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. 2025 Sep 4;16(11):5318–5332. doi: 10.1039/d5md00639b

Profile of NT-0527, a brain penetrant NLRP3 Inflammasome inhibitor suitable as an in vivo tool compound for neuroinflammatory disorders

David Harrison a,✉, Andy Billinton a,†, Mark G Bock b, Nicholas P Clarke a, Zsofia Digby a, Christopher A Gabel b, Nicola Lindsay a, Valérie Reader a, Jane Scanlon a,‡, Pamela Smolak b, Peter Thornton a, Heather Wescott b, Alan P Watt a
PMCID: PMC12409668  PMID: 40919318

Abstract

Inhibition of the NLRP3 inflammasome has emerged as a high potential treatment paradigm for the treatment of neuroinflammation, with demonstrated anti-neuroinflammatory effects in Parkinson's disease patients and a strong rationale in Alzheimer's disease and amyotrophic lateral sclerosis. To facilitate further progress in this field, brain penetrant NLRP3 inflammasome inhibitors as leads and tool compounds are required. We discovered a small molecule NLRP3 inflammasome inhibitor, NT-0527 (11), and extensively profiled this to reveal a highly potent, selective and brain penetrant compound. This was shown to be orally bioavailable, efficacious in an in vivo model of inflammation, and with good developability characteristics. However, NT-0527 exhibited CYP 2C19 time-dependent inhibition, which halted development, but this molecule could be employed as a valuable tool compound for the investigation of neuroinflammatory conditions where NLRP3 inflammasome activation is implicated.

NT-0527 is a potent, specific and brain penetrant NLRP3 inflammasome inhibitor with oral bioavailability and efficacy in an in vivo model of inflammation.graphic file with name d5md00639b-ga.jpg

Introduction

The innate immune system has evolved as the fundamental first line of defence against infection and is common to all multicellular organisms. One of the roles of the innate immune system is to recognise and react to invading pathogens and cellular damage by releasing an array of chemokines and cytokines which, in turn, recruit specific immune cell types (e.g. macrophages, neutrophils, NK cells) resulting in a local inflammatory response. In normal function this process helps to remove harmful stimuli and damaged cells and initiate the repair response.

The nucleotide-binding oligomerisation domain (NOD)-like receptor, leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome is the most widely-studied inflammasome and is a major contributor to inflammation.1 It is found in the cytosol of various innate immune cells, including macrophages and monocytes. NLRP3 is capable of sensing various danger signals, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Following activation, the NLRP3 inflammasome triggers the proteolytic cleavage of pro-caspase-1 to its active form, caspase-1. This leads to the maturation of the proinflammatory cytokines interleukin (IL)-1β and IL-18, and their subsequent release into extracellular space via a gasdermin D (GSDMD)-mediated pore forming process known as pyroptosis.

Evidence for the damaging effect that inappropriate activation of the NLRP3 inflammasome can have in humans is apparent in a series of gain-of-function mutations causing autoinflammatory disorders collectively known as cryopyrin-associated periodic syndromes (CAPS).2 These include, in order of increasing severity, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal-onset multisystem inflammatory disorder (NOMID). All of these disorders are characterised by episodic fevers and chills, recurrent rashes (particularly in response to cold) and inflammation in the joints. In MWS, the persistent inflammation can additionally lead to joint destruction, hearing loss and kidney amyloidosis. In the most severe mutations, NOMID, inflammation occurs both in the periphery and central nervous system (CNS), whereupon chronic meningitis can lead to neuronal damage and learning impairments.

Aberrant activation of the NLRP3 inflammasome is also associated with multiple human diseases, occurring in response to a wide range of endogenous DAMPs and in the absence of infection, leading to these activation signals being termed ‘sterile’ danger signals. Such disease states are accompanied by chronic low-grade inflammation, are of high unmet medical need and include type II diabetes, atherosclerosis, obesity, gout, asthma, inflammatory bowel disease, metabolic dysfunction–associated steatotic liver disease (MASLD)/metabolic dysfunction–associated steatohepatitis (MASH), and various cancers.3–12

The activation of the NLRP3 inflammasome and subsequent release of proinflammatory cytokines IL-1β and IL-18 in the central nervous system (CNS) is also common to neuroinflammatory disorders.13,14 NLRP3 is prominently expressed by microglia within the CNS, and activated microglial cells readily assemble NLRP3 inflammasomes and externalise IL-1β in response to endogenous DAMPs.15,16 These DAMPs include tau and amyloid β in Alzheimer's disease, α-synuclein in Parkinson's disease, and SOD1G93A and TDP-43 in amyotrophic lateral sclerosis (ALS).17–22 Furthermore, extracellular ATP in the brain acts as a danger signal and is a mediator of neuroinflammation.23,24

One of the first selective NLRP3 inflammasome inhibitors (NLRP3i) to be discovered, and to date the mostly widely profiled small molecule NLRP3i preclinically, is the sulfonylurea CRID3 (1, Fig. 1, also known as CP-456 773 and MCC950).25,26 This compound was progressed as far as a phase 2 study in rheumatoid arthritis, but further development was halted. CRID3 has been deployed as a tool NLRP3i in a wide range of in vitro and in vivo models, serving to further validate the potential role that molecules disrupting the activation of the NLRP3 inflammasome could have in the treatment of multiple diseases.27–29 Recently a number of specific NLRP3i have been progressed into clinical trials, including DFV890, ZYIL-1, NT-0249, VENT-01, VTX2735, AZD4144, and GDC-2394.30–36 There are several published reviews that cover these NLRP3i in further detail.37–39 Although there is great promise shown by a number of these in the earlier stages of human clinical trials, as yet none have achieved marketing approval from the relevant authorities.

Fig. 1. Structures of selected NLRP3 inflammasome inhibitors.

Fig. 1

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Amongst these specific inhibitors of the NLRP3 inflammasome, a small number have been progressed into clinical studies in patients with neuroinflammatory conditions. NT-0796 (2, Fig. 1) is an ester-based prodrug that undergoes intracellular conversion to the carboxylic acid active species.40,41 After completion of phase 1 studies that demonstrated brain penetration following oral dosing, NT-0796 was progressed into a phase 1b/2a trial in patients with Parkinson's disease.42,43 Decreases in cerebrospinal fluid (CSF) biomarkers associated with neuroinflammation, including IL-1β, IL-6, CCL2, CXCL1 and CXCL8, were seen in patients over the 28-day period.44 In addition to these observations, a reduction in the neurodegenerative biomarkers neurofilament light (NfL) and soluble triggering receptor expressed on myeloid cells 2 (sTREM2) were also detected in a number of subjects. Two further NLRP3i, VTX3232 and VENT-02, have been advanced into human clinical trials for Parkinson's disease, but structures for these compounds have not yet been disclosed.45,46 Two sulfonylurea-based NLRP3i discovered by Inflazome have been proposed as potential treatments for neuroinflammatory disorders. Following a successful phase 1 trial for emlenoflast (3, Fig. 1, IZD174) in healthy volunteers, an open label phase 1b study in patients with mild to moderate Parkinson's disease was planned.47 However, no patients were enrolled, and following the acquisition of Inflazome by Hoffmann-La Roche the trial was withdrawn and no further updates have been given on the status of clinical development for emlenoflast. The second sulfonylurea, selnoflast (4, Fig. 1, RO7486967, RG6418), was progressed into a Phase 1b, multi-centre, doubled blind, placebo-controlled study in patients with early stage idiopathic Parkinson's disease.48,49 The trial enrolled 60 patients and completed in July 2024, though no details of the results have been disclosed yet. Two previous studies using [11C]-labeled CRID3 as a positron emission tomography (PET) probe failed to detect CNS accumulation of the compound after systemic administration, suggesting limited access to the brain parenchyma. This is consistent with the physiochemical properties of sulfonylurea-based drugs, a category that CRID3, emlenoflast and selnoflast belong to, which bear a negative charge at physiological pH that limits their ability to cross the blood–brain barrier.50,51

Despite the progression of many NLRP3i into clinical studies and the encouraging data from the trial of NT-0796 in Parkinson's patients, there remains a need for further molecules in order to investigate the preclinical and clinical effects of NLRP3 inflammasome inhibition in the context of neuroinflammatory disorders. In particular, such molecules should ideally meet several hitherto challenging criteria. They need to be specific inhibitors with a clean mechanism so that pharmacological effects can be rationalised, with limited off-target activity to avoid toxicological risks. Brain penetration should be high so that resident CNS microglia are adequately exposed to drug. Ideally, a non-prodrug may help to streamline preclinical and clinical investigations, particularly when used as a tracer.

Results and discussion

During the course of a lead optimisation project seeking inhibitors of the NLRP3 inflammasome, we discovered the small molecule NT-0527 (11, Fig. 2A). Herein, the profile of NT-0527 as a potent, selective, brain penetrant NLRP3 inhibitor is discussed.

Fig. 2. (A) Structure of NT-0527 (11). (B) IL-1β output following LPS/ATP activation in human PBMCs and inhibition by NT-0527 and (C) assay controls including no stimulation (−LPS/−ATP), ATP alone (−LPS/+ATP), LPS alone (+LPS/−ATP), LPS/ATP in combination (+LPS/+ATP), LPS/ATP with CRID3 (1 μM) and LPS/ATP with BIRB-796 (100 nM; p38 inhibitor, negative control). (D) IL-1β output following LPS/MSU crystal activation in human PBMCs and inhibition by NT-0527 and (E) assay controls including no stimulation (−LPS/−MSU), MSU alone (−LPS/+MSU), LPS alone (+LPS/−MSU), LPS/MSU in combination (+LPS/+MSU), and LPS/MSU with phagocytosis inhibitor cytochalasin D (+LPS/+CPPD/+cytoD). (F) IL-1β output following CPPD (100 μg mL−1) crystal activation in human PBMCs and inhibition by NT-0527 and (G) assay controls including no stimulation (−LPS/−CPPD), CPPD crystals alone (−LPS/+CPPD), LPS alone (+LPS/−CPPD), LPS/CPPD crystals in combination (+LPS/+CPPD), LPS/CPPD crystals and phagocytosis inhibitor cytochalasin D (+LPS/+CPPD/+cytoD). Data shown are from a representative subject and expressed as mean ± SD (n = 3 per concentration point).

Fig. 2

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NT-0527 inhibits the formation of the NLRP3 inflammasome in response to various stimuli

NT-0527 is able to disrupt the formation of the NLRP3 inflammasome in human peripheral blood mononuclear cells (PBMCs) leading to a dose-dependent reduction in the output of IL-1β (Fig. 2). Cells are first treated with lipopolysaccharide (LPS) to promote transcription/translation of pro-IL-1β after which addition of an NLRP3 activating agent is introduced to promote release of mature IL-1β from these cells. NT-0527 has a potent inhibitory effect on NLRP3 inflammasome-mediated IL-1β production triggered by multiple second stimuli, with IC50 values of 0.062 ± 0.02 μM (n = 6) when activated by adenosine triphosphate (ATP) (Fig. 2B), 0.087 μM (n = 1) with monosodium urate (MSU) crystals (Fig. 2D), and 0.040 μM (n = 1) with calcium pyrophosphate dihydrate (CPPD) crystals (Fig. 2F).

NT-0527 is a potent inhibitor of IL-1β release in a human whole blood assay and does not prevent the release of IL-6 or TNFα

To determine the effectiveness in a physiologically relevant context, the potency of NT-0527 was assessed using a two-step LPS/ATP stimulated IL-1β production assay in human whole blood (huWB), as shown in Fig. 3. NT-0527 is able to reduce IL-1β production from huWB in a dose-dependent manner, with a mean IC50 value of 0.79 ± 0.4 μM from separate experiments in blood from six unique donors (Fig. 3A). The selectivity for inhibition of IL-1β release over other cytokines in huWB was also demonstrated. Whereas NLRP3 inflammasome-mediated production of IL-1β in huWB can be mediated by LPS/ATP stimulation, the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), which are NLRP3-independent, can be stimulated with LPS treatment only.52,53 Treatment with NT-0527 at various concentrations up to 40 μM has no effect on the production of IL-6 or TNFα (Fig. 3A), thus demonstrating the selectivity for the inhibition of IL-1β (and IL-18, data not shown) release over other cytokines. Furthermore, the selective inhibition of IL-1β release and lack of effect on IL-6 and TNFα release from human immune cells treated with NT-0527 indicates that this effect is not due to cytotoxicity.

Fig. 3. (A) IL-1β, IL-6 and TNFα output following LPS and LPS/ATP activation in human whole blood and inhibition by NT-0527. (B) IL-1β assay controls including no stimulation (−LPS/−ATP), ATP alone (−LPS/+ATP), LPS alone (+LPS/−ATP), LPS/ATP in combination (+LPS/+ATP), LPS/ATP with CRID3 (10 μM) and LPS/ATP with BIRB-796 (1 μM; p38 inhibitor, negative control). (C) IL-6 assay and (D) TNFα assay controls. Data shown are from a representative subject and expressed as mean ± SD (n = 3 per concentration point).

Fig. 3

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NT-0527 is selective for NLRP3 over other inflammasomes

Further demonstration of on-target selectivity was shown by assessing the effect on IL-1β production driven by an alternative inflammasome, as shown in Fig. 4. In Salmonella-infected immune cells, the bacterial type III secretion system inner rod protein PrgJ triggers activation of NLRC4 inflammasomes, resulting in the release of IL-1β.54 Fusing the PrgJ protein to the N-terminal domain of Bacillus anthracis lethal factor (LFn) and adding this to cells along with Bacillus anthracis protective antigen (PA) enables translocation of LFn-PrgJ into the cytoplasm.55 This method has been used to demonstrate that CRID3 does not inhibit NLRC4-mediated production of IL-1β.56 In these experiments, PBMCs were first primed with LPS. Addition of nigericin, a well-established NLRP3 inflammasome second (activation) signal, resulted in IL-1β release, which was inhibited by NT-0527 (Fig. 4A). However, addition of PA and LFn-PrgJ drove NLRC4 inflammasome-dependent IL-1β release, which could be blocked by a caspase-1 inhibitor, VX-765 (Fig. 4C), but not NT-0527 (Fig. 4B). This is consistent with NT-0527 being an NLRP3 inflammasome inhibitor and not a broader IL-1β inhibitor through other mechanisms.

Fig. 4. Induction of IL-1β production in LPS-primed, nigericin or PA + LFn-PrgJ stimulated human PBMCs. All samples were primed with LPS and treated as indicated on the x axis prior to addition of the indicated signal 2 trigger. The means and standard deviations of replicate measurements are plotted. Effect of NT-0527 on IL-1β production by human PBMCs primed with LPS followed by either (A) nigericin (NLRP3) or (B) PA + LFn-PrgJ (NLRC4) stimulation. (C) Controls for both experiments. VX-765 was added to demonstrate that IL-1β output in response to NLRC4 activation was sensitive to caspase-1 inhibition. Data is shown ± SD, with a minimum of n = 3 replicate wells. Statistics performed are ordinary one-way ANOVA followed by Dunnett's multiple comparisons. Significance is calculated where ns (non-significant) p > 0.05; * p < 0.05; ** p < 0.01.

Fig. 4

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NT-0527 possesses suitable pharmacokinetics for oral dosing

The pharmacokinetic properties of NT-0527 were assessed in four species – mouse, rat, cynomolgus monkey (cyno) and minipig – as summarised in Table 1. Plasma clearance following intravenous administration is low in rat and cyno, moderate in mouse and high in minipig. Steady state volume of distribution is low in rodent, but in excess of total body water in cyno and minipig. Oral bioavailability is moderate to high across species, with complete oral absorption achieved in cyno. These results show that NT-0527 exhibits pharmacokinetic properties that make it amenable to oral dosing across a range of species.

Table 1. Pharmacokinetic data for NT-0527 in mouse, rat, cyno and minipig.

Parameter Mousea Ratb Cynoc Minipigd
Half-life (iv, h) 0.19 ± 0.1 0.61 ± 0.3 2.8 ± 0.6 0.86 ± 0.4
MRT (h) 0.25 ± 0.03 0.58 ± 0.1 3.9 ± 0.7 0.93 ± 0.3
CLp (mL min−1 kg) 42 ± 4 10 ± 2 6.3 ± 1 47 ± 12
V ss (L kg−1) 0.63 ± 0.1 0.36 ± 0.01 1.4 ± 0.01 2.5 ± 0.1
AUC(0-inf), po (ng h mL−1) 470 ± 100 2400 ± 500 9700 ± 2000 680 ± 500
AUC(0-inf), iv (ng h mL−1) 1200 ± 100 4800 ± 700 8200 ± 100 1100 ± 300
F (%) 39 50 104 60
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a

Male C57/BL6J mouse, n = 3, 3 mg kg−1po/iv.

b

Male SD rat, n = 3, 3 mg kg−1po/iv.

c

Male cynomolgus monkey, n = 3, 3 mg kg−1po/iv.

d

Male Bama minipig, n = 3, 2 mg kg−1po, 1 mg kg−1iv. Dose corrected AUC values used to calculate bioavailability. Data is given ± standard deviation (SD).

NT-0527 is highly brain and CSF penetrant in rat and cynomolgus monkey studies

Brain penetration was evaluated using a rat in situ brain perfusion model. In this, a solution of the test compound at 5 μM was perfused into the right carotid artery of anaesthetised male rats for 30 seconds, then the concentration of the test compound in the left hemisphere was determined. Atenolol (infused at 50 μM concentration, then corrected to 5 μM) was used as a low permeating marker, and diazepam as the high permeating marker. The measured, dose corrected concentration of the controls following brain perfusion were 0.05 ± 0.01 μM for atenolol and 4.3 ± 0.7 μM for diazepam. As shown in Fig. 5A, the sulfonylurea compounds CRID3 (0.09 ± 0.04 μM) and emlenoflast (0.25 ± 0.05 μM) are classified as low permeating compounds. By contrast, NT-0527 is highly brain penetrant, reaching a concentration of 6.0 ± 0.9 μM in this assay. To further assess brain penetration in a higher species, NT-0527 was dosed orally at 10 mg kg−1 in three male cynomolgus monkeys (Fig. 5B). Samples of blood and cerebrospinal fluid (CSF) were taken at intervals over a period of 24 hours. CSF to blood unbound partition coefficient (Kpu,u) was calculated using the ratio of the area under the curve (AUC) in CSF versus blood (unbound), with a result of 0.8 indicative of an almost even distribution of NT-0527 between blood and CSF (Fig. 5C). Both in vivo studies demonstrate that NT-0527 freely distributes into the CNS, and is suitable for use as a compound where high brain permeability is a requirement.

Fig. 5. (A) Concentration of compounds delivered by IV bolus in the hemi-brain perfusion model measured 0.25–0.5 min post-infusion. Three studies are summarised in the graph. Compounds infused at 5 μM concentration (dotted line). Control compounds are atenolol (low brain permeability control), and diazepam (high brain permeability control). Test compounds are CRID3 (1), emlenoflast (3), and NT-0527 (11). Atenolol data are corrected from 50 μM to 5 μM to allow a direct comparison. Data are shown as mean ± SD (n = 4). (B) Blood and CSF concentrations from non-naïve male cynomolgus monkey blood and CSF (n = 3) over 24 hours after a 10 mg kg−1 oral dose. Kpu,u derived from AUC(0–24) values using Fu (0.17) and blood:plasma (0.77) measurements. (C) Numerical AUC values over 24 h, shown as mean ± SD (n = 3).

Fig. 5

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NT-0527 is efficacious in an in vivo model of inflammation

To determine in vivo efficacy, NT-0527 was evaluated in a mouse peritonitis model (Fig. 6). In this, wildtype mice (n = 8 per group) were dosed intraperitoneally with LPS to promote pro-IL-1β transcription and translation. After 90 minutes, NT-0527 was dosed orally (at 1, 3, 10, 30 and 100 mg kg−1), followed 30 minutes later by an intraperitoneal (i.p.) ATP injection (to activate the NLRP3 inflammasome). 30 minutes post ATP dosing, samples were recovered via peritoneal lavage and levels of IL-1β and IL-6 determined by ELISA. NT-0527 was able to inhibit the production of peritoneal IL-1β in a dose-dependent manner, with a significant effect measured from as low as 10 mg kg−1 (p < 0.001) (Fig. 6B and C). As expected, no inhibition of IL-6 production was detected, showing desired on-target effectiveness (Fig. 6D). These data show that NT-0527 is capable of acting as an NLRP3 inflammasome inhibitor following oral dosing in an established mouse model of in vivo efficacy.

Fig. 6. (A) Experimental schematic for mouse in vivo LPS/ATP induced peritonitis in mice. (B) IL-1β levels from peritoneal lavage (C) dose-effect relationship for IL-1β (D) IL-6 levels from peritoneal lavage. Each group consisted of 8 animals. Where error bars are shown data are represented as mean ± SEM. Statistics performed are one way ANOVA followed by Dunnett's multiple comparisons. Significance is calculated relative to LPS/ATP group, where * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Curves are fitted with GraphPad Prism v9.3 using non-linear regression.

Fig. 6

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NT-0527 in vitro physicochemical and safety profile

With NT-0527 displaying a promising profile in terms of excellent potency, on-target selectivity, good pharmacokinetics for oral dosing, high brain penetration and dose-dependent efficacy in a mouse in vivo model of NLRP3 inflammasome-driven inflammation, we wanted to further investigate its potential by profiling in a range of in vitro assays to evaluate developability, with the results summarised in Table 2. The physicochemical properties of NT-0527 (molecular weight = 360.8, one H-bond donor, topological polar surface area = 75, measured log D at pH 7.4 = 2.32) are in the ideal range for blood–brain barrier (BBB) permeability.57 In a Caco-2 monolayer assay, passive permeability is high (34 × 10−6 cm s−1) and the compound is not a substrate for efflux transporters. However, aqueous solubility at physiological pH is low (7.3 μM). Intrinsic clearance is low in human microsomes (<9.6 μL min−1 mg−1) and cryopreserved hepatocytes (<6.4 μL min−1 million cells−1), but moderate to high in rodent microsomes and hepatocytes. In a hERG patch clamp assay, the risk of hERG blockade-related cardiotoxicity was low (hERG IC50 > 30 μM). In a direct inhibition cytochrome P450 (CYP) study, NT-0527 inhibits the metabolism of 4′hydroxy-mephenytoin by CYP 2C19 (IC50 = 5.1 μM), with no effect on other CYP isoforms (1A2, 2C9, 2D6, 3A4 all >30 μM). Due to this direct inhibition of CYP 2C19, NT-0527 was profiled in a time-dependent CYP inhibition assay. In this assay, the compound of interest is preincubated with the CYP enzyme, both in the absence and presence of NADPH, before the addition of the isoform-specific substrate. NT-0527 showed a 5.6-fold shift in IC50 against the CYP 2C19 isoform in the presence of the coenzyme NADPH (0.45 μM cf. 2.5 μM without NADPH), thus showing a time-dependent inhibition (TDI) of CYP 2C19. This irreversible inhibition is a potential safety risk, since the metabolising capability of the CYP enzyme can only be restored by biosynthesis, and the reduced function of a key metabolising enzyme results in an increased risk of drug–drug interactions. Marketed drugs metabolised by CYP 2C19 include proton-pump inhibitors such as omeprazole, the antifungal agent voriconazole, the antiepileptic phenytoin, and selective serotonin reuptake inhibitors such as citalopram.58–61 Additionally, both the antiplatelet agent clopidogrel and the antiepileptic diazepam undergo CYP 2C19 metabolism resulting in the generation of active species.62,63

Table 2. Physicochemical and in vitro properties of NT-0527.

MW 360.8
tPSA 75
log D7.4 2.32
Thermodynamic solubility 7.3 μM at pH 7.4
Caco-2 Papp(A to B); ER 34 × 10−6 cm s−1; 1.2
Microsomal CLinta Human <9.6 μL min−1 mg−1
Rat 13 μL min−1 mg−1
Mouse 24 μL min−1 mg−1
Hepatocyte CLintb Human <6.4 μL min−1 million cells−1
Rat 40 μL min−1 million cells−1
Mouse 120 μL min−1 million cells−1
hERG inhibition IC50c >30 μM
CYP inhd 1A2 (acetaminophen) >50 μM
2C9 (4′-hydroxy-diclofenac) >50 μM
2C19 (4′hydroxy-mephenytoin) 5.1 μM
2D6 (dextromethorphan) >50 μM
3A4 (1′-hydroxy midazolam) >50 μM
3A4 (6β-hydroxy testosterone) >50 μM
3A4 (dehydronifedipine) >50 μM
CYP TDI (−/+ NADPH)e 2C19 2.5/0.45 μM
Others >50/>50 μM
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a

Intrinsic clearance of 1 μM test compound in presence of 0.5 mg protein/mL liver microsomes.

b

Intrinsic clearance of 1 μM test compound in presence of 5 × 105 mg cryopreserved hepatocytes per mL.

c

Manual patch clamp.

d

Inhibition of test substrate metabolism by specified CYP isoform.

e

Inhibition of test substrate metabolism by specified CYP isoform, with and without pre-incubation with NADPH. Test substrates and CYP isoforms are phenacetin (1A2), bupropion (2B6), amodiaquine (2C8), diclofenac (2C9), S-mephenytoin (2C19), dextromethorphan (2D6), midazolam (3A4), testosterone (3A4).

Synthesis

As detailed in Scheme 1, NT-0527 (11) was synthesised in six steps from commercially available starting materials. Starting from phenylacetonitrile 5, the cyclopropanation occurred on the most acidic centre using 1-bromo-2-chloroethane in the presence of catalytic benzyltriethylammonium chloride to give 6. Lithium aluminium hydride reduction furnished the amine (7), then this was coupled with methyl 2-(2-methoxycarbonylphenoxy)carbonyloxybenzoate to give carbamate 8. This underwent an intramolecular Friedel–Crafts acylation in the presence of triflic acid to form the lactam 9. Alkylation of the lactam with bromoacetic acid gave carboxylic acid 10. Finally, an amide coupling with the electron deficient 2-amino-5-fluoropyrimidine proceeded well using COMU conditions, resulting in NT-0527 (11).

Scheme 1. Synthesis of NT-0527 (11). Reagents and conditions: (a) 1-bromo-2-chloroethane, benzyltriethylammonium chloride, NaOH, H2O, N2, 50 °C, 16 h; (b) lithium aluminium hydride, THF, N2, 0 °C → rt, 1 h; (c) methyl 2-(2-methoxycarbonylphenoxy)carbonyloxybenzoate, triethylamine, THF, rt, 2 h; (d) TfOH, DCM, 0 °C, 30 min; (e) 2-bromoacetic acid, LiOtBu, THF, N2, rt → 80 °C, 2 h; (f) 2-amino-5-fluoropyrimidine, COMU, NMM, MeCN, rt → 50 °C, 8 h.

Scheme 1

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Conclusions

As part of our NLRP3 inflammasome inhibitor drug discovery project, we sought to identify a molecule with good potency and selectivity, high brain penetration and a non-prodrug mode of action to complement our brain penetrant prodrug NT-0796. NT-0527 (11) emerged as a compound worthy of further investigation. It demonstrated high and consistent potency in a PBMC assay measuring inhibition of IL-1β produced by NLRP3 inflammasome activation, whether stimulated by ATP, MSU or CPPD as the second signal. In human whole blood stimulated with LPS and ATP, NT-0527 displayed a mean half-maximal inhibitory potency of 0.79 μM ± 0.4 μM, with no effect on IL-6 or TNFα release up to 40 μM. The selectivity for NLRP3 over other inflammasomes was demonstrated by a lack of inhibition of IL-1β production driven by NLRC4 inflammasome stimulation. The pharmacokinetic properties of NT-0527 were measured in mouse, rat, cyno and minipig, with good oral bioavailability across all preclinical species. It was shown to be CNS druglike and highly brain penetrant, with high permeability and no efflux in a Caco-2 assay, high brain penetration in an in situ rat brain perfusion assay indicative of high passive diffusion through the blood–brain-barrier, and high cyno Kpu,u measured in CSF following oral dosing. To measure in vivo efficacy, NT-0527 was profiled at five doses in a mouse LPS/ATP induced peritonitis model. A dose-dependent effect on IL-1β release was observed, with significant inhibition at 10 mg kg−1po (p < 0.001) and no effect on IL-6 release. Studies looking into safety and developability revealed an inhibition of CYP 2C19, which further investigation revealed to also be time-dependent. Unfortunately, due to the greater risk of potential drug–drug interactions that CYP TDI may bring, it was determined not to advance NT-0527 any further. However, overall, the characteristics of NT-0527 make it an excellent tool compound for the preclinical investigation of NLRP3 inflammasome inhibition, particularly where brain penetration is required, and it may provide a useful starting point towards the discovery of further brain penetrant molecules.

Methods

Chemistry

All solvents and reagents were purchased from commercial suppliers and used without further purification. 1H NMR spectra were acquired using a Bruker instrument with frequency as stated. 1H NMR data was reported as follows: chemical shift (ppm), multiplicity, coupling constant (Hz) and integration. Abbreviations for multiplicity are s, singlet; br. s, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Purity of final compounds was assessed by HPLC with UV detection at 210 or 220 nm. All compounds are ≥95% purity by HPLC analysis.

1-(3-Chlorophenyl)cyclopropane-1-carbonitrile (6)

To a mixture of 2-(3-chlorophenyl)acetonitrile (5, 3.13 mL, 26.4 mmol) and 1-bromo-2-chloroethane (3.28 mL, 39.6 mmol) in H2O (8 mL) at 50 °C under N2 were added benzyltriethylammonium chloride (120 mg, 528 μmol) and NaOH (6.33 g, 158 mmol). The mixture was stirred at 50 °C for 16 h, allowed to cool to room temperature and poured into water (10 mL). The resulting suspension was extracted with chloroform (3 × 10 mL), and the combined organic layers washed with aq. 1 M HCl (3 × 10 mL), water (3 × 10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under vacuum to give the title compound as a yellow solid. Y = 96%; purity = 98%. 1H NMR (400 MHz, DMSO-d6) δ 7.45–7.31 (m, 4H), 1.80–1.75 (m, 2H), 1.59–1.54 (m, 2H).

1-[1-(3-Chlorophenyl)cyclopropyl]methanamine (7)

To a mixture of 1-(3-chlorophenyl) cyclopropane-1carbonitrile (6, 9.0 g, 50.7 mmol) in THF (200 mL) at 0 °C under N2 was added lithium aluminium hydride (1.98 g, 52.2 mmol). The reaction mixture was stirred at 25 °C for 1 h. The mixture was then cooled down and quenched with water (2 mL), then treated with 15% aq. sodium hydroxide (2 mL). The resulting suspension was filtered, and the filtrate concentrated under vacuum to give the title compound as a white solid. Y = 83%; purity = 93%. 1H NMR (400 MHz, DMSO-d6) δ 7.35–7.20 (m, 4H), 2.73 (s, 2H), 2.32 (br. s, 2H), 0.83–0.79 (m, 2H), 0.74–0.70 (m, 2H).

Methyl 2-[({[1-(3-chlorophenyl)cyclopropyl]methyl}carbamoyl)oxy]benzoate (8)

To a mixture of [1-(3-chlorophenyl)cyclopropyl]methanamine (7, 400 mg, 2.20 mmol) in THF (4 mL) were added triethylamine (306 μL, 2.20 mmol) and methyl 2-(2-methoxycarbonylphenoxy)carbonyloxybenzoate (727 mg, 2.20 mmol). The mixture was stirred at 25 °C for 2 h, then concentrated under vacuum. The resulting residue was purified by column chromatography (SiO2, 20% EtOAc in petroleum ether) to give the title compound as a colourless oil, used without further purification. Y = 99%.

6′-Chloro-2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-1′-one (9)

To a mixture of methyl 2-[({[1-(3-chlorophenyl)cyclopropyl]methyl}carbamoyl)oxy]benzoate (8, 650 mg, 1.81 mmol) in DCM (5 mL) was added triflic acid (797 μL, 9.03 mmol). The mixture was stirred at 0 °C for 30 min. To reaction mixture was treated dropwise with saturated aq. Na2CO3 to pH ∼8. The resulting mixture was extracted with ethyl acetate (3 × 5 mL). The combined organic phase was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (SiO2, 50% EtOAc in petroleum ether) to give the title compound as a white solid. Y = 67%. LC-MS m/z: 208 [(M + H)+].

2-{6′-Chloro-1′-oxo-2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-2′-yl}acetic acid (10)

To a solution of 6′-chloro-2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-1′-one (9, 0.60 g, 2.89 mmol) in THF (6 mL) at 25 °C under N2 were added lithium tert-butoxide (1.17 mL, 13.0 mmol) and 2-bromoacetic acid (270 μL, 3.76 mmol). The mixture was stirred at 80 °C for 2 h. The resulting mixture was adjusted to pH ∼4 with aqueous 2 M HCl and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound as a white solid. Y = 91%. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (s, 1H), 7.89 (d, J = 8 Hz, 1H), 7.37 (dd, J = 8, 2 Hz, 1H), 7.12 (d, J = 2 Hz, 1H), 4.19 (s, 2H), 3.46 (s, 2H), 1.20–1.15 (m, 2H), 1.06–1.00 (m, 2H).

2-{6′-Chloro-1′-oxo-2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-2′-yl}-N-(5-fluoropyrimidin-2-yl)acetamide (11)

To a solution of 2-{6′-chloro-1′-oxo-2′,3′-dihydro-1′H-spiro[cyclopropane-1,4′-isoquinolin]-2′-yl}acetic acid (10, 50 mg, 188 μmol) in acetonitrile (0.5 mL) were added 5-fluoropyrimidin-2-amine (32 mg, 282 μmol), N-methylmorpholine (20.7 μL, 188 μmol) and COMU (105 mg, 245 μmol). The mixture was stirred at 50 °C for 8 h. The reaction mixture was concentrated under reduced pressure and the resulting residue purified by prep-HPLC (column: Waters Xbridge BEH C18 100 × 30 mm × 10 μm; mobile phase: [water (10 mM NH4HCO3) – MeCN]; B: 20–50%, 8 min) and lyophilised to give the title compound as a white solid. Y = 52%; purity >99%. LC-MS m/z: 361 [(M + H)+]. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 8.76 (s, 2H), 7.88 (d, J = 8 Hz, 1H), 7.37 (dd, J = 8, 2 Hz, 1H), 7.13 (d, J = 2 Hz, 1H), 4.50 (s, 2H), 3.49 (s, 2H), 1.20–1.12 (m, 2H), 1.08–1.01 (m, 2H). 1H NMR (400 MHz, DMSO-d6 + D2O) δ 8.72 (s, 2H), 7.88 (d, J = 8 Hz, 1H), 7.36 (dd, J = 8, 2 Hz, 1H), 7.10 (d, J = 2 Hz, 1H), 4.47 (s, 2H), 3.48 (s, 2H), 1.18–1.10 (m, 2H), 1.08–1.00 (m, 2H).

PBMC IL-1β assay

Heparin-stabilised whole blood from healthy volunteers was obtained from Bloodworks NW (Seattle, WA, USA). PBMCs were prepared using Ficoll-Paque Plus centrifugation. Total cell numbers were determined using a haematocytometer and adjusted to 2.5 × 106 cells per mL. 2.5 × 105 cells were added to each well of 96-well plates, and the plates were incubated at 37 °C to allow adherence of monocytic cells. After 2 hours, medium and non-adherent cells were aspirated and 100 μL of fresh complete RPMI-1640 containing 5% FBS was added to each well and the plates were incubated overnight at 37 °C.

The following day, LPS was added to designated wells (final concentration of 100 ng mL−1) and the plates were incubated at 37 °C for 2 h to allow transcription/translation of pro-IL-1β. At this point, media were removed by aspiration and 0.143 mL of fresh RPMI GlutaMAX medium containing 1% FBS and 1% penicillin/streptomycin were added; the presence of LPS was maintained in wells previously exposed to this stimulus. Test compound at various concentrations (or 0.2% DMSO) was added to designated wells and plates were placed at 37 °C in a 5% CO2 incubator for 60 min. To designated wells, 0.0075 mL of a 100 mM ATP solution (final concentration = 5 mM) was then introduced to promote NLRP3 activation, and the plates returned to a 37 °C/5% CO2 incubator. After a 60-min stimulation, plates were subjected to centrifugation after which media supernatants were harvested for assessment of IL-1β levels by ELISA. When MSU or CPPD crystals were employed as the NLRP3 activation stimulus LPS was added to designated wells of adherent monocytes at a final concentration of 100 ng mL−1. Plates were incubated at 37 °C for 2.5 h to allow transcription/translation of pro-IL-1β. Cytochalasin D was added to designated control wells at the time of LPS addition (final concentration of 2 μM). After incubation, medium was removed by aspiration and replaced by fresh RPMI GlutaMAX medium containing 1% FBS with LPS and cytochalasin being maintained in the designated wells. Test agent or 0.2% DMSO was added to designated wells and plates were incubated at 37 °C for 0.5 h. MSU (final concentration 200 μg mL−1) or CPPD crystals (final concentration 100 μg mL−1) were added and the plates were returned to a 37 °C incubator for 4 h. Supernatants were harvested by centrifugation and levels of IL-1β were assessed by ELISA.

Human whole blood IL-1β, IL-6 and TNFα assay

Freshly isolated heparinised human whole blood was diluted with RPMI-Glutamax medium containing 20 mM HEPES, pH 7.3, 1% penicillin/streptomycin, and 1% heat-inactivated FBS (10 parts : 5 parts). Aliquots of the diluted whole blood were transferred to individual wells of a 96-well plate. Designated wells then received various concentrations of NT-0527 or DMSO (resulting in a final DMSO concentration in all wells of 0.2%) followed by addition of LPS (final of 100 ng mL−1) to activate cytokine synthesis. After a 3.5-hour incubation at 37 °C in a 5% CO2 incubator, ATP (final concentration of 5 mM) was introduced to promote NLRP3 inflammasome activation and the plate was incubated for an additional 30 minutes at 37 °C. The plate was then centrifuged and resulting plasma supernatants were recovered. Cytokine content (IL-1β, IL-6, and TNFα) within the supernatants was determined using ELISA kits selective for the individual cytokines.

NLRC4 selectivity assay

PBMCs isolated from human whole blood were resuspended in culture medium containing 1% FBS and dispensed into 96-well plates. After a 2 h incubation at 37 °C, the media and unattached cells were removed by aspiration, and culture medium containing 10% FBS was added to each well. The cells were then incubated at 37 °C overnight.

The following morning, the medium was replaced with assay medium containing 1% FBS and 100 ng mL−1 LPS, and the cells were incubated for 2.5 h at 37 °C. Next, control or test article was added to the cells. Negative-control wells received an equivalent dilution of DMSO, and the caspase-1/4 inhibitor VX-765 was used as a positive-control compound predicted to inhibit NLRC4 mediated IL-1β release. The cells were then incubated for 30 min at 37 °C.

At the end of the 30-minute incubation above, appropriate secondary stimuli were added to the cells. For activation of NLRP3, nigericin was used at a final concentration of 5 μM. PA (List Biological Laboratories) and LFn-PrgJ (expressed and isolated at NodThera) were added to yield final concentrations of 1 μg mL−1 and 4 μg mL−1, respectively. To confirm that both proteins were required for NLRC4 inflammasome activation, control wells containing each individual protein were also prepared.

After 4 hours of incubation at 37 °C, culture supernatants were harvested and stored at 4 °C. IL-1β levels in the culture supernatants were determined by ELISA (ThermoFisher) following the manufacturer's instructions.

Rat in situ brain perfusion study

Animals were group housed after arrival and during acclimatisation period until the day of the study. Animals were kept on a 12 hour light/dark cycle with food/water available ad libitum.

Under non-recovery anaesthesia, fed male SD rats (n = 4) had the right carotid artery cannulated for hemi-perfusion of the brain using a pre-prepared Ringer's buffer solution containing 5 μM of test substance, or 50 μM in the case of atenolol (as a low permeability marker). In 3 separate studies rats were hemi-perfused with 1) diazepam (5 μM; as a high permeability marker) or atenolol (50 μM; low permeability marker), 2) CRID3 (5 μM) or atenolol (50 μM) 3) emlenoflast (5 μM) or atenolol (50 μM) and 4) NT-0527 (5 μM) or atenolol (50 μM). Animals were perfused for 0.25–0.5 min after which the brain was excised and hemi-dissected and the perfused hemisphere stored for quantitative bioanalysis by LC-MS/MS. After dosing, the mean brain concentrations from 4 rats each compound were quantified.

These studies were conducted by Pharmidex Pharmaceutical Services Limited at their facility in the UK.

Cynomolgus monkey CSF exposure study

Animals were housed separately. Animals were kept on a 12 hour light/dark cycle with food/water available ad libitum. Male non-naïve cynomolgus monkeys (n = 3) were dosed with a solution of NT-0527 formulated in 0.5% methocel (400cp), 0.2% Tween 80 in purified water via oral gavage, with a dose of 10 mg kg−1, dose volume 10 mL kg−1 and target dose concentration 1 mg mL−1. Whole blood was collected and added to potassium EDTA as an anti-coagulant. Cerebrospinal fluid (CSF) was collected via catheter implanted in the Cisterna Magna. Samples were taken at pre-dosing, 0.25, 0.5, 1, 2, 4, 8 and 24 h and concentrations of NT-0527 were determined by LC-MS/MS.

This study was conducted at WuXi AppTec Co, Ltd. and in accordance with the WuXi IACUC standard animal procedures along with the IACUC guidelines that are in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals.

Mouse LPS/ATP-induced peritonitis study

Animals were group-housed (5 animals per cage) with bedding under controlled temperature (20–25 °C), noise, humidity (40–70%), and lighting (12-hour light and 12-hour dark) conditions. All animals had free access to purified water and standard certified rodent chow. C57Bl6 mice (n = 8 per group) received 1 μg of LPS (Sigma-Aldrich; Cat# L2880) in PBS (Gibco; Cat#20012-043 1000 mL) by intraperitoneal (IP) injection. After 1.5 hours, they were dosed orally with NT-0527 as a suspension in 0.5% methylcellulose (Sigma; Cat#M7140), 0.2% Tween80 (Sigma; Cat#P4780) at 1, 3, 10, 30 and 100 mg kg−1 or vehicle at a dosing volume of 10 mL kg−1. Thirty minutes later, an IP injection of 0.5 mL of 30 mM ATP, pH 7.2 (Sigma-Aldrich; Cat# 10127523001), in PBS was administered. After a further thirty minutes post-ATP administration blood was collected via cardiac puncture, and peritoneal cavities were lavaged with 3 mL of ice-cold PBS containing 25 U mL−1 heparin (SCR; Cat# 63007131) and 10% heat inactivated FBS (Biosera; Cat#FB-1058/500; Lot#015BS825). A protease inhibitor mixture (Roche; Cat# 11697498001) was added to the lavage buffer just prior to use at one tablet per 50 mL of buffer. From the recovered plasma samples levels of IL-1β, while from peritoneal lavage fluids levels of IL-1β and IL-6 were assessed by ELISA (R&D system, Cat#DY401 and R&D system, Cat#DY406, respectively).

This study was conducted by HD Biosciences (China) Co., Ltd. (HDB) at its facility in Shanghai, China. The study was conducted at AAALAC accredited facility and all animal study procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of HDB. This protocol (AUF# 181) was approved by HDB Animal Care and Use Committee before study initiation.

Ethical statement

All experiments using human whole blood were approved by NodThera, Inc. and conducted in Seattle, WA, USA, in accordance with local guidelines and national laws. Volunteers gave blood to Bloodworks Northwest, a company in Seattle, WA, USA with informed consent.

All procedures related to animal handling, care, and treatment were conducted in compliance with all applicable regulations and guidelines of, and approved by the ethics committee of, the relevant Institutional Animal Care and Use Committee (IACUC), specifically the IACUC of Pharmidex Pharmaceutical Services Limited for the rat brain perfusion studies, the IACUC of WuXi AppTec Co., Limited for the mouse, rat, cynomolgus monkey and minipig pharmacokinetic studies, and the IACUC of HD Biosciences (China) Co., Limited for the mouse peritonitis studies.

Author contributions

David Harrison: Conceptualisation, data curation, formal analysis, methodology, project administration, supervision, writing – original draft. Andy Billinton: Conceptualisation, data curation, formal analysis, methodology, project administration, supervision. Mark G. Bock: Conceptualisation, project administration, supervision. Nicholas P. Clarke: Conceptualisation, data curation, formal analysis, methodology, project administration, supervision. Zsofia Digby: Data curation, formal analysis, visualisation, writing – review & editing. Christopher A. Gabel: Conceptualisation, formal analysis, methodology, project administration, supervision, writing – review & editing. Nicola Lindsay: Data curation, formal analysis, project administration. Valérie Reader: Data curation, project administration, supervision. Jane Scanlon: Conceptualisation, data curation, supervision. Pamela Smolak: Data curation, investigation. Peter Thornton: Conceptualisation, data curation, formal analysis, methodology, supervision, writing – review & editing. Heather Wescott: Data curation, investigation. Alan P. Watt: Conceptualisation, formal analysis, methodology, project administration, supervision, writing – review & editing.

Conflicts of interest

The authors declare the following competing financial interest(s): D. H., A. B., M. G. B., N. P. C., Z. D., C. A. G., N. L., V. R., J. S., P. S., P. T., H. W. and A. P. W. were employees of NodThera, Inc. or NodThera Ltd. when this work took place, and may own shares or share options in these.

Abbreviations

CCL2

C–C motif ligand 2

CLp

Plasma clearance

COMU

(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate

CXCL

Chemokine (C–X–C motif) ligand

DCM

Dichloromethane

DMSO

Dimethyl sulfoxide

ELISA

Enzyme-linked immunosorbent assay

ER

Efflux ratio

F

Bioavailability

FBS

Fetal bovine serum

Fu

Fraction unbound

h

Hours

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

hERG

Human ether-a-go-go related gene

MeCN

Acetonitrile

min

Minutes

MRT

Mean residence time

NADPH

Nicotinamide adenine dinucleotide phosphate

NLRC4

NLR family CARD domain containing 4

NMM

N-Methylmorpholine

P2X7

P2X purinoceptor 7

SEM

Standard error of the mean

TfOH

Triflic acid

THF

Tetrahydrofuran

V ss

Volume of distribution at steady state

Supplementary Material

MD-016-D5MD00639B-s001

Acknowledgments

These studies were funded by NodThera, Inc. and NodThera Ltd. The authors would like to thank WuXi AppTec in China for the synthesis of compounds, in vitro and in vivo studies. We are also grateful to Pharmidex Pharmaceutical Services Limited in the UK for the brain perfusion studies and Cyprotex (now part of Evotec) in the UK for the Caco-2 data.

Data availability

Supplementary information: Safety statement, assay protocols for thermodynamic solubility, hERG blockade, CYP inhibition and CYP time-dependent inhibition assays, 1H NMR and LC-MS spectral data for NT-0527. See DOI: https://doi.org/10.1039/D5MD00639B.

The data supporting this article has been included in the Methods section of the publication or is contained in the accompanying SI file.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-016-D5MD00639B-s001
MD-016-D5MD00639B-s001.pdf (735.2KB, pdf)

Data Availability Statement

Supplementary information: Safety statement, assay protocols for thermodynamic solubility, hERG blockade, CYP inhibition and CYP time-dependent inhibition assays, 1H NMR and LC-MS spectral data for NT-0527. See DOI: https://doi.org/10.1039/D5MD00639B.

The data supporting this article has been included in the Methods section of the publication or is contained in the accompanying SI file.

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