4H-Thieno[3,2-c]chromene based inhibitors of Notum Pectinacetylesterase

Article abstract of DOI:10.1016/j.bmcl.2016.01.038

A group of small molecule thienochromenes inhibitors of Notum Pectinacetylesterase are described. We developed SAR on three series based on carbon, oxygen and sulfur replacement of the 5-position. In each series, highly potent Notum Pectinacetylesterase inhibitors were identified.

Full text of DOI:10.1016/j.bmcl.2016.01.038

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1184–1187
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4H-Thieno[3,2-c]chromene based inhibitors of Notum
Pectinacetylesterase
Qiang Han a, Praveen K. Pabba a, Joseph Barbosa a, Ross Mabon a, Jason P. Healy a, Michael W. Gardyan a,
Kristen M. Terranova a, Robert Brommage b, Andrea Y. Thompson b, James M. Schmidt b,
a
Alan G. E. Wilson b, Xiaolian Xu a, James E. Tarver Jr. a, , Kenneth G. Carson
⇑
a Lexicon Pharmaceuticals, 350 Carter Road, Princeton, NJ 08540, United States
b Lexicon Pharmaceuticals, 8800 Technology Forest Place, The Woodlands, TX 77381, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of small molecule thienochromenes inhibitors of Notum Pectinacetylesterase are described. We
developed SAR on three series based on carbon, oxygen and sulfur replacement of the 5-position. In each
series, highly potent Notum Pectinacetylesterase inhibitors were identified.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 13 December 2015
Revised 12 January 2016
Accepted 14 January 2016
Available online 18 January 2016
Keywords:
Notum Pectinacetylesterase
Osteroporosis
Thienochromene
SAR
Femur cortical bone thickness
Bone is a living, dynamic tissue, that is, continuously remodeled
during the adult life of all mammals. Bone mass depends on the
coordinated activities of bone-forming osteoblasts and bone-
resorbing osteoclasts. Bone turnover reflects a balance between
these anabolic and catabolic cellular functions and ensures that
the mature skeleton can repair itself when damaged and sustain
its endocrine function by release of minerals such as calcium and
phosphorous into the circulation. Modest loss of bone mineral den-
sity (BMD), referred to as osteopenia, and severe loss of bone
known as osteoporosis represent the early and late manifestations
of alterations in bone turnover that result in bone disease.1–3
The current standard of care for the treatment of osteoporosis
utilizes the bisphosphonate class of oral, small molecule antire-
sorptives. RaloxifeneTM, calcium, and vitamin D supplements are
also typically used in osteoporosis treatment.4 More recently, the
FDA approved denosumabTM as treatment of postmenopausal
osteoporosis. Longer duration of bisphosphonate therapy is associ-
ated with a higher risk of atypical femur fractures.5 Combination
therapy with teriparatide and denosumab appears to increase bone
mineral density to a greater extent than either therapy alone in
postmenopausal women at high risk for fracture.5,6 There are
several novel therapies under investigation for the treatment of
osteoporosis, which are in various stages of development.5,6 While
antiresorptive agents prevent bone loss, anabolic agents are cap-
able of increasing bone mass, improving bone quality and increas-
ing bone strength.4 In the United States, teriparatide is the only
FDA-approved anabolic agent.5,6 Because of the paucity of available
anabolic agents for osteoporosis treatment, there is an urgent need
to develop small molecule drugs to treat this disease that are non-
toxic, cost-effective, and easy to administer.1–3
Although the development of pharmacological agents that stim-
ulate bone formation is less advanced compared to antiresorptive
therapies, several pathways are known to facilitate osteoblast
function. These pathways include bone morphogenic proteins,
transforming growth factor b, parathyroid hormone, insulin-like
growth factor, fibroblast growth factor, and wingless-type MMTV
integration site (WNT) signaling.3 One of these pathways to our
interest is the WNT pathway, which is implicated in a variety
of developmental and regenerative processes.7,8 The WNT family
is comprised of 19 secreted proteins that regulate many aspects
of cell growth, differentiation, function and death, including
osteoblastogenesis and adipogenesis. Recent analysis of gene
expression data has led to the identification of new targets of
WNT signaling.9 One such target is Notum Pectinacetylesterase,
as NOTUM is
a WNT-inactivating lipase that removes the
palmitoleate essential for binding to Frizzled receptors10,12 and
inhibition of NOTUM stimulates endocortical bone formation.13
⇑
Corresponding author. Tel.: +1 215 898 4193.
E-mail address: james.tarver2@gmail.com (J.E. Tarver).
http://dx.doi.org/10.1016/j.bmcl.2016.01.038
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

Q. Han et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1184–1187
1185
No substitution tolerated
O
potency. We chose the primary amide derivatives to explore sub-
stitution on the central core, allowing us the best chance to observe
subtle potency trends. Some representative analogs are summa-
rized in Table 1. It is worth noting that while we were more con-
cerned about modulation of activity at the human enzyme,
potency at the mouse homolog was still a consideration since ini-
tial in vivo studies were planned in murine models. We anticipated
some metabolic stability challenges, particularly at positions 4 and
5 of the dihydronaphtho[1,2-b]thiophene chemotype due to high
oxidation potential of those carbons and chose to aggressively
explore modifications at these points of the molecule. Addition of
a methyl group at the 4-position (8) led to a slight impairment of
potency, while 5-methyl substitution (9) had no impact.
Compound 10 further clarified that steric modifications to the
5-position were well tolerated. Expansion of the cyclic core to a
seven-membered ring (e.g., 11, 12) retained potency. ADME profiling
of the amide derivatives revealed poor metabolic stability and fast
clearance. However, the excellent potency of most of the primary
amides did not translate to related carboxylic acids. For example,
the related carboxylic acids of 11 and 12 were 100-fold less potent.
In contrast, we observed that carboxylic acids generally had more
acceptable ADME profiles, so we decided to focus our optimization
efforts on carboxylic acids. Eventually we focused our efforts on
the six-membered ring systems without the 4,5-substitutions.
Based on the preliminary SAR, our synthetic efforts mainly con-
verged on three subseries 1a, 1b, and 1c (Fig. 1) based on carbon,
oxygen and sulfur at the 5-position. The three subtypes demon-
strated that the phenyl ring could tolerate of a range of electron
rich substituents. We wanted to probe the effect of a more electron
deficient system on activity and identify functionality that could
improve solubility and metabolic stability. The SAR of 4,5-dihy-
dronaphtho[1,2-b]thiophene-2-carboxylic acids is summarized in
Table 2. A preliminary survey of substituent position modifications
on the phenyl ring afforded the highly potent 6-cyano compound
(13). The combination of 6-cyano and 7-position substitutions
demonstrated reasonable potency gains; 7-methoxy compound
(14) improved hEC50 to 36 nM while 7-fluoro (15) and 7-methyl
(16) compounds gave 3- to 4-fold improvements in hEC50, respec-
tively. Di-substituted compounds with halide substitutions at the
6-position provided some of the most potent compounds, in partic-
ular dichloro- and difluoro-combinations. 6,7-Difluoro substitution
(23) gave a hEC50 of 17 nM, 6-chloro-7-fluoro compound 17 gave a
hEC50 of 46 nM, whereas shifting the fluoro group to the 8-position
(18) improved hEC50 to 13 nM. Subsequent replacement of the 8-F
with 8-Cl (19) gave a hEC50 of 20 nM, but moving the chloro group
to the 7-position (20) gave hEC50 of 12 nM. However, the most
potent analogs arose from modulating the 8-position substitution
of 6-fluoro compounds and 6-fluoro-8-methyl substitution (24)
produced a hEC50 of 9 nM. The best potency was achieved with
6-F, 8-Me and 9-F tri-substitution (25), which had single-digit
Amide tolerated,
primary amide
boost potency
OH
Phenyl best
2
S
9
3
No substitution tolerated
8
7
4
5
6
1
HO
O
HO
HO
O
O
S
S
S
R
R
R
1c
1b
1a
O
S
Figure 1. Lead compounds and lessons from hit-to-lead.
Herein we report
a group of small molecules, namely
thienochromenes, as potent inhibitors of Notum Pecti-
nacetylesterase. Compound EC50 values were determined using a
cell-based TCF/LEF CellSensorÒ assay having a b-lactamase reporter
gene under control of the b-catenin/LEF/TCF response element
where b-lactamase activity was measured using a FRET-based sub-
strate (GeneBLAzerÒ, Invitrogen) technology.11–13 Based on high
throughput screening, we identified 4,5-dihydronaphtho[1,2-b]
thiophene-2-carboxylic acid 1 as an interesting small molecule
lead. Compound 1 had a mouse EC50 of 1060 nM and human EC50
of 361 nM, which offered a reasonable starting point for potency
optimization. Structural modifications of lead compound 1 were
explored with focus on the carboxyl terminus, cyclic core, and sub-
stitution on the aromatic rings.
The general observations in substitutions of the carboxylic acid
were that secondary and tertiary amides were tolerated, while pri-
mary amides generally showed significant increases in potency.
Position 1 would not tolerate replacement of the sulfur atom and
no substitution for the sp2-carbon on the 3-position was tolerated,
requiring a thiophene for activity. Replacement of the phenyl ring
with hetero-aromatics generally gave much lower potency (data
not shown).
The synthesis of our derivatives was carried out following the
general synthetic methods shown in Scheme 1. The appropriate
commercially available or synthesized13 ketones 2 were treated
with phosphorus oxychloride in N,N-dimethylformamide under
Vilsmeier–Haack conditions to generate the
a,b-unsaturated
b-chloroaldehydes 3.14–16 Subsequent treatment with ethyl 2-mer-
captoacetate and sodium ethoxide effected cyclization to generate
substituted thiophenes 4. Saponification of the esters with aqueous
base yielded a range of carboxylic acids 5, which could be further
functionalized to amides 6.13
Conversion of acids 5 to amides 6 was generally tolerated, and
primary amides stood out with the biggest improvement in
nanomolar mEC50 and hEC50
.
The SAR of 4H-thieno[3,2-c]chromene-2-carboxylic acids is
summarized in Table 3. The unsubstituted 4H-thieno[3,2-c]chro-
mene-2-carboxylic acid 26 had hEC50 of 1.36 lM, about three-fold
less active than carbocyclic compound 1. We postulated that the
apparent decrease in activity might be due to the increased elec-
tron density in the phenyl ring and attempted to modulate the
electronics by probing substitution using a small electron with-
drawing group such as fluorine, a strategy which had proven effec-
tive in the dihydronaphtho-thiophene series. Preliminary SAR
showed the 9-fluoro substitution (27) gave a two-fold potency
increase to hEC50 of 706 nM, while 8-fluoro substitution (28) fur-
ther improved the potency to hEC50 of 175 nM. Changing the halo-
gen to chlorine at the 8-position (29) slightly increased the potency
to 133 nM. Subsequent efforts showed that substitution on the
9-position generally reduced potency, so our main focus was on
2
3
4
5
6
Scheme 1. Reagents and conditions: (a) POCl3, DMF; (b) HSCH2CO2Et, NaOEt;
(c) NaOH, THF/H2O; (d) HATU, R2NHR3.

1186
Q. Han et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1184–1187
Table 1
SAR of 4,5-dihydronaphtho[1,2-b]thiophene-2-carboxamide for Notum Pectinacetylesterase
Compounds
mEC50 (nM)
hEC50 (nM)
Compounds
H2N
mEC50 (nM)
hEC50 (nM)
HO
O
O
S
S
1060
361
15
7
1
10
11
H2
N
N
N
H2N
O
O
O
O
S
S
9
13
14
19
15
7
8
7
H2
H2N
O
S
S
40
O
12
8
H2
S
16
7
9
Table 2
Table 3
SAR of 4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acids for Notum
SAR of 4H-thieno[3,2-c]chromene-2-carboxylic acids for Notum Pectinacetylesterase
Pectinacetylesterase
Compounds
R6
R7
R8
R9
mEC50 (nM)
hEC50 (nM)
Compounds
R6
R7
R8
R9
mEC50 (nM)
hEC50 (nM)
26
27
28
29
30
31
32
33
34
35
36
37
38
H
H
H
H
Cl
Cl
CN
F
Cl
H
F
Cl
H
H
H
H
H
H
Cl
H
H
F
Cl
H
H
F
H
H
F
Cl
Cl
H
F
Me
H
F
Cl
F
H
F
3875
1878
662
253
173
42
75
35
126
56
59
1360
706
175
133
41
39
31
18
77
49
16
27
38
1
H
H
H
OMe
F
Me
F
H
H
Cl
OMe
H
H
H
H
H
H
H
F
Cl
H
H
Me
H
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
F
1060
82
87
70
62
72
25
34
21
30
36
61
23
8
361
67
36
21
17
46
13
20
12
23
19
17
9
13
14
15
16
17
18
19
20
21
22
23
24
25
CN
CN
CN
CN
Cl
Cl
Cl
Cl
Cl
Cl
F
H
H
H
H
H
H
H
H
H
H
F
35
67
F
H
H
F
F
F
6
thiochromene-2-carboxylic acid 39 in this series had mEC50 of
461 nM and hEC50 of 243 nM, which was somewhat more potent
than the starting points of the other two cores. Oxidation of sulfide
to sulfoxide 40 led to about 6-fold loss of potency. Further oxida-
tion to sulfone 41 led to significant improvement of potency with
hEC50 of 28 nM. However, this was not a general trend since many
of the more substituted sulfones proved to be much less potent
than their sulfide counterparts (data not shown). Apart from 41,
another outlier in this comparison is compound 42 which had sim-
ilar potency (hEC50 = 83 nM) to analog 43 (hEC50 = 53 nM). Further
substitutions at the 6-, 7-, and 8-positions. Greater potency
improvement was achieved with multiple substitutions on the
phenyl ring, similar trends to the carbon series were observed.
The best potency improvements were obtained with the 6-flu-
oro-8-methyl analog 33 (hEC50 = 18 nM) and 8-chloro-6-fluoro
compound 36 (hEC50 = 16 nM).
The SAR of 4H-thieno[3,2-c]thiochromene-2-carboxylic acids is
summarized in Table 4. The parent compound 4H-thieno[3,2-c]

Q. Han et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1184–1187
1187
Table 4
SAR
of
4H-thieno[3,2-c]thiochromene-2-carboxylic
acids
for
Notum
Pectinacetylesterase
Compounds
X
R6
R7
R8
R9
mEC50 (nM)
hEC50 (nM)
39
40
41
42
43
44
45
46
47
S
H
H
H
F
F
H
F
H
H
H
H
H
H
H
H
F
H
H
H
H
H
F
H
F
F
H
H
H
H
H
H
F
461
2309
139
183
160
186
80
243
1185
28
84
53
59
39
6
SO
SO2
SO2
S
S
S
Figure 2. Change in cortical bone thickness over 28 day study.
S
S
F
F
H
H
20
20
compound, starting at 10.5 weeks of age. The compound was
administered in the animals’ diet. Three groups of mice were used:
control (N = 15); 5 mg/kg compound (N = 12); and 20 mg/kg com-
pound (N = 12). Mice treated with the compound exhibited dose-
dependent increases in midshaft femur cortical thickness.
In summary, we have developed novel inhibitors of Notum
Pectinacetylesterase that have shown in vivo efficacy in signifi-
cantly increasing midshaft femur cortical bone thickness.
8
Table 5
Mouse pharmacokinetic data for compounds 1 and 46
Compounds Cmax M) at AUC ( M*h) at Clearance
(
l
l
Vd (L/kg) at %F
10 mg/kg po 10 mg/kg po
(mL/min/kg) 1 mg/kg iv
at 1 mg/kg iv
1
46
64
16
158
56
2.4
18.8
0.57
8.1
52
100
References and notes
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SAR explorations focused on thiochromenes. As in the other sub-
series, substitution on the phenyl ring with electron withdrawing
groups such as halogens generally boosted potency, while electron
donating groups like methoxy and methyl reduced potency (data
not shown). Among the halogens, the trend was that fluoro analogs
had better potency than their chloro counterparts. Fluoro substitu-
tion at the 6-position, compound 43, showed hEC50 of 53 nM and
8-fluoro compound 44 had hEC50 of 59 nM. Di-fluoro substitutions
generally gave better potency. The most potent compounds
observed in the series included 6,9-difluoro compound 45 which
had hEC50 of 39 nM and 6,8-difluoro compound 46 with hEC50 of
6 nM. Trifluoro compound 47 also displayed excellent potency
with a hEC50 of 8 nM.
3. Allen, J. G.; Fotsch, C.; Babij, P. J. Med. Chem. 2010, 53, 4332.
4. Guo, H.; Shao, H.; Yang, Z.; Xue, S.; Li, X.; Liu, Z.; He, X.; Jiang, J.; Zhang, Y.; Si, S.;
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11. LEF/TCF-bla FreeStyle 293F cells were incubated in medium overnight with
WNT3A conditioned medium, human or mouse NOTUM conditioned medium
and NOTUM inhibitors. Mouse WNT3A-conditioned medium was obtained
from L Wnt3A cells (ATCC #CRL-2647).
As shown in Table 5, when dosed orally to mice at 10 mg/kg,
compounds 1 and 46 achieved good Cmax and AUC while having
relatively low clearance. Compound 46 was advanced into in vivo
mouse models of bone growth and was found to increase cortical
bone thickness.10
12. Zhang, X.; Cheong, S. M.; Amado, N. G.; Reis, A. H.; MacDonald, B. T.; Zebisch,
M.; Jones, E. Y.; Abreu, J. G.; He, X. Dev Cell. 2015, 32, 719.
13. Barbosa, J.; Carson, K. G.; Gardyan, M. W.; Healy, J. P.; Han, Q.; Mabon, R.;
Pabba, P.; Tarver, J., Jr.; Terranova, K. M.; Tunoori, A. U.S. Patent 302,562, 2012.
14. Fischer, O.; Muller, A.; Vilsmeier, A. J. Prakt. Chem. 1925, 109, 69.
15. Vilsmeier, A.; Haack, A. Ber. dtsch. Chem. Ges. 1927, 60B, 119.
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Figure 2 shows the in vivo effect of acid 46 determined by
treating F1 male hybrid (129xC57) mice for 28 days with the