Design and synthesis of 3-aminophthalazine derivatives and structural analogues as PDE5 inhibitors: anti-allodynic effect against neuropathic pain in a mouse model

Article abstract of DOI:10.1016/j.ejmech.2019.05.026

Neuropathic pain is a chronic pain caused by a lesion or disease affecting the somatosensory nervous system. To date, no specific treatment has been developed to cure this pain. Antidepressants and anticonvulsant drugs are used, but they do not demonstrate universal efficacy, and they often cause detrimental adverse effects. Some studies highlighted the efficacy of sildenafil, a well-known inhibitor of phosphodiesterase 5 (PDE5, (IC₅₀ = 3.3 nM)), in models of pain. Based on these results, we focused our attention on MY 5445, another known PDE5 inhibitor. Homologues, isosteres and structural analogues of MY 5445 were designed and all synthesized compounds were evaluated for their inhibitory activity toward PDE5. Selectivity profiles towards other PDE1-4 isoenzymes, water solubility and stability in acidic medium of the most potent PDE5 inhibitors were determined and the aminophthalazine 16h and its mimetic 41n (3-aminoindazole)were evaluated in comparison to MY 5445 (4b)in vivo in a model of neuropathic pain induced by sciatic nerve cuffing in mice (3 and 0.5 mg/kg, ip twice a day). Both compounds showed the same efficacy on neuropathic allodynia as MY 5445, and thus produced a significant relief of mechanical hypersensitivity after 12 days of treatment.

Full text of DOI:10.1016/j.ejmech.2019.05.026

Accepted Manuscript
Design and synthesis of 3-aminophthalazine derivatives and structural analogues as
PDE5 inhibitors: anti-allodynic effect against neuropathic pain in a mouse model
Maud Bollenbach, Claire Lugnier, Mélanie Kremer, Eric Salvat, Salim Megat, Frédéric
Bihel, Jean-Jacques Bourguignon, Michel Barrot, Martine Schmitt
PII:
S0223-5234(19)30435-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.026
EJMECH 11336
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 January 2019
Revised Date: 30 April 2019
Accepted Date: 7 May 2019
Please cite this article as: M. Bollenbach, C. Lugnier, Mé. Kremer, E. Salvat, S. Megat, Fréé. Bihel, J.-
J. Bourguignon, M. Barrot, M. Schmitt, Design and synthesis of 3-aminophthalazine derivatives and
structural analogues as PDE5 inhibitors: anti-allodynic effect against neuropathic pain in a mouse model,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.026.
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ACCEPTED MANUSCRIPT
Design and synthesis of 3-aminophthalazine derivatives
and structural analogues as PDE5 inhibitors: anti-allodynic
effect against neuropathic pain in a mouse model
Maud Bollenbach[a], Claire Lugnier[b], Mélanie Kremer[c], Eric Salvat[c,d], Salim Megat[c],
Frédéric Bihel[a], Jean-Jacques Bourguignon[a], Michel Barrot[c] and Martine Schmitt*[a]
[a]
M. Bollenbach, Dr. F. Bihel, Dr. J.-J. Bourguignon, Dr. M. Schmitt
Université de Strasbourg, CNRS, LIT UMR 7200, Laboratory of Excellence Médalis, Illkirch, France.
E-mail: mschmitt@unistra.fr
[b]
[c]
Dr. C. Lugnier
Université de Strasbourg, CNRS, Biophysique et Pharmacologie, UMR7210, F- 67400, Illkirch
Dr. Mélanie Kremer, Dr. E. Salvat, Dr. S. Megat, Dr. M. Barrot
Centre National de la Recherche Scientifique, Université de Strasbourg,
Institut des Neurosciences Cellulaires et Intégratives,
8 allée du Général Rouvillois, 67000 Strasbourg, France
Dr. E. Salvat
[d]
Centre d’Evaluation et de Traitement de la Douleur,
Hôpitaux Universitaires de Strasbourg, Strasbourg, France
Highlights
•
•
•
Design of homologues, isosteres and structural analogues of MY 5445
Evaluation for their inhibitory activity towards PDE 5
In vivo proof of efficacy of 16h and 41n in neuropathic allodynia
Abstract: Neuropathic pain is a chronic pain caused by a lesion or disease affecting the somatosensory nervous system.
To date, no specific treatment has been developed to cure this pain. Antidepressants and anticonvulsant drugs are
used, but they do not demonstrate universal efficacy, and they often cause detrimental adverse effects. Some studies
highlighted the efficacy of sildenafil, a well-known inhibitor of phosphodiesterase 5 (PDE5, (IC50=3.3nM)), in models of
pain. Based on these results, we focused our attention on MY 5445, another known PDE5 inhibitor. Homologues,
isosteres and structural analogues of MY 5445 were designed and all synthesized compounds were evaluated for their
inhibitory activity toward PDE5. Selectivity profiles towards other PDE1-4 isoenzymes, water solubility and stability in
acidic medium of the most potent PDE5 inhibitors were determined and the aminophthalazine 16h and its mimetic 41n
(3-aminoindazole) were evaluated in comparison to MY 5445 (4b) in vivo in a model of neuropathic pain induced by
sciatic nerve cuffing in mice (3 and 0.5 mg/kg, ip twice a day). Both compounds showed the same efficacy on
neuropathic allodynia as MY 5445, and thus produced a significant relief of mechanical hypersensitivity after 12 days of
treatment.
Introduction
Cyclic AMP (cAMP) and cyclic GMP (cGMP) play major roles in normal and physio-pathological intracellular signalling.
Levels of cAMP and cGMP are influenced by cyclic nucleotide phosphodiesterase (PDE) families that specifically and
rapidly convert the cyclic nucleotide to their corresponding 5’ nucleotide. Eleven PDE families (PDE1 to PDE11) have
been characterized to date. They differ by their substrate specificities, kinetic properties, tissue and subcellular
localizations, and drug and mediator sensitivities, representing fine tuned new therapeutic targets.[1] Among these
families, the PDE5 family specifically hydrolyzes cGMP, and is characterized by two GAF allosteric domains.[2] We
characterized the first selective PDE5 inhibitor, zaprinast 1, by using purified PDE5 (also known as cGMP-PDE, or
cGMP-binding-PDE) from vascular smooth muscle [3] and pulmonary smooth muscle.[4] Since, multiple other PDE5
inhibitors has been developed, like DMPPO 2 and sildenafil 3.[1] PDE5 inhibitors constitute a new class of vasoactive
drugs that have been developed for the treatment of erectile dysfunction in patients. Inhibition of PDE5 increases cyclic
GMP (cGMP) levels,[5] and favors smooth muscle relaxation and penis erection. Sildenafil is also indicated clinically for
treatment of pulmonary hypertension [6,7] and cardiac hypertrophy.[8] In addition, further investigations showed that
vardenafil, another PDE5-inhibitor, could be effective against ischemia.[9] In the previous decade, studies in animal
models also proposed a beneficial effect of PDE5 inhibitors on various pain conditions.[10–16].This pain-relieving action
was observed in somatic [10,11] and in visceral [12,13] inflammatory pain models, as well as in models of neuropathic
pain, either lesional [15] or metabolic.[16] Moreover, a case report proposed that sildenafil may improve neuropathic pain
symptoms in patients with diabetic peripheral neuropathy. [17] Mechanistically, this analgesia mediated by PDE5
inhibitors has been related to their action on the nitric oxide (NO)/cGMP pathway.[10, 12, 14–16, 18–20]. While NO may

ACCEPTED MANUSCRIPT
contribute to neuropathic pain symptoms via an up-regulation of nitric oxide synthases [21, 22] and an activation of the
NO-cGMP-PKG pathway [23, 24] in the spinal cord and dorsal root ganglia, evidence accumulated showing that NO can
also have anti-nociceptive properties. Indeed, the NO-cGMP pathway contributes to the analgesic effect of opioid mu
receptor agonists [25] of gabapentinoïds [26, 27] as well as of PDE5 inhibitors [10, 12, 14–16, 18–20].
Zaprinast 1 [28] is the prototype of the most important series of PDE5 inhibitors deriving from pyrimidones-containing
bicyclic compounds (Figure 1, cpds 1-3). The well known sildenafil 3 (Viagra®) [29] still continued to provide novel safer
derivatives and isosteres [30, 31] (better selectivity profiles towards other PDE isoforms, water-soluble analogues).[32]
MY5445 [33] (cpd 4b), a phthalazine-deriving PDE5-I showed significant difference between both scaffolds of 1 and 3. In
addition few SAR analyses published data dealing with MY5445 series could not be clearly correlated with other
structural analogues 5 [35, 34] and 6 [36] depicted in Figure 1.
The N-benzyl phthalazine 5 could be regarded as a superior homologue of MY5445 (4b) [37]. However the respective
beneficial effects combining both homology and substitutions effects at the phthalazine nucleus are not known. We
selected in Figure 1 the N-benzyl quinazoline 6 as a putative structural equivalent (pyridazine vs pyrimidine), but with
different substitution effects at both the benzo and N-benzyl fragments.
Figure 1. Zaprinast, structurally-related compounds (2 and 3), MY 5445 4b, and structurally-related compounds 5 and 6
as selected PDE5 inhibitors.
Our general objective was the first attempts to introduce a systematic SAR analysis of series of PDE-5 belonging to a
common general formula in Figure 1. In particular, as depicting in Figure 2 and starting from MY5445, we evaluated the
importance of 1) the benzo group C, 2) the possible substitution on both phenyl rings A and B, 3) the replacement of ring
A, 4) the homologation, and 5) deletion of the imine function leading to benzamides derivatives which could be
considered as seco derivatives of both 4b and 6 subseries. Finally, starting from the benzamide structure, different semi-
rigid derivatives were designed. This large topological exploration of both the scaffold and the different substitutions of
critical fragments (rings A, B and C) should i) help to select the most potent PDE5-I, ii) determine their selectivity toward
a panel of PDE isoforms, iii) characterize the in vivo efficacy of a couple of selected compounds in a neuropathic pain
model in mice.

ACCEPTED MANUSCRIPT
Figure 2. Topologic study on MY5445.
1-Chemistry
The 4-phenyl isoquinoline-amine 10 was synthesized in a three step sequence starting from the commercially available
2-chloroisoquinoline 7 (Scheme 1). The aniline intermediate 8 was prepared in 78% yield by heating 7 at 110 °C in
presence of 3-chloroaniline in NMP.[38] Regioselective bromination was performed efficiently by treatment with
phenyltrimethylammonium tribromide,[38] yielding the desired bromoisoquinoline 9 in 99% yield. Finally a Suzuki-
Miyaura reaction of 9 with phenylboronic acid afforded the target product 10 in 52% yield.
Scheme 1. Synthesis of isoquinoline 10. (a) 3-chloroaniline (1.2 eq.), NMP, 110 °C, 15 h, 78%; (b) Me3PhNBr3 (0.90 eq.), THF, 0-25 °C, 16 h,
99%; (c) PhB(OH)2 (1.2 eq.), Pd(PPh3)4 (5 mol%), Na2CO3 (3 eq.), Tol:EtOH:H2O (5:1:1), 120 °C, 2 h, 52%.
The aminophthalazine derivatives 4a-f, 16-21 were prepared using methodologies depicted in Scheme 2. The easily
preparable 1,4-dichlorophthalazine derivatives 12a-b were treated with various amines affording intermediates 13a-d,
14a-g and 15a-d in good yields. Compounds 13-15 smoothly underwent a Suzuki-Miyaura cross-coupling reaction with
the appropriate boronic acids enabling formation of final 6-aryl amino phthalazine analogues 4a-f, 16a-g and 17a-d.
Subsequent condensation of 13b, 14e and 14h with the appropriate cyclic amines afforded the diaminophthalazine
derivatives 20 and 21a-c. Finally analogues 18-19 were obtained via a standard dehalogenation of chloro compounds
13b and 14e. 6-Substituted trifluoromethyl-aminophthalazine derivatives 16h and 21d were prepared from the readily
available trifluoromethyl 1,4-dichlorophthalazine 12b [36] in a similar fashion (Scheme 2).

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Scheme 2. Synthesis of phthalazines derivatives 4, 16-21. (a) pyridine (2 eq.), POCl3 (35 eq.), 110 °C, 1 h, 93%; (b) DIEA (1.1 eq.), PO Cl3 (25
eq.), 120°C, 3h, 75%; (c) for n=0 and R3=H : R1PhNH2 (1 eq.), iPrOH, 110 °C, 1 h, 18-100%; (d) for n=1 or 2 and R3=H : NH2(CH2)n-R1Ph (1.1
eq.), Na2CO3 (2eq.), DMF, 130 °C, 16 h, 57-96%; (e) for n=1 and R3=CF3 : 4-methoxybenzylamine (1.2 eq.), dbu (2.5 eq.), NMP, rt, 2h, 51%; (f)
R2PhB(OH)2 (1.1-1.5 eq.), Pd(PPh3)4 (5 mol%), Na2CO3 (3 eq.), Tol:EtOH:H2O (5:1:1), 120 °C, 16 h, 17-100%; (g) for n = 0 and R1 = 3-Cl : formic
acid (1.0 eq.), Pd(PPh3)4 (4 mol%), NEt3 (12.0 eq.), 110 °C, 25 min, µW, 77%; (h) for n = 1 and R1 = 4-OMe : H2 (60 psi), Pd/C (5 mol%), EtOH, rt,
16 h, 19%; (i) cyclic amine (5.18 eq.), DIPEA (5.14 eq.), NMP, 170 °C, 16 h, 26-79%.
The substituted benzamides 25-30 were carried out using standard coupling procedures starting from the corresponding
benzoic acids, as illustrated in Scheme 3. If the benzoic acids were not commercially available, they were easily
prepared by known experimental methods (see experimental part). Further structural modifications were performed on
phenyl rings of some benzamide derivatives. As an illustration, the thioanisole derivative 25f was conveniently converted
into the corresponding sulfoxide 25g at room temperature using sodium periodate as oxidant.[39] Demethylation of 25l
with BBr3 under microwave irradiation led to the corresponding phenol 25v. Alkylation of 25v with nBu-Br in DMF
afforded 25w in a satisfactory yield. Buchwald amination reaction of 25x with the help of JohnPhos allowed the formation
of 25y in 63% yield. Finally, a ligand free Ullmann N-arylation was performed starting from 26a to introduce the primary
cyclohexylamine (cpd 26c).
Scheme 3. Synthesis of benzamides 25-30. (a) HOBt.NH3 (1.5 eq.), EDC (1.2 eq.), DMF, rt, 17 h, 72%; (b) NaOH (5 eq.), MeOH:H2O (1:1), rt, 15
h, 82%; (c) SOCl2, 80 °C, 2 h; (d) R 3NH(CH2)m-PhR4 (1.2 eq.), NEt3 (2 eq.), DCM, rt, 16 h, 37-76% over 2 steps; (e) R3NH(CH2)m-R4Ph (2 eq.), 1-
HOBT (1.1 eq.), EDC.HCl (1.5 eq.), DMF, rt, 16 h, 28-99%; (f) NaIO4 (0.5M, 1 eq.), MeOH, 0-25 °C, 16 h, 90%; (g) piper idine (1 eq.), Pd2(dba)3 (10
mol%), JohnPhos (40 mol%), tBuONa (4 eq.), dioxane, 90 °C, 14 h, 63%; (h) BBr 3 (3 eq.), DCM, 100 °C, 5 mins, µW, 6 bars, 77%; (i) nBuBr (1
eq.), K2CO3 (1 eq.), DMF, 90 °C, 16 h, 65%; (j) CyNH 2 (2 eq.), CuCl (1 mol%), K3PO4 (2 eq.), DMF, 50 °C, 3 days, 76%.

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The preparation of the indazolone 37 is outlined in Scheme 4. The requisite diprotected N-methylhydrazine 33 was
prepared in two steps starting from commercially available N-Me hydrazine, following a standard literature
procedure.[40] The corresponding hydrazide 35 was synthesized easily from 2-bromobenzoyl chloride 34 and 33. Boc-
deprotection with TFA in dichloromethane afforded the hydrazide 36, which was cyclized into indazolone 37 by a copper-
mediated cross-coupling reaction using a β-diketone as the ligand.
Scheme 4. Synthesis of indazolone 37. (a) Boc2O (0.75 eq.), MeOH, rt, 15h, quant.; (b) 4-methoxybenzaldehyde (1.1 eq.), iPrOH, 90°C, 2h,
quant.; (c) NaBH3CN (1.5 eq.), AcOH (2 eq.), MeOH, rt, 39h, 63%; (d) 2-bromo-5-(trifluoromethyl)benzoyl chloride 34 (1 eq.), NEt3 (2.9 eq.), DCM,
rt, 21h, 86%; (e) TFA:DCM (1:10), rt, 1h, 99%; (f) CuI (5 mol%), 2-(2-methylpropanoyl)cyclohexan-1-one (10 mol%), Cs2CO3 (2 eq.), DMF, 90°C,
4.5h, 85%.
The functionalized aminoindazoles 41a-p were readily prepared starting from the commercially available indazole
derivatives 38 in four steps, as illustrated in Scheme 5. 3-Bromo indazoles 39 were generated from 38 after
regioselective bromination by Br2 in the presence of NaOH following a previously described method.[41] Protection of 39
by DHP afforded the N1-THP indazoles 40a-b, whereas 3-alkylindazoles 40c-h were easily obtained by N-alkylation with
the appropriate alkylhalide at 50 °C in DMF. Howeve r, when starting from secondary alkylbromides, the reaction needed
to be run at 90 °C in presence of TBAI. Likewise, N1-aryl or heteroaryl indazoles 40i-j were successfully introduced via a
copper-catalyzed N-arylation reaction using trans-cyclohexyldiamine as the ligand. Finally, the bromoindazole
derivatives 40a-j could be substituted at the C3 position by appropriate amines under Buchwald-Hartwig conditions with
the help of JosiPhos or BrettPhos (cpds 41a-n). Subsequent N-THP deprotection in TFA or HCl 1N yielded NH free
indazole derivatives 41o-p after inverse flash chromatography purification. Structures of these compounds were
established and validated by 2D RMN experiments (see SI).
Scheme 5. Synthesis of indazoles 41. (a) Br2, 1.32 eq.), NaOH, DMF, rt, 16h, 68-98%; (b) for R2 = MeI : MeI (1.5 eq.), Na2CO3 (2.5 eq.), DMF,
50°C, 12h, 66%; (c) for R2 = THP : DHP (2 eq.), PTSA (5 mol%), AcOEt, 95°C, 16h, 72 -83%; (d) for R2 = Bn : BnBr (1.5 eq.), Na2CO3 (2.5 eq.),
DMF, 55°C, 16h, 77%; (e) R2 = cPen or cHex : cPenBr or cHexBr (1.3 eq.), TBAI (3 mol%), K2CO3 (4.5 eq.), DMF, 90°C, 16h, 73-89%; (f) for R2 =
Ph or 3-pyridine : R2I (3 eq.), CuI (5 mol%), trans-N,N’- dimethylcyclohexane-1,2-diamine (20 mol%), K2CO3 (3 eq.), toluene, 130°C, 16h, 43-60%;
(g) 4-methoxybenzylamine (1.2 eq.), Pd(OAc)2 (5 mol%), JosiPhos (5 mol%), Cs2CO3 (1.5 eq.), dioxane, 120°C, 1h, 20-76%; (h) 4-
methoxybenzylamine (1.2 eq.), Pd(OAc)2 (5 mol%), BrettPhos (10 mol%), Cs2CO3 (1.5 eq.), dioxane, 120°C, 16h, 23-48%; (i) for R1 = H :
TFA:DCM (1:1), rt, 1.5h, 26%; (j) for R1 = 3-CF3 : HCl 1M (2.9 eq.), THF, rt, 20h, 26%.
The target 3-amino pyridazine 44 was prepared as outlined in Scheme 6. Acetophenone was first reacted with glyoxylic
acid and then the resulting mixture was treated with hydrazine to afford the desired pyridazinone 42 in 70% yield.
Subsequently, the pyridazinone 42 was treated with phosphorous oxychloride to afford the key intermediate 43. Finally,
the 6-chloropyridazine 43 was submitted to a Buchwald-Hartwig cross coupling reaction with the help of Pd(OAc)2 and
JosiPhos to afford the desired compound.

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Scheme 6. Synthesis of pyridazine 44 (a) glyoxylic acid hydrate (1.0 eq.), hydrazine hydrate (1.0 eq.), water, reflux, 4 h, 70%; (b) POCl3, reflux, 2
h, 99%; (c) 3-chloroaniline (1.2 eq.), Pd(OAc)2 (5 mol%), JosiPhos (5 mol%), Cs2CO3 (1.5 eq.), DMF, 50 °C, 24 h, 69%.
Condensation of the 3-trifluoromethyl benzamidine 46, readily available from the corresponding nitrile 45 by treatment
with LiHMDS,[42] was reacted with the α-bromoketone 47, and gave access to the 2,4-disubstituted imidazoles 48 in a
nearly quantitative yield (Scheme 7).
Scheme 7. Synthesis of imidazole 48. (a) LiHMDS 1M (1.5 eq.), THF, rt, 15 h, quant.; (b) NaHCO3 (4 eq.), THF:H2O (4:1), 75 °C, 3 h, 95%.
2- Results and discussion
Structure-Activity Relationship and Structural Optimization
In our experimental conditions, MY 5445 (4b) showed an IC50 value in the 1-10 µM range (6.7 µM, determined at 1 µM
cGMP concentration). This is in accordance with the original data published for MY5445, which reported an IC50 value of
0.6 µM determined at 0.4 µM cGMP concentration.[33] Under the same conditions, we obtained an IC50 value of 0.3 µM
determined at 1 µM cGMP for the reference PDE5 inhibitor, zaprinast. Table 1 shows the importance for activity of i) the
pyridazine nucleus (compare 4b and 10), ii) the presence of the benzo ring in the structure of 4b (compare 4b and the
inactive pyridazine 44 in Fig. 3), iii) a fair beneficial substituent effect could be observed by introduction of a methoxy
group in the para position (4f). However, compounds within this series had very low water solubilities.
Table 1. Importance of the scaffold and the 6-phenylring of MY 5445 a,b
PDE5
Cpd
R
Ph
X
N
%inhib at 10µM
IC50 (µM)
6.7
4b (MY 5445)
42
15
10
18
4e
4f
Ph
CH
N
>50
H
33
>10
4-ClPh
4-OMePh
N-piperidino
N
34
76c)
>10
N
3.0
20
N
28
>10
a) Zaprinast as positive control PDE5-I (inhibition at 10µM: 90%, IC50 = 0.3 µM); b) Sildenafil as a second positive control PDE5-I (inhibition at
1µM: 98%, IC50 = 3.3 nM); c) Fairly soluble in DMSO/water mixture (1%)
The superior homologues of MY 5445 (n = 1, 2) were reported in Table 2. For each homologue series, we checked
substitution effects on the N-aralkyl fragment (X1 ≠ H). A strong beneficial effect was found with specific aromatic
substitutions (m-Cl, p-OMe) in the N-benzyl phthalazine series 16 (n=1, compare 16f and 4b). However this effect could
not be clearly understood, as both the 3-Cl (16b) and 4-OMe (16e) derivatives did not show significantly improved

ACCEPTED MANUSCRIPT
activity when compared to the unsubstituted derivative 16a. As the p-methoxy benzyl derivative 16e showed a similar
potency when compared with the MY 5445, we conserved the same p-methoxy benzyl fragment in the other following
sub-series.
Table 2. Substitution on the aniline moiety and homologues (n=0, 1, 2) a, b
PDE5
Cpd
4b (MY 5445)
4a
n
0
0
0
0
1
1
1
1
1
1
1
2
2
2
2
X1
3-Cl
%inhib at 10µM
IC50 (µM)
6.7
42
5
H
nd
4c
4-Cl
31
21
43
14
25
41
68
86
73
47
14
19
28
38.1
nd
4d
4-OMe
H
16a
11.3
nd
16b
3-Cl
16c
2-OMe
3-OMe
4-OMe
3-Cl-4-OMe
3,4-O-CH2-O-
H
>10
11.4
7.2
16d
16e
16f
0.41
12.6
≥10
nd
16g
17a
17b
3-Cl
17c
4-Cl
nd
17d
4-OMe
>10
a) Zaprinast as positive control PDE5-I (inhibition at 10µM: 90%, IC50 = 0.3 µM); b) Sildenafil as a second positive control PDE5-I (inhibition
at1µM: 98%, IC50 = 3.3 nM); nd = not determined.
The phenyl ring in phthalazine 16e might be important for activity (compare 16e and 19, Table 3). However, it could be
replaced by a piperidine ring (compare 21a, b and 16e). Finally, introduction of a fully protonated basic nitrogen in N-
methyl piperazino (21c) instead of a lipophilic substituent decreased the activity significantly.
Table 3. Importance of the phenyl ring in position 6 in the N-para-methoxybenzylaminophthalazine series a, b
PDE5
Cpd
R
X1
%inhib at 10µM
68
IC50 (µM)
7.2
16e
Ph
4-OMe
19
H
4-OMe
4-OMe
35
46
>10
6.3
21a
N-piperidino
21b
60
5.3
4-OMe
21c
23
42
nd
4-OMe
3-Cl
4b (MY 5445)
Ph
6.7
a) Zaprinast as positive control PDE5-I (inhibition at 10µM: 90%, IC50 = 0.3 µM); b) Sildenafil as a second positive control PDE5-I (inhibition
at1µM: 98%, IC50 = 3.3 nM); nd = not determined
We considered the benzamides 25, 26 and 30 described in Table 4 (see also table A in SI) as seco analogues of
phthalazines 16 after removal of a phenyl imine in their structure. The aim of studying this sub-series of easily available
compounds was to identify some beneficial substitution effects on the phenyl ring (X1 ≠ H). Among the various

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substituents introduced mainly at position 3, we selected electron-withdrawing groups (CF3, NO2, SOCH3, SO2CH3, CN),
as such substituents were already showing beneficial effects in the N-benzyl phthalazine series 5.[35,36] When
compared with the almost inactive substituted derivative 25a, a fairly but significantly better activity was found with 3-
NO2 (25i), 3-CF3 (25l) and the superior homologue (n = 2, 30c). As observed in the phthalazine series (Table 2), the 3-
chloro-4-methoxyphenyl phthalazine also presented a dramatical twenty fold increase in potency (compare 25t and 25l).
However we did not observe a specific beneficial effect of the CF3 group in the meta position (compare 25t and 16f).
Based on these results, the benzamides 25 may not be considered as seco analogues of phthalazines 16.
Table 4. Effects of aromatic substitution within the N-paramethoxybenzylbenzamide series.a, b
PDE5
Cpd
n
1
1
1
1
2
-
X1
H
X2
4-OMe
4-OMe
4-OMe
3-Cl-4-OMe
3-Cl
%inhib at 10µM
IC50 (µM)
>50
25a
26
44
32
77
51
42
25i
25l
3-NO2
3-CF3
3-CF3
3-CF3
-
19.0
23.7
0.94
19.2
6.7
25t
30c
4b (MY 5445)
-
a) Zaprinast as positive control PDE5-I(inhibition at 10µM: 90%, IC50 = 0.3 µM); b) Sildenafil as a second positive control PDE5-I (inhibitor
(inhibition at1µM: 98%, IC50 = 3.3 nM).
An additional substituent X1 was introduced in benzamides 26b and 26c. An H bond accepting (6-OMe, 26b), or
donating group (6-NH-cHex, 26c) was introduced in the ortho position of the amide, establishing in both cases an
internal H bond interaction, and leading to specific conformers, as outlined in Table 5. The benzamide 26c was
particularly active with an IC50 value less than 1 µM, whereas the other benzamide 26b was inactive. These data support
a specific active conformation for benzamides that may be mimicked by the already described phthalazines 4b, 16, 17,
but also novel heterocyclics such as indazoles.
Table 5. Mimetics of internal H bond interactions in selected benzamides 26b and 26c a, b
CF3
CF3
Me
O
O
H
N
N
Me
O
N
OMe
MeO
26b
17% at 10 ꢀM
37
29% at 10 ꢀM

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PDE5
Compd
41f
n
0
1
1
1
1
1
1
1
1
1
1
1
1
-
X1
CF3
CF3
H
X2
3-Cl
R
cPen
Me
%inhib at 10µM
IC50 (µM)
1.67
15.1
>10
10.6
2.74
0.96
5.5
72
51
20
57
48
74
69
56
69
58
70
14
29
42
41c
4-OMePh
4-OMePh
4-OMePh
4-OMePh
3-Cl-4-OMePh
3-F-4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-OMePh
-
41d
cPen
cPen
cPen
cPen
cPen
cHex
Bn
41e
Cl
41g
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
41h
41i
41k
5.1
41l
1.44
7.2
41m
41n
Ph
Pyridin-3-yl
H
1.8
41o
>10
>10
6.7
41p
CF3
-
H
4b (MY 5445)
-
a) Zaprinast as positive control PDE5-I (inhibition at 10µM: 90%, IC50 = 0.3 µM); b) Sildenafil as a second positive control PDE5-I (inhibition
at1µM: 98%, IC50 = 3.3 nM).
As expected, the rigid indazolone 37 was found to be inactive. The 3-aminoindazoles 41 were particularly interesting
with compounds showing IC50 values in the 1 µM range. The presence of a lipophilic substituent (cHex) in the structure
of the amide 26c may contribute to its potency through an additional hydrophobic interaction, which is supported by data
obtained within the indazole series 41. The presence of a lipophilic group (R = cPen, cHex, Bn) increased their potency,
whereas the lack (R = H, 41p), or the presence of a small alkyl group (R = Me, 41c) led to inactive compounds. Again in
the indazole series, we observed a slight beneficial effect of a chlorine atom in the N-(p-methoxybenzyl) fragment
(compare 41h and 41g). It is also noteworthy that i) indazoles that lack a CF3 group on the benzo ring of indazoles are
inactive (41d and 41o), ii) the N-(3-chlorophenyl)aminoindazole 41f was relatively potent and may be considered as a
mimetic of MY 5445, as the cyclopentyl group could fully overlap the phenyl group in the structure of MY 5445.
Finally N-(p-methoxyphenylmethyl)phthalazines were revisited to investigate the beneficial effect of CF3 on the benzo
ring of the phthalazine scaffold (Table 6). This effect is clearly shown when comparing relative potencies of 16e and 16h
(factor 30). The chlorine atom in 14h could partially mimic lipophilicity of a phenyl ring (compare 16h and 14h). Moreover
replacement of the phenyl ring (16h) by a piperidine (21e) is also well tolerated.
Table 6. Novel benzo-substituted phthalazines a, b
PDE5
Cpd
16e
R1
H
R2
%inhib at 10µM
IC50 (µM)
19.8
Ph
68
81
82
79
42
14h
CF3
CF3
CF3
-
Cl
0.16
16h
Ph
0.065
0.35
21d
N-piperidino
4b (MY 5445)
-
6.7
a) Zaprinast as positive control PDE5-I (inhibition at 10µM: 90%, IC50 = 0.3 µM); b) Sildenafil as a second positive control PDE5-I (inhibition
at1µM: 98%, IC50 = 3.3 nM).
Some inactive compounds are represented in Figure 3. They contribute to a better knowledge for pharmacophoric
patterns for MY 5445 and other active structurally-related compounds. The lack of activity of N-methyl benzamide 27 and
the “inverso” amides 29 and 28 support the critical need in the active compounds for both an H bond acceptor and a
donor group for interaction within the pocket of PDE5.

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Figure 3. Other structurally-related compounds as inactive as PDE5 inhibitors.
PDE5 / PDE1-4 selectivity profiles
Sildenafil is a potent (IC50 close to 1nM) PDE5-I. However, it presents a moderate selectivity profile towards other PDE1-
4 isoforms in particular PDE1 and PDE6 (ratios 120 and 7 respectively) [32, 37]. In general, preliminar evaluation of the
selectivity profile towards other PDE isoenzymes (PDE 1, 2, 3 and 4) was satisfactory, as most of them did not show
enzyme inhibition by more than 30% at 10 µM (see supporting information, raw data, table D). The selectivity profiles of
the most potent PDE5 inhibitors in each chemical series are given in Table 7. We observed that MY 5445 is fairly
selective towards other PDE1-PDE4 isoforms.The novel phthalazine 16h and the amino indazole 41n showed similar
selectivity profiles when compared to MY 5445; all three compounds present a similar potency on PDE5, as well as
PDE4. Unexpectedly the more flexible benzamide 25t showed a 0.9 µM IC50 on PDE5, but presented no significant
activity on other PDE’s. Finally, MY 5445 and other selected PDE5-I congeners 16h and 41n were found fairly selective
versus PDE4 inhibition (ratio close to 5). These disappointing results did not encourage us to further check their
selectivity profiles.
Table 7. Selectivity towards other PDE 1 to 4 isoenzymes
%inhib at 10µM (IC50 µM)a
Compd
4b (MY5445)
16h
PDE1
39 (>100)
22
PDE2
25(>100)
29
PDE3
PDE4
54 (37)
(0.32)
24
PDE5
42 (6.7)b
82 (0.065)
77 (0.94)
70 (1.8)
PDE4/PDE5 ratio
14 (>100)
5
5
-
8
25t
27
26
10
20
41n
12
27
(4.4)
2
a) IC50 value determined with 1µM of cGMP. b IC50 = 0.6µM at 0.4µM of cGMP [33].
Physical properties of phthalazines and structurally-related compounds
Water solubility and comparative stabilities of phthalazines and indazoles in acidic medium of selected
compounds
It is generally well accepted that a given lead under early stage development has to present a minimal water solubility of
at least 0.1 mg/mL. Biological testing highlighted the very low water solubility of some of our compounds tested as PDE5
inhibitors. The water solubility of selected compounds representing phthalazines (MY 5445, 16e, 16h, 21e), indazoles
(41f, 41n) and benzamides (25l, 26c, 25g) has been measured using a solubility test using a HPLC/UV method (see SI,
table B). Phthalazines, indazoles and benzamides all have very low water solubility (0.01-0.06 mg/mL). Interestingly
replacing the phenyl ring of phthalazines by a piperidine significantly (ten-fold) increased the water solubility (0.33
mg/mL), in agreement with criteria needed for further in vivo investigations.

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The p-methoxy benzyl group (PMB) is a well-known protective group that can be easily removed in more or less acidic
medium.[43] A rapid evaluation of the stability of both the phthalazines and indazoles series at pH 1 has been carried
out at different reaction times (1-96 hours, see SI, Table C). The whole series of 3-aminophthalazines is stable in acidic
medium, as about 100% of the starting products are still present in the acidic medium after 96 hours exposure. The
stability of indazoles in acidic medium is moderate and strongly dependent on the nature of substituents R and R’. Most
of them bearing a substituent R ≠ H or Me are not stable (about 30 – 50% of the compounds are N-deprotected at 96
hours). Surprisingly, the N-(3-pyridyl)indazole 41n presents a good stability even after 4 days at pH 1, but no clear
explanation could be advanced. Moreover, the role of the PMB is the main source of instability of these compounds, as
supported by a completely restored chemical stability when replacing the PMB group by a substituted phenyl group
(compare 41g with 41f).
We decided to test in an in vivo model of neuropathic pain our initial hit (MY 5445, 4b) the most potent compound (based
on activity toward PDE5, water solubility and acidic stability) of both scaffold phthalazine and indazole, corresponding
respectively to 16h and 41n.
Anti-allodynic properties of selected PDE5-I (4b, 16h and 41n) in a neuropathic pain model in mice
As previously reported, cuff-implantation around the main branch of the right sciatic nerve induced an ispsilateral
mechanical allodynia in mice, while sham surgery did not affect paw withdrawal thresholds [44-46] (Figure 3; surgery x
paw x time interaction, F8,128 = 1.8, p < 0.05; post hoc: "Cuff Hydroxymethylcellulose right paw" < "Sham
Hydroxymethylcellulose right paw" at p < 0.01 on treatment days 0 to 21 and "Cuff Hydroxymethylcellulose right paw" <
"Cuff Hydroxymethylcellulose left paw" at p < 0.01 on treatment days 0 to 21). Two weeks after Cuff insertion, we started
the treatments with 4b, 16h, 41n, pregabalin or amitriptyline (3 and 0.5 mg/kg) or the control 0.1%
hydroxymethylcellulose solution. The gabapentinoid pregabalin and the tricyclic antidepressant amitriptyline were
chosen as reference treatments, as these drugs are clinically among the present first-line treaments for neuropathic
pain. [47] Mice received two injections per day for around 3 weeks, and were tested on given days in the morning before
drug injection. The 4b treatment had no effect in sham mice, but it alleviated the cuff-induced allodynia after 15 days of
treatment for the two tested doses (Figure 4; 3 mg/kg: paw x time interaction, F8,64 = 4.5, p <0.001; post hoc: "Right
paw" < "Left paw" at p < 0.05 on treatment days 0 to 12; 0.5 mg/kg: paw x time interaction, F6,48 = 3.8, p <0.01; post
hoc: "Right paw" < "Left paw" at p < 0.01 on treatment days 0 to 10). The same antiallodynic effect was also presented
with 16h (Figure 4; 3 mg/kg: group x time interaction, F8,64 = 3.5, p <0.01; post hoc: "Right paw" < "Left paw" at p <
0.05 on treatment days 0 to 12; 0.5 mg/kg: paw x time interaction, F6,48 = 3.1, p <0.05; post hoc: "Right paw" < "Left
paw" at p < 0.05 on treatment days 0 to 10) and with 41n (Figure 4; 3 mg/kg: paw x time interaction, F8,64 = 2.8, p
<0.01; post hoc: "Right paw" < "Left paw" at p < 0.05 on treatment days 0 to 12; 0.5 mg/kg: paw x time interaction, F6,48
= 4.3, p <0.01; post hoc: "Right paw" < "Left paw" at p < 0.01 on treatment days 0 to 15). For the reference treatments
pregabalin and amitriptyline, only the 3 mg/kg dose alleviated the cuff-induced allodynia, after 15 days of treatment for
pregabalin (Figure 4; 3 mg/kg: paw x time interaction, F7,70 = 6.8, p <0.001; post hoc: "Right paw" < "Left paw" at p <
0.05 on treatment days 0 to 12; 0.5 mg/kg: paw x time interaction, F7,70 = 8.2, p <0.01; post hoc: "Right paw" < "Left
paw" at p < 0.001 on treatment days 0 to 21) and at 21 days for amitriptyline (Figure 4; 3 mg/kg: paw x time interaction,
F7,70 = 3.5, p <0.005; post hoc: "Right paw" < "Left paw" at p < 0.05 on treatment days 0 to 18; 0.5 mg/kg: paw x time
interaction, F7,70 = 9.2, p <0.001; post hoc: "Right paw" < "Left paw" at p < 0.001 on treatment days 0 to 21). These
results can be summarized by the area under the curve (AUC) histogram (Figure 4), showing that the positive controls
pregabalin and amitriptyline were effective at 3 mg/kg only (F1,48 = 6.9, p < 0.001; post-hoc: “Cuff pregabalin 3 mg/kg”
and “Cuff amitriptyline 3 mg/kg” > “Cuff hydroxymethylcellulose” at p < 0.05), while the 3 PDE5 inhibitors were effective
at both doses (F1,48 = 6.9, p < 0.001; post-hoc: all groups > “Cuff hydroxymethylcellulose” at p < 0.05).

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Figure 4. Anti-allodynic properties of MY 5445 4b, of the structurally-related compounds 16h and 41n, and of reference treatments pregabalin and
amitriptyline, in a neuropathic pain model in mice. Two weeks after unilateral surgery on the right hindpaw, treatments started and were continued
until 3 weeks. The mechanical threshold of hindpaw withdrawal was evaluated using von Frey filaments before surgery (B: baseline), and on given
days during the chronic treatment. 4b, 16h and 41n treatments (3 and 0.5 mg/kg, i.p., twice a day) reversed the mechanical allodynia in mice (n =
5 per group, *p < 0.05 compared to contralateral left paw), without affecting thresholds of the controlateral paw (left paw). Pregabalin and
amitriptyline treatments were effective at dose 3 mg/kg (i.p., twice a day, n = 6 per group, *p < 0.05 compared to contralateral left paw) but not at
dose 0.5 mg/kg (i.p., twice a day). The area under the curve (AUC) above pre-treatment values is calculated over 19 treatment days and is
displayed in the last graph (*p < 0.05 compared to Cuff hydroxymethylcellulose; #p < 0.05 compared to Cuff 4b, 16h and 41n at 0.5 mg/kg; $p <
0.05 compared to Cuff 16h and pregabalin at 3 mg/kg).
The above results show that a chronic treatment with these three PDE5 inhibitors relieved mechanical allodynia in an
animal model of neuropathic pain, which is pain related to a lesion or disease of the somatosensory system.|48] The 2-
week therapeutic delay accompanying this action is in agreement with therapeutic delays that are observed for various
antidepressant and anticonvulsivant drugs [present data, 44, 47, 49-51] which are presently clinical first line treatments

ACCEPTED MANUSCRIPT
for neuropathic pain. [44, 51] Moreover, present results suggest that these PDE5-I may be effective against neuropathic
allodynia at relatively low doses, which is of therapeutic interest. In comparison, the 2 reference treatments required
higher doses to relieve neuropathic allodynia in the animal model, and were ineffective at dose 0.5 mg/kg, whis is similar
to previous report using another tricyclic antidepressant, nortriptyline [52]. Mechanistically, the action of the PDE5
inhibitors is likely related to the NO-cGMP pathway. While an increased NO production often accompanies neuropathic
pain, and while general NO donors potentiate pain-related symptoms and NOS inhibitors display pain relieving
action,[53–55] it has been evidenced that NO can have both pro- and antinociceptive functions.[56–58] In this regard,
the literature converges to point out that the NO-cGMP pathway is in fact critical to the analgesic action of PDE5
inhibitors on pain. Indeed, previous studies have highlighted anti-hyperalgesic properties and/or neuroprotective
properties of various PDE5 inhibitors, via NO and its downstream activation of the cGMP pathway, and through various
subsequent downstream effectors [10-12, 14, 16, 18-20]. Those effectors contributing to pain relief include protein
kinase G1 [11, 16], ATP-sensitive K+ channels and Ca++-activated K+ channels [11-12, 20], and Ca++ channels [18].
These pathways mediate peripheral sildenafil and vardenafil analgesia [10,14,18] as well as the systemic action of
sildenafil and of tadalafil in experimental hyperalgesia.[10,59] The action of PDE5 inhibitors on pain requires inhibitory
neuronal transmission, in particular GABA receptors [15], and it has also been associated with an attenuation of
oxidative stress and of inflammatory cytokines, such as IL-1β, IL-6, IL-18, IL-1rα and TNFα, [19,60-62] which is relevant
in the context of neuropathic pain.[47, 53, 61, 63]
3-Conclusion
In conclusion, the proof of concept supporting the therapeutic use of PDE5 inhibitors for the treatment of neuropathic
pain including sildenafil and other zaprinast structurally-related compounds has justified further work on different classes
of PDE5-I, in particular the phthalazines (MY 5445, 5 and isosteric 6). SAR data showed the pharmacophoric elements
of 3-aminophthalazines and other deriving analogues (benzamides, aminopyrazoles). Novel compounds were
demonstrated as acceptable mimetics of MY 5445 with similar potencies (versus PDE5) and selectivity profiles (PDE1-
4). Moreover, a couple of PDE5-I (16h, 41n) showed similar in vivo efficacy, when compared with MY 5445, in the
neuropathic pain model in mice. In the animal model, this action was even present for doses lower than present
reference treatments of neuropathic pain. In addition, the replacement of the phenyl ring in phthalazines (compare MY
5445 and 21d) increased significantly their water solubility. The continued discovery of novel MY 5445 structurally
related compounds will help to enhance the quality of novel leads with increased metabolic stability, water solubility and
selectivity, in particular versus PDE4.
4-Experimental Section
Chemistry
General Methods for Chemistry. All reactions were carried out under usual atmosphere unless otherwise stated. Chemicals and
solvents were purchased from Sigma-Aldrich and were used without further purification. Analytical TLC were performed using silica gel
plates Merck 60F254 and plates were visualized by exposure to ultraviolet light. Compounds were purified on silica gel Merck 60
(particle size 0.040-0.063nm) or using Armen spot flash chromatography (normal phase column: Interchim 30 SHIP 25g; reverse phase
column: AIT 50g C18). Microwave irradiation was performed with a Biotage Initiator EXP (external sensor type). Yields refer to isolated
1
compounds, estimated to be >95% pure as determined by H NMR or HPLC. 1H, 19F and 13C NMR spectra were recorded on Bruker
Avance Spectrometer operating at 400 or 500 MHz, 376 MHz and 100 or 125 MHz, respectively. All chemical shift values δ and coupling
constants J are quoted in ppm and in Hz, respectively, multiplicity (s= singulet, d= doublet, t= triplet, q= quartet, m= multiplet, br= broad).
Melting points were realized using a Büchi Melting point B-540. Analytical RP-HPLC-MS was performed using a LC-MSD 1200SL Agilent
with a Thermo Hypersilgold® column (C18, 30 mm x 1 mm; 1.9 µm) using the following parameters : 1) The solvent system: A
(acetonitrile) and B (0.05% TFA in H2O); 2) A linear gradient: t= 0 min, 98%B; t = 5 min, 5%B; t = 6 min, 5%B; t = 7 min, 98%B; t = 9 min,
98%B; 3) Flow rate of 0.3 mL/min; 4) Column temperature: 50°C; 5) The ratio of products was determinat e by integration of spectra
recorded at 210 nm or 254 nm; 6) Ionization mode : MM-ES+APCI. HPLC were performed using a Dionex UltiMate 300 using the
following parameters: Flow rate of 0.5 mL/min, column temperature: 30°C, solvent system: A (MeOH) and B (0.05% of THA in H2O), t =
0 min to 1 min: 50 to 60% of B then t = 1 min to t = 10 min: 60 to 100% of B and t = 10 min to t = 15 min: 100% of B.
Microwave irradiation was performed with a Biotage Initiator EXP (external sensor type).
Pd-Catalyzed Suzuki-Miyaura cross-coupling using Pd(PPh3)4 in a mixture toluene:EtOH:H2O: General method A for the
preparation of 4a, 4c-f, 10, 16a-h, 17a-d. A microvawe vial under argon was charged with the corresponding halogeno derivatives (1.0
equiv.), the corresponding arylboronic acid (1.1 to 1.5 equiv.), Pd(PPh3)4 (5 mol%), Na2CO3 (3.0 equiv.) and
a mixture
toluene:ethanol:water (5:1:1, 0.9 mmol/mL). The vial was capped properly, flushed with argon and heated to 120°C until complete
conversion of the starting material. After cooling at room temperature, the reaction mixture was concentrated under vacuum. The crude
residue was diluted with water. The organic phase was extracted 3 times with EtOAc. The organic layers were combined, washed with
brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography, using the appropriate heptanes:
EtOAc mixture.

ACCEPTED MANUSCRIPT
Nucleophilic substitution of dichlorophthalazine 12a with aniline derivatives: General method B for the preparation of 13a-e. A
mixture of 2,6-dichlorophthalazine 12a (1.0 equiv.) and the corresponding aniline derivative (1.0 equiv.) in iPrOH (0.3 mmol/mL) was
heated at 100°C for 1 hour. After cooling at room t emperature, the reaction media was concentrated and purified by silica gel column
chromatography using the appropriate heptanes: EtOAc mixture.
Nucleophilic substitution of dichlorophthalazine 12a with benzylamine and phenethylamine derivatives: General method C for
the preparation of compounds 14a-g and 15a-d. A mixture of 2,6-dichlorophthalazine 12a (1.0 equiv.), the appropriate benzylamine or
phenethylamine derivative (1.1 equiv.) and Na2CO3 (2.0 equiv.) in DMF (0.3 mmol/mL) was heated overnight at 130°C. After cooling, the
reaction media was diluted with water and the aqueous phase was extracted 3 times with EtOAc. The organic layers were combined,
washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography, using the appropriate
heptanes: EtOAc mixture.
Nucleophilic substitution of chlorophthalazine with NH heterocycles: General method D for the preparation of compounds 20 and
21 a-d. A mixture of 13 or 14 (1.0 equiv.), the appropriate NH heterocycles (5.2 equiv.) and DIEA (5.1 equiv.) in NMP (0.3 mmol/mL) was
heated overnight at 170°C. After cooling, the react ion media was diluted with water and the aqueous phase was extracted 3 times with
EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography using the appropriate heptanes: EtOAc mixture.
Bromation of indazole derivatives 39: General method E for the preparation of compounds 39a-c. A solution of the corresponding
indazole derivative 38 (1.0 equiv.), in an aqueous solution of NaOH 5M (16.1 equiv.) and DMF (4.8 equiv.) was cooled at 0°C and Br2
(1.3 equiv.) was added dropwise. The resulting mixture was stirred at rt for 8 hours. A saturated solution of Na2S2O3 was added to
neutralize the excess of Br2. The aqueous phase was extracted 3 times with EtOAc. The combined organic layers were washed with
water and brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography, using the appropriate
heptanes:EtOAc mixture.
Protection of indazole derivatives using a THP function: General method F for the preparation of compounds 40a-b. A solution of
the correspond indazole (1.0 equiv.), DHP (2.0 equiv.) and PTSA (5 mol%) in EtOAc (0.7 mmol/mL) was heated overnight at 95°C. After
cooling, the solution was concentrated under vacuum. The residue was diluted with water and the aqueous phase was extracted 3 times
with EtOAc. The combined organic layers were washed with water and brine, dried over Na2SO4, filtered, concentrated and purified by
silica gel column chromatography, using the appropriate heptanes:EtOAc mixture.
N-Alkylation of indazole derivatives using secondary alkyl bromides: General method G for the preparation of compounds 40d-g.
A solution of the corresponding indazole (1.0 equiv.), TBAI (3 mol%) and K2CO3 (4.5 equiv.) in DMF (1.3 mmol/mL) was heated at 90°C
and the corresponding bromoalkyl derivative (1.3 equiv.) was added dropwise. The resulting mixture was stirred overnight at 90°C. After
cooling, the solution was diluted with water and the aqueous phase was extracted 3 times with EtOAc. The combined organic layers
were washed with water and brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography, using the
appropriate heptanes:EtOAc mixture.
Cu-Catalyzed Ullmann cross-coupling: General method H for the preparation of compounds 40i-j. A microvawe vial (ovendried and
under argon) was charged with the corresponding indazole derivative (1.0 equiv.), the iodo compound (3.0 equiv.), CuI (5 mol%), trans-
N,N′-dimethylcyclohexane-1,2-diamine (20 mol%), K2CO3 (3.0 equiv.) and anhydrous toluene (0.9 mmol/mL). The vial was capped
properly, flushed with argon and heated to 130°C ov ernight. After cooling, the reaction mixture was concentrated under vacuum. The
crude residue was diluted with water. The organic phase was extracted 3 times with EtOAc. The organic layers were combined, washed
with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography using the appropriate
heptanes:EtOAc mixture.
Pd-Catalyzed Buchwald-Hartwig cross-coupling using Pd(OAc)2 and JosiPhos: General method I for the preparation of
compounds 41a-c, 41g, 41i-j, 41l. A microvawe vial (ovendried and under argon) was charged with the corresponding bromoindazole
derivative (1.0 equiv.), the appropriate amine (1.2 equiv.), Pd(OAc)2 (5 mol%), JosiPhos (5 mol%), Cs2CO3 (1.5 equiv.) and anhydrous
dioxane (0.2 mmol/mL). The vial was capped properly, flushed with argon and heated to 120°C overnight. After cooling, the reaction
mixture was concentrated under vacuum. The crude residue was diluted with water and the aqueous phase was extracted 3 times with
EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography, using the appropriate heptanes:EtOAc mixture.
Pd-Catalyzed Buchwald-Hartwig cross-coupling using Pd(OAc)2 and BrettPhos: General method J for the preparation of
compounds 41d-f, 41h-k, 41m-n. A microvawe vial (ovendried and under argon) was charged with the corresponding bromoindazole
derivative (1.0 equiv.), the appropriate amine (1.2 equiv.), Pd(OAc)2 (5 mol%), BrettPhos (10 mol%), Cs2CO3 (1.5 equiv.) and anhydrous
dioxane (0.2 mmol/mL). The vial was capped properly, flushed with argon and heated to 120°C overnight. After cooling, the reaction
mixture was concentrated under vacuum. The crude residue was diluted with water and the aqueous phase was extracted 3 times with
EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography, using the appropriate heptanes:EtOAc mixture.
N,4-Diphenylphthalazin-1-amine (4a). Following general method A and starting from 13a (15 mg, 0.059 mmol) and phenylboronic acid
(8 mg, 0.065 mmol), 4a was obtained as a white solid (14 mg, 0.046 mmol, 78%). Purity ≥ 98 %; mp = 232-234 °C; 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.05 (t, 1H, J = 7.3 Hz), 7.38 (t, 2H, J = 7.8 Hz), 7.52-7.61 (m, 3H), 7.67 (dd, 2H, J = 1.8 Hz, J = 8.2 Hz), 7.88-7.97 (m,
4H), 8.03 (td, 1H, J = 0.9 Hz, J = 7.2 Hz), 8.67 (d, 1H, J = 8.2 Hz), 9.26 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 120.7, 122.0,
122.5, 125.5, 125.8, 128.1, 128.3, 129.4, 131.3, 131.9, 134.0, 136.5, 140.4, 151.6, 161.4.

ACCEPTED MANUSCRIPT
N-(4-Chlorophenyl)-4-phenylphthalazin-1-amine (4c). Following general method A and starting from 13c (100 mg, 0.34 mmol) and
phenylboronic acid (46 mg, 0.37 mmol), 4c was obtained as a yellow solid (114 mg, 0.34 mmol, 100%). Purity = 95 %; mp = 202-206 °C;
1H NMR (400 MHz, DMSO-d6) δ ppm 7.42 (d, 2H, J = 8.9 Hz), 7.54-7.61 (m, 3H), 7.67 (dd, 2H, J = 1.4 Hz, J = 8.0 Hz), 7.89 (dd, 1H, J =
0.9 Hz, J = 8.2 Hz), 7.95 (td, 1H, J = 0.9 Hz, J = 8.2 Hz), 8.02-8.06 (m, 3H), 8.66 (d, 1H, J = 8.3 Hz), 9.39 (s, 1H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 118.4, 122.1, 122.6, 125.5, 125.7, 125.8, 128.2, 128.3, 128.5, 129.6, 131.6, 132.2, 136.6, 139.6, 151.6, 153.6.
N-(4-Methoxyphenyl)-4-phenylphthalazin-1-amine (4d). Following general method A and starting from 13d (100 mg, 0.35 mmol) and
phenylboronic acid (51 mg, 0.52 mmol), 4d was obtained as a yellow solid (102 mg, 0.31 mmol, 89%). Purity ≥ 98 %; mp = 132-134 °C;
1H NMR (400 MHz, DMSO-d6) δ ppm 3.83 (s, 3H), 7.13 (d, 2H, J = 8.3 Hz), 7.56 (d, 2H, J = 8.3 Hz), 7.62-7.71 (m, 5H), 7.98 (d, 1H, J =
7.8 Hz), 8.18 (t, 1H, J = 7.5 Hz), 8.26 (t, 1H, J = 7.5 Hz), 8.92 (d, 1H, J = 7.9 Hz), 11.14 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm
55.9, 115.5, 121.5, 125.2, 127.4, 127.5, 128.4, 128.6, 129.4, 130.2, 130.7, 133.0, 134.6, 135.9, 152.32 152.8, 158.9.
N-(3-Chlorophenyl)-4-(4-chlorophenyl)phthalazin-1-amine (4e). Following general method A and starting from 13b (100 mg, 0.34
mmol) and 4-chlorophenylboronic acid (59 mg, 0.38 mmol), 4e was obtained as a white solid (86 mg, 0.23 mmol, 68%). Purity ≥ 98 %;
mp = 219-224°C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.09 (dd, 1H, J = 1.9 Hz, J = 7.7 Hz), 7.40 (t, 1H, J = 8.2 Hz), 7.64 (d, 2H, J = 8.5
Hz), 7.72 (d, 2H, J = 8.5 Hz), 7.89 (d, 2H, J = 8.3 Hz), 7.97 (t, 1H, J = 7.7 Hz), 8.06 (t, 1H, J = 8.3 Hz), 8.27 (t, 1H, J = 1.9 Hz), 8.67 (d,
1H, J = 8.3 Hz), 9.47 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 118.4, 118.8, 119.8, 121.6, 122.6, 125.6, 125.6, 128.5, 123.0, 131.5,
131.8, 132.5, 132.8, 133.5, 135.4, 142.1, 151.7, 152.9.
N-(3-Chlorophenyl)-4-(4-methoxyphenyl)phthalazin-1-amine (4f). Following general method A and starting from 13b (100 mg, 0.34
mmol) and 4-methoxyphenylboronic acid (58 mg, 0.38 mmol), 4f was obtained as a white solid (92 mg, 0.26 mmol, 74%). Purity ≥ 98 %;
mp = 233-234°C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.03 (s, 3H), 6.24 (dd, 1H, J = 1.3 Hz, J = 7.9 Hz), 6.31 (d, 2H, J = 8.7 Hz), 6.56
(t, 1H, J = 8.2 Hz), 6.79 (d, 2H, J = 8.7 Hz), 7.05 (dd, 1H, J = 1.3 Hz, J = 8.3 Hz), 7.10-7.15 (m, 2H), 7.19-7.23 (m, 1H), 7.49 (t, 1H, J =
2.0 Hz), 7.81 (d, 1H, J = 8.3 Hz), 8.57 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 55.2, 113.9, 118.6, 118.7, 119.6, 121.4, 122.5,
125.8, 126.0, 128.8, 130.0, 131.0, 131.6, 132.2, 132.7, 142.3, 151.3, 153.6, 159.6.
N-(3-Chlorophenyl)isoquinoline (8). A mixture of 1-chloro-isoquinoline 7 (100 mg, 0.61 mmol, 1.0 equiv.) and 3-chloroaniline (78.0 µL,
0.73 mmol, 1.2 equiv.) in NMP (0.6 mL) was heated overnight at 110°C. After cooling, the reaction mixt ure was diluted with water and
the aqueous phase was extracted twice with AcOEt. The combined organic layers were washed with brine, dried over Na2SO4, filtered,
concentrated and purified by silica gel column chromatography (heptanes:EtOAc 9:1), to yield 8 as a white solid (121 mg, 0.48 mmol,
78%). 1H NMR (400 MHz, CDCl3) δ ppm 7.02 (ddd, 1H, J = 1.0 Hz, J = 2.0 Hz, J = 8.2 Hz), 7.13 (s, 1H), 7.18 (d, 1H, J = 5.8 Hz), 7.26 (t,
1H, J = 8.2 Hz), 7.49 (ddd, 1H, J = 1.0 Hz, J = 2.0 Hz, J = 8.2 Hz), 7.56 (ddd, 1H, J = 1.0 Hz, J = 6.9 Hz, J = 8.2 Hz), 7.66 (ddd, 1H, J =
1.0 Hz, J = 6.9 Hz, J = 8.2 Hz), 7.77 (d, 1H, J = 8.2 Hz), 7.87 (t, 1H, J = 2.0 Hz), 7.91 (d, 1H, J = 8.2 Hz), 8.12 (d, 1H, J = 5.8 Hz); 13
C
NMR (101 MHz, CDCl3) δ ppm 114.1, 117.9, 119.8, 121.2, 122.4, 126.7, 127.6, 129.9, 130.0, 134.6, 137.5, 140.8, 141.8.
4-Bromo-N-(3-chlorophenyl)isoquinolin-1-amine (9). A mixture of 8 (77 mg, 0.30 mmol, 1.0 equiv.) in THF (0.5 mL) was cooled at
0°C. A solution of phenyltrimethylammonium tribromi de (103 mg, 0.27 mmol, 0.9 equiv.) in THF (1.0 mL) was added dropwise in the
previous solution. The reaction media was stirred at rt for 6 hours and concentrated under vacuum. The residue was diluted with water
and the aqueous phase was extracted twice with EtOAc. The combined organic layers were washed with a saturated solution of
NaHCO3 and brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography (heptanes:EtOAc 9:1), to
1
yield 9 as an orange solid (101 mg, 0.30 mmol, 99%). H NMR (400 MHz, CDCl3) δ ppm 7.05 (d, 1H, J = 8.0 Hz), 7.11 (s, 1H), 7.17 (t,
1H, J = 8.0 Hz), 7.47 (d, 1H, J = 8.4 Hz), 7.64 (t, 1H, J = 8.0 Hz), 7.79 (t, 1H, J = 8.4 Hz), 7.83 (s, 1H), 7.90 (d, 1H, J = 8.4 Hz), 8.15 (d,
1H, J = 8.4 Hz), 8.29 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 110.3, 118.1, 120.1, 121.5, 122.9, 127.1, 127.6, 129.9, 131.2, 134.6,
135.7, 141.2, 142.2, 151.1.
N-(3-Chlorophenyl)-4-phenylisoquinolin-1-amine (10). Following general method A and starting from 9 (60 mg, 0.18 mmol) and
phenylboronic acid (26 mg, 0.22 mmol), 10 was obtained as a white solid (54 mg, 0.16 mmol, 90%). Purity = 95 %; mp = 202-204°C; 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.20 (d, 1H, J = 7.0 Hz), 7.45-7.55 (m, 6H), 7.76-7.83 (m, 5H), 8.03 (s, 1H), 8.69 (d, 1H, J = 7.7 Hz);
13C NMR (101 MHz, DMSO-d6) δ ppm 118.8, 121.0, 122.0, 123.8, 124.8, 125.3, 126.0, 127.8, 128.2, 129.2, 130.4, 131.0, 132.5, 133.7,
135.9, 136.7, 152.0, 170.8; HRMS (M+H+) 331.0992 (calcd for C21H15ClN2H+ 331.0997).
1,4-Dichlorophthalazine (12a). A mixture of phthalhydrazide 11a (2.50 g, 15.42 mmol, 1.0 equiv.) and pyridine (2.49 mL, 30.80 mmol,
2.0 equiv.) in POCl3 (50.3 mL) was heated at reflux for 1 hour. After cooling, the reaction mixture was concentrated under vacuum. The
residue was dissolved in cooled DCM and poured slowly into ice-cold water. The organic phase was separated and the aqueous phase
was extracted twice with DCM. The combined organic layers were washed with a saturated solution of NaHCO3 and brine, dried over
Na2SO4, filtered, concentrated under vacuum and purified by silica gel column chromatography (heptanes:EtOAc 3:2), to yield 12a as a
white solid (2.84 g, 14.27 mmol, 93%). 1H NMR (400 MHz, CDCl3) δ ppm 8.05-8.07 (m, 2H), 8.30-8.32 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ ppm 126.2, 127.7, 134.8, 155.4.
1,4-Dichloro-6-(trifluoromethyl)phthalazine (12b). A mixture of 11b (176 mg, 0.76 mmol, 1.0 equiv.) and pyridine (123.6 µL, 1.53
mmol, 2.0 equiv.) in POCl3 (2.5 mL) was heated at reflux for 1 hour. After cooling, the reaction mixture was concentrated under vacuum.
The residue was dissolved in cooled DCM and poured slowly into ice-cold wate. The organic phase was separated and the aqueous
phase was extracted twice with DCM. The combined organic layers were washed with a saturated solution of NaHCO3 and brine, dried
over Na2SO4, filtered, concentrated under vacuum and purified by silica gel column chromatography (heptanes:EtOAc 7:3 to yield 12b
as a white solid (186 mg, 0.70 mmol, 91%). 1H NMR (400 MHz, CDCl3) δ ppm 8.26 (dd, 1H, J = 1.8 Hz, J = 8.7 Hz), 8.49 (dt, 1H, J = 0.8
Hz, J = 8.7 Hz), 8.61 (t, 1H, J = 0.8 Hz); 19F NMR (376 MHz, CDCl3) δ ppm -63.1; 13C NMR (101 MHz, CDCl3) δ ppm 121.5, 123.9 (q, J =
4.4 Hz), 124.2, 127.2, 127.7, 128.9, 130.6 (q, J = 2.9 Hz), 136.5, 155.2 (d, J = 34.5 Hz).

ACCEPTED MANUSCRIPT
4-Chloro-N-phenylphthalazin-1-amine (13a). Following general method B and starting from 1,4-dichlorophthalazine 12a (150 mg, 0.75
1
mmol) and aniline (68.7 µL, 0.75 mmol), 13a was obtained as a white solid (34 mg, 0.13 mmol, 18%). H NMR (400 MHz, DMSO-d6) δ
ppm 7.06 (t, 1H, J = 7.4 Hz), 7.38 (t, 1H, J = 7.4 Hz), 7.87 (dd, 2H, J = 1.1 Hz, J = 8.4 Hz), 8.07-8.13 (m, 2H), 8.16-8.19 (m, 1H), 8.67
(dd, 1H, J = 2.0 Hz, J = 6.5 Hz), 9.34 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 121.0, 121.8, 123.2, 123.7, 125.2, 126.2, 128.9,
133.5, 133.8, 140.5, 146.9, 153.3.
4-Chloro-N-(3-chlorophenyl)phthalazin-1-amine (13b). Following general method B and starting from 1,4-dichlorophthalazine 12a
(100 mg, 0.50 mmol) and 3-chloroaniline (53.4 µL, 0.50 mmol), 13b was obtained as a white solid (146 mg, 0.50 mmol, 100%). 1H NMR
(400 MHz, DMSO-d6) δ ppm 7.11 (dd, 1H, J = 1.8 Hz, J = 7.8 Hz), 7.39 (t, 1H, J = 8.2 Hz), 7.82 (dd, 1H, J = 1.8 Hz, J = 7.8 Hz), 8.08-
8.16 (m, 3H), 8.19 (dd, 1H, J = 2.0 Hz, J = 7.3 Hz), 8.67 (dd, 1H, J = 2.0 Hz, J = 7.3 Hz), 9.50 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ
ppm 119.2, 120.1, 120.5, 122.1, 123.2, 124.8, 125.6, 130.0, 132.7, 133.1, 133.5, 141.5, 147.1, 152.6.
4-Chloro-N-(4-chlorophenyl)phthalazin-1-amine (13c). Following general method B and starting from 1,4-dichlorophthalazine 12a
(100 mg, 0.50 mmol) and 4-chloroaniline (64.1 mg, 0.50 mmol), 13c was obtained as a white solid (142 mg, 0.49 mmol, 98%). 1H NMR
(400 MHz, DMSO-d6) δ ppm 7.42 (d, 2H, J = 8.9 Hz), 7.93 (d, 2H, J = 8.9 Hz), 8.07-8.14 (m, 2H), 8.18 (dd, 1H, J = 2.1 Hz, J = 7.4 Hz),
8.65 (dd, 1H, J = 2.1 Hz, J = 7.4 Hz), 9.45 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 121.0, 123.1, 123.7, 125.3, 126.1, 126.7,
128.8, 133.6, 134.0, 139.5, 147.3, 153.1.
4-Chloro-N-(4-methoxyphenyl)phthalazin-1-amine (13d). Following general method B and starting from 1,4-dichlorophthalazine 12a
(100 mg, 0.50 mmol) and 4-methoxyaniline (58.4 µL, 0.50 mmol), 13d was obtained as a yellow solid (126 mg, 0.44 mmol, 88%). 1H
NMR (400 MHz, DMSO-d6) δ ppm 3.77 (s, 3H), 6.96 (d, 2H, J = 8.9 Hz), 7.72 (d, 2H, J = 8.9 Hz), 8.05-8.10 (m, 2H), 8.13 (dd, 1H, J = 2.1
Hz, J = 7.0 Hz), 8.62 (dd, 1H, J = 1.4 Hz, J = 7.2 Hz), 9.23 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 54.9, 113.4, 120.0, 122.8,
123.2, 124.4, 125.3, 132.5, 132.6, 132.9, 145.4, 152.6, 155.0.
N-Benzyl-4-chlorophthalazin-1-amine (14a). Following general method C and starting from 1,4-dichlorophthalazine 12a (100 mg, 0.50
mmol) and benzylamine (60.4 µL, 0.55 mmol), 14a was obtained as a white solid (94 mg, 0.35 mmol, 70%). 1H NMR (400 MHz, DMSO-
d6) δ ppm 4.78 (d, 2H, J = 5.8 Hz), 7.22 (t, 1H, J = 7.2 Hz), 7.31 (t, 1H, J = 7.4 Hz), 7.39 (d, 2H, J = 7.4 Hz), 7.99-8.02 (m, 2H), 8.05-8.08
(m, 1H), 8.32 (t, 1H, J = 5.5 Hz), 8.42-8.45 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.1, 119.9, 122.8, 124.5, 125.2, 126.5,
127.2, 128.1, 132.5, 132.9, 139.6, 144.3, 153.8.
4-Chloro-N-[(3-chlorophenyl)methyl]phthalazin-1-amine (14b). Following general method C and starting from 1,4-dichlorophthalazine
12a (100 mg, 0.50 mmol) and 3-chlorobenzylamine (68.1 µL, 0.55 mmol), 14b was obtained as a white solid (90 mg, 0.30 mmol, 59%).
1H NMR (400 MHz, DMSO-d6) δ ppm 4.77 (d, 2H, J = 5.9 Hz), 7.27-7.30 (m, 1H), 7.32-7.37 (m, 2H), 7.44 (s, 1H), 8.00-8.05 (m, 2H),
8.06-8.10 (m, 1H), 8.36 (t, 1H, J = 5.9 Hz), 8.40-8.43 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.2, 120.4, 123.3, 125.1, 125.7,
126.4, 127.0, 127.5, 130.6, 133.2, 133.3, 133.5, 142.9, 145.1, 154.2.
4-Chloro-N-[(2-methoxyphenyl)methyl]phthalazin-1-amine (14c). Following general method
C
and starting from 1,4-
dichlorophthalazine 12a (100 mg, 0.50 mmol) and 2-methoxybenzylamine (72.2 µL, 0.55 mmol), 14c was obtained as a yellow solid (96
mg, 0.32 mmol, 64%). 1H NMR (400 MHz, DMSO-d6) δ ppm 3.85 (s, 3H), 4.74 (d, 2H, J = 5.6 Hz), 6.84 (td, 1H, J = 0.9 Hz, J = 7.4 Hz),
7.01 (d, 1H, J = 8.0 Hz), 7.18-7.24 (m, 2H), 8.00 (m, 2H), 8.07-8.09 (m, 1H), 8.13 (t, 1H, J = 5.8 Hz), 8.46-8.48 (m, 1H); 13C NMR (101
MHz, DMSO-d6) δ ppm 39.9, 55.8, 110.9, 120.4, 120.5, 123.4, 125.0, 125.8, 127.2, 127.7, 128.2, 133.1, 133.4, 144.7, 154.5, 157.3.
4-Chloro-N-[(3-methoxyphenyl)methyl]phthalazin-1-amine (14d). Following general method
C
and starting from 1,4-
dichlorophthalazine 12a (100 mg, 0.50 mmol) and 3-methoxybenzylamine (70.9 µL, 0.55 mmol), 14d was obtained as a yellow solid (118
mg, 0.39 mmol, 79%). 1H NMR (400 MHz, DMSO-d6) δ ppm 3.71 (s, 3H), 4.75 (d, 2H, J = 5.9 Hz), 6.79 (dd, 1H, J = 2.4 Hz, J = 8.0 Hz),
6.95-6.97 (m, 2H), 7.22 (t, 1H, J = 8.0 Hz), 7.99-8.02 (m, 2H), 8.06-8.09 (m, 1H), 8.29 (t, 1H, J = 5.9 Hz), 8.42-8.45 (m, 1H); 13C NMR
(101 MHz, DMSO-d6) δ ppm 44.6, 55.4, 112.3, 113.6, 119.9, 120.4, 123.3, 125.0, 125.8, 129.7, 133.1, 133.5, 141.8, 144.8, 154.4, 159.7.
4-Chloro-N-[(4-methoxyphenyl)methyl]phthalazin-1-amine (14e). Following general method
C
and starting from 1,4-
dichlorophthalazine 12a (100 mg, 0.50 mmol) and 4-methoxybenzylamine (72.2 µL, 0.55 mmol), 14e was obtained as a white solid (128
mg, 0.43 mmol, 85%). Purity ≥ 98 %; mp = 154°C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.71 (s, 3H), 4.70 (d, 2H, J = 5.8 Hz), 6.87 (d,
2H, J = 8.7 Hz), 7.32 (d, 2H, J = 8.7 Hz), 7.98-8.03 (m, 2H), 8.06-8.09 (m, 1H), 8.23 (t, 1H, J = 5.8 Hz), 8.40-8.43 (m, 1H); 13C NMR (101
MHz, DMSO-d6) δ ppm 43.6, 54.9, 113.5, 119.9, 122.8, 124.4, 125.2, 128.6, 131.4, 132.5, 132.8, 144.1, 153.7, 158.1.
4-Chloro-N-[(3-chloro-4-methoxyphenyl)methyl]phthalazin-1-amine (14f). Following general method C and starting from 1,4-
dichlorophthalazine 12a (100 mg, 0.50 mmol) and 3-chloro-4-methoxybenzylamine (80.4 µL, 0.55 mmol), 14f was obtained as a yellow
1
solid (104 mg, 0.31 mmol, 62%). H NMR (400 MHz, DMSO-d6) δ ppm 3.81 (s, 3H), 4.69 (d, 2H, J = 5.8 Hz), 7.08 (d, 1H, J = 8.4 Hz),
7.34 (dd, 1H, J = 2.1 Hz, J = 8.4 Hz), 7.45 (d, 1H, J = 2.1 Hz), 7.99-8.02 (m, 2H), 8.06-8.08 (m, 1H), 8.28 (t, 1H, J = 5.8 Hz), 8.38-8.41
(m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 43.2, 56.0, 112.5, 119.9, 120.5, 122.8, 124.5, 125.3, 127.4, 128.9, 132.7, 132.9, 133.0,
144.4, 153.3, 153.8.
N-(2H-1,3-Benzodioxol-5-ylmethyl)-4-chlorophthalazin-1-amine (14g). Following general method
C and starting from 1,4-
dichlorophthalazine 12a (100 mg, 0.50 mmol) and piperonylamine (69.0 µL, 0.55 mmol), 14g was obtained as a white solid (151 mg,
0.48 mmol, 96%). 1H NMR (400 MHz, DMSO-d6) δ ppm 4.67 (d, 2H, J = 5.8 Hz), 5.96 (s, 2H), 6.84 (d, 1H, J = 7.9 Hz), 6.88 (dd, 1H, J =
1.4 Hz, J = 7.9 Hz), 6.97 (d, 1H, J = 1.4 Hz), 7.99-8.02 (m, 2H), 8.05-8.08 (m, 1H), 8.23 (t, 1H, J = 5.8 Hz), 8.39-8.42 (m, 1H); 13C NMR
(101 MHz, DMSO-d6) δ ppm 43.9, 100.6, 107.8, 108.0, 119.8, 120.4, 122.7, 124.4, 125.1, 132.5, 132.8, 133.4, 144.2, 145.8, 147.0,
153.7.

ACCEPTED MANUSCRIPT
4-Chloro-N-[(4-methoxyphenyl)methyl]-7-(trifluoromethyl)phthalazin-1-amine (14h). A mixture of 12b (150 mg, 0.56 mmol, 1.0
equiv.), 4-methoxybenzylamine (88.1 µL, 0.67 mmol, 1.2 equiv.) and dbu (208.9 µL, 1.40 mmol, 2.5 equiv.) in NMP (1.2 mL) was stirred
at rt for 2 hours. After cooling, the reaction mixture was diluted with water. The aqueous phase was extracted 3 times with EtOAc. The
combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography (heptanes:EtOAc 9:1 to 7:3), to yield 14h as a yellow solid (105 mg, 0.29 mmol, 51%). Purity ≥ 95 %; mp = 138-142 °C;
1H NMR (400 MHz, CDCl3) δ ppm 3.77 (s, 3H), 4.79 (d, 1H, J = 5.0 Hz), 5.67 (s, 1H), 6.85 (d, 2H, J = 8.7 Hz), 7.36 (d, 2H, J = 8.7 Hz),
8.06 (dd, 1H, J = 1.3 Hz, J = 8.7 Hz), 8.11 (s, 1H), 8.31 (d, 1H, J = 8.7 Hz); 19F NMR (376 MHz, CDCl3) δ ppm -62.7; 13C NMR (101
MHz, CDCl3) δ ppm 46.1, 55.5, 114.3, 119.2 (q, J = 3.7 Hz), 127.3, 128.5 (q, J = 3.7 Hz), 130.1, 130.2, 133.8, 134.1, 159.5; HRMS
(M+H+) 368.0769 (calcd for C17H13F3N3OH+ 368.0772).
4-Chloro-N-(2-phenylethyl)phthalazin-1-amine (15a). Following general method C and starting from 1,4-dichlorophthalazine 12a (80
1
mg, 0.40 mmol) and phenethylamine (55.7 µL, 0.44 mmol), 15a was obtained as a white solid (65 mg, 0.23 mmol, 57%). H NMR (400
MHz, DMSO-d6) δ ppm 3.01 (t, 2H, J = 7.6 Hz),3.76 (q, 2H, J = 7.6 Hz), 7.19-7.23 (m, 1H), 7.27-7.32 (m, 4H), 7.84 (t, 1H, J = 5.2 Hz),
7.98-8.02 (m, 2H), 8.06-8.08 (m, 1H), 8.32-8.35 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 34.7, 43.3, 120.5, 123.2, 125.0, 125.7,
126.5, 128.8, 129.2, 133.0, 133.3, 140.3, 144.5, 154.4.
4-Chloro-N-[2-(3-chlorophenyl)ethyl]phthalazin-1-amine (15b). Following general method C and starting from 1,4-dichlorophthalazine
12a (100 mg, 0.50 mmol) and 2-(3-chlorophenyl)ethylamine (76.9 µL, 0.55 mmol), 15b was obtained as a white solid (125 mg, 0.39
mmol, 78%). 1H NMR (400 MHz, DMSO-d6) δ ppm 3.02 (t, 2H, J = 7.0 Hz), 3.77 (q, 2H, J = 7.0 Hz), 7.23-7.27 (m, 2H), 7.31 (d, 1H, J =
7.5 Hz), 7.36 (s, 1H), 7.83 (t, 1H, J = 5.4 Hz), 7.98-8.00 (m, 2H), 8.06-8.08 (m, 1H), 8.31-8.34 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ
ppm 34.2, 42.9, 120.4, 123.2, 125.0, 125.7, 126.5, 128.0, 129.0, 130.6, 133.0, 133.4, 143.0, 144.6, 154.4.
4-Chloro-N-[2-(4-chlorophenyl)ethyl]phthalazin-1-amine (15c). Following general method C and starting from 1,4-dichlorophthalazine
12a (200 mg, 1.00 mmol) and 2-(4-chlorophenyl)ethylamine (154.7 µL, 1.11 mmol), 15c was obtained as a white solid (235 mg, 0.74
mmol, 73%). 1H NMR (400 MHz, DMSO-d6) δ ppm 3.01 (t, 2H, J = 7.2 Hz), 3.75 (q, 2H, J = 7.2 Hz), 7.31 (d, 2H, J = 8.4 Hz), 7.35 (d, 2H,
J = 8.4 Hz), 7.81 (t, 1H, J = 5.3 Hz), 7.99-8.01 (m, 2H), 8.07-8.09 (m, 1H), 8.31-8.34 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm
33.9, 43.0, 120.5, 123.2, 125.0, 125.7, 128.7, 131.1, 133.0, 133.4, 144.6, 154.5.
4-Chloro-N-[2-(4-methoxyphenyl)ethyl]phthalazin-1-amine (15d). Following general method
C
and starting from 1,4-
dichlorophthalazine 12a (100 mg, 0.50 mmol) and 2-(4-methoxyphenyl)ethylamine (81.1 µL, 0.55 mmol), 15d was obtained as a white
solid (134 mg, 0.43 mmol, 85%). 1H NMR (400 MHz, DMSO-d6) δ ppm 2.94 (t, 2H, J = 7.7 Hz), 3.68-3.73 (m, 2H), 3.71 (s, 3H), 6.85 (d,
2H, J = 8.7 Hz), 7.19 (d, 2H, J = 8.7 Hz), 7.80 (t, 1H, J = 5.3 Hz), 7.97-8.00 (m, 2H), 8.05-8.07 (m, 1H), 8.32-8.34 (m, 1H); 13C NMR (101
MHz, DMSO-d6) δ ppm 33.8, 43.5, 55.4, 114.2, 120.5, 123.2, 125.0, 125.7, 130.1, 132.1, 133.0, 133.3, 144.4, 154.5, 158.1.
N-Benzyl-4-phenylphthalazin-1-amine (16a). Following general method A and starting from 14a (75 mg, 0.28 mmol) and phenylboronic
acid (41 mg, 0.33 mmol), 16a was obtained as a white solid (55 mg, 0.18 mmol, 64%). Purity ≥ 98 %; mp = 218-220 °C; 1H NMR (400
MHz, DMSO-d6) δ ppm 4.87 (d, 2H, J = 5.9 Hz), 7.23 (t, 1H, J = 7.3 Hz), 7.33 (t, 2H, J = 7.3 Hz), 7.44 (d, 2H, J = 7.3 Hz), 7.48-7.57 (m,
3H), 7.61 (td, 2H, J = 1.4 Hz, J = 8.2 Hz), 7.81 (dd, 1H, J = 0.9 Hz, J = 8.2 Hz), 7.86 (td, 1H, J = 0.9 Hz, J = 8.2 Hz), 7.93 (td, 1H, J = 1.4
Hz, H = 8.0 Hz), 8.19 (t, 1H, J = 5.9 Hz), 8.45 (d, 1H, J = 8.2 Hz); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.5, 118.3, 122.8, 125.9,
126.0, 127.0, 127.7, 128.6, 128.7, 128.8, 130.0, 131.6, 132.2, 137.6, 140.8, 151.9, 153.3.
N-[(3-Chlorophenyl)methyl]-4-phenylphthalazin-1-amine (16b). Following general method A and starting from 14b (50 mg, 0.16
mmol) and phenylboronic acid (21 mg, 0.17 mmol), 16b was obtained as a colorless oil (10 mg, 0.03 mmol, 17%). Purity ≥ 98 %; 1H
NMR (400 MHz, DMSO-d6) δ ppm 4.90 (s, 2H), 7.37-7.46 (m, 3H), 7.57 (s, 1H), 7.62-7.69 (m, 5H), 7.95 (d, 1H, J = 8.0 Hz), 8.14 (t, 1H, J
= 8.0 Hz), 8.20 (t, 1H, J = 7.4 Hz), 8.76 (d, 1H, J = 8.0 Hz), 10.10 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 45.1, 121.1, 124.9,
126.7, 127.2, 127.9, 128.0, 128.5, 129.3, 130.2, 130.7, 130.9, 132.9, 133.7, 134.6, 135.8, 139.4, 152.3, 152.6; HRMS (M+H+) 346.1099
(calcd for C21H13ClN3H+ 346.1106).
N-[(2-Methoxyphenyl)methyl]-4-phenylphthalazin-1-amine (16c). Following general method A and starting from 14c (83 mg, 0.28
mmol) and phenylboronic acid (41 mg, 0.33 mmol), 16c was obtained as a white solid (21 mg, 0.06 mmol, 22%). Purity ≥ 98 %; mp =
161-162 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.90 (s, 3H), 4.86 (d, 2H, J = 5.6 Hz), 6.89 (t, 1H, J = 7.4 Hz), 7.04 (d, 1H, J = 8.0 Hz),
7.23-7.29 (m, 2H), 7.52-7.58 (m, 3H), 7.61-7.64 (m, 2H), 7.84 (d, 1H, J = 7.8 Hz), 7.89 (t, 1H, J = 7.4 Hz), 7.96 (t, 1H, J = 6.8 Hz), 6.03
(t, 1H, J = 5.6 Hz), 8.52 (d, 1H, J = 8.0 Hz); 13C NMR (101 MHz, DMSO-d6) δ ppm 39.9, 55.8, 110.8, 118.4, 120.5, 122.9, 125.9, 126.0,
127.6, 127.8, 128.1, 128.8, 130.0, 131.6, 132.2, 134.5, 137.6, 151.8, 153.5, 157.3; HRMS (M+H+) 342.1596 (calcd for C22H19N3OH+
342.1601).
N-[(3-Methoxyphenyl)methyl]-4-phenylphthalazin-1-amine (16d). Following general method A and starting from 14d (98 mg, 0.33
mmol) and phenylboronic acid (48 mg, 0.39 mmol), 16d was obtained as a colorless oil (55 mg, 0.16 mmol, 49%). Purity ≥ 98 %; 1H
NMR (400 MHz, DMSO-d6) δ ppm 3.72 (s, 3H), 4.83 (d, 2H, J = 5.9 Hz), 6.80 (dd, 1H, J= 2.5 Hz, J = 7.5 Hz), 7.00-7.02 (m, 2H), 7.23 (t,
1H, J = 7.9 Hz), 7.32 (t, 1H, J = 7.5 Hz), 7.50-7.56 (m, 3H), 7.59-7.61 (m, 2H), 7.77-7.82 (m, 2H), 7.86 (td, 1H, J = 1.2 Hz, J = 8.2 Hz),
7.93 (td, 1H, J = 1.2 Hz, J = 8.2 Hz), 8.02 (s, 1H), 8.17 (t, 1H, J = 5.9 Hz), 8.45 (d, 1H, J = 8.2 Hz); 13C NMR (101 MHz, DMSO-d6) δ ppm
44.6, 55.4, 112.2, 113.6, 118.4, 119.9, 122.8, 125.9, 126.0, 128.7, 128.8, 129.7, 130.0, 131.6, 132.2, 134.5, 137.6, 142.4, 151.9, 153.3,
159.7; HRMS (M+H+) 342.1599 (calcd for C22H19N3OH+ 342.1601).
N-[(4-Methoxyphenyl)methyl]-4-phenylphthalazin-1-amine (16e). Following general method A and starting from 14e (83 mg, 0.28
mmol) and phenylboronic acid (41 mg, 0.33 mmol), 16e was obtained as a white solid (70 mg, 0.20 mmol, 74%). Purity ≥ 98 %; mp = 69-
74 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.72 (s, 3H), 4.78 (d, 2H, J = 5.5 Hz), 6.89 (d, 2H, J = 8.6 Hz), 7.37 (d, 2H, J = 8.6 Hz),
7.48-7.57 (m, 3H), 7.60 (d, 2H, J = 8.0 Hz), 7.80 (d, 1H, J = 8.0 Hz), 7.85 (t, 1H, J = 8.2 Hz), 7.92 (t, 1H, J = 7.0 Hz), 8.11 (t, 1H, J = 5.5

ACCEPTED MANUSCRIPT
Hz), 8.42 (d, 1H, J = 8.2 Hz); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.0, 55.5, 114.0, 118.3, 122.8, 125.9, 126.0, 128.7, 128.8, 129.1,
130.0, 131.6, 132.2, 132.5, 137.5, 151.8, 153.3, 158.5; HRMS (M+H+) 342.1596 (calcd for C22H19N3OH+ 342.1601).
N-[(3-Chloro-4-methoxyphenyl)methyl]-4-phenylphthalazin-1-amine (16f). Following general method A and starting from 14f (80 mg,
0.24 mmol) and phenylboronic acid (31 mg, 0.25 mmol), 16f was obtained as a white solid (60 mg, 0.16 mmol, 66%). Purity ≥ 98 %; mp
= 97-99 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.86 (s, 3H), 4.85 (d, 2H, J = 4.5 Hz), 5.51 (s, 1H), 6.86 (d, 1H, J = 8.4 Hz), 7.35 (dd,
1H, J = 1.5 Hz, J = 8.4 Hz), 7.43-7.51 (m, 4H), 7.68 (d, 2H, J = 6.8 Hz), 7.72-7.77 (m, 2H), 7.84 (d, 1H, J = 7.3 Hz), 7.96 (d, 1H, J = 7.3
Hz); 13C NMR (101 MHz, DMSO-d6) δ ppm 45.3, 56.4, 112.3, 118.6, 120.8, 122.6, 126.5, 127.0, 128.1, 128.5, 128.7, 130.1, 130.3,
131.2, 131.5, 132.5, 137.2, 152.8, 153.6, 154.4; HRMS (M+H+) 376.1207 (calcd for C22H18ClN3OH+ 376.1211).
N-(2H-1,3-Benzodioxol-5-ylmethyl)-4-phenylphthalazin-1-amine (16g). Following general method A and starting from 14g (70 mg,
0.22 mmol) and phenylboronic acid (33 mg, 0.27 mmol), 16g was obtained as a white solid (53 mg, 0.15 mmol, 67%). Purity ≥ 98 %; mp
= 170-172 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 4.81 (d, 2H, J = 4.1 Hz), 6.01 (s, 2H), 6.93 (d, 1H, J = 7.9 Hz), 7.00 (d, 1H, J = 7.9
Hz), 7.10 (s, 1H), 7.64-7.67 (m, 5H), 7.93 (d, 1H, J = 7.9 Hz), 8.10-8.19 (m, 2H), 8.79 (d, 1H, J = 7.9 Hz), 10.05 (s, 1H); 13C NMR (101
MHz, DMSO-d6) δ ppm 45.5, 101.5, 108.7, 108.9, 121.1, 121.6, 125.0, 127.1, 128.2, 129.3, 130.1, 130.5, 133.5, 134.2, 135.6, 147.1,
147.8, 151.7, 152.4, 157.0; HRMS (M+H+) 356.1391 (calcd for C22H17N3O2H+ 356.1394).
N-(4-methoxybenzyl)-4-phenyl-7-(trifluoromethyl)phthalazin-1-amine (16h). Following general method A and starting from 14h (62
mg, 0.17 mmol) and phenylboronic acid (23 mg, 0.19 mmol), 16h was obtained as a yellow solid (44 mg, 0.11 mmol, 64%). Purity ≥
95 %; mp = 93-97 °C; 1H NMR (400 MHz, CDCl3) δ ppm 3.77 (s, 3H), 4.86 (d, 2H, J = 5.0 Hz), 5.65 (t, 1H, J = 5.0 Hz), 6.86 (d, 2H, J =
8.7 Hz), 7.40 (d, 2H, J = 8.7 Hz), 7.48-7.52 (m, 3H), 7.65 (dd, 2H, J = 1.9 Hz, J = 8.0 Hz), 7.89 (dd, 1H, J = 1.3 Hz, J = 8.7 Hz), 8.08 (d,
1H, J = 8.7 Hz), 8.13 (s, 1H); 19F NMR (376 MHz, CDCl3) δ ppm -62.7; 13C NMR (101 MHz, CDCl3) δ ppm 46.0, 55.5, 114.3, 119.0 (q, J
= 3.7 Hz), 124.9, 127.3 (q, J = 2.9 Hz), 127.9, 128.2, 128.7, 129.0, 130.1, 130.2, 130.8, 132.5, 132.8, 136.6, 152.8 (d, J = 3.7 Hz);
HRMS (M+H+) 410.1478 (calcd for C23H18F3N3OH+ 410.1475).
4-Phenyl-N-(2-phenylethyl)phthalazin-1-amine (17a). Following general method A and starting from 15a (57 mg, 0.20 mmol) and
phenylboronic acid (29 mg, 0.24 mmol), 17a was obtained as a white solid (41 mg, 0.13 mmol, 64%). Purity ≥ 98 %; mp = 132-134 °C;
1H NMR (500 MHz, DMSO-d6) δ ppm 3.10 (t, 2H, J = 8.0 Hz), 3.90 (q, 2H, J = 8.0 Hz), 7.24 (t, 1H, J = 7.2 Hz), 7.33 (t, 2H, J = 7.2 Hz),
7.37 (d, 2H, J = 7.2 Hz), 7.64-7.68 (m, 5H), 7.93 (d, 1H, J = 8.1 Hz), 8.14 (t, 1H, J = 7.2 Hz), 8.19 (t, 1H, J = 7.2 Hz), 9.76 (d, 1H, J = 8.1
Hz), 9.99 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ ppm 33.9, 44.1, 121.3, 125.1, 127.0, 127.1, 128.4, 128.9, 129.3, 129.4, 130.1, 130.5,
134.3, 136.0, 138.8, 152.4.
N-[2-(3-Chlorophenyl)ethyl-4-phenylphthalazin-1-amine (17b). Following general method A and starting from 15b (69 mg, 0.22 mmol)
and phenylboronic acid (32 mg, 0.26 mmol), 17b was obtained as a white solid (22 mg, 0.06 mmol, 28%). Purity ≥ 98 %; mp = 111-
112 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.09 (t, 2H, J = 7.8 Hz), 3.90 (q, 2H, J = 7.8 Hz), 7.30-7.37 (m, 3H), 3.49 (t, 1H, J = 1.5
Hz), 7.64-7.68 (m, 5H), 7.94 (dd, 1H, J = 1.5 Hz, J = 8.2 Hz), 8.14 (td, 1H, J = 1.4 Hz, J = 8.2 Hz), 8.19 (td, J = 1.5 Hz, J = 8.2 Hz), 8.72
(d, 1H, J = 7.8 Hz), 9.95 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 33.5, 43.7, 121.3, 125.1, 127.0, 127.1, 128.2, 128.4, 129.3,
129.4, 130.1, 130.5, 130.7, 133.5, 134.3, 136.0, 141.4, 152.5; HRMS (M+H+) 360.1261 (calcd for C22H18ClN3H+ 360.1262).
N-[2-(4-Chlorophenyl)ethyl-4-phenylphthalazin-1-amine (17c). Following general method A and starting from 15c (150 mg, 0.47
mmol) and phenylboronic acid (63 mg, 0.52 mmol), 17c was obtained as a white solid (113 mg, 0.24 mmol, 50%). Purity ≥ 98 %; mp =
131-133 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.09 (t, 2H, J = 7.0 Hz), 3.90 (q, 2H, J = 7.0 Hz), 7.37-7.40 (m, 4H), 7.60-7.69 (m, 5H),
7.92 (d, 1H, J = 7.7 Hz), 8.11-8.19 (m, 2H), 8.77 (d, 1H, J = 7.7 Hz), 10.07 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 33.2, 43.9,
121.4, 125.2, 127.1, 128.4, 128.8, 129.3, 130.1, 130.5, 131.3, 131.7, 133.6, 134.3, 136.0, 137.9, 151.2, 152.4; HRMS (M+H+) 360.1265
(calcd for C22H18ClN3H+ 360.1262).
N-[2-(4-Methoxyphenyl)ethyl-4-phenylphthalazin-1-amine (17d). Following general method A and starting from 15d (75 mg, 0.24
mmol) and phenylboronic acid (35 mg, 0.29 mmol), 17d was obtained as a white solid (67 mg, 0.19 mmol, 79%). Purity ≥ 98 %; mp =
139-142 °C; 1H NMR (500 MHz, DMSO-d6) δ ppm 2.99 (t, 2H, J = 7.6 Hz), 3.79 (q, 2H, J = 7.6 Hz), 6.87 (d, 2H, J = 8.5 Hz), 7.23 (d, 2H,
J = 8.5 Hz), 7.51 (d, 1H, J = 7.0 Hz), 7.55 (t, 2H, J = 7.0 Hz), 7.62 (d, 2H, J = 7.0 Hz), 7.66 (t, 1H, J = 5.5 Hz), 7.79 (d, 1H, J = 8.2 Hz),
7.83 (t, 1H, J = 7.0 Hz), 7.89 (t, 1H, J = 7.0 Hz), 8.34 (d, 1H, J = 8.2 Hz); 13C NMR (125 MHz, DMSO-d6) δ ppm 34.2, 43.5, 55.4, 114.3,
118.4, 122.8, 125.8, 125.9, 128.7, 128.8, 130.0, 130.1, 131.5, 132.1, 132.4, 137.7, 151.6, 153.5, 158.1; HRMS (M+H+) 356.1752 (calcd
for C23H21N3OH+ 356.1757).
N-(3-Chlorophenyl)phthalazin-1-amine (18). A microvawe vial (oven-dried and under argon) was charged with a solution of 13b (100
mg, 0.34 mmol, 1.0 equiv) in DMF (2.3 mL), and NEt3 (574.9 µL, 4.14 mmol, 12.0 equiv) and Pd(PPh3)4 (15.9 mg, 0.014 mmol, 4 mol%)
were added. The vial was capped properly, flushed with argon and formic acid (13.0 µL, 0.34 mmol, 1.0 equiv) was added. The resulting
mixture was microvawe heated to 110°C for 25 minute s. After it cooling, the reaction mixture was diluted with water and the aqueous
phase was extracted 3 times with EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered,
concentrated and purified by silica gel column chromatography (heptanes:EtOAc 3:2), to yield 18 as a white solid (68 mg, 0.27 mmol,
77%). Purity ≥ 98 %; mp = 216-218°C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.07 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz), 7.38 (t, 1H, J = 8.0
Hz), 7.88 (dd, 1H, J = 2.0 Hz, J = 8.2 Hz), 7.96-8.07 (m, 3H), 8.25 (t, 1H, J = 2.0 Hz), 8.59 (d, 1H, J = 8.2 Hz), 9.19 (s, 1H), 9.33 (s, 1H);
13C NMR (101 MHz, DMSO-d6) δ ppm 118.7, 119.2, 120.2, 122.0, 122.6, 127.2, 127.7, 130.5, 132.6, 132.7, 133.3, 142.7, 146.4, 152.6.
N-[(4-Methoxyphenyl)methyl]phthalazin-1-amine (19). In a hydrogenation flask, 14e (91 mg, 0.30 mmol, 1.0 equiv.) was dissolved in
ethanol (25.0 mL) then argon was bubble in through. Pd/C (1 mg, 0.013 mmol, 4 mol%) was added and the reaction media was stirred at
rt for 24 hours under a dihydrogen pressure (64 psi). The reaction media was filtered through a pad of Celite® and washed with DCM.
The filtrate was concentrated under vacuum and purified by silica gel column chromatography (heptanes:EtOAc 0:1), to yield 19 as a

ACCEPTED MANUSCRIPT
yellow solid (15 mg, 0.06 mmol, 19%). Purity ≥ 98 %; mp = 176-177 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 3.71 (s, 3H), 4.73 (d, 2H, J
= 5.8 Hz), 6.87 (d, 2H, J = 8.6 Hz), 7.33 (d, 2H, J = 8.6 Hz), 7.85-7.88 (m, 2H), 7.90-7.93 (m, 1H), 7.96 (t, 1H, J = 5.8 Hz), 8.32-8.34 (m,
1H), 8.89 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.0, 55.5, 114.0, 118.2, 122.3, 126.7, 127.4, 129.1, 131.7, 132.0, 132.6, 143.8,
153.8, 158.5.
N-(3-Chlorophenyl)-4-(piperidin-1-yl)phthalazin-1-amine (20). Following general method D and starting from 13b (200 mg, 0.62
mmol) and piperidine (317.4 µL, 3.21 mmol), 20 was obtained as a white solid (126 mg, 0.37 mmol, 60%). Purity ≥ 98 %; mp = 205-
207 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.62 (br s, 2H), 1.76 (br s, 4H), 3.20 (br s, 4H), 6.99 (d, 1H, J = 7.8 Hz), 7.32 (t, 1H, J = 7.8
Hz), 7.79 (d, 1H, J = 7.8 Hz), 7.93-7.96 (m, 2H), 8.03 (d, 1H, J = 6.5 Hz), 8.21 (s, 1H), 8.50 (d, 1H, J = 6.5 Hz), 9.10 (s, 1H); 13C NMR
(101 MHz, DMSO-d6) δ ppm 24.7, 26.2, 52.9, 118.4, 119.2, 121.1, 121.3, 123.1, 123.3, 125.0, 130.4, 131.8, 132.1, 133.3, 143.4, 150.4,
157.4; HRMS (M+H+) 339.1360 (calcd for C19H19ClN4H+ 339.1371).
N-[(4-Methoxyphenyl)methyl]-4-(piperidin-1-yl)phthalazin-1-amine (21a). Following general method D and starting from 14e (80 mg,
0.27 mmol) and piperidine (136.5 µL, 1.38 mmol), 21a was obtained as a white solid (67 mg, 0.19 mmol, 79%). Purity ≥ 98 %; mp = 89-
91 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.66 (m, 2H), 1.79-1.84 (m, 4H), 3.28 (t, 2H, J = 5.0 Hz), 4.77 (d, 2H, J = 4.4 Hz), 4.92 (s,
1H), 6.90 (d, 2H, J = 8.7 Hz), 7.39 (d, 2H, J = 8.7 Hz), 7.69 (d, 2H, J = 3.6 Hz), 7.72-7.76 (m, 1H), 8.05 (d, 1H, J = 8.0 Hz); 13C NMR
(101 MHz, DMSO-d6) δ ppm 24.9, 26.5, 46.0, 53.0, 55.5, 114.3, 121.0, 121.4, 123.8, 125.5, 129.9, 130.9, 131.0, 131.6, 151.6, 157.0,
159.3; HRMS (M+H+) 349.2019 (calcd for C21H24N4OH+ 349.2022).
1-(4-{[(4-Methoxyphenyl)methyl]amino}phtalazin-1-yl)piperidin-4-ol (21b). Following general method D and starting from 14e (150
mg, 0.50 mmol) and 4-hydroxypiperidine (262.2 µL, 2.59 mmol), 21b was obtained as a yellow solid (114 mg, 0.31 mmol, 63%). Purity ≥
98 %; mp = 92-97 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.65-1.69 (m, 2H), 1.90-1.94 (m, 2H), 2.90 (t, 2H, J = 12.7 Hz), 3.18 (d, 1H,
J = 4.9 Hz), 3.34-3.37 (m, 2H), 3.71 (s, 3H), 4.65 (d, 2H, J = 5.6 Hz), 4.72 (d, 1H, J = 3.9 Hz), 6.87 (d, 1H, J = 8.7 Hz), 7.32 (d, 2H, J =
8.7 Hz), 7.62 (t, 1H, J = 5.6 Hz), 7.83-7.86 (m, 2H), 7.94-7.97 (m, 1H), 8.29-8.31 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 35.1,
44.2, 49.7, 55.5, 66.8, 114.0, 120.7, 123.0, 123.1, 124.6, 129.0, 131.2, 131.6, 133.0, 152.0, 155.2, 158.5; HRMS (M+H+) 365.1968
(calcd for C21H24N4O2H+ 365.1972).
N-[(4-Methoxyphenyl)methyl]-4-(4-methylpiperazin-1-yl)phthalazin-1-amine (21c). Following general method D and starting from
14e (33 mg, 0.11 mmol) and 1-methylpiperazine (63.2 µL, 2.59 mmol), 21c was obtained as an orange oil (11 mg, 0.03 mmol, 26%).
Purity ≥ 98 %; mp = 92-97 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.27 (s, 3H), 2.55-2.58 (m, 4H), 3.12-3.15 (m, 4H), 3.71 (s, 3H),
4.64 (d, 2H, J = 5.8 Hz), 6.86 (d, 2H, J = 8.7 Hz), 7.31 (d, 2H, J = 8.7 Hz), 7.64 (t, 1H, J = 5.8 Hz), 7.83-7.86 (m, 2H), 7.95-7.98 (m, 1H),
8.28-8.31 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 44.1, 46.3, 51.5, 55.3, 55.5, 114.0, 120.6, 122.8, 123.1, 124.6, 129.0, 131.3,
131.6, 133.0, 152.1, 154.7; HRMS (M+H+) 364.2117 (calcd for C21H25N5OH+ 364.2132).
N-[(4-Methoxyphenyl)methyl]-4-(piperidin-1-yl)-7-(trifluoromethyl)phthalazin-1-amine (21d). Following general method
D and
starting from 14h (88 mg, 0.24 mmol) and piperidine (122.4µL, 1.24 mmol), 21d was obtained as a yellow oil (42 mg, 0.10 mmol, 42%).
Purity ≥ 98 %; 1H NMR (400 MHz, CDCl3) δ ppm δ 1.58-1.62 (m, 2H), 1.71-1.77 (m, 4H), 3.10 (t, 4H, J = 5.1 Hz), 3.72 (s, 3H), 4.65 (d,
1H, J = 5.5 Hz), 6.88 (d, 2H, J = 8.5 Hz), 7.33 (d, 2H, J = 8.5 Hz), 7.95 (t, 1H, J = 5.5 Hz), 8.14 (s, 2H), 8.81 (s, 1H); 19F NMR (376 MHz,
CDCl3) δ ppm -61.0; 13C NMR (101 MHz, CDCl3) δ ppm 24.6, 26.2, 44.3, 52.9, 55.5, 114.0, 120.2, 121.5 (q, J = 3.7 Hz), 124.9, 126.4,
127.4 (q, J = 2.9 Hz), 129.3, 131.0 (q, J = 32.3 Hz), 132.5, 151.8, 155.1, 158.6; HRMS (M+H+) 417.1895 (calcd for C22H23F3N4OH+
417.1897).
Methyl 3-carbamoylbenzoate (23). A solution of mono-methyl isophthalate 22 (150 mg, 0.83 mmol, 1.0 equiv.), HOBt.NH3 (190 mg,
1.25 mmol, 1.5 equiv.) and EDC (192 mg, 1.00 mmol, 1.2 equiv.) in DMF (1.5 mL) was stirred at rt overnight. The reaction media was
diluted with water and the aqueous phase was extracted 3 times with EtOAc. The combined organic layers were washed with a
saturated solution of NaHCO3 and brine, dried over Na2SO4, filtered and concentrated under vacuum, to yield 23 as white solid (108 mg,
0.60 mmol, 72%). 1H NMR (400 MHz, MeOD) δ ppm 3.94 (s, 3H), 7.59 (t, 1H, J = 7.8 Hz), 8.10 (ddd, 1H, J = 1.3 Hz, J = 1.9 Hz, J = 7.8
Hz), 8.17 (dt, 1H, J = 1.3 Hz, J = 7.8 Hz), 8.53 (t, 1H, J = 1.9 Hz); 13C NMR (101 MHz, MeOD) δ ppm 51.4, 128.3, 128.5, 130.4, 131.7,
132.2, 134.2, 166.3, 169.8.
3-Carbamoylbenzoic acid (24i). NaOH (107 mg, 2.68 mmol, 5.0 equiv.), in water (1.5 mL) was added in a solution of 23 (96 mg, 0.54
mmol, 1.0 equiv.) in methanol (1.5 mL) and the resulting mixture was stirred at rt overnight. The methanol was evaporated and the
aqueous phase was acidified until pH = 2 using a solution of HCl 1N. The solid was filtered, and 24i was obtained as a white solid (72
mg, 0.44 mmol, 82%). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.48 (s, 1H), 7.58 (t, 1H, J = 7.7 Hz), 8.09 (ddt, 2H, J = 1.6 Hz, J = 7.7 Hz, J
= 12.9 Hz), 8.15-8.18 (m, 1H), 8.45 (t, 1H, J = 1.6 Hz), 13.18 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ ppm 128.9, 129.1, 131.4, 132.1,
132.3, 135.2, 167.4, 167.6.
3-Bromo-1H-indazole (39a). Following general method E and starting from indazole 38a (800 mg, 6.77 mmol), 39a was obtained as a
white solid (912 mg, 4.63 mmol, 68%). 1H NMR (400 MHz, CDCl3) δ ppm 7.25 (td, 1H, J = 1.0 Hz, J = 8.4 Hz), 7.47 (td, 1H, J = 1.0 Hz, J
= 8.4 Hz), 7.57 (d, 1H, J = 8.4 Hz), 7.66 (d, 1H, J = 8.4 Hz); 13C NMR (101 MHz, CDCl3) δ ppm 110.3, 120.2, 121.9, 122.9, 128.1, 141.2.
3-Bromo-5-chloro-1H-indazole (39b). Following general method E and starting from 5-chloro-1H-indazole 38b (500 mg, 3.28 mmol),
39b was obtained as a yellow solid (592 mg, 2.56 mmol, 78%). 1H NMR (400 MHz, CDCl3) δ ppm 7.45 (dd, 1H, J = 1.9 Hz, J = 8.9 Hz),
7.62-7.64 (m, 2H), 13.62 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 113.2, 118.7, 120.1, 123.5, 126.4, 128.4, 140.0.
3-Bromo-5-(trifluoromethyl)-1H-indazole (39c). Following general method E and starting from 5-(trifluoromethyl)-1H-indazole 38c (300
mg, 1.61 mmol), 39c was obtained as a white solid (419 mg, 1.58 mmol, 98%). 1H NMR (400 MHz, CDCl3) δ ppm 7.67-7.71 (m, 2H),
7.99 (s, 1H), 11.13 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 111.1, 118.7 (q, J = 4.4 Hz), 124.1, 124.8 (q, J = 2.9 Hz).

ACCEPTED MANUSCRIPT
3-Bromo-1-(oxan-2-yl)-1H-indazole (40a). Following general method F and starting from 39a (115 mg, 0.58 mmol), 40a was obtained
as a yellow solid (119 mg, 0.42 mmol, 72%). 1H NMR (400 MHz, CDCl3) δ ppm 1.68-1.81 (m, 3H), 2.04-2.17 (m, 2H), 2.51-2.60 (m, 1H),
3.70-3.76 (m, 1H), 4.00-4.04 (m, 1H), 5.68 (dd, 1H, J = 2.6 Hz, J = 9.3 Hz), 7.23 (t, 1H, J = 7.7 Hz), 7.44 (t, 1H, J = 7.2 Hz), 7.56-7.62
(m, 2H); 13C NMR (101 MHz, CDCl3) δ ppm 22.4, 25.0, 29.3, 67.4, 85.5, 110.4, 120.4, 122.0, 122.1, 124.5, 127.7, 140.6.
3-Bromo-1-(oxan-2-yl)-5-(trifluoromethyl)-1H-indazole (40b). Following general method F and starting from 39c (215 mg, 0.81 mmol),
1
40b was obtained as a white solid (235 mg, 0.67mmol, 83%). H NMR (400 MHz, CDCl3) δ ppm 1.67-1.80 (m, 3H), 2.07-2.19 (m, 2H),
2.48-2.57 (m, 1H), 3.71-3.77 (m, 1H), 3.97-4.02 (m, 1H), 5.72 (dd, 1H, J = 2.9 Hz, J = 9.0 Hz), 7.65 (dd, 1H, J = 1.6 Hz, J = 8.9 Hz), 7.70
(d, 1H, J = 8.9 Hz), 7.93 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 22.1, 24.9, 29.3, 67.3, 85.9, 111.5, 118.7 (q, J = 4.4 Hz), 122.9,
123.9, 124.3 (q, J = 2.9 Hz).
3-Bromo-1-methyl-5-(trifluoromethyl)-1H-indazole (40c). MeI (105.7 µL, 1.70 mmol, 1.5 equiv.) was added dropwise to a solution of
39c (300 mg, 1.13 mmol, 1.0 equiv.) and Na2CO3 (300 mg, 2.83 mmol, 2.5 equiv.) in DMF (1.1 mL) and the resulting mixture was heated
at 50°C overnight. After cooling, the reaction mixt ure was diluted with water and the aqueous phase was extracted 3 times with EtOAc.
The organic layers were combined, washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography (heptanes:EtOAc 9:1), to yield 40c as a yellow solid (209 mg, 0.75 mmol, 66%). 1H NMR (400 MHz, CDCl3) δ ppm 4.09
(s, 3H), 7.47 (d, 1H, J = 8.8 Hz), 7.65 (dd, 1H, J = 1.5 Hz, J = 8.8 Hz), 7.93 (d, 1H, J = 1.5 Hz); 13C NMR (101 MHz, CDCl3) δ ppm 36.2,
110.0, 118.8 (q, J = 4.4 Hz), 121.3, 123.1, 123.8, 124.1 (q, J = 3.7 Hz).
3-Bromo-1-cyclopentyl-1H-indazole (40d). Following general method
G and starting from 39a (197 mg, 1.00 mmol) and
1
bromocyclopentane (139.4 µL, 1.30 mmol), 40d was obtained as a yellow oil (209 mg, 0.79 mmol, 79%). H NMR (400 MHz, CDCl3) δ
ppm 1.54-1.57 (m, 2H), 1.78-1.81 (m, 2H), 1.97-2.00 (m, 4H), 4.77 (quint., 1H, J = 7.4 Hz), 7.00 (ddd, 1H, J = 1.5 Hz, J = 6.1 Hz, J = 7.9
Hz), 7.20-7.26 (m, 2H), 7.42 (dt, 1H, J = 1.5 Hz, J = 7.9 Hz); 13C NMR (101 MHz, CDCl3) δ ppm 24.7, 32.4, 80.3, 109.7, 119.8, 120.6,
121.3, 124.0, 127.2, 140.7.
3-Bromo-5-chloro-1-cyclopentyl-1H-indazole (40e). Following general method G and starting from 39b (200 mg, 0.86 mmol) and
bromocyclopentane (120.4 µL, 1.12 mmol), 40e was obtained as an orange oil (202 mg, 0.67 mmol, 78%). 1H NMR (400 MHz, CDCl3) δ
ppm 1.62-1.70 (m, 2H), 1.86-1.95 (m, 2H), 2.05-2.11 (m, 4H), 4.82 (quint., 1H, J = 7.4 Hz), 7.27 (s, 2H), 7.50 (s, 1H); 13C NMR (101
MHz, CDCl3) δ ppm 24.5, 32.3, 60.4, 110.6, 118.8, 119.6, 124.6, 126.9, 127.8, 139.0.
3-Bromo-5-(trifluoromethyl)-1-cyclopentyl-1H-indazole (40f). Following general method G and starting from 39c (300 mg, 1.13 mmol)
and bromocyclopentane (157.8 µL, 1.47 mmol), 40f was obtained as a colorless oil (337 mg, 1.01 mmol, 89%). 1H NMR (400 MHz,
CDCl3) δ ppm 1.72-1.79 (m, 2H), 1.97-2.01 (m, 2H), 2.15-2.21 (m, 4H), 4.97 (quint., 1H, J = 7.4 Hz), 7.52 (d, 1H, J = 8.9 Hz), 7.61 (dd,
1H, J = 1.6 Hz, J = 8.9 Hz), 7.92 (t, 1H, J = 1.6 Hz); 13C NMR (101 MHz, CDCl3) δ ppm 24.5, 32.4, 60.4, 110.3, 118.8 (q, J = 4.4 Hz),
123.6 (q, J = 2.9 Hz), 141.3.
3-Bromo-1-cyclohexyl-5-(trifluoromethyl)-1H-indazole (40g). Following general method G and starting from 39c (80 mg, 0.30 mmol)
and bromocycloheptane (48.1 µL, 0.39 mmol), 40g was obtained as a colorless oil (77 mg, 0.22 mmol, 73%). 1H NMR (400 MHz, CDCl3)
δ ppm 1.35 (tt, 1H, J = 3.4 Hz, J = 12.7 Hz), 1.42-1.53 (m, 2H), 1.78 (dt, 1H, J = 3.4 Hz, J = 12.7 Hz), 1.86 (dt, 1H, J = 3.4 Hz, J = 13.6
Hz), 2.01-2.07 (m, 4H), 4.36-4.43 (m, 1H), 7.52 (d, 1H, J = 8.9 Hz), 7.61 (dd, 1H, J = 1.5 Hz, J = 8.9 Hz), 7.92 (d, 1H, J = 1.5 Hz); 13C
NMR (101 MHz, CDCl3) δ ppm 25.2, 25.7, 32.5, 59.1, 110.1, 118.9 (q, J = 4.4 Hz), 121.1, 123.5 (q, J = 2.9 Hz), 140.7.
1-Benzyl-3-bromo-5-(trifluoromethyl)-1H-indazole (40h). BnBr (67.7 µL, 0.57 mmol, 1.5 equiv.) was added dropwise to a solution of
39c (100 mg, 0.38 mmol, 1.0 equiv.) and Na2CO3 (100 mg, 0.94 mmol, 2.5 equiv.) in DMF (0.4 mL) and the resulting mixture was heated
at 50°C overnight. After cooling, the reaction mixt ure was diluted with water and the aqueous phase was extracted 3 times with EtOAc.
The organic layers were combined, washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography (heptanes:EtOAc 9:1), to yield 40h as a white solid (104 mg, 0.29 mmol, 77%). 1H NMR (400 MHz, CDCl3) δ ppm 5.59
(s, 2H), 7.21-7.24 (m, 2H), 7.29-7.35 (m, 3H), 7.40 (d, 1H, J = 8.8 Hz), 7.58 (dd, 1H, J = 1.6 Hz, J = 8.8 Hz), 7.95 (t, 1H, J = 1.6 Hz); 13
C
NMR (101 MHz, CDCl3) δ ppm 53.9, 110.4, 118.9 (q, J = 5.1 Hz), 124.2 (q, J = 2.9 Hz), 127.3, 128.3, 129.0, 135.6, 141.6.
3-Bromo-1-phenyl-5-(trifluoromethyl)-1H-indazole (40i). Following general method H and starting from 39c (50 mg, 0.19 mmol) and
1
iodobenzene (63.4 µL, 0.57 mmol), 40i was obtained as a yellow oil (39 mg, 0.11 mmol, 60%). H NMR (400 MHz, CDCl3) δ ppm 7.43
(tt, 1H, J = 1.1 Hz, J = 7.5 Hz), 7.57 (t, 2H, J = 7.5 Hz), 7.67-7.71 (m, 3H), 7.79 (d, 1H, J = 8.9 Hz), 8.02 (s, 1H); 13C NMR (101 MHz,
CDCl3) δ ppm 111.5, 119.1 (q, J = 4.4 Hz), 123.0, 124.7, 124.9 (q, J = 3.7 Hz), 127.8, 129.7, 138.9.
3-Bromo-1-(pyridine-3-yl)-5-(trifluoromethyl)-1H-indazole (40j). Following general method H and starting from 39c (100 mg, 0.38
mmol) and 3-iodopyridine (232 mg, 1.13 mmol), 40j was obtained as a white solid (56 mg, 0.16 mmol, 43%). 1H NMR (400 MHz, CDCl3)
δ ppm 7.53 (dd, 1H, J = 4.6 Hz, J = 8.2 Hz), 7.75 (d, 1H, J = 8.9 Hz), 7.79 (d, 1H, J = 8.9 Hz), 7.91 (s, 1H), 8.05 (d, 1H, J = 8.2 Hz), 8.68
(d, 1H, J = 4.6 Hz), 9.02 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 97.3, 110.9, 120.6 (q, J = 4.4 Hz), 124.3, 125.5 (q, J = 2.9 Hz),
129.3, 130.3, 135.8, 140.5, 143.6, 148.8.
N-[(4-Methoxyphenyl)methyl]-1-(oxan-2-yl)-1H-indazol-3-amine (41a). Following general method I and starting from 40a (50 mg, 0.18
mmol) and 4-methoxybenzylamine (27.9 µL, 0.21 mmol), 41a was obtained as a white solid (39 mg, 0.11 mmol, 65%). 1H NMR (400
MHz, CDCl3) δ ppm 1.63-1.69 (m, 3H), 1.90-1.94 (m, 1H), 2.04-2.07 (m, 1H), 2.46-2.54 (m, 1H), 3.60-3.63 (m, 1H), 3.73 (s, 3H), 3.98-
4.01 (m, 1H), 4.49 (d, 2H, J = 3.3 Hz), 5.45 (dd, 1H, J = 2.5 Hz, J = 9.8 Hz), 6.81 (d, 2H, J = 8.5 Hz), 6.93 (t, 1H, J = 8.0 Hz), 7.24-7.33
(m, 4H), 7.39 (d, 1H, J = 8.0 Hz); 13C NMR (101 MHz, CDCl3) δ ppm 23.1, 25.2, 29.4, 29.8, 47.8, 55.3, 67.7, 84.9, 109.5, 113.9, 115.9,
119.2, 119.3, 127.1, 129.5, 131.9, 141.3, 149.7, 158.9.

ACCEPTED MANUSCRIPT
N-[(4-Methoxyphenyl)methyl]-1-(oxan-2-yl)-5-(trifluoromethyl)-1H-indazol-3-amine (41b). Following general method I and starting
from 40b (350 mg, 1.00 mmol) and 4-methoxybenzylamine (157.2 µL, 1.20 mmol), 41b was obtained as a yellow solid (308 mg, 0.76
mmol, 76%). 1H NMR (400 MHz, CDCl3) δ ppm 1.72-1.76 (m, 3H), 2.01 (dq, 1H, J = 2.9 Hz, J = 12.9 Hz), 2.12-2.17 (m, 1H), 2.50-2.60
(m, 1H), 3.72 (td, 1H, J = 2.9 Hz, J = 11.0 Hz), 3.81 (s, 3H), 4.06 (dd, 1H, J = 2.3 Hz, J = 11.0 Hz), 4.55 (d, 2H, J = 5.5 Hz), 5.55 (dd, 1H,
J = 2.6 Hz, J = 9.7 Hz), 6.90 (d, 2H, J = 8.7 Hz), 7.37 (d, 2H, J = 8.7 Hz), 7.47 (d, 1H, J = 8.8 Hz), 7.54 (dd, 1H, J = 1.5 Hz, J = 8.8 Hz),
7.78 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 22.8, 25.1, 29.4, 47.7, 55.3, 67.7, 85.1, 110.1, 114.0, 115.1, 117.5 (q, J = 4.4 Hz), 123.7
(q, J = 2.9 Hz), 129.5, 131.5, 142.1, 150.0, 159.0.
N-[(4-Methoxyphenyl)methyl]-1-methyl-5-(trifluoromethyl)-1H-indazol-3-amine (41c). Following general method I and starting from
40c (195 mg, 0.70 mmol) and 4-methoxybenzylamine (109.6 µL, 0.83 mmol), 41c was obtained as a yellow solid (164 mg, 0.49 mmol,
70%). Purity ≥ 98 %; mp = 92-93°C; 1H NMR (400 MHz, CDCl3) δ ppm 3.81 (s, 3H), 3.90 (s, 3H), 4.53 (s, 2H), 6.90 (d, 2H, J = 8.5 Hz),
7.24 (d, 1H, J = 8.8 Hz), 7.37 (d, 2H, J = 8.5 Hz), 7.52 (dd, 1H, J = 1.4 Hz, J = 8.8 Hz), 7.79 (d, 1H, J = 1.4 Hz); 19F NMR (376 MHz,
CDCl3) δ ppm -60.5; 13C NMR (101 MHz, CDCl3) δ ppm 35.1, 47.9, 55.3, 108.8, 113.4, 114.0, 117.7 (q, J = 4.4 Hz), 123.5 (q, J = 3.7
Hz), 129.4, 131.3, 142.2, 149.7, 159.1; HRMS (M+H+) 336.1315 (calcd for C17H16F3N3OH+ 336.1318).
1-Cyclopentyl-N-[(4-methoxyphenyl)methyl]-1H-indazol-3-amine (41d). Following general method J and starting from 40d (150 mg,
0.56 mmol) and 4-methoxybenzylamine (88.7 µL, 0.68 mmol), 41d was obtained as colorless oil (61 mg, 0.19 mmol, 33%). Purity ≥
98 %; 1H NMR (400 MHz, CDCl3) δ ppm 1.67-1.70 (m, 2H), 1.92-1.96 (m, 2H), 2.04-2.13 (m, 4H), 3.80 (s, 3H), 4.07 (s, 1H), 4.54 (s, 2H),
4.80 (quin., 1H, J = 7.5 Hz), 6.87 (d, 2H, J = 8.7 Hz), 6.93 (ddd, 1H, J = 1.6 Hz, J = 5.9 Hz, J = 7.8 Hz), 7.24-7.27 (m, 2H), 7.38 (d, 2H, J
= 8.7 Hz), 7.46 (d, 1H, J = 8.2 Hz); 13C NMR (125 MHz, CDCl3) δ ppm 25.1, 31.9, 48.5, 55.6, 59.2, 109.2, 114.1, 115.0, 118.3, 119.6,
126.6, 129.7, 132.6, 141.2, 148.8, 159.2; HRMS (M+H+) 322.1917 (calcd for C20H23N3OH+ 322.1914).
5-Chloro-1-cyclopentyl-N-[(4-methoxyphenyl)methyl]-1H-indazol-3-amine (41e). Following general method J and starting from 40e
(150 mg, 0.50 mmol) and 4-methoxybenzylamine (78.5 µL, 0.60 mmol), 41e was obtained as a yellow oil (83 mg, 0.23 mmol, 47%).
Purity ≥ 95 %; 1H NMR (400 MHz, CDCl3) δ ppm 1.68-1.71 (m, 2H), 1.93-1.96 (m, 2H), 2.04-2.11 (m, 4H), 3.81 (s, 3H), 4.51 (s, 2H), 4.76
(quin., 1H, J = 7.3 Hz), 6.88 (d, 2H, J = 8.5 Hz), 7.18 (d, 1H, J = 8.9 Hz), 7.22 (dd, 1H, J = 1.8 Hz, J = 8.9 Hz), 7.37 (d, 2H, J = 8.5 Hz),
7.44 (d, 1H, J = 1.8 Hz); 13C NMR (101 MHz, CDCl3) δ ppm 24.6, 31.7, 48.1, 55.3, 59.2, 109.9, 114.0, 115.3, 118.8, 123.3, 126.8, 129.4,
147.8, 158.9; HRMS (M+H+) 356.1523 (calcd for C20H22ClN3OH+ 356.1524).
N-(3-Chlorophenyl)-1-cyclopentyl-5-(trifluoromethyl)-1H-indazol-3-amine (41f). Following general method J and starting from 40f
(83 mg, 0.25 mmol) and 3-chloroaniline (31.8 µL, 0.30 mmol), 41f was obtained as a white solid (34 mg, 0.09 mmol, 36%). Purity ≥
98 %; mp = 153-154 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.70-1.73 (m, 2H), 1.91-2.03 (m, 4H), 2.08-2.15 (m, 2H), 5.12-5.16 (m,
1H), 6.88 (d, 1H, J = 8.0 Hz), 7.31 (t, 1H, J = 8.0 Hz), 7.51 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.9 Hz), 7.75 (d, 1H, J = 8.9 Hz), 7.99 (s,
1H), 8.48 (s, 1H), 9.46 (s, 1H); 19F NMR (376 MHz, DMSO-d6) δ ppm -58.8; 13C NMR (101 MHz, DMSO-d6) δ ppm 24.9, 32.3, 58.6,
110.7, 114.3, 114.9, 115.7, 119.2 (q, J = 4.4 Hz), 119.3, 119.6 (q, J = 31.5 Hz), 123.5 (q, J = 2.9 Hz), 125.6 (q, J = 270.7 Hz), 130.8,
133.8, 140.6, 144.1, 144.8; HRMS (M+H+) 380.1118 (calcd for C19H17ClF3N3H+ 380.1136).
1-Cyclopentyl-N-[(4-methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41g). Following general method I and starting
from 40f (104 mg, 0.31 mmol) and 4-methoxybenzylamine (48.9 µL, 0.37 mmol), 41g was obtained as a yellow oil (24 mg, 0.06 mmol,
20%). Purity ≥ 98 %; 1H NMR (500 MHz, DMSO-d6) δ ppm 1.62-1.65 (m, 2H), 1.83-1.99 (m, 6H), 3.72 (s, 3H), 4.37 (d, 2H, J = 6.9 Hz),
4.95 (quint., 1H, J = 7.2 Hz), 6.78 (t, 1H, J = 6.0 Hz), 6.87 (d, 2H, J = 8.7 Hz), 7.35 (d, 2H, J = 8.7 Hz), 7.49 (dd, 1H, J = 1.5 Hz, J = 8.9
Hz), 7.55 (d, 1H, J = 8.9 Hz), 8.23 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ ppm 24.7, 31.7, 46.6, 55.5, 58.3, 110.0, 113.6, 113.9, 118.2
(q, J = 30.9 Hz), 119.5 (q, J = 4.5 Hz), 122.9 (q, J = 3.6 Hz), 129.7, 132.6, 141.8, 150.2, 158.6; HRMS (M+H+) 390.1785 (calcd for
C21H22F3N3OH+ 390.1788).
N-[(3-Chloro-4-methoxyphenyl)methyl]-1-cyclopentyl-5-(trifluoromethyl)-1H-indazol-3-amine (41h). Following general method J
and starting from 40f (83 mg, 0.25 mmol) and 3-chloro-4-methoxybenzylamine (43.5 µL, 0.30 mmol), 41h was obtained as a yellow oil
(47 mg, 0.11 mmol, 44%). Purity ≥ 95 %; 1H NMR (400 MHz, CDCl3) δ ppm 1.69-1.72 (m, 2H), 1.94-1.97 (m, 2H), 2.05-2.12 (m, 4H),
3.89 (s, 3H), 4.52 (s, 2H), 4.81 (quint., 1H, J = 6.7 Hz), 6.89 (d, 1H, J = 8.4 Hz), 7.30 (dd, 2H, J = 2.3 Hz, J = 8.4 Hz), 7.48 (d, 1H, J = 8.4
Hz), 7.51 (d, 1H, J = 2.3 Hz), 7.78 (s, 1H); 19F NMR (376 MHz, CDCl3) δ ppm -60.5; 13C NMR (101 MHz, CDCl3) δ ppm 24.7, 31.8, 47.4,
56.2, 59.1, 109.2, 112.0, 113.6, 117.6 (q, J = 4.5 Hz), 122.3, 123.0 (q, J = 2.7 Hz), 127.5, 130.3, 133.0, 141.5, 148.8, 154.2; HRMS
(M+H+) 424.1387 (calcd for C21H21Cl2F3N3OH+ 424.1398).
N-[(3-Fluoro-4-methoxyphenyl)methyl]-1-cyclopentyl-5-(trifluoromethyl)-1H-indazol-3-amine (41i). Following general method J and
starting from 40f (83 mg, 0.25 mmol) and 3-fluoro-4-methoxybenzylamine (41.2 µL, 0.30 mmol), 41i was obtained as a colorless oil (38
mg, 0.09 mmol, 38%). Purity ≥ 98 %; 1H NMR (500 MHz, CDCl3) δ ppm 1.69-1.72 (m, 2H), 1.94-1.96 (m, 2H), 2.05-2.10 (m, 4H), 3.88 (s,
3H), 4.23 (t, 1H, J = 5.8 Hz), 4.52 (d, 2H, J = 5.8 Hz), 4.81 (quint., 1H, J = 7.3 Hz), 6.92 (t, 1H, J = 8.4 Hz), 7.15 (d, 1H, J = 8.4 Hz), 7.21
(dd, 1H, J = 2.1 Hz, J = 12.1 Hz), 7.31 (d, 1H, J = 8.9 Hz), 7.48 (dd, 1H, J = 2.1 Hz, J = 8.9 Hz), 7.77 (s, 1H); 19F NMR (376 MHz, CDCl3)
δ ppm -60.5, -135.2; 13C NMR (125 MHz, CDCl3) δ ppm 24.7, 31.8, 47.5, 56.4, 59.1, 109.2, 113.3 (d, J = 2.7 Hz), 113.6, 116.1 (d, J =
19.1 Hz), 117.6 (q, J = 4.5 Hz), 120.1 (q, J = 31.8 Hz), 123.0 (q, J = 3.6 Hz), 123.8 (d, J = 3.6 Hz), 125.0 (q, J = 270.7 Hz), 132.9 (d, J =
5.4 Hz), 141.5, 146.8 (d, J = 10.9 Hz), 148.9, 152.3 (d, J = 246.1 Hz); HRMS (M+H+) 408.1687 (calcd for C21H21F4N3OH+ 408.1694).
1-Cyclopentyl-N-(3-methoxypropyl)-5-(trifluoromethyl)-1H-indazol-3-amine (41j). Following general method J and starting from 40f
(83 mg, 0.25 mmol) and 3-methoxypropylamine (30.6 µL, 0.30 mmol), 41j was obtained as an orange oil (19 mg, 0.06 mmol, 23%).
Purity ≥ 98 %; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.62-1.65 (m, 2H), 1.82-1.98 (m, 4H), 1.92-1.98 (m, 4H), 3.24 (s, 3H), 3.29 (q, 2H, J
= 6.4 Hz), 3.44 (t, 2H, J = 6.4 Hz), 4.94 (quint., 1H, J = 6.9 Hz), 6.34 (t, 1H, J = 5.5 Hz), 7.49 (dd, 1H, J = 1.8 Hz, J = 8.9 Hz), 7.55 (d,
1H, J = 8.9 Hz), 8.19 (s, 1H); 19F NMR (376 MHz, DMSO) δ ppm -58.6; 13C NMR (101 MHz, DMSO-d6) δ ppm 24.7, 29.4, 31.7, 58.3,

ACCEPTED MANUSCRIPT
70.4, 109.9, 113.7, 118.1 (q, J = 31.5 Hz), 119.5 (q, J = 4.4 Hz), 122.9 (q, J = 2.9 Hz), 141.7, 150.4; HRMS (M+H+) 342.1787 (calcd for
C17H22F3N3OH+ 342.1788).
1-Cyclohexyl-N-[(4-methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41k). Following general method J and starting
from 40g (62 mg, 0.18 mmol) and 4-methoxybenzylamine (28.0 µL, 0.21 mmol), 41k was obtained as yellow oil (20 mg, 0.05 mmol,
27%). Purity = 95 %; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.24 (q, 1H, J = 9.4 Hz), 1.45 (q, 2H, J = 12.8 Hz), 1.67 (d, 1H, J = 12.2 Hz),
1.82-1.90 (m, 7H), 3.72 (s, 3H), 4.36 (br s, 1H), 4.38 (d, 2H, J = 5.9 Hz), 6.71 (t, 1H, J = 5.9 Hz), 6.88 (d, 2H, J = 8.5 Hz), 7.35 (d, 2H, J =
8.5 Hz), 7.48 (d, 1H, J = 8.9 Hz), 7.58 (d, 1H, J = 8.9 Hz), 8.24 (s, 1H); 19F NMR (376 MHz, DMSO-d6) δ ppm -58.6; 13C NMR (101 MHz,
DMSO-d6) δ ppm 24.8, 31.6, 45.9, 54.8, 55.9, 109.2, 113.3, 118.9 (q, J = 2.9 Hz), 122.1 (q, J = 4.4 Hz), 129.0, 149.4; HRMS (M+H+)
404.1938 (calcd for C22H24F3N3OH+ 404.1944).
1-Benzyl-N-[(4-methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41l). Following general method I and starting from
40h (102 mg, 0.29 mmol) and 4-methoxybenzylamine (45.0 µL, 0.34 mmol), 41l was obtained as colorless oil (59 mg, 0.14 mmol, 50%).
1
Purity ≥ 98 %; H NMR (400 MHz, CDCl3) δ ppm 3.73 (s, 3H), 4.48 (s, 2H), 5.35 (s, 2H), 6.80 (d, 2H, J = 8.7 Hz), 7.09-7.14 (m, 3H),
7.17-7.22 (m, 2H), 7.28 (d, 2H, J = 8.7 Hz), 7.40 (dd, 1H, J = 1.6 Hz, J = 8.9 Hz), 7.74 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 48.0,
52.4, 55.3, 109.3, 114.0, 114.1, 117.9 (q, J = 4.4 Hz), 123.9 (q, J = 2.9 Hz), 127.1, 127.7, 128.7, 129.5, 131.2, 136.9, 142.0, 149.6,
159.1; HRMS (M+H+) 412.1626 (calcd for C23H20F3N3OH+ 412.1631).
N-[(4-Methoxyphenyl)methyl]-1-phenyl-5-(trifluoromethyl)-1H-indazol-3-amine (41m). Following general method J and starting from
40i (68 mg, 0.20 mmol) and 4-methoxybenzylamine (31.2 µL, 0.24 mmol), 41m was obtained as a yellow oil (38 mg, 0.10 mmol, 48%).
Purity ≥ 98 %; 1H NMR (500 MHz, CDCl3) δ ppm 3.82 (s, 3H), 4.62 (s, 2H), 6.92 (d, 2H, J = 8.6 Hz), 7.29 (t, 1H, J = 7.5 Hz), 7.41 (d, 2H,
J = 8.6 Hz), 7.51 (t, 1H, J = 7.5 Hz), 7.58 (dd, 1H, J = 1.7 Hz, J = 9.0 Hz), 7.68-7.72 (m, 3.0 Hz), 7.86 (s, 1H); 19F NMR (376 MHz,
CDCl3) δ ppm -60.7; 13C NMR (125 MHz, CDCl3) δ ppm 47.6, 55.3, 110.5, 114.1, 115.9, 117.7 (q, J = 4.5 Hz), 121.7, 121.8 (q, J = 32.7
Hz), 124.4 (q, J = 3.6 Hz), 124.6 (q, J = 270.7 Hz), 125.6, 127.9, 129.4, 129.5, 131.3, 140.2, 140.7, 151.0, 159.1; HRMS (M+H+)
398.1462 (calcd for C22H18F3N3OH+ 398.1475).
N-[(4-Methoxyphenyl)methyl]-1-(pyridine-3-yl)-5-(trifluoromethyl)-1H-indazol-3-amine (41n). Following general method
J and
starting from 40j (44 mg, 0.13 mmol) and 4-methoxybenzylamine (20.2 µL, 0.15 mmol), 41n was obtained as an orange solid (23 mg,
0.06 mmol, 45%). Purity ≥ 95 %; mp = 113-114 °C; 1H NMR (500 MHz, CDCl3) δ ppm 3.82 (s, 3H), 4.48 (t, 1H, J = 5.2 Hz), 4.62 (d, 1H, J
= 5.2 Hz), 6.92 (d, 2H, J = 8.5 Hz), 7.41 (d, 2H, J = 8.5 Hz), 7.44 (dd, 1H, J = 4.7 Hz, J = 8.2 Hz), 7.64 (dd, 1H, J = 1.5 Hz, J = 8.9 Hz),
7.72 (d, 1H, J = 8.9 Hz), 7.87 (s, 1H), 8.02 (ddd, 1H, J = 1.5 Hz, J = 2.4 Hz, J = 8.2 Hz), 8.51 (dd, 1H, J = 0.9 Hz, J = 4.7 Hz), 9.04 (d,
1H, J = 2.4 Hz); 19F NMR (376 MHz, CDCl3) δ ppm -60.9; 13C NMR (125 MHz, CDCl3) δ ppm 47.5, 55.3, 110.3, 114.1, 116.5, 117.8 (q, J
= 3.6 Hz), 122.5 (q, J = 32.7 Hz), 124.0, 124.5 (q, J = 271.6 Hz), 125.0 (q, J = 3.6 Hz), 128.4, 129.5, 131.0, 137.1, 140.8, 142.5, 146.3,
151.6, 159.2; HRMS (M+H+) 399.1414 (calcd for C21H17F3N4OH+ 399.1427).
N-[(4-Methoxyphenyl)methyl]-1H-indazol-3-amine (41o). A solution of 41a (70 mg, 0.21 mmol, 1.0 equiv.) in a mixture TFA:DCM (1:1
mL) was stirred at rt for 1 hour. The solution was concentrated under vacuum and purified by reverse chromatography
(MeOH/H2O+0.05%TFA), to yield 41o as a yellow oil (14 mg, 0.05 mmol, 26%). Purity ≥ 98 %; 1H NMR (400 MHz, CDCl3) δ ppm 3.76
(s, 3H), 5.22 (s, 2H), 6.81 (d, 2H, J = 8.5 Hz), 7.16-7.22 (m, 3H), 7.29 (d, 1H, J = 8.0 Hz), 7.58 (t, 1H, J = 8.0 Hz), 7.67 (d, 1H, J = 8.0
Hz); 13C NMR (101 MHz, CDCl3) δ ppm 51.9, 55.3, 110.3, 114.3, 121.5, 121.6, 129.5, 132.3; HRMS (M+H+) 254.1285 (calcd for
C15H15N3OH+ 254.1288).
N-[(4-Methoxyphenyl)methyl]-5-(trifluoromethyl)-1H-indazol-3-amine (41p). HCl 1N (0.73 mL) was added to a solution of 41b (101
mg, 0.25 mmol, 1.0 equiv.) in THF (0.4 mL) and the resulting mixture was stirred overnight at rt. The solution was basified until pH = 7
using a saturated solution of NaHCO3. The aqueous phase was extracted 3 times with EtOAc. The organic layers were combined,
washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column chromatography (heptanes:EtOAc), to
yield 41p as a white solid (21 mg, 0.07 mmol, 26%). Purity ≥ 98 %; mp = 109-112°C; 1H NMR (400 MHz, CDCl3) δ ppm 3.80 (s, 3H),
4.58 (s, 2H), 6.88 (d, 2H, J = 8.0 Hz), 7.34-7.38 (m, 3H), 7.55 (d, 1H, J = 8.4 Hz), 7.84 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 47.7,
55.3, 110.5 (q, J = 3.7 Hz), 114.1, 124.6 (q, J = 4.4 Hz), 129.2, 130.8, 159.1; HRMS (M+H+) 322.1156 (calcd for C16H14F3N3OH+
322.1162).
6-Phenyl-2,3-dihydropyridazin-3-one (42). A mixture of acetophenone (19.0 mL, 163.0 mmol, 3.0 equiv.) and glyoxylic acid hydrate
(5.0 g, 54.3 mmol, 1.0 equiv.) were heated at 100 °C for 2 hours. After it was cooled at 40 °C, water (20 mL) and ammonia (4 mL) were
added. The aqueous phase was extracted 3 times with DCM. Hydrazine hydrate (2.6 mL, 54.3 mmol, 1.0 equiv.) was added in the
aqueous phase and the resulting solution was heated at reflux four 4 hours. After cooling, the solid was filtered and washed with water,
1
to afford 42 as white solid (6.5 g, 37.5 mmol, 70%). H NMR (400 MHz, DMSO-d6) δ ppm 7.00 (d, 1H, J = 9.6 Hz), 7.45-7.52 (m, 3H),
7.87 (d, 2H, J = 7.2 Hz), 8.04 (d, 1H, J = 9.6 Hz), 13.2 (s, 1H).
2-Chloro-4-phenylpyridazine (43). A mixture of 42 (5.6 g, 32.4 mmol, 1.0 equiv.), POCl3 (30 mL) was heated at reflux for 2 hours. After
cooling, the reaction mixture was concentrated under vacuum. The residue was dissolved in cooled DCM and added slowly in ice-cooled
water. The organic phase was separated and the aqueous phase was extracted twice with DCM. The combined organic layers were
washed with a saturated solution of NaHCO3 and brine, dried over Na2SO4, filtered, concentrated under vacuum and purified by silica gel
column chromatography (heptanes:EtOAc 2:1), to afford 43 as a white solid (6.1 g, 32.0 mmol, 99%). 1H NMR (400 MHz, CDCl3) δ ppm
7.51-7.54 (m, 3H), 7.56 (d, 1H, J = 8.9 Hz), 7.83 (d, 1H, J = 8.9 Hz), 8.03-8.07 (m, 2H).
N-(3-Chlorophenyl)-6-phenylpyridazin-3-amine (44). A microvawe vial (ovendried and under argon) was charged with 3-chloro-6-
phenylpyridazine 43 (50 mg, 0.26 mmol, 1.0 equiv.), 3-chloroaniline (33.5 µL, 0.31 mmol, 1.2 equiv.), Pd(OAc)2 (3 mg, 0.01 mmol, 5
mol%), JosiPhos (7 mg, 0.01 mmol, 5 mol%), Cs2CO3 (128 mg, 0.39 mmol, 1.5 equiv.) and anhydrous DMF (0.5 mL) and heated

ACCEPTED MANUSCRIPT
overnight at 50°C. After cooling, the reaction mixt ure was diluted with water and the aqueous phase was extracted 3 times with EtOAc.
The organic layers were combined, washed with brine, dried over Na2SO4, filtered, concentrated and purified by silica gel column
chromatography (heptanes:EtOAc 7:3), to afford 44 as a brown solid (51 mg, 0.18 mmol, 69%). Purity ≥ 98 %; mp = 199-200 °C; 1H
NMR (400 MHz, DMSO-d6) δ ppm 7.02 (ddd, 1H, J = 0.6 Hz, J = 2.0 Hz, J = 7.9 Hz), 7.24 (d, 1H, J = 9.3 Hz), 7.35 (t, 1H, J = 8.2 Hz),
7.44-7.54 (m, 3H), 7.58 (ddd, 1H, J = 0.6 Hz, J = 2.0 Hz, J = 8.3 Hz), 8.05-8.08 (m, 3H), 8.20 (t, 1H, J = 2.0 Hz), 9.62 (s, 1H); 13C NMR
(101 MHz, DMSO-d6) δ ppm 116.8, 117.4, 117.6, 120.7, 125.6, 125.8, 128.8, 129.0, 130.3, 133.15 136.3, 142.2, 152.0, 156.1.
3-(Trifluoromethyl)benzene-1-carboximidamide (46).
A microvawe vial (ovendried and under argon) was charged with 3-
cyanobenzotrifluoride 45 (117.2 µL, 0.88 mmol, 1.0 equiv.) and anhydrous THF (2.2 mL). A solution of LiHMDS 1M in toluene (1.32 mL,
1.32 mmol, 1.5 equiv.) was added dropwise and the resulting mixture was stirred overnight at rt. The solution was acidified until pH = 1
using a solution of HCl 1N and then concentrated under vacuum. The residue was basified until pH =12 using a solution of NaOH. The
aqueous phase was extracted 3 times with DCM. The organic layers were combined, washed with brine, dried over Na2SO4, filtered and
1
concentrated, to yield 46 as an orange oil (168 mg, 0.88 mmol, 100%). H NMR (400 MHz, CDCl3) δ ppm 5.19 (s, 3H), 7.55 (t, 1H, J =
7.8 Hz), 7.71 (d, 1H, J = 7.8 Hz), 7.80 (d, 1H, J = 7.8 Hz), 7.87 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm 123.1 (q, J = 3.7 Hz), 125.0,
127.1 (q, J = 3.7 Hz), 129.4, 129.5, 137.3, 164.2.
5-(4-Methoxyphenyl)-2-[3-(trifluoromethyl)phenyl]-1H-imidazole (48). 46 (145 mg, 0.77 mmol, 1.0 equiv.) and NaHCO3 (259 mg,
3.08 mmol, 4.0 equiv.) were dissolved in a mixture THF:H2O (3.88:0.97 mL) and the resulting mixture was heated at reflux for 20 mins.
After it was cooled, a solution of 2-bromo-4’-methoxyacetophenone 47 (176 mg, 0.77 mmol, 1.0 equiv.) in THF (300 µL) was added
dropwise and the solution was heated at reflux for 3 hours. The solution was concentrated under vacuum and the residue was diluted
with water. The aqueous phase was extracted 3 times with EtOAc. The organic layers were combined, washed with brine, dried over
Na2SO4, filtered, concentrated and purified by silica gel column chromatography (heptane:EtOAc 7:3), to afford 48 as a yellow oil (232
mg, 0.73 mmol, 95%). Purity ≥ 98 %; 1H NMR (400 MHz, CDCl3) δ ppm 3.82 (s, 3H), 6.91 (d, 2H, J = 8.8 Hz), 7.31 (s, 1H), 7.39 (t, 1H, J
= 7.8 Hz), 7.50 (d, 1H, J = 7.8 Hz), 7.65 (d, 2H, J = 8.8 Hz), 7.96 (d, 1H, J = 7.8 Hz), 8.03 (s, 1H); 13C NMR (101 MHz, CDCl3) δ ppm
55.3, 114.3, 122.1 (q, J = 3.7 Hz), 125.0 (q, J = 3.7 Hz), 125.2, 126.4, 128.5, 129.3, 130.9, 131.2 (q, J = 33.0 Hz), 145.5, 159.1; HRMS
(M+H+) 319.1045 (calcd for C17H13F3N2OH+ 319.1053).
Biological methods
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.[3] PDE2 was isolated from human platelets following the methods indicated
in the literature.[2, 3] Purified PDEs were stored until use at -80°C in small aliquots (200 µL).
PDE activities were measured by a radioenzymatic assay as previously described in detail [64] at a substrate
concentration of 1 µM cAMP or 1 µM cGMP in the presence of 10,000 cpm [3H]-cAMP or [3H]-cGMP as tracers. The
buffer solution was of the following composition: 50 mM Tris-HCl (pH 7.5), 2 mM magnesium acetate, 50 mg BSA. PDE1
was assayed at 1 µM cGMP in calmodulin activated state (18 nM calmodulin with 10 µM CaCl2). PDE2 was evaluated at
1 µM cAMP in activated state (in presence of 5 µM cGMP). PDE3 and PDE4 were assayed at 1 µM cAMP + 1 mM
EGTA. To prevent the influence of reciprocal cross-contamination between PDE3 and PDE4, the studies were always
carried out in the presence of 50 µM rolipram (a generous gift of Schering, Berlin. Germany), for PDE3 and in presence
of 50 µM cGMP for PDE4. PDE5 activity was measured at 1 µM cGMP in the presence of 1 mM of EGTA. The
compounds were dissolved in DMSO; the final concentration of DMSO in the assay (1%) did not affect PDE activity. The
concentration of compounds that produced 50% inhibition of substrate hydrolysis (IC50) was calculated by non-linear
regression analysis (GraphPad Prism 5.00.288 software, San Diego, Ca) from concentration-response curves including
5 different concentrations of inhibitors. Maximal drug concentration used was limited to 10 µM and therefore the
percentage of inhibition obtained was given.
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 hour light/dark cycle with food and water
ad libitum. A total of 57 C57BL/6J mice were used for the experiments. All animals received proper care in agreement
with European guidelines (EU 2010/63). At the end of the experiments, mice were killed by CO2 inhalation (CO2
Euthanasia programmer 6.5 version, TEMSEGA, Pessac, France) 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. All protocols were approved by the "Comité d’Ethique en Matière
d’Expérimentation Animale de Strasbourg" (CREMEAS, CEEA35).
Neuropathic pain was induced by cuffing the right sciatic nerve.[44, 45] Surgeries were performed under ketamine (68
mg/kg) / xylazine (10 mg/kg) intraperitoneal (i.p.) anesthesia (Centravet, Tadden, France). The main branch of the right

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sciatic nerve was exposed and a cuff of PE-20 polyethylene tubing (Harvard Apparatus, Les Ulis, France) of
standardized length (2 mm) was unilaterally inserted around it (Cuff group). The shaved skin was closed using suture.
Sham-operated mice underwent the same surgical procedure without implantation of the cuff (Sham group). 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 hours after lights on. Mice were placed in clear Plexiglas boxes (7 cm x 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.[45, 65] The person who conducted the tests was
blinded to the treatments.
Treatments began fifteen days after the neuropathy was induced, and were maintained at least three weeks. During the
treatment, the mice received two intraperitoneal injections per day (morning and evening) of 4b, 16h, 41n, pregabalin
(Lyrica, Pfizer) or amitriptyline hydrochloride (Sigma-Aldrich) (3 or 0.5 mg/kg, 5 mL/kg). Drugs were prepared in 0.1%
hydroxymethylcellulose 0.9% NaCl solution that was also used for control injections.
Data were expressed as mean +/- SEM, and statistical analyses were performed using STATISTICA 7.1 (Statsoft, Tulsa,
OK, USA), with ANOVA for multiple comparisons and the Duncan test for posthoc analyses.
Acknowledgements
Maud Bollenbach was supported by a fellowship from the Ministère de l’Education Nationale, de l’Enseignement
Supérieur et de la Recherche. Experiments were supported by CNRS and Université de Strasbourg (contracts
UMR7200, UMR7213 and UPR3212), and by the Agence Nationale de la Recherche (Euridol ANR-17-EURE-0022). We
thank Dr. Ipek Yalcin for her help in conducting mice surgeries, zootechnicians from the Chronobiotron UMS3415 for
animal care, and Mr. Xavier Wurtz for support in mice testing.
Supporting information available
General procedures and experimental data for compounds 25-30, 42-44 and 46-48. 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 derivatives. These materials are available
free of charge via the Internet at http: //pubs.acs.org.
Keywords: PDE-5 inhibitors, MY5445, Structure activity relationship (SAR) studies, aminophthalazine derivatives,
neuropathic pain.
References:
[1]
T. Keravis, 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. 165 (2012) 1288–1305.
doi:10.1111/j.1476-5381.2011.01729.x.
[2]
[3]
(4)
(5)
J.F.K. Tcheudji, L. Lebeau, N. Virmaux, C.G. Maftei, R.H. Cote, C. Lugnier, P. Schultz, Molecular organization of bovine rod cGMP-
phosphodiesterase 6, J. Mol. Biol. 310 (2001) 781–791. doi:10.1006/jmbi.2001.4813.
C. Lugnier, P. Schoeffter, A. Le Bec, E. Strouthou, J.C. Stoclet, Selective inhibition of cyclic nucleotide phosphodiesterases of human,
bovine and rat aorta, Biochem. Pharmacol. 35 (1986) 1743–1751. doi:10.1016/0006-2952(86)90333-3.
R.H. Francis, T.M. Lincoln, J.D. Corbin, Characterization of a novel cGMP binding protein from rat lung., J. Biol. Chem. 255 (1980)
620–626.
J.E. Souness, R. Brazdil, B.K. Diocee, 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. 98 (1989) 725–734.
doi:10.1111/j.1476-5381.1989.tb14599.x.
(6)
(7)
(8)
N. Galiè, H.A. Ghofrani, A. Torbicki, R.J. Barst, L.J. Rubin, D. Badesch, T. Fleming, T. Parpia, G. Burgess, A. Branzi, F. Grimminger,
M. Kurzyna, G. Simonneau, Sildenafil Citrate Therapy for Pulmonary Arterial Hypertension, N. Engl. J. Med. 353 (2005) 2148–2157.
doi:10.1056/NEJMoa050010.
S. Korkmaz, T. Radovits, E. Barnucz, P. Neugebauer, R. Arif, K. Hirschberg, S. Loganathan, B. Seidel, M. Karck, G. Szabó, Dose-
dependent effects of a selective phosphodiesterase-5-inhibitor on endothelial dysfunction induced by peroxynitrite in rat aorta, Eur. J.
Pharmacol. 615 (2009) 155–162. doi:10.1016/j.ejphar.2009.05.020.
E. Takimoto, H.C. Champion, M. Li, D. Belardi, S. Ren, E.R. Rodriguez, D. Bedja, K.L. Gabrielson, Y. Wang, D.A. Kass, Chronic
inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy, Nat. Med. 11 (2005) 214–222.
doi:10.1038/nm1175.
(9)
R. Zhang, Y. Wang, L. Zhang, Z. Zhang, W. Tsang, M. Lu, L. Zhang, M. Chopp, Sildenafil (Viagra) Induces Neurogenesis and
Promotes Functional Recovery After Stroke in Rats, Stroke. 33 (2002) 2675–2680. doi:10.1161/01.STR.0000034399.95249.59.
N.K. Jain, C.S. Patil, A. Singh, S.K. Kulkarni, Sildenafil-induced peripheral analgesia and activation of the nitric oxide–cyclic GMP
pathway, Brain Res. 909 (2001) 170–178. doi:10.1016/S0006-8993(01)02673-7.
C.I. Araiza-Saldaña, G. Reyes-García, D.Y. Bermúdez-Ocaña, F. Pérez-Severiano, V. Granados-Soto, Effect of diabetes on the
mechanisms of intrathecal antinociception of sildenafil in rats, Eur. J. Pharmacol. 527 (2005) 60–70. doi:10.1016/j.ejphar.2005.10.016.
M.L. Vale, D.E. Rolim, I.F. Cavalcante, R.A. Ribeiro, M.H.L.P. Souza, Role of NO/cGMP/KATP pathway in antinociceptive effect of
sildenafil in zymosan writhing response in mice, Inflamm. Res. 56 (2007) 83–88. doi:10.1007/s00011-006-6109-8.
M.M. Bezerra, V. Lima, V.C.C. Girao, R.C. Teixeira, J.R.V. Graca, Antinociceptive activity of sildenafil and adrenergic agents in the
writhing test in mice, Pharmacol. Rep. 60 (2008) 339–344.
(10)
(11)
(12)
(13)

ACCEPTED MANUSCRIPT
(14)
(15)
(16)
C.S. Patil, N.K. Jain, V.P. Singh, S.K. Kulkarni, Cholinergic-NO-cGMP mediation of sildenafil-induced antinociception, Indian J. Exp.
Biol. 42 (2004) 361–367.
L.J. Huang, M.H. Yoon, J.I. Choi, W.M. Kim, H.G. Lee, Y.O. Kim, Effect of Sildenafil on Neuropathic Pain and Hemodynamics in Rats,
Yonsei Med. J. 51 (2010) 82–87. doi:10.3349/ymj.2010.51.1.82..
L. Wang, M. Chopp, A. Szalad, Z. Liu, M. Bolz, F.M. Ãlvarez, M. Lu, L. Zhang, Y. Cui, R.L. Zhang, Z.G. Zhang, Phosphodiesterase-5 is
a
therapeutic
target
for
peripheral
neuropathy
in
diabetic
mice,
Neuroscience.
193
(2011)
399–410.
doi:10.1016/j.neuroscience.2011.07.039.
(17)
(18)
(19)
(20)
(21)
(22)
G. Hackett, PDE5 inhibitors in diabetic peripheral neuropathy, Int. J. Clin. Pract. 60 (2006) 1123–1126. doi:10.1111/j.1742-
1241.2006.01087.x..
E.I. Gediz, C. Nacitarhan, E. Minareci, G. Sadan, Antinociceptive effect of vardenafil on carrageenan-induced hyperalgesia in rat:
involvement of nitric oxide/cyclic guanosine monophosphate/calcium channels pathway, Iran. J. Pharm. Res. 14 (2015) 1137–1143.
F. Rocha, F. Silva Jr, A. Leite, A. Leite, V. Girão, R. Castro, F. Cunha, Tadalafil analgesia in experimental arthritis involves
suppression of intra-articular TNF release, Br. J. Pharmacol. 164 (2011) 828–835. doi:10.1111/j.1476-5381.2011.01469.x.
M. Ambriz-Tututi, D.A. Velázquez-Zamora, H. Urquiza-Marín, V. Granados-Soto, Analysis of the mechanism underlying the peripheral
antinociceptive action of sildenafil in the formalin test, Eur. J. Pharmacol. 512 (2005) 121–127. doi:10.1016/j.ejphar.2005.01.055.
D. Levy, A. Höke, D. W. Zochone, Local expression of inducible nitric oxide synthase in an animal model of neuropathic pain,
Neurosci. Lett.260 (1999) 207-209. doi:10.1016/s0304-3940(98)00982-3.
A. Hervera, R. Negrete, S. Leánez, J. M. Martín-Campos, 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,
doi:10.1371/journal.pone.0014321.
5 (2010), e14321.
(23)
(24)
(25)
(26)
Y. Guan, M. Yaster, S. N. Raja, Y. X. Tao, Genetic Knockout and Pharmacologic Inhibition of Neuronal Nitric Oxide Synthase
Attenuate Nerve Injury-Induced Mechanical Hypersensitivity in Mice. Mol. Pain, 3 (2007), 1744–8069–3–29. doi:10.1186/1744-8069-3-
29.
M. Tanabe, Y. Nagatani, K. Saitoh, K.Takasu, 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, 56 (2009), 702–708. doi:10.1016/j.neuropharm.2008.12.003.
V. Granados-Soto, M. de O. Rufino, L. D. Gomes Lopes, S. H. Ferreira, Evidence for the involvement of the nitric oxide–cGMP
pathway in the antinociception of morphine in the formalin test. Eur J Clin Pharmacol., 340 (1997), 177–180. doi:10.1016/s0014-
2999(97)01399-x.
K. Takasu, M. Honda, H. Ono, M. Tanabe, Spinal α2-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., 148 (2006), 233–244. doi:10.1038/sj.bjp.0706731.
(27)
(28)
(29)
T. Mixcoatl-Zecuatl, F. J. Flores-Murrieta, V. Granados-Soto, The nitric oxide-cyclic GMP-protein kinase G-K+ channel pathway
participates in the antiallodynic effect of spinal gabapentin. Eur. J. Pharmacol., 531 (2006), 87–95. doi:10.1016/j.ejphar.2005.12.006.
A. Gibson, Phosphodiesterase 5 inhibitors and nitrergic transmission—from zaprinast to sildenafil, Eur. J. Pharmacol. 411 (2001) 1–10.
doi:10.1016/S0014-2999(00)00824-4.
A.M. Martel, A. Graul, X. Rabasseda, R. Castaner, Sildenafil. Treatment of erectile dysfunction, Phosphodiesterase V inhibitor, Drug
Future. 22 (1997) 138–143.
(30)
(31)
D. Ormrod, S.E. Easthope, D.P. Figgitt, Vardenafil, Drugs Aging. 19 (2002) 217–227. doi:10.2165/00002512-200219030-00005.
B. Dumaître, N. Dodic, Synthesis and Cyclic GMP Phosphodiesterase Inhibitory Activity of a Series of 6-Phenylpyrazolo[3,4- d
]pyrimidones. J. Med. Chem. 39, (1996) 1635–1644. doi:org/10.1021/jm950812j.
(32)
C.M.N. Allerton, C.G. Barber, K.C. Beaumont, D.G. Brown, S.M. Cole, D. Ellis, C.A.L. Lane, G.N. Maw, N.M. Mount, D.J. Rawson,
C.M. Robinson, S.D.A. Street, N.W. Summerhill,. A Novel Series of Potent and Selective PDE5 Inhibitors with Potential for High and
Dose-Independent Oral Bioavailability. J. Med. Chem. 49 (2006) 3581–3594. doi.:org/10.1021/jm060113e.
M. Hagiwara, T. Endo, T. Kanayama, 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. 228 (1984) 467–471.
N. Watanabe, H. Adachi, Y. Takase, H. Ozaki, M. Matsukura, K. Miyazaki, K. Ishibashi, H. Ishihara, K. Kodama, M. Nishino, M. Kakiki,
Y. Kabasawa, 4-(3-Chloro-4-methoxybenzyl)aminophthalazines:ꢁ Synthesis and Inhibitory Activity toward Phosphodiesterase 5, J.
Med. Chem. 43 (2000) 2523–2529. doi:10.1021/jm9905054.
(33)
(34)
(35)
M.A.J. Duncton, E.L. Piatnitski Chekler, R. Katoch-Rouse, D. Sherman, W.C. Wong, L.M. Smith II, J.K. Kawakami, A.S. Kiselyov, D.L.
Milligan, C. Balagtas, Y.R. Hadari, Y. Wang, S.N. Patel, R.L. Rolster, J.R. Tonra, D. Surguladze, S. Mitelman, P. Kussie, P. Bohlen,
J.F. Doody, Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2, Bioorg. Med. Chem. 17 (2009) 731–740.
doi:10.1016/j.bmc.2008.11.049.
(36)
(37)
Y. Takase, T. Saeki, M. Fujimoto, I. Saito, Cyclic GMP phosphodiesterase inhibitors. 1. The discovery of a novel potent inhibitor, 4-
[[3,4-(methylenedioxy)benzyl]amino]-6,7,8-trimethoxyquinazoline, J. Med. Chem. 36 (1993) 3765–3770. doi: 10.1021/jm00076a003.
G. Yu, H.J. Mason, X. Wu, J. Wang, S. Chong, G. Dorough, A. Henwood, R Pongrac, L. Seliger, B., He, D. Normandin, L Adam, J.
Krupinski, J.E. Macor, Substituted Pyrazolopyridines as Potent and Selective PDE5 Inhibitors: Potential Agents for Treatment of
Erectile Dysfunction. J. Med. Chem. 44 (2001) 1025–1027. doi:org/10.1021/jm0155042.
(38)
(39)
N. Anand, S. Brown, Z. Tesfai, C. Zaharia, Phthalazine Derivatives as Jak1 Inhibitors, WO2012037132 (A1), 2012.
https://worldwide.espacenet.com/publicationDetails/biblio?FT=D&date=20120322&DB=EPODOC&locale=en_EP&CC=WO&NR=2012
037132A1&KC=A1&ND=5 (accessed March 7, 2017).
L.T. Spada, J.G. Shiah, P.M. Hughes, T.C. Malone, G.W. Devries, J.L. Edelman, J.A. Wurster, Kinase Inhibitors, WO2010078393 (A1),
2010.https://worldwide.espacenet.com/publicationDetails/biblio?FT=D&date=20100708&DB=EPODOC&locale=en_EP&CC=WO&NR=
2010078393A1&KC=A1&ND=4 (accessed March 7, 2017).
(40)
(41)
(42)
(43)
H. Dyker, J. Scherkenbeck, D. Gondol, A. Goehrt, A. Harder, Azadepsipeptides:ꢁ Synthesis and Evaluation of a Novel Class of
Peptidomimetics, J. Org. Chem. 66 (2001) 3760–3766. doi:10.1021/jo001749v.
E. Lohou, V. Collot, S. Stiebing, S. Rault, Direct Access to 3-Aminoindazoles by Buchwald-Hartwig C-N Coupling Reaction, Synthesis.
2011 (2011) 2651–2663. doi:10.1055/s-0030-1260110.
Y. Mao, G. Tian, Z. Liu, J. Shen, J. Shen, An Improved Synthetic Route for Preparative Process of Vardenafil, Org. Process Res. Dev.
13 (2009) 1206–1208. doi:10.1021/op900235p.
J.B. Smaill, A.J. Gonzales, J.A. Spicer, H. Lee, J.E. Reed, K. Sexton, I.W. Althaus, T. Zhu, S.L. Black, A. Blaser, W.A. Denny, P.A.
Ellis, S. Fakhoury, P.J. Harvey, K. Hook, F.O.J. McCarthy, B.D. Palmer, F. Rivault, K. Schlosser, T. Ellis, A.M. Thompson, E. Trachet,
R.T. Winters, H. Tecle, A. Bridges, 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. 59 (2016) 8103–
8124. doi:10.1021/acs.jmedchem.6b00883.
(44)
(45)
(46)
M. Benbouzid, V. Pallage, M. Rajalu, E. Waltisperger, S. Doridot, P. Poisbeau, M.J. Freund-Mercier, M. Barrot, Sciatic nerve cuffing in
mice: A model of sustained neuropathic pain, Eur. J. Pain. 12 (2008) 591–599. doi:10.1016/j.ejpain.2007.10.002.
I. Yalcin, E. Waltisperger, M. Barrot, S. Megat, F. Barthas, M. Kremer, E. Salvat, The sciatic nerve cuffing model of neuropathic pain in
mice, J. Vis. Exp. JoVE. 89 (2014) e51608.doi:10.3791/51608
M. Bollenbach, E. Salvat, F.Daubeuf, P. Wagner,.I.Yalcin, M. Humo, M. Schmitt, Phenylpyridine-2-ylguanidines and rigid mimetics as
novel inhibitors of TNFα overproduction: Beneficial action in models of neuropathic pain and of acute lung inflammation. Eur. J. Med.
Chem. 147 (2018), 163–182. doi:10.1016/j.ejmech.2018.01.049.
(47)
M. Kremer, E. Salvat, A. Muller, I. Yalcin, M. Barrot, Antidepressants and gabapentinoids in neuropathic pain: Mechanistic insights,
Neuroscience. 338 (2016) 183–206. doi:10.1016/j.neuroscience.2016.06.057.

ACCEPTED MANUSCRIPT
(48)
(49)
R.-D. Treede, T.S. Jensen, J.N. Campbell, G. Cruccu, J.O. Dostrovsky, J.W. Griffin, P. Hansson, R. Hughes, T. Nurmikko, J. Serra,
Neuropathic pain Redefinition and
a grading system for clinical and research purposes, Neurology. 70 (2008) 1630–1635.
doi:10.1212/01.wnl.0000282763.29778.59.
Y. Bohren, L.-H. Tessier, S. Megat, H. Petitjean, S. Hugel, D. Daniel, M. Kremer, S. Fournel, L. Hein, R. Schlichter, M.-J. Freund-
Mercier, I. Yalcin, M. Barrot, Antidepressants suppress neuropathic pain by a peripheral β2-adrenoceptor mediated anti-TNFα
mechanism, Neurobiol. Dis. 60 (2013) 39–50. doi:10.1016/j.nbd.2013.08.012.
(50)
(51)
I. Yalcin, L.-H. Tessier, N. Petit-Demoulière, S. Doridot, L. Hein, M.-J. Freund-Mercier, M. Barrot, β2-adrenoceptors are essential for
desipramine, venlafaxine or reboxetine action in neuropathic pain, Neurobiol. Dis. 33 (2009) 386–394. doi:10.1016/j.nbd.2008.11.003.
M. Kremer, I. Yalcin, Y. Goumont, X. Wurtz, L. Nexon, D. Daniel, S. Megat, R. A. Ceredig, C. Ernst, G. Turecki, V. Chavant, J-
F.Theroux, A. Lacaud, L-E. Joganah, V. Lelivre, D. Massotte, P6E. Lutz, R. Glisbach, E. Salvat, M. Barrot, A Dual Noradrenergic
Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline . J. Neurosci. 38 (2018).
9934-9954. doi:10.1523/jneurosci.1004-18.2018.
(52)
M. Benbouzid, N. Choucair-Jaafar, I. Yalcin, E. Waltisperger, A. Muller, M. J. Freund-Mercier, M. Barrot, Chronic, but not acute,
tricyclic antidepressant treatment alleviates neuropathic allodynia after sciatic nerve cuffing in mice. European Journal of Pain, 12
(2008), 1008–1017. doi:10.1016/j.ejpain.2008.01.010.
(53)
(54)
(55)
A.K. Naik, S.K. Tandan, D. Kumar, S.P. Dudhgaonkar, Nitric oxide and its modulators in chronic constriction injury-induced neuropathic
pain in rats, Eur. J. Pharmacol. 530 (2006) 59–69. doi:10.1016/j.ejphar.2005.11.029.
D. Levy, D.W. Zochodne, NO Pain: Potential Roles of Nitric Oxide in Neuropathic Pain, Pain Pract.
doi:10.1111/j.1533-2500.2004.04002.x.
4 (2004) 11–18.
A. Hervera, R. Negrete, S. Leánez, J.M. Martín-Campos, 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.
doi:10.1371/journal.pone.0014321.
5 (2010) e14321.
(56)
(57)
(58)
(59)
(60)
I.D.G. Duarte, 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. 221 (1992) 171–174. doi:10.1016/0014-2999(92)90789-7.
G. Petho, P.W. Reeh, Sensory and Signaling Mechanisms of Bradykinin, Eicosanoids, Platelet-Activating Factor, and Nitric Oxide in
Peripheral Nociceptors, Physiol. Rev. 92 (2012) 1699–1775. doi:10.1152/physrev.00048.2010.
A. Schmidtko, Nitric Oxide-Mediated Pain Processing in the Spinal Cord, in: H.-G. Schaible (Ed.), Pain Control, Springer Berlin
Heidelberg, Berlin, Heidelberg, 2015: pp. 103–117. doi:10.1007/978-3-662-46450-2_6..
K.V. Otari, C.D. Upasani, Involvement of NO–cGMP pathway in anti-hyperalgesic effect of PDE5 inhibitor tadalafil in experimental
hyperalgesia, Inflammopharmacology. 23 (2015) 187–194. doi:10.1007/s10787-015-0240-5.
L. Garcia, S. Hlaing, R. Gutierrez, M. Sanchez, I. Kovanecz, J. Artaza, M. Ferrini, Sildenafil Attenuates Inflammation and Oxidative
Stress in Pelvic Ganglia Neurons after Bilateral Cavernosal Nerve Damage, Int. J. Mol. Sci. 15 (2014) 17204–17220.
doi:10.3390/ijms151017204.
(61)
(62)
(63)
(64)
(65)
P.J. Austin, G. Moalem-Taylor, The neuro-immune balance in neuropathic pain: Involvement of inflammatory immune cells, immune-
like glial cells and cytokines, J. Neuroimmunol. 229 (2010) 26–50. doi:10.1016/j.jneuroim.2010.08.013.
E., Rojewska, A. Piotrowska, A. Jurga, W. Makuch, J. Mika, Zaprinast diminished pain and enhanced opioid analgesia in a rat
neuropathic pain model. Eur. J. Pharmacol. 839 (2018) 21–32. doi: 10.1016/j.ejphar.2018.09.001.
A. Singh Jaggi, N. Singh, Therapeutic Targets for the Management of Peripheral Nerve Injury- Induced Neuropathic Pain, CNS Neurol.
Disord. - Drug Targets. 10 (2011) 589–609. doi:10.2174/187152711796235041.
T. Keravis, R. Thaseldar-Roumié, C. Lugnier, Assessment of Phosphodiesterase Isozyme Contribution in Cell and Tissue Extracts, in:
Phosphodiesterase Methods Protoc., Humana Press, New Jersey, 2005: pp. 063–074. doi:10.1385/1-59259-839-0:063.
M. Barrot, Tests and models of nociception and pain in rodents, Neuroscience. 211 (2012) 39–50.
doi:10.1016/j.neuroscience.2011.12.041.

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Entry for the Table of Contents
neuropathic pain model:
sciatic nerve cuff
long lasting mechanical allodynia
Design of homologues, isosteres and structural
analogues of MY5445 as PDE5 inhibitors
In vivo chronic treatment
3mg/kg i.p. (19 days)
.

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