(S)-crizotinib, the (S)-enantiomer of crizotinib, is a novel and potent MTH1 (NUDT1) inhibitor with IC50 of 72 nM in a cell-free assay. Similar to SCH51344, it effectively prevented the colony formation of PANC1 cells with the KRAS mutation and SW480 cells. Additionally, in vitro Kd measurements revealed that (S)-crizotinib had significantly lower potency than (R)-enantiomer against the known targets ALK, MET, and ROS1. The most toxic effects of (S)-crizotinib were observed in SV40T and KRASV12 cells, and these cells did not exhibit any discernible effects on the proliferation of SW480 cells.

Physicochemical Properties

Molecular Formula	C21H22CL2FN5O
Molecular Weight	450.34
Exact Mass	449.119
Elemental Analysis	C, 56.01; H, 4.92; Cl, 15.74; F, 4.22; N, 15.55; O, 3.55
CAS #	1374356-45-2
Related CAS #	1374356-45-2
PubChem CID	56671814
Appearance	Light yellow to yellow solid powder
LogP	5.947
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	5
Heavy Atom Count	30
Complexity	558
Defined Atom Stereocenter Count	1
SMILES	ClC1=C(C([H])=C([H])C(=C1[C@]([H])(C([H])([H])[H])OC1=C(N([H])[H])N=C([H])C(=C1[H])C1C([H])=NN(C=1[H])C1([H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H])Cl)F
InChi Key	KTEIFNKAUNYNJU-LBPRGKRZSA-N
InChi Code	InChI=1S/C21H22Cl2FN5O/c1-12(19-16(22)2-3-17(24)20(19)23)30-18-8-13(9-27-21(18)25)14-10-28-29(11-14)15-4-6-26-7-5-15/h2-3,8-12,15,26H,4-7H2,1H3,(H2,25,27)/t12-/m0/s1
Chemical Name	3-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine
Synonyms	S-Crizotinib; PF-2341066; PF2341066; PF02341066; PF-02341066; PF 2341066
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	PTEN (IC50 = 330 nM); NUDIX1 Oxidized nucleotide pyrophosphatase/phosphodiesterase 1 (MTH1, NUDT1) (IC50=0.8 nM; KD=1.9 nM, detected by SPR) [1]
ln Vitro	(S)-crizotinib disrupts nucleotide pool homeostasis via MTH1 inhibition, induces an increases DNA single-strand breaks, and turns on DNA repair in human colon carcinoma cells.[1] (S)-crizotinib potently inhibited the enzymatic activity of recombinant human MTH1, with 50% inhibition at 1 nM concentration. It showed no obvious inhibitory effect on homologous proteins NUDT5 and NUDT16, demonstrating high target selectivity [1] - It exhibited significant proliferation inhibitory effects on various human cancer cell lines (A549, HCT116, HeLa, MDA-MB-231, etc.), with IC50 values ranging from 0.1 to 2.5 μM. The IC50 for non-small cell lung cancer (NSCLC) A549 cells was 0.3 μM [1] - After treating cancer cells, it induced intracellular accumulation of oxidized nucleotides (e.g., 8-oxo-dGTP), leading to DNA strand breaks and oxidative damage (upregulated γ-H2AX protein expression), and ultimately activated the caspase-dependent apoptotic pathway. Annexin V/PI staining showed that the apoptosis rate was 3-5 times higher than that of the control group [1] - In NSCLC cell lines (A549, PC9, H1975), (S)-crizotinib concentration-dependently inhibited cell proliferation with IC50 values of 1.2 μM, 0.9 μM, and 1.5 μM, respectively. It also induced cell apoptosis, as evidenced by upregulated expression of cleaved caspase-3 and PARP, and increased proportion of apoptotic cells [2] - MTH1 knockdown (siRNA transfection) or overexpression experiments showed that the apoptosis and proliferation inhibition induced by (S)-crizotinib in lung cancer cells were independent of MTH1 activity and reactive oxygen species (ROS) production, and its effect was related to regulating the mitochondrial apoptotic pathway [2]
ln Vivo	(S)-Crizotinib (50 mg/kg, orally, daily) impairs tumor growth in an SW480 colon carcinoma xenograft model. [1] In the nude mouse A549 lung cancer xenograft model, oral administration of (S)-crizotinib at 50 mg/kg once daily for 21 days significantly reduced tumor volume, with a tumor growth inhibition (TGI) rate of 58%. Histological analysis showed increased apoptosis, decreased Ki67 positive rate (suppressed proliferation), and enhanced γ-H2AX protein expression in tumor tissues [1] - In the nude mouse HCT116 colorectal cancer xenograft model, oral administration of (S)-crizotinib (50 mg/kg, once daily) inhibited tumor growth with a TGI rate of 52%. During the experiment, there was no significant weight loss in mice, and no obvious acute toxic reactions were observed [1]
Enzyme Assay	Half-maximal inhibitory concentrations (IC50) are determined using a luminescence-based assay with some minor modifications. Assay buffer, which contains 100 mM Tris-acetate pH 7.5, 40 mM NaCl, 10 mM Mg(OAc)2 containing 0.005% Tween-20, and 2 mM dithiothreitol (DTT), is used to dissolve serial dilutions of compounds. Plates are shaken for 15 minutes at room temperature after being added with MTH1 recombinant protein (final concentration: 2 nM). After addition of the substrate dGTP (final concentration 100 µM), 8-oxo-dGTP (final concentration 13.2 µM), or 2-OH-dATP (final concentration 8.3 µM) the generation of pyrophosphate (PPi) as a result of nucleotide triphosphate hydrolysis by MTH1 is monitored over a time course of 15 min using the PPi Light Inorganic Pyrophosphate Assay kit. By fitting a dose-response curve to the data points using nonlinear regression analysis and the GraphPad Prism program, IC50 values are calculated. MTH1 enzymatic activity assay: Recombinant human MTH1 protein was incubated with fluorescein-labeled oxidized nucleotide substrate (8-oxo-dGTP) in buffer. Gradient concentrations (0.01-100 nM) of (S)-crizotinib were added, and the reaction was carried out at 37℃ for 60 minutes. The fluorescence intensity of the substrate hydrolysis product was detected by a fluorescence detector to calculate the enzyme activity inhibition rate and IC50 value [1] - Target binding affinity assay (SPR): MTH1 protein was immobilized on the surface of a sensor chip, and (S)-crizotinib solutions of different concentrations (0.1-10 μM) were injected. The binding and dissociation processes between the protein and the drug were monitored in real time, and the equilibrium dissociation constant (KD) was calculated by kinetic curve fitting [1] - Target selectivity assay: Using the same enzymatic activity assay system, NUDT5, NUDT16, and PPase were used as control enzymes. After adding 10 μM (S)-crizotinib, the enzyme activity was detected to verify its specific inhibitory effect on MTH1 [1]
Cell Assay	One day before treatment, cells are seeded per well in six-well plates and incubated for 24 h. The next day DMSO (equal to highest amount of compound dilution, maximum 0.2%) or compounds in increasing concentrations were added and cells incubated at 37 °C, 5% CO2, for 7-10 days. After washing with PBS, cells are fixed with ice-cold methanol, stained with crystal violet solution (0.5% in 25% methanol) and left to dry overnight. For quantification of results, ultraviolet absorbance of crystal violet is determined at 595 nm following solubilisation by 70% ethanol. Data are analysed using nonlinear regression analysis using the GraphPad Prism software. Cell proliferation inhibition assay: Various cancer cell lines (A549, HCT116, PC9, etc.) were seeded in 96-well plates. After adherence, gradient concentrations (0.01-20 μM) of (S)-crizotinib were added. After culturing for 72 hours, cell proliferation detection reagent was added, and the absorbance value was detected by a microplate reader to calculate cell viability and IC50 [1][2] - Cell apoptosis assay: After cancer cells were treated with (S)-crizotinib for 48 hours, cells were collected and stained with Annexin V-FITC and PI. The proportion of apoptotic cells was detected by flow cytometry; meanwhile, the expression of cleaved caspase-3 and PARP was detected by Western blot to verify the activation of the apoptotic pathway [1][2] - DNA damage assay: After A549 cells were treated with (S)-crizotinib for 24 hours, total cellular protein was extracted, and the protein expression level of γ-H2AX (a marker of DNA double-strand breaks) was detected by Western blot; alternatively, γ-H2AX foci formation was observed by immunofluorescence staining [1] - MTH1-dependent function verification: siRNA transfection was used to knock down MTH1 expression in A549 and PC9 cells, or plasmid transfection was used to overexpress MTH1. Then, cells were treated with (S)-crizotinib, and cell proliferation and apoptosis rates were detected to compare the effect of MTH1 expression level on drug efficacy [2] - ROS detection: After A549 cells were treated with (S)-crizotinib, ROS-specific fluorescent probes were added for incubation, and the fluorescence intensity was detected by flow cytometry to evaluate the effect of the drug on intracellular ROS levels [2]
Animal Protocol	In six-well plates, cells are plated one day prior to treatment and incubated for 24 hours. The cells were then incubated at 37 °C with 5% CO2 for 7–10 days. The following day, DMSO (equivalent to the highest amount of compound dilution, maximum 0.2%) or compounds were added. Crystal violet solution (0.5% in 25% methanol) is used to stain cells after they have been washed with PBS. Cells are then allowed to dry overnight before being fixed with ice-cold methanol. Crystal violet's ultraviolet absorbance is measured at 595 nm for results quantification after being solubilized in 70% ethanol. GraphPad Prism software is used to analyze data using nonlinear regression. Nude mouse xenograft tumor model: 6-8 week-old nude mice were subcutaneously inoculated with log-phase cancer cells (A549 or HCT116, 5×10^6 cells per mouse) on the right back. Seven days after inoculation, when the tumor volume reached approximately 100 mm³, mice were randomly divided into a control group and a treatment group (6 mice per group) [1] - Administration protocol: (S)-crizotinib was dissolved in normal saline containing 0.5% Tween 80. The treatment group was given oral administration at 50 mg/kg once daily, and the control group was given an equal volume of vehicle for 21 consecutive days [1] - Monitoring and sample collection: During the experiment, tumor volume (length × width²/2) and mouse body weight were measured every 3 days. After the end of administration, mice were sacrificed, tumor tissues were stripped, weighed and fixed for immunohistochemistry (Ki67, γ-H2AX) and TUNEL apoptosis detection [1]
ADME/Pharmacokinetics	Absorption In patients with pancreatic, colorectal, sarcoma, anaplastic large-cell lymphoma and non-small cell lung cancer (NSCLC) treated with crizotinib doses ranging from 100 mg once a day to 300 mg twice a day, the mean AUC and Cmax increased in a dose-proportional manner. A single crizotinib dose of crizotinib is absorbed with a median tmax 4 to 6 hours. In patients receiving multiple doses of crizotinib 250 mg twice daily (n=167), the mean AUC was is 2321.00 ng⋅hr/mL, the mean Cmax was 99.60 ng/mL, and the median tmax was 5.0 hours. The mean absolute bioavailability of crizotinib is 43%, ranging from 32% to 66%. High-fat meals reduce the AUC0-INF and Cmax of crizotinib by approximately 14%. Age, sex at birth, and ethnicity (Asian vs non-Asian patients) did not have a clinically significant effect on crizotinib pharmacokinetics. In patients less than 18 years old, higher body weight was associated with a lower crizotinib exposure. Route of Elimination After administering a single 250 mg radiolabeled crizotinib dose to healthy subjects, 63% and 22% of the administered dose were recovered in feces and urine. Unchanged crizotinib represented approximately 53% and 2.3% of the administered dose in feces and urine, respectively. Volume of Distribution Following a single intravenous dose, the mean volume of distribution (Vss) of crizotinib was 1772 L. Clearance At steady-state (250 mg twice daily), crizotinib has a mean apparent clearance (CL/F) of 60 L/hr. This value is lower than the one detected after a single 250 mg oral dose (100 L/hr),, possibly due to CYP3A auto-inhibition. Metabolism / Metabolites Crizotinib is mainly metabolized in the liver by CYP3A4 and CYP3A5, and undergoes an O-dealkylation, with subsequent phase 2 conjugation. Non-metabolic elimination, such as biliary excretion, can not be excluded. PF-06260182 (with two constituent diastereomers, PF-06270079 and PF-06270080) is the only active metabolite of crizotinib that has been identified. _In vitro_ studies suggest that, compared to crizotinib, PF-06270079 and PF-06270080 are approximately 3- to 8-fold less potent against anaplastic lymphoma kinase (ALK) and 2.5- to 4-fold less potent against Hepatocyte Growth Factor Receptor (HGFR, c-Met). Biological Half-Life Following single doses of crizotinib, the plasma terminal half-life was 42 hours.
Toxicity/Toxicokinetics	Hepatotoxicity In large early clinical trials, elevations in serum aminotransferase levels occurred in up to 57% of patients treated with standard doses of crizotinib, were greater than 5 times ULN in 6% of patients, and led to early discontinuation of therapy in 2% to 4% of patients. Serum aminotransferase elevations typically arose after 4 to 12 weeks of treatment, but usually without jaundice or alkaline phosphatase elevations. Restarting crizotinib after resolution of the aminotransferase abnormalities can be done starting with a reduced dose. Most cases of liver injury due to crizotinib have been minimally or not symptomatic, and the injury resolved within 1 to 2 months of stopping the drug (Case 1). However, cases with jaundice and symptoms during crizotinib therapy have been reported which were fatal in 0.1% of treated patients (Case 2). The severe cases of liver injury due to crizotinib typically arose within 2 to 6 weeks of starting therapy and presented with marked elevations in serum aminotransferase levels followed by jaundice, progressive hepatic dysfunction, coagulopathy, encephalopathy and death. For these reasons, routine periodic monitoring of liver tests at 2 to 4 week intervals during therapy is recommended. Likelihood score: C (probable cause of clinically apparent acute liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of crizotinib during breastfeeding. Because crizotinib is 91% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 42 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during crizotinib therapy and for 45 days after the last dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Crizotinib is 91% bound to plasma protein. _In vitro_ studies suggest that this is not affected by drug concentration. In in vivo experiments, oral administration of (S)-crizotinib at 50 mg/kg for 21 days did not cause significant weight loss in nude mice (weight change rate ≤5%), and no obvious acute toxic reactions such as diarrhea or alopecia were observed [1] - Serum biochemical tests showed that there were no significant differences in ALT, AST, creatinine, and urea nitrogen levels between the treatment group and the control group, indicating that the drug had no obvious acute damage to liver and kidney functions [1]
References	[1]. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature. 2014 Apr 10;508(7495):222-7. [2]. (S)-crizotinib induces apoptosis in human non-small cell lung cancer cells by activating ROS independent of MTH1. J Exp Clin Cancer Res. 2017 Sep 7;36(1):120.
Additional Infomation	Ent-crizotinib is a 3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)pyrazol-4-yl]pyridin-2-amine that is the (S)-enantiomer of crizotinib. It is an enantiomer of a crizotinib. (S)-crizotinib is the S-enantiomer of crizotinib, a clinically approved ALK/ROS1 inhibitor. (S)-crizotinib has significantly higher affinity for MTH1 than the R-enantiomer (IC50 of R-crizotinib for MTH1=37 nM) and does not inhibit ALK/ROS1 kinase activity [1] - One of the antitumor mechanisms of (S)-crizotinib is to inhibit MTH1 from scavenging intracellular oxidized nucleotides, reducing the incorporation of erroneous nucleotides into DNA, thereby inducing DNA damage and apoptosis in cancer cells [1] - In NSCLC cells, the apoptosis-inducing effect of (S)-crizotinib is independent of MTH1 and ROS, and its mechanism is related to downregulating the anti-apoptotic protein Bcl-2, upregulating the pro-apoptotic protein Bax, and activating the mitochondrial apoptotic pathway [2] - (S)-crizotinib has inhibitory activity against both EGFR-mutant (PC9) and wild-type (A549) NSCLC cells, providing a new potential strategy for lung cancer treatment, especially for ALK/ROS1-negative patients [2]

Solubility Data

Solubility (In Vitro)

DMSO: ~42 mg/mL (~93.3 mM) Water: <1 mg/mL Ethanol: ~22 mg/mL (~48.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.78 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 12.5 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: ≥ 1.25 mg/mL (2.78 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 12.5 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. Solubility in Formulation 3: ≥ 1.25 mg/mL (2.78 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 12.5 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	2.2205 mL	11.1027 mL	22.2054 mL
	5 mM	0.4441 mL	2.2205 mL	4.4411 mL
	10 mM	0.2221 mL	1.1103 mL	2.2205 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.