Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126751

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20–24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.

Full text of DOI:10.1016/j.bmcl.2019.126751

Journal Pre-proofs
Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum
inhibitors
Benjamin N. Atkinson, David Steadman, William Mahy, Yuguang Zhao, James
Sipthorp, Elliott D. Bayle, Fredrik Svensson, George Papageorgiou, Fiona
Jeganathan, Sarah Frew, Amy Monaghan, Magda Bictash, E. Yvonne Jones,
Paul V. Fish
PII:
S0960-894X(19)30714-0
DOI:
https://doi.org/10.1016/j.bmcl.2019.126751
BMCL 126751
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 September 2019
9 October 2019
10 October 2019
Please cite this article as: 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., Yvonne Jones, E., Fish, P.V., Scaffold-
hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126751
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Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors
Benjamin N. Atkinson,a David Steadman,a William Mahy,a Yuguang Zhao,b James Sipthorp,a,c Elliott D. Bayle,a,c
Fredrik Svensson,a,c George Papageorgiou,c Fiona Jeganathan,a Sarah Frew,a Amy Monaghan,a Magda Bictash,a
E. Yvonne Jones,b Paul V. Fish.a,c,
*
a Alzheimer’s Research UK UCL Drug Discovery Institute, University College London, Cruciform Building,
Gower Street, London, WC1E 6BT, U.K.
b 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.
c
The Francis Crick Institute, 1 Midland Road, Kings Cross, London NW1 1AT, U.K.
*Corresponding author: Phone: +44 (0)20 7679 6971; E-mail: p.fish@ucl.ac.uk; ORCiD 0000-0002-2117-2173.
Keywords: Notum inhibitor, Wnt signaling, CNS penetration, furano[2,3-d]pyrimidines, SBDD.
1

Abstract
The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the
depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum
suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from
thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-
one amides 20-24 as potent inhibitors of Notum with activity across three orthogonal assay formats
(biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse
microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated
efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24
showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool
suitable for cellular studies to explore the fundamental biology of Notum.
2

The Wnt signaling pathway regulates several aspects of brain development and function, and dysregulation of
Wnt signaling has been implicated to play a role in neurodegenerative diseases such as Alzheimer’s disease
(AD).1 Cognitive impairments, characteristic of AD, correlate closely with the loss of synapses and evidence
suggests that excess amyloid- (A) causes synapse dysfunction by impairing synapse maintenance, at least
in part, through causing dysfunction of Wnt signaling.2,3 Compromised Wnt signaling may also be associated
with AD through loss of blood-brain barrier (BBB) integrity4 and A generation through -secretase (BACE1)
expression.5
Signal transduction by Wnt proteins is tightly regulated by a range of mechanisms including post translational
modifications. For example, O-palmitoleoylation of Wnt proteins is required for efficient binding to Frizzled
(Fzd) receptors and the subsequent signal transduction.6 The carboxylesterase Notum is a key negative
regulator of the Wnt signaling pathway by specifically mediating the O-depalmitoleoylation of Wnt proteins.7,8
The role of Notum in the mammalian central nervous system (CNS) has yet to be established although Notum
is expressed and upregulated in endothelial cells in the hippocampus of APPPS1 mice and AD patients
compared to control.9 In a disease setting, it follows that inhibition of Notum could restore Wnt signaling with
potential benefit in disease where Wnt deficiency is an underlying cause.
Me
O
N
S
N
CO2H
S
N
CO2H
Cl
O
S
N
N
N
Cl
Me
H
S
1: LP-922056
IC50 1.1 ± 0.4 nM a
2
3
IC50 3.0 ± 0.5 nM a
IC50 85 ± 11 nM
a Notum IC50 data presented for comparison in a common assay format.
The search for Notum inhibitors has identified acids 1 and 2 which have shown utility in mouse models of bone
growth and found to increase cortical bone thickness.10,11 Although 1 demonstrates good oral bioavailability,
recent pharmacokinetic studies in mouse showed CNS penetration of 1 is very low with brain:plasma
concentration ratio of just 0.01.12 Additional compounds include irreversible inhibitor ABC99 used to show the
role of Notum in the regeneration of aged intestinal epithelium,13,14 and phenoxyacetamide 3 identified
through optimisation of an X-ray fragment screening hit.15 However, it is unlikely that these compounds will
be suitable for in vivo studies where CNS penetration is an essential requirement. Hence, our objective was to
discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in
the CNS.
3

In order to identify new small molecule inhibitors of Notum, we elected to explore if 1 and 2 could be modified
to deliver a CNS penetrant tool by capping off the acid as an amide. However, prior art had established that
similar carboxamides exhibited poor metabolic stability.10 Our initial investigations into amide derivatives of 1
somewhat confirmed this result but also showed that judicious choice of the amine partner could significantly
improve metabolic stability as measured in liver microsomes.12
At the outset, we wished to use structure based drug design (SBDD) to accelerate our progress towards the
discovery of potent inhibitors by effective binding with Notum. Crystals of C-terminal his-tagged Notum(Ser81-
Thr451 Cys330Ser) were soaked with acids 1 and 2, and the crystal structures solved to elucidate their inhibitor
binding modes (Figure 1). Notum has a well-defined, large (ca. 380 Å3), hydrophobic active-site pocket
adjacent to the catalytic triad (Ser232, His389, Asp340) that accommodates the palmitoleate group of Wnt
(PDB:4UZQ).7 Both 1 and 2 place the thienopyrimidine group into this pocket with the acid forming the only
polar interactions through a network of H-bonds to the backbone with Trp128, Gly127 and Ala233, and also a
H-bond to the sidechain of His389 (Figure S1). The position of the thiophene ring differs slightly between 1
and 2 to accommodate the substituents which sit on opposite sides of the inhibitor, but the remainder of the
molecules adopted a similar position in the pocket. Overlays of the structures of 1 and 2 with O-palmitoleoyl
serine show all three structures effectively fill this pocket (Figure S2). From a design perspective, these
structures show significant solvent exposed space at the mouth of the palmitoleate pocket to accommodate
a suitable group as an amide derivative of 1 and 2.
Figure 1: Crystal structures of 1 (yellow) and 2 (green) with the surface of the Notum palmitoleoyl binding
pocket outlined (grey). Binding site residues shown within 3Å of their respective ligands. Key hydrogen bond
interactions are shown as dashed lines. Water molecules have been removed for clarity. Atomic coordinates
have been deposited in the Protein Data Bank (PDB). PDB ID codes: 1: 6T2K; 2: 6T2H.
4

The SARs were initially directed at exploring two principle areas of the structure: (1) the amide group (4, 5)
(Tables 1, 2 and S1); and (2) the pyrimidine heterocyclic group that binds in the palmitoleate pocket (6-19)
(Table 3). Combinations of preferred amides and heterocycles were then prepared (20-24) (Table 4).
Minimising compound lipiphilicity is a well-established approach to improve overall drug-like properties,
although this would need to be tempered by the requirement for CNS penetration.16 Target compounds were
designed to have molecular and physicochemical properties consistent with CNS drug-like space and we used
the CNS MPO score to aide our design.17 In general, target compounds 4, 5 and 20-24 all demonstrated CNS
MPO scores >4.0 and had cLogP values in the range 1.5 – 3.2.
Target compounds 4-24 were prepared in two phases: advanced intermediates 4-chloropyrimidines 25 were
either purchased or prepared using a customised synthesis (see Supplementary material, Schemes S1-S16)
and then a short sequence was used to prepare 4-24 from 25 (Scheme 1). Nucleophilic displacement of the
C4-Cl of 25 by methyl thioglycolate gave ester 26 which was hydrolysed with NaOH to afford the corresponding
acid (1, 2, 6-19). Finally, activation of the acid with HBTU and subsequent reaction with the amine (HNR1R2)
afforded the amide (4, 5, 20-24).
S
N
CONR1R2
R5
Z
Y
X
R7
R5
Z
Y
X
R7
R5
Z
Y
X
R7
S
N
CO2R
R2
Cl
N
a
c
N
N
N
R6
R6
R6
R2
R2
25
26: R = Me
4 5 20 24
, ,
-
b
1, 2, 6-19: R = H
Scheme 1. Preparation of acids 1, 2, 6-19 and amides 4, 5, 20-24. Representative reagents and conditions: (a)
HSCH2CO2Me (1.2 equiv.), NEt3 (2.1 equiv.), MeOH, 0 oC to rt; (b) NaOH (1 M) (2 equiv.), THF, 0 oC, then HCl (1
M), 0 oC; (c) HBTU (1.1 equiv.), iPr2NEt (2.5 equiv.), DMF, rt, 15 min; then amine (HNR1R2)(1.05 equiv.).
Inhibition of Notum carboxylesterase activity of 4-24 (Tables 1-4) was routinely measured in a biochemical
assay where test compounds were incubated with Notum(81-451 Cys330Ser) and trisodium 8-
octanoyloxypyrene-1,3,6-trisulfonate (OPTS) as the substrate for 1 h and fluorescence recorded.15 Compounds
were then assessed for metabolic stability in mouse liver microsomes (MLM) and for cell permeability by
measuring transit performance across a MDCK-MDR1 monolayer. Selected compounds were screened for
inhibition of Notum activity in a Wnt/-catenin signaling pathway TCF/LEF Reporter (Luciferase) HEK293 cell
line and Notum occupancy in a FP-biotin competition assay.15,18
5

Initial SAR studies with amides 4 and 5 (derived from 1 and 2 resp.) suggested that the Notum activity was
largely driven by the heterocycle binding in the palmitoleate pocket with the amide moiety offering minimal
contribution, although poor choice of amine partner could disrupt the binding; this is consistent with X-ray
structures and docking studies (Table 1). 19,20 From this set of matched pairs, three amide series 4d/5d, 4e/5e
and 4f/5f emerged as having potent Notum inhibition (IC50 < 10 nM), moderate MLM stability and cell
permeability although they were all substrates for P-gp mediated efflux to some degree. The challenge then
became to retain Notum inhibition activity, further improve metabolic stability whilst developing cell
permeability without efflux.
6

Table 1:
Comparison of thieno[3,2-d]pyrimidine amides 4a with thieno[2,3-d]pyrimidine amides 5.
CONR1R2
S
N
CONR1R2
S
N
Cl
5
S
N
N
6
Cl
Me
6
S
7
4a-f
5a-f
NR1R2
Compound
Notum b
MLM c
MDCK-MDR1 c
IC50 (nM)
AB/BA Papp (x106 cm/s)
Cli (L/min/mg)
and efflux ratio (ER)
-NMe2
4a
5a
4b
5b
4c
5c
4d
5d
7.5 ± 12.4
15 ± 6
360
91 ± 67
220 ± 12
18 ± 8.7
69 ± 10
7.1 ± 4.1
5.8 ± 4.0
N
N
>500
40/38
0.95
O
O
24
19
7.9/65
12/66
8.2
5.5
N
NH
4e
5e
4f
1.5 ± 0.1
2.7 ± 0.5
1.1 ± 0.3
3.2 ± 0.1
19
65
29
13
3.8/14
14/82
3.7
5.9
98
N
O
NH
N
N
0.95/93
0.6/56
N
N
5f
93
a
b
See reference 12.
All values are geometric mean ± s.d. of n = 2-6 experiments quoted to 2 s.f. Differences of <2-fold should not be
considered significant. For details of the assay protocol, see reference 15.
c MLM, MDCK-MDR1 and additional in vitro ADME studies reported in this work were independently performed by GVK
Biosciences (Hyderabad, India. https://www.gvkbio.com/discovery-services/biology-services/dmpk-services/) or
Cyprotex (Macclesfield, UK. https://www.cyprotex.com/admepk).
7

A wider range of amides around 5d-f were then prepared in the thieno[2,3-d]pyrimidine series 5 as this
template offered the advantage of slightly lower lipophilicity when compared to 4 (5 vs 4, cLogP = -0.5).
(Tables 2 and S1). One approach to reduce P-gp mediated efflux is to remove HBD or, if the HBD is essential
for binding to the primary target, to partially mask the HBD group by placing a flanking group in close proximity.
N-Alkylation of the piperazin-2-one 5d with either a Me (5g) or Et (5h) group retained potency although impact
on efflux was inconsistent. C-Alkylation of 5d at the -position with one or two Me groups reduced potency
(5i, 5j) and combining these two modifications into ring gave 5k which was inferior to 5d in all aspects. N-
Methylation of imidazolidin-4-one 5e proved to more beneficial with 5l showing potent activity, improved
MLM stability and high cell permeability with minimal efflux (ER 1.2). N-Substitution with larger alkyl groups
such as Et (5m), cPr (5n) and CH2CF3 (5o) retained potent Notum inhibition but eroded MLM stability, and the
instability tracked with increased compound lipophilicity.
Modifications to the triazolo[4,3-a]pyrazine amide 5f proved to be detrimental. Alkylation of the available C3
position of the triazole ring with small lipophilic groups (5p-s: Me, Et, cPr, CF3) proved to be progressively
detrimental to activity. Switching to the triazole isomer triazolo[1,5-a]pyrazine amides 5t-v also reduced
potency although substitution at C2 was tolerated but offered little advantage. These triazolopyrazine amides
5p-v were at least 3-fold weaker than 5f and failed to improve MLM or the efflux ratio.
At this point, mouse pharmacokinetic data for 5l was generated in vivo to determine the extent of plasma
exposure and to check the correlation of the in vitro ADME data with in vivo outcomes. Imidazolidin-4-one 5l
(cLogP 2.6; LogD7.4 1.8) was selected as a representative example from this set as it combined good aqueous
solubility (77 g/mL; 215 M) and cell permeability with moderate microsomal stability. Following single oral
dose (p.o.) of 10 mg/kg, plasma exposure for 5l was low (Cmax 120 ng/mL; AUC(0→inf) 70 ng.h/mL) which we
attribute to high clearance and highlighted the need to further improve metabolic stability (Figure S4).
8

Table 2:
Notum inhibition, MLM stability and MDCK-MDR1 cell permeability of thieno[2,3-d]pyrimidine amides 5.a
S
CONR1R2
Cl
N
Me
S
N
5g-v
NR1R2
Compound
5g
Notum
MLM
MDCK-MDR1
IC50 (nM)
AB/BA Papp (x106 cm/s)
and efflux ratio (ER)
Cli (L/min/mg)
6.2 ± 0.5
33
23/62
2.7
O
N
N
Me
5h
5i
4.2 ± 0.4
33 ± 5
100
1.1/37
34
O
N
N
Me
O
N
NH
Me
5j
450 ± 200
20 ± 4
O
N
NH
Me Me
O
5k
63
7/51
7.3
N
N
5l
5m
5n
2.4 ± 0.4
2.6 ± 0.1
3.8 ± 1.3
25
43
49
36/42
35/51
1.2
1.5
N
N
O
Me
N
O
N
Me
O
N
N
9

5o
5p
5q
3.9 ± 0.4
16 ± 2
140
15
N
N
O
N
CF3
N
0.5/51
0.8/65
>100
81
N
3
N
Me
N
23 ± 5
38
N
N
N
Me
N
5r
5s
27 ± 3
38 ± 3
53
93
0.8/78
15/85
97
N
N
N
N
N
N
5.7
N
CF3
N
5t
5u
5v
15 ± 3
11± 6
18 ± 5
53
37
19/49
17/44
15/27
2.6
2.6
1.8
N
N
N
N
N
2
Me
N
N
N
140
N
CF3
N
N
a See footnotes Table 1.
10

The next phase of SAR was to explore the pyrimidine heterocyclic group that binds in the palmitoleate pocket
(Table 3). This series of SARs was performed with the carboxylic acids with a view to introducing the amide
group once preferred heterocycles had been identified. Thienopyrimidines 1 and 2 are potent inhibitors and
so a range of alternative substituents on the thiophene ring (6-12) were investigated. Substituents were
selected to optimise binding interactions with Notum and reduce overall lipophilicity through removal of
lipophilic groups and/or introduction of polar groups. Deletion of the 5-Cl from 2 to give 6 (5-H) resulted in a
significant drop in activity whereas direct replacement of 5-Cl with a 5-Me 7 retained activity. Further
modification of the 7 scaffold by the addition of a 2-Me group (8) led to a dramatic decrease in potency and
so substitution at C2 was not investigated further. Introduction of a CF3 group at either the 5- or 6- positions
(9, 10 resp.) proved to be beneficial whereas the application of a 6-CN as a non-traditional bioisostere for a
halogen (11, 12) was detrimental to activity on this occasion.21
Alternative fused 6,5-ring systems (13-19) were also explored with the objective of replacing the thiophene
with a more polar N or O containing heterocycle. Pyrrolopyrimidine 13 was a weak inhibitor although activity
was improved when combined with substituents at either the 5- or 7- positions (14, 15). A 7-Bn group (16)
could be accommodated but there was no significant improvement over 7-H (13), and this was at a significant
penalty in added lipophilicity. Pyrazolopyrimidine 17 proved to be the most active inhibitor from these N
heterocycles although still 1000-fold weaker than 1. In contrast, the furano[2,3-d]pyrimidines proved to be
more successful when combined with optimal substituents. 5,6-Dimethyl furan 18 was 10-fold weaker than
the corresponding thiophene analogue (18 vs 7) but replacement of the 6-Me of 18 with a 6-CF3 gave 19 which
restored potent Notum inhibition activity in this more polar template (19 vs 9; cLogP = -0.6).
11

Table 3:
Notum inhibition of thieno- (6-12), pyrrolo- (13-16), pyrazolo- (17) and furanopyrimidine acids (18-19).
R5
Z
Y
X
R7
S
CO2H
R2
N
R6
N
6-19
Compound
Notum
Compound
Notum
IC50 (nM)a
IC50 (nM)a
790 ± 70 b
77,000 ± 28,000 b
N
N
N
Me
N
N
S
N
N
H
6
13
Me
5.8 ± 0.5 b
20,000 ± 3,900
15,000 ± 1,700
Cl
N
Me
S
N
H
N
7
14
15
Me
2,100 ± 330
N
N
Me
N
S
N
N
Me
Me
8
1.0 ± 0.4
3.9 ± 0.7
50,000 ± 6,500
1,100 ± 240
Me
N
N
F3C
N
S
N
N
Ph
9
16
Me
N
F3C
N
N
Me
S
N
N
N
Me
10
17
12

50 ± 14 b
3.2 ± 0.5
Me
25 ± 4
Me
N
N
N
N
NC
NC
Me
S
O
N
N
N
18
11
Me
8.1
S
(n = 1)
F3C
O
N
19
12
a See footnotes Table 1.
b Notum IC50 values for published compounds 6, 7, 13, 18 are presented for comparison in a common assay
format and to define SAR relationships: 6, 152 nM; 7, 2 nM; 13, 15,000 nM; 18, not disclosed. See, reference
10.
13

Table 4:
Notum inhibition, MLM stability and MDCK-MDR1 cell permeability of preferred 3-methylimidazolin-4-one
amides 20-24.
O
N
O
N
Me
Me
N
N
R5
X
S
N
S
N
O
O
X
N
N
R6
R6
R7
20
21-24
Het
Compound
20
Notum
IC50 (nM)a
EC50 (nM)b
1.5 ± 0.2
110 ± 67
MLM
MDCK-MDR1
AB/BA Papp (x106 cm/s)
and efflux ratio (ER)
Cli (L/min/mg)
45
5.9/12
2.0
S
N
N
Cl
N
Me
21
22
23
24
9.4 ± 1.5
31
27
24
6.9
21/45
31/35
2.1
1.1
1300 ± 170
Me
F3C
Me
S
N
N
N
N
1.6 ± 0.1
Me
N
210 ± 130
S
F3C
7.0 ± 2.5
170 ± 31
N
S
3.9 ± 0.8
220 ± 64
23/56
2.4
Me
N
F3C
O
a Notum OPTS assay, see footnotes Table 1.
b
Notum TCF-LEF assay. All values are geometric mean ± s.d. of n = 3-4 experiments quoted to 2 s.f. For details of the
assay protocol see reference 15.
14

The strategy of combining the superior acid heterocycles (1, 7, 9, 10, 19) with the preferred 3-
methylimidazolin-4-one amine produced amides 20-24 all with potent Notum inhibition (IC50 < 10 nM) in the
biochemical OPTS assay (Table 4). In general, the Notum inhibition activity of these amides tracked closely to
the activity of their corresponding acid, and with the same rank order, again suggesting the amide moiety
offered minimal contribution (or disruption) to the binding with Notum.
These inhibitors 20-24 were screened in the cell-based TCF/LEF reporter gene (Luciferase) assay to assess their
ability to restore Wnt/-catenin signaling when activated by exogenous rWNT3a (100 ng/mL) in the presence
of Notum (500 ng/mL) (Table 4). Compounds 20, 22-24 all showed an effective activation of Wnt signaling
(EC50 <250 nM) in this model system through inhibition of Notum. In contrast, 21 showed only modest
activation of Wnt signaling.
Evaluation of 20-24 in MLM showed 20-23 to have moderate metabolic stability and offered no significant
advantage over 5l. Only 24 demonstrated high metabolic stability in MLM with the potential advantage of low
metabolic clearance in vivo. Furthermore, 24 was stable in mouse plasma with no degradation observed after
120 min and did not inhibit CYP450 enzymes (Table 5). Compound 24 displayed a modest efflux ratio (ER =
2.4) in the MDCK-MDR1 permeability assay which suggests some recognition by P-gp mediated efflux
transport. However, the ER for 24 was perceived to be within acceptable limits based on established
precedent.22
Representative inhibitors 5l and 24 were tested in a Notum occupancy assay using FP-biotin,18 a covalent
serine hydrolase activity-based probe, whereby labelling of Ser232 of Notum with FP-biotin can be blocked by
an inhibitor occupying the active site of Notum (Figures 2 and S3). As 5l and 24 are reversible, high affinity
inhibitors of Notum, it proved necessary to adapt our reported protocol15 to ensure we were in the dynamic
range of labelling by FP-biotin in the presence of 5l and 24 so that relative potencies could be determined.
Compounds 1 and 3 have been evaluated under both assay conditions and are used as standards to help bridge
results from these studies.15 Both 5l and 24 showed an ability to prevent labelling by FP-biotin, confirming
they competitively bind to Notum, with potency equivalent to 1.
15

Figure 2: Notum activity-based occupancy assay was performed with FP-biotin (FP) (2 M) and test compounds
1, 3, 5l and 24 (3 M) for 30 minutes in conditioned media from HEK293S cells stably transfected with a Notum
lentiviral construct. Relative occupancy was calculated by optical density of the fluorescent band, generated
by streptavidin linked fluorophore to detect the level of biotinylation of Notum using Image Studio Lite 5.2,
compared to the control-treated sample which was set to 1. N = 2 with S.D. UT, untreated.
Hence, on balance, 24 emerged as having a superior profile from this set and was selected for further
evaluation in mouse pharmacokinetic studies.
Table 5.
Summary of physicochemical and molecular properties, Notum inhibition and ADME data for 24
24
Physicochemical and molecular properties
mol wt
cLogP
374
2.1
LogD7.4
1.6
74.6
5.9
TPSA (Å2)
CNS MPO
Notum inhibition a
OPTS, IC50 (nM)
TCF-LEF, EC50 (nM)
3.9 ± 0.8 (n = 4)
220 ± 64 (n = 3)
ADME profile b
Aq. solubility (g/mL)/(M)
Mouse plasma protein binding (PPB) (%)
Mouse brain binding (%)
MLM, Cli (L/min/mg protein)
Mouse plasma stability, % remaining at 120 min (%)
CYP1A2 inhibition, IC50 (M)
CYP2B6 inhibition, IC50 (M)
CYP2C9 inhibition, IC50 (M)
CYP2D6 inhibition, IC50 (M)
CYP3A4 inhibition, IC50 (M)
MDCK-MDR1, AB/BA Papp (x106 cms-1)
MDCK-MDR1, efflux ratio (ER)
45/120
78.1
84.4
6.9
110
>30
>30
>30
>30
>30
23/56
2.4
a See footnotes Table 4.
b In vitro ADME studies reported in this work were performed by GVK Biosciences (Hyderabad, India)
16

Table 6.
Mouse pharmacokinetic data for 24; oral (p.o.) dose at 10 mg/kg.a
PK Parameter
T1/2
Plasma
Brain
0.6 h
0.8 h
Tmax
0.5 h
0.5 h
Cmax
3850 ng/mL
5390 ng.h/mL
5490 ng.h/mL
1210 ng/g
1550 ng.h/g
1610 ng.h/g
AUC(0-t)
AUC(0-inf)
a Male C57BL6 mice; suspension formulation in 0.1% Tween80 in water; n = 3 per time point; terminal blood and brain
levels measured at seven time points: 0.17, 0.50, 1, 2, 4, 8 and 24 h. All animals were healthy throughout the study period.
Pharmacokinetic data for 24 was generated in vivo in mouse to evaluate plasma exposure and CNS penetration
(Table 6; Figure S4). Following single oral dose (p.o.) of 10 mg/kg, plasma exposure achieved a Cmax[plasma] ≈
2.2 M (free drug) which significantly exceeds the Notum EC50 from the cell-based TCF/LEF assay. However,
the plasma half-life was moderate which was somewhat unexpected based on the in vitro MLM and mouse
plasma stability data. Compound 24 demonstrates reasonable CNS penetration with a brain:plasma
concentration ratio of 0.29 based on AUC(0→inf). The incomplete CNS penetration was probably due to some
element of P-gp efflux transport recognition as evidenced by the ER in the MDCK-MDR1 cell line. The
combination of incomplete CNS penetration along with preferential binding to brain tissue resulted in
moderate brain exposure of Cmax [brain] ≈ 0.5 M (free drug) but this still exceeds the Notum EC50 at this dose.
Hence, 24 has potential utility in mouse models of disease under carefully designed experimental protocols
where the required site of action, route of administration, dose and duration of action requirements are
understood; i.e. the pharmacokinetic-pharmacodynamic relationship is to be established.
In summary, scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure
determination, identified 3-methylimidazolin-4-one amides 20-24 as potent inhibitors of Notum with activity
across three orthogonal assay formats. A preferred example 24 demonstrated good stability in MLM and
mouse plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. PK
studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma
exposure and reasonable CNS penetration. We propose that 2423 is a new chemical tool suitable for cellular
studies to explore the fundamental biology of Notum. Amide 24 has complementary properties to CNS
excluded acid 1 and irreversible inhibitor ABC99, and so represents a valuable addition to the Notum inhibitor
chemical toolbox.
17

Acknowledgements
We thank our colleagues Veronique Birault, Jamie Bilsland, Sarah Jolly, Patricia Salinas, Ed Tate, J. P. Vincent,
Paul Whiting, and Nicky Willis of our Notum Consortium for their support and advice. 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 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) and/or Cyprotex (Macclesfield, UK).
This work was supported by Alzheimer’s Research UK (ARUK) and the Francis Crick Institute. The ARUK UCL
Drug Discovery Institute is core funded by Alzheimer’s Research UK (520909). The Francis Crick Institute
receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002),
and the Wellcome Trust (FC001002). Structural analysis was performed by Y.Z. and E.Y.J. supported by Cancer
Research UK (Programme Grant C375/A17721). The Wellcome Trust funds the Wellcome Centre for Human
Genetics, University of Oxford (Centre Grant 203141/Z/16/Z).
Supplementary material
Supplementary material: (1) Interaction maps of 1 and 2 in the Notum binding pocket; (2) Overlays of
compounds 1, 2 and O-palmitoleoyl serine in the Notum binding pocket; (3) Western blots used to generate
activity-based probe occupancy assay data; (4) Mouse PK data for 5l and 24; (5) Notum inhibition, MLM
stability and MDCK-MDR1 cell permeability of additional thieno[2,3-d]pyrimidines amides 5w-5uu; (6)
Synthetic schemes for the preparation of 4-chloropyrimidines 25.
Abbreviations
A amyloid-beta; AD, Alzheimer’s disease; ADME, absorption distribution metabolism elimination; CNS,
central nervous system; ER, efflux ratio; FP, fluorophosphonate; HBD, hydrogen bond donor; MLM, mouse
liver microsomes; MPO, multiparameter optimization; OPTS, trisodium 8-octanoyloxypyrene-1,3,6-
trisulfonate; P-gp, P-glycoprotein; SAR, structure activity relationship; SBDD, structure based drug design;
TPSA, topological polar surface area; UPLC-MS, ultra performance liquid chromatography - mass
spectrometer. HBTU, O-(1H-Benzotriazol-1-yl)-N,N,N',N'-tetra-methyluronium hexafluorophosphate.
18

References and notes
1. For reviews, see: (a) Palomer, E.; Buechler, J.; Salinas, P.C. Wnt signalling deregulation in the aging and
Alzheimer’s brain. Front. Cell. Neurosci. 2019, 13, 227; (b) Inestrosa, N.C., Varela-Nallar, L. Wnt signalling
in the nervous system and in Alzheimer’s disease. J. Mol. Cell. Biol. 2014, 6, 64-74; (c) Inestrosa, N.C.,
Toledo, E.M. The role of Wnt signalling in neuronal dysfunction in Alzheiemer’s disease. Mol.
Neurodegener. 2008, 3:9.
2. Liu, C.-C., Tsai, C.-W., Deak, F., Rogers, J., Penuliar, M., Sung, Y.M., Maher, J.N., Fu, Y., Li, X., Xu, H., Estus,
S., Hoe, H.-S., Fryer, J.D., Kanekiyo, T., Bu, G. Deficiency in LRP6-Mediated Wnt signaling contributes to
synaptic abnormalities and amyloid pathology in Alzheimer’s disease. Neuron 2014, 84, 63–77.
3. Marzo, A., Galli, S., Lopes, D., McLeod, F., Podpolny, M., Segovia-Roldan, M., Ciani, L., Purro, S., Cacucci,
F., Gibb, A., Salinas, P.C. Reversal of synapse degeneration by restoring Wnt signaling in the adult
hippocampus. Curr. Biol. 2016, 26, 2551-2561.
4. (a) Zhou, Y., Wang, Y., Tischfield, M., Williams, J., Smallwood, P.M., Rattner, A., Taketo, M.M., Nathans, J.
Canonical WNT signaling components in vascular development and barrier formation. J. Clin. Invest. 2014,
124, 3825–3846; (b) Liu, L, Wan, W, Xia, S., Kalionis, B., Li, Y. Dysfunctional Wnt/-catenin signaling
contributes to blood-brain barrier breakdown in Alzheimer's disease. Neurochem. Int. 2014, 75, 19-25.
5. Parr, C., Mirzaei, N., Christian, M., Sastre, M. Activation of the Wnt/β-catenin pathway represses the
transcription of the β -amyloid precursor protein cleaving enzyme (BACE1) via binding of T-cell factor-4
to BACE1 promoter. FASEB J. 2015, 29, 623–635.
6. Janda, C.Y., Garcia, K.C. Wnt acylation and its functional implication in Wnt signaling regulation. Biochem.
Soc. Trans. 2015, 43, 211–216.
7. 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.
8. Zhang, X., Cheong, S.M., Amado, N.G., Reis, A.H., MacDonald, B.T., Zebisch, M., Jones, E.Y., Abreu, J.G.,
He, X. NOTUM is required for neural and head induction via Wnt deacylation, oxidation, and inactivation.
Dev. Cell 2015, 32, 719–30.
9. Jolly, S., Schuhmacher, L., Palomer, E., Frew, S., Mahy, W., Monaghan, A., Jones, E.Y., Bilsland, J., Fish,
P.V., Vincent, J.P., Bictash, M., Salinas, P., Whiting, P. Notum, a negative regulator of wnt signalling, as a
therapeutic target for Alzheimer’s disease? From target validation to the development of small molecule
inhibitors. 2019 UCL Neuroscience Symposium; 21st June 2019; London UK. See:
https://www.ucl.ac.uk/research/domains/sites/research_domains/files/abstract_booklet_ucl_neurosci
ence_symposium_2019.pdf. Accessed 9th August 2019.
19

10. (a) Tarver Jr, J.E., 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; (b) Barbosa, J., Carson, K.G., Gardyan, M.W., He, W., Lombardo, V., Pabba, P., Tarver Jr,
J. Inhibitors of notum pectinacetylesterase and methods of their use. US20120065200.
11. 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 Research 2019, 7, 2.
12. 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 Arch. 2019, 201970. doi:10.3762/bxiv.2019.70.v1.
13. 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.
14. Pentinmikko, N., Iqbal, S., Mana, M., Andersson, S., Cognetta III, A.B., Suciu, R.M., Roper, J., Luopajärvi,
K., Markelin, E., Gopalakrishnan, S., Smolander, O.-P., Naranjo, S., Saarinen, T., Juuti, A., Pietiläinen, K.,
Auvinen, P., Ristimäki, 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.
15. 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.
16. (a) Leeson, P.D., Springthorpe, B. The influence of drug-like concepts on decision-making in medicinal
chemistry. Nat. Rev. Drug. Discov. 2007, 6, 881-890; (b) Rankovic, Z. CNS Drug Design: Balancing
physicochemical properties for optimal brain exposure. J. Med. Chem. 2015, 58, 2584-2608.
17. (a) 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; (b) Rankovic, Z., CNS physicochemical property space
shaped by a diverse set of molecules with experimentally determined exposure in the mouse brain. J.
Med. Chem. 2017, 60, 5943-5954.
18. Liu, Y., Patricelli, M.P., Cravatt, B.F. Activity-based protein profiling: The serine hydrolases. Proc. Natl.
Acad. Sci. USA 1999, 96, 14694-14699.
20

19. Docking experiments were used to rationalise the SAR and guide design. All docking was performed using
Schrödinger’s Glide software (Schrödinger Release 2018-3: Glide, Schrödinger, LLC, New York, NY, 2018).
20. Hydrolytic stability of representative amides, e.g. 5f and 5l, was tested in citrate buffer (0.1 M, pH 4) at
multiple time points up to 24 h and only parent was detected by UPLC-MS. Notum enzymatic stability of
5f and 5l were also tested in the Notum biochemical assay (without the OPTS substrate) up to 72 h and
again only parent was detected. Hence, 5f and 5l were stable to both aqueous hydrolysis and Notum
enzymatic activity which confirmed Notum inhibition was due to the parent amide and not from any acid
degradation product.
21. Jones, L.H., Summerhill, N.W., Swain, N.A., Mills, J.E. Aromatic chloride to nitrile transformation:
medicinal and synthetic chemistry. Med. Chem. Commun. 2010, 1, 309-318.
22. Hitchcock, S.A, Structural modifications that alter the P-glycoprotein efflux properties of compounds. J.
Med. Chem. 2012, 55, 4877-4895.
23. 3-Methyl-1-(2-((5-methyl-6-(trifluoromethyl)furo[2,3-d]pyrimidinyl-4-yl)thio)acetyl)imidazolin-4-one
(24) [SMILES: CN1CN(CC1=O)C(=O)CSc1ncnc2oc(c(c12)C)C(F)(F)F)]. Mp 209-211oC; IR νmax (neat) 1712,
1
1653, 1585, 1429, 1354, 1337, 1250, 1234, 1076, 1043, 982, 781 cm-1; H NMR; (700 MHz, DMSO-d6) δ
8.86 (seen as 2 rotameric singlets, 1H), 4.94 (seen as 2 rotameric singlets, 2H), 4.35 (seen as 2 rotameric
singlets, 2H), 4.08 (seen as 2 rotameric singlets, 2H), 2.83 (seen as 2 rotameric singlets, 3H), 2.56 (q, J =
1.9 Hz, 3H); 13C NMR; (176 MHz, DMSO-d6) δ 166.95, 166.77 (rotamer peak), 165.57, 165.54 (rotamer
peak), 164.77, 164.26 (rotamer peak), 163.34, 155.09, 135.41 (q, J = 39.9 Hz), 122.03, 119.74 (q, J = 269.1
Hz) 118.75, 115.05 63.83 (rotamer peak), 63.77, 48.01, 47.51 (rotamer peak), 32.36, 31.79 (rotamer
peak), 27.18 (rotamer peak), 27.11, 9.82; 19F NMR; (659 MHz, DMSO-d6) δ -60.82; LCMS (Basic method)
Rt 1.61, m/z 373.1 [M-H]-, purity >95 %; HRMS (TOF MS ESI+) m/z calc. for C14H14F3N4O3S [M+H]+ 375.0739,
found 375.0726.
21

Table of Contents Graphic
High definition editable figure:
O
N
S
N
CO2H
Me
N
S
N
S
N
Me
Cl
O
N
F3C
O
1
24
Notum: IC50 1.1 nM
No CNS penetration
Notum: IC50 3.9 nM
CNS penetrant
Overlay of 1 and 2 Notum structures
Low resolution image:
22