Sortilin ( IC50 = 330 nM )

AF38469 was progressed for further studies. It should be noted that Neurotensin itself exhibits a binding IC50 of 360 nM in the 3HNTS assay, thus AF38469 is essentially equipotent with this sortilin substrate. AF38469 showed no inhibition or stimulation of >50% at 10 μM in a standard selectivity panel of ca. 70 targets run at CEREP. Importantly AF38469 showed no activity against the NTR1 receptor. In addition AF38469 showed no activity against a selected panel of targets known to bind acidic molecules (δ-Opioid, GPR40, PPARδ, EP1, Angiotensin AT1, Endothelin ETA & B, MMP-12). Thus overall the selectivity profiling of AF38469 demonstrated a highly specific interaction with sortilin [1].

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A co-crystal of sortilin and AF38469 was successfully obtained and its structure determined by X-Ray crystallography to a resolution of 2.78 Å. Figure 1 shows an overlay of the crystal structure of AF38469 with that of the structure of the C-terminal region of sortilin bound Neurotensin. AF38469 makes similar interactions with sortilin as does the C-terminal Leu residue of Neurotensin. AF38469 makes a salt bridge to Arg292 via its carboxylic acid, the CF3 group occupies the same hydrophobic binding pocket as the iPr of the Leu side-chain, and the amide N–H makes a hydrogen-bond donor interaction with Tyr318. The pyridyl methyl group makes a hydrophobic π-interaction with Phe317, similar to hydrophobic interaction of Ile12 of Neurotensin. The structure also serves to rationalise many of the structure–activity relationships outlined above, for example the key role of the pthalamic acid 5 substituent, the inactivity of the 5-Ph (10f) (due to size exclusion), and the pivotal role of the carboxylic acid functionality. Figure 2 shows an overlay of the binding mode of AF38469 with the previously reported small molecule inhibitor AF40431. AF38469 and AF40431 exhibit many common interactions, including the salt bridge and leucine pocket binding features of Neurotensin, but also interestingly the pyridine ring and methyl substituent of the former are virtually congruent with the pyran ring and methyl substituent of the latter. It is anticipated that the structure of the sortilin-AF38469 complex will be used to further drive optimisation of the chemotype through classical structure based drug design [1].

AF38469 (1 mg/kg; iv) has low circulating volume (blood volume 0.7 L/kg) and clearance (4.8 L/h/kg) in sodium dodecylbenzene sulfonate with a half-life (t1/2 ) is 1.2 h [1 ].Treating Western diet - fed mice with AF38469 can decrease plasma cholesterol and hepatic cytokine expression. It is associated with reduced hepatic VLDL secretion and higher hepatic cholesterol 7α - hydrolase expression in Western diet - fed mice, but does not affect diet - induced obesity or insulin resistance [2]

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Sort1 inhibitor reduced plasma cholesterol levels, hepatic VLDL secretion, and hepatic pro-inflammatory cytokines in WD-fed mice [2]
Recently, an orally bioavailable Sort1 inhibitor, AF38469, with an IC50 value of ∼330 nM has been identified. To determine whether pharmacological inhibition of Sort1 with AF38469 might improve lipid homeostasis and inflammation, we fed mice WD supplemented with AF38469 to achieve an estimated 4 mg/kg daily dose, which was in line with the published pharmacokinetic data. AF38469 treatment did not affect the body weight gain or liver weight over a period of 8 weeks (Fig. 7A, B). Hepatic TG and cholesterol levels were not significantly different between the treated group and the controls, although we noted that hepatic TG trended ∼30% lower in the AF38469-treated mice (P = 0.19) (Fig. 7C–E). Plasma AST and ALT levels were similar between the two groups, suggesting that AF38469 treatment for 8 weeks was not associated with hepatotoxicity (Fig. 7F, G). Analysis of plasma lipid parameters revealed that AF38469 significantly decreased plasma cholesterol by ∼30% (Fig. 8A), but did not affect plasma TG levels (Fig. 8B). To gain additional insights about the cholesterol lowering effect of AF38469, we first measured hepatic VLDL secretion because previous genetic studies suggested a role of hepatic Sort1 in regulating hepatic VLDL secretion. Interestingly, AF38469-treated mice showed significantly reduced hepatic VLDL secretion (Fig. 8C). Biliary cholesterol secretion and bile acid synthesis are two major cholesterol elimination mechanisms that can impact circulating cholesterol levels. AF38469 treatment did not affect total gallbladder cholesterol content (Fig. 8D). Analysis of bile acid metabolism revealed that AF38469-treated mice showed significantly reduced hepatic bile acid levels (Fig. 8E), while gallbladder bile acid, intestinal bile acids, and the total bile acid pool size were unaltered (Fig. 8E). Analysis of the hepatic gene expression profile found that hepatic CYP7A1 was significantly induced, while other cholesterol metabolism genes (ABCG5, ABCG8, HMGCR, LDLR) were unaltered in AF38469-treated mice (Fig. 8F). Hepatic mRNA expression of chemokine MCP1 and the pro-inflammatory cytokines, IL-1β and IL-6, but not TNFα, was significantly lower in AF38469-treated mice (Fig. 8F). Lower hepatic cytokines may provide a possible explanation for higher CYP7A1 mRNA expression in AF38469-treated mice.

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Sort1 inhibitor did not affect fasting plasma glucose and glucose tolerance in WD-fed mice[2]
To gain a more comprehensive understanding of the metabolic effects of pharmacological Sort1 inhibition, we next evaluated the effect of AF38469 on insulin sensitivity in WD-fed mice. AF38469 treatment did not affect fasting plasma glucose or fasting insulin concentration (Fig. 9A, B). In addition, AF38469 treatment did not affect fasting plasma free fatty acid concentration (Fig. 9C) or glucose tolerance in a glucose tolerance test (Fig. 9D). These results suggest that AF38469 did not affect insulin sensitivity in WD-fed mice.

Sortilin was crystallized as previously described (Andersen, 2013, submitted) with a 10 molar excess of AF438469 for 1 hour. 1 μl of sortilin-AF38469 was mixed with a 1 μl reservoir and equilibrated against 500 μl reservoir using the sitting-drop vapour-diffusion method at 292 K in 24-well crystallization plates. The best diffracting crystals were obtained in 0.1 M HEPES-Tris pH 7.3, 0.4 M sodium malonate, 27 % (w/v) PEG 3350 and 4.5 % (v/v) glycerol. The crystals grew to a size of 200 x 100 x 50 μm over two weeks. Crystals were dehydrated by addition of 100 μl 80 % (v/v) PEG400 to the reservoir and mounted in Litholoops (Molecular Dimensions) from the mother liquor, excess mother liquor was dipped away by gently touching the side of the drop well with the edge of the loop and flash cooled in liquid N2. A complete single-wavelength (λ = 0.9 Å) data set of 3600 oscillation images, with 0.1o oscillation was collected at 100 K on the X06DA (PXIII) beamline at the Swiss Light Source (SLS) using the PILATUS 2M-F detector. The diffraction images were processed in XDS and scaled in SCALA. Molecular replacement was performed with the program PHASER using a search model derived from the structure of sortilin in complex with neurotensin (PDB ID 3F6K). Rigid body refinement, generation of ligand coordinates and restraints, calculation of omit maps and refinement were performed in PHENIX. Model building and analysis was performed using Coot. The final model quality was analysed using Molprobity. Superpositions were performed and structural figures were prepared using PyMol [1].

Mice: Sort1 floxed mice on a C57BL/6N background were used. The targeting strategy is illustrated in Fig. 1A. The NeoR cassette was removed by crossing Sort1 floxed founders with the FLP deleter strain on a C57BL/6J background. Cre-mediated recombination results in the deletion of exon 2 and exon 3 and subsequent frameshift of the Sort1 gene. L-Sort1 KO mice were generated by crossing Sort1 floxed mice with the albumin-cre deleter strain on a C57BL/6J background. LysM-Sort1 KO mice were generated by crossing Sort1 floxed mice with the LysM-cre deleter strain on a C57BL/6NJ mixed background. Littermates without the cre transgene were used as WT controls. Mice were housed in micro-isolator cages with corn cob bedding under a normal light-dark cycle. WT C57BL/6J mice were purchased from Jackson Laboratory. The standard chow diet was PicoLab Rodent Diet 20 containing 13% fat calories and no added cholesterol. WD (TD.88137) contained 42% fat calories and 0.2% cholesterol. Male C57BL/6J mice were used for the AF38469 study. AF38469 was mixed with powdered WD and the estimated daily dose of ∼4 mg/kg was calculated based on daily food intake of ∼4 g per mouse. The control group was given powdered WD. Powdered WD was placed in a dish inside the cage and replaced every 2 days. Only male mice were used for this study. [2]

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Glucose tolerance test: After 7 weeks of WD feeding, control and AF38469-treated mice were fasted overnight and received a single intraperitoneal injection of glucose at 2 g/kg body weight. A drop of blood was collected from a tail nick at the indicated time, and glucose was measured with a OneTouch Ultra glucometer. These mice were continued on WD or WD supplemented with AF38469 for 1 week and euthanized for tissue collection.[2]

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Measurement of VLDL secretion Male C57BL/6J mice, at 12 weeks of age, were given powdered WD or powdered WD supplemented with AF38469 to provide an estimated daily dose of ∼4 mg/kg. After 2 weeks, mice were fasted for 6 h (9:00 AM to 3:00 PM). Tyloxapol was diluted in sterile PBS and administered to mice via tail vein injection as a single dose of 300 mg/kg. Blood was collected by tail nicking at 0 h (right before injection) and at 1.5 and 3 h post injection for TG measurement.[2]

Rat pharmacokinetics: [1]
AF38469 has a low volume of distribution (relative to blood volume of 0.7 l/kg) and low clearance (relative to liver blood flow of ca. 4.8 l/h/kg), and a half-life of 1.2 h. The initial exposure in plasma is high, due in part to the low volume of distribution. The oral bioavailability is 35%. Simple calculations based on Cmax, free fraction and the sortilin potency suggest that a free plasma concentration of AF38469 ca. 2 fold higher than its IC50 is obtained at Cmax from an oral 2 mg/kg dose.

In Vitro Solubility 134 μg/ml, plasma free fraction 2% intrinsic clearance 0.4 l/h/kg
Oral (2 mg/kg) Cmax 12850 ng/ml
i.v. (1 mg/kg) Clb 0.03 L/h/Kg, t1/2 1.2 h, Vss 0.02 L/kg

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In principle a phthalimide could act as a pro-drug for an AF38469 type compound, with hydrolysis in vivo affording the corresponding phthalamic acid by an analogous process to that which lead to the liberation and identification of compound 2 itself (Scheme 1). Whilst an asymmetrically substituted phthalimide such as 1 would afford a mixture of regioisomers on hydrolysis (viz. 2 and 3), a symmetrical phthalimide would afford a single phthalamic acid (e.g. Scheme 4). To this end phthalimide 14 was investigated as a potential oral prodrug for phthalamic acid 10k. However, whilst oral administration of the phthalimide (14) did afford systemic exposure of phthalamic acid 10k, the free exposure of the acid was not an improvement on the exposure of AF38469 obtained via oral administration.

[1]. The identification of AF38469: an orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin. Bioorg Med Chem Lett. 2014 Jan 1;24(1):177-80.

[2]. Hepatocyte sortilin 1 knockout and treatment with a sortilin 1 inhibitor reduced plasma cholesterol in Western diet-fed mice. J Lipid Res. 2019 Mar;60(3):539-549.

Sortilin is a type I membrane receptor belonging to the vacuolar protein sorting 10 protein (VPS10P) family of sorting receptors. Sortilin is widely expressed in both the central nervous system and periphery. It mediates a number of important physiological functions via trafficking of, and signalling with, a variety of different protein partners. For example sortilin is involved in signalling via the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Indeed, in complex with the protein p75, sortilin has been reported to form the receptor for pro-neurotrophin-mediated apoptotic effects leading to degeneration and cell death in cellular and animal models.1 Sortilin has also been demonstrated to interact with apolipoprotein B100 in the Golgi and facilitate the export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol levels, a key contributor to atherosclerosis and ischemic heart disease. Recently, sortilin was also shown to function as a high affinity receptor for progranulin, and to mediate clearance of progranulin by binding followed by cellular uptake and distribution to lysosomes. [1]

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In summary we have identified a potent, selective and orally bioavailable inhibitor for the VPS10P family sorting receptor Sortilin. We hope and anticipate that AF38469 will serve as an important tool to further delineate the biology of Sortilin, and to facilitate evaluation of the therapeutic potential of this protein. [1]

C15H11F3N2O3

324.26

324.072

C, 55.56; H, 3.42; F, 17.58; N, 8.64; O, 14.80

1531634-31-7

1531634-31-7

72706115

Off-white to light yellow solid powder

3.432

2

7

3

23

456

0

O=C(NC1=CC=CC(C)=N1)C2=CC=C(C(F)(F)F)C=C2C(O)=O

JWCUSQCZMQIBMR-UHFFFAOYSA-N

InChI=1S/C15H11F3N2O3/c1-8-3-2-4-12(19-8)20-13(21)10-6-5-9(15(16,17)18)7-11(10)14(22)23/h2-7H,1H3,(H,22,23)(H,19,20,21)

2-[(6-methylpyridin-2-yl)carbamoyl]-5-(trifluoromethyl)benzoic acid

AF-38469; AF38469; 1531634-31-7; 2-[(6-Methylpyridin-2-Yl)carbamoyl]-5-(Trifluoromethyl)benzoic Acid; 2-((6-methylpyridin-2-yl)carbamoyl)-5-(trifluoromethyl)benzoic acid; CHEMBL3098745; MFCD28160694; 4n7e; SCHEMBL15903106; AF 38469

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: ~65 mg/mL (~200.5 mM)

Solubility in Formulation 1: 2.5 mg/mL (7.71 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5%DMSO + Corn oil: 3.25mg/ml (10.02mM)

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