Discovery of N-Acetyltransferase 8-Like (NAT8L) inhibitors based on a N-Acylated (Piperidin-3-ylmethyl)-1,2,4-Oxadiazole Scaffold

Abstract

Canavan disease (CD) is an autosomal recessive genetic disorder caused by mutations in the ASPA gene, which encodes the enzyme aspartoacylase. These mutations lead to a deficient enzymatic activity and increased concentrations of its substrate, N-acetylaspartate (NAA), in the brain and other tissues. Aspartate N-acetyltransferase, encoded by the N-acetyltransferase 8-like (NAT8L) gene, catalyzes the biosynthesis of NAA from aspartate and acetyl-CoA. Therefore, inhibition of NAT8L has been implicated as a promising therapeutic strategy for CD by normalizing NAA levels in the brain. Our high throughput screening campaign followed by a rigorous hit validation process identified 2-(2-fluorophenoxy)-1-(3-((3-(thiophen-3-yl)-1,2,4-oxadiazol-5-yl)methyl)piperidin-1-yl)ethan-1-one (4a) as a low micromolar, noncarboxylic acid inhibitor of NAT8L. Subsequent structural optimization led to the discovery of two submicromolar NAT8L inhibitors. Although these inhibitors displayed high clearance in liver microsomes, the new scaffold, devoid of a carboxylic acid moiety, could potentially lead to potent and brain-penetrant NAT8L inhibitors through further molecular refinement.

Graphical Abstract

Canavan disease (CD) is an autosomal recessive inherited leukodystrophy caused by the mutations in ASPA gene encoding the aspartoacylase (ASPA) enzyme, leading to loss of enzyme activity and elevated concentrations of its substrate N-acetylaspartate (NAA), especially in the brain.¹ Accumulation of high levels of NAA results in spongiform degeneration of white matter, aberrant myelination, brain edema, macrocephaly, and severe cognitive and motor deficits. Symptoms of CD usually appear within the first 3 to 6 months of life and progress rapidly. Death usually occurs before age 10, although some children may survive into their teens and twenties. Currently, there is no cure or disease-modifying treatment for CD.²

Aspartate N-acetyltransferase encoded by the N-acetyltransferase 8-like (NAT8L) gene is expressed in the mitochondria of neuronal cells and catalyzes the formation of NAA from aspartate and acetyl-CoA. Inhibition of NAT8L has been postulated to reduce excess brain NAA levels caused by ASPA enzyme deficiency and alleviate the NAA-driven pathology in patients with CD. Indeed, genetic disruption of NAT8L in the CD mouse model was found to reduce NAA levels in the brain and alleviate CD-like pathology, indicating the therapeutic potential of small molecule NAT8L inhibitors.^3–8 Currently, however, there are no clinically available small molecule NAT8L inhibitors. Thangavelu and et al. screened a small biased set of compounds using the recombinant human enzyme, leading to the discovery of potent NAT8L inhibitors (Figure 1; 1–3).⁹ The NAT8L inhibitors in that report include [(benzyloxy)carbonyl]-L-aspartic acid 1 with a K_i value of 17 μM and phthalate derivatives 2a–b with K_i values in the submicromolar range (K_i = 0.6–0.8 μM). Although this is important progress, none of these compounds are expected to serve as useful molecular templates for CNS drug design due to the presence of multiple carboxylic acid moieties. More recently, five potent NAT8L inhibitors were identified using a homology model and a deep convolutional neural network.¹⁰ including noncarboxylic acid 2,1,3-benzoxadiazole derivative 3, the most potent inhibitor in this report with a K_i value of 0.4 μM. 2,1,3-Benzoxadiazole derivatives similar to compound 3, however, have been reported to react with thiol trapping agents.^11–12 Indeed, our counter screening assay revealed that compound 3 interferes with the assay detection system in our fluorescent-based NAT8L assay (Supporting Information), raising concerns over its ability to directly interact with NAT8L.

Figure 1.

Representative NAT8L inhibitors 1-3 and our screening hit 4a

In this study, we screened 100,000-compound drug-like library using a fluorescence-based high throughput screening (HTS) assay developed by our team.¹³ We identified 2-(2-fluorophenoxy)-1-(3-((3-(thiophen-3-yl)-1,2,4-oxadiazol-5-yl)methyl)piperidin-1-yl)ethan-1-one (4a) as a low micromolar noncarboxylic acid NAT8L inhibitor after rigorous hit validation process. Subsequent structural optimization efforts using compound 4a as a molecular template led to the discovery of submicromolar NAT8L inhibitors, marking an important advancement toward a new treatment option for patients with CD.

We screened 100,000 compounds (preplated diversity sets PS4 and PS5 assembled by Life Chemicals) in a high throughput screening assay for NAT8L using 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) to detect the byproduct CoA as we have previously described.¹³ Primary screening at a single concentration (10 μM) identified (2-fluorophenoxy)-1-(3-((3-(thiophen-3-yl)-1,2,4-oxadiazol-5-yl)methyl)piperidin-1-yl)ethan-1-one 4a as a noncarboxylic acid hit compound. Subsequently, compound 4a was resynthesized and subjected to a series of hit confirmation assays (Table 1). Compound 4a inhibited NAT8L in a dose-dependent manner with an IC₅₀ value of 2.9 μM. To rule out assay interference, a counter assay was performed to examine the influence of 4a on the reaction of CoA and CPM. No interference was observed, eliminating the possibility of compound 4a acting as a false positive by directly interacting with CPM and/or CoA. It should be noted that compound 3 was found to interfere with the counter assay (Supporting Information), possibly due to a nucleophilic aromatic substitution reaction between 3 and CoA.^11–12 The inhibitory activity of compound 4a was further confirmed by an orthogonal radioactivity-based assay using [U-¹⁴C]-aspartate as a substrate. To rule out the possibility of non-selective inhibition of N-acetyltransferases, inhibitory activity of compound 4a was tested against arylamine N-acetyltransferase 1 (NAT1) and CREB-binding protein histone acetyltransferase (CREBBP HAT), two additional acetyl-CoA-dependent N-acetyltransferases. Compound 4a did not achieve 50% inhibition of either enzyme at the highest concentration tested (100 μM), demonstrating >30-fold selectivity for NAT8L over the two acetyl-CoA dependent N-acetyltransferases. Taken together, our hit validation studies demonstrated that compound 4a explicitly inhibits the enzymatic activity of NAT8L in a dose-dependent manner with a low probability of acting as a nonspecific inhibitor of acetyl-CoA-dependent N-acetyltransferases.

Table 1.

Preliminary Profiling of Compound 4a

Assay	Result
NAT8L Fluorescent-based assay	IC50 = 2.9 ± 1.1 μM
NAT8L Counter assay	IC50 > 100 μM
NAT8L Radiometric assay	IC50 = 5 ± 1 μM
NAT1 Fluorescent-based assay	IC50 > 100 μM
CREBBP HAT Radiometric assay	IC50 > 100 μM
Kinetic solubility in PBS (pH 7.4)	33 μM
Metabolic stability in human liver microsomes	w/o NADPH: >95% remaining after 30 min
	w/ NADPH: <5% remaining after 30 min
Metabolic stability in mouse liver microsomes	w/o NADPH: >95% remaining after 30 min
	w/ NADPH: <5% remaining after 30 min
MDCKII-MDR1 cell permeability	Papp (A-B) = 20.8 × 10−6 (cm/s)
	Papp (B-A) = 24.5 × 10−6 (cm/s)
	Efflux ratio = 1.18
CNS MPO Score	4.5
BBB Score	4.6

Subsequently, we assessed the aqueous solubility, metabolic stability in liver microsomes, and bidirectional MDCK-MDR1 permeability of 4a (Table 1). Compound 4a exhibited solubility in PBS (33 μM) and was rapidly cleared in both mouse and liver microsomes in a CYP-dependent manner. It showed good apparent permeability with a low efflux ratio, indicating a low risk for P-gp-mediated efflux. Due to the high clearance observed in liver microsomes, in vivo experiments were not performed to assess the brain distribution of compound 4a. However, compound 4a exhibits desirable CNS MPO¹⁴ and BBB¹⁵ scores (>4 on a scale from 0 to 6), suggesting favorable CNS permeability.

We next investigated the mode of inhibition of 4a by assessing the degree of inhibition at varying concentrations of aspartate or acetyl-CoA. As shown in Figure 2, Lineweaver–Burk reciprocal plots indicate that compound 4a is a competitive inhibitor with respect to aspartate with a K_i value of 3 μM while it displays noncompetitive inhibition with respect to acetyl-CoA. Previously, we identified compound 1 as a competitive inhibitor of NAT8L with respect to aspartate, as anticipated based on its structural similarity to aspartate.¹³ The similar mode of inhibition exhibited by the noncarboxylic acid compound 4a was unexpected and may account for its selectivity for NAT8L over NAT1 and CREBBP HAT.

Figure 2.

Michaelis−Menten (A) and Lineweaver−Burk (B) plots for NAT8L at varying concentrations (0−25 μM) of compound 4a and aspartate with a fixed concentration of Acetyl-CoA (50 μM). Michaelis−Menten (C) and Lineweaver−Burk (D) plots for NAT8L at varying concentrations of compound 4a and acetyl-CoA with a fixed concentration of aspartate (1 mM).

Overall, compound 4a showed a favorable profile for initiating structural optimization efforts with the primary objective of improving inhibitory potency and metabolic stability. Given the absence of the three-dimensional structure of NAT8L and the structural dissimilarity of compound 4a to aspartate, we chose to undertake unbiased yet systematic structural modifications of compound 4a. Our initial phase of chemistry focused on incorporating various acyl groups into the piperidyl nitrogen. Some of these compounds including compound 4a were prepared according to the synthetic route outlined in Scheme 1. N-Hydroxythiophene-3-carboximidamide 5 was coupled with 2-(1-(tert-butoxycarbonyl)piperidin-3-yl)acetic acid 6 to form 1,2,4-oxadiazole derivative 7. After removal of the tert-Boc group with TFA, the piperidyl nitrogen of 8 was acylated with various carboxylic acids to give compounds 4a-h. Additional analogs 4i-u were synthesized in one step from chloroacetamide intermediate 9 and various phenols as illustrated in Scheme 2.

Scheme 1.

Synthesis of Compounds 4a−h^a

^aReagents and Conditions: (a) HBTU, DIPEA, DMF, 100 °C, 12 h; (b) TFA, DCM, rt, 1–1.5 h; (c) RCO₂H, HATU, DIPEA DMF, rt, 16 h.

Scheme 2.

Synthesis of Compounds 4i−u^a

^aReagents and Conditions: (a) Chloroacetyl chloride, TEA, DCM, 0 °C to rt, 14 h; (b) ROH, K₂CO₃, ACN, rt to reflux, 2–20 h.

As illustrated in Scheme 3, several substituents are introduced as replacements for the thiophen-3-yl group of compound 4a by using N-hydroxycarboximidamide 10a–e as starting materials. Compounds 13a–e were synthesized by following the same synthetic route as depicted in Scheme 1.

Scheme 3.

Synthesis of Compounds 13a-e^a

^aReagents and Conditions: (a) HBTU, DIPEA, DMF, 100 °C to rt, 12 h; (b) TFA, DCM, rt, 1–1.5 h; (c) 2-(2-fluorophenoxy)acetic acid, HATU, DIPEA DMF, rt, 16 h.

Given that the piperidine linker of compound 4a possess a chiral center at the 3-position and exists as a racemic mixture, we synthesized both enantiomers (R)-4a and (S)-4a by using (R)-6 or (S)-6 as a starting material (Scheme 4), respectively, and following the synthetic procedure used for preparation of 4a as described in Scheme 1.

Scheme 4.

Synthesis of Compounds (R)-4a and (S)-4a^a

^aReagents and Conditions: (a) (R)-6, HBTU, DIPEA, DMF, 100 °C, 12 h; (b) (S)-6, HBTU, DIPEA, DMF, 100 °C, 12 h; (c) TFA, DCM, rt, 1h; (d) 2-(2-fluorophenoxy)acetic acid, HATU, DIEA, DMF, 0 °C to rt, 12 h

To further investigate the effects of structural modifications on the piperidine linker of 4a, we replaced the piperidine ring with other nitrogen-containing aliphatic rings by using tert-Boc-protected derivatives 14a–d (Scheme 5) as starting materials and followed the synthetic route depicted in Scheme 1.

Scheme 5.

Synthesis of Compounds 17a−d^a

^aReagents and Conditions: (a) Appropriate acid, HBTU, DIPEA, DMF, 100 °C to rt, 16 h; (b) TFA, DCM, rt, 1–1.5 h; (c) 2-(2-fluorophenoxy)acetic acid, HATU, DIPEA DMF, rt, 16 h.

The NAT8L inhibitory potency of the synthesized compounds was measured using the fluorescence-based assay as previously described.¹³ All compounds screened in the primary assay were also evaluated in a separate counter assay and showed no assay interference at concentrations up to 100 μM. The inhibitory potency of compounds 4a-u with varying acyl groups on the piperidyl nitrogen are summarized in Table 2. The conversion of the 2-fluorophenyl of 4a to a methyl group resulted in the complete loss of potency as seen in compound 4b. The removal of the 2-fluoro group (compound 4c), replacement of the 2-fluoro group with a methyl (compound 4d) or a methoxy (compound 4e) group, the addition of a methyl group to the α-position of the acyl group (compound 4f), and substitution of the ether oxygen with a sulfur (compound 4g) or methylene group (compound 4h) also led to varying degrees of loss of potency. Subsequently, we explored other fluorinated phenyl groups instead of the 2-fluorophenyl groups (compounds 4i–n). Among them, compound 4l possessing a 2,3-difluorophenyl group displayed an inhibitory activity of equal potency to compound 4a. It appears 2-susbtituted and 2,3-disubstituted phenyl rings are preferred as NAT8L inhibitors. Among additional 2-substituted phenyl rings (compounds 4o–r and 4t), the 2-chlorophenyl analog 4t exhibited an IC₅₀ value of 0.3 μM against NAT8L, a 10-fold higher potency compared to compound 4a. The 2,3-dichlorophenyl derivative 4u had a similar potency to compound 4t, following the trends observed in the corresponding fluorinated analogs 4a and 4l.

Table 2.

Inhibition of NAT8L by Compounds 4a−u.

Cmpd	R R2	IC50 (μM)a	Cmpd	R	IC50 (μM)a
4a		2.9 ± 1.1	4l		2.5 ± 0.8
4b		>100	4m		21 ± 2
4c		44 ± 1	4n		11 ± 3
4d		13 ± 2	4o		>100
4e		9.3 ± 1.1	4p		4.3 ± 0.9
4f		45 ± 8	4q		>100
4g		14 ± 2	4r		>100
4h		>100	4s		>100
4i		70 ± 7	4t		0.3 ± 0.1
4j		25 ± 4	4u		0.4 ± 0.1
4k		65 ± 6

^aValues are mean ± SD of at least three experiments.

The NAT8L inhibitory potency of analogs of 4a, in which its thiophen-3-yl group was replaced with other moieties, is summarized in Table 3. All replacements except for a thiophen-2-yl group (compound 13b) showed substantial or complete loss of inhibitory potency. There seems to be a very small degree of tolerance to structural changes at this part of the molecule as evident from compounds 13d and 13c, in which one of the thiophene carbon of 4a and 13b was replaced by a nitrogen atom.

Table 3.

Inhibition of NAT8L by Compounds 4a and 13a−e

Cmpd	R R2	IC50 (μM)a
4a		2.9 ± 1.1
13a		>100
13b		4.5 ± 0.8
13c		>100
13d		>100
13e		39 ± 8

^aValues are mean ± SD of at least three experiments.

The NAT8L inhibitory potency of analogs of 4a with modifications to the piperidine linker is summarized in Table 4. Among the two enantiomers of 4a, (S)-4a displayed >12-fold higher potency over (R)-4a, indicating that the S-enantiomer is mainly responsible for the inhibitory activity of compound 4a. Replacing the piperidine moiety with other nitrogen-containing aliphatic rings led to substantial loss of potency as seen in compounds 17a–d. It should be noted that only one carbon within the piperidine ring of 4a was replaced with an ether oxygen in compound 17d, suggesting a particular preference for a piperidine ring at the linker region.

Table 4.

Inhibition of NAT8L by Compounds 4a, (R)-4a, (S)-4a, and 17a-d.

Cmpd	Structure R2	IC50 (μM)a
4a		2.9 ± 1.1
(R)-4a		40 ± 4
(S)-4a		3.2 ± 0.9
17a		13 ± 2
17b		16 ± 5
17c		67 ± 1
17d		28 ± 2

^aValues are mean ± SD of at least three experiments.

Given that NAT8L is predominantly expressed in the brain,¹⁶ NAT8L represents an attractive therapeutic target for the treatment of CD through reduction of the brain levels of NAA. Despite its promising therapeutic potential; for treating CD, little progress has been made, to date, in development of small molecule inhibitors of NAT8L. In addition, the crucial role of NAT8L has been implicated in other disease conditions,^16–17 further highlighting the need for potent small-molecule inhibitors as probe molecules. Our NAT8L HTS hit compound 4a based on N-acylated (piperidin-3-ylmethyl)-1,2,4-oxadiazole scaffold was confirmed to be a potent noncarboxylic acid NAT8L inhibitor through rigorous hit validation process. Although it showed high clearance in liver microsomes, compound 4a displayed good permeability and a low efflux ratio, demonstrating its potential to serve as a CNS drug molecular template. Compound 4a was also found to be a competitive inhibitor with respect to aspartate, minimizing the possibility of nonselectively inhibiting other acetyl-CoA-dependent N-acetyltransferases. Systematic modifications at three distinct structural regions of compound 4a revealed important SAR trends. As for the phenoxyacetyl moiety of compound 4a, a fluorine and chlorine substituent at one of the two ortho positions appears to be favored for NAT8L inhibition as represented by compounds 4a and 4t, whereas diortho substitution is not tolerated as seen with compound 4k. Incorporation of two chlorine or fluorine substituents into the adjacent ortho and meta positions is also well-tolerated, as exemplified by compounds 4l and 4u. Additional key findings include a substantial loss of potency resulting from the incorporation of non–electron-withdrawing substituents into the phenyl ring (compounds 4d, 4e, and 4q), the replacement of the ether oxygen with a methylene group (compound 4h), and the addition of a methyl group to the acetyl moiety (compound 4f). Only a 2-thienyl group (compound 13b) was tolerated as a substitute for the 3-thienyl group of compound 4a, displaying a narrow range of tolerance for structural changes in 4a at this region. Potency changes resulting from modifications to the linker region of compound 4a appear to occur gradually, with the highest preference observed for the (S)-3-((3-(thiophen-3-yl)-1,2,4-oxadiazol-5-yl)methyl)piperidin-1-yl linker (compound (S)-4a). Although the discovery of nanomolar noncarboxylic acid NAT8L inhibitors 4t and 4u represents an important step toward our pursuit of brain-penetrant NAT8L inhibitors, both compounds were later found to show high clearance (<5% remaining after 30 min) in liver microsomes (Supporting Information). Metabolite identification (MetID) studies using microsomal samples incubated with compound 4t failed to detect any plausible metabolites, including those expected from oxidation of the thiophene ring,^18–19 suggesting multiple phases of metabolism following initial CYP-mediated oxidation. These results underscore the need for a detailed understanding of the mechanisms driving the metabolism of NAT8L inhibitors based on the N-acylated (piperidin-3-ylmethyl)-1,2,4-oxadiazole scaffold, to enable the rational design of compounds with optimized stability and preserved or enhanced potency.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.5c00623.

Synthetic procedures, ¹H and ¹³C NMR spectra, and pharmacological experimental methods

Abbreviations

CD

Canavan disease

ASPA

aspartoacylase

NAT8L

N-acetyltransferase 8-Like

NAA

N-acetylaspartate