Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine compounds targeting cysteine cathepsins K and S

Article abstract of DOI:10.1016/j.ejmech.2016.05.009

The synthesis and evaluation against various cysteine cathepsins with endopeptidase activity, of two new families of hitherto unknown 1,3,5-triazines, substituted by a nitrile function and either a cyclohexylamine moiety (5-like) or a piperazine moiety (9-like) are described. The structure-activity relationship was discussed; from 16 synthesized novel compounds, 9h was the most active and selectively inhibitor of Cat K (IC₅₀ Combining double low line 28 nM) and Cat S (IC₅₀ Combining double low line 23 nM). Molecular docking of 9h to X-ray crystal structure of cathepsins K and S confirmed a common binding mode with a crucial covalent bond with Cys25. We observed for 9h that p-trifluorophenyl group is located in S2 pocket and possess hydrophobic interactions with Tyr67 and Met68. Triazine and piperazine moieties are located in S′1 pocket and interact with Gly23, Cys63, Gly64 and Gly65. Altogether, these results indicate that the new analogs can make them effective agents against some viruses for which the glycoprotein cleavage is mediated by an array of proteases.

Full text of DOI:10.1016/j.ejmech.2016.05.009

Accepted Manuscript
Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine compounds targeting
cysteine cathepsins K and S
Elżbieta Plebanek, Florian Chevrier, Vincent Roy, Thibaut Garenne, Fabien Lecaille,
Dawid Warszycki, Andrzej J. Bojarski, Gilles Lalmanach, Luigi A. Agrofoglio
PII:
S0223-5234(16)30386-5
10.1016/j.ejmech.2016.05.009
EJMECH 8598
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 February 2016
Revised Date: 4 May 2016
Accepted Date: 5 May 2016
Please cite this article as: E. Plebanek, F. Chevrier, V. Roy, T. Garenne, F. Lecaille, D. Warszycki, A.J.
Bojarski, G. Lalmanach, L.A. Agrofoglio, Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine
compounds targeting cysteine cathepsins K and S, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.05.009.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine compounds
targeting cysteine cathepsins K and S
a
a
a
b
b
Elżbieta Plebanek, Florian Chevrier, Vincent Roy, Thibaut Garenne, Fabien Lecaille,
c
c
b
a,
Dawid Warszycki, Andrzej J. Bojarski, Gilles Lalmanach, Luigi A. Agrofoglio *
a
Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France
b
INSERM, UMR 1100, Pathologies Respiratoires: protéolyse et aérosolthérapie, Centre d’Etude des
Pathologies Respiratoires, Université François Rabelais, F-37032 Tours cedex, France
c
Medicinal Chemistry Department, Institute of Pharmacology, Polish Academy of Sciences, Kraków,
Poland
Keywords: Cysteine cathepsin; cathepsin inhibitors; 1,3,5-triazine, microwave irradiation
Abstract:
The synthesis and evaluation against various cysteine cathepsins with endopeptidase
activity, of two new families of hitherto unknown 1,3,5-triazines, substituted by a nitrile
function and either a cyclohexylamine moiety (5-like) or a piperazine moiety (9-like) are
described. The structure-activity relationship was discussed; from 16 synthesized novel
compounds, 9h was the most active and selectively inhibitor of Cat K (IC = 28 nM) and Cat
50
S (IC = 23 nM). Molecular docking of 9h to X-ray crystal structure of cathepsins K and S
50
confirmed a common binding mode with a crucial covalent bond with Cys25. We observed
for 9h that p-trifluorophenyl group is located in S2 pocket and possess hydrophobic
interactions with Tyr67 and Met68. Triazine and piperazine moieties are located in S’1 pocket
and interact with Gly23, Cys63, Gly64 and Gly65. Altogether, these results indicate that the
new analogs can make them effective agents against some viruses for which the
glycoprotein cleavage is mediated by an array of proteases.
1. Introduction
Human cathepsins K and S are lysosomal cysteine proteases that belong to the
papain family (C1) of cysteine proteases which includes eleven members for the human
1
genome. They have long been thought to be primarily involved in end-stage
degradation/recycling of endocytosed proteins within acidic lysosomal/endosomal
2
3
compartments These proteases share similar three-dimensional structures, catalytic
4,5
6,7
mechanism, and substrate specificity. They are involved in specific cellular processes
(
e.g., histone proteolysis, prohormone processing, cell-adhesion molecule cleavage and8
shedding, etc...) and in a broad spectrum of disorders (e.g., cancer progression,
inflammatory responses), making them putative targets for the development of new
9
10,11
therapies.
MHC) class II mediated immune responses and was identified as an attractive target for
drugs in autoimmune diseases (e.g. rheumatoid arthritis, multiple sclerosis allergy, psoriasis,
Especially, cathepsin (Cat) S is involved in major histocompatibility complex
(
12
diabetes and lupus erythematosus) as well neuropathic pain. Cat K that is predominantly
expressed in osteoclasts is a critical bone resorbing protease and was identified as a
13
14
clinically relevant target for the treatment of osteoporosis and bone metastasis. Since
004, over 50 patents have been filed on inhibitors of Cat K. Despite successful studies,
2
15
pharmacological inhibitors may have deleterious side effects, especially on the lungs Also,
several lead compounds directed against Cat S were active in disease models of rheumatoid

ACCEPTED MANUSCRIPT
arthritis and allergic asthma, providing a proof of principle for further development, and may
16
offer benefits during bronchopneumodysplasia (BPD) and emphysema.
From the most promising synthetic Cat K and Cat S inhibitors, the nitrile-based
heterocycles have been identified as potent and selective inhibitors and have received a lot
17,18
of attention.
The inhibition of these cysteine proteases by nitriles is achieved by covalent
but reversible formation of thioimidate intermediate originating from interaction between
cysteine-thiol moiety and carbon-nitrogen triple bond, which is particularly electrophilic due to
1
9,20
the strong electro-withdrawing nature of this class of aromatic heterocyclic rings.
In 2011,
21
Rankovic et al., looking for the treatment of osteoporosis and atherosclerosis investigated
the Cat K inhibitors, focusing on the modifications of 1,3,5-triazine scaffold; among them, the
2
-cyano-1,3,5-triazine-4,6-diamine analog (1) exhibited high potency against Cat K (IC = 1
50
nM) and moderate selectivity over Cat S (IC = 158 nM), L (IC = 1711 nM), and B (IC =
50
50
50
5
20 nM). The 2-cyano-1,3,5-triazine scaffold offers, from the druglikeness point of view,
22
many opportunities for synthetic optimization. Thus, we report herein the synthesis and
biological evaluation against various cysteine cathepsins with endopeptidase activity, of two
new families of hitherto unknown 1,3,5-triazines, substituted by a nitrile function and either a
cyclohexylamine moiety (5-like) or a piperazine moiety (9-like), (Figure 1).
[
Figure 1]
2
. Results and discussion
.1 Chemistry
The target compounds 5a-h (cyclohexylamine analogs) and 9a-h (piperazine
analogs) were obtained in a three and four steps, respectively, starting from cyanuric chloride
, (Scheme 1). Nucleophilic substitution of chlorine atoms in cyanuric chloride 2 in presence
2
2
of diisopropyl ethyl amine (DIPEA) and cyclohexylamine (for compounds 5-like) or N-Boc-
monoprotected piperazine (for compounds 9-like) in dichloromethane, provided the mono-
substituted compounds 3 and 6, respectively, in quantitative yield. The second nucleophilic
substitution was carried out with appropriated amines a-h in presence of DIPEA under
microwave activation between 60 to 90 minutes giving 4a-h and 7a-h, respectively, in
moderate to good yield. Treatment of triazine analogues 4a-h and 7a-h with potassium
cyanide (KCN) (1.1 eq.) in a presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) (0.2 equiv.),
in dimethylsulfoxide gave at room temperature the expected nitrile products 5a-h and 8a-h,
23
respectively. In the last step, the triazine derivatives 8a-h were deprotected in the presence
of trifluoro acetic acid to give the desired piperazine series 9a-h .
[
Scheme 1]
2
.2 Cathepsin inhibitory activities
Compounds 5a-h and 9a-h were evaluated for their inhibitory activities towards Cat B,
K, L and S (Table 1).
[
Table 1.]
Both series 5-like and 9-like were substituted by an amino bearing an aromatic moiety
a-h) displaying different lipophilicity, electron withdrawing effect and steric hindrance, in
(
order to establish the structure-activity relationship. Among these two triazine series,

ACCEPTED MANUSCRIPT
cyclohexylamine analogs 5a-h showed an improved activity for both Cat S and Cat K while
these compounds did not inhibit Cat B and Cat L. The –CF analog (5h), which is both more
3
electron withdrawing and lipophilic than its methoxy counterpart –OMe (5g), are more active
toward Cat K with an IC values of 34 nM and 23 nM respectively compared to Cat S (>100
50
nM). An exception was observed with the compounds 5f having a nitro electron withdrawing
group at C3 position of aromatic ring, which exhibits a high activity for all studied enzymes
(
IC50 < 80 nM). The potency enhancing effect of nitro group seems to be isomeric position
dependent for Cat B inhibition, compared with the isomer 5e, which is devoid of activity on
this enzyme. Conversely piperazine analogs 9a-h afforded a broader spectrum and inhibited
Cat B, K, L and S usually with IC50 values below 100 nM. Compared with cyclohexylamine
series, piperazine moiety is better tolerated by Cat B and Cat L. From these results it
appears that the position of nitro group on the aromatic ring is still significant for cat B
inhibition when we compare the activity of compounds 9e (no inhibition) and 9f (IC = 34nM)
50
and only inhibitor bearing hydrophobic CF group 9f maintained selective inhibitory effect on
3
both Cat K and S with an IC ˂ 30 nM. Surprisingly, 9a (with an increased distance between
50
aromatic group and triazine ring) exhibited no activity on Cat B, K, L and S, meanwhile 9c
with the same side chain substituted by hydrophilic group) was active. This could be due to
suitable H-bonding between 9c and the enzyme catalytic site, which cannot be formed with
(
9
a. Finally, 9h exhibited a selective inhibition of Cat K (IC = 28 nM) and Cat S (IC = 23
50 50
nM), respectively.
2.3 Docking studies
To define and elucidate the binding modes of 9h, molecular docking of this compound
in the active site of Cat K and S was performed. As the protein structure for docking, crystal
complexes of Cat K and S with potent inhibitor were selected from the PDB (PDB IDs: 1VSN,
resolution 2.00 Å and 3OVX, resolution 1.49 Å) and prepared in Protein Preparation
25
Wizard, under default settings. Three dimensional structure, conformation and protonation
27,28
states of 9h were generated by LigPrep26 (at pH 7.4). Finally, Glide
was used for
covalent docking (Cys25 was the reactive residue) of each conformer to enzyme model.
Each pose was ranked according to affinity score, and the highest scored pose was chosen
for further analysis.
[
[
Figure 2.]
Figure 3.]
29,30
In general, binding modes of docked compound are similar to already published.
Evaluation of binding poses of 9h (Figure 2) showed that p-trifluorophenyl group is located in
S2 pocket and possesses hydrophobic interactions with Tyr67 and Met68. Triazine and
piperazine moieties are located in S’1 pocket and interact with Gly23, Cys63, Gly64 and
Gly65. Compound 9h formed strong hydrogen bond with backbone NH of Gln19 (hydrogen
bond distance: 1.86 Å). In the binding site of Cat S (Figure 3) 9h formed three weak
hydrogen bonds: with backbones NH of Gln19 (hydrogen bond distance: 2.61 Å), Cys25
(
hydrogen bond distance: 2.44 Å) as well as Gly165 (hydrogen bond distance: 2.99 Å). p-
Trifluorophenyl group interacts weaker with S2 pocket however possess face-to-edge
stacking interaction with Phe70. Binding mode is characterized by limited number of
hydrophobic contacts with subpockets S1 and S’1.
3. Conclusion

ACCEPTED MANUSCRIPT
We synthesized a focused library of new 2-cyano-1,3,5-triazine-4,6-diamine analogs
with the aim of exploring the structure-activity relationships for some cathepsin activity
inhibition. The most promising inhibitors were found in the piperazine series; especially, 9h
exhibited high activity and selectivity for cathepsins K and S (IC50 < 30 nM), meanwhile
compounds 9f and 9g were highly active for all four investigated cathepsins (B, K, S and L)
with an IC ≤ 45nM. In the later case, this lack of selectivity and high activity can make them
50
effective agents against some viruses for which the glycoprotein cleavage is mediated by an
array of proteases.
4. Experimental section
4.1 Chemistry
General. Commercially available chemicals were of reagent grade and used as received.
The reactions were monitored by thin layer chromatography (TLC) analysis using silica gel
plates (Kieselgel 60F254, E. Merck). Column chromatography was performed on Silica Gel
1
13
6
0 M (0.040-0.063 mm, E. Merck). The H and C NMR spectra were recorded on a Varian
InovaUnity 400 spectrometer (400 MHz) using deuterated solvents as internal standard.
Chemical shifts are given in ppm and multiplicities are reported as s (singlet), d (doublet), t
(
triplet), q (quartet), bs (broad signal) and m (multiplet). High Resolution Mass spectra were
performed on a Bruker maxis mass spectrometer by the “Fédération de Recherche”
ICOA/CBM (FR2708) platform. All reactions under microwave irradiation were performed
using the Microwave Biotage Initiator in 2-5mL.sealed tubes.
4.1.1.
4,6-Dichloro-N-cyclohexyl-1,3,5-triazin-2-amine (3). The solution of 3.00g (16.27
mmol, 1equiv) of cyanuric chloride in 60 mL of CH Cl was cooled at 0 °C then the mixture of
2
2
1.86 mL (16.27 mmol, 1 equiv.) of cyclohexylamine and 3.12 mL (17.90 mmol, 1.1 equiv.) of
DIPEA in 10 mL of CH Cl was slowly added. The reaction mixture was stirred at rt for 2
2
2
hours then washed with 1M HCl and water. Collected organic phase was dried over MgSO4
and concentrated. The crude was purified by column chromatography (CH Cl ) to obtain
2
2
1
4.02g (quantitative) of the desired product as white amorphous solid. H NMR (400 MHz,
CDCl ) δ 5.70 (bs, 1H, NH), 3.99 – 3.83 (m, 1H, CH), 2.06 – 1.89 (m, 2H), 1.82 – 1.71 (m,
3
13
2
H), 1.68 – 1.61 (m, 1H), 1.50 – 1.35 (m, 2H), 1.32 – 1.15 (m, 3H); C NMR (101 MHz,
CDCl ) δ 171.12, 169.98, 165.12, 50.36 (CH), 32.60 (CH ), 25.38 (CH ), 24.54 (CH ); R:
3
2
2
2
f
+
0
.80 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H] calcd for C H Cl N 247.0512 found
9 13 2 4
247.0517.
4
2
2
1
.1.2. tert-Butyl-4-(4,6-dichloro-1,3,5-triazin-2-yl)piperazine-1-carboxylate (6). Solution of
.07g (11.2 mmol, 1 equiv.) of 17 in 32 mL of CH Cl was cooled at 0 °C then the mixture of
2
2
.09g (11.2 mmol, 1 equiv.) of tert-butyl piperazine-1-carboxylate and 1.93 mL (11.3 mmol,
.1 equiv.) of DIPEA in 10 mL of CH Cl was slowly added. The reaction mixture was stirred
2
2
at 0 °C for 30 min then washed with 1M HCl. Collecte d organic phase was dried over MgSO4
and concentrated to give 3.75g (quantitative) of 6 as white solid. CAS: 271592-49-5.
4.2.3. General procedure for compounds 4 a-h and 7 a-h
The microwave vial was charged with compound 3 or 6 (1 or 2 equiv.), appropriate amine (1
equiv.), DIPEA (1.1 equiv.) and ACN as solvent. The resulted mixture was heated under
microwave irradiation and then concentrated. The residue was dissolved in CH Cl , washed
2
2
with 2M HCl, water, then dried over MgSO and concentrated under vacuum. Purification by
4
column chromatography on silica gel gave desired product.

ACCEPTED MANUSCRIPT
4.2.1. 6-Chloro-N2-cyclohexyl-N4-phenethyl-1,3,5-triazine-2,4-diamine (4a). 3 (2 equiv.) were
used in reaction. With 1 equiv. of 3 we observed mosltly bisubstituted product. MW activation
1
time 60 min, 100 °C . Yield 96%; white solid; mp 174° C; H NMR (400 MHz, CDCl ) δ 7.38 –
3
7
.27 (m, 2H), 7.25 – 7.15 (m, 3H), 5.96 (bs, 0.54H, NH-CH ), 5.58 (bs, 0.2H, NH-CH ), 5.39 –
2
2
4
.92 (m,1.26H; 0.26H, NH-CH ; 1H NH-CH), 3.95 – 3.77 (m, 1H, CH), 3.73 – 3.57 (m, 2H,
2
NH-CH ), 2.95 – 2.80 (m, 2H, CH ), 2.07 – 1.87 (m, 2H), 1.80 – 1.57 (m, 3H), 1.45 – 1.18 (m,
2
2
13
5
H); C NMR (101 MHz, CDCl ) δ 168.50, 165.95, 165.03, 138.87, 128.89 (CH), 128.79
3
(
3
CH), 126.69 (CH), 49.87 (CH), 42.34 (d, J = 50.5 Hz, NH-CH ), 35.87 (Ar-CH ), 33.37 (CH ),
2 2 2
+
2.77 (CH ), 25.69 (CH ), 24.81 (CH ); R : 0.63 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]
2 2 2 f
calcd for C H ClN 332.1637 found 332.1635.
17
23
5
4.2.2. 6-Chloro-N2-cyclohexyl-N4-(m-tolylmethyl)-1,3,5-triazine-2,4-diamine (4b). 3 (2 equiv.)
were used in reaction. MW activation time 60 min, 100 °C . Yield quantitative; creamy solid;
1
mp 154 °C ; H NMR (400 MHz, CDCl ) δ 7.21 (t, J = 7.5 Hz, 1H), 7.16 – 7.02 (m, 3H), 6.42
3
(
bs, 0.55H, NH-CH ), 5.97 (bs, 0.20H, NH-CH ), 5.46 (bs, 0.25H, NH-CH ), 5.39 – 5.01 (m,
2 2 2
1
–
3
1
3
H, NH-CH ), 4.63 – 4.47 (m, 2H, NH-CH ), 3.95 – 3.67 (m, 1H, CH), 2.33 (s, 3H, CH ), 2.04
2
3
1.81 (m, 2H), 1.78 – 1.67 (m, 2H), 1.65 – 1.55 (m, 1H), 1.44 – 1.29 (m, 2H), 1.26 – 1.14 (m,
13
H); C NMR (101 MHz, CDCl ) δ 168.46, 166.00, 164.94, 138.56, 138.32, 128.53 (CH),
3
28.30 (CH), 128.17 (CH), 124.68 (CH), 49.86 (CH), 45.02 (NH-CH ), 33.31 (), 32.82 (CH ),
2
2
2.66 (CH ), 25.64 (CH ), 24.84 (CH ), 21.52 (CH ); R : 0.87 (PE/EtOAc 5:5); HRMS (ESI)
2
+
2
2
3
f
m/z [M+H] calcd for C H ClN 332.1637 found 332.1633.
1
7
23
5
4
.2.3. (1R)-2-[[4-Chloro-6-(cyclohexylamino)-1,3,5-triazin-2-yl]amino]-1-phenyl-ethanol (4c).
1
MW activation time 90 min, 150 °C . Yield 61%; white solid; mp 88 °C ; H NMR (400 MHz,
CDCl ) δ 7.49 – 7.31 (m, 5H), 6.77 (bs, 0.61H, NH-CH ), 6.48 (bs, 0.19H, NH-CH ), 5.97 –
3
2
2
5
3
4
1
5
.31 (m, 1.2H; 0.2H, NH-CH ; 1H NH-CH), 5.04 – 4.81 (m, 1H, CH-OH), 4.20 (bs, 1H, OH),
2
.94 – 3.75 (m, 1H, CH), 3.59 – 3.39 (m, 2H, CH ), 2.11 – 1.89 (m, 2H, CH ), 1.82 – 1.58 (m,
2
2
13
H, CH ), 1.44 – 1.16 (m, 4H, CH ); C NMR (101 MHz, CDCl ) δ 168.37, 166.31, 164.56,
2
2
3
42.02, 128.62 (CH), 127.89 (CH), 127.11 (CH), 125.97 (CH), 125.85 (CH), 73.74 (CH-OH),
0.04 (CH), 49.17 (HN-CH ), 33.22 (CH ), 32.71 (CH ), 32.65 (CH ), 25.57 (CH ), 24.77
2
2
2
2
2
+
(
CH ); R : 0.62 (PE/EtOAc 5:5); HRMS (ESI) m/z [M+H] calcd for C H ClN O 348.1586
2 f 17 24 5
found 348.1587.
4
.2.4
4-[[4-Chloro-6-(cyclohexylamino)-1,3,5-triazin-2-yl]amino]benzonitrile
(4d).
MW
1
activation time 120 min, 150 °C . Yield 64%; white so lid; mp 200 °C ; H NMR (400 MHz,
CDCl ) δ 7.98 – 7.56 (m, 4H, CH), 7.47 (d, J = 34.5 Hz, 1H, NH), 5.72 – 5.25 (m, 1H, HN-
3
CH), 4.04 – 3.70 (m, 1H, CH), 2.11 – 1.92 (m, 2H), 1.85 – 1.59 (m, 3H), 1.51 – 1.11 (m, 5H) ;
13
C NMR (101 MHz, CDCl ) δ 169.27, 165.20, 165.05, 164.09, 163.61, 142.23, 133.28 (CH),
3
1
20.02 (CH), 119.96 (CH), 119.00, 106.64, 50.29 (d, J = 69.4 Hz, CH), 33.15 (CH ), 32.57
2
+
(
CH ), 25.53 (CH ), 24.81 (CH ) ; R : 0.74 (PE/EtOAc 7:3) ; HRMS (ESI) m/z [M+H] calcd for
2
2
2
f
C H ClN 329.1276 found 329.1272.
16
18
6
4
.2.5.
6-Chloro-N2-cyclohexyl-N4-(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (4e). MW
1
activation time 120 min, 150 °C . Yield 54%; creamy s olid; mp 226 °C ; H NMR (400 MHz,
CDCl ) δ 8.32 – 8.14 (m, 2H, CH), 7.89 – 7.67 (m, 2H, CH), 5.86 – 5.39 (m, 1H, NH), 4.04 –
3
13
3
.72 (m, 1H, CH), 2.03 (bs, 2H), 1.82 – 1.58 (m, 4H), 1.50 – 1.16 (m, 5H) ; C NMR (101
MHz, CDCl ) δ 169.30, 165.06, 164.07, 144.16, 143.15, 125.16 (CH), 119.44 (CH), 119.37
3
(
(
CH), 119.28 (CH), 50.36 (d, J = 71.8 Hz ,CH), 33.13 (CH ), 32.57 (CH ), 25.53 (CH ), 24.83
2
2
2
+
CH ) ; Rf
:
0.68 (CH Cl /MeOH 98:2); HRMS (ESI) m/z [M+H]
calcd for
2
2
2
C H ClN O 349.1174 found 349.1173.
15
18
6
2

ACCEPTED MANUSCRIPT
4
.2.6.
6-Chloro-N2-cyclohexyl-N4-(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (4f). MW
1
activation time 120 min, 150 °C . Yield 71%; yellow s olid; mp 208 °C ; H NMR (250 MHz,
CDCl ) δ 9.04 (bs, 0.8H, NH), 8.62 (s, 0.2H, NH), 8.00 – 7.66 (m, 2H, CH), 7.64 – 7.36 (m,
3
2
H, CH), 5.76 – 5.40 (m, 1H, NH), 4.13 – 3.79 (m, 1H, CH), 2.14 – 1.90 (m, 2H), 1.79 – 1.59
13
(
m, 3H), 1.50 – 1.14 (m, 5H) ; C NMR (101 MHz, CDCl ) δ 169.06, 165.02, 164.16, 148.81,
3
1
39.38, 129.59 (CH), 125.38 (CH), 118.42 (CH), 115.14 (CH), 50.45 (CH), 33.16 (CH ),
2
3
2.84 (CH ), 25.57 (CH ), 24.87 (CH ), 24.65 (CH ); R : 0.46 (PE/EtOAc 8:2) ; HRMS (ESI)
2
2
2
2
f
+
m/z [M+H] calcd for C H ClN O 349.1174 found 349.1173.
15
18
6
2
4
.2.7.
6-Chloro-N2-cyclohexyl-N4-(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (4g). MW
1
activation time 90 min, 150 °C . Yield 76%; white sol id; mp 174 °C ; H NMR (400 MHz, CDCl )
3
δ 7.54 – 7.36 (m, 2H), 6.99 – 6.81 (m, 2H), 5.74 – 5.17 (m, 1H, NH), 3.99 – 3.76 (m, 4H,
13
CH , CH), 2.01 (bs, 2H), 1.84 – 1.60 (m, 3H), 1.49 – 1.09 (m, 5H); C NMR (101 MHz,
3
CDCl ) δ 165.34, 164.11, 156.73, 156.43, 130.95, 130.54, 123.43 (CH), 122.45 (CH), 114.24
3
(
(
CH), 114.11 (CH), 55.64 (CH ), 49.60 (CH), 33.23 (CH ), 32.61 (CH ), 25.62 (CH ), 24.82
3 2 2 2
+
CH ); R : 0.69 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H] calcd for C H ClN O 334.1429
2
f
16 21
5
found 334.1428.
4
.2.8. 6-Chloro-N2-cyclohexyl-N4-[4-(trifluoromethyl)phenyl]-1,3,5-triazine-2,4-diamine (4h).
1
MW activation time 90 min, 150 °C . Yield 80%; white solid; mp 186 °C ; H NMR (400 MHz,
CDCl ) δ 7.78 – 7.63 (m, 2H, CH), 7.58 (d, J = 8.3 Hz, 2H, CH), 7.52 – 7.38 (m, 1H, NH),
3
5
1
1
6
.71 – 5.32 (m, 1H, NH), 4.02 – 3.73 (m, 1H, CH), 2.14 – 1.87 (m, 2H), 1.83 – 1.60 (m, 3H),
13
.47 – 1.16 (m, 5H); C NMR (101 MHz, CDCl ) δ 169.18, 165.28, 165.10, 164.18, 163.77,
3
41.22, 126.29 (q, J = 3.7 Hz), 125.80, 125.61, 125.47, 122.92, 120.11, 119.81, 50.21 (d, J =
+
9.7 Hz), 33.19, 32.61, 25.57, 24.82; R : 0.58 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H] calcd
f
for C H ClF N 372.1197 found 372.1197.
16
18
3
5
4.2.9.
tert-Butyl-4-[4-chloro-6-(phenethylamino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
(
7a). 1.5 equiv. of 6 were used in reaction. MW activation time 60 min, 100 °C . Yield 92%;
1
1
white solid; mp 154 °C ; H NMR (400 MHz, CDCl ) δ H NMR (400 MHz, CDCl ) δ 7.29 (t, J =
3
3
7
.3 Hz, 2H, CH), 7.25 – 7.14 (m, 3H, CH), 5.84 (bs, 0.7H, NH), 5.16 (s, 0.3H, NH), 3.87 –
13
3.59 (m, 6H, CH ), 3.47 (bs, 4H, CH ), 2.87 (t, J = 7.1 Hz, 2H, CH ), 1.49 (s, 9H, CH );
C
2
2
2
3
NMR (101 MHz, CDCl ) δ 169.12, 165.61, 164.71, 154.78, 138.73, 128.85 (CH), 128.80
3
(
(
CH), 126.71 (CH), 80.39, 43.46 (CH ), 42.37 (CH ), 35.66 (CH ), 28.53 (CH ); R : 0.44
CH Cl /MeOH 98:2); HRMS (ESI) m/z [M+H] calcd for C H ClN O 419.1956 found
2 2 20 28 6 2
2 2 2 3 f
+
419.1955.
4
.2.10.
tert-Butyl-4-[4-chloro-6-(m-tolylmethylamino)-1,3,5-triazin-2-yl]piperazine-1-
1
carboxylate (7b). MW activation time 90 min, 150 °C . Yield 83%; white solid; mp 174 °C ; H
NMR (400 MHz, CDCl ) δ 7.23 (t, J = 7.4 Hz, 1H, CH), 7.17 – 7.04 (m, 3H, CH), 6.40 ( bs,
3
1
H, NH), 4.57 (d, J = 6.0 Hz, 2H, CH ), 3.79 (bs, 4H, CH ), 3.45 (bs, 4H, CH ), 2.35 (s, 3H,
2 2 2
13
CH ), 1.50 (s, 9H, CH ); C NMR (101 MHz, CDCl ) δ 169.17, 165.72, 164.66, 154.75,
3
3
3
1
38.38, 138.29, 128.61 (CH), 128.25 (CH), 124.93 (CH), 124.55 (CH), 80.36, 45.16 (CH ),
2
4
4.99 (CH ), 43.47 (CH ), 28.51 (CH ), 21.52 (CH ); R : 0.54 (PE/EtOAc 8:2); HRMS (ESI)
2
2
3
3
f
+
m/z [M+H] calcd for C H ClN O 419.1956 found 419.1957.
20
28
6
2
4.2.11.
tert-Butyl-4-[4-chloro-6-[[(2R)-2-hydroxy-2-phenyl-ethyl]amino]-1,3,5-triazin-2-
yl]piperazine-1-carboxylate (7c). MW activation time 90 min, 150 °C . Yield 78%; yellow ish oil;
1
H NMR (400 MHz, CDCl ) δ 7.52 – 7.28 (m, 5H, CH), 6.28 (bs, 0.6H, NH), 5.76 (bs, 0.2H,
3
13
NH), 5.02 – 4.81 (m, 1H, CH), 3.76 (bs, 6H), 3.46 (bs, 5H), 1.47 (s, 9H, CH ); C NMR (101
3

ACCEPTED MANUSCRIPT
MHz, CDCl ) δ 169.14, 165.95, 164.41, 154.75, 141.83, 128.72 (CH), 128.11 (CH), 128.04
3
(
(
CH), 127.12 (CH), 125.98 (CH), 80.49, 73.57 (d, J = 24.9 Hz, CH), 48.67 (CH ), 43.52
2
+
CH ), 43.35 (CH ), 28.51 (CH ); R : 0.32 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H] calcd for
2
2
3
f
C H ClN O 435.1905 found 435.1905.
20
28
6
3
4
.2.12.
tert-Butyl-4-[4-chloro-6-(4-cyanoanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
(
7d). MW activation time 120 min, 150 °C . Yield 93%; cream y solid; H NMR (400 MHz,
CDCl ) δ 7.87 – 7.54 (m, 4H), 3.96 – 3.75 (m, 4H, CH ), 3.54 (bs, 4H, CH ), 1.51 (s, 9H,
3
2
2
13
CH ); C NMR (101 MHz, CDCl ) δ 170.45, 169.63, 164.43, 163.62, 154.54, 154.43, 142.01,
3
3
1
33.21, 119.91, 118.85, 106.52, 80.69, 80.53, 53.44, 43.98 (CH ), 43.80 (CH ), 43.64 (CH ),
2 2 2
+
28.39 (CH ), 28.36 (CH ); R : 0.26 (CH Cl /MeOH 98:2); HRMS (ESI) m/z [M+H] calcd for
3
3
f
2
2
C H ClN O 416.1596 found 416.1595.
19
23
7
2
4
(
.2.13.
7e). MW activation time 90 min, 150 °C . Yield 40%; cream y amorphous solid; H NMR (400
MHz, CDCl ) δ 8.24 (d, J = 9.1 Hz, 2H, CH), 7.71 (d, J = 9.1 Hz, 2H), 7.48 (s, 1H, NH), 3.96 –
tert-Butyl-4-[4-chloro-6-(4-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
3
13
3.80 (m, 4H, CH ), 3.62 – 3.45 (m, 4H, CH ), 1.49 (s, 9H, CH ). C NMR (101 MHz, CDCl ) δ
2 2 3 3
1
69.99, 164.66, 163.83, 154.68, 143.97, 143.23, 125.27 (CH), 119.40 (CH), 80.70, 43.98
+
(
CH ), 43.84 (CH ), 28.54 (CH ); R : 0.31 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H] calcd for
2 2 3 f
C H ClN O 436.1494 found 436.1493.
18
23
7
4
4
.2.14. tert-Butyl-4-[4-chloro-6-(3-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (7f).
1
MW activation time 90 min, 150 °C . Yield 59%; creamy solid; H NMR (400 MHz, CDCl ) δ
3
8
3
1
8
.93 (bs, 1H, NH), 8.04 – 7.93 (m, 1H, CH), 7.89 – 7.42 (m, 3H, CH), 3.88 (bs, 4H, CH ),
2
13
.64 – 3.43 (m, 4H, CH ), 1.49 (s, 9H, CH ); C NMR (101 MHz, CDCl ) δ 169.78, 164.55,
2
3
3
63.89, 154.74, 148.72, 139.27, 129.73 (CH), 125.50 (CH), 118.39 (CH), 115.29 (CH),
0.65, 44.06 (CH ), 43.85 (CH ), 28.54 (CH ); R : 0.31 (PE/EtOAc 8:2); HRMS (ESI) m/z
2
2
3
f
+
[
M+H] calcd for C H ClN O 436.1494 found 436.1492.
18 23 7 4
4
.2.15. tert-Butyl-4-[4-chloro-6- (4-methoxyanilino)-
1,3,5-triazin-2-yl]
piperazine-1-
1
carboxylate (7g). MW activation time 90 min, 150 °C . Yield 79%; white solid; mp 162 °C ; H
NMR (400 MHz, CDCl ) δ 7.89 – 7.55 (m, 1H, NH), 7.44 – 7.33 (m, 2H), 6.89 – 6.82 (m, 2H),
3
13
3
1
8
.84 – 3.72 (m, 7H), 3.46 (bs, 4H), 1.47 (s, 9H); C NMR (101 MHz, CDCl ) δ 164.70,
3
64.03, 156.75, 156.50, 154.69, 130.70, 130.39, 123.38 (CH), 122.91 (CH), 114.13 (CH),
0.38, 55.56 (CH ), 43.53 (CH ), 28.48 (CH ); R : 0.24 (PE/EtOAc 8:2); HRMS (ESI) m/z
3
2
3
f
+
[
M+H] calcd for C H ClN O 421.1749 found 421.1749.
19 26 6 3
4
.2.16.
tert-Butyl-4-[4-chloro-6-[4-(trifluoromethyl)anilino]-1,3,5-triazin-2-yl]piperazine-1-
1
carboxylate (7h). MW activation time 90 min, 150 °C . Yield 98%; amorph ous beige solid; H
NMR (400 MHz, CDCl ) δ 7.66 (d, J = 8.6 Hz, 2H, CH), 7.60 (d, J = 8.8 Hz, 2H, CH), 7.51 (s,
3
13
1
H, NH), 3.94 – 3.74 (m, 4H, CH ), 3.51 (bs, 4H, CH ), 1.49 (s, 9H, CH ); C NMR (101
2 2 3
MHz, CDCl ) δ 164.67, 163.89, 154.71, 141.08, 126.39, 126.35, 125.86, 125.59, 125.54,
3
19
1
(
19.99, 80.60, 43.87, 43.73, 28.52; F NMR (376 MHz, CDCl ) δ -62.05; R : 0.69
3 f
+
CH Cl /MeOH 98:2); HRMS (ESI) m/z [M+H] calcd for C H ClF N O 459.1518 found
2 2 19 23 3 6 2
459.1516.
4.3. General procedure for compounds 5a-h and 8a-h
To the solution of appropriate compound 4 or 7 (1 equiv.) in DMSO were added DABCO (0.2
equiv.), KCN (1.1 equiv.) and then dropwise water (1 mL for 1 mmol of 4 or 7). The resulted

ACCEPTED MANUSCRIPT
solution was stirred at rt (16-48h) until the completion of reaction, monitored by TLC. After
completion of reaction, water and EtOAc were added to the mixture. The aqueous phase was
extracted with EtOAc (3x) and then the combined organic phases were washed with water,
brine, dried over MgSO and concentrated. Purification by column chromatography on silica
4
gel gave desired product.
4
.3.1. 4-(Cyclohexylamino)-6-(phenethylamino)-1,3,5-triazine-2-carbonitrile (5a). Yield 97%;
1
white solid; mp 188 °C ; H NMR (400 MHz, CDCl ) δ 7.41 – 7.30 (m, 2H), 7.28 – 7.17 (m,
3
3
H), 6.10 (bs, 0.48H, NH-CH ), 5.74 (bs, 0.23H, NH-CH ), 5.56 – 5.01 (m, 1.29H; 0.29H, NH-
2
2
CH ; 1H NH-CH), 3.99 – 3.79 (m, 1H, CH), 3.76 – 3.60 (m, 2H, NH-CH ), 3.00 – 2.80 (m, 2H,
2
2
13
CH ), 2.10 – 1.89 (m, 2H), 1.84 – 1.60 (m, 3H), 1.47 – 1.18 (m, 5H) ; C NMR (101 MHz,
2
CDCl ) δ 165.01, 164.11, 151.82, 138.71, 128.91 (CH), 128.81 (CH), 126.74 (CH), 115.24
3
(
2
CN), 49.86(CH), 42.46 (NH-CH ), 35.71 (Ar-CH ), 33.25 (CH ), 32.65 (CH ), 25.62 (CH ),
2 2 2 2 2
+
4.84 (CH ) ; R : 0.48 (PE/EtOAc 8:2) ; HRMS (ESI) m/z [M+H] calcd for C H N 323.1979
2 f 18 23 6
found 323.1977.
4
9
7
5
.3.2.
4-(Cyclohexylamino)-6-(m-tolylmethylamino)-1,3,5-triazine-2-carbonitrile (5b). Yield
1
3%; white solid; mp 116 °C ; H NMR (400 MHz, CDCl ) δ 7.24 (t, J = 7.5 Hz, 1H), 7.20 –
.07 (m, 3H), 6.32 (bs, 0.48H, NH-CH ), 5.96 (bs, 0.22H, NH-CH ), 5.58 (bs, 0.3H, NH-CH ),
.52 – 5.12 (m, 1H, NH-CH), 4.64 – 4.49 (m, 2H, NH-CH ), 3.94 – 3.65 (m, 1H, CH), 2.37 (d,
3
2
2
2
2
J = 6.6 Hz, 3H, CH ), 2.03 – 1.89 (m, 2H), 1.82 – 1.73 (m, 2H), 1.69 – 1.57 (m, 1H), 1.48 –
3
13
1
1
4
.33 (m, 2H), 1.29 – 1.17 (m, 3H); C NMR (101 MHz, CDCl ) δ 165.05, 164.10, 152.41,
3
51.89, 138.48, 137.97, 128.78 (CH), 128.65 (CH), 128.40 (CH), 124.75 (CH), 115.22 (CN),
9.84 (CH), 44.95 (NH-CH ), 33.19 (CH ), 32.69 (CH ), 32.56 (CH ), 25.57 (CH ), 24.84
2
2
2
2
2
+
(
CH ), 21.52(CH ); R : 0.75 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H] calcd for
2 3 f
C H N 323.1979 found 323.1977.
18
23
6
4
.3.3.
4-(Cyclohexylamino)- 6-[[(2R)-2-hydroxy-2-phenyl-ethyl]amino]- 1,3,5-triazine-2-
1
carbonitrile (5c). Yield 87%; amorphous creamy solid; H NMR (400 MHz, CDCl ) δ 7.62 –
3
7
5
3
.25 (m, 5H), 6.98 – 6.74 (m, 0.57H, NH), 6.50 (s, 0.2H, NH), 5.95 (bs, 0.33H, NH), 5.72 –
.29 (m, 0.9H, NH), 5.09 – 4.86 (m, 1H, CH-OH), 4.00 – 3.70 (m, 2H, CH and OH), 3.66 –
13
.35 (m, 2H, CH ), 2.26 – 1.87 (m, 2H), 1.89 – 1.59 (m, 3H), 1.49 – 1.10 (m, 5H); C NMR
2
(
(
101 MHz, CDCl ) δ 165.18, 163.60, 151.47, 141.72, 128.64 (CH), 128.58 (CH), 127.99
CH), 125.97 (CH), 125.83 (CH), 115.14 (CN), 73.35 (CH), 50.02 (CH), 48.71 (HN-CH2),
3
3
3.05 (CH ), 32.52 (CH ), 32.44 (CH ), 25.46 (CH ), 24.81 (CH ); R : 0.83 (PE/EtOAc 5:5);
2 2 2 2 2 f
+
HRMS (ESI) m/z [M+H] calcd for C H N O 339.1928 found 339.1926.
18
23
6
4
.3.4. 4-(4-Cyanoanilino)-6-(cyclohexylamino)-1,3,5-triazine-2-carbonitrile (5d). Yield 56%;
1
white solid; mp >260 °C ; H NMR (400 MHz, DMSO) δ 10.76 – 10.38 (m, 1H, NH), 8.68 –
.25 (m, 1H, HN-CH), 7.95 (dd, J = 34.5, 8.7 Hz, 2H, CH), 7.78 (dd, J = 15.0, 8.8 Hz, 2H,
8
13
CH), 3.72 (s, 1H, CH), 1.95 – 1.51 (m, 5H), 1.47 – 1.10 (m, 5H) ; C NMR (101 MHz, DMSO)
δ 163.43, 163.30, 151.65, 151.29, 143.18, 133.00 (CH), 132.96 (CH), 119.96 (CH), 119.82
(
2
CH), 119.05, 115.10, 104.54, 104.36, 49.56 (CH), 32.12 (CH ), 31.67 (CH ), 25.14 (CH ),
2 2 2
+
5.05 (CH ), 24.58 (CH ) ; R : 0.69 (PE/EtOAc 7:3) ; HRMS (ESI) m/z [M+H] calcd for
2 2 f
C H N 320.1618 found 320.1616.
17
18
7
4
.3.5.
4-(Cyclohexylamino)-6-(4-nitroanilino)-1,3,5-triazine-2-carbonitrile (5e). Yield 19%;
1
amorphous orange solid; H NMR (250 MHz, DMSO) δ 10.67 (d, J = 19.0 Hz, 0.7H, NH),
1
0.09 (d, J = 27.4 Hz, 0.3H, NH), 8.59 – 8.30 (m, 1H, NH), 8.25 – 7.87 (m, 4H, CH), 3.71 (bs,
13
1H, CH), 1.90 – 1.52 (m, 5H), 1.39 – 1.07 (m, 5H); C NMR (63 MHz, DMSO) δ 163.32,

ACCEPTED MANUSCRIPT
163.10, 162.53, 151.31, 145.26, 141.80, 124.66 (CH), 119.44 (CH), 118.65, 115.02 (CN),
4
9.57 (CH), 32.07 (CH ), 31.63 (CH ), 25.11 (CH ), 24.50 (CH ); R : 0.43 (PE/EtOAc 8:2) ;
2
2
2
2
f
+
HRMS (ESI) m/z [M+H] calcd for C H N O 340.1516 found 340.1517.
16
18
7
2
4
.3.6.
4-(Cyclohexylamino)-6-(3-nitroanilino)-1,3,5-triazine-2-carbonitrile (5f). Yield 67%;
1
amorphous creamy solid; H NMR (400 MHz, CDCl ) δ 9.23 – 8.48 (m, 1H, NH), 8.09 – 7.93
3
(
1
m, 1H, CH), 7.90 – 7.35 (m, 3H, CH), 5.95 – 5.42 (m, 1H, NH), 3.99 (bs, 1H, CH), 2.24 –
13
.92 (m, 2H), 1.82 – 1.64 (m, 3H), 1.55 – 1.23 (m, 5H) ; C NMR (101 MHz, CDCl ) δ
3
164.44, 164.18, 152.30, 148.83, 138.95, 129.88 (CH), 129.81 (CH), 118.77 (CH), 115.13
(
(
CH), 114.80 (CN), 50.39 (CH), 33.04 (CH ), 32.72 (CH ), 25.52 (CH ), 24.88 (CH ), 24.63
2 2 2 2
+
CH ) ; R : 0.59 (PE/EtOAc 9:1) ; HRMS (ESI) m/z [M+H] calcd for C H N O 340.1516
2
f
16 18
7
2
found 340.1515.
4
9
1
2
1
1
2
.3.7.
4-(Cyclohexylamino)-6-(4-methoxyanilino)-1,3,5-triazine-2-carbonitrile (5g). Yield
1
6%; creamy solid; H NMR (400 MHz, CDCl ) δ 7.57 – 7.37 (m, 2H, CH), 7.33 – 7.04 (m,
H, NH), 6.98 – 6.85 (m, 2H, CH), 5.71 – 5.29 (m, 1H, NH), 3.97 – 3.70 (m, 4H, CH , CH),
.15 – 1.92 (m, 2H), 1.86 – 1.58 (m, 3H), 1.51 – 1.13 (m, 5H) ; C NMR (101 MHz, CDCl ) δ
64.56, 164.24, 156.97, 156.67, 152.61, 152.07, 130.46, 130.04, 123.45 (CH), 122.49 (CH),
15.34 (CN), 114.34 (CH), 114.19 (CH), 55.66 (CH ), 49.95 (CH), 33.12 (CH ), 32.48 (CH ),
3
3
13
3
3
2
2
+
5.55 (CH ), 24.82 (CH ) ; R : 0.42 (PE/EtOAc 8:2) ; HRMS (ESI) m/z [M+H] calcd for
2
2
f
C H N O 325.1771 found 325.1770.
17
21
6
4
.3.8.
4-(Cyclohexylamino)-6-[4-(trifluoromethyl)anilino]-1,3,5-triazine-2-carbonitrile (5h).
1
Yield 94%; white solid; mp 198 °C ; H NMR (400 MHz, CDCl ) δ 7.92 – 7.57 (m, 4H, CH),
3
7
2
1
1
.53 – 7.32 (m, 1H, NH), 5.75 – 5.29 (m, 1H, NH), 3.98 – 3.74 (m, 1H, CH), 2.11 – 1.95 (m,
13
H), 1.86 – 1.63 (m, 3H), 1.45 – 1.23 (m, 5H); C NMR (101 MHz, CDCl ) δ 164.53, 164.24,
3
52.33, 140.79, 126.42 (CH), 126.38 (CH), 126.04 (dd, J = 1419.6, 1147.9 Hz), 120.22 (CH),
19.95 (CH), 114.88 (CN), 50.22 (CH), 33.08 (CH ), 32.50 (CH ), 25.52 (CH ), 24.82 (CH );
2
2
2
2
1
9
+
F NMR (376 MHz, CDCl ) δ -62.10; R : 0.70 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H] calcd
3
f
for C H F N 363.1540 found 363.1539.
17
18
3
6
4
.3.9.
tert-Butyl-4-[4-cyano-6-(phenethylamino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
(
8a). Yield 82%; white solid; mp 196 °C ; H NMR (400 MHz, CDCl ) δ 7.33 (t, J = 7.3 Hz, 2H,
3
CH), 7.28 – 7.19 (m, 3H, CH), 5.77 (s, 0.7H, NH), 5.25 (bs, 0.3H, NH), 3.89 – 3.63 (m, 6H,
13
CH ), 3.55 – 3.42 (m, 4H, CH ), 2.90 (t, J = 7.1 Hz, 2H, CH ), 1.51 (s, 9H, CH ); C NMR
2
2
2
3
(
101 MHz, CDCl ) δ 164.80, 163.77, 154.74, 152.00, 138.54, 128.93 (CH), 128.86 (CH),
3
1
26.82 (CH), 115.36 (CN), 80.51, 43.56 (CH ), 43.14 (CH ), 42.25 (CH ), 35.90 (CH ), 35.51
2 2 2 2
+
(
CH ), 28.53 (CH ); R : 0.56 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H] calcd for
2 3 f
C H N O 410.2299 found 410.2295.
21
28
7
2
4
.3.10.
tert-Butyl-4-[4-cyano-6-(m-tolylmethylamino)-1,3,5-triazin-2-yl]piperazine-1-
1
carboxylate (8b). Yield 88%; white solid; mp 162 °C ; H NMR (400 MHz, MeOD) δ 7.20 – 6.96
(
(
m, 4H), 4.45 (d, J = 10.2 Hz, 2H, CH ), 3.72 (bs, 4H, CH ), 3.48 – 3.33 (m, 4H, CH ), 2.27
2 2 2
13
s, 3H, CH ), 1.43 (s, 9H, CH ); C NMR (101 MHz, MeOD) δ 166.05, 164.94, 156.33,
3 3
1
8
7
53.17, 139.99, 139.16, 129.38 (CH), 129.23 (CH), 128.82 (CH), 125.62 (CH), 116.37 (CN),
1.60, 45.37 (CH ), 44.37 (CH ), 44.07 (CH ), 28.63 (CH ), 21.47 (CH ); R : 0.81 (PE/EtOAc
2
2
2
3
3
f
+
:3); HRMS (ESI) m/z [M+H] calcd for C H N O 410.2299 found 410.2297.
21
28
7
2

ACCEPTED MANUSCRIPT
4
.3.11.
tert-Butyl-4-[4-cyano-6- [[(2R)-2-hydroxy-2-phenyl-ethyl]amino]- 1,3,5-triazin-2-
1
yl]piperazine-1-carboxylate (8c). Yield 61%; white solid; mp 160 °C ; H NMR (400 MHz,
CDCl ) δ 7.53 – 7.31 (m, 5H), 6.10 (t, J = 5.5 Hz, 0.62H, NH), 5.74 (t, J = 5.5 Hz, 0.28H, NH),
3
4
1
1
8
2
.93 (bs, 1H, CH-OH), 3.96 – 3.65 (m, 5H, CH ), 3.61 – 3.33 (m, 5H, CH ), 3.16 – 2.83 (m,
2
2
13
H, OH), 1.50 (s, 9H, CH ); C NMR (101 MHz, CDCl ) δ 165.21, 163.63, 154.73, 152.09,
3
3
41.58, 128.86 (CH), 128.80 (CH), 128.37 (CH), 127.12 (CH), 125.99 (CH), 115.27 (CN),
0.58, 73.38 (d, J = 24.7 Hz, CH-OH), 48.45 (CH ), 48.28 (CH ), 43.60 (CH ), 43.20 (CH ),
2
2
2
2
+
8.53 (CH ); Rf: 0.69 (PE/EtOAc 5:5); HRMS (ESI) m/z [M+H]
calcd for
3
C H N O 426.2248 found 426.2246.
21
28
7
3
4
.3.12.
tert-Butyl-4-[4-cyano-6-(4-cyanoanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
(
8d). Yield quantitative; yellow amorphous solid; H NMR (400 MHz, CDCl ) δ 7.76-7.62 (m,
3
4
H), 7.53 (s, 1H, NH), 3.97 – 3.77 (m, 4H, CH ), 3.64 – 3.45 (m, 4H, CH ), 1.51 (s, 9H, CH );
2 2 3
1
3
C NMR (101 MHz, CDCl ) δ 163.72, 163.46, 154.66, 152.47, 141.74 (CH), 133.41 (CH),
3
1
20.20, 118.87, 114.95 (CN), 107.09, 80.78, 43.85 (CH ), 43.68 (CH ), 28.52 (CH ); R : 0.91
2
2
3
f
+
(
CH Cl /MeOH 9:1); HRMS (ESI) m/z [M+H] calcd for C H N O 407.1938 found
2 2 20 23 8 2
407.1937.
4
.3.13.
tert-Butyl-4-[4-cyano-6-(4-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
(
8e). Yield 75%; creamy solid; H NMR (400 MHz, CDCl ) δ 8.26 (d, J = 9.1 Hz, 2H, CH),
3
7
.72 (d, J = 9.1 Hz, 2H, CH), 7.42 (s, 1H, NH), 3.95 – 3.78 (m, 4H, CH ), 3.60 – 3.47 (m, 4H,
2
13
CH ), 1.50 (s, 9H, CH ); C NMR (63 MHz, CDCl ) δ 163.74, 163.47, 154.65, 152.54,
2
3
3
1
43.51, 125.29 (CH), 119.66 (CH), 114.90 (CN), 80.82, 43.92 (CH ), 43.75 (CH ), 28.54
2 2
+
(
CH ); R : 0.25 (CH Cl ); HRMS (ESI) m/z [M+H] calcd for C H N O 427.1836 found
3 f 2 2 19 23 8 4
427.1835.
4
.3.14.
tert-Butyl-4-[4-cyano-6-(3-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
(
8f). Yield 85%; yellowish solid; mp 256 °C ; H NMR (400 MHz, CDCl ) δ 8.92 (bs, 1H, NH),
3
7
3
1
.98 (dd, J = 8.7, 1.7 Hz, 1H, CH), 7.69 – 7.43 (m, 3H, CH), 4.02 – 3.76 (m, 4H, CH ), 3.62 –
2
13
.39 (m, 4H, CH ), 1.50 (s, 9H, CH ); C NMR (101 MHz, CDCl ) δ 163.67, 154.70, 152.52,
2
3
3
48.75, 138.84, 129.92, 118.77, 115.33, 114.95, 80.76, 43.96, 28.54; R : 0.25 (CH Cl );
f
2
2
+
HRMS (ESI) m/z [M+H] calcd for C H N O 427.1836 found 427.1836.
19
23
8
4
4
.3.15. tert-Butyl-4-[4-cyano-6-(4-methoxyanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate
1
(
8g). Yield quantitative; yellow amorphous solid; H NMR (250 MHz, CDCl ) δ 7.49 – 7.31 (m,
3
13
3H), 6.89 (d, J = 9.0 Hz, 2H), 3.87 – 3.70 (m, 7H), 3.54 – 3.38 (m, 4H), 1.48 (s, 9H); C NMR
(
4
63 MHz, CDCl ) δ 156.86, 154.71, 130.19, 123.29, 122.89, 114.36, 80.57, 55.67, 43.66,
3.39, 28.54; Rf: 0.78 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H] calcd for
3
+
C H N O 412.2091 found 412.2089.
20
26
7
3
4
.3.16.
tert-Butyl-4-[4-cyano-6-[4-(trifluoromethyl)anilino]-1,3,5-triazin-2-yl]piperazine-1-
1
carboxylate (8h). Yield 77%; yellow solid; mp 128 °C ; H NMR (400 MHz, CDCl ) δ 7.72 –
3
7
9
1
.56 (m, 4H, CH), 7.37 (bs, 1H, NH), 3.99 – 3.75 (m, 4H, CH ), 3.52 (bs, 4H, CH ), 1.49 (s,
2 2
13
H, CH3); C NMR (101 MHz, CDCl ) δ 163.79, 154.68, 152.48, 140.65, 126.49 (CH),
3
26.46 (CH), 126.28 (CH), 120.14 (CH), 115.03 (CN), 80.73, 43.82 (CH ), 43.62 (CH ), 28.53
2
2
1
9
(
CH ); F NMR (376 MHz, CDCl ) δ -62.13; R : 0.36 (PE/EtOAc 8:2); HRMS (ESI) m/z
3
3
f
+
[
M+H] calcd for C H F N O 450.1859 found 450.1857.
20 23 3 7 2
4.4. General procedure for compounds 9 a-h

ACCEPTED MANUSCRIPT
To the solution of 8 in CH Cl was added TFA (16 equiv). The reaction mixture was stirred for
2
2
9
0 minutes under N at room temperature. The reaction mixture was then concentrated and
2
the residue was dissolved in CH Cl and NaHCO sat. was added. The aqueous phase was
2
2
3
extracted with CH Cl . The combined organic phases were washed with water, dried over
2
2
MgSO and concentrated to give the desired product 9.
4
4
.4.1.
4-(Phenethylamino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9a). Yield 90%;
1
white solid; mp 196 °C ; H NMR (400 MHz, CDCl ) δ 7.32 (t, J = 7.6 Hz, 2H, CH), 7.27 – 7.18
3
(
3
m, 3H, CH), 5.94 (t, J = 6.0 Hz, 0.7H, NH), 5.27 (bs, 0.3H, NH), 3.99 – 3.59 (m, 6H, CH ),
2
13
.01 – 2.81 (m, 6H, CH ), 2.04 (s, 1H, NH); C NMR (101 MHz, CDCl ) δ 164.72, 163.49,
2
3
151.90, 138.68, 128.92 (CH), 128.88 (CH), 128.80 (CH), 128.76 (CH), 126.73 (CH), 115.47
(
(
CN), 46.01 (CH ), 45.88 (CH ), 44.89 (CH ), 44.37 (CH ), 42.23 (CH ), 35.51 (CH ); R 0.39
CH2Cl2/MeOH 85:15); HRMS (ESI) m/z [M+H] calcd for C H N 310.1775 found
16 20 7
2 2 2 2 2 2 f:
+
310.1774.
4
.4.2. 4-(m-Tolylmethylamino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9b). Yield 85%;
1
white solid; mp 158 °C ; H NMR (400 MHz, CDCl ) δ 7.24 (t, J = 7.4 Hz, 1H), 7.18 – 7.06 (m,
3
3
H), 5.97 (bs, 0.4H, NH), 5.50 (bs, 0.2H, NH), 4.65 – 4.49 (m, 2H, CH ), 3.96 – 3.62 (m, 4H,
2
13
CH ), 2.89 (bs, 4H, CH ), 2.36 (s, 3H, CH ), 1.85 (bs, 1H, NH); C NMR (101 MHz, CDCl ) δ
2
2
3
3
164.85, 163.54, 152.09, 138.54, 137.86, 128.73 (CH), 128.46 (CH), 128.40 (CH), 124.70
(
(
CH), 115.47 (CN), 45.99 (CH ), 45.82 (CH ), 44.96 (CH ), 44.91 (CH ), 44.78 (CH ), 44.47
2
2
2
2
2
+
CH ), 21.52 (CH ); R 0.35 (CH Cl /MeOH 9:1); HRMS (ESI) m/z [M+H] calcd for
2
3
f:
2
2
C H N 310.1775 found 310.1773.
16
20
7
4
.4.3. 4-[[(2R)-2-Hydroxy-2-phenyl-ethyl]amino]-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile
1
(
9c). Yield 54%; white amorphous solid; H NMR (400 MHz, CDCl ) δ 7.48 – 7.29 (m, 5H),
3
6
.38 (t, J = 5.7 Hz, 0.7H, NH), 5.87 (s, 0.3H, NH), 5.00 – 4.81 (m, 1H, CH), 3.94 – 3.70 (m,
13
4H, CH ), 3.56 – 3.38 (m, 2H, CH ), 2.99 – 2.34 (m, 6H, CH , NH, OH) ; C NMR (101 MHz,
2
2
2
CDCl ) δ 165.05, 163.26, 151.88, 141.89, 128.74 (CH), 128.16 (CH), 125.99 (CH), 115.36
3
(
(
CN), 73.01 (CH), 48.36 (CH ), 45.83 (CH ), 45.66 (CH ), 44.84 (CH ), 44.32 (CH ) ; R : 0.53
2 2 2 2 2 f
+
CH Cl /MeOH 8:2) ; HRMS (ESI) m/z [M+H] calcd for C H N O 326.1724 found 326.1721.
2
2
16 20
7
4
.4.4.
4-(4-Cyanoanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9d). Yield 75%;
1
creamy solid; mp 252 °C ; H NMR (400 MHz, DMSO) δ 10.60 (s, 1H, NH), 7.84 (d, J = 8.9
Hz, 2H, CH), 7.79 (d, J = 8.8 Hz, 2H, CH), 3.78 – 3.51 (m, 4H, CH ), 3.28 (bs, 1H, NH), 2.88
2
13
–
2.69 (m, 4H, CH ); C NMR (101 MHz, DMSO) δ 162.80, 162.60, 151.41, 142.91, 133.12
2
(
(
CH), 120.08 (CH), 119.05 (CN), 115.18 (CN), 104.64, 45.19 (CH ), 45.10 (CH ), 44.75
2 2
+
CH ), 44.54 (CH ); R: 0.38 (CH Cl /MeOH 85:15) ; HRMS (ESI) m/z [M+H] calcd for
2
2
f
2
2
C H N 307.1414 found 307.1413.
15
15
8
4
.4.5. 4-(4-Nitroanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9e). Yield 81%;
1
amorphous yellow solid; H NMR (400 MHz, MeOD) δ 8.24 (d, J = 9.2 Hz, 2H, CH), 7.88 (d, J
13
=
5
4
9.2 Hz, 2H, CH), 3.90 (bs, 4H, CH ), 2.96 (bs, 4H, CH ); C NMR (63 MHz, CDCl /MeOD
2 2 3
:1) δ 163.30, 152.10, 144.52, 142.65, 124.70 (CH), 119.54 (CH), 114.78 (CN), 45.00 (CH2),
4.91 (CH2), 44.19 (CH2), 43.93 (CH2); Rf: 0.27 (CH Cl /MeOH 9:1); HRMS (ESI) m/z
2
2
[
M+H]+ calcd for C14H15N8O2 327.1312 found 327.1312.
4
.4.6. 4-(3-Nitroanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9f). Yield 85%; yellow
1
solid; mp 130 °C ; H NMR (400 MHz, Acetone) δ 8.99 (bs, 1H, CH), 8.17 – 7.80 (m, 2H, CH),

ACCEPTED MANUSCRIPT
13
7
.62 (t, J = 8.2 Hz, 1H, CH), 4.08 – 3.69 (m, 4H, CH ), 3.09 – 2.58 (m, 4H, CH ); C NMR
2
2
(
(
(
101 MHz, Acetone) δ 164.07, 152.96, 149.33, 140.79, 130.67 (CH), 126.56 (CH), 118.41
CH), 116.06 (CN), 115.40 (CH), 46.45(CH2), 46.34(CH2), 46.06(CH2), 45.93(CH2); Rf: 0.42
CH2Cl2/MeOH 9:1); HRMS (ESI) m/z [M+H]+ calcd for C14H15N8O2 327.1312 found
327.1312.
4
.4.7.
4-(4-Methoxyanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9g). Yield 60%;
1
yellow amorphous solid; H NMR (400 MHz, CDCl ) δ 7.50 (bs, 1H, NH), 7.38 (d, J = 8.9 Hz,
3
2
H, CH), 6.88 (d, J = 8.9 Hz, 2H, CH), 3.87 – 3.72 (m, 7H CH , CH ), 2.97 – 2.80 (m, 4H,
3 2
13
CH ), 1.94 (bs, 1H, NH); C NMR (101 MHz, CDCl ) δ 163.50, 156.61, 152.16, 130.42,
2
3
1
22.75 (CH), 115.34 (CN), 114.24 (CH), 55.60 (CH ), 45.94 (CH ), 45.83 (CH ), 45.00 (CH ),
3 2 2 2
+
44.62 (CH ); R : 0.29 (CH Cl /MeOH 9:1); HRMS (ESI) m/z [M+H] calcd for
2
f
2
2
C H N O 312.1567 found 312.1567.
15
18
7
4
.4.8.
4-Piperazin-1-yl-6-[4-(trifluoromethyl)anilino]-1,3,5-triazine-2-carbonitrile (9h). Yield
1
83%; white solid; mp 224 °C ; H NMR (400 MHz, DMSO) δ 10.52 (s, 1H, NH), 7.86 (d, J = 8.4
Hz, 2H, CH), 7.70 (d, J = 8.6 Hz, 2H, CH), 3.78 – 3.51 (m, 4H, CH ), 3.07 – 2.63 (m, 5H,
2
13
CH , NH); C NMR (101 MHz, DMSO) δ 162.64, 151.42, 142.20, 125.99 (CH), 125.73 (CH),
2
1
23.19, 123.03, 122.87, 120.05 (CH), 115.23 (CN), 99.50, 45.22 (CH ), 45.14 (CH ), 44.74
2 2
19
(
CH ), 44.54 (CH ); F NMR (376 MHz, DMSO) δ -60.31; R : 0.31 (CH Cl /MeOH 8:2);
2
2
f
2
2
+
HRMS (ESI) m/z [M+H] calcd for C H F N 350.1336 found 350.1334.
15
15
3
7
4.5.
Kinetic assays
Human cathepsins B, L and S were purchased from Calbiochem (VWR International,
Pessac, France) while human cathepsin K was expressed in Pichia pastoris as previously
31
reported. Aspartic cathepsin D came from R & D research (Minneapolis, USA). Human
MMP-12 was a kind gift from Anne-Sophie Lamort (INSERM U1100, CEPR, University F.
Rabelais, Tours, France). Trypsin and chymotrypsin were from Euromedex (Strasbourg,
France). Human neutrophil elastase (HNE) was supplied by BioCentrum (Krakow, Poland).
Active site concentrations of cysteine cathepsins were determined using L-3-carboxy-trans-
2,3-epoxy-propionyl-leucylamide-(4-guanido)-butane (E-64) (Sigma-Aldrich, Saint-Quentin
Fallavier, France). Enzymatic assays for cathepsins B, L and K were carried out at 37 °C in
their activity buffer (0.1 M sodium acetate buffer, pH 5.5, containing 2 mM DTT and 0.01%
Brij35), using Z-Phe-Arg-AMC (benzyloxycarbonyl-phenylalanyl-arginine-4-methylcoumarin,
Bachem, Bubendorf, Switzerland) as substrate (spectromicrofluorimeter SpectraMax Gemini,
Molecular Devices, Saint Grégoire, France; λ = 350 nm, λ = 460 nm). The same protocol
ex
em
was operated for cathepsin S, except that Z-LR-AMC (Bachem) was used as substrate.
Alternatively activity buffers were 0.1 M Tris / HCl buffer, pH 8.0, 50 mM CaCl2, 100 mM
NaCl for trypsin and chymotrypsin, 0.05 M HEPES buffer, pH 7.4, NP40 0.05% 150 mM NaCl
for HNE, 0.05 M HEPES buffer, pH 7.5, 150 mM NaCl, 8 mM CaCl2 and 0.05% Brij 35 for
MMP-12, 0.1 M sodium citrate buffer, pH 4.0 for aspartic cathepsin D, respectively.
Cathepsins B, K, L and S (0.5 nM) were incubated in the activity buffer (pH 5.5) in the
presence of increasing concentrations of inhibitor (0-250 nM) for 30 minutes, before
measurement of the residual enzymatic activity. Tests were performed using three
concentrations of fluorogenic AMC-derived substrates (2.5, 5 and 10 µM). Slopes were
calculated and average values of IC determined (software Softmaxpro, Molecular Devices).
50
Assays were performed in triplicate and repeated three times.
Corresponding Author
*
Luigi A. Agrofoglio: Phone: +33-2-3849-4582. Fax: +33-2-3847-1281.

ACCEPTED MANUSCRIPT
E-mail: luigi.agrofoglio@univ-orleans.fr
Acknowlegment
E.P. is grateful to the Direction Générale de l’Armement (DGA) and Region Centre for a PhD
scholarship. D.W. received funding from the Polish Ministry of Science and Higher Education
within the Program 'Mobility Plus', decision number 1308/MOB/IV/2015/0. We thank the
LABEX SynOrg (ANR-11-LABX-0029) for partial financial support.
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DRAWING CAPTION
Figure 1. Schematic modifications of 1,3,5-triazine scaffold.
Figure 2. Binding pose of compound 9h in the active site of the human cathepsin K (PDB ID:
1VSN). The inhibitor is rendered as a stick and ball representation. Solid, transparent protein
surface was generated. Only residues situated less than 4Å from the inhibitor has been
shown. Hydrogen bonds between cathepsin K and 9h are labelled as red. Residues forming
S1 subpocket of cathepsin K binding site are labelled as red, S2 as blue and S’1 as green.
Figure 3. Binding pose of compound 9h in the active site of the human cathepsin S (PDB ID:
3OVX). The inhibitor is rendered as a stick and ball representation. Solid, transparent protein

ACCEPTED MANUSCRIPT
Scheme 1. Reaction conditions and reagents: (i) DIPEA, CH Cl ; (ii) H N-R, DIPEA, CH CN,
2
2
2
3
1
50 °C , MW; ( iii) KCN, DABCO, DMSO, H O, overnight, rt; (iv) TFA, CH Cl , rt.
2 2 2
Table 1. The IC values of the target compounds 5a-h and 9a-h against cathepsins B, K, L
50
and S
DRAWINGS
CN
CN
CN
N
N
N
N
N
N
R
R
N
H
N
N
H
N
N
N
N
H
N
N
H
NH
NH
1
9
5
Figure 1.
Figure 2.
Figure 3.

ACCEPTED MANUSCRIPT
ii
iii
i
3
4a-h
5a-h
i
ii
iii
2
6
7a-h
8a-h, R = Boc
1
9
a-h, R = H
1
NH -R =
2
a
b
c
d
e
f
g
h
Scheme 1.
IC [nM]
50
Compounds
Cat B
Cat K
80
Cat L
Cat S
23
5a
5b
5c
5d
5e
No inhibition
No inhibition
No inhibition
No inhibition
No inhibition
79
No inhibition
No inhibition
No inhibition
No inhibition
136
60
23
70
23
67
23
17
90
5f
17
23
28
5
5
9
9
9
9
9
g
h
a
b
c
d
e
No inhibition
No inhibition
23
226
136
102
34
No inhibition
No inhibition No inhibition No inhibition No inhibition
57
23
68
28
17
17
17
28
1
No inhibition
17
136
45
23
57
113
No inhibition
No inhibition
34
17
17
23
17
9
f
9
g
h
45
17
17
9
No inhibition
520
No inhibition
1711
23
2
4
1
158
Table 1.

ACCEPTED MANUSCRIPT
Highlights
Synthesis of novel 2,4,6-trisubstituted 1,3,5-triazine compounds
Human cysteine cathepsins with endopeptidase activity are the targets
Docking studies of active 9h into X-ray crystal structure of cathepsins K and S
IC50 values of some molecules were in the nanomolar range against Cat K & Cat S