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

Abstract

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). However, 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 pyrazolone (5m), equipotent with 5a. Substitution at N² or excision of N², however, removed all activity. Therefore, 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.

Graphical Abstract

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig’s disease, is a progressive and ultimately fatal neurodegenerative disease, with a worldwide prevalence of approximately 2 per 100,000. The disease generally first presents between 40–60 years of age; loss of motor neurons controlling voluntary actions 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 particular, 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 aggregation 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 investigation 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.

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 in this screen was the arylsulfanylpyrazolones.¹⁴ Although ineffective 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 in 5a (Scheme 1, Table 1), which has an EC₅₀ = 0.067 μM in our G93A-SOD1-YFP assay method previously detailedbility identified.rties.tabollic ylpyrazolone type. on of compounds active against a cellu 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.

Scheme 1.

Synthesis of lead compound 5a via Weinreb amide intermediate 3.

Table 1.

SAR studies of substitution of the pyrazolone ring.

Compound	R1	R2	EC50 (μM)
a	H	H	0.067
b	Me	H	0.67
c	Me	Me	>32a
d	Biotin	H	0.56
e	Peg4-Biotin	H	0.67
f	CH2CH2OH	H	>32
g	CH2CH2CH2OH	H	0.34
h	Ph	H	1.03
i		H	1.73
j		H	>32
k		H	>32
l		H	1.00
m	Bn	H	0.13
n		H	0.36
o		H	>32
p	CHO	H	0.49
q	Ac	H	0.27
r		H	0.42
s		H	0.31
t		H	0.33
u		H	1.71
v		H	0.93
w		H	1.32

^a>32 μM indicated EC₅₀ not reached at the highest concentration used.

All values are reported as the mean average of three determinations.

Average Z’ factor value = 0.5.

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.

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; compounds with methyl (5b, EC₅₀ = 0.67 μM), biotin (5d, EC₅₀ = 0.56 μM) and tetraethyleneglycol-linked biotin (5e, EC₅₀ = 0.67 μM) all display the same activity over a substituent length from one to 24 atoms including, in the case of biotin compounds 5d and 5e, the presence of a bulky tetrahydro-1H-thieno(3,4-d)imidazol-2(3H)-one ring.

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 6-membered intramolecular hydrogen-bonded ring and blocking the hydrogen bond donating ability of the N² position. To support this hypothesis, the hydroxypropyl-substituted compound (5g) was synthesized from known 3-hydrazinylpropan-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 this hypothesis, 5g was active (EC₅₀ = 0.34 μM). The stability of the proposed six-membered hydrogen bonded ring has been 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 activity between 5f and 5g. Explanations relating to solubility, cell permeability, or bioavailability are unlikely, given the one methylene difference from that of 5g.

An alternative possibility regarding the importance of hydrogen bonding for activity is that the tautomeric enol form of the pyrazolone 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) (Figure 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 relevant, both of those methylated compounds would have been active.

Figure 1.

Tautomeric forms of the pyrazolones

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 activity of the pyrazolone (5h, EC₅₀ = 1.03 μM), allowing further characterization 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, containing multiple strong electron-withdrawing substituents was observed. An explanation for the reduced activity seen with 5h and 5i may be the steric bulk of the introduced phenyl ring, which impedes 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 pyrazolone 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 substituted analogue (5l) is equipotent (EC₅₀ = 1.00 μM) with phenyl substituted pyrazolone 5h, illustrating that five-membered aromatics and bulky bicyclics are tolerated at this position.

The moderate activity displayed by phenyl substitution, coupled with the relatively large steric pocket that N¹ substituents inhabit, 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 pyrazolones yet synthesized, having an EC₅₀ = 0.13 μM, essentially equivalent to that of 5a (EC₅₀ = 0.067 μM) within the reproducibility 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 μM). Introduction of a para-methoxyl substituent (5o) renders the compound inactive. It is not clear why the para-methoxyl functionality exerts a deactivating 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 identification studies of these compounds phenylisothiocyanate 5t was prepared. 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 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 hydrogen 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. Lactone 9 was obtained from the nucleophilic coupling of known bromo-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.

Scheme 2.

Synthesis of lactone analogue 9.

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 biological activity.

Scheme 3.

Synthesis of cyclopentanone 13

The isoxazol-3(2H)-one analogue (17) was synthesized (Scheme 4) using a modified procedure²². 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 bromomethylisoxazole 16. Subsequent addition of 3,5-dichlorophenol provided 17, which, lacking the crucial N² hydrogen bond donating group, displayed no activity.

Scheme 4.

Synthesis of 3-bromo-4,5-dihydroisoxazole analogue 17.

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 available pyrrolidinone ester 18 to form alcohol 19, followed by mesylation 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 phenylselenyl bromide to the prepared enolate and selenoxide elimination provided unsaturated pyrrolidinone 22, identical to the most potent analogue (5m) except lacking the crucial N²-H. Again, both 21 and 22, possessing no N² hydrogen bond donor, had no activity (EC₅₀ = >32 μM).

Scheme 5.

Synthesis of protected pyrolidinone analogue 22

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 degree 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. Placement of a benzyl group β to the pyrazolone N¹ (5m) enhances its potency, suggesting the presence, within the target, of a hydrophobic pocket, possibly containing aromatic residues. The importance of the N²-H group to participate in hydrogen bond donating interactions 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.

Abbreviations

ALS

amyotrophic lateral sclerosis

FALS

familial amyotrophic lateral sclerosis

SALS

sporadic amyotrophic lateral sclerosis

SOD1

Cu/Zn superoxide dismutase 1