CDK2 13 nM (IC50) JAK2 73 nM (IC50) FLT3 56 nM (IC50)

(E/Z)-Zotiraciclib (0-10 μM) demonstrates strong inhibition of FLT3, JAK2, and CDK2, with IC50 values of 13, 73, and 56 nM, respectively[1]. Cancer cell growth is inhibited by (E/Z)-Zotiraciclib (0–10 μM; 48 h)[1]. At an IC50 value of 0.13 μM, (E/Z)-Zotiraciclib (8-1000 nM; 24 h) potently suppresses the CDK2 biomarker pRb in HCT-116 cells and potently opposes pRb in MV4-11 cells[1].

(E/Z)-Zotiraciclib (50 and 75 mg/kg; po once daily for 3 weeks) suppresses the growth of tumors[1]. Zotiraciclib (E/Z) (15 and 75 mg/kg; po once daily for two days on and five days off; ip once daily for five days on and five days off) suppresses the growth of tumors in two ways[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 Assay[1]
Cell Types: HL-60, HCT-116, RAMOS, COLO205 and DU145 cell lines
Tested Concentrations: 0-10 μM
Incubation Duration: 48 h
Experimental Results: Inhibited proliferation of HL-60, HCT-116, RAMOS, COLO205 and DU145 cells with IC50s of 0.059, 0.079, 0.033, 0.072 and 0.14 μM, respectively.

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse: with HCT-116 colon cancer cells xenografts[1]
Doses: 50 and 75 mg/kg
Route of Administration: po (oral gavage); 50 and 75 mg/kg one time/day for 3 weeks
Experimental Results: Dramatically inhibited the growth of tumors with a mean TGI of 82 %.

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Animal/Disease Models: Male balb/c (Bagg ALBino) mouse: with lymphoma Ramos cells xenografts[1]
Doses: 15 and 75 mg/kg
Route of Administration: po (oral gavage) and intraperitoneal (ip)injection; 75 mg/kg one time/day 2 days on and 5 days off (po) and 15 mg/kg one time/day 5 days on 5 days off (ip)
Experimental Results: Dramatically inhibited the growth of tumors with mean TGIs of 42% and 63% for the oral and ip delivery methods, respectively.

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 kina.

[2]. Preclinical metabolism and pharmacokinetics of SB1317 (TG02), a potent CDK/JAK2/FLT3 inhibitor. Drug Metab Lett. 2012 Mar;6(1):33-42.

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]

C23H24N4O

372.46

372.195

C, 74.17; H, 6.49; N, 15.04; O, 4.30

937270-47-8

(E/Z)-Zotiraciclib hydrochloride;1321626-25-8;Zotiraciclib;1204918-72-8; 1354567-82-0 (HCl);1204918-73-9 (citrate); 937270-47-8;(E/Z)-Zotiraciclib; 937270-47-8

16739650

Off-white to light yellow solid powder

1.1±0.1 g/cm3

577.1±60.0 °C at 760 mmHg

302.8±32.9 °C

0.0±1.6 mmHg at 25°C

1.577

4.76

1

5

0

28

499

0

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

VXBAJLGYBMTJCY-NSCUHMNNSA-N

InChI=1S/C23H24N4O/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)/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

(E/Z)-Zotiraciclib;(E/Z)-TG02; (E/Z)-SB1317; (E/Z)-TG-02; (E/Z)-SB-1317;

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 : 26.5 mg/mL (71.15 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.58 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 20.8 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: ≥ 2.08 mg/mL (5.58 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 20.8 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: ≥ 2.08 mg/mL (5.58 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 20.8 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.)