Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders

Article abstract of DOI:10.1021/acs.jmedchem.5b01208

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders.

Full text of DOI:10.1021/acs.jmedchem.5b01208

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Article
Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-
indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3
(GSK-3) Inhibitors with In Vivo Activity in Model of Mood Disorders
Guido Furlotti, Maria Alessandra Alisi, Nicola Cazzolla, Patrizia Dragone, Lucia Durando, Gabriele
Magaro, Francesca Mancini, Giorgina Mangano, Rosella Ombrato, Marco Vitiello, Andrea Armirotti,
Valeria Capurro, Massimiliano Lanfranco, Giuliana Ottonello, Maria Summa, and Angelo Reggiani
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01208 • Publication Date (Web): 20 Oct 2015
Downloaded from http://pubs.acs.org on November 2, 2015
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Hit Optimization of 5ꢀSubstitutedꢀNꢀ(piperidinꢀ4ꢀ
ylmethyl)ꢀ1Hꢀindazoleꢀ3ꢀcarboxamides: Potent
Glycogen Synthase Kinaseꢀ3 (GSKꢀ3) Inhibitors
with In Vivo Activity in Model of Mood Disorders
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*,§
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Guido Furlotti, Maria Alessandra Alisi, Nicola Cazzolla, Patrizia Dragone, Lucia
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Durando, Gabriele Magarò, Francesca Mancini, Giorgina Mangano, Rosella Ombrato,
§
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¢
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Marco Vitiello, Andrea Armirotti, Valeria Capurro, Massimiliano Lanfranco, Giuliana
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¢
*,¢
Ottonello, Maria Summa and Angelo Reggiani
§
Angelini S.p.A., Angelini Research Center, P.le della Stazione s.n.c., Santa Palombaꢀ
Pomezia (RM), (00071) Italy
¢
Istituto Italiano di Tecnologia, Drug Discovery and Development Department, Via Morego
0, (16163) Genova, Italy
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ABSTRACT
Novel treatments for bipolar disorders, with improved efficacy and broader spectrum of
activity are urgently needed. Glycogen synthase kinase 3β (GSKꢀ3β) has been suggested to
be a key player in the pathophysiology of bipolar disorders. A series of novel GSKꢀ3β
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inhibitors having the common Nꢀ[(1ꢀalkylpiperidinꢀ4ꢀyl)methyl]ꢀ1Hꢀindazoleꢀ3ꢀcarboxamide
scaffold were prepared taking advantage of an Xꢀray coꢀcrystal structure of compound 5 with
GSKꢀ3β. We probed different substitutions at the indazole 5ꢀposition and at the piperidineꢀ
nitrogen to obtain potent ATPꢀcompetitive GSKꢀ3β inhibitors with good cell activity. Among
the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing,
encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of
mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in
order to elucidate the use of ATPꢀcompetitive GSKꢀ3β inhibitors as new tools in the
development of new treatments for mood disorders.
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INTRODUCTION
Bipolar disorders are severe psychiatric conditions characterized by swings between
depressive episodes (anhedonia, cognitive disorders, and suicidal thoughts) and periods of
elevated mood (hyperarousal, irritability and hallucinations). Bipolar disorder affects about
1% of the world population, with symptoms generally emerging in young adults with
comparable prevalence in males and females. According to the current literature lithium
remains the best available mood stabilizer, but efficacy is primarily during the manic phase,
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while additional supportive therapy is required to control the depressive phase.
Anticonvulsant agents, such as valproic acid and lamotrigine or atypical antipsychotic drugs
such as olanzapine and quetiapine have some efficacy, although limited to particular patient
subꢀgroups. Moreover, current medications have little efficacy in preventing relapse (low
remission rate) and have significant sideꢀeffects. For these reasons there is a significant
medical need to identify more effective and safer drugs, acting on both the acute and global
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course of the illness.
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Recent literature has pointed out a significant role for glycogen synthase kinase 3β (GSKꢀ
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β) in the pathophysiology of bipolar disorders. Dysregulation of the enzyme has been
proposed as a potential driver of the disease and strong support for this has been the
discovery of an inherited mutation of the GSKꢀ3 gene which leads to overꢀactivation of the
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enzyme, causing psychiatric illnesses in human. Moreover both lithium, and to lesser extent
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valproic acid, are inhibitors of the GSKꢀ3β signaling cascade.
GSKꢀ3 is a constitutively active multiꢀfunctional serine/threonine protein kinase discovered
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in the late 1970s. In humans, GSKꢀ3 is encoded by two different and independent genes,
generating GSKꢀ3α and GSKꢀ3β proteins, with molecular weights of about 51 and 47 kDa,
respectively. The two isoforms share nearly identical sequences in their kinase domains (95%
homology of the catalytic domain) while differing substantially in sequence elsewhere in the
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protein. Like other kinases, GSKꢀ3β transfers phosphate groups from ATP to specific
substrates, usually proteins. After phosphorylation, the substrate undergoes a functional
change, by which kinases can mediate various biological functions. In the central nervous
system, GSKꢀ3β is widely expressed and has a fundamental role, in neuronal signaling,
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, 8
regulating the Wnt pathway (by βꢀcatenin deactivation) and tau protein function.
In the last decade, several novel classes of adenosine triphosphate (ATP) competitive and
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nonꢀATP competitive GSKꢀ3β inhibitors have been reported. Among the nonꢀATP
competitive inhibitors, 4ꢀbenzylꢀ1,2,4ꢀthiadiazolidineꢀ3,5ꢀdiones such as TDZDꢀ8 or
tideglusib (Chart 1) showed good kinase inhibition (IC50 = 2 µM and 0.06 µM, respectively)
and good efficacy in many animal models but failed to exhibit clinical benefit in Alzheimer's
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disease clinical trial. Reported ATP competitive inhibitors such as Nꢀ(1,3ꢀthiazolꢀ2ꢀyl)urea
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(
1) , 3,4ꢀdiarylꢀmaleimides and derivatives (2),
6ꢀheteroaryl pyridones (3a) and
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pyrimidinones (3b), 5ꢀarylꢀ4ꢀcarboxamideꢀ1,3ꢀoxazoles (4), shared a standard Hꢀbond
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pattern interaction with the hinge region of the binding site, involving one hydrogen bond
acceptor and one or two hydrogen bond donor sites. Despite good in vitro potency and in
some cases favorable PK properties, few of these GSKꢀ3β inhibitors have showed efficacy in
preclinical in vivo models and none of them have progressed to the clinical phase for moodꢀ
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disorder related indications.
Chart 1. Structures of GSKꢀ3β inhibitors
O
N
O
N
N
O
N
S
O
S
tideglusib
TDZD-8
O
H
N
O
N
N
O
O
NH
S
O
O
N
O
NH
NH
R1
Ar
X
R
Cl
R1
R2
+
-
N
O
OMe
OMe
3a, X = CH
, X = N
1
2
4
, R = heteroaryl
1
3
b
O
NH
N
NH
O
N
N
H
5
In the present paper, our effort to identify novel GSKꢀ3β inhibitors acting as moodꢀ
stabilizers in animal models is described. The chemical program was initiated, following the
identification of the 1Hꢀindazoleꢀ3ꢀcarboxamide hit compound 5 (after a virtual screening
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campaign described in a separate paper) . The xꢀray structure of GSKꢀ3β coꢀcrystallized
with this ligand, helped us to design novel Nꢀ[(1ꢀalkylpiperidinꢀ4ꢀyl)methyl]ꢀ1Hꢀindazoleꢀ3ꢀ
carboxamides as potent GSKꢀ3β enzyme inhibitors. Compounds demonstrated robust activity
in cellꢀbased model while selected compounds showed appropriate pharmacokinetic
properties for in vivo efficacy studies. In the absence of rodent in vivo models of bipolar
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disorders, the most promising compounds were tested in the lowꢀdose amphetamine hyperꢀ
motility test, a wellꢀestablished preclinical model of mania. Early results of these efficacy
experiments are reported here.
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RESULTS AND DISCUSSION
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Initial Hit Finding Results. Following a structureꢀbased hit discovery program, we
identified a series of Nꢀ[(1ꢀalkylpiperidinꢀ4ꢀyl)methyl]ꢀ1Hꢀindazoleꢀ3ꢀcarboxamides showing
GSKꢀ3β inhibition with low micromolar IC ’s. One of these preliminary hits (5, Chart 1)
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with IC50 = 0.64 µM was successfully coꢀcrystallised with GSKꢀ3β revealing key interaction
features between the enzyme and inhibitor. (Figure 1).
Figure 1. Xꢀray crystal structure of compound 5 in the GSKꢀ3β ATP binding site: (A) view
of the hinge region interactions, (B) view from the inner cavity toward the hinge region and
solvent accessible area.
Figures 1A and 1B show the key interactions between compound 5 and amino acid residues
of the enzyme ATP binding pocket. The indazole moiety is located at the adenine binding site
and its two nitrogen atoms form hydrogen bonds with the backbone carbonyl group of
Asp133 and with the backbone NH of Val135 of the hinge region. The backbone carbonyl of
Val135 also appears to be involved in a hydrogen bond interaction with the NH of the
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carboxamide group of compound 5 while the piperidine nitrogen is oriented toward the
guanidine group of Arg141. This configuration orients the terminal phenylacetamide moiety
toward the external solvent accessible part of the binding pocket. The apparent lack of tight
interactions between protein and ligand in this broad section of the binding site, could be
useful for modulating physicoꢀchemical properties of the molecules without significantly
influencing the inhibitory potency. Interestingly, the indazole 5ꢀmethyl group is directed
toward the inner cavity of the ATP binding pocket suggesting opportunities for additional
polar interactions with Lys85 and Asp200 or lipophilic interaction with other proximal
residues (Figure 1B).
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The encouraging initial in vitro results together with the knowledge of the protein structure
information, obtained from the Xꢀray analysis of coꢀcrystallized 5 with GSKꢀ3β, supported
an extensive hit to lead medicinal chemistry program around the hit series core scaffold. The
analysis of the crystal structure together with already available SAR information for a
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bioisosteric class of GSKꢀ3β inhibitors, suggested focus on two areas of interest: the
indazole 5ꢀsubstituent and piperidine Nꢀsubstitution. Both, as previously indicated, providing
opportunities to probe positive interaction with the protein and to modulate physicochemical
properties.
Chemistry. The compounds described in this paper were prepared according to the general
routes outlined in Schemes 1ꢀ4. The 5ꢀmethoxyindazolꢀ3ꢀcarboxamide derivatives 9a,c,f,g,oꢀ
q,u,v (Table 1) were readily prepared by a direct hydroxybenzotriazole (HOBt) ꢀ N,N'ꢀ
dicyclohexylcarbodiimide (DCC) mediated carboxylic acid ꢀ amine coupling reaction,
between 5ꢀmethoxyꢀindazolꢀ3ꢀcarboxylic acid 10a and the corresponding 1ꢀ(piperidinꢀ4ꢀ
yl)methane amines 6 (Scheme 1).
a
Scheme 1. Synthesis of indazoleꢀ3ꢀcarboxamide derivatives 9a,c,f,g,nꢀq,u,v
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O
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OH
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+
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H N
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H
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a
Reagents and conditions: a) HOBt, DCC, DMF, r.t., 39–67%; b) H
37%.
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, 10% Pd/C, 80 °C,
The remaining derivatives 9 were synthesized by piperidine Nꢀalkylation of the 5ꢀmethoxyꢀ
Hꢀindazoleꢀ3ꢀcarboxamide key intermediate 8a (Scheme 2). For carboxylic acid derivatives
b, 9d, 9x, 9z and 9ab alkaline hydrolysis of the corresponding ester produced the final
1
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compounds.
a
Scheme 2. Synthesis of indazoleꢀ3ꢀcarboxamide derivatives 9b,d,e,m,t,wꢀz,aaꢀah
O
O
OH
NH
BOC
N
R2
BOC
R2
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N
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H N
N
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H
H
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6z
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7b; R = Br
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O
NH
NH
R1
N
NH
R2
R2
b
c
7
a-b
N
N
N
H
N
H
8
a; R = MeO
9e,m,t,w,ad,af-ah
0b,d; 9y,aa,ac
b,d,x,z,ab
1
8b; R = Br
1
2
d
9
a
Reagents and conditions: a) HOBt, DCC, DMF, 0 °C, 2h ꢀ> r. t., o.n., 87 – 96%; b) 2M
1 2 3
HClꢀEtOH, MeOH, r.t., 3h, 76ꢀ89%; c) R ꢀX, K CO , DMF, 80 °C, 1h, 11ꢀ59%; d) acq.
NaOH, MeOH, ꢀ, o.n., 43ꢀ87% .
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The acyl amides 9hꢀl were prepared starting from the intermediate 8a after Nꢀalkylation
with tertꢀbutyl Nꢀ(3ꢀbromopropyl)carbamate, subsequent deꢀprotection and acylation with the
appropriate acyl chloride (Scheme 3).
a
Scheme 3. Synthesis of amide derivatives 9hꢀl
0
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O
NH
N
NH
N
BOC
O
NH
O
NH2
b
a
c
N
N
8
a
N
H
N
H
2
0l
2
1l
O
NH
N
O
NH
2
1l
N
O
N
H
R
9
h-l
a
2 3
Reagents and conditions: a) BOCꢀNH(CH ) Br, TEA, MEK, 80 °C, 1h; b) TFA, DCM, r.
t.; o.n., 63%; c) RꢀCOCl, DMSO, DCM, r. t., 2h, 17ꢀ69%.
Compounds with different indazole 5ꢀsubstituents were generally prepared following the
route depicted in Scheme 4. The key intermediate 5ꢀbromoꢀNꢀ(piperidinꢀ4ꢀylmethyl)ꢀ1Hꢀ
indazoleꢀ3ꢀcarboxamide 8b was piperidine Nꢀalkylated with the appropriate alkyl halide, to
give the 5ꢀbromoindazoleꢀ3ꢀcarboxamide intermediates 13c, 14c, 15d, 16d and 17d, which
were then coupled with a range of boronic acids or esters, to afford the target compounds 13ꢀ
17 (see Table 2 for structures).
a
Scheme 4. Synthesis of derivatives 13ꢀ17
O
O
NH
R
1
NH
R1
N
N
Br
R2
a
b
8b
N
N
N
N
H
H
13c, 14c, 15d, 16d, 17d
13-17
a
Reagents and conditions: a) R
1
ꢀX, K
2
3
CO , DMF, 80 °C, 1h, 16ꢀ62%; b) R
2
2
B(OH) or
R B(OR) , Pd(dppf)Cl , Cs CO , dioxane/H O (3:1), ꢁꢀwave (45’@ T = 160 °C, max power
2
2
2
2
3
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300 W), 15ꢀ45%.
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StructureꢀEnzymatic Activity Relationship Analysis. All the molecules prepared were
tested in the GSKꢀ3β ATPꢀase enzymatic assay where the human recombinant enzyme GSKꢀ
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β was incubated in the presence of compounds or vehicle in a reaction buffer containing
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ATP plus the unꢀphosphorylated specific substrate peptide. Substrate phosphorylation was
measured and percent inhibition calculated relative to a positive control. The half maximal
inhibitory concentration values (IC50) were determined by nonꢀlinear regression analysis of
the inhibition curves.
Focusing initially on the piperidine substitution of the 5ꢀmethoxyꢀNꢀ(piperidinꢀ4ꢀ
ylmethyl)ꢀ1Hꢀindazoleꢀ3ꢀcarboxamide derivatives (Table 1), straight chain Nꢀalkylcarboxylic
acids showed some improvement in activity with an increase in potency of twoꢀfold for the
acetyl and propionyl derivatives (9b and 9c) and 5ꢀfold for the longer butyryl derivative 9d.
Substituents bearing a terminal neutral polar group (e.g., methoxyethyl 9e, methansulfonyl
ethyl amine 9f) did not significantly affect kinase inhibitory activity relative to the parent
methyl analogue (9a). Some, particularly more lipophilic, alkyl amides such as the
derivatives 9k and 9l did give subꢀmicromolar activity.
Table 1. GSKꢀ3β activity of 5ꢀmethoxyꢀNꢀ(piperidinꢀ4ꢀylmethyl)ꢀ1Hꢀindazoleꢀ3ꢀ
carboxamide derivatives
O
NH
R
1
N
O
N
N
H
a
Inhibitory Potencies (IC ) vs Human GSK3β
50
IC50
(ꢁM)
IC50
(ꢁM)
IC50
(ꢁM)
ID
R
1
ID
R
1
ID
R
1
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Journal of Medicinal Chemistry
Page 10 of 57
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
*
*
OH
9
a
Me
1.20
9m
9n
9o
0.25 9x
0.40 9y
0.64 9z
0.31 9aa
0.35 9ab
0.32 9ac
0.27 9ad
0.35 9ae
0.56 9af
0.51 9ag
0.36 9ah
O
0.40
O
OH
*
*
OEt
9b
0.67
0.69
0.23
0.87
1.00
1.70
1.13
0.75
1.51
0.53
0.58
O
0.33
0.07
0.21
0.05
0.13
0.95
0.49
0.23
1.40
2.10
O
OH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
*
OH
O
O
*
9
c
*
*
*
*
O
CF3
N
OH
Cl
OH
O
O
*
*
9d
9p
9q
O
N
OMe
Cl
S
O
*
O
*
9
e
CH
3
CH Ph
2
N
O
OH
*
O
S
O
NH
b
*
S
OH
9f
9r
CH3
N
O
N
OMe
O
O
S
O
*
N
9
g
9s
9t
*
OH
*
*
N
N
OEt
*
N
O
O
O
9h
*
NH
N
OH
NH2
OMe
*
O
O
9
i
9u
9v
9w
*
NH
OMe
*
O
*
*
O
N
9
j
O
N
*
*
NH
NH
NH
O
*
*
N
O
9
k
O
*
9
l
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Page 11 of 57
Journal of Medicinal Chemistry
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8
9
a
IC50 values were calculated from data points obtained as averages of duplicate wells.
Table with complete list of IC and 95% confidence intervals is showed in supplementary
information file (Table S. I. 1). Sodium salt.
50
b
Alkylaryl or alkylpyridyl analogues did show some improvements in the inhibitory
potency. For example, benzyl derivatives (9mꢀq), phenylꢀethyl derivatives (9tꢀu) and
encouragingly the more polar pyridineꢀmethyl derivatives (9rꢀs) and the pyridineꢀethyl
derivative (9v), all have IC50 values between 0.64 ꢁM and 0.25 ꢁM, despite the different
nature of the inserted substituents. Esters and carboxylic acid substituted methylene 5ꢀring
heteroaryl analogues showed significantly improved target affinity, with IC50’s of 70 and 50
nM respectively with the 2ꢀmethylenꢀ1,3ꢀoxazoleꢀ4ꢀcarboxylic acid 9z and its 1,3ꢀthiazole
counterpart 9ab. All these results are in agreement with our structural hypothesis of a section
of the binding pocket without stringent electronic and steric requirements that can tolerate a
broad series of terminal groups, while also identifying specific polar interactions with Arg141
or Arg144 as proposed for compound 9z depicted in Figure 2.
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14
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17
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23
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Docking study of compound 9z in the GSKꢀ3β ATP site
Interesting is the loss of potency observed for the 4ꢀsubstituted 1,3ꢀthiazoleꢀ2ꢀcarboxylic
ethyl ester 9ad, in comparison to its methyl ester isomer 9ac, indicating a preference for the
position of the hydrogenꢀbond acceptor heteroatoms. Alkylmorpholine substitution, such as
in 9ag, was poorly tolerated.
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Journal of Medicinal Chemistry
Page 12 of 57
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1
1
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2
2
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3
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Following the investigation of the effect of substituent’s extending from the piperidine ring
we turned our attention to the 5ꢀposition of the indazole, replacing the initial methoxy group
with bromine, alkyl, aryl or heteroaryl groups, with the aim of identifying additional Hꢀbond
or van der Waals interactions. The effect of these changes on the GSKꢀ3β activity was
studied using a range of piperidine Nꢀsubstituent’s to cover potential mismatches in the
structureꢀactivity relationship (SAR) between the two positions (Table 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Based on the results obtained with the previous 5ꢀmethoxyꢀindazoleꢀ3ꢀcarboxamide
derivatives, five different piperidineꢀnitrogen substituent’s (selected on the basis of potency
and physicochemical properties) were selected for this SAR analysis: phenethyl group (13),
methoxyethyl chain (14), 5ꢀmethylenꢀfuranꢀ2ꢀcarboxylic acid (15), 2ꢀmethylenꢀ1,3ꢀoxazoleꢀ
4ꢀcarboxylic acid (16) and acetic acid (17).
Table 2. Substituent combinations used to study the GSKꢀ3β inhibitory activity of an
indazole 5ꢀsubstitieunt ꢀ R
2
O
NH
N R
1
R2
N
N
H
a
Inhibitory Potencies for Human GSKꢀ3β; IC50 , nM (compound ID)
R
1
*
O
R
2
O
*
O
*
O
*
OMe
O
N
*
OH
OH
OH
MeO
MeO
Br
870 (9e)
400 (9x)
70 (9z)
670 (9b)
c
b
330 (9y)
210 (9aa)
d
c
n.d. (13c)
n.d. (14c)
200 (14f)
210 (15c)
b
c
Br
260 (15d)
360 (16d)
530 (17d)
Et
541 (17e)
Ph
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Page 13 of 57
Journal of Medicinal Chemistry
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5
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7
8
9
F
*
1
12 (13g)
72 (14g)
17 (15g)
12 (15i)
45 (17g)
27 (17i)
H C
3
5
80 (14h)
*
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
67 (13i)
18 (14i)
6.0 (16i)
*
Cl
Cl
*
40 (17j)
OMe
5
60 (14k)
300 (15k)
9.6 (15m)
21 (15o)
*
N
N
3
9 (13l)
31 (14l)
*
*
2
0 (13m)
45 (14m)
OMe
N
N
2
6 (14n)
3 (14o)
18 (17n)
*
*
OMe
OMe
5
MeO
*
N
1400 (14p) 360 (15p)
64 (14q)
N
CH3
4
0 (13q)
*
a
IC values were calculated from data points obtained as averages of duplicate wells.
Table with complete list of IC50 and 95% confidence intervals is showed in supplementary
information file (Table S. I. 1). Methyl ester; ethylester; sodium salt. Blank cells indicate
compounds not prepared.
5
0
b
c
d
A marked increase in inhibitory potency was obtained with indazole 5ꢀaryl or 5ꢀheteroaryl
substituent’s compared with 5ꢀmethoxy or 5ꢀbromo substituted analogues. For instance the 5ꢀ
phenyl derivative 14f in comparison with its 5ꢀmethoxy counterpart 9e showed a 4ꢀfold
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Journal of Medicinal Chemistry
Page 14 of 57
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
increase in activity (IC of 200 nM and 870 nM respectively). A further 3ꢀfold increase in
5
0
potency was noted when switching to the 2ꢀfluorophenyl derivative 14g (IC50 = 72 nM) with
further improvements in activity with the 2,3ꢀdifluorophenyl derivative 14i (IC50=18 nM).
Replacing the two fluorine atoms of 17i with two chlorine atoms (17j) gave comparable
potency (IC50 of 27 and 40 nM respectively) but no advantage to compensate for the
increased lipophilicity. In contrast 2ꢀmethyl derivative 14h or 4ꢀmethoxy phenyl derivatives
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14k and 15k, showed a marked loss in potency being comparable to the corresponding 5ꢀ
methoxy indazole derivatives.
The introduction of a (pyridineꢀ3ꢀyl) indazole 5ꢀsubstituent gave potent GSKꢀ3β inhibitors
13l and 14l, with activity comparable to the 2,3ꢀdifluorophenyl analogues 13i and 14i. The
incorporation of a methoxy group at the 4ꢀ, 5ꢀ or 6ꢀposition of the pyridine ring generally was
well tolerated providing two of the most potent kinase inhibitors (15m and 17n).
Interestingly, the methoxypyridine 14o is tenꢀfold more potent than the corresponding 4ꢀ
methoxyphenyl analogue 14k, suggesting a direct effect of the pyridine nitrogen.
Figure 3. Docking study of compound 14n in the GSKꢀ3β ATP site: (A) view of the hinge
region interactions, (B) view from the inner cavity toward the hinge region and solvent
accessible area
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Page 15 of 57
Journal of Medicinal Chemistry
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9
The significant increase in potency produced by 5ꢀ(hetero)aryl indazole derivatives, could
be rationalized through docking studies. Compound 14n, for example, (Figure 3A and 3B),
with its 5ꢀmethoxyꢀ3ꢀpyridine moiety, is able to form a new Hꢀbond interaction with Lys85
(Figure 3A) and hydrophobic interactions with the residues lining the deep part of the binding
10
11
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13
14
15
16
17
18
19
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48
49
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51
52
53
54
55
56
57
58
59
60
pocket (e.g., Cys199, Val70, Phe67) (Figure 3B). The trends in activity seen with the
indazole 5ꢀsubstituent were independent of the indazole 3ꢀsubstituent, consistent with
conserved indazoleꢀhinge region interactions regardless of the piperidine substituent proximal
to the solvent accesible area of the binding site.
Finally, 2ꢀmethoxy substitution (14p and 15p) had at least a tenꢀfold drop in potency
compared with the other methoxypyridyl regioisomers (also 14m) suggesting a steric clash
between the methoxy group and the nearby amino acid residues. No significant difference in
potency was found replacing the 6ꢀmethoxy group of 14o with a methyl group (14q).
Mode of action. Two compounds (14i and 9c) were selected to characterize in more detail
their enzymatic mode of action using microfluidic technology. Their mechanism of inhibition
was determined by performing phosphorylation assays while varying inhibitor and substrate
M
concentrations around their IC50 and Michaelis constant (K ), respectively. Results were
fitted to 3 equations for competitive, noncompetitive, and uncompetitive mechanisms. The
inhibition for both compounds was found to be reversible, linear and ATP competitive (14i
Ki=15nM; 9c Ki=600nM), consistent with the ATP site binding mode used for in silico
screening and confirmed with crystallographic studies with compound 5 .
Functional Tau phosphorylation assay. A series of the most potent GSKꢀ3β inhibitors
were also evaluated, in duplicate, for their activity in a whole cell Tau phosphorylation assay
using recombinant CHO pTau expressing cells. The cells were incubated with the selected
compounds for 2 hours at 37 °C, 5% CO lysed and processed for the detection of
2
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Journal of Medicinal Chemistry
Page 16 of 57
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1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
phosphorylated tau protein (Luminex). The comparison between IC50’s obtained in the
enzyme inhibition and cellꢀbased assays is shown in Table 3.
Table 3. Cellular Tau phosphorylation inhibition results
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
NH
R
1
N
R2
N
N
H
CHO
GSKꢀ3β
IC50
pTau
a
b
Compd
9p
R
2
R
1
IC50
cLogP
(
ꢁM)
(
ꢁM)
Cl
*
MeOꢀ
0.310
1.39
4.3
Cl
O
O
9
aa
MeOꢀ
MeOꢀ
*
*
0.210
0.130
5.17
2.13
1.7
2.0
N
N
OMe
O
S
9ac
OMe
OMe
O
9
af
4i
MeOꢀ
0.230
0.018
0.82
0.24
2.4
3.3
*
O
F
F
*
*
1
OMe
N
N
N
*
*
14n
14o
15o
0.026
0.053
0.021
0.80
0.79
70.8
2.0
2.6
OMe
OMe
*
*
*
OMe
OMe
OMe
*
O
0.34
O
OH
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Page 17 of 57
Journal of Medicinal Chemistry
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7
8
9
F
O
F
*
*
O
1
6i
0.006
>100
0.43
N
OH
a
IC50 values were calculated from data points obtained as averages of duplicate wells.
Table with complete list of IC50 and 95% confidence intervals is showed in supplementary
information file (Table S. I. 3). cLogP calculated by ACD/Percepta.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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59
60
b
Selected compounds showed potent inhibitory effect in the Tau protein phosphorylation
cellular assay with IC50 in the low micromolar range reflecting a loss in potency of 10 – 40
fold between the isolated enzyme and whole cell pTau assays. Compounds 15o and 16i
showed a major loss of activity probably due to their zwitterionic nature (both cLogP < 0.5;
cLogD7.4 respectively of ꢀ0.08 and ꢀ0.01) that limits their membrane permeability.
Pharmacokinetic characterization. Compounds 9ac, 9af, 14i and 14n were assessed for
mouse plasma stability at the concentration of 5 µM. We measured the time related
percentage of remaining compound and noticed that ester 9ac was rapidly hydrolyzed to the
corresponding carboxylic acid, with elimination halfꢀlife less than 10 minutes. Other
compounds including the vinylogous carbonate 9af were stable, with percent of remaining
compounds > 80% after 2h.
On the basis of their plasma stability compounds 9af, 14i and 14n were progressed to in
vivo PK studies. Plasma and brain levels were evaluated after intraperitoneal (i.p.)
administration of 10 mg/kg of compounds. The PK parameters obtained for the selected
compounds are shown in table 4.
Table 4. In vivo PK parameters in mice (10 mg/kg, ip)
Plasma
Brain
Entry
AUC0,inf
Cmax
T
max
t
1/2
Vd
Cl
Cmax
(
ꢁg*min/ml) (ꢁg/ml)
(L/min/kg)
(ng/mg prot.)
(
min)
(min)
68±13
203±53
(L/kg)
47±9
9
af
4i
21±4
0.7±0.1
2.1±0.7
15
0.5±0.1
1.5
5.5
±
±
0.1
0.4
1
370±96
15
6
±2
0.02±0.01
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Journal of Medicinal Chemistry
Page 18 of 57
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
4n
34±11
1.3±0.2
15
48±15
20±6
0.3±0.1
0.7±0.5
Among the tested compounds, 14i displayed the highest exposures, both in plasma and in
brain tissue, reaching the maximum concentration in plasma in 15 minutes with a favorable
elimination halfꢀlife (t1/2) of 203 min and moderate clearance (Cl) of 22 ml/min/kg.
Consistent with its good PK parameters, 14i was selected for evaluation in in vivo efficacy
model of mood disorders.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Amphetamine model. This model is believed to mimic the hyperactivity component of
bipolar disorders. Both reference GSKꢀ3β inhibitors tested, i.e. LiCl and TDZDꢀ8 given
intraperitonelly, were very effective in blocking amphetamine hyperactivity with LiCl being
slightly more active than TDZDꢀ8 (Figure 4A and 4B). At the highest dose tested both
compounds also showed a trend to inhibit spontaneous locomotor activity, suggesting the
appearance of some sedative effects. Compound 14i was tested in the range of doses between
1
and 30 mg/kg and a doseꢀdependent inhibition of amphetamine induced hypermotility was
observed (Figure 4C). The first significant dose was 1 mg/kg and the effect was maximal at 3
and 10 mg/kg. The efficacy of 14i at 10 mg/kg was similar to lithium at the highest non
sedative dose and higher than TDZDꢀ8 (compare Figures 4A, B and C). Also 14i, when dosed
at 30 mg/kg, showed some sedative activity on spontaneous locomotor activity.
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Page 19 of 57
Journal of Medicinal Chemistry
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51
52
53
54
55
56
57
58
59
60
Figure 4. Effect of Lithium Chloride (A), TDZDꢀ8 (B) and 14i (C) on spontaneous motility
(
saline) and on amphetamine hyperactivity (amphetamine). Each bar is the average ± S.E.M.
n = 8 each group). °°° p<0.001 vs saline; * p<0.05, ** p<0.01, *** p<0.001 vs
(
amphetamine. 2 way ANOVA, Bonferroni post test.
Kinases selectivity assays. The kinase profile of 14i was firstly assessed in a 216 kinase
assay platform performed by CEREP, at 10 µM in duplicate. Briefly, human recombinant
kinases were incubated in the presence of specific peptide substrates plus ATP for different
times at 22 °C. The results are expressed as a percent of inhibition of control specific activity
obtained in the presence of the test compounds (See supplementary information Table S. I. 3,
for the complete list of results). Only for 34 kinases 14i showed more than 50% inhibition at
the screening concentration. For these selected kinases the IC50 determination was performed
(See supplementary information Table S. I. 4, for the complete list of results).
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Page 20 of 57
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
These experiments showed that compound 14i has a IC50 selectivity ratio versus GSKꢀ3β,
below 30 fold, only for seven additional human recombinant kinases: DYRK1a and DYRK2,
CLK1, LynB Kinase, ERK5, GRK2 (ADRBK1) and GSKꢀ3α (the isoform of GSKꢀ3β).
respectively. (Table 5).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. GSK3 kinase selectivity of compound 14i over many other kinases
IC50
Selectivity ratio
Assay
(
µM)
0.018
0.040
0.040
0.071
0.170
0.220
0.530
0.530
(vs GSKꢀ3β)
hꢀGSKꢀ3β
hꢀDYRK1a
1
2.2
hꢀGSKꢀ3α
2.2
hꢀCLK1
3.9
hꢀLynB Kinase
hꢀERK5
9.4
12.2
29.4
29.4
hꢀDYRK2 kinase
hꢀGRK2 (ADRBK1)
Based on available literature, none of the above seven kinases have been specifically linked
to mood disorders and they are not expected to affect 14i efficacy in major depression or
bipolar disorder. In fact, DYRK1A and DYRK2, have been linked to cell proliferation and
23,24
neurocognitive deficits associated to Down syndrome
while ERK5 has been also linked to
29
cell proliferation, differentiation, and death. CLK1 has been involved in neurodegeneration
2
5,26
and Parkinsonism.
Lyn kinase participates to the regulation of myeloid lineage
27,28
proliferation,
and finally, GRK2, a member of the G proteinꢀcoupled receptor kinases
(GRKs) family, participates, together with arrestins, to the regulation of G proteinꢀcoupled
30
receptors (GPCR).
CONCLUSION
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The hit to lead program described in this work for the novel class of 5ꢀsubstitutedꢀNꢀ
piperidinꢀ4ꢀylmethyl)ꢀ1Hꢀindazoleꢀ3ꢀcarboxamides identified a series of novel and potent
(
GSKꢀ3β inhibitors. Key interventions leading to an increase GSKꢀ3β inhibition potency were
the identification of additional interactions with the target from both the indazole 3ꢀposition
(e.g. Arg141) and from the indazole 5ꢀposition (probing to Lys85) while improving
hydrophobic interactions. Activity against the isolated enzyme translated well to cells, which
is indicative of good permeability. Using two compounds (9c, 14i), we demonstrated that
their mechanism of action was ATPꢀcompetitive and reversible, consistent with the binding
hypothesis studied in silico and results of Xꢀray coꢀcrystallization studies. Some indazoleꢀ3ꢀ
carboxamide derivatives showed the necessary cellular potency and plasma stability to be
progressed to an in vivo assessment. Compound 14i, showing good preliminary
pharmacokinetic profiles after i.p. administration was tested in an in vivo efficacy animal
model: the low dose amphetamine test, believed to mimic mania, a key symptom of mood
disorders. In this model, the selected compound showed excellent efficacy and maximal
effect (i.e. complete reversal of amphetamine hypermotility). Some sedative effects, at the
highest doses tested, were noticed with the positive controls (TDZDꢀ8 and LiCl) and with
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14i. As known selectivity versus other kinases is a critical factor for the potential clinic uses
of kinase inhibitors. To address this point compound 14i was tested on a panel of 216 kinases
and we found potency ratio below 30 fold only on six assays (in addition to the expected
isoform GSKꢀ3α) indicating an excellent degree of kinase selectivity.
In summary, this paper describes the synthesis and the initial pharmacological
characterization of a novel class of GSKꢀ3β inhibitors. The encouraging results of our work,
suggest that the present class of selective GSKꢀ3β inhibitor may lead to molecules providing
a significant improvement over existing mood disorders therapy.
MATERIALS AND METHODS.
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Chemistry. Reagents were purchased from SigmaꢀAldrich and were used as received.
Reaction progress was monitored by TLC or UPLCꢀqtof chromatography. For TLC was used
Merck silica gel 60 F254 (0.04ꢀ0.063 mm) with detection by UV (214 or 254 nm). Merck
silica gel 60 or aluminum oxide 90 (active neutral) were used for column chromatography.
Melting points (uncorrected) were determined in open Pyrex capillary tubes using a Buchi
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9
0
5
10 melting point apparatus. Final compound purity, always >95%, was determined both by
high pressure liquid chromatography (HPLC) and Ultra Performance Liquid Chromatography
UPLC), using the area percentage of all peaks detected. HPLC analysis were carried out with
(
a pump/autosampler Waters (2695 – Alliance model), a UV photo diode array detector
Waters (2996 model) and a Waters system data management (Empower 2). The column used
was generally Suplex pkbꢀ100 (250x4.6 mm, 5ꢁm). UPLC/QToF Cromatography (exact
mass data) were obtained by means of a SYNAPT MS ꢀ ACQUITY UPLC system, Waters.
The system was operated in positive ion mode in the “VꢀOptics” configuration. Leucineꢀ
enkephalin (200 pg/µl) was employed as the lock mass in order to provide authenticated
exact mass measurement in MS and MS/MS modes within 5 ppm RMS mass accuracy. The
column was an Acquity BEH C18 (2.1x50 mm, 1.7ꢁm). Nuclear Magnetic Resonance
1
Spectroscopy ( HꢀNMR) were obtained using a Bruker Avance system, operating at 300
1
MHz. All resonance bands were referenced to tetramethylsilane (internal standard). For Hꢀ
NMR spectroscopy: (s) = singlet; (d) = doublet; (t) = triplet; (br) = broad; (dd) = double
doublet; (dt) = double triplet; (ddd) = double double doublet ;(dtd) = double triple doublet;
(m) = multiplet; J = coupling constant; δ = chemical shift (in ppm). Preparative HPLC/MS
system consisted of a Waters 2767 Sample manager, a Waters 2478 dual λ absorbance
detector and a Waters Micromass ZQ single quadrupole mass spectrometer with an
electrospray ionization (ESI) source. The column used was a XꢀBridge Prep C18 5 ꢁm with
19x10mm (Waters) preꢀcolumn. Fraction collection was available from the system software
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TM
MassLynx v. 4.1. Detection wavelength was set to 230 nm and temperature to 25°C.
Elemental Analysis was conducted by means of a CHNSꢀO EA1108 elemental analyzer,
Carlo Erba Instruments, and the results were within +0.3% of the theoretical values, unless
otherwise noted. Compounds were purified with one of the following techniques: flash
chromatography on silica gel (Grace Reveleris flash chromatography system with 40 ꢁM
silica cartridge (generally flow was 60 ml/min); with an appropriate gradient of mixtures of
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60
CHCl
mg/ml) in DMSO/CH
CH CN + 0.1% formic acid and H O + 0.1% formic acid; flow = 40 ml/min). The starting
3
and CH
3
OH as eluents); preparative HPLC/MS system (sample was dissolved (50
3
CN in 1:1 ratio; using an appropriate gradient of the two phases
3
2
material 1ꢀ(1ꢀmethylpiperidinꢀ4ꢀyl)methanamine (6a), ethyl 3ꢀ[4ꢀ(aminomethyl)piperidinꢀ1ꢀ
yl]propanoate (6c), Nꢀ{2ꢀ[4ꢀ(aminomethyl)piperidinꢀ1ꢀyl]ethyl}methanesulfonamide (6f), 3ꢀ
[
4ꢀ(aminomethyl)piperidinꢀ1ꢀyl]ꢀN,Nꢀdimethylpropanꢀ1ꢀamine (6g), 1ꢀ{1ꢀ[2ꢀ(pyridinꢀ4ꢀ
yl)ethyl]piperidinꢀ4ꢀyl}methanamine (6v), tertꢀbutyl 4ꢀ(aminomethyl)piperidineꢀ1ꢀ
carboxylate (6z) were purchased from commercial sources.
General method for the preparation of intermediates 7, 9a,c,fꢀg,nꢀq,uꢀv
ꢀHydroxybenzotriazole (HOBt, 7.40 g, 54.8 mmoles) and N,N’ꢀdicyclohexylcarbodiimide
DCC, 11 g, 53.3 mmoles) were added to a solution of an appropriately substituted 1Hꢀ
1
(
indazoleꢀ3ꢀcarboxylic acid 10 (49.8 mmoles) in DMF (200 ml) at 0°C. After 1 hour, a
solution of an appropriate 1ꢀsubstituted [piperidinꢀ4ꢀyl]methanamine (6a,c,fꢀg,oꢀq,uꢀz) (58.1
mmoles) in DMF (100 ml) was added at the same temperature. The mixture was stirred at 0
°
C for 2 hours then it was left to reach room temperature overnight. The mixture was diluted
with AcOEt then the solid was removed by filtration. The solution was extracted three times
with hydrochloric acid (HCl) 2N. The pH of the acid phase was increased (about 13) with 5N
NaOH and solution was extracted three times with dichloromethane (DCM). The organic
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6
phase was dried with anhydrous Na
2 4
SO . The solution was filtered, evaporated under reduced
pressure and the residue was adequately purified.
5ꢀMethoxyꢀ
N
ꢀ[(1ꢀmethylpiperidinꢀ4ꢀyl)methyl]ꢀ1
H
ꢀindazoleꢀ3ꢀcarboxamide
(9a)
1
hydrochloride. (61%) H NMR (300MHz, DMSOꢀd
6
) δ = 13.55 (s, 1H), 10.27 (br. s., 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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5
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7
8
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0
1
2
3
4
5
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8
9
0
8.47 (t, J=5.8 Hz, 1H), 7.56 (d, J=2.4 Hz, 1H), 7.53 (dd, J=0.5, 9.0 Hz, 1H), 7.06 (dd, J=2.5,
9.1 Hz, 1H), 3.81 (s, 3H), 3.38 (d, J=11.0 Hz, 2H), 3.32 (s, 2H), 2.88 (t, J=11.5 Hz, 2H), 2.69
+
(s, 3H), 2.18 ꢀ 1.72 (m, 3H), 1.70 ꢀ 1.26 (m, 2H). [M+H ] exact mass found 303.1816 for
16 23 4 2
C H N O .
[4ꢀ({[(5ꢀMethoxyꢀ1
acid (9c) hydrochloride. Compound 9c was obtained by hydrolysis of the crude ethyl [4ꢀ
{[(5ꢀmethoxyꢀ1Hꢀindazolꢀ3ꢀyl)ꢀcarbonyl]amino}methyl)piperidinꢀ1ꢀyl]propionate during
the reaction alkalyne workꢀup. (42%) HꢀNMR spectra of the HCl salt. H NMR (300MHz,
DMSOꢀd ) δ = 8.24 (t, J=6.0 Hz, 1H), 7.55 (s, 1H), 7.53 (dd, J=0.5, 7.1 Hz, 1H), 7.03 (dd,
J=2.6, 8.7 Hz, 1H), 3.81 (s, 3H), 3.18 (t, J=6.3 Hz, 2H), 2.83 (d, J=11.3 Hz, 2H), 2.49 – 2.43
Hꢀindazolꢀ3ꢀyl)ꢀcarbonyl]amino}methyl)piperidinꢀ1ꢀyl]propionic
(
1
1
6
(m, 2H), 2.17 – 2.05 (m, 2H), 1.89 (d, J=12.6 Hz, 2H), 1.76 – 1.46 (m, 4H), 1.30 – 1.10 (m,
+
3
H). [M+H ] exact mass found 361.1870 for C18H25N4O4.
5ꢀMethoxyꢀ
N
ꢀ[(1ꢀ{2ꢀ[(methylsulfonyl)amino]ethyl}piperidinꢀ4ꢀyl)methyl]ꢀ1Hꢀ
1
indazoleꢀ3ꢀcarboxamide (9f). (46%) H NMR (300MHz, DMSOꢀd ) δ = 13.43 (br. s., 1H),
6
8
9
2
.27 (t, J=6.1 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.51 (dd, J=0.5, 9.0 Hz, 1H), 7.06 (dd, J=2.5,
.1 Hz, 1H), 6.81 (t, J=6.2 Hz, 1H), 3.81 (s, 3H), 3.19 (t, J=6.2 Hz, 2H), 3.04 (q, J=5.1 Hz,
H), 2.93 (s, 3H), 2.85 (d, J=11.3 Hz, 2H), 2.38 (t, J=6.8 Hz, 2H), 1.91 (t, J=10.6 Hz, 2H),
+
1.72 – 1.45 (m, 3H), 1.34 – 1.04 (m, 2H). [M+H ] exact mass found 410.1856 for
C H N O S.
18
28
5
4
N
ꢀ({1ꢀ[3ꢀ(dimethylamino)propyl]piperidinꢀ4ꢀyl}methyl)ꢀ5ꢀmethoxyꢀ1
H
ꢀindazoleꢀ3ꢀ
1
carboxamide (9g) diꢀhydrochloride. (67%) H NMR (300MHz, DMSOꢀd
6
) δ = 8.62 (t, J =
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5.9 Hz, 1 H), 8.0 (br. s., 5 H), 7.56 (d, J = 2.6 Hz, 1 H), 7.49 (d, J = 8.9 Hz, 1 H), 7.06 (dd, J
=
2.4, 9.1 Hz, 1 H), 3.80 (s, 3 H), 3.55 ꢀ 3.34 (m, 4 H), 3.16 ꢀ 3.02 (m, 4 H), 2.75 (s, 6 H),
2.15 (quin, J = 7.5 Hz, 2 H), 2.04 ꢀ 1.81 (m, 3 H), 1.61 (dtd, J = 4.3, 12.2, 12.4 Hz, 2 H).
+
[
M+H ] exact mass found 374.2550 for C H N O .
20
32
5
2
10
11
12
13
14
15
16
17
18
19
20
21
22
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60
5ꢀMethoxyꢀ
N
ꢀ({1ꢀ[4ꢀ(trifluoromethyl)benzyl]piperidinꢀ4ꢀyl}methyl)ꢀ1Hꢀindazoleꢀ3ꢀ
1
carboxamide (9o). (53%) H NMR (300MHz, DMSOꢀd
6
) δ = 13.40 (s, 1H), 8.27 (t, J = 6.10
Hz, 1H), 7.67 (d, J = 8.01 Hz, 2H), 7.47ꢀ7.57 (m, 4H), 7.05 (dd, J = 2.44, 9.06 Hz, 1H), 3.80
s, 3H), 3.53 (s, 2H), 3.20 (t, J = 6.27 Hz, 2H), 2.78 (d, J = 11.50 Hz, 2H), 1.94 (t, J = 10.80
Hz, 2H), 1.47ꢀ1.77 (m, 3H), 1.12ꢀ1.36 (m, 2H). HRMS, QꢀTOF found for C H F N O :
(
23 26
3
4
2
447.2003.
ꢀ{[1ꢀ(2,4ꢀDichlorobenzyl)piperidinꢀ4ꢀyl]methyl}ꢀ5ꢀmethoxyꢀ1
N
H
ꢀindazoleꢀ3ꢀ
1
carboxamide (9p). (Yield 68%) H NMR (DMSOꢀd
6
) δ 13.38 (br. s., 1H), 8.28 (t, J = 6.22
Hz, 1H), 7.56 (t, J = 2.38 Hz, 2H), 7.52 (d, J = 2.56 Hz, 1H), 7.50 (d, J = 1.83 Hz, 1H), 7.41
(dd, J = 2.20, 8.20 Hz, 1H), 7.06 (dd, J = 2.56 and 9.15 Hz, 1H), 3.81 (s, 3H), 3.51 (s, 2H),
3
.21 (t, J = 6.22 Hz, 2H), 2.80 (d, J = 11.34 Hz, 2H), 2.01 (t, J = 10.79 Hz, 2H), 1.48ꢀ1.78
m, 3H), 1.07ꢀ1.35 ppm (m, 2H). HRMS, QꢀTOF found C22 : 447.1357.
ꢀindazoleꢀ3ꢀ
(
2 4 2
H25Cl N O
N
ꢀ({1ꢀ[4ꢀ(Benzyloxy)benzyl]piperidinꢀ4ꢀyl}methyl)ꢀ5ꢀmethoxyꢀ1H
1
carboxamide (9q). (Yield 77%) H NMR (DMSOꢀd
6
) δ 13.44 (br. s., 1H), 8.25 (t, J = 6.22
Hz, 1H), 7.55 (d, J = 1.83 Hz, 1H), 7.50 (d, J = 9.15 Hz, 1H), 7.28ꢀ7.46 (m, 5H), 7.14ꢀ7.21
m, 2H), 7.04 (dd, J = 2.38 and 8.96 Hz, 1H), 6.88ꢀ6.98 (m, 2H), 5.07 (s, 2H), 3.79 (s, 3H),
(
3
.35 (s, 2H), 3.18 (t, J = 6.22 Hz, 2H), 2.77 (d, J = 11.34 Hz, 2H), 1.85 (t, J = 10.79 Hz, 2H),
.45ꢀ1.71 (m, 3H), 1.00ꢀ1.32 ppm (m, 2H). HRMS, QꢀTOF found for C29 : 485.2548.
ꢀ{[1ꢀ(4ꢀHydroxybenzyl)piperidinꢀ4ꢀyl]methyl}ꢀ5ꢀmethoxyꢀ1 ꢀindazoleꢀ3ꢀ
1
33 4 3
H N O
N
H
carboxamide (9n). Compound 9q (0.6 mmol) was dissolved in THF and hydrogenated in a
micro reactor continuous flow system (HꢀCube) using CartCart Pd/C 10% as cartridge. Key
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6
parameters of HꢀCube were set as follows: temperature 80 °C; pressure 1 bar; flow 1
ml/minute. The solvent was removed by evaporating under reduced pressure, and the
1
compound 9n was purified by preparative HPLCꢀMS. (Yield 47%) H NMR (DMSOꢀd
6
) δ
1
3.50 (s, 1H), 9.70 (br. s., 1H), 8.35 (d, J = 17.17 Hz, 1H), 7.55 (d, J = 2.31 Hz, 1H), 7.51 (d,
J = 8.92 Hz, 1H), 7.27 (br. s., 2H), 7.05 (dd, J = 2.31 and 8.92 Hz, 1H), 6.78 (d, J = 7.60 Hz,
H), 3.80 (s, 3H), 2.60ꢀ3.59 (m, 8H), 0.99ꢀ2.15 ppm (m, 5H). HRMS, QꢀTOF found for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
C
22
H
27
N
4
O
3
: 395.2083.
5ꢀMethoxyꢀ
N
ꢀ({1ꢀ[2ꢀ(4ꢀmethoxyphenyl)ethyl]piperidinꢀ4ꢀyl}methyl)ꢀ1Hꢀindazoleꢀ3ꢀ
1
carboxamide (9u). (Yield 55%) H NMR (DMSOꢀd ) δ 13.62 (br. s., 1H), 8.24 (t, J = 6.06
6
Hz, 1H), 7.55 (d, J = 2.42 Hz, 1H), 7.52 (dd, J = 0.61 and 8.88 Hz, 1H), 7.07 – 7.16 (m, 2H),
7
.03 (dd, J = 2.42 and 8.88 Hz, 1H), 6.75 – 6.87 (m, 2H), 3.80 (s, 3H), 3.71 (s, 3H), 3.19 (t, J
6.26 Hz, 2H), 2.90 (d, J = 11.10 Hz, 2H), 2.57 – 2.74 (m, 2H), 2.34 – 2.47 (m, 2H), 1.80 –
.98 (m, 2H), 1.46 – 1.76 (m, 3H), 1.08 – 1.30 ppm (m, 2H). HRMS, QꢀTOF found for
C H N O : 423.2402.
=
1
24
31
4
3
5ꢀMethoxyꢀ
N
ꢀ({1ꢀ[2ꢀ(pyridineꢀ4ꢀyl)ethyl]piperidinꢀ4ꢀyl}methyl)ꢀ1Hꢀindazoleꢀ3ꢀ
1
carboxamide (9v). (Yield 39%) H NMR (DMSOꢀd
6
) δ 13.43 (br. s., 1H), 8.51 ꢀ 8.36 (m,
2
H), 8.26 (t, J = 6.2 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.51 (d, J = 9.1 Hz, 1H), 7.29 ꢀ 7.21
m, 2H), 7.06 (dd, J = 2.6 and 9.1 Hz, 1H), 3.81 (s, 3H), 3.19 (t, J = 6.4 Hz, 2H), 2.90 (d, J =
11.3 Hz, 2H), 2.80 ꢀ 2.67 (m, 2H), 2.6 – 2.4 (m, 2H), 2.02 ꢀ 1.82 (m, 2H), 1.72 ꢀ 1.48 (m,
H), 1.32 ꢀ 1.03 ppm (m, 2H). HRMS, QꢀTOF found for C H N O : 394.2238.
(
3
22
28
5
2
Tertꢀbutyl
4ꢀ({[(5ꢀmethoxyꢀ1Hꢀindazolꢀ3ꢀyl)carbonyl]amino}methyl)piperidineꢀ1ꢀ
1
carboxylate (7a). (Yield 96%) H NMR (DMSOꢀd ) δ 13.47 (br. s., 1H), 8.40 (t, J = 6.1 Hz,
6
1H), 7.55 (d, J = 2.6 Hz, 1H), 7.52 (d, J = 9.2 Hz, 1H), 7.05 (dd, J = 2.6 and 8.9 Hz, 1H),
3.99 ꢀ 3.87 (m, 2H), 3.80 (s, 3H), 3.17 (t, J = 6.4 Hz, 2H), 2.78 ꢀ 2.60 (m, 2H), 1.81 ꢀ 1.61 (m,
29 4 4
3H), 1.39 (s, 9H), 1.14 ꢀ 0.92 ppm (m, 2H). HRMS, QꢀTOF found for C20H N O : 389.2183.
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9
Tertꢀbutyl
4ꢀ({[(5ꢀbromoꢀ1Hꢀindazolꢀ3ꢀyl)carbonyl]amino}methyl)piperidineꢀ1ꢀ
1
carboxylate (7b). (Yield 87%) H NMR (DMSOꢀd ) δ 13.76 (br. s., 1H), 8.49 (t, J = 6.2 Hz,
6
1
H), 8.32 (dd, J = 0.8 and 1.9 Hz, 1H), 7.61 (dd, J = 0.8 and 8.9 Hz, 1H), 7.53 (dd, J = 1.9
and 8.9 Hz, 1H), 3.92 (d, J = 12.7 Hz, 2H), 3.20 (t, J = 6.4 Hz, 2H), 2.79 ꢀ 2.59 (m, 2H), 1.93
4 3
ꢀ 1.56 (m, 3H), 1.39 (s, 9H), 1.11 ꢀ 0.95 (m, 2H). HRMS, QꢀTOF found for C19 26BrN O :
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
4
37.1182.
ꢀMethoxyꢀ
M HCl in Et
5
N
ꢀ(piperidinꢀ4ꢀylmethyl)ꢀ1
Hꢀindazoleꢀ3ꢀcarboxamide hydrochloride (8a).
2
2
O (1.8 L) was added to a solution of compound 7a (92.8 g, 0.24 moles) in
MeOH (500 mL). The mixture was stirred for 3 hours at room temperature then the resulting
solid was filtered and dried to give 5ꢀmethoxyꢀNꢀ(piperidinꢀ4ꢀylmethyl)ꢀ1Hꢀindazoleꢀ3ꢀ
1
carboxamide hydrochloride (8a) (61.1 g, 89% yield). H NMR (DMSOꢀd ) δ 13.43 (br. s.,
6
1
H), 8.27 (t, J = 6.13 Hz, 1H), 7.56 (d, J = 2.01 Hz, 1H), 7.51 (dd, J = 0.55, 8.96 Hz, 1H),
7.06 (dd, J = 2.47, 9.06 Hz, 1H), 6.81 (br. s., 1H), 3.81 (s, 3H), 3.19 (t, J = 6.22 Hz, 2H),
.85 (d, J = 11.34 Hz, 2H), 1.91 (t, J = 10.61 Hz, 2H), 1.45 – 1.72 (m, 3H), 1.04 – 1.34 ppm
m, 2H). HRMS, QꢀTOF found for C15 : 289.1648.
ꢀindazoleꢀ3ꢀcarboxamide hydrochloride (8b).
) δ 14.00 (br. s., 1H), 9.12 (br. s., 1H), 8.99 ꢀ 8.69 (m, 1H),
2
(
21 4 2
H N O
5ꢀBromoꢀ
N
ꢀ(piperidinꢀ4ꢀylmethyl)ꢀ1
H
1
(Yield 76%) H NMR (DMSOꢀd
6
8
.60 (t, J = 6.2 Hz, 1H), 8.32 (d, J = 2.2 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.53 (dd, J = 2.2
and 8.8 Hz, 1H), 3.23 (t, J = 6.4 Hz, 4H), 2.93 ꢀ 2.69 (m, 2H), 1.98 ꢀ 1.73 (m, 3H), 1.56 ꢀ 1.32
ppm (m, 2H). HRMS, QꢀTOF found for C15 : 381.0557.
4 3
H18BrN O
General method for the preparation of compounds 9 and 20.
A mixture of compound 8a or 8b (5.4 mmol) and potassium carbonate (16.6 mmol) in
DMF (45 mL) was stirred for 1 hour at 80 °C. A solution of the proper alkyl halide (8 mmol)
in DMF (5 mL) was added dropꢀwise. After 3 hours at 70 °C the reaction mixture was cooled,
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Journal of Medicinal Chemistry
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
diluted with water and extracted with EtOAc. The collected organic phases were dried over
2 4
Na SO , concentrated under vacuum and then properly purified.
Ethyl [4ꢀ({[(5ꢀmethoxyꢀ1
H
ꢀindazolꢀ3ꢀyl)carbonyl]amino}methyl)piperidinꢀ1ꢀyl]acetate
) δ 13.47 (br. s., 1 H), 8.36 (t, J = 5.9 Hz, 1H), 7.57 ꢀ
1
(
20b). (Yield 42%) H NMR (DMSOꢀd
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.49 (m, 2H), 7.07 (dd, J = 2.6 and 8.9 Hz, 1H), 4.07 (q, J = 7.0 Hz, 2H), 3.80 (s, 3H), 3.29 ꢀ
.12 (m, 4H), 2.88 ꢀ 2.76 (m, 2H), 2.19 ꢀ 2.05 (m, 2H), 1.69 ꢀ 1.48 (m, 3H), 1.34 ꢀ 1.07 ppm
m, 5H). HRMS, QꢀTOF found for C19 : 375.2027.
Ethyl 4ꢀ[4ꢀ({[(5ꢀmethoxyꢀ1 ꢀindazolꢀ3ꢀyl)carbonyl]amino}methyl)piperidinꢀ1ꢀ
yl]butanoate hydrochloride (20d). (Yield 63%) H NMR (DMSOꢀd ) δ 8.12 (br. s., 1H),
3
(
27 4 4
H N O
H
1
6
7
.71 ꢀ 7.43 (m, 2H), 7.06 (d, J = 8.6 Hz, 1H), 6.98 ꢀ 5.82 (m, 2H), 4.05 (q, J = 7.3 Hz, 2H),
.81 (s, 3H), 3.25 (br. s., 2H), 3.04 (d, J = 10.6 Hz, 2H), 2.55 (t, J = 6.9 Hz, 2H), 2.40 ꢀ 2.11
3
(
m, 4H), 2.00 ꢀ 1.53 (m, 5H), 1.48 ꢀ 1.26 (m, 2H), 1.17 ppm (t, J = 7.1 Hz, 3H). HRMS, Qꢀ
TOF found for C21 : 403.233982.
5ꢀMethoxyꢀ
31 4 4
H N O
N
ꢀ{[1ꢀ(2ꢀmethoxyethyl)ꢀpiperidinꢀ4ꢀyl]methyl}ꢀ1Hꢀindazoleꢀ3ꢀ
1
carboxamide (9e). (Yield 38%) H NMR (DMSOꢀd
6
); δ 13.41 (br. s., 1H), 8.25 (t, J = 6.07
Hz, 1H), 7.56 (d, J = 2.50 Hz, 1H), 7.51 (d, J = 9.06 Hz, 1H), 7.06 (dd, J = 2.50 and 9.05 Hz,
H), 3.81 (s, 3H), 3.41 (t, J = 5.97 Hz, 2H), 3.23 (s, 3H), 3.19 (t, J = 6.26 Hz, 2H), 2.85 (d, J
= 11.56 Hz, 2H), 2.43 (t, J = 5.97 Hz, 2H), 1.79 – 2.06 (m, 2H), 1.48 – 1.73 (m, 3H), 0.99 –
1
1
.39 ppm (m, 2H). HRMS, QꢀTOF found for C H N O : 347.2080.
18
27
4
3
N
ꢀ[(1ꢀBenzylpiperidinꢀ4ꢀyl)methyl]ꢀ5ꢀmethoxyꢀ1
H
ꢀindazoleꢀ3ꢀcarboxamide
(9m).
1
(
Yield 43%) H NMR (DMSOꢀd ) δ 13.39 (br. s., 1H), 8.25 (t, J = 6.0 Hz, 1H), 7.56 (d, J =
6
2.0 Hz, 1H), 7.50 (dd, J = 0.5 and 9.0 Hz, 1H), 7.36 ꢀ 7.17 (m, 5H), 7.05 (dd, J = 2.5 and 9.1
Hz, 1H), 3.80 (s, 3H), 3.43 (s, 2H), 3.20 (t, J = 6.1 Hz, 2H), 2.79 (d, J = 11.2 Hz, 2H), 1.89 (t,
J = 10.6 Hz, 2H), 1.74 ꢀ 1.46 (m, 3H), 1.34 ꢀ 1.07 ppm (m, 2H). HRMS, QꢀTOF found for
22 27 4 2
C H N O : 379.2124.
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
N
ꢀ({1ꢀ[2ꢀ(4ꢀHydroxyphenyl)ethyl]ꢀpiperidinꢀ4ꢀyl}methyl)ꢀ5ꢀmethoxyꢀ1Hꢀindazoleꢀ3ꢀ
1
carboxamide (9t). (Yield 41%) H NMR (DMSOꢀd ); δ 13.40 (br. s., 1H), 9.07 (s, 1H), 8.26
6
(
t, J = 6.16 Hz, 1H), 7.55 (d, J = 2.42 Hz, 1H), 7.50 (dd, J = 0.61 and 9.08 Hz, 1H), 7.05 (dd,
J = 2.50 and 9.10 Hz, 1H), 6.94 ꢀ 7.02 (m, 2H), 6.60 ꢀ 6.68 (m, 2H), 3.80 (s, 3H), 3.19 (t, J =
6.36 Hz, 2H), 2.89 (d, J = 11.30 Hz, 2H), 2.53 ꢀ 2.65 (m, 2H), 2.30 ꢀ 2.46 (m, 2H), 1.79 ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
.00 (m, 2H), 1.47 ꢀ 1.74 (m, 3H), 1.06 ꢀ 1.31 ppm (m, 2H). HRMS, QꢀTOF found for
C
23
H
29
N
4
O
3
: 409.2226.
5ꢀMethoxyꢀ ꢀ({1ꢀ[2ꢀ(3ꢀmethylcyclohexyl)ethyl]piperidinꢀ4ꢀyl}methyl)ꢀ1Hꢀindazoleꢀ3ꢀ
N
carboxamide (9w). A solution of compound 8a (1.46 mmol) in DMF (45 ml) and
triethylamine (0.61 ml, 4.4 mmol) was stirred at 80 °C for 1h and then was treated with 1ꢀ(2ꢀ
bromoethyl)ꢀ3ꢀmethylcyclohexane (300 mg, 1.46 mmol). The mixture was stirred overnight
at the same temperature. The reaction was then cooled to room temperature and the solvent
was removed by evaporation at reduced pressure. The residue was poured in water and
extracted with AcOEt. The organic phases were dried with Na SO then evaporated under
2
4
reduced pressure. 5ꢀMethoxyꢀNꢀ({1ꢀ[2ꢀ(3ꢀmethylcyclohexyl)ethyl]piperidinꢀ4ꢀyl}methyl)ꢀ
1
Hꢀindazoleꢀ3ꢀcarboxamide was purified by flash chromatography using a mixture of
1
CHCl
3
/MeOH (9:1) as eluent. (Yield = 45 mg, 18.0 %). H NMR (DMSOꢀd
6
) δ 13.41 (s, 1H),
8.30ꢀ8.20 (t, J = 6.11 Hz, 1H), 7.58ꢀ7.53 (d, J = 2.31 Hz, 1H), 7.53ꢀ7.47 (d, J = 8.59 Hz, 1H),
7
.08ꢀ7.02 (dd, J = 8.92 and 2.32 Hz, 1H), 3.80 (s, 3H), 3.23ꢀ3.13 (t, J = 6.28 Hz, 2H), 2.90ꢀ
.78 (d, J = 10.57 Hz, 2H), 2.35ꢀ2.20 (m, 2H), 1.97ꢀ1.05 (m, 17H), 0.90ꢀ0.45 ppm (m, 5H).
2
HRMS, QꢀTOF found for C24
37 4 2
H N O : 413.2910.
Ethyl 5ꢀ{[4ꢀ({[(5ꢀmethoxyꢀ1
H
ꢀindazolꢀ3ꢀyl)carbonyl]amino}methyl)piperidinꢀ1ꢀyl]
1
methyl}furanꢀ2ꢀcarboxylate (9y). (Yield 71%.) H NMR (DMSOꢀd
6
) δ 13.37 (br. s., 1H),
8
.25 (t, J = 6.04 Hz, 1H), 7.42ꢀ7.60 (m, 2H), 7.21 (d, J = 3.29 Hz, 1H), 6.97ꢀ7.12 (m, 1H),
6
.48 (d, J = 3.29 Hz, 1H), 4.26 (q, J = 7.32 Hz, 2H), 3.80 (s, 3H), 3.53 (s, 2H), 3.18 (t, J =
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