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 Profiling of Zotiraciclib/26h [1]
In the Caco-2 bidirectional permeability assays, the permeability (Papp) of 26h in the apical to basolateral (Papp,A→B) direction and in the basolateral to apical (Papp,B→A) direction was 28.0 × 10–6 and 27.4 x10–6 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 was not a substrate for efflux by intestinal P-gp transporters, suggestive of high intestinal absorption in humans (Table 6). In human liver microsomes (HLM) 26h was found to be stable with a half-life of 45 min, was moderately stable in DLM (t1/2 = 33 min), and was quite rapidly cleared in MLM (t1/2 = 12 min) and in RLM (t1/2 = 11 min). Human CYP1A2, 3A4, 2C9, and 2C19 isoforms were not inhibited by 26h at the highest tested concentration of 25 μM, but the compound inhibited CYP2D6 with IC50 = 0.95 μM, approximately at the plasma Cmax observed at the maximum tolerated dose. Compound 26h was highly bound to plasma proteins in human, mouse, and dog plasma with PPB ranging between 99.4% to 99.9%.

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Pharmacokinetics of Zotiraciclib/26h in Mice [1]
The PK properties of 26h in mice are summarized in Table 7. 26h showed high systemic clearance relative to liver blood flow and high volume of distribution at steady state, with a terminal half-life of ∼5.0 h. It showed rapid absorption (tmax = 0.5 h) and a mean Cmax and AUC of 1029 ng/mL and 2523 ng·h/mL, respectively, with a mean terminal half-life of 6.1 h following a single oral dose of 75 mg/kg. It showed an acceptable oral bioavailability of 24%. The exposures achieved in mice at the 75 mg/kg dose far exceeded the enzyme inhibiton (CDK2 IC50 = 0.013 μM, JAK2 IC50 = 0.073 μM, and FLT3 IC50 = 0.056 μM) and cell proliferation concentrations in HCT-116 (IC50 = 0.079 μM) and HL-60 (IC50 = 0.059 μM), correlating with the observed efficacy of 26h in preclinical pharmacology models at similar doses.

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Zotiraciclib/SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. 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. 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.[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 under investigation in clinical trial NCT02942264 (Zotiraciclib (TG02) Plus Dose-dense or Metronomic Temozolomide Followed by Randomized Phase II Trial of Zotiraciclib (TG02) Plus Temozolomide Versus Temozolomide Alone in Adults With Recurrent Anaplastic Astrocytoma and Glioblastoma).

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ZOTIRACICLIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 7 investigational indications.

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Herein, we describe the design, synthesis, and SAR of a series of unique small molecule macrocycles that show spectrum selective kinase inhibition of CDKs, JAK2, and FLT3. The most promising leads were assessed in vitro for their inhibition of cancer cell proliferation, solubility, CYP450 inhibition, and microsomal stability. This screening cascade revealed 26 h as a preferred compound with target IC(50) of 13, 73, and 56 nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies of 26 h in preclinical species showed good oral exposures. Oral efficacy was observed in colon (HCT-116) and lymphoma (Ramos) xenograft studies, in line with the observed PK/PD correlation. 26h (SB1317/TG02) was progressed into development in 2010 and is currently undergoing phase 1 clinical trials in advanced leukemias and multiple myeloma.[1]

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We have described the discovery of a series of small molecule macrocycles as potent inhibitors of CDKs, JAK2, and FLT3, a spectrum selective profile not previously reported. Application of a hypothesis of conformational constraint generated macrocycles that were synthesized using a RCM strategy. Screening of initial compounds in functional biochemical assays against CDK2, JAK2, and FLT3 kinases allowed selection of a preferred linker moiety containing a phenolic ether, trans double bond, and allylic/benzylic N-methyl group. SAR and broader in vitro profiling, particularly cellular assays, identified 26h, a small molecule kinase inhibitor with a distinct kinase inhibitory spectrum, as the preferred lead candidate. Further evaluation revealed excellent pharmacokinetic properties of 26h and dose-dependent efficacy in mouse models of cancer including a HCT-116 model of colon cancer and a Ramos model of lymphoma. On the basis of its favorable pharmaceutical and pharmacological profile, 26h (SB1317/TG02) was advanced into development and is currently being evaluated in phase 1 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.)