N-[6-(4-butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin- oxoethyl]-1h-indole-3-carboxamide (SAR216471), a. Acting P2Y12 Antagonist

Transcript

1 Supplementary Material N-[6-(4-butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin- 3-yl]-5-chloro-1-[2-(4-methyl-piperazin-1-yl)-2- oxoethyl]-1h-indole-3-carboxamide (SAR216471), a Novel Intravenous and Oral, Reversible and Directly Acting P2Y12 Antagonist Christophe Boldron *,#, Angélina Besse #, Marie-Françoise Bordes #, Stéphanie Tissandié #, Xavier Yvon #, Benjamin Gau #, Alain Badorc #, Tristan Rousseaux #, Guillaume Barré #, Jérôme Meneyrol #, Gernot Zech, Marc Nazare, Valérie Fossey, Anne-Marie Pflieger #, Sandrine Bonnet-Lignon #, Laurence Millet #, Christophe Briot $, Frédérique Dol #, Jean-Pascal Hérault #, Pierre Savi #, Gilbert Lassalle #, Nathalie Delesque #, Jean-Marc Herbert #, Françoise Bono #. # Sanofi R&D, 195 Route d'espagne, Toulouse Cedex, France Sanofi R&D, 1 Av. Pierre Brossolette Chilly-Mazarin Cedex, France $ Sanofi R&D, 371 Rue du professeur Joseph Blayac, Montpellier, France Sanofi R&D, Industriepark Hoechst, Frankfurt am Main, Germany AG Medizinische Chemie, Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany * Corresponding Author Adress : christophe.boldron@sanofi.com 1

2 Supplementary Material Contents I- Selectivity data for SAR II- Analytical data ( 1 H NMR) and synthetic methods for Synthetic intermediates: 1a, 1g, 1l, 1q, 1s, 1t, 1u, 2a, 2g, 2l, 2q, 2s, 2t, 2u, 3a, 3l, 3q, 3t, 3u, 4a, 4c, 4d, 4e, 4f, 4g, 4n, 4s, 5c, 5e, 5n, 6c, 6d, 6e, 6n, 7c, 7d, 7e, 7n, 8-10, 14, 15, 16h, 16x, 16ad, 17h, 17x, 17ad, 18h, 18i, 18x, 18ad, 19h, 19i, 19j, 19k, 19x, 20h, 20i, 20j, 20k, 20v, 20x, 20y, 20z, 20aa, 20ab-ad, 20af, 20ag, 20ah, 20ai, 20aj, 20ak, 23-32, 43, 44a, 44l, 44q, 44t, 44u, 45a, 45l, 45m, 45o, 45q, 45t, 45u, 45r, 46a-v, 46y-z, 46ab, 46ac, 46ad, 46af, 46ak, 47, 49, 50b-e, 50g-p, 50p, 50q-u, 50y-z, 50ab-ad, 50af, 50ak. III- Analytical data ( 1 H NMR, LCMS, Elemental Analysis, melting point) and synthetic methods for final compounds of high biological interest: 51, 52a, 52c, 52g, 52h, 52l, 52m-o, 52z, 52ad, 80, 81, 86, 87, 91, 92, 94, IV- Analytical data ( 1 H NMR, LCMS, Elemental Analysis, melting point) and synthetic methods for Final compounds of lower biological interest: 52i, 52j, 52k, 52p, 52p, 52q, 52r, 52s, 52t, 52u, 52y, 52aa, 52ab, 52ac, 52af, 52ak, 53-79, 82-85, 88-90, 93,

3 I- Selectivity data for compound 52w. Inhibition (%) of binding of cognate ligands by 10µM SAR on a panel of receptors, ion channels and transporters Targets RECEPTORS GPCRs Species % Inhibition SAR (10µM) Adenosine A1 (agonist radioligand) Human 1 A2A (agonist radioligand) Human 7 A3 (agonist radioligand) Human -10 Adrenergic α1a (antagonist radioligand) Rat -5 α2a (antagonist radioligand) Human 1 β1 (agonist radioligand) Human -3 β2 (agonist radioligand) Human 2 Angiotensin-II AT1 (antagonist radioligand) Human 24 AT2 (agonist radioligand) Human 11 Bombesin BB (non-selective) (agonist radioligand) Rat 3 Bradykinin B1 (agonist radioligand) Human 5 B2 (agonist radioligand) Human 5 Cannabinoid CB1 (agonist radioligand) Human 1 CB2 (agonist radioligand) Human -6 Chemokines CCR1 (agonist radioligand) Human -1 CCR2 (agonist radioligand) Human -6 Cholecystokinin CXCR2 (IL-8B) (agonist radioligand) CCK1 (CCKA) (agonist radioligand) CCK2 (CCKB) (agonist radioligand) Human -15 Human -81 Human 1 Dopamine D1 (antagonist radioligand) Human 2 D2S (antagonist radioligand) Human 7 Endothelin ET A (agonist radioligand) Human -4 3

4 GABA GABA B(1b) radioligand) (antagonist Human -9 Galanin GAL1 (agonist radioligand) Human 8 GAL2 (agonist radioligand) Human 7 Histamine H1 (antagonist radioligand) Human -2 H2 (antagonist radioligand) Human 1 H3 (agonist radioligand) Human 0 H4 (agonist radioligand) Human 0 Melanin hormone concentrating MCH1 (agonist radioligand) Human -21 Melanocortin MC 3 (agonist radioligand) Human 21 MC 4 (agonist radioligand) Human 10 Muscarinic M1 (antagonist radioligand) Human -6 M2 (antagonist radioligand) Human 0 M3 (antagonist radioligand) Human 1 Neurokinin NK 1 (agonist radioligand) Human 15 NK 2 (agonist radioligand) Human 22 NK 3 (antagonist radioligand) Human 5 Neuromedin-U NMU2 (agonist radioligand) Human -17 Neuropeptide Y Y 1 (agonist radioligand) Human 10 Y 2 (agonist radioligand) Human -11 Neurotensin Opioid and opioid-like NTS 1 (NT 1 ) (agonist radioligand) NOP (ORL1) (agonist radioligand) Human 9 Human -5 µ (MOP) (agonist radioligand) Human 2 κ (KOP) (agonist radioligand) Rat -27 δ2 (DOP) (agonist radioligand) Human 5 Prostanoid IP (PGI2) (agonist radioligand) Human 0 Purinergic P2X (agonist radioligand) Rat -1 P2Y (agonist radioligand) Rat -3 P2Y1 (agonist radioligand) Human 7 P2Y13 (agonist radioligand) Human -19 Serotonin 5-HT 1A (agonist radioligand) Human 4 5-HT 1D (agonist radioligand) Rat -4 4

5 5-HT 2A (antagonist radioligand) Human -9 5-HT 2B (antagonist radioligand) Human -5 5-HT 2C (antagonist radioligand) Human 3 5-HT 3 (antagonist radioligand) Human 0 5-HT 4e (antagonist radioligand) Human 8 5-HT 6 (agonist radioligand) Human 6 5-HT 7 (agonist radioligand) Human 4 Somatostatin sst (non-selective) (agonist radioligand) Mouse 5 Vasopressin V 1a (agonist radioligand) Human 2 V 1b (agonist radioligand) Human -12 V2 (agonist radioligand) Human 13 VIP Nuclear receptors VPAC 1 (VIP 1 ) (agonist radioligand) Human 0 Steroid receptors Non-steroid receptors Other receptors nuclear nuclear AR (agonist radioligand) Human -7 ERα (agonist fluoligand) Human 23 GR (agonist radioligand) Human -5 PR (agonist radioligand) Human 15 TR (TH) (agonist radioligand) Rat -4 Benzodiazepine BZD (peripheral) (antagonist radioligand) Rat 31 Cytokines TNF-α (agonist radioligand) Human -52 GABA GABA A (agonist radioligand) Rat -2 Nicotinic N muscle-type (antagonist radioligand) N neuronal α-bgtx-insensitive (α4β2) (agonist radioligand) Sigma σ (non-selective) (agonist radioligand) Human -4 Rat 12 Rat 44 ION CHANNELS Ca2+ channels Ca2+ channel (L, dihydropyridine site) (antagonist radioligand) Rat -6 Ca2+ channel (L, verapamil site) (phenylalkylamine) Rat 0 5

6 (antagonist radioligand) Ca2+ channel (N) (antagonist radioligand) Rat -38 IP3 (agonist radioligand) Rat -24 GABA channels BZD (central) (agonist radioligand) Cl- channel (GABA-gated) (antagonist radioligand) Rat 14 Rat -8 Glutamate channels Kainate (agonist radioligand) Rat -11 PCP (antagonist radioligand) Rat -4 K+ channels TRANSPORTERS Glycine (strychnine-sensitive) (antagonist radioligand) K ATP channel (antagonist radioligand) K V channel (antagonist radioligand) SK Ca channel (antagonist radioligand) Rat 8 Rat -42 Rat -7 Rat 4 Adenosine Adenosine transporter (antagonist radioligand) Guinea pig -17 Dopamine GABA Norepinephrine Dopamine transporter (antagonist radioligand) GABA transporter (antagonist radioligand) Norepinephrine transporter (antagonist radioligand) Human 2 Rat -14 Human 10 Serotonin 5-HT transporter radioligand) (antagonist Human -8 6

7 Inhibition (%) of enzymatic activity by 10µM SAR on a panel of in vitro enzyme assays from CEREP Targets Species % Inhibition SAR (10µM) ACE Human -48 Acetylcholinesteras Human -7 ATPase (Na+/K+) Porcine -7 CaMK2α Human -19 Cathepsin D Human -11 Cathepsin L Human 15 COX1 Human 7 COX2 Human 25 IRK (InsR) Human 8 12-lipoxygenase Human 25 MAO-A Human 0 MAO-B Human 2 MMP-1 Human 4 Constitutive NOS (endothelial) Human -17 PDE1B Human 5 PDE2A Human -2 PDE3A Human -1 PDE4D Human 1 PDE5 (non-selective) Human 1 PDE6 (non-selective) Bovine -3 Phosphatase1B (PTP1B) Human -11 PLC Bacillus cereus -58 Tryptase Human 19 TXA2 synthetase Human -5 7

8 Table 3 Inhibition (%) of kinase activity by SAR on a panel of in vitro serine/threonine and tyrosine kinase assays Targets Species % Inhibition SAR (10µM) % Inhibition SAR (1µM) Serine/threonine Kinases AKT1 Human 8 AKT2 Human 13 ALK4 Human 3 AMPKα1 Human 0 ASK1 Human 12 AurA Human 26 CaMK2δ Human 1 1 CaMK4 Human 6 CDK2 Human -1 CDK7/cyclinH/MAT1 Human 7 CDK9/cyclin T1 Human -40 CHK1 Human CHK2 Human 8 CK1δ Human c-raf Human 11 DAPK2 Human 25 DYRK1A Rat 3 Erk1 Human 21 Erk2 Human 12 GSK3β Human HGK/MAP4K4 Human 11 IRAK4 Human 1 MAPK1 Human -6 MAPKAPK2 Human 7 MAPKAPK5 Human 11 MARK1 Human -21 MEK1 Human -17 8

9 MLK1 Human 0 Mnk2 Human 4 MSK1 Human 7 MST2 Human -3 p38α Human 9 p70s6k Human 6 PAK2 Human -7 PI3K (p110β/p85α) Human 6 PIM2 Human 1 PKACα Human -8 PKCβ2 Human 4 11 PKCζ Human 11 PKD2 Human 21 PKG1α Human 6 PKG1β Human 29 Raf-1 Human 7 ROCK2 Human -7 RSK1 Human 6 SGK Human SIK Human -3 c-tak1 Human 8 WNK3 Human 14 Tyrosine Kinases ABL Human 19 Axl Human 5 BTK Human 2 ckit Human -23 CSK Human 10 DYRK2 Human 5 EGFR Human 12 EphB2 Human 17 Fes Human 27 FGFR1 Human -6 9

10 FLT3 Human -8 FYN Human 11 IGF1R Human -10 IR Human -8 6 IRAK1 Human 9 JAK3 Human 34 KDR Human 6 LCK Human 16 LYNa Human 8 Met Human 46 9 PDGFRα Human -12 Pyk2 Human -2 SRC Human 6 SYK Human -7 TrkB Human 8 10

11 II- Analytical data ( 1 H NMR) and synthetic methods for Synthetic intermediates: 1a, 1g, 1l, 1q, 1s, 1t, 1u, 2a, 2g, 2l, 2q, 2s, 2t, 2u, 3a, 3l, 3q, 3t, 3u, 4a, 4c, 4d, 4e, 4f, 4g, 4n, 4s, 5c, 5e, 5n, 6c, 6d, 6e, 6n, 7c, 7d, 7e, 7n, 8-10, 14, 15, 16h, 16x, 16ad, 17h, 17x, 17ad, 18h, 18i, 18x, 18ad, 19h, 19i, 19j, 19k, 19x, 20h, 20i, 20j, 20k, 20v, 20x, 20y, 20z, 20aa, 20ab-ad, 20af, 20ag, 20ah, 20ai, 20aj, 20ak, 23-32, 43, 44a, 44l, 44q, 44t, 44u, 45a, 45l, 45m, 45o, 45q, 45t, 45u, 45r, 46a-v, 46y-z, 46ab, 46ac, 46ad, 46af, 46ak, 47, 49, 50b-e, 50g-p, 50p, 50q-u, 50y-z, 50ab-ad, 50af, 50ak. 1-(5-methyl-1H-indol-3-yl)-2,2,2-trifluoroethanone (1a). Same procedure as 1f (18.2 g, 94%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.41 (s, 1H), 8.00 (s, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 2.44 (s, 3H). 1-(5-methoxy-1H-indol-3-yl)-2,2,2-trifluoroethanone (1g). Same procedure as 1f (37.7 g, 91%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.39 (s, 1H), 7.68 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 3.82 (s, 3H). 1-(5-chloro-6-fluoro-1H-indol-3-yl)-2,2,2-trifluoroethanone (1l). Same procedure as 1f (6.81 g, 87%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.58 (s, 1H), 8.23 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H). 1-(5,6-dichloro-1H-indol-3-yl)-2,2,2-trifluoroethanone (1s). Same procedure as 1f (6.65 g, 92%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.60 (q, J = 1.8 Hz, 1H), 8.29 (s, 1H), 7.85 (s, 1H). 1-(5-fluoro-1H-indol-3-yl)-2,2,2-trifluoroethanone (1t). Same procedure as 1f (6.80 g, 99%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.56 (s, 1H), 7.86 (dd, J = 9.7 Hz, J = 2.5 Hz, 1H), 7.62 (dd, J = 9.0 Hz, J = 4.5 Hz, 1H), 7.22 (td, J = 9.0 Hz, J = 2.5 Hz, 1H). 1-(6-methyl-1H-indol-3-yl)-2,2,2-trifluoroethanone (1u). Same procedure as 1f (7.80 g, 90%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.42 (s, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.38 (s, 1H), 7.17 (d, J = 8.2 Hz, 1H), 2.44 (s, 3H). 1-(6-bromo-5-methyl-1H-indol-3-yl)-2,2,2-trifluoroethanone (1q). To 1a (7.0 g, 30.8 mmol) in AcOH (192 ml) at 10 C was added Br 2 (5.91 g, 37.0 mmol). After 5 h at RT, the reaction mixture was evaporated. The residue was suspended in isopropylether, then one volume of pentane was added. The precipitate was filtered and washed with pentane. 1q was obtained as a beige powder (7.60 g, 81%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br., 1H), 8.47 (q, J = 1.9 Hz, 1H), 8.14 (s, 1H), 7.81 (s, 1H), 2.47 (s, 3H). 5-methyl-1H-indole-3-carboxylic acid (2a). Same procedure as 2f (33.8 g, 88%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (s, 1H), (s, 1H), 7.92 (s, 1H), 7.80 (d, J = 1.7 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.00 (dd, J = 8.2 Hz, J = 1.7 Hz, 1H), 2.40 (s, 3H). 5-methoxy-1H-indole-3-carboxylic acid (2g). Same procedure as 2f (16.0 g, 99%). 1H NMR: DMSOd6 (250 MHz): δ (ppm): (br, 1H), (s, 1H), 7.92 (s, 1H), 7.48 (s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 3.78 (s, 3H). 5-chloro-6-fluoro-1H-indole-3-carboxylic acid (2l). Same procedure as 2f (4.38 g, 81%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), (br, 1H), (m, 2H), 7.49 (d, J = 10.0 Hz, 1H). 11

12 6-bromo-5-methyl-1H-indole-3-carboxylic acid (2q). Same procedure as 2f (3.85 g, 62%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), (br, 1H), 7.98 (d, J = 2.3 Hz, 1H), 7.95 (s, 1H), 7.68 (s, 1H), 2.44 (s, 3H). 5,6-dichloro-1H-indole-3-carboxylic acid (2s). Same procedure as 2f (4.72 g, 87%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (m, 2H), 8.13 (s, 1H), 8.10 (s, 1H), 7.73 (s, 1H). 5-fluoro-1H-indole-3-carboxylic acid (2t). Same procedure as 2f (5.90 g, 99%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (s, 1H), (s, 1H), 8.06 (s, 1H), 7.65 (dd, J = 9.7 Hz, J = 2.5 Hz, 1H), 7.48 (dd, J = 9.0 Hz, J = 4.5 Hz, 1H), 7.04 (td, J = 9.0 Hz, J = 2.5 Hz, 1H). 6-methyl-1H-indole-3-carboxylic acid (2u). Same procedure as 2f (1.58 g, 64%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), (s, 1H), (m, 2H), 7.24 (s, 1H), 6.99 (dd, J = 8.2 Hz, J = 1.5 Hz, 1H), 2.41 (s, 3H). 1-Acetyl-5-methyl-1H-indole-3-carboxylic acid (3a). Same procedure as 3l (39.2 g, 94%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.38 (s, 1H), 8.22 (d, J = 8.5 Hz, 1H), 7.89 (s, 1H), 7.21 (d, J = 8.5 Hz, 1H), 2.72 (s, 3H), 2.43 (s, 3H). 1-Acetyl-5-chloro-6-fluoro-1H-indole-3-carboxylic acid (3l). A mixture of 2l (4.4 g, 20.5 mmol), Et 3 N (6.28 ml, 45.1 mmol) and 4-N,N-dimethylaminopyridine (0.51 g, 4.10 mmol) in DCM (100 ml) was cooled to 0 C. Acetyl chloride (1.47 ml, 20.5 mmol) was added and stirred for 3h at RT. The reaction mixture was washed with an aq. 1N HCl solution, with water and then with brine. The organic phase was dried over Na 2 SO 4 and evaporated under vacuum to afford 3l as a white powder (4.82 g, 92%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.52 (s, 1H), 8.25 (d, 1H), 8.13 (d, 1H), 2.74 (s, 3H). 1-Acetyl-6-bromo-5-methyl-1H-indole-3-carboxylic acid (3q). Same procedure as 3l (3.90 g, 88%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.53 (s, 1H), 8.42 (s, 1H), 8.01 (s, 1H), 2.72 (s, 3H), 2.43 (s, 3H). 1-Acetyl-5-fluoro-1H-indole-3-carboxylic acid (3t). Same procedure as 3l (7.20 g, 98%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.52 (s, 1H), 8.38 (dd, J = 9.2 Hz, J = 4.5 Hz, 1H); 7.76 (dd, J = 9.2 Hz, J = 2.7 Hz, 1H); 7.27 (td, J = 9.2 Hz, J = 2.7 Hz, 1H); 2.75 (s, 3H). 1-Acetyl-6-methyl-1H-indole-3-carboxylic acid (3u). Same procedure as 3l (0.68 g, 56%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): 12.7 (br, 1H), 8.35 (s, 1H), 8.20 (s, 1H); 7.96 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H); 2.73 (s, 3H); 2.49 (s, 3H). 1-(3-Ethoxycarbonyl-propyl)-5-methyl-1H-indole-3-carboxylic acid (4a ). 2-methyl-2-butene (0.46 ml, 4.40 mmol) and 10 (0.50 g, 1.83 mmol) are stirred in THF / tbuoh (50 ml, 5/1 ; v/v). An aqueous solution (20 ml) containing sodium chlorite (1.24 g, 11.0 mmol) and monosodium phosphate (1.48 g, 12.3 mmol) was added dropwise and the reaction mixture was stirred overnight under air. EtOAc (50 ml) was added and the organic phase was extracted with a saturated solution of Na 2 CO 3 (3x). The combined aqueous phases were acidified at ph 2-3 with aq. HCl 1N and extracted with EtOAc. The organic phase was dried over Na 2 SO 4 and dried over vacuum to afford 4a (0.47 g, 88%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 11.9 (s, 1H), 7.99 (s, 1H), 7.83 (s, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 4.24 (t, J = 7.2 Hz, 2H), 4.02 (q, J = 7.2 Hz, 2H), 2.42 (s, 3H), 2.27 (t, J = 7.2 Hz, 2H), 2.04 (quint, J = 7.2 Hz, 2H), 1.15 (t, J = 7.2 Hz, 3H). 12

13 1-Methoxycarbonylmethyl-5,6-dimethyl-1H-indole-3-carboxylic acid (4c). MeOH (120 ml) was poured onto Pd/C 10% (0.54 g) under a controlled argon stream. Then 7c (5.35 g, 15.2 mmol) was added followed by ammonium formate (14.8 g, 228 mmol). It was refluxed for 12h. The reaction mixture was cooled down to RT, filtered on celite and the filtrate was evaporated. The solid residue was dissolved with EtOAc and the organic phase was washed with water, with brine, dried over Na 2 SO 4 and evaporated to dryness to afford 4c (3.48 g, 88%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 7.91 (s, 1H), 7.78 (s, 1H), 7.26 (s, 1H), 5.18 (s, 2H), 3.70 (s, 3H), 2.32 (s, 6H). 1-Methoxycarbonylmethyl-4,5-dimethyl-1H-indole-3-carboxylic acid (4d). Same procedure as 4c (0.69 g, 72%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.04 (s, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 5.18 (s, 2H), 3.69 (s, 3H), 2.72 (s, 3H), 2.32 (s, 3H). 1-tert-Butoxycarbonylmethyl-2,5-dimethyl-1H-indole-3-carboxylic acid (4e). Same procedure as 4c (1.32 g, 58%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 7.80 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 6.99 (dd, J = 8.3, 1.3 Hz, 1H), 5.04 (s, 2H), 2.62 (s, 3H), 2.40 (s, 3H), 1.43 (s, 9H). 5-Bromo-1-(2-methoxy-2-oxoethyl)-1H-indole-3-carboxylic acid (4f). Same procedure as 4h (5.0 g, 64%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.16 (s, 1H), 8.12 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 5.27 (s, 2H), 3.71 (s, 3H). 5-methoxy-1-(2-methoxy-2-oxoethyl)-1H-indole-3-carboxylic acid (4g). Same procedure as 4h (12.6 g, 61%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 7.99 (s, 1H), 7.50 (s, 1H), 7.39 (d, J = 9.0 Hz, 1H), 6.86 (d, J = 9.0 Hz, 1H), 5.20 (s, 2H), 3.79 (s, 3H), 3.69 (s, 3H). 1-Methoxycarbonylmethyl-5-trifluoromethyl-1H-indole-3-carboxylic acid (4n). Same procedure as 4c (0.32 g, 74%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.36 (s, 1H), 8.26 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.57 (dd, J = 8.7, 1.6 Hz, 1H), 5.35 (s, 2H), 3.72 (s, 3H). 5,6-dichloro-1-(2-methoxy-2-oxoethyl)-1H-indole-3-carboxylic acid (4s). Same procedure as 4h (5.65 g, 92%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.16 (s, 1H), 8.14 (s, 1H), 8.00 (s, 1H), 5.27 (s, 2H), 3.71 (s, 3H). 3-[(2-Iodo-4,5-dimethylphenyl)amino]benzyl acrylate (5c). Lithium chloride (60.8 g, 1.42 mol) was added to a solution of [1,4]-benzoquinone (15.5 g, 142 mmol) in THF (350 ml). After having degassed with nitrogen, palladium acetate (3.20 g, 14.2 mmol) and benzyl acrylate (23.7 g, 142 mmol) were added and degassed again with nitrogen for about 30 minutes. Then a solution of 2-iodo-4,5-dimethylaniline (35.1 g, 142 mmol) in THF (150 ml) was added and the reaction mixture was stirred overnight at RT. It was filtered and the filtrate was evaporated. The solid residue thus obtained was triturated with diethylether and then filtered. The filtrate was washed with a solution of aq. NaOH 0.5N, with water, with brine and then evaporated. The solid residue was purified by silica gel chromatography, eluting with a cyclohexane/etoac mixture (8/2; v/v) to afford 5c as a white powder (57.6 g, 98%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (d, J = 7.7 Hz, 1H), 7.72 (dd, J = 7.7 Hz, J = 5.2 Hz, 1H), 7.59 (s, 1H), (m, 5H), 7.23 (s, 1H), 5.18 (s, 2H), 4.93 (d, J = 5.2 Hz, 1H), 2.19 (s, 3H), 2.14 (s, 3H). 3-(2-Bromo-4-methyl-phenylamino)-but-2-enoic acid benzyl ester (5e). 2-Bromo-4-methylphenylamine (5.00 g, 26.9 mmol), para-toluene-sulfonic acid monohydrate (0.051 g, mmol) and 3-oxo-butyric acid benzyl ester (5.33 g, 26.9 mmol) were stirred under reflux in toluene (50 ml) in a Dean Stark apparatus during 3h. The reaction mixture was concentrated under vacuum and the residue was purified by silica gel chromatography, eluting with a EtOAc to afford 5e as a white powder (8.51 g, 13

14 88%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (s, 1H), 7.56 (s, 1H), (m, 7H), 5.12 (s, 2H), 4.83 (s, 1H), 2.31 (s, 3H), 1.91 (s, 3H). 3-[(2-bromo-4-trifluoromethylphenyl)amino]benzyl acrylate (5n). Same procedure as 5c (4.27 g, 51%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (d, J = 12.1 Hz, 1H), 8.02 (d, J = 0.9 Hz, 1H), 7.90 (dd, J = 12.1, 8.3 Hz, 1H), (m, 1H), (m, 1H), (m, 5H), 5.21 (s, 2H), 5.15 (d, J = 8.3 Hz, 1H). Benzyl 5,6-dimethyl-1H-indole-3-carboxylate (6c). 1,4-diazabicyclo[2.2.2]octane (8.60 g, 75.1 mmol) was added to a solution of 5c (25.5 g, 62.6 mmol) in DMF (120 ml). It was degassed with nitrogen, then palladium acetate (0.703 g, 3.13 mmol) was added and the reaction mixture was heated at 120 C for 7h. EtOAc was added, then it was washed with water and with brine, dried over Na 2 SO 4 and evaporated. After trituration of the solid residue with isopropylether, 6c was obtained as a beige powder (12.3 g, 70%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 7.98 (d, J = 3.2 Hz, 1H), 7.76 (1H, s); (m, 5H), 7.24 (s, 1H), 5.32 (s, 2H), 2.30 (s, 3H), 2.29 (s, 3H). 4,5-Dimethyl-1H-indole-3-carboxylic acid benzyl ester (6d). To a solution of 4- (dimethylamino)pyridine (0.45 g, 3.68 mmol) and 9 (5.00 g, 27.9 mmol) in THF (200 ml) at 0 C, was added propynoic acid benzyl ester (6.80 g, 42.4 mmol). The reaction mixture was stirred overnight. NaOH (1.12 g, 27.9 mmol) was added and the reaction was continued for 3 h. Water (300 ml) was added followed by extraction with EtOAc. The combined organic phases were washed with water, with brine, dried over Na 2 SO 4 and evaporated under vacuum. The solid residue was purified by silica gel chromatography, eluting with a EtOAc/toluene mixture (5/95; v/v to 15/85; v/v gradient) to afford 6d as a white powder (3.10 g, 40%) and 6c as a white powder (2.25 g, 29%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.07 (s, 1H), (m, 5H), 7.20 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 5.28 (s, 2H), 2.68 (s, 3H), 2.32 (s, 3H). Benzyl 2,5-Dimethyl-1H-indole-3-carboxylate (6e). A mixture of Tri(o-tolyl)phosphine (0.118 g, mmol), Et 3 N (1.31 ml, 9.33 mmol) and 5e (2.80 g, 7.77 mmol) in acetonitrile (10 ml) were degassed under nitrogen during 30 minutes. Palladium acetate ( g, mmol) was added and the mixture was reacted during 15 minutes at 140 C using a microwave. After cooling down to RT, aq. 1N HCl was added it was extracted with EtOAc. The combined organic phases were washed with water, with brine, dried over Na 2 SO 4 and evaporated under vacuum to produce 6e (2.1 g, 97%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 7.74 (s, 1H), (m, 5H), 7.25 (d, J = 8.2 Hz, 1H), 6.95 (dd, J = 8.2, 1.6 Hz, 1H), 5.34 (s, 2H), 2.64 (s, 3H), 2.37 (s, 3H). Benzyl 5-trifluoromethyl-1H-indole-3-carboxylate (6n). Same procedure as 6c (0.74 g, 22%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.35 (s, 1H), 8.33 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), (m, 6H), 5.38 (s, 2H). 1-Methoxycarbonylmethyl-5,6-dimethyl-1H-indole-3-carboxylic acid benzyl ester (7c). To 6c (4.40 g, 15.7 mmol) and K 2 CO 3 (2.39 g, 17.3 mmol) in DMF (105 ml) was added methyl bromoacetate (2.53 g, 16.5 mmol). It was stirred at RT overnight. EtOAc (100 ml) was added, then it was washed with water and with brine, dried over Na 2 SO 4 and evaporated to afford 7c (5.35 g, 97%). 1H NMR: DMSOd6 (250 MHz): δ (ppm): 8.05 (s, 1H), 7.78 (s, 1H), (m, 6H), 5.32 (s, 2H), 5.19 (s, 2H), 3.69 (s, 3H), 2.31 (s, 6H). 1-Methoxycarbonylmethyl-4,5-dimethyl-1H-indole-3-carboxylic acid benzyl ester (7d). Same procedure as 7c (0.57 g, 96%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.16 (s, 1H), (m, 5H), 7.20 (d, J = 8.4 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.28 (s, 2H), 5.20 (s, 2H), 3.68 (s, 3H), 2.69 (s, 3H), 2.33 (s, 3H). 14

15 1-tert-Butoxycarbonylmethyl-2,5-dimethyl-1H-indole-3-carboxylic acid benzyl ester (7e). Same procedure as 7c using tert-butyl bromoacetate (3.10 g, 98%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): 7.78 (s, 1H), (m, 6H), 7.01 (d, J = 8.4 Hz, 1H), 5.37 (s, 2H), 5.07 (s, 2H), 2.63 (s, 3H), 2.37 (s, 3H), 1.42 (s, 9H). 1-Methoxycarbonylmethyl-5-trifluoromethyl-1H-indole-3-carboxylic acid benzyl ester (7n). Same procedure as 7c (0.57 g, 96%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.41 (s, 1H), 8.35 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), (m, 5H), 5.38 (s, 2H), 5.36 (s, 2H), 3.71 (s, 3H). N-Acetoxy-N-(3,4-dimethyl-phenyl)-acetamide (8). To Pd/C 10% (9.90 g) in suspension in cyclohexane (15 ml) under a nitrogen flux at 0 C, were added 1,2-Dimethyl-4-nitro-benzene (30.0 g, mmol) and hydrazine monohydrate (40.7 g, 1260 mmol). It was stirred at 0 C for 1 h. Then, it was allowed to warm to 10 C. Et 3 N (60.7 ml, 430 mmol) and acetyl chloride (296.3 ml, 4130 mmol) were added and the reaction mixture was stirred for 2 h at 10 C. After filtration over celite, the filtrate was added drop by drop over an aqueous solution of Na 2 CO 3 (8 g in 1 L). The aqueous phase was extracted with EtOAc. The organic phase was washed with aq. 0.1N HCl, with water, with brine, dried over Na 2 SO 4 and evaporated under vacuum. The solid residue was purified by silica gel chromatography, eluting with a EtOAc/toluene mixture (10/90; v/v) to afford 8 (13.0 g, 37%). N-(3,4-Dimethyl-phenyl)-N-hydroxy-acetamide (9). K 2 CO 3 (30.0 g, 217 mmol) and 8 (13.0 g, mmol) in MeOH (0.3 L) were stirred overnight at RT. The reaction mixture was concentrated then the solide residue was taken up with EtOAc. The organic phase was washed with aq. 1N HCl, with water, with brine, dried over Na 2 SO 4 and evaporated under vacuum to produce 9 (8.40 g, 80%). 1H NMR: DMSO-d6 (250 MHz): δ (ppm): (s, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.30 (dd, J = 8.3, 2.0 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 2.22 (s, 3H), 2.20 (s, 3H), 2.16 (s, 3H). 4-(3-Formyl-5-methyl-indol-1-yl)-butyric acid ethyl ester (10). 5-Methyl-1H-indole-3-carbaldehyde (1.00 g, 6.09 mmol) and potassium tert-butoxide (1.27 g, 10.8 mmol) in DMF (30 ml) at RT. 4-Bromobutyric acid ethyl ester (1.95 g, 9.82 mmol) was added and reacted for 24 h. The reaction mixture was poured onto a phosphate buffer solution (ph 7, 300 ml). It was then extracted with EtOAc and the combined organic phases were washed with water (2x), dried over Na 2 SO 4 and evaporated under vacuum to produce 10 (1.49 g, 90%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 9.88 (s, 1H), 8.25 (s, 1H), 7.93 (s, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 4.29 (t, J = 7.2 Hz, 2H), 4.02 (q, J = 7.0 Hz, 2H), 2.43 (s, 3H), 2.32 (t, J = 7.2 Hz, 2H), 2.06 (quint, J = 7.2 Hz, 2H), 1.15 (t, J = 7.0 Hz, 3H). 2-[1-Dimethylamino-methylidene]-3-oxo-butyric acid ethyl ester (14). N,N-Dimethylformamide dimethyl acetal (23.9 g, 189 mmol) and 3-Oxo-butyric acid ethyl ester (20.0 g, 158 mmol) were stirred at RT for 2h. The reaction mixture was concentrated under vacuum to produce 14 as a red oil (28.1 g, 99%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 7.59 (s, 1H), 4.11 (q, J = 7.1 Hz, 2H), (m, 6H), 2.14 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H). 5-Methyl-1-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic acid ethyl ester (15). (4-Nitro-phenyl)- hydrazine (10.0 g, 63.3 mmol) was added to a solution containing 14 (11.7 g, 63.3 mmol) in EtOH (250 ml). It was refluxed for 3h. Then, the reaction mixture was concentrated down to 50 ml and was added on water (700 ml). The precipitate formed was filtered, washed with water and dried under vacuum at 50 C to afford 15 as a beige powder (13.1 g, 76%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (m, 2H), 8.12 (s, 1H), (m, 2H), 4.28 (q, J = 7.1 Hz, 2H), 2.63 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). 15

16 5-Methyl-1-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic acid (16h). To 15 (13.0 g, 47.2 mmol) in THF/EtOH (250 ml, 1/1; v/v) was added a solution of KOH (13.3 g, 236 mmol) in water (125 ml). It was refluxed for 2h. Then, the organic solvents were evaporated and the resulting aqueous phase was neutralized by slow addition of aq. HCl 37% (19 ml). The precipitate formed was filtered, washed with water and dried under vacuum at 50 C to afford 16h as a beige powder (11.6 g, 99%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 8.40 (d, J = 9.5 Hz, 2H), 8.08 (s, 1H), 7.89 (d, J = 9.5 Hz, 2H), 2.61 (s, 3H). 5-Methyl-1-(6-nitro-pyridin-3-yl)-1H-pyrazole-4-carboxylic acid (16x). To a solution of 25 (12.5 g, 45.3 mmol) in THF (57 ml), an aq. 1N NaOH solution (45.3 ml, 45.3 mmol) was added. After 4h, LiOH (0.573 g, 23.9 mmol) was added and the reaction mixture was stirred for 14h. Then, the reaction mixture was acidified with aq. 2N HCl and the precipitated product was collected by filtration and dried under reduced pressure to afford 16x (8.2 g, 73%). Engaged directly in next step (17x) without 1 H NMR characterization. 1-(4-Benzyloxycarbonylamino-trans-cyclohexyl)-5-methyl-1H-pyrazole-4-carboxylic acid (16ad). KOH (3.27 g, 58.4 mmol) in water (20 ml) was added to a solution of 29 (4.50 g, 11.7 mmol) in THF/EtOH (20 ml, 1/1; v/v) and the reaction mixture was stirred overnight at 50 C. After cooling down, water was added and it was washed twice with isopropylether. The aqueous phase was acidified to ph 5 with aq. 37% HCl. The white precipitate formed was filtered, washed with isopropylether and dried under vacuum at 60 C to afford 16ad as a beige powder (3.80 g, 90%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), 7.73 (s, 1H), (m, 6H), 5.01 (s, 2H), 4.14 (m, 1H), 3.33 (br, 1H), 2.50 (s, 3H), (m, 6H), 1.40 (m, 2H). Ethyl 3-[5-Methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-3-oxopropanoate (17h). Same procedure as 17w (5.2 g, 67%). 1 H NMR: CDCl 3 (250 MHz): δ (ppm): 8.42 (d, J = 9.6 Hz, 2H), 8.09 (s, 1H), 7.70 (d, J = 9.6 Hz, 2H), 4.26 (q, J = 7.8 Hz, 2H), 3.88 (s, 2H), 2.70 (s, 3H), 1.28 (t, J = 7.8 Hz, 3H). 3-[5-Methyl-1-(6-nitro-pyridin-3-yl)-1H-pyrazol-4-yl]-3-oxo-propionic acid ethyl ester (17x). Same procedure as 17w (2.4 g, 58%). Engaged directly in next step (18x) without 1 H NMR characterization. 3-[1-(4-Benzyloxycarbonylamino-trans-cyclohexyl)-5-methyl-1H-pyrazol-4-yl]-3-oxo-propionic acid ethyl ester (17ad). Same procedure as 17w (2.67 g, 59%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.02 (s, 1H), (m, 5H), 5.01 (s, 2H), 4.18 (m, 1H), 4.09 (q, J = 7.0 Hz, 2H), 3.87 (s, 2H), 3.35 (m, 1H), 2.53 (s, 3H), (m, 6H), 1.41 (m, 2H), 1.18 (t, J = 7.0 Hz, 3H). Ethyl 2-{[5-Methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]carbonyl}butanoate (18h). A solution of 17h (2.44 g, 7.70 mmol) in THF (25 ml) was cooled to 0 C and NaH 60% in oil (0.34 g, 8.49 mmol) was added in portions. After stirring for 30 minutes, iodoethane (0.92 ml, 11.4 mmol) was added and reacted for 24h at RT. Water was added slowly, then the reaction mixture was concentrated under vacuum. The residue was taken up with DCM, washed with water, dried over MgSO 4 and the solvent was evaporated under vacuum. The residue was chromatographed on silica gel, eluting with EtOAc/cyclohexane mixture (gradient from 10 to 20% of EtOAc) to afford 18h as a beige powder (2.19 g, 82%). 1 H NMR: CDCl3 (250 MHz): δ (ppm): 8.42 (d, J = 9.8 Hz, 2H), 8.18 (s, 1H), 7.70 (d, J = 9.8 Hz, 2H), 4.23 (q, J = 7.8 Hz, 2H), 3.97 (t, J = 7.2 Hz, 1H), 2.72 (s, 3H), 2.06 (m, 2H), 1.28 (t, J = 7.8 Hz, 3H), 1.02 (t, J = 7.5 Hz, 3H). Ethyl 2-methyl-3-[5-methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-3-oxo-propanoate (18i). Same procedure as 18h using iodomethane (0.50 g, 96%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.43 (s, 1H), (m, 2H), (m, 2H), 4.44 (q, J = 7.0 Hz, 1H), 4.09 (q, J = 6.8 Hz, 2H), 2.61 (s, 3H), 1.32 (d, J = 7.0 Hz, 3H), 1.15 (t, J = 6.8 Hz, 3H). 16

17 2-[5-Methyl-1-(6-nitro-pyridin-3-yl)-1H-pyrazole-4-carbonyl]-butyric acid ethyl ester (18x). To a solution of 17x (890 mg, 2.80 mmol) in DMF (9 ml) were added Cs 2 CO 3 (1.00 g, 3.08 mmol) and iodoethane (224 µl, 2.80 mmol). After stirring for 1 h at RT, the reaction mixture was diluted with water and filtered through a Chem Elut cartridge by eluting with DCM. The combined organic phases were dried over MgSO 4 and the solvents were removed under reduced pressure. The residue was purified by chromatography on silica gel eluting with a gradient of n-heptane/etoac to produce 18x (0.96 g, 99%). 1 H NMR: DMSO-d6 (400 MHz): δ (ppm): 8.97 (d, J = 9.0 Hz, 1H), 8.51 (m, 3H), 4.32 (t, J = 7.2 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 2.66 (s, 3H), 1.86 (pent, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H), 0.92 (t, J = 7.2 Hz, 3H). 2-[1-(4-Benzyloxycarbonylamino-trans-cyclohexyl)-5-methyl-1H-pyrazole-4-carbonyl]-butyric acid ethyl ester (18ad). Same procedure as 18w (3.10 g of crude containing 1 eq. of tetrabutylammonium derivatives, approximative yield = 82%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.09 (s, 1H), (m, 6H), 5.01 (s, 2H), (m, 4H), 3.35 (m, 1H), 2.53 (s, 3H), (m, 8H), 1.40 (m, 2H), 1.12 (t, J = 7.0 Hz, 3H), 0.87 (t, J = 7.0 Hz, 3H). 1-[5-Methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]butan-1-one (19h). A solution of 18h (2.4 g, 6.96 mmol) in aq. 37% HCl (58 ml) was heated at 105 C for 2h. The reaction mixture was cooled down to 0 C and aq. 35% NaOH (60 ml) was added slowly under stirring. Then it was extracted with EtOAc and the combined organic phases were washed with water, dried over MgSO 4 and evaporated under vacuum to produce 19h as a beige powder (1.8 g, 95%). 1 H NMR: CDCl 3 (250 MHz): δ (ppm): 8.42 (d, J = 9.0 Hz, 2H), 8.10 (s, 1H), 7.70 (d, J = 9.0 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.75 (s, 3H), 1.78 (sext, J = 7.0 Hz, 2H), 1.02 (t, J = 7.0 Hz, 3H). 1-[5-Methyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]propane-1-one (19i). Same procedure as 19h (0.39 g, 99%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (m, 2H), 8.36 (s, 1H), (m, 2H), 2.89 (q, J = 7.2 Hz, 2H), 2.62 (s, 3H), 1.07 (t, J = 7.2 Hz, 3H). 4,4,4-Trifluoro-1-[5-methyl-1-(4-nitro-phenyl)-1H-pyrazol-4-yl]-butan-1-one (19k) and 1-[5- Methyl-1-(4-nitro-phenyl)-1H-pyrazol-4-yl]-ethanone (19j). A solution of 17h (1.00 g, 3.15 mmol) in THF (10 ml) was cooled to 0 C and NaH 60% in oil (0.139 g, 3.47 mmol) was added in portions. After stirring for 30 minutes, 2,2,2-trifluoroethyl trifluoromethanesulfonate (1.10 g, 4.73 mmol) was added and reacted for 24h at RT. Water was added slowly, then the reaction mixture was concentrated under vacuum. About 20 to 30% of starting material 17h was still present as shown by LCMS. The solide residue was taken up with aq. HCl 37% (20 ml) and the reaction mixture was heated at 105 C for 7h. The reaction mixture was cooled down to RT, then added on water (300 ml). The precipitate formed was filtered, washed with water and dried under vacuum. It was then chromatographed on silica gel, eluting with DCM to afford 19k (0.56 g, 45%) as a beige powder and 19j (0.20 g, 26%) as a beige powder. 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 19k : 8.47 (s, 1H), (m, 2H), (m, 2H), 3.19 (t, J = 7.4 Hz, 2H), (m, 5H) and 19j : 8.47 (s, 1H), (m, 2H), (m, 2H), 2.41 (s, 3H). 1-[5-Methyl-1-(6-nitro-pyridin-3-yl)-1H-pyrazol-4-yl]-butan-1-one (19x). To a solution of 18x (1.70 g, 4.91 mmol) in 1,4-dioxane (130 ml) and water (4 ml) was added Al 2 O 3 (ALOX, basic 54 g, 533 mmol) and the reaction mixture was heated to 100 C for 6 h. Then, it was cooled to RT and Al 2 O 3 was separated by filtration and washed with acetonitrile. The combined filtrates were concentrated under reduced pressure. The residue was purified by chromatography on silica gel eluting with a gradient of n- heptane/etoac to afford 19x (1.10 g, 82%). 1 H NMR: DMSO-d6 (500 MHz): δ (ppm): 8.33 (d, J = 2.6 Hz, 1H), 8.27 (s, 1H), 7.88 (dd, J = 8.8 Hz, J = 2.6 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 2.80 (t, J = 7.2 Hz, 2H), 2.48 (s, 3H), 1.62 (sext, J = 7.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H). 17

18 1-[1-(4-Aminophenyl)-5-methyl-1H-pyrazol-4-yl]butan-1-one (20h). EtOH (450 ml) was added onto Pd/C 10% (5 g) under a controlled argon stream. Then a suspension of 19h (48.7 g, mmol) in EtOH (50 ml) was added followed by cyclohexene (181 ml, 1.78 mol). The reaction mixture was heated under reflux for 4h. After cooling to RT, it was filtered on Celite and the filtrate was concentrated under vacuum. The residue was taken up in petroleum ether and the suspension was drained, washed with petroleum ether and dried under vacuum at 50 C to afford 20h as a white powder (41.3 g, 95%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.13 (s, 1H), (m, 2H), (m, 2H), 5.45 (s, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.42 (s, 3H), 1.62 (sext, J = 7.3 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). 1-[1-(4-Aminophenyl)-5-methyl-1H-pyrazol-4-yl]propane-1-one (20i). 19i (0.5 g, 1.93 mmol) was dissolved in EtOAc/MeOH (25 ml, 50/50; v/v). It was reacted using the H-Cube continuous hydrogenation apparatus (Pd/C 10% Cartridge, Mode Full H2, 50 C, flow 1 ml/min). The solvent was concentrated to dryness, the solid residue was taken up in isopropylether. The suspension was drained, washed with petroleum ether and dried under vacuum at 50 C to afford 20i as a white powder (0.25 g, 57%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.12 (s, 1H), (m, 2H), (m, 2H), 5.44 (s, 2H), 2.82 (q, J = 7.2 Hz, 2H), 2.41 (s, 3H), 1.06 (t, J = 7.2 Hz, 3H). 1-[1-(4-Aminophenyl)-5-methyl-1H-pyrazol-4-yl]ethanone (20j). Same procedure as 20i (0.10 g, 57%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.12 (s, 1H), 7.08 (m, 2H), 6.65 (m, 2H), 5.47 (br, 2H), 2.41 (s, 3H). 1-[1-(4-Amino-phenyl)-5-methyl-1H-pyrazol-4-yl]-4,4,4-trifluoro-butan-1-one (20k). Same procedure as 20i (0.44 g, 86%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.23 (s, 1H), (m, 2H), (m, 2H), 5.47 (s, 2H), 3.13 (t, J = 7.4 Hz, 2H), (m, 2H), 2.43 (s, 3H). 1-[1-(4-Amino-3-bromo-phenyl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20v). To a solution of 20h (6.00 g, 24.7 mmol) in acetonitrile (123 ml) was added N-bromosuccinimide (4.39 g, 24.7 mmol) and the reaction mixture was refluxed for 1h. After evaporation to dryness, the residue was dissolved in DCM. It was washed twice with aq. 1N NaOH, dried over Na 2 SO 4 and evaporated to dryness. The residue was purified by chromatography on silica gel eluting with DCM/MeOH (99.4/0.6 ; v/v) to afford 20v (6.03 g, 76%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.17 (s, 1H), 7.49 (d, J = 2.5 Hz, 1H), 7.19 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 5.69 (s, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.6 (sext, J = 7.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H). 1-[1-(6-Amino-pyridin-3-yl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20x). Same procedure as 20h (0.49 g, quantitative). No 1 H NMR characterization available. 1-[1-(5-Amino-pyrazin-2-yl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20y). A mixture of 36 (0.60 g, 3.94 mmol), 5-bromo-pyrazin-2-ylamine (1.37 g, 7.88 mmol), potassium carbonate (1.36 g, 9.86 mmol), proline (0.38 g, 2.37 mmol) and copper(i) iodide (0.23 g, 1.18 mmol) in DMSO (15 ml) was heated at 130 C for 20h. Water (100 ml) was added and it was extracted with DCM (3x). The combined organic phases were washed with a saturated aqueous solution of NaHCO 3, with brine, dried over Na 2 SO 4 and evaporated to dryness. The solid residue was purified by silica gel chromatography, eluting with a gradient of DCM / EtOAc mixture from (80/20; v/v) to (40/60; v/v) to afford 20y (0.17 g, 18%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (m, 2H), 7.80 (s, 1H), 6.86 (br, 2H), 2.80 (t, J = 7.0 Hz, 2H), 2.55 (s, 3H), 1.62 (sext, J = 7.0 Hz, 2H), 0.93 (t, J = 7.0 Hz, 3H). 2-Amino-5-(4-butyryl-5-methyl-pyrazol-1-yl)-benzonitrile (20z). To a solution of 20v (1.00 g, 3.11 mmol) in DMF (20 ml) in a sealed tube were successively added zinc cyanide (0.44 g 3.72 mmol) and 18

19 Tetrakis(triphenylphosphine)palladium (0.18 g, 0.15 mmol). The reaction mixture was heated at 100 C for 7h. After cooling down, it was poured into a saturated solution of NaHCO 3 and extracted with EtOAc. It is washed with water, with brine, dried over Na 2 SO 4 and evaporated to dryness. The residue was taken up in isopropylether and the precipitate that formed was drained, to afford 20z as a white powder (0.71 g, 85%). 1 H NMR: DMSO-d6 (400 MHz): δ (ppm): 8.17 (s, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.42 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.45 (br, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.44 (s, 3H), 1.61 (sext, J = 7.6 Hz, 2H), 0.92 (t, J = 7.6 Hz, 3H). 2-Amino-5-(4-butyryl-5-methyl-pyrazol-1-yl)-benzoic acid (20aa). A solution of aq. 2N NaOH (16.8 ml, 33.5 mmol) was added to 20z (1.50 g, 5.59 mmol) and it was heated at 100 C for 8h. Water was added, it was washed with DCM and then acidified with aq. 2N HCl. The precipitate was drained, then washed with water and dried in a vacuum stove to afford 20aa as a white powder (1.41 g, 87%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.17 (s, 1H), 7.69 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 2.78 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.62 (sext, J = 7.2 Hz, 2H), 0.93 (t, J = 7.2 Hz, 3H). 2-Amino-5-(4-butyryl-5-methyl-pyrazol-1-yl)-benzamide (20ab). An aq. 2N NaOH solution (2.20 ml, 4.40 mmol) was added to a solution of 20z (0.20 g, 0.75 mmol) in 1,4-dioxane (5 ml). The reaction mixture was heated at 100 C for 20h. After it returned to RT, water was added and it was extracted with DCM. It was dried over Na 2 SO 4 and evaporated to dryness. The residue was purified by silica gel chromatography, eluting with DCM/MeOH (95/5 ; v/v) to produce 20ab as a white powder (0.14 g, 66%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.16 (s, 1H), 7.81 (br, 1H), 7.65 (s, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.18 (br, 1H), 6.98 (br, 2H), 6.81 (d, J = 8.7 Hz, 1H), 2.78 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.62 (sext, J = 7.2 Hz, 2H), 0.93 (t, J = 7.2 Hz, 3H). 1-[1-(3,4-Dihydro-2H-benzo[1,4]oxazin-7-yl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20ac). A suspension of cesium carbonate (0.68 g, 2.09 mmol), 2-(Di-tert-butylphosphino)-1,1'-binaphthyl (0.11 g, 0.28 mmol) and palladium acetate (0.031 g, 0.14 mmol) in toluene (10 ml) was degazed under stirring with an argon bubbling during 10 min. Then, 32 (0.51 g, 1.39 mmol) was added and the argon bubbling was continued during 5 min. The reaction mixture was heated at 70 C overnight. After cooling down, EtOAc was added and the organic phase was washed with water, with brine, dried over Na 2 SO 4 and evaporated to dryness. The residue was purified by silica gel chromatography, eluting with DCM to produce 20ac (0.27 g, 68%). 1 H NMR: DMSO-d6 (400 MHz): δ (ppm): 8.13 (s, 1H), (m, 2H), 6.65 (d, J = 8.2 Hz, 1H), 4.16 (t, J = 4.4 Hz, 2H), 3.33 (t, J = 4.4 Hz, 2H), 2.77 (t, J = 7.2 Hz, 2H), 2.43 (s, 3H), 1.61 (sext, J = 7.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H). 1-[1-(4-Amino-trans-cyclohexyl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20ad). Same procedure as 19h from 0.60 g of the mixture containing approximatively 0.37 g of 18ad and 0.23 g of tetrabutylammonium derivatives (0.60 g of crude containing 1 eq. of tetrabutylammonium derivatives, quantitative yield). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.00 (s, 1H), 4.17 (m, 2H), 2.96 (t, J = 7.2 Hz, 2H), 2.67 (m, 1H), 2.53 (s, 3H), 2.33 (m, 1H), (m, 2H), (m, 4H), 1.48 (br, 2H), 0.89 (t, J = 7.2 Hz, 3H). 1-[1-(4-Hydroxy-phenyl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20af). 20h (1.00 g, 4.11 mmol) was suspended in water (15 ml) and H 2 SO 4 (0.81 ml, 14.4 mmol) was added. The reaction mixture was cooled down to 0 C and sodium nitrite (0.34 g, 4.93 mmol) wad added. After stirring at 0 C for 10 min, the reaction was allowed to return to RT. It was then refluxed overnight. After cooling down, it was extracted with EtOAc (3x). The combined organic phases were washed with aq. 1N HCl (2x), with water, with brine, dried over Na 2 SO 4 and evaporated to dryness to afford 20af (0.81 g, 80%). 1 H NMR: DMSO-d6 (400 MHz): δ (ppm): 9.91 (s, 1H), 8.19 (s, 1H), 7.30 (d, J = 9.0 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H), 2.78 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.63 (sext, J = 7.2 Hz, 2H), 0.94 (t, J = 7.2 Hz, 3H). 19

20 1-[1-(4-Hydroxy-phenyl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20ag). To a suspension of NaH 60% in mineral oil (0.64 g, 16.0 mmol) in DMF (32 ml) was added 36 (2.00 g, 13.2 mmol). After 10 min stirring, 2-fluoro-5-nitro-pyridine (2.20 g, 15.4 mmol) was added and the reaction mixture was stirred at RT for 16h. Water was added and the precipitate formed was collected by filtration, washed with water and dried in vacuo (2.4 g of an isomeric mixture of the nitro intermediate). A portion of this mixture (0.20 g, 0.73 mmol) was dissolved in THF (20 ml) and 10% Pd/C (0.05 g) was added under argon. The suspension was stirred at RT under an atmosphere of hydrogen (2 bars) for 3h. After filtration on celite, the filtrate was evaporated and the residue was purified by silica gel chromatography, eluting with n-heptane/etoac (1/1; v/v) to afford the right reduced isomer 20ag (0.07 g, 26% corrected yield from 36). 1 H NMR: DMSO-d6 (500 MHz): δ (ppm): 8.17 (s, 1H), 7.83 (d, J = 2.8 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 8.6 Hz, J = 2.8 Hz, 1H), 5.68 (br, 2H), 2.79 (t, J = 7.3 Hz, 2H), 2.59 (s, 3H), 1.61 (sext, J = 7.3 Hz, 2H), 0.92 (t, J = 7.3 Hz, 3H). 1-[1-(2-aminothiazol-5-yl)-5-methyl-pyrazol-4-yl]butan-1-one (20ah). To a solution of 43 (4.00 g, mmol) and 36 (2.34 g, 15.3 mmol) in DMF (100 ml) were added successively CuI (1.46 g, 7.66 mmol), K 2 CO 3 (5.30 g, mmol) and trans-n,n'-bismethyl-1,2-cyclohexane diamine (1.82 g, 12.7 mmol). The suspension was purged with argon and stirred under inert atmosphere for 12 h at 85 C. It was then filtered via a plug of Celite, washed with DMF and the combined wash solutions were concentrated. The residue thus obtained was purified by chromatography on silica gel eluting with a gradient of DCM/MeOH affording N-[5-(4-butanoyl-5-methyl-pyrazol-yl)thiazol-2-yl]carbamate (1.15 g, 23%) as N-1/N-2 isomeric mixture. It was dissolved (0.95 g, 2.47 mmol) in TFA (19.0 ml, 255 mmol) and the resulting solution was refluxed for 2.5 h. It was concentrated and the residue thus obtained was purified by preparative HPLC (C18 reverse phase column, elution with a H 2 O/MeCN gradient with 0.1% TFA). After evaporation and lyophilization, 20ah was obtained (0.10 g, 11%) as a trifluoroacetate salt. 1 H NMR: DMSO-d6 (500 MHz): δ (ppm): 8.21 (s, 1H), 7.39 (br, 2H), 7.16 (s, 1H), 2.77 (t, J = 7.2 Hz, 2H), 2.50 (s, 3H), 1.60 (sext, J = 7.2 Hz, 2H), 0.91 (t, J = 7.2 Hz, 3H). 2-Amino-5-(4-butyryl-5-methyl-pyrazol-1-yl)-N,N-dimethyl-benzamide (20ai). N-methylmorpholine (0.112 g, 1.10 mmol) and (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (0.119 g, 0.26 mmol) were successively added to a solution of 20aa (0.07 g, 0.22 mmol) in DCM (7 ml). After 2h of stirring, dimethylamine hydrochloride (0.019 g, 0.24 mmol) was added and the reaction mixture was stirred at RT overnight. After evaporation to dryness, the residue was taken up in EtOAc, washed with saturated NaHCO 3 (2x), with brine, dried over Na 2 SO 4 and evaporated to dryness. It was then purified by silica gel chromatography, eluting with DCM/MeOH (98/2 ; v/v) to produce 20ai (0.04 g, 58%). 1 H NMR: DMSO-d6 (400 MHz): δ (ppm): 8.16 (s, 1H), 7.20 (d, J = 8.6 Hz, 1H), 7.12 (s, 1H), 6.80 (d, J = 8.6 Hz, 1H), 4.70 (br, 2H), 2.92 (s, 6H), 2.78 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.62 (sext, J = 7.2 Hz, 2H), 0.94 (t, J = 7.2 Hz, 3H). 1-[1-(4-Amino-3-chloro-phenyl)-5-methyl-1H-pyrazol-4-yl]-butan-1-one (20aj). Same procedure as 20v starting from N-chlorosuccinimide (2.02 g, 88%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.20 (s, 1H), 7.48 (s, 1H), 7.19 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 5.80 (br, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.62 (sext, J = 7.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H). 2-Amino-5-(4-butyryl-5-methyl-pyrazol-1-yl)-benzoic acid methyl ester (20ak). Potassium bicarbonate (0.38 g, 3.83 mmol) and methyl iodide (0.24 ml, 3.83 mmol) were successively added to a solution of 20aa (1.00 g, 3.48 mmol) in DMF (20 ml). After 3h at RT, water was added and it was extracted with EtOAc. The organic phase was washed with water, with brine, dried over Na 2 SO 4 and evaporated to dryness to afford 20ak as a beige powder (0.89 g, 87%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 8.17 (s, 1H), 7.72 (d, J = 2.5 Hz, 1H), 7.39 (dd, J = 8.8 Hz, J = 2.5 Hz, 1H), 6.97 (br, 2H), 6.91 (d, J = 8.8 Hz, 1H), 3.81 (s, 3H), 2.78 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H), 1.61 (sext, J = 7.2 Hz, 2H), 0.93 (t, J = 7.2 Hz, 3H). 20

21 N-Benzhydrylidene-N'-(6-nitro-pyridin-3-yl)-hydrazine (23). A solution of Pd(OAc) 2 (1.1 g, 4.93 mmol), Cs 2 CO 3 (44.9 g, 138 mmol) and 2-Dicyclohexylphosphino-2'-methylbiphenyl (MePhos, 3.5 g, 9.85 mmol) in t-buoh (500 ml) was purged with argon for 30 min. Then, 5-Bromo-2-nitro-pyridine (20 g, 98.5 mmol) and Benzhydrylidene-hydrazine (19.3 g, 98.5 mmol) were successively added and the reaction mixture was heated under argon and vigorous stirring at 80 C. After 3h, the reaction mixture was cooled to RT and diluted with water. The precipitated product was collected by filtration, washed with n-heptane/ethylacetate (1/1; v/v) and dried to yield 23 as a yellow powder (23.3 g, 74%). 1H NMR: DMSO-d6 (500 MHz): δ (ppm): (br, 1H), 8.45 (s, 1H), 8.22 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.61 (m, 2H), 7.50 (m, 2H), 7.38 (m, 6H). (6-Nitro-pyridin-3-yl)-hydrazine (24). To a solution of aq. 37% HCl (27 ml) was added 23 (28.8 g, 90.5 mmol) and the reaction mixture was stirred for 2.5 h at RT. Then, water (500 ml) was added and the precipitate was filtered off. The filtrate was concentrated under reduced pressure to afford 24 as a yellow solid (12.5 g, 72%). 1H NMR: DMSO-d6 (500 MHz): δ (ppm): (br, 2H), 9.61 (br, 1H), 8.30 (d, J = 9.0 Hz, 1H), 8.18 (d, J = 2.7 Hz, 1H), 7.51 (dd, J = 9.0 Hz, J = 2.7 Hz, 1H). 5-Methyl-1-(6-nitro-pyridin-3-yl)-1H-pyrazole-4-carboxylic acid ethyl ester (25). A solution of Et 3 N (19 ml, 131 mmol) and 14 (12.5 g, 65.6 mmol) in ethanol (30 ml) was added to a suspension of 24 (12.2 g, 65.6 mmol) in ethanol (217 ml) at RT. After 3h, solvents were removed under reduced pressure and the residue was purified by chromatography on silica gel eluting with a gradient of n-heptane/etoac to afford 25 (13 g, 72%). Engaged directly in the next step (16x) without 1 H NMR characterization. N'-(4-Benzyloxycarbonylamino-cyclohexylidene)-hydrazinecarboxylic acid tert-butyl ester (26). Benzyl N-(4-oxocyclohexyl)carbamate (11.3 g, 44.3 mmol) and tert-butyl carbazate (6.6 g, 48.7 mmol) were stirred in MeOH (140 ml) during 4h at RT. The reaction mixture was evaporated under reduced pressure and the solid residue was triturated with isopropylether, then filtered and dried under vacuum to give 26 (16.4 g, quantitative). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 9.54 (br, 1H), (m, 6H), 5.01 (s, 2H), 3.60 (m, 1H), 2.74 (m, 1H), (m, 2H), (m, 2H), (m, 11H). Hydrochloride salt of N'-(4-Benzyloxycarbonylamino-trans-cyclohexyl)-hydrazinecarboxylic acid tert-butyl ester (27). To a suspension of 26 (16.3 g, 45.1 mmol) in a solution ofacoh/h 2 0 (270 ml ; 1/1 ; v/v), sodium cyanoborohydride (2.78 g, 44.2 mmol) was added portionwise and the reaction mixture was stirred overnight at RT. Aq. 35% NaOH (140 ml) was slowly poured (final ph = 6-7). The precipitate that formed was filtered, washed with water (3x) and dried under vacuum at 60 C (16.3 g, mixture containing 66% of trans isomer and 34% of cis isomer). The powder was dissolved in EtOAc (370 ml) and a 2N HCl solution in diethylether (24.7 ml, 49.3 mmol) was added drop by drop. After stirring overnight, the precipitate that formed was filtered, washed with acetone, and dried under vacuum at 60 C to afford 27, as a pure trans isomer in its hydrochloride form (11.4 g, 63%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): (br, 1H), (br, 1H), (m, 6H), 5.01 (s, 2H), 3.23 (m, 1H), 3.06 (m, 1H), 1.96 (m, 2H), 1.88 (m, 2H), (m, 11H), 1.18 (m, 2H). Hydrochloride salt of (4-Hydrazino-trans-cyclohexyl)-carbamic acid benzyl ester (28). 27 (11.3 g, 28.3 mmol) were dissolved in 1,4-dioxane (250 ml). Then a 4N HCl solution in 1,4-dioxane (93.3 ml, 373 mmol) was added drop by drop and the reaction mixture was stirred at RT during 60 h. The precipitate that formed was filtered and dried under vacuum at 60 C to afford 28 in the form of white crystals (7.61 g, 90%). 1 H NMR: DMSO-d6 (250 MHz): δ (ppm): 7.34 (5H, m), 5.00 (s, 2H), 3.24 (m, 1H), 2.84 (m, 1H), (m, 4H), 1.22 (m, 4H). 21