Abl1 (IC50 = 2.8 nM); TrkA (IC50 = 6 nM); Abl1 (IC50 = 2.8 nM); TrkB (IC50 = 9 nM); Tie-2 (IC50 = 22 nM); Aurora B (IC50 = 98 nM)
BCR–ABL1 (binding to the myristoyl pocket) [2]
ABL1 (binding to the myristoyl pocket,) [3]

ABL001 is a potent, selective BCR-ABL inhibitor with a unique, allosteric mode of action that remains active against the majority of mutations, including T315I[1]. By binding to a regulatory site that wild-type ABL normally possesses a myristoyl group occupying, ABL001 inhibits ABL kinase activity in a different way than catalytic site inhibitors[2]. The myristoylated N-terminus of ABL1 typically resides in a pocket on the BCR-ABL kinase domain, which is where it binds. This myristoylated N-terminus is responsible for autoregulating ABL1 activity; it is lost upon fusion with BCR. By taking up its empty binding site, ABL001 functionally imitates the myristoylated N-terminus'sfunctionand reinstates the kinase activity's negative regulation. BCR-ABL-negative cell lines were unaffected at concentrations 1000 times higher than ABL001, which specifically inhibits the growth of Ph+ ALL and chronic myelogenous leukemia (CML) cells at potencies ranging from 1 to 10 nM[1]. ABL001 selectively and potently binds to the myristoyl pocket of ABL1 and induces the inactive C-terminal helix conformation, as confirmed by NMR and biophysical studies (dissociation constant (Kd) = 0.5-0.8 nM). ABL001 is inactive against G-protein-coupled receptors, ion channels, nuclear receptors, and transporters, among more than 60 kinases, including SRC. ABL001 is therefore highly selective[3].

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Asciminib (ABL001) is a potent and selective allosteric inhibitor of BCR–ABL1, binding to the myristoyl pocket of ABL1 and inducing an inactive kinase conformation [2][3]
In 48-hour proliferation assays of Ba/F3 cells expressing BCR–ABL1, Asciminib inhibits cell proliferation across a dose range, with similar cellular potency to the second-generation catalytic inhibitor nilotinib; the assay uses Britelite luciferase detection with or without IL-3, performed in quadruplicate [3]
In 72-hour growth assays, KCL-22 cells show sensitivity to Asciminib, nilotinib, and dasatinib, with each compound tested in duplicate [3]
Incubation of KCL-22 cells with Asciminib for 1 hour (across a concentration range) reduces phosphorylation of STAT5 (Tyr694), BCR–ABL1 (Tyr245), and CRKL (Tyr207) as detected by western blot, while total levels of these proteins and GAPDH (loading control) remain stable [3]
Synergy studies show that Asciminib combines with imatinib, nilotinib, or dasatinib to inhibit KCL-22 cell growth; cells are incubated with compound combinations for 72 hours, and growth levels are measured relative to DMSO-treated cells [3]
KCL-22 cell clones expressing BCR–ABL1 variants (Ala337Val and Thr315Ile) exhibit different sensitivity to Asciminib compared to nilotinib; Asciminib retains activity against some variants resistant to catalytic inhibitors [3]
Mutagenesis and genetic barcoding experiments reveal that Asciminib has a resistance profile distinct from catalytic site inhibitors, with no shared resistance mutations between Asciminib and nilotinib [2][3]

ABL001 exhibits strong anti-tumor activity in the KCL-22 mouse xenograft model, as evidenced by total tumor regression and a pronounced dose-dependent relationship with pSTAT5 inhibition[1]. All species have a moderate half-life, volume of distribution, and oral absorption of ABL001. For patients with chronic myelogenous leukemia who have received a lot of pretreatment, it has been well tolerated as a single agent and has been shown to induce clinical anti-tumor activity. Regarding the pharmacokinetics, pharmacodynamics, and efficacy of ABL001, the CL (clearance) in mice, rats, and dogs following a single intravenous dose of 1 mg/kg, 2 mg/kg, and 1 mg/kg, respectively, are 12, 16, and 6 mL/min/kg. The T_1/2term for a single intravenous dose of 1 mg/kg in mice and dogs are 1.1 and 3.7 hours, respectively. Rats have a T_1/2term of 2.7 hours following a single intravenous dose of 2 mg/kg. Oral bioavailability of ABL001 at 30 mg/kg p.o. in rats and mice is 35% and 27%, respectively. On the other hand, ABL001's oral BA in dogs is 111% (15 mg/kg, p.o)[3].

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In KCL-22 xenograft models, single oral administration of Asciminib (doses 3–30 mg/kg) reduces pSTAT5 (Tyr694) levels in tumor fine needle aspirate samples, as measured by MSD assay (duplicate runs, mean ± s.d.), with pSTAT5 levels expressed as a percentage of pre-dosing (t=0) levels [3]
Asciminib shows efficacy in KCL-22 xenografts when dosed twice a day (BID) or once a day (QD) at 3–30 mg/kg, with tumor volume monitored over time (mean ± s.e.m.) [3]
In three patient-derived ALL systemic xenograft models (ALL-7015, AL-7119, AL-7155), Asciminib (7.5 mg/kg BID or 30 mg/kg BID for 3 weeks) reduces the percentage of CD45⁺ cells per live cell in blood (monitored by FACS), with a PBS control group; data are mean ± s.e.m. (n=6 per group) [3]
Single-agent Asciminib or nilotinib leads to acquired resistance in CML xenograft models, but their combination achieves complete disease control and eradicates tumors without recurrence after treatment cessation [2][3]
In KCL-22 Thr315Ile mutant xenografts, Asciminib (3–30 mg/kg BID) shows efficacy, while nilotinib (75 mg/kg BID) serves as a control; tumor volume ratios (T/C) and regression are recorded (mean ± s.e.m., n=7 per group) [3]

ABL1 Biochemical kinase assay [3]
ABL1 WT (64-515aa) protein was produced by co-expression with YopH in Sf21 cells. Cells were harvested by centrifugation and resuspended in 25mM Tris pH 7.0, 500 mM NaCl, 5% glycerol, 10 mM Imidazole, 1x complete protease inhibitor tablet, Benzonase (1:10,000 v:v) and 1 mM TCEP. Cells were lysed by dounce homogenization and cleared by centrifugation. ABL1 WT (64-515 aa) was purified by affinity chromatography using a Ni-SepharoseFF column using two sequential washing steps using the resuspension buffer described above (containing 10 mM and 35mM imidazole respectively) and eluted in the same buffer containing 250 mM imidazole. Fractions containing ABL1 were pooled and loaded onto a pre-equilibrated SEC column in 25 mM Tris pH 7.0, 200 mM NaCl, 5% glycerol and 1 mM TCEP. The activity of the enzyme and compound inhibition was tested using a DELFIA® TRF assay. The reaction mixture contained 500 nM Biotin-EAIYAAPFAKKK peptide, 10 or 2000 µM ATP and 25 pM of ABL1 WT (64-515 aa) enzyme in a reaction buffer containing 50 mM HEPES pH 7.2, 10 mM MgCl2, 2 mM DTT and 0.01% Triton-X100. Reactions were carried out for 40 min in a volume of 60 µL and quenched with 20 µL 500 mM EDTA (final concentration 125 mM). 50 µL reaction solutions were transferred to NeutroAvidin-coated 384 well plate and incubated at room temperature for 1 hour with shaking. After washing with 100 µL/well TBST buffer, 50 µL/well Eu-anti-p-Tyr was added and the plate was incubated at 4 °C overnight with shaking. 50 µL/well DELFIA® enhancement solution was added and the plate incubated at ambient temperature for 5 min. The plate was read on the EnVision using time resolved fluorescence Ex/Em: 340/615 nm. For inhibition studies, compounds were serially diluted in DMSO, using a 16-point 3-fold format, from a 5 mM top concentration. Then 100 nL per well of serial diluted compounds were transferred to Grenier polypropylene v-bottom 384-well assay plates using acoustic transfer system. The final DMSO concentration was 0.16% and the final inhibitor concentration ranged from 50 µM to 3.48E-6 µM. Each compound was tested in duplicate and the inhibitor dose response curves analyzed using normalized IC50 regression curve fitting with control-based normalization using GraphPad Prism v6.02.[3]

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Asciminib, having a dissociation constant (Kd) of 0.5-0.8 nM and selectivity to the myristoyl pocket of ABL1, is a strong and selective allosteric inhibitor of BCR-ABL1.

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Perform NMR chemical shift assays to determine the binding location of Asciminib to ABL1 [3]
Conduct NMR-based conformational assays using the resonance of Val525 to monitor helix I "bending" in the presence and absence of Asciminib [3]
Carry out isothermal calorimetry (ITC) studies to measure the binding affinity (Ka) of Asciminib to ABL1 [3]
Perform biochemical assays to evaluate the inhibitory activity of Asciminib against BCR–ABL1 kinase activity, focusing on its allosteric mechanism distinct from catalytic site inhibitors [2][3]

Ba/F3 Proliferation assay [3]
For each cell line, cell density was adjusted to 50 000cells/ml and 50ul (2500 cells) added per well of a 384 well assay plate. Test compounds were resuspended in DMSO at a concentration of 10mM. A serial three-fold dilution of each compound with DMSO was performed in 384-well plates using the Janus Liquid Dispenser. 2nL compound was delivered to the assay plates containing 2500 cells in a 50 µL volume via acoustic delivery from an ATS-100 (EDC). Cells were incubated with compound at 37°C in a humidified environment with 5% carbon dioxide for 48 hours. Britelite plus solution was prepared according to the manufacturer’s instructions and 25 µl added to each well of the assay plate. Plates were incubated for 7 minutes and the luminescence detected on an EnVision Multimode plate reader. The degree of luminescence correlates with the number of cells in each well. The effect of each inhibitor concentration can therefore be calculated and IC50’s generated.

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For 48 hours, Ba/F3 cells are exposed to asciminib at a range of concentrations (0–10,000 nM). The Britelite luciferase detection assay is used to quantify the proliferation of cells.

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Conduct 48-hour proliferation assays in Ba/F3 cells expressing BCR–ABL1, using Britelite luciferase detection with or without IL-3, testing Asciminib and nilotinib across dose ranges (quadruplicate runs) [3]
Perform 72-hour growth assays in KCL-22 cells (parental and variant clones expressing BCR–ABL1 Ala337Val/Thr315Ile) to assess sensitivity to Asciminib, nilotinib, and dasatinib (duplicate tests) [3]
Treat KCL-22 cells with Asciminib for 1 hour (concentration range), then perform western blots to detect total and phosphorylated STAT5 (Tyr694), BCR–ABL1 (Tyr245), CRKL (Tyr207), and GAPDH [3]
Carry out synergy studies by incubating KCL-22 cells with Asciminib combined with imatinib/nilotinib/dasatinib across dose ranges for 72 hours, and measure growth relative to DMSO controls [3]
Use genetic barcoding to analyze clonal dynamics and resistance mutations in cells treated with Asciminib or catalytic inhibitors [3]

Mice: Asciminib efficacy is measured using FACS monitoring of the percentage of CD45+ cells per live cell in blood samples obtained at different times following dosing with either 7.5 mg/kg BID (group 2) or 30 mg/kg BID (group 3) asciminib for three weeks in three patient-derived ALL systemic xenograft models (ALL-7015, AL-7119, and AL-7155).

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ABL001 (free base, solid dispersion form) was suspended in phosphate-buffered saline. Dosing solutions were prepared fresh every 3-4 days for dosing. ABL001 (free base, solution form) was formulated in 30% PEG 300, 6% Solutol HS15 in an acidic buffered solution. Dosing solutions were freshly prepared weekly for dosing.
Efficacy studies [3]
For efficacy studies in subcutaneous KCL-22 xenograft model, mice bearing tumors of 100- 300mm3 were randomized into treatment groups (n=6 per group) for daily compound treatment. Body weight and tumor volume were recorded twice weekly for the duration of each study. In ABL001 dose-response studies, studies were terminated when vehicle-treated animals reached 1500mm3 mean tumor volume. In ABL001 and nilotinib combination efficacy study, select randomized groups animals were dosed daily with either ABL001 or nilotinib as single agents until tumor relapse (tumor volume >500mm3), then switched to the other agent continuously until second relapse. Animals are terminated as their final tumor volume reached >600mm3 . Another randomized group received combination of both ABL001 and nilotinib daily treatment then continued monitoring post-treatment cessation. For efficacy studies in systemic primary Ph+ ALL xenograft models, mice were injected intravenously with 5x106 ALL cells. Blood was sampled weekly from tail snip to monitor tumor burden, and engrafted mice with >10% human CD45+ cells were randomized into treatment groups for compound treatment (n=6 mice per group).

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Pharmacokinetics (PK) / Pharmacodynamics (PD) studies [3]
Baseline tumor PD samples were collected from KCL-22 xenografts by fine needle biopsy before drug treatment. Animals received a single oral dose of ABL001 at 7.5 – 30 mg/kg. Blood was collected by serial tail bleed at designated time points (1-20h) for plasma PK analyses, and matching tumor PD samples were collected by fine needle biopsy at the same timepoints.

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Establish KCL-22 (parental and Thr315Ile mutant) xenograft models in mice; administer Asciminib orally at 3–30 mg/kg (BID/QD) or nilotinib at 75 mg/kg BID, monitor tumor volume over time, and calculate T/C ratios and regression [3]
Set up patient-derived ALL systemic xenograft models (ALL-7015, AL-7119, AL-7155) in mice; assign to control (PBS) or Asciminib groups (7.5 mg/kg BID or 30 mg/kg BID), treat for 3 weeks, and collect blood samples at varying time points for FACS analysis of CD45⁺ cells [3]
For pharmacokinetic/pharmacodynamic studies, administer a single oral dose of Asciminib (3–30 mg/kg) to mice bearing KCL-22 xenografts; collect plasma and tumor fine needle aspirates to measure drug concentrations and pSTAT5 (Tyr694) levels [3]
Assess the tolerability of Asciminib in mice by dosing BID at increasing concentrations, monitoring body weight 2–3 times per week (mean ± s.e.m., n=5 per group) [3]
Establish CML xenograft models; treat with single-agent Asciminib (30 mg/kg BID), nilotinib (75 mg/kg BID), or their combination, stop dosing after achieving tumor control, and monitor for recurrence [3]

Absorption, Distribution and Excretion
Following oral administration of acimenil, the median time to peak concentration (Tmax) was 2.5 hours. At a once-daily dose of 80 mg, the steady-state peak plasma concentration (Cmax) and AUCtau were 1781 ng/mL and 15112 ng·h/mL, respectively. At a twice-daily dose of 40 mg, the steady-state peak plasma concentration (Cmax) and AUCtau were 793 ng/mL and 5262 ng·h/mL, respectively. At a twice-daily dose of 200 mg (for the treatment of T315I mutants), the steady-state peak plasma concentration (Cmax) and AUCtau were 5642 ng/mL and 37547 ng·h/mL, respectively. Compared to fasting, acimenil administered with a high-fat meal reduced AUC and Cmax by 62% and 68%, respectively, while administration with a low-fat meal reduced AUC and Cmax by 30% and 35%, respectively. Acimenil is primarily excreted via bile secretion mediated by the breast cancer resistance protein (BCRP) transporter. Following oral administration, approximately 80% and 11% of the acimenil dose are excreted in feces and urine, respectively. Of the drug recovered in feces, 57% is unmetabolized parent drug, and of the drug recovered in urine, 2.5% is unmetabolized parent drug. At steady state, the apparent volume of distribution of acimenil is 151 L. At a total daily dose of 80 mg, the total apparent clearance of acimenil is 6.7 L/h; at a total daily dose of 200 mg, the total apparent clearance is 4.1 L/h. Metabolism/Metabolites
The metabolism of acimenide is negligible. The main drug component in plasma is the unmetabolized parent drug (approximately 93%), and the main drug component excreted is the parent drug (approximately 57%, primarily excreted in feces). The main circulating metabolites are M30.5, M44, and M29.5, accounting for approximately 5%, 2%, and 0.4% of the total administered dose, respectively. Oxidative metabolism of acimenide is mediated by CYP3A4, and its glucuronidation is mediated by UGT2B7 and UGT2B17.
Biological Half-Life
When administered twice daily at 40 mg, the terminal elimination half-life of acimenide is 5.5 hours; when administered twice daily at 200 mg, the terminal elimination half-life is 9.0 hours.

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In mice, rats, and dogs, asciminib demonstrated oral bioavailability (BA), with pharmacokinetic parameters including AUC (area under the curve), CL (clearance), Cₘₐₓ (maximum concentration), t₁/₂term (terminal half-life), Tₘₐₓ (time to peak concentration), and Vss (volume of distribution), which were measured after a single intravenous (IV) or oral (PO) administration [3].
In mice, asciminib reached detectable plasma concentrations after a single oral administration, and the drug concentrations were correlated with pharmacodynamic effects (pSTAT5 inhibition) in xenograft models [3].

Hepatotoxicity
In pre-marketing clinical trials of asciminib in patients with refractory and extensively treated chronic myeloid leukemia (CML), ALT elevations occurred in 13% of patients, but were generally self-limiting and mild. ALT elevations exceeding 5 times the upper limit of normal (ULN) were uncommon, occurring in only 3% of treated patients. ALT elevations were usually transient and rarely required dose interruption or adjustment. No clinically significant liver injury, liver failure, or death due to liver injury was reported in the open-label and controlled trials supporting asciminib approval. Furthermore, the incidence of transaminase elevations in patients receiving first-line and second-line BCR-ABL1 inhibitor therapy did not increase during asciminib treatment. Since the approval of asciminib in the US and Europe, there have been no reported cases of clinically significant liver injury associated with asciminib treatment. Probability Score: E (Unproven but suspected rare cause of clinically significant liver injury).

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Protein Binding
In vitro experiments showed that acimenide binds to plasma proteins at a rate of 97%, but the specific proteins it binds to are still unclear.

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In mouse tolerance studies, twice-daily (BID) escalating doses of acimenide did not cause significant weight loss, indicating acceptable safety within the tested dose range [3].

[1]. Blood (2014) 124 (21): 398.

[2]. Clin Cancer Res (2017) 23 (1_Supplement): IA01.

[1]. Nature. 2017, 543: 733-737.

Asciminib is a tyrosine kinase inhibitor (TKI) used to treat chronic-phase Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML). More specifically, it is an inhibitor of ABL1 kinase activity of the BCR-ABL1 fusion protein, a driver of cell proliferation in most CML patients. Asciminib has also shown efficacy in Ph+ CML carrying the T315I mutation, which produces a mutant BCR-ABL1 that is typically resistant to wild-type BCR-ABL1. Existing ABL inhibitors exert their effects by competing with the ATP-binding sites of these proteins and can be divided into two categories: one targeting the active conformation of the kinase domain (e.g., dasatinib, bosutinib), and the other targeting the inactive conformation of the kinase domain (e.g., imatinib, nilotinib, panatinib). Asciminib is unique in that it is an allosteric inhibitor that binds to the myristoyl pocket of the BCR-ABL1 protein and locks it in an inactive conformation. Asciminib was approved by the FDA on October 29, 2021 (Scemblix, Novartis). Asciminib is a tyrosine kinase inhibitor that specifically targets the myristoyl pocket of ABL1 for the treatment of refractory Philadelphia chromosome-positive chronic myeloid leukemia. Some patients treated with Asciminib experience elevated serum transaminases, but there have been no reports of clinically significant liver injury or jaundice due to Asciminib use. Asciminib is an orally bioavailable allosteric Bcr-Abl1 tyrosine kinase inhibitor with antitumor activity. After administration, asciminib targets and binds to the myristoyl pocket of the Bcr-Abl1 fusion protein, located at a position different from the ATP-binding domain, thereby inhibiting the activity of wild-type Bcr-Abl1 and certain mutants (including the T315I mutation). This binding leads to the inhibition of Bcr-Abl1-mediated proliferation and enhances apoptosis in Philadelphia chromosome-positive (Ph+) hematologic malignancies. Bcr-Abl1 fusion protein tyrosine kinase is an abnormal enzyme produced by leukemia cells containing the Philadelphia chromosome.
See also: Acimenil hydrochloride (salt form).
Indications
Acimenil is indicated for the treatment of adult patients with chronic-phase Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML) who have previously received ≥2 tyrosine kinase inhibitors. It is also indicated for the treatment of adult patients with Ph+ CML harboring the T315I mutation.
Scemblix is indicated for the treatment of adult patients with chronic-phase Philadelphia chromosome-positive chronic myeloid leukemia (Ph+ CML CP) who have previously received two or more tyrosine kinase inhibitors (see Section 5.1).
Treatment of Chronic Myeloid Leukemia

Mechanism of Action

In most patients with chronic myeloid leukemia (CML), disease progression is primarily driven by the Philadelphia chromosome translocation, which creates an oncogenic fusion gene, BCR-ABL1, between the BCR and ABL1 genes. This fusion gene produces the fusion protein BCR-ABL1, which possesses enhanced tyrosine kinase activity and transforming activity, thereby promoting CML cell proliferation. Acimenide is an allosteric inhibitor of the BCR-ABL1 tyrosine kinase. It binds to the myristoyl pocket of the ABL1 fusion protein and locks it in an inactive conformation, thereby inhibiting its oncogenic activity.
Pharmacodynamics

Acimenide exerts its therapeutic effect by inhibiting an oncogenic protein responsible for CML proliferation. Depending on the disease being treated, it can be taken orally once or twice daily.
A five-fold increase in the total daily dose compared to standard therapy (80 mg daily vs. 400 mg daily) can be used to treat Ph+ CML with the T315I mutation, a typical resistant CML variant. Like many other chemotherapy drugs, aciminib treatment can cause various forms of myelosuppression, including thrombocytopenia and neutropenia. Patients should undergo frequent laboratory monitoring throughout treatment, and dose adjustments may be necessary based on the severity of observed adverse reactions. Pancreatic and/or cardiovascular toxicities may also occur, both requiring frequent monitoring and potentially requiring dose adjustments based on prescribing information. Asciminib (formerly known as ABL001) is an allosteric inhibitor currently undergoing clinical development for chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph⁺) acute lymphoblastic leukemia [3]. Its mechanism of action is to bind to the myristoyl pocket of ABL1, which is different from catalytic site inhibitors (imatinib, nilotinib, dasatinib), thus enabling dual targeting of BCR-ABL1 [3]. Existing clonal populations resistant to Asciminib do not develop resistance to nilotinib, which supports combination therapy to block the development of resistance [2][3]. Asciminib has entered Phase I clinical trials and has shown safety and good monotherapy activity in CML patients. For patients who have failed prior tyrosine kinase inhibitor (TKI) therapy [3], discontinuation of TKI therapy can lead to treatment-free remission in patients with chronic myeloid leukemia (CML) who have achieved deep molecular remission; Asciminib aims to improve prognosis by eradicating CML cells [2].

C20H18CLF2N5O3

449.84

449.106

C, 53.40; H, 4.03; Cl, 7.88; F, 8.45; N, 15.57; O, 10.67

1492952-76-7

Asciminib hydrochloride;2119669-71-3

72165228

White to off-white solid powder

1.5±0.1 g/cm3

631.7±55.0 °C at 760 mmHg

335.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.662

2.1

3

8

6

31

626

1

ClC(OC1C=CC(=CC=1)NC(C1C=NC(=C(C2=CC=NN2)C=1)N1CC[C@H](C1)O)=O)(F)F

VOVZXURTCKPRDQ-CQSZACIVSA-N

InChI=1S/C20H18ClF2N5O3/c21-20(22,23)31-15-3-1-13(2-4-15)26-19(30)12-9-16(17-5-7-25-27-17)18(24-10-12)28-8-6-14(29)11-28/h1-5,7,9-10,14,29H,6,8,11H2,(H,25,27)(H,26,30)/t14-/m1/s1

N-[4-[chloro(difluoro)methoxy]phenyl]-6-[(3R)-3-hydroxypyrrolidin-1-yl]-5-(1H-pyrazol-5-yl)pyridine-3-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.56 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 (5.56 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 (5.56 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.)