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]

---

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.

---

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

---

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.

---

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.

---

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.

---

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.

---

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 aqueous solubility of 8l (ARUK3001185) could be significantly enhanced to 1 mg/mL with cosolvents bringing added flexibility to study design. Triazole 8l (ARUK3001185) has good stability in liver microsomes and hepatocytes across mouse, rat, dog, and human, which predicts for low clearance across these species. Screening in HLM including eight standard substrates of 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 not 2C8, 2D6, or 3A4 (IC50 > 50 μM). Furthermore, there was no evidence for CYP time-dependent inactivation (TDI) as assessed by the preincubation of HLM with 8l (ARUK3001185) in the presence and absence of NADPH, followed by the incubation with the discrete marker substrates (IC50 shift). There was good cell permeability across a Caco-2 monolayer consistent with good absorption from the gut. The hepatocyte data for 8l (ARUK3001185) was encouraging as this deletion of the −CH2OH group from 7y seems to have removed the liability of this metabolic soft spot. Hence, 8l (ARUK3001185) was advanced to rodent PK studies. [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]

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]

[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.

Exploration of the substituents on the aryl ring that binds in the palmitoleate pocket of Notum gave potent early lead 7y (IC50 9.5 nM) and further optimization of the triazole group delivered 8l (ARUK3001185) (IC50 6.7 nM). It is noteworthy that advanced lead 8l can be characterized as a fragment-sized molecule (MW 282; HAC 17) with high LE and LLE, and that inhibition of Notum activity was increased by over 15,000-fold by optimization of the substituents on the aryl ring (8l vs 7a). These findings highlight the benefits of FBDD when aligned with a suitable drug target. The Notum-8l X-ray structure clearly showed that 8l (ARUK3001185) has near complete occupancy of the palmitoleate pocket through the 2-Cl-3-CF3-4-Cl substituents on the aryl ring. Triazole 8l restored Wnt signaling in the presence of Notum (EC50 110 nM) in a cell-based TCF/LEF reporter assay confirming functional activity. Assessment in broader off-target and safety pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and representative drug targets. PK studies with 8l in mouse and rat showed good plasma exposure and brain penetration, and was well tolerated. In summary, triazole 8l (ARUK3001185) is a potent and selective inhibitor of Notum activity with good brain penetration suitable for oral dosing in rodent models of disease.[1]

---

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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