S100A10 protein may have potential therapeutic benefits in cancer. Virtual screening identified certain 3,4,5-trisubstituted 4H-1,2,4-triazoles as moderately potent inhibitors of this interaction. A series of analogues were synthesized based on the 1,2,4-triazole scaffold and were evaluated for inhibition of the annexin A2-S100A10 protein interaction in competitive binding assays. 2-[(5-{[(4,6-Dimethylpyrimidin-2-yl)sulfanyl]methyl}-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[4-(propan-2-yl)phenyl]acetamide (36) showed improved potency and was shown to disrupt the native complex between annexin A2 and S100A10.

Full text of DOI:10.1016/j.bmc.2014.07.043

Accepted Manuscript
Design, Synthesis and SAR Exploration of Tri-substituted 1,2,4-Triazoles as
Inhibitors of the Annexin A2-S100A10 Protein Interaction
Tummala R.K. Reddy, Chan Li, Xiaoxia Guo, Peter M. Fischer, Lodewijk.V.
Dekker
PII:
S0968-0896(14)00559-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.07.043
BMC 11738
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 June 2014
18 July 2014
26 July 2014
Please cite this article as: Reddy, T.R.K., Li, C., Guo, X., Fischer, P.M., Dekker, Lodewijk.V., Design, Synthesis
and SAR Exploration of Tri-substituted 1,2,4-Triazoles as Inhibitors of the Annexin A2-S100A10 Protein
Interaction, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.07.043
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Design, Synthesis and SAR Exploration of Tri-substituted
1,2,4-Triazoles as Inhibitors of the Annexin A2-S100A10
Protein Interaction
Tummala R. K. Reddy1, Chan Li, Xiaoxia Guo, Peter M. Fischer, Lodewijk. V. Dekker*
School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham.
Nottingham NG7 2RD, United Kingdom.
*Corresponding author: lodewijk.dekker@nottingham.ac.uk, phone +44(0)1158466327 (not for
publication)
Keywords: S100 proteins; Annexins; docking; triazole
1Current address: Department of Pharmacy, School of Life and Medical Sciences,
Hertfordshire University, Hatfield, Hertfordshire, AL10 9AB, United Kingdom

ABSTRACT
Recent target validation studies have shown that inhibition of the protein interaction between
annexin A2 and the S100A10 protein may have potential therapeutic benefits in cancer.
Virtual screening identified certain 3,4,5-trisubstituted 4H-1,2,4-triazoles as moderately
potent inhibitors of this interaction. A series of analogues were synthesized based on the
1,2,4-triazole scaffold and were evaluated for inhibition of the annexin A2–S100A10 protein
interaction in competitive binding assays. 2-[(5-{[(4,6-Dimethylpyrimidin-2-
yl)sulfanyl]methyl}-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[4-(propan-2-
yl)phenyl]acetamide (36) showed improved potency and was shown to disrupt the native
complex between annexin A2 and S100A10.

1. INTRODUCTION
Small molecule drug discovery has largely focused on drug targets within a fairly limited set
of families of enzymes and receptors that offer tractable pharmacological space. Proteomics
research has opened up a new, potentially very rich, target area consisting of the interacting
faces of protein partners. Recent studies have provided evidence that protein-protein
interactions could offer scope for small molecule intervention. For example, the interaction
between Mdm2 and p53, and the interaction between Bcl2 and Bak have both been explored
pharmacologically using small molecule inhibitors, which have subsequently shown promise
as therapeutic agents.1-3 It is of interest that both p53 and Bak contain a short helical sequence
that docks into a well-defined groove-like feature on the surface of the respective binding
partners, which in both cases constitutes a small globular protein. This suggests that some
protein interactions, characterised by these features, may be manipulated using small
molecules.
S100 proteins constitute a family of small globular adaptor proteins that regulate cell
functions by virtue of their capacity to interact with protein binding partners.4, 5 Annexins are
important binding partners of S100 proteins5 and they contain a short helical sequence feature
at the N-terminus that allows binding to several members of the S100 family. The S100A10
protein and annexin A2 (AnxA2) form a classic pairing in this way.6 The interaction between
these proteins is very well characterised and shows similarities with the tractable protein
interactions described above, in that a small helix docks into a deep well-defined binding
crevice.7, 8 Both S100A10 and AnxA2 have been implicated in cell matrix invasion, cell
movement and cell adhesion and as such play important roles in the regulation of
therapeutically relevant processes such as vascular neo-angiogenesis and tumour cell
metastastis.9-12

Using receptor-guided random docking approaches and biochemical screening, we have
previously identified several clusters of small molecule inhibitors of this protein interaction.13
Here we describe the optimisation of a cluster of substituted 1,2,4 triazole compounds
emanating from this docking strategy to achieve inhibitors with improved potency.
2. RESULTS AND DISCUSSION
2.1 Structure-based screening
We have previously reported the in silico screening of 0.7 million compounds using the
AnxA2 binding pocket of S100A10 as a receptor.13 Following detailed analysis of the binding
poses, and biochemical assessment of the ability of the top scoring compounds to inhibit the
interaction between S100A10 and the AnxA2 N-terminus, 29 potential inhibitory compounds
were identified, which were distributed over 10 different clusters. A cluster of four
trisubstituted 1,2,4-triazoles showed a degree of inhibitory activity that appeared to relate to
their chemical structure (1a-d; Figure 1). This was of interest since we have previously used
ligand-guided screening methods to identify similar 1,2,4-triazoles as moderately potent
inhibitors of this protein interaction.14 Analysis of the binding mode of 1a, predicted using
the GOLD programme15 (Figure 2b), suggested that it lies across the binding groove occupied
by the cognate AnxA2 N-terminus (Figure 2a), thereby inhibiting access of the latter.
Compound 1a interacts mainly with hydrophobic areas of this binding groove (identified as
H1 and H2 in Figure 2b). The orientation of the substituted phenyl moiety in compound 1a is
governed by hydrophobic CH-π stacking interactions mediated by Met12 in the hydrophobic
region H1. Both Phe86 and Phe13 are also predicted to contribute to the binding of this
group, through hydrophobic and van der Waals interactions (Figure 3a). The lower potency
of the compounds with more polar heterocyclic systems at the position of this phenyl moiety
(Figure 1, 1b-d) is compatible with the hypothesis that a hydrophobic binding region

accommodates this ring system. The 1,2,4-triazole core of 1a is positioned at the centre of the
AnxA2 binding groove, whereas the furfuryl substituent at the triazole N4-position is located
towards the H2 region and is predicted to make hydrophobic interactions with Leu78, Phe38
and Phe41 (Figure 3b). From this binding mode analysis it appears that the two hydrophobic
regions H1 and H2 play a dominant role in the inhibitory potency of compound 1a. The
potency of 1a (IC50 = 37 M) was low compared that of the cognate AnxA2(1-14) peptide
ligand (IC50 = 1.2 µM), but similar to that previously observed for related 1,2,4-triazoles,
which lacked the acetamide-linked aromatic ring system.14 Here we have investigated
structure-activity relationships of this series of compounds, focusing in particular on the
contribution of this aromatic system to inhibition of the protein interaction with the aim of
gaining improvements in the potency of these compounds.
2.2 Chemical synthesis
Compounds 1a, 27-56. The substituted ethyl acetate 3 (Scheme 1) was prepared by reacting
4,6-dimethyl-pyrimidine-2-thiol 2 with bromoethyl acetate in the presence of sodium acetate
as a base in ethanol. Subsequent reaction of ester 3 with excess hydrazine monohydrate
yielded acyl hydrazide 4. Condensation of the latter with various substituted aromatic or
aliphatic isothiocyanates 5 in ethanol, followed by base-catalysed cyclization, resulted in the
formation of substituted 1,2,4-triazoles (6-10). Chloro-N-aryl-acetamides 13-26 were
prepared by reacting substituted aryl amines 11 with 2-chloroactyl or propionyl chlorides 12
in the presence of Et3N as a base.
Analogues of 1a were synthesized using a 12-well Radley’s parallel synthesizer. 3-Mercapto-
1,2,4-triazole derivatives 6-10 were treated with chloro-N-aryl-acetamide derivatives 13-26 in
the presence of inorganic base in DMF to afford the desired compounds 27-56 (Scheme 1) in
moderate to good yields.16-22

Compounds 62-66. Condensation of the acetohydrazide 57 (Scheme 2) with aromatic or
aliphatic isothiocyanates 5 in ethanol followed by base-catalysed cyclization afforded the
required substituted 3-mercapto-5-methyl-1,2,4-triazoles 58-60. Finally, the target 5-methyl-
1,2,4-triazoles 62-66 were prepared by reacting substituted 3-mercapto-5-methyl-1,2,4-
triazoles 58-60 with chloro-N-aryl-acetamides 61 in the presence of potassium carbonate as a
base in DMF.23-33
2.3 Structure-activity relationships (SARs)
2.3.1 Nature of the acetamide-linked ring system
Taking compound 1a as starting point, we replaced the substituted phenyl ring side chain
with an aliphatic amide side chain to yield 27. The reduction in potency associated with this
modification suggested the importance of the phenyl ring for binding (see Figure 4 for typical
IC50 curves). Replacement of the substituted phenyl ring with the aromatic but more polar
thiazole (28) resulted in lower inhibitory activity comparable to what was observed above for
compound 1b. Introduction of a methyl substituent onto the thiazole reversed the potency
drop (29). A similar pattern was observed for the related, methyl-substituted isoxazole ring at
this position (30). The lower potency of 28 can be explained by postulating that binding
mainly relies on hydrophobic interactions, in keeping with the predicted model in which the
phenyl group of 1a occupies the hydrophobic H1 region of the AnxA2 binding groove
(Figure 2b, 3a). We therefore investigated if introduction of alternative hydrophobic
substituents into the ring system would result in better interactions (Table 1).
2.3.2 Acetamide-linked Phenyl ring substitutions
An unsubstituted phenyl ring (31) was not associated with a reduction in potency, however,
repositioning the two methyl substituents on the phenyl group increased potency, suggesting
that the substitution pattern is in fact important. Compared to an unsubstituted ring (31),
introduction of a single methyl group at either the 3- or 4-position was associated with a

small increase in potency (32 & 33). The 2,4-dimethyl substituted analogue 34 displayed
similar potency, while the 3,4-dimethyl substitution in 35 resulted in a moderate increase in
potency. Analysis of the predicted binding mode of compound 35 (Figure 2b) suggested that
the orientation of the 3,4-dimethylphenyl moiety is governed by hydrophobic interactions and
causes a change in the location of the 4,6-dimethylpyrimidine moiety, which differs from that
observed for compound 1a (Figure 2b) in that it is flipped away into a more hydrophilic
region. As a result it is involved in more polar interactions with Gly40 and Ser2. Introduction
of a bulkier hydrophobic isopropyl substitution at the para-position of the phenyl ring (36)
yielded a compound with an IC50 value of 4 µM, which is almost 10-fold greater than the
initial hit compound 1a and comparable to that of the cognate peptidic ligand (IC50 = 1 µM).
This is highly reminiscent of the SAR observed for a distinct series of inhibitors of this target
we identified previously.13 A predicted docking solution of 36 (Figure 2b) suggested that this
compound had a similar binding pose as 1a, with the para-isopropyl group projecting
towards the H1 pocket likely to be the main reason for its improved activity. We also tested
the effect of hydrophobic electron-withdrawing substitutions on the phenyl ring. Although a
CF3 substitution at the para position (37) did not provide any advantage over the
unsubstituted ring system in terms of potency, the 3,5-di-CF3 analogue 38 was comparatively
potent. Introduction of a para-chloro phenyl substituent (39) did not result in higher potency,
however, the para-bromo derivative 40 was 5-fold more potent than compound 1a.
2.3.3 Triazole N4 substituents
We next turned our attention to N4 substituents (R1) of the 1,2,4-triazole scaffold (Table 2).
For compound 35, the furfuryl substituent at this position is predicted to project towards the
hydrophobic region H2 (Figure 3b). The specific relevance of the hydrophobic, aromatic and
electronic characteristics of this group were investigated. Substitution of the furfuryl moiety
of compound 35 with a hydrophobic allyl substituent retaining π electronic characteristics

(41), a mixed aliphatic and polar (3-methoxypropyl) substituent (42), or an aromatic polar (4-
methoxypehnyl) substituent (43) and aromatic chloro (4-chlorophenyl) substituent (44), all
resulted in a reduction of potency, suggesting that the furfuryl group possessed a specific
combination of geometric and chemical properties relevant to support binding. Using
compound 35 as the starting point, extension of the sulfanyl side chain by addition of a
methylene group (45) did not appear to be advantageous in terms of activity and was not
explored further.
Following the substitution patterns of the most potent compound (36) in our series, we
combined the 4-(isopropyl)phenyl substitution with allyl (46), 3-methoxypropyl (47), 4-
methoxyphenyl (48) and 4-chlorophenyl (49) at the triazole N4 position. The resulting
isopropyl derivatives were all somewhat less active than 36 itself but were each individually
more potent than the corresponding compound from the 3,4-dimethyl series that was based on
compound 35. Thus in the context of hydrophobic electon-donating substitutions at the
phenyl ring, of the groups tested, the N4 furfuryl group appeared to be optimal.
A rather different picture emerged upon similar replacements for compound 37, which
contains a CF3 moiety at the para-position of the phenyl ring. Allyl (50), 3-methocypropyl
(51), 4-methoxyphenyl (52) and 4-chlorophenyl (53) substitutions resulted in increased
potency compared to 37. The methoxyphenyl derivative 52 was the most potent compound in
this group and is 10-fold more potent than the starting compound 1a. It was also more potent
than the triazole-N4-substituted 4-methoxyphenyl derivatives containing electron-donating
isopropyl (48) or 3,4-dimethyl (43) groups on the acetamide-linked phenyl ring. Alternative
replacements at this phenyl ring did not improve the potency achieved with that of compound
52. The 3,5-diCF3 phenyl derivative 54 was equipotent with 52 while the 4-chlorophenyl
derivative 55 and the 4-methoxy derivative 56 were less potent. Weak resonance donating
properties of the halogen may be relevant in this context, as could be the electron-donating

properties of the methoxy group. Overall, the 4-methoxyphenyl group at N4 appeared optimal
for compounds that contained a hydrophobic electron-withdrawing group at the phenyl ring,
while substitutions that increase ring electron density were not favoured in this context.
Interestingly, docking studies of the N4 4-methoxyphenyl derivatives (43, 48, 52, 54-56)
showed that most of the compounds adopted an alternative binding pose compared to that of
the N4 furfuryl compounds (Figure 2c). According to this binding pose, compounds stretch
across most of the binding area occupied by the N-terminus of the cognate AnxA2 ligand
(Figure 2c). This binding pose could be due to the introduction of bulk (para-substituted
methoxyphenyl groups) at the N4-position of the triazole ring. The 4-methoxyphenyl
substituent of the compounds appears to mimic the interactions of the Val3 side chain of the
AnxA2 peptide in the crystal structure (Figure 2a and c). The presence of AnxA2-Val3 is
essential for binding to S100A10 since its substitution leads to substantial loss of binding34.
In all these compounds, the 4,6-dimethylpyrimidine moiety extends into the hydrophilic
region, similar to what was predicted for compound 35 (Figure 2b), whereas the para-
substituted phenyl moiety is involved in hydrophobic interactions with the far end of the H1
binding pocket. In the case of compound 52 the para-CF3-phenyl moiety makes good van der
Waals and hydrophobic interactions (Figure 2c), whereas the para-(isopropyl)phenyl moiety
of compound 48 (Figure 2c) is involved in weaker hydrophobic interactions than the
corresponding para-CF3 derivative 52. The low potency of compound 43 could not be
justified based on an alternative binding mode as the 3,4-dimethylphenyl moiety appears to
form good hydrophobic and van der Waals interactions with the far end of the H1 binding
pocket.
2.3.4 Sulfanyl side chain
Finally, we examined the importance of the 4,6-dimethylpyrimidin-2-ylsulfanyl side chain on
inhibitory potency (Table 3). Inspection of the predicted binding pose of compound 1a shows

that the side chain in question interacts with the comparatively hydrophilic region of the
binding pocket (Figure 2b, 3a). Truncation of the 4,6-dimethylpyrimidin-2-ylsulfanyl side
chain of compound 35 to the corresponding methyl derivative 62 resulted in complete loss of
activity. Extending the side chain of compound 62 by an additional methylene group (63)
also abolished activity. Truncation of the 4,6-dimethylpyrimidin-2-ylsulfanyl side chain
compounds 36 to the corresponding methyl derivative 64 resulted in a 10-fold loss of activity,
whereas in the case of compounds 46 and 47 similar truncation to methyl derivatives 65 and
66 resulted in 14- and 23-fold lower activity, respectively. Thus the 4,6-dimethylpyrimidin-2-
ylsulfanyl side chain makes an important contribution to the inhibitory activity of these
compounds.
2.4 Inhibition of native complex
Although it is clear that the tri-substituted 1,2,4 triazoles act as inhibitors of the binding of the
N-terminus of AnxA2 to S100A10, it is necessary to confirm that the native complex of the
two full-length proteins, AnxA2 and S100A10, is also inhibited. Interaction between the
native proteins can be measured by co-immunoprecipitation as shown in Figure 5 and
previously.13 AnxA2 is detected by Western blotting after immunoprecipitation of the
S100A10 protein, suggesting that binding occurs between the two proteins. In the presence of
the isolated synthetic peptide AnxA2(1-14), the amount of AnxA2 in the S100A10
immunoprecipitate is reduced, due to it competing with the binding between native AnxA2
and S100A10. In the presence of compound 36, the amount of AnxA2 in the
immunoprecipitate is also reduced, indicating that it does indeed inhibit the native complex.
3. CONCLUSION
Analysis of the molecular interactions of compound 1a shows that the hydrophobic pockets
H1 and H2 of the S100A10 receptor play a crucial role in the affinity of these compounds.

Complex interactions appear to occur between the N4- and the acetamide-linked ring
systems, such that dependent on the nature of the N4 substituent, the hydrophobic character
and the electron density on the acetamide-linked ring is correlated with inhibition patterns.
Additionally, the 4,6-dimethylpyrimidin-2-ylsulfanyl side chain appears to be important for
the activity, and this needs further exploration.
Compounds 36 and 52 are the most potent in present compound series with 10-fold increase
in activity compared to the initial hit compound 1a. They also displayed higher activity than a
set of triazole analogues that lack an acetamide-linked ring that we have reported on
previously.14 Compounds 36 and 52, and various analogues described here, could thus be
used as probes further to understand the molecular interactions between S100A10 and AnxA2
and may also serve as potential starting point for further optimization.
4. EXPERIMENTAL
4.1 Protein structure preparation.
SYBYL-X 1.0 biopolymer was used for the preparation of the S100A10-AnxA2 N-terminal
peptide complex (PDB ID: 1BT6). The chain termini (Lys91, Pro1 and Ser11 of AnxA2)
were charged. Hydrogen atoms were added. AMBER 7 FF99 charges were assigned to the
complex. The side chain amides of Gln45 and Gln60 were re-oriented to maximize hydrogen
bonding. Powell’s energy minimization of the complex was achieved by a stepwise manner
using AMBER 7 FF99 parameters. Initial optimization was not performed, energy gradient of
0.5 kcal/mol was maintained and a maximum of 5,000 iterations were used. A centroid was
defined by a set of atoms within 5 Å of the AnxA2 ligand peptide and the peptide was
extracted from the complex. Water molecules were deleted before docking.
4.2 GOLD (v3.0.1) Docking.

Docking of each ligand was performed in 10 different genetic algorithm (GA) runs, and for
each of these a maximum number of 100,000 GA operations were performed on a single
population of 100 individuals. Early termination of docking runs was allowed if the top three
solutions were within 1.5 Å RMSD of each other. Crossover, mutation, migration (95, 95,
10, respectively), hydrogen bonding (4.0 Å), and van der Waals (2.5 Å) parameters were set
as default value throughout the docking. A binding site radius of 12 Å was found to be
optimal. Flipping was not allowed for those ligands that have ring-NHR and ring-NHR1R2
groups in order to avoid the addition of large torsional energy penalties to the total fitness
scores.
4.3 Fluorescence screening assay.
Assessment of compound activity was performed as described.35 Briefly, a Cy5-labelled
S100A10 tracer was developed and binding of a Cy3-labelled AnxA2(Ac1-14) peptide ligand
was assessed using a fluorescence resonance energy transfer readout. Assays were carried out
in Nunc black non-treated 384-well plates at 20 oC in 50 µL buffer C. All incubations were
performed in quadruplicate. Compounds, peptide and buffer controls were added to the wells
in a 10 µL volume in 5% DMSO. Cy5-labelled S100A10 tracer (407 nM) and Cy3-labelled
AnxA2(Ac1-14) peptide ligand (1.33 µM) were pre-incubated for 5 min at 20 oC and 40 µL
of the preformed complex was then added to the wells and mixed for 10 s to yield a final
DMSO concentration of 1%. After 5 min incubation at 20 oC, readings were taken on a
Perkin Elmer Envision plate reader by excitation at 488 nm and emission at 695 nm.
Fluorescence resonance energy transfer (FRET) was calculated by subtracting the sum of the
fluorescence emission of S100A10-Cy5 and AA2(1-14)-Cy3 individually from that measured
for the co-incubated partners. Compound binding was calculated as percentage of non-treated
control and data were analysed by non-linear regression (dose response - variable slope with

bottom and top parameters fixed at 0 and 100% respectively) using Graphpad Prism Software
to determine the IC50 of binding (primary assay).
4.4 Immunoprecipitation assay.
Human MDA-MB-231 breast cancer cells were extracted in 50 mM Tris-HCl (pH 8.0) buffer
containing 150 mM sodium chloride and 1 % (v/v) NP-40. Lysates were centrifuged at
17,000 x g and incubated at 4 °C for 16 h with 10 µL S100A10 antibody (BD transduction
Laboratories) and 50 µL protein A/G agarose (Alpha Diagnostic International Inc). Protein
A/G agarose was then recovered by centrifugation at 2,400 x g, washed three times with 10
mM phosphate buffer (pH 7.4) containing 2.7 mM potassium chloride and 137 mM sodium
chloride, resuspended in 50 µL of 160 mM Tris-HCl (pH 6.8) buffer containing 4 % (w/v)
sodium lauryl sulphate, 20 % (v/v) glycerol, 0.04 % (w/v) bromophenol blue and 10 % (v/v)
2-mercaptoethanol, boiled at 99 oC for 10 min and centrifuged at 17,000 x g for 10 min. The
supernatant was then analyzed by SDS-PAGE after which the gel was transferred to a
nitrocellulose filter. The filter was incubated with an AnxA2 monoclonal antibody (1:3,000;
BD Transduction Laboratories) followed by incubation with an anti-mouse horseradish
peroxidase IgG conjugate (1:5,000; GE Healthcare) and then developed using the ECL
detection reagent (GE Healthcare).
4.5 Synthesis.
All reagents were purchased directly from commercial sources and were used as supplied,
unless otherwise stated. Accurate mass and nominal mass measurements were performed
using a Waters 2795-Micromass LCT electrospray mass spectrometer. All NMR spectra were
recorded in deutero-DMSO in 5 mm tubes, with trimethylsilane as an internal standard, using
a Bruker ACS-120 instrument at 400 MHz (1H-NMR). Thin layer chromatography was
performed using aluminium-backed silica gel 60 plates (0.20 mm layer), the ascending

technique was used with a variety of solvents. Visualization was by UV light at either 254 or
365 nm.
(4,6-Dimethyl-pyrimidin-2-ylsulfanyl)-acetic acid ethyl ester (3). To a solution of 2 (14.2
g, 100 mmol) in EtOH (190 mL) was added NaOAc (12.3 g, 150 mmol) and ethyl
bromoacetate (11.3 mL, 100 mmol). The mixture was heated under reflux for 60 min and
EtOH was then evaporated. The residue was diluted with H2O and extracted with EtOAc. The
extract was dried over Na2SO4, filtered, and concentrated under vacuum to afford 3 as a
yellow oil (15.5 g, 69%). m/z (ES), found 227.0821 (C10H15N2O2S [M + H]+) requires
227.2954; δH/ppm (400 MHz, d6-DMSO): 6.97 (1H, s, Ar-H), 4.10 (2H, q, J = 7.1, CH2), 3.94
(2H, s, CH2), 2.33 (6H, s, 2CH3), 1.18 (3H, t, J = 7.1, CH3).
(4,6-Dimethyl-pyrimidin-2-ylsulfanyl)-acetic acid hydrazide (4). To a solution of 3 (12.3
g, 54.3 mmol) in EtOH (170 mL) was added NH2NH2.H2O (16.1 mL, 32 mmo6l) and the
mixture was heated under reflux overnight. The reaction was cooled and concentrated under
vacuum. The pale white precipitate obtained was washed with petroleum ether (PE)–EtOH
(3:1) and then dried under vacuum to afford 4 as a pale yellow powder (9.7 g, 84%). m/z
(ES), found 213.0846 (C8H13N4OS [M + H]+) requires 213.0732; δH/ppm (400 MHz, d6-
DMSO): 9.21 (1H, s, NH), 6.96 (1H, s, Ar-H), 4.17 (2H, bs, NH2), 3.79 (2H, s, CH2), 2.34
(6H, s, 2CH3).
5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazole-3-
thiol (6). To a solution of 4 (5.0 g, 23.6 mmol) in EtOH (175 mL) was added furfuryl
isothiocyanate (3.4 mL, 23.6 mmol) and the mixture was heated under reflux overnight. The
reaction was cooled and concentrated under vacuum. The white precipitate obtained was
washed with PE–EtOH (1:1). To the suspension was added 2 M aq NaOH (40 mL) and the
mixture was heated under reflux for 3 h before cooling. The reaction was neutralized with
10% aq HCl and was extracted with EtOAc. The organic layer was dried over Na2SO4,

filtered, and concentrated. The resulting yellow solid was recrystallized from EtOH to afford
6 as an off-white powder (2.4 g, 30%). m/z (ES), found 332.0606 (C14H14N5OS2 [M - H]-)
requires 332.0718; δH/ppm (400 MHz, d6-DMSO): 13.72 (1H, s, NH of thione tautomer),
7.63 (1H, t, J = 1.3/1.4, Ar-H), 6.98 (1H, s, Ar-H), 6.46-6.41 (2H, m, Ar-H), 5.37 (2H, s,
CH2), 4.53 (2H, s, CH2), 2.31 (6H, s, 2CH3).
4-Allyl-5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4H-[1,2,4]triazole-3-thiol (7). To
a solution of 4 (4.5 g, 21.2 mmol) in EtOH (150 mL) was added allyl isothiocyanate (2.2 mL,
21.2 mmol) and the reaction mixture was heated under reflux overnight. It was cooled and the
white precipitate formed was collected by filtration and was washed with EtOH. To the
suspension was added 1 M aq NaOH (25 mL) and the mixture was heated at 45 °C for 90 min
before cooling. The reaction was neutralized with 10% aq HCl. The yellow precipitate
formed was collected by filtration, washed with H2O, followed by EtOH, and then vacuum
dried to afford 7 as a pale white powder (2.9 g, 47%). m/z (ES), found 292.0616 (C12H14N5S2
[M - H]-) requires 292.0769; δH/ppm (400 MHz, d6-DMSO): 13.67 (1H, s, NH of thione
tautomer), 7.00 (1H, s, Ar-H), 5.97-5.84 (1H, m, CH), 5.21 [1H, dd, J = 1.2 + 10.4, N-CH2
(Ha)], 5.04 [1H, dd, J = 1.2 + 17.2, N-CH2 (Hb)], 4.73 (2H, d, J = 5.1, =CH2), 4.45 (2H, s,
CH2), 2.34 (6H, s, 2CH3).
5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-(3-methoxy-propyl)-4H-[1,2,4]triazole-
3-thiol (8). To a solution of 4 (4.5 g, 21.2 mmol) in EtOH (170 mL) was added 3-
methoxypropyl isothiocyanate (2.7 mL, 21.2 mmol) and the mixture was heated under reflux
overnight. The reaction was cooled and concentrated under vacuum. To the yellow crystalline
solid thus formed was added 1 M aq NaOH (25 mL) and the mixture was heated at 45 °C for
120 min before cooling. The reaction was neutralized with 10% aq HCl and was extracted
with EtOAc. The extract was dried over Na2SO4, filtered, and concentrated. The resulting
yellow solid was washed with Et2O and recrystallized from EtOH to afford 8 as an off-white

crystalline solid (2.6 g, 38%). m/z (ES), found 324.0871 (C13H18N5OS2 [M - H]-) requires
324.1031; δH/ppm (400 MHz, d6-DMSO): 13.60 (1H, s, NH of thione tautomer), 7.01 (1H, s,
Ar-H), 4.51 (2H, s, CH2), 4.07 (2H, t, J = 7.3, CH2OCH3), 3.34 (2H, t, J = 6.0, N-CH2), 3.22
(3H, s, OCH3), 2.35 (6H, s, 2CH3), 2.16-1.92 (2H, m, CH2).
5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-(4-methoxy-phenyl)-4H-
[1,2,4]triazole-3-thiol (9). To a solution of 4 (4.5 g, 21.2 mmol) in EtOH (175 mL) was
added 4-methoxyphenyl isothiocyanate (3.0 mL, 21.2 mmol) and the mixture was heated
under reflux overnight. The reaction was cooled and concentrated under vacuum. To the
yellow powder thus formed was added 1 M aq NaOH (50 mL) and the mixture was heated
under reflux for 60 min before cooling. The reaction was neutralized with 10% aq HCl. The
yellow precipitate formed was collected by filtration, was washed with EtOH–MeOH (1:1)
and then vacuum dried to afford 9 as a pale yellow powder (5.9 g, 77%). m/z (ES), found
359.9088 (C16H18N5OS2 [M + H]+) requires 360.0875; δH/ppm (400 MHz, d6-DMSO): 13.76
(1H, s, NH of thione tautomer), 7.30 (2H, d, J = 9.0, Ar-H), 6.99 (2H, d, J = 9.0, Ar-H), 6.93
(1H, s, Ar-H), 4.32 (2H, s, CH2), 3.78 (3H, s, OCH3), 2.28 (6H, s, 2CH3).
4-(4-Chloro-phenyl)-5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4H-[1,2,4]triazole-
3-thiol (10). To a solution of 4 (5.0 g, 23.6 mmol) in EtOH (175 mL) was added 4-
chlorophenyl isothiocyanate (4.0 g, 23.6 mmol). The mixture was heated under reflux for 20
min and the formation of white precipitate was observed. The reaction was cooled and the
white precipitate was collected by filtration and was washed with EtOH. To the precipitate
was added 1 M aq NaOH (50 mL) and the mixture was heated at 45 °C for 45 min before
cooling. The reaction was neutralized with 10% aq HCl. The white precipitate formed was
collected by filtration, washed with MeOH and then vacuum dried to afford 10 as an off-
white powder. In order to recover more 10 the filtrate was extracted with EtOAc. The extract
was dried over Na2SO4, filtered, and concentrated. The resulting pale white powder was

recrystallized from MeOH to afford 10 as a white powder. The combined yield of 10 was 4.9
g (57%). m/z (ES), found 363.8376 (C15H15ClN5S2 [M + H]+) requires 364.0379; δH/ppm (400
MHz, d6-DMSO): 13.86 (1H, s, NH of thione tautomer), 7.52 (2H, d, J = 8.8, Ar-H), 7.43
(2H, d, J = 8.8, Ar-H), 6.93 (1H, s, Ar-H), 4.38 (2H, s, CH2), 2.28 (6H, s, 2CH3).
2-Chloro-N-(3,4-dimethyl-phenyl)-acetamide (13). To a solution of 3,4-dimethylaniline
(1.85 g, 15 mmol) in CH2Cl2 (60 mL) was added Et3N (2.1 mL, 15.2 mmol) and the mixture
was allowed to stir at room temperature (RT) for 10 min. To this was then added chloroacetyl
chloride (1.19 mL, 15 mmol) and the reaction was left to stir at RT for 4 h. The reaction was
concentrated under vacuum. The residue was diluted with H2O and extracted with EtOAc.
The extract was washed with 1 M aq HCl, dried over Na2SO4, filtered, and concentrated
under vacuum. The yellow precipitate formed was washed with Et2O and then vacuum dried.
to afford 13 as a white powder (2.2 g, 74%). m/z (ES), found 198.0658 (C10H13 ClNO [M +
H]+) requires 198.0607; δH/ppm (400 MHz, d6-DMSO): 10.11 (1H, s, NH), 7.35 (1H, s, Ar-
H), 7.30 (1H, dd, J = 1.8 + 8.1, Ar-H), 7.07 (1H, d, J = 8.1, Ar-H), 4.21 (2H, s, CH2), 2.19
(3H, s, CH3), 2.16 (3H, s, CH3).
2-Chloro-N-phenyl-acetamide (14). Procedure as for 13 except using aniline (1.37 mL, 15
mmol) and the reaction mixture was allowed to stir at RT overnight. Pure 14 (without
washing with Et2O) was obtained as off-white crystals (963 mg, 38%). m/z (ES), found
170.0979 (C8H9ClNO [M + H]+) requires 170.0294; δH/ppm (400 MHz, d6-DMSO): 10.27
(1H, s, NH), 7.58 (2H, dd, J = 1.0 + 8.5, Ar-H), 7.36-7.30 (2H, m, Ar-H), 7.12-7.06 (1H, m,
Ar-H), 4.25 (2H, s, CH2).
2-Chloro-N-m-tolyl-acetamide (15). Procedure as for 14 except using m-toluidine (1.62 mL,
15 mmol) and the reaction mixture was allowed to stir at RT overnight. Pure 15 was obtained
as a brown powder (1.98 g, 72%). m/z (ES), found 184.0486 (C9H11ClNO [M + H]+) requires
184.0451; δH/ppm (400 MHz, d6-DMSO): 10.19 (1H, s, NH), 7.42 (1H, s, Ar-H), 7.37 (1H, d,

J = 8.4, Ar-H), 7.20 (1H, t, J = 7.8, Ar-H), 6.90 (1H, d, J = 7.5, Ar-H), 4.23 (2H, s, CH2), 2.28
(3H, s, CH3).
2-Chloro-N-p-tolyl-acetamide (16). Procedure as for 14 except using p-toluidine (1.61 g, 15
mmol) and the reaction mixture was allowed to stir at RT overnight. Pure 16 was obtained as
a brown powder (1.81 g, 66%). δH/ppm (400 MHz, d6-DMSO): 10.18 (1H, s, NH), 7.47 (2H,
d, J = 8.4, Ar-H), 7.13 (2H, d, J = 8.2, Ar-H), 4.22 (2H, s, CH2), 2.25 (3H, s, CH3).
2-Chloro-N-(2,5-dimethyl-phenyl)-acetamide (17). Procedure as for 14 except using 2,5-
dimethylaniline (1.87 mL, 15 mmol). The pale yellow precipitate formed was washed with
PE–CH2Cl2 (4:1) and then vacuum dried. Pure 17 was obtained as a white powder (1.1 g,
37%). m/z (ES), found 198.1024 (C10H13ClNO [M + H]+) requires 198.0607; δH/ppm (400
MHz, d6-DMSO): 9.57 (1H, s, NH), 7.20 (1H, s, Ar-H), 7.10 (1H, d, J = 7.7, Ar-H), 6.93 (1H,
d, J = 7.6, Ar-H), 4.28 (2H, s, CH2), 2.25 (3H, s, CH3), 2.15 (3H, s, CH3).
2-Chloro-N-(2,4-dimethyl-phenyl)-acetamide (18). To a solution of 2,4-dimethylaniline
(1.87 mL, 15 mmol) in CH2Cl2 (60 mL) was added Et3N (2.1 mL, 15 mmol) and the mixture
was allowed to stir at RT for 10 min. To this was added chloroacetyl chloride (1.19 mL, 15
mmol) and the reaction was left to stir at RT for 4 h. The reaction was concentrated under
vacuum. The residue was diluted with H2O and extracted with EtOAc. The extract was
washed with 1 M aq HCl and was dried over MgSO4, filtered, and concentrated under
vacuum. Pure 18 was obtained as a pale white powder (0.96 g, 32.5%). m/z (ES), found
198.1024 (C10H13ClNO [M + H]+) requires 198.0607; δH/ppm (400 MHz, d6-DMSO): 9.59
(1H, s, NH), 7.24 (1H, d, J = 8.0, Ar-H), 7.03 (1H, s, Ar-H), 6.98 (1H, d, J = 8.1, Ar-H), 4.28
(2H, s, CH2), 2.25 (3H, s, CH3), 2.15 (3H, s, CH3).
2-Chloro-N-(4-isopropyl-phenyl)-acetamide (19). Procedure as for 14 except using 4-
isopropylaniline (2.1 mL, 15 mmol) and the reaction mixture was allowed to stir at RT
overnight. Pure 19 was obtained as a pale yellow powder (2.0 g, 63%). m/z (ES), found

212.0961 (C11H15ClNO [M + H]+) requires 212.0764; δH/ppm (400 MHz, d6-DMSO): 10.19
(1H, s, NH), 7.49 (2H, d, J = 8.5, Ar-H), 7.19 (2H, d, J = 8.5, Ar-H), 4.22 (2H, s, CH2), 2.84
(1H, hept, CH of isopropyl), 1.18 [6H, d, J = 6.9, (CH3)2 of isopropyl].
2-Chloro-N-(4-trifluoromethyl-phenyl)-acetamide (20). Procedure as for 14 except using
4-trifluoromethylaniline (1.9 mL, 15 mmol). Pure 20 was obtained as a yellow powder (1.5 g,
42%). m/z (ES), found 235.6225 (C9H6ClF3NO [M - H]-) requires 236.0168; δH/ppm (400
MHz, d6-DMSO): 10.64 (1H, s, NH), 7.80 (2H, d, J = 8.5, Ar-H), 7.70 (2H, d, J = 8.6, Ar-H),
4.30 (2H, s, CH2).
N-(3,5-Bis-trifluoromethyl-phenyl)-2-chloro-acetamide (21). Procedure as for 14 except
using 3,5-bis(trifluoromethyl)aniline (2.4 mL, 15 mmol) and the mixture was allowed to stir
at RT overnight. The precipitate obtained was washed with PE and vacuum dried. Pure 21
was obtained as a brick-red powder (1.77 g, 39%). δH/ppm (400 MHz, d6-DMSO): 10.93 (1H,
s, NH), 8.25 (2H, s, Ar-H), 7.80 (1H, s, Ar-H), 4.34 (2H, s, CH2).
2-Chloro-N-(4-methoxy-phenyl)-acetamide (22). Procedure as for 14 except using p-
anisidine (1.85 g, 15 mmol). Pure 22 was obtained as a gray powder (1.77 g, 59%). m/z (ES),
found 200.0450 (C9H11ClNO2 [M + H]+) requires 200.0400; δH/ppm (400 MHz, d6-DMSO):
10.13 (1H, s, NH), 7.50 (2H, d, J = 9.1, Ar-H), 6.90 (2H, d, J = 9.1, Ar-H), 4.21 (2H, s, CH2),
3.72 (3H, s, OCH3).
2-Chloro-N-(4-chloro-phenyl)-acetamide (23). Procedure as for 14 except using 4-
chloroaniline (1.95 g, 15 mmol). Pure 23 was obtained as a pale yellow powder (2.26 g,
74%). m/z (ES), found 201.6550 (C8H6Cl2NO [M - H]-) requires 201.9905; δH/ppm (400
MHz, d6-DMSO): 10.41 (1H, s, NH), 7.62 (2H, d, J = 9.0, Ar-H), 7.38 (2H, d, J = 8.9, Ar-H),
4.25 (2H, s, CH2).
N-(4-Bromo-phenyl)-2-chloro-acetamide (24). To a solution of 4-bromoaniline (2.66 g, 15
mmol) in CH2Cl2 (60 mL) was added Et3N (2.1 mL, 15.2 mmol) and the mixture was allowed

to stir at RT for 10 min. To this was added chloroacetyl chloride (1.19 mL, 15 mmol) and the
reaction was left to stir at RT for 4 h. The white precipitate formed was separated by filtration
and washed with CH2Cl2. Pure 24 was obtained as a white powder (898 mg, 24%). m/z (ES),
found 247.9191 (C8H8BrClNO [M + H]+) requires 247.9400; δH/ppm (400 MHz, d6-DMSO):
10.41 (1H, s, NH), 7.56 (2H, d, J = 9.0, Ar-H), 7.51 (2H, d, J = 9.0, Ar-H), 4.25 (2H, s, CH2).
2-Chloro-N-thiazol-2-yl-acetamide (25). Procedure as for 14 except using 2-aminothiazole
(1.55 g, 15 mmol). Precipitation was observed within 15 min of the addition of chloroacetyl
chloride. The precipitate was extracted with EtOAc. Pure 25 was obtained as a pale yellow
powder (642 mg, 24%) after recrystallization from EtOH. m/z (ES), found 176.9838
(C5H6ClN2OS [M + H]+) requires 176.9811; δH/ppm (400 MHz, d6-DMSO): 12.48 (1H, s,
NH), 7.50 (1H, d, J = 3.6, Ar-H), 7.27 (1H, d, J = 3.6, Ar-H), 4.38 (2H, s, CH2).
2-Chloro-N-(4-methyl-thiazol-2-yl)-acetamide (25a). Procedure as for 14 except using 2-
amino-4-methylthiazole (1.75 g, 15 mmol). Pure 25a was obtained as a pale yellow powder
(1.4 g, 49%). m/z (ES), found 190.0078 (C6H8ClN2OS [M + H]+) requires 190.9968; δH/ppm
(400 MHz, d6-DMSO): 12.42 (1H, s, NH), 6.80 (1H, d, J = 1.0, Ar-H), 4.35 (2H, s, CH2),
2.26 (3H, d, J = 1.0, Ar-H).
2-Chloro-N-(5-methyl-isoxazol-3-yl)-acetamide (25b). Procedure as for 14 except using 3-
amino-5-methyl-isoxazole (1.5 g, 15 mmol, 1.00 eq.) and the reaction mixture was allowed to
stir at RT overnight. The precipitate obtained was washed with CH2Cl2 and then vacuum
dried. Pure 25b was obtained as a white crystalline solid (766 mg, 30%). m/z (ES), found
175.0221 (C6H8ClN2O2 [M + H]+) requires 175.0196; δH/ppm (400 MHz, d6-DMSO): 11.25
(1H, s, NH), 6.62 (1H, s, Ar-H), 4.29 (2H, s, CH2), 2.38 (3H, s, CH3).
3-Chloro-N-(3,4-dimethyl-phenyl)-propionamide (26). Procedure as for 14 except using 3-
chloropropionyl chloride (1.44 mL, 15 mmol) and the reaction mixture was allowed to stir at
RT overnight. The precipitate obtained was washed with PE and then vacuum dried. Pure 26
was obtained as a white powder (1.9 g, 60%). m/z (ES), found 212.1006 (C11H15ClNO [M +
H]+) requires 212.0764; δH/ppm (400 MHz, d6-DMSO): 9.87 (1H, s, NH), 7.37 (1H, s, Ar-H),

7.31 (1H, dd, J = 2.1 + 8.1, Ar-H), 7.04 (1H, d, J = 8.1, Ar-H), 3.86 (2H, t, J = 6.3, CH2), 2.78
(2H, t, J = 6.3, CH2), 2.19 (3H, s, CH3), 2.16 (3H, s, CH3).
N-(2,5-Dimethyl-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-
ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (1a). A solution of 6 (333 mg, 1 mmol)
in DMF (3 mL), and a solution of 17 (198 mg, 1 mmol) in DMF (2 mL) were combined in a
dry Radleys reaction tube containing K2CO3 (166 mg, 1.2 mmol). The mixture was allowed
to stir at 40 °C overnight in a Radleys parallel synthesizer. The mixture was poured on
crushed ice (75 mL) and allowed to stand for a few h. The mixture was then extracted with
EtOAc. The combined extracts were washed with distilled H2O, dried over Na2SO4, filtered,
and concentrated under vacuum. Purification of the crude product by flash column
chromatography (FCC; EtOAc, Rf = 0.26) gave pure 1a as a pale white powder (196 mg,
40%). δH/ppm (400 MHz, d6-DMSO): 9.57 (1H, s, NH), 7.63 (1H, dd, J = 0.7 + 1.7, Ar-H),
7.22 (1H, s, Ar-H), 7.05 (1H, d, J = 7.7, Ar-H), 6.97 (1H, s, Ar-H), 6.88 (1H, d, J = 7.7, Ar-
H), 6.48-6.42 (2H, m, Ar-H), 5.39 (2H, s, CH2), 4.65 (2H, s, CH2), 4.11 (2H, s, CH2), 2.33
[6H, s, (CH3)2], 2.23 (3H, s, CH3), 2.06 (3H, s, CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-acetamide (27). Procedure as for 1a except using 2-bromoacetamide (138 mg,
1 mmol) and the reaction was carried out for 48 h. The white precipitate formed was
collected by filtration, washed with H2O and lyophilized, followed by recrystallization from
EtOH. Pure 27 was obtained as a pale white powder (173 mg, 45%). m/z (ES), found
389.0885 (C16H17N6O2S2 [M - H]-) requires 389.0933; δH/ppm (400 MHz, d6-DMSO): 7.57-
7.70 (2H, m, Ar-H), 7.20 (1H, bs, OH), 6.99 (1H, s, =NH), 6.40-6.50 (2H, m, Ar-H), 5.36
(2H, s, CH2), 4.64 (2H, s, CH2), 3.85 (2H, s, CH2), 2.34 (6H, s, 2CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-thiazol-2-yl-acetamide (28). Procedure as for 1a except using 25 (177 mg, 1
mmol) and the reaction was carried out for 24 h. The precipitate formed was collected by
filtration, washed thoroughly with water and lyophilized, followed by recrystallization from
MeOH. Pure 28 (EtOAc, Rf = 0.06) was obtained as a white powder (292 mg, 62%). m/z
(ES), found 474.6843 (C19H20N7O2S3 [M + H]+) requires 474.0762; δH/ppm (400 MHz, d6-

DMSO): 12.38 (1H, s, NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 7.47 (1H, d, J = 3.6, Ar-H),
7.22 (1H, d, J = 3.6, Ar-H), 6.98 (1H, s, Ar-H), 6.52-6.40 (2H, m, Ar-H), 5.38 (2H, s, CH2),
4.63 (2H, s, CH2), 4.19 (2H, s, CH2), 2.34 (6H, s, 2CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-(4-methyl-thiazol-2-yl)-acetamide (29). Procedure as for 1a except using
25a (191 mg, 1 mmol) and the reaction was carried out for 48 h. The mixture was extracted
with EtOAc. The combined extracts were washed with H2O and dried over Na2SO4, filtered,
and concentrated under vacuum. Purification of the crude product by FCC (EtOAc–MeOH
(95:5) gave 29 as a pale white powder (219 mg, 45%). m/z (ES), found 486.0944
(C20H20N7O2S3 [M - H]-) requires 486.0919; δH/ppm (400 MHz, d6-DMSO): 12.29 (1H, s,
NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 6.98 (1H, s, Ar-H), 6.76 (1H, d, J = 1.0, Ar-H), 6.50-
6.40 (2H, m, Ar-H), 5.37 (2H, s, CH2), 4.63 (2H, s, CH2), 4.16 (2H, s, CH2), 2.34 (6H, s,
2CH3), 2.25 (3H, d, J = 0.9, CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-(5-methyl-isoxazol-3-yl)-acetamide (30). Procedure as for 1a except using
25b (175 mg, 1 mmol) and the reaction was carried out for 48 h. The precipitate was
collected by filtration, washed with H2O and lyophilized, followed by recrystallization from
MeOH. Pure 30 was obtained as a white powder (298 mg, 65%). m/z (ES), found 472.1485
(C20H22N7O3S2 [M + H]+) requires 472.1147; δH/ppm (400 MHz, d6-DMSO): 11.20 (1H, s,
NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 6.99 (1H, s, Ar-H), 6.56 (1H, s, Ar-H), 6.48-6.42
(2H, m, Ar-H), 5.37 (2H, s, CH2), 4.63 (2H, s, CH2), 4.11 (2H, s, CH2), 2.36 (3H, d, J = 0.8,
CH3), 2.34 (6H, s, 2CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-phenyl-acetamide (31). Procedure as for 1a except using 6 (600 mg, 1.8
mmol) and 14 (305 mg, 1.8 mmol). The mixture was extracted with EtOAc. The combined

extracts were washed with H2O and dried over Na2SO4, filtered, and concentrated under
vacuum. Purification of the crude product by FCC (EtOAc, Rf = 0.20), gave 31 as a pale
white powder (586 mg, 70%). m/z (ES), found 465.1360 (C22H21N6O2S2 [M - H]-) requires
465.1246; δH/ppm (400 MHz, d6-DMSO): 10.28 (1H, s, NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-
H), 7.50-7.56 (2H, m, Ar-H), 7.26-7.32 (2H, m, Ar-H), 7.05 (1H, t, J = 7.4, Ar-H), 6.98 (1H,
s, Ar-H), 6.42-6.49 (2H, m, Ar-H), 5.39 (2H, s, CH2), 4.64 (2H, s, CH2), 4.11 (2H, s, CH2),
2.34 (6H, s, 2CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-m-tolyl-acetamide (32). Procedure as for 1a except using 15 (184 mg, 1
mmol) and the reaction was carried out for 24 h. The mixture was extracted with EtOAc. The
combined extracts were washed with H2O and dried over Na2SO4, filtered, and concentrated
under vacuum. Purification of the crude product by FCC (EtOAc, Rf = 0.21), gave 32 as a
pale white powder (264 mg, 55%). m/z (ES), found 479.1382 (C23H23N6O2S2 [M - H]-)
requires 479.1402; δH/ppm (400 MHz, d6-DMSO): 10.20 (1H, s, NH), 7.63 (1H, dd, J = 0.8 +
1.8, Ar-H), 7.37 (1H, s, Ar-H), 7.31 (1H, d, J = 8.3, Ar-H), 7.17 (1H, t, J = 7.7/7.9, Ar-H),
6.98 (1H, s, Ar-H), 6.87 (1H, d, J = 7.5, Ar-H), 6.42-6.49 (2H, m, Ar-H), 5.39 (2H, s, CH2),
4.64 (2H, s, CH2), 4.09 (2H, s, CH2), 2.34 (6H, s, 2CH3), 2.27 (3H, s, CH3).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-p-tolyl-acetamide (33). Procedure as for 1a except using 16 (184 mg, 1
mmol). The mixture was extracted with EtOAc. The combined extracts were washed with
H2O and dried over Na2SO4, filtered, and concentrated under vacuum followed by
recrystallization from MeOH. Pure 33 was obtained as a white crystalline solid (195 mg,
40%). m/z (ES), found 479.1350 (C23H23N6O2S2 [M - H]-) requires 479.1402; δH/ppm (400
MHz, d6-DMSO): 10.19 (1H, s, NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 7.41 (2H, d, J = 8.5,

Ar-H), 7.09 (2H, d, J = 8.3, Ar-H), 6.98 (1H, s, Ar-H), 6.41-6.49 (2H, m, Ar-H), 5.39 (2H, s,
CH2), 4.64 (2H, s, CH2), 4.08 (2H, s, CH2), 2.34 (6H, s, 2CH3), 2.24 (3H, s, CH3).
N-(2,4-Dimethyl-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-
ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (34). Procedure as for 1a except using
6 (200 mg, 0.6 mmol), 18 (118 mg, 0.6 mmol) and the reaction was carried out for 48 h. The
mixture was extracted with EtOAc. The combined extracts were washed with H2O and dried
over Na2SO4, filtered, and concentrated under vacuum. Purification of the crude product by
FCC (EtOAc, Rf = 0.23), gave 34 as a pale white powder (147 mg, 50%). m/z (ES), found
493.1446 (C24H25N6O2S2 [M - H]-) requires 493.1559; δH/ppm (400 MHz, d6-DMSO): 9.55
(1H, s, NH), 7.65-7.62 (1H, m, Ar-H), 7.23 (1H, d, J = 8.0, Ar-H), 7.01-6.90 (3H, m, Ar-H),
6.48-6.42 (2H, m, Ar-H), 5.39 (2H, s, CH2), 4.65 (2H, s, CH2), 4.09 (2H, s, CH2), 2.33 (6H, s,
2CH3), 2.23 (3H, s, CH3), 2.06 (3H, s, CH3).
N-(3,4-Dimethyl-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-
ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (35). A solution of 6 (1.0 g, 3 mmol) in
DMF (8 mL) and a solution of 13 (593 mg, 3 mmol) in DMF (4 mL) were combined in a dry
Radleys reaction tube containing K2CO3 (442 mg, 3.2 mmol). The mixture was allowed to
stir at 40 °C overnight in a Radleys parallel synthesizer. The mixture was poured on crushed
ice (50 mL) and allowed to stand for few h. The brown precipitate formed was collected by
filtration, washed with H2O and lyophilized, followed by recrystallization from EtOH–MeOH
(1:1). Pure 35 was obtained as a pale yellow crystalline solid (952 mg, 64%). m/z (ES), found
495.1811 (C24H27N6O2S2 [M + H]+) requires 495.1559; δH/ppm (400 MHz, d6-DMSO): 10.14
(1H, s, NH), 7.64 (1H, dd, J = 0.8 + 1.8, Ar-H), 7.31 (1H, d, J = 1.8, Ar-H), 7.24 (1H, dd, J =
2.1 + 8.1, Ar-H), 7.03 (1H, d, J = 8.2, Ar-H), 6.98 (1H, s, Ar-H), 6.50-6.41 (2H, m, Ar-H),
5.39 (2H, s, CH2), 4.64 (2H, s, CH2), 4.07 (2H, s, CH2), 2.33 (6H, s, 2CH3), 2.17 (3H, s,
CH3), 2.15 (3H, s, CH3).

2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-(4-isopropyl-phenyl)-acetamide (36). Procedure as for 1a except using 19
(211 mg, 1 mmol). The mixture was extracted with EtOAc. The combined extracts were
washed with H2O and dried over Na2SO4, filtered, and concentrated under vacuum.
Purification of the crude product by FCC (EtOAc, Rf = 0.21), gave 36 as a pale white powder
(300 mg, 59%). m/z (ES), found 509.7175 (C25H29N6O2S2 [M + H]+) requires 509.1715;
δH/ppm (400 MHz, d6-DMSO): 10.20 (1H, s, NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 7.43
(2H, d, J = 8.6, Ar-H), 7.16 (2H, d, J = 8.5, Ar-H), 6.98 (1H, s, Ar-H), 6.48-6.42 (2H, m, Ar-
H), 5.39 (2H, s, CH2), 4.64 (2H, s, CH2), 4.08 (2H, s, CH2), 2.83 (1H, hept, CH of isopropyl),
2.34 (6H, s, 2CH3), 1.17 [6H, d, J = 6.9, (CH3)2].
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-ylmethyl-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-(4-trifluoromethyl-phenyl)-acetamide (37). Procedure as for 1a except
using 20 (238 mg, 1 mmol). The precipitate was collected by filtration, washed with H2O and
lyophilized, followed by recrystallization from MeOH. Pure 37 was obtained as a white
powder (314 mg, 59%). m/z (ES), found 535.6185 (C23H22F3N6O2S2 [M + H]+) requires
535.1119; δH/ppm (400 MHz, d6-DMSO): 10.65 (1H, s, NH), 7.74 (2H, d, J = 8.6, Ar-H),
7.67 (2H, d, J = 8.7, Ar-H), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 6.98 (1H, s, Ar-H), 6.48-6.42
(2H, m, Ar-H), 5.39 (2H, s, CH2), 4.64 (2H, s, CH2), 4.15 (2H, s, CH2), 2.33 (6H, s, 2CH3).
N-(3,5-Bis-trifluoromethyl-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-
furan-2-ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (38). Procedure as for 1a
except using 21 (306 mg, 1 mmol) and the reaction was carried out for 48 h. The precipitate
was collected by filtration, washed with H2O and lyophilized, followed by recrystallization
from EtOH. Pure 38 was obtained as a white powder (264 mg, 44%). m/z (ES), found
603.9979 (C24H21F6N6O2S2 [M + H]+) requires 603.0993; δH/ppm (400 MHz, d6-DMSO):
10.95 (1H, s, NH), 8.20 (2H, s, Ar-H), 7.78 (1H, s, Ar-H), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H),

6.96 (1H, s, Ar-H), 6.48-6.42 (2H, m, Ar-H), 5.39 (2H, s, CH2), 4.63 (2H, s, CH2), 4.16 (2H,
s, CH2), 2.33 (6H, s, 2CH3).
N-(4-Chloro-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-
ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (39). Procedure as for 1a except using
23 (204 mg, 1 mmol) and the reaction was carried out for 24 h. The precipitate was collected
by filtration, washed with H2O and lyophilized, followed by recrystallization from EtOH–
MeOH (1:1). Pure 39 was obtained as a white powder (227 mg, 46%). m/z (ES), found
500.6534 (C22H22ClN6O2S2 [M + H]+) requires 501.0856; δH/ppm (400 MHz, d6-DMSO):
10.42 (1H, s, NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 7.56 (2H, d, J = 8.9, Ar-H), 7.35 (2H,
d, J = 8.9, Ar-H), 6.98 (1H, s, Ar-H), 6.49-6.42 (2H, m, Ar-H), 5.39 (2H, s, CH2), 4.64 (2H, s,
CH2), 4.10 (2H, s, CH2), 2.33 (6H, s, 2CH3).
N-(4-Bromo-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-
ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (40). Procedure as for 1a except using
6 (200 mg, 0.6 mmol), 24 (149 mg, 0.6 mmol) and the reaction was carried out for 36 h. The
precipitate was collected by filtration, washed with H2O and lyophilized, followed by
recrystallization from MeOH. Pure 40 was obtained as a white powder (185 mg, 57%). m/z
(ES), found 544.9952 (C22H22BrN6O2S2 [M + H]+) requires 545.0351; δH/ppm (400 MHz, d6-
DMSO): 10.41 (1H, s, NH), 7.63 (1H, dd, J = 0.8 + 1.8, Ar-H), 7.54-7.45 (4H, m, Ar-H), 6.98
(1H, s, Ar-H), 6.48-6.42 (2H, m, Ar-H), 5.39 (2H, s, CH2), 4.63 (2H, s, CH2), 4.10 (2H, s,
CH2), 2.34 (6H, s, 2CH3).
2-[4-Allyl-5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-
N-(3,4-dimethyl-phenyl)-acetamide (41). A solution of 7 (293 mg, 1 mmol) in DMF (3
mL) and a solution of 13 (198 mg, 1 mmol) in DMF (2 mL) were combined in a dry Radleys
reaction tube containing K2CO3 (166 mg, 1.2 mmol). The mixture was allowed to stir at 40

°C overnight in a Radleys parallel synthesizer. The mixture was poured on crushed ice (50
mL) and allowed to stand for few h. The brown precipitate formed was collected by filtration,
washed with H2O and lyophilized, followed by recrystallization from EtOH. Pure 41 was
obtained as pale white crystals (96 mg, 21%). m/z (ES), found 453.1533 (C22H25N6OS2 [M -
H]-) requires 453.1610; δH/ppm (400 MHz, d6-DMSO): 10.10 (1H, s, NH), 7.30 (1H, d, J =
1.8, Ar-H), 7.24 (1H, dd, J = 2.1 + 8.1, Ar-H), 7.04 (1H, d, J = 8.2, Ar-H), 7.00 (1H, s, Ar-H),
6.04-5.83 (1H, m, CH of allyl), 5.20 [1H, dd, J = 1.0 + 10.4, N-CH2 (Ha)], 4.91 [1H, dd, J =
1.0 + 17.2, N-CH2 (Hb)], 4.73 (2H, d, J = 5.0, =CH2), 4.55 (2H, s, CH2), 4.05 (2H, s, CH2),
2.35 (6H, s, 2CH3), 2.17 (3H, s, CH3), 2.15 (3H, s, CH3).
N-(3,4-Dimethyl-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-(3-
methoxy-propyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (42). A solution of 8 (325 mg,
1 mmol) in DMF (3 mL) and a solution of 13 (198 mg, 1 mmol) in DMF (2 mL) were
combined in a dry Radleys reaction tube containing K2CO3 (166 mg, 1.2 mmol). The mixture
was allowed to stir at 40 °C overnight in a Radleys parallel synthesizer. The mixture was
poured on crushed ice (50 mL) and allowed to stand for few h. The precipitate formed was
collected by filtration, washed with H2O and lyophilized, followed by recrystallization from
EtOH. Pure 42 was obtained as a crystalline white solid (317 mg, 65%). m/z (ES), found
487.1689 (C23H31N6O2S2 [M + H]+) requires 487.1872; δH/ppm (400 MHz, d6-DMSO): 10.11
(1H, s, NH), 7.30 (1H, s, Ar-H), 7.24 (1H, d, J = 8.1, Ar-H), 7.04 (1H, d, J = 8.0, Ar-H), 7.00
(1H, s, Ar-H), 4.58 (2H, s, CH2), 4.10 (2H, t, J = 7.2, CH2OCH3), 4.08 (2H, s, CH2), 3.29
(2H, t, J = 5.8, N-CH2), 3.21 (3H, d, J = 1.7, OCH3), 2.35 (6H, s, 2CH3), 2.17 (3H, s, CH3),
2.15 (3H, s, CH3), 1.98-1.86 (2H, m, CH2).
N-(3,4-Dimethyl-phenyl)-2-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-(4-
methoxy-phenyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-acetamide (43). A solution of 9 (360 mg,
1 mmol, 1 eq.) in DMF (4 mL) and a solution of 13 (198 mg, 1 mmol) in DMF (2 mL) were

combined in a dry Radleys reaction tube containing K2CO3 (166 mg, 1.2 mmo). The mixture
was allowed to stir at 40 °C for 36 h in a Radleys parallel synthesizer. The mixture was
poured on crushed ice (25 mL) and allowed to stand for few h. The precipitate formed was
collected by filtration, washed with H2O and lyophilized. Pure 43 was obtained as a pale
yellow powder (469 mg, 90%). m/z (ES), found 519.1627 (C26H27N6O2S2 [M - H]-) requires
519.1715; δH/ppm (400 MHz, d6-DMSO): 10.13 (1H, s, NH), 7.35 (2H, d, J = 8.9, Ar-H),
7.32-7.23 (2H, m, Ar-H), 7.04 (1H, d, J = 8.2, Ar-H), 6.99 (2H, d, J = 8.9, Ar-H), 6.91 (1H, s,
Ar-H), 4.46 (2H, s, CH2), 4.08 (2H, s, CH2), 3.78 (3H, s, OCH3), 2.27 (6H, s, 2CH3), 2.17
(3H, s, CH3), 2.16 (3H, s, CH3).
2-[4-(4-Chloro-phenyl)-5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4H-[1,2,4]triazol-
3-ylsulfanyl]-N-(3,4-dimethyl-phenyl)-acetamide (44). A solution of 10 (364 mg, 1 mmol)
in DMF (3 mL) and a solution of 13 (198 mg, 1 mmol) in DMF (2 mL) were combined in a
dry Radleys reaction tube containing K2CO3 (166 mg, 1.2 mmol). The mixture was allowed
to stir at 40 °C overnight in a Radleys parallel synthesizer. The mixture was poured on
crushed ice (50 mL) and allowed to stand for few h. The brown precipitate formed was
collected by filtration, washed with H2O and lyophilized. Without any further purification, 44
was obtained as a white powder (481 mg, 92%). m/z (ES), found 523.1134 (C25H24ClN6OS2
[M - H]-) requires 523.1220; δH/ppm (400 MHz, d6-DMSO): 10.11 (1H, s, NH), 7.55-7.46
(4H, m Ar-H), 7.33-7.22 (2H, m, Ar-H), 7.04 (1H, d, J = 8.14, Ar-H), 6.92 (1H, s, Ar-H),
4.51 (2H, s, CH2), 4.08 (2H, s, CH2), 2.27 (6H, s, 2CH3), 2.18 (3H, s, CH3), 2.16 (3H, s,
CH3).
N-(3,4-Dimethyl-phenyl)-3-[5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-furan-2-
ylmethyl-4H-[1,2,4]triazol-3-ylsulfanyl]-propionamide (45). Procedure as for 1a except
using 26 (212 mg, 1 mmol) and the reaction was carried out for 36 h. The mixture was
extracted with EtOAc. The combined extracts were washed with H2O and dried over Na2SO4,

filtered, and concentrated under vacuum. Purification of the crude product by FCC (EtOAc,
Rf = 0.12), gave 45 as a pale white powder (127 mg, 25%). m/z (ES), found 509.1672
(C25H29N6O2S2 [M + H]+) requires 509.1715; δH/ppm (400 MHz, d6-DMSO): 9.83 (1H, s,
NH), 7.60 (1H, dd, J = 0.9 + 1.7, Ar-H), 7.35 (1H, d, J = 1.8, Ar-H), 7.28 (1H, dd, J = 2.1 +
8.1, Ar-H), 7.03 (1H, d, J = 8.2, Ar-H), 7.00 (1H, s, Ar-H), 6.43-6.37 (2H, m, Ar-H), 5.30
(2H, s, CH2), 4.64 (2H, s, CH2), 3.34 (2H, t, J = 6.8, CH2), 2.76 (2H, t, J = 6.7/6.8, CH2), 2.34
(6H, s, 2CH3), 2.17 (3H, s, CH3), 2.15 (3H, s, CH3).
2-[4-Allyl-5-(4,6-dimethyl-pyrimidin-2-ylsulfanylmethyl)-4H-[1,2,4]triazol-3-ylsulfanyl]-
N-(4-isopropyl-phenyl)-acetamide (46). Procedure as for 1a except using 19 (212 mg, 1
mmol) and the reaction was carried out for 22 h. The mixture was extracted with EtOAc. The
combined extracts were washed H2O and dried over Na2SO4, filtered, and concentrated under
vacuum. Purification of the crude product by FCC (EtOAc, Rf = 0.16), gave 46 as a pale
white powder (228 mg, 49%). m/z (ES), found 469.2179 (C23H29N6OS2 [M + H]+) requires
469.1766; δH/ppm (400 MHz, d6-DMSO): 10.19 (1H, s, NH), 7.43 (2H, d, J = 8.5, Ar-H),
7.16 (2H, d, J = 8.5, Ar-H), 7.00 (1H, s, Ar-H), 5.99-5.84 (1H, m, CH of allyl), 5.20 [1H, dd,
J = 1.0 + 10.4, N-CH2 (Ha)], 4.91 [1H, dd, J = 0.9 + 17.2, N-CH2 (Hb)], 4.74 (2H, d, J = 4.9,
=CH2), 4.55 (2H, s, CH2), 4.07 (2H, s, CH2), 2.83 (1H, hept, CH of isopropyl), 2.35 (6H, s,
2CH3), 1.17 (6H, d, J = 6.9, (CH3)2 of isopropyl).
2-[5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-4-(3-methoxy-propyl)-4H-
[1,2,4]triazol-3-ylsulfanyl]-N-(4-isopropyl-phenyl)-acetamide (47). Procedure as for 1a
except using 19 (212 mg, 1 mmol) and the reaction was carried out for 42 h. The precipitate
formed was collected, washed with H2O and lyophilized, followed by recrystallization from
EtOH. Pure 47 was obtained as a white powder (382 mg, 76%). m/z (ES), found 501.7603
(C24H33N6O2S2 [M + H]+) requires 501.2028; δH/ppm (400 MHz, d6-DMSO): 10.21 (1H, s,
NH), 7.43 (2H, d, J = 8.6, Ar-H), 7.16 (2H, d, J = 8.5, Ar-H), 7.00 (1H, s, Ar-H), 4.58 (2H, s,