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]
\nFour 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.\n

\nAnimals 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.\n

\nThe 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.\n

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\n\nIn vivo pharmacology [1]
\nCorrelation of GluN2B receptor occupancy with plasma drug levels [1]
\nCERC‐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.\n

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\n\nAcute depression model [1]
\nForced swim test [1]
\nYoung, 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).\n

\nEach 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.\n

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\n\nLocomotor assay [1]
\nTo 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.\n

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\n\nHemodynamic effects in telemetered rats [1]
\nTo 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.\n

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\n\nFirst‐in‐human pharmacokinetic study [1]
\nThe 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 studies [1]
CERC–301/Rislenemdaz was rapidly absorbed (Figure 5A), with the mean time to peak (T_max) reaching within 1 hour after administration at all fasting dose levels, and the terminal elimination half-life of approximately 12 to 17 hours in the 4 to 20 mg dose range. C_max and AUC were dose-proportional within the studied dose range. Postprandial administration delayed T_max by approximately 1 hour and reduced C_max by approximately 56%, but did not affect the overall extent of absorption (AUC) (Figure 5B, Table 3).

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

Non-clinical safety[1]
Neurotoxicity in rats[1]
In rats, single administration of Rislenemdaz/CERC-301 (10, 30, and 100 mg kg−1) and solvent control did not induce vacuolization or necrosis in all brain regions examined. At these doses, the mean Cmax was approximately 4, 14, and 26 μmol L−1 (1433, 5018, and 9319 ng mL−1), respectively. In contrast, vacuolization and necrosis of neurons in the cingulate gyrus were observed in all animals given MK-801 (10 mg kg−1), consistent with previous reports (Fix et al., 1995). At the 4–6 hour time point, extensive vacuolized neurons were observed in layers 3 and 4 of the cingulate gyrus of the cerebral cortex in all animals treated with MK-801 (6 males; group 5). The affected neurons were characterized by numerous tightly packed, well-defined vacuoles within their cytoplasm. On day 4, necrotic neurons were observed in layers 3 and 4 of the cingulate gyrus of the cerebral cortex in all animals treated with MK-801 (6 males; group 5) (displayed with Fluoro-Jade B staining), consistent with previous reports (Fix et al., 1995). In MK-801-treated animals, immunohistochemical staining with glial fibrillary acidic protein (GFAP) showed a slight increase in staining intensity in the cingulate gyrus region.

[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 investigating the treatment of major depressive disorder. Preclinical pharmacodynamics and pharmacokinetic characterization were performed on 4-methylbenzyl(3S,4R)-3-fluoro-4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylic acid ester (CERC-301/Liceneda), a highly bioavailable selective N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) antagonist, to develop a receptor occupancy (RO)-based conversion method to guide dose selection of CERC-301 in clinical trials for major depressive disorder. CERC-301 exhibits high affinity for GluN2B (K_i, 8.1 nmol L^-1), an IC₅₀ of 3.6 nmol L^-1, and no off-target activity. CERC-301 demonstrated efficacy in forced swimming tests, with an effective dose (ED50) of 0.3–0.7 mg kg⁻¹ (RO, 30–50%). Increased kinetic activity was observed at an ED50 of 2 mg kg⁻¹, corresponding to an RO of 75%. The predicted human 50% RO concentration (Occ50) was 400 nmol L⁻¹, similar to the predicted values in rats, dogs, and monkeys (300, 200, and 400 nmol L⁻¹, respectively). No specific safety issues were identified in safety pharmacology and neurotoxicity studies. A first-in-human study in healthy men showed dose-dependent pharmacokinetic characteristics, with a Tmax of approximately 1 hour and a t1/2 of 12–17 hours. Based on preclinical and pharmacodynamic data, it is speculated that doses ≥8 mg in humans have acceptable safety profiles and achieve clinically relevant peak plasma exposure. [1]

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Studies in awake telemetry rats showed that oral administration of ricaridin/CERC-301 transiently and dose-dependently increased arterial blood pressure in the range of 0.3 to 1 mg kg⁻¹, reaching a plateau at 1 to 10 mg kg⁻¹. Interestingly, the ED50 of the blood pressure effect was similar to that of the forced swimming test. The hemodynamic changes induced by CERC-301 were smaller than those of the broad-spectrum NMDA receptor antagonist MK-801. The results for MK-801 are consistent with previously published mechanistic data on MK-801 (Lewis et al., 1989). Changes in heart rate and blood pressure may be partly attributed to the drug-dependent enhancement of the rats' exercise capacity; dose-dependent exercise analysis was also observed in motor function studies, and this trend was supported at higher doses. This study also suggests that α1-adrenergic blockers may have a protective effect against CERC-301-mediated hypertension; however, further research is needed to elucidate its underlying mechanism. [1] Unlike some clinically used NMDA receptor antagonists (such as ketamine or memantine), CERC-301 did not show neurotoxicity in rats with a single oral dose up to 100 mg kg−1. This result is consistent with the findings of other GluN2B specific receptor antagonists (such as CP-101,606 and Ro 63-1908), none of which showed neurotoxicity. Among them, CP-101,606 may even have a neuroprotective effect on the developing brain (Gill et al., 2002; Lewis et al., 2012). GluN2B specific antagonists have not shown neurotoxic signals in non-clinical settings and have not been observed to have ketamine-like psychoactive effects in clinical settings. Therefore, if future clinical trial data are needed, the dosage of these drugs can be increased to achieve higher receptor occupancy (above the levels predicted in this paper). [1]
Pharmacokinetic data from the first human study of CERC-301 showed that the mean Cmax was 0.007 μmol L−1 (2.4 ng mL−1) to 1.65 μmol L−1 (590.3 ng mL−1) in the range of 0.1 to 20 mg single dose (fasting), and its half-life makes it suitable for once-daily administration. [1]
CERC-301 is a potent, orally bioavailable selective NMDA-GluN2B receptor antagonist with antidepressant effects. Based on preclinical pharmacokinetic and pharmacodynamic data, it is speculated that a chronic dosing regimen of 8 mg daily has acceptable safety, achieves clinically relevant plasma and brain tissue concentrations, and provides rapid antidepressant effects. [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.)