CDK2; JAK2; FLT3

SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. SB1317 was soluble, highly permeable in Caco-2 cells, and showed > 99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 μM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans.

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In general both compounds had a broadly similar pharmacological profile but Zotiraciclib was always more active in cells, particularly the prostate cancer cell line DU145 where there was a 5-fold difference between the compounds. Higher cellular potency of Zotiraciclib is probably due to its generally higher potency against the enzymes but could also be due to better solubility and permeability. Furthermore, 26g was significantly less soluble than Zotiraciclib. Taken together, these data support Zotiraciclib as the preferred compound, and as such, it was selected for advanced profiling.[1]
Intracellular pharmacodynamic marker studies showed that Zotiraciclib potently inhibited the CDK2 biomarker pRb (phospho-Rb, retinoblastoma tumor suppressor protein) in HCT-116 (Figure 8). Effects could be detected at the 40 nM with the protein phosphorylation being completely inhibited at 200 nM. Similar studies in leukemic cell lines (36) showed that Zotiraciclib was also potent against pRb in MV4-11 cells (IC50 = 0.13 μM) and also inhibited pFLT3 and pSTAT5 in the same cell line. [1]
Figure 8. HCT-116 cells were treated separately with the indicated concentrations of Zotiraciclib for 24 h prior to denaturing lysis. An amount of 30 μg of lysate from each treatment was resolved on 10% SDS–PAGE, transferred onto PVDF membrane, and probed with antibodies against phospho-Rb and β-actin. [1]
Extensive biological characterization, including kinase profiling, intracellular mechanistic studies, and antiproliferative effects on a wide range of leukemic cell lines, was reported elsewhere. These data show that Zotiraciclib has a highly novel kinase inhibitory spectrum inhibiting 17 kinases from a panel of 63, 11 of which are CDK/JAK/FLT family members. The others, Lck, Fyn, Fms, TYRO3, ERK5, and p38δ, are implicated in inflammatory and proliferative processes, and further biological studies are underway to better understand these activities in an in vivo setting.

In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution ( > 0.6 L/kg), oral bioavailability of 24%, ∼ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate.

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On the basis of its efficacy on a broad spectrum of tumor cell lines and good oral bioavailability, Zotiraciclib was selected for evaluation in human tumor xenograft mouse models. Two models were selected based on their relevance in cancer: HCT-116 colon cancer and Ramos B-cell lymphoma. Prior to conducting both experiments, dosing regimes in each model were explored and optimal schedules selected for each model that would be tolerated for the duration of the experiment. In the colon cancer model, HCT-116 cells were injected subcutaneously and tumors were established with mean group sizes of approximately 100 mm3. Treatment with Zotiraciclib at doses of 50 and 75 mg/kg po 3 times per week on a Monday, Wednesday, Friday schedule was started 8 days after cell inoculation for 15 days. Treatment with Zotiraciclib at 75 mg/kg po q.d. 3×/week significantly inhibited the growth of tumors with a mean TGI of 82%, while the lower dose of 50 mg/kg po 3×/week was marginally effective (Figure 9).[1]

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In the lymphoma model Ramos cells were injected subcutaneously and tumors were established with mean group sizes of approximately 200 mm3. Two different dosing regimens of Zotiraciclib were explored in this model: 75 mg/kg po q.d. on a 2 days on and 5 days off schedule and 15 mg/kg ip q.d. on a 5 days on 5 days off schedule were started 12 days after cell inoculation for 15 days. There were two vehicle control groups that received either MC/Tween or DMA/CRE (see Experimental Section for details). The treatment groups were compared with the corresponding vehicle control groups for assessment of percentage TGI. Treatment with Zotiraciclib using either regime significantly inhibited the growth of tumors with mean TGIs of 42% and 63% for the oral and ip delivery methods, respectively (Figure 10). Given the encouraging TGIs observed with both oral and ip dosing schedules in these challenging models, Zotiraciclib was selected for further preclinical development[1].

Enzyme Assays [1]
The recombinant enzymes (CDK2/cyclin A, JAK2, and FLT3) were used. All assays were carried out in 384-well white microtiter plates using the PKLight assay system from Cambrex. This assay platform is a luminometric assay for the detection of ATP in the reaction using a luciferase-coupled reaction. The compounds such as Zotiraciclib were tested at eight concentrations prepared from 3- or 4-fold serial dilution starting at 10 μM. For CDK2/cyclin A assay, the reaction mixture consisted of the following components in 25 μL of assay buffer (50 mM Hepes, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 5 mM BGP, 1 mM DTT, 0.1 mM sodium orthovanadate), 1.4 μg/mL of CDK2/cyclin A complex, 0.5 μM RbING substrate, and 0.5 μM ATP. The mixture was incubated at room temperature for 2 h. Then 13 μL of PKLight ATP detection reagent was added and the mixture was incubated for 10 min. Luminescence signals were detected on a multilabel plate reader. The other kinase assays were similar, with the following differences in reagents: For FLT3 assays, the mixture contained 2.0 μg/mL FLT3 enzyme, 5 μM poly(Glu,Tyr) substrate, and 4 μM ATP. For JAK2 assays, the reaction contained 0.35 μg/mL JAK2 enzyme, 10 μM poly(Glu,Ala,Tyr) substrate, and 0.15 μM ATP. The analytical software Prism 5.0 was used to generate IC50 values from the data.

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High Throughput Solubility Assay [1]
This assay measures the solubility of a compound in PBS in a high throughput mode. The assay was done using 96-well semitransparent PP microplates with V-shaped bottom and 96-well UV transparent microplates. Compound solutions (250 μM) were prepared in 10 mM phosphate buffer (pH 7.0) containing 20% DMSO in a total volume of 0.2 mL. Plates were placed on a shaker set at 600 rpm for 1.5 h, following which the plates were allowed to stand for 2 h at room temperature. The plates were centrifuged at 1500g for 15 min. The supernatants were transferred to a UV transparent microplate and analyzed by UV spectrophotometry at the appropriate absorption maxima. The concentration of the compound in the supernatant was quantified using a calibration curve. For calculated solubilities of 250 ± 30 μM, solubilities are reported as >250 μM (>150 μg/mL).

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Metabolic Stability in Liver Microsomes [1]
Compounds (5 μM) were incubated with MLM (mouse liver microsomes), RLM (rat liver microsomes), DLM (dog liver microsomes), and HLM (human liver microsomes) (final microsomal concentration of ∼0.87 mg/mL) in a reaction mix containing 50 mM potassium phosphate buffer (pH 7.4) and NADPH regeneration system, at 37 °C, in a total reaction volume of 1 mL. Reactions were terminated at 0, 15, 30, 45, and 60 min of incubation with a chilled mixture of acetonitrile and DMSO (80:20). The mixture was vortexed for 5 min, centrifuged at 13 200 rpm for 15 min at 4 °C, and the supernatants were analyzed by LC–MS/MS. Stability was assessed by plotting the percent of parent compound remaining against time on a log–linear scale, and half-life was estimated from the linear portion of the log–linear curve using the first order equation t1/2 = 0.693/k, where k is the slope of the curve (equal to the first order elimination rate constant).

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Human in Vitro CYP450 Inhibition Assay [1]
Zotiraciclib was incubated (at concentrations of 0.05, 0.25, 0.5, 2.5, 5, 25 μM in DMSO; final DMSO concentration of 0.35%) with human liver microsomes (0.25 mg/mL for CYP1A and CYP3A4, 0.5 mg/mL for CYP2C19 and CYP2D6, 1 mg/mL for CYP2C9) and NADPH (1 mM) in the presence of the probe substrate ethoxyresorufin (0.5 μM) for 5 min (CYP1A), tolbutamide (120 μM) for 60 min (CYP2C9), mephenytoin (25 μM) for 60 min (CYP2C19), dextromethorphan (5 μM) for 30 min (CYP2D6), and midazolam (2.5 μM) for 5 min (CYP3A4) at 37 °C. The selective inhibitors α-naphthoflavone, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole were used as positive controls for CYP1A, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 inhibitor, respectively. For CYP1A, the reactions were terminated by the addition of methanol, and the formation of the metabolite, resorufin, was monitored by fluorescence (excitation wavelength of 535 nm, emission wavelength of 595 nm). For the CYP2C9, CYP2C19, CYP2D6, and CYP3A4 incubations, the reactions were terminated by the addition of methanol containing an internal standard. The samples were centrifuged, and the supernatants were combined for the simultaneous analysis of 4-hydroxytolbutamide, 4-hydroxymephenytoin, dextrorphan, 1-hydroxymidazolam, and the internal standard by LC–MS/MS. Formic acid in deionized water (final concentration of 0.1%) was added to the final sample prior to analysis. A decrease in the formation of the metabolites compared to vehicle control was used to calculate an IC50 value (test compound concentration that produces 50% inhibition).

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In Vitro Plasma Protein Binding Equilibrium dialysis was performed in a Micro-Equilibrium Dialyzer with a chamber volume of 500 μL (each compartment with a volume of 250 μL). The semipermeable membrane used was rinsed with Milli-Q water and soaked for 10 min in PBS. Zotiraciclib was added to plasma (from mouse, dog, and humans) to obtain a final concentration of 1000 ng/mL. The spiked plasma was vortexed, and 250 μL was aliquoted into one chamber of the dialyzer cell. The other chamber was filled with 250 μL of PBS buffer. The assembled cell was placed into a water bath at 37 °C, and dialysis was performed for 4 h. Following dialysis, 50 μL of PBS dialyzed samples containing free Zotiraciclib was transferred into 2 mL Eppendorf tubes in triplicate for extraction. Samples were extracted with 1500 μL of MTBE (methyl tert-butyl ether) for 30 min using a mixer at motor speed setting 60 with pulsing. After 30 min, the sample tubes were centrifuged at 4 °C for 10 min at 13 000 rpm in a microcentrifuge. The supernatant (1400 μL) was transferred into fresh 2 mL Eppendorf tubes and dried in a SpeedVac at 43 °C for 35 min. The dried samples were reconstituted with 100 μL of methanol/Milli-Q H2O (60:40) and analyzed by LC–MS/MS.

Cell Proliferation Assays [1]
All cell lines were cultured according to the recommended guidelines. For proliferation assays in 96-well plates, 20 000 cells were seeded in 100 μL of medium and treated the following day with compounds (in triplicate) at concentrations up to 10 μM for 48 h. Cell viability was monitored using the CellTiter-96 Aqueous One solution cell proliferation assay. Dose–response curves were plotted to determine IC50 values for the compounds using the XL-fit software.

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Cell Pharmacodynamic Assay [1]
HCT-116 cells (2 × 105 in 5 mL of McCoy’s medium supplemented with 2 mM l-glutamine and 10% fetal bovine serum) were seeded in 60 mm dishes 16–24 h before drug treatment. Each dish was treated separately with different concentrations of Zotiraciclib or DMSO for 24 h prior to lysis using a modified radioimmunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 0.25 mM EDTA (pH 8.0), 1% Triton X-100, 0.2% NaF, and protease inhibitor cocktail. Proteins were measured using the Bradford assay, and 30 μg of lysate from each treatment was resolved on 10% SDS–PAGE and transferred onto PVDF membrane. Western blot analyses using antibodies against phospho-Rb and β-actin were performed using dilutions recommended by suppliers. Signals were detected using autoradiography with Pierce ECL Western blotting substrate.

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Caco-2 Bidirectional Permeability Assay [1]
Zotiraciclib at 5 μM in Hank’s balanced salt solution (HBSS), final DMSO concentration less than 1%, was placed in 21–28 day confluent monolayer cells in Transwell assay plates. Both apical and basolateral sides were maintained at pH 7.4. When dosed on the apical side, the permeability in the A → B direction was assessed, and when dosed on the basolateral side, the B → A direction was assessed. Both apical and basolateral sides were sampled at 2 h. The concentration of Zotiraciclib was determined by LC/MS using a four-point calibration curve. Atenolol (Papp < 0.5 × 10–6 cm/s), propranolol (15 × 10–6 cm/s < Papp < 25 × 10–6 cm/s), Lucifer yellow (Papp > 0.4 × 10–6 cm/s), and digoxin (efflux ratio of >3) were used in the quality control of the monolayer batch. The integrity of the monolayer was determined by measuring the pre-experiment TEER (between 450 and 650 Ωcm2) and using Lucifer yellow (efflux of ≤0.5%). The efflux ratio was defined as the ratio of Papp,B→A to Papp,A→B.

Pharmacokinetics [1]
\nMale BALB/c mice (aged ∼10–12 weeks and weighing 17–22 g), male Beagle dogs (∼6–7 months of age, weighing 10–14 kg), and male Wistar rats (aged 6–8 weeks, weighing 239–249 g) were used in this study. All the animal studies were performed as per approved internal protocols for animal care and use. The oral doses for mice, dogs, and rats were 75, 10, and 10 mg/kg, respectively. The doses were administered by gavage as suspensions (0.5% methylcellulose and 0.1% Tween 80) to mice and rats, and as gelatin capsules (12 Torpac) to dogs. Following oral dosing serial blood samples were collected (cardiac puncture in mice, jugular vein in dogs, and superior vena cava in rats) at different time points (0–24 h) in tubes containing K3EDTA as anticoagulant, centrifuged, and plasma was separated and stored at −70 °C until analysis. Plasma samples were processed and analyzed by LC–MS/MS. Pharmacokinetic parameters were estimated by noncompartmental methods.

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\n\nMaterials and Methods for HCT-116 and Ramos Studies [1]
\n1 Mice/Husbandry [1]
\nFemale BALB/c nude mice (ARC, West Australia), 10–12 weeks of age, were fed with sterilized tap water (ad libitum water) and irradiated standard rodent diet consisting of 19% protein, 5% fat, and 5% fiber. Mice were housed in individual ventilated cages on a 12 h light cycle at 21–22 °C and 40–60% humidity. The use of animals is compliant with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. \n

\n2 Tumor Implantation [1]
\n2.1 For HCT-116 Study [1]
\nMice were implanted subcutaneously in the right flank with 5 × 106 cells of HCT-116 human colon carcinoma. Each tumor was monitored twice per week and subsequently daily as the neoplasms reached the desired size of approximately 100 mm3. At day 8, when the tumors attained a calculated tumor volume between 75 to 144 mm3, the animals were pair-matched and distributed randomly into various treatment groups (the mean tumor volume in each group was 105 mm3). Estimated tumor volume was calculated using the formula\n\nwhere w is the width and l is the length in mm of an HCT-116 carcinoma.\n

\n2.2 For Ramos Study [1]
\nMice were implanted subcutaneously in the right flank with 7 × 106 cells of Ramos cells (100 μL). The tumor size was monitored twice per week and subsequently daily as the neoplasms reached the desired size, approximately 200 mm3. On day 12, when the tumors attained a volume of between 75 and 405 mm3, the animal were pair-matched and distributed randomly into various treatment groups (the mean tumor volume in each group was 216 mm3). Estimated tumor volume was calculated using the formula\n\nwhere w is the width and l is the length in mm of a Ramos tumor.\n

\n3 Drug [1]
\nZotiraciclib hydrochloride was synthesized at S*BIO PTE LTD and dissolved in 0.5% methyl cellulose/0.1% Tween 80 (MC/Tween) for oral (po) dosing or in 10% dimethylacetamide (DMA) and 10% Cremophor (DMA/CRE) for ip dosing. Dosing solutions were prepared weekly in a feeding volume of 10 mL per kilogram body weight and stored at 4 °C.\n

\n\n4 Treatment Plan [1]
\n4.1 For HCT-116 Study [1]
\nOn day 1, HCT-116-bearing nude mice were pair-matched and placed into 3 groups of 9–10 animals each. Treatment with all drugs was initiated on day 1. The test compound, Zotiraciclib, was administered po at the following dosing schedules: 50 and 75 mg/kg × 3 times a week (Monday, Wednesday, and Friday; 3/w). There was a vehicle control group that received vehicle (MC/Tween) on the same schedule. The study was terminated on day 15.\n4.2 For Ramos Study [1]
\nZotiraciclib was administered once daily at doses of 75 mg/kg po q.d. 2d_on–5d_off or 15 mg/kg ip q.d. 5d_on–5d_off. There were two vehicle control groups that received either MC/Tween or DMA/CRE. The treatment groups were compared with the corresponding vehicle control group for the percentage of the tumor growth inhibition (% TGI). The treatment was terminated after 14 days of dosing.

Extensive ADME analysis of Zotiraciclib/26h [1]
In the Caco-2 bidirectional permeability assay, the permeability (Papp) of 26h in the apical-to-basal lateral direction (Papp,A→B) and the basal lateral-to-apical direction (Papp,B→A) were 28.0 × 10–6 and 27.4 × 10–6 cm/s, respectively. The efflux ratio (defined as the ratio of Papp,B→A to Papp,A→B) was less than 3 (1.0), indicating that 26h is not an efflux substrate of intestinal P-gp transporters, suggesting that it has a high intestinal absorption rate in humans (Table 6). In human liver microsomes (HLM), compound 26h exhibited good stability with a half-life of 45 minutes; moderate stability in dimeric liver microsomes (DLM) (t1/2 = 33 minutes); and rapid clearance in monomeric liver microsomes (MLM) (t1/2 = 12 minutes) and recombinant liver microsomes (RLM) (t1/2 = 11 minutes). At the highest tested concentration of 25 μM, compound 26h did not inhibit human CYP1A2, 3A4, 2C9, and 2C19 isoenzymes, but it did inhibit CYP2D6, with an IC50 of 0.95 μM, which was approximately the same as the plasma Cmax value observed at the maximum tolerated dose. Compound 26h showed high binding to human, mouse, and canine plasma proteins, with plasma protein binding (PPB) ranging from 99.4% to 99.9%.

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Pharmacokinetics of Zotelacib/26h in Mice[1]
The pharmacokinetic characteristics of Zotelacib/26h in mice are summarized in Table 7. Zotelacib/26h has a high systemic clearance and steady-state volume of distribution relative to hepatic blood flow, with a terminal half-life of approximately 5.0 hours. After a single oral dose of 75 mg/kg, Zotelacib/26h was rapidly absorbed (tmax = 0.5 hours), with mean Cmax and AUC of 1029 ng/mL and 2523 ng·h/mL, respectively, and a mean terminal half-life of 6.1 hours. Its oral bioavailability is 24%, which is acceptable. In mice, drug exposure at a dose of 75 mg/kg was significantly higher than that at enzyme inhibitory concentrations (CDK2 IC50 = 0.013 μM, JAK2 IC50 = 0.073 μM, FLT3 IC50 = 0.056 μM) and HCT-116 (IC50 = 0.079 μM) and HL-60 (IC50 = 0.059 μM) cell proliferation concentrations, consistent with 26-hour efficacy observed at similar doses in preclinical pharmacological models. Zotiraciclib/SB1317 (TG02) is a novel, potent small-molecule CDK/JAK2/FLT3 inhibitor. To evaluate the full potential of this candidate drug, we conducted pharmacokinetic and pharmacodynamic studies. SB1317 is soluble, highly permeable in Caco-2 cells, and binds >99% in mouse, canine, and human plasma. Compared with mice and rats, SB1317 is metabolically stable in human and canine liver microsomes. In vitro experiments showed that SB1317 is mainly metabolized by CYP3A4 and CYP1A2. Except for CYP2D6 (IC50=1 μM), SB1317 has no inhibitory effect on any major human CYP in vitro. In vitro experiments showed that SB1317 has no significant induction effect on CYP1A and CYP3A4 in human hepatocytes. The metabolic profile in preclinical animal liver microsomes is similar in nature to that in humans. Pharmacokinetic studies showed that SB1317 has moderate to high systemic clearance (relative to hepatic blood flow), large volume of distribution (>0.6 L/kg), and oral bioavailability of 24%, approximately 4%, and 37% in mice, rats, and dogs, respectively. SB1317 has extensive tissue distribution in mice. Its good ADME properties support its preclinical development as an oral drug candidate. [2]

[1]. Discovery of kinase spectrum selective macrocycle (16E)-14-methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a potent inhibitor of cyclin dependent kinases (CDKs), Janus kinase 2 (JAK2), and fms-like tyrosine kinase-3 (FLT3) for the treatment of cancer. J Med Chem . 2012 Jan 12;55(1):169-96.

Zotiraciclib is being investigated in the clinical trial NCT02942264 (a randomized phase II trial of Zotiraciclib (TG02) in combination with high-dose or metronidazole, followed by Zotiraciclib (TG02) in combination with temozolomide versus temozolomide alone in adult patients with recurrent anaplastic astrocytoma and glioblastoma). Zotiraciclib is a small molecule drug that has completed phase II clinical trials (covering all indications) and has 7 investigational indications. This article describes the design, synthesis, and structure-activity relationships of a unique series of small molecule macrocyclic compounds with spectro-selective inhibitory activity against CDK, JAK2, and FLT3 kinases. We evaluated the most promising lead compounds in vitro, examining their inhibitory activity against cancer cell proliferation, solubility, CYP450 inhibition, and microsomal stability. The screening cascade results showed that compound 26h was the preferred compound with IC50 values of 13, 73 and 56 nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies in preclinical animal models showed that 26h had good oral exposure. Oral efficacy was observed in xenograft studies of colon cancer (HCT-116) and lymphoma (Ramos), consistent with the observed PK/PD correlation. 26h (SB1317/TG02) entered the development stage in 2010 and is currently undergoing Phase I clinical trials for advanced leukemia and multiple myeloma. [1]

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We describe the discovery of a series of small molecule macrocyclic compounds that are potent inhibitors of CDK, JAK2 and FLT3 with previously unreported spectroscopic selectivity. Based on the conformational restriction hypothesis, we synthesized these macrocyclic compounds using the RCM strategy. Through functional biochemical analysis screening of the initial compounds against CDK2, JAK2 and FLT3 kinases, a preferred linker containing phenolic ether, trans double bond and allyl/benzyl N-methyl was finally selected. Structure-activity relationship studies and more extensive in vitro analyses, especially cell experiments, identified 26h, a small molecule kinase inhibitor with a unique kinase inhibition spectrum, as the preferred lead compound. Further evaluation showed that 26h has excellent pharmacokinetic properties and showed dose-dependent efficacy in a variety of mouse cancer models, including the HCT-116 colon cancer model and the Ramos lymphoma model. Based on its good pharmaceutical and pharmacological properties, 26h (SB1317/TG02) has entered the development stage and is currently undergoing phase I clinical trials in leukemia patients. [1]

C23H25CLN4O

408.92

408.171

C, 62.03; H, 5.88; Cl, 15.92; N, 12.58; O, 3.59

1321626-25-8

(E/Z)-Zotiraciclib;937270-47-8; 1354567-82-0 (HCl);1204918-73-9 (citrate);1204918-72-8;937270-47-8;(E/Z)-Zotiraciclib

141730164

White to light yellow solid

2

5

0

29

499

0

CN1C/C=C/CCOC2=CC=CC(=C2)C3=NC(=NC=C3)NC4=CC=CC(=C4)C1.Cl

YPVRSANPCYTWDF-SQQVDAMQSA-N

InChI=1S/C23H24N4O.ClH/c1-27-13-3-2-4-14-28-21-10-6-8-19(16-21)22-11-12-24-23(26-22)25-20-9-5-7-18(15-20)17-27;/h2-3,5-12,15-16H,4,13-14,17H2,1H3,(H,24,25,26);1H/b3-2+;

(16E)-14-methyl-20-oxa-5,7,14,27-tetrazatetracyclo[19.3.1.12,6.18,12]heptacosa-1(25),2(27),3,5,8,10,12(26),16,21,23-decaene;hydrochloride

(E/Z)-Zotiraciclib HCl; TG02 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~33.3 mg/mL (~81.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.11 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 (6.11 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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 (Please use freshly prepared in vivo formulations for optimal results.)