Substituted pyrazolones require N2 hydrogen bond donating ability to protect against cytotoxicity from protein aggregation of mutant superoxide dismutase 1

Article abstract of DOI:10.1016/j.bmcl.2012.08.114

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. Although the cause remains unknown, misfolded protein aggregates are seen in neurons of sporadic ALS patients, and familial ALS mutations, including mutations in su

Full text of DOI:10.1016/j.bmcl.2012.08.114

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6647–6650
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted pyrazolones require N2 hydrogen bond donating ability
to protect against cytotoxicity from protein aggregation of mutant superoxide
dismutase 1
Paul C. Trippier a, Radhia Benmohammed b, Donald R. Kirsch b, Richard B. Silverman a,c,
⇑
a Department of Chemistry, Northwestern University, Evanston, IL, USA
b Cambria Pharmaceuticals, Cambridge, MA, USA
c Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. Although the
cause remains unknown, misfolded protein aggregates are seen in neurons of sporadic ALS patients,
and familial ALS mutations, including mutations in superoxide dismutase 1 (SOD1), produce proteins
with an increased propensity to misfold and aggregate. A structure activity relationship of a lead scaffold
exhibiting neuroprotective activity in a G93A-SOD1 mouse model for ALS has been further investigated in
a model PC12 cellular assay. Synthesis of biotinylated probes at the N1 nitrogen of the pyrazolone ring
gave compounds (5d–e) that retained activity within 10-fold of the proton-bearing lead compound
(5a) and were equipotent with a sterically less cumbersome N1-methyl substituted analogue (5b). How-
ever, when methyl substitution was introduced at N1 and N2 of the pyrazolone ring, the compound was
inactive (5c). These data led us to investigate further the pharmacophoric nature of the pyrazolone unit. A
range of N1 substitutions were tolerated, leading to the identification of an N1-benzyl substituted pyraz-
olone (5m), equipotent with 5a. Substitution at N2 or excision of N2, however, removed all activity. There-
fore, the hydrogen bond donating ability of the N2–H of the pyrazolone ring appears to be a critical part of
the structure, which will influence further analogue synthesis.
Received 28 June 2012
Revised 14 August 2012
Accepted 28 August 2012
Available online 7 September 2012
Keywords:
Amyotrophic lateral sclerosis
Superoxide dismutase 1
Neuroprotection
Pyrazolone
Pharmacophore
Ó 2012 Elsevier Ltd. All rights reserved.
Amyotrophic lateral sclerosis (ALS), commonly known as Lou
Gehrig’s disease, is a progressive and ultimately fatal neurodegen-
erative disease, with a worldwide prevalence of approximately 2
per 100,000. The disease generally first presents between 40 and
60 years of age; loss of motor neurons controlling voluntary ac-
tions results in progressive muscle paralysis and death attributed
to respiratory failure, typically within 3–5 years of diagnosis.1
Currently, the only approved drug for ALS, riluzole, extends
median survival by only 2–3 months.2 Clearly, there is a need for
new therapeutics; however, progress has been impeded because
the underlying pathology of the disease remains unknown. There
is strong evidence3 that one pathophysiological mechanism in par-
ticular, that of toxic protein misfolding and/or aggregation, may
trigger motor neuron dysfunction and loss. Mutations in familial
ALS (FALS) patients that promote protein misfolding and aggrega-
tion include Cu/Zn superoxide dismutase type 1 (SOD1),4 UBQLN2,5
TAR DNA binding protein (TDP-43),6 fused in sarcoma/translated in
liposarcoma (FUS/TLS),7 and angiogenin (ANG)8 and, in addition,
TDP-43 aggregates are seen in motor neurons of sporadic ALS
(SALS) patients.9
The clinical phenotype and pathology of SALS, which accounts
for 90% of all ALS cases, are indistinguishable from those of the
familial form.10 It has therefore been possible to make significant
advances in studying the pathology of ALS through the investiga-
tion of the familial form of the disease,11 which accounts for only
approximately 10% of all cases, 20% of which are caused by
missense mutations in the gene encoding for the enzyme SOD1.
Recent studies12 have now linked FALS to SALS through common
SOD1 containing astrocytes, demonstrating that SOD1 is a viable
target for both FALS and SALS, providing further impetus to the
identification of compounds active in mutant SOD1 disease
models.
Abbreviations: ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic
lateral sclerosis; SALS, sporadic amyotrophic lateral sclerosis; SOD1, Cu/Zn super-
oxide dismutase 1.
We previously described13 a high throughput screening method
for the identification of compounds active in a PC12 cell model in
which protein aggregation and cell death depended on the
expression of G93A SOD1. One of the chemical scaffolds identified
⇑
Corresponding author at: Department of Chemistry, Northwestern University,
2145 Sheridan Road, Evanston, IL 60208-3113, USA. Tel.: +1 847 491 5653; fax: +1
847 491 7713.
E-mail addresses: Agman@chem.northwestern.edu, r-silverman@northwestern.
edu (R.B. Silverman).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.114

6648
P. C. Trippier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6647–6650
in this screen was the arylsulfanylpyrazolones.14 Although ineffec-
tive as a potential therapeutic because of the metabolic instability
of the sulfanyl linker, this scaffold has served as a valuable lead for
detailed structure–activity relationship (SAR) studies,15 resulting
Table 1
SAR studies of substitution of the pyrazolone ring
Cl
O
O
in 5a (Scheme 1, Table 1), which has an EC50 = 0.067 lM in our
N
G93A-SOD1 cell assay and is active in a mutant SOD1 mouse model
of ALS. Methylation of 5a to 5b (Table 1) or conjugation with biotin
(5d) gave compounds with similar EC50 values, but dimethylation
to 5c gave an inactive compound. This intriguing result led us to
investigate the cause for the inactivity of 5c relative to much more
sterically-demanding active compounds, such as 5b and 5d. Here,
we describe the SAR profile of the pyrazolone ring; substitution,
excision, and replacement of one or both of the two nitrogen atoms
is described, and the pharmacophoric nature of the N2-position is
demonstrated.
N
R1
Cl
R2
Compound
R1
R2
EC50 (lM)
a
b
c
d
e
f
H
Me
Me
H
H
Me
H
H
0.067
0.67
>32a
0.56
0.67
>32
Biotin
Peg4–Biotin
CH2CH2OH
CH2CH2CH2OH
Ph
H
The synthetic route delineated in Scheme 1 was modified in
step iv to include the addition of suitably substituted hydrazine
reagents, providing access to a wide range of N1-substituted
pyrazolone analogues (Table 1). Phenyl, heteroaryl, alkyl, and
acyl-substituted pyrazolones were synthesized and their activities
determined.
g
h
H
H
0.34
1.03
i
H
H
1.73
Cl
Alkyl substitution at the N1-position of the pyrazolone ring is
tolerated, albeit with an approximately 10-fold reduction in cell
activity compared to 5a. All of the N1-substituted compounds were
active except for 5f, 5j, 5k, and 5o. Steric hindrance, apparently,
plays little or no role in the activity of the compounds synthesized;
j
>32
k
H
>32
compounds with methyl (5b, EC50 = 0.67
l
M), biotin (5d, EC50
=
=
0.56
0.67
l
l
M) and tetraethyleneglycol-linked biotin (5e, EC50
M) all display the same activity over a substituent length
N
l
H
H
1.00
0.13
from 1 to 24 atoms including, in the case of biotin compounds
5d and 5e, the presence of a bulky tetrahydro-1H-thieno(3,4-d)imi-
dazol-2(3H)-one ring.
S
m
Bn
The inactivity of 5f is intriguing because it is small and not
bulky. As an explanation for this lack of activity, we considered
the possibility that the compound exists in a bicyclic structure
with the hydroxyl hydrogen bonded to the N2 position, thereby
forming a six-membered intramolecular hydrogen-bonded ring
and blocking the hydrogen bond donating ability of the N2 posi-
tion. To support this hypothesis, the hydroxypropyl-substituted
compound (5g) was synthesized from known 3-hydrazinylpro-
pan-1-ol. 16 The corresponding hydrogen bonding in 5g would lead
to an unfavorable seven-membered ring, which therefore would
not be expected to form, allowing the hydrogen bond donating
ability of the N2 position to be unobstructed. In agreement with
n
H
0.36
OH
o
H
>32
OMe
p
q
CHO
Ac
H
H
0.49
0.27
O
r
H
0.42
CN
s
H
0.31
this hypothesis, 5g was active (EC50 = 0.34 lM). The stability of
the proposed six-membered hydrogen bonded ring has been
O
O
t
H
H
H
0.33
1.71
0.93
Cl
NCS
O
O
iii
ii
Br
O
O
R
N
S
N
u
v
Cl
1
2
Br
N(OCH3)CH3
N
O
i
3
Cl
Cl
Cl
NH2
O
O
iv
O
R
O
O
O
OEt
N
N
H
Cl
w
H
1.32
N
5a R = H
4
a
>32 lM indicated EC50 not reached at the highest concentration used. All values
Reactants and conditions: i) TEA, (Me)N(OMe).HCl, DCM (65%);
ii) 3,5-dichlorophenol, NaOEt, EtOH, heat (47%); iii) LiHMDS,
EtOAc, THF,-78 oC (56%); iv) NH2NHR, EtOH, RT (28%).
are reported as the mean average of three determinations. Average Z0 factor
value = 0.5.
estimated to be 20 kJ/mol17 (4.8 kcal/mol) and 29 kJ/mol
(6.9 kcal/mol),18 which could account for the large difference in
Scheme 1. Synthesis of lead compound 5a via Weinreb amide intermediate 3.

P. C. Trippier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6647–6650
6649
activity between 5f and 5g. Explanations relating to solubility, cell
permeability, or bioavailability are unlikely, given the one methy-
lene difference from that of 5g.
gel moiety to 5t will determine if this approach will be successful
in a protein pull-down experiment.
To further establish the pharmacophoric nature of the N2 hydro-
gen bond donor group, a series of pyrazolone ring analogues was
synthesized, which replaced N2 and other positions around the
pyrazolone ring with heteroatoms and/or methylene groups. Lac-
tone 9 was obtained from the nucleophilic coupling of known bro-
mo-substituted lactone 820 and 3,5-dichlorophenol (Scheme 2) in
analogy with compounds 5a–w. The lactone, lacking the hydrogen
bond donor ability of the pyrazolone nitrogen at N2, was inactive in
the assay, providing further evidence for the importance of the N2
position for bioactivity.
An alternative possibility regarding the importance of hydrogen
bonding for activity is that the tautomeric enol form of the pyraz-
olone is the active form, and it is the enol hydroxyl that is essential
for activity. If that were the case, there are two tautomeric forms,
one that retains the N1–H and one that retains the N2–H (both still
having the enol OH) (Fig. 1). If the OH were the important H-bond
donor, the activity would not be affected if the compound were
methylated at N1 or N2. In a related series of compounds from
our group,19 the N1-methylated analogue was active, but the
N2-methylated analogue was not active. If the enol OH were rele-
vant, both of those methylated compounds would have been
active.
Replacement of the pyrazolone ring with a cyclopentanone ring
(13) was achieved through an analogous coupling of known iodide
1221 with 3,5-dichlorophenol (Scheme 3) to produce a compound
devoid of both nitrogen atoms of the pyrazolone ring and of biolog-
ical activity.
The isoxazol-3(2H)-one analogue (17) was synthesized
(Scheme 4) using a modified procedure.22 The lithium acetylide
of propargyl bromide was treated with ethyl chloroformate to yield
bromotetrolate 15; addition of an aqueous methanolic solution of
hydroxylamine and careful control of pH afforded bromomethylis-
oxazole 16. Subsequent addition of 3,5-dichlorophenol provided
17, which, lacking the crucial N2 hydrogen bond donating group,
displayed no activity.
An increase in electron-withdrawing ability should have an
important effect on activity because of its decrease in the pKa of
N2–H and corresponding promotion of hydrogen bond donation
ability. Suprisingly, phenyl substitution at N1 attenuated the activ-
ity of the pyrazolone (5h, EC50 = 1.03 lM), allowing further charac-
terization of the relatively expansive pocket environment the N1
substituents inhabit. A further decrease in activity of analogue 5i
and total loss of activity with N1-phenyl analogues 5j and 5k, con-
taining multiple strong electron-withdrawing substituents was ob-
served. An explanation for the reduced activity seen with 5h and 5i
may be the steric bulk of the introduced phenyl ring, which im-
pedes the N2 position and prevents hydrogen bond donation. The
lack of activity of 5j and 5k may be attributed to steric crowding
by the ortho substituents, which completely block the hydrogen
bond donating ability at N2, further supporting our assertion that
this position represents an important pharmacophore of the pyraz-
olone moiety. These results support the importance of N2–H in its
activity, presumably some hydrogen-bonding interaction with a
target that is essential for activity. The N1-phenylthiazole substi-
Another ring system lacking the N2 hydrogen bond donor is
illustrated by benzyl protected unsaturated pyrolidinone 22
(Scheme 5). This was obtained by reduction of commercially avail-
able pyrrolidinone ester 18 to form alcohol 19, followed by mesy-
lation and nucleophilic addition of a bromide ion to furnish
bromide 20 in good yield (42%) relative to the alternative Appel
reaction (PBr3, pyridine; 2% yield); addition of 3,5-dichlorophenol
to 20 provided pyrrolidinone 21. Subsequent addition of phenylsel-
enyl bromide to the prepared enolate and selenoxide elimination
provided unsaturated pyrrolidinone 22, identical to the most po-
tent analogue (5m) except lacking the crucial N2–H. Again, both
21 and 22, possessing no N2 hydrogen bond donor, had no activity
tuted analogue (5l) is equipotent (EC50 = 1.00 lM) with phenyl
substituted pyrazolone 5h, illustrating that five-membered aro-
matics and bulky bicyclics are tolerated at this position.
The moderate activity displayed by phenyl substitution, cou-
pled with the relatively large steric pocket that N1 substituents in-
habit, suggested the synthesis of a benzyl-substituted pyrazolone,
providing steric relief from the phenyl moiety by the methylene
spacer. Compound 5m was the most potent of the substituted
(EC50 = >32 lM).
In conclusion, we have shown that the N2 pyrazolone position is
essential for the cellular activity of this class of compounds. While
other factors such as cell membrane penetration may play a role in
the activity patterns of some of these compounds, a sufficient de-
gree of evidence has been accumulated to support the hypothesis
that the hydrogen bond donating N2–H group is pharmacophoric
in these compounds. SAR data have provided further information
on the interactions of 5a and its analogues. Steric considerations
are of little importance around the pyrazolone N1 area, suggesting
a large open pocket or corridor within the target structure. Place-
ment of a benzyl group b to the pyrazolone N1 (5m) enhances its
pyrazolones yet synthesized, having an EC50 = 0.13 lM, essentially
equivalent to that of 5a (EC50 = 0.067 lM) within the reproducibil-
ity of the assay.13 Substitution around the benzyl ring attenuates
bioactivity; meta-hydroxybenzyl analogue 5n exhibits an almost
three-fold reduction in potency (EC50 = 0.36 lM). Introduction of
a para-methoxyl substituent (5o) renders the compound inactive.
It is not clear why the para-methoxyl functionality exerts a deacti-
vating effect.
Acyl substitution appears to be well tolerated. Pyrazolones
5p–5t have comparable EC50 values, and 5u–5w are comparable
but about one-third less potent. To aid in future target identifica-
tion studies of these compounds phenylisothiocyanate 5t was pre-
pared. It was anticipated that this compound would act as a
covalent linker, forming a bond with a suitably disposed residue
within the target of action active site for use in subsequent affinity
chromatography experiments. This was among the most potent of
the acyl-substituted pyrazolones. Appendage of a biotin or affinity
Br
Br
CO2H
CO2H
ii
i
7
6
Cl
Cl
Br
iii
O
O
O
O
8
O
9
O
OH
NH
OH
Reagents and Conditions: i) NBS, CCl4, Benzoyl
peroxide, reflux (98%); ii) 5% NaOH (61%); iii)
NaOEt, EtOH, reflux (14%)
R
R
R
NH
N
N
H
N
N
H
Figure 1. Tautomeric forms of the pyrazolones.
Scheme 2. Synthesis of lactone analogue 9.

6650
P. C. Trippier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6647–6650
potency, suggesting the presence, within the target, of a hydropho-
O
O
O
ii
bic pocket, possibly containing aromatic residues. The importance
of the N2–H group to participate in hydrogen bond donating inter-
actions with the biological target serves to promote this scaffold as
both a lead compound for further therapeutic investigation and as
a probe compound to potentially identify the biological target for
these compounds.
i
I
10
11
12
Cl
Cl
O
iii
O
Acknowledgments
13
We thank the National Institutes of Health (Grant
1R43NS057849), the ALS Association (TREAT program), and the
Department of Defense (Grant AL093052) for funding.
Reagents and Conditions: i) Me3SOI, DMSO,
NaH (33%); ii) Me3SiCl, NaI, MeCN, RT
(39%); iii) 3,5-dichlorophenol, NaOEt, EtOH,
reflux (27%)
Supplementary data
Scheme 3. Synthesis of cyclopentanone 13.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.114.
References and notes
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Scheme 4. Synthesis of 3-bromo-4,5-dihydroisoxazole analogue 17.
Br
MeO2C
HO
i)
ii)
O
O
O
N
N
N
Ph
Ph
Ph
18
19
20
Cl
Cl
Cl
iii)
iv)
O
O
O
O
Cl
N
N
Ph
Ph
17. Porath, B.; Rademacher, P.; Boese, R.; Blaser, D. Z. Naturforsch. 2002, 57, 365.
18. Ferguson, L. N. The modern structural theory of organic chemistry; Prentice-Hall:
Englewood Cliffs, NJ, 1963. p. 128.
21
22
19. Zhang, Y.; Kirsch, D.R.; Silverman, R.B. Manuscript in preparation.
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Reagents and Conditions: i) LiBH4, THF, RT (99%); ii) PBr3, Py,
DCM (2%) or MsCl, NEt3 then LiBr (42%); iii) 3,5-dichlorophenol,
NaOEt, DMF, reflux (24%); iv) a) LDA, PhSeBr, THF, -78oC, b) H2O2,
H2O, DCM (27%).
Scheme 5. Synthesis of protected pyrolidinone analogue 22.