Bcl-1 ( IC50 = 1.5 nM )

JD5037 is an inverse agonist with a Ki of 0.35 nM for peripherally restricted (PR) cannabinoid 1 (CB1) receptors. Inverse agonists, or antagonists, of the cannabinoid-1 (CB1) receptor have shown promise as novel treatments for obesity and associated metabolic disorders, including liver disease. Due to the early assumption that therapeutic benefit was primarily based on brain receptor interaction, these agents, representing different chemical series, shared the property of brain penetration. JD5037 exhibits low blood-brain barrier penetration and >700-fold selectivity for CB1 over CB2 as well as >1,000-fold selectivity over a panel of 70 receptors, transporters, and ion channels. Orally bioavailable JD5037 decreases food intake, body weight, and abnormalities related to hormones and metabolism in diet-induced obese mice.

JD5037 (3 mg/kg/day, p.o.) dramatically shrinks tumors and completely eradicates them in mice receiving DEN treatment. In mouse HCC samples, JD5037 reduces the levels of AEA. JD5037 (3 mg/kg/d, i.p.) causes comparable weight losses in obese Magel2-null mice, attenuates the hyperglycemia brought on by the HFD, and lessens the hepatic damage and steatosis brought on by the HFD.

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Peripherally restricted CB1R blockade reverses obesity and its metabolic abnormalities in Magel2-null mice [2]
As with global CB1R knockout mice, animals with a selective genetic deletion or downregulation of CB1R in CaMKIIa-containing neurons are lean and resistant to HFD-induced obesity. These findings suggest that CB1R in the hypothalamus is required for the development of obesity. However, this does not negate the role of peripheral CB1R in reversing obesity, since a globally acting CB1R blockade reduces body weight in obese but not in lean mice, and peripherally restricted CB1R antagonists ameliorate obesity and its related metabolic abnormalities in DIO mice. Therefore, we decided to test the efficacy of the peripherally restricted CB1R antagonist, JD5037, in treating obesity in Magel2-null mice. To further augment their obese phenotype, we fed both genotypes with a HFD for 12 weeks, then began daily treatment with JD5037 (3 mg/kg/d, i.p.) for 28 days. Males and females of both genotypes developed obesity under HFD conditions (Supplementary Figure 2G,H), with increased fat mass (Supplementary Figure 2I,J) and no major changes in lean body mass (Supplementary Figure 2K,L). As with Magel2-null mice on a STD (Supplementary Figure 2A,B), body weight gain was more pronounced in female knockout animals (Supplementary Figure 2G,H). [2]
Since the globally acting CB1R antagonist rimonabant reduced body weight and improved metabolic features in humans with PWS, we compared the effect of JD5037 to its brain-penetrant parent compound, SLV319. Both JD-5037 and SLV319 induced equal reductions in body weight (Figure 4A–D) and fat mass (Figure 4E,G). No major changes were documented in lean body mass (Figure 4F,H) in both genotypes and sexes. HFD-fed Magel2-null mice were hyperphagic, consuming significantly larger amounts of food in comparison with their wild-type littermates (Figure 4I,K). Both antagonists induced equal reductions in food consumption. Normalization of body weight in Magel2-null mice treated with JD5037 and SLV319 was probably due to the reversal of the hyperleptinemia (Figure 4J,L) and consequently improved leptin sensitivity, and not because of increased brain penetrance of JD5037 in Magel2-null mice, as reflected by a brain/plasma concentration ratio of <2% (Supplementary Figure 3A,B) in both strains. The two compounds were also equally effective in normalizing blood glucose (Figure 5A,C) and serum insulin (Figure 5B,D) levels, as well as in attenuating glucose intolerance (Figure 5E–H) and insulin resistance, as measured by the HOMA-IR and insulin sensitivity indices (Figure 5I–L) in both mouse genotypes and sexes.

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Equal improvements were found in the ability of the peripherally restricted CB1R blockade to reverse hepatocellular damage and hepatic steatosis, as manifested by reduced serum levels of ALT (Figure 6A,B), AST (Figure 6C,D), and hepatic TG content (Figure 6E,F), as well as a reduction in the elevated serum cholesterol levels (Figure 6G,H). As shown previously, JD5037 accumulated in the liver in both strains (Supplementary Figure 3C), an effect that may account for its high efficacy in reversing hepatic steatosis. Interestingly, neither compound normalized the HDL-to-LDL cholesterol ratio in Magel2-null mice (Figure 6I,J). Indirect calorimetry in male and female Magel2-null mice and their littermate controls revealed a similar increase in TEE (Figure 7A,B) voluntary activity (Figure 7C,D), FO (Figure 7E,F), and CHO (Figure 7G,H) in animals treated with JD5037. Taken together, these findings suggest that blocking CB1R at the periphery reverses obesity, reduces hyperphagia, and improves metabolic outcomes in obese Magel2-null mice.

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Activation of the Endocannabinoid/CB1R System in Murine and Human HCC [3]
CB1R were readily detectable by immunohistochemistry in HCC from CB1R+/+ mice, but were barely detectable in the adjacent normal liver tissue (Figure 2A). The specificity of CB1R staining was verified by its elimination by blocking peptide or in livers from CB1R−/− mice. CB1R mRNA was similarly increased in HCC versus adjacent non-tumor tissue from vehicle-treated but not from JD5037-treated mice (Figure 2B). CB1R immunoblots revealed a similar pattern of change at the protein level (Fig. 2C). Additionally, tumors from CB1R+/+ mice contained significantly more anandamide (AEA) compared to non-tumor tissue, and AEA levels were attenuated in HCC samples from JD5037-treated mice. There was no difference in AEA content in non-tumor vs HCC tissue from CB1R−/− mice (Fig. 2D). Increased AEA in tumors is likely due to increased synthesis, as indicated by the increased expression of the AEA biosynthetic enzyme N-arachidonoyl phosphatidylethanolamide-specific phospholipase D (NAPE-PLD) in both mouse and human HCC tissue (Fig. S1).

JD-5037 is a new CB1R antagonist with an IC50 of 1.5 nM that is peripherally restricted.

Mice: JD-5037 is prepared in vehicle (V; Tween80 = 1% DMSO + 95% Saline). JD5037, SLV319, or vehicle (V; 1% Tween80, 4% DMSO, 95% Saline) are administered orally to obese mice over the course of a 28-day period at a dose of 3 mg/kg. Everyday food consumption and body weight are tracked. Cervical dislocation is used to euthanize mice while they are sedated; the brain, hypothalamus, liver, and combined fat pads are removed, weighed, and frozen; trunk blood is taken in order to measure the biochemical and endocrine qualities[2].

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Animals and experimental protocol [2]
The Magel2-null mice (C57BL/6-Magel2tm1Stw/J) were maintained on a C57Bl/6J background for at least 15 generations and were genotyped as previously described. Mice carrying a paternally inherited lacZ-knockin allele were functionally null for Magel2 and were referred to as Magel2-null; littermates that were wild-type for Magel2 were used as controls. Both male and female mice from 6 to 22 weeks of age were used and maintained under a 12-h light/dark cycle and were fed ad libitum. To generate DIO mice (body weight >42 g), both genotypes were fed either a high-fat diet (HFD) (60% of calories from fat, 20% from protein, and 20% from carbohydrates) or a standard diet (STD, NIH-31 rodent diet) for 12 weeks. During the HFD or STD feeding, body weight was recorded once a week, and body composition was assessed by EchoMRI-100 every four weeks. Once the animals became obese, they were treated chronically (28 d) with vehicle (V; 1% Tween80, 4% DMSO, 95% Saline), JD5037 (J), or SLV319 (S) at a dose of 3 mg/kg, i.p. Age-matched control mice on STD were treated with V. During the treatment period, body weight and food intake were monitored daily. Mice were euthanized by cervical dislocation under anesthesia; the brain, hypothalamus, liver, and combined fat pads were removed, weighed, and snap-frozen, and trunk blood was collected for determining the endocrine and biochemical parameters.

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Tissue levels of JD5037 [2]
Mice received a single dose (3 mg/kg, i.p.) of JD5037 and were euthanized 1 h later. Blood was collected, and the mice were perfused with phosphate-buffered saline (PBS) for 1 min to remove the drug from the intravascular space before removing the brain and liver. Tissues and serum were extracted, and drug levels were determined by liquid chromatography/tandem mass spectrometry (LC–MS/MS) as described previously, with modifications. The analyses were conducted on a TSQ Quantum Access Max triple quadrupole mass spectrometer coupled with an UHPLC system, which included a Dionex Pump and an Accela Autosampler. A Kinetex™ column (C18, 2.6 μm particle size, 100A pore size, 50 × 2.1 mm) was used for separation. Gradient elution mobile phases consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). Gradient elution (0.25 mL/min) was held at 20% B for 0.7 min, followed by a linear increase to 80% B for 0.8 min, and maintained at 80% B for 5.5 min, then increased linearly to 95% B for 0.3 min, and maintained at 95% B for 2.7 min. JD5037 was detected in a positive ion mode using electron spray ionization (ESI) and the multiple reaction monitoring (MRM) mode of acquisition. The mass spectrometer parameters were set as follow: spray voltage 5000 V; vaporizer temperature 350 °C; capillary temperature 250 °C; sheath and auxiliary gases 35 and 10 arbitrary units, respectively; argon was used as the collision gas.[2]
The levels of JD5037were analyzed using [2H4]arachidonoyl ethanolamide ([2H4]AEA) as an internal standard. The molecular ions and fragments for each compound were measured as follows: m/z 572 → 555 (quantifier) and 572 → 111 (qualifier) for JD5037 (collision energy: 17 V and 49 V, respectively) and m/z 352 → 66 (quantifier) and 352 → 91 (qualifier) for [2H4]AEA (collision energy: 30 V and 45 V, respectively). TSQ Tune Software was used to optimize the tuning parameters. Data acquisition and processing were carried out using the Xcalibur program. The amounts of JD5037 in the samples were determined against a standard curve. Values are expressed as ng/g or ng/mL in wet tissue weight or serum volume, respectively.

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Mice were housed under 12 hour light-dark cycle, fed standard rodent chow and water ad libitum. Two week-old male CB1R+/+ and CB1R−/− mice were injected intraperitoneally with 25 mg/kg of diethylnitrosamine and were subjected to MRI at 8 months to verify the presence and measure the size of the tumor. DEN-treated CB1R+/+ mice were then started on the peripheral CB1R antagonist JD5037 (3mg/kg/day by oral gavage) or vehicle for 8 weeks followed by a repeat MRI scan and then sacrificed for obtaining HCC and adjacent non-tumor tissue for in vitro analyses.[3]

[1]. Peripherally restricted CB1 receptor blockers. Bioorg Med Chem Lett. 2013 Sep 1;23(17):4751-60.

[2]. Targeting the endocannabinoid/CB1 receptor system for treating obesity in Prader-Willi syndrome. Mol Metab. 2016 Oct 22;5(12):1187-1199.

[3]. Cannabinoid receptor 1 promotes hepatocellular carcinoma initiation and progression through multiple mechanisms. Hepatology. 2015 May;61(5):1615-26.

Individuals with PWS had elevated circulating levels of 2-arachidonoylglycerol and its endogenous precursor and breakdown ligand, arachidonic acid. Increased hypothalamic eCB 'tone', manifested by increased eCBs and upregulated CB1R, was associated with increased fat mass, reduced energy expenditure, and decreased voluntary activity in Magel2-null mice. Daily chronic treatment of obese Magel2-null mice and their littermate wild-type controls with JD5037 (3 mg/kg/d for 28 days) reduced body weight, reversed hyperphagia, and improved metabolic parameters related to their obese phenotype.[1]

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Antagonists (inverse agonists) of the cannabinoid-1 (CB1) receptor showed promise as new therapies for controlling obesity and related metabolic function/liver disease. These agents, representing diverse chemical series, shared the property of brain penetration due to the initial belief that therapeutic benefit was mainly based on brain receptor interaction. However, undesirable CNS-based side effects of the only marketed agent in this class, rimonabant, led to its removal, and termination of the development of other clinical candidates soon followed. Re-evaluation of this approach has focused on neutral or peripherally restricted (PR) antagonists. Supporting these strategies, pharmacological evidence indicates most if not all of the properties of globally acting agents may be captured by molecules with little brain presence. Methodology that can be used to eliminate BBB penetration and the means (in vitro assays, tissue distribution and receptor occupancy determinations, behavioral paradigms) to identify potential agents with little brain presence is discussed. Focus will be on the pharmacology supporting the contention that reported agents are truly peripherally restricted. Notable examples of these types of compounds are: TM38837 (structure not disclosed); AM6545 (8); JD5037 (15b); RTI-12 (19).[2]

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Hepatocellular carcinoma (HCC) has high mortality and no adequate treatment. Endocannabinoids interact with hepatic cannabinoid 1 receptors (CB1Rs) to promote hepatocyte proliferation in liver regeneration by inducing cell cycle proteins involved in mitotic progression, including Forkhead Box M1. Because this protein is highly expressed in HCC and contributes to its genesis and progression, we analyzed the involvement of the endocannabinoid/CB1R system in murine and human HCC. Postnatal diethylnitrosamine treatment induced HCC within 8 months in wild-type mice but fewer and smaller tumors in CB1R(-/-) mice or in wild-type mice treated with the peripheral CB1R antagonist JD5037, as monitored in vivo by serial magnetic resonance imaging. Genome-wide transcriptome analysis revealed CB1R-dependent, tumor-induced up-regulation of the hepatic expression of CB1R, its endogenous ligand anandamide, and a number of tumor-promoting genes, including the GRB2 interactome as well as Forkhead Box M1 and its downstream target, the tryptophan-catalyzing enzyme indoleamine 2,3-dioxygenase. Increased indoleamine 2,3-dioxygenase activity and consequent induction of immunosuppressive T-regulatory cells in tumor tissue promote immune tolerance.[3]

C27H27CL2N5O3S

572.505

571.121

C, 56.65; H, 4.75; Cl, 12.38; N, 12.23; O, 8.38; S, 5.60

1392116-14-1

1392116-14-1

66553204

White to off-white solid powder

1.4±0.1 g/cm3

731.3±70.0 °C at 760 mmHg

396.1±35.7 °C

0.0±2.4 mmHg at 25°C

1.662

4.58

2

5

9

38

977

2

ClC1C([H])=C([H])C(=C([H])C=1[H])C1[C@@]([H])(C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])N(/C(/N([H])S(C2C([H])=C([H])C(=C([H])C=2[H])Cl)(=O)=O)=N\[C@]([H])(C(N([H])[H])=O)C([H])(C([H])([H])[H])C([H])([H])[H])N=1

GTCSIQFTNPTSLO-RPWUZVMVSA-N

InChI=1S/C27H27Cl2N5O3S/c1-17(2)24(26(30)35)31-27(33-38(36,37)22-14-12-21(29)13-15-22)34-16-23(18-6-4-3-5-7-18)25(32-34)19-8-10-20(28)11-9-19/h3-15,17,23-24H,16H2,1-2H3,(H2,30,35)(H,31,33)/t23-,24+/m1/s1

(2S)-2-[[[(4S)-5-(4-chlorophenyl)-4-phenyl-3,4-dihydropyrazol-2-yl]-[(4-chlorophenyl)sulfonylamino]methylidene]amino]-3-methylbutanamide

JD-5037; JD 5037; ENZ75DG2Z6; CHEMBL2153670; (2S)-2-[[[(4S)-5-(4-chlorophenyl)-4-phenyl-3,4-dihydropyrazol-2-yl]-[(4-chlorophenyl)sulfonylamino]methylidene]amino]-3-methylbutanamide; (S)-2-((((S)-3-(4-Chlorophenyl)-4-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(4-chlorophenylsulfonamido)methylene)amino)-3-methylbutanamide; Butanamide, 2-((((4S)-3-(4-chlorophenyl)-4,5-dihydro-4-phenyl-1H-pyrazol-1-yl)(((4-chlorophenyl)sulfonyl)amino)methylene)amino)-3-methyl-, (2S)-; JD5037

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: 100~250 mg/mL ( 174.7~436.7 mM）
Water: Insoluble
Ethanol: < 2 mg/mL

Solubility in Formulation 1: ≥ 2.75 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
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: ≥ 2.75 mg/mL (4.80 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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