N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) [Ki = 8.1 nM; IC50 = 3.6 nM)

For agonist-stimulated NMDA-GluN1a/GluN2BL(tk-) cells, rislenemdaz (CERC-301) reduces calcium influx with an IC50 of 3.6 nM. When GluN2B receptors are compared to all other targets, including hERG potassium channels, rislenemdaz exhibits a minimum of 1000-fold selectivity. Minimal action of rislenemdaz against sigma-type receptors is also shown at 10 uM [1].

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In vitro pharmacology [1]
Affinity of Rislenemdaz/CERC‐301 for NMDA‐GluN2B receptors [1]
CERC‐301 potently inhibited radioligand ([3H]Compound‐2) binding to human NMDA‐ GluN1a/GluN2B receptors expressed in L(tk‐) cells as well as brain tissue homogenates from all tested species (rat, dog, rhesus monkey, human). The binding affinity of CERC‐301 determined using human temporal cortex homogenate yielded K i values of 3.1 nmol L−1 (0.0031 μmol L−1) and 8.1 nmol L−1 (0.0081 μmol L−1) at room temperature and 37°C, respectively. Findings in other species were consistent with the human data (Table 1).

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Functional activity and selectivity of Rislenemdaz/CERC‐301 for NMDA receptors [1]
CERC‐301 inhibited calcium influx into agonist‐stimulated NMDA‐GluN1a/GluN2B L(tk‐) cells with an IC50 of 3.6 nmol L−1 but had no effect on calcium influx into agonist‐stimulated GluN1a/GluN2A cells at concentrations up to 30,000 nmol L−1 (30 μmol L−1).

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Electrophysiology studies of Rislenemdaz/CERC‐301 effect on NMDA‐GluN2B receptors [1]
The potency of CERC‐301 was measured in the in vitro GluN2B Antagonist Voltage‐Clamp Assay. The observed on‐rates (Fig. 2A) and off‐rates (Fig. 2B) of CERC‐301 were measured in the presence of 10 μmol L−1 glutamate and 10 μmol L−1 glycine. These experiments demonstrated k on and k off rates of 1.3 × 105 mol L−1 s−1 and ~2 × 10−5 s−1, respectively, and K D of approximately 0.15 nmol L−1.

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Counter Screening of Rislenemdaz/CERC‐301 [1]
CERC‐301 exhibited no remarkable activity when tested in enzyme and radioligand binding assays at concentrations equal to and greater than 10 μmol L−1 (~3584 ng mL−1). CERC‐301 exhibited at least 1000 × selectivity for the GluN2B receptor versus all targets tested, including the hERG potassium channel. CERC‐301 also exhibited minimal activity against sigma‐type receptors at 10 μmol L−1 (sigma‐1, sigma‐2, and nonspecific).

In comparison with vehicle control, Rislenemdaz (CERC-301) (1, 3, 10 and 30 mg/kg) significantly improved swimming behavior (P<0.05 at 10 mg/kg; P<0.01 for 1, 3 and 30 mg/kg) and greatly decreased the frequency of immobility (P<0.001). Rats had plasma levels of rislenemdaz of about 15, 120, 390, 1420, 4700, and 14,110 nM (0.015, 0.120, 0.390, 1.42, 4.7, and 14.11 uM) at the time of sampling. This is equivalent to about 5, 29, 56, 83, and 94% and 98% RO, respectively. About 0.3 and 0.7 mg/kg, or roughly 30% and 50% of RO, are the estimated ED50s for increasing swimming frequency and reducing immobility, respectively. Comparing the total distance traveled with the vehicle control (1 mg/kg P < 0.01; 3, 10, and 30 mg/kg P < 0.001), rislenemdaz (1, 3, 10, and 30 mg/kg) significantly increased the distance traveled. Little to no testing [1].

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Acute depression model [1]
Forced swim test [1]
CERC‐301/Rislenemdaz (1, 3, 10, and 30 mg kg−1) significantly decreased immobility frequency (P < 0.001) and significantly increased swimming behavior (P < 0.01 for 1, 3, and 30 mg kg−1; P < 0.05 for 10 mg kg−1) compared to the vehicle control (Fig. 3), but did not affect climbing behavior except at the dose of 3 mg kg−1 (P < 0.05). Desipramine (20 mg kg−1) significantly decreased immobility (P < 0.001) and significantly increased climbing behavior (P < 0.01) compared to the vehicle control, with no change in swimming behavior. CERC‐301 plasma levels were approximately 15, 120, 390, 1420, 4700, and 14,110 nmol L−1 (0.015, 0.120, 0.390, 1.42, 4.7, and 14.11 μmol L−1) at the time of sampling, corresponding to approximately 5, 29, 56, 83, 94, and 98% RO, respectively, in rats. The ED50 for increase in frequency of swimming and decrease in immobility were ~0.3 and 0.7 mg kg−1, respectively, corresponding to RO of ~30 and 50%.

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Locomotor assay [1]
CERC‐301/Rislenemdaz (1, 3, 10, and 30 mg kg−1) significantly increased distance traveled (P < 0.01 for 1 and 3 mg kg−1; P < 0.001 for 10 and 30 mg kg−1) compared to vehicle control during the first 5 min of testing (timing correlates with time of forced swim test). CERC‐301 (1, 3, 10, and 30 mg kg−1) significantly increased total distance traveled (P < 0.01 for 1 mg kg−1; P < 0.001 for 3, 10, and 30 mg kg−1) compared to vehicle control summed over the 60‐min test. The ED50 for increase in locomotor activity was ~2 mg kg−1, translating to an RO of ~75%, which is higher than the ED50 for increase in frequency of swimming and decrease in immobility. No locomotor effects were observed for the 0.1 and 0.3 mg kg−1 dose groups (Fig. 3).

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Hemodynamic study in telemetered rats [1]
Single oral administration of Rislenemdaz/CERC‐301 increased arterial blood pressure transiently in conscious rats in a dose‐dependent manner between doses of 0.3−1 mg kg−1 (ED50≈0.7 mg kg−1), and this effect plateaued at doses 1‐10 mg kg−1 (Fig. 4). The peak effect was observed 1‐2 h postdose (Fig. 4A), consistent with PK profile of CERC‐301 in rats. The magnitude of change in hemodynamics with CERC‐301 was less than that of MK‐801 at 0.2 mg kg−1 (e.g., +36 mmHg in systolic blood pressure). The hemodynamic changes during the first 3.5 h post administration of CERC‐301 were linearly correlated with the level of activity (as estimated from telemetry recordings); in particular, activity was a predictor of HR (R 2 = 0.67), and therefore, the excitatory effects of CERC‐301 may explain the observed increases in HR (particularly at the higher dose levels) in rats. This study also demonstrated that α1‐ and/or β1‐AR blockade may provide protection from CERC‐301 mediated increases in blood pressure (Fig. 4B) and heart rate (data not shown), respectively.

In vitro pharmacology assays [1]
Affinity of Rislenemdaz/CERC‐301 for NMDA‐GluN2B receptors [1]
Radioligand binding assays were performed at room temperature (or 37°C) as described previously (Kiss et al. 2005; Liverton et al. 2007). Analogous binding experiments, as described in Appendix S1 for the cloned human receptor expressed in L(tk‐) cells, were also performed using whole brain homogenate (rat), frontal cortex homogenate (dog and rhesus monkey), and temporal cortex homogenate (human). More detailed description could be found in the Appendix S1.

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Functional activity and selectivity of Rislenemdaz/CERC‐301 for NMDA receptors [1]
The inhibition of calcium influx into L(tk‐) cells expressing either GluN1a/GluN2B or GluN1a/GluN2A human receptors was measured to determine the IC50 of CERC‐301 inhibition of NMDA receptor functions, as previously described in detail (Kiss et al. 2005; see also Appendix S1).

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Electrophysiology studies of Rislenemdaz/CERC‐301 [1]
An in vitro electrophysiological assay with cloned human NMDA‐GluN2B receptors was used to determine the binding and dissociation kinetics as well as the potency of CERC‐301 (Kiss et al. 2005; see also Appendix S1).

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Counterscreening profile of Rislenemdaz/CERC‐301 [1]
CERC‐301 was evaluated in a battery of standard receptor‐binding and enzyme assays. CERC‐301 was tested in vitro at 10, 30, or 100 μmol L−1 (~3584, 10752, or 35840 ng mL−1, respectively) for its ability to compete with reference ligands to evaluate possible undetected pharmacological activities (>150 receptors and enzymes, including sigma‐type opioid receptors, were investigated).

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Nonclinical pharmacokinetics [1]
Plasma protein binding [1]
Rat, dog, rhesus monkey, and human plasma samples (3 mL, N = 3) were incubated with 2 and 20 μmol L−1 [14C]CERC‐301 at 37°C for 30 min in a shaking water bath. Following incubation, standard ultracentrifugation methodology was used to determine the percentage of drug unbound (Pacifici and Viani 1992).

Neurotoxicity in rats [1]
Four groups of 24 Sprague–Dawley rats (12/sex) were given single doses of vehicle (0.5% methylcellulose [MC] and 0.02% sodium lauryl sulfate [SLS] in deionized water) or Rislenemdaz/CERC‐301 at 10, 30 or 100 mg kg−1 by oral gavage at a dose volume of 10 mL kg−1. An additional group of 12 male rats was given single doses of MK‐801 (a noncompetitive antagonist of the NMDA receptor; positive control) at 10 mg kg−1 by subcutaneous injection at a dose volume of 2 mL kg−1. Six rats per sex in each group were terminated and necropsied at 4 to 6 h postdose, and the remaining rats in each group were terminated and necropsied 3 days postdose (on Day 4). In‐life observations and measurements included body weight and clinical observations. At termination, rats were anesthetized and perfusion fixed. At necropsy, the brain was collected for histopathological evaluation.

Animals in Rislenemdaz/CERC‐301 and MK‐801 assessment groups were terminated at the scheduled necropsy intervals (4–6 h postdose or Day 4). All animals were anesthetized with an isoflurane/oxygen mixture and perfused via the left cardiac ventricle with heparinized 0.001% sodium nitrite in saline. The saline wash was followed by perfusion of 10% neutral buffered formalin (NBF). Brains were harvested, weighed, and stored in 10% NBF.

The brain was sectioned into 2 mm coronal sections to produce multiple sections in three blocks for each animal. The following brain regions were stained: neocortex, paleocortex, basal nuclei, limbic system, thalamus/hypothalamus, midbrain regions, cerebellum, pons region, and medulla oblongata. All brain sections from all animals sacrificed 4 to 6 h after dosing and all animals sacrificed 3 days after dosing were embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin and examined microscopically. For rats sacrificed on Day 4 (3 days after dosing), serial sections from Blocks 1 and 2 were stained with Fluoro‐Jade B (a stain increasing the sensitivity of evaluating the brain for neuronal degeneration) and glial fibrillary acidic protein (a stain for astrocyte reactions) and examined microscopically. Three additional groups of rats (four males and three females per group) were orally dosed in the same manner with CERC‐301, and 24‐h serial blood samples were obtained and analyzed for Rislenemdaz/CERC‐301 plasma concentrations and evaluated for systemic exposure.

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In vivo pharmacology [1]
Correlation of GluN2B receptor occupancy with plasma drug levels [1]
CERC‐301/Rislenemdaz was administered to rats, dogs, and rhesus monkeys, as described in detail in Appendix S1 and previously elsewhere (Liverton et al. 2007). The RO was also determined in rats following IV administration of [3H]Compound‐3. The relationship between plasma concentrations and brain RO was evaluated 15 min after IV and 60 min after oral (PO) dosing of CERC‐301, as described in Appendix S1.

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Acute depression model [1]
Forced swim test [1]
Young, adult, male Sprague–Dawley rats were randomly assigned across the treatment groups and were administered vehicle (0.5% MC/0.02% SLS), the reference compound desipramine (20 mg kg−1; a tricyclic antidepressant) dissolved in sterile water, or Rislenemdaz/CERC‐301 (0.1, 0.3, 1, 3, 10, and 30 mg kg−1) suspended in 0.5% MC/0.02% SLS, twice on Day 1 (after habituation; ~24 h prior to test, and prior to dark cycle) and once on Day 2 (30‐min pretest for desipramine and 45‐min pretest for CERC‐301 and vehicle).

Each Forced Swim chamber was constructed of clear acrylic (height, 40 cm; diameter, 20.3 cm). Rats were subjected to a predose swim test of one 15‐min session in cylinders containing water at 23°C ± 1°C, followed approximately 24 h later by the experimental 5‐min session. The water level was 16 cm deep during habituation and 30 cm deep during the test. Immobility, climbing, and swimming behaviors were recorded every 5 sec for a total of 60 counts per subject. When an animal was unable to maintain a posture with its nose above water, it was immediately removed from the water and eliminated from the study. Blood was collected at the completion of swim test procedures and plasma was analyzed for Rislenemdaz/CERC‐301 concentrations.

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Locomotor assay [1]
To confirm that the effect of Rislenemdaz/CERC‐301 in the forced swim test was not due to a general increase in activity levels, rats were subjected to a locomotor assay following oral CERC‐301 administration. Adult male Sprague–Dawley rats (N = 42) were randomly assigned across the treatment groups (vehicle or CERC‐301 at 0.1, 0.3, 1, 3, 10, and 30 mg kg−1; N = 6/group). Locomotor activity was assessed during the light cycle in photocell‐monitored cages. Each cage consisted of a standard plastic rat cage (24 × 45.5 cm) surrounded by a stainless steel frame. Infrared photocell beams were located across the long axis of the frame to measure the ambulatory distance traveled. A second set of beams was placed above the floor and was used to measure rearing activity. Photocell beam interruptions were recorded by a computer system. Filter tops were placed on top of the test enclosures during testing. Rats were administered either vehicle or test compound via oral gavage twice on Day 1 (approximately 24 h before the test and prior to dark cycle) and once on Day 2 (45 min prior to placing in the locomotor cages for a 60‐min test). Locomotor activity was captured in 5‐min bins.

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Hemodynamic effects in telemetered rats [1]
To determine the systemic hemodynamic effects, Rislenemdaz/CERC‐301 was administered as a single oral gavage dose to six (n = 6) chronically telemetered rats (implantation at least 7 days prior to the dosing day) at doses of 0.3, 0.6, 1, 3, and 10 mg kg−1 and systemic blood pressure and heart rate values were recorded. The hemodynamic effects were compared to MK‐801 at a dose of 0.2 mg kg−1 given intravenously (in 0.9% saline). In each animal, a single oral gavage dose of Rislenemdaz/CERC‐301 or vehicle (0.5% MC/0.02% SLS) was administered (volume: 5 mL kg−1). A 24‐h recording was performed prior to dosing (vehicle alone) and after each oral dose. In another set of studies, CERC‐301 (1 mg kg−1) was administered in combination with atenolol (β1‐adrenergic blocker, 1 mg kg−1, IV bolus) and prazosin (α1‐adrenergic receptor antagonist, 200 μg kg−1, IV bolus) to elucidate the underlying mechanism of hypertension. Data were analyzed and compared to baseline, with correction for 24‐h predose vehicle control.

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First‐in‐human pharmacokinetic study [1]
The study protocol was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Twenty‐four healthy, young male subjects were assigned to 1 of 3 sequential treatment panels (A, B, and C). For each panel of eight subjects, two subjects received placebo and six subjects were administered single ascending oral doses of Rislenemdaz/CERC‐301 with a minimum 7‐day washout between each dose: Panel A (0.1, 0.2, 0.5, 1, and 2 mg); Panel B (2, 4, 8, and 15 mg, and 4 mg with food); and Panel C (15 and 20 mg). Blood samples were collected pre‐dose and 0.5, 1, 1.5, 2, 3, 4, 6, 9, 12, 18, 24, 30, 48, and 72 h postdose. Plasma samples were analyzed for CERC‐301 concentrations using reversed phase high‐ performance liquid chromatography with tandem mass spectrometric detection. The analytical range was 0.5 to 500 nmol L−1 (0.180 to 180 ng mL−1).

Human pharmacokinetic study [1]
CERC‐301/Rislenemdaz was rapidly absorbed (Fig. 5A) with mean T max within 1 h postdose across all fasted dose levels and terminal elimination half‐life ranging from approximately 12 to 17 h over the 4‐ to 20‐mg dose range. C max and AUC behaved in a dose‐proportional manner over the dose range studied. Dosing in the fed state led to an approximate 1‐h delay in T max and an approximate 56% decrease in C max, but the overall extent of absorption (AUC) was not affected (Fig. 5B, Table 3).

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Plasma protein binding [1]
CERC–301/Rislenemdaz exhibited a high degree of concentration‐independent plasma protein binding at 37°C in rats (89.6%), dogs (97.2%), monkeys (96.9%), and humans (97.7%).

Nonclinical safety [1]
Neurotoxicity in rats [1]
In rats, single doses of Rislenemdaz/CERC‐301 (10, 30, and 100 mg kg−1) and vehicle control did not produce vacuolation or necrosis in all examined regions of the brain. At these doses, mean C max was approximately 4, 14, and 26 μmol L−1 (1433, 5018, and 9319 ng mL−1), respectively. By contrast, all of the MK‐801 (10 mg kg−1)‐dosed animals had vacuolation and necrosis in cingulate gyrus neurons, consistent with previous reports (Fix et al. 1995). At the 4–6 h time point, the animals treated with MK–801 (six males; Group 5) all had numerous vacuolated neurons in cortical layers 3 and 4 in the cingulate gyrus region of the cerebral cortex. Affected neurons were characterized by numerous, tightly packed, somewhat distinct, vacuoles filling the cytoplasm. On Day 4, all the animals treated with MK‐801 (six males; Group 5) had necrotic neurons (visualized using Fluoro‐Jade B stain) in cortical layers 3 and 4 in the cingulate gyrus region of the cerebral cortex, consistent with previous reports (Fix et al. 1995). In the MK‐801‐treated animals, sections stained (immunohistochemically) for glial fibrillary acidic protein showed a very slight increase in staining in the region of the cingulate gyrus.

[1]. Preclinical pharmacology and pharmacokinetics of CERC‐301, a GluN2B‐selective N‐methyl‐D‐aspartate receptor antagonist. Pharmacol Res Perspect. 2015 Dec; 3(6): e00198.

Mk 0657 has been used in trials studying the treatment of Major Depressive Disorder.

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The preclinical pharmacodynamic and pharmacokinetic properties of 4-methylbenzyl (3S, 4R)-3-fluoro-4-[(Pyrimidin-2-ylamino) methyl] piperidine-1-carboxylate (CERC-301/Rislenemdaz), an orally bioavailable selective N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) antagonist, were characterized to develop a translational approach based on receptor occupancy (RO) to guide CERC-301 dose selection in clinical trials of major depressive disorder. CERC-301 demonstrated high-binding affinity (K i, 8.1 nmol L(-1)) specific to GluN2B with an IC 50 of 3.6 nmol L(-1) and no off-target activity. CERC-301 efficacy was demonstrated in the forced swim test with an efficacy dose (ED 50) of 0.3-0.7 mg kg(-1) (RO, 30-50%); increase in locomotor activity was observed at ED 50 of 2 mg kg(-1), corresponding to an RO of 75%. The predicted 50% RO concentration (Occ50) in humans was 400 nmol L(-1), similar to that predicted for rat, dog, and monkey (300, 200, and 400 nmol L(-1), respectively). Safety pharmacology and neurotoxicity studies raised no specific safety concerns. A first-in-human study in healthy males demonstrated a dose-proportional pharmacokinetic profile, with T max of ~1 h and t 1/2 of 12-17 h. Based on the preclinical and pharmacodynamic data, doses of ≥8 mg in humans are hypothesized to have an acceptable safety profile and result in clinically relevant peak plasma exposure. [1]

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The studies in conscious telemetered rats demonstrates that Rislenemdaz/CERC‐301, when given orally, increased arterial blood pressure transiently, and in a dose‐dependent manner between 0.3 and 1 mg kg−1, and this effect plateaued at 1–10 mg kg−1. Interestingly, the ED50 for blood pressure effects was similar to ED50 for the forced swim test. The magnitude of change in hemodynamics with CERC‐301 was less than that of MK‐801, a broad NMDA receptor antagonist. The findings with MK‐801 were consistent with previously published mechanistic data on MK‐801 (Lewis et al. 1989). The changes in HR and blood pressure may be partially explained by drug‐dependent enhanced movement of rats; dose‐dependent movement analysis was also observed in the Locomotor study, and demonstrate a supporting trend at these higher doses. This study also demonstrated that, α1‐adernergic blockade may provide protection from CERC‐301 mediated increases in blood pressure; however, more studies are required to further elucidate the underlying mechanisms. [1]

Unlike some clinically utilized NMDA receptor antagonists (e.g., ketamine or memantine), CERC‐301 showed no evidence of neurotoxicity in rats given single oral doses at up to 100 mg kg−1. This result is consistent with those obtained with other GluN2B‐specific receptor antagonists (e.g., CP‐101,606 and Ro 63‐1908), which have demonstrated a lack of neurotoxicity, and in the case of CP‐101,606 paradoxically may have neuroprotective potential in the developing brain (Gill et al. 2002; Lewis et al. 2012). Lack of a nonclinical neurotoxicity signal with GluN2B‐specific antagonists, along with the absence of ketamine‐like psychotomimetic effects in the clinical setting allows administration of these agents at higher doses to achieve higher receptor occupancies (than what has been predicted herein), should the clinical data necessitate that in the future clinical trials. [1]

Pharmacokinetic data from the first‐in‐human study with CERC‐301 demonstrated mean C max ranging from 0.007 μmol L−1 (2.4 ng mL−1) to 1.65 μmol L−1 (590.3 ng mL−1) across the 0.1 to 20 mg single dose (fasted) range, with a half‐life that makes the drug suitable for once‐daily dosing in human subjects. [1]
CERC‐301 is a potent orally bioavailable, selective NMDA‐GluN2B receptor antagonist with antidepressant effects. Based on the preclinical PK and pharmacodynamic data, chronic 8‐mg daily administration in humans is hypothesized to have an acceptable safety profile, result in clinically relevant plasma and brain concentrations, and exhibit rapid onset of antidepressant activity. [1]

C19H23FN4O2

358.409927606583

358.18

C, 63.67; H, 6.47; F, 5.30; N, 15.63; O, 8.93

808732-98-1

808733-06-4 (HCl);808732-98-1;1893392-76-1 (mesylate);

11394238

White to off-white solid powder

1.2±0.1 g/cm3

527.4±60.0 °C at 760 mmHg

272.7±32.9 °C

0.0±1.4 mmHg at 25°C

1.584

2.73

1

6

6

26

439

2

CC1=CC=C(C=C1)COC(=O)N2CC[C@@H]([C@@H](C2)F)CNC3=NC=CC=N3

RECBFDWSXWAXHY-IAGOWNOFSA-N

InChI=1S/C19H23FN4O2/c1-14-3-5-15(6-4-14)13-26-19(25)24-10-7-16(17(20)12-24)11-23-18-21-8-2-9-22-18/h2-6,8-9,16-17H,7,10-13H2,1H3,(H,21,22,23)/t16-,17-/m1/s1

4-methylbenzyl (3S,4R)-3-fluoro-4-((pyrimidin-2-ylamino)methyl)piperidine-1-carboxylate

CERC-301; CERC301; Rislenemdaz; 808732-98-1; Rislenemdaz [USAN]; Rislenemdaz, (-)-; MK-0657, (-)-; 4-Methylbenzyl (3S,4R)-3-fluoro-4-((pyrimidin-2-ylamino)methyl)piperidine-1-carboxylate; CERC 301; MK-0657; MK 0657; MK0657;

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 (~279.01 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.98 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 25.0 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: ≥ 2.5 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.98 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 25.0 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.)