Discovery of a novel alpha-7 nicotinic acetylcholine receptor agonist series and characterization of the potent, selective, and orally efficacious agonist 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H- pyrazol-3-yl] amide (SEN15924, WAY-361789)

Article

pubs.acs.org/jmc

Discovery of a Novel Alpha-7 Nicotinic Acetylcholine Receptor

Agonist Series and Characterization of the Potent, Selective, and

Orally Eﬃcacious Agonist 5-(4-Acetyl[1,4]diazepan-1-yl)pentanoic

Acid [5-(4-Methoxyphenyl)-1H-pyrazol-3-yl] Amide (SEN15924, WAY-

361789)

Riccardo Zanaletti,*^,†Laura Bettinetti,^†Cristiana Castaldo,^†Giuseppe Cocconcelli,^†Thomas Comery,^‡

John Dunlop,^‡Giovanni Gaviraghi,^†Chiara Ghiron,^†Simon N. Haydar,^‡Flora Jow,^‡Laura Maccari,^†

Iolanda Micco,^†Arianna Nencini,^†Carla Scali,^†Elisa Turlizzi,^†and Michela Valacchi^†

^†Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy

^‡Neuroscience Research Unit, Pﬁzer, Eastern Point Road, Groton Connecticut 06340, United States

ABSTRACT: Alpha-7 nicotinic acetylcholine receptors (α7

nAChR) are implicated in the modulation of many cognitive

functions such as attention, working memory, and episodic

memory. For this reason, α7 nAChR agonists represent

promising therapeutic candidates for the treatment of cognitive

impairment associated with Alzheimer’s disease (AD) and

schizophrenia. A medicinal chemistry eﬀort, around our

previously reported chemical series, permitted the discovery of a novel class of α7 nAChR agonists with improved selectivity,

in particular against the α3 receptor subtype and better ADME proﬁle. The exploration of this series led to the identiﬁcation of 5-

(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H-pyrazol-3-yl] amide (25, SEN15924, WAY-361789), a

novel, full agonist of the α7 nAChR that was evaluated in vitro and in vivo. Compound 25 proved to be potent and selective, and

it demonstrated a fair pharmacokinetic proﬁle accompanied by eﬃcacy in rodent behavioral cognition models (novel object

recognition and auditory sensory gating).

and Ca²⁺. nAChRs are pentameric combinations of ﬁve

subunits that can assemble as homopentamers or hetero-

pentamers of 12 distinct subunits (α2-α10, β2-β4).

The homomeric α7 nACh receptor is one of the most

INTRODUCTION

Schizophrenia and Alzheimer's disease are both chronic

conditions characterized by dramatic and debilitating symp-

toms. Schizophrenia typically occurs in young adulthood, with a

■

prevalence of about 1.0% of the population,¹and is

abundant nicotinic receptors in the human brain and is highly

expressed in regions associated with learning and memory such

characterized by positive and negative symptoms (hallucina-

as the cerebral cortex and the hippocampus. This receptor,

tions, delusions, reduced aﬀect, social withdrawal, low

motivation, disorganized thoughts) and cognitive impairment

(decreased attention, memory deﬁcits). While positive

symptoms are partially managed with current medications,

compared to other nicotinic receptors, activates and desensi-

tizes rapidly upon agonist binding, is more permeable to

calcium, and is less sensitive to ACh and nicotine.^4,5

The link between α7 nAChR and cognition is not completely

cognitive deﬁcits and negative symptoms still remain an unmet

understood but has been associated with the high Ca²⁺

medical need.

permeability of this receptor. This is responsible for modulating

Alzheimer's disease is most often diagnosed in people over

65 years of age in industrialized countries,²although early onset

neurotransmitter pathways and for the activation of bio-

chemical signaling pathways, including phosphorylated extrac-

disease can occur much earlier. In 2006, 26.6 million people

suﬀered worldwide from this condition, and by 2050, 1 in 85

ellular-regulated kinase (pERK) and phosphorylated cAMP

response element binding protein (pCREB).^6,7The phosphor-

people globally is expected to be aﬀected, with the consequent

economic burden predicted to be unbearable.³In Alzheimer’s

ylation of CREB can increase the transcription of various genes

that may be responsible for long-lasting eﬀects on synaptic

disease, only modest, temporary, and symptomatic improve-

ment in cognitive performance is provided by standard of care

agents such as acetylcholinesterase inhibitors and 3,5-

dimethyladamantan-1-ylamine (memantine, a NMDA receptor

plasticity and memory.⁸

Several lines of experimental evidence support the

involvement of the α7 neuronal nAChR in both schizophrenia

antagonist).

Neuronal nicotinic acetylcholine receptors are a family of

Received: February 23, 2012

ligand gated ion channels permeable to cations such as Na⁺, K⁺,

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and AD.⁹⁻¹²These include reduced expression of α7 nAChR in

brain tissue from schizophrenia and AD patients, as well as

genetic linkage studies in schizophrenia, implicating the locus of

the α7 receptor gene promoter.¹³Prototypical α7 nAChR

agonists have demonstrated improved cognition in animal

models and normalized sensory gating deﬁcits, which are

believed to contribute to the cognitive fragmentation in

schizophrenia.^14,15

On the basis of these observations, there is strong hope that

selective α7 nAChR agonists will prove eﬀective in the

improvement of cognition in both schizophrenia and

AD.¹⁶⁻¹⁸In accordance with this hypothesis, the prototypical

nAChR agonist nicotine, as well as more selective α7 agonists

described recently, have been shown to ameliorate cognitive

performance in both animal models and human clinical trials. In

the past decade, there has been a continuous eﬀort to develop

new α7 nAChR agonists with better properties, and many of

them have advanced into clinical trials. Some examples are

reported in Figure 1: EVP-6124 and TC-5619, which are

The traditional pharmacophore model for α7 agonists

includes a core cationic center and a hydrogen bond

acceptor,^20,21as exempliﬁed by nicotine, acetylcholine, and

epibatidine. This two-point interaction model has been

conﬁrmed by several X-ray cocrystal structures of α7 agonists

bound to the homopentameric acetylcholine binding protein

(AChBP). AChBP is a structural homologue of the α7 agonist

binding domain and, in the absence of the α7 crystal structure,

is widely used as a surrogate of nicotinic receptors and as a

template to build theoretical receptor models.²²The agonist

binding site is located at the monomer interfaces and is mostly

buried by the Cys-loop, which delimits the site together with an

aromatic cage composed of W53, Y89, W143, Y164, Y185, and

Y192. The core pharmacophoric interaction occurs between the

basic center of the ligand and W143 of the aromatic cage via a

cation-π or hydrogen bonding contact; many cocrystal

structures also show a well-conserved water molecule that

establishes a hydrogen bond with the agonist, as in the case of

nicotine.²³

We have previously reported^24,25two chemical series of α7

agonists (series 1 and 2, Figure 3). Both are characterized by

Figure 3. Series 1, 2, 3 and pharmacophoric features: basic center

(blue); carbon chain (black); central linker (green); and biaryl system

(red).

Figure 1. α7 nAChR ligands evaluated in the clinical phase.

currently in phase 2 studies for schizophrenia and Alzheimer's

disease, are well tolerated and have shown eﬃcacy outcomes;

PHA568,487 was discontinued for cardiovascular ﬁndings of

nonsustained ventricular tachycardia in humans, while

AZD0328 and RG3487 did not meet the desired target

product proﬁle in phase 2. As highlighted in Figure 1, most of

the α7 agonists reported to date belong to the quinuclidine

family and the eﬀorts to design diﬀerent scaﬀolds have mainly

led to bicyclic amine compounds (Figure 2).¹⁹

the presence of a basic center, which is the key pharmacophoric

feature, together with a linear four carbon chain, a central

linker, and a biaryl system, typical of our series. The key

diﬀerence between the two series is the central linker: an amide

group for series 1; a urea moiety for series 2.

Series 2 shows generally better potency against α7 nAChR

compared to series 1; both suﬀer from low selectivity,

particularly against the homologous ganglionic α3 receptor.

Series 2 is also characterized by a nonoptimal hERG activity

and substantial cytochrome inhibition. We aimed at developing

a new series that combines good potency and selectivity with

no hERG and cytochrome inhibition and with the ﬁnal purpose

of developing a potential preclinical candidate. Our medicinal

chemistry eﬀort focused on exploring the ﬁrst ring of the biaryl

system in series 1 and led us to the discovery of series 3. Series

3 shows a good level of potency, better selectivity, an optimal

hERG proﬁle, and no cytochrome P450 inhibition. Interest-

ingly, series 3 retains two hydrogen bond donors, thus the

possibility of an extra interaction, compared to series 2.

Molecular modeling studies were also applied to investigate the

structural basis of ligand−receptor binding and to deﬁne the

pharmacophoric features of the described α7 nAChR agonists.

Herein we report the synthesis, SAR development, pharmaco-

logical characterization, and the in vivo eﬃcacy of this novel

series of α7 nAChR agonists.

Figure 2. Nonquinuclidine amine templates in the nAChR ﬁeld.

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Table 1. Eﬀect of First Ring Replacement on Activity

a

All functional activities were measured in a calcium ﬂux assay using a ﬂuorescence imaging plate reader. The activity at rat α7-nAChRs was

determined using a stable recombinant GH4C1 cell line expressing the receptor.³⁵Reported EC₅₀/IC₅₀values were determined in a single

experiment and obtained from triplicate data points generated within the same experimental session. Data were averaged from multiple experiments

(n) and reported as the average SEM (n). Nicotine under the same conditions had EC₅₀= 1.34 μM 0.002, E_max= 102% 1.3 (n = 62),

acetylcholine EC₅₀= 3.12 μM 0.13, E_max= 110% 4.1 (n = 6), PHA568,487 EC₅₀= 0.0362 μM 0.0006, E_max= 96% 1.9 (n = 2).

RESULTS AND DISCUSSIONS

■

The initial chemistry eﬀort concentrated on the exploration of

the ﬁrst ring of the biaryl system of series 1, as shown in Table

1. Starting from compound 1 (representative of series 1), we

replaced the ﬁrst phenyl ring with 5-membered heterocycles.

The ﬁrst objective of this modiﬁcation was to identify a novel

chemical series with acceptable potency and a full functional

response. Upon identiﬁcation of the best heterocycle, the idea

was to expand the series to evaluate its potential in terms of

activity, selectivity, and ADME proﬁling data. As appears from

the data in Table 1, replacing the phenyl ring of compound 1

with pyrazole gave 3, with a good level of potency and a full

functional response. All the other heterocycles yielded inactive

compounds. In the case of 5, the activity was in the micromolar

range but the functional response was only partial; thus, the

compound did not further progress. Interestingly, the N-

Figure 4. Compounds 2 (yellow) and 3 (pink) bound to the

methylated analogue (4) of compound 3 was completely

inactive, highlighting the importance of the hydrogen bond

donor in this position.

orthosteric site of the theoretical model of human α7 nAChR as

predicted by molecular docking studies. Residues involved in key

interactions with the ligands are reported. For the sake of clarity, part

of the Cys-loop has been removed from the image.

Previously reported docking studies²⁵have highlighted the

putative key interaction pattern of series 2 into a homology

model for the human α7 receptor (yellow structure in Figure

4). The piperidine basic nitrogen was engaged in a hydrogen

bond interaction with the backbone carbonyl of W143, while

the urea moiety was placed within the aromatic cage and stacks

over Y89. Moreover, the two NH groups were available to

further hydrogen bond to the hydroxyl oxygen of Y185. The

four carbon chain acts as a spacer between the two

pharmacophoric elements. The terminal part of the biaryl

system ﬁts into a polar pocket of the receptor which can

establish hydrogen bonding contacts with ligand acceptor

atoms. Compound 3 was predicted to have the same interaction

pattern (pink structure in Figure 4). Interestingly, the amidic-

pyrazole system of 3 was able to mimic the double H-bonding

system of the urea series with the two NHs pointing toward

Y185. This conformation is stabilized by an additional

hydrogen bond to the protein with respect to the alternative

arrangement, proposed by docking calculations, where the two

NHs are rotated by 180° and form an intramolecular hydrogen

bond. While being energetically favored, such an alternative

conformation did not ﬁt to the overall SAR of the series,

leading us to prefer the ﬁrst solution. In this case, the molecule

is oriented in order to engage both the double hydrogen

bonding system to Y185 and an additional interaction for the

hydrogen bond acceptor nitrogen of the pyrazole ring, as

described later in the text. The methylation of the pyrazole

nitrogen leads to the disruption of the productive contact to

Y185 that, together with a steric hindrance eﬀect, makes

compound 4 inactive.

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Table 2. ClogP, MPSA, Activity, Selectivity (EC₅₀for Agonist Behavior, IC₅₀for Antagonist Behavior), Solubility, Permeability

(Perm Class), Metabolic Stability (% Remaining), and P450 Subtype Percent Inhibition at 3 μM, for a Representative Selection

of Phenyl Pyrazole Derivatives in Which the Biaryl System Is Explored

a

All functional activities were measured in either calcium ﬂux or membrane potential assays using a ﬂuorescence imaging plate reader. Compounds

with E_max> 70% of nicotine were considered to be full agonists. The reported compounds gave values >90%. Only compounds with α7 activity lower

b

than 1.5 μM were considered suitable for the selectivity evaluation. NT indicates not tested. Solubilities were determined at pH 7.4 at

c

pseudothermodynamic equilibrium. Permeability was based on measuring the permeation rate of the test article through an artiﬁcial membrane. The

d

PAMPA assay uses a mixture of porcine brain lipids in dodecane (2% w/v).⁴⁰Metabolic stability was determined as percentage remaining after

e

incubation for 1 h with recombinant hCYP3A4. Percent cytochrome inhibition values <15−20% were considered low. See Experimental Section for

further details on all assays.

Compound 3 was considered a promising starting point to

begin an SAR exploration. A representative set of the molecules

synthesized is shown in Table 2, with their α7 activity,

selectivity over homologous receptor subtypes, and early

proﬁling data.

The selection of Table 2 aims to explore the eﬀects of ring

substitution in terms of steric hindrance, torsional angle of the

biaryl system, and electronic eﬀects on the aromatic rings. The

data reported in Table 2 shows that in general the modiﬁcation

of the second ring of the biaryl system has no dramatic impact

on α7 activity. Modest loss of potency, compared to our

starting molecule (compound 3), was observed in compounds

14 and 15, bearing the most electron poor rings. On the

contrary, the p-OMe-substitution in 8 confers a 5-fold increase

in potency. Compound 16, bearing a furane ring, resulted in a

loss of potency, probably due to a weaker interaction within the

binding site.

According to our docking studies (Figure 4), the biaryl

system ﬁts into a region extending from the aromatic cage

toward a receptor cavity which widens the agonist binding site,

as documented by most of the AChBP cocrystal structures.

Interestingly, the recently determined X-ray structure²⁶(2Y58

PDB code) identiﬁes a ligand-inducible subpocket of AChBP

which partly overlaps with the region occupied by the biaryl

system of series 2 compounds into our theoretical binding site.

This receptor region is rich in lysine, asparagine, and tyrosine

residues, which are prone to give hydrogen bonds to the ligand

counterpart. In particular, K137 was predicted to H-bond to the

p-OMe substituent on the end aryl ring of compound 8, while

K139 was accessible for further interactions with the acceptor

nitrogen on the pyrazole ring. Weaker EC₅₀values of

compounds 14 and 16 were likely to be due to a suboptimal

distance of the hydrogen bond with K137.

Being that compound 8 was the most active, we decided to

explore the eﬀect of the -OMe substitution in the ortho and

meta positions (compounds 9 and 10), but we observed no

beneﬁt in terms of activity. In light of this result, we opted for

substituents only in the para position for the other analogues.

The eﬀect of a methyl group on the pyrazole ring was also

investigated (compound 11) to evaluate the potential of

branching our molecules, but the loss of activity was evident.

This initial pool of molecules was also characterized in our

selectivity panel of homologous receptor and in our ADME

proﬁling assays. No agonist activity was observed on the α1, α3,

and 5HT3 receptors for any of the compounds tested.

Regarding the inhibitory activity, no problem of selectivity

was observed for the homologous α1 and 5HT3 receptors,

while all the compounds tested presented some activity against

the ganglionic α3 receptor. With no exceptions, all the

molecules showed excellent solubility, passive permeability,

metabolic stability, and no cytochrome P450 inhibition. The p-

OMe substituted compound 8 exhibited the best proﬁle in

terms of α7 activity, a selectivity ratio for the ganglionic α3

receptor (α3 IC₅₀/α7 EC₅₀) in line with the other analogues,

and an optimal overall proﬁle. For these reasons, it was

considered the best tool for further SAR studies. The next

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Table 3. ClogP, MPSA, Activity, Selectivity (EC₅₀for Agonist Behavior, IC₅₀for Antagonist Behavior), Solubility, Permeability

(Perm Class), Metabolic Stability (% Remaining), and P450 Subtype Percent Inhibition at 3 μM, for a Representative Selection

of Phenyl Pyrazole Derivatives in Which the Basic Center Is Explored

a

All functional activities were measured in either calcium ﬂux or membrane potential assays using a ﬂuorescence imaging plate reader. Compounds

with E_max> 70% of nicotine were considered to be full agonists. The reported compounds gave values >90%. Only compounds with α7 activity lower

b

than 1.5 μM were considered suitable for the selectivity evaluation. NT indicates not tested. Solubilities were determined at pH 7.4 at

c

pseudothermodynamic equilibrium. Permeability was based on measuring the permeation rate of the test article through an artiﬁcial membrane. The

d

PAMPA assay uses a mixture of porcine brain lipids in dodecane (2% w/v).⁴⁰Metabolic stability was determined as percentage remaining after

e

incubation for 1 h with recombinant hCYP3A4. Percent cytochrome inhibition values <15−20% were considered low. See Experimental Section for

further details on all assays.

exploration phase concerned the modiﬁcation of the region

bearing the basic center.

In the putative binding mode of compound 25 into the

theoretical model of α7 receptor (Figure 5), the acetyl-

diazepane moiety ﬁts the cavity hosting the nicotine structure in

the AChBP cocrystal structures. This cavity was in part

delimited by the W143 and Y192 residues of the aromatic cage

Basicity is a determinant factor for the activity on the α7

receptor and explains why most α7 agonists contain a

quinuclidine amine scaﬀold, bearing a strong basic center.

Protonated nitrogen is required to make a key pharmacophoric

interaction with the receptor binding site. The results observed

and reported in Table 3 show that the basicity of the R

substituent only appears to have a limited impact on the α7

activity. Compounds 8 and 18, for example, are comparably

active despite relatively diﬀerent pK_avalues (piperidine pK_a=

10.9, morpholine pK_a= 9.2).²⁷We believe this observation to

be valid as long as the pK_ais suﬃcient to have the basic

nitrogen protonated at physiological pH.

At the same time, the α7 activity seems to be modestly

aﬀected by the ring size, as exempliﬁed moving from piperidine

to azepine (compounds 8 and 21). These SAR studies showed

strong evidence of the detrimental eﬀect on activity, derived

from an extra hydrogen bond donor, which also confers the

molecules higher MPSA and lower ClogP values. The

additional NH moiety leads to an increase in the hydrophilicity

of the R substituents which was not tolerated in this position.

This is proven by the loss of activity in compounds 19, 20, 23,

and 24, with the exclusion of the steric hindrance eﬀect, as

demonstrated by the good activity of compound 25.

Figure 5. Predicted binding mode of compound 25. Important active

site residues are labeled. Potential hydrogen bonds are shown as

dotted lines.

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and the residues L105, Q113, and L115. The pocket was large

enough to accommodate substituted 6−7 membered rings and

has mainly a lipophilic nature, which may account for the drop

in activity of more polar compounds such as 19, 20, 23, and 24.

In the docked poses of compound 25 into the α7 homology

model, the acetyl-diazepane system mimics the pyrrolidine−

pyridine system of nicotine: the protonated nitrogen was ideally

positioned to target W143, whereas the carbonyl group was

bound to L115 and N103 through a bridging water molecule,

such as the pyridine nitrogen of nicotine and many other α7

agonists. The X-ray structures of some recently published

diazepane−pyridine α7 agonists bound to AChBP nicely agree

with the predicted interaction network established by the

acetyl−diazepane system of our molecules (3U8M, 3U8K,

3U8N, 3U8J, 3U8L PDB codes). The amide-pyrazole system

and the terminal p-methoxy substituent of compound 25

maintain the previously described contacts with the receptor,

with the former being involved in an optimal interaction with

Y185 and K139, and the latter in a hydrogen bond to K137. In

terms of selectivity, no issues were highlighted in Table 3 for

the α1 and 5HT3 receptors. Some cross-reactivity on the

ganglionic α3 receptor was present for compounds 8 and 18,

while no activity was observed on all the other analogues

present in Table 3. This better proﬁle was likely due to the

higher steric hindrance of compounds 21, 22, and 25 compared

to 8 and 18 not being tolerated by the α3 receptor.

While all the analogues presented an optimal proﬁle in terms

of solubility, metabolic stability, and cytochrome P450

inhibition, passive permeability for this series of compounds

was sometimes nonoptimal. The general trend was that

compounds with lower permeability were those with lower

ClogP and higher MPSA.

On the basis of the collective data, compound 25 was

selected to be progressed for further studies. Although its

permeability fell into the medium class, this was counter-

balanced by the excellent spectrum of activity, selectivity, and

the rest of the proﬁling data.

The physicochemical properties, additional pharmacology,

ADME properties, and pharmacokinetic proﬁle for compound

25 are summarized in Table 4, where MW, ClogP, ClogD, and

MPSA were in line for potential brain penetration. The aﬃnity

of compound 25 for the receptor was conﬁrmed in a binding

assay using the rat receptor (0.66 μM). The potency was

further demonstrated in electrophysiological studies in both rat

and human receptor expressing cell lines (4.4 and 3.0 μM,

respectively, full agonist functional responses). The hERG

inhibition was considered acceptable to forecast low potential

for cardiotoxicity issues. In addition, compound 25 did not

show any cross-activity in a broad panel of 70 receptors (%

inhibition <50 at 10 μM). In terms of ADME properties, the in

vitro intrinsic clearance in rat was optimal (13.0 μL/min/mg)

and the plasma stability was acceptable (55% after 3 h

incubation) while the in vitro permeability in the MDCK cell

line, although nonoptimal, was considered not limiting for

further progression of the molecule (P_{app A→B}1.6 × 10⁻⁶cm/s,

eﬄux ratio 4.6). Compound 25 was progressed to a PK study

and showed an overall moderate exposure, in particular low

bioavailability (12%) and moderate brain to plasma partitioning

and brain levels (0.38 B/P ratio and 3.7 ng/g, respectively).

In light of the brain levels observed, we decided to evaluate

compound 25 in behavioral animal tests.

Table 4. Selected Physicochemical Properties, Additional

Pharmacology, ADME, and Rat Pharmacokinetic Data for 25

physicochemical properties MW

cLogP

413.51 Da

1.90

cLogD at pH 7.4

MPSA

1.69

90.6 Å²

a

additional pharmacology

α7 K_i(rat)

0.66 μM

b

α7 e-phys EC₅₀(rat, hu) 4.4 μM, 3.0 μM

b

α7 e-phys E_max(rat, hu)

99%, 89%

c

hERG IC₅₀

24.2 μM

d

ADME

plasma stability 3 h

55%

e

Cl_int(rat)

13.0 μL/min/mg

1.6 × 10⁻⁶cm/s

4.6

f

MDCK AB

g

MDCK BA/AB

h

PK rat (20 mpk, po)

BBB rat (10 mpk, po)

C_max

0.18 μM

0.83 h

h

T_max

h

AUC_0‑inf

1.37 μM·h

12%

i

F

j

B/P

0.38

j

C_max

3.7 ng/g

a

Binding assay for human α7 nAChR expressed in GH4C1 cells using

b

[³H]-epibatidine as the radioligand. Whole cell patch clamp

recordings were performed on GH4C1 cells expressing rat α7

receptors or CHO cells expressing the human α7 receptor with Ric-

3. Each cell was exposed to at least one concentration of acetylcholine

c

and a full range of concentrations of 25. hERG response was obtained

from CHO cells stably expressing the channel using an IonWorks

d

system. Plasma stability was determined as percentage remaining after

e

incubation for 3 h with fresh rat plasma. In vitro intrinsic clearance

f

from rat liver microsomal stability assay. MS-based quantiﬁcation of

apical → basolateral transfer rate of a test compound at 10 μM across

g

contiguous monolayers of MDCK cells. Ratio of (basolateral →

apical) to (apical → basolateral) transfer rate of a test compound at 10

μM across contiguous monolayers of MDCK (Madin−Darby canine

h

kidney) cells. Pharmacokinetic parameters were determined following

i

a single 20 mg/kg po dose in male Long Evans rats. Oral

bioavailability (F) was determined in a separate experiment as the

ratio of AUC_0‑infnormalized for the dose, obtained following single 10

j

mg/kg iv doses in male Long Evans rats. The brain to plasma (B/P)

ratio was determined as the ratio of AUC_0‑inffollowing a single 10 mg/

kg po dose in male Long Evans rats.

vivo, during behavioral tests, is not suﬃcient to activate α7

nAChRs in vitro. This observation encouraged us to advance

our compound to in vivo eﬃcacy evaluation, even in the

presence of moderate brain exposure. Compound 25 displayed

eﬃcacy in assays of cognitive function in the rat model, namely

in a form of episodic memory measured in the novel object

recognition test (NOR, Figure 6A). In perceptual processing,

25 showed the ability to attenuate pharmacologically induced

deﬁcits via the glutamatergic system in a prepulse inhibition

model (PPI, Figure 7), a classic paradigm for evaluating

potential treatment for schizophrenia. In agreement with our

ﬁndings, very recently²⁹the positive outcome of in vivo

experiments even at in vitro subpharmacological doses has been

The estimation of the brain concentration of other α7

agonists (for example PHA-543,613)²⁸to achieve eﬃcacy in

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Figure 7. Ability of compound 25 to normalize an MK-801 induced

disruption of prepulse inhibition (PPI) of the acoustic startle response

in rats (n = 7−8 treatment group). Blue column, vehicle no. MK-801;

red columns, vehicle or compound 25 at several doses administered

orally. Statistical analysis, ANOVA and Fisher’s LDS test; * P < 0.05 vs

MK-801 plus vehicle.

memory enhancement in the NOR experiment was blocked by

the pretreatment with the α7 antagonist methyllycaconitine

(MLA), demonstrating the direct involvement of the α7

nAChR (Figure 6B). In addition, to evaluate any tolerance to

this behavioral eﬀect, 25 was administered once daily for 14

days before the NOR test. It demonstrated retention of acute

memory enhancing activity in chronically exposed animals

comparable to that of animals just given an acute treatment,

showing no loss of the in vivo signal with chronic daily dosing

and potentially no tolerance to the compound (Figure 6C).

Moreover, compound 25 administered orally reversed (+)-5-

methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine

(dizocilpine, MK-801)-induced deﬁcits in the PPI in rat with a

minimum eﬃcacious dose of 10 mg/kg (Figure 7).

The excellent in vivo pharmacological proﬁle displayed by

compound 25, despite the nonoptimal properties in the ADME

and PK areas, prompted us in the continuation of the α7

program. To further explore the chemical space around

compound 25, we performed pinpoint modiﬁcations aimed at

improving passive permeability and plasma stability. We

explored the introduction of a methyl group both on the

pyrazole ring and in the position α to the carbonyl of the amide

group (Table 5, compounds 26 and 27, respectively). We also

explored the replacement of the pyrazole ring with the

regioisomer imidazole ring (28). The idea behind the

introduction of an extra methyl group was to improve passive

permeability by increasing ClogP, and plasma stability by

hindering the amide bond (which is known to be susceptible to

hydrolysis by plasma esterases). The replacing of the pyrazole

by the imidazole ring was aimed at introducing a ﬁve

membered heterocycle with a hydrogen bond donor function

and a diﬀerent heteroatoms arrangement (with respect to

pyrazole) to potentially improve the plasma stability by altering

the electronic character of the amide bond. The biological

results showed that introduction of the methyl group on the

pyrazole ring, as noted before, was detrimental for activity as

well as the imidazole/pyrazole exchange. Interestingly, the extra

methyl group in the position α to the carbonyl of the amide

maintained a good level of potency, improved the passive

permeability, and ameliorated the plasma stability. This

modiﬁcation seemed to confer a better overall proﬁle to

compound 27 compared to compound 25, paving the way for

further exploration both of our chemical series and of

compound 27.

Figure 6. Induced memory enhancement of 25 in a novel object

recognition test (NOR) using spontaneous decay of memory (48 h

time-interval) as the amnesic factor in rats (n = 10−12 treatment

group). Blue columns, exploration time of new object; red columns,

exploration time of familiar object. (A) Eﬀects of several doses of 25

administered orally 1 hour before T1 exploration. (B) Reversion of

memory enhancement induced by 25 using pretreatment (5 min

before the orally administration of 25) with MLA. (C) Lack of

tolerance to compound 25 in NOR: the eﬀect is maintained after 14

days of intraperitoneal administration of 25 and is analogous to the

acute administration. Statistical analysis: ANOVA and Fisher’s LDS

test: * P < 0.05 vs time in exploring a familiar object.

interpreted as the coagonist activity of EVP-6124 with

acetylcholine on α7 nAChRs.

Treatment with 25 induced a memory enhancement in the

NOR at several doses when administered orally, using a 48 h

delay time as the amnesic factor, with a minimum eﬃcacious

dose of 1 mg/kg (Figure 6A). Importantly, the induced

G

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Table 5. ClogP, MPSA, Activity, Selectivity (EC₅₀for Agonist Behavior, IC₅₀for Antagonist Behavior), Solubility, Permeability

(Perm Class), Metabolic Stability (% Remaining), P450 Subtype Percent Inhibition at 3 μM, and Plasma Stability (%

Remaining, 3 h), for a Representative Selection of Phenyl Pyrazole Derivatives in Which the Cental Linker Region Is Explored

a

All functional activities were measured in either calcium ﬂux or membrane potential assays using a ﬂuorescence imaging plate reader. Compounds

with E_max> 70% of nicotine were considered to be full agonists. The reported compounds gave values >90%. Only compounds with α7 activity lower

b

than 1.5 μM were considered suitable for the selectivity evaluation. NT indicates not tested. Solubilities were determined at pH 7.4 at

c

pseudothermodynamic equilibrium. Permeability was based on measuring the permeation rate of the test article through an artiﬁcial membrane. The

d

PAMPA assay uses a mixture of porcine brain lipids in dodecane (2% w/v).⁴⁰Metabolic stability was determined as the percentage remaining after

e

f

incubation for 1 h with recombinant hCYP3A4. Percent cytochrome inhibition values <15−20% were considered low. Plasma stability was

determined as the percentage remaining after incubation for 3 h with fresh rat plasma. See the Experimental Section for further details on all assays.

a

Scheme 1. Synthesis of 5-Aminopentanoic Acid Amide via Route A

a

(a) R1R2NH, toluene, reﬂux; (b) NaOH, water, reﬂux; (c) CDI, 1,2-dichloroethane, 50 °C.

a

Scheme 2. Synthesis of 5-Aminopentanoic Acid Amide via Route B

a

(a) DIPEA, DMA, −10 °C; (b) Route B1: R1R2NH, NaI, DMA, 50 °C or Route B2: R1R2NH, NaI, DMF, 60 °C.

CHEMISTRY

Chart 1. 3-Amino-5-aryl Heterocycle Used in Route A

■

The compounds investigated, with the exception of 27, were

synthesized using one of the two general routes reported in

Schemes 1 and 2 (routes A and B, respectively).

Even though route A could be preferred for the chemical

exploration of the biaryl system and route B for the basic

moiety, the two synthetic strategies were equally eﬃcient in

terms of yields and synthetic diﬃculty. Therefore, they were

used indiﬀerently by us. In route A (Scheme 1), commercially

available ethyl 5-bromopentanoate 29 was reacted with a

secondary amine through a nucleophilic substitution reaction to

give the desired ethyl 5-aminopentanoate derivatives 30 and

31; the esters were then hydrolyzed with NaOH to give the

corresponding 5-aminopentanoic acids 32 and 33. Subse-

quently, the acids 32 and 33 were coupled to 3-amino-5-aryl

heterocycle (34−36) (Chart 1) with N,N′-carbonyldiimidazole

in dichloroethane to aﬀord the targeted products amino-

pentanoic acid amide (6, 7, and 28).

(Chart 2) in dimethylacetamide to give the corresponding 5-

bromopentanoyl amide (38a−49a).

In the case of 3-amino-5-aryl/heteroaryl pyrazoles (39−49),

the acylation on the endocyclic nitrogen of the pyrazole ring to

give products 39b−49b was also detected together with an

intramolecular cyclization of 39a−49a to give products 39c−

49c (Scheme 3). After a rapid screening of solvents,

dimethylacetamide was selected as the best medium to

minimize these side reactions (in dimethylacetamide usually

less than 10% of acylation on the endocyclic nitrogen was

observed and only traces of the intramolecular cyclization).

In route B (Scheme 2), 5-bromovaleroyl chloride (37) was

reacted with 3-amino-5-aryl/heteroaryl heterocycles (38−49)

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to aﬀord 5-aminopentanoic acid amide Boc protected 56, which

was treated with HCl in dioxane to remove Boc protection and

aﬀord the desired 5-aminopentanoic acid amide 27.

Chart 2. 3-Amino-5-aryl/Heteroaryl Heterocycles Used in

Route B

3-Amino-5-aryl/heteroaryl heterocycles, 34−36, 38−43, and

46 were found to be commercially available, while 3-amino-5-

aryl/heteroaryl pyrazoles 44, 45, and 47−49 were synthesized

according to literature procedures.³⁰⁻³²

CONCLUSIONS

■

Most of the α7 agonists reported to date belong to the

quinuclidine amine family.¹⁹Our research group concentrated

its eﬀorts in designing diﬀerent scaﬀolds in order to add novelty

to our compounds and preempt potential risks associated with

this structure. Interestingly, recent literature reported the failure

in clinical phase trials of quinuclidine containing α7 agonists

due to toxicological reasons, corroborating our initial strategic

choice. For example, PHA568,487 was discontinued for

cardiovascular ﬁndings of nonsustained ventricular tachycardia

in human; this type of toxicity is probably to be ascribed to this

structural class.³³

Our continuous eﬀorts in the α7 agonist area led us to the

identiﬁcation of a novel chemical series which, in line with our

focus, does not belong to the quinuclidine amine family. This

series emerged from a chemical exploration of our previously

reported structures and improves both selectivity and ADME

proﬁling. One of these molecules (compound 25, SEN15924,

WAY-361789) was characterized in behavioral studies in light

of the good potency, selectivity, ADME proﬁle, and acceptable

PK and was found to improve cognitive function in NOR and

to reverse a pharmacological disruption in PPI. Recent

structural modiﬁcation of compound 25 conﬁrmed the

possibility of improving its overall proﬁle and potentially oﬀers

an attractive candidate (for example, compound 27) worthy of

further investigation.

The 5-bromopentanoyl amide (38a−49a) obtained was

either reacted in situ (route B1) with the addition of a

secondary amine and NaI or isolated by precipitation with

water (route B2) and reacted with a secondary amine and NaI

in dimethylformamide to aﬀord the ﬁnal 5-aminopentanoic acid

amide (3−5, 8−26).

Compound 27 was synthesized according to route C: 2-

methyl-malonic acid dimethyl ester 50 was treated with NaH,

in tetrahydrofurane and reacted with 1,4-dibromopropane 51,

through a nucleophilic substitution reaction (Scheme 4). The

disubstituted dimethyl malonate 52, thus obtained, was

hydrolyzed and monodecarboxylated by treatment with HBr

to obtain 5-bromo-2-methylpentanoic acid (53). The acid 53

was activated with oxalyl chloride and coupled to 5-amino-3-(4-

methoxyphenyl)pyrazole-1-carboxylic acid tert-butyl ester (54)

in dichloromethane to aﬀord 5-bromopentanoyl amide 55. This

reaction required the use of the Boc protected pyrazole 54 in

order to avoid acylation on the endocyclic nitrogen of the

pyrazole. Compound 55 was then subjected to a nucleophilic

substitution reaction with acetyldiazepine in dichloromethane

EXPERIMENTAL SECTION

■

1. Chemistry. General Methods. Unless otherwise speciﬁed, all

nuclear magnetic resonance spectra were recorded using a Varian

Mercury Plus 400 MHz spectrometer equipped with a PFG ATB

broadband probe. UPLC-MS analyses were run using a Acquity

Waters UPLC equipped with a Waters SQD (ES ionization) and

Waters Acquity PDA detector, using a column BEH C18 1.7 μm, 2.1

mm × 50 mm. Gradients were run using 0.1% NH₄HCO₃water/

acetonitrile 95/5 and acetonitrile with a gradient 95/5 to 15/85, ﬂow:

0.8 mL/min over 3 min or 0.05% formic acid water/acetonitrile 95/5

and acetronitrile with a gradient 95/5 to 100, ﬂow: 0.8 mL/min as

stated in the examples. Retention times were expressed in minutes.

Temperature: 40 °C. UV detection at 215 nm and 254. ESI+ detection

in the 80−1000 m/z range. Preparative HPLC was run using a Waters

2767 system with a binary gradient module Waters 2525 pump and

coupled to a Waters Micromass ZQ (ES) or Waters 2487 DAD, using

a

Scheme 3. Acylation of Aminopyrazoles (39−49) and Side Products

a

(a) DIPEA, DMA, −10 °C.

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a

Scheme 4. Synthesis of Compound 27 via Route C

a

(a) NaH, THF, 0 °C; (b) HBr 48%, 110 °C; (c) (COCl)₂, NEt₃, DCM, RT; (d) acetyldiazepine, NEt₃, DCM, 50 °C; (e) HCl, dioxane, RT.

a X-Bridge C18 5 μm, 19 mm × 150 mm, using a 0.1% formic acid/

water and 0.1% formic acid/methanol ﬂow: 17 mL/min. The purity of

compounds submitted for screening was >95% as determined by

integrating at 215 nm the peak area of the LC chromatograms. To

further support the purity statement, all compounds were also

analyzed at a diﬀerent wavelengths (254 nm), and total ion current

(TIC) chromatogram and NMR spectra were used to further

substantiate results. HRMS data were obtained through an LTQ-

Orbitrap mass spectrometer via direct infusion of a 1 μM sample in

acetonitrile 30%/water 69.9%/formic acid 0.1% (ﬂow: 7 μL/min). The

LTQ-Orbitrap instrument was calibrated prior to analysis with the

manufacturer’s positive ion Calibration Solution in the mass range

100−2000 m/z. Each determination was the combination of ﬁve

successive scans acquired at 100.000 fwhm resolution. Elemental

composition calculations and isotopic pattern simulations were

executed using the speciﬁc tool included in the QualBrowser module

of Xcalibur (Thermoﬁsher Scientiﬁc, release 2.0.7) software, using a

tolerance of 5 ppm. All column chromatography was performed

following small modiﬁcations of the original method of Still.³⁴All TLC

analyses were performed on silica gel (Merck 60 F254) and spots

revealed by UV visualization at 254 nm and KMnO₄or ninhydrin

stain.

2. Synthesis. General Procedure for 5-Aminopentanoic Acid.

To a solution of amine (65 mmol) in toluene (15 mL) was added

ethyl 5-bromopentanoate (26 mmol), and the reaction mixture was

reﬂuxed for 10 h. The mixture was allowed to cool to room

temperature, and any solid present was ﬁltered oﬀ and washed with

ether. The ﬁltrate was concentrated under reduced pressure to give

ethyl 5-aminopentanoate, which was used in the next step without

further puriﬁcation. To a suspension of crude ethyl 5-aminopentanoate

from the previous step (about 25 mmol) in 15 mL of water was added

NaOH (1.4 g, 25 mmol), and the mixture was heated at reﬂux for 16 h.

The reaction was then allowed to cool down to room temperature, and

the solution was acidiﬁed at 0 °C with HCl 6 N and concentrated

under reduced pressure. The residue was treated with EtOH, and the

precipitated sodium chloride was ﬁltered oﬀ. Evaporation of the

solvent under reduced pressure aﬀorded the 5-aminopentanoic acid as

a white solid.

General Procedure for 5-Aminopentanoic Acid Amide. Route A.

To a suspension of 5-aminopentanoic acid (7.93 mmol) in 1,2-

dichloroethane (20 mL) was added N,N′-carbonyldiimidazole (1.2 g,

7.4 mmol), and the mixture was stirred at room temperature for 2 h

(when the activation of the amino acid was complete, dissolution of

the suspension was generally observed). The 3-amino-5-aryl/

heteroaryl heterocycle (5.29 mmol) was then added, and the reaction

was stirred for further 10 h. Upon reaction completion (as monitored

by LC-MS) if the formation of two isomers was observed, the mixture

was heated at 50 °C until the conversion of the less stable isomer to

the title compound was observed (as monitored by LC-MS). The

solvent was washed with sat. Na₂CO₃solution, extracted, and removed

under reduced pressure. The crude products were either recrystallized

from CH₃CN or puriﬁed by SiO₂column chromatography or by

preparative HPLC.

5-Piperidin-1-ylpentanoic Acid [5-(4-Chlorophenyl)[1,3,4]-

thiadiazol-2-yl] Amide (6). Following the general procedure for 5-

aminopentanoic acid amide A and starting from commercially available

5-(4-chlorophenyl)[1,3,4]thiadiazol-2-ylamine (34, 96.0 mg, 0.45

mmol), 42.0 mg of title compound as formate salt was obtained by

1

preparative HPLC (25% yield). H NMR (400 MHz, DMSO-d₆): δ

1.30−1.39 (m, 2H), 1.38−1.50 (m, 5H), 1.52−1.65 (m, 2H), 2.22−

2.36 (m, 5H), 2.48−2.53 (m, 4H), 7.54−7.60 (m, 2H), 7.90−7.97 (m,

2H), 8.17 (s, 1H), 10.30 (s, 1H). ¹³C NMR (DMSO-d₆): δ 172.37,

161.07, 159.42, 135.71, 130.06, 129.82, 129.20, 112.24, 58.64, 54.59,

35.44, 26.28, 26.18, 24.71, 23.27. Mass (ES) m/z: 379 (M + 1). UPLC

(basic method); R_t= 1.29 min; area 100%. HRMS: calcd for

C₁₈H₂₃ClN₄OS + H⁺, 379.13539; found (ESI, [M + H]⁺obsd),

379.13525.

5-Piperidin-1-ylpentanoic Acid [3-(4-Chlorophenyl)isoxazol-5-yl]

Amide (7). Following the general procedure for 5-aminopentanoic acid

amide A and starting from commercially available 3-(4-chlorophenyl)-

isoxazol-5-ylamine (35, 88.0 mg, 0.45 mmol), 28.0 mg of title

compound as formate salt was obtained as a solid by preparative

1

HPLC (18% yield). H NMR (400 MHz, CD₃OD): δ 1.5−1.90 (m,

10H), 2.50−2.57 (m, 2H), 3.05−3.12 (m, 2H), 3.03−3.28 (m, 4H),

6.70 (s, 1H), 7.47−7.51 (m, 2H), 7.76−7.82 (m, 2H), 8.49 (s, 1H).

¹³C NMR (CD₃OD): δ 170.36, 169.01, 162.55, 162.37, 136.04, 130.70,

5-Piperidin-1-ylpentanoic Acid Hydrochloride (32). Following the

general procedure for 5-aminopentanoic acid and starting from the

commercially available ethyl 5-bromopentanoate (29, 2.0 g, 9.6

mmol), 1.9 g of title compound as hydrochloride was obtained (90%

yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.39−1.58 (m, 4H), 1.61−

1.80 (m, 6H), 2.19−2.27 (m, 2H), 2.69−3.50 (m, 7H), 11.40 (br s,

1H). Mass (ES) m/z: 186 (M + 1).

129.07, 128.01, 86.09, 57.17, 53.38, 34.91, 23.89, 23.63, 22.13, 22.08.

Mass (ES) m/z: 362 (M + 1). UPLC (basic method); R_t= 1.40 min;

area 100%. HRMS: calcd for C₁₉H₂₄ClN₃O₂+ H⁺, 362.16298; found

(ESI, [M + H]⁺obsd), 362.16291.

5-(4-Acetyl[1,4]diazepan-1-yl)pentanoic Acid [5-(4-Methoxy-

phenyl)-1H-imidazol-2-yl] Amide (28). Following the general

procedure for 5-aminopentanoic acid amide A and starting from

commercially available 5-(4-methoxyphenyl)-1H-imidazol-2-ylamine

(36, 260.0 mg, 1.37 mmol), 77 mg of title compound (14%, yield)

was obtained as a solid by SiO₂column chromatography (ACN/

MeOH NH₃2 M 90:10). ¹H NMR (400 MHz, CD₃OD): δ 1.53−1.63

(m, 2H), 1.64−1.76 (m, 2H), 1.77−1.97 (m, 2H), 2.09 (s, 3H), 2.45

(t, J = 7.3 Hz, 2H), 2.49−2.59 (m, 2H), 2.63−2.77 (m, 4H), 3.52−

3.69 (m, 4H), 3.79 (s, 3H), 6.91 (d, J = 8.7 Hz, 2H), 7.04 (s, 1H), 7.57

(d, J = 8.6 Hz, 2H). ¹³C NMR (CD₃OD): δ 172.98, 171.87, 158.98,

5-(4-Acetyl[1,4]diazepan-1-yl)pentanoic Acid (33). Following the

general procedure for 5-aminopentanoic acid and starting from the

commercially available ethyl 5-bromopentanoate (29, 2.0 g, 9.6

mmol), 2.2 g of title compound was obtained by acidiﬁcation to the

1

isoelectric point (83% yield). H NMR (400 MHz, CDCl₃): δ 1.37−

1.57 (m, 4H), 1.76−1.90 (m, 2H), 2.09 (s, 3H), 2.10−2.16 (m, 2H),

2.36−2.45 (m, 2H), 2.53−2.66 (m, 4H), 3.44−3.63 (m, 4H), 11.38

(br s, 1H). Mass (ES) m/z: 241 (M − 1).

J

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141.67, 135.71, 134.74, 125.81, 113.90, 109.81, 57.15, 55.55, 54.57,

54.14, 44.29, 44.08, 35.44, 27.31, 26.13, 23.14, 20.45. Mass (ES) m/z:

414 (M + 1). UPLC (basic method); R_t= 0.92 min; area 100%.

HRMS: calcd for C₂₂H₃₁N₅O₃+ H⁺, 414.24997; found (ESI, [M +

H]⁺obsd), 414.24984.

23.51, 22.40, 20.06. Mass (ES) m/z: 371 (M + 1). UPLC (acid

method); R_t= 0.88 min; area 100%. HRMS: calcd for C₂₁H₃₀N₄O₂+

H⁺, 371.24415; found (ESI, [M + H]⁺obsd), 371.24383.

Synthesis of 5-(3-Oxopiperazin-1-yl)pentanoic Acid [5-(4-Me-

thoxyphenyl)-2H-pyrazol-3-yl] Amide (19). Following the general

procedure for 5-aminopentanoic acid amide B1 and starting from

commercially available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine

(39, 57 mg, 0.30 mmol), 28 mg of the title compound was obtained

General Procedure for 5-Aminopentanoic Acid Amide. Route B1,

Two Steps Synthesis. A solution of 5-bromovaleroyl chloride (15.7

mmol, 1 equiv) in dry DMA (35 mL) was cooled to −10 °C (ice/

water bath) under N₂; a solution of 3-amino-5-aryl/heteroaryl

heterocycle (15.7 mmol, 1 equiv) and diisopropylethylamine (15.7

mmol, 1 equiv) in dry DMA (15 mL) was added over 30′. After 2 h at

−10 °C, completion of the reaction, as monitored by LC-MS, was

generally observed (in the case of pyrazole, acylation on the pyrazole

ring was also detected). The reaction was then quenched by addition

of H₂O (ca. 50 mL); the thick white precipitate formed upon addition

of water was recovered by ﬁltration. Washing with Et₂O (3 × 10 mL)

usually eﬃciently removed the byproduct of acylation on the pyrazole

ring in the case of pyrazole.

1

by puriﬁcation with preparative HPLC (22% yield). H NMR (400

MHz, DMSO-d₆): δ 1.21−1.26 (m, 1H), 1.33−1.66 (m, 3H), 2.26−

2.32 (m, 2H), 2.47−2.53 (m, 2H), 2.78−3.02 (m, 2H), 3.08−3.23 (m,

4H), 3.76 (s, 3H), 6.73 (s, 1H), 7.05−6.90 (m, 2H), 7.71−7.43 (m,

2H), 8.14 (s, 1H), 10.32 (s, 1H), 12.61 (s, 1H). ¹³C NMR (DMSO-

d₆): δ 170.84, 167.70, 159.73, 148.77, 142.53, 126.99, 122.88, 115.02,

93.44, 56.74, 55.87, 54.24, 48.99, 36.22, 35.72, 25.37, 23.25. Mass (ES)

m/z: 372 (M + 1). UPLC (acid method); R_t= 0.64 min; area 97%.

HRMS: calcd for C₁₉H₂₅N₅O₃+ H⁺, 372.20302; found (ESI, [M +

H]⁺obsd), 372.20306.

5-Bromopentanoic acid amide (0.6 mmol, 1 equiv) was dissolved in

DMF (4 mL), and sodium iodide (0.6 mmol, 1.0 equiv) was added

followed by the secondary amine (1.5 mmol, 2.5 equiv) and

diisopropylethylamine (0.6 mmol, 1 equiv). The reaction was then

stirred under N₂at +50 °C for 18 h. Upon reaction completion (as

monitored by LC-MS), the solvent was removed at reduced pressure,

and the resulting oily residue was dissolved in DCM (20 mL), washed

with sat. Na₂CO₃(2 × 20 mL) and sat. NaCl (2 × 20 mL); the organic

layer was dried over Na₂SO₄and the solvent removed under reduced

pressure. The title compounds were puriﬁed by either SiO₂column or

preparative HPLC (standard acidic conditions).

Synthesis of 1-{4-[5-(4-Methoxyphenyl)-2H-pyrazol-3-

ylcarbamoyl]butyl}piperidine-4-carboxylic Acid Amide (20). Follow-

ing the general procedure for 5-aminopentanoic acid amide B1 and

starting from commercially available 5-(4-methoxyphenyl)-2H-pyr-

azol-3-ylamine (39, 60 mg, 0.32 mmol), 37 mg of title compound was

obtained as formate salt by puriﬁcation with preparative HPLC (28%

1

yield). H NMR (400 MHz, DMSO-d₆): δ 1.42−1.5 (m, 4H), 1.51−

1.60 (m, 4H), 1.65−1.69 (m, 2H), 2.01−2.09 (m, 3H), 2.29−2.31 (m,

2H), 2.42−2.55 (m, 2H), 2.97 (m, 2H), 3.75 (s, 3H), 6.73 (s, 1H),

6.97 (d, J = 8.9 Hz, 2H), 7.21 (s, 1H), 7.60 (d, J = 8.8 Hz, 2H), 8.11

(s, 1H), 10.31 (s, 1H). ¹³C NMR (DMSO-d₆): δ 176.35, 170.79,

164.31, 159.73, 148.20, 142.86, 126.99, 123.10, 115.02, 93.33, 57.25,

55.87, 52.48, 35.74, 35.54, 27.65, 25.15, 23.35. Mass (ES) m/z: 400

(M + 1). UPLC (acid method); R_t= 0.67 min; area 98%. HRMS: calcd

for C₂₁H₂₉N₅O₃+ H⁺, 400.23432; found (ESI, [M + H]⁺obsd),

400.23394.

5-Piperidin-1-ylpentanoic Acid [5-(4-Chlorophenyl)-2-methyl-2H-

pyrazol-3-yl] Amide (4). Following the general procedure for 5-

aminopentanoic acid amide B1 and starting from commercially

available 5-(4-chlorophenyl)-2-methyl-2H-pyrazol-3-ylamine (40, 132

mg, 0.64 mmol), 70.0 mg of title compound as the formate salt was

1

obtained by preparative HPLC (26%, yield). H NMR (400 MHz,

Synthesis of 5-[1,4]Oxazepan-4-ylpentanoic Acid [5-(4-Methox-

yphenyl)-2H-pyrazol-3-yl] Amide (22). Following the general

procedure for 5-aminopentanoic acid amide B1 and starting from

commercially available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine

(39, 90 mg, 0.48 mmol), 88 mg of title compound was obtained as

DMSO-d₆): δ 1.35−1.47 (m, 2H), 1.49−1.66 (m, 8H), 2.37−2.42 (m,

2H), 2.54−2.69 (m, 6H), 3.68 (s, 3H), 6.64 (s, 1H), 7.41 (d, J = 8.7

Hz, 2H), 7.74 (d, J = 8.7 Hz, 2H), 8.14 (s, 1H), 9.97 (s, 1H); ¹³C

NMR (DMSO-d₆): δ 171.59, 164.75, 147.62, 138.61, 132.96, 132.48,

129.30, 127.09, 96.75, 57.41, 53.55, 36.54, 35.52, 24.93, 24.59, 23.33,

23.15. Mass (ES) m/z: 375 (M + 1). UPLC (basic method); R_t= 1.22

min; area 100%. HRMS: calcd for C₂₀H₂₇ClN₄O + H⁺, 375.19462;

found (ESI, [M + H]⁺obsd), 375.19458.

1

formate salt by puriﬁcation with preparative HPLC (47% yield). H

NMR (400 MHz, DMSO-d₆): δ 1.32−1.52 (m, 2H), 1.54−1.6 (m,

2H), 1.73−1.77 (m, 2H), 2.26−2.31 (m, 2H), 2.38 −2.55 (m, 2H),

2.56−2.74 (m, 4H), 3.49 −3.73 (m, 4H), 3.78 (s, 3H), 6.73 (s, 1H),

6.98 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 8.7 Hz, 2H), 8.15 (s, 1H), 10.32

(s, 1H), 12.63 (br s, 1H). ¹³C NMR (CD₃OD): δ 171.91, 168.89,

160.22, 146.91, 144.57, 126.68, 122.73, 114.24, 93.20, 67.86, 63.72,

56.88, 56.36, 54.65, 53.13, 35.01, 26.09, 23.90, 22.40. Mass (ES) m/z:

373 (M + 1). UPLC (basic method); R_t= 0.95 min; area 97%. HRMS:

calcd for C₂₀H₂₈N₄O₃+ H⁺, 373.22342; found (ESI, [M + H]⁺obsd),

373.22346.

5-Piperidin-1-ylpentanoic Acid [5-(4-Methoxyphenyl)-2H-pyra-

zol-3-yl] Amide (8). Following the general procedure for 5-

aminopentanoic acid amide B1 and starting from commercially

available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine (39, 95 mg,

0.50 mmol), 51 mg of title compound was obtained by SiO₂column

1

chromatography (DCM/MeOH 90:10) (28% yield). H NMR (400

MHz, CD₃OD): δ 1.46−2.01 (m, 10H), 2.35−2.49 (m, 2H), 2.77−

3.58 (m, 6H), 3.78 (s, 3H), 6.72 (bs, 1H), 6.91 (d, J = 8.4 Hz, 2H),

7.51 (d, J = 8.5 Hz, 2H); ¹³C NMR (CD₃OD): δ 171.93, 160.24,

147.02, 144.52, 126.68, 122.67, 114.25, 93.28, 56.70, 54.69, 53.15,

34.98, 23.40, 23.12, 22.32, 21.52. Mass (ES) m/z: 357 (M + 1). UPLC

(acid method); R_t= 0.79 min; area 100%. HRMS: calcd for

C₂₀H₂₈N₄O₂+ H⁺, 357.22850; found (ESI, [M + H]⁺obsd),

357.22830.

Synthesis of 5-[1,4]Diazepan-1-ylpentanoic Acid [5-(4-Methox-

yphenyl)-2H-pyrazol-3-yl] Amide (23). Following the general

procedure for 5-aminopentanoic acid amide B1 and starting from

commercially available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine

(39, 110 mg, 0.58 mmol), 27 mg of title compound was obtained

by SiO₂column puriﬁcation (DCM/MeOH 90:10 to DCM/MeOH

1

NH₃2 M 80:20) (12% yield). H NMR (400 MHz, DMSO-d₆): δ

Synthesis of 5-(4-Methylpiperidin-1-yl)pentanoic Acid [5-(4-

Methoxyphenyl)-2H-pyrazol-3-yl] Amide (17). Following the general

procedure for 5-aminopentanoic acid amide B1 and starting from

commercially available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine

(39, 57 mg, 0.30 mmol), 57 mg of title compound was obtained as

the formate salt by puriﬁcation with preparative HPLC (45% yield).

¹H NMR (400 MHz, DMSO-d₆): δ 0.85 (d, J = 6.5 Hz, 3H), 0.97−

1.20 (m, 3H), 1.32 (m, 2H), 1.46−1.39 (m, 2H), 1.51 (dt, J = 14.8, 8.5

Hz, 3H), 1.96 (dd, J = 19.8, 9.1 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.34

(dd, J = 17.9, 10.7 Hz, 2H), 2.97−2.81 (m, 2H), 3.75 (s, 3H), 6.72 (s,

1H), 7.11−6.81 (m, 2H), 7.71−7.43 (m, 2H), 8.19 (s, 1H), 10.33 (s,

1H). ¹³C NMR (CD₃OD): δ 171.88, 169.02, 160.23, 146.92, 144.55,

126.66, 122.73, 114.23, 93.18, 56.32, 54.63, 52.49, 34.98, 31.07, 28.61,

1.30−1.46 (m, 2H), 1.46−1.60 (m, 2H), 1.72−1.79 (m, 2H), 2.30−

2.28 (m, 2H), 2.37−2.46 (m, 2H), 2.53−2.69 (m, 4H), 2.77−3.03 (m,

4H), 3.77 (s, 3H), 4.08 (s, 1H), 6.74 (s, 1H), 6.98 (d, J = 8.8 Hz, 2H),

7.61 (d, J = 8.8 Hz, 2H), 10.31 (s, 1H), 12.59 (s, 1H). ¹³C NMR

(CD₃OD): δ 171.83, 160.10, 146.25, 144.37, 126.55, 122.64, 113.24,

93.15, 57.26, 56.45, 56.38, 52.77, 50.15, 50.08, 36.25, 26.21, 23.17,

22.15. Mass (ES) m/z: 372 (M + 1). UPLC (acid method); R_t= 0.82

min; area 95%. HRMS: calcd for C₂₀H₂₉N₅O₂+ H⁺, 372.23940; found

(ESI, [M + H]⁺obsd), 372.23940.

Synthesis of 5-(5-Oxo[1,4]diazepan-1-yl)pentanoic Acid [5-(4-

Methoxyphenyl)-2H-pyrazol-3-yl] Amide (24). Following the general

procedure for 5-aminopentanoic acid amide B1 and starting from

commercially available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine

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(39, 57 mg, 0.30 mmol), 93 mg of title compound was obtained as the

formate salt by puriﬁcation with preparative HPLC (60% yield). H

5-Piperidin-1-ylpentanoic Acid [5-(3-Methoxyphenyl)-2H-pyra-

zol-3-yl] Amide (9). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from 5-(3-methoxyphen-

yl)-2H-pyrazol-3-ylamine hydrochloride (48, 212 mg, 0.94 mmol), 120

mg of the title compound was isolated by CH₃CN wash (35%, yield).

¹H NMR (400 MHz, DMSO-d₆): δ 1.29−1.62 (m, 10H), 2.16−2.36

(m, 8H), 3.79 (s, 3H), 6.81−6.93 (m, 2H), 7.26−7.330 (m, 2H),

7.30−7.37 (m, 1H), 10.32 (bs, 1H), 12.76 (bs, 1H). ¹³C NMR

(CD₃OD): δ 172.59, 160.43, 144.76, 143.92, 131.55, 129.90, 117.67,

113.93, 110.54, 93.92, 58.88, 54.57, 54.26, 35.77, 25.72, 25.23, 23.98,

23.69. Mass (ES) m/z: 357 (M + 1). UPLC (acid method); R_t= 0.82

min; area 94%. HRMS: calcd for C₂₀H₂₈N₄O₂+ H⁺, 357.22850; found

(ESI, [M + H]⁺obsd), 357.22839.

5-Piperidin-1-ylpentanoic Acid [5-(2-Methoxyphenyl)-1H-pyra-

zol-3-yl] Amide (10). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from 5-(2-methoxyphen-

yl)-1H-pyrazol-3-ylamine (49, 189 mg, 1.0 mmol), 24 mg of the title

compound was obtained as the formate salt by preparative HPLC and

crystallization with CH₃CN (6% yield). ¹H NMR (400 MHz, DMSO-

d₆): δ 1.34−1.60 (m, 10H), 2.30 (t, J = 6.9 Hz, 2H), 2.46−2.54 (m,

6H), 3.86 (s, 3H), 6.89 (s, 1H), 7.00 (td, J = 7.5, 1.1 Hz, 1H), 7.11

(dd, J = 8.4, 0.9 Hz, 1H), 7.23−7.38 (m, 1H), 7.62 (dd, J = 7.7, 1.6 Hz,

1H), 8.19 (s, 1H), 10.32 (s, 1H), 12.54 (br s, 1H). ¹³C NMR

(CD₃OD): δ 171.90, 168.89, 156.46, 146.80, 140.79, 129.64, 127.80,

120.89, 118.05, 111.58, 95.65, 56.70, 54.88, 53.08, 34.97, 23.42, 23.15,

22.37, 21.64. Mass (ES) m/z: 357 (M + 1). UPLC (basic method); R_t

= 0.98 min; area 100%. HRMS: calcd for C₂₀H₂₈N₄O₂+ H⁺,

357.22850; found (ESI, [M + H]⁺obsd), 357.22831.

1

NMR (400 MHz, DMSO-d₆): δ 1.08 (t, J = 12.9 Hz, 1H), 1.39−1.43

(m, 2H), 1.64−1.48 (m, 2H), 2.28 (t, J = 7.3 Hz, 2H), 2.39−2.32 (m,

4H), 2.55−2.41 (m, 2H), 3.01−3.09 (m, 2H), 3.22−3.13 (m, 2H),

3.76 (s, 3H), 6.72 (s, 1H), 7.12−6.84 (m, 2H), 7.49 (s, 1H), 7.68−

7.51 (m, 2H), 8.16 (s, 1H), 10.31 (s, 1H). ¹³C NMR (CD₃OD): δ

177.26, 172.26, 166.70, 160.23, 146.99, 144.50, 126.66, 122.71, 114.20,

93.21, 57.60, 56.19, 54.61, 49.91, 39.75, 35.27, 34.72, 24.71, 22.81.

Mass (ES) m/z: 386 (M + 1). UPLC (acid method); R_t= 0.63 min;

area 98%. HRMS: calcd for C₂₀H₂₇N₅O₃+ H⁺, 386.21867; found (ESI,

[M + H]⁺obsd), 386.21846.

5-(4-Acetyl-[1,4]diazepan-1-yl)pentanoic Acid [5-(4-Methoxy-

phenyl)-1H-pyrazol-3-yl] Amide (25). Following the general

procedure for 5-aminopentanoic acid amide B1 and starting from

commercially available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine

(39, 1.0 g, 5.56 mmol), 380 mg of title compound was obtained by

1

SiO₂column chromatography (DCM/MeOH 90:10) (14% yield). H

NMR (400 MHz, DMSO-d₆): δ 1.33−1.45 (m, 2H), 1.49−1.60 (2H,

m), 1.62−1.69 (m, 1H), 1.71−1.79 (m, 1H), 1.95 (s, 3H), 2.25−2.33

(m, 2H), 2.35−2.45 (m, 2H), 2.51−2.65 (m, 4H), 3.38−3.47 (m, 4H),

3.77 (s, 3H), 6.77 (bs, 1H), 6.98 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 8.7

Hz, 2H), 10.31 (s, 1H), 12.60 (s, 1H); ¹³C NMR (CD₃OD): δ 172.59,

171.97, 160.22, 147.57, 144.35, 126.65, 122.50, 114.19, 93.28, 57.20,

55.52, 54.60, 54.01, 43.65, 35.57, 26.91, 26.43, 25.87, 23.21, 20.33.

Mass (ES) m/z: 414 (M + 1). UPLC (acid method); R_t= 0.69 min;

area 100%. HRMS: calcd for C₂₂H₃₁N₅O₃+ H⁺, 414.24997; found

(ESI, [M + H]⁺obsd), 414.24966.

General Procedure for 5-Aminopentanoic Acid Amide. Route B2,

One-Pot Synthesis. To a solution of 5-bromovaleroyl chloride (0.94

mmol, 1 equiv) in DMA (1 mL) cooled at 0 °C was added a solution

of 3-amino-5-aryl/heteroaryl heterocycle (0.94 mmol, 1 equiv) and

diisopropylethylamine (1.88 mmol, 2 equiv) in DMA (2 mL), and the

reaction was stirred for 1 h at 0 °C. The secondary amines (2.35

mmol, 2.5 equiv) and NaI (0.94 mmol, 1 equiv) were then added. The

reaction mixture was generally heated at 60 °C for 24−48 h. Upon

complete conversion of the bromo-intermediate (as monitored by LC-

MS), the solvent was removed under reduced pressure. The residue

was taken up in DCM (2 mL) and washed with Na₂CO₃saturated

water solution. The organic phase was concentrated under reduced

pressure and the crude products were either recrystallized from

CH₃CN or puriﬁed by SiO₂column or by preparative HPLC

(standard acidic conditions).

5-Piperidin-1-ylpentanoic Acid [5-(4-Chlorophenyl)-1H-pyrazol-

3-yl] Amide (3). Following the general procedure for 5-amino-

pentanoic acid amide B2 and starting from commercially available 5-

(4-chlorophenyl)-1H-pyrazol-3-ylamine (46, 187 mg, 0.94 mmol), 40

mg of title compound was obtained by SiO₂(DCM/MeOH 90:10)

(10%, yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.28−1.56 (m, 10H),

2.20−2.42 (m, 8H), 6.88 (s, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.69 (d, J =

8.4 Hz, 2H), 10.35 (s, 1H), 12.80 (s, 1H). ¹³C NMR (CD₃OD): δ

171.07, 148.85, 141.29, 133.06, 129.63, 129.63, 127.30, 94.51, 58.44,

54.34, 35.91, 26.05, 25.68, 24.30, 23.62. Mass (ES) m/z: 361 (M + 1).

UPLC (basic method); R_t= 1.13; area 98%. HRMS: calcd for

C₁₉H₂₅ClN₄O + H⁺, 361.17897; found (ESI, [M + H]⁺obsd),

361.17897.

5-Piperidin-1-ylpentanoic Acid [5-(4-Methoxyphenyl)-4-methyl-

2H-pyrazol-3-yl] Amide (11). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from 5-(4-methoxyphen-

yl)-4-methyl-2H-pyrazol-3-ylamine (44, 100.0 mg, 0.50 mmol), 53.0

mg of the title compound as the formate salt was obtained as a solid by

1

preparative HPLC (29% yield). H NMR (400 MHz, DMSO-d₆): δ

1.30−1.63 (m, 10H), 1.92 (s, 2H), 2.24−2.45 (m, 6H), 3.15 (s, 3H),

3.78 (s, 3H), 7.02 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 8.19

(s, 1H), 9.57 (s, 1H), 12.51 (br s, 1H). ¹³C NMR (CD₃OD): δ 174.12,

159.99, 152.03, 139.95, 128.50, 123.34, 114.12, 92.30, 58.26, 54.64,

53.94, 35.22, 25.07, 24.64, 23.43, 23.30, 7.65. Mass (ES) m/z: 371 (M

+ 1). UPLC (basic method); R_t= 0.96 min; area 100%. HRMS: calcd

for C₂₁H₃₀N₄O₂+ H⁺, 371.24415; found (ESI, [M + H]⁺obsd),

371.24384.

5-Piperidin-1-ylpentanoic Acid [5-(4-Fluorophenyl)-2H-pyrazol-3-

yl] Amide (12). Following the general procedure for 5-aminopentanoic

acid amide B2 and starting from commercially available 5-(4-

ﬂuorophenyl)-2H-pyrazol-3-ylamine (42, 0.35 g, 2.00 mmol), 87.4

mg of title compound as the formate salt was obtained as a solid by

1

preparative HPLC (19%, yield). H NMR (400 MHz, DMSO-d₆): δ

1.32−1.61 (m, 10H), 2.30 (t, J = 7.0 Hz, 2H), 2.46−2.57 (m, 6H),

6.79 (s, 1H), 7.26 (t, J = 8.9 Hz, 2H), 7.82−7.64 (m, 2H), 8.23 (s,

1H), 8.23 (s, 1H), 12.49 (brs, 1H). ¹³C NMR (CD₃OD): δ 171.90,

169.08, 162.93 (d, J = 246.6 Hz), 146.47, 144.17, 127.39, 126.92,

115.67 (d, J = 22.1 Hz), 93.59, 56.61, 52.96, 34.98, 23.36, 23.08, 22.39,

21.66. Mass (ES) m/z: 345 (M + 1). UPLC (acid method); R_t= 0.82

min; area 98%. HRMS: calcd for C₁₉H₂₅FN₄O + H⁺, 345.20852; found

(ESI, [M + H]⁺obsd), 345.20835.

Synthesis of 5-Piperidin-1-ylpentanoic Acid [4-(4-Chlorophenyl)-

thiazol-2-yl] Amide (5). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from commercially

available 4-(4-chlorophenyl)-2H-thiazol-2-ylamine (38, 95 mg, 0.45

mmol), 13 mg of title compound was obtained as formate salt by

5-Piperidin-1-ylpentanoic Acid [5-(4-Bromophenyl)-2H-pyrazol-

3-yl] Amide (13). Following the general procedure for 5-amino-

pentanoic acid amide B2 and starting from commercially available 5-

(4-bromophenyl)-2H-pyrazol-3-ylamine (43, 0.50 g, 2.10 mmol),

230.0 mg of the title compound was obtained by crystallization with

1

puriﬁcation with preparative HPLC (9%, yield). H NMR (400 MHz,

ethyl acetate (27% yield). H NMR (400 MHz, DMSO-d₆): δ 1.30−

CD₃OD): δ 1.66 (s, 2H), 1.95−1.72 (m, 8H), 2.58 (t, J = 6.6 Hz, 2H),

3.16−2.95 (m, 3H), 3.30 (dt, J = 3.3, 1.6 Hz, 2H), 7.43−7.27 (m, 3H),

7.94−7.77 (m, 2H), 8.37 (s, 2H). ¹³C NMR (CD₃OD): δ 171.77,

169.11, 158.23, 148.73, 133.49, 133.31, 128.54, 127.30, 107.90, 56.84,

53.17, 34.43, 23.60, 23.33, 22.06, 21.87. Mass (ES) m/z: 378 (M + 1).

UPLC (basic method); R_t= 1.50; area 100%. HRMS: calcd for

C₁₉H₂₄ClN₃OS + H⁺, 378.14014; found (ESI, [M + H]⁺obsd),

378.13990.

1.42 (m, 1H), 1.53−1.71 (m, 7H), 1.74−1.83 (m, 2H), 2.31−2.39 (m,

2H), 2.77−2.79 (m, 2H), 2.97−3.04 (m, 2H), 3.36−3.45 (m, 2H),

6.91 (s, 1H), 7.64 (s, 4H), 8.84 (s, 1H), 12.87 (s, 1H). ¹³C NMR

(DMSO-d₆): δ 170.52, 148.89, 141.14, 132.61, 129.16, 127.59, 121.79,

94.63, 56.16, 52.66, 35.36, 23.57, 23.21, 22.80, 21.94. Mass (ES) m/z:

405 (M + 1). UPLC (acid method); R_t= 1.03 min; area 100%. HRMS:

calcd for C₁₉H₂₅BrN₄O + H⁺, 405.12845; found (ESI, [M + H]⁺

obsd), 405.12861.

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5-Piperidin-1-ylpentanoic Acid (5-Pyridin-4-yl-1H-pyrazol-3-yl)

Amide (14). Following the general procedure for 5-aminopentanoic

acid amide B2 and starting from 5-pyridin-4-yl-1H-pyrazol-3-ylamine

(45, 151 mg, 0.94 mmol) and piperidine as secondary amine, 30 mg of

title compound was obtained by SiO₂column chromatography (10%

yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.33−1.60 (m, 10H),

2.28−2.48 (m, 8H), 7.03 (bs, 1H), 7.68 (dd, J = 4.6, 1.6 Hz, 2H), 8.59

(d, J = 6.0 Hz, 2H), 10.49 (s, 1H), 13.11 (s, 1H). ¹³C NMR (DMSO-

d₆): δ 171.15, 150.99, 147.92, 141.31, 137.56, 119.78, 95.08, 58.27,

54.22, 35.85, 25.87, 25.51, 24.15, 23.54. Mass (ES) m/z: 328 (M + 1).

UPLC (basic method); R_t= 0.61 min; area 100%. HRMS: calcd for

C₁₈H₂₅N₅O + H⁺, 328.21319; found (ESI, [M + H]⁺obsd),

328.21307.

5-Piperidin-1-ylpentanoic Acid [5-(4-Triﬂuoromethylphenyl)-1H-

pyrazol-3-yl] Amide (15). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from 5-(4-triﬂuorome-

thylphenyl)-1H-pyrazol-3-ylamine (47, 214 mg, 0.94 mmol), 30 mg of

title compound was obtained by SiO₂(DCM/MeOH 90:10) (10%,

yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.29−1.59 (m, 10H),

2.15−2.33 (m, 8H), 6.98 (s, broad, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.91

(d, J = 7.9 Hz, 2H), 10.42 (s, 1H), 12.97 (s, 1H). ¹³C NMR (DMSO-

d₆) δ 171.25, 156.11, 141.54, 136.36, 128.92−128.23 (m), 126.52,

126.13, 124.87 (q, J = 271.5 Hz), 94.82, 58.96, 54.73, 36.03, 26.63,

26.26, 24.85, 23.80. Mass (ES) m/z: 395 (M + 1). UPLC (basic

method); R_t= 1.25 min; area 100%. HRMS: calcd for C₂₀H₂₅F₃N₄O +

H⁺, 395.20532; found (ESI, [M + H]⁺obsd), 395.20503.

5-Piperidin-1-ylpentanoic Acid (5-Furan-2-yl-2H-pyrazol-3-yl)

Amide (16). Following the general procedure for 5-aminopentanoic

acid amide B2 and starting from commercially available 5-furan-2-yl-

2H-pyrazol-3-ylamine (41, 0.30 g, 2.03 mmol), 96.0 mg of title

compound as the formate salt was obtained as a solid by preparative

HPLC (15%, yield). ¹H NMR (400 MHz, CD₃OD): δ 1.54−1.94 (m,

10H), 2.48 (t, J = 6.7 Hz, 2H), 3.05−3.12 (m, 2H), 3.00−3.39 (m,

4H), 6.52 (dd, J = 3.4, 1.8 Hz, 1H), 6.66 (s, 1H), 6.70 (d, J = 3.4 Hz,

1H), 7.56 (d, J = 1.7 Hz, 1H), 8.48 (s, 1H). ¹³C NMR (CD₃OD): δ

171.86, 169.12, 146.15, 145.89, 142.61, 136.72, 111.40, 106.48, 92.77,

56.63, 52.99, 34.98, 23.38, 23.10, 22.37, 21.66. Mass (ES) 317 m/z:

(M + 1). UPLC (basic method); R_t= 0.76 min; area 100%. HRMS:

calcd for C₁₇H₂₄N₄O₂+ H⁺, 317.19720; found (ESI, [M + H]⁺obsd),

317.19716.

general procedure for 5-aminopentanoic acid amide B2 and starting

from 5-(4-methoxyphenyl)-4-methyl-2H-pyrazol-3-ylamine (44, 100.0

mg, 0.50 mmol), 34.0 mg of title compound was obtained by SiO₂

column puriﬁcation (DCM/MeOH 90:10 to DCM/MeOH NH₃2 M

1

80:20) (12% yield). (16% yield). H NMR (400 MHz, CD₃OD): δ

1.43−1.62 (m, 4H), 1.67−1.77 (m, 1H), 1.80−1.88 (m, 1H), 1.92 (s,

3H), 1.98 (s, 3H), 2.26−2.34 (m, 2H), 2.53−2.70 (m, 3H), 2.72−2.83

(m, 3H), 3.40−3.54 (m, 4H), 3.78 (s, 3H), 7.02 (d, J = 8.8 Hz, 2H),

7.47 (d, J = 8.7 Hz, 2H), 8.12 (s, 1H), 9.58 (s, 1H). ¹³C NMR

(CD₃OD): δ 172.28, 168.73, 160.02, 144.12, 142.15, 128.50, 123.11,

114.12, 106.62, 57.13, 55.31, 54.62, 53.67, 43.68, 41.48, 34.82, 25.56,

24.64, 22.75, 20.44, 7.60. Mass (ES) m/z: 428 (M + 1). UPLC (basic

method); R_t= 0.96 min; area 100%. HRMS: calcd for C₂₃H₃₃N₅O₃+

H⁺, 428.26562; found (ESI, [M + H]⁺obsd), 428.26532.

Procedure for 5-(4-Acetyl[1,4]diazepan-1-yl)-2-methylpentanoic

Acid [5-(4-Methoxyphenyl)-2H-pyrazol-3-yl] Amide (27). Route C. 5-

Amino-3-(4-methoxyphenyl)pyrazole-1-carboxylic Acid tert-Butyl

Ester (54). To a vigorously stirred solution of commercially available

5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine (39, 500 mg, 2.64 mmol)

in DCM (20 mL) was added an aqueous solution of 4.5 N KOH (4.7

mL, 21.1 mmol), followed by di-tert-butyl dicarbonate (605 mg, 2.77

mmol). The mixture was stirred for 20 h, and then 20 mL of water was

added and the organic phase was collected and the solvent was

removed under reduced pressure. The crude was puriﬁed by SiO₂

column chromatography, eluting with DCM, aﬀording 720 mg of the

title product. ¹H NMR (400 MHz, DMSO-d₆): δ 1.57 (s, 9H), 3.77 (s,

3H), 5.68 (s, 1H), 6.34 (bs, 2H), 6.95 (d, J = 7.8 Hz, 2H), 7.67 (d, J =

7.8 Hz, 2H). Mass (ES) m/z: 290 (M + 1).

2-(3-Bromopropyl)-2-methylmalonic Acid (52). NaH (60%

mineral oil dispersion, 3.25 g, 81 mmol) was placed in a ﬂask under

inert atmosphere, anhydrous THF (60 mL) was added, and the

mixture was cooled down to 0 °C. A solution of 2-methylmalonic acid

dimethyl ester (50, 9.9 g, 67.7 mmol) in THF (15 mL) was added

dropwise, and the reaction was left stirring at 0 °C for 15 min. 1,3-

Dibromopropane (51, 47.8 g, 237 mmol) was added in one portion,

and after 15 min the reaction was allowed to warm up to room

temperature and left stirring for 18 h. 1 N NaOH solution was

carefully added, and the product was extracted with ethyl acetate. The

organic phase was collected and the solvent removed under reduced

pressure; the crude was puriﬁed by SiO₂column chromatography

(cyclohexane/ethyl acetate 50:50), aﬀording 15.1 g of the title

5-Morpholin-4-ylpentanoic Acid [5-(4-Methoxyphenyl)-1H-pyra-

zol-3-yl] Amide (18). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from the commercially

available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine (39, 568 mg, 3

mmol), 110 mg of the title compound as the formate salt was obtained

1

compound (84%, yield). H NMR (400 MHz, DMSO-d₆): δ 1.32 (s,

3H), 1.64−1.75 (m, 2H), 1.85−1.93 (m, 2H), 3.51 (t, J = 6.2 Hz, 2H),

3.64 (s, 6.2H).

5-Bromo-2-methylpentanoic Acid (53). 2-(3-Bromopropyl)-2-

methylmalonic acid (53, 10 g, 37.4 mmol) was mixed with 48%

aqueous HBr (80 mL, 589 mmol), and the mixture was heated at 110

°C for 24 h. The mixture was allowed to cool down to room

temperature and then to 0 °C. 100 mL of H₂O and NaOH (22 g, 550

mml) was carefully added, and the product was extracted with 200 mL

of a mixture of DCM/MeOH (95:5). The organic phase was collected

and the solvent removed under reduced pressure, aﬀording 6.6 g of

title compound (84%, yield). ¹H NMR (400 MHz, DMSO-d₆): δ 1.03

(d, J = 7.1 Hz, 3H), 1.38−1.48 (m, 1H), 1.58−1.60 (m, 1H), 1.72−

1.82 (m, 2H), 2.26−2.38 (m, 1H), 3.50 (t, J = 7.8 Hz, 2H), 12.13 (bs,

1H).

5-[5-(4-Acetyl[1,4]diazepan-1-yl)-2-methylpentanoylamino]-3-

(4-methoxyphenyl)pyrazole-1-carboxylic Acid tert-Butyl Ester (56).

To a solution of 5-bromo-2-methylpentanoic acid (53, 330 mg, 1.70

mmol) in DCM (1 mL) was added oxalyl chloride (219 μL, 2.55

mmol), and the mixture was stirred for 2 h under nitrogen ﬂow.

Solvent and excess oxalyl chloride were removed under reduced

pressure. The residue was dissolved in 1 mL of DCM and added

dropwise to a solution of 5-amino-3-(4-methoxyphenyl)pyrazole-1-

carboxylic acid tert-butyl ester (54, 490 mg, 1.70 mmol) and

triethylamine (236 μL, 1.70 mmol) in DCM (3 mL). The mixture

was stirred at room temperature for 48 h. NaHCO₃saturated solution

was added, the organic phase was collected, and the solvent was

removed under reduced pressure. 5-(5-Bromo-2-methylpentanoylami-

no)-3-(4-methoxyphenyl)pyrazole-1-carboxylic acid tert-butyl ester

1

by puriﬁcation with preparative HPLC (10% yield). H NMR (400

MHz, DMSO-d₆): δ 1.38−1.47 (m, 2H), 1.51−1.61 (m, 2H), 2.23−

2.35 (m, 8H), 3.51−3.56 (m, 4H), 3.76 (s, 3H), 6.73 (s, 1H), 6.98 (d,

J = 8.7 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 8.13 (s, 1H), 10.31 (s, 1H),

12.76 (br s, 1H). ¹³C NMR (DMSO-d₆): δ 170.99, 163.99, 159.72,

148.20, 142.79, 126.98, 123.09, 115.01, 93.32, 66.70, 58.46, 55.85,

53.87, 35.94, 25.99, 23.64. Mass (ES) m/z: 359 (M + 1). UPLC (basic

method); R_t= 1.0 min; area 95%. HRMS: calcd for C₁₉H₂₆N₄O₃+ H⁺,

359.20777; found (ESI, [M + H]⁺obsd), 359.20785.

Synthesis of 5-Azepan-1-ylpentanoic Acid [5-(4-Methoxyphenyl)-

2H-pyrazol-3-yl] Amide (21). Following the general procedure for 5-

aminopentanoic acid amide B2 and starting from the commercially

available 5-(4-methoxyphenyl)-2H-pyrazol-3-ylamine (39, 89 mg, 0.45

mmol), 46 mg of title compound as the formate salt was obtained by

puriﬁcation with preparative HPLC (27% yield). ¹H NMR (400 MHz,

CD₃OD): δ 1.69−1.96 (m, 12H), 2.49 (t, J = 7.2 Hz, 2H), 3.15−3.21

(m, 2H), 3.28−3.35 (m, 4H), 3.83 (s, 3H), 6.70 (s, 1H), 6.99 (d, J =

8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 8.46 (s, 1H). ¹³C NMR

(CD₃OD): δ 171.92, 169.00, 160.25, 146.96, 144.43, 126.64, 122.67,

114.21, 93.22, 57.10, 54.66, 54.62, 34.90, 26.23, 23.82, 23.69, 22.28.

Mass (ES) m/z: 371 (M + 1). UPLC (basic method); R_t= 1.0 min;

area 95%. HRMS: calcd for C₂₁H₃₀N₄O₂+ H⁺, 371.24415; found (ESI,

[M + H]⁺obsd), 371.24407.

5-(4-Acetyl[1,4]diazepan-1-yl)pentanoic Acid [5-(4-Methoxy-

phenyl)-4-methyl-2H-pyrazol-3-yl] Amide (26). Following the

M

dx.doi.org/10.1021/jm300247y | J. Med. Chem. XXXX, XXX, XXX−XXX

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(55, 442 mg) was obtained after SiO₂column chromatography

(DCM/cyclohexane 50:50).

General Procedure for 3-Amino-5-aryl/Heteroaryl Pyrazole.

Route D2. To a solution of dry alkanenitrile in toluene (1 mmol/

mL, 5 equiv) cooled down to −78 °C under nitrogen was added

dropwise a solution of n-butyllithium in n-hexane (1.6 N, 3.5 equiv).

The mixture was left stirring at −78 °C for 20 min, and then a solution

of the aryl or heteroaryl methyl carboxylate in toluene (0.75 mmol/

mL, 1 equiv) was added and the reaction was allowed to reach room

temperature. Upon reaction completion, after about 20 min, the

mixture was cooled down to 0 °C and HCl 2 N was added to pH 2.

The organic phase was recovered, dried over Na₂SO₄, and

concentrated under reduced pressure, aﬀording the title product,

which was generally used without further puriﬁcation.

To a solution of the β-ketonitrile (7.5 mmoL), in absolute EtOH

(15 mL), was added hydrazine monohydrate (0.44 mL, 9.0 mmol),

and the reaction was heated at reﬂux for 18 h. The reaction mixture

was allowed to cool to room temperature, and the solvent was

evaporated under reduced pressure. The residue was dissolved in

DCM and washed with water.

Triethylamine (80 μL, 0.6 mmol) and N-acetyl diazepine (158 μL,

0.6 mmol) were added to a solution of 5-(5-bromo-2-methylpenta-

noylamino)-3-(4-methoxyphenyl)pyrazole-1-carboxylic acid tert-butyl

ester (55, 280 mg, 0.6 mmol) in DCM. The reaction mixture was

stirred for 24 h at room temperature and then for 15 h at 50 °C.

NaHCO₃saturated solution was added, the organic phase was

collected, and the solvent was removed under reduced pressure. The

crude was puriﬁed by SiO₂column chromatography (DCM/MeOH,

98:2), aﬀording 181 mg of the title compound (57%, yield). ¹H NMR

(400 MHz, DMSO-d₆): δ 1.08 (m, 5H), 1.31−1.50 (m, 3H), 1.60 (s,

9H), 1.69−1.81 (m, 1H), 1.92−1.98 (m, 3H), 2.33−2.44 (m, 2H),

2.50−2.71 (m, 2H), 2.96−3.11 (m, 2H), 3.37−3.53 (m, 5H), 3.78 (s,

3H), 6.99 (d, J = 8.7 Hz, 2H), 7.04 (bs, 1H), 7.77 (d, J = 8.7 Hz, 2H),

10.04 (bs, 1H).

5-(4-Acetyl[1,4]diazepan-1-yl)-2-methylpentanoic Acid [5-(4-Me-

thoxyphenyl)-2H-pyrazol-3-yl] Amide (27). 5-[5-(4-Acetyl[1,4]-

diazepan-1-yl)-2-methylpentanoylamino]-3-(4-methoxyphenyl)-

pyrazole-1-carboxylic acid tert-butyl ester (56, 181 mg, 0.344 mmol)

was dissolved in 3 mL of DCM, and a solution of 4 N HCl in dioxane

(0.16 mL, 0.648 mmol) was added. The mixture was stirred for 3 h at

room temperature, and then NaHCO₃saturated solution was added,

the organic phase was collected, and the solvent removed under

reduced pressure. 150 mg of the title compound was isolated (82%,

yield).

The organic phase was concentrated under reduced pressure to give

a product that, if not pure enough, was puriﬁed by SiO₂column or by

precipitation from Et₂O. Yields were generally between 65 and 90%.

5-Pyridin-4-yl-2H-pyrazol-3-ylamine (45). Following the general

procedure for 3-amino-5-aryl/heteroaryl pyrazole D2 and starting from

commercially available isonicotinic acid methyl ester (3.0 g, 21.9

1

mmol), 1.8 g of the title compound was obtained (40% yield). H

NMR (400 MHz, DMSO-d₆): δ 5.00 (bs, 2H), 5.85 (s, 1H), 7.59 (dd,

J = 4.6, 1.6 Hz, 2H), 8.50 (d, J = 6.0 Hz, 2H), 11.83 (s, 1H). Mass

(ES) m/z: 161 (M + 1).

5-(4-(Triﬂuoromethyl)phenyl)-2H-pyrazol-3-ylamine (47). Follow-

ing the general procedure for 3-amino-5-aryl/heteroaryl pyrazole D2

and starting from commercially available 4-triﬂuoromethylbenzoic acid

methyl ester (5.0 g, 3.94 mL, 24.5 mmol), 5.35 g of the title compound

was obtained (40% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 5.10 (s,

broad, 2H), 5.77 (s, 1H), 7.68 (d, J = 7.1 Hz, 2H), 7.85 (d, J = 8.0 Hz,

2H), 11.92 (bs, 1H). Mass (ES) m/z: 228 (M + 1).

¹H NMR (400 MHz, DMSO-d₆): δ 1.03 (d, J = 6.6 Hz, 3H), 1.22−

1.43 (m, 3H), 1.46−1.59 (m, 1H), 1.59−1.68 (m, 1H), 1.68−1.77 (m,

1H), 1.95 (s, 3H), 2.32−2.42 (m, 2H), 2.45−2.53 (m, 2H), 2.55−2.63

(m, 2H), 3.29−3.46 (m, 5H), 3.76 (s, 3H), 6.78 (bs, 1H), 6.98 (d, J =

8.7 Hz, 2H), 7.61 (d, J = 8.7 Hz, 2H), 10.30 (bs, 1H), 12.61 (br s,

1H). ¹³C NMR (CD₃OD): δ 176.09, 171.89, 160.20, 147.33, 144.18,

126.66, 122.70, 114.21, 93.49, 57.42, 55.51, 54.62, 54.09, 44.25, 44.05,

40.79, 31.91, 27.25, 24.74, 20.41, 17.32. Mass (ES) m/z: 428 (M + 1).

UPLC (basic method); R_t= 1.02; area 100%. HRMS: calcd for

C₂₃H₃₃N₅O₃+ H⁺, 428.26562; found (ESI, [M + H]⁺obsd),

428.26549.

General Procedure for 3-Amino-5-aryl/Heteroaryl Pyrazole.

Route D1. To a solution of an aryl or heteroaryl methyl carboxylate

(6.5 mmol) in dry toluene (6 mL) under N₂was carefully added NaH

(50−60% dispersion in mineral oil, 624 mg, 13 mmol). The mixture

was heated at 80 °C, and then dry CH₃CN was added dropwise (1.6

mL, 30.8 mmol). The reaction was heated for 18 h, and generally the

product precipitated from the reaction mixture as the Na salt.

The reaction was then allowed to cool down to room temperature,

and the solid formed was ﬁltered and then dissolved in water. The

solution was then acidiﬁed with 2 N HCl solution, and at pH between

2 and 6 (depending on the ring substitution on the aryl/heteroaryl

system) the product precipitated and was ﬁltered oﬀ. If no

precipitation occurred, the product was extracted with DCM.

After workup, the products were generally used in the following step

without further puriﬁcation. The general yield was between 40 and

80%.

5-(3-Methoxyphenyl)-2H-pyrazol-3-ylamine (48). Following the

general procedure for 3-amino-5-aryl/heteroaryl pyrazole D2 and

starting from commercially available 3-methoxybenzoic acid methyl

ester (2.98 g, 18.0 mmol), 1.8 g of title compound was obtained by

1

precipitation with Et₂O (42% yield). H NMR (400 MHz, CDCl₃): δ

3.78 (s, 3H), 4.97 (bs, 4H), 5.87 (s, 1H), 6.81−6.88 (m, 1H), 7.07−

7.16 (m, 1H), 2.23−7.31 (m, 1H). Mass (ES) m/z: 190 (M + 1).

5-(2-Methoxyphenyl)-2H-pyrazol-3-ylamine (49). Following the

general procedure for 3-amino-5-aryl/heteroaryl pyrazole D2 and

starting from commercially available 3-methoxybenzoic acid methyl

ester (2.5 mL, 18.0 mmol). 1.40 g of the title compound was obtained

by precipitation from Et₂O (42% yield). ¹H NMR (400 MHz,

CD₃OD): δ 3.82 (s, 3H), 5.92 (bs, 1H), 6.85−6.90 (m, 1H), 7.16−

7.33 (m, 3H). Mass (ES) m/z: 190 (M + 1).

3. Biology. Ca²⁺-Flux and Membrane Potential Measure-

ments with a Fluorescence Imaging Plate Reader. The following

recombinant cell lines were used as speciﬁc sources of receptors:

GH4C1 cells stably transfected with pCEP4/rat α7 nAChR as

previously described,³⁵HEK293 cell lines stably expressing human 5-

HT3A receptors.³⁵Native neuroblastoma SH-SY5Y cells were used as

a source of human ganglionic nAChRs (α3), and TE671

rhabdomyosarcoma cells were used as an endogenous source of

muscle α1β1δγ receptors. GH4C1 cells expressing α7 and HEK cells

expressing 5-HT3A receptors were analyzed by Ca²⁺-ﬂux measure-

ments employing a ﬂuorometric imaging plate reader (FLIPR,

Molecular Devices) system, whereas the cells expressing the nicotinic

receptor subunits α1 and α3 were tested in the FLIPR system with a

membrane potential sensitive dye. For Ca²⁺-ﬂux analysis, cells were

plated in 96-well clear-bottom, poly ^D-lysine coated black microtiter

plates (Costar) at a density of 1 × 10⁵cells/well for α7 expressing

GH4C1 cells or 8 × 10⁴cells/well for 5-HT3A expressing HEK293

cells and cultured for 24 h prior to experiments. The medium was then

replaced with 100 μL of Hank’s balanced salt solution-HEPES 20 mM,

pH 7.4 (assay buﬀer) containing 4 μM Fluo-4 a.m., 0.02% pluronic

acid, and 5 mM probenecid. After 40 min of incubation at 37 °C, the

To a solution of the β-ketonitrile (7.5 mmoL) in absolute EtOH

(15 mL) was added hydrazine monohydrate (0.44 mL, 9.0 mmol), and

the reaction was heated at reﬂux for 18 h. The reaction mixture was

allowed to cool down to room temperature, and the solvent was

evaporated under reduced pressure. The residue was dissolved in

DCM and washed with water.

The organic phase was concentrated under reduced pressure to give

a product that, if not pure enough, was puriﬁed by SiO₂column or by

precipitation from Et₂O. Yields were generally between 65 and 90%.

5-(4-Methoxyphenyl)-4-methyl-2H-pyrazol-3-ylamine (44). Fol-

lowing the general procedure for 3-amino-5-aryl/heteroaryl pyrazole

D1 and starting from commercially available 4-methoxybenzoic acid

methyl ester (3.0 mL, 18.0 mmol) were obtained 2.1 g of title

compound by column chromatography (DCM/MeOH 90:10) (62%

1

yield). H NMR (400 MHz, CDCl₃): δ 2.03 (s, 3H), 3.58 (brs, 2H),

3.84 (s, 3H), 6.96−6.98 (m, 2H), 7.37−7.39 (m, 2H). Mass (ES) m/z:

204 (M + 1).

N

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Journal of Medicinal Chemistry

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labeling solution was replaced with 200 μL of assay buﬀer containing

2.5 mM probenecid. Plates were then transferred to the FLIPR system.

Compounds to be tested were prepared in assay buﬀer as 5×-

concentrated solutions in a separate 96-well polypropylene plate. Basal

ﬂuorescence was recorded for 30 s, followed by addition of 50 μL of

test compound (to assess agonist activity; ﬁrst addition). Measure-

ments were made at 1 s intervals for 1 min, followed by measurements

every 30 s for 10 min. Subsequently, for the second addition for alpha-

7 expressing GH4C1 cells, nicotine was added to each well except

negative controls at the EC₈₀ﬁnal concentration to assess the

antagonism of the nicotine response or at the EC₂₀ﬁnal concentration

to assess the positive modulation of the nicotine response. Assay

performance was robust, as reﬂected by a Z′ factor > 0.6. For testing 5-

HT3A receptor activity, m-chlorophenylbiguanide (CPBG, EC₈₀ﬁnal

concentration) was added to each well except negative controls.

Measurements were made at 1 s intervals for 1 min after the second

addition and at 3 s intervals for the remaining 3 min. Results were

exported from the FLIPR raw data as MAX-MIN of the ﬂuorescence

signal intensity in two intervals corresponding to the ﬁrst and the

second addition of compounds. The responses were normalized to the

positive control and EC₅₀and IC₅₀values were calculated using XlFit

version 4.2, with a sigmoidal concentration−response (variable slope)

equation: Y = bottom + (top − bottom)/(1 + (EC₅₀/X)^{Hill Slope}),

where X was the concentration, Y was the response, bottom was the

bottom plateau of the curve, and top was the top plateau. The activity

of compounds at the muscle and ganglionic type nAChR receptors was

determined using a membrane potential sensitive ﬂuorescent dye.

TE671 and SHSY5Y cells were plated at a density of 5 × 10⁴and 1 ×

10⁵cells/well, respectively, 24 h prior to assay. Growth media were

removed from the cells by ﬂicking the plates, and membrane potential

dye (Molecular Devices), reconstituted in HBSS ﬁve times more

diluted compared to the manufacturer’s instructions, was added to the

wells. Plates were incubated for 60 min at room temperature and then

directly transferred to the FLIPR system. Compounds to be tested

were prepared in assay buﬀer as 5×-concentrated solutions in a

separate 96-well polypropylene plate. Baseline ﬂuorescence was

monitored for the ﬁrst 10 s followed by the addition of compounds.

For detection of antagonist activity, agonist (epibatidine; EC₈₀ﬁnal

concentration) was added to every well except negative controls.

Signal recordings were performed as above. Results for SH-SY5Y cells

were exported as described previously whereas for TE671 were

exported from the FLIPR raw data as SUM of ﬂuorescence signal

intensity for the ﬁrst addition and MAX-MIN for the second addition

of compounds. The responses were normalized to the positive control

(epibatidine 1 μM ﬁnal concentration). The compounds tested were

found to display antagonist activity, and IC₅₀values were calculated as

described before.

Cytochrome P450 Inhibition Assay..^36,37The ﬂuorescent P450

inhibition assay was performed using the Gentest method (http://

www.gentest.com). Test compounds were dissolved in DMSO at 1.5

mM. The stock solution of 12 μL was added by a robotic system to

1488 μL of 0.1 M phosphate buﬀer at pH 7.4. The solution was mixed,

and 50 μL of the diluted samples was added to a 1 mL 96-well

polypropylene plate. Then 50 μL of cofactor with a NADPH

regenerating system was added to the wells. The plate was incubated

at 37 °C for 10 min. Enzyme−substrate mix was prepared by

prewarming the buﬀer at 37 °C for at least 10 min, and the enzymes

and substrates were added right before addition to the reaction plate.

Enzyme−substrate mix (100 μL) was added to the wells to start the

reaction. The ﬁnal substrate and isozyme concentrations and

incubation time were as follows: BFC (50 μM)/CYP3A4 (5 pmol/

mL) for 30 min, AMMC (1.5 μM)/CYP2D6 (7.5 pmol/mL) for 30

min, and MFC (75 μM)/2C9 (20 pmol/mL) for 45 min. The

reactions were stopped with 80% ACN/20% 0.5 M Tris buﬀer. The

signals were quantiﬁed using a ﬂuorescent plate reader. The ﬁnal

DMSO concentration was 0.2%. Compounds were tested in duplicate.

Percent inhibition was determined at 3 μM compound concentration.

High negative values were usually indicative of ﬂuorescent interference

from the test compounds or metabolites. The % CV obtained was

typically within 10%.

Metabolic Stability Assay. Compounds in 10 mM DMSO solution

were added to an incubation mixture in a 96-well microplate

containing 20 pmol/mL of hCYP3A4. The mixture was split in two

aliquots: one receiving a NADPH regenerating system, the other an

equal amount of phosphate buﬀer. The ﬁnal substrate concentration

was 1 μM along with 0.25% of organic solvent. Incubation proceeded

for 1 h at 37 °C and was stopped by addition of acetonitrile to

precipitate proteins. Metabolic stability was given as the percent

remaining following incubation with cofactor (NADPH) with

reference to the incubation mixture without NADPH: % remaining

= area NADPH × 100/area ctrl where area ctrl was the MS peak area

of the sample solution without NADPH and area NADPH was the MS

area of the sample solution with NADPH. The % CV obtained was

typically within 10%.

Permeability Assay. The assay was run in a PAMPA ﬁlter plate, and

compounds (10 μM in HBSS + Hepes buﬀer pH = 7.4) were added to

the donor chamber and incubated for 4 h at 37 °C and 80% humidity.

Warfarin was used in each well as control for membrane integrity.

Concentrations of reference t(0), donor, and acceptor solutions were

measured by UPLC-MS-TOF. The passive permeability was calculated

according to the following expression³⁸

⎛

⎜

⎝

⎞

⎟

⎠

⎛

⎞

⎟

⎠

1

)t

M

V + V

M

+

V_DV_A

CA(t) =

+ CA(0) −

e^−PeA(

⎜

V_D+ V_A

⎝

D

A

Solubility Assay. Standard and sample solutions were prepared

from a 10 mM DMSO stock solution using an automated dilution

procedure. For each compound, three solutions were prepared: one to

be used as standard and the other two as test solutions. Standard: 250

μM standard solution in acetonitrile/buﬀer, with a ﬁnal DMSO

content of 2.5% (v/v). Test sample for pH 3.0: 250 μM sample

solution in acetic acid 50 mM, pH = 3, with a ﬁnal DMSO content of

2.5% (v/v). Test sample for pH 7.4: a 250 μM sample solution in

ammonium acetate buﬀer 50 mM, pH = 7.4, with a ﬁnal DMSO

content of 2.5% (v/v). The 250 μM product suspensions/solutions in

the aqueous buﬀers were prepared directly in Millipore MultiScreen-

96 ﬁlter plates (0.4 μm PTCE membrane) and sealed. Plates were left

for 24 h at room temperature under orbital shaking to achieve

“pseudo-thermodynamic equilibrium” and to presaturate the mem-

brane ﬁlter. Product suspensions/solutions were then ﬁltered using

centrifugation, diluted 1:2 with the same buﬀer solution, and analyzed

by UPLC/UV/TOF-MS, using UV detection at 254 nm for

quantitation. Solubility was calculated by comparing the sample and

standard UV areas: S = (A_smp× FD × C_st)/A_st, where S was the

solubility of the compound (μM), A_smpwas the UV area of the sample

solution, FD was the dilution factor (2), C_stwas the standard

concentration (250 μM), and A_stwas the UV area of the standard

solution.

where M refers to the total amount of drug in the system minus the

amount of sample lost in membrane (and surfaces), CA(t) was the

concentration of the drug in the acceptor well at time t, CA(0) was the

concentration of the drug in the acceptor well at time 0, V_Awas the

volume of the acceptor well, V_Dwas the volume of the donor well, P_e

was the eﬀective permeability, A was the membrane area, and t was the

permeation time. Compounds were deﬁned as low, medium, or highly

permeable following the following classiﬁcation: >10 × 10⁻⁶cm/s,

high (passive permeability was unlikely to be limiting for passive

diﬀusion); between 2 and 10 × 10⁻⁶cm/s, medium (permeability may

be limiting in the case of low solubility, high metabolic turnover rate or

active secretion); between 0 and 2 × 10⁻⁶cm/s, low (high risk that

permeability was limiting for passive diﬀusion).

In Vitro Intrinsic Clearance. Test compounds were incubated

separately at 1 μM concentration in 100 mM phosphate buﬀer (pH

7.4) and 1 mM EDTA with 0.2 mg/mL rat hepatic microsomal

protein. The enzymatic reaction was initiated by addition of a NADPH

regenerating system (ﬁnal concentrations: 2 mM ß-nicotinamide

adenine dinucleotide phosphate (NADP) + 10 mM glucose-6-

phosphate (G6P) + 0.4 U/mL glucose-6-phosphate dehydrogenase

(G6PDH)). Reactions were terminated at regular time intervals (0−

5−10−20−40 min) by adding an equal volume of acetonitrile. All

O

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incubations were performed in duplicate. Verapamil as positive control

for the assay was incubated in parallel under the same conditions.

Samples were analyzed by UPLC/TOF-MS. Quantitative data were

automatically produced using the OpenLynx software. Substrate

depletion data (peak area at diﬀerent time points) were ﬁtted to a

indication of the compound being a substrate for eﬄux transporters.

The alert threshold was an eﬄux ratio >3.

Electrophysiology. GH4C1cells stably expressing rat α7-nAChR

were treated with 0.5 mM sodium butyrate added to the medium for

two days before patch clamp recordings. Patch pipets had resistances

of ∼7 MΩ when ﬁlled with (in mM): 5 EGTA, 120 K-gluconate, 5

KCl, 10 HEPES, 5 K₂ATP, 5 Na₂-phosphocreatine, 1 CaCl₂, and 2

MgCl₂. Cells were voltage-clamped at −60 mV with a HEKA EPC-9

ampliﬁer. To measure the fast activation and desensitization of the α7

current, the Dynaﬂow (Cellectricon) fast perfusion system with 16- or

48-well chips was used. Diﬀerent concentrations of acetylcholine or 25

were applied to cells in between washes with bath solution (Hanks’

Balanced Salt Solution +10 mM HEPES). Data were acquired at 1 kHz

for 2 s episodes (500 ms bath, 500 ms agonist, 1000 ms wash) with a

10 s interval between episodes. Peak current amplitude and total

charge (area under the curve) were measured with the HEKA Pulse

program. Concentration−response curves and EC₅₀values were

plotted and calculated with Origin (MicroCal).

monoexponential decay model (eq 1), with a 1/y weighting, C_(t)

=

C₀e^−kt, where C₀was the substrate concentration in the incubation

media at time 0 and k was the terminal rate constant. Under the

assumption that the concentration of 1 μM was far below the K_mof

the test compound, the in vitro Cl_intwas calculated by dividing the

elimination constant (K) for the microsomal protein concentration

(PMS), expressed in mg/μL, to obtain Cl_intin units of μL/min/mg

protein: Cl_int= K/PMS = μL/min/mg protein. Compounds were

deﬁned as low, medium, or highly metabolized based on the in vitro

Cl_intvalues: <3.4, low; 3.4−92.4, mediun; >92.4, high.

Plasma Stability. Fresh plasma (no more than 2−4 h from a blood

draw), spiked with a 250 μM solution of test item dissolved in

acetonitrile 12%, was divided into three aliquots (one for each time

point) in a 96-well plate (in duplicate) and incubated at +37 °C under

gentle agitation for up to 3 h. At time 0 and afterward at the preﬁxed

time points of 30 min and 3 h, two aliquots were transferred and three

volumes of acetonitrile were added for protein precipitation, followed

by centrifugation (4000 rpm, 15 min, 4 °C) and dilution with 0.1%

HCOOH. Analysis by LC-ESI⁺-MSMS using a fast gradient was

performed using an API4000 triple quadrupole mass spectrometer

(Applied Biosystems) in the multiple reaction monitoring mode

(MRM). Results, as average of two replicates, were obtained by

comparing the peak area at the diﬀerent time points (30 min and 3 h)

with that at time 0 and were reported as percentage remaining: percent

remaining = 100area_STM/area_{time 0}(where area_STMis the MS peak area

of the sample at the speciﬁc time points of 30 min and 3 h and

area_{time 0}is the MS area of the sample at time zero). Compounds were

deﬁned as stable, moderately unstable, or unstable based on the

percent remaining: 80−100 stable, 50−80 moderately unstable, <50

unstable.

MDCK Cellular Permeability. Madin-Darby canine kidneys were

maintained in tissue culture ﬂasks in EMEM with Glutamax added

with 1% MEM, penicillin (100 U/mL), streptomycin (100 ug/mL),

and 10% FBS. Five days before the permeability experiment, the cells

were split and placed on permeable cell culture inserts (24-well

Millipore) at a density of 250 000 cells/well. Transepithelial electrical

resistance (TEER) was measured for each well before incubation using

an EVOMX instrument (WPI) to ensure that the monolayer was

conﬂuent and the tight junctions intact. A TEER > 70 Ohm × cm²was

considered suitable for experimentation. Compounds (10 μM in

HBSS-Hepes buﬀer) were added in duplicate to the donor chamber

and buﬀer to the acceptor chamber (alternatively apical and

basolateral) and incubated for 2 h at 37 °C under gentle agitation.

Standards with a high (antipyrin), low (cimetidine), and medium

(warfarin) permeability were incubated in the same plate under the

same conditions. An aliquot (100 μL) from each well (both apical and

basolateral) at time 0 and 120 min was ﬁltered and analyzed by

UPLC/TOF-MS. Following incubation, the cell monolayer was

washed and incubated with Lucifer Yellow, a ﬂuorescent probe with

low permeability, to verify monolayer integrity after incubation. The

UPLC separation was performed using a C-18 column (Acquity UPLC

BEH C18, 1.7 μm, 2.1 mm × 50 mm, Waters). Samples were analyzed

using an LTC premier TOF (Waters). The ESI positive W mode scan

type was applied and the total ion current (TIC) scan range extended

from 100 to 800 amu, with a scan time of 0.08 s. Acquisition was from

0.3 to 1.8 min. Quantitative data were automatically produced using

the OpenLynx software. The apparent permeability (Papp) in

centimeters per second was calculated using the following equation

in both directions (apical-to-basolateral and basolateral-to-apical): P_app

= dC × V_r/dt × A × C₀(where V_rwas the volume (mL) of the receiver

chamber, A was the surface area of the cell culture insert, and dt was

the time in seconds). The mass balance in both directions was

estimated by the following equation: mass balance = (ﬁnal donor mass

+ mass transferred)/initial donor mass. The eﬄux ratio was calculated

Radioligand Binding Assay. [³H]-Epibatidine binding studies were

performed as previously described.³⁵Brieﬂy, cell membrane

preparations derived from GH4C1 cells stably expressing rat α7

nAChRs were suspended in binding buﬀer (50 mM HEPES, pH 7.4, 3

mM KCl, 70 mM NaCl, 10 mM MgCl₂), 5 nM [³H]-epibatidine (GE

Healthcare; SA = 53 Ci/mmol), and 25 to achieve a ﬁnal volume of

200 μL in a 96-well polypropylene plate. Nicotine at 300 μM was used

for determination of nonspeciﬁc binding. Following incubation at

room temperature (23 °C) for 1 h, samples were rapidly ﬁltered

through Uniﬁlter GF/B ﬁlters using a Filtermate (Perkin-Elmer) and

washed ﬁve times with ice-cold binding buﬀer. Samples were processed

and counted for radioactivity using a TopCount NXT (Perkin- Elmer).

Competition binding curves were ﬁtted with a four parameter logistic

model. K_ivalues were calculated by the Cheng-Prusoﬀ equation using

the GraphPad Prism software package.

Receptor Selectivity and hERG Activity. Interaction of 25 with ∼70

binding sites including all major classes of neurotransmitter, growth

factor, and peptide receptors (Novascreen, Caliper Biosciences,

HopkintonMA) was examined at 10 μM concentration. Activity at

the hERG ion channel was determined by employing CHO cells stably

expressing the channel and an IonWorks recording system.

Pharmacokinetics. Long Evans rats (age of 6−8 weeks, body

weight 250−550 g) were administered compound 25 as a single dose

of 20 mg/kg po (10 mg/kg po, for the brain study) at time 0 as a

solution in 2% Tween, 0.5% methocell in water (volume 2.5 mL/kg).

Levels in plasma were determined over a time period of 6 h in the po

study and in brain at 0, 0.5, 1, and 3 h. The concentration of 25 in rat

brain and plasma was measured by high performance liquid

chromatography in combination with mass spectrometry (LC-MS/

MS) with a limit of detection of 1 ng/mL in plasma and 3 ng/g in

brain. Plasma samples were prepared by protein precipitation with

acetonitrile containing 250 ng/mL of internal standard, centrifugation,

and analysis of the supernatant by LC-MS/MS. Brain samples were

prepared by homogenization and extraction with methanol. The

homogenates were subsequently centrifuged, and the supernatant was

analyzed by LC-MS/MS. Quantiﬁcation was performed in a similar

manner to that for the plasma samples.

Novel Object Recognition (NOR) Test. Male Long Evans rats

(275−250 g; Charles River Laboratories) were individually housed

with ad lib access to food and water and provided with nestlets for

environmental enrichment. All habituation, training, and testing were

performed under low illumination (approximately 10 l×) in a circular

arena (70 cm diameter, 30 cm height) constructed of plastic

surrounded by a black mesh curtain and containing corncob bedding.

The NOR task was composed of three sessions: habituation, a sample

trial (T1), and a choice trial (T2). Animal performance was tracked by

video using Noldus Ethovision XT software (Noldus Information

Technology, Inc., Leesburg, VA). Objects, constructed with Duplo

(Lego, Billund, Denmark) were placed on the arena ﬂoor in one of

four locations spaced evenly around the ﬁeld approximately 10 cm

from the ﬁeld’s edge. Rats were habituated to the arena, which

contained two identical yellow cubes for a period of 15 min 1 day prior

by comparing P_app

with P_{app A→B}. A high eﬄux ratio was an

B→A

P

dx.doi.org/10.1021/jm300247y | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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to the T1 and T2 sessions. T2 consisted of a 5 min exploration of the

ﬁeld containing both a familiar object, previously explored in T1, and a

novel object. The location of the objects, counterbalanced across

treatment groups, remained constant for each animal during the

habituation and the T1 and T2 trials. To evaluate the eﬀect of 25 in

the time delay deﬁcit model of NOR, T2 was carried out 48 h after T1,

and animals were treated with vehicle or 25 (0.3−10 mg/kg, po) 60

min prior to T1 (N = 10 per treatment group). Tolerance was assessed

in rats treated chronically with compound 25 (10 mg/kg ip per day for

14 days) prior to the experiment. The eﬀect of treatment on object

exploration during the sample trial was examined using a one-way

ANOVA on total contact time followed by Fisher’s LSD group mean

pairwise comparisons. The amount of time exploring the novel and

familiar objects across treatment groups was analyzed using repeated

measures ANOVA followed by Fisher’s LSD post hoc.

Antagonism of MK-801 Induced Prepulse Inhibition (PPI) Deﬁcits

in Rats. Each testing chamber (SR-LAB system, San Diego

Instruments, San Diego, CA) consisted of a Plexiglas cylinder (8.8

cm in diameter) mounted on a frame and held in position by four

metal pins to a base unit. Movement of the subjects (male Long Evans

rats, 200−300 g; Charles River Laboratories n = 8 per treatment

group) within the cylinder was detected by a piezoelectric

accelerometer attached below the frame. A loudspeaker mounted 24

cm above the cylinder provided background white noise, acoustic

noise bursts, and acoustic prepulses. The entire apparatus was housed

in a ventilated enclosure (39 cm × 38 cm × 56 cm). Presentation of

acoustic pulse and prepulse stimuli was controlled by the SR-LAB

software and interface system, which also digitized, rectiﬁed, and

recorded the responses from the accelerometer. Mean startle

amplitude was determined by averaging 100, 1 ms readings taken

from the beginning of the pulse stimulus onset. For calibration

purposes, sound levels were measured with a Quest sound level meter,

scale “A”, with the microphone placed inside the Plexiglas cylinder.

Test sessions consisted of 61 total trials with a 15 s intertrial interval.

Following a 5 min acclimation to a 64 dB background noise, four trial

types (20 ms 120 dBpulse, or a 69, 74, or 79 dB 20 ms prepulse paired

with a 120 dB 20 ms pulse, occurring 100 ms later onset to onset)

were presented in a pseudorandom order. Compound 25 (3−30 mg/

kg) was administered orally at 60 min prior to testing with MK-801

(0.085 mg/kg) administered sc 10 min prior to testing. Prepulse

inhibition was deﬁned as 100 − 100(startle amplitude on prepulse

trials/startle amplitude on pulse alone trials). Data from the pulse-

alone trials and average PPI values were analyzed using one-way

ANOVA followed by a least signiﬁcant diﬀerence post hoc test (p <

0.05).

4. Modeling Studies. All molecular modeling studies were

performed on a DELL PowerEdge server equipped with four

processors, Intel Xeon CPU 3.00 GHz. A homology model of the

EC domain of nAChR was built by modeling the human α7 sequence

for α7 nAChR on a 1UW6 PDB structure as a template. The amino

acid numbering reported in the text refers to the 1UW6 sequence. The

model was then used for docking purposes. Compound structures

were modeled within the Maestro software suite (Maestro version 9.0;

Schrodinger, LLC, New York). In particular, ligands were protonated

at pH 7.4 and their geometries optimized using the LigPrep module

(LigPrep 2.3; Schrodinger, LLC, New York). Docking into the

orthosteric binding site of the homology model was performed by the

docking program GOLD version 3.0.³⁹Ligands were docked using

default accurate genetic algorithm settings and the GOLDSCORE

ﬁtness function. The goodness of the docked poses was mainly

evaluated via visual inspection and supported by the scoring value. Key

pharmacophoric interaction between protonated nitrogen on the

ligand and the backbone carbonyl of W143 was considered as a

fundamental requisite to validate docking poses.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

We thank Eva Genesio for her support in the interpretation of

analytical data and Stefano Gotta for generation of HRMS.

■

ABBREVIATIONS USED

■

ACh, acetylcholine; AChBP, acetylcholine binding protein; AD,

Alzheimer’s disease; BFC, 7-benzyloxy-4-triﬂuoromethylcou-

marin; ACN, acetonitrile; B/P, brain to plasma ratio; CHO,

Chinese hamster ovary; CREB, cAMP response element-

binding; ESI, electrospray ionization; FLIPR, ﬂuorescence

imaging plate reader; HBSS, Hank’s balanced salt solution;

HEK, human embryo kidney; hERG, human ether-a-go-

gorelated gene; HPLC, high pressure liquid chromatography;

HRMS, high resolution mass spectrometry; LTQ, linear trap

quadrupole; MDCK, Madin Darby canine kidney; MFC, 7-

methoxy-4-triﬂuoromethylcoumarin; MPSA, molecular polar

surface area; MS, mass spectrometry; NMDA, N-methyl-^D-

aspartate; NOR, novel object recognition; P450, cytochrome

P450; PAMPA, parallel artiﬁcial membrane permeability assay;

PDA, photodiode array; PPI, prepulse inhibition; Rt, retention

time; SAR, structure−activity relationship; TLC, thin layer

chromatography; TOF, time-of-ﬂight; UPLC, ultraperformance

liquid chromatography

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dx.doi.org/10.1021/jm300247y | J. Med. Chem. XXXX, XXX, XXX−XXX

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Discovery of a novel alpha-7 nicotinic acetylcholine receptor agonist series and characterization of the potent, selective, and orally efficacious agonist 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H- pyrazol-3-yl] amide (SEN15924, WAY-361789)

DOI: 10.1021/jm300247y

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