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

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

DOI:

10.1016/j.ejmech.2016.05.009

Reference:

EJMECH 8598

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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Straightforward synthesis of 2,4,6-trisubstituted 1,3,5-triazine compounds targeting cysteine cathepsins K and S Elżbieta Plebanek,a Florian Chevrier,a Vincent Roy,a Thibaut Garenne,b Fabien Lecaille,b Dawid Warszycki,c Andrzej J. Bojarski,c Gilles Lalmanach,b Luigi A. Agrofoglioa,* a

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Univ. Orléans, CNRS, ICOA, UMR 7311, F-45067 Orléans, France

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INSERM, UMR 1100, Pathologies Respiratoires: protéolyse et aérosolthérapie, Centre d’Etude des Pathologies Respiratoires, Université François Rabelais, F-37032 Tours cedex, France

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Medicinal Chemistry Department, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland

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Keywords: Cysteine cathepsin; cathepsin inhibitors; 1,3,5-triazine, microwave irradiation

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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 = 28 nM) and Cat S (IC50 = 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.

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1. Introduction

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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 genome.1 They have long been thought to be primarily involved in end-stage degradation/recycling of endocytosed proteins within acidic lysosomal/endosomal compartments2 These proteases share similar three-dimensional structures,3 catalytic mechanism, and substrate specificity.4,5 They are involved in specific cellular processes6,7 (e.g., histone proteolysis, prohormone processing, cell-adhesion molecule cleavage and shedding, etc...) and in a broad spectrum of disorders (e.g., cancer progression,8 inflammatory responses),9 making them putative targets for the development of new therapies.10,11 Especially, cathepsin (Cat) S is involved in major histocompatibility complex (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, diabetes and lupus erythematosus) as well neuropathic pain.12 Cat K that is predominantly expressed in osteoclasts is a critical bone resorbing protease and was identified as a clinically relevant target for the treatment of osteoporosis13 and bone metastasis.14 Since 2004, over 50 patents have been filed on inhibitors of Cat K. Despite successful studies, pharmacological inhibitors may have deleterious side effects, especially on the lungs15 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 offer benefits during bronchopneumodysplasia (BPD) and emphysema.16

2. Results and discussion 2.1 Chemistry

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[Figure 1]

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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 of attention.17,18 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 the strong electro-withdrawing nature of this class of aromatic heterocyclic rings.19,20 In 2011, Rankovic et al.,21 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 (IC50 = 1 nM) and moderate selectivity over Cat S (IC50 = 158 nM), L (IC50 = 1711 nM), and B (IC50 = 520 nM). The 2-cyano-1,3,5-triazine scaffold offers, from the druglikeness point of view, many opportunities for synthetic optimization.22 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).

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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 2, (Scheme 1). Nucleophilic substitution of chlorine atoms in cyanuric chloride 2 in presence of diisopropyl ethyl amine (DIPEA) and cyclohexylamine (for compounds 5-like) or N-Bocmonoprotected piperazine (for compounds 9-like) in dichloromethane, provided the monosubstituted 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, respectively.23 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,

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2.3 Docking studies

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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 –CF3 analog (5h), which is both more electron withdrawing and lipophilic than its methoxy counterpart –OMe (5g), are more active toward Cat K with an IC50 values of 34 nM and 23 nM respectively compared to Cat S (>100 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 (IC50 = 34nM) and only inhibitor bearing hydrophobic CF3 group 9f maintained selective inhibitory effect on both Cat K and S with an IC50 ˂ 30 nM. Surprisingly, 9a (with an increased distance between 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 9a. Finally, 9h exhibited a selective inhibition of Cat K (IC50 = 28 nM) and Cat S (IC50 = 23 nM), respectively.

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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 Wizard,25 under default settings. Three dimensional structure, conformation and protonation states of 9h were generated by LigPrep26 (at pH 7.4). Finally, Glide27,28 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.

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[Figure 2.] [Figure 3.]

In general, binding modes of docked compound are similar to already published.29,30 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). pTrifluorophenyl 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

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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 IC50 ≤ 45nM. In the later case, this lack of selectivity and high activity can make them effective agents against some viruses for which the glycoprotein cleavage is mediated by an array of proteases. 4. Experimental section 4.1 Chemistry

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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 60 M (0.040-0.063 mm, E. Merck). The 1H and 13C 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.

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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 CH2Cl2 was cooled at 0°C then the mixture of 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 CH2Cl2 was slowly added. The reaction mixture was stirred at rt for 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 (CH2Cl2) to obtain 4.02g (quantitative) of the desired product as white amorphous solid. 1H NMR (400 MHz, CDCl3) δ 5.70 (bs, 1H, NH), 3.99 – 3.83 (m, 1H, CH), 2.06 – 1.89 (m, 2H), 1.82 – 1.71 (m, 2H), 1.68 – 1.61 (m, 1H), 1.50 – 1.35 (m, 2H), 1.32 – 1.15 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 171.12, 169.98, 165.12, 50.36 (CH), 32.60 (CH2), 25.38 (CH2), 24.54 (CH2); Rf: 0.80 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C9H13Cl2N4 247.0512 found 247.0517.

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4.1.2. tert-Butyl-4-(4,6-dichloro-1,3,5-triazin-2-yl)piperazine-1-carboxylate (6). Solution of 2.07g (11.2 mmol, 1 equiv.) of 17 in 32 mL of CH2Cl2 was cooled at 0°C then the mixture of 2.09g (11.2 mmol, 1 equiv.) of tert-butyl piperazine-1-carboxylate and 1.93 mL (11.3 mmol, 1.1 equiv.) of DIPEA in 10 mL of CH2Cl2 was slowly added. The reaction mixture was stirred 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 CH2Cl2, washed with 2M HCl, water, then dried over MgSO4 and concentrated under vacuum. Purification by column chromatography on silica gel gave desired product.

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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 time 60 min, 100°C. Yield 96%; white solid; mp 174° C; 1H NMR (400 MHz, CDCl3) δ 7.38 – 7.27 (m, 2H), 7.25 – 7.15 (m, 3H), 5.96 (bs, 0.54H, NH-CH2), 5.58 (bs, 0.2H, NH-CH2), 5.39 – 4.92 (m,1.26H; 0.26H, NH-CH2; 1H NH-CH), 3.95 – 3.77 (m, 1H, CH), 3.73 – 3.57 (m, 2H, NH-CH2), 2.95 – 2.80 (m, 2H, CH2), 2.07 – 1.87 (m, 2H), 1.80 – 1.57 (m, 3H), 1.45 – 1.18 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 168.50, 165.95, 165.03, 138.87, 128.89 (CH), 128.79 (CH), 126.69 (CH), 49.87 (CH), 42.34 (d, J = 50.5 Hz, NH-CH2), 35.87 (Ar-CH2), 33.37 (CH2), 32.77 (CH2), 25.69 (CH2), 24.81 (CH2); Rf: 0.63 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C17H23ClN5 332.1637 found 332.1635.

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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; mp 154°C; 1H NMR (400 MHz, CDCl3) δ 7.21 (t, J = 7.5 Hz, 1H), 7.16 – 7.02 (m, 3H), 6.42 (bs, 0.55H, NH-CH2), 5.97 (bs, 0.20H, NH-CH2), 5.46 (bs, 0.25H, NH-CH2), 5.39 – 5.01 (m, 1H, NH-CH ), 4.63 – 4.47 (m, 2H, NH-CH2), 3.95 – 3.67 (m, 1H, CH), 2.33 (s, 3H, CH3), 2.04 – 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, 3H); 13C NMR (101 MHz, CDCl3) δ 168.46, 166.00, 164.94, 138.56, 138.32, 128.53 (CH), 128.30 (CH), 128.17 (CH), 124.68 (CH), 49.86 (CH), 45.02 (NH-CH2), 33.31 (), 32.82 (CH2), 32.66 (CH2), 25.64 (CH2), 24.84 (CH2), 21.52 (CH3); Rf: 0.87 (PE/EtOAc 5:5); HRMS (ESI) m/z [M+H]+ calcd for C17H23ClN5 332.1637 found 332.1633.

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4.2.3. (1R)-2-[[4-Chloro-6-(cyclohexylamino)-1,3,5-triazin-2-yl]amino]-1-phenyl-ethanol (4c). MW activation time 90 min, 150°C. Yield 61%; white solid; mp 88°C; 1H NMR (400 MHz, CDCl3) δ 7.49 – 7.31 (m, 5H), 6.77 (bs, 0.61H, NH-CH2), 6.48 (bs, 0.19H, NH-CH2), 5.97 – 5.31 (m, 1.2H; 0.2H, NH-CH2; 1H NH-CH), 5.04 – 4.81 (m, 1H, CH-OH), 4.20 (bs, 1H, OH), 3.94 – 3.75 (m, 1H, CH), 3.59 – 3.39 (m, 2H, CH2), 2.11 – 1.89 (m, 2H, CH2), 1.82 – 1.58 (m, 4H, CH2), 1.44 – 1.16 (m, 4H, CH2); 13C NMR (101 MHz, CDCl3) δ 168.37, 166.31, 164.56, 142.02, 128.62 (CH), 127.89 (CH), 127.11 (CH), 125.97 (CH), 125.85 (CH), 73.74 (CH-OH), 50.04 (CH), 49.17 (HN-CH2), 33.22 (CH2), 32.71 (CH2), 32.65 (CH2), 25.57 (CH2), 24.77 (CH2); Rf : 0.62 (PE/EtOAc 5:5); HRMS (ESI) m/z [M+H]+ calcd for C17H24ClN5O 348.1586 found 348.1587.

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4.2.4 4-[[4-Chloro-6-(cyclohexylamino)-1,3,5-triazin-2-yl]amino]benzonitrile (4d). MW activation time 120 min, 150°C. Yield 64%; white so lid; mp 200°C; 1H NMR (400 MHz, CDCl3) δ 7.98 – 7.56 (m, 4H, CH), 7.47 (d, J = 34.5 Hz, 1H, NH), 5.72 – 5.25 (m, 1H, HNCH), 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, CDCl3) δ 169.27, 165.20, 165.05, 164.09, 163.61, 142.23, 133.28 (CH), 120.02 (CH), 119.96 (CH), 119.00, 106.64, 50.29 (d, J = 69.4 Hz, CH), 33.15 (CH2), 32.57 (CH2), 25.53 (CH2), 24.81 (CH2) ; Rf : 0.74 (PE/EtOAc 7:3) ; HRMS (ESI) m/z [M+H]+ calcd for C16H18ClN6 329.1276 found 329.1272. 4.2.5. 6-Chloro-N2-cyclohexyl-N4-(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (4e). MW activation time 120 min, 150°C. Yield 54%; creamy s olid; mp 226°C; 1H NMR (400 MHz, CDCl3) δ 8.32 – 8.14 (m, 2H, CH), 7.89 – 7.67 (m, 2H, CH), 5.86 – 5.39 (m, 1H, NH), 4.04 – 3.72 (m, 1H, CH), 2.03 (bs, 2H), 1.82 – 1.58 (m, 4H), 1.50 – 1.16 (m, 5H) ; 13C NMR (101 MHz, CDCl3) δ 169.30, 165.06, 164.07, 144.16, 143.15, 125.16 (CH), 119.44 (CH), 119.37 (CH), 119.28 (CH), 50.36 (d, J = 71.8 Hz ,CH), 33.13 (CH2), 32.57 (CH2), 25.53 (CH2), 24.83 (CH2) ; Rf : 0.68 (CH2Cl2/MeOH 98:2); HRMS (ESI) m/z [M+H]+ calcd for C15H18ClN6O2 349.1174 found 349.1173.

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4.2.6. 6-Chloro-N2-cyclohexyl-N4-(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (4f). MW activation time 120 min, 150°C. Yield 71%; yellow s olid; mp 208°C; 1H NMR (250 MHz, CDCl3) 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, 2H, 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 (m, 3H), 1.50 – 1.14 (m, 5H) ; 13C NMR (101 MHz, CDCl3) δ 169.06, 165.02, 164.16, 148.81, 139.38, 129.59 (CH), 125.38 (CH), 118.42 (CH), 115.14 (CH), 50.45 (CH), 33.16 (CH2), 32.84 (CH2), 25.57 (CH2), 24.87 (CH2), 24.65 (CH2); Rf : 0.46 (PE/EtOAc 8:2) ; HRMS (ESI) m/z [M+H]+ calcd for C15H18ClN6O2 349.1174 found 349.1173.

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4.2.7. 6-Chloro-N2-cyclohexyl-N4-(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (4g). MW activation time 90 min, 150°C. Yield 76%; white sol id; mp 174°C; 1H NMR (400 MHz, CDCl3) 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, CH3, CH), 2.01 (bs, 2H), 1.84 – 1.60 (m, 3H), 1.49 – 1.09 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 165.34, 164.11, 156.73, 156.43, 130.95, 130.54, 123.43 (CH), 122.45 (CH), 114.24 (CH), 114.11 (CH), 55.64 (CH3), 49.60 (CH), 33.23 (CH2), 32.61 (CH2), 25.62 (CH2), 24.82 (CH2); Rf: 0.69 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C16H21ClN5O 334.1429 found 334.1428.

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4.2.8. 6-Chloro-N2-cyclohexyl-N4-[4-(trifluoromethyl)phenyl]-1,3,5-triazine-2,4-diamine (4h). MW activation time 90 min, 150°C. Yield 80%; white solid; mp 186°C; 1H NMR (400 MHz, CDCl3) δ 7.78 – 7.63 (m, 2H, CH), 7.58 (d, J = 8.3 Hz, 2H, CH), 7.52 – 7.38 (m, 1H, NH), 5.71 – 5.32 (m, 1H, NH), 4.02 – 3.73 (m, 1H, CH), 2.14 – 1.87 (m, 2H), 1.83 – 1.60 (m, 3H), 1.47 – 1.16 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 169.18, 165.28, 165.10, 164.18, 163.77, 141.22, 126.29 (q, J = 3.7 Hz), 125.80, 125.61, 125.47, 122.92, 120.11, 119.81, 50.21 (d, J = 69.7 Hz), 33.19, 32.61, 25.57, 24.82; Rf: 0.58 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C16H18ClF3N5 372.1197 found 372.1197.

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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%; white solid; mp 154°C; 1H NMR (400 MHz, CDCl3) δ 1H NMR (400 MHz, CDCl3) δ 7.29 (t, J = 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 – 3.59 (m, 6H, CH2), 3.47 (bs, 4H, CH2), 2.87 (t, J = 7.1 Hz, 2H, CH2), 1.49 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 169.12, 165.61, 164.71, 154.78, 138.73, 128.85 (CH), 128.80 (CH), 126.71 (CH), 80.39, 43.46 (CH2), 42.37 (CH2), 35.66 (CH2), 28.53 (CH3); Rf: 0.44 (CH2Cl2/MeOH 98:2); HRMS (ESI) m/z [M+H]+ calcd for C20H28ClN6O2 419.1956 found 419.1955. 4.2.10. tert-Butyl-4-[4-chloro-6-(m-tolylmethylamino)-1,3,5-triazin-2-yl]piperazine-1carboxylate (7b). MW activation time 90 min, 150°C. Yield 83%; white solid; mp 174°C; 1H NMR (400 MHz, CDCl3) δ 7.23 (t, J = 7.4 Hz, 1H, CH), 7.17 – 7.04 (m, 3H, CH), 6.40 ( bs, 1H, NH), 4.57 (d, J = 6.0 Hz, 2H, CH2), 3.79 (bs, 4H, CH2), 3.45 (bs, 4H, CH2), 2.35 (s, 3H, CH3), 1.50 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 169.17, 165.72, 164.66, 154.75, 138.38, 138.29, 128.61 (CH), 128.25 (CH), 124.93 (CH), 124.55 (CH), 80.36, 45.16 (CH2), 44.99 (CH2), 43.47 (CH2), 28.51 (CH3), 21.52 (CH3); Rf: 0.54 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C20H28ClN6O2 419.1956 found 419.1957. 4.2.11. tert-Butyl-4-[4-chloro-6-[[(2R)-2-hydroxy-2-phenyl-ethyl]amino]-1,3,5-triazin-2yl]piperazine-1-carboxylate (7c). MW activation time 90 min, 150°C. Yield 78%; yellow ish oil; 1 H NMR (400 MHz, CDCl3) δ 7.52 – 7.28 (m, 5H, CH), 6.28 (bs, 0.6H, NH), 5.76 (bs, 0.2H, NH), 5.02 – 4.81 (m, 1H, CH), 3.76 (bs, 6H), 3.46 (bs, 5H), 1.47 (s, 9H, CH3); 13C NMR (101

ACCEPTED MANUSCRIPT MHz, CDCl3) δ 169.14, 165.95, 164.41, 154.75, 141.83, 128.72 (CH), 128.11 (CH), 128.04 (CH), 127.12 (CH), 125.98 (CH), 80.49, 73.57 (d, J = 24.9 Hz, CH), 48.67 (CH2), 43.52 (CH2), 43.35 (CH2), 28.51 (CH3); Rf: 0.32 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C20H28ClN6O3 435.1905 found 435.1905.

RI PT

4.2.12. tert-Butyl-4-[4-chloro-6-(4-cyanoanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (7d). MW activation time 120 min, 150°C. Yield 93%; cream y solid; 1H NMR (400 MHz, CDCl3) δ 7.87 – 7.54 (m, 4H), 3.96 – 3.75 (m, 4H, CH2), 3.54 (bs, 4H, CH2), 1.51 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 170.45, 169.63, 164.43, 163.62, 154.54, 154.43, 142.01, 133.21, 119.91, 118.85, 106.52, 80.69, 80.53, 53.44, 43.98 (CH2), 43.80 (CH2), 43.64 (CH2), 28.39 (CH3), 28.36 (CH3); Rf: 0.26 (CH2Cl2/MeOH 98:2); HRMS (ESI) m/z [M+H]+ calcd for C19H23ClN7O2 416.1596 found 416.1595.

M AN U

SC

4.2.13. tert-Butyl-4-[4-chloro-6-(4-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (7e). MW activation time 90 min, 150°C. Yield 40%; cream y amorphous solid; 1H NMR (400 MHz, CDCl3) 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 – 3.80 (m, 4H, CH2), 3.62 – 3.45 (m, 4H, CH2), 1.49 (s, 9H, CH3). 13C NMR (101 MHz, CDCl3) δ 169.99, 164.66, 163.83, 154.68, 143.97, 143.23, 125.27 (CH), 119.40 (CH), 80.70, 43.98 (CH2), 43.84 (CH2), 28.54 (CH3); Rf: 0.31 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C18H23ClN7O4 436.1494 found 436.1493.

TE D

4.2.14. tert-Butyl-4-[4-chloro-6-(3-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (7f). MW activation time 90 min, 150°C. Yield 59%; creamy solid; 1H NMR (400 MHz, CDCl3) δ 8.93 (bs, 1H, NH), 8.04 – 7.93 (m, 1H, CH), 7.89 – 7.42 (m, 3H, CH), 3.88 (bs, 4H, CH2), 3.64 – 3.43 (m, 4H, CH2), 1.49 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 169.78, 164.55, 163.89, 154.74, 148.72, 139.27, 129.73 (CH), 125.50 (CH), 118.39 (CH), 115.29 (CH), 80.65, 44.06 (CH2), 43.85 (CH2), 28.54 (CH3); Rf: 0.31 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C18H23ClN7O4 436.1494 found 436.1492.

AC C

EP

4.2.15. tert-Butyl-4-[4-chloro-6- (4-methoxyanilino)1,3,5-triazin-2-yl] piperazine-1carboxylate (7g). MW activation time 90 min, 150°C. Yield 79%; white solid; mp 162°C; 1H NMR (400 MHz, CDCl3) δ 7.89 – 7.55 (m, 1H, NH), 7.44 – 7.33 (m, 2H), 6.89 – 6.82 (m, 2H), 3.84 – 3.72 (m, 7H), 3.46 (bs, 4H), 1.47 (s, 9H); 13C NMR (101 MHz, CDCl3) δ 164.70, 164.03, 156.75, 156.50, 154.69, 130.70, 130.39, 123.38 (CH), 122.91 (CH), 114.13 (CH), 80.38, 55.56 (CH3), 43.53 (CH2), 28.48 (CH3); Rf: 0.24 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C19H26ClN6O3 421.1749 found 421.1749. 4.2.16. tert-Butyl-4-[4-chloro-6-[4-(trifluoromethyl)anilino]-1,3,5-triazin-2-yl]piperazine-1carboxylate (7h). MW activation time 90 min, 150°C. Yield 98%; amorph ous beige solid; 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.6 Hz, 2H, CH), 7.60 (d, J = 8.8 Hz, 2H, CH), 7.51 (s, 1H, NH), 3.94 – 3.74 (m, 4H, CH2), 3.51 (bs, 4H, CH2), 1.49 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 164.67, 163.89, 154.71, 141.08, 126.39, 126.35, 125.86, 125.59, 125.54, 119.99, 80.60, 43.87, 43.73, 28.52; 19F NMR (376 MHz, CDCl3) δ -62.05; Rf: 0.69 (CH2Cl2/MeOH 98:2); HRMS (ESI) m/z [M+H]+ calcd for C19H23ClF3N6O2 459.1518 found 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 MgSO4 and concentrated. Purification by column chromatography on silica gel gave desired product.

SC

RI PT

4.3.1. 4-(Cyclohexylamino)-6-(phenethylamino)-1,3,5-triazine-2-carbonitrile (5a). Yield 97%; white solid; mp 188°C; 1H NMR (400 MHz, CDCl3) δ 7.41 – 7.30 (m, 2H), 7.28 – 7.17 (m, 3H), 6.10 (bs, 0.48H, NH-CH2), 5.74 (bs, 0.23H, NH-CH2), 5.56 – 5.01 (m, 1.29H; 0.29H, NHCH2; 1H NH-CH), 3.99 – 3.79 (m, 1H, CH), 3.76 – 3.60 (m, 2H, NH-CH2), 3.00 – 2.80 (m, 2H, CH2), 2.10 – 1.89 (m, 2H), 1.84 – 1.60 (m, 3H), 1.47 – 1.18 (m, 5H) ; 13C NMR (101 MHz, CDCl3) δ 165.01, 164.11, 151.82, 138.71, 128.91 (CH), 128.81 (CH), 126.74 (CH), 115.24 (CN), 49.86(CH), 42.46 (NH-CH2), 35.71 (Ar-CH2), 33.25 (CH2), 32.65 (CH2), 25.62 (CH2), 24.84 (CH2) ; Rf: 0.48 (PE/EtOAc 8:2) ; HRMS (ESI) m/z [M+H]+ calcd for C18H23N6 323.1979 found 323.1977.

TE D

M AN U

4.3.2. 4-(Cyclohexylamino)-6-(m-tolylmethylamino)-1,3,5-triazine-2-carbonitrile (5b). Yield 93%; white solid; mp 116°C; 1H NMR (400 MHz, CDCl3) δ 7.24 (t, J = 7.5 Hz, 1H), 7.20 – 7.07 (m, 3H), 6.32 (bs, 0.48H, NH-CH2), 5.96 (bs, 0.22H, NH-CH2), 5.58 (bs, 0.3H, NH-CH2), 5.52 – 5.12 (m, 1H, NH-CH), 4.64 – 4.49 (m, 2H, NH-CH2), 3.94 – 3.65 (m, 1H, CH), 2.37 (d, J = 6.6 Hz, 3H, CH3), 2.03 – 1.89 (m, 2H), 1.82 – 1.73 (m, 2H), 1.69 – 1.57 (m, 1H), 1.48 – 1.33 (m, 2H), 1.29 – 1.17 (m, 3H); 13C NMR (101 MHz, CDCl3) δ 165.05, 164.10, 152.41, 151.89, 138.48, 137.97, 128.78 (CH), 128.65 (CH), 128.40 (CH), 124.75 (CH), 115.22 (CN), 49.84 (CH), 44.95 (NH-CH2), 33.19 (CH2), 32.69 (CH2), 32.56 (CH2), 25.57 (CH2), 24.84 (CH2), 21.52(CH3); Rf: 0.75 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C18H23N6 323.1979 found 323.1977.

AC C

EP

4.3.3. 4-(Cyclohexylamino)- 6-[[(2R)-2-hydroxy-2-phenyl-ethyl]amino]- 1,3,5-triazine-2carbonitrile (5c). Yield 87%; amorphous creamy solid; 1H NMR (400 MHz, CDCl3) δ 7.62 – 7.25 (m, 5H), 6.98 – 6.74 (m, 0.57H, NH), 6.50 (s, 0.2H, NH), 5.95 (bs, 0.33H, NH), 5.72 – 5.29 (m, 0.9H, NH), 5.09 – 4.86 (m, 1H, CH-OH), 4.00 – 3.70 (m, 2H, CH and OH), 3.66 – 3.35 (m, 2H, CH2 ), 2.26 – 1.87 (m, 2H), 1.89 – 1.59 (m, 3H), 1.49 – 1.10 (m, 5H); 13C NMR (101 MHz, CDCl3) 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), 33.05 (CH2), 32.52 (CH2), 32.44 (CH2), 25.46 (CH2), 24.81 (CH2); Rf: 0.83 (PE/EtOAc 5:5); HRMS (ESI) m/z [M+H]+ calcd for C18H23N6O 339.1928 found 339.1926. 4.3.4. 4-(4-Cyanoanilino)-6-(cyclohexylamino)-1,3,5-triazine-2-carbonitrile (5d). Yield 56%; white solid; mp >260°C; 1H NMR (400 MHz, DMSO) δ 10.76 – 10.38 (m, 1H, NH), 8.68 – 8.25 (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, CH), 3.72 (s, 1H, CH), 1.95 – 1.51 (m, 5H), 1.47 – 1.10 (m, 5H) ; 13C 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 (CH), 119.05, 115.10, 104.54, 104.36, 49.56 (CH), 32.12 (CH2), 31.67 (CH2), 25.14 (CH2), 25.05 (CH2), 24.58 (CH2) ; Rf: 0.69 (PE/EtOAc 7:3) ; HRMS (ESI) m/z [M+H]+ calcd for C17H18N7 320.1618 found 320.1616. 4.3.5. 4-(Cyclohexylamino)-6-(4-nitroanilino)-1,3,5-triazine-2-carbonitrile (5e). Yield 19%; amorphous orange solid; 1H NMR (250 MHz, DMSO) δ 10.67 (d, J = 19.0 Hz, 0.7H, NH), 10.09 (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, 1H, CH), 1.90 – 1.52 (m, 5H), 1.39 – 1.07 (m, 5H); 13C 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), 49.57 (CH), 32.07 (CH2), 31.63 (CH2), 25.11 (CH2), 24.50 (CH2); Rf: 0.43 (PE/EtOAc 8:2) ; HRMS (ESI) m/z [M+H]+ calcd for C16H18N7O2 340.1516 found 340.1517.

RI PT

4.3.6. 4-(Cyclohexylamino)-6-(3-nitroanilino)-1,3,5-triazine-2-carbonitrile (5f). Yield 67%; amorphous creamy solid; 1H NMR (400 MHz, CDCl3) δ 9.23 – 8.48 (m, 1H, NH), 8.09 – 7.93 (m, 1H, CH), 7.90 – 7.35 (m, 3H, CH), 5.95 – 5.42 (m, 1H, NH), 3.99 (bs, 1H, CH), 2.24 – 1.92 (m, 2H), 1.82 – 1.64 (m, 3H), 1.55 – 1.23 (m, 5H) ; 13C NMR (101 MHz, CDCl3) δ 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 (CH2), 32.72 (CH2), 25.52 (CH2), 24.88 (CH2), 24.63 (CH2) ; Rf: 0.59 (PE/EtOAc 9:1) ; HRMS (ESI) m/z [M+H]+ calcd for C16H18N7O2 340.1516 found 340.1515.

M AN U

SC

4.3.7. 4-(Cyclohexylamino)-6-(4-methoxyanilino)-1,3,5-triazine-2-carbonitrile (5g). Yield 96%; creamy solid; 1H NMR (400 MHz, CDCl3) δ 7.57 – 7.37 (m, 2H, CH), 7.33 – 7.04 (m, 1H, NH), 6.98 – 6.85 (m, 2H, CH), 5.71 – 5.29 (m, 1H, NH), 3.97 – 3.70 (m, 4H, CH3, CH), 2.15 – 1.92 (m, 2H), 1.86 – 1.58 (m, 3H), 1.51 – 1.13 (m, 5H) ; 13C NMR (101 MHz, CDCl3) δ 164.56, 164.24, 156.97, 156.67, 152.61, 152.07, 130.46, 130.04, 123.45 (CH), 122.49 (CH), 115.34 (CN), 114.34 (CH), 114.19 (CH), 55.66 (CH3), 49.95 (CH), 33.12 (CH2), 32.48 (CH2), 25.55 (CH2), 24.82 (CH2) ; Rf: 0.42 (PE/EtOAc 8:2) ; HRMS (ESI) m/z [M+H]+ calcd for C17H21N6O 325.1771 found 325.1770.

TE D

4.3.8. 4-(Cyclohexylamino)-6-[4-(trifluoromethyl)anilino]-1,3,5-triazine-2-carbonitrile (5h). Yield 94%; white solid; mp 198°C; 1H NMR (400 MHz, CDCl3) δ 7.92 – 7.57 (m, 4H, CH), 7.53 – 7.32 (m, 1H, NH), 5.75 – 5.29 (m, 1H, NH), 3.98 – 3.74 (m, 1H, CH), 2.11 – 1.95 (m, 2H), 1.86 – 1.63 (m, 3H), 1.45 – 1.23 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 164.53, 164.24, 152.33, 140.79, 126.42 (CH), 126.38 (CH), 126.04 (dd, J = 1419.6, 1147.9 Hz), 120.22 (CH), 119.95 (CH), 114.88 (CN), 50.22 (CH), 33.08 (CH2), 32.50 (CH2), 25.52 (CH2), 24.82 (CH2); 19 F NMR (376 MHz, CDCl3) δ -62.10; Rf: 0.70 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C17H18F3N6 363.1540 found 363.1539.

AC C

EP

4.3.9. tert-Butyl-4-[4-cyano-6-(phenethylamino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (8a). Yield 82%; white solid; mp 196°C; 1H NMR (400 MHz, CDCl3) δ 7.33 (t, J = 7.3 Hz, 2H, 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, CH2), 3.55 – 3.42 (m, 4H, CH2), 2.90 (t, J = 7.1 Hz, 2H, CH2), 1.51 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 164.80, 163.77, 154.74, 152.00, 138.54, 128.93 (CH), 128.86 (CH), 126.82 (CH), 115.36 (CN), 80.51, 43.56 (CH2), 43.14 (CH2), 42.25 (CH2), 35.90 (CH2), 35.51 (CH2), 28.53 (CH3); Rf: 0.56 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C21H28N7O2 410.2299 found 410.2295. 4.3.10. tert-Butyl-4-[4-cyano-6-(m-tolylmethylamino)-1,3,5-triazin-2-yl]piperazine-1carboxylate (8b). Yield 88%; white solid; mp 162°C; 1H NMR (400 MHz, MeOD) δ 7.20 – 6.96 (m, 4H), 4.45 (d, J = 10.2 Hz, 2H, CH2), 3.72 (bs, 4H, CH2), 3.48 – 3.33 (m, 4H, CH2), 2.27 (s, 3H, CH3), 1.43 (s, 9H, CH3); 13C NMR (101 MHz, MeOD) δ 166.05, 164.94, 156.33, 153.17, 139.99, 139.16, 129.38 (CH), 129.23 (CH), 128.82 (CH), 125.62 (CH), 116.37 (CN), 81.60, 45.37 (CH2), 44.37 (CH2), 44.07 (CH2), 28.63 (CH3), 21.47 (CH3); Rf: 0.81 (PE/EtOAc 7:3); HRMS (ESI) m/z [M+H]+ calcd for C21H28N7O2 410.2299 found 410.2297.

ACCEPTED MANUSCRIPT

RI PT

4.3.11. tert-Butyl-4-[4-cyano-6- [[(2R)-2-hydroxy-2-phenyl-ethyl]amino]- 1,3,5-triazin-2yl]piperazine-1-carboxylate (8c). Yield 61%; white solid; mp 160°C; 1H NMR (400 MHz, CDCl3) 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), 4.93 (bs, 1H, CH-OH), 3.96 – 3.65 (m, 5H, CH2), 3.61 – 3.33 (m, 5H, CH2), 3.16 – 2.83 (m, 1H, OH), 1.50 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 165.21, 163.63, 154.73, 152.09, 141.58, 128.86 (CH), 128.80 (CH), 128.37 (CH), 127.12 (CH), 125.99 (CH), 115.27 (CN), 80.58, 73.38 (d, J = 24.7 Hz, CH-OH), 48.45 (CH2), 48.28 (CH2), 43.60 (CH2), 43.20 (CH2), 28.53 (CH3); Rf: 0.69 (PE/EtOAc 5:5); HRMS (ESI) m/z [M+H]+ calcd for C21H28N7O3 426.2248 found 426.2246.

SC

4.3.12. tert-Butyl-4-[4-cyano-6-(4-cyanoanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (8d). Yield quantitative; yellow amorphous solid; 1H NMR (400 MHz, CDCl3) δ 7.76-7.62 (m, 4H), 7.53 (s, 1H, NH), 3.97 – 3.77 (m, 4H, CH2), 3.64 – 3.45 (m, 4H, CH2), 1.51 (s, 9H, CH3); 13 C NMR (101 MHz, CDCl3) δ 163.72, 163.46, 154.66, 152.47, 141.74 (CH), 133.41 (CH), 120.20, 118.87, 114.95 (CN), 107.09, 80.78, 43.85 (CH2), 43.68 (CH2), 28.52 (CH3); Rf: 0.91 (CH2Cl2/MeOH 9:1); HRMS (ESI) m/z [M+H]+ calcd for C20H23N8O2 407.1938 found 407.1937.

M AN U

4.3.13. tert-Butyl-4-[4-cyano-6-(4-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (8e). Yield 75%; creamy solid; 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 9.1 Hz, 2H, CH), 7.72 (d, J = 9.1 Hz, 2H, CH), 7.42 (s, 1H, NH), 3.95 – 3.78 (m, 4H, CH2), 3.60 – 3.47 (m, 4H, CH2), 1.50 (s, 9H, CH3); 13C NMR (63 MHz, CDCl3) δ 163.74, 163.47, 154.65, 152.54, 143.51, 125.29 (CH), 119.66 (CH), 114.90 (CN), 80.82, 43.92 (CH2), 43.75 (CH2), 28.54 (CH3); Rf: 0.25 (CH2Cl2); HRMS (ESI) m/z [M+H]+ calcd for C19H23N8O4 427.1836 found 427.1835.

EP

TE D

4.3.14. tert-Butyl-4-[4-cyano-6-(3-nitroanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (8f). Yield 85%; yellowish solid; mp 256°C; 1H NMR (400 MHz, CDCl3) δ 8.92 (bs, 1H, NH), 7.98 (dd, J = 8.7, 1.7 Hz, 1H, CH), 7.69 – 7.43 (m, 3H, CH), 4.02 – 3.76 (m, 4H, CH2), 3.62 – 3.39 (m, 4H, CH2), 1.50 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 163.67, 154.70, 152.52, 148.75, 138.84, 129.92, 118.77, 115.33, 114.95, 80.76, 43.96, 28.54; Rf: 0.25 (CH2Cl2); HRMS (ESI) m/z [M+H]+ calcd for C19H23N8O4 427.1836 found 427.1836.

AC C

4.3.15. tert-Butyl-4-[4-cyano-6-(4-methoxyanilino)-1,3,5-triazin-2-yl]piperazine-1-carboxylate (8g). Yield quantitative; yellow amorphous solid; 1H NMR (250 MHz, CDCl3) δ 7.49 – 7.31 (m, 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); 13C NMR (63 MHz, CDCl3) δ 156.86, 154.71, 130.19, 123.29, 122.89, 114.36, 80.57, 55.67, 43.66, 43.39, 28.54; Rf: 0.78 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C20H26N7O3 412.2091 found 412.2089. 4.3.16. tert-Butyl-4-[4-cyano-6-[4-(trifluoromethyl)anilino]-1,3,5-triazin-2-yl]piperazine-1carboxylate (8h). Yield 77%; yellow solid; mp 128°C; 1H NMR (400 MHz, CDCl3) δ 7.72 – 7.56 (m, 4H, CH), 7.37 (bs, 1H, NH), 3.99 – 3.75 (m, 4H, CH2), 3.52 (bs, 4H, CH2), 1.49 (s, 9H, CH3); 13C NMR (101 MHz, CDCl3) δ 163.79, 154.68, 152.48, 140.65, 126.49 (CH), 126.46 (CH), 126.28 (CH), 120.14 (CH), 115.03 (CN), 80.73, 43.82 (CH2), 43.62 (CH2), 28.53 (CH3); 19F NMR (376 MHz, CDCl3) δ -62.13; Rf: 0.36 (PE/EtOAc 8:2); HRMS (ESI) m/z [M+H]+ calcd for C20H23F3N7O2 450.1859 found 450.1857. 4.4. General procedure for compounds 9 a-h

ACCEPTED MANUSCRIPT To the solution of 8 in CH2Cl2 was added TFA (16 equiv). The reaction mixture was stirred for 90 minutes under N2 at room temperature. The reaction mixture was then concentrated and the residue was dissolved in CH2Cl2 and NaHCO3 sat. was added. The aqueous phase was extracted with CH2Cl2. The combined organic phases were washed with water, dried over MgSO4 and concentrated to give the desired product 9.

SC

RI PT

4.4.1. 4-(Phenethylamino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9a). Yield 90%; white solid; mp 196°C; 1H NMR (400 MHz, CDCl3) δ 7.32 (t, J = 7.6 Hz, 2H, CH), 7.27 – 7.18 (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, CH2), 3.01 – 2.81 (m, 6H, CH2), 2.04 (s, 1H, NH); 13C NMR (101 MHz, CDCl3) δ 164.72, 163.49, 151.90, 138.68, 128.92 (CH), 128.88 (CH), 128.80 (CH), 128.76 (CH), 126.73 (CH), 115.47 (CN), 46.01 (CH2), 45.88 (CH2), 44.89 (CH2), 44.37 (CH2), 42.23 (CH2), 35.51 (CH2); Rf: 0.39 (CH2Cl2/MeOH 85:15); HRMS (ESI) m/z [M+H]+ calcd for C16H20N7 310.1775 found 310.1774.

M AN U

4.4.2. 4-(m-Tolylmethylamino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9b). Yield 85%; white solid; mp 158°C; 1H NMR (400 MHz, CDCl3) δ 7.24 (t, J = 7.4 Hz, 1H), 7.18 – 7.06 (m, 3H), 5.97 (bs, 0.4H, NH), 5.50 (bs, 0.2H, NH), 4.65 – 4.49 (m, 2H, CH2), 3.96 – 3.62 (m, 4H, CH2), 2.89 (bs, 4H, CH2), 2.36 (s, 3H, CH3), 1.85 (bs, 1H, NH); 13C NMR (101 MHz, CDCl3) δ 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 (CH2), 45.82 (CH2), 44.96 (CH2), 44.91 (CH2), 44.78 (CH2), 44.47 (CH2), 21.52 (CH3); Rf: 0.35 (CH2Cl2/MeOH 9:1); HRMS (ESI) m/z [M+H]+ calcd for C16H20N7 310.1775 found 310.1773.

EP

TE D

4.4.3. 4-[[(2R)-2-Hydroxy-2-phenyl-ethyl]amino]-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9c). Yield 54%; white amorphous solid; 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.29 (m, 5H), 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, 4H, CH2), 3.56 – 3.38 (m, 2H, CH2), 2.99 – 2.34 (m, 6H, CH2, NH, OH) ; 13C NMR (101 MHz, CDCl3) δ 165.05, 163.26, 151.88, 141.89, 128.74 (CH), 128.16 (CH), 125.99 (CH), 115.36 (CN), 73.01 (CH), 48.36 (CH2), 45.83 (CH2), 45.66 (CH2), 44.84 (CH2), 44.32 (CH2) ; Rf: 0.53 (CH2Cl2/MeOH 8:2) ; HRMS (ESI) m/z [M+H]+ calcd for C16H20N7O 326.1724 found 326.1721.

AC C

4.4.4. 4-(4-Cyanoanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9d). Yield 75%; creamy solid; mp 252°C; 1H 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, CH2), 3.28 (bs, 1H, NH), 2.88 – 2.69 (m, 4H, CH2); 13C NMR (101 MHz, DMSO) δ 162.80, 162.60, 151.41, 142.91, 133.12 (CH), 120.08 (CH), 119.05 (CN), 115.18 (CN), 104.64, 45.19 (CH2), 45.10 (CH2), 44.75 (CH2), 44.54 (CH2); Rf: 0.38 (CH2Cl2/MeOH 85:15) ; HRMS (ESI) m/z [M+H]+ calcd for C15H15N8 307.1414 found 307.1413. 4.4.5. 4-(4-Nitroanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9e). Yield 81%; amorphous yellow solid; 1H NMR (400 MHz, MeOD) δ 8.24 (d, J = 9.2 Hz, 2H, CH), 7.88 (d, J = 9.2 Hz, 2H, CH), 3.90 (bs, 4H, CH2), 2.96 (bs, 4H, CH2); 13C NMR (63 MHz, CDCl3/MeOD 5:1) δ 163.30, 152.10, 144.52, 142.65, 124.70 (CH), 119.54 (CH), 114.78 (CN), 45.00 (CH2), 44.91 (CH2), 44.19 (CH2), 43.93 (CH2); Rf: 0.27 (CH2Cl2/MeOH 9:1); HRMS (ESI) m/z [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 solid; mp 130°C; 1H NMR (400 MHz, Acetone) δ 8.99 (bs, 1H, CH), 8.17 – 7.80 (m, 2H, CH),

ACCEPTED MANUSCRIPT 7.62 (t, J = 8.2 Hz, 1H, CH), 4.08 – 3.69 (m, 4H, CH2), 3.09 – 2.58 (m, 4H, CH2); 13C NMR (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.

RI PT

4.4.7. 4-(4-Methoxyanilino)-6-piperazin-1-yl-1,3,5-triazine-2-carbonitrile (9g). Yield 60%; yellow amorphous solid; 1H NMR (400 MHz, CDCl3) δ 7.50 (bs, 1H, NH), 7.38 (d, J = 8.9 Hz, 2H, CH), 6.88 (d, J = 8.9 Hz, 2H, CH), 3.87 – 3.72 (m, 7H CH3, CH2), 2.97 – 2.80 (m, 4H, CH2), 1.94 (bs, 1H, NH); 13C NMR (101 MHz, CDCl3) δ 163.50, 156.61, 152.16, 130.42, 122.75 (CH), 115.34 (CN), 114.24 (CH), 55.60 (CH3), 45.94 (CH2), 45.83 (CH2), 45.00 (CH2), 44.62 (CH2); Rf: 0.29 (CH2Cl2/MeOH 9:1); HRMS (ESI) m/z [M+H]+ calcd for C15H18N7O 312.1567 found 312.1567.

4.5.

Kinetic assays

M AN U

SC

4.4.8. 4-Piperazin-1-yl-6-[4-(trifluoromethyl)anilino]-1,3,5-triazine-2-carbonitrile (9h). Yield 83%; white solid; mp 224°C; 1H 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, CH2), 3.07 – 2.63 (m, 5H, CH2, NH); 13C NMR (101 MHz, DMSO) δ 162.64, 151.42, 142.20, 125.99 (CH), 125.73 (CH), 123.19, 123.03, 122.87, 120.05 (CH), 115.23 (CN), 99.50, 45.22 (CH2), 45.14 (CH2), 44.74 (CH2), 44.54 (CH2); 19F NMR (376 MHz, DMSO) δ -60.31; Rf: 0.31 (CH2Cl2/MeOH 8:2); HRMS (ESI) m/z [M+H]+ calcd for C15H15F3N7 350.1336 found 350.1334.

AC C

EP

TE D

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 reported.31 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-trans2,3-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; λex = 350 nm, λem = 460 nm). The same protocol 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 IC50 determined (software Softmaxpro, Molecular Devices). 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: [email protected]

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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References

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1. B. Turk, V. Turk, D. Turk, Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol. Chem. 378 (1997) 141-150. (b) J. Reiser, B. Adair, T. Reinheckel, Specialized roles for cysteine cathepsins in health and disease, J. Clin. Invest. 120 (2010) 3421–3431. (c) B. Turk, D. Turk, G. S. Salvesen, Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. Curr. Pharm. Des. 8 (2002) 1623-1637. 2. O. Vasiljeba, T. Reinheckel, C. Peters, D. Turk, V. Turk, B. Turk, Emerging roles of cysteine cathepsins in disease and their potential as drug targets. Curr. Pharm. Des. 13 (2007) 387403. 3. see MEROPS: the peptidase database: http://merops.sanger.ac.uk 4. A. Fengler, W. Brandt, Three-dimensional structures of the cysteine proteases cathepsins K and S deduced by knowledge-based modelling and active site characteristics. Protein Eng. 11 (1998) 1007-1013. 5. V. Turk, V. Stoka, O. Vasiljeva, M. Renko, T. Sun, B. Turk, D. Turk, Cysteine cathepsins: from structure, function and regulation to new frontiers. BBA-Protein Proteom. 1 (2012), 68-88. 6. Z. Khalkhali-Ellis, W. Goossens, N.V. Margaryan, M.J. Hendrix, Cleavage of histone 3 by cathepsin D in the involuting mammary gland. PLoS One 9 (2014) e103230. 7. B. Sobotič, M. Vizovišek, R. Vidmar, P. Van Damme, V. Gocheva, J. A. Joyce, K. Gevaert, V. Turk, B. Turk, M. Fonović, Proteomic identification of cysteine cathepsin substrates shed from the surface of cancer cells. Mol. Cell Proteomics. 14 (2015) 2213-2228. 8. O.C. Olson, J.A. Joyce, Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat. Rev. Cancer 15 (2015) 712-729. 9. S. Conus, H. U. Simon, Cathepsins: key modulators of cell death and inflammatory responses. Biochem. Pharmacol. 76 (2008) 1374-1382. 10. C. Palermo, J.A. Joyce, Cysteine cathepsin proteases as pharmacological targets in cancer. Trends Pharmacol. Sci. 29 (2008) 22-28. 11. F. Lecaille, J. Kaleta, D. Brömme, Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem. Rev. 102 (2002) 4459–4488) 12. R.D. Wilkinson, R. Williams, C.J. Scott, R.E. Burden, Cathepsin S: therapeutic, diagnostic and prognostic potential. Biol. Chem. 396 (2015) 867-882. (b) L. Zhang, H. Wang, J. Xu, Cathepsin S as a cancer target. Neoplasma 62 (2015) 16-26. (c) M. Perisic Nanut, J. Sabotic, A. Jewett, J. Kos, Cysteine cathepsins as regulators of the cytotoxicity of NK and T cells. Front. Immunol. 5 (2014) 616. 13. A.M. Helali, F.M. Iti, I.N. Mohamed, Cathepsin K inhibitors: a novel target but promising approach in the treatment of osteoporosis. Curr. Drug Targets 14 (2013) 1591-1600. 14. U. Verbovsek, C.J. Van Noorden, T.T. Lah, Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression. Semin. Cancer Biol. 35 (2015) 71-84. 15. (a) M. Kasabova, A. Saidi, C. Naudin, J. Sage, F. Lecaille, G. Lalmanach, Cysteine Cathepsins: Markers and Therapy Targets in Lung Disorders. Clinic. Rev. Bone. Miner. Metab. 9 (2011) 148–161. (b) D. Brömme, F. Lecaille, Cathepsin K inhibitors for osteoporosis and potential off-target effects. Expert. Opin. Investig.Drugs 18 (2009) 585–600. 16. G. Lalmanach, A. Saidi, S. Marchand-Adam, F. Lecaille, M. Kasabova, Cysteine cathepsins and cystatins: from ancillary tasks to prominent status in lung diseases. Biol. Chem. 396 (2015) 111–130. 17. M. Frizler, M. D. Mertens, M. Guetschow, Fluorescent nitrile-based inhibitors of cysteine cathepsins, Bioorg. Med. Chem. Lett. 22 (2012) 7715-7718.

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18. F. F. Fleming, L. H. Yao, P. C. Ravikumar, L. Funk, B. C. Shook, Nitrile-containing pharmaceuticals: efficacious roles of the nitrile pharmacophore, J. Med. Chem. 53 (2010) 7902-7917. 19. S. Fustero, V. Rodrigo, M. Sanchez-Rosello, C. del Pozo, J. Timoneda, M. Frizler, M. T. Sisay, J. Bajorath, L. P. Calle, F. J. Canada, J. Jimenez-Barbero, M. Gutschow, New cathepsin inhibitors to explore the fluorophilic properties of the S-2 pocket of cathepsin B: design, synthesis, and biological evaluation, Chem. Eur. J. 17 (2011), 5256-5260. 20. P. D. Greenspan, K. L. Clark, R. A. Tommasi, S. D. Cowen, L. W. McQuire, D. L. Farley, J. H. van Duzer, R. L. Goldberg, H. Zhou, Z. Du, J. J. Fitt, D. E. Coppa, Z. Fang, W. Macchia, L. Zhu, M. P. Capparelli, R. Goldstein, A. M. Wigg, J. R. Doughty, R. S. Bohacek, A. K. Knap, Identification of dipeptidyl nitriles as potent and selective inhibitors of cathepsin B through structure-based drug design, J. Med. Chem. 44 (2001) 4524-4534. 21. Z. Rankovic, J. Cai, I. Cumming, Preparation of 2-cyano-1,3,5-triazine-4,6-diamine derivatives for the treatment of osteoporosis and atherosclerosis. WO2005011703A1 (2005). 22. I. Sosic, B. Mirkovic, S. Turk, B. Stefane, J. Kos, S. Gobec, Discovery and kinetic evaluation of 6-substituted 4-benzylthio-1,3, 5-triazin-2(1H)-ones as inhibitors of cathepsin B, Eur. J. Med. Chem. 46 (2011) 4648-4656. 23. A. Ripka, G. Shapiro, A. McRiner, Preparation of imidazotriazinone derivatives for use as phosphodiesterase 9 inhibitors. WO2012040230A1 (2012). 24. Z. Rankovic, J. Cai, J. Kerr, X. Fradera, J. Robinson, A. Mistry, E. Hamilton, G. McGarry, F. Andrews, W. Caulfield, I. Cumming, M. Dempster, J. Waller, P. Scullion, I. Martin, A. Mitchell, C. Long, M. Baugh, P. Westwood, E. Kinghorn, J. Bruin, W. Hamilton, J. Uitdehaag, M. van Zeeland, D. Potin, L. Saniere, A. Fouquet, F. Chevallier, H. Deronzier, C. Dorleans, E. Nicolai, Design and optimisation of a series of novel 2-cyano-pyrimidines as cathepsin K inhibitors, Bioorg. Med. Chem. Lett. 20 (2010) 1524-1527. 25. G. M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju, W. Sherman, Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aid. Mol. Des. 27 (2013) 221-234. 26. LigPrep, version 3.0 Schrödinger, LLC, New York, NY 2014. 27. Glide, version 6.3, Schrödinger, LLC, New York, NY 2014. 28. K. Zhu, K. W. Borelli, J. R. Greenwood, T. day, R. Abel, R. S. Farid, E. Harder, Docking covalent inhibitors: a parameter free approach to pose prediction and scoring. J. Chem. Inf. Model. 54 (2014) 1932-1940. 29. X.-F. Ren, H.-W. Li, X. Fang, Y. Wu, L. Wang, S. Zou, Highly selective azadipeptide nitrile inhibitors for cathepsin K : design, synthesis and activity assays. Org. Biomol. Chem. 11 (2013) 1143-1148. 30. X.-Y. Yuan, D.-Y. Fu, X.-F. Ren, X. Fang, L. Wang, S. Zou, Y. Wu, Highly selective azadipeptide nitrile inhibitors for cathepsin K, structural optimization and molecular modeling. Org. Biomol. Chem. 11 (2013) 5847-5852. 31. F. Lecaille, E. Weidauer, M.A. Juliano, D. Brömme, G. Lalmanach, Cathepsin K activity by a selective substrate spanning its active site. Biochem. J. 375 (2003) 307-312.

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, CH2Cl2; (ii) H2N-R, DIPEA, CH3CN, 150°C, MW; ( iii) KCN, DABCO, DMSO, H2O, overnight, rt; (iv) TFA, CH2Cl2, rt.

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Table 1. The IC50 values of the target compounds 5a-h and 9a-h against cathepsins B, K, L and S

DRAWINGS CN

N H

N

N N

N H

R

N H

N 1

5

CN

N

R

N NH

EP

AC C

Figure 2.

TE D

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Figure 1.

N

Figure 3.

N H

N

N

SC

CN N

9

N

NH

ACCEPTED MANUSCRIPT ii

iii

i 3

i

iii

ii

6

7a-h

RI PT

2

5a-h

4a-h

8a-h, R = Boc 1

9a-h, R = H

NH -R =

b

e

f

Scheme 1.

d

g

h

IC50 [nM]

Compounds

Cat B No inhibition

5b

No inhibition

5c

Cat K

Cat L

Cat S

80

No inhibition

23

60

No inhibition

23

No inhibition

70

No inhibition

23

5d

No inhibition

67

No inhibition

23

5e

TE D

5a

No inhibition

17

136

90

17

23

28

No inhibition

23

226

136

No inhibition

34

5g

AC C

5h

79

EP

5f

9a

No inhibition

No inhibition

No inhibition

102

No inhibition

No inhibition

9b

57

23

9c

136

68

45

9d

57

28

113

17

17

23

9e

No inhibition

No inhibition

17 23 No inhibition

9f

34

17

17

17

9g

45

17

17

17

9h 24

1 Table 1.

c

M AN U

a

SC

2

No inhibition

28

No inhibition

23

520

1

1711

158

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

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Docking studies of active 9h into X-ray crystal structure of cathepsins K and S

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IC50 values of some molecules were in the nanomolar range against Cat K & Cat S