5-Phenyl-1,3,4-oxadiazol-2(3 H)-ones Are Potent Inhibitors of Notum Carboxylesterase Activity Identified by the Optimization of a Crystallographic Fragment Screening Hit

Article abstract of DOI:10.1021/acs.jmedchem.0c01391

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-Triazole 7 as an attractive starting point for a structure-based drug design hit-To-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.

Full text of DOI:10.1021/acs.jmedchem.0c01391

pubs.acs.org/jmc
Article
5‑Phenyl-1,3,4-oxadiazol-2(3H)‑ones Are Potent Inhibitors of Notum
Carboxylesterase Activity Identified by the Optimization of a
Crystallographic Fragment Screening Hit
William Mahy,⊥ Nicky J. Willis,⊥ Yuguang Zhao,⊥ Hannah L. Woodward, Fredrik Svensson,
James Sipthorp, Luca Vecchia, Reinis R. Ruza, James Hillier, Svend Kjær, Sarah Frew, Amy Monaghan,
Magda Bictash, Patricia C. Salinas, Paul Whiting, Jean-Paul Vincent, E. Yvonne Jones,* and Paul V. Fish*
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01391
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of
the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment
screen was performed, which identified fragment hit 1,2,3-triazole 7 as an attractive starting point for a structure-based drug design
hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-
like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the
presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and
partial blood−brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold
increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.
using both genetic and pharmacological approaches, the
inactivation of Notum secreted by osteoblasts led to an
increase in cortical bone mass, suggesting a therapeutic
opportunity in osteoporosis.6,7 In the intestinal crypt, the
expression of Notum by Paneth cells increases with age,
leading to decreased Wnt signaling and subsequent reduction
in stem cell maintenance. A small-molecule Notum inhibitor
normalized Wnt signaling and restored epithelial regeneration,
suggesting a therapeutic opportunity for the restoration of aged
or damaged tissue.8 In the brain, Wnt signaling is known to be
a key modulator of synapse number and function,9 which is
INTRODUCTION
■
The Wnt signaling system plays an essential role in both
development and adult mammalian biology.1 Its dysregulation
in a number of diseases, including cancer2 and osteoporosis,3
has led to significant interest in constituents of the Wnt
pathway as drug targets. Indeed, a number of Wnt-targeted
cancer therapies are in clinical development.4 However,
targeting the Wnt signaling system has proven to be
challenging as some of the targets would not be defined as
“classically druggable”. Notum, a secreted palmitoleoyl-protein
carboxylesterase, has recently been identified as a negative
modulator of Wnt signaling.5 Notum acts through the removal
of an essential palmiteoyl moiety from Wnt proteins, thereby
rendering them inactive. As an enzyme with a defined high-
resolution crystal structure,5 it may present a more tractable
drug target for modulating Wnt signaling.
Received: August 8, 2020
Greater understanding of the biological role of Notum
makes targeting this enzyme a potential therapeutic approach
for treating an increasing number of diseases. For example,
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
© XXXX American Chemical Society
J. Med. Chem. XXXX, XXX, XXX−XXX
A

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Figure 1. Chemical structures and IC50 values of Notum inhibitorsa,b,c,d
.
Notum IC50 data presented in Figure 1 and Tables 1−4 are for
comparison in a common assay format. This work unless otherwise stated. b Determined by competitive gel-based activity-based protein profiling
(ABPP); see ref 15. As a covalent inhibitor of Notum, the IC50 value will be time-dependent. See ref 17.
c
d
thought to be impaired in Alzheimer’s disease (AD),10
although a role for Notum in this process has yet to be
described. Notum has, however, been demonstrated to play an
important role in regulating neurogenesis in the brain,
presumably by modulating Wnt signaling.11 Thus, while
speculative, inhibiting Notum would be expected to increase
neurogenesis and may have potential as a regenerative therapy
in acute and chronic neurological and neurodegenerative
diseases.12
demonstration of modulation of AD phenotypes in cellular and
mouse models. We adopted a strategy to explore comple-
mentary hit-finding approaches to identify multiple chemo-
types to increase our chances of delivering a “fit-for-purpose”
chemical tool. Prior art has shown the success of high-
throughput screening (HTS) campaigns13 and ABPP15 to
identify inhibitors of Notum. Our own work has demonstrated
that quality hits can be identified by fragment screening.16−18
RESULTS AND DISCUSSION
There are now inhibitors of Notum carboxylesterase activity
providing chemical tools for use in target validation experi-
ments (1−6; Figure 1).13−19 A few of these inhibitors have
been evaluated in mouse pharmacodynamic (PD) and
pharmacokinetic (PK) studies and show good plasma levels
but limited brain exposure. Thieno[3,2-d]pyrimidine LP-
922056 (1) has shown utility in mouse models of bone
growth and found to increase cortical bone thickness.7,13
Although 1 demonstrates good oral bioavailability, additional
pharmacokinetic studies in mouse showed negligible brain
exposure.20 Scaffold-hopping from acid 1 identified amide 2 as
a potent inhibitor of Notum.14 Pharmacokinetic studies in
mouse with oral administration of 2 showed improved brain
penetration with a brain/plasma concentration ratio of 0.29.14
N-Hydroxyhydantoin carbamate ABC99 (3) is a potent and
selective irreversible inhibitor of Notum discovered by activity-
based protein profiling (ABPP).15 Inhibition of Notum in mice
with 3 enhanced the regenerative capacity of aged intestinal
stem cells and promoted recovery from chemotherapy-induced
damage.8 In their study, mice were injected intraperitoneally
with 3 daily for 8 days, although no drug levels were reported.8
These chemical tools are potentially suitable for use to explore
the impact of Notum inhibition in models of peripheral disease
but unlikely to be of use in models of central nervous system
(CNS) disease where blood−brain barrier (BBB) permeability
is an essential requirement.
■
To identify new small-molecule inhibitors of Notum, a
crystallographic fragment screen was performed using the
XChem platform at Diamond Light Source (Oxford,
U.K.).16,17 Crystals of C-terminal His-tagged Notum(Ser81−
Thr451 Cys330Ser) were soaked with the original DSI-Poised
library (XChem, 768 fragments, acoustically dispensed as
singletons).17,21 Notum has a well-defined, large, hydrophobic
active-site pocket adjacent to the catalytic triad (Ser232,
His389, Asp340) that accommodates the palmitoleate group of
Wnt.5 Sixty fragments were found to bind in this pocket (hit
rate: 7.8%) with a diverse mixture of chemical structures and
classes (acids, bases, and neutrals) including small clusters of
structurally related hits (Figure 2). These fragment hits were
all resupplied as solid samples by purchase or resynthesis to
establish structure and purity.
Inhibition of Notum carboxylesterase activity of these hits
was measured in a biochemical assay where test compounds
(dispensed to give 10-point concentration−response curves up
to 100 μM) were incubated with human Notum(81-451
Cys330Ser) and trisodium 8-octanoyloxypyrene-1,3,6-trisulfo-
nate (OPTS) as the substrate, and the fluorescence was
recorded; an inhibitor of Notum would suppress fluorescence
by binding to Notum and preventing hydrolysis of OPTS.
A standout hit from this set was (1-(4-chlorophenyl)-1H-
1,2,3-triazol-4-yl)methanol (7) (Figures 2 and 3). The binding
pose of 7, as identified in the crystal structure, showed
occupation of the palmitoleate binding pocket by the N-(4-
chlorophenyl) substituent, forming a π−π stacking interaction
Hence, our objective was to discover new small-molecule
inhibitors of Notum activity suitable for exploring the
regulation of Wnt signaling in the CNS and, ultimately,
B
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. Workflow for the fragment screen and summary of
properties for fragment hit 7.
with residue Phe268 (Figure 3B,C). The triazole head group
shows possible π−π stacking interactions with Trp128 and
hydrogen bonding between the triazole N2 and peptidic
backbone of Trp128. Residue Trp128 is also involved in a
hydrogen bond to the oxygen of the methyl alcohol group.
Compared to the crystal structure of O-palmitoleoyl serine
(Figure 3D), compound 7 occupies approximately the same
volume of the pocket as the palmitoleic tail. However, the
interaction with the oxyanion hole is more pronounced for the
carbonyl of the palmitoleic ester with hydrogen bonds to three
separate amino acids (Gly127, Trp128, Ala223) rather than
just Trp128 as observed with 7.
Triazole 7 was determined to be a potent inhibitor of
Notum carboxylesterase activity, IC50 11.5 3.0 μM (n = 4),
with excellent hit-like properties (mw 209; c log P 1.5; TPSA
48 Å2) and design metrics (LE = 0.49; LLE = 3.4).22,23
Furthermore, the N1-phenyl triazole structural motif is highly
chemically enabled to support the rapid exploration of
structure−activity relationships (SARs) through the applica-
tion of established synthetic methods. These features
combined to identify 7 as an attractive starting point for a
structure-based drug design (SBDD) hit-to-lead program.
The simple disconnection of hit 7 into its triazole
(heterocycle) and 4-chlorophenyl (aryl) motifs guided our
general design strategy. New compounds were designed to
improve the inhibition of Notum activity through modifica-
tions at these two principal areas: (1) the heterocycle that sits
at the mouth of the pocket near the catalytic triad 8−25
(Tables 1 and 2) and (2) the aryl ring that binds in the
palmitoleate pocket 23a−23bb (Table 3). Compound design
was further guided by the Notum crystal structure and in silico
docking of design ideas. Additional bespoke modifications of
preferred leads were then performed to further improve the
activity and tune physicochemical properties (mw, c log P, pKa)
to be consistent with good in vitro ADME property space
(Table 4). Selected compounds were screened for aqueous
solubility, transit performance in MDCK-MDR1 cell lines for
permeability, and metabolic stability in mouse and human liver
Figure 3. (A) Electron density |FO − FC| omit map. Electron density
from Notum complex with 7 (lime) shown as sky blue mesh
contoured at 3σ. (B) Notum-7 binding details (PDB 6ZUV). Notum
residues (gray) interacting with 7 (lime) are defined with distance < 4
Å. 7 is sandwiched between Phe268 in the back of the pocket and
Trp128. The backbone of Trp128 also forms a hydrogen bond with 7
(yellow dashed lines). (C) Notum-7 interaction diagram showing all
residues within 4 Å. π-Stacking interactions (green lines) and
hydrogen bonds (purple arrows) are highlighted. (D) Overlay of 7
(lime) with O-palmitoleoyl serine (teal) (PDB 4UZQ) with the
surface of the Notum palmitoleoyl binding pocket outlined (gray).
Water molecules have been removed for clarity.
C
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a b
,
Table 1. Notum Inhibition of 4-Chlorophenyl Hydroxymethyl Azoles 7−15
a
b
All values are mean s.d. of n = 2−8 experiments quoted to 2 s.f. 20−30% I @ 100 μM.
a b c
, ,
Table 2. Notum Inhibition of 4-Chlorophenyl Azoles 16−25
a
b
c
See the footnotes in Table 1. <20% I @ 100 μM. 30−50% I @ 100 μM.
microsomes (MLM and HLM, respectively) to prioritize
compounds for in vivo studies. Preferred compounds were
dosed orally in mouse to determine their pharmacokinetic
properties including BBB permeability.
Initially, the effect of modification of the heterocyclic head
group was investigated while retaining the pendant hydroxy-
methyl and the 4-chlorophenyl groups; this single group
change would allow the direct comparison of the impact of
these azoles 8−15 (Table 1). The analogues were either
sourced commercially or prepared using established methods.
Further substitution by a methyl on the C5 position of the
1,2,3-triazole 8 (and N-4 of the 1,2,4-triazole 9) seemed to be
disfavored as there was a loss of potency, although these were
the only analogues screened to test this hypothesis. It was
feasible to replace the 1,2,3-triazole ring with either an oxazole
10, isoxazoles 11 and 12, or oxadiazoles 13−15 as all showed
D
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
23 was selected for further optimization due to these factors
and, in part, because of the successful application of
oxadiazoles in drug discovery programs.25 In addition, 23
was of interest as a lead to explore the potential of weak acids
to penetrate the brain as molecules with these properties are
under-represented in the CNS drug discovery.26,27 1,2-
Isoxazol-3-one 24 and 1,3,4-thiadiazol-2-one 25 also demon-
strated a similar potency to 23, and the latter heterocycle was
revisited once an optimized aryl group had been identified.
At this point in the program, it was encouraging that two
complementary leads had been identified from one hit, i.e.,
triazole 16 and oxadiazole 23 were created from the original X-
ray fragment screening hit 7.
With the selection of 1,3,4-oxadiazol-2-one as a preferred
heterocycle, we required a reliable synthesis of substituted 5-
phenyl-1,3,4-oxadiazol-2-ones for the development of the aryl
ring SARs (Tables 3 and 4). Target compounds 23a−23ll were
readily prepared in three steps from the corresponding benzoic
acids 28 (Scheme 1). Acids 28 were either purchased or
prepared using a customized synthesis, and then esterification
with methanol gave methyl esters 29. Treatment of 29 with
excess hydrazine gave the corresponding acyl hydrazides 30,
which were then cyclized with triphosgene to afford
oxadiazolones 23a−23ll.
We first explored the SAR of the aryl ring with a variety of
single substituents at the ortho-, meta-, and para-positions
23a−23bb (Table 3). The unsubstituted phenyl 23a (IC50 41
μM) was prepared and screened as the benchmark to assess
these changes; it is interesting to note that 23a was ca. 20-fold
weaker than the 4-Cl hit 23 and proved to be the weakest
compound of all of the aryl analogues in Table 3 showing that
SAR was tolerant of a variety of groups within this pocket.
The tactic of walking a Cl, F, Me, and CF3 around the
phenyl ring showed a clear preference for a CF3 group at all
positions (23e, 23l, 23v) (Table 3). A broader range of
substituents were then prepared with a bias toward substitution
at the meta- and para-positions. In general, the preferred
groups from this exercise were compounds with small
lipophilic groups such as Et (23i, 23q), cPr (23j, 23r), CF3
(23l, 23v), NMe2 (23n, 23bb), and OiPr (23y), all achieving
IC50s < 0.5 μM. The most potent single substituent Notum
inhibitors were 4-iPr (23s; IC50 0.075 μM) and 4-tBu (23t;
IC50 0.051 μM). Incorporation of polar substitutions such as 4-
CN (23w) and 4-NO2 (23t) was detrimental to activity but
still superior to having no substituent (cf 23a).
Table 3. Notum Inhibition of Monosubstituted 5-Phenyl-
1,3,4-oxadiazol-2(3H)-one 23
a
Compound
R2; R3; R4
Notum IC50 (μM)
41
23a
H
5
2-Substituents (R2)
2-Cl
23b
23c
23d
23e
6.7 2.7
11
2-F
2-Me
2-CF3
3-Substituents (R3)
3-Cl
3
3.6 0.2
0.43 0.27
23f
1.8 0.5
23g
23h
23i
23j
23k
23l
3-F
3-Me
3-Et
3-cPr
3-CHF2
3-CF3
3-OMe
3-NMe2
4-Substituents (R4)
4-Cl
4-F
4-Me
4-Et
4-cPr
4-iPr
4-tBu
4-CHF2
4-CF3
4-CN
13
4
2.9 0.3
0.45 0.25
0.26 0.12
3.1 0.8
0.78 0.02
1.2 0.4
23m
23n
0.19 0.09
23
2.1 0.6
3.5 0.7
2.0 0.6
0.27 0.12
0.26 0.08
0.075 0.014
0.051 0.030
3.0 0.5
0.46 0.7
27 13
1.4 0.2
0.35 0.20
18
23o
23p
23q
23r
23s
23t
23u
23v
23w
23x
23y
23aa
23bb
4-OMe
4-OiPr
4-NO2
4-NMe2
3
0.19 0.02
a
See the footnotes in Table 1.
There emerged a clear trend within this set that the
inhibition of Notum activity was related to the lipophilicity of
the substituent with the most potent compounds having the
highest lipophilicity as assessed by c log P and lipophilic
fragmental constants (π) (Figure S1). Additionally, modeling
suggested that larger substituents were favorable to fully
occupy the binding pocket.
The weakly acidic nature of 5-phenyl-1,3,4-oxadiazole-
2(3H)-one (23a, calculated pKa ca. 5.8−7.128) prompted us
to consider if it would be feasible to further reduce the acidity
of the oxadiazolone head group to pKa > 7.4 and so increase
the fraction of nonionized drug at physiological pH.
A reduction in acidic pKa would potentially better align the
physicochemical properties of this template with CNS drugs as
acidic pKa values for CNS drugs rarely fall below 6.0.26,27 From
our compound collection, two disparate examples of electron-
deficient (23aa; 4-NO2) and electron-rich (23bb; 4-NMe2)
aryl substituents were selected for pKa measurement (Figure
reasonable activity. A 2- to 3-fold improvement in activity over
7 was achieved with isoxazole 11; however, this was achieved
at the expense of slightly increased lipophilicity (c log P 1.9,
LLE 3.5).
The next phase of SAR investigation was the deletion of the
hydroxymethyl group, or truncation to the directly attached
hydroxyl, in a series of 4-chlorophenyl heterocycles 16−25
(Table 2). The N1 triazole 16 (IC50 2.9 μM, c log P 2.6)
showed an improvement in activity compared to 7, as did the
C4 isomer 17, and was selected as an early lead for further
optimization; full details of this work will be reported
separately.24 In contrast, the N1 pyrazole 18 and C5 tetrazole
19 were inferior. The hydroxyl-substituted azoles 20−25 gave
a broad range in activities (1 to >100 μM) and identified
weakly acidic 1,3,4-oxadiazol-2(3H)-one 23 as a breakthrough
compound due to its improved Notum inhibition activity and
physicochemical properties (IC50 2.1 μM, c log P 2.7). Hence,
E
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 4. Notum Inhibition, Microsomal Stability, Cell Permeability, and Aqueous Solubility of 1,3,4-Oxadiazol-2(3H)-ones
a b
,
23cc−23ll, 1,3,4-Thiadiazol-2(3H)-one 26, and 1,3,4-Oxadiazole 27
a
b
See the footnotes in Table 1. MLM/HLM, MDCK-MDR1, and solubility studies reported in this work were independently performed by GVK
Biosciences (Hyderabad, India) or Cyprotex (Macclesfield, U.K.).
a
Scheme 1. Preparation of 1,3,4-Oxadiazol-2(3H)-ones 23
a
Figure 4. Measured acidic pKas of para-substituted 5-phenyl-1,3,4-
Representative reagents and conditions: (a) H2SO4 (0.5 equiv),
MeOH, RT85 °C or SOCl2 (5 equiv), MeOH, 0 °CRT; (b)
N2H4·H2O (8 equiv), EtOH-PhMe, 70 °C; and (c) triphosgene (0.4
equiv), iPr2NEt (2 equiv), CH2Cl2, 0 °CRT.
a
oxadiazol-2(3H)-onesa,b,c
.
pKa of 1,3,4-oxadiazol-2(3H)-one re-
b
ported. pKa of dimethylamino substituent measured as 2.92
c
0.06. Hammett para-substituent constant (σp).
4). This exercise would establish the likely dynamic range of
the acidity of N3-H of the 1,3,4-oxadiazol-2(3H)-one through
the influence of a single substituent at the 4-position of the aryl
ring. The results suggested that it was reasonable to modulate
the pKa of the 1,3,4-oxadiazolone within a range of 1 log-unit
and that a strongly electron-releasing substituent (4-NMe2)
could reduce the acidic pKa to 7.4.
The application of two substituents at the 3- and 4-positions
on the phenyl ring was then explored by building on earlier
SAR with a subset of preferred groups (Cl, CF3, cPr, iPr, NR2)
(23cc−23ll; Table 4). 3,4-Dichloro 23cc further improved
Notum inhibition activity when compared to the monosub-
stituted 3-Cl and 4-Cl analogues (23cc vs 23f vs 23), which
supported this approach. The combination of a 3-CF3 group
with preferred groups at the 4-position gave some of the most
potent compounds prepared so far: 3-CF3-4-Cl (23dd; IC50 18
nM), 3-CF3-4-iPr (23ff; IC50 6.2 nM), and 3-CF3-4-cPr (23gg;
IC50 1.8 nM). Switching the position of the 3- and 4-
substituents of 23gg gave 23hh (3-cPr-4-CF3; IC50 2.9 nM),
which was also a very potent inhibitor of Notum. Evaluation of
F
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
selected compounds in MLM showed that only 23dd
demonstrated high metabolic stability with the potential
advantage of low metabolic clearance in vivo. Compound
23dd also showed high cell permeability in the MDCK-MDR1
assay with a low efflux ratio (ER = 1.6), suggesting minimal
recognition by P-gp-mediated efflux transport.
Extending this approach by exploring amino substituents
(23ii) or amines incorporated into a 3,4-fused ring system
(23jj-ll) identified 23ii (3-CF3-4-NMe2; IC50 15 nM) as a
potent inhibitor of Notum but with poor metabolic stability in
MLM. It proved challenging to combine potent Notum
inhibition with metabolic stability through the application of
substituents designed to reduce the pKa of the oxadiazolone.24
Revisiting the 1,3,4-thiadiazol-2-one head group (cf 23 vs
25) in combination with the 3-Cl-4-CF3 aryl ring gave 26 (IC50
31 nM), which was also a potent inhibitor of Notum, albeit
with inferior metabolic stability. Similarly, 1,3,4-oxadiazole 27
was equipotent to 23dd but with moderate MLM stability and
offered no advantage over 23dd.
showing a preferential binding to brain tissue, and 23dd did
not significantly inhibit representative CYP450 enzymes.
Notum inhibitor 23dd was screened in a cell-based TCF/
LEF reporter gene (Luciferase) assay to assess its ability to
restore Wnt/β-catenin signaling when activated by exogenous
rWNT3a (100 ng/mL) in the presence of Notum (500 ng/
mL) (Table 5).16 Compound 23dd showed an activation of
Wnt signaling (EC50 ca. 3500
2200 nM (n = 8)) in this
model system through the inhibition of Notum in a
concentration-dependent manner. Performing this experiment
in the absence of Notum showed a maximal Wnt response at
all concentrations of 23dd tested (0.3 nM to 10 μM; (n = 4)),
confirming that the outcome was due to direct inhibition of
Notum activity and not related to assay interference or cell
toxicity (up to 10 μM).
Representative inhibitors were tested in a Notum occupancy
assay using FP-biotin, a serine hydrolase activity-based probe,
whereby labeling of Notum with FP-biotin can be blocked by
an inhibitor occupying the active site of Notum.16,31 Inhibitors
23dd and 27 showed some ability to prevent labeling by FP-
biotin, confirming that they competitively bind to Notum,
albeit with modest potency compared to 1. While target
engagement is demonstrated, this data also shows disparities
between different chemotypes with similar activity in the
Notum OPTS assay (Figure S2).
Hence, 23dd emerged as having a superior profile from this
set and was selected for further evaluation as a lead for the
program (Table 5). Compound 23dd has physicochemical and
Table 5. Summary of Physicochemical and Molecular
Properties, Notum Inhibition, and ADME Data for 23dd
a
The binding mode of 23dd is shown in Figure 5. In contrast
to the original fragment hit 7 (Figure 3), 23dd is making a
complete interaction with the oxyanion hole similar to the
palmitoleic ester while occupying an increased volume of the
palmitoleate hydrophobic pocket.
23dd
Physicochemical and Molecular Properties
mol wt
c log P
log D7.4
TPSA (Å2)
CNS MPO
pKa
Notum Inhibition
OPTS, IC50 (nM)
TCF-LEF, EC50 (nM)
ADME Profile
aq. solubility (μg/mL)/(μM)
mouse plasma protein binding (PPB) (%)
mouse brain binding (%)
MLM, Cli (μL/min/mg protein)
CYP1A2 inhibition, IC50 (μM)
CYP2C9 inhibition, IC50 (μM)
CYP2C19 inhibition, IC50 (μM)
CYP2D6 inhibition, IC50 (μM)
CYP3A4 inhibition, IC50 (μM)
MDCK-MDR1, AB/BA Papp (×10−6 cm/s)
MDCK-MDR1, efflux ratio (ER)
265
2.9
2.6
59.2
5.7
6.7
Modeling the inhibitors 23a−23ll in the active site of
Notum by molecular docking using the crystal structure of
23dd reveals that the more potent inhibitors tend to occupy
the pocket more completely with substituents also filling the
back of this pocket, as can be clearly seen for 23dd in Figure 5.
Due to the highly lipophilic nature of the palmitoleate pocket,
the preferred groups accepted also tend to be highly lipophilic.
Pharmacokinetic data for 23dd was generated in vivo in
mouse to evaluate plasma exposure and BBB penetration
(Table 6 and Figure S4). Following a single oral dose (p.o.) of
10 mg/kg, plasma exposure achieved a Cmax [plasma] ≈ 4.3
μM (free drug) with rapid absorption. Compound 23dd
demonstrated modest BBB penetration with a brain-to-plasma
concentration ratio of 0.16 based on AUC(0→inf) (total drug,
Kp), which was reduced to 0.025 (free drug, Kp,uu) when
unbound drug levels in brain and plasma were used. The
combination of incomplete BBB penetration along with
preferential binding to the brain tissue resulted in a moderate
brain exposure of Cmax [brain] ≈ 0.12 μM (free drug) from this
dose.
18 3 (n = 8)
3500 2200 (n = 8)
188/710
93.1
98.9
0.42
16.6
>25
>25
>25
>25
34/55
1.64
a
See the footnotes in Table 4 and Figure S3.
The plasma half-life and terminal clearance of 23dd were
consistent with the microsomal stability data in MLM.
However, the partial BBB penetration was somewhat
unexpected based on the design criteria, in silico, and cell
models employed leading to the in vivo assessment. The BBB
provides protection to the CNS from exposure to circulating
chemicals by maintaining tight junctions between endothelial
cells and through a range of transporter proteins that regulate
exchange between blood and the CNS. The ABC transporters
are generally regarded as the most important transporters that
restrict the permeability of toxins and therapeutic agents across
the BBB with the P-gp, BCRP, and MRP efflux transporters
having significant roles.32 These ABC transporters are able to
molecular properties consistent with small-molecule lead-like
chemical space (mw 265; c log P 2.9; log D7.4 2.6; TPSA 59.2
Å2). The oxadiazole head group of 23dd contains a weakly
acidic proton at N3 with a measured pKa 6.7, and so 23dd
would be ca. 85% ionized at pH 7.4. Assessment of 23dd by
standard design metrics (LE 0.64; LLE 4.8) showed improve-
ment over the original fragment hit 7 and properties consistent
with CNS drug-like space29 including a favorable “CNS
multiparameter optimization” score (CNS MPO = 5.7/6.0).30
Additional in vitro ADME studies measured binding to mouse
plasma proteins (fu 0.069) and mouse brain tissue (fu 0.011)
G
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 6. Mouse Pharmacokinetic Data for 23dd; Oral (p.o.)
a
Dose at 10 mg/kg
23dd
PK parameter
plasma
3.7 h
brain
2.3 h
T1/2
Tmax
0.5 h
0.17 h
Cmax
16 600 ng/mL
50 400 ng·h/mL
63 100 ng·h/mL
2990 ng/g
8870 ng·h/g
9910 ng·h/g
AUC(0→t)
AUC(0→inf)
b
Kp
0.16
0.025
c
Kp,uu
a
Male C57BL6 mice; suspension formulation in 0.1% Tween80 in
water; n = 3 per time point; terminal blood and brain levels measured
at 0.17, 0.50, 1, 2, 4, 8, and 24 h. All animals were healthy throughout
b
the study period. The brain-to-plasma ratio (Kp) was calculated by
the ratio of AUC(0→inf) of the brain concentration−time profile
(AUC(0→inf), brain) to that of the plasma concentration−time profile
c
(AUC(0→inf), plasma). Free partition coefficient of brain (Kp,uu) was
calculated by multiplying the Kp with the ratio of free fraction in brain
homogenate to plasma (fu,brain/fu,plasma).
recognize and efflux a diverse range of drugs and have, in part,
overlapping substrates belonging to different chemical classes.
The reason for the partial BBB exposure of 23dd will not be
simple to establish due to the complex nature of the BBB and
the multiple parameters that influence BBB permeability.
Although 23dd seems to have physicochemical properties
entirely consistent with passive BBB permeability29 and
excellent permeability as measured by transit performance in
MDCK-MDR1 cell lines without evidence for P-gp recognition
and efflux, 23dd may prove to be a substrate for other BBB
efflux transporters.32 P-gp substrates are usually lipophilic and
either neutral or cationic, whereas MRPs prefer anionic
substrates and BCRP transports both cations and anions. As
23dd is weakly acidic and would be predominantly anionic at
physiological pH, we propose that it may be a substrate for the
MRP or BCRP efflux transporters.
The apparent disconnection between these in vitro and in
vivo models also recommends the early assessment of a
chemical tool in rodent PK experiments to establish a
correlation (or otherwise) between the predictive models
employed and in vivo outcomes for BBB permeability.
In summary, the mouse PK experiments support the use of
23dd as a new chemical tool for exploring the role of Notum-
mediated regulation of Wnt signaling for peripheral disease.
However, its incomplete BBB penetration and modest free
drug levels in the brain at these doses mean 23dd is probably
not suitable for use in animal models of CNS disease. Recent
efforts have identified amide 214 and oxadiazole 23dd (this
work), both with partial BBB permeability (Table S2). Hence,
there is still the need for an improved inhibitor of Notum
activity for use as a chemical probe to investigate the regulation
of Wnt signaling in the CNS and demonstrate modulation of
AD phenotypes in mouse models.
Figure 5. (A) Electron density |FO − FC| omit map. Electron density
from Notum complex with 23dd (gold) shown as sky blue mesh
contoured at 3σ. (B) Notum-23dd binding details (PDB 6ZVL).
Notum residues (gray) interacting with 23dd (gold) are defined with
distance < 4 Å. Note that I291 and S232 show alternative
conformations. (C) Notum-23dd interaction diagram showing all
residues within 4 Å. π-Stacking interactions (green lines) and
hydrogen bonds (purple arrows) are highlighted. (D) Overlay of
23dd (gold) with O-palmitoleoyl serine (teal) (PDB 4UZQ) with the
surface of the Notum palmitoleoyl binding pocket outlined (gray).
Water molecules have been removed for clarity.
We propose that 23dd represents a quality addition to the
small-molecule “toolbox” of Notum inhibitors as it is a new
chemotype with a complementary profile to 1−3.
CONCLUSIONS
■
A crystallographic X-ray fragment screen with carboxylesterase
Notum identified (1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-
methanol (7) as an attractive starting point for an SBDD hit-
H
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(sext), doublet of doublets (dd), doublet of triplets (dt), triplet of
triplets (tt), multiplet (m), or broad singlet (br s).
to-lead program. Optimization of fragment hit 7 (IC50 11.5
μM) by SAR of the heterocyclic head group identified two
early lead series as potent inhibitors of Notum activity: triazole
16 (IC50 2.9 μM) and oxadiazole 23 (IC50 2.1 μM). Further
investigation of the oxadiazole series 23 by exploring
substitution on the aryl ring, which binds deep in the
palmitoleate pocket, identified 23dd as a preferred example.
Compound 23dd has physicochemical properties consistent
with the drug-like chemical space and contains a weakly acidic
proton with a measured pKa 6.7. Compound 23dd
demonstrated good metabolic stability in MLM and HLM,
and cell permeability in the MDCK-MDR1 assay with no
evidence for P-gp-mediated efflux. Compound 23dd restored
Wnt/β-catenin signaling in a cell-based TCF/LEF reporter
gene assay when activated by exogenous rWNT3a in the
presence of Notum. PK studies with 23dd were performed in
vivo in mouse with a single oral administration of 23dd
showing good plasma exposure but partial BBB penetration.
An X-ray crystallographic structure determined the binding
mode of 23dd in Notum. Inhibitor 23dd makes an effective
interaction with the oxyanion hole with hydrogen bonds to
three separate amino acids (Gly127, Trp128, Ala223) while
still filling the hydrophobic palmitoleate pocket.
Significant progress was made in developing X-ray fragment
hit 7 into lead 23dd (>600-fold increase in activity), which
showed good plasma exposure in mouse PK experiments,
making it suitable as a new chemical tool for exploring the role
of Notum-mediated regulation of Wnt signaling for non-CNS
disease. Furthermore, contemporaneous studies with alter-
native lead triazole 16 identified ARUK3001185, which was
shown to be a potent, selective, and CNS penetrant inhibitor
of Notum activity suitable for use in both cellular and in vivo
models of CNS disease.24 Full details of this research will be
published in due course.
Liquid chromatography−mass spectrometry (LCMS) analysis was
performed on a Waters Acquity H-Class UPLC system with either an
acidic (HSS C18 Column, H2O/MeCN, 0.1% TFA) or a basic (BEH
C18 Column, H2O/MeCN, 10 mM NH4OH) mobile phase.
High-resolution mass spectrometry (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.
The purity of screening compounds 7−27 was evaluated by NMR
spectroscopy and LCMS analysis. All compounds had purity ≥95%
unless otherwise stated.
General Methods. Preparation of Methyl Esters 29 (General
Method 1). Sulfuric Acid-Catalyzed. Sulfuric acid (60 μL, 1.11 mmol,
0.5 equiv) was added to a solution of carboxylic acid 28 (2.23 mmol,
1.0 equiv) in MeOH (10 mL) and heated to 85 °C for 3 days. The
reaction mixture was then cooled to RT, concentrated to
approximately one-third volume, and then diluted with EtOAc and
saturated aqueous NaHCO3. The organic phase was dried, filtered,
and concentrated under reduced pressure to give ester 29, which was
used without further purification.
Thionyl Chloride-Promoted. Thionyl chloride (0.83 mL, 7.26
mmol, 5.0 equiv) was added dropwise to a solution of carboxylic acid
28 (1.45 mmol, 1.0 equiv) in MeOH (8 mL) at 0 °C (external). The
reaction mixture was heated to reflux for 2 h, cooled to RT, and
concentrated under reduced pressure. The residue was then passed
through a short pad of silica (0−20% EtOAc in cyclohexane) to
provide ester 29, which was used without further purification.
(Trimethylsilyl)diazomethane-Promoted. A solution of
(trimethylsilyl)diazomethane (1.13 mL, 2 M in hexanes, 2.27 mmol,
1.05 equiv) was added dropwise to a precooled solution of carboxylic
acid 28 (2.16 mmol, 1.0 equiv) in MeOH at 0 °C (external) (upon
addition, mild bubbling is observed). Following complete addition,
the reaction mixture remained a faint yellow color. Acetic acid was
added dropwise until the reaction mixture turned colorless (complete
consumption of the excess TMSCH2N2). The reaction mixture was
concentrated under reduced pressure to give ester 29, which was used
without further purification.
Preparation of Acyl Hydrazides 30 (General Method 2).
Hydrazine monohydrate (350 mg, 7.0 mmol, 4.0 equiv) was added
to a solution of methyl ester 29 (1.75 mmol) in EtOH (4 mL) and
toluene (2 mL). The mixture was heated at 40 °C for 16 h, cooled to
RT, and then concentrated under reduced pressure. The residue was
azeotroped with EtOH and toluene to provide crude acyl hydrazide
30, which was used without further purification.
A few reactions were performed at higher temperatures (45−120
°C) and/or with additional equivalents of hydrazine (8−15 equiv);
these modified conditions are presented for each compound as
necessary.
EXPERIMENTAL SECTION
■
Chemistry. General Information. 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.
COware gas reactor (total volume 100 mL) was purchased from
Sigma-Aldrich (catalogue number STW5).
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.
Organic solvent layers were routinely dried with anhydrous MgSO4
Preparation of 1,3,4-Oxadiazol-2(3H)-ones 23b−23ll (General
Method 3). A solution of triphosgene (172 mg, 0.58 mmol, 0.4 equiv)
in CH2Cl2 (15 mL) was added dropwise to a solution of acyl
hydrazide 30 and iPr2NEt (0.51 mL, 2.9 mmol, 2.0 equiv) in CH2Cl2
(5 mL) at 0 °C (external). The reaction was stirred at 0 °C for 20
min, warmed to RT, and stirred for a further 40 min. The reaction
mixture was diluted with CH2Cl2 and cautiously diluted with
saturated aqueous NaHCO3. The organic phase was dried, filtered,
and concentrated under reduced pressure. The crude product was
purified by column chromatography (0−2% MeOH in CH2Cl2) to
give oxadiazolone 23b−23ll.
and concentrated using a Buchi rotary evaporator.
̈
Melting points (mp) were determined using Stuart SMP20 melting
point equipment using closed-end glass capillary tubes and are
uncorrected.
Infrared (IR) spectra were recorded on a Shimadzu IRTracer-100
FT-IR spectrometer, using a Universal ATR accessory for sampling,
with relevant absorbance quoted as νmax in cm−1.
Preparation of 1,3,4-Thiadiazol-2(3H)-ones 25 and 26 (General
Method 4). Lawesson’s reagent (360 mg, 0.88 mmol, 1.0 equiv) was
suspended in PhMe (5 mL), benzohydrazide 30 (0.88 mmol) was
1H nuclear magnetic resonance (NMR) and 13C 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
ο
added, and the mixture was heated at 110 C for 16 h. The reaction
mixture was quenched by the addition of H2O (5 mL) and stirring at
50 οC for 10 min. The biphasic mixture was then concentrated under
reduced pressure. The residue was purified by reverse-phase
chromatography (0−100% MeCN in H2O, 0.1% formic acid
I
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
modifier) to give benzothiohydrazide, which was used immediately in
the next step without further purification.
5-(4-Chlorophenyl)-2H-tetrazole (19). Purchased from Sigma-
Aldrich, CH6371382779.
3-(4-Chlorophenyl)-1-methyl-1H-pyrazol-5-ol (20). Purchased
from Fluorochem, 317356.
3-(4-Chlorophenyl)isoxazol-5-ol (21). Purchased from Activate
Scientific, AS93646.
A solution of triphosgene (52 mg, 0.18 mmol) in CH2Cl2 (10 mL)
was added dropwise to a stirred solution of benzothiohydrazide and
iPr2NEt (0.18 mL, 1.06 mmol) in CH2Cl2 (10 mL) at 0 °C (external).
The mixture was warmed to RT and stirred for 10 min. The reaction
was quenched by the addition of saturated aqueous NaHCO3, and the
organic phase was separated and concentrated under pressure. The
residue was purified by reverse-phase chromatography (0−100%,
MeCN in H2O, 0.1% NH4OH modifier) to give 1,3,4-thiadiazol-
2(3H)-one.
Notum Inhibitors. (1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)-
methanol (7). Purchased from Key Organics, 4F-329S.
(1-(4-Chlorophenyl)-5-methyl-1H-1,2,3-triazol-4-yl)methanol
(8). Purchased from Key Organics, BS-4321.
[5-(4-Chlorophenyl)-4-methyl-1,2,4-triazol-3-yl]methanol (9).
Purchased from Enamine, EN300-137908.
[5-(4-Chlorophenyl)oxazol-2-yl]methanol (10). Purchased from
Enamine, EN300-263328.
(5-(4-Chlorophenyl)isoxazol-3-yl)methanol (11). Purchased from
Key Organics, 5N-601S.
(3-(4-Chlorophenyl)isoxazol-5-yl)methanol (12). Purchased from
Key Organics, 10L-365S.
(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)methanol (13). Pur-
chased from Enamine, EN300-65731.
[5-(4-Chlorophenyl)-1,2,4-oxadiazol-3-yl]methanol (14). Pur-
chased from Enamine, EN300-105946.
(3-(4-Chlorophenyl)-1,2,4-oxadiazol-5-yl)methanol (15). Pur-
chased from Key Organics, 11X-0223.
1-(4-Chlorophenyl)-1H-1,2,3-triazole (16). Step 1: Sodium nitrite
(144 mg, 2.1 mmol) was added portionwise to a solution of 4-
chloroaniline (220 mg, 1.7 mmol) in TFA (3 mL, 3.5 mmol) at 0 οC
over 30 min. The reaction mixture was then allowed to warm to RT
for 1.5 h, H2O (0.3 mL) was added, and the mixture was re-cooled to
0 °C. Sodium azide (125 mg, 1.9 mmol) was added portionwise over
30 min, and the reaction mixture was warmed to RT over 16 h. The
mixture was basified to pH 8−9 by the dropwise addition of saturated
aqueous NaHCO3 and extracted with CH2Cl2 (2 × 30 mL). The
combined organic phase was diluted with t-BuOH (2 mL), and
CH2Cl2 was cautiously evaporated under reduced pressure at 25 °C to
provide a solution of crude 1-azido-4-chlorobenzene, which was used
without further purification.
3-(4-Chlorophenyl)-1,2,4-oxadiazol-5(4H)-one (22). Methyl
chloroformate (0.11 mL, 1.47 mmol) was added dropwise to a
suspension of 4-chloro-N-hydroxy-benzamidine (250 mg, 1.47 mmol)
and K2CO3 (200 mg, 1.47 mmol) at RT. The mixture was heated to
60 °C for 5 h, cooled to RT, concentrated under reduced pressure,
and then purified by reverse-phase chromatography (0−100% MeCN
in H2O, 0.1% formic acid modifier) to give 22 (15 mg, 5%) as a white
solid.
1H NMR (600 MHz, DMSO-d6) δ 7.79−7.74 (m, 2H), 7.46−7.40
(m, 2H); LCMS m/z 195.0 [M − H]−.
5-(4-Chlorophenyl)-1,3,4-oxadiazol-2(3H)-one (23). Purchased
from Sigma-Aldrich, JRD0393.
5-Phenyl-1,3,4-oxadiazol-2(3H)-one (23a). Purchased from Key
Organics, LE-0723.
5-(2-Chlorophenyl)-1,3,4-oxadiazol-2(3H)-one (23b). Prepared
by General Method 3 from 2-chlorobenzohydrazide. Isolated as a
white solid (84 mg, 73%).
1H NMR (400 MHz, CDCl3) δ 9.11 (br s, 1H), 7.82 (dd, J = 7.9,
1.8 Hz, 1H), 7.54 (dd, J = 7.9, 1.4 Hz, 1H), 7.46 (apparent td, J = 7.6,
1.8 Hz, 1H), 7.40 (apparent ftd, J = 7.6, 1.4 Hz, 1H); 13C NMR (175
MHz, CDCl3) δ 154.0, 153.6, 132.8, 132.5, 131.6, 130.2, 127.2, 122.6;
LCMS m/z 197.1 [M + H]+.
5-(2-Fluorophenyl)-1,3,4-oxadiazol-2(3H)-one (23c). Prepared by
General Method 3 from 2-fluorobenzohydrazide. Isolated as a white
solid (74 mg, 84%).
1H NMR (600 MHz, DMSO-d6) δ 12.71 (s, 1H), 7.81 (apparent
td, J = 7.6, 1.7 Hz, 1H), 7.63 (dddd, J = 8.4, 7.2, 5.1, 1.8 Hz, 1H), 7.43
(ddd, J = 11.2, 8.4, 0.9 Hz, 1H), 7.38 (apparent td, J = 7.7, 1.1 Hz,
1H); 13C NMR (176 MHz, DMSO) δ 159.0 (d, J = 256.1 Hz), 154.1,
150.5 (d, J = 5.0 Hz), 133.5 (d, J = 8.5 Hz), 128.5, 125.1 (d, J = 3.5
Hz), 117.0 (d, J = 20.3 Hz), 112.2 (d, J = 11.0 Hz); LCMS m/z 179.1
[M − H]−.
5-(2-Methylphenyl)-1,3,4-oxadiazol-2(3H)-one (23d). Purchased
from Combi-Blocks, WZ-9510.
5-(2-(Trifluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-one (23e).
Prepared by General Method 3 from 2-(trifluoromethyl)-
benzohydrazide. Isolated as a white solid (92 mg, 82%).
1H NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H), 7.99−7.93 (m,
2H), 7.90−7.80 (m, 2H); 13C NMR (176 MHz, DMSO) δ 154.4,
151.9, 133.2, 132.1, 131.2, 127.1 (q, J = 5.4 Hz), 126.90 (q, J = 31.8
Hz), 123.2 (q, J = 273.5 Hz), 122.1 (d, J = 1.7 Hz); LCMS m/z 229.1
[M − H]−.
Step 2: MeOH (1 mL), H2O (2 mL), sodium L-ascorbate (138 mg,
0.7 mmol), and trimethylsilylacetylene (171 mg, 1.7 mmol) were
added to the solution of crude azide from Step 1. The reaction
mixture was stirred at 50 °C for 16 h, cooled to RT, diluted with brine
(10 mL), and extracted with CH2Cl2 (2 × 50 mL). The combined
organics were dried, concentrated under reduced pressure, and the
crude product was purified by reverse-phase chromatography (0−
100% MeCN in H2O, 0.1% formic acid modifier) to give 16 (46 mg,
15%) as a tan solid.
5-(3-Chlorophenyl)-1,3,4-oxadiazol-2(3H)-one (23f). Prepared by
General Method 3 from 3-chlorobenzohydrazide. Isolated as a white
solid (121 mg, 35%).
1H NMR (600 MHz, DMSO-d6) δ 12.93−12.45 (m, 1H), 7.77−
7.73 (m, 2H), 7.65−7.63 (m, 1H), 7.59−7.56 (m, 1H); 13C NMR
(151 MHz, DMSO-d6) δ 154.3, 152.6, 133.9, 131.3, 131.2, 126.0,
124.7, 123.9; LCMS m/z 195.0 [M − H]−.
1H NMR (700 MHz, DMSO-d6) δ 8.86 (d, J = 1.2 Hz, 1H), 7.99
(d, J = 1.2 Hz, 1H), 7.98−7.94 (m, 2H), 7.71−7.66 (m, 2H); 13C
NMR (176 MHz, DMSO-d6) δ 135.5, 134.6, 132.9, 129.9, 123.3,
121.8; LCMS m/z 180.0 [M + H]+.
5-(3-Fluorophenyl)-1,3,4-oxadiazol-2(3H)-one (23g). Prepared by
General Method 3 from 3-fluorobenzohydrazide. Isolated as a white
solid (179 mg, 77%).
4-(4-Chlorophenyl)-2H-1,2,3-triazole (17). Copper (I) iodide (11
mg, 0.05 mmol, 0.045 equiv) and 1-chloro-4-ethynylbenzene (150
mg, 1.1 mmol) were dissolved in anhydrous DMF (3 mL) and MeOH
(1.5 mL) under an atmosphere of N2. Azidotrimethylsilane (0.22 mL,
1.65 mmol, 1.5 equiv) was added dropwise at RT, and the reaction
was heated to 100 °C for 15 h. The cooled solution was diluted with
MeOH and EtOAc and then concentrated under reduced pressure.
The crude product was purified by column chromatography (0−10%
MeOH in CH2Cl2) to give 17 (55 mg, 28%) as a white solid.
1H NMR (700 MHz, DMSO-d6) δ 15.69−14.82 (m, 1H), 8.75−
8.18 (m, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H);
LCMS m/z 180.1 [M + H]+.
1H NMR (400 MHz, DMSO-d6) δ 12.72 (s, 1H), 7.67−7.54 (m,
3H), 7.47−7.39 (m, 1H); 13C NMR (176 MHz, DMSO-d6) δ 162.2
(d, J = 245.1 Hz), 154.3, 152.8 (d, J = 3.5 Hz), 131.6 (d, J = 8.4 Hz),
126.1 (d, J = 8.8 Hz), 121.5 (d, J = 2.9 Hz), 118.4 (d, J = 21.2 Hz),
112.0 (d, J = 24.4 Hz); LCMS m/z 179.1 [M − H]−.
5-(3-Methylphenyl)-1,3,4-oxadiazol-2(3H)-one (23h). Purchased
from Combi-Blocks, WZ-9533.
5-(3-Ethylphenyl)-1,3,4-oxadiazol-2(3H)-one (23i). Prepared by
General Methods 1.1, 2, and 3 from 3-ethylbenzoic acid. Isolated as
an off-white solid (186 mg, 59%).
1H NMR (600 MHz, DMSO-d6) δ 12.56 (s, 1H), 7.65−7.59 (m,
2H), 7.48−7.41 (m, 2H), 2.68 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz,
1-(4-Chlorophenyl)-1H-pyrazole (18). Purchased from Key
Organics, AS-5194.
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
3H); 13C NMR (151 MHz, DMSO-d6) δ 154.5, 153.9, 144.9, 131.0,
129.3, 124.4, 124.0, 122.7, 27.9, 15.4; LCMS m/z no parent mass ion
detected.
5-(4-tert-Butylphenyl)-1,3,4-oxadiazol-2(3H)-one (23t). Prepared
by General Method 3 from 4-tert-butylbenzohydrazide. Isolated as a
white solid (123 mg, 91%).
1H NMR (600 MHz, DMSO-d6) δ 12.52 (s, 1H), 7.75−7.68 (m,
2H), 7.59−7.52 (m, 2H), 1.30 (s, 9H); 13C NMR (151 MHz, DMSO-
d6) δ 154.5, 154.3, 153.9, 126.0, 125.1, 121.3, 34.8, 30.8; LCMS m/z
219.1 [M + H]+.
5-(3-Cyclopropylphenyl)-1,3,4-oxadiazol-2(3H)-one (23j). Pre-
pared by General Methods 1.2, 2 (8 equiv at 70 °C), and 3 from
3-cyclopropylbenzoic acid. Isolated as a white solid (160 mg, 30%).
1H NMR (600 MHz, DMSO) δ 12.56 (s, 1H), 7.58−7.52 (m, 1H),
7.48 (apparent t, J = 1.7 Hz, 1H), 7.41 (apparent t, J = 7.8 Hz, 1H),
7.29−7.24 (m, 1H), 2.03 (apparent tt, J = 8.4, 5.1 Hz, 1H), 1.05−0.95
(m, 2H), 0.77−0.68 (m, 2H); 13C NMR (176 MHz, DMSO) δ 154.5,
153.9, 145.0, 129.2, 128.3, 123.9, 122.3, 122.1, 14.9, 9.7; LCMS m/z
201.1 [M + H]+.
5-(4-(Difluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-one (23u).
Prepared by General Methods 1.2, 2 (8 equiv at 70 °C), and 3
from 4-(difluoromethyl)benzoic acid. Isolated as a white solid (165
mg, 54%).
1H NMR (600 MHz, DMSO-d6) δ 12.71 (s, 1H), 7.93 (d, J = 8.1
Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.13 (t, J = 55.6 Hz, 1H); 13C
NMR (176 MHz, DMSO-d6) δ 154.3, 153.1, 136.5 (t, J = 22.3 Hz),
126.6 (t, J = 6.1 Hz), 126.2, 125.7, 114.3 (t, J = 236.5 Hz); LCMS m/
z 211.1 [M − H]−.
5-(3-(Difluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-one (23k).
Prepared by General Methods 1.1, 2, and 3 from 3-(difluoromethyl)-
benzoic acid. Isolated as an off-white solid (91 mg, 15%).
1H NMR (600 MHz, DMSO-d6) δ 12.70 (s, 1H), 7.99−7.92 (m,
2H), 7.77 (d, J = 7.6 Hz, 1H), 7.71 (apparent t, J = 7.9 Hz, 1H), 7.15
(t, J = 55.6 Hz, 1H); 13C NMR (151 MHz, DMSO-d6) δ 154.4, 153.1,
135.1 (t, J = 22.4 Hz), 130.1, 128.4, 127.7, 124.7, 122.5, 114.3 (t, J =
236.2 Hz); LCMS m/z 211.1 [M − H]−.
5-(4-(Trifluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-one (23v).
Prepared by General Method 3 from 4-(trifluoromethyl)-
benzohydrazide. Isolated as a white solid (80 mg, 30%).
1H NMR (600 MHz, DMSO-d6) δ 12.92−12.59 (m, 1H), 8.00 (d,
J = 8.6 Hz, 2H), 7.91 (d, J = 8.6 Hz, 2H); 13C NMR (151 MHz,
DMSO-d6) δ 154.4, 152.7, 131.0 (q, J = 31.8 Hz), 127.8, 126.3 (q, J =
3.7 Hz), 126.1, 123.8 (q, J = 272.4 Hz); LCMS m/z 229.1 [M − H]−.
5-(4-Cyanophenyl)-1,3,4-oxadiazol-2(3H)-one (23w). Prepared
by General Method 3 from 4-cyanobenzohydrazide. Isolated as a
white solid (79 mg, 68%).
5-(3-(Trifluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-one (23l).
Purchased from Combi-Blocks, WZ-9561.
5-(3-Methoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (23m). Prepared
by General Method 3 from 3-methoxybenzohydrazide. Isolated as a
white solid (204 mg, 82%).
1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.46 (apparent t,
J = 8.0 Hz, 1H), 7.39−7.33 (m, 1H), 7.26 (dd, J = 2.5, 1.6 Hz, 1H),
7.14 (ddd, J = 8.3, 2.6, 1.0 Hz, 1H), 3.82 (s, 3H); 13C NMR (176
MHz, DMSO-d6) δ 159.5, 154.4, 153.6, 130.5, 125.2, 117.5, 117.5,
109.9, 55.3; LCMS m/z no mass ion detected.
1H NMR (600 MHz, DMSO-d6) δ 12.85 (s, 1H), 8.03−7.98 (m,
2H), 7.97−7.92 (m, 2H); 13C NMR (151 MHz, DMSO-d6) δ 154.3,
152.5, 133.2, 128.0, 125.9, 118.1, 113.4; LCMS m/z 186.0 [M − H]−.
5-(4-Methoxy)phenyl-1,3,4-oxadiazol-2(3H)-one (23x). Prepared
by General Method 3 from 4-methoxybenzohydrazide. Isolated as a
white solid (138 mg, 60%).
5-(3-(Dimethylamino)phenyl)-1,3,4-oxadiazol-2(3H)-one (23n).
Prepared by General Method 3 from 3-(dimethylamino)-
benzohydrazide. Isolated as a white solid (81 mg, 71%).
1H NMR (600 MHz, CDCl3) δ 9.07 (br s, 1H), 7.31 (apparent t, J
= 8.3 Hz, 1H), 7.19 (apparent dt, J = 7.7, 1.3 Hz, 1H), 7.14 (dd, J =
2.7, 1.3 Hz, 1H), 6.85 (dd, J = 8.3, 2.7 Hz, 1H), 3.00 (s, 6H); 13C
NMR (151 MHz, CDCl3) δ 156.2, 154.9, 150.7, 129.8, 124.5, 115.7,
113.9, 109.1, 40.6; LCMS m/z 206.1 [M + H]+.
1H NMR (600 MHz, DMSO-d6) δ 12.42 (s, 1H), 7.90−7.52 (m,
2H), 7.32−6.95 (m, 2H), 3.83 (s, 3H); 13C NMR (176 MHz, DMSO-
d6) δ 161.6, 154.5, 153.8, 127.1, 116.3, 114.7, 55.4; LCMS m/z 193.1
[M + H]+.
5-(4-Isoproxy)phenyl-1,3,4-oxadiazol-2(3H)-one (23y). Prepared
by General Method 3 from 4-isopropoxybenzohydrazide. Isolated as a
white solid (151 mg, 89%).
1H NMR (600 MHz, DMSO-d6) δ 12.41 (s, 1H), 7.74−7.61 (m,
2H), 7.12−6.97 (m, 2H), 4.71 (hept, J = 6.0 Hz, 1H), 1.29 (d, J = 6.0
Hz, 6H); 13C NMR (176 MHz, DMSO-d6) δ 159.9, 154.5, 153.9,
127.1, 116.0, 115.9, 69.6, 21.6; LCMS m/z 221.1 [M + H]+.
5-(4-Nitrophenyl)-1,3,4-oxadiazol-2(3H)-one (23aa). Purchased
from Key Organics, GC-0606.
5-(4-Fluorophenyl)-1,3,4-oxadiazol-2(3H)-one (23o). Prepared by
General Method 3 from 4-fluorobenzohydrazide. Isolated as a white
solid (32 mg, 10%).
1H NMR (600 MHz, DMSO-d6) δ 12.58 (s, 1H), 7.89−7.80 (m,
2H), 7.44−7.34 (m, 2H); 13C NMR (151 MHz, DMSO-d6) δ 163.8
(d, J = 249.5 Hz), 154.5, 153.1, 128.0 (d, J = 9.2 Hz), 120.7, 116.5 (d,
J = 22.3 Hz); LCMS m/z 179.1 [M − H]−.
5-(4-(Dimethylamino)phenyl)-1,3,4-oxadiazol-2(3H)-one (23bb).
Prepared by General Methods 2 (8 equiv at 70 °C) and 3 from ethyl
4-(dimethylamino)benzoate. Isolated as a white solid (45 mg, 8%).
1H NMR (600 MHz, DMSO-d6) δ 12.22 (s, 1H), 7.65−7.48 (m,
2H), 6.87−6.65 (m, 2H), 2.99 (s, 6H); 13C NMR (176 MHz, CDCl3)
δ 156.4, 154.7, 152.5, 127.3, 111.6, 110.7, 40.2; LCMS m/z 206.2 [M
+ H]+.
5-(4-Methylphenyl)-1,3,4-oxadiazol-2(3H)-one (23p). Purchased
from Key Organics, LE-0722.
5-(4-Ethylphenyl)-1,3,4-oxadiazol-2(3H)-one (23q). Prepared by
General Method 3 from 4-ethylbenzohydrazide. Isolated as a white
solid (191 mg, 82%).
1H NMR (600 MHz, DMSO-d6) δ 12.51 (s, 1H), 7.75−7.64 (m,
2H), 7.38 (d, J = 8.4 Hz, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6
Hz, 3H); 13C NMR (176 MHz, DMSO-d6) δ 154.5, 153.9, 147.6,
128.6, 125.3, 121.5, 28.1, 15.2; LCMS m/z 189.1 [M − H]−.
5-(4-Cyclopropylphenyl)-1,3,4-oxadiazol-2(3H)-one (23r). Pre-
pared by General Methods 1.3, 2 (8 equiv at 70 °C), and 3 from
4-cyclopropylbenzoic acid. Isolated as a white solid (193 mg, 44%).
1H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H), 7.68−7.59 (m,
2H), 7.26−7.17 (m, 2H), 2.04−1.94 (m, 1H), 1.08−0.96 (m, 2H),
0.80−0.69 (m, 2H); 13C NMR (151 MHz, DMSO-d6) δ 154.5, 153.9,
148.0, 126.0, 125.2, 120.9, 15.2, 10.2; LCMS m/z 203.1 [M + H]+.
5-(4-Isopropylphenyl)-1,3,4-oxadiazol-2(3H)-one (23s). Prepared
by General Methods 1.2, 2 (8 equiv at 80 °C), and 3 from 4-
isopropylbenzoic acid. Isolated as a white solid (190 mg, 33%).
1H NMR (600 MHz, DMSO-d6) δ 12.51 (s, 1H), 7.76−7.66 (m,
2H), 7.41 (d, J = 8.3 Hz, 2H), 2.95 (hept, J = 6.9 Hz, 1H), 1.22 (d, J =
6.9 Hz, 6H); 13C NMR (176 MHz, DMSO-d6) δ 154.5, 153.9, 152.1,
127.2, 125.3, 121.6, 33.4, 23.5; LCMS m/z 205.0 [M + H]+.
5-(3,4-Dichlorophenyl)-1,3,4-oxadiazol-2(3H)-one (23cc). Pre-
pared by General Method 3 from 3,4-dichlorobenzohydrazide.
Isolated as a white solid (80 mg, 24%).
1H NMR (600 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.97 (d, J = 2.0
Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.76 (dd, J = 8.4, 2.0 Hz, 1H); 13C
NMR (151 MHz, DMSO-d6) δ 154.3, 152.0, 134.1, 132.2, 131.7,
127.0, 125.4, 124.6; LCMS m/z 229.0 [M − H]−.
5-(4-Chloro-3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-
one (23dd). Prepared by General Methods 1.1, 2, and 3 from 4-
chloro-3-(trifluoromethyl)benzoic acid. Isolated as a white solid (229
mg, 40%).
Mp 158−159 °C; IR νmax (film) 3078, 2824, 1178, 1748, 1609,
1
1478, 1297 cm−1; H NMR (700 MHz, DMSO-d6) δ 12.82 (s, 1H),
8.09−8.04 (m, 2H), 7.91 (d, J = 8.2 Hz, 1H); 13C NMR (176 MHz,
DMSO-d6) δ 154.2, 152.0, 133.4, 132.9, 130.6, 127.5 (q, J = 31.4 Hz),
124.2 (q, J = 5.3 Hz), 123.7, 122.3 (q, J = 273.3 Hz); LCMS m/z
263.0 [M − H]−; HRMS C9H4ClF3N2O2: calcd. 263.9908 [M]+,
found 263.99085.
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
5-(4-Methyl-3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-
one (23ee). Prepared by General Methods 2 and 3 from methyl 4-
methyl-3-(trifluoromethyl)benzoate. Isolated as a white solid (45 mg,
16%).
1H NMR (700 MHz, DMSO-d6) δ 7.91 (dd, J = 8.6, 2.0 Hz, 1H),
7.88 (d, J = 2.0 Hz, 1H), 7.46 (d, J = 8.7 Hz, 1H), 2.80 (s, 6H); 13C
NMR (176 MHz, DMSO-d6) δ 155.1, 154.3, 152.8, 129.7, 124.6 (q, J
= 5.7 Hz), 123.9 (q, J = 273.1 Hz), 122.3, 121.2 (q, J = 29.6 Hz),
117.3, 44.4, 44.4; LCMS m/z 274.2 [M + H]+.
5-(1-Methylindolin-5-yl)-1,3,4-oxadiazol-2(3H)-one (23jj). Pre-
pared by General Methods 2 (8 equiv at 45 °C) and 3 from methyl
1-methylindoline-5-carboxylate.24 Isolated as an off-white solid (60
mg, 87%).
1H NMR (600 MHz, DMSO-d6) δ 12.69 (s, 1H), 8.00−7.94 (m,
2H), 7.64 (d, J = 8.1 Hz, 1H), 2.52 (s, 3H); LCMS m/z 243.1 [M −
H]−.
5-(4-Isopropyl-3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2(3H)-
one (23ff). 5-[4-Bromo-3-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-
2(3H)-one (32) was prepared by General Methods 2 and 3 from
methyl 4-bromo-3-(trifluoromethyl)benzoate. Isolated as a white solid
(429 mg, 79%).
1H NMR (600 MHz, DMSO-d6) δ 12.19 (s, 1H), 7.44 (dd, J = 8.2,
1.7 Hz, 1H), 7.40 (d, J = 1.3 Hz, 1H), 6.53 (d, J = 8.3 Hz, 1H), 3.41
(t, J = 8.4 Hz, 2H), 2.95 (t, J = 8.4 Hz, 2H), 2.78 (s, 3H); 13C NMR
(176 MHz, DMSO-d6) δ 155.5, 154.8, 154.6, 130.6, 125.9, 120.9,
111.8, 105.7, 54.7, 34.4, 27.4; LCMS m/z 218.1 [M + H]+.
5-(1-Methylindolin-6-yl)-1,3,4-oxadiazol-2(3H)-one (23kk). Pre-
pared by General Methods 2 (15 equiv at 100 °C) and 3.2 from
methyl 1-methylindoline-6-carboxylate.24 Isolated as a white solid (63
mg, 56%).
1H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 8.08 (d, J = 8.4
Hz, 1H), 8.05 (d, J = 2.0 Hz, 1H), 7.95 (dd, J = 8.4, 2.0 Hz, 1H);
LCMS m/z 306.9 [M − H]−.
•
A thick-walled reaction vial was charged with Pd(dppf)Cl2 DCM
(26 mg, 0.03 mmol, 0.05 equiv), 2-isopropenyl-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (0.12 mL, 0.64 mmol, 1.2 equiv), and 32 (164
mg, 0.53 mmol). 1,4-Dioxane (5 mL) and an aqueous solution of
K2CO3 (0.56 mL, 2 N, 1.11 mmol) were added, the vial was purged
with Ar, and then heated at 100 οC for 16 h. The solvents were
evaporated, and the residue was dissolved in a minimum volume of
DMSO. Purification by chromatography (0−100% MeCN in H20,
0.1% NH4OH modifier) gave 5-[4-isopropenyl-3-(trifluoromethyl)-
phenyl]-1,3,4-oxadiazol-2(3H)-one (33) (84 mg, 59%) as a white
solid.
1H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 7.16 (d, J = 7.6
Hz, 1H), 7.02 (dd, J = 7.5, 1.5 Hz, 1H), 6.79 (d, J = 1.3 Hz, 1H),
3.35−3.30 (m, 2H), 2.93 (t, J = 8.3 Hz, 2H), 2.75 (s, 3H); 13C NMR
(176 MHz, DMSO-d6) δ 154.5, 154.5, 153.7, 134.2, 124.5, 123.0,
114.9, 102.3, 55.2, 35.2, 28.0; LCMS m/z 218.1 [M + H]+.
5-(1,4-Dimethyl-1,2,3,4-tetrahydroquinoxalin-6-yl)-1,3,4-oxadia-
zol-2(3H)-one (23ll). Prepared by General Methods 2 (15 equiv at
120 °C) and 3 from methyl 1,4-dimethyl-2,3-dihydroquinoxaline-6-
carboxylate.24 Isolated as a white solid (79 mg, 72%).
1H NMR (700 MHz, DMSO-d6) δ 7.99−7.92 (m, 2H), 7.54−7.50
(m, 1H), 5.33−5.28 (m, 1H), 4.88 (br s, 1H), 2.05 (s, 3H); LCMS
m/z 269.2 [M − H]−.
1H NMR (600 MHz, DMSO-d6) δ 12.18 (s, 1H), 7.02 (dd, J = 8.3,
2.0 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H), 6.50 (d, J = 8.4 Hz, 1H), 3.40
(dd, J = 5.7, 4.1 Hz, 2H), 3.26 (dd, J = 5.7, 4.1 Hz, 2H), 2.88 (s, 3H),
2.82 (s, 3H); 13C NMR (176 MHz, DMSO-d6) δ 155.0, 154.7, 139.3,
136.2, 116.4, 111.8, 109.0, 105.7, 48.9, 48.3, 38.7, 38.4; LCMS m/z
247.2 [M + H]+.
Hydrogenation of 33 to 23ff was performed in a COware gas
reactor. To chamber 1 were added 33 (80 mg, 0.30 mmol), 10% Pd/
C (32 mg, 0.03 mmol), and EtOH (4 mL). To chamber 2 were added
flakes of zinc (270 mg, 4.14 mmol). The H-Cap was secured tightly
on top of chamber 1, and the system was purged with N2. Aqueous
HCl (1.18 mL, 4 M, 4.74 mmol) was added to chamber 2, the system
was securely sealed, and the two chambers were stirred for 18 h. The
residual pressure was carefully released, and the reaction mixture from
chamber 1 was filtered and concentrated under reduced pressure. The
residue was purified by chromatography (0−100% MeCN in H20,
0.1% NH4OH modifier) to give 23ff (64 mg, 79%) as a white solid.
1H NMR (700 MHz, DMSO-d6) δ 12.65 (s, 1H), 8.01 (dd, J = 8.3,
1.7 Hz, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 3.25
(dt, J = 13.3, 6.7 Hz, 1H), 1.25 (d, J = 6.7 Hz, 6H); 13C NMR (176
MHz, DMSO-d6) δ 154.6, 152.7, 150.6, 129.3, 129.1, 126.8 (q, J =
29.6 Hz), 124.0 (q, J = 274.1 Hz), 122.3, 121.9 (q, J = 6.1 Hz), 29.2,
23.6; LCMS m/z 271.2 [M − H]−.
5-(4-Chlorophenyl)isoxazol-3(2H)-one (24). NH2OH.HCl (0.15
mL, 3.6 mmol, 3.0 equiv) was added to a solution of ethyl 3-(4-
chlorophenyl)propiolate (250 mg, 1.2 mmol) and NaOH (48 mg, 1.2
mmol, 1.0 equiv) in EtOH (4 mL) and H2O (4 mL) at RT. The
reaction mixture was stirred for 16 h and then acidified to pH 2 with
aqueous HCl (2 N). The EtOH was removed under reduced pressure,
and the aqueous residue was extracted with EtOAc (3 × 20 mL). The
combined organic extracts were dried (Na2SO4) and evaporated
under reduced pressure. The residue was purified by silica gel
chromatography (0−10% MeOH in CH2Cl2) to give 24 (12 mg, 5%)
as an off-white solid.
1H NMR (600 MHz, DMSO-d6) δ 11.46 (s, 1H), 7.92−7.74 (m,
2H), 7.65−7.47 (m, 2H), 6.62 (s, 1H); LCMS m/z 196.1 [M + H]+.
5-(4-Chlorophenyl)-1,3,4-thiadiazol-2(3H)-one (25). Prepared by
General Method 4 from 4-chlorobenzohydrazide. Isolated as a white
solid (25 mg, 13%).
5-(4-Cyclopropyl-3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-
2(3H)-one (23gg). Prepared by General Methods 1.2, 2 (15 equiv at
100 °C), and 3 from 4-cyclopropyl-3-(trifluoromethyl)benzoic acid.24
Isolated as a white solid (68 mg, 61%).
1H NMR (700 MHz, DMSO-d6) δ 13.19 (s, 1H), 7.75−7.66 (m,
2H), 7.60−7.54 (m, 2H); 13C NMR (176 MHz, DMSO-d6) δ 171.1,
150.3, 135.3, 129.4, 129.3, 127.4; LCMS m/z 211.1 [M − H]−.
5-(4-Chloro-3-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2(3H)-
one (26). Prepared by General Method 4 from 4-chloro-3-
(trifluoromethyl)benzohydrazide. Isolated as a white solid (10 mg,
8%).
1H NMR (600 MHz, DMSO-d6) δ 12.68 (s, 1H), 7.92 (m, 2H),
7.31 (d, J = 8.2 Hz, 1H), 2.16 (m, 1H), 1.17−1.10 (m, 2H), 0.93−
0.86 (m, 2H); 13C NMR (176 MHz, DMSO-d6) δ 154.3, 152.7,
145.7, 129.2, 128.5 (q, J = 30.1 Hz), 126.7, 124.1 (q, J = 274.1 Hz),
122.0 (q, J = 6.0 Hz), 121.5. 12.1 (d, J = 2.3 Hz), 10.1; LCMS m/z
269.1 [M − H]−.
5-(3-Cyclopropyl-4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-
2(3H)-one (23hh). Prepared by General Methods 1.2, 2 (8 equiv at 70
°C), and 3 from 3-cyclopropyl-4-(trifluoromethyl)benzoic acid.24
Isolated as a white solid (88 mg, 51%).
1H NMR (600 MHz, CDCl3) δ 9.50 (s, 1H), 8.01 (d, J = 2.1 Hz,
1H), 7.76 (dd, J = 8.4, 2.1 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H); LCMS
m/z 279.0 [M − H]−.
1H NMR (600 MHz, DMSO-d6) δ 12.78 (s, 1H), 7.84 (d, J = 8.3
Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.43 (s, 1H), 2.22−2.11 (m, 1H),
1.16−1.04 (m, 2H), 0.92−0.79 (m, 2H); 13C NMR (176 MHz,
DMSO-d6) δ 154.3, 152.7, 143.1, 130.0 (q, J = 29.4 Hz), 127.9, 126.8
(q, J = 5.7 Hz), 124.2 (q, J = 273.9 Hz), 122.7, 122.3, 12.0 (d, J = 2.4
Hz), 9.2; LCMS m/z 269.2 [M − H]−.
5-(4-Chloro-3-(trifluoromethyl)phenyl)-1,3,4-oxadiazole (27). p-
TsOH·H2O (28 mg, 0.15 mmol 0.3 equiv) was added to a thick-
walled reaction vial (20 mL) containing HC(OEt)3 (1.0 mL, 6.5
mmol, 15 equiv) and 4-chloro-3-(trifluoromethyl)benzohydrazide
(100 mg, 0.42 mmol) under N2. The vial was sealed, slowly warmed
to 50 oC, and then stirred for 30 min (vigorous bubbling was
observed). The mixture was cooled to RT and added dropwise to a
beaker containing a stirred solution of saturated aqueous NaHCO3
(20 mL). The aqueous mixture was extracted with CH2Cl2 (50 mL),
and the organic phase was separated, dried, and concentrated under
5-(4-(Dimethylamino)-3-(trifluoromethyl)phenyl)-1,3,4-oxadia-
zol-2(3H)-one (23ii). Prepared by General Methods 2 (4 equiv at 80
°C) and 3 from methyl 4-(dimethylamino)-3-(trifluoromethyl)-
benzoate.24 Isolated as a white solid (212 mg, 44%).
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
reduced pressure. The residue was purified by chromatography on
silica gel (30% EtOAc in cyclohexane) to give 27 (66 mg, 63%).
1H NMR (600 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.34 (d, J = 2.0
Hz, 1H), 8.31 (dd, J = 8.3, 2.0 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H);
LCMS m/z 249.1 [M + H]+.
permeability, and metabolic stability in liver microsomes as a measure
of clearance. Assay protocols and additional data are presented in
Figure S3. ADME studies reported in this work were independently
performed by GVK Biosciences (Hyderabad, India) or Cyprotex
(Macclesfield, U.K.).
pKa Determination. The pKas were determined using the
spectrometric (UV-metric) technique and were performed by
Cyprotex (Macclesfield, U.K.).
Compound 23aa. The sample was titrated in a UV-metric triple
titration from pH 2.0 to 12.0 at concentrations of 31−19 μM, under
methanol−water cosolvent conditions (the methanol mixing ratio
varied from 48.9 to 29.6% w/w). No precipitation of the sample from
solution was observed. One pKa, with an aqueous value of 6.44
0.08, was determined from the spectroscopic data collected by the
Yasuda−Shedlovsky extrapolation of the individual results obtained.
Compound 23bb. The sample was titrated in three UV-metric
single titrations from pH 1.5 to 12.5 at concentrations of 29−23 μM,
under methanol−water cosolvent conditions (the methanol mixing
ratio varied from 47.9 to 30.7% w/w). No precipitation of the sample
from solution was observed. Two pKas, with aqueous values of 2.92
0.06 and 7.42 0.01, were determined from the spectroscopic data
collected by the Yasuda−Shedlovsky extrapolation of the individual
results obtained.
Notum Protein Expression and Purification. Methods have
been described in detail in ref 18. In brief, human Notum (UniProtKB
ID: Q6P988) enzyme core sequence comprising amino acids S81−
T451 with a C330S mutation was cloned into a stable cell line vector
pNeo_sec. A stable HEK293S GNTI- cell line was obtained and used
for protein production as described previously. The cells were
expanded and grown in roller bottles (Greiner). The conditioned
media were dialyzed and passed through a 5 mL HisTrap Excel
column (GE Healthcare), followed by 20 mM imidazole PBS wash.
Notum protein was eluted with 300 mM imidazole PBS. To remove
flexible glycans, the protein was deglycosylated with endo-β-N-
acetylglucosaminidase F1 (37 °C, 1 h) and further purified by size-
exclusion chromatography (Superdex 200 16/60 column, GE
Healthcare) in 10 mM Hepes, pH 7.4, 150 mM NaCl buffer.
Notum OPTS Biochemical Assay. Methods have been described
in detail in ref 18. A Labcyte Echo 550 acoustic liquid handler was
used to dispense 500 nL of test compound into 384-well plates
(Greiner catalogue #781076). Twenty-five microliters of 2 μM
trisodium 8-octanoyloxypyrene-1,3,6-trisulfonate (OPTS, Sigma
#74875) solution in 50 mM Tris, 5 mM CaCl2, 0.5 mM MgCl2,
pH 7.4 assay buffer was added to each well, followed by 25 μL of 2.38
nM Notum carboxylesterase enzyme solution. Plates were incubated
for 40 min at room temperature, and endpoint fluorescence was
measured on a PheraSTAR FSX microplate reader with an excitation
wavelength of 485 nm and an emission wavelength of 520 nm. Plates
were quality-controlled based on a signal window greater than 3-fold
between DMSO and positive control compound, and a Z prime
greater than 0.5.
Compound 23dd. The sample was titrated in a UV-metric triple
titration from pH 2.0 to 12.0 at concentrations of 32−23 μM, under
aqueous conditions. No precipitation of the sample from solution was
observed. One pKa, with an average value of 6.72
0.01, was
determined from the spectroscopic data collected.
Mouse Pharmacokinetic Studies. In vivo mouse pharmacoki-
netic data was generated at GVK Biosciences (Hyderabad, India).
Assay protocols and additional data are presented in Figure S4.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01391.
■
Dose−Response Curve Analysis. Plate layout and measured
fluorescence values were input into Dotmatics Studies data manage-
ment tool. After implementing Quality Control with a cutoff of Z
prime values higher than 0.5, compound IC50 values were calculated
from curves using a 4PL fit and compared between technical
replicates. The final potency values implied an n ≥ 2 with IC50 within
0.5−2× between replicates. For representative curves, see Figure S6.
Data Analysis. IC50 values presented in Tables 1−4 are mean
SD (n = 2−8).
sı
X-ray structure determination: methods, data collection,
and refinement statistics; plot of Notum pIC50 vs clogP
and lipophilic fragment constant (π); Notum occupancy
assay with FP-biotin; ADME protocols and additional
data; mouse pharmacokinetic data for 23dd; analytical
and spectral data for 23dd; Notum OPTS and TCF/
LEF screening data for 23dd; and comparison of profile
of 23dd with 2 (PDF)
TCF/LEF Reporter (Luciferase) Cell-Based Assay. Methods
have been described in detail in ref 16. An aliquot of 20 μL of TCF-
LEF cells was plated in 384-well cell culture Greiner microplates
(catalogue #781098) at 10 000 cells per well and incubated overnight
o
at 37 C. An Echo liquid handler was used to acoustically dispense
Molecular formula strings (CSV)
150 nL of compounds into a V-well polypropylene Greiner microplate
(catalogue #781280) to create a 10-point curve. Twenty-five
microliters of 0.5% DMEM media (500 mL DMEM 1× GlutaMax,
4.5 g/L D-glucose, Pyruvate plus 2.5 mL of 10% fetal bovine serum
and 5 mL penicillin−streptomycin) was added to the Wnt compound
plates, and 25 μL of Notum at 1000 μg/mL was added to the Notum
compound plates and incubated at room temperature for 10 min.
Twenty-five microliters of Wnt-3A at 200 ng/mL was added to all
plates and incubated at room temperature for 1 h. Twenty microliters
of media was aspirated from the cell plates using a CyBio SELMA
followed by the addition of 40 μL from the compound plates. After an
Accession Codes
Coordinates for X-ray structures of Notum crystallized with 7
(6ZUV) and 23dd (6ZVL) have been deposited in the Protein
Data Bank.
AUTHOR INFORMATION
Corresponding Authors
■
E. Yvonne Jones − Division of Structural Biology, Wellcome
Centre for Human Genetics, University of Oxford, Oxford OX3
7BN, U.K.; Phone: +44 (0)1865 287546; Email: yvonne@
strubi.ox.ac.uk
o
overnight incubation at 37 C, GFP fluorescence was measured on a
PHERAstar FSX microplate reader with a wavelength of 458 nm and
an emission wavelength of 520 nm. Twenty microliters of the steady-
glo luciferase assay buffer (Promega #E2520) was added to the cell
plates using a CyBio, and the luminescence was measured on the
PHERAstar.
Paul V. Fish − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT, U.K.;
The Francis Crick Institute, London NW1 1AT, U.K.;
orcid.org/0000-0002-2117-2173; Phone: +44 (0) 20
7679 6971; Email: p.fish@ucl.ac.uk
Molecular Docking. Compound design was aided by molecular
̈
docking. Docking was conducted using Schrodinger Glide.
̈
̈
Schrodinger Release 2018-3: Glide, Schrodinger, LLC, New York,
NY, 2018.
Authors
William Mahy − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT, U.K.
In Vitro ADME Screens. Selected compounds were screened for
aqueous solubility, transit performance in MDCK-MDR1 cell lines for
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Notes
Nicky J. Willis − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT, U.K.
Yuguang Zhao − Division of Structural Biology, Wellcome
Centre for Human Genetics, University of Oxford, Oxford OX3
7BN, U.K.; orcid.org/0000-0001-8916-8552
Hannah L. Woodward − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London WC1E
6BT, U.K.; orcid.org/0000-0001-8429-2546
Fredrik Svensson − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London WC1E
6BT, U.K.; The Francis Crick Institute, London NW1 1AT,
U.K.; orcid.org/0000-0002-5556-8133
James Sipthorp − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London WC1E
6BT, U.K.; The Francis Crick Institute, London NW1 1AT,
U.K.
Luca Vecchia − Division of Structural Biology, Wellcome Centre
for Human Genetics, University of Oxford, Oxford OX3 7BN,
U.K.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our colleagues Stefano Benvegnu, Jamie Bilsland,
Sarah Jolly, Ernest Palomer, Morgan Roberts, and Laura
Schuhmacher for their support and advice of our Notum
Consortium. The Cell Services and Structural Biology Science
Technology Platforms (STPs) at The Francis Crick Institute
are gratefully acknowledged for their provision and purification
of recombinant Notum. We thank the staff at Diamond Light
Source for assistance with X-ray data collection and the UCL
Department of Chemistry for spectroscopic and analytical
services. ADME studies reported in this work were
independently performed by GVK Biosciences (Hyderabad,
India) or Cyprotex (Macclesfield, U.K.). Mouse PK studies
were performed by GVK Biosciences (Hyderabad, India).
ABBREVIATIONS
■
Reinis R. Ruza − Division of Structural Biology, Wellcome
Centre for Human Genetics, University of Oxford, Oxford OX3
7BN, U.K.
James Hillier − Division of Structural Biology, Wellcome Centre
for Human Genetics, University of Oxford, Oxford OX3 7BN,
U.K.
ABC transporters, ATP-binding cassette transporters; ABPP,
activity-based protein profiling; AD, Alzheimer’s disease;
ADME, absorption distribution metabolism elimination;
BBB, blood−brain barrier; BCRP, breast cancer resistance
protein; CNS, central nervous system; DMF, N,N-dimethyl-
formamide; DMSO, dimethylsulfoxide; ER, efflux ratio; HLM,
human liver microsomes; HTS, high-throughput screen; LE,
ligand efficiency; LLE, lipophilic ligand efficiency; MLM,
mouse liver microsomes; MPO, multiparameter optimization;
MRP, multidrug resistance-associated protein; OPTS, triso-
dium 8-octanoyloxypyrene-1,3,6-trisulfonate; PDB, Protein
Data Bank; P-gp, P-glycoprotein; PD, pharmacodynamics;
PK, pharmacokinetic; RT, room temperature; SAR, structure−
activity relationship; SBDD, structure-based drug design; THF,
tetrahydrofuran; TPSA, topological polar surface area
Svend Kjær − The Francis Crick Institute, London NW1 1AT,
U.K.
Sarah Frew − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT, U.K.
Amy Monaghan − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London WC1E
6BT, U.K.
Magda Bictash − Alzheimer’s Research UK UCL Drug
Discovery Institute, University College London, London WC1E
6BT, U.K.
Patricia C. Salinas − Department of Cell and Developmental
Biology, Laboratory for Molecular and Cellular Biology,
University College London, London WC1E 6BT, U.K.
Paul Whiting − Alzheimer’s Research UK UCL Drug Discovery
Institute, University College London, London WC1E 6BT, U.K.
Jean-Paul Vincent − The Francis Crick Institute, London NW1
1AT, U.K.
REFERENCES
■
(1) Clevers, H.; Nusse, R. Wnt/β-catenin signaling and disease. Cell
2012, 149, 1192−1205.
(2) Krishnamurthy, N.; Kurzrock, R. Targeting the Wnt/beta-
catenin pathway in cancer: Update on effectors and inhibitors. Cancer
Treat. Rev. 2018, 62, 50−60.
(3) Baron, R.; Gori, F. Targeting WNT signaling in the treatment of
osteoporosis. Curr. Opin. Pharmacol. 2018, 40, 134−141.
(4) Zhong, Z.; Virshup, D. M. Wnt signalling and drug resistance in
cancer. Mol. Pharmacol. 2020, 97, 72−89.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01391
(5) Kakugawa, S.; Langton, P. F.; Zebisch, M.; Howell, S.; Chang, T.
H.; Liu, Y.; Feizi, T.; Bineva, G.; O’Reilly, N.; Snijders, A. P.; Jones, E.
Y.; Vincent, J. P. NOTUM deacylates Wnt proteins to suppress
signaling activity. Nature 2015, 519, 187−192.
Author Contributions
⊥W.M., N.J.W., and Y.Z. contributed equally.
Funding
́
(6) Moverare-Skrtic, S.; Nilsson, K. H.; Henning, P.; Funck-
This work was supported by Alzheimer’s Research U.K.
(ARUK) and The Francis Crick Institute. The ARUK UCL
Drug Discovery Institute is core-funded by Alzheimer’s
Research U.K. (520909). The Francis Crick Institute receives
its core funding from Cancer Research U.K. (FC001002), the
U.K. Medical Research Council (FC001002), and the
Wellcome Trust (FC001002). Structural analysis was
performed by Y.Z., L.V., R.R.R., and E.Y.J. supported by
Cancer Research U.K. (Programme Grant C375/A17721).
The Wellcome Trust funds the Wellcome Centre for Human
Genetics, University of Oxford (Centre Grant 203141/Z/16/
Z). P.C.S. and J.-P.V. were supported by Alzheimer’s Research
U.K. (Grant ARUK-IRG2016A-7)
Brentano, T.; Nethander, M.; Rivadeneira, F.; Coletto Nunes, G.;
Koskela, A.; Tuukkanen, J.; Tuckermann, J.; Perret, C.; Souza, P. P.
C.; Lerner, U. H.; Ohlsson, C. Osteoblast-derived NOTUM reduces
cortical bone mass in mice and the NOTUM locus is associated with
bone mineral density in humans. FASEB J. 2019, 33, 11163−11179.
(7) Brommage, R.; Liu, J.; Vogel, P.; Mseeh, F.; Thompson, A. Y.;
Potter, D. G.; Shadoan, M. K.; Hansen, G. M.; Jeter-Jones, S.; Cui, J.;
Bright, D.; Bardenhagen, J. P.; Doree, D. D.; Moverare-Skrtic, S.;
Nilsson, K. H.; Henning, P.; Lerner, U. H.; Ohlsson, C.; Sands, A. T.;
Tarver, J. E.; Powell, D. R.; Zambrowicz, B.; Liu, Q. NOTUM
inhibition increases endocortical bone formation and bone strength.
Bone Res. 2019, 7, No. 2.
(8) Pentinmikko, N.; Iqbal, S.; Mana, M.; Andersson, S.; Cognetta,
̈
A. B., III; Suciu, R. M.; Roper, J.; Luopajarvi, K.; Markelin, E.;
Gopalakrishnan, S.; Smolander, O.-P.; Naranjo, S.; Saarinen, T.; Juuti,
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
̈
̈
A.; Pietilainen, K.; Auvinen, P.; Ristimaki, A.; Gupta, N.; Tammela,
T.; Jacks, T.; Sabatini, D. M.; Cravatt, B. F.; Yilmaz, O. H.; Katajisto,
P. Notum produced by Paneth cells attenuates regeneration of aged
intestinal epithelium. Nature 2019, 571, 398−402.
System and Other Disorders. WO Patent WO2020043866, Mar 5,
2020.
(25) For recent reviews, see: (a) Verma, G.; Khan, M. F.; Akhtar,
W.; Alam, M. M.; Akhtar, M.; Shaquiquzzaman, M. A review exploring
therapeutic worth of 1,3,4-oxadiazole tailored compounds. Mini Rev.
Med. Chem. 2019, 19, 477−509. (b) Sun, J.; Makawana, J. A.; Zhu, H.
L. 1,3,4-Oxadiazole derivatives as potential biological agents. Mini Rev.
Med. Chem. 2013, 13, 1725−1743.
(26) Acidic pKa values for CNS drugs rarely fall below 6.0, see:
(a) Manallack, D. T. The pKa distribution of drugs: application to
drug discovery. Perspect. Med. Chem. 2007, 1, 25−38. (b) Manallack,
D. T. The acid-base profile of a contemporary set of drugs:
implications for drug discovery. SAR QSAR Environ. Res. 2009, 20,
611−655.
(9) McLeod, F.; Salinas, P. C. Wnt proteins as modulators of
synaptic plasticity. Curr. Opin. Neurobiol. 2018, 53, 90−95.
(10) Palomer, E.; Buechler, J.; Salinas, P. C. Wnt signaling
deregulation in the aging and Alzheimer’s brain. Front. Cell. Neurosci.
2019, 13, No. 227.
(11) Mizrak, D.; Bayin, N. S.; Yuan, J.; Liu, Z.; Suciu, R. M.;
Niphakis, M. J.; Ngo, N.; Lum, K. M.; Cravatt, B. F.; Joyner, A. L.;
Sims, P. A. Single-cell profiling and SCOPE-Seq reveal lineage
dynamics of adult ventricular-subventricular zone neurogenesis and
NOTUM as a key regulator. Cell Rep. 2020, 31, No. 107805.
(12) Beckervordersandforth, R.; Rolando, C. Untangling human
neurogenesis to understand and counteract brain disorders. Curr.
Opin. Pharmacol. 2020, 50, 67−73.
(13) Tarver, J. E., Jr.; Pabba, P. K.; Barbosa, J.; Han, Q.; Gardyan, M.
W.; Brommage, R.; Thompson, A. Y.; Schmidt, J. M.; Wilson, A. G.
E.; He, W.; Lombardo, V. K.; Carson, K. G. Stimulation of cortical
bone formation with thienopyrimidine based inhibitors of NOTUM
pectinacetylesterase. Bioorg. Med. Chem. Lett. 2016, 26, 1525−1528.
(14) Atkinson, B. N.; Steadman, D.; Mahy, W.; Zhao, Y.; Sipthorp,
J.; Bayle, E. D.; Svensson, F.; Papageorgiou, G.; Jeganathan, F.; Frew,
S.; Monaghan, A.; Bictash, M.; Jones, E. Y.; Fish, P. V. Scaffold-
hopping identifies furano[2,3-d]pyrimidine amides as potent Notum
inhibitors. Bioorg. Med. Chem. Lett. 2020, 30, No. 126751.
(15) Suciu, R. M.; Cognetta, A. B.; Potter, Z. E.; Cravatt, B. F.
Selective irreversible inhibitors of the Wnt-deacylating enzyme
NOTUM developed by activity-based protein profiling. ACS Med.
Chem. Lett. 2018, 9, 563−568.
(27) There is a paucity of acids with pKa values between 6 and 7,
probably due to the dominance of carboxylic acids and phenols, and
the pKa values usually encountered for these groups, see: Manallack,
D. T.; Prankerd, R. J.; Yuriev, E.; Opera, T. I.; Chalmers, D. K. The
significance of acid/base properties in drug discovery. Chem Soc. Rev.
2013, 42, 485−496.
(28) 5-Phenyl-1,3,4-oxadiazole-2(3H)-one [1199-02-6]: predicted
pKa 5.8
0.7 (most acidic) calculated using ACD/Labs software
̈
v11.02; predicted pKa 7.1 (most acidic) calculated using Schrodinger
Release 2019-3: Jaguar pKa, Schrodinger, LLC, New York, NY, 2019.
̈
The Jaguar program calculated preference for the 1,3,4-oxadiazol-
2(3H)-one tautomer by 20.2 kcal/mol (gas phase by DFT
LACVP**B3LPY-D3).
(29) Rankovic, Z. CNS drug design: balancing physicochemical
properties for optimal brain exposure. J. Med. Chem. 2015, 58, 2584−
2608.
(30) Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Moving
beyond rules: the development of a central nervous system
multiparameter optimization (CNS MPO) approach to enable
alignment of druglike properties. ACS Chem. Neurosci. 2010, 1,
435−449.
(31) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein
profiling: The serine hydrolases. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
14694−14699.
(32) Qosa, H.; Miller, D. S.; Pasinelli, P.; Trotti, D. Regulation of
ABC efflux transporters at blood-brain barrier in health and
neurological disorders. Brain Res. 2015, 1628, 298−316.
(16) Atkinson, B. N.; Steadman, D.; Zhao, Y.; Sipthorp, J.; Vecchia,
L.; Ruza, R. R.; Jeganathan, F.; Lines, G.; Frew, S.; Monaghan, A.;
Kjaer, S.; Bictash, M.; Jones, E. Y.; Fish, P. V. Discovery of 2-
phenoxyacetamides as inhibitors of the Wnt-depalmitoleating enzyme
NOTUM from an X-ray fragment screen. Med. Chem. Commun. 2019,
10, 1361−1369.
(17) Zhao, Y.; Ren, J.; Hillier, J.; Jones, M.; Lu, W.; Jones, E. Y.
Structural characterisation of melatonin as an inhibitor of the Wnt
deacylase Notum. J. Pineal Res. 2020, 68, No. e12630.
(18) Mahy, W.; Patel, M.; Steadman, D.; Woodward, H. L.;
Atkinson, B. N.; Svensson, F.; Willis, N. J.; Flint, A.; Papatheodorou,
D.; Zhao, Y.; Vecchia, L.; Ruza, R. R.; Hillier, J.; Frew, S.; Monaghan,
A.; Costa, A.; Bictash, M.; Walter, M. W.; Jones, E. Y.; Fish, P. V.
Screening of a custom-designed acid fragment library identifies 1-
phenylpyrroles and 1-phenylpyrrolidines as inhibitors of Notum
carboxylesterase activity. J. Med. Chem. 2020, 63, 9464−9483.
(19) Zhao, Y.; Ren, J.; Hillier, J.; Lu, W.; Jones, E. Y. Caffeine
inhibits Notum activity by binding at the catalytic pocket. Commun.
Biol. 2020, 3, No. 555.
(20) Willis, N. J.; Bayle, E. D.; Papageorgiou, G.; Steadman, D.;
Atkinson, B. N.; Mahy, W.; Fish, P. V. An improved, scalable synthesis
of Notum inhibitor LP-922056 using 1-chloro-1,2-benziodoxol-3-one
as a superior electrophilic chlorinating agent. Beilstein J. Org. Chem.
2019, 15, 2790−2797.
(21) https://www.diamond.ac.uk/Instruments/Mx/Fragment-
Screening/Fragment-Libraries/DSI-poised-library.html (accessed
May 1, 2020).
(22) Hopkins, A. L.; Keseru, G. M.; Leeson, P. D.; Rees, D. C.;
Reynolds, C. H. The role of ligand efficiency metrics in drug
discovery. Nat. Rev. Drug Discovery 2014, 13, 105−121.
(23) Johnson, T. W.; Gallego, R. A.; Edwards, M. P. Lipophilic
efficiency as an important metric in drug design. J. Med. Chem. 2018,
61, 6401−6420.
(24) Fish, P. V.; Mahy, W.; Willis, N. J.; Woodward, H.; Atkinson, B.
N.; Bayle, E. D.; Sipthorp, J.; Jones, E. Y.; Zhao, Y.; Vecchia, L.; Ruza,
R. R. Inhibitors of Secreted Carboxy Esterase Notum and their use in
Increasing Wnt Protein Signalling and Treatment of Central Nervous
O
https://dx.doi.org/10.1021/acs.jmedchem.0c01391
J. Med. Chem. XXXX, XXX, XXX−XXX