Discovery of a series of 2-phenyl- N -(2-(pyrrolidin-1-yl)phenyl)acetamides as novel molecular switches that modulate modes of Kv7.2 (KCNQ2) channel pharmacology: Identification of (S)-2-phenyl- N -(2-(pyrrolidin-1-yl) phenyl)butanamide (ML252) as a potent, brain penetrant Kv7.2 channel inhibitor

Article abstract of DOI:10.1021/jm300700v

A potent and selective inhibitor of KCNQ2, (S)-5 (ML252, IC₅₀ = 69 nM), was discovered after a high-throughput screen of the MLPCN library was performed. SAR studies revealed a small structural change (ethyl group to hydrogen) caused a functional shift from antagonist to agonist activity (37, EC₅₀ = 170 nM), suggesting an interaction at a critical site for controlling gating of KCNQ2 channels.

Full text of DOI:10.1021/jm300700v

Brief Article
pubs.acs.org/jmc
Discovery of a Series of 2‑Phenyl‑N‑(2-(pyrrolidin-1-
yl)phenyl)acetamides as Novel Molecular Switches that Modulate
Modes of Kv7.2 (KCNQ2) Channel Pharmacology: Identification of
(S)‑2-Phenyl‑N‑(2-(pyrrolidin-1-yl)phenyl)butanamide (ML252) as a
Potent, Brain Penetrant Kv7.2 Channel Inhibitor
Yiu-Yin Cheung,†,‡ Haibo Yu,⊥ Kaiping Xu,⊥ Beiyan Zou,⊥ Meng Wu,⊥,# Owen B. McManus,⊥,#
Min Li,*,⊥,# Craig W. Lindsley,†,‡,§,∥ and Corey R. Hopkins*,†,‡,§,∥
‡
†Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center,
Nashville, Tennessee 37232, United States
§Department of Chemistry, ∥Vanderbilt Specialized Chemistry for Accelerated Probe Development (MLPCN), Vanderbilt University,
Nashville, Tennessee 37232, United States
#
⊥Department of Neuroscience, High Throughput Biology Center, Johns Hopkins Ion Channel Center (JHICC), Johns Hopkins
University, Baltimore, Maryland 21205, United States
S
* Supporting Information
ABSTRACT: A potent and selective inhibitor of KCNQ2, (S)-5 (ML252, IC50 = 69 nM), was discovered after a high-
throughput screen of the MLPCN library was performed. SAR studies revealed a small structural change (ethyl group to
hydrogen) caused a functional shift from antagonist to agonist activity (37, EC50 = 170 nM), suggesting an interaction at a critical
site for controlling gating of KCNQ2 channels.
(1, 1a, 24), with both 1a and 2 being advanced into clinical
INTRODUCTION
■
trials (Figure 1).5 A more recent example has been reported as
Alzheimer’s disease (AD) is the most common neuro-
degenerative disorder and the most common form of dementia,
affecting a worldwide population of >20 million people. One of
the major conditions of the disease is the decline of memory
and loss of cognitive function leading to significant burden on
the families of patients and the medical community, with an
estimated cost of more than $300 billion.1 Although there is no
known cure, nor are there drugs that slow the progression of
AD, there have been FDA approved drugs to help lessen the
symptoms although these drugs are only effective for a limited
time frame. One class of drugs that has been approved are the
cholinesterase inhibitors: compounds that prevent the break-
down of acetylcholine (ACh).2 There has been significant
research describing the loss of cholinergic tone in AD patients,
which is correlated well with the progression of dementia2 and
provides a rationale for therapeutic modulation of the
cholinergic system.
In contrast to targeting cholinesterase inhibition, one area of
research that has gained significant momentum over the past
several years is use of voltage-gated ion channel modulators,
specifically, potassium (K+) channel inhibitors, to enhance ACh
release.2,3 KCNQ (also known as Kv7) channels belong to the
voltage gated K+ channel superfamily and contain five members
(Kv7.1−Kv7.5), with each member containing six trans-
membrane segments. KCNQs are expressed in both the
peripheral and central nervous systems (PNS and CNS). A
number of compounds that modulate KCNQ2 channels have
shown promise in preclinical models of memory enhancement
Figure 1. Structures of XE-991, DMP 543, Linopirdine, and
UCL2077.
a subtype selective blocker of KCNQ channels, 3.6 Unfortu-
nately, the reported selectivity of these compounds is less than
ideal, and thus we embarked on a campaign to search for potent
and selective KCNQ2 inhibitors.
The project initiated with a screen of the >300000 NIH
Molecular Library Small Molecule Repository (MLSMR)
compound collection using a thallium influx assay (PubChem
AID: 2156) to identify inhibitors of the KCNQ2 channel.7
Thallium can be used as a surrogate ion for potassium flux as it
can permeate many potassium selective ion channels. Thallium
influx was detected with a fluorescent dye (FluxOR) in a 384-
Received: May 18, 2012
Published: July 13, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
well format.8 From the initial library screen, ∼3400 compounds
were deemed “hits” and after a round of triage, ∼1000
compounds were counterscreened against parental cells
(PubChem AID: 493029) and cells expressing KCNQ1.
From this round of results, 553 compounds were reconfirmed
against KCNQ2 channels utilizing an automated patch clamp
assay (PubChem AID: 588531) to yield 58 confirmed KCNQ2
inhibitors. From these experiments, compounds 4 and 5 were
selected as starting “lead” compounds for medicinal chemistry
(Table 1). These initial compounds displayed a significant SAR
Table 2. Initial SAR Analysis of Right-Hand Portion
Table 1. HTS Validated Hits
improvement moving from the 2-phenyl (4, 4.7 μM) to 2-
pyrrolidine (5, 0.16 μM). As KCNQ2 inhibitors have been
shown to be effective in enhancing cognition in some CNS
behavioral models, these compounds represent excellent
starting points for a CNS indication (MW ∼ 300, cLogP <
4.5, tPSA < 40).
The initial SAR assessment started with evaluation of the
right-hand portion of the molecule, keeping the 2-(pyrrolidin-1-
yl)aniline portion constant (Table 2). The initial hit, 5, was
resynthesized and reconfirmed from powder (IC50 = 163 nM).9
Modification of the ethyl side chain led to unexpected results.
Truncating the ethyl to a methyl group retained activity (6, 260
nM); however, deletion of the ethyl group or replacement with
a single fluorine led to a mode switch resulting in potent
KCNQ2 activators (7, EC50 = 743 nM; 8, EC50 = 990 nM). To
the best of our knowledge, this is the first report of a “mode
switch” in KCNQ2 channel modulators and the SAR will be
explored further (vide infra). Extending the chain into the
isopropyl group was not tolerated (9); however, activity could
be returned by cyclizing into the cyclopropyl group (10, 3500
nM) with reduced activity compared to 5. Further chain
extension into the sec-butyl group also was deleterious
compared to 5 (11, 2220 nM). Interestingly, deletion of a
methyl group, leaving the straight-chain propyl group, was well
tolerated (12, 320 nM). Cyclization of the pendant side-chain
to the phenyl group resulting in the indane structure was also
tolerated (13, 330 nM). Disubstitution on the methylene group
with either a gem-dimethyl or cyclopropyl was not tolerated (14
and 15), nor was the six-membered chromane structure (16).
Lastly, reversing the amide moiety or alkylation of the amide
resulted in complete loss of activity (17 and 18).
Next, we investigated the right-hand phenyl portion of the
molecule utilizing the ethyl substituted inhibitor scaffold as well
as the unbranched methylene activator scaffold in order to
evaluate both the SAR surrounding modes of pharmacology
(Table 3). For the inhibitor scaffold, most of the substituents
evaluated were well tolerated, leading to nanomolar com-
pounds. There were, however, a couple of exceptions. Namely,
3,4-dimethoxy substituents were not tolerated (19, 20.2 μM), a
125-fold loss of potency, and the 2-chloro substituent led to a
20-fold loss of potency (30, 3.3 μM). All of the other
substitutions (halogen, trifluoromethyl, methoxy) led to
compounds that retained activity similar (2−3-fold loss of
activity) to the unsubstituted phenyl group. Two compounds
were of equal potency to the phenyl group, 4-fluoro (26, 130
nM) and 3-methoxy (34, 120 nM). It is of note that the 3,4-
dimethoxy compound had a 125-fold loss of potency as both
the 4-methoxy (38, 380 nM) and 3-methoxy (34, 120 nM)
were individually very potent compounds. The activator SAR
did not track exactly with the inhibitor SAR; however, a
number of submicromolar compounds were discovered. The
most active compound was the 3-chloro substituent (37, 170
nM). Unlike the inhibitors, the 3-methoxy (35, 2350 nM) and
4-methoxy (39, 1750 nM) activators were much less potent
than the best compound. Although there was a diminution in
potency for some compounds, for the most part, small
substituents were tolerated on the right-hand phenyl group.
After establishing the optimal right-hand substituent for the
inhibitor (5) and activators (37), we next turned our attention
to the left-hand portion of the molecule (Table 4). The initial
HTS hits displayed a preference for the 2-pyrrolidine versus the
2-phenyl, which was confirmed upon resynthesis (4, 1320 nM;
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Table 3. SAR Evaluation of the Phenyl Substituent on the
Inhibitor and Activator Scaffolds
Table 4. SAR Evaluation of the Left-Hand Phenyl
Substituent on the Inhibitor and Activator Scaffolds
KCNQ2 (μM)10
entry
R
R1
IC50
EC50
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
3,4-dimethoxy
Et
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
Et
H
20.23 4.89
0.17 0.02
3,4-difluoro
1.73 0.34
0.49 0.12
1.32 0.29
0.70 0.82
1.07 0.34
0.95 0.27
0.35 0.21
2.35 2.25
0.17 0.12
1.75 0.59
0.80 0.31
(26.6%)
3-fluoro
0.36 0.03
0.33 0.05
0.13 0.03
0.47 0.08
3.31 0.87
0.39 0.14
0.12 0.03
0.24 0.04
0.38 0.06
0.28 0.03
0.44 0.09
3-methyl
4-fluoro
3,4-dichloro
2-chloro
3-trifluoromethyl
3-methoxy
3-chloro
4-methoxy
4-chloro
the known compounds was either midnanomolar or micro-
molar against KCNQ2 in the reported assay. However, to
compare (S)-5 with these compounds, we chose to re-evaluate
all compounds in a common assay system (PubChem AID:
588426). In this assay system, compounds 1 and 2 were more
potent (∼8−10-fold) compared to previous values; however, 3
was ∼7-fold less potent. (S)-5 and all analogues were all >40-
fold selective versus KCNQ1, whereas the previously reported
compounds were either not selective, or in some instances,
more potent versus KCNQ1. Furthermore, (S)-5 and the
previously reported compounds were further evaluated against
a panel of other Kv7.x channels and (S)-5 was more selective
versus KCNQ1/E1 and all compounds were not selective
versus KCNQ2/3 or KNCQ4, with the exception of 3. In
addition to the selectivity performed in our laboratories, 5 has
been tested in >300 assays performed within the MLPCN
network and was only active in four assays not dependent on
KCNQ2 and was weakly active (>15 μM) in each of these
assays (see Supporting Information). Lastly, to more fully
assess the selectivity of this probe molecule, (S)-5 was
evaluated on Ricerca Bioscience’s Lead Profiling Screen,
which is a radioligand binding assay profiling >68 GPCRs,
ion channels, and transporters. (S)-5 was found to bind with
only one of the 68 assays conducted (melantonin, MT1, 61%
inhibition at 10 μM). Several ion channels are included in this
profiling assay (calcium channel, L- and N-type; potassium
channel (KATP and hERG)), which were not inhibited by (S)-
5, highlighting the selectivity profile of (S)-5.
2-fluoro
5, 163 nM). Moving the 2-pyrrolidine group around the ring
produced orthogonal results when comparing the inhibitor and
activator scaffolds. The 3-pyrrolidine substituent was com-
pletely devoid of activity in the inhibitor series (44, >30000
nM), while the activator series retained some activity (although
with a ∼5-fold loss of activity, 45, 1020 nM). Further
movement to the 4-position resulted in reversal of activity,
where the inhibitor retained all activity (46, 210 nM) and the
activator lost significant activity (47, 8660 nM). Enlarging the
ring system to the 2-piperidine resulted in loss of activity in
both series (48 and 49), although the activator series did retain
weak activity (7090 nM). Further expansion of the ring to the
seven-membered ring led to total loss of activity. Any other
modification of the 2-(pyrrolidin-1-yl) ring system led to much
reduced activity or complete loss of activity in both the
inhibitor and activator series.
As the inhibitor series represents mixtures of enantiomers, we
next turned our attention to the synthesis of the discrete
enantiomers in order to determine if there is an enantiospecific
bias within this series. In the three examples analyzed, the (S)-
enantiomer proved to be more potent than the (R)-enantiomer,
with (S)-5 being the most active compound (69 nM)
(Supporting Information, Table 1). This represents the most
potent KCNQ2 inhibitor reported to date and (S)-5 was
declared an MLPCN probe (ML252).11
Lastly, we evaluated (S)-5 and other close analogues in our
tier 1 in vitro PK assays including an assessment of intrinsic
clearance (CLINT/CLHEP) in hepatic microsomes.12 Intrinsic
clearance allows for the rank ordering of compounds with
Next, we evaluated the most potent inhibitor ((S)-5), several
close analogues, and known compounds for selectivity against
KCNQ1 (Kv7.1) (Supporting Information, Table 2). Each of
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respect to their tendency to undergo oxidative metabolism,
which provides a prediction for subsequent clearance in in vivo
PK evaluation. All of the compounds evaluated in this assay
showed high clearance in both human and rat microsomes
(Supporting Information, Table 3). As the unsubstituted right-
hand phenyl group provides an attractive site for oxidation,
compounds that contain substituents meant to block this
oxidation were evaluated and all of these compounds also
showed high clearance. For a better understanding of the nature
of the instability of these compounds, we performed a
metabolic soft-spot analysis using rat hepatocytes. This analysis
showed that the major site of metabolism was on the
pyrrolidine ring, α-methylene to the nitrogen. Unfortunately,
our initial SAR evaluation showed that modification of this ring
system was not well tolerated, thus potentially limiting the in
vivo dosing regimen used for this probe molecule.
Although the metabolic stability of (S)-5 suggests it would
not be a candidate for oral administration, due to the
therapeutic importance of KCNQ2 inhibitors as CNS agents,
we further profiled (S)-5 in a snapshot in vivo PK study to
assess the plasma and brain levels after a single time point and
single dose following IP administration (Table 5). (S)-5 was
observed to be highly brain penetrant with a B:P ratio of 1.9
and absolute brain levels of 672 nM, nearly 10-fold greater than
the IC50 value.
structural changes caused a functional shift from antagonist to
agonist. Further SAR of this series identified a potent KCNQ2
activator, 37, which is equipotent to many previously reported
KCNQ2 activators, including those from our laboratories
(ML213).13 Further studies are planned to evaluate the effects
of (S)-5 on acetylcholine release and will be reported in due
course.
EXPERIMENTAL SECTION
■
Chemistry. The synthesis of (S)-5 is described below. The general
chemistry, experimental information, and syntheses of all other
compounds are supplied in the Supporting Information. Purity of all
final compounds was determined by HPLC analysis is >95%.
(S)-2-Phenyl-N-(2-(pyrrolidin-1-yl)phenyl)butanamide ((S)-
5). To a stirred solution of (S)-(−)-2-phenylbutyric acid (50 mg,
0.304 mmol) and HATU (116 mg, 0.304 mmol) in DMF (1.5 mL)
was added ethyldiisopropylamine (79 mL, 0.456 mmol) followed by 2-
(1-pyrrolidinyl)aniline (49 mg, 0.304 mmol), and the reaction mixture
was stirred at room temperature overnight. The reaction was diluted
with water (1.5 mL) and extracted with EtOAc (2× 2 mL). The
organic extracts were combined and concentrated, and the residue was
purified by RP prepHPLC eluting with 10−90% CH3CN/H2O (0.1%
TFA) to give the product as the TFA salt which was dissolved in
methylene chloride (5 mL), washed with aqueous saturated sodium
bicarbonate solution, dried (MgSO4), filtered, and concentrated. The
free base was dissolved in methylene chloride (2 mL) and treated with
4 M HCl in dioxane (0.2 mL). After 20 min, the reaction mixture was
concentrated to give the product (60 mg, 57%). 1H NMR (400 MHz,
DMSO-d6) δ 10.10 (br s, 1 H), 7.52−7.41 (m, 3 H), 7.41−7.30 (m, 3
H), 7.31−7.10 (m, 3 H), 3.76 (t, J = 7.5 Hz, 1 H), 3.72−3.42 (m, 1
H), 3.84 (br s, 4 H), 2.20−2.02 (m, 1 H), 1.88 (br s, 4 H), 1.81−1.66
(m, 1 H), 0.89 (t, J = 7.3 Hz, 3 H). LCMS: RT = 0.66 min, >98% at
215 and 254 nm, MS (ESI+) m/z = 309.3 [M + H]+. HRMS (TOF,
ES+), calcd for C20H25N2O [M + H]+, 309.1967; found, 309.1969.
Chiral HPLC: 94.2% (S)-isomer, RT = 3.97 min, 5.75% ee; (R)-isomer,
RT = 4.29 min, 88.4% ee.
Table 5. In Vitro and in Vivo Characterization of (S)-5
(S)-5
parameter
MW
TPSA
308.4
32.3
ASSOCIATED CONTENT
■
S
* Supporting Information
cLogP
4.27
in vitro pharmacology
IC50 (μM)
Experimental procedures and spectroscopic data for selected
compounds, detailed pharmacology, and DMPK methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
KCNQ2
0.07 0.01
KCNQ1
2.92 3.90
KCNQ1/E1
8.12 1.47
KCNQ2/Q3
KCNQ4
0.12 0.02
AUTHOR INFORMATION
0.20 0.06
6.1, 18.9, 3.9, 19.9
■
CYP (1A2, 2C9, 3A4, 2D6)
in vitro PK
Corresponding Author
*For C.R.H.: phone, 615-936-6892; fax, 615-936-4381; E-mail,
corey.r.hopkins@vanderbilt.edu; address, Department of Phar-
macology, Vanderbilt University Medical Center, 2213 Garland
Avenue, Nashville, Tennessee 37232, United States. For M.L.:
phone, 410-614-5131; fax. 410-614-1001; E-mail, minli@jhmi.
edu; address, Johns Hopkins University School of Medicine,
733 North Broadway, BRB 319, Baltimore, Maryland 21205,
United States.
Rat CLHEP (mL/min/kg)
rPPB (%fu)
67.3
0.6
0.3
rBHB (%fu)
in vivo PK, IP PBL
(10 mg/kg, 1 h)
Plasma (ng/mL)
Brain (ng/g)
B/P
115.8
207.5
1.9
Author Contributions
Professors Lindsley and Hopkins directed and designed the
chemistry and pharmacokinetic studies. Dr. Cheung performed
the synthetic chemistry. Drs. Yu, Wu, and Zou performed
experiments. Drs. Lindsley, Hopkins, Li, McManus, Yu, and Wu
participated in writing or editing the manuscript
CONCLUSION
■
In summary, we have discovered a potent, selective, and brain
penetrant KCNQ2 inhibitor, (S)-5, from a high-throughput
screening campaign of the MLSMR library collection. After IP
dosing, (S)-5 shows brain levels significantly higher than the in
vitro IC50 for the inhibition of KCNQ2 and thus can be used as
an in vivo tool molecule. In addition, during the course of the
SAR campaign, we discovered a site on (S)-5 where small
Funding
This work was generously supported by the NIH/MLPCN
grants R03 DA027716−01, U54 MH084691 (M.L.), and U54
MH084659 (C.W.L.).
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(12) (a) Varma, M. V. S.; Obach, R. S.; Rotter, C.; Miller, H. R.;
Chang, G.; Steyn, S. J.; El-Kattan, A.; Troutman, M. D.
Physicochemical space for optimum oral bioavailability: contribution
of human intestinal absorption and first-pass elimination. J. Med. Chem.
2010, 53 (3), 1098−1108. (b) Chang, G.; Steyn, S. J.; Umland, J. P.;
Scott, D. O. Strategic Use of Plasma and Microsome Binding To
Exploit in Vitro Clearance in Early Drug Discovery. ACS Med. Chem.
Lett. 2010, 1 (1), 50−63.
(13) Yu, H.; Wu, M.; Townsend, S. D.; Zou, B.; Long, S.; Daniels, J.
S.; McManus, O. B.; Li, M.; Lindsley, C. W.; Hopkins, C. R. Discovery,
synthesis, and structure−activity relationship of a series of N-aryl-
bicyclo[2.2.1]heptane-2-carboxamides: characterization of ML213 as a
novel KCNQ2 and KCNQ4 potassium channel opener. ACS Chem.
Neurosci. 2011, 2, 572−577.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Tammy Santomango, Frank Byers, and Ryan
Morrison for technical assistance with the PK experiments
and Nathan Kett and Sichen Chang for the purification of
compounds. Vanderbilt is a member of the MLPCN and
houses the Vanderbilt Specialized Chemistry Center for
Accelerated Probe Development. Johns Hopkins is a member
of the MLPCN and houses the Johns Hopkins Ion Channel
Center.
ABBREVIATIONS USED
■
HATU, 2-(1H-7-azabenzotriazol-yl)-1,1,3,3-tetramethyl ura-
nium hexafluorophosphate methanaminium; SID, PubChem
structure ID; CID, PubChem compound ID; AID, PubChem
assay ID; CNS, central nervous system
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(7) (a) For information on the Molecular Libraries Probe
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. (b) For information on the Vanderbilt Specialized Chemistry Center,
see: http://www.mc.vanderbilt.edu/centers/mlpcn/index.html.
(8) For assay details, see Supporting Information. Assay ID’s (AIDs)
can be viewed via the PubChem Web site.
(9) For synthetic procedures and compound characterization, please
see Supporting Information.
(10) IC50/EC50s were generated from 8-point concentration response
curves with 3-fold dilutions starting from the maximal concentration
(30 μM) with quadruplicate determinations in the automated patch
clamp assay . In cases in which a saturating response was not obtained
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SD, using estimated standard
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