, 1H, J ¼ 7.3 Hz), 6.88 (d, 2H, J ¼ 8.5 Hz), 7.18 (d, 1H, J ¼ 8.9 Hz), 7.22 (dd, 1H, J ¼ 1.8 Hz, J ¼ 8.9 Hz), 7.37 (d, 2H, J ¼ 8.5 Hz), vol.7

, 1H-indazol-3-amine (41f) Following general method J and starting from 40f (83 mg, 0.25 mmol) and 3-chloroaniline (31.8 mL, 0.30 mmol), 41f was obtained as a white solid (34 mg, 0.09 mmol, 36%)

, 46 (s, 1H); 19 F NMR (376 MHz, DMSO-d 6 ), mp ¼ 153e154 C; 1 H NMR (400 MHz, DMSO-d 6 ) d ppm 1.70e1.73 (m, 2H), 1.91e2.03 (m, 4H), 2.08e2.15 (m, 2H), 5.12e5.16 (m, 1H), 6.88 (d, 1H, J ¼ 8.0 Hz), 7.31 (t, 1H, J ¼ 8.0 Hz), 7.51 (d, 1H, J ¼ 8.0 Hz), 7.63 (d, 1H, J ¼ 8.9 Hz), 7.75 (d, 1H, J ¼ 8.9 Hz), vol.7

, HRMS (M þ H þ ) 380.1118 (calcd for C 19 H 17 ClF 3 N 3 H þ 380, 1136.

, -methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41g) Following general method I and starting from 40f, vol.104

, 4-methoxybenzylamine (48.9 mL, 0.37 mmol), 41g was obtained as a yellow oil (24 mg, 0.06 mmol, 20%)

H. Nmr, 500 MHz, DMSO-d 6 ) d ppm 1.62e1.65 (m, 2H), 1.83e1.99 (m, 6H), 3.72 (s, 3H), 4.37 (d, 2H, J ¼ 6.9 Hz), 4.95 (quint., 1H, J ¼ 7.2 Hz), 6.78 (t, 1H, J ¼ 6.0 Hz), 6.87 (d, 2H, J ¼ 8.7 Hz), 7.35 (d, 2H, J ¼ 8.7 Hz), 7.49 (dd, 1H, J ¼ 1.5 Hz, J ¼ 8.9 Hz), 7.55 (d, 1H, J ¼ 8.9 Hz), vol.8

, HRMS (M þ H þ ) 390.1785 (calcd for C 21 H 22 F 3 N 3 OH þ 390, 1788.

, Following general method J and starting from 40f (83 mg, 0.25 mmol) and 3-chloro-4-methoxybenzylamine (43.5 mL, 0.30 mmol), 41h was obtained as a yellow oil (47 mg, 0.11 mmol, 44%)

, Following general method J and starting from 40f (83 mg, 0.25 mmol) and 3-fluoro-4-methoxybenzylamine (41.2 mL, 0.30 mmol), 41i was obtained as a colorless oil (38 mg, 0.09 mmol, 38%), CDCl 3 ) d ppm 1.69e1.72 (m, 2H), 1.94e1.96 (m, 2H)

, HRMS (M þ H þ ) 408.1687

, -methoxypropyl)-5-(trifluoromethyl)-1H-indazol-3-amine (41j) Following general method J and starting from 40f, vol.83

, 24 (s, 3H), 3.29 (q, 2H, J ¼ 6, 41j was obtained as an orange oil (19 mg, 0.06 mmol, 23%). Purity !98%; 1 H NMR (400 MHz, DMSO-d 6 ) d ppm 1.62e1.65 (m, 2H), 1.82e1.98 (m, 4H), 1.92e1.98 (m, 4H), vol.3, p.3

, -methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41k) Following general method J and starting from 40g, vol.62

, 4-methoxybenzylamine (28.0 mL, 0.21 mmol), 41k was obtained as yellow oil (20 mg, 0.05 mmol, 27%)

H. Nmr, 72 (s, 3H), 4.36 (br s, 1H), 4.38 (d, 2H, 400 MHz, DMSO-d 6 ) d ppm 1.24 (q, 1H, J ¼ 9.4 Hz), 1.45 (q, 2H, J ¼ 12.8 Hz), 1.67 (d, 1H, J ¼ 12.2 Hz), 1.82e1.90 (m, 7H), vol.3

, -methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41l) Following general method I and starting from 40h, vol.102

, mL, 0.34 mmol), 41l was obtained as colorless oil (59 mg, 0.14 mmol, 50%)

H. Nmr, 74 (s, 1H); 13 C NMR (101 MHz, 400 MHz, CDCl 3 ) d ppm 3.73 (s, 3H), 4.48 (s, 2H), 5.35 (s, 2H), 6.80 (d, 2H, J ¼ 8.7 Hz), 7.09e7.14 (m, 3H), 7.17e7.22 (m, 2H), 7.28 (d, 2H, J ¼ 8.7 Hz), 7.40 (dd, 1H, J ¼ 1.6 Hz, J ¼ 8.9 Hz), vol.7

, -methoxyphenyl)methyl]-1-phenyl-5-(trifluoromethyl)-1H-indazol-3-amine (41m) Following general method J and starting from 40i, vol.68

, 4-methoxybenzylamine (31.2 mL, 0.24 mmol), 41m was obtained as a yellow oil (38 mg, 0.10 mmol, 48%)

H. Nmr,

. Hz, CDCl 3 ) d ppm 3.82 (s, 3H), 6.91 (d, 2H, J ¼ 8, Hz), 7.31 (s, 1H), 7.39 (t, 1H, J ¼ 7.8 Hz), 7.50 (d, 1H, J ¼ 7.8 Hz), 7.65 (d, 2H, J ¼ 8.8 Hz), vol.7

, 2 mM magnesium acetate, 50 mg BSA. PDE1 was assayed at 1 mM cGMP in calmodulin activated state (18 nM calmodulin with 10 mM CaCl 2 ). PDE2 was evaluated at 1 mM cAMP in activated state (in presence of 5 mM cGMP). of reciprocal cross-contamination between PDE3 and PDE4, the studies were always carried out in the presence of 50 mM rolipram (a generous gift of Schering, Cyclic nucleotide phosphodiesterase activity assay PDE1, PDE3, PDE4 and PDE5 were isolated by anion exchange chromatography from bovine aortic smooth muscle cytosolic fraction according to the literature, vol.3

, In vivo studies Experiments were performed using C57BL/6J male mice (Charles River, L'Arbresle, France) between 8 and 10 weeks old at surgery time. Mice were group-housed five per cage and kept under a 12 h light/dark cycle with food and water ad libitum, p.57

, C57BL/6J mice were used for the experiments. All animals received proper care in agreement with European guidelines

, All mice were allowed to recover from surgery for at least two weeks before starting treatments. Mechanical allodynia was tested using von Frey hairs and results were expressed in grams. Tests were done during the morning, starting at least 2 h after lights on. Mice were placed in clear Plexiglas boxes (7 cm  9 cm x 7 cm) on an elevated mesh screen. Calibrated von Frey filaments (Bioseb, Vitrolles, France) were applied to the plantar surface of each hindpaw until they just bent, in a series of ascending forces up to the mechanical threshold. Filaments were tested five times per paw and the paw withdrawal threshold (PWT) was defined as the lower of two consecutive filaments for which three or more withdrawals out of the five trials were observed, ) followed by cervical dislocation, according to the institutional ethical guidelines. The animal facilities Chronobiotron UMS3415 are registered for animal experimentation under the Animal House Agreement A67-2018-38

, 9% NaCl solution that was also used for control injections. Data were expressed as mean±SEM, and statistical analyses were performed using STATISTICA, with ANOVA for multiple comparisons and the Duncan test for posthoc analyses, vol.7

, Supporting information available General procedures and experimental data for compounds 25e30, 42e44 and 46e48. Noesy NMR of 41d, Selectivity towards other PDE 1 to 4 isoenzymes for compounds 4c, 4f, 16e, 25e, 25g, 25h, 25l, and 45. Experimental details and results for water solubility and chemical stability of phthalazines and indazoles de

T. Keravis and C. Lugnier, Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments, Br. J. Pharmacol, vol.165, pp.1288-1305, 2012.
URL : https://hal.archives-ouvertes.fr/hal-00670798

J. F. Tcheudji, L. Lebeau, N. Virmaux, C. G. Maftei, R. H. Cote et al., Molecular organization of bovine rod cGMP-phosphodiesterase 6, J. Mol. Biol, vol.310, pp.781-791, 2001.

C. Lugnier, P. Schoeffter, A. L. Bec, E. Strouthou, and J. C. Stoclet, Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta, Biochem. Pharmacol, vol.35, issue.86, pp.90333-90336, 1986.

R. H. Francis, T. M. Lincoln, and J. D. Corbin, Characterization of a novel cGMP binding protein from rat lung, J. Biol. Chem, vol.255, pp.620-626, 1980.

J. E. Souness, R. Brazdil, B. K. Diocee, and R. Jordan, Role of selective cyclic GMP phosphodiesterase inhibition in the myorelaxant actions of M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine, Br. J. Pharmacol, vol.98, 1989.

N. Gali-e, H. A. Ghofrani, A. Torbicki, R. J. Barst, L. J. Rubin et al., Sildenafil citrate therapy for pulmonary arterial hypertension, N. Engl. J. Med, vol.353, pp.2148-2157, 2005.

S. Korkmaz, T. Radovits, E. Barnucz, P. Neugebauer, R. Arif et al., Szab o, Dose-dependent effects of a selective phosphodiesterase-5-inhibitor on endothelial dysfunction induced by peroxynitrite in rat aorta, Eur. J. Pharmacol, vol.615, pp.155-162, 2009.

E. Takimoto, H. C. Champion, M. Li, D. Belardi, S. Ren et al., Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy, Nat. Med, vol.11, pp.214-222, 2005.

R. Zhang, Y. Wang, L. Zhang, Z. Zhang, W. Tsang et al., Sildenafil (viagra) induces neurogenesis and promotes functional recovery after stroke in rats, Stroke, vol.33, 2002.

N. K. Jain, C. S. Patil, A. Singh, and S. K. Kulkarni, Sildenafil-induced peripheral analgesia and activation of the nitric oxideecyclic GMP pathway, Brain Res, vol.909, pp.2673-2680, 2001.

C. I. Araiza-saldaña, G. Reyes-garcía, D. Y. Bermúdez-ocaña, F. Erez-severiano, and V. Granados-soto, Effect of diabetes on the mechanisms of intrathecal antinociception of sildenafil in rats, Eur. J. Pharmacol, vol.527, pp.60-70, 2005.

M. L. Vale, D. E. Rolim, I. F. Cavalcante, R. A. Ribeiro, and M. H. Souza, Role of NO/ cGMP/KATP pathway in antinociceptive effect of sildenafil in zymosan writhing response in mice, Inflamm. Res, vol.56, 2007.

M. M. Bezerra, V. Lima, V. C. Girao, R. C. Teixeira, and J. R. Graca, Antinociceptive activity of sildenafil and adrenergic agents in the writhing test in mice, Pharmacol. Rep, vol.60, pp.339-344, 2008.

C. S. Patil, N. K. Jain, V. P. Singh, and S. K. Kulkarni, Cholinergic-NO-cGMP mediation of sildenafil-induced antinociception, Indian J. Exp. Biol, vol.42, pp.361-367, 2004.

L. J. Huang, M. H. Yoon, J. I. Choi, W. M. Kim, H. G. Lee et al., Effect of sildenafil on neuropathic pain and hemodynamics in rats, Yonsei Med. J, vol.51, pp.82-87, 2010.

L. Wang, M. Chopp, A. Szalad, Z. Liu, M. Bolz et al., Phosphodiesterase-5 is a therapeutic target for peripheral neuropathy in diabetic mice, Neuroscience, vol.193, 2011.

G. Hackett, PDE5 inhibitors in diabetic peripheral neuropathy, Int. J. Clin. Pract, vol.60, pp.1123-1126, 2006.

E. I. Gediz, C. Nacitarhan, E. Minareci, and G. Sadan, Antinociceptive effect of vardenafil on carrageenan-induced hyperalgesia in rat: involvement of nitric oxide/cyclic guanosine monophosphate/calcium channels pathway, J. Pharm. Res, vol.14, pp.1137-1143, 2015.

F. Rocha, F. Silva, A. Leite, A. Leite, V. Girão et al., Tadalafil analgesia in experimental arthritis involves suppression of intra-articular TNF release, Br. J. Pharmacol, vol.164, 2011.

M. Ambriz-tututi, D. A. Vel-azquez-zamora, H. Urquiza-marín, and V. Granados-soto, Analysis of the mechanism underlying the peripheral antinociceptive action of sildenafil in the formalin test, Eur. J. Pharmacol, vol.512, pp.121-127, 2005.

D. Levy, A. Oke, and D. W. Zochone, Local expression of inducible nitric oxide synthase in an animal model of neuropathic pain, Neurosci. Lett, vol.260, pp.982-985, 1999.

A. Hervera, R. Negrete, S. Le-anez, J. M. Martín-campos, and O. Pol, The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice, PLoS One, vol.5, p.14321, 2010.

Y. Guan, M. Yaster, S. N. Raja, and Y. X. Tao, Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice, Mol. Pain, vol.3, 2007.

M. Tanabe, Y. Nagatani, K. Saitoh, K. Takasu, and H. Ono, Pharmacological assessments of nitric oxide synthase isoforms and downstream diversity of NO signaling in the maintenance of thermal and mechanical hypersensitivity after peripheral nerve injury in mice, Neuropharmacology, vol.56, pp.702-708, 2009.

V. Granados-soto, M. Rufino, L. D. Gomes-lopes, and S. H. Ferreira, Evidence for the involvement of the nitric oxideecGMP pathway in the antinociception of morphine in the formalin test, Eur. J. Clin. Pharmacol, vol.340, 1997.

K. Takasu, M. Honda, H. Ono, and M. Tanabe, Spinal a2-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury, Br. J. Pharmacol, vol.148, pp.233-244, 2006.

T. Mixcoatl-zecuatl, F. J. Flores-murrieta, and V. Granados-soto, The nitric oxidecyclic GMP-protein kinase G-Kþ channel pathway participates in the antiallodynic effect of spinal gabapentin, Eur. J. Pharmacol, vol.531, 2006.

A. Gibson, Phosphodiesterase 5 inhibitors and nitrergic transmissiondfrom zaprinast to sildenafil, Eur. J. Pharmacol, vol.411, pp.1-10, 2001.

A. M. Martel, A. Graul, X. Rabasseda, and R. Castaner, Treatment of erectile dysfunction, Phosphodiesterase V inhibitor, Drugs Future, vol.22, pp.138-143, 1997.

D. Ormrod, S. E. Easthope, D. P. Figgitt, and . Vardenafil, Drugs Aging, vol.19, 2002.

B. Dumaître and N. Dodic, Synthesis and cyclic GMP phosphodiesterase inhibitory activity of a series of 6-phenylpyrazolo[3,4-d ]pyrimidones, J. Med. Chem, vol.39, pp.1635-1644, 1996.

C. M. Allerton, C. G. Barber, K. C. Beaumont, D. G. Brown, S. M. Cole et al., A novel series of potent and selective PDE5 inhibitors with potential for high and dose-independent oral bioavailability, J. Med. Chem, vol.49, pp.3581-3594, 2006.

M. Hagiwara, T. Endo, T. Kanayama, and H. Hidaka, Effect of 1 -(3-Chloroanilino)-4-Phenylphthalazine (MY-5445), a specific inhibitor of cyclic GMP phosphodiesterase, on human platelet aggregation, J. Pharmacol. Exp. Ther, vol.228, pp.467-471, 1984.

N. Watanabe, H. Adachi, Y. Takase, H. Ozaki, M. Matsukura et al., 3-Chloro-4-methoxybenzyl)aminophthalazines: synthesis and inhibitory activity toward phosphodiesterase 5, J. Med. Chem, vol.43, pp.2523-2529, 2000.

M. A. Duncton, E. L. Chekler, R. Katoch-rouse, D. Sherman, W. C. Wong et al., Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2, vol.17, pp.731-740, 2009.

Y. Takase, T. Saeki, M. Fujimoto, and I. Saito, Cyclic GMP phosphodiesterase inhibitors. 1. The discovery of a novel potent inhibitor, 4, J. Med. Chem, vol.36, pp.3765-3770, 1993.

G. Yu, H. J. Mason, X. Wu, J. Wang, S. Chong et al., Substituted pyrazolopyridines as potent and selective PDE5 inhibitors: potential agents for treatment of erectile dysfunction, J. Med. Chem, vol.44, pp.1025-1027, 2001.

N. Anand, S. Brown, Z. Tesfai, and C. Zaharia, Phthalazine Derivatives as Jak1 Inhibitors, 2012. WO2012037132 (A1), 2017.

L. T. Spada, J. G. Shiah, P. M. Hughes, T. C. Malone, G. W. Devries et al., Kinase Inhibitors, 2010. WO2010078393 (A1), 2017.

H. Dyker, J. Scherkenbeck, D. Gondol, A. Goehrt, and A. Harder, Azadepsipeptides: synthesis and evaluation of a novel class of peptidomimetics, J. Org. Chem, vol.66, pp.3760-3766, 2001.

E. Lohou, V. Collot, S. Stiebing, and S. Rault, Direct Access to 3-Aminoindazoles by Buchwald-Hartwig C-N Coupling Reaction, Synthesis, 2011.

Y. Mao, G. Tian, Z. Liu, J. Shen, and J. Shen, An improved synthetic route for preparative process of vardenafil, Org. Process Res. Dev, vol.13, pp.1206-1208, 2009.

J. B. Smaill, A. J. Gonzales, J. A. Spicer, H. Lee, J. E. Reed et al., Tyrosine kinase inhibitors. 20. Optimization of substituted quinazoline and pyrido[3,4-d]pyrimidine derivatives as orally active, irreversible inhibitors of the epidermal growth factor receptor family, J. Med. Chem, vol.59, 2016.

M. Benbouzid, V. Pallage, M. Rajalu, E. Waltisperger, S. Doridot et al., Sciatic nerve cuffing in mice: a model of sustained neuropathic pain, Eur. J. Pain, vol.12, pp.591-599, 2008.
URL : https://hal.archives-ouvertes.fr/hal-00343709

I. Yalcin, S. Megat, F. Barthas, E. Waltisperger, M. Kremer et al., The sciatic nerve cuffing model of neuropathic pain in mice, J. Vis. Exp, vol.89, 2014.

M. Bollenbach, E. Salvat, F. Daubeuf, P. Wagner, I. Yalcin et al., Phenylpyridine-2-ylguanidines and rigid mimetics as novel inhibitors of TNFa overproduction: beneficial action in models of neuropathic pain and of acute lung inflammation, Eur, J. Med. Chem, vol.147, pp.163-182, 2018.

M. Kremer, E. Salvat, A. Muller, I. Yalcin, and M. Barrot, Antidepressants and gabapentinoids in neuropathic pain: mechanistic insights, Neuroscience, vol.338, pp.183-206, 2016.

R. Treede, T. S. Jensen, J. N. Campbell, G. Cruccu, J. O. Dostrovsky et al., Neuropathic pain Redefinition and a grading system for clinical and research purposes, Neurology, vol.70, pp.1630-1635, 2008.

Y. Bohren, L. Tessier, S. Megat, H. Petitjean, S. Hugel et al., Antidepressants suppress neuropathic pain by a peripheral b2-adrenoceptor mediated anti-TNFa mechanism, Neurobiol. Dis, vol.60, pp.39-50, 2013.

I. Yalcin, L. Tessier, N. Petit-demouli-ere, S. Doridot, L. Hein et al., Barrot, b2-adrenoceptors are essential for desipramine, venlafaxine or reboxetine action in neuropathic pain, Neurobiol. Dis, vol.33, pp.386-394, 2009.

M. Kremer, I. Yalcin, Y. Goumon, X. Wurtz, L. Nexon et al., A dual noradrenergic mechanism for the relief of neuropathic allodynia by the antidepressant drugs duloxetine and amitriptyline, J. Neurosci, vol.38, 2018.
URL : https://hal.archives-ouvertes.fr/hal-02108245

M. Benbouzid, N. Choucair-jaafar, I. Yalcin, E. Waltisperger, A. Muller et al., Chronic, but not acute, tricyclic antidepressant treatment alleviates neuropathic allodynia after sciatic nerve cuffing in mice, Eur. J. Pain, vol.12, pp.1008-1017, 2008.
URL : https://hal.archives-ouvertes.fr/hal-00343697

A. K. Naik, S. K. Tandan, D. Kumar, and S. P. Dudhgaonkar, Nitric oxide and its modulators in chronic constriction injury-induced neuropathic pain in rats, Eur. J. Pharmacol, vol.530, pp.59-69, 2006.

D. Levy and D. W. Zochodne, NO pain: potential roles of nitric oxide in neuropathic pain, Pain Pract, vol.4, 2004.

A. Hervera, R. Negrete, S. Le-anez, J. M. Martín-campos, and O. Pol, The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice, PLoS One, vol.5, p.14321, 2010.

I. D. Duarte and S. H. Ferreira, The molecular mechanism of central analgesia induced by morphine or carbachol and the L-arginine-nitric oxide-cGMP pathway, Eur. J. Pharmacol, vol.221, pp.90789-90796, 1992.

G. Petho and P. W. Reeh, Sensory and signaling mechanisms of bradykinin, eicosanoids, platelet-activating factor, and nitric oxide in peripheral nociceptors, Physiol. Rev, vol.92, pp.1699-1775, 2010.

A. Schmidtko, Nitric oxide-mediated pain processing in the spinal cord, Pain Control, pp.103-117, 2015.

K. V. Otari and C. D. Upasani, Involvement of NOecGMP pathway in antihyperalgesic effect of PDE5 inhibitor tadalafil in experimental hyperalgesia, Inflammopharmacology, vol.23, pp.187-194, 2015.

L. Garcia, S. Hlaing, R. Gutierrez, M. Sanchez, I. Kovanecz et al., Sildenafil attenuates inflammation and oxidative stress in pelvic ganglia neurons after bilateral cavernosal nerve damage, Int. J. Mol. Sci, vol.15, pp.17204-17220, 2014.

P. J. Austin and G. Moalem-taylor, The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, immune-like glial cells and cytokines, J. Neuroimmunol, vol.229, pp.26-50, 2010.

E. Rojewska, A. Piotrowska, A. Jurga, W. Makuch, and J. Mika, Zaprinast diminished pain and enhanced opioid analgesia in a rat neuropathic pain model, Eur. J. Pharmacol, vol.839, pp.21-32, 2018.

A. Singh-jaggi and N. Singh, Therapeutic targets for the management of peripheral nerve injury-induced neuropathic pain, CNS Neurol. Disord. -Drug Targets, vol.10, 2011.

T. Keravis, R. Thaseldar-roumi-e, and C. Lugnier, Assessment of phosphodiesterase isozyme contribution in cell and tissue extracts, Phosphodiesterase Methods Protoc, 2005.
URL : https://hal.archives-ouvertes.fr/hal-00129894

M. Barrot, Tests and models of nociception and pain in rodents, Neuroscience, vol.211, pp.39-50, 2012.