Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c00660

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition, and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, the optimization of 1-phenylpyrrolidine 8 gave acid 26. This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles.

Full text of DOI:10.1021/acs.jmedchem.0c00660

Subscriber access provided by The University of Melbourne Libraries
Article
Screening of a custom-designed acid fragment library
identifies 1-phenylpyrroles and 1-phenylpyrrolidines
as inhibitors of Notum carboxylesterase activity
William Mahy, Mikesh Patel, David Steadman, Hannah Woodward, Benjamin N.
Atkinson, Fredrik Svensson, Nicky Willis, Alister Flint, Dimitra Papatheodorou, Yuguang
Zhao, Luca Vecchia, Reinis R. Ruza, James Hillier, Sarah Frew, Amy Monaghan,
Artur Costa, Magda Bictash, Magnus Walter, E. Yvonne Jones, and Paul V. Fish
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00660 • Publication Date (Web): 29 Jul 2020
Downloaded from pubs.acs.org on August 2, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 55
Journal of Medicinal Chemistry
1
2
3
Screening of a custom-designed acid fragment library identifies 1-phenylpyrroles and 1-
phenylpyrrolidines as inhibitors of Notum carboxylesterase activity.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
1
1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
William Mahy, Mikesh Patel, David Steadman, Hannah L. Woodward, Benjamin N. Atkinson, Fredrik
Svensson,1,3 Nicky J. Willis, Alister Flint, Dimitra Papatheodorou, Yuguang Zhao, Luca Vecchia, Reinis R.
1
2
4
5
5
5
5
1
1
1
1
6
Ruza, James Hillier, Sarah Frew, Amy Monaghan, Artur Costa, Magda Bictash, Magnus W. Walter, E.
5
1,3,
Yvonne Jones, Paul V. Fish. *
1
Alzheimer’s Research UK UCL Drug Discovery Institute, University College London, Cruciform Building,
Gower Street, London, WC1E 6BT, U.K.
2
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, U.K.
3
The Francis Crick Institute, 1 Midland Road, Kings Cross, London NW1 1AT, U.K.
4
UCL School of Pharmacy, University College London, 29-39 Brunswick Square, Bloomsbury, London WC1N
1AX, U.K.
5
Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, The Henry
Wellcome Building for Genomic Medicine, Roosevelt Drive, Oxford, OX3 7BN, U.K.
6
Eli Lilly, Erl Wood Manor, Windlesham, Surrey, GU20 6PH, U.K.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions.
Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the
removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum
enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in
a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were
identified as hits for Notum inhibition and 14 of these fragments were shown to bind in the palmitoleate
pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z
as the most potent compound from this series. Similarly, optimization of 1-phenylpyrrolidine 8 gave acid 26.
This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules
possessing favorable in vitro ADME profiles.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Keywords: Notum inhibitor, Wnt signaling, fragment library, structure-based drug design, 1-phenylpyrroles,
1
-phenylpyrrolidines.
2
ACS Paragon Plus Environment

Page 3 of 55
Journal of Medicinal Chemistry
1
2
3
INTRODUCTION
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions
1
in embryonic development and adult stem cell biology. The Wnt pathways control cell proliferation, cell fate
determination, cell polarity, cell migration and apoptosis. Wnt signaling is activated by the binding of a Wnt
protein to a member of the Frizzled (FZD) family of cell surface receptors and to co-receptors such as the LDL-
receptor-related protein (LRP) family. Upon binding, an intracellular signaling cascade is triggered by the
pathway activating protein Disheveled (Dsh), which acts as a switch and branching point into multiple
downstream Wnt pathways (canonical, noncanonical planar cell polarity, and noncanonical Wnt/calcium).
Signaling by Wnt proteins is finely balanced and tightly regulated by a sophisticated network of modulators
and feedback processes including secreted inhibitory proteins and post-translational modifications (PTM).2-4
Conversely, misregulation of Wnt signaling disrupts normal development and cell homeostasis, and has been
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
associated with cancer, Alzheimer’s disease (AD), metabolic disorders and other diseases.
Recently, carboxylesterase Notum was shown to act as a negative regulator of the Wnt signaling pathway by
mediating the removal of an essential palmitoleate group from a conserved serine residue of Wnt proteins.8,9
This palimitoleate group is an important PTM required for efficient binding of the Wnt proteins to the FZD
receptors. Hence, modulation of Notum activity represents a new approach to treat disease where
dysregulation of Wnt signaling is an underlying cause and Notum has been implicated as the responsible agent.
There is an emerging understanding of the potential role Notum plays in disease. Notum is involved in the
progression of colorectal cancer (CRC) as Notum expression was increased in human metastatic CRC cells and
10
proliferation was suppressed by inhibiting expression of Notum. Osteoblast-derived Notum reduces cortical
bone mass in mice whereas Notum inhibition increases endocortical bone formation and bone strength, and
so represents a potential treatment for preventing non-vertebral fractures.11,12 Recent work has shown that
the Notum gene is expressed and upregulated in endothelial cells in the hippocampus of APP/PS1 mice and
AD patients compared to control, although the role of Notum in the mammalian central nervous system (CNS)
has yet to be established.13
3
The Notum crystal structures reveal a well-defined, large (ca. 380 Å ), hydrophobic active-site pocket adjacent
to the catalytic triad (Ser232, His389, Asp340) that accommodates the palmitoleate group of Wnt (PDB:
8
4
UZQ). The cis-C9-10 double bond of the C16 palmitoleate tail is positioned at the base of the pocket between
Ile291, Phe319 and Phe320, which helps explain substrate preference. The binding pocket can accommodate
extended carbon tails up to C8/C10; however, longer fatty acid chains need to have a bend in their structure
at C9 in order to fit. In addition, the Gly127–Trp128 amide participates in formation of the oxyanion hole along
with the canonical Ser232–Ala233 and Gly126–Gly127 amides, thereby providing extra stabilization to the
tetrahedral transition state during ester hydrolysis. The pocket entrance, lined by Ser232 and His389, is
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
relatively narrow but shows substantial flexibility. These structural features of Notum combine to render the
palmitoleate pocket to be regarded as highly druggable.14
The number of reports of inhibitors of Notum carboxylesterase activity has gradually increased in recent years
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 1). Thieno[3,2-d]pyrimidine LP-922056 (1) is a potent, orally active inhibitor of Notum that has been
1
5
found to increase cortical bone thickness in mouse models of bone growth. Scaffold-hopping from acid 1,
supported by X-ray structure determination, identified amide 2 as a potent inhibitor of Notum.16
Pharmacokinetic studies in mouse with 2 showed good plasma exposure and reasonable brain penetration; in
1
7
contrast, 1 had negligible brain exposure. N-Hydroxyhydantoin carbamate ABC99 is a potent and selective
covalent irreversible inhibitor of Notum. 18 ABC99 preserves Wnt-mediated cell signaling in the presence of
19
Notum and has been used to show the role of Notum in the regeneration of aged intestinal epithelium. A
crystallographic fragment screen using the XChem platform at Diamond Light Source identified a number of
fragment hits that bound in the palmitoleate pocket of Notum.20,21 Phenoxyacetamide 3 was identified through
the optimization of one of these hits and showed an unexpected flipped binding mode when compared to the
2
0
structures of close analogues bound to Notum. A second fragment hit from this screen was N-[2-(5-fluoro-
H-indol-3-yl)ethyl]acetamide (4), which is structurally related to the brain hormone melatonin. A detailed
1
structural analysis of the binding of 4 (and close analogues) to Notum has been disclosed that may guide the
development of new potent inhibitors.21 Several of these inhibitors (1, 3, 4 plus others) have been soaked
into crystals of Notum and the structures solved to determine the inhibitor binding modes. Hence, under
suitable conditions, Notum is very amenable to structure-based drug design (SBDD) to help guide progress
towards the discovery of potent inhibitors.
4
ACS Paragon Plus Environment

Page 5 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
S
N
CO H
2
N Me
N
S
N
S
N
Me
Cl
O
N
F3C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
1
: LP-922056
IC 1.1 ± 0.4 nM
2
a
a
IC 3.9 ± 0.8 nM
5
0
50
PDB: 6T2K
O
O
Me
O
N
N
O
N
O
N
H
N O
N
Cl
O
ABC99
IC 13 nM
IC 170 ± 20 nM
3
b
a
IC 85 ± 11 nM
5
0
50
a,c
PDB: 6R8R
5
0
Me
O
HN
F
N
H
4
d
IC 37,200 nM
5
0
PDB: 6QCQ
Figure 1: Chemical structures and PDB codes of Notum inhibitors.
a
Notum IC50 data presented for comparison in a common assay format.
b
Determined by competitive gel-based activity-based protein profiling (ABPP), see reference 18.
As a covalent inhibitor of Notum, the IC50 value will be time dependent.
See reference 21.
c
d
Our objective was to identify alternative chemical scaffolds to the prior art that efficiently inhibit Notum
enzymatic activity with the potential to deliver structurally orthogonal chemical tools for use in drug target
validation experiments. We adopted a strategy to simultaneously explore multiple chemotypes in order to
increase our chances of delivering a ‘fit-for-purpose’ chemical tool. Furthermore, diverse chemotypes, which
share the same primary pharmacology, are likely to offer the benefit of a different secondary pharmacology
fingerprint profile and so help identify any confounding off-target effects in disease models.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS & DISCUSSION
Our approach was to create a custom-designed fragment library of carboxylic acids for screening against
Notum (Figure 2). Each compound of the library would have three distinct structural features: (1) a carboxylic
acid to interact with the catalytic triad and to anchor the inhibitor through a polar interaction as observed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
previously; (2) a suitable lipophilic group to be presented into the palmiteolate binding pocket of Notum;
and (3) a linker of various lengths to join the acid with the lipophilic group (Figure 2A).
A single commercial provider was selected for ease and consistency of compound supply, and to simplify
procurement. The Enamine Carboxylic Acids Fragment Library was provisionally selected because of its size
(4350 compounds) and features of ‘novelty and core diversity’; these features were assessed through
evaluation of the library’s content.22 The full library was filtered using criteria for molecular properties and
structural parameters as described in Table 1, and this virtual collection (514 compounds) was further
manually curated by inspection to ensure a balanced representation of physicochemical property space
(Figure 2B) along with structural diversity whilst minimizing repetition of a particular core. This process of
design and selection gave an acid fragment library of 250 compounds (Supplementary Table S1), which was
screened for inhibition of Notum activity (Figure 2C).
Inhibition of Notum carboxylesterase activity of this library was measured in a biochemical assay where test
compounds (dispensed to give 10-point concentration-response-curves up to 100 M) were incubated with
Notum(81-451 Cys330Ser) and trisodium 8-octanoyloxypyrene-1,3,6-trisulfonate (OPTS) as the substrate for
4
0 minutes and fluorescence recorded. Twenty compounds showed inhibition of Notum activity with initial
IC < 25 M (n = 2), although most of these hits showed a modest decrease in activity upon routine retesting
50
(n = 5-10). All 20 of these putative hits were submitted to a Notum crystallographic screen by soaking crystals
of C-terminal his-tagged Notum(81-451 Cys330Ser) with the compounds screened at 10 mM and 14 fragments
were observed to bind in the palmitoleate pocket.
From the Notum biochemical and crystallographic screen, two preferred hit series were selected for further
investigation: pyrrole-3-carboxylic acids 5-7 and pyrrolidine-3-carboxylic acids 8-10 (Figure 3). These six hits
shared a closely related structural motif and had excellent hit-like properties (LE = 0.45 - 0.57) with no obvious
reactive groups. In addition, compounds 5-8 and 10 had been successfully crystallized with Notum to support
a SBDD program (Figure 4).
6
ACS Paragon Plus Environment

Page 7 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2: Design and screening of fragment library. (A) Compound structural features. (B) Plot of lipophilicity
(clogP) verses molecular size (MW) for the acid fragment library. (C) Flow diagram showing key activities and
results.
Table 1: Design and selection criteria for carboxylic acid fragment library.
Parameter (measure)
Criteria
Molecular size (MW)
Lipophilicity (clogP)
Polarity (TPSA)
H-bonding capacity (HBD, HBA)
Aromatic rings
170 ≤ MW ≤ 240
-2 ≤ clogP ≤ 2
≤ 90
1 ≤ HBD ≤ 2; HBA ≤ 3
≤ 2
a
Minimize no. of stereocentres
Maximize no. of sp carbons
≤ 1
3
≥ 1 (where possible)
a
Excluding the COOH group.
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CO H
CO H
CO H
2
2
2
3
F3C
Me
2
Me
Me
Me
Me
5
N
N
N
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
7
IC 6.7 ± 3.6 M
IC 6.3 ± 0.5 M
IC 12.7 ± 6.2 M
50
50
50
CO H
CO H
CO H
2
2
2
N
N
N
F
Cl
8
9
10
IC 48 ± 31 M
IC 11.3 ± 8.9 M
IC 18.6 ± 14.1 M
5
0
50
50
Figure 3: Pyrrole-3-carboxylic acids 5-7 and pyrrolidine-3-carboxylic acids 8-10 as fragment screening hits.
Notum IC (M) (n = 5-10).
5
0
The original six hits 5-10 provided some insight into preliminary structure activity relationships (SARs), along
with two structurally related inactive compounds, which were also present in the library (Figure 3 and
Supplementary Figure S1). The three pyrroles 5-7 all had 2- and 5- flanking groups either side of the N-atom
supporting the inclusion of Me and/or CF groups at these positions. There was some variation in the N1
3
substituent with both cPr and CH cPr represented and showing activity. The three pyrrolidines 8-10 had a
2
single point change in the 2-position of the N1-phenyl ring showing activity could be improved with suitable
groups (F, Cl > H). Despite these six hits providing limited SAR, it was encouraging that they represented a
series of related hits (rather than singletons) with the potential for further optimization.
8
ACS Paragon Plus Environment

Page 9 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A
B
C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
D
E
F
Figure 4: Notum structures with fragment screening hits 5-8 and 10. Crystal structures of (A) 5 (orange), (B) 6
teal), (C) 7 (gray), (D) 8 (magenta) and (E) 10 (yellow). (F) Overlay of all ligands. Binding site residues shown
within 4Å of their respective ligands. Key hydrogen bond interactions are shown as dashed lines. Water
molecules have been removed for clarity. The surface of the Notum palmitoleoate binding pocket outlined
gray) from the O-palmitoleoyl serine–Notum (S232A) structure (PDB: 4UZQ). Atomic coordinates have been
deposited in the Protein Data Bank (PDB). PDB ID codes: 5: 6YUY; 6: 6YV4; 7: 6YUW; 8: 6YV2; 10: 6YV0.
(
(
The crystal structures reveal that the inhibitors preferably occupy the core of the pocket, interacting with the
sidechains of Phe268 and Trp128, with some ligands (7, 8 and 10) also extending to the oxyanion hole
(
backbone of Trp128, Gly127, Ala233) in proximity of the catalytic triad (Figure 4). Due to the fragment’s
modest size, they fail to occupy the pocket fully. The suboptimal occupation of the pocket aligns with the
modest observed potencies.
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Our general design strategy for new compounds was initially directed at exploring three principle areas of
these hit structures: (1) the pyrrole-3-carboxylic acid ring 11-24 (Table 2); (2) the 1-phenylpyrrole ring that
binds in the palmitoleate pocket 20 (Table 3); and (3) the 1-phenylpyrrolidine ring 25-27 (Table 4). Further
bespoke modifications of preferred leads were then performed to improve activity and tune physicochemical
properties 28-30 (Table 5 and Supplementary Figure S2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Minimizing lipophilicity is a well-established approach to improve ADME properties and target compounds
2
3
were designed to have molecular and physicochemical properties consistent with drug-like space. The
palmitoleate binding pocket of Notum is a very lipophilic environment, and we were keen to avoid gains in
activity simply through increased compound lipophilicity. A helpful design metric was lipophilic ligand
efficiency (LLE),24 used to track improvements in Notum inhibition activity against lipophilicity of the
compounds as determined by calculated clogP and measured CHI logD7.4 values.25
The synthetic routes to new inhibitors of Notum in the pyrrole-3-carboxylic acid (11-24) and pyrrolidine-3-
carboxylic acid series (25-30) are presented in Schemes 1 and 2 respectively.
CO R
CO R
CO R
2
2
2
a
b
R2
R5
R2
R5
R2
N
R1
O
O O
31
33
R = Et
c
35
R = t-Bu
in situ
1
2
5
R = Et, t-Bu; R = various; R , R = Me, CF
11-24
3
R = H
Scheme 1. Preparation of pyrrole-3-carboxylic acids 11-24. Representative reagents and conditions: (a) NaH
o
5
o
1
(1.2 equiv.), THF, 0 C then R C(O)CH Cl 32 (1.0 equiv.), NaI (0.1 equiv.) 0 C – RT, 24 h; (b) Amine 34 (R NH )
2
2
o
o
(
1.0 equiv.), p-TsOH∙H O (0.1 equiv.), EtOH, 80 C; (c) LiOH∙H O (15 equiv.), 1,4-dioxane-H O, 100 C.
2
2
2
Pyrrole-3-carboxylic acid target compounds 11-24 were either purchased or prepared using a classical Paal-
5
Knorr pyrrole synthesis (Scheme 1). Alkylation of -keto ester 31 with chloromethyl ketone 32 (R C(O)CH Cl)
2
1
gave 1,4-diketo ester 33 and cyclocondensation of 33 with amine 34 (R NH ) gave pyrrole ester 35. Hydrolysis
2
with LiOH afforded the pyrrole acid. Initially, ethyl esters (31: R = Et) were used but the corresponding pyrrole
esters (35: R = Et) were found to be resilient to hydrolysis even under forcing conditions. Switching to the t-
butyl esters (31: R = t-Bu) improved the synthetic route as the pyrrole esters (35: R = t-Bu) were found to
hydrolyze under the conditions of the Paal-Knorr reaction giving directly the pyrrole-3-carboxylic acids.
1
0
ACS Paragon Plus Environment

Page 11 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CO Me
CO Me
CO H
CONRR'
2
2
2
a
b
c
N
N
N
R1
N
H
R1
R1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
38
25-27
28
O
O
N
O
NH
N
N
d, e
d, f
N
R1
N
R1
1
29
30
R = Ar and Het; NRR' = various
Scheme 2. Preparation of pyrrolidine-3-carboxylic acids 25-27, amides 28, oxadiazole 29 and oxadiazolone 30.
Representative reagents and conditions: (a) 36 (1.2 equiv.), ArI 37 (1.0 equiv.), Pd dba (2.5 mol%), Xantphos
2
3
o
(
5 mol%), Cs CO (2.7 equiv.), 1,4-dioxane, 100 C, 16 h; (b) aq. NaOH (1 M, 2.0 equiv.), MeOH, RT, 2 h; (c)
2
3
’
HATU (1.1 equiv.), DMF, RT, 15 min; then iPr NEt (2.2 equiv.), amine 39 (HNRR )(1.5 equiv.); (d) NH NH ∙H O
4 equiv.), EtOH-PhMe, 80 C, 16 h; (e) HC(OEt) (4.5 equiv.), p-TsOH∙H O (0.1 equiv.), 60 C, 5 min then RT, 1
h; (f) triphosgene (0.4 equiv.), iPr NEt (2.0 equiv.), CH Cl , 0 C - RT, 15 min.
2
2
2
2
o
o
(
3
2
o
2
2
2
Pyrrolidine-3-carboxylic acid target compounds 25-27 were prepared using a simple two-step sequence
(Scheme 2). 3-Pyrrolidine ester 36 was cross-coupled with the aryl iodide 37 under Pd-mediated catalysis to
give the corresponding 1-arylpyrrolidine 38 and then hydrolysis of the ester gave acid 25.26 The single
enantiomers (S)-26 and (R)-27 were prepared from the corresponding chiral esters, methyl (S)- and (R)-
pyrrolidine-3-carboxylate (36) respectively as described above,to provide samples of high chemical and optical
2
7
purity. Amides 28 were prepared by activation of the acid 25 with HATU and subsequent reaction with the
amine 39 (HNRR’). Heterocycles 29 and 30 were prepared from ester 38 by reaction with hydrazine to give the
acyl hydrazide and then treatment with triethyl orthoformate gave 1,3,4-oxadiazole 29, whereas treatment
with triphosgene gave 1,3,4-oxadiazol-2(3H)-one 30.
The design of new inhibitors with improved Notum activity initially focused on the SARs of the pyrrole-3-
carboxylic acids by exploring the substitution on the pyrrole ring 11-24 (Table 2). This set allowed for the direct
comparison of a number of matched pairs to identify beneficial changes. Deconstructing 5-7 to their
components in order to identify the minimum pharmacophore was achieved with a new set of fragments 11-
1
8. However, all these smaller fragments had a significant decrease in Notum activity suggesting further
1
reduction in size was not feasible (e.g. 5 v 11 v 12 v 15). In contrast, the N1 substituent (R ) proved to be quite
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
influential with the Ph group preferred: Ph (20) > cPr (6) > CH cPr (7), CH Ph (23) >> Me (18), H (17). There
2
2
1
5
was a subtle balance of interplay between the R and R groups as shown by the significant decrease in activity
1
5
1
5
when poorly partnered (e.g. R = H prefers R = CF (5) >> Me (17); whereas R = Ph prefers R = Me (20) ≥ H
3
1
5
(
19) > CF (21); and R = CH Ph prefers R = H (22) > Me (23) >> CF (24)). From this limited set, 1-phenylpyrrole
3
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20 emerged as having an attractive profile suitable for further optimization.
A wider range of N-aryls around 20 were then prepared to determine preferred substituents and substitution
patterns whilst retaining the 2,5-dimethyl groups on the pyrrole (Table 3). The standard tactic of walking a F
(20b-d), Me (20e-g) and Cl (20h-j) around the phenyl ring showed a clear preference for the 3-Cl group 20i
when compared to the unsubstituted Ph ring (20). Notum crystals were soaked with 20i and the structure
solved to show the ligand sitting deep in the lipophilic pocket with no direct interaction being made by the
acid (Figure 5A). Additional single substituents were then introduced to the 3-position (20k-q) with a general
trend emerging showing a preference for small lipophilic groups, and Notum inhibition tracking with
lipophilicity: iPr (20m) ≥ Cl (20i), tBu (20n), cPr (20l), OEt (20p) > CF (20k), OMe (20o), Me (20f), F (20c), H (20)
3
≥
CN (20q).
The combination of two substituents on the phenyl ring was initially explored by using two Cl atoms (20r-w)
as a single 3- and 4-Cl atom had proved to be favorable (20i, 20j) (Table 3) and the required dichloroanilines
3
4 were readily available. The 2,3- (20r) and 3,4-dichloro (20v) isomers were the most potent from this set
and further improved Notum inhibition activity to ca. 200-300 nM. Additional SARs were then developed using
a wider range of substituents at the 2,3- and 3,4-positions (20x-ee) by building on the earlier SAR whilst aiming
to moderate overall lipophilicity of the compounds. A combination of a 2-F with preferred groups at the 3-
position (iPr, cPr) gave some of the most potent compounds prepared so far: 2-F,3-iPr (20z) and 2-F,3-cPr
(20y).
Through an exploration of the SARs within the pyrrole series, gains in Notum inhibition activity had been
achieved when compared to the original fragment hits (e.g. for 20z v 6, 40-fold increase). However, the
molecular properties of the most active inhibitors showed these compounds to be quite lipophilic (e.g. 20z:
IC 0.15 M, clogP 5.5). Despite our efforts to moderate compound properties, Notum inhibition activity
5
0
tracked with lipophilicity in this series resulting in the most potent compounds having the highest clogP values.
Our focus then switched to the pyrrolidine-3-carboxylic acids hit series 8 as this template offered the
advantage of significantly lower lipophilicity when compared to the matched pyrrole, albeit with weaker
Notum inhibition (e.g. 8 vs 20; IC 48 vs 1.7 M; clogP = -2.4). The application of preferred aryl groups from
50
the pyrrole series to the 1-phenylpyrrolidines 25 showed the SARs were not directly transferable; there was a
significant decrease in Notum inhibition activity and a change in the rank order (Table 4). However, the 2-Cl
(
10), 3-Cl (25a) and 4-Cl (25b) were again amongst the preferred single groups (2-F, 2-Cl, 3-CF , 3-Cl, 4-F, 4-Cl;
3
1
2
ACS Paragon Plus Environment

Page 13 of 55
Journal of Medicinal Chemistry
1
2
3
IC <20 M) and used to explore the effect of two substituents on Notum activity (25j-o) with the 3,4-dichloro
5
0
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
25n as the most potent isomer within this set.
Table 2: Notum inhibition of pyrrole-3-carboxylic acids 11-24. SAR of the pyrrole ring.a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R4
CO H
2
R5
R2
N
R1
1
1-24
Compound
5
R
Notum
IC (M)
5
0
R1
R2
R4
R5
H
H
Me
H
H
H
H
Me
H
H
Me
H
H
H
H
CF3
H
H
6.7 ± 3.6
b
1
1
>>100
>>100
b
12
13
H
H
Me
H
c
c
H
ca. 100
ca. 100
>>100
ca. 100
>> 100
>100
1
1
1
1
1
1
20
21
2
2
2
4
5
6
7
8
9
H
H
H
H
Me
Ph
Ph
Ph
H
H
Me
CF3
H
Me
Me
H
Me
CF3
H
Me
CF3
b
c
Me
Me
Me
Me
Me
Me
Me
Me
Me
b
d
3.8 ± 1.4
1.7 ± 0.6
22 ± 3.1
3.1 ± 0.1
H
H
H
H
2
3
4
CH Ph
2
CH Ph
14 ± 3.7
2
d
CH Ph
>100
2
a
All values are mean ± s.d. of n = 2-4 experiments quoted to 2 s.f. Differences of <2-fold should not be considered
significant. For details of the assay protocol, see reference 20.
b
c
d
<
20 % I @ 100 M; ca. 50 % I @ 100 M; 20-40 % I @ 100 M.
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3: Notum inhibition of 2,5-dimethyl-1-phenylpyrrole-3-carboxylic acids 20. SAR of the phenyl ring.a
CO H
2
Me
Me
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R6
R2
R5
R3
R4
20
Compound
R2-R6
Notum
Compound
R2-R6
Notum
IC (M)
50
IC (M)
50
2
0
H
1.7 ± 0.6
3.3 ± 0.7
1.5 ± 0.2
20r
20s
20t
20u
20v
20w
20x
2-Cl, 3-Cl
2-Cl, 4-Cl
2-Cl, 5-Cl
2-Cl, 6-Cl
3-Cl, 4-Cl
3-Cl, 5-Cl
2-F, 3-Cl
2-F, 3-cPr
2-F, 3-iPr
2-F, 3-CN
2-OMe, 3-Cl
2-CN, 3-Cl
3-Cl, 4-CF3
0.22 ± 0.09
0.43 ± 0.17
1.7 ± 0.15
2.3 ± 0.77
0.27 ± 0.10
0.85 ± 0.30
0.38 ± 0.03
0.20 ± 0.09
0.15 ± 0.02
0.63 ± 0.23
0.80 ± 0.25
2.0 ± 0.65
1.5 ± 0.16
2.1 ± 0.12
2
2
0b
0c
2-F
3-F
4-F
2-Me
3-Me
4-Me
2-Cl
20d
20e
1.9 ± 0.6
2.2 ± 0.90
1.5 ± 0.51
2.0 ± 0.10
2.3 ± 0.74
0.43 ± 0.04
0.86 ± 0.10
1.5 ± 0.20
0.58 ± 0.19
0.36 ± 0.20
0.46 ± 0.09
1.5 ± 0.3
2
0f
0g
0h
2
2
20y
20z
2
0i
3-Cl
4-Cl
2
0j
20aa
20bb
20cc
20dd
20ee
2
0k
3-CF3
3-cPr
3-iPr
3-tBu
3-OMe
3-OEt
3-CN
20l
20m
20n
3-CF , 4-Cl
3
20o
20p
20q
0.64 ± 0.25
3.1 ± 1.1
a
See footnotes Table 2.
1
4
ACS Paragon Plus Environment

Page 15 of 55
Journal of Medicinal Chemistry
1
2
3
A
B
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
D
Figure 5: Notum structures with pyrrole and pyrrolidine inhibitors. Crystal structures of (A) 20i (emerald) and
B) 26 (green). (C) Overlay of 20i (emerald) and 26 (green) shows different binding modes. (D) Overlay of 26
green) with 1 (purple; PDB: 6T2K) shows acid groups aligned. Binding site residues shown within 4Å of their
respective ligands. Key hydrogen bond interactions are shown as dashed lines. Water molecules have been
removed for clarity. The surface of the Notum palmitoleoate binding pocket outlined (gray) from the O-
palmitoleoyl serine – Notum (S232A) structure (PDB: 4UZQ). Atomic coordinates have been deposited in the
Protein Data Bank (PDB). PDB ID codes: 20i: 6YXI; 26: 6YSK.
(
(
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4: Notum inhibition of 1-phenylpyrrolidine-3-carboxylic acids 25-27. SAR of the phenyl ring.a
CO H
CO H
3S
2
2
3
N
N
R6
R2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R5
R3
CF3
R4
Cl
2
5: 3RS
26: 3S
7: 3R
2
Compound
R2-R6
Notum
Compound
R2-R6
Notum
IC (M)
IC (M)
5
0
50
8
9
H
2-F
48 ± 31
11 ± 9
25j
25k
25l
2-Cl, 3-Cl
2-Cl, 4-Cl
2-Cl, 5-Cl
2-Cl, 6-Cl
3-Cl, 4-Cl
3-Cl, 5-Cl
3-Cl, 4-CF3
8.0 ± 1.1
10 ± 2.1
7.9 ± 1.4
26 ± 8
1.2 ± 0.2
11 ± 1.9
8.5 ± 0.77
0.20 ± 0.02
2.5 ± 0.5
6.8 ± 0.9
0.60 ± 0.23
1
0
2-Cl
3-Cl
4-Cl
3-Me
3-CF3
4-CF3
3-iPr
3-OCF3
3-CN
4-F
19 ± 14
14 ± 0.9
19 ± 4.4
49 ± 11
11 ± 0.6
50 ± 3.8
57 ± 19
63 ± 13
42 ± 12
15 ± 1.2
25a
25b
25m
25n
25o
25p
25q
25r
25s
25t
2
5c
2
5d
5e
2
3-CF , 4-Cl
3
2
5f
5g
5h
5i
3-CF , 4-F
3
2
2-F, 3-CF3
2-Cl, 3-Cl, 4-Cl
2
2
26
27
3-CF , 4-Cl
0.11 ± 0.01
0.28 ± 0.02
3
3-CF , 4-Cl
3
a
See footnotes Table 2.
1
6
ACS Paragon Plus Environment

Page 17 of 55
Journal of Medicinal Chemistry
1
2
3
Extending this approach by exploring closely related halogenated analogues (25p-s) that incorporated a 3-CF3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(cf. 25d) identified 3-CF ,4-Cl (25q) as a breakthrough compound due to its Notum inhibition activity and lower
3
lipophilicity (IC50 0.20 M, clogP 3.3). It is interesting to compare that the 3-CF ,4-Cl substituents were
3
significantly weaker in the pyrrole series (20ee, IC 2.1 M). Hence, despite the apparent structural similarity
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
between the pyrrole 20 and pyrrolidine 25 templates, the SARs proved to be divergent. This was rationalized
by the binding modes observed in the crystal structures of compounds 20i and 8 where the pyrrole series sits
deeper in the pocket compared to the pyrrolidine. The 2- and 5-methyl substituents on the pyrrole template
also change the preferred conformation of the N1-Ar group. The 2,3,4-trichloro analogue 25s (IC 0.60 M,
5
0
clogP 3.7) was also a sub-micromolar inhibitor of Notum albeit with higher lipophilicity and was not pursued
further at this time.
2
7
The two enantiomers of 25q were then prepared to give (S)-26 and (R)-27. Both enantiomers had good
Notum activity with a slight preference for 26 (IC 0.11 M, clogP 3.3). Additional structural information was
5
0
then sought to guide the next phase of design. Notum crystals were soaked with the most potent pyrrolidine
26 and the structure solved to show the compound binding in a similar mode as observed for hit 8 but with
the 3-COOH more optimally positioned for interaction with the oxyanion hole (Figure 5B and 5C). This is
possibly due to the 3- and 4-aryl substituents allowing the inhibitor to both fully occupy the lipophilic pocket
and make these interactions.
The X-ray structure of 26 in the active site of Notum was overlaid with the Notum structure of 1 (PDB: 6T2K)
and showed very good alignment of the acid groups supporting the use of carboxamides at this position in the
1
6
template (Figure 5D). Initial SAR studies with amides 28 (derived from 25q) suggested that Notum activity
was particularly sensitive to the amide substituent as most groups were detrimental (Table 5). Only the 3,3-
difluoroazetine 28e and pyrrolidine 28f amides retained activity similar to the corresponding acid 25q.
An extension to this design strategy was to explore small heterocycles as bioisosteres for the carboxamide
(Table 5). Oxadiazoles were initially selected due to their successful application in drug discovery programs
2
8
and accessibility from ester 38. However, 1,3,4-oxadiazole 29 gave a significant drop in potency compared
to acid 25q and amides 28. In contrast, switching to the corresponding weakly acidic 1,3,4-oxadiazol-2(3H)-
29
one gave 30 (IC 4.2 M, clogP 3.2)(pK ca 6.3), which restored moderate inhibition of Notum activity.
5
0
a
Oxadiazolone 30 was of particular interest as a lead to explore the potential of weak acids to penetrate the
brain as molecules with these properties are under-represented in CNS drug discovery.30,31,32
A set of preferred inhibitors (20z, 26, 28e, 30) were then assessed in in vitro ADME assays to compare their
aqueous solubility, microsomal stability (MLM) and cell permeability (MDCK-MDR1)(Table 6). These inhibitors
were selected based on their Notum activity but also their chemical structural diversity and complementary
physicochemical properties (clogP, pK ). On balance, pyrrolidine-3-acid 26 demonstrated a superior profile
a
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
consistent with its properties: good solubility; good metabolic stability in MLM that predicts for low clearance;
and good permeability without efflux by the P-glycoprotein (P-gp) transporter as measured by transit
performance in the MDCK-MDR1 cell line. Oxadiazolone 30 also had a satisfactory combination of ADME
results, whereas pyrrole-3-acid 20z suffered from moderate MLM stability probably due to its higher
lipophilicity. It is of note that there was evidence of non-CYP mediated metabolism in MLM for amide 28e (i.e.
loss of parent in an NADPH-independent manner).20
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
ACS Paragon Plus Environment

Page 19 of 55
Journal of Medicinal Chemistry
1
2
3
Table 5: Notum inhibition of 1-phenylpyrrolidine-3-carboxamides 28, oxadiazole 29 and oxadiazolone 30.a
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
N
N
O
NH
CONRR'
N
3
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CF3
CF3
CF3
Cl
Cl
Cl
28
29
30
NRR’
Compound
Notum
IC (M)
5
0
28a
8b
8c
28d
8e
28f
8g
8h
28i
NHMe
1.3 ± 0.17
3.5 ± 0.25
1.0 ± 0.05
2
OMe
N
H
2
N
H
NMe2
1.2 ± 0.15
2
F
F
0.26 ± 0.03
N
0.41 ± 0.04
9.7 ± 0.52
3.1 ± 0.01
1.4 ± 0.1
N
N
N
2
O
O
2
O
N
N
N
Me
O
2
8j
3.1 ± 0.67
N Me
2
9
-
-
24 ± 0.1
4.2 ± 0.8
30
a
See footnotes Table 2.
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6.
Summary of physicochemical and molecular properties, Notum inhibition and ADME data for 20z, 26, 28e
and 30
O
O
O
NH
F
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CO H
CO H
N
N
2
2
Me
Me
F
N
N
N
N
Me
Me
CF3
CF3
CF3
Cl
Cl
Cl
2
0z
26
28e
30
20z
26
28e
30
Physicochemical and molecular properties
mol wt
clogP
275
5.5
3.1
40.5
0.48
3.7
293
3.3
1.5
40.5
0.51
5.4
368
4.1
4.0
23.6
0.39
2.5
333
3.2
3.5
53.9
0.34
1.9
CHI LogD7.4
2
TPSA (Å )
Ligand efficiency (LE)
Lipophilic ligand efficiency (LLE) a
Notum inhibition b
OPTS, IC50 (M)
0.15 ± 0.02
0.11 ± 0.01
(n = 6)
0.26 ± 0.03
(n = 4)
4.2 ± 0.8
(n = 4)
(n = 4)
ADME profile c
Aq. solubility (g/mL)/(M)
MLM, Cl (L/min/mg protein)
i
MDCK-MDR1, AB/BA Papp (x10 cm/s)
14/51
5
>145/>595
2.5
39/43
1.1
7/19
23/69
10
13/19
1.5
d
2
21
-6
46/43
.93
8/10
1.2
0
MDCK-MDR1, efflux ratio (ER)
a
LLE based on calculation with measured CHI LogD7.4 values.
See footnotes Table 2.
Studies were performed by GVK Biosciences (Hyderabad, India).
Evidence of non-CYP mediated metabolism in MLM.
b
c
d
2
0
ACS Paragon Plus Environment

Page 21 of 55
Journal of Medicinal Chemistry
1
2
3
CONCLUSION
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We have disclosed two new chemical scaffolds that efficiently inhibit Notum enzymatic activity. Our approach
was to create a custom-designed fragment library of carboxylic acids for screening against Notum in a
biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were
identified as hits for Notum inhibition (IC50 < 25 M) with two preferred hit series selected for further
investigation; these preferred fragments were shown by crystallography to bind in the palmitoleate pocket of
Notum. Optimization of 1-phenylpyrrole 20 by SAR studies guided by SBDD identified 20z (IC 0.15 M) as the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
most potent compound from this series. Similarly, optimization of 1-phenylpyrrolidine 8 gave acid 26 (IC 0.11
5
0
M), amide 28e (IC 0.26 M) and oxadiazolone 30 (IC 4.2 M).
5
0
50
This work demonstrates that inhibition of Notum carboxylesterase activity can be achieved by small, drug-like
molecules supported by attractive in vitro ADME profiles, some of which possess structural features and
3
3
physicochemical properties consistent with CNS design space including a favorable CNS MPO scores (e.g. 30,
CNS MPO 5.6/6.0).34
OH
N
N
N
Cl
4
0: ARUK3000123
IC 11.5 ± 3.0 M (n = 9)
5
0
However, despite the significant progress in developing fragment hit 8 into lead 26 (>400-fold increase in
activity), these compounds proved to be inferior to leads discovered by complementary screening strategies.
In brief, screening of an alternative fragment library20, 21 identified (1-(4-chlorophenyl)-1H-1,2,3-triazol-4-
3
5
yl)methanol (40), which was optimized to give a potent, selective and CNS penetrant inhibitor of Notum
activity suitable for use in both cellular and in vivo models of disease.36
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL SECTION
Chemistry
General Information:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Unless preparative details are provided, all reagents were purchased from commercial suppliers and used
without further purification. Solvents were of ACS reagent grade or higher and purchased from commercial
suppliers without further purification. Anhydrous solvents were purchased as such from Acros Organics or
Sigma Aldrich. Thin layer chromatography (TLC) was carried out on aluminum backed silica plates. The plates
were visualized under UV (254 nm) light, followed by staining with phosphomolybdic acid dip or potassium
permanganate and gentle heating.
Compound purification by column chromatography was performed using a Biotage Isolera using prepacked
Biotage SNAP KP-Sil silica cartridges or Biotage SNAP Ultra C18 reverse phase cartridges. Chemical and chiral
SFC analysis were performed by Reach Separation Ltd (Nottingham, UK) using conditions described below.
Organic solvent layers were routinely dried with anhydrous MgSO and concentrated using a Büchi rotary
4
evaporator.
Melting points (mp) were determined using Stuart SMP20 melting point equipment using closed end glass
capillary tubes and are uncorrected.
Infra-red (IR) spectra were recorded on a Shimadzu IRTracer-100 FT-IR spectrometer, using a Universal
-1
ATR accessory for sampling, with relevant absorbance quoted as max in cm .
1
13
H NMR and C NMR spectra were run in deuterated (≥99.5%) solvents on either a Bruker Avance 300 (300
MHz), Bruker Avance 400 (400 MHz), Bruker Avance 600 (600 MHz) or Bruker Avance 700 (700 MHz). Chemical
shifts (δ) are reported as parts per million (ppm), coupling constants (J) are reported in Hz and signal
multiplicities are reported as singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), quintet (qu), sextet
(sext), doublet of doublets (dd), doublet of triplets (dt), triplet of triplets (tt), multiplet (m), or broad singlet
(br s).
LCMS analysis was performed on a Waters Acquity H-Class UPLC system with either an acidic (HSS C18 Column,
H O:MeCN, 0.1% TFA) or basic (BEH C18 Column, H O:MeCN, 10 mM NH OH) mobile phase.
2
2
4
HRMS data acquisition was on a Waters Micromass LCT Premier electrospray time-of-flight (ESI-TOF) mass
spectrometer. The observed mass and isotope pattern matched the corresponding theoretical values as
calculated from the expected elemental formula.
Optical rotations [] were measured on a Bellingham + Stanley ADP450 polarimeter.
D
2
2
ACS Paragon Plus Environment

Page 23 of 55
Journal of Medicinal Chemistry
1
2
3
Purity of screening compounds 11-30 was evaluated by NMR spectroscopy and LCMS analysis. All compounds
had purity ≥ 95 %.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General Methods:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.
Preparation of 1,4-diketo esters 33.
General Method 1 adapted from Kazembe et. al.37
β-Keto ester 31 (8.0 mmol, 1.0 eq.) was added to a stirred solution of NaH (385 mg, 60% in mineral oil, 9.6
mmol, 1.2 eq.) in THF (10 mL) at 0 °C, and the solution was stirred at 0 °C for 30 min. A solution of 2-
chloroacetone (0.66 mL, 8.0 mmol, 1.0 eq.) in THF (4 mL) was added dropwise and then NaI (120 mg, 0.8 mmol,
0
.1 eq.). The reaction mixture was warmed to RT and stirred for 24 h. The mixture was quenched with dilute
HCl (10 mL, 1 M) and extracted with EtOAc (3 x 30 mL). The combined organic phases were washed with
NaHCO (10 mL, 1 M), brine (10 ml), dried (MgSO ) and concentrated in vacuo. The product was then purified
3
4
by flash column chromatography on silica gel with gradient elution of EtOAc in cyclohexane.
Ethyl 2-acetyl-4-oxo-pentanoate.
Prepared by General Method 1 with ethyl acetoacetate (1.94 mL, 15.4 mmol). Purification by column
chromatography (2-20% EtOAc:cyclohexane) gave the product as a yellow oil (859 mg, 4.61 mmol, 30 %).
1
H NMR (600 MHz, CDCl ) δ 4.11 (q, J = 7.1 Hz, 2H), 3.93 (dd, J = 8.2, 5.7 Hz, 1H), 3.05 (dd, J = 18.5, 8.2 Hz, 1H),
3
13
2
.87 (dd, J = 18.5, 5.7 Hz, 1H), 2.27 (s, 3H), 2.11 (s, 3H), 1.19 (t, J = 7.1 Hz, 3H); C NMR (126 MHz, CDCl ) δ
3
205.70, 202.27, 168.87, 61.83, 53.86, 41.64, 30.17, 29.79, 14.12.
t-Butyl 2-acetyl-4-oxo-pentanoate.
Prepared by General Method 1 with tert-butyl acetoacetate (2.1 mL, 12.6 mmol). Purification by column
chromatography (5-40% EtOAc:cyclohexane) gave the product as a yellow oil (759 mg, 3.54 mmol, 28 %).
-1 1
IR max (film) 1735, 1710, 1367, 1357, 1255, 1138 cm ; H NMR (500 MHz, CDCl ) δ 3.82 (dd, J = 8.1, 5.8 Hz,
3
1
H), 2.97 (dd, J = 18.4, 8.1 Hz, 1H), 2.80 (dd, J = 18.4, 5.8 Hz, 1H), 2.23 (s, 3H), 2.07 (s, 3H), 1.40 – 1.33 (s, 9H);
13
C NMR (126 MHz, CDCl ) δ 205.71, 202.54, 167.83, 82.31, 54.88, 41.53, 29.95, 29.68, 27.85; LCMS (basic
3
-
method) R 1.65 min, m/z 214.3 [M-H] .
t
2. Paal-Knorr pyrrole synthesis 35: R = Et.
General Method 2 adapted from Kazembe et. al.37
Aniline 34 (1.2 eq.) and p-toluenesulfonic acid monohydrate (23 mg, 0.12 mmol, 0.1 eq.) were added to a
solution of ethyl 2-acetyl-4-oxopentanoate (200 mg, 1.08 mmol, 1 eq.) in EtOH (5 mL) and the reaction mixture
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
was heated at 80 C for 24 hr. The reaction was quenched with water (10 mL) and concentrated in vacuo. The
residue was dissolved in dilute HCl (10 mL, 2 M) and extracted with EtOAc (3 x 15 mL). The combined organics
were washed with NaOH (3 x 15 mL, 1 M), dried (MgSO ) and concentrated in vacuo to give the 3-pyrrole ethyl
4
ester.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
. Hydrolysis of 3-pyrrole ethyl ester.
Solid lithium hydroxide monohydrate (670 mg, 16 mmol, 15 eq.) was added to a solution of the 3-pyrrole ester
(ca. 1.1 mmol, 1.0 eq.) in 1,4-dioxane (4.0 mL) and water (1.2 mL), and the reaction mixture heated to 100 oC
for 24 h. The mixture was cooled to RT and quenched with water (5.0 mL). The mixture was acidified with conc.
HCl (1 mL) to pH 1 and extracted with EtOAc (3 x 15 mL). The combined organic phases were concentrated in
vacuo and the crude product was purified by reverse phase flash silica chromatography (0-100% MeCN:H O;
2
0
.1% formic acid modifier) to give the 3-pyrrole acid.
4
4
. Paal-Knorr pyrrole synthesis 35: R = t-Bu.
.1 DMSO as solvent:
t-Butyl 2-acetyl-4-oxopentanoate (100 mg, 0.47 mmol) was dissolved in DMSO (2.5 mL) in a thick-walled
reaction vial. The aniline 34 (0.47 mmol) and p-toluenesulfonic acid monohydrate (20 mg, 0.11 mmol) were
o
added and the vial sealed with a Teflon-lined crimp cap. The vial was heated at 120 C for 30 min under
microwave irradiation to give directly the pyrrole-3-carboxylic acid. The crude product was purified by reverse
phase flash silica chromatography (20-100% MeCN:H O; 0.1% formic acid modifier).
2
4
.2 Ethanol as solvent:
t-Butyl 2-acetyl-4-oxopentanoate (100 mg, 0.47 mmol) was dissolved in EtOH (5.0 mL) in a thick-walled
reaction vial. The aniline 34 (0.47 mmol) and p-toluenesulfonic acid monohydrate (20 mg, 0.11 mmol) were
o
added and the vial sealed with a Teflon-lined crimp cap. The vial was heated at 80 C thermally for 24 h to give
directly the pyrrole-3-carboxylic acid. DMSO was added and the EtOH was evaporated under a flow of
compressed air. The crude product was purified by reverse phase flash silica chromatography (20-100%
MeCN:H O; 0.1% formic acid modifier).
2
4.3 Xylenes as solvent:
t-Butyl 2-acetyl-4-oxopentanoate (100 mg, 0.47 mmol) was dissolved in xylenes (5.0 mL) in a thick-walled
reaction vial. The aniline 34 (0.47 mmol) and p-toluenesulfonic acid monohydrate (20 mg, 0.11 mmol) were
o
added and the vial sealed with a Teflon-lined crimp cap. The vial was heated at 110 C thermally for 24 h to
give directly the pyrrole-3-carboxylic acid. DMSO was added and the xylenes were evaporated in vacuo. The
crude product was purified by reverse phase flash silica chromatography (20-100% MeCN:H O; 0.1% formic
2
acid modifier).
2
4
ACS Paragon Plus Environment

Page 25 of 55
Journal of Medicinal Chemistry
1
2
3
5
.
N-Benzylation of 3-pyrrole esters
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General Method 5 adapted from Beard et al.38
3
1
-Pyrrole ester (0.65 mmol, 1.0 eq.) was added to a solution of NaH (ca 31 mg, 60% in mineral oil, 0.78 mmol,
.2 eq.) in THF (2.5 mL) stirring at 0 °C under nitrogen, and the solution was stirred at 0 °C for 15 min. Benzyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
bromide (93 L, 0.78 mmol, 1.2 eq.) was then added, and the reaction allowed to warm to RT and stirred for
4 h. The reaction was quenched with water (10 mL), extracted with EtOAc (3 x 15 mL), washed with brine (2
2
x 15 mL), dried (MgSO4) and concentrated in vacuo. The product was purified by column chromatography (0-
15% EtOAc:cyclohexane) to give the 1-benzyl-3-pyrrole ester.
6
.
Cross-coupling of 3-pyrrolidine ester 36 with aryl iodides 37.
6.1 Buchwald-Hartwig amination.
To a thick-walled reaction vial was added methyl pyrrolidine-3-carboxylate (36) (320 mg, 2.0 mmol),
tris(dibenzylideneacetone)dipalladium (0) (37 mg, 0.04 mmol), cesium(II) carbonate (1.4 g, 4.4 mmol)
and Xantphos (47 mg, 0.08 mmol). The vial was sealed with a Teflon-lined crimp cap and charged with a
solution of the aryl iodide 37 (1.6 mmol) in 1,4-dioxane (7.8 mL). The stirred reaction mixture was degassed
o
with Ar and then heated to 100 C for 4 h. The reaction mixture was cooled to RT and diluted with CH Cl . The
2
2
resulting suspension was filtered through a plug of sand and cotton wool and the filtrate concentrated under
a continuous flow of air to give the ester 38. This material was used directly in the next step without further
purification.
6
.2 Alternative Buchwald-Hartwig amination.
To a thick-walled reaction vial was added 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (18.4 mg, 0.03
mmol), water (0.76 uL, 0.04 mmol) and palladium acetate (2.4 mg, 0.01 mmol). The vial was purged with
o
N and the reagents dissolved in 1,4-dioxane (5mL). This mixture was stirred at 80 C under N until the
2
2
characteristic change in colour was seen (yellow to green). The catalyst mixture was then transferred to a
second thick-walled reaction vial containing the aryl iodide 37 (1.06 mmol), cesium(II) carbonate (933.0 mg,
2.86 mmol) and methyl pyrrolidine-3-carboxylate hydrochloride (36.HCl) (211 mg, 1.3 mmol) in 1,4-dioxane
o
(
5 mL). The reaction was then stirred at 110 C for 16 h under N until all the starting material was shown to
2
be consumed by TLC. Reaction work-up and purification as General Method 6.1.
7
. Hydrolysis of 3-pyrrolidine methyl ester 38.
To a solution of methyl 1-arylpyrrolidine-3-carboxylate 38 (0.32 mmol) in MeOH (3 mL) was added aqueous
NaOH (0.65 mL, 1 N, 0.65 mmol). The reaction mixture was stirred at RT for 2 h and then quenched with dilute
aqueous HCl (1 M, 2 eq.). The sample was concentrated under reduced pressure, redissolved in a minimum of
DMSO and then purified by reverse phase chromatography (0-95% MeCN:H O; 0.1% formic acid modifier).
2
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8. Preparation of 3-pyrrolidine carboxamides 28.
To a thick-walled reaction vial was added methyl 1-[4-chloro-3-(trifluoromethyl)phenyl]pyrrolidine-3-
carboxylic acid (25q) (50 mg, 0.17 mmol). Anhydrous DMF (1.0 mL) and hexafluorophosphate azabenzotriazole
tetramethyl uronium (HATU) (72 mg, 0.19 mmol) were added and the reaction mixture stirred at RT for 10
min. DIPEA (0.07 mL, 0.40 mmol) was added followed by the amine 39 (0.26 mmol) and the reaction was
stirred at RT for 1 h. The reaction mixture was directly purified by reverse phase chromatography (0-100%
MeCN:H O, 0.1% NH OH modifier).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
Notum Inhibitors:
2-Methyl-5-(trifluoromethyl)-1H-pyrrole-3-carboxylic acid (5)
Purchased from Enamine, EN300-38888
1
-Cyclopropyl-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (6)
Purchased from Enamine, EN300-13229
1
-(Cyclopropylmethyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (7)
Purchased from Enamine, EN300-69261
1
-Phenylpyrrolidine-3-carboxylic acid (8)
Purchased from Enamine, EN300-226265
1
-(2-Fluorophenyl)pyrrolidine-3-carboxylic acid (9)
Purchased from Enamine, EN300-217084
1
-(2-Chlorophenyl)pyrrolidine-3-carboxylic acid (10)
Purchased from Enamine, EN300-218252
1
H-Pyrrole-3-carboxylic acid (11)
Purchased from Fluorochem, 075258
2
-Methyl-1H-pyrrole-3-carboxylic acid (12)
Purchased from Enamine, EN300-98523
2
6
ACS Paragon Plus Environment

Page 27 of 55
Journal of Medicinal Chemistry
1
2
3
4
-Methyl-1H-pyrrole-3-carboxylic acid (13)
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Available from multiple suppliers [CAS Reg. No. 64276-66-0]. Our sample was prepared by General Method 3
from methyl 4-methyl-1H-pyrrole-3-carboxylate. Isolated as a white solid (8 mg).
1
H NMR (600 MHz, CDCl ) δ 8.23 (br s, 1H), 7.45 (m, 1H), 6.55 (m, 1H), 2.30 (s, 3H); LCMS (acidic method) Rt
3
0.54 min, m/z 126.1 [M+H]+.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-Methyl-1H-pyrrole-3-carboxylic acid (14)
Purchased from Fluorochem, 091685
5
-(Trifluoromethyl)-1H-pyrrole-3-carboxylic acid (15)
Purchased from Enamine, EN300-177045
2
,4-Dimethyl-1H-pyrrole-3-carboxylic acid (16)
Available from multiple suppliers [CAS Reg. No. 17106-13-7]. Our sample was prepared by General Method 3
from ethyl 2,4-dimethyl-1H-pyrrole-3-carboxylate. Isolated as a yellow gum (7.4 mg)
1
H NMR (600 MHz, CDCl ) δ 9.39 (br s, 1H), 7.91 (br s, 1H), 6.37 (m, 1H), 2.51 (s, 3H), 2.25 (d, J = 1.1 Hz, 3H);
3
+
LCMS (acidic method) R 0.81 min, m/z 140.1 [M+H] .
t
2
,5-Dimethyl-1H-pyrrole-3-carboxylic acid (17)
Purchased from Alfa Aesar, B20635
1
,2,5-Trimethyl-1H-pyrrole-3-carboxylic acid (18)
Purchased from Maybridge, CC08601
2
-Methyl-1-phenyl-1H-pyrrole-3-carboxylic acid (19)
Prepared by General Methods 2 and 3. Isolated as a pale pink solid (33.4 mg, 54 %).
o
1
Mp 185 C; H NMR (400 MHz, Methanol-d ) δ 7.52 (t, J = 8.0 Hz, 2H), 7.45 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0
4
Hz, 2H), 6.74 (d, J = 3.0 Hz, 1H), 6.61 (d, J = 3.0 Hz, 1H), 2.40 (s, 3H); LCMS (acidic method) R 1.55 min, m/z
t
02.2 [M+H]+.
2
2
,5-Dimethyl-1-phenyl-1H-pyrrole-3-carboxylic acid (20)
Prepared by General Methods 2 and 3. Isolated as a pink solid (7.7 mg, 32 %).
o
1
Mp 204 C; H NMR (600 MHz, CDCl ) δ 7.53 – 7.44 (m, 3H), 7.22 – 7.17 (m, 2H), 6.43 (d, J = 1.0 Hz, 1H), 2.31
3
+
(
s, 3H), 1.98 (d, J = 0.5 Hz, 3H); LCMS (acidic method) R 0.65 min, m/z 216.2 [M+H] .
t
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 55
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2,5-Dimethyl-1-(2-fluorophenyl)-1H-pyrrole-3-carboxylic acid (20b)
Prepared by General Method 4.1 with 2-fluoroaniline. Isolated as a white solid (70 mg, 64 %).
1
H NMR (700 MHz, CDCl ) δ 7.49 – 7.45 (m, 1H), 7.31 – 7.21 (m, 3H), 6.45 (d, J = 0.9 Hz, 1H), 2.31 (s, 3H), 1.98
3
(s, 3H); 13C NMR (176 MHz, CDCl ) δ 170.59, 158.32 (d, J = 251.8 Hz), 138.17, 130.87 (d, J = 7.7 Hz),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
130.49, 129.44, 125.42 (d, J = 13.2 Hz), 124.92 (d, J = 4.0 Hz), 117.01 (d, J = 19.9 Hz), 111.30, 108.48,
+
1
2.35, 12.25; LCMS (acidic method) R 1.63 min, m/z 234.2 [M+H] .
t
2
,5-Dimethyl-1-(3-fluorophenyl)-1H-pyrrole-3-carboxylic acid (20c)
Prepared by General Method 4.1 with 3-fluoroaniline. Isolated as a white solid (80 mg, 74 %).
1
H NMR (700 MHz, CDCl ) δ 7.48 (m, 1H), 7.22 – 7.16 (m, 1H), 7.03 – 6.99 (m, 1H), 6.95 (dt, J = 9.0, 2.1 Hz, 1H),
3
6.43 (s, 1H), 2.32 (s, 3H), 2.00 (s, 3H); 13C NMR (176 MHz, CDCl3) δ 170.68, 162.96 (d, J = 249.3 Hz),
1
39.18 (d, J = 9.6 Hz), 137.62, 130.78 (d, J = 9.1 Hz), 129.11, 124.26 (d, J = 3.2 Hz), 116.05 (d, J = 19.9
+
Hz), 115.87, 111.09, 108.51, 12.77, 12.63; LCMS (acidic method) R 1.70 min, m/z 234.2 [M+H] .
t
2,5-Dimethyl-1-(4-fluorophenyl)-1H-pyrrole-3-carboxylic acid (20d)
Prepared by General Method 4.1 with 4-fluoroaniline. Isolated as an off-white solid (60 mg, 55 %).
1
13
H NMR (600 MHz, CDCl ) δ 7.18 (m, 4H), 6.42 (d, J = 0.8 Hz, 1H), 2.29 (s, 3H), 1.97 (s, 3H); C NMR (151 MHz,
3
CDCl3) δ 171.11, 162.51 (d, J = 249.0 Hz), 137.88, 133.69 (d, J = 3.0 Hz), 130.02 (d, J = 8.6 Hz), 129.32, 116.61
+
(d, J = 22.9 Hz), 110.90, 108.26, 12.83, 12.68; LCMS (acidic method) R 1.71 min, m/z 234.2 [M+H] .
t
2
,5-Dimethyl-1-(2-methylphenyl)-1H-pyrrole-3-carboxylic acid (20e)
Prepared by General Method 4.2 with o-toluidine. Isolated as a light green solid (51 mg, 48 %).
1
H NMR (700 MHz, CDCl ) δ 7.35 (m, 3H), 7.12 (d, J = 7.5 Hz, 1H), 6.45 (d, J = 0.8 Hz, 1H), 2.22 (s, 3H), 1.95 (s,
3
13
3
1
H), 1.89 (s, 3H); C NMR (176 MHz, CDCl ) δ 170.87, 137.36, 136.84, 136.72, 131.15, 129.24, 128.69, 128.63,
3
27.13, 110.65, 108.08, 17.19, 12.42, 12.29; LCMS (acidic method) R 1.75 min, m/z 230.2 [M+H]+
t
2
,5-Dimethyl-1-(3-methylphenyl)-1H-pyrrole-3-carboxylic acid (20f)
Prepared by General Method 4.3 with m-toluidine. Isolated as light grey solid (97 mg, 57 %).
-1 1
Mp 195-197 °C; IR max (film) 1653, 1606, 1265, 1244, 771, 729 cm ; H NMR (500 MHz, CDCl ) δ 7.37 (t, J = 7.7
3
Hz, 1H), 7.27 (s, 1H), 6.99 (d, J = 8.4 Hz, 2H), 6.40 (d, J = 1.0 Hz, 1H), 2.42 (s, 3H), 2.30 (s, 3H), 1.97 (s, 3H); 13C
NMR (126 MHz, CDCl ) δ 169.44, 139.62, 137.75, 129.48, 129.26, 128.80, 125.25, 110.28, 108.01, 56.10, 32.10,
3
+
2
1.38, 12.78, 12.64; LCMS (acidic method) R 2.04 min, m/z 230.2 (M+H) .
t
2
8
ACS Paragon Plus Environment

Page 29 of 55
Journal of Medicinal Chemistry
1
2
3
4
5
6
2
,5-Dimethyl-1-(4-methylphenyl)-1H-pyrrole-3-carboxylic acid (20g)
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Prepared by General Methods 2 and 3 with p-toluidine. Isolated as a white solid (123 mg, 37 %).
o
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Mp 246 C; H NMR (300 MHz, CDCl ) δ 7.29 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.41 (s, 1H), 2.43 (s, 3H),
3
+
2
.30 (s, 3H), 1.97 (s, 3H); LCMS (acidic method) R 1.68 min, m/z 230.2 [M+H] .
t
1-(2-Chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (20h)
Prepared by General Method 4.2 with 2-chloroaniline. Isolated as a white solid (60 mg, 52 %).
1
H NMR (400 MHz, DMSO-d ) δ 11.73 (s, 1H), 7.77 – 7.70 (m, 1H), 7.63 – 7.45 (m, 3H), 6.25 (d, J = 1.0 Hz, 1H),
6
1
3
2.11 (s, 3H), 1.85 (s, 3H); C NMR (176 MHz, DMSO-d ) δ 166.23, 134.96, 134.72, 132.20, 131.07, 130.84,
6
+
1
30.33, 128.66, 127.82, 111.81, 107.79, 11.93, 11.66; LCMS (acidic method) R 1.73 mins, m/z 250.2 [M+H] .
t
1
-(3-Chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (20i)
Prepared by General Methods 2 and 3 with 3-chloroaniline. Isolated as an amber oil (206 mg, 76 %).
-1 1
IR max (film) 3346, 3069-3087, 2834-2990, 2582, 1646, 1378-1594, 1330, 1250, 776 cm ; H NMR (700 MHz,
13
CDCl ) δ 7.45 (m, 2H), 7.22 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.40 (s, 1H), 2.31 (s, 3H), 1.99 (s, 3H); C NMR (176
3
MHz, CDCl ) δ 169.22, 138.86, 137.70, 135.22, 130.57, 129.20, 128.63, 126.64, 110.67, 108.49, 67.22, 12.79,
3
+
1
2.63; HRMS C H ClNO : calcd. 250.0635 [M+H] , found 250.0637.
1
3
12
2
1-(4-Chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (20j)
Prepared by General Methods 2 and 3 with 4-chloroaniline. Isolated as a white solid (34 mg, 55 %).
o
1
Mp 236 C; H NMR (600 MHz, CDCl ) δ 7.48 (d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 6.42 (s, 1H), 2.31 (s, 3H),
3
-
1.98 (s, 3H); LCMS (basic method) R 1.26 min, m/z 248.1 [M-H] .
t
2
,5-Dimethyl-1-(3-(trifluromethyl)phenyl)-1H-pyrrole-3-carboxylic acid (20k)
Prepared by General Methods 2 and 3 with 3-(trifluoromethyl)aniline. Isolated as a white solid (71 mg, 70 %).
o
-1 1
Mp 243 C; IR  (powder) 3460, 2582-3080, 2857-3080, 1739, 1435-1650, 1320, 1227, 1030-1163 cm ; H
max
NMR (700 MHz, Methanol-d ) δ 7.80 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.53 (d, J = 8.0 Hz,
4
1
3
1H), 6.32 (s, 1H), 2.25 (s, 3H), 1.96 (s, 3H); C NMR (175 MHz, Methanol-d ) δ 150.9, 140.3, 135.3, 133.4, 132.8
4
(q, J = 32 Hz), 131.5, 128.8, 126.3 (q, J = 3.8 Hz), 126.2 (q, J = 3.8 Hz), 125.0 (q, J = 270 Hz), 109.8, 106.9, 12.6,
+
1
2.5; HRMS C H F NO : calcd. 284.0898 [M+H] , found 284.0893.
1
4
12
3
2
1
-(3-Cyclopropylphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (20l)
Prepared by General Method 4.2 with 3-cyclopropylaniline. Isolated as a solid (79 mg, 33 %).
2
9
ACS Paragon Plus Environment