Notum (IC50 = 6.7 nM)

1,2,3-Triazole 8l (ARUK3001185) was superior in potency to the isomeric 1,3,4-triazole 9a or the 1,2,3,4-tetrazole 9b (Table 3). These effects seem to be quite subtle as most key inhibitor–Notum interactions are available with these alternative 1-aryl azoles and may be due to a more optimal electronic interaction of 8l with Trp128 or simply that an N4 atom is detrimental.49 Modification of the phenyl ring of 8l (ARUK3001185) to the corresponding pyridine 9c was equipotent and demonstrated that some modest polarity could be accommodated in the palmitoleate pocket. Dual deuteration of 8l at the C4 and C5 positions of the triazole gave d2-8l as a tool to investigate the metabolic fate of the parent molecule. [1]

Triazole 8l (ARUK3001185) has physicochemical and molecular properties consistent with drug-like space.50 It was noteworthy that 8l (ARUK3001185) had a much lower measured distribution coefficient (LogD7.4 1.3 ± 0.05) than had been predicted by the calculated partition coefficient (clogP 3.9) despite being non-ionized at physiologically relevant pH. This disconnection could be attributed to the close proximity of the 2-Cl, 3-CF3, and 4-Cl substituents on the aromatic ring that results in a reduction in overall hydrophobic surface area when compared to summing their individual lipophilicity fragmental constants (π). These results highlight the importance of generating empirical data to correlate with calculated values. Evaluation of 8l (ARUK3001185) against standard design metrics using Notum inhibition from the biochemical assay (IC50 6.7 nM, pIC50 8.2) and measured lipophilicity (LogD7.4 1.3) calculates 8l (ARUK3001185) to have high LE (0.67), LLE (6.9), and a favorable prediction of brain penetration (CNS MPO 5.4/6.0; BBB Score 5.6/6.0). [1]

Triazole 8l (ARUK3001185) displayed satisfactory aqueous solubility in phosphate-buffered saline (PBS), good metabolic stability in both MLM and HLM, and high permeability in the MDCK-MDR1 cell transit assay with minimal evidence for recognition and efflux by the P-gp transporter (Table 3 and Supporting Information). In contrast, pyridine 9c performed poorly in the permeability assay although these results were confounded by low compound recovery. Comparison of MLM stability of 8l with d2-8l (Cli 3.2 vs 5.4 μL/min/mg protein) showed deuteration of the triazole afforded no improvement suggesting that CYP-mediated metabolism of the triazole C–H bonds was not a major route of clearance in this system. [1]

Based on these data, 8l (ARUK3001185) was selected as our nominated lead from this series for further profiling as it had the best combination of Notum activity and in vitro ADME properties including lipophilicity. [1]

Lead 8l (ARUK3001185) was then evaluated in additional in vitro pharmacology and ADME screens (Table 5). Triazole 8l restored Wnt signaling in the presence of Notum (EC50 110 nM; n = 4) in a in a cell-based TCF/LEF (Luciferase) reporter assay23,26,27 and gave standard S-shaped concentration–response curves up to 10 μM (Figure S9). Performing these experiments in the absence of Notum showed a maximal Wnt response at all concentrations tested (up to 10 μM; n = 4). The activation of Wnt signaling was due to direct on-target inhibition of Notum by 8l and not by assay interference or cell toxicity (up to 10 μM). [1]

PK data for 8l (ARUK3001185) was generated in vivo in mouse, and subsequently in rat, to evaluate plasma and brain exposure (Table 4 and Supporting Information). Following single intravenous administration of 8l (ARUK3001185) to mouse, plasma clearance was low compared to liver blood flow and volume of distribution was moderate resulting in an elimination half-life of 2.4 h (Figure S3, Table S11). Following a single oral dose to mouse, 8l (ARUK3001185) was rapidly absorbed and showed good oral bioavailability (66%) (Figure S4, Table S12). Good brain penetration was confirmed with drug levels in the brain similar to drug levels in the plasma (Kp 1.1). The brain-to-plasma ratio was 0.77 (Kp,uu) when free drug concentrations were calculated. This oral dose of 10 mg/kg achieved a concentration in the brain of Cmax ≈ 300 nM (free drug) that exceeded the Notum EC50 from the cell-based TCF/LEF assay, and brain levels were maintained above 100 nM for ∼4 h. Although encouraging, this dose, and the resulting level of brain exposure, may be insufficient to promote a sustained pharmacodynamic (PD) response, where Notum activity was reduced for an extended period of time. This will need to be determined empirically. Hence, we subsequently explored alternative dosing regimes to provide some flexibility in the determination of the required efficacious concentrations (Ceff) in rodent models of disease, and its relationship to Notum pharmacology (EC50), that is, establish the PK–PD relationship (vide infra). [1]

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PK evaluation of 8l (ARUK3001185) in rat also showed low plasma clearance and moderate volume of distribution with a half-life of 3.3 h (Table 4). Oral administration to rat showed good absorption, drug levels, and brain penetration (Kp 1.4). The oral bioavailability was supramaximal with Fo 140%. The plasma (and brain) concentration–time plots clearly show double Cmax peaks (Figure S7), which could indicate some contribution from excreted drug being reabsorbed from the gastrointestinal tract and/or an effect on gastric emptying times. On balance, these results established that 8l (ARUK3001185) has PK properties compatible with evaluation in rodent models of disease. [1]

ADME Assays [1]
Molecular properties were calculated with ChemDraw Professional v16.0.1.4(77). Distribution coefficients (LogD7.4) were measured using the shake flask method. Selected compounds were routinely screened for aqueous solubility in PBS (pH 7.4), transit performance in MDCK-MDR1 cell lines for permeability, and metabolic stability in MLM and HLM as a measure of clearance. Lead 8l (ARUK3001185) was also screened for inhibition of representative CYP450 enzymes, permeability across the Caco-2 cell monolayer, and metabolic stability in liver microsomes and hepatocytes. Assay protocols and additional data are presented in the Supporting Information. ADME studies reported in this work were independently performed by CRO companies.

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Notum OPTS Biochemical Assay [1]
Methods have been described in detail elsewhere Representative concentration–response curves are provided in the Supporting Information.

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Kinase Selectivity Panel [1]
Kinase selectivity screening was performed by Thermo Fisher Scientific in their SelectScreen Biochemical Kinase Profiling Service. Assay information and data are presented in the Supporting Information.

TCF/LEF Reporter (Luciferase) Cell-Based Assay [1]
Methods have been described in detail elsewhere.23,26,27 Concentration–response curves are provided in the Supporting Information.

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Proteomic Analysis in SW620 Cells [1]
Cell Culture [1]
SW620 cells were grown in Leibovitz’s L-15 medium supplemented with 10% FBS, Pen/Strep 10 U/mL, and 0.075% sodium bicarbonate.

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Conditioned Medium Generation [1]
SW620 cells were grown to around 80% confluence, washed three times with PBS, and then placed in serum-free media for 48 h. The medium was then removed, spun at 300G to remove any solids, and then concentrated in 10k MWCO protein concentrators by centrifugation.

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Cell Lysate Generation [1]
SW620 cells were grown to around 80% confluence, the medium was removed and cells were washed three times with PBS, and then lysed in the ice cold IP lysis buffer (Pierce). Lysates were clarified by centrifugation at 15k G for 10 min at 4 °C, then protein quantification was performed by the BCA assay (Pierce) and lysates were equalized to 1.5 mg/mL.

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The identity and quantification of each serine hydrolase identified in SW620 cells by FP-biotin ABPP for 8l (ARUK3001185) (and 1) is provided in the Supporting Information. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository56 with the dataset identifier PXD031338.

PK Studies In vivo PK data in mouse and rat was independently generated at Charles River Laboratories (Groningen, Netherlands), GVK Biosciences (Hyderabad, India), and WuXi AppTec (Shanghai, China). PK was supported by the development of suitable formulations for the route of administration, and the measurement of plasma protein binding (PPB) and brain tissue binding for the calculation of free drug levels in these compartments. Study protocols and additional data are presented in the Supporting Information

The use of a solubilizer can significantly improve the water solubility of compound 8l (ARUK3001185) to 1 mg/mL, thus providing greater flexibility in study design. The triazole compound 8l (ARUK3001185) exhibits good stability in liver microsomes and hepatocytes in mice, rats, dogs, and humans, suggesting lower clearance in these species. Screening in human liver microsomes, including standard substrates of eight cytochrome P450 (CYP450) enzymes, showed moderate/weak inhibition of 1A2 (IC50 0.96 μM), 2B6 (IC50 5.2 μM), 2C9 (IC50 15 μM), and 2C19 (IC50 8.1 μM), but no inhibition of 2C8, 2D6, and 3A4 (IC50 > 50 μM). Furthermore, pre-incubation of human liver microsomes (HLMs) with 8l (ARUK3001185) in the presence or absence of NADPH, followed by incubation with discrete labeled substrates (IC50 value variation), showed no evidence of time-dependent CYP inactivation (TDI). 8l (ARUK3001185) exhibited good cell permeability in Caco-2 monolayers, consistent with good intestinal absorption. The hepatocyte data for 8l (ARUK3001185) were encouraging, as the removal of the -CH2OH group from 7y appears to eliminate this metabolic soft spot. Therefore, 8l (ARUK3001185) was advanced to rodent pharmacokinetic (PK) studies. [1]
PK data for 8l (ARUK3001185) were generated in mice, followed by PK data in rats to assess plasma and brain exposure (Table 4 and Supplementary Information). Following a single intravenous injection of 8l (ARUK3001185) in mice, plasma clearance was lower than hepatic blood flow, with a moderate volume of distribution and an elimination half-life of 2.4 hours (Figure S3, Table S11). Following a single oral administration of 8l (ARUK3001185) in mice, the drug was rapidly absorbed with good oral bioavailability (66%) (Figure S4, Table S12). Brain tissue drug concentrations were similar to plasma drug concentrations (Kp 1.1), confirming good brain permeability. The brain-to-plasma ratio was 0.77 (Kp, uu) when calculating free drug concentration. An oral dose of 10 mg/kg resulted in a free drug concentration in brain tissue of Cmax ≈ 300 nM, exceeding the Notum EC50 value as determined by cell-based TCF/LEF assays, and the brain tissue drug concentration remained above 100 nM for approximately 4 hours. Despite the encouraging results, this dose and the resulting level of brain exposure may not be sufficient to promote a sustained pharmacodynamic (PD) response, i.e., a reduction in Notum activity over a longer period. This needs to be determined experimentally. Therefore, we subsequently explored other dosing regimens to flexibly determine the required effective concentration (Ceff) and its relationship with Notum pharmacology (EC50) in rodent disease models, i.e., to establish a pharmacokinetic-pharmacodynamic relationship (see below). [1]
Pharmacokinetic assessment of 8l (ARUK3001185) in rats also showed low plasma clearance, moderate volume of distribution, and a half-life of 3.3 hours (Table 4). After oral administration to rats, the drug was well absorbed, with good drug concentration and brain permeability (Kp 1.4). Oral bioavailability was supermax, with Fo 140%. Plasma (and brain tissue) concentration-time curves clearly showed bimodal Cmax (Fig. S7), which may indicate that some excreted drug was reabsorbed by the gastrointestinal tract and/or affected gastric emptying time. Overall, these results suggest that 8l (ARUK3001185) has pharmacokinetic properties suitable for evaluation in rodent disease models. [1]

[1]. Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit. J Med Chem. 2022 May 26;65(10):7212-7230.

Substituents on the aromatic ring bound to the Notum palmitate pocket were explored, yielding a highly efficient early lead compound, 7y (IC50 9.5 nM). Further optimization of the triazole group yielded compound 8l (ARUK3001185) (IC50 6.7 nM). Notably, the advanced lead compound 8l can be described as a fragment-sized molecule (molecular weight 282; HAC 17) with high ligand equivalent (LE) and ligand-ligand equivalent (LLE), and its inhibitory effect on Notum activity was improved by more than 15,000-fold through optimization of the substituents on the aromatic ring (8l vs 7a). These findings highlight the advantages of fragment-based drug discovery (FBDD) when combined with suitable drug targets. The X-ray crystal structure of Notum-8l clearly shows that 8l (ARUK3001185) almost completely occupies the palmitate pocket through the 2-Cl-3-CF3-4-Cl substituent on the aromatic ring. In cell-based TCF/LEF reporter gene assays, triazole 8l restored the Wnt signaling pathway in the presence of Notum (EC50 of 110 nM), confirming its functional activity. Extensive off-target and safety pharmacology screening showed that 8l was selective for serine hydrolases, kinases, and representative drug targets. Pharmacokinetic studies in mice and rats showed that 8l had good plasma exposure and brain penetration and was well tolerated. In summary, triazole 8l (ARUK3001185) is a potent and selective Notum activity inhibitor with good brain penetration suitable for oral administration in rodent disease models. [1] Notum is a carboxylesterase that inhibits the Wnt signaling pathway by deacylation of the essential palmitate group on the Wnt protein. With a growing understanding of the role of Notum in human diseases such as colorectal cancer and Alzheimer's disease, there is a need to find more effective inhibitors, especially in neurodegenerative disease models. This article describes the discovery and properties of compound 8l (ARUK3001185), a highly potent, selective, and blood-brain barrier-crossing Notum inhibitor suitable for oral administration in rodent disease models. Through crystallographic fragment screening of the Diamond-SGC Poised compound library and ranking inhibitory activity using biochemical enzyme activity assays, we identified compounds 6a and 6b as a promising pair of lead compounds. Fragment development of compound 6 yielded compound 8l, which restored the Wnt signaling pathway in the presence of Notum in a cell reporter gene assay. Pharmacological screening evaluations demonstrated that 8l exhibits selectivity for serine hydrolases, kinases, and drug targets.

C9H4CL2F3N3

282.049369812012

280.973

C, 38.33; H, 1.43; Cl, 25.14; F, 20.21; N, 14.90

2411969-39-4

146438044

Off-white to pink solid powder

3.5

0

5

1

17

277

0

N1(C2=CC=C(Cl)C(C(F)(F)F)=C2Cl)C=CN=N1

XVFXHCLMGYJAQI-UHFFFAOYSA-N

InChI=1S/C9H4Cl2F3N3/c10-5-1-2-6(17-4-3-15-16-17)8(11)7(5)9(12,13)14/h1-4H

1-[2,4-dichloro-3-(trifluoromethyl)phenyl]triazole

ARUK3001185; 2411969-39-4; 1-[2,4-Dichloro-3-(trifluoromethyl)phenyl]triazole; 1-[2,4-bis(chloranyl)-3-(trifluoromethyl)phenyl]-1,2,3-triazole; CHEMBL5178575; SCHEMBL21792325; XVFXHCLMGYJAQI-UHFFFAOYSA-N; BDBM601033;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~354.55 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)