Discovery and characterization of 2-(cyclopropanesulfonamido)-n-(2-ethoxyphenyl)benzamide, ML382: A potent and selective positive allosteric modulator of MrgX1

Article abstract of DOI:10.1002/cmdc.201402277

Previous studies have shown that the activation of mouse MrgC11, a G-protein-coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large-scale screen has been carried out to isolate small-molecule allosteric agonists of MrgX1, the human homologue of MrgC11. The goal of this study is to improve the efficacy and potency of positive allosteric modulators (PAMs) with therapeutic implications in combating chronic pain. Herein we report an iterative parallel synthesis effort and a structure-activity relationship study of a series of arylsulfonamides which led to the discovery of the first PAM of MrgX1, ML382.

Full text of DOI:10.1002/cmdc.201402277

DOI: 10.1002/cmdc.201402277
Communications
Discovery and Characterization of 2-
(Cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide,
ML382: a Potent and Selective Positive Allosteric
Modulator of MrgX1
Wandong Wen,[a] Yan Wang,[b] Zhe Li,[c] Pang-Yen Tseng,[c] Owen B. McManus,[d] Meng Wu,[d, f]
Min Li,[d, e] Craig W. Lindsley,[a] Xinzhong Dong,*[c] and Corey R. Hopkins*[a]
Previous studies have shown that the activation of mouse
MrgC11, a G-protein-coupled receptor, by its peptide ligand
BAM8-22 can inhibit chronic pain. A large-scale screen has
been carried out to isolate small-molecule allosteric agonists of
MrgX1, the human homologue of MrgC11. The goal of this
study is to improve the efficacy and potency of positive allo-
steric modulators (PAMs) with therapeutic implications in com-
bating chronic pain. Herein we report an iterative parallel syn-
thesis effort and a structure–activity relationship study of
a series of arylsulfonamides which led to the discovery of the
first PAM of MrgX1, ML382.
to their clinical use and have become a serious health threat.
The discovery of novel therapies to treat pain without the po-
tential for addiction is therefore a major goal.
Mrgs (also named Mrgprs/SNSRs) are a family of G-protein-
coupled receptors (GPCRs) consisting of more than 50 mem-
bers in the mouse genome.[1] Strikingly, the expression of
many Mrg genes, including MrgC11, is restricted to subsets of
small-diameter sensory neurons in the dorsal root ganglion
(DRG) and trigeminal ganglia, as these genes have not been
detected in the central nervous system (CNS) or in the rest of
the body.[2] Similarly, some human MrgXs such as MrgX1 are
also selectively expressed in DRG neurons.[1b] BAM8-22, a 15-
residue endogenous peptide, is a specific agonist for mouse
MrgC11 and human MrgX1.[1a,2] Based on the expression pat-
tern, ligand specificity, and similarity in signal pathways,
MrgC11 and MrgX1 are functional orthologues.
Chronic pain is difficult to treat and has become a major
health and economic burden worldwide, reaching nearly epi-
demic levels with 20–25% of the population affected. Chronic
pain is often refractory to current therapies, and the many of
the major analgesics (e.g., opioids) carry with them dose-limit-
ing adverse events and serious risk of addiction and abuse.
These risks of addiction and abuse present substantial barriers
Several complementary lines of evidence suggest that
MrgC11 functions as an endogenous regulator of persistent
pain.[3] Particularly, spinal cord application of BAM8-22 and
other MrgC11 agonists in mice significantly attenuates inflam-
matory hyperalgesia and neuropathic pain in an Mrg-depen-
dent manner.[3a,4] It has been shown recently that the activa-
tion of MrgC11 leads to inhibition high-voltage activating Ca2+
channels, which play an essential role in pain signal transmis-
sion.[5] Because of the specific expression, agonists and positive
allosteric modulators (PAMs) for MrgX1 may represent a new
class of anti-hyperalgesics for the treatment of chronic pain
without side effects in the CNS.[6,7]
[a] W. Wen,+ Prof. C. W. Lindsley, Prof. C. R. Hopkins
Department of Pharmacology
Vanderbilt Center for Neuroscience Drug Discovery
Vanderbilt Specialized Chemistry Center (MLPCN)
Vanderbilt University Medical Center, Nashville, TN 37232-6600 (USA)
E-mail: corey.r.hopkins@vanderbilt.edu
[b] Dr. Y. Wang+
State Key Laboratory of Oral Diseases and Department of Orthodontics
West China School of Stomatology
The project commenced with a screen of the NIH Molecular
Library Small-Molecule Repository (MLSMR) compound collec-
tion containing >300000 compounds using a triple addition
protocol with BAM-22 as the as the ligand (AID: 588675;
Figure 1).[8] Using a HEK293 cell line that stably expresses the
MrgX1 protein, the first addition is the drug itself (agonist
mode), the second addition is BAM-22 at EC10–EC30 for the
identification of PAMs, and the third addition uses BAM-22 at
EC90–Emax for the identification of antagonists/negative alloste-
ric modulators. From the initial screen, ~1900 compounds
were identified as hits, and after a medicinal chemistry triage,
~1100 compounds were retested and then counter-screened
against HEK293 parental cells. There were ~150 compounds
that showed no activity against the parental cells, and these
were then evaluated in a five-point concentration–response
curve (CRC), leaving 29 active compounds. These compounds
Sichuan University, Chengdu, 610041 (China)
[c] Z. Li, Dr. P.-Y. Tseng, Prof. X. Dong
Department of Neuroscience
Johns Hopkins University School of Medicine, Baltimore, MD 21205 (USA)
E-mail: xdong2@jhmi.edu
[d] Dr. O. B. McManus, Dr. M. Wu, Dr. M. Li
Johns Hopkins Ion Channel Center
Johns Hopkins University, Baltimore, MD 21205 (USA)
[e] Dr. M. Li
Glaxo Smith Kline, 709 Swedeland Road, King of Prussia, PA 19406 (USA)
[f] Dr. M. Wu
Present address: High-Throughput Screening Facility, University of Iowa
115 S. Grand Ave., 316 PHAR, Iowa City, IA 52242 (USA)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402277: experimental procedures for
medicinal chemistry, pharmacology, and drug metabolism studies, as
well as compound characterization data.
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a couple structural variants worth noting. First, the 2-ester ana-
logue was significantly less potent (3.85 mm), and second, the
2-methyl thioether was more potent than the 2-methoxy (~2-
fold). The SAR chemical optimization strategy is highlighted in
Table 1 wherein we evaluated multiple sites of the ligand in
parallel through iterative synthesis.
The synthesis of the MrgX1 compounds (generically shown
as 4) is outlined in Scheme 1. The substituted anthranilic acid
Scheme 1. Synthesis of MrgX1 allosteric agonists 4. Reagents and conditions:
a) SOCl2, DMF, Et2O, 08C!708C, 2 h; b) pyridine, Et2O, 08C!RT, 2 h;
c) RSO2Cl, pyridine, CH2Cl2, RT, 16 h.
1 was converted into the acid chloride (SOCl2, DMF) and then
coupled with aniline 2 (pyridine, Et2O) to yield the amide 3.
The sulfonamide compounds 4 were made by coupling 3 with
an appropriate sulfonyl chloride (RSO2Cl, pyridine, CH2Cl2). This
synthesis represents a modular approach to the final com-
pounds, allowing easy modification of R, R1, R2, and R3, which
permitted the synthesis of numerous analogues for this proj-
ect.
Figure 1. Flowchart of the screening paradigm for MrgX1 positive allosteric
modulators.
were then tested in 10-point CRC format, revealing 19 con-
firmed MrgX1 PAMs.
The results of the high-throughput screening (HTS) cam-
paign are summarized in Table 1. From the ~39 identified
“hits”, there were four compounds from a sulfonamide benza-
mide series. Combining the activity in the MrgX1 assay and ex-
cellent selectivity profile as observed in the PubChem Assay
and PubChem Hit rate,[9] this series was chosen for further de-
velopment toward a novel MrgX1 PAM. The initial SAR revealed
The SAR evaluation started with the re-synthesis and re-con-
firmation of the four initial hits that were identified in the HTS
campaign (Table 2). These compounds, 4a, 4b, 4g, and 4h,
were all re-confirmed within ~2–5-fold of the initial HTS values
(EC50), except for 4h, which was inactive (AID: 743016). Those
compounds with Emax >50% were considered “active” and
Table 1. Lead scaffolds from the HTS screen.
SID
4258711
4262381
3665540
3607659
MrgX1 EC50 [mm]
PubChem assays
PubChem hits
0.096
758
3
0.329
761
3
0.691
765
3
3.85
754
4
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EC50 =419 nm; 4g: EC50 =500 nm: and 4i: EC50 =
551 nm). The 2-methoxy group is less active than the
2-ethoxy or 2-methylthio groups. Also, substitution of
the sulfonamide nitrogen atom was potency-neutral
relative to the unsubstituted NH sulfonamide (e.g.,
4a vs. 4i).
Table 2. Evaluation of the right-hand phenyl group.
Compd
MrgX1 EC50 [mm][a]
Emax [%][a]
The final round of SAR used the 2-ethoxyphenyl
group as the right-hand group and evaluated a varie-
ty of small alkyl sulfonamides, as these were identi-
fied from the previous round of SAR as the optimal
substituents (Table 3). This round of SAR was far
more productive than the previous round and
showed a distinct SAR trend. Cyclopropyl sulfona-
mide 4l was the most potent (EC50 =190 nm,
Figure 2) and efficacious (Emax =195%). For this round
of SAR, the Emax values were normalized to the posi-
tive control (4i, 100%). There were several com-
pounds that showed Emax >100%. Another sub-mi-
cromolar compound was the 2-chloroethyl sulfona-
mide 4m; however, as this compound possesses the
reactive alkyl halo group, it was not progressed fur-
ther. The isopropyl sulfonamide 4o (EC50 =1.82 mm,
R
R1
H
R2
4a
4b
4c
Me
0.531ꢀ0.16
3.06ꢀ1.50
0.42ꢀ0.33
238ꢀ63
188ꢀ7
Me
Me
H
H
130ꢀ28
4d
4e
Me
Me
H
H
inactive
ND
E
max =255%) and the cyclopropylmethyl 4p (EC50 =
4.47 mm, Emax =267%) were significantly less potent
than 4l. Based on its superior potency and efficacy,
4l was declared the MLPCN probe compound for the
MrgX1 allosteric agonist project, ML382.
1.01ꢀ0.19
180ꢀ9
The dose–response studies show that ML382 en-
hances the potency of BAM8-22 on MrgX1 by >7-
fold (i.e., EC50 of BAM8-22 from 18.7 to 2.9 nm; Sup-
porting Information Figure 1). However, ML382 does
not affect the Emax of BAM8-22 if the maximum con-
centration of BAM8-22 is used (Supporting Informa-
tion Figure 1). Furthermore, in the absence of BAM8-
22, ML382 does not activate MrgX1, suggesting
ML382 itself does not has agonistic activity toward
MrgX1 (Figure 2). Pharmacological and selectivity
profiling of ML382 were performed against the close-
ly related MrgX2 (Supporting Information Figure 2),
and against a larger panel of receptors. ML382 was
evaluated in HEK293 cells expressing MrgX2 as a se-
lectivity screen. As is evident from Supporting Infor-
mation Figure 2, ML382 showed no significant effect
on the activation of MrgX2 in the presence of the
specific agonist peptide, PAMP (both EC50 and Emax).
Also, ML382 had no effect on MrgC11, the mouse ho-
mologue of MrgX1, which indicates the compound is
species selective (results not shown). In addition,
ML382 was evaluated using the Eurofins Lead Profil-
ing Screen, which is a binding assay panel of 68
4 f
Me
Me
H
2.34ꢀ1.30
0.50ꢀ0.12
241ꢀ25
184ꢀ14
4g
Me
4h
Me
Me
inactive
ND
4i
4j
4k
Me
Ph
Ph
Me
H
0.55ꢀ0.17
inactive
149ꢀ19
ND
H
inactive
ND
[a] Assays were carried out in the presence of 10 nm BAM8-22; data are the meanꢀ
SEM of n=2 experiments; ND: not determined, as Emax is too low.
were progressed to an EC50 determination. From the SAR in
Table 2, it was shown that ester groups on the R2 phenyl
group are not tolerated (4d and 4h); phenyl sulfonamides are
also not tolerated (4j and 4k). Simple methyl sulfonamides
coupled with either ether or thioether substituents on the R2
phenyl ring were well tolerated, with many of the compounds
showing nanomolar EC50 values (e.g., 4a: EC50 =531 nm; 4c:
GPCRs, ion channels, and transporters screened at 10 mm.[10]
ML382 did not inhibit 67 of the 68 targets assayed (inhibition
of radioligand binding >50% at 10 mm); the only target that
was considered active was the serotonin (5-hydroxytryptamine)
5HT2B, which showed 63% inhibition at 10 mm. Overall, ML382
displayed a very favorable selectivity profile.
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Table 3. Further SAR of the alkylsulfonamides.
Table 4. In vitro DMPK profile of ML382.
In vitro PK properties
Microsome predicted hepatic clearance [mLminꢁ1 kgꢁ1
]
Rat CLHEP
Human CLHEP
65.7
15.7
Free compound in plasma [%]
Human PPB
Rat PPB
[a]
Compd
R
MrgX1 EC50 [mm]
0.19ꢀ0.06
Emax
0.4
1.7
4l
ML382
195ꢀ8
4m
4n
0.38ꢀ0.05
0.91ꢀ0.41
204ꢀ15
204ꢀ10
oxidative metabolism and predicted to display high clearance
in both species (65.7 and 15.7 mLminꢁ1 kgꢁ1, respectively). In
addition, using an equilibrium dialysis approach, the protein
binding of ML382 was evaluated and was shown to have mod-
erate free fraction in rat (1.7%) and low free fraction (0.4%) in
human.
ethyl
4o
1.82ꢀ1.59
255ꢀ53
4p
4q
4r
4.47ꢀ3.08
ND
267ꢀ101
46.1
In summary, we have developed 4l (also known as ML382 or
VU0485891) as a potent and selective positive allosteric modu-
lator of MrgX1. This is the first report of a PAM for MrgX1. In
the presence of the known ligand BAM8-22, ML382 has an EC50
value of 190 nm with an Emax value of 148% and is inactive
against the closely related MrgX2 and against a group of se-
lected receptors from the Eurofins panel. In addition, ML382
was profiled in our Tier 1 DMPK assay and possesses favorable
free fraction in rat plasma protein binding; however, instability
in rat liver microsomes indicates the utility of ML382 as an in
vivo probe will be limited to non-oral dosing regimens (e.g.,
intraperitoneal, subcutaneous, or intrathecal). Further use of
ML382 in an in vivo animal study will be reported in due
course.
ND
29.6
4s
ND
3.78
4t
ND
ND
3.6
0.0
4u
[a] Emax as a percent of standard compound 4i; data are the meanꢀSEM
of n=2 experiments; ND: not determined, as Emax <50%.
Acknowledgements
This work was generously supported by the US National Institutes
of Health (NIH)/Molecular Libraries Probe Centers Network
(MLPCN) grants U54 MH084659 (to C.W.L.) U54 MH084691 (to
M.L.), and R03 DA033176 and R01 NS054791 (to X.D.). M.W. ac-
knowledges additional funding support from the NIH
(R03 MH090837-01, R03 MH090849-01, and R03 A031670-01).
Keywords: chronic pain · ML382 · MLPCN · molecular probes ·
MrgX1 · positive allosteric modulators
Figure 2. Pharmacological profile of ML382 on MrgX1-expressing HEK293
&
*
cells. Dose–response curve of ML382 in the presence ( ) and absence ( ) of
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Received: July 7, 2014
Published online on September 10, 2014
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