Discovery of 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl) benzenesulfonamide (A-867744) as a novel positive allosteric modulator of the α7 nicotinic acetylcholine receptor

Article abstract of DOI:10.1021/jm9003818

The discovery of a series of pyrrole-sulfonamides as positive allosteric modulators (PAM) of α7 nAChRs is described. Optimization of this series led to the identification of 19 (A-867744), a novel type II PAM with good potency and selectivity. Compound 19

Full text of DOI:10.1021/jm9003818

J. Med. Chem. 2009, 52, 3377–3384
3377
Discovery of 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide
(A-867744) as a Novel Positive Allosteric Modulator of the r7 Nicotinic Acetylcholine Receptor
Ramin Faghih,* Sujatha M. Gopalakrishnan, Jens Halvard Gronlien, John Malysz, Clark A. Briggs, Caroline Wetterstrand,
Hilde Ween, Michael P. Curtis, Kathy A. Sarris, Gregory A. Gfesser, Rachid El-Kouhen, Holly M. Robb, Richard J. Radek,
Kennan C. Marsh, William H. Bunnelle, and Murali Gopalakrishnan
Neuroscience Research, Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, Abbott Park, Illinois 60064
ReceiVed January 19, 2009
The discovery of a series of pyrrole-sulfonamides as positive allosteric modulators (PAM) of R7 nAChRs
is described. Optimization of this series led to the identification of 19 (A-867744), a novel type II PAM
with good potency and selectivity. Compound 19 showed acceptable pharmacokinetic profile across species
and brain levels sufficient to modulate R7 nAChRs. In a rodent model of sensory gating, 19 normalized
gating deficits. These results suggest that 19 represents a novel class of molecules capable of allosteric
modulation of the R7 nAChRs.
Introduction
spatially and temporally restricted manner,24 thus potentially
offering differential efficacy and adverse effect profiles com-
pared to agonists.
R7 nAChRsa play an important role in cognitive and sensory
gating processes.1-3 The R7 subtype is distinguished from other
nAChRs by its relatively high permeability to Ca2+, rapid
desensitization, and sensitivity to antagonists such as R-bun-
garotoxin and MLA.4-6 Current evidence, including validation
by subtype selective agonists and antisense and gene knockout
studies, supports the hypothesis that targeting the R7 nAChRs
offers a symptomatic approach for treating a variety of cognitive
and neurodegenerative disorders by modulating the release of
neurotransmitters and activating phosphorylation events impor-
tant for cognitive/attentive processes.7-10
In recent years, a variety of structurally distinct subtype-
selective R7 nAChR agonists have demonstrated efficacy in
preclinical models of attention and cognitive deficits relevant
to Alzheimer’s disease and schizophrenia.11-17 An alternative
approach is to enhance effects of the endogenous neurotrans-
mitter acetylcholine (ACh) at R7 nAChRs via positive allosteric
modulation, which could reinforce the endogenous cholinergic
neurotransmission without directly activating R7 nAChRs.18,19
Indeed, such a positive allosteric modulator (PAM) approach
has proven clinically successful for benzodiazepine modulation
of GABAA receptors.20 Allosteric modulators bind at sites
distinct, but as yet, undefined from the agonist-binding pocket21-23
and offer potential for better subtype selectivity than what has
been achieved with agonists, thus exquisite subtype selectivity
may be easier to achieve because the binding site(s) for PAMs
are less highly conserved than the orthosteric site. Further, in
principle, a PAM alone will not activate the receptor but will
only amplify the physiological neurotransmitter signal in a
Initially disclosed nAChR PAMs, including bovine serum
albumin,25 acetylcholinesterase derived peptide SLURP-1,26 and
small molecules such as genistein,27 galantamine,28 and 5-hy-
droxyindole,29 are generally weak and nonselective among
nAChRs. More recently, a chemically heterogeneous group of
molecules capable of differentially modulating nAChR proper-
ties have emerged (Figure 1). On the basis of the Monod-
Wyman-Changeux model of allosteric interactions, at least two
types of PAMs have been recognized: type I, which predomi-
nately affects the peak current responses,30,31 and type II, which
increases the peak current responses and changes the desensi-
tization profile of the agonist response.32-35 Both types of PAMs
have been reported to show in vivo efficacy in animal models
of cognition and gating deficit.19,31,32 However, in some cases,
in vitro properties and physiochemical properties remains to be
further optimized, as compounds are limited by relatively low
potency, low solubility, metabolic stability, and/or adequate CNS
penetration. Accordingly, identification of selective, potent, and
pharmacokinetically acceptable PAMs continues to be an active
area of discovery research.
We reasoned that inclusion of PAMs, in particular those that
affect receptor desensitization (Type II), should elicit R7 nAChR
mediated Ca2+ flux, enabling detection by conventional high-
throughput screening (HTS) approaches. Thus, after screening
of the Abbott in-house compound library, the pyrrole-sulfona-
mides 10a and 10b (Figure 2) were identified as positive
allosteric modulators of the R7 nAChR. However, initial hits
were found to have low metabolic stability in a liver microsomes
preparation and poor pharmacokinetic and CNS penetration
properties in vivo (Table 1 and Figure 4). We considered that
these shortcomings may result from metabolic liability contrib-
uted by the ester moiety. Preliminary studies on this template
demonstrated that the presence of a para-phenylsulfonamide
moiety on the pyrrole was essential for PAM activity at the R7
nAChR. We present below the synthesis and biological profile
of a novel series of pyrrole-sulfonamides with improved
microsomal stability and pharmacokinetics.
* To whom correspondence should be addressed. Phone: 847-9372972.
Fax: 847-9374143. E-mail: ramin.faghih@abbott.com.
a Abbreviations: nAChR, nicotinic acetylcholine receptor; SAR, structure-
activity relationship; MLA, methyllycaconitine; ACh, acetylcholine; PAM,
positive allosteric modulator; HTS, high-throughput screening; DMF,
dimethylformamide; TEA, triethylamine; THF, tetrahydrofuran; DMA,
dimethylacetamide; PS-DCC, polymer-supported 1,3-dicyclohexylcarbodi-
imide; HOBT, 1-hydroxybenzotriazole; CNS, central nervous system;
FLIPR, fluorometric imaging plate reader; LC-MS, liquid chromatography-
mass spectrometry; ESI-MS, electrospray ionization-mass spectroscopy;
SPE-MS, solid phase extraction-mass spectroscopy; NADPH, nicotinamide
adenine dinucleotide phosphate; HBSS, Hanks balanced salt solution;
NMDG, N-methyl-D-glucosamine; HEPES, N-2-hydroxyethylpiperazine-
N′-2-ethanesulfonic acid; POETs, parallel oocyte electrophysiology test
station; EEG, electroencephalography; PBS, phosphate buffered saline.
Chemistry. An initial objective of medicinal chemistry
approaches was to identify lead compounds with superior
10.1021/jm9003818 CCC: $40.75
2009 American Chemical Society
Published on Web 05/06/2009

3378 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Faghih et al.
Figure 1. Examples of type I and type II allosteric modulators of R7 nAChR.
in vitro potency compared to 10a, whereas the 4-chloro-3-
methylphenyl ketone (15) shows a 2-fold decline in activity.
Aryl-ketones are inactive. On the other hand, alkyl-ketones
exhibit modulator potencies equivalent or better than 10a and
offer improved efficacy as measured in the FLIPR assay.
Moreover, for alkyl-ketone derivatives, steric factors seem to
be important. Larger alkyl substituents on the carbonyl moiety
slightly decrease PAM potency (e.g., 19 vs 20 and 21). Overall,
the amides were found to be much less potent than their alkyl-
ketone counterparts except for the pyrrolidine amide 25 and
analogues possessing moieties with additional binding properties
(e.g., 28). On the basis of its potency and excellent microsomal
stability, compound 19 (A-867744) was selected for further
characterization.
Compound 19 (0.1 nM to 10 µM) by itself did not evoke
currents in Xenopus oocytes39 expressing R7 nAChRs.40 These
data suggest that 19 does not directly activate R7 nAChRs.
However, 19 enhanced acetylcholine-evoked currents in oocytes
expressing R7 nAChRs. This potentiation was concentration-
dependent, as demonstrated by measuring R7 currents evoked
by ACh (100 µM) following brief preincubation with varying
concentrations of 19 (Figure 3A). As depicted, 19 not only had
a robust effect on the peak current response but also slowed
the decay of the current response (Figure 3). Therefore, 19, like
urea 3,32 (Figure 1) represents a novel type II positive allosteric
modulator chemotype.
To further confirm the effect of 19 on R7 nAChR desensitiza-
tion, responses to a selective R7 agonist, 29,16 were measured
in rat neonatal cortical neurons (Figure 3B). Compound 29 alone
did not evoke Ca2+ fluorescence responses, which is attributable
to rapid receptor desensitization within the time scale of
detection. In the presence of 5 µM 19, however, concentration-
dependent increases in responses were observed, demonstrating
that Ca2+ flux responses can be measured when R7 nAChR
desensitization kinetics are altered by compound 19. In contrast
to effects at R7 nAChR, 19 (up to 30 µM) did not potentiate
nicotine-evoked Ca2+ transients at human recombinant R4ꢀ2
and R3ꢀ4 nAChRs expressed in HEK-293 cell lines33,40 (data
Figure 2. High-throughput screening hit.
metabolic stability while retaining in vitro modulation at the
R7 nAChR. To accomplish this, the ethyl ester in 10a was
replaced with ketones (Scheme 1) and amides (Scheme 2) as
functionalities that might lead to improved metabolic stability.
A Paal-Knorr reaction36 between the 1,4-butanedione 9 and
4-aminobenzenesulfonamide afforded compound 10a.
Treatment of ester 10a with sodium hydroxide in hot ethanol
provided the carboxylic acid 11. Compound 11 was transformed
to its acid chloride with oxalyl chloride and DMF in THF
followed by treatment with N-methyl-O-methyl hydroxylamine
hydrochloride and triethylamine in CH2Cl2 to provide the
Weinreb37 amide 13 (Scheme 1). Compound 13 when treated
with Grignard reagents provided the desired 3-keto-pyrrole-
sulfonamides.
3-Amide-pyrrolesulfonamides were prepared by treating
mixture of 11 and amines with PS-DCC39 and HOBT in DMA/
MeCN under microwave conditions (Scheme 2).
Results and Discussion
The SAR of a series of arylketones (Table 1) suggests that
the addition of electron-withdrawing or donating groups to the
aryl group does not improve in vitro potency as assessed by
changes in R7 nAChR mediated intracellular Ca2+ fluorescence
responses using FLIPR in human neuroblastoma (IMR-32) cells
expressing native R7 nAChRs. The 4-methylphenyl and 4-chlo-
rophenyl ketones (14 and 17) show at least 10-fold decrease in

DiscoVery of A-867744 as a PositiVe Allosteric Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3379
Table 1. In vitro R7 nAChR PAM Activity and Microsomal Stability
For agents targeted for treatment of CNS diseases, efficient
brain penetration is of major importance. The high throughput
screening hit 10a had a very low brain:plasma ratio and achieved
negligible brain concentrations (Figure 4A). In contrast, 19
achieved acceptable brain concentrations and favorable brain:
plasma ratios.
Compound 19 was assessed for its PK properties in rat, dog,
and monkey (Table 2). The animal pharmacokinetic profile of
compound 19 was characterized by relatively low plasma
clearance values (1.1-2.5 L/h/kg) and volumes of distribution
(1.7-4.6 L/kg) across species, with terminal elimination half-
lives in the 1.0-1.7 h. The bioavailability is 83% in rat and
somewhat lower in dog (55%) and monkey (68%).
The R7 nAChR appears to play an important role in the
filtering of afferent sensory input, a CNS function also referred
to as gating.43 For eample, abnormal hippocampal R7 nAChR
expression and function are linked to the inability of the DBA/2
mouse strain and schizophrenic patients to inhibit or “gate”
evoked potential responding to repetitive auditory stimuli.8,43,44
Sensory gating deficits are thought to be related to the poor
attention and cognitive function exhibited by schizophrenia
patients. In a paired auditory stimulus paradigm, compound 19
(0.1-10.0 µmol/kg, ip) improved sensory gating deficits when
tested in vivo in the DBA/2 mouse. Like compounds 130 and
3,33 compound 19 (Figure 5) significantly reduced the ratio of
the response to test stimulus relative to the preceding response
to conditioning stimulus (T:C ratio). Reduced T:C ratios are
indicative of improved sensory gating, and the ability to lower
T:C ratios suggests that compound 19 may be useful to improve
cognition in schizophrenia.
Brain concentrations of 19 after 0.1, 1.0, and 10.0 µmol/kg,
ip administrations were 2, 15, and 140 ng/g, respectively. Brain
levels of 19 associated with minimally effective dose after 0.1
µmol/kg ip administration is approximately 6 nM. This exposure
overlaps with the lower range of the concentration-response
determined from oocytes expressing rat R7 nAChRs (Figure
3A). These results suggest that a favorable response in vivo
can be achieved at concentrations of PAM below that necessary
to produce maximum effect in vitro.
Besides being efficacious in vivo, Compound 19 also pos-
sesses an excellent safety profile. It showed no inhibition of
major liver metabolic enzymes such as CYP3A4, CYP2C9 (IC50
> 10 µM), and CYP2D6 (IC50 ) 2 µM) and thus displays a
low potential for drug-drug interactions. Compound 19 exhib-
ited no significant hERG interactions (dofetilide binding Ki >
10 µM, hERG IC50 > 20 µM). When tested in the anesthetized
rat cardiovascular assays, compound 19 (3, 10, and 30 mg/kg
achieving mean plasma concentrations of 1.0, 5.0, and 12.4 µg/
mL) did not display any significant effects on mean arterial
pressure, heart rate, cardiac contractility (dP/dt @ 50 mmHg),
or peripheral vascular resistance (data not shown).
a Compounds were tested in a cell-based fluorescent imaging plate reader
(FLIPR) assay using human IMR-32 neuroblastoma that endogenously
express R7 nAChRs.41 Application of test compound alone (up to 10 µM)
or R7 nAChR agonists such as 29 (PNU-282987) alone did not change
intracellular Ca2+ levels. In contrast, when test compound was preapplied
at concentrations ranging from 0.3 to 30 µM, followed by subsequent
addition of a fixed concentration of R7 agonist (10 µM), concentration-
dependent increases in fluorescence responses were detected. EC50 values
are means ( standard deviation of 2-5 separate experiments, each
conducted in duplicate, except, for example, 3 where n ) 9. Numbers
between parentheses indicated efficacy ( standard deviation expressed as
% of compound 3.32 b In vitro metabolism in rat liver microsomes expressed
as percent remaining at 30 min and are shown only for active compounds
(efficacy >10%).
Conclusions
In summary, optimization of the high-throughput screening
hit 10a led to the discovery of the pyrrole-sulfonamide 19, a
potent and selective R7 nAChR positive allosteric modulator.
Compound 19 not only enhances the peak R7 nAChR current
evoked by acetylcholine but also slows the desensitization profile
of agonist responses. The excellent in vitro activity of this
compound is matched by acceptable animal pharmacokinetics
and safety profiles. Furthermore, 19 demonstrates efficacy to
improve sensory gating in a rodent model, which suggests it
may be useful in treating the cognitive deficits in schizophrenia.
Thus, the pyrrole-sulfonamide 19 represents a novel chemotype
not shown). To further investigate its pharmacological specific-
ity, 19 was tested for its ability to inhibit the binding of relevant
ligands in a panel of receptor binding assays for >70 diverse
neurotransmitter receptor and ion channel sites (CEREP,
France). At the screening concentration of 10 µM, no significant
interaction at any of the targets (>50% displacement of test
ligand) was observed. On the basis of its selectivity and potency,
along with acceptable in vitro microsomal stabilty, compound
19 was selected for further characterization.

3380 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Faghih et al.
Figure 3. (A) Compound 19 enhances rat R7 nAChR-mediated currents in Xenopus oocytes. Inset: representative recording illustrating positive
modulation of ACh-evoked currents by (a) no PAM, 0.3 (b), 1.0 (c), and 10 µM (d) of 19 at R7 nAChRs. The arrow represents compound addition.
(B) Increase in Ca2+ responses in IMR-32 cell line evoked by 19 (5 µM) in presence of varying concentrations of the R7 nAChR agonist 29.
Scheme 1. Synthesis of 3-Keto-pyrrolesulfonamide Derivativesa
a Reagents: (a) Ethylacetoacetate, K2CO3, 2-butanone, reflux, 24 h; (b) 4-aminobenzenesulfonamide, glacial acetic acid, 100 °C, 48 h; (c) NaOH (5 M),
ethanol, reflux, 2 h; (d) oxalyl chloride, DMF, CH2Cl2, 0 °C to room temperature, 3 h; (e) N,O-Dimethylhydroxylamine hydrochloride, TEA, CH2Cl2, 0 °C
to room temperature, 5 h; (f) RMgX, THF; aq HCl.
Scheme 2. Synthesis of 3-Amide-pyrrolesulfonamide
Experimental Section
Derivativesa
All solvents were purchased from Aldrich (Sure/Seal anhydrous
solvents), and commercially available reagents were used as
received.
1H NMR spectra were recorded on Varian Mercury (300 MHz)
and Varian Unity (400 MHz). NMR data are reported in parts per
million (δ) and are referenced to the tetramethylsilane as internal
standard. Electron spray ionization (ESI) mass spectra were recorded
on a Finnigin-400 instrument.
Analytical LC-MS was performed on a Finnigan Navigator mass
spectrometer and Agilent 1100 HPLC system running Xcalibur 1.2,
Open-Access 1.3, and custom login software. The mass spectrom-
eter was operated under positive APCI ionization conditions. The
HPLC system comprised an Agilent Quaternary pump, degasser,
column compartment, autosampler, and diode-array detector, with
a Sedere Sedex 75 evaporative light-scattering detector. The column
used was a Phenomenex Luna Combi-HTS C8(2) 5 µm 100 Å (2.1
mm × 30 mm). A gradient of 10-100% acetonitrile (A) and 0.1%
trifluoroacetic acid in water (B) was used at a flow rate of 2.0 mL/
min (0-0.1 min 10% A, 0.1-2.6 min 10-100% A, 2.6-2.9 min
100% A, 2.9-3.0 min 100-10% A, 0.5 min postrun delay). Or a
a Reagents: (a) PS-DCC, DMA, HOBT, Hunig’s base, amines, MeCN,
microwave, 100°C, 10 min.
useful in assessing whether a type II PAM displays advantages
when compared to type I PAMs and ultimately to agonists in
terms of efficacy, safety, and tolerability across preclinical
models.

DiscoVery of A-867744 as a PositiVe Allosteric Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3381
Figure 4. Brain and plasma concentrations of HTS hit 10a (hatched bar) and 19 (solid bar) after administration of a 5 µmol/kg ip dose in mouse.
Table 2. PK of Compound 19 in Rat, Dog, and Monkeya
and the precipitate separated by filtration. The pale-yellow solid
was washed with ether and dried. 1H NMR (CDCl3): δ 1.31 (t, J )
species
dose
t1/2
Vss
Clp
Cmax
F
8 Hz, 3H), 2.25 (s, 3H), 4.42 (m, 2H), 4.80 (m, 2H), 6.89 (s, 1H),
7.30-7.44 (m, 4H), 7.49-7.92 (m, 4H). MS (ESI) m/z 419 (M +
H)+. Anal. (C20H19ClN2O4S) C, H, N.
rat
dog
monkey
2
1.7
1.3
1.0
4.6
1.7
2.8
2.1
1.1
2.5
41
87
17
83
55
68
0.5/1
0.5/1
5-(4-Chlorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-1H-3-car-
boxylic Acid (11). The ethyl ester (4.19 g, 10 mmol) was suspended
in ethanol (50 mL) and treated with 5 M NaOH (10 mL). The
mixture was heated at reflux for 2 h. The suspension was filtered
to collect a beige solid, which was rinsed with ethanol, stirred
thoroughly in 1:1 EtOAc/CH2Cl2, recollected by filtration, and
rinsed with more 1:1 EtOAc/CH2Cl2. The solid was dried under
vacuum to give 2.18 g of the disodium salt as a light-beige powder.
1H NMR (DMSO-d6): δ 2.32 (s, 3H), 6.54 (s, 1H), 6.98 (d, J ) 8
Hz, 2H), 7.07 (d, J ) 8 Hz, 2H) 7.20 (d, J ) 8 Hz, 2H), 7.67 (d,
J ) 8 Hz, 2H). MS (ESI: negative ion detection) m/z 389/391. The
disodium salt (0.94 g) was partitioned between EtOAc and 0.5 M
aq HCl (10 mL) with stirring. Hexane was added, and the biphasic
mixture was thoroughly stirred. The organic phase was separated,
washed with water, dried (Na2SO4), and concentrated to give 744
mg of the free acid as a solid.
a Units: dose iv and po (µmol/kg), t1/2 iv (h), Vss iv (L/kg), Clp iv (L/h/
kg), Cmax po (ng/mL), F (%).
gradient of 10-100% acetonitrile (A) and 10 mM ammonium
acetate in water (B) was used at a flow rate of 2.0 mL/min (0-0.1
min 10% A, 0.1-2.6 min 10-100% A, 2.6-2.9 min 100% A,
2.9-3.0 min 100-10% A, 0.5 min postrun delay) were used.
Chromatographic separations were performed on Analogix Super
Flash columns or by preparative HPLC on a Phenomenex Luna
C8(2) 5 µm 100 Å AXIA column (30 mm × 75 mm). A gradient
of acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) was
used at a flow rate of 50 mL/min (0-0.5 min 10% A, 0.5-7.0 min
linear gradient 10-95% A, 7.0-10.0 min 95% A, 10.0-12.0 min
linear gradient 95-10% A). Samples were injected in 1.5 mL
DMSO:MeOH (1:1). A custom purification system was used,
consisting of the following modules: Waters LC4000 preparative
pump, Waters 996 diode-array detector, Waters 717+ autosampler,
Waters SAT/IN module, Alltech Varex III evaporative light-
scattering detector, Gilson 506C interface box, and two Gilson
FC204 fraction collectors. The system was controlled using Waters
Millennium32 software, automated using an Abbott developed
Visual Basic application for fraction collector control and fraction
tracking. Fractions were collected based upon UV signal threshold
and selected fractions subsequently analyzed by flow injection
analysis mass spectrometry using positive APCI ionization on a
Finnigan LCQ using 70:30 MeOH:10 mM NH4OH(aq) at a flow
rate of 0.8 mL/min. Loop-injection mass spectra were acquired
using a Finnigan LCQ running LCQ Navigator 1.2 software and a
Gilson 215 liquid handler for fraction injection controlled by an
Abbott developed Visual Basic application. Elemental combustion
analyses were obtained from Robertson Microlit Laboratories. The
purity of the compounds was analyzed by LC-MS, which showed
g95% purity for all compounds.
(E)-5-(4-Chlorophenyl)-1-(4-(N-((dimethylamino)methylene)-
sulfamoyl)phenyl)-2-methyl-1H-pyrrole-3-carbonyl Chloride (12).
Oxalyl chloride (2 M in CH2Cl2, 3.84 mL) was added dropwise at
0 °C to a solution of sodium carboxylate (1.5 g, 3.84 mmol) in
CH2Cl2 (25 mL)/DMF (0.5 mL). Mixture was allowed to come to
room temperature and stirred for 3 h. Mixture was concentrated
under reduced pressure and used directly for the next step. MS
(ESI) m/z 465 (M + H)+.
(E)-5-(4-Chlorophenyl)-1-(4-(N-((dimethylamino)methylene)-
sulfamoyl)phenyl)-N-methoxy-N-2-dimethyl-1H-Pyrrole-3-car-
boxamide (13). To a cold (0 °C) mixture of pyrrole-acylchloride
(1.9 g, 4.64 mmol) and N,O-dimethylhydroxylamine hydrochloride
(0.5 g, 5.11 mmol) in CH2Cl2 (50 mL), TEA (1.62 mL, 11.6 mmol)
was added. The reaction mixture was stirred at room temperature
for 5 h. Solvent was evaporated and the residue purified by
chromatography using a mixture of CH2Cl2:MeOH (95:5). The
desired compound was obtained in 70%. 1H NMR (CDCl3): δ 2.25
(s, 3H), 3.05 (s, 3H), 3.20 (s, 3H) 3.42 (s, 3H), 3.80 (s, 3H), 6.75
(s, 1H), 6.91(d, J ) 8 Hz, 2H), 7.12 (d, J ) 8 Hz, 2H), 7.23 (d, J
) 8 Hz, 2H), 7.93 (d, J ) 8 Hz, 2H), 8.17 (s, 1H). MS (ESI) m/z
489 (M + H)+. Anal. (C23H25ClN4O4S·H2O) C, H, N.
General Procedure for Grignard Coupling. To a solution of
13 (75 mg, 0.153 mmol) in anhydrous THF (5 mL) at room
temperature, Grignard reagent (5 equiv) was added and the mixture
heated to reflux and monitored by LC-MS. After cooling, the
reaction was quenched by careful addition of a solution of saturated
ammonium chloride (2 mL), water (5 mL), and extracted with
CH2Cl2. After drying the organic phase over sodium sulfate, solvent
was removed under reduced pressure and the residue heated in a
mixture of methanol (5 mL) and 5 N HCl (5 mL) and monitored
by LC-MS. After cooling, the mixture was concentrated and the
residue purified by reverse phase HPLC.
Ethyl 2-Acetyl-4-(4-chlorophenyl)-4-oxobutanoate (9). A mix-
ture of 2-bromo-1-(4-chlorophenyl)ethanone (3.0 g, 12.8 mmol),
ethyl acetoacetate(1.62 mL, 12.8 mmol), and potassium carbonate
(4.5 g) was heated in 2-butanone (50 mL) at reflux for 24 h. Upon
cooling, the mixture was filtered over celite and concentrated. The
residue was purified by chromatography with CH2Cl2 to give the
1
title compound in 78% yield. H NMR (CDCl3): δ 1.31 (t, J ) 8
Hz, 3H), 2.20 (s, 3H), 3.32 (m, 2H), 3.75 (m, 1H), 4.05 (m, 2H),
7.35 (d, J ) 6.7 Hz, 2H), 7.83 (d, J ) 6.7 Hz, 2H). MS (ESI) m/z
283 (M + H)+. Anal. (C8H6BrClO) C, H, N.
Ethyl 5-(4-Chlorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-
1H-pyrrole-3-carboxylate (10a). A mixture of ethyl 2-acetyl-4-
(4-chlorophenyl)-4-oxobutanoate (3.3 g, 11.7 mmol) and sulfanil-
amide (2.01 g, 11.7 mmol) in glacial acetic acid (75 mL) was heated
at 100 °C for 48 h. After cooling, the mixture was concentrated
under reduced pressure. Ether (50 mL) was added to the residue

3382 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Faghih et al.
4-(5-(4-Chlorophenyl)-2-methyl-3-(4-methylbenzoyl)-1H-pyr-
rol-1-yl)benzenesulfonamide (14). 1H NMR (CDCl3): δ 2.26 (3H),
2.35 (3H), 6.75 (s, 1H), 7.10 (d, J ) 8 Hz, 2H), 7.30 (d, J ) 8 Hz,
2H), 7.35 (d, J ) 8 Hz, 2H), 7.54 (m, 4H), 7.75 (d, J ) 8 Hz, 2H),
7.92 (d, J ) 8 Hz, 2H). MS (ESI) m/z 465 (M + H)+. Anal.
(C25H21ClN2O3S) C, H, N.
7.46 (d, J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz, 2H). MS (ESI:
negative ion detection) m/z 430 (M - H)-. Anal. (C21H22ClN3O3S)
C, H, N.
5-(4-Chlorophenyl)-N-isopropyl-N,2-dimethyl-1-(4-sulfa-
moylphenyl)-1H-pyrrole-3-carboxamide (24). 1H NMR (DMSO-
d6/D2O) δ 1.15 (d, J ) 7 Hz, 6H), 2.08 (s, 3H), 3.38 (m, 4H), 6.48
(s, 1H), 7.05 (d, J ) 8.5 Hz, 2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.46
(d, J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz, 2H). MS (ESI: negative
ion detection) m/z 444 (M - H)-. Anal. (C22H24ClN3O3S·H2O)
C, H, N.
5-(4-Chlorophenyl)-2-methyl-3-(pyrrolidine-1-carbonyl)-1H-
pyrrol-1-yl)benzenesulfonamide (25). 1H NMR (DMSO-d6/D2O)
δ 1.85 (m, 4H), 2.18 (s, 3H), 3.45 (m, 2H), 3.64 (m, 2H), 6.68 (s,
1H), 7.05 (d, J ) 8.5 Hz, 2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.46 (d,
J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz, 2H). MS (ESI: negative ion
detection) m/z 442 (M - H)-. Anal. (C22H22ClN3O3S) C, H, N.
5-(4-Chlorophenyl)-2-methyl-3-(piperidine-1-carbonyl)-1H-
pyrrol-1-yl)benzenesulfonamide (26). 1H NMR (DMSO-d6/D2O)
δ 1.59 (m, 4H), 1.62 (m, 2H), 2.09 (s, 3H), 3.56 (m, 4H), 6.46 (s,
1H), 7.05 (d, J ) 8.5 Hz, 2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.46 (d,
J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz, 2H). MS (ESI: negative ion
detection) m/z 456 (M - H)-. Anal. (C23H22ClN3O3S · H2O)
C, H, N.
5-(4-Chlorophenyl)-2-methyl-3-(morpholine-1-carbonyl)-1H-
pyrrol-1-yl)benzenesulfonamide (27). 1H NMR (DMSO-d6/D2O)
δ 2.10 (s, 3H), 3.61 (m, 8H), 6.52 (s, 1H), 7.06 (d, J ) 8.5 Hz,
2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.46 (d, J ) 8.5 Hz, 2H), 7.90 (d,
J ) 8.5 Hz, 2H). MS (ESI: negative ion detection) m/z 458 (M -
H)-. Anal. (C22H22ClN3O4S) C, H, N.
4-(3-(4-Chloro-3-methylbenzoyl)-5-(4-chlorophenyl)-2-methyl-
1
1H-pyrrol-1-yl)benzenesulfonamide (15). H NMR (CDCl3): δ
2.32 (s, 3H), 2.43 (s, 3H), 6.66 (s, 1H), 7.09 (d, J ) 8.5 Hz, 2H),
7.28 (d, J ) 8.5 Hz, 2H), 7.59-7.78 (m, 5H), 7.91 (d, J ) 8.5 Hz,
2H). MS (ESI) m/z 500 (M + H)+. Anal. (C25H20Cl2N2O3S. ·
0.5H2O) C, H, N.
4-(5-(4-Chlorophenyl)-3-(5-fluoro-2-methoxybenzoyl)-2-meth-
1
yl-1H-pyrrol-1-yl)benzenesulfonamide (16). H NMR (CDCl3):
δ 2.42 (s, 3H), 3.79 (s, 3H), 6.40 (s, 1H), 6.91 (d, J ) 8.5 Hz, 2H),
6.94 (m, 1H), 7.11 (m, 6H), 7.30 (d, J ) 8.5 Hz, 2H), 7.99 (d, J )
8.5 Hz, 2H). MS (ESI) m/z 499 (M + H)+. Anal. (C25H20ClFN2O4S)
C, H, N.
4-(3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-2-methyl-1H-pyr-
1
rol-1-yl)benzenesulfonamide (17). H NMR (CDCl3): δ 2.44 (s,
3H), 6.57 (s, 1H), 6.94 (d, J ) 8.5 Hz, 2H), 7.17 (d, J ) 8.5 Hz,
2H), 7.33 (d, J ) 8.5 Hz, 2H), 7.47 (d, J ) 8.5 Hz, 2H), 7.84 (d,
J ) 8.5 Hz, 2H), 8.01 (d, J ) 8.5 Hz, 2H). MS (ESI) m/z 486 (M
+ H)+. Anal. (C24H18Cl2N2O3S) C, H, N.
4-(3-Acetyl-5-(4-chlorophenyl)-2-methyl-1H-pyrrol-1-yl)ben-
zenesulfonamide (18). 1H NMR (DMSO-d6): δ 2.32 (s, 3H), 2.43
(s, 3H), 6.96 (s, 1H), 7.05 (d, J ) 8.5 Hz, 2H), 7.30 (d, J ) 8.5
Hz, 2H), 7.45 (d, J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz, 2H). MS
(ESI) m/z 389 (M + H)+. Anal. (C19H17ClN2O3S) C, H, N.
5-(4-Chlorophenyl)-N-(2hydroxyethyl)-2-methyl-N-propyl-1-
1
(4-sulfamoylphenyl)-1H-pyrrole-3-carboxamide (28). H NMR
4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)-
1
(DMSO-d6/D2O) δ 0.96 (t, J ) 7 Hz, 3H), 1.60 (m, 2H), 2.09 (s,
3H), 3.41-3.46 (m, 2H), 3.46-3.52 (m, 2H), 3.53-3.64 (m, 2H),
6.50 (s, 1H), 6.97-7.10 (2H), 7.05 (d, J ) 8.5 Hz, 2H), 7.27 (d, J
) 8.5 Hz, 2H), 7.46 (d, J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz, 2H).
MS (ESI: negative ion detection) m/z 474 (M - H)-. Anal.
(C23H26ClN3O4S) C, H, N.
benzenesulfonamide (19). H NMR (CDCl3): δ 1.24 (t, J ) 7
Hz, 3H), 2.47 (s, 3H), 2.87 (q, J ) 7.3 Hz, 2H), 6.74 (s, 1H),
7.17 (d, J ) 8.5 Hz, 2H), 7.28 (d, J ) 8.5 Hz, 2H), 7.98 (d, J
) 8.5 Hz, 2H). MS (ESI) m/z 403 (M + H)+. Anal.
(C20H19ClN2O3S)
C, H, N.
In Vitro Metabolism. Microsomes were distributed to 96-well
plates and warmed for 10 min at 37 °C in the on-deck incubator.
Compound and NADPH were mixed in a 96 deep-well plate and
the mixture delivered in triplicate to the warm microsomes to initiate
the reaction. In the incubation mixture, the final reagent concentra-
tions were as follows: NADPH 1 mM, compound 1 µM, and
microsomal protein 0.5 mg/mL. After incubation for 0, 10, 20, and
30 min, reactions were stopped by protein precipitation with one
volume of acetonitrile/methanol containing 0.4 µM warfarin, the
internal standard. Samples were then centrifuged at 3400 rpm for
45 min, and the clarified supernatant was transferred to 96-well
plates and divided for SPE-MS preparation and HPLC-MS analysis.
Fluorescence Measurements. Changes in intracellular Ca2+
levels were measured using calcium-4 no-wash dye as previously
described.32 Briefly, IMR-32 cells were grown as a monolayer in
black-walled clear-bottom plates coated with poly-D-lysine (BD
Biosciences, Bedford, MA). Prior to the assay, culture media was
discarded and cells loaded no-wash dye in NMDG buffer (10 mM
HEPES, pH 7.4, 140 mM N-Methyl-D-glucosamine (NMDG), 5
mM KCl, 1 mM MgCl2, 10 mM CaCl2 for 1 h at 25 °C). Primary
rat neonatal cortical neurons were isolated from newborn rat pups
(E18) and cultured,44 except that cells were plated onto poly
D-lysine-coated black-walled 384-well plates at a density of 10000
cells/well. Experiments were conducted 3 days postplating. Changes
in cellular Ca2+ levels were measured as described above, except
that assays were conducted using calcium-3 dye prepared in Hank’s
balanced salt solution buffer (HBSS) containing 20 mM HEPES
as described by the manufacturer (MDS Analytical Technologies,
Sunnyvale, CA). In both cases, after a baseline reading for 10 s,
modulator/test compounds were added to the cell plate and
incubated for 3-5 min. This was followed by the addition of
appropriate concentrations of agonist. The peak increase in
fluorescence over baseline was determined and is expressed as
relative fluorescence units (RFU), normalized to the maximal value.
4-(5-(4-Chlorophenyl)-3-(cyclopropanecarbonyl)-2-methyl-
1H-pyrrol-1-yl)benzenesulfonamide (20). H NMR (CDCl3): δ
0.96 (m, 2H), 1.21 (m, 2H), 2.47 (s, 3H), 2.49 (m, 1H), 6.90 (s,
1H), 6.95 (d, J ) 8.5 Hz, 2H), 7.18 (d, J ) 8.5 Hz, 2H), 7.28 (d,
J ) 8.5 Hz, 2H), 7.97 (d, J ) 8.5 Hz, 2H). MS (ESI) m/z 415 (M
+ H)+. Anal. (C21H19ClN2O3S·H2O) C, H, N.
1
4-(5-(4-Chlorophenyl)-2-methyl-3-(3-methylbutanoyl)-1H-
1
pyrrol-1-yl)benzenesulfonamide (21). H NMR (CDCl3): δ 1.02
(d, J ) 6 Hz, 6H), 2.31 (m, 1H), 2.44 (s, 3H), 2.69 (d, J ) 6 Hz,
2H), 6.72 (s, 1H), 6.94 (d, J ) 8.5 Hz, 2H), 7.17 (d, J ) 8.5 Hz,
2H), 7.27 (d, J ) 8.5 Hz, 2H), 7.98 (d, J ) 8.5 Hz, 2H). MS (ESI)
m/z 431 (M + H)+. Anal. (C22H23ClN2O3S·0.5H2O) C, H, N.
General Procedure for Amide Formation. In a microwave vial
containing 3 equiv of PS-DCC, a solution of 11 (29 mg, 0.06 mmol)
in DMA (1.0 mL) was added, followed in succession by a solution
of 1-hydroxybenzotriazole (8 mg, 0.06 mmol) in acetonitrile (0.6
mL), and a solution of N,N-diisopropylethylamine (23 mg, 0.18
mmol) in acetonitrile (0.6 mL). Then a solution of the amine (0.07
mmol) in acetonitrile (0.4 mL) was added. The mixture was heated
in the microwave (Emrys Optimizer/Biotage) to 100 °C for 600 s.
The reaction was filtered through a Si-carbonate cartridge (Sili-
Cycle) and concentrated to dryness. The residues were purified by
preparative HPLC.
5-(4-Chlorophenyl)-N,N,2-trimethyl-1-(4-sulfamoylphenyl)-
1H-pyrrole-3-carboxamide (22). 1H NMR (DMSO-d6/D2O) δ 2.09
(s, 3H), 3.83 (s, 6H), 6.55 (s, 1H), 7.05 (d, J ) 8.5 Hz, 2H), 7.27
(d, J ) 8.5 Hz, 2H), 7.46 (d, J ) 8.5 Hz, 2H), 7.88 (d, J ) 8.5 Hz,
2H). MS (ESI: negative ion detection) m/z 416 (M - H)-. Anal.
(C20H20ClN3O3S) C, H, N.
5-(4-Chlorophenyl)-N-ethyl-N,2-dimethyl-1-(4-sulfamoylphe-
nyl)-1H-pyrrole-3-carboxamide (23). 1H NMR (DMSO-d6/D2O)
δ 1.13 (t, J ) 7 Hz, 3H), 2.09 (s, 3H), 3.38 (s, 3H), 3.46 (m, 2H),
6.51 (s, 1H), 7.05 (d, J ) 8.5 Hz, 2H), 7.27 (d, J ) 8.5 Hz, 2H),

DiscoVery of A-867744 as a PositiVe Allosteric Modulator
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3383
ms after the initial conditioning stimulus. The software averaged
the 120 paired responses into one composite evoked potential (EP)
response. The amplitude of the auditory EP N40 wave was
determined for both the averaged conditioning (C) and test (T) EP
response, and a ratio (T:C ratio) was derived between the two
responses by dividing the test amplitude by the conditioning
amplitude. The T:C ratio was the index by which compound efficacy
was determined. The vehicle for 19 and 3 was 5% DMSO and 5%
Solutol in PBS. Compound was administered immediately before
mice were placed into the recording chambers and initiation of
auditory evoked potential recording. Recording of paired auditory
evoked potentials continued for 30 min after the recordings began.
For every experiment, each mouse was administered all treatments
including a control vehicle in random order on separate days with
at least 72 h between treatments. This within-subjects design
allowed each mouse to serve as its own control. Data are expressed
as means ( SEM. Significant differences between group means
were assessed by one-way analysis of variance (ANOVA), and
Newman-Keuls post hoc tests assessed differences between
treatment groups (GraphPad Software Inc., San Diego, CA). A p
value <0.05 was considered statistically significant.
Figure 5. Compound 19 improved sensory gating in DBA/2 mice by
significantly lowering T:C ratios. One-way repeated measures ANOVA
F(5.59) ) 6.393, p < 0.0001. *p < 0.05, ***p < 0.001 vs vehicles,
Newman-Keuls post hoc test. Compound 3 was dosed at 30.0 µmol/
kg ip. For experimental details, see ref 43.
Two-Electrode Voltage Clamp Studies. Oocytes were obtained
from adult female Xenopus laeVis frogs (Blades Biological Ltd.,
Cowden, Edenbridge, Kent, UK) and cared for in accordance with
the Institutional Animal Care Committee guidelines that meet the
guidelines of the National Institutes of Health. Chemicals and
reagents were obtained from Sigma or Fisher Scientific (Essex, UK).
Xenopus laeVis oocytes were prepared for electrophysiological
experiments as described.32 Briefly, 3-4 lobes from ovaries of
female adult Xenopus laeVis frogs were removed, manually defol-
liculated, treated with collagenase type 1A (2 mg/mL, Sigma)
prepared in low-Ca2+ Barth’s solution (in mM: 90 NaCl, 1.0 KCl,
0.66 NaNO3, 2.4 NaHCO3, 10 HEPES, 2.5 Na-pyruvate, 0.82
MgCl2, and 0.5% v/v penicillin-streptomycin solution, pH ) 7.55)
for 1.5-2 h at ∼18 °C under constant agitation to obtain isolated
oocytes. The oocytes were injected with ∼20-25 ng nAChR cRNA,
kept at 18 °C in a humidified incubator in modified Barth’s solution
(in mM: 90 NaCl, 1.0 KCl, 0.66 NaNO3, 2.4 NaHCO3, 10 HEPES,
2.5 Na-pyruvate, 0.74 CaCl2, 0.82 MgCl2, 0.5% v/v penicillin-
streptomycin solution, pH ) 7.55), and used 2-7 days after
injection. Responses were measured by two-electrode voltage clamp
techniques using POETs. During recordings, the oocytes were
bathed in Ba2+-OR2 solution (in mM: 90 NaCl, 2.5 KCl, 2.5 BaCl2,
1.0 MgCl2, 5.0 HEPES, 0.0005 atropine, pH ) 7.4) and held at
-60 mV at room temperature. Agonists were applied to the
recording chamber for 1 s at 6 mL/s with or without modulators.
Test compounds were applied for a minimum of 60 s prior to agonist
application. In two electrode voltage clamp studies, responses were
quantified by measuring apparent peak current amplitude. Apparent
peak current responses were expressed as percentage response to
100 µM ACh in modulator concentration responses. Data were
analyzed and fitted using GraphPad Prism (San Diego, CA).
Sigmoidal dose-response (variable slope) function was used to fit
the replicates. The pEC50 (-log EC50) or pIC50 (-log IC50) values
and associated standard error of the mean (SEM) were obtained
from fitted results. The maximum mean ( SEM values were
calculated from individual experiments.
N40 Gating. DBA/2 mice (16-20 g, Harlan) were used for N40
gating experiments and surgical techniques employed were similar
to those previously described.43 Briefly, mice were anesthetized
with a solution of 2.8% ketamine, 0.28% xylazine, 0.05% acepro-
mazine, and stereotaxically implanted with tripolar stainless steel
wire electrodes for EEG recordings in the CA3 region of the
hippocampus. Animals were allowed to recover for at least four
days before conducting experiments. Auditory evoked potentials
were generated by presentation of 120 sets of paired white noise
bursts (5 ms duration) from a speaker within the recording chamber
at a distance of approximately 15-20 cm from the mouse. The
first auditory stimulus of the pair, or conditioning stimulus (C),
was followed 0.5 s later by the second auditory stimulus (test
stimulus, T). The length of time between stimulus pairs was 15 s.
Data acquisition software (SciWorks, Datawave Technologies,
Berthoud, CO) recorded EEG signals 100 ms before and for 900
Supporting Information Available: LC/MS analyses data for
compounds 11 and 19 and hemodynamic evaluation of compound
19 in the rat cardiovascular model. This material is available free
of charge via the Internet at http://pubs.acs.org.
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