Structure-activity relationship studies of sulfonylpiperazine analogues as novel negative allosteric modulators of human neuronal nicotinic receptors

Article abstract of DOI:10.1021/jm201294r

Neuronal nicotinic receptors have been implicated in several diseases and disorders such as autism, Alzheimer's disease, Parkinson's disease, epilepsy, and various forms of addiction. To understand the role of nicotinic receptors in these conditions, it would be beneficial to have selective molecules that target specific nicotinic receptors in vitro and in vivo. Our laboratory has previously identified novel negative allosteric modulators of human α4β2 (Hα4β2) and human α3β4 (Hα3β4) nicotinic receptors. The effects of novel sulfonylpiperazine analogues that act as negative allosteric modulators on both Hα4β2 nAChRs and Hα3β4 nAChRs were investigated. This work, through structure-activity relationship (SAR) studies, describes the chemical features of these molecules that are important for both potency and selectivity on Hα4β2 nAChRs. (Figure presented)

Full text of DOI:10.1021/jm201294r

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies of Sulfonylpiperazine
Analogues as Novel Negative Allosteric Modulators of Human
Neuronal Nicotinic Receptors
Brandon J. Henderson,† Daniel J. Carper,∥ Tatiana F. Gonzalez-Cestari,† Bitna Yi,† Kiran Mahasenan,‡
́
Ryan E. Pavlovicz,§ Martin L. Dalefield,† Robert S. Coleman,∥ Chenglong Li,‡,§ and Dennis B. McKay*,†
‡
†Division of Pharmacology and Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, 500 West 12th
Avenue, Columbus, Ohio 43210, United States
§Biophysics Program, The Ohio State University, Columbus, Ohio 43210, United States
∥Department of Chemistry, The Ohio State University, 88 West 18th Avenue, Columbus, Ohio 43210, United States
S
* Supporting Information
ABSTRACT: Neuronal nicotinic receptors have been implicated in
several diseases and disorders such as autism, Alzheimer’s disease,
Parkinson’s disease, epilepsy, and various forms of addiction. To
understand the role of nicotinic receptors in these conditions, it
would be beneficial to have selective molecules that target specific
nicotinic receptors in vitro and in vivo. Our laboratory has previously
identified novel negative allosteric modulators of human α4β2
(Hα4β2) and human α3β4 (Hα3β4) nicotinic receptors. The effects
of novel sulfonylpiperazine analogues that act as negative allosteric
modulators on both Hα4β2 nAChRs and Hα3β4 nAChRs were
investigated. This work, through structure−activity relationship
(SAR) studies, describes the chemical features of these molecules
that are important for both potency and selectivity on Hα4β2
nAChRs.
prove beneficial for the clinical treatment of several neuro-
pathologies.
INTRODUCTION
■
Neuronal nicotinic acetylcholine receptors (nAChRs) are
associated with many important physiological mechanisms
(i.e., cognition, arousal, pain sensation), as well as a number of
neurological diseases and disorders (depression, schizophrenia,
Alzheimer’s disease, Parkinson’s disease, lung cancer, Tourette’s
syndrome, autism, and addiction).1−4 nAChRs are notable for
their implications in the addictive properties of nicotine, the
primary addictive component of tobacco products.5 nAChRs
are ligand-gated ion channels composed of five protein subunits
encoded by a family of related but distinct genes.6,7 Multiple
nAChR subtypes have been described based on subunit
(α2−α10 and β2−β4) composition, where the three most
prominent subtype compositions in the CNS are α4β2, α3β4,
and α7.8 The specific nAChR subtypes involved with most of
the above-mentioned physiological processes or diseases are
not known, and their study is challenging because of the
complex composition of some nAChRs (α4α6β2, α3α5β4,
etc.).9,10 Despite the lack of understanding concerning specific
subtypes of nAChRs in diseases and disorders, it is widely
accepted that nicotine addiction is mediated primarily by α4β2
containing nAChRs.11,12 The discovery of novel, selective
molecules that target specific subtypes of nAChRs would
contribute significantly to our understanding of the role nAChR
subtypes play in normal and pathophysiological states and
Worldwide, nicotine addiction is a significant problem.
Smoking is the primary cause of preventable death worldwide,
and roughly 90% of the people who attempt to quit are unable
to do so.13 Current FDA approved treatments for tobacco
addiction are nicotine replacement, buproprion, and vareni-
cline. Each of these therapies has a modest success of 20−30%
abstinence 1 year after quit date.14−16 The target site of many
nAChR drug discovery programs is the orthosteric (agonist
binding) site of nAChRs,9,17 and few laboratories have had
some success in identifying molecules that show some
selectivity using this approach.18,19 However, one difficulty
with this strategy is the high degree of amino acid sequence
homology among nAChR α and β subunits in the ligand
binding domain for acetylcholine, making it difficult to develop
drugs that specifically target nAChR subtypes.9,17 Thus, the
development of selective nAChR drugs directed at orthosteric
sites progresses slowly. For this reason, drugs targeting
“nonorthosteric” sites of nAChRs (e.g., allosteric, non-
competitive sites) is an approach now taken by many
laboratories.20−22
Received: September 28, 2011
Published: November 7, 2011
© 2011 American Chemical Society
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Mecamylamine, a nonselective noncompetitive nAChR
antagonist, was shown to promote 40% abstinence at the year
mark when used as an agonist−antagonist therapy in
combination with the nicotine patch.23 In addition, antagonists
of α4β2 nAChRs block nicotine self-administration on fixed
and progressive ratio schedules in rats.20,24 These data support
the use of antagonists as nicotine cessation therapies; however,
to produce new therapeutic molecules, it has widely been
accepted that nAChR subtype selectivity must be pursued.9
Selectivity is especially important for avoiding effects on α3β4
nAChRs, as they are the prominent nAChR subtype in the
peripheral nervous system.9,10 Furthermore, it has been
postulated that this nAChR subtype mediates many of the
adverse affects associated with therapeutics that target
nAChRs.14,23 Our laboratory has identified a novel allosteric
site on Hα4β2 nAChRs that is approximately 10 Å from the
orthosteric site at the interface of the α and β subunits.25 This
site was identified through a combination of homology
modeling, blind docking, and molecular dynamics studies and
was confirmed through the use of site-directed muta-
genesis.25,26 At this site, a novel class of NAMs has been
shown to have relative selectivity for Hα4β2 nAChRs (over
Hα3β4 nAChRs) principally through nonconserved amino
acids on the β2 subunit. Through SAR we have identified
several physiochemical features that mediate relative selectivity
for Hα4β2 nAChRs. These include specific placement of
aromatic residues, specific orientation of the keto group of
esters or amides, and the specific length of carbon chains. By
use of the allosteric site that was identified through computa-
tional modeling,25,26 a small library of diverse molecules were
computationally docked to identify novel chemical scaffolds
that are active as nAChR NAMs. This structure-based virtual
screening (SBVS) approach resulted in the identification of
four novel scaffolds that show preference for Hα4β2 nAChRs
and act as inhibitors of nAChRs.27 One of these SBVS hits has a
sulfonylpiperazine scaffold unique among previously described
nAChR NAMs. Here in these studies we present the SAR of
novel sulfonyl analogues on Hα4β2 and Hα3β4 nAChRs and
describe the physical and chemical features that are important
for both relative selectivity and potency on Hα4β2 nAChRs.
Scheme 1. Retrosynthetic Analysis of Lead Compound 1
Scheme 2. Synthesis of Left Hand α-Bromoamides
manner to access a variety of α-bromoamides and an α-
bromoester in unoptimized yields ranging from 26% to 96%.29
The piperazine fragment 3 was obtained in high yield from
the reaction of p-fluorobenzenesulfonyl chloride with excess
piperazine (Scheme 3).30 Four other arylsulfonyl chlorides were
CHEMISTRY
■
We have previously reported that SBVS has yielded active hits
that target Hα4β2 nAChRs.27 A hit molecule (1) has been
selected as a scaffold for the development of a focused library of
compounds. Lead compound 1 was assembled through a
convergent synthesis allowing for facile access to analogues.
Retrosynthetically, compound 1 was formed from displacement
of bromide 2 by arylsulfonylpiperazine 3 (Scheme 1). The two
coupling partners were assembled from commercially available
starting materials, where amide 2 resulted from acylation of o-
fluoroaniline (4) with bromoacetyl bromide (5) and where
piperazine 3 resulted from sulfonylation of piperazine (6) with
p-fluorobenzenesulfonyl chloride (7).
Scheme 3. Synthesis of Right Hand Arylsulfonylpiperazines
This synthetic plan allowed access to a small library of
compounds simply by using various substituted anilines and
arylsulfonyl chlorides. Analogues were thus available in two
linear steps (three total) from commercially available reagents.
In practice, the synthetic plan was executed as planned where o-
fluoroaniline (4) was acylated with bromoacetyl bromide (5) in
the presence of triethylamine to yield amide 2 (Scheme 2).28 In
addition to o-fluoroaniline (4), five other aniline derivatives, 3-
aminopyrazole, and o-phenylphenol were reacted in a similar
reacted with piperazine to obtain the arylsulfonylpiperazine
right-hand fragments needed to assemble analogues.
With the left side bromide (2) and right side piperazine (3)
in hand, the two fragments were coupled in the presence of
sodium carbonate to obtain lead compound 1 (Scheme 4). The
various other left and right side fragments were mixed and
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Scheme 4. Coupling of Left Side Bromide with Right Side
Piperazine
Scheme 5. Linear Synthesis of Aminoindazole 11
matched to obtain analogues 12−30 (Tables 1−5) in
unoptimized yields varying from 34% to 89%.
The only analogue not accessible through this convergent
method was aminoindazole analogue 11. The inability to
cleanly acylate 5-aminoindazole (10) with bromoacetyl bro-
mide (5) led to a linear approach to obtain aminoindazole 11.
To this end, bromoacetic acid (8) was reacted in the presence
of triethylamine with p-fluorobenzenesulfonylpiperazine (3) to
provide acid 9. Standard amide coupling conditions (EDCI,
HOBt) were then employed to obtain analogue 11 in modest
46% yield (Scheme 5).31
RESULTS
■
Series 1 SAR. Lead compound 1 produced inhibition on
both Hα4β2 and Hα3β4 nAChRs with IC50 values of 9.3 and
9.0 μM, respectively (Table 1). The right side p-fluorobenzene-
Table 1. Series 1 SAR Studies
a
b
c
Values represent geometric mean (confidence limits), n = 5−10. nh, Hill coefficient. No activity up to 100 μM.
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Figure 1. Concentration−response effects of epibatidine in the absence and presence of compound 1. Calcium accumulation assays were performed
as described in the methods section. The concentration−response effects of ebibatidine were investigated in the absence (■) or presence (□) of 30
μM 1 (∼IC70) by using HEK ts201 cells expressing Hα4β2 nAChRs (A) and Hα3β4 nAChRs (B). Values represent the mean ± SEM (n = 4).
Table 2. Series 2 SAR Studies
a
b
Values represent geometric mean (confidence limits), n = 5−10. nh, Hill coefficient.
sulfonylpiperazine (3) had no effect on either Hα4β2 or
Hα3β4 nAChRs (data not shown). When tested for
competitive or noncompetitive activity on Hα4β2 and Hα3β4
nAChRs, lead compound 1 reduced the maximum efficacy for
the agonist epibatidine (Figure 1). If the compounds acted at
the agonist binding site, they would compete directly with the
agonist, epibatidine, at its binding. This would produce a
parallel shift of the concentration−response curve to the right.
Compound 1 did not cause a parallel shift but instead
decreased the maximum efficacy of epibatidine. According to
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Figure 2. Concentration−response effects of compounda 1 and 16 on Hα4β2 and Hα3β4 nAChRs. Calcium accumulation assays were performed as
described in the methods section. The concentration−response effects of compound 1 (A) and compound 16 (B) were investigated on Hα4β2
nAChRs (■) and Hα3β4 nAChRs (□). Values are the mean ± SEM (n = 6−9). IC50 values of compounds 1 and 16 are reported together in Table
2.
Table 3. Series 3 SAR Studies
a
b
c
Values represent geometric mean (confidence limits), n = 6−12. nh, Hill coefficient. No activity up to 100 μM.
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Table 4. Series 4 SAR Studies
a
b
c
Values represent geometric mean (confidence limits), n = 5−10. nh, Hill coefficient. No activity up to 100 μM.
classical drug receptor theory, this result is indicative of
noncompetitive inhibition (e.g., the binding at a site other than
the agonist site). Therefore, this result suggests that compound
1 modulates nAChRs from an allosteric site. As outlined in the
section Chemistry, analogues of 1 were synthesized. None of
the compounds described here showed agonist activity on
either Hα4β2 or Hα3β4 nAChRs in the calcium accumulation
assay (data not shown).
In the first series of analogues, the amide portion of lead
compound 1 was modified using several different substitutions
(Table 1). This study was conducted to determine the synthetic
flexibility of the amide portion and to determine how these
changes effect potency on nAChRs. The pyrazole analogue
(12) produced a significant decrease in potency for both
Hα4β2 and Hα3β4 nAChRs (IC50 > 100 μM, Table 1). The
methoxy analogue (13) produced a 2-fold decrease in potency
for Hα4β2 nAChRs (IC50 = 21.1 μM, Table 1) and a 3-fold
decrease in potency for Hα3β4 nAChRs (IC50 = 29.6 μM,
Table 1). The p-fluorophenyl analogue (14) produced no
significant change in potency for either Hα4β2 or Hα3β4
nAChRs (Table 1). The benzyl analogue (15) showed no
significant change in potency on Hα4β2 nAChRs but showed a
2-fold decrease in potency for Hα3β4 nAChRs (IC50 = 18.5
μM, Table 1). The indazole analogue (11) produced a slight
increase in potency for Hα4β2 nAChRs (IC50 = 6.6 μM, Table
1) and a 3-fold decrease in potency for Hα3β4 nAChRs (IC50
23.8 μM, Table 1).
=
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Table 5. Series 5 SAR Studies
a
b
Values represent geometric mean (confidence limits), n = 5−9. nh, Hill coefficient.
Series 2 SAR. In the next series of analogues, the sulfonyl
portion of scaffold 1 was modified using several different
substitutions (Table 2). Similar to the series 1 study, this was
conducted to determine the synthetic flexibility of the sulfonyl
portion and to determine how these changes affect potency on
nAChRs. Movement of the fluorine from the para position (1)
to the ortho position (16) produced no significant change in
potency for Hα4β2 nAChRs; however, it resulted in a
significant 12-fold decrease in potency for Hα3β4 nAChRs
(IC50 = 99.8 μM, p < 0.01, Table 2). The pyridinyl analogue
(17) produced a 2-fold decrease in potency for the Hα4β2
nAChR and a 5-fold decrease in potency for the Hα3β4 nAChR
(IC50 of 20.3 and 49.6 μM, respectively, Table 2). The phenyl
analogue (18) produced a 2-fold increase in potency for Hα4β2
and Hα3β4 nAChRs (IC50 of 4.1 and 4.0 μM, respectively,
Table 2). The p-methoxyphenyl analogue (19) resulted in no
significant change in potency for both Hα4β2 and Hα3β4
nAChRs (Table 2). From examination of the inhibition curves
of 1 and 16 on nAChRs, 1 produces similar hill coefficients on
both Hα4β2 and Hα3β4 nAChRs (Figure 2 and Table 2). 16
produces a similar hill coefficient as 1 on Hα4β2 nAChRs (nh =
−1.5) but produced a statistically significant (p < 0.0005) 2-fold
decreased Hill coefficient on Hα3β4 nAChRs (nh = −0.6, Table
2).
3). Replacement of the o-fluorophenyl with a p-methoxyphenyl
(21) resulted in a 2-fold decrease in potency for Hα4β2
nAChRs (IC50 = 17.1 μM, Table 3) and a 4-fold increase in
potency for Hα3β4 nAChRs (IC50 = 19.1 μM, Table 3).
Substitution of a methyl ester at the para position (22) resulted
in no significant change for Hα4β2 and an 8-fold increase in
potency for Hα3β4 nAChRs (IC50 = 11.2 μM, Table 3).
Replacement of the fluorine with carboxylic acid (23) or
instillation of a pyrazole heterocycle in place of the
fluorophenyl (24) resulted in a loss of activity on both
subtypes (Table 3).
Series 4 SAR. Pyridinyl, phenyl, and p-methoxyphenyl
containing analogues of 1 were synthesized. The o-fluorophen-
yl, pyridinyl analogue (17) resulted in IC50 values of 20.3 and
48.9 μM on Hα4β2 and Hα3β4 nAChRs, respectively (Table
4). The pyrazole, pyridinyl analogue (25) resulted in IC50
values of 83.0 and >100 μM on Hα4β2 and Hα3β4 nAChRs,
respectively (Table 4). The p-methoxyphenyl, pyridinyl
analogue (26) resulted in a significant decrease in potency
for both Hα4β2 and Hα3β4 nAChRs (IC50 values of >100 and
95.0 μM respectively, Table 4). The o-fluorophenyl, phenyl
analogue (18) resulted in IC50 values of 4.1 and 4.6 μM on
Hα4β2 and Hα3β4 nAChRs, respectively (Table 4). The
phenyl, phenyl analogue (27) resulted in IC50 values of 9.6 and
16.4 μM on Hα4β2 and Hα3β4 nAChRs, respectively (Table
4). The o-fluorophenyl, p-methoxyphenyl analogue (19)
resulted in no significant change in potency for both Hα4β2
and Hα3β4 nAChRs (Table 4). The p-fluorophenyl, p-
methoxyphenyl analogue (28) resulted in a 2-fold decrease in
Series 3 SAR. In this series, analogue 16 (Table 2) was
used as the basis of comparison. Replacement of the o-
fluorophenyl with the p-fluorophenyl (20) resulted in no
change in potency for Hα4β2 nAChRs (Table 3) and a 6-fold
increase in potency for Hα3β4 nAChRs (IC50 = 13.6 μM, Table
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potency for both Hα4β2 and Hα3β4 nAChRs (IC50 values of
23.6 and 17.1 μM, respectively, Table 4).
position (28). This suggests that the ortho position is preferred
for Hα4β2 nAChR potency.
Series 5 SAR. Biphenyl ester analogues were made of lead
molecule 1 (29) and 16 (30). Biphenyl ester 29 showed no
change in potency for Hα4β2 nAChRs and a 2-fold decrease in
potency for Hα3β4 nAChRs (IC50 = 23.8, Table 5) compared
to 1. Biphenyl ester 30 showed a decrease in potency for
Hα4β2 nAChRs (IC50 = 20.9 μM, Table 5) and a 3-fold
increase in potency for Hα3β4 nAChRs (IC50 = 23.5, Table 5)
in comparison to 16.
Previously published data25 showed that the incorporation of
biphenyl structures is important for selectivity of molecules
targeting Hα4β2 nAChRs. Therefore, biphenyl analogues of 1
and 16 were made in series 5. It was hypothesized that the ester
carbonyl and fluorinated phenyl groups would act in a similar
way as the ester and phenylpropyl of KAB-18.25 However, these
features found in this novel scaffold lack the flexibility of the
phenylpropyl in KAB-18-like molecules and therefore may have
a different binding mode within the binding site.
DISCUSSION AND CONCLUSION
In conclusion, the SAR of sulfonylpiperizine analogues on
Hα4β2 and Hα3β4 nAChRs has been described here.
Compound 16 showed the highest relative selectivity for
Hα4β2 nAChRs (12-fold, Table 3), while 18 showed the
highest potency (Table 4) among the compounds described
here. The SAR of these compounds has identified that the
position of fluorine substitution on the sulfonyl portion (ortho
vs para) has a significant effect on relative selectivity for Hα4β2
nAChRs. In these analogues, relative selectivity for Hα4β2
nAChRs is associated with o-fluorophenyl (16) while p-
fluorobenzenes show no preference for Hα3β4 or Hα4β2
nAChRs. Additionally, the ortho substitution of halogens in the
amide portion has shown improvement in potency for Hα4β2
nAChRs. In the future discovery of novel Hα4β2 nAChR
antagonists, it would be informative to incorporate both of
these features in the design of new NAMs to improve both
potency and selectivity. Compounds 11 and 16, which have
modifications to the amide and sulfonyl positions, respectively,
both led to an increase in selectivity for Hα4β2 nAChRs.
Further studies may include making both modifications in a
single molecule to improve selectivity for Hα4β2 nAChRs.
The structural diversity of these new analogues provides
additional insight into the physiochemical features that are
important for antagonism of nAChRs at allosteric sites. As
mentioned before, the discovery of selective molecules
targeting nAChRs has been slow. Studies like these contribute
to the discovery and development of selective compounds that
can be used as novel therapeutics for nAChR related diseases
and disorders.
■
We understand that there are a multitude of nAChR subtypes
and that a study such as this, utilizing only two subtypes, cannot
use the term “selectivity” accurately. For this reason, the term
“relative selectivity” has been used to describe the difference
between only Hα4β2 and Hα3β4 nAChRs. The most
significant change in series 1 was the pyrazole (12)
modification, which abolished activity (up to 100 μM) for
both subtypes. Movement of the fluorine led to no significant
change; therefore, this suggests that the fluorine is not a
significant contributor to selectivity or potency for this scaffold.
Substitution of the indazole (11) did not affect potency on
Hα4β2 nAChRs but decreased potency on Hα3β4. This
suggests that this structure or position of the scaffold is
amenable to optimization for Hα4β2 nAChR selectivity.
The significant findings of series 2 were that the o-
fluorophenyl and pyridinyl substitutions showed an improve-
ment in the relative selectivity for Hα4β2 nAChRs (9-fold and
3-fold, respectively). This result suggests that this position of
the scaffold may improve the range of selectivity with further
modification. Moving the fluorine from the para (1) to the
ortho (16) positions may cause rotation of the ring due to
proximity to the sulfonyl oxygen groups.
Series 3 data suggest that these substitutions have little effect
on the potency of these molecules on Hα4β2 nAChRs (with
the exception of pyrazole 24); yet every change showed a
decrease in relative selectivity for Hα4β2 nAChRs. The results
of the comparison between analogues 20 and 16 (Table 3)
contradict earlier results (e.g., the comparison between
analogues 14 and 1, Table 1). The first comparison showed
that the fluorine position, ortho or para, had no effect on
potency for Hα3β4 nAChRs (Table 1). However, we noticed
that movement of the fluorine (20 and 16) makes a significant
difference on potency for Hα3β4 nAChRs (Table 3). This
suggests that this portion of the molecule is interacting at a
different position in the ligand binding domain depending on
the fluorine position on the phenyl of the sulfonyl portion.
Compound 12 (series 1) and compound 24 both contained
pyrazole moieties and resulted in a loss of activity on both
nAChR subtypes. This suggests that more hydrophilic
substitutions decrease binding and/or efficacy on nAChRs.
In pyridinyl analogues of series 4, the methoxybenzyl
substitution to the amide portion (26) produced a significant
change in potency on both subtypes (Table 4). This is
surprising considering that similar substitutions had little effect
on potency (13 in Table 1 and 21 in Table 3). These data also
show that other aromatics, such as a pyrazole, drastically reduce
potency on nAChRs (25). In both the phenyl and p-
methoxyphenyl analogues, molecules with fluorine substitutions
at the ortho position (18 and 19) showed improved Hα4β2
nAChR potency compared to molecules that lacked a fluorine
substitution (27) or had a fluorine substitution at the para
EXPERIMENTAL SECTION
■
Materials. Calcium 5NW dye was obtained from Molecular
Devices (Sunnyvale, CA). Dulbecco’s modified Eagle medium
(DMEM), penicillin, streptomycin, and L-glutamine were obtained
from Invitrogen Corporation (Grand Island, NY). Epibatidine was
purchased from Sigma-Aldrich (St. Louis, MO). All other reagents
were purchased from Fisher Scientific (Pittsburgh, PA). For
pharmacological evaluation, all compounds were initially dissolved in
100% DMSO (0.01 M stocks). Stock solutions of compounds at
concentrations less than or equal to 100 μM were made in HBK
buffer.
Calcium Accumulation Assays. A procedure previously re-
ported by our laboratory25,32,33 was used with minor modifications.
For the calcium accumulation assays, HEK ts201 cells stably expressing
either Hα4β2 nAChRs or Hα3β4 nAChRs (obtained from Professor
Jon Lindstrom, University of Pennsylvania, Philadelphia, PA) were
used. Cells were plated at a density of (2.0−2.3) × 105 cells per well in
clear 96-well culture plates previously coated with poly-L-ornithine. On
the day of the experiment (typically ∼24 h later), cells were washed
(100 μL) with HEPES-buffered Krebs (HBK) solution25,32,33 and
incubated (protected from light) for 1 h at 24 °C with 50% Calcium 5
NW dye (Molecular Devices, Sunnyvale, CA). The plates were then
placed into a fluid handling integrated fluorescence plate reader
(FlexStation II, Molecular Devices, Sunnyvale, CA). Fluorescence was
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read at excitation of 485 nm and emission of 525 nm from the bottom
of the plate, and changes in fluorescence were monitored at ∼1.5 s
intervals. Inhibition curves were obtained in the concentration−
response studies using six concentrations within a range of 0.1−100
μM for each compound reported here. Results are reported as IC50
values (see Tables 1−5). Results were expressed as a percentage of
control, 1 μM epibatidine group. Because of solubility problems,
compound concentrations greater than 100 μM were not used in our
concentration−response studies. Therefore, compounds that showed
no inhibition up to the highest concentration have been labeled with
IC50 values of “>100” (see Tables 1−4). The DMSO concentration at
this compound concentration was 1% and had no effect on basal or
agonist-induced increases in fluorescence intensity.
125.6, 125.5, 125.4, 124.7, 121.7, 115.2, 115.0, 29.4; IR (neat) λmax
3313, 1669, 1620, 1330, 1105, 758; HRMS (ESI) m/z calcd for
C8H7BrFNNaO, 253.9587; found, 253.9591.
1-([4-Fluorophenyl]sulfonyl)piperazine (3). Following general
procedure B, the crude product was crystallized (EtOAc/hexanes) to
1
give pure 3 as a white solid (93%): H NMR (CDCl3, 500 MHz) δ =
7.70−7.79 (m, 2H), 7.16−7.24 (m, 2H), 2.93−3.00 (m, 4H), 2.87−
2.93 (m, 4H), 1.52 (s, NH); 13C NMR (CDCl3, 126 MHz) δ = 166.3,
164.3, 131.8, 131.8, 130.5, 130.5, 116.4, 116.2, 46.9, 45.4; IR (neat)
λmax 2762, 1592, 1495,1341, 1170, 541, 539. Anal. Calcd for
C10H13FN2O2S: C, 49.17%; H, 5.36%; N, 11.47%. Found: C,
49.04%; H, 5.45%; N, 11.30%. HRMS (ESI) m/z calcd for
C10H14FN2O2S, 245.0755; found, 245.0758.
Calculations and Statistics. Functional data were calculated
from the number of observations (n) performed in triplicate. Curve
fitting was performed by Prism software (GraphPad, San Diego, CA)
using the equation for a single-site sigmoidal dose−response curve
with a variable slope. IC50 values are expressed as geometric mean
values (95% confidence limits). When statistical significance was
calculated, the t test was used. Where data were labeled “not
significant” or when “no significant difference” was reported, then p >
0.05.
N-(2-Fluorophenyl)-2-(4-((4-fluorophenyl)sulfonyl)-
piperazin-1-yl)acetamide (1). Following general procedure C, the
crude product was purified by flash column chromatography (silica,
1
4:6 EtOAc/hexanes) to provide 1 as a white solid (59%): H NMR
(CDCl3, 400 MHz) δ = 9.08 (br s, 1H), 8.25 (t, J = 8.0 Hz, 1H),
7.73−7.82 (m, 2H), 7.18−7.28 (m, 2H), 7.04−7.13 (m, 1H), 6.93−
7.04 (m, 2H), 3.16 (s, 2H), 3.11 (br s, 4H), 2.69 (t, J = 4.9 Hz, 4H);
13C NMR (CDCl3, 101 MHz) δ = 167.5, 166.7, 164.1, 153.7, 151.2,
131.9, 131.9, 130.5, 130.4, 125.9, 125.8, 124.7, 124.7, 124.6, 124.5,
121.5, 116.6, 116.4, 114.9, 114.7, 61.6, 52.5, 46.1; IR (neat) λmax 3504,
2831, 1699, 1531, 1173, 951, 735, 548. Anal. Calcd for
C18H19F2N3O3S: C, 54.67%; H, 4.84%; N, 10.63%. Found: C,
54.72%; H, 4.94%; N, 10.44%. HRMS (ESI) m/z calcd for
C18H20F2N3O3S, 396.1188; found, 396.1173.
2-(4-((4-Fluorophenyl)sulfonyl)piperazin-1-yl)acetic Acid
(9). Following general procedure C, using triethylamine, the crude
product was purified by crystallization (CH3OH) to provide 9 as a
white crystal (30%): 1H NMR (DMSO-d6, 500 MHz) δ = 7.80 (dd, J =
8.4, 5.2 Hz, 2H), 7.47 (br t, J = 8.7 Hz, 2H), 3.13 (s, 2H), 2.88 (br s,
4H), 2.58 (br s, 4H); 13C NMR (DMSO-d6, 126 MHz) δ = 171.2,
165.7, 163.7, 131.2, 131.1, 130.7, 130.6, 116.7, 116.5, 57.8, 50.8, 45.8;
IR (neat) λmax 3470, 1634, 1355, 1173, 935, 733, 548; HRMS (ESI)
m/z calcd for C12H16FN2O4S, 303.0809; found, 303.0794.
General Chemistry Methods. 1H (500 or 400 MHz) and 13C
(126 or 101 MHz) NMR spectra were recorded on a Bruker DRX-500
or Bruker DPX-400 spectrometer in CDCl3 using CHCl3 (1H δ 7.26)
and CDCl3 (13C δ 77.0) or DMSO-d6 using DMSO (1H δ 2.49) and
DMSO-d6 (13C δ 39.51) as internal standards. High resolution mass
spectra were recorded on a Bruker MicrOTOF ESI spectrometer
provided by OBIC. Elemental analyses were carried out by Galbraith
Laboratories, Inc. (Knoxville, TN). All reactions were conducted in
either oven-dried (120 °C) glassware or flame-dried glassware, under
an N2 atmosphere when necessary. Tetrahydrofuran (THF) was
distilled from benzophenone ketyl. Triethylamine and CH2Cl2 were
distilled from calcium hydride prior to use. All other chemicals were
used as received. All compounds were >95% pure as determined by
elemental analysis (C, H, N), unless otherwise noted.
2-(4-((4-Fluorophenyl)sulfonyl)piperazin-1-yl)-N-(1H-inda-
zol-5-yl)acetamide (11). EDCI (66.9 mg, 0.349 mmol) was added
to a solution of 9 (105.4 mg, 0.349 mmol), 5-aminoindazole (51.1 mg,
0.384 mmol), and HOBt (47.2 mg, 0349 mmol) in DMF (1.7 mL) at
0 °C. The reaction mixture was stirred for 16 h while warming to 23
°C, diluted with CH2Cl2 (10 mL), washed with H2O (3 × 5 mL),
brine (5 mL), dried (Na2SO4), and concentrated in vacuo to produce a
crude brown solid. The crude material was purified by flash column
chromatography (silica, 8:2 EtOAc/hexanes) to yield 11 as a white
Synthesis. Preparation of N-Aryl-2-bromoacetamides. Gen-
eral Procedure A. Bromoacetyl bromide (10 mmol, 1 equiv) was
added dropwise over 5 min to a solution of amine or alcohol (10
mmol, 1 equiv) and triethylamine (11 mmol, 1.1 equiv) in
dichloromethane (50 mL, 0.2 M) at 0 °C. The reaction mixture was
stirred at 0 °C for 20 min to 1 h, diluted with dichloromethane (50
mL), washed with saturated NH4Cl (3 × 30 mL), dried (Na2SO4), and
concentrated in vacuo to afford crude product. The crude product was
purified by flash column chromatography, crystallization, or trituration
to yield pure N-aryl-2-bromoacetamide (26−96%).
1
solid (67.7 mg, 46%): H NMR (DMSO-d6, 400 MHz) δ = 12.93 (br
s, NH), 9.60 (s, NH), 7.98 (s, 1H), 8.03 (s, 1H), 7.83 (dd, J = 8.7, 5.3
Hz, 2H), 7.50 (t, J = 8.7 Hz, 2H), 7.34−7.46 (m, 2H), 3.14 (s, 2H),
3.00 (br s, 4H), 2.60 (apparent t, J = 4.3 Hz, 4H); 13C NMR (DMSO-
d6, 126 MHz) δ = 167.7, 165.7, 163.7, 137.0, 133.3, 131.5, 131.5,
131.4, 130.7, 130.6, 122.6, 120.9, 116.7, 116.5, 110.5, 109.9, 61.0, 51.6,
45.7; IR (neat) λmax 3628, 1682, 1171, 948, 735, 548. HRMS (ESI) m/
z calcd for C19H20FN5NaO3S, 440.1163; found, 440.1148.
2-(4-((4-Fluorophenyl)sulfonyl)piperazin-1-yl)-N-(1H-pyra-
zol-3-yl)acetamide (12). Following general procedure C, crude
product was purified by flash column chromatography (silica, EtOAc)
to provide 12 as a white solid (47%): 1H NMR (CDCl3, 500 MHz) δ
= 10.50 (br s, NH) 9.15 (s, NH), 7.67−7.82 (m, 2H), 7.40 (d, J = 2.4
Hz, 1H), 7.15−7.30 (m, 2H), 6.64 (d, J = 2.4 Hz, 1H), 3.14 (s, 2H),
3.05 (br s, 4H), 2.65 (br t, J = 4.7 Hz, 4H); 13C NMR (CDCl3, 126
MHz) δ = 167.5, 166.5, 164.4, 146.1, 131.4, 131.4, 130.5, 130.5, 130.1,
116.7, 116.6, 96.7, 61.3, 52.6, 45.9; IR (neat) λmax 3613, 3308, 2938,
2834, 1668, 1592, 1169, 954, 836, 732, 547. Anal. Calcd for
C15H18FN5O3S: C, 49.04%; H, 4.94%. Found: C, 48.07%; H, 5.11%.
HRMS (ESI) m/z calcd for C15H19FN5O3S, 368.1187; found,
368.1170.
Preparation of Arylsulfonylpiperazines. General Procedure
B. Arylsulfonyl chloride (10 mmol, 1 equiv) was added in one portion
to a solution of piperazine (60 mmol, 6 equiv) in CH2Cl2 (100 mL, 0.1
M) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min,
diluted with CH2Cl2 (200 mL), quenched by the addition of saturated
NaHCO3(aq) (50 mL), washed with brine (50 mL), dried (Na2SO4),
and concentrated in vacuo to provide crude product. The crude
product was used directly or purified by crystallization or trituration to
yield pure arylsulfonylpiperazines (75% to quantitative).
Preparation of N-Aryl-2-((arylsulfonyl)piperazinyl)-
acetamides. General Procedure C. Sodium carbonate (0.4
mmol, 2 equiv) or triethylamine (0.4 mmol, 2 equiv) was added to
a solution of N-aryl-2-bromoacetamide (0.2 mmol, 1 equiv) and
arylsulfonylpiperazine (0.2 mmol, 1 equiv) in THF (1 mL, 0.2 M) at
23 °C. The reaction mixture was allowed to stir for 16 h, diluted with
CH2Cl2 (5 mL), filtered, and concentrated in vacuo to afford crude
product. The crude product was purified by flash column
chromatography to yield pure N-aryl-2-((arylsulfonyl)piperazinyl)-
acetamides (48−86%).
2-Bromo-N-(2-fluorophenyl)acetamide (2). Following general
procedure A, the crude product was precipitated (Et2O/hexanes) to
2-(4-((4-Fluorophenyl)sulfonyl)piperazin-1-yl)-N-(4-
methoxyphenyl)acetamide (13). Following general procedure C,
crude product was purified by flash column chromatography (silica,
1
give pure 2 as a white solid (26%): H NMR (CDCl3, 400 MHz) δ =
8.40 (br s, 1H), 8.24 (t, J = 7.8 Hz, 1H), 7.04−7.19 (m, 3H), 4.04 (s,
1
2H); 13C NMR (CDCl3, 101 MHz) δ = 163.6, 154.1, 151.6, 125.7,
1:1 EtOAc/hexanes) to provide 13 as a white solid (79%): H NMR
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Journal of Medicinal Chemistry
Article
(CDCl3, 400 MHz) δ = 8.53 (s, 1H), 7.73−7.82 (m, 2H), 7.30−7.39
(m, 2H), 7.20−7.28 (m, 2H), 6.77−6.85 (m, 2H), 3.75 (s, 3H), 3.10
(s, 2H), 3.08 (br s, 4H), 2.66 (br t, J = 4.8 Hz, 4H); 13C NMR
(CDCl3, 101 MHz) δ = 167.2, 166.6, 164.1, 156.5, 131.8, 131.8, 130.5,
130.4, 130.4, 121.5, 116.7, 116.4, 114.2, 61.6, 55.5, 52.6, 45.9; IR
(neat) λmax 2840, 1916, 1683, 1515, 1172, 952, 839, 735, 548. Anal.
Calcd for C19H22FN3O4S: C, 56.01%; H, 5.44%; N, 10.31%. Found: C,
56.29%; H, 5.64%; N, 10.22%. HRMS (ESI) m/z calcd for
C19H23FN3O4S, 408.1388; found, 408.1383.
NMR (CDCl3, 126 MHz) δ = 167.6, 153.4, 151.5, 135.9, 133.1, 129.2,
127.7, 125.9, 125.8, 124.7, 124.7, 124.6, 124.5, 121.5, 114.9, 114.7,
61.7, 52.6, 46.1; IR (neat) λmax 3302, 2829, 1918, 1693, 1172, 952, 743,
568. Anal. Calcd for C18H20FN3O3S: C, 57.28%; H, 5.34%; N, 11.13%.
Found: C, 57.61%; H, 5.51%; N, 11.16%. HRMS (ESI) m/z calcd for
C18H21FN3O3S, 378.1282; found, 378.1272.
N-(2-Fluorophenyl)-2-(4-((4-methoxyphenyl)sulfonyl)-
piperazin-1-yl)acetamide (19). Following general procedure C,
crude product was purified by flash column chromatography (silica,
1
1:1 EtOAc/hexanes) to provide 19 as a white solid (77%): H NMR
N-(4-Fluorophenyl)-2-(4-((4-fluorophenyl)sulfonyl)-
piperazin-1-yl)acetamide (14). Following general procedure C,
crude product was purified by flash column chromatography (silica,
(CDCl3, 500 MHz) δ = 9.07 (br s, 1H), 8.23 (t, J = 7.9 Hz, 1H),
7.61−7.70 (m, 2H), 7.01−7.09 (m, 1H), 6.95−7.01 (m, 4H), 3.85 (s,
4H), 3.13 (s, 2H), 3.06 (br s, 4H), 2.65 (br t, J = 4.9 Hz, 4H); 13C
NMR (CDCl3, 126 MHz) δ = 167.6, 163.2, 153.4, 151.4, 129.8, 127.2,
125.8, 125.7, 124.5, 124.5, 124.4, 124.4, 121.5, 114.8, 114.6, 114.3,
61.5, 55.6, 52.4, 46.0; IR (neat) λmax 2843, 1953, 1699, 1164, 949, 735,
559. Anal. Calcd for C19H22FN3O4S: C, 56.01%; H, 5.44%; N, 10.31%.
Found: C, 56.20%; H, 5.64%; N, 10.17%. HRMS (ESI) m/z calcd for
C19H23FN3O4S, 408.1388; found, 408.1390.
1
1:1 EtOAc/hexanes) to provide pure 14 as a white solid (86%): H
NMR (CDCl3, 500 MHz) δ = 8.63 (br s, 1H), 7.69−7.85 (m, 2H),
7.37−7.47 (m, 2H), 7.25 (apparent t, J = 8.5 Hz, 2H), 6.92−7.01 (m,
2H), 3.13 (s, 2H), 3.11 (br s, 4H), 2.69 (t, J = 4.9 Hz, 4H); 13C NMR
(CDCl3, 126 MHz) δ = 167.4, 166.5, 164.4, 160.5, 158.5, 133.4, 133.3,
132.0, 132.0, 130.6, 130.5, 121.5, 121.4, 116.7, 116.5, 115.8, 115.7,
61.7, 52.7, 46.0; IR (neat) λmax 2982, 1666, 1523, 1351, 1172, 947, 834,
740, 549. Anal. Calcd for C18H19F2N3O3S: C, 54.67%; H, 4.84%; N,
10.63%. Found: C, 55.00%; H, 5.21%; N, 10.47%. HRMS (ESI) m/z
calcd for C18H20F2N3O3S, 396.1188; found, 396.1177.
2-(4-((4-Fluorophenyl)sulfonyl)piperazin-1-yl)-N-phenylace-
tamide (15). Following general procedure C, crude product was
purified by flash column chromatography (silica, 4:6 EtOAc/hexanes)
to provide 15 as a white solid (43%): 1H NMR (CDCl3, 500 MHz) δ
= 8.65 (br s, 1H), 7.74−7.83 (m, 2H), 7.43−7.50 (m, 2H), 7.19−7.33
(m, 4H), 7.03−7.14 (m, 1H), 3.13 (s, 2H), 3.10 (br s, 4H), 2.68 (t, J =
4.9 Hz, 4H); 13C NMR (CDCl3, 126 MHz) δ = 167.4, 166.4, 164.4,
137.3, 132.0, 131.9, 130.6, 130.5, 129.1, 124.5, 119.7, 116.7, 116.5,
61.8, 52.6, 46.0; IR (neat) λmax 3270, 3059, 2944, 1677, 1524, 1173,
946, 737, 548. Anal. Calcd for C18H20FN3O3S: C, 57.28%; H, 5.34%;
N, 11.13%. Found: C, 57.39%; H, 5.54%; N, 11.00%. HRMS (ESI) m/
z calcd for C18H21FN3O3S, 378.1282; found 378.1270.
N-(4-Fluorophenyl)-2-(4-((2-fluorophenyl)sulfonyl)-
piperazin-1-yl)acetamide (20). Following general procedure C,
crude product was purified by flash column chromatography (silica,
1
4:6 EtOAc/hexanes) to provide 20 as a white solid (86%): H NMR
(CDCl3, 500 MHz) δ = 8.73 (br s, 1H), 7.75−7.87 (m, 1H), 7.54−
7.64 (m, 1H), 7.39−7.47 (m, 2H), 7.26−7.31 (m, 1H), 7.17−7.25 (m,
1H), 6.90−7.00 (m, 2H), 3.28 (br s, 4H), 3.12 (s, 2H), 2.48−2.70 (br
t, 4H); 13C NMR (CDCl3, 126 MHz) δ = 167.4, 160.3, 160.0, 158.4,
157.9, 135.4, 135.3, 133.4, 131.2, 125.3, 125.1, 124.7, 124.6, 121.4,
121.4, 117.5, 117.3, 115.7, 115.5, 61.6, 52.8, 45.6, 45.6; IR (neat) λmax
3403, 2839, 1690, 1177, 950, 836, 739, 584. Anal. Calcd for
C18H19F2N3O3S: C, 54.67%; H, 4.84%; N, 10.63%. Found: C,
54.79%; H, 5.17%; N, 10.64%. HRMS (ESI) m/z calcd for
C18H20F2N3O3S, 396.1188; found, 396.1183.
2-(4-((2-Fluorophenyl)sulfonyl)piperazin-1-yl)-N-(4-
methoxyphenyl)acetamide (21). Following general procedure C,
crude product was purified by flash column chromatography (silica,
N-(2-Fluorophenyl)-2-(4-((2-fluorophenyl)sulfonyl)-
piperazin-1-yl)acetamide (16). Following general procedure C,
crude product was purified by flash column chromatography (silica,
1
4:6 EtOAc/hexanes) to provide 21 as a white solid (89%): H NMR
1
3:7 EtOAc/hexanes) to provide 16 as a white solid (53%): H NMR
(CDCl3, 500 MHz) δ = 8.61 (br s, NH), 7.74−7.82 (m, 1H), 7.49−
7.59 (m, 1H), 7.31−7.38 (m, 2H), 7.22−7.28 (m, 1H), 7.19 (t, J = 9.3
Hz, 1H), 6.69−6.82 (m, 2H), 3.70 (s, 3H), 3.23 (br s, 4H), 3.07 (s,
2H), 2.48−2.65 (br t, 4H); 13C NMR (CDCl3, 126 MHz) δ = 167.2,
159.9, 157.8, 156.4, 135.3, 135.2, 131.1, 130.4, 125.1, 125.0, 124.6,
124.5, 121.4, 117.4, 117.2, 114.0, 61.5, 55.4, 52.7, 45.6; IR (neat) λmax
3313, 2939, 1686, 1598, 1514, 1174, 955, 828, 734. Anal. Calcd for
C19H22FN3O4S: C, 56.01%; H, 5.44%; N, 10.31%. Found: C, 55.53%;
H, 5.70%; N, 10.07%. HRMS (ESI) m/z calcd for C19H23FN3O4S,
408.1388; found, 408.1390.
Methyl 4-(2-(4-((2-Fluorophenyl)sulfonyl)piperazin-1-yl)-
acetamido)benzoate (22). Following general procedure C, crude
product was purified by flash column chromatography (silica, 1:1
EtOAc/hexanes) to provide 22 as a white solid (80%): 1H NMR
(CDCl3, 500 MHz) δ = 8.95 (br s, NH), 7.97 (d, J = 8.5 Hz, 2H), 7.85
(t, J = 6.8 Hz, 1H), 7.59−7.69 (m, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.31
(t, J = 7.6 Hz, 1H), 7.15−7.28 (m, 1H), 3.88 (s, 3H), 3.32 (br s, 4H),
3.17 (s, 2H), 2.57−2.76 (m, 4H); 13C NMR (CDCl3, 126 MHz) δ =
167.9, 166.5, 160.1, 158.0, 141.4, 135.4, 135.4, 131.3, 130.9, 125.9,
125.5, 125.4, 124.7, 124.7, 118.8, 117.6, 117.4, 61.9, 53.0, 52.1, 45.7; IR
(neat) λmax 3282, 1702, 1598, 1281, 1175, 954, 733, 584. Anal. Calcd
for C20H22FN3O5S: C, 55.16%; H, 5.09%; N, 9.65%. Found: C,
55.15%; H, 5.31%; N, 9.43%. HRMS (ESI) m/z calcd for
C20H23FN3O5S, 436.1337; found, 436.1350.
(CDCl3, 400 MHz) δ = 9.20 (br s, NH), 8.30 (t, J = 8.1 Hz, 1H),
7.80−7.92 (m, 1H), 7.54−7.69 (m, 1H), 7.27−7.35 (m, 1H), 7.23−
7.29 (m, 1H), 7.08−7.17 (m, 1H), 6.91−7.08 (m, 2H), 3.26−3.42 (m,
4H), 3.20 (s, 2H), 2.65−2.77 (m, 4H); 13C NMR (CDCl3, 101 MHz)
δ = 167.6, 160.3, 157.8, 153.7, 151.3, 135.4, 135.3, 131.3, 125.9, 125.8,
125.4, 125.3, 124.7, 124.7, 124.7, 124.6, 124.6, 124.5, 121.5, 117.6,
117.4, 114.9, 114.7, 61.8, 52.9, 45.8, 45.8; IR (neat) λmax 3308, 2857,
1700, 1185, 766, 736, 586. Anal. Calcd for C18H19F2N3O3S: C,
54.67%; H, 4.84%; N, 10.63%. Found: C, 54.82%; H, 4.77%; N,
10.49%. HRMS (ESI) m/z calcd for C18H20F2N3O3S, 396.1188; found
396.1168.
N-(2-Fluorophenyl)-2-(4-(pyridin-3-ylsulfonyl)piperazin-1-
yl)acetamide (17). Following general procedure C, crude product
was purified by flash column chromatography (silica, 8:2 EtOAc/
hexanes) to provide 17 as a white solid (66%): 1H NMR (CDCl3, 500
MHz) δ = 9.06 (br s, 1H), 9.00 (dd, J = 2.4, 0.8 Hz, 1H), 8.86 (dd, J =
5.0, 1.6 Hz, 1H), 8.21−8.30 (m, 1H), 8.03−8.10 (m, 1H), 7.52 (ddd, J
= 8.0, 4.8, 0.8 Hz, 1H), 7.07−7.12 (m, 1H), 6.96−7.05 (m, 2H), 3.18
(s, 6H), 2.72 (t, J = 4.9 Hz, 4H); 13C NMR (CDCl3, 126 MHz) δ =
167.4, 153.7, 153.4, 151.5, 148.5, 135.4, 132.8, 125.8, 125.7, 124.7,
124.7, 124.6, 124.6, 123.9, 121.5, 114.9, 114.8, 61.7, 52.5, 46.0; IR
(neat) λmax 3311, 2830, 1694, 1456, 1176, 953, 756, 583. Anal. Calcd
for C17H19FN4O3S: C, 53.96%; H, 5.06%; N, 14.81%. Found: C,
54.21%; H, 5.22%; N, 14.67%. HRMS (ESI) m/z calcd for
C17H20FN4O3S, 379.1235; found, 379.1236.
4-(2-(4-((2-Fluorophenyl)sulfonyl)piperazin-1-yl)acetamido)-
benzoic Acid (23). Following general procedure C, crude product
was purified by flash column chromatography (silica, 3% MeOH/
EtOAc) to provide 23 a white solid (44%): 1H NMR (DMSO-d6, 500
MHz) δ = 12.66 (br s, NH), 9.91 (s, NH), 7.87 (d, J = 8.7 Hz, 2H),
7.74−7.83 (m, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.46−7.53 (m, 1H), 7.44
(t, J = 7.6 Hz, 1H), 3.19 (s, 2H), 3.14 (br s, 4H), 2.60 (br s, 4H); 13C
NMR (DMSO-d6, 126 MHz) δ = 168.5, 166.9, 159.3, 157.3, 142.5,
136.1, 136.0, 130.9, 130.2, 125.5, 125.2, 125.2, 124.0, 123.9, 118.9,
N-(2-Fluorophenyl)-2-(4-(phenylsulfonyl)piperazin-1-yl)-
acetamide (18). Following general procedure C, crude product was
purified by flash column chromatography (silica, 4:6 EtOAc/hexanes)
to provide 18 as a white solid (79%): 1H NMR (CDCl3, 500 MHz) δ
= 9.09 (br s, NH), 8.26 (t, J = 8.0 Hz, 1H), 7.74−7.80 (m, 2H), 7.60−
7.67 (m, 1H), 7.50−7.60 (m, 2H), 7.06−7.15 (m, 1H), 6.91−7.06 (m,
2H), 3.16 (s, 2H), 3.13 (br s, 4H), 2.69 (br t, J = 4.9 Hz, 4H); 13C
8690
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117.7, 117.5, 61.0, 51.8, 45.4; IR (neat) λmax 2924, 2854, 1707, 1600,
1174, 952, 733, 584. Anal. Calcd for C19H20FN3O5S: C, 54.15%; H,
4.78%; N, 9.97%. Found: C, 54.52%; H, 5.20%; N, 9.40%. HRMS
(ESI) m/z calcd for C19H21FN3O5S, 422.1180; found, 422.1162.
2-(4-((2-Fluorophenyl)sulfonyl)piperazin-1-yl)-N-(1H-pyra-
zol-3-yl)acetamide (24). Following general procedure C, crude
product was purified by flash column chromatography (silica, EtOAc)
to provide 24 as a white solid (48%): 1H NMR (CDCl3, 400 MHz) δ
= 10.57 (br s, NH), 9.23 (s, NH), 7.75−7.86 (m, 1H), 7.54−7.66 (m,
1H), 7.41 (d, J = 2.3 Hz, 1H), 7.26−7.33 (m, 1H), 7.18−7.25 (m,
1H), 6.63 (d, J = 2.3 Hz, 1H), 3.23 (br s, 4H), 3.14 (s, 2H), 2.64 (br t,
J = 4.9 Hz, 4H); 13C NMR (CDCl3, 101 MHz) δ = 167.5, 160.3,
157.7, 146.1, 135.5, 135.4, 131.3, 130.2, 124.8, 124.7, 124.7, 117.7,
117.5, 96.6, 61.4, 52.8, 45.7; IR (neat) λmax 3212, 1668, 1567, 1176,
735, 583. HRMS (ESI) m/z calcd for C15H19FN5O3S: 368.1187;
found, 368.1173.
crude product was purified by flash column chromatography (silica,
4:6 EtOAc/hexanes) to provide 29 as a white solid (83%): H NMR
1
(CDCl3, 500 MHz) δ = 7.68−7.80 (m, 2H), 7.28−7.41 (m, 8H),
7.15−7.23 (m, 2H), 7.09 (dd, J = 7.7, 1.1 Hz, 1H), 3.22 (s, 2H), 2.97
(br s, 4H), 2.44 (br t, J = 4.9 Hz, 4H); 13C NMR (CDCl3, 126 MHz) δ
= 168.3, 166.3, 164.3, 147.5, 137.6, 135.1, 131.5, 131.5, 130.9, 130.6,
130.5, 129.0, 128.7, 128.4, 127.6, 126.6, 122.5, 116.4, 116.3, 58.6, 51.5,
45.8; IR (neat) λmax 3448, 1771, 1592, 1155, 1139, 737, 548. Anal.
Calcd for C24H23FN2O4S: C, 63.42%; H, 5.10%; N, 6.16%. Found: C,
60.69%; H, 5.16%; N, 5.97%. HRMS (ESI) m/z calcd for
C24H24FN2O4S, 455.1435; found, 455.1444.
N-([1,1′-Biphenyl]-2-yl)-2-(4-((2-fluorophenyl)sulfonyl)-
piperazin-1-yl)acetamide (30). Following general procedure C,
crude product was purified by flash column chromatography (silica,
1
4:6 EtOAc/hexanes) to provide 30 as a white solid (84%): H NMR
(CDCl3, 500 MHz) δ = 7.77−7.83 (m, 1H), 7.51−7.60 (m, 1H),
7.28−7.41 (m, 8H), 7.23−7.28 (m, 1H), 7.19 (td, J = 9.2, 0.8 Hz, 1H),
7.06−7.12 (m, 1H), 3.24 (s, 2H), 3.14 (br t, 4H), 2.43 (br t, 4H); 13C
NMR (CDCl3, 126 MHz) δ = 168.3, 160.1, 158.1, 147.5, 137.6, 135.2,
135.2, 135.1, 131.4, 130.9, 129.0, 128.7, 128.3, 127.6, 126.6, 124.7,
124.6, 124.5, 124.5, 122.5, 117.5, 117.3, 58.7, 51.8, 45.6; IR (neat) λmax
3370, 2860, 1770, 1599, 1130, 739, 584. Anal. Calcd for
C24H23FN2O4S: C, 63.42%; H, 5.10%; N, 6.16%. Found: C, 61.95%;
H, 5.20%; N, 5.98%. HRMS (ESI) m/z calcd for C24H24FN2O4S,
455.1435; found, 455.1433.
N-(1H-Pyrazol-3-yl)-2-(4-(pyridin-3-ylsulfonyl)piperazin-1-
yl)acetamide (25). Following general procedure C, crude product
was purified by flash column chromatography (silica, 4% MeOH/
1
EtOAc) to provide 25 as a white solid (34%): H NMR (CDCl3, 500
MHz) δ = 9.12 (s, NH), 8.99 (d, J = 1.9 Hz, 1H), 8.87 (dd, J = 4.7, 1.6
Hz, 1H), 8.05 (dt, J = 8.1, 1.9 Hz, 1H), 7.49−7.59 (m, 1H), 7.44 (d, J
= 2.5 Hz, 1H), 6.67 (d, J = 2.5 Hz, 1H), 3.16 (s, 2H), 3.14 (br s, 4H),
2.69 (br t, J = 4.7 Hz, 4H); 13C NMR (CDCl3, 126 MHz) δ = 167.3,
153.7, 148.5, 146.2, 135.5, 132.6, 130.2, 124.0, 96.8, 61.4, 52.6, 45.9; IR
(neat) λmax 3608, 3240, 2947, 2829, 1683, 1176, 951, 757, 583. Anal.
Calcd for C14H18N6O3S: C, 47.99%; H, 5.18%; N, 23.98%. Found: C,
47.61%; H, 5.54%; N, 22.48%. HRMS (ESI) m/z calcd for
C14H19N6O3S, 351.1234; found, 351.1216.
N-(4-Methoxyphenyl)-2-(4-(pyridin-3-ylsulfonyl)piperazin-1-
yl)acetamide (26). Following general procedure C, crude product
was purified by flash column chromatography (silica, 9:1 EtOAc/
hexanes) to provide 26 as a white solid (59%): 1H NMR (CDCl3, 500
MHz) δ = 8.98 (d, J = 2.2 Hz, 1H), 8.84 (dd, J = 4.8, 1.1 Hz, 1H), 8.50
(s, NH), 8.04 (dd, J = 8.2, 1.6 Hz, 1H), 7.50 (dd, J = 7.9, 4.8 Hz, 1H),
7.34 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 3.74 (s, 3H), 3.14
(br s, 4H), 3.11 (s, 2H), 2.68 (br t, J = 4.9 Hz, 4H); 13C NMR
(CDCl3, 126 MHz) δ = 167.0, 156.6, 153.7, 148.5, 135.4, 132.8, 130.4,
123.9, 121.5, 114.2, 61.6, 55.5, 52.5, 45.9; IR (neat) λmax 3320, 2834,
1679, 1514, 1175, 951, 756, 583. Anal. Calcd for C18H22N4O4S: C,
55.37%; H, 5.68%; N, 14.35%. Found: C, 55.50%; H, 5.95%; N,
13.85%. HRMS (ESI) m/z calcd for C18H23N4O4S, 391.1435; found,
391.1439.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and characterization data of pre-
cursors of compounds 12−30; NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: (614) 292-3771. Telefax: (614) 292-9083. E-mail:
Mckay.2@osu.edu.
■
ACKNOWLEDGMENTS
■
All stably transfected human cell lines were kindly provided by
Dr. Jon M. Lindstrom, Department of Neuroscience School of
Medicine, University of Pennsylvania, Philadelphia, PA. This
work was supported by the National Institutes of Health
National Institute on Drug Abuse (Grant DA029433).
Financial support for B.J.H. is from the National Institutes of
Health National Institute on Drug Abuse Diversity Supplement.
Financial support for R.E.P. is from an American Chemical
Society Predoctoral Fellowship in Medicinal Chemistry. We
thank OBIC for funding for a Bruker MicrOTOF mass
spectrometer. We thank Karl Werbovetz, Ph.D., and Rostislav
Likhotvorik for their help preparing this manuscript.
N-Phenyl-2-(4-(phenylsulfonyl)piperazin-1-yl)acetamide
(27). Following general procedure C, crude product was purified by
flash column chromatography (silica, 4:6 EtOAc/hexanes) to provide
27 as a white solid (70%): 1H NMR (CDCl3, 500 MHz) δ = 8.66 (br
s, NH), 7.77 (d, J = 7.6 Hz, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.57 (t, J =
7.6 Hz, 2H), 7.45 (d, J = 7.6 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 7.08 (t,
J = 7.6 Hz, 1H), 3.11 (s, 6H), 2.67 (br t, J = 4.7 Hz, 4H); 13C NMR
(CDCl3, 126 MHz) δ = 167.4, 137.3, 135.8, 133.1, 129.3, 129.0, 127.8,
124.5, 119.6, 61.7, 52.6, 45.9; IR (neat) λmax 3283, 2848, 1674, 1523,
1176, 945, 745, 694, 580. Anal. Calcd for C18H21N3O3S: C, 60.15%; H,
5.89%; N, 11.69%. Found: C, 60.23%; H, 6.08%; N, 11.80%. HRMS
(ESI) m/z calcd for C18H22N3O3S, 360.1376; found, 360.1361.
N-(4-Fluorophenyl)-2-(4-((4-methoxyphenyl)sulfonyl)-
piperazin-1-yl)acetamide (28). Following general procedure C,
crude product was purified by flash column chromatography (silica,
ABBREVIATIONS USED
■
NAM, negative allosteric modulator; nAChR, neuronal
nicotinic acetylcholine receptor; HBK, HEPES-buffered
Krebs; SAR, structure−activity relationship; nh, Hill coefficient;
SBVS, structure-based virtual screening
1
1:1 EtOAc/hexanes) to provide 28 as a white solid (77%): H NMR
(CDCl3, 500 MHz) δ = 8.76 (br s, 1H), 7.61−7.72 (m, J = 8.8 Hz,
2H), 7.41 (dd, J = 8.7, 4.6 Hz, 2H), 6.97−7.03 (m, J = 8.8 Hz, 2H),
6.94 (t, J = 8.7 Hz, 2H), 3.85 (s, 3H), 3.14 (br s, 2H), 3.07 (br s, 4H),
2.69 (br s, 4H); 13C NMR (CDCl3, 101 MHz) δ = 167.4, 163.3, 160.6,
158.2, 133.4, 133.4, 129.9, 127.2, 121.5, 121.4, 115.8, 115.5, 114.4,
77.3, 61.5, 55.7, 52.6, 45.9; IR (neat) λmax 3312, 2842, 1686, 1510,
1163, 949, 835, 735. Anal. Calcd for C19H22FN3O4S: C, 56.01%; H,
5.44%; N, 10.31%. Found: C, 57.30%; H, 5.73%; N, 10.32%. HRMS
(ESI) m/z calcd for C19H23FN3O4S, 408.1388; found, 408.1394.
N-([1,1′-Biphenyl]-2-yl)-2-(4-((4-fluorophenyl)sulfonyl)-
piperazin-1-yl)acetamide (29). Following general procedure C,
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