 Chemical Structure.jpg)
Zotiraciclib (formerly TG02, SB1317) is an orally bioavailable, brain penetrant and multi-kinase (CDK/JAK2/FLT3) inhibitor for the treatment of cancer (e.g. anaplastic astrocytoma and GBM, as well as DIPG, a rare pediatric cance). In Singapore, S*BIO Pte Ltd made the discovery of this small molecule macrocyclic compound. By inhibiting cyclin-dependent kinase 9 (CDK9), it depletes Myc and passes through the blood-brain barrier (BBB). It is one of several CDK inhibitors being studied for the treatment of cancer; other CDK inhibitors that target CDK9 for the treatment of acute myeloid leukemia (AML) include atuveciclib and alvocidib. Eighty percent of glioblastomas exhibit this feature, which is indicative of myc overexpression, a factor known to be involved in a variety of cancers. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have designated zotiraciclib as an orphan drug for the treatment of gliomas.
Physicochemical Properties
| Molecular Formula | C23H24N4O |
| Molecular Weight | 372.462864875793 |
| Exact Mass | 372.195 |
| Elemental Analysis | C, 74.17; H, 6.49; N, 15.04; O, 4.30 |
| CAS # | 1204918-72-8 |
| Related CAS # | 1354567-82-0 (HCl);1204918-73-9 (citrate);1204918-72-8;937270-47-8;(E/Z)-Zotiraciclib; 937270-47-8 |
| PubChem CID | 16739650 |
| Appearance | Typically exists as solid at room temperature |
| Density | 1.1±0.1 g/cm3 |
| Boiling Point | 577.1±60.0 °C at 760 mmHg |
| Flash Point | 302.8±32.9 °C |
| Vapour Pressure | 0.0±1.6 mmHg at 25°C |
| Index of Refraction | 1.577 |
| LogP | 4.76 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 0 |
| Heavy Atom Count | 28 |
| Complexity | 499 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | CN1CC=CCCOC2=CC=CC(=C2)C3=NC(=NC=C3)NC4=CC=CC(=C4)C1 |
| InChi Key | VXBAJLGYBMTJCY-NSCUHMNNSA-N |
| InChi Code | 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+ |
| Chemical Name | (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 |
| Synonyms | SB1317; TG-02; SB-1317; 937270-47-8; SB-1317 free base; TG02; 1204918-72-8; Zotiraciclib; TG02 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | CDK2; JAK2; FLT3 |
| ln Vitro |
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. 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. |
| ln Vivo |
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. 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] 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 Assay |
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. 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). 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). 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). 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 Assay |
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. 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. 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. |
| Animal Protocol |
Pharmacokinetics [1] Male 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. Materials and Methods for HCT-116 and Ramos Studies [1] 1 Mice/Husbandry [1] Female 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. 2 Tumor Implantation [1] 2.1 For HCT-116 Study [1] Mice 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 where w is the width and l is the length in mm of an HCT-116 carcinoma. 2.2 For Ramos Study [1] Mice 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 where w is the width and l is the length in mm of a Ramos tumor. 3 Drug [1] Zotiraciclib 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. 4 Treatment Plan [1] 4.1 For HCT-116 Study [1] On 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. 4.2 For Ramos Study [1] Zotiraciclib 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. |
| ADME/Pharmacokinetics |
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%. 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. 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] |
| References |
[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. [2]. Preclinical metabolism and pharmacokinetics of SB1317 (TG02), a potent CDK/JAK2/FLT3 inhibitor. Drug Metab Lett. 2012 Mar;6(1):33-42. |
| Additional Infomation |
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).
ZOTIRACICLIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 7 investigational indications. 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] 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] |
Solubility Data
| Solubility (In Vitro) | DMSO: ~50 mg/mL (~134.2 mM） |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.71 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.71 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6849 mL | 13.4243 mL | 26.8485 mL | |
| 5 mM | 0.5370 mL | 2.6849 mL | 5.3697 mL | |
| 10 mM | 0.2685 mL | 1.3424 mL | 2.6849 mL |