Optimization of a series of heterocycles as survival motor neuron gene transcription enhancers

Article abstract of DOI:10.1016/j.bmcl.2017.10.066

Spinal muscular atrophy (SMA) is a neurodegenerative disorder that results from mutations in the SMN1 gene, leading to survival motor neuron (SMN) protein deficiency. One therapeutic strategy for SMA is to identify compounds that enhance the expression of the SMN2 gene, which normally only is a minor contributor to functional SMN protein production, but which is unaffected in SMA. A recent high-throughput screening campaign identified a 3,4-dihydro-4-phenyl-2(1H)-quinolinone derivative (2) that increases the expression of SMN2 by 2-fold with an EC₅₀ = 8.3 μM. A structure-activity relationship (SAR) study revealed that the array of tolerated substituents, on either the benzo portion of the quinolinone or the 4-phenyl, was very narrow. However, the lactam ring of the quinolinone was more amenable to modifications. For example, the quinazolinone (9a) and the benzoxazepin-2(3H)-one (19) demonstrated improved potency and efficacy for increase in SMN2 expression as compared to 2.

Full text of DOI:10.1016/j.bmcl.2017.10.066

Accepted Manuscript
Optimization of a series of heterocycles as survival motor neuron gene tran-
scription enhancers
Sungwoon Choi, Alyssa N. Calder, Eliza H. Miller, Kierstyn P. Anderson,
Dawid K. Fiejtek, Anne Rietz, Hongxia Li, Jonathan J. Cherry, Kevin M. Quist,
Xuechao Xing, Marcie A. Glicksman, Gregory D. Cuny, Christian L. Lorson,
Elliot A. Androphy, Kevin J. Hodgetts
PII:
S0960-894X(17)31066-1
https://doi.org/10.1016/j.bmcl.2017.10.066
BMCL 25395
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 September 2017
22 October 2017
24 October 2017
Please cite this article as: Choi, S., Calder, A.N., Miller, E.H., Anderson, K.P., Fiejtek, D.K., Rietz, A., Li, H.,
Cherry, J.J., Quist, K.M., Xing, X., Glicksman, M.A., Cuny, G.D., Lorson, C.L., Androphy, E.A., Hodgetts, K.J.,
Optimization of a series of heterocycles as survival motor neuron gene transcription enhancers, Bioorganic &
Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.066
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Optimization of a series of heterocycles as survival motor neuron gene
transcription enhancers
a
Sungwoon Choia, Alyssa N. Calder , Eliza H. Millera, Kierstyn P. Andersona, Dawid K. Fiejteka Anne
Rietzb, Hongxia Lib, Jonathan J. Cherryb, Kevin M. Quistb, Xuechao Xinga, Marcie A. Glicksmana, Gregory
D. Cunya, Christian L. Lorsonc, Elliot A. Androphyb and Kevin J. Hodgettsa*
aLaboratory for Drug Discovery in Neurodegeneration, Brigham and Women’s Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA,
USA. bDepartment of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA. c Department of Veterinary Pathobiology, Bond Life Sciences
Center, University of Missouri, Columbia, Missouri, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received 2017
Revised
Spinal muscular atrophy (SMA) is a neurodegenerative disorder that results from mutations in the SMN1 gene, leading to
survival motor neuron (SMN) protein deficiency. One therapeutic strategy for SMA is to identify compounds that enhance
the expression of the SMN2 gene, which normally only is a minor contributor to functional SMN protein production, but
which is unaffected in SMA. A recent high-throughput screening campaign identified a 3,4-dihydro-4-phenyl-2(1H)-
quinolinone derivative (2) that increases the expression of SMN2 by 2-fold with an EC50 = 8.3 µM. A structure-activity
relationship (SAR) study revealed that the array of tolerated substituents, on either the benzo portion of the quinolinone or
the 4-phenyl, was very narrow. However, the lactam ring of the quinolinone was more amenable to modifications. For
example, the quinazolinone (9a) and the benzoxazepin-2(3H)-one (19) demonstrated improved potency and efficacy for
Accepted
Available online
Keywords:
Spinal muscular atrophy
Survival motor neuron
increase in SMN2 expression as compared to 2. 2017 Elsevier Ltd. All rights reserved
.
Spinal muscular atrophy (SMA) is a neurodegenerative disease
characterized by progressive muscle wasting, loss of motor
function and premature death in the most severe cases.1-3 Two
genes, SMN1 and SMN2, produce survival motor neuron (SMN)
protein,4-6 which is ubiquitously expressed with the highest levels
in the spinal cord7 and functions in the assembly of spliceosomal
small nuclear ribonucleoproteins.8 The majority of SMN protein
normally is produced from the SMN1 gene, while the almost
identical SMN2 gene only produces approximately 10% of
functional protein.9,10
to Phase III clinical trials for SMA, including compounds that
increased exon 7 inclusion of SMN2.19
Previously, we reported an SMN2-lucifrase reporter assay for
identifying compounds that increase SMN expression from the
SMN2 gene and its use in high-throughput screening.20 Using the
reporter assay, we discovered two hit compounds, LDN-75654
(1) and LDN-76070 (2) (Figure 1), that increase expression of
SMN2 by 2-fold, but that have different mechanisms of action
than the small molecules already in clinical trials to treat SMA.
Compound 1 increases the stability of SMN protein, whereas
compound 2 acts in a transcriptional manner.21
SMA results from mutations within exon 7 of SMN1, leading to
SMN protein deficiency.11 The clinical severity of this disease is,
therefore, indirectly proportional to the copy number of SMN2
genes, the sole source of protein in these patients.12,13 Although
the threshold level of SMN protein necessary to maintain healthy
motor neurons is not known, it has been estimated that increasing
the amount of functional protein by only 2- or 3-fold may be
clinically significant.14 Thus, the SMN2 gene has become a
therapeutic target, in which multiple strategies (i.e., small
molecules, antisense oligonucleotide) work to increase the
transcription of SMN2, increase the inclusion of exon 7 SMN2,
stabilize the full-length exon-7 included SMN2 mRNA, or
stabilize the SMN protein.15-17 As SMA is the leading heritable
cause of infant mortality, it is critical to build upon the one
recently FDA-approved treatment, Nusinersen, an antisense
oligonucleotide18 and to provide a variety of treatment options
with different modes of action. Several repurposed drugs, such
as riluzole, phenylbutyrate, valproic acid, albuterol and
hydroxyurea, have advanced into clinical trials, but none has
elicited convincing improvement in muscle function or survival
in SMA. Currently, there are several small molecules in Phase I
We recently reported the structure-activity relationship (SAR) of
analogs of the 5-isopropylisoxazole-3-carboxamide 1.22 In this
paper, we report preliminary SAR of the 3,4-dihydro-4-phenyl-
2(1H)-quinolinone 2 for increasing SMN expression.
Figure 1. Two SMN mRNA expression enhancers identified
following high-throughput screening.
The 3,4-dihydro-4-phenyl-2(1H)-quinolinone derivatives were
prepared according to the procedure outlined in Scheme 1. A
three-component coupling of an aniline (3), aromatic aldehyde
(4) and Meldrum’s acid in refluxing ethanol generated the

quinolinones 5a-m in good yield.23 These compounds were
transformed further into the methyl substituted derivatives 6a-b,
which were isolated as mixture of cis- and trans-isomers.
2-steps); (e) HCl•NH2CH2CO2Et, Py, Dean-Stark trap, reflux, 24 h
(38%).
Several oxygen-containing heterocycles (e.g., 16, 19-21) also
were prepared from the ketone 12 (Scheme 4 and Scheme 5).
Ketone 12 was reduced to the racemic alcohol 15 with sodium
borohydride in ethanol. Treatment of 15 with carbonyl
diimidazole (CDI) directly gave the oxazinone 16 in good yield.
Acylation of 15 with 2-bromoacetyl chloride or 2-
bromopropionyl chloride gave the amides 17 and 18,
respectively. Treatment of 17 with two equivalents of sodium
hydride gave the 7-membered benzoxazepin-2(3H)-one 19 in
moderate yield.25 Similarly, cyclization of 18 gave the 3-methyl-
benzoxazepin-2(3H)-one 20 as a mixture of diastereoisomers.
Finally, treatment of 19 with methyl iodide gave the N-methyl
amide 21.
R
N
R
N
O
O
R1
R1
H
N
a
b
Me
R
R1
CHO
R2
6a, 6b
R2
R2
3
5a-m
4
Scheme 1. (a) Meldrum’s acid, EtOH, 85ºC, 16 h (55-78%); (b)
BuLi, THF, 0ºC then MeI (48%).
A series of 3,4-dihydro-quinazolinone derivatives 9a-9l was
prepared according to the procedures outlined in Scheme 2.24
Anilines 3 were treated with potassium isocyanate to give the
ureas 8 (e.g., R = H). Alternatively, phenyl isocyanates 7 were
treated with primary amines to yield substituted ureas 8 (e.g., R =
alkyl). The ureas then were treated with catalytic methane
sulfonic acid in refluxing toluene, with azeotropic removal of
water, to produce the 3,4-dihydro-quinazolinones 9a-9r.
MeO
MeO
NH2
OH
H
N
MeO
MeO
O
F
a
b
12
O
MeO
Cl
F
MeO
Cl
15
16
c
NH2
R1
H
N
a
H
N
H
N
O
R
R1
R
c
R
R1
N
3
O
O
Br
H
N
O
O
MeO
MeO
MeO
MeO
NH
NCO
N
MeO
MeO
R2
8 R = H or alkyl
e
b
d
R1
R
OH
O
O
R = H
9a-9r
MeO
Cl
MeO
Cl
F
MeO
Cl
7
F
F
Scheme 2. (a) KNCO, AcOH, RT; (84-97%); (b) RNH2, THF, 0 ºC
to RT (63-89%); (c) R2PhCHO, cat. MeSO3H, toluene, reflux (31-
81%).
19 R = H
20 R = Me
17 R = H
18 R = Me
21
The synthesis of the 2-quinazolinone 13 and the seven-membered
1,4-benzodiazepin-2-one 14 is outlined in Scheme 3. The
pivolate-protected 3,4,5-trimethoxylaniline 10 was lithiated with
butyllithium in THF at -78oC, and the resulting anion was
quenched with methyl 5-chloro-2-fluorobenzoate to give the
ketone 11 in modest yield. The pivolate group was cleaved with
refluxing sulfuric acid and gave the versatile intermediate 12. For
example, the ketone 12 was treated with trichloroacetyl chloride,
and the product, upon treatment with ammonium acetate,
cyclized to 13. On the other hand, the ketone 12 also was treated
with glycine ethyl ester, with azeotropic removal of water, and
gave the 1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one 14.
Scheme 4. (a) NaBH4, EtOH, RT (82%); (b) CDI, THF, RT (76%);
(c) BrCHRC(O)Cl, Et2O, Et3N, 0ºC to RT (62-73%); (d) NaH, THF,
RT then reflux (43-72%); (e) MeI, Cs2CO3, THF, RT (81%).
The synthesis of the 5-methyl substituted benzoxazepinone 23 is
outlined in Scheme 5. Treatment of the ketone 12 with methyl
magnesium bromide gave the racemic tertiary alcohol 22 in good
yield. Reaction of 22 with 2-bromoacetyl chloride and cyclization
gave 23.
O
MeO
MeO
NH2
O
O
MeO
MeO
NH
a
b
MeO
MeO
NH
O
MeO
Cl
F
Scheme 5. (a) MeMgBr, THF, 0oC (67%); (b) BrCH2C(O)Cl, Et2O,
Et3N, 0ºC to RT; (c) NaH, THF, RT then reflux (54% over 2-steps).
MeO
Cl
F
OMe
10
11
12
Compounds were assessed for activity in the luciferase reporter
assay, as previously described, at various concentrations (0.1 –
50 µM), and were compared to DMSO (0.1%) controls.19 Dose-
response curves were generated to determine EC50 values and
percent maximum increase in SMN2 expression. Unless
otherwise stated, all values are the mean S.E.M. of 3 separate
experiments. First, the importance of the tri-methoxy group on
the aryl ring and methyl substitution at each of the 1- and 3-
positions of the dihydro-quinolinone was investigated (Figure 2).
Removal of either the 7- or 6-methoxy group (e.g., compounds
5a and 5b) resulted in the complete loss of activity. With this
information, the methoxy groups were determined to be
necessary for activity, and the tri-methoxy group was retained in
the remaining analogs described in this optimization study.
c,d
e
H
N
H
N
O
MeO
MeO
O
MeO
MeO
N
N
MeO
Cl
F
MeO
Cl
F
13
14
Scheme 3. (a) BuLi, THF -78oC then methyl 5-chloro-2-
fluorobenzoate (15%); (b) H2SO4, EtOH, reflux (57%); (c)
Cl3CCOCl, DCM, Et3N, RT; (d) NH4OAc, DMSO, 80ºC (41% over

Similarly, the 1- and 3-methyl analogs (e.g., 6a and 6b) were
inactive, and these two positions were left unsubstituted in
tolerated, and introduction of a methyl group at the benzylic
position (e.g., 23) also was not tolerated.
R1
H
N
1
H
N
1
Table 2. Effects of the heterocyclic core (Ar = 2-F,5-Cl-phenyl)
O
F
MeO
O
F
MeO
MeO
N
O
7
1
3
6
#
Compound
#
Compound
MeO
R2
F
MeO
MeO
MeO
Br
H
N
H
N
MeO
MeO
O
MeO
MeO
O
Br
5a Inactive
Br
5b Inactive
9a
9b
NH
N
6a R1 = Me, R2 = H Inactive
6b R1 = H, R2 = Me Inactive
MeO
Ar
MeO
Ar
EC50 = 4.1 µM (263%)
EC50 = 6 µM (180%)
subsequent analogs.
H
H
Figure 2. Effects of deletion of individual methoxy substituents and
methyl substitution to the dihydro-quinolinone core.
MeO
MeO
N
O
MeO
MeO
N
O
9c
14
13
16
N
N
We next investigated the effects on SMN2 expression of
substituents on the 4-aryl ring (Table 1). Compounds were
prepared following the cyclization procedure outlined in Scheme
1. Removal of either the 5-bromo (e.g., 5c) or the 2-flouro (e.g.,
5d) led to a complete loss in activity, indicating the importance
of a substituent at both positions. Therefore, we retained the 2-
flouro substituent and varied the 5-substituent. Replacing the 5-
bromo by 5-chloro gave an analog (5e) of similar activity to 2,
but other electron withdrawing groups were not active (e.g., 5f,
5g, 5h and 5i). Several other halogenated analogs also were
found to be inactive (e.g., 5j, 5k, 5l and 5m).
MeO
Ar
MeO
Ar
Inactive
Inactive
H
N
O
H
N
MeO
MeO
MeO
MeO
O
O
N
MeO
Ar
MeO
Ar
EC50 = 8.4 (125%)
EC50 =? (125%)
H
O
N
H
O
MeO
MeO
N
MeO
MeO
19
21
20
23
O
O
Table 1. Effects of substitution on the aryl ring of the 3,4-
2(1H)-quinolinone
MeO
Ar
MeO
Ar
dihydro-4-aryl-
core.
EC50 = 5.9 (150%)
EC50 = 2.6 (125%)
H
N
MeO
MeO
O
O
H
N
O
N
MeO
MeO
MeO
MeO
MeO
Ar
O
O
MeO
2, 5a-5m
Ar
MeO
Ar
Inactive
Inactive
#
Ar
EC50 (µM)
#
Ar
EC50 (µM)
IA
2
2-F,5-Br
3-Br
8.3 (186%)
5c
5e
5g
5i
2-F
Given the promising activity of the 3,4-dihydro-quinazolinone
core (e.g., 9a), we evaluated the SAR for aryl substitution with
this heterocyclic core (Table 3). Removal of either the 5-chloro
(e.g., 9b) or the 2-flouro (e.g., 9c) led to a partial, but not a
complete, loss in activity. Other mono-halogenated analogs
retained some activity (e.g., 9d, 9e and 9g), although the
methoxy analogs (9h-9j) were inactive. In the next set of analogs,
the 2-fluoro substituent was retained, and the 5-substituent was
varied. The 5-methyl analog 9k had similar potency, and the 5-
fluoro 9n was less active. The other 5-substituted analogs (9l, 9m
and 9o) were inactive but moving the 5-chloro to the 4-position
(9p) was tolerated. Although the 2-methoxy analog (9h) activity
was restored on incorporation of a 4-chloro (9q) or 5-chloro (9r).
5d
5f
5h
IA
IA
IA
2-F,5-Cl
12 (150%)
2-F,5-F
2-F,5-NO2
2-F,5-CN
IA
IA
2-F,5-
CONH2
5j
5l
2-Cl,3-Cl
3-F,4-F
IA
IA
5k
2-Cl,4-Cl
3-Cl,4-Cl
IA
IA
5m
After the disappointing finding that limited substitution was
tolerated on the 4-aryl ring, we chose to explore other cyclic
cores. The goal was to identify a more potent core and then to
revisit the SAR on the 4-aryl ring. Although the 5-bromo-2-
fluoro aryl analog 2 was the most active compound, for chemical
stability reasons, we chose to retain the 5-chloro-2-fluoro aryl
ring at the benzylic position of the new cores (Table 2). The 3,4-
dihydro-quinazolinone derivative 9a had very promising activity,
although the N-methyl (9b) was less active, and both the N-
propyl (9c) and the 2-quinazolinone (13) were inactive. The
seven-membered benzodiazepin-2-one 14 retained potency
similar to the lead 2, although its efficacy was reduced. Several
of the oxygen containing heterocycles had interesting activity.
Table 3. Effects of substitution on the aryl ring of the 3,4-
quinazolin-2(1H)-one
dihydro-4-aryl-
core.
H
N
MeO
MeO
O
NH
MeO
9a-9l
Ar
#
Ar
2-F,5-Cl
3-Cl
EC50 (µM)
4.1 (263%)
5.2 (100%)
14.0 (150%)
6.6 (150%)
#
Ar
EC50 (µM)
10 (100%)
25 (150%)
IA
9a
9c
9e
9g
9b
9d
9f
2-F
3-F
The benzoxazin-2-one 16 had encouraging activity, and the 7-
membered benzoxazepin-2(3H)-ones, 19 and 20, had similar
activities as 9a. Interestingly, the 3-methyl analog 20 was tested
as a mixture of diastereoisomers, and it is likely that one isomer
will retain all activity. N-Methylation of the amide (21) was not
4-F
2-Cl
4-Cl
9h
2-OMe
IA

Compound 9a was next tested in a preliminary study in SMN∆7
mice. Animals were injected with 20 mg/kg of compound 9a in
DMSO by intraperotineal injection once daily starting on PND 2.
Compound 9a gave a significant increase in lifespan (e.g., 15-day
median survival) compared to vehicle treated animals (e.g., 6-day
median survival). Compound 9a treated animals also displayed
an increase in the ability to right themselves when compared to
vehicle treated animals.
9i
9k
9m
9o
3-OMe
2-F,5-Me
2-F,5-CN
2-F,5-OMe
18 (100%)
4.3 (125%)
IA
9j
9l
4-OMe
2-F,5-Ph
2-F,5-F
2-F,4-Cl
IA
IA
9n
9p
17 (100%)
6.3 (150%)
3.6 (225%)
IA
9q 2-OMe,4-Cl
7.8 (200%)
9r 2-OMe,5-Cl
Racemic 9a was next separated by supercritical fluid
chromatography, using a ChiralPak IC column, into its two
individual enantiomers in > 99% enantiomeric excess. Testing of
the individual enantiomers revealed that the fastest eluting
enantiomer was inactive, whereas the second enantiomer retained
all activity in the SMN2 luciferase reporter assay (EC50 = 1.3
µM, 185%).
In the preliminary SAR of the quinazolinone series, we did not
establish the importance of the 5-methoxy group on SMN
enhancing activity (e.g., deletion of the 5-OMe). We did,
however, establish the importance of a methoxy group at the
analogous position in the benzoxazepin-2(3H)-one series. The
synthesis of 26 is outlined in Scheme 6. Palladium-catalyzed
addition of the commercially available 5-chloro-2-
fluorophenylboronic acid and 2-amino-4,5-dimethoxybenzonitril
e 24 gave the corresponding ketone, which then was reduced with
sodium borohydride in ethanol to give the racemic alcohol 25.
Reaction of 25 with 2-bromoacetyl chloride, and cyclization as
described previously, gave the 7,8-dimethoxy analog 26.
Compound 26 was determined to be inactive in the luciferase
reporter assay. Combined, the lack of activity for 5a, 5b, and 26
establishes the importance of all three methoxy groups for
In summary, we established preliminary SAR of the
quinazolinone series and discovered that most changes to the aryl
ring substituents led to a loss in activity. We discovered several
alternative cores (e.g., quinazolinone the benzoxazepin-2(3H)-
one) that had similar or better activity than 2 and the SAR of the
aryl ring was expanded. Compound 9a was identified as a more
active analog with improved solubility and stability in mouse
microsomes. In an efficacy study in SMN∆7 mice, a 2-fold
extension in survival was observed following daily treatment
with 9a.
The synthesis, determination of absolute
stereochemistry and biological evaluation of single enantiomers
of 9a and related analogs is in progress and will be reported in
due course.
Acknowledgements. The authors thank the NIH (R01
HD064850, R21 NS064349, R21 HD057402), FightSMA,
CureSMA, and Gwendolyn Strong Foundation for grant support.
activity.
Scheme 6. (a) 2-F,5-Cl-PhB(OH)2, Pd(OAc)2, DMSO, 24 h, 90ºC
(79); (b) NaBH4, EtOH, RT (77%); (c) BrCH2C(O)Cl, Et2O, Et3N,
0ºC to RT (67%); (d) NaH, THF, RT to reflux (71%).
References and Notes
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Two promising analogs, the quinazolinone (9a) and the
benzoxazepin-2(3H)-one (19), were evaluated for aqueous
solubility and stability in mouse microsomes (Figure 3). Both 9a
and 19 had improved aqueous solubility, 9a had promising
stability in mouse microsomes although 19 was poor.
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Graphical Abstract
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Optimization of a series of heterocycles as
survival motor neuron gene transcription
enhancers
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Sungwoon Choia, Alyssa N. Caldera, Eliza H. Millera, Kierstyn P. Andersona, Dawid K. Fiejteka, Anne Rietzb,
Hongxia Lib, Jonathan J. Cherryb, Kevin M. Quistb, Xuechao Xinga, Marcie A. Glicksmana, Gregory D. Cunya,
Christian L. Lorsonc, Elliot A. Androphyb and Kevin J. Hodgettsa*
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